key: cord-331827-amg309uz authors: keske, şiran; gümüş, terman; köymen, tamer; sandıkçı, sunay; tabak, levet; ergönül, önder title: human metapneumovirus infection: diagnostic impact of radiologic imaging date: 2019-02-01 journal: j med virol doi: 10.1002/jmv.25402 sha: doc_id: 331827 cord_uid: amg309uz background: human metapneumovirus (hmpv) is a recently detected virus, which can cause mild to severe respiratory tract infections. through this study, we aimed to detail the outcomes of hmpv infections. materials/methods: between january 2012 and november 2017, patients who had hmpv detected in nasopharyngeal or bronchoalveolar lavage by molecular respiratory pathogen tests were evaluated. the food and drug administration cleared multiplexed‐polymerase chain reaction system (idaho technology, salt lake city, ut) was used for diagnosis. chest radiography (cr) and computed tomography (ct) were evaluated by an expert radiologist. results: in total 100 patients were included, the mean age was 22.9 (0‐87) years, and 50% were male. the hospitalization rate was 52%. lower respiratory system infection (lrti) was diagnosed in 44 patients with clinical findings, and in 31 patients out of 44 the radiological findings supported the diagnosis. the lrti rate was significantly higher in adults than children (66.7%‐32.8%; p = 0.001). in cr, peribronchovascular infiltration (pi) was the most common feature seen in 14 out of 18 patients and was generally bilateral (13 out of 18 patients). in ct imaging, ground‐glass opacity was the most common finding seen in 11 out of 16 patients and nodular consolidation in five patients. ribavirin was given to four patients, three of whom were severe and required respiratory support. none of the patients died of hmpv infection. conclusions: the ground‐glass opacity in ct was similar to other respiratory virus infections, and pi in cr was very common and typical; however, nodular consolidation that may mimic bacterial infection was seen in one‐fourth of ct. human metapneumovirus (hmpv) was defined in 2001. it causes mild to severe respiratory system infections. [1] [2] [3] due to the increasing availability of molecular based tests, reporting of hmpv infection is increasing all over the world. [4] [5] [6] the hmpv has been reported to be one of the most commonly detected viruses that cause influenza-like illness 7 and community-acquired pneumonia 6 in both adults and children. radiologic findings of hmpv infection have not been reported to be different from other viral pathogens in studies with high sample sizes; 8 however, it has been reported that studies including radiologic findings of hmpv infections were commonly done among immunocompromised hosts. 9 hmpv infection may cause fatality in patients with hematologic malignancy. 3, 10 the infection presents with a wide variation of clinical conditions from asymptomatic to fatal. we aimed to describe the clinical course of the disease by detailing the clinical and radiological findings and the outcome. we included patients from inpatient and outpatient departments who had hmpv in nasopharyngeal or bronchoalveolar lavage, diagnosed using the molecular respiratory pathogen test. the study was conducted in a 265-bed private hospital in istanbul between january 2012 and november 2017. this was a retrospective study performed by reviewing the patient charts. the clinical features of the patients, demographic characteristics, chronic diseases including malignant disorders, diabetes mellitus, chronic kidney and liver diseases, and detailed information about antibiotic and antiviral drugs were collected by chart review. complete blood count, c-reactive protein, and liver and kidney function tests were studied among all patients. bacterial cultures (throat, sputum, blood, and urine) were obtained, and procalcitonin (pct) was studied in patients with suspicion of bacterial infection and/or in critical patients. chlamydia pneumoniae and mycoplasma pneumoniae were included in multiplex polymerase chain reaction (pcr) tests, and urine legionella antigen test was performed in case of suspicion. the food and drug administration cleared multiplexed-respiratory pcr system biofirefilmarray (idaho technology), which detects 17 viral pathogens including hmpv, and three bacterial species were used for the molecular detection of hmpv. chest radiography (cr) and computed tomography (ct) were done if lower respiratory system infection (lrsi) was suspected. the radiological imaging of patients was evaluated by a single dedicated radiologist. the upper respiratory system infection (ursi) was defined based on the modified centor score that was developed for acute tonsillitis/ pharyngitis 11 and deduced from the recommendation of the centers for disease control and prevention and infectious diseases society of america. 12, 13 the lrsi is defined as infectious inflammation of the lower respiratory tract and refers to acute pneumonia, acute bronchitis, and acute bronchiolitis. clinical lrsi is a syndrome characterized by symptoms consistent with respiratory tract infection (such as fever, cough, sputum, and dyspnea) and lung auscultation findings (crackles, rhonchus, and decreased lung sounds). 14 radiological lrsi is defined as radiological findings of the lung, including consolidation, cavitation, peribronchial ground-glass opacity (ggo), airspace consolidation, and small nodules. in the statistical analysis, the t test for continuous variables and the χ 2 test for the comparison of categorical variables were used. in the analysis, stata 11 (statacorp, college station, tx) was used, and p < 0.05 was set as significant. the institutional review board of koç university approved the study. our study included 100 patients ( figure 1 ). the mean age of the patients was 22.9 (0-87) years, and 50% of them were male (table 1) . two-third (67%) of the patients were under 18 years old. in 14 patients, more than one virus was detected. concomitant agents were as follows: seven rhino/enterovirus, five influenza, two coronavirus, and one respiratory syncytial virus. the cases were most commonly seen between november and june, and 21% of the cases were seen in december ( the antibiotics were given in 28% of the patients, despite detection of virus. the rate of antibiotic prescription was higher among lrti when compared with ursi (54%-21%; p = 0.003). the third generation cephalosporins, β-lactam/β-lactamase inhibitor combination, and quinolones were the most commonly used antibiotics (17%, 11%, and 10%, respectively). finally, ribavirin was given to four patients, three of whom were severe and required respiratory support. none of the patients died of hmpv infection. our study demonstrates the radiologic findings of hmpv infections in patients with lrsi (figures 3 and 4) . one of the most interesting radiologic findings was the presence of nodular consolidation in ct, which is generally expected to be seen in bacterial infection. the pi in cr and ggo in ct are the most common radiologic findings, which are frequently seen in viral pneumonia. in a recent review for radiologic imaging of viral agents that may cause pneumonia, the general radiologic findings of hmpv infections were followed as bilateral centrilobular nodules, ggo, and multilobar infiltrations; however, there was no information about nodular consolidation. in addition, they noted that the radiologic findings of hmpv infections were most commonly reported in patients with hematologic malignancy, but there were limited data on immunocompetent patients. 9 the time from onset of symptoms to hospital admission may affect radiologic findings. in our study, among patients with normal cr, which were lrsi-based on their clinical findings, time from onset of symptoms to hospital admission was lower (2.5 to 3.6 days; p = 0.05 antibiotic prescription rate was still high, which was 54% among patients with lrsi. there is no confirmed antiviral treatment in hmpv infection. ribavirin use in hmpv infections is limited to case reports in immunocompromised patients. 17 in a recent study, ribavirin use was not associated with a better clinical outcome. they reported that the duration of viral shedding was shorter in patients using ribavirin. 10 a recent review also concluded that there is no evidence to recommend ribavirin in immunocompromised patients. 19 the fourth european conference on infections in leukemia (ecil-4) guideline also commented that general recommendations for treatment of hmpv infection could not be made with current evidence. 20 in our study, ribavirin was used in four cases, three of whom were severe and required respiratory support and none of them died. in conclusion, ggo in ct was similar to other respiratory virus infections and the pi in cr was very common and typical; however, nodular consolidation, which may mimic bacterial infection, was a newly discovered human pneumovirus isolated from young children with respiratory tract disease the role of human metapneumovirus in the critically ill adult patient burden of human metapneumovirus infections in patients with cancer: risk factors and outcomes high seroprevalence of human metapneumovirus among young children in israel seroprevalence of human metapneumovirus in japan community-acquired pneumonia requiring hospitalization among u.s. adults the rapid diagnosis of viral respiratory tract infections and its impact on antimicrobial stewardship programs chest radiographic features of human metapneumovirus infection in pediatric patients radiographic and ct features of viral pneumonia community-acquired respiratory paramyxovirus infection after allogeneic hematopoietic cell transplantation: a single-center experience diagnosis and treatment of streptococcal pharyngitis idsa clinical practice guideline for acute bacterial rhinosinusitis in children and adults clinical practice guideline for the diagnosis and management of group a streptococcal pharyngitis: 2012 update by the infectious diseases society of america towards clinical definitions of lower respiratory tract infection (lrti) for research and primary care practice in europe: an international consensus study clinical characterization of human metapneumovirus infection among patients with cancer human metapneumovirus (hmpv) associated pulmonary infections in immunocompromised adultsinitial ct findings, disease course and comparison to respiratorysyncytial-virus (rsv) induced pulmonary infections human metapneumovirus pneumonia in patients with hematological malignancies clinical features of human metapneumovirus pneumonia in non-immunocompromised patients: an investigation of three long-term care facility outbreaks human metapneumovirus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus human metapneumovirus infection: diagnostic impact of radiologic imaging key: cord-324148-bllyruh8 authors: loubet, paul; mathieu, pauline; lenzi, nezha; galtier, florence; lainé, fabrice; lesieur, zineb; vanhems, philippe; duval, xavier; postil, deborah; amour, sélilah; rogez, sylvie; lagathu, gisèle; l'honneur, anne-sophie; foulongne, vincent; houhou, nadhira; lina, bruno; carrat, fabrice; launay, odile title: characteristics of human metapneumovirus infection in adults hospitalized for community-acquired influenza-like illness in france, 2012-2018: a retrospective observational study date: 2020-04-10 journal: clin microbiol infect doi: 10.1016/j.cmi.2020.04.005 sha: doc_id: 324148 cord_uid: bllyruh8 objectives: to describe the prevalence, clinical features and complications of human metapneumovirus (hmpv) infections in a population of adults hospitalized with influenza-like illness (ili). methods: this was a retrospective, observational, multicenter cohort study using prospectively collected data from adult patients hospitalized during influenza virus circulation, for at least 24h, for community-acquired ili (with symptom onset <7 days). data were collected from five french teaching hospitals over six consecutive winters (2012-2018). respiratory viruses were identified by multiplex rt-pcr on nasopharyngeal specimens. hmpv+ patients were compared to hmpv– patients, influenza+ and respiratory syncytial virus (rsv)+ patients using multivariate logistic regressions. primary outcome was the prevalence of hmpv in patients hospitalized for ili. results: among the 3148 patients included (1449 (46%) women, 1988 (63%) aged 65 and over; 2508 (80%) with chronic disease), at least one respiratory virus was detected in 1604 (51%, 95%ci [49-53]), including 100 cases of hmpv (100/3148, 3% 95%ci [3, 4]), of which 10 (10%) were viral co-infection. in the hmpv+ patients, mean length of stay was 7 days, 62% (56/90) developed a complication, 21% (14/68) were admitted to intensive care unit and 4% (4/90) died during hospitalization. in comparison with influenza+ patients, hmpv+ patients were more frequently > 65 years old (aor=3.3, 95%ci[1.9-6.3]) and presented more acute heart failure during hospitalization (aor=1.8, 95%ci[1.0-2.9]). compared to rsv+ patients, hmpv+ patients had less cancer (aor=0.4, 95%ci[0.2-0.9]) and were less likely to smoke (aor=0.5, 95%ci[0.2-0.9]) but had similar outcomes especially high rate of respiratory and cardiovascular complications. conclusions: adult hmpv infections mainly affect the elderly and patients with chronic conditions and are responsible for frequent cardiac and pulmonary complications similar to those of rsv infections. at-risk populations would benefit from the development of antivirals and vaccines targeting hmpv. during winter, community-acquired influenza-like illness (ili), mostly caused by respiratory 74 viruses, is very common. the most frequent viruses seen in primary care are influenza 75 viruses a/b, rhinovirus, coronavirus, respiratory syncytial virus (rsv) and human 76 metapneumovirus (hmpv) [1, 2] . in the hospital setting, adults with ili are commonly tested 77 only for influenza, resulting in limited data concerning other respiratory viruses. the use of 78 multiplex rt-pcr allows identification of multiple viruses simultaneously but remains a 79 second-line test in non-immunocompromised patients in emergency departments because of 80 its cost and the limited therapeutic options [3] . 81 human mpv, discovered in 2001, is phylogenetically similar to rsv and has been frequently 82 found associated with respiratory tract illnesses [1, 4, 5] . its circulation occurs with a seasonal 83 distribution from january to march in the northern hemisphere, often overlapping or following 84 rsv infection season [6, 7] . human mpv is a major pediatric respiratory pathogen at least one of the following respiratory symptoms: cough, sore throat or dyspnea. patient 105 with contra-indication for influenza immunization, those who had previously tested positive 106 for influenza virus in the same season and those without french social security affiliation 107 were excluded. each participant was interviewed, and nasopharyngeal samples were 108 obtained at enrolment to screen for influenza and other respiratory viruses. 109 in the present study, we included all the patients from the first six fluvac seasons 110 (2012/13, 2013/14, 2014/15, 2015/16, 2016/17, 2017/18) patients with multiple viral infections were excluded from the analyses. univariate analysis 143 was used to asses risk factors for the detection of hmpv infection, influenza infection, rsv 144 infection and acute heart failure. we performed two multivariate analyses using a backward 145 stepwise logistic regression model using hmpv test result (positive/negative) and acute heart 146 failure (yes/no) as the dependent variable in the first and second model respectively. 147 covariates with a p-value <0.2 in univariate analysis were tested in the multivariate model. 148 results from regression models are expressed as crude odds ratios (or) and adjusted ors 149 (figure 1 and table s1 ). ten of the 100 hmpv infections (10%) were coand 41% (37/90) hospitalized in the 12 months preceding the study (table 1) . 174 the median time from symptom onset to admission was similar between hmpv+ and hmpv-175 patients (2 days (iqr, 1-3)) as well as the main symptoms at inclusion except for 176 weakness/malaise that was less frequent in hmpv+ patients (13/90 (14%) vs 27%, p<.007). 177 there was no difference between hmpv+ and hmpv-groups in terms of median length of 178 stay, number of complications during hospitalization, intensive care unit (icu) admission and 179 death. however, hmpv+ patients were more likely to have an acute heart failure during 180 hospitalization (25% (22/89) vs 14% (377/2722), p<.004). 181 there was no difference in sociodemographic characteristics, clinical presentation or 182 outcomes between hmpv and viral coinfection and patients with hmpv infections alone. 183 in the multivariate analysis, when comparing hmpv+ patients to all hmpv-patients, age > 65 184 years (aor 95% ci 3.3 [1.9;6.1], p<0.001) was significantly associated with hmpv detection. 185 in contrast, the sudden onset of symptoms, defined as the occurrence of malaise/weakness, 186 was associated with the absence of hmpv infection (aor 95% ci 0.4 [0.2;0.8], p=0.008) 187 (table 1) . 188 after adjustment for chronic heart disease, age, gender, smoking status and influenza 189 vaccination, hmpv infection was significantly associated with occurrence of acute heart 190 failure during hospitalization (aor 95% ci 1.8 [1.1;3.0], p=0.02). 191 in univariate analysis, in comparison to influenza+ patients, hmpv+ patients were older 193 in our post-hoc analysis of 3148 hospitalized adult patients with community-acquired ili, 211 hmpv was found in 3% of the samples. these patients were older, had chronic conditions, 212 frequent respiratory and cardiac chronic diseases, and frequently presented complications. 213 this prevalence is consistent with several studies that found hmpv in 3 to 6% of adult 214 patients with lower respiratory tract infection in primary care [1, [14] [15] [16] and in 6% of patients 215 hospitalized for acute respiratory infection (ari) [17] . this frequency may vary according to 216 the inclusion criteria, especially temperature cut-off, as hmpv infection frequently causes 217 non-febrile illness [5] . 218 our hospitalized hmpv+ adults were mostly older and/or high risk patients as previously 219 described in the literature [5, 10, [18] [19] [20] . complications were frequent (62%), as well as icu 220 admission (17%) and death (4%). these rates were similar to those of the 91 hospitalized 221 patients with hmpv from the walsh et al. study in 2008 in the usa [10], but lower than those 222 of the 128 critically ill adults with hmpv infection (31% required icu admission and 8% died) 223 from the hasvold et al. study in 2016 [20] . 224 the majority of hmpv+ patients presented several respiratory signs (cough, dyspnea), 225 whereas sudden onset of symptoms was associated with the absence of hmpv infection. 226 there were differences in clinical presentation between hmpv+ patients and influenza+ 227 patients (less frequent constitutional symptoms (headache, weakness, myalgia) but more 228 dyspnea) but not between hmpv+ patients and rsv+ patients. these points emphasize the 229 difficulty of distinguishing respiratory viruses based on clinical signs alone and question the 230 relevance of the current ili definition to detect hmpv infection. 231 interestingly, we also found that hmpv+ patients were older and presented more chronic 232 cardiac conditions and acute heart failure during the hospitalization than influenza+ patients. 233 although, influenza [21] and rsv [22] are known to worsen heart failure, no study has 234 specifically assessed hmpv [21, 22] aetiology of lower respiratory tract infection in adults in primary care: a prospective study in 273 11 european countries baseline 275 characteristics and clinical symptoms related to respiratory viruses identified among patients 276 presenting with influenza-like illness in primary care practice guidelines by the infectious diseases society of america treatment, chemoprophylaxis, and institutional outbreak management of 282 seasonal influenzaa van den hoogen bg, osterhaus ad, fouchier ra. clinical impact and diagnosis of 310 human metapneumovirus infection respiratory virus infections in hospitalized children and adults in lao pdr. influenza 313 other respir clinical features, epidemiology, 315 and climatic impact of genotype-specific human metapneumovirus infections: long-term 316 surveillance of hospitalized patients in south korea human 320 metapneumovirus in patients hospitalized with acute respiratory infections: a meta-analysis human metapneumovirus infections on the 323 icu: a report of three cases exacerbation of chronic obstructive pulmonary disease the role of human 328 metapneumovirus in the critically ill adult patient seasonal trends 330 of heart failure hospitalizations in the united states: a national perspective from with cardiovascular disease in adults mortality in adults hospitalized for respiratory syncytial virus infections clinical 340 characteristics and outcome of respiratory syncytial virus infection among adults hospitalized 341 with influenza-like illness in france onset of symptoms > 7 days: n=124no consent obtained n=6) key: cord-317661-v93mde6l authors: vaid, shashank; cakan, caglar; bhandari, mohit title: using machine learning to estimate unobserved covid-19 infections in north america date: 2020-05-07 journal: j bone joint surg am doi: 10.2106/jbjs.20.00715 sha: doc_id: 317661 cord_uid: v93mde6l the detection of coronavirus disease 2019 (covid-19) cases remains a huge challenge. as of april 22, 2020, the covid-19 pandemic continues to take its toll, with >2.6 million confirmed infections and >183,000 deaths. dire projections are surfacing almost every day, and policymakers worldwide are using projections for critical decisions. given this background, we modeled unobserved infections to examine the extent to which we might be grossly underestimating covid-19 infections in north america. methods: we developed a machine-learning model to uncover hidden patterns based on reported cases and to predict potential infections. first, our model relied on dimensionality reduction to identify parameters that were key to uncovering hidden patterns. next, our predictive analysis used an unbiased hierarchical bayesian estimator approach to infer past infections from current fatalities. results: our analysis indicates that, when we assumed a 13-day lag time from infection to death, the united states, as of april 22, 2020, likely had at least 1.3 million undetected infections. with a longer lag time—for example, 23 days—there could have been at least 1.7 million undetected infections. given these assumptions, the number of undetected infections in canada could have ranged from 60,000 to 80,000. duarte’s elegant unbiased estimator approach suggested that, as of april 22, 2020, the united states had up to >1.6 million undetected infections and canada had at least 60,000 to 86,000 undetected infections. however, the johns hopkins university center for systems science and engineering data feed on april 22, 2020, reported only 840,476 and 41,650 confirmed cases for the united states and canada, respectively. conclusions: we have identified 2 key findings: (1) as of april 22, 2020, the united states may have had 1.5 to 2.029 times the number of reported infections and canada may have had 1.44 to 2.06 times the number of reported infections and (2) even if we assume that the fatality and growth rates in the unobservable population (undetected infections) are similar to those in the observable population (confirmed infections), the number of undetected infections may be within ranges similar to those described above. in summary, 2 different approaches indicated similar ranges of undetected infections in north america. level of evidence: prognostic level v. see instructions for authors for a complete description of levels of evidence. we have identified 2 key findings: (1) as of april 22, 2020, the united states may have had 1.5 to 2.029 times the number of reported infections and canada may have had 1.44 to 2.06 times the number of reported infections and (2) even if we assume that the fatality and growth rates in the unobservable population (undetected infections) are similar to those in the observable population (confirmed infections), the number of undetected infections may be within ranges similar to those described above. in summary, 2 different approaches indicated similar ranges of undetected infections in north america. level of evidence: prognostic level v. see instructions for authors for a complete description of levels of evidence. t he detection of coronavirus disease 2019 (covid-19) cases remains a huge challenge 1 . as of april 22, 2020, the covid-19 pandemic continues to take its toll, with close to 2.6 million confirmed infections and >183,000 deaths 2 . dire projections are surfacing almost every day, and policymakers worldwide are using projections for critical decisions. while social distancing now appears to be globally accepted, approaches vary substantially. whereas hong kong and singapore are experimenting with "suppress and lift" measures 3 , india has been estimated to be at the top of the lockdown stringency index 4 . intelligence on the number of infections and projected courses has never been more urgent as the world economy heads toward a contraction of 3% in 2020 and the world faces the worst recession since the great depression 1 . while organizations such as the world health organization (who) are establishing covid-19-detection protocols 5 , leading scientific opinion and commentaries appear to be highlighting the possibility of detection bias 6 . there also appears to be a grudging acceptance that identifying and quantifying such bias may depend largely on the number of reported cases. the challenge with reported cases is that they are dependent on the extent of testing. as of april 22 2020, the numbers of tests per 1 million population varied greatly across some of the key jurisdictions most impacted by the pandemic, including the u.s. (13,067), u.k. (8, 248) , italy (25,028), france (7,103), spain (19,896), canada (16,220), and india (335) 2 . however, the extent of testing is not just a policy matter but also is dependent on the availability of scarce public and private resources. under such circumstances, it may not be prudent for policymakers to rely only on "observable" data (i.e., confirmed covid-19 cases) as such measures are likely to under-report the extent of the problem. for example, by publicly reporting 47,676 deaths against only 840,476 cases, the united states may not be accounting for the influence of lower levels of testing (13,067 tests per million) relative to other countries. by not proactively acknowledging data that are unobservable-i.e., expected infections that have not been captured by whoestablished covid-19-detection protocols-policymakers could be grossly underestimating the true number of infections in the population. furthermore, if case fatality rates (that is, the ratio of deaths to reported cases; e.g., ;5.7% for the u.s.) do not factor in unobservable infections, models may overestimate the risk of death 7 . given this background, we modeled unobserved infections to examine the extent to which we might be grossly underestimating covid-19 infections in north america. w e developed a machine-learning model to uncover hidden patterns based on reported cases and to predict potential infections. first, our model relied on dimensionality reduction to identify parameters that were key to uncovering fig. 1-b) ; the x axis is in days. covid-19 data are also normalized for better visualization. a visual inspection indicates the temporal delay between the waves. 1, 2020 hidden patterns. next, our predictive analysis used an unbiased estimator approach to infer past infections from current fatalities. we referenced the initial rapid research and contributions by pueyo, duarte, and others [6] [7] [8] [9] [10] . broadly speaking, our analysis compared the numbers of confirmed cases, deaths, and estimated infections across north america (u.s. and canada). our data were made available thanks to the generosity of the johns hopkins university center for systems science and engineering (jhu csse), the esri living atlas team, and the johns hopkins university applied physics laboratory (jhu apl). the data were pulled from the covid-19 data repository by the jhu csse every hour. we started with exploratory data analysis. we aggregated the u.s. and canada since they were split by states and provinces. while we focused on north america, we also included countries with a minimum of 10 cases. the columns in our timeseries dataset initially included country, state, and number of deaths and confirmed cases. first, we filtered data to include at least 100 cases. as the "deaths" and "confirmed cases" data were separate in the jhu csse database, we concatenated both time series. after dropping duplicated columns, we created new columns for (1) dirty ratio = confirmed cases/(deaths 1 1) and (2) fatality = deaths/confirmed cases. we also created new columns for new deaths and new confirmed cases. next, we created a smaller dataset with at least cumulative 50 cases. we also grouped data for states (u.s.) and provinces (canada). our final dataset included the following columns: index, country, date, confirmed cases, deaths, dirty ratio, fatality, new deaths, new confirmed cases, and days since case 50. we assumed that the average time from infection to death could be 8 days (to account for older patients), 13 days, or 23 days (to account for younger patients); however, we based our results on 13 days 11 . the time from infection to death was taken as being equal to the incubation period plus the time from symptoms to death. this assumption was used to estimate the timing of the infections that led to the observed deaths. we used the fatality rate per country (total deaths/total cases) to estimate the number of infections that were responsible for the observed deaths. next, we extrapolated available information with use of duarte's elegant unbiased hierarchical bayesian estimator approach 9,10 . we inferred past infections from current daily deaths as the average fatality rate was approximated from confirmed cases 9,10,12 . in doing so, we assumed that (1) the case fatality rate (that is, the fatality rate among patients with confirmed cases) may be a good proxy for the fatality rate of the infected population, (2) the growth rate of the infected population may serve as an unbiased estimate of confirmed cases, and (3) on average, the interval between the initial symptoms and death is 8 to 23 days. o ur analysis indicated that, with a 13-day lag time from infection to death, the u.s. likely had at least 1.3 million undetected infections as of april 22, 2020 (represented by dashed bars in figs. 1-a and 1-b; covid-19 data are also normalized for better visualization). under a longer lag time of 23 days, there could have been at least 1.7 million undetected infections. given these same assumptions, the number of undetected infections in canada could have ranged from 60,000 to 80,000. we used duarte's elegant unbiased hierarchical bayesian estimator approach (which uses the case fatality rate to infer the fatality rate of the unobservable population) as a robustness check on these results. using that approach, we found that, as of april 22, 2020, the united states had up to >1.6 million undetected infections and canada had at least 60,000 to 86,000 undetected infections (figs. 2-a and 2-b) . however, the jhu csse data feed on april 22, 2020, reported only 840,476 and 41,650 confirmed cases for the united states and canada, respectively. o ur research explored the role of unobservable infections in covid-19 detection bias. we identified 2 key findings: (1) as of april 22, 2020, the united states may have had 1.5 to 2.02 times the number of reported infections and canada may have had 1.44 to 2.06 times the number of reported infections and (2) even if we assume that the fatality and growth rates in the unobservable population (undetected infections) are similar to those in the observable population (confirmed infections), the number of undetected infections may be within ranges similar to those described above. in summary, 2 different approaches indicated similar ranges of undetected infections in north america. our analysis has unique strengths. our multidimensional analysis of unobservable infections in the context of the covid-19 pandemic has helped us to uncover trends that are likely to have an impact on scientific research in 3 respects. first, we estimated the distribution of successive waves of infections (dashed bars), detections (black bars), and deaths (red bars) for both the u.s. and canada (figs. 1-a and 1-b) . with the time from infection to death being defined as the incubation period plus the time from symptoms to death, we estimated the timing of the infections that led to the observed deaths. our results indicated that, as of april 22, 2020, the u.s. may have had at least 1.3 million undetected infections and canada may have had at least 60,000 undetected infections. next, we supported these results through a robustness check that used the case fatality rate to infer the fatality rate of the infected population. assuming that the growth rate of the infected population could be an unbiased estimate of confirmed cases, we found that the numbers of undetected infections may be quite high in the u.s. (>1.6 million) and canada (60,000 to 86,000) (figs. 2-a and 2-b) . we also extended the mandate of this research to understand why a set of western countries accounted for a large number of fatalities despite high testing. as of april 9, 2020, the following countries had relatively higher testing per million population: u.s. (13,067), u.k. (8, 248) , italy (25,028), france (7,103), spain (19,896), and canada (16,220). yet, together they accounted for close to 70% of all fatalities. one reason for this finding could be that testing was late as it followed cumulative deaths and new deaths. nevertheless, this research is not without shortcomings. to some extent, our method is limited in that asymptomatic carriers (who are believed to account for 30% to 50% of all cases 13, 14 ) cannot be observed without antibody tests and thus are not factored into the derived fatality rate. this means that the number of actual infections (dashed bars) is limited to the estimation from observed deaths and cases only and serves as a lower boundary estimate 15 (figs. 1-a and 1-b) . furthermore, we relied on the extant literature, some of which may still not be peer-reviewed, to arrive at a novel framework that may point to the extent of unobservable infections across north america, specifically, the u.s. and canada. however, we hope that readers will appreciate the rapid rate at which the pandemic scenario has evolved over the past weeks and understand the limitations of this research while also acknowledging that unusual times call for unusual solutions. our goal is to contribute to the ongoing debate on detection bias and to present an alternative mechanism that can help to improve the robustness of covid-19 data being made available to the scientific community. in summary, our research adds another perspective to the ongoing debate on the e70 (4) pandemic. however, we highlight the need for more robust data. as the covid-19 pandemic progresses, it is crucial for policymakers to begin to focus on the potential for detection bias. we must be aware of the extent to which unobservable data-infections that have still not been captured by the system-can damage efforts to "flatten" the pandemic's curve. n world faces worst recession since great depression data on covid-19 coronavirus pandemic (2020). 2020. accessed suppress and lift': hong kong and singapore say they have a coronavirus strategy that works. science magazine oxford covid-19 government response tracker. blavatnik school of government world health organization. national capacities review tool for a novel coronavirus estimating case fatality rates of covid-19 lancet infect dis full spectrum of covid-19 severity still being depicted coronavirus: why you must act now report 13: estimating the number of infections and the impact of non-pharmaceutical interventions on covid-19 in 11 european countries the effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak science the next generation of the penn world table covid-19 and italy: what next? lancet up to 30% of coronavirus cases asymptomatic estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship key: cord-307144-g8d1xkub authors: monaghan, n. p. title: emerging infections – implications for dental care date: 2016-07-08 journal: br dent j doi: 10.1038/sj.bdj.2016.486 sha: doc_id: 307144 cord_uid: g8d1xkub over the last 20 years the majority of emerging infections which have spread rapidly across the globe have been respiratory infections that are spread via droplets, a trend which is likely to continue. aerosol spray generation in the dental surgery has the potential to spread such infections to staff or other patients. although the diseases may differ, some common approaches can reduce the risk of transmission. dental professionals should be aware of areas affected by emerging infections, the incubation period and the recent travel history of patients. elective dental care for those returning from areas affected by emerging infections should be delayed until the incubation period for the infection is over. if dental teams are aware of these issues they can reduce the risks to them and their patients. dentistry is often forgotten when new infections emerge; the appointment of dental emerging infection leads could ensure prompt advice is available. emerging infections -implications for dental care n. p. monaghan 1 countries, toronto, spain and the uk. 2 sars was caused by the sars cov virus, a coronavirus related to some of the viruses associated with common colds. there were 747 deaths among 9,098 reported cases. half of those over 65 who were known to have been infected died. currently there is ongoing surveillance about a different coronavirus -mers cov, which originated in the middle east. this new infection is now suspected to have originated from a single animal source, a camel, in 2011 or earlier. 3, 4 mers cov was first reported in 2012 and by july 2015 had been reported in 21 countries. while most cases have involved people from or visiting saudi arabia, there is an ongoing outbreak in the republic of korea. 5 between 2003 and 2008 it was predicted that a new form of influenza might emerge to affect humans. 6 birds in south east asia were expected to be the source. there are many strains of influenza viruses and many host species. birds, dogs, horses, pigs, humans and many other species of mammals can be affected by influenza a viruses. 7 in 2009 when a new influenza pandemic did occur it started through contact with pigs in mexico. the virus was h1n1, similar to the strain involved in the 1918 'spanish' influenza pandemic which killed about 50 million people and to the 1977/78 'russian' flu virus. 8 ebola has been known about since 1976. 9 the outbreak in west africa which commenced in december 2013 has led to cases being managed an infectious disease is regarded as emerging when the number of humans contracting an infection has increased in the past 20 years, or when there is threat of such increase in the near future. 1 there are different reasons why a disease may emerge including: • new infections from changes or evolution of existing organisms • infections affecting new areas (for example, possibly associated with ecological changes such as global warming) • old infections re-emerging because of poor public health measures or the development of antimicrobial resistance. infectious diseases that are transferred easily from person to person have scope for rapid spread. airline travel in particular can contribute to rapid spread across the globe. in 2002 a new infection emerged, severe acute respiratory syndrome (sars), initially in china but rapidly spread via international travellers to other asian over the last 20 years the majority of emerging infections which have spread rapidly across the globe have been respiratory infections that are spread via droplets, a trend which is likely to continue. aerosol spray generation in the dental surgery has the potential to spread such infections to staff or other patients. although the diseases may differ, some common approaches can reduce the risk of transmission. dental professionals should be aware of areas affected by emerging infections, the incubation period and the recent travel history of patients. elective dental care for those returning from areas affected by emerging infections should be delayed until the incubation period for the infection is over. in the usa, italy, the uk and spain. as of 20 january 2016 there have been 28,602 known cases associated with 11,301 deaths. 10 by august 2014 there had been 1,000 deaths in west africa and a british volunteer nurse had contracted the disease and been flown back to the uk for emergency treatment. 11 within the uk guidance was issued to dental teams in england (december 2014), wales (december 2014), northern ireland (january 2015) and scotland (february 2015). [12] [13] [14] [15] the emerging infection which hit the news in january 2016 is zika virus. this virus is carried by mosquito species such as aedes aegypti, which also carries yellow fever and dengue viruses. zika virus was first discovered in uganda in 1947 and since has slowly spread through south asia and pacific islands to reach brazil. 16 currently there are concerns about possible links with microcephaly. a known rare complication of zika infection is guillain barré syndrome. currently, zika infection risk in the uk would be limited to imported cases of people infected abroad, reflecting the locations where mosquitoes are carrying zika virus. aedes aegypti is currently restricted to presence in madeira, georgia and south russia, although it was recently found for the first time in the netherlands. 17 in none of these areas has zika virus been reported to date. 18 the emerging infections noted above are those which have hit the uk media. there are many other emerging infections. tb is opinion re-emerging in some areas, particularly london where it accounts for 40% of all uk cases. 19 given features of the infections which have emerged (or been expected to emerge) over the last 20 years and have potential to spread rapidly and cause significant loss of life, what is the relevance to dentistry? among the diseases which have emerged two are coronaviruses, one an influenza virus. coronaviruses and influenza viruses are spread via droplets, aerosols, or through direct contact with respiratory secretions of someone with the infection. the virus particles can survive within small droplets in the air for several hours. 20 thus when influenza is circulating there is a potential higher risk of transmission within dental practices because of the aerosol sprays generated by drills and ultrasonic scalers. for influenza it is thought that this risk of transmission is restricted to the room where dental care is given, provided the door to the room is closed. 21, 22 in the early phase of response to h1n1, when there were concerns that this form of influenza caused high mortality, the american guidance recommended negative pressure air handling and full fit-tested protective equipment. 23 in recent years a large proportion of the emerging infections have been viral infections of the respiratory tract, spread through droplets and aerosols. these have the potential to pass from person to person, and even country to country, very quickly. dental practice, involving both close contact to the airway and generation of aerosol sprays, presents a high-risk environment to catch or pass on these infections. 24 however this risk can be reduced by use of masks and gloves, pre-procedure rinses, rubber dam and high volume suction. 24 while not a 'digestive tract infection' , ebola is an infection which can be transmitted from body fluids including those affecting the digestive tract. the virus can survive for several days outside the body and a person can become infected via the mouth. 25 it is common for people to be advised not to kiss those who are symptomatic with gastrointestinal infection. although ebola virus is not believed to commonly spread as a result of droplet generation, contact with mucous membranes of an affected person is a mechanism for transmission of the virus. aerosol sprays bombardment of mucous membranes of someone with the disease is a potential mechanism to facilitate transmission of ebola (or a digestive system infection) when the causative organism may be present in the mouth of those infected. viruses are present in saliva of those very sick with ebola. droplet transmission of ebola from aerosol spray has not been demonstrated to date, although there is a report of a healthcare worker inadvertently rubbing her eyes with soiled gloves who then developed ebola disease. 26 based on the limited evidence it would seem best to avoid aerosol spray generation from the mouth of someone with the disease. by contrast zika is spread by mosquito bites. provided that normal precautions against blood-borne viruses are used there would seem to be no reason to suppose that dental care is a risk for transmission of the zika virus. with appropriate knowledge it is possible to reduce the risk of transmission of emerging infections in dental settings. the key principles in managing someone who may have been in contact with an emerging infection are: establishing whether contact with someone affected is possible, delaying non-urgent care until incubation periods are over, and use of appropriate precautions to provide care which cannot be delayed. the implications for the dental team include: • being aware of emerging infections • being aware of incubation periods • being aware of patients' recent travel history • delaying elective treatment of those from (or returning from) affected areas who may have been in contact with cases until the incubation period has passed to reduce risk of transmission • for urgent treatment of those who may have the disease or may have been in recent contact with cases, seeking advice from health protection colleagues before providing care and use of full protective equipment. while new diseases do not emerge to become a problem every year, when they do emerge and are reported on the news it would be sensible for members of the dental team to find out a little more about the infection. key information includes whether a new infection affects the respiratory or digestive tracts, awareness of affected geographical areas, awareness of incubation periods and any advice issued by health protection experts and government health departments. avoiding provision of elective dental treatment to those who may have been exposed to the disease is a key response. for many emerging infections there will be an incubation period. elective treatment can be delayed until after the incubation period has ended. similarly if a person survives an infection, is free of symptoms and shown to be free of the causative organism, elective care can be provided. not all dental care is routine and sometimes interventional treatment is needed urgently. if urgent dental care is required for symptomatic returnees or for those who have had contact with symptomatic individuals, advice should be sought from a health protection team before treatment commences. personal protective equipment and modification of cross infection precautions will be needed appropriate to the mode of transmission of the organism. 27 decontamination of the surgery and equipment will also be needed. the surgery is more likely to become widely contaminated through aerosol sprays, therefore aerosol spray generation should be minimised. experience with sars highlights appropriate responses for a highly infectious high mortality respiratory infection. 28, 29 in the absence of negative pressure operating rooms there are products which can help to manage the air in an aerosol generating environment. 30 past experience suggests that the dental team can be forgotten when new infections emerge. if we are alert and are clear about the information we need, we can ask for up to date answers and modify our practice accordingly. public health organisations should consider appointing someone with responsibility for emerging infections and dentistry with strong links to both health protection colleagues responsible for emerging infection responses and dental professionals. their role would ensure that dental care is not forgotten when new infections emerge and that early appropriate advice is available to dental professionals. predicting the future is an uncertain business. we cannot predict accurately when the next emerging infection will arise and what it will be, but we can identify features of such diseases with implications for dentistry. recent experience suggests that most emerging infections with potential to spread rapidly across the globe and cause significant loss of life will affect the respiratory tract. dental professionals should be aware of (and be made aware of) such infections because they have potential to be spread by generation of aerosol sprays in the oral cavity. awareness of emerging infections and of patient travel histories can assist us in reducing the risk of transmission of emerging infections in dental settings. if we strengthen links with health protection colleagues responsible for emerging infection responses we will be well placed to keep dental professionals informed. emerging infectious diseases sars (severe acute respiratory syndrome) molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus middle east respiratory syndrome coronavirus (mers-cov) directorate-general for health and consumer protection european commission. factsheet -avian influenza evolution and ecology of influenza a viruses influenza: the mother of all pandemics world health organization. ebola haemorrhagic fever in sudan british ebola patient arrives in uk for hospital treatment ebola guidance for dental care teams ebola dental guidance wales ebola guidance for dental care teams northern ireland ebola guidance for dental care teams zika virus: a previously slow pandemic spreads rapidly through the americas european centre for disease prevention and control. aedes aegypti european centre for disease prevention and control. epidemiological update: outbreaks of zika virus and complications potentially linked to the zika virus infection the white plague returns to london with a vengeance concentrations and size distributions of airborne influenza a viruses measured indoors at a health centre, a day-care centre and on aeroplanes prevention of 2009 h1n1 influenza transmission in dental health care settings pandemic (h1n1) 2009 iinfluenza a summary of guidance for infection control in healthcare settings prevention of swine influenza a (h1n1) in the dental healthcare setting aerosols and splatter in dentistry: a brief review of the literature and infection control implications ebola virus disease -how it spreads review of humantohuman transmission of ebola virus standard and transmission-based precautions -an update for dentistry severe acute respiratory syndrome (sars) and the gdp. part i: epidemiology, virology, pathology and general health issues severe acute respiratory syndrome (sars) and the gdp. part ii: implications for gdps a pilot study of bioaerosol reduction using an air cleaning system during dental procedures key: cord-341827-z9r5i0ky authors: macias-ordonez, r.; villasenor-amador, d. title: the misleading illusion of covid-19 confirmed case data: alternative estimates and a monitoring tool date: 2020-05-25 journal: nan doi: 10.1101/2020.05.20.20107516 sha: doc_id: 341827 cord_uid: z9r5i0ky confirmed case data have been widely cited during the current covid-19 pandemic as an estimate of the spread of the sars-cov-2 virus. however, their central role in media, official reports and decision-making may be undeserved and misleading. previously published infection fatality rates were weighted by age structure in the 50 countries with more reported deaths to obtain country-specific rates. for each country, the number of infections up to the infection date (23 days ago = incubation period + onset to death period) and the present percentage of immune population were estimated using infection fatality rate, the number of reported deaths (which is less prone to undersampling), and projecting back to infection date. we then estimated a detection index for each country as the percentage of estimated infections that confirmed cases represent. assuming that detection remains constant after infection date, we estimated the number of deaths and the estimated percentage of the population of each country expected to be immune up to 23 days into the future. estimated infection fatality rates are higher in europe. in most countries, confirmed cases currently represent less than 30% of estimated infections on infection date, and this value decreases with time. countries with flat curves throughout the pandemic show the lowest immunity percentages and these values seem unlikely to change in the near future, suggesting that they remain vulnerable to new outbreaks. estimates for some countries with low infection fatality rates suggest a still steep increase in the number of casualties in the next three weeks. countries that did not control initial outbreaks seem to have reached higher immunity percentages, although mostly still under 5%. we provide the code to monitor the trajectories of these estimates in 178 countries throughout the covid-19 pandemic. previously published infection fatality rates were weighted by age structure in the 50 27 countries with more reported deaths to obtain country-specific rates. for each country, 28 the number of infections up to the infection date (23 days ago = incubation period + 29 onset to death period) and the present percentage of immune population were 30 estimated using infection fatality rate, the number of reported deaths (which is less 31 prone to undersampling), and projecting back to infection date. we then estimated a 32 detection index for each country as the percentage of estimated infections that 33 confirmed cases represent. assuming that detection remains constant after infection 34 date, we estimated the number of deaths and the estimated percentage of the 35 population of each country expected to be immune up to 23 days into the future. 36 estimated infection fatality rates are higher in europe. in most countries, confirmed 37 cases currently represent less than 30% of estimated infections on infection date, and 38 this value decreases with time. countries with flat curves throughout the pandemic 39 show the lowest immunity percentages and these values seem unlikely to change in the 40 near future, suggesting that they remain vulnerable to new outbreaks. estimates for 41 some countries with low infection fatality rates suggest a still steep increase in the 42 number of casualties in the next three weeks. countries that did not control initial 43 outbreaks seem to have reached higher immunity percentages, although mostly still 44 . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 25, 2020. . https://doi.org/10.1101/2020.05.20.20107516 doi: medrxiv preprint introduction 50 covid-19 confirmed case data (ccd) are the central piece of information in most 51 news, official reports, conversations, forecasting efforts, and are also probably central to 52 most decisions made by authorities worldwide since the pandemic outbreak in 53 december 2019. however, it is widely known that they represent a small and unknown 54 fraction of the actual number of sars-cov-2 infections [1] [2] [3] [4] , we just do not know how 55 small. especially hard to assess is the number of asymptomatic but contagious 56 infections [5] [6] [7] [8] , as asymptomatic carriers are unlikely to seek testing. 57 elementary sampling theory and recent supported opinions [3, 6, 9] suggest that 58 ccd are highly dependent on the testing effort and sampling protocol, among other 59 factors. unless a randomized and standardized sampling protocol of the whole 60 population is carried out, there is no a priori reason to assume they are representative 61 of the magnitude or even the speed of the spread of this or any other virus. 62 furthermore, unless similar testing efforts are made in different countries, the data are 63 not comparable, and pooling them may provide an even worse picture of the spread of 64 the virus worldwide. covid-19 related deaths data are reported nearly as much, but 65 there is less focus on them. these data, however, are less prone to sampling error [2,3], 66 unless a large number of covid-19 related deaths go undetected, misdiagnosed, or 67 . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 25, 2020. provide, but see vogel [4] , and may even represent a monitoring alternative for 84 countries with little or no access to antibody testing. 85 since ifr is age dependent, its global value depends on the age structure of the 86 infected population. covid-19 is known to be more lethal in older age classes 87 [12, 15, 16, 17] , thus we can expect that the more biased the age structure of a given 88 population to such classes, the higher its ifr will be. in order to apply this value to other 89 populations, the ifr of each age class must be weighted by the relative proportion of 90 . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 25, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 25, 2020. . https://doi.org/10.1101/2020.05.20.20107516 doi: medrxiv preprint 6 modified in the form of function parameters as explained in s1. it has been recently 114 suggested [20] that sharing fully detailed code and functionality of modeling and 115 monitoring tools is more important than ever to face the current covid-19 pandemic. 116 this section describes what the script does and the parameters used as default, 117 beginning with the procedure used to obtain the data. 118 the database used for analyses is obtained from the european centre for 119 disease prevention and control (ecdc) web page [21] . the script includes code lines 120 needed to import the daily updated database with confirmed cases and deaths. the 121 ecdc keeps a daily updated database curated from over 500 sources [22] . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 25, 2020. cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 25, 2020. period. this value will be referred to as %di on idt. if a country is currently reporting no 181 deaths, this %di value is evaluated for the period ending with the last daily %di value 182 . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 25, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 25, 2020. . https://doi.org/10.1101/2020.05.20.20107516 doi: medrxiv preprint 222 estimated ifr values show a bimodal distribution with one mode around 0.5% and the 223 other slightly above 1% (fig 1) . out of the 50 countries with more reported deaths, 224 estimated ifr values are higher than 1% in all european countries (n=23) with the 225 exception of russia (0.92%), ireland (0.84%), ukraine (0.98%) and moldova (0.72%), 226 and lower than 1% in all non-european countries (n=27) with the exception of canada 227 (1.05%) and japan (1.60%) ( table 1 ). this is due to the fact that european countries 228 have age structures biased to higher age classes. this is likely one of the reasons why is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 25, 2020. ifr may also depend on country specific factors such as healthcare system, socioour results on %di on idt show great variation among countries, and suggest 266 that ccd represent less than a third of estimated infections on idt in all but 5 countries, 267 . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 25, 2020. values consistently far outside the distribution of the rest of the countries (fig 1) . other cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 25, 2020. 68-82% more deaths in the next 23 days. they would also experience an equivalent 378 increase in population immunity. 379 it has been suggested that the accumulation of herd immunity in the population 380 slows epidemic resurgence [37] . by contrast, when virtually no population immunity is 381 . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 25, 2020. . https://doi.org/10.1101/2020.05.20.20107516 doi: medrxiv preprint 19 built, such as the case of china, india, japan, bangladesh or south korea (percentage 382 of immune population below 0.1%, in table 1 ) a resurgence peak of covid-19 may be 383 nearly the same size as an uncontrolled epidemic if control policies fail [37] . the 384 numbers for japan (fig 5) , for instance, suggest a high vulnerability since it shows the 385 highest estimated ifr (1.60) of all countries, a low %di on idt (6 %), the lowest 386 percentage of immune population (0.04%), and the highest estimated increase (111%) 387 in infections from idt to the present, and thus a similar increase in deaths in the 388 following 23 days. we still don't know how long immunity to sars-cov-2 could last [4], 389 but recent modeling suggests anywhere between 40 weeks and 5 years [37] . 390 nevertheless, immunity is unlikely to end abruptly, so the rate of any further re-infection 391 with new strains should be smoother in the future as long as herd immunity has had 392 some build-up [4,16]. however, even belgium with the highest estimated percentage of 393 immune population (6.94%) seems far from values approaching herd immunity. 394 although ccd have been used to estimate the number of infections [38], we 395 believe that this will usually provide poor estimates as suggested by the short and long-396 term variation in daily %di values. in any case, we suggest that having a low %di value cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 25, 2020. . https://doi.org/10.1101/2020.05.20.20107516 doi: medrxiv preprint 20 antibodies, we suggest that estimates based on reported deaths and ifr are a more 405 reliable alternative to estimate the spread of sars-cov-2 than ccd in any country for 406 which age structure data is available and data of reported deaths is trustworthy. they 407 also illustrate the potential bias when assuming that ccd data reflect the actual spread 408 of covid-19. logistic approximations used to describe new outbreaks in the 2020 covid-19 pandemic the covid-19 infection in italy: a statistical study of an abnormally severe 421 disease as covid-19 cases, deaths 426 and fatality rates surge in italy, underlying causes require investigation first antibody surveys draw fire for quality, bias incubation period of 2019 novel 475 coronavirus (2019-ncov) infections among travellers from wuhan the epidemiological characteristics of an outbreak of 2019 483 novel coronavirus diseases (covid-19) in china impact of non-pharmaceutical interventions (npis) to reduce covid-19 mortality 488 and healthcare demand demographic science aids in understanding the spread and fatality rates of vienna: r foundation for statistical computing united nations, department of economic and social affairs european centre for disease prevention 509 and control. an agency of the european union ecdc collects and processes covid-19 data european centre for disease prevention and control an 514 agency of the european union covid-19 deaths and cases: how 522 do sources compare? the 525 incubation period of coronavirus disease 2019 (covid-19) from publicly 526 reported confirmed cases: estimation and application epidemiological data from the covid-19 outbreak, real-time case information the editorial board. the global coronavirus crisis is poised to get much, much 535 worse. the new york times prediction models for diagnosis and prognosis of covid-19 infection: 546 systematic review and critical appraisal available from: 552 . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted variación en casos por 555 reclasificación -covid19 virological assessment of hospitalized patients with covid-2019 detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr estudio ene-covid19: primera ronda estudio 575 nacional de sero-epidemiología de la infección por sars-cov-2 en españa 576 informe preliminar projecting the 580 transmission dynamics of sars-cov-2 through the postpandemic period serial interval of 585 covid-19 among publicly reported confirmed cases. emerg infect dis 412 we are grateful to isabel noriega, yareni perroni, matt draud and warren greiff for 413 proofreading and useful comments on early versions of the manuscript. it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 25, 2020. reported deaths. this spreadsheet is generated by the function cv19.tab.num() in the 604 r script (s1) and includes 10 additional columns apart from those shown in table 1 cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review)the copyright holder for this preprint this version posted may 25, 2020. . https://doi.org/10.1101/2020.05.20.20107516 doi: medrxiv preprint key: cord-344009-hm36pepp authors: nathanson, n. title: virus perpetuation in populations: biological variables that determine persistence or eradication date: 2005 journal: infectious diseases from nature: mechanisms of viral emergence and persistence doi: 10.1007/3-211-29981-5_2 sha: doc_id: 344009 cord_uid: hm36pepp in this review, i use the term “perpetuation” for persistence of a virus in a population, since this is a different phenomenon from persistence of a virus in an infected host. important variables that influence perpetuation differ in small (<1,000 individuals) and large (>10,000) populations: in small populations, two important variables are persistence in individuals, and turnover of the population, while in large populations important variables are transmissibility, generation time, and seasonality. in small populations, viruses such as poliovirus that cause acute infections cannot readily be perpetuated, in contrast to viruses such as hepatitis b virus, that cause persistent infections. however, small animal populations can turnover significantly each year, permitting the perpetuation of some viruses that cause acute infections. large populations of humans are necessary for the perpetuation of acute viruses; for instance, measles required a population of 500,000 for perpetuation in the pre-measles vaccine era. furthermore, if an acute virus, such as poliovirus, exhibits marked seasonality in large populations, then it may disappear during the seasonal trough, even in the presence of a large number of susceptible persons. eradication is the converse of perpetuation and can be used as a definitive approach to the control of a viral disease, as in the instance of smallpox. therefore, the requirements for perpetuation have significant implications for practical public health goals. from the viewpoint of the individual host, viral infections can be conveniently divided into those that are acute and those that are persistent. however, all viruses -by definition -must be able to persist in their host population, regardless of whether they cause acute or persistent infection in individual members of that population. thus, persistence in a population is a distinct phenomenon and in this discussion i will use the term "perpetuation" to distinguish it from persistence in the individual host. once a virus has infected a defined population, it may either perpetuate indefinitely or may disappear. if disappearance is a natural occurrence, it is often described as "burn out" or "fade out", while if it is induced by human intervention, it may be described as "eradication" or "elimination". eradication represents a definitive approach to prevention of a viral disease, as in the instance of smallpox. however, to develop a strategy for eradication it is necessary first to understand the requirements for perpetuation. thus, the subject has significant implications for practical public health goals. virus persistence and perpetuation has been the subject of numerous discussions, and this presentation draws heavily on some of these publications [1, 26, 30] . some of the biological variables that influence perpetuation are shown in table 1 . implicit in this table is the generality that most viruses can infect a given host only once. in the instance of an acute infection, the host acquires lifelong immunity to the infecting virus and is -from an epidemiological perspectiveno longer capable of acting as a link in the chain of infection. if the virus causes persistent infection, then the outcome varies. some persistent virus infections can be transmitted as long as the host is infected (for instance hepatitis b virus and human immunodeficiency virus [hiv] ). other viruses (such as varicella zoster and herpes simplex) persist in a latent form and are infectious only during intermittent episodes of recrudescence. virus perpetuation within a human population involves a fragile equilibrium between three different categories of hosts: those who have not been infected and are susceptible; those who are actively infected and are potentially infectious; and those who have been infected and are immune. if the infection spreads too slowly within the population (transmissibility quotient, ro <1) the virus will ultimately disappear for absence of actively infected hosts. on the other hand, if the infection spreads too rapidly (ro 1), the susceptible population will be "exhausted", also leading to disappearance of actively infected hosts. the size of the population under consideration is an important determinant of the dynamics of perpetuation, since the relative importance of other variables is different in smaller (<1,000 individuals) and larger (>10,000) groups (table 1 ). in small populations, two of the most important variables are persistence in the individual host and population turnover (the rate at which new susceptible animals are introduced into the population). in large populations, variables of high importance include transmissibility, generation time, and seasonality. transmissibility (ro) is the number of new infections that are generated by each existing infection and is a property (in part) of each virus, since under a given set of conditions, some viruses will be transmitted at a much higher rate than will others. generation time is the average time between the infection of two individuals who are successive links in an infection chain; generation time may be a short as 2-3 days in the case of influenza and as long as many years in the case of hiv or hepatitis b infection. seasonality refers to the variation in transmissibility of a given virus in a specific population at different times of year. viruses that cause acute infections are often unable to perpetuate in small populations [3] . figure 1 shows a seroepidemiological study of poliovirus in a small eskimo village in greenland, conducted in the 1950s. each of the three types of poliovirus had been introduced into this population. type 1 virus had caused an outbreak of infection 25 years prior to the study and had then disappeared; type greenland. the data show three separate introductions of types 1, 2, and 3 poliovirus, respectively. the low frequency of type 1 antibodies in persons ages 15-25 probably represents cross-reacting antibodies induced by infection with type 2 virus. it appears that this acute infection "burned out" in this small (<1,000) isolated population because it spread rapidly through persons who had not been previously infected and "exhausted" the susceptible population. after [19] 2 virus had been introduced 15 years prior to the study date and had likewise disappeared; and type 3 had been introduced within the prior 5 years and (likely) had also disappeared. in such small populations, viruses that cause acute infections spread so rapidly that they quickly exhaust the susceptible population and then fade out. conversely, hepatitis b virus, which causes both acute and persistent infections can persist in small populations as shown in fig. 2 , a study of another small eskimo population in greenland. in such populations hepatitis b virus is often transmitted during birth, from infected mothers to their newborn infants, which frequently results in persistent infections. another parameter that favors virus perpetuation is rapid turnover of the population itself. this is seen most often in animal populations some of which, in nature, may have an average lifespan of 1-2 years, so that a large fraction of the population consists of relatively young and susceptible hosts. although difficult to document in wildlife populations, this phenomenon can be more readily documented in groups of laboratory animals that are under constant surveillance. one example is a study conducted in a colony of laboratory rats that was maintained for nutritional studies [21] . this colony was infected with rat parvovirus, a small dna virus that did not cause overt disease and was only detected by serological surveillance. rat parvovirus caused an acute infection, transmitted by the enteric route, that spread rapidly through the relatively small population of about 500 young animals. based on the rate of spread, the virus might have been expected to exhaust all susceptibles by 10 months of age. however, every month about 25% of the animals aged (persons who escaped infection as children and were infected as adults). after [25] 4-5 months were removed to another room to be used for experiments and the same number of one-month susceptible weanling animals was introduced from a breeding colony. this continual introduction of young susceptible animals was sufficient to perpetuate an acute virus infection in a small population. as mentioned above, although a number of viruses cannot be maintained in small human populations, all human viruses are capable of perpetuation in large populations. important biological determinants of perpetuation include transmissibility, generation time, and seasonality, and these three may, in turn, determine the minimum size of the population required for perpetuation. transmissibility (ro) reflects in part the innate infectivity of a given virus, but is also determined by the density of the population, by the proportion of that population that is susceptible, and by the frequency of significant contact between different individuals within the population. the following examples illustrate the interaction of all these variables, and indicate the complexity of these relationships. measles has a special place as an example of virus perpetuation, since it is a rare instance where public health statistics can be used to monitor the ebb and flow of a specific virus infection in large human populations. measles has several attributes that -in the aggregate -are not seen for other common viral diseases: (i) there are longterm records of measles incidence, collected by many health departments in the united states and other countries; (ii) 95% of all measles infections manifest as illness (in contrast to 1% for poliomyelitis for example); (iii) the symptoms of measles are sufficiently pathognomonic so that it can be distinguished from other viral infections by clinical observers; and (iv) population-wide reports can be corrected for under-reporting (about 15% of measles cases were reported in most cities in the united states prior to the introduction of measles vaccine in 1963). exploiting these facts, bartlett [2] published several classical studies showing that in the pre vaccine era in the united states, measles was perpetuated in cities of 500,000 or greater population but not in cities below that size. similar observations could be made in other parts of the world. for instance, in iceland, with a population of 150,000 to 200,000, measles was introduced about 6 times during the period 1900 to 1940; each time it caused an outbreak that lasted 1-3 years, and then disappeared (tauxe, unpublished, 1979) . although these data are striking, they remained unexplained for a number of years. why was 500,000 the limiting population size, at least in the cities included in bartlett's study? a putative explanation was put forth in several papers that focused on the seasonality of measles in temperate climates [30] . data for baltimore (one of the cities included in bartlett's study), for the period 1928-1961, are shown in fig. 3 . absent seasonality, 8% of annual incidence population 500,000 measles susceptibles (estimated 10% of population) 50,000 annual measles incidence (estimated average) 10,000 cases in trough month (0.1%) 10 cases in trough generation period (12 days) 3 a an age profile for measles susceptibles was constructed from the age distribution reported for measles in baltimore, md, for 1900-1931, supplemented with serosurveys conducted prior to the introduction of measles vaccine. the average number of annual measles infections was estimated as the size of an annual birth cohort, assuming a steady state and 100% cumulative attack rate for measles. cases in trough month based on data from baltimore, md, 1928-1961, after [30] would have been expected each month; however measles peaked in march at 28% while only 0.1% was reported in september, the trough month. based on these observations, a hypothetical reconstruction for a city of 500,000 with 0.1% of measles in the trough month is shown in table 2 . in such a city, during a single trough generation period, only 3 cases of measles would be expected. under these circumstances, it is plausible that measles infection could fade out. a further test of the hypothesis that seasonality played a critical role in the fade out of measles is provided by data from new york city and baltimore, prior to and after the introduction of measles vaccine ( table 3 ). the data in table 2 imply 39 0 a the estimated number of susceptibles is based on the age distribution of measles cases and serosurveys of measles antibody, after [30] that a population of about 50,000 susceptibles (data not shown indicate that about 10% of the total population was susceptible to measles) was required to perpetuate measles in cities of north america prior to the introduction of measles vaccine. in new york city, it can be estimated that there were about 900,000 susceptibles prior to measles vaccine and about 400,000 in the late 1960s, after the introduction of measles vaccine. as table 3 shows, measles was perpetuated in new york city after the introduction of the vaccine. in baltimore, vaccination was estimated to reduce the susceptible population from 90,000 to 40,000, just below the threshold for perpetuation. in fact, measles was perpetuated in baltimore prior to measles vaccination, but showed an annual fade out each year in the late 1960s, after the introduction of measles immunization. currently, the global effort to eradicate poliovirus is moving towards its goal. in 1988, when who enunciated the eradication of polio as a goal, there were an estimated annual 350,000 cases of paralytic poliomyelitis worldwide; in 2001, there were fewer than 1,000. as we approach eradication, it is interesting to look back at the origins of this effort, the eradication of wild poliovirus in the united states in 1972 (fig. 4) . amazingly, although poliomyelitis was being tracked carefully by the centers for disease control and other public health specialists, no one anticipated eradication of wild poliovirus [16] . the explanation for this apparent paradox is not hard to find. public health surveillance was focused on poliovirus immunization surveys to determine the percent of children receiving opv, and serosurveys of immunityindicated that there was a residual susceptible population estimated at up to 10,000,000 [5, 12, 15] . it was widely assumed that this pool of susceptible hosts would continue to circulate wildtype polioviruses indefinitely, and eradication was not contemplated. under these circumstances, how could eradication occur? again, i would postulate that seasonality played a critical role in eradication [15, 16] . figure 5 shows that, as for measles, poliovirus infections were highly seasonal, particularly in the northern united states. in table 4 , the seasonal curves are used to estimate the incidence of poliovirus infection in a hypothetical metropolitan area with a population of 10,000,000, both for the northern and the southern united states. vaccine-induced reduction of susceptible individuals in such a population can be guesstimated to reduce the number of new infections per trough generation period below the threshold for virus perpetuation. when poliomyelitis incidence data for the period 1960 through 1972 are plotted by state (fig. 6) , it can be seen that each year a decreasing number of states reported paralytic polio. it can be surmised that, in area after area, the virus disappeared during the wintertime trough and was not introduced in the following summer, eventually leading to eradication. although space does not permit, it is noted a susceptible population estimates based on the age distribution of poliomyelitis and upon serosurveys of poliovirus antibodies. infections back-calculated from cases of paralytic poliomyelitis. seasonal trough based on monthly distribution of poliomyelitis cases. generation period based on studies of secondary polio cases in families. see [15] for references that a similar phenomenon occurred with measles, but measles -with a greater transmissibility than poliovirus -was reintroduced after each fade out [15] . the elimination of wild poliovirus in the united states gave credibility to the extension of eradication. major efforts were initiated in central and south [1960] [1961] [1962] [1963] [1964] [1965] [1966] [1967] [1968] [1969] [1970] [1971] [1972] [1973] , excluding imported and vaccine-associated cases. based on data in [4] america, leading to successful eradication in the 1980s. emboldened by these successes, who embarked on global eradication, a goal that appears within reach within the next several years. the principal residual sites where wild poliovirus continues to circulate are pakistan, india, and nigeria, and it is likely that the absence of seasonality [7, 28] in these semi-tropical nations has been one of the impediments to eradication. one of the salient questions regarding the biology of hiv is: how did it emerge as a human virus? i will argue that the ability of hiv to cause persistent infections likely played a key role in its emergence, and is therefore worth a brief consideration in this essay on viral perpetuation. although circumstantial, the evidence is quite persuasive that hiv arose when a simian lentivirus, sivcpz, jumped from chimpanzees to humans [9, 11, 13] . many animal viruses cause zoonotic infections of humans but very few of them are subsequently transmitted from person to person. most of those zoonotic viruses that are capable of limited human-to-human transmission exhibit marginal transmissibility, as evidenced by their containment using rudimentary quarantine measures and their fade out after a limited number of cycles. examples are crimean congo hemorrhagic fever virus [20] ; arenaviruses [14] ; ebola virus [22] ; swine influenza virus in 1976 [17, 24] ; and monkeypox virus [8] . the sars coronavirus may be another example although to date it has not established itself as a human [9, 11, 13, 18] virus, even though it underwent at least 30 human-to-human passages in china in 2003 before being controlled by quarantine measures [27] . rare indeed are those zoonotic viruses that become established permanently as human viruses. the best documented examples are influenza viruses, since avian influenza virus has on several occasions established itself in humans. it is noteworthy that, in several of these instances (such as the asian pandemic of 1957 and the hong kong pandemic of 1968) the avian virus re-assorted with a human influenza virus, to produce a genetic chimera that endowed it with novel antigenic determinants, while maintaining the capability to transmit to humans [29] . these observations raise the questions as to how sivcpz became established as a human virus. recent studies have produced a speculative reconstruction of historical events following the hypothetical transmission of sivcpz to humans (table 5) . particularly relevant to this discussion is the inference that, following transmission to humans, sivcpz was perpetuated as an unrecognized infrequent infection in rural villages in central africa during the period 1930 to 1980 [18] . different regions of the viral genomes of sivcpz and of hiv-1 differ by 10%-25% [10] and it may be assumed that many of these changes were introduced during that 50-year interval. i speculate that some of these genetic changes have led to the metamorphosis of sivcpz into hiv-1, to become an agent that can spread among humans with sufficient ease to be considered a virus of humans. if this is correct, then it would seem likely that the ability of sivcpz to persist lifelong in the humans that it first infected might have provided an essential window of opportunity for a virus of chimpanzees to evolve into a human virus. although tentative, these speculations offer interesting hypotheses for future research. persistent viral infections the critical community size for measles in the united states infectious diseases in primitive societies annual poliomyelitis summaries for the years 1960-1973 poliomyelitis summary the epidemiology of paralytic poliomyelitis in hawaii poxviruses origin of hiv-1 in the chimpanzee pan troglodytes troglodytes diversity consideration in hiv-1 vaccine selection aids as a zoonosis: scientific and public health implications the age distribution of poliomyelitis in the united states in 1955 timing the ancestor of the hiv-1 pandemic strains lassa fever: review of epidemiology and epizootology epidemiological aspects of poliomyelitis eradication. rev infectious dis 6 the epidemiology of poliomyelitis: enigmas surrounding its appearance, epidemicity, and disappearance the swine flu affair. us department of health, education, and welfare, us superintendent of documents the prevalence of infection with human immunodeficiency virus over a 10-year period in rural zaire poliomyelitis immune status in ecologically diverse populations, in relation to virus spread, clinical incidence, and virus disappearance california encephalitis, hantavirus pulmonary syndrome, and bunyavirid hemorrhagic fevers sero-epidemiological study of rat virus infection in a closed laboratory colony filoviridae: marburg and ebola viruses poliomyelitis distribution in the united states pure politics and impure science: the swine flu affair hepatitis and hepatitis b-antigen in greenland. ii. occurrence and interrelation of hepatitis b associated surface, core, and "e" antigen-antibody systems in a highly endemic area persistent viruses molecular evolution of the sars coronavirus during the course of the sars epidemic in china epidemic of poliomyelitis in puerto rico seasonality and the requirements for perpetuation and eradication of viruses in populations recurrent outbreaks of measles, chickenpox, and mumps. ii. systematic differences in contact rates and stochastic effects author's address: n. nathanson, 1600 hagys ford rd this review draws heavily upon earlier reviews that are cited in the references, particularly yorke et al., 1979, and nathanson and key: cord-353214-qo98m7jx authors: jhaveri, ravi; shapiro, eugene d. title: fever without localizing signs date: 2017-07-18 journal: principles and practice of pediatric infectious diseases doi: 10.1016/b978-0-323-40181-4.00014-1 sha: doc_id: 353214 cord_uid: qo98m7jx nan most young children with fever and no apparent focus of infection have self-limited viral infections that resolve without treatment and are not associated with significant sequelae. however, a small proportion of young children with fever who do not appear to be seriously ill may be seen early in the course of a bacterial infection that could progress to bacteremia or meningitis. despite numerous studies that have attempted to identify the febrile child who appears well but has a serious infection and to assess the effectiveness of potential interventions, no clear answers have emerged. [1] [2] [3] [4] studies show that parents usually are more willing than physicians to assume the small risk of serious adverse outcomes in exchange for avoiding the short-term adverse effects of invasive diagnostic tests and antimicrobial treatment. 5, 6 the best approach to the treatment of the febrile child combines informed estimates of risks, careful clinical evaluation and follow-up of the child, and judicious use of diagnostic tests. 7 the list of microbes that cause fever in children is extensive. relative importance of specific agents varies with age, season, and associated symptoms. the goal of this chapter is to assist in identifying the febrile child with a serious bacterial infection (sbi). table 14 .1 shows the most common causes of sbi in children younger than 3 months. [8] [9] [10] [11] [12] the division at 1 month is not absolute; considerable overlap exists. receipt of vaccines typically administered at 2 months also reduces the risk of sbi. certain viruses, notably herpes simplex, influenza, and enteroviruses, can cause serious infections in neonates, mimicking septicemia and beginning as fever with no apparent focus of infection. less serious viral infections are the most common causes of fever in children of all ages. in children between 3 and 35 months of age, most bacterial infections with no apparent focus are caused by streptococcus pneumoniae (in unimmunized children), neisseria meningitidis, or salmonella spp. infection (which often is associated with symptoms of gastroenteritis). because universal administration of pneumococcal and haemophilus influenzae type b (hib) conjugate vaccines, hib has become rare, and the incidence of infection with s. pneumoniae has fallen substantially. 13, 14 other common causes of invasive bacterial infections in these children, such as staphylococcus aureus, are usually associated with identifiable focal infections. the risk of serious bacterial infection varies with age. although longitudinal studies have shown that only 1% to 2% of children are brought to medical attention for fever during the first 3 months of life, a greater proportion of febrile infants has serious bacterial infections than older children. [15] [16] [17] [18] risk is greatest during the immediate neonatal period and through the first month of life, and risk is heightened for infants born prematurely. in a prospective study conducted by investigators at the university of rochester, factors were identified that indicate a low risk of serious bacterial infection in febrile infants younger than 3 months. 19 among 233 infants who were born at term with no perinatal complications or underlying diseases, who had not received antibiotics, and who were hospitalized for fever and possible septicemia, 144 (62%) were considered unlikely to have a serious bacterial infection and fulfilled all of the following criteria: no clinical evidence of infection of the ear, skin, bones, or joints; white blood cell (wbc) count between 5000 and 15,000/mm 3 ; less than 1500 band cells/mm 3 ; and normal urinalysis results. only 1 (0.7%) of the 144 infants had a serious bacterial infection (i.e., salmonella gastroenteritis), and none had bacteremia. in contrast, among the 89 infants who did not meet these criteria, 22 (25%) had a serious bacterial infection (p < 0.0001) and 9 (10%) had bacteremia (p < 0.0005). sedimentation rate, c-reactive protein, procalcitonin, morphologic changes in peripheral blood neutrophils, and quantitative cultures of blood. 28, [51] [52] [53] [54] [55] [56] [57] [58] clinical scales have been developed to help identify the febrile child with a serious illness. 59 unfortunately, no test has sufficient sensitivity and positive predictive value (ppv) or npv to be clinically useful for an individual patient. in the era before conjugate vaccinations for hib and s. pneumoniae, several studies examined the risk of bacteremia among children with a temperature higher than 40°c and in those with fever and a wbc count of at least 15,000/mm. 3, 41, 51, 60 the ppv of the tests for bacteremia was only about 15%. in the current era of low incidence and unconfirmed predictive value of wbc for remaining pathogens, laboratory testing no longer is justified. 1, 7, 45, 47 making a laboratory-confirmed diagnosis of a viral infection significantly reduces the likelihood that a febrile infant has a concomitant bacterial infection (with the exception of a uti). this currently holds true for influenza, rsv, and enterovirus, and in the future, it will likely be true for many other respiratory viruses (e.g., human metapneumovirus, parainfluenza virus, coronavirus) that are being diagnosed with multiplex polymerase chain reaction (pcr) technology. one study showed that incorporating viral diagnostics into the initial evaluation of febrile infants reduced the length of hospitalization and healthcare costs. 61 although there is no single correct approach to the management of febrile infants without localizing signs who appear well, studies have provided data based on which informed decisions can be made. 7 there is general agreement that febrile children who are very young (i.e., variously considered to be younger than 3, 2, or 1 month of age) should be managed differently from older children. because of the substantially greater risk of serious infections in very young infants with fever and the difficulty in assessing the degree of wellness accurately, pediatricians have approached the management of these cases conservatively. some clinicians adhere to a protocol of treating all young infants with fever and no apparent focus of infection with broad-spectrum antimicrobial agents administered intravenously in the hospital until the results of cultures of the blood, urine, and cerebrospinal fluid (csf) are known. 62 although perceived as the safe approach, it incurs considerable financial cost and risk of iatrogenic complications and of diagnostic misadventures associated with hospitalization. [63] [64] [65] these risks include errors in the type and dosage of drugs, complications of venous cannulation (e.g., phlebitis, sloughing of the skin), and nosocomial infections. hospitalization of a young infant is a major disruption for the family and may potentiate development of the vulnerable child syndrome. 66 investigators have found that selected young infants with fever can be treated expectantly without hospitalization. 4, 7, 19, 21, 27, 67 consequently, many experts think that febrile infants between 4 and 12 weeks of age with no apparent focus of infection who appear well or have a laboratorydocumented viral infection can be treated without additional laboratory tests or hospitalization, provided that careful follow-up is ensured. 7 others prefer to confirm laboratory criteria that predict low risk (sometimes including a normal csf analysis result). some providers observe the patient very closely without giving antimicrobial therapy; others treat all such infants for 2 days with a single daily dose of ceftriaxone (50 mg/ kg) administered parenterally while awaiting the results of blood, urine, and csf cultures. either approach, if careful and vigilant, is defensible. before an antimicrobial agent is administered, cultures of the blood, urine, and csf should be obtained. rapid tests for specific viral pathogens are widely available, can aid decisions about managing patients, and can reduce the duration of hospitalization or eliminate the need. 7, [31] [32] [33] 68 febrile infants at low risk for serious bacterial infection for whom adequate home observation and follow-up cannot be ensured should be hospitalized and can be observed without antimicrobial treatment. if the child appears well, doing so is reasonable and avoids the adverse side effects of antimicrobial agents and intravenous cannulation, shortens the duration of hospitalization, and saves money without placing the child at significant risk for complications. 21, 27, 67 many studies have largely corroborated results of the rochester study. 11, 12, [20] [21] [22] [23] [24] [25] although investigators have used slightly different criteria to define young febrile infants at low risk for serious bacterial infection (and some investigators excluded children younger than 1 month old), all found that the risk of a serious bacterial illness in the group defined as being at low risk is very low. approximately 10% of young febrile infants have urinary tract infections (utis), largely due to escherichia coli, and 10% of this group (1% overall) have concomitant bacteremia or meningitis. 11, 12, 26 in a meta-analysis of studies of febrile children younger than 3 months, the risks of serious bacterial illness, bacteremia, and meningitis were 24.3%, 12.8%, and 3.9%, respectively, for high-risk infants and 2.6%, 1.3%, and 0.6%, respectively, for low-risk infants. 15 the negative predictive value (npv) for serious bacterial illnesses among infants fulfilling low-risk criteria ranged from 95% to 99%, and the npv was 99% for bacteremia and 99.5% for meningitis. 15 although the risk of serious bacterial infection is high among febrile infants younger than 3 months of age with no apparent focus of infection, clinical and laboratory assessment can identify the slightly more than 50% of infants at very low risk. an observational study of more than 3000 infants younger than 3 months of age with fever greater than 38°c treated by practitioners and reported as part of the pediatric research in office settings network found that 64% were not hospitalized. 4, 27 practitioners individualized management and relied on clinical judgment; guidelines were followed in only 42% of episodes. 4, 27, 28 outcomes of the children were excellent. if the guidelines had been followed, outcomes would not have improved, but there would have been substantially more laboratory tests performed and more hospitalizations. 4 risk of serious bacterial illness in young infants has fallen further due to the marked reduction in early-onset group b streptococcal infections because of the effectiveness of peripartum antimicrobial prophylaxis of pregnant women with positive screening test results for colonization with group b streptococcus. 29 in febrile children younger than 3 months of age who have an identified viral infection such as influenza, respiratory syncytial virus (rsv), or enteroviruses, the risk of a serious bacterial infection other than a uti falls to almost zero. 24, [30] [31] [32] [33] in the 1970s and 1980s, there was concern about occult bacteremia in febrile children between 3 months and 24 to 36 months of age. 34 studies performed in that era showed that some children 3 months of age or older with fever who did not appear to be toxic and who had no apparent focus of infection had bacteremia, most often due to s. pneumoniae but occasionally due to hib or n. meningitidis. [34] [35] [36] [37] [38] [39] [40] [41] in some instances, serious focal infections such as meningitis developed in children with occult bacteremia. the concern about progression to focal infections resulted in protocols being developed to identify and intervene for infants at high risk. protocols emphasized routine blood cultures and empiric antibiotic administration for many febrile children in this age group. 28 these practices discounted several facts. first, most children with occult bacteremia have transient infection and recover without antimicrobial therapy and without developing a serious complication (e.g., meningitis, septic shock). 28, 42, 43 second, risk of meningitis complicating occult bacteremia varies with bacterial species (n. meningitidis ≫ hib ≫> s. pneumoniae). 35 third, universal vaccination against hib led to elimination in vaccinated infants of the most concerning occult bacteremia, and subsequent use of conjugate pneumococcal vaccine eliminated almost all other cases of occult bacteremia. [44] [45] [46] [47] [48] [49] the risk of uti in well-appearing febrile children in this age group has not changed significantly over the years. several studies have shown that the rate of uti varies among different populations (e.g., girls vs. boys, uncircumcised vs. circumcised) within this age group and varies with height and duration of fever. uti should be considered as a potential source of sbi in these patients. 44, 50 various diagnostic tests to quantify the risk of bacteremia and its complications have been assessed. they include the wbc count and differential count, microscopic examination of buffy coat of blood, erythrocyte if no focus is found and the child does not appear toxic, no diagnostic tests are indicated routinely. parents should be instructed to look for signs that a more serious problem is developing (e.g., persistent irritability or lethargy, inattentiveness to the environment). serial observations should be planned that permit subsequent clinical and laboratory evaluation and antimicrobial treatment as indicated. if a practitioner encounters a febrile child older than 4 months of age who is unimmunized or partially immunized, a more aggressive plan for evaluation and management may be warranted. this chapter focuses on invasive bacterial infections, particularly bacteremia, as a cause of fever without apparent focus. although other serious illnesses such as autoimmune diseases and inflammatory bowel disease can manifest as fever without a focus of infection, they are rare and come to attention because of persistence or recurrence of fever (see chapter 15) . all references are available online at www.expertconsult.com. most infants with fever who are younger than 1 month should be hospitalized and treated with antimicrobial therapy (with or without acyclovir for herpes simplex virus), although in selected instances, hospitalization without antimicrobial treatment or outpatient management after laboratory evaluations, including csf analysis, may be reasonable. if a decision is made to administer antimicrobial agents intravenously, ampicillin (100 to 200 mg/kg per day every 6 hours) plus gentamicin (7.5 mg/kg per day every 8 hours) or a third-generation cephalosporin (e.g., ceftriaxone, 50 mg/kg per day in one dose; cefotaxime, 150 mg/kg per day every 8 hours) could be chosen. ampicillin and gentamicin is a well-established combination with narrower spectrum of antimicrobial activity than ceftriaxone and excellent effectiveness against group b streptococcus, listeria monocytogenes, and many enteric gram-negative rods. because of the rising incidence of ampicillin-resistant e. coli and the rarity of listeriosis in recent large studies, a regimen with a third-generation cephalosporin without ampicillin offers coverage for the few infants who have bacteremia or meningitis with a uti due to ampicillin-resistant e. coli. 11, 12, 25 no study has directly assessed the relative risks and benefits of either regimen. before initiating antimicrobial treatment with any regimen, cultures of the blood, urine (obtained by urethral catheterization or suprapubic aspiration of the bladder), and csf should be obtained. children 3 months of age and older who appear well and have no apparent focus of infection can be evaluated clinically without laboratory tests or treatment with antimicrobial agents, with the exception of examination of the urine. in the current conjugate vaccine era, blood culture isolates are substantially more likely to be contaminants than to be pathogens. 7 substantial evidence suggests that obtaining blood cultures routinely from these children has little impact on outcome, although false-positive blood culture results lead to substantial unnecessary costs. 69, 70 the following approach seems appropriate. the febrile child should be carefully assessed for a focus of infection, including uti, and if a focus is found, the child should be treated according to likely pathogens. if the child appears toxic, appropriate cultures and diagnostic tests should be performed, and antimicrobial treatment (usually with cefotaxime, 150 mg/kg per day in divided doses every 8 hours, or ceftriaxone, 50 mg/kg once daily) should be initiated; some physicians add vancomycin (40 mg/kg per day in divided doses every 6 to 8 hours). most of these children should be hospitalized. management of the non-toxicappearing acutely febrile child: a 21st century approach changing epidemiology of serious bacterial infections in febrile infants without localizing signs changing epidemiology of bacteremia in infants aged 1 week to 3 months identification of infants unlikely to have serious bacterial infection although hospitalized for suspected sepsis serious bacterial infections in febrile infants 1 to 90 days old with and without viral infections influenza virus infection and the risk of serious bacterial infections in young febrile infants risk of serious bacterial infection in young febrile infants with respiratory syncytial virus infections diagnosis and outcomes of enterovirus infections in young infants risk factors for development of bacterial meningitis among children with occult bacteremia urinary tract infections in young febrile children management of febrile children in the age of the conjugate pneumococcal vaccine: a cost-effectiveness analysis should blood cultures be obtained in the evaluation of young febrile children without evident focus of bacterial infection? a decision analysis of diagnostic management strategies strategies for diagnosis and treatment of children at risk for occult bacteremia: clinical effectiveness and cost-effectiveness febrile infants: a 30-year odyssey ends where it started parents' versus physicians' values for clinical outcomes in young febrile children management of the young febrile child: a commentary on recent practice guidelines management of the non-toxicappearing acutely febrile child: a 21st century approach management of the febrile infant three months of age or younger viral and bacterial pathogens of suspected sepsis in young infants serious bacterial infections in febrile infants younger than 90 days of age: the importance of ampicillin-resistant pathogens changing epidemiology of serious bacterial infections in febrile infants without localizing signs changing epidemiology of bacteremia in infants aged 1 week to 3 months invasive pneumococcal disease in young children before licensure of 13-valent pneumococcal conjugate vaccine-united states progress toward eliminating haemophilus influenzae type b disease among infants and children-united states probability of bacterial infections in febrile infants less than three months of age: a meta-analysis the serious implications of high fever in infants during their first three months. six years' experience at yale-new haven hospital emergency room potential bacteremia in pediatric practice fever in the first eight weeks of life identification of infants unlikely to have serious bacterial infection although hospitalized for suspected sepsis the febrile infant outpatient management without antibiotics of fever in selected infants ambulatory care of febrile infants younger than 2 months of age classified as being at low risk for having serious bacterial infections febrile infants at low risk for serious bacterial infection-an appraisal of the rochester criteria and implications for management. febrile infant collaborative study group serious bacterial infections in febrile infants 1 to 90 days old with and without viral infections epidemiology of bacteremia in febrile infants in the united states the changing epidemiology of serious bacterial infections in young infants management and outcomes of care of fever in early infancy practice guideline for the management of infants and children 0 to 36 months of age with fever without source. agency for health care policy and research trends in perinatal group b streptococcal disease-united states influenza virus infection in infants less than three months of age influenza virus infection and the risk of serious bacterial infections in young febrile infants risk of serious bacterial infection in young febrile infants with respiratory syncytial virus infections diagnosis and outcomes of enterovirus infections in young infants bacteremia in febrile children seen in a "walk-in" pediatric clinic risk factors for development of bacterial meningitis among children with occult bacteremia complications of occult pneumococcal bacteremia in children unsuspected meningococcemia unsuspected bacteremia due to haemophilus influenzae: outcome in children not initially admitted to hospital bacteremia in febrile children under 2 years of age: results of cultures of blood of 600 consecutive febrile children seen in a "walk-in" clinic a comparative study of the prevalence, outcome, and prediction of bacteremia in children temperature greater than or equal to 40 c in children less than 24 months of age: a prospective study occult pneumococcal bacteremia: what happens to the child who appears well at reevaluation? reevaluation of outpatients with streptococcus pneumoniae bacteremia outcomes of febrile children without localising signs after pneumococcal conjugate vaccine prevalence of occult bacteremia in children aged 3 to 36 months presenting to the emergency department with fever in the postpneumococcal conjugate vaccine era an analysis of pediatric blood cultures in the postpneumococcal conjugate vaccine era in a community hospital emergency department incidence of occult bacteremia among highly febrile young children in the era of the pneumococcal conjugate vaccine: a study from a children's hospital emergency department and urgent care center occult bacteremia from a pediatric emergency department: current prevalence, time to detection, and outcome serotype prevalence of occult pneumococcal bacteremia urinary tract infections in young febrile children antimicrobial treatment of occult bacteremia: a multicenter cooperative study quantitative blood cultures in childhood bacteremia detection and quantitation of bacteremia in childhood comparison of acute-phase reactants in pediatric patients with fever early diagnosis of bacteremia by buffy-coat examinations relationship between the magnitude of bacteremia in children and the clinical disease temperature and total white blood cell count as indicators of bacteremia procalcitonin and c-reactive protein as diagnostic markers of severe bacterial infections in febrile infants and children in the emergency department history and observation variables in assessing febrile children role of the complete blood count in detecting occult focal bacterial infection in the young febrile child costs and infant outcomes after implementation of a care process model for febrile infants febrile children with no focus of infection: a survey of their management by primary care physicians hospitalization v outpatient treatment of young, febrile infants iatrogenic risks and financial costs of hospitalizing febrile infants management of young, febrile infants vulnerable children: parents' perspectives and the use of medical care the efficacy of routine outpatient management without antibiotics of fever in selected infants impact of rapid viral testing for influenza a and b viruses on management of febrile infants without signs of focal infection impact of a false positive blood culture result on the management of febrile children effects of obtaining a blood culture on subsequent management of young febrile children without an evident focus of infection key: cord-340357-gyvvcnuf authors: fallahi, hamid reza; keyhan, seied omid; zandian, dana; kim, seong-gon; cheshmi, behzad title: being a front-line dentist during the covid-19 pandemic: a literature review date: 2020-04-24 journal: maxillofac plast reconstr surg doi: 10.1186/s40902-020-00256-5 sha: doc_id: 340357 cord_uid: gyvvcnuf coronavirus is an enveloped virus with positive-sense single-stranded rna. coronavirus infection in humans mainly affects the upper respiratory tract and to a lesser extent the gastrointestinal tract. clinical symptoms of coronavirus infections can range from relatively mild (similar to the common cold) to severe (bronchitis, pneumonia, and renal involvement). the disease caused by the 2019 novel coronavirus (2019-ncov) was called covid-19 by the world health organization in february 2020. face-to-face communication and consistent exposure to body fluids such as blood and saliva predispose dental care workers at serious risk for 2019-ncov infection. as demonstrated by the recent coronavirus outbreak, information is not enough. during dental practice, blood and saliva can be scattered. accordingly, dental practice can be a potential risk for dental staff, and there is a high risk of cross-infection. this article addresses all information collected to date on the virus, in accordance with the guidelines of international health care institutions, and provides a comprehensive protocol for managing possible exposure to patients or those suspected of having coronavirus. since the first reported case in wuhan, china, in december 2019, coronavirus diseasehas widely spread to japan, korea, iran, and many european countries [1] . the world health organization (who) declared a pandemic in march 2020. as saliva is a main tool of spread, dentists are in danger of contracting covid-19. although the exact nature of this disease must be clarified in detailed studies, current knowledge of coronavirus infection should be shared without any restrictions. this article was written by an iranian team of oral and maxillofacial surgeons. as iran has many covid-19 patients, they have significant experience with this disease. maxillofacial plastic and reconstructive surgery is an open-access journal, and this type of important information can be shared via our publication platform without restrictions. coronaviruses are enveloped viruses with a positivesense single-stranded rna genome. their helical symmetry nucleocapsid is approximately 26-32 kb in size, making it the largest investigated genome among rna viruses [2, 3] . coronaviruses have a fundamental resemblance in their organization and genome expression [4] . previously, it was thought that coronaviruses only cause enzootic infections in a number of animals, including certain birds and mammals, but recent findings indicate that a variety of these viruses, including antigenic groups 1 (229e and nl63), antigenic groups 2 (oc43), and hku1, can infect humans [5, 6] . these viruses often lead to upper respiratory tract infection, frequently resulting in common cold symptoms. three specific strains of these viruses that are of zoonotic origin, including severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and 2019 novel coronavirus (2019-ncov), have recently caused lethal infections in humans [4, 6, 7] . coronavirus infections in humans mainly affect the upper respiratory tract and to a lesser extent the gastrointestinal tract. manifestations of coronavirus infections can range from relatively mild (similar to the common cold) to severe (bronchitis, pneumonia, and renal involvement) [8] (table 1) . the ability to infect humans is mainly due to the infection of peridomestic animals, which are considered intermediate hosts, nurturing recombination and mutation events as well as the development of genetic diversity among coronaviruses [29] . studies have suggested that the spike glycoprotein (s glycoprotein) plays an important role in host range restriction by attaching virions to the host cell membrane [30] . generally, coronaviruses primarily replicate in the respiratory and intestinal epithelial cells and subsequently cause cytopathic alterations [31] . since december 2019, numerous unexplained cases of pneumonia have been reported in china. the disease caused by 2019-ncov was called covid-19 by the who in february 2020 [32] . limiting the exposure of suspicious cases to the rest of society could be an effective strategy in the early outbreak phases. however, the subsequent worldwide virus spread and person-to-person transmission made the situation more complex and uncontrollable [33] . no detailed studies have been conducted to expound the pathogenicity of 2019-ncov on a molecular scale. however, exploratory data established via whole-genome sequencing and subsequent bioinformatics analyses revealed that 2019-ncov is phylogenetically related to sars-cov that was isolated for the first time in chinese horseshoe bats between 2015 and 2017 [34] [35] [36] . to a large extent, the clinical similarities of 2019-ncov infection with sars-cov infection are substantial. the incubation period of 2019-ncov has been estimated to be 1-14 days, and it has been shown that asymptomatic individuals may also be involved in the spread of this virus [26] [27] [28] . since the possibility of the transmission from asymptomatic carriers has been raised currently, checking body temperature only may not be enough to screen asymptomatic carriers. according to a recent report, temperature-based screening in the airport can detect only 46% of 2019-ncov carriers and the others were found during the self-isolation period after immigration [29] . to suppress the disease spread, a wide range of laboratory tests for immigrants and general population seems to be necessary. however, the infection rate from asymptomatic carriers has not been clarified until now. the primary non-specific reported symptoms of 2019-ncov infection at the prodromal phase are malaise, fever, and dry cough. the most commonly reported signs and symptoms are fever (98%), cough (76%), dyspnea (55%), and myalgia or fatigue (44%) [25, 26] . unlike patients with other human coronavirus infections (such as sars-cov), upper respiratory tract and intestinal manifestations such as sore throat, rhinorrhea, and diarrhea in those with 2019-ncov infection are infrequent [15, 25, 26] (fig. 1 ). the patient mean age is generally between 49 and 61 years. studies have shown that males are more likely to have this infection [25, 26, 37] . the lack of serious illness in youngsters is a characteristic of sars-cov infection, which is similarly observed in 2019-ncov infection [32] . increased exposures to 2019-ncov due to occupational requirements, for instance health care workers, maybe another factor contributing to the higher risk of infection. following the outbreak, the full 2019-ncov genomic sequence was released in public databases [38] . this facilitates the way for further pcr assays for virus detection. the who recommendations for outpatient cases and patients with more critical conditions respectively include rapid collection and nucleic acid amplification testing (naat) of respiratory samples including nasopharyngeal and oropharyngeal swabs as well as sputum and/or endotracheal aspirate or bronchoalveolar lavage [39] . table 2 presents recommended instructions that all practitioners in the field of dental care, including dentists, assistants, and others, should consider when treating patients or those suspected of having coronavirus. protocol figure 2 shows a protocol that can organize our approach to managing suspected or infected patients. the purpose of this protocol is to protect the entire dental care team, prevent any cross-infection in the office, inform health authorities active in the field of controlling and managing the disease, and ultimately provide the optimal medical and dental care for patients affected by the virus according to the cdc and the ada guidelines. the two main routes known for 2019-ncov transmission include (1) direct transmission (through coughing, sneezing, and inhalation of droplets) and (2) contact transmission (through contact with nasal, oral, and ocular mucosa) [43] . typical clinical manifestations of covid-2019 do not comprise ocular symptoms. however, conjunctival sample analysis has revealed that the transmission of 2019-ncov is not limited to the respiratory tract route [26] , but ocular exposure is also an effective method of virus transmission [44] . moreover, studies have revealed that via direct/indirect contact or course and/or droplets, respiratory viruses such as 2019-ncov may be transmitted from human to human. studies have also shown direct and indirect transmission of 2019-ncov through saliva [45] . for a comprehensive understanding of the transmission dynamics of 2019-ncov, it is also important to know that this virus is also transmissible through asymptomatic patients [34] . the remarkable feature of 2019-ncov is that its rna is detectable via quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) in stool samples after the first week of infection [46] . however, the aerosol and fecal-oral transmission routes, which carry more public concern, still need further investigation and confirmation [47] (fig. 3) . new evidence suggests that 2019-ncov may be transmitted directly from human to human via respiratory secretion containing droplets [44, 48] . virus transmission through contact and fomites is also likely [44, 48] . to et al. [44] reported that using the viral culture [49] . since 2019-ncov effectively uses ace2 receptor for cell invasion, it can promote human-tohuman transmission [50] . ace2+ cells are abundantly present all over the respiratory tract. ace2+ epithelial cells present in the salivary glands were considered one of the main targets of sars coronavirus infection. similarly, 2019-ncov may also use the same mechanism to induce infection, although definitive judgment regarding this issue needs further study [51] . due to close face-to-face contact with patients and frequent utilization of sharp devices, dental personnel are repeatedly exposed to respiratory tract secretions, blood, saliva, and other contaminated body fluids and are always at risk for 2019-ncov infection. 2019-ncov transmission in dental settings occurs through four major routes: (1) direct exposure to respiratory secretions containing droplets, blood, saliva, or other patient materials; (2) indirect contact with contaminated surfaces and/or instruments; (3) inhalation of suspending airborne viruses; and (4) mucosal (nasal, oral, and conjunctival) contact with infection-containing droplets and aerosols that are propelled by coughing and talking without a mask [51] [52] [53] [54] [55] (fig. 4) . the most important concern in dental clinics is the transmission of 2019-ncov via droplets and aerosol because, despite all of the precautions taken, it is almost impossible to reduce droplet and aerosol production to zero during dental procedures [54] . dental handpieces utilize high-speed gas to rotate with running water, which leads to the generation of a considerable amount of droplets and aerosol mixed with patients' saliva and/ or blood [55] . therefore, it can be deduced that 2019-ncov is capable of transmitting through dental practice; this transmission can be from patients to clinic staff or other patients at the clinic [56] . research has shown that coronaviruses can remain on metal, glass, and plastic surfaces for several days [52, 57] . therefore, as surfaces in dental clinics serve as venues for droplets and aerosol mixed with patients' saliva and/ or blood, they can effectively help spread infection. coronaviruses can actively maintain their virulence at room temperature from 2 h up to 9 days. their activity at 50% humidity was significantly higher than 30%. therefore, in the dental environment, it seems that keeping surfaces clean and dry will play a significant role in preventing 2019-ncov transmission [52] . table 2 standard precautions based on cdc and ada guidelines for dentists on the coronavirus disease [40] [41] [42] postponing following the announcement of disease outbreak by international or local authorities, dentists can play a significant role in disrupting the transmission chain, thereby reducing the incidence of the disease by simply postponing all non-emergency dental care for all patients. where to treat all dental care should be provided in an outpatient dental setting with a minimum of six air changes per hour, such as a hospital with dental care services or customized clinics equipped for covid-19 patients. primary non-specific reported symptoms of 2019-ncov infection at the prodromal phase are malaise, fever, and dry cough. the most commonly reported signs and symptoms are fever (98%), cough (76%), dyspnea (55%), and myalgia or fatigue (44%). they also may have traveled to one of the countries considered disease hotspots in the prior 14 days or have encountered people from those countries or people who have traveled to those countries. some patients may be asymptomatic or have unexpected symptoms such as diarrhea. since it is not possible to know the etiology of each patient's illness, it is crucial to follow the guidelines and precautions at all times during the disease outbreak. be alert, identify patients with respiratory illnesses, and provide them a disposable surgical face mask. isolate them in a room with the door closed. limit their direct contact with others. isolated patients must wear masks outside their room. isolate suspected patients before and during care to minimize their direct contact with other patients and staff and immediately report any cases to local and state public health authorities. to prevent 2019-ncov transmission, dental practices should adhere to the infection control protocol, including hand hygiene, providing tissues and no-touch receptacles, and providing face masks for coughing patients. dental health care personnel should wear white coats, gowns, head caps, goggles, face shields, masks, latex gloves, and impermeable shoe covers to prevent exposure. disposable masks should be substituted between patients or even during treatment if they get wet. since covid-19 recommendations may change rapidly with increasing information about the disease, the ada recommends checking for updates on the cdc's coronavirus infection control web page for health care professionals. the cdc strongly recommends that all health care staff, including dentists and personnel, should receive the flu vaccine and that staff with influenza must not report to work. since the fecal-oral route is considered one of the 2019-ncov transmission routes, attention to hand hygiene before, during, and after dental practice is important. dentists should exercise extreme caution to avoid contact with their own facial mucosal surfaces including their eyes, mouth, and nose. since transmission of airborne droplet is considered one of the main routes of infection spread, application of personal protective equipment such as masks, protective goggles, gowns, helmet, gloves, caps, face shields, and shoe covers is strongly recommended for all health care personnel. covid-19 patients should not be treated in a regular dental care setting without special considerations. unexpected circumstances may occur when the dentist cannot delay treatment or refer the patient to the appropriate medical institution. under such circumstances, special protective clothing such as hazardous materials (hazmat) suits are required. if hazmat suits are not available, white coats, gowns, head caps, protective eyewear, face shields, masks, latex gloves, and virus-proof shoe covers should be used [56] . the effect of chlorhexidine, which is commonly used for pre-procedural mouth washing in dental practice, has not yet been demonstrated to be capable of eliminating 2019-ncov. however, oxidative agents containing mouth rinses with 1% hydrogen peroxide or 0.2% povidone-iodine are recommended. pre-procedural use of mouthwash, especially in cases of inability to use a rubber dam, can significantly reduce the microbial load of oral cavity fluids [58] . using rubber dams due to the creation of a barrier in the oral cavity effectively reduces the generation of droplets and aerosol mixed with patient saliva and/or blood in 1 m diameter of the surgical field by 70% [59] . following the placement of the dam, extra high-volume suction is also required for maximum prevention of aerosol and spatter from spreading [60] . if it is not possible to use rubber dams for any reason, manual tools such as carisolvs or hand scalers are preferable. throughout the covid-19 pandemic, the use of any dental handpieces that do not have an anti-retraction function should be avoided. for emergency treatment, anti-retraction handpieces designed with anti-retractive valves can play an effective role in preventing the diffusion and dispersion of droplets and aerosol [60, 61] . since there is still little information available regarding 2019-ncov, relatively similar genetic features between 2019-ncov and sars-cov indicate that the novel coronavirus can be vulnerable to disinfectants such as sodium hypochlorite (1000 ppm or 0.1% for surfaces and 10,000 ppm or 1% for blood spills), 0.5% hydrogen peroxide, 62-71% ethanol, and phenolic and quaternary ammonium compounds if utilized in accordance with the manufacturer's instructions. studies show that other biocidal agents such as 0.05-0.2% benzalkonium chloride or 0.02% chlorhexidine digluconate probably have lower efficiency. in addition to the type of disinfectant, paying attention to other factors such as the duration of use, dilution rate, and especially the expiration time following the preparation of the solution according to the manufacturer's instructions is also crucial. prior to any inappropriate accumulation, dental office waste should be routinely transported to the institution's temporary storage facility. reusable tools and equipment must be properly pre-treated, cleaned, sterilized, and properly stored until the next use. dental waste resulting from the treatment of suspected or confirmed 2019-ncov patients is considered medically infectious waste that must be strictly disposed of in accordance with the official instructions using double-layer yellow medical waste package bags and "gooseneck" ligation. following the announcement of the disease outbreak by international or local authorities, dentists can play a significant role in disrupting the transmission chain, thereby reducing the incidence of disease by simply postponing all non-emergency dental care for all patients. dental professionals must be fully aware of 2019-ncov spreading modalities, how to identify patients with this infection, and, most importantly, self-protection considerations. the effect of chlorhexidine, which is commonly used for preprocedural mouth washing in dental practice, has not yet been demonstrated to be capable of eliminating 2019-ncov. however, the prescription of oxidative agents containing mouth rinses such as 1% hydrogen peroxide or 0.2% povidone is recommended. a higher rate of virus exposure because of occupational commitments in health care workers is considered a key factor associated with the increased risk of infection. yen my et al asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute 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atmospheric bacterial contamination severe acute respiratory syndrome and dentistry: a retrospective view publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations maxillofacial plastic and reconstructive surgery prof. esmaeil yazdi passed away due to coronavirus infection few days ago. he was one of the iranian society of omfs founder 60 years ago. the authors send sincere condolence to his family and colleagues. the authors also appreciate his great contribution on the progress of maxillofacial surgery. the article was written by hrf and bc. dz collected the data. illustrations were drawn by sok and bc. sok and ksg corrected the article and performed the critical review. the authors read and approved the final manuscript. not applicable.availability of data and materials not applicable.ethics approval and consent to participate not applicable. consent for publication not applicable. the authors declare that the authors have no competing interests as defined by nature research or other interests that might be perceived to influence the results and/or discussion reported in this paper. key: cord-305890-mdwjrfzp authors: bönsch, claudia; kempf, christoph; mueller, ivo; manning, laurens; laman, moses; davis, timothy m. e.; ros, carlos title: chloroquine and its derivatives exacerbate b19v-associated anemia by promoting viral replication date: 2010-04-27 journal: plos negl trop dis doi: 10.1371/journal.pntd.0000669 sha: doc_id: 305890 cord_uid: mdwjrfzp background: an unexpectedly high seroprevalence and pathogenic potential of human parvovirus b19 (b19v) have been observed in certain malaria-endemic countries in parallel with local use of chloroquine (cq) as first-line treatment for malaria. the aims of this study were to assess the effect of cq and other common antimalarial drugs on b19v infection in vitro and the possible epidemiological consequences for children from papua new guinea (png). methodology/principal findings: viral rna, dna and proteins were analyzed in different cell types following infection with b19v in the presence of a range of antimalarial drugs. relationships between b19v infection status, prior 4-aminoquinoline use and anemia were assessed in 200 png children <10 years of age participating in a case-control study of severe infections. in cq-treated cells, the synthesis of viral rna, dna and proteins was significantly higher and occurred earlier than in control cells. cq facilitates b19v infection by minimizing intracellular degradation of incoming particles. only amodiaquine amongst other antimalarial drugs had a similar effect. b19v igm seropositivity was more frequent in 111 children with severe anemia (hemoglobin <50 g/l) than in 89 healthy controls (15.3% vs 3.4%; p = 0.008). in children who were either b19v igm or pcr positive, 4-aminoquinoline use was associated with a significantly lower admission hemoglobin concentration. conclusions/significance: our data strongly suggest that 4-aminoquinoline drugs and their metabolites exacerbate b19v-associated anemia by promoting b19v replication. consideration should be given for choosing a non-4-aminoquinoline drug to partner artemisinin compounds in combination antimalarial therapy. human parvovirus b19 (b19v) is a nonenveloped icosahedral virus with a single-stranded dna genome which has been classified within the erythrovirus genus of the parvoviridae family. the virus is readily transmitted via the respiratory route and has a worldwide distribution. seroprevalence increases with age and 50%-80% of adults have detectable b19-specific antibody. since its discovery in 1975 [1] , b19v has been associated with an expanding range of clinical disorders that reflect the patient's immunologic and hematologic status. in healthy individuals, b19v typically causes a mild childhood febrile illness known as erythema infectiosum or fifth disease. more severe manifestations of b19v infection are arthropathies, aplastic anemia, hydrops fetalis and fetal death [2] . viremia occurs during the first week of infection. the virus has a predilection for bone marrow erythroid progenitor cells. at the height of viremia, there is an abrupt fall in the reticulocyte count and anemia can supervene. although this is rarely apparent in healthy patients, it can have serious clinical consequences where there is pre-existing anemia [2] . a case in point is malaria. b19v co-infection has been considered a significant risk for severe anemia in children living in malaria-endemic regions [3] . studies examining the inter-relationship between malaria, b19v infection and anemia have, however, produced inconsistent results. in a retrospective study from papua new guinea (png) [4] , 60% of children ,2 years of age and 90% of 6 year-olds were b19v seropositive. b19v infection was significantly associated with severe anemia even in the absence of risk factors including malaria. similar results were obtained in children from the republic of niger [5] . however, studies from malawi and kenya did not show evidence that b19v infection contributes to anemia in children and the seroprevalence of b19v was relatively low [6, 7] . at the time the studies were performed in png [4] and niger [5] , chloroquine (cq) was used as first-line treatment for malaria in these countries. in addition, a serosurvey performed in eritrea at a time when cq was the first-line agent also revealed an unusually high b19v seroprevalence [8] . by contrast, cq had been discontinued in malawi because of resistance of local strains of plasmodium falciparum and its use was declining in kenya when the b19v seroprevalence studies were conducted [6, 7] . although cq has, in addition to antimalarial efficacy, broad antiviral activity [9, 10] , it also enhances semliki forest virus (sfv) and encephalomyocarditis virus (emcv) infection in mice [11] , and epstein-barr virus (ebv) expression [12] . the latter effect is thought to play a role in the higher incidence of ebv-induced burkitt's lymphoma in malaria-endemic areas where cq is in common use [13] . we hypothesize, therefore, that geo-epidemiological differences in b19v seroprevalence and pathogenic potential result from cqassociated enhanced replication. to test this hypothesis, we examined the effect of cq and other commonly-used antimalarial drugs on b19v replication in three different cultured cell lines. in addition, we examined the relationship between b19v infection and use of 4-aminoquinoline drugs in a sample of children from png who were hospitalized with severe anemia. the results provide evidence that cq and aq aggravate b19v-associated anemia by promoting b19v replication. all patients were participants in a prospective observational and genetic study of severe pediatric infections (http://www.malariagen. net/home/). written informed consent was obtained from each parent/guardian. ethical approval for the study was obtained from both the png institute of medical research institutional review board and the medical research advisory committee of the png department of health. the study was conducted in accordance with the helsinki declaration. a b19v-infected plasma sample was obtained from our donation center (genotype 1; csl behring ag, charlotte, nc) and was concentrated by ultracentrifugation through 20% (w/v) sucrose. ut7/epo cells were cultured in rpmi, 10% fcs and 2 u/ml of recombinant human erythropoietin (epo; janssen-cilag, midrand, south africa). hepg2 cells were cultured in mem supplemented with 10% fcs. bone marrow mononuclear cells (bmmcs) were obtained as frozen stocks from stemcell technologies (vancouver, bc, canada) and were cultured in imdm, 10% fcs and 2 u/ml of epo. all cells were incubated at 37uc and in an atmosphere of 7.5% co 2 . all drugs were purchased from sigma (st. louis, miss). chloroquine diphosphate (cq), primaquine diphosphate (pq) and amodiaquine dihydrochloride dihydrate (aq) were dissolved in water, piperaquine (ppq) in 5% lactic acid, mefloquine hydrochloride (mq) in dmso, lumefantrine (lft) in dimethylformamide and artesunate (at) and pyrimethamine (pm) in ethanol. the final drug concentration ranges used in the b19v infectivity assay were 5-100 mm for pq and lft, aq and pm, 0.05-20 mm for mq, 2.5-100 mm for at, 1-100 mm for ppq and 0.05-100 mm for cq. the highest concentration of the drugs did not exceed 0.2% of total culture volume. b19v infectivity was assessed in two different cell lines, namely megakaryoblastic leukemia ut7/epo cells and the human hepatocellular liver carcinoma cell line hepg2, and in primary bone marrow mononuclear cells (bmmcs). ut7/epo cells are the most susceptible cell line to b19v infection [14] . although viral rna, dna and proteins can be detected in b19v-infected ut7/ epo cells, viral replication is restricted to a level that does not normally allow production of virus progeny. the hepg2 cell line was chosen because it allows virus binding and probably internalization, but it is non-permissive for b19v infection [15] . bmmcs have been shown to support b19v infection, although only a minor subset of these cells is permissive for b19v [16] . ut7/epo, hepg2 and bone marrow mononuclear cells (bmmcs) (3610 5 ) were infected with 20,000 dna-containing b19v particles per cell (5,000 for bmmcs), corresponding to an moi of approximately 20 (5 for bmmcs) in the presence of the pre-determined concentrations of the selected drugs. for viral rna and dna analysis, cells were collected at different postinfection times as indicated in the figure legends. total poly (a) + mrna was isolated and viral ns1 mrna quantified as previously described [17] . total dna was extracted and viral dna was quantified using established methods [17] . ut7/epo cells were infected as specified above in the presence of 0 or 25 mm cq. at increasing post-infection times (see figure 1c ), cells were lysed in protein loading buffer and total proteins were resolved by sodium dodecyl sulfate (sds)-10% polyacrylamide gel electrophoresis (page). after transfer to a pvdf membrane, the blot was probed with a mouse antibody against b19v structural proteins (1:2,000 dilution; us biologicals, swampscott, ma), followed by a horseradish peroxidase-conjugated secondary antibody (1:20,000 dilution). the viral structural proteins were visualized with a chemiluminescence system (pierce, rockford, il). additionally, viral protein expression was examined by immunofluorescence, as previously described [17] . human parvovirus b19 (b19v) is typically associated with a childhood febrile illness known as erythema infectiosum. the infection usually resolves without consequence in healthy individuals. however, in patients with immunologic and/or hematologic disorders, b19v can cause a significant pathology. the virus infects and kills red cell precursors but anemia rarely supervenes unless there is pre-existing anemia such as in children living in malariaendemic regions. the link between b19v infection and severe anemia has, however, only been confirmed in certain malaria-endemic countries in parallel with chloroquine (cq) usage. this raises the possibility that cq may increase the risk of severe anemia by promoting b19v infection. to test this hypothesis, we examined the direct effect of cq and other commonly used antimalarial drugs on b19v infection in cultured cell lines. additionally, we examined the correlation between b19v infection, hemoglobin levels and use of cq in children from papua new guinea hospitalized with severe anemia. the results suggest strongly that cq and its derivatives aggravate b19v-associated anemia by promoting b19v replication. hence, careful consideration should be given in choosing the drug partnering artemisinin compounds in combination antimalarial therapy in order to minimize contribution of b19v to severe anemia. chloroquine promotes b19v infection www.plosntds.org with pbs to remove unbound virus and incubated at 37uc in the presence or absence of antimalarials. at increasing post-internalization times from 1 to 7 h, the cells were washed 2 times with pbs and the amount of intact viral dna was quantified as specified above. we studied 111 children ,10 years of age with severe anemia (hemoglobin ,50 g/l) and 89 community-based age and sexmatched healthy control children with a hemoglobin .100 g/l. those with severe anemia represented a subset (15.9%) of all 697 children admitted to modilon hospital, madang province on the north coast of png with any severe illness during the period of study. modilon hospital is a referral hospital and the only provincial facility able to manage severely ill children. all such children were given treatment as recommended under png national treatment guidelines including intramuscular artemether for malaria infection. the healthy controls were recruited from the same villages as the patients and were slide-negative for malaria. as well as a hemoglobin concentration (haemocueh, angelholm, sweden) at presentation, plasma was assayed for b19v igm by eia kit (biotrin international) and, in those with severe anemia, for viral dna using two specific oligonucleotide primers [4] . we did both tests because viremia starts to decline once specific igm is produced around day 9 after inoculation, while virus-induced marrow suppression can last for another 2-3 weeks [4] . thus, although the simultaneous detection of b19v igm and dna is strongly indicative of acute infection, we did not want to exclude children with evidence of recent but resolving infection as a contributor to severe anemia. in those who were b19v igm or pcr positive, plasma was assayed for chloroquine and amodiaquine and their respective active desethyl metabolites using a validated high performance liquid chromatography assay [18] . the assay had a limit of quantitation of 1 mg/l for each analyte. effect of cq on b19v replication in ut7/epo cells cq increased the production of b19v ns1 gene transcription after 24 h incubation. at cq concentrations ranging from 10 to 75 mm, ns1 rna increased up to 1,170% ( figure 1a) . similarly, kinetic studies in the presence of 25 mm of cq showed that viral dna synthesis was more rapid and extensive than in untreated cells ( figure 1b) . the expression of structural viral proteins in extracts of infected ut7/epo cells was also increased in the presence of 25 mm cq ( figure 1c ). viral protein expression was immunofluorescence experiments showed, that in the presence of cq a larger number of cells were infected by b19v (fig. 1d ). in the absence of cq, only a minor amount of viral dna synthesis was observed starting at 120 h post-infection. no viral rna could be detected, confirming the poor permissiveness of this cell line for b19v infection. however, in the presence of increasing concentrations of cq, viral dna synthesis increased progressively reaching 2,290% at cq concentrations of 60 mm (figure 2a) . kinetic studies showed that, in the presence of cq (25 mm), viral dna was detected earlier than in untreated cells ( figure 2b ). viral ns1 rna was only detectable in cq-treated cells ( figure 2c ). the presence of cq (25 mm) accelerated b19v rna synthesis. however, in cq-treated cells, viral rna transcription ceased abruptly and was followed by progressive degradation resembling apoptosis ( figure 2d ). detection of phosphatidyl serine-anexin v complexes by fluorescence microscopy confirmed that the infected bmmcs entered the apoptotic pathway (data not shown). therefore, the effect of cq in cultured bmmcs could not be evaluated at stages later that 10 h post-infection. the apoptotic effects were not observed in the cell lines ut7/epo and hepg2 at concentrations up to 60 mm (data not shown). with the exception of the cq-analogue aq which enhanced b19v infection at concentrations above 5 mm, no other antimalarial drug had a significant effect on b19v infection ( figure 3 ). mild inhibition was observed in the presence of at, while mq inhibited the infection at concentrations .10 mm. these effects were also observed when the drugs were added 4 to 7 h post-infection (data not shown), raising the possibility that b19v infectivity was reduced by a drug-specific cytotoxicity. the enhancement of b19v infection by cq decreased progressively with increases in the time at which cq was added, with no detectable effect at 8-9 h post-infection ( figure 4a ). these data indicate that cq acts early in b19v infection. at progressive times after internalization of b19v, the cells were washed and the viral dna was quantified. in untreated cells, a progressive degradation of the incoming viral dna was evident. however, in the presence of cq (25 mm) or aq (20 mm), degradation of incoming particles was prevented or minimized ( figure 4b ). [18, 19] , this suggests that most of these children had been treated with either cq or aq within the previous 6 weeks. hemoglobin concentrations by b19v igm/pcr and 4aminoquinoline status are shown in figure 5 . the lowest concentrations were in the 5 children who were both igm and pcr positive (i.e. had acute b19 infections). these children had a similar mean age (53 vs 57 months), body weight (15 kg in both groups) and spleen size (5 vs 7 cm) to those children who were either igm or pcr positive (p.0.37 by mann-whitney u test) and the percentages with malaria were similar (40.0 vs 44.4%; p = 0.63 by fisher's exact test). in patients who were igm or pcr positive (indicating a recent but not necessarily acute infection or one which was acute but early in its course), 4-aminoquinoline use was associated with a significantly lower admission hemoglobin concentration (p = 0.037). although the number of patients treated with aq was small and restricted to children who were igm positive but pcr negative, they had some of the highest hemoglobin concentrations in this subgroup. this suggests that, consistent with the in vitro data, cq had a greater suppressive effect on bone marrow than aq in our patients. severe anemia is a common and life-threatening complication of malaria in children living in endemic areas [20] . b19v coinfection has been identified as a major factor in its pathogenesis [3] , but there are significant regional differences in its seroprevalence and resulting clinical impact [4] [5] [6] [7] [8] despite the fact that b19v infection is a common childhood illness. because b19vassociated severe anemia appears to parallel local use of cq as first-line treatment for malaria, we hypothesized that cq promotes b19v replication and that, as a consequence, it contributes indirectly to severe anemia. although a more profound clinical study would be necessary, the present results provide already a strong evidence for this hypothesis. apart from its antimalarial effects, cq has a wide antiviral activity. one of the most important mechanisms of action against viruses is the alkalinization of the endosomal vesicles. in this way, cq is active against viruses that require a low ph step for cell entry, such as flavivirus, retrovirus and coronavirus [9] . all parvoviruses studied to date also depend on endosomal acidification for cell entry because it facilitates capsid structural transitions [21, 22] , and in particular the externalization of the n-terminal region of vp1 which is required for endosomal escape and nuclear targeting [23] . accordingly, cq inhibits parvovirus infections. however, we have previously shown that b19v is unique among parvoviruses in that n-vp1 is already externalized on receptor binding [17] and thus not dependent on a low endosomal ph for this critical conformational change. b19v is also unique among parvoviruses for its higher sensitivity to acid degradation [24] . consequently, cq-associated alkalinization of endosomal vesicles would be expected to minimize the acidic degradation of incoming b19v particles. our data confirm that the intracellular degradation of b19v is prevented or minimized in the presence of cq or aq. however, other lysosomotropic drugs such as ammonium chloride or bafilomycin a1, which also raise the endosomal ph, had an inhibitory effect on b19v infection in our in vitro system (data not shown). therefore, the mechanism underlying the stabilization of b19v by cq or aq is likely to extend beyond ph-neutralizing activity to destabilization of endosome/lysosome membranes typically observed in cq-treated cells. in this way, cq would facilitate the endosomal escape of b19v before it reaches the degradative lysosomal compartment and increase the number of particles that can target the nuclei for replication. this is of particular importance since nuclear targeting chloroquine promotes b19v infection www.plosntds.org has been identified as a major limiting factor in parvovirus infections [22, 25] . the plausibility of our in vitro observations as an explanation of epidemiological data depends on the pharmacological properties of cq, especially tissue concentrations. the in vitro enhancement of b19v infection by cq was achieved at concentrations (10-75 mm) that were well above those achieved in plasma after therapeutic doses in children (typically ,5 mm) [18] . however, chloroquine promotes b19v infection www.plosntds.org b19v does not replicate in plasma but in tissues, primarily the bone marrow. cq concentrations in bone marrow are substantially higher than in plasma [26] and have been measured at approximately 100 mm in animal studies [27] . this could reflect, in part, concentration of the drug within precursor cells such as has been observed in circulating erythrocytes [28] . the long terminal elimination half-life of cq (around 10 days in children) [18] means that conditions favorable to b19v viral replication in bone marrow may persist for several weeks after dosing. in many malaria-endemic regions, antimalarial therapy is given empirically to febrile children without blood smear confirmation. ironically this might include fever due to b19v itself. the administration of frequent courses of cq may mean that a child spends long periods of each year at risk of cq-associated enhanced b19v viremia and its consequences such as anemia. aq is a long half-life 4-aminoquinoline compound like cq and also promoted b19v replication in our in vitro experiments. other drugs tested, including primaquine (an 8-aminoquinoline) and mefloquine (a methanol quinoline), did not influence b19v infection in vitro, suggesting that the effect is specific to 4aminoquinoline compounds. some other viral infections (sfv, figure 3 . effect of different antimalarial drugs on b19v infection. ut7/epo cells were infected with b19v at 4uc for 2 h. the cells were washed with pbs to remove unbound virus and incubated at 37uc in the presence of different drugs. all drugs were used in concentrations ranging from 0 to 100 mm. after 24 h, the amount of b19v ns1 rna was quantified. the data are expressed as the percentage of the value obtained in untreated cells (dotted line) averaged for two independent experiments. sd bars are shown. doi:10.1371/journal.pntd.0000669.g003 parvovirus-like particles in human sera clinical aspects of parvovirus b19 infection parvovirus infection, malaria, and anemia in the tropics -a new hidden enemy? parvovirus b19 infection contributes to severe anemia in young children in papua new guinea human parvovirus infection in children and severe anemia seen in an area endemic for malaria parvovirus b19 infection does not contribute significantly to severe anemia in children with malaria in malawi severe anemia in children living in a malaria endemic area of kenya seroprevalence of viral childhood infections in eritrea effects of chloroquine on viral infections: an old drug against today's diseases? recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the 21st century chloroquine enhances replication of semliki forest virus and encephalomyocarditis virus in mice chloroquine enhances epstein-barr virus expression antimalarial drugs and burkitt's lymphoma development of an improved method of detection of infectious parvovirus b19 hepg2 hepatocellular carcinoma cells are a non-permissive system for b19 virus infection human b19 erythrovirus in vitro replication: what's new? interaction of parvovirus b19 with human erythrocytes alters virus structure and cell membrane integrity pharmacokinetics and efficacy of piperaquine and chloroquine in melanesian children with uncomplicated malaria pharmacokinetic and pharmacodynamic study of amodiaquine and its two metabolites after a single oral dose in human volunteers pathophysiology of severe malaria in children intracellular route of canine parvovirus entry low phdependent endosomal processing of the incoming parvovirus minute virus of mice virion leads to externalization of the vp1 n-terminal sequence (n-vp1), n-vp2 cleavage, and uncoating of the full-length genome parvoviral host range and cell entry mechanisms molecular mechanism underlying b19 virus inactivation and comparison to other parvoviruses exploitation of microtubule cytoskeleton and dynein during parvoviral traffic toward the nucleus tissue distribution of chloroquine, hydroxychloroquine, and desethylchloroquine in the rat tissue distribution of subcutaneously administered chloroquine in the rat pharmacokinetics of chloroquine in thais: plasma and red-cell concentrations following an intravenous infusion to healthy subjects and patients with plasmodium vivax malaria guidelines for the treatment of malaria. geneva: world health organization we are grateful to the png children and their families for their participation, staff at modilon hospital for assistance with collection of clinical data and samples, dr. anna rosannas for pcr assays, and emeritus prof. ken ilett and dr. madhu page-sharp for 4-aminoquinoline and metabolite assays. emcv and ebv) [11, 12] are enhanced by cq but not by other 4-aminoquinoline antimalarial drugs.we were able to obtain preliminary human data that are consistent with our laboratory findings. in our 200 unselected png children who were participants in a case-control study of severe pediatric infections, we confirmed previous reports that b19v seropositivity is associated with severe anemia and that the lowest hemoglobin concentrations are in those children who had acute infections (i.e. both igm and pcr positive) [4] . although there were limited numbers, there was some evidence that prior 4aminoquinoline, especially cq, use exacerbates b19v-associated severe anemia apart from in those igm-and pcr-positive cases who were presumably at the stage of maximal viral replication and consequent bone marrow suppression. properly designed epidemiological studies in larger, non-convenience samples are, however, needed to confirm these findings.although cq is a safe and inexpensive antimalarial drug, the increasing emergence of resistant p. falciparum and p. vivax has seen its use decline throughout the tropics. our data suggest that the prevalence of severe malarial anemia should also fall as a result. however, when an effective b19v vaccine becomes available, this should be considered a priority intervention where pediatric b19v seroprevalence rates are high and other causes of anemia such as nutritional deficiency and intestinal parasitic infection are present. artemisinin-based combination therapy (act) is the current who-recommended first-line treatment for uncomplicated malaria [29] . our data suggest that, pending more definitive in vivo data including appropriately designed clinical trials, a non-4aminoquinoline drug should be preferred to partner the artemisinin derivative so that the contribution of b19v to severe anemia is minimized. conceived and designed the experiments: cb im tmed cr. performed the experiments: cb lm ml cr. analyzed the data: cb ck im tmed cr. wrote the paper: tmed cr. key: cord-345339-kyboibtq authors: steiner, israel; nisipianu, puiu; wirguin, itzhak title: infection and the etiology and pathogenesis of multiple sclerosis date: 2001 journal: curr neurol neurosci rep doi: 10.1007/s11910-001-0030-x sha: doc_id: 345339 cord_uid: kyboibtq multiple sclerosis (ms) currently defies clinical and scientific definitions, and carries a prognosis that remains practically unchanged despite many years of intensive research. although the prevailing dogma is that ms is an immune-mediated condition, it fulfills none of the criteria of an autoimmune disease. on the other hand, there is enough significant data to suggest that infectious agents(s) could be involved in either direct damage to the white matter or induce inflammatory responses that secondarily affect the brain. our goal here is to review the data supporting the possibility that infection has a critical role in the disease, examine the list of potential candidates that have been suggested, and outline an approach regarding the potential role of infectious agents in the etiology and pathogenesis of ms. despite years of investigation, no direct evidence has been found to incriminate a specific etiology and pathogenesis for multiple sclerosis (ms). based on meager evidence [1•] and on extrapolation, an autoimmune pathogenesis has been hypothesized. however, we feel that autoimmune pathogenesis is not the likely culprit; instead, we believe that infection is more likely responsible for the damage to the central nervous system (cns). we review the data for an infectious process and examine the list of potential candidates. multiple sclerosis is a chronic, inflammatory white matter disease of the cns that usually affects young adults. successive sets of diagnostic criteria, essentially requiring objective evidence of at least two distinct lesions of cns white matter (the last set almost 20 years old), allude not only to the absence of a pathognomonic test for ms, but also to the lack of adequate evidence for the reliability of the various criteria. of note is the requirement for lack of an alternative diagnosis [2] , a criterion that imposes an element of "diagnosis by exclusion." the white matter is progressively destroyed, leaving some of the victims incapacitated for life. for many years, the leading, and practically dogmatic, hypothesis has been that ms is an autoimmune condition and hence, that its symptoms and progression could be ameliorated by immunomodulating strategies. however, while the field of neuroimmunology has thrived, little progress has been made towards understanding ms. the current diagnostic criteria accommodate a wide range of conditions. indeed, the extremely variable course and prognosis of the disease suggest that what is considered today a homogenous and consistent entity of similar etiology and pathogenesis, is, in fact, a syndrome caused by various etiologies and multiple pathogenic mechanisms [1•] . pierre marie, charcot's favorite student, (but not his successor, as erroneously suggested by several reviews), was the first, at the end of the 19th century, to raise the possibility that ms is caused by an infection [3] . since then, the amount of information in this realm is overwhelming. our goal here is not to quote the copious literature, but to delineate concepts, outline an approach, and provide some food for thought regarding the possible role of infectious agents in the etiology and pathogenesis ms. basically, an infective process can damage the tissue via two major avenues: directly, due to the pathogen present in the tissue during the disease; or indirectly, where the infection is only the trigger, and the damage occurs while the infection has already been cleared and removed from the organism. each alternative has different histologic, serologic, and epidemiologic characteristics. multiple sclerosis lesions (plaques) are multifocal and mainly perivenous [4••] . their distribution, though random enough to merit conflicting generalizations and conclusions, can reflect the route of penetration of infection. the myelin loss, with neurons and axons being relatively spared, which is characteristic of the ms lesion, is associated with mononuclear inflammatory cell infiltrates within active plaques and in perivascular spaces. macrophages and t lymphocytes are present in the center of a new lesion, which also shows reduction in the number of oligodendrocytes, reactive astrocytes, and microglial cells. obviously, these findings may accompany an active ongoing infectious process. the predilection of the inflammation for the white matter is a multiple sclerosis (ms) currently defies clinical and scientific definitions, and carries a prognosis that remains practically unchanged despite many years of intensive research. although the prevailing dogma is that ms is an immune-mediated condition, it fulfills none of the criteria of an autoimmune disease. on the other hand, there is enough significant data to suggest that infectious agents(s) could be involved in either direct damage to the white matter or induce inflammatory responses that secondarily affect the brain. our goal here is to review the data supporting the possibility that infection has a critical role in the disease, examine the list of potential candidates that have been suggested, and outline an approach regarding the potential role of infectious agents in the etiology and pathogenesis of ms. relatively uncommon feature of acute viral infections of the cns, and has been noted mainly in certain chronic viral infections (see following). this discipline has provided compelling evidence for an environmental factor in ms. for example, disease distribution in different geographic regions can be classified according to climate [5•] . although differences in quality of healthcare systems and recording in different countries may temper this finding, it seems that there is a high prevalence (or risk) in temperate countries (northern europe, united states, canada, southern australia, and new zealand), a low prevalence in warm, equatorial regions (the caribbean, mexico) and a medium prevalence in between (southern europe). migration might also induce epidemics. probably the best-studied example is the outbreak of an ms epidemic in the faroe islands following stationing of british troops there during the second world war [6] . migration studies have also suggested that age at the time of migration is important. in many instances, when individuals migrate prior to 15 years of age, they exhibit the prevalence of their new home. if they migrate at a later age, they retain the disease prevalence of their native country. this has been used to argue for the presence of an infectious factor, because there are examples for the critical role of the timing of primary infection in induction of an infectious cns disorder [7] . anecdotal evidence of occurrence of ms in families has been noted for many years [8] . there are few reports of conjugal ms [9] and of ms diagnosed in children of affected parents [10] , but not enough to enable statistical analysis. whether these reports, together with other documentation of ms affecting members of the same community [11] , those who share a profession, a work place, or a residence are more than just coincidental anecdotes is not settled. with no true understanding of the cause of ms, the animal demyelinating disease models abound. there is quite a spectrum of viruses that can produce acute and chronic demyelinating conditions in a variety of experimental animals [12] . these include dna viruses such as the papova virus sv40 (rhesus monkeys) and herpes simplex virus type 1 (hsv-1) and 2 (in mice), rna viruses such as canine distemper virus (mice and hamsters), several corona virus models in mice and monkeys, the murine picorna theiler's virus, and retroviruses such as visna virus in sheep. these models prove the concept that viruses can induce demyelination. interferons for the treatment of multiple sclerosis as of today, three β-interferon preparations are licensed for the treatment of ms. they were shown to have a moderate effect upon disease relapse rate and progression of disability [13] [14] [15] . interferons are naturally occurring species-specific glycoproteins that were first discovered in 1957 because of their antiviral activity, but they also possess a variety of other biologic actions. they belong to the ever-growing group of cytokines that are capable of modulating the immune system. nevertheless, one of their major functions is to combat viral infections. they are in clinical use for treatment of conditions such as infections due to hepatitis b and c viruses, and genital warts caused by papillomavirus. although it has been argued that the β-interferons' effect upon ms is immunomodulatory, the possibility that their impact is directly antiviral is just as likely. viruses can cause a variety of naturally occurring human cns demyelinating diseases. infective and postinfective conditions have been noted: progressive multifocal leukoencephalopathy (pml) and tropical spastic paraparesis are examples of the first, whereas postinfectious encephalomyelitis occurs following diverse infections. although these disorders are clinically distinct from ms, they nevertheless suggest that viruses are capable of inducing demyelinating diseases in the human cns. cerebrospinal fluid (csf) oligoclonal bands are present in the vast majority of ms patients. although attributed to a chronic immune-mediated process, csf oligoclonal bands are also a feature of chronic cns infections [16] . in view of the current dogma of ms as an immune-mediated disorder, the possibility that the disease and/or its relapses are triggered by infections, whereas the eventual tissue damage is due to immune activation, might be more appealing than the concept that ms is a true infective process. the methodology used to examine an association between infection and clinical diseases is both epidemiologic and serologic/immunologic. some studies that examined the association between antecedent infection and occurrence of ms may suggest a sequential relationship. these included unspecified infections [17] and infections with specific agents, such as measles [18] and epstein-barr virus (ebv) [19] . there seems also to be a relationship between an infection and the occurrence of a relapse in a setup of already defined clinical ms [20] . approximately 9% of infections were temporally related to exacerbations, whereas 27% of exacerbations followed infections. this observation was confirmed by a prospective study that followed ms relapses by magnetic resonance imaging (mri) [21] . ms relapses also tended to have a seasonal increase, in association with respiratory infections. immunology/serology can serve to suggest both an ongoing infectious process and/or a previous exposure to a pathogen. antiviral antibodies were examined in the serum and the csf of ms patients. higher titers of antibodies against a large spectrum of viruses were found (but not always confirmed) in ms patients when compared with control patients. the list [12, 22] includes measles, parainfluenza, influenza, varicella zoster, hsv, rubella, ebv, mumps, human t-cell lymphotropic virus (htlv)-1 and 2, and several others. direct proof that a pathogen is responsible for the tissue damage in ms requires isolating the agent from the diseased nervous system and demonstrating that this finding is disease-specific. so far, this task has eluded researchers: no virus or any other infective agent has met these criteria. isolating a pathogen from ms patients' tissues is based on the available technology. for more than half a century, a wide spectrum of laboratory approaches has been used, from cytopathic effect produced in culture by fluids isolated from patients, to viral culture and cocultivation studies, to the present polymerase chain reaction (pcr) amplification and representational difference analysis. a long list of suspected viruses includes rabies, hsv, scrapie agent, parainfluenza, measles, simian virus v, coronavirus, htlv-1, and others. none has withstood tests of reproducibility, specific presence in a significant number of ms patients as compared with control patients, or a reasonable experimental explanation. nevertheless, based on the assumption that we are dealing with a heterogeneous entity, most likely a syndrome, it is possible that the disorder is due to many different pathogens, where in certain individual cases, an isolated pathogen is indeed responsible for the disease. therefore, several or all such pathogens might cause the disease, but would not stand out when examined on a large series of patients. as of today, four pathogens are under scrutiny, and are, therefore, discussed here in more depth. human herpes virus 6 (hhv-6) is a lately discovered dna virus that is responsible for exanthema subitum (roseola infantum) in children; up to 95% of the general population has been exposed to it [23] . it is a lymphotropic and a neurotropic virus that has been shown to infect glial cells in culture [24] . a look at control tissues, pcr amplification of hhv-6 nucleic acids, and representational difference analysis (the technology that enabled to link hhv-8 with kaposi's sarcoma) demonstrates that it resides in the nervous system of the majority of the population [25] . hhv-6 has been linked with a variety of acute and chronic neurologic disorders, such as encephalitis [26] , menin-goencephalitis [27] , and myelopathy [28] under normal or immune-compromised states. a possible association between hhv-6 and ms is suggested by a number of studies. these include studies that find higher levels of anti-hhv-6 antibodies in the sera of patients with ms as compared with control patients [29,30•] , studies that identify hhv-6 dna in a larger percentage of ms patients compared with control patients [30•] (findings which are not confirmed by other groups [31] ), and evidence for hhv-6 gene expression in the brain, in oligodendrocytes, and in plaques of ms patients [25,32•] . the latter findings, however, were not confirmed by other researchers [33] . although not unique to hhv-6, it is important to note that the timing of exposure to the virus corresponds with the exposure time to a possible infectious pathogen in ms. at present, the most intriguing finding is the association of hhv-6 with ms lesions. if confirmed, it should lead to the question whether this is an outcome of the disease (or its treatment, as the immunosuppressive and immunomodulatory therapies may induce viral reactivation with expression of viral gene products), a noncontributory factor, or indeed related to the pathogenesis of the disease. epstein-barr virus is a lymphotrophic herpes virus responsible for infectious mononucleosis that has been associated with a spectrum of neurologic disorders, including certain cns lymphomas. like other herpes viruses, it establishes latent infection and can reactivate later [34] . in several studies, ebv was found to be present or have left evidence of previous exposure in a higher percentage of ms patients compared with control patients [35] . likewise, epidemiologic studies suggest a relationship between a previous ebv infection and ms [19] . it is worth noting that, during acute ebv infections, antibodies that crossreact with neuroglial antigens are produced [36] . however, identification of the pathogen(s) in ms cannot rely on fingerprints alone. it is mandatory to detect presence of viral nucleic acids and/or antigens in the diseased tissue, which should not constitute a technical problem because ebv is already revealed and examined in certain cns lymphomas. we are not aware of any report on the presence or absence of such compounds in brain tissues of ms patients. retroviruses are rna viruses that encode proviral dna integrated into the host cell genome and can be transmitted via the germ line [37] . endogenous retroviral sequences constitute up to 20% of the human genome. the retrovirus visna, found in sheep, was one of the first animal models of a demyelinating disease, and htlv-1, which can cause a human demyelinating chronic myelopathy, was implicated, but not confirmed, as the cause of ms. in the past decade, another, yet unidentified, retrovirus has been traced. activity of the retroviral enzyme reverse transcriptase was identified in tissues and cells from ms patients [38•] , as well as retroviral sequences in the csf [38•] and serum [39] . this has come to be termed msassociated retrovirus (msrv). indeed, endogenous retrovirus can theoretically induce an immune-mediated condition [40] or an infectious process per se. unfortunately, other groups were unable to confirm these findings, and there is a similarity between the identified sequences and those of endogenous retroviruses present in the majority of the population [38•] . a collaboration between a retrovirus and ebv in inducing ms and triggering the relapses has also been suggested [41•] . the hypothesis is based on the observation that herpes viruses can transactivate retroviruses [42] , and on the ability of herpes viruses to reactivate periodically and induce recurrent disease [34] . chlamydia pneumoniae is an obligate, intracellular, gramnegative bacterium known to cause respiratory infections in humans. the organism has also been implicated in a wide variety of disorders ranging from asthma, atherosclerotic vascular disease, and arthritis to guillain-barre syndrome and encephalitis. the possible relation of chlamydia to ms was recently resurrected by a case report of a 24-year-old man who presented with a fulminant mslike disease [43] . his condition deteriorated despite corticosteroid and β-interferon therapy. although treated with cyclophosphamide, csf cultures and pcr analysis turned out to be positive for chlamydia pneumoniae, and prolonged antibiotic therapy was associated with neurologic recovery. this report was followed by analysis of 37 additional patients from the same center [44••] , showing 64% of csf samples culture-positive, and 97% pcr-positive for the organism, (compared with 11% and 18% in control patients, respectively), and 86% with csf anti-chlamydia antibodies. these findings were supported by one group [45] , but not by others [46] . although the discrepancies may be due to lack of uniform standards and technical difficulties, it seems remarkable that a bacterium with distinct morphologic features could have eluded generations of neuropathologists. furthermore, recent studies failed to detect chlamydia dna within brain lesions obtained by biopsy or autopsy of ms patients [47] . thus, although in some geographical areas some ms patients may have a chronic cns chlamydial infection, its cause and effect role in ms is questionable, because the pathogen is known to infect macrophages and could secondarily gain entrance into the cns during ms exacerbations [44••] . many details concerning the mechanism of myelin damage in ms are still obscure, but putative models for activated t-cell penetration of the cns with resultant demyelination have been put forward [48] . thus, injury might be due to a concerted effect of cytokine-and antibody-mediated injury, direct injury to oligodendrocytes by cd4+ and cd8+ t cells, and myelin digestion by macrophages. naturally, a heavy emphasis on the immune response to selfantigens is the premise of these models, but such cascades could still be easily compatible with the presence of microbial agents as the primary inciting events, or as modulatory factors during ongoing inflammation. after 50 years, no breakthrough evidence to support either a pure autoimmune pathogenesis or a direct destructive infection of white matter components has become available. infection could take part in tissue damage via a direct, pml-like infective process. although it might be argued that the attempts to treat ms with intense immunosuppression should have induced exacerbations attributed to enhanced viral proliferation (which they usually did not), there are certain nervous system persistent or latent infections that are not influenced by the host's immune state [34] . at the other end, any intercurrent infection may induce an antimyelin immune response through molecular mimicry (ie, microorganism antigens that resemble myelin antigens that are presented to the immune system under conditions which favor breakdown of self tolerance followed by the myelin being attacked by the newly primed effector cells and autoantibodies) [49] . indeed, microbial epitopes resembling myelin basic protein have been shown to induce experimental autoimmune encephalomyelitis [50••] , and fine specificities of altered epitopes capable of inhibiting the self response are under study. however, guillain-barre syndrome and paraneoplastic disorders in which molecular mimicry is thought to play a central role are either mainly monophasic or subacute progressive disorders, not characterized by frequent relapses. additionally, some viruses may act as superantigens [40] that bind outside the antigen grove, and activate whole classes of t cells, including autoreactive t cells specific for myelin antigen(s), which once activated may cross the blood-brain barrier. there are also possibilities that combine these two extremes. theoretically, latent viral infection of oligodendrocytes (capable of frequent mutations) may occasionally express novel gene products that will be recognized as foreign, and the immune system will eventually mount an inflammatory response with extensive local damage. the relative immune privilege of the cns may support such damage, because single cells harboring the virus will remain undetected and the immune response delayed, enabling tissue alteration to spread locally, causing a more extensive response later. other possibilities are even more hypothetical. for instance, double infections, in which products of one infectious process enhance a nonspecific or specific responses to a second or a latent pathogen, might be also considered. the answers are probably more complex and multifactorial. the infection could be the primary etiology, but an immune-mediated response will play an important pathogenic role. alternatively, if ms is indeed a syndrome, inter-patient differences may account for the difficulty in establishing a unifying hypothesis on ms etiology. this is supported by a recent pathologic study [4••] , where four different demyelination patterns, two showing evidence for a viral-or toxin-mediated oligodendrocytopathy, are suggested. at any rate, non-unifying approaches aimed at splitting the syndrome of ms into distinct entities are probably necessary to unravel the mystery of ms etiology. we have provided the data in favor of an infectious etiology of ms. however, as of today, there is still no evidence to incriminate a specific pathogen. the hunt is still on, and until the causative agent and the destructive scheme are be exposed, the neurologic scientific and clinical community will not give up. multiple sclerosis-in need of critical reappraisal a critical review of the immunologic hypothesis of multiple sclerosis problems of experimental trials of therapy in multiple sclerosis: report by the panel on the evaluation of experimental trials of therapy in multiple sclerosis sclerose en plaques et maladies infectieuses heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination a new look at multiple sclerosis (ms) pathology supporting the nonuniformity of the condition and the concept that ms is a syndrome epidemiologic evidence for multiple sclerosis as an infection epidemiologic studies of measles, measles vaccine, and subacute sclerosing panencephalitis sobre la clinical de la escleroisis multiple conugal multiple sclerosis. immunogenetic characterization and analysis of t-and b-cell reactivity to myelin protein familial and conjugal multiple sclerosis multiple sclerosis in research workers studying swayback in lambs: an updated report the virology of demyelinating diseases interferon beta 1b is effective in relapsing-remitting multiple sclerosis. i. clinical results of a multicenter, randomized, double blind, placebo-controlled trial impact of interferon beta-1a on neurologic disability in relapsing multiple sclerosis. the multiple sclerosis collaborative research group (mscrg) prism (prevention of relapses and disability by interferon b-1a subcutaneously in multiple sclerosis) study group.: randomized double blind placebo controlled study of interferon b-1a in relapsing/remitting multiple sclerosis virological aspects of tropical spastic paraparesis/ htlv-i associated 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antibodies by immunofluorescence analysis and of viral sequences by polymerase chain reaction association of human herpes virus 6 (hhv-6) with multiple sclerosis: increased igm response to hhv-6 early antigen and detection of serum hhv-6 dna a combined serologic/molecular analysis of possible human herpes virus-6 infection in multiple sclerosis patients eis-hubinger am: human herpesvirus 6 polymerase chain reaction findings in human immunodeficiency virus associated neurological disease and multiple sclerosis the hhv6 paradox: ubiquitous commensal or insidious pathogen? a two-step in situ pcr approach a provocative study and discussion on the possible role of human herpes virus-6 in multiple sclerosis hhv-6 and multiple sclerosis human herpes viruses latent infection in the nervous system association between clinical disease activity and epstein-barr virus reactivation in ms antibodies against epstein-barr nuclear antigen (ebna) in multiple sclerosis csf, and two pentapeptide sequence identities between ebna and myelin basic protein the viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. the collaborative research group on multiple sclerosis an important contribution to the concept that a retrovirus might be present in multiple sclerosis patients' nervous system at a higher percentage than in the normal population detection of virionassociated msrv-rna in serum of patients with multiple sclerosis a human endogenous retroviral superantigen as candidate autoimmune gene in type i diabetes the association between multiple sclerosis and infection with epstein-barr virus and retrovirus discussion on the possible combined action of more than one pathogen in inducing central nervous system damage in multiple sclerosis transactivation of human t-cell leukemia virus type 1 by herpes simplex virus type 1 multiple sclerosis associated with chlamydia pneumoniae infection of the cns chlamydia pneumoniae infection of the central nervous system in multiple sclerosis a systematic analysis of multiple sclerosis patients for evidence of a bacterial infection by a group that has first raised the possibility of chlamydia pneumoniae infection in multiple sclerosis evidence for infection with chlamydia pneumoniae in a subgroup of patients with multiple sclerosis failure to detect chlamydia pneumoniae in the central nervous system of patients with ms lack of detectable chlamydia pneumoniae in brain lesions of patients with multiple sclerosis multiple sclerosis more mayhem from molecular mimics microbial epitopes act as altered peptide ligands to prevent experimental autoimmune encephalomyelitis on the possible role of antigens of infective agents in modulating the immune response against nervous system constituents key: cord-351490-2fx0w30u authors: russell, clark d.; baillie, j. kenneth title: treatable traits and therapeutic targets: goals for systems biology in infectious disease date: 2017-04-27 journal: curr opin syst biol doi: 10.1016/j.coisb.2017.04.003 sha: doc_id: 351490 cord_uid: 2fx0w30u among the many medical applications of systems biology, we contend that infectious disease is one of the most important and tractable targets. we take the view that the complexity of the immune system is an inevitable consequence of its evolution, and this complexity has frustrated reductionist efforts to develop host-directed therapies for infection. however, since hosts vary widely in susceptibility and tolerance to infection, host-directed therapies are likely to be effective, by altering the biology of a susceptible host to induce a response more similar to a host who survives. such therapies should exert minimal selection pressure on organisms, thus greatly decreasing the probability of pathogen resistance developing. a systems medicine approach to infection has the potential to provide new solutions to old problems: to identify host traits that are potentially amenable to therapeutic intervention, and the host immune factors that could be targeted by host-directed therapies. furthermore, undiscovered sub-groups with different responses to treatment are almost certain to exist among patients presenting with life-threatening infection, since this population is markedly clinically heterogeneous. a major driving force behind high-throughput clinical phenotyping studies is the aspiration that these subgroups, hitherto opaque to observation, may be observed in the data generated by new technologies. subgroups of patients are unlikely to be static – serial clinical and biological phenotyping may reveal different trajectories through the pathophysiology of disease, in which different therapeutic approaches are required. we suggest there are two major goals for systems biology in infection medicine: (1) to identify subgroups of patients that share treatable features; and, (2) to integrate high-throughput data from clinical and in vitro sources in order to predict tractable therapeutic targets with the potential to alter disease trajectories for individual patients. infection is the largest single cause of death in humans worldwide and many infectious agents provide relevant in vitro model systems that are both amenable to study with high-throughput techniques, and recapitulate key events in disease pathogenesis. in this review, we consider how systems biology approaches may be leveraged to address the major unmet needs in infection medicine in the 21st century, with the aim of improving outcomes for patients with infection. in clinical practice we are unable to therapeutically modulate the host immune response to infection, largely due to its inevitable complexity. despite this, we contend that host-directed therapies have a high probability of success, since there is already considerable innate variation in host responses to infectious disease, ranging from extreme susceptibility, to complete resistance, and tolerance. infectious diseases are survivable if you have the right genetics. the challenge is to make the same diseases survivable for patients who would otherwise succumb. a systems medicine approach to infection has the potential to combine and integrate relevant signals from clinical, genomic, transcriptomic, proteomic and pathogen biology data to draw inferences about disease pathogenesis. below we discuss examples of aspects of this approach applied to various infectious diseases, and suggest future goals for the application of systems biology to infection medicine. more than 70 years after the discovery of penicillin [1] , this same drug is still a prominent weapon in our antibacterial armamentarium. more broadly, the concept underlying this therapeutic approach -attempting to eradicate the pathogen from a patient's body using antimicrobial drugs -remains the only effective treatment. although spectacularly successful, the focus on the pathogen has two limitations. firstly, death frequently occurs in infectious disease despite effective antimicrobial therapy. alongside the direct effects of microbial virulence factors, tissue damage is also caused by the host immune response. immune-mediated damage leading to respiratory, cardiovascular and renal failure (sepsis) continues even after eradication of the pathogen [2] . at present, no treatments exist to modify these deleterious aspects of the host immune response. secondly, antimicrobial resistance threatens to liberate pathogens from the range of our solitary weapon against them. unless something changes, deaths from infection are predicted to soar, overtaking malignant disease even in developed countries by 2050 [3] . therapies to modulate the host response to infection would have the theoretical advantage that, in addition to promoting survival in the presence of effective antimicrobials, a host-targeted therapy may exert a less powerful selection pressure on pathogens, and may be more difficult for a pathogen to evolve to overcome. in our view, the development of such therapies is wellsuited to the application of systems approaches. inevitable complexity of the immune system the human immune system is arguably the most complicated organ system in the body, encompassing numerous effectors, inter-related feedback loops and extensive redundancy. this complexity is unsurprising when considering that our immune system has evolved in the face of microbial virulence factors that directly interfere with regulatory and effector mechanisms. examples of microbial interference with host immune mechanisms are numerous and diverse. for example, one of the first innate immune mechanisms encountered by many pathogens is phagocytosis, which serves to both prime the adaptive response and eliminate invading pathogens by intracellular killing. pathogenic yersinia species, a group of facultative intracellular pathogens, encode a type three secretion system to directly inject effector proteins into the host cell cytoplasm, modulating the cytoskeleton to prevent phagocytosis, and inducing apoptosis of immune cells and blocking the mapk and nf-kb pathways to reduce cytokine production [4] . another bacterium, pseudomonas aeruginosa, secretes a protease that cleaves a host protein (corticosteroid-binding globulin) to release the corticosteroid hormone cortisol at the site of initial infection, incapacitating the local innate immune response [5] . even the relatively tiny genome of the influenza a virus encodes a protein (ns1) which is nonessential for replication and seems to be dedicated to interfering with both the induction and action of the host antiviral interferon response by sequestering viral dsrna, preventing activation of rig-1 signalling and inhibiting protein kinase r and oas/rnase l [6] . the adaptive response, mediated by t and b lymphocytes, is also a target. the human immunodeficiency virus encodes three proteins that each down-regulate cell surface mhc-1 expression by distinct mechanisms, preventing mhc-i signalling to activate the cytotoxic t-cell response to virus-infected cells [7] . to prevent b-cells mounting an antibody response to infection, the staphylococcus aureus surface protein a binds to the fc-g portion of antibodies [8] . these examples cover a few of the mechanisms pathogens have evolved to extensively interfere with the host immune response. the animal innate immune system is thought to have evolved over 1000 million years, starting with amebae able to phagocytose external material for nutrition [9] . the adaptive immune system in mammals is thought to have arisen 500 million years ago in fish [10] . since these initial events, the immune system in each host species has participated in a genetic arms race, evolving alongside relentless exposure to these microbial immune interference strategies from innumerable pathogens. furthermore, the immune system must successfully distinguish self from non-self antigens, with deleterious consequences arising (i.e. autoimmune disease) when this fails. the requirement to overcome these microbial immune interference strategies whilst preserving the recognition of self-antigens has provided the necessary pressure to drive the human immune system to evolve into a hugely complex organ system. in the context of this inevitable complexity, it is no surprise that reductionist approaches to development of hosttargeted therapies in infectious disease have largely a subgroup within a population of patients who are distinguished by a shared disease process. treatable trait the pathophysiological feature (or, in a looser sense, a biomarker or group of biomarkers for that feature) that determines whether a given therapy will improve a given patient's outcome. the same trait may be present in many different clinical syndromes or disease processes. it is reasonable to expect that as-yet undiscovered therapeutic interventions could alter the host immune response to promote survival. we infer this from the simple fact that some hosts do better than others when confronted with the same pathogen: the host response to infection is, in all cases that we know to have been studied, heterogeneous. furthermore, much of this variation is heritable [11] . we conclude that host factors exist that promote survival from specific infections, and that these must vary between individuals, and hence that it should be possible to identify and utilise these factors therapeutically to alter the biology of a susceptible host to induce a response more similar to a host who survives. the scale of the challenge of finding these targets is such that it is hard to imagine a solution being found without the power of systems approaches [12] . conceptually, this could involve promoting resistance to (suppressing pathogen replication), or tolerance of (preventing damage associated with immune response to pathogen), an infecting pathogen [13] . although theoretically attractive as a tool to limit damage to the host [14] , inducing tolerance is not without potential dangers: a sustained high pathogen load could facilitate transmission (iatrogenic super-shedders) and, perhaps more worryingly in emerging zoonotic infections, provide the time required for the selection of mutants with better host adaptation. due to their reliable and broad acting anti-inflammatory effects, corticosteroids represent an intuitively attractive strategy to reduce inflammation during infection. indeed, a survival benefit has been demonstrated in a small number of uncommon infections (bacterial meningitis, tuberculous meningitis and pericarditis, hypoxaemic pneumocystis jiroveci pneumonia) [15] . in contrast, there is uncertainty over safety and benefit in other infections (e.g. rsv bronchiolitis) and clear evidence of harm in others (viral hepatitis, cerebral malaria, influenza virus, sars coronavirus, hiv-associated cryptococcal meningitis) [15e19]. a more specific host-directed therapy, recombinant human activated protein c (rhapc), was licensed for treatment of severe sepsis based on the results of a single clinical trial [20] . rhapc has anti-inflammatory and anti-thrombotic properties, and circulating levels are low in patients with sepsis [21] . sadly, a subsequent trial in an overlapping patient group showed a trend towards increased mortality [22] . a third trial, mandated by the regulatory authorities, did not detect any mortality benefit, and the drug was quickly withdrawn from the market [23] . these negative trial results in sepsis are not necessarily conclusive. the heterogeneity of the host response, and the diverse range of microbes involved, means that amongst all patients with sepsis (itself an unrealistically broad syndrome) there are likely to be numerous biologically-different sub-groups, with distinct immune responses and more importantly, different risk: benefit balance for a given therapy. corticosteroids, for example, may save some patients but harm others. our current understanding of infection does not allow clinical differentiation of these sub-groups, so potentially beneficial therapies may be falsely rejected in clinical trials. we believe that a major output of systems approaches to infection will be the elucidation of previously hidden therapeutically-important subgroups of patients who share a 'treatable trait' (i.e. response to therapy). we have discussed two central problems in infection medicine. firstly, the development of therapies to modulate the host immune response is impeded by the inevitable complexity of the human immune system. secondly, the range and degree of characterisation of clinical syndromes in medicine is constrained by the range of observations that are available; we likely fail to identify sub-groups of patients with treatable traits due to a paucity of relevant observations. the promise of systems approaches to infection medicine is that new technologies may provide solutions to these old problems. large-scale biological data-sets from an increasing range of high-throughput technologies are becoming available, including (but not limited to) high-resolution transcriptional profiling [24] , mass spectrometry-based proteomics [25] and metabolomics, genome-scale crispr-cas9 knockout screening [26] , and whole genome sequencing/genome-wide association studies. these modalities could provide new, biologically important observations that have not previously been observed in patients. it is very likely that some of these observations will be directly relevant to clinical management e the challenge will be to identify the ones that matter. there are three broad categories of clinical utility for these new data sources in infection medicine: identifying treatable traits and therapeuticallyrelevant subgroups; identifying new therapeutic targets in the immune response; and, improving prognostication. the value of prognostication in current clinical practice is, in our view, limited by the range of therapeutic options. put simply, it is only useful to predict the future if you have some capacity to change it. particularly in the case of acute and immediately lifethreatening infection, our range of therapeutic options is very limited, regardless of the degree of certainty attached to the prognosis, therefore we suggest the first two applications should be prioritised. in contrast, the identification of syndromes (collections of clinical observations that tend to occur in patients suffering the same disease) has been the primary mechanism for progress in the understanding of human disease since long before the time of hippocrates [27] . finding a syndrome is the first step towards identifying the common biological processes that define a disease, and ultimately to identifying effective treatments. we hope that the application of systems technologies will help us define treatable traits in infection, thus providing a starting point for new therapeutic interventions ( figure 1 ). importantly, a systems model of the disease process need not predict every transcript and metabolite in the massively multi-dimensional datasets generated by new technologies. the primary challenge is instead to identify those components of inter-host variation that are amenable to intervention; the evidence of disease processes that we can change. this is agusti's concept of "treatable traits" [28] , a term coined in the field of chronic obstructive lung disease but no less relevant here. a computational model of infection could therefore include not only traditional measures of pathogen burden and evidence of systemic injury [29] , but also independent components of the immune response or the metabolic consequences, detected using highthroughput technologies. the behaviour of the whole system may be impenetrably complex, but the components required to predict the effect of an intervention may be far simpler. the trajectory followed by each patient along an informative set of vectors may reveal different groups of patients that appear clinically similar, and may even have similar outcomes, but different disease processes ( figure 2 ). by mapping the "flight path" of each patient through disease, we anticipate that summary of a systems medicine approach to infection. a wide range of data sources can be combined using various methods (see text) to achieve two fundamental goalsclinically-informative phenotyping of patients, and identification of therapeutic targets. important similarities and differences in immune response will become apparent [29] . our ability to identify groups of patients sharing therapeutically-relevant similarities is dependent on measuring the relevant biological signal that determines classification. this is the fundamental attraction of high-throughput technologies e the probability of measuring important signals is greater if more signals are measured. that such groupings of patients, or disease endotypes, exist is already clear: therapeuticallyimportant sub-classifications have recently been discovered that redefine the clinical syndromes of asthma [30, 31] , ards [32] , and acute mountain sickness [33] . in two related autoimmune conditions, anca-associated vasculitis (aav) and systemic lupus erythematosus (sle), a t-cell gene expression signature clearly delineates two distinct endotypes [34] . subsequent work elucidated the immunological process underlying this sub-classification, cd8 t-cell exhaustion [35] . this process is associated with better outcomes in autoimmune disease, but poor clearance of viral infection. in the future it may be possible to manipulate this pathway therapeutically in patients with aav or sle, to prevent relapse, or in the opposite direction in patients with chronic viral infection, to promote clearance. even in the best-case scenario, the distance between the identification of a tractable therapeutic target and successful exploitation in clinical practice is substantial, so it is no surprise that potential host-directed therapies discovered through high-dimensionality analytics have not yet been proven to be effective in clinical trials. nonetheless there are some promising leads, a few of which are described here. these exemplify different approaches: integrating cell culture, animal and human data sequentially to identify a host anti-viral factor (influenza virus); and using computational predictions from transcriptional data with proteomic and genetically modified animal studies to identify host pathways involved in pathogenesis (sars coronavirus). viruses are obligate parasites and undergo exclusively intracellular replication; properties that make them ideal for study in cell culture where host factors that affect viral replication can be expected to do the same in vivo. sirna screening has been used as a genomewide approach to identify such host factors for influenza virus infection. ifitm3 was identified as a novel host anti-viral factor by sirna screening with confirmatory in vitro work (including exogenous interferon administration and stable ifitm3 expression) demonstrating it is required for an effective interferon response to inhibit influenza virus replication [36] . work in ifitm3e/e mice was then undertaken, confirming that in vivo, influenza virus-infected mice suffer fatal viral pneumonia, even when infected with a low-pathogenicity virus [37] . the genisis/mosaic groups identified a single nucleotide polymorphism hypothetical trajectories of two groups of patients through multidimensional space. each line indicates the path taken by a single patient, with periods of organ failure highlighted in red. a superficially similar group of patients may appear clinically indistinguishable (a), but different trajectories through illness are revealed by informative vectors derived from high-throughput data (b). it is reasonable to expect that such biological differences in disease process will underlie different responses to host-directed therapies. (rs12252-c) within the ifitm3 coding region in humans that was strongly over-represented in patients hospitalised with influenza virus infection [37] , the majority of whom required invasive ventilation. metaanalysis of genomic studies of ifitm3 and influenza virus infection has confirmed this association between snp rs12252-c and increased susceptibility to infection in humans [38] . work is now underway in many groups to investigate the impact of ifitm proteins in antiviral defence, with a view to generating hosttargeted antiviral therapies. this example highlights the potential of a systems-wide approach, integrating and cross-validating results from various experimental modalities (high-throughput screening in cell culture, followed by targeted studies in mice and humans) to converge from large-scale data onto a single critical host immune factor. acute lung injury (ali) and progression to acute respiratory distress syndrome are major features of the pathophysiology of sars coronavirus (and indeed other respiratory virus) infection. pathological changes in mice are very similar to humans and there is a dose-response relationship between viral inoculum and the severity of ali. host responses to the virus that result in ali are poorly understood. to investigate host responses associated with more severe acute lung injury in a murine model of sars coronavirus (sars-cov) infection, transcriptomic profiles of mice infected with lethal and sub-lethal doses of virus were compared and correlated with pathological data at multiple time points, then subject to bioinformatic network analysis [39] . a module of genes involved in cell adhesion, extracellular matrix remodelling and wound healing was significantly up-regulated in the lethal infection model, and components of the urokinase pathway were found to be the most enriched and differentially regulated. massspectrometry proteomic analysis was then used to explore this transcriptional data further, demonstrating that sars-cov infection resulted in increased expression of fibrin b and g chains, factor viii and cytokeratins (all components of hyaline membranes), and reduced expression of surfactant proteins in the lung. these data are consistent with histological post-mortem findings in sars-cov infected humans, where extensive fibrin exudate, extensive hyaline membrane formation and alveolar collapse have been observed [40] . serpine1 is part of the urokinase pathway and contributes to ecm remodelling. to confirm this finding from transcriptional and proteomic studies, serpine1e/e knockout mice were infected with sars-cov and found to have a worse outcome compared to wild type mice. this systemsbased approach to sars-cov, sequentially applying transcriptional, proteomic then genetic modification techniques, demonstrates that the urokinase pathway contributes to ali, thus identifying a target for future experimental medicine work to determine if it can be therapeutically altered to benefit the host. these examples give us confidence that we can expect more progress along similar lines as the potential of systems medicine approaches becomes more widely appreciated, as expertise in computational methodologies grows, and as the cost of generating relevant data falls. physicians have long made progress by recognising patterns of similar observations in groups of patients, and by determining which biological features of disease are amenable to therapy. what has changed is the unprecedented rate of advance in new resources and tools with which to tackle the ancient problems of diagnosis and therapy in infectious disease. our responsibility is to ensure a similar acceleration in clinical progress. . sorensen ti, nielsen gg, andersen pk, teasdale tw: genetic and environmental influences on premature death in adult adoptees. n engl j med 1988, 318:727-732. this prospective cohort study of adoptees demonstrated that genetic background contributes more to the relative risk of death due to infection (i.e. biologic parent also died of infection) than for death due to cancer, vascular disease or natural causes, thus affirming that susceptibility to infection is a strongly heritable trait. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest first clinical use of penicillin severe sepsis and septic shock review on antimicrobial resistance. tackling drug-resistant infections globally: final report and recommendations the yersinia ysc-yop 'type iii' weaponry pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroidbinding globulin the multifunctional ns1 protein of influenza a viruses mechanisms of hiv-1 to escape from the host immune surveillance role of protein a in the evasion of host adaptive immune responses by staphylococcus aureus evolution of innate immunity: clues from invertebrates via fish to mammals origin and evolution of the adaptive immune system: genetic events and selective pressures disentangling genetic variation for resistance and tolerance to infectious diseases in animals this study utilises a murine malaria model to demonstrate that resistance to and tolerance of infection represent genetically distinct host defence mechanisms disease tolerance as a defense strategy use of corticosteroids in treating infectious diseases adjunct prednisone therapy for patients with communityacquired pneumonia: a multicentre, double-blind, randomised, placebo-controlled trial adjunctive dexamethasone in hiv-associated cryptococcal meningitis corticosteroids as adjunctive therapy in the treatment of influenza sars: systematic review of treatment effects efficacy and safety of recombinant human activated protein c for severe sepsis role of activated protein c in the pathophysiology of severe sepsis drotrecogin alfa (activated) in severe sepsis drotrecogin alfa (activated) in adults with septic shock gateways to the fantom5 promoter level mammalian expression atlas mass spectrometry-based proteomics genome-scale crispr-cas9 knockout screening in human cells genome editing using the crispr-cas9 system can be applied to genome-wide knockout screening to identify genes with an important phenotype in an in vitro model, as exemplified in this study of genes required for cell viability in cancer and genes implicated in susceptibility to a therapeutic raf inhibitor used in melanoma hippocrates on ancient medicine phenotypes and disease characterization in chronic obstructive pulmonary disease. toward the extinction of phenotypes? health trajectories reveal the dynamic contributions of host genetic resistance and tolerance to infection outcome multidimensional endotypes of asthma: topological data analysis of crosssectional clinical, pathological, and immunological data asthma endotypes: a new approach to classification of disease entities within the asthma syndrome subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials network analysis reveals distinct clinical syndromes underlying acute mountain sickness a systematic network analysis (using software originally designed for analysing microarray expression data) of symptoms associated with acute mountain sickness revealed three distinct sub-groups within the syndrome, resulting in a revision of international diagnostic criteria for a cd8+ t cell transcription signature predicts prognosis in autoimmune disease transcriptional profiling identified two distinct sub-groups (related to t-cell survival and memory) which could then be differentiated on the basis of only three genes, providing a useful biomarker for clinical trials of immunosuppressant therapies and focussing further studies on these pathways t-cell exhaustion, co-stimulation and clinical outcome in autoimmunity and infection the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus ifitm3 restricts the morbidity and mortality associated with influenza data was integrated from cell culture work, a murine infection model, human genotyping and patient clinical variables to validate ifitm3 as a critical host defence against influenza virus infection and thus a tractable therapeutic target interferon-inducible transmembrane protein 3 genetic variant rs12252 and influenza susceptibility and severity: a meta-analysis mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury multiple organ infection and the pathogenesis of sars key: cord-350928-vj5qlzpj authors: arnott, alicia; vong, sirenda; rith, sareth; naughtin, monica; ly, sowath; guillard, bertrand; deubel, vincent; buchy, philippe title: human bocavirus amongst an all‐ages population hospitalised with acute lower respiratory infections in cambodia date: 2012-04-25 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2012.00369.x sha: doc_id: 350928 cord_uid: vj5qlzpj please cite this paper as: arnott et al. (2013) human bocavirus amongst an all‐ages population hospitalised with acute lower respiratory infections in cambodia. influenza and other respiratory viruses 7(2) 201–210. background human bocavirus (hbov) is a novel parvovirus that is associated with respiratory and gastrointestinal tract disease. objectives to investigate the prevalence and genetic diversity of hbov amongst hospitalized patients with acute lower respiratory infection (alri) in cambodia. study design samples were collected from 2773 patients of all ages hospitalised with symptoms of alri between 2007 and 2009. all samples were screened by multiplex rt‐pcr/pcr for 18 respiratory viruses. all samples positive for hbov were sequenced and included in this study. results of the samples tested, 43 (1·5%) were positive for hbov. the incidence of hbov did not vary between the consecutive seasons investigated, and hbov infections were detected year‐round. the incidence of hbov infection was highest in patients aged <2 years, with pneumonia or bronchopneumonia the most common clinical diagnosis, regardless of age. a total of 19 patients (44%) were co‐infected with hbov and an additional respiratory pathogen. all isolates were classified as hbov type 1 (hbov‐1). high conservation between cambodian np1 and v1v2 gene sequences was observed. conclusions human bocavirus infection can result in serious illness, however is frequently detected in the context of viral co‐infection. specific studies are required to further understand the true pathogenesis of hbov in the context of severe respiratory illness. human bocavirus (hbov) is a novel parvovirus that was first identified in pools of nasopharyngeal aspirates obtained from individuals with respiratory tract infections in 2005. 1 the hbov genome has three open reading frames encoding the 2 non-structural proteins ns1 and np1, and the structural viral capsid proteins vp1 and vp2. 2, 3 there are four closely related hbov genotypes, hbov-1, hbov-2 (consisting of hbov-2a and hbov-2b strains), hbov-3 and hbov-4, all of which share the same genomic organisation. 3, 4 hbov-1, the genotype initially identified by schildgen et al. 5 in sweden, has since been detected worldwide. hbov-2-4 were mainly identified in stool samples. 6, 7 homology at the protein level between the four hbov genotypes is high, between 70 and 90%. 3 hbov-3 is thought by some to be the result of a recombination event between hbov-1 and hbov-2, owing to the similarity of the hbov-3 ns1 and v1v2 genes to those of hbov-1 and hbov-2, respectively. 6 indeed, hbov-3 is likely detected along with hbov-1 during routine diagnostic screening, as the conserved ns1 genes regularly targeted for testing are genetically similar. in patients where hbov is the only virus detected, the clinical symptoms reported are similar to those occurring as a result of infection with respiratory syncytial virus (rsv) and human metapneumovirus (hmpv), including bronchiolitis, bronchitis, pneumonia and exacerbation of asthma. 5 predominantly hbov-1, but also hbov-2 dna, has been detected in respiratory samples, 8 whereas all four genotypes have been detected in stool samples, demonstrating that hbov replication is not limited to the respiratory tract as first thought. 4, 8 indeed, up to 25% of patients with hbov infection report gastrointestinal symptoms. [8] [9] [10] [11] hence, the tropism of strains belonging to hbov genotypes 1-4 remains unknown. the seasonality of hbov infection is also unclear, with seasonal [12] [13] [14] and year-round [15] [16] [17] transmission reported. the clinical significance of hbov infection is also yet to be fully elucidated, as hbov is frequently detected in association with additional respiratory pathogens. in the absence of an established cell culture system or animal model, the pathogenicity and replication mechanisms of hbov remain unclear. 5, 18, 19 a recent report suggests that hbov is, however, a true respiratory pathogen capable of causing sometimes severe, and even life-threatening, illness. 20 here, we report the findings of the first study investigating the prevalence, seasonality, clinical characteristics and the molecular epidemiology of hbov in amongst an all-ages population of patients hospitalized for acute lower respiratory illness (alri) in cambodia over 3 consecutive years. during april 2007-december 2009, all patients admitted with symptoms of alri to takeo (southern cambodia) and kampong cham (central-north cambodia) provincial hospitals were recruited into this study. 21 the age-specific criteria used to diagnose alri cases and severe alri cases were defined previously. 21 samples were collected, transported and stored as described. 21 additional clinical specimens and data, including sputum samples for bacteria identification and chest x-rays, were collected from patients where possible. samples were screened by multiplex rt-pcr ⁄ pcr at the institut pasteur in cambodia for 18 respiratory viruses including rsv, human metapneumovirus (hmpv), hbov, influenza a and b viruses, coronaviruses oc43, 229e, hku1 and nl63, severe acute respiratory syndrome-associated coronavirus (sars-cov), parainfluenza viruses 1-4, adenoviruses, rhinovirus and enterovirus, as previously reported. 21, 22 samples that tested positive for hbov were included in this study. the national ethics committee of cambodia approved this study. all patients ⁄ parents of sick children who participated provided written informed consent. viral dna from nasopharyngeal samples was extracted using the qiaamp dna mini kit (qiagen, valencia, ca, usa) as per the manufacturer's instructions. dna was eluted in a final volume of 200 ll qiagen ae buffer and stored at )80°c until required. both hbov and human albumin dna were amplified in parallel from each sample. amplification of human albumin dna was performed as described. 23 to identify strains belonging to hbov groups 1, 2 and 3, strain-specific primers were used. to identify hbov-1 strains, a 354-bp fragment of the conserved hbov-1 np-1 gene was targeted using published primers. 1 the forward and reverse primers correspond to positions 2281 and 2634 of the np-1 gene of the hbov prototype strain st1 (genbank accession number dq000495). thermocycling was performed using go-taq flexi dna polymerase (promega, madisson, wi, usa) under the following conditions: pcr activation at 94°c for 2 minutes, followed by 40 cycles of denaturation at 94°c for 30 seconds, annealing at 59ae5°c for 30 seconds, extension at 72°c for 1 minute and a final extension cycle of 72°c for 10 minutes. all samples were also screened by pcr for the presence of hbov2 and hbov3 dna, as described. 8 for phylogenetic analysis, a 819-bp region of the variable hbov-1 v1 ⁄ v2 gene was amplified using published primers. 9 primers correspond to positions 4370-4387, and 5172-5189 of the hbov prototype strain st1 genome (genbank accession number dq000495). thermocycling was performed using go-taq flexi dna polymerase (promega) under the following conditions: pcr activation at 94°c for 2 minutes, followed by 40 cycles of denaturation at 94°c for 1 minute, annealing at 54°c for 1 minute, extension at 72°c for 2 minutes and a final extension cycle of 72°c for 10 minutes. each pcr contained the appropriate positive and negative controls. all pcr products were visualised using ethidium bromide under uv light on a 1ae5% agarose gel. when required, pcr products were purified using the qiaquick gel extraction protocol of the qiaquick pcr purification kit (qiagen) as per the manufacturer's instructions, prior to sequencing. single-pass sequencing reactions were performed at a contract sequencing facility using the abi big-dye terminator cycle sequencing kit on an abi 3730xl automatic dna analyser (macrogen, seoul, korea). consensus sequences were generated using clc main workbench software, version 5ae6ae1 (http://www.clcbio.com). nucleotide sequences of reference hbov strains were obtained from genbank and used to construct alignments and phylogeny (table 1) . cambodian and reference hbov nucleotide sequences were aligned using the clustal w alignment program of mega software, version 4ae0. 24 the average pairwise jukes-cantor distance was found to be 0ae01 for both of the hbov np1 and vp1 ⁄ vp2 alignments, indicating that the data were suitable to generate neighbour-joining trees. 25 neighbour-joining trees were constructed using the p-distance nucleotide substitution model, with 1000 bootstrap replicates, using mega 4ae0 software. pairwise nucleotide distances (p distance), the proportion of sites at which nucleotide sequences differ divided by the total number of nucleotides compared, were calculated using the pairwise distance function of mega software version 4ae0. pairwise amino acid distances (p distance), the proportion of sites at which amino acid sequences differ divided by the total number of residues compared, were calculated using the poisson model in the pairwise distance function of mega software version 4ae0. deduced partial amino acid sequences of cambodian np1 and vp1 ⁄ vp2 strains were generated by translating nucleotide sequences with the standard genetic code using mega software version 4ae0. potential n-glycosylation sites were predicted by the nxt motif, where x is not a proline; potential o-glycosylation sites were predicted by the kpx n motif (where x is any amino acid). [26] [27] [28] [29] nucleotide sequence accession numbers the nucleotide sequences of hbov strains obtained in this study were deposited in genbank under the accession numbers jq618248 to jo618263 (np1 sequences) and jq618264 to jq618278 (vp1 ⁄ vp2 sequences). a total of 2773 patients presenting with alri participated in the study (no refusals). nasopharyngeal swabs were collected from all patients, of which 43 (1ae5%) tested positive for hbov. hbov infections were detected year-round, and the annual incidence amongst the study patients did not vary significantly (1ae5-1ae7%) during 2007-2009 ( figure 1 ). the median age of the hbov-infected patients was 1 year (range, 0ae3-56 years), and 46ae5% were female. the incidence of hbov infection was highest amongst children aged <2 years ( table 2) . whereas bronchiolitis was only observed in patients aged <2 years, pneumonia or bronchopneumonia was the most common clinical diagnosis, regardless of age (37ae2%; table 2 ). only respiratory specimens were available for testing in this study, and no clinical information regarding gastrointestinal symptoms was recorded. nineteen (44%) patients were co-infected with an additional respiratory virus (table 3) . co-infection with hrhv was most frequently detected (21%), followed by parainfluenza virus type 3 (piv3, 4ae6%) and respiratory syncytial table 1 . reference human bocavirus (hbov) strains used in this study to construct phylogeny. shown are the name of the strain, genbank accession number, year of isolation, country of isolation and genotype. whether strains were included in figure 2 (hbov np1 phylogeny), figure virus (rsv, 4ae6%). one patient was co-infected with both rsv and hrhv (table 3 ). detection and distribution of hbov sequences were successfully obtained from 19 (44ae2%) samples: 16 np-1 gene and 15 vp1 ⁄ vp2 gene sequences. both np-1 and vp1 ⁄ vp2 sequences were successfully amplified from 12 (63%) samples. all cambodian hbov strains were classified as hbov-1 (figures 2 and 3 ). of the samples from which hbov-1 sequences were obtained, one was collected in 2007, nine in 2008 and nine in 2009 (figures 2 and 3) . twelve (63%) samples were collected in takeo province, and seven (37%) were collected in kampong cham province, but the patient recruitment was also higher in takeo than in kampong cham hospital (data not shown). analysis of hbov-1 np1 gene sequences pairwise distance (p distance) analysis revealed very high conservation of np-1 gene sequences amongst the cambodian hbov-1 strains. homology at the nucleotide (nt) level varied between 98ae9 and 100%. identity at the amino acid (aa) level was slightly lower, between 96ae7 and 100%. the np-1 gene is highly conserved between strains, and however, we identified 5 different non-synonymous mutations present in cambodian np-1 sequences relative to the prototype hbov-1 strain st1 ( figure 4 ). the observed nt mutations resulted in the following 5 aa substitutions: ser 92 (100% conservation amongst cambodian np-1 sequences), asn 44 (65%), asn 59 (12%), ser 47 (6%), arg 53 (6%) (figure 4) . we observed 1 potential n-glycosylation site, starting at amino acid position 86, which was conserved amongst all reference and cambodian hbov strains analysed (figure 4 ). analysis of hbov-1 vp1 ⁄ vp2 gene sequences homology at the nt level was also very high amongst the partial vp1 ⁄ vp2 gene sequences, between 97ae1 and 100%. homology at the aa level was slightly lower, between 96ae6 and 100%. present on the surface of the virion, the vp1 ⁄ vp2 protein is potentially subjected to strong selective pressure from the host immune response. relative to the reference strains st1 and st2, 8 non-synonymous nt mutations resulting in the following aa substitutions were observed amongst cambodian vp1 ⁄ vp2 gene sequences: his 546 (66%), leu 631 (26%), tyr 540 (26%), thr 650 (13%), ser 466 (6%), pro 490 (6%), gln 493 (6%) and lys 545 (6%) (figure 5 ). one additional nt mutation resulting in the aa substitution ser 590 , present in reference strain st2 but not st1, was conserved amongst all of the cambodian vp1 ⁄ vp2 sequences investigated ( figure 5) . a stop codon was present at position 672 in all reference and cambodian vp1 ⁄ vp2 sequences included in this study ( figure 5 ). potential n-and o-glycosylation sites amongst hbov-1 vp1 ⁄ vp2 gene sequences two potential n-glycosylation sites, located at amino acid positions 519 and 638, were conserved amongst the reference isolates and all of the cambodian vp1 ⁄ vp2 sequences investigated ( figure 5 ). one potential o-glycosylation site was identified, located at amino acid position 552, which was also conserved amongst the reference isolates and all of the cambodian vp1 ⁄ vp2 sequences analysed ( figure 5 ). here, we report the results of the first study to investigate the incidence and genetic diversity of hbov amongst globally, the incidence of hbov infection has been reported to range from 1ae5 to 19%. 30 overall, the incidence of hbov infection amongst the all-ages population of cambodian patients hospitalised with alri was 1ae5%, which did not vary annually and was lower than that reported regionally. [31] [32] [33] [34] amongst the same population, the incidence of hbov was similar to that of hmpv (1ae7%), 21 but lower than rsv (8ae2%). 35 a number of factors can influence incidence estimates, including disease severity cam2008-e244 ...........................................s.....r......................................s. cam2009-p280 ........................................n...............................................s. cam2008-e374 ........................................................................................s. cam2009-e532 ........................................n........................................... cam2008-e446 ........................................n...............................................s. cam2008-e473 ........................................n...............................................s. cam2008-e474 ........................................n............................................. amongst the population investigated, study design, testing protocols and sensitivity of diagnostic tests used, especially as low viral loads are common in hbov infections. 5 in this study, alri patient samples were screened for hbov infection using a highly sensitive multiplex pcr assay previously shown to have a lower limit of detection of 4 copies of hbov dna ⁄ ll of viral transport medium. 22 therefore, the low incidence of hbov infection observed amongst the cambodian alri population was not a result of limited sensitivity of the diagnostic test used. the proportion of hbov infections detected amongst hospitalised cambodian alri patients was significantly higher compared to that of cambodian outpatients with ili (1ae5% versus 0ae4%, p = 0ae02). 22 the higher incidence of hbov infection amongst alri patients was not thought to be a result of a high carriage rate within the population. in parallel, the incidence of hbov infection amongst cambodian ili outpatients and asymptomatic controls was investigated previously, with only one asymptomatic individual testing positive for hbov dna. 22 however, persistent and prolonged shedding is a characteristic of parvoviruses, and it is thought that low-level asymptomatic shedding can persist following hbov infection. 20, 36, 37 two independent stud-ies reported the hbov carriage rate to be 43% amongst asymptomatic children, 38 and that the incidence of hbov amongst asymptomatic children was higher than amongst symptomatic children. 39 however, these two studies were conducted amongst children aged <2 years. hence, the high carriage rate may have been biased towards the very young age of the sample population with hbov incidence highest amongst children aged <2 years. 1, 9, 40 amongst a population of slightly older children, up to 5 years of age, brieu et al. 36 reported that hbov dna was not detected following testing of asymptomatic children. in the absence of an established continuous culture system, it is currently unclear to what extent shedding occurs following hbov infection, whether shedding and carriage rates amongst children and adults are equivalent, and whether shedding is innocuous or infectious. however, the higher incidence of hbov infection amongst hospitalised cambodian alri patients observed in this study relative to outpatients with ili suggests that hbov infection can result in severe illness. twenty-four (56%) of the hbov-infected individuals tested positive for infection with hbov only, of which 14 patients were classified as severe respiratory infection, including three patients aged <1 year who presented with ..........n................................................................................................ cam2009-p280 .....s.......................p..q....................................................h........................ cam2009-p232 .....................................................................................h........................ cam2009-p231 .....................................................................................h........................ cam2009-e585 ...............................................................................y.............................. cam2009-e569 .....................................................................................h........................ cam2009-e532 ....................................................................................kh........................ cam2008-p125 .............................................................................................................. cam2008-p0202 .....................................................................................h........................ cam2008-e474 .....................................................................................h........................ cam2008-e473 .....................................................................................h........................ cam2008-e446 .....................................................................................h........................ cam2008-e374 .....................................................................................h........................ cam2008-e244 ...............................................................................y.............................. cam2008-e216 ...............................................................................y.............................. cam2007-e141 ...............................................................................y............................. . 43 recently, a severe case of hbov infection resulting in acute respiratory failure was reported, in which hbov was the only pathogen detected. 20 the possibility that clinical illness amongst the cambodian hbov-infected patients was exacerbated because of bacterial co-infection cannot, however, be excluded as testing for bacterial co-infection could not be performed because of a lack of suitable specimens obtained from young participants. whether hbov represents a true pathogen, or an opportunistic co-pathogen, has been the source of much debate. 2, 5 a primary reason that the role of hbov as a sole pathogenic agent is questioned is that hbov is commonly detected as a co-infection. globally, the frequency of hbov-1 co-infection with other respiratory viruses amongst children with lower respiratory tract infection is reported to be as high as 83%. 4, 5, 44 furthermore, it has been reported that hbov coinfection may be higher amongst hospitalised individuals compared with those with mild illness as shedding is potentially enhanced by airway inflammation resulting from coinfection with an additional respiratory pathogen, or that hbov may play a role in enhancing or aggravating symptoms of existing infections. 5 mechanisms proposed to explain the high rate of co-infection with other respiratory viruses include that hbov is either a helper virus, facilitating replication of other respiratory pathogens or that hbov may require a helper virus to facilitate productive infection. 45 it must also be considered that the more recent widespread use of highly sensitive multiplex assays enabling simultaneous detection of multiple pathogens in a single patient sample may also contribute to the high rate of coinfection with hbov and additional respiratory pathogens detected. in this study, 44% of patients were co-infected with hbov and an additional respiratory virus. regionally, the reported rate of hbov co-infections varies and does not appear to be particularly associated with hospitalisation: 42% amongst hospitalized children in singapore, 32 90% amongst out-patient children aged <5 years in rural thailand, 37 9% amongst hospitalised children in vietnam. 31 in this study, co-infection with hbov and hrhv was most frequently detected (table 3) , analogous to the findings of a similar study conducted amongst paediatric patients in rural thailand. 37 whether the high frequency of hbov and hrhv co-infection is a reflection of the high incidence of hrhv circulating in the general population 2 or whether hrhv co-infection is beneficial to hbov replication and pathogenesis remains unknown. in the absence of an established cell culture system or animal model, much remains unclear regarding the pathogenesis of hbov. all of the hbov strains obtained from respiratory specimens for analysis in this study were classified as hbov-1. globally, hbov-1 strains have been reported to show low nucleotide and protein diversity; mean genetic diversity <1% at nt and <0ae5% at aa levels. 44 comparatively, the diversity of hbov-2, -3 and -4 strains, all identified in stool species, is far greater. 8 the low level of diversity reported worldwide amongst hbov-1 strains has resulted in speculation that hbov-1 evolved recently from the enteric bocavirus species, acquiring tropism for cells of the respiratory tract. 8, 46 the organisation of the hbov genome is similar to that of other parvoviruses, namely that the non-structural proteins are encoded by conserved genomic regions, whereas diversity is concentrated in regions encoding the capsid proteins. 45 analysis of amino acid sequences obtained from cambodian hbov-1 strains revealed 0-2ae9% nt diversity and 0-3ae4% aa diversity amongst vp1 ⁄ vp2 sequences. greater variation amongst sequences encoding the vp1 ⁄ vp2 capsid proteins was anticipated as vp1 ⁄ vp2 is under pressure from host immune responses. the level of diversity amongst cambodian np1 sequences was 0-1ae1% at the nt level, which is similar to that recently reported by kapoor et al. 46 surprisingly, the level of diversity at the aa level amongst np1 sequences was equivalent to that observed for vp1 ⁄ vp2 sequences, at 0-3ae3%. five amino acid substitutions were observed amongst cambodian np1 aa sequences, including asn 599 that has been reported previously by ma et al., 47 following analysis of hbov-1 np1 sequences obtained from japanese children with alri. the observed diversity amongst cambodian np-1 sequences may have occurred as a result of immune pressure, as it was recently reported that the hbov non-structural proteins, including np1, are targeted by humoral responses. 30 the potential impact, if any, of non-synonymous mutations within this conserved region of the hbov-1 genome remains unclear and warrants further investigation. to the best of our knowledge, we present the results of the first study to investigate the circulation and diversity of hbov infection amongst hospitalised patients in cambodia. we demonstrate that similar to the findings of other studies, hbov infection can result in serious illness, however is frequently detected in the context of viral co-infection. ongoing studies are required to further understand the true pathogenesis of hbov-1 in the context of severe respiratory illness. cloning of a human parvovirus by molecular screening of respiratory tract samples the human 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and molecular virus characterization a study of the genetic variability of human respiratory syncytial virus (hrsv) in cambodia reveals the existence of a new hrsv group b genotype human bocavirus infection in children with respiratory tract disease human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand frequent and prolonged shedding of bocavirus in young children attending daycare clinical and epidemiologic characteristics of human bocavirus in danish infants: results from a prospective birth cohort study evidence of human bocavirus circulating in children and adults severe pneumonia and human bocavirus in adult serologically verified human bocavirus pneumonia in children human bocavirus: a cause of severe asthma exacerbation in children human bocavirus infections in hospitalized children and adults does human bocavirus infection depend on helper viruses? a challenging case report human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections detection of human bocavirus in japanese children with lower respiratory tract infections we would like to thank all of the technical staff in the virology and epidemiology units at the institut pasteur in cambodia for their assistance. we are also grateful to all the staff from the hospitals for their collaboration in the sisea project. key: cord-316894-zhmuzv7z authors: stetzenbach, l.d. title: airborne infectious microorganisms date: 2009-02-17 journal: encyclopedia of microbiology doi: 10.1016/b978-012373944-5.00177-2 sha: doc_id: 316894 cord_uid: zhmuzv7z inhalation exposes the upper and lower respiratory tracts of humans to a variety of airborne particles and vapors. airborne transmission of pathogenic microorganisms to humans from the environment, animals, or other humans can result in disease. inhalation is an important route of exposure as the lung is more susceptible to infection than the gastrointestinal tract. ingested microorganisms must past through the acidic environment of the stomach before they can colonize tissue while inhaled microorganisms are deposited directly on the moist surfaces of the respiratory tract. inhalation of microbial aerosols can elicit adverse human health effects including infection, allergic reaction, inflammation, and respiratory disease. following inhalation, infectious viruses, bacteria, and fungi can establish in host cells of the respiratory tract. some are translocated and infect the gastrointestinal tract and other tissues. this chapter discusses human viral, bacterial, and fungal diseases transmitted via aerosols. viral diseases presented are influenza, severe acute respiratory syndrome (sars), norwalk-like viruses (nlvs) and hantavirus disease, measles, and varicella. bacterial diseases are legionnaires’ disease, tuberculosis, and nontubercule mycobacterial disease. exposure to some gram-negative and gram-positive bacteria, endotoxin, and actinomycetes when dispersed through the air can result in disease following inhalation. fungal diseases included are histoplasmosis, coccidiomycosis, blastomycosis, cryptococcosis, and aspergillosis. the threat of bioterrorism with airborne infectious agents is also briefly presented. glossary aspergilloma pulmonary fungus ball consisting of a solid mass of hyphae growing in a lung cavity. aspergillosis disease caused by the inhalation of some species of the genus aspergillus resulting in growth of the fungus in the lungs. bioaerosol an airborne suspension of biological particles and microbial by-products. blastomycosis a symptomatic infection caused by the inhalation of the fungus blastomyces dermatitidis. chickenpox disease caused by the virus varicellazoster. coccidioidomycosis disease caused by the inhalation of coccidioides immitis arthroconidia (also called san joaquin valley fever). cryptococcosis disease caused by the fungus cryptococcus neoformans. endotoxin a lipopolysaccharide component of the gram-negative bacteria cell released during active cellular growth and after cell lysis that can cause respiratory distress when inhaled. fomite an inanimate object that transmits infectious agents to a new host. h5n1, h7n2, h9n2, and h7n3 strains of avian influenza a viruses that have been linked to respiratory disease in humans. histoplasmosis disease caused by the infectious fungus histoplasma capsulatum that can manifest as mild flu-like symptoms to chronic lung disease that resembles tuberculosis. hps hantavirus pulmonary syndrome. legionnaires' disease a severe respiratory disease resulting from the inhalation of legionella pneumophila. measles term for the disease caused by the virus of the same name. mycobacterium tuberculosis the causative agent of human tuberculosis. nlvs norwalk-like viruses. pontiac fever a mild flu-like syndrome following inhalation of nonviable legionella pneumophila. rubeola another name for the disease measles. sars severe acute respiratory syndrome. sars-cov severe acute respiratory syndrome associated coronavirus. sin nombre virus causative agent of the majority of hantavirus pulmonary syndrome cases in the united states. hantavirus pulmonary syndrome hvac heating, ventilation, and air conditioning nlvs norwalk-like viruses severe acute respiratory syndrome sars-cov sars-associated coronavirus wmd weapons of mass destruction introduction inhalation exposes the upper and lower respiratory tracts of humans to a variety of airborne particles and vapors. airborne transmission of pathogenic microorganisms to humans from the environment, animals, or other humans can result in disease. the lung is more susceptible to infection than the gastrointestinal tract as ingested microorganisms must past through the acidic environment of the stomach before they can colonize tissue, while inhaled microorganisms are deposited directly on the moist surfaces of the respiratory tract. inhalation of microbial aerosols can elicit adverse human health effects including infection, allergic reaction, inflammation, and respiratory disease. a bioaerosol is an airborne collection of biological material. bioaerosols can be comprised of bacterial cells and cellular fragments, fungal spores and fungal hyphae, viruses, and by-products of microbial metabolism. pollen grains and other biological material can also be airborne as a bioaerosol. microbial aerosols are generated in outdoor and indoor environments as a result of a variety of natural and anthroprogenic activities. wind, rain and wave splash, spray irrigation, wastewater treatment activity, cooling towers and air handling water spray systems, and agricultural processes such as harvesting and tilling are examples of activities that generate bioaerosols outdoors. indoors bioaerosols are generated and dispersed mechanical and human activity. industrial and manufacturing practices and biofermentation procedures can generate high concentrations of microbial aerosols. heating, ventilation, and air conditioning (hvac) systems, water spray devices (e.g., showerheads and humidifiers), and cleaning (e.g., dusting, sweeping, vacuuming, and mopping) result in the transport of microbial materials in the air. talking and coughing generate bioaerosols from individuals, some of which may be infectious. facilities with medical, dental, or animal care practices can generate infectious microbial aerosols. the individual particle size of particulate material in bioaerosols is generally 0.3-100 mm in diameter; larger particles tend to settle rapidly and are not readily transported in the air. virus particles are nanometer in size, bacterial cells are approximately 1 mm in diameter, and fungal spores are >1 mm. these microorganisms can be dispersed in the air as single units, but are often present as aggregate formations. the larger aggregates have different aerodynamic properties than single-cell units; therefore their dispersal may be different than singleunit particles. aggregates of biological material also afford protection from environmental stresses such as desiccation, and exposure to ultraviolet radiation ozone and other pollutants in the atmosphere. often bacterial cells and virus particles are associated with skin cells, dust, and other organic or inorganic material. during agricultural practices (e.g., during harvesting, and tilling), fungus spores are released from plant surfaces and the soil and raft on other particulate matter. this 'rafting' affects the aerodynamic characteristics and the survival of the cells in the bioaerosol. when biological material is dispersed from water sources (e.g., splash, rainfall, or cooling towers and fountains), it is generally surrounded by a thin layer of water. this moisture layer also changes the aerodynamic properties and aids in the survivability of the microorganisms while airborne. airborne particulate will remain airborne until settling occurs or they are inhaled. following inhalation, large airborne particles are lodged in the upper respiratory tract (i.e., nose and nasopharynx). particles <6 mm in diameter are transported to the lung where the 1-2 mm particles have the greatest retention in the alveoli. the average person inhales approximately 10 m 3 of air per day, which may result in an infective dose for some airborne pathogens. diseases resulting from inhalation of bioaerosols include allergic reaction, irritation, inflammation, infection, and respiratory disease. the by-products of microbial metabolism and foreign proteins of microorganisms and fragments of cells can result in allergic reactions and irritant responses. the inflammatory response and allergic reactivity can be debilitating to sensitized individuals in indoor and outdoor environments, and it has been demonstrated following exposure to airborne bacterial fragments present in airborne dust. however, the greatest adverse health impact following exposure to airborne microorganisms is associated with inhalation of infectious agents. these infectious agents include viruses, bacteria, and fungi. the following sections introduce representative pathogens of each grouping that are associated with the airborne route of exposure. table 1 provides a summary of the pathogens presented in this chapter. viruses are noncellular units consisting of either dna or rna, but not both. they do not have self-directed biosynthesis and, therefore, require a living host cell for replication. following inhalation, infectious viruses can establish in host cells of the respiratory tract. some are translocated and infect the gastrointestinal tract and other tissues. influenza is a contagious respiratory illness caused by flu viruses a, b, and c. there are subtypes of influenza type a viruses based on two proteins on the surface proteins of the virus, hemagglutinin (h) and neuraminidase (n). many animals carry influenza a viruses, while influenza b viruses circulate among humans. both influenza types a and b viruses cause epidemics of disease almost every winter. influenza type c infections cause a mild respiratory illness and are not thought to cause epidemics. while symptoms can be mild, influenza may result in severe illness and death. the centers for disease control and prevention reports that there are more than 200 000 hospital admissions and 36 000 deaths per year in the united states due to influenza. all ages can have serious illness from influenza, but those more likely to have complications are people aged 65 years and older, people with chronic medical conditions, and very young children. transmission of influenza viruses is from person to person in respiratory droplets expelled during coughing and sneezing. the droplets are propelled usually less than 3 ft through the air and deposited on people in close proximity. fomites contaminated with settled infectious droplets can also serve as a vehicle for transmission of influenza. avian influenza a viruses usually do not infect humans. however, cases of human infection with avian influenza viruses have been reported since 1997. illness caused by several strains of avian influenza a viruses (e.g., h5n1, h7n2, h9n2, and h7n3) have been reported since 2004. the majority of human avian influenza infections have resulted from inhalation during direct contact with infected poultry or contaminated surfaces such as avian cages, or from the ingestion of uncooked blood of infected birds. therefore, this influenza has been popularized as 'bird flu'. the illnesses resulting from avian influenza infection in humans range from typical mild influenza-like symptoms (e.g., fever, sore throat, cough, and muscle aches) and conjunctivitis to more serious cases of pneumonia, acute respiratory distress, and other severe and life-threatening complications. to date, there have been no sustained humanto-human transmission of avian influenza a viruses, but because of the potential for these viruses to gain the ability to spread easily between people, monitoring for human infection and person-to-person transmission is important. severe acute respiratory syndrome (sars) is a newly reported respiratory illness. it was first reported in february 2003 in asia. since then, it has also been reported in north america and europe. it is a viral respiratory illness caused by a coronavirus, termed sars-associated coronavirus (sars-cov). exposure to sars-cov results from close personal contact with infected individuals. transmission is generally the result of touching the skin of an infected person or objects contaminated with infectious droplets and then transferring the virus to the new host's eyes, nose, or mouth. coughing or sneezing by an infected individual also releases droplets into the air that are propelled a short distance (<1 m) and deposited on the mucous membranes of the mouth, nose, or eyes of persons who are nearby. it is possible that sars can be spread further through the air by very small particles, but investigations to date suggest that this type of spread is unusual. hand hygiene, use of gown, gloves, and eye protection when in contact with an infected individual, and use of an appropriate respirator minimizes the spread of sars. norwalk-like viruses (nlvs) are a group of highly infectious viruses first reported following an outbreak of gastroenteritis in 1972. the infectious dose is estimated at 10 1 -10 2 particles. outbreaks of gastrointestinal infection resulting from exposure to nlvs are characterized by diarrhea and sudden projectile vomiting. while transmission of nlvs is primarily associated with the fecaloral route, it has been estimated that over 30 million virus particles can be liberated during vomiting. therefore, airborne transmission within close contact is highly likely. hantavirus pulmonary syndrome (hps) is a life-threatening disease caused by rodent-borne hantaviruses. it is characterized by severe pulmonary illness and a mortality rate of 30-40%. the sin nombre virus causes the majority of hps cases in the united states, and peromyscus maniculatus (the deer mouse) is its predominant reservoir. the virus found in the rodent urine, saliva, and feces becomes aerosolized as mist from urine and saliva or dust from feces. inhalation is the most common route of infection. however, touching the mouth or nose after handling contaminated materials or being bitten by an infected rodent also result in infection. hantavirus is not spread from person to person. classic symptoms of disease include fever, fatigue, and muscle aches. symptoms progress in 4-10 days to cough and shortness of breath. the disease measles is contracted by inhalation of infectious droplets expelled as respiratory secretions by an infected individual. the measles virus is also known as rubeola and the only known reservoir is humans. all strains of this virus belong to a single antigenic type and immunity is long lasting following infection. fever, malaise anorexia, and respiratory symptoms mimic other respiratory infections, but the appearance of koplik's spots precedes a rash on the face that proceeds down the body to the soles and palms. severe complications may occur including a chronic degenerative neurologic disease that occurs many years after a measles infection. measles is worldwide, but the success of vaccination programs in developed countries has limited the number of cases of childhood measles. however, there are still sporadic cases of measles in the united states as a result of immigration and travel of foreign visitors who spread the infection to unvaccinated or unprotected persons. chickenpox is a highly contagious disease caused by the varicella-zoster virus. chickenpox is usually a self-limited disease lasting 4-5 days with fever, malaise, and a generalized vesicular rash of blister-like lesions. the rash covers the body, but it is usually more concentrated on the face, scalp, and trunk. serious complications including bacterial infections of skin lesions, pneumonia, cerebellar ataxia, and encephalitis may occur, but are rare. disease is spread by aerosol dissemination of the virus during coughing and sneezing by an infected person or it may become airborne directly from the skin lesions. incidence of chickenpox is highest among children 1-6 years of age, but the licensing of a vaccine in 1995 and vaccination requirements for daycare and school children greatly reduced the impact of this virus in the united states. following varicella infection, the virus becomes latent in sensory nerve ganglia and may reactivate later in life resulting in shingles. unlike viruses that require a host cell, bacteria can colonize and grow on a variety of surfaces and in liquids when physical and nutritional factors are suitable. therefore, bacteria can be released into the air from a variety of natural and anthroprogenic environments. when infectious bacteria are released into the air, they can be readily transported via bioaerosols to susceptible individuals. the first report of legionnaires' disease followed the outbreak of respiratory illness among american legion conventioneers in philadelphia in 1976. currently an estimated 10 000-15 000 cases of legionnaires' disease are reported in the united states per year. the causative agent, legionella pneumophila, is a vegetative gram-negative bacterium that is ubiquitous in freshwater environments worldwide. it survives as an intercellular parasite of protozoa thereby resulting in protection for the bacterium while in the environment. infective aerosols are generated from contaminated water spray devices such as showerheads, evaporative condensers, humidifiers, and fountains. following the inhalation, l. pneumophila replicates in host macrophage resulting in severe pneumonia. while l. pneumophila accounts for the majority of disease cases caused by this genus, 19 other legionella species are also human pathogens, often causing disease in immunosuppressed patients. legionella bacteria that cannot replicate in the lung are cited as the cause of pontiac fever, a mild flulike syndrome. often these organisms are released in aerosols from whirlpool tubs. pontiac fever results in many more cases of illness, but fewer deaths; legionnaire's disease has a much higher mortality, but lower morbidity. no human-to-human transmission has been documented. for an outbreak of legionnaires' disease to occur, a series of events must happen. a reservoir of infectious bacteria must be present, the environmental conditions must be right for the concentration of organisms to increase, the organisms must be infectious and the infectious bacteria must be dispersed resulting in human exposure, organisms must be deposited in the appropriate site in the human host, and the host must be susceptible to infection by the legionella. mycobacterium tuberculosis is recognized for its role as the causative agent of human tuberculosis. infected individuals can spread pathogenic bacilli when coughing, talking, speaking, laughing, and sneezing. overcrowding and low-socioeconomic status are often contributing factors to the spread of tuberculosis as individuals are in close association and hygiene conditions are usually poor. the confined spaces of airplane cabins have been implicated in transmissions to passengers and flight crew members. infectious aerosols resulting from the operation of suctioning instruments during medical treatment, manipulation of tuberculosis ulcers, drainage of necrotic tissue, and exposure during autopsy have also been cited as causes of nosocomial m. tuberculosis infections. also, nontuberculosis mycobacterial species can be transported via the air and result in disease. respiratory infection in immunocompromised patients has been associated with members of the mycobacterium avium complex. mycobacterium leprae bacilli have been reported from the nose blow of an untreated person with lepromatous hansen's disease (leprosy), and mycobacterium terrae isolated from a waterdamaged building demonstrated inflammatory responses in mouse lung during laboratory experiments. escherichia coli, pantoea (enterobacter) agglomerans, pseudomonas spp., and acinetobacter spp. commonly isolated in cow barns, pig houses, and poultry barns are among the airborne gram-negative bacteria associated with animal facilities that can result in human disease. disease-causing gram-negative bacteria are also dispersed into the air from wastewater treatment plants, vegetable and herb processing, and recycling facilities. these organisms can cause a variety of health problems, especially to the young, elderly, and immunocompromised persons. endotoxin is a lipopolysaccharide component of the gram-negative bacteria cell and is released during active cellular growth and after cell lysis. while it is not an infectious particle, endotoxin is biologically active material derived from bacteria that can affect many human organ systems and disrupt humoral and cellular host mediation systems. symptoms of exposure to airborne exposure include chest tightness, cough, shortness of breath, fever, and wheezing. obstruction of airflow and exacerbation of asthma has been related to airborne endotoxin concentrations, and atopic and nonatopic asthma have been associated with endotoxin exposure because of its ability to induce inflammatory reactions. repeated inhalation exposure has been shown to induce allergenspecific airway inflammation. airborne endotoxin exposure is a concern in dusty occupational facilities such as cotton processing facilities and agricultural settings, and in industrial environments that utilize water spray components (e.g., machining fluids). indoor airborne endotoxin is a concern with humidified mechanical air conditioning systems. the presence of dogs, moisture, and settled dust increase the likelihood of airborne endotoxin in the home. numerous gram-positive bacteria are commonly disseminated from the skin, oral and nasal surfaces, and hair of humans. commonly staphylococcus spp. are among the nonendospore-forming gram-positive bacteria isolated in indoor air samples. the presence of these cells in the air increases the likelihood of nosocomial infections in healthcare settings. nosocomial infections by airborne pathogens and with antibiotic-resistant bacteria in hospital environments are transmitted among patients through aerosol dispersal from patients, staff, surfaces, and aspiration from respiratory instrumentation. airborne dispersal of pathogens is also a concern in home care and assisted living environments. additionally, the gram-positive actinomycetes have been associated with illness in occupants of waterdamaged buildings. often they are associated with fungi that colonize on surfaces and building materials following water intrusion. fungi are ubiquitous in nature. they utilize organic matter for their metabolism depending on the genus they can readily disperse spores, cellular fragments, or cells into the air. infectious fungi can cause serious mycoses of the respiratory tract and some are disseminated to the bone and other systems in the body. inhalation of spores of the fungus histoplasma capsulatum can result in histoplasmosis. this disease is not transmitted from an infected person or animal to someone else but is the result of airborne transport from environmental surfaces. histoplasmosis primarily affects a person's lungs, and its symptoms vary greatly. infected people are generally asymptomatic or they experience mild flu-like symptoms not requiring medical attention. chronic lung disease resulting from infection with h. capsulatum resembles tuberculosis, and can worsen over months or years. the most severe and rarest form of histoplasmosis occurs when it is disseminated involving spreading outside the lungs. if untreated, disseminated histoplasmosis is fatal. h. capsulatum is a dimorphic fungus with a mold (mycelial phase) when it is growing in soil at ambient temperatures, and a yeast phase after being inhaled by humans or animals. the mold spores are either microconidia ranging from 2 to 5 mm in diameter or macroconidia ranging from 8 to 15 mm. the yeast cells of h. capsulatum are oval to round in shape with diameters ranging from 1 to 5 mm. inhalation of airborne coccidioides immitis arthroconidia may result in the disease coccidioidomycosis (also called san joaquin valley fever). within 48-72 h after inhalation and deposition in the lung, the arthroconidia change into spherules and develop numerous endospores. when spherules rupture, they release endospores which have the capacity to develop into a mature spherule. approximately 40% of those who become infected are symptomatic with flu-like illness with fever, cough, headaches, rash, and myalgia. some individuals fail to recover and suffer chronic pulmonary infection. others will develop widespread disseminated infection that may affect the soft tissues, joints and bone, or meninges. coccidioidomycosis meningitis may lead to permanent neurologic damage. the immunocompromised and hivinfected persons suffer severe pulmonary conditions and diffuse lung disease. c. immitis grows in the soil of semiarid areas. it is primarily found in the lower sonoran life zone and is endemic in the south-western united states; it is also found in parts of mexico and south america. exposure to airborne arthrospores occurs after disturbance of contaminated soil by humans or natural disasters (e.g., dust storms and earthquakes). persons in areas with endemic disease who have occupations exposing them to dust (e.g., construction or agricultural workers, and archeologists) are at risk. african-americans and asians, pregnant women during the third trimester, and immunocompromised persons have a higher risk than others. blastomycosis is a symptomatic infection caused by the fungus b. dermatitidis. this fungus grows in moist soil that is enriched with decomposing organic debris, and it is endemic in parts of the southcentral, southeastern, and mid-western united states. it is also present in some areas of central and south america and parts of africa. exposure is via the inhalation of airborne spores, generally after disturbance of contaminated soil. generally, individuals who frequent wooded areas with endemic disease (e.g., farmers, forestry workers, hunters, and campers) are the most exposed ones. while there are only 1 or 2 cases per 100 000 population in areas with endemic disease, infection results in a flu-like illness with fever, chills, productive cough, myalgia, arthralgia, and pleuritic chest pain. however, some infected individuals fail to recover and develop chronic pulmonary infection and permanent lung damage. other individuals develop widespread disseminated infection that may involve the skin, bones, and genitourinary tract, and occasionally the meninges of the brain are affected. the mortality rate for blastomycosis is approximately 5%. c. neoformans var. neoformans causes most cryptococcal infections in humans. infection with this yeast-like fungus can cause disease in apparently immunocompetent and immunocompromised persons, but most people do not get sick with cryptococcosis. patients with t-cell deficiencies are the most susceptible to infection, and in the united states 85% of cases occur in hiv-infected persons. while the initial pulmonary infection is usually asymptomatic, cryptococcosis can cause serious symptoms of brain and spinal cord disease, such as headaches, dizziness, sleepiness, and confusion. disseminated infection, especially meningoencephalitis, occurs in most patients. c. neoformans var. neoformans is found worldwide, and its habitat includes debris around pigeon roosts and soil contaminated with decaying pigeon or chicken droppings. exposure generally occurs when dried bird droppings are disturbed and dust containing cryptococcus disperses in the air. the exact nature of the infectious particles of c. neoformans is not known, but they are likely to be dehydrated yeast cells or basidiospores of the appropriate size range to be deposited in the lungs. once in the lung, the yeast cells become rehydrated and acquire the characteristic polysaccharide capsule. virulence of this organism is associated with the ability to produce the large capsule and shed great amounts of capsular material into the body fluids of the host. aspergillosis is an invasive pulmonary infection with fever, cough, and chest pain. a localized pulmonary infection occurs for immunocompetent persons who have an underlying lung disease. the fungus infection may disseminate to other organs, including brain, skin, and bone. exposure may also result in allergic sinusitis and allergic bronchopulmonary disease. a variety of aspergillus species have been implicated. aspergillus fumigatus and aspergillus flavus most often have been cited, but aspergillus terreus, aspergillus nidulans, and aspergillus niger have also been associated with the disease. these fungi are ubiquitous in the environment, and their conidia (spores) are disseminated from soil, compost piles and other decomposing plant matter, household dust, building materials, and ornamental plants. water-damaged buildings can also be a source for exposure, and infections have been associated with dust exposure during building renovation or construction. the threat of purposeful transmission of airborne of pathogenic microbial and microbial toxins as weapons of mass destruction (wmd) has increased the awareness of the importance of bioaerosols. the centers for disease control and prevention and the department of health and human services have listed several pathogenic microorganisms as select agents. of these, francisella tularensis, yersinia pestis, bacillus anthracis, and smallpox, the causative agents of tularemia, plague, anthrax, and smallpox, respectively, are ones that can be dispersed via aerosol and, therefore, are a concern for inhalation infection. continued researches and awareness by public health professionals are needed to recognize these diseases and minimize the risk of exposure of the public. see also: aeromicrobiology/air quality; biological warfare; emerging infections; influenza; respiratory viruses; smallpox, historical; surveillance of infectious diseases bioaerosols in the environment bacillus anthracis aerosolization associated with a contaminated mail sorting machine legionella and legionnaires' disease evaluation of exposure to airborne bacterial endotoxins and peptidoglycans in selected work environments infection due to legionella species other than l. pneumophila key: cord-328795-rs1sd42z authors: falsey, ann r.; branche, angela r. title: rhinoviruses date: 2016-10-24 journal: international encyclopedia of public health doi: 10.1016/b978-0-12-803678-5.00386-6 sha: doc_id: 328795 cord_uid: rs1sd42z human rhinoviruses (hrv) are ubiquitous pathogens and the leading cause of the common cold syndrome. hrv are very diverse with more than 100 serotypes identified which cause disease in persons of all ages with the highest incidence documented in young children. although illness is typically mild and self-limited, lost time from work and school creates a considerable economic burden. infection of the upper airways is the most common site of infection, although lower airways disease is also well documented, as is the link between hrv infection and exacerbations of asthma. unfortunately, effective specific antiviral treatments and vaccines remain elusive. human rhinoviruses (hrv) are ubiquitous pathogens which are the leading cause of the common cold syndrome. once considered mostly a nuisance, these viruses are now appreciated as the cause of medically significant illness and a substantial burden of disease. although illness is typically mild and self-limited, lost time from work and school creates a considerable economic burden. infection of the upper airways is the most common site of infection, although lower airways disease is also now well documented, as is the link between hrv infection and exacerbations of asthma. unfortunately, effective specific antiviral treatments and vaccines remain elusive. in this article, the basic virology, pathogenesis of disease, epidemiology, clinical manifestation, and current methods of treatment and prevention will be reviewed. hrv is a member of the enterovirus genus within the picornaviridae family. since it was first identified in 1956, 101 serotypes of rhinovirus have been cultured and distinguished by distinctions in neutralizing antibody. these 101 serotypes have been classified into two species, hrv-a and hrv-b, though in recent years sequencing technology has led to the identification of more than 60 genotypes of viruses which do not grow in culture and have been placed into a third species, hrv-c (bochkov et al., 2015) . the rhinoviruses are also classified on the basis of receptor specificity. the 'major' receptor group, which comprises 90% of serotypes a and b, uses the intercellular adhesion molecule 1 (icam-1) as the receptor for infecting host cells (staunton et al., 1989) . twelve hrv-a serotypes use an alternative site, the lowdensity lipoprotein receptor (ldlr), and the receptor for the hrv-c viruses is human cadherin-related family member 3 (bochkov et al., 2015) . rhinoviruses are small, nonenveloped viruses with a single strand of positive-sense rna (greenberg, 2003) . each virion is composed of 7.2 kb of genomic material packed in a 30-nm icosahedric protein shell known as a capsid (royston and tapparel, 2016 ; figure 1 (a)). the capsid is, in turn, comprised of 12 pentamers each with five protein subunits called protomers containing four viral proteins, vp1, vp2, vp3, and vp4. the capsid is comprised of 12 pentamers with a central shared depression or 'canyon.' (c) protomers are protein subunits of the pentamer containing four viral proteins, vp1, vp2, vp3, and vp4. vp1 proteins form a hydrophobic pocket in the canyon region which serves as the binding site for intercellular adhesion molecule 1 (icam-1) receptors in host cells. each pentamer contains a central shared depression or 'canyon' in the five vp1 proteins of the protomer subunits, which serves as the binding site for icam-1 receptors in host cells (figure 1 (b) and 1(c)). once attachment to the host cell is achieved, viral replication occurs in the host cell cytoplasm. antibody neutralization occurs when igg binds to the viral surface and obstructs access of the host-cell receptor to the viral attachment site at the base of the canyon (turner, 2015) . vp2 and vp3 are also found on the surface of the capsid and have antigenic sites important for the host immune response. vp4 is located on the internal side of the capsid and interacts with the viral genome. the genomic data that code for vp1 are somewhat conserved among the various subtypes of hrv, though variations in vp1, vp2, and vp3 account for antigenic diversity among serotypes and impede the development of effective vaccines and antiviral agents. for infection to occur hrv must reach the nasal mucosa of the susceptible individual. transmission from infected to susceptible individuals most likely occurs when viral load is highest and secretions are plentiful and difficult to control. under experimental conditions, a viral titer of 10 3 tcid 50 (tissue culture infective dose) was needed to transmit infection and most often occurred on the 2nd or 3rd day of a cold during peak symptoms (turner, 2015) . epidemiologic evidence suggests that direct contact and self-inoculation account for the majority of hrv transmissions. classic transmission studies of infected and susceptible subjects demonstrated that hand to hand or from hands to intermediary surfaces with recipient self-inoculation efficiently transmits hrv (hendley et al., 1973) . more recent experimental studies have shown that transmission of hrv is possible by small and large particle aerosols, but how frequently this occurs in natural settings is unclear. once the virus reaches the nasal mucosa, attachment to the epithelial cells occurs via the icam-1 receptor in 90% of infections or by the ldlr receptor depending on the serotype (pitkaranta and hayden, 1998) . hrv grows best at 33 c in cell culture and primarily in the upper respiratory tract, although infection of the lower airways and sinuses, at higher body temperatures, is possible. both ciliated and nonciliated epithelial cells can be infected, and as infection progresses, affected areas may be patchy ( figure 2 ). hrv infection is not cytopathic, and it is generally accepted that the majority of symptoms are due to the host inflammatory response. infection is limited to the respiratory tract with only rare reports of invasive disease (brownlee and turner, 2008) . although hrv is rarely cultured from blood, hrv rna has been detected in the plasma in 10% of normal children with hrv infection and was more common in those with severe illness and asthma exacerbations. however, it is unclear if detection of viral rna in plasma truly represents systemic infection. once infection has been established, a complex interplay of neurogenic and host responses occurs. early infection is characterized by a vigorous inflammatory response brought about by the elaboration of a variety of proinflammatory cytokines and chemokines, including interleukin (il)-1b, il-6, il-8, and interferon-induced protein 10 (pitkaranta and hayden, 1998; turner, 2015) . the concentration of these inflammatory mediators correlates with the severity of symptoms and results in an influx of neutrophils, increased vascular permeability, swelling of the nasal mucosa, and leakage of serum into nasal secretions. the kinins, bradykinin, and lysyl bradykinin can also be found in the secretions of hrv-infected individuals, and their presence and concentrations also correlate with symptoms. yet, treatment by blocking kinins has been ineffectual in alleviating symptoms raising questions as to the relevance of kinins in hrv disease pathogenesis. moreover, despite the frequency of antihistamines in many cold treatments, there is no evidence of histamine release in hrv infection. this may be due to the fact that neurogenic reflexes also appear to play a role in disease pathogenesis, with parasympathetic nerves controlling the flow of secretions from the nasal seromucous glands. finally, the importance of hrv infection and asthma exacerbations is now well recognized (gern and busse, 1999 ). the precise mechanism by which this occurs has yet to be fully investigated but appears to involve genetic predisposition and allergic airway factors measured by immunoglobulin e (ige). hrv-specific neutralizing antibody has been shown to be protective against infection and symptoms with homologous viruses (gwaltney et al., 1966) . infection results in the production of both serum igg and nasal iga beginning around 2 weeks after infection and results in long-lasting protection to the specific infecting hrv serotype. resolution of symptoms and clearance of virus occur before the induction of nasal or serum antibodies, indicating a different mechanism for viral clearance. the inflammatory response produced during initial infection with hrv does not appear responsible for clearance of the virus. although the inflammatory response correlates with symptoms, asymptomatic individuals with little or no inflammatory response can efficiently clear hrv infection. the mechanism by which hrv clearance occurs likely involves the cellular immune response. both cd4 and cd8 cells can recognize heterologous hrv antigens and represent memory cells in persons with prior hrv infection (kennedy et al., 2012; steinke et al., 2015) . these cells function as immune surveillance and are able to produce a rapid adaptive immune response. hrv have worldwide distribution and circulate throughout the year. in temperate climates, peaks of viral activity are seen in the late summer and fall as well as the late spring (pitkaranta and hayden, 1998) . although hrv activity is relatively low during the summer months, it remains the most common cause of the common cold throughout the year. multiple serotypes can circulate simultaneously in the same geographic area, and no clear pattern of reappearance has been noted. households and schools are the most common places for spread of hrv infection. hrv affects persons of all ages with the highest incidence documented in young children. the incidence of hrv infection in children during the first 2 years of life was noted to be 0.7-2 infections per year in older studies using cell culture for viral detection (brownlee and turner, 2008) . more recently, winther et al. reported an average incidence of six picornavirus infections per year in a cohort of children aged 3 months to 15 years. colds remain common among working adults averaging two per year with hrv accounting for 23-50% of illnesses and then appear to decline in frequency toward middle age (gwaltney et al., 1966) . although the incidence of acute respiratory tract infection declines with age, hrv infection can be a problem for older adults. in studies of community-dwelling older adults hrv was identified in 24% of illnesses reported by 533 elderly persons followed for two winters in the united kingdom (nicholson et al., 1997) . additionally, hrv has been described as a cause of infection in older adults in day cares, long-term care facilities, and hospitalized with acute respiratory tract illnesses. asymptomatic hrv infection was recognized in the era of diagnosis by viral culture, but the widespread use of reverse transcription polymerase chain reaction (rt-pcr) for detection in recent studies demonstrates a high incidence of asymptomatic infection. approximately 12-22% of asymptomatic subjects sampled are positive for hrv and approximately 20% of all hrv infections detected by rt-pcr are asymptomatic (brownlee and turner, 2008) . these findings make assessing the casualty of hrv with illness in epidemiologic studies challenging without the use of control subjects. the most frequent clinical syndrome ascribed to hrv is the common cold, an aggravating but usually self-limited illness. sir william osler characterized the illness particularly well in the principles and practice of medicine published in 1925: "the patient feels indisposed, perhaps chilly, has a slight headache, and sneezes frequently. at first the mucous membranes of the nose is swollen, "stuffed up," and the patient has to breathe through the mouth. a thin, clear, irritating secretion flows, and makes the edges of the nostrils sore . usually within 36 hours the nasal secretions become turbid and more profuse. and gradually, within 4-5 days, the symptoms resolve" (gwaltney et al., 1967) . in his studies, gwaltney et al. (1967) had detailed the symptoms and time course of the typical hrv upper respiratory infection (uri). rhinorrhea (65%) and sneezing (50%) were the most common complaints in the first 3 days. scratchy or mild sore throat and headaches are also common in the first few days of illness. fever is uncommon, although some patients complained of feverishness (15%) and chilly sensations (10%). lower respiratory tract symptoms such as hoarseness and cough are less common occurring in one-fourth to one-third of cases although if present, symptoms tend to linger throughout the illness. when compared with other viral respiratory illnesses, significantly more sneezing occurred with hrv infections. the mean length of illness was 8.9 days with a range of 1-33 days. respiratory viral infection is felt to be a common predisposing factor for acute otitis media (aom). middle ear pressures have been shown to be abnormal in up to 75% of individuals with either experimentally induced or natural hrv infection (mcbride et al., 1989) . these abnormalities gradually abate over a 2-week period; however, swelling and obstruction of the eustachian tube may lead to the development of aom. a number of studies indicate that direct viral infection of the middle ear fluid is possible as hrv can be detected by viral culture (1-8%) and pcr (24%) of middle ear fluids in infected individuals. thus, aom may be due to virus, secondary bacterial infection, or coinfection (greenberg, 2003) . primary viral rhinosinusitis appears to be a common phenomenon during uncomplicated natural infection with hrv. sinus abnormalities were observed on computed tomography (ct) scans in over 85% of adults with colds (gwaltney et al., 1994) . most commonly affected were the maxillary (85%) and ethmoid sinuses (65%), though the sphenoid (39%) and frontal sinuses (32%) could also be affected. hrv can often be detected by culture and rt-pcr in sinus brushings or aspirates from patients with viral rhinosinusitis, though the pathogenesis remains incompletely defined. the pressure created by nose blowing, sneezing, and coughing is felt to be an important factor in propelling hrv into the sinuses (greenberg, 2003) . importantly, in most patients, the abnormalities found on ct scans resolve within 2 weeks without antibiotic treatment. because hrv replicates most efficiently at 33 c, for many years infection of the lower respiratory tract at higher body temperatures was felt to be unlikely. recent studies have established hrv replication in the lower airways in natural and experimental infection. after experimental infection with hrv by inoculation of the upper respiratory tract, hrv was detected by in situ hybridization on bronchial biopsy during the peak of infection and 6-8 weeks later (papdopoulos et al., 2000) . consistent with this observation, hrv infection has been linked to bronchiolitis, exacerbations of asthma and chronic obstructive pulmonary disease (copd), and rarely pneumonia. hrv is an important trigger for asthma exacerbations in children and adults (gern and busse, 1999) . the peak of asthma exacerbations in the fall coincides with the increase in hrv activity as well as the start of the school year and seasonal allergen exposures. using rt-pcr in addition to standard testing, johnson et al. demonstrated that over 80% of the asthma exacerbations in children aged 9-11 years were associated with viral infections of which hrv accounted for approximately two-thirds. in addition to epidemiologic links of hrv and asthma, hrv human challenge models have provided insights into the mechanisms by which hrv infection may worsen asthma (greenberg, 2003; papdopoulos et al., 2000) . intranasal and inhaled inoculations of hrv induced bronchoconstriction after methacholine challenge in subjects with mild to moderate asthma (gern and busse, 1999) . the host immune response to infection induces proinflammatory mediators leading to a cascade of inflammation, activation of neural pathways to enhance airway hyperresponsiveness, and obstruction ( figure 3 ). in addition to asthma, hrv has been associated with 40% of acute exacerbations of copd (greenberg, 2003) . in longitudinal studies of figure 3 mechanisms of inflammation and asthma exacerbation triggered by human rhinovirus (hrv) infection. the host immune response to infection induces proinflammatory mediators leading to a cascade of inflammation that triggers airway hyperresponsiveness and obstruction. respiratory illness, hrv were the most commonly identified pathogens. for patients with moderate to severe copd, viral infections frequently lead to emergency room visits (12%) and hospitalizations (19%). the role of hrv as a cause of bronchitis or pneumonia in immunocompetent persons is unclear. a number of studies using either culture or rt-pcr have detected hrv in the upper airways in a small percentage of young children hospitalized with bronchiolitis or pneumonia (brownlee and turner, 2008) . hrv was detected in 21% of children <3 years of age hospitalized with bronchiolitis but was the sole pathogen in only 2%. similarly, 24% of children hospitalized with pneumonia had hrv found in secretions but over half had evidence of simultaneous bacterial infection. while there may be an association of hrv with these types of lower respiratory tract disease, the frequency of hrv in the general population makes assessing causality difficult. the evidence of lower respiratory tract infections with hrv in immunocompromised patients is more convincing (greenberg, 2003) . in a study of 22 hospitalized immunocompromised patients, 32% developed fatal pneumonia. in six of seven total cases hrv was isolated from bronchiolar lavage fluid or endotracheal aspirates. persistent hrv in the lower respiratory tract of lung transplant patients has also been documented (brownlee and turner, 2008; ison et al., 2003) . although symptoms associated with 'the common cold' syndrome are often attributed to hrv disease, the clinical findings of rhinovirus infections are indistinguishable from those of other viral pathogens. a specific microbiologic diagnosis is most often made by detecting hrv in culture or with molecular assay and rarely using immunofluorescence or serotype-specific antibody assays. a variety of sample types from the upper airways have been used to confirm infection including nasal washes, nasopharyngeal swabs, and combined nose and throat swabs, though a few studies now convincingly report lower respiratory tract disease with virus detected in sputum and bronchoalveolar lavage samples from immunocompromised hosts (ison et al., 2003; papadopoulos et al., 2000) . for most patients, the timing of collection also plays a role in where virus is detected, with the highest concentration of virus typically detected in nasal fluids in the first 2-7 days when symptoms also peak (turner, 2015) . traditionally, viral culture has been used to identify hrv infection. viral isolation is accomplished by inoculation of virus on human embryonic fibroblast cells, incubation at 33 c, and demonstration of cytopathic effect in the cell monolayers. in clinical practice, however, this proves to be a time-consuming method of diagnosis, requiring several days for virus isolation and yielding results usually after the acute phase of infection. consequently, microbiologic confirmation is rarely sought using culture methods. in contrast, the advent of new molecular techniques has led to a better understanding of the burden of disease associated with hrv infection with some studies demonstrating an increase in yield of three-to fivefold compared to standard virus culture (pitkaranta and hayden, 1998) . while not necessarily widely used in practice, rt-pcr has become the standard diagnostic tool and proven to be efficient, sensitive, and specific for detecting rhinoviruses. single-target hrv rt-pcr assays use primers that probe a conserved region of the hrv genome and are able to detect most serotypes (pitkaranta and hayden, 1998; turner, 2015) . however, hrv rt-pcr assays are currently only available commercially as part of multiplexed real-time pcr platforms for respiratory viruses such as the filmarray respiratory panel (biofire, utah). this has resulted in some loss of sensitivity and specificity since most of these assays are unable to distinguish between hrv and other enteroviruses. however, one potential benefit of using rt-pcr in practice is the ability to identify low-level viremia and asymptomatic shedding which may be important for infection control purposes in hospital wards with immunocompromised patients. rhinovirus antigen detection using elisa assays has been attempted but requires serotype-specific antibody and is therefore only of use in research settings. a single immunoassay has been developed which utilizes antibody against a conserved protein, the 3c protease of rhinovirus, but is unfortunately too insensitive for diagnostic use (turner, 2015) . similarly detection of serotype-specific neutralizing antibodies has been useful to diagnose natural hrv infections in family cohort and experimental virus challenge studies but is impractical for clinical use. treatment of hrv infections is supportive. the use of over-thecounter therapies may reduce symptoms of the common cold syndrome but are not specific to hrv infections. these remedies include antihistamines and nonsteroidal anti-inflammatory drugs which may alleviate symptoms without shortening duration of viral shedding and are also associated with sedation. other therapies reported to decrease symptoms, such as decongestants, saline nasal spray, expectorants, zinc sulfate lozenges, and high-dose vitamin c, have not been shown to be of significant benefit in randomized controlled trials (turner, 2015) . currently, there are no antiviral drugs approved for clinical use in hrv infections although a few agents have been advanced to clinical trials and shown modest results in decreasing either symptom severity or viral activity. leukocyte interferon was the first agent tested for activity against rhinovirus infection and demonstrated efficacy in preventing infection but not as treatment. however, the development of leukocyte interferon for prophylaxis was impeded by its prohibitively high cost and association with nasal toxicity (turner, 2015) . conversely, monoclonal antibody blockade of the icam-1 receptor, the site of cellular attachment for the majority of hrv-a and hrv-b serotypes, has also been studied and demonstrated a reduction in the severity of symptoms and viral shedding but failed to prevent infection in the rhinovirus challenge model (greenberg, 2003) . moreover, with the discovery of hrv-c viruses, which utilize a different cellular receptor, interest in this approach has waned. the hrv 3c protease cleaves the rhinovirus genome into individual structural and enzymatic components and is important to viral replication (turner, 2015) . it is highly conserved among rhinovirus serotypes and consequently a target for antiviral therapy. ruprintrivir is a 3c protease inhibitor which in experimental trial did not prevent rhinovirus infection but in clinical trial modestly reduced illness severity when treatment was initiated within 1 day of infection (patick et al., 1999) . finally, the hydrophobic pocket formed by vp1 in the canyon region of the capsid, which facilitates attachment to the icam-1 receptors of host cells, has also been a target for drug development. capsid-binding agents bind to the hydrophobic pocket and prevent viral attachment to the cellular receptor and therefore viral replication. pleconaril is a capsid-binding agent with demonstrated antiviral activity against enteroviruses. in two large randomized, double-blind, placebo-controlled trials, patients with common cold symptoms were treated with pleconaril versus placebo within 24 h of symptom onset greenberg, 2003) . compared to placebo, pleconaril treatment resulted in reduced illness duration by 1 day and an overall decrease in the severity of symptoms. however, it was found to induce cytochrome p-450 3a enzymes and therefore was not approved by the food and drug administration due to the potential for severe drug-drug interactions (greenberg, 2003) . new formulations of this agent are currently being considered for clinical trial. efforts to develop an effective vaccine for hrv infections have been unsuccessful to date, and strategies for preventing hrv infections are focused primarily on the interruption of transmission. since transmission occurs from direct contact, standard precautions including appropriate handwashing practices are likely the most effective preventive tool. in high-risk patients such as patients who have undergone recent stem cell transplantation, more stringent infection control practices including screening of symptomatic patients, early implementation of droplet and contact precautions, and restriction of visitors with upper respiratory tract symptoms have been shown to reduce rates of nosocomial infections (ison et al., 2003) . a number of handwashing agents have been assessed for their potential virucidal activity such as hand sanitizers containing 62% ethanol and treatments with acidic compounds turner, 2015) . none have demonstrated efficacy for the prevention of infection and are likely to be as effective as the use of soap and water. shortly after the discovery of rhinoviruses, serum serotypespecific neutralizing antibody and nasal hrv-binding immunoglobulin iga were shown to be protective against infection. efforts to develop hrv vaccinations soon followed, and initial clinical trials in humans involved the use of formalin-inactivated single serotype hrv vaccines. results of these trials demonstrated significant reduction in the rate of symptomatic colds (glanville and johnston, 2015) . however, with the discovery of hrv antigenic diversity resulting in more than 100 different serotypes isolated, this approach was abandoned in favor of multivalent vaccines incorporating 10 serotypes which unfortunately failed to demonstrate significant cross-protection among hrv serotypes. consequently hrv vaccine research was largely abandoned for more than 20 years. recent advances in the development of a mouse modelfor hrv infection have permitted exploration of alternative approaches to immunization including induction of t cell-mediated immunity. in one recent report, recombinant vp0 protein (vp4 ã¾ vp2 precursor), which appears to be highly conserved in hrv-a and hrv-b serotypes, was used as an immunogen in mice and shown to induce cross-serotype t cell recruitment and activation as well as neutralizing antibody production which resulted in accelerated clearance of virus in vivo (mclean, 2014) . this approach suggests that cross-serotype protection is possible, and while it is unlikely that a single immunogen will generate protection against all hrv serotypes, vp0 may be a useful candidate for a broadly protective subunit vaccine. hrv infect persons of all ages. infections in children can occur two to seven times a year and continue through life resulting in two to five symptomatic illnesses per year in adults. while most illnesses are mild and self-limited, in at-risk populations hrv infections may result in severe illnesses. consequently, though innocuously referred to as 'the common cold,' hrv infections constitute a significant burden with associated health care and economic costs. further study is needed to understand the pathogenesis of disease and adaptive immune responses associated with hrv infections which may also provide new targets for antiviral agents and broadly cross-protective vaccines. see also: influenza; pneumonia; respiratory infections, acute; respiratory syncytial virus; viral infections, an overview with a focus on prevention of transmission. cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus c binding and replication new developments in the epidemiology and clinical spectrum of rhinovirus infections association of rhinovirus infections with asthma challenges in developing a cross-serotype rhinovirus vaccine respiratory consequences of rhinovirus infection rhinovirus infections in an industrial population. i. the occurrence of illness rhinovirus infections in an industrial population. ii. characteristics of illness and antibody response computed tomographic study of the common cold efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials transmission of rhinovirus colds by self-inoculation rhinovirus infections in hematopoietic stem cell transplant recipients with pneumonia pathogenesis of rhinovirus infection alterations of the eustachian tube, middle ear, and nose in rhinovirus infection developing a vaccine for human rhinoviruses acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden rhinoviruses infect the lower airways in vitro antiviral activity of ag7088, a potent inhibitor of human rhinovirus 3c protease rhinoviruses: important respiratory pathogens rhinoviruses and respiratory enteroviruses: not as simple as abc a cell adhesion molecule, icam-1, is the major surface receptor for rhinoviruses immune surveillance by rhinovirus-specific circulating cd4 ã¾ and cd8 ã¾ t lymphocytes rhinovirus a randomized trial of the efficacy of hand disinfection for prevention of rhinovirus infection further reading how rhinovirus infections cause exacerbations of asthma key: cord-339039-6gyo9rya authors: bonvehí, pablo e.; temporiti, elena r. title: transmission and control of respiratory viral infections in the healthcare setting date: 2018-04-30 journal: curr treat options infect dis doi: 10.1007/s40506-018-0163-y sha: doc_id: 339039 cord_uid: 6gyo9rya purpose of the review: viral respiratory infections have been recognized as a cause of severe illness in immunocompromised and non-immunocompromised hosts. this acknowledgement is a consequence of improvement in diagnosis and better understanding of transmission. available vaccines and antiviral drugs for prophylaxis and treatment have been developed accordingly. viral respiratory pathogens are increasingly recognized as nosocomial pathogens as well. the purpose of this review is to describe the most frequent and relevant nosocomial viral respiratory infections, their mechanisms of transmission and the infection control measures to prevent their spread in the healthcare setting. recent findings: although most mechanisms of transmission and control measures of nosocomial viral infections are already known, improved diagnostic tools allow better characterization of these infections and also lead to the discovery of new viruses such as the coronavirus, which is the cause of the middle east respiratory syndrome, or the human bocavirus. also, the ability to understand better the impact, dissemination and prevention of these viruses, allows us to improve the measures to prevent these infections. summary: healthcare viral respiratory infections increase patient morbidity. each virus has a different mechanism of transmission; therefore, early detection and prompt implementation of infection control measures are very important in order to avoid their transmission in the hospital setting. healthcare-associated viral infections cause an increase in morbidity and mortality among hospitalized patients and also in healthcare-associated costs. viral respiratory pathogens are increasingly recognized as etiological agents of nosocomial infections. they can be acquired in different settings inside the hospital and they can produce upper (uri) or lower respiratory tract infections (lri) [1•]. transmission can occur by air through aerosolization, by drops of respiratory secretions or by inoculation after touching contaminated surfaces [2] . among immunocompromised hosts, these types of infections can present themselves with subclinical manifestations and can be associated with high morbidity and mortality. for this reason, a high suspicion vigilance must be kept to confirm viral infections in these hosts. it is also known that viral shedding is more prolonged [3, 4•] . there are several etiological agents that cause these infections, with different ways of transmission depending on each virus. for this reason, it is mandatory to suspect and correctly identify the etiological agent and promptly implement isolation precautions. molecular methods have improved sensitivity for the diagnosis of viral infections and have also facilitated the identification of other viral pathogens that cause respiratory tract infections [5] . in the clinical setting, it is sometimes difficult to establish if a respiratory viral infection was acquired in the hospital or in the community. this situation is most commonly observed when the length of hospitalization is less than or equal to the incubation period of the disease. in some cases, virological studies such as next generation sequencing can be used to differentiate between these situations [6] . as a measure to establish the magnitude of respiratory viral infections in the healthcare setting, it must be noted that 20% of all nosocomial pneumonias are of viral origin. in this context, the incidence of each virus is related to its circulation in the community [2, 7] . the objective of this revision is to describe the means of transmission and the measures to control the dissemination of respiratory viral infections in the healthcare setting. there are several viruses associated with upper and lower respiratory infections in humans. we describe the most relevant ones for the clinician. this virus belongs to the mononegavirales order, paramyxoviridae family and pneumovirinae subfamily. main characteristics of rsv include the number and order of genes and lack of hemagglutinin and neuraminidase activity [8] . rsv is the main cause of respiratory tract disease in small children. viral propagation is so efficient that humans can acquire this infection in their first years of life. immunity achieved after rsv infection is incomplete and reinfections are frequent. although life threatening infections occur mainly during the first 2 years of life, rsv infections can cause a high morbidity due to acute upper and lower respiratory tract infections. in children, rsv causes bronchiolitis and pneumonia and it is a common pathogen in older adults and immunocompromised hosts, where it can either cause upper respiratory infections or pneumonia [9] . influenza viruses are single-stranded, negative-sense rna viruses belonging to the orthomyxoviridae family and are classified into three main types, a, b and c, based on their antigenic different characteristics. these three types show significant differences related to their genetic organization, structure, possible hosts, epidemiology and clinical manifestations, with influenza a and b the most common human pathogens. these viruses share certain features such as the presence of an envelope derived from the host cell and surface glycoproteins essential for replication. they have a spheric or filamentous shape and measure 8 to 120 nm with surface projections that are spicules of hemagglutinins (ha) and neuraminidases (na), also important for their antigenic mutations, as they are used as antigens for the influenza vaccine [10] . this virus is an important cause of infections in immunocompetent and in immunocompromised hosts causing mild upper respiratory infections (pharyngitis, coryza, conjunctivitis) and complicated infections (pneumonia, hepatitis, hemorrhagic cystitis or disseminated diseases), respectively. there are seven species of hadv (a to g) and an increasing number of different serotypes were identified through genomic assays. besides this, the improved understanding of viral persistence sites and reactivation requires continuous adaptations of diagnostic tools in order to facilitate timely detection and control of hadv infections. hadv infections are more frequent in closed communities such as the armed forces, nurseries and hospitals with immunocompromised hosts [11••] . this is a single-stranded rna virus from the paramyxoviridae family classified into five types: respirovirus genus (piv 1 and 3) and rubulavirus (piv 2, 4a, and 4b). [12] piv serotypes may cause bronchiolitis, laryngotracheobronchitis (croup) and pneumonia in different age groups in immunocompetent hosts, with piv 3 more frequently associated with lower respiratory tract infections. among immunocompromised hosts, piv has been found in upper and lower respiratory tract infections in 2.2 -7.1% of hsct recipients. in solid organ transplant patients, lung recipients have demonstrated to be at risk for piv disease and this infection was also associated to allograft rejection or dysfunction [13] . these viruses are non-segmented rna viruses, belonging to the mononegavirales order, paramyoviridae family, pneumovirinae subfamily and metapneumovirus genus. initially described as a pathogen in children, where it can cause upper respiratory disease, bronchiolitis and pneumonia, has also been later identified as an adult respiratory viral pathogen [14] . in immunocompromised hosts, hmpn has also been associated to produce upper and lower respiratory tract infections among hsct patients and solid organ recipients, mainly lung transplant patients [13] . picornaviridae family includes nine genuses, four of which (rhinovirus, enterovirus, parechovirus and hepatovirus) can infect humans. human rhinoviruses (hrv) were classified based on immune serotypes, type of receptor, antiviral sensitivity and genotype. rhinovirus infectivity can be neutralized by specific antisera and the new serotypes are defined based on the absence of cross reactivity with known specific antisera. hrv are rna viruses and more than 160 serotypes have been identified. clinical importance of hrv infections is well demonstrated in children where they are responsible for more than 70% of asthma exacerbations [15] . in immunocompetent adults, these are the most common cause of selflimited upper respiratory tract infections and could be responsible for more than 80% of common colds in autumn and spring seasons [16] . immunocompromised hosts are particularly prone to severe infections. in lung transplant patients can be a common infection and lead to graft dysfunction. among hematological malignancies hrv can produce lower respiratory tract infections with potential adverse consequences [11••, 17, 18] . hcov are positive-stranded rna viruses from the coronaviridae family, causing common colds; in children, they have also been associated with bronchiolitis and croup [19] . there are five hcov identified as hcov-oc43, hcov-229e, hcov-hku1, hcov-nl63 and hcov-sars. the latter virus was responsible of an epidemic of severe respiratory disease syndrome in some countries in asia (china, hong kong sar, taiwan, singapore, vietnam) and canada in 2003, with approximately 10% mortality and high attack rate in healthcare workers and family members; it was detected in humans for the last time in 2004 [19, 20] . more recently, one coronavirus was able to cause severe respiratory disease in humans in the middle east. this coronavirus was named as mers-cov and was isolated in saudi arabia in 2012 from respiratory secretions in a man who died from pneumonia. this finding, combined with a retrospective assessment of stored samples from an outbreak observed in healthcare workers in jordan, allowed the recognition of mers-cov as a cause of this new severe respiratory disease [21• ]. the origin of mers-cov has been widely debated. it was originally believed that the reservoir was bats due to the similarity between mers-cov and bat coronavirus. however, the strain found in humans was not related to the one described in bats. another possible source for this type of coronavirus is the camel because they have antibodies against mers-cov similar to those found in humans. most human cases of mers-cov were described in saudi arabia and, to a lesser degree, in the united arabs emirates. later on, other cases outside the arabian peninsula were notified, but all of them were imported from endemic countries in the middle east, and were related to close contact with sick people. most of the patients were healthy males around 50 years [22] . this is a single-stranded dna virus that has recently been found as a human pathogen. it belongs to the parvoviridae family, parvovirinae subfamily, bocaparvovirus genus; four genotypes have been described (hbov 1 -4) [23, 24] . hbov has been associated with upper and lower respiratory tract infections, sometimes as a single agent or as a co-pathogen with other viruses. it is also found in high proportion in fecal samples of children who had the virus already detected in respiratory samples. it has also been reported in stool and blood of immunosuppressed hosts [13] . this virus is transmitted through droplets or by direct contact (table 1) [25] . droplet transmission is possible when the source is within 1 m. the size of droplets, produced by coughing, sneezing or any other procedure that moves respiratory secretions, is larger than 5 μm. recent data has demonstrated the transmission of rsv through aerosolized particles based on the fact that they appear close to hospitalized children with bronchiolitis and rsv infections. these particles can remain suspended in the air for prolonged periods of time and can infect human epithelial cells [26] . the survival of rsv in the environment seems to be partly dependent on drying time and dew point. at room temperature rsv in patients' secretions can be viable on polished surfaces for 3 to 30 h. on porous surfaces such as cloth or tissue papers viability is shorter, usually less than an hour. viral infectivity in the hands varies from person to person, generally less than an hour [27] . the risk of transmission is not the same in all the hospital areas: in pediatric and neonatal units, risk of transmission occurs between 6% and 56%, in transplant and oncohematological units fluctuates between 6% and 12%, and in other adult wards between 30% and 32% [28] . it must also be considered that healthcare workers can be a source of transmission for rsv, considering that shedding occurs between 15% to 20% of them, even without symptoms [29] . in high transmission sites, as in oncohematological adult patient wards, it is recommended to manage these infections in ambulatory care to decrease the risk of transmission within the hospital. nevertheless, it has been demonstrated that this group of patients can acquire rsv and piv infections in spite of their short time of hospitalization [30, 31] . influenza virus can be transmitted through infectious droplets eliminated by patients when coughing or sneezing, or through direct contact with surfaces contaminated by respiratory secretions from symptomatic infected subjects (table 1 ) [32] . airborne transmission was observed with 2009 influenza a h1n1p strain; therefore, airborne precautions should also be implemented in case of a pandemic situation where high risk of aerosolization can occur (e.g. intubation) [13] . in closed areas with high risk patients, such as bone marrow transplant units about 50% of the infections come from healthcare personnel or infected visitors [33•] . another source of nosocomial transmission of influenza are hospitalized patients with prolonged viral shedding, sometimes for weeks or months. high risk patients for prolonged shedding include those under high doses of steroids, oncohematological patients and children [13, 28, 32] . another potential source of transmission at the hospital are those individuals not yet symptomatic, but within 1-3 days prior the infection. these cases are difficult to detect, and hence, preventive control measures are usually not implemented [34] . different studies show that healthcare workers are one of the main vehicles of transmission of influenza within the hospital. there is data showing that many of them continue to work being ill, which certainly amplifies the rate of transmission [35] . it is transmitted through direct contact with a contaminated patient's environment and also by fomites containing the virus (table 1) . adenovirus can also be transmitted through the fecal-oral route and by organ transplantation from infected donors. it can infect an individual, remain latent for a period of time and later cause disease during immunosuppression [36] . the ability of adenovirus to survive in the environment on certain surfaces like non-porous ones can be as prolonged as 49 days, facilitating its transmission [37] . it is transmitted through direct contact with patients' secretions or by direct contact with the contaminated environment (table 1) . this virus can survive more than 4 h on porous surfaces and more than 10 h on non-porous surfaces. however, it is difficult to recover it from the hands and only 5% has been detected after 10 min of contact with contaminated secretions [38] . piv can also be transmitted through droplets, and nosocomial outbreaks have been described [13, 39, 40] . nosocomial transmission has been reported in pediatric and neonatal emergency wards, as well as in nurseries [41••] . immunosuppressed hosts, particularly hsct and oncohematological patients, are more prone to present piv infections with an incidence of 4% and 2%, respectively [42] . viral asymptomatic excretion has been described in hsct and oncohematological patients, further increasing transmission in the hospital setting [43] . hmpn this virus is transmitted by respiratory secretions from coughing, sneezing or through direct contact with contaminated surfaces (table 1) . it is transmitted mainly within the community, but a few reports of nosocomial outbreaks have been described [44] . autoinoculation is the most common way of transmission after contact with contaminated objects. aerosolization also contributes to viral spread (table 1 ) [45] . some epidemiologic data support the possibility of transmission by contact or by air (table 1) . however, means of transmission of mers-cov have not been elucidated yet. the epidemiology of mers-cov infection since isolation of the pathogen in 2012 is consistent with multiple introductions of the virus into humans from an animal reservoir, with no long-term sustained human-to human transmission [46] . hbov is probably transmitted through respiratory droplets; however; nosocomial transmission of hbov has not been reported (table 1 ) [13] . education of healthcare workers, close monitoring of compliance to preventive measures, and infection surveillance are useful tools to reduce the risk of transmission for most respiratory viruses that cause nosocomial infections [2] . in the following section, preventive and control measures for each respiratory virus are described. the precautions recommended by the cdc to avoid rsv transmission include standard and contact precautions (handwashing with water and soap or alcohol hand rub; appropriate use of gloves in order to decrease autoinoculation and use of gown) [2] . use of facemask (droplet precautions) and goggles when direct contact with the patient are also recommended by others (table 2 ) [29, 47] . eye protection should always be worn when performing procedures that might generate sprays from respiratory secretions, considering that nose and eyes are the main sites for virus inoculation [2, 47] . cdc also recommends that infected patients be hospitalized in single rooms or kept in cohort isolation if no individual isolation rooms are available. when cohorting, there should be at least 0.9 m of separation between beds [2] . another measure to be considered is the use of individual equipment for each patient, including toys. other strategies that help prevent rsv transmission in the healthcare setting include: reducing the quantity of persons in contact with hospitalized patients (especially oncohematological), avoiding the presence of children under 12 years of age in the hospital when rsv circulates either in the community or the hospital, and using single-bed rooms for infected patients [2, 29, 48] . some studies have demonstrated that healthcare workers use of eye protection might more efficient than contact precautions in decreasing the risk of rsv transmission within the hospital setting [28] . other recommendations, with very little or no evidence at all, suggest that healthcare personnel caring for rsv infected patients be exclusive for these individuals and that hospital visitor restrictions be taken during rsv season. the use of drugs for prophylaxis is limited to palivizumab in the high risk pediatric population although there are some data that demonstrates its effectiveness in the prevention of rsv in hsct adult patients [13, [49] [50] [51] . annual influenza vaccination of healthcare workers and high-risk patients for influenza complications and their households is the most effective measure to prevent the disease and its complications. it also contributes to decrease viral transmission in the healthcare setting [2] . vaccination of healthcare workers is the second most important strategy, just behind handwashing, to prevent nosocomial dissemination of influenza [41••] . several studies have demonstrated a reduction in morbidity and mortality among patients in different healthcare settings when healthcare workers have been vaccinated against influenza [41••] . different strategies were successfully implemented in order to increase vaccine uptake in healthcare personnel. among these were educational campaigns regarding benefits of the vaccine for patients and personnel, actions addressed to remove administrative barriers for vaccination, implementation of accessible locations for vaccine administration and of signed declination statements, and mandatory vaccination policies for healthcare workers [52] . however, the scientific support for this last strategy has been questioned recently [53] . antivirals against influenza have proved effective to reduce influenza viral transmission. the ones now available are the neuraminidase inhibitors such as oseltamivir, zanamivir and peramivir. oseltamivir which is administered through the oral route has proved to effectively prevent secondary cases of influenza among household contacts if it is given within 48 h of symptoms initiation of the index case. zanamivir, which is administered by inhalation per mouth, has also proved effective in preventing secondary influenza cases in households and healthcare workers [54, 55] , but caution is advised, as its efficacy has only been demonstrated in particular circumstances. during a nosocomial outbreak, in high-risk patients and non-vaccinated healthcare workers, antiviral administration should be implemented. when a mismatch between the circulating influenza strain and the vaccine strain is identified, antiviral prophylaxis should be given to all healthcare workers if an outbreak occurs. duration of antiviral administration during a nosocomial outbreak of influenza should be two weeks or one week after the outbreak control [2] . besides vaccination and antivirals, infection control measures are of utmost importance in order to prevent influenza virus transmission and dissemination in the hospital environment. these measures include handwashing, limitation of visitors and of health personnel in contact with infected patients, cohort or single bed rooms isolation, and droplet precautions. precautions to avoid aerosolization should also be implemented in case of a new pandemic strain or when performing high risk procedures (e.g. intubation) ( table 2 ) [13] . preventive measures to avoid adenovirus nosocomial infections include patient cohorting, reduction of visitors and contact and droplet precautions, along with the exclusion of infected healthcare workers from clinical duties (table 2 ) [13, 36] . handwashing seems to be non-effective in removing adenovirus from contaminated fingers [56] . there are no available vaccines or antiviral prophylaxis to help prevent adenovirus infection [33•, 57, 58] . in order to prevent nosocomial transmission of piv several measures are proposed. they include contact precautions, reinforcement of hand hygiene and standard precautions in all patients to avoid an outbreak in the hospital setting (table 2 ) [2] . there are some vaccines under the initial phases of development that take advantage of mucosal immunity, but there are no efficacy data yet [59] . hmpv infection control measures to prevent nosocomial transmission of this virus include handwashing and contact precautions ( table 2 ) [42] . there is no consensus regarding recommendations to prevent mers-cov transmission. while who recommends contact isolation and droplet precautions for any suspected case and respiratory precautions only for aerosol generating procedures, the cdc promotes air and contact precautions for all the activities related to patient care (table 2 ). other centers recommend intensification of handwashing, contact precautions, ocular protection and the use of facemasks. currently, any suspected or confirmed mers-cov case should be treated in a medical facility. transfer and transportation of an infected patient in the hospital environment should include a minimum crew, all wearing surgical facemasks. healthcare workers with direct patient contact should follow the recommendations mentioned before and keep updated on new recommendations. any transference of mers-cov infected patients from one healthcare setting to another should be coordinated with public health authorities [22, 57, 60] . although nosocomial transmission has not been reported, it seems prudent to implement at least contact precautions in patients when this virus is detected (table 2) . the epidemiology of viral respiratory infections has been better understood and modified in the last years for multiple reasons, including vaccination, improvement in diagnostic assays such as molecular techniques and better knowledge and understanding of mechanisms of transmission. cdc/nhsn surveillance definitions for specific types of infections surveillance definitions janu healthcare infection control practices advisory committee. guidelines for preventing health-careassociated pneumonia, 2003: recommendations of cdc and the healthcare infection control practices advisory committee viral pneumonia in patients with hematologic malignancy or hematopoietic stem cell transplantation respiratory viral infections in solid organ and hematopoietic stem cell transplantation reference is important because nosocomial respiratory tract infections are frequent and severe in this group of patients. this article allows the reader to get updated on respiratory viral infections in immunosuppressed hosts detection of respiratory viruses and legionella spp. by real-time polymerase chain reaction in patients with community acquired pneumonia the role of next generation sequencing in infection prevention in human parainfluenza virus 3 infections in immunocompromised patients nosocomial viral respiratory infections: perennial weeds on pediatric wards adenovirus 1784-1793. in: mandell, douglas, and bennett's principles and practice of infectious diseases a case control study assessing the impact of non-ventilated hospital-acquired pneumonia on patient outcome treanor: influenza (including avian influenza and swine influenza) 2272-2296. in: mandell, douglas, and bennett's principles and practice of infectious diseases this reference reviews all the aspects related to respiratory viral infections including those which are of nosocomial origin. it is a very detailed and exhaustive description of the most common and frequent viral respiratory tract infections. it describes each virus, their mechanisms of transmission ison parainfluenza viruses. 2580-87. in: mandell, douglas, and bennett's principles and practice of infectious diseases respiratory viral infections in hematopoietic stem cell and solid organ transplant recipients. semin respir crit care med rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses frequency and natural history of rhinovirus infections in adults during autumn chronic rhinoviral infection in lung transplant recipients clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy world health organization. summary of probable sars cases with onset of illness from 1 middle east respiratory syndrome is a severe and emerging respiratory viral infection that can also be transmitted in the health care setting. this article describes in detail the latest advances in the research of mers-cov, its epidemiology, mechanisms of transmission, virus characteristics middle east respiratory syndrome coronavirus: review of the current situation in the world cloning of a human parvovirus by molecular screening of respiratory tract samples risk of acute gastroenteritis associated with human bocavirus infection in children: a systematic review and metaanalysis outbreak of respiratory syncytial virus (rsv) infection in immunocompromised adults on a hematology ward evidence of respiratory syncytial virus spread by aerosol. time to revisit infection control strategies? breese hall respiratory syncytial virus (rsv) risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review. influenza other respir viruses nosocomial respiratory syncytial virus infections: the "cold war" has not ended prolonged outbreak of human parainfluenza virus 3 infection in a stem cell transplant outpatient department: insights from molecular epidemiologic analysis molecular characterization of strains of respiratory syncytial virus identified in hematopoyetic stem cell transplant outpatient unit over 2 years: community or nosocomial infection guidelines for preventing infectious complications among hematopoietic cell transplant recipients: a global perspective the authors of this article address the importance of rsv, adenovirus, pai, hrv, influenza and hmpn in both sot and hsct patients. they point out the main characteristics of these viruses in immunosuppressed hosts such as prolonged excertion and higher morbidity and mortality in relation to normal hosts the influenza viruses and influenza incidence and recall of influenza in a cohort of glasgow healthcare workers during the 1993-4 epidemic: results of serum testing and questionnaire adenovirus in solid organ transplant recipients prolonged recovery of desiccated adenoviral serotypes 5, 8, and 19 from plastic and metal surfaces in vitro potential role of hands in the spread of respiratory viral infections: studies with human parainfluenza virus 3 and rhinovirus 14 infection control of nosocomial respiratory viral disease in the immunocompetent host an outbreak of human parainfluenza virus 3 infection in an outpatient hematopoietic stem cell transplantation clinic dare and talbot make a very good description of health care acquired viral respiratory infections, their different control measures(education, hand-washing, isolation, ppe, cohorting of patients and personnel) and other measures such as influenza vaccination parainfluenza virus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection viral respiratory tract infections in transplant patients: epidemiology, recognition and management how contagious are common respiratory tract infections? clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission an outbreak of respiratory syncytial virus in a bone marrow transplant center detection and control of a nosocomial respiratory syncytial virus outbreak in a stem cell transplantation unit: the role of palivizumab respiratory syncytial virus infection in adults immunization of health-care personnel: recommendations of the advisory committee on immunization practices (acip) influenza vaccination of healthcare workers: critical analysis of the evidence for patient benefit underpinning policies of enforcement safety and effectiveness of neuraminidase inhibitors for influenza treatment, prophylaxis, and outbreak control: a systematic review of systematic reviews and/or meta-analyses oseltamivir and inhaled zanamivir as influenza prophylaxis in thai heaith workers: a randomized, double-blind, placebo-controlled safety trial over 16 weeks adenovirus type 8 epidemic keratoconjunctivitis in an eye clinic: risk factors and control adenovirus: current epidemiology and emerging approaches to prevention and treatment emerging viral respiratory tract infections-environmental risk factors and transmission safety and immunogenicity of an intranasal sendai virus-based human parainfluenza virus type 1 vaccine in 3-to 6-year-old children an opportunistic pathogen afforded ample opportunities: middle east respiratory syndrome coronavirus this is also observed in the healthcare setting where respiratory viruses are recognized as a cause of uri and lri. these infections are not only limited to children and may cause severe infections in immunocompromised patients.knowing the mechanisms of respiratory viral transmission in the healthcare setting allows to implement infection control measures to prevent its dissemination and reduce the risk of a hospital outbreak.early detection and strict compliance to infection control measures limit the spread of nosocomial viral respiratory infections. other measures, such as healthcare workers receiving an annual influenza vaccination, should be encouraged. the authors declare that they have no competing interests. this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord-349396-a6zyioc1 authors: tsurumi, amy; flaherty, patrick j.; que, yok-ai; ryan, colleen m.; mendoza, april e.; almpani, marianna; bandyopadhaya, arunava; ogura, asako; dhole, yashoda v.; goodfield, laura f.; tompkins, ronald g.; rahme, laurence g. title: multi-biomarker prediction models for multiple infection episodes following blunt trauma date: 2020-10-07 journal: iscience doi: 10.1016/j.isci.2020.101659 sha: doc_id: 349396 cord_uid: a6zyioc1 severe trauma predisposes patients to multiple independent infection episodes (miie), leading to augmented morbidity and mortality. we developed a method to identify increased miie risk before clinical signs appear, which is fundamentally different from existing approaches entailing infections’ detection after their establishment. applying machine learning algorithms to genome-wide transcriptome data from 128 adult blunt trauma patients’ (42 miie cases and 85 non-cases) leukocytes collected ≤48 hours of injury and ≥3 days before any infection, we constructed a 15-transcript and a 26-transcript multi-biomarker panel model with the least absolute shrinkage and selection operator (lasso) and elastic net, respectively, which accurately predicted miie (auroc [95% ci]: 0.90 [0.84-0.96] and 0.92 [0.86-0.96]), and significantly outperformed clinical models. gene ontology and network analyses found various pathways to be relevant. external validation found our model to be generalizable. our unique precision medicine approach can be applied to a wide range of patient populations and outcomes. severe trauma predisposes patients to multiple independent infection episodes (miie), leading to augmented morbidity and mortality. we developed a method to identify increased miie risk before clinical signs appear, which is fundamentally different from existing approaches entailing infections' detection after their establishment. applying machine learning algorithms to genomewide transcriptome data from 128 adult blunt trauma patients' (42 miie cases and 85 noncases) leukocytes collected ≤48 hours of injury and ≥3 days before any infection, we the high value of a tailored approaches in the care of patients is increasingly appreciated (chaussabel, 2015; parikh et al., 2016; sweeney and wong, 2016) . a method to expedite the timeline of threat detection to before infection happens could yield valuable time for early prophylactic and therapeutic interventions. moreover, the ability to identify patients at high risk for repeated infections, or infection-related morbidity and mortality, might be considered an important measure to fairly allocate resources such as medication, personal protective equipment, or another high-value scarce intervention (massachusetts, 2020; medicine, 2013; medicine, 2020) . trauma is one of the leading causes of morbidity and mortality worldwide (heron, 2018; krug et al., 2000) . severe trauma induces various immune-related responses acutely -it can trigger a state of immunosuppression (islam et al., 2016; ward, 2005) , prolonged inappropriate immune response (heffernan et al., 2012; huber-lang et al., 2018) , leukocytosis (paladino et al., 2010) , and the elevation of specific subpopulations of myeloid cells (cuenca et al., 2011) . among trauma patients, infections and infections-related complications contribute to substantial mortality and morbidity, and prolonged hospital stay, significantly adding to health care costs (cole et al., 2014; dutton et al., 2010; glance et al., 2011; hashmi et al., 2014) . infections and infections-related outcomes vary across individuals, suggesting the importance of considering individual patients' underlying susceptibility and the degree of immunosuppression, or inappropriate immune response experienced. methods to identify trauma patients with particularly increased risk of infection could be advantageous for ensuring timely and appropriate delivery of preventative measures (such as early immune-modulating nutrition, microbiome modulation, early mobilization, early removal of lines/tubes, taking all transmission-based precautions), improving surveillance and promoting antibiotic stewardship to limit the emergence of multi-drug resistance bacteria, reduce toxicity to patients and decrease health care costs. previous studies have evaluated the use of injury severity scores, such as the acute physiology and chronic health evaluation (apache) ii j o u r n a l p r e -p r o o f (knaus et al., 1985) injury severity score (iss) (baker et al., 1974) and new injury severity score (niss) (osler et al., 2010) as predictors of infection, in addition to their intended use to predict mortality (cheadle et al., 1989; jamulitrat et al., 2002) . using genome-wide transcriptomic information from leukocytes provided at triage to assess the underlying susceptibility, well before the onset of infections, is expected to significantly improve the accuracy of identifying patients who are most at-risk of multiple independent infection episodes. a recent study described that employing a combination of predictors could be more effective than using a single predictor with strong statistical significance, further suggesting that multibiomarker panels could be highly effective (lo et al., 2015) . previous studies have utilized transcriptome data in the trauma setting to find transcripts that correlate with poor outcome (desai et al., 2011) or sepsis (sweeney et al., 2015) . the objective of this study is distinct, as we aim to develop a method to predict multiple infections prior to classic clinical signs of infection. and thus, our approach focuses on the prevention of infections, aiming to predict the outcome before its onset, using early blood samples. in a previous study among burn trauma patients, we developed a blood transcriptomic multi-biomarker panel for predicting multiple independent infection episodes (miie) outcome during the course of recovery (yan et al., 2015) . we employed the least absolute shrinkage and selection operator (lasso) machine learning algorithm to select probe sets that together (i.e., multi-transcriptome panel) resulted in highly accurate prediction. this model performed significantly better than those based on injury severity assessments at triage and demographic information (yan et al., 2015) . here, we employed a new approach of combining the use of two algorithms, lasso and elastic net regression, to investigate miie outcome. lasso and elastic net were used to reduce the complexity of regression models, in conjunction with crossvalidation to select the optimal parameter for reducing the number of predictors. these techniques are highly beneficial in cases such as transcriptome data where the number of potential predictors is extremely large, to overcome the problem of overfitting. lasso j o u r n a l p r e -p r o o f regression reduces highly correlated predictors and selects a minimal panel of predictors, compared to elastic net that includes some correlated predictors. we investigated blunt trauma patients in the multi-center inflammation and host response to injury ("glue grant") cohort. this cohort enrolled a high number of patients, generated genome-wide transcriptome data, and collected data longitudinally, allowing us to effectively build a predictive model. our approach employing unbiased analyses of genome-wide information in the identification of patients at increased risk for miie before clinical signs of infection may also be advantageous for providing new insights into the molecular pathways that characterize the patho-physiology underlying hypersusceptibility to infections. baseline characteristics of the 128 blunt trauma patients included in the study ( figure 1a , b) are presented in table 1 while ais (highest score in any body region) was comparable. as expected, orthopedic procedures were the most frequent surgical interventions that patients received overall (76.6%), followed by laparotomy (47.7%), vascular procedures (23.4%), thoracotomy (6.3%) and craniotomy (1.6%) ( table 1) . apart from the proportion of patients having undergone laparotomy, which was significantly higher among miie-cases j o u r n a l p r e -p r o o f compared to non-cases (41.2% vs. 60.5%, p=0.04), other procedures were similar between non-cases and miie-cases. there were five total patients who did not survive, and the cause of death was different for each (supplementary table s2 ). mortality was similar between non-cases (3.5%) and miiecases (4.7%, p=1.00). among survivors, miie-cases had significantly longer hospital stay than (table 2) . among all 128 patients, 36 (28.1%) experienced surgical site infections, compared to 80 (62.5%) who experienced other nosocomial infections (table 2 ). comparing specific subtypes of j o u r n a l p r e -p r o o f nosocomial infections, pneumonia (39.1% overall) was highest, followed by urinary tract infection (18.8%), blood infection (17.2%), pseudomembranous colitis (3.9%), catheter-related bloodstream infection (3.9%), empyema (2.3%), and other unspecified infections (5.5%). when comparing the incidence of various microorganisms among non-cases with one infection episode versus miie-cases, relatively higher proportion was found for miie-cases specifically for gram positives of staphylococcus aureus (18.2% vs. 39.5%), enterococcus species (11.4% vs. 25.6%), coagulase negative staphylococci (2.3% vs. 16.3%), and streptococcus pneumoniae and viridans (2.3% vs. 4.7% for both). the incidence of the gram positive, clostridium species was the same for non-cases and miie-cases (both 7.0%) ( table 2 ). for gram negative bacteria, the incidences of the following microorganism were higher for miie-cases compared to non-cases: enterobacter species (11.4% vs. 37.2%), acinetobacter species (9.1% vs. 30.2%), pseudomonas aeruginosa (11.4% vs. 16.3%), haemophilus influenza (4.5% vs. 14.0%), bacteroides species (0% vs. 9.3%), klebsiella pneumoniae (2.3% vs. 7.0%), neisseria (0% vs. 7.0%), proteus (2.3% vs. 4.7%), serratia marcescens (2.3% vs. 4.7%), and gram negative, not otherwise specified (nos) (2.3% vs. 11.6%). the incidence was higher among non-cases than miie-cases for escherichia coli (11.4% vs. 4.7%), and stenotrophomonas (4.6% vs. 0%). fungi incidences were higher among miie-cases compared to non-cases: candida species (9.1% vs. 11.6%) and unspecified fungi (0% vs. 2.3%). the median time to the first day of detection of different microorganisms ranged widely ( figure 2 ). the gram positives, streptococcus pneumoniae, and streptococcus viridans were found first (at median day 4 and 5, respectively), followed by various gram negatives and the gram positive, clostridium species, between median day 7 and 9.5. microorganisms that appeared relatively later (day 11 to 12) include candida species, enterobacter species, and j o u r n a l p r e -p r o o f serratia marcescens, stenotrophomonas, enterococcus, coagulase negative staphylococci and staphylococcus aureus. we identified 137 probe sets showing at least 1.5-fold up-or down-regulated difference in expression levels between non-cases and miie-cases ( figure 3a ), and performed gene ontology (go) analyses to find enriched biological processes and kegg pathway terms (supplementary table s3 ). as expect, terms relevant to various immune response pathways were among the top fold enrichment. they included interleukin-4 secretion, regulation of interferon-gamma secretion, cytolysis, regulation of natural killer cell-mediated cytotoxicity, viral genome replication, cellular defense response, adaptive immune response, humoral immune response, t cell co-stimulation, regulation of immune response, t cell receptor signaling pathway, response to tumor necrosis factor, response to virus, immune response and innate immune response. other biological processes terms with high fold enrichment were signaling pathways with relevance to cell proliferation and differentiation, including regulation of p38 mapk kinase, calcium-mediated signaling, regulation of fat cell differentiation, organ regeneration, map kinase activity, and phosphatidylinositol 3-kinase (pi3k) signaling. similarly, kegg pathway terms with high fold enrichment included those relevant to immune response, including natural killer cell-mediated cytotoxicity, malaria, hematopoietic cell lineage, and t cell receptor signaling pathway. additionally, regulation of cancer, which may have overlapping signaling pathways with infections-related terms, and related to tissue regeneration, also appeared among the enriched terms. although according to the fdr-adjusted p-values, statistical significance in enrichment was detected only for a small number of terms, the fold enrichment ranking consistently points to the relevance of immune response terms, as expected. to identify a multi-biomarker panel that collectively predicts the outcome of miie, we analyzed the 137 differentially-regulated probe sets by employing a machine learning pipeline that we previously developed and successfully used in our previous study among burn patients (yan et al., 2015) . in the current study, we further added to our analyses pipeline by utilizing a combination of lasso and elastic net regression methods. the lasso regression that reduces redundancy in predictor selection would allow for a narrow selection of a minimal biomarker panel, which is expected to be more practical. elastic net regression that includes correlated predictors would allow for a more comprehensive discovery of additional probe sets that are potentially biologically relevant. with lasso, 15 probe sets were selected, mostly relevant to immune functions and signaling cascades for cellular proliferation and differentiation we assessed the molecular network connection among the 26 probe sets that were selected by elastic net ( figure 3b ). the major nodes with the most extensive edges that were central to the connections consisted of major signaling pathway components that are key regulators of inflammation, mitogentic response, and tissue regeneration. these notable nodes included tumor necrosis factor (tnf), transforming growth factor beta-1 (tgfβ-1), and various chemokine (c-x-c motif) ligand (cxcl) members and chemokine (c-c motif) ligand (ccl) members. tnf and tgfβ-1 are major cytokines that can act both synergistically or antagonistically to each other, depending on the cell context. further substantiating the involvement of these factors, we found p38 mitogen-activated protein kinase (mapk), which can act downstream of both tnf-α and tgfβ-1 pathways. the other major nodes, extracellular signal-regulated kinases 1/2 (erk1/2), and mothers against decapentaplegic homolog 3 (smad3) are also key downstream components of the tgfβ pathway. a key downstream transcriptional regulator for the canonical wnt pathway, β-catenin (ctnnb1), another major pathway known to cross-talk with tgfβ, was also found as a major node in this network. table s8 ). for the various injury severity scores (apacheii, iss, niss), the sensitivity, specificity, ppv, and npv were generally lower compared to the multi-biomarker panel models (supplementary table s8 our study shows that employing novel prognostic models based on early blood transcriptome profiling following severe trauma is an effective method for identifying patients who are particularly at high risk for miie and thus, hypersusceptible to infections. that the transcriptome information provides much better prediction than injury severity information, argues for the importance of considering each patient's underlying susceptibility and of elucidating relevant molecular mechanisms. the comprehensive dataset we used had genomewide transcriptome and clinical data collected longitudinally from a large number of patients, providing the opportunity to assess early susceptibility. notably, our results suggest that by j o u r n a l p r e -p r o o f measuring the biomarkers among our panel at admission, patients at increased risk for miie could be identified before any clinical signs of infection appear. the biomarker panel models in our study had particularly high specificity and npv measures, while also exerting good sensitivity and ppv. moreover, when applied to an external validation cohort, it still performed decently. on the other hand, none of the injury severity scores used often at triage in the trauma setting were effective in predicting the miie. the objective of the study was to provide proof-of-concept results for developing a method to gain additional insights into patients' course of recovery from a simple blood draw at admission. further prospective studies that entail blood draws at admission and measuring the biomarkers we describe here to compare with the severity scores and physiological measures taken normally, would provide additional confirmation of the notion that transcriptome data has the potential to improve outcome predictions in the clinical setting. this study focused on the outcome of miie and the potential prevention of infections, however it is conceivable that the biomarker discovery method described here can be applied to develop prediction methods for other outcomes. a personalized medicine approach and rapid identification of patients with high risk of specific outcomes based on a simple blood draw at admission, is expected to improve surveillance, facilitate decision-making and adequate resource allocation, and improve prevention and management before outcomes occur. our findings could potentially facilitate clinical decision-making by effectively discriminating between those who are expected to develop multiple infections and those who are not. a proposed approach could be that patients who are found not to be hypersusceptible to miie could continue to receive the currently established standard of care, whereas those who are identified to be at high risk could benefit from increased surveillance and additional preventative measures to be taken early. additional interventions for the high risk group may include increased surveillance for early mobilization and removal of lines/tubes, coating iv lines and urine catheters with antimicrobials and/or antibiotics, immunomodulatory nutrition therapies (aghaeepour et al., 2017; lorenz et al., 2015) , and microbiome alterations (harris et al., 2017; tosh and mcdonald, 2012) . such additional measures would incur unnecessary costs if implemented in all trauma patients, however, could be cost-effective when used in this targeted set of patients. efforts aimed at increased prevention have the potential to contribute to alleviating the current antibiotic resistance crisis, toxicity of antibiotics, and the imposed burden on healthcare costs. it is also conceivable that accurate outcome prediction and risk stratification methodologies, such as one we describe here, could be valuable amid crisis situations that result in severe hospital overload with critically ill patients and scarcity of medical resources. the ability to identify patients at low risk for specific morbidity and mortality could aid in informed prioritization of resource allocation to patients with better potential for recovery and survival (massachusetts, 2020; medicine, 2013; medicine, 2020) . having applied both lasso and elastic net regression methods allowed us to construct a highly predictive model from a minimal set of predictors, meanwhile also more comprehensively assess underlying biological mechanisms by allowing additional transcripts to be included. the lasso approach selects a stringent set of predictors with less redundancy, which is advantageous in the clinical setting, where a device requiring less measurements is more practical and easier to implement. the elastic net approach that allows for correlated predictors to be selected found additional transcripts for a more comprehensive discovery of biological mechanisms. the probe sets selected consisted of transcripts with go terms relevant to infections, as expected, and signaling pertinent to oncogenesis and cancer progression. in our study, hgf was the transcript showing the highest upregulation among miie patient blood and in both the 15 probe set and 26 probe set panels. hgf and met expression levels have been suggested as a putative biomarker for monitoring infections, as it is wellestablished that the hgf-met signaling pathway deregulation promotes the growth and invasion by various pathogens (imamura and matsumoto, 2017) . another upregulated transcript, adora3, has also been implicated in the clinical setting, and agonists have been developed and shown to induce anti-inflammatory effects by altering the wnt and nf-κb pathways. as such, the agonists are considered for purposes of treating cancers, and inflammatory diseases such as rheumatoid arthritis and psoriasis (fishman et al., 2012) . neat1 is a non-coding rna that is shown to colocalize with malat1, a long non-coding rna often associated with metastatic cancer, at many genomic sites to transcriptionally regulate target genes (west et al., 2014) . cd96 is highly expressed in t and nk cells and well-established to be a regulator of immune responses during infection and cancer (georgiev et al., 2018) . elastic net selected two probe sets corresponding to mme, providing further support for its importance in miie outcome. studies on its molecular mechanisms and clinical use of inhibitors to its protein product, neprilysin has been conducted widely, including in alzheimer's, heart failures, hypertension, and renal diseases (riddell and vader, 2017) . our study suggests that its potential role in immunity among patients warrants further investigation. klrk1 and klrf1, both killer cell lectin-like receptor subfamily members, were found by elastic net, providing evidence of their relevance in infections in the blunt trauma setting. these receptors are abundant on nk cells, and it is well-established that they play crucial roles in innate immunity (barten et al., 2001) . these previous findings provide additional confidence in the relevance of our methodology. the pathway analysis found key signaling pathway components among the central nodes having extensive edges, including the major cytokines, tnf, tgfβ-1, ccls, and cxcls, as well as key signaling components, p38 mapk, erk1/2, smad3, and ctnnb1. these components represent the chief signaling pathways that regulate inflammation, mitogenic response, and tissue regeneration, which are also often dysregulated in cancer. notably, our results suggest that the tnf, tgfβ-1, and wnt signaling pathways, which are known to also cross-talk with one another through downstream cascades, may be important central pathways that explain the interconnection between the prognostic biomarkers identified. these results may suggest that these signaling pathways may represent new host j o u r n a l p r e -p r o o f immunomodulatory targets that warrant future mechanistic studies. follow-up studies in model organisms and controlled studies would aid in establishing whether the genes identified in this study drive susceptibility, and in uncovering further mechanistic insights. it is noteworthy that when comparing the current biomarker panels in the blunt trauma setting with that from our previous study among burn patients (yan et al., 2015) , we observed that none of the transcripts in the panels were shared. these differences may indicate that increased risk depends on the interaction of the type of trauma with each patient's underlying susceptibility to miie. such observation may suggest the need for developing different multibiomarker panels catered to different types of trauma. our study describes methods towards the development of precision medicine tools and offers the possibility of analyses also for outcomes other than multiple infections. the failure of drug trials targeting sepsis (marshall, 2014; mitka, 2011) highlights the importance of further studies elucidating the underlying molecular mechanisms and components of heterogeneity in susceptibility to infection and infection-related morbidity within a population. it is conceivable that measuring our biomarker panel to triage patients according to susceptibility to multiple infections will strategically guide prophylactic patient management and help reduce the incidence of infections to limit sepsis (boomer et al., 2011; chaussabel, 2015; parikh et al., 2016) . moreover, the analyses process we describe in this study can potentially also be applied to towards biomarker development for sepsis outcome. this study provides for the first time, prediction models for hypersusceptibility to infections, which is highly relevant for critically injured trauma patients, using a machine learning approach. a concern in general for prediction model building is that models may overfit to a specific dataset, making them less generalizable. however, using the multi-biomarker panel dataset was very small in sample size. despite these differences, our multi-biomarker panels still conferred prediction, providing additional assurance in the validity of our results and evidence for the generalizability of our model. additional large prospective studies would more rigorously test the validity and generalizability of the multi-biomarker panel identified in this study. nevertheless, this study provides the first step towards the idea of developing novel approaches for predicting outcomes from blood transcriptome information at admissions. the value of early miie identification, prior to any clinical sign of infection, could be an indispensable tool in other types of trauma and to a wide range of clinical settings. uncovering biomarkers of increased susceptibility to infections may open new avenues for novel therapeutic targets, as well as contribute to standardizing populations in clinical trials. although predictive algorithms cannot eliminate medical uncertainty, our analysis method is expected to be widely applicable to other susceptible populations, such as those with diabetes or cardiac disease, the frail elderly population, those treated with immunosuppressive medication, as well as others. the described methodology of multi-biomarker panel development has the potential to be applied to outcomes and clinical contexts other than miie and trauma, providing additional value. this study entailed a secondary analysis, with external validation using a small dataset. additional external datasets with larger sample sizes, and moreover, a large prospective study would provide additional concrete evidence for the validity and utility of our biomarker panel. j o u r n a l p r e -p r o o f further information and requests for resources should be directed to and will be fulfilled by the lead contact, laurence g. rahme (rahme@molbio.mgh.harvard.edu). this study did not generate new unique reagents dataset requests should be made to the glue grant consortium, due to human study irb restrictions. the code for the analyses is available from the lead contact upon request. deep immune profiling of an arginine-enriched nutritional intervention in patients undergoing surgery the injury severity score: a method for describing patients with multiple injuries and evaluating emergency care divergent and convergent evolution of nk-cell receptors immunosuppression in patients who die of sepsis and multiple organ failure signatures of inflammation and impending multiple organ dysfunction in the hyperacute phase of trauma: a prospective cohort study assessment of immune status using blood transcriptomics and potential implications for global health comparison of trauma assessment scores and their use in prediction of infection and death the burden of infection in severely injured trauma patients and the relationship with admission shock severity a paradoxical role for myeloid-derived suppressor cells in sepsis and trauma dissecting inflammatory complications in 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model hospital policy for allocating scarce critical care resources drug for severe sepsis is withdrawn from market, fails to reduce mortality simplified estimates of the probability of death after burn injuries: extending and updating the baux score leukocytosis as prognostic indicator of major injury integrating predictive analytics into high-value care: the dawn of precision delivery potential expanded indications for neprilysin inhibitors limma powers differential expression analyses for rna-sequencing and microarray studies proc: an open-source package for r and s+ to analyze and compare roc curves ema -a r package for easy microarray data analysis epir: tools for the analysis of epidemiological data a comprehensive time-coursebased multicohort analysis of sepsis and sterile inflammation reveals a robust diagnostic gene set risk stratification and prognosis in sepsis: what have we learned from microarrays? infection control in the multidrug-resistant era: tending the human microbiome immunosuppression after trauma the long noncoding rnas neat1 and malat1 bind active chromatin sites gcrma: background adjustment using sequence information. r package version 2500 prediction of multiple infections after severe burn trauma: a prospective cohort study gene ontology biological processes and kegg pathway term fold enrichment of the 137 probe sets that had 1.5-fold change, using the database for annotation, visualization and integrated discovery (david) functional annotation analyses shriners hospitals grant #71008 to lgr. key: cord-324923-29kudfjp authors: sarma, u.; ghosh, b. title: quantitative modeling and analysis show country-specific quarantine measures can circumvent covid19 infection spread post lockdown date: 2020-05-26 journal: nan doi: 10.1101/2020.05.20.20107169 sha: doc_id: 324923 cord_uid: 29kudfjp the outbreak of covid19 has been declared a global pandemic by who which started in wuhan last november and now has spread to more than 200 countries with 4.5 million cases and a death toll of more than 300 thousand. in response, many countries have implemented lock down to ensure social distancing and started rigorously quarantining the infected subjects. here we utilized the infection dynamics available from who and quantitatively calibrated the confirmed, recovered, and dead populations from 23 different countries. the chosen countries chosen are in three stages of infection 1. where the first wave of infection is significantly diminished 2. infection peak is reached but daily infection still persists significantly 3. the infection peak is not yet reached. the model successfully captured the daily trajectories of countries with both early and late phase of infection and determined incubation time, transmission rate, quarantine and recovery rates. our analysis shows, the reduction in the estimated reproduction number with time is significantly correlated to the testing rate and medical facility of a country. further, our model identifies that an increase in quarantine rate through more testing could be the most potent strategy to substantially reduce the undetected infection, accelerate the time to infection peak and facilitate faster recovery of a nation from the first infection wave, which could perhaps have direct social and economic implications. our model also shows, that post lockdown infection spread towards a much larger second wave can be controlled via rigorous increase in the quarantine rates which could be tailored in a country specific manner; for instance, our simulations suggest that usa or spain would require a 10 fold more increase in quarantine rates compared to india to control the second wave post lockdown. our data driven modeling and analysis of the trajectories from multiple countries thus pave a way to understand the infection dynamics during and post lockdown phases in various countries and it can help strategize the testing and quarantine processes and influence the spread of the disease in future. declaration of the coronavirus pandemic by who severely overhauled global economic and social endeavors for last few weeks [1] . with the first case encountered in wuhan, china, in november 2019 and subsequent outbreak in hubei province, the virus now has spread to more than 200 countries globally. western europe and the united states are severely affected with more than 4.5 million infected population and global death toll rising over 300 thousands [1] . many european countries already started imposing strong mitigation measures through nationwide lock-down in order to maintain effective social distancing within the population. even with nationwide lock-down, many countries are struggling to contain growth of the infection [2] . hospitals are getting overwhelmed with patients and are running out of necessary equipment and medicines [3, 4] . the health-care personnel were in severe crisis of medicines, masks, testing kits, ventilators etc. the vaccine preparation is already on it's way at a breakneck pace [5] but a fully operational vaccine after clinical trial is expected to take at-least a year from now. until then, isolating the infected population by aggressive testing and maintaining strong social distancing measures are adopted as the two most effective ways to deal with the current situation [6] . from the experience in wuhan, we learned that the outbreak can be effectively contained with strong social distancing measures [2, 7] . on the other hand singapore, hong kong and south korea took a different strategy by aggressive testing and isolating the infected population without imposing nationwide lock-down [6] . however, at this moment both the strategies are argued as equally essential to deal with the situation, especially in europe, us and countries with high population density like india. thus in this global emergency scenario, and in the absence of vaccines, model driven strategies to contain the infection rate could be of immense use. hence, in general, if we can predict the spread of the infection and project possible numbers as well as estimate the social and medical factors influencing the spread of the disease, it would help policy makers in considering multiple strategies to address the state of infection that would also have far reaching socio-economic implications. there indeed is an upsurge in epidemic models to predict possible projections of the current situation in different countries [8] [9] [10] [11] which aims to help the policy makers and the medical practitioners to prepare for upcoming situations. along with prediction, analysis of the models should also inform us with possible quantitative measures to deal with the current and subsequent waves of the infection. in this paper, we present a dynamic epidemic model for the spread of coronavirus. by quantitatively calibrating the time series data(data from who [1]) for confirmed, recovered and dead population for 23 different countries with various stages of infection, we made an estimate of different important parameters like incubation time, transmission rate, rate of quarantine, recovery and death rate, that controls the infection dynamics in a given country. we introduced lock-down in our model to observe the effect of social distancing and also estimated the effectiveness of implementation of lock down in individual countries. using the best fit parameter sets, a prediction of the infected numbers for different countries has been projected. variations in the reproduction number as well as variation in reduction of reproduction number with time is also observed . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 26, 2020. . https://doi.org/10.1101/2020.05.20.20107169 doi: medrxiv preprint to be correlated with different demographics and socioeconomic quantities, including health-care facilities, which we show by building statistical regression models. the key insights from our study are 1. lock-down start time following infection is the most sensitive parameter determining the final infection status w.r.t the first wave of infection 2. at any stage of infection, rigorous and country specific tailoring of testing and quarantining can accelerate the time to peak and should eventually contain the infection 3. lock-down removal would most likely start the second wave irrespective of the time of early lockdown to contain the first wave. this is because even in countries with a minimal number of asymptotic infected people, the susceptible subjects can get exposed and infected eventually 4. immediate early lock-down and rigorous testing coupled to systematic quarantining could be the most effective way to rapidly contain the second wave of infection and hence reduce the time of lockdown as well as size of infected population in a country. in order to explore the dynamics of the covid19 infection spread, we took the daily confirmed infection time course data from who and clustered the region-wise data according to their dynamic pattern. a hierarchical clustering algorithm (hierarchical clustering from pheatmap package in r) is used to analyze the dynamics of 93 countries selected (each country with at least 1000 infections per day). the provinces in china clearly are clustered together since infection spread happened at the earliest times. then the infection spreads to different parts of western europe and the united states. the number of new cases in western europe and the united states are much higher compared to other parts of the world which can also be seen as close proximity of these countries in the clustering analysis ( figure 1a , color bar represents log transformed value of the daily confirmed cases). next, we calculated the doubling rate from the time series of the countries. the doubling rate is defined as the inverse of the doubling time, i.e., how much time does the population take to double the number of infections ( figure 1b ) . the clustering analysis shows similar patterns, although, there are large region to region variations in the doubling rate. using a stochastic nearest neighbor algorithm (tsne), we projected the 93 dimensional time-course data onto two dimensions ( figure 1c ). tsne is a machine learning technique for dimension reduction of high dimensional data [12] extensively utilized for visualization of high dimensional data in diverse fields such as computer graphics [13] , neuroscience [14] , medicine [15] to protein structure [16] or embryonic development [17] . the analysis provides us with clearer visual information about different countries and their proximities according to their respective infection trajectories. here also, we observed that the provinces in china are very closely placed and countries in europe form a different cluster. similar analysis was conducted for the doubling rate time courses( figure 1d ). an estimate of reproduction number is calculated from the doubling rate rate using an incubation time of 4 days. we observed a 20% cv in the reproduction number among countries. these results indicate that there is a substantial country-wise . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 26, 2020. . https://doi.org/10.1101/2020.05.20.20107169 doi: medrxiv preprint variability in the infection spread. these variations may stem from each region's different demography, health-care facility, general health or factors implicit to a given country. we now extracted the doubling rate at the start of the infection and after 30 days. although, the initial doubling rate does not show any correlation with any of the factors,( figure 1e ) the doubling rate at 30 days exhibits significant correlation with number of tests per million ( figure 1f ). this indicates the possibility that countries performing more tests on the population are able to manage the infection rate much more efficiently. the total population size also displays significant correlation. this could be due to the fact that countries with higher population size usually tend to have lower test per million rate. in order to estimate different parameters controlling the infection dynamics in different countries we next constructed dynamic epidemiological models for multiple countries and. from the group of 93 countries, we selected 23 countries, comprising a combination of early, mid and late stage of infection, and fitted their confirmed (co), recovered(re) and dead(de) population trajectories (methods for details). the standard susceptible, infected, recovered (sir), or seir(e = exposed) model did not simultaneously fit the trajectories co/re/de in most of the countries (data not shown), but, implementing a quarantine compartment (q), which provided a time delay between infected and removed compartments, dramatically improved the fit quality. the q compartment also ensured a distinction between infected and identified (q) and infected but unidentified(i) subjected in a population. the seiqr model divides the population in five compartments namely susceptible (s), exposed (e), infected (i), quarantined (q) and removed (r), which contain both recovered and dead population [12] . figure 2a shows the structure of the seiqr model. here a susceptible person can be exposed to the infection through transmission from an infected person. after exposure, the symptoms are exhibited within an incubation time and the infected individual either recovers or dies after a time, represented by a recovery or death rate. figure 2c shows cartoon trajectories of confirmed, recovered and dead population which depicts the key qualitative feature -confirmed > recovered > dead. dynamics of the system are captured by a set of 6 coupled ordinary differential equations (details in methods). the calibration data for each country comprises the time courses of the number of confirmed, recovered and dead people. through model fitting we estimated the parameters that best explains the co/re/de trajectories simultaneously of each country. model fitting also includes a lockdown function ( figure 2b ). the lock down is introduced in the model through a reduction in the transmission rate that follows an inverse sigmoid function. the process of lockdown is controlled by three variables-time of lockdown (start time of lockdown implementation) , strength of lockdown (the extent of lockdown in a country, 0.1 would mean 90% lockdown implemented) and the effectiveness of lockdown (how fast the maximum lockdown is . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) achieved from the lockdown starting time point); during the calibration these parameters were also estimated for each country. the assumption is rooted in the fact that once an infected person is tested positive he/she will be quarantined, thus, only the infected but untested subjects can further infect the susceptible. next we extracted the daily confirmed cases from the fitted cumulative trajectories from the model and compared it with data. figure further, we also observed a large variability in effective lockdown among countries (cv=126%), suggesting plausible influence of different social structures within countries in the implementation. this may result from multiple implicit factors specific to a country such as socio-economic structure, state healthcare, population size or other similar factors. as observed from the model, the infection rates substantially vary from country to country. here, we ask the pertinent question why the infection rate and it's reduction vary from one country to another. the socio-economic factors like health infrastructure, demography or population size may influence the infection rate depending on how quickly and efficiently a country's health care facilities react to emergencies. similarly, the reaction can also be limited by the country's population density or demography with respect to age distribution, as it is in general expected that the younger population would be able to react to a new infection . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 26, 2020. . https://doi.org/10.1101/2020.05.20.20107169 doi: medrxiv preprint more efficiently compared to the older population. in fact, the death rate indeed is substantially high for older people with covid19 infection [21] . here, in order to explore social, economic and demographic factors which may influence infection rate of the model, we extracted medical infrastructure, health care spending, demography and population density datasets for all the 23 countries and a correlation analysis based on linear correlation were performed. using the fitted trajectories from the model, values were first calculated (methods) over time for all the countries r 0 ( figure 5a ) and the initial , after 30 days ( ) were extracted from the time course. correlation analysis of , with the health care and demographic factors were performed separately. although was not found to be correlated with any of the parameters, values show significant correlation with r 30 doctors/100, life expectancy and test per million ( figure 5b ). other factors like hospital beds/1000 and health care spending tend to show some negative correlation as noticed from the scatter plot, however, the correlations are not significant due to the relatively small number of samples/country number used for fitting. the initial outbreak although does not depend on the heath-care facility, in absence of early lockdown, how effectively the countries would react to contain the reproduction number, can be correlated to their health infrastructure. however, importantly, the pandemic has affected almost all the countries in the world, irrespective of their healthcare or socio-economic structure, and the need of the moment is to devise scientifically informed strategies to manipulate the spread of infection and minimize the loss of human life which can perhaps be adopted by most countries tailored to their infection status. in an attempt to look for such general strategies to circumvent the infection we systematically explored the calibrated seiqr models further. to understand the relative sensitivity of the model parameters such as î²( susceptible â�� exposed), î± 1 cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 26, 2020. . implementation window for the 'time of lockdown' is already missed, but , although not the most sensitive î± 2 among all parameters can still be tuned anytime by changing the testing and quarantine strategies. notably, the effect of variation (in the range chosen for sensitivity analysis, [0.75-1.25]* fitted) has diverse î± 2 î± 2 sensitivity profile in the group of countries; countries like south korea is extremely sensitive to variation î± 2 where it is known that the testing and quarantining is one of the most rigorous. to understand how the manipulation of might change the long term infection dynamics in the present scenario, we next simulated î± 2 the models for 300 days, with lock down. from the simulated time course (figure 6a ,b, shown for india and korea, two representative countries with high differences in quarantine rate and time to infection peak positions), peak position from the start of the infection, the shift between undetected infection(i) peak and the tested and 'confirmed' peak were estimated for the 23 countries. our analysis showed peak position (tp) is negatively correlated with the quarantine rate, indicating that the more quarantine facilitates an earlier peak ( figure 6c ). this suggests the infection time can be manipulated by quarantining more people. the peak shift(ts), or the time between the confirmed and undetected infected peak position, is also smaller if the quarantine rate is high, indicated by the negative correlation between ts and quarantine rate (figure 6,d) . subsequently, the fraction of undetected infection is also low if the quarantine rate is high for a given country( figure 6e ). these results suggest that by quarantining a lot of infected people through testing, the infection dynamics and time to peak can be closely predicted with lesser error. additionally, this may lead to faster exit from the first wave of infection, as seen in countries like korea. we also observed correlation of tp, ts and fraction of undetected infection with the incubation rates ( figure 6e , f, g). this is due to the correlation between incubation and quarantine rates in the calibrated models. testing and quarantine rate the trajectory remains practically unchanged( figure 7a ). however, as the lockdown is lifted, 5 fold increase in (sapin, usa) appears not sufficient to contain the infection and a much larger 2nd î± 2 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 26, 2020. . https://doi.org/10.1101/2020.05.20.20107169 doi: medrxiv preprint wave can emerge if testing rate is ramped up only 5 fold( figure 7d-e) ; india, on the other hand doesn't show dramatic changes in total number of infection post lockdown with the same increase in value ( figure 7f ). î± 2 we also explored how the total susceptible population size (absolute number of succestable subjects in a population is typically unknown in reality, hence, here for each country the susceptible population size is determined by fitting a population size around the total number of tests carried out in that country) might affect the observed result. figure s5 -s7 shows that the peak of daily confirmed cases for a country and its time to exit from the first wave can robustly be captured in the models for a range of susceptible population size (tested for [0.2-5 fold] of fitted population size). typically, when lock down is removed, the transmission rate (î²) would increase substantially. the long term simulation for india, usa and spain predicted various features of their confirmed time series by the end of june, for varying degrees of quarantine rate, with or without lockdown. for instance, for india the expected number of confirmed cases are estimated to be 450 thousands by the end of june when the lockdown continues and 100 thousands more cases will be added if lock down is removed ( figure 8a , b).however, for both spain and the usa a substantial change in total confirmed cases could be observed (figure 8 a,b) . the daily confirmed cases show a similar trend for both usa and spain, but for india, the post lockdown changes in daily cases are relatively less ( figure 8c ). this indicates different countries may have different infection spread dynamics after the lockdown is lifted. as seen in figure 8d , a 5 fold increase in quarantine dramatically reduces the fraction of undetected infections in the (model)population, both pre and post lockdown, for all the countries. the increased quarantine rates can facilitate the arrival of the peak a bit earlier for countries like india where peak is yet to arrive, especially, when the lock down is removed( figure 8e ). we also calculated the value just after the lock down is removed. for these 3 countries, an increase in the value of is observed r 0 r 0 as the lockdown is lifted but the value can be substantially suppressed by five fold increase in the r 0 quarantine rate for india. where the 10% increase in r0 due to removal of lock down can be effectively circumvented by 40% reduction in r0 by five fold increase in the quarantine rate ( figure 8f ). but, both for spain and usa, a dramatic increase in is observed which can not be compensated by a five fold increase in the r 0 quarantine rate ( figure 8f ). thus, for spain and the usa, we explored if much higher quarantine rates would be required to compensate for the enhanced resulting from the lock down release. we performed a set of simulations by r 0 systematically increasing the value to 50 times of it's fitted value for each country. now, for both spain and î± 2 the usa a 50 fold increase in quarantine rate is able to drastically reduce the peak of the second wave ( figure 9 ). quantitatively, our simulations suggest that both the usa and spain require different degrees of change in this paper, using an epidemic model coupled with statistical regression models we quantitatively explored the time series of infection spread in different countries for the covid19 outbreak and its relation with quarantine measures as well as medical infrastructure. the reproduction numbers of this pandemic are found to be comparable to the sars-cov values [22, 23] and much higher than the mers infection [24, 25] . we employed the mathematical model and fitted the confirmed, recovered and dead population trajectories from 23 countries where the countries are a combination of early, mid and late stage infections for the first wave of covid19 infection. the fitted mathematical models display large variabilities in the infection rate among countries as well as the reduction in their infection rates over time, primarily, due to implementation of the social distancing measures. we show that the variabilities can be correlated to some extent by disparate healthcare infrastructure. in the european countries, the infection has spread faster either due to strong airline connection with wuhan or due to the cold climate but they could control the reproduction number( ) with r 0 time and the first wave of infection is almost over for many such countries (figure 4 ). the lockdown measure to implement social distancing which is implemented in almost every country infected with covid19, is a necessary measure to reduce the infection spread, but how well a country is sampling its population for testing and further how well they quarantine the infected population are also important factors during the lockdown. lockdown is a preparatory measure for the health care system to reorganize itself to deal with the situation since long term lock down would be detrimental to the economy of any country [26] . . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 26, 2020. . in line with the other epidemic models, such as sir or seir models, we assumed a constant initial susceptible population in the fitting process which calculated based on the tests per million conducted in a country. however the actual susceptible population size is unknown and it may be different from the tested population size in either direction. as a result, the number of infected people from the data only captures the infected people out of the tested sample. so to understand the effect of population size on infection peak time and time of completion of the first infection wave, we varied the initial population size in [0. [2] [3] [4] [5] fold of its fitted population size. absolute population size does impact the height of peak in each country tested, but the time to peak and the time for completion of the first wave could robustly be captured by varying size of initial susceptible population. typically the actual infected number of people is expected to be higher than the sampled one's. this is one of the reasons why increased testing rate is so important in capturing the real magnitude of the infection (and not only the dynamics), in addition to the need of quarantining infectious people to reduce infection spread. however, the projection can provide us with a lower bound on the estimated time to contain the infection so that we can remain prepared. in conclusion, in developing countries like india with high population density and size, early implementation of lockdown was critical where the delay in lockdown such as in italy or spain could have had a much more serious impact due to inadequate health infrastructure. however, reopening the economy is also an impending necessity in many countries under lock down. thus, to minimize the health hazards of social proximity while being able to continue economic activities will require careful planning and implementation. we propose strategies where rigorous quarantining of the infected subjects is argued as the only effective measure to effectively deal with infection spread post lockdown. as a policy measure, our model suggests that quarantine and testing should be increased substantially after the lockdown is lifted, in order to contain the infection in the coming months. the effective increase in quarantine measure is found to be country specific, depending on the transmission or incubation rates. in our analysis, we assumed a full lockdown removal. a partial or periodic lockdown removal coupled with increased quarantine rate can also be explored to deal with the situation as studied elsewhere [20] . as there are uncertain components like the number of subjects comprising the susceptible population size in such sir/seir/seiqr models, we also need to be careful about the possible acceleration in the disease spread due to lockdown removal, as a small unidentified fraction of infected population during the lockdown removal can potentially remain unidentified due to the long incubation period characteristic to this infection. the spanish flu pandemic in 1918 in fact came back again in a few weeks after the first wave was apparently contained and the second wave was much bigger than the first one [27] . so, even if the first wave of the current corona pandemic is over for many countries, the global population and policy makers need to remain pragmatically careful for possible subsequent waves [9] and should stratize to maximally quarantine the early susceptible population. in the fight against covid19, it seems critical to act early and act with full force; at the same time, . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 26, 2020. . https://doi.org/10.1101/2020.05.20.20107169 doi: medrxiv preprint controlling overhyped panic stemming from either political polarization or media misinformation [28] could also be prioritized to bring forward a robust and collaborative global effort to fight the pandemic. the seiqr model: the model comprises of susceptible(s), exposed(e), infected(i), quarantined(q), removed(r, contains two compartments 'recovered' and 'dead' ) the equations are the lockdown is opened by modifying the ï�(t) function such that ï�(t) returns to 1 from its lockdown status to no-lockdown(1) status in a designated time . model calibration involves minimizing an objective function that gives best fit parameter sets for confirmed, recovered and dead populations for a given country simultaneously. we fitted the time series provided by jhu csse at github [30] to the seiqr model developed in the study and minimized the objective function using the . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 26, 2020. . lsqnonlin function of matlab which minimizes differences in the sum of squares between model and data. to fit the observed cumulative confirmed trajectory for a given country, [quarantined + recovered + dead] from the model is fitted against the confirmed data. the objective function is thus minimized to achieve the best fits for all three trajectory types simultaneously. this was repeated for all the 23 countries independently. the value is calculated using the r0 package in r [29] . we took the time course data of the daily confirmed r 0 cases with a sliding window of 5 days in order to calculate the value locally with time. r0 package takes the log (2) . the value of incubation time is generated from a gamma distribution with mean as and a standard t 1 deviation of 2.5 in order to perform the calculation of . the authors declare no conflict of interest. the description of the seiqr model: the model utilized to fit the cumulative confirmed, recovered and dead cases comprises susceptible, exposed, infected, quarantined, recovered and dead compartments. the . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 26, 2020. . https://doi.org/10.1101/2020.05.20.20107169 doi: medrxiv preprint lockdown is implemented through a sigmoid function as indicated. the quarantined, recovered and dead cases together comprise the confirmed cases. the seiqr models fit the data for 8 countries as indicated for the cumulative confirmed, recovery and death cases. the figures indicate the cumulative data and the corresponding fit based on the seiqr model. the fitted model also reproduces the daily cases. the daily infected cases were calculated from the fitted confirmed trajectories and the data was calculated from the respective cumulative data trajectories of the respective countries. the effect of lock down removal in india, spain and the usa. the simulation trajectories for daily confirmed cases are shown for three countries and 4 different quarantine rates with lockdown and lockdown removal, as indicated. the quarantine rates are increased on the 115th day (after 22nd jan, 2020, the first respored day of infection in wuhan) and lockdown removal on the same day is considered for these simulations.. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 26, 2020. . https://doi.org/10.1101/2020.05.20.20107169 doi: medrxiv preprint figure 9 : optimal, country specific change in quarantine rate can potentially circumvent the infection spread post lockdown. the simulated trajectories for daily confirmed cases when quarantine rate is increased on 115th day and lockdown is lifted on 130the day for the three countries. similar results were obtained when both quarantine rate and lockdown removal times are chosen as the same. the three panels depict the current situation, the situation with quarantine rate increased on 115th day in presence of lockdown, and lockdown removed 15 days after changes in the quarantine rates, respectively, as indicated. the simulated trajectories for lockdown, presence and absence, coupled to varying quarantaine rates, and, with different population sizes, is shown for spain. the simulated trajectories for lockdown, presence and absence, coupled to quarantaine rates, and, with different population sizes, is shown for usa. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 26, 2020. . https://doi.org/10.1101/2020.05.20.20107169 doi: medrxiv preprint figure 57 : the simulated trajectories for lockdown, presence and absence, coupled to quarantaine rates, and, with different population sizes, is shown for india. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 26, 2020. . https://doi.org/10.1101/2020.05.20.20107169 doi: medrxiv preprint . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 26, 2020. . https://doi.org/10.1101/2020.05.20.20107169 doi: medrxiv preprint . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 26, 2020. . https://doi.org/10.1101/2020.05.20.20107169 doi: medrxiv preprint the demand for inpatient and icu beds for covid19 in the us: lessons from chinese cities italy hospitals at virus limit covid19 infections rise in new york with peak weeks away draft landscape of covid19 candidate vaccines how will country-based mitigation measures influence the course of the covid19 epidemic? epidemiology and transmission of covid19 in shenzhen china: analysis of 391 cases and 1,286 of their close contacts data-driven modeling reveals a universal dynamic underlying the covid19 pandemic under social distancing projecting the transmission dynamics of sars-cov-2 through the postpandemic period science epidemic situation and forecasting of covid19 in and outside china yubei huanga age-structured impact of social distancing on the covid19 epidemic in visualizing data using t-sne" (pdf) maaten laurens van der approximated and user steerable tsne for progressive visual analytics nonlinear dimension reduction for eeg-based epileptic seizure detection exploring nonlinear feature space dimension reduction and data representation in breast cadx with laplacian eigenmaps and t-sne the protein-small-molecule database, a non-redundant structural resource for the analysis of protein-ligand binding single-cell transcriptomics reveals gene expression dynamics of human fetal kidney development a seiqr model for pandemic influenza and its parameter identification population-level covid19 mortality risk for non-elderly individuals overall and for nonelderly individuals without underlying diseases in pandemic epicenters john p.a. ioannidis transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions transmission dynamics and control of severe acute respiratory syndrome transmission characteristics of mers and sars in the healthcare setting: a comparative study the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission cross-protection between successive waves of the 1918-1919 influenza pandemic: epidemiological evidence from us army camps and from britain using social and behavioural science to support covid-19 pandemic response the r0 package: a toolbox to estimate reproduction numbers for epidemic outbreaks key: cord-316500-vik30moa authors: cardillo, lorena; piegari, giuseppe; iovane, valentina; viscardi, maurizio; alfano, flora; cerrone, anna; pagnini, ugo; montagnaro, serena; galiero, giorgio; pisanelli, giuseppe; fusco, giovanna title: lifestyle as risk factor for infectious causes of death in young dogs: a retrospective study in southern italy (2015–2017) date: 2020-06-05 journal: vet med int doi: 10.1155/2020/6207297 sha: doc_id: 316500 cord_uid: vik30moa infectious diseases are a common cause of death in young dogs. several factors are thought to predispose young dogs to microbiological infections. identifying the cause of death is often a challenge, and broad diagnostic analysis is often needed. here, we aimed to determine the infectious causes of death in young dogs aged up to 1 year, examining how it relates to age (under and over 6 months), lifestyle (owned versus ownerless), breed (purebred and crossbreed), and gender. a retrospective study was conducted in a 3-year period (2015–2017) on 138 dead dogs that had undergone necropsy and microbiological diagnostics. enteritis and pneumonia were the most commonly observed lesions. polymicrobism was more prevalent (62.3%) than single-agent infections and associated with a higher rate of generalised lesions. ownerless dogs showed over a three-fold higher predisposition to viral coinfections than owned dogs. above all, canine parvovirus was the most prevalent agent (77.5%), followed by canine coronavirus (31.1%) and canine adenovirus (23.9%); ownerless pups had a higher predisposition to these viruses. escherichia coli (23.9%), clostridium perfringens type a (18.1%), and enterococcus spp. (8.7%) were the most commonly identified bacteria, which mostly involved in coinfections. a lower prevalence of cdv and clostridium perfringens type a was observed in puppies under 6 months of age. in conclusion, this study is the first comprehensive survey on a wide panel of microbiological agents related to necropsy lesions. it lays the groundwork for future studies attempting to understand the circulation of infectious agents in a determined area. a correct and complete determination of the cause of death in young dogs is a challenge for veterinarian practitioners. a mere anatomopathological examination is often not sufficient to define a lesion's etiology. puppies are susceptible to several viral and bacterial pathogens owing to the incomplete ability of their immature immune system [1] . in the first days of life, bacterial infections are described to be the prevalent cause of neonatal disease and death [2] ; in contrast, at other ages, many factors have been attributed for outbreaks of viral diseases, including age, vaccination status, breed [3, 4] , habitat [5] , and seasons [6] . stressful conditions due to overpopulation, high environmental contamination [7] , lengthy travel for illegal importations, and lack of vaccination can create immune deficiency [8] . in this context, viral infections and bacterial superinfections can occur, and mixed infections are frequently detected [9, 10] . polymicrobial infection recognises several etiopathogenetic mechanisms: (1) universally recognised virus-induced immunosuppression created by some agents such as canine parvovirus (cpv) leukopenia and disruption of the gastrointestinal barrier [1] or canine distemper virus (cdv) lymphopenia [11] , which create a niche for the growth of other opportunistic pathogens; (2) the so-called primary with secondary infections, where the first agent creates the ideal condition needed for the colonisation and replication of the second one, e.g., "kennel cough," where some respiratory viruses such as cdv, canine adenovirus type 2 (cav-2), canid herpesvirus-1 (chv-1), and others precede the secondary bacterial infection [12, 13] ; (3) a condition characterised by concurrent infection of multiple agents to induce the disease [8] . in addition, an immune-compromised system can play a key role for systemic spread of localised infections in various ways, including extraintestinal diffusion of enteric pathogens, because of gut dysbiosis, either through the choledochus or via bacterial translocation from the lymphhematic route [14] [15] [16] , as described for coliform septicemia in dogs with viral-induced damage of intestinal epithelial cells during parvoviral infections [17] . in many cases, coinfectants can exacerbate clinical signs; thus, normally mild pathogens can cause severe diseases [18, 20] . although vaccines for some pathogens that cause high mortality in pups have been produced, a failure of vaccination can occur, due to interference of high titres of maternal-derived antibodies (mdas), incorrect vaccination protocols, high environmental contamination, or stressful conditions [21, 22] . e identification of specific causes of death has a fundamental epidemiologic role. several studies have been conducted in the past, based only on anatomopathological lesions [23] [24] [25] [26] , and, more recently, a retrospective study was reported in the province of rome (italy), based on anatomohistopathological examinations and collateral exams [27] . to the authors' knowledge, there are no epidemiologic surveys on the causes of death for infectious diseases in young dogs performed in southern italy. us, the aim of this study was to identify infections and coinfections associated with macroscopic lesions in deceased dogs under 1 year of age, related to their age and lifestyle. our study is a retrospective survey carried out at the istituto zooprofilattico del mezzogiorno (izsm) of portici, naples (southern italy), as part of our routine diagnostic activities aimed to verify the causes of death in owned and ownerless young dogs. from 511 dead dogs brought to the izsm by veterinary practitioners, owners, or law enforcement officers in order to assess the cause of death, 138 fulfilled the criteria to be included in the study. ese criteria included signs of death due to infectious diseases, confirmed by anatomopathological and chemical exams, complete panel for necropsy, and microbiological examinations; ages ranged from 0 to 12 months. data included signalment, anamnesis, necropsy, and microbiologic reports, which were carried out over a 3-year period (january 2015 to december 2017) from the informative system for analysis laboratory management (s.i.g.l.a.) database of the izsm. for the content of this survey, the authorisation of the ethical committee was deemed unnecessary, according to national regulations. diagnosis of cause of death. all necropsies were performed in the necropsy room in the izsm with a standard necropsy protocol codified by piegari et al. [28, 29] . approximatively, 2 cm 2 of tissue samples and swabs were collected from deeper structures of the liver, lungs, brain, heart, and small intestine. fecal swabs were collected from rectal ampulla. specimens were processed for microbiological examinations as soon as possible. approximately, 2 mg of each tissue sample was collected and suspended in 2 ml of sterile phosphate buffer saline (pbs) in 2-ml tubes, homogenised with glass beads with the tissuelyser (qiagen), and clarified by centrifugation for 5 min at 4000 rpm. fecal specimens were subjected to a previously described treatment, in which 100 mg of feces was suspended in 900 µl of tissue lysis buffer (atl), vortexed for 1-2 min, and incubated for 10 min at room temperature to obtain sedimentation of greater particles; finally, 600 µl of supernatant was transferred to a 1.5-ml tube and incubated at 70°c for 10 min in a thermomixer (eppendorf ). aliquots of 200 µl of both tissue and fecal supernatants were collected for nucleic acid extraction using the qiasymphony automated system (qiagen) and processed according to the manufacturer's protocol, eluted in 60 µl, and stored at −80°c until use. samples and fecal swabs were tested for the most common viruses that cause diarrhea, pneumonia, hepatitis, encephalitis, and myocarditis in young dogs, using pcr protocols reported in table 1 . for molecular detection and characterisation of cpv variants (2a-2b-2c), real-time pcr was performed using different sets of primers and probes to amplify fragments encoding for capsid protein vp2, as described previously [30] . for ccovs, the molecular detection and genotyping of ccov-i and ii were performed using a set of primers and probes that amplify fragments of orf5 as described by decaro et al. [32] , and the 2 subtypes ccov-iia and iib were characterised by the amplification of the gene encoding the spike (s) protein using the rt-pcr protocol reported by decaro et al. [33] . finally, for cavs, the protocol used was based on a duplex real-time pcr assay for the simultaneous detection of types 1 and 2, amplifying a fragment of the hexon gene, as described by dowgier et al. [40] . e characterisation of cdv samples was performed on the hemagglutinin (h) gene sequence, according to an et al. [41] . next, 1 µl of the amplification product was applied to the tapestation 2200 (agilent technologies) using d1000 screen tape and reagents (agilent technologies). e pcr products were purified using the minielute reaction cleanup kit (qiagen) according to the manufacturer's protocol. reactions were subjected to sanger sequencing carried out by bigdye terminator cycle sequencing kit v.1.1 (applied biosystems). e amplification conditions used for the sequencing reactions included an initial denaturation at 95°c for 1 min, followed by 25 cycles of denaturation at 96°c for 10 s, annealing at 50°c for 5 s, and extension at 60°c for 4 min. amplicons were then cleaned up using the dyeex 2.0 spin kit (qiagen) following the manufacture's indications. e sequencing reactions were applied to a 3500 genetic analyzer capillary electrophoresis system (applied biosystems). e forward and reverse sequences were assembled using the geneious r9 software package (biomatter) and compared to analogous sequences in the blast genetic database (http://www.ncbi.nlm.nih.gov/blast.cgi). bacteriological analyses were performed by validated standard methods. briefly, swab samples from the first isolation of the liver, lungs, brain, and small intestine were plated on macconkey agar, aerobically incubated at 37 ± 1°c for 24-48 h and 5% blood sheep agar, incubated at 37 ± 1°c for up to 72 h in an aerobic or anaerobic atmosphere. for campylobacter spp., skirrow agar was used, and plates were incubated at 42 ± 1°c for 24-48 h in microaerophilic atmosphere, containing 6% o 2 , 10% co 2 , and 84% n 2 . anaerobic atmosphere was created by adding a 2.5-l oxoid anaerogen sachets to an oxoid anaerojar (2.5 l, ermo fisher scientific). when appropriate, enrichment broths were used. salmonella spp. were isolated using preenrichment müller-kauffmann tetreathionatenovobicin broth (mkttn) incubated at 37 ± 1°c for 24 ± 3 h and rappaport-vassalidis soy broth (rvs) at 41.5 ± 1°c for 24 ± 3 h and subsequently plated on xylose lysine desoxycholate agar (xld) and brilliance salmonella agar base (bsa), both incubated at 37 ± 1°c for 24-48 h. single colonies were selected and plated on specific media according to the findings. for biochemical identification, the vitek2 system was used (biomérieux). finally, pcr methods were used in order to characterise c. perfringens toxins [42] . according to the identified etiologic agent, 5 categories were created: no agent detected, pure viral infections, mixed viral infections, bacterial infections, and mixed viral-bacterial infections. univariate models were used. e variables considered were lifestyle, age, gender, and breed. data were analyzed with an online software for statistical computation (vassarstats, vassar college). prevalence was calculated at a 95% confidence level. a chi-squared test of association was used to obtain the statistical significance level between groups, and risk assessment was performed with the odds ratio (or) test to confirm the difference between groups. results were considered statistically significant with a p value <0.05 and an or > 1.0. from january 2015 to december 2017, 511 necropsies and microbiological analyses were recorded. data were obtained from the izsm database, and a total of 138 dogs were identified to fulfil the inclusion criteria. ese dogs were under 1 year of age and comprised of 53 females (38.4%), 78 males (56.5%), and 7 of unknown sex (5.1%). ey belonged to 30 different breeds: 37 crossbreeds (26.8%) and 93 purebreds (67.3%). of the purebreds, the following breeds were recorded: 12 pomeranians (8.7%), 11 maltese (7.9%), 8 chow chows (5.8%), 6 each english bulldogs and chihuahuas (4.3% for each), 5 each labrador retrievers and yorkshire terriers (3.6%), 4 neapolitan mastiffs (2.9%), 3 each for fox terriers, german shepherds, husky, italian mastiffs, pointers and poodles (2.1%), 2 each for cavalier king charles spaniel, pinscher and shiba inu (1.4%), 1 each for maremma shepherd, pitbull, jack russell terrier, great dane, english setter, beagle, dogue de bordeaux, argentine mastiff, akita inu, and pug and dachshund (0.7%). for 8 reports (5.8%), however, no information about the breed was available. four variables were examined: "lifestyle," which included 70 ownerless dogs (50.7%), 33 strays that were housed in shelters (47.1%), and 37 that were confiscated by law enforcement for illegal importation (52.8%), and 68 dogs who lived in a house with their owner (49.2%). e "age" variable included 109 dogs under 6 months (78.9%) and 29 dogs from 6-12 months old (21%). real-time pcr, screening cpv/fplv [30] real-time pcr, cpv-2 based vaccine/cpv field strain [31] real-time pcr, antigenic variants cpv-2a/2b and cpv-2b/2c [32] canine coronaviruses (ccovs) real-time rt-pcr, screening ccovs [33] real-time rt-pcr, genotypes ccov-i/ccov-ii [34] real-time rt-pcr, subtypes ccov-iia/ccov-iib [35] canid herpesvirus 1 (chv-1) real-time pcr [36] canine distemper virus (cdv) real-time rt-pcr [37] rt-pcr [41] rotavirus (rv) real-time rt-pcr [38] canine adenoviruses (cav-1 and cav-2) pcr [39] real-time pcr [40] veterinary medicine international 3.1. necropsy examination. evaluation of the necropsy examination reports showed that the most frequently observed lesion was enteritis, which was found in 115 cases (83.3%), followed by pneumonia in 101 cases (73.1%), hepatitis in 65 cases (47.1%), encephalitis in 46 cases (33.3%), and finally myocarditis in 8 cases (5.8%). multiorgan lesions were found to be more prevalent than those on a single organ; these lesion types were observed in 116 (84%) and 16 cases (11.6%), respectively ( figure 1 ). in 6 cases, no lesions were observed (4.3%). e organs were submitted to a broad microbiological analysis, and the chi-square analysis showed that there was a different trend in the distribution of the lesions in the organs related to microbiologic categories (p < 0.0001). us, while in single-organ lesions there was a higher prevalence of pure viral infections (43.7%), for cases with lesions in 2 or more organs, mixed viral-bacterial infections were more prevalent. e distribution of the lesions for the variables-lifestyle, age, gender, and breed-was investigated too (table 2) , and a different trend was observed between ownerless and owned dogs (p � 0.013) as well as between crossbreed and purebred dogs (p � 0.0026). cases in which one or two organs were affected were more frequently observed in owned dogs, whereas instances with three-or-four-organ lesions were prevalent in the ownerless group of dogs. regarding the breed variable, in crossbreeds, two organs were more commonly affected (56.7%); in contrast, purebreds had more diffuse lesions on three (30.1%) and four organs (21.5%). no difference was observed for age (p � 0.1936) or gender variables (p � 0.2328). a univariate analysis of the correlation of microbiological categories with the four variables considered in this study was conducted (table 3) . a significant difference was observed in ownerless dogs, which were approximatively 3fold more predisposed to viral mixed infections (or � 3.24; p � 0.0117). in contrast, ownerless dogs were less frequently affected by bacterial infections than owned dogs (or � 0.2; p � 0.0120). for the breed variable, a statistically significant difference was observed in pure viral infections; thus, purebreds exhibited lower rates of pure viral infections than crossbreeds (or � 0.22; p � 0.0012). no difference was observed in the age or gender variables for any of the microbiological categories that were examined. our analysis of pathogens showed high prevalence of cpv (107/138; 77.5%), and it was frequently the causative agent of pure viral infections (27/33; 81.8%). furthermore, it was also the most prevalent virus involved in viral coinfections (26/26; 100%) and viral-bacterial coinfections (55/ 58; 94.8%). high prevalence of canine coronaviruses was also observed (43/138; 31.1%). although it was rarely observed in pure viral infections (4/33; 12.1%), canine coronaviruses were the second most prevalent agent involved in viral (18/ 26; 69.2%) and viral-bacterial (21/58; 36.2%) coinfections. a frequent association of this virus with cpv was observed in 79% (34/43) of ccovs-positive samples. a modest circulation of cavs and cdv was found in 23 (16.6%) and 19 (13.7%) pups, respectively. in contrast, chv-1 and canine rotavirus (rv) had a very low prevalence: 5 (3.6%) and 2 cases (1.4%), respectively. bacterial infections were observed in the 52.9% of cases (73/138), mainly associated with viral pathogens (58/138; 42%). e following bacteria were found to be most prevalent: escherichia coli was detected in 33 cases (23.9%), clostridium perfringens type a (cpa) in 25 cases (18.1%), and enterococcus spp. in 12 cases (8.7%). ey were rarely identified as single pathogens but were often involved in mixed infections, mostly associated with cpv. us, e. coli, cpa, and enterococcus spp. showed mixed infections with cpv in 75.7% (25/33), 80% (20/25), and in 100% of the cases, respectively. a very low prevalence was detected for other bacteria, which were mostly identified as opportunistic agents. after examining bacterial and viral prevalence, infection risk was examined for the most frequently detected pathogens related to the four variables considered in the study (table 4) . ownerless dogs showed a higher risk of infection to viral pathogens than owned dogs. e highest hazard risk was due to canine coronaviruses, with an odds ratio of 14.96 (p < 0.0001). when considering age as the variable, younger dogs, under 6 months, were less predisposed to cpv, cdv, and cpa than older dogs (with p values of 0.0444, 0.0007, and 0.0470, respectively). finally, purebred dogs showed a higher prevalence for e. coli infections than crossbreeds (or � 4.88; p � 0.0137). e relationship between pathogen and single/multiple organ (s) being affected was also investigated ( table 5 ). in single infections, cpv and ccovs were associated with a higher degree of lesion generalisation: 51.8% (14/27) in 2 organs and 100% (4/4) of the cases in 3 organs, respectively, whereas e. coli was more associated with localised infections in 42.8% of the cases (3/7). association of pathogens was observed to higher dissemination of the lesions. lastly, cpv, ccov, and cav variants were investigated. in 16 dogs (11.6%), a generalised cpv-vaccine strain was detected. in 15 of them, there was the copresence of other agents, and in 14 of them, there was also the cpv field strain. within the 38.4% of positive samples (53/107), cpv-2a was found to be the prevalent antigenic variant, followed by cpv-2b, which was found in 28.2% of the cases (39/107). in contrast, no cpv-2c alone was detected. an association of 2 variants was observed in 15/107 cases (10.8%). more specifically, we found 8 cases of cpv-2b and 2c, 5 cases of cpv-2a and 2b, and 2 cases of cpv-2a and 2c. for canine coronaviruses, type ii was the most prevalent, with 72.1% cases (31/43); of these, the ccov-iia variant was most common (28/31; 90.3%). finally, cav-2 had a higher prevalence than cav-1, with 87.8% (29/33) and 12.1% veterinary medicine international (4/33) of the cav infections, respectively; 1 case (3%) showed copresence of both strains. furthermore, in 16 cdv-positive samples, genetic characterisation was conducted on the hemagglutinin (h) glycoprotein, which showed a close match to arctic-like lineages, both in owned and ownerless dogs. breed, age, habitat, and stress are some of the risk factors that are known to predispose dogs to infections [3, 5, 8] . a compromised immune system, due to stress from overpopulation, long travels, or high levels of environmental contamination, can create conditions for viral infections and bacterial superinfections [9, 10] . defining the causative agent of death on the basis of clinical and/or necropsy data without ancillary tests is quite difficult because of the similarity of symptoms and lesions between pathogens [43] , and the presence of superinfectants can modify the original pathological findings of the diseases [44] . to assess infectious causes of death in dogs aged under 1 year of age, the circulation of agents in southern italy and whether age, lifestyle, gender, or breed can influence infections and relative lesion generalisation, a survey was conducted on 138 deceased pups. a strong association between generalisation of lesions and microbiological infections was observed in this study (p < 0.0001). necropsy examination showed that multipleveterinary medicine international organ lesions were more frequently detected (84%) than single-organ lesions (11.6%), mostly due to viral-bacterial coinfections. our results showed that multiple agents, with 2 or more pathogens detected, represented 62.31% of the cases. e presence of multiple agents creates conditions suitable for generalisation, because polymicrobism is reported to enhance other agent symptoms [18, 46] and diffusion of localised pathogens, as for coliform septicemia during parvoviral infection in puppies [17] . enteritis was the most prevalent lesion (83.3%) due to the high detection of enteric agents. in 6 necropsies (4.3%), no macroscopic sign was observed. it is likely that sudden death occurred (unpublished data), and histologic alterations could have been present, given that in 5 of these 6 cases (83.3%) one or more pathogens were detected, but this aspect was not investigated in our study. our study highlights the importance of lifestyle [1] ; indeed, in dogs of 0-1 year of age, a higher rate of coinfection is observed than in any other age group [9] . ownerless dogs showed a higher trend in lesion diffusion than dogs with owners (p � 0.013). environmental microbial contamination, lack of vaccination, overpopulation, and habitat are some of the risk factors reported to cause stress and predisposition to infections and coinfections [7, 8] . ese conditions are frequently identified in shelters. inappropriately constructed areas, dietary changes, and transport [47, 48] , together with continuous introduction of new animals, make kennels a place where exposure, transmission, and susceptibility to infections are more evident [8, 49] . e same stressful conditions are identified in other contexts too, such as with the illegal trading of puppies or in stray dogs. ownerless pups, in fact, showed a 3-fold higher risk for mixed viral infections (p � 0.0117), but they had lower susceptibility to bacterial infections (or � 0.2; p � 0.0120). e breed was also identified as a significant risk factor for the generalisation of the lesions (p � 0.0026). indeed, purebreds showed a higher prevalence for multiple-organ lesions than crossbreeds, in which lesions were found with 56.7% of double-organ afflictions but a lower rate of pure viral infections (or � 0.22; p � 0.0012). our survey showed high prevalence of cpv (77.5%) and canine coronaviruses (31.1%) that are described to be the main causative agent of enteritis worldwide [50] . eleni and colleagues in central-south italy demonstrated that cpv was the main causative agent of death in dogs under 1 year of age [27] . other studies in diarrheic dogs showed lower prevalence of this virus, with a range from the 16% up to 54.3% [10, 51, 52] . cpv is characterised by high morbidity (100%) and mortality (up to 91%) [53] ; thus, it is likely that the inclusion criteria of living dogs could be the main cause of difference with the other studies [10] , making it challenging to compare the results. it was also observed that all the three parvoviral antigenic variants are circulating in southern italy; cpv-2a was the most prominent genotype, followed by cpv-2b and 2c [54] . cpv was also the most common virus involved in coinfections and detected in 100% of mixed viral infections and in 94.8% of viral-bacterial ones. e second most prevalent agent was canine coronavirus (31.15%), which is consistent with earlier studies [10, 50] . as described earlier, a strong association of ccovs and cpv was observed [10] in 79% of the cases of coronavirus infection. when associated with other agents, coronavirus can cause either diarrhea by itself or exacerbate the symptoms of other viruses [18, 19, 55] . cav was found in 23.9% of cases, and the cav-2 strain was involved in 87.8% of the positive samples. lower prevalence of cdv (13.7%), chv-1 (3.6%), and rotavirus (1.4%) was observed. molecular typing of cdvs was conducted on the h glycoprotein, which is considered the best target for the determination of the lineages due to its high variability [56] . in italy, the europe-1/south africa-1 lineage is historically connected to cdv outbreaks; however, in the last 2 decades, the arctic-like lineage is spreading throughout the country among both domestic and wild animals [57] [58] [59] [60] . our study corroborates these data, since the arctic-like lineage was detected in cdv samples in both owned and ownerless dogs. is spread is speculated to be caused by dogs trading from east europe, where the lineage was first detected [57, 59] . lifestyle has been found to be an important risk factor for viral infections. ownerless dogs showed a higher predisposition to cpv than dogs with owners (or: 2.68; p � 0.0331), cav (or: 4.16; p � 0.0019), and ccov, which represented the highest hazard for this variable (or: 14.96; p < 0.0001), as assessed in other studies [8, 61, 62] . for the age variable, we did not observe the typical age trend of cpv infection, because these viruses are described to have the highest range of susceptibility from 3 to 6 months of age and are related to the decline of mda [56, 63] . in our study, younger dogs were less involved in parvoviral infections (p � 0.0444), as for cdv (p � 0.0007) [4] . another difference was observed for the breed and gender variable, which were, in general, associated with a higher risk for cpv and ccov infections [4, 10] . we thus speculated about the role of lifestyle, which could be a more important risk factor than other factors, and the low immune system defense caused by [63] [64] [65] . bacteria, mainly represented by enteropathogens, were rarely detected in pure infections (10.8%), and they were mostly associated with viruses in mixed infections (42%). above all, e. coli (23.9%), cpa (18.1%), and enterococcus spp. (8.7%) were the most common bacteria, followed by low percentages of other opportunistic bacteria. since many of them belong to normal enteric microflora, they were considered only when toxigenic or in cases of localisation other than the gut. dismicrobism, immunosuppression, and drug resistance are the predisposing causes for the passage of the bacteria from the gut to bile ducts and via the lymph-hematic route [14] [15] [16] that can lead to bacteremia [66] , as for overpopulation, high environmental contamination, and viral copresence. is causes young dogs to have more exposure to environmental bacteria; moreover, especially when vaccination is not provided, they are predisposed to infection and superinfection, given a lack of a completely competent immune system [9] . to the authors' knowledge, this is the first comprehensive survey on the circulation of several infectious agents in southern italy related to necropsy examination. our results indicate that lifestyle is an important risk factor for several viral pathogens and their generalisation, mostly identified in ownerless dogs that live in overcrowded habitat or are in contact with a stressful environment and intense conditions due to long travels. high circulation of enteric pathogens has been detected, with relative enteritis lesions in necropsy examination, mostly identified in cpv and ccov and frequently involved in coinfections. cpv has been found as the most common pathogen involved in viral and viralbacterial coinfections, highlighting its role in the suppression of the immune system. in conclusion, the application of a broad microbiologic diagnostic panels for the identification of infectious agents that are responsible for the death of young dogs is an important tool for understanding the presence of infectious agents in a determined area and to clarify their epidemiological role in the genesis of diseases that lead to death. in addition, it can also be used as support for intra vitam diagnosis and in the therapeutic approach of veterinarian practitioners. e data used to support the findings of this study are included within the supplementary information file. e authors declare that there are no conflicts of interest regarding the publication of this paper. immunodeficiencies caused by infectious diseases e pathological newborn in small animals: the neonate is not a small adult a comparative study on canine parvovirus infection of dog in bangladesh and india risk factors associated with parvovirus enteritis in dogs: 283 cases (1982-1991 canine parvovirus 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prior habituation enteropathogens identified in dogs entering a florida animal shelter with veterinary medicine international 9 normal feces or diarrhea western european epidemiological survey for parvovirus and coronavirus infections in dogs comparison of the prevalence of enteric viruses in healthy dogs and those with acute hemorrhagic diarrhea by electron microscopy identification of enteric viruses circulating in a dog population with low vaccine coverage status-report-canine viral-enteritis molecular epidemiology of canine parvovirus type 2 in italy from 1994 to 2017: recurrence of the cpv-2b variant infectious canine hepatitis: an "old" disease remerging in italy canine distemper virus heterogeneity within the hemagglutinin genes of canine distemper virus (cdv) strains detected in italy a distinct cdv genotype causing a major endemic in alpine wildlife update on canine distemper virus (cdv) strains of arctic-like lineage detected in dogs in italy occurrence of different canine distemper virus lineages in italian dogs canine parvovirus: current perspective risk factors for delays between intake and veterinary approval for adoption on medical grounds in shelter puppies and kittens canine parvoviral enteritis; an update on clinical diagnosis, treatment and prevention effect of canine parvovirus strain variations on diagnostic test results and clinical management of enteritis in dogs factors affecting the occurrence, duration of hospitalization and final outcome in canine parvovirus infection role of the gut in multiple organ failure acknowledgments e authors would like to thank mr. domenico giudice for informatics support. e supplementary material contains raw data on age, breed, gender, and lifestyle of the dogs and the results of microbiological and necropsy examination. (supplementary materials) key: cord-333950-e0hd3iuu authors: maillard, jean-yves; bloomfield, sally f.; courvalin, patrice; essack, sabiha y.; gandra, sumanth; gerba, charles p.; rubino, joseph r.; scott, elizabeth a. title: reducing antibiotic prescribing and addressing the global problem of antibiotic resistance by targeted hygiene in the home and everyday life settings: a position paper date: 2020-04-18 journal: am j infect control doi: 10.1016/j.ajic.2020.04.011 sha: doc_id: 333950 cord_uid: e0hd3iuu antimicrobial resistance (amr) continues to threaten global health. although global and national amr action plans are in place, infection prevention and control is primarily discussed in the context of healthcare facilities with home and everyday life settings barely addressed. as seen with the recent global sars-cov-2 pandemic, everyday hygiene measures can play an important role in containing the threat from infectious microorganisms. this position paper has been developed following a meeting of global experts in london, 2019. it presents evidence that home and community settings are important for infection transmission and also the acquisition and spread of amr. it also demonstrates that the targeted hygiene approach offers a framework for maximizing protection against colonization and infections, thereby reducing antibiotic prescribing and minimizing selection pressure for the development of antibiotic resistance. if combined with the provision of clean water and sanitation, targeted hygiene can reduce the circulation of resistant bacteria in homes and communities, regardless of a country's human development index (overall social and economic development). achieving a reduction of amr strains in healthcare settings requires a mirrored reduction in the community. the authors call upon national and international policy makers, health agencies and healthcare professionals to further recognize the importance of targeted hygiene in the home and everyday life settings for preventing and controlling infection, in a unified quest to tackle amr. the global impact is already profound and expected to intensify, particularly among the poorest nations. 3, 4 the main driver is overuse and misuse of antibiotics in medicine and agriculture including unregulated over-the-counter sales, while global spread of resistant bacteria or resistance genes is attributed to poor infection prevention and control in healthcare facilities, and sub-optimal hygiene and sanitation in communities, confounded by poor infrastructure and weak governance. 5 in the us, between 80-90% of the volume of human antibiotic use occurs in the outpatient setting, with nearly 50% considered to be inappropriate or unnecessary. 6 without prompt action, it is estimated that rates of amr to commonly-used antibiotics could exceed 40-60% in some countries by 2030, 7 and by 2050, around 10 million people could die each year as a result of resistance to antibiotics and other antimicrobial agents. 8 almost 9 million of these will be in africa and asia. 8 in 2015, an alliance of the who, the food and agriculture organization of the united nations interventions." 9 the gap emphasizes the need for society-wide engagement, with a clear focus on "prevention first." 9 one of the five strategic objectives is a reduction in the incidence of infection through improved sanitation, hygiene, and infection prevention. 9 at least 120 countries have finalized national action plans, with the plans of more than 60 other countries under development. 10 what is striking is that the gap and national plans discuss infection prevention and control primarily in the context of healthcare facilities. (see https://www.who.int/antimicrobialresistance/national-action-plans/library/en/). by contrast, the latest 2019 uk national action plan, which sets out a 20-year vision 11 and a 5-year plan 12 for how the uk will contribute to controlling amr by 2040, offers guidelines on infection prevention in healthcare settings, but also highlights the role of the community, noting that, when it comes to infections in the community, the public have a huge part to play. 12 in recent years, demographic changes and changes in health service structure mean that the number of people living in the community needing special care, because they are at greater risk of infection, has significantly increased. the largest proportion of these are the elderly, who generally have reduced immunity to infection which is often exacerbated by other illnesses like diabetes and malignant illnesses. a decrease in immunity usually starts from 50 years old. other infection-susceptible groups include the very young, patients recently discharged from hospital, and family members with invasive devices such as catheters, as well as those whose immune competence is impaired as a result of chronic and degenerative illnesses (including hiv/aids) or because they are receiving immunosuppressant drugs or other therapies. immunosuppressed individuals are often also on other medications such as antibiotics, to help protect them from infection but can further increase susceptibility to infections such as clostridium difficile. home and everyday life settings provide multiple opportunities for spread of infection. everyday life settings include locations where normally there is no mandated hygiene policy as is typically found in clinical and educational settings; for example: work places, public transport, gyms, child day-care facilities, and shopping centers. poor hygiene is considered a major factor in the transmission of community-based infections, including gastrointestinal (gi) and respiratory tract (rt) infections such as colds and influenza, and skin infections caused by s. aureus. 14 for the elderly, communal living environments, combined with problems of fecal incontinence, create an environment in which enteric and foodborne pathogens are easily spread. as a result, the incidence of salmonellosis and campylobacter diarrhea appears to be higher among the elderly in these situations. more vulnerable 'at risk' members of society are now being looked after outside hospital settings. for example, in germany, it is estimated that approximately three quarters of all people in need of care are currently being cared for at home. 15 in the community the immunocompromised are also at risk from opportunistic pathogens such as e. coli, klebsiella spp., and pseudomonas aeruginosa, which are considered as hospital related. 15 the key steps in preventing the spread of infection, known as breaking the chain of infection, are the same regardless of setting. in the home, pathogens may have been brought home from hospital settings or enter the home via colonized or infected people, pets/domestic animals, or through contaminated food and water. 15, 16 pathogens and other microbes are shed constantly from these sources, with rapid transmission around the home mainly via hands, hand and food contact surfaces, cleaning utensils and in the air ( figure 1 ). 15 respectively. for campylobacter, counts of >100 and >1000 were isolated from 5 and 1.7% respectively. this is a concern, since it is estimated that 80% of salmonella infections originate in the home, 32 and a uk study detected campylobacter spp. in 56% of chilled retail chickens, with 7% of samples containing >1000 colony forming units (cfu)/g of skin. 33 the infectious dose of campylobacter is estimated at <500 cfu. 34 chaidez et al. 35 demonstrated that the risk of salmonella transmission from cleaning cloths via hands to mouth was far higher than the guideline levels for acceptable risk. since most pathogenic organisms die relatively rapidly, particularly on dry surfaces, the greatest risk of human exposure presents immediately after shedding from an infected or contaminated source. however some species, including s. aureus, e. coli, and other organisms such as fungal species, rhinovirus, and norovirus can survive for long periods even on dry surfaces. 36 audit studies suggest that some gram-negative organisms can form permanent reservoirs or secondary sources of contamination, particularly where moisture is present such as in sinks and drains, kitchen cleaning cloths and sponges. 37 44 the dose also depends on host susceptibility and mode of entry, and may be lower for at-risk groups in the community such as children, the elderly, and people with compromised immunity. 44 although care of increasing numbers of patients in the community, including at home can help alleviate over-burdened health systems, it can be undermined by inadequate infection control in the home and urgent focus is now needed on infection transmission in homes and community settings in addition to healthcare settings. although multidrug-resistant (mdr) bacteria (i.e. bacteria that have acquired resistance to at least one agent in three or more antimicrobial classes) are typically hospital-acquired, 50 since 2000, we have seen the emergence of new "community acquired" strains of mrsa (ca-mrsa). while healthcare-associated strains are mainly a risk to vulnerable people, for ca-mrsa, any family member is at risk and it is more prevalent among children and young adults where they cause infections of cuts, wounds and abrasions. us experience suggests the risk is greatest among those engaging skin-to-skin contact activities and contact with contaminated objects such as towels, sheets and sports equipment. transmission is common in settings such as prisons, schools and sports teams. 42 a study assessing the transmission of ca-mrsa in a university in the us, found multidrug resistant usa300 responsible for diseases including necrotizing pneumonia, severe sepsis and necrotizing fasciitis, on common touch surfaces at the university, student homes and local community settings. this suggests transfer between different locations within the community. 61 enterobacterales are a common cause of community-associated infections, including urinary tract infections and bacteremia as well as gastrointestinal infections. 51 67 kitchen sponges not only act as reservoirs of microorganisms, but also as disseminators over domestic surfaces, which can lead to cross-contamination of hands and food, which is considered a main cause of foodborne disease outbreaks. carbapenem-resistant enterobacteriaceae (cre) are also on the rise globally, but, to date, most cre infections in the us and europe have been healthcare-associated. 68, 70 although data from asia is sparse, carbapenemases have been found in bacteria recovered from drinking water in india and in food-producing animals in china. 69, 71, 72 in european studies during the 1990s, vancomycin-resistant enterococci (vre) were detected in the stools of healthy volunteers. [73] [74] [75] [76] [77] however, rates of vre, carbapenem resistance in acinetobacter infections, and mdr p. aeruginosa are thought to be low in individuals living in the community. 51 overall, the evidence suggests that mdr strains of bacteria, like any other strains of bacteria, can enter the home or other settings via people who are infected or colonized or via contaminated food and can be spread to other members of the family via hands and contaminated surfaces. if implemented effectively, home and everyday life hygiene has the potential to reduce rates of infection and the need for antibiotic prescriptions, thereby reducing the selective pressure for the development and subsequent dissemination of resistance. 15 microbiological data 14, 15 suggest that the surfaces that are most often responsible for spread of harmful microbes, at key moments include the hands themselves, hand contact surfaces, food contact surfaces, and cleaning cloths and other cleaning items ( figure 2 ). these surfaces are referred to as critical surfaces or critical control points. clothing, household linen, toilets, sinks and bath surfaces may also contribute to establishing a chain of infection, however, the risks associated with these surfaces are typically lower as they rely on the hands and other "chain links" to disseminate infectious microbes to cause human exposure. an important aspect of targeted hygiene is hygienic cleaning -as opposed to visible cleaningto break the chain of infection. this is achieved using hygiene procedures (products plus process) to reduce pathogenic microorganisms on critical surfaces to a level where they are no longer harmful to health -thereby preventing ongoing spread. 15, 84 several methods exist to achieve such reduction in potential pathogens: mechanical/physical removal using dry wiping, soap or detergent-based cleaning together with adequate rinsing, inactivation or eradication using a disinfectant on hard surfaces or an alcohol-based sanitizer on the hands, or a physical process such as heating (to ≥60°c/140°f) or ultraviolet treatment. most frequently, a combination of these approaches is likely to be used. 15, 84 when developing hygiene procedures aimed at breaking the chain of infection, the goal should be to ensure that each procedure is appropriate to its intended use. in recent years, risk modelling has been developed in order to achieve this. 80 quantitative microbial risk assessment (qmra) was originally developed for ensuring water quality and is increasingly being used to develop infection prevention control strategies in other settings, including healthcare. 85, 86 qmra is a scientifically-validated approach that uses published data to model the chain of infection and estimate safe residual level of contamination at critical points in the chain. 84 , 87 this information is then used to estimate the log reduction required to reduce contamination to a safe level. based on these estimates, tests modelling use conditions can be used to develop effective hygiene procedures to achieve the required reduction. the approach is set out in more detail by bloomfield et al. 84 in the past, recommendations on selection of hygiene procedures for home and everyday life were based on the health status of family members, and it is still argued by some that disinfectants should only be used in situations where people are infected or at increased risk of infection. 87 although there is data to show that hygiene is important in preventing transmission of mrsa colonization and infection in the domestic environment, further investigation is required to demonstrate the full extent to which poor home hygiene may contribute to the burden of foodborne infection associated with antibiotic resistant strains. quantifying the impact of hygiene on the burden of infection in home and everyday life is challenging because of the large population sizes required to generate significant results, and difficulties in conducting studies involving multiple interventions. most data have been generated from single intervention studies -primarily hand hygiene -where meta-analyses show a positive impact on gi and rt infections. [91] [92] [93] children who attend day-care centers have significantly more infections than those who do not. the most common are rt and gi infections, and the risk of otitis media is almost twice that of children remaining at home. 94 studies in day-care centers and schools in which hand hygiene was combined with cleaning and/or disinfection of environmental surfaces indicate a positive impact on illness rates and reduction in the use of antibiotics. [94] [95] [96] [97] in an intervention study 96 reduction of antibiotic prescriptions for rt infections in a group who used hand sanitizers compared with a control group. 98 another 2018 intervention study 99 found that children were prescribed antibiotics for significantly fewer weeks in day care centers using specific disinfecting products and cleaning protocols than centers that continued to use their standard procedures and products (rr=0.68 [95% ci 0.54, 0.86]; p=0.001) -a relative risk reduction of almost onethird. to the best of our knowledge, only one study on the impact of targeted hygiene in the home has been conducted. 100 this study, conducted among low-income communities in cape town, south africa, evaluated the impact of hygiene education alone and education in combination with hand washing with soap at critical times, bathing at least three times a week, cleaning/disinfecting household surfaces at critical times, and proper waste disposal. 100 qmra is also now being used to estimate the impact of hygiene interventions on infection in community settings. 102 haas et al. 103 concern has been expressed as to whether expanding use of microbicidal products, in the home and everyday life may contribute to the rise in amr. 105 sub-lethal levels of microbicides can induce stress on bacterial cells, causing expression of mechanisms that reduce the biocide concentration at the bacterial target site further and allow the bacterial cell to repair. 106, 107 these include overexpression of an efflux system, membrane regulatory changes, and changes in membrane permeability and composition. 107 these same mechanisms can produce changes in the susceptibility profile to unrelated antimicrobials. 107 in other words, the use of microbicides may cross-select for antibiotic resistance and be associated with reduced antibiotic susceptibility to clinically significant levels (recently reviewed by maillard 107 ). factors inherent to the microbicide (i.e. concentration, formulation, mechanism of action), the microorganisms (i.e. type/strain, metabolism, resistance mechanisms), and product usage (e.g. concentration, exposure time), all impact on product efficacy. 107 decreases in efficacy, for example, following shorter contact time or product dilution, will lead to bacterial survivalantimicrobial damage caused by a sub-lethal concentration of a microbicide is likely to be repairable. 107 a number of expert reports commissioned in the last 10 years have highlighted laboratory studies linking microbicide use with reduced antibiotic susceptibility. however, these reports conclude that there is little evidence for this effect occurring in real-life clinical practice, and have called for further research into whether microbicide use influences antibiotic resistance in the community. 108-111 rutala et al. (2000) found that the frequency of occurrence of antibiotic resistance in environmental isolates from homes was much lower than for clinical isolates from a hospital intensive care unit and an outpatient setting where there was routine extensive use of antibiotics. 112 two studies were carried out to investigate whether antibiotic resistant strains were more likely to be found in homes where antibacterial products were used, compared with homes where they were not. 113, 114 samples were collected from houses in the usa and uk of 30 users and nonusers of antibacterials. susceptibility tests against antibiotics and antibacterial agents (triclosan, pine oil, bac and para-chloro-meta-xylenol) were carried out on the bacteria isolated. the authors concluded that there was no evidence that antibiotic resistant strains occurred more frequently in user homes compared with non-user homes. a 1-year study by aiello et al (2005) also showed that household use of antibacterial cleaning products was not a significant risk factor for occurrence of antibiotic resistant isolates from hands. 115 despite more than 20 years of research, there is still no conclusive resolution to the question of whether and to what extent; microbicides might contribute to amr in clinical practice. in light of laboratory data, which indicates that microbicide-induced amr is biologically plausible for some types of microbicides, it is concluded that use of microbicides needs to be prudent and appropriate and that the products containing them must be used at recommended concentrations and with the appropriate contact time. targeted hygiene works to ensure that use of disinfectants and hand sanitizers (i.e. microbicides used at the correct concentration and contact time) are confined to situations where there is identifiable risk of spread of harmful microorganisms, ensuring that they play an essential role in tackling amr. the need for antibiotic prescribing may in fact increase if disinfectants and hand sanitizers are not used as indicated, due to the increased risk of infection and survival of bacteria bearing amr determinants. these could potentially spread to other areas in the home and on into the community. it is important to note also that preventing viral infections as well as bacterial infections, such as those that cause respiratory and gi infections, can also have a role in reducing amr as this will eliminate the potential for mis-prescribing or misuse of antibiotics. in in 2015, an estimated 663 million people around the world were drinking from unimproved water sources, and 2.4 billion had no access to improved sanitation -the vast majority of these were in sub-saharan africa and south asia. 119 it is estimated that 2.3 billion people lack the use of sanitation facilities which are not shared with other households, 120 42 as with studies conducted in hics, the highest levels of contamination in lmics are typically found in moist locations such as kitchen sponges and dishcloths. [122] [123] [124] the key question, however, is whether, and to what extent, the incidence and levels of potentially harmful pathogens (and thus infection risks) are higher in homes without access to adequate water and sanitation. sinclair and gerba 124 monitored fecal coliforms, total coliforms, e. coli and heterotrophic plate count bacteria on household surfaces in 8 homes that had improved latrines (i.e. a pour-flush latrine) in a rural village of cambodia, and compared the results with similar data from homes in the us 38 and japan. 39 fecal coliform levels in cambodia were found to be highest in moist locations such as the plastic ladle used for sink water, the toilet seat surface, and the cutting board surface. 124 for e. coli, the mean log cfu per 4 cm 2 ranged from 0.5 to 4.0, with highest counts found on the top of the squat toilet, the wash basin, and the floor around the toilet. fecal coliform levels were 100-fold higher on these surfaces in cambodia than on equivalent surfaces in the us and japanese studies. in lmics, due to a lack of basic sanitation, good hand hygiene is of vital importance. 116 globally, it has been estimated that only 19% of the population washes its hands with soap after contact with excreta. 93 observations show that hand washing with soap is undertaken in an ad hoc manner, 116 with many households having no access to handwashing facilities. 125 unsurprisingly, studies in lmics have reported high levels of fecal indicator bacteria on the hands of household members, [126] [127] [128] with one study correlating presence of fecal contamination on the hands with the prevalence of gastrointestinal and respiratory symptoms within the household. 126 a cochrane review showed that improving hand washing practices probably reduces diarrhea episodes in child day-care centers in both high income countries and among communities living in low to middle income countries by as much as 30%. 92 the evidence set out in this paper suggests that, if combined with measures ensuring clean water and adequate sanitation, targeted hygiene practices in home and everyday life settings could make a significant contribution to tackling amr through infection prevention and a consequential reduction in antibiotic prescribing. this is true in all areas of the world including low-income countries. additionally, the evidence suggests that hygiene promotion would contribute to preventing the transmission of resistant bacteria from the home and everyday life settings, into healthcare settings, and back into the community. further research is still needed to evaluate the extent to which this might occur, especially in communities in low income countries. to be effective, hygiene interventions need to consider all aspects that are likely to affect the outcome. this includes a reduction of antibiotics from the food chain and the environment, improved hygiene education and availability of appropriate products as well as the provision of clean water and improved sanitation. based on these findings, the authors of this paper issue a call to action to national and international health policy makers, health agencies, and healthcare professionals to give greater recognition to the importance of hygiene in the home and everyday life and development and promotion or more effective codes of practice for hygiene in the home and everyday life as part of national action plans to tackle amr. although the precise impact of hygiene on transmission of infection between community and healthcare settings needs further investigation, it is important to recognise, that reducing the need for antibiotic prescribing and the circulation of amr strains in healthcare settings cannot be achieved without also reducing circulation of infections and amr strains in the community. we cannot allow hygiene in home and everyday life settings to become the weak link in the chain. the development of this position paper was supported by an educational grant 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cord-333024-1yrmun3z authors: von lilienfeld-toal, marie; berger, annemarie; christopeit, maximilian; hentrich, marcus; heussel, claus peter; kalkreuth, jana; klein, michael; kochanek, matthias; penack, olaf; hauf, elke; rieger, christina; silling, gerda; vehreschild, maria; weber, thomas; wolf, hans-heinrich; lehners, nicola; schalk, enrico; mayer, karin title: community acquired respiratory virus infections in cancer patients—guideline on diagnosis and management by the infectious diseases working party of the german society for haematology and medical oncology date: 2016-09-25 journal: eur j cancer doi: 10.1016/j.ejca.2016.08.015 sha: doc_id: 333024 cord_uid: 1yrmun3z background: community acquired viruses (crvs) may cause severe disease in cancer patients. thus, efforts should be made to diagnose crv rapidly and manage crv infections accordingly. methods: a panel of 18 clinicians from the infectious diseases working party of the german society for haematology and medical oncology have convened to assess the available literature and provide recommendations on the management of crv infections including influenza, respiratory syncytial virus, parainfluenza virus, human metapneumovirus and adenovirus. results: crv infections in cancer patients may lead to pneumonia in approximately 30% of the cases, with an associated mortality of around 25%. for diagnosis of a crv infection, combined nasal/throat swabs or washes/aspirates give the best results and nucleic acid amplification based-techniques (nat) should be used to detect the pathogen. hand hygiene, contact isolation and face masks have been shown to be of benefit as general infection management. causal treatment can be given for influenza, using a neuraminidase inhibitor, and respiratory syncytial virus, using ribavirin in addition to intravenous immunoglobulins. ribavirin has also been used to treat parainfluenza virus and human metapneumovirus, but data are inconclusive in this setting. cidofovir is used to treat adenovirus pneumonitis. conclusions: crv infections may pose a vital threat to patients with underlying malignancy. this guideline provides information on diagnosis and treatment to improve the outcome. the importance of community acquired respiratory virus (crv) infections is increasingly recognised. crv are responsible for respiratory infections, which usually present as a common cold in the immunocompetent individual but may be life-threatening in the immunocompromised host. usually, orthomyxoviridae (influenza a, b and c), paramyxoviridae (including parainfluenza 1e4 [piv], respiratory syncytial virus a and b [rsv] , and human metapneumovirus [hmpv]), coronaviridae, picornaviridae (including >100 different serotypes of rhinovirus and enterovirus), adenoviridae, polyomavirus type 1 and bocavirus are regarded as potential causes of crv infection. this guideline is intended to give haematologists and oncologists a broad overview with regard to clinical relevance and diagnosis of crv infection and management of cancer patients affected by crv. detailed information on respective viruses including emerging resistance is not the scope of this guideline. most data on this topic originate from patients following allogeneic stem cell transplantation (allo-sct), and we know little about crv infections in cancer patients outside the setting of allo-sct. however, in recent years increasing evidence has been gathered about other cancer patients, revealing clinical relevance of crv infections in non-transplant patients. therefore, this guideline discusses crv infections in all cancer patients with ongoing relevant immunosuppression. it is left to the treating physician to assess the degree and relevance of immunosuppression in the individual patient. this guideline has been developed by a panel from the infectious diseases working party (agiho) of the german society of haematology and medical oncology including 17 experts certified in internal medicine, haematology/oncology, infectious diseases, microbiology/virology or radiology and one medical student. first, predefined topics were delivered by the designated coordinator (mvlt) to all participants of the panel to form subgroups. data were extracted and tabulated after a systematic literature search by subgroup members and revised in several steps by the members of the panel on the basis of an email-based discussion process and a face-to-face meeting. finally, preliminary recommendations of the panel were discussed, revised and approved by the agiho assembly. in may 2014, the first literature search was performed for crv and immunosuppression using the terms '-virus' and 'immunocompromised' (for example: 'adenovirus immunocompromised'). this search was performed for adenovirus, bocavirus, coronavirus, enterovirus, hmpv, influenza, piv, parechovirus, rsv and rhinovirus. the references were then screened by the subgroup members and relevant articles retrieved as full papers. wherever applicable, additional papers were identified in the reference lists and treated as described. crvs cause respiratory tract infections, which can be divided into upper respiratory tract infection (urti), influenza-like illness (ili) and lower respiratory tract infection/pneumonia (lrti). commonly, urti can be assumed, if a patient has a new onset of symptoms including at least one of cough, coryza, sore throat or shortness of breath which is deemed to be due to an infection by the treating physician. lrti requires clinical or radiological evidence of pneumonia [2] . ili is diagnosed in patients with a sudden onset of new symptoms including at least one of fever or feverishness, malaise, headache or myalgia and at least one of the respiratory symptoms cough, sore throat or shortness of breath [3] . to be certain of the viral origin, the detection of the virus from respiratory samples like swabs, nasopharyngeal aspirates or bronchoalveolar lavage fluid is required. of note, surveillance studies showed some patients to be asymptomatic but still shedding the virus [2,4e7] . for that reason, some authors distinguish between respiratory infection (detection of virus independent of symptoms) and respiratory infection disease (detection of virus and respective symptoms) [8] . however, for the purpose of this guideline, we omit this distinction and define urti, lrti and ili as described above. some crv like influenza or rsv have a seasonality with most infections occurring during the winter months [2, 9, 10] . others like rhinovirus or parainfluenza are independent of seasonality [11] . thus, an appropriate diagnostic work-up and clinical management is warranted in any patient presenting with typical symptoms regardless of the time of the year. as may be expected considering the nature of the disease, crv frequently cause outbreaks in health care settings [7,12e15] . importantly, outbreaks may also occur in outpatient settings [16] emphasising the need for awareness during all periods of cancer treatment. generally, viral urti in cancer patients has some impact on the clinical course because the patients are symptomatic to a degree that frequently requires postponement of chemotherapy [17] . however, critical table 1 grading of evidence as suggested by the escmid [1] . strongly support a recommendation for use b moderately support a recommendation for use c marginally support a recommendation for use d support a recommendation against use quality of evidence for interventionsdlevel i evidence from at least one properly designed randomized, controlled trial ii* evidence from at least one well-designed clinical trial, without randomization; from cohort-or case-control analytic studies (preferably from more than one centre); from multiple time series; or from dramatic results from uncontrolled experiments. (1) at least one well-designed prospective singlecentre cross-sectional or cohort study or (2) a properly designed retrospective multicentre crosssectional or cohort study or (3) from case-control studies iii evidence from opinion of respected authorities, based on clinical experience, descriptive case studies, or report of expert committees illness and mortality due to viral urti are rare. in contrast, most patients who died were suffering from lrti, which thus poses the biggest threat to cancer patients. rates of lrti and mortality differ amongst the respective crv [18] and exact estimation is hampered by the fact that fatal cases are probably overreported. we have tried to deduce reliable information on lrti and mortality from the literature for various crv: influenza appears to have a high rate of lrti with approximately 30% and an associated mortality rate of approximately 25% [19e22] . rsv appears to be at least as dangerous with a rate of lrti of approximately 33% and an associated mortality rate of 27% [16] . however, it has to be kept in mind, that most data regarding rsv originate from sct-recipients and very little is known regarding patients with other forms of malignancy. on the other hand, there are several reports of outbreaks in general haematology/oncology units, which showed a significant disease burden even in patients not undergoing stem cell transplantation [12] . with regard to hmpv and piv, exact information on the clinical relevance is even more difficult to obtain. however, although both viruses may cause asymptomatic infection [6, 23] , the available evidence suggests a similar overall rate of lrti and mortality [7, 11, 13 ,24e28] compared with influenza and rsv. in contrast, despite case reports of fatal outcomes of infections with rhinovirus and coronavirus, these viruses as well as bocavirus appear to be rarely the cause of lrti and dangerous only when patients are coinfected with other pathogens [2, 5, 29] . herpesviridae like herpes simplex virus, human herpes virus 6, cytomegalovirus, varicella zoster and epsteinebarr virus as well as polyomaviruses or parechoviruses usually do not cause crv infection. pneumonia due to reactivation of herpesviridae in severely immunocompromised patients is not an infection by crv and thus not covered by this guideline. in crv infection, coinfection with bacteria, fungi or even other viruses appears to occur in approximately 30% of the patients [10, 11, 28] . they play a vital role with regard to outcome of patients since patients with bacterial or fungal superinfection have a dramatically higher mortality rate than those with viral infection only [11, 28] . therefore, possible co-or superinfection should be considered when managing cancer patients with crv. in addition to lrti and bacterial or fungal superinfection, other risk factors for adverse outcome include haematological malignancy [22] , severe immunosuppression such as steroid use or graft versus host disease or cytopenias [30e32] or low immunoglobulins [12] . epidemiology of adenoviruses is somewhat different from the other crv: often, the source of infection is a childhood infection in children under 5 years [33] and reactivation as well as new infection have been described to be the cause of disease [34, 35] . other than crvs like rsv or influenza, adenoviruses can cause a variety of symptoms such as conjunctivitis, haemorrhagic cystitis, gastroenteritis, and urti in immunocompetent patients and hepatitis, colitis, nephritis, meningoencephalitis and lrti in the immunocompromised host. in adult allo-sct recipients, dnaemia occurs in up to 20% [36e38], but symptomatic disease by adenovirus is much less common with t-cell suppression being the predominant risk factor [36] . again, coinfection is an important risk factor for severe illness [39] . cancer patients presenting with symptoms consistent with crv infection should be diagnosed using material from the respiratory tract (table 2) . serology is not useful to detect ongoing crv infection and thus not recommended (d iii). regarding material used for microbiological diagnosis, a variety of approaches are used in various centres. as a general rule, a close collaboration with the local microbiology laboratory is highly recommended because this may determine which material should be used preferably since commercially available test kits are licensed for specific materials. for example testing for viral antigens usually requires more thorough sampling like combined nasal/throat swabs than testing for viral nucleic acids which can often be performed reliably on gargles alone. it is therefore essential for the clinician to be aware of the tests used in the respective laboratory. the overall evidence in the literature is best for combined nasal/throat swabs and nasopharyngeal aspirates (a iit, table 2 ). the best evidence for reliable detection of a crv present in respiratory samples exists for nucleic acid amplification based-techniques (nat) like pcr. therefore, the use of nat is highly recommended (a ii, table 2 ) and any methods involving the detection of antigen appear to be second best in immunosuppressed cancer patients (c ii [40e42]). also, culture methods are not commonly used anymore and cannot be recommended for general diagnosis, but they are essential in individual cases in which no known virus can be detected or results of pcr-analysis are inconclusive (a ii, [43] ). in the era of multiplex-test kits, it is difficult to make a definite recommendation with regard to which viruses should be looked for. in the absence of any reliable data regarding this question, the panel feels that it is wise to search for influenza, rsv, piv and viruses currently prevalent in the local environment in all immunosuppressed cancer patients presenting with symptoms. patients with more severe disease (for example pneumonia or critical illness) may have the panel broadened to include hmpv and adenovirus and even viruses that only rarely cause lrti like rhinovirus and coronavirus. however, evidence for this approach is low and it is strongly advisable to define local guidelines on this topic. it should be kept in mind, that herpesviridae are not the cause of crv infection. therefore, there is no rationale to look for cytomegalovirus, eps-teinebarr virus, herpes simplex virus or varicella zoster in patients with typical symptoms of crv infection only. in patients with symptoms of lrti, it is essential to determine the degree of pulmonary involvement. crv can affect the tracheobronchial system or the lung parenchyma [44] . generally, a chest x-ray has been proven to be unhelpful to diagnose pathologic changes in this setting because of lack of sensitivity. it is therefore not recommended (d ii, see table 2 ). in contrast, there is good evidence to recommend a ct scan of the chest to detect lrti in patients with crv infection (a ii, see table 2 ). bronchial wall thickening as well as interstitial infiltrates presenting as ground-glass opacities may be detected. these are defined as increased lung density, whereas underlying lung architecture is still detectable. ground-glass opacities may be patchy or diffuse [44e47] and can be well distinguished from consolidations, which show higher density obscuring e.g. the pulmonary vessels and which are typical for other differential diagnoses including bacterial infection. affection of the terminal bronchioles might lead to visibility of those usually invisible structures at ct as small centrilobular nodules or 'tree-in-bud' sign [45] or evidence of bronchiolitis causing air-trapping. to reliably detect airtrapping while inspiratory ct is normal, a ct scan in expiration is necessary [44] . in addition to good diagnostic accuracy with regard to the diagnosis of a viral lrti, the ct scan may also reveal evidence for an outbreak, since specific viruses tend to present with a typical pattern in the ct scan [45] . for an example see fig. 1 . in the light of the danger of outbreaks with fatal consequences, the most important measure in the management of cancer patients with crv infection is infection control ( table 3) . local authorities should give exact guidance on the necessary precautions in the respective institutions. the following statements intend to give a general overview. there is sufficient evidence to recommend stringent hand hygiene (a iit), the use of face masks (b iit) and contact isolation (a iii, table 3 ). importantly, shedding of crv in cancer patients often lasts 2 weeks or longer [5, 10, 48] . it is therefore wise to perform follow-up testing of respiratory material in index patients and stop contact isolation only when they became negative. of note, early implementation of infection control appears to be more effective than late implementation [49] which gives reason to recommend infection control as soon as symptoms appear and not only after evidence of crv. almost anybody who catches a cold applies some form of home remedies convinced that they ease the symptoms and positively influence the course of the disease. in contrast to this widespread use, there is very little evidence to recommend any such measures. in particular with regard to cancer patients, evidence is too poor to give a sound recommendation in favour of the use of vitamin c [50] , echinacea [51] , garlic [52] , zinc [53] , humidified hot air [54] or chinese herbal medicine [55] . surprisingly, even painkillers [56] and non-steroidal anti-inflammatory drugs [57] may ease the pain but have little influence on severity and duration of the crv infection. however, as there is some evidence towards a considerable placebo effect [58] , it can be argued that patients should be allowed to continue their home remedies provided there is no reason to assume harmfulness as may be the case for some chinese herbal medicines contaminated with heavy metals [59] or the possibility to contract invasive fungal infection from inhaling contaminated air. needless to say, there is little to be gained from treating a viral infection with antibiotics [60] , which is also true for cancer patients (d iir,t for treating viral infections with antibiotics). however, having in mind the high rate of superinfection, cancer patients with viral lrti and suspected or proven bacterial/fungal coinfection have to be treated accordingly (a iii). in cancer patients who present with symptoms consistent with crv infection prior to initiation of chemotherapy, delaying treatment should be considered. since a retrospective study with 2 groups showed a benefit, if treatment was delayed in patients undergoing allo-sct, we clearly recommend delaying conditioning in those patients who are scheduled for allo-sct and have evidence of crv infection (a ii, see table 3 ). the situation is less clear for patients with less aggressive treatment, since there have been reports of uneventful courses of even high-dose chemotherapy in patients with initial crv infection [61] . therefore, the recommendation to delay the chemotherapy if possible to be on the safe side is merely a weak one (table 3) . again, ground-glass opacities can be found but are of a more patchy character (fig. 1c) . they are often combined with centrilobular nodules (tree-in-bud, fig. 1d ). in some cases, only nodules with a ground-glass character are detected (fig. 1e) . rsv, respiratory syncytial virus. similarly, reducing immunosuppression in patients with lrti due to adenovirus-infection after allo-sct is well founded [36, 62] on retrospective cohort studies with 2 groups and is thus clearly recommended (a ii, table 3 ). data can be transferred to the situation of other lrti caused by crv, therefore, reduction of immunosuppression is also recommended in allo-sct recipients with lrti caused by other crv (a iit, table 3 ). in contrast, the situation is less clear in patients with urti and therefore only a very weak recommendation can be made (c iii, table 3 ). with regard to supportive application of systemic medication other than antivirals, no recommendation can be made for the use of steroids, since they show no effect and prolong viral shedding (d iii, table 3 ). in contrast, intravenous immunoglobulins (ivigs) are a therapeutic option in rsv infection (b iii) and may also be beneficial in influenza, piv and hmpv infection (c iii, table 3 ). because of lack of data, no recommendation can be made regarding the use of ivig in other crv infections. if causal treatment was deemed necessary, influenza a was traditionally treated with amantadine or rimantadine. nowadays, resistance rates are so high that neither can be recommended (d ii [63e66]). in contrast, despite ongoing discussion regarding the balance of efficacy and side effects [67, 68] the treatment of choice appears to be a neuraminidase inhibitor, be it oseltamivir, zanamivir or peramivir. they are recommended as prophylaxis as well as for treatment (for example http://www.rki.de/ de/content/infaz/i/influenza/ipv/ipv_node.html or [63, 69, 70] ). however, data regarding the efficacy of prophylactic use of neuraminidase inhibitors in cancer patients are very weak and almost exclusively in the setting of stem cell transplantation [71, 72] . therefore, the authors believe it is not justified to give any recommendation in favour of or against their use in cancer patients in general. thus, prevention of influenza by application of neuraminidase inhibitors remains one of the unresolved issues requiring further study. still, we have included information on dose and duration in table 5 . treatment of influenza is usually recommended in symptomatic patients at high risk preferably <48h after the onset of symptoms [63] . cancer patients might be regarded as high-risk patients per se, which is why neuraminidase inhibitors (usually oseltamivir) are often routinely given to patients with malignancies and influenza [69, 70, 73] , since mostly retrospective data show a benefit of (early) antiviral treatment with regard to development of lrti or further complications [10,20,74e76 ]. thus, we do recommend the use of oseltamivir or zanamivir (b ii, tables 4 and 5 ). there is evidence to recommend early initiation of treatment, but that does not necessarily mean later treatment is futile [77] and therefore many authors recommend treatment regardless of timepoint [70] . however, we do not think either dose (150e300 mg/d) nor duration (5e10 d or longer) are well defined from the available evidence and need determination by local specialists. the reasons usually given for higher doses and longer treatment duration in high-risk patients is the prolonged viral shedding observed in cancer patients and a thus deduced susceptibility to develop resistances if not treated effectively [69, 70] . peramivir has received fda approval during the 2009 pandemic and is recommended for patients with h1n1 infection unable to take oral medication [78] . it is not available in germany but included in this guideline for the sake of completeness ( table 5 ). the authors advise its use in severe cases, when oral intake or inhalation is not possible (ciii). another salvage option may be the combination of zanamivir or oseltamivir with ribavirin, since that has shown some efficacy in older studies [79] . rsv is usually treated with intravenous immunoglobulins (ivig, b iii, table 3 ) and ribavirin. in europe, the monoclonal antibody palivizumab is licensed for prevention of rsv in children only. in addition, the benefit over polyclonal ivig is not entirely conclusive [80] . for these reasons, we do not make a clear recommendation for or against its use in cancer patients, since we regard table 4 recommendations regarding causal treatment of influenza, rsv, parainfluenza and adenovirus. the question whether to use palivizumab instead of ivig as an unresolved question requiring further study. ribavirin is the agent of choice in the treatment of rsv infection. most available data concern allo-sct recipients [80] , but recent evidence also suggests a benefit in less severely immunosuppressed cancer patients [12, 81] . it appears to lower the progression rate to lrti [82] and is reported to have a positive influence on survival [12] . however, some authors report favourable outcome of rsv infections without any causal treatment [61, 83, 84] . traditionally, it is used as an aerosol (see table 5 ), but this application mode is cumbersome and may be associated with a teratogeneic effect [85] . also, patients may not be able to inhale for such a long time or they may react with bronchospasm. thus, oral application has been used increasingly with a similar efficacy [12, 30, 81, 86, 87] and even intravenous application is reported [87] . despite some reports with a good outcome without treatment, we believe the available evidence justifies a recommendation for the use of ribavirin in cancer patients with rsv infection (b ii, table 4 ). also, at least in high-risk patients the treatment should be given at the stage of urti, since this has shown a benefit (b ii [82] ). experience with antiviral therapy (generally ribavirin) in patients with parainfluenza infection is not very large and the efficacy is not entirely convincing [11, 24, 79, 86, 88, 89] . this may be partly because causal treatment is started too late in the course of the disease and partly because the cause of death often is a coinfection requiring antibiotic therapy [7, 28] . nonetheless, it may be reasonable to attempt therapy with ribavirin in patients with parainfluenza infection (c iii, table 4 ). causal therapy with cidofovir is justified in immunosuppressed cancer patients with lrti caused by adenovirus (b ii, tables 4 and 5). more experimental therapies, which are employed in the setting of allo-sct include donor-lymphocyte infusions [90] or adaptive transfer of specific t-cells [91] . however, to date evidence is too weak to justify a recommendation in favour of or against the use of these treatment modalities. causal therapy with ribavirin has been attempted in patients with infections caused by hmpv [92, 93] , albeit with unconvincing results. there is not enough evidence to make a definitive recommendation for or against the use of any specific antiviral drug or other causal treatment approaches like interferon for any of the crv other than the ones discussed above. early diagnosis and general infection prevention may improve the outcome of cancer patients with crv infections. despite some data regarding some viruses (influenza, rsv) and patient populations (hsct-recipients), there is still a lack of information on most crv and on other patient populations (for example those with solid tumours). also, almost no prospective randomised trials have been performed for the treatment of crv infections in cancer patients. thus, most recommendations have to be deduced from other populations and further study is urgently needed. none. travel expenses and costs for group meetings were reimbursed by the german society for haematology and medical oncology. mvlt has received honoraria and travel support from gilead, msd, pfizer, celgene and janssen cilag, has received travel support from astellas pharma and has received research support from msd. she is member of the advisory board to msd. mc has received research funding from deutsche forschungsgemeinschaft (dfg) and erich und gertrud roggenbuck stiftung, been a speaker for msd and basilea, has been a consultant for msd and basilea, received travel grants from celgene, takeda, gilead and msd and is a recipient of the msd stipend oncology 2013. mh served on advisory boards of gilead, roche pharma and takeda and served on the speakers' bureau for celgene, novartis, janssen and amgen. cph is a stock owner of stada and gsk and has received consultation fees and/or honoraria from schering-plough, pfizer, basilea, boehringer ingelheim, novartis, roche, astellas, gilead, msd, lilly, intermune, fresenius, olympus, gilead, astrazeneca, bracco, meda pharma, chiesi, siemens, covidien, pierre fabre, grifols and research funding from siemens, pfizer, mevis and boehringer ingelheim. mk has received honoraria and travel support and served on the speakers' bureau for gilead, msd, pfizer. op has received honoraria and travel support from astellas, gilead, jazz, msd, neovii biotech and pfizer. he has received research support from bio rad, gilead, jazz, neovii biotech, pierre fabre, sanofi and takeda. he is member of the advisory board to alexion, jazz, gilead and msd. mjgtv is a consultant to: berlin chemie, msd/merck and astellas pharma; has served at the speakers' bureau of: pfizer, merck/msd, gilead sciences, organobalance and astellas pharma; received research funding from: 3m, astellas pharma, davolterra and gilead sciences. gs has received grant/research support from: msd sharp & dohme gmbh, haar, germany, pfizer, berlin, germany, gilead sciences, martinsried, germany and astellas pharma gmbh, mü nchen, germany. she has served as a consultant to: msd sharp & dohme gmbh, haar, germany and basilea pharmaceutical internatio ltd; switzerland. all remaining authors have declared no conflicts of interest. escmid* guideline for the diagnosis and management of candida diseases 2012: developing european guidelines in clinical microbiology and infectious diseases epidemiology of viral respiratory tract infections in an outpatient haematology facility european centre for disease prevention and control. ecdc influenza case definitions detection, control, and management of a respiratory syncytial virus outbreak in a pediatric hematology-oncology department human rhinovirus and coronavirus detection among allogeneic hematopoietic stem cell transplantation recipients respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection epidemiology and clinical 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the infectious diseases working party of the european group for blood and marrow transplantation disseminated adenovirus infections after allogeneic hematopoietic stem cell transplantation: incidence, risk factors and outcome early diagnosis of adenovirus infection and treatment with cidofovir after bone marrow transplantation in children description of an adenovirus a31 outbreak in a paediatric haematology unit biopsy-proven adenoviral diarrhea responding to low-dose cidofovir the authors thank ramona kraft for technical assistance with the retrieval of full papers. key: cord-333355-ykmp4ven authors: kuchar, e.; miśkiewicz, k.; nitsch-osuch, aneta; szenborn, l. title: pathophysiology of clinical symptoms in acute viral respiratory tract infections date: 2015-03-19 journal: pulmonary infection doi: 10.1007/5584_2015_110 sha: doc_id: 333355 cord_uid: ykmp4ven in this article we discuss the pathophysiology of common symptoms of acute viral respiratory infections (e.g., sneezing, nasal discharge, sore throat, cough, muscle pains, malaise, and mood changes). since clinical symptoms are not sufficient to determine the etiology of viral respiratory tract infections, we believe that the host defense mechanisms are critical for the symptomatology. consequently, this review of literature is focused on the pathophysiology of respiratory symptoms regardless of their etiology. we assume that despite a high prevalence of symptoms of respiratory infection, their pathogenesis is not widely known. a better understanding of the symptoms’ pathogenesis could improve the quality of care for patients with respiratory tract infections. 1 introduction the literature reports that each year up to 25 million people in the us visit their doctor because of respiratory tract infections. the upper respiratory tract infections, better known as common colds, are the most common clinical presentation of said infections ). viral lower respiratory tract infections, bronchitis, bronchiolitis, and pneumonia; e.g., with respiratory syncytial virus (rsv) or influenza are generally more common in infants, young children, and in patients with chronic medical conditions, whereas older children and healthy adults usually suffer from milder upper respiratory tract infections (eccles 2005) . common cold was the third most common diagnosis made during ambulatory care visits by patients of all ages in the us (hsiao et al. 2010 ). it has been estimated that an average adult suffers from 2 to 4 colds per year and a schoolchild suffers from 6 to 10 colds per year (spector 1995; johnston and holgate 1996; winther et al. 1998; eccles 2005) . therefore, respiratory tract infections represent a significant clinical and therapeutic problem, and an economic burden (wat 2004) . upper respiratory tract infections are generally mild, selflimiting, and viral in their origin (johnston and holgate 1996; snow et al. 2001; turner 2011; kennedy et al. 2012) . in experimental studies, the infections have been defined as a short mild illness, with the early symptoms being headaches, chills, sneezing, and a sore throat, and delayed symptoms being nasal obstruction or discharge, cough, and malaise (jackson et al. 1958) . the duration of symptoms varies from 7 to 10 days, with a peak occurring on the 2-3rd day. however, some symptoms may be observed up to 3 weeks after the onset of the infection (heikkinen and järvinen 2003; eccles 2005) . common colds are triggered by rhinovirus (rv) coronavirus, influenza and parainfluenza viruses, and rsv (wat 2004; eccles 2005; kennedy et al. 2012) . the diagnosis of upper respiratory tract infections is based on symptoms and, with the exception of antivirals which may be used in influenza, treatment remains symptomatic. studies on different viruses responsible for the upper respiratory tract infections have shown that it is not possible to identify the virus based on the symptoms (johnston and holgate 1996; eccles 2002) . not only is the clinical presentation of these infections similar regardless of their etiology, but the order in which the symptoms develop is also similar. if the etiology of infections cannot be determined on the basis of clinical symptoms, the host reaction must play a major role in their pathogenesis. the similarities in the clinical presentation of viral upper respiratory tract infections stems from the same immune response pattern to different etiologic agents (eccles 2005) . furthermore, in acute respiratory tract infections a routine diagnosis to determine the etiology is not usually carried out in primary care settings. advances in molecular biology help explain the mechanisms that generate the symptoms of viral respiratory tract infections. nevertheless, the practical use of the pathophysiology of common symptoms seems relatively poor compared with the amount of scientific knowledge available. clinical manifestations of respiratory tract infections are familiar and well-known (eccles 2005; turner 2011 ). although the symptomatology depends, to some extent, on the type and location (e.g., pharyngitis, rhinitis, sinusitis, and bronchitis), the etiology of respiratory infection (viral or bacterial), patient's age, general health, co-morbidities, immunity, and on whether the infection is primary or secondary, e.g., in rsv or influenza there is a great amount of variation and overlap in both etiology and symptoms of individual infections. consequently, even defining the exact syndrome, like common cold or influenza-like morbidity, is difficult and problematic (eccles 2005) . the most significant signs of viral respiratory tract infections include sneezing, rhinorrhea (runny nose and nasal discharge), nasal congestion, cough, tachypnea, and fever. subjective symptoms include a sore throat, malaise, shivering (chills), shortness of breath, muscle aches and weakness, fatigue, and a loss of appetite and headaches (snow et al. 2001; wat 2004; eccles 2005; kennedy et al. 2012) . febrile seizures are a rare but important symptom in young children up to 6 years of age (schuchmann et al. 2011) . symptoms of upper respiratory tract infections have been traditionally classified as early or late (jackson et al. 1958; eccles 2005) . the early symptoms are those that develop quickly and resolve rapidly after 1-2 days, like headaches, sneezing, chills, sore throat, and malaise. in children a high fever may be observed, complicated by seizures in some cases (monto et al. 2000; schuchmann et al. 2011) . late symptoms include nasal discharge, nasal obstruction, and cough. the later symptoms develop over several days and are present one week after experimental infection (jackson et al. 1958; eccles 2005) . the development of sneezing before coughing in patients with a common cold may be partly explained by the involvement of the upper airways first and the infection subsequent spread to the lower respiratory tract (eccles 2005) . the aim of this review is to discuss the pathophysiology of symptoms of respiratory tract infections. we focused on the most significant symptoms of acute viral respiratory infections: sneezing, nasal discharge and obstruction, sore throat, coughing, muscle pains, malaise and mood changes, fever, and febrile seizures in children. we believe that despite a high prevalence of the symptoms, their pathogenesis is not widely known and a better understanding should have a beneficial effect on the therapeutic approach and should improve the quality of patient care. references from relevant articles were also identified. studies were included if they met the following two criteria: published in english and containing valid, consistent, and relevant data. the first two authors of the present review independently assessed all titles and abstracts to determine whether the inclusion criteria were satisfied. if either of the assessors considered the abstract potentially suitable, full-text articles were retrieved and then assessed by both assessors for their suitability for inclusion. eventually, 129 publications were selected and studied by each of the authors before the manuscript was prepared. 3 results agents of upper respiratory tract infections rhinoviruses are the most common etiologic agents of common cold. they are responsible for one-third to half of all upper respiratory tract infections reported in adults annually (proud et al. 1990; hendley 1998; heikkinen and järvinen 2003; ruuskanen et al. 2013) . improved knowledge about the structure and function of rhinoviruses has been acquired in recent years using virus culture techniques and new molecular genetics methods available. currently, more than 100 serotypes have been identified with hrv-a and hrv-b being the most important of them all (heymann et al. 2005; peltola et al. 2008; bochkov et al. 2011; kennedy et al. 2012 ). it has been demonstrated that the pathomechanism of symptoms in rhinoviral respiratory tract infections does not result from the cell damage, unlike influenza virus or rsv action (winther et al. 1990 ). rather, rhinovirus disrupts the tight junctions of the epithelial barrier, which facilitates the translocation of pathogens and stimulates the host's innate and adaptive immune responses (rezaee et al. 2011; kennedy et al. 2012) . over 90 % of rhinovirus serotypes enter the nasal epithelial cells through the host receptor with the intercellular adhesion molecule-1 (icam-1) being a glycoprotein immunoglobulin expressed on non-ciliated epithelial cells and on the basal surface of the ciliated epithelium of nasopharyngeal mucosa, while only around ten rhinovirus serotypes use a minor group of receptors, low-density lipoprotein (ldl) (bardin et al. 1994; winther et al. 1997; whiteman et al. 2003; bella and rossmann 2000; vlasak et al. 2005; kennedy et al. 2012) . newly discovered and sequenced human rhinovirus-c (hrv-c) is somehow unique as the virus isolates do not grow in typically used cell cultures, e.g., embryonic lung fibroblasts. in vitro growth of hrv-c was successfully performed using the sinus mucosal tissue as a substrate (bochkov et al. 2011; kennedy et al. 2012) . furthermore, studies on the structure and function of hrv-c have shown that the virus enters the cells using neither the icam-1 nor ldl receptor and the pathomechanism of the hrv-c infection remains unclear (arden and mackay 2010; simmonds et al. 2010; bochkov and gern 2012; lee et al. 2012; ruuskanen et al. 2013) . in otherwise healthy individuals, rhinovirus infections are generally limited to the upper respiratory tract with rhinorrhea and nasal obstruction being the most prominent symptoms (kennedy et al. 2012 ). entry of the rhinovirus triggers the release of interleukin-8 (il-8) a major cytokine in the recruitment of polymorphonuclear cells (hendley 1998) . il-8, which is produced locally, increases the production of nasal secretions and causes an influx of neutrophils (douglass et al. 1994) . oxidative stress caused by viral infection is probably responsible for the cellular mechanisms that lead to the production and release of il-8 (zhu et al. 1997) . studies of volunteers infected with a rhinovirus show a local infection in a small proportion (1-2 %) of nasopharyngeal epithelial cells (turner et al. 1982; bardin et al. 1994; arruda et al. 1995) . the higher the nasopharyngeal viral load the more severe is the disease (esposito et al. 2014) . vasoactive kinin peptides are other important mediators produced on-site in nasal mucosa and submucosa in rhinovirus-infected humans. kinins released in the nose following plasma exudation increase the symptoms of rhinoviral infection and cause an increase in vascular permeability, vasodilatation, and glandular secretion. bradykinin applied to the nasal mucosa causes symptoms that mimic the common cold, including rhinitis, nasal obstruction, and sore throat (proud et al. , 1990 . symptom scores correlate with an increase in the kinin concentration. while nasal secretion in adults with symptomatic experimentally-induced rhinovirus infection contain significantly higher concentrations of bradykinin and lysyl-bradykinin, asymptomatic infections do not result in increased kinin concentrations ). interestingly, the presence of rhinovirus has been detected by rt-pcr in 20 % of asymptomatic people who had an infected family member (jartti et al. 2008 ). the coronavirus (cov) is the second etiologic agent of upper respiratory tract infections (eccles 2005; mesel-lemoine et al. 2012) . the most common human-infecting coronaviruses include 229e, oc43, sars-cov, and the recently discovered nl63 and hku1 (arden et al. 2005; esper et al. 2005; vabret et al. 2005; pyrc et al. 2007 ). the virus is transmitted by aerosol inhalation and reinfections often occur due to a short-lived immunity (callow et al. 1990; wat 2004) . as a result, coronavirus accounts for 15-30 % of upper respiratory tract infections in humans. these infections are limited predominantly to the upper respiratory tract and rarely spread to lower airways and lungs (mesel-lemoine et al. 2012) . the coronavirus infection can occasionally involve other organs (arbour et al. 2000; collins 2002; desforges et al. 2006 ). it has been reported that the two new members of the coronavirus family, nl63 and hku1, especially the nl63, could also trigger severe lower respiratory tract infections and abdominal disorders such as pain and diarrhea (arden et al. 2005; esper et al. 2005; vabret et al. 2005; pyrc et al. 2007) . epidemics caused by cov-nl63 and cov-hku1 have been observed every 2-3 years (kahn 2006; jartti et al. 2012) . studies have confirmed that the infection with cov-nl63 is associated with croup and acute respiratory disease mostly in children, the elderly, and patients with chronic diseases, with some fatal cases being reported han et al. 2007; wu et al. 2008; oosterhof et al. 2010; sung et al. 2010; milewska et al. 2013) . the growth of coronaviruses using standard tissue cultures is poor and advanced molecular methods are needed to isolate the virus, so that most infections may remain undiagnosed (walsh et al. 2013) . human aminopeptidase n (hapn), a zinc-binding protein with endopeptidase activity, is used by cov-229e for entry into the epithelial cells, whereas cov-oc43 uses hemagglutinin-esterase (he) and spike (s) -its own surface glycoproteins -to enter the cell (tyrrell et al. 1993; künkel and herrler 1996; wat 2004) . recent studies have shown the ability of cov-229e to destroy the dendritic cells, which are essential components of the respiratory tract's immune system, which may explain multiple reinfections with the same type of cov (mesel-lemoine et al. 2012) . although the pathogenesis of infection caused by the two main groups of cov -229e and oc43 -is different, the clinical symptomatology is similar (tyrrell et al. 1993; wat 2004 ). respiratory syncytial virus (rsv) is responsible for many flu-like illnesses (zambon et al. 2001) . rsv replication starts in the nasopharynx and then the virus infects the bronchiolar epithelium presumably by cell-to-cell spread or aspiration of secretions. the virus spares the basal cells and subsequently extends to the alveolar pneumocytes. the pathologic findings of rsv are characterized by necrosis of epithelial cells, infiltration with t cells and monocytes around arterioles and with neutrophils between the vascular structures and small airways (johnson et al. 2007 ). this leads to airway obstruction, air trapping, increased airway resistance, and is also associated with neutrophilia in bronchoalveolar lavage (everard et al. 1994 ). rsv has never been isolated from blood (peebles and graham 2005) . the immune response to rsv, especially cytokines and chemokines, seems to be responsible for the symptoms and severity of bronchiolitis (garofalo et al. 2001; legg et al. 2003 ). the cytokines il-8, il-6, tnf-alpha, and il-1 beta were detected in respiratory secretions from infected children and high il-6 concentrations are associated with severe manifestations of the disease (matsuda et al. 1995; noah et al. 1995; smyth et al. 1997) . respiratory secretions from infected children contain chemokines expressed and secreted by t-cells (chemokine ligand 3 -ccl3, i.e. macrophage inflammatory protein-1 -mip-1 alpha; chemokine ligand 2 -ccl2, i.e. monocyte chemoattractant protein-1 -mcp-1; chemokine ligand 11 -ccl11 -eotaxin, and chemokine ligand 5 -ccl5, i.e. rantes; regulated on activation). mip-1 alpha, and to a lesser extent other beta-chemokines, primarily secreted by activated immune cells, are associated with severe manifestations of the disease (noah et al. 1995; welliver et al. 2002; garofalo et al. 2005) . experimental infection of explanted polarized respiratory epithelium in tissue culture generates il-8 and ccl5 (mellow et al. 2004 ). nonetheless, it is unknown whether the cytokines and chemokines are the cause of disease or are by-products of enhanced inflammatory responses (barr and graham 2014). whether respiratory tract infections caused by influenza viruses present as common colds with typical symptoms or as severe lower respiratory tract diseases depends on the type of virus, pre-existing immunity, the patient's underlying disorders, and multiple other factors (wat 2004) . the phenomena such as antigenic shift or drift have led to the formation of more recent and increasingly virulent variations of the influenza virus and, consequently, to more serious clinical manifestations (gething et al. 1980; treanor 2004) . as an example, pandemic influenza a (h1n1)pdm09 affected all age groups, but it was more prevalent in younger patients and children in whom there was the highest rate of hospitalization and pneumonia (kuchar et al. 2013 ). it has previously been shown that coughing and fever are the best predictive factors of influenza infections having a positive predictive value (ppv) of 79 % (monto et al. 2000) . however, neither symptom is sufficiently predictive in children aged 1-4 (ohmit and monto 2006) . overall, influenza viruses are generally responsible for 5-15 % of acute upper respiratory tract infections in humans (wat 2004 ). the influenza virus causes damage to the epithelial cells and its replication occurs in the airways with predilection to the lower respiratory tract (hers and mulder 1961; hers 1966; wat 2004 ). the two main glycoproteins of the influenza virus surface -hemagglutinin (ha) and neuraminidase (na)play an essential role in the infection spread. ha targets cells for infection binding to the epithelial sialylated glycans (specific for upper or lower airways) (shinya et al. 2006; de wit et al. 2010; fukuyama and kawaoka 2011) , while na is responsible for effective viral replication (pappas et al. 2008) . another viral protein, nonstructural protein 1 (ns1) is also important due to its counteracting ifn-α production in infected cells (fukuyama and kawaoka 2011) . viral replication is possible in host cells due to activation of nuclear factor kappa b (nf-κb) and the raf/mek/erk cascade, and then proinflammatory cytokines are produced with interleukin 6 (il-6) being the most important of them (kaiser et al. 2001; pinto et al. 2011; wine and alper 2012) . il-6, tumor necrosis factor-α (tnf-α), interferon-α (ifn-α), il-8, and il-1β increase significantly in response to the viral invasion resulting in the development of fever, nasopharyngeal mucous production, and respiratory and systemic symptoms. viral replication and the intensity of the main influenza symptoms are correlated with the level of cytokines, particularly with il-6 and tnf-α (hayden et al. 1998; kaiser et al. 2001 ). evidence presented in our review of the pathophysiology of signs and symptoms in the four most common viral upper respiratory tract infections suggest that the immune system's response to infection rather than the virusspecific damage to the respiratory tract is responsible for the symptomatology (turner 1997; hendley 1998; eccles 2005) . studies on different viruses responsible for upper respiratory tract infections have shown that it is not possible to identify the virus based on the symptoms (eccles 2005) . the pathology of rhinovirus infections consists of the influx of polymorphonuclear leukocytes at the beginning of the infection (winther et al. 1984) . macrophages play a key role in triggering an acute phase response with the production of cytokines (beutler 2003) , while the release of proinflammatory cytokines and other mediators cause upper respiratory tract infection symptoms (eccles 2000a, b) . cytokines are responsible for the systemic symptoms (e.g., fever) and bradykinin plays a major role in local symptoms of respiratory tract infections (e.g., sore throat and nasal congestion) shibayama et al. 1996; conti et al. 2004 ). a sore throat, irritation, and pain in the pharynx usually appear at the beginning of a respiratory tract infection. a sore throat is most likely caused by the action of prostaglandins and bradykinin on sensory nerve endings in the upper respiratory tract. intranasal administration of bradykinin causes symptoms of rhinitis and a sore throat, so it is likely to be responsible for these symptoms (rees and eccles 1994; proud 1998) . the sensation of pain is mediated by the cranial nerves supplying the nasopharynx. similar symptoms have been observed in bacterial upper respiratory tract infections, pharyngitis, and tonsillitis (georgitis 1993 ). nasal congestion is a subsequent symptom of respiratory infection that develops over the first week of symptoms (tyrrell et al. 1993) . the mechanism of nasal congestion relies on the dilation of the venous sinuses in the nasal epithelium in response to the vasodilator mediators such as bradykinin (proud et al. 1990; widdicombe 1997) . symptom scores increase as kinin concentrations rise (proud et al. 1990 ). dilatation of the sinuses in the narrow nasal valve region causes obstruction of the nasal airway (eccles 2000b) . the so-called nasal cycle (alternating congestion and decongestion of the nasal passages controlled by the sympathetic vasoconstrictor nerves) is accentuated and the asymmetry of nasal airflow is more pronounced during respiratory infection (eccles et al. 1996) . a watery nasal secretion often accompanied by sneezing is an early symptom of a respiratory tract infection. nasal discharge in respiratory infections is a complex mixture of plasma and glandular exudates with cellular elements (e.g., goblet cells, plasma cells, and neutrophils) of variable composition that changes over the course of the infection and severity of the inflammatory response (eccles 1983 ). the first phase of nasal discharge consists of a glandular secretion reflex caused by stimulation of the upper airway's trigeminal nerves. studies have demonstrated that intranasal administration of ipratropium inhibits nasal secretions in the first 4 days of a common cold (e.g., when it is caused by coronavirus) (akerlund et al. 1993; hayden et al. 1996) . the color of the nasal discharge may change from watery clear to yellow and green during the course of the respiratory tract infection and this reflects the severity of the inflammatory response rather than the etiology of the infection (stockley et al. 2001) . the green or yellow color of nasal discharge is often regarded as a clinical marker of bacterial superinfection and clinical indication to antibiotic treatment, but there is no evidence that supports this concept (murray et al. 2000) . the color change is related to the recruitment of leukocytes into the airway lumen (stockley et al. 2001) . neutrophils and activated monocytes contain chromatic, green granules (azurophil granules) containing myeloperoxidase with heme pigment. the more leukocytes present in nasal discharge the more colorful the nasal discharge appears (stockley et al. 2001) . although the literature is related to sputum color changes, the same mechanisms apply to nasal discharge. a watery nasal secretion in the infection's early stage is often accompanied by sneezing. sneezing together with a sore throat are the early symptoms of respiratory tract infections. sneezing is a reflex mediated by the trigeminal nerves which supply the nasal epithelium (leung and robson 1994; eccles 2005) . the sneeze center in the brainstem coordinates the sneeze reflex. sneezing is related to inflammatory responses in the nose and nasopharynx that stimulate the trigeminal nerves (eccles 2005) . as intranasal administration of histamine causes sneezing, sneezing is probably mediated by histamine receptors on the trigeminal nerves (mygind et al. 1983; eccles 2005) . coughing is the most common clinical symptom and the most frequent reason for visits to see a doctor (mcgarvey and morice 2006) . coughing is a protective reflex that prevents the aspiration of food and fluids into the airway and cleans the respiratory tract of mucus and other foreign bodies. the reflex is mediated exclusively by the vagus nerve (eccles 2005) . coughing is initiated in the airway through stimulation of the sensory nerves in the larynx or below (widdicombe 1995) . the airway inflammation associated with rhinitis must reach the larynx to cause coughing. the external ear and esophagus are also supplied by the vagus nerve and coughing can also be triggered by gastroesophageal reflux (morice 2002) . respiratory tract infections are often accompanied by redundant, dry, and unproductive coughing in the first days. the unproductive coughing may be caused by the inflammatory process spreading to the larynx since nasal inflammation causes sneezing rather than coughing. coughing in respiratory tract infections is believed to be mediated by hyperreactivity of the cough reflex due to the effects of inflammatory mediators on the airway's sensory nerve endings (lee et al. 2002; eccles and lee 2004) . when the larynx is inflamed and hyperreactive, coughing may occur spontaneously or in response to stimuli that would not normally cause coughing, e.g., cold air. it may persist for three weeks or longer. some coughs may be voluntary and related to airway irritation (lee et al. 2002) . productive coughing usually occurs later in the course of respiratory tract infection and is related to the mucus production associated with inflammation of the lower airways (eccles 2005) . rhinovirus and coronavirus do not usually cause significant damage to the airway cells and the common cold is typically associated with little, if any, coughing while the influenza virus may cause substantial cellular damage to the respiratory epithelium and an influenza infection is usually associated with coughing (monto et al. 2000) . respiratory tract infections are associated with impaired psychomotor function (smith et al. 1998) . mood changes and malaise may be explained by the unpleasant objective symptoms of respiratory tract infections such as nasal congestion, rhinorrhea, and coughing (eccles 2005) . these symptoms may cause discomfort and lower the patient's quality of life. however, there is increasing evidence that mood changes may also be caused by the effects of cytokines on the central nervous system (mahoney and ball 2002) . interferon alpha treatment for chronic hepatitis b and c is associated with flu-like adverse effects similar to those observed in respiratory tract infections: malaise, fever, myalgia, and mood changes (schaefer et al. 2002) . psychiatric adverse effects such as depression, irritability, impaired concentration, and psychoses have been reported with interferon alpha therapy. it has been reported that cytokines, e.g., tumor necrosis factor-α (tnf-α) and interleukins 1, 2, and 6 cause mood changes with anhedonia, cognitive dysfunction, anxiety, irritability, psychomotor slowing, fatigue, anorexia, sleep alterations, and a lower pain threshold (capuron and miller 2004) . the production of these cytokines is also associated with respiratory tract infections which may mediate mood changes associated with these infections. the exact mechanisms of cytokine action in the brain are poorly understood, but there is a growing body of evidence suggesting that anorexia associated with respiratory infections is mediated by cytokines that act directly on the feeding center in the hypothalamus (langhans 2000) . headaches are a common early symptom of respiratory tract infections. the majority (60 %) of patients with respiratory tract infections with a sore throat reported headaches in a clinical trial (eccles et al. 2003) . the mechanism of a headache associated with a respiratory tract infection is unknown but headaches may be triggered by cytokines released in response to a viral infection (smith 1992) . it has been shown that the administration of some cytokines such as tumor necrosis factor and interferons cause headaches (smith 1992; gold et al. 2005; van zonneveld et al. 2005 ). myalgia is a common symptom of respiratory tract infections. around half of patients with a common cold complain about muscle pain (eccles et al. 2003; eccles 2005) . myalgia occurs in the acute immune response to infection phase and is related to the effects of cytokines on skeletal muscles (baracos et al. 1983 ). proinflammatory cytokines including tnf-α have been implicated in the breakdown of muscle proteins (kotler 2000) . fever and myalgia associated with respiratory tract infection may be caused by the production of prostaglandin e2 in response to cytokines (baracos et al. 1983 ). the cytokine-related synthesis of prostaglandin e2 and the breakdown of skeletal muscle has been inhibited in vitro by non-steroidal anti-inflammatory agents and similarly fever and myalgia accompanying acute respiratory infection are relieved with acetylsalicylic acid, a classic anti-inflammatory agent (eccles et al. 2003) . since prostaglandin e2 is a pain mediator, its increased synthesis may explain the myalgia associated with acute respiratory tract infections. fever is a classic response to infection. it is a manifestation of cytokine release in response to a variety of stimuli. it is believed to be beneficial for the host's response to infection (cabanac 1990; eccles 2005) and is usually associated with novel or severe viral infections such as influenza (monto et al. 2000) . consequently, fever is a common symptom in infants, probably because viruses responsible for acute respiratory tract infections are new to the infant and induce a strong immune response. however, in adults who had been exposed to numerous common cold viruses in the past, subsequent infections do not elicit a strong cytokine response and fever is a rare symptom of a common cold in adults (eccles 2005) . on the contrary, some patients experience a transient fall in body temperature during the early stages of acute benign respiratory tract infection and about 1/3 of all patients experience chills (eccles et al. 2003) . chills associated with a fall in skin temperature related to vasoconstriction of the skin's blood vessels may be explained as an initial stage of fever, but chills may also be unrelated to changes in skin temperature. chills have developed after administration of exogenous pyrogens, even if the subjects maintained a neutral skin temperature (34.5 c in water) in experimental human studies (guieu and hellon 1980) . chills occur together with shivering and the latter symptom is probably related to the cerebral cortex influence over the shivering control. both chills and shivering may be caused by cytokines acting on the temperature control center of the hypothalamus. many cytokines act as endogenous pyrogens and are released from leukocytes in response to infection (conti et al. 2004 ). the proinflammatory cytokines (il-1 and il-6) are regarded as the most important cytokines for causing fever (netea et al. 2000; leon 2002) . they are believed to cross the blood-brain barrier and increase the thermal set point in the temperature control center. the hypothalamus then induces shivering, constriction of the skin's blood vessels, and chills (eccles 2005) . febrile seizures are a rare but significant symptom of acute viral respiratory infections in children. they occur in 2-5 % of all children and the majority of febrile seizures are triggered by respiratory tract infection (schuchmann et al. 2011) . febrile seizures are defined as occurring in children aged 6-60 months with a temperature 38.0 c with no central nervous system infection, metabolic disturbance, or history of afebrile seizure. they are the most common type of seizure in children under 60 months (american academy of pediatrics 2011; graves et al. 2012) . cytokines seem to play a crucial role in febrile seizures, however there is a lot of confusion about the relationship between proinflammatory and anti-inflammatory cytokines and the febrile seizure risk. is it generally accepted that the genotype il-1α-889 1/1 and il-1β-511 t/t homozygote as well as the serum concentration of il-6 are associated with an increased risk of febrile seizures (saghazadeh et al. 2014 ). the treatment of acute viral respiratory tract infections remains primarily supportive. there is evidence that medications like acetaminophen (paracetamol) and non-steroidal anti-inflammatory agents such as ibuprofen or aspirin relieve some symptoms of acute respiratory tract infections (fever, sore throat, pain, and malaise), but it is debatable whether symptomatic treatment could speed up recovery (eccles 2005) . many other common advices like drinking plenty of fluids or steam inhalation have not been scientifically proven. some new agents seem to be promising. for example in a study by asada et al. (2012) l-carbocysteine reduced the baseline and rs virus infection-induced secretion of pro-inflammatory cytokines, including il-1β, il-6, and il-8 as well as virus titers in the supernatant of human tracheal epithelial cells culture. although a virus-orientated approach and the development of anti-viral agents should be more beneficial, the diverse etiology makes the development of universal antivirals highly unlikely (passioti et al. 2014 ). since clinical symptoms are not sufficient to determine the etiology of acute viral respiratory tract infections, we believe that the host defense mechanisms are critical to the symptomatology. immune response seems to be fundamental for understanding the pathomechanisms of these infections. inflammatory mediators such as prostaglandins and bradykinin are responsible for the local symptoms of nasal congestion and rhinorrhea, while cytokines are responsible for systemic symptoms. a better understanding of the immune response including cytokines 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nasal mucosa during experimental rhinovirus colds respiratory virus infection of monolayer cultures of human nasal epithelial cells surface expression of intercellular adhesion molecule 1 on epithelial cells in the human adenoid viral-induced rhinitis clinical manifestations of human coronavirus nl63 infection in children in taiwan contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study rhinovirus stimulation of interleukin-8 in vivo and in vitro: role of nf-kappab the authors have no funding or conflicts of interest to disclose. key: cord-324333-huris8br authors: lee, na hyun; choi, hee joung; kim, yeo hyang title: clinical usefulness of serum procalcitonin level in distinguishing between kawasaki disease and other infections in febrile children date: 2017-04-25 journal: korean j pediatr doi: 10.3345/kjp.2017.60.4.112 sha: doc_id: 324333 cord_uid: huris8br purpose: the aims of this study were to compare serum procalcitonin (pct) levels between febrile children with kawasaki disease (kd) and those with bacterial or viral infections, and assess the clinical usefulness of pct level in predicting kd. methods: serum pct levels were examined in febrile pediatric patients admitted between august 2013 and august 2014. the patients were divided into 3 groups as follows: 49 with kd, 111 with viral infections, and 24 with bacterial infections. results: the mean pct level in the kd group was significantly lower than that in the bacterial infection group (0.82±1.73 ng/ml vs. 3.11±6.10 ng/ml, p=0.002) and insignificantly different from that in the viral infection group (0.23±0.34 ng/ml,p=0.457). the mean erythrocyte sedimentation rate (esr) and c-reactive protein (crp) level in the kd group were significantly higher than those in the viral and bacterial infection groups (p<0.001 and p<0.001 for esr, p<0.001 and p=0.005 for crp, respectively). the proportion of patients in the kd group with pct levels of >1.0 ng/ml was significantly higher in the nonresponders to the initial intravenous immunoglobulin treatment than in the responders (36% vs. 8%, p=0.01). conclusion: pct levels may help to differentiate kd from bacterial infections. a combination of disease markers, including esr, crp, and pct, may be useful for differentiating between kd and viral/bacterial infections. procalcitonin (pct), the prehormone of calcitonin, is released in response to proinflam matory stimuli, particularly bacteriaassociated mediators 1) . recently, pct has been more frequently used as a biomarker of sepsis than creactive protein (crp), another acutephase reactant 2) . in a study of pct levels in adult patients in various inflammatory states, pct levels were found to be significantly elevated in patients with bacterial or fungal infections, but normal or slightly elevated in those with several inflammatory diseases 3) . furthermore, in a study of pct levels in pediatric patients, pct was found to be a significant biomarker for diagnosing pediatric urinary tract infection and neonatal sepsis, and for identifying patients without serious bacterial infections in order to prevent antimicrobial overuse in the pediatric intensive care unit 46) . many children present to hospitals with fevers of varying etiologies. one of the most important causes of fever in children younger than 5 years is kawasaki disease (kd). kd is a wellknown systemic vasculitic disorder, and the systemic in flammation associated with the condition causes elevations in white blood cell (wbc) counts and crp levels, in addition to ele vation in the erythrocyte sedimentation rate (esr) 7, 8) . before a diagnosis of kd is made and intravenous immunoglobulin (ivig) therapy is initiated, many patients with kd receive antibiotic (oral/intravenous) therapy and a diagnosis of kd is delayed because high esr and crp levels are commonly thought to indi cate bacterial infections. the first study of pct levels in patients with kd compared with pct levels in patients with other diseases was reported in 2004 9) . although the clinical impact of serum pct levels in patients with kd has not been determined, the relation between pct and kd severity, and the comparison of pct levels between complete and incomplete kd were also reported 10, 11) . the aim of this study was to report our experience about pct levels in febrile children with kd and those with bacterial or viral infections, and the clinical usefulness of pct level in predicting kd. we evaluated the serum pct levels of 400 febrile pediatric patients admitted from august 2013 to august 2014 at single tertiary center. fever was defined as a body temperature of ≥38°c, and the age range of the enrolled patients was from 29 days to <5 years. the enrolled patients were divided into 3 groups-the kd, viral infection, and bacterial infection groups-according to the definitions described below. complete kd was diagnosed in cases in which a fever persisted for ≥5 days, and at least 4 of the 5 typical clinical manifestations were observed, based on the 2004 american heart association criteria 7, 8) . we also included patients with incomplete kd, which was diagnosed when fewer than four of the 5 typical clinical manifestations were observed based on the criteria 12) . responders to ivig treatment included patients with kd whose fever subsided within 48 hours after the initial treatment, whereas nonrespon ders included patients with kd whose fever persisted beyond 48 hours and required additional ivig treatment. to identify viral pathogens, virus reverse transcription poly merase chain reaction (rtpcr; 16 ace detection, seegene, seoul, korea) was performed on nasal or throat swab specimens obtained on the day of admission. rtpcr included tests for metapneumovirus, adenovirus (a-f), coronavirus 229e/oc43, parainfluenza virus 1/2/3, influenza a/b virus, rhinovirus, re spiratory syncytial virus a/b, and bocavirus. viral infections were diagnosed on the basis of positive rtpcr results. patients with clinical diagnostic features of viral infections, such as hand, foot, and mouth disease, or herpangina, were also included. bacterial infections were confirmed by positive culture results in various samples from each patient (i.e., blood, urine, or cerebrospinal fluid). kd with viral infection confirmed by rtpcr or bacterial infection confirmed by culture was excluded. we retrospectively reviewed the patients' characteristics and clinical courses by using medical records. peripheral blood samples were analyzed on the day of admis sion for measurement of markers including pct. serum pct levels were measured by using electrochemiluminescence immu noassays (elecsys brahms pct, roche, henningsdorf, germany) with a detection range of 0.02-100 ng/ml. the following cutoff levels of pct were used to determine whether antibiotic treatment would be used: pct<0.25 ng/ml, antibiotics strongly discourag ed; pct<0.5 ng/ml, antibiotics discouraged; and pct>1.0 ng/ml, antibiotics strongly encouraged 13) . we also obtained complete blood cell counts, esr, and crp levels. serum crp levels were measured by using latexenhanced immunoturbidimetry (advia chemistry xpt system, siemens, erlangen, germany). this study was approved by the institutional review board of the keimyung university dongsan medical center (approval number: 201501003). all statistical analyses were performed with ibm spss statistics ver. 21.0 (ibm co., armonk, ny, usa), and collected data were described as frequencies and means with the standard deviations. the chisquare test was used for comparisons of categorical data, and 1way analysis of variance followed by the bonferroni post hoc test for multiple comparisons was used for comparisons of continuous data among the 3 groups. in patients with kd, the unpaired t test and logistic regression analysis were applied to compare the pct, esr, and crp levels. a p value of <0.05 was considered statistically significant. a total of 184 patients were enrolled in this study, including 49 with kd, 111 with viral infections, and 24 with bacterial infec tions. the characteristics of patients in each group are shown in table 1 . kd and incomplete kd were observed in 40 and 9 patients, respectively. all patients with kd were initially treated with 2 g/ kg ivig and 60-100 mg/kg oral aspirin, and 38 patients were classified as responders and 11 patients as nonresponders. of the 111 patients with viral infections, 108 showed positive rtpcr results. the most commonly detected virus was respira tory syncytial virus a/b (40 of 108, 37%), followed by rhinovirus (22 of 108, 20%) and parainfluenza (17 of 108, 16%). thirteen patients (13 of 108, 12%) had mixed infection with 2 kinds of viruses. among the patients with viral infection, the most com mon diagnosis was acute lower respiratory tract infection. acute bronchiolitis was diagnosed in 65 patients (60%) and pneumonia in 18 patients (18 of 108, 17%), followed by croup in 8, influenza in 5, and asthma in 5 patients. the other diagnoses were bronchi tis in 3; hand, foot, and mouth disease in 1; herpangina in 1; influenza in 1; and fever without localizing signs in 1. bacterial infections were confirmed in 10, 13, and 1 patient, respectively, on the basis of positive results of blood, urine, and both blood and urine cultures. of the 24 patients with bacterial infec tions, the causative organisms were staphylococcus or strep tococcus in blood, and enterococcus, escherichia coli, or klebsiella pneumoniae in urine. the final diagnoses of bacterial infections were septicemia, pneumonia, cellulitis, and urinary tract infec tions. the mean age of the kd group was significantly higher than that of the viral infection group (p=0.006). patients in the kd group had a significantly higher mean body weight than those in the viral and bacterial infection groups (p=0.035 and p=0.014); however, the mean body mass index was not significantly different among the groups. the sex distributions were not differ ent among the 3 groups. the duration of fever before admission was a mean of 4.4, 2.6, and 1.7 days in the kd, viral infection, and bacterial infection groups, respectively. patients in the kd group had a significantly longer fever duration before admission than those in the viral and bacterial infection groups (p=0.001 and p< 0.001). the bacterial infection group had a significantly longer duration of admission than the viral infection group (p=0.006). the laboratory findings of all patients included in the study are shown in table 2 . the mean wbc count in the kd and bacterial infection groups was significantly higher than that in the viral infection group (p=0.044 and p=0.005), and the mean neutrophil proportion in the kd group was significantly higher than that in the viral infec tion group (p<0.001). the mean esr in the kd group was signifi cantly higher than that in the viral and bacterial infection groups (p<0.001 and p<0.001). the mean crp level in the kd infection group was significantly higher than that in the viral and bacterial groups (p<0.001 and p<0.001). the mean pct levels in the kd and viral and bacterial infection groups were 0.82±1.73, 0.23±0.34, and 3.11±6.10 ng/ml, res pectively. the mean pct level in the bacterial infection group was significantly higher than that in the kd and viral infection groups table 2 . the number of patients with pct levels <0.25 ng/ml and >1.0 ng/ml was significantly different between the kd and viral infection groups (p=0.001) and between the viral infection and bacterial infection groups (p<0.001), but not between the kd and bacterial infection groups (p=0.099). in the kd group, pct levels were not significantly correlated with fever durations, esr, or crp levels (p=0.792, p=0.994, and p=0.516, respectively). there was no significant difference in pct levels between patients with complete and incomplete kd (p=0.477) (fig. 1a) . among the nine patients with incomplete kd, only one had a pct level >0.5 ng/ml. between ivig responders and nonresponders among patients with kd, there was also no significant difference in pct level, esr, and crp level (p=0.434, p=0.052, and p=0.107, respectively) . however, after adjustment for age by using analysis of covariance (ancova), a significant difference was observed in pct and crp between responders and nonresponders in the kd group (p=0.023 and p=0.012). of the 11 ivig nonresponders, 2 (18%) had a pct level <0.25 ng/ml and 4 (36%) had a pct level >1.0 ng/ml. in comparison, of the 38 ivig responders, 25 (66%) had a pct level <0.25 ng/ml and 3 (8%) had a pct level >1.0 ng/ml. a lower proportion of nonresponders than responders had pct levels <0.25 ng/ml (p= 0.01). a higher proportion of nonresponders than responders had pct levels >1.0 ng/ml (p=0.01) (fig. 1b) . the results of this study demonstrated that mean pct level was beneficial in differentiating febrile children with kd from those with bacterial infections. moreover, patients with kd with pct levels >1.0 ng/ml tended to be nonresponders to initial ivig treat ment. in addition, esr and crp levels were useful in differ entiating between patients with kd and those with viral infec tions. among many clinical markers of inflammation and sepsis, pct is more accurate than crp in differentiating bacterial infections from noninfectious inflammation 1) . this is because the peak pct level is reached more rapidly than the peak crp level, and the synthesis mechanisms of pct and crp are somewhat different. pct is mainly produced in the liver and monocytes, and its secre tion is modulated by lipopolysaccharides and sepsisrelated cytokines such as tumor necrosis factor (tnf)α. crp is produced in the liver, mainly in response to interleukin (il) 6 2,14) . in pedia tric research, pct has been used in the study of neutropenia, fever in young infants, pediatric urinary tract infections, pediatric communityacquired pneumonia, and appendicitis 4, 1517) . kd is a systemic vasculitic disorder that leads to endothelial cell activation and damage caused by increased levels of inflamma tory cytokines such as tnfα, il1, and il6 18) . these inflamma tory cytokines increase the levels of acutephase reactants such as crp and pct. increased crp level in patients with kd plays a crucial role in diagnosis and treatment, and is also a risk factor for coronary arteryrelated complications 12, 19, 20) . however, the clinical impact of serum pct levels in patients with kd has not been determined. fever is the first symptom to appear in kd, and the other symp toms do not usually develop simultaneously 7, 8) . the initial high esr and crp levels associated with fever are suspected to be indi cative of bacterial infection; hence, oral or intravenous antibiotic therapy may be administered, which usually continues until the symptoms fulfill the criteria of kd. however, patients with kd do not respond to antibiotic therapy and require ivig treatment in stead. because kd is a difficult inflammatory disease to diagnose and pct is a known biomarker of sepsis to diagnose bacterial infection and decide antibiotic treatment, we hypothesized that serum pct level may differentiate kd from other infections. the first study of pct levels in patients with kd was reported in 2004. in this report, patients with kd had a higher mean pct level than patients with viral and autoimmune diseases and healthy children, whereas patients with kd and those with bac terial infections showed a similar pct level 9) . the present study also showed similar results: (1) the mean pct level in patients with kd and in those with bacterial infection was significantly higher than that in patients with viral infection. (2) the mean pct level in patients with kd was lower than that in patients with bacterial infection; however, this difference was not significant. we speculated that immune responses to bacterial infections may occur in patients with kd, although the causative microorganism of kd has not yet been confirmed. the definite cause of kd is currently unknown; however, it is generally accepted that kd de velops as a result of a genetic predisposition combined with an infection with an undefined trigger or an autoimmune mecha nism. many bacterial and viral agents have been suggested but not confirmed as triggers of kd infection. recently, bacterial toxins or intracytoplasmic inclusion bodies associated with viral infections have been newly hypothesized as causes for the development of kd 18) . in the present study, pct levels were not significantly different between patients with complete kd and those with incomplete kd. however, more nonresponders than responders had a pct level >1.0 ng/ml, and there was a significant difference in pct between responders and nonresponders in the kd group after adjustment for age by using ancova. another study investiga ting responders and nonresponders to initial ivig treatment demonstrated that pct levels were significantly higher in non responders than in responders 10) . this result was similar to the findings of our study. a recent study enrolled 77 patients with complete kd and 24 patients with incomplete kd, and showed that the pct levels of patients with complete kd were signifi cantly higher than those of patients with incomplete kd 11) . the authors described that nonresponders to initial ivig more frequently had complete kd than incomplete kd (16% vs. 4%). we suspect that more nonresponders with complete kd had higher pct levels than those with incomplete kd. the pct level seems to be useful in predicting ivig response in patients with kd. recent guidelines for determining whether antibiotic therapy should be implemented use the following criteria: pct<0.25 ng/ ml, antibiotics strongly discouraged; pct<0.5 ng/ml, antibiotics discouraged; and pct>1.0 ng/ml, antibiotics strongly enco uraged 13) . of the 49 kd patients in the present study, 37 (76%) would be recommended to not receive antibiotics (pct<0.5 ng/ ml). in comparison, 99 patients (89%) with viral infections and 16 patients (67%) with bacterial infections would be recommend ed to not receive antibiotics. as stated above, pct levels were significantly different between patients with kd and those with viral infection, and between patients with viral infection and those with bacterial infection, but not between patients with kd and those with bacterial infection. although pct levels were not useful in differentiating between kd and bacterial infections, esr and cpr levels were significantly higher in patients with kd than in those with bacterial infection. therefore, not only pct but also esr and crp levels should be considered in febrile pediatric patients. if febrile patients have high esr and crp levels and low pct levels in addition to 2 or 3 symptoms of kd, kd should be suspected rather than bacterial infection. the limitations of the present study were the small sample size, especially patients with incomplete kd and patients with kd who were ivig nonresponders, and the lack of comparisons with other systemic inflammatory conditions such as autoimmune disease. further studies on cytokines, including tnfα and il6, will help in determining the role of pct in kd. in conclusion, the pct levels may help differentiate kd from bacterial infections. a combination of disease markers including esr, crp, and pct may be useful for differentiating between kd and viral or bacterial infections. in addition, the pct level may be somewhat useful in predicting responses to ivig treatment. update on procalcitonin measurements serum pro calcitonin and creactive protein levels as markers of bacterial infec tion: a systematic review and metaanalysis can procalcitonin measurement help in differen tiating between bacterial infection and other kinds of inflamma tory processes? procalcitonin and creactive protein in urinary tract infection diagnosis serum procalcitonin as a diagnostic marker for neo natal sepsis: a systematic review and metaanalysis procalcitonin use in a pediatric intensive care unit kawasaki disease diagnosis, treatment, and longterm management of kawasaki disease: a statement for health professionals from the committee on rheumatic fever, endocarditis and kawasaki dis ease, council on cardiovascular disease in the young serum procalcitonin concentration in patients with kawasaki disease serum procalcitonin value is useful for predicting severity of kawasaki disease procalcitonin levels in patients with complete and incomplete kawasaki disease diagnosis, treatment, and longterm management of kawasaki disease: a statement for health professionals from the committee on rheumatic fever, endocarditis, and kawasaki dis ease, council on cardiovascular disease in the young use of procalcitonin to reduce patients' exposure to antibio tics in intensive care units (prorata trial): a multicentre rando mised controlled trial procalcitonin behaves as a fast responding acute phase protein in vivo and in vitro society of pediatric emergencies. procalcitonin in pediatric emergency departments for the early diagnosis of in vasive bacterial infections in febrile infants: results of a multicen ter study and utility of a rapid qualitative test for this marker procalcitonin measurements for guiding antibiotic treatment in pediatric pneumonia procalci tonin as an early marker of bacterial infection in neutropenic fe brile children with acute lymphoblastic leukemia kawasaki disease: aetiopathogenesis and therapeutic utility of intravenous immuno globulin parameters to guide retreatment after initial intravenous immunoglobulin therapy in kawasaki dis ease evolution of laboratory values in patients with kawasaki disease no potential conflict of interest relevant to this article was reported. key: cord-355906-yeaw9nr8 authors: nedjadi, taoufik; el-kafrawy, sherif; sohrab, sayed s.; desprès, philippe; damanhouri, ghazi; azhar, esam title: tackling dengue fever: current status and challenges date: 2015-12-09 journal: virol j doi: 10.1186/s12985-015-0444-8 sha: doc_id: 355906 cord_uid: yeaw9nr8 according to recent statistics, 96 million apparent dengue infections were estimated worldwide in 2010. this figure is by far greater than the who prediction which indicates the rapid spread of this disease posing a growing threat to the economy and a major challenge to clinicians and health care services across the globe particularly in the affected areas. this article aims at bringing to light the current epidemiological and clinical status of the dengue fever. the relationship between genetic mutations, single nucleotide polymorphism (snp) and the pathophysiology of disease progression will be put into perspective. it will also highlight the recent advances in dengue vaccine development. thus far, a significant progress has been made in unraveling the risk factors and understanding the molecular pathogenesis associated with the disease. however, further insights in molecular features of the disease and the development of animal models will enormously help improving the therapeutic interventions and potentially contribute to finding new preventive measures for population at risk. dengue fever is a major cause of illness and death worldwide. the disease is caused by dengue virus which gets transmitted to humans by the bites of infected mosquitoes, aedes (ae.) aegypti and ae. albopictus [1] . the disease represents a global health issue as it is endemic in around 100 countries, most of which are in tropical and sub-tropical areas. over the last decades, the incidence rate and the geographic distribution of dengue have rapidly increased (almost 30-fold). data from the world health organization (who) estimates up to 100 million cases of dengue fever each year [2] . however, a recent published work by bhatt et al. (2013) suggested that the burden of dengue is far more than the who estimation and indicated that 390 million infections of dengue virus could have happened every year [3] . changes in dengue epidemiology and the increase in incidence rates (with and without co-morbidities) have led the who to propose a new dengue classification system according to disease severity ( fig. 1 ) [2] . dengue fever is caused by infection with dengue virus (denv). the denv is a vector-borne virus transmitted to humans primarily by bites from two mosquito species, ae. aegypti or ae. albopictus. denv is a single positivestranded rna virus belonging to flavivirus genus of the flaviviridae family and has 4 major serotypes (denv 1-4) that are antigenically distinct from each other. each denv serotype is phylogenetically distinct suggesting that each serotype could be considered a separate virus [4] . three dengue serotypes out of four (denv 1-3) have been found in middle eastern countries including saudi arabia and yemen. interestingly, denv-1 strain isolated in saudi arabia exhibited a high genetic similarity with denv-1 strain isolated from asian population, suggesting a widespread of the asian genotype, probably through asian pilgrims [5, 6] . a recently published article has unveiled a new serotype , to be added to the existing ones [7] . this discovery is still controversial and little-known enough to conclude how the 5 th dengue serotype might add to the burden associated with dengue infection. mosquitoes transmit the virus by feeding on blood of infected persons. at first, the virus infects and replicates in the mid-gut epithelium of the mosquito and then spreads to other organs until it reaches the salivary glands after 10-14 days where it can be inoculated to another person during subsequent blood meal. vertical transmission of denv in mosquitoes, i.e. from mosquito to larvae has been reported by a number of research groups. in india, angel & joshi (2008) reported the detection of dengue virus by indirect fluorescence antibody test (ifat) in laboratory reared mosquitoes originating from larvae collected from urban and rural areas [8] . a similar study was conducted in brazil by martins et al. (2012) and confirmed the isolation of denv-type 3 in ae. albopictus larvae and denv-type 2 in ae. aegypti larvae [9] . similar findings were also reported in mexico [10] and indonesia [11] . on the other hand, mother-to-infant transmission of dengue virus via cord blood or breast milk remains controversial [12] [13] [14] . based on the results from several studies, the who has launched a new dengue classification. this classification divides dengue cases into a) cases with/without warning signs and b) severe dengue cases [2] . however, it is important to note that numerous research groups have debated the rational of this classification as it does not fit their unique local settings. the criteria for dengue case classification are presented in fig. 1 . clinically, dengue infection has a broad spectrum of features. the vast majority of cases are asymptomatic and passes unnoticed. typically, the symptoms start to be prominent after an incubation period of 3-10 days [15] . the severity of the clinical manifestations varies from mild symptoms to severe life threatening symptoms in the case of dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) [16] . predicting the progression of the mild signs to a severe dhf/dss remains a challenge due to non-specificity of clinical presentation and the incomplete understanding of pathophysiology of the disease and its underlying molecular mechanisms. the early signs of the disease are non-specific. according to the who classification (2009), df is characterized by febrile episode (≥40°c for 2-7 days) frequently associated with rash, nausea, vomiting, and headache. although the disease affects people of all ages from infancy through to adulthood [17] , epidemiological data showed that children tend to tolerate this phase of illness better than adults [18] . the persistence of the aforementioned symptoms and appearance of other symptoms, such as abdominal pain, mucosal bleed, and lethargy and restlessness can be seen 3-7 days later. laboratory analysis of mild dengue fever cases usually shows abnormal leukocyte counts and moderate elevation of the hepatic amino-transferase enzyme activity [19] . the emergence of these symptoms is a warning sign for disease progression to severe form (dhf/dss) if therapeutic intervention is not undertaken. at this stage clinical intervention and continuous surveillance are imperative to prevent vascular leakage, especially in an endemic area. this form of dengue infection can be attributed to any of the four known serotypes denv 1-4. the likelihood of developing dhf/dss is high in patients who have experienced dengue infection in the past with heterogeneous serotype [20] . about 5-10 % of patients progress to develop a severe dhf/dss which can be fatal unless treated promptly [21] . this form develops at a late stage of df, where patients may go through defervescence phase characterized by a sudden drop of body's temperature. this phase is also distinguished by severe bleeding, particularly bleeding from the gastrointestinal tract (black, tarry stool), and thrombocytopenia (<50,000/mm3), which may affect up to 50 % of dhf cases [22] . interestingly, there was an observed negative correlation between the severity of dhf and the level of platelets in the blood. the exact mechanism of this correlation has yet to be delineated. the drop of platelet counts and the loss of their functionality lead to a vascular fragility increasing the risk of hemorrhage and plasma leakage [23] . it has been suggested that during acute phase of the infection denv replicates quickly in platelets, as this is very critical for virus survival and dissemination [24, 25] . the existence of other symptoms such as retro-orbital pain, maculopapular rash, petechiae, or bleeding from the nose or gums will help making definitive diagnosis for df [25] . evidence of plasma leakage in various body cavities such as the pleural cavity and the peritoneal cavity, associated with profuse perspiration, adynamia, and sometimes fainting are signs of rapid progression to shock. subsidence in systolic pressure and hypotension may result in profound shock, known as dengue shock syndrome (dss). the duration of dss for a long time might predispose to further complications such as massive bleeding, disseminated intravascular coagulopathy (dic), respiratory failure, multi-organ failure, and infrequently encephalopathy leading to death [26, 27] . it has been proposed that case fatality related to dhf may reach 15 % of all cases, however, proper medical care and symptomatic management can reduce mortality rate to less than 1 % [28] . an early and accurate laboratory diagnosis of dengue infection is of paramount importance in the management of the disease. it has been estimated that the number of misdiagnosed dengue cases could reach a record ratio of 50 % of all cases, mainly due to a large disparity of dengue signs and symptoms which overlap with the symptoms of other viral infections, especially for persons living in or traveling to endemic areas of tropical infectious diseases. dengue fever should be distinguished from other illnesses which share similar symptoms such as chikungunya, mayaro fever, ross river fever, west nile fever, zika fever, yellow fever and viral hemorrhagic fevers [4] . until the antiviral vaccine becomes available, the prevention of severe cases and cut-down of the economic burden of the disease rely enormously on early and accurate diagnosis. the latter is made possible through the availability of several diagnostic laboratory and virological tests. the onset of later stage symptoms of the illness can be overwhelming and more pathognomonic. nonetheless, based on who classification schemes, the appearance of leukopenia in patients with febrile illness is a major consideration in making diagnosis of dengue infection [29] . overall, there is an urgent need to reduce dengue morbidity and mortality by improving the diagnosis and molecular analysis of emerging dengue virus. thus far, two diagnostic modalities have been applied to detect the disease at an early stage. the first one is a direct method targeting the acute phase of dengue disease, which is based upon detection of genomic rna by rt-qpcr or soluble ns1 by antigen capture in blood samples from viremic patients. the second is the indirect method that relies on serological tests to detect denguerelated immunoglobulins par mac-elisa for the capture of specific igm or indirect elisa for the capture of anti-den iggs [30] [31] [32] [33] [34] . several risk factors have been associated with dengue infection and its progression to severe dhf/dss forms. recent advances in molecular biology have revealed that the genetic makeup of the three elements of dengue infection (the virus, the vector, and the host) plays a primordial role in the pathogenesis of the disease and could potentially contribute to the dhf progression [19, 24, 35] . hence, an in-depth analysis of genetic variability including polymorphism and mutations could be beneficial in identifying the possible factors and mechanisms of disease development [36] . the list of host's genetic factors that confer susceptibility or resistance to dengue infection is summarized in table 1 . like most arboviruses, denv infect different organs of the mosquito, including the salivary glands and the central nervous system. mosquito infection elicit behavioral changes including increase of the probing time which lead to host interruption that might lead to wider spread of the virus [37] . it has been demonstrated that denv infection induced the expression of cathepsin-b, a putative cystatin, and a hypothetical ankyrin repeat-containing protein genes [38] . the latter could alter the efficiency of virus replication in the salivary gland. this study has shown that modulation of obp10 and obp22 genes expression as well as denv infection-responsive odorant-binding protein genes increase the time length for initiation of probing before a successful blood meal, resulting in changes in the host seeking behavior of the mosquito. comparative analysis of the salivary gland transcriptomes of native and denv-infected ae. aegypti identified a number of differentially expressed genes related to sugar/protein digestion enzymes, immunity related genes and blood meal acquisition enzymes that might have an impact on the efficiency of viral replication or mosquito feeding behavior. this study showed that denv infection alter the expression of key host-seeking genes in the mosquito's main olfactory organs and the antennae [38] . recent updates have indicated that resistance of ae. aegypti to conventional insecticides is related to different mechanisms, one of which is associated with genetic abnormalities within the vector's genome. single point mutation in the voltage-gated sodium channel gene at position 1534 (f1534c) resulting in phenylalanine to cysteine substitution in ae. aegypti confers resistance to permethrin. this mutation is widespread in this vector in southeast asia and latin america [39, 40] . it has also been reported that a single amino acid substitution valine to glycine at position 1016 in domain ii, segment 6 of the voltage-gated sodium channel gene was associated with less sensibility of ae. aegypti to deltamethrin in thailand [41] . numerous multi-disciplinary studies confirmed that race, young age, virus strain, female sex and high body-mass index correlate well with increased burden of dengue [42, 43] . thus, a closer consideration of human genes regulating the severity of dengue infection, especially genes associated with the immune response, might help in controlling disease spread and improve the acute symptoms of the infection. a number of studies have investigated the relationship between the host genetic polymorphisms and denv infection ( table 1) . a single nucleotide polymorphism (snp) in the promoter of cd209/dc-sign was associated to increased risk of developing dengue fever [44] . association studies have successfully identified a link between polymorphisms in the human major-histocompatibility-complex (hla) class i/ii genes and non-hla host genetic factors and severity of dengue disease [45] [46] [47] . polymorphisms of the tap1 and tap2 genes could be directly associated with the risk of developing dengue disease among the primary-infected individuals [48] . both tap1 and tap2 are located within the mhc class ii region and homozygosity of the tap1 at position 1333 and 1637 and for tap2 at position 2379, respectively, was found to protect against developing severe forms of dengue [46] . in an independent study [49] , the authors showed that single nucleotide polymorphism of the oligoadenylate synthetase genes (oas1, 2 and 3), of the oas/rnase l antiviral immune system, enhance susceptibility to clinical outcomes of dengue infection. an association between the severity of the disease and other genes including human leukocyte antigen class i and class ii genes, tumor necrosis factor-alpha, fcgriia, vitamin d receptor, transporters associated with antigen presentation, and jak1 has also been proposed [50] . the importance of vit-d in denv pathogenesis was concluded from newly-gathered data showing that vit-d impairs denv replication and polymorphism of vit-d gene increases the expression of both cd209/dc-sign and fcgriia receptors that enhance denv entry in the target cells [51, 52] . in another study [53] , the authors have successfully applied genome-wide association study (gwas) approach to identify loci that confer susceptibility to severe forms of dengue disease. the investigators used samples from 2008 children affected with severe dengue infection against 2018 population control cases in vietnam. the data showed that snps at two loci, micb and plce1, significantly increased the likelihood of developing dss in children. this finding was further validated in an independent cohort of 1737 cases and 2934 controls [53] . a snp in the micb gene coding for the mhc class i polypeptide-related sequence b, an inducible activating ligand for the nkg2d type ii receptor of immune cells could alter the protective role of natural killer and cd8 + t cells in the host responsiveness to denv at the early stage of infection [54, 55] . on the other hand, plce1 plays a primordial role in maintaining intact vascular endothelial cell barrier function, hence, polymorphism of the plce1 gene may lead to blood vessels leakage and circulatory hypovolemia during dss [56] . other host candidate genes have also been associated with early onset dengue disease. among these genes, there were receptors/attachment factors for denv linked to immune system and inflammatory response. the chemokines cxcl10, cxcl11 and its respective chemokine receptor cxcr3 were reported as biomarkers for severe form of dengue infection [57] . these results are in agreement with recent emerging data indicating strong association between cxcl10, cxcl11 and cxcr3 and vascular permeability [58] . the three genes are components of the nf-kb pathway and are involved in the pathogenesis of sars and west nile virus encephalitis [59, 60] . [61] . this process depends enormously on vector-derived salivary factors inoculated on the skin cells [62] . till-date, there is no effective, commercially available, therapy/vaccine for dengue virus. numerous groups have already made intensive efforts and made good progress to develop a safe, affordable and effective vaccine against all serotypes for global public health [63] [64] [65] [66] [67] [68] [69] . vaccines which are being developed use various approaches such as live attenuated viruses, inactivated viruses, subunit vaccines, dna vaccines, and chimeric viruses using yellow fever vaccine and attenuated dengue viruses as backbones ( table 2) . currently, only one tetravalent vaccine against dengue virus, developed by sanofi-pasteur (france) has reached phase iii clinical trial and is expected to be launched in 2015. this vaccine is based on the production of four chimeric live dengue-yellow fever viruses in which the yellow fever (yf) 17d vaccine sequences encoding the envelope proteins prm and e genes were substituted by the prm and e genes from dv of serotype 1, 2, 3, or 4 in a molecular clone of yf-17d [69] . this vaccine was produced and tested over 6000 people using four dengue virus isolates from indonesia and thailand. this candidate vaccine was found to be attenuated and stable in animal models with respect to plaque size and yellow fever virus neurotropism [70] . results of the clinical trials showed no adverse effects except moderate injection site pain, headache, and myalgia. another randomized, controlled trial was launched using a total of 4002 thai school children to investigate the efficacy of a recombinant, tetravalent vaccine for dengue virus and only 134 dengue cases were reported [71] . phase i trial of the vaccine in the philippines showed that the seropositivity increased gradually (53, 72 & 92 %) after 1-3 vaccinations against all four serotypes as compared to control group. the most promising results were observed in children 2-5 years old who exhibited high levels of reactivity of 91, 100, 96, 100 % for denv 1-4; respectively [72] . another placebo-controlled trial was conducted on 10,275 children from vietnam (vaccine, n = 6851 vs placebo, n = 3424) to determine the clinical efficacy and safety of cyd-tdv. the results demonstrated virologically-confirmed cases in 47 % of the vaccine group as compared to the control group (53 %). the efficacy was achieved in up to 56.5 % (95 % ci 43.8-66.4). these findings indicated that the vaccine is highly efficacious with good safety profile when three injections were given to children with age group 2-14 years at 0, 6 and 12 months intervals [73] . the data emerging from another randomized phase ii trial in india indicated that the vaccine has no serious adverse events and the immunogenicity and safety of cyd-tdv were satisfactory [74] . a pilot study carried out in five latin american countries where more than 20,000 children aged 9-16 were recruited to receive either the cyd-tdv vaccine or placebo. the results on efficacy (60.8 %) and safety profiles were consistent with the previous findings [74, 75] . interestingly, the vaccine efficacy (80.3 %) against hospitalization for dengue was promising and represented a step forward to developing an effective dengue vaccine [75] . other candidate dengue vaccines have been developed in usa by the johns hopkins university and national institute of allergy and infectious diseases (niaid) and have reached advanced clinical trials [65] . four liveattenuated denv/delta-30 were generated each containing 30 nucleotides deletion of the 3'-untranslated region of genomic rna (delta-30). these vaccines efficiently impaired viral growth in human liver carcinoma cells [76] . to improve the attenuation of denv-2/delta-30 and denv-3/delta-30, chimeric denv were developed by substitution of the prm-e gene region of denv-4/ delta-30 virus with the prm-e genes of denv-2 and denv-3 [72, 77] . the results from phase i clinical trial showed that all four live-attenuated denv/ delta-30 are safe and immunogenic with minor side effects such as faint rash and transient leucopenia only after higher dose [78, 79] . dengue virus serotype-1 antigen was expressed in a vector based on pediatric live-attenuated schwarz measles vaccine (mv) by using the envelope domain iii (ediii) fused with the ectodomain of the membrane protein (ectom). after immunization, long-term production of denv-1 serotype-specific neutralizing antibodies was observed in measles virus susceptible mice [80] . a new strategy was evaluated based on single minimal tetravalent denv antigen expression using viral vector derived from pediatric live-attenuated measles vaccine (mv). a recombinant mv vaccine construct was developed using envelope domain iii (ediii) and ectodomain of the membrane protein. the neutralizing antibodies were induced against all four serotypes of dengue virus after two injections in mice susceptible to mv infection. a strong memory neutralizing response was observed against all four serotypes in immunized mice after inoculation with live denv from each serotype [81] . a naked dna-based candidate vaccine against denv has been developed by the naval medical research center [67, 82, 83] . the genes encoding prm and e of denv were cloned into a shuttle vector under the transcriptional control of human cytomegalovirus (cmv) promoter. the results of phase i clinical trial showed no adverse effects except mild injection site pain, swelling, and fatigue. after second dose, strong igm and igg antibody response was observed which favors the safety profile of this vaccine. to get a better immunogenicity profile, a vaccine based on lipid adjuvant vaxfectin (vical incorporated, san diego, usa), was developed and the results demonstrated good protection profile against denv compared to dna alone [84] . based on this technology, different groups have developed other candidate vaccines and achieved good protection in mouse models using envelope glycoproteins prm and e, the non-structural protein ns1 and the helicase/protease ns3 as vaccine antigens [85] [86] [87] . the first purified inactivated vaccine was developed with aluminum hydroxide (alum) adjuvant and tested in mice and rhesus macaques in the mid-1990s, by walter reed army institute of research against dengue 2 serotype and good virus protection was reported after two doses [88, 89] . using similar technology, second generation japanese encephalitis (je) piv vaccine was developed [90, 91] . currently, a new je vaccine (ixiaro; novartis vaccines) has been approved for use in many countries, including the usa [92] . another dengue vaccine (dengue 1 piv), recombinant subunit dengue e glycoprotein antigen (r80e) was also developed and has entered phase i clinical trial [93] [94] [95] . the centers for disease control and prevention (usa) have also developed a live-attenuated vaccine named denvax, which was found to be highly immunogenic in both children and adults and has currently entered phase i clinical trial in the united states [96, 97] . recently, a novel third generation approach is being used to develop a vaccine containing recombinant subunit e domain iii (ed3) and the results of laboratory tests have shown the development of potent neutralizing antibodies in a mouse model [98] [99] [100] . using the same technology, a tetravalent vaccine was developed and expressed in pichia pastoris by splicing and using flexible pentaglycyl linkers of the four ediii. the observed results showed that this antigen elicit specific antibodies against all four denv serotypes in balb/c mice [101] . animal models are very useful for vaccine test development. the lack of animal models significantly hampered the development and efficacy testing of dengue vaccine. currently only rhesus macaques and aotus monkeys are being used for testing the vaccine before clinical trials are initiated [62] . the d1me100 vaccine was evaluated in both aotus monkeys and rhesus monkeys, and found to be immunogenic with 80-95 % protection against dengue infection [102, 103] . porter et al. (2012) demonstrated that injection of non-human primate with three doses on day 1, 28 and 84, with tetravalent dengue dna vaccine vaxfectin-adjuvanted, was more efficient against live dengue-2 virus compared to control animals. this finding support initiation of vaxfectin-adjuvanted phase i clinical trial [84] . successful induction of immune response was obtained in mice and rhesus monkeys to the vaccines developed using dengue 4 prm-e, dengue 1 prm-e-nonstructural (ns)1, and dengue 2 ns3 antigens, and piv adjuvanted with alum [85, 86] . centers for disease control and prevention (fort collins, co), hawaii biotech, and simmons developed different vaccines that showed good immunogenicity in animal models [104] . similarly, the psoralen/uv inactivation dengue vaccine was found to be more immunogenic and protective against dengue serotype 1 virus in aotus monkeys [105] . thus far, there are no antiviral drugs available to treat dengue fever; therefore the community will continue to depend on the control of the mosquito vector as the main route to prevent the spread of disease. alternative approaches have been utilized against flaviviruses by targeting and inhibiting virus entry and the essential elements used in virus replication, nonstructural proteins, rna polymerase, and proteases. the most important target elements include ns3 helicase nucleoside triphosphatase (ntpase/ rna 5' triphosphatase (rtpase), ns5 methyl transferase/ rna-dependent rna polymerase, and ns3/ns2b protease [106] [107] [108] . rna interference (rnai) technology is also being used to impair virus replication against respiratory syncytial virus, hepatitis viruses, influenza virus, poliovirus and hiv [109, 110] . low molecular weight phenolic compounds such as flavonoids and phytochemicals isolated from plants were previously tested and are being used for anti-dengue therapy [111, 112] . an anti-viral inhibitory effect ranging from 50-75 % against denv replication was observed when methanolic extracts of momordica charantia and andrographis paniculata were used in cultured primate cells [113] . several attempts have been made in the past to tackle dengue through elimination of ae. aegypti. the most successful experiences were related to vector control programs adopted in cuba and singapore. the programs were based on intensive insecticidal treatment and reduction of the availability of aedes larval habitats [18, 114] . unfortunately, lack of sustainability of these stringent measures led to reappearance of dengue outbreaks. recently, a novel form of biological control of dengue transmission has been developed and is currently being applied. this is based on the development of genetically modified (gm) mosquitoes infected with a bacterium known as wolbachia to combat dengue infection. this bacterium blocks replication of the virus inside the mosquito and prevents its transmission to humans [115] . in 2012, 10 million gm male mosquitoes were released in the wild to decrease the number of aedes mosquitoes and reduce the rate of dengue transmission. a closer monitoring of the insects revealed that over 85 % of the eggs were wolbachia-positive which indicated that gmmosquitoes were overriding wild-mosquitoes resulting in decreased virus transmission [116] . in an initiative to eradicate dengue fever, scientists from australia, are leading eliminate dengue (ed) program which involves community engagement as a key component in this program. since the program kicked off in 2011, millions of wolbachia mosquitoes were released across the north queensland city-australia. based on the promising results obtained from local trial, eliminate dengue became an international research program across countries affected by dengue including australia, vietnam, indonesia, brazil and colombia [117, 118] . dengue infection can be prevented by alternative approaches. the first one includes blocking virus entry into cells which is mediated by the viral envelope glycoprotein e via receptor-mediated endocytosis [119] . dendritic cells, monocytes, and macrophages are the main targets of denv infectious entry. the second approach involves blocking virus attachment to specific cellular receptors expressed on immune cells, liver cells, and endothelial cells. small molecules and peptides targeting the hydrophobic pocket of the envelope e glycoprotein are characterized as inhibitors of virus entry. nicholson et al. (2011) explored the inhibitory effects of dn59 and 1oan1, peptide entry inhibitors. the authors demonstrated that dn59 and 1oan1 can effectively block antibody dependent enhancement (ade) in-vitro suggesting that entry inhibitors are potential candidates to prevent development of dhf/ dss [120] . two other compounds have also been shown to qualify as potent inhibitors of dengue virus infection are imino-sugars deoxynojirimycin and castanospermine [121] . these compounds are natural alkaloids derived from the black bean and act as inhibitors against all 4 dengue serotypes by disrupting the folding pathways of the envelope glycoproteins prm and e [122] . various types of carbohydrate-binding agents, isolated from different organisms, have been shown to have antiviral activities. three plant lectins, hippeastrum hybrid agglutinin, galanthus nivalis agglutinin and urtica dioica agglutinin isolated from amaryllis, snowdrop and stinging nettle respectively were found to be potent inhibitors of denv-2 infection by inhibiting viral replication [123] . heparan sulfate (hs) is a putative receptor for denv which interacts with domain iii of the e-protein. virus entry can be blocked by targeting the e-protein-hs interaction with soluble gags and other highly charged hs [124] . fucoidan was isolated from marine algae and showed antiviral activity against denv-2 in bhk cells [125] . similarly, carrageenan and dl galactan, sulfated polysaccharides from red seaweeds, exhibited strong antiviral activity against denv-2 and denv-3 but a very weak activity against denv-4 and denv-1. furthermore, two α-d-glucans were isolated from a chinese herb and demonstrated high anti-denv-2 activities in bhk cells [112, 126] . dengue fever represents a real economic burden especially in affected countries. extensive efforts are needed to tackle disease spread and reduce the mortality rates and the associated healthcare cost. there is a need for more scientific research which we believe is a key route to provide further insight in the pathogenesis of dengue infection and help understanding the underlying molecular mechanisms associated with progression to the severe forms of the disease (dhf/dss). this will be a step forward to develop an adequate preventive vaccine and effective treatment. the authors disclose that there is no conflict of interest. authors' contributions tn participated in the review design, coordination and helped to draft the manuscript. sk and ss participated in the review design and helped to draft the manuscript. pd participated in the article revision. gd and ea participated in the review design. read and approved the final manuscript. this project is funded by the king abdulaziz city for science and technology (kacst) under grant number ( ‫ﺍ‬ ‫ﺕ‬ -33 -26 ). the authors are also grateful to the diagnosis of dengue: an update guidelines for diagnosis, treatment prevention and control the global distribution and burden of dengue dengue viral infections 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anti-dengue virus activity of sulfated polysaccharide from a marine alga structure elucidation and sulfated derivatives preparation of two α-dglucans from gastrodia elata bl. and their anti-dengue virus bioactivities submit your next manuscript to biomed central and we will help you at every step: key: cord-320454-dhfl92et authors: srivastava, s.; shetty, n. title: healthcare-associated infections in neonatal units: lessons from contrasting worlds date: 2007-03-12 journal: j hosp infect doi: 10.1016/j.jhin.2007.01.014 sha: doc_id: 320454 cord_uid: dhfl92et neonatal intensive care units are vulnerable to outbreaks and sporadic incidents of healthcare-associated infections (hais). the incidence and outcome of these infections are determined by the degree of immaturity of the neonatal immune system, invasive procedures involved, the aetiological agent and its antimicrobial susceptibility pattern and, above all, infection control policies practised by the unit. it is important to raise awareness of infection control practices in resource-limited settings, since overdependence upon antimicrobial agents and co-existing lack of awareness of infection control is encouraging the emergence of multi-drug-resistant nosocomial pathogens. we reviewed 125 articles regarding hais from both advanced and resource-limited neonatal units in order to study risk factors, aetiological agents, antimicrobial susceptibility patterns and reported successes in infection control interventions. the articles include surveillance studies, outbreaks and sporadic incidents. gram-positive cocci, viruses and fungi predominate in reports from the advanced units, while gram-negative enteric rods, non-fermenters and fungi are commonly reported from resource-limited settings. antimicrobial susceptibility patterns from surveillance studies determined the empirical therapy used in each neonatal unit. most outbreaks, irrespective of the technical facilities available, were traced to specific lack of infection control practices. we discuss infection control interventions, with special emphasis on their applicability in resource-limited settings. cost-effective measures for implementing these interventions, with particular reference to the recognition of the role of the microbiologist, the infection control team and antibiotic policies are presented. summary neonatal intensive care units are vulnerable to outbreaks and sporadic incidents of healthcare-associated infections (hais). the incidence and outcome of these infections are determined by the degree of immaturity of the neonatal immune system, invasive procedures involved, the aetiological agent and its antimicrobial susceptibility pattern and, above all, infection control policies practised by the unit. it is important to raise awareness of infection control practices in resource-limited settings, since overdependence upon antimicrobial agents and co-existing lack of awareness of infection control is encouraging the emergence of multi-drug-resistant nosocomial pathogens. we reviewed 125 articles regarding hais from both advanced and resource-limited neonatal units in order to study risk factors, aetiological agents, antimicrobial susceptibility patterns and reported successes in infection control interventions. the articles include surveillance studies, outbreaks and sporadic incidents. gram-positive cocci, viruses and fungi predominate in reports from the advanced units, while gram-negative enteric rods, non-fermenters and fungi are commonly reported from resource-limited settings. antimicrobial susceptibility patterns from surveillance studies determined the empirical therapy used in each neonatal unit. most outbreaks, irrespective of the technical facilities available, were traced to specific lack of infection control practices. we discuss infection control interventions, with special emphasis on their applicability in resource-limited settings. cost-effective measures for implementing these interventions, with particular reference to the recognition of the role of the microbiologist, the infection control team and antibiotic policies are presented. ª 2007 the hospital infection society. published by elsevier ltd. all rights reserved. the neonatal intensive care unit is an ideal situation to incorporate good infection control policy and practice, since it lends itself not only to the spread of severe infections but also to successful interventions. a collaborative effort between neonatologists and clinical microbiologists who take on the role of infection control can successfully mount a defence against healthcare-associated infections (hais) . clinical liaison between microbiologist and clinician is well established in developed countries, whereas in the developing countries such practices are yet to be widely recognized. one reason could be that microbiology results are often delayed in less technologically advanced laboratories, thus forcing the clinician to make empirical treatment decisions without consulting or depending on the microbiologist. however, technical advancement is not a prerequisite for appropriate selection of empirical antimicrobial agents, infection control practices or formulating antibiotic policies. in an environment where resources are scarce, it only requires determination and professional cooperation for suitable interventions to work. this review on healthcare-associated neonatal infections studies the definitions, associated risk factors and the aetiological agents involved with their antimicrobial susceptibility patterns in two contrasting worlds. we discuss the microbiological and infection control intervention strategies that might help, even in resource-limited settings, to prevent the morbidity and mortality associated with hai. levels of neonatal care may be classified as shown in box 1. 1 a large proportion of neonates in developing countries (63%) and in rural india (83%) are born at home, with poor facilities for safe and clean delivery by unskilled 'dais' or village health workers. 2, 3 even larger hospitals with a high delivery rate do not have access to level ii neonatal care. 3 no sick newborn care unit (scnu), government or private, is available at district level in many provinces. the equipment and infrastructure are often limited and doctors are forced to select which babies will be admitted and offered facilities such as ventilators. 4 few state-owned centres are equipped with neonatal intensive care units (nicus) and these are scattered across the country. thus, in developing countries we are dealing with neonates with completely different demographic characteristics. whereas the minimum gestational age of live-born babies managed in a nicu in developed countries is 25 weeks with birthweights as low as 300 g, the average gestational age of live-born babies in developing countries is !30 weeks with birthweights !1000 g. in a study on anthropometry and body composition of south indian babies at birth, the mean ae standard deviation (sd) birthweight of all newborns was 2.80 ae 0.44 kg. 5 financial constraints in developing countries limit the use of technical interventions, due to which very few neonates undergo invasive medical or surgical procedures, unlike reports from developed countries. the available microbiological diagnostic facilities also vary from centre to centre. semiautomated and automated culture systems are available only in a handful of tertiary care centres. the cost of providing these services to patients is borne by the family and is often prohibitive; most clinicians treat patients empirically. microbiological results are particularly important in neonates as signs of sepsis are often non-specific. hence, while financial constraints are difficult to resolve, we still have the option to utilize cost-effective, alternative interventions such as infection control, which by reducing the incidence of infection will decrease the overall morbidity and mortality in sick neonates. successful field trials for home-based neonatal care have already been reported. 2 we need to extend these achievements to healthcare settings. we searched for articles on the pubmed database, using the index terms 'hospital acquired infection', 'neonate', 'nosocomial infection neonate', 'neonatal care level', 'neonatal care india'. reference lists of all articles retrieved were searched to obtain literature for the review. the articles were scrutinized to obtain a comparable and standard definition of nosocomial infection in neonates, inclusion and exclusion criteria used, aetiological agents involved, antimicrobial susceptibility patterns and infection control interventions. within these search results, we reviewed articles mentioning infection control and antibiotic policies, with special reference to neonatal units in developing countries. full text articles were scrutinized for a majority of english language papers; for a small number of articles we relied only on the published abstract. for foreign language studies we were able to quote only from the abstract published in english. the foetus is exposed to a sterile environment in utero, provided no invasive procedures have been carried out on the mother, the membranes are intact until the onset of labour and there is no prolonged rupture of membranes. during the process of delivery, the neonate is exposed to several sources of microbes. these include the maternal genital tract followed by ambient air or water depending on the type of delivery, handling by healthcare personnel and the instruments used at resuscitation. rotimi and duerden studied the development of bacterial flora of neonates during the first week of life. 6 the predominant organisms in the gut, by the end of the first week, were anaerobes. bifidobacteria were isolated from all the neonates. bacteroides and clostridia were isolated from 78.3%. enterococci were isolated from all neonates, enterobacteria from 82.6%, anaerobic cocci from 52.2%. staphylococcus aureus was the predominant species isolated from the umbilicus; it was isolated from 21.7% of neonates on the first day rising to 87% by the sixth day and represented 49% of isolates from this site. viridans streptococci (31.4% of isolates) were the commonest species recovered from the mouth. they were present from 8 h after birth. the authors also studied the development of microbial flora of preterm neonates. 7 the numbers of infants studied were too small to draw any firm conclusions; their flora predominantly reflected the maternal genital tract. in contrast, preterm and full-term babies born by caesarean section were slow to acquire colonizing flora as compared to those born vaginally. the skin of infants born by caesarean section is sterile soon after birth compared to neonates born by vaginal delivery. 8 bowel colonization of infants born by caesarean delivery is also delayed. 9 colonization with bifidobacterium-like bacteria and lactobacillus-like bacteria reached levels similar to vaginally delivered infants at 1 month and 10 days, respectively. many hais result directly or indirectly from patient colonization; studies have shown that hospitalized patients are colonized rapidly with hospital flora. 10 colonizing flora such as candida albicans in the gastrointestinal tract, vagina or perineal area, can precede infection when normal body defences are impaired through underlying disease, immunomodulating therapy, the use of invasive devices, or when the delicate balance of the normal flora is altered through antimicrobial therapy. however, antimicrobial therapy to eradicate colonizing micro-organisms such as pseudomonas aeruginosa is not beneficial and can propagate drug-resistant pathogens. 11 immune status of the neonate a newborn infant, particularly the preterm infant and to some extent the low birthweight infant, does not have a mature immune system and is often unable to mount an effective immune response. 12 natural barriers, such as the acidity of the stomach or the production of pepsin and trypsin that maintain sterility of the small intestine, are not fully developed until 3e4 weeks after birth. membrane protective iga is missing from the respiratory and urinary tracts, and unless the newborn is breast-fed, is absent from the gastrointestinal tract as well. on a cellular level, there is decreased ability of leukocytes to concentrate where necessary. these leukocytes are less bactericidal and phagocytic. at the humoral level, the newborn has low or non-existent levels of the immunoglobulin antibodies igm, ige and iga. the neonate is born with igg antibodies acquired from the mother. however, it is important to note that passive transfer of maternal antibodies does not take place till 29 weeks of gestation. this has implications for preterm infants born 25e29 weeks of gestation; they are susceptible to infection despite the mother's antibody status. there is a slow rise of immunoglobulin levels after 3 months of age to levels of older children. before embarking on a review of nosocomial infections, we reviewed the definitions of nosocomial infections used in various studies. at the outset, the national nosocomial infections surveillance system (nnis) of the centers for disease control and prevention (cdc), usa defines nosocomial infection as a localized or systemic condition (1) that results from adverse reaction to the presence of an infectious agent(s) or its toxin(s) and (2) that was not present or incubating at the time of admission to the hospital. 13 limitations encountered during the review process in the neonate, the definition of an hai is complicated by the fact that neonates can acquire infection from the maternal genital tract during birth. for this reason, neonatal infections are often classified as early onset (usually 0e7 days after birth) and late onset (>7 days after birth). 14 some authors also classify them as 72 h after birth and >72 h after birth. 12 it is interesting to note that the cdc includes infections acquired from the maternal genital tract in their surveillance of nosocomial neonatal infection. several investigators have found these criteria unsatisfactory. for the purposes of this review, we have considered only those studies that have excluded infections acquired directly from the maternal genital tract; we found that the definition of nosocomial infections varied between studies and were often related to time of acquisition. the dutch group have modified the cdc criteria to include infections occurring in the neonatal unit 24e48 h after admission. 15 in some studies, infections which manifested after the patient was in the hospital for !48 h and those infections which developed within a period of 7 days after discharge from the hospital were considered nosocomial. 16e21 in other studies, all neonates residing for !3 days in a hospital unit were included. 22e25 nosocomial transmission of candida in neonates was considered if the neonate showed negative surveillance cultures at birth and positive cultures from one week later, until death or discharge. 26 shankar et al. also recommend using surveillance cultures to differentiate endogenous colonization from nosocomial acquisition. 21 we believe that a consistent and universally accepted case definition of hai in the neonate is important because it offers uniformity of data across centres and facilitates a standardized measurement of outcomes. many studies that we reviewed did not have clear-cut case definitions with clearly stated inclusion and exclusion criteria; when they did, they varied from centre to centre. other common limitations were inadequate sample size, 17,24,26e31 or variability of denominator data wherein some authors reported number of infections per 100 patients (attack rate) 32 or the number of infections per 1000 patient-days (incidence density). 16, 32 annual incidence per 100 000 live births and per 100 nicu discharges have also been used. 24 absence of robust statistical analysis and the inclusion of anecdotal case reports were also limitations. 28,33e37 some authors acknowledge the lack of technical equipment to report viral, fungal and parasitic causes of hais. 38 neonates present with their own unique risk factors that predispose them to acquisition of hai. the vulnerability of the neonate, particularly the preterm neonate, is directly linked to an immature immune system. this is the single most important host-related factor that predisposes them to infection. neonatal age itself is a risk factor for hai [odds ratio (or) 5.89; 95% confidence interval (ci): 2.96e 11.58; p < 0.05]. 18 in another study, admission to the neonatal unit, rather than age at admission, was associated with increased risk of hai (p < 0.001). 39 the overall nosocomial infection rate was positively correlated with average length of stay in high-risk nurseries (r ¼ 0.6, p < 0.05). 40 preterm gestational age (<32 weeks) was a risk factor in 26e60% neonates with bacterial, viral and fungal hai. 19,41e46 the percentage of neonates with low birthweight (1.5e2.5 kg) and with very low birthweight (1.0e1.5 kg) who acquired hai was 55.5 and 28.2 to 29.6% respectively. 47, 48 infection, including hai, was the most common cause of death in extremely low birthweight (<1.0 kg) neonates and septicaemia (bacterial and fungal) was the most common presentation (68.4%). 25, 49 male sex was a predisposing factor for nosocomial infections (p < 0.05). 16, 50 the male predominance in neonatal sepsis has suggested the possibility of an x-linked factor in host susceptibility. 12, 51 underlying medical conditions such as chronic lung disease, gastro-oesophageal reflux, history of neonatal respiratory distress, maternal infection and congenital heart disease predisposed to hais in 4.3e26.1% of neonates. 52e55 a high complexity score, which categorizes procedures by severity of illness and technical complexity, was associated with increased incidence of hai in neonates after cardiac surgery (or: 4.03; 95% ci: 1.87e8.43; p < 0.05). 18 prism (pediatric risk of mortality) score of >25 was also related to neonatal hai (crude or: 8.90; 95% ci: 3.49e22.76; p < 0.001). 56 the clinical risk index for babies (crib) score shows that nosocomial bacteraemia is independently associated with low birthweight and preterm neonates. 19 lack of maternal antibodies was a risk factor for infection with unusual rotavirus strains. 57 factors relating to healthcare personnel, practices and the environment are often overlooked, and yet remain the most obvious and inexpensive area of intervention. indeed, the most common route of spread of nosocomial pathogens is personto-person transmission within the unit and during transfer of patients between units. such incidents have been linked with outbreaks of bacterial and viral infection in the nicu. 55,58e60 the most common iatrogenic factor contributing to neonatal hais is hands of healthcare workers. 22,53,58,61e65 intervention in the form of simple handwashing procedures and infection control practices has prevented outbreaks, as reported in many studies. 22,42,55,58e60,63,66 during the process of delivery, the neonate is exposed to several sources of microbes. medical devices such as umbilical catheters, central venous catheters, urinary catheters and endotracheal tubes are commonly used in the nicu. 23, 28, 53, 54, 67, 68 central venous catheters contributed to 48.9% of hais in one study 69 and was a significant risk factor (p < 0.05) in others. 20, 21, 23, 39 the nosocomial infection rate was higher in neonates subjected to device use (r ¼ 0.26, p < 0.02). 40 about 10.8% of catheterized patients developed hospital-acquired urinary tract infection (uti). 70 the duration of ventilation was also related to the acquisition of hai. 71 reuse of single-use items, a common though unsound practice in many units, has led to outbreaks of hai. endotracheal tubes and mucous extraction suction catheters soaked in hibitane were associated with hai in the labour room and the special care baby unit. 47 baby placement services, resuscitation equipment and cleansing solutions have also been implicated in hai. 72 an environmental risk factor often overlooked is related to the seasonal variation in the incidence of neonatal hai. factors such as warm climate have been associated with a rise in colonization rates with enterobacter spp. 73 increased humidity and increased environmental dew point at the time of use of nursery air conditioners propagates airborne dissemination of acinetobacter spp. and has been associated with acinetobacter-related bloodstream infections. 67 bacteria in ambient air have been reported to colonize the conjunctiva in neonates. 65 agent factors contributing to hai relate to the aetiological agents implicated in infection. infection with drug-resistant organisms plays a significant role in the outcome of hai in all patients, irrespective of their gestational age and underlying condition. hospitalization leads to colonization of the skin and gastrointestinal tract with resistant flora found in hospitals and subsequent bloodstream infection, when the skin or mucosa is abraded. studies have reported that administration of prophylactic antibiotics to neonates can increase the incidence of hai with drug-resistant pathogenic micro-organisms. about 64.8e100% of neonates presenting with hai had received prior broadspectrum antibiotics. 16, 30, 54 clinical presentation of hais in neonates a summary of the most commonly reported neonatal hai is described in table i . 15, 16, 18, 21, 25, 31, 39, 40, 44, 61, 74, 75 a review of the findings of these studies is hampered by the variation and sometimes lack of denominator data. the reader is advised to study these reports with caution, taking into consideration the limitations mentioned earlier. healthcare-associated infections in the neonatal unit cover the entire spectrum of organisms: bacterial, fungal, viral and rarely parasitic. a review of healthcare-associated bacterial (table ii) , 15,17,18,21, 23,25,30,33,40,61,76e79 fungal (table iii) 21,25,26,32,46, 80e84 and viral (table iv) 42,43,52,57,60,63,85e94 infections is summarized in the relevant tables. fortunately parasitic nosocomial infections are rare. there have been isolated reports of babesiosis transmitted by blood transfusion in neonates. 37 among four neonates transfused with blood from asymptomatic babesia-infected donors, two (50%) became parasitaemic, of whom only one developed symptoms of babesiosis. it is interesting to note that gram-negative fermenters (e. coli, klebsiella spp.) and nonfermenting gram-negative rods such as acinetobacter spp. and pseudomonas spp. have established themselves as predominant causes of serious neonatal infections in the indian subcontinent (table ii) . in contrast, the predominant organisms isolated from invasive neonatal infections in technologically advanced countries are gram-positive cocci (coagulase-negative staphylococci, group b streptococcus). 95 the reason for this difference is probably multifactorial and could be due to gestational age of the babies involved, the use of invasive devices (central vascular catheters and shunts), ambient moisture, humidity and the prevalent flora in the unit. evidence supporting these risk factors has been discussed elsewhere in the review and probably merits further evaluation. of all the fungal infections reported in neonatal patients, candida spp. cause significant mortality and morbidity in the neonatal unit (table iii) and will be discussed in some detail here. although the source of c. albicans infection in the nicu is often considered to be endogenous, molecular typing studies have shown that nosocomial transmission of c. albicans is the predominant mode of acquisition. 26, 96 the nosocomial acquisition of c. albicans is related to cross-contamination via the hands of healthcare workers or parents and the use of contaminated equipment. 26, 97 in one study, retrograde medication syringe fluids were significantly more likely to be contaminated with candida than other fluids being administered to the infants (p < 0.001). candidaemia was significantly associated with total parenteral nutrition (p ¼ 0.04) and retrograde medication administration (p ¼ 0.02). 98 central vascular catheters, steroid administration, endotracheal intubation and h2-blockers have also been reported as risk factors for systemic fungal infections in neonates. 66, 82 other risk factors include prematurity, low birthweight and use of broad-spectrum antibiotics. 36 complications of candidaemia such as endocarditis and uveitis have been reported in neonates. the onset of endocarditis was related to persistant candidaemia. fungal endocarditis was present in 13.7% neonates with persistent disease (>5 days of candidaemia) and 3.7% patients with non-persistent disease (or: 4.19), while uveitis developed in 3.4% patients. mortality in neonates with persistent disease was comparable to the mortality in neonates with non-persistent disease. 81 viruses account for about 1% of infections in hospitalized neonates. 85 the most common viral infections are due to enterovirus/parechovirus (table iv) . enteroviruses were responsible for the highest mortality and development of serious sequelae. 85 respiratory syncytial virus (rsv) is the second most common virus causing infections in hospitalized neonates (14e37%). 42,52,60,85e89 respiratory viruses were diagnosed in 29.5% of neonates on mechanical ventilators; the most frequent was rsv (14.1%), followed by influenza a virus (10.2%). 88 in another study, nosocomially acquired rsv infection was present in 37% of neonates, 54.3% had an underlying condition predisposing to severe disease and 13% died. 52 human parainfluenza type 3 is the most common cause of bronchiolitis and pneumonia after respiratory syncytial virus. parainfluenza type 3 virus was isolated in six of 17 neonates cultured (five symptomatic patients and one asymptomatic patient). eighteen of 52 nursing personnel had been ill during the previous week, concomitantly with cough and nasal congestion. 93 nosocomial transmission of rotavirus in neonates has been reported. 43, 57, 90 the onset of acute diarrhoea due to rotavirus in two neonates was followed by five neonates developing gastroenteritis with the same strain of rotavirus. 90 in another study, in 51% of inpatients with nosocomial gastroenteritis, the causative agent was rotavirus and 26% of those were premature neonates. 43 hais and resistance to antimicrobial agents compared to community-acquired infections, hais are often caused by multi-drug-resistant pathogens. in this section we concentrate on reports from the subcontinent and other resource-poor settings. in a retrospective study of bacterial isolates from cases of neonatal septicaemia over a period of 5 years, there was a significant rise in the incidence of drugresistant acinetobacter spp. and p. aeruginosa. 99 the incidence rate of acinetobacter septicaemia in another study was 11.1/1000 live births. 100 other studies have also documented acinetobacter spp. as emerging neonatal pathogens. 23, 33, 101 susceptibility tests showed that acinetobacter isolates were resistant to two or more antibiotics, most notably to ampicillin (82.5%), cephalexin (69.6%), gentamicin (66.5%) and cefotaxime (47.8%). most isolates were susceptible to amikacin (82.6%), ciprofloxacin (73.9%) and piperacillin (69.6%). 33 only about 30% of bacterial aetiological agents of neonatal hai would be covered by an empirical regimen of ampicillin and gentamicin. 102 gramnegative organisms causing hai in neonates were cause of candidaemia, endophthalmitis, endocarditis, meningitis, peritonitis. source of infection was central venous catheter. 82 rhodotorula mucilaginosa 83 outbreak (n ¼ 4) of indwelling catheter-related septicaemia in nicu. related to birthweight, gestational age, duration of parenteral nutrition, antibiotic therapy and prophylactic fluconazole. rhizopus microsporus 84 outbreak of cutaneous infection in preterm neonates (n ¼ 4) . source traced to wooden tongue depressors used in the nursery as splints for intravenous and arterial cannulation site. the combination of warm, humid conditions in neonatal incubators, particularly in association with occlusive dressings, also favours cutaneous fungal infections. nicu, neonatal intensive care unit. a non-albicans candida spp. included c. parapsilosis, c. tropicalis, c. lusitaniae, c. glabrata, c. krusei, c. guillermondii. less susceptible to the commonly used antibiotics, such as ampicillin (20.7%), amoxicillin (25.4%), gentamicin (56.8%), ceftazidime (28.4%) and cefotaxime (44.8%). these organisms were more susceptible to imipinem (76.4%), amikacin (77.7%), ofloxacin and ciprofloxacin (88.1%). 14,103 other workers found third-generation cephalosporins and aminoglycosides such as netilmicin to be effective in the treatment of neonatal sepsis. 104 at the same time, studies have also shown that administration of antimicrobial prophylaxis, presumed to prevent hais, can be a putative risk factor in itself for hai. 16, 54, 78 single-centre studies have shown that probiotics containing anaerobic bacteria may reduce the rate and severity of necrotizing enterocolitis. 105 antifungal agents fluconazole has been recommended as prophylaxis against systemic fungal infections in preterm low birthweight infants. 106, 107 however, other workers have found no resurgence of fungal infection after cessation of prophylactic fluconazole use. 108 there is also concern about emergence of resistance to fluconazole. in an investigation into the resurgence of bloodstream infections due to c. parapsilosis in one unit, after the institution of fluconazole prophylaxis, primary resistance to fluconazole was not detected. 109 others propose a twice weekly dosing of prophylactic fluconazole to decrease candida colonization, invasive infection, cost and patient exposure in high-risk preterm infants weighing <1000 g at birth; the lower and less frequent dosing may even delay or prevent the emergence of antifungal resistance. 110 there are reports of c. albicans resistance to fluconazole (12.5%) and amphotericin-b (25%) in studies from india. 80 newer antifungal agents, including voriconazole and caspofungin, show promise in the treatment of potentially fatal fungal infections in neonates and additional controlled studies are indicated to evaluate their role. 111 the existing evidence base for infection control practices specifically for the neonatal unit is described in table v . 22,29,39,48,55,58e60,63,72,86,87,93,112e118 important lessons in infection control can be learnt from published accounts of specific outbreaks. in addition to the outbreaks documented in tables iii and iv , we have selected other outbreak reports that we believe reinforce the infection control message (table vi) . 30,31,58,59,113,114,119e124 environmental surveillance is not routinely recommended since pathogens present in the inanimate nicu environment, e.g. floors, walls, sink-drains or furniture are not associated with 42,52,60,85e89 nosocomially acquired infection among 2/4 neonates with rsv. infection control measures successful. 86 rotavirus 43, 57, 85, 87, 90, 91 nosocomial transmission of rotavirus in 5/9 neonates with diarrhoea. 90 winter peak. high morbidity. among patients with nosocomial rotavirus diarrhoea, 26% were preterm neonates. 43 cytomegalovirus (cmv) 85, 92 transfusion-acquired cmv in 2/21 neonates. 92 adenovirus 85 gastroenteritis was the main clinical presentation in preterm infants. parainfluenza, type 3 85, 93 outbreak in nicu (n ¼ 6). linked to hcw. controlled by glove, gown and cohorting. 93 herpes simplex virus, rhinovirus, rubellavirus 85 infections reported in the nicu. influenza a virus 88 neonates on mechanical ventilation were nosocomially infected with influenza a virus. human coronaviruses 63 patient-to-staff and staff-to-patient transmission in nicu. universal precaution with surface disinfection and handwashing prevent spread of infection. echovirus type 7 coxsackie b3 94 nosocomial outbreak (n ¼ 6) in special care nursery. transmission by staff. nicu, neonatal intensive care unit; hcw, healthcare worker. hais. only three nicu sites, namely baby placements, resuscitation equipment and various cleansing solutions, were found to be significantly associated with hais (p < 0.001) in one study. 72 the relative risk of infection was greatest if baby placement sites were colonized (odds ratio ¼ 7.48; p < 0.01). this reinforces the need for scrupulous cleaning regimens rather than adopting a policy of routine environmental surveillance. however, environmental cultures may play a role in specific outbreak situations. outbreak strains of salmonella worthington were isolated from the baby warmer mattress, baby cot, suction machine bottle and wall of the refrigerator. 114 the role of surveillance cultures to predict the onset of nosocomial infections in neonates undergoing invasive procedures, such as exchange transfusion, has been studied. 29 the authors found that except for staphylococci, the flora from umbilical stump and umbilical vein blood in asymptomatic neonates was similar to the flora from infected neonates. 'intensive care' need not be synonymous with 'invasive care'. 3 in the presence of constraints such as lack of trained staff, intermittent power supply or lack of disinfection between their use, incubators and other medical devices can be a risk factor for hai. in these situations kangaroo care provided by the mother has emerged as a cost-effective and widely accepted style of caring for an infant in hospital. in a study from india, there was significant improvement among the kangaroo care group compared with the conventional group, in terms of hypothermia (10/44 vs 21/45, p < 0.01), higher oxygen saturations (95.7 vs 94.8%, p < 0.01) and decrease in respiratory rates (36.2 vs 40.7, p < 0.01). however, there was no statistically significant difference in the incidence of hyperthermia, sepsis, apnoea, onset of breastfeeding and hospital stay in the two groups. 125 further studies are needed to evaluate the role of kangaroo care and the incidence of hai in neonates. the role of microbiology in the detection, epidemiological analyses and prevention of hais cannot be overemphasized, whether the unit is one that benefits from being resource rich or resource poor. in a setting where most physicians are reluctant to use first-line agents, due to misleading or lack of sufficient susceptibility data, a qualified microbiologist is indispensable. communication between microbiologist and neonatologist helps in deciding the most probable pathogen and in initiating the most appropriate antimicrobial therapy. the formulation of a mutually agreed antibiotic policy at community, institutional and national levels is imperative. an infection control team (ict) comprising an infection control nurse or an infection control trained link nurse in the nicu, a neonatologist/ physician and a microbiologist must actively participate in outbreak management and infection control policy issues. in turn, it is mandatory that microbiologists balance their focus equally on diagnostic as well as clinical microbiology. microbiological influence and involvement can be enhanced if the microbiologist joins regular clinical ward rounds and helps to raise awareness among healthcare professionals regarding all aspects of infectious disease management. education and training is an important remit of the ict. besides training of healthcare staff we believe it is important to provide training to empower the mother. as the main carer in the family her education is vital; if she can be made aware of the rationale behind the microbiologist or neonatologist's advice, she will be in a stronger position to participate in the wellbeing of herself and the baby. even as huge efforts are underway to halt the misuse of antimicrobial agents, issues regarding antimicrobial resistance in pathogens are less important to the lay public. as long as these essential drugs are available over-the-counter in many countries, all efforts in any other part of the world toward preventing their misuse will be undermined. in addition, a number of privately funded laboratories have sprung up in several cities and towns in developing countries. they lack quality assurance and the personnel who work in identifying pathogens and reporting susceptibility are not trained adequately in quality control methods. in resource-limited settings, as in technologically advanced units, advising that we wash our hands and use the most appropriate antimicrobial agent may be more valuable than suggesting expensive tools for molecular testing. we provide a simple, resource-efficient template for the instigation and maintenance of infection control in the clinical setting (box 2). in the present era of global information sharing, professionals working in the area of infection control need not feel isolated. there are several useful web tools that provide practical information and guidance; our own outbreak investigation klebsiella spp. outbreak of septic arthritis (n ¼ 17) linked to contaminated cover sheets. 31 epidemiological evidence of an association between acquiring p. aeruginosa bloodstream infection in neonates and exposure to nurses with long and artificial fingernails. short natural fingernails is a policy that is essential to reduce the incidence of hai in neonates. 113 an outbreak of invasive s. marcescens in the nicu (n ¼ 14) . 119 molecular tests showed that a vast majority of clinical and environmental isolates (from hands of nurse, handwashes and disinfectants) belonged to the same clonal type. cohorting of non-infected neonates, isolation of colonized and infected neonates, glove use and handwashing controlled the outbreak. outbreak (n ¼ 9) of s. marcescens in the nicu. 58 epidemic strain isolated from handwashes and doors of incubators. strict handwashing, disinfection of incubators, cohorting and isolating patients controlled further transmission. acinetobacter spp. during an outbreak, isolates with similar antibiogram were recovered from intravenous catheter and washbasin. 30, 120 neonatal cross-infection due to contaminated equipment resulted in sepsis and central nervous system disease. 121 outbreak of seven cases, six fatalities. equipment and environment were the source of outbreak. outbreak was controlled through cleaning and fumigation. 114 transmission among nursery staff. 59 enterotoxigenic e. coli (etec) outbreak involved preterm neonates (n ¼ 16); surveillance cultures of swabs from the utensils used to prepare milk feed, culture of the formula feed and all items handled by one particular cook were undertaken. the cook's hand swabs and faecal sample yielded growth of etec. the outbreak was controlled by appropriate therapy and institution of proper measures of hygiene. 122 enterobacter spp. outbreak (n ¼ 30 and n ¼10) of enterobacter cloacae septicaemia traced to preceding bladder catheterization and/or parenteral nutrition solution, respectively. 123, 124 policy is available free of charge at www.infectioncontrolservices.co.uk/. 1. ensure a strict protocol for hygienic handwashing and provision of clinical handwash basins or sinks 2. involve the microbiologist in the planning stages or when refurbishing the unit; advice on physical setting of the unit and general layout of cots, bays, sinks will impact on infection control 3. provision of side rooms and bays for the isolation of infected babies or protection of healthy neonates 4. provide training and advice regarding environmental cleaning; ensuring that all surfaces are maintained clean and dry 5. create an infection control policy document and a rational antibiotic policy that is constantly reviewed 6. appoint an infection control team (ict) comprising a microbiologist, neonatologist, infection control nurse/liaison nurse trained in infection control 7. support the ict in the management of infectious diseases and in promoting infection control practices 8. provide education and training of unit staff in infection control 9. take the lead in outbreak 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disease transmission? outbreak of salmonella worthington meningitis & septicaemia in a hospital at chandigarh (north india) listeriosis e a review of eighty-four cases the bacterial flora of neonates in intensive care-monitoring and manipulation computerized detection of nosocomial infections in newborns neonatal sepsis: the antibiotic crisis molecular epidemiology of an outbreak of serratia marcescens in a neonatal intensive care unit outbreak of acinetobacter spp septicemia in a neonatal icu neonatal crossinfection with listeria monocytogenes nosocomial outbreak of diarrhoea by enterotoxigenic escherichia coli among preterm neonates in a tertiary care hospital in india: pitfalls in healthcare risk factors for enterobacter septicemia in a neonatal unit: caseecontrol study enterobacter cloacae sepsis outbreak in a newborn unit caused by contaminated total parenteral nutrition solution feasibility of kangaroo mother care in mumbai key: cord-347460-9vechh4x authors: chang, feng-yee; chen, hsiang-cheng; chen, pei-jer; ho, mei-shang; hsieh, shie-liang; lin, jung-chung; liu, fu-tong; sytwu, huey-kang title: immunologic aspects of characteristics, diagnosis, and treatment of coronavirus disease 2019 (covid-19) date: 2020-06-04 journal: j biomed sci doi: 10.1186/s12929-020-00663-w sha: doc_id: 347460 cord_uid: 9vechh4x on march 11, 2020, the world health organization declared the worldwide spread of the infectious disease covid-19, caused by a new strain of coronavirus, sars-cov-2, as a pandemic. like in all other infectious diseases, the host immune system plays a key role in our defense against sars-cov-2 infection. however, viruses are able to evade the immune attack and proliferate and, in susceptible individuals, cause severe inflammatory response known as cytokine storm, particularly in the lungs. the advancement in our understanding of the mechanisms underlying the host immune responses promises to facilitate the development of approaches for prevention or treatment of diseases. components of immune system, such as antibodies, can also be used to develop sensitive and specific diagnostic methods as well as novel therapeutic agents. in this review, we summarize our knowledge about how the host mounts immune responses to infection by sars-cov-2. we also describe the diagnostic methods being used for covid-19 identification and summarize the current status of various therapeutic strategies, including vaccination, being considered for treatment of the disease. on december 31, 2019, a cluster of cases of pneumonia was announced in wuhan, hubei province, china. subsequently, on january 7, 2020, the chinese health authorities confirmed that this cluster was associated with a novel coronavirus, ncov, which was later named as sars-cov-2, and the ensuing disease was named covid-19. the covid-19 outbreak by the new coronavirus strain was recognized as a pandemic by the world health organization (who) on march 11, 2020. throughout history, there have been a number of pandemic diseases; the more notable and recent ones caused by viruses include the influenza pandemic (spanish flu) in 1918 and another by the influenza virus h1n1 in 2009. the immune system clearly plays a key role in the host defense against the infectious agents during these pandemics. the host is able to mount immune responses upon infection by viruses, as well as other microbes, and control the spread of these pathogens within the body. however, some viral strains are capable of evading the immune attack and proliferate in the body, as well as elicit inflammatory responses, in particular in the lungs, resulting in pneumonia. more importantly, in susceptible individuals, viruses can cause massive inflammatory responses, known as "cytokine storm", resulting in a severe pathological consequence. the advancement in our understanding of the mechanisms of the host immune response are crucial to development of approaches for prevention and treatment of these fast spreading and devastating infectious diseases. the components derived from our immune systems, such as antibodies, can be used to develop sensitive and specific methods for the diagnosis of infectious diseases, as well as novel therapeutic modalities. in this review, we briefly summarize our knowledge about the host immune response upon infection by sars-cov-2. we also discuss the epidemiological aspects of the outbreak, and the potential mechanism of the severe host response, such as cytokine storm. we also describe the antibody-based approaches for diagnosis of covid-19 infection and summarize the current status of various preventive and therapeutic modalities for treatment of the infection. coronaviruses are single-stranded enveloped rna viruses that cause diseases in mammals and birds. in humans, the low pathogenicity strains, including hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku, infect the upper respiratory tract and cause mild to moderate common cold-like symptoms in healthy individuals. they are responsible for 15-30% of all common cold cases. the highly pathogenic strains, including those causing severe acute respiratory syndrome [sars-cov] , middle east respiratory syndrome [mers-cov] , and covid-19 [new sars-cov-2], infect the lower respiratory tract and can cause severe pneumonia [1] . in addition to their rna genetic material, coronaviruses are composed of nucleocapsid (n) and spike (s) proteins, which participate in viral genome assembly, transcription and replication, or mediate viral entry and cause cytopathic effect [2, 3] . the s protein mediates the fusion of viral and host membrane [4] and contains a receptor-binding domain (rbd) that attaches to cells during viral entry. angiotensin-converting enzyme 2 (ace-2) is the receptor for both sars-cov and sars-cov-2 [5] . notably, the four human coronaviruses that cause common cold like symptoms show limited sequence homology in their n (30-67%) and s proteins (9-57%) compared with those of sars-cov-2 [6] . mers-cov also exhibits more distal relationship to sars-cov and sars-cov-2. however, the latter two are more closely related, with their n and s proteins sharing high homology (70-90%) . in 2003, the sars-cov infection, which started in southern china, led to an epidemic; in total, over 8400 cases were reported, which included close to 900 deaths with a case fatality rate of 11% [7] . in 2012, the first case of mers took place in saudi arabia. from that moment on, close to 2500 cases have been reported globally, which included close to 860 deaths, with an estimated case fatality rate of approximately 34% [8] . evidence is mounting that covid-19 spreads via human-to-human transmission of the virus [9] . after exposure to sars-cov-2, the majority of patients recover with little or mild symptoms that include cough and fever [10] . however, it is estimated that approximately 20% of infected individuals develop severe disease, including acute respiratory distress syndrome (ards). according to who, as of april 22, 2020, a total of 2,503, 072 confirmed cases of covid-19 have been detected and 171,791 deaths resulting from the infection have been confirmed worldwide. the case fatality rates in wuhan and worldwide were approximately 4.5 and 6.9%, respectively. the numbers are lower in some countries, for example approximately 5.4% in the united states, 2.2% in south korea, 3.3% in germany, and 1.4% in taiwan. these numbers are likely affected by the extent of screening, thereby implying that covid-19 cases might be underdiagnosed in many other countries. the availability and infrastructure of medical facilities in afflicted countries, especially those seriously affected ones, also likely affect the overall death rates. using public and published information, wu et al. estimated that the overall "symptomatic case fatality risk" (the probability of dying after developing symptoms) associated with covid-19 was 1.4% [11] . the rate of development of severe symptoms and death are clearly associated with the age. it is to be noted this is based on all tested and confirmed cases of covid19, and the true fatality risk is likely lower than 1.4%, since many mildly symptomatic/asymptomatic people might have never got tested. nevertheless, the risk is still higher than that associated with seasonal influenza virus, which is approximately 0.1%. with regard to the spectrum of the severity of the disease, chinese center for disease control and prevention reported of the 44,672 confirmed cases (with the age distribution of > 80 years: 3%; 30-79 years: 87%; 20-29 years: 8%; 10-19 years: 1%; < 10 years: 1%), the spectrum of disease were: mild: 81%; severe: 14%, critical: 5%; and case fatality rate: 2.3% [12] . finally, the basic reproduction number (ro) of the virus has been estimated to be between 1.4 and 3.9, meaning each infection from the virus can result in 1.4 to 3.9 new infections, when no members of the community are immune and no preventative measures, such as vaccination, are taken. by comparison, the median ro value for sars-cov was in the range of 2 to 4 and for 1918 influenza was 1.80. the outcome of clinical infection likely largely depends on the capacity in mounting effective antiviral immune responses in time, to control viral spreading, to limit organ injuries and to speed up recovery. here we summarize the immune response induced by cov. three components are crucial for sars-cov induced diseases: 1) the role of cd8+ t cells in defense against the virus, which causes apoptosis in the infected cells, 2) interactions of the virus with macrophages and dendritic cells, which initiate the early innate and subsequent adaptive immune responses, and 3) type i interferon (ifn) system, an innate response against viral infections, which can inhibit virus replication in the early phase. firstly, the central part of the body's anti-viral immunity is based on the interaction between antigen and antigen presentation cells (apc) when the virus enters the cells. the infected cells are recognized by virus-specific cytotoxic t lymphocytes (ctls) via viral peptides as the antigen presented by major histocompatibility complex (mhc). the antigen presentation of virus mostly depends on mhc i molecules, but mhc ii also has its contribution in some cases. the mhc i molecules display pieces of virus proteins on the surface of infected cells, which creates a signal to activate nearby cd8+ t cells to induce apoptosis in the infected cells. there are many reports on the relationship between various mhc polymorphisms and the susceptibility to sars-cov [13] [14] [15] , but little is known about this association in covid-19. such information could provide beneficial aspects of personalized medicine for treatment or prevention of covid-19. secondly, dendritic cells and macrophages are other first patrolling components of innate immune network, which play important roles in driving both innate and adaptive immune responses to the viral pathogens [16] . the invasion of viruses can be recognized by innate immune cells via pathogen-associated molecular patterns (pamps). in the case of cov, pamps are viral genomic rna, which are recognized by endosomal rna receptors such as tlr3, tr7, tr8, and tlr7 [17] . this can cause rapid responses of the innate immune cells to viruses, resulting in production of a large amount of type i ifn with antiviral functions. lastly, efficient innate immune responses against viruses also depend on type i ifn responses and downstream cascade. type i ifn, by directly interfering with the viruses' replication ability, can prevent reproduction of viruses in infected cells. by mounting type i ifn responses successfully, viral replication and dissemination in an early stage are suppressed. sars-cov and mers-cov use several strategies to avoid the innate immune response, these are probably also employed by sars-cov-2. these include the inhibition of type i ifn recognition and signaling, as well as downregulation of mhc class i and class ii molecules in infected macrophages or dendritic cells, resulting in impaired antigen presentation and diminished t cell activation. moreover, some proteins encoded by sars-cov can interact with the signaling cascades downstream of the pattern recognition receptors. after exposure to sars-cov-2, patients respond to the virus by generating specific igm antibodies within a few days, followed by specific igg production within a week [3, 6, 18] . in the case of sars-cov infection, although the serum anti-viral igm antibody levels decline in a few months, the antiviral igg antibody titers can persist for years. among the many structural and non-structural proteins encoded by sars-cov-2, the n and s proteins are the most immunogenic antigens. antibodies against the n protein are the first to appear and thus can serve as an early and reliable serum marker for virus exposure, whereas antibodies against the s protein develop later and can bind to the viral envelope. recent studies indicated that the convalescent serum contains antibodies that can neutralize sars-cov-2 in cell cultures [2, 3] . therefore, igg against the s protein is both a marker for viral exposure and an indicator of recovery. the potential risk of disease exacerbation by ade, a phenomenon in which pre-existing poorly neutralizing antibodies lead to enhanced infection, has been a serious concern for vaccine development and antibody-based therapeutic strategy. compared to the ade in dengue viral infections, which was supported by a great deal of epidemiological and clinical evidence in the past four decades [19] , this phenomenon in coronaviruses has mainly been observed in cell-based experimental models [20, 21] . to illustrate this further by also using dengue virus as an example: while more severe symptoms, such as dengue hemorrhagic fever (dhf)/dengue shock syndrome (dss), can be observed during primary infection, they are much more frequently developed following a secondary infection with a different serotype (out of the four existing serotypes) [19] . furthermore, it has been well documented that the high level of virus replication seen during secondary infection with a heterotypic virus is a direct consequence of ade of viral replication. this is mediated primarily by the pre-existing, non-neutralizing, or sub-neutralizing antibodies to the virion surface antigens, resulting in enhanced access to target cells, through binding of the virion-antibody complexes to igg fc receptors (fcγr) on these cells [19] . this common underlying theme of ade-based magnification of virus replication also indicates that severe disease is not merely attributable to inherent virulence of virus [22] . interestingly, as described in a later section, several available evidence from the use of convalescent sera in patients with sars, mers [23] and 245 cases with covid-19 [24] suggest the feasibility and safety of convalescent serum trials. here, caution and vigilance to identify any evidence of enhanced coronavirus infection by ade will be required. despite the fact epidemiological and clinical observations supportive of existence of ade in coronavirus infection is not available, a molecular mechanism behind ade of coronavirus has currently been provided [21] . the authors demonstrated that a neutralizing antibody binds to the s protein of coronaviruses like a viral receptor, triggering a conformational change of the spike and mediating a viral entry into fcγr -expressing cells through canonical viralreceptor-dependent pathways. however, an enhanced entry of these pseudovirus-based approaches does not support directly the magnification of viral replication in these cells. compared to dengue viruses, these fcγrbearing cells such as dendritic cells, monocytes and macrophages, even if infected through ade process, would presumably not be so "permissive" for coronaviruses, in terms of their replication and assembly. however, these viewpoints need further investigation. apparently, many host factors could also exacerbate disease during secondary infection. these host factors need to be identified by combined epidemiological and genetic analyses of appropriate patients, and the contribution of underlying host factors to the control of coronavirus replication needs to be determined. for example, ade of replication can possibly occur with the vaccine strains of viruses in the endemic populations, such as attenuated or recombinant coronavirus vaccines. however, the level of replication will likely remain low, and thus this small enhancement of replication will probably not augment the disease. in addition, this could result in heightened vaccine immunogenicity due to a small increase in the virus load. however, further investigation on correlations between immunological responses and disease outcome and the validation of these findings in vaccine trials will be invaluable for developing safe and effective sars-cov2 vaccines (see below). while sars-cov can evade innate immune system, they can also induce intensive inflammatory reactions through innate immune cells. in fact, sars-cov and sars-cov-2 infections are known to activate a massive over-production of cytokines by the host immune systema phenomenon known as "cytokine storm", which usually occurs a few days after the onset of the illness. this also results in increased local and systemic vascular permeability in major organs. cytokine storm is reported in many viral infections, and contributes significantly to the pathogenesis and severity of acute viral infections. the tissue tropism of each virus determines the cytokine profiles of virus-induced cytokine storm (reviewed in [25] ). for example, macrophages produce a higher amount of proinflammatory cytokines than endothelial cells, while virus-infected endothelial cells are the major source of chemokines. however, this may not be generalizable, and it is crucial to elucidate the tropism of sars-cov-2 in order to interpret the data. most proinflammatory cytokines are released from macrophages and severe acute infections are usually associated with the activation of macrophages by enveloped viruses. in addition, activation of neutrophils may also be involved. as mentioned above, viral nucleic acids can induce the production of interferons and proinflammatory cytokines, through engaging endosomal tlrs. these intracellular nucleic acid receptors/sensors have been defined as "protective host factors", as they are critical for host defense against viral infections. however, the identity and contribution of "pathogenic host factors" to virus-induced severe inflammatory reactions and lethality, and how different viruses cause distinct clinical symptoms, remain unclear. some available information related to the cytokine storm induced by sars-cov and mers-cov is summarized below. it is to be noted that while both sars-cov and sars-cov-2 utilize ace-2 as their receptors, mers-cov binds to the receptor dipeptidyl peptidase 4 (dpp4/cd26). the differences in receptor usage may account for the differences in disease patterns, including the organs involved and the extent of the cytokine storm induced. [26] [27] [28] . the clinical course of this infection has three phases: 1) robust virus replication accompanied by fever in the first few days; 2) high fever and pneumonia with progressive decline of virus titers; 3) ards resulting from active host immune responses in the absence of detectable viruses [1] . in addition to infecting and proliferating in the airways and alveolar epithelial cells, sars-cov can also infect dendritic cells, monocytes, and macrophages, without undergoing proliferation (i.e., abortive infection) [29] . sars-cov-infected epithelial cells produce high levels of chemokines such as ccl2, ccl3, ccl5, and cxcl10. in addition, sars-cov-infected dendritic cells [30, 31] and macrophages [29] secrete high levels of proinflammatory cytokines tnf and il-6, and significant amounts of chemokines. it is interesting to note that higher levels of il-1, il-12, ifn-gamma, il-8, and cxcl9, in addition to the cytokines and chemokines mentioned above, were also observed in sars patients with severe diseases. this suggests that other cell types also contribute to sars-cov-induced cytokine storm. the typical pathological changes in the lungs include focal hemorrhage and mucopurulent materials in bronchial trees with diffuse alveolar damage. histological examination shows extensive macrophage and neutrophil infiltration with lower levels of t lymphocytes. existing information suggests that the sars-cov-infected airways and alveolar epithelial cells secrete abundant chemokines to attract immune cell infiltrations to the lungs, including macrophages and neutrophils, thereby causing damage due to high levels of proinflammatory cytokines and other mediators secreted by these cell types. in addition to the airway epithelial cells, mers-cov can also replicate in human monocytes, macrophages, dendritic cells, and activated t cells. the typical lung pathological changes caused by mers-cov is diffuse alveolar damage. in addition, pleural and pericardial effusions associated with generalized congestion and consolidation of lungs have been noted [32] , and the severity of lung lesions were noted to be correlated with extensive infiltration of neutrophils and macrophages [32] . similar to sars-cov, mers-cov can induce high levels of proinflammatory cytokines and chemokines in human monocyte-derived macrophages and dendritic cells. mers-cov infection was also reported to induce increased concentrations of proinflammatory cytokines (ifn-γ, tnf-α, il15, and il17), [33] . the high serum cytokine and chemokine levels in mers patients were correlated with increased infiltration of neutrophil and monocytes along with severe tissue damage in the lungs [32, 34, 35] . thus, the pathological change in the lungs is similar between sars-cov and mers-cov. whereas, the higher mortality rate in mers-cov-infected patients may be due to the higher incidence of pericarditis in infected patients. the first autopsy of covid-19 victims along with immuno-histological staining revealed the presence of sars-cov-2 in the airway epithelia and macrophages, suggesting that the virus can infect both epithelial cells and macrophages [36] . the majority of infiltrating cells are macrophages and monocytes with moderate amounts of multinucleated giant cells and neutrophils. increased levels of cytokines and chemokines, including il-2, il-7, g-csf, m-csf, ifn-γ, ip-10, mcp-1, mip-1α, and tnf-α, were detected in the plasma of covid-19 patients [37] . the most significant predictors of mortality in these patients are serum ferritin level and il-6, suggesting that mortality is due to virus-induced hyperinflammation and cytokine storm during viral infection [38, 39] . compared to the low pathogenic coronaviruses, the common features of high pathogenic coronaviruses include extensive infiltration of leukocytes, which secrete abundant proinflammatory cytokines and other chemical mediators to cause diffuse alveolar damage. also, high pathogenic viruses are associated with abortive infection; for example, in contrast to the less pathogenic strain of influenza virus h1n1, the highly pathogenic influenza virus h5n1 does not replicate in macrophages; the latter is also a more potent inducer of the chemokine cxcl10 [40] the key innate immunity receptors/sensors responsible for high pathogenic coronavirus-induced proinflammatory cytokines are still unclear. finally, notably, more deaths from covid-19 have been caused by multiple organ dysfunction syndrome rather than respiratory failure, which is different from infections caused by sars-cov and mers-cov; the basis for this remains unknown. detection of viral rna in the secretions from the respiratory tract of infected patients by reverse transcription-polymerase chain reaction (rt-pcr) test is currently the standard method for diagnosis of covid-19. they have some limitations, including long turnaround time (typically 2-4 h) and the requirement of specialized facilities. scientists around the world have been devoting effort to developing improved nucleic acid-based, simpler and faster methods. the us fda issued an emergency-use authorization to cepheid's xpert xpress sars-cov-2 test, which became the first pointof-care covid-19 diagnostic test to receive this designation in the us. the test is designed to use the company's automated genexpert systems and has a turnaround time of approximately 45 min. another prominent example is a method for detection of sars-cov-2 by using crispr technologies [41] . these newly developed platforms will clearly require clinical testing before approval for routine use. tests based on antibodies are obviously another option for diagnosis and screening. immediately after infections, viral genome and proteins start to increase, becoming the earliest markers for diagnosis within days. as the host immune responses gear up to confront and reduce viral replications, the viral rna or antigen level declines, but the antiviral igm and igg titers rise up. as patients usually present themselves with cough, fever or shortness of breath, and are already beyond the early stage of infection [10] ; here, nucleic acid tests are expected to pick up only a proportion of patients. a complementary serological test for specific igm or igg will help identify the rest. one report from shenzhen studied 173 patients within 7 days of illness, who were later diagnosed with covid-19 [42] ; in this study rt-pcr could detect two-thirds of the patients, but only 45% tested positive even after 15 days. in contrast, although the serological positive rate within 1 week was less than 40%, the rate increased to 100% after day 15 of disease onset. a combination of nucleic acid and serological test significantly increased the diagnosis rate from 66 to 78% even within 1 week of illness [42] . if these results can be validated, such a combination may become a standard clinical practice in the future. in this regard, li et al., developed an immunoassay that can detect igm and igg antibodies against sars-cov-2 in human blood within 15 min. they tested samples from close to 400 confirmed patients and over 100 negatively-tested patients at 8 different clinical sites and reported a sensitivity of over 88% and specificity of over 90% [43] . in the early stage (containment) of covid-19 pandemic, there was a strong interest for rapid diagnosis and thus prototypes of rapid viral antigen or antibody tests were being developed for point-of-care use. these platforms have the advantage of convenience and a fast turnaround time, but suffer from inadequate sensitivity and specificity, as compared with standard rt-pcr. hence, their results need to be interpreted with caution. preparation of high-quality antibodies and antigens requires years in perfecting such point-of-care tests, judging from the experience in developing such tests for influenza viruses. eventually, the more stringent criteria for a definitive diagnosis will need paired serum samples to demonstrate a true seroconversion [6] . finally, the issue of antibody cross-reactivity with other human coronaviruses warrants discussion. as mentioned above, serological assays usually adopt viral n or s protein as antigens and the protein components from all four human coronaviruses that cause common cold show very limited sequence homology with those of sars-cov-2. thus, despite the fact the majority of the populations have been exposed to the four low pathogenic human coronaviruses, their sera do not react positively in sars-cov-2 elisa [6, 18] . while mers-cov is also more distally related and presents no concerns, the situation for sars-cov-exposed patients is different. as the n and s proteins of the sars-cov strain share high homology (70-90%) with those of sars-cov-2, serum from sars patients can actually cross-react with sars-cov-2 n or s protein in immunoblot or viral neutralization assays [2, 3] . however, as sars-cov epidemic was only transient with a very small proportion of the populations being exposed, this cross-reactivity should not be an important issue. as with many vaccine-preventable viral diseases like measles and chicken pox, the newly emerged sars-cov-2 infection assumes an epidemiological characteristic capable of evading containment measures and facilitating pandemic potential. thus, a high proportion of undetected infections with mild or no symptoms can efficiently sustain viral transmission [44] . while containment and lockdown can serve as temporary control measures, effective vaccines or therapeutic agents are much needed for the ultimate control of the disease. the vaccine research and development thus far have progressed at an unprecedented speed; the first dose of rna-based sars-cov-2 vaccine was administered to test its safety in humans on march 16, 2020, only 2 months after the new virus was first identified. such rapid progress was facilitated by a combination of multiple factors, including advances in vaccine research on sars and mers, as recently reviewed [45] , progress in a number of vaccine technology platforms to the early stage of human trial [46] [47] [48] , and readily available support from the well-orchestrated international collective effort of the coalition for epidemic preparedness innovations (cepi) [49] . the aforementioned innovative aspects that could potentially drive sars-cov-2 vaccine development to market launch within a year or two are summarized below. the coronavirus s protein is a critical target for antiviral neutralizing antibodies and functions to mediate entry into mammalian cell expressing the viral receptor ace2 [3, 50] . moreover, the target neutralizing epitope of sars-cov was further narrowed to a smaller fragment of the s protein, later termed receptor binding domain (rbd) [51] . building on these paradigmatic scientific advances that the s protein is the putative target antigen, sars-cov-2 vaccine candidates are designed to include full or various lengths of the s protein focusing on the rbd. vaccine based on the whole virus may be less preferred due to its association with eosinophilic pulmonary pathology [52, 53] . a number of novel vaccine platforms, including vector-, dna-, and rna-based vaccines, are being developed or improved with innovative technology specifically to combat pandemic-prone outbreaks and have been recently reviewed [54] . nucleic acid vaccines, including both dna and rna, offer the potential to accurately express any protein antigen in host cells and to present the antigen closely resembling antigen expression and presentation during viral infection. dna vaccines against mers and rna vaccine against h7n9 have completed phase i trials that showed these platforms to be safe [46] [47] [48] . the nucleic acid vaccine can be completely synthetic and formulated within a few weeks at sufficient quantities to support clinical trialsa valuable feature when facing potential pandemic. vector-based vaccine consists of a target antigen inserted into a viral genome to render faithful antigen generation, targeting and processing in vivo after vaccination. the first ebola vaccine approved by us fda is a vector-based vaccine using vesicular stomatitis virus expressing a surface glycoprotein of ebola [55] , thus supporting the applicability of this platform in combating emerging infectious diseases. these platforms offer versatile adaptation for antigen of new diseases, and the process development for production is relatively simple and quick, indicating the value of these platforms for the urgent response to new diseases. the rapid infusion of funding from and coordination by cepi in january 2020 was the major driver for the speed of sars-cov-2 vaccine r&d progress (fig. 1) . with its mission being "to stimulate, finance, and coordinate the development of vaccines for epidemic diseases" especially aiming to drive vaccine innovation for highpriority public health threats, cepi has supported a number of "technology platforms". these included a vaccine printer, molecular clamp technology for protein production, and a self-amplifying rna vaccine platform since 2017 (cepi web page https://cepi.net/research_ dev/technology/). an innovated vaccine platform technology pertains to a system that uses the same basic core technological components as a backbone and can be adapted by inserting new genetic or protein sequences to target newly emerging pathogens. it is with the application of this concept that an rna-based sars-cov-2 vaccine, built on the avian flu vaccine platform [46] , is expected to be developed and quickly proceed to human trial within only a few months since covid-19 became an epidemic, and a dna-based vaccine candidate modeling that of mers is soon to follow [46] . the cepi coordinated effort encompassing a wide range of available technologies from industry and research institutes globally has shown preliminary success toward an urgent response to control a pandemic. while coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines also present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology [53, 56] ; this seems to be either exacerbated or eliminated by the formulation of adjuvant selection depending on the th1/th2 bias and induction of durable ifn-γ responses, respectively [57] . in addition, ade, described above, was seen in the lungs of macaques after administration of inactivated sars-cov vaccine or vaccine composed of certain s antigen fragments [58] . these studies highlight the importance of designing the target antigen and selection of adjuvants to ensure both efficacy and safety. considering the novel nature of sars-cov-2 and that an animal model has yet to be established for testing of the vaccine to especially focus on the immunopathological perspectives, the safety concern is anticipated to present most of the hurdles in the development process. herd immunity refers to a state when sufficient proportion of a population becomes immune to sars-cov-2, via natural infection or vaccination, so as to eventually halt further spread of disease, and thus individuals not immune to the virus are protected. in the case of covid-19, the herd immunity was estimated to be 60% of the population. before vaccines are available for mass immunization, the strategy of achieving herd immunity via natural infection has been considered and deemed not advisable when a number of factors were considered. thus, self-isolation and social distancing remain crucial in combating this pandemic so that the initial pressure on our healthcare systems is reduced, and more time is given to us to develop vaccines or effective therapies. the disease spectrum of covid-19 can be divided into mild infection, pneumonia, ards, and even multiple organ failure [37] . after a decade of research on coronavirus, unfortunately, still there are no licensed vaccines, effective specific antivirals, nor drug combinations supported by high-level evidence to treat the infection, especially for newly emerging strains such as sars-cov-2 [59] . several strategies are being considered for the treatment of covid-19, including the use of antimicrobial agents, immunotherapy with virus-specific antibodies in convalescent plasma, monoclonal and polyclonal antibodies produced in vitro or genetically modified antibodies, and interferons. here we focus on immunebased therapies, but for the sake of completeness, we also include therapies using antimicrobial agents as 7supplementary information. the potential interventions for sars-cov infection are summarized in table 1 . biologic drugs composed of monoclonal antibodies (mabs) have been developed for treatment of a variety of diseases. it is thus not surprising that this approach is being considered for the treatment of sars-cov infection and shows promise. a human igg1 mab, cr3022, that binds to sars-cov s protein has been developed [70] . sui et al. found one recombinant human mab (single-chain variable region fragment, scfv, 80r) against the s1 domain of s protein of sars-cov from two nonimmune human antibody libraries. the mab could efficiently neutralize sars-cov and inhibit syncytia formation between cells expressing s protein and those expressing the sars-cov receptor ace2 [71] . a human igg1 mab, cr3014, has been generated and found to be able to neutralize sars-cov and shown to be able to prevent sars-cov infection in ferrets [72] . more recently, ju et al. reported the isolation and characterization of 206 rbd-specific mabs derived from single b cells of eight sars-cov-2 infected individuals [79] . for clones from one patient they demonstrated their ability to neutralize live sars-cov-2. none of these antibodies cross-reacted with rbds from either sars-cov or mers-cov, although the patient plasma exhibited such cross-reactivity. these neutralizing antibodies have the potential to be used for prophylaxis for or treatment of sars-cov-2 infection. agents that directly block the binding of s protein to the functional receptor ace2 also have the potential to be used for prevention of covid-19. guillon et al. demonstrated that binding of sars-cov s protein to ace2 could be inhibited by anti-histo-blood group antibodies, presumably because the virus carries histo-blood group antigen structures of the host [4] . while whether this approach can be developed into effective treatment strategies is uncertain, the findings have a bearing on the effect of the naturally occurring anti-histo-blood group antibodies on the individual variations in susceptibility to sars-cov infection. convalescent plasma can be employed for passive immunotherapy and is usually chosen when there are no specific vaccines or drugs available for emerging infection-related diseases. yeh et al. reported a favorable outcome in the use of convalescent plasma for treatment of sars-cov-infected healthcare workers [73] . arabi et al. tested the feasibility of convalescent plasma therapy as well as its safety and clinical efficacy in critically ill patients suffering from mers-cov infection [74, 80] . if available, convalescent plasma could certainly be considered for the treatment of sars-cov-2-infected critically ill patients. interferons (ifns), including ifn-α and ifn-β, are produced during the innate immune response to virus infection and they are able to inhibit the replication of virus in vitro [75, 81] . as mentioned above, ifn transcription was blocked in tissue cells infected with sars-cov. recombinant ifn-α given on 3 days before the infection could reduce viral replication and lung damage, as compared with the control in monkeys and in a pilot clinical trial [82] . ifn-α inhalation can also be considered. combination of interferon-α-2a with ribavirin was used in treatment of patients with severe mers-cov infection and the survival of these patients was improved [76, 83] . these findings suggest that these fda-approved ifn's could be used for the treatment of covid-19. as mentioned above, cytokine storm is the major underlying pathology in severe cases of covid-19. thus, neutralization of some of the major cytokines are considered as a novel approach for treatment of severely ill cases and reducing morbidity and mortality. huang c et al. reported that increased il-1ß, ifn-γ, ip-10, and mcp-1 in sars-cov-2 infection and higher concentrations of g-csf, ip-10, mcp-1, mip-1a, and tnf-α were found in patients requiring treatment at icu than those not treated at icu [37] . they also noted that cytokine storm was associated with disease severity. conti p et al. reported that pro-inflammatory cytokines of interleukin (il)-1β and il-6 in mild and acute respiratory syndrome are associated with development of lung fibrosis in covid-19 [77] . thus, suppression of proinflammatory il-1 family members and il-6 might have a potential therapeutic effect. il-37, an immunosuppressive cytokine, acts on mtor and increases the activity of adenosine monophosphate kinase, which inhibits protease inhibitors nelfinavir cell a selective post-translational inhibitor [65] nucleotide analog prodrug remdesivir cell and clinical use (first case of covid-19 in the united states) possible inhibitor of rna replication [66, 67] indole-derivative molecule arbidol cell inhibits fusion between viral envelope and cellular membranes [68] immunosuppressive agent cyclosporine a cell block replication via inhibition of nucleocapsid protein [69] monoclonal antibody cr3022 cell and clinical use potently binds the receptor binding domain of s protein [70] monoclonal antibody single-chain variable region fragments, scfv, 80r cell acts against the s1 domain of s protein [71] monoclonal antibody cr3014 cell neutralization of viral infectivity [72] immunotherapeutic potential convalescent plasma cell and clinical use neutralization of viral infectivity [73, 74] interferons ifn-α and ifn-β cell induction of interferon-stimulated genes to suppress viral replication [75, 76] cytokine blocker cytokine il-37 cell inhibits inflammation, by acting on mtor and increasing the activity of adenosine monophosphate kinase [77] cytokine blocker lianhuaqingwen cell anti-inflammation; inhibits il-6 receptor [78] cytokine blocker antibody against il-6 receptor clinical use anti-inflammation; inhibits il-6 receptor inflammation by suppressing production of multiple cytokines downstream of myd88, including il-1β, il-6, tnf and ccl2. il-37 might be a potential therapeutic cytokine for inhibition of inflammation in covid-19 [77] . runfeng l et al. demonstrated that lianhuaqingwen, a traditional chinese medicine, significantly inhibited sars-cov-2 replication by suppressing mrna of il-6 and other pro-inflammatory cytokines in vero e6 cells [78] . cytokine blocker that target interleukin 6 receptor in covid-19 could be potentially developed as therapeutic agents in future. in fact, fda approved mab against il-6 receptor (il-6r) is available for treatment of rheumatoid arthritis. the society for immunotherapy of cancer has issued a statement on access to il-6-targeting therapies for covid-19. it is encouraging that pharmaceutical companies have in fact initiated clinical trials of anti-il-6r for treatment of patients with severe covid-19. there are still a large number of unanswered questions. how fast sars-cov-2 would mutate and would the mutated virus become more infectious or invasive. according to andersen et al. [84] , viruses constantly mutate, but those mutations do not typically make the virus more virulent or cause more serious disease. in fact, most mutations are detrimental to the virus or have no effect. there was a study of the sars-cov in primate cells suggesting that a mutation in this viral strain acquired during the 2003 sars outbreak probably reduced virulence of the virus [85] . another issue is whether sars-cov-2, unlike sars-cov and mers-cov will continue to cause epidemic or even behave like seasonal flu. in fact, we have already witnessed the second wave of outbreak occurring in north america and europe, after the first wave that occurred in asia, and covid-19 may bounce back-and-forth between north and south hemispheres, as influenza virus does each year. finally, factors that determine the individual susceptibility to covid-19 remain to be elucidated. as mentioned above, there are many reports on the relationship between various mhc polymorphisms and the susceptibility to sars-cov. also, what governs the development of severe illness, including cytokine storm, besides the pre-existence of certain diseases and age factor, awaits clarification. despite these uncertainties, scientists in academia and industry around the world have moved at an unprecedented speed to develop improved methods for detection of the virus and treatment of the disease. advancement in immunology over the years has certainly facilitated many of these developments. we shall witness some of the recent advancements in development of vaccines and biologics for treatment of various other serious illnesses being used for fighting against covid-19. we are hopeful these efforts will be sustained even after the pandemic is over, allowing us to be even more ready in the unfortunate event that another epidemic or pandemic, like covid-19, takes place in the future. supplementary information accompanies this paper at https://doi.org/10. 1186/s12929-020-00663-w. additional file 1. supplemental 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a study protocol prevention of experimental coronavirus colds with intranasal alpha-2b interferon interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review the proximal origin of sars-cov-2 attenuation of replication by a 29 nucleotide deletion in sars-coronavirus acquired during the early stages of human-to-human transmission publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank dr. ming-hsiang hong for his assistance in preparation of the manuscript. authors' information none. not applicable.availability of data and materials not applicable. consent for publication not applicable. the authors declare that they have no competing interests. key: cord-308201-lavcsqov authors: desforges, marc; le coupanec, alain; dubeau, philippe; bourgouin, andréanne; lajoie, louise; dubé, mathieu; talbot, pierre j. title: human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? date: 2019-12-20 journal: viruses doi: 10.3390/v12010014 sha: doc_id: 308201 cord_uid: lavcsqov respiratory viruses infect the human upper respiratory tract, mostly causing mild diseases. however, in vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, causing a more severe disease (e.g., pneumonia). respiratory viruses can also exacerbate asthma and lead to various types of respiratory distress syndromes. furthermore, as they can adapt fast and cross the species barrier, some of these pathogens, like influenza a and sars-cov, have occasionally caused epidemics or pandemics, and were associated with more serious clinical diseases and even mortality. for a few decades now, data reported in the scientific literature has also demonstrated that several respiratory viruses have neuroinvasive capacities, since they can spread from the respiratory tract to the central nervous system (cns). viruses infecting human cns cells could then cause different types of encephalopathy, including encephalitis, and long-term neurological diseases. like other well-recognized neuroinvasive human viruses, respiratory viruses may damage the cns as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuro-immunopathology) and/or viral replication, which directly causes damage to cns cells (virus-induced neuropathology). the etiological agent of several neurological disorders remains unidentified. opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of these disorders whose etiology remains poorly understood. herein, we present a global portrait of some of the most prevalent or emerging human respiratory viruses that have been associated with possible pathogenic processes in cns infection, with a special emphasis on human coronaviruses. the central nervous system (cns), a marvel of intricate cellular and molecular interactions, maintains life and orchestrates homeostasis. unfortunately, the cns is not immune to alterations that lead to neurological disease, some resulting from acute, persistent or latent viral infections. several viruses have the ability to invade the cns, where they can infect resident cells, including the neurons [1] . although rare, viral infections of the cns do occur [2] . however, their incidence in clinical practice common virus, is associated with febrile illness, fever, cough and congestion [97, 98] , as well as a characteristic rash and koplik's spots [99] . in rare circumstances, significant long-term cns diseases, such as [99] post-infectious encephalomyelitis (pie) or acute disseminated encephalomyelitis (adem), occur in children and adolescents. other examples of rare but devastating neurological disorders are measles inclusion body encephalitis (mibe), mostly observed in immune-compromised patients, and subacute sclerosing panencephalitis (sspe) that appears 6-10 years after infection [100] . yet, with the exception of hiv, no specific virus has been constantly associated with specific human neurodegenerative disease. on the other hand, different human herpes viruses have been associated with alzheimer's disease (ad), multiple sclerosis (ms) and other types of long-term cns disorders [101] [102] [103] . as accurately stated by majde [104] , long-term neurodegenerative disorders may represent a "hit-and-run" type of pathology, since some symptoms are triggered by innate immunity associated with glial cell activation. different forms of long-term sequelae (cognitive deficits and behavior changes, decreased memory/learning, hearing loss, neuromuscular outcomes/muscular weakness) were also observed following arboviral infections [83, 103, [105] [106] [107] . including the few examples listed above, more than one hundred infectious agents (much of them being viruses) have been described as potentially encephalitogenic and an increasing number of positive viral identifications are now made with the help of modern molecular diagnostic methods [8, 70, [108] [109] [110] . however, even after almost two decades into the 21st century and despite tremendous advances in clinical microbiology, the precise cause of cns viral infections often remains unknown. indeed, even though very important technical improvements were made in the capacity to detect the etiological agent, identification is still not possible in at least half of the cases [110, 111] . among all the reported cases of encephalitis and other encephalopathies and even neurodegenerative processes, respiratory viruses could represent an underestimated part of etiological agents [104] . respiratory syncytial virus (rsv), a member of the orthopneumovirus genus [112] , infects approximately 70% of infants before the age of 1 and almost 100% by the age of 2 years old [113] , making it the most common pathogen to cause lower respiratory tract infection such as bronchiolitis and pneumonia in infants worldwide [32, 114] . recent evidence also indicates that severe respiratory diseases related to rsv are also frequent in immunocompromised adult patients [8, 115] and that the virus can also present neuroinvasive properties [8] . over the last five decades, a number of clinical cases have potentially associated the virus with cns pathologies. rsv has been detected in the cerebrospinal fluid (csf) of patients (mainly infants) and was associated with convulsions, febrile seizures and different types of encephalopathy, including clinical signs of ataxia and hormonal problems [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] . furthermore, rsv is now known to be able to infect sensory neurons in the lungs and to spread from the airways to the cns in mice after intranasal inoculation, and to induce long-term sequelae such as behavioral and cognitive impairments [127] . an additional highly prevalent human respiratory pathogen with neuroinvasive and neurovirulent potential is the human metapneumovirus (hmpv). discovered at the beginning of the 21st century in the netherlands [128] , it mainly causes respiratory diseases in newborns, infants and immunocompromised individuals [129] . during the last two decades, sporadic cases of febrile seizures, encephalitis and encephalopathies (associated with epileptic symptoms) have been described. viral material was detected within the cns in some clinical cases of encephalitis/encephalopathy [130] [131] [132] [133] [134] but, at present, no experimental data from any animal model exist that would help to understand the underlying mechanism associated with hmpv neuroinvasion and potential neurovirulence. hendra virus (hev) and nipah virus (niv) are both highly pathogenic zoonotic members of the henipavirus genus and represent important emerging viruses discovered in the late 1990s in australia and southern asia. they are the etiological agents of acute and severe respiratory disease in humans, including pneumonia, pulmonary edema and necrotizing alveolitis with hemorrhage [135] [136] [137] [138] . although very similar at the genomic level, both viruses infect different intermediate animal reservoirs: the horse for hev and the pig for niv as a first step before crossing the barrier species towards humans [135] . in humans, it can lead to different types of encephalitis, as several types of cns resident cells (including neurons) can be infected [139, 140] . the neurological signs can include confusion, motor deficits, seizures, febrile encephalitic syndrome and a reduced level of consciousness. even neuropsychiatric sequelae have been reported but it remains unclear whether a post-infectious encephalo-myelitis occurs following infection [141] [142] [143] . the use of animal models showed that the main route of entry into the cns is the olfactory nerve [144] and that the nipah virus may persist in different regions of the brain of grivets/green monkeys [145] , reminiscent of relapsing and late-onset encephalitis observed in humans [146] . influenza viruses are classified in four types: a, b, c and d. all are endemic viruses with types a and b being the most prevalent and causing the flu syndrome, characterized by chills, fever, headache, sore throat and muscle pain. they are responsible for seasonal epidemics that affect 3 to 5 million humans, among which 500,000 to 1 million cases are lethal each year [147, 148] . associated with all major pandemics since the beginning of the 20th century, circulating influenza a presents the greatest threat to human health. most influenza virus infections remain confined to the upper respiratory tract, although some can lead to severe cases and may result in pneumonia, acute respiratory distress syndrome (ards) [30, 149] and complications involving the cns [150] [151] [152] . several studies have shown that influenza a can be associated with encephalitis, reye's syndrome, febrile seizure, guillain-barré syndrome, acute necrotizing encephalopathy and possibly acute disseminated encephalomyelitis (adem) [153] [154] [155] [156] [157] [158] . animal models have shown that, using either the olfactory route or vagus nerve, influenza a virus may have access to the cns and alter the hippocampus and the regulation of neurotransmission, while affecting cognition and behavior as long-term sequelae [8, 69, [159] [160] [161] [162] . the influenza a virus has also been associated with the risk of developing parkinson's disease (pd) [151] and has recently been shown to exacerbate experimental autoimmune encephalomyelitis (eae), which is reminiscent of the observation that multiple sclerosis (ms) relapses have been associated with viral infections (including influenza a) of the upper respiratory tract [163] [164] [165] . another source of concern when considering human respiratory pathogens associated with potential neuroinvasion and neurovirulence is the enterovirus genus, which comprises hundreds of different serotypes, including polioviruses (pv), coxsackieviruses (cv), echoviruses, human rhinoviruses (hrv) and enteroviruses (ev). this genus constitutes one of the most common cause of respiratory infections (going from common cold to more severe illnesses) and some members (pv, ev-a71 and -d68, and to a lesser extent hrv) can invade and infect the cns, with detrimental consequences [166] [167] [168] [169] . even though extremely rare, hrv-induced meningitis and cerebellitis have been described [170] . although ev infections are mostly asymptomatic, outbreaks of ev-a71 and d68 have also been reported in different parts of the world during the last decade. ev-a71 is an etiological agent of the hand-foot-mouth disease (hfmd) and has occasionally been associated with upper respiratory tract infections. ev-d68 causes different types of upper and lower respiratory tract infections, including severe respiratory syndromes [171] . both serotypes have been associated with neurological disorders like acute flaccid paralysis (afp), myelitis (afm), meningitis and encephalitis [166, [172] [173] [174] [175] . last but not least, human coronaviruses (hcov) are another group of respiratory viruses that can naturally reach the cns in humans and could potentially be associated with neurological symptoms. these ubiquitous human pathogens are molecularly related in structure and mode of replication with neuroinvasive animal coronaviruses [176] like phev (porcine hemagglutinating encephalitis virus) [177] , fcov (feline coronavirus) [178, 179] and the mhv (mouse hepatitis virus) strains of mucov [180] , which can all reach the cns and induce different types of neuropathologies. mhv represents the best described coronavirus involved in short-and long-term neurological disorders (a model for demyelinating ms-like diseases) [181] [182] [183] . taken together, all these data bring us to consider a plausible involvement of hcov in neurological diseases. the first strains of hcov were isolated in the mid-60s from patients presenting an upper respiratory tract disease [184] [185] [186] [187] . before the severe acute respiratory syndrome (sars) appeared in 2002 and was associated with sars-cov [188] [189] [190] , only two groups of hcov, namely hcov-229e (previous group 1, now classified as alphacoronavirus) and hcov-oc43 (previous group 2, now classified as betacoronavirus) were known. several new coronaviruses have now been identified, including three that infect humans: alphacoronavirus hcov-nl63 [191] and betacoronaviruses hcov-hku1 and mers-cov [192, 193] . the hcov-229e, -oc43, -nl63 and -hku1 strains are endemic worldwide [31, 184, [194] [195] [196] [197] [198] [199] and exist in different genotypes [200] [201] [202] [203] [204] [205] [206] [207] . in immunocompetent individuals they usually infect the upper respiratory tract, where they are mainly associated with 15-30% of upper respiratory tract infections (uri): rhinitis, laryngitis/pharyngitis as well as otitis. being highly opportunistic pathogens [14] , hcov can reach the lower respiratory tract and be associated with more severe illnesses, such as bronchitis, bronchiolitis, pneumonia, exacerbations of asthma and respiratory distress syndrome [17, 31, [208] [209] [210] [211] [212] [213] [214] . the 2002-2003 sars pandemic was caused by a coronavirus that emerged from bats (first reservoir) [25] to infect palm civets (intermediary reservoir) and then humans [215] . a total of 8096 probable cases were reported and almost 10% (774 cases in more than 30 countries) of these resulted in death [216] [217] [218] . the clinical portrait was described as an initial flu-like syndrome, followed by a respiratory syndrome associated with cough and dyspnea, complicated with the "real" severe acute respiratory syndrome (sars) in about 20% of the patients [31,219]. in addition, multiple organ failure was observed in several sars-cov-infected patients [220] . in the fall of 2012, individuals travelling from the arabian peninsula to the united kingdom were affected by the middle-east respiratory syndrome (mers), a severe lower respiratory tract infection that resembled sars, leading also to gastrointestinal symptoms and renal failure among some patients [221] . molecular sequencing rapidly showed that the new epidemic was caused by a new coronavirus: the mers-cov [193, 222, 223] . mers-cov most probably originated from bats before infecting an intermediary reservoir (the dromedary camel), and also represented a zoonotic transmission to humans. phylogenetic analyses suggest that there have been multiple independent zoonotic introductions of the virus in the human population. moreover, nosocomial transmission was observed in multiple hospitals in saudi arabia [221, [224] [225] [226] [227] [228] [229] [230] . although possible, human-to-human mers-cov transmission appears inefficient as it requires extended close contact with an infected individual. consequently, most transmission have occurred among patients' families and healthcare workers (clusters of transmission). a more efficient human-to-human transmission was observed in south korea, during the 2015 outbreak of mers-cov [231, 232] . even though it has propagated to a few thousand people and possesses a high degree of virulence, mers-cov seems mostly restricted to the arabic peninsula and is not currently considered an important pandemic threat. however, virus surveillance and better characterization are warranted, in order to be prompt to respond to any change in that matter [23, [233] [234] [235] . as of october 8, 2019, the world health organization (who) reported that mers-cov had spread to at least 27 different countries, where 2468 laboratory-confirmed human cases have been identified with 851 being fatal (https://www.who.int/emergencies/mers-cov/en/). as observed for the four circulating strains of hcov [31, 194] , both sars-cov and mers-cov usually induce more [228, 236] severe illnesses, and strike stronger in vulnerable populations such as the elderly, infants, immune-compromised individuals or patients with comorbidities [31,237]. over the years, like sars-and mers-cov, the four endemic hcov have also been identified as possible etiological agents for pathologies outside the respiratory tract. indeed, myocarditis, meningitis, severe diarrhea (and other gastrointestinal problems) and multi-organ failure [220, 221, [238] [239] [240] [241] have been reported, especially in children. recent investigations on hcov as enteric pathogens demonstrated that all hcov strains can be found in stool samples of children with acute gastroenteritis; however, no evidence of association could yet be clearly demonstrated with disease etiology [242, 243] . different reports also presented a possible link between the presence of hcov within the human central nervous system (cns) and some neurological disorders among patients examined [244] [245] [246] [247] [248] [249] . like all viruses, hcov may enter the cns through the hematogenous or neuronal retrograde route. in the human airways, hcov infection may lead to the disruption of the nasal epithelium [250] and, although they bud and are released mostly on the apical side of the epithelial cells, a significant amount of viruses is also released from the basolateral side [251] . thus, although hcov infections are, most of the time, restricted to the airways, they may under poorly understood conditions pass through the epithelium barrier and reach the bloodstream or lymph and propagate towards other tissues, including the cns [33, 38, 208, 252] ; this was also suggested for other respiratory viruses that can reach the human cns, namely, rsv [8, 53] [257, 258] . moreover, persistently-infected leukocytes [252] may serve as a reservoir and vector for neuroinvasive hcov [245] . therefore, neuroinvasive hcov could use the hematogenous route to penetrate into the cns. the second form of any viral spread towards the cns is through neuronal dissemination, where a given virus infects neurons in the periphery and uses the machinery of active transport within those cells in order to gain access to the cns [35, 36] . although the olfactory bulb is highly efficient at controlling neuroinvasion, several viruses have been shown to enter cns through the olfactory route [259, 260] . after an intranasal infection, both hcov-oc43 and sars-cov were shown to infect the respiratory tract in mice and to be neuroinvasive [261] [262] [263] [264] [265] . over the years, we and others have gathered data showing that hcov-oc43 is naturally neuroinvasive in both mice and humans [244, 245, 261, 263, 266] . experimental intranasal infections of susceptible mice also indicate that, once it has invaded the cns, the virus disseminated to several regions of the brain and the brainstem before it eventually reaches the spinal cord [266] [267] [268] . furthermore, based on more recent work [269] , figure 1 illustrates the olfactory route, which is clearly the main route of neuroinvasion used by hcov-oc43, as well as the early steps of subsequent neuropropagation within the cns in susceptible mice and recapitulates the suggested equivalent pathway in humans. nevertheless, our data suggest that hcov-oc43 may also invade the cns from the external environment through other pathways involving other cranial peripheral nerves [269] , reminiscent of what was shown for other human respiratory viruses such as rsv and influenza virus [8] . therefore, on the one hand, an apparently innocuous human respiratory pathogen such as the hcov may reach the cns by different routes and induce short-term illnesses, such as encephalitis. on the other hand, it may persist in resident cells of the human cns and may become a factor or co-factor of neuropathogenesis associated with long-term neurological sequelae in genetically or otherwise predisposed individuals. because of their natural neuroinvasive potential in humans and animals, a possible association between the presence of ubiquitous human coronaviruses in the triggering or exacerbation of neurological human pathologies has often been suggested over the years. it is now accepted that hcov are not always confined to the upper respiratory tract and that they can invade the cns [220, 245, 248, 270] . as other viruses listed herein, hcov are neurotropic and potentially neurovirulent. even though no clear cause and effect link has ever been made with the onset of human neurological diseases, their neuropathogenicity is being increasingly recognized in humans, as several recent reports associated cases of encephalitis [244] , acute flaccid paralysis [271] and other neurological symptoms, including possible complications of hcov infection such as guillain-barré syndrome or adem [249, [272] [273] [274] [275] [276] [277] [278] [279] . the presence and persistence of hcov in human brains was proposed to cause long-term sequelae related to the development or aggravation of chronic neurological diseases [245] [246] [247] [248] [280] [281] [282] . given their high prevalence [31, 283] , long-term persistence and newly recognized neuropathogenesis, hcov disease burden could currently be underestimated. this suggest that better surveillance, diagnoses and deepened virus-host interactions studies are warranted in order to gather more knowledge that will make possible the development of therapeutic strategies to prevent or treat occurrences. potential short-term neuropathologies sars-cov, hcov-oc43 and -229e are naturally neuroinvasive and neurotropic in humans and therefore potentially neurovirulent [220, 244, 245, 249, 270, 271] . furthermore, animal models showed that sars-cov could invade the cns primarily through the olfactory route [265] or even after an intra-peritoneal infection [284] , and induce neuronal cell death [265, 284] . to our knowledge, no reports on the presence of the three other coronaviruses that infect humans in the cns have been published. however, neurological symptoms have been described in patients infected by all three viruses [276, 277, 285] . making use of our in vivo model of hcov neuropathogenesis, relying on the natural susceptibility of mice to hcov-oc43-the most prevalent strain among endemic hcov [210, 286] -encephalitis and transient flaccid paralysis associated with propagation towards the spinal cord and demyelination and long-term persistence in surviving mice were observed [261, [266] [267] [268] [287] [288] [289] ; thus, recapitulating the neurological afflictions reported in some patients infected by hcov [244, 249, 271, 272, [275] [276] [277] 290] . although we must interpret data obtained in rodents with all the caution dictated by the use of a non-human host, it is likely that the underlying mechanisms described will have relevance to the human situation or at least provide leads to investigate neurotropic hcov in humans. in susceptible mice, hcov-oc43 has a selective tropism for neurons in which it is able to use axonal transport as a way of neuron-to-neuron propagation [269] . these results, together with data harvested with the use of microfluidic devices (xona microfluidic), helped to elaborate a putative model of propagation adapted from tomishima and enquist [291] , in which infectious hcov-oc43 could either be assembled in the cell body or at different points along the axon using the anterograde axonal transport to propagate between neurons or from neurons to glial cells surrounding neurons in the cns (figure 2 ). furthermore, based on previous data using different mutant recombinant viruses harboring mutation in the s protein [266, 268, 269] and making use of a luciferase expressing recombinant hcov-oc43 [292] [293] [294] , we are now showing that the rate and success of virus propagation towards the spinal cord, in part through the neuron-to-neuron pathway, correlates with the exacerbation of neurovirulence ( figure 3 ). [269] and adapted from tomishima and enquist [291] . in this model, solid arrows represent fully assembled virus transport and dashed arrows represent subvirion assemblies [291] . schematic representations were assembled with the motifolio neuroscience toolkit, 2007. [292] was injected intra-nasally (i.n.) into mice. virus spread was assessed by bioluminescence imaging (bli) with the xenogen vivo vision ivis 100 imaging system (perkin-elmer) in infected anaesthetized mice placed in a light proof specimen chamber after intraperitoneal injection of d-luciferin. images were taken with a ccd camera mounted in a light-tight imaging chamber, using the acquisition software living image version 4.3.1 (caliper-lifesciences). evaluation of associated clinical scores: (levels 0 to 4: 0 is asymptomatic; 1 is mice with early hunched back; 2 is mice presenting slight social isolation, weight loss and abnormal gait; 3 is mice presenting total social isolation, ruffled fur, hunched back, weight loss and almost no movement; and 4 is mice moribund or dead (presented elsewhere; [266] ), indicate that only mice with a positive signal at both the level of the brain and spinal cord were evaluated to be at level 2 to 3. hcov-oc43 structural and accessory proteins are important for infection and some clearly represent virulence factors [38, [266] [267] [268] [269] 289, 295, 296] . using neuronal cell cultures and our murine model, we gathered data indicating that some of these proteins also play a significant role in viral dissemination [269, 296] and now aim to exploit these promising leads to fully understand the course and determinants of propagation to and through the cns and complete the neurologic portrait of short term hcov neuropathogenesis. the presence of hcov rna in the human cns establishes the natural neuroinvasive properties of these respiratory viral agents. moreover, it also suggests that they persist in human cns [245] as they do in human neural cells [297, 298] and in the cns of mice that survive acute encephalitis. these surviving mice exhibited long-term sequelae associated with decreased activity in an open field test and a reduced hippocampus with neuronal loss in the ca1 and ca3 layers [287] , reminiscent of what was observed after infection by the influenza a virus and rsv [127, 162] and to the significant loss of synapses within the ca3 region after infection by wnv [299, 300] . the precise and complete etiology of several long-term neurological pathologies still represent a conundrum. multiple sclerosis (ms) represents one such neurological disease for which an infectious agent or agents may play a triggering role, with viruses the most likely culprit in genetically predisposed individuals [301] . it has been suggested that several neurotropic viruses could be involved in ms pathogenesis but that they may do so through similar direct and/or indirect mechanisms [302] [303] [304] [305] [306] . however, although research has not yet led to a direct link to any specific virus, association of coronaviruses with ms has been suggested [307] . even though hcov-oc43 and -229e were detected in some control brains and in some brains coming from patients with different neurological diseases, there was a significantly higher prevalence of hcov-oc43 in brains of ms patients [245] . moreover, autoreactive t cells were able to recognize both viral and myelin antigens in ms patients but not in controls during infection by hcov-oc43 and hcov-229e [308, 309] . thus, the immune response may participate in the induction or exacerbation of long-term neuropathologies such as ms in genetically or otherwise susceptible individuals. furthermore, it was shown that in recombination activation gene (rag) knock-out mice, hcov-oc43-induced encephalitis could be partially mediated by the t-cell response to infection [263] . this underlines the possibility that, like its murine counterpart mhv, long term infection of the cns by hcov [245] may participate in the induction of demyelinating ms-like lesions. immune cell infiltration and cytokine production were observed in the mouse cns after infection by hcov-oc43. this immune response was significantly increased after infection by viral variants, which harbor mutations in the viral glycoprotein (s) [267] . these variants also induced glutamate excitotoxicity [268, 289] , thus increasing damage to neurons [310] and/or disturbing glutamate homeostasis [311] and thereby contributing to neuronal degeneration and hind-limb paralysis and possible demyelination [266] [267] [268] [269] . the degeneration of neurons may eventually lead to death of these essential cells by directly generating a cytotoxic insult related to viral replication and/or to the induction of different regulated cell death (rcd) pathways [312] [313] [314] . our results indicate that the underlying mechanisms appear to involve different cellular factors and pathways of rcd, described and reviewed elsewhere [38, 315] . virus-cell interactions are always important in the regulation of cell response to infection. for hcov-oc43, we clearly showed that the viral s and e proteins are important factors of neurovirulence, neuropropagation and neurodegeneration of infected cells [267] [268] [269] 296, 312] . we have also demonstrated that the he protein is important for the production of infectious hcov-oc43 and for efficient spreading between neuronal cells, suggesting an attenuation of the eventual spread into the cns of viruses made deficient in fully active he protein, potentially associated with a reduced neurovirulence [269, 295] . coronavirus accessory proteins have been extensively studied and are now considered as important viral factors of virulence implicated in pathogenesis while counteracting innate immunity [316] [317] [318] [319] . two of these accessory proteins (ns2 and ns5) produced during infection by hcov-oc43 play a significant role in virulence and pathogenesis in the mouse cns [38] . like for several other respiratory viruses, accumulating evidence now indicate that hcov are neuroinvasive in humans and we hypothesize that these recognized respiratory pathogens are potentially neurovirulent as well, as they could participate in short-and long-term neurological disorders either as a result of inadequate host immune responses and/or viral propagation in the cns, which directly induces damage to resident cells. with that in mind, one can envisage that, under the right circumstances, hcov may successfully reach and colonize the cns, an issue largely deserted and possibly underestimated by the scientific community that has impacted or will impact the life of several unknowing individuals. in acute encephalitis, viral replication occurs in the brain tissue itself, possibly causing destructive lesions of the nervous tissue with different outcomes depending on the infected regions [320] . as previously mentioned, hcov may persist in the human cns as it does in mice [245, 287] and potentially be associated with different types of long-term sequelae and chronic human neurological diseases. in their famous review on cns viral infection, published a few years ago, koyuncu et al. [35] insisted that, under the right conditions, all viruses can have access to the cns. what "under the right conditions" means certainly represents a subject of debate among virologist and physicians. nevertheless, as stated in the introduction of this review, viral factors (mutations in specific virulence genes), host factors (immunodepression, age) or a mixture of both (underlining the importance of virus-host interactions), are all good candidates to refer to if one intends to find the beginning of an explanation. a fast and accurate diagnosis would certainly improve prognosis for patients with a suspected cns infection. identification of a specific virus provides relevant information on how to treat a patient; therefore, the development of modern technologies, such as high throughput sequencing (next generation sequencing) are warranted as it represents a potentially unbiased marvelous tool for rapid and robust diagnosis of unexplained encephalitis or other types of encephalopathies or neuronal manifestations, especially in the context where more traditional techniques have failed to identify the etiological agent [21, 108, 111, 244, 321, 322] . therefore, although our attention is mainly on a few different viruses such as hsv, arboviruses and enteroviruses, it may now be the time to look at cns viral infection from another perspective. these viruses truly represent an important proportion of cns viral infection associated with encephalitis, meningitis, myelitis and long-term neurological disorders. nevertheless, accumulating evidence in the scientific literature strongly suggest that many other viral candidates could be underestimated in that matter. several human respiratory viruses are neuroinvasive and neurotropic, with potential neuropathological consequences in vulnerable populations. understanding the underpinning mechanisms of neuroinvasion and interaction of respiratory viruses (including hcov) with the nervous system is essential to evaluate potentially pathological short-and long-term consequences. however, viral infections related to diseases that are rare manifestations of an infection (like long term chronic neurological diseases), represent situations where koch's postulates [323] need to be modified. a series of new criteria, adapted from sir austin bradford hill, for causation [324, 325] was elaborated by giovannoni and collaborators concerning the plausible viral hypothesis in ms [326] . these criteria certainly represent a pertinent tool to evaluate the involvement of human respiratory viruses as a factor that could influence long-term human neurological diseases. to continue the gathering of epidemiological data is justified to evaluate the clear cause and effect link between neuroinvasive respiratory viruses and short-and long-term human neurological diseases. understanding mechanisms of virus neuroinvasion and interactions with the central nervous system is essential for different reasons. first, to 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writing of the manuscript, or in the decision to publish the results. key: cord-318984-8m9ygzn5 authors: chen, yin-yin; chen, liang-yu; lin, seng-yi; chou, pesus; liao, shu-yuan; wang, fu-der title: surveillance on secular trends of incidence and mortality for device–associated infection in the intensive care unit setting at a tertiary medical center in taiwan, 2000–2008: a retrospective observational study date: 2012-09-10 journal: bmc infect dis doi: 10.1186/1471-2334-12-209 sha: doc_id: 318984 cord_uid: 8m9ygzn5 background: device–associated infection (dai) plays an important part in nosocomial infection. active surveillance and infection control are needed to disclose the specific situation in each hospital and to cope with this problem effectively. we examined the rates of dai by antimicrobial-resistant pathogens, and 30–day and in–hospital mortality in the intensive care unit (icu). methods: prospective surveillance was conducted in a mixed medical and surgical icu at a major teaching hospital from 2000 through 2008. trend analysis was performed and logistic regression was used to assess prognostic factors of mortality. results: the overall rate of dais was 3.03 episodes per 1000 device–days. the most common dai type was catheter–associated urinary tract infection (3.76 per 1000 urinary catheter–days). there was a decrease in dai rates in 2005 and rates of ventilator–associated pneumonia (vap, 3.18 per 1000 ventilator–days) have remained low since then (p < 0.001). the crude rates of 30–day (33.6%) and in–hospital (52.3%) mortality, as well as infection by antibiotic-resistant vap pathogens also decreased. the most common antimicrobial-resistant pathogens were methicillin–resistant staphylococcus aureus (94.9%) and imipenem–resistant acinetobacter baumannii (p < 0.001), which also increased at the most rapid rate. the rate of antimicrobial resistance among enterobacteriaceae also increased significantly (p < 0.05). after controlling for potentially confounding factors, the dai was an independent prognostic factor for both 30–day mortality (or 2.51, 95% confidence interval [ci] 1.99–3.17, p = 0.001) and in–hospital mortality (or 3.61, 95% ci 2.10–3.25, p < 0.001). conclusions: the decrease in the rate of dai and infection by resistant bacteria on the impact of severe acute respiratory syndrome can be attributed to active infection control and improved adherence after 2003. surveillance of nosocomial infections (nis) has become an integral part of infection control and quality assurance in many countries. gastmeier et al. reported that effective surveillance could reduce the ni rate on average about 20-30% [1, 2] . surveillance programs provide data on the microbes causing specific nis and their resistance to antibiotics. moreover, such programs can guide clinical practices and ni prevention efforts in different geographic regions and clinical settings. the surveillance of device-associated infections (dais) in intensive care units (icus) has become more important owing to the more frequent employment of invasive advanced life support devices, especially after the introduction in 2004 of surviving sepsis bundles [3, 4] . nevertheless, according to the three largest surveillance systems, the pooled mean rates of dais were: ventilator-associated pneumonia (vap), 1.3-13.6 per 1000 ventilator-days; central line-associated bloodstream infection (clabsi), 2.0-7.6 per 1000 catheterdays; and catheter-associated urinary tract infection (cauti), 2.0-6.3 per 1000 catheter-days [5] [6] [7] . in addition, dais have been associated with significant cost and mortality [3, 5, 6] . the crude mortality rates of icu patients with dai were 32.9-43.7% [5] . moreover, as indicated by the message "bad bugs, no drugs" released by the infectious disease society of america in 2004, the emergence of antibiotic resistance threatens to exacerbate the problem of nis in critically ill patients. decreased susceptibility of both gram-positive and gram-negative microbes to antibiotics has been well described in several surveillance studies over the past decade, and increases in the rate of bloodstream infection caused by multi-drug resistant (mdr) gramnegative bacteria have been reported to be 16-fold [5, [8] [9] [10] [11] . in addition, both the morbidity and mortality rates have increased [12] [13] [14] . in this study, prospective surveillance was conducted to determine the dai rate and prevalence of antibiotic-resistant isolates at an adult medical-surgical icu (ms icu). our aim was to analyze the secular trend of incidence for different types of dais, determine the common pathogens involved, and determine the rates of antimicrobial resistance and overall 30-day and in-hospital mortality during the period 2000-2008. this study was conducted in a 42-bed adult medicalsurgical icu with more than 1500 admissions (age 18 years or older) per year located in a 2900-bed major teaching hospital in the northern part of taiwan. the hospital-wide infection surveillance and control program was established in 1982, with one infection control practitioner (icp) for every 250 beds. all patients admitted to the icu in the period 2000-2008 who developed infections more than 48 hours after admission (i.e., nosocomial infections) were eligible for the study. the protocol of this study was approved by the institutional review board of our teaching hospital. this icu-based surveillance was conducted according to the us centers for disease control and prevention (cdc) procedures. all patients in the unit were monitored for nis that affected particular body sites. infections at more than one site in the same patient were counted as separate infections. the antibiotic susceptibility of each pathogen involved was analyzed. the data were prospectively collected at least once a week in the icu by trained icps according to standardized protocols and definitions of the us cdc national healthcare safety network (nhsn; formerly the national nosocomial infection surveillance system [nnis]) [15] . all dais of the outcome surveillance component were categorized using standard us cdc nhsn definitions that included laboratory and clinical criteria [16] . the involved patient demographic information, the dates and sites of infection, device-utilization (du) ratio, pathogens, antimicrobial susceptibilities, invasive procedures, and overall 30-day mortality and in-hospital crude mortality were recorded. reports of cases of dai were also verified by an infectious disease specialist. data were also collected for each exposed patient in the icu from the prospective hospital database, including demographics and clinical characteristics. pneumonia was defined when a patient had a new or progressive infiltrate, consolidation, cavitation, or pleural effusion on chest radiograph and had the following signs or symptoms: new onset of purulent sputum or change in character of sputum. a vap was categorized as ventilator associated if the patient had been intubated and received ventilation for more than 48 hours prior to the development of pneumonia. to detect vap microorganisms, tracheal aspirates obtained via endotracheal tube suction or tracheostomy tube suction methods were cultured. laboratory-confirmed bloodstream infection (bsi) was defined when a patient had a recognized pathogen cultured from one or more blood cultures and the microorganism cultured from blood was not related to an infection at another site. common skin contaminants (e.g., coagulase-negative staphylococcus [cons]) required culture from two or more blood cultures drawn on separate occasions or at least one blood culture for a patient with intravascular devices and microorganisms of the tip culture identical to those of the blood culture. a clabsi was considered central catheter-associated if a catheter had been in place for more than 48 hours and a secondary site of infection was not present. to detect clabsi micro-organisms, a central catheter were removed aseptically and a 5-cm segment from the most distal end of the tip of the catheter along with paired peripheral blood samples were cultured. central catheter-tip colonization was defined as isolation of 15 colony-forming units from a central catheter tip by using the roll-plate semiquantitative maki's culture technique. symptomatic uti was defined when a patient had one or more of the following signs or symptoms with no other recognized cause: fever (>38°c), dysuria, urgency, frequency, or suprapubic tenderness and (1) the patient had a positive urine culture, that is, ≥10 5 microorganisms per cm 3 , or urine with no more than two species of microorganisms, or (2) pyuria (urine specimen with ≥10 white blood cells /mm 3 and a positive urine culture of ≥10 3 and <10 5 cfu/ml with no more than 2 species of microorganisms. a cauti was a symptomatic uti that occurred in a patient who had an indwelling urinary catheter in place within the 48 hour period before the onset of the uti. to detect cauti organisms, a urine sample was aseptically aspirated from the sampling port of a urinary catheter and cultured quantitatively. pathogens were isolated from blood cultures using the bactec w nr-660 system (becton dickinson diagnostic instrument systems, spark, md, usa) between 2000 and 2001 and using the bact/alert 3d system (bio-mérieux, inc., marcy l'étoile, france) between 2001 and 2008. pathogens were isolated from other specimens using standard methods specified by the clinical laboratory standard institute (clsi) 2008 [17] . antibiotic susceptibilities were determined using disk diffusion tests and interpreted according to the criteria specified by the clsi 2008. the ni rate was defined as the number of nis per 1,000 patient-days. patient days were calculated as the number of icu days of the non-ni cohort or the number of icu days after the onset of ni. device-associated infection rates were calculated as the number of deviceassociated infections for a specified body site per 1,000 device days. the du ratio was calculated as the number of device-days per number of patient-days. secular trends of du ratio, antimicrobial resistant rates, 30-day mortality and in-hospital mortality rates were analyzed by chi-square test for linear trend. the overall and site-specific dai rates were analyzed by poisson regression analysis. logistic regression with a stepwise forward approach was used to assess prognostic factors of mortality, while controlling for potentially confounding variables (i.e., demographics, invasive devices, and laboratory data) [12] . odds ratios (or) and 95% confidence intervals (ci) were calculated. a p-value < 0.05 was defined as statistically significant. statistical analysis was conducted using epi info tm version 3.5.3 released by us cdc. graphs of secular trends, 30-day mortality and inhospital mortality rates were created using sigma-plot version 10.0 (systat inc., san jose, ca, usa). during the study period, 126,315 patient-days and 275,067 device-days were evaluated, and 2,054 nis and 833 dais occurred in 14,734 patients admitted to ms icus with a mean apache ii score of 23.6 ± 7.2. the crude mortality rate was 14.4% during the study period. those patients who were admitted to ms icus had a mean age of 69.5 ± 16.9 years, and male gender accounted for 71.8%. the length of icu stay was 9.6 ± 7.5 days in average. most patients were admitted due to major medical conditions (59%), such as neoplasms (22.2%), digestive system problems (20.7%), and respiratory system problems (17.8%). patients with serum albumin <2.5 g/dl were 44.4% and blood creatinine >1.5 mg/dl were 67.1%. there were 13.2% patients undergoing hemodialysis during their icu stay. the overall rates of nis and dais were 16.26 episodes per 1000 patient-days and 3.03 episodes per 1000 device-days, respectively. the most common dai type was cauti (mean 3 figure 1) . a total of 1,290 pathogens were isolated from clinical specimens ( table 2) . acinetobacter baumannii (23%), pseudomonas aeruginosa (18.2%), and staphylococcus aureus (17.4%) were the three most common pathogens associated with vap, while s. aureus (20.4%), a. baumannii (14.9%), and candida albicans (14.6%) accounted for the majority of clabsis. in contrast, non-albicans candida (nac) spp. (31%) rather than bacteria were the most common cauti pathogens, followed by enterococci (10.1%) and escherichia coli (9.9%). the rate of antibiotic resistance every year is presented in table 3 (figures 3 and 4) . dai was an independent factor for 30-day mortality (or 2.51, 95% ci 1.99-3.17, p = 0.001) and in-hospital mortality (or 2.61, 95% ci 2.10-3.25, p < 0.001) by multiple regression analysis. other significant prognostic factors (p < 0.001) for mortality included apache ii score, service, length of stay after the onset of infection, serum albumin, blood creatinine, neoplasms and hemodialysis (table 4 ). the mean rates of ni and dai in our adult ms icu during the study period were much lower than those reported by the inicc as well as for 173 icus in developing countries [5] , were similar to those reported by 1,545 hospitals in the us through the cdc nhsn [3, 6] , and were slightly higher than those indicated by 586 icus in the german surveillance system (icu-kiss) [7] . reasons for these high dai rates in the inicc report and developing countries may include resource limitations, lack of legal enforcement of the infection control program, and poor adherence to infection control guidelines [5] . the prospective hospital-wide surveillance and infection control program has been established for nearly 30 years in our hospital, which made a great effort to control infection by implementing infection control bundles and educational programs. the increase of device-related infections is not obvious after 2004, except for cauti. these strategies showed effectiveness in controlling dai rates and suggest the necessity of infection control bundles implementation. the common device-associated pathogens show geographic variation in distribution. a. baumannii, s. aureus, and p. aeruginosa were the three most common vap pathogens in our study, the us cdc nhsn study, and the sentry antimicrobial surveillance study, although their percentages differed between studies [18, 19] . the percentage of isolates of mrsa (p = 0.678) and irpa (p = 0.953), but not isolates of irab (p < 0.001) remained relatively constant. however, any variation in these percentages would not be statistically significant and might rather be due to chance than to an actual variation. in contrast, higher rates of a. baumannii and c. albicans isolation compensated for the relatively low rates of cons and enterococcus spp. isolation in cases of clabsi, while c. albicans was replaced by nac spp in cases of cauti. differences in clinical setting, institution, study period, target population, and specific infection type might account in part for differences between studies. cons was less frequently identified in clabsi, because our criteria were slightly different from the us cdc definition for laboratory-confirmed bsi. the cdc defined skin contaminant bsi in 1998 and 2004 as 'the common skin contaminant (e.g., cons) is cultured from at least one blood culture from a patient with an intravascular line, and the physician institutes appropriate antimicrobial therapy'. however, cons bsis in our study were enrolled if the patient had only one blood culture of cons that was positive but then microorganisms cultured from the tip of the intravascular device that were also cons. the percentage of cons isolates was expected to be at least that of s. aureus isolates reported in previous studies [15, 16, 18, 20] . however, the frequency of a. baumannii and candida spp. in specimens from patients with clabsi was also reported to be increasing in other hospitals in taiwan as well as several asian countries such as turkey and thailand [20] [21] [22] [23] [24] , although the frequency of nac spp. represented by only one candida spp. has also been rising in specimens from patients with cauti. early and empirical usage of broad spectrum antibacterial agents in critically ill patients and preemptive administration of fluconazole are common factors contributing to the increase in frequency of isolation of these relatively resistant pathogens [22, 23, 25] . use of indwelling catheters increases susceptibility to those multi-drug resistant pathogens and is associated with biofilm formation [26, 27] . the high prevalence of mrsa is a common problem worldwide, and this situation was much more severe in our institute. our data showed a lower incidence density of s. aureus but a higher proportion of mrsa. the percentage of mrsa infections was 74.4-84.1% in the inicc report [5] , 54.4-65.2% in the us cdc nhsn report [18] , and, in the asia-pacific region, it was 38.2% in the sentry antimicrobial surveillance program report (2003) (2004) [28] and 20-85% in the tigecycline evaluation and surveillance trial (test) report [29] . mrsa rates were decreasing in many european countries but not in usa [18, 29] . the more severe illnesses of patients and more frequent use of broad-spectrum antibiotics might account in part for the high rate of mrsa isolation from patients in icu at major teaching hospitals [29, 30] . another possible explanation is the clonal spread of resistance genes or resistant strains [31, 32] , but molecular analysis will be needed to prove this hypothesis. according to the infection control policies in our icu, when a patient was admitted to the icu, a multi-drug resistant (mdr) checklist was used to inquire about mdr pathogens including mrsa infection or colonization. if mrsa had been isolated, then contact precautions were implemented. furthermore, we have promoted hospital-wide hand-washing activity from 2006 to the present. the infection control team also carries on the non-warning investigation of hand-washing and of isolation precautions in each season, and gives feedback of the results to the unit. infectious disease doctors assist in carrying on the infectious disease treatment and the antibiotic use in the icu. mrsa infection rates have been reduced by year from 2006. interestingly, the rates of antibiotic resistance for pathogens other than mrsa was lower at our hospital than those in previously published reports and lower than those for all nis reported by the test and sentry antimicrobial surveillance programs [8, 19, 28, 33] . however, despite the carbapenems being the most active antimicrobials against acinetobacter species, the increasing development of significant carbapenem resistance among acinetobacter species has been reported [3, 5, 34] . in the present study, the average percentage of a. baumannii isolates resistant to imipenem was 22.2%. the rate of icu patients with irab dai has been rapidly rising (from 6.1% to 34.3%). among enterobacteriaceae, ciprofloxacin-r e. coli and ceftazidime-r k. pneumoniae from 2003, and ceftazidime-r e. coli from 2004, had significant increases. this finding revealed that the resistance of gram-negative bacteria has increased, the development of which should require closely monitored. aside from the fact that dai is an important prognostic factor of mortality., several previous studies have shown that the mortality rate attributed to dai is 1.65-2.75 times higher than that attributed to no infection [13, 35, 36] . our study supports the findings of these published reports. in the present study, the multiple regression analysis indicated that patients with dais (compared to patients with no hai) had significantly increased likelihood of mortality (p < 0.05). moreover, the annual 30-day mortality rates of cautis and clabsis had significant changes over the period 2000 through 2008. these results may be caused by chance, because this study period did not change substantially in terms of medical care, novelty medical technology, and patient disease severity. in addition to the above-mentioned findings, we used a multiple regression analysis approach to adjust covariables, in addition to demographics, invasive devices, and laboratory investigations. we also identified severity of illness using apache ii scores as a predictor of mortality, with the results indicating that the hazard of mortality is associated with increasing scores. patients who died with dai infection were usually already severely ill and their existing illness, rather than the dai, was often the main cause of death. thus, an important prognostic factor was the severity of their illness, which resulted in an increased likelihood of mortality [14, 25] . we also found that patients with blood creatinine over 1.5 mg/dl were the highest risk group for dying. excluding an endogenous effect, the reason may be that many patients in this group were receiving hemodialysis with cvcs inserted. the rates of dais of all types decreased during the period 2005-2006, but this decrease was maintained [37] [38] [39] . some limitations of the present study should be noted. the study was performed at a single medical center. however, the results could be provided to the hospital as a part of the teaching or research mission. this study was a retrospective nine-year survey which might have some potential biases. in the analysis of long-term changes in infection rates or mortality rates, we must consider whether changes in the population, advances in laboratory diagnostic techniques, changes in exposure to risk factors, microbial culture and other factors lead to increased or decreased rates. however, there did not occur any outbreaks of dais during the study period, except for the sars outbreak. we have presented here the secular trend of dais at our institution in northern taiwan, and the great achievement of our infection control and surveillance program, which was the maintenance of a low dai incidence despite high device-utilization ratios. the incidence of dais decreased in 2005. the incidence of vap remained low, and the rate of antimicrobial resistance of the three most common pathogens causing vap decreased. implementation of infection control and traffic control bundles improved adherence to hand hygiene practices and antibiotic stewardship, and the impact of the sars pandemic on adherence to these practices might explain the decrease in dai incidence and rate of antibiotic resistance in 2005. this study also demonstrated that dai was an important independent prognostic factor of mortality. effectiveness of a nationwide nosocomial infection surveillance system for reducing nosocomial infections reproducibility of the surveillance effect to decrease nosocomial infection rates system report, data summary from surviving sepsis campaign guidelines for management of severe sepsis and septic shock international nosocomial infection control consortium (inicc) report, data summary for national healthcare safety 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teicoplanin: a sentry program risk factors associated with nosocomial methicillin-resistant staphylococcus aureus (mrsa) infection including previous use of antimicrobials relationship between spread of methicillin-resistant staphylococcus aureus and antimicrobial use in a french university hospital prevalence and nosocomial spread of methicillin-resistant staphylococcus aureus in a long-term-care facility in slovenia antimicrobial susceptibility among organisms from the asia/pacific rim, europe and latin and north america collected as part of test and the in vitro activity of tigecycline appropriate antimicrobial treatment in nosocomial infections-the clinical challenges device-associated infection rates and mortality in intensive care units of peruvian hospitals: findings of the international nosocomial infection control consortium device-associated infection rate and mortality in intensive care units of 9 colombian hospitals: findings of the international nosocomial infection control consortium secular trends of healthcare-associated infections at a teaching hospital in taiwan antimicrobial stewardship taiwan's traffic control bundle and the elimination of nosocomial severe acute respiratory syndrome among healthcare workers submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank the icps in the department of infection control, taipei veterans general hospital, for data collection during the study period. this study was supported by a part of research grant from taipei veterans general hospital (v99b2-006), taipei, taiwan. the funding institutes did not have any role in study design, data collection/analysis, the writing of the manuscript and the decision to submit the manuscript for publication. the authors declare that they have no competing interests.authors' contributions yyc participated in the design, data collection and analysis, and drafted the manuscript. lyc participated in the analysis and drafted the manuscript. syl and pc commented on drafts of the manuscript. syl participated in the data collection. fdw conceived of the project, participated in the design and helped to draft the manuscript. all authors approved the final manuscript. key: cord-354656-9ao33rq8 authors: cossart, yvonne e title: the rise and fall of infectious diseases: australian perspectives, 1914‐2014 date: 2014-07-07 journal: med j aust doi: 10.5694/mja14.00112 sha: doc_id: 354656 cord_uid: 9ao33rq8 australia has been fortunate in its experience with infectious diseases over the past century. by the 1960s, many communicable diseases were controlled through a combination of high living standards, progressive adoption of vaccines and antimicrobial treatment. australian medical scientists have made substantial contributions to the understanding of many historically significant communicable diseases and global initiatives for control. new challenges have emerged as previously unrecognised viral infections have emerged, and microbial resistance to antibiotics has developed in many old pathogens. ongoing evolutionary forces, both environmental and social, change the balance between humans and microbes. the effects of these forces are most sorely felt in poor countries and communities. i n 1914, when the british medical association launched the medical journal of australia, the medical profession and the general public believed that infectious diseases would soon be conquered. acrimonious 19th century disputes between the contagionists and the sanitarians 1 had given way to an alliance which was steadily improving health. rising living standards reduced infant deaths from gastroenteritis, through better food and water hygiene, and reduced deaths from "consumption" (tuberculosis), because of better nutrition. 2 the success of rat extermination in controlling plague in sydney 3 provided a triumphant validation of new microbiological theories; joseph lister's carbolic spray was adopted by local surgeons; 4 emil von behring's antitoxin treatment reduced mortality from diphtheria; 5 and paul ehrlich's vision of a magic bullet to cure all infections was given credibility by the effi cacy of salvarsan (arsphenamine, an arsenic-containing compound) against syphilis. 6 maritime quarantine provided a signifi cant, if not impregnable, barrier to the introduction of epidemic diseases. 7 the mja refl ected the progressive mentality of australian doctors through reports on new international discoveries and leading articles. in reality though, infection still caused at least a quarter of all deaths, 8 10% due to tuberculosis alone. many of the victims were children or young adults. eradication of bovine tuberculosis produced a welcome fall in infant cases, but about 4000 cases continued to be notifi ed annually between 1917 and 1950. the fi ne sanatorium buildings in "healthy" locations such as the blue mountains are reminders of the desperate attempts to combine sanitary and microbiological principles by isolating patients to prevent spread of the disease while they were treated with rest and diet. 9,10 9,10 during world war ii (wwii), intensive screening of australian troops by miniature x-ray was followed up with bacteriological testing to identify patients with active infection, for whom treatment was compulsory. the success of this program prompted a postwar attempt to eradicate the disease from the civilian population, and the advent of streptomycin and sickness benefi t payments made compulsory treatment acceptable to the community. 11 11 tuberculosis has again become resurgent in many countries. the high expense and long duration of triple therapy test the resources of the poorest countries. undertreatment allows emergence of multidrug-resistant strains for which treatment reverts to pre-antibiotic options. in australia, such strains are, mercifully, still uncommon. syphilis also imposed a high burden of chronic disease on society. in 1914, about 5% of admissions to mental institutions and 60% of cases of aortic aneurysm in victoria were due to tertiary syphilis, while 10% of pregnant women had positive wassermann test results. 12 12 the social stigma of sexually transmitted diseases and the heroic nature of combined mercurial and salvarsan therapy deterred many asymptomatic patients from seeking treatment. notifi cation of acute cases and compulsory treatment were not successfully implemented, even in the army. in civilian settings, policies of testing only female prostitutes doomed any hope of eradication. 6 many acute infections also remained endemic. there were dedicated wards for patients with typhoid in general hospitals, and the prevalence and mortality of typhoid remained high until the advent of antibiotics. bedside vigils to await the crisis of pneumonia were all too familiar, and childhood infections claimed many young lives. in 1911, gastroenteritis, diphtheria, scarlet fever, whooping cough and measles between them killed one of every 30 live-born children. 13 13 about as many more died from pneumonia and meningitis. in the interwar decades, diphtheria and pertussis vaccines were produced in the newly established commonwealth serum laboratories and school-based vaccination began. but in 1928, public confi dence was shaken by the bundaberg tragedy, in which a multidose vial of diphtheria toxin-antitoxin became contaminated with staphylococci from the skin of a vaccinee. twelve other children died. 14 14 this galvanised regulation of vaccine manufacture, safety testing and surveillance for adverse effects. however, almost a century on, the antivaccination movement still opposes mass vaccination of children in early life. during its fi rst few years of publication, the mja reported on mortality due to infection among wwi troops, which exceeded combat deaths. 15 15 but the battlefi elds were also a clinical laboratory where the effi cacy of tetanus antitoxin was proven and typhoid vaccination was introduced. the mja also reported episodes which, with hindsight, showed that evolutionary forces were altering the balance between microbes and their human hosts. in 1918, infl uenza killed more europeans than had perished in the war. the source of the pandemic strain remains obscure, but there is no doubt about the role of returning troops in its global spread. australia's isolation and its quarantine system protected it for some time, but eventually the country experienced a catastrophic outbreak. 12 12 australia became an active participant in the subsequent international efforts to maintain surveillance and produce effective infl uenza vaccines. pivotal infl uenza studies led by frank macfarlane burnet at the walter and eliza hall institute were conducted during this period, and the institute's interests soon broadened into basic scientifi c research on many important infections. in the postwar period, the establishment of the australian national university strengthened the national capability in infectious diseases research. poliomyelitis was on the increase in the most "hygienic" countries of scandinavia, north america and australasia: a paradox for an infection spread by the faecal-oral route. this was the penalty for the delay in the average age when infection occurred, which was a consequence of improved hygiene. this in turn increased the numbers of clinical cases because the chance of neurological involvement increased spectacularly with age. 16 16 the crippling legacy of infection made the sight of children with leg braces all too familiar in schools nationwide, while the invention of the iron lung enabled many patients with respiratory paralysis to survive. norman gregg's pivotal 1941 discovery of the role of rubella in causing congenital defects 17 17 altered scientifi c attitudes to infections during pregnancy. it was almost two decades before cell culture techniques paved the way for vaccines against polio, and then other childhood infections. the growing list of vaccines demanded new combinations to reduce the number of injections needed, and the cost became an issue even in wealthy countries where the plummeting prevalence of vaccine-preventable diseases justifi ed the investment. 18 18 in the second half of the 20th century, even the basic triple antigen (diphtheria, tetanus, pertussis) was unaffordable in many developing countries, but international philanthropy plus political support have progressively been mobilised, resulting in signifi cant reduction of vaccine-preventable disease. money is not the only problem. the projected global eradication of polio has stalled because confl icts in africa and pakistan have disrupted infrastructure and fanned ideological doubts about the political motivations of governments and charities. 19 19 in the optimistic political climate of the post-wwii years, the world health organization undertook an unprecedented program to achieve global eradication of smallpox. frank fenner was chairman of the project's management commission. using edward jenner's 18th century vaccination technique and an international army of fi eld workers, smallpox eradication was achieved in 1980. 20 20 it remains the only example of intentional eradication of a human infectious disease. not all microbial evolution resulted in populations of organisms with increased virulence for humans. scarlet fever, after causing devastating epidemics in the late 19th century, declined in terms of incidence and mortality by the 1950s. this was attributed to the loss of toxin-encoding genes from streptococcus pyogenes. conversely, recent resurgence of severe streptococcal infections underlines the mutability of "old" pathogens. 21 21 antimicrobial drugs sulfonamides made their spectacular entry into medicine just before wwii and cut the mortality from pneumonia and puerperal fever dramatically. prontosil, a forerunner of all sulfonamide drugs, was not patentable, and manufacturers fl ooded the market with sulfonamide-like drugs. penicillin soon followed -with australian scientist howard florey being a key fi gure in its development -and was quickly adopted in military medicine. the antibiotic era had begun; as discovery followed discovery, it seemed that no bacterial infection would remain untreatable. osteomyelitis, empyema, rheumatic fever and subacute bacterial endocarditis disappeared from the wards, and gonorrhoea and syphilis notifi cations plummeted. patients expected to recover from septicaemia, pneumonia and even meningitis; but it did not take long for the fi rst drug-resistant organisms to appear. sophisticated medical technology has offered unprecedented opportunities to microorganisms. drug resistance has rapidly developed owing to selective pressure resulting from profl igate use of antibiotics in medicine and agriculture. australian hospitals experienced some of the earliest outbreaks of antibiotic-resistant staphylococci, 22 22 which led to radical improvements in infection control. although most of the resistant organisms were no more (and often less) virulent than the original susceptible strains, their competitive advantage meant that acute urinary infections could no longer be reliably treated with ampicillin, nor gonorrhoea with penicillin, by the mid 1980s. discovery of new antibiotics could not keep pace, and regulatory attempts to restrict their use have proved diffi cult. 23 23 transfusion-associated hepatitis b infection was discovered during wwii. 24 24 after the virus was identifi ed in 1968, it was found that about 0.1% of most western populations were chronically infected, but carrier rates of 10% or more were common in asia and africa. 25 25 moreover, these rates diptheria* patients expected to recover from septicaemia, pneumonia and even meningitis; but it did not take long for the fi rst drug-resistant organisms to appear were maintained by silent transmission -from mother to infant during delivery, rather than by intravenous drug use or blood transfusion. 26 26 before blood banks started screening donated blood for hepatitis b, transfusion was a major route of transmission of acute hepatitis b infection in western countries. the need for mass screening prompted development of commercial testing kits, which have revolutionised laboratory diagnostic services. the most concerning feature of hepatitis b infection was the recognition during the 1970s that adult carriers often developed liver cancer, which was then the leading cause of cancer deaths in asia. 27 27 rather than a rarity, hepatitis b was a major global health problem; this justifi ed universal infant vaccination, which was made possible by recombinant dna technology in the 1980s. the hepatitis b vaccine was the fi rst human recombinant dna vaccine and the fi rst human cancer vaccine. other agents soon emerged from obscurity, often in response to changes in human activity. aids fi rst appeared in the gay communities of "world cities" in the early 1980s -the downside of sexual liberation. the inexorable rise in prevalence and apparently inevitable mortality of hiv infection spurred public health initiatives and scientifi c investigation. in australia, safe-sex campaigns had an almost immediate effect in reducing numbers of new cases, as did controversial needle-exchange and harm-minimisation strategies for injecting drug users. 28 28 global investment in research led to effective antiviral drugs and greatly extended the healthy lifespan of infected individuals in western countries. however, in the populous countries of africa, south america and asia, heterosexual transmission dominated and led to epidemics of neonatal infection via transplacental transmission. 29 29 this social and economic catastrophe reawakened respect for infection, and also fuelled fear of nosocomial transmission of bloodborne viruses. the shadowy entity of non-a, non-b hepatitis unexpectedly proved to cause both liver cirrhosis and cancer. acute hepatitis c infection causes only minor symptoms, but the hepatitis c virus often establishes chronic infection with sinister consequences. tests were developed to screen donated blood and it soon became apparent that injecting drug use had silently amplifi ed prevalence of hepatitis c infection in young people in western countries. 30 30 hepatitis c infection became the commonest indication for liver transplant in australia, 31 31 and health authorities struggled to fi nd an effective control strategy. treatment was protracted and beset by the adverse effects of interferon, a key component of drug regimens that were initially used. newer drugs achieve high cure rates, but cost puts them out of reach for patients in poor countries, where the reservoir of infection remains high. human papillomavirus was the fourth "new" infection to engage late 20th century society. genital warts were well known to the ancient romans, and for centuries they were regarded as an embarrassing but harmless sexually transmissible disease. this attitude changed dramatically when their association with cervical cancer was established in the 1980s. 32 32 the high-risk types of papillomavirus produce "fl at" penile warts which are easily ignored, but results of cervical cytology testing made the cancer connection -it was noted that cells with telltale warty changes were often seen in cervical smears which had malignant or premalignant changes. in australia, preventive human papillomavirus vaccination was pioneered by ian frazer and viral dna detection has been added to screening by pap smear, but neither of these approaches are affordable in poor countries, where death rates from cervical cancer are highest. 33 33 the challenges of demographic and environmental change in 2014, old infections still lurk, while human and environmental changes create new opportunities. malaria -which depends on humans as a reservoir for the parasite and mosquitoes for transmission -was vulnerable to a combination of treating patients with drugs and spraying the environment with dichlorodiphenyltrichloroethane (ddt) to kill mosquitoes. large areas of the temperate zone and even the tropics became malaria free but, because mosquito eradication measures were inconsistently applied, drug-resistant parasites and ddt-resistant mosquitoes soon emerged. in the absence of an effective vaccine, prevention now relies on avoidance of mosquito bites by use of repellents, protective clothing and screens, plus an ever-diminishing number of effective prophylactic drugs. malaria has been holding its own in many countries, aided by global warming. 34 34 military deployment of troops in exotic areas and largescale movement of refugees are associated with outbreaks of communicable diseases. cholera has been a recurrent problem when people seek shelter from war or natural disaster, most recently in haiti. 35 35 hantaan virus 36 36 caused over 3000 cases of korean haemorrhagic fever and almost 300 deaths among american troops during the korean war. the soldiers were exposed through inhalation of aerosolised rodent faeces when camped in wilderness areas. other haemorrhagic fevers -such as ebola virus disease and lassa fever, for which native wildlife act as reservoirs -have caused human outbreaks with high mortality, particularly in sub-saharan africa. australia has several indigenous arboviruses, including murray valley encephalitis, and ross river and barmah forest viruses. as global population pressure drives clearance of forested areas for agriculture, humans have become targets for many infections carried by wild animals. in australia, outbreaks of hendra virus infection and the emergence of the australian lyssavirus have been linked to contact with fl ying foxes. 37 37 severe acute respiratory syndrome (sars), which caused a deadly outbreak of respiratory disease centred in southern 38 38 but was transmitted to humans via infected palm civets, which were often for sale in chinese markets. control of sars was achieved through international cooperation in identifying the new coronavirus and applying strict isolation procedures. 39 39 even so, the high mortality brought home to the world the potential threat of contagious disease. memories of the 1918 infl uenza pandemic lent renewed vigour to the who surveillance system for respiratory infections that emerge from reservoirs of infl uenza viruses in pigs, horses, poultry and wild birds (the latter two in particular). alas, the ability to identify novel strains has not been matched by the ability to predict infectivity and severity of disease. intensive agricultural production also provides new routes of infection. mad cow disease resulted from the use of inadequately rendered animal-based food supplements for cattle which allowed the variant creutzfeldt-jakob disease agent to survive. 40 40 infected cattle often reached maturity and were slaughtered for human consumption before they developed clinical disease, and then humans became infected. the ramifi cations of this outbreak were economic and social. more new infections will undoubtedly emerge as humans change their environment. these pressures also affect old infections such as tuberculosis, malaria, cholera and even plague. the lessons of the past should not be forgotten. for a hundred years, the mja has reported on the overall decline of most infections in our "lucky country" -the result of our high standard of living combined with rational treatment and control measures. aboriginal australians who endure poverty and limited access to medical resources have not shared this luck. 41 41 this mirrors the disparity in communicable diseases mortality between industrialised and developing countries. abolishing this gap is the immediate priority for the forthcoming century. provenance: commissioned; externally peer reviewed anticontagionism between 1821 and 1867 the australian mortality decline: all-cause mortality 1788-1990 on the epidemiology of plague the beginning of antiseptic surgery in australia the diphtheria prophylactic of e. von behring sex, disease and society: a comparative history of sexually transmitted diseases and hiv/aids in asia and the pacifi c australian quarantine service. maritime quarantine administration. melbourne: arthur j mullett, government printer tuberculosis in new south wales: a statistical analysis of the mortality from tubercular diseases during the last thirty-three years consumption: report of a conference of principal medical offi cers on uniform measures for the control of consumption in the states of australia tuberculosis: its nature, prevention, and treatment, with special reference to the open air treatment of phthisis the epidemiology, mortality and morbidity of tuberculosis in australia: 1850-94 health and disease in australia: a history the mortality in australia from measles, scarlatina and diphtheria the hazards of immunization the australian army medical services in the war of 1914-1918. volume ii. canberra: australian war memorial natural history of infectious disease congenital cataract following german measles in the mother immunisation: a public health success progress toward global polio eradication -africa a successful eradication campaign. global eradication of smallpox severe streptococcal infections in historical perspective history of staphylococcal infection in australia control of fl uoroquinolone resistance through successful regulation jaundice occurring one to four months after transfusion of blood or plasma recent advances in the study of the epidemiology of hepatitis b vertical transmission of hepatitis b antigen in taiwan hepatitis b virus. the major etiology of hepatocellular carcinoma clinical and immunologic sequelae of aids retrovirus infection in and out of africa epidemiology of hepatitis c virus infection among injecting drug users in australia liver transplantation for hepatitis c-associated cirrhosis in a single australian centre: referral patterns and transplant outcomes papillomaviruses and cancer: from basic studies to clinical application hpv immunisation: a signifi cant advance in cancer control global malaria mortality between 1980 and 2010: a systematic analysis cholera surveillance during the haiti epidemic -the fi rst 2 years hemorrhagic fever with renal syndrome in korea emerging viral infections in australia bats are natural reservoirs of sars-like coronaviruses world health organization multicentre collaborative network for severe acute respiratory syndrome diagnosis. a multicentre collaboration to investigate the cause of severe acute respiratory syndrome an overview of bovine spongiform encephalopathy (bse) in britain indigenous disparities in disease-specifi c mortality, a cross country comparison: new zealand, australia, canada, and the united states key: cord-347064-ljd121no authors: josé, ricardo j.; brown, jeremy s. title: opportunistic bacterial, viral and fungal infections of the lung date: 2016-05-05 journal: medicine (abingdon) doi: 10.1016/j.mpmed.2016.03.015 sha: doc_id: 347064 cord_uid: ljd121no opportunistic infections are a major cause of morbidity and mortality in severely immunocompromised patients, such as those receiving chemotherapy or biological therapies, patients with haematological malignancy, aplastic anaemia or hiv infection, and recipients of solid-organ or stem cell transplants. the type and degree of the immune defect dictate the profile of potential opportunistic pathogens; t-cell-mediated defects increase the risk of viral (cytomegalovirus, respiratory viruses) and pneumocystis jirovecii infections, whereas neutrophil defects are associated with bacterial pneumonia and invasive aspergillosis. however, patients often have combinations of immune defects, and a wide range of other opportunistic infections can cause pneumonia. importantly, conventional non-opportunistic pathogens are frequently encountered in immunocompromised hosts and should not be overlooked. the radiological pattern of disease (best assessed by computed tomography) and speed of onset help to identify the likely pathogen(s); radiological imaging can subsequently be supported by targeted investigation including the early use of bronchoscopy in selected patients. rapid and expert clinical assessment can identify the most likely pathogens, which can then be treated aggressively, providing the best opportunity for a positive clinical outcome. opportunistic infections occur when a loss of established innate or adaptive immune responses allows an organism that is normally weakly virulent to cause infection. the type and degree of immune defect dictate which potential opportunistic pathogens are likely (table 1) . opportunistic lung infections are a major cause of morbidity and mortality for patients immunocompromised because of hiv infection, haematological malignancy, aplastic anaemia or chemotherapy treatment, or who are recipients of solid-organ or stem cell transplants, and also can complicate treatment with the new biological therapies for inflammatory conditions. immunocompromised patients also have an increased risk of infections caused by more conventional pathogens, which should be considered in the differential diagnosis. expert clinical assessment with early diagnosis and aggressive treatment are required for a positive outcome. computed tomography (ct) is more sensitive than plain chest radiography for defining the predominant pattern(s) of lung involvement. combined with knowledge of the patient's immune status (loss of t-cell or antibody-mediated immunity, or defects in neutrophil-mediated immunity), this can often identify the most likely pathogens. this article provides a concise overview of the most common opportunistic lung infections. conventional bacterial pathogens although the risk of opportunistic infection is high in immunocompromised patients, most pneumonias are related to the more key points c knowledge of the immune defect helps to narrow down the potential pathogens c computed tomography of the chest is better than radiographs at defining the radiological pattern of disease in immunocompromised hosts c in selected patients, early bronchoscopy increases the yield of microbiological identification of a potential pathogen c prolonged high-dose glucocorticoids (>20 mg/day for >21 days) predispose to pneumocystis jirovecii pneumonia (pjp) c biological agents are associated with specific immune defects that increase the risk of opportunistic lung infections (e.g. tumour necrosis factor-a inhibitors and risk of mycobacterial disease, endemic fungi and legionella pneumophila; anti-cd20 drugs and mycobacterial disease, cytomegalovirus pneumonitis and pjp) c due to the increase in azole resistance of aspergillus fumigatus, combination of an azole with an echinocandin antifungal agent is recommended in immunocompromised hosts with severe invasive pulmonary aspergillosis conventional bacterial pathogens. these are particularly common after a viral illness. they usually present similarly to pneumonia in immunocompetent individuals 1 with fever, respiratory symptoms, focal consolidation and rapid rises in inflammatory markers. the major risk factors are neutropenia, antibody deficiencies and high-dose corticosteroids. the organisms involved are more diverse than those seen in conventional pneumonia and are more likely to be resistant to first-line antibiotics. they include both gram-positive (streptococcus pneumoniae, staphylococcus aureus) and gram-negative (e.g. pseudomonas aeruginosa, proteus species, escherichia coli, other enteric pathogens) organisms. reactivation of latent tuberculosis occurs in patients with t-cell immune defects. mycobacterium tuberculosis cultures and polymerase chain reaction (pcr) should be carried out on respiratory samples from immunocompromised individuals with pulmonary infiltrates who were born or live in countries with a high prevalence of tuberculosis. non-tuberculous mycobacterial infections can present similarly. mycobacterial infections are usually readily diagnosed by microscopy and culture or by biopsy of affected lung tissue. nocardiosis is an uncommon gram-positive bacterial infection with a high mortality when disseminated. there are over 80 nocardia species, but human disease is usually caused by the nocardia asteroides complex. nocardia is found in soil, decaying vegetable matter and stagnant water. inhalation is the most common route of entry so pneumonia is the most common infection. the main risk factors are defects in t-cell-mediated immunity (e.g. after transplantation), prolonged glucocorticoid therapy, malignancy, graft-versus-host disease (gvhd), diabetes mellitus, chronic granulomatous disease and alveolar proteinosis. nocardia pneumonia usually develops over weeks with cough, haemoptysis, weight loss, fever and night sweats but can be more acute. common radiological features are patches of dense consolidation or macronodules, frequently pleurally based. cavitation and pleural effusions are common. these appearances can be mistaken for metastases, mycobacterial infection or invasive aspergillosis. local spread to the pericardium and mediastinum, and haematogenous spread to brain, joints and soft tissue, occur in about half of patients. a rapid diagnosis can be made by identifying the characteristic beaded, branching gram-positive and weakly acid-fast filaments on microscopy of tissue or respiratory samples. blood and sputum cultures can be positive but require prolonged aerobic culture. susceptibility to antibiotics varies among the nocardia species, and treatment with two or three intravenous antibiotics may initially be necessary in immunocompromised individuals. trimethoprimesulphamethoxazole is firstline therapy, with carbapenems, amikacin, third-generation cephalosporins, tetracyclines or amoxicillineclavulanate as alternatives. the duration of treatment is prolonged (up to 12 months) in immunocompromised patients and with central nervous system (cns) disease. lower respiratory tract infections with the respiratory viruses (respiratory syncytial virus, parainfluenza, influenza, adenovirus, metapneumovirus, coronavirus, rhinovirus) are relatively common in immunocompromised patients with defects in t-cellmediated immunity. respiratory viruses usually cause bronchiolitis that presents with coryzal symptoms, cough, fever and dyspnoea. in a minority of patients, auscultation of the lungs reveals characteristic squeaks or wheeze. the chest radiograph is often normal or non-specific. ct classically demonstrates 'tree-in-bud' changes suggestive of small airways inflammation or diffuse ground-glass opacities. the diagnosis is confirmed rapidly using nasopharyngeal aspirate samples for viral antigen immunofluorescence or pcr for viral nucleic acids, the latter being favoured in immunocompromised hosts. if nasopharyngeal aspirate findings are negative, immunofluorescence or pcr of bronchoalveolar lavage fluid (balf) has a higher sensitivity. in the absence of pneumonia, mortality from respiratory virus infection is relatively low, although infection can persist for several weeks. treatment is supportive, but in immunocompromised hosts specific antiviral treatment is recommended ( table 2 ). in cases of severe infection, combination with intravenous immunoglobulin should be considered. viral infection, particularly influenza (including h1n1) has effects on lung host defences and predisposes to secondary bacterial infection, 1, 2 which in immunocompromised hosts (particularly after chronic glucocorticoid use, chemotherapy for cancer and haemopoietic stem cell transplantation (hsct)) can lead to more severe illness. clinically, this is suspected when there is relapse of fever, increased oxygen requirements and c-reactive protein concentration, and radiographic evidence of more dense consolidation or infiltrates. antibiotic treatment for secondary bacterial infection should cover the organisms most commonly encountered after influenza, including strep. pneumoniae, staph. aureus and haemophilus influenzae. novel viruses that have recently emerged, such as middle east respiratory syndrome coronavirus and avian influenza a strain h7n9, are potential rare causes of severe pneumonias in immunocompromised patients. 1 the herpesvirus cmv is an important cause of lung infection in patients with impaired t-cell-mediated immunity, such as transplant recipients. cmv infection is defined as active cmv replication irrespective of symptoms or signs, while cmv disease is infection associated with evidence of organ-specific disease. cmv infection in immunocompromised patients is usually caused by reactivation of latent cmv acquired in early life, but can also be primary infection in previously uninfected individuals, in whom it is often more severe. pneumonitis is an important complication and commonly presents with an insidious onset of fever, malaise, cough and dyspnoea with hypoxia. classic features on ct are symmetrical peribronchovascular and alveolar infiltrates predominantly affecting the lower lobes, but asymmetrical changes, consolidation and effusions are not uncommon. in suspected cmv infection/disease, cmv replication can easily be identified and the viral load determined by pcr or cmv pp65 antigen testing of blood or balf. cmv infection is also identified by culture of urine, throat and balf specimens. evidence of cmv reactivation does not always mean that concurrent lung disease is caused by cmv, and conversely cmv viraemia can occasionally be absent in patients with cmv pneumonitis. cmv pneumonitis is more likely with high-level viraemia, especially if the viral load increased rapidly. cmv pneumonitis can be confirmed by finding inclusion bodies in balf cells or transbronchial or video-assisted thoracic surgery (vats) biopsy samples. first-line treatment of cmv pneumonitis is intravenous ganciclovir or oral valganciclovir (unlicensed indication). secondline treatments include foscarnet (unlicensed indication), cidofovir (unlicensed indication) and maribavir. cmv immunoglobulin can be used as an adjunct to therapy in immunocompromised individuals. treatment efficacy is monitored by measuring blood cmv viral load, with treatment usually continued for at least 2 weeks after resolution of viraemia. other herpesviruses, such as herpes simplex virus (hsv), varicella zoster (vzv) (both associated with the characteristic rash) and human herpesvirus (hhv) 6, are rare causes of diffuse pneumonitis similar to cmv in the immunocompromised host. firstline treatment of hsv and vzv is with aciclovir, but valaciclovir, famciclovir (licensed for hzv, unlicensed for hsv), cidofovir (licensed for hsv) and foscarnet (licensed for hsv, unlicensed indication for hzv) can also be used. no drug has been specifically approved for the treatment of hhv-6, but ganciclovir and foscarnet are recommended by experts for the treatment of severe hhv-6 infection. 3 treatment options for fungal pneumonias are listed in table 3 . pneumocystis jirovecii (formerly p. carinii) pneumocystis jirovecii pneumonia (pjp) is the most common aids-defining illness (cd4 counts <200 cells/mm 3 ) but is also important in non-hiv immunocompromised patients with defects in t-cell-mediated immunity or who are taking prolonged high-dose systemic glucocorticoids. clinical presentation is classically with slowly increasing dyspnoea, dry cough and hypoxaemia, with few physical or radiological findings, but can be more rapidly progressive fulminant disease. exercise-induced oxygen desaturation is a sensitive clinical marker. chest radiographs may show diffuse, bilateral, interstitial infiltrates but can be normal, whereas high-resolution ct is much more sensitive and often shows extensive ground-glass opacities with an apical distribution and peripheral sparing. pneumatocoeles are not uncommon, and chronic infection can lead to bizarre-looking cystic changes. p. jirovecii cannot be cultured, and diagnosis requires identification of the organism in induced sputum or balf by microscopy with giemsa and grocott staining. immunofluorescence and pcr techniques increase the diagnostic yield, but false-positive pcr can occur because of lung colonization by p. jirovecii. p. jirovecii can be found in balf for 48e72 hours after starting empirical treatment. first-line treatment is high-dose trimethoprim esulphamethoxazole for 21 days, with adjunctive corticosteroids for severe hypoxaemia (po 2 <8 kpa) ( table 3) . second-line therapies include clindamycin plus primaquine, pentamidine, atovaquone, or trimethoprim plus dapsone. prophylaxis with trimethoprimesulphamethoxazole or nebulized pentamidine is recommended in patients with hiv infection (cd4 count <200 cells/mm 3 ), transplant recipients (solid-organ and hsct) and patients receiving prolonged high-dose glucocorticoids (>20 mg/ day for 21 days or longer). mortality is approximately 10%. aspergillus species are ubiquitous and are continuously inhaled but usually establish infection only when there are major defects in phagocyte function, such as severe and prolonged neutropenia (e.g. after hsct or aplastic anaemia), in patients taking highdose glucocorticoids, or with haematological malignancy or chronic granulomatous disease. chronic gvhd is also a significant risk factor and, rarely, patients with chronic lung disease or milder forms of immunosuppression develop semi-invasive forms of aspergillosis. the most common infective species is aspergillus fumigatus. the respiratory tract (including the sinuses) is most often affected, although blood-borne spread to internal organs (especially the cns) and skin can occur. the classic presenting triad in invasive pulmonary aspergillosis (ipa) is fever, chest pain and haemoptysis, although fever alone or various respiratory symptoms can occur. aspergillus has a predilection for growing into blood vessels, potentially causing fatal massive haemorrhage. chest radiographs show patchy infiltrates or nodules that can cavitate. ct features include macronodules (single or multiple, with or without cavitation) or patchy consolidation. nodules can show the 'halo' (surrounding ground-glass infiltrates caused by haemorrhage) or 'air-crescent' (cavitation around a fungal ball) signs. when the patient's immune function recovers, fungal balls can form in a walled-off cavity created by the invasive phase of the disease. other manifestations of invasive aspergillus infections affecting the lung include: aspergillus tracheobronchitis, in which infection is restricted to the tracheobronchial tree, causing a relentless cough. ct may show focal bronchial wall thickening and 'tree-in-bud' changes. bronchoscopy is diagnostic, identifying highly inflamed mucosa with necrotic white slough that is positive for aspergillus on culture and histology. chronic necrotizing pulmonary aspergillosis (cnpa) or chronic cavitary pulmonary aspergillosis (ccpa), which are more indolent forms of invasive aspergillosis associated with mild immunosuppression or chronic lung disease. these present with a long history of cough and frequently with marked systemic symptoms. there is also a slowly progressive patch of consolidation with or without cavitation (cnpa) or an expanding dry upper lobe cavity with a thickened wall (ccpa). diagnosis of ipa is suggested by detection of galactomannan (a relatively specific cell wall component) or b-d-glucan (a cell wall component of many fungi including aspergillus and pneumocystis) antigen in blood or balf. false-positives galactomannan antigen results occur with concomitant treatment with b-lactam antibiotics. definitive diagnosis of ipa is made by positive culture for aspergillus and histopathological demonstration of tissue invasion on ct-guided or vats biopsy specimens. histology is highly sensitive, showing dichotomous (45 ) branching of septated hyphae on gomori methenamine silver or periodic acideschiff staining. however, histology specimens are often unavailable, and culture is relatively insensitive, so diagnosis is frequently made on clinical grounds (suggestive ct appearances, high-risk patient, with or without a positive galactomannan test). aspergillus antibodies have no role in the diagnosis of ipa but are positive in ccpa and sometimes in cnpa. there has been a worldwide increase in a. fumigatus resistance to azoles, 4 and treatment with a combination of an azole and an echinocandin antifungal agent may be necessary in immunocompromised hosts with severe ipa. non-aspergillus filamentous fungi filamentous fungi, including fusarium, zygomycetes, scedosporium and penicillium, can cause invasive pulmonary infections in immunocompromised patients with a clinical presentation similar to ipa. diagnosis is made by culture from respiratory samples or lung biopsy, and is important as some species are resistant to conventional antifungal agents. galactomannan and b-d-glucan cell wall antigen tests are negative in zygomycetes infections. mortality is high. direct pulmonary invasion by candida species is rare even in immunocompromised patients, despite frequent isolation from sputum. pulmonary infection usually occurs in neutropenic patients as haematogenous spread from infected indwelling vascular catheters or infections related to transplant surgery. lung nodules are often peripheral and sometimes very large. candida albicans is most commonly identified, but a range of non-albicans candida (e.g. candida parapsilosis, candida tropicalis, candida glabrata, candida krusei) can cause disease. a novel culture-independent test allows for the rapid detection of candida in blood, with a particularly high negative predictive value e positive results require confirmation by culture. 5 infected indwelling lines should be removed. cryptococcus neoformans pneumonia almost always affects only immunocompromised patients and can present with dyspnoea, cough and fever. hiv/aids (cd4 count <200 cells/mm 3 ) is the most common risk factor but cryptococcal pneumonia also occurs in other defects of t-cell-mediated immunity (especially after solid organ transplantation). radiological features include diffuse interstitial infiltrates, focal consolidation, discrete nodules and hilar lymphadenopathy. diagnosis is by microscopic identification (indian ink stain) or culture from respiratory tract samples. the lung is the port of entry for disseminated infection (usually cns), and neurological symptoms should prompt a lumbar puncture and cerebrospinal fluid culture. endemic fungi are found in specific geographical areas and cause primary infection by inhalation or inoculation of contaminated material (e.g. bat faeces). reactivation of latent infection can occur in immunocompromised patients, especially with defects in t-cell-mediated immunity, so a history of travel or residence in a high-risk area can be relevant. common endemic fungi causing pulmonary infections include histoplasma capsulatum, coccidioides (candida immitis, candida posadasii), blastomyces dermatitidis and sporothrix schenckii. presentation varies by pathogen but tends to mimic tuberculosis, with cavitating pneumonias, pulmonary nodules, enlarged mediastinal and hilar lymph nodes, or a miliary pattern. systemic dissemination is not uncommon in immunocompromised patients. diagnosis requires identification of the fungus in respiratory samples or biopsy material, including bone marrow aspirates. culture can take 6 weeks. hist. capsulatum can be rapidly detected with an antigen detection assay but this can cross-react with other endemic fungi. serology identifies patients with previous exposure for most fungi but is not reliable in immunocompromised patients. mortality is high without timely appropriate treatment. community-acquired pneumonia viral pneumonia prevention and treatment of cancer-related infections genomic context of azole resistance mutations in aspergillus fumigatus determined using whole-genome sequencing t2mr and t2candida: novel technology for the rapid diagnosis of candidemia and invasive candidiasis key: cord-352230-8mazd3eu authors: beeraka, narasimha m.; sadhu, surya p.; madhunapantula, subbarao v.; rao pragada, rajeswara; svistunov, andrey a.; nikolenko, vladimir n.; mikhaleva, liudmila m.; aliev, gjumrakch title: strategies for targeting sars cov-2: small molecule inhibitors—the current status date: 2020-09-18 journal: front immunol doi: 10.3389/fimmu.2020.552925 sha: doc_id: 352230 cord_uid: 8mazd3eu severe acute respiratory syndrome-corona virus-2 (sars-cov-2) induced coronavirus disease 19 (covid-19) cases have been increasing at an alarming rate (7.4 million positive cases as on june 11 2020), causing high mortality (4,17,956 deaths as on june 11 2020) and economic loss (a 3.2% shrink in global economy in 2020) across 212 countries globally. the clinical manifestations of this disease are pneumonia, lung injury, inflammation, and severe acute respiratory syndrome (sars). currently, there is no vaccine or effective pharmacological agents available for the prevention/treatment of sars-cov2 infections. moreover, development of a suitable vaccine is a challenging task due to antibody-dependent enhancement (ade) and th-2 immunopathology, which aggravates infection with sars-cov-2. furthermore, the emerging sars-cov-2 strain exhibits several distinct genomic and structural patterns compared to other coronavirus strains, making the development of a suitable vaccine even more difficult. therefore, the identification of novel small molecule inhibitors (nsmis) that can interfere with viral entry or viral propagation is of special interest and is vital in managing already infected cases. sars-cov-2 infection is mediated by the binding of viral spike proteins (s-protein) to human cells through a 2-step process, which involves angiotensin converting enzyme-2 (ace2) and transmembrane serine protease (tmprss)-2. therefore, the development of novel inhibitors of ace2/tmprss2 is likely to be beneficial in combating sars-cov-2 infections. however, the usage of ace-2 inhibitors to block the sars-cov-2 viral entry requires additional studies as there are conflicting findings and severe health complications reported for these inhibitors in patients. hence, the current interest is shifted toward the development of nsmis, which includes natural antiviral phytochemicals and nrf-2 activators to manage a sars-cov-2 infection. it is imperative to investigate the efficacy of existing antiviral phytochemicals and nrf-2 activators to mitigate the sars-cov-2-mediated oxidative stress. therefore, in this review, we have reviewed structural features of sars-cov-2 with special emphasis on key molecular targets and their known modulators that can be considered for the development of nsmis. covid-19 is a devastating disease caused by a coronavirus related to the one that caused outbreaks of severe acute respiratory syndrome (sars) in the year 2002 (1, 2) . middle east respiratory syndrome (mers)-related coronavirus is an infamous member of this cohort. covid-19, which is caused by the sars-cov-2 infection, was detected in wuhan, china in december 2019. the world health organization (who) declared this infection a pandemic on march 11 2020 due to its severity and rapid spread across the globe. as of june 11 2020, sars-cov-2 had infected 7.4 million individuals, and caused 4,17,956 deaths across 212 countries worldwide ( table 1) . coronaviruses (cov) belongs to a family of single-stranded rna viruses (+rna) that can infect a variety of mammals such as bats and humans (3) . sars-cov-2 contains rna of 29,891nucleotide length, which codes for 9,860 amino acids (4) . the rna has a 5' cap and 3' poly-a tail and produces a poly-protein 1a/1ab (pp1a/pp1ab) in the host (4) . sars-cov-2 belongs to beta cov category and appears in a crown shape with a size of ∼60-140 nm (figure 1) . gene sequencing data revealed that sars-cov-2 has 89 and 82% sequence similarity with bat sars-like-cov-zxc21 and human sars-cov, respectively (4, 5) . the spike (s) proteincoding gene mutation in the nsp2 and nsp3 regions results in the replacement of glycine (g) with serine (s) at 723 position (g723s), and an isoleucine (i) replaced with proline (p) at 1010 amino acid position (i1010p). due to these mutations, the invading potential of sars-cov-2 has increased significantly toward host tissues. this virus can also be transmitted through the respiratory droplets from coughs and sneezes of infected individuals (4) . this mode of aerosol transmission is possible, especially, when protracted exposure occurs in closed areas (4) . the incubation time of the virus varies significantly from individual to individual. in general it takes about 6 days from the day of infection to the first appearance of symptoms. however, in a few cases the symptoms may appear only after 2 weeks (6) . members of coronaviridae are known to induce respiratory complications in humans (7, 8) . at first, sars-cov, mers-cov, and sars-cov-2 varieties were transmitted from animals to humans which triggered severe respiratory diseases (9) (10) (11) . however, subsequent transmission occurred among humans primarily due to physical contact. hence, conventional preventive measures such as physical isolation were implemented to avoid propagation of early infection across the human population (1, 12) . similar to the sars-cov, the pathological manifestations of sars-cov-2 could induce lung malfunction in humans as indicated by the severe acute respiratory syndrome and pneumonia (12) . recent studies reported that sars-cov-2 infection can induce mild, moderate, and severe illness in infected patients (4) . clinical manifestations of this infection include chronic pneumonia, sepsis, septic shock, fever, and dry cough (4) . a progressive respiratory failure during this infection may lead to sudden death (4) . mild illness resulting from a sars-cov-2 infection is characterized by the presence of malaise, headache, low fever and dyspnea. in the case of moderate illness from sars-cov-2, the complication is manifested by the presence of cough and mild pneumonia. severe illness from sars-cov-2 is associated with chronic pneumonia, cough, sars, hypoxia, and tachypnea (in children) followed by respiratory, and cardiovascular system failure (4). the autopsy and biopsy reports of sars-cov-2 patients revealed severe edema with pulmonary tissue exudates, focal reactive hyperplasia, damage to pneumocytes as well as alveolar macrophages, and patchy cellular infiltration (13) . coronavirus-induced lung damage has been demonstrated experimentally by several investigators in animal models (14) . for instance, the sialodacryoadenitis virus and parker's rcov were shown to induce damage to alveolar type-i cells through the expression of pro-inflammatory cytokines, and chemokines such as cinc-2, cinc-3, lix, mip-3α, and fractalkines (15) (16) (17) (18) (19) (20) (21) . for example, fractalkine promotes the infiltration of cytotoxic lymphocytes in the alveolar epithelium thereby inducing a severe inflammatory response (15, 22) . similarly, mip-3α confers the chemotaxis of immune cells via il-1β and tnf-α inflammatory mediators (17, (22) (23) (24) (25) . therefore, these animal models could be used to develop effective pharmacological agents against sars-cov-2 infections. studies from several laboratories have demonstrated that the entry of sars-cov-2 into human cells is facilitated by ace-2 (26) . ace-2 is a member of the renin-angiotensin system (ras), which plays a vital role in cardiovascular and renal homeostasis. ace-2 and tmprss2 facilitates the entry of the virus into host cells during sars-cov-2 infection (7). in addition, there are other proteases such as aminopeptidase n (apn) which plays a prominent role for the entry of hcov-nl63 and hcov-229e into host cells (27) (28) (29) (30) . apn is a membrane-bound glycoprotein that mediates the zincdependent protease activity during the entry and or replication of coronavirus strains into host cells (29, 31, 32) . hence, the ace-2 receptor's down-modulation may prevent sars-cov-2 viral entry/replication (33) . the s-protein of sars-cov and other coronavirus strains are different in their structural and functional domains (3). s-protein can bind to the n-terminus of ace-2 receptors on the outer surface of host cells including respiratory epithelium of the lungs (34) (35) (36) . identifying the key amino acid residues in s-protein of the sars-cov-2 strain may benefit virologists and medical scientists to develop better therapeutic agents. however, to date these details are not known, hence, there is an immediate requirement to identify the amino acids involved in binding s-proteins to ace-2 receptors on host cell surfaces. furthermore, investigations should also focus on establishing the structural similarities of s-protein motifs that are interacting with the ace-2 receptors of other coronavirus strains (37) (38) (39) (40) (41) . these investigations might help in deciphering molecular strategies to target receptor binding sites of ace-2 proteins with sars-cov-2 using novel therapeutics and vaccines to avoid membrane fusion process and viral entry (7) . the tmprss2 protease can foster the entry of the sars-cov-2 virus by activating the s-protein for virus-host cell membrane fusion, consequently enhancing viral replication in the host cells (7, (42) (43) (44) (45) (46) . tmprss2 plays a vital role in generating inflammatory cytokines and chemokines in lung epithelial cells by cleaving s-protein during coronavirus infections including sars-cov-2. hence, tmprss2 is another potential therapeutic target to consider for the novel drug development against sars-cov-2 (46) (47) (48) . prevention and treatment of sars-cov-2 infections are achieved at different levels (49) . the primary approach involves physical isolation to prevent the spread of virus from individual to individual; the second approach involves inhibiting the entry of virus into human cells and the third method includes treating the infected individuals to minimize inflammatory reactions and blocks cathepsin l required for sars-cov processing note: yet to be examined against sars-cov-2 infection blocks sars-cov interaction with ace-2 note: yet to be examined against sars-cov-2 infection [n-(9,10-dioxo-9,10-dihydroanthracen-2yl)benzamide] blocks sars-cov fusion to host cell membrane note: yet to be examined against sars-cov-2 infection (67) nct numbers were obtained from https://clinicaltrials.gov/. pulmonary damage. although physical isolation is the ideal way of limiting the spread, in reality this approach is difficult to execute, hence, many pharmacological companies are actively involved in developing small molecule inhibitors to prevent the entry of the virus into human hosts (7, 49) . in this regard several nsmis have been investigated to treat sars-cov; but, significant breakthroughs are yet to come for treating sars-cov-2 (48) ( table 2) . adedeji et al. (49) reported the discovery and characterization of novel inhibitors to block sars-cov replication via different mechanisms. one mechanism uses screening of small molecule inhibitors using "hiv-1 pseudotyped with sars-cov surface glycoprotein s (sars-s)" (49, 68) . "ssaa09e2" is a novel small molecule inhibitor, which blocks the interaction of cov sars-s with ace-2 receptors, thus blocking the viral entry (49) . another nsmi is "ssaa09e1" reported to be involved in blocking the cathepsin l, which is required for cov-sars-s processing to mediate viral entry into the host cell (49) . ssaa09e3 is another nsmi, which can block the fusion of viral membranes with host cell surfaces (49) (figure 2 ). since the pathological aspects and genomic similarity of sars-cov-2 virus with sars-cov, the above strategies of inhibition may figure 2 | molecular pathogenesis of sars-cov-2 in human lung cells. binding of s-protein of sars-cov-2 to the ace-2 receptors triggers the processing of ace-2 through adam-17/tnf-α-converting enzyme and induces the "ace-2 shedding" into the extracellular space and facilitates uptake of sars-cov-2 followed by the development of sars. alternatively, the entry of sars-cov-2 by membrane tmprss2 serine protease'/hat (human airway trypsin-like protease)-mediated cleavage of ace2 can facilitate sars-cov s-glycoprotein-mediated virus entry. even though, several nsmis targeting these processes were described and their mode of action against coronavirus were delineated, their efficacy against sars-cov-2 is yet to be tested. be considered for developing potent pharmacological agents to prevent sars-cov-2 infections (49) . however, the prospective research should address the efficacy of these inhibitors against sars-cov-2 infections. cytokine storm was predominantly reported during sars-cov-2 infection. targeting cytokine-mediated inflammatory responses induced by sars-cov-2 is another viable approach for mitigating the complications of viral infection. in this regard, chang et al. (35) , documented the inflammatory cascades mediated through intracellular signaling pathways conferred by the sars-cov in both lung epithelial cells and fibroblasts. authors of this study have reported that s-protein of sars-cov efficiently mediate the il-8 release in the infected lung cells by activating mapkinases, and activator protein-1 (ap-1) without intervention of nf-kb cascade (35) . this study suggested a promising lead for novel rational drug design through the identification of a "specific sequence motif of sprotein functional domain, " which is responsible for inducing il-8-mediated inflammatory response in lungs (35) . baricitinib is a pharmacological agent, which was reported to block the sars-cov-2 viral entry and inflammation through the inhibition of ap2-associated protein kinase 1 (aak1), cyclin g-associated kinase, and janus kinase-1 and 2 (69) . chloroquine (cq) and hydroxychloroquine (hcq) were reported to be effective in mitigating the coronaviral load (70, 71) . cq and hcq not only inhibit the entry of sars-cov-2 but also change the ph of acidic intracellular organelles such as endosomes and lysosomes thereby preventing membrane fusion reactions. however, many contradictions and queries prevail pertaining to the use of hcq for the treatment of covid-19. at the time of the submission of this review, results of many clinical trials are yet to be announced, hence, the efficacy of hcq for inhibiting sars-cov-2 infection is still a possibility. prospective studies should focus on testing the fda approved inhibitors of "abl-1 kinases, " "pi3k/akt/mtor" signaling, and "mapkinase" pathways against sars cov-2. since these pathways are involved in cell survival, inflammatory cytokines production, and proliferation of cells, targeted downregulation of these pathways is likely to mitigate the exacerbations induced by coronavirus. in this direction, many of these inhibitors are currently being tested against sars-cov-2 ( table 3 ) (72) (73) (74) . for instance, sorafenib, which inhibits raf, is being experimented in preclinical models and early clinical trials (72) . likewise, the efficacy of il-1 receptor antagonists and tnf-α receptor antagonists for blocking the rat coronavirus-mediated chemokine production was already proven effective in animal models (15, 75, 76) . further studies testing the safety and efficacy are warranted before considering these inhibitory agents for treating individuals infected with sars-cov-2 (76). however, the concept of "one drug to treat all" should be followed to combat several devastating viral infections (77, 78) . for instance, the ebola, marburg, and sars-cov-2 are undoubtedly devastating viral pathogens, which can induce high mortality as they transmit rapidly via air and body fluids (78) . outbreaks of these viruses occur sporadically and currently there are no clinically approved nsmis available to combat these viruses. a recent report by taylor et al. (79) demonstrated the efficacy of a synthetic adenosine analog, bcx4430 in blocking a broad spectrum of viral species viz., "coronaviruses, paramyxoviruses, and bunyaviruses" as these viruses could induce sars, measles, and mumps. bcx4430 could efficiently block both ebola and marburg viral titers in non-human primate models by targeting viral rna polymerase (78, 79) . hence, this molecule should be tested for further studies against sars-cov2 infections in humans. targeting the membrane protease involved in viral s-protein processing and the viral entry into host cells is another approach in mitigating sars-cov-2. the host cellular proteases viz., "trypsin, ", "miniplasmin, " "human airway trypsin like protease, " "tryptase clara, " and "tmprss2" could cleave the ha glycoprotein located in influenza a virus and thereby promote viral entry into lung cells (80) . the usage of serine protease inhibitors such as camostat and aprotinin significantly blocked the replication of influenza virus in epithelial cells of lungs and bronchioles (81) . in addition, these nsmis could block the release of inflammatory mediators such as cytokines, il-6 and tnf-α, during this infection (81) . tmprss2 is a key protein involved in the pathogenesis of several seasonal viral infections including influenza, h1n1, h3n2, and h7n9 (82) (83) (84) (85) . tmprss2 cleaves the s-protein of coronavirus to produce unlocked, fusion-catalyzing viral forms and binds to the host cell surface thereby enhancing rapid viral entry (43, 44, (86) (87) (88) (89) (90) . both sars-cov and mers-cov could rapidly enter into the host cells as tmprss2 can facilitate viral binding to the cell surface (42, 43, 45, 87, 91, 92) . tmprss2 also plays a vital role in the immuno-pathology of coronavirus infections including sars-cov-2 across lungs by inducing lung fibrosis (46) . hence, the emerging research should promote the development of nsmis to target these proteases thereby hindering the entry of sars-cov-2 into host cells. a proof-of-concept study by iwata-yoshikawa et al. (46) reported that sars-cov failed to replicate in the bronchioles and lungs of tmprss2 knockout mice. authors of this study reported elevated expression of tlr3-mrna expression in the lungs of "sars-cov-inoculated tmprss2-deficient mice" and showed enhanced tlr-3 mediated localization of dsrna into endosomes (46) . in this study, tmprss2 knockout has resulted in downregulation of inflammatory cytokines and chemokine expression, which are involved in the bronchiolitis obliterans organizing pneumonia (boop), sars, and pulmonary fibrosis in sars-cov infection (46, 93, 94) . the intricate sars-cov-2 pathogenesis is similar to that of sars-cov. studies have reported the efficacy of ifns to block sars-cov in cell line models but not against sars-cov-2. among ifnα/ -β/ and -γ, the ifn-β was reported to be the most potent blocker of sars-cov growth (3, (95) (96) (97) (98) . furthermore, ifn-β and-γ have a synergistic effect in blocking sars-cov viral replication (62, 63) . however, the effect of this combination against sars-cov-2 is not yet reported. therefore, future studies should focus on determining the efficacy of ifnα/ -β/ and -γ against sars-cov-2 infections. unlike small molecule inhibitors, sirnas are specific and can be designed to mitigate sars-cov associated structural proteins by targeting orf4 (99, 100), orf5 (101, 102), orf9a (50, 103, 104) , and orf7a (50, (105) (106) (107) . for example, sirnas sisc2 and sisc5 have shown success in cultured cells as well as in preclinical mouse models in inhibiting the sars infection without causing toxicity (108) . several other reports have also recently demonstrated the efficacy of sirnas to inhibit the expression of sars-cov genes coding for 3cl protease in cell line models (108) (109) (110) (111) (112) (113) (114) . the activity of sars-cov 3cl protease is essential for viral replication as this protein is involved in the processing of viral proteins (114) . selective optimization and screening of hexa-chlorophene analogs can be "active 3cl protease inhibitors" during a sars-cov infection (114) . hence, the pharmacological agents/sirnas targeting these pathways may likely produce effective clinical outcomes in sars-cov-2 infections. however, clinical studies should test the utility of these agents/sirna in reducing the burden of infections caused by sars-cov-2 (111). the genome of coronaviruses is reported to be significantly involved in coding both structural proteins, and non-structural proteins (nsp's) for the effective viral replication (115) . the nsp's (nsp8c and nsp7) are required for novice cov viral particle formation through viral orf 1ab polyprotein processing (115) . several nsmis were reported to target these non-structural proteins in coronavirus infections to treat sars (115) . for instance, grl0617, a bendioxolane derivative, could target papain-like proteinases like nsp3 (51, 52, 116, 117) , whereas 5choloropyridinyl indolecarboxylate targets nsp5 (53, (118) (119) (120) and a "combination of zinc derivatives with pyrithione" targets nsp12 (121, 122) ; ranitidine bismuth citrate targets nsp13 (123) (124) (125) (126) (127) . monoclonal antibodies; cr3014 (128), mab-201 (129), mdef-201 (130) , ampligen (131), polyiclc (61, 132) , stinging nettle lectin (131) , and tapi-2 (a tace-inhibitor) (59) are anticoronaviral agents tested in vivo models of sars. for instance, a study showed that amiodarone (a known anti-arrhythmic agent) effectively targets coronaviral spreading in in vitro models (55) . working in a similar fashion, 2878/10 humanized antibodies can neutralize coronaviruses thereby reduce the complications caused by viral infections (54) . however, the above nsmis should be tested against sars-cov-2 viral associated proteins and against the activity of nsp's to derive an effective therapeutic intervention. prospective research must focus on the development of novel "helicase inhibitors, viral attachment inhibitors, and activity of rhesus θ -defensin" that block sars-cov-2 infection using in vitro, in vivo, and clinical studies (115) . hence, the development of nsmis to target the synthesis of nsp's in sars-cov-2 may deliver cellular antiviral responses by blocking their replication in host cells (115, 133, 134) . repurposing existing drugs is another strategy widely under consideration to target key proteins involved in the sars-cov-2 infection. in this regard, the existing nsmis viz., antivirals (umefenovir, remdesivir, nitazoxanide, favipiravir, ritonavir, lopinavir, ifns), anticytokines, antimalaria drugs (chloroquine, hydroxychloroquine), and passive antibody therapies are currently being evaluated to improve clinical outcomes in sars-cov-2 infected patients (3, 47, 64, 135, 136) . however, these agents require additional experimental and clinical validations before being tested in sars-cov-2 infections. for example, hydroxychloroquine (anti-malarial drug) and the tocilizumab (immunosuppressive drug) are preferred currently to mitigate viral entry and cytokine production in the sars-cov-2 infection. these drugs are being tested in ongoing trails in china and italy (135, 137) . priming the spike (s)-protein of coronavirus by host cells using membrane proteases is a necessary process for viral entry and replication, which further determines zoonotic potential of coronaviruses (138) . a recent report by markus hoffmann et al. (7) investigated the protease dependence of sars-cov-2 for its entry into cells. for example, sars-cov-2 uses the tmprss2 protease for its priming (7) . inhibition of tmprss2 using camostat mesylate retarded the viral entry into caco-2 cells (7). camostat mesylate could be recommended as an nsmi for human clinical trials to combat the sars-cov-2 virus (7). this report delineated the ability of neutralizing antibody responses against s-protein to block the sars-cov-2 entry into host cells (139) . the serum antibody responses raised to combat the "sars-s protein/ace-2 interface" during the sars-cov-2 infection indicates that the vaccination strategy may be an effective therapeutic modality against the covid-19 infection (7). ace-2 catalytic efficacy is significantly higher than ace for angiotensin-ii (140) . several compounds, such as mln-4760, were screened according to structure-based/substrate-based studies through virtual screening for inhibiting ace-2 activity (140) (141) (142) (143) . ace-2 is predominantly expressed in lungs, brain, heart, blood vessels, and renal organs (144, 145) . ace-2 is essential for cardiovascular homeostasis, and cns homeostasis as ace-2 confer redox homeostasis by mitigating ang-ii-induced oxidative stress (146) . however, in covid-19, ace-2 acts as receptor on human respiratory epithelial cells for sars-cov-2 binding (7). a recent report by markus hoffmann et al. (7) provided evidence that the sars-cov-2 strain use its spike (s)-protein to bind to ace-2. authors of this paper have also demonstrated the efficiency of tmprss2 in sars-cov-2 viral strain priming in host cells (7) . therefore, targeting ace-2 could be a viable strategy to prevent the entry of sars-cov-2 into the human system. however, a recent report by guan et al. (147) cautioned that the administration of ace inhibitors significantly induced adverse clinical outcomes in covid-19 patients due to severe hypertension, coronary artery disease, and chronic renal failure; hence, further use of ace inhibitors to treat covid-19 infections was halted (147) (148) (149) . in another report diaz (149) hypothesized that covid-19 patients receiving i.v. infusions of aceis and arbs (at1-receptor blockers) are at a higher risk of attaining severe disease pathogenesis. hence, they supported the development of nsmis such as "tmprss2 inhibitors to treat sars-cov-2 infections (7)". the failure of disease management and lack of selective therapies could be due to the intricate covid-19 pathogenesis induced by the sars-cov-2 infection. hence, the early recognition of disease is essential for effective management of covid-19 (48) . although, several reports delineated the efficacy of certain nsmis viz., ribavirin, promazine, and imp dehydrogenase inhibitors to inhibit in vivo models of sars-cov replication, later, they were proven ineffective (60, (150) (151) (152) . a report by reghunathan et al. (153) showed that the immune response produced against sars-cov may be different from other viral infections as indicated by the lack of upregulation in mhc-i genes, cytokines, and ifns or complementmediated cytolysis in peripheral blood mononuclear cells (pbmcs). the failure in the development of a vaccine is due to antibody-dependent enhancement and th-2 immunopathology (154) (155) (156) . pegylated ifn-α inhibits viral replication of sars-cov and offers protection against type i pneumocytes in lungs (2) . a significant reason for the failure or lack of selective therapies against sars-cov-2-induced sars is the intricate immune system mediated pathophysiology (4) . other reports by law et al., also detailed similar mechanisms (157, 158) . sars-cov can evade host ifn-mediated viral growth inhibition by activating ifn-regulatory factor 3 (157) . furthermore, sars-cov could induce apoptosis in lymphocytes in vitro using "orf 7a, orf 3a, and orf 3b, e protein, and n protein" (159) (160) (161) (162) . for instance, the sars-cov can evade immunity as indicated by the decline in cd4 and cd8 t cells (163) . therefore, it is necessary to uncover the complement-based cytolysis in human patients in response to the sars-cov-2 strain as this virus executes unusual mechanisms to evade the human immune system consequently inducing pathogenesis and mortality. the prospective research should focus on this viral-mediated immune signaling with respect to sars-cov for developing effective nsmis. intravenous (iv) hyperimmune globulin therapy is one of the immunotherapies known to downmodulate pro-inflammatory cytokines and mitigate the severity of infection in covid-19 patients. iv infusion of immunoglobulins composed of a high dose of antibodies, which can bind to a number of inhibitory receptors viz., fc gamma receptor iib (fcγriib) (164, 165) and fcγriic (166) and confer anti-inflammatory responses against sars-cov-2 (completed clinical trials: hyperimmune plasma nct04321421). oxidative stress is significantly induced by several viral infections inside the lungs through the downregulation of redox regulator nuclear factor-erythroid 2 related factor 2 (nrf-2) (167). nrf-2 is a leucine-zipper transcription factor (167) expressed predominantly in nasal epithelium, epithelial cells of lungs, and alveolar macrophages (168, 169) . disruption of nrf-2 and keap1 interaction triggers the activation of the anti-oxidant defense mechanism (169) . for instance, nrf-2 activation offers protection against inflammation and lung injury induced by influenza viral infections and respiratory syncytial virus (rsv) through the anti-oxidant defense pathway (170) . several viral proteins in the host cells can foster optimum levels of ros-mediated oxidative stress to facilitate viral metabolism and the viral replication cycle without killing host cells (168, (171) (172) (173) (174) . recent seminal studies described the active role of viruses in inhibiting the nrf-2 pathway (175) (176) (177) . for instance, the positive regulation of nrf-2 in modulating the thiol redox system and oxidative stress for the survival of infected astrocytes was observed in moloney murine leukemia virus and hiv virus (178) . the hcv virus could induce the downregulation of nrf-2 dependent nqo1, gclc, and gpx and modulate oxidative stress (179, 180) . an rsv infection mediates proteasomal degradation, deacetylation, and sumoylation of nrf-2 consequently causing the downregulation of nqo1, cat, and sod1 gene expression (181) . hence, nrf-2 activators are potential anti-viral agents, which can be tested against the sars-cov-2 infection (167) . future research is highly imperative in unraveling the underlying activity of nrf-2 for emerging sars-cov-2 survival by analyzing nrf2 target genes nqo1, gclc, and gpx. in addition, the sars-cov-2 mediated expression of serine and cysteine proteases in different cell lines should be investigated in relation to nrf-2 activation, which is a beneficial strategy to combating sars-cov-2 pathogenesis. however, in the case of certain viral infections, it is imperative to develop nrf-2 inhibitors to protect the host cells (182) . for instance, the marburg virus (a causative agent for lethal hemorrhagic fever) can modulate oxidative stress by activating nrf-2 dependent signaling through the blockade of "vp-24 viral protein" binding to keap1 (183) . therefore, vp-24 dependent nrf2 activation can mediate the upregulation of genes ho (heme oxygenase)-1, nqo1, and gclm (183) . in the case of dengue virus, the viral particles could induce er stress and activate nrf-2 signaling, which then lead to tnf-α secretion (184) . in this scenario, it is crucial to uncover any underlying mechanisms of emerging sars-cov-2 survival through the modulation of oxidative stress via nrf-2 signaling in different cells of different organs including lungs (183) . the prospective lindera erythrocarpa makino (195) celastrol (quinone methide triterpene) inhibits tat-induced hiv-1 infection tripterygium wilfordii (197) bakuchiol (phenolic isoprenoid) psoralea corylifolia l. (199) rupestonic acid (sesquiterpene) artemisia rupestris l. (continued) frontiers in immunology | www.frontiersin.org research studies should focus on the development of nrf-2 modulators against sars-cov-2. natural products were proven to offer protection against virusinduced oxidative stress by modulating anti-oxidant defense pathways (185, 186) . for instance, the administration of egcg has mitigated viral replication of "influenza a/bangkok/1/79 infection" by activating nrf-2 to attenuate virus-induced oxidative stress, inflammation, and apoptosis in lung cells (186) . similarly, the cytoprotective and antioxidant efficacy of nrf-2 was reported against pr8 influenza-a viral infection in at-i and at-ii cells (186) . prospective research should focus on testing the efficacy of several natural products to block sars-cov-2 viral replication by ascertaining nrf-2 mediated antioxidant responses. studies have also shown the activation of host cellular transmembrane proteases (for example, serine proteases, cysteine proteases), which can further foster a prompt viral entry and viral replication in host cells by reducing nrf-2 expression (4). decline in proteolysis of the above proteases can actuate the propagation of several human viruses viz., influenza, hiv, nipah, ebola, and coronaviruses (sars-cov, mers-cov, sars-cov-2) (42, 90, (187) (188) (189) . in this scenario, similar to influenza-a virus (190) , it is highly important to unravel the influence of nrf-2 expression on tmprss2, and human airway trypsin-like protease during sars-cov-2-mediated inflammatory conditions and oxidative stress in lungs. the downregulation of the nrf-2 gene is correlated to serine protease activity and consequent influenza viral entry (185) . recent studies have demonstrated the efficacy of natural nrf-2 activators viz., egcg and sulforaphane (sfn) for blocking viral entry/viral replication as well as promoting antiviral mediators rig-i, ifn-β, and mxa (185) . in this context, it is essential to demonstrate the effects of nutritional interventions like sfn and egcg against sars-cov-2 induced oxidative stress by modulating nrf-2 signaling. evidence has demonstrated the use of naturally occurring nrf2 activators for mitigating viral infections/post-viral infection induced complications. for example, α-luminol is a natural nrf-2 activator, which confers the protection of astrocytes against the momul virus (191) . egcg enhances nuclear nrf-2 levels during tat-induced hiv-1 infection and offers protection against virus induced oxidative stress (192) . tanshinone ii a can induce upregulation of nrf-2 expression and mitigates ros production during tat-induced hiv-1 infection via modulating ampk/nampt/sirt1 signaling in host cells (193) . sfn enhances the phagocytic function of "hiv-infected alveolar macrophages in lungs" by activating nrf-2 signaling, which further induces downstream antioxidant cascades (194) . lucidone is effective for the nrf-2 mediated blockade of dengue virus by inducing heme oxygenase-1 (195) ; rographolide could induce nrf-2 induced antioxidant defenses against influenza a in lung cells (196) ; celastrol can mediate nrf-2 induced antioxidant defenses against hiv-1 tat-induced inflammation (197) . broccoli sprouts containing sfn acts as a nrf-2 activator to reduce influenza-induced infection in lung cells (198) . bakuchiol and rupestonic acid are phytoconstituents that confer nrf-2 activation thereby promoting nqo1 gene expression and ho-1-mediated interferon activity to enhance antioxidant response against influenza virus in lung cells (199, 200) . curcumin is another significant compound that can modulate nrf-2 signaling and enhance the generation of ifn-β to offer protection against the influenza virus (201) . curcumin can mitigate this viral infection by modulating tlr2/4, p38/jnk mapk, and nf-κb pathways (201) . however, studies are required to decipher the activity of these phytochemicals against sars-cov-2 ( table 4) . a recent report by drȃgoi (209) hypothesized that the potent natural nrf-2 activators viz., resveratrol, sfn, curcumin, and asea redox should be evaluated in different combinations with conventional drugs against sars-cov-2 infection in both in vitro and in vivo models and to further deduce a correlation between nrf-2 activity and sars-cov-2 viral entry/replication. sars-cov-2 is progressively inducing a high mortality rate across the globe due to the lack of selective therapeutic interventions or vaccination. recent reports by lu et al. (210) and xu et al. (148) delineated that the s-protein of sars-cov-2 and sars co-v exhibit similar 3-d pharmacophore in the receptor binding domain (rbd) of ace-2 of human cells. covid-19 patients are characterized by the severe viral pathogenesis due to extensive cytokine storm viz., tnf-α, il-1β, il-10, ifnγ, and mcp-1 in infected lung tissues (48) . a report by chen and du (211) hypothesized that the phyto-constituents such as "baicalin, scutellarin, hesperetin, nicotianamine, and glycyrrhizin" may deliver anti-sars-cov-2 effects. hesperetin glycoside abundant in citrus fruits, which can inhibit the sars-cov 3clpro (212) . the activity of this molecule must be examined against serine/cysteine proteases, which support sars-cov-2 viral entry/replication. traditional citrus flavonoids were frontiers in immunology | www.frontiersin.org reported to have a potential to act against sars-cov-2 as studied by molecular docking studies. molecular docking simulations, lc-ms studies described the efficacy of citrus flavonoids (ex. naringenin) in binding to ace-2, and mitigating inflammationinduced lung injury by the sars-cov-2 virus (213) . further studies should evaluate these compounds in preclinical models to determine the safety and efficacy against the sars-cov-2 infection. natural products such as di/tri-terpenoids, lignoids were proven to inhibit the viral replication of coronaviruses in vitro (214) ; griffithsin could block coronaviral entry by binding to the sars-cov spike glycoprotein (215) . tsl-1 can block coronaviral entry/replication; leaf extracts of toona sinesis roem effectively blocked sars-cov replication (57) . betulinic acid, savinin can act as competitive inhibitors against sars-cov 3cl protease to block viral entry (214) . the research gap must be filled to develop nutritional therapeutic interventions by investigating the efficacy of these phytochemicals against sars-cov-2 viral entry. seeds of psorelia corylifolia exhibit inhibitory effects against the sars-cov papain-like protease required for coronavirus entry/replication. the efficacy of these molecules should be examined against sars-cov-2 (216) ( table 5 ). the active site pockets of main proteases such as 6lu7 and 2 gtb in sars-cov-2 are reported to be involved in conferring viral entry/fusion; hence, these sites should be considered as the potential drug targets against sars-cov-2 (66) . a molecular docking study by khaerunnisa et al. (65) reported the predicted efficacy of bioactive compounds against above sars-cov-2 main protease (mpro) sites viz., "nelfinavir, lopinavir, kaempferol, quercetin, luteolin-7glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, epicatechin-gallate, zingerol, gingerol, and allicin" ( table 5) . the life-threatening consequences of the covid-19 pandemic remain high due to lack of selective targeted therapies and vaccination strategies. this is primarily due to extreme genomic variability of rna viruses as well as variations in the host-cell invading mechanisms. hence, this review benefits virologists, medical scientists, and cell biologists to ascertain and develop nsmis, nrf-2 modulators, and clinically viable vaccines to combat this devastating sars-cov-2 strain. however, many more preclinical and clinical studies are required to uncover the therapeutic efficacy of potential phytochemicals, natural nrf2 modulators, and several nsmis against the sars-cov-2 infection. furthermore, studies are also warranted to overcome ade responses, and th-2 immunopathology for the development of safe and efficacious vaccines against sars-cov-2. in summary, this review provides an overview on the existing knowledge and shows directions to various areas that require immediate attention. nb, ss, and sm: idea development, data collection, manuscript preparation, writing, and proof reading. as, vn, and lm: cross referencing, data collection, and proof reading. sm, ga, and rr: editing, literature search, and proof reading. ss: execution of the literature search. all authors contributed to the article and approved the submitted version. isolation and characterization of viruses related to the sars coronavirus from animals in southern china pegylated interferon-α protects type 1 pneumocytes against sars coronavirus infection in macaques coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus evaluation and 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supplement" [ars]) should be clinically tested as adjuvants in all types of medium and severe cases of aggressive respiratory viral infections (including influenza a/b/c, sars, mers, covid-19, measles, avian influenza etc.) based on their extrapolated cytoprotective antioxidant effects (especially on vital organs), including the cytoprotection offered by ars on the cardiac muscle of dmd patients which can be extrapolated to the lungs -a very short medical communication genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding anti-sars coronavirus 3c-like protease effects of isatis indigotica root and plant-derived phenolic compounds citrus fruits are rich in flavonoids for immunoregulation and potential targeting ace2 specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia baicalin, a metabolite of baicalein with antiviral activity against dengue virus dual effect of glucuronidation of a pyrogallol-type phytophenol antioxidant: a comparison between scutellarein and scutellarin co-occurrence of nicotianamine and avenic acids in avena sativa and oryza sativa azetidine-2-carboxylic acid derivatives from seeds of fagus silvatica l. and a revised structure for nicotianamine betulin and betulinic acid: triterpenoids derivatives with a powerful biological potential the griffithsin dimer is required for high-potency inhibition of hiv-1: evidence for manipulation of the structure of gp120 as part of the griffithsin dimer mechanism research & consulting llc.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 beeraka, sadhu, madhunapantula, rao pragada, svistunov, nikolenko, mikhaleva and aliev. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-342133-khrljehj authors: principi, nicola; piralla, antonio; zampiero, alberto; bianchini, sonia; umbrello, giulia; scala, alessia; bosis, samantha; fossali, emilio; baldanti, fausto; esposito, susanna title: bocavirus infection in otherwise healthy children with respiratory disease date: 2015-08-12 journal: plos one doi: 10.1371/journal.pone.0135640 sha: doc_id: 342133 cord_uid: khrljehj to evaluate the role of human bocavirus (hbov) as a causative agent of respiratory disease, the importance of the viral load in respiratory disease type and severity and the pathogenicity of the different hbov species, we studied all hbov-positive nasopharyngeal samples collected from children who attended an emergency room for a respiratory tract infection during three winters (2009–2010, 2011–2012, and 2013–2014). human bocavirus was detected using the respiratory virus panel fast assay and real-time pcr. of the 1,823 nasopharyngeal samples, 104 (5.7%) were positive for hbov; a similar prevalence was observed in all three periods studied. among hbov-infected children, 53.8% were between 1–2 years old, and hbov was detected alone in 57/104 (54.8%) cases. all of the detected hbov strains belonged to genotype 1. the median hbov load was significantly higher in samples containing strains with both the n546h and t590s mutations compared to other samples (p<0.05). children with a single hbov-1 infection more frequently had upper respiratory tract infections (urtis) than those who were co-infected (37.0% vs 17.8%, respectively, p = 0.04). the duration of hospitalization was longer among children with high viral loads than that observed among children with low viral loads (8.0 ±2.2 days vs 5.0 ±1.5 days, respectively, p = 0.03), and the use of aerosol therapy was more frequent among children with high viral loads than among those with low viral loads (77.1% vs 55.7%, respectively, p = 0.04). this study shows that hbov is a relatively uncommon but stable infectious agent in children and that hbov1 seems to be the only strain detected in italy in respiratory samples. from a clinical point of view, hbov1 seems to have in the majority of healthy children relatively low clinical relevance. moreover, the viral load influences only the duration of hospitalization and the use of aerosol therapy without any association with the site of the respiratory disease. human bocavirus (hbov) is a recently identified viral agent that belongs to the family parvoviridae and contains a single linear positive-sense or negative-sense single-stranded deoxyribonucleic acid genome [1] . this virus has been detected mainly in younger children, in nasopharyngeal secretions, in sera and blood samples of patients with upper (urti) and lower (lrti) respiratory tract infections and in faecal specimens of subjects with gastroenteritis [2] . currently, hbovs are classified into species 1 through 4; hbov1 is predominantly found in the respiratory tract, and hbov2, hbov3, and hbov4 are found mainly in stool [3] . despite there are studies suggesting that hbov is able to infect the lower airways causing severe infections in both children and adults, the role of hbov as a causative agent of respiratory disease is frequently questioned due to its common detection with other potential pathogens [4] and the evidence that in some studies co-infections can have a significantly greater clinical and socioeconomic impact on infected children and their households than hbov infection alone [5] . moreover, the importance of the viral load in determining the type and severity of respiratory disease as well as the pathogenicity of the different hbov species [6] are not precisely defined. the main aim of this study was to contribute to resolving these problems. the circulation of hbov during several winter seasons in italy was investigated, and a phylogenetic analysis of detected strains was performed. in addition, correlations between different hbov strains and the severity of disease in cases with infections due to hbov alone or due to co-infections were studied. finally, the role of the viral load was analysed. to evaluate the circulation of the different hbov types and the possible relationships between viral load, virus genetic characteristics, and the severity of infection, nasopharyngeal swabs were collected from otherwise healthy children attending the emergency room of the fondazione irccs ca' granda ospedale maggiore policlinico, university of milan, italy, due to a respiratory tract infection arising between november 1 and march 31 during 3 winters (2009-2010, 2011-2012, and 2013-2014) . the study was approved by the ethics committee of the fondazione irccs ca' granda ospedale maggiore policlinico, milan, italy. written informed consent of a parent or legal guardian was required, and children 8 years of age were asked to give their written assent. patients' demographic characteristics and medical histories were retrieved from hospital charts and were systematically recorded before and after the first visit to the emergency room using standardized written questionnaires [7] . the study patients were classified into disease groups (i.e., acute otitis media, rhinosinusitis, pharyngitis, croup, infectious wheezing, acute bronchitis, pneumonia) on the basis of signs and/or symptoms using well-established criteria and were finally subdivided into two subgroups: upper (urtis) and lower respiratory tract infections (lrtis) [8] . nasopharyngeal secretions were collected from all of the children immediately after admission to the emergency room using a paranasal flocked swab (1 swab per child), which was stored in a tube containing 1 ml of universal transport medium (kit cat. no. 360c, copan italia, brescia, italy). viral nucleic acids were extracted from each swab by means of a nuclisens easymag automated extraction system (biomeriéux, craponne, france), and the extract was tested for respiratory viruses using the respiratory virus panel xtag rvp fast v2 (luminex molecular diagnostics, inc., toronto, canada), which simultaneously detects influenza a virus (subtypes h1 or h3); influenza b virus; respiratory syncytial virus (rsv) types a and b; human parainfluenza virus types 1-4 (hpiv1-4); adenovirus (adv); human metapneumovirus (hmpv); coronaviruses (hcov) 229e, nl63, oc43 and hku1, enterovirus/rhinovirus (ev/hrv); and hbov, in accordance with the manufacturer's instructions [9, 10] . samples that were positive for hbov were stored at -80°c. hbov real-time polymerase chain reaction (pcr) viral nucleic acid extracts previously testing positive for hbov were re-tested for confirmation by two different singleplex real-time pcrs using taqman universal master mix ii (applied biosystems, california, usa). amplification and detection of viral dna were performed with a 7900ht real-time pcr system machine (applied biosystems, california, usa). conserved regions for rt-pcr primers and probes were identified in the hbov ns-1 and np-1 genes from the nucleotide sequence alignments available from genbank (for ns1, dq206700-08, dq000495-96, and dq200648, and for np-1, dq000495-96, ab243566-72, dq296618-35, dq353695-99, dq299885, dq267760-75, dq284856, dq295844, and am109958-66; http:// www.ncbi.nlm.nih.gov/genbank/). each 25 μl singleplex reaction mixture consisted of 0.5 μl of forward primer 5'-tgcagacaacgcytagttgttt-3' and reverse primer 5'-ctgtcccg cccaagataca-3' for the 88 base pair ns-1 target or forward primer 5'-agcatcgctcctacaaaagaaaag-3' and reverse primer 5'-tcttcatcacttggtctga ggtct-3' for the np-1 target, 0.125 μl of probe 5'-ccaggattgggtggaacctgcaaa-3' or 5'-aggctcgggctcatatcatcaggaaca-3', and 2.5 μl of sample viral dna. pcrs were conducted at 50°c for 2 min and then at 95°c for 10 min, followed by 45 cycles at 95°c for 15 sec and at 60°c for 1 min. taqman probes were labelled at the 5' ends with the reporter molecule 6-carboxyfluorescein and at the 3' ends with black hole quencher 1 (biosearch technologies, inc., novato, ca). each run included one synthetic template control and one no-template control for each target. specimen extracts were also tested by real-time pcr for the human rnase-p gene to monitor specimen quality and the presence of pcr inhibitors. a positive test for both the ns1 and np-1 targets or for a single target confirmed from a second extraction from a new sample aliquot was considered definitive evidence of hbov infection. the viral load was obtained using real-time pcr with the ns1 primers and probe previously described and a dna plasmid used as the standard calibrator. the amplified target fragment of the plasmid was verified by sequencing. plasmid dna concentrations were detected with an nd-1000 spectrophotometer (nanodrop products, wilmington, de, usa). each run included plasmid and negative controls. standard precautions were taken throughout the pcr process to avoid cross-contamination. negative controls and serial dilutions of the positive controls were included in every pcr assay. finally, quantitative results were reported as dna copies/ ml of respiratory samples. the viral load was defined as low for values 10 6 log (copies/ml) and as high for values >10 6 log (copies/ml). for genotyping, the viral vp-1/2 gene was amplified using a conventional pcr assay. briefly, 4 sets of forward and reverse primers (5'-cacagacagaagcagacgagat-3' and 5'-ggtg agaagtgacagctgtattg-3'; 5'-ttcagaatggtcacctctaca-3' and 5'-ctgtgc ttccgttttgtctta-3'; 5'-aactttgactgtgaatgggtta-3' and 5'-aaatagtgcc tggaggatgat-3'; 5'-ctatcaccagagaaaatccaatc-3' and 5'-gagacggtaaca ccacta-3') were used in pcr amplification and the quantitect probe master mix (qia-gen, venlo, netherlands) was used as the basis for the reaction mix. viral products were analysed by electrophoresis on a 1.5% agarose gel and purified with the qiaquick gel extraction kit (qiagen, venlo, netherlands). sequencing reactions were set up with purified dna, one of the specific primers used in the pcr and bigdye terminator v3.1 cycle sequencing kit (applied biosystems, california, usa) according to the protocol recommended by the manufacturer. sequencing and sequence analysis were performed on a 3130 genetic analyser (applied biosystems, california, usa). all alignments were performed using clustalx 2.1 and bioedit (version 7.1.3.0) software (ibis biosciences, carlsbad, ca). phylogenetic trees of the vp-1/2 protein gene were generated using the neighbour-joining method and p-distance model of the molecular evolutionary genetics analyses (mega) software, version 5.05 [11] . bootstrap probabilities for 1,000 iterations were calculated to evaluate confidence estimates. the graphs were made using graphpad prism version 5.01 for windows (graphpad software, san diego, ca). all genotyped sequences of the hbov vp-1/2 gene were submitted to genbank (accession numbers kr014412-kr014516). tests for positive selection were conducted on the datamonkey server [12] using the singlelikelihood ancestor (slac), and the fixed-effects likelihood (fel) [13] , the internal branch fixed-effects likelihood (ifel) [14] , the mixed effects model of evolution (meme) [15] , and fast unconstrained bayesian approximation methods (fubar). the dn/ds ratios were calculated using the slac and fel codon-based maximum likelihood approaches. the slac approach counts the number of non-synonymous changes per non-synonymous site (dn) and tests whether it is significantly different from the number of synonymous changes per synonymous site (ds). the fel approach estimates the ratios of non-synonymous to synonymous changes for each site in an alignment. the ifel method is similar to the fel, but tests site-bysite selection along only the internal branches of the phylogeny. in order to avoid an excessive false-positive rate, sites with slac, fel, ifel and meme p-values of <0.1 and a fubar posterior probability of >0.90 were accepted as candidates for selection. descriptive statistics of the responses were generated. continuous variables were presented as mean values and standard deviations (sds) and categorical variables as numbers and percentages. for categorical data, comparisons between groups were performed using a contingency table analysis with the χ 2 or fisher's exact test when appropriate. for ordered categorical data, a cochran-armitage test for trend was used to compare the groups. continuous data were analysed using a two-sided student's t-test after ensuring the data were normally distributed (based on the shapiro-wilk statistic) or using a two-sided wilcoxon's rank-sum test if the data were non-normal. all analyses were two tailed, and p-values of 0.05 or less were considered to be statistically significant. all analyses were conducted using sas version 9.2 (cary, nc, usa). during the three study periods, 1,823 nasopharyngeal samples were collected in the emergency room. of these, 104 (5.7%) tested positive for hbov (table 1) . among hbov infected children, 53.8% were between 1-2 years old, whereas 28.8% and 17.3% were aged <1 and 3 years, respectively. the prevalence of hbov detection was quite similar in the three studied periods; hbov was the only virus detected in 57/ 104 (54.8%) cases and was detected in association with one (89.5%) or more (10.5%) viruses in 47 (45.2%) cases. ev/hrv and rsv were the most common co-infecting viral agents and were found, respectively, in 20 and 18 samples. subjects with co-infection were younger than those without (p = 0.03). considering 10 6 dna copies/ml as a cut-off, the viral load was classified as low in 66 (63.5%) cases and as high in 38 (36.5%) cases. the phylogenetic tree constructed using the vp1/vp2 sequences showed that all of the italian hbov strains detected during the three study periods belonged to hbov genotype 1 (fig 1) . no unusual clustering was observed among the identified strains; hbovs circulating in 2009 were closely related to strains circulating in 2014. the sequence identity matrix of the vp1/vp2 gene showed minimum to maximum identity ranges of 97.8-100% between the italian strains and 98.4-99.7% with respect to hbov st1 reference strains (dq000495). in comparison to the reference strain, 8/105 (7.6%) strains had only one amino acid difference, 32/105 (30.4%) strains had two amino acid differences, and the remaining strains (65/105; 61.9%) had at least three amino acid changes. a total of 61/672 (9.1%) amino acid positions were observed to have at least one change in the vp1/vp2 sequence alignments (fig 2) . of these, 7/61 (11.5%) changes occurred within the vp1 unique (vp1u) region corresponding to the first 129 amino acids at the n-terminus of the vp1 gene. specifically, the following changes were observed: r17k, g28d, e29k, l40s, h43q, d72n, and g126e (fig 2) . the vp1u region includes the conserved phospholipase a 2 (pla 2 ) motif (nt 21-63). the vp1u sequences of all hbov isolates identified in this study revealed conserved yxgxg (nt [16] [17] [18] [19] [20] and hdxxy (nt 41-45) motifs in the catalytic site of secreted pla 2 . in addition, the amino acid residues at positions 21, 41, 42 and 63 have been hypothesized to form the catalytic network for enzymatic activity. in our hbov strains, all the sequences had amino acids associated with efficient enzymatic activity (p21, h41, d42, and d63). of note, two hbov strains (mi-267-jan2014 and mi-272-jan2014) had a peculiar amino acid sequence in the 19-amino acid segment starting at amino acid 411 (kvptrrvqpyirqtnwkhr), which has not been previously reported in hbov strains included in the genbank database (in red, fig 1) . overall, these two strains had 13 and 14 amino acid changes compared to the hbov st1 strain, and almost all these changes were included in the region described below. the origin of this highly divergent region, which occurred in spite of the conservation displayed in the rest of the hbov dna genome, remains to be defined. a global analysis of selective pressure made using the slac model indicated an estimated overall dn/ds ratio of 0.18. overall, the site-specific analyses identified three sites (411, 536, and 546) as under positive selection by at least two methods used (slac, fel, fubar, and meme). the ifel model was used to determine the selection pressure acting on the vp1/vp2 codons along the internal branches of the tree. two positively selected codons (411 and 546) were identified. the selected sites, highlighted with arrows in fig 2, were presumably located in of these strains, 6/13 (46.1%) were also characterized by the n534k, q535p, and q535d mutations. several negatively selected sites were identified by different methods (table 2 ). regarding the viral load, a wide range of hbov dna levels from 3.5x10 2 to 7.5x10 9 copies/ml were found in the clinical samples. in fig 2 (right side) , the viral load of each italian hbov strain is reported near the aligned mutations. in the group of strains (n = 70; 66.6%) harbouring at least 2 mutations in addition to a149t, the values of the hbov load greater than 1x10 8 dna copies/ml are reported in grey. a total of 16 strains had a very high viral load, and 13/16 (81.3%) harboured the t590s mutation. this percentage is nearly significantly different than the overall frequency (42/70; 53.8%) of t590s in the group of strains described below (p = 0.08). seven of the 13 strains with the t590s mutation had an additional mutation in one of the sites under positive selection (reported in fig 2 with an asterisk) . in detail, 4/13 (30.8%) had the n546h change, 2/13 (15.4%) had the a411d change, and 1/13 (7.7%) had the a411t change. based on the observed data, we hypothesize that the double mutation of n546h with t590s may positively affect viral replication and specific immune response. as shown in table 3 , the median hbov load was significantly higher in samples of strains with the n546h and t590s changes than that in samples of wild type t590 strains, strains with only the t590s mutation, and strains with only the n546h mutation (p-values of 0.0078, 0.016 and 0.018, respectively). finally, the two divergent strains (mi-267-jan2014 and mi272-jan2014) with unusual amino acid changes were observed with viral loads lower than 10 6 dna copies/ml. this could suggest that these mutations do not confer a replicative advantage in these virus strains. in table 4 , data regarding demographic, clinical and laboratory characteristics of children infected by hbov-1 alone or co-infected with hbov-1 and one or more other respiratory viruses are reported. because a preliminary evaluation did not find any differences among subjects co-infected with ev/hrv, rsv or other viruses, all co-infections were considered together. as shown, children infected with only hbov-1 had urtis more frequently than those with a co-infection (37.0% vs 17.8%, respectively, p = 0.04). moreover, a similar illness within the family in the 7 days since patient enrolment was significantly more common among co-infected children than among those with a single infection (48.9% vs 26.5%, respectively, p = 0.02). no other significant differences between the groups were observed. table 5 shows the demographic, clinical, and laboratory characteristics of the enrolled subjects according to the hbov load. subjects with low and high viral loads were quite similar. the only significant differences were found in the duration of hospitalization, which was longer among children with a high viral load than among those with a low viral load (8.0 ±2.2 days vs 5.0 ±1.5 days, respectively, p = 0.03), and in the use of aerosol therapy, which was more frequent among children with a high viral load than among those with a low viral load (77.1% vs 55.7%, respectively, p = 0.04). moreover, mutations leading to a high or low viral load were not associated with atypical clinical characteristics. this study shows that in italy during the winter periods 2009-2010, 2011-2012, and 2013-2014, the incidence of hbov infection among children with respiratory disease was relatively low, limited to approximately 5% of cases, and did not significantly vary from year to year. the phylogenetic analysis showed that all of the strains detected in this study belonged to hbov genotype 1 and were closely related to the prototype strain identified by allander et al. [1] . this was expected because this genotype is the most common among hbovs associated with respiratory infections [1] . most of the patients in whom hbov1 was identified were younger than 3 years of age, further highlighting that younger children are the individuals most frequently infected by this viral agent [1] . serological studies have shown evidence that the number of subjects positive for anti-hbov1 antibodies continuously increases with increasing age group from the ages of 6 months to 6 years, and by the age of 2 years approximately 80% of children have been infected with hbov1 [16, 17] . more than 50% of the children infected by hbov1 in this study were coinfected with at least one other respiratory virus. moreover, co-infected patients had lrti more frequently than those infected by hbov alone. these findings are not surprising because simultaneous detection of hbov1 and other viruses in children with respiratory disease and greater severity in co-infected cases have been already reported in studies in which it was also demonstrated that hbov1 can frequently be identified in the respiratory secretions of asymptomatic subjects [18] [19] [20] [21] [22] [23] . recently, it has been reported that hbov1 can be shed for several days or months after a previous infection [24] , which could explain the simultaneous identification of hbov1 and other respiratory viruses, the frequency of asymptomatic infections and the generally greater severity of infections in co-infected individuals compared to those with hbov1 alone. in most of the co-infected cases, detection of hbov in the respiratory secretions with the new sensitive molecular methods able to identify very low viral loads might be a consequence of a previous clinically resolved disease, and a virus other than hbov was therefore the real cause of the disease. however, reports of severe clinical manifestations in patients infected with only hbov have been published [25] , highlighting that the assessment of the real importance of hbov infection in a single patient remains very difficult. studies on children with severe pneumonia, acute wheezing, asthma and/or bronchiolitis suggested that hbov1 is able to infect the lower airways down to the bronchioles [26] [27] [28] [29] [30] [31] [32] . moreover, hbov1 has been found as the only infectious agent in adult lung transplant recipients with severe lrti, whereas it was not detected in respiratory secretions of asymptomatic transplanted subjects [33] . this would indicate that hbov1 is not always a bystander or the cause of mild respiratory problems but rather a real, although relatively rare, causative agent of severe disease in both children and adults, particularly when they are immunocompromised. on the other hand, hbov can cause serious neurological infections [34] and contribute to chronic disease in adult patients mainly because it can persist after childhood infection and reactivate [35] . evaluation of the viral load has been considered a possible method to define when this virus is the real cause of a respiratory disease and when it is only a secondary infection. unfortunately, this approach has had no success because although some studies have shown evidence for a strict correlation between high viral load and severe lrti in children with a single hbov infection [36] [37] [38] , others, including the present study, did not show a clear relationship between these two variables [39] . however, the evolution of virulence appears to involve a variety of mechanisms in different viral systems, including mutations in regulatory regions and viral adaptation for utilization of alternative or expanded repertoires of cellular receptors. an alternative hypothesis to evaluate the importance of hbov1 concerns the correlation between viral load levels and the presence of specific mutations. however, mutations associated with increased or reduced replication are rarely reported for hbov. recently, hao et al. have reported that few nucleotide changes were correlated with a lower viral load [40] . in the present study, a double mutant (n546h and t590s) was observed in samples with a significantly higher viral load. however, further phenotypic validation studies are required to draw major conclusions regarding the impact of these mutations on viral replication. furthermore, as reported by qu et al. [41] , it seems that nucleotide changes in the vp1u region could affect the replication efficiency of hbov. likewise, in our strains all the amino acids of the catalytic site were conserved, and no mutations that affect spla 2 activity were identified. in agreement with others [42] , the phylogenetic analysis of this study confirmed the very low degree of variability in the hbov genomic region encoding proteins that are exposed to the virus surface and are therefore under immunologic pressure. only 9% (62 codons) of amino acids were found to have at least one change in the vp1/vp2 gene, a finding not substantially different from that reported by hao et al. in a different geographic area [40] . in our study, several amino acid changes were observed in strains circulating in almost all the respiratory seasons. this finding provides evidence that the selection of those variants best adapted to each particular environment might select for variants with an evolutionary advantage. seven of these mutations were located in a genomic region (i.e., vp1u) previously reported to be involved in the mechanisms of virus replication. for instance, the vp1u amino acid variations r17k and l40s have been previously reported [43] , whereas the remaining variations have not yet been described. interestingly, two hbov strains identified in respiratory samples collected in january 2014 had unusual amino acid sequences in a somewhat conserved genomic region. the reason for this genetic diversity is still undefined. however, as described for hbov, other parvoviruses [44] , and enteroviruses, a series of α-helices and β-barrels in the vp2 protein were intercalated by an external loop, in which the majority of the genetic variability accumulated. nevertheless, these two divergent strains were found in samples with low viral loads, and we might hypothesize a loss of replication advantage for these strains. in the present study, the dn/ds ratios for all pairwise comparisons were <1, which is in line with previous results showing that positive selection was extremely limited in parvoviruses [45] . in fact, selective pressure analyses have identified 3 codons under positive selection. in a previous paper, a different codon (l40) was identified as being under positive selection [46] . nevertheless, the great majority of codons were under negative or neutral selection, which has also been confirmed by others [47] . this finding suggests that only a few amino acids of the vp1/vp2 proteins present on the surface of the virion are potentially subjected to a strong selective pressure by the host immune response. in conclusion, this study confirms that hbov is less common than other respiratory viruses but that the frequency of its detection in children with respiratory disease is in time stable. it was detected with a prevalence of about 5% in several consecutive seasons and no unusual clustering was observed among identified strains, with strains circulating in 2009 being closely related to those circulating in 2014. moreover, only a minority of virus sites were found to be under positive selective pressure, and all the strains detected in respiratory tract infections of this italian study belonged to genotype 1. from a clinical point of view, this study highlights that in otherwise healthy children, hbov1 seems to have relatively low clinical relevance, because patients infected with hbov alone mainly suffered from an urti. the viral load was not associated with clinical characteristics of the infection, and viral mutations, despite affecting viral replication, did not affect the conditions or severity of the clinical presentation. further studies are needed to clarify the clinical relevance of hbov in children, particularly in those at risk for severe chronic underlying disease, and to evaluate the role of viral modification in conditioning the degree of viral virulence and the specific immune response. conceived and designed the experiments: np se. performed the experiments: ap az as. analyzed the data: fb. contributed reagents/materials/analysis tools: sbi gu as sbo ef. wrote the paper: np ap se. cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infections human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses impact of human bocavirus on children and their families the human bocavirus role in acute respiratory tract infections of pediatric patients as defined by viral load quantification clinical and socioeconomic impact of different types and subtypes of seasonal influenza viruses in children during influenza seasons textbook of pediatric infectious diseases comparison of the luminex respiratory virus panel fast assay with in-house realtime pcr for respiratory viral infection diagnosis comparison of the luminex xtag 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vp1 unique region human bocavirus amongst an allages population hospitalised with acute lower respiratory infections in cambodia complete coding sequences and phylogenetic analysis of human bocavirus (hbov) human bocavirus capsid structure: insights into the structural repertoire of the parvoviridae evolutionary relationships among parvoviruses: virus-host coevolution among autonomous primate parvoviruses and links between adeno-associated and avian parvoviruses epidemic and molecular evolution of human bocavirus in hospitalized children with acute respiratory tract infection rapid molecular evolution of human bocavirus revealed by bayesian coalescent inference key: cord-348844-4rpbsj48 authors: wessel, lindsay; hua, yi; wu, jianhong; moghadas, seyed m title: public health interventions for epidemics: implications for multiple infection waves date: 2011-02-25 journal: bmc public health doi: 10.1186/1471-2458-11-s1-s2 sha: doc_id: 348844 cord_uid: 4rpbsj48 background: epidemics with multiple infection waves have been documented for some human diseases, most notably during past influenza pandemics. while pathogen evolution, co-infection, and behavioural changes have been proposed as possible mechanisms for the occurrence of subsequent outbreaks, the effect of public health interventions remains undetermined. methods: we develop mean-field and stochastic epidemiological models for disease transmission, and perform simulations to show how control measures, such as drug treatment and isolation of ill individuals, can influence the epidemic profile and generate sequences of infection waves with different characteristics. results: we demonstrate the impact of parameters representing the effectiveness and adverse consequences of intervention measures, such as treatment and emergence of drug resistance, on the spread of a pathogen in the population. if pathogen resistant strains evolve under drug pressure, multiple outbreaks are possible with variability in their characteristics, magnitude, and timing. in this context, the level of drug use and isolation capacity play an important role in the occurrence of subsequent outbreaks. our simulations for influenza infection as a case study indicate that the intensive use of these interventions during the early stages of the epidemic could delay the spread of disease, but it may also result in later infection waves with possibly larger magnitudes. conclusions: the findings highlight the importance of intervention parameters in the process of public health decision-making, and in evaluating control measures when facing substantial uncertainty regarding the epidemiological characteristics of an emerging infectious pathogen. critical factors that influence population health including evolutionary responses of the pathogen under the pressure of different intervention measures during an epidemic should be considered for the design of effective strategies that address short-term targets compatible with long-term disease outcomes. the findings highlight the importance of intervention parameters in the process of public health decision-making, and in evaluating control measures when facing substantial uncertainty regarding the epidemiological characteristics of an emerging infectious pathogen. critical factors that influence population health including evolutionary responses of the pathogen under the pressure of different intervention measures during an epidemic should be considered for the design of effective strategies that address short-term targets compatible with long-term disease outcomes. epidemics of infectious diseases have been observed throughout history, with substantial variability in their dynamical patterns. the 1918 influenza pandemic is a notorious case documented as the most devastating epidemic with over 50 million deaths and multiple outbreaks in many geographic areas worldwide [1, 2] . distinct pandemic infection waves were recorded with an 8 to 15 week interval; the latter were more severe than the first and were associated with the majority of deaths [2, 3] . although several factors may be involved, such as the effect of seasonal changes, demographics, and evolution of the virus, the true mechanism by which subsequent waves occur is not fully understood. nor is it clearly understood how different control measures and strategies for deployment of limited health resources may interfere with disease dynamics and the occurrence of later infection waves. recent epidemiological and modelling studies have attempted to provide explanatory theories for the mechanisms of multiple outbreaks of an infectious pathogen capable of establishing an epidemic [2, [4] [5] [6] [7] [8] [9] [10] . spontaneous behavioural changes (e.g., a change in the number of contacts due to modified behaviour of susceptible individuals) have been shown to affect the course of infection events and produce subsequent outbreaks in an epidemic episode [9] . this has been further investigated through modelling "concerned awareness" of individuals that may result in contagion dynamics of fear and disease [6] , and the implementation of public health control measures (e.g., social distancing) that may interfere with the individuals' contact patterns during the epidemic [5] . co-infection has also been suggested as a possible explanation for multiple infection outbreaks as a result of increased transmissibility in coinfected individuals and non-synchronicity in the time course of the two co-circulating infections [8] . other possible mechanisms include transient post-infection immunity and evolutionary changes that may occur in the characteristics of the infectious pathogens [2, 4, 10] . in this study, we consider the occurrence of multiple infection waves of a pathogen from a public health perspective, and develop mathematical models to investigate how intervention measures may affect the transmission dynamics in a population. specifically, we are interested in exploring the impact of changes in policy-relevant parameters on the patterns of disease spread during the course of an epidemic. these parameters may reflect the effectiveness of intervention strategies (e.g., treatment or isolation of infected cases) in reducing disease transmission, or their epidemiological consequences (e.g., emergence of drug resistance), and may therefore play an important role in determining the outcome of disease control activities. the significance of this work thus relates to the process of public health decision-making, in particular when confronting the emergence of a novel infectious disease with substantial uncertainty regarding the epidemiological characteristics of the invading pathogen. for the purpose of this investigation, we develop both mean-field and stochastic epidemiological models that describe the transmission dynamics of a disease in the population, and incorporate treatment and isolation of infected cases as control measures. we parameterize these models to simulate the spread of influenza as a case study, and determine the impact of control parameters on disease dynamics. we illustrate the occurrence of multiple infection waves associated with different treatment levels and the development of drug resistance in the population under the scenario of limited capacity for treatment and isolation of infectious individuals. we compare the results obtained by simulating the mean-field model with those observed in the stochastic model, and discuss our findings in the context of epidemiology and public health. to formulate the models for describing disease epidemic, we assume that the population is initially entirely susceptible to the infectious pathogen. it is assumed that the infection can be treated with drugs, but the pathogen may develop resistance during the course of treatment with potential for transmission. since resistance emergence may impose fitness cost on pathogen replication and transmission [11] , we assume that the drug-resistant pathogen is less transmissible than the drug-sensitive pathogen. treatment is assumed to reduce transmissibility of the drug-sensitive infection, but remains ineffective against drug-resistant infection. we also assume that the recovery from infection confers immunity to re-infection with either drug-sensitive or resistant pathogens. considering epidemics with relatively short time-courses, we ignore the effect of recruitment, natural death, and other demographic variables of the population. with the assumption of homogeneous mixing, we divide the population into classes of susceptibles (s); individuals exposed (not yet infectious) to sensitive (e) and resistant (e r ) infections; untreated individuals infected with sensitive (i) and resistant (i r ) infections; treated individuals infected with sensitive (i t ) and resistant (i t,r ) infections; isolated individuals infected with either sensitive or resistant infection (j); and recovered individuals (r). figure 1 shows the movements of individuals between these classes during the course of an epidemic. with parameters described in table 1 , the dynamics of the mean-field model can be mathematically expressed by the following system of differential equations: ). (1) details of the model in its stochastic form are provided in the appendix. a key parameter in disease epidemiology is the basic reproduction number of the invading pathogen, commonly denoted by r 0 , which is the average number of new infections generated by a single infected case introduced into an entirely susceptible (non-immune) population [12] . the quantity r 0 can be used to estimate the growth rate of an epidemic (during the initial phase) and the total number of infections (final size of the epidemic) [13] . when public health interventions are implemented, the reproduction number of disease is affected by parameters that determine the effectiveness of control measures; and we therefore introduce the control reproduction number ( r c ) to evaluate the impact of such parameters on transmissibility of the pathogen and epidemic dynamics. applying a previously established method [12, 14] , for model (1) where s 0 is the size of the susceptible population at the onset of the outbreak. in the absence of treatment and isolation, r c reduces to the basic reproduction number of the sensitive pathogen, given by r 0 = bs 0 /g. using the expression for r c s in (2), one can easily calculate the critical value p * at which r c s = 1 , and therefore the spread of the sensitive infection can be contained for p >p * . rewriting r c s in terms of r 0 , the value p * is given by however, the spread of disease caused by the sensitive pathogen cannot be controlled if r 0 exceeds the threshold r * = (g + a)/δ t qg, which results in p * > 1. since 0 ≤ q ≤ 1, for parameter values used in simulations (table 1) , disease control becomes infeasible if r 0 > 2.5. similarly, there is a critical value p r * at which r c r = 1 , and the spread of the resistant pathogen is contained if which highlights the importance of isolation for controlling the spread of resistant infection. to simulate the models, we considered influenza infection as a case study, for which emergence and spread of drug-resistance during an outbreak can result from treatment of infected individuals. we assumed that the epidemic is triggered by a drug-sensitive influenza virus, and investigated the role of several key model parameters in changing the epidemic patterns and generating multiple waves of infection. these parameters include the fractions of infected individuals identified for treatment or isolation, and the basic reproduction number of disease which varies within the estimated range published in the literature (table 1) . since public health resources may be limited during an epidemic, we [3, 19, 20] baseline values of the parameters used for simulations of the models with sources from published literature. for a given value of r 0 , the baseline transmission rate b can be calculated using the expression r 0 = bs 0 /g. also defined a parameter (t c ) as the capacity for treatment of infected individuals including those who are isolated (i.e., the percentage of the total population that can be treated). to illustrate various scenarios, we initially seeded a susceptible population of size s 0 = 10,000 with e 0 = 10 individuals exposed to the sensitive virus, and assumed that treatment can result in the emergence of resistance with the relative transmissibility δ r = 0.8 during the outbreak. other parameter values are given in table 1 . the mean-field model was simulated for a number of scenarios to show the occurrence of multiple infection waves during an epidemic episode ( figure 2 ). these simulations indicate that variation in the transmissibility of the pathogen (determined by r 0 ), as well as parameters that govern the effectiveness of control measures can significantly impact the epidemic profile, leading to sequences of infection waves with different magnitudes and time-courses. to explore the causes of these multiple outbreaks, we plotted time-courses of treated and untreated sensitive (black curves) and resistant infections (red curves), corresponding to epidemic profiles in figures 2a-2d. as illustrated in figures 2a-2b , a large scale use of treatment (combined with isolation) suppresses the spread of the sensitive infection quickly, but leads to the emergence and spread of resistance that causes the first wave of infection. due to the limited capacity of treatment and isolation (run-out scenario), a second wave of infection follows as a result of wide-spread resistance (red curves), which declines once a sizable portion of the susceptible population is infected and the level of susceptibility reduces below a threshold that is sufficient to block the transmission of the resistant pathogen with reduced fitness. however, this level of susceptibility may still be above the threshold required for disease containment, and therefore the sensitive pathogen can cause the third wave of infection (black curves). as the reproduction number of the sensitive infection increases (figures 2c-2d) , higher treatment levels are required for the resistant infection to prevail and cause a significant outbreak [18] . for a reduced level of treatment and a higher transmissibility of the sensitive virus, corresponding to the epidemic profile in figure 2c , we observed two infection waves, both of which are caused by the spread of the sensitive virus, with generation of very few cases of resistant infection. in this scenario, run-out occurs before epidemic is contained, and a second infection wave takes place. similar dynamics can occur with two subsequent waves of resistant infections for a significantly higher treatment level (figure 2d) . however, the second wave that occurs after the treatment capacity is fully dispensed (run-out scenario) leads to a major reduction in susceptibility of the population; thereby ending the epidemic. these simulations indicate that multiple infection waves could occur due to limited resources for treatment/isolation of infected cases, the ways that such resources are deployed during the outbreak, the evolutionary responses of the pathogen to control measures (e.g., emergence of drug resistance), or a combination thereof. we performed further experiments with small changes in these parameters, and observed significant influences on the epidemic dynamics that can be associated with the elimination or creation of an infection wave. it is worth noting that the above scenarios can take place even for sufficient drug stockpiles for which run-out does not occur, if a policy for adaptation (e.g., reduction) of treatment at the population level is implemented due to wide-spread drugresistance [15] . for comparison purposes, we simulated the stochastic version of the model using a markov chain monte carlo method and observed sequences of infection waves for different sets of parameter values (see appendix). consistent with previous observations [4] , the stochastic model displays a later peak time of infection waves (with lower magnitudes) than the homogeneous mean-field model. this depends not only on the treatment level, but also on other parameters involved in the spread of sensitive and resistant infections, such as the reduction in the potentially infectious contacts and the fitness of resistance. furthermore, stochastic effects can play a significant role in determining disease dynamics even during the outbreak well past the initial establishment phase of the epidemic. this is illustrated in figure 4c of the appendix that the epidemic dies out after the first outbreak in the stochastic model; whereas a second wave of infection takes place in the mean-field model with a larger magnitude compared to the first outbreak. in addition to parameters pertaining to the nature of disease and effectiveness of interventions, the number of infected cases at the onset of an epidemic can greatly influence the dynamics of disease. our simulations (figure 3 ) indicate that small changes in the initial number of infections may result in different epidemic profiles exhibiting more than one infection wave. this suggests that the true dynamics of an emerging disease (with unknown initial number of infections) may not be predicted with certainty, even when reliable estimates of other pathogen-related and intervention parameters are available. stellar advances in the prevention and management of infectious diseases have been achieved since the great influenza pandemic of 1918. yet, emerging pathogens often inflict incalculable devastation to humanity. the global mobilization with rapid international transportation between populations makes the impact of such diseases even more dramatic with potential socioeconomic upheaval. this was recognized in 2003 with the appearance of severe acute respiratory syndrome (sars) as the first major infectious disease threat of the 21 st century [21] , and was recently experienced with the worldwide spread of a swine-origin influenza a virus h1n1, that led the world health organization to declare this virus as the cause of an influenza pandemic on june 11, 2009 [22] . public health responses to the emergence of new diseases often involve difficult decisions on optimal use of health resources over very short timelines. such decisions are further confounded by substantial uncertainties regarding the epidemiological characteristics of the novel infectious pathogen, the effectiveness of public health intervention strategies, and the evolutionary responses of the pathogen under the pressure of control measures [23] . from a population health perspective, it is therefore imperative to look beyond short-term targets and account for long-term disease outcomes in strategy development and implementation. this is particularly important for preventing multiple infection outbreaks that may result from imprudent use of resources or unintended adverse consequences of disease containment strategies. given the historical evidence for the occurrence of multiple infection waves [2, 3, 7] , several modelling studies have attempted to provide explanatory theories for these events in a single epidemic course [2, 5, 6, [8] [9] [10] . in this study, we developed mean-field and stochastic models to investigate possible causes of sequential outbreaks from a public health perspective. our results show that epidemic dynamics can be substantially affected by factors that influence policy design and implementation (e.g., treatment level or isolation of infected individuals), and parameters that determine the effectiveness and consequences of control measures (e.g., reduction in infectiousness due to treatment or emergence of drug-resistance). furthermore, the initial number of infections can influence disease outcomes. while mean-field and stochastic models may exhibit similar epidemic behaviour, we also observed differences in their predictions in terms of the speed with which disease spreads through the population (with further delay in the peak time of outbreaks in the stochastic model); the magnitudes of infection outbreaks; and more importantly, the occurrence of infection waves (see appendix). the latter is particularly influenced by stochastic effects, in addition to the structure of contact patterns and heterogeneity in population interactions [4] . previous work [4, 24] provides a solid foundation for extension of this study through the development of network dynamical models of disease transmission in which heterogeneous contacts between individuals are accounted for. in this study, we simplified the models and included compartments corresponding to some possible stages of a disease; yet we understand that different pathogens may cause infections with different clinical manifestations and infectiousness periods. for example, influenza is known to have a short latent period of less than 2 days before becoming infectious [17] , followed by a pre-symptomatic infection during which disease can be transmitted without showing clinical symptoms; however, the latent period of sars is estimated to be longer and may be comparable to the duration of a complete course of influenza infection [17] . it is also well-documented that influenza can be table 1 of the main text with: (a) r 0 = 1.9, p = 0.65, q = 0.72, t c = 19.5% (three infection waves); (b) r 0 = 1.9, p = 0.5, q = 0.65, t c = 15% (two infection waves); and (c) r 0 = 1.9, p = 0.5, q = 0.66, t c = 16% (one infection wave). black and red curves correspond respectively to the sensitive (untreated and treated: i + i τ ) and resistant (untreated and treated: i r + i t,r ) infections. blue curves illustrate the corresponding scenarios for the total number of infections (i + i t + i r + i t,r ) during epidemic simulated in the mean-field model. in all simulations, initial number of infected cases is e 0 = 10. transmitted in asymptomatic form without developing clinical symptoms [25] ; while evidence for asymptomatic transmission of sars is rather scant. these discrepancies in infection stages of human diseases, combined with the ability of the pathogens to overcome the pressures that are applied to limit their replication and spread, can profoundly impact not only the feasibility and effectiveness of control measures, but also the dynamics of disease over the course of an epidemic. our study highlights these considerations for further investigation, while demonstrating possible mechanisms for the occurrence of multiple infection waves in a single epidemic. future research in this direction should address some limitations of the present study, including a systematic exploration of parameter space to characterize which intervention parameter regimes are more likely to give rise to sequences of infection outbreaks, and to determine the sensitivity of model outputs (epidemic dynamics) on parameter changes. although models considered here are simulated for influenza infection as a case study, understanding the interplay between intervention parameters, evolutionary responses of the pathogens, and epidemic dynamics remains a critical objective of public health for many diseases [26] , including hiv, tuberculosis, malaria, and several bacterial infections. such diseases often share common features, including the emergence and prevalence of drug resistant pathogens under the pressure of drug treatment. the initial rise of resistance is generally associated with fitness costs that make the resistant pathogen less capable of competing with the sensitive pathogen (as the dominant competitor) in a given host population [11] . however, evolutionary mechanisms (e.g., compensatory mutations [27] ) may improve the fitness of resistant pathogens, and therefore intervention measures may result in further selection of resistance, as has been documented for the global spread of seasonal influenza drug resistance that appears to be associated with fitness enhancement processes [28] . this suggests that future modelling efforts should integrate factors that govern pathogen-host interactions with the mechanisms of disease epidemiology to guide public health in devising novel and effective means of infection control. with the same population compartments as defined in the mean-field model described in the main text, we develop a stochastic model for disease transmission dynamics to investigate the epidemic patterns with random effects. we consider time t as a continuous variable, and define the following random vector for ) that represents changes that occur to the random vector at δt units of time. we define the transition probability as , the function θ(·) describes the status of an individual in a subpopulation (i.e., θ(·) = -1: an individual leaves the subpopulation; θ(·) = 0: no changes occur to the individuals' status in the subpopulation; θ(·) = 1: an individual enters the subpopulation). we assume that δt is sufficiently small, so that at most one change of status can occur during the time interval δt, which can be viewed as a markov chain process. the resulting stochastic model can be described as a continuous time markov model, with the transition probabilities given in table 2 . for simulating the stochastic model, we used the markov chain monte carlo method, with an initial e(0) = 10 exposed individuals to sensitive infection in a population of s 0 = 10, 000 susceptibles. a key parameter in these simulations is the step-size of the monte carlo method. using a fixed step-size requires a large number of steps to guarantee that the transitions between subpopulations take place and disease transmission can occur, which is computationally very demanding in terms of both timing and resources. to reduce such a computational load, we implemented an adaptive step-size method [29] to estimate the transition time to the next event (δt) by calculating the sum of the frequencies of all possible events, given by h = b(i + δ t i t )s(t) + δ r b(i r + i t,r )s(t) + (1p)µ e (e + e r ) + pqµ e (e + e r ) + ai t + p(1q)µ e (e + e r ) + g(i + i r + i t,r + j + i t ). then, by choosing δt = u 1 /h, where u 1 is uniform distribution in the interval [0,1], we ordered all possible events as an increasing fraction of h and generated another uniform deviate (u 2 [0,1]) to determine the nature of the next event. for the convergence of the results, we ran these simulations for 1000 samples, and considered the average of sample realizations of the stochastic process to generate infection curves. we ran stochastic simulations with parameter values given in table 1 to illustrate the possibility of multiple infection waves for different scenarios with variation in the basic reproduction number, fractions of treated and isolated ill individuals, and the capacity for treatment and isolation. figure 4a shows that, since the transmission of the sensitive infection is largely blocked by a high treatment level, resistance emerges and causes the first infection wave of the outbreak. the second wave of resistant infections follows after the capacity of treatment and isolation (t c ) is exhausted, and declines when susceptibility of the population falls below a certain threshold that is sufficient to end the resistant outbreak (red curve). however, due to higher fitness of the sensitive infection, a third wave of outbreak occurs which results in depletion of the susceptible population to levels sufficient for ending the epidemic (black curve). we observed similar behaviour in the mean-field model, as illustrated by the blue curve in figure 4a . when treatment level is reduced by a significant margin, generated resistant infection is out-competed by the sensitive infection which has a higher fitness advantage (figure 4b) , and only outbreaks of the sensitive infection occur; the second wave takes place after the capacity of treatment is fully dispensed (black curve). while, the mean-field model also produces similar results (blue curve), we observed differences in the behaviour of the stochastic model. a small reduction in the fraction of isolated individuals leads to the elimination of the second wave in the stochastic model, while mean-field model still produces a second wave with even a larger magnitude than the first wave of the outbreak ( figure 4c ). this suggests that not only are stochastic effects important during the early stages of disease outset, but they also can play a critical role in shaping the epidemic well beyond the establishment phase of the disease. influenza pandemic planning influenza: the mother of all pandemics potential impact of antiviral drug use during influenza pandemic a comparative evaluation of modelling strategies for the effect of treatment and host interactions on the spread of drug resistance quantifying social distancing arising from pandemic influenza coupled contagion dynamics of fear and disease: mathematical and computational explorations turning points, reproduction number, and impact of climatological events for multi-wave dengue outbreaks coinfection can trigger multiple pandemic waves spontaneous behavioural changes in response to epidemics qualitative analysis of the level of crossprotection between epidemic waves of the 1918-1919 influenza pandemic rna viruses mutations and fitness for survival verlag; 2001. 14. van den driessche p, watmough j: reproduction numbers and subthreshold endemic equilibria for compartmental models of disease transmission antiviral resistance during pandemic influenza: implications for stockpiling and drug use management of drug resistance in the population: influenza as a case study incubation periods of acute respiratory viral infections: a systematic review population-wide emergence of antiviral resistance during pandemic influenza containing pandemic influenza at the source strategies for containing an emerging influenza pandemic in southeast asia modelling strategies for controlling sars outbreaks world now at the start of 2009 influenza pandemic world health organization modelling of pandemic influenza: a guide for the perplexed the effect of population structure on the emergence of rug resistance during influenza pandemics factors that make an infectious disease outbreak controllable gaining insights into human viral diseases through mathematics the role of compensatory mutations in the emergence of drug resistance the genesis and spread of reassortment human influenza a/h3n2 viruses conferring adamantane resistance the interplay between deterministic and stochasticity in childhood diseases ):s2. submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution developed mean-field model and performed simulations: lw, sm. developed stochastic model and performed simulations: yh, sm. designed the study and wrote the paper: jw, sm. all the authors have read the final version of the paper and approved it. the authors declare that they have no competing interests. recovery from treated infectionrecovery from treated sensitive infectionincrease in treated resistant infection key: cord-327888-4g3x5dje authors: yuan, c. t.; dembry, l. m.; higa, b.; fu, m.; wang, h.; bradley, e. h. title: perceptions of hand hygiene practices in china date: 2009-02-28 journal: journal of hospital infection doi: 10.1016/j.jhin.2008.09.017 sha: doc_id: 327888 cord_uid: 4g3x5dje summary hand hygiene is considered one of the most important infection control measures for preventing healthcare-associated infections. however, compliance rates with recommended hand hygiene practices in hospitals remain low. previous literature on ways to improve hand hygiene practices has focused on the usa and europe, whereas studies from developing countries are less common. in this study, we sought to identify common issues and potential strategies for improving hand hygiene practices in hospitals in china. we used a qualitative survey design based on in-depth interviews with 25 key hospital and public health staff in eight hospitals selected by the chinese ministry of health. we found that hospital workers viewed hand hygiene as paramount to effective infection control and had adequate knowledge about proper hand hygiene practices. despite these positive attitudes and adequate knowledge, critical challenges to improving rates of proper hand hygiene practices were identified. these included lack of needed resources, limited organisational authority of hospital infection control departments, and ineffective use of data monitoring and feedback to motivate improvements. our study suggests that a pivotal issue for improving hand hygiene practice in china is providing infection control departments adequate attention, priority, and influence within the hospital, with a clear line of authority to senior management. elevating the place of infection control on the hospital organisational chart and changing the paradigm of surveillance to continuous monitoring and effective data feedback are central to achieving improved hand hygiene practices and quality of care. healthcare-associated infections (hcais) are a significant cause of morbidity and mortality among hospitalised patients, affecting more than 1.4 million people worldwide at any time. 1 although hand hygiene (i.e. hand washing with soap and water or the use of a waterless, alcohol-based hand rub) has long been considered one of the most important infection control measures for preventing hcais, compliance rates by healthcare workers with recommended hand hygiene procedures generally fall below 50%. 2 poor adherence to recommended hand hygiene procedures by healthcare workers has been shown to be related to system constraints as well as to individual, group and community behaviour. 3e5 experts in quality improvement have suggested that a multidisciplinary strategy is necessary to improve hand hygiene, including improved training, protocols, engineering controls and equipment, and routine observation and feedback. 6, 7 despite the extensive discussion in the literature about potential interventions to improve hand hygiene in the usa and europe, research from developing countries is less common. given the limited resources available in many hospital settings in developing countries, improving hand hygiene, while critical to reducing hospital-acquired infection rates, may be particularly challenging. 8 specific barriers and hence potential strategies to change healthcare workers' behaviour regarding hand hygiene in resource-poor settings remain unclear. therefore we undertook the current study to identify common issues and potential strategies for improving hand hygiene practices in hospitals in china as an example of a developing country. improving hand hygiene practices globally is a priority of the world health organization, which recently highlighted the worldwide problem of insufficient hand hygiene practices in hospitals and the need for implementation of guidelines. 1 as a developing country, improving hand hygiene in china may be particularly challenging due to resource constraints. by conducting in-depth interviews of infection control directors and key staff, we sought to describe issues of adherence to current people's republic of china's hand hygiene guidelines. we also aimed to describe potential ways of improving hand hygiene practices in the hospital from the perspectives of staff who had been involved with improvement efforts in infection control. we used a qualitative study design based on indepth interviews with key hospital and public health staff. we chose a qualitative study design because it is well-suited to exploratory studies when there is limited previous literature, and to studies seeking to describe in detail causal factors in human behaviour and organisational change that are central to improving hand hygiene in the hospital setting. 9, 10 we conducted in-depth interviews and site visits with a purposeful sample of eight hospitals identified by the chinese ministry of health (moh) as having extensive experience with improvement efforts aimed at infection control and hand hygiene. the interviewees were identified by directors of infection control as having been involved with improvement efforts. we selected hospitals and staff with experience in improvement efforts as 'information rich' sites and individuals who had adequate experience and personal knowledge of the barriers and challenges to improving hand hygiene practices, as recommended by experts in in-depth interviewing. 9 the hospitals were located in beijing (n ¼ 2), shanghai (n ¼ 3), and guangdong (n ¼ 3). a total of 25 healthcare workers of various disciplines were interviewed, including 17 formal, in-depth interviews and eight less formal discussions with physicians and nurses on patient care units. as recommended by experts in qualitative research, in-depth interviews were conducted using a standardised discussion guide consisting of openended questions and probes to encourage greater detail or clarity. 9, 11, 12 examples of questions included: 'how would you characterise hand hygiene practices among the nurses and patient care assistants here at the hospital?'; 'what about among the physicians?'; 'what have you found to be the biggest challenges in improving hand hygiene here at the hospital?'; 'how have you addressed those challenges?'. the interviews were conducted on-site by a single person who was fluent in both mandarin and english. interviews lasted between 120 and 180 min and were transcribed, translated, and then typed to facilitate formal analysis by the research team. we employed the constant comparative method of qualitative data analysis to summarise key themes that emerged from the interview and site visit data. 13, 14 data from the transcribed interviews were reviewed line-by-line by all members of the research team and coded into key concepts. all transcripts were coded by two or more researchers, first independently and then jointly, with differences resolved through negotiated consensus. the code structure was developed iteratively and was reviewed three times by the full research team to ensure its breadth and comprehensibility. hospital staff viewed proper hand hygiene as important for quality and safety both hospital and government officials stated that infection control practices were of primary importance to the quality of their hospital care. furthermore, all participants recognised the fundamental role of hand hygiene in infection prevention. hospital workers noted that hand hygiene was important for their own safety as well as the patients' well-being. as one hospital president reflected: hands are a major link to transmission of antibioticresistant microorganisms, which then cause difficulty in clinical treatment, and also waste medication and money. therefore, infection control management is very important. hospital staff also viewed the process of improving hand hygiene as having ancillary benefits, stating that the systems and strategies for improving hand hygiene could have positive 'spillover' effects for other infection control practices. these staff suggested that hand hygiene might bring about greater focus on additional behaviour changes such as use of personal protective equipment and more regular and thorough equipment decontamination practices by healthcare workers. hospital workers were knowledgeable about hand hygiene practices participants were generally well-informed about recommended hand hygiene practices, and several officials described that this knowledge had improved substantially since the severe acute respiratory syndrome (sars) outbreak. as one hospital infection control director commented: during the sars outbreak and avian flu prevention period, we had dedicated orientations for educating healthcare workers about hand hygiene. since sars, there are many more hand hygiene training sessions, and people's consciousness in this area has increased. although there was some variation among healthcare workers in their knowledge and practices regarding hand hygiene, in general, interviewees suggested that compliance had improved substantially in the previous few years since the sars outbreak. some suggested that the knowledge of hand hygiene guidelines was greater among young hospital staff or among the more educated public. whereas healthcare workers both appreciated the importance of hand hygiene and understood the recommended practices, many reported that proper practices often did not occur due to limited equipment to support hand hygiene efforts. staff remarked on inadequate budgets for the infection control department, which was viewed as a source of cost rather than as a source of revenue generation. equipment gaps included running water and soap, clean towels, and gloves. the issue of inadequate equipment and resources was highlighted by this hospital infection control director who said: even if healthcare workers' sense to do hand hygiene is strong, facilities and equipment need to improve, like having paper towels for instance. no one is willing to use publicly shared towels. they all wipe their hands on their lab coats. a primary issue that healthcare workers reported as hindering improvements in hand hygiene practices was inadequate organisational authority vested in hospital infection control departments. limited numbers and qualification of hospital infection control staff was also cited as a challenge, but the lack of organisational influence of the departments and their staff were most problematic. typically, infection control departments were managed by nurses or junior physicians, who were viewed as 'outside' the more powerful spheres of senior physicians and hospital management. despite the participants' acknowledgement of the importance of having links to senior management, in many hospitals infection control directors did not report organisationally to hospital administration. like hospital staff interviewed, several government officials also stated that infection control staff needed to have the authority to monitor and then follow up on problems with other staff in order to be effective. without adequate senior level commitment to these goals, infection control functions were unlikely to be able to make any substantial improvements in practices. as one city quality control association representative said: infection control personnel do not have power to enforce infection control guidelines: no funding, no administrative power, and no financial budgeting power. in addition to statements about infection control having inadequate access to senior management, participants also highlighted the importance of having physicians, not just nurses, represented in hospital infection control departments. participants noted the importance of physician-tophysician communication about hand hygiene in order to change behaviour. they indicated that having nurses, who may be informed about infection control, talk with physicians was unsuccessful in influencing changes in physician hand hygiene practices due to the power differentials between physicians and nurses. the physician perspective was described as important for all aspects of infection control: developing hospital policies about hand hygiene, monitoring staff adherence with policies, and providing surveillance data about hand hygiene performance to physicians. for example, one hospital infection control director commented: most infection control personnel are nurses. the hierarchical gap between physicians and nurses is important. physicians would not follow nurses' suggestions, and don't even mention their criticism. despite the obvious need for physicians in infection control departments, physician participants stated that they were not eager to work in or with infection control departments due to the lack of respect afforded to infection control clinicians by other physicians and the lower pay in such positions. an added challenge for infection control departments was their lack of autonomy from other clinical departments in the hospital. although the moh guidelines were described as recommending that the infection control departments should be independent from hospital clinical departments, many infection control staff were housed within other clinical departments and hence faced some conflicts of interest in monitoring and reporting non-adherence to hand hygiene guidelines by colleagues. one hospital infection control director made this conflict clear: we have an independent infection control department now, but soon it will be allocated to another department. it will be difficult to manage surveillance and criticism of this other department because they pay our salary and our bonus. although all hospitals had surveillance systems for monitoring hand hygiene, no system was designed to enhance adherence to recommended hand hygiene practices. several aspects of both the moh and hospital internal surveillance approaches were problematic. first, surveillance focused on outcomes such as infection rates and bacteria counts, rather than on the process of proper hand hygiene. some participants noted that data on adherence to recommended hand hygiene processes which were known to improve outcomes (reduced hcais) would provide concrete targets for improvement. however, participants also said that monitoring of hand hygiene practices was not done routinely in any hospital. the focus was on bacterial counts on sampled hands of workers. second, data were rarely actionable as they were produced from random checks and reported in unidentified ways months after the measurement. subsequently, while the data could show trends in infection rates at the hospital, they were too generalised to foster quality improvement efforts on particular patient care units. as a chinese center for disease control and prevention (cdc) representative commented: the surveillance system is primordial. the outcomes are not very sensitive to result-based surveillance of disinfection agents and sample of hands. it is more useful to get results from process-based surveillance, but it is difficult to implement in hospitals. participants also said that individual hospitals would be unlikely and perhaps unable to initiate improved data monitoring unless directed by the government, citing that there was limited understanding of quality improvement and data feedback techniques in their hospitals. participants suggested that a government mandate to do increased surveillance at the hospital level would be the best strategy to change behaviour. as one hospital infection control director noted: in china, the government's mandate is the most powerful tool. if anything has to be promoted or implemented fast, you must obtain government's administrative order first. a small minority of participants supported the development of a hospital accreditation system that might include surveillance of infection control practices, although details of how this might dovetail with existing governmental monitoring by moh or by increased government mandates were not discussed. findings from this study suggest that the primary challenges in improving hand hygiene in china are the limited authority of infection control departments in hospitals, the lack of essential resources, and the ineffective use of data monitoring and feedback to hospital staff. these insights are important as previous studies have attributed poor hand hygiene practices to individuals' knowledge and attitudes, and typical strategies to improve hand hygiene involve staff training. 15, 16 based on our study, the reasons for inadequate hand hygiene are more complicated, and strategies to address this behaviour require greater understanding of the organisational culture and systems of accountability that exist in hospitals in china. a major decision is where the hospital infection control department is on the organisational chart. in our study hospitals, infection control staff often reported within general medicine and not to senior administration. this was problematic for two reasons. first, access to senior management, who set overall goals for the hospital and who determined the allocation of resources in part, was limited or non-existent. the infection control budget was typically under the departmental level. as department heads were evaluated in part by their financial outcomes, they were less willing to allocate budget to infection control since it did not generate revenue. as a result, infection control was not included in the strategic or financial discussions of priorities in the hospital, and the infection control department director was unable to discuss directly with senior management in order to request resources for the department. second, as a result of its reporting to the same department that it was monitoring, often general medicine, some infection control directors had conflicts of interest in performing surveillance on peers within the same department, especially when in some cases they depended on the head of general medicine for their departmental budget. while this could be effective in integrating infection control in a clinical department, given that many infection control staff were nurses, their ability to influence physician behaviour was limited. based on our findings, having the infection control director report to senior management and allowing the department some independence from medicine might provide the context in which the necessary resources and organisational attention could be directed at improving hand hygiene practices. in addition to building greater management support for infection control, more modern methods of hand hygiene surveillance were needed. data monitoring and feedback is central to quality improvement techniques and has been shown to be effective in a number of clinical areas including hand hygiene. 17e19 previous studies on hand hygiene adherence have shown that, with respect to processes that staff can control, consistent and timely data feedback provide for greater accountability and improvements in the monitored process. 20e23 however, the participants in this study indicated that surveillance for hand hygiene was focused on bacterial counts on hand samples, which were randomly checked in the hospital. monitoring of observed hand hygiene practices was not generally conducted, limiting the ability to provide timely data on the action needing improvement. instruments exist to facilitate a simple process of observation and data feedback to staff on hand hygiene. 24 using the principles of quality improvement, hospitals could set targets for hand hygiene practices, implement observation-based data monitoring, and provide feedback to staff about performance. although such monitoring does require resources and attention, it can be far more effective than random checks of hand bacteria. if done in a non-punitive, learning environment, such data feedback can drive substantial and sustained improvements in healthcare worker practices. these findings should be interpreted in light of the study limitations. this was a qualitative, exploratory study in which we sought to understand in depth the reasons for inadequate hand hygiene practices in china. interviews and observations were conducted at hospitals that had had previous experience in improvement efforts directed at infection control practices and therefore may not represent the experiences in other hospitals. in addition, although we ensured that the interviewer was fluent in mandarin and was embedded in the hospitals to reduce misunderstandings and mistrust, participants may have withheld information. given the types of responses received and the guaranteed anonymity, however, we believe that participants were forthcoming. furthermore, the sample was relatively small, which is common with qualitative studies. 9 we did achieve theoretical saturation, suggesting that we obtained a comprehensive view of the issues. 9, 13 however, a broader sample with more diverse participants may have generated additional themes. finally, this was a hypothesis-generated study about the possible causes of inadequate hand hygiene in hospitals. we did nonetheless employ several strategies recommended by experts to enhance the rigor and validity of qualitative studies, including the consistent use of a discussion guide, use of researchers from diverse disciplines to conduct the analysis, and sampling until the point of theoretical saturation. 9e14 future research should test whether changes in these factors result in significant improvements in hand hygiene practices. as we strive to improve quality of hospital care, resource-poor settings present particular challenges. china, with all its economic growth, is on the verge of enormous expansion and the quality of hospital care will be a critical factor in supporting a healthy and productive population. infection control practices are of critical importance to overall quality of care and safety of healthcare workers and their patients, as well as the communities we share. despite international engagement in improving hand hygiene, all countries struggle to sustain proper hand hygiene practices in healthcare. 1 our study suggests that the core issues are about the degree to which the infection control department and its staff are given adequate attention, priority and influence within the hospital with a clear line of authority to senior management. elevating the place of infection control on the hospital organisational chart and changing the paradigm of surveillance to continuous monitoring and effective data feedback are central to achieving improved hand hygiene practices and quality of care. who guidelines on hand hygiene in health care (advanced draft): a summary e clean hands are safer hands. world alliance for patient safety. geneva: world health organization influence of role models and hospital design on hand hygiene of healthcare workers hand hygiene among physicians: performance, beliefs, and perceptions determinants of good adherence to hand hygiene among healthcare workers who have extensive exposure to hand hygiene campaigns behavioural considerations for hand hygiene practices: the basic building blocks improving adherence to hand hygiene practice: a multidisciplinary approach guideline for hand hygiene in health-care settings. recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force. society for healthcare epidemiology of america/association for professionals in infection control/infectious diseases society of america the evolution: handwashing to hand hygiene guidance qualitative research and evaluation methods qualitative methods: what are they and why use them? the long interview basics of qualitative research: techniques and procedures for developing grounded theory the discovery of grounded research: strategies for qualitative research qualitative data analysis for health services research: developing taxonomy, themes, and theory behavioral interventions to improve infection control practices why healthcare workers don't wash their hands: a behavioral explanation out of crisis quality control handbook data feedback efforts in quality improvement: lessons learned from us hospitals increasing handwashing in an intensive care unit increasing icu staff handwashing: effects of education and group feedback teaching hospital medical staff to handwash effectiveness of a hospital-wide programme to improve compliance with hand hygiene promotion of hand hygiene techniques through use of a surveillance tool none declared. key: cord-341987-lsvifqyo authors: kalyanasundaram, sridhar; krishnamurthy, kandamaran; sridhar, aparna; narayanan, vidya kanamkote; rajendra santosh, arvind babu; rahman, sayeeda title: novel corona virus pandemic and neonatal care: it’s too early to speculate on impact! date: 2020-08-03 journal: sn compr clin med doi: 10.1007/s42399-020-00440-8 sha: doc_id: 341987 cord_uid: lsvifqyo the entire world is reeling under the effects of the novel corona virus pandemic. as it is a new infection, our knowledge is evolving constantly. there is limited information about impact of corona virus on neonatal care in relation to newborns with confirmed or suspected covid-19. in this article, we summarize the current approach to this infection in relation to newborn babies. we discuss the basic aspects of the infection, the approach of care to novel corona virus disease 2019 (covid-19) in positive pregnant women, the likely presentation in newborns (as per current knowledge), and the approach to the management of neonates with infection or at risk of the infection. children are less susceptible to covid-19 infection and generally have a mild course. there is a lower risk of severe disease among pregnant women and neonates. it was recommended to follow the current protocols for management of symptomatic newborn with isolation precautions, antibiotics, and respiratory support. since december 2019, when the novel corona virus-related infections were reported in the wuhan province in china, the world has witnessed a situation never seen before. the virus this article is part of the topical collection on covid -19 has now been reported in most countries around the world and since march 11, 2020 , has been declared a pandemic by the world health organization (who) [1] . there has been a high case fatality rate, and as of now (mid-june 2020), close to 8 million cases and nearly 440,000 deaths have been reported [2] . as it is a new infection and disease characteristics are still being elucidated in many settings, the exact protocols that we follow in different age groups will need regular updates. the knowledge on covid-19 in neonates is only based on a recent experience over the past 6 months or so. children are less susceptible to covid-19 infection and generally have a mild course in newborns, and children experience with significantly lower death rates [3, 4] . moreover, there is limited information about the impact of corona virus on neonatal care in relation to newborns with confirmed or suspected covid-19 [5, 6] . in this article, we discuss the basic aspects of the infection, the approach of care to novel corona virus disease 2019 in positive pregnant women, the likely presentation in newborns (as per current knowledge), and the approach to the management of neonates with infection or at risk of the infection. the novel coronavirus, named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), belongs to the family of viruses called beta coronavirus, similar to the one causing sars-1 outbreak in 2002-2003 [7] . it is a single-stranded rna virus with a helical capsid with radiating spikes (hence the name corona), and the disease is referred to as coronavirus disease 2019 . later on, on february 2020, the coronavirus study group of the international committee on taxonomy of viruses issued a statement announcing an official designation for the novel virus: severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [8] . the virus spreads by airborne (aerosol and droplet generation by atomization while coughing, sneezing, or even talking) and droplet contact spread and has a high attack rate [2, 9] . although the elderly people with co-morbidities are at high risk, so far, newborns and children are relatively spared from serious complications [10] . symptoms of covid-19 appear to be less severe in infants and children in comparison than that of adult patients [1, [11] [12] [13] . the diagnosis is made by positive reverse transcriptase-polymerase chain reaction (rt-pcr) test result for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) usually on swabs taken from deep intranasal and oropharyngeal swabs [2, 9] . the antibody testing and other tests to assess immune status are not fully developed yet. pregnant women with covid-19: impact on maternal/neonatal health currently, there is no evidence of higher risk of severe disease and complications among pregnant women with covid-19 compared with healthy non-pregnant adults [14] [15] [16] [17] [18] . there is only limited data on the impact of the current covid-19 outbreak on women affected during pregnancy and newborns. currently, no data was suggesting an increased risk of miscarriage in pregnant women with covid-19 infection. in women with symptomatic covid-19, there may be an increased risk of fetal compromise in active labor [19] . women have been advised to avoid water births to prevent the risk of disease transmission through feces. data from china found severe complications in 8% of pregnant women with covid-19 [14] . however, the high rate of cesarean section deliveries (csd) in chinese reports is concerning, and subsequent reports from different countries have not confirmed any need to consider csd apart from the obstetric and maternal conditionbased decisions. martinez et al. [15] reported delivery details of 72 covid-19 positive mothers in spain, and the csd decision was based on obstetric decision (just over 40% csd rate). the study could not demonstrate the presence of coronavirus in placenta, amniotic fluid, or cord blood in the cases [16] . the maternal outcome was slightly worse (in terms of needing respiratory support) for mothers undergoing csd, and the newborn outcome was not different. only 3 of the 72 newborns were positive on the initial test, and all of these were negative on the repeat test at 48 h. two of the babies developed infection after 2 days, likely acquired from the mother, but all babies were well and asymptomatic [15] . besides, initial reports on the covid-19 infected pregnant women in wuhan indicated that most of them were in their third trimester, few on second trimester, and none identified at first trimester [20] . however, the study showed that the fetus of the sars-cov infected mother in the first trimester of pregnancy would develop intrauterine growth restriction (iugr); therefore, more attention should be paid on the prevention of covid-19 in the first trimester of pregnancy [21] . there is no evidence that covid-19 has an effect on fetal development [22] ; however, increased potential risk of preterm delivery has been emphasized [23] . an analysis of 23 studies involving pregnant mothers with covid-19 demonstrated a preterm delivery rate of 47% [24] . one of the major complications of preterm deliveries, necrotizing enterocolitis, may overburden the obstetrics and neonatal services [23] . recent studies from the uk and other countries confirmed that vertical transmission due to covid-19 can occur, although the rate is low [14] [15] [16] [17] [18] . it is encouraging that horizontally infected neonates had shown a mild clinical profile with good outcomes [10, 25] . early chinese reports suggested that vertical transmission of sars-cov-2 does not occur, as amniotic fluid, vaginal mucus, placenta, umbilical cord, cord blood, and neonatal stool specimens tested negative for the virus [19, 26] . congenital sars-cov-2 infection, with virus present in a neonate's nasopharynx at the time of birth, may occur, with a frequency not yet defined. there are reports of perinatal spread especially where the mother is symptomatic just prior to delivery; this could be explained by the relatively high viral load in symptomatic mothers [15, 19, 27] . pcr is a highly sensitive test, and even vaginal secretions in the baby's nose can cause positivity. results from cord sample and liquor samples are not clear-cut. in another series of 7 neonates born by csd in zhongnan hospital of wuhan university, 3 newborns showed high igm levels to covid 19, suggesting antenatal infection (as igm does not cross placenta); however, false positive tests cannot be ruled out [28] . recent ukoss (uk) study confirmed that vertical transmission due to covid-19 can occur; however, the rate is low [29] . the study followed up 427 pregnant women admitted to the hospital with confirmed covid-19 infection, and 243women had given birth. among the infants delivered, 5% (n = 12) tested positive, and six infants tested positive within the first 12 h of birth. igm antibodies were detected (cord blood serum) in 3 infants who were tested negative and were all asymptomatic. postnatal transmission from parents or carers who have the infection (or are asymptomatic carriers) is the commonest reason a baby may get infected. the role of breast milk in spreading is also being debated, as there have been reports of breast milk being positive for the virus where the mother was symptomatic around delivery [30] . however, the world health organization (who) as well as other bodies like the canadian pediatric society encourage breast feeding either directly or as expressed milk after parents have been explained the risk and benefits [31, 32] . a recent study has reported that neonatal covid-19 infection is uncommon, uncommonly symptomatic, and the rate of infection is no greater when the baby is born vaginally, breastfed, or allowed contact with the mother [33] . another recent case study published in nature communication reported transplacental transmission of covid-19 from a positive pregnant mother during the last trimester to her offspring which occurred due to maternal viremia, placental infection, and neonatal viremia following placental infection [34] . during this crisis, obstetrics and neonatal departments in most hospitals need to plan major changes in their daily health professional team as there may be shortages of staff due to the infection or replacement in other positions [18] . further, stress for being infected and possibly infecting families, overwork with long shifts, isolation, restrictions on socialization, and death of relatives or colleagues may decrease their performance [18, 35] . strict precautions should be maintained by using personal protective equipments (ppe) with social distancing measures in the obstetric and neonatal wards to minimize staff exposure [18, 36] . breaks should be spread out so colleagues do not eat or drink with each other without mask. if any team member has symptoms or has an infected family member, he/she should be self-isolated and tested. asymptomatic contacts of this team member should be tested as well, and self-isolation in these cases depends on exposure risk and unit policy [35] . the ppe shortages are reported worldwide, and this should be considered in decision-making. some hospitals have devised their own policies to reuse n95 or equivalent masks on a rotational basis. this is an increasingly common scenario as the virus spreads in the local community and the pregnant women are likely to get infected. thankfully, thus far the disease has not been reported to be any more severe in pregnant women compared with other healthy adults, though mothers with co-morbidities will still be at high risk. routine separation of the mother and baby is not promoted, and guidance on individualized care is recommended in pregnancy and delivery [12] . this has been well discussed in a recent article published by chandrasekharan et al. [32] . from the obstetric and neonatal team's point of view, a clear plan has to be put in place preferably including the parents in the discussion-the following should be discussed [18, 32, 35, 37] : 1. full ppe as per guidelines for all healthcare personnel involved in delivery process and attending the delivery (resuscitation of newborn). be careful when baby needs suction, intubation, or mask ventilation as these are aerosol generating procedures. it is recommended to move babies between areas in an incubator as far as feasible. 2. avoid close contact with the mother soon after birth (skin to skin care not given); delayed cord clamping should be done as per protocol. 3. if the mother is not symptomatic, the baby can be roomed in with the mother with a safe distance of 6 ft (2 m) between her bed and the crib. except while feeding and during cares, the baby should be in the crib. the mother should wear gloves while handling the baby (preferably) and should wear a mask while approaching or holding the baby or during feeding. 5. breast feeding options should be discussed. if the mother is symptomatic, we could consider giving a formula and expressing and discarding the milk during the symptomatic phase (when the likelihood of viral shedding in milk is more, and also since the mother may be on antiviral treatment some of which may be relative contraindications during breast feeding). if mother is asymptomatic, she could either breast feed directly wearing a mask or express breast milk wearing a mask and an unaffected relative/carer could feed the baby. 6. where the mother is symptomatic, especially if she is unwell to look after the baby, it is better to consider separation until the mother is asymptomatic. the baby can then be transferred to her and would use the approach to feeding as above. due consideration should be given for formula feeds if expressed milk is not adequate or the mother is unable to express due to her condition. depending on family circumstances and resource availability, the baby can be kept away from the mother either in a nursery setting or in a separate room and looked after by an unaffected family member, with plan to discharge home with them once stable. this option is not preferred if mother is symptomatic but can be offered in an asymptomatic mother if family prefers this option and resources permit the same. the parents should be involved in this decision, and they should be aware that despite these precautions, there is a small risk of the baby getting the infection, and this discussion should be documented. 7. the current recommendation is for the baby to get the test on day 1 (preferably before any direct contact with mother), and if positive, the test could be repeated on days 3-5 and further repeat if still positive. if initial test is negative, we could consider testing again after 48 h in case of a false negative first test. if the baby and mother are well, and the baby is negative, the baby could be discharged home. if the baby is positive for covid-19, because of the unpredictable course, we could consider monitoring in the hospital until negative result is documented. as the condition evolves and we see more cases, testing to confirm in asymptomatic babies may not be needed-it should be feasible to manage asymptomatic covid-19 positive babies at home as well after a period of initial stability, and routine repeat tests may not be needed as long as they are isolated and monitored adequately. in some cases, the babies have presented with fever, loose stools, and respiratory distress, but majority is asymptomatic [25] . since community spread is noted in most countries, any symptomatic baby presenting with fever, diarrhea, unexplained respiratory distress, and other manifestations should have a covid-19 pcr test sent. even if the baby's test is positive, management is according to current protocols for symptomatic newborn with isolation precautions, antibiotics and respiratory support as indicated. such babies should be nursed in incubators. though the clinical outcome has been good, the neonates with covid-19 are more likely symptomatic than older children who get the infection. babies who are asymptomatic could be managed with the parents (rooming in). babies are unlikely to be infectious unless aerosol generating events like crying or sneezing, but healthcare workers should wear full ppe while handling them. stool may be infective as well, and precautions are essential while handling stools. antipyretics like paracetamol can be used as normally indicated. if the baby is unwell with respiratory distress, antibiotic cover as per unit policy would be indicated. in babies presenting with gastrointestinal concerns, a period on intravenous (iv) fluids may be needed, but most of these symptoms appear to resolve over 2-3 days. some babies present like acute bronchiolitis and may need a period of respiratory support. as high flow nasal cannula therapy and nasal continuous positive airway pressure (cpap) are aerosol generating procedures, such babies should be in incubators, with expiratory flow tubing preferably within the incubator. the severe disease in adults is a result of an uncontrollable host inflammatory response, a cytokine storm [38] , and luckily, this is less pronounced in children as a group including neonates and that could be a factor behind the milder manifestations in this age group. the kawasaki like inflammatory syndrome described in older children has not been noted in newborns, but we should be alerted to record and publish such presentations if we encounter them. there are no reports so far regarding experience with antivirals and use of immunomodulators like hydroxychloroquine in neonates so far. the recent recovery study [33] in a mainly adult population (unpublished as of now) has reported improvement in patients needing oxygen or ventilatory support with the use of steroids, and if a newborn is sick with covid-19-related complications, this could be a factor to consider, though not evidence based yet. all suspected or confirmed covid-19 neonates are required to be admitted to neonatal intensive care units (nicus) [39, 40] . since community transmission places any individual at risk of being asymptomatic and carrying the virus, it is advisable to minimize visiting hours (and allow only parents to visit) [41] . skin-to-skin care and direct breast feeding while in nicu may need to be minimized in open layout nicus [33] . unfortunately, one of the negative effects of this practice would be exposure to bottle feeding, as cup feeding or syringe feeding needs closer contact and possible aerosol exposure. it was suggested that covid-19 negative results of respiratory specimens or anal swabs should be obtained at least 48 h before discharge [39] . general oral care recommendations such as oral hygiene care by gentle wiping of oral cavity using sterile gauze dipped in drinking water should be followed. colostrum can serve as a beneficial oral care in newborn especially for preterm infants. dental procedures are usually indicated when neonates have the presence of natal or neonatal teeth. dental extraction is indicated when natal or neonatal teeth are associated with the following conditions: (i) mobility, (ii) inconvenience during sucking/breast feeding, (iii) oral ulceration, and (iv) supernumerary teeth [33] . during covid-19 pandemic, the dentist must clinically evaluate oral cavity and history associated with feeding discomfort. the dentist should prefer early appointment during the beginning of weekday. early appointment will prevent the neonate to expose to the patient crowd in the dental office and preventing cross-infection. telephone conversation and teledentistry should be preferred mode of communication with dentists. dental practices are considered the focal points for cross-infection, and dental care professionals must take precautions to minimize the risk of infection by adopting national/international infection control and prevention guidelines [42] . since this is a novel infection, an ongoing coordinated multinational data collection [43] the current crisis is a unique situation faced by the medical fraternity worldwide. it is very important to share clinical and research information and disseminate unique presentations, as well as contribute wholeheartedly to the data collected by registries as mentioned above. as more neonates are affected with acute disease, it is possible that we will see a broader spectrum of problems and we should be alert to new presentations. more challenges will be faced like payment systems and insurance-related issues restricting more frequent testing (when governments scale down testing), and local teams should work together to formulate guidelines suitable to their system, so they can overcome such challenges by working together in a team. authors' contributions sk: conceptualized the review, conducted literature review, extracted relevant information, and drafted the manuscript. kk: conceptualized the review, conducted literature review, extracted relevant information, and drafted the manuscript. as: conducted literature review, extracted relevant information, and reviewed and revised the manuscript. vkn: conducted literature review, extracted relevant information, and reviewed and revised the manuscript. as: conducted literature review, extracted relevant information, and reviewed and revised the manuscript. sr: conducted literature review, extracted relevant information, and critically reviewed the manuscript for important intellectual content. conflict of interest the authors declare that they have no conflict of interest. ethical approval this article does not contain any studies with human participants or animals performed by any of the authors. consent for publication all authors reviewed and approved the final version and have agreed to be accountable for all aspects of the 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so far the royal college of paediatrics and child health (rcpch) covid-19 -guidance for neonatal settings royal college of obstetricians & gynaecologists. coronavirus (covid-19) infection in pregnancy. information for healthcare professionals. version 3. london: rcog analysis of clinical features of 29 patients with 2019 novel coronavirus pneumonia national clinical research center for child health and disorders and pediatric committee of medical association of chinese people's liberation army. a contingency plan for the management of the 2019 novel coronavirus outbreak in neonatal intensive care units working group for the prevention and control of neonatal sars-cov-2 infection in the perinatal period of the editorial committee of chinese journal of contemporary pediatrics maternal transmission of sars-cov-2 to the neonate, and possible routes for such transmission: a systematic review and critical analysis coronavirus disease (covid-19): characteristics in children and considerations for dentists providing their care publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-349298-8s69wprh authors: munywoki, p. k.; koech, d. c.; agoti, c. n.; kibirige, n.; kipkoech, j.; cane, p. a.; medley, g. f.; nokes, d. j. title: influence of age, severity of infection, and co-infection on the duration of respiratory syncytial virus (rsv) shedding date: 2014-06-05 journal: epidemiol infect doi: 10.1017/s0950268814001393 sha: doc_id: 349298 cord_uid: 8s69wprh rsv is the most important viral cause of pneumonia and bronchiolitis in children worldwide and has been associated with significant disease burden. with the renewed interest in rsv vaccines, we provide realistic estimates on duration, and influencing factors on rsv shedding which are required to better understand the impact of vaccination on the virus transmission dynamics. the data arise from a prospective study of 47 households (493 individuals) in rural kenya, followed through a 6-month period of an rsv seasonal outbreak. deep nasopharyngeal swabs were collected twice each week from all household members, irrespective of symptoms, and tested for rsv by multiplex pcr. the rsv g gene was sequenced. a total of 205 rsv infection episodes were detected in 179 individuals from 40 different households. the infection data were interval censored and assuming a random event time between observations, the average duration of virus shedding was 11·2 (95% confidence interval 10·1–12·3) days. the shedding durations were longer than previous estimates (3·9–7·4 days) based on immunofluorescence antigen detection or viral culture, and were shown to be strongly associated with age, severity of infection, and revealed potential interaction with other respiratory viruses. these findings are key to our understanding of the spread of this important virus and are relevant in the design of control programmes. respiratory syncytial virus (rsv) is a major viral cause of lower respiratory tract infection in children worldwide [1] with the key risk group being young infants [2] . no vaccine is currently available for this age group. development of alternative control strategies depends on the mechanisms of transmission, which are intrinsically related to viral shedding [3, 4] . detailed data on shedding in individuals in relation to age, infecting subtype (groups a or b), infection severity, and gender would help in identifying the source of infant infection. such data from the natural setting unaffected by sampling bias are limited and absent in resource-poor settings. additionally, this study assesses the impact of the presence of other respiratory viruses, prior to or concomitant with rsv, on rsv infection duration. any interaction might be mediated through direct interference or host immune and physiological responses. these possibilities have received very little attention in the literature. previous studies on rsv shedding have been mainly in the hospital setting limiting the generalizability of the results [3, 5, 6] . hospital studies are biased to young children with severe rsv disease and fail to precisely establish the start and, often, the end of shedding, particularly when symptoms do not coincide with virus shedding which is common for rsv [6] . community-based studies are likely to provide a more complete representation of the rsv shedding patterns. such studies require frequent nasopharyngeal swabbing regardless of symptoms and use of sensitive molecular techniques for viral testing in order to minimize the likelihood of missing infection episodes especially in older age groups. the current prospective study utilizes the above approach, with intensive sampling (every 3-4 days), molecular testing, and follow-up of individuals of all ages for one complete rsv season in a rural kenyan community [7] . this provides for more realistic estimates on duration of and influencing factors on rsv shedding which are required in designing rsv prevention strategies and to better understand the impact of vaccination on rsv transmission dynamics. the study was undertaken in rural coastal kenya within the kilifi health and demographic surveillance system (khdss) [8] . the study methods have been described elsewhere [7] . briefly, a prospective cohort study was undertaken with a recruitment target of 50 rsv-naive infants and their household members. a household was defined as a group of individuals living in the same compound and with common cooking arrangements. households were eligible if they contained a child born after the end of the 2008-2009 rsv epidemic, and one or more older siblings (aged <13 years). sampling visits were timed to begin and end coincident with the start and finish of the expected rsv season of 2009-2010 [7] . trained field assistants made twice weekly household visits, collecting deep nasopharyngeal swabs (nps) irrespective of symptoms and recording clinical illness data from all participants. nps were collected and tested for rsv (groups a and b) and other respiratory viruses [adenoviruses, rhinoviruses and human coronaviruses (nl63, 229e, oc43)] using real-time multiplex pcr as described previously [7] . in order to establish the genetic similarity of the rsv strains in suspected repeat infections, the ectodomains of the rsv attachment (g) protein gene were sequenced and analysed phylogenetically [9] . the authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the helsinki declaration of 1975, as revised in 2008. data were analysed using stata version 11.2 (statacorp, usa). the infection data were intervalcensored and three possible durations of viral shedding were estimated, i.e. minimum, midpoint and maximum, as described in the supplementary online material and shown in figure s1 . an rsv infection episode was defined as the period within which an individual provided specimens which were pcr positive for the same infecting rsv group with no more than 14 days separating any two positive samples. episodes where the first sample was positive for both rsv groups a and b counted as one infection episode. episodes where no samples were collected for >7 days before or after the infection episode were considered left-or right-censored, respectively. symptomatic infection was defined as the presence of one or more of the following symptoms: cough, nasal discharge/blocked nose, or difficulty in breathing at any time during the infection episode. co-infection was assigned when within the rsv episode any sample was pcr positive for another virus, i.e. coronavirus, rhinovirus, or adenovirus. presence of these viruses in the samples collected in the period of 14 days prior to start of rsv episodes was also defined. household outbreak was defined as a period within which more than one individual episodes occurred in members of the same household without an interval of 514 days in which a pcrpositive specimen was absent from the household. the proportion of the household members infected during household outbreaks measured the intensity of the outbreak. cox proportional hazards models were used to identify factors influencing the rate of loss of virus detection (hereafter referred as the recovery rate). the effect of left-censoring was accounted for in the multivariate model by including a dummy variable or by excluding the left-censored episodes. of the 493 individuals in the 47 households followed, 179 (36·3%) had at least one rsv infection from 40 (85·1%) different households. the median age (interquartile range; iqr) at the start of the first observed infection was 6·5 (iqr 2·4-14·5) years, and females numbered 96 (53·6%) ( table 1) . a total of 205 infection episodes were observed with 155 individuals experiencing one episode, 22 with two episodes and two individuals experiencing three episodes (fig. 1 ). rsv group a was associated with 88 infection episodes, rsv group b with 113 while seven episodes were co-infections. there were 177 (86·3%) fully observed episodes while 11 and 15 infection episodes were left-and right-censored, respectively, and two episodes were both left-and right-censored. of the 24 individuals with two or more episodes (suspected repeat infections), 17 (70·8%) were infected with the same rsv group and otherwise all except one group a infection followed group b. the mean age at the first infection for individuals with rsv group a and group b was 2·3 and 7·2 years, respectively. the duration between the episodes ranged from 17 to 54 days with median of 28 days. for the 17 homologous infections, sequencing of the rsv g gene was successful in 10 (59%) of the 18 possible pairs of samples (one individual had three suspected rsv episodes). the failure to sequence was mainly in samples with a pcr cycle threshold (c t ) value of > 28·0, an indicator of low viral load. only one of the successfully sequenced paired samples showed nucleotide differences: 13 nucleotide differences associated with three non-synonymous changes and a change in the stop codon position. the episodes in this individual (id no. 1803 in fig. 2 ) occurred 54 days apart. for the purposes of estimation of the shedding duration, all the episodes were considered distinct. from the 205 infection episodes, the mean duration of shedding based on minimum, midpoint and maximum estimates were 8·6 [95% confidence interval (ci) 7·5-9·7], 11·2 (95% ci 10·1-12·3) and 14·0 (95% ci 12·8-15·2) days, respectively, for all rsv episodes (fig. 3) . the corresponding mean durations of shedding for the 'fully observed' episodes were 8·2 (95% ci 7·1-9·4), 10·9 (95% ci 9·8-12·1) and 13·6 (95% ci 12·4-14·8) days, respectively ( table 2) . twenty-four individuals shed rsv for 521 days, and of these 10 (41·7%) were aged <1 year, six (25·0%) were aged 1-4 years, and eight (29·2%) aged 5-17 years. twenty-two (91·7%) of these infection episodes were symptomatic throughout or at some time point during the shedding. the prolonged shedders contributed 647·5 days of rsv shedding which was 29·7% of the cumulative shedding duration for all episodes based on the midpoint estimation. in 14 infected individuals, one or more samples were identified to contain both rsv groups a and b. the timing of these co-detections is shown in figure 4 . in most (12/14) episodes, rsv group a was shed for longer duration relative to group b. the hazard ratios (hrs) for the various factors from univariate cox regression were similar for minimum, midpoint and maximum estimates data (supplementary table s1 ). the midpoint data were taken forward for the multivariate analysis and the final model is reported in table 3 . the results were similar without and with inclusion of the left-censored rsv episodes (table 3 and supplementary table s2 , respectively). the proportionality assumption in the cox regression model was not violated based on the test of the schoenfeld residuals (supplementary table s3 ). the rate of recovery from rsv infection was age-dependent. the adjusted hr (ahr) were 1·98 (95% 1·30-3·02), 1·82 (95% ci 1·16-2·87), 2·10 (95% ci 1·20-3·66) and 1·31 (95% ci 0·36-4·81) in the 1-4, 5-14, 15-39 and 540 years age groups, respectively, relative to infants (<1 year). the rate of recovery was lower by 44% in symptomatic infections relative to asymptomatic infections (ahr 0·56, 95% ci 0·40-0·79). the presence of one or more additional viruses (rhinovirus, coronavirus, adenovirus) was detected in 86 rsv infection episodes. the rate of rsv recovery was lower (i.e. shedding duration increased) by 65% in episodes with co-infection compared to those without (ahr 0·35, 95% ci 0·23-0·51), with a similar result for each virus individually. detection of infection with any one or more of rhinovirus, adenovirus or coronavirus, in the 2 weeks preceding the start of rsv infection, but not during the rsv episode itself, was associated with a 56% increase in the rate of recovery (i.e. reduced shedding duration) from the rsv infection (ahr 1·56, 95% ci 1·02-2·39). in contrast, rsv episodes associated with detection of other viruses in the 2 weeks prior to and also during the rsv infection were associated with a 52% decrease in the rate of recovery relative to those with no other virus prior to and during the rsv episode (ahr 0·48, 95% ci 0·32-0·73). the rate of recovery of rsv episodes associated with spread in the household (outbreaks) was 42% lower than the single household episodes (ahr 0·58, 95% ci 0·43-0·78). a variable denoting the proportion of individuals infected during the household outbreak improved the model fit and was used in the multivariate analyses (likelihood ratio test p = 0·0229). the rate of recovery did not differ significantly by gender and infecting rsv group (ahr 0·97, 95% ci 0·77-1·23 and ahr 1·07, 95% ci 0·83-1·39, respectively). recovery rate was similar in suspected repeat infections compared to the first observed episodes (ahr 0·91, 95% ci 0·53-1·56). we observed 205 infections with rsv during one epidemic with a most realistic estimate of 11·2 (95% ci 10·1-12·3) days of shedding. the most conservative and least conservative estimates were 8·6 days and 14·0 days, respectively. the duration of shedding based on the most realistic estimate decreased with age: 18 days in infants and 9 days in adults (aged 515 years). symptomatic infections on average had longer virus shedding of 13·5 days compared to 7·8 days in asymptomatic episodes. the presented average durations of virus shedding are longer than published estimates of 6·7 days [4] , 3·4-7·4 days [10] , 3·9 days [11] , and 4·5 days [12] which could be attributed to differences in study methods. the present study was informed by critical review of the previous studies and it incorporated frequent sampling regardless of symptoms and screening by highly sensitive pcr methods. the specimen collection procedure was acceptable, recording a good compliance across all ages [7, 13] . the use of the sensitive viral detection method (pcr) results is likely to result in longer estimates of shedding. a community study nested within a birth cohort in coastal kenya targeting symptomatic rsv infections by okiro and colleagues reported a mean duration of shedding of 4·5 days [12] . our corresponding estimate in symptomatic cases was 13·5 days. in a subset of the children whose start of symptoms could be established from the clinic records, the okiro et al. study reported a longer duration of 7·7 days [12] . given that rsv shedding has been reported to start before illness [6] , the actual duration in the symptomatic children would have been an underestimate and our estimate of 13·5 days is likely to be more accurate. a rochester family study, in the usa, with similar design as the present study (collecting samples every 3-4 days regardless of symptoms) reported lower estimates of duration of shedding of 3·4-7·4 days [10] . the okiro et al. and rochester study used the immunofluorescent antibody test (ifat) and culture, respectively, which are less sensitive methods [14] . as a counter argument, it is not known to what degree pcr positivity equates with shedding of viable and infectious virus. thus, while the molecular methods might be more sensitive, the resultant increase in duration of shedding over more traditional methods such as culture (which directly measures viral infectivity) may not necessarily translate to increased period of infectivity. further work relating virus infectiousness and detectability is warranted. in the okiro et al. study, sampling started when participants were symptomatic and stopped at the first negative follow-up sample. the present study revealed instances where negative samples arose within rsv infection episodes. even though this observation raises, again, questions on the relationship between infectivity and shedding duration, accounting for periods of rsv-negative samples would still result to longer shedding duration compared to previous estimates. alternative estimation of the shedding patterns by calculating the area under the c t (viral load) curve would have some additional advantages and will be explored in future. prolonged shedders of > 3 weeks' duration have been reported [6, 10] . in the present study, 24 (13·4%) episodes in the 179 infected individuals involved shedding rsv for >3 weeks. most (22, 91·7%) of these episodes were symptomatic, and occurred in young children (median age 14·7 months). individuals with compromised immunity have been known to shed rsv for longer [15] but participants in the current study were not tested for hiv. the hiv prevalence in women and men aged 15-49 years in coastal kenya was 4·2% according to the kenyan demographic and health survey of 2008/2009 [16] . in settings where hiv prevalence is high, the effect of the poor viral clearance might influence the temporal epidemiology as was observed in south africa [15] . there was no obvious malnourished participant in the study cohort based on midupper arm circumference measurements. more than two rsv episodes were observed in 24 (13·4%) individuals. on average, the episodes were 4 weeks apart. most (70·8%) of the suspected repeat infections were with homologous rsv group. sequencing of the most variable region of the rsv genome, the ectodomain of the g gene, did not greatly assist in resolving the infection episodes as most (9/10) had identical sequences. it is, thus, not clear whether the two phases of rsv shedding were repeat infections with the same variant or were persistent infection with periods of low viral load that was undetectable by the methods used. using post-mortem lung tissue from infants, rsv rna has been detected even in children dying during inter-epidemic periods suggesting the persistence of rsv in the lungs of these infants [17] . similar observations have been made in experimental infection with rsv [18] and measles viruses [19] . the lack of variability in the virus identified in the two phases of infections suggests virus mutation might not be the primary mechanism for virus persistence or re-infection. regardless of whether it is re-infection within a short period or persistence, the observation represents an interesting phenomenon of rsv which has potential importance on our existing view of acute rsv infection, the development of immunity and effects on viral transmission. the age of the individual, infection severity, detection of other viruses before and during the rsv infection, and presence of concurrent rsv infections in the same household were all associated with virus shedding. the rate of recovery increased with increasing age, with individuals in 1-4 years, 5-15 years and 515 years age groups recovering 1·98, 1·82 and 1·97 times faster than the infants, respectively. the rochester family study reported similar findings where longer shedding was observed for children aged <2 years compared to those aged 2-16 years 14·8 -ci, confidence interval; rsv, respiratory syncytial virus. a all rsv infection episodes; b fully observed episodes; c left-censored episodes only; d right-censored episodes only; e episodes with both left and right censoring; f intervals between nasopharyngeal swab collection before, during, and after the rsv infection episode in days; g age (in years) at the start of the episode; h shedding duration (in days) based on the midpoint estimation. (9 vs. 4 days). the okiro et al. study did not find any association with age but found that children with previous rsv infection (using the assumption that those aged >3 years were by default experiencing a repeat infection) had 1·37 times faster rate of recovery compared to those without a history of infection [12] . in the present study, a subsequent rsv infection during the same rsv season was not significantly associated with reduced shedding duration, but such infections were few (n = 24). the current study is for one epidemic only, so age must act as a proxy of exposure to rsv in earlier epidemics. prolonged shedding enhances the possibility of personto-person transmission and makes young children a potential source of community spread of infection, both of which have important implications in the control and prevention of rsv infection. a study involving 23 hospitalized children (aged <2 years) with sampling extended beyond discharge reported an association of duration of shedding and symptom severity [4] . children with lower respiratory tract infection shed for longer than those with upper respiratory tract infection (8·4 vs. 1·4 days). duration of shedding may be related to severity of disease but evidence is controversial on the link between disease severity and viral load [5, [20] [21] [22] . an interaction between detection of other viruses (i.e. coronavirus, rhinovirus, adenovirus) in the nasopharynx and rate of recovery from rsv infection was observed. recovery from a viral infection just prior to rsv might have led to up-regulation of innate viral immunity or non-specific cross-reactivity that reduced subsequent rsv shedding. presence of co-infections might be a marker of low immunity associated with poor viral clearance. rsv episodes with concurrent spread in the household were associated with increased recovery rate. however, any conclusions other than association cannot be made since the extended duration of shedding increases the risk of both co-infection with other viruses and multiple rsv infections in the household. a different estimation framework is required to untangle these results. the problems of ascertainment and analysis (i.e. censoring and test sensitivity) are not completely eliminated by the careful study design, but did not seem to affect the association of the examined factors with virus shedding (supplementary table s1 ). short rsv infections occurring between specimen collections particularly in older individuals might have been missed but this is likely to be at random and bias hazard ratios towards null. in conclusion, this study defines rsv shedding patterns in the natural setting with significant potential for improved understanding of the spread of this important virus and with relevance the design of control programmes. for supplementary material accompanying this paper visit http://dx.doi.org/10.1017/s0950268814001393. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis risk of primary infection and reinfection with respiratory syncytial virus shedding of infectious virus and virus antigen during acute infection with respiratory syncytial virus respiratory syncytial virus infections in infants: quantitation and duration of shedding quantitative shedding patterns of respiratory syncytial virus in infants patterns of shedding of myxoviruses and paramyxoviruses in children the source of respiratory syncytial virus infection in infants: a household cohort study in rural kenya profile: the kilifi health and demographic surveillance system (khdss) genetic relatedness of infecting and reinfecting respiratory syncytial virus strains identified in a birth cohort from rural kenya respiratory syncytial virus infections within families respiratory syncytial virus infections in previously healthy working adults duration of shedding of respiratory syncytial virus in a community study of kenyan children improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real-time pcr using nasopharyngeal flocked swabs comparison of direct immunofluorescence, conventional cell culture and polymerase chain reaction techniques for detecting respiratory syncytial virus in nasopharyngeal aspirates from infants increased burden of respiratory viral associated severe lower respiratory tract infections in children infected with human immunodeficiency virus type-1 kenya demograpghic and health survey 2008-09 detection of respiratory syncytial virus nucleic acid in archival postmortem tissue from infants latency and persistence of respiratory syncytial virus despite t cell immunity prolonged persistence of measles virus rna is characteristic of primary infection dynamics respiratory syncytial virus load predicts disease severity in previously healthy infants evaluation of quantitative and type-specific real-time rt-pcr assays for detection of respiratory syncytial virus in respiratory specimens from children natural infection of infants with respiratory syncytial virus subgroups a and b: a study of frequency, disease severity, and viral load we thank the field and laboratory teams for their dedication in sample collection and testing, respectively. the article is published with the permission of the director of the kenya medical research institute. this work was supported by the wellcome trust (090853 and 084633). none. key: cord-324950-ux7shvji authors: saade, georges; deblanc, céline; bougon, juliette; marois-créhan, corinne; fablet, christelle; auray, gaël; belloc, catherine; leblanc-maridor, mily; gagnon, carl a.; zhu, jianzhong; gottschalk, marcelo; summerfield, artur; simon, gaëlle; bertho, nicolas; meurens, françois title: coinfections and their molecular consequences in the porcine respiratory tract date: 2020-06-16 journal: vet res doi: 10.1186/s13567-020-00807-8 sha: doc_id: 324950 cord_uid: ux7shvji understudied, coinfections are more frequent in pig farms than single infections. in pigs, the term “porcine respiratory disease complex” (prdc) is often used to describe coinfections involving viruses such as swine influenza a virus (swiav), porcine reproductive and respiratory syndrome virus (prrsv), and porcine circovirus type 2 (pcv2) as well as bacteria like actinobacillus pleuropneumoniae, mycoplasma hyopneumoniae and bordetella bronchiseptica. the clinical outcome of the various coinfection or superinfection situations is usually assessed in the studies while in most of cases there is no clear elucidation of the fine mechanisms shaping the complex interactions occurring between microorganisms. in this comprehensive review, we aimed at identifying the studies dealing with coinfections or superinfections in the pig respiratory tract and at presenting the interactions between pathogens and, when possible, the mechanisms controlling them. coinfections and superinfections involving viruses and bacteria were considered while research articles including protozoan and fungi were excluded. we discuss the main limitations complicating the interpretation of coinfection/superinfection studies, and the high potential perspectives in this fascinating research field, which is expecting to gain more and more interest in the next years for the obvious benefit of animal health. bacterial and viral respiratory diseases are a major health issue in species reared under confined conditions in large groups. most often multiple infectious agents are involved in the development of these clinical conditions making unsuited the common reductionist approach of host-pathogen interactions by the study of single infection [1] . infection by more than one type of pathogen (viruses, bacteria and parasites amongst others) is described as a mixed infection. however, the term coinfection is frequently used to describe concomitant infection of a cell or a host by separate pathogens [2] . since in the literature the definitions of coinfection and mixed infection have been both used to describe the same events, we will use the term "coinfection" in the current review. additionally, in virology, the term superinfection is used if one virus infects the cell or the host before infection by the second superinfecting virus. we will also use the term "superinfection" in the review. finally, an opportunistic pathogen is usually considered as a pathogen that would not have infected animals in absence of the primary infection, or alternatively, "pathogen" that would have been asymptomatic in the absence of the primary infection. in some studies, however, the use of the terms "coinfection" is not suitable and "superinfection" should be used instead, as we will see later. this semantic point is responsible for a lot of confusion and makes comparisons between studies sometimes tricky. the outcome of any coinfection or superinfection can be affected by the interactions taking place between the infectious agents, the nature of the cell/host, adverse environmental and management conditions, intestinal and respiratory microbiomes, and the triggered immune response-innate and adaptive-developed afterwards [2, 3] . when occurring at the same time or with a delay, infections can impact the virulence of causative pathogens with subsequent consequences on the host immune response and its ability to clear the infections [2] . the first contact with a pathogen can change the cell/host response against any other second pathogen, possibly causing a more virulent infection, reducing its severity or suppressing it completely [4] . thus, different scenarios concerning the pathogen interactions can be observed, the first infectious agent can promote the second one, attenuate its effects or simply prevent its establishment. conversely, the second pathogen may also influence the first one directly or indirectly. coinfections have been described in both humans and animals [1, 2] . moreover, bacterial and viral infections might be followed by secondary bacterial or viral infections, which in some cases are responsible for the pathology development and the observed clinical signs. in this review, the current knowledge regarding frequent coinfections that occur in the porcine respiratory tract and particularly in the lungs are reviewed. when possible, we focused on the interactions between the mentioned pathogens and the various mechanisms justifying these interactions and their consequences on the host's response. we especially discussed coinfections involving main bacteria and viruses associated with the so-called porcine respiratory diseases, excluding coinfections involving parasites and fungi (including their metabolites, such as mycotoxins). moreover, we do not discuss the impact of adverse environmental and management conditions which have been shown to be of major importance in the modulation of respiratory infections' severity [3]. respiratory diseases have been formally described in pigs as early as the 1960′s [5] and several studies have been carried out to identify associated agents. the role of the infectious pathogens has been assessed by using two main approaches: direct research of the pathogens (by culture or polymerase chain reaction-pcr for instance) from tissue samples of diseased (acute or chronic stage) and non-diseased pigs or indirect detection by serological tests to look for antibodies produced after exposure to specific pathogens. these studies indicated that frequently under field conditions, several infectious pathogens are simultaneously detected from lung lesions (see [6] [7] [8] causative respiratory infectious agents can be divided into primary and secondary or opportunistic pathogens. primary pathogen being defined here as pathogen that can infect the animal as first unique pathogen and then facilitate secondary or opportunistic coinfection. these primary pathogens include common bacteria such as highly virulent actinobacillus (a.) pleuropneumoniae, m. hyopneumoniae, bordetella (b.) bronchiseptica in young piglets and common viruses such as swiav [1] . prrsv and pcv2 are not strictly respiratory pathogens as swiav, however, since they also frequently affect the respiratory system and since they can act as facilitators of secondary respiratory infections, they must be considered too. other primary pathogens such as aujeszky's disease virus (adv) and prcov are reported but they are far less frequently encountered today or they have less impact on porcine health [1]. then, some viruses like the porcine cytomegalovirus can also inhibit host immune functions-particularly the action of t lymphocytes-and promote respiratory diseases such as the porcine reproductive and respiratory syndrome [9] . among the secondary pathogens common bacteria such as lower virulence strains of a. pleuropneumoniae, a. suis, glaesserella parasuis, pasteurella multocida, and streptococcus (s.) suis are reported. together primary and secondary pathogens are involved in the "porcine respiratory disease complex" (prdc) [10] . several studies have assessed the nature of the infectious agents directly or indirectly associated with respiratory diseases in pigs [7, 8, 11, 12] . in one of these studies involving breeding sows in five french farrow-to-finish herds [12] , results indicated that s. suis, a secondary pathogen, was quite widespread among sows-67.1% of the animals being positive using a pcr assay-and pcv2 and swiav infections were highly prevalent (75% of the sows with antibodies against pcv2 and between 91.7% and 100% of the sows with antibodies against swiav). other infectious agents such as a. pleuropneumoniae, g. parasuis and p. multocida were detected in 31%, 25%, and 23% of the sows, respectively [12] . in another study evaluating infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds in france, it has been shown that m. hyopneumoniae, prrsv, and swiav subtype h1n1 were the major pathogens involved in pneumonia-like gross lesions [8] . for extensive pleuritis, prrsv was frequently associated with a. pleuropneumoniae [8, 12] . regarding bacteria associated with lung lesions in 3731 french slaughter pigs [8] , a report mentioned lesions of pneumonia and pleuritis as the most frequent lesions. in these lesions, bacteria such as m. hyopneumoniae, p. multocida, a. pleuropneumoniae, s. suis, and g. parasuis were detected in 69.3%, 36.9%, 20.7%, 6.4%, and 0.99% of the lungs, respectively [13] . in a retrospective analysis of the etiologic agents associated with respiratory diseases in pigs in usa, two or more infectious agents were identified in 88.2% of the analyzed cases [7] . prrsv (35.4% of the samples), p. multocida (31.6%), m. hyopneumoniae (27%), swiav (22.2%), g. parasuis (22.0%) and pcv2 (18.6%) were the infectious agents most frequently encountered [7] . in korean pigs, prrsv and pcv2 were frequently identified associated or not to various bacteria such as s. suis (25.2%), m. hyopneumoniae (20.1%), p. multocida (12.9%), and a. pleuropneumoniae (5%) [11] . below we review the main primary pathogens as defined above, common viruses such as prrsv, pcv2, swiav, prcov and adv as well as bacteria like a. pleuropneumoniae, m. hyopneumoniae and b. bronchiseptica. conversely, other pathogens involved in the prdc are not presented in the following sections while considered in additional file 1 presenting the different coinfections' situations. prrsv is an enveloped single stranded positive rna virus belonging to the arteriviridae family. two different species, prrsv-1 (also known as betaarterivirus suid 1), from european origin, and prrsv-2 from american origin, are now distinguished [14] . this enveloped virus replicates mainly or exclusively in macrophages such as alveolar macrophages (ams), but also macrophages from the nasal mucosa and pulmonary intravascular macrophages (pims) [15, 16] . in vitro, prrsv can also replicate in cultured monocytes and monocyte-derived cells including macrophages [17] and in vitro-derived dendritic cells (dcs) generated either from bone marrow hematopoietic cells (bmdcs) or blood monocytes (modcs), depending on the in vitro culture conditions [18, 19] . however, such in vitro generated dcs are not representative of in vivo primary dcs which do not seem to be permissive to viral replication [20] . in fact, modc and bmdc (at least when generated using granulocyte macrophage colony-stimulating factor, gm-csf) although possessing functional overlaps with the dc family, do not represent bona fide dcs, which represent an own lineage of hematopoietic cells distinct from the monocytic lineage [21] . different cell surface molecules are involved in prrsv entry and infection of cells: heparan sulfate, porcine sialoadhesin-also known as sialic acid-binding immunoglobulin-type lectin 1 (siglec-1), siglec-10, cd151 and cd163 [22, 23] . heparan sulfate is a glycosaminoglycan (gag) that seems to play a modest or secondary role in prrsv infection since the blocking of this receptor on ams induced only a mild decrease in prrsv infectivity. moreover, this effect was not observed with all the prrsv isolates tested, suggesting that the involvement of heparan sulfate depends on the antigenic diversity of prrsv [22] . siglec-1/cd169 is a member of the sialic acid-binding lectins (siglecs) family and is expressed on macrophages [22] and siglec-10 has been identified as an alternative receptor to siglec-1 [23] . binding of prrsv to siglecs induces its internalisation by clathrin-mediated endocytosis. expression of recombinant porcine sialoadhesin is sufficient to induce the internalisation of prrsv by non-permissive cells, but not replication [24] . cd163 is a scavenger receptor involved in prrsv infection [22] . its expression on nonpermissive cells makes them susceptible to infection with prrsv and allows productive replication of the virus [22] . moreover, cd169-ko animals are still susceptible to prrsv-2 infection [22] , whereas cd163-ko animals are resistant to prrsv-1 and prrsv-2 [25, 26] . finally, myh9 has been recently identified as an indispensable partner of cd163 for prrsv cell entry for both prrsv-1 and prrsv-2 [27] . prrs clinical signs can be nearly absent to severe depending on the considered prrsv species and strains. when observed, there are, amongst the most frequent, lethargy, dyspnea, tachypnea, as well as a reproductive disease [16] . prrsv can persist in infected pigs for several months after the initial infection particularly in lymphoid tissues and has the ability to alter the host's immune system to escape it (for review see [16] ). prrsv interferes with the porcine innate immune response through downregulation of type i interferons (ifns-ifnα and ifnβ mostly), which are cytokines known for their antiviral properties [28] . prrsv-infected macrophages also had a reduced capacity to produce the pro-inflammatory cytokines tnfα and il1β [28] and the production of the anti-inflammatory cytokine il10 was found enhanced during infection [29] . nevertheless, the role of cytokine modulation during prrs is unclear considering that the effects appeared to depend on the prrsv species, as well as on the prrsv isolates, since opposite results can be found with different prrsv strains [30, 31] . in fact, some prrsv-2 isolates were shown to enhance ifnα production while other prrsv-1 isolates suppressed it. results seemed also very variable for the immunoregulatory il10 along different isolates of prrsv-1 [30, 31] , making general conclusions about how prrsv alters innate immune responses difficult. prrsv impact on adaptive cellular immunity seems also to be highly variable according to the species and the strain [20] . conversely, whereas non-protective antibody response against the viral nucleocapsid is found within a week post-infection, neutralizing antibodies appearance is highly delayed for all prrsv species and strains, appearing only after 3 or 4 weeks of infection and peaking even later [16] . pcv2 is a naked circular single stranded dna virus belonging to the circoviridae family and responsible for porcine circovirus disease (pcvd). the attachment of pcv2 to target cells occurs through chondroitin sulfate b and probably other receptors [32] . internalisation is not fully known but it does not seem to involve a specific receptor and the gags could mediate internalisation and binding to the target cells [33] . most of the time the infection is subclinical but in some circumstances such as coinfections with other respiratory pathogens it can cause the post-weaning multisystemic wasting syndrome (pmws), clinically characterized by wasting respiratory disease, and enteritis [34] . infection with pcv2 can occur in utero, resulting in stillborn piglets and mummified fetuses, or death at different ages after birth [34] . in young and older animals, pcv2 was found in cells expressing monocytes (cd14 + ), and t and b cells (cd4 + , cd8 + , igm + ) markers [35] . further results showed that active replication of the virus was supported by t and b cells, with enhanced replication in proliferative cells [36] . in vitro, pcv2 can also infect many other cell types including endothelial cells, gut epithelial cells, fibrocytes, and dcs [37] . in dcs the virus seems to persist and remain infective for a prolonged period without replication indicating that these cells might serve as a vehicle for virus spread in the host [38] . pmws is characterized by the depletion of lymphoid cells affecting t cells, b cells, and nk cells [39] . this lymphopenia was also associated with impaired responses of peripheral blood mononuclear cells (pbmcs) to mitogen stimulation with lower levels of il2, ifnγ, and il4 production compared to pbmcs from non-infected pigs [40] . another feature of pmws is an elevated level of il10 found in lymphoid organs, especially in the t cells rich areas [41] . il10-mediated immunosuppression could play an important role in the pcv2 infection and the development of pmws. pcv2 has also the ability to alter the innate immune response [42] . even though the virus does not productively infect dcs, evidence shows that it can interfere with the normal plasmacytoid dcs (pdcs) response. upon stimulation with cpg-odn, pdcs' ability to produce ifnα and tnfα was impaired in cells previously infected with pcv2 [43] . pcv2 dna isolated from infected cells induced the suppression of pdc ifnα production [43] . influenza a viruses are enveloped single stranded negative rna viruses belonging to the orthomyxoviridae family. these enveloped viruses can infect a broad range of hosts, with pigs being one of their natural hosts (for a review see [44] ). the three main iav subtypes encountered in pigs are h1n1, h1n2, and h3n2 [44] , but many genetic lineages and antigenic variants within these subtypes are co-circulating in the pig population worldwide. subclinical infections with swiavs are common in pigs, but they can also induce a disease similar to what is observed in humans, with upper respiratory tract distress associated with fever, cough, rhinitis, high morbidity, and low mortality [44] . the main targets of swiavs are epithelial cells of the respiratory tract but iavs can also non-productively infect alveolar macrophages [45] . two major glycoproteins are present at the surface of the virus: hemagglutinin (ha) and neuraminidase (na). binding of ha with the sialic acid molecules at the surface of the host cells will induce the endocytosis of the viral particle [44] . the na molecule plays the main role in the budding of the virus by removing the sialic acid, allowing the release of neoformed virus particles from the infected cell [44] . the innate response against the virus includes production of high levels of pro-inflammatory cytokines such as ifnα, tnfα, and il6. dcs, in particular pdcs play an important role in this response [46] . an important observation was that the production of these cytokines correlated to the viral loads and the severity of the disease. infection with swiav induces cellular and humoral specific immune responses in pigs recovering from the disease and the serum igg and the mucosal iga can protect the animal from re-infection [44] . ns1 and pa-x are the main viral proteins that alter the innate immune response, mainly by blocking the type i ifn response [47] as well as the nlrp3 inflammasome activation [48] in infected-epithelial cells and alveolar macrophages. finally, the main mechanisms through which the swiav escapes the adaptive host immune system are the antigenic drift and the antigenic shift concerning mainly ha and na which are also the two major antigenic proteins expressed on the surface of the virus and against which the neutralizing humoral response is directed [44] . prcov is an enveloped single stranded positive rna virus belonging to the coronaviridae family. in pigs, four alphacoronavirus, one betacoronavirus and one deltacoronavirus have been described [49, 50] . thus, most of the porcine coronaviruses are from the genus alphacoronavirus. the only respiratory porcine coronavirus, prcov, is a variant of transmissible gastroenteritis virus (tgev) where a large 5′ region deletion (nucleotides 621-681) in the spike gene of the virus altered the tropism and the virulence. even if pigs have been shown to be susceptible to the first sars-cov (serological evidence and isolation of the virus in a pig farm in the xiqing county of tianjin, china) [51] they have not been successfully experimentally infected, at this stage, by sars-cov-2 [52] . prcov uses aminopeptidase-n (cd13) domain iv to enter cells [53] and replicates to high titers in the lungs (1 × 10 7 -10 8 tissue culture infectious dose 50-tcid 50 ) specifically in type 1 and 2 pneumocytes. moreover, it can infect epithelial cells of the nares, trachea, bronchi, bronchioles, alveoli, and, occasionally, alveolar macrophages [49] . infections with the prcov are usually subclinical, but there is variation between strains and some can induce a more severe disease. prcov can infect pigs of all ages by direct contact transmission or aerosol [49] . the clinical signs are associated to the respiratory system and are mild to severebronchointerstitial pneumonia-depending the strain and the context (environmental and management factors as well as the presence of other pathogens). suid herpesvirus 1, usually known as prv or adv is the responsible agent of aujeszky's disease in pigs. it is a double stranded enveloped dna virus from the herpesviridae family and alphaherpesvirinae subfamily targeting respiratory and/or genital mucosae for its replication [54] . adv has a very broad host range varying from domestic animals like pigs, cattle, goats, sheep, cats and dogs to wild animals such as ferrets, foxes, hares, raccoons, and wild deer, and where it induces different diseases [54] . infected animals usually show fever, sneezing, coughing and vomiting accompanied occasionally with typical nervous manifestations like convulsions, aggressiveness and lack of coordination. mortality rate can reach 100% in suckling piglets while in mature pigs the infection is inapparent or mild [54] . adv possesses eleven types of envelope glycoproteins playing major roles in the interaction with host cells and the induction of immune response [54] . viral binding and fusion with the plasma membrane of the target cell-epithelial cells, neurons and alveolar macrophages-are controlled by a cascade of events orchestred by glycoproteins c (gc), gb, gd, gh and gl. the binding process starts with an interaction of gc with heparin sulfate proteoglycans [54, 55] . stabilization of this interaction is then assured by the binding of gd to specific cellular receptors known as herpesvirus entry mediators such as hvea (tnfrsf14), hveb (prr2, nectin 2), hvec (prr1, nectin 1), hved (pvr, cd55), and 3-o-sulfated heparin sulfate [54, 56] . at this stage, tyrosine-based or dileucine-based endocytosis in parallel with clathrin-mediated endocytosis occur by the mediation of gb, gh and gl, leading to the penetration of the capsid and the tegument into the cellular cytoplasm. finally, the interaction of the capsid with dynein leads to the release of viral dna into the cellular nucleus after a transport along microtubules from the periphery to the nuclear pores [55] . porcine humoral immune response is induced by adv and neutralizing antibodies are mainly directed against gc [57] . specific cell mediated immune responses are also triggered and mhc class i restricted, gc-specific, cytotoxic cells are induced. adv also alters the ifn signaling pathway by suppressing stat1 tyrosine phosphorylation leading to an inhibition of ifn-stimulated genes (isgs) expression [54, 57] . adv may be involved in the prdc and can be isolated alone or with other pathogens. accordingly, a study conducted in taiwan reported the association of adv with pcv2 in 10.3% of the evaluated pigs using a multiplex pcr [58] . animals affected with this gram negative bacterium develop a pleuropneumonia characterized by fibrinohemorrhagic necrotizing bronchopneumonia and fibrinous pleuritis which can reach a high mortality rate [59, 60] . although the disease is best known in its acute/peracute forms, subacute and/or chronic presentations with low or no mortality are highly prevalent, especially in the presence of antibiotic treatments. many herds are subclinically infected without previous or present episodes of clinical disease and in the absence of suggestive lesions at the slaughter house. animals are, nevertheless, carriers of the pathogen. this happens in several conventional herds which may be simultaneously infected not only with several low/intermediate virulent strains, but also, in some cases, with strains highly likely to cause disease. in the latter case, outbreaks may suddenly appear in the presence of concomitant diseases or as a consequence of changes in management and/or environment [59, 60] . eighteen serotypes of the bacterium have been described, which can all induce disease, although clear differences in virulence have been described [59, 60] . these bacteria can be found mainly in tonsils of carrier animals; virulent strains have a tropism for the lower respiratory tract where they preferentially bind to ciliated cells of the terminal bronchioli and pneumocytes [59, 60] . different virulence factors expressed by a. pleuropneumoniae are involved in the colonization and the development of the disease. adhesion to cells could be mediated by type iv fimbriae that are expressed upon contact with respiratory epithelial cells in vitro and during lung infection [59, 60] . adhesion of a. pleuropneumoniae to respiratory epithelial cells also involves the binding of bacterial lipopolysaccharides to glycosphingolipids on the surface of the cells [59, 60] . the formation of biofilm by the bacteria is likely to play an important role in the colonization of the host [61] . after attachment to the target cells, the bacteria can produce four different pore-forming exotoxins (apx i, ii, iii and iv) inducing the lysis of alveolar epithelial cells, thus allowing the acquisition of nutrients by the bacteria, but also participating in the development of the lesions [60, 62] . some of the virulence factors expressed by a. pleuropneumoniae interfere with the host's immune response. the toxins apx i, ii and iii induce the lysis of not only respiratory epithelial cells, but also of cells involved in the innate immune response such as macrophages and neutrophils [60, 63] . at lower concentrations, these toxins lose their lytic properties but can still impair macrophages chemotactic activity and their phagocytic abilities [64] . the capsular polysaccharides of a. pleuropneumoniae interfere with macrophage phagocytosis and enable resistance to complement-mediated killing [60] . a. pleuropneumoniae may also interfere with the antibody response by producing proteases that can degrade porcine iga and igg [59, 60] . this cell wall-free bacterium is considered to play a primary role in prdc and is the causative agent of porcine enzootic pneumonia (ep), a disease with high morbidity but low mortality rates [65] . the main pathological mechanisms involved in m. hyopneumoniae infections are: (i) adhesion to the ciliated cells of the tracheal epithelium inducing ciliostasis, loss of cilia and exfoliation, dysregulation of cellular homeostasis (with increased intracellular calcium concentration) and secretion of cytotoxic factors, (ii) alteration of the mucociliary tract, (iii) inflammatory reactions sometimes exacerbated and prolonged, and (iv) manipulation of the innate and adaptive immune responses [65, 66] . among the adhesins described in m. hyopneumoniae, p97 is reported to be a major determinant of cell adhesion [65] [66] [67] . several other adhesins were reported: p102 linked to p97, lpps, lppt, mgpa, p65, p76, p110, p146, p159, and p216 [65, 66] . most adhesins are transcribed and translated during m. hyopneumoniae infection and then undergo posttranslational cleavage to result in diverse products on the membrane surface [65, 67, 68] . the diversity of surface proteins can also derive from the variation in the number of repeats in genes encoding adhesins [69] . these mechanisms of antigenic variation enable the bacterium to escape from immune system recognition and to invade the host [66] . adhesins can also recruit extracellular matrix components (plasminogen, fibronectin and actin amongst others), and therefore can promote invasion and inflammatory response [65, 70] . the immune response induced against m. hyopneumoniae, may have a double action: over-activation of the local immune response resulting in a pathologic inflammatory reaction or local immunosuppression explaining the chronic nature of the associated pathologies [65, 66] . acute m. hyopneumoniae infection leads to the recruitment and activation of various innate immune cells, essentially through the involvement of a large range of cytokines: il1, il6 and tnfα in lungs; cxcl8, il1, il2, il4, il6, tnfα and il10 in bronchus-associated lymphoid tissue (balt) or tracheobronchial lavage fluid (tblf) [65, 71, 72] . some of these inflammatory cytokines (tnfα, cxcl8, il1β, il6) are produced chronically in the lungs and play powerful roles in apoptosis (tnfα), differentiation and chemotaxis of neutrophils (respectively, il6 and il8), and macrophage activation (tnfα, il1β). chronic infections are typically associated with intense lymphoid hyperplasia [71] and are characterized by an accumulation of igg-and iga-expressing plasma cells, cd4 + t cells, macrophages and dcs in the balt of inflamed lung tissue [73] . involvement of t cell activation in chronic inflammation is also supported by the presence of t-cell cytokines such as il-2 and il-4 in bronchoalveolar exudates [72] . in vitro studies conducted with macrophages cocultured with m. hyopneumoniae highlighted a strong activation of inflammatory pathways inducing the production of cytokines and chemokines, and expression of receptors or pathways inducing cell apoptosis [65, 66, 74, 75] . moreover, m. hyopneumoniae is described as an inhibitor of macrophages phagocytic activity, which may explain the chronicity of m. hyopneumoniae infections and the greater host susceptibility to other pathogens [65, 66, 74] . mycoplasma hyopneumoniae was found to activate costimulatory molecule expression on bona fide dcs with poor tnfα production, contrasting with monocytes. interestingly, a strong mitogenic activity for b cells was observed [76] . altogether, these data indicate that m. hyopneumoniae is well sensed by the innate immune system, but the presence of immune evasion mechanisms targeting antigen presenting cells remains a possibility that needs further investigations. antibody responses after infection develop slowly and do not appear to correlate with protection [65, 66] . the literature on m. hyopneumoniae infections coupled with information from mouse models indicates that adaptive immune responses represent a fragile balance between pathogenic and protective th-cell responses, probably belonging to the th1 or th17 types [65, 66] . this aerobic gram-negative bacterium can be found in the respiratory tract of several animal species and it presents a worldwide distribution in the porcine rearing [77] . b. bronchiseptica has a strong tropism for ciliated cells from the respiratory tissue and is mostly detected in the apical portion of the ciliated cells of turbinates, trachea and lungs [77, 78] . it can also be found in the cytoplasm of neutrophils and macrophages and rarely in the alveolar lumen associated with small tufts of cilia [77, 78] . hence, infected pigs show cilia loss in the bronchial and bronchiolar epithelium associated with multifocal erosion, fibrosis, and hyperplasia. neutrophil infiltrates are noted in the peri-conchal meatus and the submucosa of the bronchioles and alveoli, while lymphocyte and plasma cell infiltrations occur at the level of the lamina propria [77, 78] . cell adhesion of b. bronchiseptica is a multifactorial process involving two main virulence factors; filamentous hemagglutinin (fha) and pertactin (prn) [77, 79] . the expression of both adhesins is controlled by the bordetella virulence genes (bvg)as signal transduction system. fha is an adhesin with several binding domains including a carbohydrate-recognition domain responsible of the adhesion to macrophages and ciliated epithelial cells, a heparin-binding domain that mediates the binding to sulfated polysaccharides, and an arg-gly-asp domain (rgd) regulating the intercellular adhesion molecule 1 (icam1) by epithelial cells after interaction with the nf-κb signalling pathways [79] . this rgd domain is also present in the structure of prn and contributes to the binding process [77, 79] . on the other hand, non-opsonic adhesion mechanisms play a role in binding to the host cells such as carbohydrate-specific mechanisms and those involving sialic acid-containing compounds [77] . virulence of the bacteria depends on the strains; therefore, clinical signs can be different going from sneezing and transient nasal discharge for moderate and non-toxic strains to bronchopneumonia and atrophy of the nasal turbinate bones for virulent strains, especially if they are associated to other bacteria such as p. multocida [77] . thus, b. bronchiseptica is usually described as primary lung pathogen in young pigs where it causes necrohemorrhagic bronchopneumonia whereas in older pigs this bacterium is mostly known as an opportunistic pathogen contributing to the prdc [77] . the immune response against b. bronchiseptica is mainly triggered by the different toxins expressed such as adenylate cyclase, tracheal cytotoxin and dermonecrotic toxin (dnt). in the following section and additional file 1, we have used the published studies evaluating multiple infections including viral-viral, bacterial-viral, viral-bacterial and bacterial-bacterial respiratory coinfections and superinfections in swine. both in vivo and in vitro studies comparing single to multiple infections were included. studies evaluating vaccinations and the development of diagnostic techniques such as elisa or qpcr were excluded as well as trials testing antiviral or antibacterial molecules when there was no clear comparison between single and multiple infections. an attempt to present a synthetic view of coinfections is depicted in the heat maps (see figures 1, 2, 3) . however, we recommend readers to refer to additional file 1 for each coinfection couple to get a more detailed view. in these heat maps, we were interested in the effect of the first pathogen on the multiplication of the second one (named "assessed pathogen" in the figures) and on the host immune response and/or clinical signs. these effects were evaluated and a grade from − 5 to + 5 was given to every pathogen depending on the intensity of its impact on the multiplication of the second agent and on the immune response or on the clinical signs. negative grades were given to pathogens decreasing the multiplication of other pathogens, while positive grades were given to those inducing an increase. similarly, negative grades were attributed to pathogens with a tendency to decrease clinical signs or immune response related to the other pathogen. positive grades were given in case of an increase. the sum of the grades was calculated if the same pathogen combination was evaluated in several studies except in the case of discordant results. this grading was represented in the following heat maps and the number of the identified studies for the same pathogen combination is shown on the maps. in the heat maps, other pathogens, that are less associated to prdc such as g. parasuis, m. hyorhinis, m. floculare, p. multocida, and s. suis or even not considered as respiratory pathogens like staphylococcus aureus, classical swine fever virus, hepatitis e virus, porcine rubulavirus, ppv, and torque teno sus virus 1 have also been included. indeed, these pathogens can also impact the outcome of respiratory infections and deserve, at least, to be mentioned. viral/viral respiratory coinfections have always had an important role in the porcine respiratory disease complex [1] . several studies assessed the presence of two or more viral pathogens in pigs showing respiratory clinical signs in farms located in endemic regions [7, 8, 13] . the main viruses contributing to the porcine respiratory disease are swiav, prrsv, pcv2, and to a lower extent the prcov and the adv. due to their fast-spreading and their economic consequences, some viruses were more studied than others in the last 20 years, especially pcv2, prrsv, and swiav as shown in additional file 1a and b. we will thus put more emphasis on these three viruses as causes of primary infections. many in vivo studies were carried out to assess the severity of the clinical signs and the development of the microscopic/macroscopic lesions. this approach enabled a comparison between coinfection/superinfection and single-infection conditions. then, viral interference was progressively more frequently measured as a way to better understand the consequences of coinfections. in the last decades, the strong development of molecular biology and various tools enabled the evaluation of the immune response developed following polyviral infections. in additional file 1, the selected studies that were carried out on viral coinfections are presented from the oldest in vivo experiments to the latest in vitro and ex vivo experiments (additional file 1a and b). this data synthesis highlights the major impact of prrsv primary infection, which can both increase the titre of the following virus (pcv2, hepatitis e virus-hev) in vitro [80] and in vivo [81] [82] [83] (figure 1a ), but can also worsen the clinical score associated to the disease ( figure 3a ). interestingly, even when the prrsv does not increase the viral production of the other virus, as observed in coinfections involving swiav [84] or prcov [85] (figure 1a) , it can also worsen the associated clinical signs. swiav and pcv2 as primary infectious agents have been less studied. however, it can be observed that swiav can interfere with other virus productions (prrsv and prcov) [85, 86] whereas pcv2 has some detrimental impact on the clinical outcome of secondary viral infections (prrsv, swiav, and ppv) [87] [88] [89] (see additional file 1 and figure 1a) . then, regarding the inflammation induced in coinfection conditions, various outcomes were observed depending which viruses were considered (figure 2a) . many in vitro and in vivo experiments, with different bacterium-virus and virus-bacterium combinations, have been performed to identify the underlying mechanisms of the prdc (see figures 1b, c, 2b , c, and 3b). the main studies are presented in additional file 1c and d. bacterium-viral coinfections can also involve various primary respiratory pathogens. among them, the most frequently studied bacterium is m. hyopneumoniae, a pathogen that induces a chronic respiratory disease and can influence the outcome of a subsequent viral infection. however, mycoplasma infection needs to be already well established in the respiratory tract at the time of the viral infection to potentiate it. indeed, m. hyopneumoniae inoculated to pigs simultaneously or shortly before the virus did not strongly impact the severity of the viral infections (pcv2, swiav, prrsv) [90] [91] [92] , while its impact was clearly evidenced when inoculated 3 weeks before viral infections [93] . it is well-known that viral infections can induce an ideal environment for a bacterial superinfection through different mechanisms such as the destruction of the epithelial barrier, the over-expression of the receptors involved in the bacterial adhesion to the cells, and the alteration of the host immune response [1, 2, 94, 95] . the swiav infection has been shown, for instance, as a way to facilitate the colonization of epithelial cells by s. suis, but only for the serotypes containing sialic-acid in their capsule [96] . the swiav infection induces a loss of ciliated cells leading to the impairment of the mucociliary clearance function, but induces also the presence of the viral ha on the surface of infected cells that interacts with the sialic acid of the bacterial capsule, leading to increased adherence of s. suis [96, 97] . although these swiav effects on s. suis have been clearly shown in vitro, no clear in vivo impact of swiav infection on s. suis pulmonary load has been described [98] . it was clearly shown that the presence of both pathogens significantly induces more inflammation than single infections [98, 99] . overall, studies carried out in pigs showed that a bacterium-virus or a virus-bacterium coinfection frequently induces an aggravation of pulmonary lesions ( figure 3b ) and a higher inflammation ( figure 2b ) and immune response, with increased production of pro-inflammatory cytokines. in many bacterium-virus and virus-bacterium associations, this worsened outcome seems to be the result of additive effects from both pathogens rather than a real synergy [100, 101] . however, a potentiation of the viral infection by bacteria can also be observed in other cases, such as in the m. hyopneumoniae-prrsv coinfection [102] . in that case, higher amounts of prrsv genomes were detected in lymphoid tissue and blood [102] and a slower viral clearance was observed [75] (figure 1b) , suggesting that the recruitment of immune cells in the lung parenchyma upon established m. hyopneumoniae infection may provide a steady supply of susceptible cells for prrsv [1] . then, in porcine ams and in the "african green monkey" (originally described as porcine origin) st-jude porcine lung (sjpl) cell line, prrsv infection has been shown to be blocked by a pre-infection with a. pleuropneupmoniae, this antiviral activity being due to the a. pleuropneumoniae metabolites [103] ( figure 1b) . given the fact that in vivo studies involving prrsv and a. pleuropneumoniae did not always investigate the impact of an a. pleuropneumoniae pre-infection on the subsequent prrsv infection [104, 105] , as done in experiments performed in vitro [103] , it cannot be easily concluded if this interference would be observed in the target species. however, in vivo, prrsv-a. pleuropneumoniae interactions were reported as absent or mild [104, 105] (figure 3b ). in virus-bacterium coinfections, the dogma usually encountered is that viruses play an immunomodulatory role, which favors bacterial superinfections. nevertheless, a pre-disposing effect is also described for m. hyopneumoniae, which promotes viral but also bacterial superinfections [65] (figure 1d ). few studies of experimental coinfections or superinfections with m. hyopneumoniae and/or other bacteria involved in prdc were performed compared to coinfections involving viruses. these studies are reported in additional file 1e. overall, these coinfections or superinfections induce more clinical signs and lung lesions and poorer technical performances when compared to single infections with the same infectious pathogens ( figure 3c ). the bacterialbacterial coinfections are also responsible for immune response alterations (for reviews see [106, 107] ). for example, macrophages from pigs infected by m. hyopneumoniae decrease their phagocytosis capacity against a. pleuropneumoniae [60, 65] . m. hyorhinis and m. flocculare, two mycoplasmas commonly co-isolated with m. hyopneumoniae in gross pneumonia-like lesions, may also impact the immune response by inducing the cytotoxicity of immune cells and/or the secretion of cytokines affecting its outcome [108] . co-stimulation of porcine bmdcs with m. hyopneumoniae and m. hyorhinis induces a strong il12 production. in this last in vitro model, m. hyopneumoniae associated with m. flocculare reduces tnfα production compared to bmdcs stimulation by m. flocculare alone producing a tnfα concentration greater than that observed after stimulation with m. hyopneumoniae alone and m. hyorhinis alone [108] . therefore, m. flocculare might play an initial role in pulmonary inflammation by inducing the production of tnfα by resident myeloid cells. supplementary investigations will be needed to elucidate the role of this cytokine in the pathogenesis of the disease [108] . other examples of bacterium-bacterium in vivo coinfections are presented in additional file 1e. regarding coinfections and superinfections, most studies assessed the clinical outcome of the process but less is known about the mechanisms of interactions between pathogens and the consequences for the pathogens themselves, the infected cells and more generally for the host. the outcome of dual infection is variable depending on the antagonism, neutrality or synergy between the infectious agents. on the host side, coinfection can make the host response ineffective, and vice versa. if we look now at the possible interactions that can occur between pathogens we have to consider the nature of the infectious agents (summary provided in figure 4) . different situations can be observed and coinfections can involve virus with virus, bacterium with virus and vice versa, and bacterium with bacterium. regarding virus-virus interactions, consequences are diversified and many studies looking at virus replication in coinfection situations have been carried out [2]. the first consequence of coinfection could be the so-called viral interference, a situation whereby one virus interferes with the replication of the other one making the cells resistant to the superinfecting virus [109] . the most common way for viral interference is indirect and based on the production of type 1 and 3 ifns which induce the expression of isgs after interacting with their cognate receptors [110, 111] . these proteins then activate numerous mediators of the cellular antiviral system that may non-specifically block the replication of viruses. they may also interfere, to a certain extent, with bacterial multiplication since ifn can also be induced by intracellular bacterial pathogens (ibps) or some extracellular bacteria [107, 112] . nevertheless, in some situations type 1 ifns can also increase the host susceptibility to subsequent bacterial infection [113] through impaired macrophage recruitment with a reduced cxcl1 and cxcl2 transcription [114] and a reduced il17 [115] production. then, there is also the non-interferon-mediated viral interference (or intrinsic interference) which is a cellular state of resistance induced by the virus to new viral infection by closely related or unrelated viruses [116] . various mechanisms are described to explain this cellular state of direct or indirect resistance (for examples see [2]). in this type of interference, which can occur between viruses but also between viruses and bacteria [107, 117] , there is a competition between pathogens for the metabolites and all the host factors that allow their multiplication. besides the mechanisms involving a competition for common cellular factors, there are also several other mechanisms of interference described. these relies on viral defective interfering (di) particles [118] , rna interference (rnai) [119] , non-specific double stranded rna (dsrna) [120] as well as trans-acting proteins [121] . interference can occur at specific steps or multiple steps of the viral replication cycle (attachment, penetration, genome replication and/or budding) and can be direct or indirect. inhibition of superinfection (superinfection exclusion and superinfection suppression) is one of several consequences that can be observed in the interference between related and unrelated microorganisms. in superinfection exclusion, an established infection interferes with a subsequent, closely related infection [2, 122] . an example of this phenomenon in pigs is the exclusion of highly virulent classical swine fever virus strain margarita in wild boars persistently infected with this virus upon a challenge infection with the same margarita strain [123] . superinfection suppression is a quite close concept where this time persistently infected cells resist to a challenge with a heterologous virus [2] . furthermore, when the host immune response-innate or adaptive-is considered in the study of the complex interactions taking place in viral coinfections, additional mechanisms of indirect interference linked to cellular and humoral cross-protection-resulting from a first viral contact with a wild-type or a vaccine strain-can be described. conversely, in some situations, viral coinfections can directly or indirectly result in enhanced replication and virulence for one or both pathogens as observed in several studies involving porcine viruses [80, 83, [124] [125] [126] . in other cases, coinfection/superinfection has no effects on virus replications and the viruses can coexist in a relation called accommodation [2] . besides consequences in terms of viral replication, there are also consequences for the genetic of the viruses and their evolution through events of recombination between closely related viral genomes. recombination, the parameters influencing it and its consequences were reviewed in rna and dna viruses [127, 128] . then, as a result of all these possible interactions between viruses, the severity of the resulting disease and its epidemiology can be altered as exemplified in additional file 1. in the pig studies, most often, however, the exact mechanisms controlling interactions between viruses were not elucidated. several mechanisms explaining bacterium-virus and virus-bacterium interactions have been identified (for reviews see [1, 94, 117] ). the interactions can have either a positive or a negative impact on both pathogens depending on the bacterial and viral species involved. usually, when the interactions are direct they promote viral infection without affecting the bacterial species [1, 94, 117] . examples of these direct interactions are (i) direct binding of the virus to a bacterium or (ii) the utilization of a bacterial product by the virus. an example of direct interactions in the respiratory tract is the cleavage of the iav ha into ha1 and ha2 by a staphylococcus aureus protease helping the viral particle to become infectious [129] . on the contrary, when interactions are indirect they often provide an advantage to bacterial infections. four mechanisms dealing with indirect interactions have been described: (i) viral alteration of the epithelial barrier, (ii) reduction or suppression of the host immune response, (iii) viral alteration/displacement of the microbiota, and (iv) virus-induced alteration of bacterial cellular receptor expression [94] . all these mechanisms can operate together for the benefit of the superinfecting bacteria. a typical example of these indirect interactions is provided by pcv2 and swiav and porcine pathogenic bacteria such as a. pleuropneumoniae [130] and s. suis [96, 97, 131] where the bacteria benefit from the prior viral infections. however, bacteria can also directly benefit from a previous viral infection as observed in a study demonstrating that staphylococcus aureus was able to bind viral ha [132] . the consequence of that binding was an enhanced bacterial internalisation by two mechanisms: (i) binding to ha exposed at the surface of infected cells, and (ii) binding to free extracellular virions. in some other situations, non-pathogenic bacteria can also directly or indirectly protect the host from viral infection as typically observed with probiotic bacteria which can show antiviral activity through the binding/ capture of the viruses and/or the competition for cell adhesion (for a review see [117] ). this type of interaction has been frequently observed with enteric bacteria [117] and an example in pigs is the reduced infection of ipec-j2 cells by vesicular stomatitis virus after pre-incubation of the cells with multiple probiotic bacteria [133] . an intriguing relationship is occurring between ibp and viruses where metabolic reprogramming in host cells triggered by viruses might support or conversely limit coinfection by an intracellular bacterial partner (for a review see [107] ). different possibilities can be identified in that type of interaction [107] : (i) the first pathogen can reprogram cellular metabolism related to cellular immunity and decrease the defense against the other pathogen, (ii) the metabolic changes triggered by the first pathogen can facilitate the adhesion, the penetration, and the replication of the other, and (iii) the coinfection transform the active replicative state of the first pathogen into a stable persistent state. the first possibility associated to a decrease of the cellular defense is a commonly accepted mechanism [134] while the second and the third possibilities are less experimentally demonstrated [107] . looking at bacterium-bacterium interactions, they are extremely complex to assess because of the large diversity of the bacterial world and because little is known about the mechanisms underpinning these interactions during infections. moreover, it is now also clear that intestinal and respiratory microbiomes affect the interactions between pathogenic bacteria and the porcine host [135] . some examples of the complex interactions occurring in bacteria-bacteria coinfections are presented in additional file 1e, but little is known about the mechanisms controlling these interactions. however, some mechanisms were provided above and interesting reviews dealing with that subject were published recently [106, 107] discussing the possible direct interactions between bacteria-mainly chemical and physical. indirect interactions between bacteria were not reviewed in these articles but were discussed to some extent in other review papers focusing on polymicrobial infections [1, 136]. the first observation coming from this review must be, even if several studies have been carried out on the subject, a lack of data about some specific coinfections and many discrepancies between studies. for instance, there are only a few in vivo studies about pcv2 in virus/virus coinfections and about pcv2 and prrsv in virus/bacterium coinfections (additional file 1 and heat maps in figures 1, 2, 3) . discrepancies are not surprising because of the definition of coinfection is not always the same between studies in addition to huge variations in the coinfection parameters amongst studies. in this review we focused on experimental (in vivo and in vitro) coinfections, it is worth to underline that these studies are inspired by field veterinarians and epidemiologists observations. however, the definition of epidemiologist coinfection can also vary between studies. indeed, in some cases there is concomitant direct identification of two microorganisms in the same animals, sometimes in the same farms, while in other cases it is just an indirect identification of the microorganisms' presence at some points through indirect serological assays. moreover, as stated before, the term coinfection is sometimes used to describe some situations of superinfections where the delay can be significant. regarding the experimental parameters, the multiplicities of infection (moi), the strains, the potential delays between successive infections, the routes of inoculations, the types of cellular hosts considered (more or less susceptible to one of the microorganism), the genetic background (breeds) and the sanitary status (specific pathogen free or conventional breeding) of the pigs, and the readout to assess coinfection outcome varied a lot between studies. to fully compare studies, a standardization of the assays would be needed. interestingly also, whereas in vitro studies' usefulness is not in question, it is important to underline here that in vitro observed interplay between pathogens cannot be automatically applied in vivo. for instance, whereas m. hyopneumoniae decreases the prrsv titre in vitro [75] , it increases prrsv shedding in vivo and indeed worsens the clinical signs upon coinfection [102] . consequently, the use of intermediary settings, such as co-culture of different cell types (see [2] for examples), precision cut lung slices (pcls) [137] or organoids [138] , could help to understand the complexity of coinfections in the respiratory tissue. in vitro approaches usually consider one or a few cell types with some limitations during the evaluation process of the coinfection consequences. some viruses can contribute to the elimination of other viruses just because of their ability to replicate faster on a particular cell type [139] . thus, results obtained in vitro cannot mimic the field situation when both agents coinfect the same pig, providing inaccurate conclusions about the coinfection dynamics. under such circumstances, pathogens may simply have different host cells and no longer be under direct competition for resources [140] . besides the different interactions that infecting agents can have between them through a competition to resources, studies showed clearly that the immune system and the immunological responses can highly affect these interactions by inducing the competitive power of a pathogen or abolishing it and making it less competitive on the resource [141, 142] . the effects of the immune system (especially humoral parameters) are often not taken into consideration in selected in vitro models [140] . on the other hand, in vivo coinfection experiments have to deal with numerous constraints (health status of the animals used, cost, husbandry, and ethics amongst others) and therefore are not always easy to perform. hence, although in vivo experiments are required in this very complex field, they surely need to be combined to in silico/in vitro/ex vivo analyses of potential interactions between pathogens. moreover, multiple parameters of the coinfection protocols appeared difficult to set without any a priori such as the choice of the pathogen that will be inoculated first and the delay between infections. one possibility to deal with multiple parameters is to use intra-host infection mathematical modelling [143] allowing to play, at limited cost, with the different parameters of the coinfections. however, these models need to be fed with data coming from conventional in vitro experiments as well as more complex in vivo studies. the other possibility is to rely on field prevalence studies monitoring the very presence of the pathogens (isolation, pcr) instead of the sero-conversion, in order to have a clear epidemiological picture of when and where coinfections occur. consequently, ex vivo models such as pcls generated from freshly sacrificed pigs [137] or organoids [138] are developing. these models are closer to mimic the in vivo situation than usual in vitro approaches, combining different types of cells and providing the pathogens with a wider range of cell hosts. however, the contribution of the immune response to the interaction between different pathogens is rarely considered [97] . furthermore, the moi cannot be controlled because the number of infected cells in the slice or the organoid cannot be monitored easily either. another limiting factor in coinfection studies is the cell regeneration, which can vary between in vivo and in vitro models. cell regeneration can highly affect the dynamics of a coinfection, giving some pathogens extra target cells guarantying their longer existence while contributing to the clearance of others [140] . finally, other potential technical limitations could always be discussed such as the lack of precision or sensitivity in the different diagnostic techniques especially in the presence of multiple agents. hence, the detection of coinfecting pathogens could be compromised or reduced as compared to their detection level in the context of single infections. as shown in this review many works have been dedicated to the study of coinfections and superinfections in pigs. usually, when the experiments were carried out in vivo, the researchers were more interested in the clinical outcome than in the interactions occurring between pathogens. indeed, in most of the cases the fine interactions between pathogens and especially the mechanisms behind these interactions and its potential consequences, at the molecular level, on the immune response were not studied for several reasons including technical limitations. also, in the studies assessing the occurrence of coinfections/superinfections in the fields, coinfection identification based on molecular tools such as pcr would be more accurate than sero-prevalence approaches which are less prone to identify currently present pathogens and thus coinfective pathogens. then, a better knowledge of each pathogen involved is crucial. we thus would like to make recommendations for future studies dealing with respiratory coinfections in pigs: (i) authors should clearly summarize their coinfection or superinfection experimental setup-doses of pathogens, delays between infections-in their materials and methods section; (ii) in this summary they would need to clearly present the pathogens they use and they should, as often as possible, select well-characterized strains; (iii) environmental and management conditions would need a strict control and monitoring; (iv) animal genetic and sanitary status would need to be carefully described and monitored during the study; and (v) the multiplications of all the pathogens shall be followed during the experiment using highly sensitive and specific assays. a clear description of all these parameters would help the scientific community to compare studies and progress in the understanding of the complex interactions between microorganisms. in the last years, the concept of innate immune memory or trained immunity has gained a lot of interest. this concept is coming from old observation, in 1946 [144] , recognizing that the bacterial vaccine strain "bacille de calmette et guérin" (bcg) was protecting not only against mycobacterium tuberculosis but also against antigenically different microorganism causing childhood mortality, suggesting an "adaptation" of the cellular innate immune system. since then, many interesting studies about innate immune memory or trained immunity have been published (for a review see [145] ) and it is recognized that cells such as myeloid cells, nk cells, and even epithelial cells [146] can have a higher and quicker response upon re-exposure to a pathogen. trained immunity is accompanied by epigenetic changes and most often associated with modifications in cellular metabolism. a close look at potential epigenetic changes and cellular metabolism modifications would be of high interest in respiratory coinfection studies in the porcine species. recently an alternative to the mechanism of trained immunity in resident lung innate immune cells named "epigenetic legacy" has been described [147] . in that study, the authors demonstrated that following iav clearance and clinical recovery (1-month post-infection), mice were better protected from streptococcus pneumoniae infection by adult bone-marrow-derived ams displaying transient transcriptional and epigenetic distinct profiles. this newly described consequence of a first viral infection also needs additional studies about prdc with an identification of the mechanisms shaping the complex interactions between pathogens. supplementary information accompanies this paper at https ://doi. org/10.1186/s1356 7-020-00807 -8. additional file 1. studies about 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herpesviruses 1 and 5 plos one 11:e0155589 research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year ready to submit your research ? choose bmc and benefit from dominance of hepatitis c virus (hcv) is associated with lower quantitative hepatitis b surface antigen and higher serum interferon-γ-induced protein 10 levels in hbv/hcv-coinfected patients dual infections of cd163 expressing nptr epithelial cells with influenza a virus and prrsv why, when and how should exposure be considered at the within-host scale? a modelling contribution to prrsv infection results of bcg immunization in new york city trained immunity: a program of innate immune memory in health and disease trained immunity carried by non-immune cells influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the colleagues who shared their experience regarding coinfections in pigs. fm is supported by an establishment grant from the région pays de la loire (rfi food for tomorrow-cap aliment). gs phd is funded by foundation philippe jabre (liban). all the authors were involved in the writing of the review. gs, nb, and fm generated the figures. gs, cd, jb, cf, cm-c, gsi, nb and fm prepared the additional file 1. all the authors read and approved the final manuscript. key: cord-326138-16kpn9db authors: weinstein, robert a.; singh, kamaljit title: laboratory-acquired infections date: 2009-07-01 journal: clin infect dis doi: 10.1086/599104 sha: doc_id: 326138 cord_uid: 16kpn9db laboratory-acquired infections due to a wide variety of bacteria, viruses, fungi, and parasites have been described. although the precise risk of infection after an exposure remains poorly defined, surveys of laboratory-acquired infections suggest that brucella species, shigella species, salmonella species, mycobacterium tuberculosis, and neisseria meningitidis are the most common causes. infections due to the bloodborne pathogens (hepatitis b virus, hepatitis c virus, and human immunodeficiency virus) remain the most common reported viral infections, whereas the dimorphic fungi are responsible for the greatest number of fungal infections. because of the increasing attention on the role of the laboratory in bioterrorism preparation, i discuss the risk of laboratory-acquired infection with uncommon agents, such as francisella tularensis and bacillus anthracis. physicians who care for a sick laboratory worker need to consider the likelihood of an occupationally acquired infection while advising exposed laboratory workers about postexposure prophylaxis. in addition, physicians should be aware of the importance of alerting the laboratory if infection with a high-risk agent is suspected. formed with a sputum specimen and returned a positive result for sars coronavirus. additional epidemiologic investigation revealed that the laboratory where he worked was also involved in research on sars coronavirus and that one of the cell cultures of west nile virus was contaminated with the same infecting strain of sars coronavirus. although this case represents an exceptional event, it serves to highlight the inherent risk posed to laboratory workers by virtue of their occupation. infectious diseases specialists may be asked to evaluate an ill laboratory worker. this article provides a framework for assessment of such patients by reviewing the published literature on infections acquired in the clinical diagnostic laboratory. laboratory infections due to a wide variety of bacteria, viruses, rickettsiae, fungi, and parasites have been described in the literature. the largest survey of infections was reported in 1976 by pike [3] , who found that 4079 laboratory-acquired infections were due to 159 agents, although 10 agents accounted for 150% of the cases (table 1) [3, 4] . at least 173 deaths have resulted from laboratory-acquired infection [5, 6] . however, care should be taken in the interpretation of historical surveys, because some infections (e.g., q fever, venezuelan equine encephalitis, and dermatomycoses) occurred predominantly in research and animal laboratories, and many of these infections (e.g., psittacosis and typhoid) were reported before 1955 [1, 3] . surveys of diagnostic laboratory workers in the united kingdom conducted since 1971 have reported that tuberculosis and enteric infections (especially shigellosis) were the most common laboratory-acquired infections [7, 8] . a follow-up survey of uk laboratories from 1994-1995 reported that gastrointestinal infections predominated, particularly shigellosis [9] . similar results were obtained from a survey of clinical microbiology laboratories in utah from the period 1978-1992, with shigellosis reported to be the most common laboratory-acquired infection [10] . these results suggest a shift in the pattern of laboratoryacquired infections, with enteric infections predominating. however, no denominator data have been provided that would help determine the actual risk or incidence of infection for laboratory workers. in a 2002-2004 survey of clinical laboratory directors who participate in clinmicronet, an online forum sponsored by the american society of microbiology, 33% of laboratories reported the occurrence of at least 1 laboratory-associated infection (table 2) [11] . the 3 most common laboratory-acquired infections were shigellosis, brucellosis, and salmonellosis. in contrast, the highest incidences of infection were associated with brucella species (641 cases per 100,000 laboratory technologists, compared with 0.08 cases per 100,000 persons in the general population) and neisseria meningitidis (25.3 cases per 100,000 laboratory technologists, compared with 0.62 cases per 100,000 persons in the general population). of note, the annual number of laboratory-acquired infections has steadily decreased since 1965 [3, 5] . for example, survey results from the united kingdom from the period 1988-1989 found an infection incidence of 82.7 cases per 100,000 person-years, compared with an incidence of 16.2 cases per 100,000 person-years during the period 1994-1995 [8, 9] . this finding undoubtedly reflects an improved awareness of the hazards of working with infectious agents and placement of a greater emphasis on laboratory safety, such as through the use of personal protective equipment. in addition, there have been improvements in laboratory design, such as the use of laminarflow biological safety cabinets (bscs), which provide unidirectional airflow that entraps any aerosolized particles in the airstream and subsequently into air filters [12] . bacteria account for the largest proportion of infections (43%) in diagnostic laboratories, with over 37 different species reported [3] . below, i highlight common causes of infection that are currently of most concern. brucella species. brucellosis continues to be the most frequently reported laboratory-associated bacterial infection [13] [14] [15] [16] [17] [18] [19] . in the united states, brucella infection is one of the most common laboratory-acquired infections, accounting for 24% of laboratory-acquired bacterial infections and 11% of deaths due to laboratory infection [20] . aerosolization is the major source of transmission, but the bacterium can also be transmitted via direct contact. however, in many reported cases, it has not been possible to accurately determine the mechanism for transmission. the disease has also affected janitors and persons who have made brief visits to the laboratory [21] . person-to-person transmission is rare, although a case of brucella infection transmitted from a laboratory worker to a spouse has been documented, presumably through sexual intercourse [22] . although no controlled studies have been performed to assess the benefit of postexposure prophylaxis (pep), it should be considered for laboratory workers who have high-risk exposure to brucella species (e.g., because of direct manipulation of brucella cultures outside of laminar-flow bscs). doxycycline (or trimethoprim-sulfamethoxazole for pregnant women) and rifampin have been frequently used for pep [13, 23] . in a report from canada, 26 laboratory workers were exposed to brucella melitensis, which had been isolated from a patient from india with a draining chest sinus. six laboratory workers were offered pep with doxycycline (100 mg twice daily) and rifampin (600 mg daily) for 3 weeks. only 1 person declined pep; 10 weeks after exposure, the technologist developed fever (temperature, р40њc), and 2 sets of blood cultures confirmed brucellosis. none of the other laboratory workers developed infection or evidence of seroconversion. follow-up serologic tests should be performed for all exposed individuals, probably every fortnight for the first 3 months, then every month for an additional 6-9 months [13, 18] . n. meningitidis. sejvar et al. [24] examined the risk of laboratory-acquired n. meningitidis infection using postings on listservs, to obtain reports of laboratory-acquired meningococcal disease occurring worldwide during the period 1985-2001. sixteen cases of probable laboratory-acquired meningococcal disease were identified, including 6 cases in the united states. nine cases (56%) were due to serogroup b, and 7 (44%) were due to serogroup c. overall, 8 cases (50%) were fatal. all cases occurred among clinical microbiologists and were likely due to exposure to aerosols containing n. meningitidis. the calculated attack rate was 13 cases per 100,000 microbiologists, compared with an attack rate of 0.3 cases per 100,000 persons among the general population. the results of this analysis suggest that laboratory-acquired meningococcal disease represents a significant occupational hazard to clinical microbiologists. although primary prevention of laboratory-acquired meningococcal disease should focus on appropriate handling and manipulation of cultures in a laminar-flow bsc, all laboratory microbiologists should be offered the tetravalent vaccine [25] . it will decrease-but not eliminate-the risk of infection, because it is less than 100% effective and does not provide protection against serogroup b [26] . microbiologists who inadvertently manipulate invasive n. meningitidis isolates on an open bench-top in a manner that could result in aerosolization should consider pep with either a single 500-mg dose of ciprofloxacin or 600 mg of rifampin given twice daily for 2 days [24] . mycobacterium tuberculosis. early surveys of laboratoryacquired tuberculosis found an incidence of tuberculosis among laboratory personnel 3-9 times greater than that in the general population [7, 27] . however, unless there is some accident to which the infection can be traced, it is difficult to state with certainty that tuberculosis was laboratory acquired, because of the potential for exposure outside of the workplace and the long incubation period before symptomatic disease develops. m. tuberculosis can be isolated from a variety of clinical spec-imens, and manipulation of specimens or cultures that generate aerosols is the most important risk factor for acquiring tuberculosis in the laboratory. the high infectivity of m. tuberculosis is related to the low infective dose for humans (50% infective dose, !10 bacilli) [28] . the use of laminar-flow bscs for aerosol-generating manipulations with biosafety level у2 practices and fit-tested respirators with n-95 rating should be routinely used [12, 29] . laboratory personnel should undergo an annual mantoux purified protein derivative skin test or an interferong release assay to demonstrate conversion. persons with positive test results should be evaluated for active tuberculosis by chest radiography. in the event of accidental exposure, laboratory workers should be tested 3 and 6 months after the accident, and persons with new conversion should be offered prophylaxis [29] . francisella tularensis. f. tularensis is a fastidious, gramnegative coccobacillus that is infrequently encountered in the clinical microbiology laboratory, but it has gained increased importance because of its possible use as a bioterrorism agent [30] . the greatest hazard to laboratory workers is from exposure to infectious aerosols from manipulation of cultures. clinicians should be aware that, although patients with tularemia, brucellosis, or endemic mycoses do not pose a communicable disease risk to health care workers, specimens obtained from these patients pose a significant threat to laboratory workers. shapiro et al. [31] described 12 laboratory workers who were exposed to f. tularensis after clinicians failed to notify the laboratory about a suspected case of pneumonic tularemia in a 43-year-old man who eventually died. blood cultures, sputum cultures, and autopsy pleural fluid were all positive for gramnegative coccobacilli, which failed to grow on sheep blood agar and macconkey agar and were initially misidentified as haemophilus species. eleven laboratory workers and 2 autopsy personnel with high-risk exposure to f. tularensis received pep with doxycycline (100 mg twice daily for 14 days), with no resulting infections. to minimize the risk of exposure of laboratory workers, any suspicion about infection with a high-risk pathogen should be immediately communicated to the laboratory. this practice not only protects the staff but also benefits the patient, because a faster and more directed laboratory evaluation can be initiated. bacillus anthracis. before the 2001 outbreak of bioterrorism-related anthrax in the united states, anthrax was an uncommon illness in the united states [32] . in march 2002, the centers for disease control and prevention were alerted about a laboratory worker who had received a diagnosis of cutaneous anthrax [33] . one day after he had cut himself over the right jaw while shaving, the patient assisted a coworker in moving vials of b. anthracis from the laminar-flow bsc in the main laboratory to a freezer. the patient had handled the vials without gloves but washed his hands with soap and water. over the next 3 days, the cut over the laboratory worker's jaw increased in size, and he developed low-grade fever, cervical lymphadenopathy, and cellulitis around the scab. cultures of specimens from beneath the scab were positive for b. anthracis, and the patient was treated with intravenous ciprofloxacin and doxycycline and discharged while receiving ciprofloxacin. epidemiologic investigation of this case revealed that the tops of the vials tested positive for b. anthracis. although all specimen processing surfaces were decontaminated with 10% bleach solution, storage vials were sprayed with 70% isopropyl, because bleach caused labels to become dislodged. this case brings the number of cases of bioterrorism-related anthrax identified since 3 october 2001 to 23 and is the first laboratory-acquired case of bioterrorism-related anthrax. enteric pathogens. salmonellosis is one of the most common reported infections in published surveys [3, 5, 6, 8, 11] . blaser et al. [34] reported 32 cases of laboratory-acquired typhoid fever in the united states over a 42-month period from 1977 to 1980, representing 11.2% of the sporadic cases of typhoid fever reported in the united states. of particular concern was that a number of cases occurred in individuals who had not directly worked in the microbiology laboratory, including cases in 2 family members of a microbiologist who worked with salmonella culture, 1 of which proved to be fatal. in fact, ty-phoid fever has accounted for more reported fatalities than any other laboratory-acquired infection [5] . of note, many earlier reported cases of typhoid fever were associated with mouth pipetting and handling of proficiency test strains-practices which are now avoided [1, 35, 36] . in recent surveys, shigella species was the most frequently identified agent of laboratory-acquired infection [9] [10] [11] . one explanation for the large number of reported cases of laboratory-acquired shigellosis is that shigella species are more virulent and require a much lower inoculum to cause illness. however, it is also probably true that microbiology laboratory staff who develop diarrhea are more likely to attempt to establish a cause for their illness, compared with the general population. a number of other enteric pathogens have also been identified as less common causes of laboratory-acquired infection, including clostridium difficile and escherichia coli o157:h7 [11, 37] . viral agents transmitted through blood and bodily fluids cause most of the laboratory-acquired infections in diagnostic laboratories and among health care workers [1] . although the viral hemorrhagic fevers incite the most fear and dominate the imagination of the media and public, the viruses responsible are rare causes of laboratory infection [3, 4] . however, there is always the possibility that an agent not previously seen may be encountered. this occurred in 1967, when 31 workers were infected while handling tissue specimens from african green monkeys, with 7 deaths resulting [38] . the causative agent was named marburg virus, after the town in germany where most cases occurred. of the common blood-associated viruses, hepatitis b virus (hbv) is the most common cause of laboratory-acquired infection [1] . the incidence of hbv infection among all health care workers in the united states is estimated to be 3.5-4.6 infections per 1000 workers, which is 2-4 times than the level for the general population [39] . it is encouraging that, in the 2 most recent surveys of laboratory-acquired infections in the united kingdom, there were no reported cases of hbv infection among laboratory workers [8, 9] . this finding is probably related to the use of universal precautions when handling blood specimens, improvements in needleless devices, and the availability of immunization. because hepatitis b is a vaccine-preventable disease, all laboratory workers should be offered the hepatitis b vaccine without charge. nonimmunized laboratory workers who have percutaneous, ocular, or mucous membrane exposure to contaminated blood should receive pep with hepatitis b immunoglobulin and vaccine [39] . during 2005-2006, there were 802 confirmed cases of acute hepatitis c reported to the centers for disease control and prevention, with 5 occupational exposures (1.5%) to blood [40] . however, there are few data on the incidence of hepatitis c among laboratory workers, and only single case reports in surveys have been performed in the united states and the united kingdom [8] [9] [10] . human immunodeficiency virus (hiv) infection associated with exposure to contaminated blood or body fluids probably causes the greatest concern. the risk of hiv transmission after a percutaneous exposure to hiv-infected blood has been estimated to be ∼0.3%, and the risk has been estimated to be ∼0.09% after exposure to a mucous membrane [41, 42] . data on occupational transmission of hiv from the period 1981-1992 revealed a total of 32 health care workers in the united states with occupationally acquired hiv infection; 25% of these health care workers were laboratory workers [43] . in the event of laboratory-based exposure from a source person with hiv infection, or if information suggests the likelihood that the source person is hiv infected, immediate referral to employee health for hiv pep should be sought [44] . the dimorphic fungi blastomyces dermatitidis, coccidioides immitis, and histoplasma caspsulatum are responsible for the majority of laboratory-acquired fungal infections in the united states (table 1) [1, 3, 4] . although cutaneous infections due to accidental inoculation are documented, most laboratory-acquired infections are caused by inhalation of infectious conidia from the mold form, resulting in pulmonary infection. the mere lifting of a culture plate lid often suffices to cause the release of large numbers of conidia, and should a sporulating culture be dropped, millions of conidia would be dispersed. the risk of infection in the mycology laboratory probably is low, because handling of specimens is done in laminar-flow bscs, and culture plates are secured with shrink seal to prevent accidental opening. however, a greater risk of infection is likely on the aerobic culture bench, because colonies of b. dermatitidis and c. immitis can grow on routine media and may be visible within 2-3 days. it cannot be overemphasized that clinicians who suspect a dimorphic fungal infection should immediately alert the microbiology laboratory. laboratory-acquired parasitic infections are uncommon in the diagnostic microbiology laboratory [1, 3, 6] . approximately 313 cases of laboratory-acquired infection, with a variety of blood and intestinal protozoa, have been reported (table 3) [3, 45] . most of these cases occurred in research and reference laboratories. readers are referred to the review by herwaldt [45] . fifty-two cases of malaria among laboratory workers and health care workers have been reported, with 34 cases reviewed by herwaldt [45] ; 10 cases were due to plasmodium cynomolgi, 9 cases were due to plasmodium vivax, and 15 cases were due to plasmodium falciparum [3, 45] . most of the cases of p. vivax and p. falciparum infection occurred among health care workers and laboratory workers rather than among researchers and resulted from needlestick injuries that occurred while obtaining blood or preparing blood smears from patients [45] . infection due to intestinal protozoa are uncommon in clinical diagnostic laboratories [45] . one case of giardiasis was reported in a clinical laboratory technologist who processed specimens, many of which were in leaky containers. one case of isospora belli infection occurred in a technologist who examined numerous stool specimens from a patient infected with i. belli. laboratory-acquired infection represents an occupational hazard unique to laboratory workers, especially those in the microbiology laboratory. exposures may occur inadvertently, may not even be recalled, or may result from lapses in technique leading to accidental inoculation. however, not every exposure results in infection. a risk assessment for infection based on the host's immune system, mechanism of the exposure, infectious dose of the exposure, virulence of the agent, use of personal protective equipment, and immunization status needs to be performed. the accurate quantification of such risk is unfortunately difficult, because there is no systematic reporting system that monitors the number of laboratory-related exposures and infections. the centers for disease control and prevention has recently convened a committee to address these issues that will, i hope, provide evidence-based guidelines on exposure risk and use of pep. laboratory associated infections and biosafety laboratory-acquired severe acute respiratory syndrome laboratory-associated infections: summary and analysis of 3921 cases past and present hazards of 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1979 suspected cutaneous anthrax in a laboratory worker-texas fatal salmonellosis originating in a clinical microbiology laboratory acquisition of typhoid fever from proficiency testing specimens acquisition of typhoid fever from proficiency testing specimens nosocomial and laboratory-acquired infection with escherichia coli o157 zur atiologie einer unbekanten, von affen ausgegangen menschlichen infektionskrankheit the risk of hepatitis b infection among health care professionals in the united states: a review surveillance for acute viral hepatitis-united states occupational risk of human immunodeficiency virus infection in healthcare workers: an overview italian study group on occupational risk of hiv infection. the risk of occupational human immunodeficiency virus in health care workers surveillance for occupationally acquired hiv infection-united states updated us public health service guidelines for the management of occupational exposures to hiv and recommendations for postexposure prophylaxis laboratory-acquired parasitic infections form accidental exposures potential conflicts of interest. k.s. has served on the speakers' bureau for wyeth. key: cord-304251-dohglrm1 authors: scully, c; samaranayake, lp title: emerging and changing viral diseases in the new millennium date: 2015-08-06 journal: oral dis doi: 10.1111/odi.12356 sha: doc_id: 304251 cord_uid: dohglrm1 most viral infections encountered in resource‐rich countries are relatively trivial and transient with perhaps fever, malaise, myalgia, rash (exanthema) and sometimes mucosal manifestations (enanthema), including oral in some. however, the apparent benignity may be illusory as some viral infections have unexpected consequences – such as the oncogenicity of some herpesviruses and human papillomaviruses. infections are transmitted from various human or animal vectors, especially by close proximity, and the increasing movements of peoples across the globe, mean that infections hitherto confined largely to the tropics now appear worldwide. global warming also increases the range of movement of vectors such as mosquitoes. thus recent decades have seen a most dramatic change with the emergence globally also of new viral infections – notably human immunodeficiency viruses (hiv) – and the appearance of some other dangerous and sometimes lethal infections formerly seen mainly in, and reported from, resource‐poor areas especially in parts of asia, latin america and africa. this study offers a brief update of the most salient new aspects of the important viral infections, especially those with known orofacial manifestations or other implications for oral health care. professor jens pindborg, in a lecture in the 1970s, spoke of the very few viral infections then known, most of which were regarded as relatively trivial, transient and with few or no serious sequelae and which were largely clinical phenomena with few therapies available (scully, 1979) . indeed, most viral infections encountered in resource-rich countries were and still are, usually relatively trivial and transient with perhaps fever, malaise, myalgia, rash (exanthema) and sometimes mucosal manifestationsan enanthema, and these have been well documented (scully and samaranayake, 1992) . gradually, however, the unexpected consequences of some oral viral infections have emerged and been recognised, not without some surprise (scully, 1983) especially the oncogenicity of some herpesviruses (eglin et al, 1983) and human papillomaviruses (hpvs) which we (eglin et al, 1983; maitland et al, 1987; cox et al, 1993 ) and many others (e.g. lind et al, 1986) have explored, culminating in the appreciation of unanticipated transmission routes for some cancers, such as sexual (scully, 2002) . viruses are increasingly appreciated to cause a wide range of human diseases, ranging from acute self-resolving conditions to acute or chronic fatal diseases. effects that arise long after the primary infection can increase the propensity for chronic conditions (e.g. erythema multiforme) or in some, lead to the development of cancers other than oral (herrington et al, 2015) . in addition, other viruses such as hepatitis c virus (hcv) have been implicated in common but diverse oral lesions such as lichen planus/lichenoid lesions and sicca syndrome (baccaglini et al, 2013) , the latter also sometimes arising after some other viral infections (youinou et al, 2005) although the full aetiopathogenesis of sjogren syndrome remains unclear (kivity et al, 2014) . the possible viral associations in many other oral conditions including periodontitis (vincent-bugnas et al, 2013; ambili et al, 2014; contreras et al, 2014; ly et al, 2014) and several conditions of unknown aetiology, however, remain enigmatic, and associations are not necessarily causal. the recent several decades have also seen a most dramatic change with the emergence globally of new viral infectionsnotably human immunodeficiency viruses (hiv)and the appearance also in resource-rich countries, of some other dangerous and sometimes lethal infections hitherto latent, unrecognised or unappreciated in resource-poor areas. rare lethal and other dangerous viral infections were formerly seen mainly in, and reported from, resourcepoor areas of asia, latin america and africa. pandemics of severe acute respiratory syndrome (sars) and influenza viruses (both h5n1 and h1n1), heralded the surge of zoonotic virus outbreaks. laboratory diagnosis of viral infections is traditionally based on the isolation of the virus, detection of viral nucleic acid or antigens, or serological confirmation (generally presence of igm antibodies), and it is clear that the rate of discovery of new viruses is accelerating, due to a combination of true emergence of new pathogens, increasing awareness and the advance of new mainly molecular biology technologies making rapid detection and characterisation possible (wang, 2011) . various respiratory viruses and ebola virus disease (evd) in particular have served to awaken humanity to the related social inequalities and challenges. this study discusses the importance of emergent new infections and recent findings in relation mainly to those viral infections that may manifest orally or may affect oral health care. fuller descriptions of ebola virus, marburg virus, coronaviruses, nipah virus, noroviruses, enteroviruses, hiv, measles, mumps, respiratory syncytial virus (rsv), influenza, cytomegalovirus (cmv), varicella zoster virus (vzv), epstein-barr virus (ebv), hpvs and kaposi's sarcoma-associated herpesvirus (kshv) are available elsewhere (e.g. herrington et al, 2015) . it is increasingly evident that there is now an amazing range of viral agents, many unidentified, which may be new or newly recognised or old, re-emerging. risk factors for infection include more risk • from greater exposure, • if defences are down, • if agents evade defences, • generally in warm, moist places. emerging viral diseases have arisen mainly because of increasing and changing air travel habits, but other factors that have also increased exposure to the vectors of these infections include conflicts, displacement and migration, as well as changing climate and vector distribution, changing farming/manufacturing, changing lifestyles such as promiscuity and changing clinical practices (mathis et al, 2015) . transmission of respiratory and enteric viruses is highwhile sexually shared infections (ssi) are of low infectivitygenerally requiring the close apposition of infected mucosae, increased when there is epithelial discontinuity. however, with the existence of transnational sexual networks in many countries including usa (hughes, 2001) and europe, with high rates of migration and travel between, for example, amsterdam, barcelona, berlin, london and paris, there have been ssi outbreaks among sexually exploited people of both genders but especially in hiv-positive men. such close encounters, as well as large gatherings of humans, and/or prolonged periods in confined spaces, can enhance transmission of many agents; this has led to the development of a new area of medicine -'mass gathering medicine (mgm)' (memish et al, 2012) . mass gatherings consist of large numbers of people attending an event at a specific site for a finite time. some of the largest mass gatherings are spiritual in nature but other examples include olympics, rock concerts and political rallies. the public health issues associated with the hajj (the annual muslim pilgrimage to mecca, saudi arabia) is the best reported and has international or even intercontinental implications in terms of infection spread. hajj routinely attracts 2.5 million muslims (memish et al, 2012) , and the unavoidable overcrowding raises the risk of respiratory infections. 'hajj cough' is the most frequently reported but influenza (shafi et al, 2008; haworth et al, 2013) and bacterial meningococcal w135 strains16 are more seriousas are severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronavirusesnew viruses that cause severe acute lower respiratory infections (ari), with 10% and 35% mortality rates, respectively, and >50% mortality rates seen in older and immunosuppressed people (gralinski and baric, 2015) . the past decade has also seen the emergence of several other novel viruses that have appeared in resource-rich countriesoften in travelling peopleincluding an h7n9 influenza a virus, a swine-like influenza h3n2 variant virus and a human adenovirus 14p1 (gautret et al, 2014) . a number of zoonotic and vector borne viral diseases have emerged in south-east asia and the western pacific including japanese encephalitis, barmah forest and ross river viruses. australia sees ross river and barmah viruses now appearing regularly. nipah virus causes limited but deadly epidemics in south-east asia. finally, infections by lyssa viruses, kunjin, murray valley and zika viruses are also emerging (bhutta et al, 2014; carson et al, 2015) . in usa, the centers for disease control and prevention (cdc) recognise such threats and have a mission cri-tical (frieden et al, 2015) which focuses on antibiotic resistance and viral infections as shown in box 1. coronaviruses cause illnesses ranging from the common cold to sars. mers-cov has an especially high mortality and has recently spread widely outside the middle east to asia. dr frieden, director of cdc, had stated 'in this interconnected world we live in, we expected mers-cov to make its way to the united states. we have been preparing since 2012 for this possibility (frieden et al, 2015) '. avian influenza a (h5n1) and a (h7n9) viruses have continued to circulate widely in some poultry populations and infect humans sporadically. sporadic human cases of emerging and changing viral diseases c scully and lp samaranayake avian a(h5n6), a(h10n8) and a(h6n1) have also emerged. closure of live poultry markets in china has reduced the risk of a(h7n9) infection (hui and zumla, 2015) . standard infection control precautions are mandatory to prevent cross-transmission from recognised and unrecognised sources of infection. these sources of (potential) infection include blood and other body fluid secretions or excretions (excluding sweat, non-intact skin or mucous membranes) and any equipment or items in the care environment which are likely to become contaminated (booty et al, 2010) . rna viruses, with their high potential for mutation and epidemic spread, are the most common new viral causes of human disease. most emerging viruses are zoonoses; they have jumped from mammals or birds to humans. the annual rate at which novel viruses have been found remains at 2-3 a small number which is an artefact of inadequate surveillance in resource-poor countries, where even established endemic pathogens are often misdiagnosed. indeed, many of the emerging viruses of the future are already infecting humans (rosenberg, 2015) . more awareness, internet communications, advances in diagnostic technology [high-throughput sequencing and specific polymerase chain reaction (pcr) assays] now help assists recognition. promed-mail is one robust and sensitive mechanism for the discovery of emerging disease outbreaks and for rapid dissemination of information to the public health community (madoff and woodall, 2005) . for example, severe fever with thrombocytopenia syndrome (sfts) was recognised as associated with a novel sfts bunyavirus (sftsv) (bao et al, 2011; jiang et al, 2015) using real-time reverse transcription pcr (rt-pcr), viral culture, genetic sequencing, microneutralisation assay (mna) and indirect immunofluorescence assay (ifa). another example is a new arenavirus related to lymphocytic choriomeningitis viruses that was recognised in a cluster of fatal transplant-associated diseases (palacios et al, 2008) . specific pcr assays showed the virus in the kidneys, liver, blood and cerebrospinal fluid of the transplant recipients. awareness of the infections possible and likely, their epidemiology, and a history of travel, contact and relevant vaccinations is crucial to diagnose an infection, but the history is often overlooked. virus infections typically manifest with a fever, but this is neither always present nor measured by the clinician. clinical features are often non-specific and may include malaise, myalgia, rashes and mucosal lesions such as ulceration or bleeding. these are not always major features, not always checked by a clinician, or may often be seen by non-orally trained healthcare workers. examination conditions in the field may be less than ideal; thus, underdiagnoses or misdiagnoses are likely to be quite common. although evd has been recently discussed fully elsewhere (samaranayake et al, 1996 (samaranayake et al, , 2015 scully et al, 2014) , we provide a brief overview account of its oral manifestations below. the three cardinal oral signs and symptoms of evd are gingival bleeding, mucosal lesions (see below) and pain (odynophagia). other important features typical of early and mild forms of disease, which may help oral health care providers suspect evd, include epistaxis, bleeding from injection sites, conjunctivitis and rash. bleeding is very frequent in advanced forms of evd, but it is only relatively frequent in mild or early evd forms; thus, it is likely that ebola-infected patients seeking care may not show such a sign. typically, gingival bleeding is concomitant with other forms of bleeding, particularly epistaxis and bleeding from injection sites. concurrent bleeding at disparate sites is a discriminatory sign of evd. mucosal lesions such as white or red patches, aphthouslike ulceration and greyish exudative lesions may be seen in evd, but these remain to be more accurately defined. odynophagia (discomfort), the consequence of oedema and mucosal lesions, may range from sore throat to severe dysphagia, when mucosal lesions are ulcerated. ebola virus disease is readily transmitted by body fluids, so universal infection control is essential. there is no reliably effective antiviral against this agent yet available. dengue viruses (denvs) cause denguethe most common arthropod-borne viral disease in humansnow seen in more than 100 tropical countries. the word dengue is obtained from swahili phrase ka-dinga pepo meaning 'cramp-like seizure'. it is also called break-bone fever. dengue virus infection can be asymptomatic or a selflimited, acute febrile disease ranging in severity. the classical form is characterised by high fever, headache, abdominal pain, morbilliform rash, myalgia and arthralgia. in a small proportion of cases, the disease progresses to life-threatening dengue haemorrhagic fever, which results in non-infectious bleeding, thrombocytopenia and leakage of plasma, or dengue shock syndrome. there is no reliably effective antiviral against this agent yet available. in the absence of a vaccine and antiviral drugs, the sole control measure is limiting the mosquito vectors. the main mosquitoes, aedes aegypti and aedes albopictus, are potential vectors of numerous arthropod-borne viruses (arboviruses), and have now expanded from the tropics and subtropics, although a. albopictus still plays a relatively minor role compared to a. aegypti in denv transmission. oral manifestations in dengue are rarely reported; gingival bleeding is the most common (lambrechts et al, 2010; mithra et al, 2013; roopashri et al, 2015) . postoperative haemorrhage (dubey et al, 2013) , gingival and lip swelling (pontes et al, 2014) or mucous membrane involvement with no further details (desruelles et al, 1997) have also been noted. neurological sequelae include hypoglossal palsy (jaganathan and raman, 2014) and taste emerging and changing viral diseases c scully and lp samaranayake changes (scully, 2013) . of interest in this context is that saliva has been attempted to be used as a diagnostic fluid for dengue fever (ravi banavar and vidya, 2014) . chikungunya virus (chikv), a togaviridae family alphavirus has emerged as a worldwide threat, causing fever and devitalising arthritis (the name reflects the condition of many of the stricken, 'bent down or become contorted', in the tanzanian makonda language) in a range of countries including many holiday destinations (hasan et al, 2015) . chikungunya virus is a mosquito-borne virus, like dengue transmitted by aedes mosquitoes and has features of fever, headache, rash, nausea, vomiting, myalgia and arthralgia. the presentation differs in adults and childrenthe latter may present with fever, a rash on the face and arms and intra-oral lesions reported as 'koplik spots' (centers for disease c & prevention, 2014). oral manifestations reported also include ulceration (macdonald-ottevanger et al, 2015) and candidiasis (bandyopadhyay and ghosh, 2010) . thus, chikungunya mimics dengue and like dengue, there is no reliably effective antiviral against this agent yet available. indigenous to tropical africa, recent large chikungunya outbreaks have been reported in south-east asia, the indian ocean islands, the caribbean, and the united states and europe (balkans, france, greece, ireland, italy, madeira, the netherlands and spain), mainly in travellers (kumar et al, 2010) . the rapid spread of a. albopictus into europe and the americas coupled with high viraemia in infected travellers returning from endemic areas increases the risk that chikv could establish itself in new endemic regions (thiboutot et al, 2010) . a mutation in chikv (e1-a226v) appears to improve virus survival in a. albopictus and increase its virulence (burt et al, 2012 ). the majority of virus infections of the oral mucosa are due to the herpes group, which are dna viruses. the classical oral manifestations of these virus infections ranging from herpes simplex, herpes zoster to kaposi`s sarcoma, to infections caused by ebv are adequately described elsewhere (balasubramaniam et al, 2014) . the description below refers to recent developments in this regard. herpes simplex virus infections. herpes simplex virus (hsv) infections are increasingly seen in adults, are sometimes caused by hsv-2 and can be an ssi with stomatitis or pharyngitis (looker and garnett, 2005) . herpes labialis recurrences are now recognised to be significantly common in immune defects and to occur where the innate antiviral immune response involving the interferon (ifn-k) promoter is lacking due to polymorphisms within the ifn-k gene (griffiths et al, 2013) . therapy, despite many studies, still involves antivirals such as aciclovir or penciclovir, but hydrocolloid patches may have a place (karlsmark et al, 2008; stoopler and balasubramaniam, 2013) . herpes simplex virus has long been associated with cranial neuropathies and now has also been associated with alzheimer disease (l€ ovheim et al, 2015) . human papillomavirus infections. one of most common ssi in world, hpv can cause cutaneous or mucosal papillomas, common warts (verruca vulgaris), genital warts (condyloma acuminatum) and multifocal epithelial hyperplasia (heck disease). some hpv types (oncogenic or 'high-risk' hpv types such as hpv16) are now implicated in some mouth cancer, especially oropharyngeal cancer (opc). opc incidence has significantly increased, predominantly in economically developed countries (scully et al, 1988; scully, 2002; chaturvedi et al, 2013; brewer and calo, 2015) . hpv is a strong and independent prognostic factor for better survival of opc (ang, 2010; lowy and munger, 2010; o'rorke et al, 2012) , human papillomaviruses infection does not necessarily lead to opc. a study of 1626 males in brazil, mexico, usa; 1 year showed that 0.4% acquired incident oral hpv, 1.7% acquired oral oncogenic hpv infection and 0.6% acquired oral hpv16 infection. new oral oncogenic hpv infections are rare and most are cleared spontaneously within 1 year (kreimer et al, 2013) . the united states food and drug administration (fda) approved the quadrivalent hpv vaccine for girls in 2006 and for boys in 2011 aimed at genital, hpv-related lesions. vaccination has proved to be successful at preventing hpv and associated cervical and other anogenital tumours. hpv vaccines are effective and also against the hpv strains that are most commonly found in the oropharynx (wierzbicka et al, 2014) and have reduced infections (herrero et al, 2013) , but vaccination is yet to be universally recommended for all adolescents. there are two vaccines; both regarded as safe and usually given as a three dose series. • cervarix: recommended for females from 10 to 25 years of age and protects against hpv16 and hpv18. • gardasil: recommended for 11-and 12 year-old girls and also females 13 to 26-year-old. gardasil is also recommended for 9-to 26-year-old males to protect against some genital warts. this vaccine protects against hpv6, hpv11, hpv16 and hpv18. human parvoviruses. the only known parvovirus to infect humans is b19, which is transmitted by droplets, touch and occasionally in blood, and infects rapidly dividing tissues, most commonly the foetus, intestinal epithelium or haematopoietic system. b19 infection in pregnancy is associated with early foetal loss, although the probability of this is low (<10%). b19 also acutely depresses erythrocyte production, which is of little clinical significance, except in patients with other haematological diseases, particularly sickle cell disease, when haemolytic crises may be precipitated. emerging and changing viral diseases parvovirus commonly causes 'fifth disease' (erythema infectiosum; slapped cheek syndrome), a mild illness with a lace-like rash on the face, trunk and extremities, usually in children. papular-purpuric glove-and-sock syndrome is pruritus, oedema and symmetrical erythema, with a 'gloves-and-socks' distribution and oral blisters, erosions and ulcers; 50% of published cases are related to parvovirus b19 infection (segura saint-gerons et al, 2007) . cranial neuropathies have also been reported (soares-fernandes and mar e, 2006) . in many patients (80%) infected with b19, there is also arthropathy, particularly in adults. as the vaccination induced disappearance of rubella, parvovirus is the commonest cause of infection-related transitory arthritis, particularly if it affects the hands. there is no specific therapy available for parvovirus infections. the new century has seen the emergence of several novel viruses that cause respiratory tract infections in humans including sars coronavirus infection mainly in china, mers-cov in saudi arabia and in asia, an h7n9 influenza a virus in eastern china, a swine-like influenza h3n2 variant virus in the usa and a human adenovirus 14p1 also in the usa. mers-cov and h7n9 viruses are still a major worldwide public health concern, but the pathogenesis and mode of transmission of mers-cov and h7n9 influenza a virus are poorly understood and their oral manifestations, if any, ill-defined (gautret et al, 2014) . there are no reliably effective antiviral agents available for these agents. hand, foot and mouth disease. hand, foot and mouth disease (hfmd) is a common childhood illness characterised by fever and vesicular eruptions on hands and feet and in the mouth. complications are rare, but pneumonia, meningitis or encephalitis may occur. hand, foot and mouth disease is not associated with one single agent; it is caused by members of the family picornaviridae in the genus enterovirus; there are over 100 serotypes of enterovirus species a-d, which are the common cause of hfmd and illnesses in infants, such as meningitis and encephalitis . outbreaks of hfmd have been mainly caused by two types of enterovirus a species, coxsackievirus (cv) a16 (cva16), or enterovirus 71 (ev71), and only sporadic cases involve other members of the enterovirus a species have been reported (osterback et al, 2009 ). most cva16associated infections cause only mild symptoms; however, some cva16 infections can lead to severe complications and even death (chen et al, 2014a) . a chinese study of hfmd showed many patients were infected with cva16, fewer with ev71, some were co-infected with cva16 and ev71, and a few were infected with other enteroviruses. upper respiratory tract infection was significantly higher in cva16-associated patients, while neurological complications and hyperglycaemia were significantly higher in ev71-infected patients (liu et al, 2014) . enterovirus infections remain an important public health problem, and other enteroviruses and other agents are emerging as major causative agents of hfmd in some epidemics. serotypes causing severe symptoms such as hfmd including ca16 and ev71 are decreasing, while the proportion of unidentified ev serotypes causing herpangina and viral encephalitis are on the rise . there may be an upward trend in cocirculation of the two pathogens globally and a new role that recombinants play in the emergence of new enterovirus variants. in 2008, a large, hfmd outbreak in fuyang city of anhui province in south-eastern china resulted in a large number of fatalities. phylogenetic analyses of the entire vp1 capsid protein sequence showed isolates belonging to the c4a cluster of the c4 subgenotype, and additionally, genetic recombinations were found between the fuyang hev71 strain and cv-a16, resulting in a recombination virus. in 2008, another nationwide outbreak of hfmd was reported in day care centres and schools in finland (osterback et al, 2009) . from vesicle fluid specimens of hospitalised children, the authors identified the aetiological agent as coxsackievirus a6. enterovirus d68 (ev-d68) appears to cause severe respiratory illness in childrenaffecting most severely those with asthma in the 2014 usa epidemic, and may cause paralyses, including affecting cranial nerves (chen et al, 2014b; foster et al, 2015; maloney et al, 2015) . there is no reliably effective antiviral against these agents yet available. an assay using multilocus pcr and reverse transcription pcr coupled with electrospray ionisation mass spectrometry (rt-pcr/esi-ms), which simultaneously detects and identifies human enterovirus a-d, adenovirus a-f, human herpesvirus 1-8, parvovirus b19 and polyomavirus, detected not only enteroviruses in hfmd, but also herpesviruses, polyomaviruses, adenoviruses and human rhinoviruses in 36% hfmd specimens (chen et al, 2014a) . virus and the orofacial implications of infection have been extensively scrutinised and reported (greenspan, 1998; nokta, 2008; scully, 2014) . hiv infection appeared in the 1920s in the congo from simian origins but was not recognised until the 1980s. the pan epidemic continues to expand with worldwide latest figures of new reported infections (2014) at 2.3 m, a total of over 34 m, and infection in sexually active people now at 1 in 100 (http://www.who.int/hiv/data/en/). worldwide, hiv is mainly heterosexually transmitted and, in the over 50s, there is a threefold increase. africa has a known infected rate >20%, and in eastern europe and central asia, there has been a known increase of 250% in 10 years. uk has among highest rates of new hiv in europe, outstripped by portugal, ukraine, estonia and russia. most hiv infections are in heterosexuals, on vacation but many men having sex with men have hiv. hiv infection is undiagnosed in one in three affected. multidrug-resistant hiv has appeared. the two main viruses, hiv-1 (most common) and hiv-2, infect cells with cd4 surface receptors, mainly t-helper lymphocytes and brain glial cells, and replicate within and damage them, causing hiv disease which may be emerging and changing viral diseases c scully and lp samaranayake symptomless but, over time, ultimately damages cd4+ cells crucial to host defences against fungi, viruses, mycobacteria and parasites, thus causing hiv disease and ultimately producing symptoms (mainly infections and tumours) and the acquired immune deficiency syndrome (aids) and a range of lesions. hiv/aids common manifestations are infections, neoplasms, neurological and autoimmune disorders. infections with viruses, mycobacteria, fungi and parasites, particularly pneumocystis carinii (jiroveci) pneumonia and mucosal candidiasis, are common. loss of weight and wasting ('slim disease') is common. aids is a lethal infection, defined as hiv infection plus 1 or more aids-defining illnesses and a cd4 t lymphocyte count <200 9 10 6 /l. without antiretroviral treatment (art), all eventually develop aids within 5-10 years. candidiasis is universal especially in hiv subtype b strain crf19 infection, but other infections in hiv/ aids depend also upon their environmental exposure; thus, tb is particularly common in people from africa and in urban iv drug users in usa; leishmaniasis is common in persons from around the mediterranean; mycoses such as penicillosis are seen mainly in northern thailand. neoplasms may include virally related kaposi sarcoma, lymphomas or carcinomas (jose et al, 2013; mthethwa et al, 2013; meless et al, 2014; nair et al, 2014) . body fluids such as semen may contain hiv as may saliva, breast milk and blood. hiv transmission is sexual mainly: most new cases are via heterosexual intercourse. hiv can also be transmitted by infected blood or blood products, including plasma or tissues. hiv transmission by contaminated needles and syringes is an important route in injecting drug users. cross-placental transfer is not uncommon. transmission by needlestick ('sharps') injury is an occasional risk for healthcare workers. there is no reliable evidence for hiv transmission by normal social contact or by biting insects. pre-exposure prophylaxis using safe sex practices plus tenofovir and emtricitabine is highly effective to prevent hiv infection. as for treatment, art is typically effective although drugs are costly and often associated with resistance and/ or adverse reactions. immune reconstitution inflammatory syndrome (iris) may follow art and can include exacerbation of some lesions such as tuberculosis, mycobacterium avium complex infections, zoster, hpv, cmv, cryptococcosis and kaposi sarcoma (tsang and samaranayake, 2010) . every effort must be made to avoid needlestick (sharps) injuries, as these could transmit hiv, hepatitis viruses or other infections. in the event of such an injury, speed is of the essence, and where appropriate, counselling and postexposure prophylaxis (pep). current pep for hiv (and other blood-borne agents such as hepatitis b and c) viruses in uk consists of (samaranayake and scully, 2013); • hiv: tenovir, emtricitabine, lopinavir, ritonavir, • hbv: hepatitis immunoglobulin plus hbv vaccine or booster, • hcv: interferon plus ribavirin. mumps. mumps is a common childhood infection caused by the mumps virus (muv), a member of the paramyxoviridae family of enveloped, non-segmented, negative-sense rna viruses. the defining feature of classical mumps is swelling of the parotid gland, but this is not present in all cases and it can also occur in various other disorders. only mumps causes epidemic parotitis. other causes of parotitis include infection by parainfluenza virus types 1 and 3, influenza a virus, coxsackie a virus, echovirus, lymphocytic choriomeningitis virus, human immunodeficiency virus, human t lymphotropic virus 1 and non-infectious causes (e.g. drugs, tumours, immunologic diseases and salivary duct obstruction). mumps virus not only affects salivary glands but also other glands including reproductive glands and pancreas, leading to orchitis or oophoritis (hviid et al, 2008; ternavasio-de la vega et al, 2010) . acute hormonal disturbances are common including decreased testosterone and inhibin b levels with low or normal levels of gonadotropins in 35% and a high incidence of sperm disturbance. importantly, mumps is highly neurotropic, and central nervous system infection ensues in approximately half of cases and may lead to aseptic meningitis, viral encephalitis, and rarely deafness and pancreatitis (rubin et al, 2015) . mumps is vaccine preventable, and one dose of mumps vaccine is about 80% effective against the disease. routine vaccination has proven highly effective in reducing the incidence of mumps and is presently used by most developed countries; however, there have been outbreaks of disease in vaccinated populations (hviid et al, 2008) .since the introduction of the mumps vaccine, the age of appearance of mumps infection has shifted from children to adolescents and young adults, groups with a higher incidence of disease complications and sequelae. in 2005, a large epidemic peaked in the uk, and, in 2006, the american mid-west had several outbreaks. in both countries, the largest proportion of cases was in young adults. in the uk, susceptible cohorts too old to have been vaccinated and too young to have been exposed to natural infections were the primary cause of the mumps epidemic. in the usa, effectiveness and uptake in combination appear not to have been sufficient to obtain herd immunity for mumps in populations such as college students. severe fever with thrombocytopenia syndrome virus (sftsv) infection. an emerging infectious disease, sfts, was identified to be associated with a novel sfts rna bunyavirus (sftsv). transmission of the disease among humans has been described, but clinical impact factors and transmission mechanisms still need further study (bao et al, 2011; li, 2015) . risk factors assessment of the person-to-person transmission revealed that the major exposure factor was blood contact without personal protection equipment. information from this study provided solid references of sfts incubation time, clinical and laboratory parameters related to sfts severity and outcome, and biosafety issues for preventing personto-person transmission or nosocomial infection of sftsv. emerging and changing viral diseases there is no reliably effective antiviral against this agent yet available. viruses are ubiquitous, and they cause a wide range of human diseases, ranging from acute self-resolving conditions to fatal diseases. we continue to witness the emergence of new viruses and infections and there appear to be no signs of abeyance in the march of these elusive enemies. apart from the acute infections with either mild or severe symptoms, a number of new and old virus infections could leave a legacy of secondary illnesses long after the primary infection has subsided. these secondary effects can also increase the propensity for chronic conditions or lead to the development of cancer. the discussion above on emerging viral diseases and old viral diseases appearing in new guises indicates the impact of such infections on the practice of dentistry not only from the perspective of oral manifestations and oral healthcare sciences, diagnosis and management but also from an equally important aspects related to infection control. viruses: are they really culprits for periodontal disease? a critical review human papillomavirus and survival of patients with oropharyngeal cancer urban legends series: lichen planus update on oral herpes virus infections mucocutaneous manifestations of chikungunya fever a family cluster of infections by a newly recognized bunyavirus in eastern china, 2007: further evidence of person-to-person transmission global burden, distribution, and interventions for infectious diseases of poverty standard infection control precautions hpv transmission in adolescent men who have sex with men chikungunya: a re-emerging virus global participation in core data sets for emerging pathogens update on emerging infections: news from the centers for disease control and prevention. notes from the field: chikungunya virus spreads in the americas-caribbean and south america worldwide trends in incidence rates for oral cavity and oropharyngeal cancers detection and identification of viral pathogens in patients with hand, foot, and mouth disease by multilocus pcr, reverse-transcription pcr and electrospray ionization mass spectrometry biology and pathogenesis of cytomegalovirus in periodontal disease human herpes simplex-1 and papillomavirus type 16 homologous dna sequences in normal, potentially malignant and malignant oral mucosa postextraction bleeding following a fever: a case report. oral surg oral med oral pathol oral radiol detection of rna complementary to herpes simplex virus in human oral squamous cell carcinoma enterovirus d68: a clinically important respiratory enterovirus mission: critical -2014 year in review emerging viral respiratory tract infections-environmental risk factors and transmission molecular pathology of emerging coronavirus infections oral manifestations of hiv. hiv insite knowledge base chapter a systematic analysis of host factors reveals a med23-interferon-lambda regulatory axis against herpes simplex virus type 1 replication a comprehensive immunoinformatics and target site study revealed the corner-stone toward chikungunya virus treatment prevention of influenza at hajj: applications for mass gatherings reduced prevalence of oral human papillomavirus (hpv) 4 years after bivalent hpv vaccination in a randomized clinical trial in costa rica viruses and disease: emerging concepts for prevention, diagnosis and treatment the "natasha" trade: transnational sex trafficking emerging respiratory tract viral infections hypoglossal nerve palsy: a rare consequence of dengue fever a cluster of person-toperson transmission cases caused by sfts virus in penglai, china prevalence of oral and systemic manifestations in pediatric hiv cohorts with and without drug therapy randomized clinical study comparing compeed cold sore patch to acyclovir cream 5% in the treatment of herpes simplex labialis infection and autoimmunity in sjogren's syndrome: a clinical study and comprehensive review incidence and clearance of oral human papillomavirus infection in men: the him cohort study oral candidiasis in chikungunya viral fever: a case report consequences of the expanding global distribution of aedes albopictus for dengue virus transmission severe fever with thrombocytopenia syndrome: a newly discovered emerging infectious disease epidemiology of childhood enterovirus infections in hangzhou local immunoreactivity and human papillomavirus (hpv) in oral precancer and cancer lesions co-circulation and genomic recombination of coxsackievirus a16 and enterovirus 71 during a large outbreak of hand, foot, and mouth disease in central china a systematic review of the epidemiology and interaction of herpes simplex virus types 1 and 2 herpes simplex infection and the risk of alzheimer's disease: a nested case-control study prognostic implications of hpv in oropharyngeal cancer altered oral viral ecology in association with periodontal disease the internet and the global monitoring of emerging diseases: lessons from the first 10 years of promed-mail detection of human papillomavirus dna in biopsies of human oral tissue mri findings in children with acute flaccid paralysis and cranial nerve dysfunction occurring during the 2014 enterovirus d68 outbreak emerging and re-emerging infectious threats in the 21st century oral lesions among hivinfected children on antiretroviral treatment in west africa emergence of medicine for mass gatherings: lessons from the hajj oral presentation in dengue hemorrhagic fever: a rare entity the prevalence of hiv associated oral lesions among adults in the era of haart orofacial viral infections-an update for clinicians oral manifestations associated with hiv infection human papillomavirus related head and neck cancer survival: a systematic review and meta-analysis coxsackievirus a6 and hand, foot, and mouth disease a new arenavirus in a cluster of fatal transplant-associated diseases severe oral manifestation of dengue viral infection: a rare clinical description diagnostic efficacy of saliva for dengue -a reality in near future? a piloting initiative clinical and oral implications of dengue fever: a review detecting the emergence of novel, zoonotic viruses pathogenic to humans molecular biology, pathogenesis and pathology of mumps virus needlestick and occupational exposure to infections: a compendium of current guidelines ebola virus infection: an overview viral haemorrhagic fevers with emphasis on ebola virus disease and orodental healthcare fungal and viral infections, skeletal disorders and malignancies. hospital update viruses and cancer: herpesviruses and tumors in the head and neck. a review oral squamous cell carcinoma; from an hypothesis about a virus, to concern about possible sexual transmission persistent metallic taste churchill livingstone: london, uk. oral diseases emerging and changing viral diseases c scully and lp samaranayake clinical virology in oral medicine and dentistry papillomaviruses: the current status in relation to oral disease infection control: ebola aware; ebola beware; ebola healthcare papular purpuric gloves and socks syndrome. presentation of a clinical case hajj: health lessons for mass gatherings isolated velopalatine paralysis associated with parvovirus b19 infection topical and systemic therapies for oral and perioral herpes simplex virus infections mumps orchitis in the post-vaccine era (1967-2009): a singlecenter series of 67 patients and review of clinical outcome and trends chikungunya: a potentially emerging epidemic? immune reconstitution inflammatory syndrome after highly active antiretroviral therapy: a review ebv infection is common in gingival epithelial cells of the periodontium and worsens during chronic periodontitis discovering novel zoonotic viruses hpv vaccination in head and neck hpv-related pathologies viruses contribute to the development of sj€ ogren's syndrome an epidemic analysis of hand, foot, and mouth disease in zunyi we are grateful to dr nihal bandara for the editorial assistance rendered and to the royal college of surgeons of edinburgh for support of professorships (king james iv) for both authors. both authors have equally contributed to the review. key: cord-314505-7qh8dsew authors: stegelmeier, ashley a.; van vloten, jacob p.; mould, robert c.; klafuric, elaine m.; minott, jessica a.; wootton, sarah k.; bridle, byram w.; karimi, khalil title: myeloid cells during viral infections and inflammation date: 2019-02-19 journal: viruses doi: 10.3390/v11020168 sha: doc_id: 314505 cord_uid: 7qh8dsew myeloid cells represent a diverse range of innate leukocytes that are crucial for mounting successful immune responses against viruses. these cells are responsible for detecting pathogen-associated molecular patterns, thereby initiating a signaling cascade that results in the production of cytokines such as interferons to mitigate infections. the aim of this review is to outline recent advances in our knowledge of the roles that neutrophils and inflammatory monocytes play in initiating and coordinating host responses against viral infections. a focus is placed on myeloid cell development, trafficking and antiviral mechanisms. although known for promoting inflammation, there is a growing body of literature which demonstrates that myeloid cells can also play critical regulatory or immunosuppressive roles, especially following the elimination of viruses. additionally, the ability of myeloid cells to control other innate and adaptive leukocytes during viral infections situates these cells as key, yet under-appreciated mediators of pathogenic inflammation that can sometimes trigger cytokine storms. the information presented here should assist researchers in integrating myeloid cell biology into the design of novel and more effective virus-targeted therapies. the ability of the immune system to recognize invading pathogens and tissue damage, and subsequently respond in a targeted and reproducible manner bestows longevity to our existence. within the diverse cellular network of the immune system, recent research has shown that myeloid cells deserve new-found attention due to their ability to detect and mitigate viral infections and promote inflammation. upon viral infection, there are a number of myeloid cell subsets that play various roles in the subsequent inflammatory, cellular, and humoral responses. myeloid cells are granulocytic and phagocytic leukocytes that traverse blood and solid tissues. when they recognize virus-infected cells or tissues damaged by viruses, these sentinels rapidly initiate an innate immune response [1] . this multifaceted response involves cellular activation [2] , signaling cascades [3] , and the release of cytokines [4] to guide leukocytes to mount an effective response. evidence is accumulating that two myeloid cell subsets, in particular, are playing a larger role in recognizing and halting viral infections than was previously thought. researchers are discovering that both neutrophils and inflammatory monocytes are intertwined in the immune system's anti-viral response. moreover, they play unique immuno-regulatory roles post-infection, and are critical for restoring homeostasis. neutrophils are the most abundant leukocyte subset in mammals, ranging from 40-70% of white blood cell counts [5] . they are responsible for both pro-inflammatory and anti-viral responses, and, therefore, constitute a first line of defense against invading pathogens and cell damage [6] . neutrophils are effector innate cells that live for a relatively brief five days [7] and exist in one of three states: quiescent, primed, or active. although they are predominately considered cells that target extracellular organisms such as bacteria via phagocytic uptake, their control of other cell subsets enables them to play important indirect roles in clearing viral infections and modulating inflammation. monocytes are large mononuclear leukocytes that are involved with the inflammation and clearance of pathogens. these non-dividing cells are able to further differentiate into other myeloid subsets such as dendritic cells (dcs) and macrophages. monocytes constitute a heterogeneous population that is endowed with a high degree of plasticity, allowing them to respond to environmental cues in tissues. current research is uncovering the role that inflammatory monocytes play during inflammation and viral infections. this subset preferentially traffics to inflamed regions, where they secrete inflammatory cytokines [8] . however, they can also function as regulatory cells [9] . for example, alveolar macrophages have been shown to recruit inflammatory monocytes through a type i interferon (ifn)-mediated mechanism [8] . these monocytes can then provide protection against virus-induced pathology. evidence exists that both neutrophils and monocytes can contribute to viral clearance or exacerbate pathological damage depending on the context of the infection ( figure 1 ). in terms of myeloid cells contributing to virus-induced pathologies, a linkage can be made between the induction of cytokine storms and dysregulated type i ifn responses. in cases where these cells are beneficial, they can be therapeutically boosted, whereas they can also be depleted when viruses have commandeered them towards destructive fates. exploring the pronounced involvement that myeloid subsets have in mitigating viral replication and pathology, therefore, has the potential to create novel therapeutics that are more efficacious against viral infections. the aim of this review is to explore recent advances in our understanding of the roles that neutrophils and inflammatory monocytes play during viral infections. although previous reviews have provided comprehensive coverage on the impact that these myeloid subsets have during bacterial infections [1, 10, 11] , there is no current review with an extensive focus on their contributions to mitigating viral infections. further, this review has a novel focus on the expanding literature discussing the regulatory roles of these cell types during viral infections, as well as a possible link between the virus-mediated blockade of type i interferon signaling and virus-induced cytokine storms. figure 1 . schematic of myeloid cells highlighting their ability to respond to pulmonary viral infections via the initiation and modulation of anti-viral inflammatory activity. lung-resident myeloid cells, such as alveolar macrophages, utilize a complex sensory system to integrate disturbances of pulmonary tissues by viruses such as respiratory syncytial virus (rsv) into the activation of local effector leukocytes. (a) rsv enters and infects the lungs. viral pathogen-associated molecular patterns (pamps), such as double-stranded rna or danger associated molecular patterns (damps), are detected by pattern recognition receptors (prrs) in or on sentinel cells in the lungs, such as tlr3 in the endosomes of lung-resident macrophages. tlr stimulation activates the nf-κß signaling cascade, resulting in the release of chemokines and inflammatory cytokines. a chemokine gradient forms between the lungs and bone marrow. (b) homeostatic bone marrow tends to retain cxcr4 + neutrophils and monocytes through endogenous expression of high levels of cxcl12. however, the release of pamps, as well as the secretion of cytokines and chemokines as a consequence of pulmonary rsv infections, is sensed by cells in the bone marrow, which in turn allow recruitment of new neutrophils and monocytes from the bone marrow into the lungs. specifically, g-csf downregulates cxcr4 on neutrophils, triggering their release. similarly, ccl2 is produced in the bone marrow by endothelial cells following tlr signaling in infected lungs, which is crucial for inflammatory monocyte release into the bloodstream. once in the bloodstream, these cells sense disrupted endothelium from the viral infection, which triggers a complex adhesion cascade. activated ly6c hi inflammatory monocytes are recruited to the site of infection by a variety of chemokine receptors including ccr1, 5 and 6, as well as cxcr2 binding to their respective ligands. (c) once at the site of infection, they differentiate into dendritic cells and macrophages that initiate an inflammatory cascade that includes copious amounts of inflammatory cytokines, in particular il-12 and ifn-γ, which are potent inducers of th1-biased immune responses. once these dendritic cells and macrophages acquire viral antigens, they home to lymph nodes via chemokine receptors, including ccr7. monocyte-derived dendritic cells that home to lymph nodes present viral antigens to naïve cd4 + and cd8 + t-cells that are required to kill infected cells. (d) the basic neutrophil function of clearing an inflamed area by removing killed pathogens and host cells contributes to reduced inflammation and wound debridement. neutrophils are also capable of promoting tissue repair and increased angiogenesis. further, monocytes can suppress lymphocytes in various clinical scenarios. in lungs, myeloid cells are able to inhibit pro-inflammatory tissue-resident leukocytes through direct cell-to-cell contact through galectin9/tim3 and the effect of tgf-β on nkp30 in order to regulate tcells and nk cells, respectively. myeloid cells can also exert suppressive functions through secretion of soluble factors such as il-10, arginase-1 and indoleamine 2,3-dioxygenase. (e) we speculate that disruption of the cellular sensing of type i ifn responses can result in excessive production of proinflammatory cytokines, including ifn-γ, il-1, il-6, and tnf-α, leading to a toxic cytokine storm. figure 1 . schematic of myeloid cells highlighting their ability to respond to pulmonary viral infections via the initiation and modulation of anti-viral inflammatory activity. lung-resident myeloid cells, such as alveolar macrophages, utilize a complex sensory system to integrate disturbances of pulmonary tissues by viruses such as respiratory syncytial virus (rsv) into the activation of local effector leukocytes. (a) rsv enters and infects the lungs. viral pathogen-associated molecular patterns (pamps), such as double-stranded rna or danger associated molecular patterns (damps), are detected by pattern recognition receptors (prrs) in or on sentinel cells in the lungs, such as tlr3 in the endosomes of lung-resident macrophages. tlr stimulation activates the nf-κß signaling cascade, resulting in the release of chemokines and inflammatory cytokines. a chemokine gradient forms between the lungs and bone marrow. (b) homeostatic bone marrow tends to retain cxcr4 + neutrophils and monocytes through endogenous expression of high levels of cxcl12. however, the release of pamps, as well as the secretion of cytokines and chemokines as a consequence of pulmonary rsv infections, is sensed by cells in the bone marrow, which in turn allow recruitment of new neutrophils and monocytes from the bone marrow into the lungs. specifically, g-csf downregulates cxcr4 on neutrophils, triggering their release. similarly, ccl2 is produced in the bone marrow by endothelial cells following tlr signaling in infected lungs, which is crucial for inflammatory monocyte release into the bloodstream. once in the bloodstream, these cells sense disrupted endothelium from the viral infection, which triggers a complex adhesion cascade. activated ly6c hi inflammatory monocytes are recruited to the site of infection by a variety of chemokine receptors including ccr1, 5 and 6, as well as cxcr2 binding to their respective ligands. (c) once at the site of infection, they differentiate into dendritic cells and macrophages that initiate an inflammatory cascade that includes copious amounts of inflammatory cytokines, in particular il-12 and ifn-γ, which are potent inducers of th1-biased immune responses. once these dendritic cells and macrophages acquire viral antigens, they home to lymph nodes via chemokine receptors, including ccr7. monocyte-derived dendritic cells that home to lymph nodes present viral antigens to naïve cd4 + and cd8 + t-cells that are required to kill infected cells. (d) the basic neutrophil function of clearing an inflamed area by removing killed pathogens and host cells contributes to reduced inflammation and wound debridement. neutrophils are also capable of promoting tissue repair and increased angiogenesis. further, monocytes can suppress lymphocytes in various clinical scenarios. in lungs, myeloid cells are able to inhibit pro-inflammatory tissue-resident leukocytes through direct cell-to-cell contact through galectin9/tim3 and the effect of tgf-β on nkp30 in order to regulate t-cells and nk cells, respectively. myeloid cells can also exert suppressive functions through secretion of soluble factors such as il-10, arginase-1 and indoleamine 2,3-dioxygenase. (e) we speculate that disruption of the cellular sensing of type i ifn responses can result in excessive production of pro-inflammatory cytokines, including ifn-γ, il-1, il-6, and tnf-α, leading to a toxic cytokine storm. the fatal outcome of severe lung infections is shown to be correlated with the early persistent production of inflammatory cytokines and chemokines that recruit neutrophils and monocytes. while inflammatory cytokines and chemokines are essential for effective control of viral infections, they can also contribute to the severity of disease and tissue damage. multiple progenitor cell types arise from self-renewing multi-potent hematopoietic stem cells that become committed in the bone marrow to lineage-specific myeloid cells of the immune system [12] . clonogenic myeloid-primed precursors (cmps) give rise to myeloid cells, which then further differentiate into granulocyte/monocyte progenitors (gmps) [13] . subsequently, gmps undergo multiple stages of differentiation before they are terminally differentiated into neutrophils or monocytes in the bone marrow [14] . monocytes require the growth factor colony-stimulating factor-1 to develop [15] and high levels of the transcription factor pu.1 to steer gmps to commit to a monocyte lineage [16] . monocyte-dc progenitors (mdps) create descendants that are destined to become either dcs or monocytes [17] . recent discoveries have expanded our understanding of neutrophil development. advances in isolation techniques have elucidated that neutrophils are derived from unique cd11b + ly6g lo ly6b int cd115 − precursors that possess proliferation capabilities [18] . indeed, transcriptional profiling coupled with mass cytometry has provided additional information on the process required for gmps to differentiate into neutrophils [19] . researchers determined the bone marrow possesses three distinct subsets: the aforementioned proliferative precursor cells, as well as non-proliferative immature and non-proliferative mature neutrophils. precursors required the transcription factor c/ebpε to differentiate from gmps. as precursors further shift into non-proliferative populations, they exchange proliferation capacity for increases in effector function and migration [19] . further experiments on neutrophil precursors demonstrated their ability to expand in the presence of cancers such as melanoma and suppress regulatory t cells [20] . the role of precursor neutrophils during viral infections has not been determined and represents a novel avenue of research. once viruses such as influenza virus or respiratory syncytial virus (rsv) manage to infect a tissue ( figure 1a ), type i ifns are released from the infected cells and stimulate the expression of hundreds of genes [21] , appropriately known as ifn-stimulated genes (isgs), in neighboring cells. this induces an antiviral state within minutes to hours that is characterized by reduced transcription and translation [22] , the induction of enzymes that degrade viral rnas and proteins, and even the sensitization of cells to apoptosis [23] . products of isgs, including cytokines and chemokines, also recruit leukocytes, including neutrophils and monocytes to the virally infected tissue [24] . the induction of the ifn response following viral infections fundamentally changes the bone marrow microenvironment ( figure 1b) , leading to the enhanced differentiation of myeloid cells [24] and emigration of neutrophils and monocytes to the site of infection, which is facilitated by chemokine gradients interacting with their cognate receptors ( figure 1a ) [25] . murine ly6c hi monocytes originate from the bone marrow and travel to sites such as skin, lungs, and lymph nodes [26] , whereas ly6c low monocytes typically scan vasculature and the endothelial cells lining the lumen for damage. the human counterparts to these subsets are cd14 + cd16 − and cd14 low cd16 + monocytes, respectively [27] . monocytes require kruppel-like factor-4 to differentiate into inflammatory monocytes in vivo [28] . a recent advance by yáñez and colleagues demonstrated that gmps and mdps can independently generate functionally distinct monocytes [29] . gmps and mdps are both derived from cmp-flt3 + progenitors but differentiate into the above subsets when either the toll-like receptor (tlr)-4 agonist lipopolysaccharide (lps) or the tlr9 agonist cpg dna, respectively, are injected into mice. ly6c hi monocytes can be derived from either subset [29] . therefore, the innate pathogen-associated molecular patterns (pamps) and their cognate receptors, known as pattern recognition receptors (prrs), will dictate which monocyte subset is preferentially generated. infections can affect hematopoiesis and influence the proportions of cell subsets. human immunodeficiency virus (hiv) is capable of infecting bone marrow microvascular endothelial cells and provoking hematopoietic dysfunction [30] . a plethora of regulatory signals required to differentiate and release myeloid cells, including granulocyte-colony-stimulating factor and interleukin (il)-6, can be suppressed by hiv. human t-cell leukemia virus (htlv)-1 has recently been shown to infect several lineages of hematopoietic stem cells in addition to t-cells [31] . both neutrophil and monocyte lineages were permissive to infection, as evidenced by the viral tax protein in neutrophils and the ability of monocyte progenitors to become infected. indeed, these infected monocytes were capable of differentiating into dcs and spreading the infection to t-cells. moreover, four subsets of neutrophils have been characterized in infants with viral respiratory infections [32] . these subsets include suppressive, progenitor, mature, and immature neutrophils, which are present in the blood of infected individuals. however, cd16 high cd62l low suppressive neutrophils were only observed in patients with bacterial co-infections. strikingly, the viral dysregulation of hematopoiesis can lead to numerous diseases [24] . for example, the epstein-barr virus (ebv) causes infectious mononucleosis, characterized by a dramatic increase in white blood cells in the bloodstream. in rare instances, this virus can cause pancytopenia, which is a severe reduction in the number of platelets, red, and white blood cells [33] . pancytopenia has also been found in patients who have contracted hepatitis c virus (hcv) [34] . reducing the number of progenitor cells available to differentiate into lymphoid and myeloid cells may be a reasonably common viral strategy to avoid clearance by the immune system. the functional capacity of myeloid cells to respond to viral particles is influenced by the origin of their precursors. defining the molecular and cellular mechanisms underlying myeloid cell precursor development in viral illnesses will provide a better understanding of the susceptibility of patients to different viruses and the immunological events that may ultimately be exploited for therapeutic benefit. indeed, progenitor cells constitute a promising gene therapy target to treat hiv infections because they can differentiate into multiple cell lineages, all possessing a therapeutic transgene such as an anti-hiv ribozyme [35] . other viral infections that, in theory, may be successfully treated by targeting hscs with gene therapies include viruses that dysregulate hematopoiesis, such as hcv or ebv. the human body relies on a robust innate sensory system to quickly eliminate many viruses. prrs are present in and on a variety of cells including neutrophils and monocytes to recognize pamps ( figure 1a ). tlrs are a subset of prrs that recognize pamps. there are multiple tlrs in and on neutrophils and monocytes that specifically recognize viral pamps or danger associated molecular patterns (damps) released from virus-damaged cells. the nucleic acid from rna and dna viruses constitutes a predominant source of viral pamps that can be recognized either via phagocytosis of cellular debris such as epithelial cells, or in cases where viruses infect myeloid cells. within endosomes, tlr3 recognizes dsrna from viruses (dsrna constitutes the genome of one family of viruses, but is also generated during the life cycle of many viruses) [36] , ssrna is recognized by tlr7 and tlr8 [37] , whereas tlr9 recognizes dna viruses while distinguishing from host dna [38] . monocytes are activated via signaling through surface-bound tlr2 during varicella-zoster virus [4] , measles [39] , and type 1 and 2 herpes simplex virus (hsv) infections [40] . tlr2 can recognize a wide range of viral pamps including the glycoproteins gb and gh/gl from hsv [41] and hemagglutinin from measles [39] . tlr stimulation after phagocytosis activates the nf-κb signaling cascade, resulting in the release of inflammatory cytokines such as tnf-α, il-1, and il-6 from monocytes [4] to control virus infections by direct antiviral mechanisms and the recruitment of other leukocytes. direct antiviral mechanisms of monocytes and neutrophils, including phagocytosis and oxidative burst, were reduced in patients who had contracted hcv and were taking ifn-based therapies [42] . neutrophils also use tlrs to conduct anti-viral surveillance, and express ten out of eleven known human tlrs (they lack tlr3) [43] . the endosomal tlr7 is essential for recognition of influenza viruses by neutrophils via sensing viral single-stranded rna when they phagocytose cell debris [2] . lack of tlrs is associated with increased mortality during viral infections. for example, blocking tlr4 leads to increased mortalities associated with influenza virus infections by disrupting phagocytosis of infected cells [44] . although influenza viruses do not contain lps, tlr4 activation is also involved with delaying fusion between lysosomes and phagosomes, thereby preventing virus entry, and thus has an additional role in innate immunity besides recognition of pamps [45] . the multifaceted functions of tlrs should, therefore, be studied in greater detail to determine whether additional tlrs have unappreciated mechanisms to mitigate viral infections. another method for host recognition of viruses involves retinoic acid inducible gene-i (rig-i) and melanoma differentiation factor 5 (mda5) [46] . to clear viral infections, rig-i-like receptors and mda5 recognize cytosolic viral rnas via the helicase domain [47] . in contrast to tlrs that are predominately present in leukocyte subsets, these receptors are ubiquitous in human cells. neutrophils and monocytes themselves can become infected by viruses [48] and therefore possess cytoplasmic and endosomal mechanisms to recognize them, including rig-i and mda5 signaling cascades in the cytoplasm and endosomal tlrs. in fact, the double-stranded rna mimetic poly(i:c) stimulates neutrophils to increase many antiviral genes, including type i ifn mrna transcripts, ifn-responsive genes, tnf-α, and ifn regulatory factor (irf)7 [49] . when infected with encephalomyocarditis virus (emcv), mda5-deficient mice mount significantly reduced tnf-α and ifn-β responses [49] . similar results were also observed after infections with coxsackie b virus (cvb) [50] and west nile virus (wnv) [51] . notably, tnf-α and ifn-β have the capacity to upregulate the expression of major histocompatibility complex molecules on antigen-presenting cells, which would make viruses more susceptible to t-cell-mediated clearance. damps are endogenous molecules that are released in response to tissue damage from trauma, including cells killed by viruses, and, like pamps, trigger an immune response ( figure 1a ). damps can be derived from a variety of cellular components, including the nucleus, cytoplasm, exosomes, plasma, or the extracellular matrix [52] . damps that promote inflammation and immunogenic cell death [53] include the chromatin protein high mobility group box 1 (hmgb1) and mitochondrial damps such as mitochondrial dna and formyl peptides [54] . hmgb1 interacts with neutrophils and monocytes [55] by binding to the inflammatory receptor for advanced glycation end-products (rage). this damp causes monocytes to secrete pro-inflammatory cytokines, including il-1 and tnf-α, reorganize their cytoskeleton, and increases migration across epithelial barriers. monocytes are also capable of secreting hmgb1 themselves when lysosome exocytosis is induced by the inflammatory lipid lysophosphatidylcholine [56] . neutrophils, in turn, have upregulated transcription of genes for pro-inflammatory molecules involving the nf-κb, p38 mapk, and erk1/2 pathways in response to recognition of hmgb1 [57] . cell damage from viral infections leads to a release of damps and subsequent detection by myeloid cells. for example, infection of epithelial cells with dengue viruses results in the release of hmgb1 from necrotic cells [58] . the interaction between viral pamps and prrs in or on myeloid cells can play an essential survival role in the response to viral infections but may, simultaneously, be responsible for tissue injury associated with severe virus-induced inflammation. in theory, mechanisms involved in the recognition of danger signals by neutrophils and monocytes could be targeted selectively to enhance protection against detrimental viral infections while, simultaneously, preventing exaggerated, pathological innate immune responses. chemokines and their receptors play a critical role in dictating the migration and positioning of myeloid cells. an extensive list of chemokines, their receptors, and their various functions has been described [59] . neutrophils and monocytes begin their journey to a site of infection by first leaving the bone marrow ( figure 1b ). neutrophil and monocyte retention in the bone marrow is dictated by steady signaling between the chemokine (c-x-c motif) receptor 4 (cxcr4) and its ligand cxcl12 expressed on bone marrow stromal cells. during maturation, these cells downregulate cxcr4 and become less sensitive to cxcl12, causing their release into the bloodstream [60] . cxcr1, and mainly cxcr2 expression, on neutrophils grants an additional form of chemotaxis away from the bone marrow via their respective ligands, cxcl1 and cxcl2 [25] , which are produced by macrophages and mast cells at the site of infection [61] . however, retention is typically favored in the steady state, as cxcl12 appears to be constitutively expressed in the bone marrow. inflammation mediated by viral infections that induce g-csf enhances cxcl2 release and decreases cxcr4 expression on bone marrow-resident neutrophils, tipping the balance in favor of neutrophil release [25] . ly6c hi inflammatory monocytes appear to require chemokine receptor 2 (ccr2) signaling to efficiently exit the bone marrow and travel to sites of inflammation, whereas ccr2 signaling appears to be contextually dependent for monocyte emigration from circulation into virus-infected tissues [62] . more research needs to be conducted to ascertain if ccr2 signaling is required to respond to viral infections of various tissues. ccr2 signaling, via ccl2 binding to the receptors on monocytes, causes the downregulation of cxcr4 and renders the monocytes less sensitive to cxc12, causing their release from the bone marrow [63] . interestingly, low concentrations of circulating tlrs cause rapid ccl2 release by mesenchymal stem cells and their progeny in the bone marrow, which triggers the release of monocytes [64] . the dissemination of neutrophils and monocytes to virally infected tissues involves many complex processes ( figure 1b) [59, 65, 66] . generally speaking, myeloid cell migration to infected tissues relies on transmigration through vascular endothelium from the blood. this transmigration is dictated by a milieu of cytokines, and chemokines produced by tissue injury and resident sentinel cells in response to damps and viral pamps. the disruption of homeostasis confers a change to the vascular endothelium near sites of infection [67] . the multitude of changes to the endothelium can happen rapidly, and have been reviewed extensively elsewhere [68] . in brief, endothelial changes that start and subside within minutes are known as type i activation and can be mediated by factors such as histamine [69] ). alternatively, type ii activation can last hours to days with substantial changes in gene expression profiles mediated by tumor necrosis factor (tnf)-α [70] . both forms of activation cause increased blood flow, vascular leakage of plasma proteins, and the recruitment of leukocytes [70] . these disruptions in endothelium homeostasis can trigger a leukocyte adhesion cascade [71] that, in harmony with various cytokines released by inflamed endothelium, such as il-8 and monocyte chemoattractant protein (mcp)-1 [72] , initiates the selectin-mediated rolling of leukocytes along the surface of endothelial cells. trafficking of neutrophils and monocytes through the endothelium towards the site of infection is then facilitated by crawling via macrophage-antigen-1 (mac-1/cd11b) expressed on monocytes and the intercellular adhesion molecule-1 (icam-1/cd54) expressed on endothelial cells [73] . crawling appears to facilitate the paracellular (between cells) transmigration of neutrophils and monocytes, which is generally the preferred method of trafficking (occurring 70-90% of the time), as opposed to transcellular (through cells) transmigration [71] . the dissemination of neutrophils and monocytes from the vasculature into infected tissues is critical for viral clearance. neutrophils are initially recruited to sites of infection by their ability to recognize tissue damage via sensing of h 2 o 2 , dna, n-formyl peptides, adenosine triphosphate, uric acid, and other damps [74] . further guidance to sites of infection is provided by a family of cxcl8 chemokines originating from concentrated sites of pamps and damps, including cxcl1, cxcl2, cxcl3, cxcl5, cxcl6, cxcl7, and cxcl8 (il-8), which are sensed by cxcr1 and cxcr2 on neutrophils and monocytes [74] . within the context of viral infections, experimental data from mice infected with theiler murine encephalomyelitis virus have demonstrated that cxcl1 released from epithelial cells, macrophages, and neutrophils recruits both neutrophils and monocytes to sites of infection [75] . macrophages infected with rotaviruses release cxcl2 to recruit neutrophils [76] and nipah virus c protein is capable of inducing the release of numerous chemokines, including cxcl2, cxcl3, and cxcl6, from endothelial cells [77] . pamps from viruses tend to amplify neutrophil recruitment. the inflammatory ly6c hi subset and the "patrolling" ly6c low cx3cr1 hi subset migrate along luminal and endothelial cell surfaces, with the latter being able to respond rapidly to infections in a cx3cr1-dependent fashion [78] . the migration of inflammatory monocytes to tissues is ccr2-dependent. however, as mentioned above, this tends to only be required to exit the bone marrow. nonetheless, ccr2 signaling appears to be critical for inflammatory monocyte recruitment in cases of west nile virus-induced encephalopathies, and influenza virus infections [1, 66] . in summary, a range of trafficking signals and endothelial barrier regulatory molecules shape myeloid cell recruitment to virally infected and inflamed tissues. neutrophils are able to lyse and phagocytose virus-infected cells [44] , and are one of the first leukocyte subsets to enter inflamed tissues ( figure 1c ). the magnitude of the neutrophil response is a predictor of the host's ability to clear an influenza virus infection with minimal damage [79] . depleting neutrophils causes greater viral spread and host mortality [79] , and neutrophils are also crucial to mitigate hsv type-1 corneal infections in a murine model [80] . moreover, tate and colleagues demonstrated that neutrophils are critical for limiting the replication of influenza viruses [81] and that a loss of neutrophils increases disease severity. thus, the antiviral response of neutrophils contributes to clearing viral infections. neutrophils can directly mediate innate immune responses, activate adaptive immunity and recruit lymphoid cells to sites of viral infections [82, 83] . a key mechanism of action that enables neutrophils to neutralize invading viruses is the production of neutrophil extracellular traps (nets) [82] . nets are strands of dna and granule proteins secreted by neutrophils that form around viral particles, preventing their spread [84] . poxvirus infections in mice were mitigated in liver microvasculature via this mechanism [82] . in addition to the physical containment of infections, nets are coated with antiviral enzymes that enable neutrophils to concentrate lethal antimicrobial proteins such as histones at sites of infection [84] . neutrophils are also capable of mediating antibody-dependent cellular cytotoxicity (adcc) or antibody-dependent phagocytosis, which involve the release of cytolytic granules or phagocytosis, respectively, after binding antibodies via fc receptors [85] . these antibody-dependent processes are critical in the clearance and neutralization of certain viruses such as hiv [85] . adcc responses peak quickly (i.e., within four hours) and are controlled by the fcγr family of receptors and can also utilize the extracellular release of reactive oxygen intermediates [85] . reactive oxygen intermediates are also involved in other pathological responses, including exocytosis. exocytosis is a cellular active transport process whereby membrane-bound vesicles transport molecules to the cell surface. neutrophils emit an array of compounds including myeloperoxidase to control sepsis [86] , antiviral lysozyme with anti-hiv properties [87] , and n-formyl-methionyl-leucyl phenylalanine (fmlf)-stimulated superoxide release in the presence of periodontitis pathogens [88] . exocytosis, therefore, expands the neutrophil arsenal to neutralize the array of pathogens they encounter. neutrophils are incredibly diverse in their functions. in addition to trafficking to sites of infection to phagocytize viruses and form nets, they also stimulate virus-specific adaptive immune responses [83] . neutrophils that have detected viral antigens can home to draining lymph nodes dependent on il-1r, where they can act as antigen-presenting cells [83, 89] . neutrophils present processed viral antigens to naïve cd8 + t-cells via the major histocompatibility complex i and t-cell receptor interactions, along with the expression of cd80 and cd86 to provide co-stimulation, thereby providing the two signals required to activate t-cells [83] . furthermore, neutrophils are responsible for the recruitment of effector cd8 + t-cells to sites of viral infections. the mechanism by which they recruit t-cells during influenza virus infections has been linked to cxcl12 deposits left behind like a "trail of breadcrumbs". cd8 + t-cells follow this chemoattractant trail left behind by neutrophil uropods to the sites of influenza virus infections [90] . rsv causes lung infections that are characterized by neutrophils contributing to host damage [91] . rsv is capable of delaying the apoptosis of neutrophils and eosinophils, which is hypothesized to delay antigen presentation and increase tissue damage. il-6 and tlr7/8 binding was determined to contribute to this delay and depended on nf-κb and pi3k activation. the authors of this study did not directly examine whether this delay resulted in an increase in host tissue damage in their model, but hypothesized this was the case, constituting an area of future study. during an rsv infection, neutrophils migrate through infected airway epithelial cells [92] . these neutrophils are characterized by the increased expression of myeloperoxidase and cd1b, and their migration promotes epithelial shedding and airway tissue damage. aside from delaying apoptosis, rsv infection has also been shown to increase eosinophil recruitment and degranulation based on the macrophage inflammatory protein (mip)1-α and eosinophil cationic protein concentrations measured in lower respiratory airway secretions [93] . ly6c hi monocytes migrate to injured sites, induce inflammation, and eliminate the cause of tissue injury ( figure 1c ) [94] . for instance, type i ifns amplify the production of mcp-1, the primary chemokine responsible for recruiting inflammatory monocytes to the lungs during influenza virus infections [95] . these monocytes have been implicated in influenza virus-induced lung injury [96] . importantly, elevated mcp-1 levels have been associated with severity of illness in pediatric influenza virus infections [97] . in mice, the recruitment of monocytes to lungs was shown to be accompanied with an increase in type i ifn production, nlrp3 inflammasome activation, and alveolar epithelial barrier dysfunction [98] . it has been identified that increased pro-inflammatory monocytes are a major immunological determinant of severity of disease in previously healthy adults with life-threatening influenza virus infections [99] . this provides a possible mechanistic cause for disease severity in these patients, a potential early identifier and a modifiable immune pathway for therapeutic targeting. however, there is no role for recruited monocytes in the lungs of mice infected with the natural rodent pathogen, pneumonia virus of mice [100] , indicating the pathogen-specific functions of these cells. interestingly, monocytes have been proposed to be educated in the bone marrow to promote their tissue-specific functions at sites of persistent challenge [101] . long-lasting epigenetic alterations in monocyte precursors may account for the "trained immunity" phenomena [102] . indeed, monocytes have an immunological memory of past insults. thus, this evidence shows that neutrophils and inflammatory monocytes participate in inflammation that is needed for an effective immune response against viruses. a shared feature of neutrophils and monocytes is their ability to synthesize pro-inflammatory cytokines that help the host overcome viral diseases. however, these responses can also be overly robust, thereby contributing to virus-induced tissue damage. future research directions should include a focus on furthering our understanding of the diverse antiviral arsenal of myeloid cells. robust immune responses are critical for protecting hosts against lethal viral infections. it is equally important that immune responses are of adequate magnitude and duration. the capacity for a host to resolve inflammation and return to homeostasis has important consequences for health ( figure 1d ). the induction of an immune response that is too severe or the failure to return to homeostasis can result in immunopathology [103] , including tissue and organ damage [65] , cytokine storms ( figure 1d ) [104] , chronic inflammation [105] , and autoimmune diseases [106] . as innate immune responders, myeloid cells are key players in orchestrating appropriate inflammatory responses and the return to homeostasis following virus infections. the role of myeloid cells in the regulation of immune responses is complex and involves specialized cellular subsets, suppressive receptors, and cytokines. in addition, much of what we know about the regulatory and immunosuppressive effects of myeloid cells originates from research investigating bacterial, fungal, and sterile inflammation models, but has implications for virus infections. neutrophils possess multiple mechanisms to control inflammation, despite their predominately pro-inflammatory role ( figure 1d ) [107] . one mechanism involves the formation of aforementioned nets [108] . these nets function via serine proteases to degrade excess cytokines and chemokines in areas with high densities of neutrophils [108] . neutrophils are also capable of reducing lung injury during influenza virus infections [79] . a neutrophil depletion study in a h3n2 murine model demonstrated that their absence led to weight loss, viremic spread, and increased inflammation. the basic neutrophil function of clearing an inflamed area by removing killed pathogens and host cells contributes to reduced inflammation and wound debridement [107] . they are also capable of healing mucosal regions of the intestine [109] , and increasing angiogenesis [109] . a recent advance in our knowledge of neutrophils concerns their ability to de-prime [110] . originally considered an irreversible process, neutrophils are capable of returning to quiescence. neutrophils can be spontaneously de-primed in the circulatory system via the degradation of a superoxide anion response [111] , with a de-priming half-life of approximately forty minutes [112] , or retained in the bone marrow [113] to limit the number of primed cells that can traverse the body and cause damaging effects such as lung injury [114] . recent experimental data have demonstrated that inflammatory monocytes are capable of exhibiting suppressive properties. inflammatory monocytes are recruited to sites of vaccine-mediated inflammation via mcp-1 [115] . within the vaccine draining lymph node, monocytes sequester cysteine, resulting in t-cell suppression [115] . blocking monocyte suppression in this context may prove to be an effective mechanism to improve vaccine effectiveness. monocytes are also capable of suppressing b cells. in vitro studies have demonstrated that monocytes suppress b cell differentiation, proliferation, and ig class distribution [116] . monocytes, therefore, represent a prime example of a cell type that can be both pro-inflammatory and suppressive, depending on the context. the resolution of immune response is an active regulatory process, which is initiated via the release of soluble mediators such as cytokines and chemokines, as well as through cell-to-cell interactions mediated by surface-expressed ligands and receptors [117] . evidence has revealed that monocytes that are part of inflammation also can be reprogrammed to cells that are highly anti-inflammatory and contribute to resolution of inflammation [117] . moreover, during sepsis, human monocytes have been shown to undergo a transition from a pro-inflammatory to an anti-inflammatory status [118] , although it remains unclear whether the conversion of monocytes from pro-inflammatory to a regulatory phenotype occurs in viral diseases. further studies are needed to understand the mechanisms to explain how monocytes can be switched into suppressor/anti-inflammatory cells during a viral infection, which in turn would allow intervention with targeted therapeutics to control and down-modulate excessive inflammation in viral diseases. an additional myeloid subset of interest is the myeloid-derived suppressor cells (mdscs). mdscs can suppress immune responses in numerous anatomical locations, including tumor microenvironments, virally infected tissues, and sites of inflammation. the subset of mdscs with neutrophil-like properties have been designated polymorphonuclear (pmn)-mdscs or granulocytic (g)-mdscs, while their myeloid counterparts have the nomenclature m-mdscs. viral infections can induce mdscs, as is the case with hcv [119] . cd33 + mdscs were upregulated upon co-culture with hcv infected hepatocytes, resulting in t cell suppression mediated by reactive oxygen species. moreover, nk cells are also suppressed by mdscs during hcv infection [120] . the production of mdsc-derived arginase-1 resulted in a decrease in ifn-γ production by nk cells. the suppression of key effector cells contributes to viral persistence. hcv is not the only virus to control mdscs to evade the immune system. patients with hiv-1 have m-mdsc populations that suppress helper t cells [121] , and elevated levels of these myeloid cells were correlated with increased viral loads. future research should focus on determining whether other viruses engage mdscs to prolong infections. additionally, more research is required to fully determine the position these subclasses have in myeloid cell differentiation. although a recent review concluded that mdscs constitute bona fide alternate lineages [122] , future studies will be required to cement their status within the field of immunology. myeloid cells are able to translate micro-environmental cues into an effector profile that initiates lymphocyte responses [123] . innate lymphoid cells (ilcs) react to pathogens indirectly through myeloid or epithelial cell-derived cytokines and other inflammatory mediators including il-12, il-23, and il-33 [124] . ilcs are derived from a lymphoid progenitor but do not contain either a b or t-cell receptor due to the absence of the recombination-activating gene [125] . there are three major subsets of ilcs: groups 1, 2, and 3. group 1 includes cells that produce ifn-γ and tnf-α and is predominately composed of classical natural killer (nk) cells. ilcs that require gata3 and rorα to develop and express the cytokines il-5 and il-13 are denoted as group 2, while intestinal ilcs that express nkp46 and depend on rorγ comprise group 3 [126] . since evidence shows that ilcs are tissue-resident cell types with limited capacity to directly recognize pamps [123] , myeloid cells may play a crucial role in controlling ilc homeostasis and function [127] . in the steady state, monocytes enter tissues and replenish macrophages and dcs [128] . however, during viral infections they are recruited to infected tissues and mediate direct antiviral activities [129] . for instance, in mice infected with murine cytomegalovirus, inflammatory monocytes are recruited to the liver and produce mip-1a, which recruits nk cells [130] . nk cells are relevant to viral infections because they target infected cells for destruction. nk cells are cytotoxic ilcs that require il-15 to develop, differentiate, and survive [131] . il-15 is secreted by several cell types, including monocytes after viral recognition [132] , which therefore places nk cells under the control of myeloid cells. expression of the activating receptor nkg2d is upregulated on nk cells in response to il-15. il-15-activated nk cells show preferential expression of the tnf-related apoptosis-inducing ligand (trail) as well as activation and phosphorylation of erk1 and 2, and increases in perforin production [133] . the increased expression of these activating receptors and effector compounds increases the killing potential of nk cells. many viruses down-regulate the expression of mhc on infected cells to escape detection by cd8 + t-cells [134] . therefore, il-15 secretion by monocytes constitutes a mechanism to upregulate multiple cell receptors. changes in granzyme regulation were not documented in these studies, but represent an area of future investigation due to the role of this compound in the apoptosis of virus-infected cells. human monocytes express membrane-bound il-15 constitutively, with its expression increased in the presence of ifn-γ [135] . the monocyte-mediated production of il-15 was increased in the presence of the anti-inflammatory cytokine il-10, but was unaffected by il-4 or il-13 [135] . il-15 also influences monocytes and can transform them into dcs in airway epithelia [136] , which has implications for improving the presentation of viral antigens, suggesting a cross-talk between nk cells and myeloid cells under viral inflammatory conditions. recently, ashkar and colleagues [137] showed that type i ifns produced during a viral infection stimulated vaginal mcp-1 production, which is a chemoattractant that is responsible for inflammatory monocyte migration to inflamed sites. once recruited, type i ifns stimulate inflammatory monocytes to produce il-18, which then signals through the il-18 receptor expressed by nk cells to induce their production of ifn-γ. interestingly, cytokine il-12 also promotes the secretion of ifn-γ by nk cells [138] and neutrophils [139] . neutrophils can also increase ifn-γ production by nk cells using multiple pathways. the first method is to interact with dcs via icam-1 to further upregulate il-12p70 [140] , creating a positive feedback loop. the direct co-stimulation of nk cells also occurs with cd18 and icam-3 binding on neutrophils and nk cells, respectively [140] . our unpublished data (personal observation by karimi k and bridle b) have demonstrated that the induction of viremia in mice, which induces the release of high concentrations of inflammatory cytokines into the circulation, is accompanied by increased numbers of pulmonary ilc subsets and the accumulation of multiple myeloid cell subsets that, interestingly, were type i ifn-dependent (data not shown). additionally, we demonstrated that the induction of inflammation by concanavalin a in mice, which occurs due to macrophage activation downstream of the rapid stimulation of t-cells, led to increased numbers of ilc2 populations in all organs examined, including the bone marrow, spleen, and liver [141] (unpublished data). recently, mortha and burrows [123] discussed how the feedback communication between ilcs and myeloid cells contributes to stabilize immunological homeostasis. further studies are needed to dissect cell-to-cell interactions between myeloid cells and ilcs other than nk cells in viral inflammatory conditions. the concept that neutrophils can initiate, amplify and/or suppress adaptive immune effector responses by establishing direct bidirectional cross-talk with t-cells has garnered attention in the past few years [142] . a th1 response can be induced by neutrophils in a murine model [143] , which increases the number of cd8 + cytotoxic t-cells available to lyse virally infected cells. indeed, in vivo murine studies have demonstrated that neutrophils can cross-present ovalbumin to cd8 + t-cells in a tap-and proteasome-dependent manner [144] . neutrophils can further impact the adaptive immune response by inducing dc maturation, which in turn increases antigen presentation to adaptive cells [145] . neutrophils have been observed to cluster with immature dcs and bind their mac-1 to dc-specific intercellular adhesion molecule-3-grabbing non-integrin (dc-sign). dc-sign is also referred to as cd209 and is a prr that recognizes and binds to mannose residues, a conserved pamp associated with a variety of viral infections. however, neutrophil depletion studies have demonstrated an increase in antigen presentation to cd8 + t-cells. the mechanism by which this phenomenon occurs is thought to be a reduction in competition for viral antigens between neutrophils and dcs [146] . there are extensive demonstrations that neutrophils in humans and mice can also suppress t-cell responses ( figure 1d ). suppressive neutrophils that express low levels of cd62l are induced after acute inflammation arising from either viral infections or tissue injury [147] . they have been shown to impair t-cells by releasing hydrogen peroxide into an immunological synapse, which impairs t-cell migration via the cxcl11 chemokine gradient. ball and colleagues have shown that cxcl11-induced migration to sites of infection decreases as the concentration of hydrogen peroxide released into the immunological synapse is increased. results demonstrate the impaired recruitment of th1 and cd8 + t-cells to the periphery. ultimately, the mechanistic consequence pertains to defective migration mechanisms rather than tcr:mhc signal transduction. it is also important to note that this interaction required mac-1 (cd11b). additional research has demonstrated that mac-1-expressing neutrophils are crucial in limiting pathology caused by t-cells in a murine model of infection with influenza virus, presumably by suppressing t-cell proliferation [148] . we have demonstrated that a subset of neutrophils function as negative regulators of excessive cytokine production in a mouse model of viremia, in which type i ifn signaling has been disrupted (karimi k and bridle b, unpublished data). altogether, these findings allow us to envision the therapeutic potential of subsets of neutrophils. however, one of the major challenges would be the heterogeneity of immunosuppressive or regulatory neutrophils. future studies taking advantage of flow cytometry technology and next-generation sequencing to phenotypically and functionally define neutrophil subsets will extend our knowledge about the immunoregulatory role neutrophils play in viral infections and inflammation. neutrophils also have an indirect mechanism to modulate t cells during a viral infection. the bacteria mycobacterium tuberculosis is capable of delaying neutrophil apoptosis, which delays an adaptive cd4 + t-cell response [149] . although this has not been demonstrated via a viral infection, it nonetheless demonstrates a key effect neutrophils have on controlling a cd4 + t helper cell response. this response may be delayed because dcs ingest whole infected neutrophils [150] to acquire antigens and present them to t-cells. additionally, dcs that ingest neutrophils possessing pathogen-derived antigens can migrate to lymph nodes more efficiently [151] . the differentiation of inflammatory monocytes into cd11b + pulmonary dcs is triggered by the presence of respiratory viruses such as influenza virus [152] . defects in this differentiation delay the clearance of influenza viruses and significantly reduce the activation of cd8 + t-cells [1] . while inflammatory monocytes are key regulatory cells in maintaining macrophage and dc populations in healthy tissues, a function of homeostasis, they are quintessential in the clearance of infections due to their ability to induce adaptive immunity and prime a variety of lymphocytes, including t-cells ( figure 1c ) [152] . upon viral infection, inflammatory monocytes in the blood are recruited to the primary site of infection or the draining lymph node. cells that traffic to the primary site of infection play a critical role in the recruitment of t-cells and, thereby, the activation of inflammatory responses and cellular immunity [153] . however, inflammatory monocytes that traffic to draining lymph nodes acquire a dc phenotype that enables them to present viral antigens to naïve t-cells [153] . in particular, studies have shown that inflammatory monocytes stimulate a th1-biased immune response via production of il-12 that promotes production of ifn-γ by t cells primed in lymph nodes [153] . this th1 immunity is critical in the defense against intracellular pathogens, such as viruses [153] . although memory is traditionally considered a hallmark of the adaptive immune response, recent advances have shed light on the contributions of innate memory. innate memory, also referred to as trained immunity, is a multifaceted response. a recent component of trained immunity involves its modulation of hematopoiesis [154] . although myeloid cells have a short lifespan in circulation, the administration of the agonist ß-glucan resulted in myeloid progenitor expansion and subsequent improved responses to a secondary challenge with the agonist lps. trained immunity was able to reduce myelosuppression from chemotherapy, and was associated with metabolic shifts in cholesterol biosynthesis and glucose metabolism [154] . other benefits of innate myeloid memory have been elegantly reviewed by netea and colleagues [155] . in brief, monocytes are influenced by vaccination and viral infections, and are more responsive upon re-challenge. this innate memory response helps mitigate pathogens via upregulated cytokine production and enhanced pathogen elimination response times. this exciting new field may allow vaccines to be optimized for viruses by targeting the innate memory response. clearly, the cross-talk that is occurring between monocytes, neutrophils, and t-cells constitutes a crucial bridge between innate and adaptive immunity. future investigations are encouraged to examine the full extent of communication between these cells, further elucidate the mechanisms, and the anatomical locations of these interactions. depletion assays will be beneficial to determine which cell subsets can mount effective anti-viral responses, not just by t-cell and apc interactions, but also by direct interactions with neutrophils and monocytes. extensive studies have highlighted the role type i ifns play in initiating an anti-viral state in cells through the inhibition of viral replication [156] . in some cases, the disruption of this response results in the excessive production of cytokines, leading to a so-called cytokine storm that can be very toxic ( figure 1e ) [157] . this is a cause of mortality in cases of severe acute respiratory syndrome (sars) [158] , infection with some strains of influenza viruses [3] , ebola virus [159] , and dengue virus [104] . during viral infections, the regulation of cytokine networks and the mechanisms by which the cytokines may interact with neutrophils and monocytes are poorly documented. the fatal outcome of severe influenza infections is shown to be correlated with the early persistent production of inflammatory cytokines and chemokines that recruit neutrophils and monocytes [65, 160] . lethal outcomes of h5n1 influenza infections in humans correlated with early excessive innate immune response, involving type i ifns followed by prolonged inflammatory responses, and were associated with high viral loads and hypercytokinemia [65, 160] . while inflammatory cytokines and chemokines are absolutely essential for the effective control of viral infections, they can also contribute to the severity of disease [161, 162] . other fatal viral infections that are hallmarked by dysregulated type i ifn responses and cytokine storms are hantaviruses [163] and wnv [103, 164] . given the dynamic nature of cytokines, the complexity of signaling pathways they interact with, and the fact that their excessive production is often associated with some of the worst clinical outcomes of viral infections, there is a need for much more research into the mechanisms by which virus-induced cytokine storms are triggered or controlled. investigation into the mechanisms involved in host responses to viral infections demonstrates a complex and carefully balanced interaction between type i ifns and inflammatory neutrophils and monocytes. recent analysis of mrnas in the blood of humans responding to infections with influenza viruses revealed that early gene expression patterns of anti-viral molecules, such as the genes encoding for myxovirus resistance protein-1 (mx1) and isg-15, are correlated with the heightened production and activation of type i ifns after viral infections [165] . late gene expression patterns were also induced by type i ifns, but in contrast to patterns of antiviral molecules being observed, the transcriptional profiles of patients in the late stages of infections were highly reflective of neutrophil and inflammatory molecule activation [165] , suggesting an important interplay between the secretion of type i ifns and the activation of neutrophils and inflammatory monocytes. it is important to study the receptors mediating the neutrophil antiviral response to reduce aberrant host responses and damage. nlrp12 is a nucleotide-binding domain leucine-rich repeat protein that is expressed on blood-derived leukocytes, including monocytes, and modulates neutrophil recruitment by increasing the chemokine cxcl1 through the il-17-nlrp12 axis and increasing vascular permeability [166] . another activator and recruiter of neutrophils is produced by liver cells and is entitled serum amyloid a (saa) [167] . injections of saa increased phagocytosis of influenza viruses by neutrophils, resulting in the release of il-8. modulating these protein concentrations might represent a promising therapeutic strategy to achieve ideal neutrophil responses to promote elimination of influenza viruses without excessive bystander damage to tissues. neutrophil-mediated antiviral responses have varying effects on the outcome of influenza virus infections, depending on the strain of virus [168] . neutrophils contributed to terminating infections with h3n2 influenza virus strains of intermediate virulence and h1n1 strains that were highly virulent, while they did not limit the severity of disease during infection with an h3n2 strain of low virulence. the early production of virus-induced type i ifns has been observed to upregulate genes in neutrophils that encode pro-apoptotic molecules, such as ifn-induced dsrna-activated protein kinase, and the oligoadenylate synthase-like proteins and the rnase l system [165] . experiments with irf-3 -/x irf-7 -/double-knockout mice and wnv [169] concluded that the viral induction of cellular ifn-β secretion depends on interferon-β promoter stimulator-1-mediated signaling without requiring the ifn transcription factors irf3/7, suggesting the essentiality of the immediate and optimal activation of the type i ifn response. sars-coronaviruses are highly pathogenic and cause alveolar damage, fibrin deposition, and tissue necrosis [170] . the delayed expression of the type i ifn response in mice infected with sars-coronaviruses was implicated in the promotion of inappropriate and chronic inflammatory responses, such as excessive inflammatory monocyte, neutrophil and cytokine accumulation, and impaired virus-specific t-cell responses due to augmented t-cell apoptosis, leading to lung damage [171] . in contrast, an early type i ifn response reduced the immunopathological damage observed, linking the early activation of the type i ifn response to the control of overly robust inflammation. additionally, type i ifns have been implicated in the regulation of myeloid cell migration during initial exposure to viral infections, heightening inflammatory and virus-specific b and t-cell responses [8, 90] . the production of type i ifns by sentinel leukocytes, in particular that of plasmacytoid dcs that serve as a potent source of ifns, upon viral infection initiates a type i ifn-dependent secretion of neutrophil and inflammatory monocyte chemoattractants such as il-1α, cxcl1 and cxcl2 [61, 172] , highlighting the role of virus-induced type i ifns in the regulation of neutrophil and monocyte trafficking. pollara et al. [173] demonstrated that the secretion of type i ifns by hsv-1-infected myeloid dcs results in the activation of uninfected dcs. this process enables the adaptive immune system to become activated even during a viral infection that targets myeloid cells and prevents their maturation, such as in the case of hsv. the protective functions of type i ifns have been associated not only with the recruitment of neutrophils and inflammatory ly6c hi monocytes to sites of viral infections, but also with the prevention of excessive monocyte and neutrophil activation, thereby controlling inflammation caused by type ii ifns, such as ifn-γ [172] . the interplay between type i and ii ifns was crucial for mitigating damage stemming from influenza a virus-induced inflammation in rag2 -/-, ifnar1 -/-, ifngr1 -/and stat1 -/-c57bl/6 mice [172] . both ifns were required to prevent excessive numbers of neutrophils trafficking into lungs. stat1 was experimentally determined to coordinate inflammation via type i and ii ifn receptors. when type i ifns were absent, ly6c lo monocytes transitioned to being more inflammatory than ly6c hi monocytes. in the absence of type i ifn signaling, ly6c lo monocytes traditionally associated with tissue re-modeling became phenotypically and functionally more pro-inflammatory during infection with influenza a viruses [172] . notably, infection of trophoblasts with zika virus induced a lower secretion of type i ifns, and higher immunopathological inflammatory immune responses when compared to trophoblasts infected with yellow fever virus and dengue virus [174] . measurement of immune mediators in nasal fluids from rsv-infected infants indicated that severe disease caused by heightened inflammatory responses was also associated with diminished type i ifn responses [175] , furthering the idea that a link between type i ifns and the promotion versus suppression of virus-induced inflammation exists. taken together, these findings suggest that type i ifn signaling drives a balance of pro-and anti-inflammatory effects on the functions of monocytes and neutrophils in response to viral infections; providing protective immunity while simultaneously limiting immunopathology. these results suggest that the administration of type i ifns at optimized time points and doses could prove beneficial in the limitation of toxic cytokine storm onset and the control of excessive immunopathological damage. indeed, in vitro evidence suggests that the administration of exogenous type i ifns can mitigate excessive cytokine production induced by sars-coronaviruses [176] . determining the means by which type i ifns control excessive inflammation while ensuring effective anti-viral responses is required. neutrophils, inflammatory monocytes, and their roles in mitigating bacterial infections have been extensively studied and well characterized. exciting new research in immunology and virology has demonstrated that these first responders of the innate immune system are also crucial in limiting viral infections, replication, and associated off-target pathological damage. a multifaceted range of tactics is utilized to combat an equally diverse range of viruses, including phagocytosis, the formation of extracellular traps, the production of cytokines such as ifns, and modulation of ilcs and lymphocytes. despite rapid advances in the field, many exciting unknown aspects of the involvement of neutrophils and inflammatory monocytes in combating viral infections remain to be clarified. current research has documented the impact of neutrophil/monocyte retention in the bone marrow as it pertains to viral infections, but we still do not completely understand all mechanisms by which myeloid cells are recruited from the blood stream to the primary sites of infection. future studies should aim to elucidate the specific signaling cascades that recruit myeloid cells into infected tissues and the mechanistic consequences of disruptions in these cascades via the chemokine gradient as well as depletions of specific ligands. if the scientific community can determine how different cell subsets can influence the production of chemokine populations and hone in on the essential ligands required for migration into the primary sites of infection, drugs could potentially be developed to exploit this localized production of chemokines. the discovery of pharmaceuticals that could fine-tune myeloid cell trafficking could prove beneficial to inducing rapid antiviral responses. differential ligation versus the blockade of prrs associated with protective versus pathological inflammation constitutes another strategy to balance rapid viral clearance and minimize host damage. current knowledge from myeloid cell studies in bacterial diseases demonstrated that neutrophils are essential for monocyte recruitment and function. additionally, it has been shown that the ratio of neutrophils to lymphocytes is higher in bacterial than viral infections among patients hospitalized for fevers [177] . it is clear that neutrophils and monocytes work in concert to enhance immune responses against bacterial pathogens. however, future studies are needed to explore the mechanisms by which these myeloid cells collaborate with each other to control viral infections, with the aim of gaining new insights into how they function in virus-infected microenvironments to regulate cell-to-cell communication within the innate and adaptive arms of the immune system. gaining a better understanding of the role of myeloid cells in the pathogenesis of viral diseases will facilitate the design of better therapies. importantly, viruses and virus-mediated tissue damage stimulate both neutrophils and monocytes, triggering a cascade of cytokine/chemokine-mediated innate immune responses. this antiviral activity is not always beneficial for a host and, when improperly regulated, may contribute to immunopathologies such as cytokine storms that have been observed in many severe 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10.1016/b978-0-12-801496-7.00009-5 sha: doc_id: 314841 cord_uid: b5l6epy3 molecular analysis of respiratory viruses and the host response to both infection and vaccination have transformed our understanding of these ubiquitous pathogens. polymerase chain reaction for the rapid and accurate diagnosis of viral infections has led to a better understanding of the epidemiology and impact of many common respiratory viruses and resulted in better patient care. over the past decade a number of new respiratory viruses including human metapneumovirus and new coronaviruses have been discovered using molecular techniques such as random primer amplification, pan-viral array and next generation sequencing. analysis of the host transcriptional response during respiratory viral infection using in-vitro, animal models and natural and experimental human challenge have furthered the understanding of the mechanisms and predictors of severe disease and may identify potential therapeutic targets to prevent and ameliorate illness. respiratory viruses are a major cause of morbidity and mortality throughout the world and affect persons of all ages [1] [2] [3] [4] . in addition to >100 million office visits for upper respiratory infections each winter, hospitals fill to capacity with admissions due to community acquired pneumonia, acute exacerbations of chronic obstructive pulmonary disease, asthma and bronchitis and many of these illnesses are due to viral infection. "pneumonia and influenza" consistently ranks as the fourth most common discharge diagnosis, and each year, 270,000 to 540,000 hospitalizations and 7600 to 72,000 deaths in the united states are attributable to influenza [3, [5] [6] [7] [8] . due to their epidemic nature influenza and rsv are widely recognized as pathogens in adults and children, respectively. however, the true burden of disease and the contributions of other viruses such parainfluenza viruses (piv), human metapneumoviruses (hmpv), coronaviruses (cov), and rhinoviruses (hrv) now being more fully recognized using modern molecular detection methods [9] [10] [11] [12] [13] . in addition to sensitive and rapid diagnostic testing, new molecular techniques allow an understanding of viral evolution, mechanisms and predictors of severe disease, interrogation of vaccine responses, improved bacterial and viral diagnostics and associations of viral infections with non-respiratory medical events. in this chapter the many ways molecular and precision medicine have impacted the field of respiratory viral disease will be reviewed. in the past defining the epidemiology and impact of viral respiratory pathogens was significantly hampered by slow and/or insensitive diagnostic techniques such as cell culture and antigen detection [13, 14] . polymerase chain reaction (pcr) has revolutionized the study of respiratory viruses and provides extremely sensitive, specific and rapid means for the detection of fastidious and non-cultivatable respiratory viruses [13] . pcr based epidemiologic studies now provide a more complete understanding of the clinical spectrum and age ranges of populations affected [15] [16] [17] [18] [19] [20] . in one study, conventional methods yielded a viral diagnosis in 14% of pneumonia cases, while use of pcr increased the yield to 56% [21] . technology has rapidly evolved from single-plex pcr and gel electrophoresis to multiplex real time assays where products are detected by luminescent signals proportional to the target amplified [14] . there are currently a variety of commercially available assays that detect from 2 to 20 viral respiratory pathogens and maintain excellent sensitivity [14] . many clinical microbiology laboratories are now moving to primarily molecular methods for viral detection and pcr formats are becoming increasingly simple so that nucleic acid extraction and pcr is fully automated with little operator input. molecular point of care assays will soon be feasible [22] . in addition to providing more sensitive means of detecting known viruses, molecular methods are extremely useful for viral discovery [23] [24] [25] . over the past several decades a number of new respiratory viruses or variants have been identified including hmpv, novel strains of coronaviruses (hku1, nl63, sars-cov, mers-cov), rhinovirus c, human boca virus, parechoviruses and new strains of avian influenza viruses. molecular methods have been critical for the rapid identification of new viruses associated with dramatic lethal outbreaks but also for pathogen discovery for routine respiratory illnesses. despite intensive investigation, in 12-62% of lower respiratory illnesses no pathogen can be identified suggesting additional agents may yet be discovered [26] [27] [28] . several different genomic approaches for pathogen discovery have been used successfully and include random primer amplification, pan-viral dna microarray and next generation sequencing ( fig. 1 ) [24] . if a viral class of an unknown pathogen or variant virus is suspected, consensus pcr using degenerate primers to detect sequences broadly conserved between members of a group can be used as was done to identify two new coronaviruses, hku1 and mers-cov [29, 30] . another technique for viral discovery is random primer amplification with conventional shotgun sequencing of pcr products [31] [32] [33] . such was the case when van den hoogen discovered a new respiratory virus in 2001, in young children with bronchiolitis who tested negative for rsv [31] . after detecting paramyxovirus like particles in cell culture, rna was subjected to random primer pcr and viral sequences were compared to all known pathogens. the new virus most closely aligned to avian pneumovirus but was determined to be a unique human pathogen and named human metapneumovirus (hmpv). similarly, in 2003 peiris identified a novel coronavirus as the cause of severe acute respiratory syndrome (sars-cov) using degenerate/random primers pcr amplification [32] . using pan viral micro array investigators at the center for disease control and prevention independently identified the same sars-cov [34] . in this technique, after random primer amplification, pcr products are hybridized to microarrays consisting of 70mer oligonucleotides derived from every fully sequenced viral genome. hybridized sequences are scraped from the microspot, amplified, cloned and finally sequenced [25] . identification of completely novel infectious agents requires unbiased and sequence independent methods for universal amplification [23, 24, 35] . conventional sanger sequencing may have poor sensitivity for genomes at low quantity. next generation sequencing (ngs) involves the analysis of millions of sequences and can detect small amounts of novel nucleic acid sequences in clinical samples. continuous sequences are assembled, host sequences are subtracted and the residual sequences are analyzed for similarity to known microbial sequences. ngs has led to the discovery a numerous novel human and animal pathogens [24] . a recent study of nasopharyngeal aspirates of thai children with respiratory illness using ngs identified a number of mammalian viral sequences belonging to newly described families of viruses such anelloviridae as well as novel strains of hrv, enteroviruses and hbov [35] . a critical step in viral discovery is the availability of bioinformatic tools to efficiently identify unique viral sequences in complex mixtures of host, bacterial and fungal sequences. new computational tools for analysis of the virome such as "virusseeker" are being developed [36] . of note, detection does equate with causation and after discovery further studies are necessary to infer more than association. the genetic and antigenic evolution of error prone rna respiratory viruses, particularly influenza, has been of interest for several decades [37, 38] . understanding the selective pressure exerted by pre-existing immunity on viral evolution may help design more effective influenza vaccines and surveillance of animal populations can be critical for early identification of emerging influenza viruses [39] . advances in deep sequencing make it possible to measure low frequency within host viral diversity and factors such as antigenic diversity, antiviral resistance, and tissue specificity can now be studied to understand the complexities of viral evolution [40] . influenza evolution at a population level has been studied years, yet, new antigenic variants are initially generated and selected at the level of the individual infected host. within a host, influenza viruses exist as a "swarm" of genetically distinct viruses [41] . sanger sequencing defines consensus sequences and cannot resolve minority variants below 20% of the viral population. deep sequencing has been used in natural infection and human challenge studies to characterize between and within host genetic diversity [41, 42] . the identification of low frequency mutations in the hemagglutinin (ha) antigenic sites or near the receptor-binding domain in vaccinated and unvaccinated influenza infected persons highlight viral evolution within a host due to selective immune pressure [41] . similarly, ngs can reveal the rapid evolution of drug resistant variants during therapy [43] . using samples collected over time, the mutational spectrum of h3n2 influenza a virus in an immunocompromised child was delineated [44] . individual resistance mutations appeared weeks before they became dominant, evolved independently on cocirculating lineages. the within host evolution of antiviral resistance reflected a combination of frequent mutation, natural selection, and a complex pattern of segment linkage and reassortment. within host sequencing diversity has also been examined in an infant with severe combined immune deficiency with persistent rsv infection [45] . ngs was performed on 26 samples obtained before and after bone marrow transplantation. the viral population appeared to diversify after engraftment with most variation occurring in the attachment protein (g). in addition, minority viral populations with palivizumab resistance mutations emerged after its administration. deep sequencing of hrv during human challenge studies has shown that hrv generates new variants rapidly during the course of infection with accumulation of changes in "hot spots" in the capsid, 2c, and 3c genes [46] . a genome-wide association study (gwas) involves rapidly scanning sets of dna, or genomes, of many people to find genetic variations associated with a particular disease. typically, the genomes of cases are compared to non-affected controls and search for single nucleotide polymorphisms (snps) or polygenic changes that are associated with risk or protection from susceptibility or severity of the condition. gwas have been useful to find genetic variations and risk for asthma, cancer, diabetes, heart disease and autoimmune illnesses with relatively limited studies relating to infectious diseases [47, 48] . recent studies examining host genetic factors conferring susceptibility to respiratory viruses such as pandemic h1n1 2009 influenza a, sars-cov and rsv now provide some insight into host genetic factors for respiratory viral infections [49] [50] [51] . previously most influenza research focused on viral genetics of novel viruses, yet experience with h1n1 2009 and h5n1 clearly indicate host factors also influence disease severity [49, 50] . a number of candidate genes influencing respiratory virus susceptibility have been identified in animal and human studies and involve host virus interactions, innate immune signaling, interferon related pathways and cytokine responses (table 1) [49] [50] [51] [69] [70] [71] [72] [73] [74] [75] . over 20 studies have evaluated genetic polymorphisms associated with severe rsv disease and none demonstrates dramatic results [51] . most focused on one or a few candidate genes resulting in only modestly increased odds ratios of severe illness. a relatively large study of almost 500 hospitalized children that examined 384 snps in 220 candidate genes demonstrated that susceptibility to rsv is complex with a several associations to a few innate immunity genes. these included a vitamin d receptor gene associated with down regulating interleukin 12 (il-12), gamma interferon (ifn-γ), nitrous oxide synthase (nos2a), the jun oncogene, an important transcriptional regulator for innate immune pathways, and ifn-α (ifna5) an antiviral cytokine [68] . the host transcriptional response can be analyzed to investigate disease pathogenesis using a variety of methods including in-vitro studies of bronchial epithelial cells (bec), animal models and infection both natural and experimental challenge [76] [77] [78] [79] . in addition, two compartments, the respiratory epithelium and blood can be sampled in human studies and interrogated using different viruses or viral strains to develop gene signatures for prognosis, as indicators of severity and to identify potential therapeutic targets. most respiratory viral mechanistic studies have been performed using influenza viruses, rsv, hrv and coronaviruses [80] [81] [82] [83] . using bec, the common and (h5n1, h7n7, h3n2 and h7n9 ) and analyzed cellular responses using microarray [83] . common proinflammatory cytokines and antigen presentation were identified although each viral response was unique and notably, h7n9 responses were most similar to h3n2. the response of different clinical isolates of rsv in a549 cells, and monocyte derived human macrophages demonstrated that the pattern of innate immune activation was both host cell and viral strain specific [85] . using rna seq, differences in il-6 and ccl5 were noted among the responses to different clinical isolates suggesting different rsv strains may vary in inherent virulence. human studies have shown significant differences in the blood transcriptional profiles which change over time and differ depending on the infecting respiratory virus. mejias and colleagues were able to differentiate rsv, hrv and influenza in young children based on the blood gene profile (fig. 3) . hrv infection exhibited the mildest innate and adaptive responses compared to rsv and influenza and neutrophil gene expression was greatest in rsv infection with marked suppression of b and t cell and lymphoid responses [79] . notably, gene expression changes persisted up to 1 month after infection. similarly, studies of h7n9 infected patients showed transcriptional profile changes persisting up to 1 month with a transition from innate to adaptive immunity [86] . because of the association of hrv and exacerbations of asthma, the host response to hrv has been of particular interest [87] [88] [89] [90] . studies using becs from asthmatic and healthy donors demonstrate different transcriptional profiles when infected with hrv [87] . hrv, similar to other picornaviruses induces gene expression down regulation by the 2a and 2c proteins. in both asthmatic and healthy control derived cells the majority of genes were down regulated after exposure to hrv. however, some significant expression differences in inflammatory, tumor suppressor, airway remodeling and metallopeptidase pathways have been noted in asthmatic derived cells. asymptomatic hrv infection is quite common and its role in asthma pathogenesis has been questioned. interestingly, heinonen et al. did not find a difference in the blood transcriptome of asymptomatic hrv infected children compared to non-infected controls [91] . whereas, wesolowska-anderson and colleagues demonstrated over 100 differentially expressed genes in the nasal epithelium of asymptomatic infected hrv patients [90] . thus, the blood transcriptome may not be as informative as the nasal epithelial transcriptional response for asymptomatic hrv infection. given the significant host response to asymptomatic infection, hrv may play a role in asthma exacerbations in the absence of clinically evident disease. lastly, it may be possible to identify patients with asthma who are prone to frequent hrv related exacerbations by examining the gene expression response of their pbmcs stimulated with hrv [89] . gene expression studies focusing on illness severity may enhance our understanding of disease pathogenesis, can identify potential therapies to modulate harmful host responses and can be used to develop biomarkers for predicting life threatening disease [79, [92] [93] [94] . a number of studies have been undertaken to understand the pathogenesis of severe rsv in young children and have identified a variety of gene expression patterns in blood including under expression of t cell cytotoxicity/nk cells and plasma cell genes, as well as upregulation of jak/stat, prolactin, il-9 signaling, cell to cell signaling, and immune activation pathways [79, 92] . using nasal epithelial gene expression analysis, van den kieboom identified 5 differentially expressed genes in 30 children with mild, moderate and severe rsv infection [81] . ubiquitin d, tetraspanin 8, mucin 13, β microseminoprotein, chemokine ligand 7 were up regulated and differentiated mild from severe illness. lastly, nasal gene expression is complicated by interactions of the nasal microbiota and host cell gene responses [95] . in nasal samples from children with rsv infection, h. influenzae and s. pneumoniae dominated microbiota, toll like receptors and neutrophil/macrophage signaling were over expressed and the presence of h. influenzae and s. pneumoniae along with age and sex were predictive of risk of hospitalization due to rsv. transcriptional profiling related to severity has been analyzed in seasonal influenza as well as emerging avian pathogens with a recognition that disease is not only due to an infection with a novel virus in a non-immune host but may also be due to an exaggerated host immune response [78, 96] . in a study of primarily seasonal influenza (h1n1, h3n2), influenza infection was associated with a significantly stronger antiviral, cytokine, attenuation of t/nk cell response compared to patients with respiratory illnesses of unknown etiology regardless of severity [96] . notably, ifn and ubiquitination was significantly down regulated in those with severe vs. mild to moderate disease. in a study of the lethality of 1918 h1n1 influenza and h5n1 vietnam influenza virus in macaques, upregulation of key components of the innate immune response and cell death pathways were noted were noted with 1918 h1n1 infection but were down regulated with h5n1 [78] . early up regulation of the inflammasome likely resulted in some of the severe tissue damage noted with the 1918 h1n1 influenza infections. in vitro, animal and human challenge studies have been used to identify new strategies control or prevent symptomatic or severe infection [82, 97] . in hrv challenge studies, virperin expression correlated with rhinorrhea and chilliness. knockdown of expression resulted in increased viral replication in becs suggesting virperin has antiviral actions and might have potential therapeutic use. influenza challenge studies clearly show a definable transcriptomic profile in the blood prior to the onset of symptoms offering the possibility of earlier and more effective oseltamivir treatment [77, 98] . lastly, host gene expression studies may allow investigation into links between respiratory viral infections and specific non-respiratory events. there is ample epidemiologic evidence that influenza epidemics are linked with increased rates of strokes and myocardial infarction (mi) [99, 100] . increased rates of falls and functional decline in nursing homes have also been associated with increased influenza activity [101, 102] . however, direct links of events to viral infection are scarce in part due the event of interest may follow the infection by several weeks when the virus is no longer detectable by traditional testing. several gene profiling studies have identified viral infection signatures that may persist up to 1-month post infection [79, 86] . thus, it might be possible to study patients with falls or cardiac events for evidence of recent viral infection using a host response viral signature. in addition, evaluating the host response can provide information on mechanisms of disease. a viral gene signature was used to evaluate patients undergoing cardiac catheterization [103] . notably, 25% vs. 12%, p = 0.04 of those with a viral gene signature present vs. those without viral signatures, suffered an mi. furthermore, h1n1 infected patients showed an increased gene platelet expression signature providing insight into how infection may induce a prothrombotic state. given the availability of rapid and accurate multiplex pcr for viral detection, host-based diagnostics might seem unnecessary. however, current pcr assays use conserved known viral sequences but can miss novel or significantly mutated viruses. this issue was seen in 2009 with pandemic h1n1 when influenza pcr assays had to be adapted to optimally detect the new influenza strain [104] . the emergence of novel respiratory viruses are a persistent threat and methods to detect a "viral signature" in the setting of clusters of severe pneumonia cases could be very useful. zaas and colleagues developed an acute respiratory viral gene signature using microarray analysis of the blood from volunteers experimentally infected with influenza a, hrv or rsv [105] . the signature was subsequently 89% sensitive and 94% specific in classifying as viral 25 influenza and 3 hrv infected patients presenting to an emergency room. additionally, a distinct blood transcriptome signature was noted in patients with severe h1n1 pneumonia [106] . upregulated genes included those related to cell cycle, dna damage, apoptosis, protein degradation, and t helper cells. down regulated genes were primarily in immune response pathways suggesting immunosuppression as a mechanism of severe influenza pneumonia. investigators developed a 29 gene classifier which predicted h1n1 influenza a regardless of concomitant bacterial infection and such a predicator could guide antiviral therapy in the face of negative pathogen detection methods. in most cases of respiratory infection, the precise microbial etiology is unknown and antibiotics are frequently administered empirically [27, 107] . although sensitive molecular diagnostics (pcr) now allow rapid diagnosis of a wide variety of respiratory viruses, their impact on patient management and antibiotic prescription has been modest primarily due to concern about bacterial co-infection [108] [109] [110] . approximately 40% of adults hospitalized with a documented viral respiratory infection have evidence of concomitant bacterial infection and thus clinician concerns are reasonable [109] . importantly, sensitive and specific diagnostic tests for bacterial lung infection are currently lacking [111, 112] . although the site of infection is the respiratory tract, blood is a convenient sample comprised of components of the innate immune system (neutrophils, natural killer cells), as well as the adaptive immune system (b and t lymphocytes) [113] . recent studies indicate that viral and bacterial infections trigger pathogen specific host transcriptional patterns in blood, yielding unique "bio-signatures" that may discriminate viral from bacterial causes of infection [114] [115] [116] [117] . in the largest study to date, tsalik et al. used gene expression in blood to discriminate bacterial from viral infection or non-infectious illness in 273 subjects with respiratory illness [118] . these investigators defined 130 predictor genes in a model with an accuracy of 87% to discriminate clinically adjudicated bacterial, viral, and non-infectious illness. most studies to date have used micro array but recently rnaseq has been used to differentiate viral and bacterial respiratory illness and in one study 141 genes were noted to be differentially expressed [119] . three pathways (lymphocyte, α-linoleic acid metabolism, igf regulation pathways) which included 11 genes as predictors for bacterial infection from non-bacterial infection (naïve auc = 0.94; nested cv-auc = 0.86). to date, a number of gene expression studies of adults and children have developed predictors with similar accuracy (auc ranging from 78% to 94%), yet there has been little overlap in classifying genes identified [105, 106, [114] [115] [116] [118] [119] [120] [121] [122] . diverse populations, types of infection, plus alternate analytic tools used, likely explain the different genes identified. more work needs to be done to refine predictive gene sets including patients with mixed viral-bacterial respiratory tract infection. most studies to date have focused on blood; however, analysis of the nasal respiratory epithelium which is the site of infection might offer advantages. although data are limited, several recent papers demonstrate that nasopharyngeal host response can also be used as a diagnostic for respiratory viruses [93, 123, 124] . immune response to influenza vaccine is variable and influenced by a variety of factors including prior vaccinations and infections, age, the presence of underlying conditions and the type of vaccine administered. yet, even among a relatively homogeneous cohort of young healthy adults, antibody responses to vaccine can be variable [125] . transcriptional profiling of whole blood provide insights into the mechanisms of variability, the effects of age, and vaccine types. the ability to predict vaccine response at baseline based on a transcriptomic signature would have significant clinical implications. to understand the biologic effects of live attenuated influenza vaccine (laiv) compared to trivalent inactivated vaccine (tiv) blood transcription profiles from 85 young children were assessed by microarray at day 7 post vaccination [126] . many more genes were differentially expressed in children receiving laiv compared to tiv (245 vs. 49, respectively) and many modulated type 1 ifn. the efficacy of laiv has been problematic in recent years and assessing stimulation of type 1 ifn genes could represent a potential biomarker for response to laiv [126] . bucasas and colleagues evaluated gene expression at multiple time points after vaccination of healthy young men with tiv [127] . they noted marked up regulation of gene expression of ifn signaling, il-6 regulation, antigen processing and presentation genes within 24 h of vaccination and were able to define a 494 gene expression signature that correlated with the magnitude of antibody response. in another study, a gene profile predictive of antibody response 28 days after influenza vaccination of young and older adults was developed [128] . notably, the predictive genes were the same in young and old as well as a subgroup of subjects with diabetes suggesting similar pathways were involved despite differences in age and underlying medical conditions. additionally, transcriptional profiling has been used to signatures in blood associated with b cell memory responses to vaccination. in a study of 150 older and middle aged adults vaccinated with tiv including an h1n12009 antigen, metabolic, cell migration/adhesion, map kinase and nf-ᵏb cell genes correlated with peak memory b cell elispots [129] . finally, in a study of over 500 subjects vaccinated over several seasons, a predictive signature of nine genes and three gene modules were significantly associated with the magnitude of the serum antibody response (fig. 4 ) [130] . interestingly and in contrast to a previous study, the signature was distinct to the younger cohort. for example, inflammatory genes were associated with better response in the young but a worse response in the elderly. in summary, gene expression studies could be used to evaluate new vaccines and develop predictors of vaccine response in different subgroups of patients based on age and disease state allowing for individualized vaccine regimens. molecular analysis of respiratory viruses and the host response to both infection and vaccination have transformed our understanding of these ubiquitous pathogens. the ability to accurately diagnosis viral infections has not only impacted patient care but also changed our perceptions of the burden of disease and populations effected. transcriptional profiling of blood and nasal epithelium may provide therapeutic targets to prevent and ameliorate illness as well as offer predictors of severe disease. 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influenza vaccines in young children early patterns of gene expression correlate with the humoral immune response to influenza vaccination in humans systems biology of vaccination for seasonal influenza in humans transcriptional signatures of influenza a/h1n1-specific igg memory-like b cell response in older individuals multicohort analysis reveals baseline transcriptional predictors of influenza vaccination responses key: cord-349821-5ykwwq75 authors: ippolito, g.; puro, v.; heptonstall, j. title: biological weapons: hospital preparedness to bioterrorism and other infectious disease emergencies date: 2006-09-09 journal: cell mol life sci doi: 10.1007/s00018-006-6309-y sha: doc_id: 349821 cord_uid: 5ykwwq75 in the last 2 decades, successive outbreaks caused by new, newly recognised and resurgent pathogens, and the risk that high-consequence pathogens might be used as bioterrorism agents amply demonstrated the need to enhance capacity in clinical and public health management of highly infectious diseases. in this article we review these recent and current threats to public health, whether naturally occurring or caused by accidental or intentional release. moreover, we discuss some components of hospital preparedness for, and response to, infectious disease of the emergencies in developed countries. the issues of clinical awareness and education, initial investigation and management, surge capacity, communication, and caring for staff and others affected by the emergency are discussed. we also emphasise the importance of improving the everyday practice of infection control by healthcare professionals. the global eradication of smallpox, arguably the greatest international public health achievement of the twentieth century, was certified in 1980 at a time of almost untrammelled optimism that the fight against infectious diseases had been won. in the following 2 decades, successive outbreaks of infectious diseases caused by new, newly recognised and resurgent pathogens -which have been described as a series of 'perfect microbial storms' -amply demonstrated that such optimism was misplaced, and that, far from winding down capacity in clinical and public health management of highly infectious diseases, it was necessary to enhance it [1, 2] . the risk that highconsequence pathogens, including smallpox (variola) virus, might be used as biological weapons or bioterrorism agents had been recognised, and policy makers and planners were encouraged to ensure that that health and other services were adequately prepared to deal with the threat, even before the attacks on the world trade center and the pentagon in september 2001 [3, 4] . these attacks, coupled with the deliberate release of letters containing anthrax spores via the us postal service a month later [5] , showed that the threat was real, and that work to improve preparedness and response was urgently needed at local, national, and international levels. in this article we review recent and current threats to public health in developed countries from bioterrorism and other highly infectious diseases, and discuss some of the components of hospital preparedness for, and response to, infectious disease emergencies. we also emphasise the importance of improving the every-day practice of infection control by healthcare professionals and of taking a generic, 'all-hazards' approach to hospital preparedness, integrating planning for response to infectious disease emergencies, whether naturally occurring or caused by accidental or intentional release, with planning for major incidents and natural disasters. the concepts used in developing laboratory biosafety guidelines forms the basis for categorisation of biological agents by risk group and the designation of appropriate biosafety levels. these concepts rely on expert risk assessment of the severity of human infection, the potential for transmission to exposed individuals and to the wider community or environment, and the availability of effective prophylaxis or treatment. additional assessments include likely ease of dissemination by terrorists and the estimated overall impact of any dissemination to generate lists of 'critical agents' [6, 7] . the lists then rank the biological agents that might be used in deliberate release by priority, and identify measures needed to ensure public health preparedness. critical agent lists are also being used to set priorities in biodefence research, including basic research on biology and pathogenesis, and development and evaluation of molecular diagnostic assays, vaccines, antivirals, and other preventive or therapeutic interventions [8] . although critical agent lists generally make provision for inclusion of newly recognised or recently emergent pathogens, they were developed specifically to improve preparedness for bioterror events. so though all categorise smallpox virus as a high-priority, 'category a pathogen', none accords high-priority to highly pathogenic influenza viruses, or severe acute respiratory syndrome (sars) coronavirus, despite the fact that these viruses are epidemic prone, and capable of rapid global spread and enormous public health impact. the term 'highly infectious diseases' describes infections caused by pathogens that are transmissible from person to person, cause severe or life-threatening illness; present a serious hazard in healthcare settings and in the community; and require specific control measures, which may include management of cases in a highly secure isolation unit. it thus includes some, but not all, of the infections caused by category a critical agents (including smallpox, lassa, marburg, ebola and congo crimean haemorrhagic fevers, and pneumonic plague, but excluding anthrax, bubonic plague, tularaemia and botulism because these are not transmissible from person to person), and also includes sars, influenza caused by avian influenza virus h5n1 or other highly pathogenic influenza virus, and unusual illness of unknown, but possibly infectious, aetiology. incidents caused by the intentional release of pathogens are rare: in the last 50 years, five such incidents have been recognised and reported. three of the first four incidents involved gastrointestinal pathogens (salmonella typhi, salmonella enteritidis, shigella dysenteriae); in the fourth a group of students developed asthma and pulmonary eosinophilia after being fed ascaris suum [9] [10] [11] [12] . in the largest of the outbreaks of gastrointestinal infection, over 700 people developed symptoms after eating from salad bars in two restaurants in dalles, oregon, in 1984. this was a 'covert' deliberate release: unannounced, without any warning or indication of the organism involved, or of the population affected, and it was not until late in 1985 that it was discovered that followers of baghwan shree rajneesh had deliberately contaminated the salad bars with cultures of s. enteritidis, nicely illustrating that intentional and naturally occurring outbreaks may be indistinguishable. in the united states, the intentional dissemination of anthrax through the postal service in 2001 led to 22 cases of anthrax (11 pulmonary, 11 cutaneous) among residents of seven states along the eastern seaboard. five of the pulmonary infections were fatal [13] [14] [15] [16] [17] [18] [19] . this was an example of an 'overt deliberate release', insofar as explicitly threatening notes were enclosed in the envelopes containing the anthrax spores, although the diagnosis of the first cases preceded recognition of the risk. most (18) cases occurred in postal service employees or employees of the media companies targeted in the attacks; environmental sampling detected widespread anthrax contamination of the postal system. anthrax, along with all other infections caused by category a pathogens, is uncommon in developed countries, and lack of familiarity with the disease, coupled with failure to include it in the differential diagnosis of an unusual skin lesion or of sudden onset of serious sepsis and respiratory failure led to delays in diagnosis: the median time from onset to diagnosis of the cases of cutaneous anthrax in the first cluster was 10 days [20] . many of these had already occurred by the time that the index case of pulmonary anthrax was diagnosed in florida by an alert clinician who had recently undergone bioterrorism preparedness training. although the number of cases was small, the overall impact of the incident on an already stretched public health system was considerable. in new york alone, over 700 'suspect' cases, identified as a result of intensive case finding in hospitals and through clinician networks, or by self-referral by calls to telephone hotlines, required clinical assessment. all those who had potentially been exposed to anthrax required assessment for prophylaxis; completion of a 60-day course of post-exposure antibiotics was recommended for over 10,000 persons [21] . laboratories within the laboratory response network tested over 125,000 clinical specimens. the incident highlighted the need for coordination and clear command structures at local and national levels; for stronger linkages between clinicians, clinical microbiologists, hospitals and public health departments; for information, communications and laboratory systems with inbuilt 'redundancy', readily capable of expansion to meet surges in requirements, and for coordinated and effective communication with clinicians, the media and the public [22] . in the oregon outbreak, the organism had been obtained from a commercial source; in two of the three other outbreaks caused by gastrointestinal pathogens, the per-cell. mol. life sci. vol. 63, 2006 multi-author review article 2215 petrators, a bacteriologist and a laboratory worker, had access to organisms from their laboratories. the source of the b. anthracis used in the united states in 2001 remains uncertain. recent changes intended to strengthen containment of critical agents within laboratories include more stringent regulation of work on, and transfer of, high-consequence pathogens, and updated guidance that recommends that all clinical, diagnostic and research laboratories develop threat and risk assessment based site-specific biosecurity plans covering personnel selection, access and inventory control, specimen accountability, reporting of incidents, injuries and breaches, and response to an emergency [23] [24] [25] . the epidemic of sars in 2002-2003, with over 8000 cases in 29 countries, illustrated how a new infection can, given the speed and reach of international air travel, spread globally within weeks [26] . transmission was amplified within hospitals, as early cases were cared for without effective infection control measures; 22% of sars cases in hong kong and nearly half (43%) of sars cases in toronto and singapore (41%) occurred in healthcare workers. overall, 20% of hospitalised patients required mechanical ventilation, and 15% of hospitalised cases died. sars coronavirus, although a newly emergent virus, was transmitted in the same way as more common respiratory infections, mainly by respiratory droplet spread, and the sars epidemic was controlled by the efficient application of long-recognised public health control measures: rapid identification and early isolation of cases, and stringent adherence to infection control precautions. in canada, where sars 'paralysed the greater toronto area healthcare system for weeks' [27] , and the toronto public health department investigated 2132 potential cases of sars, identified over 23,000 contacts as requiring quarantine and logged more than 316,000 calls on its sars hotline [28] , a national review commission identified systemic deficiencies in response capacity, including 'inadequacies in institutional outbreak management protocols, infection control and infectious disease surveillance', and found that these deficiencies resulted at least in part from failure to implement lessons learned from earlier public health emergencies [22] . global travel and global trade expose industrialised countries to other infectious disease threats. human monkeypox is a zoonosis, normally geographically confined to west and central africa, which is clinically similar to smallpox in that a vesiculopustular rash follows a febrile prodromal illness. the illness tends to be milder than smallpox; in contrast with smallpox, lymphadenopathy is often a prominent feature, and person-toperson transmission is uncommon. in 2003, the first clus-ter of human cases (37 confirmed, 72 suspected) of community-acquired monkeypox in the western hemisphere occurred in the united states [29, 30] . infection followed exposure to infected pet prairie dogs that had been housed or transported with african rodents imported from ghana. although pox virions were seen on electron microscopy of clinical samples from the index case, the diagnosis of smallpox was excluded because the development of pocks in the case followed, and was localised to, the site of a bite by a sick pet prairie dog. the diagnosis of monkeypox was made by specialist testing of referred samples in the national laboratory. this incident again highlighted the role of the astute clinician in outbreak recognition; the value of maintaining close working relationships between clinicians working in healthcare facilities and in public health departments, and the need for multi-level, multi-agency cooperation, including collaboration between animal and human health experts, in outbreak management. in 2005, an outbreak of marburg haemorrhagic fever in angola, and the potential for exported cases prompted the rapid development of national guidelines for risk assessment and case management in countries that had not previously published such guidelines [31, 32] . marburg viral haemorrhagic fever, and ebola, congo-crimean, and lassa haemorrhagic fevers are of particular concern in healthcare settings because there is a high risk of person-to-person transmission through percutaneous or mucocutaneous exposure to blood. lassa fever is endemic in west africa, where estimates suggest that around 300,000 cases occur each year [33] ; congo-crimean haemorrhagic fever has a wide geographic range that includes greece, albania and pakistan; and outbreaks of the more geographically restricted ebola and marburg haemorrhagic fevers have recently occurred with apparently increasing frequency [34, 35] . despite this, imported cases of viral haemorrhagic fever are uncommon: 5 laboratory confirmed cases of lassa fever (likely to be the most frequent importation) have been reported from the united states, and fewer than 20 from other industrialised countries since the disease was recognised in 1969 [36] [37] [38] [39] . this is perhaps because transmission and outbreaks of haemorrhagic fever viruses occur mostly in rural areas, which thus limits the opportunities of most business travellers or tourists for exposure. nevertheless, and because there have been reports of weaponisation of marburg, ebola and lassa viruses [40] , all of which are category a pathogens, clinicians should remain alert to the possibility of these infections, maintain an awareness of current outbreaks, and should know how to conduct a risk assessment of febrile illness compatible with a diagnosis of viral haemorrhagic fever, how to safely undertake initial management and apply appropriate infection control measures, and, most important, know whom to contact for further advice on diagnosis and further management [33, 41, 42] . infections emergencies and hospital preparedness one of the consequences of the resurgence in biodefence-related research is that more laboratories, and more laboratory workers, are now working with category a pathogens, which increases the potential for occupational exposure, for occupationally acquired infection and, for some pathogens, for onward transmission to others. laboratory workers may also be exposed to high-consequence or newly emerging pathogens whilst working on diagnostic or surveillance-related samples. since 2000, cases of laboratory acquired glanders (1 case, us military research laboratory, the first case reported in the united states since 1945) [43] ; the wr strain of vaccinia (1 case, research laboratory, brazil) [44] ; recombinant vaccinia virus (4 cases, research laboratories in germany, united kingdom, canada, and united states) [45] [46] [47] [48] , tularaemia (3 cases, us research laboratory) [49] ; sars (4 cases, in research laboratories in taiwan, singapore, and china; all occurred after the end of the sars epidemic, infection spread from the 2 laboratory workers in china to a further 7 people, 1 of whom died) [50] [51] [52] ; ebola viral haemorrhagic fever (1 fatal case, russia, research laboratory) [53]; anthrax (1 cutaneous case, us laboratory) [54] ; brucellosis (2 linked cases, us clinical microbiology laboratory) [55] ; and west nile virus (2 cases, us laboratories) [56] have been reported. in several of these cases, diagnosis was delayed because the possibility of occupationally acquired infection was not considered, and/or because of difficulties in identifying the organism in the clinical microbiology laboratory. in only 5 of these 19 cases was the exposure that led to infection identifiable. guidelines on laboratory biosafety advise that laboratory workers should have access to expert occupational health advice, including, where appropriate, pre-exposure prophylaxis, and that those working in bsl3 or bsl4 facilities should carry 'medical contact cards' [23] . clinicians should take an occupational history as a routine, and, if a laboratory or animal house worker presents with an unexplained febrile illness, a senior clinician should obtain further information from the laboratory director about the agents to which the patient may have been exposed, regardless of whether the worker can recall a specific exposure. since 1997, a new, highly pathogenic strain of avian influenza virus, a/h5n1, has emerged, initially in se asia, but with more recent spread to countries in europe, the middle east, central asia, and africa. the first human cases were reported from vietnam in 2003; to date (may 2006), 206 laboratory-confirmed cases, including 115 deaths (case fatality rate 56%) have been reported to the world health organisation from 10 countries (azerbaijan, cambodia, china, djibouti, egypt, indonesia, iraq, thailand and turkey) [57] . there is limited evidence of human-to-human transmission of the virus [58] ; most cases have followed close contact with infected birds (of-ten from household or 'backyard' flocks) or their faeces, other body fluids or carcasses. it is not known whether, and if so, when, how or where, influenza virus a/h5n1 will evolve to become more easily transmissible between humans. nor is it known whether an increase in transmissibility would be accompanied by a change in lethality. however, the world health organisation believes that the threat of pandemic human influenza is now greater than at any time since 1968, when the last influenza pandemic occurred [59] . the world health organisation uses a series of six alert levels to inform the world of the seriousness of the threat, and to recommend progressively more intense preparedness activities. at present (pre-pandemic threat level 3) [59] , clinicians need to be aware of the potential for infection in travellers returning from affected countries, and in those who may have had occupational (e.g. poultry farmers, veterinarians, animal cullers) or other contact with infected domestic, commercially farmed or wild birds, a human case or virus in the laboratory. advice, algorithms and response protocols for investigation and management of possible cases or case clusters have been published, and give details of reporting mechanisms, diagnostic specimens, infection control measures and other containment responses [60] [61] [62] . assessments of preparedness plans in europe and the united states suggest that, at best, most countries remain only moderately prepared for pandemic influenza; furthermore, the degree to which planning at the national level has been translated into increased preparedness at the local level within healthcare facilities remains unknown [63] [64] [65] . it would be prudent, however, for planners within hospitals to review existing influenza pandemic contingency plans in conjunction with the relevant national preparedness plan, with the aim of ensuring preparedness to provide supportive medical care for influenza cases, prevent transmission of infection and at the same time continue to provide essential medical services to their community. where concerns arise about issues (e.g. criteria for hospital admission; prioritisation of antiviral use; prioritisation of admission to intensive care units; responsibility for decisions to defer elective surgical admissions; sourcing of additional supplies e.g. of personal protective equipment; use of volunteer personnel) that are not clearly dealt with within the national plan, urgent clarification should be sought from the relevant national authority. the overall aim of hospital preparedness for an infectious disease emergency is to be able to provide adequate medical care to those affected whilst at the same time continuing to provide essential medical services to the community. cell. mol. life sci. vol. 63, 2006 multi-author review article 2217 the phases of the traditional disaster management cycle (preparation, response, recovery and mitigation) are paralleled in infectious disease emergency management by preparedness (activities undertaken before an event, including planning, training, and undertaking practice drills and exercises to test the plans); surveillance and detection (recognising that an infectious disease emergency is occurring); and response, control and containment (the clinical, public health and other measures that minimise the health, social and economic consequences of the incident). effective preparedness planning requires a multidisciplinary approach, involving emergency planners, clinical practitioners, laboratorians, managers and administrators, emergency responders, pharmacists, voluntary agencies, mental health and occupational health services, religious and spiritual advisors, support staff including catering, housekeeping, portering and security, medical records, communications, information technology and transport/ courier services; with clear, pre-event designation of roles and responsibilities and clear chains of command, control and communication, and regular testing and evaluation of 'major incident' or emergency operations' plans by drills and exercises. hospital preparedness for infectious disease emergencies needs to be sufficiently versatile to encompass response to incidents that range from those of high/moderate probability-low/moderate consequence (e.g. a local, but severe point-source outbreak of norovirus infection), through low probability-moderate consequence (e.g. managing a single imported case of viral haemorrhagic fever or a hospital-associated outbreak of legionellosis), to low probability-high consequence (e.g. pandemic influenza; bioterrorist attack). early diagnosis and prompt institution of effective control measures are critical determinants of the eventual impact of any infectious disease emergency [66] . nine of the 11 cases of pulmonary anthrax in the us outbreak in 2001 presented direct to hospitals or emergency rooms. these clinicians are also likely to be the first to see cases of newly emergent highly pathogenic influenza, re-emergent sars or imported viral haemorrhagic fever. clinicians need, therefore, to maintain their awareness of current infectious disease threats by daily review of national, regional and international web-based alerting systems, or by ensuring that their department receives national cascade alerts, and to incorporate relevant epidemiological information into their daily practice (e.g. by using knowledge of areas currently affected by avian influenza h5n1 coupled with travel and occupational histories to exclude the diagnosis in patients with febrile respiratory illness). useful and reliable open-access sources of medical intelligence include the web sites developed by the infectious diseases society of america (promed; http://www. promedmail.org) [67] , the world health organization (http://www.who.int/csr/don/en/) [68] ; further links to additional sources can be found at http://www.ecdc.eu.int/. all clinicians must remain open to the possibility that they may be the first person to recognise a deliberate release or other infectious disease emergency; must be prepared to consult urgently with their local infectious disease specialist, clinical microbiologist and public health department on suspicion alone, without waiting for a definitive diagnosis, and must remain alert to the unusual, the unexpected and the case that 'just doesn't fit'. examples of the unusual include unusual illness (e.g. a sudden, unexplained febrile death, critical illness or pneumonia in a previously healthy adult); an unusual number of patients with the same symptoms presenting within a short time frame; illness unusual for the time of year (e.g. 'flu in summer'); an illness unusual for the patient's age group (e.g. chicken pox in a middle-aged adult); illness in an unusual patient (e.g. cutaneous anthrax in a patient with no history of contact with animals, animal hides or products); an illness acquired in an unusual place (e.g. tularaemia acquired in the united kingdom); unusual clinical signs (e.g. mediastinal widening on chest x-ray; symmetrical flaccid paralysis of sudden onset; 'chickenpox' rash predominantly on the extremities); and unusual progression of illness (e.g. lack of response to usually effective antibiotics) [69] . most of the illnesses caused by high-consequence pathogens are uncommon in industrialised countries (though some e.g. plague, anthrax remain endemic elsewhere) so few clinicians have direct experience of them; similarly, few of those now practising have ever seen a case of smallpox. considerable resources have therefore been invested since 2001 in training clinicians and emergency responders to recognise illnesses caused by these pathogens, and in developing web-based and other training materials, guidelines, fact sheets, and incident response check lists for health care and emergency response professionals. these can be found on, or through, national authorities' web sites (e.g. http://www.bt.cdc.gov; http://www.hpa. org.uk) and used, where formal face-to-face training programmes are not accessible, as the basis for self-directed learning. decision-based algorithms for diagnosis and clinical management pathways have also been developed, and can be used to guide initial responses to suspected cases of smallpox, sars, viral haemorrhagic fever or avian influenza [62, [69] [70] [71] [72] . effective infection control saves lives. all healthcare workers have a responsibility to ensure that their clinical infections emergencies and hospital preparedness practice prevents transmission of infection, and puts neither their own health, nor that of their patients, coworkers or others at risk. all healthcare workers should be trained in standard and transmission-based infection control precautions at induction [73] . overall standards might be improved if an annual demonstration of competence was made a requirement for re-accreditation, and if a more stringent approach was taken to any recognised breach of infection control practice. infection control guidelines, updated after the sars epidemic, now stress the importance of incorporating 'respiratory hygiene' or 'cough etiquette' -simple measures designed to prevent transmission of respiratory infections -into standard infection control precautions [74, 75] . this is particularly applicable to emergency departments, outpatient clinics and day-care centres -and includes training staff to identify and segregate or spatially separate patients with signs and symptoms of respiratory tract infection from others; offering a surgical mask to symptomatic patients; instructing all patients to cover their nose and mouth with tissues when coughing or sneezing, to dispose of used tissues safely and to clean their hands frequently, and providing tissues and tissue-disposal and hand-cleaning facilities for patients [74, 75] . emergency departments should review their existing infection control practices and consider whether these are adequate to prevent intra-hospital transmission of infection, from the moment that a patient with an unrecognised but highly infectious disease arrives in the department, through the initial evaluation and investigation, to the point when the patient is admitted or transferred elsewhere. this should include identifying a space (ideally a negative pressure room) suitable for airborne infection and respiratory isolation and ensuring that staff understand when and how it should be used; review of available personal protective equipment (ppe: gloves, gowns, face and eye protection, surgical masks or other respiratory protection e.g. n95-type respirators), hand-cleaning facilities, and sharps safety and disposal arrangements; assessment of staff competency in choosing, and safely using, removing and disposing of the ppe available, ensuring that, if n95-type respirators are to be used, staff have been fit-tested and know how and when to perform fit checks; reinforcement of the importance of hand hygiene; arrangements for cleaning and ensuring environmental hygiene; and setting triggers for notifying the infection control team and public health department, and for seeking further expert advice. departmental competency can and should be tested by regular drills and simulation exercises. infection control planning for infectious disease emergencies should also consider the number of isolation or single rooms available, and determine when, where and how cases posing a risk of transmission to others could be cohort-nursed once the supply of single rooms is exhausted. the aim of the initial investigation and management of a patient suspected of having a highly infectious disease, or a patient who presents with an unusual, and possibly highly transmissible, illness is to provide life-sustaining medical care to the patient whilst ensuring staff safety. this implies placing the patient in a side room, limiting the number of staff exposed to the patient to the minimum necessary, evaluation of the patient by a senior clinician, using appropriate personal protective equipment during the evaluation (gloves, gown, face and eye protection, surgical mask or n95-type respirator for staff, surgical mask for patient), and urgently seeking expert advice about management and diagnostic testing before taking diagnostic samples. expert advice should also be sought on the desirability and mechanism of transfer of the patient to a highly secure infectious disease unit. detailed, disease-specific national guidance on the management and investigation of highly infectious diseases has been produced by many countries, and can usually be found on the website of the relevant national authority [32] ; planners should download this guidance, incorporate relevant points (e.g. contact details for national or regional laboratories that will undertake specialised laboratory diagnostic testing; smallpox response team) into emergency plans, and designate the task of ensuring that the locally available version is up-to-date to a specific jobholder. laboratorians should be involved in planning, and protocols for the safe collection, transport and external referral of clinical specimens should be available, and should comply with international transport regulations and international and national guidance on biosafety and biocontainment. robust systems for information management and specimen tracking should also be in place pre-event. if the event is linked to deliberate release or criminal action, or there are other forensic considerations, chain of custody (sometimes called 'chain of evidence') documentation of samples, and close liaison with the police or security forces will be required. surge capacity is the ability to expand healthcare provisions to respond to an increased number of patients that exceeds usual capacity, including the provision of specialised or unusual medical care (e.g. paediatric care; intensive care requiring mechanical ventilation; haemodialysis or haemofiltration). this is sometimes split into 'surge' capacity -the expansion of healthcare provision whilst retaining near-normal care standards, and 'supersurge' capacity -further expansion, requiring the use of alternative care facilities (e.g. schools, church halls) and/ cell. mol. life sci. vol. 63, 2006 multi-author review article 2219 or significant changes in standards of care, sometimes referred to as 'planned degradation of care' [65] . infectious disease emergencies may range from providing care to a single, seriously ill, highly infectious patient, to providing care to a community affected by a bioterror event or pandemic influenza. surge requirements might include not only the ability to increase bed and personnel capacity to cope with an increased number of acute admissions, but also, for example, the ability to manage an increased number of laboratory samples, increased clinical waste for disposal, increased communication and information technology requirements (e.g. networked computers, cell phones, telephone lines connected to automatic routing systems), and increased requirements for supplies of ppe. planners need also to consider, and formalise, with nearby healthcare providers, arrangements for collaborative working and mutual aid, and to consider also how essential functions (e.g. providing ventilatory support; communications) might be maintained if utility supplies (e.g. electricity, fuel) were compromised [76, 77] . modelling estimates for pandemic influenza in the united states suggest that, although many patients could be cared for at home, if the pandemic was severe, and numbers affected paralleled those of the 1918 pandemic, at the peak, hospitals would need 191% of available non-intensive care beds; 461% of available intensive care beds; and 198% of available ventilators for influenza alone [65, 78] . thus, for influenza, the ability to provide intensive care might well be the rate-limiting step in surge capacity, and criteria for admission to intensive care and for continuation of intensive support might need to be different from those that would normally be used. this raises complex ethical and legal questions, which are best thought through, preferably at the national level, in advance of, rather than during, the event. effective communication is essential to management of infectious disease emergencies, and a communication plan is an integral part of any emergency management plan. 'communication' is a broad term that encompasses provision of accurate, timely, complete, easily understood information to the community about what the emergency is, what is being done to control it and what people can do to make themselves safer; provision of information to healthcare professionals about diagnosis, investigation, and pre-or post-exposure interventions; communication with families and others close to those affected by the emergency; communication with the media; and communication within and between all levels of all those involved in managing the emergency. recent international guidelines on risk communication, and on communicating with the media and the public during an infectious disease emergency provide greater detail, and highlight the importance of communicating in ways that build or maintain trust, of planning and testing outbreak communications strategies, and of providing media communications training for all public officials as part of professional development [79, 80] . occupational health services should be involved in hospital preparedness planning. this involvement will help to ensure that staff are as well protected before the event as is possible (e.g. by ensuring uptake of seasonal influenza vaccine, pneumococcal vaccine and hepatitis b vaccine by all those who are eligible under existing national policies, and of vaccines specifically relevant to laboratory staff). occupational health services should also participate in development of systems for surveillance of infection in health care workers, which are needed both pre-event, as a means of detecting that an infectious disease emergency is occurring [81] , and during an event, to monitor the outcome for potentially exposed workers, and of infection specific protocols for post-exposure management [32, 82] . any traumatic incident, emergency or disaster, whether natural or man-made, has a psychological impact on those involved -survivors, the bereaved, witnesses, rescuers, responders and health professionals, and their families, relatives, friends and workmates. planning should ensure that, whatever the emergency, staffing levels will be sufficient for time on duty to be limited to no more than 12 h a day, and should make provision for staff rotation from highly taxing to less taxing functions. staff will need somewhere quiet, safe and private 'off-scene' to eat, drink and rest without interruption, and facilities will also need to be such that staff are able to stay in touch with friends and family. staff should also be made aware of other sources of support (e.g. their family doctor, hospital chaplain, and other religious and spiritual advisors), and should be provided with details of how to contact confidential listening or counselling services [69, 83] . it is impossible to predict when, where or how another deliberate release of a biological agent will occur, and equally impossible to predict which emergent infection will next threaten global public health, or whether the influenza pandemic will occur this year, next or in some years time. it is, however, possible to predict that infectious disease emergencies will continue to occur 2220 g. ippolito, v. puro and j. heptonstall infections emergencies and hospital preparedness with regularity, and it is possible, with appropriate planning, to be prepared to meet them in a way that ensures that they cause as little social disruption as possible. the greatest danger is complacency -the belief that 'it cannot happen', or that ' it could happen, but it will happen somewhere else'. it is not clear to what extent the efforts made at the international and national level and the long lists of lessons learned have translated into improved and sustainable hospital preparedness at the local level; we hope this article will provoke you to prepare, plan and practice, now. microbial threats to health: emergence, detection, and response the challenge of emerging and re-emerging infectious diseases 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guidelines for laboratory exposures to agents of bioterrorism emergency preparedness and response. disaster mental health resources acknowledgement. this work was performed within european commission project eunid, and within ricerca finalizzata and ricerca corrente irccs projects, italian ministry of health. key: cord-354492-6r6qs4pp authors: messina, giovanni; polito, rita; monda, vincenzo; cipolloni, luigi; di nunno, nunzio; di mizio, giulio; murabito, paolo; carotenuto, marco; messina, antonietta; pisanelli, daniela; valenzano, anna; cibelli, giuseppe; scarinci, alessia; monda, marcellino; sessa, francesco title: functional role of dietary intervention to improve the outcome of covid-19: a hypothesis of work date: 2020-04-28 journal: int j mol sci doi: 10.3390/ijms21093104 sha: doc_id: 354492 cord_uid: 6r6qs4pp background: on the 31 december 2019, the world health organization (who) was informed of a cluster of cases of pneumonia of unknown origin detected in wuhan city, hubei province, china. the infection spread first in china and then in the rest of the world, and on the 11th of march, the who declared that covid-19 was a pandemic. taking into consideration the mortality rate of covid-19, about 5–7%, and the percentage of positive patients admitted to intensive care units being 9–11%, it should be mandatory to consider and take all necessary measures to contain the covid-19 infection. moreover, given the recent evidence in different hospitals suggesting il-6 and tnf-α inhibitor drugs as a possible therapy for covid-19, we aimed to highlight that a dietary intervention could be useful to prevent the infection and/or to ameliorate the outcomes during therapy. considering that the covid-19 infection can generate a mild or highly acute respiratory syndrome with a consequent release of pro-inflammatory cytokines, including il-6 and tnf-α, a dietary regimen modification in order to improve the levels of adiponectin could be very useful both to prevent the infection and to take care of patients, improving their outcomes. month later (on 8 january 2020), the chinese authorities declared the identification of a new type of coronavirus, informing the who a few days later that the outbreak was associated with exposure in a seafood market in wuhan city. the infection spread firstly in china and then in the rest of the world, and on the 11th of march, the who declared that covid-19 was a pandemic. coronaviruses (covs) belong to the subfamily orthocoronavirinae in the family of coronaviridae in the order nidovirales, and this subfamily includes α-coronavirus, β-coronavirus, γ-coronavirus, and delta-coronavirus [1] . coronaviruses primarily cause enzootic infections in birds and mammals and, in the last few decades, have shown to be capable of infecting humans as well [2] . in human infections with highly virulent respiratory viruses-such as avian influenza h5n1, h7n9, severe acute respiratory syndrome (sars) coronavirus, and coronavirus disease-19 (covid-19)-immunopathogenesis caused by the overproduction of pro-inflammatory cytokines may play an essential role in disease progression and mortality [3] . several recent studies have reported that covid-19 caused the destruction of the pulmonary parenchyma, including interstitial inflammation and extensive consolidation, similarly to the previously reported coronavirus infection [4, 5] . during coronavirus infection, it was observed that the lungs increased in weight, with a mild pleural effusion of clear serous fluid, named pulmonary edema, and extensive consolidation [6, 7] . in some areas, there was interstitial thickening, with mild-to-moderate fibrosis, but a disproportionately sparse infiltrate of inflammatory cells (mainly histiocytes, including multinucleated forms, and lymphocytes) [8] . a dilatation of the airspaces was observed, as was focal honeycombing fibrosis. an intra-alveolar organization of exudates was described, and the formation of granulation tissues in the small airways and airspaces was reported. these lesions were typically located in the sub-pleural region, and the cellular component mainly consisted of histiocytes, as reported in a previous paper [9] . xu et al. described in their case report the pathological findings of covid-19 associated with acute respiratory distress syndrome. at the x-ray investigation, they detected a rapid progression of bilateral pneumonia. the biopsy samples were taken from the lung; the histological examination showed bilateral diffuse alveolar damage with cellular fibromyxoid exudates [6] . considering that the mortality rate of covid-19, about 5-7% [10] , and the percentage of positive patients admitted to intensive care units being 9-11% [11] , it should be mandatory to consider and take all necessary measures intended to contain the viral infection. a recent study analyzed the data of 150 covid-19 patients, with the aim of defining the clinical predictors of mortality. the results obtained from this study suggest that covid-19 mortality might be due to virus-activated "cytokine storm syndrome", considering that the plasma levels of il-6 were higher in deceased patients compared to in discharged subjects [12] . considering that a detailed study has not been performed on the immunological response to covid-19, the only way to discuss this thematic is to refer to previous knowledge about sars-cov and mers-cov. the first response is obtained through pattern recognition receptors (prrs) including c-type lectin-like receptors, toll-like receptors (tlr), nod-like receptors (nlr), and rig-i-like receptors (rlr). moreover, several inflammatory factors are expressed such as il-6 and tnf-α; moreover, the synthesis of type i interferons (ifns) is activated, and these exert their actions against virus diffusion, accelerating macrophage phagocytosis [13] (figure 1 ). in the light of these considerations and the recent evidence in different hospitals suggesting il-6 and tnf-α inhibitor drugs as a possible therapy for covid-19, this review aims to highlight how a dietary intervention could be useful to prevent the infection and/or to ameliorate the outcome during therapy. the first laboratory report about covid-19 patients indicated several parameters that were found to be altered in blood samples; for example, d-dimer, neutrophil count, blood urea, and creatinine levels were significantly higher. in the same way, several cytokines such as il-6 and tnfα were overexpressed, indicating the immune status of the patients [14] . il-6 represents pro-inflammatory signaling produced by adipose tissue; for this reason, this endocrine cytokine could be important in regulating the host response during acute infection [15] . several papers have described the essential role of il-6 in generating a proper immune response during different kinds of viral infection in the pulmonary tract. others link this cytokine to an exacerbation of viral disease. these latter findings support the hypothesis that il-6 upregulation during viral infections may promote virus survival and the exacerbation of the clinical disease [16, 17] . indeed, il-6 has a pleiotropic function, and it is produced in response to tissue damage and infection. in particular, at the pulmonary level, innate and adaptative immune cell proliferation is strongly influenced by this cytokine. after targeting its specific receptor, il-6 starts a cascade of signaling events mainly associated with the jak/stat3 activation pathway, promoting the transcription of multiple downstream genes related to cellular signaling processes, including cytokines, receptors, adaptor proteins, and protein kinase [15] . furthermore, it has been reported that il-6 is an essential factor for the survival of mice with a viral infection. this cytokine promotes the optimal regulation of the t-cell response, inflammatory resolution, tissue remodeling promoting lung repair, cell migration, and the phagocytic activities of macrophages, as well as preventing virus-induced apoptosis in lung epithelial cells. however, experimental scientific evidence also suggests potential adverse consequences that increased levels of il-6 might have on the cellular immune response against viruses. in this context, different possible mechanisms involving this cytokine might affect viral clearance, ultimately favoring the establishment of a persistent viral state in infected hosts [18, 19] . tumor necrosis factor is a cell-signaling protein (cytokine) involved in systemic inflammation, released predominately from macrophages, but it is also released from a variety of other immune cells. it has been well described that during infection with the influenza virus, the expression of tnfα in lung epithelial cells was higher, exerting powerful anti-influenza virus activity [20] . in an animal model, it has been demonstrated that tnf-α plays a pivotal role in the development of pulmonary fibrosis. tnf-α signals via two receptors, tnf-ri and tnf-rii; the first receptor (tnf-ri) promotes the first laboratory report about covid-19 patients indicated several parameters that were found to be altered in blood samples; for example, d-dimer, neutrophil count, blood urea, and creatinine levels were significantly higher. in the same way, several cytokines such as il-6 and tnf-α were overexpressed, indicating the immune status of the patients [14] . il-6 represents pro-inflammatory signaling produced by adipose tissue; for this reason, this endocrine cytokine could be important in regulating the host response during acute infection [15] . several papers have described the essential role of il-6 in generating a proper immune response during different kinds of viral infection in the pulmonary tract. others link this cytokine to an exacerbation of viral disease. these latter findings support the hypothesis that il-6 upregulation during viral infections may promote virus survival and the exacerbation of the clinical disease [16, 17] . indeed, il-6 has a pleiotropic function, and it is produced in response to tissue damage and infection. in particular, at the pulmonary level, innate and adaptative immune cell proliferation is strongly influenced by this cytokine. after targeting its specific receptor, il-6 starts a cascade of signaling events mainly associated with the jak/stat3 activation pathway, promoting the transcription of multiple downstream genes related to cellular signaling processes, including cytokines, receptors, adaptor proteins, and protein kinase [15] . furthermore, it has been reported that il-6 is an essential factor for the survival of mice with a viral infection. this cytokine promotes the optimal regulation of the t-cell response, inflammatory resolution, tissue remodeling promoting lung repair, cell migration, and the phagocytic activities of macrophages, as well as preventing virus-induced apoptosis in lung epithelial cells. however, experimental scientific evidence also suggests potential adverse consequences that increased levels of il-6 might have on the cellular immune response against viruses. in this context, different possible mechanisms involving this cytokine might affect viral clearance, ultimately favoring the establishment of a persistent viral state in infected hosts [18, 19] . tumor necrosis factor is a cell-signaling protein (cytokine) involved in systemic inflammation, released predominately from macrophages, but it is also released from a variety of other immune cells. it has been well described that during infection with the influenza virus, the expression of tnf-α in lung epithelial cells was higher, exerting powerful anti-influenza virus activity [20] . in an animal model, it has been demonstrated that tnf-α plays a pivotal role in the development of pulmonary fibrosis. tnf-α signals via two receptors, tnf-ri and tnf-rii; the first receptor (tnf-ri) promotes intracellular signaling involving c-jun n-terminal kinase (jnk) and nuclear factor (nf)-κb, while the other receptor, tnf-rii, promotes tnf-ri-dependent cell death, without directly inducing apoptosis. although both receptors are broadly expressed, it is known that the majority of inflammatory signaling is elicited through tnf-ri [21] . in an in vitro model, it has been described that serine/threonine kinases can phosphorylate tnf-ri and its molecules, preventing tyrosine phosphorylation [22] [23] [24] . in patients with covid-19, the high serum levels of il-6 and tnf-α are negatively correlated to t cells; contrariwise, it has been demonstrated that t cell levels were restored by reducing il-6 and tnf-α concentrations [25] . these findings suggested that these cytokines could represent important targets of anti-covid-19 therapies. through the secretion of adipokines, adipose tissue participates in the regulation of several pathophysiological processes in many organs and tissues. among the adipokines, adiponectin is the most relevant. adiponectin is one of the most abundant circulating adipocytokines, accounting for 0.01% of total serum protein. adiponectin is an important regulator of cytokine responses, and this effect is isoform-specific. it is involved in a wide variety of physiological processes, including energy metabolism, inflammation, and vascular physiology. these effects are mediated by two atypical, widely expressed seven-transmembrane receptors, adipor1 and adipor2 [26] . adiponectin has beneficial effects in cardiovascular systems and blood vessels, protecting these tissues through the inhibition of pro-inflammatory and hypertrophic responses and stimulation of endothelial cell responses [27] . adiponectin circulates as three different isoforms (low molecular weight-lmw, medium molecular weight-mmw, and high molecular weight-hmw) [28] . infectious diseases are characterized by an increased production of adiponectin. several papers suggest that adiponectin may be related to disease activity and/or severity in different conditions such as rheumatoid arthritis, osteoarthritis, and systemic lupus erythematosus. since adiponectin has been found to display both pro-and anti-inflammatory activities, controversial findings have been observed regarding the role of total adiponectin in systemic autoimmune and inflammatory joint diseases. for this reason, the relative contribution of each adiponectin isoform to the inflammatory response and joint and/or tissue damage requires further study [29] . it is reported that adiponectin is regulated by transcription factors in adipose tissue, such as peroxisome proliferator-activated receptor-γ (ppar-γ) [30] . during viral infections, it has been reported that the role of the predisposition of hosts is also important, as well as their state of health and nutrition. indeed, it is well known that white adipose tissue is considered an endocrine source of biologically active substances with local and/or systemic action, called adipokines. the inappropriate secretion of adipokines seems to participate in the pathogenesis of obesity-related diseases, including endothelial dysfunction, inflammation, and atherosclerosis [31] [32] [33] . the biological function of adipokines in lung diseases seems to be mainly related to the inflammatory process. in particular, the intercorrelation between adipose tissue and the lung has become evident as the involvement of adiponectin has been demonstrated in several lung diseases such as chronic obstructive pulmonary disease (copd), emphysema, and cancer [34] . in fact, with specific regard to copd, a low-grade inflammatory state has been demonstrated [35] [36] [37] . moreover, increasing evidence suggests that adiponectin also exerts a crucial role in the vascular endothelium, maintaining vascular homeostasis and protecting against vascular dysfunctions. altogether, these findings support the anti-inflammatory role of adiponectin in copd and, in general, in other lung diseases [38] . the critical role of adiponectin in the pathophysiological conditions of the lung is also supported by the modulation of adipors with the downregulation of adipor2. it has been described that the adiponectin oligomerization state is altered in copd; moreover, the presence of adipor1 and adipor2, with a lower expression of adipor2 compared to adipor1, in lung tissue [39] has been demonstrated. the low expression of adipor2 could suggest a specific role of this receptor, mainly implicated in adiponectin's effects on inflammation and oxidative stress. mainly, it has been observed that higher levels of adiponectin are associated with a significant and specific increase in hmw adiponectin, representing the most biologically active forms. thus, hmw adiponectin increases il-6 secretion in human monocytes and human monocytic leukemia cell lines but does not suppress lipopolysaccharide (lps)-induced il-6 secretion. byn contrast, lmw adiponectin reduces lps-mediated il-6 release and also stimulates il-10 secretion [40] . furthermore, several in vitro studies have demonstrated that adiponectin in the a549 adenocarcinoma human alveolar basal epithelial cell line has an essential apoptotic effect and also reduces the production of pro-inflammatory cytokines such as tnf-α, blocking nf-κb nuclear translocation [41, 42] . indeed, adiponectin can reduce innate and adaptive immune cell proliferation and polarization, also blocking the production of pro-inflammatory cytokines such as tnf-α, il-2, and il-6, and enhancing that of anti-inflammatory cytokines such as il-10, with a decrease in the phosphorylation of ampk, p38, erk1/2, and c-jnk [43] [44] [45] [46] . data from in vitro studies on lung cells were consistent with an anti-inflammatory function of adiponectin, and adiponectin-deficient mouse models developed lung function impairments and systemic inflammation [47] . the possible role of adiponectin in inflammatory pulmonary diseases, such as asthma and chronic obstructive pulmonary disease (copd), and in critical illnesses has been the subject of recent investigations. particularly, the hmw isoform has a specific role in pulmonary diseases and critical illnesses, even if its role should be better clarified [48, 49] . an interesting study reported that systemic adiponectin concentrations in humans fall during the acute phase of lung infection: particularly, during the early phase, the pro-inflammatory state is generated by the high systemic tnf-α and il-6 concentrations, with the subsequent inhibition of adiponectin production. contrariwise, it has been described that the reduction in tnf-α and il-6 factors generates a corresponding bounce-back in systemic adiponectin concentrations [50] . although it is still unclear whether the modulation of systemic adiponectin or its signaling pathways has any therapeutic benefit in pulmonary or critical illnesses, it may serve as a novel therapeutic or preventative tool for these illnesses in the future. one obvious pharmaceutical treatment would be the exogenous administration of adiponectin by the inhalational or intravenous route. although this has been tried in mouse models [51] , the problems to be overcome prior to human administration include establishing what the biologically active molecule is and what role post-translational modifications have upon its function, and the associated difficulties in generating biologically active molecules on a large scale. considering the difficulty linked to the direct administration of adiponectin, in the last few years, other drugs have been used that indirectly improve adiponectin production. for example, a synthetic ligand of peroxisome proliferator-activated receptors can increase adiponectin mrna in adipocytes, improving the production and secretion of adiponectin [52] [53] [54] [55] . moreover, other drugs such as fibrates can increase systemic adiponectin levels by enhancing ppar-γ activity [56, 57] . another way to improve adiponectin levels is the use of angiotensin converting enzyme inhibitors [58] [59] [60] . furthermore, it is possible to stimulate adipocyte differentiation [61] and the activation of ppar [62] . finally, it has been described that calcium channel blockers [63] and a central-acting anti-hypertensive agent [64] also increase systemic adiponectin concentrations [65] . the possibility to improve the action of adiponectin through diet is intriguing; it has been described that nutritional interventions may help to regulate systemic adiponectin concentrations. in an animal model, it has been demonstrated that a diet with a high concentration of polyunsaturated fatty acids and supplemented with ω-3 can improve the plasma levels of adiponectin, increasing gene expression [66] . on the other hand, in humans, adiponectin levels are positively associated with a healthy lifestyle and the mediterranean diet, even if the mechanisms of action are not completely known [66] . finally, in light of these considerations, in covid-19 therapy, it could be very useful to combine drug therapy with a specific diet regimen. another important mediator involved in the immune response and influenced by nutrition are fatty acids, in particular, ω-3 pufas [67, 68] . in fact, during bacterial and viral infections, they are able to act on immune cells and regulate diverse inflammatory processes. ω-3 pufas are known to have anti-inflammatory properties and play an essential role in the resolution of inflammation [69] . in several lung infections, the administration of pufa can ameliorate the outcome of the patient in acute pneumonia. sharma et al. reported in their study that the dietary supplementation of ω-3 pufa can exert an overall beneficial effect against acute pneumonia through the upregulation of the host's specific and nonspecific immune defenses [70] . ω-3 polyunsaturated fatty acids (pufa, ω-3-fatty acids), the key components of fish and flaxseed oils, are increasingly consumed by the public because of their potential health benefits and can be used clinically for the treatment of metabolic, cardiac, inflammatory, and autoimmune diseases [71] . however, numerous studies have shown that these compounds are immunoregulatory and immunosuppressive and thus may increase susceptibility to infection. while reports suggest that ω-3 pufas may have beneficial effects against extracellular pathogens, few studies have been performed on systemic viral infections in mammals. jones and roper described in their study that a diet rich in ω-3 pufas did not significantly lower survival of the vaccinia virus infection, at least with short-term (~6 week) feeding in mice [71] . ω-3 pufas are metabolized into various mediators possessing anti-inflammatory properties such as resolvins and protectins. it is known that ω-3 pufas can reduce nf-κb activation by preventing nuclear p65 nf-κb translocation. furthermore, ω-3 pufas minimize the activation of erk1/2 mapk, also reducing cox-2 production. the ω-3 pufa-derived lipid mediator could markedly attenuate influenza virus replication via the rna export machinery. in addition, the treatment of protectin d1 with peramivir could completely stop mouse mortality [72] . ω-3 supplementation was previously studied in acute respiratory distress syndrome (ards). singer and shapiro suggested that the enteral administration of natural antioxidant substances could improve oxygenation and clinical outcomes in icu patients [73] . a systematic review performed in 2015 reported a positive effect only for patients suffering from ards with high mortality [74] . a more recent meta-analysis highlighted the importance of clinical trials in order to clarify the use of ω-3 fatty acids and antioxidants in patients with ards to ascertain the positive effects in order to reduce the lengths of icu stays and the numbers of days spent on ventilators [75] . although the role of ω-3 supplementation in ards should be better clarified, its pivotal role in reducing reactive oxygen species and pro-inflammatory cytokines, such as tnf-α, il-1β, il-6, and il-8 [76] , is well known. therefore, ω-3 pufas, including protectin d1, which is a novel antiviral drug, could be considered for potential interventions for covid-19. as previously described, other dietary constituents can be used to improve the patients' outcomes during lung infection, regulating the inflammatory response. among these, antioxidants play an important role in protecting lung cells against viruses and bacteria. viral infection leads to an increase in the intrapulmonary oxidative burden. in many diseases, the balance between oxidants and antioxidants (redox balance) is altered, with severe consequences [77] . the pathophysiological mechanisms by which free radicals generate various types of stress-such as oxidative, nitrative, carbonyl, inflammatory, and endoplasmic reticulum stress-lead to lung inflammation and an altered lung immune response. in this scenario, dietary antioxidants may play an important role against lung oxidative stress [77] . several studies reported the protective role of the antioxidants in lung infection and in lung inflammation [78, 79] . in particular, vitamin c, polyphenols, and flavonoids can play a protective role in lung infections, being immune modulators and inflammatory mediators. indeed, as reported by carr et al., during infection, vitamin c levels may become depleted; for this reason, vitamin c supplementation can attenuate infection. based on this evidence, these authors suggested a clinical trial with vitamin c infusion for the treatment of severe covid-19 patients [80] . among polyphenols, epigallo-catechin 3 gallate (egcg) is the most potent ingredient in green tea and exhibits antibacterial, antiviral, antioxidative, anticancer, and chemo-preventive activities. recently, numerous studies have investigated the protective effects of egcg against asthma and other lung diseases such as copd and lung pneumonia. egcg may suppress inflammation and inflammatory cell infiltration into the lungs of asthmatic mice, and may also inhibit epithelial-mesenchymal transition emt via the pi3k/akt signaling pathway through upregulating the expression of phosphatase and tensin homolog (pten), both in vivo and in vitro [81] . moreover, flavonoids can be used to attenuate lung injury in mice; it has been reported that they inhibit influenza virus and toll-like receptor signaling, blocking nf-κb translocation [82] . therefore, as summarized in table 1 , supplementation with vitamin c, flavonoids, and polyphenols can be tested in covid-19 patients, both in order to prevent viral infection and to improve patients' outcomes. table 1 . the principal antioxidants involved in lung infection and the immune-inflammatory response. red wine, oranges, red fruits and vegetables reduces inflammation and immune response, blocking nuclear nf-κb translocation polyphenols green tea, broccoli, apples vitamin c oranges, lemons, mangoes during pulmonary infections, and particularly in covid-19 patients, intracellular signaling leads to the production of pro-inflammatory cytokines, such as tnf-α and il-6, which act in concert with chemoattractants, such as cxcl1 and cxcl2, to recruit polymorphonuclear leukocytes (pmns) to the lungs, killing pathogens but generating fibrosis [83] . another important consideration during covid-19 infection is related to the modification of the secretory products of the upper and lower airways, which usually include mucin and pulmonary surfactant. during infection, mucin production is upregulated, with the function of preventing microbes from binding to and infecting epithelial cells [84] . the primary source of phospholipids (pls) in the lung is pulmonary surfactant, synthesized and released by alveolar epithelial type ii cells. the surfactant contains approximately 80-90% pls, with fatty acid chains that can be oxidized during different challenges in the lung [85] . the oxidation of these pls in the lung can occur in the setting of an increased oxidative stress situation, such as infection and inflammation [86] . the immune effects of oxidized phospholipids oxpls during infectious diseases are inevitably dictated by the balance among activation, degradation, and scavenging. it has been shown that oxpls are generated in the lung during several pulmonary infections, including influenza and avian influenza (h5n1), as well as sars coronavirus, even if the mechanisms of action are not well known [87] [88] [89] . as reported by imai et al., oxpl-induced inflammation is mediated by tlr4 and trif, driving an increase in il-6 production [89] . it is intriguing to consider that oxpl-dependent defects in phagocytosis and ros generation may lead to an increased susceptibility to respiratory infections [90] . cholesterol is the major neutral lipid in pulmonary surfactant, in which it is thought to promote the spreading, mobility, and adsorption of surfactant films [91] . as previously documented, modulating adiponectin levels can be considered an important way to reduce cytokines levels; in this way, the adverse effects related to the covid-19 infection should be attenuated. it is well described in animal models that the consumption of hyperlipidemic diets, rich in saturated fat, reduces the levels of adiponectin, while diets rich in polyunsaturated fatty acids and supplemented with ω-3 pufa increase adiponectin levels, reducing pro-inflammatory cytokines [66] . innate and adaptive immune responses are influenced not only by oxpls and cholesterol but also by the fatty acid profiles of tissues in response to pharmacological agents and diet [92] . several studies performed in animal models demonstrated how ω-3 pufa uptake into the lung tissue influences outcomes associated with infection, promoting the resolution of inflammation [93] . in another study, ω-3 pufas reduced the levels of pmns and lowered il-6 levels in lung infections [94] . these positive effects remain controversial; for example, jones and roper reported that in their experimental model, no statistically significant differences were found among the diet regimens, with and without ω-3 pufas, with respect to the susceptibility of mice to viral infection, morbidity, viral organ titers, recovery time, or mortality [71] . in conclusion, it is well known that general treatments are very important to enhance the host immune response against rna viral infection. in addition, the immune response has often been shown to be weakened by inadequate nutrition in many model systems as well as in human studies. however, the nutritional status of the host, until recently, has not been considered as a contributing factor to the emergence of viral infectious diseases. the recent reports about the pathogenesis of covid-19 suggested that one of the most important consequences of this infection is the cytokine storm syndrome [95] , which could be strictly linked with coagulopathy, generating acute pulmonary embolism caused by in-situ thrombosis [96, 97] . therefore, a great number of clinical trials are ongoing to define a useful therapy to attenuate cytokine storms [98] . for these reasons, an adequate ω-3 pufa intake may be a valid strategy against viral infection. indeed, following the recommended intake of ω-3 pufa, in the range of 0.5% and 2% of total calories (250 mg/day), may be important to protect against an excessive inflammatory response, also reducing il-6 levels. this theory found important support in a recent study that demonstrated that ω-3 pufa-derived lipid mediator protectins can suppress influenza virus replication through a mechanism that blocks the export of viral mrna. moreover, imai demonstrated that this mediator can be used in combination with the antiviral peramivir, even at late time points in infection [99] . nevertheless, the efficacy of ω-3 pufas at the clinical level is under investigation; for example, hecker et al. described a beneficial effect for a diet regimen with ω-3 pufas, describing that the pro-inflammatory cytokine levels decreased after this diet regimen [100] . the suggested positive role in the outcome and prevention of the covid-19 infection is summarized in figure 2 . innate and adaptive immune responses are influenced not only by oxpls and cholesterol but also by the fatty acid profiles of tissues in response to pharmacological agents and diet [92] . several studies performed in animal models demonstrated how ω-3 pufa uptake into the lung tissue influences outcomes associated with infection, promoting the resolution of inflammation [93] . in another study, ω-3 pufas reduced the levels of pmns and lowered il-6 levels in lung infections [94] . these positive effects remain controversial; for example, jones and roper reported that in their experimental model, no statistically significant differences were found among the diet regimens, with and without ω-3 pufas, with respect to the susceptibility of mice to viral infection, morbidity, viral organ titers, recovery time, or mortality [71] .s in conclusion, it is well known that general treatments are very important to enhance the host immune response against rna viral infection. in addition, the immune response has often been shown to be weakened by inadequate nutrition in many model systems as well as in human studies. however, the nutritional status of the host, until recently, has not been considered as a contributing factor to the emergence of viral infectious diseases. the recent reports about the pathogenesis of covid-19 suggested that one of the most important consequences of this infection is the cytokine storm syndrome [95] , which could be strictly linked with coagulopathy, generating acute pulmonary embolism caused by in-situ thrombosis [96, 97] . therefore, a great number of clinical trials are ongoing to define a useful therapy to attenuate cytokine storms [98] . for these reasons, an adequate ω-3 pufa intake may be a valid strategy against viral infection. indeed, following the recommended intake of ω-3 pufa, in the range of 0.5% and 2% of total calories (250 mg/day), may be important to protect against an excessive inflammatory response, also reducing il-6 levels. this theory found important support in a recent study that demonstrated that ω-3 pufaderived lipid mediator protectins can suppress influenza virus replication through a mechanism that blocks the export of viral mrna. moreover, imai demonstrated that this mediator can be used in combination with the antiviral peramivir, even at late time points in infection [99] . nevertheless, the efficacy of ω-3 pufas at the clinical level is under investigation; for example, hecker et al. described a beneficial effect for a diet regimen with ω-3 pufas, describing that the pro-inflammatory cytokine levels decreased after this diet regimen [100] . the suggested positive role in the outcome and prevention of the covid-19 infection is summarized in figure 2 . in addition, adiponectin plays a role in lung diseases and obesity; in the development and progression of lung disease and cancer, a pathogenic role of adiponectin was defined by both in vivo and in vitro studies. recently, immunometabolic pathomechanisms have been identified as important factors determining and modulating lung function and disease. particularly, adiponectin levels have been found to be greater in patients with copd compared with in control patients, and adiponectin-deficient mice are protected from several lung diseases [101] . moreover, it has been in addition, adiponectin plays a role in lung diseases and obesity; in the development and progression of lung disease and cancer, a pathogenic role of adiponectin was defined by both in vivo and in vitro studies. recently, immunometabolic pathomechanisms have been identified as important factors determining and modulating lung function and disease. particularly, adiponectin levels have been found to be greater in patients with copd compared with in control patients, and adiponectin-deficient mice are protected from several lung diseases [101] . moreover, it has been reported that adherence to the mediterranean diet was associated with an increase in adiponectin levels, improving cardiovascular system functionality [102] , particularly in elderly people [103] . these findings are only apparently contradictory to the first data about the mortality rate from covid-19 infections in the mediterranean area (such as in italy and spain) [104] . first of all, the data have been referred only to the tested population; moreover, it is well described that the presence of several comorbidities such as hypertension, diabetes, and cardiovascular diseases severely influenced the mortality rate reported in this area [105] . all these comorbidities can be counteracted with a correct dietary regimen. therefore, both adiponectin and ω-3 pufas appear to be attractive biomarkers for monitoring lung disease progression. finally, considering that the covid-19 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randomized controlled trial elevated plasma adiponectin levels in patients with chronic obstructive pulmonary disease the association between adherence to the mediterranean diet and adiponectin levels among healthy adults: the attica study ageing and plasma adiponectin concentration in apparently healthy males and females at the epicenter of the covid-19 pandemic and humanitarian crises in italy: changing perspectives on preparation and mitigation. catal. non comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we wish to thank the scientific bureau of the university of catania for language support. the authors declare no conflict of interest. key: cord-321481-vrfwczve authors: watashi, koichi; urban, stephan; li, wenhui; wakita, takaji title: ntcp and beyond: opening the door to unveil hepatitis b virus entry date: 2014-02-19 journal: int j mol sci doi: 10.3390/ijms15022892 sha: doc_id: 321481 cord_uid: vrfwczve chronic hepatitis b virus (hbv) infection, affecting approximately 240 million people worldwide, is a major public health problem that elevates the risk of developing liver cirrhosis and hepatocellular carcinoma. given that current anti-hbv drugs are limited to interferon-based regimens and nucleos(t)ide analogs, the development of new anti-hbv agents is urgently needed. the viral entry process is generally an attractive target implicated in antiviral strategies. using primary cells from humans and tupaia belangeri, as well as heparg cells, important determinants of viral entry have been achieved. recently, sodium taurocholate cotransporting polypeptide (ntcp) was identified as an hbv entry receptor and enabled the establishment of a susceptible cell line that can efficiently support hbv infection. this finding will allow a deeper understanding of the requirements for efficient hbv infection, including the elucidation of the molecular entry mechanism. in addition, pharmacological studies suggest that ntcp is able to serve as a therapeutic target. this article summarizes our current knowledge on the mechanisms of hbv entry and the role of ntcp in this process. hepatitis b virus (hbv) infection constitutes a serious public health problem, affecting approximately 240 million carriers worldwide [1] . chronic hbv infection significantly elevates the risk for developing liver cirrhosis and hepatocellular carcinoma. currently, conventional interferon-α (ifnα) or pegylated-ifnα and nucleos(t)ide analogs are available as anti-hbv agents [2, 3] . however, ifn-based therapies, which cause significant side effects, yields long-term clinical benefits in less than 40% of treated patients [4] . nucleos(t)ide analogs suppress an essential step in virus replication and thereby provide biochemical and histological improvement, but some of the early drugs give rise to drug-resistant viruses, which adversely affect long-term clinical outcome. thus, in order to approach curative treatments, new anti-hbv agents targeting different molecules involved in hbv infection and propagation are needed. nucleos(t)ide analogs suppress hbv replication mainly by inhibiting the reverse transcription process in the viral lifecycle ( figure 1 ) [3] . ifn functions as an immunomodulator and is also reported to directly interfere with hbv replication at multiple steps of the lifecycle [5] . given that hbv encodes only one viral protein carrying enzymatic activity, polymerase, in its genome, strategies for inhibiting viral enzyme are limited. although capsid or envelope protein assembly and the regulatory x-protein are possible future targets, it is critical for developing new classes of anti-hbv agents to identify cellular factors serving as possible drug targets. in general, the viral entry step is an attractive target for the development of antiviral agents [6] [7] [8] . the early hbv lifecycle, including the entry step, has gained significant attention very recently with regard to molecular mechanisms, triggered by the identification of sodium taurocholate cotransporting polypeptide (ntcp) as a cellular entry receptor [9] . this article summarizes the molecular evidence related to hbv entry, mainly focusing on recent findings, and its implications. hbv infection into host hepatocytes follows a multiple step process: (1) initially, hbv reversibly attaches to host cell surface proteoglycans with a low affinity; (2) this is followed by the process involving more specific receptor(s) with high affinity to mediate the early entry step; and (3) after endocytosis-mediated internalization, the virus fuses with the cellular membrane compartment, probably in an endosomal compartment, although the mechanisms are not fully understood. both initial attachment and, probably more importantly, specific receptor recognition contribute to host specificity and tissue tropism [10] . the initial attachment step is at least partly mediated by heparan sulfate proteoglycans [11] [12] [13] [14] . the third internalization step is reported to involve caveolae-, clathrin-or macropinocytosis-dependent endocytosis, depending on the cell types and experimental systems [15] [16] [17] [18] . however, cellular factors involved in the high-affinity binding and the early entry process remained to be elucidated until recently. the hbv surface proteins are composed of three proteins, termed the large (lhbs), middle (mhbs) and small (shbs) surface proteins, and include the pres1, pres2 and s regions: lhbs encompasses the pres1, pres2 and s regions; mhbs encompasses the pres2 and s; and shbs comprise the s region [19, 20] . the molecular requirement of hbv envelope proteins for hbv infection has been studied for more than a decade using primary hepatocytes from humans and tupaia belangeri, as well as heparg cells [21] . a series of analyses using neutralizing antibodies and introduced point mutations suggested that the s and pres1, but not the pres2, regions play a significant role in hbv infection [22] [23] [24] [25] [26] [27] . in a direct approach, the pres1 region in the lhbs has been shown to be essentially involved in the hbv infection process. this was demonstrated by the introduction of mutations in the viral context, infection competition with anti-pres1 antibodies and with peptides mimicking this region [28] [29] [30] [31] [32] [33] [34] . a myristoylated peptide encompassing amino acids 2-48 of the pres1 region turned out to be the most efficient in infection inhibition of hbv and also the envelope protein-related hepatitis d virus (hdv) [30, 31] . such a peptide has been used as a tool for characterizing the early infection step, including the identification of ntcp as an entry receptor [9] and as a lead substance (myrcludex-b) presently in the clinical development (see below) [10, 35, 36] . one of the recent milestones in the field in hbv molecular biology is the identification of ntcp as a host entry receptor, as reported by yan and zhong et al. in late 2012 [9] . by affinity purification and mass spectrometry analysis using an hbv pres1-derived lipopeptide as bait, they identified tupaia belangeri ntcp (tsntcp) as a cellular factor interacting with this lipopeptide. ntcp is a transporter residing in the basolateral membrane of hepatocytes and is involved in the hepatic uptake of mostly conjugated bile salts (see below). the lipopeptide was confirmed to specifically bind human ntcp (hntcp), as well as tsntcp, but surprisingly not crab-eating monkey ntcp (mkntcp), which correlated with the species specificity of hbv infection: hbv is able to efficiently infect humans and tupaia, but not crab-eating monkey [9] . interestingly, this result also correlated with the in vitro binding activity of the peptide to the respective primary hepatocytes [10] and their in vivo hepatotropism [37] . the role of ntcp in the viral infection of hbv, its satellite virus, hdv, and a closely related primate hepadnavirus wooly monkey hbv was further examined by knockdown and overexpression analyses [9, 38, 39] . sirna-mediated knockdown of ntcp in primary human hepatocytes (phh), primary tupaia hepatocytes and differentiated heparg cells reduced hbv and hdv infection, while ectopic expression of ntcp conferred hbv susceptibility in hepg2 cells, which originally did not support efficient infection [9] . this strongly argues that ntcp is an essential factor for hbv infection. the expression of ntcp in different cells was consistent with the hbv susceptibility, as it was significantly expressed in hbv-susceptible cells, phh and differentiated heparg cells, but was weakly expressed or absent in hepg2, huh-7, flc4 and hela cells, which show little to no infection [40] [41] [42] . the introduction of ntcp into huh-7 and undifferentiated heparg cells conferred hbv infection to these cells to some extent [38] . although the total expressions in these transduced cells were comparable, hntcp-expressing hepg2 cells showed much higher infection efficiency when compared with other human hepatocyte cell lines [38, 43, 44] . in the initial study, infection efficiency was ~10% in ntcp-overexpressing hepg2 cells cultured with medium containing 2% dimethyl sulfoxide (dmso) [9] . subsequent analysis showed that increasing the dmso concentration to more than 2.5%~3% augmented infection efficiency to 50%~70%, as evaluated by immunofluorescence of hbv proteins, although the virus inoculum was different in these studies [38, 43] . the speculations include that dmso augmented the gene expression of ntcp, promoted the membrane localization of ntcp and changed the post-translational modification of ntcp, but the detailed molecular mechanisms for dmso-mediated promotion of hbv infection is open for further studies. it remains unknown why not all of the cells were infected with hbv in these reports, but it is possible that the ntcp function for supporting hbv entry is reflected by post-translational modification, subcellular localization or other factors that are governed by cell conditions or by more general conditions, such as the cell cycle, cellular microenvironment or architecture. another open question is on the high susceptibility for hdv, but not hbv, in huh-7 cells overexpressing hntcp [9, 38] . future analysis of this issue is necessary in order to establish a cell culture model that is 100% susceptible to hbv infection. crucial amino acid sequences in ntcp involved in hbv infection have been analyzed. by sequence comparison between hntcp and mkntcp, replacement of amino acids 157-165 of hntcp with the respective sequence from mkntcp abrogated the ability to support hbv pres1-binding and, subsequently, infection, while mkntcp carrying a conversion to this region from hntcp conferred hbv susceptibility. thus, amino acids 157-165 of ntcp are crucial for ntcp-mediated hbv binding and infection [9, 45] . it has also been shown that hntcp bearing a substitution of the 84-87 aa from the mouse counterpart was able to bind pres1, but was not functional for hbv infection, while replacing these residues in mouse ntcp (mntcp) with the human counterparts supported the infection [38, 44] . these data indicate that the 84-87 aa residues are a determinant for ntcp function as an hbv entry receptor. it remains to be elucidated why mntcp does not support hbv infection, but mntcp was shown to support specific binding of the pres1-lipopeptide on the cell surface, although the binding capacity of mntcp to the pres1 region appears to be weaker than that of hntcp [44] . it is possible that the binding of hbv to ntcp is not sufficient and requires an additional molecule or mechanism to trigger the following early infection process. hdv is a virusoid-like particle, which depends on hbv for assembly and propagation [46] . hdv shares the hbv envelope proteins, lhbs, mhbs and shbs, and its attachment/early entry mechanism seems to be very similar to that for hbv. due to its completely different replication strategy, it is very likely that it depends on different cellular factors and follows different pathways after membrane fusion. intriguingly, hdv infection can be observed by complementing hntcp in either mouse-derived hepa1-6, mmhd3 and hep56.1d cells, rat hepatocyte tc5123 cells or non-hepatocyte hela, cho and vero cells. this is in stark contrast to hbv, which cannot infect these cells [38, 44] . this suggests that hbv requires additional host factors for infection or is restricted at a post-entry step prior to covalently closed circular dna (cccdna) formation. it is of particular interest to clarify the molecular mechanisms underlying the different cellular requirements between infection by hbv and hdv, especially when trying to establish a susceptible mouse model in the future. it is presently unclear whether there are additional cellular factors besides ntcp required for viral infection and determining the tissue and species tropism of hbv. these include factors essentially involved in the viral lifecycle during attachment, internalization, endocytosis, membrane fusion, uncoating, nuclear translocation and cccdna formation and those affecting post-entry restriction. overexpression of hntcp in mouse hepatocyte cell lines, such as hepa1-6 and mmhd3 cells, did not confer susceptibility to hbv infection, in contrast to the hbv infection observed after ntcp introduction into hepg2 cells [44] . hntcp conferred efficient hbv infection in hepg2 cells, but only a low efficiency of infection was observed in huh-7 and undifferentiated heparg cells and no detectable infection to mouse and rat hepatoma cells, including hep56.1d, hepa1-6 and tc5123 cells [38] . we also showed that different hepg2 clone isolates that similarly expressed high levels of ectopic ntcp, but were likely to have different cellular genetic backgrounds, had diverse efficiencies of hbv infection [43] . these observations favor the existence of additional host factors determining susceptibility to hbv infection. for hepatitis c virus (hcv) infection, multiple cellular factors are required for efficient viral entry, including low density lipoprotein receptor (ldlr), scavenger receptor class b type i (sr-bi), cd81, occludin (ocln) and claudin-1 (cldn-1) as viral entry receptors and niemann-pick c1-like 1 (npc1l1), epidermal growth factor receptor (egfr) and ephrin a2 (epha2) as other factors involved in entry [21, 47, 48] . it has been reported that the complementation of both hcd81 and hocln are required for rendering high hcv susceptibility in mice [49] . furthermore, in the case of duck hepatitis b virus (dhbv), multiple factors are suggested to be essential for efficient viral infection. carboxypeptidase d was confirmed to bind the dhbv envelope and function in viral attachment and entry [50] . however, overexpression of this protein alone in huh-7 cells did not support dhbv infection [51] . carboxypeptidase d was able to bind to dhbv and heron hbv, which did not infect primary duck hepatocytes, and this protein is also expressed in non-liver tissues [52] . thus, additional factors are likely to be required to explain dhbv susceptibility, one candidate of which can include duck ntcp [53] . these examples in viruses that utilize multiple receptors favor pursuing the identification of additional cellular factors crucial for hbv entry. ntcp, also designated as solute carrier family 10a1 (slc10a1), is a member of the slc10 transporter gene family. the slc10 family consists of seven members (slc10a1-7). among these, ntcp and apical sodium-dependent bile salt transporter (asbt), also known as slc10a2, are sodium-dependent transporters for bile acids [54] . ntcp is mainly distributed at the basolateral membrane of hepatocytes and plays a major role in the hepatic influx of conjugated bile salts from portal circulation [55, 56] . ntcp on the plasma membranes in hepatocytes binds two sodium ions together with one molecule of preferentially conjugated bile salt for uptake. in addition to bile salts, ntcp, like other transporters, binds and/or transports other molecules, including steroid hormones, thyroid hormones, drug-conjugated bile salt and a variety of xenobiotics [57, 58] . hntcp is a 349 aa protein with an apparent mass of 56 kda and includes a putative seven or nine transmembrane domains with a predicted topology of n-terminal extracellular and c-terminal intracellular ends [59] [60] [61] . while the structure of ntcp has not been resolved, the crystal structures of the asbts from neisseria meningitis (asbt nm ) and yersinia frederiksenii (asbt yf ) were recently reported [62, 63] . asbt nm shows a ten transmembrane domain and a hydrophobic inward-facing binding cavity. this structure is different from the model for hasbt currently favored based on bioinformatic prediction and experimental data, which carry seven to nine transmembrane helices with n-terminal extracellular and the c-terminal cytoplasmic domain [60, 64, 65] . a structural analysis of asbt yf proposed two conformations, inward-and outward-open structures of bile salt transporters by rotating two core helices transmembrane (tm)-4 and tm9 [63] . because asbt nm and asbt yf has only 26% and 22% homology, respectively, with hasbt and even lower homology with hntcp [62, 63] , it is uncertain whether the structural features of asbt nm or asbt yf are useful for designing drugs targeting hasbt and hntcp. several single nucleotide polymorphisms (snps) that alter the transporter activity of ntcp have been reported [66, 67] . as non-synonymous snps, i223t, a variant seen in 5.5% of allele frequencies in african americans, decreased plasma membrane-localized ntcp and reduced its transporter activity. the s267f variant, seen in 7.5% of allele frequencies in chinese americans, exhibited almost complete loss of function for bile acid uptake, but possessed normal transport activity for the non-bile acid substrate, estrone sulfate. another report showed that the a64t and s267f variants, carried by 1.0% and 3.1% of allele frequencies in koreans, respectively, decreased the uptake of taurocholic acid. these polymorphisms are dependent on ethnicity. however, there have been no reports of serious diseases associated with defects in the ntcp gene. no reports describing ntcp knockout mice have been published to date. thus, it is difficult to draw conclusions on whether the physiological roles of ntcp are complemented by other factors that share the redundant physiological function and whether ntcp inhibition is able to safely serve as an anti-hbv drug target. importantly, it was very recently reported that molecular determinants for the transporter function of ntcp overlapped with those for the ability to support hbv entry [68] . ntcp mutations in amino acids that were critical for bile salt binding (n262a, q293a/l294a) abrogated both the binding to pres1 peptide and the infection of hbv. the s267f variant of ntcp could neither bind to the pres1 region nor support hbv infection in cell culture. in general, the viral entry process is an attractive target for the development of antiviral agents. as noted above, the 2-48 aa region of pres1 in the lhbs protein is important for hbv infection [31] . myrcludex-b, which is an optimized synthetic lipopeptide consisting of the myristoylated 2-48 aa region of pres1, is able to strongly inhibit hbv infection in both cell culture and an in vivo mouse model [36] . the ic 50 in a cell culture model was reported to be approximately 100 pm [35] . following the successful clinical development of enfuvirtide as the first peptidic hiv entry inhibitor mimicking the region derived from the viral gp41 envelope glycoprotein [69] , myrcludex-b is now under clinical development in phase ib/iia [70] . mechanistically, myrcludex-b binds hntcp and inactivates its receptor function for hbv and hdv ( figure 1 ). remarkably, ic 50 to the transporter activity of ntcp was approximately 4 nm [38] , showing that binding saturation is not required for receptor inactivation, thus allowing a therapeutic window for infection inhibition without a complete abrogation of bile salt transportation [38] . thus, agents targeting ntcp are expected to be potent candidates that act as anti-hbv drugs. cyclosporin a (csa) is the first line of such compounds revealed to inhibit hbv infection by targeting ntcp [42, 45] . csa is known to be an immunosuppressant classified as a calcineurin inhibitor and is clinically used for the suppression of the immunological failure of xenografts after tissue transplantation [71] . in cell culture analyses, csa was also reported to suppress the replication of numerous viruses, including hiv, hcv, influenza virus, severe acute respiratory syndrome coronavirus, human papillomavirus, flaviviruses and hbv [72] [73] [74] [75] [76] [77] [78] [79] . in most of these cases, cyclophilins (cyps), cellular peptidyl prolyl cis-trans isomerases that catalyze conformational changes in proteins and are the primary cellular target for csa, were critical for efficient viral replication, and cyp inhibition by csa was responsible for antiviral activity. however, the anti-hbv entry activity of csa was not mediated by the inhibition of cyp, but rather, via direct targeting of ntcp. csa bound to ntcp on the plasma membrane and inhibited transporter activity ( figure 1 ) [42, 45] . it also inhibited binding between lhbs and ntcp in vitro (figure 1 ) [42] . this suggests that csa interacted with ntcp, thus inhibiting the recruitment of lhbs of incoming hbv to ntcp on the plasma membrane and blocking hbv entry. the anti-hbv activity of csa was pan-genotypic [42] . moreover, our derivative analysis identified a series of csa analogs having a stronger anti-hbv entry activity with a submicromolar ic 50 [42] . notably, non-immunosuppressive csa analogs may be potent anti-hbv agents. given that non-immunosuppressive csa analogs, including alisporivir (debio 025) and scy-635, have significant activity in decreasing hcv viral load in clinical trials and are regarded as promising anti-hcv drug candidates [80, 81] , further derivative analysis of csa may be a reasonable approach for drug development. as other examples, compounds known to be ntcp inhibitors, including progesterone, propranolol and bosentan, have been shown to block hbv infection (figure 1 ) [42] . ntcp substrates, such as taurocholate, tauroursodeoxycholate and bromosulfophthalein, also inhibited hbv infection [38, 42, 68 ]. an anticholesteremic drug, ezetimibe, has been shown to block hbv entry [82] , and this drug was reported to inhibit the ntcp transporter [83] . these results indicate that compounds modulating ntcp function could substantially inhibit hbv infection. hepg2 cells engineered to overexpress ntcp are also useful for high-throughput screening to identify compounds targeting ntcp and inhibiting hbv infection. one example identified in such chemical screening is the oxysterols, which are oxidized derivatives of cholesterol or by-products of cholesterol biosynthesis [43] . host-targeting antivirals are generally expected to have significant advantages, including a much lower frequency drug resistance, universal antiviral effects beyond viral genotypes and complementary mechanisms of action that might act in a synergistic manner with currently available antiviral agents [48] . more importantly, they offer an additional therapeutic choice, given that only ifns and nucleoside analogs are currently available as anti-hbv agents. identification of ntcp as an hbv entry receptor has accelerated the understanding of hbv molecular biology and offered useful experimental systems to analyze the hbv and hdv lifecycle, including the identification of host restriction and dependency factors. ntcp also represents a new therapeutic target in the development of new 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polypeptide (ntcp) the authors are grateful to all of the members of the department of virology ii, national institute of infectious diseases, the department of infectious diseases, molecular virology, university hospital heidelberg, and the li lab at the national institute of biological sciences, beijing, for their research, technical support and discussions. funding was provided by the ministry of health, labor and welfare, japan, the ministry of education, culture, sports, science and technology, japan, the japan society for the promotion of science, the deutsche zentrum für infektionsforschung (dzif), the deutsche krebshilfe, the deutsche forschungsgemeinschaft (dfg) ur72/7-1, for1202/ur72/5-1, the ministry of science and technology, china (2010cb530101), and the national science and technology major project, china (2013zx09509102). all of the authors wrote the paper. the authors declare no conflict of interest. key: cord-333286-lr32e0w4 authors: lehtoranta, liisa; latvala, sinikka; lehtinen, markus j. title: role of probiotics in stimulating the immune system in viral respiratory tract infections: a narrative review date: 2020-10-16 journal: nutrients doi: 10.3390/nu12103163 sha: doc_id: 333286 cord_uid: lr32e0w4 viral respiratory tract infection (rti) is the most frequent cause of infectious illnesses including the common cold. pharmacological solutions for treating or preventing viral rtis are so far limited and thus several self-care products are available in the market. some dietary supplements such as probiotics have been shown to modulate immune system function and their role in reducing the risk and the course of rtis has been investigated extensively within the past decade. however, the mechanism of action and the efficacy of probiotics against viral rtis remains unclear. we searched pubmed, google scholar, and web of knowledge for pre-clinical and clinical studies investigating the effect of probiotics on respiratory virus infections, immune response, and the course of upper and lower respiratory tract illness. the literature summarized in this narrative review points out that specific probiotic strains seem effective in pre-clinical models, through stimulating the immune system and inhibiting viral replication. clinical studies indicate variable efficacy on upper respiratory illnesses and lack proof of diagnosed viral infections. however, meta-analyses of clinical studies indicate that probiotics could be beneficial in upper respiratory illnesses without specific etiology. further studies aiming at discovering the mechanisms of action of probiotics and clinical efficacy are warranted. respiratory viruses cause the most common infectious illnesses in humans-acute rtis that can be divided into upper rtis (urti), e.g., the common cold, and lower rtis (lrti), e.g., bronchitis and pneumonia. these illnesses affect all age groups annually and cause a high burden on health care systems and global economics due to absenteeism from daycares, school, and work. over 200 virus types have been identified as causative agents for respiratory illnesses [1, 2] . in most cases, especially illnesses of the upper respiratory tract are mild to moderate and often self-limiting. on the other hand, lrtis leading to pneumonia can be especially fatal among children and the elderly, or immunocompromised subjects [2] . in the past decade, studies linking the microbiota and immune system function have laid the foundation for the opportunities in microbiota modulation and bacterial therapeutics in health and disease. further, studies have associated the gut and airway microbiota with upper and lower respiratory tract health and immunity [3, 4] . thus, modulation of the gut microbiota and immunity by dietary supplements or pharmaceuticals is of increasing interest in finding novel solutions to manage rtis. among the promising candidates are probiotics that have been studied for immune function modulation and viral infections. meta-analyses suggest that probiotics could be beneficial in the context of acute urti typically caused by viruses [5, 6] . viruses that cause rtis are found in various virus families, which differ in virulence and utilize variable strategies to infect the host cells and to evade the host immune system [14, 15] . respiratory viruses spread via nasal secretions that can be transmitted through the air or by hand-to hand and surface-to-hand contact [16] . infection requires penetration of the virus through the host mucus layer, including the microbiota, and antiviral molecules in the mucus, such as antibodies and collectins. once on the mucosal epithelial cells, respiratory viruses attach to specific receptors, such as intercellular adhesion molecule (icam)-1 (rhinoviruses), peptidases (coronaviruses), or sialic acids (influenza viruses), that mediate the internalization of the virus by endocytosis [17, 18] . the viral receptors are differentially expressed on host cells resulting in virus-specific host cell tropism that is one key factor in viral pathogenesis. for example, influenza viruses typically infect bronchial cells, whereas rhinoviruses infect the epithelial cells of the upper airways, resulting in differences in illness presentation. viral genomic structure can be, for example, positive-or negative-sense single-stranded (ss) or double-stranded (ds) rna or dna [19] . most rti causing viruses, picornaviruses, influenza viruses, and coronaviruses are ssrna viruses, whereas adenoviruses are dsdna viruses. the genomic structures are recognized by different receptors in the host and activate different types of immune responses. some respiratory viruses, e.g., coronaviruses, are surrounded by a viral envelope which confers additional protection from the host immune system. respiratory viruses have further developed molecules that help in evading the immune response, for example, by disrupting the interferon (ifn) response and hijacking the host's cellular machinery for the production of virus copies [15] . once the viruses have penetrated into the host cells, the epithelial and immune cells detect the viral structures by pattern recognition receptors (prr) of which toll-like receptors (tlrs) and retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) play a central role. tlr3, tlr7, tlr8, and tlr9 are located in the endosomes and can identify viral ss (tlr7 and 8) and ds (tlr3) rna structures, and dna (tlr9) [18] . the recognition by tlrs leads to activation of transcription factor nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb) and ifn regulatory factors (irf) 3, 5, and 7 [12, 20, 21] , resulting in expression of pro-inflammatory cytokines and type i ifns, ifn-α and ifn-β. type i ifns are broadly secreted by cells, but epithelial cells further secrete type iii lambda ifns in response to viral infections. cytoplasmic rnas, on the other hand, are recognized by rlrs, of which rig-i recognizes ssrna and melanoma differentiation associated 5 (mda-5) dsrna. the activation of rlrs leads to type i (and type iii in epithelial cells) ifn production via mitochondrial antiviral signaling protein (mavs). type i and iii ifns induce an antiviral state in the surrounding cells which is not, however, necessarily sufficient to resist the infection, but delays the spreading of the infection [12, 22] . the activation of rlrs, and tlrs by viral infection and cellular stress, leads to formation of nucleotide-binding oligomerization domain (nod)-, leucine-rich repeat (lrr)-, and pyrin domain-containing protein 3 (nlrp3) inflammasome [23] . although the role of nlrp3 is still somewhat unclear in viral rtis, it seems to play a role at least in rhinovirus, influenza, adenovirus, and rsv infections. nlrp3 inflammasome activation drives caspase 1-dependent il-1β and il-18 cytokine response and inflammatory programmed cell death (pyroptosis) [24] . epithelium-derived pro-inflammatory cytokines tumor necrosis factor (tnf)-α, interleukin (il)-1β, il-6, chemokine (c-c motif) ligand (ccl) 2, ccl5, chemokine (c-x-c motif) ligand (cxcl)8, and cxcl10 induce innate cellular responses by attracting and activating natural killer (nk) cells, macrophages, and neutrophils that further amplify the innate cytokine and chemokine response [17] . the role of other innate cells like, for example, intraepithelial lymphocytes, γδt cells, mucosa associated invariant t cells (mait), and innate lymphoid cells (ilc) is less well described in viral infections, however, they are likely to contribute to innate and adaptive responses against viral infections, as exemplified by the nk cells [25, 26] and by the role of ilc2 cells in overcoming an influenza infection [27] . if the innate immune or memory responses cannot clear the pathogen effectively and the adaptive immune system is unexperienced with the virus, an adaptive immune response is initiated and required. key are dendritic cells (dcs) that present the viral antigens and induce b and t cell responses against the pathogen in the secondary lymph nodes. b and t cell responses initiate within four-six days post-infection and peak later at days 7-14 depending on the respiratory virus [28] [29] [30] . typically, common respiratory viruses, such as rhinovirus and influenza virus, are cleared before adaptive immune responses are activated [22, 30] indicating that memory responses and innate immunity are essential in viral eradication. however, the induction of cytotoxic cd8 t cells, cd4 t cells, and antibody responses is key for virus eradication by adaptive immunity and for establishing protective immunity for secondary infections. the activation of the epithelium, innate immune cells, and adaptive responses is important for defense against respiratory viruses, but on the other hand, the host inflammatory response is the major cause of symptoms and more severe pathologies [12, 18, 31] . chronic activation of cd8 t cell responses and adaptive immunity may lead to pulmonary damage and acute respiratory distress syndrome, like in severe cases of coronavirus infections (e.g., sars-cov or sars cov-2) and pandemic influenza virus infections [18, 29, 32] . in milder colds, rhinoviruses are not cytolytic and do not actually cause considerable damage to host cells and may pass asymptomatically. presentation of cold symptom severity seems to correlate with host inflammatory response. specifically, the early expression of pro-inflammatory il-8 [33] and high levels of neutrophils in nasal aspirates [34] have been shown to correlate with symptom severity of rhinovirus and influenza infection [35, 36] . production of anti-inflammatory il-10, resolvins, and regulatory t cell responses acts as a natural mechanism to control lung inflammation during acute influenza virus (and others) infection [37] [38] [39] . virus-host immune interactions are key to viral pathogenesis and to ultimately determine the outcome of the infection. probiotics, by definition, are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host [40] . most probiotics are lactic acid bacteria, belonging to lactobacillus spp., (now with new taxonomy including lacticaseibacillus spp., lactiplantibacillus spp., levilactobacillus spp., ligilactobacillus spp., limosilactibacillus spp. [41] ) or bifidobacterium spp. furthermore, some strains of other microbial genera, such as propionibacterium spp., and bacillus spp., have been reported to have probiotic properties. traditional lactic acid bacteria have long been considered safe and suitable for human consumption as very few instances of infection have been associated with these bacteria, and several published studies have specifically addressed their safety (reviewed, e.g., by [42] [43] [44] ). regarding probiotics safety in rtis, meta-analyses conclude that the reported side effects related to the consumption of probiotics were minor [5, 6, 45] . probiotics are mostly consumed orally in the form of dietary supplements and food (e.g., yoghurt). therefore, their primary site of action is in the gastrointestinal (gi) tract [46] . however, probiotics have been detected with pcr-based methods from nasopharyngeal mucosa, adenoids, and tonsils after oral consumption [13, 47, 48] , but it is unclear what the contribution to upper respiratory tract immune stimulation against viral rti by probiotics is, as oral ingestion results in the stimulation of the intestinal immune system as well. the small intestine, that is naturally exposed to microbes and nutrients due to the thin mucosal layer, seems to play an important role in immune stimulation by probiotics [49, 50] . however, dissecting the relative contributions of the small and large intestine and upper gi tract on immune stimulation against rti is challenging with available research data. independent of the actual mucosal inductor site of the probiotic, it has been shown that lymphocytes circulate between mucosal tissues [51] . thus, local mucosal stimulatory effects may influence immune responses at other mucosal tissues and contribute to antiviral immunity. even very closely related bacteria have differences in their antigenic structures and thus influence the immune system uniquely. probiotics are thought to influence immune function primarily in a strain-specific manner [40] . although these effects in general are strain-specific, probiotics also share common mechanisms of immune stimulation, such as the secretion of metabolites. for instance, short chain fatty acids are known to have immunomodulatory effects [52] . direct effects of the probiotics on immune function are driven by interactions of bacterial structures or metabolites with receptors, like tlrs, on the host epithelial and immune cells. on the other hand, probiotics may influence immune function indirectly by changing the composition and/or activity of the host microbiota [53] . for example, in the small intestine where the number of endogenous bacteria is lower than in the large intestine, ingestion of probiotics temporally changes the microbiota composition and influences the host immune response [49, 50] . in rtis, orally consumed probiotics may elicit systemic effects from the gi tract via the "gut-lung axis" by modulating mucosal immune function [54, 55] . probiotics are taken up by m cells or by cx3c chemokine receptor 1 (cx3cr1)+ macrophages located in the gut epithelium and then transferred to dcs in the subepithelial tissue. probiotics are able to modulate dc polarization and function [56] that influence the subsequent t and b cell responses [57] in the inductive sites (peyer's patch, and mesenteric lymph nodes). t and b cells can also enter the circulation and migrate to extraintestinal sites, such as the respiratory tract [51, 58] . however, the exact mechanisms of probiotics (and their metabolites) in respiratory infections has not been clearly established and may be influenced by the investigated probiotic strain, microbiota composition, and immunological status of an individual. in the following chapters, we review the current pre-clinical evidence of immunomodulatory and antiviral mechanisms of probiotics against respiratory virus infections with a focus on the direct stimulatory effects of probiotics on virus-infected immune cells and animal models. probiotic bacteria can engage and activate tlrs leading to the activation of nf-κb and irfs in immune cells that are essential in antiviral defense. for example, it has been demonstrated in murine bone marrow-derived dcs that lactobacillus acidophilus ncfm and l. acidophilus x37 induce the upregulation of tlr3, il-12, and ifn-β in a tlr2-dependent manner [59] . in macrophage-derived raw264.1 cells, lactobacillus gasseri sbt2055 upregulated ifn-β and myxovirus resistance (mx)1 mrna expression [60] and in human monocyte-derived macrophages, two lacticaseibacillus rhamnosus strains, gg and lc705, induced type i ifn-dependent gene activation [61] . pro-inflammatory and ifn-regulated genes including il-6, il-12, il-1β, il-8, ccl20, cxcl10, mx1, and mx2 were induced by both lacticaseibacillus strains. in addition, the gene expression of tlr3 and tlr7, receptors recognizing viral dsrna and ssrna, respectively, was upregulated by these strains. tlr3 gene expression was upregulated only by l. rhamnosus lc705, the strain with higher antiviral potential, while tlr7 was moderately upregulated by both strains. in vitro studies with immune cells have also shown the ability of probiotics and their components to restrict viral replication. in human monocyte-derived macrophages, lacticaseibacillus strains showed the ability to prevent influenza a virus replication which correlated with the ability to activate type i ifn-dependent antiviral genes [61] . similarly, mouse adapted influenza a virus (pr8) titer was reduced in raw264.1 cells by l. gasseri sbt2055 [60] . in mouse bone marrow dcs, the inhibition of viral replication by l. acidophilus atcc4356 s-layer protein was demonstrated [62, 63] . priming of cells with s-layer protein prior to h9n2 avian influenza virus infection inhibited the invasion and replication of the virus, stimulated the type i ifn signaling pathway, increased il-10 mrna, and decreased tnf-α mrna expression [62] . polyinosinic/polycytidylic acid (poly i/c), a synthetic mimic of dsrna, is widely used in in vitro studies to stimulate tlr3. it induces characteristic inflammatory responses associated with virus infections such as increased production of inflammatory cytokines. stimulation of airway epithelial cells with poly i/c has been found to closely mimic inflammatory responses associated with respiratory virus infections [64] . poly i/c challenge in peripheral blood mononuclear cells (pbmcs) induced changes in the gene expression of tlr3, ifn, and nf-kb-dependent pathways similar to acute viral infections [63] . moreover, poly i/c was able to induce a pulmonary dysfunction similar to rsv in a mouse model [65] . probiotic bacteria have shown the ability to modulate poly i/c-induced responses. studies with heat-killed lacticaseibacillus casei crl431 showed that the strain reduced tnf-α, ifn-γ, and il-17 levels when it was introduced before or simultaneously with poly i/c challenge in pbmcs alone and in a co-culture system with alveolar epithelial cell line a549 [66] . il-10 and il-29 (also known as ifn-λ1) were induced in response to poly i/c together with heat-killed l. casei crl431, indicating a boost in pro-inflammatory responses and the activation of anti-inflammatory and antiviral mechanisms. in the intestinal human colon cell line (hct116), regulation of poly i/c response by lactiplantibacillus plantarum subsp. plantarum du1, latilactobacillus sakei du2, and weissella cibaria du1 was examined [67] . these strains modified poly i/c-induced expression of cytokines and antiviral genes by upregulating ifn-β, tlr3, and rig-i while dampening the inflammatory response. moreover, the probiotic strains induced ifn-α, ifn-β, and il-10 and reduced the expression of inflammatory cytokines il-1β and tnf-α in human monocytic thp-1 macrophages [67] . in addition to the pro-inflammatory and antiviral gene activation described above, also direct interactions of viruses and probiotic bacteria have been demonstrated between porcine influenza a virus and enterococcus faecium in vitro [68] . similar upregulation of ifn response by probiotics has been shown in several studies in intestinal epithelial cells [69] [70] [71] and macrophages [68, 72] . overall, the in vitro studies indicate that probiotics may stimulate similar innate immune pathways to respiratory viruses and potentially modulate virus-induced immune responses. several mouse studies have shown that the administration of probiotics can help to fight against viral rtis. the beneficial effects of oral probiotic supplementation on mouse survival and health status is well demonstrated [60, [73] [74] [75] [76] [77] . for example, oral administration of lacticaseibacillus paracasei subsp. paracasei cncm-i-1518 [74] , l. gasseri lg2055 [60] , and bifidobacterium longum mm-2 [75] reduced mortality and improved immune control in influenza-infected mice. probiotic administration resulted in better health status of the mice and lower virus loads in the lungs after influenza infection. in addition, the immune response to viruses was modulated by probiotic administration. for instance, l. paracasei cncm-i-1518 modified the pro-and anti-inflammatory cytokine release in the lungs before and after influenza infection and affected total cell counts in the lungs after probiotic treatment [74] . similarly, b. longum mm-2 suppressed inflammation in the lower respiratory tract through the decrease in influenza virus proliferation and pulmonary il-6 and tnf-α cytokine production [75] . activation of host defense systems by increased ifn-γ, il-2, il-12, and il-18 gene expression and nk cell activation in lungs was also demonstrated. in non-infected mice, b. longum mm-2 significantly enhanced ifn-γ production by peyer's patch cells and splenic nk cell activity. in the infected mice, nk cell activity was significantly enhanced both in the spleen and lungs by the probiotic strain. another bifidobacterium (b. bifidum) improved anti-influenza immune responses by inducing both humoral and cellular immunities [76] . decreased il-6 levels were detected in the lung and higher igg1 and igg2 levels in the sera of probiotic-treated mice compared with control mice. furthermore, l. gasseri sbt2055 has been found effective in preventing both influenza a virus [60] and rsv [78] infections in mice. pre-treatment with l. gasseri sbt2055 induced the expression of antiviral genes mx1 and 2 -5 oligoadenylate synthase (oas)1a in lung tissues before viral infection and reduced lung inflammatory responses after viral infection [60] . the same l. gasseri strain was found effective in preventing rsv infection in mice by reducing lung viral loads and pro-inflammatory cytokines and by stimulating ifns and ifn responsive gene expression such as ifn-β1, ifn-γ, interferon-inducible transmembrane protein (ifitm)3, oas1a, and interferon-stimulated gene (isg)15 [78] . in addition to live probiotics, also orally administered heat-killed bacteria seem to confer protection against viral rtis in mice [73] . heat-killed l. paracasei mcc1849 reduced symptom scores and lung virus titers in influenza-infected mice and induced antigen-specific iga production in the small intestine, serum, and lungs. the proportion of iga+ b cells and follicular helper t cells (tfh) in peyer's patches was increased as was the gene expression of il-12p40, il-10, il-21, signal transducer and activator of transcription (stat)4, and b cell lymphoma protein (bcl)-6, which are associated with tfh cell differentiation. to conclude, different probiotic strains have been shown effective in inhibiting the replication of various respiratory viruses including influenza viruses and rsv in vitro. similar effects have been demonstrated in several mouse studies with the ability to reduce virus titers in lung tissues and modulation of antiviral and pro-inflammatory gene expression before and after viral infection. accumulating clinical evidence suggests that probiotics in general may have favorable effects against rtis. for instance, several systematic reviews and/or meta-analyses have evaluated the effects of prophylactic ingestion of probiotics on the rti-associated outcomes, e.g., either only in children [79, 80] , or both in children and adults [5, 6, 45] (table 1) . of note, the majority of the outcomes in these analyses are related to urti, and data on lrti outcomes are either not available or are very limited. therefore, in the below chapter, we primarily focus on clinical trials on probiotics' effects on urti symptoms/episodes/duration. probiotic consumption had no effect on the duration of rtis (9 rcts, n = 3529, md -0. 81 in children (below 18 years), the meta-analysis by wang et al., 2016 , reported that probiotic use compared with placebo significantly decreased the number of subjects having at least one rti episode, had fewer numbers of days of rtis per person, and had fewer numbers of days absent from daycare or school [80] . however, the meta-analysis did not find a statistically significant difference on the illness episode duration between the probiotic and the placebo. laursen and hojsak [79] limited the analysis to children up to 7 years old and reported that probiotic use was associated with reduced risk of at least one urti and reduced the risk of antibiotic use, but the use was not associated with a reduction in rti duration or missed days of daycare due to rti [79] . this meta-analysis also discussed the effects of the individual probiotic strains on rti outcomes. the results of the analysis showed that the most effective probiotic strains on rti-related outcomes were l. rhamnosus gg (rti duration) and l. acidophilus ncfm as a single supplement and in combination with b. lactis bi-07 (rti duration and antibiotic use). interestingly, these strains have shown in vitro the ability to induce antiviral ifn signaling pathways (see section 4.2) which may potentially explain their beneficial effects observed in rtis. however, as multiple studies with probiotic strains other than l. rhamnosus gg are limited or lacking, comparison and interpretation of the strain specific results should be made carefully. meta-analyses that pool data from clinical trials conducted with children, adults, and the elderly show that probiotic use is more beneficial over placebo in reducing the number of participants experiencing episodes of acute urti [5, 6] , reducing antibiotic prescription rates for acute urtis [5, 6] , and reducing the mean duration of an episode of an acute urti as well as cold-related school absences [5, 45] . when the literature search was conducted, meta-analyses were not found in the databases searched on probiotic effects on respiratory infections restricted to the elderly population, potentially due to the fact that data are fairly limited regarding this age group. while there is consensus that probiotics could have potential in reducing the risk for rtis, it should be noted that clinical trials in the meta-analyses have been conducted in populations of different ages and genetic backgrounds, with various strains and/or their combinations, supplementation matrices, and doses. moreover, the measured outcomes and data collection procedures between the trials (i.e., infection episode definition) are not harmonized and therefore may vary considerably. consequently, pooling all the data creates a bias, as the probiotic effect is generally dependent on the dose, population, and strain. moreover, as discussed above, the probiotics effects on the immune system are strain-specific which affects the interpretation of the results. with regard to probiotics effects to specific respiratory viruses in clinical settings, several trials have characterized the respiratory infection etiology in infants [81] , in children [82] [83] [84] , in adults [85] , and in the elderly [86] . in addition, two clinical trials have investigated the efficacy of probiotics in an experimental rhinovirus challenge model [87] [88] [89] (table 2) . children had less days with respiratory symptoms per month (6.5 vs. 7.2, p < 0.001). no effect on the occurrence of respiratory viruses during the study or respiratory symptoms associated with viral findings. l. rhamnosus gg 10 9 cfu of live or heat-inactivated (by spray-drying) in 100 ml of fruit juice or control juice daily for 6 weeks. in the clinical trials conducted in free-living subjects in the community, no consistent data exist that show that specific probiotics would reduce the incidence of laboratory-confirmed respiratory virus infections as such. in preterm infants, the use of l. rhamnosus gg for 60 days was associated with lower incidence of rhinovirus-induced episodes (comprising 80% of all rti episodes) compared with the placebo. however, l. rhamnosus gg had no effect on rhinovirus rna load during infections, duration of rhinovirus rna shedding, duration or severity of rhinovirus infection, or the occurrence of rhinovirus rna in asymptomatic infants. in children attending daycare, l. rhamnosus gg [83] consumption for 28 weeks did not reduce the occurrence of any of the common respiratory viruses either. in otitis-prone children, supplementation of a combination of l. rhamnosus gg, l. rhamnosus lc705, b. breve 99, and propionibacterium jensenii js, for six months, reduced the number of human bocavirus-positive nasopharyngeal samples when compared with placebo, but not the number of rhino/enterovirus-positive samples [84] . furthermore, in schoolchildren, the consumption of levilactobacillus brevis kb290 during influenza season was associated with lower incidence of physician-diagnosed influenza virus cases [82] . in adults attending military service, the use of a combination of l. rhamnosus gg and b. lactis bb-12 for either 90 or 150 days was not overall associated with lower occurrence of common respiratory viruses upon presentation of cold symptoms [85] . however, in a subgroup, there was a lower occurrence of rhino/enteroviruses after three months in the probiotic group when compared with the placebo. in nursing home residents, wang et al., 2018 , reported that the use of l. rhamnosus gg for six months was not associated with the reduction in occurrence of confirmed viral respiratory infections [86] . the differences between the findings in these trials may be explained by the fact that these studies were conducted in various age groups with different immune system statuses (infants vs. children vs. healthy adults vs. the elderly), different seasons, as well as variable probiotic strains, strain combinations, doses, and variable lengths of intervention. furthermore, most of the studies were not designed for analyzing the viral infection etiology as the primary outcome and the diagnosis for the identification of the viral agent was not applied. since over 200 respiratory virus types can cause respiratory infections and, in many cases, the infections and symptoms overlap, or the etiology is undiagnosed, the potential antiviral effects of probiotics against specific viruses can be difficult to determine in clinical trials targeting free-living subjects within the community. to overcome this caveat, two probiotics have been investigated in an experimental rhinovirus challenge model that allows investigation of the effect of a probiotic strain to a specific viral pathogen. in a rhinovirus (type 39) challenge model, b. lactis bl-04 was administered for 28 days prior and during five days of experimental rhinovirus infection to healthy volunteers [89] . b. lactis bl-04 supplementation resulted in significantly lower rhinovirus titers in nasal washes during the infection as well as in a lower number of infected participants shedding the virus compared with the placebo. moreover, b. lactis bl-04 induced a significantly higher concentration of il-8 in nasal washes after 28 days of supplementation and prior to infection. given the reduced viral titer, an increase in il-8 could indicate priming of the mucosal immune system prior to infection. this hypothesis is in line with a clinical study conducted in healthy active adults, where supplementation of b. lactis bl-04 reduced the risk of urti episodes compared with placebo [90] . in another similar experimental rhinovirus type 39 challenge pilot trial, no significant antiviral effect was seen with live or inactivated l. rhamnosus gg supplementation compared with placebo [87, 88] , suggesting potential strain-specific differences on the efficacy of probiotics in respiratory virus infections. nevertheless, further adequately powered trials with harmonized study designs are necessary to draw conclusions on the efficacy of probiotics against specific respiratory viruses. viral rtis are the most common infections of mankind and the health and financial impact of seasonal epidemics and global pandemics on society is high. due to the large number of various respiratory viruses, the development of efficient therapies, such as vaccines, is challenging. when preventative measures are scarce or lacking, the role of a well-functioning immune system becomes crucial for providing resistance to an infection. within the past decade, research highlighting the importance of the microbiota on immune system function has raised interest in understanding the role of microbiota modulation and bacterial therapeutics by dietary and pharmaceutical solutions in health and disease. of the available solutions, probiotic bacteria have been studied for immune function modulation in the context of respiratory viral infections. in this review, we have summarized the current evidence on the effects of probiotics on antiviral immune function in vitro and in vivo, and clinical evidence on the effect of probiotics on viral rtis and on the course of rti (figure 1 ). importance of the microbiota on immune system function has raised interest in understanding the role of microbiota modulation and bacterial therapeutics by dietary and pharmaceutical solutions in health and disease. of the available solutions, probiotic bacteria have been studied for immune function modulation in the context of respiratory viral infections. in this review, we have summarized the current evidence on the effects of probiotics on antiviral immune function in vitro and in vivo, and clinical evidence on the effect of probiotics on viral rtis and on the course of rti (figure 1 ). in vitro data indicate that probiotics have strain-specific immunomodulatory effects on the host and immune cells by engaging tlrs that stimulate ifn pathways. the upregulation of ifn response seems to prime cells for better resistance against virus infection as probiotics were shown effective in inhibiting the replication of various respiratory viruses, including influenza viruses and rsv. similar effects have been demonstrated in mice with the ability of the probiotics to reduce virus titers in lung tissues and to modulate antiviral and pro-inflammatory gene expression before and after viral infection. interestingly, some studies in mice show an increase in il-10 response, suggesting control of the pro-inflammatory response that typically drives lung pathology in severe infections. most likely probiotics' effects in the gut are transferred into the respiratory tract via the gut-respiratory tract axis, however, this mechanism of action remains to be studied in more detail. the pre-clinical studies further show improvement in the symptom scores of mice, suggesting potential clinical benefits. indeed, some evidence exists for specific probiotic strains, e.g., from the species of l. rhamnosus, l. acidophilus, and b. lactis for their ability to induce antiviral immune responses in preclinical models, which is in agreement with their effects observed in clinical trials in reducing the risk of rti-associated outcomes. however, translation of probiotic effects from cell culture and animal studies to humans can be challenging and variable confounding factors, e.g., age, diet, microbiome, genetic and epigenetic immune status of an individual, study season, and variable viral epidemiology, all have an impact on the study outcome and are difficult to standardize. the clinical studies that have diagnosed and characterized viral etiology are limited, nevertheless, the metaanalyses investigating probiotic clinical interventions on rtis show that probiotic use is associated with lower incidence and duration of mild rtis, both in children and in adults. further studies aiming at discovering the mechanism of action of probiotics and establishing the association of immune system function stimulation and clinical efficacy are warranted. in vitro data indicate that probiotics have strain-specific immunomodulatory effects on the host and immune cells by engaging tlrs that stimulate ifn pathways. the upregulation of ifn response seems to prime cells for better resistance against virus infection as probiotics were shown effective in inhibiting the replication of various respiratory viruses, including influenza viruses and rsv. similar effects have been demonstrated in mice with the ability of the probiotics to reduce virus titers in lung tissues and to modulate antiviral and pro-inflammatory gene expression before and after viral infection. interestingly, some studies in mice show an increase in il-10 response, suggesting control of the pro-inflammatory response that typically drives lung pathology in severe infections. most likely probiotics' effects in the gut are transferred into the respiratory tract via the gut-respiratory tract axis, however, this mechanism of action remains to be studied in more detail. the pre-clinical studies further show improvement in the symptom scores of mice, suggesting potential clinical benefits. indeed, some evidence exists for specific probiotic strains, e.g., from the species of l. rhamnosus, l. acidophilus, and b. lactis for their ability to induce antiviral immune responses in pre-clinical models, which is in agreement with their effects observed in clinical trials in reducing the risk of rti-associated outcomes. however, translation of probiotic effects from cell culture and animal studies to humans can be challenging and variable confounding factors, e.g., age, diet, microbiome, genetic and epigenetic immune status of an individual, study season, and variable viral epidemiology, all have an impact on the study outcome and are difficult to standardize. the clinical studies that have diagnosed and characterized viral etiology are limited, nevertheless, the meta-analyses investigating probiotic clinical interventions on rtis show that probiotic use is associated with lower incidence and duration of mild rtis, both in children and in adults. further studies aiming at discovering the mechanism of action of probiotics and establishing the association of immune system function stimulation and clinical efficacy are warranted. the common cold epidemiology of viral pneumonia. clin microbiome and disease in the upper airway the influence of the microbiome on respiratory health probiotics for preventing acute upper respiratory tract infections probiotics for preventing acute upper respiratory tract infections respiratory virus infections emerging respiratory viruses other than influenza sars-cov-2 viral load in upper respiratory specimens of infected patients epidemiology of viral respiratory infections epidemiologic, clinical, and virologic characteristics of human rhinovirus infection among otherwise healthy children and adults: rhinovirus among 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individuals key: cord-320663-xypg6evo authors: market, marisa; angka, leonard; martel, andre b.; bastin, donald; olanubi, oladunni; tennakoon, gayashan; boucher, dominique m.; ng, juliana; ardolino, michele; auer, rebecca c. title: flattening the covid-19 curve with natural killer cell based immunotherapies date: 2020-06-23 journal: front immunol doi: 10.3389/fimmu.2020.01512 sha: doc_id: 320663 cord_uid: xypg6evo natural killer (nk) cells are innate immune responders critical for viral clearance and immunomodulation. despite their vital role in viral infection, the contribution of nk cells in fighting sars-cov-2 has not yet been directly investigated. insights into pathophysiology and therapeutic opportunities can therefore be inferred from studies assessing nk cell phenotype and function during sars, mers, and covid-19. these studies suggest a reduction in circulating nk cell numbers and/or an exhausted phenotype following infection and hint toward the dampening of nk cell responses by coronaviruses. reduced circulating nk cell levels and exhaustion may be directly responsible for the progression and severity of covid-19. conversely, in light of data linking inflammation with coronavirus disease severity, it is necessary to examine nk cell potential in mediating immunopathology. a common feature of coronavirus infections is that significant morbidity and mortality is associated with lung injury and acute respiratory distress syndrome resulting from an exaggerated immune response, of which nk cells are an important component. in this review, we summarize the current understanding of how nk cells respond in both early and late coronavirus infections, and the implication for ongoing covid-19 clinical trials. using this immunological lens, we outline recommendations for therapeutic strategies against covid-19 in clearing the virus while preventing the harm of immunopathological responses. natural killer (nk) cells are a key component of the innate immune system and are critical in the response to many viral infections in humans and animal models (1) (2) (3) . in addition to their beneficial antiviral role, nk cells have also been associated with immunopathology in infections such as respiratory syncytial virus (rsv) (4), influenza a virus (5) (6) (7) (8) , and hepatitis b (9) . additionally, in the context of non-respiratory viral infections by hiv and hcv, nk cells appear to act as a rheostat by eliminating activated cd4+ and cd8+ t cells, thus preventing t cell-mediated autoimmunity (10) . the etiologic agent of the 2019 outbreak of pneumonia in wuhan, china, was identified as belonging to the coronaviridae family and named severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this virus causes the coronavirus disease 2019 (covid19) which was declared a pandemic by the world health organization (who) on march 11th, 2020 (11, 12) . with the paucity of information currently available, there is a lack of consensus on the role played by nk cells in the response to coronavirus (cov) infection. in this review, we will explore evidence for both the protective and pathological role that nk cells may play in cov infection. based on this knowledge we will comment on immune modulating treatment options that are being developed for the current covid-19 crisis. first discovered in the 1960s, covs are part of the coronaviridae family of enveloped positive single-strand rna viruses (13, 14) . the subfamily orthocoronaviridae includes four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (15) . alpha-and betacoronaviruses circulate in mammals, including bats, gammacoronaviruses infect mostly avian species, and deltacoronaviruses infect birds and mammals (15) . low pathogenic human covs (hcovs), such as hcov-299e (16) , infect upper airways and etiological studies suggest they account for 15-30% of common colds (17, 18) . on the other hand, highly pathogenic covs infect the lower respiratory tract and can cause severe pneumonia (19) . these highly pathogenic covs include sars-cov-1, the virus responsible for the 2002-2004 severe acute respiratory syndrome (sars) epidemic, and mers-cov, the virus responsible for the outbreak of middle eastern respiratory syndrome (mers) in 2015 (19) (20) (21) . while highly pathogenic covs have become a relatively recent issue for humans; feline, canine, and bovine covs have long been recognized as significant pathogens with implications in veterinary medicine and agriculture (22, 23) . all covs have a roughly 30 kb genome packed into an enveloped helical capsid ranging from 80 to 120 nm (24) . at minimum, coronaviridae members encode 4 structural and 16 non-structural proteins (14) with the family owing its name to the crown-like appearance produced by their spike (s) proteins (25) . mutations in the s protein have allowed sars-cov1/2 to co-opt ace2 or mers-cov to co-opt dipeptidyl peptidase 4 (dpp4) receptor/cd26 as viral entry receptors, thus facilitating the zoonosis of nonhuman covs (15, (26) (27) (28) . in addition, another mechanism that may have allowed these viruses to adapt to human hosts is through s protein cleavage by host cell proteases to expose the s2 domain fusion peptide, which induces viral and cellular membrane fusion and results in the release of viral genome into the cytoplasm (15) . genetic sequencing revealed sars-cov-2 to be a betacoronavirus that shares 79.0% nucleotide identity with sars-cov-1 and 51.8% identity to mers-cov (29) . the epidemic of sars in 2002-2004 caused by sars-cov-1 illustrated the devastating potential of coronaviruses to cause serious disease in humans (24) . sars ultimately reached 29 countries and 5 continents causing over 8,000 infections and over 900 deaths. the basic reproductive rate (r 0 ) or the number of expected cases arising from one infected individual, ranges from 2 to 4 (20, 30, 31) . with its reservoir in bats, sars-cov-1 is a zoonosis that was transmitted to humans by palm civets (24, 32, 33) . sars-cov-1 infects lung pneumocytes (34) and enterocytes in the digestive tract (35) most often producing flulike symptoms (36, 37) . more severe presentations including pneumonia, pronounced lymphopenia, liver abnormalities, and acute respiratory distress syndrome (ards) were also reported, with most fatalities due to respiratory failure (19, (36) (37) (38) (39) . the subsequent mers-cov outbreak in 2015 also originated in bats, with dromedary camels being the intermediary host (14, 40, 41) . the r 0 for mers-cov is estimated to be under 1 (21) . the extent of mers-cov transmission was more limited than sars-cov-1, but its case fatality rate was greater with 2,494 cases over 27 countries and 858 deaths being reported at the end of 2019 (21) . common presentations for mers-cov include fever, dyspnea, muscle pain, and digestive tract symptoms and disease progression is more likely in those with comorbidities (42) . like sars-cov-1 and mers-cov, sars-cov-2 is thought to have originated in bats through an unknown intermediary host (43) . at the time of writing, the number of global infections is estimated to be over 5,000,000 with over 340,000 deaths (44) and the r 0 is roughly 2.2 (45) . like other diseases caused by infectious covs, most patients present with flu-like symptoms including fever, cough, and lethargy, with the development of pneumonia and ards often proving fatal (46) . furthermore, patients with underlying conditions are at risk for further complications if infected with covid-19, such as those with cardiovascular disease (47) . sars-cov-2 has been posthumously detected in not only the lungs, but the pharynx, heart, liver, brain, and kidneys (48) . transmission of sars-cov-2 is thought to mainly occur through direct contact/inhalation of respiratory droplets and aerosols from infected carriers, but indirect transmission by fomites has also been reported, although less efficient (49, 50) . sars-cov-2 viral entrance is thought to be mediated by binding of the s protein to the ace2 receptor (51, 52) , although this is still under debate (53) . while direct cytopathic effects are thought to play a major role in cov pathology, studies have suggested that a dysregulated immune response resulting in pathological inflammation is also partly responsible (19) . with the current pandemic already surpassing the previous cov outbreaks (54) , rapid deployment of novel approaches to understanding and treating coronavirus infections are needed. innate immunity is essential in disease prevention and viral clearance. among the first responders to viral infections, tissueresident macrophages and dendritic cells (dcs) (55) recognize evolutionarily conserved microbial structures termed pathogenassociated molecular patterns (pamps) via germline-encoded pattern recognition receptors (prrs) (56). in the context of respiratory rna viruses, airway epithelial cells, that also express some prrs (57) , are often infected and have a major role in the first line of defense. tlr3, tlr7, tlr8, mda-5, and rig-i are prr expressed by immune and non-immune cells that are especially relevant in fighting respiratory rna viruses, such as coronaviruses (57) . sensing through prrs results in the transcription of genes involved in the inflammatory response, with type i interferons (ifns) (ifn-α/β) production being a critical part of the antiviral response (58) . type i ifns are produced by many immune and non-immune cells (55, 57, 59) and in addition to eliciting intrinsic antiviral responses (60), they are also essential to prime innate and adaptive lymphocytes, including nk cells (61) . nk cells are cytotoxic lymphocytes that directly target infected, stressed, or transformed cells and play a critical role in bridging the innate and the adaptive immune responses (62) . in humans, mature nk cells comprise 10-15% of total peripheral blood leukocytes and are described phenotypically as cd3 − cd14 − cd19 − cd56 + cd16 +/− (63). nk cells do not undergo clonal selection but instead express several germline-encoded receptors that regulate their activity (62, 64, 65) . upon viral infection, host cells become more susceptible to nk cell killing through: (i) upregulation of self-encoded molecules induced by infection/cellular stress (66, 67) that bind activating nk cell receptors such as natural cytotoxicity receptors (ncrs) (nkp30, nkp44, and nkp46) (68), c-type lectin-like receptors nkg2d and nkp80 (69) , and co-activating receptors such as dnam-1 (70); (ii) downregulation of ligands for inhibitory receptors such as killer immunoglobulin-like receptors (kirs) (71) (72) (73) and the c-type lectin-like receptor cd94-nkg2a (74, 75) which suppress nk cell activation, and; (iii) direct recognition of viral moieties, via engagement of pamps (76) or transmembrane activating receptors such as mouse ly49h (77) or human nkg2c (78) . moreover, nk cells can eliminate virus-infected cells via cd16-mediated antibody-dependent cellmediated cytotoxicity (adcc), which has been shown to be particularly important for herpesvirus clearance (79) . finally, nk cell activity is modulated by cytokines, including, but not limited to, the activating cytokines interleukin (il)-2/12/15/18 (80) and type i ifn, which can be produced by virally infected cells or activated antigen presenting cells (81, 82) . il-2/12/15/18, alone or in combination, promotes nk cell survival, proliferation, cytotoxicity, and cytokine production, including ifn-γ (80) . therefore, nk cells are uniquely equipped to sense and quickly respond to viral infections. nk cells are found in circulation and in peripheral tissues (63) and can be quickly recruited to sites of infection where they facilitate and accelerate viral clearance. in fact, nk cells are not thought to have permanent tissue residency but instead move dynamically between the blood and tissues, such as the lungs (83) . nk recruitment is regulated by chemokine gradients that are sensed via chemokine receptors (84, 85) . activated nk cells induce the apoptosis of target cells through the engagement of death receptors, such as trail and fas (86) or via direct cytotoxicity through ca 2+ -dependent exocytosis of cytolytic granules (perforin and granzymes) (87) . moreover, nk cells secrete cytokines, including ifn-γ, which have key anti-viral properties (88) . in addition to being essential first responders to viral infection, nk cells can elicit a stronger secondary response resembling the memory features of adaptive lymphocytes (89, 90) . nk cell memory has been initially described in mice infected by mcmv, where ly49h + nk cells quickly expand and have stronger responses after a secondary encounter with the virus (91) . interestingly, a similar nk cell subset has been identified in humans, where nk cells expressing nkg2c are expanded and persist in cmv infected patients (92) . both ly49h and nkg2c bind viral determinants, highlighting how nk cell memory is linked with the ability of nk cells to directly recognize viruses (93, 94) . in addition to direct recognition of viral molecules, longlasting changes in nk cells are induced by the cytokine milieu (89, 95) , which can be elicited by viral infection. the relevance of nk cells in fighting viral infections has been highlighted by several studies where nk cells, in mice and humans, were not present or had compromised functions (96) . for example, individuals with nk cell deficiencies (nkd), a subset of primary immunodeficiency diseases, are highly susceptible to viral infection, particularly by herpesvirus and papillomavirus families (96) . the seminal 1989 case of nkd in an adolescent female with severe herpesvirus infections (varicella pneumonia, disseminated cmv, and disseminated hsv) revealed how functional nk cell deficiencies have clinical consequences in terms of viral infections (97) . cancer patients are also at risk of viral infections (98) , which may be explained, at least in part, by an impairment of nk cell responses often observed in humans and in murine tumor models (99) (100) (101) (102) (103) (104) . unsurprisingly, cancer patients are at a significantly increased risk of severe covid-19 (105, 106) . elderly patients are also more susceptible to viral infections (107) . mouse studies highlighted how a decreased number of circulating mature nk cells in aged animals paralleled with increased susceptibility to viral infections (108) . studies in humans suggest that although nk cell numbers can actually increase with aging, nk cell activity declines significantly (109, 110) . przemska-kosicka et al. investigated nk cell function in response to seasonal influenza vaccination in young and old populations and observed quantitative and qualitative changes associated with impaired responses in the nk cell population and this was associated with poor seroconversion in the older population (111) . additionally, obesity, which has been shown to cause systemic nk cell dysfunction (112, 113) , has also been linked to increased covid-19 severity and could be the reason behind the high prevalence of severe covid-19 in younger people (113) . in short, nkd and individuals with reduced nk cell numbers or function are more susceptible to viral infections. unsurprisingly, the cdc has already highlighted a higher risk of infection and severity of covid-19 in older individuals and individuals with comorbidities such as obesity and cancer (114) . however, this point is still controversial as a systematic review showed that primary immunodeficiencies are not linked with increased covid-19 severity (115) , but these data have to be interpreted keeping in mind that a large part of covid-19 pathology is caused by excessive immune activation, which is arguably harder to reach in immunocompromised individuals. given the paradoxical role of the immune response in covid-19 patients, it would be extremely useful to be able to rely on immunological functional biomarkers that could predict the outcome of disease severity. such assays are readily available for determining nk cell activity, e.g., nkvue tm , and there is therefore an opportunity to conduct studies that would link nk cell functions to disease severity. evasion of host immune responses is necessary for the successful propagation of a virus. mechanisms employed by covs to evade the immune response could provide insights into how the immune system, and nk cells in particular, responds to sars-cov-2. covs have been shown to target components of the innate ifn response, employing non-structural proteins (nsps), structural proteins, and accessory proteins to achieve this goal. nsp16 methylates viral rna therefore preventing recognition by mda5 and dampening type i ifn production (116) . nsps also suppress type i ifn responses via the inhibition of the transcription factor stat1 mrna transcription (nsp1) and deubiquitination of transcription factors like interferon regulatory transcription factor (irf)3 (nsp3) (116) . moreover, viral-encoded accessory proteins from sars-cov-1 open reading frame (orf)3b and mers-cov orf4a/4b also block ifn production and signaling (116) . in addition, the mers-cov orf6-encoded protein blocks p-stat1 import, thus blocking ifn signaling (116) . finally, the structural m protein of mers-cov (27) physically sequesters kinase proteins rig-i, tbk1, ikke, and traf3 and the sars-cov-1 n protein inhibits activator protein (ap)-1 signaling, protein kinase r function, and nfκb activation, all of which act to impede ifn responses (117) . in vivo murine studies report young mice rapidly clear sars-cov-1 infection, while old mice do not and that this discrepancy is due to a delay in type i ifn. furthermore, early administration of ifn-β induces a stronger immune response and reduces mortality in old mice (118) . since type i ifns are critical for nk cell activation and effector functions, it is possible that nk cell-mediated clearance of sars-cov-2 is being subverted by these mechanisms. further research into the role of nk cells in cov clearance and potential immune evasion mechanisms are necessary to inform therapeutic development and use. there is currently a paucity of studies into the role of nk cells not only in covid-19 pathophysiology, but also in other coronavirus infections. an in vivo study reported that beige mice on a b6 background cleared sars-cov-1 normally, indicating that functional lymphocytes, including nk cells, may not be required to eliminate sars-cov-1 in murine models (119) . however, in a more recent study characterizing the cellular immune response to sars-cov-1 in 12-14-month old balb/c mice, t cell depletion did not prevent control of sars-cov-1 replication (120), suggesting a role for the innate immune system, and nk cells, in viral clearance. importantly, in this study cd4-depletion resulted in enhanced lung immunopathology and delayed viral clearance, while cd8-depletion did not affect viral replication or clearance, thus highlighting an important role for cd4 + t cells in coronavirus infection. these conflicting results may be due to the inherent limitations of cov murine models. in 4-8 week-old mice, sars-cov-1 is associated only with mild pneumonitis and cytokines are not detectable in the lungs (119, 121, 122) . a sars-cov-1 isolate (ma-15) replicates to a high titer and is associated with viremia and mortality, however the model lacks significant inflammatory cell infiltration into the lungs (123) . thus, mouse models developed for the study of sars fell short in terms of reproducing the clinical and histopathological signs of disease (119, (121) (122) (123) . it is therefore necessary to develop a usable animal model that is capable of reproducing the clinical and histopathological signs on covid-19. israelow et al. recently described a sars-cov-2 murine model based on adeno associated virus (aav)9-mediated expression of human (h)ace2, which replicated the pathologic findings found in covid-19 patients (124) . this model, which overcame the inability of murine (m)ace2 to support sars-cov-2 infection, was used to show the inability of type i ifn to control sars-cov-2 replication (124) . in a similar attempt to overcome the lack of infectability through mace2, dinnon et al. recently described a recombinant virus (sars-cov-2 ma) with a remodeled s protein mace2 interface, which replicated in upper and lower airways in young and aged mice with disease being more severe in aged mice. the authors used this model to screen therapeutics from vaccine challenge studies and assessed pegylated ifn-λ-1 as a promising therapeutic. the authors suggested that this model has greater ease of use, cost, and utility over transgenic hace2 models (125) to evaluate vaccine and therapeutic efficacy in mice (126) . a preliminary analysis of nk cell function and phenotype has been performed by zheng et al. using peripheral blood from covid-19 patients (127). on admission, nk cell levels in the peripheral blood inversely correlated with disease severity. furthermore, covid-19 patients with severe disease had significantly lower numbers of circulating nk cells, as compared to mild disease (p < 0.05) (127) . additionally, circulating nk cells in severe disease displayed increased expression of the inhibitory receptor nkg2a and had an hyporesponsive phenotype with lower levels of ifn-γ, tumor necrosis factor (tnf)-α, il-2, and granzyme b, although degranulation was maintained (127) . finally, as compared to patients with active disease, patients recovering from covid-19 had higher numbers of nk cells and lower nkg2a expression (127) . liao et al. performed singlecell rnaseq on the cells obtained from bronchoalveolar lavage fluid of severe and mild covid-19 patients and found that covid-19 patients had significantly more nk cell infiltrates into the lungs, however patients with severe disease had reduced proportions of nk cells (128) . in addition, klrc1 (nkg2a) and klrd1 (cd94) were highly expressed by nk cells (128) . carvelli et al. analyzed myeloid and lymphoid populations by immunophenotyping from blood and bronchoalveolar lavage fluid (balf) in 10 healthy controls, 10 paucisymptomatic covid-19 patients, 34 pneumonia patients, and 28 patients with ards due to sars-cov-2 and found that absolute numbers of peripheral blood lymphocytes, including nk cells, were significantly reduced in the pneumonia and ards groups compared to healthy controls. furthermore, the proportion of mature nk cells was reduced in patients with ards and nk cells showed increased nkg2a, pd-1, and cd39 (129) . finally, wilk et al. performed single-cell rna-sequencing on 7 covid-19 patients and 6 healthy controls and found that the cd56 bright population was depleted in all covid-19 patients but the cd56 dim population was depleted only in patients with severe covid-19. furthermore, nk cells had increased expression of the exhaustion markers lag3 and havcr2 (130) . nk cell cytopenia seems to be a consistent characteristic among sars-cov-2 infected patients (131). altogether, these data indicate alterations in the nk cell phenotype and functional profile that are consistent with the hypothesis that to establish a productive and lasting infection, sars-cov-2 needs to dampen the nk cell response. nk cell dysfunctions were also observed in patients from the previous cov outbreaks. wang et al. assessed nk cell number and phenotype using peripheral blood from 221 sars patients admitted to hospitals in beijing, china (132) . nk cell proportion and absolute number were significantly reduced in sars patients as compared to healthy donors and patients infected with the bacterium mycoplasma pneumoniae (131). nk cell number correlated inversely with disease severity and patients with anti-sars cov-specific igg or igm antibodies had significantly fewer nk cells (132) . the patients assessed had varied disease duration from 4 to 72 days (mean 31.7 days) and this allowed for patient stratification by disease duration. within the first 10 days of sars-cov-1 infection, nk cell numbers remained high but this period was followed by the development of lymphopenia with levels recovering only around day 40 (132) . dong et al. also observed a reduction of nk cell numbers in sars patients, and these levels were lower in patients with severe, as compared to mild, sars (133) . in addition, mers infection is strongly associated with leuko-and lymphopenia (42, (134) (135) (136) . the mechanisms underlying the reduction of circulating nk cells in patients infected with covs are still unclear. as most studies have focused on peripheral blood nk cells, it is possible that the reduced number of circulating nk cells is due to redistribution of blood nk cells into the infected tissues (137) . while it is hard to assess nk cell migration to infected tissues in covid-19 patients, this hypothesis was corroborated by mouse studies, where nk cells have been shown to migrate to the lungs in cov infected animals (120) . an abundance of inhibitory factors, such as tgf-β, may be partially responsible for the nk cell hyporesponsiveness observed in covid-19 patients. in support of this hypothesis, huang et al. found significantly higher tgf-β levels in sars patients compared to healthy controls and this positively correlated with length of stay (138) . given the importance of tgf-β in suppressing nk cell functions, it is possible that the higher levels of tgf-β (as well as other inhibitory cytokines) in cov patients leads to suppression of nk cell antiviral activity (138) . early studies of covid-19 patients report secondary (super-) infections, including nosocomial pneumonia or bacteremia as a complication of sars-cov-2 infection (138) . since nk cells are critical first responders that play a role in preventing and clearing infections (139) , a poor nk cell count or exhausted phenotype, in addition to negatively influencing covid-19 patient outcomes, could facilitate the development of secondary infections and have a significant negative impact on patient outcomes. one of the main barriers in studying the role of nk cell activation in the early clearance of cov infection in asymptomatic or mildly symptomatic patients is the fact that these individuals are rarely diagnosed in the clinic and therefore an opportunity to collect samples for research does not exist. thus, while there is currently no direct evidence to support a role for nk cells in the clearance of sars-cov-2, evidence showing that viral infection has a negative effect on the nk cell compartment is accumulating. given the importance of nk cell activity in early viral clearance and late immunopathology, having a rapid and reliable test to predict nk cell function, such as nkvue tm (atgen canada/nkmax), whereby whole blood is stimulated by an nk cell-specific activating cytokine mix and activity is measured via ifn-γ production, might allow researchers to predict who will mount an adequate response with asymptomatic or minimally symptomatic viral clearance and who will need icu admission, as has been shown with cancer patients (140) . further research will be required into the innate immune response to cov infection to more fully understand nk cell contributions to viral clearance. in the context of covs, the significant morbidity and mortality associated with severe disease is due to acute lung injury (ali) and the development of ards (19, 141) . pathological analysis of tissues obtained from sars and mers patients showed edematous lungs with areas of consolidation, bronchial epithelial denudation, loss of cilia, squamous metaplasia, pneumocyte hyperplasia, and bronchial submucosal gland necrosis (19, 29) . histological features include diffuse alveolar damage and acute fibrinous and organizing pneumonia (29) . a heightened inflammatory response in the lungs resulting in tissue damage has been hypothesized to explain the development of ali. there are several key factors that may be responsible for the induction of this dangerous inflammation (138) . both sars-cov-1 and mers-cov replicate to high titers early in infection, which could lead to enhanced cytopathic effects and increased production of pro-inflammatory cytokines/chemokines by infected cells. chen et al. developed a pneumonia model where pulmonary replication of sars-cov-1 was associated with histopathological evidence of disease, including bronchiolitis, interstitial pneumonitis, diffuse alveolar damage, and fibrotic scarring (120) . they identified a biphasic cellular immune response in which cytokines (tnf-α and il-6) and chemokines [interferon gamma-induced protein (ip)-10, monocyte chemoattractant protein (mcp)-1, macrophage inflammatory protein (mip)-1a, rantes] were produced early, likely by infected airway epithelial cells, alveolar macrophages, and recruited inflammatory monocyte-macrophages and neutrophils, which have been shown to replace resident alveolar macrophages (19, 142) . sars-cov-1 and mers-cov encode structural and non-structural proteins that antagonize the interferon response, which may initially delay the innate immune response but eventually potentiate inflammatory monocyte-macrophage responses (19) . in covid-19 patients, liao et al. reported increased lung infiltration by macrophages identified via rnaseq analysis of bronchoalveolar lavage fluid. patients with mild cases exhibited infiltration by alveolar macrophages [fatty acid binding protein (fabp)4 + ] while patients with severe ards exhibited infiltration by highly inflammatory [ficolin (fcn1) + ] monocyte-derived macrophages (128) . in the sars-cov-1 pneumonia model, the first wave of cytokines and chemokines induced an accumulation of nk cells, as well as plasmacytoid (p)dcs, macrophages, cd4 + t cells and nkt cells in the lungs. a second wave of inflammatory mediators was detected later on day 7 post-infection [cytokines tnf-α, il-6, ifn-γ, il-2, il-5, and chemokines mcp-1, mip-1a, rantes, monokine induced by gamma interferon (mig), ip-10] and correlated with lung infiltration of t cells and neutrophils (120) . these findings are consistent with studies that have shown increased levels of activating and inhibitory cytokines and chemokines in the blood and lungs of sars patients, as well as histological studies of sars and mersinfected lungs which show extensive cell infiltrates (19, 29, (143) (144) (145) . when huang et al. investigated the cytokine/chemokine profile in the acute phase of sars infection in a cohort of taiwanese patients, they observed an ifn-γ-led cytokine storm (138) . they assessed sera from hospitalized patients prior to the administration of immunomodulators and found significantly increased levels of ifn-γ, il-18, ip-10, mcp-1, mig, and il-8 (138) , which returned to basal levels in convalescent sera. ip-10, mig, mcp-1, and il-18 levels were all significantly increased in death vs. survival groups. interestingly, they found an inverse relationship between ifn-γ levels and lymphocyte numbers and suggested this could either be due to ifn-γ-induced lymphocyte apoptosis or sequestration of chemokine-recruited lymphocytes in the lungs (138) . indeed, this hyper-cytokinemia has been consistently observed in sars-infected patients (146) . however, a recent study found that levels of six pro-inflammatory cytokines (il-1b, il-1ra, il-6, il-8, il-18, and tnf-α) implicated in the cytokine storm in covid-19 patients did not differ significantly from levels in cytokine storms caused by other conditions. they suggest that it is therefore possible that increased levels of proinflammatory cytokines in the context of severe covid-19 may simply reflect an increased viral burden rather than an exuberant immune response and suggest that immunotherapies should therefore be used with caution (147) . altogether these studies show that during acute cov infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce cytokines and chemokines that induce the activation and migration of lymphocytes, including nk cells, to the lungs, where they could be one of the main producers of ifn-γ (148). under normal conditions, human lung nk cells are typically hyporesponsive but dynamically migrate in and out of pulmonary tissues (83) . this supports the hypothesis that during infectious respiratory diseases, an increased recruitment of hyperresponsive nk cells would worsen the festering immunopathology (8) . in fact, through viral-track scanning of unmapped single-cell rnasequencing data, bost et al. showed that patients with severe covid-19 exhibited a hyperinflammatory response with an enriched and highly proliferative nk cell compartment (142) . high levels of ifn-γ leads to epithelial and endothelial cell apoptosis and vascular leakage, suboptimal t cell response, accumulation of alternatively activated macrophages and altered tissue homeostasis, and ards (19) , all of which may contribute to covid-19 disease severity. in summary, the evidence is consistent with the hypothesis that nk cells are involved in the cytokine storm associated with cov infection and that this hyper-cytokinemia contributes significantly to disease severity via inflammation-mediated lung damage (figure 1) . interestingly, this duality of nk cell roles mirrors what is seen in critically ill patients with sepsis. studies suggest that while early nk cell stimulation and ifn-γ production is beneficial to combat infections, excessive and prolonged stimulation of nk cells leads to reduced nk cell numbers and an exhausted phenotype and was associated with increased systemic inflammation in systemic inflammatory response syndrome (sirs)/sepsis and increased mortality (149) (150) (151) (152) . this review of the literature suggests that nk cells may play an important role in both cov clearance and immunopathology. the continued probing of nk cell involvement is essential for a more complete understanding of cov pathophysiology and for the deployment of immunotherapeutics. depending on the patient, the stage of disease, and other still poorly understood factors, it may be necessary to either boost nk cell activity to ensure viral clearance, e.g., at exposure or during early infection, or to finely tune nk cell effector functions in late stage infections to prevent hyper-cytokinemia and inflammatory lung damage. indeed, all covs that infect humans are zoonoses and there is an extensive reservoir of covs that could serve as a source for future pandemics (14, 153) . therefore, a broader understanding of the immune response to coronaviruses and insights into therapeutic implications will be of significant value not only for the current covid-19 pandemic, but also for potential future pandemics. the race to vaccinate and find a cure for covid-19 has resulted in a spectacular effort from researchers and medical practitioners around the world. early attempts at creating targeted therapeutics have mostly relied on historical evidence from related, but not identical, coronaviruses and on the paucity of studies investigating sars-cov-2. these strategies have attempted to combat the virus by targeting various stages of its life cycle starting with neutralizing sars-cov-2 virions using monoclonal antibodies or plasma from convalescent patients (154) . the entry mechanism of covs has been shown to rely on binding the ace2 receptor and using proteases such as tmprss2 for s protein priming (52) . thus, preventing ace2 receptor binding through blocking antibodies or competitive binding with soluble ace2 and tmprss2 protease inhibitors (camostat mesylate) are being tested (155) . upon viral entry, the viral proteolysis or replication cycle can be targeted with protease inhibitors (lopinovir and ritonavir) (156) or rna-dependent rna polymerase inhibitors (remdesivir and ribavirin) (157) . at the time of writing this review, the results of these trials have not been released or are still preliminary and will require further evaluation to assess their clinical efficacy in larger cohort studies. as nk cell activity is critical for viral clearance and may be involved in disease immunopathology, a rapid and reliable predictor of nk cell function may allow for the prediction of clinical progression and the stratification of patients to receive therapeutic intervention. the remainder of this review will discuss the various ways immunotherapies are being deployed to tackle covid-19, with a focus on therapies that use nk cells (table 1) . lastly, while nk cells play an important role in combating viral infections, we also need to be fully cognizant of the potential damage immunotherapies could have in severe cases of covid-19, and how these adverse effects may need to be attenuated ( table 2) . in the absence of a clinically approved vaccine against sars-cov-2, scientists have begun developing therapeutics to halt the spread of covid-19 by alternative strategies. studies have reported that patients infected with sars-cov-2 have lower levels of circulating nk cells and these express a greater level of inhibitory receptors (e.g., nkg2a) while producing less ifn-γ (127, 129, 130) . these findings provide a rationale for pursuing nk cell-based therapies as a tool to fight covid-19. although nk cell-based therapies have mostly been developed for use against cancer, similar concepts and mechanisms could provide guidance in the fight against viruses. therapeutic nk cell products can be thought of as "living drugs" as they generally use either primary nk cells isolated from peripheral blood mononuclear cells (pbmcs) or are generated from stem cell precursors or genetically engineered immortalized human nk cell lines (158) . primary nk cell products are often pre-treated and expanded in vitro with cytokines or via co-culture with target cells before being infused into patients. patients can also receive immune stimulants [e.g. recombinant il-2 (159) or il-15 (160) ] with the goal of improving the in vivo activity and persistence of the nk cell products (161) as is being tested in this covid-19 trial (nct04344548). the first cell-based investigational drug to be approved by the fda for clinical testing in covid-19 patients is an allogeneic, off-the-shelf, cryopreserved nk cell therapy made by celularity (cynk-001), originally developed for cancer immunotherapy (162) . the trial (nct04365101) is split into two phases. phase i will assess the frequency and severity of adverse events in mild, non-icu covid-19 patients (n = 14) following infusion of nk cells derived from placental cd34 + cells. the subsequent phase ii trial will recruit up to 72 patients and include a standard of care comparator at a 1:1 allocation. genetically modified nk cells are also being investigated for efficacy against covid-19. chimeric antigen receptor nk cells (car-nk cells) are engineered to express virtually any receptor(s) of interest and were originally designed to enhance the ability of nk cells to eliminate cancer cells via receptors targeting egfr (163) or cd19 (164) , which are present on many cancer types and b cell hematological malignancies, respectively (164) . although the efficacy of car-nk cells to control viral infections has yet to be rigorously tested in large scale clinical trials, the promising safety profile of car-nk cells in cancer patients, who are often immunocompromised, suggests that car-nk therapy can be well-tolerated in early phase/mild covid-19 patients. notably, car-nk cells are considered "safe" largely because they are less likely to lead to cytokine release syndrome (crs), a severe adverse event of car-t cell therapy (165) . but as these are unchartered waters, it is critical that car-nk cells are used cautiously and not given to late/severe covid-19 patients. a phase i/ii study in early stage covid-19 patients (within 14 days of illness) employing car-nk cell therapy is currently being tested using off-the-shelf nk cells derived from human umbilical cord blood expressing nkg2d and ace2 cars (nct04324996). this complex five-arm study will compare the efficacy of different car-nk constructs: (i) nk cells, (ii) nk cells secreting il-15, (iii) nkg2d car-nk cells, (iv) ace2 car-nk cells, and (v) nkg2d-ace2 car-nk cells. nkg2d car-nk cells have shown promising preclinical results in cancer studies (166) , and although not proven for sars-cov-2, the rationale for expressing nkg2d derives from work showing that nkg2d-ligands (nkg2dl) are upregulated on virally infected cells (167) . similarly, the investigators hypothesize that expressing ace2 on nk cells will facilitate the elimination of sars-cov-2 virions and infected cells by binding the viral spike proteins-but it is unknown whether or not car-nk cells can eliminate virions or if infected cells display sufficient levels of spike protein to be recognized by ace2-nk cells upon viral infection. the investigators also suggest that expressing ace2 on nk cells may also have a secondary benefit as a decoy cell that will be infected by the virus thereby indirectly protecting lung epithelial cells. as described previously, it is unclear whether this strategy will work to stop viral spread to healthy epithelial cells or if it will serve to perpetuate viral spread if the virus can replicate in nk cells. in arms ii-v of this trial, the car-nk cells have been engineered to secrete il-15 based on studies showing improved in vivo persistence of car-nk cells in cancer patients (168) . however, the addition of the proinflammatory cytokine il-15 to this treatment strategy should be monitored closely for life-threatening toxicities, as elevated il-15 has been previously reported to accompany chronic pulmonary inflammatory diseases (169) and mers-cov infection (170) even if no correlation has been reported for sars-cov-2. interestingly, a study compared il-15 levels from lung tissue homogenates following sars-cov infection in aged vs. juvenile monkeys and showed that il-15 concentrations were only elevated in juvenile monkeys 10 days post-infection (171) . this study would suggest that il-15 therapy may be tolerated and effective in older covid-19 patients that may not be able to produce il-15, however this has not been confirmed. lastly, all the car-nk cells in this trial secrete gm-csf neutralizing scfv antibodies, since this cytokine has a known role in crs in cancer patients treated with car-t cells (172) , and has been shown to be correlated with covid-19 disease severity in association with pathogenic cd4 + th1 cells (173) . although nk cell based therapies are versatile, have shown safety and efficacy in cancer patients, and can be utilized in immunocompromised individuals, their potential has yet to be fully realized as an antiviral therapy. furthermore, the logistics of manufacturing nk cell products (cost and time) may pose limitations and barriers to access. for this reason, therapies focused on stimulating a patient's own nk cells offer many advantages over adoptive transfer of nk cells. the importance of the interferon pathway is underscored by the fact that many viruses actively interfere with host interferon responses, for which coronaviruses are a prime example. as described above, covs utilize numerous tactics to avoid elimination by disrupting the host type i ifn response (174) . therefore, since the majority of covs fail to induce any detectable type i ifn response, eliciting a type i ifn response is a very attractive therapeutic strategy (118, 175) . given the robust immunomodulatory nature of type i ifns, uninfected or early symptomatic patients would benefit the most from this therapy to prevent exacerbating immunopathology at later stages of disease. numerous clinical trials have been initiated investigating type i ifns ( table 1) . a large study (nct04320238) of ∼3,000 medical staff allocated participants to two trial arms: (i) low-risk (non-isolated wards or laboratories) or (ii) highrisk (isolated wards in direct contact with covid-19 patients). in addition to the ifn-α-1b nasal drops, high-risk medical staff will also receive the immune-modulating tlr activator, thymosin α1, which indirectly activates nk cells through pdcs (176, 177) . interestingly, reports in sars-cov-1 studies showed that ifn-β therapy had a 50-fold greater anti-viral activity in vero cells than ifn-α treatment (178) . promising results have been published from a phase ii study (nct04276688) (179) , showing that complementing lopinavir-ritonavir and ribavirin with subcutaneous ifn-β-1b in mild-to-moderate covid-19 patients is safe with no serious adverse events reported in the triple combination therapy group, and highly effective, with significant and clinically meaningful reductions in time to complete alleviation of symptoms, hospital length of stay, and time to negative viral load (179) . despite our best efforts in timing type i ifn therapy to mitigate immunopathology, these treatments still increase the risk of excessive activation of proinflammatory signals, which could damage host tissues and perpetuate immunopathology (180, 181) . for this reason, alternative therapeutic avenues to direct type i ifn administration are being explored. type iii ifns can be a valid alternative to type i ifns, because they maintain antiviral functions yet are less toxic and less prone to mediate immunopathology (182) . the type iii ifn, ifn-λ, activates nk cells indirectly (compared to type i ifns which directly act on nk cells), resulting in a less potent and slower immune response (183, 184) . ifn-λ activates nk cells by stimulating macrophages to produce il-12 which in turn induce nk cells to produce ifn-γ (185) . pegylated ifnλ is being tested in covid-19 positive patients with mild symptoms in the absence of respiratory distress (nct04331899). while ifn-λ can lead to the eventual activation of nk cells, its primary utility is in preventing the tissue damaging potential of neutrophils at mucosal surfaces, such as the lungs. however, ifn-λ also has been shown to reduce the rate of tissue repair, which in the context of covid-19 which has a long disease course, could mean greater risk of secondary infections. since exogenous administration of any ifn therapy poses the risk of tipping the balance toward severe covid-19 immunopathology, broggi et al. assessed the levels of ifns in upper and lower respiratory samples from healthy and covid-19 patients. in this preprint, they report that while the upper airway swabs showed similar mrna expression levels of type i and iii ifn compared to healthy controls, the balf samples of severe covid-19 patients had significantly elevated type i and iii ifn levels (186) . therefore, as with all of the therapies discussed in this review, careful consideration about safe and effective timing should guide our design of clinical trials. in addition to ifn cytokine therapy, interleukin cytokine therapy can enhance the effector functions of nk cells (158) . the use of whole, unmodified recombinant cytokines as a monotherapy has resulted in minimal success in humans in cancer immunotherapy. the earliest cytokine therapies to gain fda-approval were ifn-α and recombinant il-2, approved for renal cell carcinoma and metastatic melanoma (187) . although approved, they were limited by their in vivo half-life, marginal anti-tumor activity, and associated toxicities. the next generation of cytokine therapies were created to address these issues by first improving their biological stability through pegylation and fusion to chaperone molecules and secondly improving their specificity by fusing cytokines with antibodies or intratumoral administration. these advances in the field have allowed for the reassessment of the therapeutic potential of specific cytokines (187) . given the importance of il-15 signaling and nk cell function, researchers have developed il-15 "superagonists" which are il-15:il-15r heterodimers that have better in vivo stability and bioactivity compared to monomeric il-15 (168) . although at the time of writing il-15 superagonists are not being studied for their efficacy in covid-19 patients, il-15 superagonists, such as alt-803, are safe in humans (188) and have been used in conjunction with many of the therapies being discussed in this review including: car-nk cell therapy, adoptive nk cell transfers, checkpoint inhibitors, and the bcg vaccine in cancer (189). it should be noted that although the therapeutic potential of cytokine therapy to specifically stimulate nk cells is enticing, exogenous cytokine therapy has a high risk for exacerbating crs if given at the incorrect time. some viruses are known to induce a state of functional hyporesponsiveness in t cells that is essential for the productive establishment of chronic viral infections (190) . a vast body of literature has identified inhibitory checkpoint receptors, including ctla4 and pd-1, as key regulators of this process (191) . interestingly, cancer exploits similar mechanisms to escape the immune response, which provided the rationale for the introduction of antibodies targeting checkpoint receptors for cancer immunotherapy (192) . ctla4 and pd-1/pd-l1 blockade have revolutionized cancer immunotherapy, and their success provides a strong rationale for the use of these drugs in covid-19 patients, where emerging evidence suggests that the immune response is also subverted. a clinical trial (nct04268537) is currently assessing the efficacy of pd-1 blocking antibodies in severe covid-19 patients within 48 h of reported respiratory distress. pd-1 has also been shown to play a role in regulating nk cell responses, in addition to modulating t cell functions (193) (194) (195) (196) (197) , and has been reportedly increased in covid-19 patients (129) . inhibitory receptors on the surface of nk cells regulate nk cell activation and can be targeted by antibody therapy. one of the most promising is certainly the inhibitory receptor nkg2a, which binds to hla-e (74, 198, 199) . nkg2a expression is increased in circulating (127) and balf nk cells from covid-19 patients, in contrast to nkg2c, an activating receptor closely related to nkg2a, which remains unchanged (129) . however, it is unclear whether the observed increase in nkg2a + nk cells is due selective proliferation of nkg2a + cells or if it is the result of nkg2a negative cells migrating out of circulation to infected tissues. circulating nk cells from patients with active hepatitis b disease had higher levels of nkg2a compared to patients without active disease, however antiviral administration was associated with a reduction in nkg2a expression. additionally, blocking nkg2a in vitro with nkg2a monoclonal antibodies led to improved nk cytotoxicity (200) . given the association between nkg2a expression in patients with severe covid-19 (127, 201) , a promising avenue of investigation would be anti-nkg2a therapy, even in light of results showing that nkg2a + nk cells are tuned to present a higher level of responsiveness to stimulation (202) . while nk cells can be stimulated directly by cytokines such as interferons and interleukins, their activity can also be enhanced through a by-stander effect following stimulation of other innate immune cells, such as macrophages and pdcs ( table 1 ). this type of coordinated innate immune response may be more effective at cov viral clearance and mitigation of severe covid-19. trained immunity has been recently described as an epigenetic re-wiring occurring in myeloid cells and progenitors upon stimulation that primes for a stronger response to subsequent stimuli, even of a different nature (90, 203, 204) . whereas, the consensus is that myeloid cells are primarily responsible for trained immunity (205) , it is likely that the resulting alteration in the cytokine milieu also has an effect on nk cells (204, 206) . this is the case for the bcg vaccine, which has been shown to provide non-specific protection against yellow fever viral infection (90, 207, 208) . the bcg vaccine is composed of a live attenuated strain of mycobacterium bovis originally given to young children to protect against tuberculosis (m. tuberculosis) (209) . this vaccine provides an initial boost to innate immunity, but more importantly, results in the secretion of il-1β from monocytes/macrophages, which feeds back to further stimulate the innate response (204) . the use of a heterologous vaccine to provide enhanced protection against non-specific/new pathogens makes this a compelling strategy against covid-19 that warrants thorough investigation in randomized controlled trials (209, 210) . the bcg vaccine is undergoing clinical trials in healthcare workers in the netherlands (nct04328441), australia (nct04327206), egypt (nct04350931), and the usa (nct04348370) to enhance overall innate immunity and provide heterologous protection against sars-cov-2. interestingly, an association was found that linked lower covid-19-attributable mortality rates in countries using bcg in their national immunization schedules (211) . on the contrary, a study that assessed the association of childhood bcg vaccination in adults living in israel did not show a beneficial difference in covid-19 infection rates. the discrepancy between these two reports likely stem from the fact that the latter study only included adults who were previously vaccinated during childhood, supporting the fact that heterologous vaccination may not result in long-term protection (212) . childhood bcg immunization has a limited window of opportunity to protect younger individuals from infection (213) , but it is hypothesized that reducing the number of infected children can have a meaningful impact on curbing the spread of covid-19 to the rest of the population (206, 211) . another heterologous vaccine in the process of clinical trial development for covid-19 studies is imm-101 (cctg id# ic8). created by immodulon therapeutics ltd, imm-101 is composed of heatkilled mycobacterium obuense and may have an improved safety profile over the bcg vaccine (214) . imm-101 has been studied in multiple clinical trials for its non-specific immune stimulating properties as a cancer immunotherapy in pancreatic (215) and melanoma patients (216, 217) . agonists of toll-like receptors (tlrs) have been shown to broadly activate different immune populations and have had both preclinical and clinical success as adjuvants in vaccination and in the treatment of a variety of viral pathogens (218) . for example, cpg oligodeoxynucleotides (cpg odns) are short dna sequences that contain unmethylated cpg dinucleotides which activate tlr9 particularly on dcs and b cells (219). bao et al. showed that their cpg odn construct, bw001, had protective effects against sars-cov-1 in a mechanism that relied on nk cell activation likely through a dc intermediate (220) . amidst the ongoing sars-cov-2 pandemic, two clinical trials (nct04313023, nct04312997) have opened using the tlr2/6/9 agonist, pul-042, in order to prevent infection. ascorbic acid, more commonly known as vitamin c, has been shown to exhibit potent immunomodulatory, antioxidant, and antimicrobial effects (221) . vitamin c has been shown to restore nk cell cytotoxicity in individuals exposed to toxic chemicals through protein kinase c expression, a critical component in lymphocyte metabolism (222) . additional reports have shown that vitamin c also enhances the expression of nkp46, cd69, cd25 and ifn-γ production by nk cells (223) and can increase the expression of irf3 in lung tissues of influenza infected, pneumonia-induced mice (224) . vitamin c also harbors potent antioxidant attributes which can scavenge reactive oxygen species (ros) and prevent lung injury (225, 226) . although ros production is an important component in the host defense response to viruses, they can be harmful to cells and lead to the pathogenesis of viral-induced host injury (227) . the underlying rationale to investigate the therapeutic potential of vitamin c has been based on two key observations: (i) critically ill patients have lower levels of vitamin c (228) (229) (230) and (ii) vitamin c has pleiotropic immunomodulatory, antioxidant, and antiviral effects (221) . it is important to underscore that reports on the clinical outcomes of vitamin c treatment in humans are mixed and context dependent. a thorough metaanalysis on vitamin c supplementation for the common cold has been reported by hemilä and chalker (231) . briefly, they concluded that while the incidence of colds was not reduced, the duration and severity of colds was reduced when assessing studies of regular vitamin c intake (231). interestingly, a separate metaanalysis on vitamin c and cardiac surgery showed a reduction in the length of icu stay and shortened the need for mechanical ventilation (232) . this is an important correlation as clinical trials are currently investigating the efficacy of vitamin c to reduce mortality and hospital burden in covid-19 patients ( table 1) . a phase ii clinical trial (nct04264533) was initiated in wuhan where covid-19 patients will be given a high dose intravenous infusion of vitamin c. lastly, whether oral dosing of vitamin c can achieve therapeutically relevant concentrations, as described in the above studies, is currently unknown, thus caution should be taken as exceeding the recommended dietary allowance of 100-200 mg/day may lead to mild toxicities including abdominal discomfort and diarrhea (231, 233). the main cause of death for covid-19 patients has been pulmonary complications and respiratory failure often as a result of an unregulated cytokine storm (234) . it is unclear whether the hyperinflammation seen in severe cases of covid-19 is the result of the viral replication within pulmonary epithelial cells or an overactive/avalanching immune response. however, studies in sars-cov-1 reported hyperinflammation in later stages of disease progression, despite reduced viral titers, suggesting that the damage was immune-mediated (19) . the most appropriate course of therapy can only be determined by elucidating the pathophysiology of disease progression. scientists and physicians, however, have had to respond quickly to the growing number of severe covid-19 cases and this has resulted in therapy mainly through a combination of anti-inflammatory and anti-viral interventions ( table 2) . as described above, there is a potential for nk cells to contribute to the cytokine storm and therefore the development of ali. a possible explanation for the observed lymphopenia in covid-19 patients is that nk cells and other lymphocytes migrate out of the circulation and into pulmonary tissues to aid in the elimination of infected epithelial cells (235) . this could be the premise for the large, unintended, amount of tissue damage that worsen the respiratory distress (148). for this reason, therapeutics that dampen the immune response have been effective in mitigating immunopathology in severe covid-19 patients. the following review papers have thoroughly discussed many of these immunotherapies already (236) (237) (238) (239) (240) (241) , therefore, this section will focus on immunotherapies and their potential implications on nk cells. the main cytokines responsible for the life threatening respiratory distress seen in reported cases of severe covid-19 are il-2, il-6, il-7, il-10, g-csf, ip-10, mcp-1, mip1a, and tnf-α (234) . many clinical trials have focused on targeting il-6 signaling with anti-il-6r monoclonal antibodies (e.g., tocilizumab, sarilumab, siltuximab) because of the important role il-6 has in propagating crs (242) . tocilizumab, in particular, is being used as the primary therapy in the majority of these trials, likely owing to its fda approved status as a therapeutic for crs in car-t cell therapy (243) . a case report demonstrated the potential for tocilizumab therapy in treating severe covid-19 illness, where a single dose on day 24 of symptoms led to progressive reduction in il-6 levels and resolution of symptoms (244) . a phase iii study (nct04320615) led by hoffman-la roche is recruiting patients to study the safety and efficacy of tocilizumab therapy in a randomized, double-blind, placebocontrolled, multicenter study in over 300 patients with severe covid-19 pneumonia ( table 2) . targeting the il-6 axis in severe covid-19 patients may also serve to improve nk cell functions as cifaldi et al. showed that increased il-6 negatively impacts nk cell function (245) . they also showed that tocilizumab treatment improved nk cell function in vitro (245) . mazzoni et al. recently reported that serum il-6 levels were inversely correlated (p = 0.01) with nk cell function in covid-19 icu patients. additionally, in a small subset of covid-19 icu patients (n = 5), nk cells displayed improved markers of activation (granzyme a and perforin) after tocilizumab treatment (246) . similar therapies have emerged in the fight against covid-19 including an il-1r antagonist (anakinra; nct04330638) (247) and cytosorb (nct04324528) (248) . high dose anakinra therapy has shown promising safety and efficacy in a small retrospective study, as part of the covid-19 biobank study (nct04318366) (247) . cytosorb therapy is used in conjunction with conventional dialysis through a whole blood cartridgebased filtration system designed to remove middle molecular weight molecules (which include inflammatory cytokines <75 kda) through extracorporeal cytokine adsorption (248) . it is reported to be effective at removing ferritin and il-6 in a case study of a 14-year-old with severe crs following car-t cell therapy (249) . jak1/2 inhibitors (jaki) are also undergoing clinical trials in moderate-severe covid-19 patients, such as baricitinib (nct04320277). in addition to their ability to impede the production of il-6, thus curb the excessive inflammation, they may also block clathrin mediated endocytosis-indicating a dual role for jaki (241) . however, jaki can also lead to the transient increase in nk cells as shown in baricitinib treated rheumatoid arthritis patients (250) , which could be detrimental for severe covid-19 patients. corticosteroids have played a key role in the treatment of auto-immune diseases over the past 70 years (251, 252) . whether endogenous or exogenous, corticosteroids decrease the number of circulating monocytes and lymphocytes and decrease synthesis of pro-inflammatory cytokines (il-2, il-6, tnf-α) (251) . their strong anti-inflammatory and immunosuppressive effects make them good candidates for rapidly suppressing inflammation during early auto-immune disease or viral infections. corticosteroids have been shown to inhibit nk cells in ex vivo experiments (253, 254) . while corticosteroids may delay clearance of infections, their major benefit lies in suppressing excessive innate immune responses, thus preventing lung damage and ards commonly present in severe viral infections (255) (256) (257) . in fact, this was the main rationale for the widespread use of corticosteroids during mers and sars infections (255, 256) . specific to covid-19, some groups have advocated for the use of low-dose corticosteroids in a specific subset of critically-ill patients with refractory ards, sepsis, or septic shock ( table 2 ) (257) . there is one known ongoing randomized clinical trial examining the effect of the corticosteroid ciclesonide in adults with mild covid-19 infections (nct04330586). this trial is based on preclinical studies showing in vitro antiviral activity of ciclesonide against sars-cov-2. while there may be a benefit to using corticosteroids in a subset of critically-ill patients with refractory ards or sepsis (257) , their routine use in covid-19 is not recommended outside of clinical trials, based on expert opinion and who recommendations (258) (259) (260) . corticosteroids also cause a multitude of side effects, most notably diabetes mellitus, osteoporosis, and increased risk of infections (251) . controversially, a 2019 systematic review of over 6,500 influenza patients showed that corticosteroids actually led to increased mortality, length of icu stay, and secondary infections (261) . additionally, one retrospective observational study examined the use of corticosteroids in 31 covid-19 patients, and reported no significant association between corticosteroids and viral clearance time, hospital length of stay, or duration of symptoms (262) . these studies highlight the need to be vigilant in our attempts to fight covid-19. healthy, uninfected individuals, who are at a high risk of becoming infected (through situational circumstances such as healthcare workers) would be most fit and suitable to receive investigational prophylactic therapies such as exogenous ifns and heterologous vaccines. (b) individuals who have tested positive for covid-19 that are asymptomatic or have mild to moderate disease progression may benefit from receiving investigational immune stimulating therapies, including nk cell-based therapies. it is critical that investigators must be vigilant to assess the safety profile and potential immunopathologies associated with these immunotherapies. (c) in severe covid-19 patients, the most appropriate therapies to investigate would be those that mitigate immunopathologies, such as anti-inflammatory and immunosuppressive therapies. given the relatively low chance of toxicity and the wide range of beneficial immune effects, natural health products such as vitamin c and vitamin d can be suitable for investigation at all categories of covid-19 patients. non-steroidal anti-inflammatory drugs, or nsaids, are one of the most commonly prescribed drugs for treating fever, pain, and inflammation. nsaids include over-the-counter household names such as ibuprofen, naproxen, and aspirin. given the widespread use of these medications it is appropriate that researchers have investigated the potential benefits and harms of nsaids in patients diagnosed with covid-19. thus far, the evidence for using nsaids in the context of covs are mixed and might not be generalizable to all nsaids as reports tended to focus on specific nsaids. these studies also focused on the potential for nsaids to act as an antiviral, with a potential added benefit of being able to treat inflammatory symptoms. one report showed that the nsaid indomethacin could directly inhibit sars-cov replication in vero cell monolayers in a dosedependent manner (263) . the antiviral properties of naproxen have been described in the context of influenza virus (264, 265) and has prompted the initiation of a clinical trial investigating the efficacy of naproxen as a treatment for critically ill covid-19 infected patients (nct04325633). nsaid therapy should be used with caution as they have been shown to interfere with immune responses and ability to produce antibodies, with ibuprofen having the greatest suppressive effect (266) . furthermore, ibuprofen has been reported to increase the expression of the ace2 receptor (267) which could facilitate sars-cov-2 viral entry. this finding should be considered for any current (nct04334629) and potential covid-19 clinical trial assessing ibuprofen therapy. nsaids also have been shown to have a direct suppressive effect on nk cell ifn-γ and tnf-α production (268) which may be beneficial for late stage covid-19 patients. the relevance of nk cells as antiviral first responders is highlighted in patients with nkd and immunocompromised individuals who show increased susceptibility to viral infections. while there is currently little direct evidence to support a role for nk cells in the clearance of sars-cov-2 there is a paucity of research in this field. however, studies in admitted covid-19 patients with mild and severe disease reported a reduction in circulating nk cell levels and function as compared to healthy individuals. furthermore, reduced nk cell levels and function were inversely correlated with disease severity, suggesting that nk cells may be involved in some capacity. one of the potential mechanisms by which nk cells may become hyporesponsive is via sars-cov-2 interference with type i ifn pathways. in investigating the pathogenesis of other cov infections, namely sars and mers, studies suggest that during acute cov infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce chemokines and cytokines that induce nk cell migration and activation. as nk cells are one of the main producers of ifn-γ, they may be involved in the ifn-γ-led cytokine storm that is responsible for the induction of inflammation-mediated ali, ards, and subsequent mortality associated with covid-19. inarguably, more research into the role of nk cells in covid-19 is required. despite the knowledge gaps in covid-19 pathophysiology, there has been a surge of clinical trials as the fda continues to fast-track the approval of investigational therapeutics (269). here we have outlined potential therapeutics with a focus on mediating nk cell activity, including prophylactic treatments that could boost innate immunity in addition to therapeutics that could mitigate the 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hypertension and diabetes mellitus at increased risk for covid-19 infection? office of the commissioner. coronavirus (covid-19) update: fda continues to accelerate development of novel therapies for covid-19. us food and drug administration the authors would like to thank dr. doug gray for his thorough editing, proofreading, and thoughtful suggestions. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 market, angka, martel, bastin, olanubi, tennakoon, boucher, ng, ardolino and auer. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-320909-p93gxjm2 authors: natoli, s.; oliveira, v.; calabresi, p.; maia, l. f.; pisani, a. title: does sars‐cov‐2 invade the brain? translational lessons from animal models date: 2020-05-22 journal: eur j neurol doi: 10.1111/ene.14277 sha: doc_id: 320909 cord_uid: p93gxjm2 the current coronavirus disease (covid‐19) outbreak, caused by the novel severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2), has raised the possibility of potential neurotropic properties of this virus. indeed, neurological sequelae of sars‐cov‐2 infection have already been reported and highlight the relevance of considering the neurological impact of coronavirus (cov) from a translational perspective. animal models of sars and middle east respiratory syndrome, caused by structurally similar covs during the 2002 and 2012 epidemics, have provided valuable data on nervous system involvement by covs and the potential for central nervous system spread of sars‐cov‐2. one key finding that may unify these pathogens is that all require angiotensin‐converting enzyme 2 as a cell entry receptor. the cov spike glycoprotein, by which sars‐cov‐2 binds to cell membranes, binds angiotensin‐converting enzyme 2 with a higher affinity compared with sars‐cov. the expression of this receptor in neurons and endothelial cells hints that sars‐cov‐2 may have higher neuroinvasive potential compared with previous covs. however, it remains to be determined how such invasiveness might contribute to respiratory failure or cause direct neurological damage. both direct and indirect mechanisms may be of relevance. clinical heterogeneity potentially driven by differential host immune‐mediated responses will require extensive investigation. development of disease models to anticipate emerging neurological complications and to explore mechanisms of direct or immune‐mediated pathogenicity in the short and medium term is therefore of great importance. in this brief review, we describe the current knowledge from models of previous cov infections and discuss their potential relevance to covid‐19. highly pathogenic coronavirus (cov) infections are well-established sources of previous epidemics in humans, i.e. severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov). the novel cov named sars-cov-2, which shares a highly homological sequence with sars-cov, is responsible for the current covid-19 outbreak with more than 2 million patients diagnosed and over 146 000 deaths, which exceeds by far the total of sars and mers in 2002 and 2012, respectively [1] [2] [3] . despite the short duration of the current pandemic outbreak, several neurological and neuroradiological phenotypes have been reported [4, 5] , requiring urgent investigation into the mechanisms and etiology underlying the interplay between sars-cov-2 and the central nervous system (cns). a translational neuroscience approach is mandatory to explore the possible cns involvement in cov infections, accelerate scientific knowledge transfer to the clinical frontline and test new disease-oriented treatments. indeed, both clinical features of the previous cov epidemics (sars and mers) and lessons from animal models used in the study of sars and mers constitute valuable tools to understand the viral pathogenesis in the host and to characterize mechanisms of viral access and dissemination in the cns. meanwhile, several laboratories are rushing to study sars-cov-2 in a number of different animals, including primates, mice, rats, hamsters and ferrets [6] . here, we will provide a neurological perspective by analysing the main features of these models and point out relevant similarities and specificities in comparison to sars-cov-2. a comprehensive systematic search of medline, scopus, web of science and https://www.who.int/emerge ncies/diseases/novel-coronavirus-2019/situationreports/ was performed. in 2002, the outbreak of sars in guangdong province, china led to the discovery of sars-cov, a highly pathogenic cov, as the causative pathogen of the epidemic [7] . although the virus is primarily a respiratory pathogen, there are reports of neurological manifestations, such as epileptic seizures and encephalitis, that may suggest a cns involvement of the infection [8, 9] (table 1) . complementing these reports, post-mortem neuropathological studies have detected the sars-cov n protein and rna polymerase gene fragment in neurons of infected patients and pathological changes such as brain tissue edema and vasculitis of cerebral veins [10] . compelling evidence demonstrates that sars-cov attaches to the cell membrane by binding to human angiotensin-converting enzyme 2 (hace2), now also known to be the sars-cov-2 functional receptor [11] . human tissue studies have shown an abundant presence of these receptors not only in the epithelia of the lung and small intestine, but also in arterial and venous endothelial cells and arterial smooth muscle cells in all organs studied, including the brain [12] . based on a transgenic mouse expressing hace2, it was possible to show that angiotensin-converting enzyme 2 (ace2) is also expressed at neuronal level, namely in the cytoplasm of cell bodies [13] . distinctive properties in the structure of mouse ace2 (as compared with hace2 proteins) significantly reduce the virus tropism for mouse tissues. hence, in order to overcome this species-related difference, a transgenic model has been generated in which a vector carrying a hace2-coding sequence was introduced in wild-type mice under control of the human cytokeratin 18 (k18) promoter [14] . notably, when k18-hace2 transgenic mice were infected with sars-cov, the infection would start in the respiratory epithelium and rapidly spread to the alveoli. more importantly, neuroinvasive routes were later explored using the same model, by monitoring the kinetic profile of viral antigen [15] . strikingly, the authors showed that the viral spread started in the olfactory bulb and progressively invaded subcortical and cortical regions. such a trans-neuronal hypothesis could not apply for other infected regions, such as those brainstem nuclei that are not directly connected to the olfactory bulb. the authors raise the possibility that, once the virus is established in the brain, it might spread along specific neurotransmitter pathways or via non-neuronal routes (blood or virchow-robin spaces) [15] . overall, their results showed that, in this model, sars-cov primarily entered the brain via the olfactory nerve. alternatively, other authors theorize that cov can primarily use a hematogenous route to penetrate the cns using dendritic or white blood cells as reservoirs [16] . this presumption is based on pathological studies that have shown that monocytes and macrophages can be infected by sars-cov [17] and on cell-line studies that revealed that dendritic cells (regulators of immune responses) can be infected and impaired by this virus [18] (table 1) . multiple animal models have been explored in the context of sars, including non-human primates, hamsters, ferrets and mice ( table 2) . a comprehensive descriptive review of all suitable models is beyond the scope of this review and we refer the reader to a number of excellent reviews [19] [20] [21] . it can be inferred that there is no single ideal animal model for sars, although the evidence collected so far has significantly contributed to advancing the field. in particular, it is well established that models range from those in which only virus replication is observed (young balb/c, b6 mice) to those in which replication is accompanied by minimal signs and histopathology (such as non-human primates, ferrets and hamsters) [19] . curiously, old immunodeficient balb/c mice exhibit a clinical syndrome, supporting age as a risk factor for more severe clinical phenotypes [19] . transgenic k18-hace2 mice infected with sars-cov develop a severe pulmonary phenotype, starting in the respiratory epithelium with rapid alveolar dysfunction [14] . in this model there is a massive infiltration of macrophages and lymphocytes in the lungs, promoting a release of pro-inflammatory cytokines not only at pulmonary level, but also in the brain. in a relatively short time frame (within 5 days), k18-hace2 mice develop a severe phenotype, that includes a lethargic-like state, suggesting cns involvement. in follow-up studies in the same mouse strain, k18-hace2 [15] , the authors demonstrated an extensive involvement of the transgenic mouse brain. sars-cov produced a widespread infection involving vital brainstem nuclei, such as dorsal motor nucleus of the vagus, nucleus tractus solitarii and area postrema. this model also raised questions as to the cause of neuronal destruction and death in these animals [15] . as there was no pathological evidence of inflammation, the authors considered the possibility of apoptosis as the cause of neuronal death, although this was not confirmed [terminal deoxynucleotidyl transferase (tdt) dutp nick-end labeling (tunel)-positive cells were not detected]. it was proposed that a dysregulated cytokine response could be the cause of death in these animals. at day 4 postinfection, infected k18-hace2 mice had an upregulation of the proinflammatory cytokines interleukin (il)-1, tumor necrosis factor alpha and il-6. the authors also propose a possible direct involvement of the dorsal vagal complex, a vital region of the brain that plays an important role in orchestrating cardiorespiratory function. in fact, animals intracranially inoculated with low-dose virus exhibited limited viral spreading but succumbed rapidly [15] . overall, these data show the relevance of the transgenic approach in converting the mouse response to infection from mild to severe leading to cns human neurons are infectible [53] and ace2 neuronal expression has been identified in human cns [54] capable of infecting human neuronal cells in in-vitro cell lines [55] . ddp4 has a low expression in the brain [56] -neuropathology sars genome sequences detected in the brain in autopsies; also, edema and scattered red degeneration of neurons [17] samples not available for investigation [22, 23] . currently, mers-cov is still a relevant threat for populations in the middle east, with a high lethality (close to 35%) [2] . patients exhibit predominantly pulmonary clinical involvement in contrast to fewer patients presenting neurological manifestations such as coma, ataxia, focal motor deficits and peripheral nerve symptoms [24, 25] . unfortunately, there are no published data regarding human neuropathological findings (table 1) . the mers-cov ex-vivo models supported the clinical tropism for the pulmonary tract by showing that the virus can replicate in human lung cultures (in bronchial, bronchiolar and alveolar epithelial cells) [26] . this cell line susceptibility study also revealed that, although presenting a lower viral expression and no cytopathic effects, mers can infect human neuronal lines. dipeptidyl peptidase-4 (dpp4), also known as cd26, was identified as a functional receptor for mers-cov. dpp4 is generally expressed in human bronchiolar epithelial cells and bronchial lung tissue [27] . it can also be found in the intravascular portion of vascular endothelial cells and in the cerebrospinal fluid [28] . after identification of dpp4 as a functional receptor, which is expressed in the airway epithelia of rodents, it was expected that rodents would have been vulnerable to infection. this turned out to be wrong, as the human binding domain differs from that of rodents [29] . this limitation was overcome by developing mice expressing human dpp4 that exhibited high susceptibility to infection and displayed the features of human disease [30] , including a lethargic state, and showing high mortality and extrapulmonary involvement ( table 2 ). the authors detected a severe lung infection, but brain invasion was not seen until day 4 of infection, suggesting substantially different kinetics of mers-cov infection in the lung and brain [30] . a different animal model using human dpp4 transgenic mice studied the differences in viral replication in animals infected by a clinical aerosol transmission simulator compared with intranasal instillation-inoculated mice [31] . they found that the disease onset, lung lesion and viral replication progression were slower in the mers-cov aerosol-infected mice than in the mers-cov instillation-inoculated mice. furthermore, after aerosol infection, they detected high viral loads after 3-9 days in the lungs versus 7-9 days in the brain. again, although both lungs and brain are infected, the timing is different, with a later infection of the brain [31] . such different kinetics could suggest a hematogenous route of infection. indeed, neuroinvasive routes were not explored in either of these models. in addition, similarly to sars-cov, mers-cov has been shown to replicate in human dendritic cells and macrophages, which would support the hematogenous hypothesis [32] . a number of models have been developed and discussed in detailed review articles [33, 34] (table 2 ). in a non-human primate model of mers, de wit and colleagues inoculated rhesus macaques with mers-cov, which primarily affected the epithelium of the lower respiratory tract, giving rise to a mild-to-moderate interstitial pneumonia [35] . this model was able to replicate virus shedding and replication in tissues, as well as gene expression and cytokine and chemokine profiles. however, despite the mild clinical syndrome, no neurological signs and symptoms were reported. thus, the self-limiting nature of mers-cov infection, as transient patterns at various levels of the model, suggests that this model does not fully resemble the lethal infection observed in humans [35] . it is of note that, when macaques were immunocompromised by immunosuppressive agents, the mers-cov replicated to significantly higher titers and disseminated in other organs (cns not examined). surprisingly, histopathological alterations were reduced in the immunosuppressed animals [36] . together, these data suggest a prominent role of the host response in the manifestation of the disease. the macaque model allowed the testing of a number of potential drugs as novel therapeutics. remdesivir, an antiviral agent used also for covid-19, was able to prevent/treat the histological and radiological signatures of the disease [37] . in studies using transgenic mice expressing human dpp4, it was possible to induce features of human disease in the animals [30] . from the studied cells, pneumocytes, brain microglia, astrocytes and neuronal cells all presented high titers of virus. with regard to pathology, whereas infected mice presented an extensive pulmonary inflammatory infiltrate, the only findings in the brain were a mild perivascular cuffing [30] . however, in a different study using human dpp4 transgenic mice, a few days after the appearance of pulmonary lesions, pathological changes were documented in the brain, with dilatation and congestion of the cerebral vessels and few areas of cellular necrosis in the cerebral cortex, hippocampus and thalamus [31] . as in sars-cov-2, mers-cov infection was also shown to induce a profound acute inflammatory response within the lungs and brain of hcd26 tg mice, with upregulation of multiple genes related to the inflammatory response [30] . clinical and neuropathological features in human patients covid-19 is the most recent and dramatic pandemic, caused by sars-cov-2. registered lethality varies between european countries ranging from 1.5% in germany to over 10% in italy [1] . as in sars and mers, pulmonary clinical involvement is most prominent. however, more recently, neurological phenotypes involving central and peripheral nervous system have emerged and are being increasingly recognized, i.e. anosmia, ageusia, necro-hemorrhagic encephalitis and guillain-barr e syndrome [4] [5] 38] . so far there are no published human neuropathological findings (table 1) . the sars-cov-2 ultrastructure was recently characterized by high-resolution cryo-electron microscopy [39] . remarkably cov spike glycoprotein, by which the virus binds the cell membrane, binds ace2 with a higher affinity compared with sars-cov. in addition, most of the available antibodies to sars-cov targeting acebinding domain were unable to bind the sars-cov-2 spike protein, indicating that binding sites differ between sars-cov and sars-cov-2. such a finding indicates the urgent need for generating specific antibodies for sars-cov-2 binding domain, but might also explain the distinct pathogenic properties of sars-cov-2 [40] . in addition to ace2 receptor, sars-cov-2 uses the serine protease type ii transmembrane serine protease (tmprss2) for spike protein priming [41, 42] . very recently, in a preliminary report, brann et al. took advantage of bulk mouse whole olfactory mucosa (wom) rna sequence data derived from macaque, marmoset and human and found in both mouse and human datasets that olfactory sensory neurons do not express two key genes involved in cov-2 entry, i.e. ace2 and tmprss2 [43] . in contrast, olfactory epithelial support cells and stem cells express both of these genes, as do cells in the nasal respiratory epithelium. taken together, these findings suggest possible mechanisms through which cov-2 infection could lead to anosmia or other forms of olfactory dysfunction. moreover, these findings may question the olfactory bulb as an entry route for covs into the cns [43] . to our knowledge, no study has evaluated, so far, any type of pathway targeted at the cns or peripheral structures. clinical and pathological lessons from animal models several laboratories worldwide are accelerating attempts to develop a suitable animal model for covid-19. experimental infection with sars-cov-2 in these models provides basic information to address a number of fundamental questions regarding its pathogenicity, the interaction with the different hosts and, hopefully, establishing the criteria for prevention and care. in line with observations in sars models, non-human primates and wild-type mice infected with sars-cov-2 exhibit a relatively mild clinical disease, in spite of the evidence that quantitative reverse transcription polymerase chain reaction (rt-qpcr) revealed a massive infection of the respiratory tract [44, 45] (table 2) . rhesus macaques infected with bronchoalveolar lavage fluid obtained from an affected patient developed a histopathologically confirmed interstitial pneumonia, associated with a widespread presence of sars-cov-2 in the respiratory tract. clinical signs were mild and no viral rna was detectable by means of rt-qpcr in the blood of the primates during the whole course of infection (14 days) [44] . these findings demonstrate the causal relationship between sars-cov-2 and interstitial pneumonia, reminiscent of covid-19. moreover, and consistent with observations in sars models, bao et al. [45] used the hace2 transgenic mice and infected them with sars-cov-2 inducing interstitial pneumonia, with typical histopathological elements and, accordingly, viral antigens were found in airway epithelia. these lines of experimental evidence are relevant as they demonstrate the causal relationship between sars-cov-2 and pulmonary involvement, but, unlike in sars models, nervous system involvement was not documented in these experiments. however, it is unclear if brain tissue was systematically assessed and the most susceptible brain regions explored for direct or indirect viral presence. overall, the pathogenicity of sars-cov-2 is lower as compared with sars-cov in mice. indeed, as discussed above, hace2 transgenic mice infected with sars-cov exhibited widespread organ damage, whereas sars-cov-2, at least in this model, was confined to lungs, indicating a differential pathogenicity [45] . these studies reveal important commonalities between sars-cov-2 and sars-cov infection and identify a potential target for antiviral intervention. in fact, very recently, a tmprss2 inhibitor approved for clinical use (camostat mesylate) was tested and blocked sars-cov-2 entry into lung cells [42] . finally, the same authors were able to show that the sera from convalescent patients with sars cross-neutralized sars-2-spike-driven entry [42] . if the same effect occurred in pre-clinical models, then we would be closer to both a preventive and a diseaseoriented treatment. • major routes of cns infection: (i) spread via olfactory bulb and/or (ii) synapse-connected route to the medullary cardiorespiratory center from the mechanoreceptors and chemoreceptors in the lung and lower respiratory airways; and/or (iii) hematogenic via brain endothelial ace2 receptors. • major pathogenic pathways for cns involvement: (i) direct viral pathogenicity; and/or (ii) immunemediated pathogenicity targeting brain tissue; and/ or (iii) inflammatory involvement of brain blood vessels; and/or (iv) intravascular coagulation secondary to the systemic inflammatory response as a mayor cause of thrombosis, hemorrhage and stroke. • host individual susceptibility factors that underlie the variable severity of the disease in human patients. however, a relevant issue is also represented by gender. the clinical observation of a specific involvement of males might suggest a specific protective estrogenic effect. unravelling these points could clarify if cns contributes to respiratory failure in patients with covid-19 [15, 46] and may provide a rationale to preventive and therapeutic strategies for major neurological events such as stroke, encephalitis or other reported complications. established ex-vivo and animal models of cov infection may help to dissect pathogenicity, infective routes and nervous system targets of covs. if we manage to mimic the pathological hallmarks of covid-19 we may have the tools to test treatment efficacy and evaluate the efficacy of vaccines and therapeutics. among limitations, the following emerge as of primary importance. • neurological subtle clinical phenotypes are not easily reproducible in animal models. • neurological severe phenotypes are dependent on using specific transgenic approaches to enhance virulence, which limit direct translation to humans. • severity of the clinical features does not always parallel either the viral replication level or the histopathological findings, hinting at indirect disease mechanisms (such as inflammation and prothrombotic states) that have not been attained in the present models. • innate animal characteristics seem to influence viral infection kinetics leading to faster virus clearance. the ongoing outbreak of sars-cov-2 confirms that human covs are primarily respiratory pathogens and koch postulates have already been fulfilled in this regard [45] . previous reports suggest that sars-cov and mers-cov can occasionally cause clinically relevant cns infections. in fact, animal models suggest invasiveness of these viruses through the cns, either via the olfactory bulb or through blood dissemination of infected and activated monocytes passing through a permeable blood-brain barrier as a consequence of the systemic inflammatory response. with regard to the pathogenesis of immunemediated cns pathology, data derive broadly from mice infected with murine hepatitis virus strains, a beta-cov genetically related to human cov-oc43 [47] . briefly, three mechanisms of immune-mediated cns lesions can be recognized. (i) an excessive host response to the infection can occur resulting in a systemic inflammatory response syndrome that causes a multiple organ dysfunction (including cns). the main pathogenic mechanism in this case includes tissue 'dysoxia' due to intravascular coagulation and dysfunction of the microcirculation homeostasis. (ii) direct viral infection of immune cells, including macrophages, microglia and astrocytes in the cns, may activate glial cells that locally produce pro-inflammatory cytokines, including il-6, tumor necrosis factor alpha, il-1b and il-12 [48] . moreover, activated immune cells may contribute to tissue damage by producing toxic agents, recruiting and activating further immune cells and inducing apoptosis. immune-mediated events, either through t-cells or by means of other cytokine and chemokine pathways, may also eventually lead to demyelination. (iii) an autoimmune reaction is generated by an adaptive immune response directed against host epitopes or proteins either misrecognized by pathogen-directed antibodies or expressed by damaged tissues (and previously cryptic to the adaptive immune system) [49, 50] . in order to speed up clinically useful discoveries, it would be desirable to follow some indications such as: (i) to build a systematic, consecutive, prospective registry including epidemiological data in patients with covid-19 with attention to neurological manifestation to fully understand if sars-cov-2 infections can cause cns involvement and to what extent; (ii) to measure sars-cov-2 rna in the cerebrospinal fluid of symptomatic vs. asymptomatic patients; and (iii) to perform autoptic investigations of patients with covid-19 in order to find and characterize virus distribution across tissues (cerebral blood vessels, endothelia, glia and neurons) and neuropathological consequences such as antibody-based neuroinflammatory responses in gray and white matter, vasculitis, neuroglial death or apoptosis and ischaemic or hemorrhagic events. taking into account the fact that other covs are prone to infecting neurons in animal models as well as in humans [16, 50] we must keep an open mind regarding medium-to long-term sequelae and consequences of the acute infection. therefore, despite immune-mediated control of acute infection being attained, host-mediated immune regulatory mechanisms may fail to clear the virus potentially leading to 'chronic infections' and hence impact chronic neurological diseases, such as parkinson's disease and multiple sclerosis [51] as well as acute disseminated encephalomyelitis [52] . this calls for long-term patient follow-up in the clinics and also exploring the effect of sars-cov-2 in mouse models of neurodegenerative disorders to anticipate the occurrence of chronic sars-cov-2 cns infection. world health organization -coronavirus disease (covid-2019) situation reports world 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sars-coronavirus quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation unravelling the immunological roles of dipeptidyl peptidase 4 (dpp4) activity and/or structure homologue (dash) proteins we are grateful to drs magdalena mroczek and andrea mancini for sharing the literature on covid-19. this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. the authors declare no financial or other conflicts of interest that relate to the research covered in this article. data sharing is not applicable to this article as no new data were created or analyzed in this study. key: cord-335614-qh98622y authors: xu, puzhi; liu, ping; zhou, changming; shi, yan; wu, qingpeng; yang, yitian; li, guyue; hu, guoliang; guo, xiaoquan title: a multi-omics study of chicken infected by nephropathogenic infectious bronchitis virus date: 2019-11-16 journal: viruses doi: 10.3390/v11111070 sha: doc_id: 335614 cord_uid: qh98622y chicken gout resulting from nephropathogenic infectious bronchitis virus (nibv) has become a serious kidney disease problem in chicken worldwide with alterations of the metabolic phenotypes in multiple metabolic pathways. to investigate the mechanisms in chicken responding to nibv infection, we examined the global transcriptomic and metabolomic profiles of the chicken’s kidney using rna-seq and gc–tof/ms, respectively. furthermore, we analyzed the alterations in cecal microorganism composition in chickens using 16s rrna-seq. integrated analysis of these three phenotypic datasets further managed to create correlations between the altered kidney transcriptomes and metabolome, and between kidney metabolome and gut microbiome. we found that 2868 genes and 160 metabolites were deferentially expressed or accumulated in the kidney during nibv infection processes. these genes and metabolites were linked to nibv-infection related processes, including immune response, signal transduction, peroxisome, purine, and amino acid metabolism. in addition, the comprehensive correlations between the kidney metabolome and cecal microbial community showed contributions of gut microbiota in the progression of nibv-infection. taken together, our research comprehensively describes the host responses during nibv infection and provides new clues for further dissection of specific gene functions, metabolite affections, and the role of gut microbiota during chicken gout. gout is a urate crystal deposition disease that occurs when supersaturation of individual tissues with urate arises, leading to the construction of monosodium urate (msu) crystals in and around the joints, abdomen, and organs [1, 2] . it is worth noting that in addition to its occurrence in humans, gout is one of the common diseases that plague poultry and causes huge economic losses to the poultry industry worldwide [3] . avian gout is commonly divided into visceral gout and joint gout, and the typical clinical pathology of visceral gout is hyperuricemia. according to reports, various aviaries from all over the world have visceral gout, which has become one of the most commonly diagnosed causes of fatality in poultry [4, 5] . in the poultry industry, visceral gout can be caused by many factors, including vitamin a deficiency, high dietary calcium, renal insufficiency, chicken diseased room (dis, n = 120). birds in each experimental room were randomly divided into three subgroups (30 birds for each subgroup), with ad libitum access to food and water. each chicken of dis groups was intranasally injected with 0.2 ml 10 5 median embryo lethal doses of strain sx9 at 28 days of age [24] , while the con group intranasally received 0.2 ml of sterile physiological saline. at 38 days of age, four chicken randomly chosen per subgroups were euthanized by carbon dioxide inhalation, then dislocated their cervical vertebra. the samples in a group were pooled and dead birds were not used for analysis. ten serum samples were randomly collected from surviving chickens in the con and dis groups before euthanasia that were used for uric acid test. six biological replicates of kidney samples were extracted from each group making a total of 12 samples that were used for gc-tof/ms analysis. four biological replicates of kidney samples were collected from each group giving a total of eight samples that were used for rna-seq analysis. six biological replicates of cecal contents from each group were collected giving a total of 12 samples that were used for 16s rrna gene sequencing analysis ( figure 1a shows the experimental design). the isolated kidney tissues were fixed by immersion in 10% neutral formalin at room temperature for over 48 h. tissues were then routinely processed; h&e staining was performed and a section per chicken was observed under the optical microscope. metabolite extraction, metabolite derivatization, metabolite detection, and data analysis followed those of yang et al. [25] . first, methanol (v methanol :v chlorofrom = 3:1) was used as an extraction liquid, and l-2-chlorophenylalanine (1 mg/ml stock in dh 2 o) was added as an internal standard. the metabolites are then derivatized with the methoxy amination hydrochloride (20 mg/ml in pyridine) and the stfa regent (1% tmcs, v/v). finally, gc-tof/ms analysis was performed using an agilent 7890 gas chromatograph system coupled with a pegasus ht time-of-flight mass spectrometer. the energy was -70 ev in electron impact mode. after 6.04 min of solvent delay, mass spectrometry data were acquired in full-scan mode with an m/z range of 50-500 at a rate of 20 spectra per second. chroma tof4.3x software (leco corporation, st. joseph, mi, usa) and the leco-fiehn rtx5 database were used for raw peak exacting, data baseline filtering and calibration, peak alignment, deconvolution analysis, peak identification, and integration of the peak area. simca14 software package (umetrics, umea, sweden) was used for further data analysis, including principal component analysis (pca) and orthogonal projections to latent structures-discriminate analysis (opls-da). in addition, commercial databases including kyoto encyclopedia of genes and genomes (kegg, http://www.genome.jp/kegg/) and national institute of standards and technology (nist, http:// www.nist.gov/index.html) were utilized to search for metabolic pathways. metaboanalyst (http: //www.metaboanalyst.ca) was used for pathway enrichment analysis. rna preparation, library preparation, rna-sequencing, and data analysis followed those of yang et al. [26] . first, total rna was extracted from each kidney sample individually using trizol reagent (invitrogen, burlington, on, canada). we further used the nanodrop 2000 (thermo, waltham, ma, usa) to measure the rna concentration, and the agilent bioanalyzer 2100 system (agilent technologies, ca, usa) to assess the rna integrity. library preparation was performed as described by li et al. [27] . briefly, a total amount of 1 µg rna per sample was used for the rna sample preparations. sequencing libraries were generated using nebnext ultratm rna library prep kit for illumina (neb, ipswich, ma, usa) and index codes were added to attribute sequences to each sample. the library fragments were purified with ampure xp system (beckman coulter, beverly, ca, usa). at last, pcr products were purified (ampure xp system) and library quality was assessed on the agilent bioanalyzer 2100 system. finally, the constructed cdna libraries were sequenced by an illumina hiseq xten platform. clean data of high quality were obtained from the raw data through in-house scripts by removing those containing adapters or poly-n sequences. all clean reads were aligned to the reference genome (https://www.ncbi.nlm.nih.gov/assembly/gcf_000002315.4/) using tophat2 [28] . cufflinks was used to calculate and analyze the gene expression levels, and fpkm (fragments per kilobase of exon per million fragments mapped) values of each gene were calculated based on the length of the gene and the fragments count mapped to this gene. differential expression genes (degs) analysis was performed by using the deseq r package (1.10.1). only those genes with a fc (fold change) ≥ 2 and fdr (false discovery rate) < 0.01 were defined as degs. furthermore, gene ontology (go) enrichment analysis of degs was implemented by the goseq r packages [29] and the enrichment analysis of degs in kegg pathways was performed using kobas software [30] . in addition, the target seven degs in response to nibv infection were chosen for validation using real-time quantitative pcr (rt-qpcr). the primer pairs for the selected genes were designed using primer 5 and are shown in table s1 . total genome dna from samples was extracted from the cecal contents using ctab/sds method. dna concentration and quality were determined and diluted to 1 ng/µl using sterile water. the 16s rrna genes of each sample v3-v4 region were amplified. the primer set corresponding to primers 341f-806r with the unique barcode was applied for amplification (forward 341f: cctaygggrbgcascag and reverse 806r: ggactacnngggtatctaat). all pcr reactions were carried out with phusion ® high-fidelity pcr master mix (new england biolabs, usa). the pcr products were detected on 2% agarose gel electrophoresis and bands of the desired size (approximately 400-450 bp) were chosen for further experiments. sequencing libraries were generated using trueseq dna pcr-free sample preparation kit (illumina, san diego, ca, usa) and index codes were added. next, sequencing was performed on an illumina hiseq 2500 platform (novogene company, beijing, china). based on their unique barcode, the paired-end reads were granted to samples. those files were demultiplexed and quality filtered with quantitative insights into microbial ecology (qiime) software (version 1.7.0) [31] . the chimera sequences were removed by using uchime algorithm and then the effective tags finally obtained [32] . sequences were clustered to the same otus at 97% similarity by uparse software (uparse v7.0.1001). moreover, the greengene database was used to annotate taxonomic information and the multiple sequence alignment was conducted using the muscle software (version 3.8.31). further, otus abundance information was normalized and subsequent analysis were all performed based on this output normalized data including alpha diversity, beta diversity, and linear discriminant analysis (lda) effect size (lefse) analysis [33] . heatmap for metabolomics, transcriptomics, and microbiomics data was generated using the pheatmap package. corrplot package in r was used to visualize the correlations coefficient (spearman method). availability of data and materials: rna-seq raw data are accessible through ncbi' database: bioproject: prjna510170; 16s rrna gene sequencing raw data are accessible through ncbi' database: bioproject: prjna510025. the institutional animal care and use committee of jiangxi agricultural university approved these animal experiments and all animal experiments adhered rigorously to the animal care guidelines of jiangxi agricultural university (approval id: jxaull-2017003; approval date: 8 march 2017). all the birds were sacrificed using carbon dioxide euthanasia, and all attempts were carried out to minimize the suffering of the animals. obvious enlargement and urate deposition in the kidney of a chick infected with nibv at 10 dpi (right). (d) histopathological changes in the kidney of chickens infected with nibv (h&e staining). the black arrow shows the shedding of kidney tubular epithelial cells and the white arrow shows the interstitial expansion and prominent inflammatory cell infiltration. the black asterisk shows the brush border that was lost in some segments of proximal tubules. the black delimited area shows the loss of the kidney tubular structure. to explore the metabolic pathway alterations associated with nibv infection, we used a gc-tof/ms-based metabolomics method to examine metabolite alterations in the kidney. a total of 519 valid peaks were identified in the total ion current profiles. to compare the metabolite composition of the con and dis groups, pca models were tested (figure 2a ). opls-da was conducted to determine whether nibv infection influenced the metabolic pattern ( figure 2b ) and a 7-fold cross validation was further applied to estimate the robustness and predictive ability to validate the model (figure 2c ). the results of pca and opla-da analysis showed that there was an obvious separation between the content of the con and dis groups, obvious enlargement and urate deposition in the kidney of a chick infected with nibv at 10 dpi (right). (d) histopathological changes in the kidney of chickens infected with nibv (h&e staining). the black arrow shows the shedding of kidney tubular epithelial cells and the white arrow shows the interstitial expansion and prominent inflammatory cell infiltration. the black asterisk shows the brush border that was lost in some segments of proximal tubules. the black delimited area shows the loss of the kidney tubular structure. the chicks inoculated with strain sx9 showed obvious clinical signs from 3 to 9 "day post-infection" (dpi). the nibv-infected chickens were listless, huddled together, and displayed ruffled feathers, while no clinical symptoms similar to the above were observed in the con group. the 10 dpi survival rate of all nibv-infected chickens under analysis was 80%, and the uric acid level in the serum of the dis group was much higher than that of the con group (~1352 vs.~85 µmol/ml, n = 10, p < 0.001, student's t-test, figure 1b ). we observed that kidney lesions were present in all dis group chickens infected with sx9. at 10 dpi (mortality peak), the kidney parenchyma of the dead birds were pale, swollen, and mottled ( figure 1c ). histological examination revealed remarkable injuries in the kidney, including tubular epithelial cell detachment, loss of the kidney tubular structure, as well as interstitial expansion and prominent inflammatory cell infiltration ( figure 1d ). these results indicate that the sx9 strain has strong renal tissue tropism and successfully replicates the chicken visceral gout model. to explore the metabolic pathway alterations associated with nibv infection, we used a gc-tof/ms-based metabolomics method to examine metabolite alterations in the kidney. a total of 519 valid peaks were identified in the total ion current profiles. to compare the metabolite composition of the con and dis groups, pca models were tested (figure 2a ). opls-da was conducted to determine whether nibv infection influenced the metabolic pattern ( figure 2b ) and a 7-fold cross validation was further applied to estimate the robustness and predictive ability to validate the model (figure 2c ). the results of pca and opla-da analysis showed that there was an obvious separation between the content of the con and dis groups, revealing significant changes in the concentrations of metabolites in the kidney induced by nibv infection. all samples fell within the 95% (hotelling's t-squared ellipse) confidence interval. the differential metabolites between dis and con groups were the key to explaining the occurrence of gout in chickens under nibv infection. a total of 160 metabolites displayed significantly different levels based on vip > 1 (variable importance for the projection, opls-da model) and p-value < 0.05 (student's t-test). the lists of differential metabolites are shown in table s2 and its volcano plot are shown in figure 2d . there were 90 annotated metabolites in all 160 differential metabolites, and its categories are shown in figure 2e . as figure 2e shows, the differential metabolites were mainly classified as amino acids, carbohydrates, fatty acids, and conjugates. among those annotated differential metabolite profiles which were displayed in heat maps ( figure s1 ), 65 metabolites were significantly upregulated and 25 were significantly downregulated in the dis group compared to the con group. in addition, the pathway enrichment results are shown in figure 2f . the analysis revealed that the differential metabolites participated in six target pathways (p < 0.05), including valine, leucine and isoleucine biosynthesis, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, d-glutamine and d-glutamate metabolism, aminoacyl-trna biosynthesis, and beta-alanine metabolism. among them, there are two pathways with an impact factor > 0, including valine, leucine and isoleucine biosynthesis (impact value = 0.43), and arginine and proline metabolism (impact value = 0.17). these pathways are related to amino acid metabolism and glycometabolism, which are involved in protein synthesis and energy production, respectively. moreover, metabolomics highlighted n-formyl-l-methionine, a well-known agent for kidney injury for its effects on the increase of the vascular tone/resistance and reduction of renal perfusion [34] , as the most relevant metabolite alteration in nibv-infected chickens (vip = 1.894). undoubtedly, the uric acid level in the dis group was significantly upregulated compared with the con group (vip = 1.798, 4.62-fold). to study the gene expression alterations of chicken's kidney under nibv infection, cdna libraries from two groups were subjected to illumina sequencing. a total of 81.18 gb clean data was obtained, and the q30 base percentage of each sample was not less than 90.37%. from the mapping results, the mapping efficiency between the reads and the reference genome of each to study the gene expression alterations of chicken's kidney under nibv infection, cdna libraries from two groups were subjected to illumina sequencing. a total of 81.18 gb clean data was obtained, and the q30 base percentage of each sample was not less than 90.37%. from the mapping results, the mapping efficiency between the reads and the reference genome of each sample was between 75.59% and 79.29%. the detail of the sequencing and mapping results is provided in table s3 . pca analysis of rna-seq replicates from the kidneys of dis and con group revealed a great deal of variability (figure 3a) . a total of 2868 genes were differentially expressed in the chicken's kidney with nibv infection compared to con group, in which 1521 genes were upregulated and 1347 genes were downregulated. then, we screened seven degs for rt-qpcr analysis to validate the accuracy of the rna-seq data, and the result (figure 3b) showed that the general trends of the selected genes were consistent which proved the reliability of our rna sequencing profiling. go (http://www.geneontology.org) project was applied to explore the potential biological functions of degs. in this study, the 20 most enriched go terms classified by molecular function (mf), cellular components (cc), and biological processes (bp) terms were listed in figure 3c . among them, heparin binding, oxidoreductase activity, external side of plasma membrane, extracellular space, membrane, integral component of membrane, oxidation-reduction process, and immune response were the significantly enriched go terms (p adj < 0.05) in chickens with nibv infection. to further study the biochemical metabolic pathway and signal transduction pathways related to nibv infection, kegg analysis and pathway enrichment analysis were conducted in the kegg pathway database. there were 901 of the 2868 degs that were annotated to 177 pathways in kegg analysis. the level-2 kegg classes are shown in figure 3d , and the 11 significant enriched pathways (corrected p-value < 0.05 as determined by a fisher's exact test) are marked with red number. in environmental information processing pathways which include cytokine-cytokine receptor interaction and cell adhesion molecules (cams) were dramatically regulated. in cellular processes pathway, peroxisome was dramatically affected. in metabolism pathways, metabolism of xenobiotics by cytochrome p450, drug metabolism-cytochrome p450, pyruvate metabolism, arginine and proline metabolism, glycolysis/gluconeogenesis, and fatty acid degradation were significantly regulated. in organismal systems pathways, intestinal immune network for iga production and toll-like receptor signaling pathway were significantly regulated. furthermore, most of these pathways of the upregulated genes enriched were immune system-related pathways, and "cytokine-cytokine receptor interaction" was the most represented pathway. among these pathways we found upregulated, "toll-like receptor signalling pathway", "nod-like receptor signalling pathway" and "rig-i-like receptor signalling pathway" are pattern recognition signal receptor pathways involved in innate immunity. simultaneously, the transcriptome showed that the "peroxisome" and amino acid metabolite pathways were suppressed in chickens infected with nibv. based on the above results, we predicted a schematic diagram of important pathways for chickens in the nibv infection processes (figure 3e) . signalling is activated by monosodium urate (msu) and reactive oxygen species (ros). the activation of tlrs and the ros accumulation activates the nlrp3 inflammasome, resulting in proteolytic cleavage of caspase-1 and the maturation of il-18 and il-1β, and msu has been identified to activate inflammasome complex. considering that the intestinal tract is an important organ for lowering serum uric acid concentrations, 16s rrna sequencing was performed and demonstrated marked alterations of the gut microbial communities in the dis group. the rarefaction analysis curves for each group were near saturation, revealing that the sequencing data had a great quality and that certainly few new species were present ( figure s2 ). nmds (nonmetric multidimensional scaling, figure 4a (figure 4g,h) . lefse was performed to identify significant microbiota composition differences between the groups. seven differentially represented core major groups were identified (figure 4i,j) . differentially abundant phylum detected showed that proteobacteria (lda = 4.7644853231) phylum was most dominantly present in dis group. at the genus level, the microbiota of the con group was enriched with bacteroides (lda = 4.80848153017) from the bacteroidetes phylum and lactobacillus (lda = 4.22406025945) from the firmicutes phylum. at the species level, the microbiota of the con group was enriched with bacteroides vulgatus (lda = 4.79658975077) from the bacteroidetes phylum. the above data describe that nibv infection alters kidney gene expression and metabolic pathways (figure 5a ). we filtered out five overlapping pathways in the transcriptome and metabolome pathway enrichment analysis, including valine, leucine and isoleucine biosynthesis, arginine and proline metabolism, taurine and hypotaurine metabolism, alanine, aspartate and glutamate metabolism, and glutathione metabolism. in addition, the degs in significantly enriched pathways associated with innate immunity and differential metabolites with vip > 1.8 were also chosen for correlation analysis (tables s4 and s5 , respectively). differences in those pathways-associated with degs and differential metabolites were shown with heatmap in figure 5b . then, we analyzed the spearman correlation between genes and metabolites in those pathways, and the result is shown in figure 5c . as shown in figure 5c , there was a positive correlation between tlr7 (tlr, toll-like receptor) and proline (r = 1, p = 0), isoleucine (r = 0.9524, p = 0.0098), threonine (r = 0.9762, p = 0.0035), and valine (r = 0.9762, p = 0.0035). conversely, strong negative correlations were found between glutathione synthetase (gss) and glutamine (r = −1, p = 0). interestingly, we observed that most genes related to innate immune responses had strong positive correlations with differentially abundant metabolites, such as amino acids and fatty acids. the gut microbiota is deliberated a massive "organ" able to perform complex functions and thereby produce a myriad of differentially abundant metabolites. to investigate the functional correlation between the gut microbiome changes and host metabolome alterations, a correlation plot was visualized by calculating spearman's correlation coefficients (figure 5d ). the result indicated that clear correlations could be identified between altered metabolic profiles and modulated gut microbiomes (table s6) . of particular note, some metabolites, including trans-4-hydroxy-l-proline, guanine, and 3,6-anhydro-d-galactose, which decreased in the kidney of nibv-infected chickens, were negatively correlated with the presence of the phylum proteobacteria. furthermore, other metabolites, including canavanine, 2,4-diaminobutyric acid, 5,6-dihydrouracil, malonamide, thymine, phenylalanine, 1,3-diaminopropane, 4-acetamidobutyric acid, proline, threonine, isoleucine, valine, and oxalacetic acid, which increased in the kidney of nibv-infected chickens, were positively correlated with the presence of the phylum proteobacteria. these observations indicated that the significantly modulated gut microbiota was correlated with host metabolic disorders. in our study, the three pathways included in the activation of the innate immune system, the toll-like receptor signalling, rig-i-like receptor signalling, and nod-like receptor signalling pathways, that are the most important three parts of the pattern-recognition receptor (prr) signalling pathway are usually activated in response to infections to stimulate inflammatory responses [35] [36] [37] . in the toll-like receptor signalling pathway (figure 3f, signal 1) , a series of upregulated genes were noticed, including tlr4, tlr7, myd88, irf5, traf3, and irf7. it was already reported that tlr7 primarily recognizes single-stranded rna (ssrna) sequences of rna viruses that enter endosomes by endocytosis [38] [39] [40] . nibv used in this experiment is a single-stranded positive sense rna virus. thus, the expansion of tlr7 is pivotal for the recognition of nibv and variable functions of the toll-like receptor signalling pathway. moreover, a number of viral glycoproteins have been shown to act as viral pamps (pathogen-associated molecular pattern) that bind to and activate tlr4, leading to ifn-β and/or proinflammatory cytokine expression (such as sars coronavirus) [41] . ru liu-bryan's study has established that host expression of tlr2, tlr4, and their intracellular adapter protein myd88 is a major mediator of msu crystal-induced inflammation [42] . this explains the reason for the increase in tlr4 transcription levels in this experiment. in addition, the transcriptomic analysis showed that nibv infection also activated the rig-i-like receptor signalling pathway (figure 3f , signal 2), which included the transcriptional upregulation of genes such as mda5, ips-1, traf3, and iκb. this induction may be due to mda5 acting as a double-stranded rna (dsrna) sensor to trigger an innate immune response against viral infection [43] [44] [45] , while coronaviruses can produce dsrna intermediates during replication [46] . rlrs can not only be expressed in cells infected with various viruses but also directly recognize and perceive virus products and virus particles present in the cytosol outside of the endosomes [47] . therefore, we suspect that the rig-i-like receptor signalling pathway has a greater role than toll-like receptor signalling in recognizing nibv infection. the activation of the toll-like receptor signalling and rig-i-like receptor signalling pathways results in the production of chemokines and other cytokines that induce a proinflammatory response and tissue destruction. in our study, several features that are familiar to chickens infected with nibv are consistent with the immunopathological disease. these features include the pathological damage of kidney and the presence of increased transcription of chemokines and other cytokines, such as il-8 (interleukin 8), il-18 (interleukin 18), and tgf-β (transforming growth factor β). peroxisomes act to eliminate a microbial infection by modulating the canonical innate immunity pathways through ros signalling [48] . several enzymes with antioxidant activity in the peroxisome are involved in neutralizing ros to protect the cells from ros damage. among these enzymes, catalase (cat), superoxide dismutase 1 (sod1), peroxiredoxin 5 (prdx5), glutathione s-transferase kappa 1 (gstk1), dehydrogenase/reductase member 4 (dhrs4), and epoxide hydrolase 2 (ephx2) are included [49, 50] . multiple viruses have been shown to use different mechanisms to reduce peroxisome numbers or interfere with their functions. in our study, the "peroxisome" was the most important pathway according to the downregulated gene enrichment analysis, which includes cat, sod1, dhrs4, and ephx2 that belong to the peroxisome antioxidant defence system, while the catalase activity was decreased both in kidney and serum ( figure s3 ). thus, decreased abundance and/or impaired function of the peroxisome could potentially cause endogenous elevation of ros. ros may either straightly trigger nlrp3 inflammasome assemblage or be indirectly sensed through cytoplasmic proteins that modulate inflammasome activity (figure 3f, signal 3) . the nlrp3 inflammasome senses pathogens or danger signals to promote the maturation of cytokines such as il-18 [51] . the release of active il-18 engages il-18 receptor-harbouring cells and promotes inflammatory responses [52] . in addition, uric acid has been reported as another well-established activator of nlrp3 that is usually generated via xanthine oxidase (xod), accompanying the generation of o 2 •− [53, 54] . consistently, in this study, we detected a significant increase in xod activity in serum ( figure s3 ) and a significant increase in serum uric acid levels (figure 1b) . these results indicate that nibv infection can activate the inflammatory response by inducing severe ros accumulation. the kidney is responsible for the elimination of 70% of the daily ua production [55] . atp-binding cassette transporter, sub-family g, member 2 (abcg2) is a high-capacity urate exporter that is located in the brush border membrane of kidney proximal tubule cells (s3 segment). abcg2 dysfunction results in extra-renal urate under-excretion and is a common mechanism of hyperuricemia [56] [57] [58] . in the present study, the abcg2 mrna was downregulated in the model group chicken kidneys, partially explaining the significantly increased uric acid levels caused by nibv infection. in addition to being caused by insufficient urate excretion, hyperuricemia can also be caused by excessive production of uric acid [59] . we found a high level of glutamine, which enter the purine metabolic pathway as a raw material for uric acid synthesis. the high level of glutamine can be explained by two reasons. first, the lack of mrna abundance of the gene gss in the kidney tissue of the model group inhibited the conversion of glutamine to glutathione. a significant negative correlation between gss and glutamine has confirmed this finding. second, the transcription level of the gene glutamic pyruvate transaminase 2 (gpt2) in the model group chickens' kidneys increased significantly. gpt2 is a pyridoxal enzyme that promotes the conversion of α-ketoglutarate to glutamate [60] . of note, α-ketoglutarate is an intermediate in the tca cycle, and glutamate can be reversibly converted to glutamine by glutamine synthetase. together, these data point to key genes and metabolites related to elevated uric acid synthesis, which provides us with new insights into host response to nibv infection. the present study highlights the correlation between differential expression of genes and differential abundance of metabolites in significantly enriched pathways, and the results showed that those differentially abundant metabolites that map in amino acid metabolism pathways have strongly positive correlations with degs related to innate immune responses. it is well known that organisms fuel or instruct the immune response to pathogen threats by modulating metabolic pathways [61] . innate and acquired immune systems are regulated by a highly interactive network of chemical communications, which include the synthesis of the antigen-presenting machinery, immunoglobulins, and cytokines. this network is highly dependent on the sufficient availability of amino acids. thus, amino acids affect immune responses either directly or indirectly through their metabolites [62] . in our study, the correlation analysis highlighted a significant positive correlation between tlr7 and proline, isoleucine, threonine, and valine. according to reports, isoleucine could maintain the development of immune organs and cells and stimulate the secretion of immune molecule substances in humans and animals [63, 64] , valine could improve dendritic cell function [65] , and it has been proved in vitro that threonine plays a key role in lymphocyte proliferation and immunoglobulin production [66] . we observed an increase in the level of amino acids enriched in proline, leucine, and isoleucine biosynthesis pathway, including isoleucine, valine, and threonine. therefore, changes in these amino acid metabolism pathways may occur through the activation of innate immunity to enhance the body's antiviral response. although there are many studies on amino acids and immunity, the modulation of amino acid metabolism in innate immune responses is still poorly known and deserves further study. previous research has confirmed that the dysfunction of the gut microbiome is tightly associated with kidney and liver diseases, including liver cirrhosis [67] , liver cancer [68] , and chronic kidney disease [69] . the gut microbiome incorporates a wide variety of commensal microbiota and has a large effect on the health of individuals. a community-wide effect involving the gain and loss of microbial populations and changes in general metabolic functions always occurs under pathological conditions. our results showed that the abundance of bacteroides, lactobacillus, and bacteroides vulgatus were significantly reduced in the cecal microbiota of nibv-infected chickens based on lefse analysis. similar to many other bacteroides species, especially bacteroides vulgatus which was shown to promote intestinal homeostasis, the bacteroides vulgatus-mediated induction of semimature dendritic cells is associated with inflammation silencing [70, 71] . thus, decreased abundance of bacteroides vulgatus promotes intestinal flora imbalance and activation of the immune system. furthermore, it is well accepted that lactobacillus may lower serum uric acid levels by reducing intestinal absorption of purines in humans [72] . therefore, a decrease in the abundance of lactobacillus in the caecum of the model group infected with nibv further promoted an increase in uric acid levels in serum. the results of our correlation analysis further confirm this point, that is, the negative relationship between the abundance of bacteroides vulgatus, lactobacillus and the uric acid concentration. however, the mechanism of nibv infection leading to a decrease in their abundance still needs further study. taken together, multitudinous studies to date endorse the concept that a bloom of proteobacteria in the gut reflects gut dysbiosis or an unstable gut microbial community [73] . in view of balanced gut microbiota with high stabilization that has symbiotic relationships with the immune system of the host, which is capable of suppressing the uncontrolled expansion of proteobacteria, the increase in the abundance of proteobacteria in this experiment may be closely related to the immune and metabolic changes caused by nibv infection. this report is the first time that multi-omics approach was employed to profile the metabolic changes and immune responses in kidney, as well as the effects on the intestinal microbiome during nibv infection. our results showed that nibv significantly increased uric acid synthesis, inhibited the function of peroxisomes, and significantly elevated the pattern recognition receptor signalling pathways. in summary, our study comprehensively describes the host responses during nibv infection and provides new clues for further dissection of specific gene functions, metabolite affections, and the role of gut microbiota during chicken gout. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/11/11/1070/s1. figure s1 : heat map of annotated significant differential metabolites. note: the increasing gradient of red color indicate higher upregulation, while an increasing gradient of blue color indicate higher downregulation. figure s2 : rarefaction curves of the species number in the con and dis groups. the curve in each group is nearly smooth when the sequencing data set is sufficiently large (n = 6 per group). figure s3 : the catalase activity (a, b), xanthine oxidase activity (c, d) in kidney and serum. table s1: primers of selected genes for rt-qpcr. table s2 : differentially abundant metabolites identified by gc-tof/ms. significant differences were declared at the level of p-value < 0.05 and vip > 1. table s3 : basic summary of sequence and sequencing reads mapping to reference genome. table s4 : degs in enriched pathways for correlation analysis. table s5 : differentially abundant metabolites in enriched pathways for correlation analysis. table s6 : the relative abundance of seven discriminative features (lda score ≥ 4) identified by lefse analyse. author contributions: conceptualization, x.g.; methodology, x.g. and p.l.; software, c.z. and y.s.; validation, q.w., p.x., and y.y.; formal analysis, p.x., c.z., and p.l.; resources, g.l., p.l., and x.g.; data curation, p.x. and x.g.; writing-original draft preparation, p.x. and p.l.; writing-review and editing, x.g., p.l., g.l., and g.h.; visualization, p.x., c.z., and y.s.; supervision, g.h.; project administration, x.g.; funding acquisition, x.g. funding: this research was funded by the national natural science foundation of china, grant number 31860723. acknowledgments: all authors thank all members of the team for their help in the experimental process in clinical veterinary medicine laboratory in the college of animal science and technology, jiangxi agricultural university. we also thank vincent latigo for the language help rendered in reviewing the revised manuscript. the authors declare no conflict of interest. gout induced by intoxication of sodium bicarbonate in korean native 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into pathogenesis of hypouricemia in siadh decreased extra-renal urate excretion is a common cause of hyperuricemia glutamic pyruvate transaminase gpt2 promotes tumorigenesis of breast cancer cells by activating sonic hedgehog signaling proline catabolism modulates innate immunity in caenorhabditis elegans amino acids and immune function isoleucine administration alleviates rotavirus infection and immune response in the weaned piglet model induction of beta-defensins by l-isoleucine as novel immunotherapy in experimental murine tuberculosis extracellular branched-chain amino acids, especially valine, regulate maturation and function of monocyte-derived dendritic cells geahel, i. factors controlling cell proliferation and antibody production in mouse hybridoma cells: i. influence of the amino acid supply altered profile of human gut microbiome is associated with cirrhosis and its complications obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome altered gut microbiota and microbial biomarkers associated with chronic kidney disease extensive mobilome-driven genome diversification in mouse gut-associatedbacteroides vulgatusmpk outer membrane vesicles blebbing contributes to b. vulgatus mpk-mediated immune response silencing evaluation of purine utilization by lactobacillus gasseri strains with potential to decrease the absorption of food-derived purines in the human intestine proteobacteria: microbial signature of dysbiosis in gut microbiota this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-354931-0bwf8f1i authors: song, jae-hyoung; shim, aeri; kim, yeon-jeong; ahn, jae-hee; kwon, bo-eun; pham, thuy trang; lee, jongkook; chang, sun-young; ko, hyun-jeong title: antiviral and anti-inflammatory activities of pochonin d, a heat shock protein 90 inhibitor, against rhinovirus infection date: 2018-05-02 journal: biomol ther (seoul) doi: 10.4062/biomolther.2017.233 sha: doc_id: 354931 cord_uid: 0bwf8f1i human rhinoviruses (hrv) are one of the major causes of common cold in humans and are also associated with acute asthma and bronchial illness. heat-shock protein 90 (hsp90), a molecular chaperone, is an important host factor for the replication of single-strand rna viruses. in the current study, we examined the effect of the hsp90 inhibitor pochonin d, in vitro and in vivo, using a murine model of human rhinovirus type 1b (hrv1b) infection. our data suggested that hsp90 inhibition significantly reduced the inflammatory cytokine production and lung damage caused by hrv1b infection. the viral titer was significantly lowered in hrv1b-infected lungs and in hela cells upon treatment with pochonin d. infiltration of innate immune cells including granulocytes and monocytes was also reduced in the bronchoalveolar lavage (bal) by pochonin d treatment after hrv1b infection. histological analysis of the lung and respiratory tract showed that pochonin d protected the mice from hrv1b infection. collectively, our results suggest that the hsp90 inhibitor, pochonin d, could be an attractive antiviral therapeutic for treating hrv infection. human rhinoviruses (hrv) are positive single-stranded rna viruses belonging to the family piconaviridae. hrv infection in humans usually causes common cold and mild illnesses, but is sometimes associated with asthma exacerbation and viral upper respiratory tract infection (bartlett et al., 2008) . hrvs are divided into three distinct species including type a, type b, and type c, with over 100 immunologically noncross reactive hrv serotypes (park et al., 2012) . among the hrv serotypes, the majority accounting for 90%, use human intercellular adhesion molecule-1 (icam-1) as their cellular receptor and do not bind mouse icam-1, whereas the remaining 10% of the minor serotypes including hrv1b use a member of the low-density lipoprotein (ldl-1) receptor family and can bind the mouse counterpart (bartlett et al., 2008) . thus, hrv1b is thought to be a suitable model for murine infection. despite showing the highest incidence and occasional complications of chronic bronchitis as well as reactive airway disease exacerbation, there is no approved medication for the treatment of rhinovirus infection. many efforts have been made to produce vaccines to prevent rhinovirus infection, but it is very difficult to produce appropriate ones because of the existence of more than 100 immunologically non-crossreactive rhinovirus serotypes (ledford et al., 2005) . therefore, studies concentrating on the development of effective antiviral agents for treating rhinovirus infections are now underway (al-nakib and tyrrell, 1992) . hsp90, a 90 kda heat shock protein, is a highly abundant, essential, and evolutionarily conserved molecular chaperone at the center of a large protein-folding network (geller et al., 2013) . there are two cytoplasmic isoforms of hsp90 in mammals. hsp90α is an inducible isoform, whereas hsp90β is expressed constitutively. hsp90 function is regulated by a cohort of co-chaperones that modulate its atpase cycle, enabling it to acquire and select client proteins, and to provide a link with other chaperone systems including proteasome degradation systems for protein degradation (geller et al., 2012) . hsp90 inhibitors are known to have broad-spectrum anti-cancer effects via blocking diverse pathways in cancer cells, and they also inhibit the growth and survival of cancer stem cells (li et al., 2009) . in addition, a recent study has shown that hsp90 is practically required for viral protein homeostasis and is critical for viral replication, folding, and assembly (nagy et al., 2011) . hsp90 has been shown to play an important role in the replication of various viruses including dna and rna viruses, with both positive-and negative-sense genomes, and also double-stranded rna viruses, suggesting that hsp90 inhibitors may have broad-spectrum antiviral effects (geller et al., 2012) . in this regard, several hsp90 inhibitors have been studied for the development of antivirals, in vitro, against influenza, sars-cov, hcv, hiv (li et al., 2004) , and herpes viruses (hsv1/2, cmv, vzv) (sun et al., 2013) , as well as against picornaviruses including poliovirus, coxsackievirus, and rhinovirus (geller et al., 2012) . hsp90 inhibitors are very attractive antiviral agents for infections lacking antiviral therapies and for an urgent response to the outbreak of novel viral diseases. in addition, application of hsp90 inhibitors to several animal models of infectious diseases was demonstrated to decrease viral replication in case of poliovirus and hcv infections (geller et al., 2007; nakagawa et al., 2007) . these experiments emphasize the possibility of using these inhibitors as human therapeutic agents. several inhibitors of hsp90 are known and their potential as therapeutic drugs have been tested at both preclinical and clinical stages (neckers and workman, 2012) . radicicol is a natural product and is reported as one of the most potent hsp90 inhibitors (moulin et al., 2005) . it can selectively block the function of atpase and the chaperone function of hsp90 (roe et al., 1999; zhou et al., 2010) . despite its potent activity, radicicol has labile moieties that are rapidly inactivated in vivo, leading to poor efficacy in practice (zhou et al., 2010) . pochonin d was originally isolated from pochonia chlamydosporia and is a radicicol analog with potential hsp90 inhibitory ability (moulin et al., 2005; wang et al., 2016; choe et al., 2017) . it has been shown to inhibit the replication of herpes simplex virus 1 (hsv1) and the parasitic protozoan eimeria tenella (hellwig et al., 2003) . collectively, we assessed the anti-rhinoviral activity of pochonin d both in vitro and in vivo. we found that pochonin d has a significant antiviral effect against hrv1b by inhibiting virus replication and attenuating hrv1b infection-associated lung inflammation. pochonin d was synthesized and purified as recently reported (choe et al., 2017) . ribavirin and sulforhodamine b (srb) were purchased from sigma-aldrich (st. louis, mo, usa). hrv1b, hrv14, and hrv15 viruses were obtained from atcc (american type culture collection, manassas, va, usa), and were cultured at 32°c in hela cells, a human cervical cancer cell line (song et al., 2013) . hela cells were maintained in minimal essential medium (mem) supplemented with 10% fetal bovine serum (fbs) and 1% antibiotic-antimycotic solution. mem, fbs, trypsin-edta, and antibiotic-antimycotic solution were purchased from gibco brl (thermo fisher scientific, waltham, ma, usa). wild type (wt) balb/c mice were purchased from spl laboratory animal company (koatech, pyeong-taek, korea). all mice used in these experiments were between 4 and 5 weeks of age. mice were intranasally infected with 1×10 8 pfu/30 μl of hrv1b. mice were maintained in an experimental facility at the kangwon national university. the animal experiments were approved by the institutional animal care and use committees of kangwon national university. antiviral activity was assessed by the srb method using cytopathic effect (cpe) reduction as reported previously (song et al., 2014) . briefly, one day prior to infection, hela cells (2×10 4 cells/well) were seeded onto a 96-well culture plate (bd biosciences, san jose, ca, usa). on the next day, medium was replaced with medium containing 30 mm of mgcl2, 1% fbs, diluted virus suspension containing a 50% cell culture infective dose (ccid50) of the virus, and an appropriate concentration of the test compounds. the culture plates were incubated at 32°c in 5% co2 for 2 days until the appropriate cpe was achieved. after incubation in ice-cold 70% acetone for 30 min, cells were stained with 0.4% (w/v) srb in 1% acetic acid solution. cell morphology was observed using an axiovert microscope (axiovert 10; carl zeiss, oberkochen, germany) to examine the effect of the compounds on hrv-induced cpe. bound srb was then solubilized with 10 mm unbuffered tris-based solution, and absorbance was read at 562 nm using a versamax microplate reader (molecular devices, palo alto, ca, usa) with reference absorbance measured at 620 nm. the percentage of cell viability was calculated for comparison based on the measured absorbance. in addition, the cell morphology was observed under a microscope at 32×10 magnification (st ernst-leitz, wetzlar, germany), and images were recorded. we performed elisa for assessing the cytokine and chemokine levels. elisa kits for tnf-α, il-1β, and ccl2 (mcp1) were purchased from e-bioscience, and the elisa kit for cxcl1 was purchased from r&d systems. bronchoalveolar lavage fluid (balf) was obtained as described previously (seo et al., 2010) . lungs from mice infected with hrv1b were dissected and homogenized. the levels of cytokines and chemokines in the lung homogenates were evaluated according to the manufacturer's instructions (seo et al., 2011) . absorbance was then read at 450 nm using spectra max 340 (molecular devices). total rna was isolated from each group using the qiaamp viral rna mini kit (qiagen, hilden, germany). reverse transcription was performed using rnase inhibitor, m-mlv rt 5× buffer, m-mlv reverse transcriptase, oligo(dt)15 primer, and dntp mixture manufactured by promega (madison, wi, usa), and quantitative real-time pcr was performed using the thunderbird tm sybr qpcr mix (toyobo, osaka, japan) on the cfx96 tm optics module (bio-rad, hercules, ca, usa), with the following primers: hrv5′-ncr-up: 5′-tcc tcc ggc ccc tga atg-3′, hrv5′-ncr-down: 5′-gaa aca cgg aca ccc aaa g-3′, ccl2-up: 5′-tta aaa acc tgg atc gga acc aa-3′, ccl2-down: 5′-gca tta gct tca gat tta cgg gt-3′, il-6 up: 5′-ctg gag tca cag aag gag tgg-3′, il-6 down: 5′-ggt ttg ccg agt aga tct caa cells from the bronchial alveolar lavage fluid (balf) in the lungs were collected and stained with the following antibodies: fluorescein isothiocyanate conjugated anti-cd11b, phycoerythrin: cy-7 conjugated anti-ly6g, allophycocyanin conjugated anti-ly6c, and phycoerythrin conjugated anti-f4/80. all antibodies used for flow cytometric analysis were purchased from bd biosciences. the data were acquired on a facs verse system (bd bioscience) and analyzed using the bd facsuite software. to evaluate the effect of pochonin d on lung histology in hrv1b-infected mice, we dissected the lung tissue and performed histological analysis as reported previously (ahn et al., 2008) . briefly, lung tissues were fixed in 4% formaldehyde (masked formalin, dana korea, incheon, korea), dehydrated in graded concentrations of ethanol, washed in xylene, and embedded in paraffin. paraffin blocks were sliced to obtain 10-μm-thick sections, which were then stained with hematoxylin and eosin (h&e). the sections were observed by a pathologist in a blind test under a light microscope. each sample was scored based on the extent of edema, hemorrhage, and cell infiltration. to analyze the mouse respiratory tract, we excised the mouse respiratory tract after hrv1b infection with or without pochonin d treatment. the tissues were washed with pbs, and then fixed in 4% glutaraldehyde (ted pella, redding, ca, usa) and 1% paraformaldehyde solution (electron microscopy sciences, hatfield, pa, usa) in 0.1 m cacodylate buffer (ph 7.4) for 4 h. after fixation, the tissues were rinsed thrice in 0.1 m cacodylate buffer (sigma-aldrich) (ph 7.4), for 10 min. samples were then sequentially immersed in 60, 70, 80, 90, and 100% ethanol (merck, kenilworth, nj, usa) for 20 min each. samples were then immersed in ethanol and isoamyl acetate (sigma-aldrich) buffer, and dried. the dried samples were observed using a supra55v vp-fesem (carl zeiss) at the korean basic science institute, chuncheon. we used student's t-test to compare differences between two groups. to compare multiple groups, we carried out oneway anova followed by the newman-keuls test. values of p<0.05 were considered significant at a 95% confidence interval. to identify the anti-hrv1b effect of pochonin d, we per-biomol ther 26 (6) to evaluate the effect of pochonin d on hrv1b-induced cpe, we observed the morphology of hrv1b-infected cells. two days after infection of hela cells with hrv1b, mock cells ( fig. 1d -a) and cells treated with 10 μm pochonin d (fig. 1d -b) or 2 μm pochonin d (fig. 1d-c) showed typical spread-out shape and normal morphology. at the tested concentration, no signs of cytotoxicity with pochonin d were observed ( fig. 1d -b, 1d-c). infection with hrv1b in the absence of pochonin d resulted in severe cpe (fig. 1d-d) . however, addition of 10 μm pochonin d or 2 μm pochonin d to infected hela cells inhibited formation of visible cpe (fig. 1d-e, 1d-f) . thus, the cpe induced by virus infection is prevented in the presence of pochonin d. to assess and verify the in vivo antiviral activity of pochonin d against hrv1b, we first determined the pathological phenotype of mice after intranasal hrv1b infection. balb/c mice were intranasally infected with 1×10 8 pfu/30 μl of hrv1b. mice were killed at 8 h, 1 day, 3 days, and 5 days after virus inoculation, and the levels of pro-inflammatory cytokines including ccl2, cxcl1, il-1β, tnf-α, and il-6, and virus titers in the lungs were assessed. mice infected with hrv1b produced significantly higher levels of ccl2, cxcl1, il-1β, tnfα, and il-6 (supplementary fig. 2a-2e) with elevated virus titers ( supplementary fig. 2f ) at 8 h and 1 day after infection than the uninfected control mice. the levels of pro-inflammatory cytokines and virus titers peaked at 8 h after infection, and were reduced by day 5 to those observed in uninfected mice as reported previously (bartlett et al., 2008) . we therefore decided to monitor the lung cytokine levels and virus titers at 8 h after hrv1b infection for evaluating the antiviral activity of pochonin d in mice. to assess the antiviral activity of pochonin d against hrv1b, mice were intraperitoneally administered 200 μg/kg of pochonin d, at 1 h prior and 4 h after intranasal hrv1b infection. we performed placebo infection in control mice, and administered vehicle intraperitoneally in hrv1b-infected mice as a negative control. after 8 h of infection, we sacrificed the mice and obtained lung samples. real-time pcr analysis of viral mrna in lung tissues revealed that the virus titer was significantly reduced in pochonin d-treated mice compared to that of vehicle-treated mice after hrv1b infection ( fig. 2a) . we thus confirmed that pochonin d has an anti-hrv activity in vitro as well as in vivo when administered systemically via the intraperitoneal route. virus-induced cytokines and chemokines were secreted in the alveolar spaces and balf at 8 h after hrv1b infection. virus infection increases the secretion of cxcl1, ccl2, il-6, il-1β, and tnf-α from leukocytes and activates innate immune responses to induce inflammation (subauste et al., 1995; saklatvala et al., 1996; bartlett et al., 2008) . to evaluate the effect of pochonin d on pulmonary cytokines and chemokines induced by hrv infection, mice were infected and treated with 200 μg/kg of pochonin d twice, 1 h before and 4 h after intranasal hrv1b infection. at 8 h after hrv1b infection, lungs and balf were obtained from the mice, and subjected to elisa for measuring cytokines and chemokines. in the lungs, the levels of ccl2, il-1β, tnf-α, il-6, and cxcl1 were significantly increases by hrv1b infection, and these were decreased by pochonin d treatment (fig. 2b-2f ). similarly, the levels of il-1β, tnf-α, il-6 and cxcl in the balf were significantly increased by hrv1b infection and reduced by pochonin d treatment; however, the level of ccl2 in balf was not significantly altered after hrv1b infection (fig. 3) . these data indicate that increased secretion of pro-inflammatory cytokines and chemokines induced by rhinovirus infection was mitigated by pochonin d treatment. to identify the mechanisms underlying the anti-inflammatory effects of pochonin d in rhinovirus infection, we analyzed cellular infiltrates in the balf of rhinovirus-infected mice after pochonin d or vehicle treatment. a few changes in the total cell numbers in the lung and balf were observed after treatment of hrv1b-infected mice with pochonin d (supplementary fig. 3) . recent studies suggest that myeloid-derived suppressor cells (mdscs), which expand during cancer, inflammation, and infection, might be involved in increasing the level of cytokines and chemokines in influenza-induced pulmonary inflammation (jeisy-scott et al., 2011; atretkhany and drutskaya, 2016) . we investigated whether the same was true for rhinovirus. we therefore assessed cellular infiltration of neutrophils into the balf of hrv1b-infected mice treated with pochonin d or vehicle (fig. 4) . cd11c + f4/80 + alveolar macrophages were excluded from the gating for neutrophils. the number of mdscs in balf showing the phenotype of granulocytic neutrophils was significantly higher in rhinovirus-infected mice than in non-infected mice. moreover, the number of these cells decreased with pochonin d treatment in virus-infected mice (fig. 4b) . similarly, the percentage of mdsc cells in balf from rhinovirus-infected mice was higher than that in control mice, and the percentage of granulocytic neutrophils in balf from hrv1b-infected mice was reduced by pochonin d treatment. thus, we could confirm that inflammatory cell infiltrates in the lung were significantly increased by hrv1b infection, and could be significantly inhibited by administration of pochonin d. next, we assessed the histological changes in lungs of hrv1b-infected mice. lungs from uninfected mice exhibited typical normal pulmonary tissue, whereas rhinovirus-infected mice demonstrated characteristic inflammatory lesions with viral infection, including necrotizing bronchiolitis and interstitial pneumonia (fig. 5a) . on the other hand, rhinovirus-infected biomol ther 26 (6) mice treated with pochonin d showed moderate inflammation with reduced necrosis, inflammatory cell infiltrates, and pulmonary edema compared to those in rhinovirus-infected mice without treatment. we scored the lung sections based on the extent of edema, hemorrhage, and cell infiltration (fig. 5b ). hrv1b-infected mice showed serious hemorrhage, edema, and cell infiltration compared to control mice, and the pochonin d treatment group showed reduced damage induced by hrv1b infection. we also observed the changes in the airway cilia of the trachea by sem (fig. 5c ). uninfected mice showed abundant cilia in the trachea with regular arrangement and intact septum. however, the trachea of rhinovirus-infected mice was damaged, and we could observe abnormal cilia with some phlegm. on the contrary, the condition of the trachea in hrv1b-infected mice treated with pochonin d showed amelioration of damage caused by hrv1b infection compared to that in vehicle-treated mice. despite the presence of phlegm and decreased number of cilia, the cilia in pochonin d-treated mice after hrv1b infection were relatively intact. these results suggest that the lung and trachea were significantly damaged by rhinovirus infection, and treatment with pochonin d protected the mice from histological damage induced by hrv1b. generally, virus-specific proteins have drawn attention for the treatment of viral infection as targets. however, the focus of antiviral approaches has recently started to move toward targeting host factors essential to virus multiplication. hsp90, a molecular chaperone that regulates the function, turnover, and trafficking of several proteins including signaling and regulatory proteins, is one of the important host factors that play critical roles in the viral life cycle. hsp90 inhibitors have been reported to inhibit ebola virus (ebov) replication, and cause degradation of the viral polymerase (smith et al., 2010) . however, the exact mechanism underlying the anti-ebov activity of hsp90 inhibitors remains unknown. in influenza virus infection, hsp90 is required for viral genome replication. as hsp90 associates with subunits of the influenza virus, inhibition of hsp90 leads to degradation of viral subunits. besides, hsp90 inhibitors reduce the levels of the assembled polymerase complex, resulting in decreased viral rna levels (momose et al., 2002) . a recent study showed that hsp90 is also required for the replication of beta-herpesviruses (burch and weller, 2005) . in the human cytomegalovirus infection model, hsp90 inhibition resulted in degradation of the viral polymerase and reduction of viral gene expression via downregulation of the pi3-kinase pathway (basha et al., 2005) . similarly, in the flock house virus, hsp90 influences rna polymerase stability (kampmueller and miller, 2005) . collectively, pharmacological inhibitors of hsp90 have potential as broad spectrum antiviral agents. in addition to their universal activity against diverse viral infections, hsp90 inhibitors show the possibility of overcoming viral drug resistance. most antiviral agents lead to generation of drug-resistant variants, which is one of the major issues in the development of effective antiviral therapy (zur wiesch et al., 2011) . interestingly, hsp90 inhibitors are not reported to induce viral drug resistance till date. therefore, they might be particularly useful for antiviral therapy against viruses prone to develop drug resistance (geller et al., 2012) . hsp90 inhibitors also have potent anti-inflammatory and anti-oxidative actions in vascular tissues (hsu et al., 2007) . hsp90 inhibitors were shown to extend survival, attenuate inflammation, and reduce lung injury in murine sepsis (chatterjee et al., 2007) . hsp90 was also suggested to participate induced by hrv1b infection. mice were infected with 10 8 pfu/30 μl pfu hrv1b intranasally. pochonin d treatment was performed twice, before and after virus infection. eight hours after infection, the lung was isolated and histological analysis was performed as described in the materials and methods section. (a) representative h&e stained sections were prepared from hrv1b-infected mice treated with pochonin d or vehicle. (b) histological scores based on edema, hemorrhage, and cell infiltration. scores from 1 to 5 were given based on the criteria. bar graphs show the mean ± sem. *p<0.05, **p<0.01, and ***p<0.001, newman-keuls multiple comparison test (anova). (c) airway cilia in trachea isolated from hrv1b-infected mice were observed by sem. the trachea of control, hrv1b-infected, and hrv1b-infected pochonin d-treated groups by sem. lower magnification ×1000 and higher magnification ×4000. in viral capsid protein folding and in the assembly of various picornaviruses including poliovirus, rhinovirus, and coxsackievirus, which renders hsp90 an attractive candidate for the development of antiviral vaccines (brenner and wainberg, 1999) . hsp90 is also important for subcellular localization of specific mrnas in regions neighboring the mitochondria, which could explain the inhibitory effect of hsp90 inhibitors on rna polymerase. human rhinoviruses cause common cold in humans, and can sometimes accelerate airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis (zaheer et al., 2014) . as an important human respiratory virus, hrv is a non-enveloped positive-sense single-strand rna virus involved in 50-80% of upper respiratory tract infections and has also been associated with lower respiratory tract disease in high-risk populations, for example in patients with asthma or other airway inflammations (gern and busse, 1999) . generally, symptoms of rhinovirus in mice are not severe. however, our present data showed that the levels of pro-inflammatory cytokines such as tnf-α and il-6 in the lung and balf of mice were increased upon intranasal hrv1b infection, which is reported to contribute to the pathogenesis of asthma during long-term infection (liebhart et al., 2002; jartti and korppi, 2011; rincon and irvin, 2012) . ribavirin is the only antiviral drug approved by the fda for treatment of rsv infection (molinos-quintana et al., 2013) , and is also a broad-spectrum antiviral drug for rna viruses including flu-a, hrv 14, rsv, and cvb3 (shi et al., 2007) . although ribavirin is known to have a broad-spectrum antiviral activity against several respiratory viruses, it has limitations due to its controversial efficacy and toxicity (kneyber et al., 2000) . indeed, ribavirin did not show efficient antiviral activity against hrv1b infection in our experiment, and 50 μg/ml of ribavirin showed only marginal antiviral activity in hela cells infected with hrv1b (data not shown). in the present study, we analyzed the antiviral activity of pochonin d against hrv infection. although pochonin d is a well-known hsp90 inhibitor (moulin et al., 2005; wang et al., 2016; choe et al., 2017) , it is still uncertain that the inhibition of hsp90 by pochonin d is directly associated with the antiviral activity of it. we found that treatment with pochonin d lowered the level of pro-inflammatory cytokines in the lung and balf of mice, which were increased by rhinovirus infection. furthermore, the virus titers of hrv-infected mice treated with pochonin d were significantly decreased to levels similar to those in naïve mice. we also examined the levels of pro-inflammatory chemokines/cytokines (ccl2, cxcl1, tnf-α, il-6, and il-1β) in lung lysates and lung rna. their concentrations were decreased by pochonin d treatment in hrv1b-infected mice, and were comparable to the chemokines/cytokines levels in naïve mice. these data suggest that pochonin d may reduce inflammatory damage in rhinovirus-infected mice. we also found that neutrophil infiltration into the inflammatory site was reduced by pochonin d treatment in hrv1b-infected mice. this reduction may be due to the mild viral infection and inflammation in pochonin d-treated group. finally, we observed the histopathology of the lung and airway, and found that pochonin d treatment ameliorated the damage induced by rhinovirus infection in the lung and airway. in vitro, 10 μm of pochonin d did not influence cell viability; however, slight toxicity was observed at pochonin d concentrations greater than 50 μm (data not shown). adverse ef-fects were also observed in mice treated with pochonin d at 1.75 mg/kg and 600 μg/kg, but not with 200 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hsp90-kinase interactions human rhinovirus-induced isg15 selectively modulates epithelial antiviral immunity insights into radicicol biosynthesis via heterologous synthesis of intermediates and analogs population biological principles of drug-resistance evolution in infectious diseases key: cord-312964-vsrqmmv7 authors: doyle, william j.; alper, cuneyt m. title: prevention of otitis media caused by viral upper respiratory tract infection: vaccines, antivirals, and other approaches date: 2003 journal: curr allergy asthma rep doi: 10.1007/s11882-003-0093-7 sha: doc_id: 312964 cord_uid: vsrqmmv7 otitis media (om) imposes significant morbidity on the pediatric age group and a large financial burden on the general population. because standard medical treatments are not highly efficacious in resolving the accompanying middle ear (me) inflammation, a goal of current research is om prevention. past studies show that new episodes of om are usually a complication of viral upper respiratory infection (vuri), and therefore, a rational approach to achieving that goal is to develop intervention strategies that target vuriassociated om. however, past experiences with antibiotics show that, in the absence of well-defined treatment protocols that maximize expected efficacy, the adoption of prophylactic or active treatments for om can have negative consequences for the patient and for the general population. in this review, we discuss the hypothesized mechanisms by which a vuri is translated into an acute om episode and describe different strategies for aborting that process. limitations to deployment of each strategy are outlined. otitis media (om) is a very common disease in infants and young children and occurs, albeit less frequently, in older children and adults as well [1] . the hallmark feature of om is an inflammation of the middle-ear (me) mucosa with or without the presence of effusion in the me airspace. new om episodes (ie, acute om [aom]) can be accompanied by local symptoms of pain and overt signs (eg, erythema and bulging of the tympanic membrane) interpretable as evidencing an in situ bacterial infection or can be relatively asymptomatic in presentation. as yet, it is unclear if these presentations represent different underlying etiologies (eg, local infection versus eustachian-tube dysfunction) or differ-ent expressions of a common etiology (eg, by stage, pathogen, host response). aom can progress and/or predispose to a persistent inflammatory condition of the me mucosa; om with effusion (ome, synonyms: secretory om, glue ear) that is recalcitrant to standard medical therapies including antibiotics and anti-inflammatories [2, 3] . because of the conductive hearing loss that usually accompanies that disease expression, ome in infants and young children can cause delayed speech and language acquisition, educational deficits, and poor social adjustment [4] . effusion recovered from symptomatic aom is usually culture-positive for bacterial pathogens of which streptococcus pneumoniae, hemophilis influenzae (non-typeable), and branhamella catarrhalis are the most common [5] . in the pre-antibiotic era, aom was associated causally with a number of potentially life-threatening complications, including meningitis and mastoiditis, as well as with sensorineural hearing loss. current practice in the united states is to treat symptomatic aom with antibiotics, which was shown in clinical studies to eradicate the bacterial pathogens and decrease the duration of pain-related symptoms [6] . nonetheless, it is not established that antibiotic treatment promotes earlier resolution of the me mucosal inflammation or aborts disease progression to ome. moreover, the experience of physicians who practice "watchful waiting" (ie, antibiotic treatment reserved for aom episodes for which signs/symptoms fail to resolve within a period of days) shows that most episodes of aom are self-limiting and that antibiotics can be withheld without obvious, immediate consequence in as many as 80% of the episodes [7] . although wide adoption of this treatment strategy is expected to reduce antibiotic use and perhaps decrease the selective pressures driving the increasing prevalence of antibiotic-resistant pathogens, ad hoc identification of patients in need of antibiotic treatment is not possible at present, and the long-term consequences of this practice, with respect to possible increased risks of complications, sequelae, and recurrences are not known. in the united states, the direct and indirect costs of treating om are estimated at $5 billion per year [8] . because of this large economic burden and existing controversies over current treatments for aom and ome, disease prevention otitis media (om) imposes significant morbidity on the pediatric age group and a large financial burden on the general population. because standard medical treatments are not highly efficacious in resolving the accompanying middle ear (me) inflammation, a goal of current research is om prevention. past studies show that new episodes of om are usually a complication of viral upper respiratory infection (vuri), and therefore, a rational approach to achieving that goal is to develop intervention strategies that target vuriassociated om. however, past experiences with antibiotics show that, in the absence of well-defined treatment protocols that maximize expected efficacy, the adoption of prophylactic or active treatments for om can have negative consequences for the patient and for the general population. in this review, we discuss the hypothesized mechanisms by which a vuri is translated into an acute om episode and describe different strategies for aborting that process. limitations to deployment of each strategy are outlined. has been a primary goal. in that regard, a number of prophylactic strategies were reported to be effective in preventing aom or recurrences of aom in "high-risk" children. these include: tympanostomy tube insertion [9] , breast-feeding [10] , antibiotic prophylaxis [9] , passive immunization with high-titer serum [11] , vaccination against bacterial pathogens (eg, s. pneumoniae [12•]) , and other treatments that modify the bacterial flora of the nasopharynx (eg, adenoidectomy, xylitol chewing gum or lozenges, nasopharyngeal seeding with probiotics [13] [14] [15] ). however, general deployment of some of these strategies had (or is suspected of having) unanticipated consequences that curtailed enthusiasm for their use. this is best exemplified by seasonal, antibacterial prophylaxis in high-risk children, an intervention believed by many to contribute to the selection and dissemination of pathogenic bacteria resistant to common (and increasingly less common) antibiotics [16] . of note, one well-controlled, double-blind clinical study [12•] reported that conjugated pneumococcal vaccines decreased the frequency of aom episodes caused by pneumococcal types of bacteria included in the vaccine but increased the frequency of om caused by nonrepresented types. because the ecology of the nasopharynx with respect to bacterial adaptation, niche availability, species competition, and species turnover is poorly understood, implementation of any strategy that targets specific (eg, antibacterial immunization) or nonspecific (eg, antibiotics) bacterial species (types, strains) at that site will have unintended consequences, some of which might be detrimental to the host and/or general population [16] . although bacteria can usually be recovered from the me during aom, it is well accepted that the onset of most of these episodes is temporally associated with a viral upper respiratory tract infection (vuri). specifically, more than 60% of new om episodes are diagnosed immediately following or concurrent with a symptomatic vuri, and conversely, aom occurs in approximately 25% of otherwise healthy infants and young children with naturally acquired vuris [17,18,19•] . a causal relationship between these diseases is supported by epidemiologic studies that document a similar seasonal patterning for vuri and aom [20] and by others that reported vuri to be a significant risk factor for om [21] . also, upper respiratory viruses, viral proteins, and/ or viral genomic sequences were shown to be present in a high percentage of me effusions recovered from aom episodes [22, 23] . disagreements exist concerning the relative causal importance for aom of the different upper respiratory viruses, including, among others, respiratory syncytial virus (rsv), rhinovirus, adenovirus, influenza and parainfluenza virus, and coxsackie virus, but the evidences presented favoring any one virus are not convincing, given the biases introduced by sampling season, differential sensitiv-ity of employed viral assays, and the relative skills of the laboratories. it seems that most, if not all, upper respiratory viruses can cause om as well as provoke eustachian-tube dysfunction and me underpressure, which are considered to be preconditions for om pathogenesis [24] [25] [26] . direct support for vuri causality of aom is provided by animal studies that reported om to result from adenovirus and influenza virus infection of the nose and nasopharynx [27, 28] and by studies in adult volunteers that reported om to be a complication of experimental infection with rhinovirus and influenza a virus [24, 25] . as mentioned, pathogenic bacteria are recovered from the me during aom, but this does not preclude a primary viral etiology. active synergy between certain upper respiratory viruses and nasopharyngeal pathogens was demonstrated for om pathogenesis in chinchillas and humans [27] [28] [29] , and pre-existing or concurrent vuri in infants and children with acute, bacterial om is frequently observed [24, 25] . given these evidences of causality, interventions that prevent vuris and/or prevent the development of om during vuris offer a rational and, perhaps, preferential alternative to antibiotic treatment or prophylactic strategies that target bacterial pathogens. in this review, we discuss the potential targets for this strategy and outline the practicality of the various options. others have published reviews of this topic with a different emphasis and focus [30• ]. the specific mechanism of om pathogenesis during a vuri is debated. although virus is not usually recovered by culture from the me during vuri-associated om, viral protein and/ or nucleic acids can be detected in high percentages of effusions using sensitive assays [21, 22] . the interpretation of this observation as evidencing local viral infection can be questioned given the lack of proof that these viruses replicate within the me mucosa. recent work showed that viral genomic sequences are confined to the effusion compartment and are not recoverable from mucosal biopsies [31] . of interest, hiv was detected by polymerase chain reaction in me effusions recovered from adult patients with om, who were also seropositive for that virus [32•] . most reasonably, this can be interpreted as delivery to the me of virus sequestered within infiltrating leukocytes during acute inflammation. a similar mechanism can account for the detection of virus protein and nucleic acids in me effusions during or immediately following a vuri. there, inflammatory cells that have engulfed virus on encounter at the nose and nasopharynx "home" to the me in response to inflammation initiated by bacterial infection, neurogenic inflammation (ngi), hydrops ex vacuo, or other causes [33] . this hypothesis is consistent with existing data including the observation that bacteria-positive me effusions also containing viral genomic sequences are less likely to resolve with antibiotic therapy than are those with bacteria alone [34] . there, the presence of viral genomes in the effusion evidences concurrent or pre-existing vuri with attendant eustachian-tube obstruction and the consequent inhibition of effusion clearance. a review of existing data suggests a more complicated mechanism of om pathogenesis during a vuri. in such a case, a cascade of events is precipitated by exposure of the nasal mucosa (with probable localized cellular infection) to a virus. upon virus detection by local antigen-presenting cells (eg, dendritic cells, resident macrophages) and virus interaction with epithelial cells, chemical components of the innate immune system are produced and/or upregulated (eg, defensins, increased nk activity [35] ), and the host-alert cytokines, tumor necrosis factor-α (tnf-α), and interferon are synthesized [36, 37] . if this initial response does not abort viral replication, the sustained levels of chemical signals are processed and amplified by genetically programmed (eg, cytokine genotypes [38•]) and environmentally tuned (eg, chronic stress [39] ) biologic filters, whose outputs first, cause the synthesis and release of inflammatory mediators (eg, histamine, bradykinin, arachidonic acid metabolites) that enhance local influx of effector chemicals (eg, serum antibodies) and leukocytes [40, 41] ; second, moderate production of tnf-α and interferon that initiate ngi and coordinate the immune response; and third, modulate the upregulation of more downstream cytokines such as interleukin (il)-1, il-6, il-8, and il-10 that control the magnitude and duration of the immune/ inflammatory response. the synthesized and released inflammatory mediators, neurokines, and cytokines interact in both positive and negative feedback loops to moderate the degree of inflammation and to specify the type (primary th1 or th2) and magnitude of the host immune response [42] . the primary effect of these responses is to neutralize free virus and kill virus-infected cells, events that signal the production of "anti-inflammatory" cytokines and chemokines. these "late" signals in turn downregulate inflammation, eliminate cellular debris, and promote mucosal healing. the local tissue damage caused by virus infection, the nasal inflammatory response attributable to host defense, and the local and systemic effects of the various chemical signals are expressed as symptoms and signs of illness. extension of the inflammation with or without disseminated virus infection to anatomically contiguous structures (eg, the eustachian tube, paranasal sinuses, lungs) can cause complications including om, sinusitis, and asthmatic exacerbations. also, ngi is initiated by tissue damage, upregulated by tnf-α, and modulated by other inflammatory mediators [43, 44] . ngi amplifies the degree of local inflammation (thus promoting effector component influx to the site of infection) and causes reflexive inflammation at more distal target tissues (perhaps in anticipation/preparation for disseminated infection). because the distal effects of ngi can include eustachian-tube dysfunction and increased me mucosal perfusion, possible consequences of ngi activation are destabilized me pressure regulation, the subsequent development of significant me underpressures, and, if prolonged, om by hydrops ex vacuo [45] . a secondary consequence of nasal infection with certain upper respiratory viruses is a more favorable environment for the nasopharyngeal growth of specific bacterial pathogens (eg, influenza virus infection and s. pneumoniae colonization [27, 29] ). the results of other studies suggest that upregulated antiviral defense is accompanied by downregulated antibacterial defense [46] [47] [48] . both of these effects serve to free resident nasopharyngeal pathogens from physiologic sequestration, thereby allowing them to effectively compete with and replace commensal species. intermittent relief of viral-initiated me underpressure by eustachian-tube openings can aspirate the nasopharyngeal pathogens into the me and cause in situ bacterial infection. infection at that site causes the early, local synthesis of tnf-α by the me mucosa and a cascading production of other cytokines/ mediators within the me [49] . in turn, these chemical signals provoke the me mucosal inflammation, which characterizes om. although incomplete, the previous description highlights the various checkpoints that can be targeted to prevent om during a vuri (fig. 1) . broadly, these checkpoints can be subdivided into two general classes on the basis of temporal dynamics; ie, those existing before (i, ii) and after (iii-v) established virus infection of the nose/nasopharynx. an obvious, although somewhat impractical (given current demographic patterns and economic realities in the united states and other countries) option to prevent om caused by a vuri is to reduce the probability of virus exposure, and thus the risk for infection. this can be accomplished by: maintaining good hygienic behaviors in all social situations (eg, frequent hand washing, daily nasal lavage [50] ); withdrawing children at high risk for om from day care or preschool (or enforced absence during virus epidemics); strict adherence to rules that "send home" children (and teachers) presenting to day care or primary school with signs/symptoms of a cold/flu; and early antiviral treatment of ill parents, siblings, and other frequent contacts. to date, none of these options has been explored in clinical trials, and their potential impact on om incidence is not known. one inherent limitation to their expected efficacy is the absence of symptoms/signs in a high frequency of persons with confirmed upper respiratory virus infection [51•,52] , and thus the inability to identify all infected contacts for avoidance and/or treatment. alternatively, om could be prevented by priming the host to resist virus infection. past studies show that high homotypic serum igg antibody titer, high mucosal secretory iga-specific antibody titer, and upregulated immuno-logic surveillance (eg, natural killer, cd8 function) can prevent infection, reduce viral load, diminish the magnitude of the signal chemical production, and decrease the magnitudes and frequencies of overt signs and symptoms of a vuri [53, 54] . therefore, increasing the pre-exposure, host levels of those antibodies, and/or functional activation of t-cell subsets by vaccination and/or immunoglobulin therapy could limit viral replication and abort the pathogenesis of a vuri and its complications. recent clinical studies confirmed the efficacy of this overall strategy. specifically, a number of studies reported a significantly lesser incidence of infection with the target virus and fewer associated om episodes in infants and children immunized with influenza virus vaccines (by both parenteral and intranasal routes [55, 56, 57 •]) or pretreated with anti-rsv-enriched serum [58] . unfortunately, although vaccines for other upper respiratory viruses are in development and testing (although a rhinovirus vaccine is not anticipated, given the large number of circulating types), only influenza vaccines are currently deployed, and their overall reduction of om burden is limited (approximately 10%). also, an interpretation of the results for passive immunization with enhanced rsv antibody serum is not clear-cut because the observed om reduction was not restricted to the rsv season, and a study that used monoclonal anti-rsv antibodies (palivizumab; synagis, medimmune, gaithersburg, md) did not reduce om episodes [59] . this leaves open the possibility that om prevention was mediated by coexistent antibodies directed against pathogenic bacteria within the immunization serum. an untested possibility is the prophylactic use of antivirals in children at high risk for om during those seasons typically characterized by infectious spread of the target virus or after the virus is identified in family members and/or regular contacts (eg, classmates, family members). however, prophylactic antiviral treatment during a viral season might endanger the health of treated patients (as experience with long-term use of these potent chemicals is limited) and might represent a societal hazard by selecting for resistant virus strains or, perhaps of greater concern, virus strains with altered infectious and/or trophic properties [60] . also of interest is the possible, prophylactic use of nonspecific, immune stimulants that upregulate components of innate immunity and/or increase immune surveillance [61] , although given the controversial status and/or poorly rationalized mechanism of action for available treatments (eg, homeopathic medicines), the practical application of this approach lies in the future. [24-26,40,51•] . also, the extent of symptoms and signs of illness during vuris does not predict the temporal dynamics of infection resolution (ie, both asymptomatic and symptomatic persons resolve the infection), and although most available treatments target known chemical mediators and are effective in promoting symptom relief, they do not significantly prolong the period of viral shedding [62] . these results suggest that the host response to common upper respiratory viruses (black box, fig. 1) includes: virus-specific, immunologic defense pathways; requisite inflammatory pathways; and nonspecific, coincidental inflammatory pathways. although the requisite and virus-specific responses are important for preventing virus dissemination and re-establishing mucosal health, the nonspecific, coincidental inflammatory responses can cause unnecessary symptoms and provoke complications. therefore, an ideal vuri treatment would reduce illness and decrease complications by attenuating the nonspecific, coincidental responses while sparing the requisite responses; ie, pharmacologically tune the host response. this approach requires a much better understanding of the complex signaling involved in activating the effector components of these different pathways, but the promise of generalized applicability across viruses makes the pursuit of such knowledge a research priority. because viral load modulates the production of the chemical signals that provoke inflammation, early antiviral treatment might prevent the necessary preconditions for om. one study of experimental influenza virus infection in adults reported a significantly lesser frequency of otologic manifestations (ie, abnormal me pressure) for the group pretreated with the neuraminidase inhibitor, zanamivir, when compared with those treated with placebo [63] . in contrast, a second study in that model of infection used rimantadine and placebo treatments begun at the time of initial symptoms and reported less influenza virus shedding, symptoms of illness, nasal secretions, and nasal pro-inflammatory cytokine levels for the rimantadine group, but no between treatment differences in otologic complications, including the frequency of om [64] . the discrepant results of these two studies with respect to antiviral efficacy for preventing the otologic complications of influenza a infection are explicable by the timing of the intervention (ie, prophylaxis vs treatment). therefore, this strategy might require introduction of the antiviral treatment soon after infection, and, consequently, the pre-existence of an early, overt signal (ie, symptom/sign) of infection and, given the specificity of antivirals, a rapid method to identify the virus. other targets for om prevention during a vuri are the specific pathways unique to om pathogenesis. as discussed earlier, clinical and experimental data support eustachiantube dysfunction (ie, failure to effectively open) and altered nasopharyngeal bacterial flora as requisite components of the mechanism by which a vuri translates into an otologic disease. the cause of eustachian-tube dysfunction during a vuri is debated. histologic studies in chinchillas document extension of the virus infection to the luminal mucosa, but eustachian-tube function tests in infected ferrets, children, and adults evidence mucosal swelling, possibly caused by ngi [65] [66] [67] . attempts to preserve adequate eustachiantube function (and ambient me pressure) during a vuri using oral decongestants in children and adults have not been successful [68] . in one double-blind clinical study, intranasal steroid (fluticasone propionate) was administered for 7 days immediately after onset of vuri symptoms in an attempt to decrease nasopharyngeal inflammation (and possible eustachian-tube obstruction), but was not efficacious in preventing aom and might have increased om incidence during rhinovirus infection [69] . nonetheless, some promise for this strategy is demonstrated by the results of a randomized study that compared no treatment, influenza vaccination, and tympanostomy-tube insertion (which bypasses the eustachian tube to maintain ambient me pressure) for preventing om during the influenza season [55] . there, the group treated with tympanostomy tubes had less om than the group treated with the influenza vaccine, which in turn had less om than the untreated group. however, a significant limitation to the pharmacologic manipulation of eustachian-tube function is the paucity of data regarding drug effects on that function. this is a focus of active research by groups in sweden, japan, and the united states. alternatively, vuri-associated om could be prevented by targeting emergent or extant nasopharyngeal pathogens. as mentioned, vaccination against s. pneumoniae, extended antibiotic prophylaxis, seeding the nasopharynx with probiotics, and nasopharyngeal exposure to xylitol (by lozenge or chewing gum) reduced the frequency of om episodes [9-11,12•,13-15] . in an adaptation of this strategy, clinical studies were designed to evaluate prophylactic antibiotics and xylitol administered for the limited period immediately after symptom onset during a vuri. the one published, doubleblind, placebo-controlled, clinical study of xylitol did not show efficacy in preventing the development of om during a vuri [70] , and the three studies that used antibiotics (penicillin v, amoxicillin clavulanate, and sultamicillin) in a double-blind, placebo-controlled format did not demonstrate protective efficacy [71] [72] [73] . however, it is not known if these treatments shorten the course of the om episodes, or alternatively, if antivirals administered at the time would have such an effect. possible strategies to prevent om caused by vuri include interventions that reduce the risk of virus exposure, reduce the risk of infection given virus exposure, and reduce the risk of om given a viral infection. as noted, reducing exposure to the many types of circulating upper respiratory viruses is difficult under most circumstances, but the listed options (such as attention to hygiene within the family and withdrawal from day care) should be given serious consideration for infants and children with a history of or at high risk for recurrent om. breast-feeding is well established as lessening the latter two risks and should be encouraged for all infants, with perhaps particular enthusiasm for those with a family history of om or at high risk for vuri (eg, prematurity). passive immunization with high-titer antiviral/antibacterial serum might have a limited role in preventing om caused by viral and/or bacterial infection. however, because this treatment option is expensive, requires monthly administration by injection, is associated with pain and possible side effects, and might be redundant to breast-feeding (in young infants) and active immunization (in older infants and children), it is best reserved for immunocompromised infants and children who are at high risk for more serious, invasive diseases (eg, rsv-associated pneumonia). effective vaccinations targeting upper respiratory virus will decrease the incidence of vuri and reduce the incidence of om but, currently, this option is restricted to influenza. vaccines against other viruses are in development and/or testing but their efficacy remains to be demonstrated, and regulatory issues might delay their introduction for many years. because the attack rates for the different viruses that cause vuri and the conditional incidence of om per infection with a given viral species are not known, effective immu-nization with any number of these anticipated viral vaccines might or might not have a significant impact on om incidence. this information is extremely important in developing strategies to prevent om caused by vuris because parental resistance to multiple vaccinations (in addition to those currently required or recommended) of their infants and children is an expected impediment to strategy deployment. also, the documented synergism between bacterial pathogens that cause om and specific viruses suggests that immunization against some viruses might be redundant to immunization against bacteria species. for example, the efficiency (operationally defined as cases of om prevented/ number of individuals treated) of influenza vaccination for om prevention can be expected to decrease as multivalent pneumococcal vaccination becomes universal (given that both will prevent pneumococcal om). similar redundancies might exist for other bacteria/virus combinations. om prevention by viral vaccination is not expected to be a strategy applicable to rhinovirus infection for which the development of a vaccine with reasonable valence is unlikely because of the large number of circulating rhinovirus types, the typespecific host immune response, and the inability of neutralizing antibodies to access and bind with conserved capsid domains [74] . this appreciably limits the overall efficiency of the vaccination for om prevention because rhinoviruses are the most common cause of vuris and are recognized as a frequent cause of om. for rhinovirus and perhaps other viruses, prevention of infection (and/or presymptomatic treatment to abort infection) might require the prophylactic use of antivirals. a series of candidate drugs are in testing, and some of these were shown to be effective in reducing viral load during documented infection. however, deployment of this strategy for the purpose of preventing om shares the same concerns described for antibiotic prophylaxis; ie, toxicity and sideeffects for the patient and the selection of drug-resistant virus stains with unpredictable properties. because of the relatively long rhinovirus season and the continuous exposure to different rhinovirus strains, such concerns make this strategy unacceptable for general use. as discussed, there is a wide variety of other treatment possibilities that could decrease om during a vuri. however, all of these options require that an identifiable signal indicative of infection precedes the development of om. this temporal relationship is depicted in figure 2 . ideally, for strategies that target om during a vuri, all viral infections would be associated with early symptom/sign expression, the magnitudes of those expressions would be sufficient to be identified as a vuri illness, and om and other complications would develop days after detection of the illness signal, thereby allowing for diagnosis of the specific virus and introduction of the designated intervention. studies have shown that the real-life situation falls far short of this ideal. for example, experimental infection in adults shows that the time between viral infection and symptom/sign development is highly variable across viruses (eg, 2-3 days for influenza a and hrv, >5 days for rsv infection) and that the magnitudes of symptoms and signs of cold (flu)-like illness might or might not achieve a level sufficient to be perceived as signaling an infection [24] [25] [26] . in that experimental model, the absence of illness does not preclude the development of otologic complications, and there is no fixed temporal relationship between those complications and sign/ symptom presentation [51•] . similarly, in young children (2-5 years) with natural, symptomatic vuris, approximately 50% of new om episodes developed within 7 days before or on the day of parent identification of a cold episode [14] . for these reasons, the expected efficiency of any intervention for om prevention begun during a vuri infection is low. for all proposed strategies, nontargeted application is expected to have rather low efficiency. therefore, the ad hoc identification of subpopulations and, preferably, individuals at high risk for om as a complication of vuris would allow for targeted introduction of the various strategies for om prevention, thereby increasing the efficiency and decreasing both the risks of adverse events and the total cost of om prevention. because a high heritability for om was reported, family history is important in making such risk assignments [75•] . also, in a preliminary study, a significant relationship was reported for om during natural rsv infection and the genotype for an interferon-α promoter polymorphism [76] . these results are encouraging, and continued study of the genetics of om during a vuri is a promising avenue to pursue. many members of the research community are expressing optimism that the introduction of vaccines and antivirals effective against most of the viruses that predispose to om will soon assign om to the category of a preventable disease [30•] . our position is more cautious, and we emphasize that effective deployment of these and other related strategies requires careful consideration of their possible impact on individual and societal health. because most episodes of om (and vuris) are self-limiting and without significant long-term consequence, some of these strategies should be reserved for those individuals who are predisposed for frequent disease recurrence and/or ome. as yet, the epidemiology of om caused by vuri is not completely developed (eg, which viruses are most important? what is the time lapse between infection, symptoms, and complications?), the mechanism underlying om pathogenesis is not fully understood (eg, are there specific chemical signals that can be modulated to preserve host defense but prevent om? does virus infect the me mucosa?), and the specific target population for aggressive intervention is not well characterized. these remain research questions whose answers might or might not support the optimism of our colleagues with respect to making om a preventable disease. ambulatory health care visits by children: principal diagnosis and place of visit. national center for health statistics efficacy of amoxicillin with and without 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vaccination prevents om caused by types included in the vaccine, but that om caused by the nonincluded pneumococcal types is increased adenoidectomy and adenotonsillectomy for recurrent acute otitis media: parallel randomized clinical trials in children not previously treated with tympanostomy tubes xylitol chewing gum in prevention of acute otitis media: double blind randomized trial effect of recolonisation with "interfering" alpha streptococci on recurrences of acute and secretory otitis media in children: randomised placebo controlled trial impact of antimicrobial therapy on nasopharyngeal carriage of streptococcus pneumoniae, haemophilus influenzae, and branhamella catarrhalis in children with respiratory tract infections temporal development of acute otitis media and respiratory virus infections time to development of acute otitis media during an upper respiratory tract infection in children alper cm: daily home tympanometry to study the pathogenesis of otitis media this study followed children by daily tympanometry through the cold/flu season. most episodes of om were related temporally to a symptomatic vuri, but approximately 50% of those episodes occurred within 7 days before or on the day that parents identified first symptoms of vuri. parental assessment of symptoms might not be useful in signaling the time for intervention to prevent om a longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion a meta-analytic review of the risk factors for acute otitis media detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction prevalence of various respiratory viruses in the middle ear during acute otitis media otologic manifestations of experimental rhinovirus infection nasal and otological effects of experimental respiratory syncytial infection in adults influenza a virusinduced acute otitis media otitis media: the chinchilla model synergistic effect of adenovirus type 1 and nontypeable haemophilus influenzae in a chinchilla model of experimental otitis media effect of experimental influenza a virus infection on the isolation of streptococcus pneumoniae and other aerobic bacteria from the oropharynx of allergic and non-allergic adult subjects otitis media: a preventable disease presentations at this conference by well-recognized researchers who study om prevention were published as a series of papers. the included papers address om prevention from a variety of perspectives and strategies. they present alternative and viral rna in middle ear mucosa and exudates in patients with chronic otitis media with effusion analysis of adult otitis media: polymerase chain reaction versus culture for bacteria and viruses the investigators report that hiv genomic sequences can be detected in the me of adult patients with om who are also infected with that virus. they do not suggest that hiv is causal for the om episode. this observation cautions our interpretations of the significance of viral genomic sequences in the me during om and leaves open the possibility that those sequences are not evidence of in situ viral infection ccr5(+) t lymphocytes: a lymphocyte phenotype found in the middle ear effusion respiratory viruses interfere with bacteriologic response to antibiotics in children with acute otitis media innate immunity: ancient system gets new respect. science local and systemic cytokine responses during experimental human influenza a virus infection: relation to symptom formation and host defense development of il-6 and tnf-␣ activity in nasopharyngeal secretions of infants and children during infection with respiratory syncytial virus cytokine gene polymorphisms moderate responses to respiratory syncytial virus in adults this paper relates the severity of an experimental rsv infection in adults to the individual's genotype for an il-6 promoter and relates characteristics of the adaptive immune response to the individual's genotype for an interferon-␥ promoter. these results suggest that the various responses to virus infection (including om) are partly controlled by genetics. they hold promise that identification of genotypic markers for om susceptibility would allow targeted application of some of the intervention strate social ties and susceptibility to the common cold pattern of nasal secretions during influenza virus infection urine histamine metabolite elevations during experimental colds update on cytokines dose-dependent effects of capsaicin nasal challenge: in vivo evidence of human airway neurogenic inflammation in vivo observation with mri of middle ear effusion in response to experimental underpressures depression of monocyte and polymorphonuclear leukocyte oxidative metabolism and bactericidal capacity by influenza a virus effect of rhinovirus 39 infection on immune and inflammatory parameters in allergic and non-allergic subjects effect of influenza a virus infection on lymphocyte phenotype and function upregulation of mrna for inflammatory mediators in middle ear mucosa in a rat model of infectious otitis media the nose knows: sinusitis, digestion and appropriate sanum therapy illness and otological change provoked by experimental upper respiratory virus infection in adults with experimental influenza a or rhinovirus infection, the presence of symptoms is not required for the development of otologic changes such as me underpressure. approximately 30% of infected persons with otologic changes did not report symptoms of illness. this observation calls into question the use of sign/symptom signaling for introducing treatments to prevent om during a vuri human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms nasal and otologic effects of experimental influenza a virus infection pre-challenge antibodies moderate disease expression in adults experimentally exposed to rhinovirus strain hanks influenza vaccination in the prevention of acute otitis media in children influenza a vaccine decreases the incidence of otitis media in 6-to 30-month-old children in day care the efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children a large, double-blind study that convincingly demonstrates a reduced incidence of om in children vaccinated with an intranasal influenza vaccine respiratory syncytial virus-enriched globulin for the prevention of acute otitis media in high risk children the impact-rsv study group: palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in highrisk infants fda rejects picovir for lack of drug interaction data what's new with common colds? complications and management effects of the neuraminidase inhibitor zanamavir on otologic manifestations of experimental human influenza effect of rimantadine treatment on clinical manifestations and otological complications in adults experimentally infected with influenza a (h1n1) virus eustachian tube histopathology during experimental influenza a virus infection in the chinchilla otologic and systemic manifestations of experimental influenza a virus infection in the ferret effect of upper respiratory tract infection on eustachian tube ventilatory function in the preschool child effect of topical adrenergic decongestants on middle ear pressure in infants with common colds intranasal fluticasone propionate does not prevent acute otitis media during viral upper respiratory infection in children xylitol administered only during respiratory infections failed to prevent acute otitis media short-term penicillin-v prophylaxis did not prevent acute otitis media in infants efficacy of antibiotics against influenza-like illness in an influenza epidemic short-term use of amoxicillin-clavulanate during upper respiratory tract infection for prevention of acute otitis media structure of human rhinovirus serotype 2 (hrv2) the heritability of otitis media: a twin and triplet study a large, prospective study of om in twins and triplets. the results document a high om heritability (.73) when measured as the percent of time with effusion modulation of rsv infection in infants by cytokine genotype supported in part by a grant from the national institutes of health no. dc05832. key: cord-306111-wn1gxhk9 authors: dommett, r. m.; klein, n; turner, m. w. title: mannose‐binding lectin in innate immunity: past, present and future date: 2006-09-01 journal: tissue antigens doi: 10.1111/j.1399-0039.2006.00649.x sha: doc_id: 306111 cord_uid: wn1gxhk9 the human collectin, mannose‐binding lectin (mbl), is an important protein of the humoral innate immune system. with multiple carbohydrate‐recognition domains, it is able to bind to sugar groups displayed on the surfaces of a wide range of microorganisms and thereby provide first‐line defence. importantly, it also activates the complement system through a distinctive third pathway, independent of both antibody and the c1 complex. three single point mutations in exon 1 of the expressed human mbl‐2 gene appear to impair the generation of functional oligomers. such deficiencies of functional protein are common in certain populations, e.g. in sub‐saharan africa, but virtually absent in others, e.g. indigenous australians. mbl disease association studies have been a fruitful area of research and implicate a role for mbl in infective, inflammatory and autoimmune disease processes. overall, there appears to be a genetic balance in which individuals generally benefit from high levels of the protein. however, in certain situations, reduced levels of circulating mbl may be beneficial to the host and this may explain the persistence of the deleterious gene polymorphisms in many population groups. it is now 60 years since the australian nobel prize winner sir frank macfarlane burnet, together with john mccrea, identified three inhibitors in serum (called a, b and g), which were able to inactivate influenza virus (1) . we now know that the b inhibitor was, in fact, a protein called mannosebinding lectin (mbl), a component of the innate immune system (2) . during the past 30 years, our understanding of this protein has steadily increased as a result of extensive research activity in three main areas: (a) bio/immunochemistry (including molecular genetics), (b) microbiology and (c) immunodeficiency. work in these areas initially proceeded independently as evidence for both an inexplicable biological function and a clinical deficiency state emerged. the isolation and characterization of the protein were necessary in order to illuminate the observations of the so-called rarf bactericidal activity (3, 4) in the microbiology area and the opsonic deficiency reported in many paediatric populations. some of the main developments are summarized in table 1 . this review briefly addresses issues relating to the early history of mbl, its structure, function, genetics and disease associations. finally, future developments including the potential use of both plasma-derived and recombinant mbl are discussed. the existence of mammalian serum lectins was first predicted in 1975 by robinson et al. (5) , and the protein was first isolated in 1978 from cytosolic fractions of rabbit liver by kawasaki et al. (6) . subsequently, wild et al. (7) were able to isolate mbl from both human and rat liver. more recently, extrahepatic transcription of mbl has been reported and this may have implications regarding its role in localized host defence (8) . mbl belongs to a family of proteins called the collectins, which possess both collagenous regions and lectin domains. the other major human collectins, surfactant protein a and surfactant protein d, possess structural characteristics similar to those of mbl and are found predominantly in the lung and other mucosal sites (9) . plasma-associated phagocytic defect (28) 1975 existence of mammalian serum ôlectin-like proteins specific for mannoseõ predicted (5) 1976 association of opsonic defect with frequent infections in infancy, but deficiency also present in 5% of the general population (29) 1978 mbl isolated from rabbit liver (6) 1980 opsonic deficiency in infants with chronic diarrhoea (30) 1981 association of yeast opsonization defect with suboptimal c3b deposition (31) 1982 description of mouse rarf: a complement-activating bactericidal protein (3) 1983 human mbl isolated from liver (7); human serum mbl isolated (121) prospective study of opsonic deficiency in infancy (122) 1984 rarf activity present in vertebrate classes (4) 1985 bovine serum mbl described (123) opsonic defect linked to absence of an unidentified co-factor of the complement system (124) 1987 mbl activation of classical complement pathway (18) 1988 rat serum mbl a and c described (125) ; description of c-type crd (126) 1989 gene for human mbl cloned (33, 34) ; human mbl has bactericidal activity (127) ; opsonic nature of mbl demonstrated (35) mbl inhibits in vitro infection by hiv (85) correlation of opsonic defect with low serum mbl levels (32) 1990 bovine and mouse serum b inhibitors of influenza a virus identified as mbl (2) correlation of mbl levels with classical complement pathway activation at low serum concentrations (128) 1991 mouse mbl a and c described (129) opsonic deficiency and low mbl levels linked to single point mutation in codon 54 (variant b) (50) 1992 human mbl levels in acute-phase responses (59) ; crystallography of mbl crd (130) ; novel protease (masp-1) and complement activation by mbl (19) human rarf identical to mbl-masp (131) low mbl levels in africans linked to codon 57 (variant c) mutation in the mbl gene (51) 1994 third mbl mutation in codon 52 (variant d) described (52) 1995 polymorphisms found in promoter region of mbl gene (55) 1997 second masp found to activate complement (20) mbl mutations are an important risk factor for infections in children (132) 1998 reconstitution of opsonizing activity by infusion of purified mbl into mbl-deficient humans (112) 1999 truncated form of masp-2 -map19 (21) 2000 complement-activating complex of ficolins and masp (133) mbl shown to bind to clinically relevant organisms (15) structural aspects of mbl the protein structure of mbl has been studied extensively, and aspects are presented in figures 1 and 2 . the protein consists of multimers of an identical polypeptide chain of 32 kda. each chain comprises four distinct regions encoded by different exons of the mbl-2 gene, as will be discussed in more detail later. each chain has a c-terminal, calcium-dependent carbohydrate-recognition domain (crd); a short, a-helical, hydrophobic neck region (in the so-called coiled-coil configuration); a collagenous region containing 19 gly-xaa-xaa triplets and a cysteine-rich n-terminal region. three polypeptide chains form a triple helix within the collagenous region, stabilized by hydrophobic interactions and interchain disulphide bonds within the n-terminal cysteine-rich region. this is the basic building block of all circulating molecular forms of mbl. in serum, mbl consists of oligomers ranging from dimers to hexamers, and x-ray crystallographic studies/electron micrographs have revealed that these oligomers have a sertiform or a bouquetlike structure due to an interruption in the collagenous region, giving rise to a kink/hinge. the ability of the protein to bind effectively to microorganisms and activate complement appears to depend on the presence of higher order oligomers (tetramers and above). work by drickamer and colleagues (10, 11) and also by ezekowitz and colleagues (12) has provided an insight into the structure of the crd. each crd binds a calcium ion, enabling it to form co-ordination bonds with the 3-and 4-hydroxyl groups of specific sugars including mannose, n-acetyl-d-glucosamine, n-acetyl-mannosamine, fucose and glucose. the three crds in each structural subunit are separated by a constant 45-å distance (12) . clustering of the structural subunits provides a flat platform, permitting binding of mbl to the arrays of repeating sugar groups on microbial surfaces. although the binding affinity of each individual crd-sugar interaction is relatively low at 10 23 m (13), the formation of higher order oligomers provides multiple crds, which are able to bind simultaneously with high avidity. mbl is a major pattern-recognition molecule of the innate immune system. it primarily recognizes specific sugar groups (as above) on the surface of microorganisms, enabling it to distinguish self from non-self. it can also bind to phospholipids, nucleic acids (14) and non-glycosylated proteins. mbl has been shown to bind promiscuously to a wide range of bacteria, viruses, fungi and protozoa and some selected examples are listed in table 2 . neth et al. used flow cytometry to demonstrate mbl binding to clinically relevant bacterial isolates from immunocompromised children and noted differences in binding within some species such that one isolate might show strong binding, whereas another was much weaker (15) . the role of specific structural features of microorganisms (e.g. the capsule), which permit or prevent binding to mbl, has been explored in several studies. the earliest work was probably by kawakami et al. on the socalled rarf complex (which was later identified as mbl) and its interaction with salmonella enterica serovar typhimurium (3). this suggested that the structure and composition of lipopolysaccharide play a crucial role in mbl binding and function. other mechanisms that enable microorganisms to avoid recognition and killing by mbl include lipooligosaccharide sialyation (16, 17) . despite much progress in this area, many puzzles remain to be addressed, mostly related to the exact disposition of sugars on microbial surfaces. our understanding of mbl function has grown rapidly over the past three decades. it is now recognized to have a role in processes as diverse as complement activation, promotion of complement-independent opsonophagocytosis, modulation of inflammation, recognition of altered self-structures and apoptotic cell clearance. a role for mbl in host defence was first proposed in 1987 when ikeda et al. observed that the protein was able to activate the classical pathway of complement (18) . however, it is now clear that mbl activates a novel third pathway of complement, often termed the mbl pathway, in an antibody-and c1-independent fashion as illustrated in figure 3 . this functional activity reflects the fact that mbl circulates in association with a group of mbl-associated serine proteases (the so-called masps). in 1992, matsushita and fujita demonstrated the presence of a novel complement enzyme in serum, which was thought to generate the c3 convertase (c4bc2a), associated with classical pathway activation (19) . however, this activity was later found to be mediated by masp-2 (20) , and the original enzyme is now known as masp-1 and may activate c3 directly. subsequently, a small separately synthesized fragment of masp-2 termed smap or map19 was identified (21, 22) and a third masp (masp-3) with no known function was also described (23) . current understanding suggests that on binding to microorganisms, autoactivation of masp-2 occurs, permitting cleavage of c4 and c2 to form a c3 convertase, which is indistinguishable in specificity from the convertases found in the other two activation pathways of complement (24) . it should be noted that the so-called mbl pathway is also activated by another family of proteins called ficolins. the ficolins are structurally similar to collectins, with collagenous domains linked to fibrinogen-like domains having sugar-binding properties. l-and h-ficolins are humoral factors synthesized by hepatocytes, although h-ficolin has also been observed in bronchial/alveolar fluid and in bile (25) . in contrast, m-ficolin is found on peripheral blood mononuclear cells, polymorphonuclear cells and type ii lung epithelial cells (26) . ficolins are also found in complexes with the masps and are considered to have different binding specificities compared with mbl (27) . in 1968, miller et al. reported a plasma-associated defect of phagocytosis in a child with severe recurrent infections, failure to thrive and diarrhoea (28) . in vitro work revealed a failure of the childõs plasma to opsonize heat-killed bakers yeast (saccharomyces cerevisiae). this defect was later detected in the sera of children with recurrent unexplained infections (29) and chronic diarrhoea of infancy (30), but, interestingly, studies in the general population also revealed a relatively high frequency of the defect (5%). in 1981, studies linked this opsonic deficiency to the complement figure 3 complement activation pathway. the lectin pathway of complement is activated by mbl and ficolins. on binding to appropriate targets, mbl-masp-2 complexes cleave c4 and c2 to form c3 convertase (c4bc2a). mbl-masp-1 complexes may activate c3 directly. ficolins also work in combination with the masps. the classical and alternative pathways also generate c3 convertase enzymes, which cleave c3. the lytic pathway (c5-c9) is common to all three routes of c3 cleavage. mbl, mannose-binding lectin; masp, mbl-associated serine proteases; masp-1, mbl-associated serine protease-2; masp-2, mblassociated serine protease-2. system by demonstrating that sera with the deficiency deposited less c3b on yeast surfaces (31) . however, it was not until 1989 that the common opsonic defect was found to be associated with low levels of the mannose-binding protein, which we now refer to as mbl (32) . in that same year, the gene for mbl was cloned (33, 34) (genetics of human mbl). in a study of mbl-coated salmonella montevideo, kuhlman et al. reported that mbl was able to interact directly with cell surface receptors and promote opsonophagocytosis (35) . subsequently, a number of putative mbl-binding proteins/receptors have been proposed including cc1qr/ calreticulin (36), c1qrp (37) and cr1 (38, 39) . however, it is unclear whether mbl is acting as a direct opsonin or is merely enhancing other complement pathways and/or antibody-mediated phagocytosis. the role of mbl as a modulator of inflammation appears to be complex and, accordingly, its mechanism of action remains unexplained. one possible explanation is that mbl is able to trigger proinflammatory cytokine release from monocytes (40, 41) . this concept was addressed in studies by jack et al. using neisseria meningitidis incubated with increasing concentrations of mbl before being added to mbl-deficient whole blood. release of tumour necrosis factor a, interleukin (il)-1b and il-6 from monocytes was enhanced at mbl concentrations below 4 mg/ml but suppressed at higher concentrations (42) . clinical studies in this area are discussed later. the role of mbl in the recognition of altered self and apoptosis a role for mbl in the clearance of apoptotic cells was first proposed by ogden et al. in 2001 (43) . mbl was found to bind directly to apoptotic cells that expose terminal sugars of cytoskeletal proteins, thereby permitting their recognition and directly facilitating their phagocytosis by macrophages. defects in the clearance of apoptotic cells have been implicated in the pathogenesis of certain autoimmune conditions, although the precise role of mbl, if any, remains elusive. for example, in 2005, stuart et al. reported that although mbl-deficient mice displayed defective apoptotic cell clearance, they did not develop autoimmune diseases (44) . in animal studies, mbl has been implicated in the pathophysiology of ischaemia reperfusion injury due to its ability to recognize altered self-structures. stahl and colleagues have proposed the lectin pathway as a mediator of this process in certain organs, and the absence of mbl/masp pathway activation appears to afford protection in these disease models (45, 46) . however, the relevance of these findings to human health needs to be established. changes in cell surface structures during oncogenic transformation appear to promote binding of mbl to cancer cells (47) where the protein can mediate cytotoxic effects including mbl-dependent cell mediated cytotoxicity (48, 49) . the relative importance of such mechanisms in tumour immunology is, at present, unknown. there are two human mbl genes, but mbl-1 is a pseudogene and only mbl-2 encodes a protein product. the functional mbl-2 gene is located on chromosome 10 (q11.2-q21) and comprises four exons as illustrated in figure 1 . exon 1 encodes the signal peptide, a cysteine-rich region and part of the glycine-rich collagenous region. exon 2 encodes the remainder of the collagenous region and exon 3 encodes an a-helical coiled-coil structure, which is known as the ôneckõ region. exon 4 encodes the crd, which adopts a globular configuration. the promoter region of the mbl gene contains a number of regulatory elements, which affect transcription of the protein. in 1991, the complete nucleotide sequence of all four exons of the human mbl-2 gene was determined by sumiya et al. in two british children with recurrent infections and low mbl levels (50) . in both individuals, a point mutation was observed in codon 54, changing the codon sequence from ggc to gac and substituting aspartic acid for glycine in the translated protein. familial studies confirmed that the defect was inherited in an autosomal dominant fashion. in 1992, lipscombe et al. identified a second exon 1 mutation in codon 57 (gly / glu), when studying a sub-saharan african population (51) , and in 1994, madsen et al. reported a mutation in codon 52 (arg / cys) (52) . these point mutations are now commonly referred to as variants b, c and d respectively, with variant a indicating the wild type. the b variant mutation occurs at a gene frequency of approximately 25% in eurasian populations. in contrast, the c variant is rare in eurasians but is commonly seen in sub-saharan african populations, with frequencies of 50%-60%. population studies suggest that the b variant mutation may have arisen between 50,000 and 20,000 years ago (53) since no structural gene mutations have been identified in studies of indigenous australian populations who arrived on the continent approximately 50,000 years ago, whereas the b variant mutation was probably introduced into both north and south america at the time of the last glaciation approximately 20,000 years ago. the effect of these exon 1 mutations on the protein product continues to be the focus of study. they are believed to impair oligomerization and lead to a functional deficiency. the b and c mutations result in the replacement of critical axial glycines in the triple helix by dicarboxylic acids, resulting in distortion of this important part of the protein (50) . in contrast, the d mutation results in the replacement of arginine with cysteine. this extra cysteine has been proposed to cause formation of adventitious disulphide bonds that hinder higher oligomer formation (54) . several polymorphisms have also been reported in the promoter region of the gene. studies by madsen et al. investigating the large interindividual variation in serum mbl levels revealed three polymorphisms, h/l, x/y and p/q at positions 2550, 2221 and 14 of the mbl gene (55, 56) . subsequently, four common haplotypes were identified, namely lxp, lyp, lyq and hyp. of these, hyp, which is associated with medium to high levels of mbl and lxp, which is associated with low levels of the protein, appear to be most important. these promoter haplotypes are in strong linkage disequilibrium with the exon 1 mutations, resulting in seven common extended haplotypes, namely hypa, lypa, lyqa, lxpa, hypd, lypb and lyqc. other rare haplotypes have also been described (57) . figure 4 illustrates the frequency of these various haplotypes in selected populations and highlights the degree of ethnic variation. the combination of structural gene and promoter polymorphisms results in a dramatic variation in mbl concentration in apparently healthy individuals of up to 1000-fold (caucasian: range <20-10,000 ng/ml). in addition, ezekowitz and colleagues presented evidence in 1988 that mbl was an acute-phase reactant (58) . in these investigations, rna was isolated from a ônormalõ liver taken as part of a staging biopsy for hodgkins disease and was compared with rna isolated from a fresh post-mortem liver of a victim with severe trauma. the authors found that mbl messenger rna transcripts were barely detectable in normal liver but that induction was seen in liver exposed to acute stress. subsequent studies have shown that mbl levels can increase between 1.5 and threefold during the acute phase, but this response is variable between individuals (59) . it should also be noted that even during an acutephase response, individuals heterozygous or homozygous for mbl mutations appear unable to achieve the protein levels of those possessing a wild-type genotype. approximately one-third of the caucasian population possess genotypes conferring low levels of mbl, with approximately 5% having very low levels. no absolute level of mbl deficiency has been defined. genotype and phenotype show a relatively strong correlation and studies often use just one measure to infer deficiency. however, there is ôadded valueõ in performing both measures and we would strongly advocate this approach whenever possible. mbl occurs in two distinct forms in rodents and rhesus monkeys (60), but only one form is found in humans and chickens. as discussed previously, there are two human mbl genes, which are most likely due to a gene duplication event (61) . however, mbl-1 is a pseudogene and the potential mechanisms responsible for silencing the mbl-1 (63). such substitutions were also found in other higher primates including chimpanzees and gorillas but not in more distant primates such as the rhesus monkey. the authors concluded that both the mbl-1 and the mbl-2 genes have been selectively silenced by the same molecular mechanisms, but skewed in time resulting in overall downregulation of mbl levels in the present human population. the high frequency of variant alleles observed in certain populations was initially puzzling since it suggests that functional mbl deficiency may well be advantageous. similarities have been proposed between the mbl genetic system and the role of the sickle cell gene in protection against malaria as occurs in carriers of the sickle cell haemoglobin allele (64) . the argument runs as follows: certain intracellular parasites use c3 opsonization and c3 receptors on monocytes/macrophages to enter their host. therefore, any reduction in complement-activating function of the host may reduce the probability of parasitization. in support of this notion is a study on patients with visceral leishmaniasis, which revealed that such patients are more likely to have high mbl levels than uninfected controls (65) . a small study of ethiopian patients with lepromatous or borderline lepromatous leprosy also found that their mbl levels were significantly higher than those of healthy blood donors (66) . an alternative explanation of the unexpectedly high frequency of low mbl phenotype individuals found in many tropical regions is that excessive complement activation can result in immunopathologically mediated host damage; therefore, any mechanism that reduces complement activation may be beneficial (51) . the identification of mbl deficiency as the cause of the so-called common opsonic defect has been followed by a plethora of disease association studies aimed at defining the precise role of this protein. a number of the early studies concentrated on paediatric populations and mbl was suggested to provide substitute ôantibodyõ-like activity during the ôwindow of vulnerabilityõ (approximately 6-24 months), when maternal immunoglobulin g (igg) antibody levels have waned but the infantõs own adaptive immune response is still immature (32) . nevertheless, studies in adults suggested that there might be a role for mbl throughout life (67) . notwithstanding these reports, the majority of individuals possessing a variant mbl allele apparently suffer no ill effects and remain essentially healthy. in a study that apparently confirms this, dahl et al. monitored 9245 adults in a danish caucasian population and found no evidence for significant differences in infectious disease or mortality in mbl-deficient individuals compared with controls (68) . similar findings were reported by tacx et al. in unselected adults admitted to hospital with infections (69) . nevertheless, these studies should not be regarded as proof that mbl levels have no clinical relevance. many groups have undertaken case-control studies, which do indeed suggest that mbl is an important immunological modulator. in some cases, there is evidence that the significance of mbl deficiency is more readily appreciated when there is another co-existing defect (70), as we first proposed in 1991 (71). space does not permit a comprehensive review of all the mbl clinical studies that have been undertaken to date, and the topics covered below have been selected in order to illustrate examples of possible roles for mbl in a variety of clinical situations. most studies have explored the role of mbl in relation to the acquisition of an infectious organism (susceptibility) and the nature of the associated clinical course (severity). in clinical practice, this distinction can be difficult. however, for the purposes of this review, we will highlight examples of infections in which mbl appears to have an influence on one or other of these two aspects of infectious diseases. hamvas et al. have recently shown a role for mbl in mycoplasma infection (72) . they studied cases of infection in patients with primary antibody deficiencies (pad) that are known to be particularly susceptible to such organisms and compared them with a control population. more than two-thirds of pad patients with mycoplasma infections were mbl deficient (in possession of an exon 1 variant allele) compared with one-third of the control group. in the same study, they were able to demonstrate binding of mbl to three strains of mycoplasma using flow cytometry and proposed a role for mbl in prevention of invasive disease. in 2003, severe acute respiratory syndrome (sars) emerged as a highly infectious disease caused by a novel coronavirus (sars-cov). it provided a new challenge to previously unexposed individuals predominantly in asia. specific antibodies to sars-cov could be detected 10 days after the onset of symptoms, making sufferers reliant on innate immune mechanisms during the early phase of infection. since the structure of the virus was rapidly established (73, 74) , it also became clear that this novel infectious agent was rich in the sugars known to be targeted by mbl and it was hypothesized that this lectin might well be involved in first-line defence against this infection. subsequent studies found significant differences in the distribution of mbl-deficient genotypes in patients with sars compared with those in controls (75, 76) . these studies suggested that mbl plays a role in susceptibility to the infection but does not influence subsequent severity. in their investigations, ip et al. were also able to demonstrate binding of mbl to the virus and its ability to inhibit infection (75) . (77). the b variant allele was found more commonly in patients with symptomatic hepatitis b cirrhosis and in those with spontaneous bacterial peritonitis. it was also noted that mbl levels were lower in this patient cohort with chronic infection. screening for mbl mutations in such patients was suggested in order to enable identification of those at increased risk of complications who may benefit from prophylactic antibiotic treatment. in 2005, chong et al. also reported that mbl genotypes correlating with low protein levels were associated with the occurrence of cirrhosis and also hepatocellular carcinoma in hepatitis b carriers (78) . they also demonstrated that mbl is able to bind hepatitis b surface antigen. in the same year, thio et al. published the results of a nested case-control study of 527 patients who had either naturally recovered from hepatitis b (n ¼ 338) or had persistent infection (n ¼ 189). they found that mbl genotypes correlating with high serum levels were associated with recovery from infection, whereas those correlating with lower levels were associated with persistence of the virus (79) . it should be noted that approximately half of the subjects were also infected with human immunodeficiency virus (hiv), but the authors concluded that this did not influence the results obtained. matsushita et al. investigated the influence of mbl mutations in hepatitis c infection and found that sufferers who were homozygous for b variant alleles were less likely to respond to interferon treatment (80) . further work would be warranted in order to define the role of mbl in the pathogenesis of hepatitis infection. secondary immunodeficiencies due to disease or treatment have provided interesting patient populations within which to study the role of mbl. one such group comprises those receiving chemotherapy for malignancy. these patients are rendered neutropenic by their treatment (or underlying disease process) and are subsequently at increased risk of infectious complications. in 2001, two studies were published reporting an effect of mbl deficiency in such patients. neth et al. studied 100 children and measured mbl levels and genotype. children in possession of mbl variant alleles spent twice as many days in hospital with febrile neutropenia during the first 6 months of their treatment compared with wild-type individuals (81) . in the other study, peterslund et al. followed 54 adults undergoing chemotherapy for various haematological malignancies and found that those who developed ôsignificantõ infections (bacteraemia, pneumonia or both) in the 3-week periods post-treatment had significantly lower levels of mbl compared with those without significant infections (82) . subsequent studies have shown differing results, but drawing comparisons between them is inherently difficult. these patients are a highly heterogeneous population, with different underlying disease processes, undergoing treatment regimens of differing intensity, resulting in various degrees of immunosuppression. in one contrasting study, bergmann et al. followed 80 adults undergoing therapy for acute myeloid leukaemia, which involves intense highly myelosuppressive treatment. they found no effect of mbl deficiency on frequency, severity or duration of fever and suggested that the nature of the treatment overwhelmed any potential influence of mbl (83) . further clinical studies in such patients are required in order to delineate the exact role of mbl. an mbl double-knockout mouse model has been used to explore the above clinical conundrum. in 2004, shi et al. demonstrated that mbl null mice were highly susceptible to intravenous inoculation with staphylococcus aureus, all dying within 48 h, compared with 55% survival of mbl wild-type mice. however, when the mice were inoculated via the intraperitoneal route and rendered neutropenic (using cyclophosphamide), neutropenic mbl null mice were found to have higher accumulations of bacteria in the blood and organs compared with neutropenic wild-type mice. by day 8 post-infection, the neutropenic wild-type mice had cleared their blood, but the neutropenic mbl null mice had persistent bacteraemia. the authors were able to reverse the phenotype by treating the mbl null mice with recombinant mbl (84) . to date, nearly 40 million humans have been infected with hiv. the clinical consequences of viral exposure are variable. some individuals can be repeatedly exposed to the virus but remain free from infection. others can be infected but remain free from clinical disease. while numerous viral and host factors will determine the fate of an individual exposed to hiv, there are data to indicate that mbl can influence both susceptibility and severity of hiv infection. the likely target for hiv binding is the heavily glycosylated glycoprotein, gp120. while mbl can be readily demonstrated to bind to purified gp120 (85) , the capacity of mbl to neutralize primary hiv isolates is less convincing. recent data indicate the mbl can opsonize hiv but does not induce neutralization at the levels at which it is normally present in serum. however, binding and opsonization of hiv by mbl may alter virus trafficking and viral antigen presentation during hiv infection. mbl may influence uptake by dendritic cells (dc), which express a cell surface lectin called ôdc-specific intracellular adhesion molecule 3-grabbing non-integrinõ (dc-sign). dc-sign has been shown to mediate a type of infection called ôtransõ-infection, where dc bind hiv and efficiently transfer the virus to t cells. preincubation of hiv strains with mbl prevents dc-sign-mediated trans-infection of t cells and indicates that at least in vitro, mbl may inhibit dc-sign-mediated uptake and spread of hiv (86) . whatever the mechanism of mbl interactions with hiv, a number of clinical studies have suggested that deficiency of mbl is a risk factor for acquiring hiv infection. mbl deficiency appears to increase the acquisition of hiv infection by between three-and eightfold (87) (88) (89) (90) . there is also an increased risk of vertical transmission from infected mothers to their offspring (91) . however, these findings have not been replicated in all populations, with some studies failing to demonstrate a role for mbl in hiv infection (92) (93) (94) . there is even less clarity with regard to the role of mbl in hiv disease progression. garred et al. (87) demonstrated that men with mbl variant alleles had a shorter survival time following the onset of acquired immune deficiency syndrome (aids) than did patients with wild-type mbl alleles. however, in a well-characterized cohort of homosexual men, variant mbl alleles had an insignificant effect on survival following the diagnosis of aids (95) . in this latter study, there appeared to be a protective effect of mbl variant alleles, with a delay in the development of aids from the time of hiv seroconversion. patients with mbl variant alleles had lower cd4 counts at the time of developing aids, indicating that mbl deficiency may influence the onset of aids for any given cd4 count. furthermore, mbl mutations appeared to protect against the development of kaposi sarcoma, a finding that was difficult to explain (95) . in another study, prohaszka et al. (90) found that mbl levels were lower in asymptomatic hiv-positive individuals compared with hiv-negative controls. however, the protective effect of mbl was lost in patients with an aids diagnosis; patients with high mbl levels had significantly lower numbers of cd4 cells. a possible explanation is that enhanced proinflammatory cytokine production in advanced hiv disease acts to increase mbl synthesis (96) , elevating levels in patients with late-stage disease. indeed, a recent study has shown in vitro that mbl can enhance proinflammatory cytokine production and viral replication (97) . in the light of studies indicating a role for mbl in inflammatory modulation, it is tempting to suggest that under some circumstances, mbl may act to promote inflammatory cell activation, thereby accelerating the rate of cd41 t-cell depletion. few studies have assessed the impact of mbl in the context of effective antiviral therapy. however, one study has attempted to relate mbl status and hiv-infected longterm non-progressors (ltnps) (98) . mbl levels were consistent with a wild-type genotype in the six ltnps studied. amoroso and colleagues had also suggested such an effect in a study showing that children with rapidly progressing disease were more likely to have mbl variant alleles (codon 54) than slower progressors (99) . cystic fibrosis provides an example of a clinical condition where mbl appears to be exerting its role as an infection susceptibility gene and inflammatory modulator. garred et al. were the first group to report that patients with mbl variant alleles have significantly impaired lung function and decreased life expectancy in comparison with wild-type individuals (100) . the effect of mbl deficiency on the severity of lung disease was most apparent in patients with chronic pseudomonas aeruginosa infection and it was also found that burkholderia cepacia infection was more common in patients with mbl deficiency. in 2004, davies et al. reported that an effect of mbl was only seen in adults homozygous for mbl mutations. these patients had significantly reduced lung function, more frequent hospital admissions and raised systemic inflammatory markers. however, there was no evidence of increased susceptibility to burkholderia cepacia and pseudomonas aeruginosa (101) . whether mbl has an effect on early colonization with burkholderia cepacia and pseudomonas aeruginosa or subsequent secondary viral infections or whether there is an (anti)inflammatory effect on subsequent lung damage remains unclear. clinical studies of critically ill patients requiring intensive care management have shown that individuals who are mbl deficient are more likely to develop the systemic inflammatory response syndrome (sirs) ( figure 5 ) and progress to septic shock and death (102, 103) , findings which may well relate to the proinflammatory cytokine response. it should also be noted that chronic inflammation is now increasingly accepted to be a risk factor for myocardial infarction (mi), and a recent study by saevarsdottir et al. has found that patients with high mbl levels have a decreased likelihood of suffering a mi -again suggesting a potential role for mbl in modulating the inflammatory response (104). as a component of the complement system with similarities to c1q, but also as a player in infectious and inflammatory processes, the structure and function of mbl have prompted studies exploring a possible role in autoimmune conditions. systemic lupus erythematosus (sle) has been the focus of a number of mbl genotyping studies, but the results have been somewhat inconsistent. nevertheless, a recent meta-analysis has reviewed studies in this area and found that mbl variant alleles are indeed sle risk factors (105) . as with infectious disease, there is some evidence that the risk of pathology increases if there is another co-existing immune defect. for example, in a cohort of spanish patients, the odds ratio for developing sle was 2.4 for individuals with mbl deficiency, but this increased to 3.2 when there was also a co-existing partial c4 deficiency (106) . studies in patients with sle have reported that mbl deficiency also influences their risk of developing certain complications, which include arterial thromboses (107) and respiratory tract infections (108, 109) . a role for mbl in the pathogenesis of rheumatoid arthritis has also been suggested. malhotra et al. reported that changes in igg glycosylation secondary to the underlying disease results in mbl-associated complement activation (110) . such complement activation then contributes to chronic inflammation of the synovial membrane. however, graudal et al. found that patients with lower mbl levels experienced earlier, more severe, symptoms and had more rapid joint destruction as visualized radiologically (111) . several recent research publications suggest the directions in which future work on this collectin and its associated molecules may proceed. these include therapeutic interventions, functional assays and the evaluation of the importance of mbl in disease. these are considered briefly below. therapeutic potential of mbl mbl replacement was first attempted (without any knowledge of the deficiency) when fresh frozen plasma was given to patients and found to correct the opsonic defect (28, 29) . since then, affinity-purified, plasma-derived mbl has been safely given to many patients, resulting in normalization of enzyme-linked immunosorbent assay detectable mbl and complement-mediated opsonic activity (112) . a phase 1 study showed the half-life of the protein to range between 18 and 115 h (113) . the development of recombinant mbl is also at the phase 1 trial stage and such developments provide exciting prospects for the future exploration of the therapeutic potential of mbl. exactly who would benefit from replacement therapy is under debate and the importance of targeting well-defined patient groups will be vital to its success. the discovery of other components of the lectin pathway including the ficolins and the masps indicates that this limb of the immune system is complex and extends beyond mbl and masp-2 alone. this knowledge enables us to question the impact of these molecules either in isolation or in combination. functional assessment of the lectin pathway may be a far more accurate and clinically relevant measurement than mbl level and/or genotype alone. a number of different assays have been reported, which assess activity at different stages of the functional pathway; therefore, the results must be interpreted accordingly (114, 115) . the impact of deficiencies of the various adjunctive components is also the subject of much current research. in 2003, stengaard-pedersen et al. reported the first identified case of masp-2 deficiency (116) . functional analysis of the ability of mbl to activate the lectin pathway, estimating c4b deposition on a mannan surface, was performed on a group of patients with suspected immunodeficiency. one patient was found to have deficient pathway activity despite having sufficient mbl. no masp-2 or map19 was found in the plasma, and genetic analysis indicated that the patient was homozygous for a point mutation in exon 3 of the gene (d105g). clinically, the patient suffered from recurrent infections and autoimmune symptoms. subsequently, the frequency of this mutation has been assessed in a small number of populations and values range from 1.3% to 6.3% (117) . as discussed previously, the contribution of masp-1 and masp-3 in the pathway remains unexplained. the role of ficolins is now beginning to be addressed in clinical studies. like mbl, no absolute levels of deficiency have yet been defined. atkinson et al. studied more than 300 children with recurrent respiratory tract infections and measured l-ficolin levels (118) . an association with mbl deficiency in the same patient cohort had already been reported (119) . in this study, low levels of l-ficolin were more common in patients than in controls and most common in patients with co-existing atopic disorders, suggesting a role for l-ficolin in protection from microorganisms complicating allergic disease. polymorphisms in the ficolins have been identified, although their clinical significance is as yet unknown. mbl is an ancient molecule, which has probably been subject to a large number of evolutionary pressures. the last 50,000 years of human evolution have been associated with major changes as hominids moved from an essentially nomadic lifestyle to increasingly crowded living arrangements in large settled communities. associated with these changes, the spectrum of common infectious diseases would also have changed. more recently, the introduction of antibiotics, the emergence of novel infections and increasing use of immunosuppressive therapies have provided new challenges to our innate host defence system. despite all these changing evolutionary pressures, mbl gene polymorphisms persist at high frequencies, suggesting that they offer potential advantages to the host. thus, there exists a balance in which certain individuals benefit from the expression of high levels of the protein, whereas others (living in differing environments, eg. the tropics) may benefit from reduced levels of circulating mbl ( figure 6 ). mbl status may also be either advantageous or disadvantageous when considered from the viewpoint of the severity of a particular illness. thus, it is known that those with higher levels of mbl are better able to modulate inflammation, probably through an effect on cytokine responses. in contrast, those deficient in mbl appear to be at risk of sepsis and sirs. for these reasons, we believe that analyses of the relevance of mbl (120) should be extended beyond its role in infectious disease and include clinical areas such as autoimmunity and inflammatory disorders. inhibitory and inactivating action of normal ferret sera against an influenza virus strain bovine and mouse serum beta inhibitors of influenza a viruses are mannose-binding lectins properties of a new complement-dependent bactericidal factor specific for ra chemotype salmonella in sera of conventional and germ-free mice a group of bactericidal factors conserved by vertebrates for more than 300 million years affinity chromatography of human liver alpha-d-mannosidase isolation and characterization of a mannan-binding protein from rabbit liver isolation of mannosebinding proteins from human and rat liver extra-hepatic transcription of the human mannose-binding lectin gene (mbl2) and the mbl-associated serine protease 1-3 genes collections and ficolins: humoral lectins of the innate immune defense structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by mad phasing trimeric structure of a c-type mannose-binding protein human mannose-binding protein carbohydrate recognition domain trimerizes through a triple alpha-helical coiled-coil binding of sugar ligands to ca(21)-dependent animal lectins analysis of mannose binding by site-directed mutagenesis and nmr nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins a and d and mannose-binding lectin mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition activation of complement by mannose-binding lectin on isogenic mutants of neisseria meningitidis serogroup b the lipopolysaccharide structures of salmonella enterica serovar typhimurium and neisseria gonorrhoeae determine the attachment of human mannose-binding lectin to intact organisms serum lectin with known structure activates complement through the classical pathway activation of the classical complement pathway by mannose-binding protein in association with a novel c1s-like serine protease a second serine protease associated with mannan-binding lectin that activates complement a truncated form of mannose-binding lectin-associated serine protease (masp)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene masp-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway crystal structure of the cub1-egf-cub2 region of mannose-binding protein associated serine protease-2 hakata antigen, a new member of the ficolin/opsonin p35 family, is a novel human lectin secreted into bronchus/alveolus and bile human m-ficolin is a secretory protein that activates the lectin complement pathway l-ficolin specifically binds to lipoteichoic acid, a cell wall constituent of gram-positive bacteria, and activates the lectin pathway of complement a familial plasma-associated defect of phagocytosis defective opsonization. a common immunity deficiency yeast opsonisation in children with chronic diarrhoeal states a study of c3b deposition on yeast surfaces by sera of known opsonic potential association of low levels of mannan-binding protein with a common defect of opsonisation exon structure reveals its evolutionary relationship to a human pulmonary surfactant gene and localization to chromosome 10 structure and evolutionary origin of the gene encoding a human serum mannose-binding protein the human mannose-binding protein functions as an opsonin human leukocyte c1q receptor binds other soluble proteins with collagen domains mannose binding protein (mbp) enhances mononuclear phagocyte function via a receptor that contains the 126,000 m(r) component of the c1q receptor complement receptor 1/cd35 is a receptor for mannan-binding lectin complement receptor type 1 (cr1, cd35) is a receptor for c1q activation of human monocytes by streptococcal rhamnose glucose polymers is mediated by cd14 antigen, and mannan binding protein inhibits tnf-alpha release induction of tnf-alpha in human peripheral blood mononuclear cells by the mannoprotein of cryptococcus neoformans involves human mannose binding protein mannose-binding lectin regulates the inflammatory response of human professional phagocytes to neisseria meningitidis serogroup b c1q and mannose binding lectin engagement of cell surface calreticulin and cd91 initiates macropinocytosis and uptake of apoptotic cells mannose-binding lectin-deficient mice display defective apoptotic cell clearance but no autoimmune phenotype gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving c1q mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: mannan-binding proteindependent cell-mediated cytotoxicity antitumor activity of mannan-binding protein molecular basis of opsonic defect in immunodeficient children high frequencies in african and non-african populations of independent mutations in the mannose binding protein gene a new frequent allele is the missing link in the structural polymorphism of the human mannan-binding protein restricted polymorphism of the mannose-binding lectin gene of indigenous australians molecular determinants of oligomer formation and complement fixation in mannose-binding proteins interplay between promoter and structural gene variants control basal serum level of mannan-binding protein different molecular events result in low protein levels of mannan-binding lectin in populations from southeast africa and south america a new strategy for mannosebinding lectin gene haplotyping a human mannose-binding protein is an acute-phase reactant that shares sequence homology with other vertebrate lectins the concentration of the c-type lectin, mannan-binding protein, in human plasma increases during an acute phase response characterization of two mannose-binding protein cdnas from rhesus monkey (macaca mulatta): structure and evolutionary implications characterization of murine mannose-binding protein genes mbl1 and mbl2 reveals features common to other collectin genes the human ortholog of rhesus mannose-binding protein-a gene is an expressed pseudogene that localizes to chromosome 10 the ôinvolutionõ of mannose-binding lectin protection afforded by sickle-cell trait against subtertian malareal infection mannan-binding lectin enhances susceptibility to visceral leishmaniasis dual role of mannan-binding protein in infections: another case of heterosis? mannose binding protein gene mutations associated with unusual and severe infections in adults a population-based study of morbidity and mortality in mannose-binding lectin deficiency mannan binding lectin in febrile adults: no correlation with microbial infection and complement activation mannan binding lectin deficiency and concomitant immunodefects the molecular basis of a common defect of opsonization role for mannose binding lectin in the prevention of mycoplasma infection characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus mannose-binding lectin in severe acute respiratory syndrome coronavirus infection association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection mannose binding lectin gene mutations are associated with progression of liver disease in chronic hepatitis b infection mannose-binding lectin in chronic hepatitis b virus infection mannose binding lectin genotypes influence recovery from hepatitis b virus infection hepatitis c virus infection and mutations of mannose-binding lectin gene mbl deficiency of mannose-binding lectin and burden of infection in children with malignancy: a prospective study association between deficiency of mannose-binding lectin and severe infections after chemotherapy low levels of mannose-binding lectin do not affect occurrence of severe infections or duration of fever in acute myeloid leukaemia during remission induction therapy mannose-binding lectin-deficient mice are susceptible to infection with staphylococcus aureus a human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus interaction of mannose-binding lectin with hiv type 1 is sufficient for virus opsonization but not neutralization susceptibility to hiv infection and progression of aids in relation to variant alleles of mannose-binding lectin mannan-binding lectin in the sub-saharan hiv and tuberculosis epidemics the level of the serum opsonin, mannan-binding protein in hiv-1 antibody-positive patients mannan-binding lectin serum concentrations in hiv-infected patients are influenced by the stage of disease polymorphisms in the mbl2 promoter correlated with risk of hiv-1 vertical transmission and aids progression absence of association between mannose-binding lectin gene polymorphisms and hiv-1 infection in a colombian population mannose-binding protein in hiv-seropositive patients does not contribute to disease progression or bacterial infections circulating levels of mannose binding protein in human immunodeficiency virus infection presence of the variant mannose-binding lectin alleles associated with slower progression to aids human mannose-binding protein gene is regulated by interleukins, dexamethasone and heat shock modulatory effect of mannose-binding lectin on cytokine responses: possible roles in hiv infection low mannose-binding lectin serum concentrations in hiv long-term nonprogressors? polymorphism at codon 54 of mannose-binding protein gene influences aids progression but not hiv infection in exposed children association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis impaired pulmonary status in cystic fibrosis adults with two mutated mbl-2 alleles association of mannose-binding lectin polymorphisms with sepsis and fatal outcome, in patients with systemic inflammatory response syndrome increased incidence and severity of the systemic inflammatory response syndrome in patients deficient in mannose-binding lectin mannan binding lectin as an adjunct to risk assessment for myocardial infarction in individuals with enhanced risk the mannose-binding lectin gene polymorphisms and systemic lupus erythematosus: two case-control studies and a meta-analysis a dysfunctional allele of the mannose binding protein gene associates with systemic lupus erythematosus in a spanish population mannose-binding lectin variant alleles and the risk of arterial thrombosis in systemic lupus erythematosus association of mannose-binding lectin gene variation with disease severity and infections in a population-based cohort of systemic lupus erythematosus patients association of mannose binding lectin (mbl) gene polymorphism and serum mbl concentration with characteristics and progression of systemic lupus erythematosus glycosylation changes of igg associated with rheumatoid arthritis can activate complement via the mannose-binding protein mannan binding lectin in rheumatoid arthritis. a longitudinal study reconstitution of opsonizing activity by infusion of mannan-binding lectin (mbl) to mbl-deficient humans human plasma-derived mannose-binding lectin: a phase i safety and pharmacokinetic study an assay for the mannan-binding lectin pathway of complement activation functional analysis of the classical, alternative, and mbl pathways of the complement system: standardization and validation of a simple elisa inherited deficiency of mannan-binding lectin-associated serine protease 2 deficiency of the mannan-binding lectin pathway of complement and poor outcome in cystic fibrosis: bacterial colonization may be decisive for a relationship l-ficolin in children with recurrent respiratory infections mannan-binding lectin insufficiency in children with recurrent infections of the respiratory system human mannose-binding lectin in immunity: friend, foe, or both? isolation and characterization of a mannan-binding protein from human serum a common congenital immunodeficiency predisposing to infection and atopy in infancy mannan-binding protein and conglutinin in bovine serum suboptimal c3b/c3bi deposition and defective yeast opsonization. i. evidence for the absence of essential co-factor activity isolation and characterization of two distinct mannan-binding proteins from rat serum two distinct classes of carbohydrate-recognition domains in animal lectins a serum lectin (mannan-binding protein) has complement-dependent bactericidal activity the level of mannan-binding protein regulates the binding of complement-derived opsonins to mannan and zymosan at low serum concentrations molecular characterization of the mouse mannose-binding proteins. the mannose-binding protein a but not c is an acute phase reactant structure of a c-type mannose-binding protein complexed with an oligosaccharide human mannose-binding protein is identical to a component of ra-reactive factor association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series cutting edge: complementactivating complex of ficolin and mannose-binding lectin-associated serine protease mannose-binding lectin accelerates complement activation and increases serum killing of neisseria meningitidis serogroup c activation of the lectin complement pathway by h-ficolin (hakata antigen) differential recognition of obligate anaerobic bacteria by human mannose-binding lectin differential binding of mannose-binding lectin to respiratory pathogens in cystic fibrosis human mannose-binding protein inhibits infection of hela cells by chlamydia trachomatis binding of mannan-binding protein to various bacterial pathogens of meningitis interaction of human mannose-binding protein with mycobacterium avium interaction of mannose-binding lectin with primary isolates of human immunodeficiency virus type 1 high mannose glycans and sialic acid on gp120 regulate binding of mannose-binding lectin (mbl) to hiv type 1 mannose binding lectin (mbl) and hiv mannan-binding protein and bovine conglutinin mediate enhancement of herpes simplex virus type 2 infection in mice mannan-binding lectin modulates the response to hsv-2 infection mannose binding protein is involved in first-line host defence: evidence from transgenic mice binding of host collectins to the pathogenic yeast cryptococcus neoformans: human surfactant protein d acts as an agglutinin for acapsular yeast cells mannose-binding lectin is a component of innate mucosal defense against cryptosporidium parvum in aids recognition of plasmodium falciparum proteins by mannan-binding lectin, a component of the human innate immune system the major surface glycoprotein of trypanosoma cruzi amastigotes are ligands of the human serum mannose-binding protein novel masp2 variants detected among north african and sub-saharan individuals analysis of mannose-binding lectin 2 (mbl2) genotype and the serum protein levels in the korean population association of mannose-binding lectin gene haplotype lxpa and lypb with interferon-resistant hepatitis c virus infection in japanese patients key: cord-314359-fw14b5cv authors: bajaj, satish kumar; tombach, bernd title: respiratory infections in immunocompromised patients: lung findings using chest computed tomography date: 2016-11-23 journal: radiol infect dis doi: 10.1016/j.jrid.2016.11.001 sha: doc_id: 314359 cord_uid: fw14b5cv respiratory infections and subsequent complications are one of the leading causes of high mortality in immunocompromised patients. although chest radiograph and computed tomography are the commonly used diagnostic tools for the early diagnosis of lung manifestations of infections, they lack the specificity for the wide range of chest infections which can occur in immunocompromised patients. systematic analysis of the imaging findings in correlation with the clinical settings along with comparison with the old images can expedite early and accurate diagnosis for subsequent appropriate management. computer tomography findings in immunocompromised patients with respiratory infections, with regards to various clinical settings, will be discussed here. infections are very common in an immunocompromised host (ich), which includes patients on chemotherapy (cancer), on immunosuppressive therapy (post transplantation patients, rheumatologic disorders) or acquired immunodeficiency diseases (aids, post-splenectomy), and their population is expanding globally. pneumonitis implies inflammation of the lung and in an immunocompromised patient (icp) may occur due to disease progression, infections or secondary to non-infectious causes like drug induced toxicities [1e5,21] . lung inflammations due to infections are known as pneumonia. infection in ich may be primary or secondary due to the underlying noninfectious inflammatory condition of the lung, which further compromises the immune system of the body. respiratory infections, along with drug toxicity, are major concerns in more than two thirds of the icps. these are leading causes of therapy failure of the primary diseases, are often life threatening and are associated with high mortality after reading this article the reader will be able to: (a) understand the classification, patterns and imaging features of the respiratory infections (b) understand the role of clinical settings while interpreting computer tomography findings (c) understand some of the mimics of chest infections and morbidity. therefore, an accurate and quick diagnosis is a major cornerstone for the management team. pneumonia can be classified, on the basis of different modes of acquirement of the infections, into community-acquired pneumonia (cap), hospital acquired pneumonia (also referred as nosocomial pneumonia, hap) or ventilator associated pneumonia (vap) ( table 1) . pneumonia can be further stratified as mild, moderate or severe on the basis of clinical parameters and scoring systems like pneumonia severity index (psi) or curb-65 [6] . these validated scoring systems assist healthcare professionals in determination of the clinical outcomes. another method of classification divides patients into typical or atypical groups, based on the clinical manifestations and the expected pathogens differ accordingly. patient who present with classical symptoms like fever, rigors, chills, cough with expectoration, chest pain, dyspnea and whose chest radiographic findings are suggestive of common bacterial infections is considered to have typical pneumonia. on the other hand, atypical pneumonia usually presents with persistent low grade fever without the typical pneumonia symptoms and signs. the etiologic pathogen could be either a bacterial, viral, fungal or any other opportunistic organism. the most common pathogens of cap are streptococcus pneumoniae, mycoplasma pneumoniae, chlamydia pneumoniae, haemophilus influenzae, legionella pneumoniae and respiratory viruses. vap are likely due to gram negative infections such as pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae and acinetobacter as well as gram positive pathogens like staphylococcus aureus. in the early stages of immunosuppression, such as in the induction phase of chemotherapy, infections are most likely bacterial in origin. common isolated bacterial pathogens in ich are legionella, mycoplasma and chlamydia ( table 2 ). the predominant viral agents are rhinoviruses, coronaviruses, influenza virus, respiratory syncytial virus (rsv), adenovirus and parainfluenza virus. icps as a result of organ transplantation are vulnerable to infections with cytomegalovirus (cmv) and rarely herpes virus. coronavirus and hantavirus infections are commonly observed during seasonal and non-seasonal outbreaks. pneumonia due to multidrug-resistant organisms such as s. pneumoniae (drsp) or s. aureus (mrsa) are often seen in icps. viral infections can be also be complicated by secondary bacterial super-infections. fungal infections are very common in ich with leukemia, cancer chemotherapy or organ transplant, mainly seen in later stages of immunosuppression either as a primary infection or as a result of a super-infection. pneumocystis jiroveci pneumonia (pjp) constitutes the most frequent opportunistic fungal infection. other common fungal infections reported are aspergillus fumigatus, candida albicans, and cryptococcus neoformans. histoplasmosis, blastomycosis and mucormycosis are not so common globally but are isolated in endemic areas (table 2) . non-infectious etiologies like drug toxicities, cardiac causes and organizing pneumonias should always be considered in the differential diagnosis of atypical and non-resolving pneumonias. on the basis of imaging patterns, pneumonia is classified as lobar pneumonia, bronchopneumonia and interstitial or atypical pneumonia. lobar and bronchopneumonia are usually bacterial in origin, whereas, viruses, parasites and fungi are likely pathogens in interstitial pneumonia. however, one should always keep in mind overlapping imaging features. radiographically, lobar pneumonia is manifested by homogeneous consolidation with air bronchogram involving one, or less commonly, multiple lobes. segmental pneumonia shows consolidation involving one or more segments of a lobe and may occasionally manifest as a round opacity simulating a pulmonary mass, called round pneumonia. frank consolidation and air bronchogram have been associated with a higher incidence of bacteremia. klebsiella infection may show radiographic evidence of lobar expansion with bulging of interlobular fissures due to voluminous inflammatory exudates. cavitation may also be present and there is a tendency to occur in the upper lobes. legionella has a predilection for the lower lung fields. radiologic resolution tends to lag far behind the clinical improvement by six to eight weeks (fig. 1 ). bronchopneumonia, also known as multifocal or lobular pneumonia, is radiographically identified by its patchy appearance with peri-bronchial thickening and poorly defined air-space opacities. as the illness becomes more severe, consolidation involving respiratory bronchioles and alveoli results in the development of centrilobular nodular opacities or air-space nodules. the consolidation can progress further and coalesce to give a lobular or lobar pattern of involvement. typically, air bronchogram are absent. the pathogens known to cause this pattern of pneumonia are particularly destructive. thus, abscesses, pneumatoceles, and pulmonary gangrene may develop. pathologically, bronchopneumonia stems from inflammation of large airways (bronchitis) with patchy (lobular) involvement. in severe staphylococcus infection, lobar enlargement with bulging of interlobular fissures can be seen. abscess, cavitation (with air-fluid levels), and pneumatocele are not uncommon and 30e50% of patients develop pleural effusions, half of which are empyema. it may be noted that cavitation and associated pleural effusions are also observed in cases of anaerobic infections, gram-negative infections and tuberculosis. in pseudomonas infection, the radiographic findings tend to be nonspecific and are difficult to differentiate from the underlying lung disease. all the lobes are involved, with a predilection for the lower lobes. necrosis and cavitation may occur. invasive aspergillosis as well as pulmonary vasculitis with infarction can resemble bronchopneumonic pattern. interstitial pneumonia is classified into focal and diffuse types. the radiological appearance results from that the edema and inflammatory cellular infiltrate into the interstitial tissue of the lung. the pathological development of interstitial pneumonia generally takes 1 of 2 forms: (1) an insidious infectious course that results in lymphatic infiltration of alveolar septa without parenchymal abnormality; (2) acute or rapidly progressive disease that results in diffuse alveolar damage affecting the interstitial and air spaces. radiographically, this disease manifest with a reticular or reticulo-nodular pattern (table 4) . legionella, mycoplasma and chlamydial infections cause this pattern of pneumonia commonly. legionella species usually cause a patchy, localized infiltrate in the lower lobes. associated hilar adenopathy may be present. pleural effusion is seen in up to 30% of cases. in rare instances, it may be associated with cavitation and a mass-like appearance (fig. 2) . the infiltrates in mycoplasma pneumoniae can be unilateral, multi-lobular, or bilateral. in about 20% of patients, pleural effusion or hilar adenopathy may be present. chlamydia pneumonia may be sub-segmental or more extensive in elderly patients; pleural effusions are rarely seen. chest radiograph (cxr) reveals 50% resolution in four weeks. in 20% of cases, resolution takes longer than 9 weeks. aspergillosis is a mycotic disease caused by aspergillus species, usually aspergillus fumigatus. pulmonary aspergillosis can be subdivided into five categories: (a) saprophytic aspergillosis (aspergilloma), (b) hypersensitivity reaction (allergic broncho-pulmonary aspergillosis), (c) semi-invasive (chronic necrotizing) aspergillosis, (d) airway-invasive aspergillosis (acute trachea-bronchitis, bronchiolitis, bronchopneumonia, obstructing bronchopulmonary aspergillosis), and (e) invasive pulmonary aspergillosis (ipa). ipa usually affects . clinically, viral pneumonia in adults can be divided into two groups: atypical pneumonia in otherwise normal hosts and viral pneumonia in ich. influenza virus types a and b account for the majority of viral pneumonia in immunecompetent adults. ichs are susceptible to pneumonia caused by cmv and hsv, as well as measles and adenovirus ( table 2 ). the radiologic findings of viral pneumonia are variable and overlapping (table 4 ). specific etiological diagnosis of a viral pneumonia cannot be made on the basis of imaging features alone (fig. 3) . clinical features such as patient age, immune status, time of year, illness in other family members, community outbreaks, different stages of the underlying disease at onset, severity and duration of symptoms, and presence of a rash remain important in diagnosing viral causes of atypical pneumonia in immune-competent as well as icps. cxr is an essential tool for rapid diagnosis of lung changes and may also be help in follow up of the treatment response. however, it lacks specificity and often fails to show early and subtle findings of lung infections. computed tomography (ct) of chest has become a mainstay tool for detecting early lung changes. there is better delineation and characterization of the patho-mechanism of the findings, enabling proper differential diagnosis including infection mimics [7] . the accuracy of radiological diagnosis can be improved significantly if it is correlated with the clinical background of the patient. hence detailed information about the particular clinical setting including clinical presentation, past medical history such as exposure to tuberculosis et al., treatment history, and overall clinical condition is essential for better evaluation and proper diagnosis [8] . pneumonia in a vulnerable ich is influenced by several environmental and epidemiological factors like mode of exposure, i.e., cap or hap, history of previous infections and nature of ongoing drug therapy (cytotoxic, immunosuppressive) and stage of the disease. cytotoxic drugs causes neutropenia which is further complicated by increased risk of gram negative infections whereas immunosuppressive or immune-modulating drugs lead to increased risk of infections by causing defects in cell mediated immunity and phagocytosis. infections caused by mycobacterium tuberculosis (fig. 4) , pneumocystis jiroveci (pcp), toxoplasma gondii and varicella zoster virus are often on account of reactivation of past infections. therefore, inquiring about past medical history suggestive of these infections is important. similarly, hap or nosocomial infections are caused by a specific group of organisms. the risk is increased by intubation, use of broad spectrum antibiotic, use of strong immunosuppressive drugs combined with histamine 2 receptor blockers which reduce gastric acidity which in turn leads to increased colonization by gram negative bacilli, such as pseudomonas, klebsiella (fig. 2 ), e. coli and acinetobacter. ventilators and central air-conditioning system are associated with legionella and gram negative infections. catheter and drainages increase the risk of s. aureus, p. aeruginosa and candida in vulnerable hosts. the type of immune defect also determines the specific pathogen to a larger extent. complement system defects lead to infection by extracellular or encapsulated bacteria. phagocytosis failure is mainly associated with bacterial and fungal infections. ich with humoral immunodeficiency is at risk of getting infected with encapsulated bacteria like s. aureus, s. pneumoniae, h. influenza and pjp. whereas, cell mediated immunodeficiency or t-cell defect, due to underlying primary disease or secondarily due to viral infections like cmv or epsteinebarr virus, pose a high risk of opportunistic infections. in patients with aids, pathogenic agents are determined by the cd4 cell count-whether below 100 (viral and fungal) or above 100 (pcp and mycobacterium). immune defects due to splenectomy or hypo-splenismp redisposes to infection with encapsulated microorganisms such as s. pneumoniae, h. influenza and s. aureus [13e15]. duration of immunosuppression also determines the specific pattern of chest infection. gram-negative infections and s. aureus are often seen in the early phase of neutropenia, whereas opportunistic organisms like fungus are found in the later phases of neutropenia. organ transplanted ich are susceptible to infections with likely causative pathogen depends on the post-transplant phase. in the early phase, common bacterial infections are observed, whereas viral or fungal infections predominate in later phases (usually 6 months posttransplant). the likelihood of a particular pathogen depends on the type of immune defect, which is governed by the type of immunosuppressive therapy. the radiological diagnosis should be corroborated by special laboratory tests like polymerase chain reactions (pcr), serology, immunoassays (elisa) and galactomannan test, whenever necessary. bronchoscopy with broncho-alveolar lavage aids in detection and confirmation of pcp, candida and other infections. correct interpretation of abnormal radiological findings is limited by several factors, such as insufficient clinical information and failure in obtaining old radiological imaging for comparison. furthermore, overlapping imaging features of different organisms, lack of experience of the radiologist, subtle findings which are difficult to correlate and co-morbidities (such as congestive heart failure, cor pulmonale, radiation induced changes etc.) pose further difficulty in correct assessment of radiological changes. hence, it is suggested to adopt a comprehensive and holistic approach to the radiological diagnosis. cxr and chest ct are the frontline diagnostic tools in early detection of respiratory infections. chest ultrasonography is useful in evaluating para-pneumonic effusions and can be used to evaluate complications like pneumothorax or as an assist tool for thoracocentesis. non-enhanced ct chest is the modality of choice when plain cxr is inconclusive or inadequate for interpretation. there are certain radiological features suggestive of pneumonia and when accompanied with presence of certain "special imaging signs" can clinch the diagnosis [9] . these characteristic findings with probable differential diagnosis will be discussed below (table 3) . consolidation is the most common and easily interpretable findings on cxr and ct. it occurs mainly due to opacification of the air spaces by exudates and is usually bacterial in origin. air tracks (bronchioles) are visualized when the walls are effaced by surrounding consolidation. this is known as air bronchogram sign and a reliable sign of lung infection. focal consolidation can also be seen in other conditions like non-obstructive atelectasis, neoplasia, aspiration and organizing pneumonia. hence, these should be closely analyzed to differentiate from each other. for instance, lung volume loss with subsequent elevation of diaphragm, vascular crowding and mediastinal shift are normally due to atelectasis, whereas bulging fissure (bulging fissure sign) along with air bronchogram suggests pneumonia. bulging fissure sign is most often seen in upper lobe pneumonia caused by klebsiella and pneumococcal infections. neoplasia, large abscesses, infected bullae and other lesions can also present with this sign and a comparison with old films is utmostly helpful. silhouette sign is another very useful sign in detecting subtle changes of chest infection. this sign is characterized by obscuring of normal air interfaces of the thorax. thoracic aperture, thoracic wall, para-mediastinal spaces and pericardiac spaces are the areas where this sign is well illustrated. it can also be seen in space occupying lesions, atelectasis and localized effusions. feeding vessel sign is a very useful sign of septic emboli in ct scan when cavitating or non-cavitating nodules are associated with a pulmonary vessel [10] . air fluid level sign is suggestive of abscess and empyema and mainly caused by s. aureus and klebsiella. the wall of this cavitation may enhance in homogeneously on ct scan with contrast. a chest wall or fissure based focal opacity, also called split pleural sign, and is normally suggestive of empyema. it may also be illustrated in case of hemothorax, pleurodesis and post lobectomy. ground glass opacities (ggo) are defined as nonspecific high attenuation of the lung parenchyma in ct scan due to decrease in air content or partial effacement resulting from exudates in the alveolar space (fig. 5a) . hence normal lung markings are not obscured in contrast to frank alveolar consolidations. this is not a very specific sign for infection and can be seen in other conditions like pulmonary congestion, interstitial lung disease, vasculitis, etc. the pattern of distribution of ggo with other accompanying signs may suggest a particular type of pneumonia. for example ggo confined to central and upper lobes with sparing of the subpleural spaces is very specific for pcp [16] . tree-in-bud sign (fig. 5b) is a well-appreciated sign seen in various conditions such as infectious bronchiolitis, aspiration and cystic fibrosis. terminal bronchioles are normally not perceived on ct scan because of their very thin walls and caliber (less than 1 mm). when peripheral areas of lung are opacified or plugged by exudates, they cause the appearance of a v or y shaped branching tree pattern accompanied by bud-like nodules. normally this sign spares the subpleural and peri-fissure areas (fig. 5b) . halo sign is a well-appreciated ct scan sign in a clinical setting of fever with neutropenia and is always suggestive of invasive aspergillosis (fig. 5c) [12] . the focal infarction with surrounding hemorrhage is perceived as a halo. halo sign is a good prognostic predictor for response to therapy. reverse halo sign is seen seldom in aspergillus or mucormycosis infection where the lesion shows consolidation around a central area of ground glass like changes with interception lines (fig. 5d) . air crescent and monad sign are other signs suggestive of fungal infections (fig. 5e) . separation of the necrotic infective mass by a crescent air space in response to therapy is known as air crescent sign. it implicates good response to treatment. this is to be differentiated from an air crescent space resulting from secondary fungal infection with mycetoma (fungal ball) in a preexisting cavity, known as monad sign. this is a bad sign and usually causes hemoptysis and needs surgical or interventional management. crazy paving sign is due to alveolar opacity resulting from exudates accompanied by septal thickening leading to a characteristic picture of pedestrian path pattern, normally seen in alveolar proteinosis (fig. 5f ). pcp and viral infections may seldom manifest this sign. miliary pattern consists of widespread distribution of tiny nodules of less than 3 mm in size. random type of military pattern is centrilobular and is characteristic of hematogenous spread of either mycobacterium infection or lung metastases. on the other hand, non-random type is peri-lymphatic in distribution and is a common feature of sarcoidosis [13, 14] . as mentioned above, it is not always easy to pin point a specific pathogen for the cause of the pneumonia on the basis of imaging findings alone. furthermore, old pulmonary lung changes, cardiac insufficiency, atelectasis and pleural effusions can mask the new changes resulting from the current infection. hence, we recommend an algorithm (fig. 6 ) to be followed, for proper evaluation of the various lung changes in order to have a conclusive diagnosis and follow up. due to regular follow up regime of our icps (many western countries), pneumonia with frank lobar consolidations are hardly observed these days. patchy, segmental or nonsegmental consolidations are the usual radiological presentation of typical bacterial pneumonia in case of ich with acute onset of fever. however, in hospitalized patients with similar radiological features without any relevant clinical and laboratory findings consistent with lung infection, a possible diagnosis of atelectasis, old changes and organizing pneumonias following a course of antibiotics should be considered. other accompanying features like loss of lung volume and vascular crowding may assist in the interpretation. basal infiltrates and atelectasis are difficult to differentiate from each other. in addition, a likely pneumonic infiltrate overlying a basal lung collapse should always be considered, if clinically suspected. in our experience, tree-in-bud phenomenon is often seen either as a footprint of old infection which can be verified by comparing with old films or as a sign of a fresh infective bronchiolitis usually caused by bacterial or fungal infections (fig. 7) . in our clinical setting, tuberculosis is not very common in ich. it is mainly seen in patients with aids or as reactivation of an old tuberculosis infection in the migrant subgroups from the endemic areas. in aids patients, tuberculosis may cause minimal lung changes (with or without effusion), and necrotic mediastinal lymphadenopathy is often seen. cavitating and military lung lesions are observed in the advanced disease (fig. 4) . nodular opacities (with or without cavitation) are commonly a manifestation of septic emboli from infected catheter, endocarditis, etc. and patients should be screened for such sources of infection (fig. 2) . rarely, wegener's granulomatosis can present in a similar fashion. in cases with nodular lung changes with mediastinal lymphadenopathy, one should always consider sarcoidosis as an alternative coincidental diagnosis, particularly in a younger age group. nodular opacities may be seen in fungal infections, occurring secondary to virus infections. typical pneumonia, due to bacterial infections, is common in early stages of immunosuppression and is easy to diagnose based on clinical and laboratory parameters and their response to therapy. atypical pneumonia is not uncommon as well and can lead to serious complications with high morbidity and mortality, if not timely diagnosed and treated. generally, viral pneumonia manifests as ground glass opacities in early stages, mainly in upper and middle lung fields and a tendency of consolidation in later stages (figs. 3 and 7 ). reactive small mediastinal lymph nodes can be seen on ct scan. influenza virus shows midfield changes more than upper lobe changes as well as early consolidations. pneumatoceles and pneumothorax are not observed in viral infections, although these are frequent features of pcp (fig. 8) . a complicated viral infection may rapidly proceed to acute respiratory distress syndrome (ards). pjp seems more frequent compared to legionella, mycoplasma and chlamydial lung infections in our ichs. fungal infections comprise the third major group of pneumonias. ipa (with the characteristic halo sign) is likely to be more frequent than candida infections. reverse halo sign is not a commonly seen entity in our practice. it is very important and imperative that radiologists be aware of lung changes, which may mimic lung infection. these are caused by various conditions like cardiogenic conditions, cryptogenic organizing pneumonia (cop) (fig. 9) , progressive cancer disease with lymphangitis and chemotherapy associated lung changes. hence recognizing the relevant findings, correlating with the clinical findings and course of management can provide a complete differential diagnosis and thus play an important role in patient care. ggo due to fluid retention in bedridden ich is common. chf in old patients may present with typical findings of central and lower lobe dominant ggo accompanying with small effusions (fig. 10) . atelectasis is inadvertently found in such conditions. progressive dyspnea without fever may be a feature of fig. 7 . ct scan of 49 years old male with bone marrow transplantation with viral infection followed by aspergillosis. lymphangitis and normally shows ct findings of perihilar and segmental reticular or reticulo-nodular opacities. cop is not very uncommon, and should be considered when consolidation persists or increases during the course of the treatment whereby the laboratory findings are not suggestive of active infection [11] . in case of viral infections, increasing nodular opacities or consolidation, and superinfection (such as accompanied with bacterial, pneumocystis or fungal infections) should be considered. the effect of drug toxicities on the lung can manifest as infiltrates (interstitial, alveolar or both), pulmonary edema and hypersensitivity pattern and changes secondary to cardiotoxic chf. they may be asymptomatic coincidental findings or may present with progressive dyspnea. some of the newer drugs may also be accompanied with neutropenia and fever [20] . chemotherapy induced patchy pneumonitis is not uncommon and should be considered in differential diagnosis when approaching lung parenchymal changes [21] . these are anticipated changes in later stages of chemotherapy regime. however, paclitaxel and anti egfr agents may show similar changes in the earlier cycles of chemotherapy. many cytotoxic agents and the new agents may show ggo and consolidation (mtor antagonist like everolimus, temsirolimus, bleomycin) or a cop like image. some drugs (gemcitabine) may cause capillary leakage syndrome and few agents may cause interstitial changes due to heart failure (doxorubicin). ct chest is, in particular, a very sensitive radiological tool for detection of the early signs of respiratory infections in sick icps. the specificity of the interpretation can be improved significantly through a comprehensive approach taking into consideration of the clinical setting, past infections and treatment history. used appropriately, ct scan is a very valuable tool for early detection and differential diagnosis of lung infections in icps and therefore for the subsequent reduction of morbidity and mortality highly vulnerable patients. none. understanding and diagnosing infectious complications in the immunocompromised host evolving risk factors for infectious complications of cancer therapy imaging infection early detection of pneumonia in febrile neutropenic patients: use of thin-section ct acute lung disease in the immunocompromised host: differential diagnosis at high-resolution ct severity assessment tools for predicting mortality in hap ct findings in immnunocompromised patients with pulmonary infections pulmonary infections in immnunocompromised hosts: the importance of correlating the conventional radiological appearance with clinical setting imaging pulmonary infection: classic signs & patterns pulmonary septic emboli: diagnosis with ct reversed halo sign on high-resolution ct of cryptogenic organizing pneumonia: diagnostic implications invasive pulmonary aspergillosis in acute leukemia: characteristic findings on ct, the ct halo sign, and the role of ct in early diagnosis pattern recognition of the pulmonary manifestations of aids on ct scans tuberculosis: a radiologic review cd4 counts as predictors of opportunistic pneumonias in hiv infection pneumocystis jiroveci pneumonia: high-resolution ct findings in patients with and without hiv infection spectrum of pulmonary aspergillosis: histologic, clinical, and radiologic findings thoracic mycoses from opportunistic fungi: radiologicpathologic correlation pulmonary fungal infection: imaging findings in immunocompetent and immnunocompromised patients viral pneumonias in adults: radiologic and pathologic findings ct findings of chemotherapy -induced toxicity: what radiologists need to know about the clinical and radiologic manifestations of chemotherapy toxicity cancer patient, with dyspnea and without fever showing ggo due to mild chf key: cord-344297-qqohijqi authors: smith, jacqueline; sadeyen, jean-remy; cavanagh, david; kaiser, pete; burt, david w. title: the early immune response to infection of chickens with infectious bronchitis virus (ibv) in susceptible and resistant birds date: 2015-10-09 journal: bmc vet res doi: 10.1186/s12917-015-0575-6 sha: doc_id: 344297 cord_uid: qqohijqi background: infectious bronchitis is a highly contagious respiratory disease which causes tracheal lesions and also affects the reproductive tract and is responsible for large economic losses to the poultry industry every year. this is due to both mortality (either directly provoked by ibv itself or due to subsequent bacterial infection) and lost egg production. the virus is difficult to control by vaccination, so new methods to curb the impact of the disease need to be sought. here, we seek to identify genes conferring resistance to this coronavirus, which could help in selective breeding programs to rear chickens which do not succumb to the effects of this disease. methods: whole genome gene expression microarrays were used to analyse the gene expression differences, which occur upon infection of birds with infectious bronchitis virus (ibv). tracheal tissue was examined from control and infected birds at 2, 3 and 4 days post-infection in birds known to be either susceptible or resistant to the virus. the host innate immune response was evaluated over these 3 days and differences between the susceptible and resistant lines examined. results: genes and biological pathways involved in the early host response to ibv infection were determined andgene expression differences between susceptible and resistant birds were identified. potential candidate genes for resistance to ibv are highlighted. conclusions: the early host response to ibv is analysed and potential candidate genes for disease resistance are identified. these putative resistance genes can be used as targets for future genetic and functional studies to prove a causative link with resistance to ibv. electronic supplementary material: the online version of this article (doi:10.1186/s12917-015-0575-6) contains supplementary material, which is available to authorized users. infectious bronchitis (ib) is a highly contagious respiratory disease of chickens first described in the usa in the 1930's [1] [2] [3] . clinical signs include: coughing, sneezing, rales and nasal discharge. the disease can also affect the reproductive organs, which leads to a decrease in egg quality and production, thus making it a major cause of economic losses within the poultry industry [4] . the causative virus, infectious bronchitis virus (ibv) is a coronavirus, which is an enveloped virus with a single positive-stranded rna genome, which replicates in the host cell cytoplasm [5] . proteins encoded by ibv include the viral rna polymerase, structural spike proteins, membrane and nucleocapsid and various other regulatory proteins. the spike glycoprotein mediates cell attachment and plays a significant role in host cell specificity [6] . the existence of many different ibv serotypes, which are not cross-protective means that control of ib, is very difficult. mortality is usually fairly low (~5 %), however some strains of the virus can also cause nephritis meaning that, depending on strain, mortality can be greater than 50 % [7, 8] or even up to 80 % with some australian isolates [9] . ibv infection leaves birds more susceptible to colibacillosis [10] and subsequent bacterial infections can also lead to a high level of mortality [11] . currently, attenuated live vaccines are used in broilers and pullets, and killed vaccines are used in layers and breeders [12] . however, virus control is very difficult, as there are only a few vaccine types and many different strains of ibv. the virus also continues to mutate rapidly, generating more virulent strains of the disease [13] [14] [15] . coronaviruses have now also been detected in other avian species such as turkey, duck, goose, pheasant, guinea fowl, teal, pigeon, peafowl and partridge [4] . the extent to which the virus affects the host is highly dependent on the chicken breed [4] and the mhc b locus is known to play a role in susceptibility to the virus [16] . in this study we attempt to identify non-mhc genes, which may be involved in resistance to ibv. no genetic analyses have thus far been undertaken in order to try and do this and no quantitative trait loci or genes associated with resistance have been determined, so far. based on differential gene expression in susceptible and resistant lines of chickens, we identify potential candidate genes for disease resistance towards ibv (virulent m41 strain). building on the previous work by dar et al. [17] and wang et al. [18] we used affymetrix wholegenome chicken microarrays to examine the tracheal gene expression profiles of a line of birds known to be susceptible to ibv infection (line 15i) and a line known to show resistance (line n). we determined the early host response to infection and propose possible candidate genes for involvement in disease resistance towards ibv. understanding how coronaviruses infect the host and identifying genes involved in resistance is important not only for the poultry industry but also has important implications for human health, as diseases such as sars are also caused by coronaviruses [19, 20] . all animal work was conducted according to uk home office guidelines and approved by the roslin institute animal welfare and ethical review body. the lines used in these experiments are an ibv susceptible lineline 15i (inbred white leghorn strain) [21] and an ibv resistant lineline n (non-inbred cornell strain). line 15i was developed at east lansing in the usa in the 1940s [22] and line n at cornell, usa in the 1960s [23] . the lines have since been maintained at the institute for animal health in compton, uk. twoweek-old chicks from each line (15i and n) were separated into two experimental rooms, with ad libitum access to food and water. in one room, 54 birds (27 from each line) were infected with 4 log 10 cid 50 (10 4 cid 50 ) of virulent ibv-m41 strain in a total of 100 μl of 0.2 % bsa in pbs equally by intra nasal and ocular routes. in the other room, 54 control birds (27 from each line) received 100ul pbs via the same route. trachea samples (upper half ) were collected at 2, 3 and 4 days postinfection (9 individual birds from each line at each time point). the trachea of infected and control birds from each line were analysed for viral load using taqman real-time quantitative rt-pcr assays. tissue samples (~30 mg) were stabilized in rnalater (ambion, life technologies, paisley, uk) and disrupted using a bead mill (retsch mm 300, retsch, haan, germany) at 20 hz for 4 min. total rna was prepared using an rneasy kit (qiagen, crawley, uk) extraction method as per the manufacturer's protocol. samples were resuspended in a final volume of 50 μl of rnasefree water. concentrations of the samples were calculated by measuring od 260 and od 280 on a spectrophotometer (nanodrop, thermo scientific, paisley, uk). quality of the rna was checked on a bioanalyser (agilent technologies, south queensferry, uk). an rna integrity number (rin) > 8 proved the integrity of the rna. biotinylated fragmented crna was hybridized to the affymetrix chicken genome array. this array contains comprehensive coverage of 32,773 transcripts corresponding to over 28,000 chicken genes. the chicken genome array also contains 689 probe sets for detecting 684 transcripts from 17 avian viruses. for each experimental group (control and infected birds in each of the two lines at each of 2, 3 and 4 dpi), three biological replicates (3 rna pools from 3 birds) were hybridized. thus, 36 arrays were used in total. hybridization was performed at 45°c for 16 hours in a hybridization oven with constant rotation (60 rpm). the microarrays were then automatically washed and stained with streptavidin-phycoerythrin conjugate (sape; invitrogen, paisley, uk) in a genechip fluidics station (affymetrix, santa clara, ca). fluorescence intensities were scanned with a genearray scanner 3000 (affymetrix, santa clara, ca). the scanned images were inspected and analyzed using established quality control measures. array data have been submitted to array express (http://www.ebi.ac.uk/arrayexpress/) under the accession number e-tabm-1128. gene expression data generated from the genechip operating software (gcos) was normalised using the plier (probe logarithmic intensity error) method [24] within the affymetrix expression console software package. this normalised data was then analysed using the limma and farms [25] packages within r in bioconductor [26] . probes with a false discovery rate (fdr) value <0.05 and a fold change ≥1.5 were deemed to be biologically significant. in order to determine which biological pathways are involved in the responses to viral infection, we analysed our differentially-expressed (de) genes using pathway express [27, 28] which uses kegg pathways [29] to pictorially display up/down regulation of genes. (nb. these diagrams are based on the human pathways and so are not completely representative of the chicken pathways). genes differentially expressed during the host response (fdr <0.05) were analysed against a reference background consisting of all genes expressed in the experiment. factors considered by pathway express include the magnitude of a gene's expression change and its position and interactions in any given pathway, thus including an 'impact factor' when calculating statistically significant pathways. anything with a p-value <0.25 is deemed significant when using this software. use of the ingenuity pathway analysis (ipa) program [30] revealed which canonical pathways are being switched on by ibv infection in the host (with benjamini-hochberg multiple testing correction) and allowed us to analyze the gene interaction networks involved in the host response. genes were clustered by similar expression pattern and analysed for enriched go-terms and transcription factor binding sites (tfbs) using expander (v5.2) [31]. normalised expression data from control samples were compared with infected samples to examine the host response to ibv infection. enrichment analysis of particular go terms or tfbs within clusters was done using the tango and prima functions, respectively, within the expander package. taqman real-time quantitative rt-pcr (qrt-pcr) was used to quantify viral rna levels and for confirmation of the microarray results for the mrna levels of selected genes. this was performed on 3 replicate pools of 3 samples (9 birds). primers (sigma) and probe (pe applied biosystems, warrington, uk) ( table 1) were designed using primer express (pe applied biosystems). briefly, the assays were performed using 2 μl of total rna and the taqman fast universal pcr master mix and one-step rt-pcr mastermix reagents (pe applied biosystems) in a 10 μl reaction. amplification and detection of specific products were performed using the applied biosystems 7500 fast real-time pcr system with the following cycle profile: one cycle at 48°c for 30 min and 95°c for 20 sec, followed by 40 cycles at 95°c for 3 sec and 60°c for 30 sec. data are expressed in terms of the cycle threshold (ct) value, normalised for each sample using the ct value of 28s rrna product for the same sample, as well described previously [32] [33] [34] . final results are shown as 40-ct using the normalised value, or as fold-change from uninfected controls. taqman real-time quantitative rt-pcr analysis was used to measure viral load in trachea samples from both control and infected birds from both lines 15i and n. tracheal tissue was chosen for examination in this study as the target of ibv is the epithelial surface of the respiratory tract. viral rna was detected in infected birds, but no significant difference in viral load was detected between lines at any of the days 2, 3 or 4 post infection (fig. 1) . this would indicate that the resistance to the virus seen in line n is due to how the birds respond to the virus once it has entered the body and is not a measure of how the birds can prevent initial infection by the virus itself. when resistance to ibv infection was originally determined in these lines, it was noted that they were equally susceptible to infection, but a variation in outcome was seen. in line n, 33 % of birds showed air sac lesions whereas 73 % of 15i birds presented lesions. mortality was 0 in line n, but 47 % within line 15i birds. it was hypothesized that the different lines were producing different immunological responses upon infection [21] . gene expression differences found in the susceptible 15i line between infected and control birds over days 2, 3 and 4 post infection were analysed, with a view to examining the innate host response to infection by ibv. genes seen to be induced during the host response to infection include c1s, irf1, stat1, mx1, tlr3 and ctss as previously recognised by guo et al. [35] . we also identified ifit5, oasl, sca2, lyg2, isg12-2, ddx60, ifih1, irf7, table s1 . to elucidate which biological pathways are being perturbed during the host response to ibv infection, we analysed our data using pathway express [36] . the resulting pathway diagrams are extremely useful in establishing which gene networks are involved in a particular experimental response. as seen in fig. 2 , genes involved in antigen presentation and the toll-like receptor (tlr) pathway are up-regulated. tlrs identify pathogen associated molecular patterns (pamps) and are crucial to the innate immune system. in this study tlr3 is shown to be induced at 3 dpi. tlr3 recognizes double-stranded rna intermediates produced during viral replication and has previously been shown to be induced in the trachea at this time after ibv infection [37] . another pathway involved is the phosphatidylinositol signalling pathway (table 2) . phosphatidylinositol kinases are known to play an important role in the viral life cycle after infection of the host and pi4kb is known to be exploited by coronaviruses for viral entry. the product of pi4kb catalysis is phosphatidylinositol 4-phosphate (pi4p) and coronavirus entry into the host is mediated by the pi4p lipid microenvironment [38] . genes involved in the complement system are also highlighted as being up-regulated in response to ibv infection. complement-mediated lysis of viruses is an important facet of the host innate immune system and its role in defence against viral infection [39] as reflected in the induction of these genes in this study. use of ingenuity pathway analysis (ipa) software also allowed us to determine which biological systems are active during the host response. up-regulated genes are seen to be part of the canonical biological pathways shown in fig. 3a . biological processes involving pattern recognition receptors and interferon signalling feature heavily. the interferon response is a powerful antiviral mechanism, which has previously been shown to be involved in the host response after ibv infection. a very early induction of ifn-γ has been reported in splenocytes [40] , and in peripheral blood mononuclear cells (pbmcs) and lung leukocytes [41] . ifnb expression has also been reported in trachea between 1 and 2 dpi [42] . we do not see this increase in expression of interferon genes (due to the absence of data earlier than 2 dpi), but we do see the downstream effects, with increased expression of many interferon-induced genes. specific physiological processes activated upon ibv infection can also be seen in fig. 3b . the stimulation of various different immune cells is seen along with the indication of reproductive abnormality, which would reflect the problems seen with egg-laying upon ibv infection. in order to cluster genes seen to be involved in the host response to infectious bronchitis into groups with similar expression profiles and probably sharing similar functions or gene regulatory pathways, we utilised the click algorithm within the expander program [43] . figure 4a shows the expression profile of genes upregulated during the response to virus. the expander program was also used to analyse the gene ontology (go) functional annotations of the genes being differentially expressed. figure 4b shows the biological process terms, which are significantly enriched in the genes responding during the host response to infection. as would be expected, these include terms like 'innate immune response' and 'antigen processing and presentation'. 'nad + adp-ribosyltransferase activity' and 'phosphoinositide binding' are also highlighted. transcription factor binding sites present in de genes which are significantly over-represented were also predicted. figure 4c shows that genes up-regulated during the host response have a high proportion of irf7 and isre binding sites. irf7 is a transcriptional activator, which binds to the interferon-stimulated response element (isre) in ifn promoters and functions as a molecular switch for antiviral activity. analysis of the gene expression differences between infected and control birds across the two lines has provided us with information on how these lines differ in their response to infection. examination of the gene expression profiles in the control birds of the two different lines also allowed us to identify genes, which are inherently different between the susceptible and resistant birds. it can be seen that there are numerous genes, which show large expression differences between the two lines, even before infection. dramatic differences in gene expression of certain genes, including ddt, sri, blb1, hscb, bf1, bf2, suclg2, mx1 and sri, which are more highly expressed in the resistant n line table s2 shows all 1930 de probes) so, it can be seen that these are genes which have inherently different expression levels between susceptible and resistant birds, even before infection occurs. we therefore postulate that some of these genes may play an important role in disease resistance. the potential interactome of ibv has recently been investigated by stable isotope labelling with amino acids in cell culture (silac) coupled to a green fluorescent proteinnanotrap pull-down methodology [44] . host proteins, which bind to the ibv n protein were identified, some of the genes for which, we see as being inherently expressed at higher levels in susceptible birds in this study. these genes include myh9, caprin1, dhx57, hnrnph3, rpl27a, fmr1, c22orf28, hnrpdl, sfrs3, rpl31, npm1 and rpsa. this may therefore be one of the reasons why line 15i is more susceptible to ibv infectionthere are more host proteins to which the virus binds, compared with the resistant line n. upon infection, differences in response are also seen between the two lines. interestingly, apart from cd38 and cd4 at 3 dpi and fkbp5 at 4 dpi, all other differential gene expression between the lines is seen at 2 dpi in this study (additional file 3: table s3 ). cd38 is a glycoprotein found on the surface of many immune cells including cd4+, cd8+, b lymphocytes and natural killer cells and is a marker of cell activation. it functions in cell adhesion, signal transduction and calcium signalling. cd4 is found on the surface of immune cells such as t helper cells, monocytes, macrophages and dendritic cells. it is a membrane glycoprotein which interacts with mhcii antigens. the protein functions to initiate or augment the early phase of t-cell activation. the protein encoded by fkbp5 is a member of the immunophilin protein family, which play a role in immuno-regulation and basic cellular processes involving protein folding and trafficking. early defence by the host is a key mechanism for combatting viral infection, and induction of ifnb and other innate genes in response to ibv infection has been shown to peak around 18-36 hr post infection [42] . in this study, genes more highly expressed (or less down-regulated) in the resistant n line at 2 dpi include a number of collagen genes (col3a1, col1a2, col9a1, col9a2, col6a1 and col4a1) and other genes such as acan, fstl1, comp, eif3a, stat3 and igfbp5. genes seen to be more highly expressed (or less down-regulated) in the susceptible 15i line include rbm39, mafb, nnk2, ccn1, mgat5 and thrap3. one consequence of ibv infection is the production of poor quality, misshapen eggs by infected birds [45] . some of the genes previously identified as being important for the creation of a healthy eggshell are seen to be more highly expressed by the resistant n line birds after infection in this study. these genes include col1a2, creld2, hsp90b1, p4hb and erp29 [46] . for a full list of genes differentially expressed between the two lines in trachea (409 de probes) see additional file 3: table s3 . ipa analysis of genes showing different inherent expression between lines 15i and n shows that the molecular functions of these genes is primarily concerned with their involvement in cell death and cell adhesion (fig. 5) , two processes previously shown to be significant in infected kidneys [47] . when the differential host responses to infection are examined, it is seen that genes involved in proliferation of t-lymphocytes and genes concerned with cell attachment and cytoplasmic organization are more highly expressed in the resistant line n. other processes significantly involved are apoptosis and necrosis (fig. 6a) , which have been previously documented in ibv-infected vero cells by liu et al. [48] . one of the most perturbed biological networks noted in this analysis is that involving genes related to connective tissue disorders and involve many collagen genes. these genes are more highly expressed in susceptible line 15i birds compared to resistant line n birds (fig. 6b) suggesting that ibv infection might cause more disorder of eggshell formation in this line [49] . the production of poor quality eggs by ibv infected birds may, in part be a reflection of the expression of these kinds of gene networks compared to that seen in resistant birds. twenty-one genes were selected for qrt-pcr validation ( table 3) . these genes were chosen based on their involvement in the host response and whether they were differentially expressed between the susceptible and resistant lines (either inherently or during the course of infection). of the 21 genes tested, 19 showed comparable higher expression in response to infection in the resistant than in the susceptible line c inherently higher expression in the resistant line differential expression to that determined by the arrays. however, the results for ifnar2 and igfbp5 were not confirmed (additional file 4: figure s1 ). besides knowing that the mhc b locus has a bearing on disease resistance, the lack of any genetic information or identified qtl meant that we had to rely upon the gene expression differences we saw between susceptible and resistant lines to give us clues as to genes potentially involved in resistance to ibv infection. identifying genes which were expressed at different levels in the two lines of birds highlighted b-locus genes (blb1, bf1, bf2 , b-g) as well as bringing to our attention various other non-mhc genes which, due to their known biology, could be candidates for being involved in resistance to ibv infection (table 4) . mx1, c1s, irf7, tlr3, c1r, ccli7, isg12-2 and ifitm3 are all strongly induced during the host response to ibv infection. they are all innate immune genes which could potentially have a role in determining susceptibility to the virus. mx1 and ifitm3 are already established as anti-viral molecules [50] [51] [52] . cd38, cd4, fkbp5 and stat3 all show a higher level of expression during the host response in the resistant birds compared to that of the susceptible birds, indicating their involvement in the host defence mechanism. cd38 and cd4, with their role as receptors on immune cells, as described above, are obvious candidates, along with fkbp5 as an immune-regulator. stat3 is activated by various cytokines and growth factors and functions in cellular processes such as cell growth and apoptosis. even before infection, many genes are seen to be highly differentially expressed between lines 15i and n. oasl is an interferon-induced molecule known to have anti-viral activity against certain viruses such as hepatitis c virus. ddt is highly homologous to the macrophage migration inhibition factor, mif. we have also shown it to be highly differentially expressed in other chicken lines, which are susceptible or resistant to marek's disease virus [53] . ifnar2 is an obvious candidate prediction, as the interferon response is central to the host's defence against ibv infection. tpd52l1, bcl2l1, faim2 and ciapin1 are all known to be involved in regulation of apoptosis, a process seen to be important during ibv infection. hscb, sri, and suclg2, although not having an obvious potential biological role in disease resistance, are highly differentially expressed between susceptible and resistant lines and should thus be considered as potential candidates. resistance to ibv infection is brought about by the immune response after the virus has entered the host and is not due to prevention of initial viral infection. there is a small initial innate response at 2 dpi, with much more gene expression seen at 3 and 4 dpi. analysis of genes being activated or inhibited upon infection shows that the biological pathways primarily affected during ibv infection include mapk signalling, those involved in the interferon response and those involving pattern recognition receptors. susceptible and resistant lines show a differential host response mostly at 2 dpi. there are also genes which are inherently different between the two lines studied, including many genes, which control the apoptotic potential of the host. these differences seen in gene expression levels, allow us to postulate on many candidate genes for disease resistance. some potential candidates for involvement in disease resistance include genes already known to confer resistance to other viral infections (mhc-b locus genes, mx1, oasl and ifitm3), genes involved in apoptotic processes (tpd52l1, bcl2l1, faim2 and ciapin1) and others which could be potential candidates due to their known biology (e.g. ddt and cd4). array data have been submitted to array express (http://www.ebi.ac.uk/arrayexpress/) under the accession number e-tabm-1128. additional file 1: table s1 . gene expression seen during the host response to ibv infection in the trachea of susceptible birds. (xlsx 24 kb) additional file 2: table s2 . gene expression differences found to be inherent between susceptible and resistant lines in the trachea. (xls 386 kb) additional file 3: table s3 . genes found to be differentially expressed between susceptible and resistant lines in response to ibv infection in the trachea. (xls 107 kb) additional file 4: figure s1 . the authors declare no conflicts of interest and no competing financial interests. an apparently new respiratory disease in baby chicks studies of infectious coryza of chickens with special reference to its etiology cultivation of the virus of infectious bronchitis coronavirus avian infectious bronchitis virus coronavirus replication and interaction with host recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism interleukin-6 expression after infectious bronchitis virus infection in chickens review of infectious bronchitis virus around the world nephropathogenic avian infectious bronchitis viruses the role of phagocytic cells in enhanced susceptibility of broilers to colibacillosis after infectious bronchitis virus infection ability of massachusetts-type infectious bronchitis virus to increase colibacillosis susceptibility in commercial broilers: 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supply of minerals or in the shell mineralization process transcriptome analysis of chicken kidney tissues following coronavirus avian infectious bronchitis virus infection induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus effects of avian infectious bronchitis virus antigen on eggshell formation and immunoreaction in hen oviduct interferons: cell signalling, immune modulation, antiviral response and virus countermeasures mx proteins: mediators of innate resistance to rna viruses ifitms restrict the replication of multiple pathogenic viruses submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was supported by the biotechnology and biological science research council (bbsrc), as part of grant numbers bb/d013704/1, bb/ d013704/2 and bb/d010705/1. the authors would like to thank alison downing (edinburgh genomics, edinburgh, uk) for excellent technical assistance with the affymetrix microarray experiments. authors' contributions js performed the arrays, analysed the results and wrote the manuscript; dc carried out challenge experiments, j-rs prepared rna, measured viral load and performed qrt-pcr; db and pk conceived and supervised the project and revised the manuscript. all authors read and approved the final manuscript. key: cord-347246-0vofftmj authors: everitt, j i; richter, c b title: infectious diseases of the upper respiratory tract: implications for toxicology studies. date: 1990-04-17 journal: environ health perspect doi: nan sha: doc_id: 347246 cord_uid: 0vofftmj the consequences of adventitious infectious agents upon the interpretation of toxicology studies performed in rats and mice are incompletely understood. several prevalent murine pathogens cause alterations of the respiratory system that can confuse the assessment of chemically induced airway injury. in some instances the pathogenesis of infection with these agents has been relatively well studied in the lower respiratory tract. however, there are few well-controlled studies that have examined the upper respiratory region, which result in interpretive problems for toxicologic pathologists. the conduct and interpretation of both short-term and chronic rodent bioassays can be compromised by both the clinical and subclinical manifestations of infectious diseases. this paper reviews several important infectious diseases of the upper airway of rats and mice and discusses the potential influence of these conditions on the results of toxicology studies. the validity and reproducibility of rodent toxicology studies depend, in part, on the interaction of numerous environmental, microbial, and genetic factors. control of important animal and extrinsic factors forms the basis of laboratory animal science programs in toxicology. despite numerous advances and sophistication in the field of laboratory animal medicine, adventitious murine microbial agents continue to pose a threat to both shortand long-term studies that use rats and mice. programs for prevention, detection, elimination, and control of rodent infectious agents are necessary in toxicology research and testing. these programs are of particular importance to both toxicologists and pathologists responsible for conducting and interpreting rodent bioassays. specific procedures and equipment used in inhalation toxicity experiments can contribute significantly to the spread of disease and the interaction of infectious agents with inhaled toxicants. many factors, such as inhalation caging; cage rotation schemes; inhalation chamber microenvironments; food and water deprivation during exposures; and stresses from crowding, frequent handling, and servicing of animals, all play important roles in the interplay between pathogen and chemical exposure. the complications of rodent infectious agents to toxicology research and testing has been the subject of increasing awareness and review (1, 2) . recent rodent serology surveys indicate that sialodacryoadenitis virus (sdav), sendai virus, and mycoplasrna pulmonis are three of the most prevalent murine pathogens (3) . all three agents cause significant rodent respiratory disease, with lesions in the upper airways, including the nasal passages. in addition to being a common site for microbial-induced disease, the upper respiratory tract is a target organ of chemically induced injury. this paper will review the respiratory pathology of several important murine pathogens and discuss their importance in toxicology and carcinogenicity studies. sialodacryoadenitis virus is a common, highly infectious coronavirus affecting rats and causing a selflimiting necrosis and inflammation of mixed or serous salivary glands, lacrimal glands, and upper airway epithelium (4, 5) . respiratory tract lesions are generally confined to the upper airway and usually precede the inflammatory changes that occur in the exocrine tissues of the head (6). gross lesions consisting of edematous and/or inflammatory changes are sometimes noted in extrarespiratory tissue in mixed or serous salivary glands, exorbital lacrimal glands, harderian glands, periglandular connective tissue, cervical lymph nodes, or the thymus. everi1t and richter microscopic lesions in experimental sdav infection begin in the nasal respiratory epithelium approximately 48 hr postinfection. initial respiratory epithelial necrosis accompanied by congestion and edema is rapidly followed by a mixed inflammatory cell infiltrate of the lamina propria. necrosis primarily occurs in the respiratory epithelium lining the ventral turbinates and lateral wall of the nasal cavity but usually spares the olfactory mucosa. the nasal meatuses may be filled with exudate composed of necrotic epithelial cells admixed with inflammatory cells and mucus. serous mucosal glands of the nasopharynx generally sustain relatively mild injury (6) , although necrosis of ducts and acini does occur. lesions similar to those in the nasopharynx, but milder and less uniform, are found in the trachea. the rhinotracheitis of sdav is usually self-limiting and it is resolved by the end of the second week of infection (6) . although not as prevalent or as severe as upper respiratory tract lesions, microscopic changes do occur in the lower respiratory tract in experimental infections of rats (7), consisting of focal nonsuppurative bronchiolitis and peribronchial lymphocytic infiltration. although sdav infection is generally a self-limiting disease without significant mortality, it can cause marked effects on toxicology studies through clinical disease or subclinical manifestations. rats with sdav infection often have reduced food consumption, weight loss, and reduced breeding performance all of which can affect toxicology studies. the interactions of sdav with other respiratory pathogens and chemical agents is not well established. recent experimental evidence suggests that sdav infection depletes salivary epidermal growth factor (egf) and may thereby affect egfdependent cell growth processes and experimental carcinogenesis studies in rats (8) . results of a 2-year inhalation toxicity study of methylene chloride were clouded by sdav infection of rats early in the study (9) . in this study, a low number of male rats exposed to the two highest doses of the chemical had an increased incidence of sarcomas in the ventral neck region. the relevance and toxicological significance of these neoplasms was questionable because the species and sex specificity of the response was inconsistent with the body of knowledge on the toxicity of the chemical. it was postulated that the tumor response may have been due to a combination of viral infection and exposure to high concentrations of methylene chloride. this case exemplifies the problems that may occur in toxicologic studies when unusual and unexpected lesions are found in animals that had infectious processes within the target organ of toxicity. sendai virus is a very common respiratory pathogen of laboratory rodents that has complicated numerous toxicology and carcinogenicity studies (10) . in mice and rats, this paramyxovirus causes clinical and subclinical changes with great strain variability in disease expression and resultant pathologic lesions. the virus has a marked tropism for the respiratory tract, including the nasal cavity. sendai virus infection has been well described for mice (11) (12) (13) , and recently the course of experimental infection in the rat has been reported (14) . sendai virus infection causes rhinitis, tracheobronchitis, bronchiolitis, and varying degrees of alveolitis in both rats and mice. there is marked strain variability in qualitative, quantitative, and chronological aspects oflesion development, which causes difficulty in making generlizations regardingthe pathology ofthe respiratory tract. although there are many excellent descriptive studies ofthe histogenesis of sendai virus-induced lesions within the lower respiratory tract, few pathology reports include a description of lesions in the nasal cavity and upper airway. nasal lesions have been well described in experimental infections of the sprague-dawley rat (14) . in this model, a rapid development of severe rhinitis with marked infiltration of the epithelium and lamina propria with lymphocytes and some neutrophils occurred at 1 day postinfection. lesions were most severe in the respiratory epithelium of the naso-and maxilloturbinates, compared to minimal lesions in the olfactory epithelium of the ethmoid region. over the next 4 days postinfection, there was a progressive accumulation of inflammatory cells, chiefly of lymphocytes, in the lamina propria. the lesions expanded to involve the middle portions of the nasal septum. respiratory epithelial cells exhibited pyknosis and karyorrhexis, particularly in the more basilar regions of the mucosa. exudate was noted in the ethmoid region, although there was little inflammation in that area. between 5 and 21 days postinfection, the severity of the nasal epithelial necrosis, inflammatory cell infiltrate, and lumenal exudate decreased. by day 28 postinfection, no discernable lesions were noted in experimentally infected rats. there are numerous systemic effects (thble 1) that toxicologists and pathologists need to consider when interpreting the impact of sendai virus infection upon study results (10, 15) . studies can be compromised through reduction in animal numbers, changes in immune function, xenobiotic metabolism, and physiology (10) . sendai virus infection has altered the course of chemically induced pulmonary carcinogenesis in strain a mice by reducing the number of resultant lung tumors (16) . in addition, the pulmonary carcinogenic responses have been altered in sendai virus infected balb/c mice following exposure to urethane (17 murine respiratory mycoplasmosis (mrm), due to mycoplasrna pulmonis is a naturally occurring, slowly progressing, chronic disease in rats and mice. numerous respiratory responses are associated with m. pulmonis infection of rodents (table 2 ) (18) . the prevalence of the pathogen and the numerous respiratory and systemic effects of infection make this disease one of the most important entities for pathologists and toxicologists during studies of the respiratory tract. respiratory mycoplasmosis is often clinically silent, although lesions of the upper respiratory tract commonly occur (19) . the principal lesions of mrm in rats include rhinitis, otitis media, laryngitis, and tracheitis (20) . gross lesions in the upper respiratory tract are generally not discernable, although rats may occasionally show mucopurulent nasal exudate and/or porphyrin-tinted oculonasal discharge. the microscopic pathology of mrm is characterized by epithelial changes including cellular hypertrophy and hyperplasia, as well as metaplastic changes. neutrophilic exudation and lymphoplasmacytic infiltrates are common throughout the upper airway. subepithelial lymphoid accumulations can occur in mrm-associated rhinitis (plates 1 and 2). ciliary loss and epithelial hyperplasias can be severe and quite extensive. the relative importance of direct mycoplasmal damage, as opposed to immune and nonimmune inflammatory reactions form. pulmonwn has yet to be completely discerned (19) . it is known that cytolysis follows attachment of m. pulmonis to upper airway epithelial cells. ciliastasis, loss of cilia, distension of intercellular spaces, cytoplasmic vacuolization, disruption of mitochondria, epithelial hyperplasia and metaplasia, and syncytial cell formation have also resulted following attachment of this organism (19) . the tympanic cavity of the ear may be completely filled with a neutrophilic exudate. frequently in mrminduced otitis media, the lining epithelium is hyperplastic and the lumenal cavity may become filled with collagenous connective tissue. thickened connective tissue lining the tympanic cavity may remain as a chronic sequellum of the infection. epithelial hyperplasias and lymphoid cell accumulations are commonly found in the certain chemical agents, including ammonia, which is commonly found in the cage environment from soiled bedding, can exacerbate mrm in rats. inhalation of the important industrial compound hexamethylphosphoramide (hmpa) can cause a synergistic enhancement of the progression and severity of mrm (21, 22) . in hmpa toxicity studies, nasal tumors, rhinitis, nasal epithelial degeneration, metaplasia, and dysplasia were noted in cornunction with an enhanced mortality from chronic pneumonia in infected rats. the increased mortality was possibly due to a chemically induced destruction of the mucociliary apparatus in the upper airway, which may have contributed to fatal lower respiratory infections. a chronic inhalation bioassay of propylene oxide revealed dose-dependent nasal epithelial proliferative lesions and two nasal adenomas in rats with intercurrent mrm (23) . the significance of the proliferative nasal lesions, which appeared to be treatment related, was difficult to interpret, since their development may have been influenced by intercurrent infectious inflammatory disease. although numerous bacteria can infect the upper airway of the rat and mouse, they are not generally prevalent in well-conducted toxicology studies begun with animals free of adventitious murine pathogens and maintained with modern methods of laboratory animal husbandry. however, the cilia-associated respiratory (car) bacillus has recently received attention. this gramnegative, filamentous, rod-shaped bacillus (24, 25) infects both rats and mice and causes lesions morphologically similar to those of m. pulmonis infections. the car bacillus was generally overlooked in the past because it often occurred as a dual infection with m. pulmonis. lesions typical of mycoplasmal chronic respiratory disease have been noted following natural and experimental infection with the car bacillus. these lesions include massive accumulations of predominantly mononuclear inflammatory cells surrounding bronchi and bronchioles (plate 3), as well as various degrees of suppurative bronchopneumonia, squamous metaplasia, and bronchiectasis. the ciliated epithelium of the airway is usually heavily colonized with filamentous bacteria, which can be readily demonstrated with warthin-starry silver impregnation techniques (plate 4). little is known regarding the prevalence of this organism as a subclinical infection. although lesions induced by the car bacillus have been described in the lungs of both rats and mice, there have been no descriptions of pathology in the upper airway where, presumably, the organism can also grow. elucidation of the significance of this pathogen for rodent toxicity awaits further studies and characterization. descriptions of naturally occurring fungal infections of the respiratory tract of rodents are extremely rare. the histological features of fungal rhinitis, which occurred in high incidence in two separate chronic carcinogenicity studies in male wistar rats (129/597 animals), have been described (26) . since these animals had titers to both sendai and sdav viruses, the authors suggested that viral-induced inflammation of the upper respiratory mucosa rendered the epithelium susceptible to the fungal infection. in addition to the 129 animals with fungal infection, 80 animals had suppurative rhinitis without evident fungal growth. an etiologic diagnosis ofaspergillusfumigatus was based upon characteristic morphology of the fruiting heads found in a small percentage of cases. aspergillus rhinitis was generally noninvasive and limited to the naso-and maxilloturbinates. although purulent inflammation was noted in the olfactory epithelium of some cases, fungal growth was rarely noted in this region. hyphal conglomerates were usually associated with foreign bodies (hair and plant material) and were surrounded by polymorphonuclear leukocytes, bacteria, debris, and nasal secretions. the underlying respiratory epithelium was usually hyperplastic, hypertrophic, or revealed squamous metaplasia. subepithelial connective tissue and submucosal glands were often infiltrated by aggregates of lymphocytes, plasma cells, and neutrophils. a relatively small number of cases had epithelial necrosis associated with fungal invasion. a review of cases of fungal rhinitis in the national tbxicology program (ntp) archives revealed the occurrence of fungal rhinitis in eight bioassays. in the affected studies that encompassed animals that were both serologically negative as well as some bearing viral titers, cases occurred in both sexes of fischer 344 rats in relatively low incidence (up to 10% of a dose group). a few scattered cases were noted in b6c3f1 mice. the histologic appearance (plates 5 and 6) was identical to that described in the literature in the wistar rat. in the affected bioassays the chemical was administered by inhalation, feed, or gavage in several different laboratories. foreign material was noted in most of the cases suggesting that particles from food or elsewhere may serve as local irritants and carriers for the fungus. one of the principal ways in which infectious diseases complicate toxicologic studies is by interference with the interpretation of the pathology data. in addition to alterations in the morphologic appearance of target organs, there can be changes in clinical signs of toxicity, food consumption, body weight gain, hematology, urinalysis, and serum chemistry parameters (27) . body weight gain depression of greater than 10% is often considered to be a sign of toxicity in treated groups of animals. the combined effects of infection and treatment influence food consumption and body weight gain to a greater extent than infection or chemical treatment alone. this synergism can confound data interpretation (27) . it has recently become apparent that infectious agents can markedly affect the metabolism of foreign compounds by impairing hepatic mixed-function monoxygenases (cytochrome p-450-dependent monoxygenase system) (28) . alteration of xenobiotic metabolism has important ramifications for toxicologists who administer compounds that are subject to bioactivation and biotransformation. the mechanistic basis for the microbialinduced inhibition of biotransformation has not yet been completely elucidated. it appears that interferon induction as a result of infection can act as a mediator in depressing p-450, and it may have a direct action on the hemoprotein itself (28) . many of the important murine viral agents including sendai virus are potent inducers of interferon (29) . infectious diseases can also modulate hepatic cytochrome p-450 by effects on the reticuloendothelial system. furthermore, microbial agents that perturb kupffer cells decrease drug biotransformation activity (28) . effects on nonhepatic xenobiotic metabolism by infectious agents have received little attention. several upper respiratory viral infections in man, including rhinoviruses and influenza viruses, are known to impair drug biotransformations by affecting the cytochrome p-450 system (28) . similar viral effects have been demonstrated in pr8 influenza-infected mice (30) . mice infected with this virus have significantly decreased pulmonary benzo[a]pyrene hydroxylase activity. nasal cytochrome p-450-dependent monooxygenases may be extremely important in the bioactivation and biotransformation of inhaled xenobiotics. in most species, the nasal olfactory concentration of these enzymes is second only to that found in the liver (31) . mouse hepatitis virus, a common mouse coronavirus infection, is reported to depress hepatic p-450 levels (32) . strains of this pathogen cause nasal cavity infections with necrotizing lesions in the olfactory mucosa (33) . it is therefore possible that extrahepatic alterations of p-450 may result and be of importance to toxicology studies. in addition to alterations of xenobiotic metabolism, infectious diseases cause other cellular effects that can modulate toxic and carcinogenic responses. the association between enhanced cell replication and cancer induction has gained the interests of many toxicologists (34) . gross and co-workers found that sites of cell replication correlated well with sites of tumor induction in the nasal cavities from rats exposed to formaldehyde in acute and chronic inhalation studies (35) . although it is uncertain how enhanced cell replication affects the carcinogenic process, such correlations suggest that a cause and effect relationship may exist. infectious diseases that cause epithelial necrosis and repair lead to significant alterations in cell turnover. microbial agents such as m. pulmonis are known to cause significant alterations in cell cycle kinetics in populations of upper airway epithelial cells (36) . this clearly has profound implications for carcinogenesis studies in which cell turnover may be an important contributing factor in the multistage process. not only would the microbial infection and degree of cell turnover be important, but the chronology of the infection with regard to the chemical administration could prove critical. a variety of important microbial pathogens including viruses, mycoplasmas, bacteria, and fungi infect the upper respiratory tract of the mouse and rat and result in significant pathologic alterations. the rodent upper respiratory tract may also be an important target organ in chronic bioassays. lbxicologists and pathologists need to understand the etiopathogenesis of common pathogen-related diseases in the murine respiratory tract, so that the effects of natural disease processes may be separated from chemically induced injury. additional studies are needed to assess the impact of several of the more prevalent adventitious pathogens on the results of rodent bioassays. the examination of the rodent nasal cavity has been overlooked in many previous experimental models of murine infectious disease. descriptive pathology studies, which carefully examine the histogenesis of upper airway injury, are warranted for many of the common pathogens that complicate toxicology studies. in addition, the effects of murine infectious diseases on xenobiotic metabolism and cytokinetics in the upper airway should be further investigated. the authors wish to thank j. griffith for slides of car bacillus. viral and mycoplasmal infections of laboratory rodents: effects on biomedical research complications of viral and mycoplasmal infections in rodents to toxicology research and testing prevalence of pathogenic murine viruses and mycoplasma that are currently a problem to research biology and diseases of rats pathogenesis of sialodacryoadenitis in gnotobiotic rats sialodacyoadenitis virus infection, upper respiratory tract, rat comparison of strain susceptibility to experimental sialodacryoadenitis in rats effects of sialodacryoadenitis virus on experimental carcinogenesis in the rat (abstract) methylene chloride: a two-year inhalation toxicity and oncogenicity study in rats and hamsters sendai virus-disease processes and research complications the pathogenesis of sendai virus infection in the mouse lung susceptibility of inbred and outbred mouse strains to sendai virus and prevalence of infection in laboratory rodents naturally occurring sendai virus infection of athymic nude mice experimental sendai virus infection in laboratory rats ii. pathology and immunohistochemistry sendai virus influence of sendai virus on carcinogenesis in strain a mice respiratory infections and the pathogenesis of lung cancer. recent results cancer res mycoplasmal infections: disease pathogenesis, implications for biomedical research, and control the pathogenic potential of mycoplasmas: mycoplasma pulmonis as a model murine respiratory mycoplasmosis, upper respiratory tract, rat enhancement of natural and experimental respiratory mycoplasmosis in rats by hexamethylphosphoramide pulmonary response to inhaled hexamethylphosphoramide in rats y carcinogenic and toxicologic effects of inhaled ethylene oxide and propylene oxide in f344 rats isolation, propagation, and characterization of a newly recognized pathogen, cilia-associated respiratory bacillus of rats, an etiological agent of chronic respiratory disease cilia-associated respiratory (car) bacillus infection of obese mice aspergillus rhinitis in wistar (crl:(wi)br) rats toxicology: complications caused by murine viruses and mycoplasmas the influence of infection on the metabolism of foreign compounds the effect of cyclophosphamide on sendai virus infection of mice effect of pr-8 viral respiratory infection on benzo possible consequences of cytochrome p-450-dependent monooxygenases in nasal tissues research complications and state of knowledge of rodent coronaviruses mouse hepatitis s in weanling mice following intranasal inoculation the value of measuring cell replication as a predictive index of tissuespecific tumorigenic potential formaldehydeinduced neoplasia, acute toxic responses and cell turnover in the nasal passages of f-344 rats (abstract) the kinetics of cell proliferation in the tracheobronchial epithelia of rats with and without chronic respiratory disease photomicrograph of the nasal cavity from a f344 rat with aspergillus rhinitis. the respiratory epithelium has early squamous metaplasia. a diffuse inflammatory infiltrate is present in the underlying lamina propria. the nasal cavity is filled with debris and polymorphonuclear leukocytes plate 6. fungal hyphae within nasal cavity debris of f344 rat with aspergillus rhinitis. gomori-methenamine silver key: cord-332379-340wczmq authors: pennington, matthew r.; saha, amrita; painter, david f.; gavazzi, christina; ismail, ashrafali m.; zhou, xiaohong; chodosh, james; rajaiya, jaya title: disparate entry of adenoviruses dictates differential innate immune responses on the ocular surface date: 2019-09-13 journal: microorganisms doi: 10.3390/microorganisms7090351 sha: doc_id: 332379 cord_uid: 340wczmq human adenovirus infection of the ocular surface is associated with severe keratoconjunctivitis and the formation of subepithelial corneal infiltrates, which may persist and impair vision for months to years following infection. long term pathology persists well beyond the resolution of viral replication, indicating that the prolonged immune response is not virus-mediated. however, it is not clear how these responses are sustained or even initiated following infection. this review discusses recent work from our laboratory and others which demonstrates different entry pathways specific to both adenovirus and cell type. these findings suggest that adenoviruses may stimulate specific pattern recognition receptors in an entry/trafficking-dependent manner, leading to distinct immune responses dependent on the virus/cell type combination. additional work is needed to understand the specific connections between adenoviral entry and the stimulation of innate immune responses by the various cell types present on the ocular surface. conjunctivitis, a common ocular condition with a range of etiologies, is highly prevalent, affecting approximately 6 million people annually in the united states and accounting for 1% of all primary care office visits [1] [2] [3] [4] . viruses are responsible for up to 80% of conjunctivitis, and human adenoviruses (hadvs) are implicated in up to 65% of all viral cases [5, 6] . adenoviruses are small, non-enveloped viruses with a linear, double-stranded dna genome of approximately 36 kilobase pairs. the seven species (a-g) and more than one hundred genotypes currently in genbank exhibit a broad range of tropisms across the various mucosal surfaces of the body, including those within the respiratory, gastrointestinal, and genitourinary tracts, in addition to cells on the ocular surface. while adenovirus infections are generally acute and self-limiting in immunocompetent patients, they can be fatal in children and immunocompromised individuals [7] [8] [9] . the most severe adenovirus infections of the ocular surface are associated with hadvs of species d (hadv-d) [10] , the species with the largest number of described viruses. the term "ocular surface" broadly refers to the cornea and the conjunctiva (figure 1 ). the principal function of the cornea is to refract incoming light to the lens, where the light is then focused onto the retina for visual discrimination. the cornea is composed of five distinct layers (from anterior to posterior): the epithelium, bowman's layer, stroma, descemet's membrane, and endothelium [11] . the stroma, sometimes called the corneal perhaps due to the relatively self-limiting nature of most conjunctivitis presentations, in which symptoms usually resolve within two to three weeks of onset, very few studies have investigated conjunctival immune responses to virus infection [17] . clinically, however, ocular hadv infection generally presents as one of three highly contagious syndromes: follicular conjunctivitis, pharyngoconjunctival fever (pcf), or epidemic keratoconjunctivitis (ekc) [18] . follicular conjunctivitis is characterized by bulbar conjunctival injection and chemosis, follicular hyperplasia, preauricular adenopathy, and sometimes conjunctival petechiae or frank subconjunctival hemorrhages. pcf appears similar, however, in addition to the ocular signs, is associated with a systemic, flu-like illness [19, 20] . ekc, which is most commonly caused by hadv-d8, -37, -53, -54, -56, and -64, is a severe, hyperacute, and particularly contagious infection [21, 22] . ekc is characterized by acute membranous keratoconjunctivitis and delayed-onset subepithelial corneal infiltrates (seis) (figure 2 ). perhaps due to the relatively self-limiting nature of most conjunctivitis presentations, in which symptoms usually resolve within two to three weeks of onset, very few studies have investigated conjunctival immune responses to virus infection [17] . clinically, however, ocular hadv infection generally presents as one of three highly contagious syndromes: follicular conjunctivitis, pharyngoconjunctival fever (pcf), or epidemic keratoconjunctivitis (ekc) [18] . follicular conjunctivitis is characterized by bulbar conjunctival injection and chemosis, follicular hyperplasia, preauricular adenopathy, and sometimes conjunctival petechiae or frank subconjunctival hemorrhages. pcf appears similar, however, in addition to the ocular signs, is associated with a systemic, flu-like illness [19, 20] . ekc, which is most commonly caused by hadv-d8, -37, -53, -54, -56, and -64, is a severe, hyperacute, and particularly contagious infection [21, 22] . ekc is characterized by acute membranous keratoconjunctivitis and delayed-onset subepithelial corneal infiltrates (seis) (figure 2) . seis, the hallmark feature of ekc, occur in approximately one-third of all ekc cases and may persist or recur for months to years following infection [23] [24] [25] [26] . seis impair vision by physically blocking the passage of light and by disrupting the arrangement of collagen fibrils and other extracellular matrix components of the meticulously organized and normally transparent corneal stroma [27] [28] [29] [30] [31] [32] . clinically, this manifests as reduced vision, foreign body sensation, and photophobia [33] [34] [35] . based on both experimental and clinical evidence, seis form as a consequence of infiltrating leukocytes, recruited from the corneal limbus to the superficial corneal stroma [31, 32] . sei appearance is delayed by up to three weeks from the onset of infection, a time when active viral replication has ceased [36] , which suggests that long term morbidity associated with infection is immune-mediated rather than a result of virus-associated tissue damage. unfortunately, despite frequent outbreaks of adenoviral conjunctivitis microorganisms 2019, 7, 351 3 of 24 and the substantial economic impacts-due to lost work time and expenses associated with medical visits and diagnostic testing-there are currently no specific antiviral therapies for adenovirus ocular infections. further, due to their apparent immunological origin, seis are unresponsive to direct-acting antivirals. the elucidation of the immunopathogenesis of infection and its relationship with the virus replication cycle is crucial to the potential development of future immunomodulatory therapies. seis, the hallmark feature of ekc, occur in approximately one-third of all ekc cases and may persist or recur for months to years following infection [23] [24] [25] [26] . seis impair vision by physically blocking the passage of light and by disrupting the arrangement of collagen fibrils and other extracellular matrix components of the meticulously organized and normally transparent corneal stroma [27] [28] [29] [30] [31] [32] . clinically, this manifests as reduced vision, foreign body sensation, and photophobia [33] [34] [35] . based on both experimental and clinical evidence, seis form as a consequence of infiltrating leukocytes, recruited from the corneal limbus to the superficial corneal stroma [31, 32] . sei appearance is delayed by up to three weeks from the onset of infection, a time when active viral replication has ceased [36] , which suggests that long term morbidity associated with infection is immune-mediated rather than a result of virus-associated tissue damage. unfortunately, despite frequent outbreaks of adenoviral conjunctivitis and the substantial economic impacts -due to lost work time and expenses associated with medical visits and diagnostic testing -there are currently no specific antiviral therapies for adenovirus ocular infections. further, due to their apparent immunological origin, seis are unresponsive to direct-acting antivirals. the elucidation of the immunopathogenesis of infection and its relationship with the virus replication cycle is crucial to the potential development of future immunomodulatory therapies. dogma maintains that hadvs enter host cells via dynamin-dependent, clathrin-mediated endocytosis before trafficking along microtubules to the nucleus for replication [37, 38] . however, recent work has demonstrated that adenoviruses utilize several entry mechanisms, including macropinocytosis [39] [40] [41] and caveolin-mediated pathways [42] . the specific mechanism of entry appears to depend most on the specific pairing of cell and virus type. in some cell types, viruses may exploit more than one pathway with no apparent preference. furthermore, a predominant pathway may be supplanted by another pathway if the former is blocked. redundancy in both entry and subsequent immune responses may be the rule rather than the exception. furthermore, analyses of viral entry may be complicated by the finding that some host signaling proteins that were initially identified as specific to a particular pathway are in fact shared by disparate pathways. innate immune responses to adenoviruses rely on the detection of pathogen-associated molecular patterns (pamps): distinct ligands present on the external surfaces, and nucleic acids of pathogens (but absent in the host) that feature molecular signatures able to be recognized by pattern recognition receptors (prr) on or in infected host cells [43] [44] [45] [46] . due to the specific distribution of these prrs on the cell surface, in endosomes, and in the cytosol, it is expected that adenoviruses utilizing disparate entry and trafficking mechanisms may stimulate specific and unique subsets of prrs, ultimately resulting in unique immune response signatures. consistent with this hypothesis, it was shown that rapidly trafficking adenoviruses replicate more efficiently [47] , but may not stimulate host cytokine responses as effectively as a virus that enters and traffics more slowly [22] . dogma maintains that hadvs enter host cells via dynamin-dependent, clathrin-mediated endocytosis before trafficking along microtubules to the nucleus for replication [37, 38] . however, recent work has demonstrated that adenoviruses utilize several entry mechanisms, including macropinocytosis [39] [40] [41] and caveolin-mediated pathways [42] . the specific mechanism of entry appears to depend most on the specific pairing of cell and virus type. in some cell types, viruses may exploit more than one pathway with no apparent preference. furthermore, a predominant pathway may be supplanted by another pathway if the former is blocked. redundancy in both entry and subsequent immune responses may be the rule rather than the exception. furthermore, analyses of viral entry may be complicated by the finding that some host signaling proteins that were initially identified as specific to a particular pathway are in fact shared by disparate pathways. innate immune responses to adenoviruses rely on the detection of pathogen-associated molecular patterns (pamps): distinct ligands present on the external surfaces, and nucleic acids of pathogens (but absent in the host) that feature molecular signatures able to be recognized by pattern recognition receptors (prr) on or in infected host cells [43] [44] [45] [46] . due to the specific distribution of these prrs on the cell surface, in endosomes, and in the cytosol, it is expected that adenoviruses utilizing disparate entry and trafficking mechanisms may stimulate specific and unique subsets of prrs, ultimately resulting in unique immune response signatures. consistent with this hypothesis, it was shown that rapidly trafficking adenoviruses replicate more efficiently [47] , but may not stimulate host cytokine responses as effectively as a virus that enters and traffics more slowly [22] . however, such a relationship has yet to be fully defined in the context of ocular surface cells. this review will focus broadly on the mechanisms of adenoviral entry and trafficking, the immune responses to adenovirus infection of the ocular surface, and the possible connection between the two. the hadv genome is encapsidated by an icosahedral protein shell (capsid) composed of three major capsid proteins (hexon, penton base, and fiber) and four minor/cement proteins (iiia, vi, viii, and ix). the hexon protein is the most abundant adenovirus protein, with 240 hexon trimers (720 individual hexon proteins) forming the bulk of the capsid structure. each of the 12 capsid vertices is formed by a ring of five penton base proteins, from which a trimeric fiber protein protrudes, ending in its distal fiber knob (reviewed in [48] ). the infection cycle begins with the binding of the fiber knob to a cell surface receptor. most hadvs, except hadv-b and some members of hadv-d, utilize the coxsackievirus-adenovirus receptor (car), which is expressed in most human tissues [49] [50] [51] . typically, hadv-bs bind cd46, a complement regulatory protein, as the primary receptor [52] . the ekc-associated viruses also bind cd46 and additionally bind ganglioside sialic acids, notably gd1a [50, [53] [54] [55] [56] [57] [58] . receptor binding draws the capsid toward the cell membrane, enabling an interaction between an arginine-glycine-aspartic acid (rgd) motif in the outer loop of each penton base protein and cell surface integrins, which serve as secondary receptors. specifically, for hadv-d37, it has been shown that αvβ1 and αvβ3 are utilized for the infection of corneal epithelial cells [59, 60] . the binding of the rgd motif induces conformational changes in the integrins, which mediate downstream intracellular signaling to promote viral entry into the host cell. it is generally thought that specific fiber knob amino acids and the availability of the necessary cellular receptors and integrins determine the tropism of hadvs for the ocular surface [59, 61] . indeed, we recently illustrated positive selection pressure on one particular amino acid in the fiber knob, specifically a lysine or alanine at residue 240, which is critical for corneal tropism and differentiates ekc adenoviruses from non-ekc viruses [62] . for non-ocular, car-utilizing viruses, expression levels of car control entry and nuclear trafficking efficiency (reviewed in [63] ). however, it is important to note that entry into a specific cell type does not guarantee successful trafficking or viral replication. few studies to date have specifically sought to characterize the entry mechanisms for ekc-associated hadvs in ocular cells. most have focused on the non-ekc-associated, species c, specifically hadv-c2 and -5, as well as species b, hadv-b3, -7,-9, and -35 [63] . in addition, immortalized human cell lines, including hela cells, kb cells (subcloned from hela cells), and a549 cells, have been the preferred cell types for hadv-c2 and -5 entry studies due to their support for rapid and robust viral replication observed in these cells [63] . these studies are frequently cited as support for clathrin-mediated endocytosis as the sole or primary means by which adenoviruses enter cells [10] . further work with hadv-cs has led to the idea that subsequent trafficking to the nucleus occurs along the microtubule network in a dynamin-dependent manner [64] [65] [66] [67] . however, recent experimental evidence supports viral utilization of several entry mechanisms, including clathrin-mediated endocytosis [64] , macropinocytosis [39] , and caveolin-mediated pathways [42] . clathrin-mediated endocytosis is the best elucidated route for viral entry, including for adenovirus [64, [68] [69] [70] [71] . this entry process involves the internalization of virions into a double membrane coated pit with triskelion-shaped clathrin proteins, which collectively interact to form a polyhedral lattice surrounding the endocytosed vesicle [64, 72, 73] . for cornea-tropic adenoviruses, there is a general paucity of data on the entry mechanism. however, in agreement with previous studies, we found that hadv-d37 infection of immortalized human corneal epithelial cells was predominantly clathrin-mediated (figure 3a (i)), with a lesser contribution from macropinocytosis (figure 3a (ii)). factors beyond just the clathrin-coated pit are required for clathrin-mediated endocytosis [39] . for example, dynamin, a 100-kda gtpase, plays a critical role in the fission of newly formed vesicles, without which clathrin-mediated endocytosis does not occur [74] . of the three dynamin isoforms, dynamin 2 is expressed in most cell types. it also functions as a microtubule binding protein [75] and as a negative regulator of microtubule stability [76] . the binding of dynamin 2 to microtubules activates its gtpase function, resulting in endosome migration along the cytoskeletal network [77] . clathrin-mediated endocytosis of hadv-c2 and -5 has been shown to require dynamin activity [37, 64, 78] . however, dynamin is not required for the entry of all adenoviruses. for example, hadv-b3 utilizes dynamin-independent endocytosis for rapid entry into epithelial and hematopoietic cells [79] . adenovirus infection of human corneal epithelial cells is highly restricted [42, 80] , and in vitro tropism of specific hadv types for immortalized human corneal epithelial cells very closely matches the known associations between specific hadv types and clinical infection in ekc [22, 81, 82] . corneal epithelial cell infection by tropic hadvs also occurs via a dynamin 2-independent pathway (manuscript in preparation). further, in fibroblasts derived from human corneal stromal keratocytes-also known as human corneal fibroblasts (hcfs)-dynamin 2 activity had no impact on the cellular entry of hadv-d37 but did influence the delivery of viral dna to cell nuclei. specifically, the knockdown of dynamin 2 resulted in increased microtubule acetylation, closer proximity of microtubule organizing centers (mtocs) to cell nuclei, and increased perinuclear hadv-d37 localization [38] . consistent with others' findings that adenovirus accumulation around the mtoc is a prerequisite to nuclear entry [83] , dyamin 2 knockdown in corneal fibroblasts resulted in greater delivery of adenoviral dna into cell nuclei. in parallel experiments, overexpression of dynamin 2 resulted in the opposite effects, including reduced nuclear entry of viral dna [38] . these data confirm a dichotomous role for dynamin in adenoviral entry, dependent on both cell and virus type. dynamin 2 is expressed in most cell types. it also functions as a microtubule binding protein [75] and as a negative regulator of microtubule stability [76] . the binding of dynamin 2 to microtubules activates its gtpase function, resulting in endosome migration along the cytoskeletal network [77] . clathrin-mediated endocytosis of hadv-c2 and -5 has been shown to require dynamin activity [37, 64, 78] . however, dynamin is not required for the entry of all adenoviruses. for example, hadv-b3 utilizes dynamin-independent endocytosis for rapid entry into epithelial and hematopoietic cells [79] . adenovirus infection of human corneal epithelial cells is highly restricted [42, 80] , and in vitro tropism of specific hadv types for immortalized human corneal epithelial cells very closely matches the known associations between specific hadv types and clinical infection in ekc [22, 81, 82] . corneal epithelial cell infection by tropic hadvs also occurs via a dynamin 2-independent pathway (manuscript in preparation). further, in fibroblasts derived from human corneal stromal keratocytes -also known as human corneal fibroblasts (hcfs) -dynamin 2 activity had no impact on the cellular entry of hadv-d37 but did influence the delivery of viral dna to cell nuclei. specifically, the knockdown of dynamin 2 resulted in increased microtubule acetylation, closer proximity of microtubule organizing centers (mtocs) to cell nuclei, and increased perinuclear hadv-d37 localization [38] . consistent with others' findings that adenovirus accumulation around the mtoc is a prerequisite to nuclear entry [83] , dyamin 2 knockdown in corneal fibroblasts resulted in greater delivery of adenoviral dna into cell nuclei. in parallel experiments, overexpression of dynamin 2 resulted in the opposite effects, including reduced nuclear entry of viral dna [38] . these data confirm a dichotomous role for dynamin in adenoviral entry, dependent on both cell and virus type. corneal keratocytes or tert-immortalized human corneal epithelial cells were infected with moi = 10 of cesium chloride purified hadv-d37 for 1 hour at room temperature. cells were washed with pbs, fixed in a fixative solution (2% paraformaldehyde containing 2.5% glutaraldehyde, 0.1 m cacodylate, and 2.5 mm cacl2) for 1 hour, and collected in 2% agarose. the cell pellet was further fixed for 1.5 hours in 2% aqueous oso4 and dehydrated. after dehydration, the cell pellet was embedded in epon and sectioned into 70-90 nm thin sections. the sections were stained with saturated aqueous uranyl acetate, sato's lead stain, and micrographs were taken on a philips cm-10 electron microscope operating at 80 kv and fitted to a ccd camera. (a) electron micrographs of infected tert-immortalized human corneal epithelial cells show that hadv-d37 (white arrows) can enter via clathrin-mediated endocytosis (i) or macropinocytosis (ii). (b) electron micrographs of infected keratocytes showing caveolae (black arrows) associated with hadv-d37 (white arrows) at the cell membrane (i) and inside a caveosome (ii). (c) immunogold staining for caveolin in uninfected (i) and hadv-d37 infected (ii) keratocytes (white arrows indicate virus). figure 3 . electron micrographs of hadv-d37 entering ocular surface cells. corneal keratocytes or tert-immortalized human corneal epithelial cells were infected with moi = 10 of cesium chloride purified hadv-d37 for 1 hour at room temperature. cells were washed with pbs, fixed in a fixative solution (2% paraformaldehyde containing 2.5% glutaraldehyde, 0.1 m cacodylate, and 2.5 mm cacl 2 ) for 1 hour, and collected in 2% agarose. the cell pellet was further fixed for 1.5 hours in 2% aqueous oso4 and dehydrated. after dehydration, the cell pellet was embedded in epon and sectioned into 70-90 nm thin sections. the sections were stained with saturated aqueous uranyl acetate, sato's lead stain, and micrographs were taken on a philips cm-10 electron microscope operating at 80 kv and fitted to a ccd camera. (a) electron micrographs of infected tert-immortalized human corneal epithelial cells show that hadv-d37 (white arrows) can enter via clathrin-mediated endocytosis (i) or macropinocytosis (ii). (b) electron micrographs of infected keratocytes showing caveolae (black arrows) associated with hadv-d37 (white arrows) at the cell membrane (i) and inside a caveosome (ii). (c) immunogold staining for caveolin in uninfected (i) and hadv-d37 infected (ii) keratocytes (white arrows indicate virus). caveolae-dependent cell entry of adenovirus is controversial. caveolae are flask-or omega-shaped lipid raft invaginations of the plasma membrane, with an average diameter of 50-100 nm [84] [85] [86] . caveloae are abundantly present in many cell types including fibroblasts, cardiomyocytes, and adipocytes, but are not common to all eukaryotic cell membranes [87] . caveolins are the major integral membrane proteins of caveolae and include three types: caveolin-1 and -2 are found in most cell types, while caveolin-3 is present only in myocytes [88, 89] . caveolae exist stably at the plasma membrane, but following internalization, they fuse with other caveolae to form larger structures called caveosomes, or they fuse with endosomes in a rab5-dependent manner [90] . while many molecules are needed to initiate the internalization of caveolae, dynamin 2, and src, pkc activation and recruitment of actin appear to be important [91] [92] [93] [94] [95] [96] . caveolae have been implicated in potential entry pathways for a diverse range of viruses, including coronavirus [97] , hepatitis b [98] , polyomavirus [99] , papillomavirus [100] , simian virus 40 [101] , filoviruses [102] , and human immunodeficiency virus [103, 104] , among others [105] [106] [107] [108] [109] [110] [111] . our work has indicated that hadv-d37 enters human keratocytes in vitro using caveolae [42] , while the non-cornea tropic hadv-c2 fails to infect keratocytes altogether. confocal analysis of infected cells revealed robust colocalization of hadv-d37 with caveolin-1, but not lamp1, the latter of which is a late endosomal marker. a general increase in caveolin-1 in lipid rafts as well as increased src phosphorylation was noted in the infected cells. caveolin-rich endosomal fractions were found to contain higher levels of viral dna compared to fractions rich in lamp1. il-8, a cytokine rapidly induced following hadv-d37 infection, was found to be reduced following caveolin-1 knockdown. electron microscopy (em) of the infected cells found multiple flask-shaped vesicles resembling caveolae and caveosome-like structures both contain hadv-d37 virions (figure 3b ). using immunoelectron microscopy, these virus-containing invaginations were also found to express caveolin-1 (figure 3c) . utilizing a novel mouse model of infection, caveolin-1 deficient mice were found to accumulate virus on the cell membranes of corneal stromal cells and exhibit delayed viral entry compared to wild type mice. further, src phosphorylation and expression of cxcl1 (a murine analog of il-8) were both reduced in infected, caveolin-1 knockout mice compared to control mice. collectively, these data support a caveolin-dependent entry pathway for hadv-d37 in the cornea stromal cells. in contrast, in a549 cells, hadv-d37 colocalized with lamp1, which is consistent with an endosomal trafficking pathway in these particular epithelial cells. macropinocytosis is an endocytic process that begins with interactions between a ligand and its host cell receptors, activating a cascade of intracellular signaling and actin rearrangement which drives the formation of cell membrane ruffles on the host cell surface. these ruffles enclose the cargo, forming a large (>250 nm) vesicle known as a macropinosome near the plasma membrane, which then releases the cargo into the cytosol [112, 113] . macropinocytosis may either be constitutively active, as in dendritic cells, or induced. many families of viruses, including herpesviruses [114, 115] , vaccinia viruses [116, 117] , picornaviruses [118] , and some adenoviruses [39] [40] [41] , are known to exploit macropinocytosis for their entry. while clathrin-mediated endocytosis is the predominant entry pathway for hadv-c2 and -5, as discussed above, these viruses can simultaneously utilize macropinocytosis as an alternative entry mechanism. additionally, if clathrin-mediated endocytosis is blocked, macropinocytosis becomes the primary means of entry [39] . in contrast, hadv-b3 and -35 utilize macropinocytosis as their primary entry pathway. macropinocytosis is also variably dependent on dynamin activity. hadv-b3 entry via macropinocytosis does not require dynamin, whereas macropinocytosis of hadv-b35 requires dynamin for entry into the hela-kyoto clonal derivation, but not into the parental hela-atcc cells or wi-38 cells, a diploid human fibroblast cell line derived from lung tissue [41, 79] . in a tert-immortalized human corneal epithelial cell line, as shown by em, macropinocytosis was also utilized for adenovirus entry (figure 3a(ii) ). macropinocytosis is regulated by p21-activated kinase (pak), a serine/threonine kinase, and pretreatment with the pak inhibitor ipa-3 reduced viral gene expression in a dose-dependent manner. interestingly, the sodium-proton exchange inhibitor 5-(n-ethyl-n-isopropyl) amiloride did not block the endocytic uptake of hadv-d37 (manuscript in preparation). further, clathrin knockdown completely abrogated viral entry, while macropinocytosis blocking agents did not. this strongly suggests that clathrin-mediated endocytosis is the dominant pathway in corneal epithelial cells, with macropinocytosis playing a secondary role. additional studies are needed in order to define the molecular entry mechanisms of hadv-d37 and other ekc-associated adenoviruses into ocular surface cells. following adenoviral entry into the cell, for viral replication to occur, the viral genome must reach the nucleus. because intact adenovirions are too large to enter the nucleus through the nuclear pore complex, they must first uncoat to release their genome [119] . mechanical and chemical forces drive this dynamic, tightly-regulated, and irreversible process (reviewed in [120] [121] [122] [123] [124] ). for viruses utilizing clathrin-mediated endocytosis, such as hadv-c2 and -5, uncoating begins with the interaction between the penton base rgd motifs and cellular integrins. subsequent fiber-shedding exposes protein vi (pvi) and the virion core is endocytosed. further, pvi, a 22 kda cement protein, functions as the primary lytic factor for penetration of the endosomal membrane [125] . in contrast, for caveolin-dependent endocytosis and macropinocytosis, the specific adenovirus uncoating pathway is not well-defined. once in the cytosol, the adenovirus capsid core associates with microtubule-based motors, including dynein and kinesin, and is transported to the nucleus via the mtoc [126] [127] [128] . beyond their role in virion transport, dynein and kinesin also exert mechanical forces on the capsid, completing the uncoating process [128] . finally, the viral core particle binds to the nucleoporin nup214 at the nuclear membrane, leading to kinesin-1 mediated disassembly and enabling the viral genome to enter the nucleus [129] [130] [131] . the observation that adenoviral entry, uncoating, and trafficking may vary based on virus and cell type has interesting potential implications for host cell immune responses. all cells are capable, at least to some degree, of stimulating local innate immune and antiviral responses. as discussed above, this is largely mediated through the detection of conserved pamps on or in invading pathogens by host cell prrs, inducing both divergent and convergent signaling cascades and leading to the production of inflammatory mediators [43] [44] [45] [46] . prrs are known to have specific distributions within each cell, unique to cell type, to facilitate the detection of pathogen components based on their cellular location. for example, toll-like receptors (tlrs) 1, 2, 4, 5, and 6 are expressed on the cell surface, while tlrs 3, 7, 8, and 9 are expressed on intracellular endosomal membranes [132] . other prrs, such as the nucleic acid sensors rig-i, aim2, cgas, and the nlrp3 inflammasome complex, are expressed in the cytosol [133] . many of these prrs have been shown to function as sentries to detect adenovirus infection [134] . it follows that the specific pathway of adenoviral entry and trafficking could expose adenoviral pamps to different host cell prrs, inducing cell-and virus-type specific responses. studies have sought to elucidate the connections between entry, trafficking, and innate immune responses by infected cells [44, [135] [136] [137] . in a549 cells, hadv-c5 was shown to move relatively quickly (within 1 hour of infection) from early endosomes to the cell nucleus. in contrast, hadv-d26 and -b35 remained within late endosomes at 2-6 h post-infection. the persistence of virions in late endosomes in human peripheral blood mononuclear cells (pbmcs) was associated with higher expression of ifnα2, il-1β, il-6, mip-1β, and tnfα. pre-treatment with inhibitors of endosomal acidification reduced expression of these factors, supporting viral accumulation in late endosomes as a strong stimulator of innate immune responses [135] . the same findings were reproduced in vivo. vaccination of rhesus macaques with hadv-d26 and hadv-b35 was associated with higher serum levels of pro-inflammatory cytokines and chemokines than with hadv-c5 [136] . in other studies, it was shown that divergent prr activation in different types of immune cells can lead to a convergent interferon (ifn) response. plasmacytoid dendritic cells, a minor subset of the monocyte population found in blood, recognize adenoviral dna in the late endosome in a tlr9 and myd88-dependent manner, leading to the production of ifnα [43] . in conventional dendritic cells, hepatic kupffer cells, and peritoneal macrophages, all of which are more prevalent than plasmacytoid dendritic cells, recombinant adenoviral vectors utilized a tlr-independent pathway to detect cytosolic viral dna and drive ifnα production [44] . while the cytosolic dna sensor has yet to be defined, it was shown that this pathway requires endosomal escape, signaling via sapk/jnk and irf3 [137] . further work is needed to establish specific connections between viral pamps and host sensors during adenoviral infection of ocular surface cells. while the connection between entry, trafficking, and immune responses has not been specifically addressed in the cell types present on the ocular surface, general immune responses to adenovirus have been studied. since the 1940s, it has been understood that the cornea is an immune privileged site capable of mounting immune responses that both protect against insult and preserve the anatomical integrity and visual function of the eye (reviewed in [138, 139] ). as ekc is the ocular adenovirus-associated disease most associated with long term morbidity and vision loss [19, 140] , it is not surprising that the majority of research over the past 70 years has largely focused on immune responses within the cornea. these studies, discussed below, suggest that different ocular surface cells respond in immunologically distinct ways. primary viral replication in corneal epithelial cells results in punctate and geographic epithelial keratitis [141] [142] [143] [144] . corneal epithelial cells produce a variety of cytokines, including ccl20, egf, il-1α, il-6, il-8, and tgf-β1 [145] [146] [147] [148] [149] , in response to different stimuli, though typically at lower levels relative to other corneal cell types. few studies have examined the cytokine responses of corneal epithelial cells to ocular adenoviruses. one study showed that il-1α secreted by hadv-d37 infected epithelial cells can enhance icam-1 expression on endothelial cells to promote lymphocyte entry into the infected cornea, thereby promoting sei formation [150] . in immortalized corneal epithelial cells, although hadv-d37 was able to enter and replicate, our attempts to profile cytokine induction using cytokine arrays failed to identify any elevation of commonly studied cytokines (unpublished data). this is consistent with similar studies performed on corneal epithelial cells with human alphaherpesvirus 1, in which chemokine induction by infected cells was meager in comparison to that by infected corneal fibroblasts [146] . perhaps the study of monolayer epithelial cell cultures in isolation from other cell types, those that are normally present in vivo, is misleading. it is also possible that use of primary corneal epithelial cell cultures and/or a more sensitive cytokine detection methodology would lead to a different conclusion. since the 1950s, it has become clear that the adenovirus-infected corneal stroma is more than just a bystander or mere target of corneal inflammation, as famously suggested by barrie jones [151] . instead, immune responses to adenovirus infection involve the active participation of stromal resident cells, including keratocytes, which are capable of inducing and orchestrating the secretion of specific immune factors. keratocytes are the major cell population of the stroma (figure 1 ) and play important roles in preserving the clarity and highly ordered structure of the stroma [28, 152] . in vitro, cultured keratocytes resemble fibroblasts and have been shown to produce a broad and robust spectrum of cytokines, including ccl11 [153, 154] , ccl20 [149] , cxcl9 [155, 156] , g-csf [157] , gro-alpha/cxcl1 [158] , ifn-γ [154] , il-1α/β [148, 159] , il-6 [147, 154, 160, 161] , il-8 [145, 146, 153, 154, [162] [163] [164] [165] [166] [167] , il-12 [154] , ip-10 [154, 155] , mcp-1/ccl2, rantes [154, 161, 163, [167] [168] [169] [170] , tgf-β1 [148] , and tnfα [160] . additionally, the aforementioned studies found that keratocytes are often more potent producers of pro-inflammatory cytokines than corneal epithelial cells, indicating that they have a considerable role in the orchestration of corneal immune responses. in vitro, adenoviral infected keratocytes express the pro-inflammatory chemokines il-6, il-8/cxcl8, and mcp-1/ccl2, which promote subsequent immune cell infiltration to the cornea and sei formation [31, 164, 171, 172] . however, during natural infection of the eye, it has not yet been demonstrated if adenovirus is capable of reaching the corneal stroma. work from our laboratory has defined the signaling pathways by which these cytokines are stimulated in keratocytes (figure 4) . the prrs tlr2 and tlr9 were shown to mediate cytokine responses to ekc-associated adenoviruses [172, 173] . tlr2 is expressed on the cell surface and may recognize adenoviruses [174, 175] . tlr9 is expressed in the endosome and classically detects unmethylated cpg regions of viral dna [176] . tlr2 and tlr9 act synergistically and the knock-down of both was required to reduce keratitis in a mouse model [173] . however, pro-inflammatory cytokine production, immune cell recruitment, and keratitis, although reduced, were still noted, indicating that there are likely other, as of yet undefined, prrs responsible for mediating the innate immune responses to adenoviruses in hcfs. animal and human clinical studies have shown that seis form in the superficial stroma just below bowman's layer and including the corneal epithelial basement membrane (figure 1 ), areas which are comprised of a variety of immune cells. acutely, sei are comprised of polymorphonuclear leukocytes, but t lymphocytes and dendritic cells have been identified at later time points [24, 25, 29, 30, 32, 184, 185] . neutrophils are the first and by far the most abundant cell type recruited to the cornea, typically within the first 24 hours post-infection in the mouse model, and are a critical pathogenic event in this infection [31, 32, 172, 181] . il-8 is a well-described and highly potent neutrophil chemoattractant and its expression in the adenovirus infected cornea closely correlates to the rapid infiltration of neutrophils [164, 186] . in a three-dimensional culture system incorporating figure 4 . overview of human adenovirus induced cell signaling and downstream immune responses in human keratocytes, highlighting the centrality of src kinase. following engagement with the primary receptor (cd46 or gd1α) by the viral fiber protein and secondary engagement of the penton base with the αvβ1 or αvβ3 integrins, group d adenoviruses are internalized via src-dependent, caveolin-mediated endocytosis. following uncoating and fiber shedding, virions traffic along the microtubule network, through the microtubule organizing center (mtoc), and the viral genome enters the nucleus for replication. adenovirus stimulates cell surface tlr2 and endosomal tlr9, which synergistically activate myd88. myd88 further activates src, which then mediates multiple downstream kinases, leading to nfκb activation. this culminates in the inhibition of apoptosis and expression of pro-inflammatory genes, including il-8 and mcp-1. a similar signaling pathway in human corneal epithelial cells has yet to be elucidated. following the stimulation of tlr2 and tlr9, myd88 is recruited and initiates a signaling cascade that results in the activation of src kinase [173] , a protein-tyrosine kinase which plays key roles in cell growth, division, migration, and survival pathways (figure 4 ) (reviewed in [177] ). src is also a central signaling molecule in keratocytes in response to adenovirus infection [165] and appears to be phosphorylated within minutes of infection by at least two mechanisms. first, myd88 was shown to physically interact with and phosphorylate src, suggesting that tlr activation may directly activate this kinase [173] . second, virus-integrin binding can also lead to src activation [165] . myd88 downstream signaling via irak1/4 and traf6 can also lead to the activation of the same downstream signaling pathways and pro-inflammatory responses as in src activation [178] . src promotes a cell survival pathway by activating the p85 subunit of the phosphoinositide 3-kinase (pi3k), which then activates akt to drive translocation of nfκb p65 to the nucleus. this pathway inhibits caspase 3/7-dependent apoptosis and thereby promotes cell survival, corresponding with increased viral titers, presumably due to prolongation of cell viability [179] . src also activates focal adhesion kinase (fak) within 15 minutes of infection, leading to pi3k activation. it is not known if pi3k activation is dependent on fak or is directly activated by src [180] . src activation induces the expression of il-8 and mcp-1 by activating the mitogen-activated kinases (mapks), including p38, jnk, and erk1/2 [165] . this results in translocation of the p65 and p50 subunits of nfκb to the nucleus, with subsequent binding to and transcriptional activation of the il-8 promoter [181] . src kinase phosphorylates the p38 mapk kinase, leading to iκb phosphorylation, with convergence to the same pathway as erk1/2, resulting in p65/p50 translocation and il-8 expression within 1 hour post-infection [182] . src also phosphorylates mmk7 shortly after infection, leading to jnk and c-jun activation, and subsequent mcp-1 expression [183] . inhibitors of jnk decrease crel protein binding to mcp-1 promoters and reduce mcp-1 expression at 4 hours post-infection [181] . il-8 and mcp-1 are therefore mediated by two distinct pathways, explaining why mcp-1 expression occurs later in infection compared to il-8 [181, 182] . animal and human clinical studies have shown that seis form in the superficial stroma just below bowman's layer and including the corneal epithelial basement membrane (figure 1 ), areas which are comprised of a variety of immune cells. acutely, sei are comprised of polymorphonuclear leukocytes, but t lymphocytes and dendritic cells have been identified at later time points [24, 25, 29, 30, 32, 184, 185] . neutrophils are the first and by far the most abundant cell type recruited to the cornea, typically within the first 24 hours post-infection in the mouse model, and are a critical pathogenic event in this infection [31, 32, 172, 181] . il-8 is a well-described and highly potent neutrophil chemoattractant and its expression in the adenovirus infected cornea closely correlates to the rapid infiltration of neutrophils [164, 186] . in a three-dimensional culture system incorporating primary corneal fibroblasts and extracellular matrix, we were able to mimic human corneal infection by adenoviruses. il-8 was produced by virus infected stromal cells within the cornea facsimile and was bound to heparin sulfate in the facsimile basement membrane, thus presenting a reservoir of chemoattractant for subsequent leukocyte infiltration [187] . similarly, mcp-1 is a chemoattractant for inflammatory monocytes [188] and is thought to be the chemokine responsible for the delayed migration of monocytes in the adenovirus infected cornea [31] . keratocytes are also known to upregulate adhesion molecules following infection, including icam-1/cd54, which are hypothesized to further promote the extravasation of leukocytes into the cornea [165, 167, 189] . in addition, as mentioned earlier, corneal epithelial cell-derived interleukin-1 alpha (il-1α) promotes the expression of icam-1 and vcam-1 on endothelial cells following infection. this allows for transendothelial leukocyte migration and recruitment to the infected cornea [150] . the combination of these cytokines promotes a rapid infiltration of immune cells to the cornea. studies evaluating the effects of corticosteroid treatment on corneal seis have shown that immune cell infiltrate/sei formation is reduced, however both the duration and magnitude of virus shedding are increased [190] [191] [192] [193] [194] . these data suggest that the immune response also serves to limit viral replication. paradoxically, keratitis does not appear to require active viral replication. in the mouse model, uv-inactivated virus was found to be sufficient to induce keratitis, while heat denatured capsid was unable to induce keratitis. this study showed that hadv-d37 viral dna played only a minor role in innate immune responses, while implicating capsid penton base rgd in the onset of clinical keratitis. in further support of this finding, injection of empty capsid directly into the stroma provoked similar cytokine responses, leukocyte infiltration, and clinical disease as intact virus [172] . unadulterated adenovirus does not replicate in the mouse cornea, further supporting the notion that intact and replicating virus is not required for inflammation [32, 195] . it is not known why seis can persist and/or recur for months to years following infection. their presence does not appear to be mediated by viral antigens because, years after infection, adenoviral particles are not apparent by em [184] and adenoviral antigen is not detected by immunofluorescence [185] . other prrs, viral ligands, and cytokines are likely critical to corneal inflammation after adenoviral infection. for example, microarray analysis of hadv-64 infected keratocytes showed upregulation of genes related to cell growth and differentiation, apoptosis and oncogenesis, cell signaling, transcription, and immune responses, including fra1, g-β, lif, mip-2α, thymosin beta 4, and il-8, among others [165, 196] . microarray analysis and transcriptome sequencing of non-ocular cell types infected with different adenovirus types have defined robust changes in innate immune responses. these included the tlr signaling, cell cycle progression, apoptotic, rna binding and processing, and nfκb signaling pathways [197] [198] [199] [200] . it is not known if other cell types present on the ocular surface respond to adenovirus infection in a similar fashion. healthy, uninfected corneas contain several distinct resident populations of antigen presenting cells (apcs). conventional dendritic cells (dcs) and possibly also plasmacytoid dcs are present at the level of the corneal epithelium/basement membrane [13] . macrophages are also found in the anterior corneal stroma [12, 13, 201] . the limbus and corneal periphery contain mature and immature langerhans cells (figure 1 ) [202] . these resident cells stimulate the recruitment of systemic innate (neutrophil, macrophage, and natural killer cells) and adaptive (t-cells) immune responses to the eye [201, [203] [204] [205] [206] [207] . few studies on the role of ocular surface apcs during adenovirus infection have been performed. we recently demonstrated the involvement of myeloid-derived cells in infection using the macrophage fas-induced apoptosis (mafia) mouse. this mouse expresses a membrane bound suicide protein under the control of the myeloid-lineage specific c-fms promoter. following treatment with the fk506 dimerizer ap20187, myeloid cells, including those in the cornea, then undergo apoptosis [208] . ap20187 treated mice were found to have clinically normal corneas; however, following hadv-d37 infection and in comparison to control and vehicle treated mice, ap20187 treated mice showed reduced immune cell infiltration and reduced myeloperoxidase expression, the latter if which is a correlate for neutrophil infiltration [209] . these data suggest that dcs and macrophages are critical in the development of the immunopathology of keratitis following adenovirus infection. despite strong evidence for the role of corneal stromal cells in the pathogenesis of adenovirus keratitis in the mouse model, an understanding of their participation in human adenovirus keratitis remains incomplete. the conjunctiva (figure 1 ) plays critical roles in protecting the ocular surface from infection. it provides antimicrobial protection and lubrication to the ocular surface via the production of tears and mucins, which collectively form the 3 µm thick tear film. the tear film consists of three layers, an innermost mucin layer, consisting of epithelial cell and conjunctival goblet cell-secreted mucins, an aqueous layer secreted by the lacrimal and accessory lacrimal glands, and an outermost lipid layer, secreted by meibomian glands at the eyelid margin. the aqueous and mucin layers mix in a gradient, with the greatest mucin concentration at the epithelial surface and the greatest aqueous concentration just posterior to the lipid layer. the tear film provides an effective chemical and physical barrier due to mucins, lysozymes, soluble iga, and antimicrobial peptides that can entrap and/or destroy invading pathogens, though these functions have primarily been studied for bacteria [210] . as discussed earlier, the conjunctiva also houses a variety of immune cells that are capable of responding rapidly to insults on the ocular surface. the conjunctiva appears to be more susceptible to adenovirus infection than the cornea-many adenoviruses cause conjunctivitis, while only a limited number cause keratitis. additionally, in ekc, conjunctivitis typically precedes keratitis. the evidence is clear that ocular-tropic adenoviruses can infect and replicate in the cells of the human conjunctiva. both fully infectious adenovirus and viral dna are frequently isolated from conjunctival scrapings during human outbreaks [211] [212] [213] [214] . one report showed the persistence of the virus in the tear film and conjunctiva of a subset of patients for up to a decade following primary infection [215] , although the site of viral persistence was not established. interestingly, studies using a rabbit model of human adenovirus infection have demonstrated that hadv-c5 is able to infect and replicate in the acinar epithelial cells of the lacrimal gland, possibly contributing to its detection in the tear film [216] . hadv-d37 replicates efficiently in the chang c conjunctival cell line in vitro [61] , although it is now known that this cell line was established via hela contamination, based on isoenzyme analysis, hela marker chromosomes, and dna fingerprinting [217] . a few studies have focused on the conjunctival immune responses to adenovirus infection. it was shown by oligonucleotide microarray analysis that the infection of primary human conjunctival epithelial cells with low multiplicities of infection of hadv-c5 resulted in the upregulation of cxcl2, cxcl5, cxcl10, cxcl11, and several interferon induced signaling molecules, including irf7 and stat1. this suggests that the infection of conjunctival epithelial cells with adenovirus initiates signaling that would be expected to drive the recruitment of neutrophils, monocytes/macrophages, t cells, nk cells, and dendritic cells to the site of infection [218] . however, these experiments were only performed with hadv-c5, a respiratory virus, and, as discussed, these results cannot be extrapolated to infection by ekc-associated adenoviruses. nevertheless, in ekc, immune rich conjunctival membranes form in response to adenoviral induced inflammation. these membranes contain macrophages, neutrophils, cd4+ and cd8+ t cells, b cells, langerhans cells, and activated dendritic cells, in proportion to the degree and intensity of the inflammation [219] . conjunctival membranes seen in ekc also contain intact adenoviruses and are therefore infectious, as with other ocular secretions in ekc. in the microarray study mentioned above [218] , in addition to their participation in recruiting leukocytes during inflammation, an anti-microbial peptide (defensin)-like role for cxcl10 and cxcl11 was demonstrated against hadv-b3 and hadv-c5, but not against hadv-d8 or hadv-d64. likewise, the β-defensins were found to inhibit respiratory, but not ocular genotypes. it has been suggested that ocular-tropic adenovirus types have evolved immune evasion strategies to avoid host defensins, which are abundantly expressed on the ocular surface [220] . in adenoviral conjunctivitis, it was previously shown that the number of conjunctival lymphocytes, natural killer (nk) cells, and monocytes increases significantly during the acute phase of the infection [221] . generally, nk cells respond rapidly to viral infection by killing virus infected cells and producing cytokines which promote crosstalk between dendritic cells and t cells, thereby inducing t cell differentiation (reviewed in [222] ). mature cd56 dim nk cells that are capable of producing ifnγ are present in the epithelium of healthy conjunctiva [221] . however, upon infection with hadv-d types, mature nk cells are replaced by cd56 bright , immature nk cells. this correlates with the detection of ccl2, ccl3, ccl4, ccl5, cxcl9, and cxcl10 in the tear film, all of which are potent chemokines for immature nk cells. the upregulation of the inhibitory ligand human leukocyte antigen-e on infected epithelial cells and the overall impairment of nk cell responses upon infection with hadv-d types by reducing the expression of activating ligands on the surface of infected epithelial cells) represent additional mechanisms by which hadv-d types actively promote immune escape. notably, non-species d adenoviruses, such as hadv-c3, -e4, and -c5, were unable to induce these responses. this dampening of antiviral responses likely enables enhanced virus replication in the conjunctiva and, thus, subsequent spread to other ocular surface cells [221] . conjunctival epithelial cells express the membrane-associated mucins muc1, muc4, and muc16, which form the protective glycocalyx [223] . mucins are high molecular weight, heavily glycosylated proteins that have been shown to prevent the penetrance of invading pathogens to the eye. further, ocular surface mucins have complex o-and n-glycans with sialyated cores [224] . as mentioned, sialic acid residues on the gd1α ganglioside are one of the primary receptors for cornea-tropic hadv-ds [53, 56] . therefore, sialic acid containing mucins in the tear film and glycocalyx might act as decoy receptors to reduce subsequent infection of ocular surface cells. while sialic acid-based decoy receptors have been evaluated as a therapy for adenovirus infection [225, 226] , the potential for sialic acids present in the normal tear film to limit natural infection has not been evaluated to our knowledge. a study from our laboratory found that hadv-d37 was able to induce ectodomain release of muc16, resulting in decreased glycocalyx barrier function in cultured corneal and conjunctival epithelial cells. in contrast, hadv-d19, which is not associated with ekc, was unable to cleave muc16. this suggests that specific hadv-d types have evolved in their capacity to penetrate the mucin layer to infect the eye. conjunctival goblet cells are another potential mediator of ocular surface immune responses. goblet cells are important for proper tissue homeostasis through the secretion of the major gel-forming mucin muc5ac, but they also produce cytokines [227, 228] . in response to staphylococcus aureus infection, conjunctival goblet cells produce both mucins and il-1β, the latter of which is dependent on the nlrp3 inflammasome [229] . intriguingly, these cells appear to distinguish between commensal and non-toxigenic bacteria, the latter of which does not induce conjunctival goblet cells to initiate an inflammatory response [230] . paradoxically, it was recently shown that the respiratory pathogen hadv-c5, but not the enteric pathogen hadv-f41, preferentially infects goblet cells in human enteroid cultures, suggesting type-specific tropisms for intestinal goblet cells [231] . however, such type-specific tropism and associated immune responses have yet to be investigated for conjunctival goblet cells. dogma maintains that adenoviruses enter host cells by clathrin-mediated endocytosis, uncoat within endosomes, and rapidly traffic to the nucleus for subsequent replication. however, this paradigm was established based on studies utilizing a limited number of virus types and using tumor-derived cell lines. recent work has demonstrated that entry may not always be this straightforward, with adenoviruses entering via diverse and overlapping mechanisms, depending on cell and virus type pairing. due to the cell-specific and varied pattern of the expression of host prrs, divergent entry mechanisms have the potential to lead to the stimulation of immune responses that are distinguishable based on the specific cell and virus pair. such a connection has not been studied for ocular surface cells. we have demonstrated that ekc-associated hadvs enter and traffic differently in corneal epithelial cells than in stromal keratocytes. furthermore, hadvs are known to induce a more diverse and robust cytokine response in stromal keratocytes than in epithelial cells. however, substantial work remains to support or refute the hypothesis that differential entry of adenoviruses to ocular surface cells dictates subsequent immune responses. first, the entry mechanisms of ekc-associated hadvs would need to be defined across a multitude of cell types, including corneal epithelial cells, keratocytes, conjunctival epithelial cells, conjunctival goblet cells, conjunctival fibroblasts, dendritic cells, macrophages, and other immune cell types that are present on the normal ocular surface. it will be important to determine whether ocular-tropic hadvs can productively replicate in these cells. then, the specific immune responses to infection with hadv will need to be defined, along with the connection between cytokine production and entry pathways. such experiments would greatly increase our knowledge of ocular surface immunology and may permit the development of therapies that manipulate immune responses to infection to improve the clinical outcomes of infection. author contributions: m.r.p., j.c., and j.r. conceived the original idea for the manuscript. m.r.p., a.s., and d.f.p. reviewed the literature and prepared the original draft. m.r.p., a.s., d.f.p., c.g., a.m.i., x.z., j.c., and j.r. all assisted in editing and revisions. funding: this work was funded by national institutes of health grants ey013124, ey021558, ey014104, and 5t32ey007145-20, and a senior scientific investigator award grant (to j.c.) from research to prevent 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antiviral innate immunity vaccination with adenovirus serotypes 35, 26, and 48 elicits higher levels of innate cytokine responses than adenovirus serotype 5 in rhesus monkeys key role of splenic myeloid dcs in the ifn-alphabeta response to adenoviruses in vivo ocular immune privilege ocular immune privilege and transplantation epidemic keratoconjunctivitis: the current situation and recommendations for prevention and treatment chronic adenovirus type 2 keratitis in man adenovirus type 8 infections in the united states. iv. observations on the pathogenesis of lesions in severe eye disease cytopathology of adenovirus keratitis by replica technique adenovirus epithelial keratitis induction of interleukin-8 gene expression is associated with herpes simplex virus infection of human corneal keratocytes but not human corneal epithelial cells il-8 gene expression in cultures of human corneal epithelial cells and keratocytes differences in interleukin-6 gene expression between cultured human 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fibroblasts tlr2 activation in corneal stromal cells by staphylococcus aureus-induced keratitis corneal fibroblasts as sentinel cells and local immune modulators in infectious keratitis proinflammatory cytokines induce rantes and mcp-1 synthesis in human corneal keratocytes but not in corneal epithelial cells. beta-chemokine synthesis in corneal cells involvement of c-c chemokine ligand 2-ccr2 interaction in monocyte-lineage cell recruitment of normal human corneal stroma mcp-1 derived from stromal keratocyte induces corneal infiltration of cd4 + t cells in herpetic stromal keratitis chemokine cxcl1/kc and its receptor cxcr2 are responsible for neutrophil chemotaxis in adenoviral keratitis viral capsid is a pathogen-associated molecular pattern in adenovirus keratitis role of myd88 in adenovirus keratitis adenovirus vector-induced innate inflammatory mediators, mapk signaling, as well as adaptive immune responses are dependent upon both tlr2 and tlr9 in vivo defects in innate immunity render breast cancer initiating cells permissive to oncolytic adenovirus adenovirus efficiently transduces plasmacytoid dendritic cells resulting in tlr9-dependent maturation and ifn-α production src protein-tyrosine kinase structure, mechanism, and small molecule inhibitors corneal cell survival in adenovirus type 19 infection requires phosphoinositide 3-kinase/akt activation activation of focal adhesion kinase in adenovirus-infected human corneal fibroblasts specific nfkappab subunit activation and kinetics of cytokine induction in adenoviral keratitis human adenovirus type 19 infection of corneal cells induces p38 mapk-dependent interleukin-8 expression jnk regulates mcp-1 expression in adenovirus type 19-infected human corneal fibroblasts corneal histology after epidemic keratoconjunctivitis histopathology of adenovirus type 8 keratitis overexpression of il-8 in the cornea induces ulcer formation in the scid mouse novel model of innate immunity in corneal infection ifn-α-driven 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and c-fms conditional ablation (mafia) mice reveal an essential role for resident myeloid cells in lipopolysaccharide/tlr4-induced corneal inflammation resident corneal c-fms + macrophages and dendritic cells mediate early cellular infiltration in adenovirus keratitis complexity of the tear film: importance in homeostasis and dysfunction during disease monitoring of adenovirus from conjunctival scrapings in japan during 2005-2006 outbreaks of epidemic keratoconjunctivitis caused by human adenovirus type 8 in the tibet autonomous region of china in 2016 evaluation of adenovirus amplified detection of immunochromatographic test using tears including conjunctival exudate in patients with adenoviral keratoconjunctivitis. graefe's arch immunofluorescent detection of adenovirus antigen in epidemic keratoconjunctivitis evidence for persistence of adenovirus in the tear film a decade following conjunctivitis adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland survey of atcc stocks of human cell lines for hela contamination adenovirus-directed ocular innate immunity: the role of conjunctival defensin-like chemokines (ip-10, i-tac) and phagocytic human defensin-α cellular and tissue architecture of conjunctival membranes in epidemic keratoconjunctivitis defensins and other antimicrobial peptides at the ocular surface dynamic change in natural killer cell type in the human ocular mucosa in situ as means of immune evasion by adenovirus infection evolutionary struggles between nk cells and viruses ocular surface membrane-associated mucins glycosylation pathways of human corneal and conjunctival epithelial cell mucins sulfated glycosaminoglycans as viral decoy receptors for human adenovirus type 37 decoy receptor interactions as novel drug targets against ekc-causing human adenovirus secreted mucins on the ocular surface goblet cells of the conjunctiva: a review of recent findings staphylococcus aureus activates the nlrp3 inflammasome in human and rat conjunctival goblet cells neither non-toxigenic staphylococcus aureus nor commensal s. epidermidi activates nlrp3 inflammasomes in human conjunctival goblet cells adenovirus infection of human enteroids reveals interferon sensitivity and preferential infection of goblet cells key: cord-324301-bzrh2fni authors: zambon, maria title: influenza, respiratory syncytial virus and sars date: 2005-05-01 journal: medicine doi: 10.1383/medc.33.5.130.64960 sha: doc_id: 324301 cord_uid: bzrh2fni abstract acute lower respiratory tract infections (lrtis) are a major worldwide health problem, particularly in childhood, and are ranked first among the conditions contributing to the global burden of disease. about 30–50% of acute lrtis are viral in origin; of these, influenza and respiratory syncytial virus (rsv) are associated with the greatest disease burden in humans. vaccination against circulating human influenza strains and the use of neuraminidase inhibitor drugs have improved the options for control of influenza, but as yet there are no successful vaccines or antiviral drugs for use against rsv infection. the recent emergence of the sars coronavirus in the human population in 2003, with an ensuing global epidemic affecting more than 8000 individuals with a case fatality of about 10%, underlines the fact that respiratory viral infections of humans may originate in animals, and that many different influenza a viruses also occur naturally in animal reservoirs, representing a constant threat of zoonotic infections of humans and ensuing global pandemics. avian influenza viruses have transmitted directly to humans from domestic poultry on several occasions in the last decade, and the current extensive burden of disease from avian influenza in south east asia provides a real possibility for the emergence of a novel influenza virus pathogenic in humans. acute lower respiratory tract infections (lrtis) are a major worldwide health problem, particularly in childhood. about 30-50% of acute lrtis are viral in origin; of these, influenza and respiratory syncytial virus (rsv) are associated with the greatest disease burden in humans. the emergence of the severe acute respiratory syndrome (sars) coronavirus in 2003, and the ensuing worldwide epidemic, highlights the fact that respiratory viral infections in humans may originate in animals. many different influenza a viruses occur naturally in animal reservoirs, and present a constant threat of zoonotic infections and global pandemics. influenza viruses are small (80-120 nm diameter), contain rna and are enveloped. there are three types -a, b, and c. type a is further classified according to the properties of the surface proteins haemagglutinin and neuraminidase. all a subtypes are found in aquatic birds, which are the natural reservoir ( figure 1) ; only a few subtypes circulate in humans and other mammals. type b and c influenza have only one subtype and are restricted to humans. seasonal illness, epidemics and pandemics -influenza viruses circulating in humans (a h1n1, h3n2, b and c) cause respiratory tract disease. influenza a is generally considered to be clinically more severe than influenza b; influenza c causes only a mild illness confined to the upper respiratory tract. circulation of influenza a and b viruses in humans ( figure 2 ) causes unpredictable seasonal epidemics of disease in temperate climates, with excess population morbidity and mortality, usually occurring between october and march in the northern hemisphere and lasting about 6-8 weeks. 1 in the uk, 5000-10,000 deaths are associated with influenza a and b epidemics every year, and more than 20,000 in severe years. widespread pandemics of severe disease occur less frequently, occurring on at least three occasions in the last century (1918, 1957, 1965) , and have been associated with high mortality. clinical features -influenza a and b illness in humans ranges from subclinical or mild upper respiratory tract symptoms to more severe illness including laryngotracheitis and pneumonia or, less commonly, death from respiratory system failure. the most common presenting symptoms are cough, high temperature, joint pain and general malaise ( figure 3 ). the rapid onset and short incubation period (about 48 hours) are characteristic, though incubation can last up to 4 days. individuals at greatest risk of complications are those with pre-existing cardiac and respiratory disease, the elderly, and those with impaired immune systems ( figure 4 ). the severity of the illness reflects pre-existing host immunity and the prevailing virus strain. virus variability -protective immunity to influenza is conferred by antibodies. the ability of influenza to cause re-infections is related to the genetic mutability of the virus. in every replication round, mutant viruses are generated, some of which have a growth advantage because they can partially evade host immune responses. variants capable of causing epidemics in susceptible populations emerge by a process termed 'antigenic drift'. new influenza a drift variants arise every 2-3 years; influenza b drift variants arise every 4-5 years. influenza as a zoonosis -the segmented nature of the influenza genome allows reassortment of segments when a single host is infected with more than one virus. this occurs regularly in aquatic birds, in which almost all combinations of influenza a virus segments can be detected, but is less common in mammals. it was previously thought that, for a novel subtype of influenza to arise in humans, reassortment of two virus subtypes had to occur in a mammal that could then transmit to humans. the pig was considered a suitable 'intermediate host', because both avian and human viruses, which differ slightly in their surface receptor requirements, can replicate in pigs. however, following transmission of h5n1 directly from birds to humans on 18 occasions figure 5 ), it is evident that cross-species barriers to transmission may be less stringent than was thought. nevertheless, the requirements for adaptation of avian viruses to mammals are poorly understood. cross-species transmission of novel subtypes into susceptible human populations (antigenic shift) are thought to be the source of pandemics of influenza. zoonotic infections involving several different subtypes of influenza have occurred in the last few years, indicating the pandemic potential of influenza viruses circulating in domestic poultry. control -the presence of a large, mobile animal reservoir of influenza a virus suggests that eradication of this agent will be impossible. control strategies focus on limiting the opportunities for cross-species transmission of novel subtypes; for example: • housing domestic poultry in shelters to avoid contact with overflying migratory birds • eliminating/reducing live bird markets • housing aquatic birds and domestic poultry separately • slaughtering domestic flocks infected with highly pathogenic influenza a viruses. these measures may achieve some success in preventing zoonotic transmission of influenza a to humans, but have little impact on its annual cycle. unprecedented levels of h5 circulating in domestic poultry in south east asia present a particularly high risk for emergence of a novel pandemic influenza a strain. immunization -antibodies against haemagglutinin (and, to a limited extent, neuraminidase) can prevent disease caused by the same strain of virus. this is the basis of vaccination for influenza. currently, most vaccines used worldwide are subunit vaccines. however, the high variability of influenza virus means that antibodies to one strain confer only limited protection against drift variants. thus, influenza vaccines are given annually before the influenza season to boost pre-existing immunity, and the composition of the vaccine is updated regularly. in developed countries, the benefit of immunization has led to expansion of age-related vaccination policies. in the uk in 2000, vaccination was introduced for all individuals over 65 years of age, irrespective of pre-existing illness. vaccination rates vary considerably between countries. there is increasing interest in vaccination of children. 2 they induce broader, long-lasting immunity. child vaccination may also help to prevent transmission in the community in general. antiviral drugs -despite the preventive efficacy of vaccination, the need for treatment of severe influenza remains. amantadine (or the related compound rimantadine) was, until recently, the only anti-influenza drug available. it selectively targets a viral protein (m2) and inhibits viral replication, but its use has been limited in the last 30 years, partly because of side-effects (dizziness, confusion) that particularly affect the elderly, and also because drug-resistant mutants arise frequently and can be readily transmitted. neuraminidase inhibitors are a more recently developed, novel class of anti-influenza compounds ( figure 6 ). 3 they act on viral neuraminidase, prevent release of virus particles from infected cells, and are likely to be most efficacious when given early in illness. since 2000, the uk national institute for clinical excellence has recommended that neuraminidase inhibitors may be used for treatment and prophylaxis, with certain restrictions. mutations conferring resistance to neuraminidase inhibitors do not arise very frequently in vivo, and viruses containing them generally lose reproductive fitness and are at a disadvantage in transmission. this suggests that widespread resistance to antiviral drugs is unlikely to emerge and is most likely in treated children, probably because the viral load is highest and therefore the capacity for viral replication in the presence of drugs is greatest. rsv (figure 7) is a negative-sense, non-segmented, enveloped rna virus of 100-300 nm diameter. it is best known as a cause of bronchiolitis in infants, but can cause respiratory tract infection in all age groups. upper respiratory tract infections (urtis) and lrtis range in severity from subclinical infection to pneumonia and death. more than 60% of children have been infected with rsv by their first birthday and more than 80% by 2 years of age; thereafter, individuals are infected approximately every 3 years. rsv infections in adults are probably under-recognized, and the severity of rsv infection in the elderly may be much underestimated as a cause of pneumonia. about 5% of elderly individuals are thought to become infected with rsv every year. transmission of rsv is primarily through large aerosol droplet or secretions, causing widespread nosocomial infection. outbreaks in adult and paediatric facilities are difficult to control. rsv infection in immunocompromised individuals is severe and lifethreatening; mortality may be 50-70% in adult bone marrow transplant recipients, in whom viral shedding may be prolonged. pathophysiology -rsv infects the respiratory epithelium, leading to increased goblet cell production of mucus. dying infected ciliated epithelial cells combine with mucus to form plugs that block the airways; the consequences are most severe in very small babies with narrow airways. this leads to the characteristic signs and symptoms of atelectasis and the clinical syndrome of bronchiolitis. in rsv bronchitis and pneumonia, the peribronchiolar and interstitial infiltrate is characteristically lymphocytic. clinical features -the severity of rsv infection is related to age. in young infants, the illness is seldom asymptomatic and lasts for 1-3 weeks. early signs of infection may include difficulty in feeding, nasal congestion, cough and otitis media compatible with urti. fever is often but not invariably present in rsv infection. abnormal breath sounds, tachypnoea and hypoxaemia suggest lower respiratory tract involvement. bronchiolitis and pneumonia are the two primary manifestations of progression to lrti; they may be difficult to distinguish and can occur simultaneously. the clinical features of bronchiolitis are wheezing and hyperaeration, and these are characteristic of infants with rsv infection. in the usa, rsv is estimated to cause 4500 deaths per year in children under 2 years of age. the risk of hospitalization in otherwise healthy under-2s is 0.5-2%, and 10-20% of children admitted to hospital require mechanical ventilation. these rates are higher in the first 6 months, and may be higher still in children with underlying acquired or congenital cardiopulmonary disease. older children and adults with rsv infection or re-infection usually have a milder or asymptomatic respiratory infection with a lower likelihood of lrti. adults with rsv-associated respiratory tract infections may experience prolonged symptoms. disease in the elderly may be particularly severe; up to 50% develop pneumonia. investigations -rsv infection is often diagnosed on the basis of the clinical features. the certainty of the diagnosis is increased when rsv is known to be circulating in a seasonal epidemic. chest radiography findings are nonspecific and commonly include hyperaeration and peribronchial thickening, with areas of consolidation and interstitial infiltrates in patients with rsv pneumonia. there is a range of respiratory findings in immunocompromised adults, including pleural effusions. laboratory diagnosis depends on detection of viral antigen in respiratory secretions by immunofluorescence, rapid antigen tests or culture of the virus. serological tests are of little help in diagnosis of rsv infection, because they rely on the use of paired acute and convalescent sera. complications -premature and very young infants are more likely to suffer acute apnoeic episodes and require assisted ventilation. bronchiolitis and pneumonia are the major complications of rsv disease in young children. children with congenital heart disease or chronic lung disease, and immunocompromised children and adults, are also at risk of severe disease. pneumonia is the major complication in adults and the elderly. estimates of the incidence range from 10% in nursing homes to 55% in a hospital in-patient population; estimated mortalities in the elderly are 3-5% in the former and 10-20% in the latter. rsv infection has very high mortality (50-70%) in severely immunocompromised individuals. management -supportive care is the mainstay of management of rsv disease in infancy. maintenance of oxygenation, hydration and nutrition is essential in hospitalized patients, and ventilatory support may be necessary in severe cases. a trial of bronchodilators may be beneficial. controlled trials of corticosteroids and vitamin a supplementation have not proved efficacy in infant rsv disease. immunization -there are two subtypes of rsv -a and b. the surface glycoproteins f and g are the major antigens of the virus to which neutralizing antibodies are directed. protective immunity to rsv is complex. antibodies generated during natural infection are not necessarily protective. a high proportion of primary rsv infections occur before 6 months of age, when maternal antibody levels are highest. overall, current (controversial) data suggest that neutralizing antibody to rsv is beneficial. neutralizing antibody titres in human sera correlate inversely with the likelihood of hospitalization as a result of rsv infection, and neutralizing antibody titre correlates with a reduced risk of re-infection. immunoglobulin infusions with high neutralizing titres of antibody to rsv (rsvig, figure 8 ) have been used to treat rsv illness in normal-risk and high-risk infants. in normal infants, there is little evidence to justify rsvig for treatment of rsv infection. however, prophylaxis may be useful in high-risk infants (e.g. those with bronchopulmonary dysplasia). assessment of risk is important, because intravenous rsvig is contraindicated in congenital cyanotic heart disease. recombinant humanized rsv monoclonal antibodies are now available for intramuscular treatment and prophylaxis of rsv. early clinical data suggest that this preparation of passive antibodies may have wider application, with fewer limitations than rsvig; however, it is extremely expensive. few antiviral drugs are available for rsv. ribavirin is a guanosine analogue that has been used widely, though its precise mode of action is uncertain and many clinical studies show conflicting results. several studies have raised further doubts about the clinical effectiveness of ribavirin in infants and children at risk of severe rsv disease, and in ventilated children. in most centres, its use is now restricted to the immunocompromised or severely ill. several rsv vaccines (live attenuated, subunit and recombinant) are undergoing clinical trials. development of safe vaccines has been impeded by poor understanding of the factors governing immune protection in different age groups, 4 and early vaccination attempts in which more severe disease was seen in vaccinees. it is likely that there will be significant progress in future and that the types of vaccines suitable for different age groups may differ. transmission -sars cov spread worldwide within weeks in early 2003. the major route of transmission was respiratory, primarily through droplet, secretions and aerosol formation. about 60% of cases resulted from infection in health-care settings, emphasizing the importance of close contact and exposure to bodily secretions. (the syndrome was noticed through unusual clusters of severe respiratory illness in hospital settings.) peak virus shedding is 7-12 days after the onset of illness (figure 9 ). the virus is found in various body fluids. infectious virus has not been recovered later than 21 days after illness onset. neutralizing antibody is detected from about 10 days post-onset. risk factors for sars cov infection are: • close contact with civet cats/racoon dogs • eating/preparing civet meat • laboratory work with sars cov • contact with a known case of sars. clinical features -presentation is with fever and respiratory illness with cough and shortness of breath, progressing to acute respiratory distress syndrome and death in 10% of cases. fatalities increase significantly over the age of 40 years; mortality is up to 40% in the over-50s. onset of illness is 2-10 days post-infection, with a mean of about 5-6 days. about 60% of patients suffer later gastrointestinal symptoms of diarrhoea and vomiting. management and control -specific control measures have not yet been developed, though work is in progress on antiviral drugs and suitable vaccines. during the 2003 epidemic, various nonspecific therapeutic measures were used with variable success: • corticosteroids • antimicrobials to prevent secondary bacterial infection • positive-pressure ventilatory support • anti-inflammatory drugs • infusion of antisera from convalescent patients. the epidemic was controlled mainly through public health measures such as contact-tracing and quarantining. this policy was effective because there is little evidence of transmission before symptom onset, so infected individuals are easily identified.  • consider for prophylaxis in infants < 2 years receiving oxygen therapy for bronchopulmonary dysplasia • infants born < 32 weeks with bronchopulmonary dysplasia are likely to benefit from 6-12 months' prophylaxis • infants born > 32 weeks with bronchopulmonary dysplasia may not benefit • should not be used in cyanotic congenital heart disease • not evaluated in paediatric or adult immunocompromised patients • main emphasis in nosocomial outbreaks should be infection control; efficacy is improved in such settings • initiate treatment before onset of respiratory syncytial virus season • defer live virus vaccines (e.g. mmr) until last dose the contribution of influenza to acute respiratory infections, hospital admissions and deaths in winter the japanese experience with vaccinating school children against influenza neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir age related differences in humoral immune response to respiratory syncytial virus infection in adults confirmation of a novel corona virus as the primary cause of severe acute respiratory syndrome key: cord-332533-iqe6sdq2 authors: grant, william b.; lahore, henry; mcdonnell, sharon l.; baggerly, carole a.; french, christine b.; aliano, jennifer l.; bhattoa, harjit p. title: evidence that vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths date: 2020-04-02 journal: nutrients doi: 10.3390/nu12040988 sha: doc_id: 332533 cord_uid: iqe6sdq2 the world is in the grip of the covid-19 pandemic. public health measures that can reduce the risk of infection and death in addition to quarantines are desperately needed. this article reviews the roles of vitamin d in reducing the risk of respiratory tract infections, knowledge about the epidemiology of influenza and covid-19, and how vitamin d supplementation might be a useful measure to reduce risk. through several mechanisms, vitamin d can reduce risk of infections. those mechanisms include inducing cathelicidins and defensins that can lower viral replication rates and reducing concentrations of pro-inflammatory cytokines that produce the inflammation that injures the lining of the lungs, leading to pneumonia, as well as increasing concentrations of anti-inflammatory cytokines. several observational studies and clinical trials reported that vitamin d supplementation reduced the risk of influenza, whereas others did not. evidence supporting the role of vitamin d in reducing risk of covid-19 includes that the outbreak occurred in winter, a time when 25-hydroxyvitamin d (25(oh)d) concentrations are lowest; that the number of cases in the southern hemisphere near the end of summer are low; that vitamin d deficiency has been found to contribute to acute respiratory distress syndrome; and that case-fatality rates increase with age and with chronic disease comorbidity, both of which are associated with lower 25(oh)d concentration. to reduce the risk of infection, it is recommended that people at risk of influenza and/or covid-19 consider taking 10,000 iu/d of vitamin d(3) for a few weeks to rapidly raise 25(oh)d concentrations, followed by 5000 iu/d. the goal should be to raise 25(oh)d concentrations above 40–60 ng/ml (100–150 nmol/l). for treatment of people who become infected with covid-19, higher vitamin d(3) doses might be useful. randomized controlled trials and large population studies should be conducted to evaluate these recommendations. the world is now experiencing its third major epidemic of coronavirus (cov) infections. a new cov infection epidemic began in wuhan, hubei, china, in late 2019, originally called 2019-ncov [1] the general metabolism and actions of vitamin d are well known [7] . vitamin d 3 is produced in the skin through the action of uvb radiation reaching 7-dehydrocholesterol in the skin, followed by a thermal reaction. that vitamin d 3 or oral vitamin d is converted to 25(oh)d in the liver and then to the hormonal metabolite, 1,25(oh) 2 d (calcitriol), in the kidneys or other organs as needed. most of vitamin d's effect arises from calcitriol entering the nuclear vitamin d receptor, a dna binding protein that interacts directly with regulatory sequences near target genes and that recruits chromatin active complexes that participate genetically and epigenetically in modifying transcriptional output [8] . a well-known function of calcitriol is to help regulate serum calcium concentrations, which it does in a feedback loop with parathyroid hormone (pth), which itself has many important functions in the body [7] . several reviews consider the ways in which vitamin d reduces the risk of viral infections [9] [10] [11] [12] [13] [14] [15] [16] [17] . vitamin d has many mechanisms by which it reduces the risk of microbial infection and death. a recent review regarding the role of vitamin d in reducing the risk of the common cold grouped those mechanisms into three categories: physical barrier, cellular natural immunity, and adaptive immunity [16] . vitamin d helps maintain tight junctions, gap junctions, and adherens junctions (e.g., by e-cadherin) [18] . several articles discussed how viruses disturb junction integrity, increasing infection by the virus and other microorganisms [19] [20] [21] . vitamin d enhances cellular innate immunity partly through the induction of antimicrobial peptides, including human cathelicidin, ll-37, by 1,25-dihdroxyvitamin d [22, 23] , and defensins [24] . cathelicidins exhibit direct antimicrobial activities against a spectrum of microbes, including gram-positive and gram-negative bacteria, enveloped and nonenveloped viruses, and fungi [25] . those host-derived peptides kill the invading pathogens by perturbing their cell membranes and can neutralize the biological activities of endotoxins [26] . they have many more important functions, as described therein. in a mouse model, ll-37 reduced influenza a virus replication [27] . in another laboratory study, 1,25(oh) 2 d reduced the replication of rotavirus both in vitro and in vivo by another process [28] . a clinical trial reported that supplementation with 4000 iu/d of vitamin d decreased dengue virus infection [29] . vitamin d also enhances cellular immunity, in part by reducing the cytokine storm induced by the innate immune system. the innate immune system generates both pro-inflammatory and anti-inflammatory cytokines in response to viral and bacterial infections, as observed in covid-19 patients [30] . vitamin d can reduce the production of pro-inflammatory th1 cytokines, such as tumor necrosis factor α and interferon γ [31] . administering vitamin d reduces the expression of pro-inflammatory cytokines and increases the expression of anti-inflammatory cytokines by macrophages ( [17] and references therein). vitamin d is a modulator of adaptive immunity [16, 32] ; 1,25(oh) 2 d 3 suppresses responses mediated by the t helper cell type 1 (th1), by primarily repressing production of inflammatory cytokines il-2 and interferon gamma (infγ) [33] . additionally, 1,25(oh) 2 d 3 promotes cytokine production by the t helper type 2 (th2) cells, which helps enhance the indirect suppression of th1 cells by complementing this with actions mediated by a multitude of cell types [34] . furthermore, 1,25(oh) 2 d 3 promotes induction of the t regulatory cells, thereby inhibiting inflammatory processes [35] . serum 25(oh)d concentrations tend to decrease with age [36] , which may be important for covid-19 because case-fatality rates (cfrs) increase with age [37] . reasons include less time spent in the sun and reduced production of vitamin d as a result of lower levels of 7-dehydrocholesterol in the skin [38] . in addition, some pharmaceutical drugs reduce serum 25(oh)d concentrations by activating the pregnane-x receptor [39] . such drugs include antiepileptics, antineoplastics, antibiotics, anti-inflammatory agents, antihypertensives, antiretrovirals, endocrine drugs, and some herbal medicines. pharmaceutical drug use typically increases with age. vitamin d supplementation also enhances the expression of genes related to antioxidation (glutathione reductase and glutamate-cysteine ligase modifier subunit) [40] . the increased glutathione production spares the use of ascorbic acid (vitamin c), which has antimicrobial activities [41, 42] , and has been proposed to prevent and treat covid-19 [43] . moreover, a former director of the center for disease control and prevention, dr. tom frieden, proposed using vitamin d to combat the covid-19 pandemic on 23 march 2020 (https://www.foxnews.com/opinion/former-cdc-chief-tomfrieden-coronavirus-risk-may-be-reduced-with-vitamin-d). influenza virus affects the respiratory tract by direct viral infection or by damage to the immune system response. the proximate cause of death is usually from the ensuing pneumonia. patients who develop pneumonia are more likely to be < 5 years old, > 65 years old, white, and nursing home residents, to have chronic lung or heart disease and a history of smoking, and to be immunocompromised [44] . seasonal influenza infections generally peak in winter [45] . cannell et al. hypothesized that the winter peak was due in part to the conjunction with the season when solar uvb doses, and thus 25(oh)d concentrations, are lowest in most mid-and high-latitude countries [46] , extended in [47] . mean serum 25(oh)d concentrations in north and central regions of the united states are near 21 ng/ml in winter and 28 ng/ml in summer, whereas in the south region, they are near 24 ng/ml in winter and 28 ng/ml in summer [48] . in addition, the winter peak of influenza also coincides with weather conditions of low temperature and relative humidity that allow the influenza virus to survive longer outside the body than under warmer conditions [49] [50] [51] . ecological studies suggest that raising 25(oh)d concentrations through vitamin d supplementation in winter would reduce the risk of developing influenza. table 1 presents results from randomized controlled trials (rcts) investigating how vitamin d supplementation affects risk of influenza. the rcts included confirmed that the respiratory tract infection was indeed derived from influenza. only two rcts reported beneficial effects: one among schoolchildren in japan [52] , the other among infants in china [53] . an rct in japan that reported no beneficial effect did not measure baseline 25(oh)d concentration [54] and included many participants who had been vaccinated against influenza (m. urashima; private communication). the two most recent rcts included participants with above average mean baseline 25(oh)d concentrations [55, 56] . a comprehensive review of the role of vitamin d and influenza was published in 2018 [15] . it concluded that the evidence of vitamin d's effects on the immune system suggest that vitamin d should reduce the risk of influenza, but that more studies are required to evaluate that possibility. large population studies would also be useful, in which vitamin d supplementation is also related to changes in serum 25(oh)d concentration. note: 95% confidence interval (95% ci); day (d); hazard ratio (hr); inflammatory bowel disease (ibd); months (mos); not available (n/a); relative risk (rr); upper respiratory tract infection (urti); week (wk); years (yrs). an observational study conducted in connecticut on 198 healthy adults in the fall and winter of 2009-2010 examined the relationship between serum 25(oh)d concentration and incidence of acute rtis (artis) [57] . only 17% of people who maintained 25(oh)d >38 ng/ml throughout the study developed artis, whereas 45% of those with 25(oh)d < 38 ng/ml did. concentrations of 38 ng/ml or more were associated with a significant (p < 0.0001) twofold reduction in risk of developing artis and with a marked reduction in the percentage of days ill. eight influenza-like illnesses (ilis) occurred, seven of which were the 2009 h1n1 influenza. the first step in developing a hypothesis is to outline the epidemiological and clinical findings regarding the disease of interest and their relationship with 25(oh)d concentrations. from the recent journal literature, it is known that covid-19 infection is associated with the increased production of pro-inflammatory cytokines [58] , c-reactive protein [30] , increased risk of pneumonia [58] , sepsis [59] , acute respiratory distress syndrome [59] , and heart failure [59] . cfrs in china were 6%-10% for those with cardiovascular disease, chronic respiratory tract disease, diabetes, and hypertension [37] . two regions hard hit by covid-19 are regions of high air pollution in china [60] and northern italy [61] . the possible roles of vitamin d for the clinical and epidemiological characteristics of diseases associated with the increased risk of covid-19 cfr are given in table 2 . most of the beneficial effects of vitamin d given in table 2 are from observational studies of disease incidence or prevalence with respect to serum 25(oh)d concentrations. rcts comparing outcomes for participants treated or given a placebo are preferred to establish causality related to health outcomes. however, most vitamin d rcts have not reported that vitamin d supplementation reduced the risk of disease [62, 63] . reasons for the lack of agreement between observational studies and rcts seems to be due to several factors, including enrolling participants with relatively high 25(oh)d concentrations and using low vitamin d doses and not measuring baseline and achieved 25(oh)d concentrations. previous studies proposed that rcts of nutrients such as vitamin d be based on nutrient status, such as 25(oh)d concentration, seeking to enroll participants with low values, supplementing them with enough agent to raise the concentration to values associated with good health, and measuring achieved concentrations as well as cofactors such as vitamin c, omega-3 fatty acids, and magnesium [64, 65] ,. two recently completed rcts reported significantly reduced incidence in the secondary analyses for cancer [66] and diabetes mellitus [67] . table 2 . how vitamin d is related to the clinical and epidemiological findings for incidence and case-fatality rates. clinical severe cases associated with pneumonia inverse correlation for cap [68, 69] increased production of pro-inflammatory cytokines such as il-6 inverse correlation [70, 71] increased crp inverse correlation [72, 73] increased risk of sepsis inverse correlation [74, 75] risk of ards inverse correlation [76, 77] risk of heart failure inverse correlation [78, 79] risk of diabetes mellitus inverse correlation [67, 80] epidemiological began in december 2019 in china, spread mainly to northern midlatitude countries low 25(oh)d values in winter [48, 81] males have higher incidence and much higher cfrs than females smoking reduces 25(oh)d [82] cfr increases with age chronic disease rates increase with age; vitamin d plays a role in reducing risk of chronic diseases [83] higher cfr for diabetics diabetics may have lower 25(oh)d [84] higher cfr for diabetics lower 25(oh)d associated with increased risk of incidence [85] higher cfr for hypertension lower 25(oh)d may be associated with increased risk of incidence [86] higher cfr for cardiovascular disease lower 25(oh)d associated with increased risk of incidence and death [87] higher cfr for chronic respiratory disease for copd patients, 25(oh)d inversely correlated with risk, severity, and exacerbation [88] found at higher rates in regions with elevated air pollution air pollution associated with lower 25(oh)d concentrations [89] note: 25-hydroxyvitamin d ((25(oh)d); acute respiratory distress syndrome (ards); community-acquired pneumonia (cap); case-fatality rate (cfr); interleukin 6 (il-6); chronic obstructive pulmonary disease (copd); c-reactive protein (crp); vitamin d deficiency (vdd). table 3 lists some findings for vitamin d supplementation in reducing the clinical effects of covid-19 infection found from treating other diseases. treatment of cap with vitamin d did not significantly result in complete resolution. baseline 25(oh)d was 20 ng/ml. achieved 25(oh)d in the treatment arm was 40 ng/ml. [90] increased production of pro-inflammatory cytokines such as il-6 reduces concentration of il-6 [11] increased crp reduces crp in diabetic patients [91] increased risk of sepsis no reduction in mortality rate found for adults with sepsis supplemented with vitamin d. most trials included participants with 25(oh)d <20 ng/ml; vitamin d 3 doses between 250 and 600 thousand iu. [92] risk of ards vitamin d deficiency contributes to development of ards [77, 93] acute respiratory distress syndrome (ards); community-acquired pneumonia (cap); case-fatality rate (cfr); interleukin 6 (il-6); chronic obstructive pulmonary disease (copd); c-reactive protein (crp); vitamin d deficiency (vdd). a possible reason for the monotonic increase in cfr with increasing age could be that the presence of chronic diseases increases with age. for example, the global prevalence of diabetes mellitus increases from about 1% below the age of 20 years, to~10% at 45 years and to 19% at 65 years, decreasing to 14% by 95 years [94] . invasive lung cancer incidence rates for females in the united states in 2015 increased from 1.1/100,000 for those aged 30-34 years, to 51.0/100,000 for those aged 50-54 years, 204.1/100,000 for those aged 65-79 years, and 347.3 for those aged 75-79 years [95] . several studies report that people with chronic diseases have lower 25(oh)d concentrations than healthy people. a study in italy reported that male chronic obstructive pulmonary disease patients had mean 25(oh)d concentrations of 16 (95% ci, 13-18) ng/ml, whereas female patients had concentrations of 13 (95% ci, 11-15) ng/ml [96] . a study in south korea reported that community-acquired pneumonia (cap) patients had a mean 25(oh)d concentration at admission of 14 ± 8 ng/ml [97] . a study in iran reported that hypertensive patients had lower 25(oh)d concentrations than control subjects: males, 13 ± 11 vs. 21 ± 11 ng/ml; females, 13 ± 10 vs. 20 ± 11 ng/ml [98] . another factor that affects immune response with age is reduced 1,25-dihydroxyvitamin d (1,25(oh) 2 d, or calcitriol), the active vitamin d metabolite, with increased age. parathyroid hormone (pth) concentration increases with age. a u.s. study was based on 312,962 paired serum pth and 25(oh)d concentration measurements from july 2010 to june 2011. for participants with 20-ng/ml 25(oh)d concentration, pth increased from 27 pg/ml for those <20 years to 54 pg/ml for those >60 years [99] . serum calcitriol concentrations are inversely related to pth concentrations. in a study conducted in norway on patients with a mean age of 50 (sd, 21) years, calcitriol decreased from 140 pmol/l for those aged 20-39 years to 98 pmol/l for those >80 years despite an increase in serum 25(oh)d from 24 ng/ml for those 20-39 years to 27 ng/ml for those >80 years [100] . the seasonality of many viral infections is associated with low 25(oh)d concentrations, as a result of low uvb doses owing to the winter in temperate climates and the rainy season in tropical climates-such as respiratory syncytial virus (rsv) infection [101, 102] ,. this is the case for influenza [45, 46] , and sars-cov [103] . however, mers showed a peak in the april-june quarter [104] , probably affected by both hajj pilgrims gathering and the fact that 25(oh)d concentrations show little seasonal variation in the middle east [105] . in the tropics, seasonality is related more to rainy periods (low uvb doses), for example, for influenza [106] . considerable indirect evidence is inferred from effects found for other enveloped viruses. table 4 presents the findings from various studies. table 4 . findings regarding the associations and effects of vitamin d on enveloped viral infections. dengue vitamin d mechanisms discussed [107] dengue inverse association between 25(oh)d concentration and progression of disease state [108] dengue vitamin d supplementation trial with 1000 and 4000 iu/d. 4000 iu/d resulted in higher resistance to denv-2 infection. mddcs from those supplemented with 4000 iu/d showed decreased mrna expression of tlr3, 7, and 9; downregulation of il-12/il-8 production; and increased il-10 secretion in response to denv-2 infection [29] hepatitis c 1,25-hydroxyvitamin-d3-24-hydroxylase, encoded by cyp24a1 gene, is a key enzyme that neutralizes 1,25(oh) 2 d. this study found that alleles of cyp24a1 had different effects on risk of chronic hepatitis c infection. [109] chb 25(oh)d concentrations were lower in chb patients than that of healthy controls and inversely correlated with hbv viral loads [110] kshv found that cathelicidin significantly reduced ksvh by disrupting the viral envelope. [111] hiv-1 review of 29 clinical studies of vitamin d supplementation showed there was a decrease in inflammation. in 3 of 7 studies, cd4+ t cell count increased, but effect on viral load was inconclusive since most patients were on cart. [112] in a lung epithelial cell study, calcitriol treatment prior to and post infection with h9n2 influenza significantly decreased expression of the influenza m gene, il-6, and ifn-β in a549 cells, but did not affect virus replication. [113] rsv demonstrated that the human cathelicidin ll-37 has effective antiviral activity against rsv in vitro and prevented virus-induced cell death in epithelial cultures, [114] rsv performed a laboratory study that identified the mechanism by which vitamin d reduced risk of rsv. [28] rsv found that the t-allele of the vitamin d receptor has a lower prevalence in african populations and runs parallel to the lower incidence of rsv-associated severe alri in african children, 1 year. [115] rotaviral diarrhea found serum 25(oh)d <20 ng/ml associated with an odds ratio of 6.3 (95% ci, 3.6 to 10.9) for rotaviral diarrhea [116] note: acute respiratory tract infection (alri); combination antiretroviral therapy (cart); chronic hepatitis b (chb); dengue virus-2 (denv-2). human immunodeficiency virus 1 (hiv-1); kaposi's sarcoma-associated herpesvirus (kshv); monocyte-derived dendritic cells (mddcs); respiratory syncytial virus (rsv). one way that covs injure the lung epithelial cells and facilitate pneumonia is through increased production of th1-type cytokines as part of the innate immune response to viral infections, giving rise to the cytokine storm. a laboratory cell study reported that interferon γ is responsible for acute lung injury during the late phase of the sars-cov pathology [117] . pro-inflammatory cytokine storms from cov infections have resulted in the most severe cases for sars-cov [118] and mers-cov [119] . however, covid-19 infection also initiated increased secretion of the th2 cytokines (e.g., interleukins 4 and 10) that suppress inflammation, which differs from sars-cov infection [30] . an example of the role of vitamin d in reducing the risk of death from pandemic respiratory tract infections is found in a study of cfrs resulting from the 1918-1919 influenza pandemic in the united states [120] . the u.s. public health service conducted door-to-door surveys of 12 communities from new haven, connecticut, to san francisco, california, to ascertain incidence and cfrs. the canvasses were made as soon as possible after the autumn 1918 wave of the epidemic subsided in each locality. a total of 146,203 people, 42,920 cases, and 730 deaths were found. as shown in their table 25 , fatality rates averaged 1.70 per 100 influenza cases but averaged 25.5 per 100 cases of pneumonia. the percentage of influenza complicated by pneumonia was 6.8%. the pneumonia cfr (excluding charles county, md, because of inconsistencies in recording cause of death) was 28.8 per 100 for whites and 39.8 per 100 for "coloreds". as shown in table 23 , "coloreds" in the southeastern states had between a 27% and 80% higher rate of pneumonia compared to whites. as discussed in an ecological study using those cfr data, communities in the southwest had lower cfr than those in the northeast because of higher summertime and wintertime solar uvb doses [121] . previous work suggested that higher uvb doses were associated with higher 25(oh)d concentrations, leading to reductions in the cytokine storm and the killing of bacteria and viruses that participate in pneumonia. african americans had much higher mortality rates than white americans for the period 1900-1948 [122] . the reasons cfrs were higher for "coloreds" than whites may include that they have higher rates of chronic diseases, are more likely to live in regions impacted by air pollution, and that with darker skin pigmentation, blacks have lower 25(oh)d concentrations. a clinical trial involving postmenopausal women living on long island, ny with mean baseline 25(oh)d concentration 19 ± 8 ng/ml found that supplementation with 2000 iu/d resulted in significantly fewer upper respiratory tract infections, including influenza, than a placebo or supplementation with 800 iu/d [123] . see, also, references in [11] . an analysis of serum 25(oh)d concentrations by race for 2001-2004 indicated mean 25(oh)d concentrations for people over 40 years: non-hispanic whites,~25-26 ng/ml; non-hispanic blacks, 14-17 ng/ml; mexican-americans, 18-22 ng/ml [124] . a reason proposed for the higher mortality rates in some communities during the 1918-1919 influenza pandemic was that they were near to coal-fired electricity generating plants [125] . recent studies have confirmed that air pollution, from combustion sources, increases the risk of influenza [126, 127] . the highest concentration of these plants is in the northeast, where solar uvb doses are lowest. [93] . in a follow-on pilot trial involving 30 mechanically ventilated critically ill patients, 500,000 iu of vitamin d 3 supplementation significantly increased hemoglobin concentrations and lowered hepcidin concentrations, improving iron metabolism and the blood's ability to transport oxygen [128] . hospitals are a source of rtis for both patients and medical personnel. for example, during the sars-cov epidemic, a woman returned to toronto from hong kong with sars-cov in 2003 and went to a hospital. the disease was transmitted to other people, leading to an outbreak among 257 people in several greater toronto area hospitals [129] . during the 2014-2015 influenza season, 36% of health care workers in a german hospital developed influenza infection [130] . working in a hospital dealing with covid-19 patients is associated with increased risk of covid-19 infection. for example, 40 of 138 hospitalized covid-19 patients in wuhan in the zhongnan hospital from 1 to 28 january were medical staff, and 17 more were infected while in the hospital [58] . it was announced on february 14, 2020, that more than 1700 chinese health workers were infected by covid-19 and six had died (https://www.huffpost.com/entry/chinese-health-workers-infected-by virus_n_5e46a0fec5b64d860fc97c1b). vitamin d supplementation to raise serum 25(oh)d concentrations can help reduce hospitalassociated infections [131] . concentrations of at least 40-50 ng/ml (100-125 nmol/l) are indicated on the basis of observational studies [132, 133] . during the covid-19 epidemic, all people in the hospital, including patients and staff, should take vitamin d supplements to raise 25(oh)d concentrations as an important step in preventing infection and spread. trials on that hypothesis would be worth conducting. the data reviewed here supports the role of higher 25(oh)d concentrations in reducing risk of infection and death from artis, including those from influenza, cov, and pneumonia. the peak season for artis is generally when 25(oh)d concentrations are lowest. thus, vitamin d 3 supplementation should be started or increased several months before winter to raise 25(oh)d concentrations to the range necessary to prevent artis. studies reviewed here generally reported that 25(oh)d concentrations of 20-30 ng/ml reduced the risk of artis [134] . one reason for that result may be that the studies included few participants with higher 25(oh)d concentrations. however, one observational study reported that 38 ng/ml was the appropriate concentration to reduce the risk of cap [57] . although the degree of protection generally increases as 25(oh)d concentration increases, the optimal range appears to be in the range of 40-60 ng/ml (100-150 nmol/l). to achieve those levels, approximately half the population could take at least 2000-5000 iu/d of vitamin d 3 [135] . various loading doses have been studied for achieving a 25(oh)d concentration of 30 ng/ml. for example, one study used a weekly or fortnightly dose totaling 100,000-200,000 iu over 8 weeks (1800 or 3600 iu/d) [136] . however, to achieve 40-60 ng/ml would take higher loading doses. a trial involving canadian breast cancer patients with bone metastases treated with bisphosphonates but without comorbid conditions reported that doses of 10,000 iu/d of vitamin d 3 over a four-month period showed no adverse effects, but did unmask two cases of primary hyperparathyroidism [137] . a study involving 33 participants, including seven taking 4000 iu/d of vitamin d 3 and six who took 10,000 iu/d of vitamin d 3 for 8 weeks, reported that 25(oh)d concentrations increased from 20 ± 6 to 39 ± 9 for 4000 iu/d and from 19 ± 4 to 67 ± 3 for 10,000 iu/d and improved gut microbiota with no adverse effects [138] . thus, from the literature, it is reasonable to suggest taking 10,000 iu/d for a month, which is effective in rapidly increasing circulating levels of 25(oh)d into the preferred range of 40-60 ng/ml. to maintain that level after that first month, the dose can be decreased to 5000 iu/d [135, 139, 140] . when high doses of vitamin d are taken, calcium supplementation should not be high to reduce risk of hypercalcemia. a recent review suggested using vitamin d loading doses of 200,000-300,000 iu in 50,000-iu capsules to reduce the risk and severity of covid-19 [43] . the efficacy and safety of high-dose vitamin d supplementation has been demonstrated in a psychiatric hospital in cincinnati, ohio [141] . the age range was from 18 to 90 years. half of the patients were black, and nearly half were white. all patients entering since 2011 were offered supplementation of 5000 or 10,000 iu/d vitamin d 3 . for 36 patients who received 5000 iu/d for 12 months or longer, mean serum 25(oh)d concentration rose from 24 to 68 ng/ml, whereas for the 78 patients who received 10,000 iu/d, mean concentrations increased from 25 to 96 ng/ml. no cases of vitamin d-induced hypercalcemia were reported. this article includes a brief review of other high-dose vitamin d studies, including the fact that vitamin d doses of 60,000-600,000 iu/d were found to treat and control such diseases as asthma, rheumatoid arthritis, rickets, and tuberculosis in the 1930s and 1940s. those doses are much higher than the 10,000-25,000 iu/d of vitamin d 3 that can be made from solar uvb exposure [142] . however, after reports of hypercalcemia associated with use of supra-physiological doses of vitamin d surfaced, e.g., [143] , high-dose vitamin d supplementation fell out of favor. a recent article on a high-dose vitamin d supplementation trial in new zealand involving 5110 participants reported that, over a median of 3.3 years, monthly supplementation with 100,000 iu of vitamin d 3 did not affect the incidence rate of kidney stone events or hypercalcemia [144] . unfortunately, most countries do not have guidelines supporting vitamin d supplementation doses and desirable serum 25(oh)d concentrations that would deal with wintertime rtis. guidelines for many countries consider 20 ng/ml (50 nmol/l) adequate. according to the statement from the european society for clinical and economic aspects of osteoporosis, osteoarthritis, and musculoskeletal diseases, "attainment of serum 25-hydroxyvitamin d levels well above the threshold desired for bone health cannot be recommended based on current evidence, since safety has yet to be confirmed" [145] . this statement, published in 2017, is no longer correct since a number of vitamin d supplementation studies have reported that long-term vitamin d supplementation has health benefits without adverse health effects, e.g., 2000 iu/d for cancer risk reduction [66, 146] and 4000 iu/d for reduced progression from prediabetes to diabetes [67] . a recent review on the status of vitamin d deficiency worldwide stated that because of inadequate evidence from clinical trials, "a 25(oh)d level of >50 nmol/l or 20 ng/ml is, therefore, the primary treatment goal, although some data suggest a benefit for a higher threshold" [147] . a companion article in the same issue of the journal stated, "although 20 ng/ml seems adequate to reduce risk of skeletal problems and artis, concentrations above 30 ng/ml have been associated with reduced risk of cancer, type 2 diabetes mellitus, and adverse pregnancy and birth outcomes" [148] . however, on the basis of the findings in several studies discussed here, as well as recommendations for breast and colorectal cancer prevention [149] , the desirable concentration should be at least 40-60 ng/ml. the u.s. institute of medicine issued vitamin d and calcium guidelines in 2011 [150] . the institute recommended vitamin d supplementation of 600 iu/d for people younger than 70 years, 800 iu/d for those older than 70 years, and a serum 25(oh)d concentration of 20 ng/ml (50 nmol/l) or higher. that recommendation was based on the effects of vitamin d for bone health. the institute recognized that no studies had reported adverse effects of supplementation with less than 10,000 iu/d of vitamin d, but set the upper intake level at 4000 iu/d, partly out of concerns stemming from observational studies that found u-shaped 25(oh)d concentration-health outcome relationships. however, later investigation determined that most reports of j-or u-shaped relationships were from observational studies that did not measure serum 25(oh)d concentrations and that the likely reason for those relationships was a result of enrolling some participants who had started taking vitamin d supplements shortly before enrolling [151] . moreover, in 2011, the endocrine society recommended supplementation of 1000-4000 iu/d of vitamin d and a serum 25(oh)d concentration of 30 ng/ml or higher [152] . those guidelines were for patients. it appears that anyone with chronic disease should be considered in that category. the u.s. institute of medicine noted that no adverse effects of vitamin d supplementation had been reported for daily doses <10,000 iu/d [150] . measuring serum 25(oh)d concentration would be useful to determine baseline and achieved 25(oh)d concentrations. a recent article recommended testing for groups of people who were likely to have low concentrations and could benefit from higher concentrations, such as pregnant women, the obese, people with chronic diseases, and the elderly [148] . part of the rationale for testing was to increase awareness of actual 25(oh)d concentrations and the benefits of higher concentrations. in addition, increases in 25(oh)d concentration with respect to vitamin d supplementation depend on various personal factors, including genetics, digestive system health, weight, and baseline 25(oh)d concentration. for about half the population, taking 5000 iu/d of vitamin d 3 or 30,000-35,000 iu/wk would raise 25(oh)d concentration to 40 ng/ml. taking 6235-7248 iu/d as proposed to ensure that 97.5% of the population has concentrations >20 ng/ml [153] would not exceed the 10,000-iu/d threshold. vitamin d supplementation is required for many individuals to reach 25(oh)d concentrations above 30 ng/ml, especially in winter [154] . however, vitamin d fortification of basic foods such as dairy and flour products [83, 155] can raise serum 25(oh)d concentrations of those members of various populations with the lowest concentrations by a few ng/ml. doing so can result in reduced risk of artis for individuals with extreme vitamin d deficiency [134, 156] . however, for greater benefits, daily or weekly vitamin d supplementation is recommended [134] , as is the annual determination of serum 25(oh)d concentration for those with health risks [148] . magnesium supplementation is recommended when taking vitamin d supplements. magnesium helps activate vitamin d, which in turn helps regulate calcium and phosphate homeostasis to influence the growth and maintenance of bones. all the enzymes that metabolize vitamin d seem to require magnesium, which acts as a cofactor in the enzymatic reactions in the liver and kidneys [157] . the dose of magnesium should be in the range of 250-500 mg/d, along with twice that dose of calcium. the hypothesis that vitamin d supplementation can reduce the risk of influenza and covid-19 incidence and death should be investigated in trials to determine the appropriate doses, serum 25(oh)d concentrations, and the presence of any safety issues. the rct on vitamin d supplementation for ventilated icu patients conducted in atlanta, georgia, is a good model [93] . a recent review stated: "although contradictory data exist, available evidence indicates that supplementation with multiple micronutrients with immune-supporting roles may modulate immune function and reduce the risk of infection. micronutrients with the strongest evidence for immune support are vitamins c and d and zinc. better design of human clinical studies addressing dosage and combinations of micronutrients in different populations are required to substantiate the benefits of micronutrient supplementation against infection." 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2009-10-24 journal: hot topics in infection and immunity in children vi doi: 10.1007/978-1-4419-0981-7_1 sha: doc_id: 347761 cord_uid: wgodcsav infections in the immunocompromised differ significantly from those in the immunocompetent. they can be more serious, more often life threatening, more difficult to diagnose and are caused by more unusual organisms. children can be immunocompromised for a variety of reasons and the numbers, worldwide, are growing. cryptosporidium and aspergillus species. b lymphocyte defects can result in infections by bacteria such as streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus, as well as echo virus and protozoa such as giardia, whereas phagocytic defects will result in infections by staphylococcus, pseudomonas and aspergillus species. when considering infections in those undergoing hsct, it is also important to note that there is a recognized sequence of risk for different infections at different times after hsct, which equates with different aspects of the immune system compromise at these times (fig. 1) . within the first 30 days, when the patient is neutropenic, there is a significant risk of infection by both gram-negative and grampositive bacteria, along with herpes-simplex virus. between 30 and 90 days after transplant, when t-cell immunity is still limited, there is a rise in the numbers of fungal and cmv infections. later infections are more commonly caused by varicella zoster virus (vzv) or s. pneumoniae. whatever the cause of the immunocompromise, possible infection requires a different approach to investigation and management from those in an immunocompetent child. although an understanding of the type of immunocompromise is helpful to predict the likely organism, infection in the immunocompromised also needs to be considered by the system affected. infections can occur in any system of the body, but the respiratory system and gastrointestinal tract are especially vulnerable, as they have large surface areas and their barrier defences are, of necessity, compromised by the need to transport oxygen and nutrients, respectively. disseminated viral and fungal infections are another important risk, whilst central venous catheter (cvc) infections also constitute a frequent problem in the immunocompromised. each of these will be discussed in turn. the respiratory tract can be exposed to a wide variety of different organisms. pneumocystis jiroveci pneumonia (pcp), cmv and aspergillus are particularly important and well recognized sources of infection in the immunocompromised host; however, other significant pathogens have more recently been identified. these include respiratory syncytial virus (rsv); influenza; parainfluenza; adenovirus; picornaviruses; measles; human metapneumovirus; cocavirus; coronaviruses nl63, and hku1 and polyomaviruses wu and k1. pneumonitis and bronchiolitis are the most common presentations of respiratory infection, but lobar pneumonia may also occur. a defective immune/inflammatory response means that patients may have few respiratory symptoms, so there should be a low threshold for investigation. in one study where broncho-alveolar lavage (bal) was performed in 69 children with immunodeficiency pre-hsct, pathogens were isolated in 26 of these, six of whom were asymptomatic. pcp and bacteria were the most commonly identified organisms, followed by parainfluenza virus, cmv, rsv, influenza b and human herpes virus-6 (hhv6) (slatter et al., 2007) . accurate diagnosis depends on collecting the right samples and using appropriate diagnostic techniques. these include throat swabs, nasopharyngeal aspirates (npa), bal fluid and even lung biopsy, as deemed appropriate. samples must be sent to look specifically for bacteria, fungi and viruses. some respiratory pathogens will not be isolated from the upper respiratory tract; for example, pcp will not be identified on npa, whilst other organisms found on npa may not be found in the lower respiratory tract. this highlights the importance of bal as a diagnostic procedure. lung biopsy may be particularly important in the diagnosis of fungal infection, especially when there is a negative bal in patients with persistent signs, symptoms or chest x-ray changes. diagnosis may require culture of organisms (bacteria, mycobacteria or viruses), immunofluorescence (viruses), polymerase chain reaction (bacteria, viruses and fungi) or antigen testing (e.g. galactomannan for aspergillus). serological testing is often ineffective, as immunodeficient children may not mount an antibody response or may be receiving intravenous immunoglobulin (ivig), which will make results impossible to interpret. it is important to know what tests are available in your local laboratory. discussion with the local microbiologist or virologist is essential to ensure the right samples are sent for appropriate investigations, so as not to miss a serious infection. high resolution computerized tomography (hrct) of the chest is more sensitive than chest x-ray, aiming to classify a disease as interstitial, airway or involving airspace, which may aid diagnosis. pcp has historically been associated with hiv but is also a significant cause of morbidity in other groups of immunocompromised patients, particularly those with haematological malignancies, brain tumours requiring prolonged courses of steroids, prolonged neutropaenia or lymphopaenia, and those undergoing hsct. therefore pcp prophylaxis is important, as recommended by a recent cochrane review (green et al., 2007) . this treatment is generally in the form of cotrimoxazole given three times per week. in children that cannot tolerate cotrimoxazole, either dapsone or aerosolized pentamidine can be used. p. jiroveci infection commonly presents with tachypnoea, non-productive cough and fever, but the severity can vary. there is usually a sub-acute diffuse pneumonitis and chest x-ray changes can be subtle. these often take the form of bilateral diffuse interstitial changes, although lobar, miliary or nodular changes can be seen. hrct may show ground glass attenuation, consolidation, nodules, thickening of interlobular septa and thin walled cysts. mortality ranges between 5 and 40%, if treated, but can reach nearly 100% if left untreated. identification of pcp can be difficult. definitive diagnosis depends on identifying the organism in respiratory tract secretions or lung tissue, usually from tracheal secretions, bronchial secretions or from lung biopsy. more recently, pcr technology has been developed for identifying pcp from secretions. in a review of children diagnosed with severe combined immune deficiency (scid) treated at a supra-regional center, 10 out of 50 were identified as having pcp. one was diagnosed on bal prior to transfer to the supra-regional center, one was diagnosed on nasopharyngeal secretions and bal, seven were diagnosed on bal alone, and in one diagnosis was not made until lung biopsy was performed (berrington et al., 2000) . recommended first line pcp treatment is high dose cotrimoxazole. this can, however cause a number of adverse effects, for example, neutropenia, anaemia, renal dysfunction, rash, vomiting and diarrhea. those that cannot tolerate cotrimoxazole or those that have not improved after 5-7 days of treatment should be changed to a different agent. choices include pentamidine, atovaquone, clindamycin/primaquine or dapsone, but experience with these agents in children is limited. corticosteroids should be given as an adjunctive therapy in moderate and severe pcp. a number of studies have shown a reduction in acute respiratory failure, decreased need for ventilation and decreased mortality (sleasman et al., 1993; bye et al., 1994; mclaughlin et al., 1995) . a recent cochrane review supports the use of corticosteroids in hiv-infected patients with pcp, especially in those with substantial hypoxaemia (briel et al., 2006) . a wide variety of respiratory viruses will also cause significant morbidity and mortality in the immunocompromised. measles is an important respiratory pathogen in the immunocompromised host and it must be remembered that the typical rash may not develop. mortality can be high, especially amongst patients with leukaemia and those undergoing hsct. a prospective multi-center review of patients undergoing hsct found direct rsv-associated mortality to be 17.4%, and mortality directly attributable to influenza a to be 15.3% (ljungman et al., 2001) . respiratory viruses often present with non-specific symptoms but progress to a significant lower respiratory tract infection. chest x-ray will often show a pneumonitis picture with diffuse interstitial changes. hrct may show peri-bronchial thickening and ground glass attenuation without consolidation in a lobular distribution. diagnosis requires identification of the organism from respiratory secretions. this may be possible on nasopharyngeal secretions or throat swab but may require more invasive testing, such as bronchoscopy and bal. laboratory techniques include immunofluorescence, pcr and viral culture. treatment is mainly supportive, but specific treatment options are evolving, making rapid and accurate diagnosis increasingly important. appropriate isolation and infection-control measures are essential to prevent transmission between immunocompromised patients, as these viruses can be easily spread. one uk study in a hsct unit identified 10 cases of rsv over one winter season, and eight of the nine rsv strains that could be tested by molecular methods were found to be identical (taylor et al., 2001) . specific treatments for rsv infection include ribavirin and rsv monoclonal antibody (palivizumab). ribavirin can be given orally, intravenously or via inhalation; however, the aerosolized route has been used most frequently for rsv infection. historically, pooled hyperimmune rsv immunoglobulin has been proposed as an additional treatment, but this has been superseded by the anti-rsv monoclonal antibody, palivizumab. combinations of inhaled ribavirin and intravenous palivizumab have shown encouraging results. palivizumab has been shown to be safe and well tolerated in patients undergoing hsct, with a suggestion of better outcome (improved survival) when compared to ribavirin alone (boeckh et al., 2001; chavez-bueno et al., 2007) . there are two groups of drugs available for the treatment of influenza -namely the adamantanes (effective against influenza a, e.g. rimantadine) and the neuraminidase inhibitors (effective against both influenza a & b, e.g. oseltamivir). in recent years, there has developed increasing resistance to adamantanes. the neuraminidase inhibitors have been shown to reduce the duration of illness by one day when given to an immunocompetent host within 48 h of onset of symptoms. although there are few data on the benefit of treating influenza in an immunocompromised patient with a neuraminidase inhibitor, their use appears sensible and safe. there is, thus far, no specific treatment available for rhinovirus, coronavirus or human metapneumovirus. ribavirin has been proposed as a treatment for parainfluenza virus infection but evidence, so far, of benefit is disappointing. although there is little clinical data on the use of ribavirin for measles pneumonitis, it does have in vitro activity and therefore, due to the high level of mortality with this condition, should be considered. a review of respiratory viral infection in children with primary immune deficiencies in a hsct unit found 22 of 73 patients admitted for hsct had respiratory viral infection. of these, 11 had paramyxoviruses (rsv or parainfluenza i-iv), and were treated with aerosolized ribavirin and ivig. five of these patients also received nebulized immunoglobulin and corticosteroid. three of these five survived, compared to two out of the six who did not receive nebulized treatment. it was concluded that the nebulized treatment was well tolerated and could be a useful adjunctive therapy (crooks et al., 2000) . in children who have undergone hsct, infection and inflammation can become inextricably interwoven to generate pneumonitis. in this case, in addition to the need for anti-infective agents, immunomodulation will be required through agents such as steroids, ivig and anti-tumour necrosis factor monoclonal antibodies. the gastrointestinal tract is also exposed to a wide variety of organisms and viruses which are of particular concern in the immunocompromised child, notably enteroviruses, adenovirus, rotavirus, caliciviruses, but also protozoa, mainly cryptosporidium and giardia. presentation is most commonly with diarrhea and vomiting, which may protracted. cryptosporidium can also be responsible for ascending cholangitis and liver disease. in some cases, identification of the causative organism can be difficult. culture may be required to identify some viruses. pcr can be useful, for example, for adenovirus and is more sensitive than microscopy alone in detecting cryptosporidium. prevention of transmission between immunocompromised patients is essential. there must be strict adherence to infection-control policies to prevent hospital wards from becoming sources of infection. one study looking at the extent of gastroenteric virus contamination in a pediatric-primary immunodeficiency ward and a general pediatric ward found viruses on 17 and 19% of environmental swabs, respectively. interestingly, these were contaminating objects used by parents rather than stafffor example the parents' room television, the parents' toilet tap and the microwave used by parents on the pediatric-primary immunodeficiency ward (gallimore et al., 2008) . this highlights the importance of ensuring that parents and visitors, as well as staff, comply with hand washing and infection control measures. rotavirus infection, which is usually relatively mild and self-limiting in the immunocompetent, can lead to persistent vomiting and diarrhea and, if untreated, severe malnutrition, in the immunocompromised. it can be identified in stool by using enzyme immunoassay and may also be identified on electron microscopy. there is no specific treatment. fluid and electrolyte management is important. orally administered immunoglobulin has been used in some cases. caliciviruses, namely noroviruses and sapoviruses can also cause significant problems in the immunocompromised. symptomatic infection and virus shedding can be prolonged; for example, one case report of a child undergoing hsct for cartilage hair hypoplasia demonstrated norovirus shedding for 156 days following transplant, during the period of immune reconstitution. the child was symptomatic throughout this time (gallimore et al., 2004) . again, there is no specific treatment but meticulous management of fluids, electrolytes and nutritional support is essential, allowing time for immune reconstitution and consequent viral clearance. adenovirus will be discussed in more detail in the section on disseminated infection in the immunocompromised. cryptosporidium species are oocyst-forming protozoa that cause watery diarrhea which can result in severe dehydration and even death, if not treated. disease is normally confined to the gastrointestinal tract, but there is a risk of biliary tree, pulmonary or even disseminated disease in the immunocompromised. infection may be diagnosed on identification of oocysts by microscopy. enzyme immunoassays have also been used and pcr, too, can be helpful. treatment of cryptosporidium infection can be difficult and a number of agents have been proposed, including nitazoxanide, paromomycin, rifabutin and the macrolides. evidence is limited but a recent review has indicated that nitazoxanide may reduce parasite load and therefore be useful (abubakar et al., 2007) . in the authors' experience, azithromycin and nitazoxanide are safer options in post-hsct patients, as paromomycin has been associated with significant hearing loss, particularly when given with ciclosporin. supportive care remains essential. in those with hiv, anti-retroviral therapy, with its associated improvement in cd4 count, can result in improvement in the cryptosporidium infection. giardia intestinalis is a flagellate protozoan that exists in trophozoite or cyst forms. the cysts are the infective form. children with humoral immunodeficiencies are particularly at risk of chronic symptomatic infection, with foul-smelling stool, abdominal distension and anorexia. cysts may be identified on stool microscopy or by using immunofluorescent antibody testing. treatment is with metronidazole, tinidazole or nitazoxanide. it may be necessary to use combination therapy in the immunocompromised if they have failed to respond to single-agent treatment. disseminated viral infection in the immunocompromised is of particular concern. the most significant culprits are adenovirus and members of the human herpes virus: cmv, ebv, hhv6, hsv and vzv. these can affect the lungs, gastrointestinal tract and brain, resulting in a variety of symptoms. reactivation of latent herpes viral infection is more common than primary infection after sot or hsct. investigation using pcr techniques allows early diagnosis and quantification of viral load, and is now possible for adenovirus, cmv, ebv and hhv6. prophylaxis to prevent cmv and hsv reactivation is used for children undergoing hsct and many sots. surveillance in high-risk patients enables pre-emptive treatment to be given before damaging disease occurs. treatment will depend on the causative virus. adenovirus is usually responsible for relatively minor upper respiratory tract or gastrointestinal infection but can result in life-threatening pneumonia, meningitis, encephalitis and disseminated disease in the immunocompromised. those most at risk are patients who receive allogeneic bone marrow transplant, those with active graft versus host disease and those who receive total body irradiation. there are a number of different species of adenovirus, and these are divided into serotypes, some of which are primarily associated with the respiratory tract, while others have a predilection for the gastrointestinal tract. young children are particularly vulnerable, as they often carry adenovirus in their gastrointestinal tract, predisposing them to reactivation and dissemination when they become immunocompromised. in view of this, screening can be important in the immunocompromised and adenovirus is usually identified in urine, stool, or sometimes respiratory secretions prior to being identified in blood. a study of 132 patients undergoing hsct were screened for adenovirus in stool, urine, on throat swab and in peripheral blood during the post transplant period. 27% had a positive adenoviral pcr on at least one screening test, but this was not associated with clinical signs unless it was detected in peripheral blood and, even then, there was a median delay of 3 weeks from first detection of adenovirus until the patient demonstrated clinical signs. in one study, mortality was as high as 82% in those with adenovirus detected on peripheral blood. this highlights the importance of early recognition and consideration of pre-emptive use of antivirals (lion et al., 2003) . successful treatment of adenovirus infection has so far been limited. the most widely used agents are cidofovir or ribavirin, which may be given together with ivig. although cidofovir has potent nephrotoxic effects, these can be greatly reduced by the concurrent use of intravenous hyperhydration and probenecid. cidofovir has been shown to be more effective in adenovirus and is now considered the best first-line treatment. data on the clinical effectiveness of ribavirin in adenoviral infections are more conflicting. in vitro data suggest that ribavirin alone has activity against subgenus c serotypes. in a post-hsct patient with adenoviral infection, immune suppression should be reduced as much as possible, as t-cell immune reconstitution is very important for viral elimination. cmv infection is often asymptomatic in the immunocompetent; however, in the immunocompromised it can lead to pneumonia, colitis and retinitis. cmv persists in a latent form after primary infection and can result in reactivation in someone who later becomes immunosuppressed -for example, when undergoing hsct. cmv can be identified from respiratory secretions, urine and blood. as with adenovirus, pcr screening may be useful in identifying the virus before a child becomes symptomatic, especially in cases where reactivation is likely with immunosuppression. treatment is usually with intravenous ganciclovir, with foscarnet or cidofovir as second-line treatment. oral valganciclovir is very well absorbed and is also now an option for treatment. foscarnet has also been used in cases of children undergoing hsct to avoid the myelosuppressive effects of ganciclovir. ivig should be used alongside antiviral therapy. there has been one case report of ganciclovir-and foscarnet-resistant cmv being successfully treated with artesunate (shapira et al., 2008) . there is also interest in the new antiviral agent maribavir for resistant cmv. ebv is associated with lymphoproliferative disorders in the immunocompromised. replication of ebv in b cells is usually inhibited by natural killer cells, antibodydependent cell cytotoxicity and t-cell cytotoxic responses. therefore, children with cellular immune deficiencies are at risk of uncontrolled lymphoproliferation. those at particular risk are children who are transplant recipients, both sot or hsct, and those with hiv. ebv can be detected in blood by pcr and viral load can be monitored. alongside monitoring of the virus, it is important to monitor for signs of lymphoproliferation, both clinically and biochemically. biopsy of suspicious lesions is often needed to make a diagnosis. ebv infection requires treatment if it causes b lymphoproliferation or posttransplant lymphoproliferative disease (ptld). this may take the form of the anti-cd20 monoclonal antibody rituximab, chemotherapy or radiotherapy. decreasing immunosuppression whenever possible in a post-transplant patient is very important. more recently there have been encouraging results from work with cytotoxic t-cell therapy in ptld. this involves the infusion of ebv-specific cytotoxic t lymphocytes (ctls) generated from ebv sero-positive blood donors. in one recent multi-center study, 33 patients who had failed conventional therapy were recruited and monitored for response: 14 patients achieved complete remission while three showed a partial response (haque et al., 2007) . primary hhv6 infection in the immunocompetent host leads to the typical clinical picture of roseola or a non-specific febrile illness. the virus remains latent after primary infection and therefore, similar to cmv, can reactivate in immunocompromised states. the importance of hhv6 as a pathogen in the immunocompromised is probably underestimated, and many labs do not screen for infection; thus, many infections may not be recognized. hhv6 can cause fever, rash, hepatitis, pneumonia and encephalitis, as well as bone marrow suppression. hhv6 also appears to have synergistic effects and interactions with other infectious agents, such as cmv, adenovirus and fungi. it can be identified and quantified on blood samples by pcr. treatment, where necessary, is with intravenous ganciclovir or foscarnet. primary varicella infection results in chickenpox, a common and generally selflimiting childhood illness. in the immunocompromised, there is a significant risk of both primary or reactivated disease becoming disseminated. this is particularly associated with t lymphocyte defects. vzv is the second most common cause of viral pneumonitis in children with aids. it should be remembered that fatal vzv infection has been reported in cases where the only immunosuppressant medication has been corticosteroids at a dose of 1 mg/kg/day of prednisolone for 2 weeks. the virus can be identified from vesicular fluid. treatment is usually in the form of intravenous aciclovir, but, oral valaciclovir is a useful alternative in older children. an important area to consider in relation to vzv infection is that of postexposure prophylaxis. although long-term prophylaxis for vzv is not usually recommended, post-exposure prophylaxis in non-immune immunocompromised children is important. two options are available. the most widely used is varicella zoster immunoglobulin (vzig). however, due to a shortage of vzig a few years ago, oral aciclovir was reconsidered and has been shown to be effective. it must be remembered, however, that aciclovir has low bioavailability when given orally and requires multiple daily dosing. it may be more appropriate to consider the oral pro-drug valaciclovir, which has been shown to be effective and well tolerated (nadal et al., 2002) . further work to clarify the best prophylactic and pre-emptive treatment regimens is needed. fungal infections must be considered in specific circumstances; for example, in those who are neutropenic (where risk increases exponentially with duration of neutropenia), those on steroids and those with graft versus host disease. candida and aspergillus are of particular interest in children who have undergone hsct. symptoms that should raise the suspicion of fungal infection are persistent fevers unresponsive to antibiotics, skin nodules, chest pain and radiological evidence of infection crossing tissue planes. candida is most commonly associated with cvc infection but can also cause disseminated disease. aspergillus infection can have an insidious onset, frequently affecting the respiratory tract but then spreading to involve other areas such as the spine and intracranial cavity. investigation and diagnosis remain difficult and may require antigen testing, pcr, cross-sectional imaging and biopsy of suspicious lesions/areas. persistent mucocutaneous candidiasis is seen in patients with defects in t-cell function and may be a presenting feature for hiv infection or primary immune deficiency. disseminated infection can involve almost any organ or any anatomical site and can be rapidly fatal. it is a particular concern in patients with cvc, especially those receiving multiple infusions and/or parenteral nutrition. there are a number of different candida species that can result in disseminated infection. candia albicans is the most common but c. parapsilosis, c. glabrata, c. tropicalis and c. krusei are increasingly common (fig. 2) . diagnosis may be difficult, as blood cultures are not always positive. however, identification can be made by microscopy of biopsy specimens. suspicious lesions, which are often found in organs such as the liver, kidney, spleen and brain, are best identified by cross-section imaging. pcr techniques have been developed, as well as detection of antigen from the fungal cell wall (mannan). however, these techniques are not as yet wholly reliable. there are a number of agents available for treatment, including amphotericin b, caspofugin or an azole, such as voriconazole. prolonged treatment is usually required and if there is a cvc invasive aspergillus infection in the immunocompromised usually involves lungs, sinuses, brain or skin and commonly crosses tissue planes. less commonly, it can cause endocarditis, osteomyelitis, meningitis and infection around the eye or orbit. it can cause angio-invasion, resulting in thrombosis and, occasionally, erosion of the blood vessel wall, often with catastrophic hemorrhage as a consequence. there are a number of aspergillus species that cause invasive disease. most commonly it is due to aspergillus fumigatus, but a. flavus, a. terreus, a. nidulans and a. niger are also responsible for invasive infection. diagnosis can be challenging. crosssectional imaging is very important in identifying suspicious lesions. aspergillus is infrequently identified from blood and is most commonly indicated from biopsy specimens. galactomannan, a complex sugar molecule found in the cell wall of the aspergillus species, may also be identified from blood and can be useful in aiding diagnosis. treatment is usually with amphotericin b, voriconazole or caspofungin and requires a prolonged course. surgical excision of fungal lesions may be required, especially if there are significant areas of necrotic tissue into which antifungal agents will not penetrate effectively. there is also an important association between aspergillus infection and building work on a hospital site. one study in an italian hematology unit found three cases of proven aspergillosis in patients with acute leukemia that coincided with renovation work on the hospital site and high levels of a. fumigatus in the corridors (pini et al., 2008) . this highlights the importance for high-risk patients (e.g. after hsct) of sterile isolation in cubicles maintained at positive pressure with highly purified air. extra attention must be paid to reducing exposure of immunocompromised patients when there is building work on any hospital site. many immunocompromised children will have indwelling cvc for treatment, be this an external broviac or hickman line, or an internal portacath. although very beneficial they, unfortunately, provide a site for infection. catheter-related blood stream infections can be serious and in some cases life-threatening. clinical features of catheter-related blood stream infection can be very non-specific. diagnosis is often made on identification of organisms from blood culture along with lack of focal infective symptoms/signs. organisms causing cvc infection are often those that would be non-virulent normal flora in an immunocompetent host; for example, coagulase negative staphylococci, enterococci and viridans streptococci. however, mycobacterial cvc infections also occur (hawkins et al., 2008) , as do candida cvc infections. prevention has to be the priority. lines should be inserted under strict aseptic technique and, once in place, access should be by fully trained staff using aseptic technique. local policies should be followed for accessing and flushing cvcs. historically, cvcs were often removed when infection was identified; however, many patients were left in the difficult situation of poor venous access and in need of a further general anaesthetic to replace the line. many catheter-related blood stream infections can be treated with antibiotics, without requiring cvc removal. if there is clinical suspicion of catheter-related blood stream infection, antibiotics for both coagulase negative staphylococcus and gram negative organisms should be introduced. once organisms are identified from blood culture, antibiotics can be tailored appropriately. antibiotic "locks" can be used alongside systemic antibiotics to reduce colonization within the cvc. antibiotic "locking" involves instilling 1-2 ml of concentrated antibiotic solution in to the cvc and leaving it for a pre-determined time before removal. antibiotics used in studies to treat cvc colonization have included vancomycin, amikacin and minocycline. there is also limited evidence on the use of amphotericin locks. studies have attempted to look at whether using locks alone or in combination with systemic antibiotics has benefits. the results are variable and, at this stage it must be concluded that locks are a useful adjunct to systemic treatment. there is not enough evidence to suggest they can be used alone in immunocompromised children with cvcs (berrington and gould 2001) . in an attempt to present cvc infection, antibiotic-impregnated cvcs have also been developed. a recent systematic review found significant reductions in catheterrelated blood stream infections in heparin-coated or antibiotic-impregnated cvcs, when compared to standard cvcs, as well as those coated with chlorhexidine, silver sulphadiazine, or silver-impregnated. there were, however, some concerns about the development of antibiotic resistance and further study is required before recommendations can be made about the most appropriate cvc to be used (gilbert and harden, 2008) . it must be remembered that catheter-related blood stream infection can be life threatening and there should be a low threshold for removal of the cvc if there are signs of clinical deterioration on treatment or if blood cultures drawn from cvcs are repeatedly positive, despite ongoing appropriate antibiotic treatment. there is increased mortality associated with delayed catheter removal in s. aureus and fungal infections, and so removal must be considered urgent if these organs are isolated. the benefits of removing the cvc if gram-negative organisms are identified is slightly more difficult to assess due to scarcity of data; however, it is likely that immediate removal does contribute to increased survival. in all infections the risk/benefit ratio of removing or retaining cvcs should be carefully considered. in children receiving treatment for malignancy, febrile neutropenia is a significant cause of morbidity and mortality. over time, outcome has improved dramatically but it still remains a frequent reason for hospitalization. it has been shown that (schmipff et al., 1971) ; hence, empiric antibiotics have become a standard part of treatment for children and adults with febrile neutropenia. fever with neutropenia in any immunocompromised child should be acted on promptly. however, exactly how this is defined and what is appropriate management varies widely. this was highlighted by a recent review of febrile neutropenia management in the united kingdom children's cancer study group centers (phillips et al., 2007) . there was wide variation in the definition of fever (from persistent temperature higher than 37.5 â�¢ c to a single reading of 39 â�¢ c) and neutropenia (absolute neutrophil count <1 ã� 10 9 , < 0.75 ã� 10 9 or < 0.5 ã� 10 9 ). empirical antibiotic regimes also varied greatly, including aminoglycosides plus a second agent (piperacillin based, cephalosporin or carbapenem), carbapenem alone or, in two cases, cefuroxime plus flucloxacillin and ciprofloxacin plus ceftazidime. timing of the anti-fungal therapy was even more variable, in terms of when to start and the duration of empirical treatment. some of this variation can be explained by variations in organisms isolated and antibiotic sensitivity from unit to unit, but this does not seem to account for all the differences in practice. therefore, although local findings should influence presenting patterns, further work is required to devise a framework within which local policies that target specific patient populations and microbiological flora are implemented. a specimen protocol is shown in fig. 3 infections in immunocompromised children offer a variety of challenges in both diagnosis and management. organisms that result in mild, self-limiting illness in an immunocompetent host can have catastrophic effects on an immunocompromised child. signs and symptoms are often less specific and finding a causative organism can be more difficult. it is important to have a low threshold for thinking about infections and looking for them. negative tests should not be taken to be reassuring if there is clinical suspicion and it may be necessary to look further and more closely. it is important to develop a good relationship with local microbiology and virology laboratories to aid this process. once an infection is identified, it must be acted upon quickly as delay may be disastrous. treatment of any infection in an immunocompromised child is likely to be more intense and prolonged than in a child with a fully functioning immune system. it is also important to consider prophylaxis for specific patient groups in specific situations (e.g. post hsct) and each unit should have defined policies and guidelines to follow for these patients. in summary, when dealing with an immunocompromised child, for whatever reason, when there is suspicion about infection, think early, look carefully and treat now! treatment of crypotspodiiosis in immunocompromised individuals: systematic review and meta-analysis infectious complications in children with cancer and children with human immunodeficiency virus infection. clinical approach to infection in the compromised host adenovirus. mandell, douglas and bennet's principles & practice of infectious disease second line salvage treatment of aids-associated pneumocystis jiroveci pneumonia: a case series and systematic review use of antibiotic locks to treat colonized central venous catheters unsuspected pneumocystis carinii pneumonia at presentation of severe primary immunodeficiency antiviral drugs for cytomegalovirus diseases phase 1 evaluation of the respiratory syncytial virus specific monoclonal antibody palivizumab in recipients of hematopoietic stem cell transplants prevention of vzv infection in immunosuppressed patients using antiviral agents adjunctive corticosteroids for pneumocystis jiroveci pneumonia in patients with hiv infection markedly reduced mortality associated with corticosteroid therapy of pneumocystis carinii pneumonia in children with acquired immune deficiency syndrome treating opportunistic infections in hiv infected children. guidelines for the children's hiv association (chiva) intravenous palivizumab and ribavirin combination for respiratory syncytial virus disease in high-risk paediatric patients adenoviruses. textbook of pediatric infectious diseases respiratory viral infections in primary immune deficiencies: significance and relevance to clinical outcome in a single bmt unit maribavir sensitivity of cytomegalovirus isolates resistant to ganciclovir, cidofovir or foscarnet contamination of the hospital environment with gastroenteric viruses: comparison of two pediatric wards over a winter season oral valganciclovir leads to higher exposure to ganciclovir than intravenous ganciclovir in patients following allogeneoic stem cell transplantation chronic excretion of a norovirus in a child with cartilage hair hypoplasia(chh) respiratory viruses other than influenza virus: impact and therapeutic advances identification of a novel polyomavirus from patients with acute respiratory tract infections prophylaxis for pneumocystis pneumonia (pcp) in non-hiv immunocompromised patients. cochrane database syst rev effectiveness of impregnated central venous catheters for catheter related blood stream infection: a systematic review allogeneic cytotoxic t cell therapy for ebv positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial catheter related bloodstream infections caused by rapidly growing nontuberculous mycobacteria: a case series including rare species diagnosis, prevention and management of catheter related bloodstream infection during long term parenteral nutrition investigation of blood cultures (for organisms other than mycobacterium species) viral respiratory tract infections in transplant patients antiviral therapy for adenovirus infections molecular monitoring of adenovirus in peripheral blood after allogeneic bone marrow transplantation permits early diagnosis of disseminated disease respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation effect of corticosteroids on survival of children with acquired immune deficiency syndrome and pneumocystis carinii-related respiratory failure in investigation of the steady-state pharmacokinetics of oral valaciclovir in immunocompromised children epidemiology of invasive candidiasis: a persistent public health problem variation in policies for the management of febrile neutropaenia in united kingdom children's cancer study group centres invasive pulmonary aspergillosis in neutropenic patients and the influence of hospital renovation human bocavirus: passenger of pathogen in acute respiratory tract infections? empiric therapy with carbenicillin and gentamicin for febrile patients with cancer and granulocytopenia management of pneumocystis jiroveci pneumonia in children receiving chemotherapy artesunate as a potent antiviral agent in a patient with late drug-resistant cytomegalovirus infection after hematopoietic stem cell transplantation value of bronchoalveolar lavage before haematopoietic stem cell transplantation for primary immunodeficiency or autoimmune diseases corticosteroids improve survival of children with aids and pneumocystis carinii pneumonia respiratory virus infections in the immunocompromised host molecular epidemiology of outbreak of respiratory syncytial virus within bone marrow transplant unit infections in recipients of blood and marrow transplantation. mandell, douglas and bennet's principles & practice of infectious disease adenovirus: an increasingly important pathogen in paediatric bone marrow transplant patients beta-herpesviruses in febrile children with cancer key: cord-302403-kahi8cbc authors: miller, robert f.; lipman, marc c.i. title: pulmonary infections date: 2009-05-15 journal: clinical respiratory medicine doi: 10.1016/b978-032304825-5.10034-0 sha: doc_id: 302403 cord_uid: kahi8cbc nan it is hard to believe that the first consistent reports of acquired immunodeficiency syndrome (aids) were only 25 years ago. since then, respiratory and general physicians have become accustomed to dealing with an extraordinary range of esoteric and previously unheard of conditions. pneumocystis carinii (now known as pneumocystis jirovecii) pneumonia is frequently part of the differential diagnosis of treatment-nonresponsive pneumonic illness. human immunodeficiency virus (hiv) testing is almost routinely offered as a "rule out" test in clinical cases that defy simple diagnosis. in many parts of the developing world, advanced hiv disease is unfortunately the likeliest reason for seeking medical care. the change this has wrought on people, countries and their economies is huge and depressing. the isolation of hiv from patients with aids in the mid 1980s paved the way for intensive research with the ultimate development of drugs directed against this chronic infection. however, despite such advances, the methods by which hiv infection leads to severe immune dysregulation and clinical disease are still not fully defined. the introduction in 1996, in the developed world, of highly active antiretroviral therapy, also known as combination antiretroviral therapy (cart) hereto referred to as haart, has altered the natural history of this extraordinary condition. before haart, defined as a combination of medications that usually includes at least three potent anti-hiv agents, treatment largely consisted of specific opportunistic infection management and less effective antiretroviral therapy. the clinical consequences of this change are enormous. the relative hazard for development of pneumocystis pneumonia (pcp) in an hivinfected individual has fallen by more than 80%. drug therapy does have a down side. it has significant unwanted effects, as well as major interactions with other medication (e.g., rifamycins used in treating tuberculosis). the profound change in immunity induced by haart may also lead to disease (the immune reconstitution inflammatory syndrome [iris] ). notwithstanding haart, respiratory disease remains an important cause of morbidity and mortality. much of the world cannot afford such medication, and more than two thirds of hiv-infected individuals have at least one respiratory episode during the course of their illness. in the early stages of hiv infection, when patients have relatively preserved immune responses, individuals have the same infections found in the general population, although at a greater incidence. with progressive hiv disease, subjects are at an increased risk of opportunistic disease. for example, the north american prospective study of pulmonary complications of hiv infection (pspc), a multicenter cohort drawn from all hiv risk groups at various stages of immunosuppression, revealed over an 18-month study period that of approximately 1000 subjects who were not using haart: 33% reported an upper respiratory tract infection 16% had an episode of acute bronchitis 5% had acute sinusitis 5% had bacterial pneumonia 4% had pcp develop the immune dysregulation that arises from hiv infection means that bacteria, mycobacteria, fungi, viruses, and protozoa can all cause disease in subjects with advanced infection. table 34 -1 shows the organisms that typically infect the lung in hiv disease. of these, bacterial infections, tuberculosis, and pcp are the most important. in the west, 40% of aids diagnoses are due to pcp. this chapter provides a brief general overview of the epidemiology and pathogenesis of hiv infection before concentrating on hiv and its infectious pulmonary complications. it is reported that by the end of 2006, 39.5 million individuals worldwide had acquired hiv infection (figure 34 -1). of these, more than 40% are thought to have had aids develop (for definition of aids, see tables 34-2 and 34-3, and box 34-1). globally, 4.3 million individuals acquired hiv infection in 2006, and over this time 2.9 million died of aids. the developing world has been most affected. sub-saharan africa is the current epicenter of the pandemic (two thirds of all infections); here, one in five adults is hiv infected. south and southeast asia are responsible for almost a fifth of the estimated hiv global burden. in central-eastern europe and central asia, there are currently 1.7 million hivinfected individuals. in the developed world, north america and western europe account for approximately 1.4 million and 740,000 infections, respectively. most of these are spread through sexual contact, although vertical (mother-to-child) and bloodborne infections are also common. in the developing world, heterosexual transmission is the norm; however, in north america and europe, homosexual and bisexual men constitute the largest group of infected individuals. hiv was first isolated in 1983 from patients with symptoms and signs of immune dysfunction. two subtypes (hiv-1 and hiv-2) have subsequently been identified. hiv-1 (hereafter referred to as hiv) is responsible for most infections, has a more aggressive clinical course, and is the focus of this chapter. hiv is a human retrovirus belonging to the lentivirus family. cell-free or cell-associated hiv infects through attachment of its viral envelope protein (gp120) to the cd4 antigen complex on host cells. the cd4 receptor is found on several cell types, although the t-helper lymphocyte is the main site of hiv infection in the body. hiv gp120 must also bind to a cell surface protein coreceptor called chemokine receptor 5 (ccr5) or to other co-receptors, including cxcr4, depending on the host cell type. polymorphisms in genes for ccr5 may affect disease progression by reducing the ability of hiv to enter and infect cells. however, at a population level, this effect is small. once hiv is inside the cell, it can, by use of the enzyme reverse transcriptase (rna-dependent dna polymerase), transcribe its hiv rna into a dna copy that can translocate to the nucleus and integrate with host cell dna by use of its viral integrase. the virus (as proviral dna) remains latent in many cells until the cell itself becomes activated. this may arise from cytokine or antigen stimulation. the viral genetic material is then transcribed into new rna, which, in the form of a newly created virion, buds from the cell surface and is free to infect other cd4-bearing cells. hiv infection directly attacks the immune system and in particular the t-helper cells that are central to a coordinated immune response. this leads to progressive immune dysfunction and an inability to resist opportunistic disease. the pathogenic process is not well defined, although it is thought that at the time of primary infection, hiv spreads to the lymph nodes, circulating immune cells, and thymus. this is a massive viral infection of the human host; and although there seems to be a relatively potent immune response, in fact, this initial onslaught is so devastating (targeting as it does specific memory t cells responsible for sustaining long-term protective immunity) that the scene is set for progressive immune failure. this occurs through a combination of direct cell killing by hiv replicating within cells, as well as the negative effects of chronic immune activation. ultimately, these lead in most individuals to immune system destruction and dysfunction. this is reflected not only by clinical disease indicating profound immunosuppression but also by a measurable reduction in the circulating absolute cd4 cell count, the percentage of t cells expressing cd4 markers, and in the progressive reduction in cd4/cd8 t-cell ratio. the use of haart, as well as preventative (prophylactic) therapies for opportunistic infections, has changed the clinical picture of hiv disease in countries where these interventions are available. death rates have fallen to one sixth of their previous levels. however, in the absence of such treatments, the median interval between hiv seroconversion and progression to aids in the developed world has been estimated to be 10 years, although rather less in resource-poor countries. almost all individuals have aids develop if untreated, and without haart, 95% of these will die within 5 years. in many parts of the world, the main causes of death in patients with hiv infection include bacterial pneumonia, tuberculosis, and pcp. the course of hiv infection can be divided clinically into several distinct periods: acquisition of the virus seroconversion, with or without a clinical illness (primary hiv infection) clinically silent period, lasting several months to years development of symptoms and signs indicating some degree of immunosuppression aids (where the subject has opportunistic disease implying profound immunosuppression [e.g., pcp]) the time from acquisition of hiv infection to development of detectable antibodies (the "window" period) is usually approximately 6-8 weeks. between 30 and 70% of individuals who become infected will have a seroconversion illness. hiv antibody is normally present within 2-3 weeks of these symptoms, although this can take longer. hiv rna in peripheral blood is detectable before this and is often used to confirm infection. the nonspecific features of primary hiv infection are almost always self-limiting, and typically seroconversion mimics a "flulike" illness or glandular fever. most individuals with primary hiv infection recover from the acute symptoms within 4 weeks. a proportion may have persistent symmetric generalized lymphadenopathy. there is no difference in prognosis in this group compared with asymptomatic hiv-positive individuals. although a proportion of individuals remain completely well without any treatment for an extended period (approximately 20% after 10 years), many hiv-infected individuals have minor symptoms and signs suggesting immune dysfunction. examples of these include new or worsening rashes (including herpes simplex), tiredness, cough, and low-grade anemia. certain clinical symptoms and signs provide important prognostic information. most studies have shown that oral candidiasis and constitutional symptoms (e.g., malaise, idiopathic fever, night sweats, diarrhea, and weight loss) are the strongest clinical predictors of progression to aids. the term aids was created as an epidemiologic tool to capture those conditions that early in the hiv epidemic seemed to suggest significant immune destruction. over time, it has been modified to incorporate the expanding spectrum of recognized diseases affecting immunosuppressed individuals, such as cervical carcinoma and recurrent bacterial pneumonia (see box 34-1). the 1993 centers for disease control and prevention (cdc) classification included an immunologic criterion for aids (cd4 count <200 cells/ml or cd4 percentage <14% of total lymphocytes) regardless of clinical symptoms (see table 34 -3). these data are used to define a point at which the risk of severe opportunistic infection rises dramatically. an example of this can be seen in the multicenter aids cohort study (macs) of homosexual and bisexual men without aids, which found that the incidence of pcp in subjects who did not use prophylaxis rose from 0.5% at 6 months in men with a baseline cd4 count >200 cells/ml to 8.4% in those with a cd4 count <200 cells/ml. apart from cervical carcinoma, aids indicator diseases differ little between men and women. injecting drug users have a high incidence of wasting syndrome, recurrent bacterial pneumonia, and tuberculosis. geographic differences in diseases occur that reflect the opportunistic pathogens present in the local environment (e.g., histoplasmosis or visceral leishmaniasis usually occur only in patients from endemic areas). in the developed world, sexual, racial, and hiv risk factor survival differences after an aids diagnosis mainly arise from variation in ease of access to medical care. it is certainly the case, however, that better treatment outcomes are associated with genuine specialist care provided by people with extensive experience in the field. in countries where haart is available, the spectrum of hiv-related disease has changed. in the eurosida cohort (a pan-european prospective study of hiv infection), between 1994 and 2002, opportunistic infections associated with very low cd4 counts (e.g., cytomegalovirus [cmv] retinitis and mycobacterium avium-intracellulare complex [mac] infection) were observed less frequently over time. malignant disease, such as non-hodgkin lymphoma, increased as an aids-defining event between 1994 and 2004. although death rates have fallen in haart-treated populations, there has been a rise in the proportion of non-aids deaths. in some series, this accounts for most events. causes include liver disease (often caused by viral hepatitis) and cancer, as well as cardiovascular disease and drug-related toxicity. in such circumstances, aids deaths usually occur among patients who have not accessed medical care regularly and who are initially seen with advanced hiv disease. a new manifestation of opportunistic infection has been described in patients commencing haart. the immune reconstitution disease, iris, may cause severe, if temporary, clinical illness as the individual's immunity recovers. patients will appear to develop a relapse of their original (and partially treated) disease. this is often seen in mac infection, tuberculosis, hepatitis b, cmv retinitis, and herpesviral infection. metabolic complications of haart, such as ischemic heart disease and diabetes, are a potential problem in hiv practice in the developed world. a significant number of individuals taking haart also experience drug toxicity. an increasing number of patients are also surviving to manifest symptoms associated with chronic hepatitis b and c infection. hivassociated nephropathy (often with chronic kidney disease) is common in black africans and is a significant cause of long-term morbidity. laboratory markers and clinical symptoms (e.g., oral thrush) can independently reflect the immune changes that lead to serious disease. staging systems have been developed that can predict the risk of progression to severe opportunistic disease (aids). the fall in absolute blood cd4 t-lymphocyte count is the most widely used prognostic marker, although cd4 counts may be affected by a number of factors apart from hiv, including intercurrent infection, cigarette smoking, exercise, time of day, and laboratory variation. the percentage of cd4 cells and ratio of cd4/cd8 cells are more stable measures and may be used if the cd4 absolute counts seem to vary widely from visit to visit. measurement of plasma hiv rna "viral load" provides important prognostic information that can both guide therapy and suggest long-term outcome. it has a particular value in subjects who are clinically well and have high cd4 counts, because it can give some indication of the expected speed of clinical progression. it is clear from the frequency with which hiv-related respiratory disease occurs that the pulmonary immune response is profoundly dysregulated. however, the mechanism underlying this has not been fully explained. in part, this is because the alterations that arise reflect the complex interplay between systemically derived hiv and other circulating antigens trafficking through the pulmonary vasculature, local immune cells, and airborne antigen. most studies investigating the pulmonary immune response have used in vitro cell culture systems that seek to mimic the pulmonary environment or in some cases bronchoscopy and bronchoalveolar lavage (bal) to recover lung cells from infected individuals. until recently, these have been performed on symptomatic patients who required bronchoscopy as a diagnostic procedure. such subjects generally have advanced hiv infection, are often taking a number of different drugs (including antiretrovirals), and may have any number of different pathogens causing their pulmonary disease-which by themselves can influence the immune findings. finally, the question of whether bal fluid truly represents the site of the immune response (the lung parenchyma) remains unclear. for all these reasons, reported data should be interpreted with some care. an individual's risk for respiratory disease is determined by his or her medical history (e.g., receipt of effective preventative or antiretroviral therapy), place of residence and travel history (e.g., the influence of geography on mycobacterial and fungal disease), and state of host immunity. falling blood cd4 counts or high plasma rna "viral loads" increase the chance of respiratory infection, with an increased spectrum of potential organisms responsible for infection in the more immunosuppressed individual. for example, hiv-infected individuals with a cd4 count <200 cells/ml are four times more likely to have one episode of bacterial pneumonia per year than those with higher cd4 cell counts. more exotic organisms are found in subjects with very low cd4 counts. these include bacteria such as rhodococcus equi and nocardia asteroides and fungi such as aspergillus species and penicillium marneffei. just as with p. jirovecii, this reflects the importance of t-cell depletion and macrophage dysfunction in the loss of host immunity (a process that has been confirmed by animal experiments). among hiv-infected patients, injecting drug users are at greatest risk for development of bacterial pneumonia and tuberculosis. individuals who have had previous respiratory episodes (pcp or bacterial pneumonia) seem to be at increased risk of further disease. whether this relates to host or environmental factors is not certain, although it seems likely that structural lung damage and abnormal pulmonary physiology would, in part, contribute to this. this argument is supported by the increased rates of pneumonia in hiv-infected smokers compared with nonsmokers. recent work has shown chronic obstructive pulmonary disease (copd) and lung cancer occur more frequently among hiv-infected individuals compared with the general population. given that a large number of hiv-infected individuals smoke heavily, there is a pressing need to target this population for smoking cessation. this is reinforced by the association demonstrated in some (but not all) studies between smoking and a more rapid progression to first aids illness and death. the presentation mimics bacterial exacerbations of chronic obstructive lung disease; most patients have a productive cough and fever. the pathogens commonly identified are similar to those in the general population (i.e., streptococcus pneumoniae and haemophilus influenzae). however, patients with advanced disease may be infected with pseudomonas aeruginosa or staphylococcus aureus. response to appropriate antibiotic therapy in conventional doses is good, although relapses frequently occur. bronchiectasis is increasingly recognized in hiv-infected patients with advanced hiv disease and low cd4 lymphocyte counts. it probably arises secondary to recurrent bacterial or p. jirovecii infections. the diagnosis is most often made by high-resolution/fine-cut computed tomography (ct) scanning. its prevalence has not been accurately determined, although with improved survival from both opportunistic infections and hiv disease, it is likely that it will be increasingly common in clinical practice. the pathogens isolated in patients with bronchiectasis are those seen in bronchitis. in addition, pseudomonas cepacia and moraxella catarrhalis have been described. community-acquired bacterial pneumonia occurs more frequently in hiv-infected patients than in the general population. it is especially common in hiv-infected injecting drug users. the spectrum of bacterial pathogens is similar to that in non-hiv-infected individuals (see table 34 -1). s. pneumoniae is the most common cause, followed by h. influenzae. hiv-infected individuals with s. pneumoniae pneumonia are frequently bacteremic. in one study, the rate of pneumococcal bacteremia in hiv-infected individuals was 100 times that of an hiv-negative population. more recent work has confirmed this to be the case for all causes of hiv-related bacterial pneumonia. typically, blood cultures have a 40-fold increased pickup rate in hiv-positive patients. the widespread use of haart has led to some decrease in rates of bacterial pneumonia and bacteremia, although they are still considerably higher than those seen in a non-hiv-infected population. bacterial pneumonia has a similar presentation in hivinfected and uninfected individuals. chest radiographs are frequently atypical, mimicking pcp in up to 50% of cases ( figure 34 -2). by contrast, radiographic lobar or segmental consolidation may also be seen in a wide range of bacterial organisms (figure 34 -3); these include s. pneumoniae, p. aeruginosa, h. influenzae, and mycobacterium tuberculosis. pcp may also present with lobar or segmental consolidation. in subjects with more advanced hiv disease and low cd4 lymphocyte counts, p. aeruginosa and s. aureus also cause pneumonia. complications of bacterial pneumonia frequently occur, and pleural effusions are twice as likely in hiv infection (often occurring with s. aureus infection); empyema and intrapulmonary abscess formation are present in up to 10% of patients. inevitably, the mortality rate is high (approximately 10%). nocardia asteroides infection. this has been reported in patients with advanced hiv disease and low cd4 lymphocyte counts. the widespread use of trimethoprim/sulfamethoxazole (tmp/smx) for prophylaxis of pcp may have reduced the incidence of infection. the clinical presentation is often indistinguishable from that of other bacterial infections. chest radiographic appearances may mimic tuberculosis (see later), with upper lobe consolidation, cavitation, interstitial infiltrates, pleural effusion, and hilar lymphadenopathy. the diagnosis is made by identification of the organism in sputum/bal fluid or lung tissue. rhodococcus equi. r. equi usually produces pneumonia in patients who have advanced hiv infection and have been in contact with farm animals or with soil from fields or barns where animals are housed. the presentation is subacute, with 2-3 weeks of cough, dyspnea, fever, and pleuritic chest pain. the chest radiograph typically shows consolidation with cavitation. pleural effusions are common. the diagnosis is usually made by culture of sputum or blood; bronchoscopy with bal or pleural aspiration may be necessary in some cases. bartonella henselae. b. henselae is a gram-negative bacillus that causes bacillary angiomatosis in hiv-infected patients. clinically, the cutaneous lesions may mimic kaposi sarcoma, from which they may be distinguished by demonstration of organisms in tissue with warthin-starry silver stain. bacillary angiomatosis may also infect the lungs, where it produces endobronchial red or violet polypoid angiomatous lesions, which may resemble kaposi sarcoma. biopsy is necessary to confirm a diagnosis. tuberculosis hiv infection is associated with at least a 40-fold increased risk of an individual having active tuberculosis develop compared with noninfected subjects. taken together with its ability to infect both the immunosuppressed and immunocompetent, tuberculosis is perhaps, therefore, the single most important disease associated with hiv infection. it is estimated that there are at least 13 million individuals with hiv-tuberculosis coinfection. as such, tuberculosis is a major cause of hiv-related morbidity and mortality. it is also a major driver in both resource-rich and resource-poor countries for the current overall increase in tuberculosis rates. where hiv infection is endemic, tuberculosis control at a population level is almost impossible if treatment for both infections is not available. in the united kingdom, many centers routinely offer hiv antibody testing to all patients with tuberculosis, regardless of risk factors for hiv infection. in the united states, the cdc now recommends hiv testing as a routine part of health care for all patients aged 13-64 accessing medical services. the advantage of this is that individuals who are found to be hiv infected can be given haart. furthermore, strategies to modify high-risk behavior and reduce ongoing hiv transmission may be offered. active tuberculosis can occur at any stage of hiv infection, and unlike almost every other hiv-related infection, may do so despite effective antiretroviral therapy. in the united states, united kingdom, and most european countries, reporting of tuberculosis in both hiv-infected and non-hiv-infected individuals is mandatory. clinical disease in hiv-infected patients may arise in several different ways: by reactivation of latent tuberculosis, by rapid progression of pulmonary infection, and by reinfection from an exogenous source. pulmonary disease is the most common presentation; and clinical manifestations are related to the level of an individual's cell-mediated immunity. for example, subjects with early hiv disease have clinical features similar to "normal" adult postprimary disease (table 34 -4). symptoms typically include weight loss, fever with sweats, cough, sputum, dyspnea, hemoptysis, and chest pain. these patients may have no clinical features to suggest associated hiv infection. the chest radiograph frequently shows upper lobe consolidation, and cavitary change is common (figure 34-4) . the tuberculin skin test (purified protein derivative [ppd] ) is usually positive, and the likelihood of spontaneously expectorated sputum or bal fluid being smear positive for acid-fast bacilli is high. in individuals with advanced hiv disease (i.e., low cd4 lymphocyte counts and clinically apparent immunosuppression), it may be difficult to diagnose tuberculosis. the clinical presentation here is often with nonspecific symptoms. fever, weight loss, fatigue, and malaise may be mistakenly ascribed to hiv infection itself. in this context, pulmonary tuberculosis is often similar to primary infection, with the chest radiograph showing diffuse or military-type shadowing ( figure 34 -5), hilar or mediastinal lymphadenopathy, or pleural effusion; cavitation is unusual. in up to 10% of patients the chest radiograph may appear normal; in others, the pulmonary infiltrate can be bilateral, diffuse, and interstitial in pattern, thus mimicking pcp. hilar lymphadenopathy and pleural effusion may also be produced by pulmonary kaposi sarcoma or lymphoma, with which m. tuberculosis may coexist. the tuberculin skin test is usually negative, and spontaneously expectorated sputum and bal fluid are often smear negative (but culture positive). in addition to pulmonary tuberculosis, extrapulmonary disease occurs in a high proportion of hiv-infected individuals with low cd4 lymphocyte counts (<150 cells/ml). mycobacteremia and lymph node infection ( figure 34 -6) are common, but involvement of bone marrow, liver, pericardium, meninges, and brain also occurs. evidence of extrapulmonary tuberculosis should be sought in any hiv-infected patient with suspected or confirmed pulmonary tuberculosis, by culture of stool, urine, and blood or bone marrow. traditional solid phase culture and speciation techniques may take 6-10 weeks. liquid culture methods (e.g., bactec, becton dickinson) that detect early growth may provide a diagnosis in only 2-3 weeks. molecular diagnostic tests that use m. tuberculosis genome detection (e.g., by polymerase chain reaction [pcr] ) offer the possibility of yet more rapid diagnosis (within hours), but are not yet in routine clinical use. they are also less useful in primary samples with low bacterial load (e.g., smear negative sputum)-which is often when they will be most needed in hiv-coinfected patients. the recent description of simple, but highly sensitive and specific, methods that use the inoculation of large quantities of, for example, sputum onto microscopic plates with subsequent rapid detection (in days) of both mycobacterial growth and resistance patterns (mods) is of great potential significance. until the results of culture and speciation are known, acidfast bacilli identified in respiratory samples, biopsy tissue, an aspirate, or blood in an hiv-infected individual, regardless of the cd4 lymphocyte count, should be regarded as being m. tuberculosis, and conventional antituberculosis therapy should be commenced. if culture fails to demonstrate m. tuberculosis and instead another mycobacterium (see later) is identified, treatment can be modified. multiple drug-resistant (mdr) tuberculosis-that is, m. tuberculosis that is resistant to isoniazid and rifampicin (rifampin), with or without other drugs, is now an important clinical problem in hiv-infected individuals in the united states, where it is responsible for approximately 3% of all tuberculosis in hiv-infected patients. outbreaks of mdr tuberculosis have occurred in both hiv-infected and non-hiv-infected individuals in the united states in prison facilities, hostels, and hospitals. similar outbreaks have also been documented among hiv-infected patients in europe. inadequate treatment (including case management and supervision of medication) of tuberculosis and poor patient compliance with antituberculosis therapy are the most important risk factors for development of mdr tuberculosis. other cases have arisen because of exogenous reinfection of profoundly immunosuppressed hiv-infected patients who are already receiving treatment for drug-sensitive disease. despite antituberculosis therapy, the median survival in hiv-infected individuals with mdr-tuberculosis was initially only 2-3 months. recently this has improved, largely because of an increased awareness of the condition with early initiation of suitable therapy as determined by drug sensitivity testing. extensively drug-resistant (xdr) tuberculosis-that is, m. tuberculosis resistant to isoniazid and rifampicin (rifampin), plus any fluoroquinolone and one or more of the three injectable second-line drugs (capreomycin, kanamycin and amikacin)-is an increasingly important clinical problem. originally described in south africa in association with hiv infection, xdr tuberculosis has also been identified in most parts of the world. as of march 2007, 35 countries had reported at least one case; although in many places, testing for fluoroquinolone sensitivity is not standard practice; this number may be, in fact, a huge underestimate. what is of concern about xdr tuberculosis is that, despite specific therapy, mortality is high among hiv-infected individuals. the current picture seems to mirror early reports of mdr tuberculosis in hiv infection: in the original south african study from kwazulu natal, survival was less than 3 weeks from the time of receipt of the first sputum sample. mycobacterium avium-intracellulare complex. before the widespread availability of haart, disseminated mac infection developed in up to 50% of hiv-infected patients. it remains a problem in patients with advanced hiv disease not receiving antiretroviral therapy and who have cd4 lymphocyte counts <50 cells/ml. clinical presentation is nonspecific and may be confused with the effects of hiv itself. fever, night sweats, weight loss, anorexia, and malaise are common. anemia, hepatosplenomegaly, abdominal pain, and chronic diarrhea are frequent findings. the diagnosis of disseminated mac infection is based on culture of the organism from blood, bone marrow, lymph node, or liver biopsy specimens. also, mac is frequently identified in bal fluid, sputum, stool, and urine, but detection of the organism at these sites is not diagnostic of disseminated infection. evidence of pulmonary mac infection is not usually obtained from a chest radiograph, which may be negative or show nonspecific infiltrates. rarely, focal consolidation, nodular infiltrates, and apical cavitation (resembling m. tuberculosis) have been reported. mycobacterium kansasii. mycobacterium kansasii is the second most common nontuberculous opportunistic mycobacterial infection in hiv-infected individuals and usually appears late in the course of hiv infection in patients with cd4 lymphocyte counts <100 cells/ml. the most frequent presentation is with fever, cough, and dyspnea. in approximately two thirds of those who have m. kansasii infection, the disease is localized to the lungs; the remainder have disseminated disease that affects bone marrow, lymph node, skin, and lungs. the diagnosis is made by culture of the organism from respiratory secretions or from bone marrow, lymph node aspirate, or skin biopsy. focal upper lobe infiltrates with diffuse interstitial infiltrates are the most common radiographic abnormalities; thin-walled cavitary lesions and hilar adenopathy have also been reported. mycobacterium xenopi. mycobacterium xenopi may occasionally be isolated from sputum or bal fluid samples, but its significance is uncertain. patients have low cd4 counts, and m. xenopi is usually accompanied by isolation of a copathogen, such as p. jirovecii. treatment of the latter condition is associated in most cases with resolution of symptoms. there is some evidence that starting haart prevents disease recurrence, provided there is an adequate immune response. pneumocystis jirovecii pneumonia. the development of pcp is largely related to underlying states of immunosuppression induced by malignancy or treatment thereof, organ transplantation, or hiv infection. in 2007 in the united states, united kingdom, europe, and australasia, pcp is largely seen only in hiv-infected individuals unaware of their serostatus or in those who are intolerant of, or noncompliant with, anti-p. jirovecii prophylaxis and haart. until recently, p. jirovecii was regarded taxonomically as a protozoan, on the basis of its morphology and the lack of response to antifungal agents such as amphotericin b. the organism has now been ascribed to the fungal kingdom. the demonstration of antibodies against p. jirovecii in most healthy children/adults suggests that organisms are acquired in childhood and persist in the lungs in a dormant phase. subsequent immunosuppression (e.g., as a result of hiv infection) allows the fungus to propagate in the lung and cause clinical disease. however, this "latency" hypothesis is challenged by several observations: p. jirovecii cannot be identified in the lungs of immunocompetent individuals. "case clusters" of pcp in health care facilities suggest recent transmission. different genotypes of p. jirovecii are identified in each episode in hiv-infected patients who have recurrent pcp. genotypes of p. jirovecii in patients who have pcp correlate with place of diagnosis and not with their place of birthsuggesting infection has been recently acquired. taken together, these data suggest that pcp arises by reinfection from an exogenous source. the clinical presentation of pcp is nonspecific, with an onset of progressive exertional dyspnea over days or weeks, together with a dry cough with or without expectoration of minimal quantities of mucoid sputum. patients often complain of an inability to take a deep breath, which is not due to pleurisy (table 34-5) . fever is common, yet patients rarely complain of temperatures or sweats. in hiv-infected patients, the presentation is usually more insidious than in patients receiving immunosuppressive therapy, with a median time to diagnosis from onset of symptoms of more than 3 weeks in those with hiv compared with less than 1 week in non-hiv-infected patients. in a small proportion of hiv-positive individuals, the disease course of pcp is fulminant, with an interval of only 5-7 days between onset of symptoms and progression to development of respiratory failure. in others, it may be much more indolent, with respiratory symptoms that worsen almost imperceptibly over several months. rarely, pcp may present without respiratory symptoms as a fever of undetermined origin. clinical examination is usually remarkable only for the absence of physical signs; occasionally, fine, basal, end-inspiratory crackles are audible. features that would suggest an alternative diagnosis include a cough productive of purulent sputum or hemoptysis, chest pain (particularly pleural pain), and signs of focal consolidation or pleural effusion (see table 34 -5). it should be noted that infection with more than one pathogen occurs in almost one fifth of individuals, and thus symptoms may be the product of several agents. the chest radiograph in pcp is typically unremarkable initially. later, diffuse reticular shadowing, especially in the perihilar regions, is seen and may progress to diffuse alveolar consolidation that resembles pulmonary edema if untreated or if the patient is seen late in disease. at this stage, the lung may be massively consolidated and almost airless (figure 34-7) . up to 20% of chest radiographs are atypical, showing lobar consolidation, honeycomb lung, multiple thin-walled cystic air space formation (pneumatoceles), intrapulmonary nodules, cavitary lesions, pneumothorax, and hilar and mediastinal lymphadenopathy. predominantly apical changes, resembling tuberculosis, may occur in patients who have pcp develop having received anti-p. jirovecii prophylaxis with nebulized pentamidine (figure 34 -8). all these radiographic changes chest radiograph early: perihilar "haze" or bilateral interstitial shadowing late: alveolar-interstitial changes or "white out" (marked alveolar consolidation with sparing of apices and costophrenic angles) arterial blood gases pao 2 : early, normal: late, low paco 2 : early, normal or low; late, normal or high are nonspecific, and similar changes occur with other pulmonary pathogens, including pyogenic bacterial, mycobacterial, and fungal infection, as well as kaposi sarcoma and nonspecific interstitial pneumonitis. respiratory symptoms in an immunosuppressed, hiv-infected individual with a negative chest radiograph should not be discounted, because over an interval of 2-3 days radiographic abnormalities may appear. the diagnosis of pcp is made by demonstration of the organism in induced sputum, bal fluid, or lung biopsy material by use of histochemical or immunofluorescence techniques. the early promise of molecular diagnostic techniques has not been borne out. many fungal infections of the lung are confined to specific geographic regions, although with widespread travel, they may present in patients outside these areas. candida, aspergillus, and cryptococcus species are ubiquitous and occur worldwide. in contrast to infections of the oropharynx and esophagus, candidal infection of the trachea, bronchi, and lungs is rare in hiv-infected patients, as are candidemia, disseminated candidiasis, and deep focal candidiasis. the clinical presentation of pulmonary candidal infection has no specific features. chest radiography is equally nonspecific-it may be negative or show patchy infiltrates. isolation of candida from sputum may simply represent colonization and does not mean the patient has candidal pneumonia. indirect evidence may be obtained from positive cultures or rising antibody titers. however, in hivinfected patients, a high antibody titer alone is a less reliable indicator, and antibodies may be absent in proven cases of invasive candidal infection. some correlation occurs between identification of large quantities of candida species in bal fluid and candida species as the cause of pneumonia. definitive diagnosis is made by lung biopsy. by contrast with patients immunosuppressed and rendered neutropenic by systemic chemotherapy, infection with aspergillus species is relatively rare in hiv-positive individuals. risk factors for aspergillosis are neutropenia, which is commonly drug induced (zidovudine or ganciclovir), or patient's receipt of corticosteroids. fever, cough, and dyspnea are the most common presenting symptoms, but pleuritic chest pain and hemoptysis are found in approximately one third of patients. patterns of pulmonary disease include cavitating upper lobe disease, focal radiographic opacities resembling bacterial pneumonia, bilateral diffuse and patchy opacities (nodular or reticular-nodular in pattern), pseudomembranous aspergillosis, which may obstruct the lumen of airways, and tracheobronchitis. diagnosis of pulmonary aspergillosis is made by the identification of fungus in sputum, sputum casts, or bal fluid associated with respiratory tract tissue invasion ( figure 34 -9). the role of antigen testing (such as galactomannan assays), which is commonplace in hematology patients at risk of invasive aspergillus, has not been clearly defined in hiv-infected individuals. infection may present in one of two ways: either as primary cryptococcosis or complicating cryptococcal meningitis as part of disseminated infection with cryptococcemia, pneumonia, and cutaneous disease (umbilicated papules mimicking molluscum contagiosum; figure 34 -10). primary pulmonary cryptococcosis presents in a very nonspecific way and is frequently indistinguishable from other pulmonary infections. in disseminated infection, the presentation is frequently overshadowed by headache, fever, and malaise (caused by meningitis). the duration of onset may range from only a few days to several weeks. examination may reveal skin lesions, lymphadenopathy, and meningism. in the chest, signs may be absent or crackles may be audible. arterial blood gas tensions may be normal or show hypoxemia. the most common abnormality on the chest radiograph is focal or diffuse interstitial infiltrates. less frequently, masses, mediastinal or hilar lymphadenopathy, nodules, and effusion are noted. the diagnosis of cryptococcal pulmonary infection ( figure 34 -11) is made by identification of cryptococcus neoformans (by staining with india ink or mucicarmine, and by culture) in sputum, bal fluid, pleural fluid, or lung biopsy. cryptococcal antigen may be detected in serum by use of the cryptococcal latex agglutination (crag) test. titers are usually high but may be negative in primary pulmonary cryptococcosis, in which case bal fluid (crag) is positive. in patients with disseminated infection, c. neoformans may also be cultured from blood and cerebrospinal fluid. the mortality rate is high in this disseminated form (up to 80%). the endemic mycoses caused by histoplasma capsulatum, coccidioides immitis, and blastomyces dermatitidis are found in hiv-infected patients living in north america (especially the mississippi and ohio river valleys). histoplasmosis is also found in southeast asia, the caribbean islands, and south america. coccidioidomycosis is endemic in the southwest united states (southern california), northern mexico, and in parts of argentina and brazil. blastomycosis has a similar distribution, with an extension north into canada. progressive, disseminated histoplasmosis in patients with hiv typically presents with a subacute onset of fever and weight loss; approximately 50% of patients have mild respiratory symptoms with a nonproductive cough and dyspnea. hepatosplenomegaly is frequently found on examination, and a rash (similar to that produced by cryptococcus species) may be seen. rarely, the presentation may be rapidly fulminant, with clinical features of the sepsis syndrome, including anemia or disseminated intravascular coagulation. the chest radiograph may be unremarkable (in up to one third of patients), although characteristic abnormalities are bilateral, widespread nodules 2-4 mm in size. other radiographic features are nonspecific and include interstitial infiltrates, reticular nodular shadowing, and alveolar consolidation. histoplasmosis may disseminate to the central nervous system and produce meningoencephalitis or mass lesions. the diagnosis is made reliably by identification of the organism in wright-stained peripheral blood or by giemsa staining of bone marrow, lymph node, skin, sputum, bal fluid, or lung tissue. it is important that identification is confirmed by detection of h. capsulatum var. capsulatum polysaccharide antigen by radioimmunoassay, which has a high sensitivity. false-positive results may occur in patients infected with blastomycosis and coccidioides species. tests for histoplasma antibodies by complement fixation or immunodiffusion may be negative in immunosuppressed, hiv-positive patients. the clinical presentation of coccidioidomycosis is variable. the chest radiograph may show focal pulmonary disease with focal alveolar infiltrates, adenopathy, and intrapulmonary cavities or, alternately, diffuse reticular infiltrates. diagnosis is made by isolation of the organism in sputum or bal fluid. disseminated disease is identified by isolating the fungus in blood, urine, or cerebrospinal fluid. serologic tests may also be used for diagnosis. blastomycosis presents in patients who have advanced hiv infection, when cd4 lymphocyte counts are usually less than 200 cells/ml. clinical symptoms include cough, fever, dyspnea, and weight loss. patients may present late in respiratory failure. disseminated disease can occur with both pulmonary and extrapulmonary features. there is frequently multiple involvement of the skin, liver, brain, and meninges. chest radiographic abnormalities include focal pneumonic change, miliary shadowing, or diffuse interstitial infiltrates. diagnosis is made by culture from bal fluid, skin, and blood. in this infection, cytologic or histologic diagnosis is important for early diagnosis, because culture of the organism may take 2-4 weeks. the mortality rate is high in patients with disseminated infection. p. marneffei infection is particularly common in southeast asia. most hiv-infected patients present with disseminated infection and solitary skin or oral mucosal lesions, or with multiple infiltrates in the liver or spleen, or bone marrow (leading to presentation with pancytopenia). pulmonary infection has no specific clinical features, and chest radiographs may be negative or show diffuse, small nodular infiltrates. diagnosis is made by identifying the organism in bone marrow, skin biopsy samples, blood films, or bal fluid. the differential diagnosis of p. marneffei infection includes both pcp and tuberculosis. these occur with equal frequency in hiv-infected and non-hiv-infected patients; however, respiratory complications after influenza infection are increased in patients affected with underlying conditions such as cardiac or pulmonary disease and immunosuppression. in prospective studies of hivinfected patients undergoing bronchoscopy for evaluation of suspected lower respiratory tract disease, the communityacquired respiratory viral infections (i.e., influenza, parainfluenza, respiratory syncytial virus, rhinovirus, coronavirus and adenovirus) are found only rarely, if at all. cmv chronically infects most hiv-infected individuals, and up to 90% of homosexual hiv-infected men shed cmv intermittently in urine, semen, and saliva. clinical disease may be caused by cmv in patients who have advanced hiv infection and cd4 counts <100 cells/ml. chorioretinitis is most frequently encountered, but encephalitis, adrenalitis, esophagitis, and colitis are also seen. frequently, cmv is isolated from bal fluid, being found in 40% of samples from patients with cd4 counts <100 cells/ml. however, the role of cmv in causing disease in this context is unclear (see later). in patients who have cmv as the sole identified pathogen, clinical presentation and chest radiographic abnormalities (usually diffuse interstitial infiltrates) are nonspecific. diagnosis of cmv pneumonitis is made by identifying characteristic intranuclear and intracytoplasmic inclusions, not only in cells in bal fluid but also in lung biopsy specimens (figure 34-12 ). pulmonary involvement with leishmania species may rarely occur as part of the syndrome of visceral leishmaniasis in hiv-infected patients. patients usually have advanced hiv disease with cd4 lymphocyte counts less than 300 cells/ml and present with unexplained fever, splenomegaly, and leukopenia. respiratory symptoms are often absent. diagnosis of visceral leishmaniasis is most often made by staining a splenic or bone marrow aspirate and subsequent culture. occasionally, the parasite is found by chance in a skin or rectal biopsy or bal fluid taken for other purposes. the chest radiograph may be negative or show reticular-nodular infiltrates. toxoplasma gondii infection in patients who have aids usually occurs as a result of reactivation of latent, intracellular protozoa acquired in a primary infection. patients are invariably systemically unwell, with malaise and pyrexia. clinical disease in association with hiv infection is most commonly seen in the central nervous system, where it produces single or multiple abscesses. multisystem infection with t. gondii is uncommon in patients who have hiv infection. toxoplasmic pneumonia is frequently difficult to distinguish from pcp. nonproductive cough and dyspnea are the symptoms most commonly reported. chest radiographic abnormalities include diffuse interstitial infiltrates indistinguishable from those of pcp ( figure 34-13) , as well as micronodular infiltrates, a coarse nodular infiltrate, cavitary change, and lobar consolidation. the diagnosis is made by hematoxylin-eosin or giemsa staining of bal fluid that reveals cysts and trophozoites of t. gondii. staining of bal fluid is not always positive; the diagnostic yield is increased either by staining of transbronchial biopsy material or by performing nucleic acid amplification procedures such as pcr to detect t. gondii dna in bal fluid. the most frequent manifestation of infection with cryptosporidium species in hiv infection is a noninflammatory diarrhea that may be of high volume, intractable, and life threatening. cryptosporidium species may colonize epithelial surfaces, including the trachea and lungs, occasionally resulting in pulmonary infection. most cases of pulmonary cryptosporidiosis have co-pathology such as pcp or bacterial pneumonia; ascertaining the exact role of cryptosporidiosis as the cause of respiratory symptoms may be difficult. diagnosis is made by ziehl-neelsen or auramine-rhodamine staining of bal fluid or transbronchial biopsy specimens. pulmonary microsporidia infection may occur as part of systemic dissemination from gastrointestinal infection with septata intestinalis or encephalitozoon hellem. the organism may be identified by conventional staining in bal fluid. electron microscopy is necessary to distinguish the two species. the nematode strong yloides stercoralis is endemic in warm countries worldwide. in immunosuppressed patients, the organism has an increased ability to reproduce parthenogenetically in the gastrointestinal tract without the need for repeated exposure to new infection-so-called autoinfection. this results in a great increase in worm load and a hyperinfective state ensues; massive acute dissemination with s. stercoralis may occur in the lungs, kidneys, pancreas, and brain. although infection with s. stercoralis is more severe in immunocompromised patients, it is no more common in patients who have hiv infection. presentation with hyperinfection may be with fever, hypotension secondary to bacterial sepsis, or disseminated intravascular coagulation. the clinical features of respiratory s. stercoralis infection are nonspecific. s. stercoralis in sputum or bal fluid (figure 34 -14) may be identified in hiv-positive patients in the absence of symptoms elsewhere; this can predate disseminated infection and, as such, requires prompt treatment. it is apparent from the foregoing discussion that hiv-related pneumonia of any cause may present in a similar manner. a wide range of investigations is available to aid diagnosis. these are listed in table 34 -6. if the subject is producing sputum, it is important to obtain samples for bacterial and mycobacterial detection. in up to one third of cases, these will assist in diagnosis. three samples on consecutive days (preferably either with overnight or early morning production) is the critical first step in the diagnosis of pulmonary tuberculosis. this is considerably easier and safer for health care personnel than obtaining hypertonic saline-induced sputum or bal fluid. blood cultures are also important, because very high rates of bacteremia have been reported in both bacterial and mycobacterial disease (see earlier). a patient who is initially seen with symptoms and signs consistent with pneumonia should have chest radiography and arterial oxygen assessments performed at his or her first consultation. the question at this stage is usually whether this infectious episode is due to bacterial infection, tuberculosis, or pcp. in general, alveolar and interstitial shadowing is taken as evidence for pcp, although important caveats apply. transcutaneous pulse oximetry and arterial blood gas analysis are useful tests for hypoxemia. they can be used to distinguish an alveolar condition (i.e., pcp) from bacterial pneumonia. the alveolitis produces a greater impairment of oxygen transfer (especially during exercise), such that for a given clinical situation there will be more hypoxemia and a wider alveolararterial oxygen gradient (a-ao 2 ) in those with pcp. with pulse oximetry, this manifests as low oxygen saturations at rest that decrease further with exercise. in general, more information can be obtained from arterial blood gas analysis, although this advantage is offset by the need for direct arterial puncture. of patients with pcp, fewer than 10% have a normal pao 2 and a normal a-ao 2 . these measures are sensitive, although not particularly specific for pcp, and similar results may occur with bacterial pneumonia, pulmonary kaposi sarcoma, and m. tuberculosis infection. the diagnostic value of identifying exercise-induced desaturation, measured by transcutaneous oximetry, has been validated only in hiv-infected patients who have pcp and a normal or "near normal" appearance on chest radiographs. the test's value has not been confirmed in patients with abnormal chest radiographs because of pcp or other pathogens. exercise-induced desaturation may persist for many weeks after treatment and recovery from pcp, even in the absence of active pulmonary disease. abnormalities of lung function are well documented with hiv infection. the most common of these relate to tests measuring gas exchange, rather than the size of the conducting airways. in general, an overall reduction in diffusing capacity for carbon monoxide (dlco) occurs at all stages of hiv infection, with the largest changes found in hiv-infected patients with pcp. thus, to some extent, patients who have probable pcp can be differentiated and treatment guided. a normal dlco in an individual who has symptoms but a negative or unchanged chest radiograph makes the diagnosis of pcp extremely unlikely. data from the north american pspc cohort study suggest that individuals with rapid rates of decline in dlco are at an increased risk for development of pcp. recent work suggests that hiv-infected smokers are at increased risk of early-onset emphysema. smoking history must, therefore, be taken into account when assessing a patient's lung function results in the context of possible pcp. high-resolution (fine-cut) ct scanning of the chest may be helpful when the chest radiograph is normal, unchanged, or equivocal. the characteristic appearance of an alveolitis (i.e., areas of ground-glass attenuation through which the pulmonary vessels can be clearly identified) may be present, which indicates active pulmonary disease (figure 34-15 ). this feature, however, is neither sensitive nor specific for pcp, although its sensitivity can be improved if evidence for reticulation and/or small cystic lesions is added to this. hence, a negative test result implies an alternative diagnosis. in the context of an hiv-infected patient who is seen with an acute or subacute pneumonitis, an elevated serum lactate dehydrogenase (ldh) enzyme level is strongly suggestive of pcp. when interpreting such results, it is important to remember that other pulmonary disease processes (e.g., pulmonary embolism, nonspecific pneumonitis, and bacterial and mycobacterial pneumonia) and extrapulmonary disease (castleman disease and lymphoma) may also cause elevations of ldh and may need to be considered in the correct clinical context. from the previous information, it is evident that noninvasive tests cannot reliably distinguish the different infecting agents from each other but may be useful in excluding acute opportunistic disease. thus, the clinician is left with either proceeding to diagnostic lung fluid or tissue sampling (by either induced sputum collection or bronchoscopy and bal with or without transbronchial biopsy table 34-7) or treating an unknown condition empirically. haart has also altered the investigation of respiratory disease. the numbers of invasive procedures performed are falling and tend to be in patients spontaneously expectorated sputum is inadequate for diagnosis of pcp. sputum induction by inhalation of ultrasonically nebulized hypertonic saline may provide a suitable specimen (see table 34 -7). the technique requires close attention to detail and is much less useful when samples are purulent. sputum induction must be carried out away from other immunosuppressed patients and health care workers, ideally in a room with separate negative-pressure ventilation, to reduce the risk of nosocomial transmission of tuberculosis. although very specific (>95%), the sensitivity of induced sputum varies widely (55-90%), and therefore a negative result for p. jirovecii prompts further diagnostic studies. the use of immunofluorescence staining enhances the yield of induced sputum compared with standard cytochemistry. fiberoptic bronchoscopy with bal is commonly used to diagnose hiv-related pulmonary disease. when a good "wedged" sample is obtained, the test has a sensitivity of more than 90% for detection of p. jirovecii (figure 34-16 ). just as with induced sputum, fluorescent staining methods increase the diagnostic yield, which makes it the procedure of choice in most centers. more technically demanding (both of the patient and the operator) than induced sputum collection, bronchoscopy and bal have the advantage that direct inspection of the upper airway and bronchial tree can be performed and, if necessary, biopsies taken. transbronchial biopsies may marginally increase the diagnostic yield of the procedure. this is relevant for the diagnosis of mycobacterial disease, although the relatively high complication rate in hiv-infected individuals (pneumothorax and the possibility of significant pulmonary hemorrhage in up to 10%) outweighs the advantages of the technique for routine purposes. samples of bal fluid are examined for bacteria, mycobacteria, viruses, fungi, and protozoa. inspection of the cellular component may also provide etiologic clues-cooperation of a pathology department with experience in opportunistic infection diagnosis is vital. the drug interactions associated with antiretroviral protease inhibitor (pi) therapy mean that special care should be exercised when sedation is used with either benzodiazepine or opiate drugs. prolonged sedation and life-threatening arrhythmias have been reported. a diagnostic strategy, therefore, includes sputum induction and, if results are nondiagnostic or if the test is unavailable, bronchoscopy and bal. if this does not yield a result, consideration is given to either a repeat bronchoscopy and bal with transbronchial biopsies or surgical biopsy. the latter can be performed as either an open lung or thoracoscopic procedure. surgical biopsy has a high sensitivity. although empirical therapy is usually reserved for the management of presumed bacterial pneumonias, and at first sight may seem unwise when dealing with possible opportunistic infection, in reality pcp is almost invariably a diagnosis of exclusion, and certain clinical and laboratory features may guide the assessment of an hiv-infected individual's risk for this condition. the likelihood that p. jirovecii is the causative organism increases if the subject is not taking effective anti-pneumocystis drug prophylaxis or has a previous medical history with clinical or laboratory features that suggest systemic immunosuppression (i.e., recurrent oral thrush, longstanding fever of unknown cause, clinical aids, or blood cd4 count <200 cells/ml). hence, some centers advocate use of empirical therapy for hiv-infected patients who are seen with symptoms and chest radiographic and blood gas abnormalities typical of mild pcp, without the need for bronchoscopy. invasive measures are reserved for those with an atypical radiographic presentation, those who fail to respond to empirical therapy by day 5, and those who deteriorate at any stage. most clinicians in north america and the united kingdom would seek to obtain a confirmed diagnosis in every case of suspected pcp. in practice, both strategies seem to be equally effective, although a number of caveats should be borne in mind when empirical treatment is given for pcp. patients who have pcp typically take 4-7 days to show clinical signs of improvement, so a bronchoscopically proven diagnosis ensures that the treatment being given is correct, particularly in the first few days of therapy, when it may not be well tolerated. in addition, the diagnosis of pcp has implications for the infected individual, because it may influence the decision to start either haart or anti-pneumocystis prophylaxis. finally, empirical therapy requires the patient to be maximally adherent to treatment, because nonresolution of symptoms may be seen as failure of therapy rather than of compliance. molecular biologic techniques (such as pcr) are increasingly used in the diagnosis of respiratory disease. two examples of this are dna amplification of loci of the p. jirovecii and m. tuberculosis genomes. the advantages of molecular methods are that the diagnosis may be made by use of samples that are more readily obtained than bal fluid (i.e., expectorated sputum or nasopharyngeal secretions) and also that these methods are rapid (the answer may be available within a working day, compared with conventional mycobacterial culture, which may take weeks). despite encouraging results in the research setting (sensitivity and specificity have been reported as 60-100% and 70-100%, respectively), problems persist when these techniques are applied to routine diagnostic samples. these include extraction of nucleic acid from clinical material, cross-contamination with the products of previous assays, and clinical interpretation of a test result. currently, treatment individuals infected with hiv, compared with the non-hivinfected general population, have an increased likelihood of adverse reactions to therapy. this includes tmp/smx (see later) and other antibacterial and antimycobacterial agents. in addition, there are complex drug interactions with other medications, particularly components of haart. before instituting therapy for any infectious complication in an hivinfected individual, it is important to consult with a physician experienced in the care of patients with hiv infection and to seek advice from a specialist pharmacist. the main organisms causing pneumonia in hiv-infected individuals are similar to those found in the general population with community-acquired pneumonia. thus, bacterial pneumonia in hiv-infected patients should be treated in a similar manner to that in hiv-negative individuals, by use of the published american thoracic society (ats) and british thoracic society (bts) guidelines. in addition, expert advice on local antibiotic resistance patterns should be sought from infectious disease or microbiology colleagues, because treatment is usually begun on an empirical basis before the causative organism is identified and antibiotic sensitivities known. the same clinical and laboratory prognostic indices that are described for the general population apply to hiv-infected patients and should be documented on presentation. response to appropriate antibiotic therapy is usually rapid and is similar to that seen in the non-hiv-infected individual. early relapse of infection after successful treatment is well described. those hiv-infected patients who have presumed pcp and are being treated empirically with high-dose tmp/ smx, and who have infection with either s. pneumoniae or h. influenzae rather than p. jirovecii, may also improve. in addition, in those patients who are treated with benzylpenicillin for proven s. pneumoniae pneumonia but do not respond, and penicillin resistance can be discounted as the cause, it is important to consider whether there is a second pathologic process, such as pcp. co-pathogens are reported in up to 20% of cases of pneumonia. before instituting treatment, assessment of the severity of pcp should be performed on the basis of history, findings on examination, arterial blood gas estimations, and chest radiographic abnormalities. patients can then be stratified into those with mild, moderate, or severe disease (table 34-8) . this is important, because some drugs are of unproven benefit and others are known to be ineffective for the treatment of severe disease. in addition, adjuvant glucocorticoid therapy may be given to patients with moderate or severe pneumonia. patients with glucose-6-phosphate dehydrogenase deficiency should not receive tmp/smx, dapsone, or primaquine, because these drugs increase the risk of hemolysis. several drugs are effective in the treatment of pcp. tmp/ smx is the drug of first choice (tables 34-9 and 34-10). overall it is effective in 70-80% of individuals when used as firstline therapy. adverse reactions to tmp/smx are common and usually become apparent between days 6 and 14 of treatment. neutropenia and anemia (in up to 40% of patients), rash and fever (up to 30%), and biochemical abnormalities of liver function (up to 15%) are the most frequent adverse reactions. hematologic toxicity induced by tmp/smx is neither attenuated nor prevented by coadministration of folic or folinic acid. furthermore, the use of these agents may be associated with reduced therapeutic success. during treatment with tmp/ smx, full blood count, liver function, and urea and electrolytes should be monitored at least twice weekly. it is not known why hiv-infected individuals, especially those with higher cd4 counts, have such a high frequency of adverse reactions to tmp/smx. the optimum strategy for an hiv-infected patient who has pcp and who becomes intolerant of high-dose tmp/smx has not been established. many physicians "treat through" minor rash, often adding an antihistamine and a short course of oral prednisolone (30 mg every 24 h, reducing to zero over 5 days). if treatment with tmp/smx fails, or is not tolerated by the patient, several alternative therapies are available (see tables 34-9 and 34-10). the combination of clindamycin and primaquine is widely used for treatment of pcp whatever the severity, although there is no license in the united kingdom or united states for this indication. the combination is as effective as oral tmp/smx and oral trimethoprim-dapsone for the treatment of mild and moderate-severity disease. as a second-line treatment it is effective in up to 90% of patients. methemoglobinemia caused by primaquine occurs in up to 40% of patients. if 15 mg four times daily of primaquine is used, rather than 30 mg four times daily, the likelihood of methemoglobinemia is reduced. diarrhea develops in up to 33% of patients receiving clindamycin. if this occurs, stool samples should be analyzed for the presence of clostridium difficile toxin. this oral combination is as effective as oral tmp/smx and oral clindamycin plus primaquine (see earlier) for treatment of mild and moderate-severity pcp. the combination has not been shown to be effective in patients who have severe pcp. most patients experience methemoglobinemia (caused by dapsone), which is usually asymptomatic. up to one half of patients have mild hyperkalemia (<6.1 mmol/l) caused by trimethoprim. a methotrexate analog, trimetrexate, is given intravenously together with folinic acid "rescue" to protect human cells from trimetrexate-induced toxicity. in patients who have moderate regimen recommended by cdc/ nih/idsa, widely used in usa methylprednisolone iv at 75% of dose given above for prednisolone methylprednisolone prednisolone 1 iv q24h, days 1-3 0.5 iv days 4-6 then 40 g po q24h reducing to 0, days 7-16 nb, none of these regimens for adjuvant glucocorticoid therapy have been compared in prospective clinical trials. to severe disease, trimetrexate-folinic acid is less effective than high-dose tmp/smx, but serious treatment-limiting hematologic toxicity occurs less frequently with trimetrexate-folinic acid. atovaquone is licensed for the treatment of mild and moderate-severity pcp in patients who are intolerant of tmp/ smx. in tablet formulation (no longer available), this drug was less effective but was better tolerated than tmp/smx or intravenous pentamidine for treatment of mild or moderateseverity pcp (see tables 34-9 and 34-10). there are no data from prospective studies that compare the liquid formulation (which has better bioavailability) with other treatment regimens. common adverse reactions include rash, fever, nausea and vomiting, and constipation. absorption of atovaquone is increased if it is taken with food. intravenous pentamidine is now seldom used for the treatment of mild or moderate-severity pcp because of its toxicity. intravenous pentamidine may be used in patients who have severe pcp, despite its toxicity, if other agents have failed (see tables 34-9 and 34-10). nephrotoxicity develops in almost 60% of patients given intravenous pentamidine (indicated by elevation in serum creatinine), leukopenia develops in approximately half, and up to 25% have symptomatic hypotension or nausea and vomiting. hypoglycemia occurs in approximately 20% of patients. given the long half-life of the drug, this may occur up to several days after the discontinuation of treatment. pancreatitis is also a recognized side effect. for patients who have moderate and severe pcp, adjuvant glucocorticoid therapy reduces the risk of respiratory failure by up to half and the risk of death by up to one third (see tables 34-9 and 34-10). glucocorticoids are given to hivinfected patients with confirmed or suspected pcp who have a pao 2 <9.3 kpa (<70 mmhg) or an a-ao 2 of >4.7 kpa (<33 mmhg). oral or intravenous adjunctive therapy is given at the same time as (or within 72 h of starting) specific anti-p. jirovecii therapy. clearly, in some patients treatment is commenced on a presumptive basis, pending confirmation of the diagnosis. in prospective studies, adjuvant glucocorticoids have not been shown to be of benefit in patients with mild pcp. however, it would be difficult to demonstrate this, given that survival in such cases approaches 95% with standard treatment. patients with mild pcp may be treated with oral tmp/smx as outpatients if they are able to manage at home, willing to attend the outpatient clinic for regular review, and that there is clinical and radiographic evidence of recovery. if the patient is intolerant of oral tmp/smx despite clinical recovery, either the treatment is given intravenously or treatment may be changed to oral clindamycin plus primaquine. all patients with moderate and severe pcp should be hospitalized and given intravenous tmp/smx or intravenous clindamycin and oral primaquine (plus adjuvant steroids). patients with moderate or severe disease who show clinical and radiographic response by day 7-10 of therapy may be switched to oral tmp/smx to complete the remaining 14 days of treatment. if the patient has failed to respond within 7-10 days or deteriorates before this time while receiving tmp/smx, then treatment should be changed to clindamycin and primaquine or trimetrexate plus folinic acid. deterioration in a patient who is receiving anti-p. jirovecii therapy may occur for several reasons (table 34-11) . before ascribing deterioration to treatment failure and considering a change in therapy, these alternatives should be evaluated carefully. it is also important to consider treating any co-pathogens present in bal fluid, to perform bronchoscopy if the diagnosis was made empirically, to repeat the procedure, or to carry out open lung biopsy to confirm that the diagnosis is correct. most centers advocate admission to the icu for pcp with respiratory failure and for acute severe deterioration after bronchoscopy. the prognosis for severe pcp in such circumstances has improved over the past decade. this is likely because of a greater understanding of successful general icu management of respiratory failure and acute respiratory distress syndrome (ards) rather than specific improvements in pcp care. factors associated with poor outcome include increasing patient age, need for mechanical ventilation, and development of a pneumothorax. the latter reflects both the association between this complication and pcp, as well as the subsequent difficulty in successful mechanical ventilation of such individuals. the treatment of hiv-related mycobacterial disease is complex. not only do individuals have to take prolonged courses of relatively toxic agents, but also these antimycobacterial drugs have side effects similar to those of other prescribed in the developed world, isoniazid-related peripheral neuropathy is rare in hiv-negative subjects taking pyridoxine. the nucleoside reverse transcriptase inhibitors (rti) didanosine and stavudine, which are now less frequently used in the united states and the united kingdom but which remain a mainstay of haart in the developing world, can also cause a painful peripheral neuropathy. this complication develops in up to 30% of patients if stavudine and isoniazid are coadministered. rash, fever, and biochemical hepatitis are common adverse events with rifamycins, pyrazinamide, and isoniazid (occurring more frequently in patients with tuberculosis who have hiv infection with hepatitis c coinfection). the nonnucleoside rti drugs (e.g., nevirapine) have a similar toxicity profile. if treatment for both hiv and tuberculosis is co-administered, ascribing a cause may be problematic. drug-drug interactions between medications used to treat tuberculosis and hiv infection occur because of their common pathway of metabolism through the hepatic cytochrome p-450 enzyme system. rifampin is a potent inducer of this enzyme (rifabutin less so), which may result in subtherapeutic levels of nonnucleoside rti and pi antiretroviral drugs, with the potential for inadequate suppression of hiv replication and the development of resistance to hiv. in addition, the pi class of antiretroviral drugs inhibits the metabolism of rifamycins, which leads to increases in their plasma concentration and is associated with increased drug toxicity. the nonnucleoside rti drugs are inducers of this enzyme pathway. coadministration of rifabutin with efavirenz requires an increase in the dose of rifabutin to compensate for the increase in its metabolism induced by efavirenz (see later). the optimal duration of treatment of tuberculosis, by use of a rifamycin-based regimen, in a patient who has hiv infection is unknown. current recommendations (joint tuberculosis committee of the bts and the ats/cdc/infectious disease society of north america [idsa]) are to treat tuberculosis in hiv-infected patients in the same way as for the general population (i.e., for 6 months for drug-sensitive pulmonary tb). in addition, ats/cdc/idsa guidelines recommend that treatment be extended to 9 months in those who have cavitation on the original radiograph, continuing signs, or a positive culture after 2 months of therapy. recent work has highlighted the increased risk for development of rifampin monoresistance in hiv-infected individuals on treatment. this is especially so if intermittent regimens are used and may arise from a lack of efficacy of the other drugs present in the combination (e.g., intermittent isoniazid). hence, daily medication regimens are recommended and should be closely supervised in all hiv-positive patients. it should be remembered that although rifabutin is usually given three times a week with ritonavir-boosted protease inhibitors, this seems to achieve adequate rifamycin levels; there have been no reports of this leading to rifamycin resistance in patients who are appropriately adherent. directly observed therapy (dot) is an important, although fairly labor-intensive, strategy that has the support of the world health organization. the best time to start therapy in patients being treated for tuberculosis is unknown. decision analyses show that early treatment with antiretroviral therapy leads to a marked reduction in further opportunistic disease. against this is balanced the risk of needing to discontinue antituberculosis therapy or hiv therapy because of drug toxicity or drug-drug interactions. iris is reported to be more likely if the treatments are started at the same time as each other. pragmatically, delaying the start of antiretroviral therapy simplifies patient management and may reduce or prevent adverse drug reactions and drug-drug interactions and may also reduce the risk of iris. on the basis of current evidence, patients with cd4 counts >200 cells/ml have a low risk of hiv disease progression or death during 6 months of treatment for tuberculosis. in these patients, the cd4 count should be closely monitored, and antiretroviral therapy may be deferred until treatment for tuberculosis is completed. in patients who have cd4 counts from 199-100 cells/ml, many centers currently delay starting antiretroviral therapy until after the first 2 months of treatment for tuberculosis have been completed; patients are given concomitant pcp prophylaxis. in patients who have cd4 counts of <99 cells/ml, antiretroviral therapy is started as soon as possible after beginning treatment for tuberculosis. this is based on evidence that shows a significant short-term risk of hiv disease progression and death in this patient group if antiretroviral therapy is delayed. two options exist for starting antiretroviral therapy in a patient already being treated for tuberculosis. first, the rifampin-based regimen is continued, and antiretroviral therapy is commenced, for example, with a combination of two nucleoside rtis and a non-nucleoside rti, such as efavirenz (if the patient weighs <50 kg, the efavirenz dose is often increased to 800 mg once daily to compensate for rifampin-induced metabolism of efavirenz). alternately, the rifampin is stopped and rifabutin is started: antiretroviral therapy is given, with a combination of two nucleoside rti drugs and either a single ritonavir-boosted pi or a nonnucleoside rti. here the dose of rifabutin is adjusted to take into account the pharmacokinetic effect of the co-administered drug. with a boosted pi, it is usually prescribed at a dose of 150 mg three times weekly and with efavirenz it is increased to 450 mg once a day. before the advent of antiretroviral therapy, it was recognized by tuberculosis physicians that patients who were apparently responding to their antimycobacterial treatment would sometimes have a short period of clinical deterioration develop. this "paradoxical reaction" (in the face of overall treatment response) was seen as an interesting and probable immunebased phenomenon of generally little consequence. the widespread introduction of haart has led to an increased awareness by clinicians of similar, but generally more severe, events in hiv-infected individuals. in the context of hiv, these are termed iris, or immune reconstitution disease. they can present in a number of ways and with a range of opportunistic conditions. perhaps the most common of these is similar to a paradoxical reaction. here, after initiation of antiretroviral therapy in a patient being treated for tuberculosis, for example, there arises the return of the original or the development of new symptoms and signs. these are often of a systemic nature and may be associated with marked radiographic changes. examples of this include fever, dyspnea, lymphadenopathy, effusions, parenchymal pulmonary infiltrates, or expansion of cerebral tuberculomas. this form of iris is seen most frequently with mycobacteria (commonly tuberculosis or mac), fungi (notably, cryptococcus), and viruses (hepatitis and herpes viridae). iris develops in up to one third of hiv-infected patients being treated for tuberculosis when antiretroviral therapy is started. the median onset of tuberculosis-related iris is approximately 4 weeks from beginning antituberculosis treatment or 2 weeks from commencing haart. it seems to be more likely in patients who have disseminated tuberculosis (and hence presumably more antigen present as well as more potential for significant inflammatory reactions) and a lower baseline blood cd4 count. a rapid fall in hiv load, as well as a large increase in cd4 counts in response to haart, may also predict iris. the relationship between early use of haart and low blood cd4 counts suggests that care must be taken when starting antiretrovirals in patients with tb at sites where rapid expansion of an inflammatory mass could be life threatening. examples of this would include cerebral, pericardial, or peritracheal disease (figure 34-17) . it is important to note that iris is currently a diagnosis of exclusion. there is no laboratory test available to assist with this; it should be made only after progressive or (multi) drug-resistant tuberculosis, poor drug adherence (to either antituberculosis or antiretroviral agents) and drug absorption, or an alternative pathologic process have been excluded as an explanation for the presentation. criteria have been drawn up that seek to provide clinical diagnostic criteria (table 34-12) . the mechanism leading to iris is unclear. it is not due to failure of treatment of tuberculosis or to another disease process; if anything, it is most likely to represent an exuberant and uncontrolled response to mycobacterial antigens (from both dead and live organisms). current treatments include nonsteroidal antiinflammatory drugs or glucocorticoids. the latter are undoubtedly effective, although they can lead to hyperglycemia and hypertension. recent preliminary data suggest that the leukotriene receptor antagonist montelukast may be of benefit in iris (this drug is unlicensed for this indication). recurrent aspiration of lymph nodes or effusion may also be needed. although iris is often self-limiting, it may persist for several months. rarely, temporary discontinuation of antiretroviral therapy is required. in this situation there may be precipitous falls in cd4 counts; patients are at risk of other opportunistic infections. attention has also focused on what is possibly more of a concern-the form of iris referred to as "unmasking phenomenon." here, individuals with presumably latent tuberculosis infection who start haart have systemic active (and often infectious) tuberculosis develop within a 3-month period. although it is likely that the patient's disease would have presented in time anyway and that some of the reported cases may, in fact, represent ascertainment bias, the current view is that this is real and represents an adverse effect of haart. given that the people most at risk live in countries with limited facilities for pre-haart screening, this has major implications for antiretroviral therapy roll-out programs in resource-poor areas. combination antimycobacterial therapy by itself does not cure mac infection. a commonly used regimen is oral rifabutin, 300 mg once daily, with oral ethambutol, 15 mg/kg once daily, and oral clarithromycin, 500 mg once daily or every 12 h. if clarithromycin is not used, oral rifabutin, 600 mg once daily, is given-the lower dose adjusting for yet more drug-drug interactions. use of three drugs has no impact on overall outcome, although it reduces the risk of resistance and possibly enhances early mycobacterial killing. in patients severely compromised by symptoms, intravenous amikacin, 7.5 mg/kg once daily for 2-4 weeks, is also given. trough blood levels must be measured to ensure toxic accumulation of amikacin does not occur. fluoroquinolones such as moxifloxacin or levofloxacin may be extremely useful, because they have good antimycobacterial activity with limited side effects. at present, many of these agents are not licensed for this indication. given the concerns over xdr tuberculosis, it is important to ensure that patients are adherent to such treatments, and hence reduce the risk of fluoroquinolone resistance developing. a frequently used regimen includes rifampin, isoniazid, and ethambutol in conventional doses; all drugs are given by mouth. the treatment regimens for fungal infections complicating hiv infection are shown in table 34 -13. after initial treatment of cryptococcal infection, there is a high likelihood of relapse of infection; hence, lifelong secondary preventative therapy is needed unless antiretroviral therapy is commenced and results in sustained improvements in cd4 counts (>250 cells/ml) and suppression of hiv load in peripheral blood. secondary prophylaxis is most often oral fluconazole 200-400 mg four times daily. just as with mycobacterial disease, "late" iris events can occur after months or even years. these should be investigated to exclude active disease and other conditions. oral itraconazole, 200 mg twice daily, is the current treatment of choice. the dose is adjusted to achieve blood trough drug levels that are above the standard lowest effective concentration. there are no data on the impact of antiretroviral therapy on which to base decisions about discontinuation of secondary prophylaxis. treatment of this infection is difficult. after initial treatment with amphotericin b, itraconazole or fluconazole may be given for long-term suppression. the overall prognosis is poor, with a 40% mortality rate despite therapy. there are no data on the impact of antiretroviral therapy on which to base decisions about discontinuation of secondary prophylaxis. oral itraconazole has now replaced amphotericin b as the treatment of choice for p. marneffei infection, apart from the subgroup who are acutely unwell. fluconazole is less effective than itraconazole. after initial treatment, lifelong suppressive therapy with itraconazole is needed. there are no data on the impact of antiretroviral therapy on which to base decisions about discontinuation of secondary prophylaxis. the treatment regimens are shown in table 34 -14. a combination of sulfadiazine and pyrimethamine is the regimen of choice for t. gondii infection. the most frequent dose-limiting side effects are rash and fever. adequate hydration must be maintained to avoid the risk of sulfadiazine crystalluria and obstructive uropathy. alternate regimens are given in table 34 -14. once treatment is completed, lifelong maintenance is necessary to prevent relapse, unless antiretroviral therapy achieves adequate immune restoration (blood cd4 count >250 cells/ml and undetectable hiv load). visceral leishmaniasis is usually treated with liposomal amphotericin b, although this is still associated with a high rate of relapse. second-line therapy (or first-line in resourcepoor environments) is to use sodium stibogluconate (see table 34-14) . phenomena temporally associated with starting haart. this includes, but is not limited to: 1. new or enlarging lymphadenopathy, cold abscesses, or other focal tissue involvement 2. new or worsening central nervous system disease 3. new or worsening radiological features of tuberculosis 4. new or worsening serositis (pleural effusion, ascites, pericardial effusion, or arthritis. 5. new or worsening constitutional symptoms such as fever, night sweats, and/or weight loss 6. retrospective review indicating that a clinical or radiologic deterioration occurred with no change having been made to tuberculosis treatment c. immune restoration, e.g., a rise in cd4 lymphocyte count in response to haart d. a fall in hiv "viral load" in response to haart alternative diagnoses to be excluded progressive underlying infection treatment failure due to drug resistance (mdr or xdr) treatment failure from poor adherence adverse drug reaction another diagnosis coexisting (e.g., non-hodgkin lymphoma) the treatment of choice is ivermectin. risk of treatment failure with thiabendazole in hiv-infected individuals is higher than that in non-hiv-infected patients. cytomegalovirus pneumonitis is treated with intravenous ganciclovir, 5 mg/kg every 12 h, for 14 days. drug-induced neutropenia is managed with granulocyte colony-stimulating factor. some centers use valganciclovir, an oral formulation of ganciclovir, at a dose of 900 mg orally every 12 h, to treat cmv pneumonitis. side effects and their management are as for ganciclovir. there are no data that demonstrate efficacy for cidofovir for treatment of cmv pneumonitis, but this agent is used as second-line therapy in many centers. phosphonoformate (foscarnet) can be used for treatment of cmv endorgan disease (e.g., pneumonitis), although it has an extensive toxicity profile. it is also a moderately effective antiretroviral agent. this effect is occasionally used as an adjunct in controlling nonresponsive viral infections. within the past few years, drug therapy has radically altered the depressingly predictable nature of progressive hiv infection. combinations of specific opportunistic infection prophylaxis and antiretroviral therapy can reduce both the incidence and the mortality associated with common conditions. the observational north american macs cohort demonstrated that the risk of pcp in individuals with blood cd4 counts of <100 cells/ml can be reduced almost fourfold if both specific prophylaxis and haart are taken (from 47% to 13%). however, as common conditions are prevented, so other less treatable illnesses may arise. the initial impact of p. jirovecii prophylaxis was a reduction in the incidence of pcp at the expense of an increase in cases of disseminated mac infection, cmv infection, esophageal candidiasis, and wasting syndrome. new prophylactic therapies targeting those conditions associated with high morbidity and mortality (in particular mac) have further improved survival. it has become apparent that specific infection prophylaxis may also confer protection against other agents. this "crossprophylaxis" is particularly seen with the use of tmp/smx for pneumocystis, which also provides cover against cerebral toxoplasmosis and several common bacterial infections (although not s. pneumoniae) and with macrolides for mac infection, which further reduce the incidence of bacterial disease and also pcp. use of large amounts of antibiotic raises the possibility of future widespread drug resistance. this is clearly of concern, and recent reports suggest that, indeed, in some parts of the world the incidence of pneumococcal tmp/smx resistance is rising. current preventive therapies pertinent to lung disease focus on p. jirovecii, mac, m. tuberculosis, and certain bacteria (table 34-15 ). numerous studies have demonstrated the greatly increased risk in subjects who do not take adequate drug therapy with blood cd4 counts <200 cells/ml. clinical symptoms are also an independent risk factor for pcp, and hence the current guidelines recommend lifelong prophylaxis against p. jirovecii in hiv-infected adults who have had prior pcp, cd4 counts <200 cells/ml, constitutional symptoms (documented oral thrush or fever of unknown cause of <37.8 c that persists for more than 2 weeks), or clinical aids. the importance of secondary prophylaxis (i.e., used after an episode of pcp) becomes clear from historical data, which indicate a 60% risk of relapse in the first 12 months after infection. the increase in systemic and local immunity that occurs with haart has led to several studies evaluating the need for prolonged prophylaxis in individuals with sustained elevations in blood cd4 counts and low hiv rna load. in summary, it seems that both primary and secondary pcp prophylaxis can be discontinued once cd4 counts are >200 cells/ml for more than 3 months. a caveat to this is that the patient should have a low or undetectable hiv rna load, that the cd4 percentage is stable or rising and is >14%, and that the individual plans to continue haart long term with good adherence. the risk of pcp recurrence is real if the cd4 count falls below 200 cells/ml. if this does happen, pcp prophylaxis should be restarted. similar algorithms have been successfully used for all the major infections except tuberculosis. they all rely on an estimation of the general blood cd4 count above which clinical disease is highly unlikely. for example, secondary prophylaxis of mac may be discontinued once the blood cd4 count is consistently >100 cells/ml. this is a general guideline, however, and patients must be assessed on an individual basis. as with treatment strategies, tmp/smx is the drug of choice for prophylaxis (table 3416) . it has the advantages of being highly effective for both primary and secondary prophylaxis (with 1-year risk of pcp while on the drug being 1.5 and 3.5%, respectively). it is cheap, can be taken orally, acts systemically, and provides some cross-prophylaxis against other infections, such as toxoplasmosis, salmonella species, staphylococcus species, and h. influenzae. its main disadvantage is that adverse reactions are common (see earlier), occurring in up to 50% of individuals taking the prophylactic dose. the standard dose of tmp/smx is one double-strength tablet (160 mg trimethoprim, 800 mg sulfamethoxazole) per day. other regimens have been tried; these include one "double-strength" tablet three times weekly and one single-strength tablet per day. in general, when used for primary prophylaxis, these regimens are tolerated well (if not better than the standard) and seem as efficacious as one double-strength tablet per day. the data are less clear on secondary prophylaxis, in which subjects are at a much higher risk of recurrent pcp. attempts to desensitize patients who are intolerant of tmp/ smx have met with some success. in patients who cannot tolerate tmp/smx, dapsone is a safe and inexpensive alternative. it has been studied in a number of trials as both primary and secondary prophylaxis and is effective at an oral dose of 100 mg/day. when combined with pyrimethamine (25 mg three times weekly), it provides a degree of cross-prophylaxis against toxoplasmosis. before starting dapsone, patients are tested for glucose-6-phosphate dehydrogenase deficiency. nebulized pentamidine has largely fallen from use as a prophylactic agent. this is despite it being better tolerated and having a similar efficacy to tmp/smx for primary preventive therapy. however, its breakthrough rate is higher in subjects who have lower cd4 counts (i.e., <100 cells/ml) and in those who take it as secondary prophylaxis. other disadvantages include equipment costs and complexity (alveolar deposition is crucial, and hence the nebulizer system used is important), the risk of transmission of respiratory disease (e.g., tuberculosis) to other patients and staff during the nebulization procedure, an alteration in the clinical presentation of pcp while on pentamidine (increased frequency of radiographic upper zone shadowing, increased incidence of pneumothorax), and a lack of systemic protection against pneumocystis and other infectious agents. there is also an acute bronchoconstriction effect during nebulization. long-term follow-up studies have not demonstrated any significant negative effect on lung function. atovaquone oral suspension is used as a second-line prophylactic agent in subjects intolerant of tmp/smx. it seems to have similar efficacy to dapsone (given together with weekly pyrimethamine), with a reduced incidence of side effects, of which the most frequent are rash, fever, and gastrointestinal disturbance. azithromycin is used in many centers as a third-line prophylactic agent. it is given at a dose of 1250 mg once weekly, and may provide protection against some bacterial infections, as well as mac. a low blood cd4 count (<50 cells/ml) is the current best laboratory predictor of prophylaxis failure. this is not particularly surprising given that the median blood cd4 count of subjects not on prophylaxis who have pcp develop is below 50 cells/ml. persistent fever of unknown cause is an important clinical risk factor for pcp. used as preventive therapy, tmp/ smx significantly reduces the chance for development of pneumocystis. it is, therefore, vital that subjects who are most vulnerable be encouraged to use this drug on a regular basis. the pspc cohort study revealed that 21% of subjects with a cd4 count <200 cells/ml were not receiving any form of pcp prophylaxis. the effective and safe (i.e., replication incompetent) bacterial vaccines that are available would be expected to be widely used to prevent hiv-related disease. in fact, uptake of both pneumococcal and the h. influenzae type b (hib) vaccines is poor (current estimates for the former are at most only 40% of the infected population with the recommended 23-valent vaccine). one reason for this may be that the protection conferred by vaccination (90%) in the general population is not seen in immunosuppressed hiv-infected individuals, reflecting their inability to generate adequate memory b-cell responses (especially those subjects with cd4 counts <200 cells/ml). however, in north america, cdc/idsa recommend the pneumococcal vaccine as a single dose as soon as hiv infection is diagnosed, with a booster at 5 years, or if an individual's blood cd4 count was <200 cells/ml and subsequently increased on haart. several studies show pneumococcal immunization reduces the risk of invasive pneumococcal infection in this population. this does not seem to be the case in a developing world setting, where not only is the 23-valent vaccine ineffective against both invasive and noninvasive pneumococcal disease, but the overall incidence of pneumonia is increased. infection with h. influenzae type b is much less common in hiv-infected adults and, therefore, immunization with hib vaccine is not routinely recommended. there is little evidence to suggest that the high frequency of bacterial infections in the hiv population is related to bacterial colonization. therefore, continuous antibiotics are rarely indicated, although both tmp/smx and the macrolides (clarithromycin and azithromycin) given as long-term prophylaxis for opportunistic infections have been shown to reduce the incidence of bacterial pneumonia, sinusitis/otitis media, and infectious diarrhea. the use of tmp/smx also confers a survival advantage in many studies performed in resource-poor settings. there is little evidence, however, that tmp/smx protects against pneumococcal infection. the interaction between hiv and tuberculosis is of fundamental importance, because the annual risk for the development of clinical tuberculosis in a given individual is estimated to be 5-15% (i.e., similar to a non-hiv-infected subject's lifetime risk). hiv-infected individuals with pulmonary tuberculosis are less likely to be smear positive than their hiv-negative counterparts, although they can still transmit tuberculosis. thus, within a community, tuberculosis prevention involves case finding and treatment of active disease, as well as specific prophylactic drug therapies for those exposed. if possible, hiv-positive subjects should make every effort to avoid encountering tuberculosis (e.g., at work, homeless shelter, health care facility). one of the problems with standard methods of tuberculosis contact tracing in hiv infection is that both tuberculin skin test results and chest radiology may be unreliable. however, in the absence of bacillus calmette-guã©rin (bcg) immunization, a positive ppd (e.g., <5 mm induration with 5 tuberculin units) indicates a greatly increased risk (6-to 23-fold compared with nonanergic, ppd-negative, hiv-infected subjects) of future active disease. the chance that hiv-infected subjects may contract disseminated infection if given bcg means that (having excluded active infection) the only option in these circumstances is to use a preventative drug regimen. options include at least 6 months of isoniazid (together with pyridoxine to prevent peripheral neuropathy). this is safe and well tolerated, although compliance is a problem (especially with regimens longer than 6 months), and dot may need to be instituted (e.g., 900 mg isoniazid twice weekly). there is little evidence to suggest that this single-agent regimen leads to isoniazid resistance, which probably reflects the low mycobacterial load present in such individuals. attempts to shorten the length of treatment for latent infection have produced variable results. recent studies used rifampin and pyrazinamide for short-course prophylaxis (2 months). this was as effective as 12 months of isoniazid in hiv-coinfected individuals, although it was associated with fatal hepatotoxicity (almost exclusively in the hiv-negative population). hence, it is currently out of favor. if used, liver function should be closely monitored, and it is recommended that this regimen not be given to patients with preexisting liver disease (e.g., because of alcohol or viral hepatitis). because rifampin should not be used by subjects taking pis, this may also limit widespread application of the two-drug regimen. the same applies to combinations of isoniazid and rifampin taken for at least 3 months, which are also effective in hivnegative individuals. alternate protocols also exist for subjects thought to be resistant to first-line prophylactic agents. these have not been widely clinically evaluated. it is recommended that hiv-infected subjects who have had close contact with an active case of tuberculosis should also receive prophylaxis. there is little evidence to suggest that anergy confers an increased risk for development of clinical disease. however, patients who have not had bcg, have a negative skin test, and have started haart may benefit from regular skin tests, because some studies suggest that cutaneous responses may return with increasing cd4 counts, and that this may help in identifying newly infected individuals requiring prophylaxis. in populations where the prevalence of tuberculosis is low and bcg may be given during childhood or adolescence (e.g., the united kingdom), the value of ppd testing is more limited. here, an arbitrary cutoff of 10 mm for tuberculin reactions is used to define who should receive preventive therapy. the introduction of immune-based blood tests that can accurately distinguish between bcg vaccination and tuberculosis infection may be helpful when screening for evidence of latent tuberculosis. the two currently available commercial tests use fairly specific cd4-directed mycobacterial antigen responses with consequent production of detectable interferon-g. the need for reasonably intact cd4 function means that they may, in fact, be less useful in hiv infection, especially in those subjects with very low blood cd4 counts, who are possibly at greatest risk of developing active tuberculosis. secondary tuberculosis prophylaxis may be important, because studies indicate a high rate of relapse in endemic areas. here, no specific guidelines exist, although 6 months of isoniazid and rifampin after a full treatment course shows a greatly reduced risk of relapse within the subsequent 2 years. whether this is enough to prevent clinical disease (which may also arise from reinfection in areas of high tuberculosis prevalence) without concomitant antiretroviral therapy is unclear. in the developed world, secondary prophylaxis is usually not recommended. the use of haart also can reduce the risk of tuberculosis in endemic areas. work in south africa indicates that this is most beneficial in patients with advanced disease and leads to a reduction in rr of at least 80%. data from north america indicate that the prevention of disseminated mac infection has an effect on survival (25% reduction in mortality rate in subjects taking clarithromycin). the us guidelines advise prophylaxis with a macrolide (either clarithromycin, 500 mg orally twice per day, or azithromycin, 1250 mg orally, once a week) in all hiv-infected individuals with blood cd4 counts >50 cells/ml. in europe, where the prevalence of disseminated mac infection is probably lower (perhaps because of previous bcg vaccination), this may be less relevant. here, surveillance cultures of blood may be more cost-effective in the at-risk hiv population with low cd4 counts. routine stool and sputum cultures probably do not add much to this strategy, because disseminated mac is much more common than isolated organ disease. single-agent prophylaxis may lead to antibiotic resistance. this does not seem to be reduced by the addition of a second drug (rifabutin) to the prophylactic regimen. the latter is now a second-line prophylactic agent, largely as a result of its rather worse protective effect and its adverse interaction profile with pis. as mentioned earlier, if an individual sustains a rise in cd4 count >100 cells/ml for >6 months, it is safe to discontinue prophylaxis. several clinical and laboratory features have prognostic significance in hiv-infected individuals with pcp (box 34-2). a severity score on the basis of the serum ldh levels, the a-ao 2 , and the percentage of neutrophils in the bal fluid can predict survival reasonably accurately, with the highest scores indicating the worst outcome. other workers have shown that increased age (<50 years) leads to an increased mortality rate-in part as a result of late, "unsuspected" diagnosis. the overall mortality rate from an episode of pcp is approximately 14% and has not changed since the advent of haart. among individuals with access to haart, post-pcp survival has improved. in 1981, the median survival after pcp was 9 months. by 1995, this had risen to 20 months. the introduction of haart has led to a further improvement, with survival in the period up to year 2000 of 40 months. in those without access to haart and/or prophylaxis, survival post-pcp, unfortunately, remains poor. in general, mortality from bacterial respiratory infection in hiv-infected individuals is similar to that seen in the general population. clinical and laboratory markers of disease severity that have been defined in the adult general population (e.g., those described in the ats or the bts guidelines for the management of community-acquired pneumonia in adults) apply to hiv-infected patients. these are confusion, raised respiratory rate, abnormal renal function, and low blood pressure. recurrent pneumonia is common (reported in up to 55% of cases) and may lead to chronic pulmonary disease (see earlier). although tuberculosis normally responds to standard multipledrug therapy, work from africa has highlighted the increased mortality rate in hiv-infected compared with non-hivinfected individuals. a relationship has also been described between mortality and declining blood cd4 count: hivinfected patients with cd4 counts <200 cells/ml have a mortality rate of 10% compared with 4% in those with cd4 counts from 200-499 cells/ml. compared with hiv-infected individuals without tuberculosis, the main effect on mortality is seen in patients with higher cd4 counts (>200 cells/ml), where the relative risk of death is three times that of the nontuberculous population. several case-controlled studies have indicated that in the absence of effective treatments, mac-infected patients have a reduced survival compared with blood cd4 level-matched control subjects (approximately 4 months vs 9 months, respectively). currently available treatment regimens may reduce this difference, although severe anemia seems to be an independent predictor of mortality. the presence of cmv in bal fluid also containing p. jirovecii has been related to outcome (see earlier). the mortality rate at 3 and 6 months after bronchoscopy is greater in those with cmv detected at bronchoscopy. however, cmv recovered as a sole pathogen does not impact on survival. the effect of antiretroviral therapy on opportunistic infections the introduction of haart, together with the wide availability of accurate methods of determining plasma rna viral load, has led to profound changes in both clinical practice and hiv outcome. although it is still the case that respiratory disease remains above non-hiv infected background levels, in particular, bacterial pneumonia, tb, and lung cancer are more common, despite apparently effective haart, in hiv-infected subjects. overall data indicate that clinical progression is rare in subjects who are able to adhere rigorously to at least 95% of their antiretroviral drug regimen. mortality rates have fallen by 80% for almost all conditions, and it seems that a damaged immune system can, to a clinically significant extent, be reconstituted for a period of at least several years. hence, clinicians need to consider not only opportunistic infection or malignancy within their diagnostic workup but also the effects of drug therapy itself. the side effect profile of haart (e.g., metabolic and mitochondrial toxicities, liver damage, and neuropsychiatric disorders), as well as the large number of drug-drug interactions, makes this a very complex area of management. the best example of this is hiv-related tuberculosis. here, not only is there overlapping toxicity and pharmacologic interaction, but iris is common. research is needed to address this area. studies should inform the decision on when to start haart in patients already on antituberculosis medication. other work needs to focus on understanding box 34-2 prognostic factors associated with poor outcome in pneumocystis jirovecii pneumonia on admission patient's age no previous knowledge of hiv status tachypnea (respiratory rate >30/min) second or subsequent episode of pneumocystis jirovecii pneumonia poor oxygenation ã� pao 2 <7.0 kpa (<53 mmhg) or a-ao 2 >4.0 kpa (>30 mmhg) low serum albumin (<35 g/dl) low hemoglobin (<12.0 g/dl) peripheral blood leukocytosis (>10.8 ã� 10 9 /l) elevated serum lactate dehydrogenase levels (>300 iu/l) cd4 count <50 cells/ml marked chest radiographic abnormalitiesdiffuse bilateral interstitial infiltrates with or without alveolar consolidation medical comorbidity (e.g., pregnancy) at bronchoscopy 1. in bronchoalveolar lavage fluid detection of a. copathology cmv bacteria b. neutrophilia (>5%) 2. detection of pulmonary kaposi sarcoma serum lactate dehydrogenase levels that remain elevated development of pneumothorax high apache ii score on admission to the icu need for mechanical ventilation why full pulmonary immunity is not restored. this may reflect abnormalities in the innate immune response, which is currently poorly described in hiv infection. despite the benefits of haart, it is likely that in the long term many patients will progress to severe disease. there is currently little research in this area. research should focus on correlating clinical and laboratory findings. an example of this would be assessing the risk of an individual for development of active tuberculosis. it is clear that much of the excess mortality in hiv-tuberculosis coinfection occurs early in hiv infection. thus, if tests can be devised that indicate who has latent tuberculosis infection (and who is, therefore, most likely to have clinical disease develop), steps can be taken to prevent illness. as discussed previously, immune-based tests have shown promise in immunocompetent individuals with tuberculosis infection. if these can be refined to work consistently in patients with hiv infection at a reasonable cost, there is the possibility of targeting those at risk of future tuberculosis, or of tuberculosis "unmasking" after starting haart. the other role for a test such as this would be in rapid diagnosis of active tuberculosis. it is common to be faced with a patient who has nonspecific symptoms and a wide differential diagnosis. often treatment is multiple and empirical. a quantitative test would help resolve some of these dilemmas by indicating the chance of the condition being caused by a particular disease. an example would be the patient from an endemic tuberculosis area, with low cd4 counts, who has both pulmonary and central nervous system disease. is this tuberculosis, toxoplasmosis, cryptococcosis, or viral or bacterial infection? any such test for tuberculosis would also have to distinguish between the different states of old (treated), old (inactive), old (latent), and active. although not insurmountable, at present, this is not possible. rapid diagnostic assays that assess organism viability are also important. if a clinician can receive early feedback on whether treatment is producing a suitable killing effect, therapy can be tailored to the individual. this enables regimens to be "dose adjusted" as needed and removes the element of concern that is often present when patients are slow to respond. examples of this would be in the treatment of pcp or mycobacterial disease. the frequency of bacterial infection (often recurrent) with its attendant sequelae makes effective strategies for vaccination an important priority. it is uncertain why there is a differential response to vaccination; even in the united states, african americans do not seem to derive the same benefit as whites. this needs further research, together with more emphasis on identifying the local immune response present in the lung in such individuals. bacterial infections may be clinically indistinguishable from other pathogens, and only two thirds of all respiratory infections are formally diagnosed. there is a need for improved methods to assist with this. the use of rapid antigen tests may be one way forward. this is especially so given the high incidence of (potentially fatal) bacteremia present in such populations. for maximum benefit, this needs to use a system that is simple and cheap, and hence suitable to both resourcerich and resource-poor countries. m. tuberculosis is globally the most important hiv-related pathogen. strategies of control and prevention are vital to ensure that millions of people do not become coinfected and that those who are do not go on to have clinical disease develop. rapid diagnostics are critical. the encouraging reports of the simple and cheap method of mods to both diagnose tuberculosis and then provide resistance data in field settings (see earlier) argues for large-scale roll out and evaluation. beyond public health measures, such as dot, fixed-dose combination drugs, case management, and education, research needs to improve on current drug therapy. long-acting preparations such as rifapentine show promise but, as the problem with rifampicin monoresistance demonstrates, there is still much work to be done. for the first time in many years, there are several antimycobacterial drugs that are in various stages of clinical trials. all are promising, and several have novel mechanisms of action. the global alliance and the who "stop tb" campaigns have been crucial in this regard. the fluoroquinolones, moxifloxacin and gatifloxacin, are closest to the market. they are potent drugs with considerable ability both to kill and also sterilize mycobacteria-infected sites. trials of treatment-shortening regimens are ongoing worldwide. vaccination against m. tuberculosis with bcg has understandably not been widely used in an immunosuppressed hiv-infected population. however, a safe vaccine may be the only affordable way of protecting large parts of the world from tuberculosis. so far there seems to be more success in vaccines to either enhance or replace the primary protective effects of bcg. the use of immunotherapy (e.g., with heat-killed mycobacterium vaccae) in combination with chemotherapy has been disappointing in clinical trials. newer methods of diagnosis (e.g., pcr tests on saliva) may prove invaluable for quick and easy disease confirmation, although their applicability to routine samples needs further evaluation. p. jirovecii prophylaxis was the first important hiv treatment widely available. however, despite the efficacy of tmp/smx, compliance remains a problem. regimens that use a gradual increase in dosage when starting prophylaxis may help. one concern with widespread use of prophylaxis is that resistance will start to occur to tmp/smx. reports have indicated that there are mutations in the p. jirovecii dihydropteroate synthase gene that confer resistance. these seem to be increasing over time, although they do not seem to be present in many patients who fail treatment for pcp with tmp/smx. the implications of this are uncertain but could include a greater likelihood of treatment failure and the possibility of worsening patterns of global bacterial drug resistance. hiv-infected populations in the developed world have high rates of smoking. the evidence that this is harmful above those effects seen in the general population continues to accrue. the accelerated course of both obstructive lung disease and cancer, together with the increased risk of respiratory infection in smokers, persuasively argues the case for targeted smoking cessation. that hiv infection and haart have profound (and probably negative) effects on blood lipids and insulin resistance further support the need to reduce smoking rates in this population. it seems that we are starting to see increased rates of cardiovascular disease in this now aging population. the natural history of hiv-related respiratory disease continues to evolve. haart and newer therapeutic strategies have made a significant impact on morbidity and mortality. yet individuals continue to become hiv infected, progress, and die from an ever-expanding range of conditions. p. jirovecii remains the most common aids-defining event in the developed world, whereas m. tuberculosis is globally the most common cause of death. bacterial respiratory infection is not far behind. given the huge number of individuals with hiv infection, the only effective way to manage this disease is to find simple ways of treating hiv itself, and thus contain the worst ravages of this illness. treating opportunistic infections among hiv-infected adults and adolescents revised recommendations for hiv testing of adults, adolescents, and pregnant women in healthcare settings treatment of hiv-related tuberculosis in the era of effective antiretroviral therapy treatment and prophylaxis of pneumocystis carinii pneumonia immune reconstitution disease associated with mycobacterial infections in hiv-infected individuals starting antiretroviral therapy immune reconstitution inflammatory syndrome in hiv guidelines for preventing opportunistic infections among hiv-infected persons-2002 on behalf of the bhiva guidelines writing committee pneumocystis and trypanosoma cruzi: nomenclature and typifications oxford: blackwell scientific aids and respiratory medicine key: cord-346318-d8oq3dyw authors: fang, yeqing; deng, qiwen; yu, zhijian; wang, hongyan; zhang, songrong title: reply: practical experiences on the prevention and treatment strategies to fight against covid-19 in hospital date: 2020-05-05 journal: qjm doi: 10.1093/qjmed/hcaa158 sha: doc_id: 346318 cord_uid: d8oq3dyw nan we appreciate the concerns and comments from dr jianhui peng et al. regarding our recent article, shenzhen' experience on containing 2019 novel coronavirus-infected pneumonia transmission [1] , which was published on april 3, 2020. we read their letter describing their recent experience with interest. as we know that the covid-19 has spread all over the world. on march 19, 2020, when many countries in the world, especially u.s. were hobbled by the epidemic, the number of new patients in hubei province, china approached zero [2] . as a doctor, i am deeply concerned with the world outbreak. the author of the letter described their treatment strategies to contain the epidemic and improve clinical outcomes: increase the medics' protective gear to ensure double-zero infection: no nosocomial infection, no developed into critical or death case; timely control and regulate the inpatient area by adopting the ai and infection control observing system; "two early, three changes and three strictness"; early use of traditional chinese medicine according to characteristics of different persons, etc. as for the world pandemic, i believe these measures are crucial：first, all countries should enhance the covid-19 detectability, give timely diagnosis and treatment to positive patients and build the fangcang shelter hospitals or temporary hospitals to treat patients with mild symptoms [3] . second, increase the stockpile of protective supplies, and guarantee the protection of medical workers. standard personal protective equipment should be applied in special high-risk posts to avoid infection of medical staff. next, during the outbreak, hospitals need to redeploy the working arrangement of healthcare workers, suspend or close non-emergency departments and selective operations to add staff to supplementary emergency, fever clinic and infection wards, so that there are sufficient medical workers to respond to the increasing coronavirus patients efficiently. last but not least, recommend patients using internet hospitals by seeing a doctor online, to reduce the crowds in the hospital and avoid nosocomial infection. shenzhen' experience on containing 2019 novel coronavirus-infected pneumonia transmission hospitals: a novel concept for responding to public health emergencies in a word, timely and rapid diagnosis and treatment of patients, interrupting the infection source, as well as immediate testing, isolation and contact tracing are crucial measures to tackle the coronavirus pandemic. we will definitely win this battle with concerted efforts, scientific containment and targeted policies. key: cord-328720-o9h1vquo authors: davis, cristina e.; hill, jane e.; frank, matthias; mccartney, mitchell m.; schivo, michael; bean, heather d. title: breath analysis for respiratory infections date: 2020-09-18 journal: breathborne biomarkers and the human volatilome doi: 10.1016/b978-0-12-819967-1.00021-9 sha: doc_id: 328720 cord_uid: o9h1vquo one of the most logical applications of modern breath analysis techniques is to provide information on respiratory infections. ongoing work in various types of pulmonary infections has begun to denote candidate breath biomarkers of bacterial, viral, and fungal lung infections. groundbreaking studies have been performed in naturally occurring cases with humans and with animal models of the disease. this has been coupled with cell culture work to understand the nature of the origins of breath biomarkers generated from the pathogen itself as it proliferates. much work remains to be done, and the published studies described in this chapter are helping to set a foundation for this research area. respiratory pathogen diagnostics are an important area of current breath research, and researchers have taken various tactics to approach studies on this topic. 1 any studies of infectious diseases in humans have relied on subjects in preidentified cohorts who tested positive for the diseases under normal standards of care, using gold-standard confirmatory methods. breath samples, collected with informed consent, have been concentrated and/or directly analyzed in a variety of ways. room air analytical controls are sometimes used as a best practice. a "control" cohort of subjects is usually employed as a comparison and typically is comprised of uninfected age-matched and gender-matched healthy individuals, although in more ideal circumstances, this would be a group that was suspected of having the infection but was later ruled out (see chapter 35 for further details on clinical study design). indeed, recruiting those with the same symptoms in the control groups, including noninfectious disease subjects, such as sarcoidosis patients in the case of breath sampling for tuberculosis, is an increasingly essential parameter in a study design. once breath samples are obtained by an appropriate sampling method (see chapter 2), a common theme has been to use different forms of mass spectrometry (ms) analysis of the breath samples to provide complete metabolomic chemical identification of the sample contents. using an untargeted approach to statistical analysis, researchers then applied various data analysis techniques to try to identify candidate breath biomarkers of that specific pulmonary infection. parallel animal model studies have provided insight into human breath biomarker studies. the animals may be chosen from natural disease occurrence, similar to the human studies, or laboratory animals in carefully controlled studies that target a specific pathogen at different doses of inoculation. these animal models should bedand often aredof high human relevance and have included macaques, mice, ferrets, and swine. animal model studies have played a key role in developing and maturing biomarker identification, as well as providing fundamental information, such as how biomarkers change over an infection time course postinoculation. breath biomarkers of infection from human and animal studies can arise from the host, generated by the immune system 2 or by cellular responses of infected tissues, or they can arise from the microbial pathogen as a function of virulence or as a consequence of normal cell cycles and proliferation. thus, there is also a role for in vitro cell culture models, which can produce translatable biomarkers or be used to understand the origin of biomarkers during disease progression (see chapter 26) . together, this triad of approaches (human, animal, and in vitro cell culture studies) has allowed researchers to identify candidate breath biomarkers that can be carried forward into larger studies. a summary of these advances is outlined in this chapter. tuberculosis (tb) is the largest infectious disease killer in the world and the biggest killer of people with human immunodeficiency virus (hiv) infection and/or acquired immune deficiency syndrome (aids). tb is one of the respiratory diseases for which breath analysis studies have been performed in both humans and animal models. traditional means of tb detection, still used in many countries, 3 are slow and require weeks-long sputum culturing to confirm diagnosis and assess antibiotic resistance. nucleic acid amplification approaches generate useful diagnostic data faster, typically in three days, but this timeline is problematic as many patients do not return for their result and thus are lost to follow up. and, children and immunodeficient patients generally do not produce sputum. thus, breath diagnostics are poised to provide a much more rapid timeline for physicians, especially to allow patient treatment to begin rapidly. breath studies to diagnose bacterial infections caused by mycobacterium tuberculosis (mtb) are more likely to succeed than any other approach due to the extensive groundwork done by the belgian-tanzanian group apopo (anti-persoonsmijnen ontmijnende product ontwikkeling) who have evaluated the volatiles from tens of thousands of sputum samples using trained giant gambian rats. 4 morozov and colleagues performed a proof-of-principle study aimed at developing noninvasive diagnostics for pulmonary tb based on potential tb biomarkers in microdroplets exhaled by tb patients and collected on electrospun fibers. 5 this study involved 42 tb patients (including recent and chronic tb cases) and 13 healthy volunteers. samples were tested for the presence of mtb cells, mtb dna, and protein biomarkers. while no mtb cells or mtb dna could be detected, an ultrasensitive immunoassay was developed that detected immunoglobulin a (iga) in >90% of samples from both tb patients and at higher rates than healthy volunteers. antigen-specific iga was detected at higher rates in the patient samples compared with the healthy control samples. although the authors report and comment on the relatively low sensitivity and specificity, they suggest that expanding this method to include inflammation-specific biomarkers in addition to the tb-specific antibodies promises to increase the level of discrimination in the future. beccaria and colleagues conducted two studies evaluating the use of human breath collected and stored on thermal desorption tubes and analyzed by comprehensive gas chromatographyetime-of-flight mass spectrometry (gcâgc-tofms) to diagnose active tb in subjects with confirmed mtb infection. 6, 7 the control samples used in each study included room air, as well as either a group presenting to the clinic with the same symptoms (south africa) or in subjects with nonrespiratory infectious diseases (haiti). about 84 subjects were evaluated, of which 34 were patient controls. both studies present a chemometric pathway, utilizing statistical and machine learning tools to translate thousands of analytical signals to a putative biomarker set in the context of an underpowered study design. hill and colleagues also investigated the potential of breath analysis for detecting mycobacterial infections using a murine model and mycobacterium bovis bacillus calmetteeguerin (bcg), 8 as well as mtb in macaques. 9, 10 this was the first report of breath being collected from mice or nonhuman primates infected with a pathogen from the mycobacterium tuberculosis complex (mtbc). a related study on mice used breath collected at two time points for eight mice infected with m. bovis bcg and eight healthy mouse controls exposed to instilled phosphate-buffered saline (pbs). room air samples were collected as controls. the breath of ventilated mice was collected in tedlar bags and then concentrated onto thermal desorption tubes. analysis of the breath samples was performed using gcâgc-tofms. statistical analysis using random forest identified 23 features in the data that discriminated between the breath samples of infected mice and the controls. tentative identification of these 23 compounds was provided by mass spectral matches to a reference library. four of these markers were also reportedly found in previous breath studies on animals with mtbc infections. this study showed that this overall methodology can differentiate between healthy and infected mice using breath analysis. it also indicated that a mouse breath model might be useful to study tb pathogenesis and evaluate preclinical drug regimen efficacy. a design for the collection of breath in gently anesthetized macaques in a biological safety level three laboratory was published by mellors et al., in 2017. 8 a chemometric process for evaluating the repeatability of breath samples in terms of chemical composition was given, and a pilot level determination of 37 putative biomarkers to distinguish mtb lung infections from healthy animals using breath was generated. in a follow-up study, 10 the same group looked for and found substantial synergy between cell culture volatile molecules of mtb in the breath of nine cynomolgus macaques with infection by the same strain (erdman). thirtyseven molecules found in culture could distinguish the breath of infected and healthy macaques with an area under the receiver operating characteristic curve (auroc) of 87%. the authors point out, however, that the origin of the molecules in the breath of the macaques was unknown; therefore, the translation from culture to breath should be considered precarious unless supported by unique metabolism, as can be found in some fungal infections (see section 21.4). bergmann and colleagues investigated the in vivo volatile organic compound (voc) signatures of mycobacterium avium ssp. paratuberculosis (map) in goats 11 in a study that involved 26 map-inoculated animals and 16 healthy controls. volatile molecules were collected from the animals' breath using an automated alveolar sampling device and needle trap microextraction, as well as from feces using solid-phase microextraction (spme), with sampling performed over various time points spanning up to 48 weeks after inoculation. all samples were analyzed by gc-ms, with tentative substance identification accomplished using a reference library. in parallel, blood was sampled and analyzed for map-specific antibodies and map-specific interferon-g response to correlate measured breath molecule patterns to measurements of these blood-based biomarkers. the observed voc patterns differed between map-inoculated and noninoculated animals. although the measured voc patterns evolved during the infection, notable differences in observed vocs between inoculated and noninoculated animals remained throughout the course of infection. a total of 28 vocs were identified as potential markers for map infection in vivo, some of which have been found in previous in vitro studies in the headspace of map cultures. 12 some of the observed voc markers in breath were attributed to host response, and the authors cautioned that results from in vitro voc studies (bacterial headspace sampling) may not simply transfer to in vivo conditions. the results of this work also indicated the potential usefulness of voc profiles from feces to detect the presence of mycobacteria in the gut of ruminants. further details of this and other studies with ruminants are given in chapter 27. several human studies have targeted chronic pseudomonas aeruginosa (pa) in subjects with cystic fibrosis. cystic fibrosis (cf) is a genetic disease that results in polymicrobial lung infections, with the presence of pa, in particular, being linked to poorer outcomes. 13 extricating a breathprint from the milieu of complex microbial ecology and host comorbidities is one of the most complicated undertakings in breath research. gilchrist and colleagues evaluated hydrogen cyanide (hcn) in breath using selected ion flow tube-mass spectrometry (sift-ms) as an early biomarker for pa infection in 233 patients over 2 years (2055 total breath samples). 14 grounded in microbiology confirmation, which revealed 71 pa-positive cultures, the specificity of the approach was estimated to be 99%, although the low sensitivity (estimated at 33%) makes the current test inadequate as a screening tool. span el and colleagues evaluated the breath of 20 cf patients with confirmed p. aeruginosa infection and contrasted those with 38 cf patients who were pa-free. using a panel of 16 molecules obtained from a linear logistic model, they achieved an auroc of 0.91. further details of these studies with sift-ms are given in chapter 9. nasir and colleagues evaluated a lung sample, bronchoalveolar lavage (bal), from patients with cf, seeking potential biomarkers for p. aeruginosa and staphylococcus aureus. 15 using 154 samples, they identified breathprints for each infection type, with good negative predictive values for p. aeruginosa (0.92) and good positive predictive values for s. aureus (0.86). two mouse studies also provided insights into lung infections by bacterial pathogens. purcaro and colleagues evaluated a core breathprint for pa in mouse lung infections, the contribution of the host to the breathprint, and explored which molecules could be used to distinguish mice infected with the major clades of p. aeruginosa (pao1, pa14, pak, pa7). 16 sham infection controls (pbs) were employed. nine compounds from the headspace of those cultures were sufficiently consistent and frequently observed, and were thereby considered core in the in vivo model at 24 and 48 h infection time points. a breathprint containing ten molecules for mice infected with different strains could somewhat reflect the phylogenetic groups to which the strains belong. barbour and colleagues developed a method to sample nose-only breath from mice and performed voc analysis of breath from mice with systemic bacterial infections caused by borrelia hermsii. 17 while inflammatory and antiinflammatory cytokines were found to be elevated in infected mice compared with uninfected controls, no significant difference was found in the profiles of 72 different vocs from nasal sampling, although carbon monoxide (co) was notably elevated in the breath of infected mice. control experiments of headspace sampling from the cultures of b. hermsii indicated that bacteria themselves did not produce co and thus were unlikely to be the source of elevated co in mouse breath; however, in vivo verification is still required to confirm this hypothesis. the findings of this work indicate the potential utility of co concentration in exhaled breath during systemic infection and inflammation. exhaled co in inflammatory pulmonary diseases is treated at length in chapter 6. numerous viruses can infect the human airway, with common examples being influenza viruses and respiratory syncytial virus (rsv). most respiratory viruses cause similar symptoms of a fever and aches, but can be quite severe for patients of certain ages or who are immunocompromised, leading to more developed maladies, such as pneumonia or bronchitis. rsv leads to nearly 14,000 deaths annually in us adults, a number that is likely underestimated. as with bacteria, there have been significant advancements in breath tests for viral infections. while bacteria and fungi can generate their own metabolite signatures, viruses rely on the metabolic machinery of their host to propagate. thus, viral detection often looks for direct evidence of the virus, accomplished by hunting for nucleic acid segments specific to a virus in a bodily fluid sample, such as blood or mucous. this technique of nucleic acid screening, known as polymerase chain reaction (pcr), is very sensitive but often includes a lag time with samples being shipped to offsite laboratories and additional time for the analysis to be conducted. more rapid tests for viral infections are needed, as correct diagnoses by even a day sooner could save lives. in addition, specifying between viral versus bacterial infection is rather important for treatment, as prescription of antibiotics is ineffective against viruses. finally, methods to collect bodily fluids for pcr can be quite invasive, especially for respiratory infections. bronchoscopies are a common technique, in which an instrument is threaded through the mouth or nose and down into the lungs, for instance, to collect liquid biosamples for analysis, as in bal. as breath is collected noninvasively, it is a much more attractive technique, especially for those already suffering from pulmonary disorders. around the turn of the millennium, pigs in industrial farm settings were contracting aujeszky's disease, caused by suid herpesvirus 1 (shv-1). the disease starts with respiratory symptoms that often become severe, leading to death. mortality rates were as high as 100% for piglets, and at the time it was not understood how the virus spread. gillespie et al. posited that as infected pigs coughed or sneezed, they would produce aerosols that could travel long distances, carrying the virus and infecting nearby pigs. in their study, they inoculated a set of pigs with aujeszky's disease. once they showed symptoms, an infected pig was paired with a healthy "recipient" pig. a mouthpiece was placed over each pig's snout, which was then connected with tubing. this allowed the recipient pig to inspire the exhaled breath of the infected pig, without physical contact between animals. afterward, nasal mucus was collected from all pigs. each infected pig had detectable levels of shv-1 in their mucus samples, but no virus was detected from the recipient pigs. yet, all recipient pigs developed clinical symptoms of aujeszky's disease a the authors made two conclusions, namely (1) that viruses can be carried by respiratory aerosols and infect others nearby and (2) that pigs are much more sensitive indicators of airborne shv-1 virions than laboratory aerosol detection methods at the time. 18 this work demonstrates what is commonly known todayd that respiratory viruses can be spread through exhaled aerosols. influenza infection is a major cause of seasonal morbidity and mortality in humans worldwide, and efforts to detect influenza viruses and virus infections rapidly and specifically are evolving. phillips and colleagues conducted a before-and-after study looking at exhaled metabolites emitted by subjects receiving live attenuated influenza vaccine (laiv). 19 confounding biologic a since this study was first reported, a vaccine has been developed for aujeszky's disease. variables were likely minimized because each subject was his/her own control. laiv breath metabolites were measured and compared pre-and postvaccination. the authors hypothesized that oxidative stress products (e.g., 2,8-dimethyl-undecane and 4,8-dimethyl-undecane) were seen in postelaiv-treated subjects because influenza virus characteristically causes increased reactive nitrogen oxide species. indeed, oxidative stress products were observed with changing concentrations over time post-laiv inoculation, and these products did not appear in the laiv headspace. the breath test performances in classifying subjects as pre-or post-laiv inoculation ranged from a c-statistic of 0.82 (day 2) to 0.95 (day 7) to 0.95 (day 14), thereby demonstrating the feasibility of this approach. this study provides an important framework for future studies assessing influenza virus infection in its elegant design. aksenov and colleagues assessed metabolites from several different influenza strains that infected cultured human b-lymphoblastoid cells. 20 lymphoblastoid cells were used to minimize the contribution of apoptosis to metabolite production. several metabolite patterns were increased significantly over background and were used to identify specific influenza virus strains (e.g., h1n1 vs. h6n2) and strength of virus inoculation (e.g., h1n1 multiplicity of infection [moi] 1 vs. moi 10). this proof-of-concept study has many implications for influenza detection at both an individual level and for epidemiologic studies (consider pandemic influenza strain testing). importantly, there were many overlapping alkene and other compounds seen in the aksenov and phillips studies. this provides phenomenological significance in the metabolites generated from influenza-infected cells, despite not knowing the exact mechanism of metabolite production. zoonotic reservoirs are common for influenza a viruses, and efforts to determine breath metabolites from animals may shed light on understanding emerging pandemic influenza strains. traxler and colleagues assessed breath metabolites from swine during a complete h1n1 infection cycle. 21 the study reported six metabolites that classified swine as infected, as measured against standard diagnostic testing. although the animals showed biochemical evidence of infection, they did not show clinical evidence of infection, such as decreased food or water intake, lethargy, and so on. this suggests that breath metabolite analysis may identify subclinical influenza a infection in animals, which may ultimately become more virulent when they bridge from animals to humans. as mentioned, a limitation of these studies is that they do not address the biologic origin or mechanism(s) of metabolite production, which will be important considerations of future studies. lower respiratory tract infections, such as pneumonia, are more challenging to diagnose compared with upper respiratory infections. doctors are required to order invasive tests for the patients with suspect infections, including oropharyngeal swabs, induced sputum collection, and bal. toward less invasive techniques for lower respiratory tract diagnoses, pneumoniacheck is a commercially available tool to collect aerosol particles from exhaled breath patients. users simply cough into a handheld device, which diverts away breath exhaled from the upper respiratory tract and allows air from the lower respiratory tract to collect onto a filter. the filter can then undergo molecular and biochemical analyses for disease detection. while previously demonstrated as a successful tool for bacterial infections, patrucco et al. investigated its utility for viral diagnostics. using pneumoniacheck, samples were collected from persons with pneumonia infections, in addition to bal. using pcr, both sets of specimens were screened for a panel of common respiratory viruses, such as influenza, rhinovirus, and bocavirus. a high concordance was observed among the paired samples collected by bal and pneumonia-check. specificity rates were 100%, but sensitivity was 66%. thus, while more investigation needs to be done, this work shows that this approach has high potential for noninvasive respiratory viral diagnostics. 22 other ways in which breath could be used as a viral detection matrix have also been explored. etiological studies have recently demonstrated a relationship between certain viruses and the development of lung cancers. specifically, two herpes viruses have been of great interest. the epsteinebarr virus (ebv), a herpes virus, is a recognized carcinogen. another herpes virus, cytomegalovirus (cmv), has been associated with maladies of the brain, lung, and colon. early diagnosis of these viral infections could lead to lifestyle changes to reduce the associated cancer risk. carpagnano et al. investigated breath as a sample matrix for detection of ebv and cmv. pcr was used to investigate if ebv and/or cmv dna were present in exhaled breath condensate (ebc) from 110 patients, among which 70 had some type of lung cancer and 40 were healthy controls. these studies demonstrated that these two viruses were directly detectable in breath samples and increased in positivity among lung cancer patients, especially those in advanced stages. this technique could be a huge relief from the current gold-standard approaches, whereby viral nucleic acid is screened from bronchial brushings during fiber-optic bronchoscopies. 23 detection paradigms using breath have shown specificity to certain pathogens. bacterial infections of pa, for example, are especially problematic to patients when co-infected with rsv. purcaro et al. modeled the human airway by culturing human epithelial cells, a technique covered more in depth in chapter 26. cultures were inoculated into four classes: (1) pa only, (2) rsv only, (3) coinfection with pa and rsv, and (4) no infection (healthy controls). detection of vocs released by the cells was used to create a volatile profile of each category. using higher statistics, the study demonstrated that a coinfection of the virus did not inhibit the ability to identify cells with the bacterial infection. in this particular case, however, cells inoculated with just rsv did not yield a sufficient change to the volatile profile for accurate diagnoses, providing evidence that each respiratory viral infection may have to be independently evaluated for its ability to be detected directly in breath samples. 24 over 150 million people worldwide have serious fungal disease and more than 1.6 million people die from fungal infections annually, a rate similar to tb. 25 risk factors for fungal infections include comorbidities, such as hiv/aids, tb, cancer, asthma, and chronic obstructive pulmonary disease (copd), as well as immunosuppressive treatments for chronic conditions, such as transplant rejection and rheumatoid arthritis. 25 as the incidences of these risk factors and comorbidities are rising globally, so too are the incidences of fungal infections. 25 in the clinic, the diagnosis of a fungal infection is usually made based on fungal growth on selective media or the detection of fungal antigens or antibodies in patient specimens. while these tests can be highly sensitive, if a good-quality lung specimen is obtained, e.g., via biopsy, bronchoscopy, or sputum induction, these procedures are contraindicated in many of the patients who are at high risk for fungal pneumonias. therefore, the detection of fungal infections via biomarkers in breath or ebc would significantly enhance the surveillance and detection of mycoses. one of the most prevalent causes of pulmonary fungal infections, globally, is aspergillus spp., 25 which can cause invasive disease in immunosuppressed individuals. koo and colleagues designed a study to identify putative breath biomarkers of invasive aspergillosis (ia) by taking an "in vitroeinformed" approach to selecting classes of secondary metabolites to analyze in their clinical experiments. 26 the majority of cases of ia are caused by aspergillus fumigatus, and therefore they focused their efforts on identifying vocs that discriminate a. fumigatus from other aspergillus spp., first by culturing the fungi in vitro. the study design included two important steps for the in vitro experiments to enhance the translation of results to clinical samples. first, oxygen and temperature conditions that favor hyphal growth (the dominant aspergillus infection morphology) over conidia formation (the dominant environmental morphology) were established and benchmarked by comparing the in vitro transcriptome to the murine lung infection transcriptome. second, aspergillus spp. vocs were characterized in a wide range of different media, from an all-purpose rich fungal medium (yeast extract peptone dextrose (ypd) broth) to media that are low iron, low nitrogen, alkaline stress, and aspergillus minimal media. a. fumigatusespecific vocs that were produced in most of these conditions were identified to enhance robustness of the approach, with several terpenes named as putative a. fumigatus ia biomarkers. to validate the ia biomarkers, koo et al. prospectively collected breath from 64 patients with suspected ia and analyzed the samples by gc-ms, focusing on the detection and identification of the terpenes observed in the in vitro studies. blinded to the breath sample data, two clinicians reviewed the clinical data to classify the subjects as ia (having proven or probable aspergillosis, based on signs and symptoms) or non-ia (having possible ia, or another cause of invasive fungal disease), resulting in 34 ia and 30 non-ia breath samples. several of the aspergillus in vitro terpenes were observed in the breath of patients with or without ia, which were thus eliminated as possible biomarkers. nevertheless, four terpenes and terpenoids specific to in vivo samples were identified that distinguished ia from non-ia with 94% sensitivity and 93% specificity. in one case, of the two false-positive samples, one was from a patient whose respiratory specimens were fungal culture negative and antigen negative, but upon autopsy, pulmonary nodules were recovered that stained positive for aspergillus. this not only highlights the difficulty in benchmarking new biomarkers against a clinical diagnostic with poor sensitivity but also underscores the potential impact of adding breath tests to the diagnostic armamentarium. fungi can also play a significant role in noninfectious respiratory disease. fungi are implicated in more than 11 million annual cases of allergic asthma and 12 million annual cases of rhinosinusitis globally, 25 but studying the causal relationships between fungal colonization and disease severity is impeded by respiratory specimen access. carpagnano and colleagues sought to determine whether fungi could be cultured from ebc from asthma patients, with the ultimate goal of supplanting sputum induction, which is not advised for asthmatics with moderate-to-severe disease. 27 ebc was collected from 28 atopic (i.e., allergic) asthma patients, 19 nonatopic asthma patients, and 20 controls, and these samples were paired with induced sputum, where clinically permissible. in addition, data on disease severity, anthropometric variables (body mass index, age, sex, etc.), and fraction of exhaled nitric oxide (f e no) were collected. using fungal growth on selective media as a positive result, 70% of ebc from asthmatic subjects were found to be positive for fungi, whereas none of the control subjects were fungal-positive. where a paired sputum sample was available, the same culture results were obtained from induced sputum and ebc, yielding a 100% sensitivity for the ebc test. interestingly, ebc fungal positivity correlated with disease severity, with 100% of moderate-to-severe asthma patients culturing fungi versus 90% of mild and 63% of intermittent asthmatics, but there was no correlation to f e no. a weakness in this study was the lack of gold-standard specimens (induced sputum) in the moderate-to-severe cases of disease, but this again highlights the significant clinical need that ebc can fill for tracking fungal disease and colonization. while not specific to the airway, malaria is a well-known viral infection, with hundreds of millions of worldwide cases each year. early diagnosis is crucial to prevent death, but diagnosis is still contingent on a century-old test: visualization of the parasite in stained blood films. berna et al. collected breath samples from subjects involved in controlled malaria studies for the purposes of developing vaccines and treatments. 28 astonishingly, nine vocs were not only elevated in the breath of the malaria cohort but also the concentration of these volatiles tracked with parasitemia levels measured in their blood. when subjects began their antimalarial treatments 8 days after being infected, the levels of these vocs decreased, still correlating with the measured level of blood parasites. thus, the interaction of the parasite with the body produced volatile compounds in human breath that could be used for malaria diagnostics. detection of malaria infection via breath is covered in depth in the next chapter. over the last decade, there have been tremendous advances in breath analysis techniques applied to respiratory infection and colonization, which represents one of the most clinically relevant applications for the field of breath diagnostics and monitoring. the studies outlined in this chapter present a paradigm of untargeted biomarker discovery, when study design is employed to uncover breath compounds that are statistically correlated with specific clinical conditions. as with all breath studies, pitfalls exist. in most human studies in this area, there have been limited subject numbers, and the breath biomarkers have not been confirmed in independent cohorts. currently, there is no adequate understanding of how confounding factors or other unrelated clinical conditions may affect biomarker profiles. it is possible that breathborne biomarker signals may change over time and may vary with pathogenicity and microbial load. it is also possible that animal studies could be performed to inform pilot human studies, but that is not yet a common practice, and there have not yet been exhaustive comparisons of animal and human trials to cross-correlate and confirm results between the two. there is a tremendous amount of ongoing parallel research to elucidate biomarker signals that emanate from in vitro cell culture modelsdin addition to the human/animal studies work (as outlined in chapter 26). most of this in vivo and in vitro work has been accomplished using various types of confirmatory analytical chemistry methods (e.g., mass spectrometry). while bringing novel sensors into this research area will eventually be an important step toward clinical translation, unequivocal chemical identification is critical during this untargeted exploratory phase of biomarker discovery. the field is now on the cusp of profiling candidate breath biomarkers of infection that can be pursued further. the best clinical studies to date have compared human/animal breath data paired with studies of in vitro organism cell models, especially those that examined biomarkers from the actual microbial clinical isolates involved in the clinical work. applications to aid modern emerging infectious disease epidemics or pandemics truly represent a holy grail of breath researchdthe chance to provide meaningful early, real-time, noninvasive diagnostics and distinguish trivial conditions from grave infectious pathogens. given that so many respiratory infections result in nonspecific clinical presentations (e.g., elevated body temperature, malaise, etc.), it would be extremely valuable to differentiate patients who are critically ill with a specific circulating pathogen of interest. it would also allow clinicians to focus on truly ill patients and distinguish them from the "worried well" in western nations who may descend on hospital emergency departments during an infectious disease outbreak. in the far future, it would also allow public health officials to use potential technology to help limit or prevent disease spread and could provide information to help during quarantine endeavors in extreme situations. for epidemiologists, asymptomatic biomarker detection of infection could be very valuable while tracing contacts during an outbreak. while the breath research community works toward these goals, more research is needed before any of these scenarios is possible. ideally, researchers would have immediately available high-fidelity in vivo cell culture and animal models to generate candidate breath biomarkers without the need for parallel human discovery efforts. this would save the most time during an infectious disease outbreak or in the unlikely event of intentional or engineered biological pathogen release. none of this is possible at present, but as in vitro cell culture models advance and begin to increase in fidelity, it is possible this could ultimately occur. this paradigm would have been very beneficial three times since the turn of the century, during the 2002 severe acute respiratory syndrome (sars) pandemic, the 2009 swine influenza h1n1 pandemic, and the 2019 coronavirus disease (covid-19) pandemic currently underway. the potential role of exhaled breath analysis in the diagnostic process of pneumoniada systematic review breathprints of model murine bacterial lung infections are linked with immune response tuberculosis diagnostics: state of the art and future directions using giant african pouched rats to detect human tuberculosis: a review non-invasive approach to diagnosis of pulmonary tuberculosis using microdroplets collected from exhaled air exhaled human breath analysis in active pulmonary tuberculosis diagnostics by comprehensive gas chromatography-mass spectrometry and chemometric techniques preliminary investigation of human exhaled breath for tuberculosis diagnosis by multidimensional gas chromatography e time of flight mass spectrometry and machine learning towards the use of breath for detecting mycobacterial infection: a case study in a murine model a new method to evaluate macaque health using exhaled breath: a case study of m. tuberculosis in a bsl-3 setting identification of mycobacterium tuberculosis using volatile biomarkers in culture and exhaled breath vivo volatile organic compound signatures of mycobacterium avium subsp. paratuberculosis volatile emissions from mycobacterium avium subsp. paratuberculosis mirror bacterial growth and enable distinction of different strains clinical outcome after early pseudomonas aeruginosa infection in cystic fibrosis exhaled breath hydrogen cyanide as a marker of early pseudomonas aeruginosa infection in children with cystic fibrosis volatile molecules from bronchoalveolar lavage fluid can 'rule-in' pseudomonas aeruginosa and 'ruleout' staphylococcus aureus infections in cystic fibrosis patients breath metabolome of mice infected with pseudomonas aeruginosa elevated carbon monoxide in the exhaled breath of mice during a systemic bacterial infection infection of pigs by aujeszky's disease virus via the breath of intranasally inoculated pigs effect of influenza vaccination on oxidative stress products in breath cellular scent of influenza virus infection voc breath profile in spontaneously breathing awake swine during influenza a infection use of an innovative and non-invasive device for virologic sampling of cough aerosols in patients with community and hospital acquired pneumonia: a pilot study viral colonization in exhaled breath condensate of lung cancer patients: possible role of ebv and cmv volatile fingerprinting of pseudomonas aeruginosa and respiratory syncytial virus infection in an in vitro cystic fibrosis co-infection model global and multi-national prevalence of fungal diseases-estimate precision a breath fungal secondary metabolite signature to diagnose invasive aspergillosis analysis of the fungal microbiome in exhaled breath condensate of patients with asthma. allergy analysis of breath specimens for biomarkers of plasmodium falciparum infection key: cord-342915-r9kv67we authors: hayden, frederick g. title: advances in antivirals for non‐influenza respiratory virus infections date: 2013-11-01 journal: influenza and other respiratory viruses doi: 10.1111/irv.12173 sha: doc_id: 342915 cord_uid: r9kv67we progress in the development of antivirals for non‐influenza respiratory viruses has been slow with the result that many unmet medical needs and few approved agents currently exist. this commentary selectively reviews examples of where specific agents have provided promising clinical benefits in selected target populations and also considers potential therapeutics for emerging threats like the sars and middle east respiratory syndrome coronaviruses. recent studies have provided encouraging results in treating respiratory syncytial virus infections in lung transplant recipients, serious parainfluenza virus and adenovirus infections in immunocompromised hosts, and rhinovirus colds in outpatient asthmatics. while additional studies are needed to confirm the efficacy and safety of the specific agents tested, these observations offer the opportunity to expand therapeutic studies to other patient populations. considerable progress has been made in the development of influenza antivirals with two major classes of drugs, the adamantanes and neuraminidase inhibitors, being available for clinical use in most countries and a variety of agents in various stages of investigational development. 1, 2 the widescale use of nais in some countries during response to the 2009 a(h1n1) pandemic provided much new observational data on the effectiveness of oseltamivir treatment in reducing the risk of severe influenza outcomes like pneumonia and hospitalization and of mortality in those hospitalized. [3] [4] [5] [6] [7] such observations confirm the principle that selective inhibitors of influenza replication are associated with both symptom relief and complications reduction and suggest that timely antiviral therapy of other respiratory viral infections might provide similar benefits. unfortunately, we have only a few approved agents for other respiratory viruses and the use of these agents is typically limited to specific risk groups like infants and immunocompromised hosts. one challenge in developing both vaccines and therapeutics for non-influenza respiratory viruses is the diversity of these pathogens, representing six major virus families, primarily rna but also dna viruses, and numerous serotypes and genotypes. the pathogenesis of these infections ranges broadly depending on the virus, host, and clinical setting, and for many of these illnesses, it remains uncertain to what extent inhibition of viral replication would be associated with clinical benefits. most of the treatment data regarding antivirals for non-influenza respiratory viruses have been derived from observational studies in immunocompromised hosts, and sometimes, infants, but recent randomized, controlled trials in specific target populations have helped to address the potential value of antiviral interventions. the following commentary, based on a presentations by the author at the 2nd meeting of the international society of influenza and other respiratory viruses antiviral interest group, hanoi, november 2012, and at the xvth international symposium on respiratory viral infections, rotterdam, march 2013, is a highly selective review of antiviral treatment interventions that show particular promise for specific respiratory viruses. it focuses primarily on clinical reports but also includes some observations from pre-clinical studies. potential treatments and the lessons learned in therapeutic studies of severe acute respiratory syndrome (sars) covinfected patients. in sars patients, the role of antiviral therapy was supported by finding that viral load was positively correlated with the development of organ dysfunction and death. 12 sars was characterized by protracted virus replication, peaking during the second week of illness, prominent host pro-inflammatory responses, and histologic evidence for diffuse alveolar damage. few data regarding viral replication patterns and disease pathogenesis are currently available for mers-cov infections, although prolonged viral replication in the lower respiratory tract, extrapulmonary virus detection, lung injury, respiratory failure and often renal failure are notable features in reported cases. [8] [9] [10] [11] a wide range of agents were reported to have anti-sars-cov activity in pre-clinical studies, [12] [13] [14] [15] [16] and a considerable number of therapeutic interventions directed at inhibiting cov replication or modifying the host responses to infection were attempted in sars patients. although corticosteroids were widely used for treating those with progressive sarsrelated pneumonia in efforts to reduce excessive pro-inflammatory responses and presumed immune-mediated tissue damage, their benefits were not conclusively demonstrated, and various reports documented increases in the plasma viral loads, opportunistic infections, and both immediate and delayed (e.g., osteonecrosis) side effects. 12, [17] [18] [19] in addition, systematic reviews of the observational reports concluded that the common use of multiple agents in combination, varying dose regimens, paucity of studies with systematic data collection, complications from immunosuppressive therapy, and the lack of randomized, controlled trials meant that existing data were inconclusive with regard to putative antivirals and thus inadequate to determine appropriate management of sars infections. 12, 18, 19 the use of available agents like ribavirin and hiv protease inhibitors was not associated with proven benefit in sars patients, although retrospective studies reported that severe outcomes (ards or death) occurred less often in those receiving a combination of lopinavir/ritonavir and ribavirin with corticosteroids compared with historic controls receiving only ribavirin with corticosteroids. 20, 21 early combination treatment was also associated with fewer nosocomial infections, less use of pulse corticosteroids for progressive disease, and lower nasopharyngeal viral loads over time, perhaps related to corticosteroid-sparing effects. 21 of note, in mice, high ribavirin dosing at 75 mg/kg starting 4 hour prior to sars virus exposure and then given twice daily for 3 days was found to increase virus lung titers and prolong the duration of virus detectability, 22 and it is unclear whether ribavirin might have had similar effects in treated humans. one small pediatric study found no correlation between ribavirin administration and plasma sars rna levels. 23 in sars patients, ribavirin was associated also with significant toxicities, including hemolytic anemia and metabolic disturbances like hypocalcaemia and hypomagnesaemia. 24 one recent in vitro study found that very high ribavirin concentrations were inhibitory for mers-cov in vero and llc-mk2 cells but that when combined with recombinant human interferon-alfa2b, lower concentrations were inhibitory. 25 consequently, ribavirin's possible role in treating cov infections remains uncertain. sars-cov impairs the induction of interferons and their associated antiviral effects but is inhibited by exogenous interferon in cell culture studies. 15 injection of pegylated interferon-alfa2b was highly protective given 3 days before and reduced lung viral levels and histopathology when initiated 1 day post-infection in a cynomolgus macaque model of sars-cov, 26 and both a hybrid interferon and an interferon inducer, mismatched double-stranded poly(i:c), reduced lung viral titers in a murine model. 14 intranasal application of interferons or the interferon inducer poly(i:c) was protective and reduced virus replication in an aged mouse model of sars. 27 interferons also have immunomodulatory effects, and in an aged macaque model of sars, treatment with type i interferon reduced pathology and diminished pro-inflammatory gene expression, including interleukin-8 (il-8) levels, without affecting virus replication in the lungs. 28 one observational canadian study in sars patients with progressive illness found that synthetic interferon alfacon-1 in conjunction with corticosteroids appeared to reduce time to resolution of pulmonary infiltrates, improve oxygen saturation, reduce the duration of supplemental oxygen use, and sped resolution of serum ldh elevations compared with corticosteroids alone. 29 interferon alfacon-1 was generally well tolerated, although associated with transient reductions in neutrophil counts and elevations in transaminase levels. of note, the mers-cov has been shown to be readily inhibited by type i and iii interferons in human bronchial epithelium ex vivo. 30, 31 such observations are sufficiently encouraging to support controlled studies of systemic interferon in affected patients. in addition, one approved agent for selected parasitic infections, oral nitazoxanide, may have interferon-inducing properties, is inhibitory for various respiratory viruses including influenza and a canine cov in vitro, 32 and has shown promising dose-related activity in a phase 2, placebo-controlled, randomized trial in treating uncomplicated influenza 33 consequently, nitazoxanide would be an interesting agent to test alone and in combination with other antivirals for cov infections. the protective efficacy of sars-cov-neutralizing antibodies, including humanized and human monoclonals directed to the spike protein, has been demonstrated in various animal models. 15, 34, 35 these studies found potent antiviral effects without evidence for the immune enhancement. passive immunotherapy with convalescent plasma from sars patients was used in treating sars patients in advances in antivirals for non-influenza rvis ª 2013 blackwell publishing ltd hong kong and taiwan in open-label fashion with apparent clinical benefits and rapid decreases in plasma viral load (from up to >10e5 copies/ml to undetectable levels 24 hour after infusion). 36, 37 among 80 patients given convalescent plasma in hong kong, a higher day 22 hospital discharge rate was observed among patients who were received plasma before day 14 of illness compared with later (58á3% versus 15á6%; p < 0á001) and among those who were viral rt-pcr positive but sars-cov seronegative compared with those seropositive at the time of plasma infusion. 36 efforts to develop neutralizing human monoclonals for mers-cov are in progress, as is screening for other inhibitors. other inhibitors of the spike (s) protein and its receptor interactions have been described. for example, in a rhesus macaque sars model, sirna inhibitors targeting the sars-cov genome at s protein-and nsp12-coding regions reduced sars-cov-induced fever, numbers of infected cells, and histologic findings of diffuse alveolar damage, compared with control or a non-specific sirna, when given intranasally as prophylaxis or post-infection. 38 pre-clinical reports suggest that inhibitors of angiotensin-converting enzyme-2 (ace-2), one of the attachment sites for the sars s protein, and other s protein inhibitors like mannose-binding lectin (mbl) might provide therapeutic benefit in sars. however, the mers-cov does not use ace2 but rather dipeptidyl peptidase 4 (dpp4 or cd26) to initiate infection 39 and has a much broader spectrum of cell infectivity, including human, swine, and bats cells than sars-cov. 40 consequently, ace2 inhibitors would be unlikely to exert specific inhibition of hcov-emc. identification of the receptor used by mers-cov may provide selective inhibitors of this interaction, but available agents that inhibit the enzymatic function of dpp4 apparently do not. 39 one study of sars patients found that they had snps affecting mbl expression and reduced levels of serum mbl compared to controls without sars, 41 although serum mbl levels did not correlate with disease severity. serum mbl concentrations vary widely due to mutations of the promoter and coding regions of the human mbl gene. recombinant and plasma-derived human mbl bind to the s protein through one or more n-linked glycosylation sites and inhibit sars-cov replication in susceptible cell lines at concentrations below those observed in the serum of healthy individuals. 42 the cell entry inhibitory effect appears to be mediated in part by blocking viral binding to c-type lectins but apparently not to the ace2 receptor. 42 no in vivo therapeutic studies have been reported in sars, but high doses of recombinant human mbl showed activity in a lethal ebola virus model in mice. 43 because mbl binds to carbohydrates on the surface of a wide range of respiratory viruses, including influenza, paramyxoviruses, and coronavirus, and other important pathogens, mbl is a potential therapeutic intervention of general interest. respiratory syncytial virus is a well-recognized cause of morbidity and, in those at the extremes of age or with severe immunocompromising conditions, mortality. it is the leading cause of bronchiolitis in infants and young children, and one analysis estimated that in 2005, rsv infections were associated with approximately 34 million episodes of acute lower respiratory illness (alri), or about 22% of alri, in children below the age of 5 years. 44 these illnesses led to hospitalization in about 10% of cases and to between 66 000 and 200 000 deaths globally, with 99% of these deaths in low-and middleincome countries. 44 in contrast, in developed countries like the united states, rsv is associated with many more deaths in adults aged 65 years and older than in children. 45 the available antiviral options for rsv are quite limited. much work has been carried out on developing antibody preparations, and the anti-f protein monoclonal palivizumab is currently approved for use for prophylaxis in high-risk children. 46 while effective in reducing rsv-related hospitalization by about 50%, its very high cost limits availability to well-resourced countries. aerosolized ribavirin is approved for treating hospitalized children with serious rsv illness, but it is used very infrequently in this population due to its difficult delivery method, modest antiviral and clinical effects, cost, and concerns about healthcare worker exposure to a potential teratogen. the american academy of pediatrics recommends against its routine use. 47 however, ribavirin has been used frequently in hematopoietic stem cell transplant (hsct) recipients, because of their high mortality risk when rsv lower respiratory tract illness (lrti) develops. 48 a systematic review of observational studies across multiple of centers found that compared with no ribavirin use, ribavirin treatment, regardless of route or whether combined with an immunomodulatory regimen (intravenous immunoglobulin [ivig], rsv immunoglobulin, or palivizumab), was associated with reduced frequencies of progression to lrti. 49 less frequent progression to lrti and significantly fewer deaths in those with lrti were observed among patients treated with combinations of aerosolized ribavirin and an immunomodulatory agent than in those treated with ribavirin alone. however, a recent retrospective analysis of one center's experience concluded that the addition of palivizumab to aerosolized ribavirn did not improve outcomes in rsv-infected hsct recipients. 49a a recent randomized controlled trial comparing two aerosolized ribavirin regimens found that intermittent delivery (2 g over 2 hours every 8 hours) appeared to be more effective in preventing lower respiratory progression than a standard continuous one (6 g over 18 hoursr) and was easier to administer. 50 while data on oral ribavirin are very limited, one study suggested favorable outcomes in hsct recipients with upper respiratory rsv infection when used in combina-tion with ivig and palivizumab. 51 however, a recent retrospective analysis of hematologic malignancy and hsct patients with rsv or other paramyxovirus infections concluded that oral ribavirin was not clinically effective in preventing mortality. 52 in lung transplant recipients with rsv or human metapneumovirus infections, observational studies suggest that ribavirin given intravenously or orally may be beneficial with regard to improving clinical outcomes and recovery of lung function, 53,54 but the uncontrolled nature of these findings precludes firm conclusions. further controlled studies of oral ribavirin, which is less expensive and easier to administer than aerosolized ribavirin, are warranted in these important patient groups. a number of rsv investigational agents have received some degree of recent clinical testing, although development of several apparently has been stopped at this time. for example, a potent anti-f antibody, motavizumab, was shown to have antiviral effects in hospitalized children 55 but was not superior to palivizumab in seasonal rsv prophylaxis in atrisk children and had increased cutaneous adverse events relative to palivizumab. 56 a placebo-controlled rct of a single motavizumab injection did not find reductions in upper respiratory tract viral titers or illness measures in infants aged < 12 months and hospitalized with rsv illness [ramilo et al.,r presented at the 8th annual respiratory syncytial virus symposium, september 27-30, 2012, santa fe, nm]. more potent monoclonals to f protein are in development. among those in active clinical development, there is a polyclonal high-titer rsv immunoglobulin (ri-001; adma biologics inc., hackensack, nj, usa) being tested in immunocompromised patients to prevent progression from upper to lower respiratory tract illness, a topically applied f protein inhibitor (mdt-637; microdose therapeutx, monmouth junction, nj, usa) and an orally administered f protein inhibitor (gs-5806; gilead sciences, inc., foster city, ca, usa). the results of double-blinded, placebo-controlled studies with gs-506 to test efficacy in experimental rsv infection in adult volunteers (nct01756482) and safety in young children hospitalized for rsv illness (nct01797419) are expected to be available shortly. because the envelope glycoprotein f plays an important role in rsv fusion with and entry into the host cell, it serves as an attractive target for developing rsv entry inhibitors, although resistance emergence has been a notable limitation in earlier studies. 57 the agent that has progressed furthest in testing is a double-stranded oligonucleotide directed against the viral n gene (aln-rsv01; alnylam pharmaceuticals, cambridge, ma, usa). this sirna, complementary to the mrna that encodes the n protein, showed robust antiviral effects both in vitro and in a murine model, with over a 1000-fold reduction in lung virus titers, 58 and was active when given intranasally to volunteers experimentally infected with rsv. 59, 60 in a placebo-controlled rct of 24 lung transplant recipients with rsv infection, aerosolized aln-rsv01 (0á6 mg/kg) given daily for 3 days was generally well tolerated and significantly reduced the risk of new or progressive bronchiolitis obliterans syndrome (bos) at day 90 compared with placebo (6á3% versus 50%). 61 there were no differences in upper respiratory tract viral loads, but those in the lower could not be tested. consequently, a larger phase 2b rct was conducted at 33 sites in six countries at the same dose but extended to 5 days administration, again added to standard of care. 62 the primary endpoint of bos at day 180 among the 77 lung transplant patients in the intent-to-treat-infected population tended to be lower with inhaled aln-rsv01 (30á3% versus 13á6%; p = 0á058) and was significantly lower among the 73 patients in the per-protocol analysis (28á1% versus 9á8%). deaths occurred in 2 placebo and 1 sirna recipient that were unrelated to treatment. these encouraging results warrant further studies of this novel sirna therapeutic in other risk populations and support the study of sirna inhibitors for other respiratory viral infections. 59 parainfluenza virus (piv) illness can be very severe in immunocompromised hosts, particularly hsct recipients. the results with ribavirin have been quite mixed. aerosolized ribavirin has not resulted in reductions in viral shedding or survival benefit in hsct with piv pneumonia, while oral or intravenous ribavirin has provided apparent benefit in individual hsct recipients and hematologic malignancy patients with piv illness. 48 a recent retrospective analysis concluded that oral ribavirin did not reduce mortality in hematologic malignancy patients with paramyxovirus infections compared with supportive care alone, 52 whereas oral ribavirin seems to have been associated with benefit in lung transplant patients with paramyxovirus infections. 54 a new investigational agent for severe piv infection is das181, a fusion construct that includes a sialidase from actinomycosis viscosus that cleaves both a2,6-and a2,3-linked sialic acid receptors on host cells. 63 consequently, its antiviral spectrum includes influenza viruses and it is active when topically applied in animal influenza models, including avian h5n1 and h1n1pdm09 viruses, [63] [64] [65] and has shown antiviral effects in a phase 2 rct in uncomplicated human influenza. 66 of note, the h-n protein of piv also binds to sialic acid receptors, and das181 is inhibitory for piv in cell culture, in human airway epithelium, and given intranasally in a cotton rat model. 67 das181 has been administered on a compassionate use basis for treating hsct and lung transplant patients with severe piv illness with apparent clinical benefit and antiviral effects in some cases. [68] [69] [70] this host-directed agent is available on compassionate use from its sponsor (formerly nexbio, now ansun biopharma, inc., san diego, advances in antivirals for non-influenza rvis ª 2013 blackwell publishing ltd ca, usa), and an open-label study is being undertaken at the nih for this particular problem. adenovirus infections are often very severe in immunocompromised hosts but occasionally in those in the community as well. 71, 72 there are currently no approved antivirals for adenovirus treatment, but a number of agents have some degree of in vitro inhibitory activity. 73 ribavirin has variable in vitro activity at high concentrations 74 and proven largely ineffective in treatment of serious infections. intravenous cidofovir, a nucleoside phosphonate analog, has become the standard treatment for most immunocompromised patients with adenovirus disease, but its use is associated with nephrotoxicity in up to 50% and neutropenia in 20% of patients. 75, 76 donor leukocyte infusion and use of in vitroexpanded adenovirus-specific t cells have been used in some patients. 76 the most promising anti-adenovirus antiviral in clinical development is the orally administered, lipid ester derivative of cidofovir, designated cmx001or brincidofovir (chimerix, durham, nc, usa). in vitro cmx001 provides higher intracellular levels compared with the parent molecule and is over 50-fold more active than cidofovir for adenoviruses. 77 it was shown to be inhibitory for lethal adenovirus infection in a syrian hamster model. 78 in addition to the advantage of being orally bioavailable, cmx001 also has lower risk of nephrotoxicity compared with cidofovir during clinical use. one case series of 13 immunocompromised patients, the majority of whom were hsct recipients, described its use in severe adenovirus disease. 79 all had adenoviremia, and six had disseminated disease involving other organs. following onset of the adenoviral disease at a median of 75 days posttransplant, they were initially treated with cidofovir, but then switched to cmx001 because of either persistent viral replication or nephrotoxicity. prolonged courses of cmx001 were associated with good virologic responses, including at least 100-fold reductions in plasma adenoviral dna levels in nine patients. some delay in mortality was also observed among those with virologic responses. two current cmx001 studies will provide further insights regarding its clinical utility for adenoviral infections in high-risk populations. one is a controlled trial of pre-emptive therapy in hsct patients with adenoviremia that has finished enrollment and is under analysis (nct01241344), and the other is an open-label study of treating a variety of serious dna viral diseases, including those due to adenoviruses, in immunocompromised hosts (nct01143181). an initial press release (14 august 2013) indciated that cmx001 100 mg biw decreased levels of adenoviremia and showed potential benefits in reducing both progression to adenovirus disease and all-cause mortality, compared to subjects who received placebo or cmx001 given once weekly. phase 1 safety and pharmacology studies have been completed in healthy volunteers. 80 cmx001 is a promising agent that also warrants testing in non-immunocompromised hosts with severe adenovirus illness. human rhinovirus (hrv) infections are the most frequent infections that humans experience and are implicated in causing a wide range of respiratory tract syndromes. in addition to being the most frequent cause of colds, hrv infections are the leading cause of exacerbations of asthma and chronic obstructive pulmonary disease. past studies of the capsid-binding anti-hrv agent pleconaril showed that early treatment exerted antiviral effects and reduced the duration of uncomplicated hrv colds by about 1 day. 81 symptomatic improvement in pleconaril-treated subjects was related to the drug susceptibility of the infecting hrv, and strains with >10fold reduced susceptibility emerged in about 11% of recipients. 82 however, it was not approved for clinical use largely because of potential drug interaction concerns. recently, another capsid-binding anti-hrv agent designated bta798 (or vapendavir) (biota holdings ltd, notting hill, vic., australia) showed dose-related antiviral effects in experimentally infected volunteers 83 and beneficial effects in a phase 2 placebo-controlled rct in asthmatics with cold symptoms (nct01175226). 84 among the 300 persons enrolled, 93 had a documented hrv infection. oral vapendavir (400 mg twice daily for 6 days) was associated with significantly lower upper respiratory symptom scores early in the illness and continuing up to 2 weeks compared with placebo. vapendavir recipients also had significant improvements in secondary outcomes including higher peak expiratory flow rates on day 5, reduced overall use of asthma relief medications, and less frequent hrv rna detection on day 3 (74% versus 91%). this modest degree of antiviral effect raises the possibility that more potent inhibition might be able to provide even greater clinical benefits. intranasal recombinant interferon-alpha2b was shown to be effective in preventing hrv colds when used for postexposure prophylaxis 85, 86 but ineffective for treatment of established colds 85 and also associated with local side effects. recently, 14 days treatment with an inhaled interferon-beta designated sng001 (synairgen plc, southampton, england) was associated with therapeutic efficacy in a phase 2, placebocontrolled rct of adult asthmatics receiving inhaled corticosteroids who had a history of deterioration with colds (nct01126177). 87 among 147 enrolled, 134 met the criteria for a cold; hrvs represented 68% of the respiratory viruses detected. although the trial did not meet its primary endpoint (changes in the shortened asthma control questionnaire) in the overall population, among the subset of "difficult to treat" asthma patients representing about onehalf of those enrolled, inhaled sng001 was associated with significant improvements in asthma symptoms, 65% fewer moderate exacerbations, improved morning peak expiratory flow rates, and reduced use of relief bronchodilators. while the findings in the oral vapendavir and inhaled interferonbeta studies need to be confirmed in larger trials, the results suggest that early antiviral treatment of colds in asthmatics can moderate asthma exacerbations. in summary, currently, there are many unmet medical needs and few approved agents for the non-influenza respiratory virus infections. recent studies have provided encouraging results in serious respiratory viral infections in hospitalized immunocompromised hosts and in outpatient asthmatics with colds. while additional trials are needed to confirm the efficacy and safety of the agents discussed above, these or other selective antivirals offer the opportunity to expand therapeutic studies to other patient populations. one of the challenges in developing more effective therapeutics is the diversity of respiratory viruses and the differences in disease pathogenesis across viruses and patient groups. in addition to das181 for piv, several agents in clinical development for influenza (e.g., favipiravir, nitazoxanide) have in vitro activity against several other respiratory viruses. 32, 88 in addition, analogous to work in influenza, 89 one forward-looking strategy is understanding how respiratory viruses interact with host cellular pathways during replication and identifying potential viral-host protein interactions to target. for example, one large mrna gene expression database search identified 67 common pathways among seven different respiratory viruses; of the top five pathways, 53 had differentially expressed genes affected by at least five of the seven viruses. 90 such studies might lead to identifying existing drugs that could be repurposed to target these pathways and eventually to broader spectrum 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patients how i treat adenovirus in hematopoietic stem cell transplant recipients ether lipid-ester prodrugs of acyclic nucleoside phosphonates: activity against adenovirus replication in vitro hexadecyloxypropyl-cidofovir, cmx001, prevents adenovirus-induced mortality in a permissive, immunosuppressed animal model safety and efficacy of cmx001 as salvage therapy for severe adenovirus infections in immunocompromised patients first pharmacokinetic and safety study in humans of the novel lipid antiviral conjugate cmx001, a broad-spectrum oral drug active against double-stranded dna viruses efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses rhinovirus both in vitro and in an experimental human infection model biota press release. hrv phase iib study achieves primary endpoint prevention of natural colds by contact prophylaxis with intranasal alpha 2-interferon prophylactic efficacy of intranasal alpha 2-interferon against rhinovirus infections in the family setting positive phase ii asthma clinical trial data t-705 (favipiravir) and related compounds: novel broad-spectrum inhibitors of rna viral infections genetic screens for the control of influenza virus replication: from meta-analysis to drug discovery identification of common biological pathways and drug targets across multiple respiratory viruses based on human host gene expression analysis acknowledgements i thank dr. michael ison for helpful comments on the draft manuscript and lisa cook for assistance with manuscript preparation. since 2008 fgh has served as an unpaid consultant to multiple companies involved in developing antivirals for respiratory viral infections including several investigational agents discussed in this article (synairgen; nexbio, now ansun). key: cord-315794-se0sq3c3 authors: lamps, l w title: infective disorders of the gastrointestinal tract date: 2006-12-14 journal: histopathology doi: 10.1111/j.1365-2559.2006.02544.x sha: doc_id: 315794 cord_uid: se0sq3c3 gastrointestinal infections are a major cause of morbidity and mortality worldwide. infectious organisms are often recovered by microbiological methods, but surgical pathologists may play a very valuable role in diagnosis. this review will focus on infective disorders of the gastrointestinal tract with an emphasis on enterocolitides caused by food‐ and water‐borne pathogens. diagnostic histological features of selected enteric infections will be emphasized, including those that mimic other inflammatory conditions of the gut (such as ischaemia or idiopathic inflammatory bowel disease), along with available diagnostic methods that can aid in diagnosis. infective disorders of the gastrointestinal tract are a major cause of morbidity and mortality worldwide. as numbers of transplant patients and those with other immunocompromising conditions increase, and as global urbanization and transcontinental travel become more frequent, the surgical pathologist must be familiar with infectious diseases that were previously encountered only infrequently. the pathologist's goal in evaluating a gastrointestinal biopsy for infectious colitis is twofold. first, acute self-limited and ⁄ or infectious processes must be differentiated from chronic idiopathic inflammatory bowel disease (ulcerative colitis or crohn's disease). [1] [2] [3] [4] second, dedicated attempts must be made to identify the infecting organism(s). the surgical pathologist's ability to diagnose infectious processes in tissue sections has grown exponentially with the advent of new histochemical stains, immunohistochemistry, in situ hybridization and numerous other molecular methodologies. as these techniques have developed, our understanding of the correlation between histological patterns of inflammation and specific organisms or groups of organisms has also increased. the majority of enteric infections are self-limited. those patients that undergo endoscopic biopsy often have chronic or debilitating diarrhoea, systemic symptoms, or a history of immunocompromise or other significant clinical scenarios. a discussion with the gastroenterologist regarding exact symptomatology and colonoscopic findings, as well as facts including travel history, food intake (such as sushi or poorly cooked beef), sexual practices and immune status, can greatly aid in the evaluation of a biopsy for infectious diseases. 5 as many infective disorders of the gastrointestinal tract are acquired through contaminated food, this review will focus primarily on enterocolitides caused by food-borne bacteria. in addition, infectious processes that mimic or cause ischaemia and those that mimic chronic idiopathic inflammatory bowel disease will be emphasized, along with ancillary techniques that aid in diagnosis. despite the vast number of food-borne infections that affect the gastrointestinal tract, the spectrum of histological findings produced by a given organism can be generally categorized in one of the following ways (see table 1 ): 1 those producing minimal or no histological changes (e.g. vibrio species and many enteric viruses). 2 those producing a non-specific acute self-limited ⁄ infectious colitis (aslc) pattern (e.g. campylobacter jejuni); this is one of the most common inflammatory patterns in enteric infections. [1] [2] [3] [4] 3 those producing suggestive or diagnostic histological features (e.g. pseudomembranes, granulomas, or visible organisms). acute infectious-type colitis characteristically features intact crypt architecture with neutrophilic infiltrates in the crypt epithelium. the lamina propria may be hypercellular, containing a mix of lymphocytes, histiocytes and neutrophils; plasma cells are generally not prominent, and basal plasma cells should not be seen in acute infectious-type colitis as these are a marker of chronicity. crypt abscesses and granulomas associated with damaged crypts may also be seen. there is often damage also to the surface epithelium. [2] [3] [4] since patients often do not come to endoscopy until several weeks after onset of symptoms, pathologists frequently do not see the classic histological features of acute infectious-type colitis. this is important, as the resolving phase of infectious colitis is more challenging to diagnose, as one may find only occasional foci of neutrophilic cryptitis with a patchy increase in lamina propria inflammation, which may contain numerous plasma cells and increased intraepithelial lymphocytes ( figure 1 ). as these features can also be seen in smoldering crohn's disease and lymphocytic colitis, it is important to know the patient's symptoms and, ideally, culture results as this differential diagnosis may be difficult to resolve on histological grounds alone. selected specific food-and ⁄ or water-borne gastrointestinal infective disorders escherichia coli, campylobacter, salmonella, yersinia, shigella and enteric viruses represent the most common food-borne pathogens worldwide. 5 it is important to note, when reviewing a patient's history, that many organisms associated with food-borne illness can also be transmitted through water or unpasteurized milk. 5 naturally occurring water-borne outbreaks of sa. typhi, enterohaemorrhagic e. coli and norwalk virus have been reported, usually due to improperly operated water treatment plants. several other organisms (including anthrax, francisella tularensis and shigella) are particularly stable in water due to chlorine tolerance. 6 although these organisms are sometimes recovered by culture, surgical pathologists may play a very valuable role in diagnosis, particularly when clinicians have failed to obtain cultures prior to initiation of antibiotic therapy. in addition, many of the food-and water-borne gastrointestinal infective diseases discussed below mimic other entities that are commonly encountered in surgical pathology practice, such as ischaemic colitis or idiopathic inflammatory bowel disease. these gram-negative bacteria are a major cause of naturally occurring diarrhoea worldwide. campylobacter jejuni and c. coli are most frequently associated with human enteric disease. 7, 8 campylobacter may contaminate poultry, beef, veal, pork, water and milk, and are also transmitted by the faecal-oral route. the infective dose is low (as few as 500 organisms can cause symptomatic disease). patients typically present with fever, malaise, abdominal pain and watery diarrhoea that often contains blood and faecal leucocytes. [8] [9] [10] symptoms generally present within 1-5 days of exposure and last for 4-10 days. most infections are self-limited, particularly in otherwise healthy patients, although relapse is common. of note, both reactive arthropathy and guillain-barré syndrome may be associated with campylobacter infection. 8 endoscopic findings are non-specific and include friable colonic mucosa with associated erythema and haemorrhage. histological examination shows features of acute self-limited colitis, including a neutrophilic infiltrate in the lamina propria, often most prominent in the mid-to-upper crypts; cryptitis; and scattered crypt abscesses ( figure 2 ). architecture overall is preserved, although mild crypt distortion is occasionally noted. 7-10 the histological differential diagnosis includes other infectious entities that produce the aslc pattern. the mainstay of laboratory diagnosis is culture from stool or blood. preliminary diagnosis may be made by detection of organisms in fresh stool smears by darkfield microscopy. excellent molecular techniques exist, but are not widely available for clinical use. 7 these gram-negative bacilli are particularly prevalent in underdeveloped countries where sanitation is poor and dairy and water supplies are contaminated with the organism. however, salmonella are also an important cause of sporadic food poisoning in developed countries. they are present in water, meat, dairy products, egg products and occasionally vegetables and fruits; they may survive partial cooking. 5 the discussion of salmonella species is historically divided into typhoid and non-typhoid species. patients with typhoid (enteric) fever, usually caused by sa. typhi, typically present with fever that rises over several days, abdominal pain and headache. abdominal rash ('rose spots'), delirium, hepatosplenomegaly and leukopenia are also fairly common. the diarrhoea, which begins in the second or third week of infection, is initially watery but may progress to severe gastrointestinal bleeding and perforation. non-typhoid species (e.g. sa. enteritidis, sa. typhimurium, sa. muenchen, sa. anatum, sa. paratyphi and sa. give) generally cause a milder, often self-limited gastroenteritis with vomiting, nausea, fever and watery diarrhoea. [11] [12] [13] [14] [15] the infective dose is relatively low (approximately 10 2 )10 3 organisms may cause human disease). although most salmonella infections in developed countries resolve with antibiotics and supportive care, enteric infection may progress to septicaemia and death, particularly in the elderly, the very young or patients who are debilitated. delayed treatment is associated with higher mortality. 12 any level of the gastrointestinal tract may be affected, but the ileum, appendix and right colon are preferentially involved. the bowel wall is thickened, with raised nodules corresponding to hyperplastic peyer patches. apthoid ulcers overlying peyer patches, linear ulcers, discoid ulcers or full thickness ulceration and necrosis often occur as disease progresses. perforation and toxic megacolon may be seen, as can suppurative mesenteric lymphadenitis. peyer patches become hyperplastic, followed by acute inflammation of the superficial overlying epithelium. eventually the lymphoid follicles are infiltrated and obliterated by macrophages. the histiocyte is the predominant inflammatory cell in typhoid fever; admixed lymphocytes and plasma cells are seen, but neutrophils are typically not prominent. the ulcers are characteristically very deep, with the base at the muscularis propria. [11] [12] [13] [14] [15] architectural distortion severe enough to mimic ulcerative colitis or crohn's disease may be present ( figure 3) . with non-typhoid species, the gross findings are often milder. the lesions can be focal, and occasionally the mucosa is grossly normal or only mildly hyperaemic and oedematous. the pathological features are most often those of acute self-limited colitis, although severe cases may have significant crypt distortion. 14 cultures are the mainstay of salmonella diagnosis. the differential diagnosis includes other enteric bacterial pathogens as well as crohn's disease and ulcerative colitis. 13, 14 clinically, the incubation period of salmonella infection is longer (10-15 days) than with similar enteric pathogens. salmonella infection often lacks significant numbers of neutrophils in comparison with other pathogens (as well as idiopathic inflammatory bowel disease), and granulomas are unusual in salmonellosis. crypt distortion is generally more pronounced in ulcerative colitis than in salmonellosis. clinical presentation and stool cultures may be very helpful in resolving the differential. shigella are virulent, invasive gram-negative bacilli that cause severe bloody diarrhoea. shigella dysenteriae is the most common species isolated, although sh. sonnei and sh. flexneri are increasingly reported in the usa. they are generally ingested from faecally contaminated water, but person-to-person transmission is also possible. the infective dose is very low (as few as 10-100 organisms in sh. dysenteriae type 1). 16 infants and small children, homosexual males and malnourished or debilitated patients are most commonly affected in developed countries. like salmonellosis, the gross and microscopic features of shigellosis may mimic chronic idiopathic inflammatory bowel disease. symptoms include abdominal pain, fever and watery diarrhoea, followed by bloody diarrhoea that is often mucoid and ⁄ or purulent. constitutional symptoms are common. 16, 17 perforation and haemolytic-uraemic syndrome have been described. most studies of mortality in shigellosis were performed in underdeveloped nations, and figures range from 2 to 10%. mortality is significantly higher (> 20%) if patients develop sepsis. 16 the large bowel is typically affected (often the left colon most severely), but the ileum may also be involved. the mucosa is haemorrhagic, and variably ulcerated, sometimes with pseudomembranous exudates. 18 early shigellosis has features of aslc with cryptitis, crypt abscesses (often superficial) and ulceration. apthoid ulcers similar to crohn's disease are variably present. as disease continues, mucosal destruction develops with many neutrophils (as well as other inflammatory cells) in the lamina propria. marked architectural distortion is common, leading to diagnostic confusion with chronic idiopathic inflammatory bowel disease. 17 stool culture is essential to diagnosis, but multiple cultures may be necessary to recover the organism, for it is fastidious and dies off quickly. polymerase chain reaction (pcr), dna probes and serological studies are also available. 16 the differential diagnosis of early shigellosis is primarily that of other infections, particularly enteroinvasive e. coli and clostridium difficile. later on in the course of the disease, it may be extremely difficult to distinguish shigellosis from crohn's disease or ulcerative colitis both endoscopically and histologically. 1 stool cultures and clinical history may be very helpful in resolving the differential. yersinia enterocolitica (ye) and y. pseudotuberculosis (yp) are the most common causes of bacterial enteritis in western and northern europe, and increasing numbers of cases have been documented in north america and australia. yersinia have been found in chicken, fish, shellfish, swine, cattle, milk, ice cream, beef, lamb, poultry, pork, ham and water. 5 infection with either species may cause abdominal pain, diarrhoea and symptoms and signs of appendicitis, enterocolitis or an acute abdomen. associated mesenteric lymphadenopathy is frequent. most infections are selflimited; however, immunocompromised and debilitated patients, as well as patients on deferoxamine or with iron overload, are at risk of more serious disease. 19 yersinia preferentially involves the ileum, right colon and appendix. grossly, involved bowel has a thickened, oedematous wall with nodular inflammatory masses centred around peyer patches. apthoid and linear ulcers may be present. both suppurative and granulomatous patterns of inflammation can be seen, and a mixture of the two is common. [20] [21] [22] recent studies have shown that there is significant overlap between the histological features of ye and yp infection and that either species may show lymphoid hyperplasia, epithelioid granulomas with prominent lymphoid cuffing (figure 4) , transmural lymphoid aggregates, giant cells, mucosal ulceration, cryptitis and concomitant lymph node involvement. 20 special stains are not helpful in the diagnosis of yersinia, for the organisms are small, may be present in low numbers and are difficult to distinguish from normal non-pathogenic colonic flora. culture, serological studies and pcr assays may be helpful in confirming the diagnosis. 20,23 the major differential diagnoses for yersiniosis include other infectious processes, particularly mycobacteria and salmonella. acidfast stains and culture results should help to distinguish mycobacterial infection; clinical features and the presence of greater numbers of neutrophils, microabscesses and granulomas may help to distinguish yersiniosis from salmonellosis. crohn's disease and yersiniosis may be very difficult if not impossible to distinguish from one another histologically. features that may favour crohn's include cobblestoning of mucosa and creeping fat grossly, and changes of chronicity microscopically, including crypt distortion, thickening of the muscularis mucosa and prominent neural hyperplasia. however, some cases are indistinguishable on histological grounds. 20 aeromonas species, initially thought to be non-pathogenic gram-negative bacteria, are increasingly recognized as causes of gastroenteritis in both children and adults. the motile a. hydrophila and a. sobria most often cause gastrointestinal disease in humans. the typical presentation is bloody diarrhoea, sometimes chronic, accompanied by nausea, vomiting and cramping pain. the diarrhoea may contain mucus as well as blood. the duration of illness varies widely, ranging from a few days to several years, indicating that aeromonas infection can cause a chronic colitis. [24] [25] [26] [27] [28] endoscopically, signs of colitis may be seen, including oedema, friability, erosions, exudates and loss of vascular pattern; the features are often segmental and may mimic ischaemic colitis or crohn's disease. a pancolitis mimicking ulcerative colitis has also been described. the histological features are usually those of acute self-limited colitis. however, ulceration and focal architectural distortion may be seen ( figure 5 ). stool cultures are critical to diagnosis. the differential diagnosis includes other infectious processes, ischaemic colitis and chronic idiopathic inflammatory bowel disease. culture should help exclude other infections, and typical features of ischaemia (crypt withering, mucosal necrosis) are not present with aeromonas. when architectural distortion is present in a patient with chronic symptoms, it may be very difficult to resolve the issue of aeromonas infection versus crohn's disease or ulcerative colitis. [24] [25] [26] [27] [28] some authorities recommend culturing for aeromonas in all patients with refractory chronic inflammatory bowel disease. approximately 10% of the world's population are infected with this parasite, and the prevalence is much higher in tropical and subtropical locations. increasingly, male homosexuals are also found to harbour this pathogen. although some patients suffer a severe, dysentery-like fulminant colitis, many others are asymptomatic or have only vague gastrointestinal symptoms (abdominal pain, diarrhoea with or without blood, and malaise). 29 rarely, large inflammatory masses (amoebomas) may be formed. grossly, small ulcers are the early lesion, which may coalesce to form large, irregular ulcers that are often geographical or serpiginous. the ulcers often have associated inflammatory exudate or inflammatory polyps. intervening mucosa is often normal. ulcers may undermine adjacent mucosa to produce the classic 'flask-shaped' lesion. the caecum is most commonly involved, but any level of the large bowel or appendix may be involved. fulminant colitis resembling ulcerative colitis, pseudomembranous colitis resembling that caused by c. difficile, and toxic megacolon have been described. 30 colonoscopy may be normal in asymptomatic patients or those with mild disease. 29 histologically, the earliest lesion is a mild neutrophilic infiltrate. in more advanced disease, ulcers are often deep, extending at least into the submucosa, with undermining of adjacent normal mucosa. there is associated necroinflammatory debris; the organisms are generally found within this purulent material. invasive amoebae are occasionally seen within the bowel wall. the adjacent mucosa is usually normal, but may show gland distortion and inflammation. the organisms, which may be very few in number, resemble macrophages with foamy cytoplasm and a round, eccentric nucleus; the presence of ingested red blood cells is pathognomonic of entamoeba histolytica. 30 in patients who are asymptomatic or have mild symptoms, histological changes may be subtle and organisms may be particularly difficult (if not impossible) to find. invasive amoebiasis is not generally seen in patients with mild or absent symptoms. 29 the differential diagnosis most often is that of amoebae versus macrophages within an inflammatory exudate. amoebae are trichrome positive; in addition, macrophages stain with immunostains for a 1 -antitrypsin and chymotrypsin, whereas amoebae do not. given the not infrequent presence of skip lesions and mucosal architectural distortion, amoebiasis may also be confused with crohn's disease or ulcerative colitis, as well as other types of infectious colitis. for the diagnostic pathologist, it cannot be overemphasized that yersinia (enterocolitica and pseudotuberculosis), salmonella (both non-typhoid and typhoid fever-producing strains), shigella, aeromonas and e. histolytica, in addition to the aslc pattern of inflammation, can produce both gross and histological features that mimic idiopathic inflammatory bowel disease (table 1) . correlation with clinical presentation, food and water exposure, and culture results can be invaluable tools in resolving this differential diagnosis. enterohaemorrhagic e. coli the most common strain of enterohaemorrhagic e. coli (ehec) is o157:h7, although there are others. this pathogen gained national attention in 1993 when a massive outbreak in the western usa was linked to contaminated hamburger patties. the organism binds intestinal epithelial cells and produces a cytotoxin similar to that produced by sh. dysenteriae, although there is no invasion. contaminated meat is the most frequent mode of transmission, but infection may also occur through contaminated water, milk, produce and person-toperson contact. 31, 32 diarrhoea is usually bloody, with severe abdominal cramps and mild or no fever. non-bloody, watery diarrhoea may occur, however. only one-third of patients have faecal leucocytes. children and the elderly are at particular risk of serious illness, including the haemolytic-uraemic syndrome or thrombotic thrombocytopenic purpura. 31, 32 endoscopically, patients may have bowel oedema, erosions, ulcers and haemorrhage, and the right colon is usually more severely affected. the oedema may be so marked as to cause obstruction, and surgical resection may be required to relieve this or to control bleeding. the lamina propria and submucosa contain marked oedema and haemorrhage, with associated mucosal acute inflammation, cryptitis, crypt abscesses, ulceration and necrosis. crypt withering, such as that seen in other causes of ischaemia, is often seen as well. microthrombi may be seen within small vessels and pseudomembranes are occasionally present ( figure 6 ). 31, 32 infection with ehec is probably markedly underdiagnosed. routine stool cultures will not distinguish pathogenic strains from normal intestinal flora. if ehec strains are suspected, the clinical laboratory should be notified to search for them specifically. ehec are cleared rapidly from stool, often within 4-7 days, thus cultures should be taken as early as possible. culture and serotyping are the mainstay of laboratory diagnosis; although immunohistochemical stains and molecular assays for ehec have been described, they are not widely available. the differential diagnosis of ehec includes c. difficilerelated colitis, idiopathic inflammatory bowel disease, and especially ischaemic colitis. culture and food intake history may be invaluable. in general, ehec does not produce the level of mucosal distortion seen in idiopathic inflammatory bowel disease, and lacks granulomas. the histological picture may be very difficult to distinguish from c. difficile, and the c. difficile antigen test may be very useful. as the cytotoxin produced by ehec causes ischaemia, the histological features are indistinguishable from ischaemia of other causes. biopsies from patients with enteric viral infections seldom if ever cross the stage of the surgical pathologist, as they are detected in stool samples rather than biopsy specimens. some common enteric viruses known to cause diarrhoea include, but are not limited to adenovirus, rotavirus, coronavirus, echovirus, enterovirus, astrovirus and norwalk virus. rotavirus is the most common cause of severe childhood diarrhoea and diarrhoeal mortality worldwide, followed by adenoviruses. 33 rotavirus infections occur predominantly in infants < 2 years old. the diarrhoea is usually accompanied by fever and vomiting. biopsy changes are very non-specific and include increased inflammatory cells in the lamina propria, degenerative epithelial changes and swollen villous tips. the diagnosis of rotavirus is generally made by stool immunoassay and ⁄ or culture, and the disease is rarely biopsied. adenovirus infection is second only to rotavirus as a cause of childhood diarrhoea. however, it has also gained much attention in recent years as a cause of diarrhoea in immunocompromised patients, especially those with hiv and aids. virtually all patients present with diarrhoea, sometimes accompanied by fever, weight loss and abdominal pain. histological features of adenovirus infection include epithelial changes such as cellular disorder, loss of orientation and degeneration. 34 characteristic inclusions may be seen, especially in immunocompromised patients, within the nuclei of surface epithelium (particularly goblet cells). useful aids to diagnosis of adenovirus infection include immunohistochemistry, stool examination by electron microscopy and viral culture. other, less common but well-recognized causes of food and water-related gastrointestinal disease include brucellosis, bacillus cereus and listeria monocytogenes. it is also important to remember that many serious food-borne gastrointestinal pathogens may produce little or no inflammatory infiltrate at all, particularly in immunocompromised patients, even in the face of grave clinical disease. these include vibrio cholerae and non-cholera vibrio infections, enteropathogenic and enteroadherent e. coli and infection with many enteric viruses (table 1 ). surgical pathologists may play a very valuable role in the diagnosis of gastrointestinal infectious processes. once it is determined that an infectious process is in the differential diagnosis, it is very important to alert the clinician, and often the microbiology laboratory, that this is a consideration. in addition to valuable culture techniques, the surgical pathologist's ability to diagnose infectious diseases in tissue sections has grown exponentially in recent years with the advent of new histochemical stains, immunohistochemistry, in situ hybridization and pcr analysis (table 2) . 7, 20, 23, 35 in summary, infective (including food-and waterborne) gastrointestinal disorders are common, although they are in general underdiagnosed and under-reported. many of these infections mimic other inflammatory gastrointestinal disease processes, such as ischaemic colitis, crohn's disease and ulcerative colitis. it is important for surgical pathologists to consider infectious agents in the differential diagnosis as they evaluate inflammatory lesions. the availability of pcr assays and immunohistochemical antibodies for infectious agents is increasing, and these techniques may prove invaluable in associating specific infectious microorganisms with histological patterns of disease, thus facilitating the diagnosis of infectious processes. the role of rectal biopsy in infectious colitis surgical pathology of the infected gut the clinical significance of focal active colitis the histopathologic spectrum of acute self-limited colitis (acute infectious-type colitis) enteric infections associated with exposure to animals or animal products precautions against biological and chemical terrorism directed at food and water supplies molecular detection of campylobacter infection in cases of focal active colitis campylobacter jejuni acute colitis caused by campylobacter fetus ss. jejuni acute diarrhoea: campylobacter colitis and the role of rectal biopsy bacterial diarrheas and dystenteries salmonella including s. typhi histopathology of typhoid enteritis: morphologic and immunophenotypic findings pathology of salmonella colitis pathology of the alimentary tract in salmonella typhimurium food poisoning infections of the gastrointestinal tract morphology of rectal mucosa of patients with shigellosis distribution and spread of colonic lesions in shigellosis: a colonoscopic study yersinia enterocolitica and yersina pseudotuberculosis the role of y. enterocolitica and y. pseudotuberculosis in granulomatous appendicitis: a histologic and molecular study the pathology of yersinia enterocolitica ileocolitis the histopathology of enteric infection with yersinia pseudotuberculosis molecular biogrouping of pathogenic yersinia enterocolitica: development of a diagnostic pcr assay with histological correlation aeromonas sobria associated left sided segmental colitis the clinical significance of stool isolates of aeromonas aeromonas as a cause of segmental colitis aeromonas-associated gastroenteritis aeromonasrelated diarrhea in adults nondysenteric intestinal amebiasis: colonic morphology and search for entamoeba histolytica adherence and invasion pathology of human amebiasis the colonic pathology of e. coli 0157:h7 infection escherichia coli 0157:h7-associated colitis: a clinical and histological study of 11 cases diarrhoeal disease: current concepts and future challenges adenovirus colitis in human immunodeficiency virus infection: an underdiagnosed entity infections involving lymph nodes key: cord-336510-qzm9wgde authors: ellermann-eriksen, svend title: macrophages and cytokines in the early defence against herpes simplex virus date: 2005-08-03 journal: virol j doi: 10.1186/1743-422x-2-59 sha: doc_id: 336510 cord_uid: qzm9wgde herpes simplex virus (hsv) type 1 and 2 are old viruses, with a history of evolution shared with humans. thus, it is generally well-adapted viruses, infecting many of us without doing much harm, and with the capacity to hide in our neurons for life. in rare situations, however, the primary infection becomes generalized or involves the brain. normally, the primary hsv infection is asymptomatic, and a crucial element in the early restriction of virus replication and thus avoidance of symptoms from the infection is the concerted action of different arms of the innate immune response. an early and light struggle inhibiting some hsv replication will spare the host from the real war against huge amounts of virus later in infection. as far as such a war will jeopardize the life of the host, it will be in both interests, including the virus, to settle the conflict amicably. some important weapons of the unspecific defence and the early strikes and beginning battle during the first days of a hsv infection are discussed in this review. generally, macrophages are orchestrating a multitude of anti-herpetic actions during the first hours of the attack. in a first wave of responses, cytokines, primarily type i interferons (ifn) and tumour necrosis factor are produced and exert a direct antiviral effect and activate the macrophages themselves. in the next wave, interleukin (il)-12 together with the above and other cytokines induce production of ifn-γ in mainly nk cells. many positive feed-back mechanisms and synergistic interactions intensify these systems and give rise to heavy antiviral weapons such as reactive oxygen species and nitric oxide. this results in the generation of an alliance against the viral enemy. however, these heavy weapons have to be controlled to avoid too much harm to the host. by il-4 and others, these reactions are hampered, but they are still allowed in foci of hsv replication, thus focusing the activity to only relevant sites. so, no hero does it alone. rather, an alliance of cytokines, macrophages and other cells seems to play a central role. implications of this for future treatment modalities are shortly considered. virus-host interactions are crucial for the outcome of infections. several strategies have been utilized by viruses to overcome the host defence. for the virus to be successful, these evasive strategies have to be balanced with the pathology induced and the possibilities of transmission to new susceptible individuals. the mammalian host utilizes ubiquitous and redundant antiviral defence mechanisms. in different viral infections, different parts of the host defence seem to be crucial. however, the redundancy ensures that other systems are ready to take over, if one of them fails. the final outcome of a viral infection depends on a delicate regulation and timing of these antiviral effector mechanisms in response to the invading virus. a viral infection of an individual thus involves a conflict between the virus and the host, which could conceptually be viewed upon as a human controversy escalating to invasion and armed struggle. to understand the resulting course of events it is important to know each party of the conflict and to conduct an analysis of the powerful weapons held by each of the combatants. the present review analyzes the early non-specific events in the conflict upon herpes simplex virus (hsv) infection. initially, each participant of the conflict, the infecting hsv and the non-specific antiviral weapons of the host, are described. subsequently, the early events of the conflict, the armament, early strikes and the opening battle between hsv and the host are discussed. insight into the early non-specific defence mechanisms are important for our understanding of the conflict and may indicate how to intervene during serious systemic infections. herpesviruses are ubiquitous viruses generally infecting humans early in life. the majority of humans has had a primary infection with one or more herpesviruses and harbour these viruses in a latent state for the rest of their lives. the initial infection is most often asymptomatic, but can be symptomatic depending on the herpesvirus in question and the age and immune status of the host. the viruses are phylogenetically old and humans and herpesviruses have evolved together [1] . this co-evolution has created viruses which are well adapted to the human host and environment. thus, herpesviruses are capable of coping with the human immune defence in a balanced manner generally without serious threads to the life of the host. infection with a foreign herpesvirus, normally hosted by another species, does not always hold this balance, and the pathology is unpredictable. this is seen when humans are infected with the simian b virus, which often shows serious clinical outcome [2] . the human herpes simplex viruses were initially identified by lowenstein, who passed it onto rabbits in 1919, and found it to be sensitive to alcohol and higher temperatures [3] . the viruses were classified into two serologically different types by schneweiss in 1962 [4] , and these are now known to belong to the subfamily of alphaherpes-virinae together with varicella-zoster virus. these alphaherpesviruses all show neurotropic latency, and mucosal or skin lesions are frequently seen as a result of viral reactivation from sensory nerves. the two types of herpes simplex virus confer the genera simplexvirus 1 and -2, which were formally designated by the international committee on taxonomy of viruses as human herpesvirus (hhv) 1 and 2 [5] . herpes simplex virus (hsv) type 1 and type 2 are very closely related, showing a homology at the dna level of 83% in protein coding regions and less in noncoding regions [6] . the genetic map of the two herpes simplex viruses is colinear [6] , and the genomes are of approximately the same size, hsv-1 of 152 kbp [7] and hsv-2 of 155 kbp, and code for corresponding genes [6] . the minor sequence variations give different cleavage sites for restriction endonucleases, which has been used intensively as an important epidemiological tool [8] [9] [10] . as all other herpesviruses the herpes simplex viruses are enveloped, icosahedral dna viruses with a capsid of approximately 100 nm ( fig. 1 ) [1] . the envelope holds at least 10 different glycoproteins protruding from the outer side (gb, gc, gd, ge, gg, gh, gi, gk, gl, and gm). the glycoproteins are primarily responsible for attachment to cellular receptors and fusion of membranes (especially gb and gd) [11] [12] [13] [14] . in addition, there are two unglycosylated proteins in the viral envelope. the glycoproteins of the envelope have several immunoregulatory effects besides their primary more mechanical functions in viral attachment and entry [15] [16] [17] [18] [19] . in the space between the envelope and the capsid, the complete viral particles posses an almost amorphous structure which was termed the tegument by roizman and furlong [20] . the tegument consists of several viral proteins involved in the initial phases of viral infection and replication such as transport of the viral dna out of the capsid [21] , early shutoff of cellular protein synthesis (vhs) [22] , and initiation of transcription of viral genes (α-trans-inducing factors) [23] . besides the tegument seen in complete viral particles, tegument-like structures are seen in enveloped particles lacking a capsid and dna, the so called light particles [24, 25] . the capsid is composed of a complex icosahedral structure of 162 capsomeres, each with a central channel running from the outside to the interior of the capsid. inside the capsid the double stranded linear dna is packed as a spool with the ends in close proximity [21, 26, 27] . the genome consists of a long (l) and a short (s) segment which are covalently linked [28] , and contains a high density of genetic information with about 94 open reading frames (orf) and encodes approximately 84 polypeptides [7, 29] , of which only 37 are required for replication of the virus in cultured cells [30, 31] . the viral genes are expressed in a cascade in groups classified as immediate early (ie, α), early (β), early late (γ 1 ) and late (γ 2 ) genes, each with a certain characteristic group of promoters regulating the sequential expression [29, 32] . generally, the α-gene products are transcription inducers, the β-gene products are viral enzymes such as the thymidine kinase and the viral dna polymerase, and the products of γgenes are the structural proteins of the viral particle [33] . the viral transcriptional chain is closed by some of the tegument proteins (e.g. vp16/vmw65) which are γ-gene products with structural properties in the tegument of the viral particle and besides this harbour transcriptioninducing capacity upon α-gene promoters crucial in the induction of the next replication cycle of the virus [32, 34] . the hsv infection is initiated by adsorption of the viral particle via gb or gc to a cellular receptor, which is a heparan sulphate chain on cellular proteoglycans [35] . thus hsv adsorption can be inhibited by heparin and soluble heparan sulfates [36, 37] . this initial binding, in which gc is important but dispensable, is of greater significance for hsv-1 than for hsv-2, a divergence which could have implications for the different pathogenic patterns of the two strains [38, 39] . furthermore, trapping of hsv to heparan sulfate motives in the tissues, e.g. basal laminas, may be of importance for containment of the infection at a specific site [40] . binding to the heparan sulfate-containing cellular receptors, which are in size with the hsv particle itself, is reversible, and serves to concentrate the viral particle in near proximity to the cell ( fig. 1 ) [35, 39, 41] . a crucial step is then conducted by gd binding to an entry receptor, of which three classes has been described [42] . these include herpes virus entry mediator (hvem), later designated as herpes virus entry protein a (hvea), which is a member of the tumour necrosis factor receptor family, nectin-1 (hvec) and nectin-2 (hveb), both members of the immunoglobulin superfamily, and heparan sulfate sites modified by 3-o-sulfotransferases [43] [44] [45] [46] . the differential use of these receptors is of importance for hsv entry of different cell types and infection of polarized cells [47] [48] [49] [50] [51] , exemplified by nectin-1, which is of importance in infection of the vaginal mucosa [52] . upon binding to one of these entry receptors, conformational changes in gd lead to interaction with gb or gh-gl dimer, which results in membrane fusion by a mechanism not known in detail ( fig. 1) [41, 53] . the membrane fusion can take place both with the plasma membrane on the surface of the target cell and with an endosomal membrane after intraluminal ph-reduction, as it is seen for some other enveloped viruses [50,54-56]. following these initial steps of infection several immunomodulatory cellular events are induced, but the potential importance of signalling through receptors involved in adsorption and membrane fusion is only scarcely analysed [57] . the receptor molecule hvem is by its normal ligand capable of inducing activation of nuclear factor κb (nf-κb) and activation of t cells. by interaction with hsv-gd these receptor responses are inhibited. thus, the hsv interaction with at least one of its receptors has multiple potentials for modulation of the host response to the infection [58, 59] . upon fusion, the hsv nucleocapsid is transported by microtubules to a nuclear membrane pore where the viral dna is released into the nucleus [60,61]. both viral tegument products and cellular kinases are responsible for the initiation of α-gene transcription [62] . in these initial events the determination of whether it will lead to a lytic infection cycle or a latent infection seems to be directed largely by the infected cell type in question [63, 64] . a key event in this seems to be early induction of latency-associated transcripts (lats) with sequences antisense to the infected cell protein null (icp0) and icp4 [65] [66] [67] . in the hsv composition and entry figure 1 hsv composition and entry. electron micrograph of negatively stained hsv particle with indications of major structural elements. important mediators of adsorption to cells (1), receptor binding (2) and fusion of membranes (3) during the process of infection are drawn stylistically. initial phase of the lytic replication cycle, the ie-gene products, besides being transcription factors for the next wave of viral proteins, intimately regulate cellular functions in favour of viral replication and immune evasion [33, 68] . of these, the icp0, a promiscuous transactivator without much dna-binding capacity, forces the cell to a pre-dividing state optimal for viral protein synthesis [69, 70] . furthermore, icp0 is active in inhibiting immune mechanisms such as interferon production and antiviral effects of interferons [71-73] and induces degradation of cellular proteins, involving the proteasome [74,75]. very early in infection, the first transcriptional activity is seen just inside the nuclear membrane at the site were the viral dna enters the nucleus [76] . the produced icp0 colocalizes with the promyelocytic leukaemia (pml) nuclear bodies and initiates degradation of these, an event which seems to be important for productive replication of the virus [77, 78] . icp4 binds to parental viral dna which is juxta-localized to the pml bodies, and later, when the bodies are degraded, replication compartments are formed, in which also icp27 can be found [76, 79, 80] . icp27 affects the posttranscriptional polyadenylation and splicing of rna, and it is thus an element of the delayed host protein shutoff [81] . immune evasion is additionally induced by the ie protein icp47 which binds to transporter associated with antigen processing, tap1/tap2 and blocks the presentation of viral peptides by the major histocompatibility complex (mhc)-i [82]. the hsv progeny is formed in the nucleus of the infected cell, where the viral dna is packed into preformed capsids. these are assembled with the tegument proteins and bud through the inner nuclear membrane to the perinuclear space [11] . the route of virus from here to the external side of the cell is controversial. apparently two routes of viral egress are possible [11] . one way is by continuous passage through vesicles and the golgi apparatus, where the membrane proteins are modified. the other route is by fusion of the newly acquired envelope with the outer nuclear membrane or the membrane of a vesicle, generating naked nucleocapsids in the cytoplasm. from here a new budding event should take place, for instance into the golgi apparatus. the progeny virus thus acquires the envelope from other membranes, than the inner nuclear lamella, as it is indicated by analysis of membrane lipids [83] . increasing evidence is pointing at this latter possibility of de-envelopment and re-envelopment as the dominating route of hsv egress [84-86]. the progress of hsv infection in tissues is influenced by the capacity of hsv to infect adjacent cells directly through cell junctions. the virus is thus avoiding exposure to extracellular substances such as antibodies and complement. the glycoproteins ge and gi are crucial for this kind of polarised transmission which primarily takes place in epithelial infections [47, 87] . as it is the case at the molecular level, the two herpes simplex viruses show similarities in their clinical appearance, both giving rise to primary infections of mucosal membranes and showing latency in sensory nerve ganglia [1] . the primary infections with hsv are often asymptomatic, especially at young age, but in a minority of cases vesicular or ulcerative lesions are seen. although hsv-1 and -2 can give rise to indistinguishable clinical infections, there are differences in the anatomical distribution of these infections, as described in 1967 by dowdle et al. [88] . hsv-1 is predominantly giving rise to infections above the waist, and hsv-2 to infections below the waist. this pattern is, however, not as straightforward as primarily described. in the last decades changes in both prevalence and distribution of hsv infections have been seen. the overall prevalence of hsv infection is very different in different countries and ethnic and social populations [89] [90] [91] . a decline in hsv prevalence has been observed in the western countries, probably because of improved socioeconomic conditions [92] [93] [94] . in parallel to the decline in prevalence, the aetiology of herpes genitalis has changed in several countries, presumably because of altered human habits and conditions of life [92] . in some areas of the world the proportion of genital infections caused by hsv-1 is still low (4-20 %) the aetiology of a genital infection is not insignificant, in that the frequency of recurrence is higher in hsv type 2infected individuals than in those infected with type 1 [89, 95] . the frequency of primary and recurrent infections with both hsv-1 and -2 has been reported to be higher among women than men [97, 103, 105] . overall, these epidemiological changes could have implications for the risk of neonatal infection from vaginal delivery, in that more women are seronegative at delivery and thus a higher number have the risk of caching a primary hsv infection. on the other hand, less hsv is circulating, reducing the risk of those who are susceptible. clinical appearance and pathogenesis as described above, primary infection with hsv is most often asymptomatic, especially in younger children [106] . however, some individuals experience a symptomatic primary infection with vesicular herpetic gingivostomatitis or in adolescence more often a pharyngitis [107] . as it is the case with orofacial infections, a primary genital hsv infection can be both asymptomatic and symptomatic with ulcerative lesions and with or without generalized symptoms such as fever, headache etc. [108, 109] . rarely, the infection disseminates to one or several organs giving rise to infections such as necrotising hepatitis, meningitis, encephalitis or to disseminated intravascular coagulopathy [110] [111] [112] [113] . such a clinical course, although uncommon, is most often seen in immunosuppressed patients e.g. transplant patients, neonates or pregnant women [114] [115] [116] . in pregnancy, primary infection with hsv without previous seroconversion at the time of delivery seems to be the main risk factor for infection of the newborn [109, 117] . genital hsv reactivations at labour only seem to posses a minor risk for neonatal infection of the baby [117, 118] , but in spite of this, approximately 70% of neonates infected are born by asymptomatic women [63] . the amount of virus in vaginal secretions during reactivations is much lower than the amount of virus in primary infections, and in reactivated cases maternal antibodies furthermore seems to be protective for the neonate [117, [119] [120] [121] [122] . when transmitted, the course of hsv infection in the newborn varies. in the pre-acyclovir era about one third of cases were mucocutaneus infections only involving the skin, mouth and eyes, one third were infections of the central nervous system (cns) with or without mucocutaneus involvement, and the last third were disseminated infections involving multiple organs, including the liver, lungs, adrenals, and often the cns [119] . of these, neonates with a generalized infection had a one-year mortality of approximately 60%, those with cns-infections had intermediary mortality, and nearly no mortality was seen in the group of patients with only mucocutaneus involvement [119] . in infected with multi-organ involvement the deaths are often set off by infection of liver or lungs or by coagulopathy. sequelae, such as mental or neurological disabilities are seen in some of those with cns involvement [123] . now a day, after initiation of high-dose acyclovir treatment, the mortality and sequela rates have dropped [124] . the clinical pattern of neonatal hsv infections has changed in that less of the mucocutaneus infections disseminate to generalized infections when treated [123] . even with high-dose acyclovir, improvements in treatment protocols are still needed, because the mortality is still as high as 30% in disseminated infections. reduction in the time from debut of symptoms to initiation of therapy is vital and passive immunotherapy with hsv-specific antibodies could posses a potential as adjuvant to the antiviral treatment [123, 125, 126] . other adjuvant treatment modalities are still needed in both neonatal infections and in generalized infections at later ages. the pathology of hsv infections is mainly caused by a direct cytopathic effect of the virus, resulting in cellular lysis and focal necrosis of the infected area [119, 127, 128] . in tissues capable of regeneration, this is not devastating, provided that the lesions do not totally destroy the organ or result in functional disability during the infection. in the brain, however, the capacity for regeneration is small, and larger necroses induced by viral infection will result in life-long sequelae [119, 123] . a delicate balance exists between the direct hsv-induced pathology and the immunopathology induced by immune reactions to the virus and the toxic and functional side effects of these reactions [129] . immunopathogenesis seems to be the main aspect of hsv stromal keratitis, which often leads to blindness [130, 131] . the scarification from this infection has even been attributed to autoimmunity by molecular mimicry [132] . weak immune response to the virus leads to severe infections because of massive viral replication and dissemination. an immense immune reaction, especially with high amounts of virus to trigger a response, can bring about increased symptoms of infection, local symptoms such as high intra-cerebral pressure or pulmonary complications, as well as generalized or septic symptoms [129, [133] [134] [135] [136] . it is thus clear that early control of hsv replication in the initial phases of infection is crucial for the host. early containment or at least inhibition of viral replication can prevent dissemination of the infection, and the early nonspecific immune reactions thus have the potential to inhibit development of a symptomatic infection. obviously the host will benefit from an attenuated or asymptomatic course of infection, but hsv -with the potential of subsequent reactivation from a latent site -could also benefit from such a course of infection, in that the host will survive and the activity of the host in society will not be hampered by symptoms from infection. thus, the hsv has excellent chances to reach new susceptible hosts which bring the virus and the host in a situation of mutual benefit [33] . with phagocytic activity. in 1892 metchnikoff named them macrophages (large eaters) in contrast to microphages (the polymorphonuclear leukocytes) [137] , and in 1924 aschoff defined the reticuloendothelial system by the criteria of uptake of vital dye [138] . the macrophages are now more precisely defined as an important member of the mononuclear phagocyte system, defined in 1969 by van furth and colleagues [139] . in the tissues they constitute a dynamic pool of cells with many functional capabilities, among which the capacity of phagocytosis, microbial killing, motility, and adherence to surfaces are classic [139] . the macrophages originate from the bone marrow, where proliferating promonocytes give rise to monocytes which enter the blood stream [140] . after a mean circulation time of approximately 11/2 day, the blood monocytes migrate to the tissues [140] . in the tissues the monocytes differentiate into macrophages with characteristics determined by the environment of the tissue in question [141] . the tissue macrophages in the major organs are represented by kupffer cells in the liver, alveolar and interstitial lung macrophages, spleenic and sinusoidal lymph node macrophages, microglia in the brain, osteoclasts in bone, and langerhans cells of the skin. thus, macrophages are strategically situated all over the body taking care of debris from the organism itself and foreign material, among others invading microorganisms, including viruses [142, 143] . macrophages in different organs have different characteristics and functional capabilities and can not totally substitute one another in studies on macrophages [141, [144] [145] [146] [147] . likewise, macrophages from different species can possess differences in their functional capability, e.g. the capacity for nitric oxide (no) production [148, 149] . macrophages in tissues are, as described above, in part originating directly from monocytes, but they are also in part originating from local proliferation. this local proliferation in the tissues is performed by newly recruited monocytes, and in the steady state situation they only constitute a small fraction of the mononuclear phagocytes present [150] . of the monocytes produced in the bone marrow of mice and passing through the blood, approximately half are targeting the liver, 15 % are going to the lungs, 25 % to the spleen and 7 % to the peritoneal cavity [150] [151] [152] . in the lungs, 70% of tissue macrophages in the steady-state originate from monocyte influx and 30% from local proliferation [153] . this proportion might vary between different tissues, as the lifespan of tissue macrophages in different organs also varies from around 6 days in mouse spleen to approximately one month for alveolar macrophages [151, 152] . in the skin, langerhans cells are a very stable and long-lived population of cells staying there for at least 18 month in the steady-state situation. however, in inflammation the langerhans cells are within 2 weeks replaced and supplemented by circulating mononuclear cells [154] . when an inflammatory process is initiated, the dynamics of monocytes and macrophages are changed. monocytes and other white blood cells are produced and recruited from the bone marrow, and the white blood cell count in the circulation is increased. the monocytes are mainly passing through the blood to become tissue macrophages, and the number of macrophages in the inflamed tissue can be increased by more than ten times [155] . in inflamed tissue the local proliferation of macrophages does not seem to increase, although the number of newly recruited cells is high, indicating that the differentiation of monocytes in the tissues is accelerated [155] . the differentiation of monocytes and activation of macrophages have been a focus of interest for many years because of the observation that macrophage activation is crucial in the defence against many intracellular pathogens [156] [157] [158] [159] . it became clear relatively early that lymphocytes and soluble factors secreted by these (lymphokines) are important in activation of macrophages for killing of intracellular bacteria, e.g. listeria [160] . in the killing of bacteria, interferon (ifn)-γ was shown to be an important stimulator of macrophage activation [161] . as mechanisms in performance of the killing simple toxic substances of reactive oxygen species (ros) and nitric oxide were identified and seem to conduct their action in synergy [162] [163] [164] . the toxic substances are chemically simple, but their production and regulation in macrophages are very complex and still a matter of intense studies [149] . the state of the activated macrophage has changed conceptually from being viewed as one specific condition of the cell towards a more dynamic picture, provoked by the fact that macrophages activated by different means show different phenotypical characteristics [163, 165] . the activated macrophage is now viewed as a cell with floating characteristics of many functional capacities regulated by a multitude of stimulating substances, such as the cytokine environment, hormones, and pathogenic and foreign substances [147, 166] . among variables, controlling macrophage activity in infected individuals, are the genetic constitutions of the host. the genetic background has been shown to be of importance for the regulation of both basic proliferation and function of macrophages and for the more specific antimicrobial responses [167, 168] . soluble mediators of lymphocyte activities were described as early as 1953, but the first lymphokines/cytokines found and characterized were the type i interferons. soon after, many other soluble mediators of lymphocyte and monocyte/macrophage activities were found [169] [170] [171] . the term lymphokine was introduced by dumonde et al. in 1969, to describe lymphocyte derived factors, and the term monokine was used as a description of factors coming from the mononuclear phagocyte system, both acting on many cells, primarily leukocytes [172] . because of a broader view on origin and function of these factors, the term cytokine is now more often used. each cytokine was originally named according to biological activity in a functional assay, which often gave several different names to one cytokine, and thus confusion at the molecular level. to straighten this out, a numerical nomenclature of interleukins (between leukocytes) was introduced in 1979 [173] . this numbering system has clarified the field, but since it has no mnemonic functional anchorage it has drawn critique since then [174] [175] [176] . the cytokines are generally smaller proteins, some composed of two subunits, utilizing specific receptors on target cells for induction of their functional effects. they are structurally related in three families, with the prototypes being il-1, il-2 and il-17 [176] . functionally, cytokines are highly potent regulatory proteins acting in a paracrine or autocrine manner at picomolar concentrations [177] . the cytokine receptors are also structurally clustered in families, and functionally utilize a battery of overlapping kinases and nuclear binding proteins in their signalling pathway and thus have overlapping functions [178] . the final functional capacity of the effector cell thus reflects the cytokine environment experienced by the cell [177] . thus the cytokines comprise a network of factors inducing or inhibiting each others secretion and function in different cells, giving rise to a constantly floating landscape of a large array of functional capacities [177] . in the early hours of a viral infection, the cytokines produced by cells infected or coming into contact with viral products are vital in conduction of the innate immune response to the infection [168, 179] . the interferons (ifns) were described and named in 1957 by isaacs and lindenmann [170] , who characterized the substances involved in the previously described interference of one virus with the replication of another unrelated virus, and the interfering activity of inactivated influenza virus with the subsequent infection of chorioallantoic membranes [180] [181] [182] . the ifns were the first cytokines described in detail, and thus provided the fundamental basis for the understanding of the cytokine concept [183] . the ifns are divided into three major groups. the two original groups of ifns are designated type i and type ii, type i being the so called non-immune ifn, and type ii the immune ifn. type ii (ifn-γ) is produced in high amounts as part of a specific immune reaction, whereas the type i ifns can be produced by many cell types in response to, in immunological terms, non-spe-cific stimulation. the many functions of ifns and the growing understanding of signalling and regulation indicate that ifn analogues may play a major role in the next generation of new antiviral compounds [171] . the type i ifns are a diverse group of cytokines, consisting of ifn-α, ifn-β, ifn-ε, ifn-κ, ifn-ω, ifn-δ, ifn-τ, and ifn-ξ/limitin [171, 184] . the first five of these are expressed in humans, and their relative production depends on the stimulus and the cell type in question. the ifn-α family consists of multiple species and some of these in different allelic forms in both humans and mice. in humans 13 ifn-α genes and one pseudogene and in mice 14 ifn-α genes and 3 pseudogenes have been identified, clustering on chromosome 4 in mouse and chromosome 9 in man [185] . the functional importance of such a diversity is largely unknown. the subtypes differ in potency and have previously been shown to vary in their profile of activities [186, 187] , but new studies show correlation between antiproliferative and antiviral effects of various ifn-α species [185] . thus, it seems that the importance of the diversity could come from varying expression patterns of the different ifn-α species. most of the α ifns are n-glycosylated, but glycosylation does not correlate with activity of the molecule, but rather with in vivo stability, and recombinant ifns are shown to have activity comparable with that of the naturally produced molecules [185, 188] . only one ifn-β species exists, coded by a gene situated in the ifn type i cluster on chromosome 4 in mouse and chromosome 9 in man, as described above [185] . the natural ifn-α and -β have a molecular weight of 19 -26 kda and most species retain stability at ph 2 [189] . all type i ifns bind to one common receptor composed of two subunits, ifn-α-receptor(r)1 and ifn-αr2. the ifnα/β receptor (ifnar) signal through the jak/stat-pathway by phosphorylation of the janus kinase (jak)1, tyrosine kinase (tyk)2, signal transducer and activator of transcription (stat)1 and stat2, and induces genes with an ifn-stimulated response element (isre) in their promoter [171, 190] . generally the type i ifns exhibit a huge range of biological effects, such as antiviral and antiproliferative effects, stimulation of immune cells such as t cells, natural killer (nk) cells, monocytes, macrophages, and dendritic cells, increased expression of mhc-i, activation of pro-apoptotic genes and inhibition of anti-apoptotic mechanisms, modulation of cellular differentiation, and inhibition of angiogenesis [171] . the newly discovered ifn-ξ/limitin also interacts with the ifn-α/β receptor, and is regarded as a type i ifn [184, 191] . antiviral activity of ifn-ξ has been shown against many viruses including hsv, and it exhibits both immunomodulatory and anti-tumour effects, but the lymphosuppressive activity is less than that of ifn-α [184, 192] . a human homolog of ifn-ξ could thus have interesting potential in the therapy of tumours and viral infections. the type ii ifn is represented by only one member, the ifn-γ [193] . structurally, ifn-γ is distinct from the type i ifns, and it signals through a different receptor. for many years ifn-γ was thought only to be expressed by t cells. later the large granular lymphocytes (nk cells) were recognised as important producers by the fact that ia-antigen (mhc-ii) expression on mouse macrophages could be induced by listeria monocytogenes infection in scid mice lacking t cells [194] [195] [196] . in recent years it has, however, been clear that other cell types, originally thought not to be producers of ifn-γ, are in fact capable of ifn-γ expression. so now macrophages, b cells, nkt cells and professional antigen-presenting cells are also recognized as ifnγ producers in certain situations [197] [198] [199] [200] [201] [202] . induction and production of ifn-γ in antigen-presenting cells and nk cells seem to be vital in the early non-specific response to infections and of importance in the linkage to the adaptive specific responses coming up later [202] [203] [204] . the induction of ifn-γ production in non-t cells (e.g. nk cells) is conducted by cytokines, especially il-12 in synergy with other proinflammatory cytokines, largely produced by mononuclear phagocytes [205, 206] . ifn-γ exerts its effects through a distinct class ii cytokine receptor, the ifn-γ receptor (ifngr), composed of two subunits, ifn-γr1 and ifn-γr2. upon binding of a homodimer of ifn-γ to the receptor complex, jak2 autophosphorylates and then transphosphorylates jak1. activated jak1 in turn phosphorylates ifn-γr1, which allows binding of the stat1 homodimer to the receptor and subsequent phosphorylation of stat1 [204] . the ifngr and stat1 are preformed as hetero-and homo-dimers, and upon receptor binding, the ifn-γ-ifn-γr1-stat1 complex seems to be internalized and translocated to the nucleus, where the activated stat1 homodimer binds to dna at gas elements and induces the first wave of responses [204, [207] [208] [209] [210] [211] . many of these initial ifn-γ induced products are transcription factors participating in further regulation of the many ifn-induced cellular response. among these products are the ifn regulatory factors (irfs) which stimulate or inhibit transcription of genes possessing an isre in the promoter region [204, 212] . for many years the key mediator of macrophage activation during antigen-induced processes was recognised as macrophage activating factor (maf) [213] . only later, the crucial importance of these effects was attributed to ifn-γ [214, 215] . ifn-γ has antiviral activity, but the most important effects of ifn-γ seem to be activation of macro-phages, antigen-presenting cells, and nk cells and inhibition of t-helper type 2 (th2) cells, resulting in a th1driven cell-mediated response to infection [204] . experiments in knock out (ko) mice with deficient ifn-γ, ifngr, or stat1 expression have shown that this system is of major importance, but not vital, in the host response to viral infections [216] [217] [218] [219] . besides the two traditional groups of ifns, a new group of ifn-like cytokines has been described in various species and named il-28a (ifn-λ2), il-28b (ifn-λ3), and il-29 (ifn-λ1) [171, 220] . these cytokines are antiviral proteins interacting with a distinct heterodimeric class ii cytokine receptor composed of ifn-λr1 and il-10r2, but sharing with the type i ifns some intracellular signalling pathways through the isre [221] . thus, they have a largely similar antiviral effect as the type i ifns [220] . tumour necrosis factor (tnf, former designated tnf-α) and lymphotoxin (lt; former tnf-β) were for many years also known as cachectin from their involvement in cachexia of cancer patients [222] . tnf is a prototype and the second member of the tnf ligand superfamily (tnfsf2), now encompassing over 40 known signalling molecules, among which the ltα, ltβ, and light (ltlike, exhibits inducible expression, and competes with hsv glycoprotein d for hvem, a receptor expressed by t lymphocytes) are some of the more prominent ligands [58, 223] . each member is the ligand of one or two distinct receptors of the tnf receptor family sharing a high degree of homology. the current nomenclature of these ligands and receptors has now been gathered on the internet [224] . tnf is a type ii transmembrane glycoprotein coded from the human chromosome 6 and from chromosome 17 in mice [223] . it is synthesized as a 26 kda transmembrane pro-tnf, primarily located in the membranes of the golgi apparatus [225] . the pro-tnf is cleaved by a metalloprotease releasing the 17 kda extracellular portion of the molecule [222, 226] . production and release of tnf from the cell is regulated at both the transcriptional and translational level and by post translational modification as described above [227] . during hsv infection both preand post-transcriptional regulatory mechanisms are involved in tnf production [228] . tnf is produced by many cell types of immune origin, primarily mononuclear phagocytes, neutrophils, nk cells and t cells, and has diverse effects on different cells [222] . both membrane bound and soluble tnf interact as homotrimers with two different receptors, the p55 tnfr1 (tnfrsf1a) and the p75 tnfr2 (tnfrsf1b) [222] . as most other receptors of this family, tnfr1 holds a death domain important in the pro-apoptotic pathway. tnfr1 is expressed virtually on every cell type except erythrocytes, whereas tnfr2 is mostly expressed on endothelial and bone marrow derived cells [227] . the tnfr2 activates nf-κb (p50, p65/rela, and p52/relb) by ubiquitin-mediated degradation of inhibitor-κb (iκb) after phosphorylation by an iκb kinase (ikk). besides inducing apoptosis, tnfr1 also activates nf-κb (p50/ p65) [229, 230] . furthermore, the activator protein 1 (ap-1) is activated by mitogen-activated protein kinases (mapks) and together with nf-κb primarily acts in the proinflammatory pathways. thus, signalling from the tnf receptor family induces a delicate balance between life and death (apoptosis) of the cell. both of the tnf receptors can by proteolytic cleavage be converted to soluble receptors with the capacity to compete with their signalling ancestors, but also act to stabilize the trimeric tnf and thus maintain its activity [227, 231] . the tnf superfamily seems to have evolved with the adaptive immune system in vertebrates and is crucial for the embryonic development of lymphoid tissue [223] . furthermore, tnf is, as a proinflammatory cytokine, involved in activation of many immune cells and is thus an important factor of both the early non-specific and the specific immune response [232] . the importance of the tnf superfamily in antiviral defence is illustrated by the fact that different viruses have developed mechanisms for interference with nearly every step of activity of this system [227, 229] . il-12 is the prime member of a small group of heterodimeric cytokines, all with the capacity to induce production of ifn-γ in a variety of cells. il-12 was first described as an nk cell stimulatory factor (nksf) and identified as a heterodimeric molecule composed of a p40 and a p35 subunit, which are covalently linked [233] . the p35 subunit has homologies to il-6, and p40 is homologous to the extracellular domain of the haematopoietin receptor family, particularly the il-6rα chain [234] . the two il-12 subunits are coded from different chromosomes, i.e. the human chromosomes 3 and 5 and the mouse chromosomes 6 and 11, respectively [235] . these genes are regulated separately, and coordinated induction in the same cell is required for secretion of the biologically active il-12p70 heterodimer [236] . il-12 is produced by monocytes, macrophages, dendritic cells, neutrophils and b cells [235, 237] . in the initial response of spleen cells in mice injected in vivo with extracts of toxoplasma gondii or with lipopolysaccharide (lps), the cellular source was found to be dendritic cells, but cultured macrophages have by themselves also been shown to produce il-12p40 upon hsv-2 infection [238, 239] . such differences could depend on variations in the signalling mechanisms involved, which is also illustrated by the observation that the production in dendritic cells and macrophages has dif-ferent kinetics. this difference could be brought about by differences in the requirement for co-stimulation with ifn-γ [240] . a collaborative action of dendritic cells and macrophages could be important, as indicated for il-12 induction by influenza virus and other inducers [241] . the receptor for il-12 is found on nk cells, t cells and dendritic cells and consists of two subunits (β1 and β2), which signal by the β2 subunit through the jak/stat pathway, primarily by activated stat4 [235] . the primary effect of il-12 is induction of ifn-γ production in nk cells and t cells, and il-12 activates the cytotoxic potential of these cells. the ifn-γ locus in nk cells is constitutively demethylated and is thus ready for transcription of the gene, which is in contrast to that of t cells, [242] . macrophages and nk cells are then stimulated by ifn-γ, resulting in activation for enhanced antimicrobial capacity [243, 244] . il-12 and ifn-γ in conjunction are the main responsible factors for activation of a th1-driven adaptive cellular immune response, important for the long-term control of intracellular pathogens [235] . il-12 stimulates proliferation of naïve t cells, and in conjunction with ifnγ inhibits th2 cell differentiation and the production of th2 cytokines (e.g. il-4, il-5, and il-13) [235] . thus il-12 holds a key position in induction and control of the th1 response. the il-12-induced ifn-γ production is synergistically enhanced by other cytokines such as tnf and il-1 [240] , and ifn-γ production can even be induced in macrophages by co-stimulation with il-18 [197, 245, 246 ], a cytokine which by itself does not possess major ifn-γinducing capacity [240] . a positive feed-back loop is initiated by the il-12-induced production of ifn-γ, in that ifn-γ is an important primer of il-12 production, thus accelerating the system [247]. furthermore, t cells enhance il-12 production through signals of the proinflammatory tnf family [240] . in virus-infected macrophages a similar autocrine feed-back loop involving il-12, il-18, ifn-α/β, and ifn-γ could be speculated [248] . this potentially harmful situation, with accelerating ifnγ production, regulated in a positive feed-back loop by il-12, is inhibited by cytokines possessing anti-inflammatory properties. among these il-10 holds a crucial position as an inhibitor of il-12 production, an effect which is also conducted by transforming growth factor-β (tgf-β) [249] [250] [251] .the th2 cytokines of the other side of the adaptive response, il-4 and il-13, inhibit il-12 induction in the early phases of stimulation, but later they can be potent inducers of il-12 production, although they still inhibit many of the ifn-γ-induced activities [212, 252, 253] . phagocytosis of apoptotic cells by macrophages inhibits production of il-12, a regulatory mechanism which seems to be important in restriction of the damages induced by uncontrolled defence mechanisms [254] . injection of high doses of il-12 to virus-infected mice is toxic, and leads to death with the pathology of tnf-related toxic shock, an effect which was explained by increased sensitivity to the toxic effects of tnf, and found to be dependent on the genetic constitution of the host [255, 256] . the small il-12 cytokine family also includes two other heterodimeric cytokines, il-23 and il-27, and a homodimer of il-12p40. the latter is found in vivo in mice and functions as an antagonist of il-12, but it is debated whether it exists in humans [257, 258] . il-23 is composed of the il-12p40 and a p19 subunit and likewise binds to a receptor with one of the il-12 receptor subunits (il-12rβ1) and a distinct il-23r subunit [240, 259] . the production and function of il-23 is quite similar to that of il-12, but il-23 has a unique capacity to induce proliferation of memory t cells [235] , and it has been found in nervous ganglia of hsv-infected mice on day 3 of infection [260] . il-23 drives il-17 production of nk cells, which mobilizes neutrophils and promotes production of the proinflammatory cytokines il-1, il-6, and tnf [261] . il-27 is the newest recognized member of the family, constructed of two distinct subunits (ebi3 and p28), but still with functional capacities alike those of il-12 [262] . the functional implications of these later discovered members of the il-12 family is not yet clear, but it seems as if they are contributors to the overall effects of the il-12 family and fine-tune the system [235, [263] [264] [265] [266] . the induction of ifn-γ and activation of nk cells is not only mastered by members of the il-12 cytokine family. other cytokines, like il-15, are also implicated in development, function, and activation of these cells [267, 268] . generally, the il-12 cytokine family has shown itself of importance in early defence against several viral infections, and as a vital inducer and regulator of the adaptive immune response against viruses and other intracellular pathogens [219, 256, 261, 269] . upon an accelerating pro-inflammatory response induced by initial viral replication the organism has to embank the ifn-γ-activated potentially harmful actions of macrophages and nk cells. important mediators of this embankment are il-4 and il-13, which as described above repress the induction of il-12, and thus put a brake on the positive feed-back loop of ifn-γ production [249, 252] . furthermore, il-4 suppresses the production of other proinflammatory cytokines such as tnf and il-1 [270] . most importantly, il-4 and il-13 are potent inhibitors of the efferent arm of the pro-inflammatory system, and thus inhibit production of reactive oxygen species and nitric oxide. the production of these two potentially harmful effector mechanisms of activated macrophages is hampered by inhibition of production of the responsible enzymes in these reactions, the nadph oxidase and the inducible nitric oxide synthase (inos) [271] [272] [273] . the primary producer cells of il-4 and il-13 are the th2 cells, but these cytokines are also produced by basophils and mast cells [274] [275] [276] . the receptors for il-4 and il-13 are expressed on most cells and are composed as dimers of four different chains. il-4 is the ligand of two receptors: a high-affinity heterodimer of il-4rα and the il-2r common γ-chain and another heterodimeric receptor composed of il-4rα and il-13rα1. il-13 binds to three complexes: a high-affinity heterodimer of il-13rα and il-4rα and two homodimers composed of either il-13rα1 or il-13rα2, which are both coded from genes on the human x-chromosome [276] . the immunomodulatory signalling is conducted through the jak/stat-pathway utilizing jak1, jak3 and stat6. phosphorylated and homodimerized stat6 binds to stat binding elements (sbe), which includes gas, and either trans-activates or inhibits transcription of the adjacent genes [212] . the functions of il-4 and il-13 are nearly overlapping with only discreet discrepancies [276, 277] . il-4 was discovered in 1982 on the basis of another important effect of the cytokine, namely the ability to induce proliferation of b cells, and it was from this effect in the early years called b cell growth factor [278] . as this, some other effects of il-4 are stimulating, in that it furthermore activates other th2-like effects such as b cell class-switching and expression of mannose receptor and fc receptor for ige on macrophages [276] . despite the anti-inflammatory profile il-4 has in vivo been shown to confer some resistance to hsv infection [279, 280] . il-4 is thus not only an inhibiting cytokine but essentially an immunomodulatory cytokine with regulatory effects on macrophages as well. the early innate defence mechanisms have for many years been regarded as important for the course of many viral infections, including infections with hsv [281] . the control of viral replication and dissemination during the first days of an hsv infection seems to be vital for the final outcome. if the viral replication is not halted by natural defence mechanisms during induction and maturation of the antigen-specific immune response, the adaptive immune system can be overwhelmed by massive viral infection at the dawn of activity of the specific reactions. the mechanisms of the anti-herpetic natural defence have been analysed extensively. it became relatively early clear that antiviral activity of macrophages [281] and nk cells [282] and early activity of the ifn-system [283] were important mediators of innate resistance to hsv. the relative contribution of each of these players in the early defence has been much debated, and as more interactions and molecular mechanisms are now elucidated, it seems clear that all of these players each hold a crucial position in an integrated antiviral natural defence system. an important model used in the study of resistance mechanisms in defence against generalized infection with hsv is a mouse model, where mice infected intra-peritoneally or intra-venously experience a generalized infection with hsv replication in most organs, including the liver, spleen, and eventually the brain [284] . the dissemination of infection to the brain and the severity of infection of the peripheral organs depend in part on the age of the mice, as is the case in humans, where neonates have difficulties in controlling a hsv infection [281, [285] [286] [287] . the course of infection in mice also depends on the type of hsv in question. furthermore, in 1975 lopez described a differential susceptibility of inbred mice to generalized infection with hsv, and this genetic difference in sensitivity has since been used for analysis of resistance factors of importance for the anti-herpetic defence [288] . in generalized infections, the genetics of the relative resistance to hsv-2 was shown to segregate with the x-chromosome [289] . this pattern of resistance to the generalized infection was for both hsv-1 and -2 attributed to a genetically determined difference in the capacity for ifn-α/β production [179, 290, 291] , and it was shown that the x-linked pattern of resistance segregated with the hsv-2-induced production of ifn-α/β in macrophages during the first hours of infection [168] . furthermore, macrophages from female mice respond to hsv with higher ifn-α/β production than macrophages from male mice [168] . this observation is in line with female mice being more resistant to hsv infection in vivo [291] . early production of ifn-α/β has been correlated to resistance of hsv infections in several other studies. treatment of mice with antibodies to ifn-α/β increases and accelerates mortality of a generalized hsv-1 infection and with higher doses of virus, mice are dying already after three to four days, a period where antigen-specific mechanisms are still in the induction and proliferation phase [292] . furthermore, mice treated with mercuric chloride showed higher titres of hsv-2 in the first days of infection, an effect which could be correlated to impaired production of ifn-α/β [293, 294] . in studies on peripheral hsv infections, such as cutaneous or corneal infections, ifn-α/β has been shown to be produced locally and to restrict the local replication of hsv and infection of nervous ganglia cells of the area, an effect which has also been correlated to the genetic constitution of the host [295] [296] [297] [298] . the genetic background for the x-linked trait of hsv resistance and ifn-α/β production of macrophages remains unravelled. induction of ifn-α/β upon hsv infection seems to be governed by different mechanisms in different cells [299] . ifn-α/β can be induced early by both infectious and uv-inactivated hsv in various cells, with the infectious virus being the more potent inducer in mouse peritoneal macrophages, whereas the uv-inactivated virus showed most potency in human peripheral blood mononuclear cells (pbmc) [168, [299] [300] [301] [302] . production of ifn-α/β was induced by gd of hsv-1 in pbmc, but not in murine macrophages [17, 303, 304] . in pbmcderived dendritic cells, however, the cellular mannose receptor was shown to be involved [299, 305] . furthermore, different toll-like receptors (tlrs) have been shown to react with hsv [306] . tlrs are transmembrane pattern recognition receptors (prrs) that detect redundant microbial molecular motives and induce antiviral and proinflammatory cytokines in response to alerting signals. in dendritic cells, tlr9-signalling, induced by the gc-rich hsv genome, has been shown to govern the induction of ifn-α/β, but tlr9-ko mice are still capable of controlling hsv infections in vivo [307] [308] [309] . however, in mouse macrophages the tlrs do not seem to be crucial for ifn-α/β induction upon hsv infection [304] . this is in agreement with the observation that the majority of ifn-α/β produced by spleen cells and dendritic cells and the total production from bone marrow macrophages was independent of tlr9 or myd88, which is necessary for signalling by most tlrs [308] . in this study, heat inactivated virus was shown still to induce ifn-α/β in cells utilizing tlr9. as resident peritoneal macrophages do not produce ifn-α/β in response to even high doses of heat inactivated hsv, this gives an additional indication of independency from tlr9 of ifn-α/β production in macrophages [300] . moreover, efficient induction of ifn-α/β by hsv in macrophages required dsrna-activated protein kinase (pkr) activity and infectivity of the virus [304] . this is in agreement with the observation that dsrna, which is produced by most viruses during replication, induces ifn through pkr, and not through tlr3, which also binds dsrna [310] . furthermore, another mechanism of ifn induction by dsrna through a rna helicase has been proposed [311] . the different induction patterns in different cells types, and the fact that ifn-α/β seems largely to be induced by other mechanisms than tlrs, explain the fact that knocking out tlr-signalling by myd88 did not influence the in vivo infection with hsv in mice [309] . other tlrs have also been shown to mediate signals in hsv infections. in hsv encephalitis in tlr2-ko mice, viral replication seemed unchanged or slightly increased during the first 4 days of infection, and the production of il-6 and monocyte chemoattractant protein 1 were impaired, but interestingly pathological changes and mortality were reduced [134] . in relation to the x-linked resistance pattern of hsv infection and ifn production upon hsv infection, it is interesting that some of the tlrs are coded from the xchromosome [312] . these are the tlr7 and tlr8, which are triggered by guanosine-or uridine-rich ssrna in the endosomal compartment of cells [313, 314] . there are, however, no indications that this pathway is implicated in ifn induction in cells during hsv infection, but the question has still not been directly addressed. regulation of the ifn-α/β gene induction is in part governed by activation of the transcription factors irf-3 and -7, which are induced by ifn-α/β itself, resulting in a positive feed back loop, an effect which has been known for years without knowledge of the signalling mechanisms [315] [316] [317] . thus, one possible explanation for the genetic differences in hsv-induced ifn-α/β production could be an elevated physiological level of this ifn self-stimulating system [318] [319] [320] [321] . an analysis of the levels of irf-3 and -7 in normal macrophages from these mice could be of interest. analysis of the levels of the ifn-induced enzyme 2'-5'-oligoadenylate synthetase (oas) in uninfected cells showed low but slightly higher levels in cells from relatively resistant mice [322] . with lps, cells from the relatively resistant (c57bl/6) mice show an early induction pattern of ifn-α/β, peaking within 2 hours, whereas cells from the susceptible balb/c mice demonstrate a delayed response, peaking 7 hours after induction [323] . among other transcription factors involved in induction of the various ifn-α/β genes are the heterodimeric nf-κb family, which is activated by tlrs, il-1r, and tnfr [324] . during a hsv infection nf-κb is activated and translocated to the nucleus [325] . many regulatory mechanisms of nf-κb activation exist, one of them exerted through tnf, which is produced by macrophages very early during hsv infection ( fig. 2) [293, 300, 325, 326] . thus, the responsible mechanisms might be exerted by other regulatory signals, influencing the magnitude of the hsv-triggered ifn-α/β induction pathway, and perhaps not by this pathway in itself [327] . a number of x-linked immunodeficiencies have been described, one of them being the wiskott-aldrich syndrome with defects in a protein expressed in haematopoietic cells, facilitating reorganization of the actin cytoskeleton, and thus influencing the mobility of immune cells and chemotaxis of macrophages. patients with this x-linked immunodeficiency show aggravated herpetic infections, and cells from some patients seem to produce lower amounts of ifn in response to hsv [328] [329] [330] . cells from patients with another x-linked immunodeficiency with mutations in the cd40-ligand, a member of the tnf family, showed decreased ifn-α/β production when infected with hsv-1, but these patients apparently show a normal response to viral infections [331, 332] . this supports the notion above that other regulatory signals might be involved. the overall effects of the ifn-α/β system, besides the production as described above, are determined by the sensitivity of cells to the secreted ifn-α/β. the effector mechanisms of ifn-α/β on hsv replication are not fully elucidated. several ifn-α/β-activated systems are involved, including the dsrna-activated pkr, which phosphorylates, and thereby inhibits, the elongation initiation factor (eif)-2α, resulting in inhibition of translation [333] . another important mediator of the antiviral activity is the oas system, which activates 2'-5'oligoadenylate-dependent rnase l with the capacity to degrade single-stranded rna [333] . lately, the pml bodies have been described as crucial for the anti-hsv effect of ifn-α/ β [334] . in mice exhibiting a relatively hsv-resistant phenotype, the direct antiviral effect of ifn-α/β in embryonic cells was found to be approximately three-fold higher than in cells from susceptible mice [322] . data from another study showed comparable results on ifn-α/β sensitivity concerning the replication of encephalomyocarditis virus (emcv) in cells from the same mouse strains [335] . this phenomenon was inherited as a co-dominant autosomal trait without any apparent influence of x-linked genes [322, 336] . further studies in mouse fibroblasts have revealed that tnf intensify the antiviral effect of ifn-α/β and, thus, the in vivo situation seems more complicated ( fig. 2) [300, 337] . in the original publication on genetics of hsv susceptibility in inbred mice, lopez reported fibroblasts from the different mice to replicate hsv equally, and the same was found in the cells showing differential sensitivity to ifn-α/β [288, 322] . in line with these results, the ifn-activated oas, an inhibitor of hsv replication, was induced to a higher degree in cells from the resistant mice upon ifn-α/β treatment [322, 333, 338] . furthermore, the level of stimulated and unstimulated oas was generally found to vary between different inbred mouse strains [339] . thus, the genetic difference in antiviral action of type i ifns seems to affect the replication of several different viruses and to correlate with resistance to hsv. the viral host protein synthesis shutoff, exerted by the hsv vhs-protein of the tegument, has major effects on the cytokine production of infected cells and reduces the effect of ifn-α/β on hsv replication [340] . furthermore, the tegument proteins have been shown to induce cellular inhibitors of the jak/stat pathway, resulting in inhibition of both ifn signalling and production [341, 342] . the ie protein icp0 inhibits activation of irf-3 and thereby also restricts ifn-induced pathways [71-73], and icp0, icp4 and icp27 induce late shutoff of protein synthesis with decreased mrna stability and thus reduced cytokine production [81,343]. as outlined, it thus seems hsv has evolved several mechanisms to evade the consequences of the ifn-α/β system, which underline the importance of these cytokines in the antiviral defence. during hsv infection macrophages are activated and possess an increased antiviral potential [281, 344] . classically, the macrophage antiviral activity has been described as intrinsic or extrinsic [345] . resting macrophages possess a high degree of intrinsic activity against hsv, generally being non-permissive to viral replication. the macrophages are thus a blind end for the hsv infection, and they can in that way protect other cells from infection, for example as a barrier lining the liver sinusoids [344] . the extrinsic antiviral activity refers to the ability of macrophages to inactivate virus outside the macrophage itself or to inhibit viral replication in other cells [346] . the intrinsic antiviral activity depends among other factors on macrophage differentiation and has been correlated to ifn activity, either physiological levels of "spontaneous" preinfection-synthesized or rapidly acting autocrine ifn-α/β [344] . in that respect, macrophages from mice of the resistant phenotype showed higher intrinsic activity by being less permissive to hsv replication [281, 347] . one potential antiviral mechanism of macrophages may be the production of ros. these were originally assigned to bacterial killing, but the effect of ros has also been correlated to antiviral functions, although they might not be of major importance [348] . the ros are mainly produced by nadph-oxidases (nox), which are membrane-bound multi-component enzymes primarily situated in the phagolysosome [349] . activation of the nahpd-oxidase, by phosphorylation and fusion of the enzyme subunits, primarily results in production of superoxide anion (o 2 -), which by superoxide dismutase can be converted to hydrogen peroxide (h 2 o 2 ). the h 2 o 2 in turn is then by fe 2+ (fenton reaction) or by fe 3+ and o 2 -(haber-weiss reaction) converted to hydroxyl radical (·oh), hydroxyl anion (oh -) and singlet oxygen ( 1 o 2 ), or by the myeloperoxidase to hypochlorous acid (hocl) [349, 350] . small amounts of ros are also produced by the mitochondria and may be of importance as signalling molecules from tnf [351, 352] . during hsv infection in vivo, macrophages are activated and achieve an increased capacity to react with a respiratory burst of ros when appropriately triggered, i.e. by phorbol esters (fig. 2) [353] . this macrophage activation is induced early in response to hsv infection, reaching a plateau within the first 12 hours of i.p. infection [353] . in vitro, macrophages were shown to be the cell type responding with an oxidative burst, and this capacity peaked after only 8 hours of infection with hsv [353] . this hsv-induced capacity for an increased respiratory burst was shown to be governed by autocrine ifn-α/β as a sine qua non phenomenon [300, 353] . nevertheless, tnf was also found to influence the macrophage activation. by itself, tnf reduced the macrophage capacity for a respiratory burst, but in combination with ifn-α/β it synergistically enhanced the ifn-induced activation [293, 300] . interestingly, a secreted portion of the hsv-gg acts as a phagocyte chemoattractant and induces production of ros by signalling through the receptor activated by the phorbol esters [354] . the hsv-induced activation of macrophages in vivo is influenced by the genetic constitution of the host, with the most pronounced activation of macrophages originating from resistant mice, as expected on the basis of the genetics of ifn-α/β production in response to the infection. furthermore, the genetics of the efferent part of the ifn-α/β-mediated hsv-induced activation of macrophages, displayed a co-dominant autosomal trait, as was the case with the antiviral effect of ifn-α/β in fibroblasts [336] . thus, the genetically-determined sensitivity to ifnα/β seems to be expressed in different cell-types. the influence of tnf on the genetics of this phenomenon has not been addressed. in contrast to these observations, the genetics concerning the antiproliferative effect of ifn-α/β in bone marrow cells seems to be reversed [335, 355] . this might, however, be linked, in that ros are shown to activate various signalling molecules, mediate apoptosis, and exhibit antiproliferative effects depending on the dose and time of exposure [352] . little is known on the potential antiviral effect of ros. by examining peroxidized lipids, which is an oxidative product from ros in tissues, it has been documented that these are produced during the acute hsv infection in vivo, and speculations on antiviral mechanisms have focused on induction of apoptosis [348, 356, 357] . hsv triggers apoptosis of infected cells by several pathways, and the importance of this phenomenon is indicated by the fact that the virus has evolved mechanisms to counteract each of these pathways [358] . macrophages generally suppress apoptosis in hsv infections, as seen by increased apoptosis in macrophage-depleted mice [359] . several studies on the mechanisms involved in the early battle against hsv, performed in in vivo animal models, have pointed to ifn-α/β as a crucial player. in adoptive transfer experiments, the effect of adult mouse spleen cells on the initial phase of a generalized hsv infection in suckling mice was conducted by ifn-α/β [360] . furthermore, administration of a hematopoetic growth factor to neonatal mice increases the number of dendritic cells, b cells and nk cells, and confers resistance in a cutaneous model of hsv infection. the effect in this model could also largely be attributed to the actions of ifn-α/β, with some additional contributions by ifn-γ [361, 362] . in ko-mice ifn-α/β was able to control the initial phase of a generalized hsv infection without contributions from nk, t-or b cells, but these latter players were necessary for survival and long term control of the infection [363] . the importance of an early, local ifn-response in models including in vivo progression and evaluation of final outcome of infection is more unclear, in that many other viral and host factors are of importance in these more complicated models with several stages of infection and involvement of different organs. such models are, however, more close to the normal human hsv infection, starting at an epithelial surface, but to expect that one resistance factor in such a complicated system will come out clear as the responsible factor for the outcome downstream the sequence of events, is too simplistic. nevertheless, induced expression of ifn-α/β in the eye by plasmid dna or an adenovirus vector was shown to inhibit early local replication of hsv and the concomitant spread of virus to the brain and death from encephalitis [333, 364] , and in ifn-α/βr ko-mice hsv replicated to much higher titres than in normal mice [297] . this tells us that the innate and adaptive immune systems exhibit much redundancy, and that ifn-α/β is of vital importance in local inhibition of hsv replication. the multitude of antiviral mechanisms, be it innate or adaptive, have varying effects and importance in the different phases of infection, such as initial local infection, dissemination to other organs, establishment of latency and reactivation, and conclusions can not be drawn from one situation to another. the reactions discussed above, involving production of ifn-α/β and tnf, take place within the first 6 to 12 hours of a hsv infection, and thus are reactions, which can execute an effect within the first replication cycle of the virus. a little later, other cytokines such as il-12, il-18 and ifn-γ are produced and give rise to other weapons in the battle against the virus. they will, in turn, within the next replication cycle execute their actions, with potential harmful consequences for either parts of the conflict. a few hours after the type i ifn and tnf response, macrophages react upon hsv infection with production of il-12, which is seen from 8 to 12 hours after infection and on [238, 365] . the same was found with other viruses 12 to 24 hours after infection [366] . in these and other studies, the producers of il-12p40 during viral infection seem to be inflammatory cells, including macrophages, and not the infected stromal cells [365, 367] . the il-12 induction during hsv infection requires infectious virus, and it was shown to be regulated at the transcriptional level [238] , as it is also the case when it is induced by lps [247, 367] . the dependence on infectivity is, however, in conflict with results from in vivo production of il-12p40 and ifn-γ in draining lymph nodes from sites injected with uv-inactivated hsv [302] . high doses of uv-inactivated virus were used, and some minimal transcription of viral genes could have taken place, although the virus was not replication competent. transcription of the il-12p40 gene in macrophages requires de novo protein synthesis during the inducing hsv infection, which could explain the relatively late appearance of il-12 production [238, 367] . the κb-sequence of the il-12p40 promoter binds nf-κb in hsv-infected cells, and the production of il-12p40 was found to be repressed by an inhibitor of nf-κb activation [238] . both these observations indicate that signalling through nf-κb is of significance in hsv-induced il-12 production. in human macrophages, tnf has been shown to inhibit il-12p40 production, but not p35 production, by a mechanism not involving nf-κb [368] . furthermore, il-12 has in a mouse model been shown to stimulate tnf expression [255] , indicating that tnf can participate in a negative feed-back loop in the regulation of the il-12 system [369] . likewise, ifn-α/β has been shown to inhibit il-12 production in both humans and mice [370] [371] [372] . the implication of such inhibition by ifn-α/β and tnf, which are secreted very early in hsv infections, well before the production of il-12, has so far not been elucidated. as described earlier, the il-12p40 induction is influenced by ifn-γ in a positive feed back loop. ifn-γ could activate il-12 transcription through binding of irf-1, -2, and -8 to an isre site in the promoter-region of il-12 [373, 374] . upon hsv infection, ifn-γ is produced as part of the nonspecific response to the virus. a marked synergism between hsv and ifn-γ in il-12 induction has been demonstrated [238] , indicating that the il-12 / ifn-γ autoaccelerating system is of importance during hsv infections. the ifn-γ-inducing activity of the produced il-12 is pronounced in mouse peritoneal cells after 24 hours of infection with hsv [238] . in a study by kirchner et al. ifn-γ was detected as early as on day 3 of in vivo hsv infection, and the ifn-γ production was correlated to the genetics of hsv resistance [375] . during hsv infection, the production of ifn-γ is mainly induced as a concerted action of several factors and not by il-12 alone. ifn-α/β by itself was shown to be a weak inducer of ifn-γ production by nk cells, but in synergy with il-12 the production of ifnγ was markedly enhanced [376] . in elicited peritoneal macrophages, hsv induced efficient ifn-γ production through cooperation of il-12, ifn-α/β and il-18 [377] . in such a proinflammatory environment even other cells than nk and t cells, e.g. macrophages, might produce lower levels of ifn-γ [200, 202, 245] . il-12 signals through stat4, but stat4 translocation to the nucleus of nk cells has also been seen after ifn-α/β stimulation [206, 378] . likewise, ifn-α/β induces stat4 phosphorylation in t cells [379] , indicating that il-12 and ifn-α/β at this point act through a shared signalling pathway. furthermore, the synergistic action of il-12 and il-18 in ifn-γ production by macrophages was shown to be dependent on stat4 [197] . in addition to these factors, tnf and il-1 have also been shown to act in synergy with il-12 in ifn-γ induction [206, 380, 381] and vice versa, ifn-γ has been shown to synergize with hsv in induction of tnf production [325] . this further emphasizes the concept of positive feed-back mechanisms in the regulation of early ifn-γ production. the important direct effect of ifn-α/β on hsv replication was found to be enhanced synergistically by ifn-γ in both cell culture and in vivo in mice [363, [382] [383] [384] . this is, however, in conflict with an early study, which could not reveal any synergism between ifn-α/β and ifn-γ on the replication of hsv in human blood mononuclear cells [385] . synergistic action of the two types of ifn is further supported by the observation of synergism between ifn-γ and tnf on hsv replication in corneal cells, and the fact that this was exerted through production of ifn-β [386] [387] [388] . the effect was, however, greatly dependent on the cell type examined, which could explain the above-mentioned inconsistency. synergism between tnf and ifn-γ in inhibition of hsv replication has now been shown to be mediated by activation of a tryptophan-depleting enzyme [389] . thus, relatively small amounts of early ifn-γ produced by nk cells in response to il-12, ifn-α/β, tnf, and il-18 could in collaboration with the already present ifn-α/β and tnf have important local effect on hsv replication in permissive cells ( fig. 3 ). this conclusion is further supported by observations in ko mice, indicating that collaborated action of ifn-α/β and ifn-γ is of importance in control of subcutaneous hsv infections [362] . in vivo studies on hsv infections in immunodeficient, ko, and antibody-treated mice have shown that the il-12, second early wave of response figure 3 second early wave of response. regulatory pathways controlling production and action of ifn-γ during early hsv infection. when infected with hsv macrophages (mφ) produce several cytokines, including il-12, which stimulate production of ifn-γ, primarily in nk cells. ifn-γ then induces no production in macrophages and stimulate the direct antiviral activity of ifn-α/β in other cells. stimulatory pathways are indicated by green arrows (→), and inhibitory pathways are drawn in red. -23 / ifn-γ system is able to control the infection, affecting both the survival rate and the hsv titres early in infection [390, 391] . the effect of il-12 in hsv infections seems to be conducted in synergy with il-18 [390] , as it has also been shown for vaccinia virus [392] . in hsv corneal infections in ko mice, il-12 was shown to participate in the immune pathogenesis [393] , but in another study utilizing il-12 encoding plasmid dna, corneal expression of il-12 reduced the angiogenesis, and thus the pathology of the infection [394] . however, both studies agreed that il-12 does not affect the local titres of hsv in the eye. after a thermal injury, wide-spread hsv infections are an important risk, and treatment of injured mice with il-12 combined with soluble il-4r results in augmentation of the ifn-γ production and decreased viral replication and mortality [395] . in mice infected with murine cytomegalovirus (mcmv) production of il-12-induced ifn-γ by nk cells has been demonstrated in vivo, and the system was further shown to lower the viral titres [396, 397] . the il-12 / ifn-γ system seems, however, not to be of importance in all viral infections, in that the latter study could not detect any production of early il-12 or ifn-γ in a model of infection with the arenavirus lymphocytic choriomeningitis virus. analyses of the il-12, -23 / ifn-γ system in humans with genetic defects and in ko-mice reveal more redundancy in man than in mouse and indicate that the system is of more importance in dna-than in rna-virus infections [219] . the producers of early ifn-γ, the nk and nkt cells, and the cytokine il-15 and the transcription factor t-bet, which are both crucial for the differentiation and function of these cells, have all been shown to be decisive for the early control of hsv infection in vivo [268, [398] [399] [400] . although nk cells but not ifn-γ was shown to be decisive for survival from ocular infections [401] , such an effect of ifn-γ has been seen by others [402] . furthermore, a review of genetic functional nk cell defects found nk cells and their innate ifn-γ production to be of central importance in herpesvirus infections [403] . overall, it can be concluded that the il-12 / ifn-γ system is active in hsv infections and possesses an important antiviral potential, capable of controlling viral replication during the early phases of infection. in macrophages exposed to ifn-γ, the enzyme inducible nitric oxide synthase is induced, which eventually results in production of no from molecular oxygen and a guanidino nitrogen by conversion of l-arginine to l-citrulline [404] . upon hsv infection, the inos gene is induced, as shown by detection of inos-mrna in infected mouse peritoneal cells and corneal neutrophils [405, 406] . the production of no in hsv-infected cultures of resting mouse peritoneal cells, which comprise a mixed population of macrophages, lymphocytes, nk cells etc., is dependent on the virus being infectious [405] . this is in line with the requirement of infectious hsv for il-12 production and thus for production of ifn-γ as described previously [238] . no could itself be involved in a positive feed-back, in that signalling of il-12 utilizing tyk2 requires the activity of no [407] . when exogenous ifn-γ is added to virus-infected cells, a marked synergism is seen. this synergistic effect of hsv on the ifn-γ-induced no production in macrophages was shown to be mediated by autocrine secretion of tnf [325, 405] . in line with this, mice with a targeted disruption of the tnf gene showed impaired resistance to hsv and increased viral replication within the first days of infection [408] , and antibodies to tnf and an inhibitor of no production impaired early control of hsv infection in peripheral nervous tissue [409] . the induction of inos and the following production of no in response to ifn-γ and hsv is a relatively slow reaction, coming up after about 18 hours of infection [405] . in in vivo vaginal hsv infections inos mrna could be detected after 24 hours of infection [410] . thus, the production of this relatively toxic substance is part of the second wave of innate defence mechanisms. the retarded production of no and the requirement for two or more signals for induction of inos are logic considering the in hsv-infected macrophages exposed to ifn-γ, inos is induced synergistically though tnf-induced nf-κb activation and translocation to the nucleus, as shown by binding of a heterodimeric complex of p55/p65 and a homodimer of p55 to the κb-site of the inos promoter during infection [325] . the crucial position of nf-κb in the induction of inos and production of no is also indicated by experiments showing that antibodies to tnf inhibit activation of nf-κb and production of no in hsvinfected cells and abolish the synergism between the virus and ifn-γ, an observation which was also seen with inhibitors of nf-κb activation [325] . further analysis of the signalling mechanism has revealed that the synergism upon hsv infection is influenced by physical interaction of irf-1 and the nf-κb subunit p65 and controlled by the isresite and the distal κb-site of the inos promoter ( fig. 5 ) [411] . a further support for this notion comes from the observation that the dna-binding capacity of nf-κb and the nuclear translocation of irf-1 have similar kinetics upon hsv infection [411] and the fact that irf-1 is essential for inos induction [412] . induction of other genes such as ifn-β and vascular cell adhesion molecule 1 also involve physical interaction of irf-1 and nf-κb [413] , and both irf-1 and irf-2 have in other cells types been shown to form complexes with nf-κb [414, 415] . another potential mechanism in the synergistic induction of inos could involve complex formation of ifn consensus sequence-binding protein (icsbp or irf-8) and irf-1, which is also important for high-output no production but has still not been studied in hsv infections [416] . thus, high-output no production from activated macrophages is controlled by a "double-lock" signalling mechanism restricting the production of this antiviral toxic substance to sites of active viral replication, and sparing uninfected tissue from the detrimental effects ( fig. 4 ). the antiviral effects of no have been documented in several viral infections, although there clearly exist viruses and conditions where no does not exhibit major antiviral properties. no is thus not a magic bullet against virus infections [417] . in hsv infections, no has been shown to confer a substantial part of the antiviral activity induced by ifn-γ in a macrophage cell line and to participate in the extrinsic anti-hsv effect of macrophages [418] [419] [420] [421] . an exogenously added donor of no has in several cell lines been shown to reduce the replication of hsv [422] . in vivo, analysis of mice treated with an inhibitor of no production showed higher titres of hsv in the lungs but increased survival rates due to reduced inflammation [135] . recently, a study using another inhibitor of no production has confirmed the anti-herpetic effect of no during a hsv respiratory infection, but in this study mice with inhibited no production showed increased inflammatory responses, symptoms of infection, and mortality ifn-γ il-4 (at high ifn-γ / stat1) [423] . replication of hsv during vaginal infection was increased in the presence of an inhibitor of no production, and this enhanced viral replication was most prominent during the first 24 hours of infection [410] . in inos-ko mice, the herpes virus mcmv replicates to higher titres in various organs and in macrophages, and this results in impaired survival of the animals [424] . weanling mice with a targeted disruption of the inos gene showed increased hsv replication, but apparently without differences in hsv titres during the first days of infection [425] , and in adult ko mice, we could not detect any significant effect of no during the early days of a generalised hsv infection (ellermann-eriksen, unpublished results). probably, these in vivo results are due to redundancy of the antiviral system. [426] . the final effects of no on hsv infections therefore appear to be balanced between antiviral versus toxic effects, and the final outcome seems to depend on the timing, infectious dose, and tissues involved. thus no production in the early phases of hsv infection is one of the effector mechanisms of the innate immune response inhibiting hsv replication, but when overproduced, no might itself result in pathology, as discussed in the following section. as outlined above, positive feed-back mechanisms exist at the afferent side of the early cytokine response, involving especially the production of ifn-γ, il-12, ifn-α/β and tnf, and synergisms at the efferent side, resulting in highoutput no production. as a result of coordinated induction of the inos gene by several transcription factors, activated by especially ifn-γ and tnf, a potent early antiviral system is activated. however, no causes damage to dna, proteins and lipids in cells and tissues and could thus be deleterious for the host [427] [428] [429] [430] . a study in ko-mice indicates that no can be responsible for inflammation and life-threatening symptoms to hsv infection of the lungs [135] . this effect of no on pulmonary symptoms is also observed in influenza virus infections [431] , although no inhibits replication of both influenza virus and severe acute respiratory syndrome coronavirus [432, 433] . consequently, when this system is activated, it has to be controlled and eventually closed down, as it would otherwise induce unnecessary harm to the host. such negative regulations of the inos gene induction in ifn-γ activated macrophages is conducted by il-4 and il-13 [272, 273, 434] . furthermore, tgf-β can exhibit downregulation of no production through several post-transcriptional regulatory mechanisms, but the contribution of these pathways have not been analysed in hsv infections [435, 436] . il-4 production during hsv infection has in vaginal and cns infections been demonstrated on day 2 of infection and to increase for the next days [437, 438] . in peritoneal cells from mice infected i.p. pro-duction of il-4 could be detected at day 5 of infection [439] . at low ifn-γ concentrations, il-4 has been shown to inhibit inos induction through stat6 competition with stat1 binding to the gas element of the irf-1 promoter region. this results in reduced expression of the transcription factor irf-1, which is crucial for induction of inos [440] . generally, stat6 was shown to be a key factor in il-4-and il-13-induced inhibition of inos gene transcription induced by ifn-γ (fig. 5 ) [441] . at higher ifn-γ concentrations, activated stat6 is no longer able to compete with the high amounts of activated stat1 dimer [440] . however, in this situation il-4 is still able to inhibit the production of no from ifn-γ-stimulated macrophages [272, 273, 434] . in the presence of high levels of ifn-γ, il-4 is not able to alter the induction of irf-1, but the production of irf-2 is increased [434] . the human promoter region of irf-2 contains a sbe, and the induction of irf-2 could thus potentially be mediated by stat6 binding to this element [442] . this is in agreement with the fact that irf-2 is known to compete with the binding of irf-1 to isre sites and to antagonize the transactivating activity of irf-1 in the regulation of other ifninduced genes [443] [444] [445] . inhibition of inos expression by high concentrations of irf-2 relative to irf-1 has thus been proposed as a controlling mechanism in situations with high levels of ifn-γ ( fig. 5 ) [434] . furthermore, another mechanism could evolve from the observation that il-4 signalling can result in disruption of the complex formation of icsbp and irf-1 and thereby inhibit inos induction [416] . other mediators of il-4-induced repression of inos induction might exist, in that another dnabinding transcriptional repressor competing with irf-1 has been described [446] . in ifn-γ activated macrophages the il-4-and il-13induced inhibition of inos induction can thus be overruled by hsv infection, leading to a sustained no production ( fig. 4) [439, 447] . this effect of hsv infection is mediated through tnf production and nf-κb activation [439, 447] . however, pre-treatment with il-4 has in a theiler's murine encephalomyelitis virus model showed inhibition of nf-κb activation [448] . in thioglycollateinduced peritoneal cells, lps and tnf could only overcome the inhibiting effect of il-4 in situations, where il-4 was added simultaneously or after the stimulators [272] , a sequence of events which, however, is in agreement with the sequence of cytokine production in hsv infections. when activated, the nf-κb p65 physically interacts with irf-1 and trans-activate inos transcription in hsvinfected and tnf-treated cells [411, 449] . it is thus tempting to speculate that the nf-κb-irf-1 complex has higher affinity for the combined dna-binding site and thus is able to obstruct the binding of irf-2 to the isre site of the inos promoter and in that way turn the competition towards transcriptional activity ( fig. 5 ) [411] . this will block the inhibiting effect of il-4 in foci of hsv replication and open up for no production at sites where the antiviral effect is of more importance than the potential toxicity. in treatment of hsv infections, we have for many years had a very powerful tool in the antiherpetic drug acyclovir and related compounds. but there are still therapeutic problems in the group of patients with generalized or cns infections, and therefore it is tempting and timely to hypothesize on possible future treatment strategies. as described, it is clear that relatively discrete but early actions of the non-specific defence systems are crucial for the long term outcome of the infection. the same holds for the antiviral therapy, and early presumptive therapy and rapid diagnostics could thus potentially improve the final outcome. in the seeking for improved antiviral treatment, adjuvant therapy with anti-hsv antibodies could potentially accelerate the clearance of viral particles, and block viremic dissemination in patients, who are still seronegative at the time of treatment. immunomodulatory treatment modalities imitating the early non-specific antiviral defence, working as described in this review, could be considered. the key players exhibiting the least toxicity by themselves could be used, taking advantage of potential synergy with other cytokines in the foci of hsv infection. in the future, molecules with affinity for various receptors are expected to be produced, and when we know the signalling mechanisms in detail and all the potential interactions, molecular signalling could be addressed directly by pharmaceuticals. in consequence of the crucial position of the type i ifns in innate response to hsv, future analogues of ifn-α/β seem obvious as candidates for adjuvant treatment of severe hsv infections. this could be supplemented with il-12, which would give 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maturation the role of stat4 in species-specific regulation of th cell development by type i ifns interleukin 12 and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist il-1 alpha and tnf-alpha are required for il-12-induced development of th1 cells producing high levels of ifn-gamma in balb/c but not c57bl/ 6 mice inhibition of replication of herpes simplex virus in mouse macrophages by interferon alpha/beta interferon and gamma interferon synergize to inhibit the replication of herpes simplex virus type 1 mathematical analysis demonstrates that interferons-beta and -gamma interact in a multiplicative manner to disrupt herpes simplex virus replication effect of interferon on replication of herpes simplex virus types 1 and 2 in human macrophages mechanism of inhibition of hsv-1 replication by tumor necrosis factor and interferon gamma synergistic anti-hsv effect of tumor necrosis factor alpha and interferon gamma in human corneal fibroblasts is associated with interferon beta induction synergistic anti-herpes effect of tnf-alpha and ifn-gamma in human corneal epithelial cells compared with that in corneal fibroblasts daubener w: inhibition of human herpes simplex virus type 2 by interferon gamma and tumor necrosis factor alpha is mediated by indoleamine 2,3-dioxygenase interleukin-12 (il-12) and il-18 are important in innate defense against genital herpes simplex virus type 2 infection in mice but are not required for the development of acquired gamma interferon-mediated protective immunity interleukin-12-and gamma interferon-dependent innate immunity are essential and sufficient for long-term survival of passively immunized mice infected with herpes simplex virus type 1 il-12 and il-18 act in synergy to clear vaccinia virus infection: involvement of innate and adaptive components of the immune system reduced severity of hsv-1-induced corneal scarring in il-12-deficient mice il-12 suppresses the expression of ocular immunoinflammatory lesions by effects on angiogenesis therapeutic protective effects of il-12 combined with soluble il-4 receptor against established infections of herpes simplex virus type 1 in thermally injured mice requirement for natural killer cell-produced interferon gamma in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration an absolute and restricted requirement for il-12 in natural killer cell ifn-gamma production and antiviral defense. studies of natural killer and t cell responses in contrasting viral infections interleukin-15 and natural killer and nkt cells play a critical role in innate protection against genital herpes simplex virus type 2 infection in the absence of t cells, natural killer cells protect from mortality due to hsv-1 encephalitis protective immunity to genital herpes simpex virus type 2 infection is mediated by t-bet the role of natural killer cells in protection of mice against death and corneal scarring following ocular hsv-1 infection kinetics of cytokine production in the cornea and trigeminal ganglion of c57bl/6 mice after corneal hsv-1 infection human natural killer cell deficiencies and susceptibility to infection nitric oxide as a secretory product of mammalian cells herpes simplex virus type 2 synergizes with interferon-gamma in the induction of nitric oxide production in mouse macrophages through autocrine secretion of tumour necrosis factor-alpha production of key molecules by ocular neutrophils early after herpetic infection of the cornea requirement for type 2 no synthase for il-12 signaling in innate immunity absence of tumour necrosis factor facilitates primary and recurrent herpes simplex virus-1 infections macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system nitric oxide and hsv vaginal infection in balb/c mice interferon (ifn)-gamma and herpes simplex virus/tumor necrosis factor-alpha synergistically induce nitric oxide synthase 2 in macrophages through cooperative action of nuclear factor-kappa b and ifn regulatory factor-1 requirement for transcription factor irf-1 in no synthase induction in macrophages endothelial interferon regulatory factor 1 cooperates with nf-kappa b as a transcriptional activator of vascular cell adhesion molecule 1 nf kappa b and interferon regulatory factor 1 physically interact and synergistically induce major histocompatibility class i gene expression interferon regulatory factor-2 physically interacts with nf-kappa b in vitro and inhibits nf-kappa b induction of major histocompatibility class i and beta 2-microglobulin gene expression in transfected human neuroblastoma cells complex formation of the interferon (ifn) consensus sequence-binding protein with irf-1 is essential for murine macrophage ifn-gamma-induced inos gene expression does nitric oxide play a critical role in viral infections? inhibition of viral replication by interferon-gammainduced nitric oxide synthase interferon-gamma induced type i nitric oxide synthase activity inhibits viral replication in neurons inhibition of viral replication by nitric oxide and its reversal by ferrous sulfate and tricarboxylic acid cycle metabolites nitric oxide and macrophage antiviral extrinsic activity evidence for antiviral effect of nitric oxide. inhibition of herpes simplex virus type 1 replication early inhibition of nitric oxide production increases hsv-1 intranasal infection role of nitric oxide synthase type 2 in acute infection with murine cytomegalovirus mice lacking inducible nitric-oxide synthase are more susceptible to herpes simplex virus infection despite enhanced th1 cell responses phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase epr characterization of molecular targets for no in mammalian cells and organelles dna strand breakage, activation of poly (adp-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. formation of novel nitrogen-containing oxidized lipid derivatives altered immune responses in mice lacking inducible nitric oxide synthase rapid interferon gamma-dependent clearance of influenza a virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice osterhaus ad: inhibition of influenza virus replication by nitric oxide nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus interleukin-4-mediated inhibition of nitric oxide production in interferon-gamma-treated and virus-infected macrophages mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta spontaneously increased production of nitric oxide and aberrant expression of the inducible nitric oxide synthase in vivo in the transforming growth factor beta 1 null mouse effect of the deletion of us2 and us3 from herpes simplex virus type 2 on immune responses in the murine vagina following intravaginal infection small amounts of exogenous il-4 increase the severity of encephalitis induced in mice by the intranasal infection of herpes simplex virus type 1 herpes simplex virus type 2 infection of macrophages impairs il-4-mediated inhibition of no production through tnfalpha-induced activation of nf-kappab il-4-induced stat6 suppresses ifngamma-stimulated stat1-dependent transcription in mouse macrophages impaired il-13-mediated functions of macrophages in stat6-deficient mice structure and regulation of the human interferon regulatory factor 1 (irf-1) and irf-2 genes: implications for a gene network in the interferon system structurally similar but functionally distinct factors, irf-1 and irf-2, bind to the same regulatory elements of ifn and ifn-inducible genes absence of the type i ifn system in ec cells: transcriptional activator (irf-1) and repressor (irf-2) genes are developmentally regulated recognition dna sequences of interferon regulatory factor 1 (irf-1) and irf-2, regulators of cell growth and the interferon system b lymphocyte-induced maturation protein (blimp)-1, ifn regulatory factor (irf)-1, and irf-2 can bind to the same regulatory sites inhibition of no production in macrophages by il-13 is counteracted by herpes simplex virus infection through tumor necrosis factor-alpha-induced activation of nk-kappa b interleukin-4 and interleukin-10 modulate nuclear factor kappab activity and nitric oxide synthase-2 expression in theiler's virus-infected brain astrocytes interaction of interferon regulatory factor-1 and nuclear factor kap-pab during activation of inducible nitric oxide synthase transcription i wish to thank all members of the group for highly constructive discussions and especially søren c. mogensen for critical review of the manuscript. for excellent technical help with the electron microscopy i thank ruth nielsen. furthermore, i wish to thank the department of clinical microbiology, aarhus university hospital, skejby and department of medical microbiology and immunology, university of aarhus for their hospitality and support. key: cord-322104-f1dukpso authors: niederman, m.s. title: pneumonia | community acquired pneumonia, bacterial and other common pathogens date: 2006-05-13 journal: encyclopedia of respiratory medicine doi: 10.1016/b0-12-370879-6/00310-0 sha: doc_id: 322104 cord_uid: f1dukpso community-acquired pneumonia (cap) is the number one cause of death from infectious diseases in the us, and the patient population that is affected is becoming increasingly more complex due to the presence of chronic illness which is commonly managed in outpatients who are at risk for pneumonia. the number one pathogen causing cap is pneumococcus, which is commonly resistant to multiple antibiotics, thus complicating management. other common pathogens include atypical organisms (chlamydophila pneumoniae, legionella pneumophila, mycoplasma pneumoniae), hemophilus influenzae, enteric gram-negatives (especially in those with chronic illness and aspiration risk factors), and staphylococcus aureus. successful management requires careful assessment of disease severity so that a site-of-care decision can be made (outpatient, inpatient, intensive care unit), appropriate samples for diagnostic testing collected, and antibiotic therapy initiated in a timely and accurate fashion. initial antibiotic therapy is empiric, but even with extensive diagnostic testing, less than half of all patients have an etiologic pathogen identified. all patients with cap require therapy for pneumococcus, atypical pathogens, and other organisms, as dictated by the presence of specific risk factors. because pneumonia has both short-term and long-term impact on mortality, it is also important to focus on prevention of this illness, which requires smoking cessation, and giving at-risk individuals both pneumococal and influenza vaccines. pneumonia, a respiratory infection of the alveolar space, can vary from a mild outpatient illness to a severe illness necessitating hospitalization and intensive care. it is the sixth leading cause of death in the us and the number one cause of death from infectious diseases. when the infection occurs in patients who are living in the community it is termed community-acquired pneumonia (cap), while it is called nosocomial pneumonia if it arises in patients who are already in the hospital. presently, the distinction between community-acquired and nosocomial infection is less clear because the 'community' includes complex patients such as those who have recently been hospitalized, those in nursing homes, and those with chronic diseases who are commonly managed in such facilities as dialysis centers or nursing homes. when this latter group develops pneumonia, it has been termed healthcare associated pneumonia (hcap) and this illness shares clinical features with cap, but the etiologic pathogens may overlap with those seen in more traditional nosocomial pneumonia. thus, the relationship between bacteriology and the site of origin of infection is a reflection of several factors, including: the types of patients who develop the illness, their host defense related predisposition to infection with specific pathogens, and their environmental exposure to certain organisms. this discussion focuses on pneumonia arising out of the hospital in immune-competent individuals, but excludes discussion of patients with human immunodeficiency virus (hiv) infection or traditional immune suppression (cancer chemotherapy, immune suppressive medications). in 1994, over 5.6 million people were diagnosed with cap in the us, but the majority, 4.6 million, were treated out of the hospital. although the majority of cases of cap are managed in the outpatient setting, the greater part of the cost of treatment is focused on hospitalized patients. those who are admitted to the hospital commonly tend to be older and have a high frequency of comorbid illness. in the us, the population of elderly patients is increasing, and those aged over 65 make up about one-third of all cap patients, but this group accounts for more than half of the cost because of the frequent need for elderly cap patients to be hospitalized. the reported mortality of cap varies with the population being evaluated, ranging from less than 5% among outpatients, to 12% among all hospitalized cap patients, but rising to over 30% among those admitted to the intensive care unit (icu). while most studies have focused on the in-hospital mortality of cap, one recent evaluation of cap patients over the age of 65 found that while the short-term risk (in-hospital mortality) of illness was an 11% death rate, the mortality rate at 1 year was over 40%, emphasizing that for many patients, cap is a marker of underlying serious comorbidity, and a predictor of poor outcome, even after hospital discharge, for a variety of reasons. these findings add to the emphasis on pneumonia prevention, especially through the use of available vaccines. the complexity of cap management is also increasing because the etiologic pathogens are changing. historically, cap was regarded as a bacterial illness caused by one pathogen, streptococcus pneumoniae. today, the number of etiologic pathogens has mushroomed to include not only bacteria, but also viruses (influenza, severe acute respiratory syndrome (sars)), fungi, and a number of other recently identified organisms (such as legionella, chlamydophila pneumoniae). not only is the number of pathogens expanding, but our ability to treat is being challenged by the rising frequency of resistance among bacteria to a wide range of commonly used, and often overused, antimicrobial agents. pneumonia is an infection of the gas-exchanging units of the lung, most commonly caused by bacteria, but occasionally caused by viruses, fungi, parasites, and other infectious agents. in the immunocompetent individual, it is characterized by a brisk filling of the alveolar space with inflammatory cells and fluid. if the alveolar infection involves an entire anatomic lobe of the lung, it is termed 'lobar pneumonia', and multilobar illness can be present in some instances. when the alveolar process occurs in a distribution that is patchy, and adjacent to bronchi, without filling an entire lobe, it is termed as 'bronchopneumonia'. pneumonia occurs when a patient's host defenses are overwhelmed by an infectious pathogen. this can happen because the patient has an inadequate immune response, often as a result of underlying comorbid illness (congestive heart failure, diabetes, renal failure, chronic obstructive lung disease, malnutrition), because of anatomic abnormalities (endobronchial obstruction, bronchiectasis), as a result of acute illness-associated immune dysfunction (as can occur with sepsis or acute lung injury), or because of therapy-induced dysfunction of the immune system (corticosteroids, endotracheal intubation). pneumonia can also occur in patients who have an adequate immune system, if the host defense system is overwhelmed by a large inoculum of microorganisms, which can occur in a patient with massive aspiration of gastric contents. in patients outside the hospital, a normal immune system can be overcome by a particularly virulent organism, to which the patient has no pre-existing immunity (such as certain bacteria or viruses) or to which the patient has an inability to form an adequate acute immune response. bacteria can enter the lung via several routes, but aspiration from a previously colonized oropharynx is the most common way that organisms lead to pneumonia. although most pneumonias result from microaspiration, patients can also aspirate large volumes of bacteria if they have impaired neurologic protection of the upper airway (stroke, seizure), or if they have intestinal illnesses that predispose to vomiting. other routes of entry include inhalation, which applies primarily to viruses, legionella pneumophila and mycobacterium tuberculosis; hematogenous dissemination from extrapulmonary sites of infection (right-sided endocarditis); and direct extension from contiguous sites of infection (such as liver abscess). keeping these concepts in mind, it is easy to understand why previously healthy individuals develop infection with virulent pathogens such as viruses, l. pneumophila, mycoplasma pneumoniae, c. pneumoniae, and s. pneumoniae. on the other hand, chronically ill patients can be infected by these organisms, as well as by organisms that commonly colonize patients, but only cause infection when immune responses are inadequate. these organisms include enteric gram-negative bacteria (escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter spp.) and fungi. recent studies have evaluated the normal lung immune response to infection and have shown that it is generally 'compartmentalized', and thus most patients with unilateral pneumonia have an inflammatory response that is limited to the site of infection, not spilling over to the uninvolved lung or to the systemic circulation. in patients with localized pneumonia, tumor necrosis factor (tnf) and interleukins 6 and 8 (il-6, il-8) levels were increased in the pneumonic lung and generally not increased in the uninvolved lung or in the serum. in patients with severe pneumonia, the immune response is characterized by a spillover of the immune response into the systemic circulation, reflected by increases in serum levels of tnf and il-6. it remains uncertain why localization does not occur in all individuals, and why some patients develop diffuse lung injury (acute respiratory distress sydrome, ards) or systemic sepsis as a consequence of pneumonia. recent studies have suggested that genetic polymorphisms may explain some of these differences, with patients who have certain inherited patterns of immune response being more prone than others to severe forms of pneumonia, and even mortality from this illness. for example, certain genetic polymorphisms are associated with a greater risk of death from sepsis but not cap. specifically, tnf-308 polymorphisms increase sepsis mortality, but not mortality from cap. in addition, cap severity is increased with genetic changes in the il-10-1082 locus, which are often present along with changes in the tnf-308 locus. another genetic change associated with an increased risk of septic shock from cap is a modification in heat shock protein 70-2. currently, we are aware of the large number of genes that can affect the severity and outcome of cap, but much more must be learned to put these findings into a true clinical context. even with extensive diagnostic testing, an etiologic agent is defined in only about half of all patients with cap, pointing out the limited value of diagnostic testing, and the possibility that we do not know all the organisms that can cause cap. for example, in the past three decades, a variety of new pathogens for this illness have been identified, including l. pneumophila, c. pneumoniae, hantavirus, and the sars virus (a coronavirus). in addition, antibiotic resistant variants of common pathogens such as s. pneumoniae (pneumococcus) have become increasingly common and are referred to as drug-resistant s. pneumoniae (drsp). for all patients with cap, pneumococcus (including drsp) is the most common pathogen, and some studies have suggested that it may be responsible for many of the patients with no established etiologic diagnosis, using standard diagnostic methodology. recent studies have also suggested that atypical pathogens (m. pneumoniae, c. pneumoniae, and l. pneumocphila) are common in patients with cap, often as copathogens, along with bacterial organisms. viruses may be present in up to 20% of all patients, but specialized diagnostic testing, usually involving acute and convalescent titers, is needed, thus explaining the ordinary low frequency of documenting these organisms. hemophilus influenzae is a common organism in patients who smoke cigarettes, and in those with chronic obstructive lung disease. enteric gramnegatives are not common causes of cap, but can be present in up to 10% of hospitalized patients, particularly those with advanced age, comorbid illness, or a high likelihood of aspiration. in general, certain specific patients are at risk for infection with specific pathogens, in different frequencies. table 1 summarizes the common pathogens causing cap in both outpatients and inpatients. the classification is based on the severity of illness and the presence of clinical risk factors for specific pathogens, referred to as modifying factors. patients with severe cap may have a slightly different spectrum of organisms than less severely affected individuals, being commonly infected with pneumococcus, atypical pathogens, enteric gram-negatives (including p. aeruginosa), staphylococcus aureus, and h. influenzae. the modifying factors that increase the risk of infection with drsp are: age over 65 years, beta-lactam therapy within the past 3 months, alcoholism, immune suppressive illness (including therapy with corticosteroids), multiple medical comorbidities, and exposure to a child in a day care setting. the modifying factors for enteric gram-negatives include: residence in a nursing home, underlying cardiopulmonary disease, multiple medical comorbidities, and recent antibiotic therapy. the risk factors for p. aeruginosa infection are: structural lung disease (bronchiectasis), corticosteroid therapy (410 mg day à 1 prednisone), broad-spectrum antibiotic therapy for 47 days in the past month, and malnutrition. table 2 shows that certain clinical conditions are associated with specific pathogens, and these associations should be considered in all patients when obtaining a history. as mentioned, s. pneumoniae is the most common pathogen for cap, in any patient population, possibly even among those without an etiology recognized by routine diagnostic testing. in one study, transthoracic needle aspirates were used to define the etiology of cap in patients with no identified organisms by conventional diagnostic testing, using a polymerase chain reaction (pcr) probe analysis of the samples, and in half of the patients in whom the needle provided a diagnosis when other methods had failed, pneumococcus was identified. the organism is a gram-positive, lancet-shaped diplococcus, of which there are 84 different serotypes, each with a distinct antigenic polysaccharide capsule, but 85% of all infections are caused by one of 23 serotypes, which are now included in a vaccine. infection is most common in the winter and early severe cap, with risks for p. aeruginosa all of the pathogens above, plus p. aeruginosa pneumococcus resistant to the class of agents to which the patient was recently exposed spring, which may relate to the finding that up to 70% of patients have a preceding viral illness. the organism spreads from person to person, and commonly colonizes the oropharynx before it leads to pneumonia. pneumonia develops when colonizing organisms are aspirated into a lung that is unable to contain the aspirated inoculum. infection is more common in the elderly; those with asplenia, multiple myeloma, congestive heart failure, or alcoholism; after influenza; and in patients with chronic lung disease. in patients with hiv infection, pneumococcal pneumonia with bacteremia is more common than in healthy populations of the same age. the classic radiographic pattern is a lobar consolidation, but bronchopneumonia can also occur, and in some series, this is the most common pattern. bacteremia is present in up to 20% of hospitalized patients with this infection, but the impact of this finding on mortality is uncertain. extrapulmonary complications include meningitis, empyema, arthritis, endocarditis, and brain abscess. since the mid-1990s, antibiotic resistance among pneumococci has become increasingly common, and penicillin resistance, along with resistance to other common antibiotics (macrolides, trimethoprim/sulfamethoxizole, selected cephalosporins), is present in over 40% of these organisms. fortunately, in the us, a large number of penicillin-resistant organisms are of the intermediate type (penicillin minimum inhibitory concentration, or mic, of 0.1-1.0 mg l à 1 ) and not of the high-level type (penicillin mic of 2.0 mg l à 1 or more). it is difficult to show a clinical impact of in vitro resistance on outcomes such as mortality in large numbers of patients, but most experts believe that organisms with a penicillin mic of x4 mg l à 1 , still an uncommon finding, can lead to an increased risk of death. in an early study of the topic, there was no impact of resistance on mortality, after adjusting for severity of illness in a population with nearly a 30% frequency of in vitro resistance. more recently, some studies have shown that resistance can affect outcome. in a group of patients with pneumococcal bacteremia, of which more than half were hiv-positive, high-level resistance was a predictor of mortality. other investigators did not find an increased risk of death from infection with resistant organisms, but did find an enhanced likelihood of suppurative complications (empyema), and a more prolonged length of stay in hospital. these conflicting data may have been the result of studying relatively few patients, many of whom did not have high levels of in vitro resistance. one large study evaluated more than 5000 patients with pneumococcal bacteremia and cap and found an increased mortality for patients with a penicillin mic of at least 4 mg l à 1 or greater, or with a cefotaxime mic of 2.0 mg l à 1 or more. however, this increased mortality was only present if patients who died in the first 4 days of therapy were excluded from analysis. fortunately, very few organisms are currently at this level of resistance, which may explain the conflicting findings in various studies. more recently, another study using both a cohort and matched control methods found that severity of illness, and not resistance or accuracy of therapy, was the most important predictor of mortality. in some studies, severity of illness was greater in patients without resistant organisms, implying a loss of virulence among organisms that become resistant, a finding echoed in other studies that have found that the absence of invasive illness is a risk factor for pneumoccal resistance. there are also conflicting data on the impact of discordant therapy in patients infected with drsp. in one study of bacteremic infection, the only antibiotic associated with a poor outcome, in the presence of in vitro resistance, was cefuroxime. in another study, discordant therapy in general was associated with an increased risk of mortality, as was multilobar illness, underlying chronic obstructive pulmonary disease (copd), and hospitalization within the past 3 months, but these latter factors did not have as much of a risk of death as did discordant therapy. also in this study, discordant therapy was less likely if patients were treated with ceftriaxone or cefotaxime, compared to other therapies. macrolide-resistant pneumococcus is also occurring with increasing frequency, and up to 40% of organisms may be resistant to these agents in vitro. however, it is important to understand that most macrolide resistance in the us is low level, and due to an efflux mechanism, and thus it is a type of resistance that may not be clinically relevant, because local concentrations of macrolides at respiratory sites of infection may be adequate for effective therapy. however, some resistance is higher level and due to an inability of the antibiotic to bind to its ribosomal site of action, and this form of resistance may be much higher level and more likely to be clinically relevant. there are however, very few reports of macrolide failures in cap, especially considering the widespread use of these agents. reports of breakthrough bacteremia have appeared, but have been due to organisms that were resistant by either the efflux or ribosomal mechanism. it is likely that higher levels of resistance will become more likely in the future, and the impact on outcome is likely to be more apparent. resistance of pneumococcus has even been reported to the quinolones, which are ordinarily a reliable class of antibiotics for these organisms. in general, one important risk factor for resistance is repeated use of these agents in the same patient. in fact, pneumococcal resistance to beta-lactams (penicillins and cephalosporins), macrolides, and quinolones is more likely if a patient has received the same agent in the recent past. it remains uncertain how long after antibiotic exposure that there is still risk of resistance, but 3-6 months has been reported for beta-lactams, up to 6 months for macrolides, and up to 12 months for quinolones. with these data in mind, new guidelines have suggested that cap patients should not receive the same antibiotic as in the recent past, with a relatively arbitrary cutoff of defining this time interval as 'within the past 3 months'. originally the term 'atypical' was used to describe the unusual clinical features of infection with certain organisms, but recent studies have suggested that the term does not accurately describe a unique clinical pneumonia syndrome related to specific pathogens. however, the term has been retained to refer to a group of organisms which includes m. pneumoniae, c. pneumoniae, and legionella, and this group of organisms cannot be reliably eradicated by betalactam therapy (penicillins and cephalosporins), but must be treated with a macrolide, ketolide (telithromycin is the only one currently available), tetracycline, or a quinolone. some recent studies have shown that these infections are common in patients of all ages, not just young and healthy individuals as originally described, and these organisms have even been reported among the elderly in nursing homes. in addition, they can occur as either primary pathogens, or may be part of a mixed infection, along with traditional bacterial pathogens. when mixed infection is present, it may lead to a more complex course and a longer length of stay than if a single pathogen is present. there may be a particular synergy between c. pneumoniae and pneumococcus, with either sequential, or mixed infection with c. pneumoniae leading to a more severe course for pneumococcus. the frequency of atypical pathogens can be as high as 60% of all cap patients, in some series, with as many as 40% of patients having mixed infection identified. although these high incidence numbers have been derived with serologic testing, which is of uncertain accuracy, the importance of these organisms is suggested by studies of inpatients, which have shown a reduced mortality and length of stay when patients receive empiric therapy that accounts for these organisms, compared to regimens that do not account for these organisms. in fact, several studies of patients with pneumococcal bacteremia have even suggested a mortality benefit to combination therapy that provides coverage for atypical pathogens, as well as pneumococcus. atypical organism pneumonia may not be a constant phenomenon, and the frequency of infection may vary over the course of time and with geographical location. in one study, the benefit of providing empiric therapy directed at atypical pathogens was variable, being more important in some calendar years than in others. the incidence of legionella infection among admitted patients has varied from 1% to 15% or more, and is also a reflection of geographic and seasonal variability in infection rates, as well as a reflection of the extent of diagnostic testing. for legionella to be identified, it is necessary to collect both acute and convalescent serologic studies. in patients with severe cap, atypical pathogens can be present in almost 25% of all patients, but the responsible organism may vary over time. in one series, legionella was the most common atypical pathogen leading to severe cap, but in the same hospital a decade later, it had been replaced by mycoplasma and chlamydophila infection. l. pneumophila is a small, weakly staining, gramnegative bacillus first characterized after an epidemic in 1976, and can occur either sporadically or in epidemic form. at present, although multiple serogroups of the species l. pneumophila have been described, serogroup 1 causes the most cases of pneumonia. the other species that commonly causes human illness is legionella micdadei. legionella is a water-borne pathogen and can emanate from air-conditioning equipment, drinking water, lakes and river banks, water faucets, saunas, and shower heads. infection is more common in the summer and early fall, and is generally caused by inhalation of an infected aerosol generated by a contaminated water source. when a water system becomes infected in an institution, endemic outbreaks may occur, as has been the case in some hospitals, particularly in patients who are receiving corticosteroid therapy. in its sporadic form, legionella may account for 7-15% of all cases of cap, being a particular concern in patients with severe forms of illness. as mentioned, it is very difficult to use clinical features to predict the microbial etiology of cap; however, the classic legionella syndrome is characterized by high fever, chills, headache, myalgias, and leukocytosis. the diagnosis is also suggested by the presence of a pneumonia with preceding diarrhea, along with early onset of mental confusion, hyponatremia, relative bradycardia, and liver function abnormalities, but this syndrome is usually not present. symptoms are rapidly progressive, and the patient may appear to be quite toxic, so this diagnosis should always be considered in patients admitted to the icu with cap. m. pneumoniae can cause cap year-round, with a slight increase in the fall and winter. all age groups are affected, and although it is common in those less than 20 years of age, it is also a common cause of cap, even in older adults. respiratory infection occurs after the organism is inhaled and then binds via neuraminic acid receptors to the airway epithelium. an inflammatory response with neutrophils, lymphocytes, and macrophages then follows, accompanied by the formation of immunoglobulin m (igm) and then igg antibody. some of the observed pneumonitis may be mediated by the host response to the organism rather than by direct tissue injury by the mycoplasma. up to 40% of infected individuals will have circulating immune complexes. although mycoplasma causes pneumonia, infection is often characterized by its extrapulmonary manifestations. up to half of patients will have upper respiratory tract symptoms, including sore throat and earache (with hemorrhagic or bullous myringitis). pleural effusion is quite common, being seen in at least 20% of patients with pneumonia, although it may be small. other extrapulmonary manifestations include neurologic illness such as meningoencephalitis, meningitis, transverse myelitis, and cranial nerve palsies. the most common extrapulmonary finding is an igm autoantibody that is directed against the i antigen on the red blood cell and causes cold agglutination of the erythrocyte. although up to 75% of patients may have this antibody and a positive coombs' test, clinically significant autoimmune hemolytic anemia is uncommon. other systemic complications include myocarditis, pericarditis, hepatitis, gastroenteritis, erythema multiforme, arthralgias, pancreatitis, generalized lymphadenopathy, and glomerulonephritis. the extrapulmonary manifestations may follow the respiratory symptoms by as long as 3 weeks. the most common gram-negative organism causing cap is h. influenzae (see below). enteric gram-negatives are generally not common in cap, unless the patient is elderly and has chronic cardiac or pulmonary disease, has healthcare associated pneumonia, or is alcoholic. in these patients, organisms such as e. coli and k. pneumoniae can be found. p. aeruginosa is an uncommon cause of cap, but can be isolated from patients with cap and bronchiectasis, and in those with severe forms of cap, particularly in the elderly patient aged over 75. controversy has persisted about how commonly enteric gram-negative bacteria are a cause of cap. however, in one large study of hospitalized cap patients, careful diagnostic testing identified gram-negative enterics in 11%, with more than half of these being p. aeruginosa. identified risk factors for gramnegative infection were: probable aspiration, previous hospital admission within 30 days of admission, previous antibiotics within 30 days of admission, and presence of pulmonary comorbidity. risk factors for p. aeruginosa were pulmonary comorbidity and previous hospitalization. infection with a gramnegative increased the chance of dying by more than threefold, with a mortality rate of 32%, and these patients also need icu admission and mechanical ventilation more often than patients with other organisms. these organisms have always been a concern in patients with poor dentition who aspirate oral contents, and those at risk have been patients with neurologic or swallowing disorders, as well as individuals who abuse alcohol and opiate drugs. several recent studies have questioned whether these organisms are really common in patients with risk factors for aspiration. in one evaluation of residents of longterm care facilities who had severe aspiration pneumonia (defined by the presence of risk factors for oropharyngeal aspiration), requiring icu admission, bacteriology was determined by protected bronchoalveolar lavage (bal) within 4 h of admission. when a pathogen was identified, the organisms were: enteric gram-negatives in 49%, anaerobes in 16%, and s. aureus in 12%. many of the anaerobes were recovered with aerobic gram-negatives, and their presence did not correlate with oral hygiene, but the presence of gram-negatives did correlate with functional status, being more common in patients who were totally dependent. many patients received inadequate therapy for anaerobes, yet most recovered, raising a question about whether infection with these organisms really needs to be treated. these findings suggest that anaerobes may not always be pathogens, but may be colonizers in the institutionalized elderly, including those with aspiration risk factors. this gram-negative coccobacillary rod can occur in either a typable, encapsulated form or a nontypable, unencapsulated form. the encapsulated organism can be one of seven types, but type b accounts for 95% of all invasive infections. encapsulated organisms require a more elaborate host response, and thus they are more virulent than unencapsulated organisms. however several studies have shown that in adults, particularly those with copd, infection with unencapsulated bacteria is more common than infection with encapsulated organisms. the organism may cause bacteremic pneumonia in some patients, particularly in those with segmental pneumonias as opposed to those with bronchopneumonia. the encapsulated type b organism is more common in patients with segmental pneumonia than in those with bronchopneumonia. most patients with this infection have some underlying illness such as alcoholism, smoking history, or copd. cap can also be caused by s. aureus, which can lead to severe illness and to cavitary lung infection. this organism can also seed the lung hematogenously from a vegetation in patients with right-sided endocarditis or from septic venous thrombophlebitis (from central venous catheter or jugular vein infection). when a patient develops post influenza pneumonia s. aureus can lead to secondary bacterial infection, along with pneumococcus and h. influenzae. most recently, there have been reports of cap caused by methicillin-resistant s. aureus (mrsa), and if this becomes a more common occurrence, it may change the way this disease is treated empirically. to date, most patients with mrsa cap have had a severe necrotizing pneumonia, generally following influenza or another viral infection. the organism responsible has had a specific virulence factor, the panton-valentine leukocidin, and all the organisms causing pneumonia appear to be genetically related. the incidence of viral pneumonia is difficult to define, but during epidemic times, influenza should be considered, and it can lead to a primary viral pneumonia, or to secondary bacterial pneumonia. one careful study of over 300 non-immune-compromised cap patients collected paired sera for respiratory viruses, and found that 18% had viral pneumonia, with about half being pure viral infection and the others being mixed with bacterial pneumonia. patients with congestive heart failure were at more risk of pure viral pneumonia than they were of pneumococcal infection. influenza (a more than b), parainfluenza, and adenovirus were the most commonly identified viral causes of cap. while influenza a and b still remain the most common causes of viral pneumonia, vigilance to new agents is essential as evidenced by the recent experience with sars, which demonstrated the potential of epidemic, person-to-person spread of virulent respiratory viral infection. continued concern about epidemic viral pneumonia remains, with the current worry being focused on avian influenza, and bioterrorism with agents such as smallpox. patients with an intact immune system who develop cap generally have respiratory symptoms such as cough, sputum production, and dyspnea, along with fever and other complaints. cough is the most common finding, and is present in up to 80% of all patients, but is less common in those who are elderly, those with serious comorbidity, or individuals coming from nursing homes. the elderly generally have fewer respiratory symptoms than a younger population, and the absence of clear-cut respiratory symptoms and an afebrile status have themselves been predictors of an increased risk of death. this may be the consequence of nonrespiratory presentations being an indication of an impaired immune response, as well as a factor leading to delayed presentation to medical attention and recognition of the correct diagnosis. pleuritic chest pain is also commonly seen in patients with cap, but its absence has been identified as a poor prognostic finding. in the elderly patient, pneumonia can have a nonrespiratory presentation with symptoms of confusion, falling, failure to thrive, altered functional capacity, or deterioration in a pre-existing medical illness, such as congestive heart failure. in general, overall symptoms are less prominent in those above age 65 than in those who are younger. patients with advanced age generally also have a longer duration of symptoms such as cough, sputum production, dyspnea, fatigue, anorexia, myalgia, and abdominal pain than younger patients. studies have found no association between the type of etiologic microorganisms and the clinical presentation of cap, except for pleuritic chest pain, which is likely to be more common in pneumonia caused by bacterial pathogens such as s. pneumoniae than in nonbacterial pneumonia. delirium or acute confusion can be more frequent in the elderly patients with pneumonia than in age-matched controls who do not have pneumonia. very few elderly patients with pneumonia are considered well nourished, with kwashiorkor-like malnutrition being the predominant type of nutritional defect, and the one associated with delirium on initial presentation. physical findings of pneumonia include tachypnea, crackles, rhonchi, and signs of consolidation (egophony, bronchial breath sounds, dullness to percussion). patients should also be examined for signs of pleural effusion. in addition, extrapulmonary findings should be sought to rule out metastatic infection (arthritis, endocarditis, meningitis), or to add to the suspicion of an 'atypical' pathogen such as m. pneumoniae or c. pneumoniae. one of the most important evaluations in any patient suspected of having pneumonia is a measurement of respiratory rate. in the elderly, an elevation of respiratory rate may be the initial presenting sign of pneumonia, preceding other clinical findings by as much as 1-2 days. in prospective evaluations in a long-term care setting, most patients who were diagnosed with lower respiratory tract infection had a respiratory rate above the normal range of 16-25 min à 1 , and in general the elevated rate preceded other clinical findings. although this finding is certainly not specific, it appears to be a very sensitive indicator of the presence of respiratory infection. in general, tachypnea is the most common finding in elderly patients with pneumonia, being present in over 60% of all patients, and being present more often in the elderly than in younger patients with pneumonia. measurement of respiratory rate not only has diagnostic value, but also prognostic significance. in evaluating patients with cap, the finding of a respiratory rate 430 min à 1 is one of several factors associated with increased mortality. in the past, the clinical and radiographic features of cap have been characterized as fitting into a pattern of either 'typical' or 'atypical' symptoms, and the pattern was used to predict a specific etiologic agent. recent studies have shown that this approach is not highly accurate, and there is only a weak relationship between clinical features and the etiologic pathogen, primarily because host as well as pathogen factors play a role in defining patient symptoms. the typical pneumonia syndrome is characterized by sudden onset of high fever, shaking chills, pleuritic chest pain, lobar consolidation, a toxic appearing patient, with the production of purulent sputum. although this pattern has been attributed to pneumococcus and other bacterial pathogens, these organisms do not always lead to such classic symptoms, especially in the elderly, as discussed above. the atypical pneumonia syndrome, which is characterized by a subacute illness, nonproductive cough, headache, diarrhea, or other systemic complaints, is usually the result of infection with m. pneumoniae, c. pneumoniae, legionella sp., or viruses. however, patients with impaired immune responses (especially the elderly with chronic illness) may present in this fashion, even with bacterial pneumonia. clinical features have been shown to be only about 40% accurate in differentiating pneumococcus, m. pneumoniae, and other pathogens from one another. in addition, careful comparisons of patients with s. pneumoniae, h. influenzae, l. pneumophila, and c. pneumoniae have shown no significant differences in their clinical presentations. the limitations of clinical features in defining the microbial etiology also apply to evaluations of radiographic pattern. one of the most important patient assessments is to define severity of illness, which can be used to guide decisions about whether to hospitalize a patient, and if so, whether to admit the patient to the icu. while a number of prediction models have been developed to predict severity of illness and to guide the admission decision, no rule is absolute and the decision to admit a patient should be based on social as well as medical considerations, and remains an 'art of medicine' determination. in general the hospital should be used to observe patients who have multiple risk factors for a poor outcome, those who have decompensation of a chronic illness, or those who need therapies not easily administered at home (oxygen, intravenous fluids, cardiac monitoring). risk factors for a poor outcome include: a respiratory rate x30 min à 1 , age x65 years, systolic blood pressure o90 mmhg, diastolic blood pressure p60 mmhg, multilobar pneumonia, confusion, blood urea nitrogen 419.6 mg dl à 1 , pao 2 o60 mmhg, paco 2 450 mmhg, respiratory or metabolic acidosis, or signs of systemic sepsis. the two best-studied and widely regarded prediction rules for pneumonia severity are the pneumonia severity index (psi) and the curb-65 rule, a modification of a prognostic model developed by the british thoracic society. the psi uses a number of demographic and historical findings, physical findings, and laboratory data to assign a score to each patient, and the score is used to categorize patients into one of five classes, each with a different risk of death. this tool has worked very well to define mortality risk, but has had variable success in predicting need for admission, is cumbersome to use, and does not discriminate very well among the most severely ill patients. the curb-65 rule is simpler, using only five assessments: % confusion, % urea47 mmol l à1 , % respiratory ratex30 min à1 , % blood pressureo90 mmhg systolic or p60 mm diastolic, and age x65 years. each of the five criteria is scored and as the score rises from 0 to 5, mortality risk rises. in recent studies, both tools have worked equally well to identify patients at low risk of dying, but the curb-65 has been more discriminating in recognizing patients who need icu care (score of at least 3) and who have the highest risk of death. a major difference between the two models is that the psi weights advanced age and chronic illness very heavily, while the curb-65 model includes age as only one of several risk factors, and comorbid illness is not measured, but instead most of the score is based on acute physiologic abnormalities. none of the prediction models includes social factors in the scoring system, and clearly these issues need to be included in patient assessment, paying attention to whether the patient has a stable home environment for outpatient care, an ability to take oral medications, the absence of acute alcohol or drug intoxication, and stability of other acute and chronic medical problems. there is no specific rule for who should be admitted to the icu, but in general the icu is used for approximately 10% of all cap patients, and this population has a mortality rate of at least 30%, compared to a mortality rate of 12% for all admitted patients, and a 1-5% mortality rate for outpatients. there is some debate about the benefit of icu care for patients with cap, but the benefit seems most certain if patients are admitted early in the course of severe illness, making assessment of mortality risk an important clinical assessment. criteria for icu admission, in addition to need for mechanical ventilation and septic shock, are the presence of at least two of the following: pao 2 /fio 2 ratio o250, multilobar infiltrates, systolic blood pressure o90 mmhg. the entry point into most treatment algorithms for cap is the presence of a new radiographic infiltrate, but not all patients with this illness will have this finding when first evaluated. even when the radiograph is negative, if the patient has appropriate symptoms and focal physical findings, pneumonia may still be present. in one study, nearly 50 patients with clinical signs and symptoms of cap were evaluated with both a chest radiograph and a high-resolution computed tomography (ct) scan of the chest and there were almost 20% of patients identified by ct scan to have pneumonia who had a negative chest radiograph. in addition, more extensive abnormalities were found on ct scan in many patients than were present on the chest radiograph. the findings of this study confirm the need to repeat the chest film after 24-48 h in certain symptomatic patients with an initially negative chest film. the reason for an initially negative chest film is not clear, but some studies have suggested that febrile and dehydrated patients can have a normal radiograph when first admitted, although the idea of hydrating a pneumonia is in the realm of 'conventional wisdom' and anecdotal reports. although a variety of radiographic patterns can be seen in pneumonia, and radiographic findings cannot generally be used to predict microbial etiology in cap, there are certain patterns that have been associated with specific pathogens. focal consolidation can be seen with infections caused by pneumococcus, k. pneumoniae (with upper lobe consolidation and the classic bulging down of the upper lobe fissure), aspiration (especially if in the lower lobes or other dependent segments), s. aureus, h. influenzae, m. pneumoniae, and c. pneumoniae. interstitial infiltrates should suggest viral pneumonia as well as infection due to m. pneumoniae, c. pneumoniae, chlamydia psittaci, and pneumocystis jerovici. lymphadenopathy with an interstitial pattern should raise concerns about anthrax, francisella tularensis, and c. psittaci, while adenopathy can be seen with focal infiltrates in tuberculosis, fungal pneumonia, and bacterial pneumonia. cavitation can be the result of an aspiration lung abscess, or infection with s. aureus, aerobic gram-negatives (including p. aeruginosa), tuberculosis, fungal infection, nocardia, and actinomycosis. pleural effusion may appear on the initial chest radiograph and if present, it is necessary to distinguish empyema from a simple parapneumonic effusion by sampling the pleural fluid. pneumococcal pneumonia is the infection most commonly complicated by effusion (36-57% of patients), but other pathogens causing effusion include h. influenzae, m. pneumoniae, legionella, and tuberculosis. once the clinical evaluation suggests the presence of pneumonia, the diagnosis should be confirmed by chest radiograph. although some patients may have clinical findings of pneumonia (focal crackles, bronchial breath sounds), and a negative chest radiograph, the need for antibiotic therapy of cap has been established in studies of patients with a radiographic infiltrate. in some populations, such as the elderly and chronically ill, the clinical diagnosis is difficult, and for these individuals, a chest radiograph is essential to define the presence of parenchymal lung infection. although a radiograph is recommended in all outpatients and inpatients, it may be impractical in some settings outside of the hospital. a chest radiograph not only confirms the presence of pneumonia, but can be used to identify complicated illness and to grade severity of disease, by noting such findings as pleural effusion and multilobar illness. as mentioned above, there is no specific radiographic pattern that can be used to define the etiologic pathogen of cap, but certain findings can be used to suggest specific organisms, such as anaerobes if a cavitary infiltrate is found, or tuberculosis if a posterior upper lobe infiltrate is present. even with extensive testing, most patients do not have a specific pathogen identified, and many who do, have this diagnosis made days or weeks later, as the results of cultures or serologic testing become available. in addition, recent studies have emphasized the mortality benefit of prompt administration of effective antibiotic therapy, with a goal of administering intravenous antibiotics within 4 h of admission to the hospital, for those with moderate to severe illness. thus, therapy should never be delayed for the purpose of diagnostic testing, and the diagnostic workup should be streamlined, with all patients receiving empiric therapy based on predicting the most likely pathogens, as soon as possible. recommended testing for outpatients is limited to a chest radiograph and pulse oximetry, if available, with sputum culture being considered in patients suspected of having an unusual or drug-resistant pathogen. for admitted patients, diagnostic testing should include a chest radiograph, assessment of oxygenation (pulse oximetry or blood gas, the latter if retention of carbon dioxide is suspected), and routine admission blood work. if the patient has a pleural effusion, this should be tapped and the fluid sent for culture and biochemical analysis. although blood cultures are positive in only 10-20% of cap patients, they can be used to define a specific diagnosis and to define the presence of drugresistant pneumococci. one concern with blood cultures is that they be limited to patients with a reasonable likelihood of being positive. if low-risk patients routinely have blood cultures, it is possible that the frequency of false positives may exceed the true positives, and lead to inaccurate and unnecessary therapy. one study of a large medicare database found that predictors of bacteremia, among admitted patients, were: absence of prior antibiotics, comorbid liver disease, systolic blood pressure o90 mmhg, fever o35 or 4401c, pulse 4125 min à 1 , blood urea nitrogen 430 mg dl à 1 , serum sodium o130, white blood cell count o5000 or 420 000. based on these findings, blood cultures will have the greatest yield of true positive results in patients who have at least one of these risk factors above, or if none, when there is also no history of receiving antibiotics prior to admission. sputum culture should be limited to patients suspected of infection with a drug-resistant or unusual pathogen. the role of gram's stain of sputum to guide initial antibiotic therapy is controversial, but this test has its greatest value in guiding the interpretation of sputum culture, and can be used to define the predominant organism present in the sample. the role of gram's stain in focusing initial antibiotic therapy is uncertain since the accuracy of the test to predict the culture recovery of an organism such as pneumococcus depends on the criteria used. investigators have shown the practical limitations of the test, because fewer than half of all patients can even produce a sputum sample, only about half of these are valid, and very few are diagnostic, and thus it is uncommon to choose an antibiotic directed to the diagnostic result. even if gram's stain findings are used to focus antibiotic therapy, this would not allow for empiric coverage of atypical pathogens which might be present with pneumococcus, as part of a mixed infection. in spite of these limitations, gram's stain can be used to broaden initial empiric therapy by enhancing the suspicion for organisms that are not covered in routine empiric therapy (such as s. aureus being suggested by the presence of clusters of gram-positive cocci, especially during a time of epidemic influenza). routine serologic testing is not recommended. however, in patients with severe illness, the diagnosis of legionella can be made by urinary antigen testing, which is the test that is most likely to be positive at the time of admission, but a test that is specific only for serogroup 1 infection. commercially available tests for pneumococcal urinary antigen have been developed, but their role in the clinical management of cap is still being defined. bronchoscopy is not indicated as a routine diagnostic test, and should be restricted to immune-compromised patients, and to selected individuals with severe forms of cap. the initial therapy for patients with cap should be focused on the provision of antibiotics and supportive care. antibiotics are given on a empiric basis, since it is virtually impossible to rely on clinical or laboratory data to provide an exact etiologic pathogen, at the time of initial diagnosis, and thus therapy must be focused on the pathogens most likely to be present for a given type of patient. supportive care includes oxygen as needed, hydration, possibly chest physiotherapy, as well as bronchodilators and expectorants. for more severely ill patients, it may be necessary to support the blood pressure with pressors, use corticosteroids for possible relative adrenal insufficiency, and provide other therapies directed at signs of sepsis (such as drotrecogin alpha in selected patients). there may also be benefit from the routine use of corticosteroids in severe pneumonia, for unclear reasons, because some studies have shown a survival benefit to this intervention. initial empiric therapy is selected by categorizing patients on the basis of place of therapy (outpatient, inpatient, icu), severity of illness and the presence or absence of cardiopulmonary disease or specific modifying factors that make certain pathogens more likely. by using these factors, a set of likely pathogens can be predicted for each type of patient (table 1) , and this information can be used to guide therapy. if a specific pathogen is subsequently identified by diagnostic testing, then therapy can be focused. in choosing empiric therapy of cap, certain principles should be followed ( table 3) . empiric therapy for outpatients with no cardiopulmonary disease or modifying factors should be with a new oral macrolide (azithromycin or clarithromycin) or a tetracycline. although erythromycin has been used for these patients, its value is limited by its lack of coverage of h. influenzae, and a higher frequency of intestinal complications (nausea, vomiting) than with the newer macrolides. therapy with an antipneumococcal quinolone (gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin) is not necessary in these outpatients, because they are not at risk for organisms such as drsp and enteric gram-negatives. however, outpatients with cardiopulmonary disease and/or modifying factors, should not receive macrolide monotherapy, but should be treated with either a selected oral beta-lactam (table 3 ) with a macrolide or with monotherapy using an oral antipneumococcal quinolone (gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin) alone. the ketolide telithromycin can also be used in this population as oral monotherapy for patients at risk for drsp, but with no risk factors for aspiration or for enteric gramnegatives. for the non-icu inpatient, therapy can be with an intravenous macrolide (azithromycin) alone, provided that the patient has no underlying cardiopulmonary disease, and no risk factors for infection with drsp, enteric gram-negatives, or anaerobes. although very few patients of this type are admitted to the hospital, macrolide monotherapy has been documented to be effective in this population. the majority of inpatients will have cardiopulmonary disease and/or modifying factors, and they can be treated with either a selected intravenous beta-lactam (table 3) combined with a macrolide, or they can receive an intravenous antipneumococcal quinolone (gatifloxacin, levofloxacin, moxifloxacin) alone. from the available data, it appears that either regimen is therapeutically equivalent, and although not proven, it may be useful to use these two types of regimens interchangeably, striving for 'antibiotic heterogeneity', selecting an agent in a different class from what the patient received in the past 3-6 months. although oral quinolones may be as effective as intravenous quinolones for admitted patients table 3 principles of antibiotic therapy administer initial antibiotic therapy within 4 h of arrival to the hospital treat all patients for pneumococcus and for the possibility of atypical pathogen coinfection: use either a macrolide alone (selected patients with no cardiopulmonary disease or modifying factors) or for those outpatients with cardiopulmonary disease or modifying factors: use monotherapy with a quinolone, a ketolide (if no risk factors for enteric gram-negatives), or the combination of a selected beta-lactam with a macrolide or ketolide or tetracycline limit macrolide monotherapy to outpatients or inpatients with no risk factors for drsp, enteric gram-negatives, or aspiration limit ketolide monotherapy to outpatients with risk factors for drsp, but no risk factors for enteric gram-negatives provide initial therapy for hospitalized patients with an intravenous agent, or if oral only, use a quinolone for patients at risk for drsp, acceptable oral beta-lactams are: cefpodoxime, cefuroxime, high-dose ampicillin (3 g day à 1 ) or amoxicillin/clavulanate (up to 4 g day à 1 ) for inpatients at risk for drsp, the selected acceptable intravenous beta-lactams include: ceftriaxone, cefotaxime, ertapenem, ampicillin/sulbactam limit antipseudomonal therapy to patients with risk factors: antipseudomonal agents include: beta-lactams such as cefepime, imipenem, meropenem, piperacillin/tazobactam; quinolones such as ciprofloxacin or high-dose levofloxacin (750 mg day à 1 ); and aminoglycosides such as amikacin, gentamicin, or tobramycin; aztreonam is a monobactam that is also active, but cannot be used alone the new antipneumococcal quinolones, in order of decreasing antipneumococcal activity are: gemifloxacin (oral only), moxifloxacin (oral and intravenous), gatifloxacin (oral and intravenous), levofloxacin (oral and intravenous) never use monotherapy for patients with severe cap. if no pseudomonal risk factors use a selected beta-lactam (above) plus a macrolide or antipneumococcal quinolone. for those with pseudomonal risk factors, use an antipseudomonal beta-lactam (above) plus either ciprofloxacin/high-dose levofloxacin or the combination of an aminoglycoside with either a macrolide or antipneumococcal quinolone (above) vancomyin and linezolid should be used rarely and only in those with severe cap and either meningitis (vancomycin) or severe necrotizing pneumonia after influenza (either agent) with moderately severe illness, most admitted patients should receive initial therapy intravenously to be sure that the medication has been absorbed. once the patient shows a good clinical response, oral therapy can be started. selected inpatients with mild to moderate disease can initially be treated with the combination of an intravenous beta-lactam and an oral macrolide, switching to exclusively oral therapy once the patient shows a good clinical response. in the icu population, all individuals should be treated for drsp and atypical pathogens, but only those with appropriate risk factors (above) should have coverage for p. aeruginosa. since the efficacy, dosing, and safety of quinolone monotherapy has not been established for icu-admitted cap patients, the therapy for such patients, in the absence of pseudomonal risk factors, should be with a selected intravenous beta-lactam, combined with either an intravenous macrolide or an intravenous quinolone. for patients with pseudomonal risk factors, therapy can be with a two-drug regimen, using an anti-pseudomonal beta-lactam (cefepime, imipenem, meropenem, piperacillin/tazobactam) plus ciprofloxacin or highdose levofloxacin, or alternatively, with a three-drug regimen, using an anti-pseudomonal beta-lactam plus an aminoglycoside plus either an intravenous nonpseudomonal quinolone or macrolide. the antipneumococcal quinolones are being widely used in both inpatients and outpatients as monotherapy because as a single drug, given once daily, it is possible to cover pneumococcus (including drsp), gram-negatives, and atypical pathogens. quinolones penetrate well into respiratory secretions, and are highly bioavailable, achieving the same serum levels with oral or intravenous therapy, thereby allowing moderately ill outpatients to be managed effectively with oral antibiotics. there are differences among the available agents in their intrinsic activity against pneumococcus, and, based on mic data, these agents can be ranked from most to least active as: gemifloxacin (available only in oral form), moxifloxacin, gatifloxacin, and levofloxacin. some data suggest a lower likelihood of both clinical failures and the induction of pneumoccal resistance to quinolones, if the more active agents are used in place of the less active agents. in addition to the general approach to therapy outlined above, there are several other therapeutic issues in the management of cap, which are highlighted in table 3 . these include the need for timely administration of initial antibiotic therapy, the limited use of therapy directed at methicillin-resistant s. aureus, the need for routine atypical pathogen coverage for all patients, and the emphasis on using highly active agents in all patients with risk factors for infection with drsp. the reason for this last recommendation is because if a patient is at risk for infection with drsp, use of a highly active agent is not only likely to minimize the risk of treatment failure, but may also rapidly and reliably eradicate pneumococcal organisms that have low levels of resistance, so that there is less selection pressure for emergence of organisms with high level of resistance. the majority of outpatients and inpatients will respond rapidly to the empiric therapy regimens suggested above, with clinical response usually occurring within 24-72 h. clinical response for inpatients is defined as improvement in symptoms of cough, sputum production, and dyspnea, along with ability to take medications by mouth, and an afebrile status for at least two occasions 8 h apart. when a patient has met these criteria for clinical response, it is appropriate to switch to an oral therapy regimen and to discharge the patient, if he is otherwise medically and socially stable. radiographic improvement lags behind clinical improvement, and in a responding patient, a chest radiograph is not necessary until 2-4 weeks after starting therapy. if the patient fails to respond to therapy in the expected time interval, then it is necessary to consider infection with a drug-resistant or unusual pathogen (tuberculosis, anthrax, c. burnetii, burkholderia pseudomallei, pasteurella multocida, endemic fungi, or hantavirus); a pneumonic complication (lung abscess, endocarditis, empyema); or a noninfectious process that mimics pneumonia (bronchiolitis obliterans with organizing pneumonia, hypersensitivity pneumonitis, pulmonary vasculitis, bronchoalveolar cell carcinoma, lymphoma, pulmonary embolus). the evaluation of the nonresponding patient should be individualized but may include ct scanning of the chest, pulmonary angiography, bronchoscopy, and occasionally open lung biopsy. prevention of cap is important for all groups of patients, but especially the elderly, who are at risk for both a higher frequency of infection and a more severe course of illness. appropriate patients should be vaccinated with both pneumococcal and influenza vaccines, and cigarette smoking should be stopped in all at-risk patients. even for the patient who is recovering from cap, immunization while in the hospital is appropriate to prevent future episodes of infection, and the evaluation of all patients for vaccination need and the provision of information about smoking cessarion are now performance standards used to evaluate the hospital care of cap patients. pneumococcal vaccine pneumococcal capsular polysaccharide vaccine can prevent pneumonia in otherwise healthy populations, as was initially demonstrated in south african gold miners and american military recruits. the benefits in those of advanced age or with underlying conditions in nonepidemic environments are less clearly defined. in immunocompetent patients over the age of 65, effectiveness has been documented to be 75%. the vaccine efficacy has ranged from 65% to 84% in patients with diabetes mellitus, coronary artery disease, congestive heart failure, chronic pulmonary disease, and anatomic asplenia. its effectiveness has not been proven in immune-deficient populations such as those with sickle cell disease, chronic renal failure, immunoglobulin deficiency, hodgkin's disease, lymphoma, leukemia, and multiple myeloma. a single revaccination is indicated in a person who is aged over 65 years who initially received the vaccine 46 years earlier and was less than 65 years old on first vaccination. if the initial vaccination was given at age 65 or older, repeat is only indicated (after 6 years), if the patient has anatomic or functional asplenia, or has one of the immune compromising conditions listed above. the available pneumococcal vaccine is widely underutilized, and the 23-valent pneumococcal vaccine carries the pneumococcal serotypes causing the majority of clinical infection seen in the us. a proteinconjugated pneumococcal vaccine has been licensed and it appears more immunogenic than the older vaccine, but it contains only seven serotypes, and is recommended for healthy children, and has not yet been shown to be effective in adults for preventing both pneumococcal bacteremia and hospitalization for pneumonia. hospital-based immunization for most admitted patients could be highly effective, since over 60% of all patients with cap have been admitted to the hospital, for some indication, in the preceding 4 years, and hospitalization could be defined as an appropriate time for vaccination. the current vaccine includes three strains: two influenza a strains (h3n2 and h1n1) and one influenza b strain. vaccination is recommended for all patients aged over 65, and to those with chronic medical illness (including nursing home residents), and to those who provide healthcare to patients at risk for complicated influenza. it is given yearly, usually between september and mid november (in the northern hemisphere). when the vaccine matches the circulating strain of influenza, it can prevent illness in 70-90% of healthy persons aged over 65. for older persons with chronic illness, the efficacy is less, but the vaccine can still attenuate the influenza infection and lead to fewer lower respiratory tract infections and the associated morbidity and mortality that follow influenza. endotoxins. leukocytes: neutrophils. pleural effusions: pleural fluid analysis, thoracentesis, biopsy, and chest tube community-acquired pneumonia due to gram-negative bacteria and pseudomonas aeruginosa: incidence, risk and prognosis microbiology of severe aspiration pneumonia in institutionalized elderly validation of predictive rules and indices of severity for community acquired pneumonia mortality from invasive pneumococcal pneumonia in the era of antibiotic resistance antimicrobial therapy of community-acquired pneumonia a prediction rule to identify low-risk patients with community-acquired pneumonia associations between initial antimicrobial therapy and medical outcomes for hospitalized elderly patients with pneumonia timing of antibiotic administration and outcomes for medicare patients hospitalized with community-acquried pneumonia empiric antibiotic therapy and mortality among medicare pneumonia inpatients in 10 western states pneumonia: still the old man's friend? multiple pathogens in adult patients admitted with community-acquired pneumonia: a one year prospective study of 346 consecutive patients defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study addition of a macrolide to a b-lactam-based empirical antibiotic regimen is associated with lower in-patient mortality for patients with bacteremic pneumococcal pneumonia testing strategies in the initial management of patients with community-acquired pneumonia predicting bacteremia in patients with community-acquired pneumonia guidelines for the management of adults with community-acquired lower respiratory tract infections: diagnosis, assessment of severity, antimicrobial therapy and prevention high resolution computed tomography for the diagnosis of community-acquired pneumonia fungal infection generally originates from an exogenous, environmental source acquired by inhalation, ingestion, or trauma. fungi are rarely associated with significant disease in the normal host, although many more cause serious disease in the immunocompromised host. opportunistic fungal infections have become increasingly common, especially in aids patients, and constitute a major cause of morbidity and mortality in this group. pathogenicity depends on the interplay between components of the host immune system and specific features of the fungal strain. considerable efforts are underway to conduct genetic characterization of fungal virulence and host susceptibility factors in disease. genome projects have been undertaken for a number of the key fungal pathogens. here we consider the etiology, pathology, clinical features, management, and molecular mycology of blastomycosis, coccidioidomycocis, histoplasmosis, paracoccidioidomycosis, aspergillosis, candidosis, cryptococcosis, and mucormycosis. fungi are a diverse group of eukaryotic organisms that have cell walls and no chlorophyll. they exist in nature as parasites or saprobes, dependent on living or dead organic matter for nutrition. about 250 000 species of fungi have been described, but less than 200 have been associated with human disease. fungal infection usually originates from an exogenous, environmental source acquired by inhalation, ingestion, or trauma. very few fungi cause significant disease in the normal host, but many more can cause disease in the immunocompromised host. thus, pathogenicity can be considered to depend on the interplay between aspects of the host immune system and specific features of the fungal strain. considerable efforts are underway to conduct genetic characterization of fungal virulence and host susceptibility factors in disease. genome projects have been undertaken for a number of key pathogens (see 'relevant websites').systemic mycoses often start in the lung, but may spread to other organs. they are usually acquired by inhaling spores of organisms growing as saprobes in the soil or decomposing organic material, or as plant pathogens. the organisms that cause systemic fungal infection in humans can be divided into two groups, true pathogens and opportunists. true pathogens, able to invade and grow in tissues of a normal host, include histoplasma capsulatum, coccidoides immitis, blastomyces dermatidis and paracoccidioides braziliensis ( table 1 ). in general, infections with true pathogenic fungi are asymptomatic or mild, of short duration, occur in regions endemic for the fungus, and follow inhalation of spores from the environment. individuals either recover from infection with protective immunity to re-infection or sometimes develop chronic granulomatous disease. in the immunocompromised host, however, true pathogenic fungi may cause significant, relapsing, life-threatening disease that is difficult to treat. opportunist fungi, such as aspergillus fumigatus, are less virulent and less well-adapted organisms that are only able to invade the tissues of a debilitated or immunocompromised host. resolution of infection does not confer protection and re-infection or reactivation may occur. many of these organisms are ubiquitous saprobes found in the soil, decomposing organic key: cord-307016-4hdsb5oq authors: allen, upton; green, michael title: prevention and treatment of infectious complications after solid organ transplantation in children date: 2010-04-30 journal: pediatric clinics of north america doi: 10.1016/j.pcl.2010.01.005 sha: doc_id: 307016 cord_uid: 4hdsb5oq effective prevention, diagnosis, and treatment of infectious diseases after transplantation are key factors contributing to the success of organ transplantation. most transplant patients experience different kinds of infections during the first year after transplantation. children are at particular risk of developing some types of infections by virtue of lack of immunity although they may be at risk for other types due the effect of immunosuppressive regimens necessary to prevent rejection. direct consequences of infections result in syndromes such as mononucleosis, pneumonia, gastroenteritis, hepatitis, among other entities. indirect consequences are mediated through cytokines, chemokines, and growth factors elaborated by the transplant recipient in response to microbial replication and invasion, which contribute to the net state of immunosuppression among other effects. this review summarizes the major infections that occur after pediatric organ transplantation, highlighting the current treatment and prevention strategies, based on the available data and/or consensus. upton allen, mbbs, msc, frcpc a,b,c, *, michael green, md, mph d,e organ transplantation is the most practical means of rehabilitating patients with a variety of forms of end organ dysfunction. this procedure is arguably the outstanding clinical biomedical accomplishment of the last 3 decades. potent immunosuppressive drugs have dramatically reduced the incidence of rejection of transplanted organs, but have also increased the susceptibility of patients to opportunistic infections. 1 thus, the success of organ transplantation is dependent in part on effective prevention, diagnosis, and treatment of infectious diseases after transplantation. to this end, emphasis is increasingly being placed on prevention. most transplant patients will have evidence of microbial invasion in the first year after transplant. the effects of this microbial invasion are diverse, resulting in direct and indirect consequences. the direct consequences result in a variety of clinical infectious disease syndromes such as mononucleosis, pneumonia, gastroenteritis, hepatitis, among other entities. the indirect consequences are mediated through cytokines, chemokines, and growth factors elaborated by the transplant recipient in response to microbial replication and invasion, which contribute to the net state of immunosuppression, the pathogenesis of acute and chronic allograft injury, and in some cases, the development of lymphoproliferative or malignant disorders. the risk of infection in the solid organ transplant patient is largely determined by the interaction of 3 factors: technical/anatomic factors that involve the transplant procedure itself, and the perioperative aspects of care such as the management of vascular access, drains, and the endotracheal tube; environmental exposures (box 1); and the patient's net state of immunosuppression (box 2). in the case of technical/anatomic mishaps, the best way to prevent infection is to correct the anatomic abnormality under coverage of appropriate antimicrobial therapy as antimicrobial treatment alone will not eliminate the risk of developing recurrent infections related to the uncorrected problem. as a consequence, the transplant recipient remains at high risk of subsequent infections with an increased risk of developing antimicrobial resistance until successful correction of the underlying abnormality. 1, 2 when one is considering therapy in the transplant patient, the concept of the therapeutic prescription package is useful. this package has 2 major components: an immunosuppressive component to prevent and treat rejection and an antimicrobial component to make it safe. thus, the nature of the antimicrobial program being administered must be closely linked to the nature and intensity of the immunosuppressive program required and the resulting net state of immunosuppression. 1, 2 there are 3 modes in which antimicrobial agents can be administered to the transplant recipient: a therapeutic mode, in which antimicrobial agents are administered in the treatment of established clinical infection; a prophylactic mode, in which antimicrobial agents are administered to an entire population before an event to prevent the occurrence of an infection important enough to justify this intervention: and a preemptive mode, in which antimicrobial agents are administered to a subpopulation noted to be at particular risk of clinically important infection based on clinical, epidemiologic, or laboratory markers. this review focus on preventive strategies (prophylactic and preemptive) and on the diagnosis and management of established infection. infection in the posttransplant period has a stereotyped temporal pattern, a timetable. although some clinical syndromes, such as pneumonia, can occur at any time point after transplant, the causes may be very different at different time points. fig. 1 delineates the timetable for the onset of infections after organ transplantation in the absence of effective preventative strategies. when preventative antimicrobial therapy fails to completely protect the patient, a common clinical effect is to extend the time period in which the infectious complication will likely appear. for example, in the case of cytomegalovirus (cmv) infection, in the absence of prophylaxis cmvinduced clinical disease is most common 1 to 3 months after transplantation. when prophylaxis is used, but fails, it is common for the disease to occur 4 to 8 months after transplantation (depending on the nature and duration of the prophylaxis and the immunosuppressive regimen). 2, 3 like all patients, the transplant recipient is at risk of acquiring infections in the health care and community settings. such infections are not necessarily transplant specific. fig. 1 is a graphical representation of the timing of infections during the posttransplant period. 2 in general, 3 time periods are recognized, each with differing forms of infection: [1] [2] [3] in the first month, there are 3 major causes of infection: (1) infection that was present in the recipient before transplant, with its effects now increased as a result of surgery, anesthesia, and immunosuppressive therapy; (2) infection conveyed with a contaminated allograft; and (3) the same bacterial and candidal infections of the wound, lungs, drainage catheters, and vascular access devices that are seen in nonimmunosuppressed patients undergoing comparable surgery. most (more than 95%) of the infections occurring in the first month after transplant fall into this last category; the main factor determining the incidence of such infections is the technical aspects of surgery as well as specific aspects of perioperative and postoperative care. this second time period is when the effect of immune suppression is most notable on the risk of infection. during this period, 2 major classes of infection predominate. the first of these is attributable to a group of viral pathogens that are associated with latent and/or chronic infections. examples include cmv, epstein-barr virus (ebv), human herpes virus 6 (hhv-6), and the hepatitis viruses (b and c); all of which may cause disease through acquisition of primary infection (typically from the donor) or secondary infection within the recipient under the pressure of immune suppression (secondary infection includes reactivation of latent pathogens and reinfection with a new strain). the second set of pathogens observed in this time period cause socalled opportunistic infections and include organisms such as listeria monocytogenes, aspergillus fumigatus, and pneumocystis jiroveci. development of infection with these opportunistic pathogens is attributable to the combination of sustained immunosuppression, which is often combined with the immunomodulating effects of viral infection creating a net state of immunosuppression great enough that these opportunistic infections can occur without an especially intensive environmental exposure. information describing infections occurring in children more than 6 months after transplant is limited because transplant recipients commonly return to their homes, which are often far from their transplant centers. accordingly, details regarding infectious complications occurring in this time period may be biased to include more significant infections resulting in hospitalization. despite this limitation, experience supports dividing individuals with infections during this last time period into 2 main categories: (1) most patients with a good result from transplantation (maintenance immunosuppression, good allograft function) are at greatest risk from typical community-acquired infections (such as influenza, parainfluenza, and respiratory syncytial virus); (2) a smaller group of patients with poorer outcomes from transplantation (excessive acute and chronic immunosuppression, poor allograft function, and, often, chronic viral infection). these patients remain at high risk for recurrent infections related to uncorrected mechanical problems as well as opportunistic infections attributable to organisms like pneumocystis jiroveci, listeria monocytogenes, cryptococcus neoformans, and nocardia asteroides. the time line of infections after transplantation outlines the wide spectrum of infections that occur after transplantation. among these infections, the major burden is represented by bacteria, candida species, cmv, ebv, adenovirus, varicella zoster virus, and community-acquired respiratory viruses. in addition, certain infections represent challenges for specific organ groups (eg, bk virus infection in renal transplant recipients and toxoplasma infection in heart/heart-lung transplant recipients). selected aspects of these infections are summarized later. as indicated earlier, bacterial infections are most commonly seen during the early posttransplant period. however, bacterial infections can occur at any time after transplantation. risk factors include the presence of indwelling catheter devices, including endotracheal tubes, foley catheters and central venous catheters. in this regard, hospital-acquired gram-negative organisms, coagulase-negative staphylococci and staphylococcus aureus are often encountered. the nature of these infections and the specific pathogens involved vary according to the organ transplanted, sites of infection, the microbiologic flora of the institution, and the pretransplant status of the patient. in general, the most common site of bacterial infection is at or near the site of transplantation. urinary tract infection, notably pyelonephritis, has been recognized as the most common infectious complication among renal transplant recipients. 4 infections after organ transplantation liver transplant recipients, the most frequent site of bacterial infection is within the intraabdominal space, often accompanied by bacteremia. 5, 6 intraabdominal and wound infections are also commonly seen in intestinal transplant recipients. bacteremia, which can be partly explained by disruption of the mucosal barrier associated with harvest injury or rejection, is commonly seen. 7, 8 infection of the lower respiratory tract (including pneumonia and lung abscess) is the most common site of infection reported in most, but not all, series of pediatric heart transplant recipients. 9-12 mediastinitis is another important infection after thoracic transplantation, particularly if reexploration of the chest is required. pathogens associated with mediastinitis include s aureus and gram-negative enteric bacilli. children undergoing lung transplantation because of cystic fibrosis experience a high rate of infectious complications as they often have preexisting colonization with resistant organisms, including pseudomonas species, burkholderia species and other bacterial pathogens. [13] [14] [15] [16] given the importance and difficulty in treating these often resistant organisms, transplant centers usually recommend a thorough microbiologic evaluation of heart-lung or lung transplant candidates before transplantation. the transplant patient is also at risk of developing infection as a result of community-acquired bacterial pathogens, the most important of which is streptococcus pneumoniae (pneumococcus). transplant recipients are known to be at increased risk of pneumococcal sepsis. 17 among these patients, heart recipients who have been transplanted at a young age seem to be at an increased risk compared with other pediatric organ recipients. 17 the frequency of fungal infections varies according to the type of organ transplanted. [18] [19] [20] for example, invasive fungal infections are uncommon after renal transplantation. for these patients, the most frequently encountered entity is candida urinary tract infection. similarly for liver, heart, and intestinal transplant recipients, the major fungal infections are also caused by candida species. for all of these patients, invasive aspergillosis and other mycoses occur uncommonly. the consequences of invasive aspergillosis and other noncandidal mycoses associated with invasive infections are frequently devastating. lung transplant recipients are unique in that they experience proportionately more infections with aspergillus species compared with other organ recipients. these infections are often seen in children undergoing transplantation as treatment of cystic fibrosis and reflect infection with aspergillus that was present in the recipient before transplantation. however, aspergillus is also frequently recovered from the lungs of transplant recipients with obliterative bronchiolitis (chronic rejection of the lung) regardless of the cause of their original lung disease leading to transplantation. 21 cmv cmv infection and disease remain important causes of mortality and morbidity among pediatric organ transplant recipients. 22 data on the precise burden in pediatric organ transplant recipients are limited, however, by wide differences in data collection and reporting. in addition, nonuniform approaches to the laboratory diagnosis and definition of cmv disease applied in retrospective studies affects the ability to interpret available data. in 5 centers in the united states, 10% to 20% of liver transplant patients experienced cmv disease within 2 years after transplantation. 23 a review of first-time pediatric lung transplant patients indicated that among at-risk subjects, the incidence of cmv viremia was 29% to 32%, whereas the incidence of cmv pneumonitis was 20% during the first year after transplantation. 24, 25 cmv disease is often associated with fever and hematologic abnormalities, including leucopenia, atypical lymphocytosis, and thrombocytopenia. visceral sites affected may include the gastrointestinal tract, lungs, and liver. central nervous system involvement, including chorioretinitis, is rare in organ transplant recipients. the diagnosis of cmv infection and disease in organ transplant recipients can be affected by the variable lack of sensitivity and/or specificity of different diagnostic tests. serology has no role in the diagnosis of active cmv disease after transplantation as it does not differentiate between prior infection and active disease. the interpretation of serologic results is further confused by the potential presence of passive antibody from blood products provided during or after the transplant procedure. in addition, the altered immune responses after transplantation might impair the patient's ability to mount predictable humoral responses. viral culture of blood for cmv has limited clinical usefulness for diagnosis of disease caused by poor sensitivity. there is no role for cmv urine culture in the diagnosis of disease caused by poor specificity. 26 a positive culture from bronchoalveolar lavage specimens may not correlate with disease. 27, 28 the presence of a positive measurement of cmv load in the peripheral blood (measured by either nucleic acid amplification techniques (nat) or pp65 antigenemia assay) in a patient with a compatible cmv clinical syndrome is strongly suggestive of cmv disease. however, the cmv load may be positive before the onset or in the absence of clinical disease and may be seen in the presence of disease from other causes. further, the cmv load in the peripheral blood may be negative in some patients with tissue invasive disease, especially cmv involving the gastrointestinal tract. given the variable usefulness of these tests, histopathologic examination of involved organs is essential to confirm the presence of cmv when the diagnosis of invasive cmv disease is being considered. intravenous ganciclovir (10 mg/kg/d, given twice daily) remains the preferred drug for the treatment of cmv disease in pediatric transplant recipients. reduction of immunosuppression is desirable unless concurrent evidence of rejection precludes this. ganciclovir therapy is sometimes accompanied by cmv hyperimmune globulin therapy in some centers. typically, a clinical response to treatments is expected in 5 to 7 days after treatment has been initiated. foscarnet and cidofovir may be considered in the setting of ganciclovir resistance. the optimal length of treatment should be determined by monitoring viral loads weekly. 22 treatment is typically continued until 2 consecutive negative samples are obtained. in cases of serious disease and in tissue invasive disease without viremia, longer treatment periods with clinical monitoring of the specific disease manifestation are recommended. data are emerging on the use of valganciclovir in the prevention and treatment of cmv infection/disease among adult transplant recipients. 29, 30 considerably less data are available for children. 31 a summary of the approach to prophylaxis is outlined in table 1 , including the roles of ganciclovir with or without immune globulin and suggestions on duration of their use, where indicated. although the most feared ebv-associated disease after transplantation is posttransplant lymphoproliferative disorder (ptld), patients may experience a broad range of clinical symptoms that do not meet the definitions of ptld. these might include the manifestations of infectious mononucleosis (fever, malaise, exudative pharyngitis, lymphadenopathy, hepatosplenomegaly, and atypical lymphocytosis), specific organ diseases such as hepatitis, pneumonitis, gastrointestinal symptoms, and hematological manifestations such as leucopenia, thrombocytopenia, hemolytic anemia, and haemophagocytosis. 32 ebv-associated leiomyosarcoma has also been described. 33 ebv disease is seen most frequently in patients experiencing primary ebv infections following transplantation. rates of ebv disease and ptld vary according to the organ transplanted with recipients of intestines and lungs being at the highest risk and those receiving liver, kidney, and heart at lower risk. as for cmv disease, serology is not useful for diagnosis in the posttransplant period. the presence of increased ebv viral load in the peripheral blood as determined by quantitative polymerase chain reaction (pcr) is widely accepted as an assay to predict or indicate the likely presence of ptld. however, these assays are limited in specificity and may remain persistently elevated in asymptomatic patients. the definitive diagnosis of ebv diseases, including ptld requires histopathologic examination of biopsy material. the use of ebv-specific assays (eg, ebv encoded rna [eber] staining) enhances the sensitivity and specificity of histologic examination in these patients. the approach to the treatment of ebv disease and ptld remains somewhat controversial. reduction of immune suppression is widely accepted as critical in the management of patients with these complications. the role of the antiviral agents acyclovir and ganciclovir are unproven, although many transplant clinicians use them in the treatment of ebv infection. 34, 35 treatment approaches are often modified from regimens used to treat cmv disease. currently, when antiviral agents are used to treat ebv, the agent of choice is ganciclovir, as in vitro it is 10 times more active against ebv compared with acyclovir. the controversy on the use of these agents for ebv/ptld arises because although these agents can suppress ebv lytic infection, infections after organ transplantation they seem to be of limited value in treatment nonlytic ebv proliferation, which is believed to be the dominant component of ebv-related ptld. increasing evidence (albeit anecdotal) supports the use of the anti-cd20 monoclonal rituximab in the treatment of ebv disease and ptld. however, the optimal timing and treatment strategy for this agent remain to be defined. additional alternative strategies such as the use of chemotherapy require collaborative input from oncologists familiar with the management of ebv-related disease in organ transplant recipients. the prevention of posttransplant ebv diseases, including ptld remains controversial. antiviral regimens have been modeled from the cmv scenario. to date, preemptive reduction in immunosuppression in the setting of increasing viral load may have the most supportive data and is increasingly being used (see table 1 ). adenovirus infection may be acquired by exogenous means or endogenously as a result of reactivation of latent infection. the clinical spectrum of infection and disease in pediatric transplant recipients is variable. 36 there are more than 51 serotypes that generally show some fidelity as this relates to the types of organs affected and the resultant syndromes. 37 among liver transplant patients, disease manifestations include self-limited fever, gastroenteritis, cystitis, hepatitis, and pneumonitis. these manifestations may occur in other transplant recipients, depending on the level of immunosuppression. adenovirus dna can be detected in the peripheral blood using qualitative or quantitative pcr techniques. in the appropriate clinical setting, the presence of adenovirus dna in the blood provides presumptive evidence of infection, with examination of tissue by histopathology providing more definitive evidence of infection. the management of adenovirus infection poses challenges because of limited effective treatment options. cidofovir is currently accepted as the drug of choice. however, this conclusion is primarily based on a retrospective review of historical experience and the agent is not approved for this indication by the us food and drug administration or similar agencies. nonetheless, ongoing experience continues to support a role for the treatment of adenoviral infections with this agent. before the advent of cidofovir, intravenous ribavirin was used with anecdotal reports of successes and failures. 38 although the major burden of bk virus infection is among adult renal transplant patients, the role of this virus in pediatric organ transplantation is becoming more clearly defined. most infections are as a result of reactivation in adults. primary infection may occur, notably among pediatric transplant recipients. the major clinical manifestation in the renal transplant recipient is tubulointerstitial nephritis. renal biopsy is required for definitive diagnosis. noninvasive testing modalities include screening of blood and urine for bk dna using pcr. 39 there is no firm consensus on the preferred approach to the management of bk nephropathy. early detection is a desired goal. to that end, quantitative pcr monitoring for bk dna is performed in some centers. this often provides opportunities to modulate immunosuppression. in situations where antiviral therapy is used, the agent most often used is cidofovir, for which there are reports of success. 40 however, at present no consensus exists supporting the therapeutic efficacy of this agent. varicella zoster virus (vzv) is a major threat to pediatric transplant patients and many individuals enter transplantation without immunity to this virus. 41 immunosuppressed individuals are at risk of severe outcomes from vzv infection. visceral involvement may accompany severe infection and clinicians should be reminded that disseminated disease can rarely occur in the absence of typical cutaneous vesicles. 42 pretransplant vaccination has been shown to provide sustained humoral immunity for at least 2 years after transplantation. 43 it is strongly recommended that transplant candidates be vaccinated before transplantation. given that this is a live vaccine, the minimum interval between vaccination and transplantation is recommended to be 4 to 6 weeks. although some centers have selectively considered the use of vzv vaccine in susceptible children after transplantation, this approach cannot be recommended at this time because of the lack of safety data, given the known risk of live vaccines in immunosuppressed individuals. families of transplant patients should be educated to be alert to potential exposures in settings such as schools and should report them promptly to health care providers to allow for postexposure prophylaxis. varicella-susceptible transplant recipients should receive varicella zoster immune globulin within 96 hours after a varicella exposure. 44 if this window has passed or if varicella zoster immune globulin is not available, there is the option for the use of postexposure chemoprophylaxis with acyclovir (80 mg/kg/d, given 4 times daily for 7 days; maximum dose 800 mg, 4 times daily) starting at day 7 to 10 after exposure. 44 in the absence of profound immunosuppression, no prophylaxis is usually necessary for exposed organ recipients who are immune to vzv as a result of prior infection or vaccination before transplantation. treatment of the transplant patient with vzv infection is usually initiated with intravenous acyclovir until there is evidence of clinical improvement (fever abates, no new lesions, lesions starting to crust, no visceral disease). outpatient treatment with oral acyclovir or valacyclovir has been used in children with mild infection, low levels of immunosuppression, and when there are no concerns regarding the adequacy of follow-up. famciclovir and valacyclovir are approved for use in adults. famciclovir is the prodrug of penciclovir, which has an extended half-life in infected cells. valayclovir is the prodrug of acyclovir and produces fourfold greater serum levels than those produced by acyclovir. pediatric formulations are current not available. most children who have undergone organ transplantation experience communityacquired viral infections and have no significant problems. however, it is well recognized that children who are significantly immunocompromised can have severe disease caused by these viruses, including respiratory syncytial virus infection, parainfluenza, and influenza viruses. 45, 46 for pediatric transplant recipients, the likelihood of more severe outcomes is greater during the early months after transplantation or during periods of peak immunosuppression. in 2009, the advent of a pandemic strain of influenza a (pandemic h1n1 2009) has been cause for concern. 47, 48 in general, the principles that govern the prevention and treatment of pandemic h1n1 in pediatric transplant patients are similar to those for seasonal influenza. transplant patients are among those who are known to be at increased risk of severe outcomes from pandemic h1n1. they are candidates for treatment with oseltamivir or zanamivir (where appropriate) if they have acute respiratory illness that is suspected or confirmed to be caused by h1n1. 49 like other immunocompromised patients, they are at an increased risk of having prolonged shedding of virus and the harboring of drug-resistant strains of influenza a, including pandemic h1n1. pediatric transplant patients are candidates for vaccination against this virus (as they are for seasonal influenza a) if they are greater than 6 months of age. most infections after organ transplantation experts currently delay vaccination until after the first months following organ transplantation. 49 pneumocystis pneumonia (pcp) is a recognized threat in the posttransplant period. 50, 51 the risk is greatest during the first 6 to 12 months after transplantation, with the time of onset being usually after the first month. trimethoprim-sulfamethoxazole remains the prophylactic agent of choice. 52 this agent is also preferred for initiation of therapy in individuals who develop pcp. although the optimal duration of pcp prophylaxis remains unclear, most experts provide pcp prophylaxis for a minimum of 6 to 12 months, with some recommending indefinite use, especially for solid organ transplant recipients requiring more prolonged periods of higher levels of immunosuppression. intravenous pentamidine is an alternative for treatment of pcp for patients who are intolerant of trimethoprim-sulfamethoxazole or whose disease has not responded to this agent after 5 to 7 days. 52 however, pentamidine is associated with a relatively high incidence of adverse events, including pancreatitis, renal dysfunction, hypoglycemia, and hyperglycemia. atovaquone may be used to treat milder forms of pcp among adults; however, pediatric data are limited. alternatives to trimethoprim-sulfamethoxazole for prophylaxis include oral atovaquone or dapsone. aerosolized pentamidine is recommended if children cannot tolerate these oral agents. another alternative is intravenous pentamidine, albeit at the risk of greater toxicity. 52 toxoplasma gondii infection is of greatest concern among heart transplant patients, but infection can occur in other categories of transplant recipients, including kidney and liver recipients. 53, 54 toxoplasma organisms can remain encysted within muscle tissue, such as cardiac muscle. thus, infection is acquired as a result of the reactivation of cysts that remain dormant in the donor hearts of toxoplasma seronegative children. clinical manifestations can occur as early as 2 weeks after transplantation. manifestations include pneumonia, fever syndrome, myocarditis, chorioretinitis, and central nervous system disease. current prophylaxis includes pyrimethamine/sulfadiazine for d1rã� patients. trimethoprim-sulfamethoxazole is typically used in r1 patients. however, some experts also recommend trimethoprim-sulfamethoxazole for d1rã� patients. the duration of prophylaxis is usually 6 months. infection with this parasitic worm is of relevance to individuals who previously acquired infection following a period of residence in endemic regions. 55, 56 donorassociated transmission of stronygloides has also occurred. asymptomatic immunocompromised persons, including transplant recipients are at risk of strongyloides hyperinfection, which results from dissemination of larvae via the systemic circulation, resulting in abdominal pain, diffuse pulmonary infiltrates, and septicemia or meningitis from enteric gram-negative bacilli. serologic screening is recommended for individuals from endemic regions ( table 2 ). ivermectin treatment is indicated for screenpositive individuals. tuberculosis (tb) is always a concern for immunocompromised hosts. 57-60 incidence rates are low in most transplant centers in the developed world, but outcomes of tb can be devastating in organ transplant recipients. before transplantation a careful history for tb exposure or infection, mantoux test screening, and a chest radiograph can assists in establishing the diagnosis of latent tuberculosis infection. the interferon-gamma release assays are currently being evaluated to define their role in settings where the tb skin test has poor utility. 61, 62 the use of antituberculous agents in transplant patients poses challenges because of the interaction between isoniazid and rifampin with immunosuppressive medications. however, this should not be seen as a contraindication to the use of antituberculous agents, which have to be used when warranted by the clinical situation. the pretransplant phase is arguably the most important phase of transplantation. a detailed history and physical examination are necessary to identify conditions that influence the risk or management of infections after transplantation. this assessment allows for the identification of preexisting conditions that require treatment or prophylaxis in the period before or after transplantation. table 2 summarizes screening tests that should be performed in the pretransplant period. immunizations represent an important strategy for preventing infections in the transplant patient. [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] wherever possible, vaccines should be administered in the pretransplant period to improve the chances of optimal immunologic take. a guideline on vaccinations for the transplant candidate/patient has recently been published. 73 in some situations, accelerated vaccination schedules may be used for selected vaccines. given differences in childhood vaccination schedules in different jurisdictions, clinicians should acquaint themselves with the appropriate schedules and the circumstances under which accelerated schedules could be used. when using vaccines after transplantation, one needs to be concerned about safety as well as efficacy. in general, all live virus vaccines should be avoided in the transplant recipient. the oral polio, yellow fever, and oral typhoid vaccines are live and are contraindicated in immunosuppressed patients. the live attenuated intranasal influenza vaccine is also contraindicated. measles, mumps, and rubella vaccines are somewhat contraindicated and their use should be limited to outbreak scenarios. the varicella vaccine is also somewhat contraindicated and is not approved for use in transplant patents. although limited published data support the potential use of this vaccine in transplant recipients, 72 most experts continue to advise against this practice. in the cases of the nonlive vaccines, the major concern is not safety, but efficacy. thus, in general, it is advisable to give nonlive vaccines at times when the level of immunosuppression would allow for immunogenicity. table 3 summarizes the vaccines that are indicated and contraindicated in transplant recipients. given the relative burden and importance of invasive pneumococcal disease in pediatric transplant recipients, the importance of pneumococcal vaccination should not be underestimated. 17, 63, 69 donor organ screening the organ donor is a frequent source of exposure to pathogens in the organ transplant recipient. accordingly, screening of the donor organ is a crucial aspect of the preventive strategies aimed at minimizing adverse outcomes from infections in the posttransplant period. despite a long-standing recognition of the importance of donor-derived infections, increased concern about this problem has emerged because of recent donor-related transmission of human immunodeficiency virus (hiv). this case, as well as concerns about the lack of sensitivity of serologic testing and the relatively long time period until seroconversion against hiv, hepatitis b virus (hbv), and hepatitis c virus (hcv), have led to interest in the use of nat-based testing for the pathogens hiv, hcv, and hbv. although arguments exist for and against the use of nat testing, a final international consensus addressing if and when to use these tests is only beginning to emerge. 74 decisions relating to the use of such tests must consider not only the reliability of this technology but also the feasibility of universal implementation of these testing procedures for all procurement organizations. recent cases of donor-associated transmission of lymphocytic choriomeningitis virus and west nile virus have also raised questions on whether the panel of routine tests performed on potential donors should be expanded to include these and other potential donorderived pathogens. to date, a consensus has not been reached on whether or not screening against these pathogens should be routinely included in donor testing panels. it is to be hoped that the implementation of working groups and committees focusing on the problem of donor-derived infections in north america and europe will lead to improved data to better inform subsequent recommendations regarding donor testing. current requirements for screening of nonliving donors are shown in table 4 . at present, no specific requirements have been implemented for screening allen & green of live donors. in general, testing strategies that are in place for deceased donors are applied to the use of these organs. the importance of documenting the presence of potential donor-transmissible pathogens is imperative not only to inform decision making regarding the use of potential donor organs but also because results of donor testing can inform specific preventative strategies even when donor-associated exposure to pathogens is unavoidable. various prophylaxis regimens are used in the posttransplant period. although there are common basic principles, the specific regimens vary across centers and by the type of organ transplanted. for most patients, the major targets of prophylaxis are infections after organ transplantation bacterial pathogens, herpes group viruses, and fungal pathogens, including pneumocystis. perioperative antibiotics are typically used for 48 to 72 hours to provide prophylaxis against surgical contamination. the burden of cmv infection in transplant patients is such that it represents the major focus of prevention in the posttransplant period, when intravenous ganciclovir is usually used with or without cmv hyperimmune globulin in selected patient groups. table 1 summarizes various pathogens and the regimens that are often used for prevention of infection in the posttransplant period. in the evaluation of the febrile transplant patient, clinicians should consider if the child's fever is related to common childhood infections or infections that are unique to the immunosuppressed transplant recipient. to this end, the timing of infections after transplantation (see fig. 1 ) provides guidance regarding the most likely pathogens. for example, as discussed earlier, the most likely causes of infection within the first month after transplantation are often bacterial or candidal and are largely similar to what is seen in nonimmunosuppressed patients who have undergone comparable surgery. the nature of the evaluation will depend on the clinical status of the patient and whether or not a source of infection has been identified. examination abnormal, focus of infection defined. admission to hospital may be indicated depending on the clinical status of the patient and the site of the infection. the diagnostic evaluation varies, but should include a minimum of a complete blood count and differential, blood, and urine cultures. additional investigations depend on the clinical assessment and the timing of presentation after transplantation. examination normal, no focus of infection defined. patients who are clinically unwell typically require admission for evaluation and treatment. the diagnostic evaluation should consider the likely differential diagnoses. consultation with infectious diseases is recommended. patients who are well may not necessarily require admission. however, this depends on several factors, including the adequacy of follow-up, the degree of immune suppression and the suspected diagnoses. the diagnostic evaluation should include a minimum of a complete blood count and differential, blood, and urine cultures. in all of these situations, clinicians need to be aware of the spectrum of viral infections that are associated with febrile syndromes without necessarily having a readily apparent organ focus of infection (eg, cmv virus). infection in organ-transplant recipients infection in the renal transplant patient the therapeutic prescription for the organ transplant recipient: the linkage of immunosuppression and antimicrobial strategies urinary tract infection in pediatric renal transplantation pediatric liver transplantation: patient evaluation and selection, infectious complications, and life-style after transplantation infectious complications in liver transplantation bacteremia after intestinal transplantation in children correlates temporally with rejection or gastrointestinal lymphoproliferative disease unique aspects of the infectious complications of intestinal transplantation infections in pediatric orthotopic heart transplant recipients pediatric heart transplantation at stanford: results of a 15-year experience heart transplantation during the first 12 years of life. loma linda university pediatric heart transplant group rejection and infection after pediatric cardiac transplantation pseudomonas cepacia empyema necessitatis after lung transplantation in two patients with cystic fibrosis burkholderia cepacia complex genomovars and pulmonary transplantation outcomes in patients with cystic fibrosis lung transplantation for cystic fibrosis, patients with burkholderia cepacia complex. survival linked to genomovar type update on the burkholderia cepacia complex invasive pneumococcal disease in pediatric organ transplant recipients: a high-risk population infections after organ transplantation risk factors for fungal infection in paediatric liver transplant recipients trends in invasive disease due to candida species following heart and lung transplantation australian candidemia study. candidemia following solid organ transplantation in the era of 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transplant patients by pcr compared with traditional laboratory criteria long-term outcomes of cmv disease treatment with valganciclovir versus iv ganciclovir in solid organ transplant recipients a multicenter study of valganciclovir prophylaxis up to day 120 in cmv-seropositive lung transplant recipients valganciclovir dosing according to body surface area and renal function in pediatric solid organ transplant recipients epstein-barr virus infections epstein-barr virus-associated multifocal leiomyosarcomas arising in a cardiac transplant recipient: autopsy case report and review of the literature management of epstein-barr virus-induced posttransplant lymphoproliferative disease in recipients of solid organ transplantation new developments in the diagnosis and management of posttransplantation lympholiferative disorders in solid organ transplant recipients adenoviral infections in pediatric transplant recipients: a hospital-based study adenovirus infections adenoviral infections and a prospective trial of cidofovir in pediatric hematopoietic stem cell transplantation sustained bk viruria as an early marker for the development of bkv-associated nephropathy: analysis of 4128 urine and serum samples quantitative viral load monitoring and cidofovir therapy for the management of bk virus-associated nephropathy in children and adults varicella zoster infection in solid organ transplant recipients: a hospital-based retrospective study varicella zoster virus sustainability of humoral responses to varicella vaccine in pediatric transplant recipients following a pre-transplantation immunization strategy varicella-zoster infections respiratory syncytial virus infections in pediatric liver transplant recipients parainfluenza and influenza virus infections in pediatric organ transplant recipients the first pandemic of the 21st century: a review of the 2009 pandemic variant influenza a (h1n1) virus outbreak of swine-origin influenza a (h1n1) virus infection -mexico guidance on novel influenza a/h1n1 in solid organ transplant recipients pneumocystis carinii pneumonia following heart transplantation unexpectedly high incidence of pneumocystis carinii infection after lung-heart transplantation. implications for lung defense and allograft survival pneumocystis jirovecii infections primary and reactivated toxoplasma infection in patients with cardiac transplants. clinical spectrum and problems in diagnosis in a defined population toxoplasmosis in pediatric recipients of heart transplants acute respiratory failure due to disseminated strongyloidiasis in a renal transplant recipient tacrolimus allows autoinfective development of the parasitic nematode strongyloides stercoralis mycobacterial infection after liver transplantation: a report of three cases and review of the literature mycobacterium tuberculosis after liver transplantation: management and guidelines for prevention mycobacterium tuberculosis infection in solid-organ transplant recipients: impact and implications for management american society of transplantation infectious diseases community of practice. mycobacterium tuberculosis interferon-gamma release assays improve the diagnosis of tuberculosis and nontuberculous mycobacterial disease in children in a country with a low incidence of tuberculosis performance of an interferon-gamma release assay for diagnosing latent tuberculosis infection in children seven-valent pneumococcal conjugate vaccine in pediatric solid organ transplant recipients: a prospective study of safety and immunogenicity a prospective, comparative study of the immune response to inactivated influenza vaccine in pediatric liver transplant recipients and their healthy siblings double-dose accelerated hepatitis b vaccine in patients with end-stage liver disease failure of hepatitis b immunization in liver transplant recipients: results of a prospective trial immunogenicity and safety of hepatitis a vaccine in liver and renal transplant recipients immunity to poliomyelitis, diphtheria and tetanus in pediatric patients before and after renal or liver transplantation safety and immunogenicity of the american academy of pediatrics-recommended sequential pneumococcal conjugate and polysaccharide vaccine schedule in pediatric solid organ transplant recipients report from the advisory committee on immunization practices (acip): decision not to recommend routine vaccination of all children aged 2-10 years with quadrivalent meningococcal conjugate vaccine (mcv4) pretransplant varicella vaccination is cost-effective in pediatric renal transplantation ast infectious diseases community of practice. guidelines for vaccination of solid organ transplant candidates and recipients safety and immunogenicity of varicella-zoster virus vaccine in pediatric liver and intestine transplant recipients special report. nucleic acid testing (nat) of organ donors: is the ''best'' test the right test? key: cord-350618-rtilfnzi authors: lambelet, valentine; vouga, manon; pomar, léo; favre, guillaume; gerbier, eva; panchaud, alice; baud, david title: sars‐cov‐2 in the context of past coronaviruses epidemics: consideration for prenatal care date: 2020-05-26 journal: prenat diagn doi: 10.1002/pd.5759 sha: doc_id: 350618 cord_uid: rtilfnzi since december 2019, the novel sars‐cov‐2 outbreak has resulted in millions of cases and more than 200,000 deaths worldwide. the clinical course among non‐pregnant women has been described but data about potential risks for women and their fetus remain scarce. the sars and mers epidemics were responsible for miscarriages, adverse fetal and neonatal outcomes and maternal deaths. for covid‐19 infection, only 9 cases of maternal death have been reported as of april 22, 2020 and pregnant women seem to develop the same clinical presentation as the general population. however, severe maternal cases, as well as prematurity, fetal distress and stillbirth among newborns have been reported. the sars‐cov‐2 pandemic greatly impacts prenatal management and surveillance and raise the need for clear unanimous guidelines. in this narrative review, we describe the current knowledge about coronaviruses (sars, mers and sars‐cov‐2) risks and consequences on pregnancies and we summarize available current candidate therapeutic options for pregnant women. finally, we compare current guidance proposed by rcog, acog and the who to give an overview of prenatal management which should be utilized until future data appear. this article is protected by copyright. all rights reserved. severe cases of covid-19 infection in pregnant women are not frequent as with the previous coronavirus infections described above. nevertheless, nine (2.7%) maternal deaths have been reported among eleven iranian patients with 3 rd trimester infections (44-46). all women presented with typical symptoms, including dyspnea. they were previously healthy except two patients known for hypothyroidism and one patient with suspected gestational diabetes. maternal age was between 22 and 49 y.o. and two women had dichorionic/diamniotic twin gestations. all women were admitted to the icu, intubated and ventilated and died from cardiopulmonary collapse or multiple organ failure (mof). one had septic shock and disseminated intravascular coagulation (dic) before progressing to heart failure. intrauter ine fetal death (iufd) were described among four patients (4/9) with gestational ages between 24 and 30 wg. cs were performed in other cases (5/9) with gestational age between 30 and 38 wg. cohort studies have reported a rate of severe disease requiring icu admission of 6.9-8% (n=9/118; n=8/116), including 3 requiring mechanical ventilation among chinese patients. in the italian cohort, a total of 17% of pregnant women (n=7/42) required either oxygen supplementation through continuous positive airway pressure (cpap) or icu admission, while in the american cohort, 4 (9.3%) presented with severe disease and 2 (4.7%) required icu admission without mechanical ventilation. severe complications in pregnant women are similar to what has been described in the general population and include multiple organ failure, respiratory failure requiring mechanica l ventilation, and even extracorporeal membrane oxygenation, as described in a patient at 35 wg (47). in the latter, the patient required an emergency cesarean section for maternal resuscitation and the newborn unfortunately died due to an intrauterine asphyxia. the mother had multiple organ failure, needed mechanical ventilation before extracorporeal membrane oxygenation for a total of 7 days. she was discharged from hospital 6 weeks later. two cases of cardiomyopathy related to covid19 were reported by juusela and al. (48) . the first pregnant woman was 45 y.o., had a bmi of 44.6 m 2 /kg and was diagnosed with dietcontrolled gestational diabetes. she delivered via cesarean at 39 wg for severe preeclamps ia and tested positive to sars-cov-2 on postpartum day 1 with evidence of fever, tachypnea and suspicious chest imaging. she was then diagnosed with acute heart failure after an echocardiogram was performed, showing a moderately reduced left ventricular ejection fraction (lvef) of 40% with global hypokinesis. on day 5 postpartum, the mother required this article is protected by copyright. all rights reserved. mechanical ventilation and was still intubated at the time of publication. the second patient was a 26 y.o. women with no relevant medical history. she was admitted for respiratory symptoms necessitating nasal oxygen support. due to the previous experience, an echocardiogram was completed and showed a moderately reduced lvef of 40-45% with global hypokinesis. she rapidly developed severe features leading to a cesarean section at 34 wg. at the time of publication, she was postpartum day 1 and did not require oxygen support. though current data suggest that most pregnant women with covid-19 will have an uncomplicated clinical course, severe complications must be anticipated. nevertheless, the observed rates appear similar to those for non-pregnant patients between 20-40 years old. in a recent analysis based on a chinese cohort, the actual rate of severe disease was 173/1170 (9.8%) among 20-39 y.o. patients; after adjusting for demographic factors, the expected rate of severe disease was 0.6-8.6% (21) . breslin et al. also reported a similar rate of complications in pregnant women compared to nonpregnant adults (11) : 86 vs 80 % with mild clinical symptoms, 9.3 vs 15 % with severe symptoms and 4.7 vs 5% requiring icu admission respectively. we should point out, however, that complications in the general population mainly impacted the elderly and patients with comorbidities. when comparing pregnant woman to their typical age group, they qualify as a high-risk group for adverse maternal outcomes (49). therefore, although the majority of infected pregnant women seems to demonstrate a mild clinical course, pregnancies should be approached with caution considering the potential critica l complications reported in several cases published so far report. more exhaustive data, however, this article is protected by copyright. all rights reserved. are needed to understand the additional risk pregnancy may pose to women with a covid-19 infection. sars-cov-1 and mers-cov infections during pregnancy were associated with adverse fetal and neonatal outcomes. a report on twelve pregnant women suffering from sars-cov-1 (2002-03 pandemic) was published (50) and the rate of adverse fetal / neonatal outcomes was 66% (8/12) in this series. four of the seven patients (57%) infected during the first trimester experienced miscarriages. two others decided to terminate their pregnancy after recovering from sars, and the last had an uncomplicated pregnancy. among the five patients infected during the second or third trimester, four (4/5, 80%) had a preterm delivery, including one for fetal distress (1/5, 20%) . two neonates exhibited respiratory distress syndrome and other complicatio ns related to prematurity (necrotizing enterocolitis). all placentas of these patients (5/5, 100%) weighed below the 5 th percentile, of which 2 had abnormal anatomo-pathology results (thrombotic vasculopathy with avascular fibrotic villi and / or placental infarct) (51). when the infection occurred during the week before birth, no fetal growth restriction was noted (0/2). when the infection occurred one month or more before birth, two fetuses (2/3, 33%) had fetal growth restriction (fgr) with oligohydramnios, related to the abnormal placentas presented above. another chinese series (52) reported fetal demise in one of five (20%) fetuses exposed to sars-cov-1 during the second or third trimester of pregnancy. this article is protected by copyright. all rights reserved. eleven fetuses / neonates from mothers infected with mers-cov have been described (53)(54)(55). among them, 3 (3/11, 27%) had fetal or neonatal demise: two intrauterine fetal deaths at 20 and 34 weeks, and one neonatal demise at 24 weeks due to extreme prematurity (55)(56). abruption was identified on placental examination from these fetuses, and from another liveborn neonate who presented with fetal distress at 37 weeks (57). with regards to sars-cov-2 infection during pregnancy, several case-series and case reports show that similar adverse fetal and neonatal outcomes could occur. overall, we included 142 cases with fetal and/or neonatal outcomes available at the time of this review. among them, 40 (28%) were born prematurely (<37w) and 20 (14%) had adverse outcomes (fgr, fetal or neonatal demise, severe symptoms at birth). congenital or perinatal transmiss io n was suspected in 6 of 115 (5%) newborns tested. details of all cases available are presented in table 1 . in a case-control study (58), among 17 fetuses from sars-cov-2 infected mothers, 3 exhibited fgr (3/17, 18%), 2 had fetal distress (2/17, 12%), and four were born prematurely (4/17, 24%) due to prom or placental bleeding. the rates of low birth weight and premature birth were significantly higher when compared to the control groups. one of these fetuses also exhibited sinus tachycardia that persisted after birth. zhu and colleagues (59) described the outcomes of 10 neonates from sars-cov-2 infected mothers. two of them were small for gestational age (2/10, 20%), and 6 had a pediatric critical illness score (pcis) below 90 with shortness of breath (6/10, 60%), fever (2/10, 20%), thrombocytopenia accompanied by abnormal liver this article is protected by copyright. all rights reserved. function (2/10, 20%), tachycardia (1/10, 10%), vomiting (1/10, 10%), and pneumothorax (1/10, 10%). neonatal radiography showed abnormalities in 7 of them (7/10, 70%): 4 had signs of infection, 2 respiratory distress syndrome and 1 pneumothorax. among these neonates, two (2/10, 20%) had disseminated intravascular coagulation and one (1/10, 10%) refractory shock with multiple organ failure leading to death at day 8 of life. liu y. (60) presented the outcomes of 10 other newborns exposed during pregnancy: none which were positive for sars-cov-2 at birth, 6 (6/10, 60%) were premature (for fetal distress in 3 cases, 3/10, 30%), and one was stillborn (1/10, 10%). chen and colleagues (61) described a series of 9 newborns from infected mothers during the third trimester. two (2/9, 22%) had a low birthweight and four (4/9, 44%) were premature (for fetal distress in 2 cases), none experienced a severe adverse outcome. yu and colleagues also reported a series of 7 newborns from infected mothers during the third trimester, without adverse outcomes. one of these neonates had a positive sars-cov-2 pcr 36 hours after birth, leading to the suspicion of a perinatal transmission. liu d. and colleague s (62) described briefly the outcomes of 13 newborns from infected mothers. induced prematurity was noted in 54% (7/13), but none had neonatal complications. in the new-york series (42), which presented the outcomes of 18 infants from infected mothers, all but one had negative neonatal testing for sars-cov-2. one infant had an 'indeterminate' test result, which was clinically managed as a 'presumptive negative' diagnosis, as this result may reflect low level detection. in this series, 3 (3/18, 17%) instances of fetal distresses were noticed, one infant (1/18, 6%) was premature and one (1/18, 6%) presented with rds with a concern for sepsis. zeng and colleagues (63) reported the largest series to date, with 33 newborns included. a perinatal infection was suspected in three of them (3/33, 9%), with a positive pcr at day 2 and 4 of life. infected newborns presented with higher rates of fgr, prematurity and complicatio ns at birth (fever, pneumonia, rds, shortness of breath) than non-infected newborns: 33% vs 7%, 33% vs 10 %, 100% vs 10%, respectively. wang (64) reported one case with a positive pcr in both the mother and her newborn (whereas placental and umbilical blood samples were negative). this newborn had lymphocytopenia, abnormal liver function and elevated creatine kinase, although was clinically stable. congenital or perinatal transmission was also suspected in three other cases (65) (66) . sars-cov-2 igm antibodies were elevated in these three newborns, although their nasopharyngeal pcrs were negative. in an editorial related to these cases, kimberlin (67) pointed out that false-positive results due to cross-reactivity of igm could occur and perinatal testing remains a challenge. interestingly, zamaniyan and al.(68) , described a case of positive sars-cov-2 amniot ic sample from a newborn, raising concern about potential vertical transmission in mothers with serious illness. indeed, possible vertical transmission has been questioned by other authors (65, 66) and remain unclear. a case of second trimester miscarriage was reported by baud and al.(69) in a patient at 19 wg positive for sars-cov-2. virological findings confirmed the presence of the virus in the placenta, but not in fetal tissue or maternal samples, suggesting a potential impact of sars-cov-2 early in the pregnancy. in other cov infections during the second or third trimester of pregnancy, it is interesting to note that placental changes seem to precede fgr. severe maternal respiratory illness related to cov infection may lead to a circulatory insufficiency in both the placenta and the fetus. thus, this article is protected by copyright. all rights reserved. a maternal covid infection could affect the oxygen supply, leading to placental insufficienc y, iugr, fetal distress and / or fetal demise. a direct impact of the virus itself, by increasing fibrin deposits or thrombo-embolic events in the placenta, cannot be excluded and warrants further investigation. similarly, maternal sars related to cov-2 infection during the first trimester of pregnancy could disrupt the uterine placental flow, leading to miscarriage. although the risk of miscarria ge has been described with sars-cov-1 infection, no cases have yet been reported with sars-cov-2 infection. currently, no curative agent has been found for covid-19. studies conducted so far (includ ing randomized controlled trials = rcts) have been plagued by poor methods and reporting, such as exclusion of patients with worse outcome from the treated group, different endpoints between protocols and published reports, premature stopping of rct (leading to lack of statistical power), use of endpoints of no clinical value (such as viral load), degrees of severity of enrolled patients (so that the benefit of a treatment or lack thereof in a cohort of patients may not generalizable to patients with different degrees of severity, lack of optimization of treatment dose or duration of treatment, to name but a few). this article is protected by copyright. all rights reserved. several drugs are currently being evaluated as potential treatment for sars-cov-2 includ i ng hydroxychloroquine, lopinavir-ritonavir combination, remdesivir, oseltamivir, interferon alpha, darunavir, baricitinib, tocilizumab and immunoglobulin therapy. hydroxychloroquine use in pregnant women has raised concerns in the past especially for an increased risk of cardiac malformation (70) and its retinal and ototoxicity (71)(72), related to the use of chloroquine and not hydroxychloroquine, findings which were not confirmed in more recent case series (73)(74)(75) (76) . in the most recent systematic review and meta-analys is conducted in 2016, kaplan yc et al (77) , found no increase "in the rates of major congenita l craniofacial and cardiovascular, nervous system and genitourinary malformations in the infants." however, there was a significant increase in the spontaneous abortion rate, which could be associated with the underlying disease activity rather than the treatment. that being said, (hydroxy)chloroquine is one of the antimalarial drugs considered compatible with pregnancy in all trimesters for prophylaxis and treatment of malaria (78, 79) . a recent article gathered evidence on its use during lactation and found that it was compatible with breastfeeding (80), concluding that hydroxychloroquine could be used for the treatment of covid-19 infection, in usual rheumatological doses (200-400 mg/day) if proven to be effective. the lopinavir ritonavir combination is used as part of the haart regimen to treat hiv infected women during pregnancy (81) . in a systematic review that included 4,864 lpv/r-exposed pregnancies, the authors reported the rate of congenital abnormalities to be similar to that of the general population. however, the stillbirth rate was higher than in the general population in the uk (9.2 per 1000 infants against 4.7 per 1000 infants in 2013) (82). there has been general concern regarding protease inhibitor exposure in utero and its association with an increased risk of preterm birth (83), however, to our knowledge this risk has not been evaluated specifica lly for lopinavir and ritonavir alone, and could be associated with the underlying disease activity rather than the treatment. finally, moderate adverse events such as gastro-intestinal symptoms (84) and an increased risk for alteration in fasting glycemia (85) were reported. lopinavir and ritonavir are drugs considered compatible with pregnancy in all trimesters for hiv treatment and has been associated with very low excretion into breastmilk (78, 79) . regarding remdesivir, no adverse effect was reported in pregnant participants in a randomized controlled trial on ebola virus (86). safety data on remdesivir in pregnancy are still scarce. oseltamivir was used during the 2009 influenza a/h1n1 pandemic and notably in pregnant mothers. in the most recent population-based study (87) conducted on 946,176 pregnancies in denmark from 2002 to 2013 of which 1898 were exposed to oseltamivir during pregnancy, ehrenstein.v and colleagues found no increased risk of any major congenital malformatio n, fetal death, preterm birth, sga or low 5-min apgar score. this confirmed previous observations from the european registry study (88) and the roche global safety database (89). oseltamivir could be considered compatible with pregnancy in all trimesters if proven effective in covid-19 treatment and has been associated with very low excretion into breastmilk (78, 79) . the interferon alpha drug (infα) is used to treat essential thrombocythemia, chronic myelocytic leukemia or hepatitis b and c in pregnant women. in a recent review including 43 exposed women, sakai k et al. found that no adverse event had required discontinuation of the this article is protected by copyright. all rights reserved. treatment but alerted physicians to "pay attention to (…) rare adverse events, such as impaired liver function, interstitial pneumonia, and attempts at suicide" (90). safety data on infα in pregnancy are scarce but its similarity to beta interferon, of which safety data during pregnancy are substantial and reassuring, makes it compatible in pregnancy if proven effective for covid-19 infection. regarding darunavir, no embryotoxicity or teratogenicity of this molecule was found in anima l studies (91). in a brief review of darunavir use in pregnant women, the authors concluded that it is a well-tolerated molecule which has few minor adverse effects (92). darunavir is considered compatible with pregnancy in all trimesters for hiv treatment despite its lack of safety data in pregnancy as its maternal benefit outweighs the potential unknown risks (78, 79) . animal studies have demonstrated embryotoxicity of baricitinib (93) and no safety data are available in human. analysis of the roche global safety database does not suggest a substantially increased risk of malformations with the use of tocilizumab. however, an increased rate of preterm birth and low birth weight children was possibly associated with tcz exposure and could be associated with the underlying disease activity rather than the treatment (94). safety data in pregnancy are limited and due to treatment-induced immunosuppression, an increased risk of maternal-feta l infections is theoretically possible in pregnant women treated with tocilizumab. in a systematic review assessing the benefits and safety of hyperimmune globulins to prevent hbv mother to child transmission in 2440 pregnant women, only one study mentioned adverse events consisting in swelling in two women (96). more recently, convalescent serum to treat the ebola virus disease was evaluated in a non randomized comparative study of 99 patients which included eight pregnant women. no serious adverse reaction were associated with the transfusion (98). two cases of pregnant women report the use of convalescent serum to treat sars-cov-2 infection (47,99). in the first case of a 31 years old pregnant woman, no serious adverse event related to the use of convalescent plasma was reported but its relative contribution to surviva l could not be determined due to other concomitant treatments. the authors concluded that its clinical benefit remained unknown (47). in the second case of a 35 year old pregnant woman with severe co morbidities who received both convalescent serum and remdesivir, no conclusion regarding safety or benefit of convalescent plasma could be drawn by the authors (99). data on the use of specific hyper immunoglobulins to prevent infections in pregnant women seem reassuring as well as those on the use of convalescent serum although they are more scarce. if they proved to be effective in covid-19 treatment, convalescent serum and specific hyper immunoglobulins directed against sars-cov-2 could be considered compatible with pregnancy in all trimesters. this article is protected by copyright. all rights reserved. (table 2) regarding potential asymptomatic infected pregnant women, the who recommends careful monitoring of patients with epidemiological history of contact with infected individuals, while the american college of obstetricians and gynecologists (acog) suggests routine antenatal care in this situation. an algorithm for assessment and management of symptomatic parturients has been proposed by acog, classifying them in three categories of risk: low, moderate and elevated. for mild presentations, women without comorbidities (low risk) should self-isolate at home, whereas those with health problems, obstetrical issues or the inability to care for themselves (moderate risk) should be seen in an ambulatory setting. according to the royal college of obstetricians and gynaecologists (rcog), pregnant women with moderate symptoms should self-isolate, unless they attend a maternity unit where patients in the 2 nd or 3 rd trimester meeting phe criteria ( ≥ 1 of: (1) clinical/radiological evidence of pneumonia, (2) acute respiratory distress syndrome (ards), (3) fever ≥37.8 and at least one of acute persistent cough, hoarseness, nasal discharge/congestion, shortness of breath, sore throat, wheezing or sneezing) should be tested for covid-19 and treated as infected until results are available. when pregnant women present with severe symptoms (high risk), they should immediately go to an emergency department according to acog algorithm. all guideline s agree that administration of corticosteroids for fetal lung maturity is still recommended per protocol for in the setting of a high risk of preterm birth when the mother's condition is stable. regarding fetal growth surveillance, rcog recommends an antenatal ultrasound fourteen days after acute illness resolution for hospitalized patients, while acog suggests a 3 rd trimester ultrasound for covid-19 pregnant women infected in 2 nd and 3 rd trimester. a detailed anatomy ultrasound could be considered for 1 st trimester infections (acog). data suggest that covid-19 may be associated with an increased thromboembolic risk with a rate of venous thromboembolism (vte) of 39% in icu patients (100). therefore, routine vte other coronavirus epidemics, such as sars-cov-1 had a higher impact on pregnant women encompassing 40% of icu admissions and 12% of mortalities. the mers-cov epidemic was even more lethal with a 40% mortality without significant difference of severity between pregnant and non-pregnant women. in this review, we gathered more than 150 cases of sars-cov-2 in pregnancy and identified a maternal mortality of 2.7% (9 cases) among those described in the literature. icu admissions were between 6.9% and 8%. the proportion of this article is protected by copyright. all rights reserved. severe complications seem to be equal to the non-pregnant population, however these must still be anticipated in pregnant women. these rates will have to be reviewed when the true denominator (number of infected pregnant women) is known, as a significant proportion of patients remain asymptomatic. past coronavirus epidemics were associated with adverse outcomes for the fetus and/or newborns including miscarriages (57%), preterm birth, fetal distress and fgr with sars-cov-1 infection during the 2 nd and 3 rd trimesters. also, mers-cov infection resulted in fetal and neonatal demise in 27% of cases. in this review, we found that of 142 cases of sars-cov-2 infections in pregnancy, 28% experienced preterm birth and 14% had adverse fetal/neonata l outcomes (fgr, fetal/neonatal demise, severe symptoms at birth). potential mechanis ms include placental changes, as observed with sars-cov-1, and severe respiratory maternal illness, which could lead to placental insufficiency, iugr and fetal distress/demise. the role of sars-cov-2 in early adverse pregnancy outcomes needs further investigation. with regards to pharmacological management, most agents currently tried are safe in pregnancy. as of april 22, 2020, prenatal management should be adapted to the patient's condition as indicated by acog and other algorithms (109). there is currently no agreement on specific prenatal ultrasound surveillance, but due to the potential risk of iugr, it would seem reasonable to assess fetal growth surveillance during the third trimester of pregnancy. administration of corticosteroids in pregnant women at risk of preterm birth should be administered per protocol, with consideration for the patient's condition. we recommend this article is protected by copyright. all rights reserved. considering parental preferences, the severity of illness and obstetrical indications when addressing the mode of delivery. guidelines for pregnancy management will continue to be updated and professionals should stay informed about new guidelines. the acquisition of robust data on the impact of emergent pathogens on pregnant women is often lacking or only available after a considerable delay (4) this article is protected by copyright. all rights reserved. 102. covid-19 and vte-anticoagulation -hematology.org [internet] . this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. suspected perinatal infection * 0 (0%) * 0/5 * (0%) 0/9 * (0%) 0 (0%) 1 (6%) 1/3 * (33%) * 0/6 * (0%) 3 (9%) 0 (0%) 0/10 * (0%) 0 (0%) 0 (0%) 2/10 * (20%) the nucleic acid test from the mother's amniotic fluid, vaginal secretions, cord blood, placenta, serum, anal swab, and breast milk were also negative. the most comprehensively tested case reported to date confirmed that the vertical transmission of covid is unlikely. the current data show that the infection of sars-cov-2 in late pregnant women does not cause adverse outcomes in their newborns, however, it is necessary to separate newborns from mothers immediately to avoid the potential threats. 9 vlachodimitropoul ou koumoutsea e, covid19 and acute coagulopathy in pregnancy. journal of thrombosis and haemostasis none the laboratory derangements may be reminiscent of hellp syndrome, and thus knowledge of the covid19 relationship is paramount for appropriate diagnosis and management. in addition to routine measurements of d-dimers, prothrombin time, and platelet count in all patients presenting with covid19 as per isth guidance, monitoring of aptt and fibrinogen levels should be considered in pregnancy, as highlighted in this report. 10 li l, [2] and their efforts to point out the error in table 3 . after consideration of the information presented by moradi et al., we have corrected the contents of although vertical transmission of sars-cov2 has been excluded thus far and the outcome for mothers and fetuses has been generally good, the high rate of preterm cesarean delivery is a reason for concern. these interventions were typically elective, and it is reasonable to question whether they were warranted or not. in mothers infected with coronavirus infections, including covid-19, >90% of whom also had pneumonia, ptb is the most common adverse pregnancy outcome. miscarriage, preeclampsia, cesarean, and perinatal death (7-11%) were also more common than in the general population. it seems that the data in the second and third columns in table 3 have been transposed, which needs correction. we present here the best evidence available to address many of these challenges, from making the diagnosis in symptomatic cases, to the debate between nucleic acid testing and chest imaging, to the management of the unwell patient in labor. this article is protected by copyright. all rights reserved. khan s, impact of covid-19 infection on pregnancy outcomes and the risk of maternal-toneonatal intrapartum transmission of covid-19 during natural birth. infection control and hospital epidemiology 3 we report a case report study of 3 pregnant women with laboratory-confirmed covid-19 pneumonia. all 3 pregnant women had vaginal deliveries. these patients presented with symptoms manifested by people with covid-19.2 of 3 patients, only 1 patient delivered a preterm baby. saccone g, the novel coronavirus (2019-ncov) in pregnancy: what we need to know. european journal of obstetrics, gynecology, and reproductive biology none in conclusion, strict monitoring of women with suspected 2019-ncov is firmly recommended. obstetricians should promptly recognize the symptoms of 2019-ncov, and adequately assess severity and fetal well-being. with interest, we read the guidelines by guillaume favre and colleagues1 on the management of pregnant women with suspected severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. therefore, we propose a translated algorithm for spanish-speaking countries (appendix). we also suggest that the new breastfeeding recommendations and the option to use dexamethasone as an alternative to betamethasone are adopted in latin america. this article is protected by copyright. all rights reserved. khan s, association of covid-19 infection with pregnancy outcomes in healthcare workers and general women. clinical microbiology and infection 17 in summary, we found two neonates suspected for covid-19 infection and five neonates with neonatal pneumonia, suggesting the possibility that adverse pregnancy outcomes may be linked to covid-19 infection. shah ps, classification system and case definition for sars-cov-2 infection in pregnant women, fetuses, and neonates. acta obstetricia et gynecologica scandinavica none at present the evidence for intrauterine transmission from mother to fetus or intrapartum transmission from mother to the neonate is sparse. there are limitations associated with sensitivity and specificity of diagnostic tests used and classification of patients based on test results has also been questioned. deprest j, feto-placental surgeries during the covid-19 pandemic: starting the discussion. prenatal diagnosis none fetal diagnosis and pregnancy care need to be maintained, and we should strive to preotect the vulnerable population of pregnant women as well as their fetus, as much as possible. this includes both sars-cov2-negative and positive patients with fetal anomalies that may benefit from prenatal intervention. mimouni f, perinatal aspects on the covid-19 pandemic: a practical resource for perinatalneonatal specialists. journal of perinatalogy none vertical transmission from maternal infection during the third trimester probably does not occur or likely it occurs very rarely. consequences of covid-19 infection among women during early pregnancy remain unknown. we cannot conclude if pregnancy is a risk factor for more severe disease in women with covid-19. little is known about disease severity in neonates, and from very few samples, the presence of sars-cov-2 has not been documented in human milk. this article is protected by copyright. all rights reserved. wilson an, caring for the carers: ensuring the provision of quality maternity care during a global pandemic. women and birth none this article provides an overview of important considerations for supporting the emotional, mental and physical health needs of maternity care providers in the context of the unprecedented crisis that covid-19 presents. cooperation, planning ahead and adequate availability of ppe is critical. thinking about the needs of maternity providers to prevent stress and burnout is essential. palatnik a, protecting labor and delivery personnel from covid-19 during the second stage of labor. american journal of perinatology none we recommend that labor and delivery personnel have the utmost caution and be granted the protection they need to protect themselves and other patients. this includes providing labor and delivery personnel full ppe including n95 for the second stage of labor. this is critical to ensure the adequate protection for health care workers and to prevent spread to other health care workers and patients. in summary, at present it must be stated that the general care recommendations that also apply to non-covid-19 patients are initially valid with regard to obstetric anesthesia. nevertheless, the special requirements on the part of hygiene and infection protection result in special circumstances that should be taken into account when caring for pregnant patients from an anesthetic point of view. mcintosh jj, corticosteroid guidance for pregnancy during covid-19 pandemic. american journal of perinatology none it is necessary that obstetricians adjust practice to carefully weigh the fetal benefits with maternal risks. therefore, our institution has examined the risks and benefits and altered our corticosteroid recommendations. gonzalez-brown vm, operating room guide for confirmed or suspected covid19 pregnant patients requiring cesarean delivery. american journal of perinatology none this is a suggested protocol which may not be applicable to all health care settings but can be adapted to local resources and limitations of individual l&d units. this article is protected by copyright. all rights reserved. parazzini f, delivery in pregnant women infected with sars-cov-2: a fast review. international journal of gynaecology and obstetrics 64 the rate of vertical or peripartum transmission of sars-cov-2 is low, if any, for cesarean delivery; no data are available for vaginal delivery. low frequency of spontaneous preterm birth and general favorable immediate neonatal outcome are reassuring. huang x, epidemiology and clinical characteristics of covid-19. archives of iranian medicine none the basic strategy for controlling the epidemic is early detection, early isolation, early diagnosis and early treatment. covid-19 cases are insidious and transmissible in the incubation period, and multiple clusters have been reported in china. the causal role of covid-19 in these cases is therefore uncertain and larger studies are needed in the future to describe the prevalence, clinical characteristics and course of the disease. pérez-lópez fr, severe acute respiratory syndrome coronavirus 19 and human pregnancy. gynecological endocrinology none outcomes of pregnants delivering in the upcoming months will provide more information on this particular new disease and its relation to pregnancy. in the meantime, it seems best that women should be encouraged to delay becoming pregnant until more evidence related to risks associated to covid-19 infection during pregnancy is available. in addition, women susceptible to be submitted to assisted reproductive technology should take some additional precautions as recently recommended by la marca et al. pregnant women in shanghai critically concern about the risk of 2019-ncov infections, and highly demand knowledge and measures on prevention and protection from covid-19. they ask for having time-lapse appointments for anc and online access to health information and services. maternal and child care institutes should understand the demands of pregnant women, optimize the means of anc service, and provide tailored and accessible health education and service for the safety of mother and child. the manuscript outlines the precautions and steps to be taken before, during, and after resuscitation of a newborn born to a covid-19 mother, including three optional variations of current standards involving shared-decision making with parents for perinatal management, resuscitation of the newborn, disposition, nutrition, and postdischarge care. the availability of resources may also drive the application of these guidelines. this article is protected by copyright. all rights reserved. bourne lancet . infectious diseases 7 the maternal, fetal, and neonatal outcomes of patients who were infected in late pregnancy appeared very good, and these outcomes were achieved with intensive, active management that might be the best practice in the absence of more robust data. the clinical characteristics of these patients with covid-19 during pregnancy were similar to those of nonpregnant adults with covid-19 that have been reported in the literature. given that 420 patients were diagnosed by mar. 11th in shenzhen and tens of cases was diagnosed in our hospital, yet no nosocomial infection has occurred and none of the pregnant woman registered in our hospital was reported to be infected, this management should be effective to an extant, however mathematical model may be needed to quantify the effectiveness of these methods. on january 28, we published "guidance for maternal and fetal management during pneumonia epidemics of novel coronavirus infection in the wuhan tongji hospital (first edition)" [4] . based on the clinical characteristics, diagnosis and treatment progress of the recently discovered diseases, we offered an updated clinical management for pregnant women and newborns with ncp. for ordinary covid-19 patients intraspinal anesthesia is preferred in cesarean section, and the influence on respiration and circulation in both maternal and infant should be reduced; while for severe or critically ill patients general anesthesia with endotracheal intubation should be adopted. the safety of medical environment should be ensured, and level-ⅲ standard protection should be taken for anesthetists. special attention and support should be given to maternal psychology. we therefore updated the guidelines according to the data available at the beginning of march, 2020 (appendix). it is our responsibility, as specialists working in different fields of perinatology, to improve our own recommendations and that of others for the benefit of our patients. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. the lancet a novel coronavirus from patients with pneumonia in china zika virus infection -after the pandemic sars and mers: recent insights into emerging coronaviruses aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 first experience of covid-19 screening of health-care workers in england. the lancet who | summary table of sars cases by country this article is protected by copyright. all rights reserved who | middle east respiratory syndrome coronavirus (mers-cov) early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the hospitalized patients with 2019 novel coronavirus-infected pneumonia in coronavirus disease 2019 (covid-19) and pregnancy: what obstetricians need to know guillain-barré syndrome related to covid-19 infection presumed asymptomatic carrier transmission of covid-19 a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. the lancet real estimates of mortality following covid-19 infection authors' reply. the lancet infectious diseases estimates of the severity of coronavirus disease 2019: a model-based analysis. the lancet infectious diseases clinical manifestations and outcome of sars-cov-2 infection during pregnancy clinical characteristics and records. the lancet pregnancy and perinatal outcomes of women with coronavirus disease (covid-19) pneumonia: a preliminary analysis neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan coronavirus disease in china mothers with covid-19 pneumonia possible vertical transmission of sars-cov-2 from an infected mother to her newborn available from: this article is protected by copyright. all rights reserved can sars-cov-2 infection be acquired in utero?: more definitive evidence is needed preterm delivery in pregnant woman with critical covid-19 pneumonia and vertical transmission profound childhood deafness. inner ear pathology ototoxicity of chloroquine hydroxychloroquine (hcq) in lupus pregnancy: double-blind and placebo-controlled study electroretinograms of children born to mothers treated with hydroxychloroquine during pregnancy and breast-feeding use of hydroxychloroquine during pregnancy and breastfeeding: an update for the recent coronavirus pandemic (covid-19) drugs in pregnancy and lactation drugs during pregnancy and lactation -3rd edition role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings safety and efficacy of society of critical care medicine (sccm) should we stop aspirin prophylaxis in pregnant women diagnosed with covid-19? covid-19: ibuprofen should not be used for managing symptoms, say doctors and scientists middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases & review of the literature middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia stillbirth during infection with middle east respiratory syndrome coronavirus pregnancy and perinatal outcomes of women with severe acute respiratory syndrome maternal and neonatal outcomes of pregnant women with covid-19 pneumonia: a case-control study clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records. the lancet clinical features and obstetric and neonatal outcomes of pregnant patients with covid-19 in wuhan, china: a retrospective, single-centre, descriptive study. the lancet infectious diseases clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia pregnancy and perinatal outcomes of women with coronavirus disease (covid-19) pneumonia: a preliminary analysis neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan, china. jama pediatr clinical manifestations and outcome of sars-cov-2 infection during pregnancy clinical features and outcomes of pregnant women suspected of coronavirus disease perinatal transmission of covid-19 associated sars-cov-2: should we worry? a case report of neonatal covid-19 infection in china a case of 2019 novel coronavirus in a pregnant woman with preterm delivery covid-19 in pregnancy with comorbidities: more liberal testing strategy is needed an uncomplicated delivery in a patient with covid-19 in the united states emergency cesarean section on severe acute respiratory syndrome coronavirus 2 (sars-cov-2) confirmed patient covid-19), practice advisory. the american college of obstetricians and gynecologists covid-19) infection in pregnancy, information for healthcare professionals version 7. royal college of obstetricians & gynaecologists covid-19 -guidance for neonatal settings. royal college of paediatrics and child health current criteria phe criteria(correct at the time of publishing this update) are: women who are being/are admitted to hospital with one of the following: • clinical/radiological evidence of pneumonia, • acute respiratory distress syndrome (ards), • fever ≥37.8 and at least one of acute persistent cough, hoarseness, nasal discharge/congestion, shortness of breath national institute for health and care excellence this article is protected by copyright. all rights reserved.accepted article this article is protected by copyright. all rights reserved. this article is protected by copyright. all ten key recommendations were provided for the management of covid-19 infections in pregnancy.this article is protected by copyright. all rights reserved.accepted article 92 zhou d, covid-19: a recommendation to examine the effect of hydroxychloroquin e in preventing infection and progression.review article the journal of antimicrobial chemotherapy none in summary, we propose that hydroxychloroquine (hcq) could serve as a better therapeutic approach than chloroquine (cq) for the treatment of sars-cov-2 infection. there are three major reasons for this: (i) hcq is likely to attenuate the severe progression of covid-19 through inhibiting the cytokine storm by reducing cd154 expression in t cells; (ii) hcq may confer a similar antiviral effect at both pre-and post-infection stages, as found with cq; (iii) hcq has fewer side effects, is safe in pregnancy and is cheaper and more highly available in china. if there is an indication for obstetric surgery or critical illness of covid-19 in pregnant women, timely termination of pregnancy will not increase the risk of premature birth and asphyxia of the newborn, but it is beneficial to the treatment and rehabilitation of maternal pneumonia. preventive use of long-acting uterotonic agents could reduce the incidence of postpartum hemorrhage during surgery. 2019-ncov infection has not been found in neonates deliverd from pregnant women with covid-19. journal of infection 13 the report showed pregnant women are also susceptible to sars-cov-2 infection. sars-cov-2 may increase health risks to both mothers and infants during pregnancy. efforts should be taken to reduce the infection rate of sars-cov-2 both in pregnant and perinatal period, and more intensive attention should be paid to pregnant patients. review article international nursing review none the next decade is likely to produce any number of global challenges that will affect health and health care, including pan-national infections such as the new coronavirus covid-19 and others that will be related to global warming. nurses will be required to react to these events, even though they will also be affected as ordinary citizens. the future resilience of healthcare services will depend on having sufficient numbers of nurses who are adequately resourced to face the coming challenges. none not about coronavirus -2 therefore, p100c4 potentially could be tested as a priming vaccine or be further modified using reverse genetics. it also can be administered in multiple doses or be combined with inactivated or subunit vaccines and adjuvants as a pedv vaccination regimen, whose efficacy can be tested in the future.this article is protected by copyright. all rights reserved. not about coronavirus -2 mers-cov remains an uncommon disease among children, and its course follows a milder path among children than those of adults. majority of cases were asymptomatic and were diagnosed during the course of contact investigation. not about coronavirus -2 ifn-based drugs enhance the protective effect of vaccination against associative infections in the newborn calves. they stimulate a rise in the titer of antibodies to rotavirus, coronavirus, vd, and mucosal disease complex as well as an increase in immunoglobulins a, m, and g. key: cord-352178-irjhmxsg authors: saxton-shaw, kali d.; ledermann, jeremy p.; borland, erin m.; stovall, janae l.; mossel, eric c.; singh, amber j.; wilusz, jeffrey; powers, ann m. title: o'nyong nyong virus molecular determinants of unique vector specificity reside in non-structural protein 3 date: 2013-01-24 journal: plos negl trop dis doi: 10.1371/journal.pntd.0001931 sha: doc_id: 352178 cord_uid: irjhmxsg o'nyong nyong virus (onnv) and chikungunya virus (chikv) are two closely related alphaviruses with very different infection patterns in the mosquito, anopheles gambiae. onnv is the only alphavirus transmitted by anopheline mosquitoes, but specific molecular determinants of infection of this unique vector specificity remain unidentified. fifteen distinct chimeric viruses were constructed to evaluate both structural and non-structural regions of the genome and infection patterns were determined through artificial infectious feeds in an. gambiae with each of these chimeras. only one region, non-structural protein 3 (nsp3), was sufficient to up-regulate infection to rates similar to those seen with parental onnv. when onnv non-structural protein 3 (nsp3) replaced nsp3 from chikv virus in one of the chimeric viruses, infection rates in an. gambiae went from 0% to 63.5%. no other single gene or viral region addition was able to restore infection rates. thus, we have shown that a non-structural genome element involved in viral replication is a major element involved in onnv's unique vector specificity. o'nyong nyong virus (onnv) is an arthropod-borne virus (arbovirus) associated with a small number of large-scale epidemics. one such epidemic began in 1959 in uganda, lasted three years and affected over 2 million people [1] . serological evidence of onnv transmission indicated circulation in kenya until the late 1960s [2] , additional serological surveys in [1974] [1975] showed circulation in west africa [3] , but onnv did not cause another epidemic until 1996, when 400 people were sickened in the rakai district in southern uganda [4] . the known distribution of onnv mirrors that of the mosquito vectors that transmit the virus, anopheles gambiae and anopheles funestus [5] . humans are the only currently known reservoir of onnv [6] . onnv infection in humans is usually selflimiting, but does cause a low grade-fever, joint pain, lymphadenopathy, and a generalized papular or maculopapular rash [7] . chikungunya (chikv) virus is a closely related alphavirus which has caused millions of cases of disease throughout countries in and surrounding the indian ocean since its re-emergence in 2004 [8] [9] [10] . additional cases occurred in travelers returning from affected areas to asia, north america, and to europe, where a few small epidemics have since occurred due to autochthonous transmission [11] [12] [13] [14] . humans are infected with chikv when bitten by infected aedes aegypti or, during epidemics, aedes albopictus mosquitoes. patients infected with chikv suffer from clinical symptoms similar to those infected with onnv except that the fever is a higher, there is typically no lymphadenopathy, and the arthralgia is both incapacitating and chronic. chikv and onnv diverged from a common ancestor thousands of years ago [15] and despite their genetic similarity, onnv and chikv have distinct ecologies. in particular, they are not transmitted by the same mosquito vectors [6] . in fact, onnv is the only alphavirus to be vectored by anopheline mosquitoes [5] while chikv is transmitted by culicine mosquitoes. the differential mosquito infectivities of chikv and onnv have been characterized in the laboratory where an. gambiae mosquitoes have been shown to be highly susceptible to onnv infection and refractory to chikv infection [6] . thus, this mosquito serves as an ideal system to examine the molecular determinants of infection using hybrids of the two viruses. members of the genus alphavirus contain a single-stranded, positive-sense rna genome (,11.7 kb) that contains two regions, a non-structural domain making up the 59 two-thirds of the rna and a structural domain at the 39 end making up the remaining one-third of the genome [16] . the non-structural domain encodes four viral non-structural proteins: nsp1, nsp2, nsp3, and nsp4 which are essential for replication and polyprotein processing. in addition to copying the rna genome, the non-structural proteins synthesize 26s subgenomic mrna which is capped and polyadenylated and which ultimately produces five individual structural proteins (capsid, e3, e2, 6k, e1) [17] . previous studies with chimeric alphaviruses have shown that viral components in both the structural and non-structural portions of the venezuelan equine encephalitis genome contribute to infection phenotypes in guinea pigs [18] . similar results were seen with chimeric eastern equine encephalitis viruses, with both structural and nonstructural proteins contributing to neurovirulence and viral tissue tropism in mice [19] . chimeric viruses are also useful for studying virusvector interactions as seen in a study that mapped mosquito infection determinants specifically to the e2 envelope glycoprotein region of venezuelan equine encephalitis [20] . earlier studies using chimeric onnv suggested that all of the viral structural proteins are necessary for onnv to infect an. gambiae mosquitoes [21] ; however these studies used limited sample sizes and looked only at the structural regions of the genome. the current study expanded upon this earlier work to examine the contribution of each specific viral region or gene to virus-vector specificity. all plasmid clones used in this study were designed and constructed in house. the full length clone ponn.ap3 was constructed from onnv strain sg650 [5] (genbank accession number af079456) while pchik.b was constructed from chikv strain 37997 (genbank accession number ay726732). these two full -length parental clones were used to construct 15 chimeric viruses as shown in figures 1 and 2 . all plasmid clones designed to evaluate structural regions of the genome were constructed in a similar fashion, with the substituted region produced from a pcr product and the backbone region produced from the parental plasmid clones described previously. for example, to construct pchik/onn e2, the e2 region of onnv was amplified from parental ponn.ap3 by pcr with pfu turbo polymerase (stratagene, la jolla, ca). the onnv amplicon and pchik.b were digested with the same restriction enzymes (table 1) . when necessary, appropriate restriction enzyme sites were added to pchik.b using a quikchange xl site-directed mutagenesis kit (stratagene) according to the manufacturer's instructions. modifications were performed so that no amino acid changes were introduced and all viruses generated from constructs with introduced mutations were tested to insure they replicated in a manner comparable to the parental viruses. both restriction digests were run on an agarose gel at low voltage for at least 8 hours. the doubly-digested insert and plasmid backbone were cut from the gel and purified from the agarose using the minelute gel extraction kit (qiagen, valencia, ca). the backbone vector was treated with antarctic phosphatase (neb, beverly, ma) to remove the 59 phosphate groups, thus preventing self-ligation of the plasmid. prepared plasmid backbone and insert were ligated overnight with t4 dna ligase (neb) and then electroporated into xl-1 blue electrocompetent cells (stratagene). transformed cells were grown on yt plates with 50 mg/ml ampicillin and incubated overnight at 37uc. colonies were picked and screened for confirmation of onnv insert by pcr. plasmid clones were confirmed by sequencing the entire construct. plasmid clones designed to evaluate non-structural regions of the genome were engineered as exact gene substitutions by using a series of subclones, as described in the protocol s1 and in figures s1, s2, s3, s4, s5, s6. briefly, chikv regions flanking the desired non-structural protein, and containing convenient restriction sites were amplified from pchik.b by pcr with pfu turbo polymerase (stratagene). primers used for amplification added a type ii restriction enzyme site to the outside of each desired insert product. these two pcr products and a modified cloning vector, puc19m2 were each double digested using type i enzymes exterior to the type ii engineered sites. a 3-way ligation then produced the first subclone (puc19m2 with the chikv sequence flanking where the desired non-structural protein sequence would subsequently be inserted). the desired onnv non-structural protein was amplified using primers which added the same type ii enzyme site as was added to the pcr products. the first subclone and the onnv pcr product were both digested with the same type ii enzyme, which cuts itself out upon digestion. ligation of the two digested products produced a second subclone (puc19m2 with the entire and exact onnv nonstructural protein, flanked by chikv sequence). this second subclone and pchik.b were then digested using the convenient restriction sites already present in the flanking chikv sequence. ligation of the doubly-digested pchik.b backbone and the insert obtained from the second subclone produced the final construct. colonies were screened and verified by complete genome sequencing and plasmid dna was prepared as described above. templates for in vitro transcription were generated by linearizing each full-length clone with a unique not i restriction site present downstream of the poly (a) tail. linearized plasmids were treated with proteinase k (invitrogen, carlsbad, ca) to digest any endogenous rnases or dnases. dna was purified using a phenolchloroform extraction followed by an ethanol precipitation. a 20 ml aliquot of linearized and treated dna was then transcribed in vitro by incubating the dna with 0.4 ml (100 mm) each rntp (promega, madison, wi), 0.4 ml bsa (10 mg/ml) (neb, beverly, ma), 2 ml dtt (100 mm) (promega, madison, wi), 8 ml (5x) transcription buffer (promega, madison, wi), 1.3 ml (15 u/ml) t7 rna polymerase (promega, madison, wi), and 4 ml (10 mm) acap-structure analog (neb, beverly, ma) for one hour at 39uc. transcribed rna (10 ml) was mixed with 400 ml bhk-21 cells (1610 7 cells/ml) in a 2 mm gap cuvette (btx:harvard apparatus, inc., holliston, ma) and electroporated twice using a btx electrocell manipulator with the following settings: 460volts, 725ohms, 75 mf [22] . after electroporation, the cells were transferred to a t-25 tissue-culture flask. dulbecco's modified eagle medium (dmem) with 10% by volume fetal bovine serum and 1% by volume penicillin/streptomycin was added to the flask o'nyong nyong virus (onnv) is unique in that it is the only alphavirus, and one of few viruses in general, to be transmitted to humans by the bite of an anopheline mosquito. the genetics responsible for this unique vector specificity would be useful information in helping to develop antivirals, vaccines, and other methods for interrupting virus transmission. previous research using other arboviruses has shown that specific viral genomic regions, amino acid sequences, or even single nucleotide mutations can have a profound effect on virus growth, infection, and virulence characteristics. using chimeric viruses that substitute a gene from one virus with a gene from a closely related virus is a proven method of evaluating the relative contribution of each gene to a given phenotype. our study analyzed both structural and non-structural regions of the onnv genome using chimeric viruses and artificially infected anopheles gambiae mosquitoes. when onnv non-structural protein 3 (nsp3) replaced nsp3 from chikungunya virus in one of the chimeric viruses, infection rates in an. gambiae went from 0% to 63.5%. no other single gene or viral region addition was able to restore infection rates. that onnv nsp3 is largely responsible for onnv's unique ability to infect an. gambiae is especially interesting since the exact mechanisms and functions of this highly-variable protein remain poorly understood. before incubation at 37uc. tissue culture supernatant was harvested approximately 72 hours post-transfection or when cytopathic effects (cpe) were observed. supernatant was aliquoted and stored at 280uc until later use. each time a virus was generated, the entire virus was sequenced to verify fidelity to the original sequence. viral rna was extracted using qiaamp viral rna mini kit (qiagen). extracted rna was then added to a reverse-transcriptase pcr reaction using the titan one tube rt-pcr system (roche, indianapolis, in). complementary dna for sequencing the 59 end of each viral genome was generated using a firstchoice rlm-race kit (ambion, austin, tx). this complementary dna was then sequenced using virus-specific primers with the big dyev3.1 kit on an abi 3130xl genetic analyzer (applied biosystems, foster city, ca). sequence files were aligned and analyzed for sequence quality and genome coverage using lasergene suite software (dnastar, madison, wi). virus rescued from clones was titered by plaque assay. ten-fold virus dilutions from 10 21 to 10 27 were added to individual well of 6-well plates covered in monolayers of vero cells. plates were incubated at 37uc, with 5% co 2 . cells were fixed 48-72 hours later using a solution of 40% methanol and 0.25% crystal violet in water. plaques were then counted and titers were calculated as plaque forming units per milliliter (pfu/ml). the ability of each chimeric virus to infect mosquitoes was evaluated using the g3 strain of an. gambiae, originally obtained from the national institute of health. this strain has been maintained as a colony in our lab with rearing conditions that include a 12:12 hour light:dark cycle in chambers maintained at 28uc with approximately 95% humidity [23] . infectious blood meals were prepared from equal volumes of packed, calf erythrocytes, 10% sucrose in fetal bovine serum, and 4.4-6 log 10 pfu/ml of virus. mosquitoes were allowed to feed on the warmed infectious blood meal for one hour through an artificial membrane feeder (hemotek, accrington, uk). fully engorged females were separated and maintained for an incubation period of up to 12 days. mosquitoes were sacrificed at days 4, 8, and 11 or 12 post-infectious-blood meal. heads and bodies were separated into individual tubes and stored at 280uc until subsequent processing. infection rates were determined using results from the 2-3 replicate feeds were combined and the titer (log 10 pfu/ml) shown is an average (for titers that were within 0.5 log 10 pfu/ml of one another). infection rate is determined using mosquito bodies while dissemination rate is derived from heads. doi:10.1371/journal.pntd.0001931.g001 individual bodies and dissemination rates were calculated as the number of positive heads among the positive bodies. at least two replicate infectious feeds were done for each chimeric virus, with replicate feeds performed entirely independent of one another. no less than 140 mosquitoes were tested for any one chimeric virus. individual frozen mosquito bodies and heads were triturated in 300 ml of dmem supplemented with (by volume): 10% heatinactivated fetal bovine serum, 1% penicillin/streptomycin, 0.1% gentamicin, and 0.1% fungizone. the mosquito homogenates were passed through a 0.2 mm gelman acrodisc filter (krackeler scientific inc., albany, ny) to remove potential bacterial or fungal contaminates. filtrate from each body or head was added to a single well of a 96-well flat-bottom tissue-culture plate, along with 50 ml of prepared bhk-21 cell suspension (approximately 4.6 log 10 cells/well). inoculated tissue-culture plates were incubated at 37uc for 5 days. cells were observed daily for cpe due to virus replication. virus replication in mosquito body samples indicated that virus had infected the mosquito's midgut, while replication in the mosquito heads showed a disseminated infection. to confirm that all constructed viruses were comparably replication competent, growth curves were performed in cell culture on all rescued viruses. briefly, 24-well plates (corning, corning, ny) were seeded with vero (african green monkey) cells. monolayers at 90% confluency were infected with virus at a multiplicity of infection (moi) of 0.1. at specified times post infection, supernatant was removed from two wells for each virus and placed in a screw-cap cryovial at 270uc until titration by plaque assay. titration results for each virus were compared at all time points by the student t-test. to confirm virus replication (and not just persistence of the input virus) within the mosquito, five females that had fed on the onnv infectious blood meal were sacrificed every other day post-infectious feed. each body and head was processed separately, as described earlier. rna was extracted from homogenized mosquitoes using qiaamp viral rna mini kit (qiagen). the amount of rna in each head and body was determined using the quanti tect probe rt-pcr kit (qiagen) and a taqman real-time pcr assay as previously described [24] , except that onnv sg650 specific primers were used (10692 fwd-59 gca ggg agg cca gga cag t, 10840 rev-59 gcc cct ttt tcy ttg agc cag ta) . the real-time probe was labeled with a 59 end hex reporter dye and a 39 end bhq1 quencher dye (10759 fwd-59 aaa gac cag cgg cag gag caa tac ac) and pcr results are reported here as pfu-equivalents/mosquito by comparison with known concentration standards. fisher's exact probability test was employed to evaluate whether infection rates with chimeric viruses were statistically different from those with parental viruses. the infection rate was defined as significantly different from parental chikv if the two-tailed pvalue was ,0.007. the two-tailed p-value had to be ,0.01 to be statistically different from parental onnv infection rates. both adjusted alphas were obtained using the bonferroni correction for multiple comparisons to ensure an overall type i error of 0.05. computations were made using freely-available software [25] . this study built upon previously established disparate infectivity patterns for two closely related alphaviruses, chikv and onnv, in an. gambiae [5, 6] and infection rates with the parental viruses generated from our full-length infectious clones were concordant with those previously reported. by day 12, up to 91% of an. gambiae mosquitoes were infected with onnv, whereas a maximum of only 6% were infected with chikv. these values are similar to previously published work [6] . with this highly significant difference (p,0.0001) between the two viruses, characterization of individual viral gene substitutions was likely to reveal which elements were involved in mosquito infection. prior to initiating these experiments with chimeric viruses in an. gambiae, viral replication (and not just persistence of input virus) within both cell culture and in the mosquito was confirmed. cell culture growth curves of all of the chimeras were performed in vero cells to confirm that all viruses were indeed replication competent and replicated in a manner similar to their parental viruses ( figure 3 ). the structural change viruses all replicated efficiently and replication was virtually identical among all chimeras sharing non-structural genes. in general, those viruses with the onnv non-structural genes grew to peak titers of 10 6.5 -10 7 pfu/ ml while those with chikv non-structural genes had peak titers of 10 7.5 -10 8 pfu/ml. all non-structural chimeric viruses grew similarly well, rapidly increasing in titer from 1000 pfu/ml to 10 7 -10 8.5 pfu/ml. no consistent statistical differences were observed among the non-structural substitution viruses. the quantity of onnv rna present in individual mosquito bodies and heads through 11 days post-infectious feed adhered to the expected pattern of decrease during the extrinsic incubation period followed by a rise in virus replication at later time points as determined by qrt-pcr. moreover, after 5 days post infection, the five mosquito bodies tested at each of the subsequent time points had more rna copies than could have been initially imbibed in the blood meal indicating replication of the virus was indeed occurring (figure 4) . nine unique chimeric hybrids of chikv and onnv were constructed using convenient restriction enzyme sites to produce substitutions in the structural region of the viral genome and to examine the contribution of each of these specific regions to virusvector specificity (figure 1 ). six additional non-structural chimeric viruses were also constructed using a novel type ii restriction enzyme cloning strategy to examine the broader genome with respect to onnv's unique vector specificity for an. gambiae mosquitoes (figure 2) . having confirmed viral replication and infections rates of parental onnv in an. gambiae, infection and dissemination rates with each of the 15 chimeric viruses were determined. each time a virus was generated through in vitro transcription for this study, it was sequenced completely prior to use in an infectious feed. mosquitoes containing replicating virus in the body, as shown by cpe analysis, were defined as being positive for viral infection. mosquito heads were analyzed separately from bodies to determine dissemination rates. each chimeric virus constructed from the parental chikv genome maintained a chikv-like infection profile (,10% infection rate), with one exception. when allowed to feed on a blood meal containing approximately 5.5 log 10 pfu/ml of chik/onn nsp3 virus, 63.5% (n = 85) of mosquitoes had replicating virus when harvested on day 8 post infection ( figure 2) . none of the onnv substitutions made to the structural regions of the chikv parental genome produced infection results deviating from those seen with the complete chikv parental genome ( figure 1 ). three of the 5 chimeric viruses constructed from the parental onnv genome retained onnv-like infection rates at day 8 in an. gambiae, while the remaining two viruses showed significantly lower infection rates. only 11.1% (n = 135) of mosquitoes feeding on onn/chik 39str and 53.2% (n = 77) of onn/chik 59str were shown to be infected at day 8. infection rates for mosquitoes sacrificed at days 4, 11, or 12 corroborate day 8 results (data not shown). dissemination rates for each of the viruses in an. gambiae was very low and all were comparable for both day 8 and day 11 samples. only 5 viruses showed any dissemination (figures 1 and 2): parental onn.ap3,onn/chik e2, onn/chik estr, onn/ chik 39nsp4-59c, chik/onn 39str. the rest of the viruses showed no dissemination. a panel of 15 chimeric viruses were developed here to study specific elements of the onnv genome and to determine which of these regions are necessary for onnv to infect an. gambiae mosquitoes. as chikv virus primarily infects aedes species and onnv primarily infects anopheles species, these two closely related viruses provide an ideal opportunity to study these viral genetic determinants of infection. this study is the first to look at the importance of onnv non-structural proteins in an. gambiae infection. of the ten chikv-backbone chimeras constructed and tested, only the one containing onnv nsp3 produced infection rates closer to parental onnv than to the parental chikv. the ability of onnv nsp3 to up-regulate infection rates so substantially shows that onnv nsp3 is the main determinant of onnv vector specificity for an. gambiae. interestingly, the reciprocal chimeric virus (full length-onnv with the chikv nsp3) was not able to be rescued from cdna in either mammalian or insect cells. this would further suggest that nsp3 plays a critical role in viral replication that is distinct in these two closely related viruses that exhibit 81% and 72% amino acid and nucleotide identity respectively in nsp3. that nsp3 should be found to be essential to infection is especially interesting given the fact that the precise functions of this protein are not fully defined. it is required for the correct formation and localization of replication complexes and does provide essential functions in both minus strand and subgenomic rna synthesis, but specific mechanisms are not yet resolved [21] [22] [23] . to further add to the intrigue of this protein, it has been shown that some members of the alphavirus family actually contain inserts of foreign genetic material within nsp3. an eight amino acid sequence from the carboxyl-terminus of chikv nsp3 maps to a putative zinc finger protein in ae. aegypti, the main vertebrate vector for that virus [26] . in semliki forest virus, a 7 amino acid sequence corresponds to elements found in a wide-range of cellular proteins [26] . numerous other examples of what may be inserts of foreign genetic material been shown by sequencing nsp3 from the following alphaviruses: chikv, eastern equine encephalitis virus, semliki forest virus, and venezuelan equine encephalitis virus [26] . alphavirus nsp3 can be clearly divided into two distinct domains. the macro domain, or amino-terminal region, is highly conserved, not just among alphaviruses but also among coronaviruses, hepatitis e virus, rubella virus and even cellular proteins [27, 28] . the carboxyl-terminus domain of the alphavirus nsp3 is highly variable in size and sequence and is devoid of any predicted secondary structure [29, 30] . chimeric viruses were constructed using the natural division between the conserved and nonconserved regions of nsp3 to engineer two additional chimeric viruses to determine if the region conferring specificity to an. gambiae could be attributed solely to either of the distinct domains within nsp3. interestingly, the addition of just the carboxylterminus of onnv nsp3 did produce a small, although not a statistically significant, increase in infection rates as compared with parental chikv in an. gambiae. the carboxyl-terminus of nsp3, which has been subject to rapid alteration during alphavirus evolution, may also be involved in the optimization of replication in diverse host cell types [29] . studies with sindbis showed that deletions in the carboxyl-terminus rendered mutants defective at initiating a productive infection, generating plaques in mosquito cells at only 1-2% the efficiency of the parental virus [31] . a recent study noted a carboxyl-terminus, proline-rich sequence motif, the pipppr motif, shared by many alphavirus nsp3 proteins and demonstrated that even a single mutation in this region of semliki forest virus or sindbis virus greatly impaired rna synthesis by disrupting binding with host cell amphiphysins [32] . it is possible that this motif also modulates onnv vector specificity since onnv and chikv do differ from one another by one amino acid in this pipppr region. attenuated virulence and reduced rates of rna synthesis and virus replication were also seen in vertebrate cells with semliki forest virus mutants lacking some portion of the carboxyl-terminus of nsp3 [33] . yet, studies in mammalian cell lines showed that a 34 amino acid deletion in this region of nsp3 in venezuelan equine encephalitis had no detectable effect on replication [34] . collectively, these studies support the current finding that nsp3 can be vital for productive infection, but in a manner that is host and virus specific. another interesting characteristic of the carboxyl-terminus of nsp3 is that it is phosphorylated at multiple serine and threonine residues [35, 36] . the role of this phosphorylation is not exactly clear, except that it does seem to modulate the efficiency of minusstrand rna synthesis [37, 38] . determination of the exact mechanisms of this modulation and the mechanisms for the host-specific effects seen with nsp3 mutants in this and other studies would be extremely valuable information and allow for design of further studies. furthermore, our studies show that an intact onnv nsp3 is required for onnv-like infection rates, and that dividing the region either disrupts a vital interaction between the two or removes an element necessary for an. gambiae infection. the former seems more probable since substituting chikv for either half of onnv nsp3 results in infection rates not significantly different from rates with parental chikv. while molecular determinants residing in nsp3 did turn out to be the most dramatic finding of our study, we did also examine the structural regions of the genome. previously published studies by another group had suggested that all of the viral structural proteins are necessary for onnv to infect an. gambiae mosquitoes [21] . our study was able to provide critical fine tuning to this conclusion. in our experiments with chikv-backbone chimeras containing various regions of the onnv structural proteins, each maintained parental chikv-like infection profiles despite containing portions of the onnv genome. in fact, even an intact onnv structural region was not sufficient for infection of an. gambiae, as shown with the chimera chik/onn estr (figure 1 ). the reciprocal chimeras, substituting sections of chikv structural regions for the like section of onnv structural genes, in most cases, did not greatly reduce mosquito infection rates. the notable exceptions were in the chimeras that divided the onnv structural region in half. both onn/chik 59str and onn/ chik 39str were significantly less infectious to an. gambiae than was parental onnv. however, since the reciprocal chimeras, chik/onn 59str and chik/onn 39str, did not show upregulated infection rates, the drop in infection with the chimeric viruses is likely due to disruption of one or more virus-virus or virus-host interactions. in alphaviruses, the extreme 39 terminus of the genome, just preceding the poly(a) tail, has a sequence which is highly conserved among all alphaviruses and which is absolutely required for efficient virus replication [39, 40] . this 19-nucleotide sequence is identical in chikv and onnv so this could not have played a role in the decreased infection rates seen with onn/chik 39str. however, studies using sindbis mutants with large deletions in the 39non-translated region (ntr) have shown that the rest of the 39 ntr is also important for virus replication in a host-specific manner [40] . onnv is 156 additional nucleotides shorter in the 39ntr when compared to chikv; this size difference alone could result in conformational changes resulting in the inability of the virus to interact with itself or with host proteins. of note is the design of our estr and 39str structural clones, which contain the indicated structural region as well as the 39 ntr from the non-parental virus ( figure 1) ; this design is different from those previously described [21] and may suggest the possibility of multiple interactions within the proteins or gene sequences of the virus itself that may have a minor role in the overall ability of a chimeric virus to replicate within the mosquito. it is further possible that the differences in chikv and onnv conserved sequence elements [41] are sufficient to undercut rna stability, resulting in greatly reduced mosquito infection patterns. studies with chimeric viruses must be viewed in the overall context of the virus' life cycle. when substitutions are made to construct chimeric viruses, numerous aspects of the virus-host interactions and virus-virus regulatory functions can be disrupted resulting in reduced infection rates. reduced infection rates may be a direct consequence of missing the essential genomic region or may be an indirect result of disrupting an essential regulatory interaction. conversely, when the addition of a specific region increases mosquito infection rates, we must conclude the region itself to be essential for infection. interestingly, because there was such a low dissemination rate of all viruses within this study, elements involved in dissemination throughout the mosquito may be distinct from those important in initial infection. however, this study has shown that onnv nsp3 is directly responsible for onnv infection of an. gambiae. there are also numerous interactions within nsp3 itself, within the two halves of the structural region, and possibly the 39 ntr which, when disrupted, can eliminate mosquito infection. figure s1 illustration of exact nsp3 substitution made to create chik/onn nsp3. (tiff) figure s2 construction of chikv nsp3 receiving plasmid. pcr primers were designed to generate two amplicons flanking the dna insertion sites and extend outward to include unique restriction enzyme sites and inward to include a unique type ii restriction site. amplification with these primers, subsequent digestion with pcii/saci or ecori/saci, followed by a 3-part ligation produce a puc-based vector containing chikv sequence flanking the site where onnv nsp3 will later be inserted. (tiff) figure s3 amplifying onnv nsp3. pcr primers were designed to amplify the desired dna insert, with the addition of type ii restriction enzyme sites to the termini. type ii sites were oriented such that they will be removed upon later digestion. (tiff) figure s4 expanded sequence of assembled chikv nsp3 receiving plasmid (top). termini of onnv nsp3 amplicon (bottom). the lines indicate the cut sites for the type ii restriction enzymes. (tiff) figure s5 products produced after digestion with appropriate type ii restriction enzymes. these products were ligated to build the chikv/onnv nsp3 cassette plasmid. (tiff) figure s6 construction of final clone, chik/onn nsp3. chikv/onnv nsp3 cassette plasmid and pchik.b were digested with spei and avrii. the resulting products were ligated to generate the final clone with the complete onnv nsp3 gene replacing the like gene in chikv. protocol s1 methods for construction of gene specific clones. (docx) o'nyong-nyong fever: an epidemic virus disease in east africa. 8. virus isolations from anopheles mosquitoes the epidemiology of o'nyong-nyong in the kano plain viral infections in travellers from tropical africa emergence of epidemic o'nyong-nyong fever in southwestern uganda infection patterns of o'nyong nyong virus in the malaria-transmitting mosquito, anopheles gambiae differential infectivities of o'nyong-nyong and chikungunya virus isolates in anopheles gambiae and aedes aegypti mosquitoes o'nyong nyong fever: an epidemic virus disease in east africa. iii. some clinical and epidemiological observations in the northern province of uganda seroprevalence of chikungunya virus (chikv) infection on lamu island seroprevalence of chikungunya virus infection on grande comore island, union of the comoros chikungunya fever: an epidemiological review of a re-emerging infectious disease infection with chikungunya virus in italy: an outbreak in a temperate region autochthonous chikungunya virus transmission may have occurred in bologna, italy, during the summer 2007 outbreak first cases of autochthonous dengue fever and chikungunya fever in france: from bad dream to reality re-emergence of chikungunya and o'nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships the alphaviruses: gene expression, replication, and evolution a new role for ns polyprotein cleavage in sindbis virus replication the use of chimeric venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence vector infection determinants of venezuelan equine encephalitis virus reside within the e2 envelope glycoprotein determinants of vector specificity of o'nyong nyong and chikungunya viruses in anopheles and aedes mosquitoes intracellular immunization of mosquito cells to lacrosse virus using a recombinant sindbis virus vector manual for mosquito rearing and experimental techniques virulence variation among isolates of western equine encephalitis virus in an outbred mouse model lineage replacement accompanying duplication and rapid fixation of an rna element in the nsp3 gene in a species of alphavirus evolution and taxonomy of positive-strand rna viruses: implications of comparative analysis of amino acid sequences evolutionary conservation of histone macroh2a subtypes and domains functions of alphavirus nonstructural proteins in rna replication amino acid mutations in the replicase protein nsp3 of semliki forest virus cumulatively affect neurovirulence deletion and duplication mutations in the c-terminal nonconserved region of sindbis virus nsp3: effects on phosphorylation and on virus replication in vertebrate and invertebrate cells sh3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsp3 promotes viral rna replication deletions in the hypervariable domain of the nsp3 gene attenuate semliki forest virus virulence in vitro synthesis of infectious venezuelan equine encephalitis virus rna from a cdna clone: analysis of a viable deletion mutant semliki forest virusspecific non-structural protein nsp3 is a phosphoprotein phosphorylation of sindbis virus nsp3 in vivo and in vitro elimination of phosphorylation sites of semliki forest virus replicase protein nsp3 phosphorylation site analysis of semliki forest virus nonstructural protein 3 deletion mapping of sindbis virus di rnas derived from cdnas defines the sequences essential for replication and packaging mutagenesis of the 39 nontranslated region of sindbis virus rna the 39 untranslated region of sindbis virus represses deadenylation of viral transcripts in mosquito and mammalian cells we would like to thank andrea peterson for maintaining the an. gambiae, g3 colony used in this study. we would also like to thank dr. kimberly keene for modifying infectious clones. thanks to dr. mark delorey for his guidance in choosing appropriate statistical analysis tools. a special thanks to dr. carol wilusz and dr. susan knudson for their valuable insight. the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. key: cord-323643-lu3ngt6r authors: chow, c.b. title: post-sars infection control in the hospital and clinic date: 2004-11-05 journal: paediatr respir rev doi: 10.1016/j.prrv.2004.07.006 sha: doc_id: 323643 cord_uid: lu3ngt6r the recent severe acute respiratory syndrome (sars) outbreak has almost mandated a re-evaluation of infection control practices in hospitals, clinics, schools and domestic environments, especially for patients with respiratory tract symptoms. triage, early case detection followed by prompt isolation and quarantine are major preventive measures. respiratory tract infections are the most common childhood illnesses and paediatric sars poses special problems in diagnosis because of its non-specific presentation. the main lessons learnt from the outbreak were: (1) despite well established guidelines on infection control precautions, poor understanding of underlying principles and deficiencies in compliance are common among healthcare professionals, especially during emergencies; (2) even a slight lapse can be fatal; and (3) over-protection can be counterproductive. hence it is important to: (1) be protected to protect others; (2) be vigilant and prepared for emerging infections; (3) be proficient and scrupulous in infection control measures; (4) be apposite and practical on personal protective equipments to ensure sustainability; and (5) be dutiful and prompt in informing of potential threats and work closely with others. sars is a new devastating disease, the understanding of which is still evolving and includes its clinical syndromes, infectivity and transmissibility, optimal treatment and prognosis. children are less commonly infected than adults, they have milder disease, are less infectious and present with non-specific clinical features. however, severe illness can develop in adolescents and young infants. 1,2 detailed infection control guidances on sars have been prepared by who, us center for disease control, health canada, health protection agency of uk, ministry of health singapore, cdc of china, cdc of taiwan and the hospital authority of hong kong. a comprehensive review and account of infection control measures will not be attempted. this article makes special reference to children, is based on personal experience and on local systems developed in hong kong. it may need modifying to suit local situations and will need to be updated as new knowledge arises. the incubation as reported by most countries is said to be 4-6 days, with a mean of 4.6 days. 3 analysis of cases in hong kong revealed that the incubation period lies from 4-10 days with a mean of 6.4 days. 4 the main mode of transmission differed in different countries. using mathematical and statistical models it was estimated that 71.1% and 74.8% of sars infections in hong kong and singapore were attributable to super-spreading events. 5 it is not known whether the route of transmission affects the incubation period. paediatric summary the recent severe acute respiratory syndrome (sars) outbreak has almost mandated a re-evaluation of infection control practices in hospitals, clinics, schools and domestic environments, especially for patients with respiratory tract symptoms. triage, early case detection followed by prompt isolation and quarantine are major preventive measures. respiratory tract infections are the most common childhood illnesses and paediatric sars poses special problems in diagnosis because of its non-specific presentation. the main lessons learnt from the outbreak were: (1) despite well established guidelines on infection control precautions, poor understanding of underlying principles and deficiencies in compliance are common among healthcare professionals, especially during emergencies; (2) even a slight lapse can be fatal; and (3) over-protection can be counterproductive. hence it is important to: (1) be protected to protect others; (2) be vigilant and prepared for emerging infections; (3) be proficient and scrupulous in infection control measures; (4) be apposite and practical on personal protective equipments to ensure sustainability; and (5) be dutiful and prompt in informing of potential threats and work closely with others. ß 2004 published by elsevier ltd. sars-coronavirus (cov) can be found in respiratory secretions, saliva, tears, blood, urine and faeces of sars patients. sars-cov is stable in the environment for up to 2 days at room temperature and longer at a lower temperature. survival in a variety of stool suspensions varies depending on the ph, consistency of the stool and possibly other factors (up to 4 days in alkaline, diarrhoeal stool, 6 h in normal stool and 3 h in normal, acidic baby stool). the virus loses infectivity after exposure to different commonly used disinfectants (including alcohol and hypochlorite), and heating at 56 8c for 15 min. based on rt-pcr data, 36% of nasopharyngeal aspirate (npa)/nasal throat swabs tested positive for virus on days 0-2, peaking at 61% positive on days 9-11, declining to 35% on days 15-17 and are 0% by day 23. however, 0-22% of stools tested positive for the virus on days 0-2, peaking at 100% on days 12-14 and falling to 50% by days 21-23. detection of viral rna has a much lower yield from serum, with only 19% testing positive on days 0-2, peaking at 39% on days 6-8 of illness and being undetectable by day 12. viral excretion in npa and stool peaked on days 12-14 but viral load in npa specimen was at two orders of magnitude lower than viral excretion in stools. hence, respiratory specimens including nasopharyngeal aspirates, throat swabs or sputum samples were the most useful clinical specimens in the first 5 days of illness, after this stools would be the best choice. 6 infectivity is greatest in the second week of the illness, including those with severe illnesses, but patients can be infective within the first 1-2 days. there is no reported instance of transmission before the onset of symptoms of the disease and transmission after the second week of illness is rare. the size of coronavirus is about 0.1 mm. sneezing could have an ejection velocity of up to 30 metres per sec and cough 35 metres per sec. the number of droplets produced from coughing is about 40 000, talking produces 10 000 and singing 1000. droplet size varies from 50 mm to 3 mm. large droplets do not travel far and usually settle within 2 metres but droplets of 3 mm can stay in the air for 3 h. with forced ejection a good portion of these droplets can be evaporated rapidly, especially under low humidity and they may suspend in air longer. using fluorescentstained water, it was demonstrated that toilet seats had significant contamination by droplets after flushing and the aerosol effect can be up to the standing height of a child. 7 huge amounts of submicron (0.3-0.5 mm) aerosols can be generated from drainage flow, in the order of 60 000 per litre of air in the domestic sewage drainage system of high rises, suggesting that airborne transmission is possible from the empty u-traps and pipes leaking and containing infectious agents. 8 the primary mode of transmission is by direct mucous membrane (eyes, nose and mouth) contact with infectious agents. the main routes are through contaminated hands or direct exposure to respiratory droplets -contact and droplet. the basic reproduction ratio of three is consistent with the main mode of transmission by droplets. however, airborne transmission of sars can indeed occur in hospital and in community housing complexes. 9 infected cases occurred primarily in persons in close contact with ill sars patients in healthcare and household settings. transmission to casual and social contacts has occasionally occurred, especially as a result of intense exposure to sars patients eg. in lifts, in the workplace, on airlines or in taxis. transmission from children to adults is uncommon. 3,10 the attack rate for children was found to be lower than adults among quarantined close contacts in beijing (5% in children <10 years of age, 11.4% in those between 30 and 39 years of age and 27.6% in those aged between 60 and 69 years) 11 there was no report of sars transmission in schools both in hong kong and china where the outbreak was most extensive. a cluster of nine mild paediatric patients had been reported in a private boarding school for 820 students. all lived in the same building and ate daily meals together in the school canteen. 12 congenital and perinatal infection have not been documented in the 12 pregnancies reported in hong kong, 13 nine in china 14 and one in the usa. 15 in hospital settings, aerosolised respiratory secretions and direct contact with patients' secretion, excreta and fomites are other amplifying events. the role of faecal-oral transmission is unknown but is probably of some significance as profuse watery diarrhoea is common and large amounts of virus are found in stool. there have been no reports of food or waterborne transmission. the role of contaminated fomites in transmission is uncertain but must not be under estimated as the virus can survive for days at room temperature on most surfaces. in one report of 193 emergency department workers exposed to sars, nine (4.7%) were infected. pneumonia developed in six, two had mild illness and one remained asymptomatic. 16 the emergency department is a high-risk area because of its nature of trauma, heavy workload, crowded environment and lack of isolation facilities. there were at least two outbreaks in community clinic settings in hong kong. in one, a nurse was first infected by a sars patient attending the clinic and subsequently infected two other nurses, the doctor and his wife. in the second, a doctor was found to be infected on contact tracing of a household cluster of four sars cases. on further case tracing two more of the doctor's patients were found to be infected. 17 despite great concerns, compliance to infection control precautions by community general practitioners in hong kong lagged behind their hospital counterparts -97.7% had not worn masks at all times, a third did not wash their hands after seeing/examining a patient and half did not wear gowns. three-quarters did not wear goggles during patient encounters, just over half insisted patients wore masks during their consultation and over 10 doctors (12.4%) who were diagnosed with suspected or probable sars had closed their clinics. 18 however, the sample size is small and may not be representative of community doctors in hong kong. healthcare workers (hcws) working in small clinics are of particular concern because of their small size and lack of adequate decontamination facilities and a good ventilation system. ingenious designs have been developed by some to overcome this. the rate of transmition of the disease to their household contacts were 26.1%, 9.8% and 0% in non-healthcare workers, healthcare workers infected before the use of protective equipments and healthcare workers infected after the use of protective equipments respectively, indicating that use of protective equipments, adhering to infection control precautions and early quarantine were effective in stopping transmission. 19 a similar study in singapore also found that hcws had a lower rate of household transmission but that their secondary household transmission rate was higher (6.2%). 20 phasing of illnesses is probably the reason for a much higher transmission rate in healthcare facilities. in a study of 1192 patients with probable sars reported in hong kong, 26.6% were hospital workers, 16.1% were members of the same household as sars patients, 14.3% were amoy garden residents, 4.9% were in-patients and 9.9% were contacts of sars patients who were not family members. using multivariate analysis of 1: 2 matched casecontrols of the remaining cases of undefined sources of infection, it was found that having visited mainland china, hospitals or the amoy gardens were significant risk factors. in addition, frequent mask use in public venues, frequent hand washing and disinfection of living quarters were significant protective factors. 21 a similar study conducted in beijing on 94 unlinked probable sars cases also showed that clinical sars was associated with visits to fever clinics. 22 these indicate that household transmission is much less common and should allay public anxiety and panic. the main infection control measures are droplet and contact precautions. practices in paediatric and neonatal wards in hong kong that were utilised during the outbreak were well described. 23, 24 it is important that strict hand hygiene and adequate decontamination be performed after each direct or potential exposure to patients and at any time that body parts are perceived to be contaminated by patients' bodily fluids. a shower after high-risk procedures and before leaving duty would have been most desirable. the employment of a 'policing nurse' had been found to be very effective in ensuring compliance to infection control precautions and procedures during the sars epidemic. while n95 masks have higher filtration efficiency compared with surgical masks, they have lower breathability, higher thermal stress, more discomfort and cause more fatigue. 25 cdc recommended the use of n95 (95% filtration of 0.1 mm sodium chloride particles at a flow rate of 85 l/min), 26 the eu recommended the use of ffp2 or ffp3 masks (which had a filtering efficiency of 92% and 98% respectively, tested at 95 l/min with 0.1 mm sodium chloride particles) and canada n100 respirators (filtering efficiency of 99.97% for mono-dispersed particles of size 0.12 mm). 27 a full face respirator with an ultra-low penetrating air filter has also been recommended for its higher efficiency, good fit and protection of the mucous membranes but the disadvantage is cost, cleaning, disinfection and maintenance. 28 n95 masks should be test-fitted and the same model used whenever possible. a check fit should be performed each time one puts on the respirator and before entering the patient's room. in a study looking into factors affecting nosocomial infection in hong kong, it was found that all hcws consistently used n95s or surgical masks and perceived that the inadequacy of personal protective equipment (ppe) supply, infection control training <2 h and inconsistent use of goggles, gowns, gloves and caps were significant independent risk factors for sars infection. 29 the wearing of masks, gowns and goggles does pose considerable stress and fatigue to hcws. comfort and usability are other important issues to be considered. masks can also affect visibility and patient rapport. the psychological impact of masking on children has not been studied. in low-risk times and areas, surgical masks would probably be sufficient. it would be useful to have children wearing a surgical mask of appropriate size when they have respiratory symptoms, though the risk of transmission is considered to be lower than in adults. the associated discomfort may make it difficult to continue wearing the masks for a long period of time. with education, most children can be taught to put on a mask, at least when being examined or nursed and when outside the room. prescribed eye glass is not sufficient to protect against splashes. face shields should be sufficient for most pro-cedures unless excess splashes or direct coughing is expected, in these cases goggles should be worn. full face masks and hoods are more cumbersome alternatives. ppc are essential elements of infection control precautions, but which type of the available ppc provide better protection in terms of water repellency, water resistance, risk of environmental contamination, usability and comfort has not been determined. it is important to identify risk factors for non-compliance and design interventions and routines that are sustainable and practicable. in a study comparing different types of ppc available in hong kong, the use of a surgical gown in ordinary work procedures was recommended. when heavy splashes or droplets are expected, an additional plastic apron should be worn to protect the trunk. 30 it is important that ppc should be removed when soiled. due care should be taken to avoid contamination of the environment and ppc should only be worn when needed and removed immediately on leaving isolation rooms. great care should be taken when removing ppc. lack of appropriate ppc removal procedures can lead to lapses in infection control measures. this should be done outside the patient's area and with adequate spacing to avoid cross contamination and contamination of the environment. mirrors would be helpful so that one can observe the whole procedure. one must avoid contamination of the nose, mouth and eyes while removing the cap, gown, gloves, mask and eye protectors. there are several sets of recommendations on the sequence of removing ppc. the one recommended by the national institute for infectious diseases in italy is probably the safest. 31 the essentials are that the procedures are clear, consistent and simple to follow. the use of shoe covers is controversial and was not used in several hospitals in hong kong during the epidemics. stringent infection precautions, especially for high-risk procedures, appropriate triage and prompt isolation of potential sars patients will contribute to the control of nosocomial spread and acquisition of hcw in hospital settings. 23, 24, 32 in a retrospective case control study of 91 intubations, risk factors for infection included difficult intubation (or 8.8), extensive bagging (or 25.9), intubation in a general ward environment (or 8.2) and extensive droplet contamination. 33 before performing high-risk pro-cedures including cpr, intubation etc, one must ensure adequate protection in appropriate and properly equipped isolation facilities. call for help if alone and choose the right technique before embarking on the procedure. 34 the analogy of putting on your own oxygen mask before attending to others while in air flight emergencies should be remembered. neubulisation, bronchoscopy induced sputum collection and face mask ventilation should be avoided as far a possible. if medically indicated, they should be undertaken in a negative pressure room with minimal but adequate staffing. all staff should be in ppc covering the torso, arms and hands as well as eyes, nose and mouth. n95s, n-99s or n-100s are adequate but full face masks are desirable. however, the use of powered air purifying respirators is not recommended because of risk to self and environmental contamination. the use of a face mask with a good fit and attached valved manifold may reduce the risk of transmission. 35 no infection has been attributed to the taking of nasopharyngeal aspirate from sars patients in hong kong. when performed it should be taken in a single room while wearing full ppc. a new upper respiratory tract irrigation method has been devised to replace nasopharyngeal aspirate testing, which should be safer. 36 the disadvantage of this method is that it cannot be used in young children. early recognition followed by prompt initiation of isolation and infection control precautions are the most important strategies for controlling sars and other emerging infectious diseases. clinical features alone cannot reliably distinguish sars from other respiratory illnesses. having an epidemiological linkage was the most consistent finding (95.5%) in children infected with sars in hong kong. 37 combining clinical findings and epidemiological linkage or clustering of cases and interpreting clinical findings with key epidemiological risk factors serves as a good framework for triage, especially for children. precise and timely information about these epidemiological risks should be provided, coupled with proper training of frontline healthcare professionals on its interpretation. a predictive model basing on a four-item clinical score of cough before or concomitant with fever, myalgia, diarrhoea and rhinorrhoea or sore throat had a 100% sensitivity and 75.9% specificity of early detection of probable sars. the addition of lymphopaenia and thrombocytopaenia increased the specificity to 86.2%. 38 in another model, a scoring system of attributing 11, 10, 3, 3 and 3 points to the presence of independent risk factors of epidemiological link, radiographic deterioration, myalgia, lymphopaenia and elevated alt respectively, generated high(11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) and low-(0-10) risk scores for sars. the sensitivity and specificity of this prediction rule in positively identifying a sars patient were 97.7% and 81.3% respectively. 39 the prediction rule could be useful at the bedside. however, these studies were conducted in adult patients and would need to be validated in paediatric patients. other clinical guidance has also been developed but again is probably only applicable to the adult population. 40 the case definition for clinical sars used by leung et al. in hong kong was: fever (rectal temperature of 38.58c or oral temperature of 388c); chest radiograph (cxr) findings of pulmonary infiltrates or acute respiratory distress syndrome; and suspected or probable contact with a person under investigation for sars or exposure to a locality with suspected or documented community transmission of sars through either travel or residence within 10 days of the onset of symptoms, as well as 1 of the following: chills, malaise, myalgia, muscle fatigue, cough, dyspnoea, tachypnoea, hypoxia, lymphopenia, decreasing lymphocyte count, or failure to respond, in terms of fever and general well-being, to antibiotics covering the usual pathogens of community-acquired pneumonia (e.g. a broad-spectrum lactam plus a macrolide) after 2 days of therapy. this case definition had a sensitivity and specificity of 97.8% and 92.7% respectively in identifying paediatric sars during the sars outbreak. 36 while almost all reported patients with laboratory evidence of sars have radiographic evidence of pneumonia at some point during their illness, paediatric sars have non-specific radiographic features, making it difficult for radiological differentiation. 41 a private general clinic participating in the sars-screening programme in hong kong during the sars epidemic -by using telephone triage followed by chest radiograph of cases with 'flu-like' illness, the author successfully and safely screened 1161 attendees, x-rayed 151 patients and diagnosed one case of sars. therefore, a chest x-ray (cxr) would be a useful screening tool during outbreaks. 42 a sars and avian influenza algorithm for early recognition and investigation of potential paediatric cases, modified from the uk health protection agency's algorithm, is suggested in the appendix. 40 negative pressure rooms are recommended for the isolation of patients with sars. however, it should be noted that negative pressure rooms only prevent the virus from travelling outside the room and may not reduce viral load or environmental contamination inside the room. several designs such as low level suction and laminar flow have tried to reduce the viral load inside the room but the effectiveness is unproven. 43 various devices such as portable/mobile local exhaust ventilation devices, tents and personal isolation systems have been designed and tested but the usability, risk of contamination of staff and effectiveness are still under study. 44, 45 a rethink on the best design for effective infection control which also improves clinical and psychological care of patients is very much needed. no matter how good the design this cannot replace preparedness, a good clinical routine and appropriate personal protection. elaborate ventilation designs and negative pressure systems would be difficult in most clinical settings. exhaust fans and mobile local exhaust ventilation devices with hepa filters have been used in hospitals and clinics in hong kong. their efficacy has not been tested. several ingenious barrier precaution designs have been made by local medical practitioners: a torch mounted to a face shield for throat examination; cling film wrapping of telephones, keyboards and medical instrument to facilitate cleaning; and home-made air powered helmet hoods or tents for high-risk patients. these have not been tested and cannot replace hand hygiene, appropriate ppc and regular decontamination. while having adequate infection control equipment and facilities are important, overcrowding or inadequate bed/ clinic spacing or triage rooms and insufficient manpower are two major risk factors for hospital cross infection. having clear clinical guidelines and timely information are essential but it is it even more important that everyone has adequate information and proper training, practice and enforcement on infections and infection control starting in schools and in the community. panic and fear can be more harmful than the disease itself. sars and avian flu have taught us that infections are not just a problem for healthcare professionals, they involve everyone of all ages within communities throughout the world. adolescent twin sisters with severe acute respiratory syndrome a young infant with severe acute respiratory syndrome consensus document of the epidemiology of severe acute respiratory syndrome (sars) epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong capturing super spreading events during the 2003 sars epidemics in hong kong and singapore severe acute respiratory syndrome and biology, air quality, physics and mechanical enginerring spreading of disease through the drainage system evidence of airborne transmission of the severe acute respiratory syndrome virus epidemiological linkage and public health implication of a cluster of sars in an extended family evaluation of control measures implemented in the severe acute respiratory syndrome outbreak in beijing sars-associated coronavirus infection in teenagers severe acute respiratory syndrome and pregnancy clinical analysis of pregnancy in second and third trimester complicated severe acute respiratory syndrome sars and pregnancy: a case report report of sars expert committee how did general practitioners protect themselves, their family and staff during the sars epidemic in hong kong? risk of transmission of sars to household contacts from infected health care workers and patients heng eh, mark a, ma ea, tan oh. secondary household transmission of sars sars transmission, risk factors, and prevention in hong kong risk factors for sars among persons without known contact with sars patients infection control for sars in a tertiary paediatric centre in hong kong infection control for sars in a tertiary neonatal centre scientific evaluation of protection, heat stress, comfort and usability of facemasks cdc public health guidance for community-level preparedness and response to sars version 2. supplement i: infection control in healthcare, home and community setting sars and masks sars respiratory protection: update sars transmission among hospital workers in hong kong effective personal protective clothing for health care workers attending patients with sars sars and removal of personal protective equipment current concepts: the severe acute respiratory syndrome risk factors for sars in hcws following intubation of sars patients -a retrospective multi-centre study. sars clinical management workshop a practical approach to airway management in patients with sars dispersal of respiratory droplets with open vs. closed oxygen delivery masks -implications for the transmission of severe acute respiratory syndrome conjunctivaupper respiratory tract irrigation for early diagnosis of severe acute respiratory syndrome severe acute respiratory symptom among children predictive model of diagnosing probable cases of severe acute respiratory syndrome in febrile patients with exposure risk gold standard for sars and clinical diagnosis models sars and avian influenza algorithm: recognition, investigation and initial management of potential cases. uk health protection agency severe acute respiratory syndrome: chest radiographic features in children an outcome analysis of chest x-ray examination for detecting severe acute respiratory syndrome in general practice sars busters validating air distribution design and performance controlling biohazards at the source personal isolation system key: cord-325436-pp3q022y authors: alkhatib, ahmad title: antiviral functional foods and exercise lifestyle prevention of coronavirus date: 2020-08-28 journal: nutrients doi: 10.3390/nu12092633 sha: doc_id: 325436 cord_uid: pp3q022y novel coronavirus (covid-19) is causing global mortality and lockdown burdens. a compromised immune system is a known risk factor for all viral influenza infections. functional foods optimize the immune system capacity to prevent and control pathogenic viral infections, while physical activity augments such protective benefits. exercise enhances innate and adaptive immune systems through acute, transient, and long-term adaptations to physical activity in a dose-response relationship. functional foods prevention of non-communicable disease can be translated into protecting against respiratory viral infections and covid-19. functional foods and nutraceuticals within popular diets contain immune-boosting nutraceuticals, polyphenols, terpenoids, flavonoids, alkaloids, sterols, pigments, unsaturated fatty-acids, micronutrient vitamins and minerals, including vitamin a, b6, b12, c, d, e, and folate, and trace elements, including zinc, iron, selenium, magnesium, and copper. foods with antiviral properties include fruits, vegetables, fermented foods and probiotics, olive oil, fish, nuts and seeds, herbs, roots, fungi, amino acids, peptides, and cyclotides. regular moderate exercise may contribute to reduce viral risk and enhance sleep quality during quarantine, in combination with appropriate dietary habits and functional foods. lifestyle and appropriate nutrition with functional compounds may offer further antiviral approaches for public health. viral infections are responsible for significant global morbidity and mortality rates across the world, and viral outbreaks such as novel coronavirus (covid-19) [1] . reports from the world health organization (who) estimate 3-5 million hospitalized cases of seasonal influenza severe illness, resulting in 290,000-650,000 annual deaths [2] . currently, covid-19 is causing a health crisis across the world. limiting the spread of infections in the short and medium terms involves a number of preventative public health practices including regular hand washing, covering coughs, lockdown, and social distancing measures. vaccines have been implemented for preventing and controlling several viruses over the past century and have also been used for preventing common influenza [3, 4] . however, influenza vaccine development takes a significant amount of time [5] , which necessitates alternative complementary remedies for covid-19. furthermore, antiviral medication treatments face continuous challenges in terms of drug dose and selection and intervention phase, especially during acute respiratory infections [6] . lifestyle approaches could play an essential antiviral long-term preventative role. the antiviral role of nutrition and exercise as the two lifestyle prevention pillars has received little research attention. in particular, how the antiviral immunological defence capacity could be enhanced using functional foods, nutraceuticals, and physical activity behaviors, whether such behaviors are alone or combined. functional foods and nutraceuticals can be safe and cost-effective strategies to enhance the immune system and provide protection from pathogenic viral infections. for example, optimal intake of selected micronutrients has been highlighted in controlling the impact of virulent strain infections, including lower and upper respiratory tract infections, through optimizing a well-functioning immune system [7] . on the other hand, the role of physical activity and exercise in enhancing the immune system is well established [8] . nutrition and lifestyle modifications may not be definitive measures to absolutely prevent persons from contracting covid-19 when exposed. however, they could help as an adjuvant therapy to reduce the risk through enhanced immunity. this review presents key evidence on how functional foods and lifestyle approaches, including physical activity, effective for cardiometabolic disease prevention outcomes [9] , can also optimize the immune system response to viral infection, especially respiratory tract infections and covid-19. the review also makes specific and practical evidence-based recommendations for the use of antiviral functional foods and lifestyle approaches. in terms of physical activity prevention of chronic disease morbidity and mortality risks, a dose response relationship is well established for non-communicable disease (ncd) prevention [10] . exercise induces cardiovascular, respiratory, and metabolic adaptations, which result in higher maximal oxygen uptake ( . v o 2max ), carbon dioxide production, minute ventilation, breathing frequency, stroke volume, and cardiac output [11] . improvement in whole-body cardiometabolic and respiratory functions boosts the immune system defence through several acute and long-term mechanisms, which have been well highlighted recently [12, 13] . however, less is reported about how such exercise-dependent mechanisms protect against communicable diseases (cds), especially viral infections such as influenza and the recent covid-19 outbreak. exercise impacts all immune cells within both innate and adaptive immune systems, particularly elevating the activity of natural killer (nk) cells, neutrophils, and macrophages following moderate exercise (less than 60% of . v o 2max ) [8] . for example, acute aerobic exercise (running, cycling) have been shown to increase monocyte number [14] . monocytes play an integral antiviral role, and it has been shown that in a variety of influenza a viruses (including the circulating swine-origin virus, similar to covid-type viruses), exercise induces monocytes to differentiate within 18 hours into cd16(-)cd83(+) mature dendritic cells with enhanced capacity to activate t-cells [15] . long-term moderate exercise training increases the expression of t-helper (th) cells and associated th balances [16] . enhanced t-cell proliferation was particularly found following prolonged moderate exercise training in high-risk populations such as postmenopausal women cancer survivors and the very old. an increase in t-cell proliferation (by 218 per dpm × 106 cells) has been found in post-menopausal breast cancer survivors who exercised for 15-30 min for 15 weeks at 50% and 70% of . v o 2max [17] , while increased cd4+ t-cells were also found in older adults aged 80 years, walking 30 km per day [18] . those high-risk groups have been found to be particularly vulnerable to viral infections and more serious symptoms as reflected by recent governmental advice regarding covid-19 [19, 20] . exploring the exercise role in protecting and controlling covid-19 and other novel viral infections is essential. severe exercise intensity, whether acute or chronic, can be counter-productive in terms of the susceptibility to infections, since it is linked with higher upper respiratory tract infection rates among elite endurance athletes [21, 22] . mucosal immunology antibodies such as salivary immunoglobulin a (iga), and immunoglobin m (igm) concentrations have been shown to decline immediately after a bout of intense exercise in elite swimmers, but usually recover within 24 h [22] . however, modulating the intensity and duration of exercise can optimize the immune system response outcomes acutely and chronically [8, 23] . higher exercise intensities (above 70% . v o 2max ) and supramaximal exercise (100% or above) induce a transient oxidative stress and muscle damage response of oxidative stress, cell integrity, and homeostasis biomarkers, especially during the first 24 hour post exercise [24, 25] . both young and older adults have shown an increase in recombinant interleukin-2 (ril-2) stimulation of nk cells immediately (15 min) following an acute intense bout of maximal cycling exercise [26] . when followed for 12 days, it was found that neutrophil mobilization was concurrent with enzyme efflux, particularly those related to cell damage such as creatine kinase (ck) and antioxidative capacity such as superoxide dismutase (sod) [27] . acute repeated exercise bouts have also been implicated in the removal and regeneration of aged immune cells, especially cell senescent naïve, memory cd4+ and cd8+ t-lymphocytes, and an elevated apoptotic lymphocyte in peripheral blood [28] . the immunological transient response includes a temporal stress (e.g., disturbance of cell homeostasis and oxidative stress) induced by an acute exercise challenge, and may play a role in long-term enhanced immune system, especially when exercise is repeated chronically (i.e., exercise training). these entropic exercise-induced effects on the immune system may act as a natural vaccine against viral infections such as covid-19. in fact, eccentric exercise has already been demonstrated to act as an adjuvant to influenza vaccination in humans [29] . the trial randomized 60 healthy men and women who performed upper body eccentric exercise (deltoid and biceps brachii muscles) 6 hours prior to receiving influenza vaccination, and were monitored for antibody titers up to 20 weeks. the results showed that interferon-gamma responses were enhanced by exercise in men, whereas antibody titres were enhanced in women, which were concurrent with improved arm circumference (i.e., physical outcome benefit). the interferon-gamma response was positively associated with the percentage increase in arm circumference. the study suggested that eccentric exercise of the muscle at the site of vaccine administration could act as a behavioral adjuvant to vaccination. therefore, exercise immunological benefits alone or as an adjuvant antiviral treatment should be further investigated for preventing and controlling covid-19. the immune function response to exercise is influenced by several factors including nutritional status, body weight, hygiene, and mental health. the immune function is known to be superior in highly conditioned versus sedentary individuals. sedentary lifestyle and insufficient physical activity levels induce several physiological impairments, which reflect reduced cardiovascular and respiratory capacity, obesity, and associated cardiometabolic chronic diseases [30, 31] . consequences of sedentary lifestyle and physical inactivity include a compromised immune system due to manifestation of systemic inflammation, oxidative stress, and associated immunosuppressive mechanisms [10, 31] . prevalence of sedentary behavior and low physical activity levels have been reported in those with obesity, diabetes, and underlying insulin resistance and oxidative stress, and have been linked with increased susceptibility to contracting viral infections, including pandemic influenzas such as h1n1 and covid-19 [32] . conversely, higher physical activity and fitness levels in adults are associated with an optimized immunity indicated by reduced white blood cell count, c-reactive protein (crp), interleukins (il-6, and il-18), tumor necrosis factor alpha (tnf-α), and other inflammatory biomarkers [33] . therefore, any physical activity or exercise dose is considered beneficial compared to being sedentary, especially during and after covid-19-related lockdown, social distancing, or quarantine measures introduced in several countries. moderate exercise intensity is recommended, especially during and after a social distancing lockdown, which requires an avoidance of severe intensities. a practical method of achieving moderate exercise intensity is using 40-70% of maximum heart rate (hr max = 220-age). exercising at home, especially to perform resistance type activities using own body weight, and to interrupt sedentary behavior by reducing sitting times are particularly recommended for older and high-risk individuals with chronic conditions such as diabetes [31, 34] . exercise at home is also suited for the avoidance of the airborne coronavirus, especially during quarantine, and may include strengthening, balance and control, stretching, or a combination of these (walking, lifting and carrying, lunges, stair climbing, stand-to-sit and sit-to-stand using house items, squats, sit-ups, yoga) [35] . a volume increase in weekly exercise is recommended under the covid-19 quarantine from 150 min to 200-400 min aerobic exercise distributed across 5-7 days, with at least 2-3 resistance sessions, to compensate for the decreased mobility during lockdown [36] . this results in achieving an increase in . v o 2max as a practical aim. enhanced . v o 2max is particularly important for those who are considered at high-risk of covid-19 such as those who are overweight or those with obesity, insulin resistance, and diabetes, who typically have chronic low-grade inflammation characterized by increased levels of several pro-inflammatory cytokines and the inflammasome, and who are predisposed to greater risks for infection along with more adverse outcomes [37] . it is recommended that exercise is performed as part of a multicomponent personalized lifestyle approach (personalized nutrition, exercise intensities, technology, behavior, mental wellbeing) especially for high-risk individuals such as those with diabetes [38] . functional foods naturally possess active ingredients or "nutraceuticals" that are associated with disease preventative health benefits are now widely accepted for the prevention and management of major ncds, especially those characterized by inflammatory and oxidative stress disorders such as diabetes and cardiovascular disease [9, 39] . however, less is known about the role of functional foods in communicable diseases (cds), especially on the immune system defence against viral infections such as covid-19. a variety of fruits, vegetables, oily fish, olive oil, nuts, legumes are all considered functional foods based on their natural contents of nutraceuticals, including polyphenols, terpenoids, flavonoids, alkaloids, sterols, pigments, and unsaturated fatty acids [9, 40] . polyphenol-rich herbs, especially coffee, differently fermented teas (green, black)and yerba maté, have also shown to have various effectiveness on metabolic and microvascular activities, cholesterol and fasting glucose lowering, anti-inflammation and anti-oxidation in high-risk populations [9, 41] . bioactive peptides, naturally present in food proteins or formulated as nutraceuticals based on their molecular weight, amino acid chain length, or peptide composition, have also been postulated to elicit versatile physiological responses associated with immunological, antimicrobial, cardiovascular, gastrointestinal, neurological, and other hormonal activities of the human system [42] . such functional food benefits can be translated to protect against viral infections and covid-19. viral infections are characterized by a compromised immune function and deficient micronutrient stores, particularly vitamins, including vitamins a, b6, b12, c, d, e, and folate, and trace elements, including zinc, iron, selenium, magnesium, and copper [7] . evidence already supports an efficient function of the immune system through consuming those various nutraceuticals within a variety of functional foods including essential fatty acids, linoleic acids, essential amino acids, and the aforementioned vitamins and minerals, especially where forms of immunity may be affected by deficiencies in one or more of these nutraceuticals [43, 44] . adequate dietary intake, and supplementation of such functional foods, contribute to maintaining optimal levels in the human body, which enhances several aspects of the immune system [7, 45] , and provides an important antiviral prevention of covid-19 [46] . conversely, less robust immune responses have been shown to be the primary risk factor for covid-19 [47] , which makes it timely to describe the protective role of functional food component benefits in the context of preventing covid-19 and seasonal infections. in terms of jointly addressing ncd and cd prevention within high-risk populations, investigating the functional foods effects on cds including covid-19 is particularly important. higher infection and mortality rates related to covid-19 have been documented among older adults and patients with obesity, cardiac diseases, hypertension, or diabetes [48] . for example, covid-19 statistics in england showed that almost a third (31.3%) of covid-19-related mortalities had type-2 diabetes [49] , while there was a two-fold increase (86%) in requiring mechanical ventilation among covid-19 infected obese individuals compared with (47%) of infected healthy weight individuals [50] . the prevalence of ncds, especially diabetes amongst high-risk groups, is becoming a matter of emerging importance, and diabetes is now considered a risk factor for the progression and prognosis of covid-19 [51, 52] . therefore, optimal "immune-enhancing" functional foods combined with behavioral lifestyle approaches (especially exercise) could provide an optimal prevention of the double burdens of ncd and cd multimorbidity. various dietary patterns contain functional food components that have been promoted in the past for ncd prevention, especially the vegetarian diet, the nordic diet, or the mediterranean diet (md), and its combination with other lifestyle approaches [9, 39, 53, 54] . common functional foods within those diets include plant-based fruit and vegetables such as olive oil and tree nuts, seeds, fish, dairy products, and herbs, teas, and fermented products, which contain key nutraceuticals with disease protective anti-inflammatory and anti-oxidation properties [9, 54, 55] . established health protective functional components include monounsaturated fatty acids (mufa) such as oleic acid in olive oil, omega-3 polyunsaturated fatty acids (e.g., alpha-linolenic acid) found in tree nuts such as walnuts, eicosapentaenoic acid (epa), and docosahexaenoic acid (dha) found in oily fish, high amounts of polyphenol flavonoids and antioxidants found in fruit and vegetables, and high amounts of fiber found mainly in cereal and whole-grain foods [54] . consuming those functional foods, and their components vary across geographical global regions [9, 41, 54, [56] [57] [58] , but what is agreed on is their cardiometabolic protective benefits of reducing major ncds and mortality risks [9, 53, 59] . the challenge is to translate such functional effects towards enhancing and protecting the immune system and its antiviral defence response into the prevention of emerging cds such as covid-19. enhancing the antiviral immune defence can benefit from the functional food intake of a considerable variety of plant, animal, and fungi species, consumed across different diets and cultural practices including traditional herbal medicine such as teas, roots, mushrooms, and fermented plants and leaves; md components such as olive-based products, oily fish, seeds, fruits, and vegetables; popular beverages such as coffee; and protein-rich foods such as chicken extract and soybean peptides. the majority of such foods contain naturally occurring vitamins and minerals (e.g., vitamins c, d, b 6 , b 12 , a, e, and minerals of zinc, copper, iron, and selenium), and other phenolic compounds that are immunoprotective particularly through antioxidation and anti-inflammation properties [41, 43] . other foods such as oily fish omega-3 fatty acids contain monounsaturated fatty acids such as omega-3 fatty acids (epa and dha) in oily fish, which can be enzymatically converted to specialized pro-resolving mediators (spms) known as resolvins, protectins, and maresins, which are molecules that support inflammatory resolution and healing of infected sites including the respiratory tract, which could prevent acute lung injury [7] . fermented food products (e.g., yoghurt, pickles, fermented fruits, vegetables, plant, and drinks) contain probiotics, and have also been shown to enhance gut bacteria profile and gut-lung axis-related respiratory fitness [60, 61] . a summary of systematic reviews and randomized controlled trials reported reduced incidence and severity of upper and lower respiratory tract infections (odds ratio ∼ 0.8-0.5) by using different probiotics, especially lactobacilli and bifidobacteria [61] . the efficacy of probiotics in reducing covid-19 infected patients has not yet been established, but the prophylactic benefits for enhancing the immune system are supportive of their long term use [60] , especially considering that improving gut microbiota profile has been recently implicated in preventing covid-19 in older and high-risk individuals with compromised immune systems [62] . selected food supplements and micronutrient vitamins and trace elements have been reviewed elsewhere in terms of optimizing the immune responses [7, 63, 64] . other reviews have highlighted the importance of key vitamins (e.g., vitamin d) for regulating sleep patterns during quarantine or lockdown measures [65] [66] [67] . given the promising role of popular functional foods, such as those within the md including olive oil, and asian and african herbal teas and fermented foods and popular beverages as part of lifestyle disease prevention [11, 40] , it is important to contextualize the antiviral mechanisms of such functional foods. below is a review of popular foods within various dietary patterns, including olive oil nutraceuticals, popular vitamins such as vitamin d, traditional medicinal herbs and roots, and protein peptides for preventing viral infections including covid-19, especially when they are adopted as part of an active lifestyle. olive oil (oo), and its constituents (leaves and bark), form an important immune-enhancing functional food due to the significant ncd preventative benefits, especially of cardiovascular disease, diabetes, and cancer, which have been reviewed elsewhere [40, 68] . oo, especially extra-virgin oo (evoo) contains monounsaturated fatty acids, and several polyphenols including oleuropein and hydroxytyrosol, which have several antioxidative and anti-inflammatory properties, which can be linked with significant antiviral and antibacterial potential. oleuropein has shown a potential antiviral activity against respiratory syncytial virus (rsv), a common upper respiratory infection (uri) virus [69] . this effect has been attributed to the antioxidative property of elenolic acid as a main fragment in oleuropein, which has long been shown to have potent antiviral activities against herpes, influenza a and b, and parainfluenza 1, 2, and 3 viruses [70] . antioxidant capacity of oo was later shown to be independent of the size of the antiviral effect, with oleuropein showing superior antiviral effects compared with other secoiridoid glucosides isolated from ligustrum lucidum [69] . however, antioxidant properties can vary among oo phenolics. a more recent study by paiva-martins et al. (2010) [71] compared the capacity of four important oo phenolic compounds, oleuropein, hydroxytyrosol, and the oleuropein aglycones 3,4-dihydroxyphenylethanol-elenolic acid (3,4-dhpea-ea) and 3,4-ihydroxyphenylethanol-elenolic acid dialdehyde (3,4-dhpea-eda) for their protection of red blood cells (rbcs) from oxidative haemolysis induced by the physiological initiator h2o2. the study tested the amount of haemolysis by spectrophotometry, and the compounds were also tested in the presence and absence of the naturally occurring antioxidant ascorbic acid. all compounds were revealed to significantly protect rbcs from oxidative haemolysis induced by h2o2 at 40 and 80 µm, with the order of activity being 3,4-dhpea-eda>3,4-dhpea-ea>hydroxytyrosol=oleuropein. however, at 20, 10, and 5 µm, only 3,4-dhpea-eda showed a significant protection against the oxidative injury, suggesting that 3,4-dhpea-eda plays an important protective role against reactive oxygen species-induced oxidative injury in rbcs, and this effect is more potent than the one evidenced by hydroxytyrosol or oleuropein. the antioxidation protective benefits of oo, especially evoo, which has a higher phenolic content [40] promotes its role for enhancing the immune system defence against viruses. hydroxytyrosol antiviral mechanisms were showed through its inactivation effects on influenza a viruses, especially during the virus morphological changes, such as the presence of a viral envelope which is an integral membrane protein involved in several aspects of the virus life cycle including its assembly, budding, and pathogenesis [72] . the mechanisms of which oo nutraceuticals protect against viral infections have often focused on the hydroxytyrosol preventative effects on hiv from entering the host cell and binding the catalytic site of the hiv-1, and its inhibitory effect on both viral entry and integration [73] . regular intake of olive leaf extracts, rich in polyphenol flavonoids, have been shown to be responsible for a 33% reduction in uri [74] . such promising antiviral potential was attributed to the following antioxidation actions of oleuropein with dose-dependent inhibition of the copper sulphate-induced oxidation of low-density lipoproteins (ldls), and induced increase in nitric oxide production in macrophages and functional activity. in another study among high-school athletes who were prone to uri, olive leaf extract supplementation (20 g, containing 100 mg oleuropein) was shown to reduce the duration of infection (28% reduction in sick days) but not the incidence rate [75] . thus, olive polyphenols (both in oo and leaf), especially oleuropein and hydroxytyrosol, seem to promote antiviral defence and can be an adjacent prevention to control uris. exploring oo mechanisms for protecting against novel viruses such as covid-19, especially its protein viral envelop function and interaction with host cells would also be important. the benefits of oo intake, especially as part of a balanced diet such as the md can be further augmented via physical activity, especially strength and resistance type exercise [40] . such an approach is likely to be an effective prevention of viral infections. in terms of the recommended oo dose, a moderate dose of 20-30 g/day (especially polyphenol-rich evoo) in combination with other dietary functional foods can be recommended for enhancing the immune system, which is in line with recent ncd prevention recommendations [68] . the role of vitamin d in ameliorating the effects of both ncds and cds is now well accepted. vitamin d reduces acute respiratory tract infection, and its deficiency is linked with susceptibility to viral infections, and also with various cancers, diabetes, and cvd [64, 66] . research into the role of vitamin d in preventing covid-19 and influenza viral infections has gained recent momentum through various reviews and meta-analyses, especially given that individuals who are susceptible to covid-19 infections are mainly high-risk individuals with various ncds such as diabetes and cvd, suggesting that vitamin d could be the missing link between ncds and viral cds [46, 66] . based on the latter [66] , several mechanisms of how adequate vitamin d availability can reduce the risk of viral infections and covid-19 through the following mechanisms: (a) lowering viral replication rates through cathelicidins and defensins, and preventing lung injures that lead to pneumonia through its anti-inflammatory cytokines; (b) potential for vitamin d supplementation effectiveness in reducing the risk of influenza especially in deficient individuals; (c) documented reduced risk of covid-19 during the summer indicated by a lower number of cases in the southern hemisphere, compared with higher number of cases in the winter months when 25-hydroxyvitamin d (25(oh)d) concentrations are lowest; (d) vitamin d deficiency has been found to contribute to acute respiratory distress syndrome; and that case-fatality rates increase with age and with chronic disease comorbidity, both of which are associated with lower 25(oh)d concentration. sleep disorders during covid-19 quarantine could also be ameliorated through maintaining adequate vitamin d levels within the body. this could mainly be done through sun exposure [76] , and to a smaller extent from dietary intake or supplementation [77] . vitamin d plays an important role in regulating sleep patterns, circadian rhythm, enhancing sleep quality, and indirectly ameliorating sleep apnea [78] . for example, vitamin d receptors and the enzymes that control its activation and degradation have been found in brain regions involved in sleep regulation; and vitamin d is also involved in melatonine production pathways, and can affect restless legs syndrome and obstructive sleep apnea syndrome [67] . the best source of vitamin d is sunshine exposure, but it is abundant in several foods including oily fish, tuna, dairy, and egg yolk. grant et al. (2020) [46] recommended supplementation in individuals with highest risk of covid-19 infection or vitamin d deficiency (10,000 iu/d of vitamin d3) to raise 25(oh)d concentrations above 40-60 ng/ml (100-150 nmol/l). for treatment of people who become infected with covid-19, higher vitamin d3 doses are recommended. traditional antiviral medicinal therapies across different cultures are essentially based on a combination of several functional foods and nutraceuticals with active immunomodulators, polyphenols, anti-inflammatory, and anti-oxidation components. armeniacae semen (apricot seeds), cinnamomi cortex (chinese cinnamon), glycyrrhizae radix (liquorice root), and ephedrae herba form a japanese traditional medicine called "maoto", which is often administered orally as granules to adults with seasonal influenza [79] . it has been shown to be well tolerated and associated with equivalent clinical and virological efficacy to neuraminidase inhibitors in helping progeny influenza viruses to leave without re-infecting the host cell [80] . licorice roots which contain the active component glycyrrhizin, have been shown to inhibit influenza a virus uptake into the cell, and reduced ccid50 by 90% [81] . licorice and curcumin have recently been reviewed for their postulated antiviral potential [82] . other common traditional herbal remedies for respiratory viruses that have been supported by scientific evidence include berries' extracts, echinacea, clinacanthus siamensis, punica granatum (pomegranate), psidium guajava linn. (guava tea), epimedium koreanum nakai; scutellaria baicalensis georgi (baicalin), and paeonia lactiflora pall. (bai shao) [79] . examples of their antiviral role include reduction in viral replication, enhancement of anti-influenza virus igg and iga antibody production, improvement of t-cell function (e.g., stimulation of interferon-gamma production by t-cells), neuraminidase inhibitor, virus budding prevention, inhibition of viral rna and viral protein synthesis, viral haemagglutination, viral binding to and penetration into host cells [79] . fungi are also commonly used in asian and chinese medicine to enhance the immune system. for example, cordyceps militaris is a mushroom traditionally used for diverse pharmaceutical purposes in east asia, including china, for enhancing immunity. in a human study 1.5g/day of cordyceps militaris for 4 weeks enhanced the nk cell activity and lymphocyte proliferation and partially increased th cytokine secretion [83] . immune enhancing antiviral mechanisms of traditional medicine especially roots and fungi are important preventing and controlling novel influenza viruses, including covid-19. promising evidence has been shown about the restorative and antioxidative role of traditional medicinal herbs and peptides post trauma or physical challenges, which may be important in lung injury pathology. for example, the chinese ginseng rg1 herb has been shown to restore satellite cell depletion following an exercise challenge, through enhancing glutathione (gsh), and gssg [84] . gsh is considered important in immune modulation, remodeling of the extracellular matrix, apoptosis, and mitochondrial respiration through its gamma-glutamylcysteine synthetase heavy and light subunits oxidant/antioxidant response to phenolic antioxidants, and is considered key to the development of an oxidant/antioxidant imbalance in lung inflammation [85] . in another study it was shown that anserine, beta-alanyl-3-methyl-l-histidine, a dipeptide, replenished the free radical scavenging enzymes sod (superoxide dismutase) and preserved catalase and gsh cofactors, while preserving cell integrity and homeostasis, together with a haematological increase in red blood cell volume-to-concentration and an attenuated white blood cellelevation following muscular challenge in healthy men [24] . dietary intake of anserine, carnosine dipeptides, and other animal-based amino acids including taurine, creatine, and 4-hydroxyproline promote the immunological defence of humans against infections by bacteria, fungi, parasites, and viruses (including coronavirus) through enhancing the metabolism and functions of monocytes, macrophages, and other cells of the immune system [86, 87] . plant-based peptides from soybean have also been shown to modulate cellular immune systems (increased lymphocytes and granulocytes number, increased cd11b(+) cells and cd56(+) natural killer cells), regulate neurotransmitters (decreased adrenaline and increased dopamine), and boost brain function [88] . however, fish, meat, and poultry are the primary sources of immunomodulatory peptides and amino acids, and hence they have long been considered functional foods taken to alleviate fatigue, respiratory, and cold symptoms in older individuals, especially in asia [86, 87, 89] . plant cyclotides are well-studied antivirals, since they can be mimicked for antiviral drug development, given their stable chemical structure [90] . they have been originally extracted from african tea used in traditional african medicine to accelerate childbirth because of their postulated uteroactive antiviral hiv properties [90] . the protective mechanisms of plant cyclotides against infections and pathogens are postulated through preventing malfunctioning of the immune cells by growth-inhibiting growth effects on the human immune system especially on lymphocytes (e.g., t-cells), which can cause an over-reactivity of this defence machinery during infections [91] . cyclotides can be obtained from various plants including violaceae and rubiaceae, but are abundant in several other plant families, especially cucurbitaceae (e.g., squash, pumpkin, zucchini), fabaceae (legumes, peas, beans), and solanaceae (eggplant, tomato, potato, pepper). therefore, it is likely that consumption of such foods, especially seasonal intake plays a protective role in enhancing the immune system and enhances antiviral defence mechanisms. coffee, caffeine, and naturally caffeinated beverages are well known to induce various health benefits and prevention of disease. all forms of coffee consumption (differently roast beans, fermented or non-fermented leaves) are common across various cultures across the world for centuries [92, 93] . epidemiological evidence suggests that consuming 2-3 cups of coffee daily is associated with reduced incidence of metabolic diseases which are often concurrent with a compromised immune system such as diabetes [41, 94] . therefore, it is plausible to imply a positive role for caffeine as a useful immunomodulator. nutraceuticals within coffee have shown different antiviral outcomes, with caffeic acid inhibiting the multiplication of influenza a virus in vitro, whereas caffeine, quinic acid, and chlorogenic acid do not [95] . caffeic acid has also been shown to have antiviral activity against herpes simplex virus (dna virus) and polio virus (rna virus), and to decrease the progeny virus yield (especially within 3 h post-infection) and suppresses the degeneration of the virus-infected cells [95, 96] . however, caffeine reported immuno-protective mechanisms from laboratory in vivo and in vitro trials have been equivocal [97, 98] . positive caffeine effects on innate immunity involve suppression of neutrophil and monocyte chemotaxis, and pro-inflammatory cytokines (such as tnf-α) from human blood, but caffeine has also been reported to suppress antibody production and human lymphocyte function as indicated by reduced t-cell proliferation and impaired production of th1 (il-2 and interferon-gamma), th2 (il-4, il-5), and th3 (il-10) cytokines [99] . some of the immunomodulatory actions of caffeine have been explained by its inhibitions of cyclic adenosine monophosphate (camp)-phosphodiesterase, and consequential increase in intracellular camp concentrations [99] . however, recent in vitro evidence suggests that caffeine may suppress endotoxins lipopolysaccharide (lps)-induced inflammatory responses by regulating nuclear factor nf-κb activation and mapk phosphorylation [100] . lps activation of nf-κb triggers mucin transcriptors (e.g., muc2 gene) and respiratory tract mucus in response to respiratory pathogens including influenza viruses [101] . caffeine suppression of lps has also been reported in a recent human study in females with obesity [102] . the latter study also found that caffeine ameliorates the obesity-induced metabolic side-effects following intense exercise lifestyle intervention including elevated lps, insulin action, glucose homeostasis, and androgen levels. this suggests that caffeine optimizes the metabolic and immunoprotective benefits when combined with other lifestyle components, especially exercise. future research is needed to determine caffeine antiviral effects for the prevention and management of covid-19. exercise and physical activity enhance the immune system and reduce susceptibility to infections, especially respiratory infections including covid-19. moderate intensity exercise can be adopted by the large population including high-risk groups with ncds such as those with diabetes and cardiovascular disease. functional foods may provide a further effective diverse antiviral approach and could have a joint prevention of both ncds and cds among diverse populations. dietary intake of foods rich in vitamins and minerals can be increased to provide an immune boost, especially in individuals with deficiency in these micronutrients. increased intake of probiotics, omega-3 from fish, protein peptides from chicken and fish, and olive-based products are also recommended (table 1, figure 1 ). there is no specific model to follow to enhance the immune system against covid-19. however, the more varied the dietary sources, the better the protection is against all viral infections. adopting exercise together with an enhanced dietary intake of functional compounds may contribute as a preventative medicine against emerging viral infections. promote antioxidation and anti-inflammation properties, protect the respiratory system, and reduce risks of infection and re-infection [44] . cyclotides protect against infections and pathogens by preventing malfunctioning of the immune cells (t-cell lymphocytes), which reduces over-reactivity of this defence machinery during infections [91] . intake is highly recommended as part of a balanced diet. complements an active lifestyle, supports circadian rhythm, and sleep quality dairy products vitamins d, a, & e vitamin d reduces the risk of contracting respiratory infections and covid-19 [46, 64] . lowers viral replication rates through cathelicidins and defensins, and prevents lung injures that lead to pneumonia through anti-inflammatory cytokines [66] . dietary intake is preferred. supplements (zinc, selenium, and vitamin d) are recommended in older adults and the most deficient. enhances sleep quality. seeds and nuts zinc, selenium, copper, trace minerals contain phenolic compounds that are immunoprotective particularly through antioxidative and anti-inflammatory properties in high-risk adults [9] . supplementation is recommended when dietary intake is low, especially in older and high-risk individuals support inflammatory resolution and healing of infected sites including the respiratory tract, which could prevent acute lung injury, mainly through pro-resolving mediators (spms) such as resolvins, protectins, and maresins [7] . increased intake is recommended in high-risk individuals protein rich foods (e.g., red meat, chicken, seafood) amino acids and peptides: anserine, carnosine, taurine, creatine, and 4-hydroxyproline, vitamins, iron, copper dietary intake of anserine and carnosine promote immunological defence against infections by bacteria, fungi, parasites, and viruses (and coronavirus) through enhanced immune cell functions of monocytes and macrophages [24, 86] . plant peptides (e.g., soybean) increase lymphocytes and granulocytes; enhance natural killer activity [88] . dietary intake is sufficient, but an increased intake is recommended in high-risk individuals and infected patients. can be obtained from both animal and plant sources. olive based products (olive oil, olive leaves) oleuropein, hydroxytyrosol, elenolic acid, vitamin e reduced upper respiratory infection attributed to antioxidative property of oleanolic acid in oleuropein, especially influenza a and b, parainfluenza 1, 2, and 3 viruses, and herpes [69] . dietary intake (20-30 g/day), especially from extra-virgin olive oil, which is high in polyphenol content. increase benefits with physical activity. coffee (coffee leaves, differently fermented) caffeic acid, caffeine, polyphenols, chlorogenic acid caffeic acid decreases the progeny virus yield (especially within 3 h post-infection) and suppresses the degeneration of the virus-infected cells; caffeine can suppress of neutrophil and monocyte chemotaxis, and pro-inflammatory cytokines (e.g., tnf-α) [96] . it suppresses endotoxins lps-induced inflammatory responses (regulates nf-κb activation and mapk phosphorylation) [102] , and prevents mucosal response to pathogens infecting the respiratory tract and influenza viruses [101] . coffee intake (2-3 cups/daily) is recommended and has superior immunological benefit to caffeine supplementation since it is more wholesome (contains both caffeic acid and caffeine). roots & fungi, traditional herbs, and medicinal plants maoto, licorice roots, cordyceps mushrooms, chinese mushrooms, ginseng herbs and roots prevent viral replication, enhance anti-influenza virus igg and iga antibodies production, and improve t-cell function [80] . glycyrrhizin (in maoto) helps progeny influenza viruses to leave without re-infecting, inhibits influenza a virus uptake into the cell and reduces ccid50 by 90% [82] . ginseng and cordyceps have antioxidative (gsh, sod) and cell senescence angiogenesis properties [84] . dietary intake is highly recommended. supplement when dietary intake is low (e.g., cordyceps, 1.5 g/day). microbiota especially lactobacilli and bifidobacterial enhance gut bacteria profile and gut-lung axis-related respiratory fitness [61, 62] . dietary intake of fermented foods is recommended covid-19, novel corona virus-19; epa, eicosapentaenoic acid; dha, docosahexaenoic acid; gsh, glutathione; sod, superoxide dismutase; igg, immunoglobulin g; iga immunoglobulin a; lps, lipopolysaccharides; tnf-α, tumor necrosis factor-alpha; nf-κb, nuclear factor-κb; mapk, mitogen-activated protein kinase. world health organization (who). fact sheets. influenza (seasonal) editors of the journal of 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potential as immunosuppressant peptides? effects of processing method and age of leaves on phytochemical profiles and bioactivity of coffee leaves a historical study of coffee in japanese and asian countries: focusing the medicinal uses in asian traditional medicines coffee consumption and risk of type 2 diabetes mellitus among middle-aged finnish men and women antiviral activities of coffee extracts in vitro inhibition by caffeic acid of the influenza a virus multiplication in vitro coffee and the immune system caffeine: well-known as psychotropic substance, but little as immunomodulator immunomodulatory effects of caffeine: friend or foe? caffeine prevents lps-induced inflammatory responses in raw264.7 cells and zebrafish the interaction between respiratory pathogens and mucus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license author contributions: a.a. performed all parts of this work including concept, design, literature scoping and synthesis, and writing all parts of the manuscript. the author have read and agreed to the published version of the manuscript. the authors declare no conflict of interest. key: cord-306278-c4q4la5c authors: esposito, susanna; zampiero, alberto; bianchini, sonia; mori, alessandro; scala, alessia; tagliabue, claudia; sciarrabba, calogero sathya; fossali, emilio; piralla, antonio; principi, nicola title: epidemiology and clinical characteristics of respiratory infections due to adenovirus in children living in milan, italy, during 2013 and 2014 date: 2016-04-05 journal: plos one doi: 10.1371/journal.pone.0152375 sha: doc_id: 306278 cord_uid: c4q4la5c to evaluate the predominant human adenovirus (hadv) species and types associated with pediatric respiratory infections, nasopharyngeal swabs were collected from otherwise healthy children attending an emergency room in milan, italy, due to a respiratory tract infection from january 1 to february 28 of two subsequent years, 2013 and 2014. the hadvs were detected using a respiratory virus panel fast assay (xtag rvp fast v2) and with a hadv-specific real-time polymerase chain reaction; their nucleotides were sequenced, and they were tested for positive selection. among 307 nasopharyngeal samples, 61 (19.9%) tested positive for hadv. hadv was the only virus detected in 31/61 (50.8%) cases, whereas it was found in association with one other virus in 25 (41.0%) cases and with two or more viruses in 5 (8.2%) cases. human enterovirus/human rhinovirus and respiratory syncytial virus were the most common co-infecting viral agents and were found in 12 (19.7%) and 7 (11.5%) samples, respectively. overall, the hadv strain sequences analyzed were highly conserved. in comparison to hadv-negative children, those infected with hadv had a reduced frequency of lower respiratory tract involvement (36.1% vs 55.2%; p = 0.007), wheezing (0.0% vs 12.5%; p = 0.004), and hospitalization (27.9% vs 56.1%; p<0.001). antibiotic therapy and white blood cell counts were more frequently prescribed (91.9% vs 57.1%; p = 0.04) and higher (17,244 ± 7,737 vs 9,565 ± 3,211 cells/μl; p = 0.04), respectively, in children infected by hadv-c than among those infected by hadv-b. on the contrary, those infected by hadv-b had more frequently lower respiratory tract involvement (57.1% vs 29.7%) but difference did not reach statistical significant (p = 0.21). children with high viral load were absent from child care attendance for a longer period of time (14.5 ± 7.5 vs 5.5 ± 3.2 days; p = 0.002) and had higher c reactive protein levels (41.3 ± 78.5 vs 5.4 ± 9.6 μg/dl; p = 0.03). this study has shown that hadv infections are diagnosed more commonly than usually thought and that hadvs are stable infectious agents that do not frequently cause severe diseases. a trend toward more complex disease in cases due to hadv species c and in those with higher viral load was demonstrated. however, further studies are needed to clarify factors contributing to disease severity to understand how to develop adequate preventive and therapeutic measures. frequently cause severe diseases. a trend toward more complex disease in cases due to hadv species c and in those with higher viral load was demonstrated. however, further studies are needed to clarify factors contributing to disease severity to understand how to develop adequate preventive and therapeutic measures. human adenoviruses (hadvs) are a group of at least 68 non-enveloped viruses containing double-stranded linear dna [1] . they belong to the family adenoviridae, genus mastadenovirus and are categorized into seven species (a-g) according to their biophysical, biochemical, and genetic characteristics. moreover, in each of these species, several types have been identified [1] . species identity strongly correlates with antigenicity, epidemiologic characteristics, clinical manifestations of hadv infection, and in vitro response to some antiviral drugs [2, 3] . from a clinical point of view, species d (hadv-d8, hadv-d19, and hadv-d37) is usually associated with the development of conjunctivitis; species f (hadv-f40 and hadv-f41) is usually associated with gastroenteritis; and species b (hadv-b3 and hadv-b7), c (hadv-c1, hadv-c2, and hadv-c5), and e (hadv-e4) are usually associated with respiratory diseases [2] . recombination between members of the same species and between members of different species has been frequently described [4] . as a result, certain new types may acquire different pathogenicity and have strong potential for widespread and epidemic outbreaks. consequently, surveillance of hadv circulation with an early evaluation of the relationships between clinical manifestations and molecular characteristics of new infecting strains may be important for the development of adequate diagnostic, prophylactic, and therapeutic measures against hadv infection. hadvs play an important role in the determination of respiratory infections, particularly in children. hadvs are responsible for a number of lower respiratory tract diseases in children, including community-acquired pneumonia (cap). wo et al. reported that among 3,089 nasopharyngeal aspirates collected in children with cap in china, 186 (6.0%) tested positive for hadv [5] . although hadvs are associated with mild to moderate disease in most cases, lifethreatening disease can occur in some patients, particularly if they are immunocompromised, [2] . overall, little data on hadv circulation have been collected in europe and no recent data regarding the epidemiology, molecular characterization, and clinical features of respiratory hadv infections in children have been collected in italy. the main aim of this study was to evaluate the predominant hadv species and types associated with pediatric respiratory infections in milan, italy, during two consecutive winter seasons. clinical features related to hadv types and genetic characteristics were also studied. to evaluate the circulation of the different hadv types and the possible relationship between viral load, viral genetic characteristics, and the severity of infection, nasopharyngeal swabs were collected from otherwise healthy children consecutively attending the emergency room of the fondazione irccs ca' granda ospedale maggiore policlinico, university of milan, italy, due to a respiratory tract infection. the study was carried out during the period from january 1 to february 28 in two subsequent years, 2013 and 2014, and was approved by the ethics committee of the fondazione irccs ca' granda ospedale maggiore policlinico, milan, italy. written informed consent from a parent or legal guardian was required, and children 8 years of age were asked to give their written assent. patient demographic characteristics and medical histories were systematically recorded before the visit to the emergency room using standardized written questionnaires. the study patients were classified into disease groups (i.e., acute otitis media, rhinosinusitis, pharyngitis, croup, infectious wheezing, acute bronchitis, pneumonia) on the basis of signs and/or symptoms using well-established criteria and were subdivided into two subgroups: upper respiratory tract infections (urtis) and lower respiratory tract infections (lrtis) [6] . nasopharyngeal secretions were collected from all of the children immediately after admission to the emergency room using a paranasal flocked swab (1 swab per child), which was stored in a tube containing 1 ml of universal transport medium (kit cat. no. 360c, copan italia, brescia, italy). viral nucleic acids were extracted from the swab by means of a nuclisens easymag automated extraction system (biomeriéux, craponne, france), and the extract was tested for respiratory viruses using the respiratory virus panel (xtag rvp fast v2) (luminex molecular diagnostics, inc., toronto, canada) fast assay in accordance with the manufacturer's instructions (luminex molecular diagnostics inc.) this assay simultaneously detects the following viruses: influenza a virus (flua subtypes h1 or h3); influenza b virus (flub); respiratory syncytial virus (rsv); parainfluenzaviruses (hpiv) 1-4; adenoviruses (hadv); human metapneumovirus (hmpv); coronaviruses (hcov) -229e, -nl63, -oc43, and -hku1; enterovirus/rhinovirus (hev/hrv) and human bocavirus (hbov). moreover, considering the risk of false negative results reported for the rvp fast v2 assay [7] , the negative samples were also tested with an alternative hadvspecific real time-pcr [8] . positive results were also quantified with a hadv-specific real-time polymerase chain reaction (pcr), as previously described [9] . viral nucleic acid extracts were tested using a specific hadv plasmid by a single-plex realtime pcr using taqman universal master mix ii (applied biosystems, foster city, california, usa). amplification and detection of viral dna was performed with a 7900ht real-time pcr system instrument (applied biosystems). the real-time hadv-specific primer sequences were as follows: 5'-gccacggtggggtttctaaactt-3', adenoquant 1 (aq1) and 5'-gcccc agtggtcttacatgcacatc-3', adenoquant 2 (aq2). the sequence of the probe was 5'-tgcaccagacccgggctcaggtactccga-3' (adenoprobe) labeled with fam on the 5'end as a fluorescent dye and labeled with tamra on the 3'-end as a fluorescence quencher dye. cycling conditions were as follows: 50°c for 2 min, 95°c for 8 min and 50 cycles of 95°c for 15 sec and 59°c for 1 min. the plasmid amplified target fragment was verified by sequencing [7] . plasmid dna concentrations were detected using an nd-1000 spectrophotometer (nanodrop products, wilmington, de). real-time fluorescence quantitative pcr was carried out in a total reaction volume of 20 μl consisting of 10 μl of taqman universal master mix (applied biosystems), 0.8 μl (0.4 mm) of each primer, 0.6 μl (0.3 mm) of the probe, 5 μl of template, and 2.8 μl of double-distilled water. the real-time pcr thermal cycling reaction and quantitative measurement were performed in a stepone real-time pcr instrument (applied biosystems) using the following conditions: one cycle at 50°c for 2 min, one cycle at 95°c for 10 min, 45 cycles at 95°c for 15 s, and one cycle at 60°c for 1 min [9] . each run included plasmid and negative controls. standard precautions were taken throughout the pcr process to avoid cross-contamination. negative controls and serial dilutions of the plasmid positive control were included in every pcr assay. finally, quantitative results were reported as dna copies/ml of respiratory samples. hadv typing was performed by sequencing the hypervariable region (1-6) of loop 1 of the hexon protein using a protocol proposed by lu and erdman [10] . pcr products ranging in size from 764 to 896 bp (first pcr) and 688 to 821 bp (nested pcr). first-round amplification was carried out using primers for adhexf1 (nt 19135-19160 geneamp pcr system 9700 (applied biosystems) with the following settings: 94°c for 2 min denaturation followed by 35 cycles of 94°c for 1 min, 45°c for 1 min, and 72°c for 2 min, with a final extension of 72°c for 5 min. for the nested reaction, 0.5 μl of the first pcr product was amplified as above. amplified products were separated on 1% agarose gels and purified with the qiaquick pcr purification kit (qiagen, chatsworth, california, usa). sequencing was performed in both directions using adhexf2/adhexr2 primers and the abi prism bigdyetm terminator cycle sequencing ready reaction kit ver. 3.1 on an abi 3100 dna sequencer (applied biosystems). sequencher 3.1.1 software (gene codes, ann arbor, minnesota, usa) was used for sequence assembly and editing. all sequences were aligned using clustalx 2.1 and bioedit (version 7.1.3.0) software (ibis biosciences, carlsbad, california, usa). phylogenetic trees were generated using the maximum likelihood method with molecular evolutionary genetics analyses (mega) software, version 5.05 [11] , and adenovirus prototype strains. bootstrap probabilities for 1,000 iterations were calculated to evaluate confidence estimates. the graphs were made using graphpad prism version 5.01 for windows (graphpad software, san diego, california, usa). all the hadv sequences originated from this study were submitted to genbank (accession numbers kt963953-kt964000). descriptive statistics of the responses were generated. continuous variables were presented as the mean values and standard deviations (sds), and categorical variables were presented as numbers and percentages. for categorical data, comparisons between groups were performed using a contingency table analysis with a χ 2 or fisher's exact test when appropriate. for ordered categorical data, a cochran-armitage test for trends was used to compare the groups. continuous data were analyzed using a two-sided student's t-test after ensuring the data were normally distributed (based on the shapiro-wilk statistic) or using a two-sided wilcoxon's rank-sum test if the data were non-normal. all analyses were two tailed, and p-values of 0.05 or less were considered to be statistically significant. all analyses were conducted using sas version 9.2 (cary, nc, usa). during the two study periods, a total of 307 nasopharyngeal samples were collected in the emergency room. of these, 61 (19.9%) tested positive for hadv. the luminex xtag rvp fast v2 assay identified 30 cases, all confirmed by real-time pcr. this method revealed 31 positive cases that tested negative with the rvp fast v2 assay. among the hadv infected children, 14.8% were <1 year old, whereas 42.6% and 42.6% were 1-2 and 3 years old, respectively ( table 1) . the prevalence of hadv detection was similar in the two studied periods: 28 (45.9%) and 33 (54.1%) positive samples were collected in 2013 and 2014, respectively. hadv was the only virus detected in 31/61 (50.8%) cases, whereas it was found in association with one other virus in 25 (41.0%) cases and with two or more viruses in 5 (8.2%) cases. hev/hrv and rsv were the most common co-infecting viral agents and were found in 12 (19.7%) and 7 (11.5%) samples, respectively. molecular typing assignments were based on the identity of the closest matching sequences after both blast and phylogenetic analysis. the 61 hadvs belonged to species b in 7 cases (11.5%; all hadv-b3), species c in 37 cases (60.7%; 10 hadv-c1, 25 hadv-c2, and 2 hadv-c5), species d in 1 case (1.7%; hadv-d26), species e in 2 cases (3.4%; hadv-e4), and species f in 1 case (1.6%; hadv-f41) (fig 1) . it was not possible to identify the species and type of 13 (18.6%) samples due to inadequate sample volume. no peculiar clustering was observed among hadv strains. hadvs circulating in 2013 were closely related to strains identified in 2014. however, among hadv-c2 sequences, two distinctive branches were observed with 15 and 10 italian strains (fig 1) . similarly, in the branch of the tree corresponding to the hadv-c1 strains, two strains appeared to cluster separately from the other 8 strains. using 10 6 dna copies/ml as a cut-off, the viral load was classified as low in 37 (62.7%) and as high in 22 (37.3%) cases (2 hadv-b, 14 hadv-c, and 7 of 11 without species identification). for hadv-b, viral load varied from 1.4 x 10 2 to 3.9 x 10 8 copies/ml, whereas for hadv-c it was between 3.4 x 10 3 and 3.0 x 10 9 copies/ml. no significant difference in viral load was observed between each hadv species the sequence identity matrix of the hadv partial hexon gene for groups with at least 7 sequences (hadv-c2, -c1 and -b3) showed a minimum to maximum identity range of 97.6-100.0% for hadv-c2, 98.7-100% for hadv-c1, and 99.4-100% for hadv-b3. overall, the hadv strain sequences analyzed were highly conserved and only few amino acid changes were observed. in detail, among the hadv-c2 sequences, 15/25 strains (60.0%) had an insertion of one glutamic acid (e) in position 151 and the m305l change. in two of serotype was not available for 13 positive subjects, and viral load was not available for 2 positive subjects. viral load was categorized in two groups and was considered "low" for values <6 log(copies/ml) and "high" for values 6 log(copies/ml). no statistically significant result emerged for the relationship between adenovirus types and age or between viral load and age. doi:10.1371/journal.pone.0152375.t001 these strains, the additional change s195t was identified. among the hadv-c1 sequences, only one amino acid change (a190t) was evidenced in 2/10 strains (20.0%). finally, in 5/7 strains (71.4%) belonging to the hadv-b3 group, two changes, g205v and t254i, were observed. in table 2 , demographic, clinical, and laboratory characteristics of children infected by hadv alone or co-infected with hadv and one or more other respiratory viruses are compared with those of children with respiratory infection due to other agents. a preliminary analysis revealed that no statistically significant difference between cases infected by hadv alone or co-infected with other viruses, in particular rsv or rhinovirus could be evidenced, all the children with hadv infection were considered together. as shown, in comparison to hadv-negative children, those infected with hadv were younger (4.3 ± 3.3 vs 3.2 ± 2.5 years; p = 0.01) and had high-grade fever more frequently (56.4% vs 72.4%; p = 0.03). moreover, children infected with hadv had lower respiratory tract involvement less frequently (55.2% vs 36.1%; p = 0.007) and never suffered from wheezing unlike children with disease due to other etiologic agents. crp, c reactive protein; sd, standard deviation; spo 2 , peripheral oxygen saturation."38.0°c or more any time during the illness (before or at enrolment, or during follow-up);°39.0°c or more any time during the illness (before or at enrollment or during follow-up). a data were extracted from datasets of different studies that collected different information; therefore, the denominators vary across characteristics. however, when not indicated the reported number refers to the whole enrolled sample. children infected with other agents wheezed in 12.5% of the cases (p = 0.004) and were hospitalized more frequently (56.1% vs 27.9%; p<0.001). no other significant differences between groups were observed. in table 3 , comparisons based on demographic, clinical, and laboratory variables between subjects with hadv b and c species are shown. two significant differences were found between the groups: antibiotic therapy was more frequently prescribed (91.9% vs 57.1%; p = 0.04) and white blood cell count was higher (17,244 ± 7,737 vs 9,565 ± 3,211; p = 0.04) in children infected by hadv-c. table 4 shows data regarding characteristics of children with hadv infection according to viral load. children with high viral load were younger, had high-grade fever more frequently, were more frequently hospitalized, were absent from the community for a longer period of time, and had a higher c reactive protein (crp) level. however, differences were statistically significant only for absence from the community (14.5 ± 7.5 vs 5.5 ± 3.2 days; p = 0.002) and crp level (41.3 ± 78.5 vs 5.4 ± 9.6 μg/dl; p = 0.03). several previous epidemiological studies have shown that hadvs are considered the cause of respiratory infections in otherwise healthy children in approximately 4-10% of the cases [12] [13] [14] [15] . in this study, similarly to what has been found in asia by other authors [16] , a prevalence of approximately 20% was found, suggesting that the relevance of this infectious agent in the determination of respiratory problems could be higher than previously thought. the methods used to identify hadvs might partially explain this finding. in the past, most of the epidemiological studies of viral respiratory infections were carried out using methods that could underestimate viral presence in respiratory secretions, such as viral culture, antigen detection by immunofluorescence, and visualization by electron microscopy [17] . to overcome this problem, molecular methods were suggested. multiplex assays, including the rvp fast v2 assay, were developed to obtain the simultaneous identification of all the most common respiratory viruses and are now commonly used in routine practice. as previously reported [8] and confirmed by this study, the rvp fast v2 assay has poor sensitivity for hadv and can lead to undervaluation of the real importance of these viruses in the determination of respiratory infections. the addition of a specific real-time pcr can solve this issue. moreover, the higher than expected prevalence of hadv infection evidenced by this study could be strictly related to the period during which it was carried out. samples were collected in two winter months of two consecutive years. although hadvs circulate during the whole year, peak periods are in winter and early spring [1] . consequently, it is possible that the study was carried out during epidemics leading to the higher prevalence values reported here. in this study, the most commonly identified species were b and c, types 3, 2, and 1. this is not surprising because, despite possible temporal and regional changes in predominant type [18] , these types are more commonly reported as the cause of respiratory infection worldwide. hadv-b3 has been identified in successive outbreaks of severe acute respiratory illnesses in korea [19, 20] , brazil [21] , and taiwan [22, 23] , where this virus was the predominant type for respiratory hadv infection from 1981 to 2002. moreover, together with other hadv types, it has been the causative agent in epidemic outbreaks of respiratory diseases in europe, america, and oceania [24] [25] [26] . finally, hadv-b3 is known to be a causative agent of a characteristic syndrome of acute pharyngo-conjunctival fever in older children and adults, especially in summer camps and swimming pools [27] . types c1 and c2 have been more frequently reported as the cause of endemic or sporadic cases [28] , although there have been reports of epidemics [19] . in italy, a survey carried out approximately 10 years ago found that hadv-c1 and -c2 were the most common hadvs isolated in patients with infection due to these agents [28] . the same was shown by this study, showing that epidemics of infection due to the same hadv in a given geographic area can be prolonged compared with outbreaks of rsv, parainfluenza virus, and influenza viruses, which are well defined and have duration limited to some months [29, 30] . the severity of hadv infection varies according to age, socioeconomic status, environmental status, and above all, the immunological characteristics of the patient. detection of hadv in severely immunocompromised children has been implicated as a risk factor for poor outcome [31] . however, severe cases have been frequently described in otherwise healthy children [5, 19, 20] . in this study, most of the children with hadv infection had a mild infection and, globally, the severity of respiratory infection of children in whom hadv alone or in association with other respiratory viruses was identified was lower than that due to other infectious agents considered together. both the prevalence of lrtis and hospitalization rate were significantly lower in hadv-infected children than in children not infected by hadv. the long-term circulation of hadvs with similar genetic characteristics could partially explain the generally poor clinical relevance of infections due to the strains identified in this study. in italy, the most common hadvs identified in children during the periods of this study were of the same species and of the same types of those identified several years before. moreover, despite sequencing analyses that were focused on one of the more variable genes (hexon) [32] , no significant variation in hadv genetic characteristics was seen and no recombination between viruses was found. hadv-c2 strains were the only strains to have two slightly different clusters, suggesting the circulation of two different hadv-c2 strains with indistinct pathogenetic roles. as a result, it is possible that many of the children had previously had contact with these viruses and developed sufficiently high immunity to limit the clinical expression of subsequent infections. however, the number of non-hadv infection children included in this study is significantly higher than that of hadv infected patients and this difference could have led comparison to wrong results. a longer period and expanded surveillance may help to construct the complete picture on the hadv circulation, other infections and related clinical features. prevalence of lrti was higher in children infected by type b3, although the difference compared with type c was not statistically significant and the total number of patients with this type of infection is too small to draw definitive conclusions. potentially increased virulence of type b3 in comparison to other hadvs is not surprising because this virus has already been associated with a number of severe lrtis [5, 19, 20] and to the development of acute meningo-encephalopathy [33] . in this study, higher viral load was more common in children with some markers of more severe disease, such as higher fever, higher hospitalization rate, higher crp values, and delayed return to normal activities, independent of the infecting hadv type. however, clinical differences between patients with high or low viral load were not always significant, and it is not possible to state that hadv load can be a marker for severity of infection. by contrast, hadv load was found to be significantly higher in patients developing severe hadv infection after transplantation, especially in pediatric stem cell transplant recipients [31] . consequently, the measurement of hadv load is considered a possible method for an early diagnosis of disseminated hadv disease and for the initiation and monitoring of antiviral therapy in these subjects [34] . further studies are needed to evaluate whether high viral load could indicate which subjects might have to receive antiviral therapy to avoid negative evolution of the infection even if they are not immunocompromised. in conclusion, this study has shown that when adequately investigated, hadv infections are diagnosed more commonly than usually thought. moreover, these data seem to indicate that hadvs are stable infectious agents that do not frequently incur genetic variations and, for this reason, do not cause frequently severe diseases. it seems that there are some differences in the severity of disease outcome between types and according to viral load, with hadv type c and high viral load apparently associated with a more severe disease. however, further studies are needed to identify the potential pathogenetic role of the different species and types of hadv and the importance of viral load in the severity of infection. clarification of these unsolved problems may be useful for deciding how to develop adequate preventive and therapeutic measures for immunocompromised and otherwise healthy children who suffer from hadv infection. family adenoviridae adenovirus infections in immunocompetent and immunocompromised patients differential susceptibility of adenovirus clinical isolates to cidofovir and ribavirin is not related to species alone evidence of frequent recombination among human adenoviruses epidemical features of hadv-3 and hadv-7 in pediatric pneumonia in chongqing textbook of pediatric infectious diseases comparison of the luminex respiratory virus panel fast assay with in-house real-time pcr for respiratory viral infection diagnosis detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real-time pcr assay rapid and quantitative detection of human adenovirus dna by real-time pcr molecular typing of human adenoviruses by pcr and sequencing of a partial region of 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phylogenetic, taxonomic, and clinical implications adenovirus infection associated with central nervous system dysfunction in children adenoviral load diagnostics by quantitative polymerase chain reaction: techniques and application key: cord-340629-1fle5fpz authors: o’shea, helen; blacklaws, barbara a.; collins, patrick j.; mckillen, john; fitzgerald, rose title: viruses associated with foodborne infections date: 2019-05-21 journal: reference module in life sciences doi: 10.1016/b978-0-12-809633-8.90273-5 sha: doc_id: 340629 cord_uid: 1fle5fpz foodborne pathogens cause acute and chronic health outcomes of very different durations, severity and mortality, resulting in high costs and burdens to society. the issues of food safety and food poisoning are being increasingly emphasised, particularly in developed countries. infection/contamination with many agents i.e., bacterial, parasitic and viral entities can result in foodborne illness. this article will focus mainly on viral agents of infection. a range of different viruses can cause food poisoning/foodborne infection, and infection can result in a myriad of symptoms, ranging from mild, acute disease to chronic, debilitating disease and even death. due to the inherent differences between bacteria and viruses, namely the fact that viruses do not replicate in food, while bacteria do, viruses are frequently difficult to detect. this is compounded by the fact that many of the viruses associated with enteric disease do not replicate in cell culture. these factors can lead to a lag between reporting, detection and analysis of foodborne viruses versus bacterial agents. despite these constraints, it is now evident that there are both well-established and emerging viruses implicated in foodborne infections, and the role of molecular detection and characterisation is becoming increasingly important. in recent years, there has been increasing awareness regarding food safety in terms of food poisoning that results in viral gastroenteritis i.e., stomach flu. it is important to clearly define these terms, for the purpose of this article, prior to focusing on the smaller group of diverse viruses associated with food borne illness. food safety encompasses the handling, preparation, and storage of food to prevent food-borne illness. food poisoning (also referred to as foodborne illness) is caused by eating contaminated food. infectious organisms, which include bacteria, viruses and parasites (or their toxins), are the most common causes of food poisoning. infectious organisms or their toxins can contaminate food at any point of processing or production. contamination can also occur in the home and eateries, if food is incorrectly handled, stored, or cooked. viral gastroenteritis (also known as stomach flu) is an intestinal infection with symptoms including watery (usually nonbloody) diarrhea, abdominal cramps, pain, nausea or vomiting, or both, and sometimes headaches, muscle aches and fever. the most common way to develop viral gastroenteritis (stomach flu) is through contact with an infected person or by ingesting contaminated food, water and water products such as ice. there is a huge annual cost of illness, disease burden and quality-adjusted life year (qaly) loss globally, caused by food-borne pathogens, which has been reported on by many investigators (scallan et al., 2011) . in the western world, the viruses frequently reported as having the highest total cost-of-illness, to date, are norovirus and rotavirus. these viruses will be discussed first. this profile could change, however, as new virus threats are constantly emerging. a variety of different foods and different agents are implicated in foodborne illnesses, and we will elaborate on these in the following sections. many agents cause gastroenteritis, the most common outcome being diarrhea. this can range from mild, self-limiting illness to fatal infection. diarrheal disease is the major cause of illness and death in children in developing countries, while in the developed world it is usually mild, except in the very young, the elderly and the immunocompromised. gastroenteritis (acute gastroenteritis, age) is caused by a variety of different pathogens, including parasites, bacteria and viruses ( table 1) . every day we swallow large numbers of microorganisms in our food and beverages, and from contact with our environment e.g., from fingers. our body's defense mechanisms, [innate (non-specific) and adaptive (specific) defense systems] are very efficient, and the microorganisms rarely succeed in surviving passage to the intestine in sufficient numbers to cause infection. the body's innate (non-specific) defense is the first line of protection and does not distinguish between infectious agents. it includes physical (e.g., skin and mucous membranes) and chemical barriers (e.g., sweat, gastric juices), cellular (e.g., natural killer cells and phagocytosis) and modular defenses (e.g., interferon) and bodily responses (e.g., inflammation and fever). with regard to protection against potential enteric pathogens, the intestinal tract has an abundant microbiota that competes with invading pathogens for space and nutrients. the peristaltic action of the digestive tract encourages movement through the system, deterring colonisation by invading pathogens, with vomiting and diarrhea flushing harmful microbes and their chemical products from the digestive tract. the extremely acidic stomach environment discourages pathogen replication. however, these fortifications are not impenetrable e.g., the enteric virus hepatitis a has the ability to survive and penetrate the body through the gastrointestinal tract (campbell et al., 1999) . in addition, the epithelial barrier of the intestine is embedded with mucous-producing goblet cells. these simple columnar cells secrete gel-forming glycoproteins (mucins), to produce a transparent, impervious barrier (johansson and hannsson, 2013) on the luminal surface. the prevailing mucin of the intestinal tract is muc2 (mucin 2), which is a major structural component of intestinal mucus layer. goblet cells emerge during foetal development at 9-10 weeks of gestation (kim and khan, 2013) and are continuously secreted, migrating from the intestinal glands; crypts of lieberkühn to the lumen of the intestinal tract via the apex of the digestive villi. non-bacterial gastroenteritis and diarrhea is usually caused by viruses. acute viral gastroenteritis (age) is seen in all parts of the world, especially in infants and young children. age ranges from mild and self-limiting to severe, debilitating diarrhea, occasionally resulting in mortality. these illnesses have a huge impact, particularly in parts of asia, africa and latin america, with over 3 million fatalities recorded per annum. age also has major effects on nutritional status and growth. viruses appear to be the commonest causes of gastroenteritis in infants and young children but are not distinguishable clinically from other types of gastroenteritis. some of these viruses are specific for humans, while others are implicated in zoonotic disease. in most instances, infection follows the general rules for faecal-oral transmission. enteric viruses are transmissible by food and water and enter the body through the gastrointestinal tract, thus the viruses are commonly acquired after ingestion of sewage contaminated food or water or from poor hygiene when preparing food. the viruses are shed in high concentrations in faeces and vomit and can remain infectious in the environment for several months. many viruses are difficult or impossible to cultivate in vitro, and tests are unreliable or not developed, thus many cases of non-bacterial acute gastroenteritis viruses are under reported. well established viral pathogens will be discussed in this section. later in the article, emerging viral pathogens associated with food poisoning/foodborne illness/foodborne infections will also be discussed. there are several different viruses involved in foodborne outbreaks. some of these are human viruses that infect and cause illness following ingestion. virus particles are shed in the faeces. to date, the most notable examples in this category are noroviruses and hepatitis a virus. these viruses are different, belonging to different families ( table 2) : noroviruses (formerly called small round structured viruses or srsvs) belong to the family caliciviridae and are small, non-segmented, positive sense, single stranded nonenveloped, icosahedral rna viruses. hepatitis a virus is also a small, non-segmented, positive sense, single stranded nonenveloped icosahedral rna virus, but belonging to the family picornaviridae. hepatitis a virus is slightly smaller and contains a linear genome. is one the five pathogens (namely, salmonella, campylobacter, listeria, toxoplasma, and norovirus) responsible for causing roughly 90% of mean loss due to foodborne illness in the united states (hoffman et al., 2012; scallan et al., 2011) . as outlined above, noroviruses are small, non-enveloped, positive sense rna viruses, within the family caliciviridae. noroviruses have been classified into seven genogroups (gi-gvii) and over 30 genotypes. gii.4 has long been established as a predominant variant, but new variants are continually emerging (lennon et al., 2014) . transmission of the virus may occur through sewage contamination early in the food chain or lack of personal hygiene later in the food chain, particularly when 'ready-to-eat' (rte) food-items are involved. transmission can also occur via by person-to-person contact, environmental contamination, as well as via food-and water-borne transmission. typically, noroviruses cause problems in individuals who have consumed contaminated raw oysters. other implicated foods are fruit and vegetables that have, for example, been irrigated with contaminated water. outbreaks frequently occur in hospitals, nursing homes, restaurants, cruise ships, schools, summer camps, and even at family dinners. in these cases, large numbers of people often eat food handled or prepared by others. the symptoms of norovirus infection vary, but may include nausea, vomiting, abdominal pain or cramps, watery or loose diarrhea, malaise, low-grade fever, muscle pain. signs and symptoms usually begin 12-48 h after first exposure to the virus and last one to three days. shedding of virus can continue for up to two weeks post recovery. some people with norovirus infection may show no signs or symptoms but are still contagious and can spread the virus. there are several recommendations concerning food preparation, disinfection and decontamination, outlined on e.g., the centres for disease control and prevention (cdc) website (see "relevant websites section"). these measures involve practicing good hand hygiene, washing fruit and vegetables and cooking seafood, especially shellfish e.g., oysters, thoroughly, individuals who are ill avoiding contact with others, also food preparation, for at least 48 h after symptoms abate, and decontamination using suitable disinfectants and detergents. some people are particularly vulnerable and should take measure to avoid norovirus infection, especially infants, older adults and people with underlying disease where vomiting and diarrhea can be severely dehydrating and require medical attention. preventative measures e.g., avoiding raw shellfish should be taken in these instances. for many years, rotavirus (rv) has been recognized as a major cause of gastroenteritis in the young of many animal species, including humans (bishop et al., 1973; estes and kapikian, 2007; flewett et al., 1975) . in humans, rv associated disease typically occurs in children less than 5 years of age but rotavirus can infect all ages, including adults (anderson et al., 2012; collins et al., 2015; gunn et al., 2012) . in 2008, an estimated 453,000 deaths worldwide were attributed to rotavirus infection, with most deaths occurring in resource-poor countries (o'shea et al., 2016; tate et al., 2012) . rotavirus belong to the family reoviridae and are medium sized, segmented, double stranded non-enveloped, icosahedral rna viruses. the 11 segments encode six virion proteins (vp1-4, 6-7) and six non-structural proteins (nsp1-6). rotaviruses are classified into 8 antigenically distinct groups and one proposed group (a-i) (mihalov-kovács et al., 2015) , using immunological and phylogenetic analysis of the vp6 gene. rotavirus groups a, b, c, h and i have been identified in mammals, including humans, while rotavirus d-g to date have only been identified in non-human mammal and avian species. a tenth novel rotavirus species has been identified in schreiber's bats (miniopterus schreibersii), provisionally known as rotavirus j (bányai et al., 2018) . within the rva group, the viruses are classified antigenically and genetically, based on the main antigenic determinants, the outer capsid proteins, vp7 and vp4, which specify the g and p serotypes/genotypes, respectively. a whole genome classification system has been adopted for classification of all 11 segments of the rotavirus genome, applying nucleotide homology cutoff values to distinguish genotypes. transmission of rotavirus is predominantly faecal-oral. in humans, rotaviruses are ubiquitous, with 95% of children worldwide being infected by three to five years of age. in infants, prior to the introduction of rotavirus vaccines, rvas could be detected in up to 50%-60% of all childhood hospitalisations due to acute gastroenteritis each year, were estimated to cause 138 million cases of gastroenteritis annually, and 527,000 deaths in children o5 years of age living in developing countries. in other animals, rotavirus disease also occurs in the young of the species and in farm animals, leads to significant economic losses. the symptoms are usually characterised by watery dehydrating diarrhea and vomiting, often accompanied by abdominal cramps and low-grade fever, lasting 6-10 days. subsequent reinfections are associated with mild or subclinical presentations (bishop et al., 1973; velázquez et al., 1996) ; however, such reinfections are important in boosting immunity and maintaining long-term protection from rotavirus disease. due, in part, to the segmented nature of the genome, accumulation of point mutations (genetic drift) and re-assortment (genetic shift) are responsible for the huge genetic heterogeneity of rotaviruses. these mechanisms, in combination with the potential for interspecies (zoonotic and anthroponotic) transmission, can also lead to the emergence of "novel" strains in a given species, with a potential for epidemic or epizootic spread (martella et al., 2010; matthijnssens et al., 2008) . the zoonotic potential of rva has been documented on several occasions (martella et al., 2010) . uncommon human rva genotypes include g5, g6, g8, g10 and g11, in combination with p[3], p[9], p[10], p[11] and p[14] p types, and are generally considered to be animal to human reassortment variants. however, certain animal-like genotypes have become established in human circulation, particularly in developing countries. since its discovery, many attempts have been made to produce effective rotavirus vaccines, with an emphasis on those directed to the prevention of human disease. today, two oral live attenuated vaccines are being used in rotavirus immunisation programmes globally; rotateq™, which is a pentavalent human-bovine reassortment containing human rva derived g1 to g4 and p [8] types within the backbone of the wc3 bovine strain, and rotarix™, a monovalent human g1p[8] live attenuated vaccine. both vaccines are efficacious and endorsed by the who, which recommends the implementation of these vaccines in national immunisation programs (who, 2009) . the introduction of universal mass vaccination (umv) has resulted in a significant decrease in childhood rotavirus infection morbidity and mortality (curns et al., 2010; pendleton et al., 2013; tate et al., 2012; usonis et al., 2012; zeller et al., 2010) . hepatitis or inflammation of the liver can have a number of causes, including medications, toxins, alcohol use and viral infection. there are 5 hepatitis viruses, a-e, but only a and e are transmitted by the oro-faecal route. both viruses are major causes of infectious disease with associated socio-economic consequences caused by morbidity and, in vulnerable groups, mortality. hepatitis a virus (hav), also known as hepatovirus a, belongs to the order picornavirales in the family picornaviridae and is the type species of the genus hepatovirus. it is an icosahedral, non-enveloped virus with a monopartite, positive, single-stranded rna genome. humans and vertebrates serve as natural hosts. there is a single serotype of hav but several genotypes: ia, ib, iia, iib, iiia, iiib primarily in humans and iv-vi, primarily found in non-human primates (cristina and costa-mattioli, 2007) . the virus infects hepatocytes and kupffer cells (liver macrophages). transmission: hav is spread in food, in the water system, by touching contaminated surfaces or by direct contact with an infected person. contamination of food can occur at any stage from farm to fork. according to the who, approximately 1.5 million people are infected each year, although the true infection rate is probably much higher due to the asymptomatic nature of many infections (hepatitis a fact sheet. in: world health organisation: media centre). due to the prolonged infection times, the economic consequences of an outbreak through absenteeism from work can be significant. in developing countries where sanitation is poor, most children are infected while young and are asymptomatic. this gives them immunity and, as such, they cannot become reinfected as adults. in such areas, epidemics of the virus are uncommon but unvaccinated adult visitors from areas where hav is not commonplace are vulnerable. in economically developed countries, epidemics are rare, because good hygiene practices will prevent person-to-person spread. however, in transitional economies, the introduction of improved sanitation may mean that the population is not exposed as children. as such, the introduction of the virus to these susceptible populations of adults can result in significant outbreaks (lemon et al., 2018) . the symptoms: the virus is normally mild without permanent repercussions and is typified by fever, fatigue, loss of appetite, nausea, vomiting, abdominal pain, dark urine, clay-coloured stools, joint pain and jaundice (yellowing of the skin and eyes). the development of fulminant hepatitis is rare (less than 0.5% of cases) and can lead to more serious illness, including liver failure and death. severe illness is more common in older people, while in children around 70% are asymptomatic and in those with clinical manifestations the symptoms are generally mild. infected persons may shed infectious virus for several weeks before they show symptoms. in those individuals where symptoms occur, they usually start appearing 4 weeks after exposure, but can occur as early as 2 and as late as 7 weeks following exposure. symptoms usually develop over a period of several days and last less than 2 months, although some people (10%-15%) with hepatitis a can have symptoms for as long as 6 months (jeong and lee, 2010) . hea can survive outside a host cell for several weeks in groundwater and it has been shown to retain infectivity after 92 days at 25°c in seawater. the virus is tolerant of desiccation and freezing and will survive on vegetables throughout the production process until consumption (a critical review of the effect of heat, ph and water activity on the survival of hepatitis a and e viruses -a report to the united kingdom food standards agency july 2014). hea is more resistant to heat than other picornaviruses and the centre for disease control states that temperatures of 85°c for a least 1 min are required for inactivation. hep a is vaccine preventable (ott et al., 2012) . monovalent formalin killed vaccines are available worldwide and live attenuated versions are available in a number of countries. a double dose of vaccine can provide long-term protection and single dose immunisation strategies have been shown to be effective at least in the short and medium term. as well as for active immunisation, the vaccine can be used prophylactically in the first few weeks of infection. human immunoglobulins can also be used for both preventative and post-exposure prophylactic treatment (liu et al., 2009) . post-exposure prophylaxis provides high levels of protection (80%-90%) if provided in the first 2 weeks of exposure. otherwise standard methods for the prevention of food-borne infections apply: ensuring good sanitation and a clean water supply, hand washing and food safety. hepatitis e virus (hev) is a small non-enveloped icosahedral virus, 27-34 nm in size, with a single-stranded positive sense rna genome. the virus belongs to the family hepeviridae, genus orthohepevirus. there are four species in this genus, and the viruses that infect humans belong to the species orthohepevirus a. currently, eight genotypes of orthohepevirus a have been identified. hev1 and hev2 infect humans only, hev3 has a range of known host animals including humans and swine, hev4 infects humans and swine, hev5 and hev6 have been detected in wild boar and hev7 has been detected in camels (sridhar et al., 2017) . transmission: hepatitis e virus (hev) is the most common cause of acute viral hepatitis worldwide (sridhar et al., 2015) . the virus is responsible for endemics and epidemic outbreaks and the who estimate there are 20 million infections and 3.3 million symptomatic cases worldwide per year; in 2015 these led to 44,000 deaths (world health organization, 2017). hev is primarily transmitted through the faecal-oral route via shedding in the faces of infected individuals and subsequent contamination of drinking water. other routes of transmission are consuming raw or undercooked foods from infected animals or shellfish, blood transfusions or vertical transmission from pregnant women to the foetus (wulffen et al., 2018) . the virus has a worldwide distribution but is most prevalent in south and east asia and is associated with areas where sanitation is poor and contaminated faeces can enter the water system. in regions where the virus is endemic, outbreaks are common, usually associated with contaminated drinking water, recurring at intervals of years and may involve thousands of people. such infections are predominantly genotype 1 or 2 (melgaço et al., 2018) . in non-endemic regions, infections were previously associated with international travel, but now the majority of cases are zoonotic genotype 3 or 4 infections from swine or deer. handling of swine and manure of porcine origin is now a public health concern and infectious hev has been isolated from meat products (yugo and meng, 2013) . the symptoms: while hev is primarily a disease of the liver, it has also been associated with non-hepatic diseases such as subacute and monophasic neurological disorders of the peripheral nervous system, acute pancreatitis, glomerulonephritis, mixed cryoglobulinemia, severe thrombocytopenia and haemolytic anaemia (pischke et al., 2017) . the hepatic symptoms of hev are indistinguishable from other forms of acute hepatitis and laboratory diagnosis is required for a definitive diagnosis. the incubation period is approximately 2-6 weeks. hev is characterised by fever, reduced appetite, nausea and vomiting, abdominal pain, and a slightly enlarged, tender liver (hepatomegaly) (kamar et al., 2017) . in rare cases, acute hepatitis e can be severe, resulting in liver failure and death. pregnant women and immunosuppressed people are at high risk of these severe symptoms. in pregnant women mortality rates of 30% have been recorded (perez-gracia et al., 2017) . like hav, infection of young children is often mild or asymptomatic. hev is self-limiting and most cases do not require treatment. for those with acute symptoms or for high risk individuals, hospitalisation is required. the antiviral drug ribavirin may be given, although in pregnant women this must be carefully considered, due to the teratogenic nature of this antiviral compound (krzowska-firych et al., 2017) . a recombinant subunit vaccine to prevent hepatitis e virus infection exists in china but is not licenced elsewhere (nan et al., 2018) . the family adenoviridae contains 5 genera; atadenovirus (8 species) infecting birds, lizards and mammals; aviadenovirus (14 species) infecting birds; ichtadenovirus (1 species) isolated from sturgeon; mastadenovirus (45 species) infecting mammals only, including humans and; siadenovirus (6 species), mostly infecting birds and 1 frog species. virions are non-enveloped, 70-90 nm in diameter with a double-stranded dna genome and an icosahedral capsid. there are 7 human species of human adenoviruses, a-g (lennon et al., 2007) . within these species there are at least 79 subtypes distinguished either by serological differences of by genotypic classification (chen and tian, 2018) . transmission: adenovirus-associated gastroenteritis often occurs in clusters in schools, hospitals or military camps. adenoviruses are spread person-person contact, by coughing and sneezing, by touching contaminated surfaces or by the faecal-oral route (eckardt and baumgart, 2011). adenoviruses infections are not associated with contaminated food, but transmission can occur in water, through public water systems or in swimming pools; the latter are predominately associated with conjunctivitis (rodríguez-lázaro et al., 2012) . symptoms: adenoviruses in humans cause respiratory infections, gastrointestinal disease and conjunctivitis with hemorrhagic cystitis, hepatitis, hemorrhagic colitis, pancreatitis, nephritis, or encephalitis (lynch and kajon, 2016) . adenoviruses infections are often asymptomatic or mild and self-limiting. however, they can be associated with severe morbidity or mortality, with immunocompromised and the young being more susceptible. however, novel or emerging strains have been found to cause mortality in people of all ages such as ad14, which was responsible for fatal respiratory illness in the usa (centres for disease control, 2007) . a number of human adenovirus subtypes can cause gastrointestinal symptoms, but subtypes 40 and 41 from species f are the most commonly associated with age and have been reported to be responsible for 5%-20% of acute gastroenteritis in children (ziros et al., 2015) . adenovirus-associated gastroenteritis is most common in children under 2 years old and is uncommon in adults, accounting for 1.5%-5.4% of cases (eckardt and baumgart, 2011) . the incubation period is around 8-10 days after which diarrhea develops and, in some cases, mild vomiting. fever lasting 2-3 days may develop; severe dehydration is rare (wood, 1988) . currently vaccines against human adenovirus 4 and 7 are being used by the us military, but no vaccines are available for general use (chen and tian, 2018) . as the virus is usually self-limiting in most cases, treatment of adenoviral gastroenteritis is not required. in severe cases, hospitalisation and rehydration may be needed. prevention of adenovirus infection is by standard antiviral hygiene methods; avoiding sharing of eating and drinking utensils, hand washing and avoiding contact with sick individuals, especially in at risk environments such as hospitals. adenoviruses are susceptible to chemical disinfectants but can be resistant to uv irradiation (rodríguez-lázaro et al., 2012) ; in swimming pools, chlorine levels must be adequate. the family astroviridae consists of 2 genera; avastrovirus and mamastrovirus infecting birds and mammals, respectively. virions are non-enveloped, approximately 28-40 nm in diameter with a single-stranded positive sense genome and an icosahedral capsid. the international committee for the taxonomy of viruses (ictv) currently identifies 19 species in the genus mamastrovirus infecting felines, canines, cattle, cervids, rodents, swine, sheep, mink, bats, rabbits, sea lions and dolphins. the viruses that infect humans (referred to as classical hastvs) have traditionally been classified into 8 serotypes, but recently a number of viruses have been detected in humans that are more similar to those from other animals, suggesting cross species transmission (bosch et al., 2014) . transmission: hastvs are predominantly transmitted through the faecal-oral route as well as through drinking water and in sewage. recreational activities in sewage contaminated water bodies is a risk of infection. hastvs are considered to be food-borne viruses with molluscs grown in, or fruits or vegetables irrigated with contaminated water, representing the biggest threat (vu et al., 2017) . as would be expected, poor food hygiene practices may play a role in outbreaks, particularly as asymptomatic carriers are more likely to contribute to infection in the food industry than symptomatic workers. contaminated fomites and surfaces can represent a threat in institutions such as schools and hospitals (abad et al., 2001) . as we are now aware that inter-species infections with astroviruses can occur, and, as such, the zoonotic route should be considered as a potential source of astrovirus infection (bosch et al., 2014) . the symptoms: classical human astroviruses are known to cause mild gastrointestinal disease in children, but symptoms and prevalence are serotype dependant. immunocompromised and elderly patients are also susceptible. serotype 1 is the most common, with a reported level of seropositivity of 90%-100% having been reported in children by 5 years of age (koopmans et al., 1998) . the symptoms are usually watery diarrhea, vomiting, fever, abdominal pain, anorexia, and headache but these are usually mild in nature and self-limiting, typically lasting for 2-4 days (vu et al., 2016) . however, hastvs do cause asymptomatic infections (méndez-toss et al., 2004) and the association of these viruses with disease is not fully understood. while the symptoms are mild, hastvs do contribute to a large proportion of outbreaks of diarrhea, 0.5%-15% of all cases, and often occur as coinfections with other viruses such as noroviruses and rotavirus (de benedictis et al., 2011) . rarer astroviruses have been reported as being responsible for fatal cases of central nervous system infection (fremond et al., 2015) . as with most gastrointestinal infections control of hastvs is by prevention of contamination of food and water and prevention of person to person, or fomite to person spread through hygiene and handwashing. while survival of astroviruses in drinking water is known to be high, chlorination has been shown to be effective in reducing astrovirus viability by alteration of the capsid, resulting in non-infectious virions (abad et al., 1997) and 90% alcohol has been shown to be effective for decontamination of surfaces and hands (kurtz et al., 1980) . no vaccines are available for hastvs. as infections are usually mild and self-limiting, no treatment is normally required. however, those with severe gastroenteritis may require oral or intravenous rehydration. recent emerging epidemic and pandemic virus infections that cause severe disease in humans and that are associated with food production, preparation and food contamination include the coronavirus, severe acute respiratory syndrome (sars-cov), nipah virus, ebola virus and some of the highly pathogenic influenza virus strains, such as the h5n1 subtype. transmission may be by a variety of routes, but often the emergent epidemic is started by contamination of a food source by saliva, urine or faeces from a wild reservoir species or use of a wild animal (bush meat) as the food source and infection through the capture and butchering process (e.g., hiv is thought to have entered the human population by this route). these infections have a low probability of occurring, but a very high impact if they establish infection in domestic animals and humans. the coronaviridae belong to the order nidovirales of single stranded, positive sense rna viruses. within the coronaviridae there are 2 sub-families, of which the coronavirinae contain 4 genera. phylogenetically, sars-cov is within the betacoronavirus genus (ictv, 2017) . in older literature, sars-like covs were assigned to group 2b coronaviruses (lau et al., 2005) . the viruses are roughly spherical (120-140 nm diameter), with an envelope that contains many surface glycoproteins that form a corona (crown) under electron microscopy (masters and perlman, 2013) . although enveloped, the virus is relatively stable especially in faeces and urine for 1-2 days (or longer) at room temperature, and up to 4 days if stool is from diarrhea patients (the ph is higher) (see "relevant websites section"). however, they are sensitive to heat, lipid solvents, oxidizing agents and non-ionic detergents (markey et al., 2013a) . many coronaviruses are associated with respiratory and intestinal disease and can cause severe epidemic gastrointestinal disease in agriculturally important species such as porcine epidemic diarrhea virus in pigs (masters and perlman, 2013) . transmission: is faecal-oral, respiratory by aerosolised secretions. sars-cov was first discovered in 2003, after a pandemic of severe respiratory disease that originated in guangdong province, china. it caused disease in 8098 people and of these, 774 died (see "relevant websites section"). it is now believed that chinese horseshoe bats are the original reservoir host of sars-like covs (lau et al., 2005; li et al., 2005) . bats transmit these viruses, probably via faecal contamination of food sources directly to humans or to intermediate hosts, most notably palm civets and raccoon dogs, and these then transmit virus to humans (guan et al., 2003) . these animals are traded in live animal markets as food and it is thought that aerosolisation of faeces and other body fluids caused respiratory infection of humans (wang et al., 2005) . once sars-cov entered humans, it was spread by respiratory droplets or contamination of surfaces by droplets that were then transferred to the mouth, nose or eyes by touching. there was also a cluster of cases in an apartment block in hong kong that probably arose from sewage aerosolisation into the ventilation system that proceeded to cause respiratory infection of many of the inhabitants (tilgner et al., 2003) . the incubation period is 2-7 days but can be up to 10 days. infection results in symptoms of a high fever, chills, and headache. some people have mild respiratory symptoms from the beginning that progress to lower respiratory involvement with a nonproductive cough, which can lead to hypoxemia and pneumonia. a low proportion of patients (10%-20%) had diarrhea. as stated above, approximately 10% of patients died from the severe respiratory complications, but these were usually older patients or those with other health problems. up to 20% of patients needed mechanical ventilation to survive. the severe sequela could take many days and weeks to develop. shedding in faeces occurs for a long time after symptoms are cleared so contact from soiled material from past patients can be a source of infection (masters and perlman, 2013) . there have been no known cases of sars since 2004 (see "relevant websites section"). however, sars-cov is not eradicated, as similar viruses are still present in their wild-life hosts. therefore, continued vigilance must be used to stop the spread of these viruses from their zoonotic hosts to humans. control of live animal markets and a ban on sales of exotic animals is required (see "relevant websites section"), but difficult to introduce due to the cultural background of the communities in which they are found. there is no vaccine or treatment against the virus (coleman and frieman, 2014) , although several drugs have been developed that could be used in the future (masters and perlman, 2013). nipah virus is a member of the viral family, paramyxoviridae in the genus henipavirus (ictv, 2017). it was originally identified in 1999 in malaysia during an outbreak of encephalitis and respiratory disease in pig farmers and those who had close contact with pigs. its virions are roughly spherical of about 150 nm diameter but filamentous forms can be seen (wang et al., 2013) . it is a relatively unstable virion (markey et al., 2013c) , but can survive well in ph-neutral fruit bat urine (44 days at 22°c), on fruit (up to 2 days) and in artificial date palm sap (de wit et al., 2014; fogarty et al., 2008) . it is however susceptible to desiccation (fogarty et al., 2008) . it is an enveloped virus that is inactivated by most common disinfectants, lipid solvents, and non-ionic detergents (markey et al., 2013c) . transmission is respiratory and oral. the wild-life reservoir host of nipah virus are pteropus fruit bats (chua et al., 2002; yadav et al., 2012) and transmission may be by direct infection of humans, after the virus has passed through an intermediate host e.g., pigs, or from fomites. it is thought that bat saliva or urine is the major source of virus and so food such as partially eaten fruit contaminated with saliva or contamination by faeces and urine may be the source of infection for other hosts. in the malaysian outbreak of 1998-99, it is thought that contaminated fruit from trees adjacent to pig farms encroaching into forest were dropped into enclosures where they were eaten by the pigs or that there was direct contamination of enclosures with urine. the pigs amplified the virus (they also showed clinical respiratory and neurological disease), so that farmers and workers in direct/close contact were infected and were the population where the majority of cases was seen (parashar et al., 2000) . however, in the indian sub-continent, especially bangladesh, where nipah virus infection was first recognized in 2001, the major risk factor for contracting nipah virus is drinking raw palm sap (luby et al., 2006) . there is photographic evidence of fruit bats licking sap from the cuts in the trees from which the sap is collected, and contamination by faeces is seen in or on the pots (khan et al., 2010; rahman et al., 2012) . human infection may be asymptomatic, but there can be acute respiratory disease with fatal encephalitis, leading to mortality rates of 40%-75%. the incubation period is believed to be 4-14 days or longer (see "relevant websites section"). the malaysian outbreak saw more cases of encephalitis than respiratory disease and there was little evidence for human to human transmission. in the indian sub-continent, especially bangladesh, the symptoms of infection manifested more with respiratory disease with fewer cases of encephalitis (luby et al., 2009) . the aerosolisation of respiratory secretions (droplets) and close contact with the sufferer allows respiratory transmission to lead to human to human transmission chains (gurley et al., 2007; yadav et al., 2012) . nipah virus will be maintained in its bat reservoir and so it is unlikely to be eradicated. slaughter and burial of pigs in the affected regions controlled the malaysian and singapore outbreak. routine cleaning and disinfection on pig pens with detergents may reduce exposure of pigs but if an outbreak is suspected, quarantine and culling can reduce the spread of disease (wang et al., 2013) . in bangladesh, the use of bamboo skirts to restrict access of bats to the sites on trees where sap is collected reduces the risk of contamination (khan et al., 2010) . heat treatment of sap also inactivates the virus and this, along with stopping the feeding of raw palm sap to humans and domestic animals reduces the risk of infection and transmission (luby et al., 2009) . continual surveillance for re-occurrences must be in place to monitor possible incursions into humans. there is no vaccine and no specific treatment at present (see "relevant websites section"). influenza viruses are members of the orthomyxoviridae family, and the h5n1 genotype is in the alphainfluenzavirus genus and is of the influenza a species (ictv, 2017). influenza viruses are negative stranded rna viruses that have a segmented genome (shaw and palese, 2013) . influenza a nomenclature uses the 2 genomic segments that encode the envelope glycoproteins, haemagglutinin (h) and neuraminidase (n) because these proteins are major contributors to the pathogenicity of the viruses. the wildlife reservoir for these viruses is wild birds, especially waterfowl, in which a subclinical gastroenteric infection is usually seen. thus, these viruses are often called avian influenza. there are several high pathogenicity avian influenza a (hpai) virus genotypes but one of the most pathogenic for humans and other animals are those in the h5n1 genotype (wright et al., 2013) . with widespread sequencing, the ability to group viruses as 'clades', i.e., those viruses that are derived from a common ancestor, has now allowed a numerical naming system to be instituted. the eurasian-african h5n1 viruses that are circulating and causing human and animal disease can be split into several (≥20) clades (see "relevant websites section"). within all the different clades, the original h5 genotype has remained, despite re-assortment of the other gene segments. the virions are spherical (80-120 nm diameter) or filamentous (up to 20µm long) (noda, 2011) . the virus is present in the faeces of wild waterfowl, water contaminated by their faeces, domestic poultry secretions (respiratory and faeces) and aerosolised respiratory secretions from infected animals. the virion is usually very labile (markey et al., 2013b) but it has been shown that avian influenza virus is infectious for months in low temperature water and for over a week in water at 22°c (hinshaw et al., 1979; markwell and shortridge, 1982) with increased survival when water has neutral to basic ph (7.0-8.5) and low ammonia concentrations (keeler et al., 2014) . it is also stable in frozen lakes (shoham et al., 2012) , allowing maintenance in the environment and infection of waterfowl from year to year. the seasonality of epidemics in humans is affected by temperature and humidity with the virus surviving better at 20°c in low humidity conditions but at 30°c requiring higher humidity. thus, the cool, dry conditions of winter favour transmission in temperate zones and humid, rainy conditions favour transmission in tropical and sub-tropical zones. the presence of salts and proteins in respiratory droplets allows virus survival in aerosolised droplets for up to 1-24 hr (sooryanarain and elankumaran, 2015) . contamination of fomites may also occur allowing transfer by hands. transmission is via inhalation of small aerosol droplets, faecal contamination from poultry and water borne infection. in asia, contamination of the environment by the faeces of waterfowl leads to infection of domestic poultry including chickens, ducks and geese which are often kept in the same environment. this usually causes severe disease and high titres of virus to be secreted from the domestic birds promoting infection of those working with or in contact with them, or in the food chain. farming of poultry and pigs together increases the risk of transmission to pigs and they can amplify and increase the risk of human infection. influenza virus a causes seasonal outbreaks with occasional severe epidemics/pandemics in humans and animals. the asian-african h5n1 lineage is thought to have originated from commercial geese in the guangdong province of china in 1996. this has since been spread across the globe by wild birds and infected people. there is evidence that h5n1 has infected humans directly by drinking duck blood or eating duck meat, however, aerosolisation and inhalation is the more common route (shao et al., 2011; tumpey et al., 2002) . this virus has evidence of direct bird to human transmission without the need for adaptation through a secondary species such as pigs (wright et al., 2013) . in humans, the symptoms it causes are an acute respiratory tract infection with fever and sore throat, cough and malaise. if the virus becomes systemic there may also be vomiting and abdominal pain. h5n1 viruses are highly pathogenic and there is a high case mortality rate with these infections (wright et al., 2013) . reducing the exposure of domestic poultry to wild waterfowl i.e., increased biosecurity reduces the risk of infection of these poultry and so exposure of workers to the virus. quarantine and culling of premises as well as closing live bird markets also reduces the risk of spreading the infection (see "relevant websites section"). in humans, there is a killed vaccine against annual strains of influenza a and b, and a live attenuated influenza a and b nasal spray vaccine. however, none is specifically against the h5n1 strains. there are several antiviral drugs (amantadine, rimantadine, zanamivir, oseltamivir) available to treat those infected with influenza a, but there is the risk of resistance occurring, with some of the circulating h5n1 strains showing resistance to amantadine and rimantadine and oseltamivir resistant strains have been isolated from patients treated with the drug (wright et al., 2013) . infections by severe acute respiratory syndrome (sars) virus, nipah virus (niv), h5n1 virus, hepatitis a virus (hav), hepatitis e virus (hev), adenovirus, astrovirus, norovirus (nov) and rotavirus (rva) in humans and animals are detected by nucleic acid amplification tests and serologic tests. for example, a standardised method based on quantitative real-time pcr (rt-qpcr) for the detection of nov and hep a in food has been developed by the european committee for standardisation (cen) working group (tc 275/wg6/tag 4 -detection of viruses in food) (lees and cen wg6 tag4, 2010) and provides a tool to quantify nov concentration in shellfish. it has been used to demonstrate that the risk of gastrointestinal illness associated with consumption of oysters increases with increasing concentrations of nov genome copies present (lowther et al., 2012) . costantini et al. (2010) demonstrated that a commercially available nov enzyme immunoassay showed excellent specificity but low sensitivity both for outbreaks as well as for samples from sporadic cases. the assay detected 18 of the 21 genotypes evaluated and that at least 10 7 virus particles g −1 of faecal sample were required for a positive signal. this assay may be useful for rapid screening of faecal samples collected during an outbreak of acute gastroenteritis. to detect and quantify hev virus present in environmental and food samples, a rt-qpcr method was developed by jothikumar et al. (2006) . this taqman assay was designed to target a conserved region in orf3, allowing the detection of four different genotypes of hev. a number of commercially elisa (enzyme-linked immunosorbent assay) kits for the hev detection of igm and igg antibodies which can be used early diagnosis of patients suspected for infection with hev, for the screening of blood units and the follow-up of hev-infected patients are also available. a taqman ). this method, when applied to environmental samples, was able to detect rotavirus at a level 2.6 â 10 4 and 2.6 â 10 5 particles/ml in tap water and environmental water, respectively. a competitive rt-pcr sybr green assay was designed based on conserved regions of the vp6 gene of group a rotaviruses, producing a 433 base pair fragment (schwarz et al., 2002) . an in vitro synthesised rna with a 43-base deletion with respect to the wild-type sequence of this fragment was used as an internal control. using these transcripts as templates, 10 rna molecules were amplified and reproducibly detected. using this protocol, the assay could be used to investigate the presence of rotavirus in environmental, food and water samples. immunochromatography tests are also available for detecting rotaviruses in stool samples. a study undertaken by de grazia et al. (2017) compared the performance of two commercially available one-step chromatographic immunoassays that detect both rotavirus and adenovirus. both tests were able to detect the wide range of rva genotypes circulating over the study period (including g1p[8] , g2p[4] , g3, g4, g9 and g12p[8] ). the results of the present study showed a satisfactory efficacy of the two diagnostic tests analyzed using real-time pcr as a reference test. the case fatality rate for niv is estimated to be 40%-75%. in addition, there is no treatment or vaccine available for either people or animals. the main tests used are real time rt-pcr from bodily fluids and antibody detection via enzyme-linked immunosorbent assay elisa (see "relevant websites section"). for example, a taqman assay as described by guillaume et al. (2004) for niv detected a wide range of virus concentrations from 1.2 â l0 5 pfu to 1.2 pfu per reaction, corresponding to a threshold of 200 pfu/ml for rapid, accurate and quantitative diagnosis. the specificity of the niv taqman assay was determined by the absence of amplification using measles and hendra paramyxovirus rna. an antigen capture elisa was developed by chiang et al. (2010) for the viral detection of henipavirus and for the differentiation between niv and hendra virus. this assay allows for the rapid detection and differentiation between the henipaviruses and could be used in any future outbreaks of henipaviruses. astroviruses are classified into two genera: mammalian viruses (mamastroviruses, mastvs) and avian viruses (avastroviruses, aastvs). human astroviruses are found in four mastv species (mastv 1, 6, 8, 9) (pérot et al., 2017) . an rt-pcr assay was designed by finkbeiner et al. (2009) , based on the astrovirus rna polymerase (orf 1 b) that allows for the detection of the eight human astroviruses serotypes found in mastv 1, a common cause of viral gastroenteritis in children. this primer set also detects viruses in mastv 6. these two groups also contain astroviruses from cats, pigs, dogs and rabbits (pérot et al., 2017) . to date, there is no universal pan-astrovirus rt-pcr assay. immunochromatography (ic) tests are also available for detecting astroviruses. an ic test for the detection of astrovirus was evaluated in 44 stool samples of pediatric patients with acute gastroenteritis in japan, during january to march 2007, and it is a rapid method for the detection of astroviruses and may be useful for screening astroviruses during outbreaks of food-borne and person-to-person transmission (khamrin et al., 2010) . a broad-spectrum pcr assay was developed by sibley et al. (2011) for the detection of mastadenovirus (maadv) and atadenovirus (atadv), based on the adenovirus hexon gene. maadv, which comprises human and bovine adenoviruses and a large variety of mammalian adenoviruses. atadv includes bovine adenovirus (badv) as well as adenovirus infecting ducks, goats, sheep, deer, and reptiles (sibley et al., 2011) . this assay has been shown to demonstrate natural badv excretion in urine, badv detection in groundwater, and recombination in adv of livestock origin (sibley et al., 2011) and has the potential to be used in screening samples during outbreaks of food-borne disease. for the detection of h5n1 influenza viruses, the world health organisation (who) (see "relevant websites section") provides updated information on the molecular detection/diagnostic protocols for the surveillance of influenza viruses in humans. for example, the who provides primers and probes sequences for real-time rt-pcr procedures for the detection of: (1) influenza type a viruses (matrix gene). a taqman based assay was designed for the detection of sars and middle east respiratory syndrome (mers) coronaviruses (covs) by noh et al. (2017) , based on the conserved spike s2 region of human sars-cov, mers-cov, and their related bat covs. this assay can detect sars-cov and mers-cov in humans but also several bat covs that are closely related to these viruses in bats. a monoclonal antibody-based capture enzyme immunoassay for the detection of nucleocapsid antigen in sera from patients with sars was developed by che et al. (2004) . this assay used a mixture of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen. the sensitivity of the assay was 84.6% in 13 serologically confirmed sars patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). the specificity of the assay was 98.5% in 1272 healthy individuals (1253 of 1272). there was no cross-reaction with other human and animal coronaviruses in this assay. • food can become contaminated by viruses at source and through contaminated food handlers and environments. • good food hygiene and personal hygiene, especially hand washing, are essential to help minimise the spread of these viruses within the food chain. • since foodborne viruses tend to be more resistant to physical and chemical treatments than bacteria, their control represents a challenge for the food industry. • ongoing research is required, for example, current laboratory detection methods can be modified to allow differentiation between e.g., infectious and non-infectious viruses. • there are a number of knowledge gaps in terms of how, for example, nov, hav and hev are transmitted through the food chain, the contribution they make to overall foodborne illness and their survival and elimination from food. • emerging trends indicate that viruses play an important role in foodborne illness. this has implications for the whole of the food chain. potential role of fomites in the vesicular transmission of human astroviruses astrovirus survival in drinking water rotavirus in adults requiring hospitalization virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis human astroviruses acute respiratory disease associated with adenovirus serotype 14 -four states alarmins: awaiting a clinical response sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome vaccine development for human mastadenovirus use of monoclonal antibodies against hendra and nipah viruses in an antigen capture elisa isolation of nipah virus from malaysian island flying-foxes immunology: a short course coronaviruses: important emerging human pathogens changing pattern of rotavirus strains circulating in ireland: re-emergence of g2p [4] and identification of novel genotypes in ireland diagnostic accuracy and analytical sensitivity of ideia norovirus assay for routine screening of human norovirus genetic variability and molecular evolution of hepatitis a virus reduction in acute gastroenteritis hospitalizations among us children after introduction of rotavirus vaccine: analysis of hospital discharge data from 18 us states astrovirus infections in humans and animals -molecular biology, genetic diversity, and interspecies transmissions performance analysis of two immunochromatographic assays for the diagnosis of rotavirus infection foodborne transmission of nipah virus in syrian hamsters fields virology. 2 detection of newly described astrovirus mlb1 in stool samples from children letter: virus diarrhea in foals and other animals henipavirus susceptibility to environmental variables next-generation sequencing for diagnosis and tailored therapy: a case report of astrovirus-associated progressive encephalitis isolation and characterization of viruses related to the sars coronavirus from animals in southern china specific detection of nipah virus using real-time rt-pcr (taqman) molecular characterization of group a rotavirus found in elderly patients in ireland person-to-person transmission of nipah virus in a bangladeshi community sensitive detection of multiple rotavirus genotypes with a single reverse transcription-real-time quantitative pcr assay water-bone transmission of influenza a viruses annual cost of illness and quality-adjusted life year losses in the united states due to 14 foodborne pathogens hepatitis a: clinical manifestations and management a broadly reactive one-step real-time rt-pcr assay for rapid and sensitive detection of hepatitis e virus hepatitis e virus infection abiotic factors affecting the persistence of avian influenza virus in surface waters of waterfowl habitats evaluation of a rapid immunochromatography strip test for detection of astrovirus in stool specimens use of infrared camera to understand bats' access to date palm sap: implications for preventing nipah virus transmission goblet cells and mucins: role in innate defense in enteric infections age-stratified seroprevalence of neutralizing antibodies to astrovirus types 1 to 7 in humans in the netherlands hepatitis e -a new era in understanding the action of alcohols on rotavirus, astrovirus and enterovirus severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats international standardisation of a method for detection of human pathogenic viruses in molluscan shellfish type a viral hepatitis: a summary and update on the molecular virology, epidemiology, pathogenesis and prevention a comparison of the efficiency of elisa and selected primer sets to detect norovirus isolates in southern ireland over a fouryear period (2002-2006): variation in detection rates and evidence for continuing predominance of nov gii.4 genotype prevalence and characterization of enteric adenoviruses in the south of ireland bats are natural reservoirs of sars-like coronaviruses immunoglobulins for preventing hepatitis a comparison of norovirus rna levels in outbreak-related oysters with background environmental levels foodborne transmission of nipah virus transmission of human infection with nipah virus adenovirus: epidemiology, global spread of novel serotypes, and advances in treatment and prevention clinical veterinary microbiology clinical veterinary microbiology clinical veterinary microbiology possible waterborne transmission and maintenance of influenza viruses in domestic ducks zoonotic aspects of rotaviruses full genome-based classification of rotaviruses reveals a common origin between human wa-like and porcine rotavirus strains and human ds-1-like and bovine rotavirus strains prevalence and genetic diversity of human astroviruses in mexican children with symptomatic and asymptomatic infections candidate new rotavirus species in sheltered dogs vaccine development against zoonotic hepatitis e virus: open questions and remaining challenges native morphology of influenza virions simultaneous detection of severe acute respiratory syndrome, middle east respiratory syndrome, and related bat coronaviruses by real-time reverse transcription pcr molecular detection of animal viral pathogens long-term protective effects of hepatitis a vaccines. a systematic review case-control study of risk factors for human infection with a new zoonotic paramyxovirus, nipah virus, during a 1998-1999 outbreak of severe encephalitis in malaysia impact of rotavirus vaccination in australian children below 5 years of age hepatitis e and pregnancy: current state hepatitis e virus: infection beyond the liver date palm sap linked to nipah virus outbreak in bangladesh virus hazards from food, water and other contaminated environments foodborne illness acquired in the united states-major pathogens detection and quantitation of group a rotaviruses by competitive and real-time reverse transcriptionpolymerase chain reaction a brief review of foodborne zoonoses in china orthomyxoviridae persistence of avian influenza viruses in various artificially frozen environmental water types detection of known and novel adenoviruses in cattle wastes via broad-spectrum primers environmental role in influenza virus outbreaks hepatitis e: a disease of reemerging importance hepatitis e virus genotypes and evolution: emergence of camel hepatitis e variants estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis world health organization environmental team characterization of a highly pathogenic h5n1 avian influenza a virus isolated from duck meat the unpredictable diversity of co-circulating rotavirus types in europe and the possible impact of universal mass vaccination programmes on rotavirus genotype incidence rotavirus infection in infants as protection against subsequent infections novel human astroviruses: novel human diseases? epidemiology of classic and novel human astrovirus: gastroenteritis and beyond henipaviruses sars-cov infection in a restaurant from palm civet adenovirus gastroenteritis global use of rotavirus vaccines recommended. who orthomyxoviruses detection of nipah virus rna in fruit bat (pteropus giganteus) from india hepatitis e virus: foodborne, waterborne and zoonotic transmission rotavirus incidence and genotype distribution before and after national rotavirus vaccine introduction in belgium development and evaluation of a loop-mediated isothermal amplification assay for the detection of adenovirus 40 and 41 prevention and control: oie -world organisation for animal health who -first data on stability and resistance of sars coronavirus compiled by members of who laboratory network who information for the molecular detection of influenza viruses who -updated unified nomenclature system for the highly pathogenic h5n1 avian influenza viruses key: cord-318826-l922zqci authors: holschbach, chelsea l.; peek, simon f. title: salmonella in dairy cattle date: 2018-03-31 journal: veterinary clinics of north america: food animal practice doi: 10.1016/j.cvfa.2017.10.005 sha: doc_id: 318826 cord_uid: l922zqci as an infectious, contagious pathogen, salmonella is probably rivaled by only bovine viral diarrhea virus in its ability to cause clinical disease, such as enteritis, septicemia, pneumonia, and reproductive losses. the increasing prevalence of salmonella, particularly salmonella dublin, on dairies presents new challenges to producers and veterinarians. no current discussion of bovine salmonellosis is complete without acknowledging the increasing public health concern. increasing antimicrobial resistance among enteric pathogens brings the use of antimicrobials by veterinarians and producers under ever stricter scrutiny. this article provides a comprehensive review of salmonella etiology, prevalence, pathogenesis, diagnostics, treatment, and control. facet to salmonellosis on many modern dairies. the ability to establish lifelong infection, characterized by an asymptomatic carrier status, with intermittent periods of bacteremia and intermittent shedding, challenges control of this serotype. enteric infection with other non-host-adapted serotypes, particularly in calves, can also be associated with true bacteremia, sepsis, and high mortality rates. no current discussion of bovine salmonellosis could be complete without acknowledging the increasing public health concern regarding its relevance as an important zoonosis, the risk that contaminated dairy and dairy beef products can pose to human health, and, just as important, the reality that increasing antimicrobial resistance among zoonotic enteric pathogens such as salmonella brings the use of antimicrobials by veterinarians and producers under ever stricter scrutiny. salmonella is a genus of gram-negative, facultative anaerobic bacteria that belong to the family of enterobacteriaceae. there are 2 recognized species within the genus: s enterica and salmonella bongori. s enterica can be further divided into 6 subspecies, s enterica subspecies enterica being the most relevant in dairy cattle. 1 more than 2500 serovars (serotypes), differentiated by their antigenic composition, have been identified. serovars are based on the somatic (o), flagellar (h), and capsular (vi) antigens. 2 most human and veterinary diagnostic laboratories have phenotypically divided salmonella isolates into serogroups based on detection of the o lipopolysaccharide and h flagellar antigens, historically by agglutination methods. 2, 3 although these traditional serotyping techniques have formed the basis of human and veterinary diagnostic practice for salmonellosis for several decades, they are labor intensive and time consuming, typically taking at least 48 hours. 4 with the advent of more advanced molecular diagnostic methods, genetic approaches to serotyping are beginning to supercede traditional tests. in general, these methods use 1 of 2 types of targets for serotype determination, the first are indirect targets, which use random surrogate genomic markers known to be associated with certain serotypes, and the second method uses direct targets requiring the use of highly specific genetic determinants of a particular serotype. 5 the latter typically involve the rfb gene cluster responsible for o somatic group antigen synthesis 6 and the flic and flib genes encoding the 2 flagellar antigens of salmonella. 7 genomic sequencing is becoming increasingly common for the identification and serotyping of salmonella isolates. 4, 5 the hope is that, with diminishing costs and continued refinement, more rapid, accurate genoserotyping will improve diagnostic and surveillance efforts for both public health and veterinary purposes. 8 most commonly, clinical bovine isolates have been divided by their o antigens, and serovars are further grouped into serogroups assigned to an early letter of the alphabet (eg, a, b, c, d, and e). 9 by current convention, salmonella isolates are referred to by their serovar/serogroup classification (eg, s enterica subspecies enterica serovar typhimurium, is abbreviated to salmonella typhimurium). despite the diversity of serovars, relatively few are of clinical importance among cattle. the majority of cattle isolates are salmonella of types b, c, and e, which are non-host specific, or salmonella dublin (type d), which is the host-adapted serovar in cattle. 9 the isolation of salmonella from the feces of dairy cows or calves as well as the environment on dairy farms is increasingly common. as part of the united states department of agriculture's national animal health monitoring system (nahms) dairy 2007 holschbach & peek study, 10 fecal samples were collected from approximately 30 healthy cows on each of 121 dairy operations across 17 states. forty percent of the dairy operations had at least 1 cow that was salmonella positive on fecal culture. of the roughly 3800 healthy cows sampled, 14% were fecal culture positive. compared with the dairy 1996 nahms study, 11 the percentage of salmonella-positive operations had doubled and the percentage of positive cows had more than doubled. 10 for the 2007 study, when environmental sampling was performed in conjunction with individual cow sampling, the number of dairies with a positive salmonella culture increased to nearly 50%. 10 within the 2007 study, the most frequently isolated salmonella serotypes included salmonella cerro, salmonella kentucky, salmonella montevideo, and salmonella muenster. these serotypes fall within groups k, c3, c1, and e, respectively. in a comprehensive study of more than 800 dairy herds in the northeastern united states in 2009, fecal samples were collected from female dairy cattle for salmonella culture based on a suspicion of clinical disease. 12 salmonella was found in 11% of the dairy herds monitored for approximately 1 year over the course of the study. the herd-level incidence rate was approximately 9 positive herds per 100 herdyears; however, just 17% of the positive study herds accounted for more than 70% of the clinical salmonella cases. 12 the predominate serotype identified was salmonella newport, accounting for 41% of the cases, followed by salmonella typhimurium, accounting for nearly 20% of cases. 12 clustering of disease among herds was consistent with another us prevalence study that found that 25% of the enrolled dairy farms accounted for more than 75% of the salmonella-positive fecal and environmental samples. 13 in this study, sampling of conventional and organic herds on 5 occasions over a period of 1 year resulted in detection at least 1 salmonella-positive fecal sample on more than 90% of farms (100/110). serogroup e1 was the most commonly identified serogroup in fecal samples, although serogroup b was the most common isolate across farms, with 43% of fecal-positive farms having at least 1 serogroup b isolate. data from a more recent 2013 study demonstrated that of the nearly 1800 salmonella isolates identified at the national veterinary services laboratory from clinical and nonclinical case submissions, the most common serotype was salmonella dublin (18%) followed by salmonella cerro (16%) and salmonella typhimurium (13%). 14 a retrospective study of s enterica isolates submitted to the wisconsin veterinary diagnostic laboratory from 2006 to 2015 parallels the findings from the national veterinary services laboratory. of the nearly 5000 isolates identified, salmonella dublin was the most prevalent serotype identified, accounting for a total of 1153 isolates (23% of total). along with dublin, salmonella cerro (16%), newport (14%), montevideo (8%), kentucky (8%), and typhimurium (4%) comprised the top 6 most commonly isolated sertotypes. 15 the emergence of salmonella dublin as one of the most commonly isolated serotypes is of major concern for the dairy industry. as the host-adapted strain of salmonella in cattle, animals infected with salmonella dublin can become chronic, subclinical carriers that have the potential to shed large numbers of organisms into the environment. these carriers also play an important role in maintaining infection within a herd by shedding not only in feces, but also in milk and colostrum. salmonella infections are well-known for their association with clinical signs of enterocolitis, septicemia, and abortion in dairy cattle. 9 pneumonia is an increasingly common manifestation of salmonella dublin infection in calves 16, 17 and worth bearing in mind when dealing with mild, moderate, or severe respiratory disease on heifer rearing facilities. whether or not this merely represents hematogenous localization of the salmonella in dairy cattle organism to the lungs in much the same way that is seen with septic arthritis, for example, or a more specific organ tropism for the lungs by this serovar is uncertain. however, personal observations by one of the authors and many others suggest that this particular clinical manifestation of salmonella dublin infection is increasingly common during the late nursing and postweaning period. salmonella infection is most commonly transmitted by fecal-oral contamination from other livestock, rodents, birds, or by feeding contaminated protein source animal byproducts. 1 given the increased frequency with which the organism can be isolated on dairy farms, from both symptomatic and asymptomatic cattle, it is reasonable to assume that fecal-oral spread from other cattle is the most common means of spread on modern dairies. older literature establishing that aerosol transmission was possible in closely confined, penned calves would also seem to be currently relevant with respect to the spread of certain salmonella serotypes, especially salmonella dublin, on endemic heifer rearing facilities. 18, 19 in both calves and adults, those factors that determine pathogenicity and whether or not clinical disease is seen include virulence of the serotype, dose of inoculum, degree of immunity (passive or adaptive) or previous exposure of host to the serotype, and other stressors currently affecting the host. 20 the organism will less frequently penetrate ocular or nasal mucous membranes. the most detailed studies of the pathogenesis of bovine salmonellosis infection come from the literature describing enteric infection via the oral route, mainly in calves. [21] [22] [23] once ingested, salmonella attaches to mucosal cells and is capable of destroying enterocytes. attachment is increased if gastrointestinal stasis is present or the normal flora has been disturbed or is not yet established, as is the case in neonates. 1 the organism penetrates through the enterocytes to the lamina propria of the distal small intestine and colon, where they stimulate an inflammatory response or are engulfed by macrophages and neutrophils. 1 once salmonellae have gained entry to mononuclear phagocytes, they can be rapidly disseminated throughout the body. salmonellae have a predilection for lymphoid tissues, invading through m-cells, and are found in the highest numbers in the peyer patches and mesenteric lymph nodes. from here, the organism often enters the lymphatics and may eventually lead to bacteremia. 9, 23 experimental studies have also shown that oral exposure can lead to infection and systemic dissemination via pharyngeal lymphoid tissue (tonsils) without the need for true enteric infection. 24 salmonellae are capable of surviving and multiplying in numerous host tissues, often as facultative intracellular bacteria in macrophages and reticuloendothelial cells. 1 these characteristics guard them against the hosts' normal defense mechanisms and potentially facilitate true bacteremia. the virulence mechanisms of salmonellae are, therefore, composed of their ability to invade the intestinal mucosa, locate to and multiply within the lymphoid tissues, and to evade host defense mechanisms. enterocolitis caused by salmonella spp. is due to inflammation with subsequent maldigestion and malabsorption, and to a lesser extent from secretory mechanisms. 9, 23 inflammation in the colon leads to the commonly observed fresh blood in the feces of both adults and calves. the diarrhea caused by salmonella spp. is principally mediated by the host inflammatory reaction to the infection. to establish infection, enteropathogens such as salmonella must first be able to overcome those host factors that resist colonization of the gut, principle among these being a fairly dense gut microbiota, 25 which secrete a variety of bacteriocins, antibiotics, and colicins that hinder enteropathogen growth. 26 there is increasing evidence that many enteropathogens, including salmonellae, are not able to colonize the gut in the face of a normal microbiota 25 and hence factors that negatively influence this key component of resistance are important in the predisposition to enteric disease. once salmonella density reaches a critical threshold (about 10 8 colony-forming units per gram in the case of salmonella typhimurium in mice), then a sufficient number of organisms can invade the gut epithelium by first docking with and then invading the epithelial cells. 25 at a molecular level, salmonella typhimurium does this by specific bacterial adhesins for attachment and then a secretion system that injects a cocktail of bacterial toxins (the type iii secretion system) that enables the bacterium to reach the lamina propria. 27 damaged gut cells are expelled into the lumen, as part of the host defense system, giving rise to some of the clinical signs of salmonellosis and a profound inflammatory response is initiated via interleukin-18 within 10 to 18 hours after infection. 27 there are molecular reasons that underscore the clinical observation that differences in pathogenicity between serotypes exist. some strains of salmonella dublin and salmonella typhimurium, for example, have a virulence plasmid (carrying the spv gene) that facilitates survival of the organism within phagocytes, partly perhaps explaining the increased association of these 2 serotypes with clinical disease in calves and adults. the ease with which genes can be transferred between salmonella and other members of the enterobacteriaceae also provides a rational explanation for the transfer of antimicrobial resistance. 20 precise and eloquent experimental data on the mechanisms by which salmonella infection can lead to reproductive loss and abortion are hard to find. clinically, abortions are most common when serotypes b, c, or d are involved and it makes intuitive sense that abortion in cattle infected with salmonella spp. could arise through several different mechanisms. septicemia could lead to seeding of the fetus and uterus, causing fetal infection and death. 1, 9 the fact that diagnostic post mortem investigations of aborted fetuses can often recover the organism from fetal samples supports this possibility. endotoxemia leading to inflammatory mediator release might also cause luteolysis secondary to prostaglandin release. high fevers or hyperthermia could also play a role in prostaglandin release or cause abortion through more direct fetal injury. cows may abort at any stage of gestation, but expulsion of the fetus is most common at 5 to 9 months of gestation. 1,9 a definitive diagnosis of salmonella infection in the live animal involves detection of the organism, most commonly by aerobic culture. although a clinical history of febrile illness accompanied by hemorrhagic enteritis and anorexia may be suggestive in either calves or adults, there is a sufficient differential diagnosis list in both age groups that diagnostic sampling must be performed. when reproductive losses are encountered in pregnant cattle, unless there are concurrent cases of bloody diarrhea, the clinical signs are even less definitive for salmonellosis and the differential list even longer. for hemorrhagic enteritis in adults, the differential list principally includes winter dysentery and bovine viral diarrhea virus infection; in calves, depending on age, such a presentation merits consideration of several viral (rotavirus, coronavirus), protozoal (cryptosporidium, eimeria), and bacterial causes (escherichia coli, clostridium perfringens). however, one should not rely on the presence of blood in the stool; many cases of enteric salmonellosis present without this clinical finding. remarkable variation in clinical severity will occur based on serovar virulence, host immunologic status, and inoculating dose. in calves, death may occur owing to septicemia before diarrhea salmonella in dairy cattle becomes obvious or a significant clinical abnormality. in large free stall dairies, it is increasingly common to encounter salmonella infection as an endemic challenge with clinical presentations that are highly variable, ranging from the classic textbook description of reproductive losses and enteric disease in adult cattle through to lower impact problems with fevers of unknown origin, little to no diarrhea, and only modest consequences in terms of appetite and milk yield reduction. because salmonella organisms are easily and rapidly out competed by other fecal gram negatives, the majority of diagnostic laboratories use enrichment media such as tetrathionate or selenite broth to improve the chances of salmonella growth and then plate these enriched samples onto selective media such as brilliant green or xylose lysine deso-oxycholate agar. 28 veterinarians working in the field are advised to contact their local diagnostic laboratory for assistance with sample handling, processing, and submission before investigating either individual or group problems with enteric disease suspicious for salmonella infection. it is frequently worthwhile to place samples directly into enrichment media before submission to improve the chances of positive culture and to keep samples chilled until they arrive at the diagnostic laboratory. disadvantages of fecal culture include the fact that shedding can be sporadic, even in true infections (certainly when one considers the sensitivity of bacterial culture) and that, in the face of an ongoing outbreak, one can occasionally encounter clinically normal calves and adults who shed the organism but never develop any clinical signs. 20 the latter situation may still provide useful information, however, both from the perspective of deciding which animals merit treatment but also from the broader standpoint of identifying an enteric pathogen that should never be trivialized or considered a commensal. however, the general pattern is that subclinically or persistently infected cattle shed low numbers of organisms, whereas clinically ill or acutely infected animals may excrete higher numbers in feces. 17 when the clinical suspicion of salmonella is high, a single negative culture is not sufficient to rule out infection. as mentioned, fecal samples should be submitted to qualified diagnostic laboratories that are equipped to culture enteric pathogens and with careful attention to sample handling. 9 although culturing of individual cow fecal samples is the most common method used to assess individual and herd salmonella status, it can be expensive and time consuming, especially in larger herds. in a study comparing individual, pooled, and composite fecal samples, it was found that composite fecal sampling was more sensitive at the sample level than the other 2 methods, primarily because of the increased number of cattle sampled indirectly through this method. 29 hence, if one is merely trying to obtain a yes or no answer or identify and track specific serovars, or antimicrobial susceptibility patterns over time, composite fecal samples are typically collected from areas on dairy operations where manure accumulates from a majority of adult animals, such as holding pens, alleyways, and lagoons. 29 newer techniques for diagnosing salmonella are based on detection of genetic material from the bacteria, that is, polymerase chain reaction (pcr) techniques. 30, 31 these techniques are generally thought to be more sensitive than culture, but have the disadvantage that subsequent serotyping is not always possible. 17 both species-specific (s enterica) and individual serotype pcr tests are available at some, but not all, veterinary diagnostic laboratories within the united states. there are 2 main pcr methods: the traditional pcr and the real-time pcr. in the traditional pcr method, the test result is qualitative (yes or no). in real-time pcr, a threshold cycle (ct-value) gives a quantitative value of dna in the sample; the ct-value is inversely correlated with the starting concentration of the target dna; hence, the lower the ct number the more salmonella dna there will be in the sample. at the current point in time, only a few veterinary diagnostic laboratories offer both species-specific and serotype (usually salmonella dublin) assays for use with biological samples such as feces, milk, or tracheal and bronchoalveolar lavage fluid. the advantage is a quicker turnaround time and the potential for greater sensitivity, although parallel cultures are still necessary for in vitro antibiograms to be performed to aid treatment decisions (dr keith poulsen, wisconsin state veterinary diagnostic laboratory, personal communication, 2017). it is possible that, in the near future, pcr assays may become used for environmental samples although these can contain so many potential pcr inhibitors and out-competing organisms that sensitivity and specificity may be lost. 32 the use of pcr methodology to investigate contamination of milk is also of increasing relevance, potentially for veterinarians, but also from the public health perspective. certain serovars, notoriously salmonella dublin, but also to include salmonella typhimurium and newport, can be found in the milk or colostrum of infected lactating animals. 9 although conventional pasteurization should kill the organism, there is an understandable desire for food safety reasons to use highly sensitive methods to detect the organism after harvest. 33 although fecal culture remains the gold standard at most laboratories, blood culture, a culture of transtracheal wash or bronchoalveolar lavage fluid, and joint fluid may all be useful choices for individuals experiencing bacteremic salmonellosis. the propensity for bacteremia in neonatal calves with salmonellosis makes aseptically obtained aerobic blood cultures a particularly useful diagnostic sample to consider in valuable animals. 1, 9 culture of these nonfecal samples is far less likely to be diagnostically valuable in adults, although pcr methods on such samples may potentially improve sensitivity in the future. although gross post mortem findings of severe, diffuse, fibrinonecrotic ileotyphlocolitis with watery, often bloody content are highly suggestive, they are neither consistent enough or definitive for enteric salmonella infection in calves or adults. 20 however, in both calves and adults, necropsy material can provide excellent diagnostic material for the definitive diagnosis of salmonella infection. in all age groups, it is advised to obtain numerous samples from the gastrointestinal tract (ileum, cecum, colon), mesenteric lymph node, and gall bladder (bile is a particularly useful sample), as well as lung tissue, especially when consideration of salmonella dublin is warranted, as increasingly is the case. because veterinarians are rarely only interested in the diagnosis of salmonella infection during a field necropsy, one may need to take multiple samples from such sites and handle the samples specifically as described to enhance the chances of a positive salmonella culture. culture remains the most common method used by most diagnostic laboratories to confirm salmonella infection in post mortem samples. samples from abortion cases that may have been caused by salmonella, should include fluid or tissue from both the dam and the fetus. most salmonella-associated abortions are in the last trimester so there will be a fetus to work with, preferably relatively fresh depending on the delay before the fetus is discovered. samples from the dam might include milk or colostrum, serum, and feces. feces and milk can be screened via culture or pcr, whereas the serum sample can be used for salmonella dublin serology (described elsewhere in this article). providing the fetus is not severely autolyzed, heart blood, abomasal contents, and intestinal or biliary samples might be useful but diagnostically veterinarians are all too commonly challenged by the "freshness" of an abortus. as is true of many enteritis investigations, with abortion cases veterinarians are typically attempting to submit samples that might reveal one of many possible infectious etiologies and it may be simpler to submit the entire fetus if this can be done in a timely manner. environmental sampling on dairy farms and heifer rearing facilities has largely been a research tool rather than a clinically applicable procedure. however, quite a lot of information has been learned regarding areas of large free stall facilities where positive salmonella cultures can often be repeatedly obtained either in herds with or without known clinical disease. 34, 35 not surprisingly, areas of high traffic use and density and where sick cows and cows soon to calve are located are frequently discovered to yield positive cultures. 34 just as was discussed under individual cow fecal sampling, veterinarians are advised to seek the input of the laboratory to which they are going to submit samples before obtaining on-farm environmental specimens. the use of buffered peptone water or more specific enrichment broths before submission may improve chances of salmonella being isolated from heavily contaminated samples. 34 drag swabs, milk filters, and even absorbent socks worn over shoes, as have been used for environmental sampling in poultry houses, can be used. proof of current infection with salmonella dublin can be achieved via conventional culture with serotyping or pcr methodologies if available. 9, 17 in addition, both in the united states and several countries in europe it is also currently possible to use an enzymelinked immunosorbent assay (elisa) to measure the level of antibodies directed against o-antigens from salmonella dublin in blood and milk. in this way, one can measure the humoral immune response as an indicator of current or previous infection. 36, 37 some laboratories report the elisa result as a semiquantitative percentage value, giving an optical density reading referable to a standard set of controls. in addition, elisa tests can also be used for individual or bulk tank milk sample screening, 38 and have come to be used quite extensively in countries such as denmark, where active surveillance programs for this serovar are in effect. 17, 39 sensitivity for the serum elisa is considerably higher than fecal culture for the identification of salmonella dublin infected cattle, 17 and as a diagnostic test the serum elisa is reported to perform best when used in animals between 3 and 10 months of age (box 1). 36 treatment fluid therapy is the mainstay of treatment for cattle with enteric salmonellosis. 40 the type of fluid and route of administration is based on the severity of clinical signs box 1 salmonella diagnostic testing options individual animal fecal culture using enrichment and selective media. composite fecal sampling. salmonella polymerase chain reaction (feces, milk, tracheal or bronchoalveolar lavage fluid). blood, transtracheal wash, bronchoalveolar lavage, or joint fluid culture when bacteremia is suspected in calves. culture of post mortem samples: gastrointestinal tract, mesenteric lymph node, bile, and lung. salmonella dublin enzyme-linked immunosorbent assay: serum or milk. and the economic value of the animal. in calves with acute, severe diarrhea showing signs of hypovolemic shock, intravenous fluid therapy using a balanced electrolyte solution, such as lactated ringers, is necessary. 20, 40 in severely depressed or comatose animals, resuscitative fluids, such as hypertonic saline, are indicated. if administered, hypertonic saline, dosed at 2-4 ml/kg, should always be followed with isotonic crystalloids or water to replace the "borrowed" water from the intracellular space. dextrose supplementation can be a critical part of the intravenous fluid therapy plan for calves with salmonellosis, not only because of poor feed intake, but because of the increased risk of hypoglycemia that may accompany septicemia. calves that are ambulatory, have a suckle, and are only moderately dehydrated can often be managed with oral fluids. 40 calves and even adult cattle can develop severe metabolic acidosis with peracute salmonella infections and intravenous bicarbonate-rich fluids should be considered when profound depression or shocklike signs accompany diarrhea. oral electrolyte solutions have proven to be helpful in correcting mild to moderate dehydration; however, depending on the degree of bowel inflammation, fluid absorption and digestion may be altered. fluid therapy for adult cattle in the field setting can prove to be more challenging owing to the sheer volume of fluid needed in cases of severe dehydration. hypertonic saline followed by at least 10 gallons of oral electrolytes or water, either consumed voluntarily or given by orogastric tube, is a highly efficient method of fluid resuscitation in adult cattle. in valuable calves or adults, colloids (plasma or hetastarch) are often indicated as a result of hypoproteinemia secondary to albumin loss from the gastrointestinal tract. synthetic colloids, such as hetastarch, are a more reasonably priced option, but only augment colloidal pressure. plasma has the added benefit of immunoglobulins and acute phase proteins, which provide therapeutic benefits in septic or inflammatory conditions. 9 antimicrobial therapy for the treatment of salmonellosis was, is, and probably always will be, controversial. of utmost concern is the potential for the creation of antibiotic-resistant strains of salmonella that may present a risk to humans or animals in the future. although antimicrobial therapy may aid in clinical recovery, it has also been criticized as failing to limit fecal shedding or to impart a positive effect on the duration of fecal shedding. in truth, this criticism is largely extrapolated from research in other species. in cattle, the effect of prior antibiotic use on fecal shedding may be age variable, with research identifying that the risk of fecal shedding after antibiotic treatment is greater for adults and heifers than in calves. 41 however, the risk of true bacteremia in calves with enteric salmonellosis is substantial, justifying the use of antimicrobials in patients of this age. 1 bacteremic spread of the organism can result in concurrent disease in multiple organs, such as pneumonia, arthritis, and meningitis. the presence of these clinical infections should always merit antimicrobial administration. the comparative risks for such systemic complications in adults are less than in calves, making the routine use of antimicrobials in mature animals less justifiable. if possible, antimicrobial selection should be based on culture and susceptibility of the salmonella isolate. the dilemma faced by practitioners is frequently that real-time decisions regarding antimicrobial use and selection have to be made in advance of any definitive microbiologic data. some guidelines regarding salmonella susceptibility can be provided, however. according to the nahms 2007 study, isolates were found to be most resistant to tetracycline, streptomycin, ampicillin, and ceftiofur, but were frequently sensitive to aminoglycosides, fluoroquinolones, and trimethoprim-sulfas. 10 to the us readership, these lists will not provide much comfort because of restrictions on antimicrobial use under the current animal medicinal drug use clarification act. fluoroquinolones and certain sulfonamides may not be used extra-label in the united states. additionally, there is a voluntary ban on the use of aminoglycosides, such as gentamicin and amikacin, in food-producing animals because of long-term tissue residues. as of 2012, the extra-label use of ceftiofur in regard to dose, route, and frequency of administration is also prohibited. owing to the facultative intracellular nature of the organism, it is also worth bearing in mind that antimicrobial penetration into the cell can be limited, even for antimicrobials that show in vitro efficacy. when chosen, antibiotic therapy should be continued for at least 5 to 7 days in cases of acute or peracute salmonellosis. 9 appropriate withdrawal times should be observed for all antimicrobial usage and animal medicinal drug use clarification act guidelines followed at all times. for questions regarding extended withdrawal times and extralabel use of antimicrobials, us readers are advised to contact the food animal residue avoidance database. in addition to crystalloid fluid therapy, colloid administration when indicated by hypoproteinemia, and responsible, legal, and signalment appropriate selection of antibiotics, the third and final component of therapy for salmonellosis is antiinflammatory use. the inflammatory cascade triggered by local or systemic infection with salmonella is a critical component of the pathogenesis of this organism and culminates in many of the clinical signs observed. direct endotoxin-mediated effects alongside the host systemic inflammatory response are major components of many calf and adult salmonella infections that can be mitigated, at least in part, by the use of nonsteroidal antiinflammatory drugs. 1 cattle may be dosed with flunixin meglumine at 1.1 mg/kg of body weight intravenously every 24 hours and then tapered to 0.5 mg/ kg every 24 hours, or the medication discontinued after the patient stabilizes. 9 label use of flunixin meglumine includes dosages of up to 2.2 mg/kg in the united states. prolonged administration of nonsteroidal antiinflammatory drugs, particularly at the higher dose or in the face of dehydration, can lead to abomasal ulceration and renal papillary necrosis. 1, 9, 20 in rare circumstances, some clinicians elect to administer "shock" doses of corticosteroids, but this measure would be uncommon in either general or referral practice. soluble prednisolone sodium succinate would be the preferred agent in such circumstances. from both the literature and personal experience, it seems that not only are herd epidemics becoming more common, but perhaps more worryingly the disease has become endemic on an increasing number of facilities. endemicity is obviously problematic with any serovar, but is inevitable when the herd prevalence of salmonella dublin infection increases. frequently, the disease becomes a cyclical problem responsible for a spectrum of illness that varies from the more classic presentations described through to milder illness perhaps characterized by fever, looser than normal stool, and mild production loss. depending on the interaction of general cow health, other concurrent stressors, climatologic stress, and the level of fecal-oral challenge at any one time, adult cows may or may not become clinically ill. transition cow management becomes an important factor in whether or not new infections are acquired and subsequently result in clinical illness in the late dry and early lactation period, a time when cattle may be at their most susceptible to infectious disease. 9 as with any fecally-orally spread organism, control strategies are broadly speaking simple to describe, but not necessarily so easy to put into place for many dairies. larger herd size, crowded husbandry, and free stall housing all contribute to an increased propensity for exposure to contaminated manure, and although purchased feedstuffs are still occasionally incriminated as a means by which new salmonella infections are introduced onto farms, as are rodent and bird populations, the major source of infection are other cattle shedding the organism in their feces. the high likelihood of feces being contaminated with salmonella organisms on many diaries should mitigate against the spreading of manure on fields that are to be used for forages, or common use equipment for manure handling and feed distribution. evidence suggests that heating of manure to greater than 45 c for more than 3 days, alongside aeration of composted manure using straw, markedly and significantly reduces the number of salmonella organisms, although it is uncertain how practical this information is to larger dairies with modern large-volume manure handling systems. 42 peculiarly, and perhaps rather worryingly, 1 study looking at risk factors for increased antimicrobial resistance among salmonella isolates on dairy farms identified the use of composted manure for bedding as a significant problem. 43 the most directly applicable research regarding modern manure handling systems and survival of salmonella organisms under natural rather than laboratory conditions demonstrated that a multiple-drug-resistant strain of salmonella newport survived for less than 24 hours in a compost pile at 64 c, but would survive for more than 4 months and more than 9 months in an effluent lagoon and field soil, respectively. 44 once salmonellosis has been confirmed in adult cattle, there are a number of further investigative and control measures that may be implemented. these measures do not differ according to serotype, but there are some specific challenges concerning the host adapted serovar salmonella dublin that will be discussed in a later section. it is prudent to consider the possible source(s) of the infection. although commodities, especially protein feed sources, and wild bird and rodent populations have been incriminated in many texts over the years, it seems quite uncommon these days for a single point source event to have introduced the infection onto a dairy de novo. environmental sampling of feed, water, and storage facilities can be helpful in identifying contamination in this regard, but if, as is commonly the case on larger dairies, management continues to purchase replacement animals or expand from other herds, it seems inevitable from prevalence data that the infection will be introduced via infected cattle and their feces. in all probability, many "new" outbreaks are likely surges in clinical disease and new infections in a herd where the infection already existed but hitherto had remained subclinical. factors in transition cow management that reduce immunologic competence or increase exposure risk, are likely to contribute to the onset of clinical disease in such circumstances. the isolation of affected animals and strict attention to hygiene are pieces of advice routinely given but difficult to implement on large dairies. the numbers of affected animals can be overwhelming and lactating cows have to be milked at least twice a day, requiring them to walk and congregate in frequently trafficked areas and holding pens for the parlor into which they release enormous numbers of organism whenever they defecate. avoidance of common use equipment for manure handling and feed distribution have already been mentioned, but should be in place on well-managed dairies anyway. sick, transition, and maternity animals should never be housed together, but unfortunately are for convenience on many occasions; this condition merely ensures exposure of the most susceptible animals to those most likely to be contagious. cleaning and disinfection of the environment are also important, but again somewhat intimidating in the context of a larger dairy. proper cleaning and disinfection of the environment and equipment after a salmonella outbreak can, however, be critically important in decreasing the risk of disease transmission to both cattle and humans. cleaning is defined as the removal of all visible debris and is arguably the most important step in decontamination of animal environments. even the best disinfectants will be minimally effective when used in the presence of organic matter, such as feces and bedding material. 1 not only does cleaning remove the physical barrier between disinfectants and the organism, but it also removes a majority of the organisms so that fewer need to be killed by the disinfectants. this is especially helpful with fecally-orally spread infections like salmonella. where the infectious dose is relatively high (often in the order of 10 6 -10 8 organisms 9,17 ). livestock trailers, maternity and calf pens, feeding equipment, and other areas suspect of being contaminated with salmonella should be the main focus for cleaning and disinfection. although high-power washing can be quite helpful in removing organic debris, its use is not recommended because of the risk of cross-contamination of the environment, and splashing and aerosolization of contaminated material, which can lead to human and animal infection. 9, 45 power washing also fails to remove biofilm, which is an essential and vital component to proper cleaning. in place of power washing, hand-held foamers can be used to apply alkaline detergent and acid rinses for cleaning. the wisconsin veterinary diagnostic laboratory has formulated a cleaning and disinfecting protocol specifically for premises with confirmed salmonella, which can be found at www.wvdl.wisc.edu. a recent paper examining disinfection efficacy against several common bacterial pathogens in a large animal hospital environment showed an approximately 90% reduction in colony-forming units per milliliter of s enterica when either an accelerated hydrogen peroxide or peroxy monosulfate disinfectant product was used via a mist application technique, provided adequate cleaning was performed first. 46 as with antimicrobial drugs, disinfectants have a spectrum of activity that can be highly variable between disinfectant classes. 1 examples of disinfectants commonly used in veterinary medicine include bleach (sodium hypochlorite), quaternary ammonium, phenols, and peroxides. bleach is rapidly inactivated by organic debris, but has a broad spectrum of activity. quaternary ammonium has moderate activity in organic debris and is effective against gram-negative bacteria, such as salmonella. the principle advantage of phenols is better activity in organic debris. peroxides are increasingly used for environmental disinfection, footbaths, and environmental misting and fogging, 1, 46 and are perceived as being more environmentally friendly than chemicals such as phenols and bleach. chlorine dioxide is a powerful oxidant as well as disinfectant, and it can be used to remove and prevent biofilm formation. its use in the dairy industry is becoming more common. current recommendations from the wisconsin state veterinary diagnostic laboratory are for its use in solution at 250 ppm. although rarely done on farm, the effectiveness of environmental cleaning and subsequent disinfection for salmonella control can be assessed by postdisinfection sampling. ongoing efforts at animal isolation and environmental hygiene will be important because shedding of salmonella will continue for many weeks after the initial cases have seemingly resolved. with respect to control, shedding continues periodically for the life of the animal in the case of salmonella dublin. once salmonella has been identified on a farm, veterinarians and management should increase awareness of the public health risk among workers and revisit personal hygiene, protective clothing, and appropriate disinfectant footbath use for employees. if time and labor resources are limited, then concentrating cleaning and disinfection efforts toward highrisk groups (transition cows, maternity pen) and high use traffic areas may be a reasonable compromise. inevitably, the identification of salmonella infection in adult cows or calves will lead to a conversation about vaccine use as a preventative strategy. many farms have at one time or another tried a commercially available or autogenous salmonella vaccine as an adjunct component of control. the safety and efficacy of autogenous products are questioned by many academicians, but individual experiences are sometimes compelling, at least in the short term in the face of an outbreak. as with other infectious contagious diseases such as infectious bovine keratoconjunctivitis, when any vaccine product is used during an outbreak it is impossible to know whether improvement was associated with vaccine use or natural immunologic exposure and protective antibody responses. the most commonly used product in the united states currently for the control of salmonellosis in adults is a siderophore receptor/porin vaccine derived from salmonella newport (salmonella newport bacterial extract, zoetis animal health, parsippany, nj). it is administered to dry cows as an initial 2 injection series and boostered annually. it can, however, be given at any stage of lactation or to heifers. it will not prevent infection, but has been associated with an amelioration in disease severity. it does result in demonstrable antibody levels in colostrum when administered twice during the dry period, although the protective effect of these antibodies against challenge postnatally in calves at this time is unknown. 47 the efficacy of other gram-negative core vaccines to prevent or decrease salmonella disease, such as the j5 product (enviracor, zoetis animal health) or endovac-bovi (immvac, columbia, mo), which are specifically marketed for protection against coliform mastitis, is highly debatable. the maintenance of good general health, excellent hygiene, and particular attention to the well-being of late gestation and early lactation animals are all critical components of salmonella control. a closed herd is ideal, but rarely achieved, making exposure to the organism inevitable on most dairies. prompt diagnosis, treatment, and isolation are important during an outbreak in adult cattle and environmental sampling to include bulk tank milk and high-risk housing areas should now be considered a routine part of disease prevention and surveillance. many of the important components of adult cow control programs mentioned in the previous section overlap with specific measures recommended for calves. an article in a previous volume of this journal provided an excellent review of control measures specific to calves. 20 as in adult herds, endemic disease is increasingly common among calves. commercial heifer rearing facilities that manage preweaned calves from as young as a few hours of age onward, sourced and transported from multiple farms of origin, create a high-risk environment for the acquisition and spread of neonatal salmonellosis. adequate passive transfer, although imperative for rearing healthy calves, is not an absolute guarantee for protection from salmonella infection. fecal-oral transmission is a prime means of spread for enteric and septicemic salmonella infection in calves, but one must be mindful of the risk posed by other secretions such as colostrum, unpasteurized milk, and respiratory secretions, especially in the case of salmonella dublin. hygiene, isolation, and treatment principles for calves, calf housing, and personnel working with calves are very similar to those discussed in the adult section. special consideration should be given to fecal contamination of milk, milk replacer, colostrum, feeding equipment, and starter rations as a means of cross-infection. periodic environmental sampling of equipment such as nipple feeders, buckets, and housing can be valuable tools to trouble shoot outbreaks and improve quality control and prevention efforts. milk and colostrum are effective enrichment media for salmonella, so sampling these sources should be done "as fed" rather than as initially mixed or prepared. 9 the increased availability of colostrum pasteurizers has added a very helpful tool to control not only salmonella dublin, but also other serotypes that can also be found in colostrum. maternity area hygiene and management are extremely important salmonella in dairy cattle in the control of neonatal salmonellosis. decreasing the postpartum exposure to the dam reduces the chances of immediate infection. a rather alarming recent publication has identified that true vertical transmission in newborn calves is documented with several serovars common to cattle in the united states. 48 if further studies confirm this finding, it would add yet another serious challenge to the control of salmonellosis in calves. because exposure of calves to salmonella is very likely in the commercial dairy environment, management efforts should be directed toward limiting dose and maximizing health and disease resistance in the young replacement animal population. there are no revelations within this advice, but just as occurs with adult cattle, the degree to which farms are able to dedicate personnel and time may only be prioritized in the midst of, or immediately after, an outbreak of clinical disease. prompt diagnosis, separation, and treatment are important, but group housing of calves can quickly create a "perfect storm" for contagious disease spread. as with adults, vaccination and immunization with modified live or killed (autogenous or commercially available) products is often part of the control and prevention measures instituted. there is very little evidence to support effective control of salmonella infection in calves via passive transfer from immunized dams with any type of vaccine although the siderophore/porin product mentioned in the previous section in adults will stimulate colostral antibody. 47 salmonella is predominantly cleared by cellular immune responses and humoral antibody alone may not provide satisfactory protection. vaccine use in calves is best considered when management efforts at control and prevention have already been put in place, or if these have been implemented but found to make little difference in the pattern or severity of disease. autogenous products derived from a specific serovar isolated from clinical cases must be used very carefully owing to the risk of anaphylactic reactions, and only from reputable biologic manufacturers. similarly, caution is advised regarding modified live vaccine use in calves owing to the potential for adverse reactions. killed vaccines have performed inconsistently in the small number of trials carried out in the past in calves. 49, 50 comments regarding salmonella dublin control the increasing prevalence of salmonella dublin infection in the us dairy industry 14, 15 and its unique status as the host adapted serovar of s enterica subspecies enterica in cattle merit some more specific attention. for readers who wish more, and a greater in-depth discussion of this serovar, we refer you to the excellent primary sources and review paper authored by dr liza nielsen from denmark who, together with her international collaborators, has published a great deal of excellent work, particularly as it applies to disease impact as well as control and surveillance strategies. 17, 36, 38, 39, [51] [52] [53] within the european community, especially within the scandinavian countries, there are currently several active surveillance and certification programs that are designed to control, and potentially eradicate salmonella dublin infection in cattle herds. it is doubtful whether the immediate future holds much promise for such coordinated efforts within the us dairy industry, but there are undoubtedly useful lessons to be learned from experiences in other countries. all of the control measures described in this article for adults and calves can be applied to salmonella dublin infection, just as they can to other serovars. however, the serologic response to salmonella dublin, and the ability to measure that as a potential surrogate marker of the carrier status, opens up possibilities for identification and control. currently within the united states, the serologic test for salmonella dublin is available commercially through the animal health diagnostic center at cornell university and can be applied to either blood (serum) or bulk tank milk samples. it is important to recognize that a single time point positive test result does not confirm the carrier status, but indicates an antibody response owing to previous exposure, current infection, or passively derived antibody in a calf less than 3 months of age. 17 repeated individual animal sampling at specified intervals can be used during surveillance programs to identify animals that are likely to be carriers based on the persistence of an elisa positive result with a high optical density reading. 17, 36, 39 using the data generated by nielsen as a guide, the animal health diagnostic center at cornell university categorizes a carrier as any animal that has 3 strong positive serum elisa results over an 8-month period (dr belinda thompson, personal communication). from the currently available literature it does not seem to be possible to predict or estimate what percentage of infected calves or adults will go on to become true carriers, although the number is probably quite low. in herds classified as being endemic for salmonella dublin in denmark, the seroprevalence is highly variable but may only be at 15% of the whole herd, with a higher proportion of infection in young stock compared with adults. 17 reinfection of previously infected and seemingly recovered animals also seems to be possible when individuals are followed over long periods of time. some of these subsequent infections may also result in the development of carrier status (dr belinda thompson, personal communication). bulk tank samples can be used for periodic milking herd surveillance, or, if applied to selected milking groups, to identify whether salmonella dublin has been introduced into a herd or is present in a particular population of cattle within the herd. 17 from epidemiologic data, it seems that the risk of becoming a carrier after infection is greater for calves and for adults infected around the time of calving. 54 another study shows that salmonella dublin infection in endemic herds can be reduced when an individual employee was dedicated to colostrum administration to newborn calves and calving cows were moved into a specific maternity pen before calving. 51 a number of epidemiologic investigations in endemic salmonella dublin herds in scandinavia have identified risk factors and important control points for eradication of infection. 51, [54] [55] [56] [57] many of the risk factors and management tools demonstrated to improve control of salmonella dublin infection are intuitively sensible and relevant to other salmonella serovars. improving the likelihood of control is associated with avoiding cattle purchases from other farms and ensuring good calving area management and individual calf-rearing practices with solid, not permeable, barriers between calves. 51 aggressive culling programs are not practical in situations where prevalence is high and may only become reasonable once new calf infections are serologically proven to decline to very low, or absent, levels. 17, 56 it may be difficult for some producers and heifer rearers to instigate all of the management changes and practices that have been successful in european countries, but readers are directed to information available through the animal health diagnostic center at cornell university website for very helpful guidelines concerning control of salmonella dublin. 58 in the united states, there is a commercial live salmonella dublin vaccine (entervene d, boehringer ingelheim vetmedica, st. joseph, mo) that is being used as a component of salmonella dublin control on many farms. the product is administered parenterally to newborn calves to stimulate an immune response before initial exposure to the pathogen. the goal is to prevent the serious health consequences of natural infection as well as the development of the carrier status in what is the most susceptible population of animals within endemic herds. however, when given according to label instructions the product will interfere with serologic testing, giving a false-positive result at up to 8 months of life. 9 furthermore, the product can be associated with fatal anaphylactic reactions in some recipient calves. these reactions seem to be more common in endemic herds than in naã¯ve ones. 9 this product can stimulate colostral antibody production when given to dry cows and was not associated with any adverse reactions when given to late pregnant animals. 59 the vaccinated cohort in this study were from a farm with no clinical history of salmonellosis in recent years. 59 whether this colostral antibody might provide protection against neonatal infection is currently unknown. herd biosecurity? maintain a closed herd. if purchasing cattle, ensure a negative serologic test from individual animals or a negative bulk tank milk test from the herd of origin within the last 6 months. maintain separate maternity and sick cow pens. have separate equipment for feed and manure handling. dedicate personnel to solely work with high-risk or sick cattle versus neonates. salmonellosis not only can cause severe disease in cattle, but also poses a significant zoonotic risk. farm workers, calf handlers, and their families are clearly at risk of becoming infected by salmonella spp. during outbreaks of clinical illness, but the risk of exposure goes far beyond farm workers or veterinarians with direct animal contact during outbreaks of disease. asymptomatic shedding of salmonella, a characteristic of salmonella dublin infection, but also an issue with many other common bovine serovars such as newport and typhimurium, creates risk for people in direct contact with the animal, its feces, or milk. 9,60,61 however, the majority of human salmonellosis cases do not derive from direct animal contact, but are instead acquired through foodborne exposure. 62 so-called nontyphoidal salmonellosis is one of the leading causes of acute bacterial gastroenteritis in humans in the united states, responsible for an estimated 1.4 million cases of illness annually. 63 the predominant risk for zoonotic salmonellosis from cattle lies in exposure to contaminated meat from beef, which would include dairy beef and cull dairy cows, typically via fecal contamination of the carcass at the time of slaughter. [63] [64] [65] although salmonella mastitis is extremely uncommon, shedding of the organism in milk is not, and its presence has been documented in bulk tank milk in several studies. 66-69 a positive bulk tank or milk filter sample may represent fecal contamination, true lactational shedding, or a combination of both. conventional pasteurization should kill the organism, provided effective temperature and duration are reached. it is important to consider the diagnostic procedure performed to identify the salmonella in bulk tank or milk filter samples when interpreting these studies. studies using pcr 66, 67, 69 rather than culture will detect a greater prevalence of salmonella-contaminated samples because of genomic material from both live and dead organisms in the sample. side-by-side comparisons of conventional culture and pcr using the same samples have been performed and show that approximately one-quarter (2.6% vs 11.2%) of those bulk tank samples that are pcr positive for s enterica will be positive by culture. true "dairy" products actually account for only a small percentage of human salmonellosis in the united states, and many of these outbreaks are due to the consumption of raw milk and raw milk products. 67, 70 bacterial antimicrobial resistance represents an important current and future problem in infectious disease public health. concerns regarding zoonotic salmonella infections have been amplified in recent years by the emergence of multiple drug-resistant strains of several s enterica serovars associated with cattle. [71] [72] [73] [74] it is generally accepted that antimicrobial-resistant bacteria are produced, maintained, and disseminated as a result of selection pressure introduced by the use of antimicrobial drugs. 75 suspected principal foci of selection pressure include use of antimicrobials for the treatment of humans and in food-producing animals for treatment or prevention of disease and growth promotion. 75, 76 modern molecular methods combined with other conventional techniques such as pulse field gel electrophoresis can be used to investigate the origins of foodborne human enteric disease and the role of antimicrobial use in cattle with the occurrence of multiple drug-resistant salmonella infection in humans. at this point in time, there are few published studies establishing such links from "farm to fork." 73 a recent extensive systematic literature review of 858 publications on the effect of antimicrobial use in agricultural animals on drug-resistant foodborne salmonellosis in humans from 2010 to 2014 concluded that, although antibiotic use in cattle increased the likelihood of colonization in the host, there were no studies that traced antimicrobial-resistant salmonella in humans back to the farm. 76 the antimicrobials of choice for treating bacterial gastroenteritis in humans are generally the fluoroquinolone, ciprofloxacin, for adults and the cephalosporin, ceftriaxone, for children. 63, 77 at issue today is whether the veterinary analogs of these drugs may be responsible for the emergence of antimicrobial resistance in foodborne pathogens like salmonella. the mechanism by which s enterica typically acquires antimicrobial resistance to fluoroquinolones differs quite markedly and, importantly, from that by which resistance to cephalosporins develops. specifically, fluoroquinolone resistance is usually acquired through clonal dissemination of salmonella isolates with mutations in chromosomally encoded resistance genes. cephalosporin resistance usually is obtained via independent acquisition of mobile genetic elements via plasmids and transposons. 78 further work is needed in this area to determine whether there is a connection between veterinary use of ceftiofur and the emergence of ceftriaxone resistance in salmonella spp. 63 although ceftriaxone-resistant salmonella typhimurium has been documented in cattle, 73 other larger studies have demonstrated little to no resistance to this particular third-generation cephalosporin in cattle sourced serovars despite more common resistance to other cephalosporins. 72, 79 although it is now 8 years old, interested readers are directed to the excellent review of antimicrobial resistant salmonella in dairy cattle by alexander and colleagues. 79 in a more recent publication, a significant decrease was observed in antimicrobial resistance among dairy cattle salmonella isolates in the northeastern united states. 71 many practitioners and diagnostic laboratories will be very familiar with the wide variety of antimicrobial sensitivity patterns demonstrated by different s enterica serovars obtained from individual animal and environmental samples. certain serovars seem to be more commonly associated with greater in vitro resistance than others. the paper by cummings and colleagues 71 demonstrated a decrease in resistance trends between 2004 and 2011. it was postulated that this might have been related to an increase in the prevalence of the serovar cerro in fecal samples from their study population. the biggest concern arises with serovars that have historically been more common in dairy cattle and that are associated with human disease outbreaks, such as newport and typhimurium. in particular, several human foodborne outbreaks caused by salmonella typhimurium dt104 of dairy or beef origin that are characteristically resistant to the antibiotics ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline have been reported. 63, 80 an interesting and rigorously investigated example of zoonotic multiple drug resistant salmonella from cattle is provided by the wisconsin experience with salmonella heidelberg over the last 2 years. since 2015, the wisconsin state veterinary diagnostic laboratory (wvdl), in conjunction with human and veterinary health organizations throughout wisconsin, have been tracking a multidrug-resistant strain of salmonella heidelberg, a group b serovar (dr keith poulsen, wvdl personal communication). as of november 2016, there were 12 confirmed human infections from 7 different wisconsin counties. upon questioning, more than 90% of the infected individuals reported purchasing holstein bull calves from livestock dealers or sale barns. during 2015 and 2016, the wvdl also isolated several multidrug-resistant salmonella heidelberg isolates from calves located mostly in wisconsin. pulse-field gel electrophoresis and whole genome sequencing of isolates indicated that the human and bovine isolates were very closely related. this strain of salmonella heidelberg is highly pathogenic and multidrug resistant. only 1 antimicrobial drug is an effective treatment option for human cases and no effective, legal (united states) options exist for cattle (dr keith poulsen, wvdl, personal communication). as the application of modern molecular techniques becomes more commonplace, it is probable that diagnostic and surveillance efforts will place food animal species and production methods under greater scrutiny with respect to zoonotic enteric diseases. increased awareness, rigor, and possibly limitations regarding antimicrobial use in food animals should not be surprising outcomes. large animal internal medicine escherichia, shigella, and salmonella methodologies for salmonella enterica subsp. enterica subtyping, gold standards and new methodologies the validation and implications of using whole genome sequencing as a replacement for traditional serotyping for a national salmonella reference laboratory salmonella serotype 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resistance patterns of bovine salmonella enterica isolates submitted to the wisconsin veterinary diagnostic laboratory: 2006-2015 histopathology case definition of naturally acquired salmonella enterica serovar dublin infection in young holstein cattle in the northeastern united states review of pathogenesis and diagnostic methods of immediate relevance for epidemiology and control of salmonella dublin in cattle transmission of salmonellae among calves penned individually aerosol infection of calves and mice with salmonella typhimurium salmonella in calves cross protective immunity conferred by a dna adenine methylase deficient salmonella eneterica serovar typhimurium vaccine in calves challenged with salmonella serovar newport contribution of salmonella typhimurium virulence factors to diarrheal disease in calves salmonella infections in cattle salmonellosis in calves -the effect of dose rate and other factors on the transmission salmonella typhimurium diarrhea reveals basic principles of enteropathogen infection and disease-promoted dna exchange the roles of inflammation, nutrient availability and the commensal microbiota in enteric pathogen infection an nk cell perforin response elicited via il-18 controls mucosal inflammation kinetics during salmonella gut infection methods for the cultural isolation of salmonella comparison of individual, pooled, and composite fecal sampling methods for detection of salmonella on u.s. dairy operations development and evaluation of a real time multiplex polymerase chain reaction procedure to clinically type prevalent salmonella enterica serovars validation of a 20h real time pcr method for screening of salmonella in poultry fecal samples real time pcr method for detection of salmonella spp. in environmental samples recombinant plasmid based quantitative real time pcr analysis of salmonella enterica serotypes and its application to milk samples isolation of salmonella spp. from the environment of dairies without any clinical history of salmonellosis cattle and environmental sample level factors associated with the presence of salmonella in a multi-state study of conventional and organic dairy farms age stratified validation of an indirect salmonella dublin serum elisa for individual diagnosis in cattle humoral antibody responses to experimental and spontaneous salmonella infections in cattle measured by elisa factors associated with variation in bulk tank milk salmonella dublin elisa odc% in dairy herds simulation model estimates of test accuracy and predictive values for the danish salmonella surveillance program in dairy herds disease management of dairy calves and heifers effect of previous antimicrobial treatment on fecal shedding of salmonella enterica subsp. enterica serogroup b in new york dairy herds with recent salmonellosis pathogen reduction in minimally managed composting of bovine manure survival characteristics of salmonella enterica serovar newport in the dairy farm environment farm level associations with the shedding of salmonella and antimicrobial resistant salmonella in us dairy cattle salmonella transmission through splash exposure during a bovine necropsy comparison of disinfectant efficacy when using high-volume directed mist application of accelerated hydrogen peroxide and peroxymonosulfate disinfectants in a large animal hospital passive immunity stimulated by vaccination of dry cows with a salmonella bacterial extract evidence supporting vertical transmission of salmonella in dairy cattle salmonella typhimurium infection in calves: protection and survival of virulent challenge bacteria after immunization with live or inactivated vaccines immunization of calves against salmonellosis effect of prevention of salmonella dublin exposure of calves during a one-year control programme in 84 danish dairy herds dynamic changes in antibody levels as an early warning of salmonella dublin in bovine dairy herds gross margin losses due to salmonella dublin infection in danish dairy cattle herds estimated by simulation modelling salmonella dublin infection in dairy cattle: risk factors for becoming a carrier a questionnaire study of associations between potential risk factors and salmonella status in swedish dairy herds culling decisions of dairy farmers during a 3-year salmonella control study salmonella dublin infection in young dairy calves: transmission parameters estimated from field data and an sir model nyschap recommendations for the control of salmonella dublin in dairy calf and heifer raising operations characterization of the serologic response induced by vaccination of late gestation cows with a salmonella dublin vaccine the effect of clinical outbreaks of salmonellosis on the prevalence of fecal shedding among dairy cattle in new york human multi-drug resistant salmonella newport infections food related illness and death in the united states animal sources of salmonellosis in humans outbreak of multi-drug-resistant salmonella enterica serotype typhimurium definitive phage type 104 infection linked to commercial ground beef, northeastern united states highly resistant salmonella newport-mdrampc transmitted through the domestic us food supply: a foodnet case -control study of sporadic salmonella newport infections antimicrobial resistance of salmonella enterica isolates from bulk tank milk and milk filters in the united states prevalence of salmonella enterica, listeria monocytogenes and escherichia coli virulence factors in bulk tank milk and in line filters from us dairies prevalence of salmonella enterica in bulk tank milk from us dairies as determined by polymerase chain reaction factors associated with salmonella presence in environmental samples and bulk tank milk from us dairies a survey of foodborne pathogens in bulk tank milk and raw milk consumption among farm families in pennsylvania antimicrobial resistance trends among salmonella isolates obtained from dairy cattle in the northeastern united states prevalence of antimicrobial resistance among salmonella on midwest and northeast dairy farms ceftriaxone-resistant salmonella infection acquired by a child from cattle antimicrobial resistance and serotype prevalence of salmonella isolated from dairy cattle in the southwestern united states antimicrobial resistance of commensal escherichia coli from dairy cattle associated with recent multiresistant salmonellosis outbreaks effects of antimicrobial use in agricultural animals on drug resistant foodborne salmonellosis in humans. a systematic literature review nontyphoidal salmonellosis ceftiofur resistant salmonella strains isolated from dairy farms represent multiple widely distributed subtypes that evolved by independent horizontal gene transfer antimicrobial resistant salmonella in dairy cattle in the united states analysis of antimicrobial resistance genes detected in multi-drug resistant salmonella enterica serovar typhimurium isolated from food animals key: cord-354068-4qlk6y7h authors: friedrich, brian m.; trefry, john c.; biggins, julia e.; hensley, lisa e.; honko, anna n.; smith, darci r.; olinger, gene g. title: potential vaccines and post-exposure treatments for filovirus infections date: 2012-09-21 journal: viruses doi: 10.3390/v4091619 sha: doc_id: 354068 cord_uid: 4qlk6y7h viruses of the family filoviridae represent significant health risks as emerging infectious diseases as well as potentially engineered biothreats. while many research efforts have been published offering possibilities toward the mitigation of filoviral infection, there remain no sanctioned therapeutic or vaccine strategies. current progress in the development of filovirus therapeutics and vaccines is outlined herein with respect to their current level of testing, evaluation, and proximity toward human implementation, specifically with regard to human clinical trials, nonhuman primate studies, small animal studies, and in vitro development. contemporary methods of supportive care and previous treatment approaches for human patients are also discussed. the family filoviridae includes two accepted genera, ebolavirus and marburgvirus. the genus ebolavirus includes five species (each represented by a single virus): zaire ebolavirus (ebola virus, ebov), sudan ebolavirus (sudan virus, sudv), reston ebolavirus (reston virus, restv), taï forest ebolavirus (tai forest virus, tafv), and bundibugyo ebolavirus (bundibugyo virus, bdbv). the genus marburgvirus includes a single species, marburg marburgvirus, which has two members: marburg virus (marv) and ravn virus (ravv) [1, 2] . in 1967, the first cases of filoviral infection were documented in three simultaneous outbreaks in west germany and yugoslavia. the virus responsible for the outbreaks was named "marburg virus" after the german city of marburg in which it was first recognized [3, 4] . from documented instances of infection, it seems that the members of the filovirus genera may exist in quite opposite climates of africa with marburgvirus infections occurring more frequently in the dry woodlands, while ebolavirus infections occur more frequently in rain forests [5] . more than 40 years of effort have focused on the search for the reservoir of these viruses in africa, and while the search is still ongoing, recent evidence indicates that bats may be reservoirs for both marburgviruses and ebolaviruses [1, [6] [7] [8] [9] [10] [11] . however, the recent outbreak of restv in domestic pigs in the philippines demonstrated the potential for animals other than primates and bats to be infected and potentially spread or amplify outbreaks [12] . filoviruses are named for their long, filamentous shape which can been seen on the order of micrometers in length, while their width is more narrow (usually around 80 nm) with little fluctuation [13] . contained within this filamentous virus is a single, 19-kb negative-sense rna genome that encodes seven proteins [14, 15] . the seven filoviral proteins are the glycoprotein (gp), the polymerase (l), the nucleoprotein (np), a secondary matrix protein (vp24), the transcriptional activator (vp30), the polymerase cofactor (vp35), and the matrix protein (vp40) [16, 17] . homotrimers of the viral gp cover the surface of the virion, and this viral gp is believed to be the sole host attachment factor for filoviruses [6, 18] . candidates for filoviral receptor and co-factors include transferrin, dc-sign, tim-1, and npc1 [16, [19] [20] [21] [22] . after entry, filoviruses replicate their genomes and viral proteins in the cytoplasm using a rna-dependent rna-polymerase which is carried in with the virus. wild-type filovirus infection has been associated with severe case fatality rates in humans, as high as 90% [15] . in humans, filovirus infection is characterized by an abrupt onset of flu-like illness, after an initial incubation period of 2-21 days. following this initial illness, signs and symptoms of disease include anorexia, nausea, vomiting, chest pain, cough, edema, postural hypotension, neurologic complications, petechiae, and mucosal hemorrhage. there have also been several observed wild-type filovirus outbreaks among great apes in africa that have demonstrated similarly high mortality rates [23] . in an effort to create cost and time-effective models of filoviral disease for the development of vaccines and therapeutics, small animals, such as mice and guinea pigs, are often used. however, these animals usually demonstrate significant resistance to wild-type filovirus infection, and only demonstrate mortality rates similar to primates when the filovirus in question has been adapted to the model species [24] . due to the difficulties in evaluating wild-type filovirus infection in small animals and the generally high level of immune protection correlates derived from non-human primate (nhp) models of infection, therapeutics and vaccines are ultimately evaluated in nhp species for efficacy against filovirus. of the nhp models available for filovirus study, rhesus and cynomolgus macaques have been the most highly characterized and utilized for therapeutic and vaccine development, respectively. however, the starting point of vaccine and therapeutic development remains small animal models due to the cost, ethical, and time-associated benefits [17] . this review will highlight the current research into filovirus vaccines and therapeutics. the current clinical standard for filoviral infection is supportive care as there are currently no fda-approved treatment strategies. supportive care consists of oral fluid rehydration, oral medication, nutritional supplementation, and psychosocial support [25] . nasogastric feeding tubes and i.v. administration of both fluids and medication are increasingly considered supportive care where possible during outbreak scenarios to prevent dehydration and facilitate support of blood pressure [25, 26] . however, given the limited equipment and laboratory support during outbreaks, care must be taken to prevent overaggressive fluid administration [27] . fluid replacement was evaluated briefly in rhesus macaques, and while there was no significant benefit to survival, a less severe renal compromise was observed [28] . while supportive care may (or may not) reduce the overall case fatality rate in humans, the true impact of simple interventions such as fluid management has yet to be fully evaluated and the potential for benefit in combination with direct antiviral measures has yet to be assessed [29] . treatment of filovirus infection with passive transfer of antibodies is an attractive therapy. while there have been conflicting results in vitro, in animals, and in humans, recent breakthroughs have solidified the potential for this strategy of intervention. in addition, there have been a number of immunotherapies developed for other agents, such as respiratory syncytial virus (rsv), which can provide potential roadmaps or precedence to facilitate the advancement of these products through the necessary regulatory hurdles [30, 31] . transfer of immune serum for the treatment of filovirus infection in humans has previously been attempted. however, interpretation of these results has been cautious due to the study conditions as well as the uncertainty of the disease stage at which the individuals were treated [6] . as a result, much attention has focused on animal studies evaluating candidate products. while many of the early studies in mice and guinea pigs were successful, these successes did not translate to nhp studies and tempered the enthusiasm for further evaluation of candidate products. [6, [32] [33] [34] [35] . when similar passive transfer strategies were attempted in nhps, viremia onset and outward signs of disease were reduced, but the treatment did not affect survivorship [36] [37] [38] . in addition, the suggestion that antibodies could enhance filovirus infections in vitro caused further concern [39] [40] [41] . however, recently a series of experiments have made researchers, developers, and funding agencies reconsider the potential of this category of products for filoviruses. both polyclonal and monoclonal passive therapies have been shown to be efficacious in rodents for filovirus infection [42] [43] [44] . furthermore, evidence of enhancing antibodies exists in the antibody response to ebov [38] . more recent studies have demonstrated protection in macaques with polyclonal and monoclonal passive therapy [45] [46] [47] . these sources of monoclonal antibodies have ranged from murine monoclonal antibodies to recombinant-derived cloned human monoclonal antibodies from survivors of filovirus infection [37, 43] . development of new antibodies to be used for post-exposure treatment is on-going. in one study, an antibody (13f6) targeting the ebov gp mucin-like domain was generated and subsequently shown to protect 100% of mice against a lethal ebov challenge when given 2 days post-exposure [44] . this antibody was then modified to generate h-13f6, a human recombinant antibody. this human recombinant antibody also significantly protected mice against a lethal challenge of ebov [48] . in another method, a recombinant vsvδg/ebovgp was used to generate a total of 8 monoclonal antibodies which were subsequently characterized. all 8 monoclonal antibodies improved survival rate of mice (33%-100%) against a high-dose lethal challenge by mouse-adapted ebov [49] . another antibody, kz52, was isolated from the bone marrow of a human survivor of ebov infection and is specific for the complex of gp1and gp2 [50] . kz52 neutralized ebov in vitro and offered protection from lethal ebov challenge in a rodent model [43] , but was non-protective in nhps [37] . table 1 3.1.1. dna vaccines the first clinical trials involving filovirus vaccines were based off of plasmids expressing ebov np and gp as well as sudv gp [51] . this strategy proved safe in 27 subjects involved with phase i testing. however, the prime/boost dna vaccine strategy covering four separate plasmid doses administered three times each was ineffective at creating durable immunity as evidenced by the near non-existent antibody titer in these subjects after 1 year [51] . while clinical trials with this vaccine have halted, it may be possible that this strategy can supplement another vaccine technology in a prime/boost capacity. while no vaccines or therapeutics are currently licensed for use by the fda, phase i clinical trial safety tests have been performed on one particular platform for an ebov vaccine. this vaccine is based on a replication deficient, recombinant adenovirus serotype 5 (rad5) vector genetically engineered to carry the genes for ebov glycoprotein (gp (z)) and sudv glycoprotein (gp (s/g)). as a common human pathogen, this vector vaccine utilized the broad-tropism of the adenovirus vector to infect cells and once inside the inserted ebolavirus glycoproteins are expressed. upon expression of these inserted ebolavirus genes, the host immune system will recognize them as foreign and mount a response against them. the advancement of this vaccine technology to phase i trials manifested from its ability to provide 100% protection among cynomolgus macaques vaccinated 28-35 days prior to challenge and its ability to generate potent humoral and cell-mediated immune responses [52, 53] . in the clinical trial participants remained asymptomatic after a single vaccination with either 2 × 10 9 or 2 × 10 10 viral particles [54] . furthermore, for both doses of the vaccine significant antibody titers were observed at 4 weeks post-vaccination with 100% and 55% of participants receiving 2 × 10 10 viral particles being positive for gp (s/g) and gp (z), respectively. significant antibody titers were observed again at 48 weeks post-vaccination and, while decreased from 4 weeks, demonstrated the potential durability of this vaccine over time [54] . t-cell activation was also examined for these individuals and found to directly correlate with the dose administered, but to a lesser extent than the previously mentioned antibody response. cd4 + activation was observed with greater frequency than cd8 + activation in those receiving the vaccine. importantly, these results were obtained in the context of significant pre-existing immunity to ad5 as pre-entry evaluation of antibody titers revealed that 50% of participants were positive against ad5, showing that pre-existing immunity to ad5 still resulted in protection against ebov [54] . while this study shows great promise, further development and additional studies in nhps and humans are needed. table 1 3. in addition to the adenovirus platform in clinical trials, additional variations of the rad5 vaccine are also in development and have been evaluated in nhp models. based on the adenovirus vector platform, the complex adenovirus (cadvax) technology substantially increased the genetic payload capacity of the vector, up to 7 kb. additionally, this strategy involved the blending of four separate vectors expressing the glycoproteins of ebov, sudv, and marv along with the nucleoproteins of ebov and marv. when administered in a prime/boost strategy, this technology offered 100% protection against ebov, sudv, and marv [55] . another variation of the cadvax system designed to express modified ebov glycoprotein and sudv glycoprotein was effective in protecting against both parenteral and aerosol challenge when administered in a prime/boost strategy [56] . both implementations of the cadvax technology demonstrated significant antibody titers. further improvement upon the adenovirus-based ebov vaccine technology is ongoing. richardson et al. reformatted the genetic insert for the vector which included the addition of a cytomegalovirus (cmv)-chicken β-actin hybrid promoter, optimized codons and a consensus kozak sequence [57] . these improvements led to three-to seven-fold increases in ebov glycoprotein expression. neutralizing antibody titers were found at doses as low as 10 4 infectious forming units (ifu) with comparable titers requiring 10 7 ifu of the unmodified vaccine in mice. these modifications demonstrated 100% protection of mice at doses two orders of magnitude lower than the unmodified vaccine. interestingly, at 30 min post-challenge, the modified ad-cmvzgp/ad-cagoptzgp offered 100% protection compared to the 22% protection of mice offered from the original vaccine [57] . this vector format has also recently showed promise when administered sublingually in mice, therefore eliminating the complexities of parenteral administration such as the necessity for sterile tools, aseptic chemicals, and the risks of potential blood-borne pathogen exposure [58] . while this adenovirus vector vaccine technology is promising, demonstrations that pre-existing immunity to the ad5 vector depressed the desired immune response may impede its implementation. in efforts to circumvent issues of pre-existing immunity to ad5, geisbert et al. sought out a less prevalent serovariation [59] . in their study, a heterologous prime/boost strategy with recombinant adenovirus serotypes 26 and 35 carrying gp (z) and gp (s/g) demonstrated complete protection among nhps. each of these vectors was capable of stimulating humoral and cell-mediated immune responses in the context of nhps pre-vaccinated with rad5 as evidenced by antibody titers reaching an order of magnitude above those achieved in rad5 vaccinated subjects (1:32,000 compared to 1:6,800), and cd8 + intracellular cytokine staining was 4.7-fold greater among heterologous prime/boosted subjects (0.41% compared to 0.09%) [59] . rhabdoviruses have recently offered unique vaccine platforms to generate both genus/species specific immunity as well as potential for cross-protective immunity for filoviruses. for example, based on an attenuated recombinant vesicular stomatitis virus (rvsv), the replication-competent virus expresses the glycoprotein of the target filovirus in place of its wild-type membrane glycoprotein. as this virus is primarily an agricultural pathogen, pre-existing immunity interfering with the desired immune response and subsequent protection is unlikely [60] . several studies have begun to address the safety of the filovirus vsv platforms. evaluation of this platform in immunocompromised nhps has suggested that this technology may be safe among similarly immunocompromised humans [61] . further encouragement for the safety of this live-attenuated vaccine came recently from mire et al. who showed that ebov and marv rvsv showed no signs of neurovirulence associated with vsv [62] . the utility of the vsv-based vaccine for protection against filoviral hemorrhagic fever was highlighted by geisbert et al. [63] . using a blended vaccine consisting of equal amounts of three different vsv vectors each carrying the ebov, sudv or marv glycoprotein, they were able to generate 100% protection of nhps against challenges with ebov, sudv, tafv, and marv with no observed ill effects from this replication-competent vaccine. of all vaccinated nhps, only one showed signs of viremia as assayed by rt-pcr. each of the vaccinated nhps also demonstrated elevated antibody responses after vaccination, with titers ranging from 1:32 to 1:100 for all three glycoprotein components of the blended vaccine [63] . in addition to providing such high levels of protection as a prophylactic vaccine strategy, the vsv-based technology has demonstrated post-exposure protection for both ebov and marv when administered via intramuscular (i.m.) injection [64] . when marv-rvsv was administered i.m. 20-30 min post-challenge with marv, 100% of nhps survived. in this study viral rna was observed in the blood on day three post-challenge when assayed by rt-pcr, but active virus was unobservable by traditional plaque assay. clinical chemistry results demonstrated that these surviving nhps experienced significant rises in aspartate aminotransferase, gamma-glutamyl transferase, total bilirubin, and blood urea nitrogen indicating that, while protective, the post-exposure treatment did not completely prevent typical pathogenic events associated with marv infection. similar experiments demonstrated sudv-rvsv delivered 20-30 min after challenge offered 100% protection [65] . post-exposure protection for ebov-rvsv was less effective at 20-30 min but still afforded 50% protection to eight nhps [66] . as a post-exposure treatment, ebov glycoprotein rvsv was used recently 48 h after a suspected human exposure via needlestick in the laboratory. while there is no direct evidence the laboratory worker was indeed exposed, that person survived the experience with no discernible sequelae from the treatment outside of a transient fever occurring 12 h after an injection of 5 × 10 7 plaque forming units (pfu) [67] . also of note for rhabdovirus-based filoviral vaccines was a recent report that generated dual immunity for both ebov and rabies virus infection. ebov gp was efficiently expressed from an attenuated vaccine used for wildlife against rabies virus in place of the wild-type rabies envelope glycoprotein, g [68] . this vaccine vector was capable of inducing protective immunity to ebov infection as well as to rabies virus infection in both live-attenuated format and β-propriolactone inactivated vaccine. neurovirulence of the recombinant vector was unobserved in suckling mice when compared to the unaltered vaccine [68] . this versatility offers increased storage options with an inactivated vaccine as well as the opportunity to vaccinate for each disease where they are both endemic. alphavirus particle-based vaccines also provide high levels of protection to nhps against lethal filovirus challenge. these vaccines for ebov, sudv, and marv are composed of venezuelan equine encephalitis virus (veev)-based replicon particles (vrps) that express the viral glycoprotein of interest [69] . replicon particles are replication-defective, single-cycle vectors which cannot spread from cell-to-cell. the vrps are composed of an attenuated veev replicon that contains veev non-structural genes and the filovirus glycoprotein. vrps are generated when the replicon is co-transfected into cells with helper plasmids containing the veev structural genes. the resulting single-cycle propagation defective particles are then administered to the appropriate animal model for efficacy testing [69, 70] . this technology offers several advantages, including high expression levels of heterologous genes, dendritic cell tropism, induction of robust cellular and antibody immune responses, rapid construction into single and multivalent vaccines, and relative resistance to anti-vector immunity [69, 70] . in vivo studies with the marv vrp offered the first demonstration of a fully protective filovirus vaccine. nhps exhibited 100% survival after vaccination with 10 7 focus forming units (ffu) of vrp in three consecutive doses spaced at 28 day intervals prior to challenge with marv [71] . this protection was offered when the vrps were constructed to express gp alone or gp + np; however, the nhps vaccinated with np alone all exhibited clinical symptoms of illness and only two out of three survived the challenge. substantial antibody titers were found in each of the vaccinated nhps. additionally, no conspicuous elevations in clinical chemistries were observed in nhps throughout the experiment. experiments performed on mice and guinea pigs supported the ability of vrps expressing gp to mediate complete protection from lethal marv and ebov challenge [71] . in mice, adoptive transfer of cd8 + cells, but not cd4 + cells or passive antibody transfer, from vrp-np-immunized mice was protective, suggesting this vaccine may be most protective by stimulating the host cell-mediated immune response [72] . additionally, adoptive transfer of cd8 + t-cells after activation via specific ebov peptides provided mice complete protection indicating a mechanism for vrp-based immunity [73] . recent studies indicate that a vrp-based vaccine is fully protective in cynomolgus macaques against ebov, sudv, and marv parenteral and aerosol virus challenges (unpublished observations, olinger). paramyxovirus-based vectors for vaccination against filoviral threats have recently demonstrated the capacity to protect nhps from infection and stimulate strong immune responses. paramyxoviruses have a natural tropism for the respiratory tract and, as filoviruses are both emerging diseases and potential weaponized threats, the idea of targeting vaccines to this area is ideal. two candidates for this category of vaccines have been investigated to date: human parainfluenza virus 3 (hpiv-3) and newcastle disease virus (ndv). of these two systems, the hpiv-3 system has been evaluated in nhp models of ebov infection. combinations of ebov gp alone, ebov gp + np, and ebov gp + human granulocyte macrophage colony stimulating factor (gm-csf) were inserted into the genome of hpiv-3. each of these vaccine vectors was used to vaccinate nhps both intranasally (i.n.) and intratracheally (i.t.) as initial studies offered complete protection of guinea pigs vaccinated via the respiratory route [74] . at least one nhp in all three vaccine groups receiving 4 × 10 6 tcid 50 (median tissue culture infective dose) of their respective vector displayed clinical signs of illness after challenge during the study. each group held two nhps and out of the three groups only one vaccinated animal from the ebov gp + np succumbed to the disease. immune responses from these subjects, prior to challenge, revealed antibody titers ranging from 1:400 to 1:1,600 [75] . by manipulating dose and administration strategies, bukreyev et al. were able to achieve complete protection of nhps after two successive doses of 2 × 10 7 tcid 50 given at day zero and again at day 28 with challenge occurring on day 67 [75] . the two-dose strategy produced igg titers ranging between 1:1,600 and 1:25,600, much higher than the single dose. each of these experiments highlights the potential of the hpiv-3 platform for ebov vaccination but the known prevalence of pre-existing immunity to hpiv-3 in humans could hinder the generation of targeted immunity [76] . to address these concerns, bukreyev et al. compared the immunogenicity of ebov gp expressing hpiv-3 vector among naïve and pre-immune nhps [77] . in these experiments ebov-specific igg levels were substantially decreased among hpiv-3 pre-immune nhps; however, this hindrance was overcome when the nhps were vaccinated with two doses of recombinant vector which was previously shown to offer complete protection against ebov challenge [77] . in efforts to diversify the paramyxovirus-based vectors and avoid issues surrounding the pre-existing immunity found for hpiv-3, a new vector design based on ndv was established. ndv is an avian paramyxovirus that infects the respiratory tract. this virus has been shown to be highly attenuated in nhps due to natural host restriction processes [78] . additionally, this vector system has proven successful as a vaccine platform for severe acute respiratory syndrome-associated coronavirus (sars-cov) and influenza h5n1 in nhps [79] . although this system has yet to be evaluated in the context of nhp models of ebov infection and disease, it was recently shown to be immunogenic in nhps. single vaccination with ndv expressing ebov gp produced lower titers than the hpiv-3 platform, demonstrating this vector is less immunogenic; however, in a homologous prime/boost vaccination strategy ebov-specific mucosal iga levels reached those similar to the hpiv-3 homologous prime boost vaccination strategy [80] . igg specific for ebov did not reach levels comparable to the previous hpiv-3 platform. these reports support the potential use of paramyxoviruses as vaccine candidates, but further examination of immunostimulatory effects and pre-existing immunity will require investigation. the vlp technology works by generating non-replicating virus particles that do not contain any filoviral genetic material. immune responses generated in response to exposure to vlps are derived from the filoviral protein shell that is the vlp itself. vlps are constructed through the matrix protein vp40's ability to drive the budding process. by simple transfection and expression of vp40 into target cells, filamentous structures can be generated [81] . co-expression of additional filoviral proteins, such as gp and np, can dramatically enhance and stabilize the production of vlps from target cells [82] . when traditional target/production cells were swapped out for insect cells and filoviral protein expression was driven from a baculovirus vector, vlps were generated and found to have filamentous structures resembling wild-type virus. vlps have demonstrated immunogenicity and the ability to protect nhps from lethal challenge. the first such study involving nhps utilized the baculovirusproduced vlps containing ebov gp, np, and vp40 [83] . five cynomolgus macaques were vaccinated three times at 42-day intervals with 250 µg of vlp with ribi adjuvant. after the full vaccination schedule, 100% survived a lethal ebov challenge, and there was a three-to ten-fold increase in ebov specific antibodies which possessed an 80% plaque reduction at titers between 1:20 and 1:160 [83] . additionally, these antibodies were shown to have both compliment-mediated and antibody-dependent cell-mediated cytotoxic properties [83] . using this same vlp technology, swenson et al. were able to show a similar effect for marv. three i.m. injections of 1 mg of vlp in 0.1 ml of qs-21 adjuvant spaced 42 days apart offered 100% protection against marv and ravv; however, one animal challenged with ravv did exhibit slight morbidity [84] . all animals had no detectable viremia as determined by plaque assay at days 0, 3, 5, 7, 10, 14, and 21 post-exposure. additionally, two injections of the vlps correlated with a peak in homologous antibody titer while three injections correlated to peak heterologous antibody titer [84] . the ability of vlps to generate protective immunity against filoviral challenge has been demonstrated in guinea pigs as well [85] . table 2 3. immunogenic virus-like particles (vlps) of ebov and marv can be easily generated in mammalian systems. ebov-like particles (vlps) can be efficiently generated in mammalian cells after expression of vp40 alone, but other filovirus proteins can be co-expressed as well [85] [86] [87] . baculovirus-derived vlps have also been successfully generated in insect cells and stimulated both cellular and antibody immune responses against hepatitis e virus, human papilloma virus, rotavirus, and simian immunodeficiency virus [88] [89] [90] [91] . several groups have made and tested baculovirus-generated filovirus-vlps as vaccines in small animal models. warfield et al. generated ebola-vlps (evlp) and marburg-vlp (mvlp) containing vp40, np, and gp. this vaccine had up to 100% survival following a lethal infection in mice (vaccine dose-dependent) [85] . sun et al. produced an evlp containing vp40 and gp. this vaccine was also up to 100% effective following a lethal ebov infection in mice (vaccine dose-dependent) [92, 93] . many reports document the ability of filoviral gp to act as a potent immunogen, and as such, viral vectors are a popular method of vaccinating through the expression of gp by the host and subsequent immune recognition. however, a recent report sought to utilize gp itself as the vaccination agent. in order to efficiently produce the protein, an expression vector was constructed such that the fc portion of a human igg was fused to ebov-gp [94] [95] [96] . this gp-fc fusion protein induced both cell-mediated and humoral immune responses, and mice vaccinated with zebovgp-fc demonstrated 90% protection against a lethal ebov challenge. [97] . while further studies are required, these results show that vaccination with ebovgp-fc alone is effective against a lethal ebov infection, and thus fusion proteins could potentially be used as an effective vaccine against filoviruses [97] . the use of plants to produce vaccine antigens and antibodies has been demonstrated previously and is attractive for several reasons, including low manufacturing cost, efficient production, minimal risk of contamination with human pathogens or toxins, and the fact that plants have similar secretory pathways and endosomal systems as mammalian cells [98] [99] [100] [101] . recently, a bean yellow dwarf virus (beydv)-derived replicon system was developed and shown to efficiently produce antibodies against ebov in nicotiana benthamiana leaves [100] . poolcharoen et al. used this system to develop and fuse ebov-gp1 to the c-terminus of an igg heavy chain [102] . these ebov immune complexes (eic) were then used as a vaccine to immunize mice. serum antibody response tests showed that this eic was highly immunogenic in mice and produced antibody levels similar to mice protected from a lethal ebov challenge [102] . while antibody titers alone do not fully correlated with protection from lethal challenge, there is potential for further development of this vaccine. a replicating vaccine that could spread through the target population after initial inoculation would be an attractive, alternative approach to filovirus development. this approach could provide high coverage with minimal initial vaccinations. a cmv-based vaccine would allow for this type of spread [103] . cmv, a member of the family betaherpesvirinae, induces a high "effector" memory t-cell (t em ) response before establishing a low-level persistent infection [104] [105] [106] . this high immunogenicity makes cmv a good potential vaccine vector, and the cmv vector has been previously shown to be effective as a vaccine for siv in rhesus macaques [104, 107] . tsuda et al. constructed a mouse cmv vector expressing a cd8 + t cell epitope from the nucleoprotein (np) of ebov (mcmv/ebov-npctl). this vaccine induced high levels of ebov-specific cd8 + t cells, and subsequently, protected 100% of mice against a lethal ebov challenge. this shows a proof-ofconcept for cmv as a potential vaccine vector for ebov [103] . as discussed previously, paramyxovirus-based vectors have demonstrated the capacity to induce strong immune responses and protect animals from infection. as such, a chimeric hpiv3 was developed where all the hpiv3 surface markers/receptors were deleted and replaced with ebov-gp [108] . this chimeric virus can be amplified and recovered easily. additionally, this chimeric virus has 2-fold greater incorporation of ebov-gp into the virion due to the lack of competition with hpiv3 surface proteins [108] . testing in guinea pigs showed that hpiv3/δf-hn/ebogp is highly attenuated, as compared to both hpiv3 and hpiv3/ebogp, and immunogenic, as 67% developed neutralizing antibodies against ebov. finally, a 4 × 10 6 pfu i.n. inoculation of hpiv3/δf-hn/ebogp was able to protect 100% of guinea pigs against a lethal ebov challenge [108] . table 3 4.1.1. rnapc2 severe coagulation disorders are one of the most prominent features of filoviral infection. in the event of a breach in vascular integrity a strict balance of pro-and anti-coagulant host factors must be maintained to successfully clot the breach and to prevent too much or too little clot formation. in the instance of filovirus infection, sustained microvascular injury in effected organs results in host coagulation inhibitor depletion which results in disseminated intravascular coagulation (dic). in dic, tissue factor (tf), a clotting factor normally present on cells not exposed to blood, complexes with circulating factor vii leading to clot formation and fibrin deposition through the extrinsic pathway [109, 110] . as numerous studies have demonstrated a clear link with dic and resultant organ failure, geisbert et al, sought to eliminate potential tf pathogenesis during filovirus infection by using recombinant nematode anticoagulant protein c2 (rnapc2) [111] . they demonstrated that rnapc2, an inhibitor of the tf pathway, provided partial post-exposure protection to rhesus macaques during filovirus infection [111] . previous studies with rnapc2 have already gone through phase ii trials in orthopedic surgery [112] and coronary revascularization [113] . geisbert et al. showed a 33% survival rate, in addition to a 3.4-day increase in mean time-to-death, for ebov-infected rhesus macaques and a 17% survival rate, with a 1.7-day increase in mean time-to-death, in marv-infected rhesus macaques, when treated with rnapc2 post-exposure [111, 114] . in a normally 100% lethal model of filovirus infection, rnapc2 demonstrated a clear benefit to survival as an increase in the mean time-todeath was observed. rnapc2 has completed phase ii clinical trials with a good safety record. primates. comparison of current drug candidates at the highest level of development, either in human clinical trials or those that have shown promise in nhps. also listed are the afforded levels of protection in nhps, the type of drug used to induce immunity and the dosing paradigm used to achieve the listed results. phosphorodiamidate morpholino oligomers (pmos) exert their anti-translation effects through steric hindrance of the translation machinery. this steric hindrance is possible due to a morpholino group, similar to a ribose base in rna, and a methylene phosphorodiamidate linking moiety that physically bind to mrna and prevent the translation machinery from accessing the mrna [115] . once the antisense pmos bind to their target mrna, they are highly stable and highly soluble which would allow high levels of translation inhibition and predictably low levels of potential cytotoxicity [115] [116] [117] [118] . pmos have previously demonstrated effective antiviral activity against coronaviruses and flaviviruses [119, 120] . swenson et al. initially utilized pmos targeting ebov vp24 and ebov vp35 to highly protect mice and guinea pigs against a lethal challenge with ebov and marv [121, 122] . subsequently, avi-6002 (a combination of pmos against both ebov vp24 and vp35) and avi-6003 (a combination of pmos against both marv vp24 and np) were developed and tested in a nhp post-exposure scenario. these pmos, delivered 30-60 min post-exposure, protected >60% of rhesus macaques against lethal ebov infection and 100% of cynomolgus macaques against marv infection [123] . both the ease of controlled manufacture and their efficacy in nhp models to combat filovirus infection, pmos represent a viable therapeutic strategy [123] . currently, avi-6002 and avi-6003 are in phase i clinical trials. table 3 4.2.1. recombinant human activated protein c ebolavirus disease (evd) and severe sepsis (or septic shock) share many clinical features including fever, hypotension, increased production of tissue factor, elevated levels of nitric oxide, and elevated levels of d-dimers [124] [125] [126] . in addition, the most prominent and consistent finding in severe sepsis is severe protein c deficiency [127, 128] . it was shown that treatment of patients with severe sepsis with recombinant human activated protein c (rhapc) resulted in improved survival [129] . later experiments in nhps demonstrated that ebov infection results in rapid reduction of circulating protein c levels [111, 130] . therefore, it was tested if treatment with rhapc could protect against lethal ebov infection in rhesus macaques. fourteen rhesus macaques were infected with a lethal dose of ebov; eleven were then treated with iv rhapc 30-60 min after challenge, continuing for 7 days. all control animals died on day 8 post-exposure; however, 2 of the 11 rhapc-treated animals survived (~20% survival). additionally, the mean time-to-death for rhapc-treated animals was 12.6 days, which is significant compared to the 8.3 days observed in placebo and historical controls [125] . this product was pulled as a single post-exposure treatment, but given that this intervention is not directly targeting the virus, there may be additional merit in assessing this product in conjunction with a direct antiviral. rna interference (rnai) represents a powerful, naturally occurring biological strategy for inhibiting gene expression. rnai interferes with the translation of mrna to protein products by either sterically blocking mrna or by triggering rnase h-mediated cleavage of the dna/rna duplex, resulting in inhibition of gene expression [122] . for many years rnai as demonstrated clear efficacy in preventing viral replication in vitro against a number of viruses, including coxsackieviruses, hepatitis b virus (hbv), hepatitis c virus (hcv), herpesviruses, human immunodeficiency virus 1 (hiv), human papillomavirus, rsv, influenza a virus, lymphocytic choriomeningitis virus, polioviruses, and sars-cov [131] [132] [133] [134] . fowler et al. demonstrated that small-interfering rna (sirna) downregulation of various marv mrna transcripts was able to significantly decrease viral protein production and subsequent viral release in cell culture [135] . unfortunately, efficient delivery vehicles providing effective drug targeting and stability have hindered the application of rnai in the clinical setting. however, recent developments in the field of nanotechnology have made nanoparticles the solution to increasing pharmacokinetic profiles for rnai therapies [136] . additionally, tekmira, inc. developed proprietary lipid encapsulation as a means of improving the pharmacology of sirna targeting the ebola rna polymerase l protein, as demonstrated by geisbert et al. [137, 138] . to efficiently deliver the sirna to target cells, a mixture of lipids forming a bilayered liposome, or stable nucleic acid-lipid particles (snalp), was designed. the snalp ensures cell entry by preferential fusing with the endosomal membrane upon exposure to the decreasing ph of the endosome. the snalps were further modified by conjugation with polyethylene glycol (peg) ensuring stability and efficient delivery of the sirna payload by neutralizing surface charges and presenting a hydrophilic exterior. this encapsulation was initially demonstrated to significantly increase the stability, half-life, and effectiveness of sirna directed against hbv [139] . the snalp-encapsulation of sirna targeting the ebov l protein was initially shown to completely protect guinea pigs when administered shortly after a lethal ebov challenge [137] . this treatment was then assessed for efficacy in rhesus macaques. snalp-encapsulated sirnas targeting ebov l polymerase, vp24, and vp35 were given to rhesus monkeys either four or seven times following a lethal challenge of ebov. two of the three monkeys given four doses survived lethal infection, while all four monkeys given seven doses survived infection [138] . the enhanced survivorship among the snalp-treated group highlights the efficacy of this potential therapeutic. additionally, there was little to no evidence of side effects associated with the treatment group, aside from mildly altered liver enzyme levels (which could have been an artifact separate from the challenge course) [138] . table 4 4. innate immunity is often the first line of defense against invading pathogens. one mechanism by which innate immunity functions relies on the identification of pathogen-associated molecular patterns (pamps). pamps consist of unique carbohydrate moieties on the external surfaces of foreign microbes, such as hexose and mannose, which are not expressed on the surfaces of the host cells. these pamps are then recognized by host proteins such as mannose-binding lectins (mbl), which recognize these high hexose and mannose contents [140] . filoviruses have large amounts of mannose comprising their glycoproteins and thus are a target of mbl [141] . upon exposure, host mbl targets filoviruses for compliment-dependent virus neutralization through the lectin pathway of the compliment cascade [142] . when administered in supraphysiological doses before or after lethal challenge with ebov, recombinant mbl treatment protected 40% of mice [140] . these studies showed that mbl may be a potential post-exposure prophylactic. table 4 . post-exposure treatments effective in small animal models. comparison of current treatment candidates at the small animal model level of development, specifically mouse models. also listed are the afforded levels of protection, the type of drug used to induce immunity and the dosing paradigm used to achieve the listed results. high-throughput screening (hts) is a significant tool for novel drug discovery. hts involves screening libraries consisting of thousands to hundreds of thousands of unique molecules against specific targets. available libraries used in hts have included natural compounds [143, 144] , peptides [145] , drugs [146] , and synthetic compounds [144, 147] . recently, compound fgi-103 was identified during a screen with an ebov-gfp pseudotyped virus and has shown strong antiviral activity in vitro against high doses of ebov and marv. fgi-103 was subsequently shown to protect mice against lethal challenges of both ebov and marv [148] . additionally, compound fgi-106 was initially identified in a similar manner and was shown to exhibit strong antiviral activity in vitro against ebov, rift valley fever virus (rvfv), all four types of dengue virus (denv), hcv, and hiv-1. fgi-106 also protected mice against a lethal challenge of ebov when given postexposure [149] . taken together, this suggests that fgi-106 probably acts on a conserved pathway common to these four viruses, and potentially makes for a very intriguing antiviral. a second screen was done using a collection of 1,990 small molecule compounds obtained from the nci and the ebov-gfp pseudotyped virus [150] . as a result, nsc 62914, a reactive oxygen species scavenger, was identified and shown to have high antiviral activity against ebov, marv, lassa virus, rvfv, and veev. in vivo studies demonstrated that this compound protected mice against a lethal challenge of ebov and marv when given either pre-or post-exposure [151] . metal ion-based therapeutics are a new potential class of drugs because they differ from carbon-based compounds due to the charged central ion which determines the molecular geometry of the compound. through these unique molecular geometries, specific compounds can be isolated that inhibit biological processes, and are unlike traditional carbon-based compounds because of their unique geometry. this difference allows these compounds to form octahedral and square planar molecular geometries. hexamminecobalt (iii) chloride (cohex) is a complex of a cobalt (iii) ion surrounded by six ammonia ligands in a full octahedral coordination. cohex was initially reported to have antiviral activity against adenovirus and sindbis virus, and was subsequently thought to have potential broad-spectrum antiviral activities [152] . cohex was shown to be well-tolerated in mice with no apparent toxicity. mice were treated with cohex daily and infected with a lethal dose of ebov. cohex-treated mice had a significant increase in mean time-to-death, and the highest concentration treatment group had a 20% survival rate [152] . this suggests that cohex has the potential to be an effective treatment against ebov infection. niemann-pick c1 (npc1), a cholesterol transporter found in endosomes and lysosomes, was recently identified as being required for ebov replication during a gene trap screen in hap1 cells using a replication-competent vsv bearing the ebov glycoprotein (rvsv-gp-ebov). in these experiments, cells with non-functional npc1 demonstrated decreased infectivity by rvsv-gp-ebov; however, expression of a functional npc1 rescued the normal infectivity of these viruses [20] . npc1 is known to affect calcium homeostasis as well as endosomal and lysosomal fission and fusion [153] [154] [155] . it has also been shown to be involved in hiv-1 release [156] . loss of npc1 causes a neurological disorder called niemann-pick disease, which is characterized by cholesterol accumulation in lysosomes [153] . while heterozygous npc1 knockout mice (npc1 +/− ) do not show evidence of niemann-pick disease, most npc1 +/− knockout mice were protected against a lethal challenge of mouse adapted ebov (80% survival) and marv (100% survival) [20] . additionally, small molecules, such as u18666a, have been identified that interfere with npc1 and cause a cellular phenotype similar to npc1 deficiency [157] . as such, u18666a was subsequently shown to block infection of ebov in vitro [21] . heat-shock protein 90 (hsp90) is a molecular chaperone that aids in the folding, trafficking, and proteolytic processing of many proteins [158, 159] . as a result of their many functionalities, hsp90 inhibitors have been designed to combat diseases such as cancer, and there are currently several drugs now in phase i and ii clinical trials [160, 161] additionally, hsp90 has shown to be important for replication of negative-strand viruses, as well as hcv, hbv, and polio [162] [163] [164] [165] . the effects of several natural and synthetic hsp90 inhibitors on ebov replication were tested in vitro. results of this study demonstrated that three hsp90 inhibitors significantly inhibited the replication of ebov in vero cells and primary human monocytes, suggesting their use as a potential therapeutic [158] . ebolaviruses express two secreted glycoproteins, soluble gp (sgp) and small soluble gp (ssgp) [166] . sgp has been associated with stabilization of the endothelial barrier function and reduction of endothelial barrier permeability by opposing the effects of tnf-α. these effects are in direct opposition to the observed roles for gp, which has been associated with endothelial cell destruction [167] . ssgp has yet to have a clear role during infection [166] . each of the gp forms contains identical n-termini but differ in the structure of their c-termini. during the differentiation process of the c-termini, the homodimer sgp is cleaved by furin to yield the mature sgp and a δ-peptide which is retained in cells longer as compared to sgp. when these δ-peptides from ebov, sudv, and tafv were fused with fc tags and transfected into cells before infection, they were capable of inhibiting both ebov and marv infection in a dose dependent fashion [166] . these observations indicated that δ-peptides might play an important role in filovirus pathogenesis, and could be exploited as a novel anti-filoviral therapeutic [166] . while many events in filovirus entry remain undiscovered, a fusion mechanism similar to hiv and sars-cov is thought to occur such that a conformational rearrangement of glycoproteins on the viral surface results in viral fusion with the cellular membrane. inhibitors of viral fusion have been developed for hiv-1 and sars-cov. these inhibitors prevent the c-terminal heptad repeat in the envelope protein from interacting with the cellular membrane proteins by directly competing and blocking its interaction with the n-terminal heptad repeat, which normally would result in the formation of the six-helix bundle required for membrane fusion. c-peptides, which are inhibitors of viral envelope fusion, have had limited success against filoviruses in the past, most likely due to the suggestive evidence that filovirus fusion occurs far along in the endosomal maturation process [168] [169] [170] [171] [172] . however, when these c-peptides were conjugated with an argenine-rich segment of hiv-1's tat protein (a protein known for its endosomal localization) the conjugated c-peptide exhibited marked antiviral effects, up to 99% inhibition of ebov and marv in vitro [172] [173] [174] . unfortunately, the concentrations used to generate this inhibition in vitro were not possible in the clinical setting, but this report indicates that future c-peptide research may result in filovirus entry prevention for future therapeutics. a recent report that screened 2,200 molecules demonstrated that chlorophyllide was able to decrease the section of hbv dna in a hbv antiviral assay. these results were obtained at compound concentrations which exhibited no cytotoxic effects. this molecule is an alkylated porphyrin containing copper and as such this compound is carries a charge at neutral phs [175] . during these screens, the chlorin e6 compound, a metal-free chlorophyllide-like molecule, was found to be the most potent and was subsequently tested against other viruses, including marv. during testing, the chlorine e6 compound showed significant antiviral activity in vitro against marv. this compound also inhibited junin virus, denv, hcv, and hiv-1 [175] . another recent study that examined a library of 52,500 compounds yielded 57 candidates capable of generating ≥90% inhibition of a hiv-1/ebov-gp pseudotyped virus, while not interfering with the hiv-1/vsv-g control virus. from these candidates, compound 7, a benzodiazepine derivative, showed an ability to inhibit both ebov and marv to a similar degree in vitro [176] . results from these experiments suggested that compound 7 acts at an early stage of viral entry, preventing infection by an unknown mechanism [176] . lj001, an aryl methyldiene rhodanine derivative, was identified during a high-throughput screening of inhibitors for nipah virus entry in the context of a vsv-pseudotyped vector [177] . this compound was subsequently shown to inhibit a variety of enveloped viruses, including marv, ebov, nipah, rvfv, hiv-1, hcv, wnv, etc. [177] . however, it did not inhibit nonenveloped viruses such as adenovirus and reovirus. further testing demonstrated that lj001 binds to the viral membrane and prevents virus-cell fusion. while initial testing in mice did not show antiviral efficacy, further development of this compound to improve pharmacokinetics and potency is distinctly possible [177] . development of medical countermeasures for ebov and marv remain a high priority and substantial progress has been made over the past decade. we have moved from the inability to protect from infection in various animal models of disease to a realm of medical countermeasures that protect prophylactically and more recently successful treatments that can be employed following known exposure to the viruses. initial efforts, focused on preventing the disease with vaccination strategies, ranged from subunit vaccines to vlps, vectored systems, dna vaccines, and live-attenuated virus systems that express the ebov or marv glycoproteins. to that aim, vaccine efficacy has been achieved by multiple vaccines against parenteral and aerosol routes of exposure. with the success of these new vaccine platforms, the attention of the past 5 years has focused on the ability to treat infected patients. in the animal models, success has been demonstrated with traditional small molecules and antibodies directed against the virus or critical host proteins or pathways associated with pathogenesis. the ability to utilize various rna silencing technologies has been a focus for therapeutics that could be beneficial for filovirus infection, other infectious diseases and cancer therapy. despite these successes, there is much work to do to adequately prepare for this infectious threat. the ability to provide a beneficial therapeutic impact at a point when patients experience clinical symptoms and seek relief from caregivers remains a hurdle for the medical countermeasure development. moreover, the quality of life of patients following infection and treatment may require additional development efforts or the combination of multiple therapeutic approaches. as seen in outbreaks, the clinical sequelae observed in patients that survive infection are severe and life changing. these observations emphasize the need for medical countermeasures that not only provide survival but also decrease morbidity and long-term pathological outcomes following infection. lastly, the funding resources fortitude and the ability to navigate regulatory pathways will be essential to reaching either emergency use authorization (eua) status or licensed drug status for therapeutics and vaccines. however, the field remains optimistic that medical countermeasure solutions for human use are possible. proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations virus taxonomy-ninth report of the international committee on taxonomy of viruses a hitherto unknown infectious disease contracted from monkeys filoviridae: a 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cynomolgus macaques with monoclonal antibodies enhanced potency of a fucose-free monoclonal antibody being developed as an ebola virus immunoprotectant characterization of zaire ebolavirus glycoprotein-specific monoclonal antibodies neutralizing ebolavirus: structural insights into the envelope glycoprotein and antibodies targeted against it a dna vaccine for ebola virus is safe and immunogenic in a phase i clinical trial immune protection of nonhuman primates against ebola virus with single low-dose adenovirus vectors encoding modified gps development of a preventive vaccine for ebola virus infection in primates a replication defective recombinant ad5 vaccine expressing ebola virus gp is safe and immunogenic in healthy adults vaccine to confer to nonhuman primates complete protection against multistrain ebola and marburg virus infections protection of nonhuman primates against two species of ebola virus infection with a single complex adenovirus vector enhanced protection against ebola virus mediated by an improved adenovirusbased vaccine a single sublingual dose of an adenovirus-based vaccine protects against lethal ebola challenge in mice and guinea pigs recombinant adenovirus serotype 26 (ad26) and ad35 vaccine vectors bypass immunity to ad5 and protect nonhuman primates against ebolavirus challenge rhabdoviridae: the viruses and their replication vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates single-injection vaccine protects nonhuman primates against infection with marburg virus and three species of ebola virus cross-protection against marburg virus strains by using a live, attenuated recombinant vaccine recombinant vesicular stomatitis virus vector mediates postexposure protection against sudan ebola hemorrhagic fever in nonhuman primates effective 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mechanism and therapeutic prospects cellular uptake of unconjugated tat peptide involves clathrin-dependent endocytosis and heparan sulfate receptors alkylated porphyrins have broad antiviral activity against hepadnaviruses, flaviviruses, filoviruses, and arenaviruses identification of a small-molecule entry inhibitor for filoviruses a broad-spectrum antiviral targeting entry of enveloped viruses the authors declare no conflict of interest. key: cord-354200-51wk3h75 authors: miller, a. c.; hannah, l.; futoma, j.; foti, n. j.; fox, e. b.; d'amour, a.; sandler, m.; saurous, r. a.; lewnard, j. title: statistical deconvolution for inference of infection time series date: 2020-10-20 journal: nan doi: 10.1101/2020.10.16.20212753 sha: doc_id: 354200 cord_uid: 51wk3h75 accurate measurement of daily infection incidence is crucial to epidemic response. however, delays in symptom onset, testing, and reporting obscure the dynamics of transmission, necessitating methods to remove the effects of stochastic delays from observed data. existing estimators can be sensitive to model misspecification and censored observations; many analysts have instead used methods that exhibit strong bias or do not account for delays. we develop an estimator with a regularization scheme to cope with these sources of noise, which we term the robust incidence deconvolution estimator (ride). we validate ride on synthetic data, comparing accuracy and stability to existing approaches. we then use ride to study covid-19 records in the united states, and find evidence that infection estimates from reported cases can be more informative than estimates from mortality data. to implement these methods, we release incidental, a ready-to-use r implementation of our estimator that can aid ongoing efforts to monitor the covid-19 pandemic. information on the progress of an ongoing epidemic arrives with delays. new cases, hospitalizations, and deaths are reported potentially weeks after individuals are infected. this stochastic delay obscures patterns in transmission. accurate estimation of daily infections is crucial for understanding the dynamics of disease transmission, and assessing the impacts of interventions [11, 14, 29] . however, as we show, currently available methods for reconstructing infection curves exhibit bias and instability. mathematically, observations such as daily reported cases can be described as a convolution of the underlying time series of new infections with a delay distribution -the probability distribution that describes time from infection to reporting. thus, recovering the infections curve from delayed reports is a deconvolution operation ( figure 1 ). unfortunately, deconvolution of noisy data presents a well-known ill-posed inverse problem, in which signal and noise cannot be separated, even when the delay distribution is known perfectly [24] . practically, this ill-posedness manifests as instability in estimates, both when the data and assumptions about the delay distribution are perturbed. this instability is compounded in infection estimation by right-censoring -recent infections have a smaller probability of being reported in the observation period. in general, instability in deconvolution problems is addressed by regularization, which imposes structure on the signal that is recovered from noisy data [28] . day -is perturbed by a delay distribution (center) that is measured with other data or assumed known. each infection date is stochastically delayed, resulting in the reporting curve (red ×'s) -new reported cases per day. bottom: the estimation procedure aims to undo this stochastic delay. given observed report curve (left) we use a statistical estimator with the delay distribution to recover the underlying curve. in this paper, we propose a statistically robust method to infer infection time series from delayed data, which we call the robust incidence deconvolution estimator (ride). ride incorporates a specific form of regularization that yields stable infection estimates, even in the presence of right censoring. in a simulation study we validate ride and compare it to existing methods. we then use ride to study transmission dynamics of sars-cov-2 at state and local levels. we compare ride to existing estimators on epidemic data from different regions in the united states, qualitatively showing its stability and robustness to censoring. we show that infection estimates from reported cases are often well-aligned with infection estimates from new hospitalizations -a more reliable but less widely available source of epidemic information. our findings also demonstrate how the implementation and phased lifting of non-pharmaceutical interventions contributed to reductions and increases, respectively, in transmission intensity. in our simulated and empirical examples, we compare ride to two classes of existing methods. the first class, which we term re-convolution estimators, estimate the infection curve by sampling from an assumed delay distribution and shifting observed case reports backward in timeeffectively, applying a convolution operation in reverse. this heuristic has been applied in a number of public tools for tracking the covid-19 pandemic [1, 2, 21, 26, 27] , but exhibits biases because it is not a deconvolution operation. the second class of estimators perform regularized deconvolution, but yield unstable estimates, in part because their regularization is inadequate in the presence of right-censoring. these include back projection (or back calculation) estimators developed to analyze the aids epidemic [7, 8, 9, 10] , and the richardson lucy (rl) algorithm, a model-free deconvolution method that has been used to analyze influenza [13] . we first give a brief overview of the statistical estimation problem, existing approaches, and our proposed method, ride; we compare these methods in a simulation study. we then study transmission dynamics of sars-cov-2, compare infection incidence estimators on real data, and analyze infection time series in relation to non-pharmaceutical interventions. method overview. given a time series of delayed observations for t days, y = (y 1 , . . . , y t ) (e.g., counts of daily new reported cases) and a delay distribution θ = (θ 1 , . . . , θ p ) (e.g., the distribution of time from infection to reporting) up to p days, the goal is to infer the time series of new infections x = (x 1 , . . . , x t ). the expected value of the observed data y is a convolution of the infection time series x with the delay distribution θ; estimation of x involves the deconvolution of y and θ. to produce an estimator that is robust to noise in y , we propose a model-based estimator using a cubic spline [15] to describe the underlying infections, x, and a poisson likelihood to describe the observed cases. we set the degrees of freedom of the spline basis using aic [3] . additionally, we add a regularization penalty on the second difference of the spline parameters, encouraging smoothness and select regularization strength with out-of-sample log likelihood. finally, we include an additional regularization to stabilize estimates in the presence of right censoring. due to delayed reporting, right censoring effectively reduces the number of observations in the most recent time windows, making estimates less stable. we address this issue as a missing data problem, and use a strategy similar to multiple imputation techniques for missing data [22] . specifically, we sample many extrapolations of the observed time series from a random walk that exhibits the same autocorrelation as the observed data. this random walk encodes the assumption that the autocorrelation in the observed data, which is a direct result of the convolution of infections with the delay distribution, will remain in future observations. for each extrapolation, we condition on the simulated counts to form the incidence estimate, and average estimates across these replicates. we find that this random walk regularization forms much more stable estimates when the report curve is censored. complete methodological details are given in the methods section and si a. simulation study. we examine the stability and reconstruction accuracy of estimators on a set of synthetic examples designed to mimic statistical issues associated with covid-19 data: right censoring and model misspecification. in this simulation study we include re-convolution, richardson-lucy, back projection [7, 32] implemented in the surveillance package in r [17] , and our proposed approach ride. we compare methods on four synthetic infection curves to study varying levels of complexity: a steep curve with a slow decay, a symmetric curve, a double peaked curve (representing two waves), and a pathological curve that has a sharp climb followed by a total drop-off in cases. for each curve, we consider different observation windows -from highly censored to fully observed -and data simulated under a misspecified delay distribution that approximates processing delays that are common with commercial testing facilities and recording offices. see si b for a detailed description of synthetic data and results. in general, we find that the model-based approaches more accurately infer the infection time series than the re-convolution and richardson-lucy estimators (as measured by mean squared error). additionally, we find that ride's regularization scheme is crucial when the model is misspecified; random walk extrapolation is key to stabilizing estimates for highly censored data. figure 7 summarizes the simulation study. 3 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 20, 2020. application to covid-19. monitoring the covid-19 pandemic presents numerous data challenges [14, 18] . disease transmission can be studied using different sources of data, each with unique benefits and drawbacks. data on cases are widely available, with a time delay as short as 12.8 days, on average, from infection to diagnosis [16, 31] ; however, such data are greatly affected by widespread variation in testing levels. data from hospitalizations may be more robust to changes in testing effort [21] , but are associated with longer delays, represent a smaller proportion of total cases, and are not widely available in the united states. deaths suffer the longest delay in both occurrence and reporting, and represent an even smaller proportion of infections. additionally, improved treatment strategies will likely cause both the infection fatality rate and the duration between infection and death to vary over time. we aggregated data for daily sars-cov-2 tests and new reported covid-19 cases, hospital admissions, and deaths for four selected regions with high-quality hospital data: arizona, new york, ohio, texas, and virginia. hospital admission data is readily available in new york city and houston within new york and texas, respectively. in addition to incidence data, we assembled dates for the following policy interventions at the state and regional levels: school closures, bans of gatherings of 10 people or more, stay-at-home orders, reopening of low risk non-essential businesses, reopening of hair salons, and reopening of indoor dining and/or bars. see si section c for data sources. delay distributions for infection to event are computed by estimating a series of delay times. we estimate time from infection to symptom onset by matching quantiles of a gamma distribution to the data from [19] ; from symptom onset to positive test by fitting a gamma distribution to non-zero delay times from florida for all cases through july 14, 2020 1 ; from symptom onset to hospitalization, as fitted by [20] using data from [30, 33] ; and from hospitalization to death [20] . see si section d for full delay distribution specification. comparison with existing estimation methods. we apply existing estimation methods and the proposed ride procedure to covid-19 case data from queens, new york, staten island, new york, the austin, texas metro area, and the state of arizona in figure 2 . we compare the ride procedure output to three existing approaches: back projection [8] 2 , richardson-lucy deconvolution [13] , and re-convolution [1, 2, 21, 26, 27] . all estimators are described in the methods section in further detail. the re-convolution procedure is a biased estimator and leads to over-smoothing of the infections curve and under estimating the peak. back projection and richardson-lucy are highly sensitive to noise, leading to large oscillations. further, back projection is sensitive to right censoring -recent infections estimates in austin and arizona are highly unstable. the ride estimator, on the other hand, is robust to noise in reporting and right censoring. comparison of inferences by data source. we next apply ride to case, hospitalization, and mortality data to estimate infections in arizona, ohio, virginia, houston, and new york city boroughs -locations chosen for their readily available hospitalization data. figure 3 depicts inferred daily infections (scaled by estimates of the proportion of cases resulting in confirmation, hospitalization, and death, respectively). note that the peak in estimated infections precedes the peak in reported cases, and has steeper upward and downward trajectories than the observed data time series. additionally, uncertainty regions near the end of the time series are much larger for daily infections inferred from deaths data. this is due to the longer lag between infection and death relative to cases and hospitalizations. reproductive numbers fitted using the method of cori and colleagues [11] , based on the inferred infection time series, are given in figure 4 . inferred infections from deaths consistently peaked earlier by about a week than inferred infections from hospitalizations in new york city. this may be due to nascent treatment regimes and overwhelmed hospitals in new york city during march and april. we note that r t estimates based on hospitalization and death inferences track closely in most areas. infections inferred from raw case data early in the epidemic (february through april) is often misaligned with hospitalization inferred infections due to low testing levels. there are some changes in testing levels from may onward, but changes in positive rates swamp changes in testing levels for many areas. for some areas, case upswings in june and july 2020 produce lower levels of hospitalization than earlier in the pandemic, likely due to the infected age skewing younger in later months. reproductive number estimates computed based on infections inferred from hospitalizations and cases are quite close in all areas examined. we quantitatively evaluate infection estimates from mortality and case data by measuring alignment with infection estimates from hospitalization data. we use two metrics: (i) the distance between the inferred infections curve using hospitalizations and the inferred infections from cases and deaths, and (ii) the distance between observed hospitalizations and imputed hospitalizations from the infections curve inferred from cases, deaths, and hospitalizations -the infections time series convolved with the hospitalization delay distribution. the first metric is a direct comparison of the infections time series from hospitalizations to infections inferred from others sources. the second metric measures a baseline accuracy of the inferred incidence, where no estimate is expected to have lower error than the curve derived from hospitalizations data. results are given in figure 5 . case infections generally, but not always, have lower error in the two metrics. case infections has much lower error than death incidence in new york, while deaths infections is a better representation of hospitalization incidence in ohio. this may be due to a cluster of about 4,000 cases related to ohio prisons in mid-april, which did not result in a corresponding spike in deaths or hospitalizations. when there are changes in testing regimes or population demographics of the infected, inferred curves from differing data sources may diverge. see si section e for further details. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. estimating infections at state and local scales. covid-19 data -cases, deaths, and hospitalizations -are often reported at the state or national level, which obscures local variation in prevalence and response to policy interventions. we compare inferred infections from cases in the five new york city boroughs with new york state; the austin, dallas/fort worth, and houston metros with the state of texas; and virginia health regions (central, eastern, northern, northwest, and southwest) to statewide across virginia. inferred infections are shown in figure 6 . for each region and state, we also include the following policy decisions: school closures, gathering bans for 10 people or more, stay-at-home orders, reopening of low risk non-essential businesses, and reopening of bars or restaurants with indoor seating. given the length of the time series fitted and the noise of the case data, slope changes in infections are accurate to within a few days due to estimator smoothing. our estimator strongly suggests an association between indoor bar/restaurant openings and an increase in transmission in texas and the southwestern and eastern regions of virginia. additionally, we find that in new york city, estimates suggest that the infections incidence decline postdated school closures, but was aligned with statewide stay-at-home orders. likewise, statewide -but not county level -stay-at-home orders coincided with a local incidence decline in houston in early april. earlier stay-at-home orders in harris county were not aligned with the decline ( figure 6 ). . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. covid-19 transmission dynamics are obscured by the incubation period, testing delays, reporting delays, and time to hospitalization or death. accurate estimation of the underlying infection time series is a pressing problem rife with challenges and complications, including observation noise, censoring, and model misspecification. existing methods are either statistically flawed (e.g., re-convolution) or sensitive to noise in reported data (e.g., richardson-lucy and back projection). our proposed estimator, ride, is statistically rigorous and robust to some of the data challenges that are present with covid-19 reported data, including high noise levels and right censoring. despite concerns that variation in testing effort may make hospitalizations data superior to data from all cases for monitoring infection dynamics, we found that our inferences from case data provided a reasonable proxy for those fit from hospitalization data, which are much less widely available and pose signal-to-noise ratio problems in smaller regions. case data allow effective estimation of infections at county and regional levels, rather than requiring entire states or countries. one of the most promising uses for regional infections estimation is as a tool in the evaluation of public policy. accurate reconstruction of infection time series is necessary to assess how policies influenced transmission over time, in particular when reporting is lagged and multiple interventions may have been undertaken in succession. as we have demonstrated, local covid-19 dynamics may differ from state-level patterns, and policy decisions are often implemented to mitigate effects in the areas with the highest case loads. only looking at state-level responses to policy decisions can blur policy effects as areas with different responses are aggregated. there remains room to improve these estimators. one salient aspect of the ongoing covid-19 pandemic are day-of-week reporting effects -it is common to see a smaller volume reported on saturday, sunday, and monday in many locations, likely due to increased processing time. this 7 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. comparison to hospitalization via convolution figure 5 : hospitalization error metrics. the top row is root mean squared error (rmse) between the normalized hospitalization incidence curve and death/case incidence curves by region. the bottom row is rmse between the case, hospitalization, and death incidence curves convolved with the hospitalization delay distribution and daily hospitalizations by region. source of error can be incorporated into the likelihood model with additional parameters, introducing a new regularization problem that warrants further study. additionally, reporting delays can vary by region and change over time. one potential approach for coping with such variation is to jointly model case and hospitalization data and use the relative stability of hospitalization delays to identify changes in the case reporting delay distribution. a joint approach has the potential to more efficiently use all available information, but would be limited to regions where hospitalization data is relatively available. lastly, it is worth noting that the delay distribution is the single-most important hyperparameter for estimating the infection time series [6] . this delay may change due to reporting habits, improvements in care, or shifting demographics of the infected population, among other factors [4] . improving estimates hinges on better characterization of these non-stationary effects. our results suggest that the method chosen to estimate infection counts influences the estimate itself and conclusions drawn. providing a stable, accurate, and consistent way to estimate infection time series can enable more accurate characterization of real-time transmissibility (i.e., the effective reproductive number) and ultimately may help policy-makers assess the effectiveness of public health interventions at the state and local levels. 8 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. we consider the following observation model of individual infected and reported dates. each individual n ∈ {1, . . . , n } who becomes infected on day i n is confirmed on day c n ∈ {1, . . . , t }, and we assume that there were no infections prior to the initial time, denoted t = 1. the date of confirmation is stochastically delayed from the date of infection i n ∈ {1, . . . , t }, where d n is a random number of days sampled from a discrete distribution with probability vector θ = (θ 1 , . . . , θ p ). the infection curve is a time series of daily infection counts, denoted x = (x 1 , . . . , x t ) where x t = n n 1(i n = t) (and 1 is the indicator function). similarly, the observed data is a time series of daily reported cases, denoted y = (y 1 , . . . , y t ), defined analogously using c n . our goal is to reconstruct the incidence curve x from an observed realization of y = y = (y 1 , . . . , y t ). we study the performance of different estimators that consider θ fixed and known. practically, covid-19 line lists track the number of days between symptom onset and the reporting of individual cases, which can be used to form an estimate of the distribution θ [16, 31] . we note that this model is a simplification -in practice we observe day-of-week effects in reporting and non-stationarity in θ. we examine robustness to model misspecification in our simulation study. the report curve is related to the incidence curve by a discrete convolution -the expected value 9 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . of y given x can be written as the convolution of x and the delay distribution θ, where θ s = 0 when s < 0 or s > p . the discrete convolution against θ can be expressed as a matrix, p θ , where (p θ ) t,s θ t−s . the expectation can be written as a matrix multiply e [y | x] = p θ x (see si section a for more details). intuitively, this indicates that to de-convolve the observed signal, an estimator needs to invert p θ . "re-convolution" incidence reconstruction. a popular method for incidence estimation attempts to undo the stochastic delays by sampling from the delay distribution and subtracting the value from each observed time [2, 21, 26, 27] . for each case n, the re-convolution estimator samples a delayd n ∼ categorical(θ) and computesî n = c n −d n , aggregating these into the incidence curve estimatex t = n n 1(î n = t). we call this the re-convolution estimator, as it amounts to a convolution of the already convolved report curve. the linear relationship between x and y makes clear the conceptual error of the re-convolution method. the re-convolution estimator has the expectation which will be inconsistent in general, as p θ = p −1 θ . this conceptual error motivates the use of methods developed for deconvolving signals (see si section a). deconvolution estimators. deconvolving signals is a well-studied problem in signal processing. one such deconvolution method is the richardson-lucy estimator [23, 25] , an iterative algorithm. while flexible, this approach can be highly sensitive to observation noise. nevertheless, the richardson-lucy estimator has been used to reconstruct incidence curves for infectious disease [13] , and we include the method in our simulation study. an alternative class of methods uses statistical models to form deconvolved incidence estimates. back projection (or back calculation) methods are model-based estimators that were developed to infer hiv/aids infection incidence [6, 7, 8, 9, 10, 32] . in the hiv/aids setting, the incubation period is on the order of months -much longer than the incubation period for seasonal influenza or sars-cov-2, necessitating a more statistically rigorous approach. back projection is closely related to empirical bayes methods for deconvolution [12] . these approaches form estimates by maximizing the marginal likelihood of observed data given a model for x 1 , . . . , x t and some form of regularization. parameterizing the incidence time series as x 1 , . . . , x t = x(β) (e.g., with smoothing splines or a step function), a model-based objective function takes the form where the likelihood function varies from method to method. both multinomial [9] and poisson [7] observation models have been considered; both model-based and post-hoc methods of smoothing have been studied [32] . ill-posedness and regularization. incidence estimation is a deconvolution exercise -a classic ill-posed inverse problem. specifically, the true incidence curve x is just identified -the number of free parameters is equal to the number of observed data points [24] . without observation noise, the convolution matrix p θ can be inverted, and the true incidence x can be identified. with observation noise, however, there may be many plausible incidence curves that 10 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . explain the observed data. the purpose of regularization is to bias the optimization objective toward estimates with properties we believe to be true, a priori. smoothness -the belief that incidence should not vary wildly day to day -is the main property induced by regularization. here, we devise a model-based estimate using a cubic spline [15] to describe the underlying incidence, x(β), and a poisson likelihood to describe the observed cases. locations with low case counts can be prone to over-fitting due to small sample size. to address this issue, we first select the degrees of freedom of the spline basis using aic [3] . additionally, we add an l2 regularization penalty on the second difference of the β parameters. to select λ, we split the observed data and use out-of-sample log likelihood. by default, we use 25% of the data to estimate out-of-sample log likelihood, and average over four random splits. the stability of estimates in the most recent time window before t is another practical concern. in this window, the effective number of observations is small due to right censoring, and incidence estimates can be unstable without a sensible prior. in this epidemiological setting, the convolution of the incidence and delay distribution results in a time series with significant autocorrelation. we exploit this structure by extrapolating the report curve forward in time with a random walk and condition on these simulated counts to form the incidence estimate. we first apply an anscombe transform [5] to the observed report curve to stabilize the variance and use the empirical singlelag autocorrelation to simulate random walk extrapolations. we average estimates over replicates of extrapolated random walks in a style similar to common techniques for handling missing data [22] . we find that this random walk regularization forms much more stable estimates when the reported data are censored. we term this procedure the robust incidence deconvolution estimator (ride); further details are given in si section a. reconstructing known incidence curves from simulated case reports. we study the accuracy of various incidence estimators described herein, including re-convolution, richardson-lucy, back projection [7, 32] implemented in the surveillance package in r [17] , and our proposed approach ride. the back projection method also employs a poisson likelihood, but regularizes by injecting a rolling window smoothing step into an expectation maximization fitting procedure. in general, we find that the model-based approaches for incidence estimation are more accurate than the re-convolution and richardson-lucy estimators. additionally, we find that regularization is crucial when the model is misspecified. we examine four synthetic incidence curves to study varying levels of complexity: a steep incidence curve with a slow decay, a symmetric curve, a double peaked curve (representing two waves), and a pathological curve that has a sharp climb followed by a total drop-off in cases. additionally, we consider different observation windows from highly censored to fully observed. we assume a delay distribution gamma(k = 10, θ = 1), with a mean delay of 10 days. additionally, we study incidence reconstruction for data simulated under a misspecified delay distribution. in the misspecified setting, we mimic weekly reporting delays where on every sixth and seventh day a uniform random proportion of the cases between 30% and 50% are reported two days later. this approximates reporting delays that are common with commercial testing facilities and recording offices. see si section b for a detailed description of synthetic data and results. synthetic data are depicted in figure 7a . inference quality of the methods are evaluated by root mean squared error (rmse) between the inferred incidence curve and the true incidence curve. additional metrics are available in the supplement. performance of each estimator for the four fully observed time series are presented in figure 7d . the model-based estimators fare better in both the correctly specified and misspecified setting -we observe a consistent improvement in inferred incidence over re-convolution and richardson-lucy estimates. back projection as implemented in the surveillance package is competitive in the well-specified setting, but in the misspecified setting accuracy suffers in 11 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020. 10.16.20212753 doi: medrxiv preprint comparison to our regularization scheme. we also compare different censoring windows t = 40, 50, 60, and 80. results for the "slowdecay" curve are depicted in figure 7e . for censored data, random walk regularization stabilizes incidence reconstruction. in the misspecified setting, the poisson model (and back projection) catastrophically fail for t = 50, due to instability in the maximum likelihood solution. the random walk regularization mitigates this pathology while remaining as accurate (or competitive) in all other regimes. we see that both the re-convolution and back projection methods struggle with right censoring. the root of the issue is that neither method accounts for the fact that the reported curve -due to the delayed convolution -is smooth. the incidence curve, therefore, must describe observations we have yet to see. we directly incorporate this into our scheme by sampling potential extrapolations of the report curve, modeled with a simple autoregressive (ar) process. additionally, averaging over random walk extrapolations yields more realistic uncertainty toward the right of the curve. figure 8, 9 , and 10, illustrate these points in both the correct and misspecified settings. we use the inferred incidence curves to compute time-varying effective reproductive numbers using the estimator proposed in [11] . richardson-lucy deconvolution produces unstable and noisy estimates, leading to reproductive numbers that vary wildly. the re-convolution method over-smooths the incidence curve, leading to systematically biased estimates and typically lower reproductive number estimates in the beginning of the growth cycle. we observe that the modelbased estimates more accurately reconstruct the ground truth incidence curve, which translates into more accurate characterization of the time-varying reproductive number. additional methodology details are available in si a. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. am and lh led the development of the proposed algorithm and its implementation, with input from ad. am designed and ran the synthetic experiments. lh aggregated publicly available epidemic data, and designed and ran the empirical study. am led the writing of the manuscript. all authors contributed to designing the research and writing the manuscript. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.16.20212753 doi: medrxiv preprint we consider the following observation model for infections and confirmed cases. each individual n ∈ {1, . . . , n } who eventually becomes infected is confirmed to be infected on day c n ∈ {1, . . . , t }, and we assume that there were no infections prior to the initial time, denoted 1. the time of a confirmed case is stochastically delayed from the true date of infection i n ∈ {1, . . . , t }, where d n is a random number of days sampled from a discrete distribution with probability vector θ = (θ 1 , . . . , θ p ). we assume that θ is available as external knowledge from an auxiliary data source and consider it fixed throughout. for example, covid-19 line lists track the number of days between symptom development and reported case in individual cases can be used to form an estimate of the distribution θ [16, 31] . while the methods considered condition on a specific θ, uncertainty in θ can be explored by a sensitivity analysis or averaging over a distribution of θ estimates. daily incidence is a summary of all cases from a given day. we write confirmed case incidence on day t as y t = n n 1(c n = t), where 1(c n = t) takes the value one when c n is equal to t and zero otherwise. similarly, the unobserved infection incidence curve is defined x t = n n 1(i n = t). the infection incidence curve is the target of our estimator. we denote realizations of observed daily confirmed case counts as c = (c (1) , . . . , c (t ) ), and realizations of underlying infection incidence as i = (i (1) , . . . , i (t ) ). we depict an example of a delay distribution θ and the resulting infection incidence and observed incidence pair in figure 1a . observed case likelihood. the likelihood corresponding to the observed time series, y is a function of the convolution of the infection incidence x = x 1 , . . . , x t and the delay distribution θ. to see this, we view each marginal y t as the sum of n poisson processes. consider the infection date i n for individual n. we can view the value c n as the realization of a poisson process with intensity function θ offset to start at the value of i n , conditioned on a single realization. take all x s individuals infected on day s. due to the additivity of poisson processes, the number of cases reported on day t that originated on day s will follow a poisson distribution, which we denote where we define θ s = 0 for s < 0 and s > p . the total number of reported cases on day t is a sum of x(s, t) over its first argument up to day t, which is again poisson where the mean is the familiar convolution operator. conditioning on the total number infected, n , the vector c (1) , . . . , c (t ) is jointly multinomial the expected number of reported cases, is related to the underlying infection time series x = (x 1 , . . . , x t ) by the convolution this is equivalent to a linear mapping of x, e[c] = p θ x for the matrix p θ constructed to correspond to the discrete convolution with vector θ all unidentified entries of p θ are 0. intuitively, recovering x from y involves the inverse of this mapping. however, a popular reconstruction approach does not correspond to inversion. an intuitive approach to infection incidence reconstruction is to stochastically "undo" the results of the delay random variable, d n . one type of estimator for this is to simply sample a new d n ∼ cat(θ) from the delay distribution and subtract this new value from the observed case date a related approach is to "undo" the convolution of θ with i by running the convolution backward over the observed case counts, resulting in the estimator where * indicates reversing the direction of the convolution operator. intuitively, this operation distributes observed cases backward in time according to the delay distribution. this has become a popular approach to cope with delayed observations [1, 2, 21, 26, 27] . this reverse convolution corresponds to multiplication by the transpose of p θ which effectively applies another convolution to the already convolved time series y , further smoothing the already smoothed observation. given this characterization, a straightforward estimator is to simply invert p θ to form an estimate ofx.x 18 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . this estimator, however, is not well-defined in the presence of noise in the daily case counts y , rendering it impractical for applied analysis. intuitively, this inverted convolution estimator is similar to fitting a linear regression model where the number of observations is equal to the number of covariates (i.e., n = p) -an ill-posed inverse problem [24] . using the "re-convolution" estimator to impute the incidence curve results in an even smoother imputed curve than the already smoothed reported case incidence curve. intuitively that makes sense -the "re-convolution" method convolves the already convolved reported case curve with the delay distribution once more, again smoothing (but backward in time). using the noiseless (but unobserved) expected reported incidence would recover the underlying daily infections, however applying this estimator to the noisy observed reported incidence yields highly unstable estimates -we see the estimate vary between −10 20 and 10 20 cases. this instability motivates our development of a practical deconvolution estimator. as stated in the methods section, model-based estimators start with a likelihood model for observed case data, conditioned on the underlying incidence curve. concretely, model-based methods define an objective where the form of the log-likelihood is specified by a probabilistic model, and the regularization penalty enforces smoothness in the incidence curve. back calculation and back projection methods have examined the use of poisson and multinomial likelihoods [9, 7] . for ride, we use a poisson likelihood and splines for the mean where β ∈ r p and b(t) is the p -dimensional spline basis value at time t. we also note that the model-based method is closely related to a set of techniques known as empirical bayes. in fact, the empirical bayesian method g-modeling was developed to de-noise (or deconvolve) noisy observed data [12] . the basic idea behind g-modeling is to fit a flexible prior distribution over the unobserved quantity using marginal maximum likelihood. the persubject unobserved values are the infection times i n for n = 1, . . . , n , and the noisy observations are confirmed case dates c n . the g-modeling approach parameterizes the prior (i.e., models the g-function), and numerically optimizes this parameter by maximizing the marginal likelihood (i.e., the f -function). specifically we define a prior over i n parameterized by β g β (s) ≡ p r(i n = s) = arg max β n ln g β (s)p(c n | s)ds . 19 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . given this estimate, we can compute the de-noised incidence curve via bayes rule. as a concrete example, g β (s) can be parameterized by a set of basis functions g β (s) ∝ exp(β b(s)) (26) b(s) = (b 1 (s), . . . , b p (s)) e.g., p -dimensional spline basis. here, the distribution g β (s) is determined by a log-linear function of splines, and is normalized. this empirical bayes approach leads to a slightly different likelihood -a multinomial. while performance can be similar, we found the poisson likelihood model easier to fit. to form estimates, we must choose a set of hyperparameters, including the number of spline basis functions (i.e., the degree of freedom (dof) parameter, which controls how complex the sample paths can be) and the regularization strength λ. additionally, to handle censoring, we treat data to the right of the observation window as missing, and impute plausible sample paths, averaging over their uncertainty [22] . because the smoothing of the stochastic delay typically smooths observations and induces a temporal autocorrelation among reported cases, we extrapolate the report curve with a simple random walk. we first apply an anscombe transform [5] (i.e., a variance stabilizing transform) and use the empirical single-lag autocorrelation to simulate a set of random walk imputations. we use a data-driven selection procedure: • select spline dof: -set the default λ to be 1/ t y t -compute theβ estimate for a grid of dof -select the dof parameter with the lowest aic (or bic) • select λ by data splitting -repeat the following four times (by default) and average -split the data into (by default) 25% validation and 75% training by randomly thinning each observed count -compute theβ estimate for a grid of candidate λ values (with selected dof value) -select the largest (i.e., smoothest) λ within (by default) 2% of the best observed held out log likelihood the rest of the procedure is as follows: for each imputed time-series, form a set of samples • compute theβ estimate given the selected λ and dof parameters • sample β from a laplace approximation formed at theβ estimate • compute the sample path x 1:t for this β sample this procedure yields a collection of x 1:t samples, which we use to form the incidence estimate. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.16.20212753 doi: medrxiv preprint the synthetic models are specified as follows. all of the shapes are normalized to have density 1 and then re-scaled to have 5,000 infections distributed over 100 time steps. all delay distributions are gamma (10, 1) . for the well-specified model, each case on the incidence curve is randomly propagated forward s days according to the delay distribution. for the noisy (non-well-specified) case, every sixth and seventh day a uniform random number between 0.3 and 0.5 is drawn and that proportion of cases are recorded two days later (e.g. cases from day six move to day eight). this is done to approximate reporting delays for testing and death records. symmetric. incidence has a gaussian shape, for t = 0, . . . , 89. symmetric censored. incidence is the same as symmetric, except that the incidence curve and reporting stops at t = 65. slow decay. incidence increases quickly then declines slowly, for t = 0, . . . , 88. pathological. incidence increases quickly then stops entirely, for t = 0, . . . , 79. double peak. incidence has two symmetric peaks in succession, for t = 0, . . . , 100. all case and death data for regions other than new york city is obtained from the new york times database 3 . hospitalization data are gathered from other sources in all regions. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.16.20212753 doi: medrxiv preprint texas. houston hospitalization data is from the texas medical center 6 , which has about 9,200 beds in houston out of slightly more than 19 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 20, 2020. delay distribution is the symptom delay distribution plus a delay distribution between symptoms and hospitalization, which [20] modeled as a gamma distribution with shape = 5.078 and rate = 1.307. finally, we fit a gamma distribution to the time from hospital admission until death for the distributions presented in [20] using moment matching. we compute two methods: 1. distance between case, death incidence curves with the hospitalization incidence curve, and 2. distance between observed hospitalizations and the inferred incidence curve from case, death, and hospitalization data convolved with the hospitalization delay distribution. in both methods, we: • subset all data to 14 days after hospitalization records begin and 14 days before they end to eliminate ramp up/ramp down effects, and then • normalize all incidence curves to have a sum of 1 within those ranges. in the first method, we compute root mean squared error between the normalized hospitalization 23 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 20, 2020. . https://doi.org/10.1101/2020.10.16.20212753 doi: medrxiv preprint estimating the time-varying reproduction number of sars-cov-2 using national and subnational case counts temporal variation in transmission during the covid-19 outbreak a new look at the statistical model identification serial interval of sars-cov-2 was shortened over time by nonpharmaceutical interventions the transformation of poisson, binomial and negative-binomial data backcalculation of hiv infection rates a method of non-parametric backprojection and its application to aids data reconstruction and future trends of the aids epidemic in the united states a method for obtaining short-term projections and lower bounds on the size of the aids epidemic minimum size of the acquired immunodeficiency syndrome (aids) epidemic in the united states a new framework and software to estimate time-varying reproduction numbers during epidemics empirical bayes deconvolution estimates reconstructing influenza incidence by deconvolution of daily mortality time series nonparametric regression and generalized linear models: a roughness penalty approach detailed epidemiological data from the covid-19 outbreak the r-package surveillance caution warranted: using the institute for health metrics and evaluation model for predicting the course of the covid-19 pandemic the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application incidence, clinical outcomes, and transmission dynamics of severe coronavirus disease 2019 in california and washington: prospective cohort study incidence, clinical outcomes, and transmission dynamics of hospitalized 2019 coronavirus disease among 9,596,321 individuals residing in california and washington statistical analysis with missing data an iterative technique for the rectification of observed distributions a statistical perspective on ill-posed inverse problems bayesian-based iterative method of image restoration estimating the infection and case fatality ratio for coronavirus disease (covid-19) using age-adjusted data from the outbreak on the diamond princess cruise ship solutions of ill-posed problems. scripta series in mathematics different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan epidemiological data from the covid-19 outbreak, real-time case information reconstruction of the infection curve for sars epidemic in beijing, china using a back-projection method time incidence curve and the normalized method incidence curve,whereĩ method t is the normalized incidence curve for a given method on day t.in the second method, we convolve a scaled version of the incidence curve to create an estimate of hospitalizations,where n hosp is the total number of hospitalizations. letting h t be the observed number of hospitalizations at time t,note that the second method can be used to measure the quality of the incidence curve inferred from hospitalizations. this value should be viewed as a lower bound for other incidence curves. key: cord-329904-e05ywn5e authors: jose, merin; desai, krishna title: fatal superimposed bacterial sepsis in a healthy coronavirus (covid-19) patient date: 2020-05-29 journal: cureus doi: 10.7759/cureus.8350 sha: doc_id: 329904 cord_uid: e05ywn5e coronavirus disease 2019 (covid-19) is a highly infectious disease caused by the newly discovered coronavirus, sars-cov-2 (severe acute respiratory syndrome coronavirus 2). the novel coronavirus first emerged in wuhan, china, in december 2019 and has led to a global pandemic. the virus mainly spreads through respiratory droplets from an infected person, but environmental contamination can also act as a source of infection, making social distancing an important key in containing the spread of infection. those with underlying health conditions are more susceptible to complications such as acute respiratory distress syndrome, which can be fatal. however, healthy individuals experience a mild flu-like illness or may be asymptomatic, recuperating from the infection even without any particular intervention. we present a case of a healthy covid positive individual, with no underlying comorbidities, who rapidly deteriorated overnight on readmission to the hospital after initial discharge and succumbed to this disease due to a superimposed bacterial infection with covid pneumonia. this case report highlights the importance of educating covid-19 positive patients about the precautions, as well as signs and symptoms of a superimposed bacterial infection, when their plan of care is in a home setting. it also emphasizes the potential role of checking procalcitonin levels as a part of routine laboratory investigation at initial presentation in all suspected as well as confirmed covid-19 cases to rule out an on-going bacterial infection that can prove fatal in the course of the disease. coronaviruses can cause an array of respiratory conditions, ranging from common cold to severe acute respiratory syndrome (sars) to the middle east respiratory syndrome (mers). in december 2019, a new betacoronavirus was identified, which is now known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] . the initial clusters of pneumonia cases caused by this sars-cov-2 were traced in wuhan, china [2] . in march 2020, the world health organization declared the outbreak to be a pandemic, with more than four million infections worldwide and still counting [3] . the spectrum of the presentation and clinical course of the infection may vary from mild to critical, with 81% of the infections being asymptomatic to mild, 14% patients developing severe disease such as dyspnea, hypoxia, or more than 50% lung involvement on imaging, and only 5% of the total infected cases progressing to a critical stage with respiratory failure and multiorgan dysfunction. overall case fatality rate was 2.3%, with no deaths reported among non-critical cases [4] . the most common presenting symptoms are fever, dry cough (few cases with reported concurrent sputum production), dyspnea, lethargy, myalgia, and, seldom, diarrhea, hemoptysis, dysgeusia, and anosmia. the lower respiratory tract involvement can offset pneumonia, which can rapidly progress into acute respiratory distress syndrome (ards) and is often associated with multiorgan failure, which appears to be the most dreaded complication, as it attributes to most fatalities from the infection [1, 2] . patients with underlying comorbidities are more vulnerable to more severe complications and a poorer prognosis, as compared with healthy patients [5] . we present a case of a healthy male with no underlying comorbidities who was clinically stable on presentation and was therefore discharged for home care. five days later, he presented again to the emergency department (ed) with worsening shortness of breath and diarrhea for three days and was found to have a superimposed bacterial infection. to our understanding, this caused a rapid deterioration in his clinical status and led to his early demise on the same day of his readmission. superimposed bacterial infection has not been a frequently reported feature of this infection so far. our emphasis from this case report is to highlight the risk of superimposed bacterial infection in covid-19 patients. our aim is to focus on the need to educate the patients on precautions to be taken during home care and using procalcitonin as a routine investigation for covid-positive patients even in the absence of clinical instability [6] . a 62-year-old healthy male presented to the ed with complaints of cough, bodyache, and fever. the symptoms first appeared five days back. he had no chest pain, shortness of breath, or abdominal pain. the patient was in good health up until now, with no significant medical or surgical history. he worked as a packet distributor and was active at his baseline. he is a nonsmoker and has no known addictions. he had no history of recent travel, but had a history of contact with two covid-positive individuals (grandson and friend), and therefore covid was suspected, for which he tested positive. on examination, he was not in distress and his vitals were stable. chest x-ray showed clear lung and pleural spaces, a normal heart with minimally tortuous aorta, and no evidence of any active cardiopulmonary, disease, or mild hypertrophic changes in the spine, as shown in figure 1 . given his stable clinical condition, normal x-ray findings, and no underlying comorbidities, he was discharged home with symptomatic treatment. following his discharge, in the next couple of days, he developed worsening shortness of breath, spiking fever with intermittent chills, diaphoresis, and diarrhea for three days. he presented to the ed again after five days of his initial presentation for these complaints. on examination, he was hypotensive with blood pressure of 94/44 mm hg and was hypoxic at room air saturating to 82%, which improved to 94% on 15 l of non-rebreather. he was in distress with tachypnea. he was admitted and immediately intubated, and a nasogastric tube was placed. on initial investigations, his labs were as shown in table 1 , and blood cultures were sent. wbc, white blood cell; rbc, red blood cell; bun, blood urea nitrogen; alp, alkaline phosphatase; ast, aspartate aminotransferase; alt, alanine aminotransferase figure 2 shows chest x-rays from initial the ed visit on day 1 and subsequent admission on day 5, comparing the drastic changes in a short span of five days. findings of the second chest x-ray were concerning for low lung volumes and development of infectious/inflammatory process with interval development of bilateral airspace opacities, including a focal consolidation in the left upper lung. ekg showed atrial fibrillation with rapid ventricular response and non-specific t wave abnormality. he was given 3.5 l of fluid bolus to address his hypotension. sodium bicarbonate and norepinephrine drip was also started for pressor support. digoxin 0.25 mg was given intravenously for atrial fibrillation and was started on amiodarone drip as options for rate control were limited, considering his hypotension. with the concern for possible superimposed bacterial infection, he was empirically started on cefepime and vancomycin intravenously (both renally dosed). he was also started on hydroxychloroquine and doxycycline. over the next six to seven hours, he continued to worsen clinically. blood cultures were followed up and it grew imipenem-resistant escherichia coli (it was sensitive to cefazolin and piperacillin/tazobactam). from being stable on day 1 of presentation, our patient continued to deteriorate overnight on subsequent admission. he eventually became bradycardic and was asystolic within one minute. the patient had an advance directive of "do not resuscitate" and died on the same night of admission due to septic shock with multiorgan dysfunction secondary to superimposed bacterial infection. he developed multifocal pneumonia due to covid-19, which was possibly accelerated by superimposed e. coli infection or vice-versa. the possible sources of his e. coli bacteremia were either translocation from the bowel, given he was having diarrhea for three days, or a genitourinary source, as no symptoms pertaining to it were reported. covid-19 is a systemic disease caused by the highly pathogenic sars-cov-2, which led to a pandemic, globally infecting more than 4,525,497 cases and causing 307,395 deaths worldwide as of now, with the united states of america having one-third of it [3] . the incubation period for the infection is 14 days after the exposure to the virus, but most cases show symptoms by the fifth day, with an average incubation period of four days [1] . sars-cov-2 and previous betacoronavirus infections have overlapping clinical features. secondary bacterial and fungal infections are common coinfections in viral illness. other coronaviruses outbreaks such as sars and mers, as well as influenza, had concurrent superimposed infections [7] . bacterial co-infection contributed to a significant amount of mortality during previous flu pandemics in 1918 and 2009 [8] . these co-infections are associated with increased intensive care unit admissions and mortality. superimposed bacterial infection in influenza is reported to occur in approximately 0.5% of healthy young patients and at least 2.5% of older patients. a systematic review that was performed during the 2009 pandemic reported that one out of four h1n1-infected patients had a bacterial or fungi coinfection [7] . secondary infection was found in 50% of non-surviving covid-19 patients [8] . despite its commonality in prevalence and the extensive adversities it causes in sars-cov-2 infection, it seems to be a fairly inadequately researched topic. furthermore, the main focus of the published papers in the literature with respect to secondary infections with other pathogens is revolving around the prevention and cross-transmission [7] . with this case report, we aim to accentuate the importance of meticulously identifying the presence of superimposed bacterial infections and attributing an adequate weightage along with other predictors for determining prognosis in a patient with sars-cov-2 infection. ards with multiorgan failure is the most dreaded and the most common complication attributable to mortality [9] . cardiovascular complications such as an arrhythmia, heart failure, and myocardial ischemia can also be fatal complications in patients with pneumonia [10] . patients suffering from chronic ailments such as longstanding hypertension, diabetes, cardiovascular diseases, immunosuppressive conditions (such as malignancies, chronic lung, and kidney diseases), chronic smoking history, and advanced age comparatively had relatively poorer outcomes [10, 11] . they fall under the "high-risk population" and extra caution should be maintained to keep them from acquiring the infection. according to a study, the following four predictors of higher mortality in patients with covid-19 pneumonia were identified: (1) age ≥ 65 years, (2) preexisting concurrent cardiovascular or cerebrovascular diseases, (3) cd3+cd8+ t cells ≤ 75 cell·μl −1 , and (4) cardiac troponin i ≥ 0.05 ng·ml −1 [12] . another study suggests age, sequential organ failure assessment (sofa) score, and d-dimer as the determinants of prognosis. the same study also highlighted that sars-cov-2 directly causes sepsis, even in the absence of concurrent infection with a different pathogen, but the mechanism was not well understood [10] . however, our patient with superimposed e. coli bacteremia succumbed to septic shock with multiorgan failure. he was otherwise a healthy individual in his sixties, indicating that bacterial superinfection is a risk factor even in healthy individuals without any prior hospitalization history. a meta-analysis recommended that serial procalcitonin measurement holds an important role in predicting the development of a more critical course of the disease. given the fact that the production of procalcitonin is ramped up in extra-thyroidal tissues in the presence of an underlying bacterial infection, its levels are also curtailed in viral infections by interferongamma, solidifying its correlation to complicated versus non-complicated disease processes [6] . superimposed bacterial infections are important predictors of prognosis in sars-cov-2 infection. it can accelerate the deterioration and can prove to be fatal despite prior optimum health of a covid-positive patient. if the patient is clinically stable with low risk of mortality and if home care is planned, appropriate precautionary measures and warnings should be issued against bacterial and/or fungal infections and the need for immediate attention in case of any worsening of symptoms. also, through this case, we would like to report a case of bacterial coinfection in a patient with covid, which will help in tracking and recognizing the extent of co-or superinfection. serial procalcitonin measurements should be routinely performed at initial presentation and thereafter to monitor and predict the prognosis of the disease. clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet. 2020 coronavirus disease (covid-19): situation report -118 characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china procalcitonin in patients with severe coronavirus disease 2019 (covid-19): a meta-analysis bacterial and fungal infections in covid-19 patients: a matter of concern co-infections: potentially lethal and unexplored in covid-19 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study case-fatality rate and characteristics of patients dying in relation to covid-19 in italy predictors of mortality for patients with covid-19 pneumonia caused by sars-cov-2: a prospective cohort study human subjects: consent was obtained by all participants in this study. in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. key: cord-336045-8qcj5uiy authors: langlois, isabelle title: viral diseases of ferrets date: 2005-03-01 journal: vet clin north am exot anim pract doi: 10.1016/j.cvex.2004.09.008 sha: doc_id: 336045 cord_uid: 8qcj5uiy distemper and rabies vaccination are highly recommended because of the almost invariable fatal outcome of these conditions. vaccination should constitute an important part of a ferret's preventative medicine program. with the current and anticipated development and licensing of new vaccines, practitioners are invited to gain awareness of the latest vaccine information. establishment of a practice vaccination protocol with regards to the site of administration of rabies and distemper vaccines is paramount to document any future abnormal tissue reactions. influenza is the most common zoonotic disease that is seen in ferrets. although it generally is benign in most ferrets, veterinarians must take this condition seriously. the characteristic continuous antigenic variation of this virus may lead to more virulent strains; the recent emergence of avian influenza virus outbreaks; and the increased susceptibility of elderly, young, and immunosuppressed individuals. shedding begins approximately 7 days postinfection [3] . the primary site of viral replication is the respiratory epithelium and lymphoid tissue of the nasopharynx [1, 4] . the virus disseminates by way of peripheral blood leukocytes to the liver, kidneys, gastrointestinal tract, urinary bladder, and brain. viremia has been documented 2 days postinoculation or infection and persists until the virus is neutralized by antibodies or the animal dies [5] . gastric hypochlorhydria has been associated with cdv infection in ferrets [6] . the lack of gastric acid was hypothesized to be due to direct action of the virus on gastric mucosa or was secondary to the viral effect on the central nervous system. infected ferrets become symptomatic after an incubation period of 7 to 10 days [1, 7, 8] . in ferrets, natural cdv infection most often consists of a catarrhal phase followed by a fatal neurotropic phase [9] . the initial phase is characterized by anorexia, pyrexia, conjunctivitis, and serous nasal discharge. an erythematous, pruritic rash appears on the chin (fig. 1 ) and eventually spreads to the inguinal area [1, 9] . hyperkeratosis of the footpads (fig. 2 ) occurs inconsistently in ferrets [1] . melena may be observed early in the course of the disease [1, 9] . some ferrets die during the catarrhal phase secondary to bacterial infections, such as pneumonia. clinical signs that are seen in the neurotropic phase include hyperexcitability, muscle tremors, hypersalivation, seizures, and coma. ferrets die 12 to 16 days after being infected with ferret-adapted cdv [1, 8] . the disease has a longer course when ferrets are infected with a wild canine strain; death occurs 21 to 35 days postinfection [1] . sometimes a decrease in temperature is observed by the time the animals become moribund [4] . a tentative diagnosis of canine distemper is based on the presence of typical clinical signs, severe leukopenia, a history of potential exposure to the virus, and questionable vaccination. a study demonstrated that virulent and attenuated cdv strains caused a severe leukopenia 1 week after infection [4] . leukocyte counts from animals that were infected with virulent strains remained low, whereas those of animals that received attenuated strains progressively increased to reach preinfection, or near preinfection, values 35 days postinfection. the diagnosis of canine distemper is confirmed routinely by an immunofluorescence test on peripheral blood smear, buffy coat, or conjunctival scrapings in live animals. in experimentally-infected ferrets, a reverse transcriptase-polymerase chain reaction (rt-pcr) has been used to detect the virus in peripheral blood [10] . recently, nested-pcr was found to be a sensitive and specific method for diagnosis of cdv infection [11] . on postmortem examination, the gross lesions correspond to the aforementioned clinical signs. on hematoxylin and eosin-stained sections, round eosinophilic intracytoplasmic and occasional intranuclear inclusion bodies are present in epithelial cells of the trachea, bronchi, and urinary tract [1] . other tissues, such as skin, gastrointestinal tract, salivary and adrenal glands, spleen, lymph nodes and brain, have been reported to contain inclusions [1] . ferrets that have signs that are suggestive of canine distemper infection should be placed in isolation. supportive care, including fluid therapy, systemic and ophthalmic antibiotics, gavage feeding, and bathing with antipruritic shampoo, may be initiated. administration of immune anti-cdv serum also may be considered. thorough daily disinfection of the environment is indicated. in all cases, prognosis is grave. the virus is inactivated by heat, visible light, and commonly used disinfectants (0.75% phenol, 0.2% roccal, 2%-5% sodium hydroxide, and 0.1% formalin) [1, 3] . some investigators reported that out of more than 1000 cases of distemper, not a single ferret survived the infection [8] . blair et al [12] treated distemper in a 7-month-old ferret while in the catarrhal phase. the ferret's life span and quality of life were improved with treatments, but the neurotropic phase could not be prevented and the animal was euthanized. when faced with an outbreak of canine distemper in a ferret colony, euthanasia and repopulation following disinfection is recommended. distemper is prevented best by vaccination. two vaccines are approved for use in ferrets in north america-fervac-d canine distemper vaccine (united vaccines inc., madison, wisconsin) and purevax ferret distemper vaccine (merial, athens, georgia and montre´al, quebec, canada). fervac-d is a modified-live virus vaccine of chick cell origin. a 5.9% incidence of anaphylactic reaction has been reported with this vaccine [13] . adverse events developed within 25 minutes and were characterized by hyperemia, hypersalivation, and vomiting. purevax is a lyophilized vaccine of a recombinant canary pox vector that expresses the ha and f glycoproteins of cdv. the manufacturer uses the term ''ferret distemper'' vaccine on the product label to prevent confusion with their canine products; however, there is no such thing as a ''ferret distemper virus'' and ferrets are infected by cdv. the absence of adjuvant or entire distemper virus decreased postvaccination risks and the manufacturer reports an incidence of 0.3% reversible anaphylactic reactions in its field safety trials. another modified-live canine distemper vaccine attenuated in primate cell line (galaxy d, schering plough animal health, omaha, nebraska) has been studied in ferrets. this vaccine was effective in preventing canine distemper in young ferrets that were challenged with virulent cdv after two vaccine inoculations [14] ; however, the duration of immunity and the incidence of vaccine reactions are unknown. clinical use of this vaccine is extra label (not approved for use in ferrets by usda) and requires informed owner consent. postvaccinal distemper infections were reported in black-footed ferrets that were vaccinated with chicken embryotissue culture-origin cdv vaccine and in domestic ferrets that were vaccinated with canine cell products [15, 16] . therefore, vaccination of ferrets with these products must not be performed. most ferrets that are obtained from pet stores at a young age received only one dose of cdv vaccine before leaving the breeding facility. repeated inoculations are required because maternal antibodies may interfere with proper immune response to cdv vaccine antigens. ferret kits should be vaccinated at 8 weeks of age and every 3 weeks for a total of 3 vaccinations [17, 18] . yearly booster vaccines are recommended [17] . observation of the ferret for 25 minutes following vaccination is advisable. in the event that a vaccine reaction occurs, administration of fluids, oxygen, antihistamine, and epinephrine may be indicated. parvovirus strains of varying virulence and immunogenicity cause aleutian disease. the disease was first reported in mink in the 1940s and got its name because mink that were homozygous for the aleutian (blue) gene were affected most severely [1] . the infection was documented in ferrets in the late 1960s [19] . at least three separate strains of aleutian disease virus (adv)-distinct from the mink strains-have been identified in ferrets [20, 21] . the ferret strains are believed to be mutant strains of the mink parvovirus; the hypervariable capsid region of the ferret strains of adv is similar to that of the mink parvovirus [22] . ferrets can be infected with the mink virus [20, 21] and ferret adv can infect mink; however, the virulence is lower compared with mink that are infected with mink strains [21, 23, 24] . transmission of the virus may occur by aerosolization; by direct contact with urine, feces, saliva, and blood; or by contact with fomites [1, 24] . vertical transmission of adv occurs in mink [1, 25] . the lesions that are caused by adv infection are immune mediated, but the mechanism by which adv interferes with the immune system is unknown. the severity of disease depends on the origin (mink or ferret) of the adv strain that is involved as well as the immune status and genotype of the infected individual [25] . minks that are infected with mink adv strains deposit immune complexes in various organs that result in glomerulonephritis, bile duct proliferation, and arteritis [26] . mink kits from antibody-free jills die from acute interstitial pneumonia when infected with virulent adv [27] . affected individuals are immunosuppressed, and therefore, are more susceptible to influenza, viral enteritis, and distemper [1, 26] . ferrets that are inoculated with ferret-adapted adv exhibit marked, persistent hyperglobulinemia and periportal lymphocytic infiltrates of the liver [21] ; however, immunocompetent adult ferrets that are infected experimentally can develop a persistent infection without clinical disease [21] . mink strains cause milder lesions and only a moderate elevation in gamma globulins in ferrets [1] . most ferrets that show clinical signs are between 2 and 4 years old. ferrets can be infected for years before clinical symptoms are noted [26] . any situation that leads to immunosuppression may play a role in the development of clinical disease. the clinical presentation of infected ferrets varies. some ferrets that are infected with adv die without clinical signs in good body condition [24] . generally, ferrets show signs of a chronic wasting disease with progressive weight loss, malaise, and melena [1] . acute dyspnea was described in one report [28] . central nervous system signs, such as tremors, ataxia, paralysis, and convulsions, also have been reported [25, [29] [30] [31] [32] . affected animal also may present with fecal and urinary incontinence [25, 29] . a presumptive diagnosis can be made based on a high gamma globulin concentration, the history, and clinical signs. although hypergammaglobulinemia is considered to be pathognomonic in mink, this feature is not always present in infected ferrets [28, 29] . serum protein electrophoresis often shows that gamma globulins account for more than 20% of the total protein concentration [1, 21] . other clinical pathologic findings of infected ferrets are variable. anemia, possibly attributable to hemolysis and decreased erythrocyte production, may be present [25] . biochemical abnormalities, such as azotemia and elevated liver enzymes, may be seen according to damage that results from immune complex deposition. proteinuria and urinary casts that are secondary to kidney damage also may be observed [28] . diagnosis of aleutian disease is confirmed antemortem with a positive serum titer coupled with hypergammaglobulinemia or lymphoplasmacytic inflammation in tissue biopsy samples. two serologic tests are available for adv testing-counterimmunoelectrophoresis (cep or ciep) (united vaccines, madison, wisconsin) and elisa (avecon diagnostics, bath, pennsylvania). the specificity and sensitivity of the elisa test has not been investigated in ferrets. therefore, results must be interpreted with caution. the cep test is used routinely in ferrets and is an effective method for identifying ferrets that have adv antibodies [25, [30] [31] [32] [33] . presence of antibodies without clinical disease for extended periods is possible [34] . ferrets probably develop persistent and nonpersistent, nonprogressive forms of adv infection similar to mink [25, 35] . detection of adv dna by in situ hybridization has been performed, but this method is not practical to screen for this condition [36] . recently, polymerase chain reaction amplification of part of the capsid gene that is specific to adv and restriction fragment length polymorphism to distinguish the ferret types of adv from the mink types of adv are valuable, time-saving assets for diagnosis of this infection in ferrets [20] . at necropsy, infected ferrets may have few or no gross lesions. hepatosplenomegaly, splenomegaly, and mesenteric lymphadenopathy have been reported [23, 24] . the most consistent histologic findings of adv infection in ferrets are periportal infiltration of the liver by plasma cells, lymphocytes, and macrophages with stimulated lymphoid tissues [23, 24] . bile duct hyperplasia, periportal fibrosis, and membranous glomerulonephrosis have been documented [23, 24, 28] . in individuals that present with neurologic signs, perivascular lymphocytic cuffing in the brain and spinal cord (fig. 3 ) and lymphoplasmacytic meningitis may be observed [25, 30, 31] . there is no definitive treatment of aleutian disease in ferrets. symptomatic ferrets may benefit from use of anti-inflammatory medication or immunosuppressive drugs, such as prednisone and cyclophosphamide. in mink, treatment with cyclophosphamide has been used to control infections for up to 16 weeks, but virus titers did not decrease [37] . administration of gamma globulin-containing adv antibody may be considered because it contributed to decreased mortality rates in mink kits [38] . control of aleutian disease is dependent on testing, cessation of breeding, and isolation of seropositive ferrets. all seropositive ferrets should be considered to be potential sources of adv and should be isolated from seronegative ferrets [1] . in mink farms, testing by ciep and removing all individuals that have adv antibodies has been an efficient method to eradicate the disease [39] . this approach, coupled with thorough disinfection, should be considered in any facility with a large number of ferrets. formalin, sodium hydroxide, and a phenolic disinfectant were efficacious against adv in the presence of organic material [40] . there is no vaccine to prevent aleutian disease and vaccination probably is contraindicated because of the immune-mediated nature of this condition. in mink, vaccination exacerbated the severity of aleutian disease [41] . in the late 1980s, a novel diarrheal disease that affected domestic ferrets was reported by pet owners and ferret breeders in the mid-atlantic area of the united states. since that time, this condition has been diagnosed throughout north america and in several other countries. the disease was named epizootic catarrhal enteritis (ece) on the basis of similarities to the epizootic catarrhal gastroenteritis of mink [42] . ece of mink is caused by a coronavirus that is related to transmissible gastroenteritis virus of pigs [43] . research strongly implicates a coronavirus as the causative agent of ece because: (1) microscopic lesions that were consistent with intestinal coronavirus infection were detected consistently in diseased ferrets; (2) coronavirus particles were identified in the feces and enterocytes, but no other viruses could be identified; and (3) immunohistochemical staining of jejunum showed coronavirus antigens in affected ferrets, but not in healthy individuals [44] . the disease is characterized by high transmissibility, high morbidity, and a low mortality rate. ferrets show signs of lethargy and anorexia within 48 to 72 hours postinfection. vomiting is the first sign of gastrointestinal disease in most ferrets, but it may go unnoticed by some owners because it subsides within hours [44] . subsequently, profuse green, bile-tinged diarrhea with a variable amount of mucus develops (fig. 4) . the stool's appearance is responsible for the term ''green slime disease'' that also is used to describe this condition [45] . the severity of clinical signs is highly variable; older ferrets that have concurrent diseases, such as insulinoma, long-standing infection with helicobacter mustelae, or adrenal neoplasia, are prone to develop severe clinical signs. ulcerations of the intestinal wall may occur which leads to the presence of blood in the feces. young ferrets tend to have mild or subclinical infection. the hypersecretory phase of uncomplicated ece often resolves within 5 to 7 days in healthy young animals. in some ferrets, this phase may be followed by a period of maldigestion or malabsorption of variable duration secondary to persistent lymphocytic inflammation of the intestinal wall. the feces become yellowish in color and contain grainy material that resembles bird seed. ece often can be diagnosed solely on the basis of characteristic historical findings and clinical signs [44] . thorough collection of the history data often reveals exposure to an asymptomatic ferret 48 to 72 hours before the onset of clinical signs. generally, clinicopathologic findings are nonspecific. inanition may cause increased serum activity of alanine aminotransferase and alkaline phosphatase secondary to mobilization of peripheral fat stores to the liver, with resultant hepatocellular swelling [1, 44, 45] . hypoalbuminemia may develop as a result of enteritis and malabsorption in chronically affected individuals. leukocytosis may be present in ferrets that have concurrent bacterial infection or gastric ulcers. definitive diagnosis of coronavirus infection often is difficult. coronavirus-like particles may be identified in the feces by electron microscopy during the acute phase of the disease process. characteristic histologic lesions that are seen in intestinal coronavirus infection, such as lymphocytic enteritis with villus atrophy, fusion, blunting and vacuolar degeneration or necrosis of the apical epithelium, may be identified on intestinal biopsy or necropsy specimens [44] . ferrets may become dehydrated rapidly during the hypersecretory phase of infection. dehydration and electrolyte imbalances need to be addressed. fluids that are supplemented with dextrose and electrolytes may be administered orally, subcutaneously, or intravenously, according to the degree of dehydration. antibiotic may be indicated to prevent secondary bacterial infection, particularly in cases with suspected intestinal ulcerations. in these cases, administration of sucralfate and an h 2 antagonist (eg, cimetidine) also are beneficial. sucralfate requires an acidic environment to be effective, so it should be given at least 30 minutes before an h 2 antagonist. syringe feeding with a highly digestible diet (science diet a/d, hill's prescription diet) may be indicated if anorexia persists. if clinical signs that are suggestive of maldigestion develop, oral administration of prednisone may be indicated. use of an anti-inflammatory dose of prednisone for 1 to 4 weeks, was followed by gradual tapering of the dose successfully. influenza viruses belong to the class orthomyxoviridae. human influenza types a and b are pathogenic to ferrets [1] . ferrets also are susceptible to avian, seal, equine, and swine influenza a viruses, although only human, avian, and swine strains induce clinical signs [46] [47] [48] [49] [50] . infection with influenza b virus less frequently results in illness and is associated with a milder clinical course [1] . transmission of influenza virus from human to ferrets and from ferrets to humans was documented in the 1930s [51, 52] . ferrets are used extensively as an animal model for influenza virus pathogenesis and immunity studies because their biologic response to influenza infection is similar to that of humans [53, 54] . influenza virus is transmitted by aerosol droplets from an infected individual, either to a human or a ferret. after intranasal inoculation, the virus localizes and replicates in great numbers within the nasal mucosa [55] . following a short incubation period, the body temperature increases and then decreases approximately 48 hours later [56] . transmission of the virus begins at the height of pyrexia and continues for 3 to 4 days [53] . as in humans, the disease is characterized by upper respiratory signs. clinical signs appear 48 hours postinfection and include anorexia, malaise, fever, sneezing, and serous nasal discharge [1, 56] . infection usually is mild in adult ferrets compared with neonates who can be severely ill [57] . conjunctivitis, photosensitivity, and unilateral otitis also may be seen [1, 58] . influenza infection may involve the lower respiratory tract in some susceptible animals [1, 59] . usually, influenza virus is confined to the bronchial and bronchiolar tissues [53, 60] . the disease may be fatal in 1-to 2-day-old ferret kits secondary to bronchiolitis, pneumonia, and aspiration of material from the upper respiratory tract [57, 61, 62] . lancefield group c hemolytic streptococci have been involved in secondary bacterial pneumonia [56] . influenza virus may infect the intestinal epithelium and cause limited enteritis [50, 63] . hepatic dysfunction also has been reported in ferrets that were infected experimentally with influenza [64] . neurologic symptoms, including ataxia, hind-limb paresis, and torticollis, were reported in ferrets that were infected experimentally with avian influenza a (h5n1) viruses that were isolated from the 1997 outbreaks of disease in domestic poultry markets in hong kong [50, 65] . generally, the diagnosis of influenza infection is based on the presence of compatible clinical signs, a history of exposure to infected individuals, and recovery from illness within 7 to 10 days. the differential diagnosis of any ferrets that has upper respiratory signs should include canine distemper. the usually mild and brief nature of influenza infections help to distinguish it from distemper. the use of virus isolation or hemagglutinin-inhibiting antibody titers on acute and convalescent serum samples rarely is needed for a diagnosis [1] . an enzyme-linked immunosorbent assay has been used to detect antibodies against influenza a and may be used to obtain a diagnosis rapidly [66] . clinical pathologic findings may present abnormalities. studies demonstrated an elevation in the neutrophil:lymphocyte ratio in the peripheral blood [50, 67] . transient lymphopenia, with a loss of 60% to 65% of peripheral blood lymphocytes 3 days postinfection was reported experimentally with avian influenza a (h5n1) [50] . plasma biochemical values generally are within reference ranges, but increases in concentrations of creatinine, blood urea nitrogen, potassium, albumin, and alanine aminotransferase were reported in some infected ferrets [64] . in most cases, infected ferrets can be treated at home. owners should be instructed to let their ferret rest until fully recovered. offering affected animals their favorite diet, highly palatable food (chicken baby food, beef baby food), or a highly energetic diet (science diet a/d, hill's prescription diet) is indicated. force feeding and offering water by syringe can be performed as needed. treatments to relieve clinical symptoms should be done on a case by case basis. if coughing is persistent, a pediatric cough suppressant without alcohol (at the pediatric dosage on a per weight basis) has been used [59] . to alleviate nasal congestion, the use of an antihistamine, such as diphenhydramine (2-4 mg/kg, by mouth, every 8 to 12 hours) [8, 59] , or intranasal delivery of phenylephrine may help [68] . antibiotics may be indicated to control secondary bacterial infections of the respiratory tract. neonates typically succumb to secondary bacterial infections. therefore, antibiotics may be useful in these patients to reduce mortality [69] . the use of non steroidal anti-inflammatory drugs to control fever is of questionable benefit because fever seems to be important in restricting the severity of infection [70] . experimentally, ferrets who received aspirin had cooler body temperature, but they shed more virus and their viral levels decreased less rapidly compared with ferrets that were not treated with an antipyretic. administration of antiviral medication has been studied in ferrets. amantadine hydrochloride (6 mg/kg, by mouth, every 12 hours) (symmetrel, bristol-myers squibb canada, montreal, quebec, canada) has been effective in treating ferrets that have influenza [71] . zanamivir (12.5 mg/kg) (relenza, glaxo wellcome, mississauga, ontario, canada), given as a onetime intranasal dose was able to prevent influenza infection [72] . administration of amantadine in ferrets rapidly produces antiviral resistance, but use of zanamivir does not [73] . vaccination of ferrets against influenza virus generally is not recommended because it is usually a mild disease and the antigenic variation of the virus complicates vaccination [1, 59] . experimentally, ferrets who recovered from influenza infection remained resistant to infection with the same strain for 5 weeks following initial infection [74] . ferret kits are protected from disease by milk-derived antibodies in immunized females [75] . controlling influenza infection resides in avoiding exposure of susceptible ferrets to infected individuals, either ferret or human. owners should be advised to minimize contact with their ferrets if they have a respiratory infection and should be sure to wash their hands thoroughly before changing the animal's cage, food, and water. veterinarians who have respiratory infections may consider wearing a mask and gloves. rhabdovirus causes rabies, an acute and almost invariably fatal disease that affects many mammals and humans. over the past several years, there have been numerous reports of ferret bite injuries, including unprovoked attacks on infants and small children [76] [77] [78] . these reports brought great controversy over the acceptability of keeping ferrets as pets with regard to the potential risk for rabies. this was because the period of viral shedding in the animal's saliva-before the onset of recognizable signs-was unknown at that time [1, 79, 80] . to the author's knowledge, there is no reported case of human rabies secondary to a ferret bite. since 1958, less than 30 cases of rabies in domestic ferrets have been reported to the u.s. centers for disease control [81, 82] . one of theses cases was attributed to vaccinating a ferret with modified-live virus rabies vaccine [1] . rabies virus must contact nerve endings and enter nerve fibers before infection occurs. exposure to rabies virus does not always lead to productive infection [81] [82] [83] . host response to rabies virus is influenced by the rabies virus variant, the viral dose, the route of transmission, the host species, and individual variations [84] . infection occurs primarily by contact of nerve endings with infected saliva from a rabid animal as a result of a bite wound. contact with the conjunctiva or olfactory mucosa also can result in transmission [84] . transmission of rabies through ingestion of an infected mouse was unsuccessful [85] . the pathogenesis of rabies in ferrets has been studied using european red fox rabies variant, north central skunk rabies variant, and raccoon rabies variant [81] [82] [83] . the mean incubation period is approximately 1 month [81, 83] . clinical signs reported include ascending paralysis, ataxia, tremors, paresthesia, hyperactivity, anorexia, cachexia, bladder atony, constipation, fever, and hypothermia [81] [82] [83] . two of 19 rabid ferrets that were inoculated with raccoon rabies variant showed aggressive behavior [83] . signs were reported to be mild in ferrets that were inoculated with the european red fox rabies variant [82] . mean morbidity period was 4 to 5 days [81, 83] . mortality in ferrets that were inoculated with the european red fox variant was dose dependent [82] . in the study that used skunk variant, ferret susceptibility was dose dependent and the incubation period was inversely proportional to dose [81] . in contrast, ferrets that were inoculated with raccoon variant were only moderately susceptible-regardless of dose-and there was no correlation found between viral dose and incubation period [83] . ferrets who survived experimental infection remained clinically normal except for one ferret that was given skunk rabies variant and had severe paralytic sequelae [81] [82] [83] . the immunologic response of ferrets to rabies virus infection seems to vary depending on the rabies virus variant that was inoculated. virus neutralizing antibodies were demonstrated in 14 of 33 (42.4%) rabid ferrets that were inoculated with skunk rabies variant compared with only 2 of 19 (10.5%) rabid ferrets that were given raccoon rabies variant [81, 83] . also, among ferrets that survived the infection, the proportion of seropositive cases was greater in ferrets that were given skunk rabies variant [81, 83] . ferrets may or may not excrete rabies virus in their saliva depending on the virus variant to which they have been exposed. viral shedding was not documented in the saliva 120 days postinoculation with european red fox variant [82] . rabies virus was not detected in the saliva of any ferrets that were given skunk rabies variant, but it was isolated from the submaxillary salivary gland of one rabid ferret that was euthanized [81] . shedding of rabies virus in the saliva was documented in ferrets that were inoculated with the raccoon rabies variant [83] . rabies virus was isolated from the salivary glands of 63% of rabid ferrets and 47% shed rabies virus in their saliva. initial viral excretion ranged from 2 days before the onset of clinical signs to 6 days after the onset. viral shedding also was documented in 1 of 23 ferrets that were inoculated with a rodent strain of rabies virus [86] . rabies should be included in the differential diagnoses of any ferret that has an unexpected onset of paralysis or acute personality change, especially if the ferret is unvaccinated, has access to outdoors, or lives in an area that is experiencing an epizootic of rabies. a ferret that is suspected of having rabies should be euthanized and its head should be submitted to the appropriate laboratory. generally, the diagnosis is based on direct immunofluorescent antibody testing of brain tissue [1, 81, 83] . confirmation may be established by intracerebral inoculation of suckling mice or inoculation of tissue culture with homogenized brain tissue [1] . the protective efficacy of killed rabies vaccine was demonstrated in ferrets that were vaccinated once subcutaneously with an inactivated rabies virus and were challenged 1 year later with street virus of fox origin. vaccinated ferrets had a survival rate of 89% compared with less than 6% for unvaccinated controls [87] . domestic ferrets that were vaccinated with a commercially inactivated rabies vaccine showed rapid induction of virusneutralizing antibody-a seroconversion that persisted at least 7 months [88] . based on these studies, killed rabies vaccines were approved for immunizing ferrets [89, 90] (table 2 ). ferrets should be vaccinated once at 3 months of age or older and then annually thereafter [87] . an animal is considered to be immunized if the primary vaccination was administered at least 28 days previously [89] . because a rapid anamnestic response is expected, an animal is considered to be vaccinated currently immediately after booster vaccination [89] . local injection site reactions were reported to develop frequently with rabies vaccine [91] . studies in cats showed that rabies vaccines more consistently produce granulomatous inflammation at vaccine sites, although leukemia virus vaccines are incriminated more often in the pathogenesis of vaccine-associated sarcomas [92] [93] [94] . there is only one report of vaccineassociated sarcoma in a ferret [91] . the ferret had been vaccinated for distemper and rabies on an annual basis in the dorsal area of the neck or interscapular area, so it is impossible to determine from which vaccine the tumor arose. practitioners should consider establishing a protocol in regard to the site of administration of rabies and distemper vaccines. this would allow the determination of which vaccine may be involved in the event that a tumor develops. a study demonstrated that the cellular response to the canary poxvectored rabies vaccine in ferrets was much milder than to the adjuvanted rabies vaccines [95] . consequently, its future use may be associated with a decreasing number of local vaccine reactions. also, because persistence of lymphocytes and macrophages has been suggested to play a role in the pathogenesis of vaccine-associated sarcomas, the canary pox-vectored vaccine would be less likely to be involved in the oncogenesis of these tumors. although it shows great promise, practitioners should remember that canary pox-vectored rabies vaccine is not approved for use in ferrets in north america at this time. to the author's knowledge, there is only one report of anaphylactic reaction in a ferret following administration of an inactivated rabies vaccine [13, 96] . this ferret previously had an anaphylactic reaction after receiving a distemper and rabies vaccine simultaneously. observation of ferrets for 25 minutes following rabies vaccination is advisable. unvaccinated ferrets that are exposed to a rabid animal should be euthanized immediately [89, 97] . if the owner refuses, the animal should be quarantined strictly for 6 months and vaccinated 1 month before being released [89, 97] . ferrets that are vaccinated currently should be revaccinated immediately [89, 97] . if a healthy ferret bites a person, it should be confined and observed for 10 days [89, 97] . the animal needs to be evaluated by a veterinarian at the first sign of illness during confinement. if signs that are suggestive of rabies develop, the animal should be euthanized and tested for rabies [89, 97] . rotavirus belongs to the family reoviridae. all rotaviruses are unique because they possess double-stranded rna genome. generally, these viruses cause diarrhea in young animals and children, but they also can occur in older individuals [98] . an atypical rotavirus was isolated from neonatal ferrets (mustela putorius furo) that had diarrhea at a large commercial farm in the united states. the disease was recognized at the ferret farm for several years and was referred as ''ferret kit disease'' [1] . partial characterization identified this virus as an atypical rotavirus, based on the lack of rotavirus group a common antigen and on its distinct double-stranded rna electropherotype pattern in polyacrylamide gels. in finland, a rotavirus outbreak in ferret kits had a mortality rate that approached 100% [1] . clinical signs of rotavirus infection occur in 2-to 6-week-old ferrets. soft yellow to green diarrhea is present with associated fecal staining or matted hair on their bodies. erythema of the anus and perineum also is reported [98] . the disease was prevalent throughout the year at the american commercial farm, with a increased incidence in colder months. morbidity among primiparous jills was high (up to 90%); the morbidity rate decreased with each gestation to range between 10% to 25% in multiparous jills [98] . the condition could be reproduced in 2-to 3-week-old ferret kits that were inoculated orally with viral preparations that were obtained from diarrheic ferrets; however, mortalities nor histologic lesions were observed in individuals that were infected experimentally [1, 98] . antemortem diagnosis is difficult. viral particles may be detected by electron microscopy in clarified, ultracentrifuged fecal suspensions, following negative staining in symptomatic ferrets. in a study, 58% of diarrheic ferrets that were infected naturally were positive for rotavirus particles in their feces. the ferret atypical rotavirus does not react with the rotazyme test (abbott laboratory, chicago, illinois), a commercially available enzyme immunoassay [1, 98] . the prevalence of this viral infection is unknown in ferrets because of the absence of reliable serologic tests. on postmortem examination, gross lesions are limited to the gastrointestinal tract. subtle histologic lesions that consist of mild blunting of the tips of the intestinal villi of the small intestine with enterocyte metaplasia to cuboidal cells may be observed. secondary bacterial infections may be a significant factor in the severity of the diarrhea. therefore, antibiotics are indicated and may contribute to accelerated recovery and decreased mortality rates. additional supportive care, including appropriate fluid therapy and force feeding, are indicated in most cases. in piglets, colostral antibodies play a key role in the protection against rotavirus [1] . this also may be true for ferrets. ferret kits acquire most of their passive immune globulins from their mothers by intestinal transmucosal absorption from colostrum and milk; they acquire all of their iga from their mother's milk [99] . the observed decrease in morbidity from ferrets of primiparous jills to ferrets of multiparous jills may be secondary to build-up of colony immunity with increasing age and exposure to the virus [98] . oral vaccination of primiparous jills was attempted at a ferret breeding farm; however, this procedure was ineffective. atypical rotaviruses have not been cultivated successfully in cell culture [1] . the ability to propagate the virus in cell culture in sufficient numbers will play a key role in the development of a vaccine. the infectious bovine rhinotracheitis virus (ibr) belongs to the family herpesviridae. there is only one published case report of spontaneous ibr infection in a ferret [100] . the virus was isolated from the liver, the spleen, and the lungs of a clinically normal ferret. its diet consisted of 5% raw beef by-products; it was hypothesized that virus-laden raw beef was the source of infection. the pathogenesis by which the virus disseminated to the liver, spleen, and lung tissue has not been elucidated. in contrast to naturallyoccurring infection, experimental infection of ferrets with ibr virus by intranasal and intraperitoneal inoculations induced acute and chronic respiratory disease [101] . considering that ibr virus can cause pathology in ferrets, these animals should not be fed raw meat or meat products [1] . distemper and rabies vaccination are highly recommended because of the almost invariable fatal outcome of these conditions. vaccination should constitute an important part of a ferret's preventative medicine program. with the current and anticipated development and licensing of new vaccines, practitioners are invited to gain awareness of the latest vaccine information. establishment of a practice vaccination protocol with regards to the site of administration of rabies and distemper vaccines is paramount to document any future abnormal tissue reactions. influenza is the most common zoonotic disease that is seen in ferrets. although it generally is benign in most ferrets, veterinarians must take this condition seriously. the characteristic continuous antigenic variation of this virus may lead to more virulent strains; the recent emergence of avian influenza virus outbreaks; and the increased susceptibility of elderly, young, and immunosuppressed individuals. viral diseases canine distemper in black-footed ferrets (mustela nigripes) from wyoming pathogenicity of morbilliviruses for terrestrial carnivores a ferret model of canine distemper virus virulence and immunosuppression experimental distemper in mink and ferrets. i. pathogenesis gastric hypochlorhydria in ferret distemper a clinical guide to the pet ferret practical exotic animal medicine, the compendium collection canine distemper virus infection in the domestic ferret canine distemper virus (cdv) infection of ferrets as a model for testing morbillivirus vaccine strategies: nyvac-and alvac-based cdv recombinants protect against symptomatic infection application of n-pcr for diagnosis of distemper in dogs and fur animals treating distemper in a young ferret incidence of adverse events in ferrets vaccinated with distemper or rabies vaccine:143 cases serologic evaluation, efficacy, and safety of a commercial modified-live canine distemper vaccine in domestic ferrets fatal vaccine-induced canine distemper virus infection in black-footed ferrets distemper virus infection in ferrets: an animal model of measles-induced immunosuppression ferrets, rabbits and rodents clinical medicine and surgery. philadelphia: wb saunders basic approach to veterinary care hyperglobulinemia in ferrets with lymphoproliferative lesions (aleutian disease) nucleotide sequence and polymerase chain reaction/restriction fragment length polymorphism analyses of aleutian disease virus in ferrets in japan aleutian disease in ferrets identification of a dna segment in ferret aleutian disease virus similar to a hypervariable capsid region in mink aleutian disease parvovirus comparison of the lesions of aleutian disease in mink and hypergammaglobulinemia in ferrets spontaneous aleutian disease in ferrets parvovirus-associated syndrome (aleutian disease) in two ferrets other diseases studies on the sequential development of acute interstitial pneumonia caused by aleutian disease virus in mink kits spontaneous aleutian disease in a ferret aleutian disease in the ferret aleutian disease in the ferret aleutian disease in domestic ferrets: diagnostic findings and survey results aleutian disease in laboratory ferrets aleutian disease in ferrets protides of the mustelidae immunoresponse of mustelids to aleutian mink disease virus identification of a non-virion protein of aleutian disease virus: mink with aleutian disease have antibody to both virion and nonvirion proteins detection of aleutian disease virus dna in tissues of naturally infected mink aleutian disease of mink: prevention of lesions by immunosuppression treatment of neonatally aleutian disease virus (adv) infected mink kits with gamma-globulin containing antibodies to adv reduces death rate of mink kits eradication of aleutian disease of mink by eliminating positive counter immunoelectrophoresis reactors evaluation of chemical disinfectants for aleutian disease virus of mink the pathogenesis of aleutian disease of mink. ii. response of mink to formalin treated diseased tissue and to subsequent challenge with virulent inoculum detection of coronavirus-like particles from mink serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus coronavirus-associated epizootic catarrhal enteritis in ferrets gastrointestinal diseases viral disease of pet ferrets: part ii. aleutian disease, influenza, and rabies response of ferrets and monkeys to intranasal infection with human, equine, and avian influenza viruses the infection of ferrets with swine influenza virus pathogenesis of avian influenza a (h5n1) viruses in ferrets the virus obtained from influenza patients influenza infection of man from the ferret lessons for human influenza from pathogenicity studies in ferrets the ferret as an animal model of influenza virus infection quantitative studies on the tissue localization of influenza virus in ferrets after intranasal and intravenous or extracordial inoculation proven and potential zoonotic diseases of ferrets studies of influenza infection in newborn ferrets otologic and systemic manifestations of experimental influenza a virus infection in the ferret philadelphia: wb saunders differential distribution of virus and histological damage in the lower respiratory tract of ferrets infected with influenza viruses of differing virulence the role of lung development in the agerelated susceptibility of ferrets to influenza virus role of upper respiratory tract infection in the deaths occurring in neonatal ferrets infected with influenza virus intestinal influenza infection in ferrets potential for hepatic and renal dysfunction during influenza b infection, convalescence, and after induction of secondary viremia neurological manifestations of avian influenza viruses in mammals an elisa for detection of antibodies against influenza a nucleoprotein in humans and various animal species the local origin of the febrile response induced in ferrets during respiratory infection with a virulent influenza virus induction and relief of nasal congestion in ferrets infected with influenza virus the role of naturally-acquired bacterial infection in influenza-related death in neonatal ferrets role of maternal immunity in the protection of newborn ferrets against infection with a virulent influenza virus the effects of per oral or local aerosol administration of 1-aminoadamantane hydrochloride (amantadine hydrochloride) on influenza infections of the ferret chemoprophylaxis of influenza a virus infections, with single doses of zanamivir, demonstrates that zanamivir is cleared slowly from the respiratory tract assessment of development of resistance to antivirals in the ferret model of influenza virus infection immunity to influenza infection in ferrets. i. response to live and killed virus elevation of nasal viral levels by suppression of fever in ferrets infected with influenza viruses of differing virulence severe facial injuries to infants due to unprovoked attacks by pet ferrets reported human injuries or health threats attributed to wild and exotic animals kept as pets (1971-1981) childhood risks from the ferret national association of state public health veterinarians. compendium of animal rabies control epidemiologists and public health veterinarians issue statement on ferrets viral diseases: ferret rabies. rabies surveillance, annual summary pathogenesis of experimentally induced rabies in domestic ferrets viral excretion in domestic ferrets (mustela putorius furo) inoculated with a raccoon rabies isolate the pathogenesis of rabies and other lyssaviral infections: recent studies susceptibility of carnivore to rabies virus administered orally zur frage der adaptationsfa¨higkeit von zwei in mitteleuropa isolierten tollwutvirusstaˆmmen an eine domestizierte und zwei wildlebende spezies. ein beitrag zur epidemiologic der tollwut 4. mitteilung: u¨bertragsversuche an frettchen mit einem nagerisolat. [the adaptability of two rabies virus strains isolated in central europe to one domesticated and two wild-living species: a contribution to epidemiology of rabies evaluation of an inactivated rabies virus vaccine in domestic ferrets serologic response of domestic ferrets (mustela putorius furo) to canine distemper and rabies virus vaccines national association of state public health veterinarians. compendium of animal rabies prevention and control canadian food inspection agency, animal health and production division, veterinary biologics section vaccine injection-site sarcoma in a ferret historical review and current knowledge of risk factors involved in feline vaccine-associated sarcomas feline vaccine-associated sarcomas focal necrotizing granulomatous panniculitis associated with subcutaneous injection of rabies vaccine in cats and dogs: 10 cases (1988-1989) cats differ from mink and ferrets in their response to commercial vaccines: a histologic comparison of early vaccine reactions another interpretation of ferret's reaction to vaccination rabies postexposure prophylaxis isolation of an atypical rotavirus causing diarrhea in neonatal ferrets ontogeny of the ferret humoral response isolation of infectious bovine rhinotracheitis virus from mustelidae experimental infectious bovine rhinotracheitis virus infection of english ferrets (mustela putorius furo l) key: cord-305207-fgvbrg8d authors: ohara, hiroshi; pokhrel, bharat m.; dahal, rajan k.; mishra, shyam k.; kattel, hari p.; shrestha, dharma l.; haneishi, yumiko; sherchand, jeevan b. title: fact-finding survey of nosocomial infection control in hospitals in kathmandu, nepal—a basis for improvement date: 2013-06-29 journal: trop med health doi: 10.2149/tmh.2013-03 sha: doc_id: 305207 cord_uid: fgvbrg8d the purpose of this study was to investigate the actual conditions of nosocomial infection control in kathmandu city, nepal as a basis for the possible contribution to its improvement. the survey was conducted at 17 hospitals and the methods included a questionnaire, site visits and interviews. nine hospitals had manuals on nosocomial infection control, and seven had an infection control committee (icc). the number of hospitals that met the required amount of personal protective equipment preparation was as follows: gowns (13), gloves (13), surgical masks (12). six hospitals had carried out in-service training over the past one year, but seven hospitals responded that no staff had been trained. eight hospitals were conducting surveillance based on the results of bacteriological testing. the major problems included inadequate management of icc, insufficient training opportunities for hospital staff, and lack of essential equipment. moreover, increasing bacterial resistance to antibiotics was recognized as a growing issue. in comparison with the results conducted in 2003 targeting five governmental hospitals, a steady improvement was observed, but further improvements are needed in terms of the provision of high quality medical care. particularly, dissemination of appropriate manuals, enhancement of basic techniques, and strengthening of the infection control system should be given priority. recently, nosocomial infections have become a global concern recognized as a major patient safety issue. they not only cause a significant burden on patients but also lower the quality of medical care. in addition, prolonged hospitalization due to nosocomial infections increases costs and unnecessary expenses for the hospital [1, 2] . in the healthcare setting, particularly in developed countries, various measures including the organization of infection control teams (icts), preparation of manuals, strengthening of surveillance systems, and training of staff have been taken to assure effective control. however, it is only some decades ago that importance was attached to nosocomial infection control and effective measures were employed, even in developed countries [3] . in developing countries, where the incidence of infectious diseases is high and environmental conditions of healthcare facilities are poor, nosocomial infections may frequently occur, and some studies have reported a high incidence at healthcare facilities in these countries [4] [5] [6] . effective nosocomial infection control is crucial in the healthcare facilities of developing countries, but in actual fact, attention to it is still limited and control measures are not functioning well in many facilities. furthermore, as implementation of control measures seems to be costly and to consume resources, nosocomial infection control is often given a low priority. severe acute respiratory syndrome (sars), which originated in guangdong province, china in november 2002, spread to more than 30 countries. in many hospitals where sars cases were encountered, nosocomial infections also broke out, causing many casualties along with economic havoc [7, 8] . it is not overstatement to say that such outbreaks have heightened awareness regarding nosocomial infection control even in developing countries. in more recent years, epidemics of novel influenza have also posed a threat of nosocomial infections [9] . these facts made many people realize again the importance of strengthening nosocomial infection control at hospitals in developing countries. some of the authors of the present paper have been engaged in technical cooperation for nosocomial infection control with hospitals in developing countries, recognizing the importance of strengthening control measures in order to enhance the quality of medical care. between 2000 and 2009, they have contributed to the promotion of nosocomial infection control in vietnam in collaboration with leading hospitals [10, 11] . since 2010, in response to the growing concern regarding nosocomial infections, we have focused our efforts in nepal through collaboration with tribhuvan university teaching hospital (tuth) in kathmandu city, where a technical cooperation project by japan international cooperation agency (jica) had been implemented to strengthen the hospital. following studies including those on hospitalacquired diarrheal diseases [12] and the prevalence of multiple drug-resistant pathogens [13] , this survey was carried out as a baseline study aiming to contribute to the improvement of nosocomial infection control at tuth and consequently in kathmandu city. the primary purpose of this study was to evaluate nosocomial infection control conditions and to prepare the basic information needed to provide technical guidance. the subjects of this survey are 17 leading hospitals in kathmandu city (five national hospitals, nine private hospitals, and three other hospitals). the national hospitals included three general hospitals (one out of three was a university hospital; i.e. tuth), one pediatric hospital and one obstetric hospital. all the private hospitals were general hospitals, while three other hospitals included one semigovernmental hospital and two non-profit organization hospitals (these three hospitals were general hospitals). the 17 hospitals play a crucial role in medical care in kathmandu city. a questionnaire was developed based on the form used in the previous surveys in vietnam [11] . the form consisted of the following items: "general information of the hospitals, control system including manual and infection control committees (icc), equipment and facility preparedness, training conditions, surveillance conditions, expectation for international cooperation and current problems. the contents of each item in the questionnaire are shown in table 1 . the questionnaire was distributed to the 17 hospitals in october 2011 and filled out by the hospital staff members who were responsible for nosocomial infection control or the director of the hospital. the recovered data were processed using spss ver19 for windows. in some hospitals, to determine the actual situation of icc, manuals, current problems and awareness level of hospital staff regarding nosocomial infection control, direct observations were conducted along with a brief interview with the hospital staff responsible for nosocomial infection control or hospital dicurrent problems requested the hospital to describe the current problems. rector in addition to the information obtained by the questionnaire. in 2003, a questionnaire survey was conducted at five national hospitals in kathmandu city [10] . these five hospitals were included in this study (2011) . the results of the 2003 questionnaire were compared with those of this study (2011), including manuals, iccs, in-service training and preparedness of personal protective equipment (ppe). a comparative statistical analysis of the 2003 and 2011 results was carried out by the fisher's exact method using spss ver19 for windows. tuth was established in 1980 with the assistance of a grant-aid from the japanese government as the first medical school in nepal, followed by the implementation of a technical cooperation project supported by the jica from 1980 to 1996 (the corresponding author participated as a team leader). the purpose of the project was to strengthen medical and educational services at tuth. during the above period, technical guidance was conducted in the field of hospital management, clinical medicine, nursing management, laboratory management and medical education. however, nosocomial infection control was not included in the project, probably because awareness regarding nosocomial infection control was still poor in those days even in developed countries including japan. currently tuth is playing a leading role in medical care as well as human resource development in nepal as the oldest and one of the most advanced medical schools. in this study, the current situation of nosocomial infection control at tuth was investigated in detail as a basis for further improvement. during the jica project period, technical guidance was provided, not on nosocomial infection control, but on bacteriological testing as a priority subject. in this study, investigation was performed by direct observation and interviews with heads of the departments of clinical microbiology and pharmacology and doctors of internal medicines, focusing on whether bacteriological testing was utilized for implementation of nosocomial infection control, in addition to detailed observation of the hospital and the questionnaire survey. ethical approval was obtained from the institute of medicine, kathmandu, nepal prior to using the questionnaire in the target hospitals. the 17 hospitals responded to most of the questionnaire items, but for some items, a response was obtained from only 16 hospitals. the average number of beds in the surveyed hospitals was as follows: national hospitals; 372 (150-497), private hospitals; 206 (50-750), other hospitals 328 (156-500). the average number of clinical departments was as follows: national hospitals (excluding the two specialized hospitals); 18.0 (14-22), private hospitals 9.7 (6-15); other hospitals 17.5 (15) (16) (17) (18) (19) (20) . tuth, which is one of the national hospitals, had 468 beds and 22 clinical departments. manuals for infection control were used in 52.9% (9/17) of the hospitals (national 4/5, private 3/9, and other hospitals 2/3). however, most of these manuals were more than five years old and some of their contents were not considered suitable for recent infectious diseases and antibiotic use. the manuals at three hospitals were considered obsolete. two national hospitals had good manuals with up-todate contents. only three hospitals had manuals for novel influenza. an infection control committee (icc) was established in 41.2% (7/17) of the hospitals (fig. 1) . however, a regular icc meeting was held in only two hospitals (once a month, and every three months) and the remaining hospitals held meetings when requested. it was noted that the operations of these committees were far from adequate. no hospitals had an infection control team (ict). equipment and facility preparedness: the number of hospitals which met the standard quantity requirements for disinfectants and personal protective fig. 2 . no hospital was equipped with a sufficient quantity of n95 masks and goggles. eleven and 12 hospitals responded that n95 masks and goggles were unavailable, respectively. a total of 81.3% (13/16) of hospitals responded that the preparation level for novel influenza was poor or slight. four hospitals responded that they could prepare isolation rooms to deal with novel influenza/ sars, but no hospital was equipped with negative pressure rooms. only one hospital had a plan of zoning formulated according to the risk of infection. training conditions: current training conditions are summarized in table 2 . six hospitals (four national hospitals, one private hospital and one other hospital) were organizing training programs for their staff (in-service training). regarding future plan, five hospitals responded that they planned to conduct inservice training, and eight hospitals responded that they did not have any plans at the present time but hoped to in the future. among all the hospitals, one had already conducted a training program on sars and/or novel influenza and three hospitals intended to conduct training. surveillance conditions: bacteriological testing was regularly performed for nosocomial infection cases at 62.5% (10/16) of the hospitals and 6.3% (1/16) of the hospitals for some cases. surveillance of nosocomial infections according to reports from clinical departments on clinical signs such as fever, respiratory signs, diarrhea, etc. was regularly carried out in 43.8% (7/16) of the hospitals in the survey (fig. 3) . seven hospitals had a strong interest in cooperating with foreign hospitals. a particularly strong expectation was observed regarding research support, information supply, ppe provision, and guidance in constructing an effective control system (table 3 ). among the problems observed in the study were weak icc function, few training opportunities among the hospital staff, inadequate use of antibiotics, shortage of infection control staff, shortage of doctors and nurses and their overload in daily medical practice, shortage of fundamental equipment including ppe, inadequate practice of basic tech comparison of nosocomial infection control conditions between 2003 and 2011 at five national hospitals showed an improvement trend. particularly, preparation of ppe and disinfectants remarkably improved as shown in figure 4 (p = 0.0238 and p = 0.004, respectively), categories in which all five hospitals met the standard quantity. in 2011, four out of five hospitals (except for one specialized hospital) were conducting in-service training, while only one hospital was conducting such training in 2003 (p = 0.099). among these four hospitals, manuals were on hand and an icc was established (p = 0.4167). the first icc in nepal was established in 1988 at tuth. since then, an icc meeting has been held once a month. a comparatively good infection control manual was prepared and has been revised according to necessity. inservice training has been conducted for most of the staff at tuth. this study showed a good situation regarding equipment preparedness including disinfectant (sufficient amount), ppe (sufficient amount of ordinary masks, disposable gloves and gowns) along with preparation of isolation rooms. however, incomplete observance of basic techniques such as standard precautions, as well as the need to further strengthen the function of icc, have been pointed out as challenges. performance of bacteriological testing was well carried out in the clinical microbiology department of tuth, and the results were passed on to the clinical side through the drug information office. however, the interview suggested that increased bacterial resistance to antibiotics was a growing issue at tuth. appropriate nosocomial infection control is a key strategy in providing high quality medical care, and effective measures are particularly required in developing countries, where the frequency of infectious diseases is high and environmental conditions of hospitals are poor [14, 15] . however, nosocomial infection control is generally not given high priority, and awareness among medical practitioners is still low, a situation that jeopardizes health care functions. in this survey in kathmandu city, steady progress was observed in national hospitals in comparison with the results in 2003. it is particularly noteworthy that awareness among staff and the level of training activities increased with an improvement in the preparedness of essential infection control equipment such as ppe and disinfectants. regarding private hospitals and other hospitals, a comparative study was not conducted using this survey, but an improvement in infection control similar to that of the national hospitals is assumed. however, further efforts to strengthen nosocomial infection control at the target hospitals are still considered necessary. the results showed that the majority of hospitals did not have an up-to-date nosocomial infection control manual, that the surveillance system was not established sufficiently, and that preparations against sars and novel influenza were poor. it is crucial to improve these fundamental systems. moreover, special emphasis should be placed on observance of basic techniques (standard precautions) such as hand hygiene, effective use of ppe and appropriate practice of disinfection [16] [17] [18] . enlightenment activities, such as distribution of manuals and teaching materials and the organization of training courses for medical staff, are very useful and effective for the improvement of nosocomial infection control. an increasing number of hospitals have been establishing iccs in recent years, but the management and implementation of activities need further improvement to achieve effective control measures. hereafter, icts also need to be set up in leading hospitals. furthermore, the detailed status of nosocomial infections and their causative agents should be strictly monitored and properly utilized in clinical practice. among the targeted hospitals in this survey, tuth showed comparatively good results. bacteriological testing, supervised by the jica project, was functioning well and contributing to the surveillance of nosocomial infection based on bacteriological examination and reports from clinical departments for suspected nosocomial infection cases. however, our previous study on pathogens associated with nosocomial lower respiratory infections showed a high frequency of gram negative bacilli such as escherichia coli, pseudomonas aerginosa, acinetobactor baumanii, klebsiella pneumoniae, as well as a high multiple drug resistance rate for isolated bacteria. in addition, a high rate of extended stratum beta lactamase (esbl) producing bacteria was observed [13] . the spread of multi-resistant bacteria reported by many developing countries is considered to be a facilitating factor in nosocomial infection [19] [20] [21] . methallo β lactamase (mbl) producing bacteria, which originated from india, is also suspected to be spreading to nepal [13, 22] . these findings suggest the need for more aggressive measures to tackle this global threat. the appropriate use of antibiotics based on accurate bacteriological testing, along with appropriate guidelines, is a worldwide challenge. nepal, fortunately, has not experienced a sars outbreak, and no human case of avian influenza has been reported to date. on the other hand, awareness of nosocomial infection control seems to be lagging behind countries where a sars outbreak did occur as shown in the 2003 study [10] . when a novel influenza becomes an epidemic and human to human infection is common, nosocomial infections may easily occur as seen in the spanish influenza pandemic of 1918-1919. appropriate nosocomial infection control is also considered useful for novel influenza control. special importance should be placed on setting up a foundation for appropriate nosocomial infection control in daily practice, training medical staff and establishing a control system, before nosocomial infections become a frequent occurrence. nosocomial infection control is crucial in providing high quality medical care. greater efforts should be focused on training medical staff to enhance basic techniques and establish control systems at ordinary times, not waiting until after an outbreak or epidemic. with such a foundation, it will be possible to promptly apply stringent nosocomial infection control in the event of an outbreak of novel influenza, sars or other emerging infectious disease. these measures will contribute to the reduction of unnecessary costs and can improve the financial condition of the hospital. based on the results of this survey, the authors intend to collaborate with nepalese authorities and further contribute to the improvement of nosocomial infection control. currently, our collaborative activities at tuth are related to basic studies on bacterial resistance to antibiotics and the appropriate use of antibiotics. in addition, guidance on the promotion of standard precautions and surveillance systems is currently being prepared. the results of the present survey are expected to provide baseline data for monitoring the progress of the nosocomial infection control situation at tuth as well as that in hospitals in kathmandu. in this survey, only hospitals in kathmandu city were investigated. infection control conditions are improving in these hospitals but further improvement in the software aspect is still needed to assure high quality medical care. in nepal as well as other developing countries, a significant disparity in the conditions of medical care and the health system exists between major cities and rural areas. in the future, the expansion of nosocomial infection control to hospitals in remote areas will be needed along with the implementation of guidance for hospitals in those areas. hospital-acquired infections in belgian acute-care hospital: an estimation of their global impact on mortality, length of stay and healthcare costs the cost of hospital-acquired infection and the value of infection control what can we learn from each other in infection control? experience in europe compare with the usa health-care-associated infections in developing countries paediatric hospital-acquired bacteraemia in developing countries evaluation of surveillance for surgical site infections in thika hospital canada; canadian severe acute respiratory syndrome study team. identification of severe acute respiratory sundrome in canada experience and review of sars control in vietnam and china prevention of nosocomial transmission of swine-origin pandemic influenza virus a/h1n1 by infection 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threat for critically ill patients contribution of acquired carbapenem-hydrolyzing oxacillinase to carbapenem resistance in acinetobacter baumannii extended spectrum beta-lactamase (esbl) medicated resistance to third generation cephalosporins among klebsiella pneumonia in chhenai new delhi metallo-beta-lactamase (ndm-1): towards a new pandemia? the authors would like to express thanks to the 17 hospitals in kathmandu city for their cooperation during the implementation of this study. this survey was conducted with the support of grants from the national center for global health and medicine, japan. none. key: cord-343074-dsubeaso authors: lee, wan‐ji; jung, hee‐dong; cheong, hyang‐min; kim, kisoon title: molecular epidemiology of a post‐influenza pandemic outbreak of acute respiratory infections in korea caused by human adenovirus type 3 date: 2014-06-01 journal: j med virol doi: 10.1002/jmv.23984 sha: doc_id: 343074 cord_uid: dsubeaso an outbreak of upper respiratory tract infections associated with human adenovirus (hadv) occurred on a national scale in korea from september to december 2010, following a major h1n1 influenza pandemic. data from the korea influenza and respiratory surveillance system (kinress) showed an unusually high positive rate accounting for up to 20% of all diagnosed cases. to determine the principal cause of the outbreak, direct polymerase chain reaction (pcr) amplification followed by sequence analysis targeting parts of the hexon gene of hadv was performed. serotypes of 1,007 pcr‐diagnosed hadv‐positive samples from patients with an acute upper respiratory tract illness were determined and epidemiological characteristics including major aged group and clinical symptoms were analyzed. the principal symptom of hadv infections was fever and the vulnerable aged group was 1–5 years old. based on sequence analysis, hadv‐3 was the predominant serotype in the outbreak, with an incidence of 74.3%. from the beginning of 2010 until may, the major serotypes were hadv‐1, 2, and 5 (70–100%) in any given period. however, an outbreak dominated by hadv‐3 started between july and august and peaked in september. phylogenetic analysis revealed that there was no genetic variation in hadv‐3. the results demonstrated that an outbreak of upper respiratory illness followed by h1n1 influenza pandemic in korea was caused mainly by emerged hadv‐3. j. med. virol. 87: 10–17, 2015. © 2014 wiley periodicals, inc. human adenoviruses (hadvs) in the family adenoviridae, genus mastadenovirus are responsible for various illnesses, including respiratory tract infections, conjunctivitis, cystitis, and gastroenteritis [kunz and ottolini, 2010] . typically, hadv-b species (types 3, 7, 12, 14, and 55) , hadv-c (types 1, 2, 5, and 6), and hadv-e (type 4) are closely associated with respiratory tract infections [metzger et al., 2007; kajon et al., 2010; walsh et al., 2010; tang et al., 2013] . identification of serotypes is important because the pathogenesis of hadv infection is determined mainly by serotypes. based on nucleotide and amino acid sequences of the hypervariable region in the hexon gene, phylogenetic analysis has been used for the clustering and typing of hadvs [crawford-miksza and schnurr, 1996a; biere and schweiger, 2010] . accordingly, phylogenetic analysis can facilitate molecular epidemiological investigations of hadv outbreaks. respiratory hadv infections have been closely associated with acute respiratory infections such as febrile illness, pharyngitis, and coryza (common cold). repeated outbreaks of such diseases have been reported worldwide [ryan et al., 2002; kim et al., 2003; chang et al., 2008] . in the same context, respiratory hadv infections can be identified from patients with an upper respiratory tract infection throughout the entire year, with an average detection rate of 4% in korea [lee et al., 2010] . however, in 2010-after a major h1n1 influenza pandemic in 2009-an outbreak of hadv infections occurred in korea. this study aimed to identify the major type of hadv involved through direct pcr amplification followed by hexon gene sequence analysis. the public health impact based on clinical features of the outbreak is also emphasized. this is believed to be the first report of a nationwide outbreak of respiratory adenovirus infections in korea. in 2010, 13,502 throat swabs from patients with an acute respiratory infections exhibiting a fever (>38˚c), coughing, sore throat, and runny nose were collected and used to detect viral causative agents through the korea influenza and respiratory viruses surveillance system (kinress), which obtains routinely clinical specimens from outpatients with acute respiratory infections who seek primary clinical care from about 100 hospitals nationwide in korea. specimens were placed in virus transport medium (bd, sparks, md) and sent to 17 local institutes of health and environmental research maintained at 4˚c to be tested for major respiratory viruses including hadvs. the remained samples were stored at à70˚c until use. because an abnormally high positive rate of hadv was observed nationwide from july 2010 until the end of the year, all positive cases (n ¼ 1,007) of hadvs were selected and then subjected to differentiate serotypes retrospectively. to compare any difference in hadv prevalence between the pre-and postpandemic phases, 91 samples of nasal aspirates taken in 2008 before the h1n1 influenza pandemic were collected through the acute respiratory infection network. the study was approved by the ethics committee of the korean national institute of health (approval # 2008; 2012 -09con-03-4c, 2010 ; 2010-03exp-1-r). characteristics of the clinical symptoms associated with hadv infections as well as records of all enrolled patients with acute respiratory infections were analyzed from the clinical records compiled via acute respiratory infections network for 2008 and kinress for 2010. logistic regression analysis with odds ratio values were performed to test the relationships between hadv infection and clinical symptoms using sas software (version 9.2; sas institute, cary, nc). total viral dna was extracted from 140 ml aliquots of specimens using quickgene-810 (fujifilm, tokyo, japan) following the manufacturer's protocol. to amplify part of the hexon gene, primer pairs av3r (5 0 -atgtggaaicaggcigtigacag-3 0 ) and av5l (5 0 -cggtggtgittiaaiggittiacittgtccat-3 0 ) [craford-miksza et al., 1999] were used to produce a 458 base pair (bp) fragment (position 19,552-20,009; accession # dq086466). the pcr was performed using sp-taq polymerase (cosmogenetech, seoul, korea) to maximize fidelity of dna amplification. the amplification products were analyzed by electrophoresis in 2% agarose gels (gendepot, barker, tx) and they were visualized using sybr safe dna gel stain (invitrogen, carlsbad, ca) under ultraviolet light. the pcr products were then purified using a qiaquick spin column purification system (qiagen, hilden, germany) to remove any trace of primers and analyzed directly on an abi3730 sequencer (applied biosystems, foster city, ca). partial hexon gene sequences were analyzed using dnastar 8.0 (lasergene, madison, wi) and aligned against other available hadv sequences using clus-talw. to type hadv, the nucleotide sequence homology was inferred from the identity scores obtained using the blastn program (national center for biotechnology information, bethesda, md). mega4 was used to create phylogenetic trees through the neighbor-joining method based on the kimura 2parameter distance matrix listed in the software, and bootstrap values were obtained from a random sampling of 1,000 replicates [tamura et al., 2007] . reference hadv sequences were obtained from genbank (http://www.ncbi.nlm.nih.gov/genbank/) and used for phylogenetic analysis. the nucleotide sequences of the partial hexon genes for strains identified in this study have been deposited in the genbank nucleotide sequence database under serial accession numbers from kc747631 to kc747649. reference sequences with accession number used in this study were given in supplementary table si . from the beginning of 2010 to the end of the year, an outbreak of respiratory infections associated with hadv in korea was examined retrospectively because of the significantly high positive rate of hadv found in samples. the monthly positive rates of hadv from throat swabs at the 17 local institutes of health and environmental research units during the period are shown in figure 1 and compared with those taken in 2008 as a baseline. the median positive rate of hadv in patients with an acute respiratory infection in the baseline period (previous 3-year average) was 2.0% (range 1.0-5.0%); however, during the outbreak period from june to december 2010, the rate increased significantly to 16.7% (range 3.8-31.3%; p < 0.001; fig. 2 ). the majority of these hadv positive cases (up to 77.9%) were identified from children (1-5 years old aged group; fig. 3 ) and the highest positive rate was observed at 2 years old aged group (24.2% in 1-5 years old aged group) in 2010. no gender bias was identified in a given aged group. human respiratory syncytial viruses (hrsvs), human rhinoviruses (hrvs), human metapneumovirus (hmpv), human para-influenzaviruses (hpivs), human coronaviruses (hcovs), human bocaviruses (hbovs), and influenza viruses were also detected through the kinress databank. of these, hrvs was the leading co-infecting agent among the cases of hadv infection with a positive rate of 25.3% in 2008 and 6.6% in 2010. other respiratory viruses were also identified as coor multi-infecting agents with positive rates ranging from 1.1% to 2.2%. the serotype of each adenovirus was determined by sequencing analysis followed by direct pcr of a part of the hexon gene. from the beginning of 2010 until may, the major serotypes were types 1, 2, and 5 (70-100%) in any given period. however, an outbreak dominated by type 3 started between july and august and peaked in september. based on the sequence analysis, hadv-3 was the predominant serotype, in this phase of the outbreak, at 74.3%. besides hadv-3, 12 more serotypes were also identified: hadv-2 (10.1%), hadv-1 (8.0%), hadv-5 (3.2%), hadv-4 (2.3%), hadv-6 (0.8%), hadv-8 (0.6%), hadv-7 (0.2%), hadv-11 (0.1%), hadv-19 (0.1%), hadv-34 (0.1%), hadv-41 (0.1%), and hadv-55 (0.1%). by contrast, only six serotypes (hadv-1, hadv-2, hadv-3, hadv-4, hadv-5, and hadv-6) were identified in samples from 2008. hadv-3 was also the major serotype at that time at 40.7%. however, hadv-2 (25.3%) and hadv-1 (23.1%) comprised larger proportions in 2008 than in 2010 (fig. 4) . interestingly, hadv-41, which is known to be a gastroenteritic cause of hadv infection, was also confirmed from a patient with acute respiratory infections. the clinical features of overall adenovirus infections are summarized in table i . most of the patients had a fever, coughing, runny nose, nasal congestion, and/or sore throat. over 80% of patients had a febrile illness and approximately half of them had a cough and a runny nose. symptomatic comparison of patients who were infected with hadv-3 in the baseline period (2008), there was no significantly elevated proportion of patients with fever and severe infections in the outbreak period (83.8% vs. 89.8%, p ¼ 0.24). instead, coughing, a runny nose, and nasal congestion were significantly lower in 2010 (p < 0.001; fig. 5 ). the partial hexon gene sequences of 1,007 pcrpositive cases of hadv from patients with acute respiratory infections were determined. in terms of species a through g, hadv sequences from the outbreak period did not form a cluster differing from reference sequences. sequence alignment was also performed to determine the sequence similarities for the leading serotype, hadv-3. all the hadv-3 strains isolated in the outbreak period showed high sequence identity (98.15-100%) compared with those in most other years (fig. 6 ). one isolate of the gastroenteritis strain hadv-41 also showed no significant variation from formerly reported sequence information. there were no sequence differences between the hadvs that were isolated in 2008 and 2010 for all types (hadv-1, hadv-2, hadv-3, hadv-4, hadv-5, and hadv-6). hadv infection is one of the leading causes of respiratory illness with typical clinical features, seen in community-based surveillance [cao et al., 2014] . to date, over 57 serotypes of hadv have been identified, and different serotypes have been correlated with different clinical manifestations [chu and pavan-langston, 1979; wood, 1988; gonçalves and de vries, 2006 ]. frequent outbreaks of acute respiratory infections caused by hadv have been described worldwide. the most frequent serotypes associated with respiratory outbreaks in various countries listed below have been mainly classified into subspecies b (hadv-11: china, hadv-7: korea, hadv-3: taiwan), c (hadv-1 and 2: malaysia), and e (hadv-4: usa) chang et al., 2008; yang et al., 2009; abd-jamil et al., 2010; lee et al., 2010] . symptoms caused by hadv infection range from mild symptoms to severe pneumonia. furthermore, respiratory illness associated with hadv can be confused with an influenza-like illness (ili) including fever, coughing, sore throat, and muscle pain [tohma et al., 2012] . to avoid the misdiagnosis of hadv infection from patients with an ili, a molecular biology technique such as direct pcr is used for discriminating possible causative viral agents. in korea, the laboratory surveillance system named kinress has been targeting outpatients with acute respiratory infections since 2005. annually, in 1-5% of patients with acute respiratory illness the cause is infection with hadv and this proportion is consistent with previous reports [lee et al., 2010a,b] . in the first post-influenza pandemic period, an abrupt outbreak of hadv was identified starting in june 2010. the median positive rate of adenovirus infections in the baseline period (previous 3-year average) was 2.0%. however, during the hadv outbreak-from june to december 2010-the mean positive rate increased significantly to 16.7%, ranging from 3.8% to 31.3%. a total of 1,007 hadv-positive cases were diagnosed from 13,502 clinical specimens (7.5%) in 2010. among these, hadv-3 (74.3% of positive cases) was identified as the major causative agent responsible for this outbreak of respiratory illness. interestingly, hadv-41 belonging to subspecies f was isolated from a patient with respiratory illness without diarrhea. because the hadv-41 is known to be a causative agent for viral gastroenteritis [uhnoo et al., 1984] rather than respiratory illness, it remains to be clarified whether this finding solely caused by pathological changes of the virus or not. phylogenetic analysis revealed that the outbreak caused by hadv type 3 during 2010 in korea seemed not to be related to any specific changes in viral genotypes. instead, in 2010 this strain played a unique role as a causative agent of respiratory illness. similar to other reports regarding hadv infection in patients with respiratory illness, the most affected age group was children aged 1-5 years old (77.9%). although 1-5 years old aged group took a higher proportion than other aged groups, the positive rate of hadv-3 in each age group also higher than other aged groups. these result was consistent with previous report that the positive rate of hadv was the most higher among children less than 5 years of age [cooper et al., 2000; chang et al., 2008; cheng et al., 2008] . to confirm hadv-3 as the sole causative agent for this outbreak, multiple co-infections by other respiratory viruses together with hadvs were analyzed. dual or more multiple infections with hrsvs, hrvs, hmpv, hpivs, hcovs, hbovs, and influenza viruses were studied. the results showed that hrvs were the leading cause of co-infection with hadv in 2008 (25.3%) and 2010 (6.6%). the incidence of this single infection rate was significantly higher in 2010 (86.4%) than in 2008 (70.3%) (p < 0.001). thus, it is highly feasible that hadv-3 infection was the sole cause of the outbreak in 2010. epidemiological and molecular data presented in this study confirmed that the outbreak in 2010 was not associated with genetic alterations causing a change in the pathology of the major causative agent, hadv-3, nor with multiple infections with other respiratory viruses. because the present study focused on outpatients with acute respiratory infections, severe manifestations such as bronchitis, pneumonia, and bronchiolitis were not scored in this study. nevertheless, several symptoms associated with hadv-3 infection such as nasal congestion, runny nose, and coughing were fewer in 2010 when compared with 2008 (p < 0.001). because little is known about the association between disease severity and the virulence-determining factors of hadv, it is unclear whether these reduced clinical symptoms arose from a particular change in virulence. in recent years, pcr and sequence analyses targeting the hadv hexon gene encoding a serotype-specific epitope have enabled rapid identification and classification of the genotypes and serotypes of hadvs [craford-miksza et al., 1999] . the strategy of serotype analysis used in this study was also based on a hexon gene sequence and allowed basic molecular biological information on circulating hadvs to be obtained. even though hadvs have dna as their genetic material, recombination is very frequently reported for this virus, which could be important for hadv evolution resulting in shifts in its pathology and virulence [craford-miksza and schnurr, 1996b; walsh et al., 2009; rebelo-de-andrade et al., 2010] . one recent report indicated that recombinant hadvs introduced into the community could produce highly virulent strains with epidemic and lethal potential [halstead et al., 2010] . therefore, there might be a limit to explaining the relationship between clinical manifestations and hadv infections through pcrbased molecular typing targeting just one gene. it would be better to use two or more genomic point comparisons for serotype determination to obtain better evidence for the pathogenicity and disease severity of particular serotypes of hadvs. the unusually high incidence of hadv-3 in 2010 following a pandemic of influenza (h1n1) in 2009 might have had several causes. one possibility is there was a micro-environmental change in hosts following the pandemic era. as presented in this study, the outbreak caused by hadv-3 following the pandemic era seemed not to be associated with specific changes in the viral hexon gene, which is known to encode major antigenic sites of hadv. instead, extensive changes in the overall host population's immune status resulting from the primary introduction of h1n1 influenza into the community could have mediated this unusually high incidence of hadv. there was no significant changes were observed in the hadv genome supports this hypothesis. the other possibility is that the original introduction of hadv could lead to fluctuations in population immunity triennially, regardless of the influence of the influenza pandemic. during the preparation of this manuscript, repetitive hadv outbreaks caused by hadv-3 were also detected in 2013 (data not shown). again, no significant mutations were observed using hexon sequence analysis targeting randomly sampled hadv-3 viruses. this repetitive outbreak caused by the hadv-3 suggests that when hadv-3 was the major circulating virus, population immunity might have been diluted. thus, newborn infants might be a newly susceptible group to hadv-3 and this might have contributed to the triennial outbreak of hadv-3 infection. to validate this hypothesis, a birth cohort-derived seroepidemiological study targeting specific antibodies against hadvs should be carried out to test the present data further. there are published data about the prevalence and distribution of hadv serotypes in korea [kim et al., 2003; lee et al., 2010a] , but those descriptions are limited by being hospital based and not representative of the general korean population. this study is believed to be the first nationwide report regarding an outbreak of hadv infection based on the kinress data. in conclusion, through a nationwide surveillance system during 2010 in korea, this study has documented that in an outbreak of hadv infection following a pandemic era, hadv-3 was the predominant serotype based on the hexon gene. further investigations to verify which factor(s) were associated with this abrupt outbreak following the h1n1 influenza virus pandemic in 2010 are needed to understand the major causes. molecular identification of adenovirus causing respiratory tract infection in pediatric patients at the university of malaya medical center human adenoviruses in respiratory infections: sequencing of the hexon hypervariable region reveals high sequence variability emergence of community-acquired adenovirus type 55 as a cause of community-onset pneumonia a community-derived outbreak of adenovirus type 3 in children in taiwan between molecular and clinical characteristics of adenoviral infections in taiwanese children in 2004-2005 ocular surface manifestations of the major viruses the epidemiology of adenovirus infections in greater manchester, uk 1982-96 adenovirus serotype evolution is driven by illegitimate recombination in the hypervariable regions of the hexon protein strain variation in adenovirus serotypes 4 and 7a causing acute respiratory disease analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues adenovirus: from foe to friend recombinant adenovirus type 3 and type 14 isolated from a fatal case of pneumonia molecular epidemiology of adenovirus type 4 infections in us military recruits in the postvaccination era molecular epidemiology and brief history of emerging adenovirus 140associated respiratory disease in the united states genome type analysis of adenovirus types and 7 isolated during successive outbreaks of lower respiratory tract infections in children the role of adenovirus in respiratory tract infection molecular classification of human adenovirus type 7 isolated from acute respiratory disease outbreak (ard) in korea comprehensive serotyping and epidemiology of human adenovirus isolated from the respiratory tract of korean children over 17 consecutive years (1991-2007) clinical severity of respiratory adenoviral infection by serotypes in korean children over 17 consecutive years abrupt emergence of diverse species b adenoviruses at us military recruit training centers outbreak of acute respiratory infection among infants in large epidemic of respiratory illness due to adenovirus types 7 and 3 in healthy young adults meg a4: molecular evolutionary genetics analysis (mega) software version 4.0 genome and bioinformatic analysis of a hadv-b14p1 virus isolated from a baby with pneumonia in beijing detection and serotyping of human adenoviruses from patients with influenza-like illness in mongolia importance of enteric adenovirus 40 and 41 in acute gastroenteritis in infants and young children evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis computational analysis identifies human adenovirus type 55 as a re-emergent acute respiratory disease pathogen adenovirus gastroenteritis genomic analyses of recombinant adenovirus type 11a in china outbreak of acute respiratory disease in china caused by b2 species of adenovirus type 11 we thank 17 local institutes of health and environmental research for supporting kinress. we thank you-jin kim for help in preparation of this paper. additional supporting information may be found in the online version of this article at the publisher's web-site. key: cord-350749-ihkxouz8 authors: panda, aditya k; tripathy, rina; das, bidyut k title: plasmodium falciparum infection may protect a population from severe acute respiratory syndrome coronavirus 2 infection date: 2020-07-29 journal: j infect dis doi: 10.1093/infdis/jiaa455 sha: doc_id: 350749 cord_uid: ihkxouz8 nan to the editor-we read with great interest the article published by nickbakhsh et al [1] describing epidemiological evidence of interaction between seasonal coronaviruses and other co-circulating viruses in a united kingdom population. the authors have suggested that prior exposure of children to coronavirus oc43 offers protection against severe covid-19 phenotype by possible crossimmunity. these observations encouraged us to investigate the possible role of plasmodium infection on coronavirus disease 2019 (covid-19) infection or severity. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection and mortality rates are variable in different countries. several factors may help account for the variability in the virus infection or mortality rate, such as age, sex, comorbidity, or genetic makeup. recent studies indicated that protozoan infections may offer some protection against various positive-strand rna viruses. prior exposure to plasmodia significantly suppressed chikungunya virus-associated pathogenesis, characterized by reduced viral load and improved joint inflammation [2] . furthermore, coinfections of a rodent plasmodium strain and lactate dehydrogenase-elevating virus offered protection against experimental cerebral malaria and experimental autoimmune encephalomyelitis [3] . based on these observations on plasmodium infection and positive-strand rna viruses, we hypothesized that there could be a possible association between malaria and sars-cov-2 infection. to validate our observation, we investigated the prevalence of covid-19 in the plasmodium falciparum-endemic area of odisha, india, odisha is highly endemic for p. falciparum infection. we obtained the annual parasite index (api) of p. falciparum for the last 10 years (2010-2019) from the national vector borne disease control program and covid-19 infection status in odisha from the government of odisha website (see https://health. odisha.gov.in/covid19-dashboard. html). api and covid-19 data from 30 districts were analyzed and shown in figure 1 . a significant negative correlation (spearman r = -0.37; p = .04; n = 30) was observed between 10-year average api scores and the number of covid-19 cases detected. malaria is known to stimulate b cells resulting in hyper-gammaglobulinemia [4] and multiple cross-reactive antibodies are often produced, which could be protective. a recent study further highlighted the production of immunoglobulin g (igg) and immunoglobulin m (igm) against p. falciparum merozoite, which persists for an extended period and protects the host from clinical malaria [5] . it has also been well established that infection by some enveloped viruses triggers naturally occurring antibodies to activate the complement system, leading to lysis of the virus [6] . antibodies against disaccharide galactose α-(1,3)-galactose (α-gal) are most prevalent and constitute about 2% of total igg and igm. previously, we have demonstrated high levels of antibodies to α-gal (igg and igm) in healthy subjects in malaria-endemic areas (igg: 144.62 ± 47.23; igm: 19.93 ± 15.08 enzyme-linked immunosorbent assay [elisa] units) compared to residents of nonendemic regions (igg: 30.15 ± 9.3; igm: 8.5 ± 6.1 elisa units) [7] . these are polyspecific antibodies capable of interacting with multiple antigens. although the presence of α-gal on the surface of sars-cov-2 has not been reported, the anti-gal antibodies, being polyspecific, can cross-react with multiple epitopic determinants [8] . furthermore, in our earlier study (unpublished), we observed high levels of specific antimalarial antibodies (pfp0, pfemp, resa, msp-1, and msp-3) in residents of malaria-endemic areas. the cross-reactivity of these antibodies, along with anti-gal antibodies and covid-19, needs to be investigated for possibly demonstrating a cause-effect relationship. further validation of this hypothesis might be established from other p. falciparum-endemic areas of the world. epidemiology of seasonal coronaviruses: establishing the context for the emergence of coronavirus disease 2019 plasmodium co-infection protects against chikungunya virus-induced pathologies a virus hosted in malaria-infected blood protects against t cell-mediated inflammatory diseases by impairing dc function in a type i ifn-dependent manner to b or not to b: understanding b cell responses in the development of malaria infection igm in human immunity to plasmodium falciparum malaria natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity immunological correlates in plasmodium falciparum infection with special reference to cerebral malaria naturally-occurring anti-alphagalactosyl antibodies in human plasmodium falciparum infections-a possible role for autoantibodies in malaria covid-19 coronavirus pandemic department of health and family welfare, government of india key: cord-333724-a3dufzxt authors: wong, t. e.; thurston, g. m.; barlow, n.; cahill, n.; carichino, l.; maki, k.; ross, d.; schneider, j. title: evaluating the sensitivity of sars-cov-2 infection rates on college campuses to wastewater surveillance date: 2020-10-11 journal: nan doi: 10.1101/2020.10.09.20210245 sha: doc_id: 333724 cord_uid: a3dufzxt as college campuses reopen, we are in the midst of a large-scale experiment on the efficacy of various strategies to contain the sars-cov-2 virus. traditional individual surveillance testing via nasal swabs and/or saliva is among the measures that colleges are pursuing to reduce the spread of the virus on campus. additionally, some colleges are testing wastewater on their campuses for signs of infection, which can provide an early warning signal for campuses to locate covid-positive individuals. however, a representation of wastewater surveillance has not yet been incorporated into epidemiological models for college campuses, nor has the efficacy of wastewater screening been evaluated relative to traditional individual surveillance testing, within the structure of these models. here, we implement a new model component for wastewater surveillance within an established epidemiological model for college campuses. we use a hypothetical residential university to evaluate the efficacy of wastewater surveillance to maintain low infection rates. we find that wastewater sampling with a 1-day lag to initiate individual screening tests, plus completing the subsequent tests within a 4-day period can keep overall infections within 5% of the infection rates seen with traditional individual surveillance testing. our results also indicate that wastewater surveillance can be an effective way to dramatically reduce the number of false positive cases by identifying subpopulations for surveillance testing where infectious individuals are more likely to be found. through a monte carlo risk analysis, we find that surveillance testing that relies solely on wastewater sampling can be fragile against scenarios with high viral reproductive numbers and high rates of infection of campus community members by outside sources. these results point to the practical importance of additional surveillance measures to limit the spread of the virus on campus and the necessity of a proactive response to the initial signs of outbreak. infected individuals. some colleges are monitoring wastewater on their campuses for signs of the virus, which has been found to be capable of detecting viral rna. if a wastewater sample shows signs of viral rna, then screening tests are administered to the individuals who live or work in the buildings that contribute to the sewer in question. we present a model for such wastewater surveillance within a larger model for the spread of sars-cov-2 on a college campus. we show that wastewater surveillance can reduce the number of false positive cases and the associated disruptions to student life, while maintaining similar overall numbers of infections. however, we find that surveillance testing strategies that rely solely on wastewater sampling may be less effective if the local transmission rate of the virus is high, or if the rate of infection of members of the campus community by outside sources is high. as colleges put into action their reopening plans for fall 2020, a natural experiment in epidemic management is unfolding. this experiment is stress-testing colleges' strategies for reducing the spread of sars-cov-2 on their campuses. these strategies include reducing the capacities of classrooms and residence halls, mandating the use of face masks, implementing extensive sanitization and cleaning protocols between classes, putting in place social distancing requirements, and banning large gatherings. additionally, many colleges are using traditional individual screenings to test students for the virus [1-3] at periodic intervals or by random sampling throughout the fall term. previous work has found that this type of surveillance testing is critical for controlling the spread of virus on campus [4] [5] [6] , and that surveillance testing the campus population every 2-10 days is a minimum requirement to keep the overall number of infections manageable. such frequent screenings of all of a university's students places a high financial and logistical burden on universities, as well as an intrusion on the lives and comfort of students. as an alternative or supplementary form of viral surveillance, many municipalities and universities are turning to collecting and testing wastewater from their campuses for signs of viral ribonucleic acid (rna) [7] . viral presence in wastewater can be a leading indicator for positive cases in traditional individual screening tests [8] [9] [10] . in this approach, wastewater is collected at sewer locations around campus and tested in a laboratory, typically by a testing method that incorporates a polymerase-chain reaction (pcr) assay [11] . if the wastewater sample from a particular sewer shows signs of viral presence, then the individuals in the building(s) that the wastewater sampling location serves can be given traditional individual screening tests for sars-cov-2. in this way, wastewater surveillance can be an effective tool to reduce the overall number of randomized screening tests required in order to maintain low infection rates. however, the epidemiological models used to inform college campus reopening strategies do not yet include a representation of wastewater surveillance. in this work, we present a wastewater module for the susceptible-exposed-infected-recovered (seir) model of paltiel et al. [4] . the seir modeling approach [12] employs a type of compartment model, wherein all individuals are assumed to be in one of four general categories: susceptible to infection, exposed (but not yet infectious or symptomatic), infected (either symptomatic or asymptomatic), and removed/recovered (recovered from the infection or deceased) [13] . these models do not track individual persons (i.e., are not agent-based), and as such seir models are simple and stylized in their representation of persons and processes. however, this simplicity leads to high computational efficiency, making seir models ideal for the large numbers of model simulations required for uncertainty and sensitivity analyses, and for informing decision-making to manage campus community health under uncertain conditions. such decision support is critical when campus officials must manipulate decision levers including the rate at which to administer screening tests to the campus population and what share of courses should be held on-campus versus online. previous efforts to account for the sensitivity of projected infection rates have focused on simple monte carlo sensitivity analyses, wherein each parameter is sampled from a probability distribution and the effects of these changes on the infection rates is compared (e.g., 5 ). these foundational approaches assess the sensitivity of infection counts to uncertainties in model parameters, which represent real, on-the-ground uncertainties. however, previous work provides a largely qualitative view of these sensitivities; a formal global sensitivity analysis is still needed. such a global analysis would quantify how the variation in infection rates is attributable to each uncertain model parameter and potential decision lever, including, for example, the viral reproduction rate and the rate at which screening tests are administered to the campus population. here, we address these issues by assigning marginal prior probability distributions to each uncertain model input parameter. we sample from these prior distributions to examine the sensitivity of infection rate to uncertainty in the model parameters. by considering changes in these parameters in combination, we conduct a formal global sensitivity analysis to attribute the variance in the total number of infections to variation in each of the input parameters (including decision levers) and interactions among the parameters. we use a previously published seir model to incorporate a model component to represent wastewater surveillance testing. we use our new model to assess the ability of a wastewater surveillance system to prevent numbers of infections from exceeding a desired maximum. we examine the vulnerability of a wastewater surveillance system to failures in underlying assumptions, including increases in the viral reproductive rate, larger numbers of new infections of campus members from outside sources, and higher rates of noncompliance with quarantine procedures. our model is a modified version of the seir model of paltiel et al. [4] . the interested reader is referred to that work for a more detailed description of their original model, but we provide an overview here for convenience. the original model code is based on the source code provided accompanying the online dashboard for running simulations using the model of paltiel et al. ([14] ; https://data-viz.it.wisc.edu/covid-19-screening/). the model parameters discussed below are summarized in table 1 . the overall campus population is divided into three groups. the first group contains individuals who are circulating on campus and are capable of becoming infected by others, or capable of infecting others. this includes individuals who are uninfected and susceptible (u), those who have been exposed to an infectious individual but are not symptomatic and not infectious (e), and those who have been infected and are asymptomatic but infectious (a). the second group contains individuals who are in quarantine/isolation, including those who have been tested and received either a false or true positive result (fp and tp, respectively) and those who are symptomatic (s). the third group contains individuals who have been removed from the pool of potentially infected/infectious persons, including those who have been infected but have since recovered (r) and those who were infected and later died (d). the model tracks the total number of individuals in each of these model compartments, but not the individuals themselves. fig 1 provides a schematic of these model components and the exchanges of individuals between them. we assume that the overall size of the campus population is 12,500 individuals, and that initially a(0)=10 of them are asymptomatic and infectious. susceptible individuals (u) become exposed (e) at a rate determined by the effective viral reproduction rate (rt) and the overall prevalence of asymptomatic infectious individuals (a) in the circulating group. we take rt as an uncertain input parameter, with a default value of 1.1, which (as of this writing) is appropriate for upstate new york where our home institution is located [15, 16] . we consider uncertainty in rt and the impacts of incorrect assumptions about the value of this key parameter in the sensitivity and risk analyses described in sections 2.3 and 2.4. susceptible individuals (u) also become exposed due to exogenous shocks, such as infections of campus community members by outside individuals or "superspreader" events. the time between exogenous shocks (texo) and number of new exposures from each shock (nexo) are both taken as uncertain input parameters (see table 1 ). exposed individuals (e) become infectious and transition into the asymptomatic and infectious (but still circulating on campus) group (a) at a rate governed by the incubation period, tinc. we take tinc=5 days as a default value [4, 17] , but also consider uncertainty in this parameter in our subsequent analyses. we assume that asymptomatic infectious individuals (a) develop symptoms at rate σ (transition to s), and individuals who are infectious recover at a rate ρ (transition to r), regardless of whether or not they exhibit symptoms. following paltiel et al. [4] , we assume that the percentage of infected individuals that eventually develop symptoms (psymptoms) is 30%. thus, psymptoms=0.3=σ/(σ+ρ). letting trec be the recovery time from the infection, then ρ=1/trec. we take trec=14 days as a default parameter value, which leads to σ=3/98 days -1 as a default. individuals who are symptomatic perish at rate δ, which depends on the recovery rate (ρ) and the symptomatic case fatality ratio, ffatal. we take ffatal=0.05% as a default value. we note that values for this parameter are likely to be higher for certain subpopulations of the campus population (for example, groups designated as high-risk by the united states centers for disease control and prevention (cdc) [18, 19] . we assume that recovered individuals (r) are no longer susceptible to infection. as our model simulations are for a single semester (about 14 weeks), this is in line with the current thinking on the length of protection offered by sars-cov-2 antibodies [20, 21] . traditional individual surveillance testing is carried out with period ts so that the entire circulating campus population is screening once every ts days. note that the ts parameter represents how long it would take to test the entire campus population, but this testing is divided up so that some portion occurs in each model time step. the screening test sensitivity (se, true positive rate) and specificity (sp, true negative rate) are taken as uncertain input parameters. we take se=70% and sp=98% (false negative and false positive rates of 30% and 2%, respectively) as default values, together with the prior distributions described below. following the original work of paltiel et al. [4] , we assume that false positives are detected via a confirmatory test afterward with 100% specificity. the time to return a false positive to the susceptible circulating group is an input parameter that we take to be 1 day as a default. also following paltiel et al. [4] , testing of exposed, but not infectious or symptomatic, individuals (e) is assumed to always lead to a negative screening result. this assumption is made in light of the practical difficulty involved in tracking how long each member of the exposed group has spent in that model compartment, as the proportions of false positives and false negatives are affected by the prevalence of viral rna in the affected individuals. additionally, as screening tests are carried out, some of the exposed individuals will transition into the asymptomatic (infectious) compartment, where they will potentially be screened as true positives. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint in the original formulation of paltiel et al. [4] , it is assumed that individuals who develop symptoms or receive a positive screening test result immediately are moved into isolation/quarantine. we have added a parameter to represent noncompliance with isolation/quarantine procedures. this parameter, fnc, is a fraction between 0 and 1 that represents the proportion of the asymptomatic and circulating population that does not go into quarantine even after either developing symptoms or testing positive for sars-cov-2. fnc can also represent the event that an individual develops symptoms but those symptoms are so mild that they do not realize they are infected. we take this parameter to be equal to 0 by default. however, in light of recent news of student noncompliance with quarantine/isolation procedures [22] [23] [24] , it is important to evaluate the impacts of student noncompliance on the efficacy of campus strategies to manage the spread of sars-cov-2. we note that fnc only represents noncompliance with quarantine/isolation rules, and does not represent noncompliance with limitations on large gatherings or mask-wearing, for example. restrictions on gathering sizes and mask mandates are represented in the effective reproductive rate, rt. we begin with a brief overview of the wastewater module that we have added to the seir model of paltiel et al. [4] . then, in section 2.2.2, we provide specific details regarding the implementation in the model structure. wastewater from a handful of different sewer sites around campus is tested for signs of sars-cov-2 every few days. in the real world, if there are signs that the virus is present in one . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint or more of these wastewater sampling locations, then the relevant wastewater sample is considered to be "positive" and screening tests will be administered to the subpopulations on campus that would have contributed to those wastewater samples. the limit of detection will vary between campuses and the specific wastewater collection and testing systems employed [25] . in our model, a positive wastewater sample is triggered when the number of asymptomatic or exposed (but not yet infectious) individuals who contribute to the wastewater samples exceeds a threshold parameter. we assume that a set of wastewater samples is drawn from sewer systems around campus and sent to a laboratory for testing every at regular intervals, tww. we assume that wastewater sample results return from the laboratory every tww days as well. thus, at time t=n⨉tww, the wastewater results from time t=(n-1)⨉tww return from the laboratory, where n is a positive integer. as a default parameter value, we take tww=3 days. we assume that a proportion, fww, of the overall population is contributing to wastewater samples, and that the population is well-mixed. this subpopulation consists of students who live on campus, and students, faculty, and staff who work or study on campus. a positive wastewater sample result is returned in the model if the number of asymptomatic and exposed individuals contributing to the wastewater samples at the time that the wastewater sample is drawn exceeds a threshold parameter, w (i.e., wastewater sample is found to indicate the presence of viral rna). based on results from university of arizona [26] and preliminary analyses at our home institution, we take w=2 as a default value. we include uncertainty in w in the sensitivity and risk analyses described in sections 2.3 and 2.4. for every college campus that is implementing a wastewater surveillance system, the building(s) that each sewage sample represents will be different. thus, if there is evidence of viral rna found in the wastewater sample from a particular sewer draw, it is unknown exactly how many individuals contributed to that particular sample. in light of this uncertainty, we use a parameter, nbuilding, as a representative number of individuals that would contribute to an arbitrary wastewater sample. this can represent a single large residence hall or a collection of several smaller buildings. uncertainty in nbuilding also arises from the fact that different sewers from which a campus might sample will represent (collections of) buildings of different capacities, and multiple sewers can yield positive results simultaneously. we use a default value of nbuilding=750 persons. 750 is toward the upper end of the range of building capacities for our home institution. if a positive wastewater signal leads to the testing of, on average, fewer than nbuilding individuals, then choosing higher values for nbuilding reflects the belief that sometimes multiple sewer systems will give positive wastewater results simultaneously. we assume that the wastewater-triggered individual screening tests begin some time, tlag, after receiving the positive wastewater result. by default, we take tlag=1 day. there are practical reasons to potentially delay initiating screening tests after a positive wastewater result. for example, a large portion of the exposed individuals who are not yet infectious might go undetected if screening tests are administered before they have transitioned into the asymptomatic (and infectious) model compartment. each time that a wastewater sample is returned from testing (i.e., at intervals of tww), the model checks if the wastewater result is positive. if a positive wastewater result occurs and screening tests begin, then we assume that all fww⨉(e(t)+a(t)) exposed and asymptomatic individuals contributing to wastewater sampling reside within the same representative building (or set of buildings) of size nbuilding. it is important to note that this might represent a collection of . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint actual buildings; hence, our choice to use a value for nbuilding that is toward the upper end of the set of values for our campus. here, t represents the current day in the simulated semester (tww+tlag days since the wastewater sample in question was drawn), e(t) represents the number of exposed individuals at time t, and a(t) represents the number of asymptomatic individuals at time t. we assume that the remaining nbuilding -fww⨉(e(t)+a(t)) individuals are divided between the susceptible (u(t)) and recovered (r(t)) groups. the relative proportions of this remaining building subpopulation that are susceptible and recovered are the same as those proportions in the general campus population. following the original implementation of paltiel et al. [4] , we assume that the exposed group (e(t)) will always yield a true negative result when tested for sars-cov-2, but they can contribute to a positive wastewater signal [9, 25] . the time required to administer tests and receive results for the size nbuilding subpopulation that contributes to the wastewater samples is ts,ww. as a default, we assume that all individuals contributing to a positive wastewater result will be screened within ts,ww=4 days. similarly to our modification of the traditional individual surveillance testing, the false and true positive results from the wastewater-based screenings are modulated by a noncompliant proportion parameter, fnc. after wastewater-triggered individual screening tests remove detected infections from the wastewater-contributing subpopulation, the relative proportions of asymptomatic individuals in the wastewater-contributing subpopulation can be lower than the proportion among the general campus population. our assumption of a well-mixed general population means that asymptomatic cases from outside the wastewater-contributing population will redistribute into the wastewater-contributing population. this may be viewed as the advent of a new outbreak in a different set of buildings than the set that was just administered the screening tests. an agent-based model would offer an opportunity to investigate these dynamics further, but is beyond the scope of the present work. we use the sobol' method for global sensitivity analysis [27] to evaluate the sensitivity of our model to each of its input parameters and their interactions with one another. global sensitivity analyses are preferable to one-at-a-time sensitivity analyses in the sense that a oneat-a-time analysis risks missing important interactions among uncertain parameters. however, global analyses are more computationally expensive due to the larger number of effects to explore. here, our simple seir model is sufficiently inexpensive that running hundreds of thousands of simulations is possible on a time-scale of hours, so a global sensitivity analysis is feasible. the sobol' method examines how the variance in a model output of interest changes when the model input parameters are all varied simultaneously. via our sobol' analysis, we decompose the variance in the total cumulative number of infections over a 100-day semester into portions attributable to each input parameter, and to each parameter interaction. the portion of variance in the model output that is directly attributable to changes to an individual parameter is that parameter's first-order sensitivity index. the second-order sobol' sensitivity indices are the proportions of variance in the model output that are attributable to pairs of model input parameters that are varied together. higher-order indices may be computed as well, but are typically not presented due to challenges of visualization and the large number of possible three-parameter combinations (for our model, there are over 800 such triplets). however, we also compute the total sensitivity index for each parameter, which is the proportion of variance attributable to that parameter and all of its interactions with other parameters, including these higher-order interactions. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. "tri" denotes a triangular distribution, which is defined by its range parameters (a and b) and their mode parameter (c). "bin" denotes a binomial distribution, which is defined by a number of bernoulli trials (n) and the probability of "success" of one of those trials (p). "nbin" denotes a negative binomial distribution, which is defined by a number of bernoulli successes (r) and the probability of success (p). "dunif" and "unif" denote discrete uniform and continuous uniform distributions, respectively. we assign each model input parameter a marginal prior probability distribution (table 1 and supplementary material). we sample from these prior distributions to create two independent ensembles of 10,000 simulations using a latin hypercube sampling approach [28] . the sobol' method computes the parameters' sensitivity indices by constructing new simulations by swapping values for each parameter, and combinations of parameters, from one . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint ensemble to the other. for example, the second-order sensitivity to rt and nexo would be computed by swapping all of the values for rt and nexo from the second ensemble into the first, and observing the change in model output variance from when all parameters were from the first ensemble. to estimate all of the first-order, second-order, and total sensitivity indices, a grand total of 380,000 model simulations are required. we use bootstrap resampling with 1,000 replicates to compute 95% confidence intervals for each sensitivity index. we diagnose convergence when the widest confidence interval has a width that is less than 10% of the highest total sensitivity index (e.g., 29). we report only sensitivities that constitute at least 1% of the total variance in the modeled total cumulative number of infections, and whose 95% confidence interval excludes 0. in the sensitivity analysis, we assume our hypothetical campus pursues a testing strategy that relies primarily on wastewater surveillance, but is complemented with a small amount of traditional individual surveillance testing. campus decision-makers would seek to optimize obvious objectives such as minimizing the total number of infections or minimizing the total number of screening tests needed. public health mandates may also provide additional objectives that decision-makers aim to satisfy. here, we consider the ability of our university to maintain fewer than 100 new infections across any 14-day period. this is the threshold beyond which universities in new york state would need to switch to all-online classes for at least two weeks [30] . we note that there are specific 14-day periods in which infection counts are tabulated, but examine the objective of maintaining fewer than 100 new infections across any such period, as this will ensure that the state requirement is met. additionally, we note that smaller institutions would need to close upon reaching a new infection count equal to 5% of their population, and that local health departments may elect to keep the institution closed for in-person classes longer as the situation demands. for brevity's sake, for a given model simulation, we denote the maximum number of new infections across any 14-day window as imax,14. while a testing strategy (wastewater and/or traditional individual surveillance) might satisfy the imax,14<100 objective under nominal conditions (default parameter values), incorporating uncertainty in the model parameters by sampling from their prior distributions will lead to a corresponding distribution of imax,14 values for the given testing strategy. we define the reliability of maintaining imax,14<100 as the probability pr(imax,14<100). this probability, of course, is conditioned on the testing strategy as well as the underlying parameter prior distributions and other model structural assumptions. with these caveats in mind, we examine which wastewater testing strategies provide the highest reliability of maintaining imax,14<100 infections. guided by our hypothesis that uncertainty in model input parameters will diminish the ability of our hypothetical university to satisfy its objectives for containment of the sars-cov-2 virus on campus, we conduct a monte carlo risk analysis. we consider surveillance testing strategies that involve only wastewater surveillance, as traditional surveillance testing (e.g., nasal swabs and/or saliva tests) has been considered extensively elsewhere in the literature [4, 5] . we consider testing strategies with a time lag to begin wastewater screenings after finding a positive result in the wastewater samples of tlag=1 day. we consider times to complete all wastewater-triggered screening tests, which begin after the 1-day lag, of ts,ww=1, 2, …, 7, or 8 days. preliminary experiments (see supplemental material) suggest that the lag time to initiate wastewater screenings did not substantially (>1%) affect the resulting infection rates. this is . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint because eventually, infections are so prevalent on campus that the wastewater-triggered screenings become a nearly continuous process. we evaluate the sensitivity of these eight testing strategies, corresponding to the eight values of ts,ww above, to our assumptions about three critical parameters: the effective reproductive rate, rt, the number of new infections from exogenous sources each week, nexo, and the fraction of individuals who are not compliant with quarantine/isolation procedures, fnc. specifically, we consider a control scenario in which we assume that rt=1.1, nexo=15 on a weekly time-scale (texo=7 days), and fnc=0.01 (1%). in the risk analysis, we suppose that the hypothetical campus uses only wastewater-triggered individual testing and does not conduct any additional traditional individual testing (e.g., nasal swab or saliva). we let ts,ww vary at its values stated above, we keep the parameters tlag, rt, nexo, texo, and fnc fixed, and we sample all of the other model parameters from their prior distributions (table 1) . we assume that the total campus population is fixed (12,500 individuals), and do not consider uncertainty in the time to return false positive results (1 day). we estimate the reliability of maintaining imax,14<100, pr(imax,14<100), as the proportion of simulations in which the maximum number of new infections across any 14-day window does not exceed 100. for each testing strategy and combination of rt, nexo, and fnc, we generate 3,000 sets of the other parameters from their marginal prior distributions using a latin hypercube sampling approach [28] . we find that at least about 2,000 samples are required in order to stabilize our estimates of pr(imax,14<100), subject to the uncertainties in the other model parameters (see supplemental material). we then consider how "breaking" our assumptions about the parameters rt, nexo, and fnc affects the reliability pr(imax,14<100). we construct seven additional ensembles by increasing rt to 1.5, increasing the number of new exposures for each weekly shock to 30, and increasing the fraction of noncompliant individuals to fnc=0.1, as well as all combinations of these increases. by observing the decrease in pr(imax,14<100), we quantify the fragility of each testing time-scale to broken assumptions about the rate of viral transmission on campus, the influence of infections of campus community members by outside sources, and noncompliance by students. importantly, in the simulations that we present here, there are two total infection counts. first, there is the number of true infections, including asymptomatic cases (a(t)), symptomatic (s(t)), and true positive cases (tp(t)). the number of true infections is generally unknown in real life. by contrast, our model also computes the number of perceived -or detected -infections. the number of perceived infections includes symptomatic cases (s(t)), true positives (tp(t)) and false positives (fp(t)), but misses asymptomatic cases. we thus present two sets of reliabilities: one using the true number of infections and one using the perceived infections only. this distinction, and the fact that in the "model world" the true number of infections is known, enables us to characterize how a lack of timely testing can obscure the true infection count. so, we hypothesize that as the testing time ts,ww increases, the perceived reliability will increase, while the true reliability will decrease. however, we hypothesize that beyond a certain length of time for testing, the perceived reliability will begin to decrease because the number of symptomatic cases becomes overwhelming. before embarking on our monte carlo sensitivity and risk analysis, we first examine individual simulations under the control scenario as described in sec. 2.4. we use the default . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. (fig 2a, solid lines) . the total cumulative numbers of infections under the wastewater and traditional surveillance approaches were 410 and 399 infections, respectively, at the end of the 100-day model simulations. this difference constitutes an increase of 2.8% of the number of true infections under the traditional screening approach. under the control scenario, the projected numbers of deaths for the semester are 0.043 and 0.042 for the wastewater and traditional screening cases, respectively. these differences increase slightly in the moderate (rt=1.5, nexo=20, and fnc=0.05; fig 2c) and severe (rt=2, nexo=30, and fnc=0.1; fig 2e) scenarios. in the moderate scenario, there are 860 total infections (0.082 deaths) projected under the wastewater surveillance program and 828 infections (0.079 deaths) under the traditional surveillance testing program. in the severe scenario, there are 3,825 infections (0.30 deaths) and 3,655 infections (0.29 deaths) using the wastewater and traditional surveillance approaches, respectively. the numbers of true infections when using wastewater surveillance constitute increases of 3.9% and 4.7% over the 1-week traditional surveillance plan in the moderate and severe risk cases, respectively. while the numbers of true infections are similar, the numbers of perceived infections are quite different. under the control scenario, our model predicts that using wastewater surveillance leads to a dramatic underestimation of the number of true infections: at the end of the semester, there are about 45 perceived infections and 66 true infections. this is the result of wastewater surveillance testing a sicker subpopulation resulting in many fewer false positive individuals than traditional surveillance testing (fig 2b) . traditional surveillance overestimates the number of true infections under the control scenario (fig 2a, green lines) , but this overestimation is the result of a larger portion of false positive individuals and not from detecting more asymptomatic cases (fig 2b) . the sizable number of false positive cases ensnared by the traditional individual surveillance testing strategy can be understood by a quick heuristic calculation: out of a campus population of 12,500 individuals, 12,500/7≅1,786 of them are administered a test on the first day of the simulated semester. with a nominal false positive rate of 2% (1-specificity), we expect to see about 1,786⨉0.02≅36 false positive cases. this roughly matches the false positive census by day 20 seen in fig 2b. however, using traditional surveillance in the moderate scenario initially overestimates the number of true infections, then at day 37, the number of true infections becomes larger than the number of perceived infections (fig 2c, green lines) , which thus underestimate the number of true infections after day 37. for the severe risk scenario, this switch occurs after only 15 days (fig 2e) . one of the reasons a campus might pursue a traditional surveillance testing approach is to err on the side of a type 1 error (false positive), when the potential drawbacks to missing positive covid-19 cases are high (e.g., an outbreak). these results suggest that the utility of this traditional approach relies on the testing strategy being sufficiently aggressive as to match the local viral prevalence and spread. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint day . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. the model prediction that wastewater surveillance does not result in a high portion of false positives is not surprising. the structure of our model assumes only a certain fraction of the campus population contributes to the wastewater samples (fww=0.55 is the nominal parameter value). indeed, not all members of the campus community will be equally represented in wastewater samples. wastewater surveillance enables the model to identify a subpopulation of sicker individuals and focus screening tests on this subpopulation. of course, this also means that many individuals will not be within the group that is administered wastewater-triggered screening tests. in practice, wastewater sampling should be used in tandem with some traditional individual surveillance testing to address this issue. however, even in the moderate and severe scenarios, the number of overall infections is never more than 5% higher when using wastewater-based surveillance testing as opposed to traditional individual surveillance testing (fig 2c and 2e) . thus, it appears that for the scenarios considered here, the infections that wastewater sampling misses do not lead to large outbreaks of infections. for the set of model input parameters and prior distributions chosen to reflect conditions with low rates of viral transmission and exogenous exposures, we find that nine parameters, out of 18 total, contribute significantly to variance in the overall number of infections (fig 3) . the criteria for "significance" is for the sensitivity index to be at least 1% of the total variance in estimated cumulative infections, and for the 95% confidence interval to exclude 0. the nine sensitive parameters, in order of their first-order sensitivity indices, are: the effective reproductive rate (rt, 35%), the number of new exposures per exogenous shock (nexo, 12%), the frequency of exogenous shocks (texo, 6%), the fraction of the population contributing to wastewater samples (fww, 4%), the amount of time between wastewater sample results return (tww, 4%), the amount of time for an infected individual to recover (trec, 4%), the fraction of infected individuals who exhibit symptoms (psymptoms, 3%), the amount of time for an exposed individual to become infectious (tinc, 3%), and the amount of time required to complete screening tests after a positive wastewater result (ts,ww, 2%). we find a total sensitivity to rt and its interactions with other parameters of 63%. this highlights the importance of practical measures to reduce rt, including reduced classroom and residence hall capacities, limits on large gatherings, and mask mandates, for example. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint the fact that two out of the top three parameters to which the total infection count is most sensitive are related to infections of campus community members through interactions with external individuals (e.g., social events and interactions off-campus) highlights the importance of campus and municipal public health measures to limit the spread of the virus. this result also illustrates the practical impacts of members of the campus community making (or not making) safe and responsible choices. we emphasize that these results employ prior distributions for the model input parameters that have been chosen to reflect the conditions and characteristics of our home institution in upstate new york. we include analogous sensitivity analyses for hypothetical scenarios at a larger university situated within a community with higher . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint rates of transmission and a smaller college with a much lower student population and less active interactions with its surrounding community (see supplemental material). we now turn to the question: how well can different wastewater surveillance strategies be expected to help maintain low rates of infection on campus? in light of the model's sensitivities to rt and nexo, we examine the fragility of different wastewater surveillance strategies using hypothetical scenarios in which the values for those parameters, and the noncompliance parameter, fnc, are more severe than the default values from table 1 . we use a set of eight ensembles to examine the reliability of maintaining fewer than 100 infections during any 14-day period (imax,14<100) under different policies for wastewater-triggered surveillance testing (fig 4) . all scenarios use a lag time to initiate screenings of tlag=1 day. rt, nexo, and fnc are held fixed at either their nominal values (rt=1.1, nexo=15 new infections per week, and fnc=0.01 (1%) noncompliance) or their high-risk values (moderate/severe cases: rt=1.5, nexo=30, and fnc=0.1). we assume that no additional traditional individual screening tests beyond those triggered by wastewater sampling are performed, and all other model parameters are sampled from their prior distributions. under nominal conditions, the reliability is about 89% when screenings are completed within 1 day (ts,ww =1), excluding the 1-day lag time to initiate the screenings (fig 4a) . as the time to complete the screening tests increases, the perceived reliability increases to 100% ( fig 4a, gray shading) while the actual reliability decreases to about 65% with a screening time of 8 days (orange shading). that this discrepancy between the perceived and actual reliability as screening time increases can be seen in all of the higher-risk scenarios as well (fig 4b-h) . this demonstrates how failing to react in a timely fashion to signs of viral rna can lead to the perception of safety, while in reality the infection count is growing. additionally, even though both the control scenario and the high-noncompliance scenario yield perceived reliabilities of 100% for screening times longer than 3 days (fig 4a, c) , higher rates of noncompliance lead to about 20-30 additional infections beyond the control scenario (see supplemental material). when rt or nexo are increased to their high-risk values, the actual reliability of maintaining imax,14<100 is never greater than 50% (fig 4b, d-h) ; the perceived reliability can be up to 76%, with a screening time of 5 days. this reveals the fragility of a surveillance testing strategy that relies on wastewater sampling. scenarios in which both rt=1.5 and nexo=30 lead to outcomes in which even the perceived reliabilities do not exceed 20% (fig 4f, h) . for these scenarios, the actual reliability is 0% if the time to receive screening results is longer than 2 days. this highlights the compound hazard associated with multiple drivers of risk on college campuses. holiday parties, reuniting with friends after winter break, spending more time indoors during winter, and (at some universities) spring break can all serve to increase both rt and nexo. this means that these high-risk scenarios must be considered as possible, if not probable, realworld cases as colleges plan for their spring semesters. . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint we find that across all scenarios, completing the wastewater-triggered screening tests within 1 day of initiating the tests maximizes the actual reliability of maintaining imax,14<100. however, in addition to improving student health conditions by maximizing the actual reliability for imax,14<100, campus decision-makers in our hypothetical situation would want to also maximize the perceived reliability (based on the perceived number of infections). we emphasize that in practice, the perceived number of infections is the only information known to decisionmakers; the actual number of infections is information only known to us here in our model experimental setting. in all scenarios except those with higher rt combined with higher nexo, the perceived reliability is maximized by using a screening time of 4-5 days (fig 4a-e, g) . in the two scenarios with both higher rt and higher nexo, the perceived reliability is maximized by using a screening time of 2 days (fig 4f, h) . a time to receive screening results of 2 days appears to be a suitable compromise between (i) guarding against high-risk scenarios (i.e., higher rt and nexo simultaneously) and (ii) balancing minimizing true infections against the practical concern of minimizing the number of perceived (detected) infections. we have tuned a seir compartment model to represent scenarios of wastewater screening for sars-cov-2 at a hypothetical university of 12,500 students during a time period in which the area is experiencing relatively low viral presence and transmission, as well as low exposure of campus community members from the surrounding community. by developing and implementing a model component for wastewater surveillance, we find that wastewater surveillance testing can be an effective strategy to maintain a similar number of overall infections on campus to traditional individual surveillance testing (fig 2) . this result is conditioned on the assumed values and prior distributions for our parameters, which have been selected to represent situations that might plausibly face our home institution, and is conditioned on the campus health decision-makers responding to signs of infection in a timely manner. the discrepancy between the true infection count and the number of infections that campus health officials would know about (the perceived number of infections) is striking (figs 2 and 4) . guarding against the worst-case scenarios that include higher reproductive rate, rt, simultaneous with higher exogenous infections, nexo, suggests that a time to screening results of ts,ww=2 days (plus a 1-day lag to initiate the screenings) is optimal for maximizing the perceived reliability of maintaining imax,14<100 (fig 4f, h) . however, in the other higher-risk scenarios, the perceived reliability is maximized by taking ts,ww=4 or 5 days (fig 4a-e, g) . across all scenarios, the actual reliability is maximized by taking ts,ww=1 day, the fastest time-toresults considered here. thus, we suggest that a time of ts,ww=2 days to complete wastewatertriggered screening tests balances decision-makers' desire to avoid a strategy that is fragile against higher-than-nominal risk factors, to minimize the overall number of infections, and the practical importance of maintaining fewer than 100 perceived infections across any 14-day period (fig 4) . exogenous infections and rt are the parameters with the highest direct influence on overall number of infections (fig 3) . these results reflect the importance of taking practical steps to mitigate viral spread on campus and within the local community. additionally, the fact that rt and the frequency and rate of exogenous infections have a stronger influence on overall infection count than the characteristics of the surveillance testing protocol (wastewater or traditional) is likely attributable to the relatively low prevalence and spread of virus in our main test case. in a supplementary experiment, we change the parameters' prior distributions to represent the case of a large university (20,000 individuals) in an area with higher rates of . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint transmission and exogenous shocks and a small college (3,000 individuals) in an area with low transmission and exogenous shocks (see supplementary material). in the large university case, we find that the characteristics of the surveillance testing protocol become more important than the frequency and rate of exogenous infections. the results for the small college case more closely resemble our results for the hypothetical campus of sec 3.2, with the exception that exogenous infections contribute a relatively smaller portion to the variability in overall numbers of infections. while our sensitivity analysis and preliminary risk analysis (see supplementary material) may appear to indicate that the lag time to initiate screening tests based on a positive wastewater return is inconsequential for improving the reliability, the total number of infections suffers with greater lag times. furthermore, in many simulations, the prevalence of the virus is high enough that once wastewater-based screening tests are initiated, they continue until the end of the semester (fig 1) . thus, initiating screening tests within one day, in response to a positive wastewater signal, appears to be the best option to mitigate higher numbers of subsequent infections. we find that wastewater sampling leads to 410 infections over the course of the entire 100-day semester in the control (low) forcing scenario, in which rt=1.1, nexo=15 new exogenous exposures each week, and fnc=0.01. in the control scenario, the traditional individual surveillance testing approach leads to 399 total infections. thus, the 11 additional infections under the wastewater surveillance plan represents an increase of about 2.8%. this difference is larger in the moderate (3.9%) and severe (4.7%) forcing scenarios, but remains within 5%. in practice, deploying a blend of wastewater and traditional individual screening approaches would mitigate the effects of this potential issue. given these general similarities in outcomes, our results suggest that, under nominal or moderate risk conditions, wastewater surveillance is a viable and noninvasive option to monitor the campus population for signs of infection. our risk analysis results (sec 3.3) suggest that perceived reliability -which is the only reliability that will be known in practice -will generally be higher than the actual reliability of maintaining imax,14<100. this is driven by the result that wastewater sampling is targeted to a portion of asymptomatic cases (fig 2) . thus, in practice, we expect that a wastewater sampling plan may appear to keep the overall infection count low, but asymptomatic cases will go unnoticed. by contrast, traditional individual screening tests appear to overestimate the actual number of infections for low-or moderate-risk scenarios (fig 2a, c) . these results have profound practical implications. this result implies that universities that exceed the new york state 100-infection limit and were using a traditional individual surveillance testing approach may not have actually exceeded the 100-infection threshold due to a prevalence of false positive results. on the other hand, universities that employ a wastewater surveillance approach may exceed the 100-infection threshold even if they have an apparent 14-day running total infection count below 100 infections. consistent with the results of paltiel et al. [4] , through our global sensitivity analysis, we find that the sensitivity and specificity of the screening tests do not play a central role in contributing to the uncertainty in the overall number of infections. this is, of course, conditioned on the prior distributions that we have chosen for the sensitivity and specificity parameters, which exclude sensitivities less than 70% and specificities less than 95%. as new testing methods become available, it may be necessary to update these prior distributions. uncertainties in the testing sensitivity and specificity are dwarfed by the uncertainties in on-theground conditions and campus health response, including the effective reproductive rate (rt) and the frequency and rate of infections of campus community members from off-campus . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint interactions (fig 3) . additionally, we have chosen the prior distributions for all of the model parameters in order to best represent the conditions facing our home institution in new york state. the choices for the specific forms and parameterizations of these distributions involves many subjective choices, which offer opportunities for further exploration. for example, the times required to carry out surveillance tests (ts and ts,ww) could be reparameterized as rate parameters and assigned gamma distributions as their conjugate priors. here, we retain the time lengths as parameters to reflect the fact that these time-scales are the decision levers that would be directly manipulated by campus decision-makers by prescribing, for example, weekly surveillance testing. there are, of course, practical steps that a university may take to enhance the efficacy of a wastewater screening protocol beyond what we have illustrated here in our stylized model university. for example, after a wastewater sample result shows signs of infection, our model assumes that all asymptomatic and exposed individuals who triggered the positive result are within an identifiable group of size nbuilding, but this group still interacts with other members of the campus community as wastewater-triggered screening tests are administered. spread of virus may be further mitigated by quarantining the affected building(s). additionally, our risk analysis focuses on examining how wastewater testing alone can be an effective tool to keep overall infection numbers low. our results from sec 3.1 indicate that the reliability of maintaining imax,14<100 infections can likely be improved by complementing wastewater screening with traditional individual surveillance testing. as with any model, the coarse nature of our model and the original model of paltiel et al. [4] means that the results should not be taken as a prediction of specific future behavior or infection rates. rather, these models serve as a tool to evaluate the efficacy of various policy decisions to mitigate the spread of covid-19 on college campuses, and the impacts of uncertainties on infection rates. our model for wastewater surveillance assumes that some constant fraction, fww, of individuals in the exposed and asymptomatic compartments can contribute to a positive wastewater signal. however, this is an approximation of the reality that the levels of viral shedding in wastewater contributions varies with time during an infection. additionally, time-variability in the reproductive rate, rt, may become important as the northern hemisphere enters winter and, in cold and rainy areas, people will spend more time indoors. specifically, previous work has estimated a winter rt of about 2.2, in contrast to a summer rt of 1.3 [31] . our risk analysis indicates that such an increase of rt would dramatically diminish the ability of universities to maintain low infection rates (fig 4b) . taken together, these findings underscore the importance of mask and physical distancing mandates, moratoriums on large gatherings, reduced classroom capacities, and striking a careful balance between in-person and online learning modalities, which can reduce the number of students and faculty circulating on campus at any given time. this balance will of course be different for different campuses, and should be driven by science and evidence that is specific to the unique conditions faced by each university. decision-makers must carefully consider how the distribution of class modalities affects the numbers of contacts among students each day and how, in turn, this affects transmission rates on college campuses. we have presented a model to represent wastewater screening and incorporated it into an existing seir compartment model [4] to track the spread of an infection within a college campus community. we find that wastewater surveillance is an effective approach to detect and remove infected individuals from circulating among the campus community. under nominal or moderate-risk conditions, the model predicts that wastewater surveillance can maintain an . cc-by-nc 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 11, 2020. . https://doi.org/10.1101/2020.10.09.20210245 doi: medrxiv preprint overall number of infections similar to the performance of weekly traditional surveillance testing, while also dramatically reducing the number of false positive cases (fig 2) . a period of one day to receive a positive wastewater result, interpret it, and initiate a plan for screening tests, combined with a period of 2 days to complete the screening tests, appears to form the most robust strategy to maintain low infection rates. however, complementing a wastewater surveillance program with conventional surveillance testing via nasal swabs would offer improvements. these results have practical importance for developing strategies to mitigate the spread of covid-19 on college campuses. markedly heterogeneous covid-19 testing plans among us colleges and universities testing, screening, and outbreak 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with certain medical conditions cdc updates, expands list of people at risk of severe covid-19 illness seasonal coronavirus protective immunity is short-lasting northern illinois university. changes to undergraduate courses and in-person activities police break up fsu off-campus party with over 1,000 people wastewater surveillance for population-wide covid-19: the present and future wastewater testing at uarizona stops coronavirus spread; garners national attention global sensitivity indices for nonlinear mathematical models and their monte carlo estimates comparison of three methods for selecting values of input variables in the analysis of output from a computer code deep uncertainty surrounding coastal flood risk projections: a case study for new orleans governor cuomo issues guidance for infection rates on college campuses following reports of students at large gatherings kissler et al 2020 projecting the transmission dynamics through the postpandemic period we thank enid cardinal, wendy gelbard and lindsay phillips for informative conversations about the surveillance testing plans for the rochester institute of technology. we thank a. david paltiel for assistance interpreting the original seir model. we thank srikanth aravamuthan, sean kent, steve goldstein, and brian yandell for sharing the source code for the original seir model and providing the seir model dashboard (https://dataviz.it.wisc.edu/covid-19-screening/). any views expressed in this work are solely those of the authors and do not necessarily reflect the views of our home institution. all model codes, analysis codes, input files, and output data sets are freely available at https://github.com/tonyewong/seir-ww under the mit open-source license. gt and tw conceived the study. gt, dr and nb wrote the original exploratory model for the rit test case. tw wrote the modified model for wastewater sampling, ran the simulations, and wrote the first draft of the manuscript. all authors analyzed the model output, developed the figures, and contributed to the final versions of the model and the manuscript. key: cord-325172-a8ntxnmm authors: yip, ming shum; leung, nancy hiu lan; cheung, chung yan; li, ping hung; lee, horace hok yeung; daëron, marc; peiris, joseph sriyal malik; bruzzone, roberto; jaume, martial title: antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus date: 2014-05-06 journal: virol j doi: 10.1186/1743-422x-11-82 sha: doc_id: 325172 cord_uid: a8ntxnmm background: public health risks associated to infection by human coronaviruses remain considerable and vaccination is a key option for preventing the resurgence of severe acute respiratory syndrome coronavirus (sars-cov). we have previously reported that antibodies elicited by a sars-cov vaccine candidate based on recombinant, full-length sars-cov spike-protein trimers, trigger infection of immune cell lines. these observations prompted us to investigate the molecular mechanisms and responses to antibody-mediated infection in human macrophages. methods: we have used primary human immune cells to evaluate their susceptibility to infection by sars-cov in the presence of anti-spike antibodies. fluorescence microscopy and real-time quantitative reverse transcriptase polymerase chain reaction (rt-pcr) were utilized to assess occurrence and consequences of infection. to gain insight into the underlying molecular mechanism, we performed mutational analysis with a series of truncated and chimeric constructs of fragment crystallizable γ receptors (fcγr), which bind antibody-coated pathogens. results: we show here that anti-spike immune serum increased infection of human monocyte-derived macrophages by replication-competent sars-cov as well as spike-pseudotyped lentiviral particles (sars-covpp). macrophages infected with sars-cov, however, did not support productive replication of the virus. purified anti-viral iggs, but not other soluble factor(s) from heat-inactivated mouse immune serum, were sufficient to enhance infection. antibody-mediated infection was dependent on signaling-competent members of the human fcγrii family, which were shown to confer susceptibility to otherwise naïve st486 cells, as binding of immune complexes to cell surface fcγrii was necessary but not sufficient to trigger antibody-dependent enhancement (ade) of infection. furthermore, only fcγrii with intact cytoplasmic signaling domains were competent to sustain ade of sars-covpp infection, thus providing additional information on the role of downstream signaling by fcγrii. conclusions: these results demonstrate that human macrophages can be infected by sars-cov as a result of igg-mediated ade and indicate that this infection route requires signaling pathways activated downstream of binding to fcγrii receptors. the continuous threat of respiratory viruses to public health was exemplified by the global impact of the sars-cov outbreak in 2003 [1] , by the occurrence since 2003 of confirmed human cases of h5n1 avian influenza in many countries, particularly across asia [2] , and the 2009 h1n1 influenza pandemic [3] . the recent emergence in the arab peninsula of a novel coronavirus responsible for the middle east respiratory syndrome (mers-cov), [4, 5] and the new h7n9 strain of avian influenza that has jumped into humans [6, 7] in china underscore the need to continue work in this direction. it is now agreed that sars-cov can infect not only the respiratory tract, but can also affect other organ systems and several reports have demonstrated infection of hematopoietic cells [8] [9] [10] ; however, the mechanism by which sars-cov enters into immune cells, which do not express the sars-cov receptor angiotensin-converting enzyme 2 (ace2) [11, 12] has remained poorly understood. both c-type lectin receptors such as liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (l-sign) or dendritic cell specific intercellular adhesion molecule 3-grabbing non-integrin (dc-sign) [13, 14] , as well as antibody-mediated infection may provide sars-cov with an opportunity to modify its tropism. because of the lack of effective antiviral strategies to control coronaviruses infections, vaccination is still regarded as a major option for preventing resurgence of sars and related diseases. we previously showed that a sars-cov vaccine candidate based on recombinant, full-length sars-cov spike-protein trimers triggered infection of human b cell lines despite eliciting in vivo a neutralizing and protective immune response in rodents [15] . more recently, we demonstrated that anti-spike antibody potentiates infection of both monocytic and lymphoid immune cell lines, not only by sars-covpp but also by replication-competent sars-coronavirus [16] , thus providing evidence for a novel and versatile mechanism by which sars-cov can enter into target cells that do not express the conventional ace2 virus receptor and are otherwise refractory to the virus. such infection pathway may have implications for understanding the tropism and pathogenesis of the virus and, therefore, we further investigated the molecular and cellular mechanisms underlying ade of sars-cov infection. by monitoring the susceptibility of human bulk primary immune cells (i.e. peripheral blood mononuclear cells) we have established the occurrence of ade of sars-covpp infection in different circulating immune cell types, among which the monocytic lineage (cd68 + cells) was the primary target. in addition to monocytes, human macrophages were also infected by sars-cov in presence of anti-spike antibodies only. ade-mediated infection of macrophages, however, did not support productive replication of the virus. finally, we have provided evidence that the intracellular signaling motifbut not the igg binding motifof the fcγr is the key molecular determinant for triggering ade of sars-covpp. our findings conclusively demonstrate that anti-spike serum promotes internalization of sars-cov by human macrophages. primary human macrophages are susceptible to sars-cov infection through antibody-mediated pathway because our previous observations revealed that a human monocytic cell line thp-1 was susceptible to ade of infection [16] , we investigated the occurrence of ade of infection in primary human macrophages in vitro, firstly by taking advantage of sars-covpp that can be safely used to mimic the viral entry process [15, 17] . we found that sars-covpp opsonized with a 1:1000 dilution of anti-spike serum readily infected over 80% of primary human macrophages, as determined by immunofluorescence staining of firefly luciferase at 72 hours post infection (h.p.i.). by contrast, cells exposed to sars-covpp opsonized with control serum did not show any positive staining of the luciferase reporter protein (figure 1 ). these experiments extend our previous observations and indicate that anti-spike antibodies facilitate infection of sars-covpp into human macrophages. we next tested whether this altered tropism was also displayed by replicationcompetent sars-cov. human macrophages were infected at a multiplicity of infection (moi) of 1 with the same dilutions of anti-spike or control immune serum and the infection pattern was examined by both immunofluorescence staining of sars-cov nucleocapsid protein and real-time quantitative pcr measurement of viral rnas. positive immunofluorescence signals were detected only at 6 h.p.i. when sars-cov was opsonized with both control and anti-spike serum ( figure 2 ). interestingly, whereas presence of control serum led to only modest infection by sars-cov (~5% of cells), a 4-fold increase in the percentage of positive cells was noted in the presence of anti-spike serum for two out of three donors ( figure 2 ). such enhanced infection pattern was paralleled by the number of viral gene copies measured ( figure 3 ). thus, compared to inoculums containing control serum, there was a 2 to 3-fold increase in the detection of both positive and negative strands of viral genes in macrophages infected in presence of anti-spike serum, which was more pronounced at 1 and 6 h.p.i. there was a rapid decline in viral rna from 6 to 24 h.p.i. for both conditions and no further changes were detected at later time points ( figure 3 ). however, it should be noted that the primer set used for the nucleocapsid gene could also detect the negative strand of the viral genomic rna and could not distinguish it from sub-genomic material. in addition, we measured by real-time quantitative pcr copies of both viral nucleocapsid and orf1b genes in culture supernatants to determine the release of sars-cov particles from the infected macrophages. copy numbers of both viral rnas, however, remained unchanged at all the selected time points during the course of the experiment (less than 200 copies/μl), regardless of whether macrophages had been infected in the presence of either control or anti-spike serum (data not shown). we have previously demonstrated that mouse anti-spike serum could trigger infection of raji cells [16] . in the context of ade, antibodies against viral proteins are considered as the major players of enhancement [18, 19] ; see for review [20, 21] . to eliminate the possibility of the participation of other soluble factors during ade of sars, in this section we investigated the capability of anti-spike antibodies alone in enhancing infection of immune cells. we purified igg by protein g-sepharose chromatography from mouse anti-spike and control serum, and used 2fold serial dilutions from 10 to 0.125 μg/ml of the purified portion to form immune complex with sars-covpp and then infected raji cells. our results show that purified igg from mouse anti-spike serum triggered infection in raji cells, which was more pronounced with increasing immunoglobulin concentrations ( figure 4 ). the flow-through (ft) from the same serum, which was diluted by the appropriate factor to be comparable to the final igg concentration used, elicited no detectable infection at all concentrations (data not shown). significant differences between ade of infection with purified mouse anti-spike igg and flow-through were observed at concentrations 10 and 2.5 μg/ml, and marginal difference was observed at 0.625 μg/ml. as expected, neither purified igg ( figure 4 ) nor flow-through from mouse control serum triggered significant ade of infection at all concentrations. our recent work had revealed the predominant role of human fcγrii (cd32) in mediating ade of sars-cov [16] . to gain further insight into the underlying molecular mechanism we have investigated the involvement of different domains of fcγrii in mediating ade. to this end we produced a series of truncated constructs that only carried ectodomain and transmembrane domains of fcγrii, and chimeric constructs with the ectodomain of one receptor and transmembrane and endodomain of another ( figure 5a ). we then verified the expression of these constructs on st486 cells by flow cytometry using a specific monoclonal antibody (clone fli8.26) that binds to the second ig-like domain d2γ of fcγrii. of note, the fc binding portion of the fcγrii group is very similar among fcγriia-h131, fcγriia-r131, fcγriib1 [22] and also fcγriib2, as it harbors identical extracellular structures as fcγriib1. our findings indicate that all fcγrii constructs exhibited detectable expression of fcγrii (dark grey) in st486 cells in comparison to isotype control (light grey) ( figure 5b ). we then tested the ability of the various constructs to bind purified anti-spike igg-sars-covpp immune complexes and observed that all st486 transfectants were able to bind immune complexes ( figure 5c ). finally we investigated whether any difference in susceptibility to sars-covpp ade of infection was conferred by different fcγrii constructs. when immune complexes formed by 5 μg/ml of purified mouse anti-spike igg (the concentration at which the highest infection level was observed in raji cells) and sars-covpp were added to fcγrii-expressing st486 cells, all four transfectants expressing wildtype fcγrii forms (cf. fcγriia-h vs. fcγriia-r, and fcγriib1 vs. fcγriib2, corresponding to constructs 1, 5, 9, 10, respectively) were infected ( figure 5d ), with fcγriia-expressing st486 being more prone to infection than fcγriib (cf, constructs 1 and 5 with 9). all the endodomain-truncated constructs (fcγriia-h.δic, fcγriia-r.δic and fcγriib.δic, corresponding to constructs 2, 6, 11 respectively) were not susceptible to ade of infection, indicating that binding of anti-spike igg-sars-covpp immune complexes was not sufficient to mediate entry and that the signaling-competent endodomain was required. however, not all chimeric constructs were able to sustain ade of infection, suggesting that domain swapping may have partially interfered with signal transduction. thus only chimeras with fcγriia-h ectodomain and fcγriib1 endomain, or with fcγriib ectodomain and fcγriia endomain, exhibited a statistically significant ade of infection ( figure 5d ). this cannot be explained by differences in surface expression or binding of opsonized pseudoparticles, as fcγriib.ec/ iia.ic (viz., construct 12) showed robust ade despite one of the lowest binding ability of immune-complexes. the possibility that immune response to pathogens may have also deleterious effects on the host homeostasis has been the focus of several studies. for example, the hyper-induction of cytokines following avian influenza infection has been implicated in the severity of the disease [23] and infection of cells by ade has been known to occur for several viral diseases [20, 21] . here we demonstrate that anti-spike antibody potentiates infection although we unambiguously obtained evidence of ongoing infection (e.g., de novo synthesis of the structural viral protein n), ade-infected macrophages did not support productive replication of sars-cov and, after initiation of viral gene transcription and viral protein synthesis, the replication process stalled ultimately ending in an abortive viral cycle without detectable release of progeny virus. abortive replication of sars-cov into macrophages has already been documented [24] but, at variance with this previous report in which 90% of the macrophages were infected by sars-cov in the absence of immune-serum (moi = 1-2), we observed a much lower infection rate (about 5-7%). one possibility is that such discrepancy may be due to difference of time of sampling (6 hours in our study versus 15 hours in cheung's study) and the protocol used for in vitro differentiation (viz., macrophages were differentiated in the presence of fetal calf serum in our study, and autologous plasma was removed two days prior to infection in ref. [8] ), leading to difference in infectivity of the cells observed in the studies. alternatively, we should also consider that the readout of the pseudo-particle experiments was the expression of the luciferase reporter gene, which is under the control of the hiv backbone. this may lead to higher level of protein expression when compared to the abortive replication that follows sars-cov infection of macrophages. thus, the difference may be, in part due to the inability to detect low amounts of spike protein by immunofluorescence and the difference in sensitivity of the two methods. of note, the anti-spike mediated entry is specific for spike-pseudotyped particles, as shown in figure 1 of [16] . because clinical observations have reported poor disease outcomes in early seroconverted sars patients [25] [26] [27] , it would be of interest to test sars patient sera collected at different time-points after sars onset. however, we have been unable thus far to conduct conclusive assays on a well characterized serum library; moreover, we have to be cognizant of the possibility that ade may only occur within a narrow window during the course of an infection and only in a subset of infected patients. the alternative possibility that internalization by macrophages may in fact represent an additional mechanism to control viral spread requires further investigation. in general, studies aiming at better understanding antibody-dependent enhancement of viral infections are focusing on either identifying the (immune) receptor(s) and/or serum component(s) allowing penetration of the pathogen into the target cell, also known as extrinsic ade, or the outcome response(s) of the target cell downstream to ade of infectionor intrinsic ade [20, 21, 28] . our previous results demonstrated that only fcγriia and, to a lesser extent, fcγriib1 triggered infection by sars-covpp in presence of anti-spike serum [16] . although it would have been desirable to perform fcγr blocking experiments also in macrophages, coexpression of all fcγrs in these cells [29, 30] however, the relationship between internalization of immune complexes and ade of infection by sars-cov via fcγriis appears to be a complex process. thus, fcγriib2 has been shown to mediate internalization of immune complexes at a faster rate than fcγriia [33] , whereas we found that ade of infection via fcγriia was more prominent than with fcγriib. recently, involvement of downstream signaling triggered by fcγrs activation has been evaluated with respect to ade of dengue virus infection [34] . thus, abrogation of fcγri and fcγrii signaling competency was associated with significant impairment of phagocytosis, but only the signaling-incompetent fcγri become unable to trigger ade of dengue virus. conversely, no discernible effect on dengue virus immune complex infectivity was observed for both wild type and signaling incompetent fcγriia. these findings point to fundamental differences between fcγria and fcγriia with respect to their immuneenhancing capabilities and suggest that different mechanisms of dengue virus immune complex internalization may operate between these fcγrs [34] . altogether our results demonstrate that, in presence of vaccine-elicited antiviral antibodies, sars-cov displays an altered tropism toward primary human immune cells, which do not express the conventional virus receptor and are otherwise refractory to the virus. a number of sars vaccine candidates have been tested in experimental animal models [35, 36] , many of them based on the viral spike glycoprotein previously identified as the most immunogenic antigen inducing neutralizing and protective antibodies [14, 15, 37, 38] . of note, some vaccines against animal coronaviruses have been also generated, but their development has proven difficult due to immune [39] [40] [41] . despite the fact that this alternative infection pathway appears to have a limited impact, it remains of interest to appreciate the cost of antibody-mediated sars-cov infection on the functionality of the target cells, in order to have a broad understanding of the tropism and pathogenesis of the virus and evaluate potential pitfalls associated with immunization against human coronaviruses. this aspect is gaining more relevance as the emergence of the mers-cov further indicates that the availability of vaccines targeting this group of viruses, which have demonstrated the ability to jump species, is one of the few options available to prevent the spread of infections causing severe diseases with high mortality in humans [42] . the following cell lines used in the study were obtained from the american type culture collection (atcc; manassas, va, usa): hek293t (human kidney epithelial cells; crl-11268), raji (burkitt's lymphoma/b lymphoblast), st486 (burkitt's lymphoma/b lymphoblast lacking expression of fcγr; crl-1647). hek293t cells were cultured in dmem supplemented with 10% heat-inactivated fetal bovine serum (fbs), 0.6 mg/l penicillin and 60 mg/l (hfcγriib2) . b) surface expression in stably transduced st486 cell lines was evaluated by flow cytometry using a mouse monoclonal anti-fcγrii antibodies (dark grey) or isotype control (light grey), and expressed as mean fluorescence intensity in arbitrary units (au) as described under materials and methods. constructs are identified by arabic numbers and grouped from top to bottom as indicated in a. c) immune complex-binding ability of wild-type, truncated and chimeric fcγr receptors. sars-covpp were incubated with purified mouse anti-spike igg (dark grey) or control igg (light grey) to form immune complexes, which were then added to the st486 transfectants (see materials and methods). after washing and fixation, bound immune complexes were detected by flow cytometry with an fitc-conjugated goat anti-mouse f(ab')2, and results shown as in b. constructs are identified by arabic numbers and grouped from top to bottom as indicated in a. d) susceptibility of st486 transfectants to ade of infection by sars-covpp. sars-covpp were incubated with purified mouse anti-spike igg (dark grey) or control igg (light grey) and then used to infect cells (see materials and methods). cells were washed and three days post infection incubation was stopped by adding luciferase substrate to measure enzymatic activity, which was expressed as relative luminescence units (rlu). constructs are identified by arabic numbers and grouped from top to bottom as indicated in a. results are shown as the means ± sem of three (b and c) or 4-5 independent experiments (d). *p < 0.05; **p < 0.01 by the unpaired student's t test. streptomycin, whereas hematopoietic cells were grown in rpmi-1640 supplemented with 10% heat-inactivated fbs, 1% non-essential amino-acids, 4 mm l-glutamine, 1 mm sodium pyruvate, 0.6 mg/l penicillin, 60 mg/l streptomycin, and 20 μm 2-ß-mercaptoethanol (all from invitrogen life technologies, carlsbad, ca, usa). all cells were maintained at 37°c in a humidified atmosphere with 5% co 2 supply. the research protocol was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw09-375). blood samples of healthy donors were obtained from the hong kong red cross blood transfusion service. human blood cells were isolated from buffy coats and monocyte-derived macrophages were generated in vitro using a modified protocol as previously described [24, 43] . briefly, mononuclear cells were isolated by a ficoll-paque density gradient (pharmacia biotech, uppsala, sweden) to remove erythrocytes, granulocytes, and cell debris. monocytes were enriched by plastic adherence, harvested, seeded (10 6 cells/ml) on tissue culture plates and allowed to differentiate for 14 days in the presence of 5% autologous human plasma and 1% fetal calf serum before use. the purity of macrophages was consistently above 90%, as ascertained by immunofluorescence staining for human cd68, a lysosomal glycoprotein that is highly expressed by macrophages and monocytes [44, 45] . balb/c mice were immunized with recombinant sars-cov spike proteins adjuvanted with alum, as previously described [15, 16] . saline-injected mice served as controls. serum was collected at day 55 post-immunization, heatinactivated for 30 min at 56°c and stored at −20°c for subsequent use. the protocol to produce sars-cov pseudotyped lentiviral particles expressing a luciferase reporter gene (sars-covpp) has been described elsewhere [17] . following a purification step on 20% sucrose cushion, the concentrated viral particles were titrated by elisa for lentivirus-associated hiv-1 p24 protein according to the manufacturer's instruction (cell biolabs, inc., san diego, ca, usa), and stored at −80°c until use. for ade assays, heat-inactivated mouse anti-spike or control serum was incubated for 1 hr at 37°c with sars-covpp. this inoculum was deposited on cells and infection proceeded for 1 hr at 37°c. following repeated washing cells were incubated for additional 60-65 hours and then fixed in 4% paraformaldehyde (sigma-aldrich inc., st. louis, mo, usa) for immunofluorescence microscopy. laboratory procedures involving replication-competent viruses were performed in biosafety level-3 containment (state key laboratory of emerging infectious diseases, the university of hong kong). the sars-cov strain used (hk39849) is a clinical isolate [46] , which was cultured in fetal rhesus kidney-4 (frhk-4, atcc code crl-1688) cells. in ade assays, heat-inactivated mouse anti-spike or control serum was incubated for 1 h at 37°c with sars-cov and human monocyte-derived macrophages were infected at an moi of 1 for 60 min at 37°c. after repeated washings, cells were cultured with fresh medium (time 0) supplemented with 2% fbs for the indicated time points (hours) post infection (p.i.) and eventually either fixed in 4% paraformaldehyde (sigma-aldrich inc.) for immunofluorescence microscopy, or resuspended in lysis buffer (rlt buffer, rneasy rna mini kit; qia-gen, germantown, md, usa) for real-time quantitative rt-pcr. samples of cell culture supernatants harvested at different h.p.i. were also mixed with rlt buffer and processed as above for rt-pcr. infectivity of replication-competent sars-cov was determined by indirect immunofluorescence with a mouse monoclonal antibody directed against sars-cov nucleocapsid protein (clone 4d11; a gift from dr. kwok-hung chan, department of clinical microbiology, queen mary hospital, hong kong), at a dilution of 1:200. staining was revealed with a tetramethylrhodamine-5-(and-6)isothiocyanate (tritc) conjugated goat anti-mouse antibody (invitrogen life technologies) on an axioobserver z1 microscope (carl zeiss, inc., thornwood, new york, usa). pictures taken from 5 randomly chosen fields at a magnification of 400x were acquired with an axiocam mrm camera and analyzed with metamorph software (molecular devices, sunnyvale, ca, usa). infectivity was determined as the percentage of cells expressing viral antigen. similar staining procedures were employed for assessing infection by sars-covpp, except for the use of direct immunofluorescence with a fluorescein isothiocyanate (fitc) conjugated goat monoclonal antibody specific for firefly luciferase (rockland, gilbertsville, pa, usa) at a dilution of 1:100. rna was extracted with the rneasy rna mini kit (qiagen), according to the manufacturer's recommendations; concentration and purity of rna were measured by standard optical methods. reverse transcription was performed on total rnas with superscript iii reverse transcriptase as specified by the manufacturer (invitrogen life technologies). for quantification of sars-cov gene copies, cdna was generated in separate reactions in presence of either forward or reverse primers specific for the nucleocapsid and orf1b genes ( table 1 ) that amplified negative and positive strand viral rnas, respectively. this assay allows distinguishing between signals generated by entry of input virus from downstream events reflecting an active replication process [47] . specific primers (table 1) and taqman minor groove binder probes used for detection of sars-cov nucleocapsid gene have been previously described [48] . however, it should be noted that the primer set used for the nucleocapsid gene could also detect the negative strand of the viral genomic rna and could not distinguish it from sub-genomic material. the qpcr assay was carried out in a final volume of 20 μl and the fluorescence signal was detected with a lightcycler 480-ii (roche applied science, mannheim, germany) programmed as follows: 95°c for 10 min, followed by 45 cycles of 95°c for 10 sec, 60°c for 30 sec, and 72°c for 1 sec. results were expressed as the number of target copies per 10 8 copies of the 18s rrna gene, which was used to normalize results. to ensure the consistency of qpcr measurements over the time period of the study, the same batches of primers and probes were employed. the qpcr results were considered valid when the efficiency of the standard curve was between 1.9 to 2.1 and the r 2 value was greater than 0.99. iggs was purified from immunized and control mice using protein g-sepharose (ge healthcare, little chalfont, united kingdom) according to the manufacturer's recommendations. the purity of the igg fraction was checked by silver staining following gel electrophoresis and the concentration of anti-spike igg determined by elisa. construction of lentiviral vectors for wildtype, endodomain-truncated and chimeric forms of fcγrii (hcd32) the strategy to produce plasmids for wild-type human igg receptors (fcγrs) consisted in replacing the enhanced green fluorescent protein (egfp) gene from the bicistronic vector pchmws-egfp_ires_hygromycin (a kind gift from drs. rik gijsbers and zeger debyser, katholieke universiteit leuven, belgium) with the coding sequences for fcγriia (hcd32a) isoforms (fcγriia.r131 and fgriia. h131 genbank accession no. nm_021642) and fcγriib1 (hcd32b; genbank accession no. af543826) flanked by bglii and sali sites, as described elsewhere [16] . the synthetic sequences (geneart, regensburg, germany) were inserted into the original transfer plasmid to yield wildtype constructs. pchmws-fcγriib2_ires_hygromycin was constructed by swapping the intracellular domain of fcγriib2, obtained from rt-pcr amplification of the desired sequence from polyclonal raji cdna, with the corresponding region of the pchmws-fcγriib1_ires_hygromycin construct. wild-type constructs were subsequently used to generate truncated and chimeric forms of fcγriis by standard techniques (see figure 5a for a schematic representation of the mutants tested). all plasmids were sequenced by the centre for genomic sciences of the university of hong kong. generation of stable st486 cell lines expressing wild-type and mutated fcγrs using lentiviral particle-based gene transduction cell lines were generated by transduction of monoclonal st486 cells with vesicular stomatitis virus (vsv) g pseudotyped lentiviral particles bearing the specified transgene as previously described [16] . at 2 days post-infection, cell surface expression of the fcγrs was monitored by flow cytometry and cells were subsequently cultured in selective medium containing 250 μg/ml hygromycin (invitrogen life technologies) when appropriate. finally, several monoclonal cell lines for each construct were isolated and expression of the transgene was confirmed by flow cytometry. expression of fcγriis was evaluated by flow cytometry as described in [16] . the following mouse mabs were table 1 sequences of primers and probes used in real-time qpcr assay and cdna synthesis used: 3g8 anti-hcd16, fli8.26 for hcd32, and 10.1 anti-hcd64 (all from bd pharmingen, frankin lakes, nj, usa); mopc-21 (igg1, κ) or mpc-11 (igg2b, κ) isotype controls (both from biolegend, san diego, ca, usa). cells were washed and primary antibody binding was revealed by staining on ice for 30 min with fitcconjugated goat anti-mouse antibodies (jackson immu-noresearch, west grove, pa, usa). data were collected from at least 10,000 singlet living cells on a lsrii flow cytometer (bd biosciences, san jose, ca, usa) and analyzed using flowjo software (treestar, ashland, or, usa). sars-covpp-igg immune complexes were obtained by incubating sars-covpp with 30 μg/ml of either purified mouse anti-spike or control igg at 37°c for 1 hr. the mixture was then quickly chilled on ice for 10 min and added (100 μl/well) to st486 cells (3×10 5 cells/well), which had been previously stained with 0.1% fixable viability dye efluor 660 (ebioscience, san diego, ca, usa). following a 1 hr incubation on ice, cells were washed twice with cold pbs, fixed with 1% paraformaldehyde for 20 min on ice and immune complex binding was revealed by staining with 5 μg/ml fitc-conjugated goat anti-mouse f(ab') 2 (jackson immunoresearch) at 4°c for 30 min. data were collected and analysed 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transcription-pcr detection of sars coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-pcr assays antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus the hku-pasteur research pole is a member of the institut pasteur international network. we wish to thank lewis siu (hku-pasteur research pole) for sharing with us his stock of sars-cov spike lentiviral particles when it was most needed. we are grateful to kwok hung chan (queen mary hospital, pokfulam, hong kong) for the gift of the mouse monoclonal antibody (4d11) specific for the sars-cov nucleoprotein, and rik gijsbers and zeger debyser (katholieke universiteit leuven, belgium) for providing the bicistronic retroviral vector used to establish the fcγr-expressing stable cell lines. many thanks to suki lee, chris mok and members of the hku-pasteur research pole for their expert advice and critical reading of the manuscript. m.s. yip was a phd and nancy h.l. leung was an mphil student supported in part by the university of hong kong. this work was supported by the research fund for the control of infectious disease (rfcid; project no. 09080872) of the hong kong government and by the respari project of the institut pasteur international network. the authors declare that they have no competing interests. key: cord-346673-kyc1wks5 authors: nickbakhsh, s.; thorburn, f.; von wissmann, b.; mcmenamin, j.; gunson, r. n.; murcia, p. r. title: extensive multiplex pcr diagnostics reveal new insights into the epidemiology of viral respiratory infections date: 2016-03-02 journal: epidemiol infect doi: 10.1017/s0950268816000339 sha: doc_id: 346673 cord_uid: kyc1wks5 viral respiratory infections continue to pose a major global healthcare burden. at the community level, the co-circulation of respiratory viruses is common and yet studies generally focus on single aetiologies. we conducted the first comprehensive epidemiological analysis to encompass all major respiratory viruses in a single population. using extensive multiplex pcr diagnostic data generated by the largest nhs board in scotland, we analysed 44230 patient episodes of respiratory illness that were simultaneously tested for 11 virus groups between 2005 and 2013, spanning the 2009 influenza a pandemic. we measured viral infection prevalence, described co-infections, and identified factors independently associated with viral infection using multivariable logistic regression. our study provides baseline measures and reveals new insights that will direct future research into the epidemiological consequences of virus co-circulation. in particular, our study shows that (i) human coronavirus infections are more common during influenza seasons and in co-infections than previously recognized, (ii) factors associated with co-infection differ from those associated with viral infection overall, (iii) virus prevalence has increased over time especially in infants aged <1 year, and (iv) viral infection risk is greater in the post-2009 pandemic era, likely reflecting a widespread change in the viral population that warrants further investigation. acute respiratory infections are the commonest cause of illness in all ages, and a leading cause of mortality in children aged <5 years, creating a significant global healthcare burden [1] [2] [3] . various aetiological pathogens (viruses, bacteria and some fungi) are recognized, causing largely indistinguishable symptoms. in most settings, viruses are the most frequently detected agent [4, 5] . although most infections are mild, respiratory viruses have the potential to cause severe illness in high-risk groups. although influenza is a major research focus [6] , the advent of polymerase chain reaction (pcr) technology has led to improved awareness that non-influenza viruses are also important contributors to disease burden, and of the role of viral subtype in clinical severity [7] [8] [9] . the use of pcr testing as part of routine diagnostics provides an important resource for monitoring respiratory viruses as part of national surveillance [10] . multiplex pcr methods in particular provide a valuable resource for epidemiological enquiry [11] . all patients requiring microbiological diagnosis are tested for all pathogens included in the panel, ensuring consistency in testing across patients. the collation of multiplex diagnostic data from a large patient population and over an extended time-frame therefore enables robust comparisons of infection trends temporally and across patient subgroups. furthermore, when testing is implemented over multiple years, sufficient data can be accrued to investigate the clinical relevance of co-infections and their epidemiological patterns [12] . although the utility of diagnostic data in the epidemiology of respiratory infections has been demonstrated [11, [13] [14] [15] [16] , studies that cover all major viruses, patient age and illness severity groups, and that span multiple years, are lacking. the largest nhs health board in scotland, greater glasgow and clyde (nhsggc), has used multiplex pcr testing as part of their routine diagnostic services since 2005. this health board serves ∼1·1 million people, representing ∼1·7% of the total uk population [17] . the resultant accumulation of data provides a novel opportunity to investigate viral respiratory infections in a more comprehensive fashion than previously possible. these data also provide a unique opportunity to compare the periods before and after the introduction of the novel pandemic influenza virus [a(h1n1) pdm09] into scotland (see [18] ). we analysed diagnostic data generated by nhsggc using multiplex pcr from 2005 to 2013 with the following objectives: (i) to describe testing and virus prevalence trends, (ii) to examine temporal and patient subgroup distributions for each individual virus, and (iii) to compare factors associated with overall viral infection and co-infection using statistical modelling, in order to provide robust and timely estimates of who is most at risk of viral-associated respiratory illness, and when, within a major urban uk population. in this study we used virological diagnostic data generated by the west of scotland specialist virology centre (wossvc) for nhsggc during 2005-2013 [19] . during this period, a total of 61 427 clinical samples were received from 40 962 patients attending primary and secondary healthcare services for respiratory diagnostic purposes (i.e. excluding pathology-origin samples). most (98%) clinical samples were taken from the upper or lower respiratory tract: primarily nasal and/or throat swabs (67%), gargles (13%), nasopharyngeal aspirates (7%), sputum (5%), bronchoalveolar lavage (3%) and nasopharyngeal/ endotracheal secretions (2%). in a minority of cases (n = 142 samples), plasma was additionally taken for follow-up investigation; most (89%) of these samples related to the 2009 influenza a pandemic period which was excluded from statistical modelling analyses. each sample was tested by real-time rt-pcr for 11 groups of respiratory viruses: human rhinovirus (rv); influenza a virus [iav; a generic assay detecting seasonal h3n2 and h1n1 subtypes and one specific to a(h1n1)pdm09], influenza b virus (ibv), human respiratory syncytial virus (rsv), human coronavirus (cov; aggregating 229e, nl63, hku1 and oc43 species), adenovirus (adv), human metapneumovirus (mpv) and human parainfluenza types 1-4 (piv1-4). details of nucleic acid extraction methods and the real-time pcr assays are provided elsewhere [20] . complete testing coverage across viruses was largely maintained throughout the study period. however, high frequencies of partial testing did arise due to the burden placed on laboratory resources during the major waves of a(h1n1)pdm09 virus circulation. the laboratory protocols were consistent throughout the study period, with the exception of the rv assay which was modified during 2009 to detect a wider array of rv and enteroviruses (including d68), and the cov-hku1 assay which was discontinued in 2012. for each of the 61 427 clinical samples, positive/negative pcr test results were recorded by the laboratory for each virus group. information was also provided on the sampling date, patient's age at sampling, gender, and the origin of the sample [whether the patient had attended a general practice (gp), hospital outpatient or non-critical-care inpatient services, or was admitted to a critical care ward]. in the case of inconclusive/absent test results or other patient information, the corresponding data were coded as missing. all patient identifiers were anonymized. of the 40 962 patients, 8394 had multiple samples submitted for virological testing during the study period (range 1-37 samples, median 1, s.d. = 1·22). for 70% of these patients, the samples were received within a 30-day window. we aggregated the pcr test results to within this time-frame generating single 'episodes' of respiratory illness, using the collection date of the first sample when assigning temporal information. episodes were classified as positive for a given virus if at least one sample tested positive. following data exclusions, 44 230 patient episodes, representing 36 157 individual patients, were retained for analysis of temporal distributions. we conducted descriptive statistical analyses of viral infection prevalence in the patient population providing time-and agestratified estimates. by the end of april 2009, scotland was afflicted by the influenza pandemic [20] . figure 1a highlights the resultant upsurge in testing frequencies during the summer and autumn waves of 2009, and during a third wave of a(h1n1)pdm09 virus circulation in the winter of 2010/2011. during these periods, testing was primarily directed towards iav and only subsets of iav-negative patients were tested for other viruses. due to this disruption in regular testing procedures, we focused our description of viral infection distributions across patient subgroups on the 26 974 patient episodes tested outside this period, and refer readers to a previous report for details of viruses detected during the 2009 pandemic [20] . for each virus group, we compared the frequency of mono-infection episodes (one virus group detected) and co-infection episodes (more than one virus group detected). to correctly classify episodes into these subgroups, we excluded all partially tested patients. in more detailed analyses, we counted the frequency of each possible virus pair and quantified the statistical correlation between mono-infection and co-infection frequencies across viruses. we investigated statistical associations between time period, season, patient age, gender, and gp/general hospital/critical care origin (a proxy for illness severity), and two outcomes: (i) virus-positive vs. virusnegative episodes, and (ii) co-infection vs. monoinfection episodes. with respect to time, we split sampling dates into two major periods either side of the influenza pandemic and periods of high partial testing: pre-pandemic [prior to may 2009 when the a(h1n1)pdm09 virus was established in scotland] and post-pandemic [following subsidence of the third major wave of the a(h1n1)pdm09 virus in january 2011]. associations with each factor were first assessed by crude unadjusted odds ratios, and then adjusted for confounding using multivariable logistic regression models that included all factors to assess their independence. statistical interactions were examined using mantel-haenszel stratification methods (based on p < 0·05, results not shown). the potential interactions were added to the main effects models and their significance assessed based on an interaction parameter p < 0·05. model fit was assessed by le cessie van houwelingen global goodness-of-fit tests [21] . all statistical analyses were carried out in r v. 3·1·1 [22] . to correctly classify patients into outcome groups, all partially tested patients were excluded. of the 36 157 fully tested patients, 90% sought healthcare facilities once during the study period thereby contributing a single episode. however, 4218 patients had attended healthcare facilities more than once, providing information for multiple episodes (range 2-26 episodes, median 2, s.d. = 2·04). we retained the first observed episode per patient in the statistical analyses to ensure the patient-level interpretation of statistical associations was not influenced by the nonindependence of data relating to the same individual. see supplementary figure s1 for full details of data preparation. we analysed 44 230 episodes of respiratory illness tested by wossvc during 2005 to 2013. full details of patient distributions across subgroups and per study year are provided in supplementary table s1. patients' median age was 27 years (range 0-98 years, s.d. = 25·5 years) and 49% were male. excluding the three major waves of influenza a(h1n1)pdm09 virus circulation, episode frequencies increased year-by-year from 2472 cases tested in 2005 to 6149 cases tested in 2013. however, the age patterns were not consistent over this period; the percentage of adult episodes was greater in 2013 than in 2005 (e.g. 21% vs. 8% in patients aged 565 years), while the percentage of child episodes was fewer in 2013 than 2005 (e.g. 16% vs. 26% in patients aged 1-5 years) ( fig. 1b) . at least one virus was detected in 35% (15 302/44 230) of tested patients; these patients had a median age of 17 years (range 0-96 years, s.d. = 25 years) and 49% were male. the prevalence of confirmed viral infection in the patient population was greater in the 2013 influenza season than in 2005 in all age groups (fig. 1c) ; the absolute difference in prevalences was 22% (infants aged <1 year), 12% (1-5 years), 14% (6-16 years), 18% (17-45 years), 12% (46-64 years) and 17% (565 years). overall virus-specific prevalences in the patient population were ranked as follows: rv (14%, n = 4847); iav (9·7%, n = 4244); rsv (4·9%, n = 1786); cov (4·1%, n = 1339); adv (3·6%, n = 1221); ibv (3%, n = 1019); mpv (2·6%, n = 345); piv-3 (2·2%, n = 757); piv-4 (0·86%, n = 286); piv-1 (0·84%, n = 295) and piv-2 (0·35%, n = 122). age distributions for each viral infection group are presented in supplementary table s2 . the most common infection in each 6-month period (excluding 2009) was rv, constituting a low of 19% of infections during the typical influenza period of 2005/2006, to a high of 59% during the typical non-influenza period of 2010 (fig. 1d) . for most virus groups, detections were most frequent in the 1-5 years group (with the exception of iav, ibv and cov), males, and hospital attendees not admitted to a critical care ward (fig. 2) . seasonally, virus detections were most common in december (45% in gp attendees, 43% in hospital attendees) and least common in august (11% in gp attendees, 22% in hospital attendees) (fig. 3a,c) . the most commonly detected viral infection in each month was rv, peaking in september in both gp and hospital attendees (fig. 3b,d) . influenza a and b were the most common detections in december-march in gp attendees (combined proportion: range 31-45%), and in january-february in hospital attendees (combined proportion of 30%). of the remaining non-influenza viral infections, a large proportion was attributed to rsv, rv and cov during periods of high influenza activity; their combined proportions ranged from 39% to 52% in gp attendees (december-march) and from 51% to 55% in hospital attendees (january-february). of 9094 positive patients (in 26 974 patients outside of the pandemic period), 1952 were gp attendees, 6560 were general hospital attendees (outpatients and non-critical-care inpatients), and 1282 were inpatients admitted to a critical care ward [an intensive care unit (icu), intensive therapy unit (itu), high dependency unit (hdu), or coronary care unit (ccu)]. the latter group provided a proxy for classifying episodes of severe respiratory illness. eighty-eight percent (n = 4443) of gp attendees and 69% (n = 15 027) of hospital attendees were aged >5 years. as shown in figure 4 , the prevalence of severe episodes in all viruspositive patients, regardless of origin, was greater in patients with rv (7·5%), rsv (7·5%), piv1 (11·8%) and piv4 (7·4%) infections than in virus-negative patients or other viral infections including iav (5·5%) and ibv (4·1%). investigating further the rv/ iav and rv/piv1 comparisons, we found the observed difference in prevalence was statistically significant based on pearson's χ 2 tests (p = 0·036 and p = 0·05, respectively). age-specific prevalence of severe episodes was greatest at the extremes of age (<5 and 565 years) for all viruses except hpiv2 (we note the particularly small sample size for this virus group). of 9654 virus-positive patients from 27 284 episodes tested for all 11 viruses, 11% (1086/9654) had a co-infection. the median age in co-infected patients was 3 years (range 0-91 years, s.d. = 22 years) and 58% were male. co-infections were more commonly detected in those aged ≤5 years overall (18% compared to 7% in the >5 years group) and for each viral infection, particularly rv, rsv, adv and cov (detected in 6%, 3%, 3% and 2% of these infections, respectively, in those aged >5 years) (fig. 5a,b) . a total of 1389 virus pairs were detected in 1086 episodes of co-infection; most episodes involved two viruses (87%, 964/1086), the remaining involved three (n = 105), four (n = 15) and five (n = 2) viruses. all viruses were detected with most others at least once (fig. 5c) ; however, a clustering pattern was evident in which rv, adv, rsv and cov were frequently detected with one another. the most common virus detection in a co-infection was rv (56% of coinfections), the majority of which were with adv (n = 195, 25%) and rsv (n = 181, 23%). other viruses relatively frequently detected in co-infections were adv, rsv and cov; constituting 31%, 30% and 28% of co-infections, respectively. we found a significant positive correlation between virus detection frequencies in mono-infections and coinfections [pearson's product-moment correlation = 0·88 (95% ci 0·60-0·97, p < 0·001) and fitted linear regression model slope = 0·85 (p < 0·001)] (fig. 5d) . however, iav and ibv were identified in co-infections at relatively low frequencies (n = 121 and n = 68, respectively) compared to non-influenza viruses (e.g. rv, n = 678) (fig. 5d ). table 1 summarizes the results of univariable and multivariable logistic regression analyses for associations with viral infection. season, age group, and patient origin were significantly associated with the odds of viral infection based on unadjusted odds ratio estimates. in the multivariable analysis, several independently significant factors were identified based on the adjusted odds ratios. viral respiratory infections were more likely to be detected in winter, in children aged 1-5 years, and in gp attendees, irrespective of the other factors. following adjustment for multiple factors, time period was also a significant predictor (because of a negative confounding by age): the odds of viral infection were significantly greater postpandemic than pre-pandemic. significant statistical interactions (based on p < 0·05) revealed that the effect of age was not homogeneous across gender or patient-origin subgroups. this variation in age association across other factors is shown in figure 6a ,b where age-specific infection prevalences are stratified by the third factor. these figures show that the age distribution of infection differed according to gender and patient-origin subgroups. table 2 summarizes the results of univariable and multivariable logistic regression analyses for associations with co-infection. several differences were found in comparison with viral infections overall. based on unadjusted odds ratio estimates, time period, season (autumn only), age group, gender and patient origin were significantly associated with co-infection. however, in the multivariable analysis time period and gender were confounded by age and were therefore not identified as significant independent factors. in contrast to viral infection overall, co-infections were equally likely to be detected in spring and winter, were less likely to be detected in the 1-5 years age group than infants, and were more likely to be detected in general hospital attendees (outpatients and those not admitted to critical care wards) than gp attendees. significant statistical interactions (based on p < 0·05) revealed that the effect of age on co-infection status was not homogeneous across gender and patient-origin groups. in contrast to viral infection overall, co-infections were relatively more common in males than females in those aged 46-64 years and hospital attendees in all age groups (fig. 6c-d) . there was no evidence of a poor model fit based on the global goodness-of-fit tests: (i) p values = 0·147, 0·07, 0·07 for the main effect model and two models with interaction terms, respectively, for associations with viral infection overall, and (ii) p values = 0·940, 0·985, 0·746 for the main effect model and two models with interaction terms, respectively, for associations with co-infection. the advent of multiplex pcr as part of routine diagnostics provides an unprecedented opportunity for studying the epidemiology of multiple respiratory viruses simultaneously within a single population. previous uk-based studies have highlighted the utility of laboratory-based surveillance for monitoring respiratory infection trends, and in comparing the relative burdens between viruses [10, 13, 23] . our study is the first to compare the epidemiologies of different respiratory virus groups utilizing extensive diagnostic data generated by multiplex rt-pcr from patients attending both primary and secondary healthcare services. the collation of test-negative results by diagnostic laboratories provides valuable denominator information for measuring disease occurrence, to estimate the relative contribution of different pathogens to healthcare usage (such as gp consultations) and to provide an early warning for periods of increased healthcare pressures. importantly, the diagnostic test data utilized in this study were generated by a single laboratory, permitting a more consistent comparison of trends across patient and virus groups because testing methods were on the whole standardized throughout the study. our study has revealed changes in the frequency of virological testing of respiratory illnesses in the nhsggc health board during 2005-2013, with adults representing an increasingly greater percentage of episodes. however, age-specific prevalences were greater in the 2013 influenza season than in 2005 for all age groups. it is possible that there is raised awareness in the public and/or clinicians, and consequently greater healthcare seeking and/or sampling behaviour in adults. alternatively these results could reflect a true increase in non-viral causes of respiratory illness in this age group. we note that a shift in the demography of the glasgow population has been reported [24] . our observations might indicate the impact of an ageing population on respiratory-related healthcare services, through an increase in gp/hospital consultations, or a genuine increase in the incidence of adult respiratory infections. rv was the most prevalent virus overall, corroborating previous uk-based studies that include patients attending both primary and secondary healthcare services [10, 12] . the clinical significance of rv is disputed, although severe cases of disease are recognized depending on virus species, patient subgroups, and season [7, [25] [26] [27] . in additional analyses (fig. 4) we found the prevalence of severe respiratory illness (patients located in critical care wards) was significantly greater in rv infections than iav, supporting the proposition that rv is associated with more severe disease than traditionally accepted. of the other non-influenza viruses, rsv and cov were relatively highly prevalent. we note that the extent of research into the commonly circulating covs is small compared to iav and rsv, although severe clinical cases are recognized [28] . our study is the first comparative analysis in the uk to include cov, providing an important opportunity to quantify its temporal and patient subgroup distributions and co-infection patterns in comparison to the other common virus groups. we confirm that cov contributes a large fraction of infections during periods of high influenza activity and that cov is relatively frequently co-detected with other viruses. the contribution of different respiratory viruses to the healthcare burden in scotland has previously been studied [23] . further investigation on a seasonal basis is needed to help elucidate the public health relevance of rv and cov, particularly since cov has a similar age distribution as the influenza viruses. the remaining viruses (adv, mpv, piv1-4) were detected in comparatively smaller numbers on a yearly basis and during months of high influenza activity. the 9-year study period provided a novel opportunity to compare the epidemiology of respiratory viruses before and after the 2009 influenza a pandemic [18] . in our multivariable statistical analysis we found viral infections to be more likely in the post-pandemic era. this result was independent of other factors such as patient's age implying non-patient factors, such as a change in the underlying virus population, have increased the likelihood that a patient seeking healthcare services will have a viral infection (as opposed to non-viral causes). whether this is a direct consequence of the pandemic virus, its impact on the epidemiologies of others viruses, or a consequence of long-term changes in the non-influenza virus population, remains to be elucidated. seasonal and patient-related factors corroborate existing knowledge and were independent of time, indicating the generality of these factors as predictors of viral infection. it is well recognized that the burden of viral respiratory illness lies predominantly in young children [29] . we found that in patients with respiratory illness attending healthcare facilities, those aged 1-5 years were more likely than other age groups to have a viral infection independent of season or time period. the most commonly detected viruses in this age group were rv, rsv, adv and mpv (20%, 9·3%, 9·1% and 4·7% of infections, respectively) corroborating previous reports [23, 30] . together with a recent study that found bacterial-viral co-infections were relatively uncommon in children with pneumonia [31] , these findings support the concern regarding the overprescription of antibiotics in children [32] . that the increasing trend in virus prevalence was most notable in infants (<1 year) also warrants further attention. while it is possible that these findings are influenced by changes in clinical testing decisions, we note that this trend is particularly pertinent in relation to recent european outbreaks of enterovirus d68 in children [33] ; investigation into the contribution of individual viruses will be the focus of future work. we further note that, based on the multivariable statistical analyses, the increasing trend in prevalence in children explained why co-infections were more likely detected in the post-2009 pandemic era. there are very few studies describing co-infection patterns in respiratory viruses. our study provides the largest examination to date, confirming that around 11% of viral infections in patients attending healthcare services in an urban setting involve more than one virus, similar to the 10·4% reported by a previous uk-based study [12] . that nearly all respiratory viruses were co-detected with all other viruses highlights the sufficient opportunities for co-infections. we would expect co-infection frequencies to reflect individual virus prevalences. indeed, in line with the aforementioned study [12] , rv was the most common detection in co-infections, rv/rsv was a frequent pairing, and most co-infections were in children aged <5 years. our study also reveals that covs are relatively frequently involved in co-infections. however, co-infections with influenza viruses were relatively few, perhaps explained by differences in their age and seasonal distributions, or an inter-viral interference [34] . we found that the average age of co-infection was 3 years, compared to 17 years for viral infections overall, and co-infections were more likely in infants than in those aged 1-5 years. that co-infections were more likely in young children is probably explained by (i) a greater opportunity for co-infection due to a shorter exposure lifetime and consequently greater susceptibility to a wider array of viruses, and (ii) a greater chance of co-infections being detected because children tend to shed virus for longer periods. in adults, the age distribution of co-infections differed according to gender and patient origin; the prevalence was greatest in males and in general hospital attendees not admitted to critical care wards for those aged 46-64 years (fig. 6c,d) . this result provides insight into an age-dependent factor in co-infection patterns in adults but must be viewed with some caution; it is potentially influenced by a bias in multiple specimens submitted in relation to single episodes of illness in adults, most likely as a result of comorbidities. interestingly, co-infections were more likely in general hospital attendees not admitted to critical care wards than gp attendees, supporting the potential role of co-infections in illness severity [35] . there are several limitations to our study to be noted. detection of viral nucleic acid may not represent active infection for all viruses in all cases [36] , potentially introducing detection biases temporally and across patient groups. furthermore, the timing of infection events, and variation in shedding duration across virus and patient groups [37, 38] , could potentially bias the observed co-infection patterns. we also note that our study lacked information on the presence/absence of bacterial pathogens which are also significant contributors to respiratory infections. one further important consideration is that laboratory diagnostic data cannot inform on the epidemiology of asymptomatic infections in the community, or in symptomatic people who do not attend healthcare services. furthermore, that viral populations are not static could also impact on the generalisability of the observed trends and associations; the introduction of new strains can alter disease outcomes, and consequently healthcare seeking behaviour, influencing the stability of healthcare consultation rates in patient subgroups. given the dynamic nature of virus populations, the epidemiological information generated through surveillance must be maintained to ensure future vaccine and antiviral developments are directed to where they are most needed [39, 40] . 1·15 (0·75-1·79, p = 0·521) or, odds ratio; ci, confidence interval. * distribution of patient numbers, with corresponding % in parentheses, across factor levels for all patients (summary) and for co-infection and mono-infection groups. † unadjusted or based on univariable logistic regression. ‡ adjusted or based on multivariable logistic regression. § patients' location corresponding with first clinical sample: gp, general practitioner's surgery; hospital (general), outpatients and non-critical-care patients; hospital (critical care), patients admitted to an intensive care, intensive therapy, high dependency, or coronary care unit. our study provides the most comprehensive description of viral respiratory infections in the uk to date, revealing new epidemiological insights with public health relevance. of particular concern is a greater viral prevalence in 2013 compared to 2005, particularly in infants, and a greater risk of viral infection in the post-2009 pandemic era. further investigation into the long-term temporal dynamics of individual viruses and the epidemiological consequences of virus cocirculation is needed. for supplementary material accompanying this paper visit http://dx.doi.org/10.1017/s0950268816000339. global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis disability-adjusted life years (dalys) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the global burden of disease study viruses and bacteria in the etiology of the common cold adults hospitalised with acute respiratory illness rarely have detectable bacteria in the absence of copd or pneumonia; viral infection predominates in a large prospective uk sample investments in respiratory infectious disease research 1997-2010: a systematic analysis of uk funding association between human rhinovirus c and severity of acute asthma in children human metapneumovirus: review of an important respiratory pathogen newly discovered coronavirus as the primary cause of severe acute respiratory syndrome a new laboratory-based surveillance system (respiratory datamart system) for influenza and other respiratory viruses in england: results and experience from seasonal variations of 15 respiratory agents illustrated by the application of a multiplex polymerase chain reaction assay single, dual and multiple respiratory virus infections and risk of hospitalization and mortality assessing the burden of influenza and other respiratory infections in england and wales epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern china viral etiologies of hospitalized acute lower respiratory infection patients in china epidemiology and viral etiology of the influenza-like illness in corsica during the 2012-2013 winter: an analysis of several sentinel surveillance systems impact of the 2009 h1n1 pandemic on age-specific epidemic curves of other respiratory viruses: a comparison of pre-pandemic, pandemic and postpandemic periods in a subtropical city during the summer 2009 outbreak of 'swine flu' in scotland what respiratory pathogens were diagnosed as h1n1 a goodness-of-fit test for binary regression models, based on smoothing methods r: a language and environment for statistical computing. r foundation for statistical computing disease burden of the most commonly detected respiratory viruses in hospitalized patients calculated using the disability adjusted life year (daly) model population trends by age group in glasgow human rhinovirus species and season of infection determine illness severity clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children the significance of rhinovirus detection in hospitalized children: clinical, epidemiological and virological features epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology respiratory viral infections during the 2009-2010 winter season in central england, uk: incidence and patterns of multiple virus co-infections community-acquired pneumonia requiring hospitalization among u.s. children respiratory virus infections in febrile children presenting to a general practice out-of-hours service emergence and epidemic occurrence of enterovirus 68 respiratory infections in the netherlands in 2010 do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? single and multiple respiratory virus infections and severity of respiratory disease: a systematic review clinical utility of pcr for common viruses in acute respiratory illness influence of age, severity of infection, and co-infection on the duration of respiratory syncytial virus (rsv) shedding epidemiology of multiple respiratory viruses in childcare attendees emerging respiratory viruses: challenges and vaccine strategies respiratory viruses other than influenza virus: impact and therapeutic advances the authors are grateful to dominic mellor, emma thomson, louise matthews and richard reeve for their helpful critique of the manuscript and discussions. a subset of the clinical samples was provided by health protection scotland as part of the scottish enhanced respiratory viral infection surveillance programme.this work was supported by the medical research council uk (grant g0801822). none. key: cord-333730-qsx0m68e authors: tsai, y. c.; tsai, t. f. title: oral disease-modifying antirheumatic drugs and immunosuppressants with antiviral potential, including sars-cov-2 infection: a review date: 2020-09-03 journal: ther adv musculoskelet dis doi: 10.1177/1759720x20947296 sha: doc_id: 333730 cord_uid: qsx0m68e there have been several episodes of viral infection evolving into epidemics in recent decades, and severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the latest example. its high infectivity and moderate mortality have resulted in an urgent need to find an effective treatment modality. although the category of immunosuppressive drugs usually poses a risk of infection due to interference of the immune system, some of them have been found to exert antiviral properties and are already used in daily practice. recently, hydroxychloroquine and baricitinib have been proposed as potential drugs for sars-cov-2. in fact, there are other immunosuppressants known with antiviral activities, including cyclosporine a, hydroxyurea, minocycline, mycophenolic acid, mycophenolate mofetil, leflunomide, tofacitinib, and thalidomide. the inherent antiviral activity could be a treatment choice for patients with coexisting rheumatological disorders and infections. clinical evidence, their possible mode of actions and spectrum of antiviral activities are included in this review article. lay summary: immunosuppressants often raise the concern of infection risks, especially for patients with underlying immune disorders. however, some disease-modifying antirheumatic drugs (dmards) with inherent antiviral activity would be a reasonable choice in the situation of concomitant viral infections and flare up of autoimmune diseases. this review covers dmards of treatment potential for sars-cov-2 in part i, and antiviral mechanisms plus trial evidence for viruses other than sars-cov-2 in part ii. infections are a common concern of immunosuppressive drugs. however, some immunosuppressants or disease-modifying antirheumatic drugs (dmards) show antiviral activity and may be safely used or even beneficial in patients with selected concomitant viral infections. certain dmards may even be considered as an alternative treatment for recalcitrant infections. moreover, the concomitant use of immunosuppressants and antiviral agents was proved to be more effective than antiviral agent monotherapy in some reports. 1 the antiviral property of immunosuppressants may act through (a) direct virucidal activity, (b) blockage of receptors, (c) inhibition of necessary molecules for viral replication in the hosts, or (d) amelioration of inflammatory symptoms. also, control of inflammation may decrease the susceptibility or enhance host ability to defend against viral infection. the are indicated to treat and prevent malaria. they are also used as dmards for rheumatoid arthritis, lupus erythematosus, and porphyria cutanea tarda. in addition, the application for viral infections in off-label use has recently been investigated vigorously. the antiviral activity is through blocking the virus/cell fusion via increasing endosomal ph and hindering the glycosylation of cellular receptors ( figure 2 ). 4 in vitro cq revealed low half-maximal effective concentration (ec50) and high half-cytotoxic concentration (cc50) for covid-19. 5 a preliminary study conducted in china showed benefits in pneumonia image, shortening of disease course, and promoting a virus-negative conversion compared with control group. 6 then, four completed clinical studies demonstrated favorable outcomes in clinical and radiologic amelioration, while another two randomized controlled trials (rcts) illustrated no statistically significant change compared with control arms. [7] [8] [9] [10] [11] [12] based on the inhibitory effect of azithromycin against ebola and zika viruses in vitro, and the possibility of preventing from progressing to severe respiratory tract infections, two french trials which combined the use of azithromycin and hcq revealed better efficacy. 7, 9 however, further studies are still needed to draw conclusions because most of these studies bear limitations including selection bias, allocation bias, or insufficient case numbers. several multicenter, double-blind, and well-designed controlled trials are already underway to assess the efficacy and safety of cq or hcq in the treatment of covid-19 pneumonia. in the absence of other confirmed effective therapy specific to sars-cov-2, both drugs are currently still listed in the treatment guidelines (table 1) . baricitinib, blocking janus kinase (jak)1 and jak2, is approved for rheumatoid arthritis and has been investigated in atopic dermatitis. sars-cov-2 binds on the angiotensin-converting enzyme 2 (ace2) receptors and enters lung cells through receptor-mediated endocytosis. some of the numb-associated kinase (nak) family members, ap2-associated protein kinase 1 (aak1) and cyclin g-associated kinase (gak), are hypothesized to regulate the ace2-mediated endocytosis. baricitinib demonstrated high affinity to aak1 and in one controlled open-label study (n = 24), patients were given either baricitinib 4 mg/day plus lopinavir-ritonavir or antiretroviral plus hydroxychloroquine (control group) for 2 weeks. significant improvement of symptoms and laboratory results, no intensive care unit transfer (versus 33% transfer in control cases), and 58% discharge from wards (versus 8% in control) was shown among the baricitinib-treated individuals. 14 in addition to antiviral property, baricitinib has been suggested as an approach for a cytokine storm syndrome, which features hypercytokinemia and multi-organ failure. elevated ferritin and il-6 in covid-19 cases were predictive of a high mortality rate according to a china retrospective study. 17 baricitinib inhibits cytokines including il-2, il-6, il-10, interferon gamma (ifn-γ), and granulocytecolony-stimulating factor (g-csf) 13, 18 and may bring the benefit of immune reconstruction which could be used in rapidly progressive diseases. however, there are competing ideas about the interference of jak inhibitors with ifn-mediated antiviral activities. ifns prohibit viral spreading in the early phase of infections. in animal models of sars and middle east respiratory syndrome (mers), ifn-α and ifn-β showed benefit at the early stage but were harmful at the late phase. patients with severe sars who died of hypoxemia revealed high ifn-α, -γ, while those discharged dmards, disease-modifying anti-rheumatic drugs. journals.sagepub.com/home/tab 5 from hospital had low ifn-α, -γ. therefore, some experts suggested baricitinib's use in the situation of hyperinflammation and cytokine syndrome, rather than in those with mild diseases. in fact, clinical trials have commenced to evaluate the optimal timing, duration, and safety of baricitinib in viral infections, including sars-cov-2. [19] [20] [21] cyclosporine a cyclosporine a (csa) is indicated for rheumatoid arthritis, psoriasis, organ transplants to prevent injection, and keratoconjunctivitis sicca. it is also used in severe atopic dermatitis, chronic urticaria, pyoderma gangrenosum kimura disease, acute systemic mastocytosis, and ulcerative colitis. csa inhibits lymphocyte function, mainly t cells, by forming a complex with cyclophilin. cyclophilin-csa complex binds on the calcineurin, which blocks the dephosphorylation of nuclear factor of activated t cells (nf-at). this interferes with entry of nf-at into the t-cell nucleus and further suppresses cytokine production such as il-2. severe covid-19. experimentally, csa targets cyclophilin d to inhibit mitochondrial permeability transition pore (mptp) opening and rescues mitochondria from apoptosis. [22] [23] [24] moreover, melanoma-differentiation-activated protein 5 (mda5), an rlr helicase and putative cytoplasmic receptor of sars-cov-2, is also the target antigen of clinically amyopathic dermatomyositis (cadm). patients with mda5 plus cadm have higher risks of developing rapidly progressive interstitial lung diseases and respiratory failure, while this could be reversed by calcineurin inhibitors. based on these hypothetical functions, csa was proposed as a modulator for cytokine storm syndrome in covid-19 infections. 25 mycophenolic acid (mpa), an active metabolite of mycophenolate mofetil (mmf), inhibits inosine monophosphate dehydrogenase (impdh), an essential enzyme in the de novo purine synthesis pathway. impdh inhibition especially influences t and b lymphocytes because they use almost a de novo pathway to synthesize (minimally use a salvage pathway). mmf and mpa are utilized in organ transplantation, crohn's disease, and as steroid-sparing agents for conditions such as pemphigus, behçet's disease, and lupus erythematosus. although they were associated with higher risk of opportunistic infections including herpes zoster, cytomegalovirus (cmv), and bk virus (bkv) nephropathy, literature also revealed its possible benefit for hiv and influenza virus. 26, 27 in vitro: mmf showed low ec50 (0.47 μmol/l) in sars-cov-2-infected vero e6 cells, while the ec50 of remdesivir, as a positive control, was 0.77 μmol/l. besides, mmf probably inhibited sars-cov-2 through impdh and especially dihydroorotate dehydrogenase (dhodh). dhodh is another essential enzyme for pyrimidine synthesis, and mmf might control viral infection by depleting the intracellular pyrimidine pools. 28 thalidomide thalidomide, a derivative of glutamic acid, is approved for erythema nodosum leprosum and is also used in many conditions such as prurigo nodularis, pyoderma gangrenosum, bechet's disease, lupus erythematosus and erythema multiforme. it exerts anti-inflammatory effect through cereblon e3 ubiquitin ligase as the primary target and thus inhibits chemotaxis of leukocytes, monocytes as well as the production of tumor necrosis factor (tnf)-alpha, il-8, and il-12. case report: a 45-year-old woman with critical symptoms of covid-19 was treated by thalidomide 100 mg every 24 h. after the first day use of thalidomide, clinical conditions including oxygen index improved. cytokines such as il-6, il-10, ifn-γ all decreased to normal range. 15 proposed mechanisms are as follows: thalidomide inhibits nf-κb, which further suppresses the production of pro-inflammatory cytokines such as tumor necrosis factor alpha (tnf-α) and il-8, and prevents the cytokine surge. it also regulates immune function by activating t cells and t-cell receptors. moreover, the sedative and antiemetic property of thalidomide helps anxious patients calm down, which reduces oxygen consumption. 15 now, at least one clinical trial has been conducted to investigate the efficacy and safety of thalidomide as an adjuvant therapy for covid-19 pneumonia. 29 part ii: dmards with antiviral potential other than sars-cov-2 many oral dmards have inherent antiviral activity and could be the treatment of choice for patients with coexisting immune-based diseases and infections. especially when the infection is still in progression, choosing dmards with anti-microbial evidence would bring double benefits for better infection control without sacrificing underlying disease management. the antimicrobial mechanisms of dmards are often distinct from their immunomodulatory pathway, and the efficacy is different in viral species (tables 2, 3 leflunomide is approved for rheumatoid arthritis and psoriatic arthritis (not in the united states). leflunomide inhibits the synthesis of pyrimidine via acting on the mitochondrial enzyme dhodh; therefore, rapidly dividing cells, especially lymphocytes, are suppressed. on the other hand, leflunomide showed antiviral activity at least for cmv, bkv, and hiv. it works by teriflunomide, the active metabolite of leflunomide, which disrupts nucleocapsid tegumentation, and thus prevents virion assembling, rather than influences the de novo pyrimidine synthesis pathway. 97 herpes simplex virus a case report with perianal hsv-2 lesions in an acyclovir-resistant hiv patient significantly improved with leflunomide 40 mg, twice a day. 98 another hiv patient with hsv-1/hsv-2 pseudo-tumors on the perineum and scrotum only slightly improved with valacyclovir, foscarnet, and imiquimod. after 9 months of leflunomide, complete regression of the lesions was noted. leflunomide has both immunomodulation and antiviral activities in the hsv pseudotumors because pseudotumor formation is an immune reconstruction phenomenon in hiv patients. 99 human immunodeficiency virus an rct (n = 18) demonstrated leflunomide decreased the activation and cycling of cd4+ t cells. the expression of hiv co-receptors ccr5 and cxcr4 was also reduced compared with placebo. 102 three patients with atopic dermatitis treated with azathioprine developed multiple verrucae and molluscum contagiosum. due to treatment resistance, azathioprine was switched to leflunomide (100 mg loading 3 days, then 20 mg/day). all the lesions subsided in three patients within 2 months of leflunomide treatment. 103 multiple recalcitrant verrucae in three and molluscum in one of renal allograft recipients cleared after switching from mmf to leflunomide. 104 leflunomide can serve as a potential option for patients with skin warts or molluscum concomitant with immune conditions that require immunosuppressants. leflunomide was regarded as an add-on treatment for multi-drug-resistance cmv infection. in vitro anti-cmv properties of leflunomide were not through blocking the replication of viral dna, so it is effective even in patients with direct antiviral drug-resistance history. 105 disseminates to the urinary-tract system and lives there persistently. a sudden increase of bk-virusassociated nephropathy is related to the administration of potent immunosuppressants such as mmf and tacrolimus. leflunomide is now generally accepted as a second choice after reduction of immunosuppressive agents. however, in a phase ii rct (n = 46), viremia was decreased in the group of leflunomide active metabolites, but no significant improvement of renal function was noted. 108 treatment options for rsv are limited to supportive care or ribavirin with only marginal effectiveness. leflunomide showed a potent, dose-dependent anti-rsv activity in cell cultures. 92 also, pulmonary viral loads were prominently reduced in cotton rats, even if there was a 3-day delay of leflunomide administration after viral inoculation. 109 leflunomide offers a dual benefit of both viral-load reduction and anti-inflammatory effects that attenuate the destruction of cytokine-related diseases. tofacitinib, a jak1 and jak3 inhibitor, is indicated in rheumatoid arthritis, ulcerative colitis, and psoriatic arthritis. it is also used off label for vitiligo and alopecia areata. tofacitinib treats inflammatory diseases by interfering with the activation of the jak/signal transducers and activators of transcription (stat) pathway, which inhibits gene transcription, and cytokine production is thereby reduced. htlv-1, a retrovirus, has been linked to diseases such as adult t-cell lymphoma/leukemia (atll), htlv-1-associated myelopathy (ham), and uveitis. ex vivo and animal studies revealed positive results of tofacitinib for atll and ham. 110 htlv-1-encoded tax protein activates il-2, -9, -15, which further trigger jak3-stat5 pathway. accumulating data demonstrated a major role of jak3 in the pathophysiology of atll. 114 as a result, tofacitinib targeting jak3 has been suggested as a therapeutic strategy in future studies. hydroxyurea, a deoxyribonucleic acid (dna)synthesis inhibitor, belongs to the antineoplastic medications. however, it may be used as a second-line drug for psoriasis and palmoplantar pustulosis 115 based on the ability to slow down the rapid division of keratinocytes. bone marrow suppression is the major and common adverse effect of hydroxyurea. hydroxyurea demonstrated promising results in reducing hiv rna viral loads in five placebocontrolled clinical trials. among all the trials, hydroxyurea was combined with didanosine, a nucleoside analog reverse-transcriptase inhibitor (nrti). however, one should be reminded that decreased cd4 counts were noted in some studies. therefore, close follow up of hematologic change is required in daily practice. [65] [66] [67] [68] [69] in vitro studies demonstrated the antiviral modes of hydroxyurea. first, hydroxyurea depletes deoxynucleoside triphosphate (dntp) pools, which impedes dna synthesis and in turn slows down the production of viral dna. second, hydroxyurea enhances nrti phosphorylation and reduces resistance to nrtis. this may partially explain the benefits of adding hydroxyurea to nrti for viral control. finally, cytotoxic effect of hydroxyurea makes cellular division of cd4+ t cells decline. this enables hydroxyurea to block hiv proliferation, because hiv could only replicate in dividing cd4+ t cells. 64 although the mode of action of hydroxyurea for hcv, hbv, hsv, and b19v is unknown, viral replications were inhibited by hydroxyurea in in vitro studies. 70, [74] [75] [76] small-scaled clinical trials showed significant reduction of hcv rna levels and hbv viral loads in chronic hcv and hbv carriers, respectively. 71, 72 however, there is a case report of an elderly patient with essential thrombocythemia experiencing reactivation of hbv during treatment with hydroxyurea. 73 a retrospective review of children with sickle cell anemia demonstrated decreased requirement of blood transfusion and attenuation of clinical symptoms when using hydroxyurea in patients with b19v infection. 77 minocycline, a second-generation of tetracyclines, is frequently used for bacterial infections, acne, and rheumatoid arthritis. the small size and lipophilic nature facilitate its penetration into blood-brain barrier easily. the neuroprotection and anti-inflammation effects 116, 117 brought interest in the treatment of virus-induced encephalitis such as hiv, japanese encephalitis virus (jev), and reovirus. the antiviral property is not clearly known but seems to be diverse, including neuroprotective, antiapoptotic, interference of viral protein expression, and anti-inflammatory effects. in microglial cell culture, minocycline reduced viral replication by 71-96%. 78 in vivo, macaque monkeys treated with minocycline showed less destruction of axons and less replication of viruses in the central nervous system. the experiment suggested that the antiviral effect of minocycline was through reducing the activation of monocytes and hence, viral replication was blocked. 79 nevertheless, two double-blind, randomized, placebo-controlled human studies revealed that under minocycline 100 mg twice daily, there was no difference in cognitive function compared with placebo. 80, 81 japanese encephalitis virus minocycline showed high efficacy in animal models and in vitro studies for the treatment of jev. 82 33, 34 four clinical trials showed positive results of hiv control, either in the reduction of immune activation or lowering the vertical transmission. [35] [36] [37] [38] however, another two trials (one rct, one single-arm) revealed no efficacy. 39, 40 as for dengue, 41-44 chikungunya, [45] [46] [47] influenza a viruses, [48] [49] [50] [51] and hcv, 52,53 paradoxical outcomes were found in the literature. therefore, the utilization of cq in these viral diseases still needs further investigation. the major concern of zika virus infection is that it can transmit from placenta to fetus and cause microcephaly or congenital defects. cq prevented zika virus internalization in cell cultures and reduced morbidity or mortality in mice. in addition, it prevented fetal mice from microcephaly. 54 therefore, cq might be a potential treatment waiting for clinical verification. hcq had been reported to downregulate the expression of ifn genes and reduce the production of type i ifns. this phenomenon was noted in vitro 118 and in human studies of autoimmune diseases. 119 since ifns are crucial in innate immunity to defend viral infections, the usage of hcq may raise concerns about the counter effects in viral control. nevertheless, opposite results were also presented: hcq activated ifnβ signaling pathways in cell studies of dengue virus. 120 betaretrovirus is regarded as one of the environmental factors triggering the recurrence of primary biliary cirrhosis (pbc) after liver transplantation. earlier and more severe recurrence of pbc occurred with tacrolimus compared with csa as an immunosuppressant. this may be partially explained by the antiviral activity of csa. according to an in vitro study, it was suggested that csa interrupted viral replication through inhibiting viral protein synthesis, gag and envelope assembly, and particle budding. 63 the combination of mmf and highly active antiretroviral therapy improved the control of viral replication and delayed viral-load rebound in a randomized pilot study (n = 17 the effectiveness of thalidomide for ks might be related to anti-angiogenesis, and experts hypothesized the modulation of the immune system to trigger an antiviral action. the treatment of immune-based diseases has been revolutionized by the introduction of target therapy, mainly biologics. compared with biologics, conventional synthetic dmards exert broad-spectrum functionality. dmards work through immunosuppressive and antiinflammatory effects with the possibility of higher infection risk. however, many none-biologic dmards demonstrate antiviral activities instead. although in most instances, the antiviral activity of dmards is based on in vitro or small-scale controlled studies, this property would be useful in the choice of dmards for patients with concomitant viral infections. also, the combinational use of antiviral drugs and dmards has been shown to be more effective and less resistant in the control of some viral infections. furthermore, in the face of novel viral infection, such as sars-cov-2, screening of existing chemicals, including dmards, may prove to be fruitful. dr tsen-fang tsai has conducted clinical trials or received honoraria for serving as a consultant for abbvie, boehringer ingelheim, bristol-myers squibb, celgene, elililly, galderma, gsk-stiefel, janssen-cilag, leo-pharma, merck, novartis, pfizer inc., and ucb pharma. dr ya-chu tsai has delivered speeches held by abbvie, elililly, janssen-cilag, leo-pharma, novartis, pfizer. the authors received no financial support for the research, authorship, and/or publication of this article. efficacy of combination therapy of antiviral and immunosuppressive drugs for the treatment of severe acute exacerbation of chronic hepatitis b antiviral, immunosuppressive and antitumour effects of ribavirin analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with moderate covid-19 clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial. medrxiv. epub ahead of print 2020 treating covid-19 with chloroquine hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial mechanism of baricitinib supports artificial intelligence-predicted testing in covid-19 patients baricitinib therapy in covid-19: a pilot study on safety and clinical impact thalidomide combined with low-dose short-term glucocorticoid in the treatment of critical coronvirus disease baricitinib as potential treatment for 2019-ncov acute respiratory disease covid-19: consider cytokine storm syndromes and immunosuppression jak1/2 inhibition with baricitinib in the treatment of autoinflammatory interferonopathies baricitinib for covid-19: a suitable treatment? janus kinase inhibitor baricitinib is not an ideal option for management of covid-19 baricitinib for covid-19: a suitable treatment?-authors' reply why choose cyclosporin a as first-line therapy in covid-19 pneumonia. reumatol clin. epub ahead of print 16 a novel in vitro cypd-mediated p53 aggregation assay suggests a model for mitochondrial permeability transition by chaperone systems loss of cyclophilin d reveals a critical role for mitochondrial permeability transition in cell death chloroquine is a potent inhibitor of sars coronavirus infection and spread anti-hiv effects of chloroquine: mechanisms of inhibition and spectrum of activity the additive in vitro anti-hiv-1 effect of chloroquine, when combined with zidovudine and hydroxyurea hydroxychloroquine treatment of patients with human immunodeficiency virus type 1 effect of chloroquine on human immunodeficiency virus (hiv) vertical transmission reduction of immune activation with chloroquine therapy during chronic hiv infection hydroxychloroquine drastically reduces immune activation in hiv-infected, antiretroviral therapy-treated immunologic nonresponders effects of hydroxychloroquine on immune activation and disease progression among hiv-infected patients not receiving antiretroviral therapy: a randomized controlled trial assessment of chloroquine as a modulator of immune activation to improve cd4 recovery in immune nonresponding hiv-infected patients receiving antiretroviral therapy chloroquine inhibits dengue virus type 2 replication in vero cells but not in c6/36 cells antiviral activity of chloroquine against dengue virus type 2 replication in aotus monkeys chloroquine use improves dengue-related symptoms a randomized controlled trial of chloroquine for the treatment of dengue in vietnamese adults chikungunya virus: an update on antiviral development and challenges chloroquine phosphate treatment of chronic chikungunya arthritis. an open pilot study on chikungunya acute infection and chloroquine treatment confronting an influenza pandemic with inexpensive generic agents: can it be done? anti-malaria drug chloroquine is highly effective in treating avian influenza a h5n1 virus infection in an animal model influenza a/h1n1 vaccination of patients with sle: can antimalarial drugs restore diminished response under immunosuppressive therapy? chloroquine for influenza prevention: a randomised, doubleblind, placebo controlled trial lysosomotropic agents as hcv entry inhibitors porphyria cutanea tarda in an hcv-positive liver transplant patient: a case report fdaapproved drug, prevents zika virus infection and its associated congenital microcephaly in mice functional association of cyclophilin a with hiv-1 virions long-term follow-up of hiv positive asymptomatic patients having received cyclosporin a placebo-controlled trial of cyclosporin-a in hiv-1 disease: implications for solid organ transplantation critical role of cyclophilin a and its prolylpeptidyl isomerase activity in the structure and function of the hepatitis c virus replication complex cyclophilin b is a functional regulator of hepatitis c virus rna polymerase combined interferon alpha2b and cyclosporin a in the treatment of chronic hepatitis c: controlled trial cyclosporine inhibits flavivirus replication through blocking the interaction between host cyclophilins and viral ns5 protein a screen of fda-approved drugs for inhibitors of zika virus infection cyclosporine a inhibits in vitro replication of betaretrovirus associated with primary biliary cirrhosis rationale for the use of hydroxyurea as an anti-human immunodeficiency virus drug combination of a drug targeting the cell with a drug targeting the virus controls human immunodeficiency virus type 1 resistance a placebo-controlled trial of didanosine plus stavudine, with and without hydroxyurea, for hiv infection. the swiss hiv cohort study the hydile trial: efficacy and tolerance of a quadruple combination of reverse transcriptase inhibitors versus the same regimen plus hydroxyurea or hydroxyurea and interleukin-2 in hiv-infected patients failing protease inhibitorbased combinations activity, safety, and immunological effects of hydroxyurea added to didanosine in antiretroviral-naive and experienced hiv type 1-infected subjects: a randomized, placebo-controlled trial, actg 307 a randomized trial to investigate the recycling of stavudine and didanosine with and without hydroxyurea in salvage therapy (restart) hydroxyurea as an inhibitor of hepatitis c virus rna replication hydroxyurea suppresses hcv replication in humans: a phase i trial of oral hydroxyurea in chronic hepatitis c patients amazing results with hydroxyurea therapy in chronic hepatitis b: a preliminary report reactivation of hepatitis b virus during treatment with hydroxyurea in an elderly patient with essential thrombocythemia reversible inhibition of herpes simplex virus replication by hydroxyurea hydroxyurea enhances the activity of acyclovir and cidofovir against herpes simplex virus type 1 resistant strains harboring mutations in the thymidine kinase and/or the dna polymerase genes hydroxyurea inhibits parvovirus b19 replication in erythroid progenitor cells original research: parvovirus b19 infection in children with sickle cell disease in the hydroxyurea era a novel action of minocycline inhibition of human immunodeficiency virus type 1 infection in microglia neuroprotective and anti-human immunodeficiency virus activity of minocycline minocycline treatment for hiv-associated 20 journals.sagepub.com/home/tab cognitive impairment: results from a randomized trial randomized trial of minocycline in the treatment of hiv-associated cognitive impairment minocycline neuroprotects, reduces microglial activation, inhibits caspase 3 induction, and viral replication following japanese encephalitis minocycline differentially modulates viral infection and persistence in an experimental model of japanese encephalitis minocycline trial in japanese encephalitis: a double blind, randomized placebo study role of oral minocycline in acute encephalitis syndrome in india: a randomized controlled trial drug repurposing of minocycline against dengue virus infection antibiotic minocycline prevents respiratory syncytial virus infection antiinflammatory and antiviral effects of minocycline in enterovirus 71 infections transcriptomic characterization of the novel avian-origin influenza a (h7n9) virus: specific host response and responses intermediate between avian (h5n1 and h7n7) and human (h3n2) viruses and implications for treatment options minocycline inhibits west nile virus replication and apoptosis in human neuronal cells minocycline delays disease onset and mortality in reovirus encephalitis therapy with minocycline aggravates experimental rabies in mice effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo mycophenolic mofetil, an alternative antiviral and immunomodulator for the highly pathogenic avian influenza h5n1 virus infection broadspectrum antivirals for the emerging middle east respiratory syndrome coronavirus treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset inhibition of herpes simplex virus type 1 by the experimental immunosuppressive agent leflunomide successful treatment of acyclovirresistant herpes simplex virus type 2 proctitis with leflunomide in an hiv-infected man leflunomide in the treatment of a pseudotumoral genital herpes simplex virus infection in an hiv patient inhibition of hiv replication by a77 1726, the active metabolite of leflunomide, in combination with pyrimidine nucleoside reverse transcriptase inhibitors anti-hiv-1 activity of leflunomide: a comparison with mycophenolic acid and hydroxyurea the effect of leflunomide on cycling and activation of t-cells in hiv-1-infected participants leflunomide: an immune modulating drug that may have a role in controlling secondary infections with review of its mechanisms of action conversion from tacrolimus/mycophenolic acid to tacrolimus/leflunomide to treat cutaneous of four pediatric renal allograft recipients leflunomide for cytomegalovirus: bench to bedside concurrent antiviral and immunosuppressive activities of leflunomide in vivo leflunomide as part of the treatment for multidrug-resistant cytomegalovirus disease after lung transplantation: case report and review of the literature assessment of efficacy and safety of fk778 in comparison with standard care in renal transplant recipients with untreated bk nephropathy inhibition of respiratory syncytial virus in vitro and in vivo by the immunosuppressive agent leflunomide cp-690,550, a therapeutic agent, inhibits cytokine-mediated jak3 activation and proliferation of t cells from patients with atl and ham/tsp activity of thalidomide in aids-related kaposi's sarcoma and correlation with hhv8 titre activity of thalidomide in aids-related kaposi's sarcoma a prospective evaluation of leflunomide therapy for cytomegalovirus disease in renal transplant recipients ferm domain mutations induce gain of function in jak3 in adult t-cell leukemia/ lymphoma pustulosis palmaris et plantaris treated with hydroxyurea inhibition of autoimmune encephalomyelitis by a tetracycline minocycline protects pc12 cells from ischemic-like injury and inhibits 5-lipoxygenase activation hydroxychloroquine is associated with impaired interferon-alpha and tumor necrosis factor-alpha production by plasmacytoid dendritic cells in systemic lupus erythematosus hydroxychloroquine treatment downregulates systemic interferon activation in primary sjögren's syndrome in the joquer randomized trial hydroxychloroquine-inhibited dengue virus is associated with host defense machinery predictors of major infections in systemic lupus erythematosus previous antimalarial therapy in patients diagnosed with lupus nephritis: influence on outcomes and survival infection risk in systemic lupus erythematosus patients: susceptibility factors and preventive strategies thalidomide in the treatment of kaposi's sarcoma visit sage journals online journals.sagepub.com/ home/tab key: cord-346253-0mnsm6s4 authors: ahanchian, hamid; jones, carmen m; chen, yueh-sheng; sly, peter d title: respiratory viral infections in children with asthma: do they matter and can we prevent them? date: 2012-09-13 journal: bmc pediatr doi: 10.1186/1471-2431-12-147 sha: doc_id: 346253 cord_uid: 0mnsm6s4 background: asthma is a major public health problem with a huge social and economic burden affecting 300 million people worldwide. viral respiratory infections are the major cause of acute asthma exacerbations and may contribute to asthma inception in high risk young children with susceptible genetic background. acute exacerbations are associated with decreased lung growth or accelerated loss of lung function and, as such, add substantially to both the cost and morbidity associated with asthma. discussion: while the importance of preventing viral infection is well established, preventive strategies have not been well explored. good personal hygiene, hand-washing and avoidance of cigarette smoke are likely to reduce respiratory viral infections. eating a healthy balanced diet, active probiotic supplements and bacterial-derived products, such as om-85, may reduce recurrent infections in susceptible children. there are no practical anti-viral therapies currently available that are suitable for widespread use. summary: hand hygiene is the best measure to prevent the common cold. a healthy balanced diet, active probiotic supplements and immunostimulant om-85 may reduce recurrent infections in asthmatic children. asthma is a major public health problem with a huge social and economic burden affecting 300 million people worldwide [1] . viral respiratory infections are the major cause of acute asthma exacerbations and contribute to asthma inception in high risk young children with susceptible genetic background. a history of wheeze associated with respiratory viral infections early in life is one of the major risk factors for the later development of asthma [2] [3] [4] [5] [6] [7] , together with sensitization to aeroallergens in early life and a family history of asthma and allergies, reflecting a genetic predisposition. respiratory viral infections are also the principal cause of asthma exacerbations in children and adults [8] [9] [10] [11] [12] [13] . however, the question of whether viral infections "select" susceptible hosts or whether viral infections may induce asthma de novo by "damaging" airways is not settled. in other words, do viruses cause or simply unmask asthma? respiratory viruses first infect nasal epithelial cells which triggers an antiviral response. this response is driven by type i (α/β) and iii (λ) interferons (ifn) that are induced following recognition of viral ribonucleic acid (rna) by pattern recognition receptors (prrs). toll-like receptors (tlrs) are cell surface and endosomal prrs, whilst the rna helicase receptors (rig-i and mda-5) and nodlike receptors (nod2), detect viral rna in the cytoplasm. signalling via the prrs activates transcription factors (irf-3, irf-7, nf-κb), which lead to the production and secretion of type i and iii ifn. the ifns then bind to cell surface receptors to activate a separate pathway leading to the production of interferon stimulated genes (isgs) which encode antiviral proteins that combat infection, as well as prrs and transcriptional factors which further amplify ifn production. the respiratory syncytial virus (rsv), human meta-pneumovirus (hmpv) and human rhinovirus (hrv) are all single stranded rna viruses but engage differently with cell signalling pathways. in airway epithelial cells rsv and hmpv rna are primarily detected by rig-i in the cytoplasm [14, 15] . rsv can also be detected by nod2 [16] . hrv is endocytosed by epithelial cells, and is therefore primarily detected by tlr3 in the endosome early in the infection process and by rig-i and mda-5 later in infection following upregulation of these prrs [17] . the fusion (f) protein of rsv is recognised by tlr4 at the epithelial cell surface [18] . a successful antiviral response would see the infection limited to the upper airway, as is the case clinically with the majority of viral infections in healthy individuals. should such a response be deficient, then predominantly upper-airway viral infections, such as hrv, may spread to the lower airways, causing lower respiratory symptoms and an exacerbation of asthma in predisposed individuals. while definitive data are yet to be produced, experimental hrv infections in adult volunteers initially suggested that asthmatics were more likely to develop lower respiratory infections (lri) than healthy adults, i. e. less likely to be able to limit viral replication to the upper airways [19, 20] . subsequent in vitro infection of primary airway epithelial cells from asthmatic and healthy adults with hrv have demonstrated that asthmatic cells produce less ifn-β [21] and ifn-λ [22] making them potentially more susceptible to infection, slower to clear infection, and more susceptible to virus-induced cell cytotoxicity. deficiencies in the ifnα response of peripheral blood mononuclear cells and plasmacytoid dendritic cells from asthmatic adults and children has also been observed, in these particular studies, in response to rsv, hrv [14, 15] and influenza a [23] . it is likely that the overall impaired innate immune response of the asthmatic airway epithelium is a result of deficiencies in the antiviral response of both epithelial cells and immune cells. childhood, especially infancy, is characterized by developmentally-regulated deficiencies in innate and adaptive immunity [24] . such deficiencies are likely to increase the risk of viral lri in children, especially in those at high risk for asthma and allergies. each year, at the end of summer, parents of asthmatic children are concerned about acute asthma exacerbations following a common cold, asking how to minimize the risk during the winter viral season. it is a valid concern as up to 70% of asthmatic children have an intermittent or wheeze which is mostly symptomatic after viral infections [25, 26] . asthmatics with exacerbationprone phenotype are susceptible to acute exacerbations requiring hospitalization or an unscheduled visit for medical attention. major risk factors for acute exacerbations include previous acute exacerbation, allergy, young age, poorly controlled asthma, and, in particular, viral respiratory infections. moreover, recent data suggests an interaction between allergies and viral infections occurs to increase the risk of asthma exacerbation [27] . acute exacerbations are associated with decreased lung growth or accelerated loss of lung function and, as such, add substantially to both the cost and morbidity associated with asthma [28, 29] . viral respiratory infections are the main cause of asthma exacerbations in children (80-85%) and are a major risk factor for admission in hospital every autumn [30] [31] [32] . hrv are the most common viral agents [33] ; other respiratory tract viruses detected in children with an asthma exacerbation include rsv, influenza, coronavirus, hmpv, parainfluenza virus, adenovirus, and bocavirus [34] [35] [36] . current drugs for the prevention and treatment of virus-induced exacerbation of asthma are poorly effective and novel alternative therapies are needed. much research interest has focused on the potential role respiratory viral infections play in the inception of asthma. it is well established that hospitalization for rsv bronchiolitis is a risk factor for asthma during childhood [37, 38] . epidemiological studies have shown an increased risk of asthma with lri caused by hrv. in the childhood origin of asthma (coast) birth cohort study, wheezing with rsv (odds ratio [or], 2.6), hrv (or, 9.8), or both hrv and rsv (or, 10) was associated with increased asthma risk at age six years [7] . the childhood asthma study (cas) in perth, australia showed that wheezing with hrv or rsv in the first year of life was a risk factor (or, 2.5) for current wheeze at five years of age [4] . infant birth about four months before the winter virus peak carried the highest risk of developing asthma compared with birth 12 months before the peak [39] . the risk of asthma is increased by severe lri (slri), especially in the presence of allergic sensitization in early life [4, 25] . there appears to be a synergistic interaction between viral infection and allergic sensitization, suggesting a "two hit" model for induction of persistent asthma. these data also provide a series of novel strategies for the primary prevention of asthma by prevention of either allergic sensitization or of slri in high risk children. this strategy is also supported in a study by simoes et al. [40] , in which the use of palivizumab to prevent rsv infection decreased the risk of recurrent wheezing in nonatopic premature infants. the crucial period, with respect to asthma initiation, appears to be the first two to three years of life during which the growth and remodelling of lung and airways proceeds at maximum rates. pulmonary inflammation resulting from atopy and slri occurring during this vulnerable time is hypothesized to perturb underlying tissue differentiation programs, resulting in deleterious long term effects on respiratory functions. as a result, there is widespread belief amongst the paediatric respiratory community that intervention measures that can lower the frequency and/or intensity of slri in early life amongst the high risk atopic subgroup of children are likely to be successful at preventing asthma. if successful, these strategies would have major implications for reducing the high impact of this chronic disease on the community [17, 41, 42] . recent studies using culture-independent techniques have challenged the long-held dogma that lungs are sterile and have demonstrated that a microbiota community exists in the lung [43] [44] [45] . the implications of these new data are not clear, however new concepts and more research is required. the resident microbiome is different in the presence of respiratory disease [45, 46] ; therefore interactions between respiratory viruses and the resident pulmonary microbiome are postulated. the pulmonary and gastrointestinal microbiota influence the immune system and interventional approaches (by bacterial immunostimulants, prebiotics and/or probiotics) to create a healthy gut and respiratory microbiota are potential strategies for the prevention of viral infections [45] . children are important vectors for hrv transmission to family members particularly siblings [47, 48] . hrv shedding peaks two to four days after infection and decreases sharply thereafter, although nasal samples can be positive for rhinovirus for up to five weeks after a symptomatic infection [49] . there are three ways of common cold transmission in children. first, inhalation of small particles aerosolized by coughing; second, large particle droplets from saliva expelled while sneezing; and third, self-inoculation of one's own conjunctivae or nasal mucosa after touching a person or object contaminated with the cold viruses. the first two methods are inefficient [50] , while the third is the most important method of transmission. the mode of transmission could differ with age of the index case, duration of contact, and other factors. moreover, there is some evidence that the daily activities of infected people can lead to the contamination of environmental surfaces with hrv e.g. light switches, telephone dial buttons and handsets [48] . meticulous hand hygiene is the best measure to prevent the common cold; frequent hand washing and avoid touching one's nose and eyes [51] [52] [53] . the use of alcohol-based hand sanitizers is also effective [54, 55] . the promotion of handwashing was associated with a 12-34% reduction in respiratory-tract infections and colds in child-care centres in the usa [56] canada [57] and australia [58] and a 21% decrease in absences due to respiratory illness in the school setting [56] . hand hygiene campaigns were also successful in reducing absenteeism caused by influenza-like illnesses among schoolchildren in egypt [59] . similar programs within families would be expected to reduce transmission of hrv between family members. a recent cochrane review which included data from 67 randomised controlled trials and observational studies, investigated the effectiveness of physical interventions to reduce the spread of respiratory viruses. the authors concluded that respiratory virus spread can be reduced by hygiene measures (such as handwashing), especially around younger children and can reduce transmission from children to other family members [51] . controversy still exists and a newly published study showed that an antiviral hand treatment used by adult volunteers, recruited from a university community, did not significantly reduce rv infection or rv-related common cold illnesses [60] . asthmatic children should avoid close contact with people who have colds especially during the first three days of their illness. there is little evidence to support the effectiveness of face masks to reduce the risk of viral respiratory infections and consequently, the use of mask is generally not recommended for prevention of common cold [51, 61] . immune function and anti-viral defenses have a number of components, both specific and non-specific. asthmatic children can improve their immune function by following some simple advice including a healthy life style with regular exercise, a balanced diet, adequate sleep and avoiding environmental tobacco smoke, stress and unnecessary antibiotics. exercise has anti-inflammatory effects and in the long term can protect the development of chronic diseases and obesity [62] . regular exercise of moderate-intensity is associated with a reduced incidence of upper respiratory tract infection. however, long hours of intensive training appear to make children more susceptible to upper respiratory tract infections [63] [64] [65] . the recommended means of aerobic exercise is walking, with an optimal frequency of three to five days a week and an optimal duration of 20 to 30 minutes of continuous activity [66] . in a recent study, the iga secretion rate was negatively correlated with the incidence of infections [67] . a recent randomized trial comparing meditation and exercise with wait-list control among adults aged 50 years and older found significant reductions in ari illness [68] . malnutrition is the most common cause of immune deficiency worldwide and a balanced diet is fundamental for a healthy immune system. vitamin d deficiency has been associated with increased risk of infections, earlylife wheeze and reduced asthma control [69, 70] . vitamin a derivatives are involved in the regulation of the immune system and tissue inflammation as well as prevention of respiratory infections [71] . zinc, selenium and other trace elements are necessary for function of both innate and adaptive immune function. a high intake of fruit and vegetables ensures adequate consumption of nutrients and antioxidants and appears to be beneficial for asthma. although recent reviews have shown that zinc [72] , garlic [73] , echinacea purpurea [74] or ginseng [75] supplementation for several months may reduce cold incidence, there is insufficient evidence to recommend any vitamin or mineral supplementation in the management of asthmatic children without nutrient deficiency [76, 77] . however, a large controlled trial showed echinacea was ineffective in reducing infection rate or symptom severity of hrv infection in healthy young adult volunteers [78] . vitamin c supplementation failed to reduce the incidence of colds in the general population except in those exposed to short periods of extreme physical stress [79] . finally, it is worth remembering that infants who are not breastfed have significantly higher risk of respiratory, gastrointestinal, and other infections, as breast milk is a biologically active substance containing antimicrobial and immunomodulatory elements [80] [81] [82] . sleep and the circadian system exert a regulatory influence on immune functions. sleep deprivation can affect immune function in several ways including reduced natural killer cell activity, suppressed interleukin-2 production and increased levels of circulating proinflammatory cytokines [83, 84] . there is also evidence for an enhanced susceptibility to the common cold and pneumonia with poor sleep efficiency [85, 86] . air pollutants (nitrogen dioxide, ozone, particulate matter) and environmental tobacco smoke (ets) have long been correlated with multiple adverse effects on the immune system and susceptibility to viral respiratory tract infections in children [87] [88] [89] [90] . studies in europe and the united states have shown that 40% of children live with a smoker [34] and they have approximately twice the risk of contracting a serious respiratory tract infection in early life [91] . cigarette smoking leads to a longer duration of cough, greater frequency of abnormal auscultatory findings during acute respiratory tract illness [92, 93] and higher risk for severe exacerbations [94] . urinary leukotriene e4 levels identify children exposed to ets at high risk for asthma exacerbation [94] . there is strong evidence that some pharmacological preparations can help prevent viral infection by specific effects on immune system. these results have been promising with a hope that using these strategies can attenuate the role of viruses in asthma inception. ancient physicians of the middle east prescribed yogurt for curing disorders of the stomach, intestines and for stimulation of appetite. it is written in the old persian testament that "abraham owed his longevity to the consumption of sour milk" [95] . the popularity of probiotics and intestinal microbiota significantly increased when the nobel prize-winning russian scientist eli metchnikoff suggested in 1908 that the long life of bulgarian peasants resulted from their consumption of fermented milk products [96] . the term probiotic, meaning for life, is used for live micro-organisms (typically of the bifidobacterium and lactobacillus species) administered in adequate amounts which confer a beneficial physiological effect on the host. prebiotics are nutrients, in particular oligosaccharides, which foster the growth of probiotics in the colon. the term synbiotics is used when a product contains both probiotics and prebiotics [97] . up to 100 trillion bacteria from different species colonize the human gut [98] . this microbiota participates in: host metabolism, vitamin synthesis, control of epithelial cell growth, protection from infectious microbes, and helps proper development and function of the immune system. there is constant cross-talk between microbiota and gut-associated lymphoid tissue (the largest lymphoid tissue of the human body which contains more than 60% of all body lymphocytes) to establish mucosal immune tolerance in the gut. common mucosal immunity describes the phenomenon where immune cells, especially regulatory t-cells, traffic to and influence responses at other mucosal surfaces, including the lungs [99] . alteration in the microbiota composition (dysbiosis) results in immunological dysregulation that may underlie many human diseases such as inflammatory diseases [100] , obesity [101] , allergy [102] and autoimmunity [103] . reduced bacterial diversity in the infant's gastrointestinal tract has been associated with an increased risk of allergic sensitization and allergic rhinitis but not asthma or atopic dermatitis [102] . in the first year of life, especially the first few weeks, the microbiota of the newborn is highly variable during this critical time of post-natal maturation of the immune system. microbiota is shaped by genetic and environmental factors including: mode of delivery, neonates born by means of vaginal delivery are exposed to mothers gut, skin, and vaginal flora [104] ; breast feeding and diet [105] ; farm or urban living [106] ; vitamin d status [107] ; and antibiotic consumption [98, 108] . this knowledge stimulated interest in the use of probiotics and prebiotics as the intentional introduction or encouragement of specific microbes to shape immune system development. specifically, the microbiota can activate distinct tolerogenic dendritic cells in the gut and through this interaction can drive regulatory t-cell differentiation that modulates both th1 and th2 responses inside and outside the gut [109] [110] [111] . probiotics have been successfully used for the treatment of several gastrointestinal disorders (viral and antibiotic-associated diarrhea, inflammatory bowel disease) [112, 113] . however, attempts to prevent or treat allergic disorders such as eczema, asthma and allergic rhinitis have had inconsistent results [99, 109, [114] [115] [116] . there are a growing number of clinical trials using probiotics for the prevention and management of respiratory infections. while the precise mechanisms are largely unknown, speculations include: probiotics compete against pathogens; increase the barrier function in respiratory epithelium; immunostimulatory effects by enhancing cellular immunity with increased activity of natural killer cells and macrophages in airways [117] . probiotics reduce the frequency of gastrointestinal and respiratory tract infections in children who attend day care centres [118] . they have also been found to reduce the incidence of ventilator-associated pneumonia, respiratory infections in healthy and hospitalized children, and reduce the duration of common cold symptoms [119] [120] [121] [122] . one study demonstrated that that daily probiotic supplementation for six winter months in children three to five years of age reduced the incidence of fever, coughing and rhinorrhea by 32-43% with no notable adverse events [123] . probiotic combination with vitamins and minerals also reduced the duration and severity of common cold [124] . a recent cochrane review of 14 randomised controlled trials showed that probiotics were better than placebo in reducing the number of episodes of acute upper respiratory infections (uris) and reducing antibiotic use, while there were no differences in the mean duration of an episode and no increase in adverse events [125] . probiotic foods such as probiotic milk or yogurt (functional foods) containing well-defined probiotic strains may reduce the risk of catching the common cold and represent a simple, safe, effective, available and affordable method for preventing respiratory infections in children [112, 120, [126] [127] [128] [129] [130] [131] . although there are several clinical trials that showed the preventive effect of probiotic, prebiotic [132] or synbiotics treatments [133] on respiratory infections, not all studies are positive with some failing to show any significant preventive effect [134] . to explain the different results in clinical trials, it is of particular importance to point out that the immunomodulatory capabilities of probiotics are strain-dependent. difference in dosage, duration of intervention, population and environmental background may also affect the results. one major limitation in this field is that it is not possible to test just how "probiotic" a particular preparation is. technical advances will be required before some of the apparent discrepant results of studies can be resolved. several immunostimulants, including herbal extracts, bacterial extracts, synthetic compounds, have been promoted as increasing the immune defences of the respiratory tract. a recent cochrane review included data from 35 placebo-controlled trials including 4060 participants below the age of 18 years in which various types of "immunostimulants" were used to reduce acute respiratory tract infections, involving either upper or lower airways. the authors concluded that immunostimulants reduced the incidence of acute respiratory infections by 40% on average in susceptible children, but that trial quality was generally poor and a "high level of statistical heterogeneity was evident". a subgroup analysis focusing on bacterial immunostimulants, including om85, produced similar results with lower statistical heterogeneity [135] . om-85 bv (broncho-vaxom) is an immunostimulant extracted from eight common bacterial pathogens of the upper respiratory tract: haemophilus influenzae, diplococcus pneumoniae, klebsiella pneumoniae and ozaenae, staphylococcus aureus, streptococcus pyogenes and viridans, neisseria catarrhalis and has been used in several countries around the world for as long as 20 years [136] . recent studies showed that om-85 bv can reduce the number of acute respiratory infections by 25% to 50% compared with placebo in children with a history of recurrent infection [137] . of particular interest, razi et al. showed that children between the age one and six years with recurrent wheezing who were given om-85 bv had a 40% reduction in the rates of wheezing over the subsequent 12 months, compared to placebo (p < 0.001). in addition, the duration of each wheezing attack was two days shorter in the group given om-85 bv than in the group given placebo (p = 0.001) [138] . again, direct evidence of the mechanisms involved are lacking from human studies. however, recent data from rodents shows that baseline regulatory t lymphocyte activity in the airways can be boosted by microbe-derived stimulation of the gut [139] . bacterial immunostimulants were also shown to enhance innate immunity (i.e. intensification of phagocytosis) and adaptive immunity [140] . as discussed above, evidence exists for an impaired innate immune response to respiratory viral infections in asthmatics [141] . entry of rhinovirus into normal epithelial cells initiates a vigorous innate immune response with ifn-β secretion and apoptosis induction. in asthma, ifn-β and ifn-λ responses are impaired, resulting in viral replication, cell cytotoxicity, enhanced virion shedding and increased susceptibility to common cold [17, 142] . epithelial cells of asthmatic patients responded to exogenous treatment with ifn-β exhibiting reduced rhinovirus release (cakebread, xu et al. 2011; jackson, sykes et al. 2011). if the proposed deficiency of type i and iii contribute to asthma exacerbations [21, 22, 143] , correcting this deficiency with exogenous interferons would be a logical approach. the advantages of interferon application include the broad spectrum of activity with low risk of resistance development [47] . prophylactic intranasal recombinant ifn-α and ifn-β have been shown to be effective against rhinovirus infection in humans [144] [145] [146] . the results of these clinical trials are awaited with interest [147, 148] . however, the systemic symptoms associated with severe viral infections, e.g. influenza, are associated with interferons, so careful dosing may be required. considering the occurrence of the local side effects, neutropenia and cost, the use of long-term prophylaxis with daily, intranasal administration of interferons is not feasible [144] . however, randomized clinical trials using similar strategies are currently underway in adults with chronic respiratory disease and the results are keenly awaited. vitamin d deficiency is a common worldwide problem [149] [150] . beside importance for bone health, vitamin d plays an important role in adequate function of both the innate and adaptive immune systems including development of dendritic cells and regulatory t lymphocytes [151, 152] production of antimicrobial proteins by airway epithelium [153] , modifying the effect of intestinal flora on inflammatory disorders [107] , and modulation of the inflammatory response to viral infections [154] . recent reports suggest that vitamin d might play a role in the recent increase in allergic disease [155] [156] [157] . vitamin d insufficiency has been associated with a higher incidence of respiratory tract infection, wheezing illness in children [158] , reduced asthma control [159] , emergency department visits, severe asthma exacerbations and hospitalizations [70, 160] . in a recent study of 48 children from five to 18 years of age, with newly diagnosed asthma, vitamin d supplementation during the northern hemisphere winter months (september to july) prevented declining serum concentrations of 25(oh) d and reduced the risk of asthma exacerbation triggered by acute respiratory tract infections [161] . macrolides possess anti-inflammatory and immunomodulatory properties extending beyond their antibacterial activity [162] . indeed, they can attenuate pro-inflammatory cytokine production by bronchial epithelial cells, neutrophils and macrophages that may contribute to clinical improvement in many patients with chronic airway inflammation [163] [164] [165] . azithromycin has anti-rhinoviral activity and can reduce hrv replication and release by increasing interferon production from epithelial cells [42, 166, 167] . macrolide antibiotics inhibit rsv infection in human airway epithelial cells [168] . a three weeks treatment with clarithromycin in rsv bronchiolitis had statistically significant effects on hospital length of stay and rate of readmission to the hospital within six months after discharge [169] . however, direct evidence of macrolides preventing respiratory viral infection in children is lacking. as the majority of respiratory viral infections in young children are caused by hrv or rsv, we will briefly discuss anti-viral strategies to prevent hrv or rsv infections in asthmatic children. because there are more than 100 serotypes of hrv, antiviral drugs are considered to be more effective than vaccination. antiviral agents have been designed to inhibit rhinovirus attachment, entry to the cell, viral uncoating, and rna and protein synthesis [47] . table 1 shows how intervention strategies can be targeted to various steps in the infective process. hrv has the icosahedrally shaped capsid formed by 60 identical copies of viral capsid structural proteins vp1-4. the capsid protects the single-stranded, positive sense rna genome. while hrv-a and -b most often induce a self-limited upper respiratory infection, the recently discovered hrv-c was associated with slris in infants, bronchiolitis, and asthma exacerbations in children [170, 171] . prevention of attachment, entry and uncoating hrv deposits on nasal or conjunctival mucosa and is transported to the posterior nasopharynx by mucociliary action of epithelial cells [172] . the so-called major group of hrv uses intercellular adhesion molecule-1 (icam-1) as their receptor [173] and the minor group attach to low density lipoprotein (ldl) receptor and very-ldl (vldl) receptors on epithelial cells in the adenoid area to bind and enter cells [174, 175] . viral attachment can be prevented by specific anti-hrv neutralizing antibodies, anti-receptor antibodies and soluble receptor molecules. endothelial cells express histamine receptors and increased adhesion molecule expression, such as icam-1, was demonstrated by histamine infusion. second-generation h1-antihistamines decrease expression of icam-1 on cultured bronchial epithelial cells [176] . zinc may also act as an antiviral agent by reducing icam-1 levels [177] . the monoclonal antibody to the cellular icam-1 was not effective. cfy196 (coldsol) is a nasal spray multivalent fab fusion proteins against icam-1 with a better avidity and in vitro potency against hrv [178] . tremacamra, a soluble intercellular adhesion molecule 1 reduced the severity of experimental rhinovirus infection [179] . pleconaril, an orally administered antiviral drug, acts by binding to a hydrophobic pocket in viral protein 1, and stabilizes the protein capsid so that the virus cannot release its rna genome into the target cell. outcomes of clinical trials with pleconaril have revealed mixed results and new compounds are currently being developed [180] . despite extensive research, no agent has been approved for prevention and/or therapy of rhinovirus-induced diseases so far. ruprintrivir selectively inhibits hrv 3c protease and shows potent, broad-spectrum anti-hrv activity in vitro. ruprintrivir nasal spray (2% solution) prophylaxis reduced the proportion of subjects with positive viral culture by 26% and reduce viral titers, but did not decrease the frequency of colds [181] . hrv rna synthesis during replication can be blocked by deoxyribozymes [182] , morpholino oligomers [183] , and small interfering ribonucleic acids [184] . the novel antiviral therapies that have been discovered recently, may one day add significantly to the armamentarium of antiviral agents, against respiratory viral infections in asthmatic children. maternally-derived rsv neutralizing antibodies help to protect infants against rsv hospitalization [185] . palivizumab, a humanised monoclonal antibody against the rsv fusion protein is effective against rsv and wheezing in children and reduces hospitalization in high-risk individuals [185, 187] . rsv prophylaxis with palivizumab significantly reduced the relative risk of subsequent recurrent wheezing in nonatopic premature infants [40] . motavizumab is another monoclonal antibody against rsv, with an approximately 20-fold increase in ability to neutralize rsv and 100 fold increase in ability to reduce viral titers compared to palivizumab [188, 189] . motavizumab was also found to be superior to palivizumab in reducing outpatient medically attended lower respiratory illness by 50% [190] . vaccination against hrv and rsv have been in development for quite some time, but there are no safe and effective vaccines at present [33, 191] . high rates of exposure to viruses in early life, presence of more than 100 serotypes of hrv, the presence of maternal antibodies, the risk of vaccine induced disease and relative immaturity of the infant immune system make effective vaccination difficult [186, 192, 193] . respiratory viral infections are major contributors to the global burden imposed by asthma. in early life, they contribute to the inception of asthma and are responsible for most of the acute exacerbations for asthma in childhood. while the debate is not completely settled, children at high risk of developing asthma and those with established asthma may be at increased risk of acquiring respiratory viral infections and may be less able to contain these to the upper airway. several simple general strategies can be used to help prevent respiratory viral infections in asthmatic children (table 2) , with good personal hygiene, hand-washing and avoidance of cigarette smoke likely to reduce respiratory viral infections. general immuno-stimulatory strategies, such as eating a healthy balanced diet, active probiotic supplements and bacterial-derived products, e.g. om-85, may reduce recurrent infections in susceptible children. while research continues on specific anti-viral therapies, including vaccination, there are no currently available practical therapies that are suitable for widespread use. the role of preventative strategies in primary prevention of asthma in high risk children is of considerable academic interest and a number of studies are currently in the pipeline. the results are awaited with interest. the authors declare they have no competing interests. author's contribution ha and pds conceived and designed the review. all authors reviewed the articles, abstracted data, and participated in the data synthesis. ha, pds, ysc drafted the current manuscript, with critical review by pds and cmj. all authors contributed, 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recurrent wheezing development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract a review of palivizumab and emerging therapies for respiratory syncytial virus motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial vaccination for asthma exacerbations viral respiratory tract infections and asthma: the course ahead association between human rhinovirus c and severity of acute asthma in children submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors wish to acknowledge dr. catherine gangell for reviewing the manuscript. key: cord-314449-ukqux772 authors: curtis, l.t. title: prevention of hospital-acquired infections: review of non-pharmacological interventions date: 2008-06-02 journal: j hosp infect doi: 10.1016/j.jhin.2008.03.018 sha: doc_id: 314449 cord_uid: ukqux772 hospital-acquired (nosocomial) infections (hais) increase morbidity, mortality and medical costs. in the usa alone, nosocomial infections cause about 1.7 million infections and 99 000 deaths per year. hais are spread by numerous routes including surfaces (especially hands), air, water, intravenous routes, oral routes and through surgery. interventions such as proper hand and surface cleaning, better nutrition, sufficient numbers of nurses, better ventilator management, use of coated urinary and central venous catheters and use of high-efficiency particulate air (hepa) filters have all been associated with significantly lower nosocomial infection rates. multiple infection control techniques and strategies simultaneously (‘bundling’) may offer the best opportunity to reduce the morbidity and mortality toll of hais. most of these infection control strategies will more than pay for themselves by saving the medical costs associated with nosocomial infections. many non-pharmacological interventions to prevent many hais will also reduce the need for long or multiple-drug antibiotic courses for patients. lower antibiotic drug usage will reduce risk of antibiotic-resistant organisms and should improve efficacy of antibiotics given to patients who do acquire infections. much of the recent research on nosocomial infections has dealt with the need for new antibiotics, better antibiotic management and better diagnostic techniques to detect infections earlier. better drug treatment and earlier infection diagnosis can certainly play a major role in reducing morbidity and mortality from hospital-acquired infections (hais). however, there are many nonpharmacological interventions that can significantly reduce the incidence of hais, but these are often overlooked in practice. this review is not exhaustive and will not attempt mathematical data analysis but will examine recent research that examines non-pharmacological interventions for reducing hais. it will also include a brief description of the morbidity, mortality and medical costs associated with nosocomial infections, along with a brief discussion of the routes by which hais spread. a bibliographic search between january 1995 and january 2008 was conducted in databases including pubmed, medline and google scholar. additional research articles were collected from conference proceedings, books and pre-1995 journal articles as appropriate. many terms were used in the literature searches including nosocomial, hospital acquired, mrsa (meticillin-resistant staphylococcus aureus), staphyloccoccus, streptococcus, vre (vancomycinresistant enterococcus), clostridium difficile, legionella, klebsiella, tuberculosis, airborne infection, waterborne infection, hand washing, hospital cleaning, urinary catheters, central catheters, haemodialysis, ultraviolet light, hepa (high-efficiency particulate air) filtration and many others. a total of 160 articles was included in this review. care was taken to see that a balanced representation of articles was presented. hais cause a huge amount of morbidity and mortality. klevens et al. used data from the national nosocomial infections surveillance (nnis) system, and data from the national hospital discharge survey and the american hospital association survey to estimate nosocomial morbidity and mortality. 1 this study estimated that there were 1 737 125 nosocomial infections in the usa in 2002, of which 561 667 were due to urinary tract infections, 290 485 to surgical site infections, 250 205 to pneumonia, 248 678 to bloodstream infections and 386 090 to other causes. it is difficult to obtain a precise estimate of deaths from nosocomial infections since patients often die from several causes and infection is often not mentioned on death certificates of patients who die of a combination of a chronic illness (such as cancer) and acute infection(s). 1 estimated annual deaths in the usa due to hais in 2002 was 98 987. 1 in 2005, an estimated 18 650 died in the usa of mrsa infections, of which most were nosocomial. 2 a study of 524 consecutive deaths in a spanish 800-bed tertiary care hospital reported that 21.3% of the deaths of patients which occurred more than 48 h after admission were due to nosocomial infections. 3 while it has been long known that hais are very expensive to treat, cost estimates of nosocomial infections vary. a us study of 1 355 347 admissions in 55 us hospitals from 2001 to 2006 estimated that each nosocomial infection increased medical costs by $12,197 . 4 a french study reported that hospital-acquired sepsis increased medical costs by a mean of v39,500. 5 various studies have estimated that the average cost of ventilator associated nosocomial pneumonia from us$10,019 to $50,000 per case. 6, 7 hospital-acquired bacteraemia has been estimated to increase medical costs in a belgian study by an average of v12,853 and in a michigan study by $34,508. 8, 9 a british study reported that the average increased medical cost for each central venous catheter infection was £6,200. 10 traditionally, it has been believed that most nosocomial infections, with a few exceptions such as tuberculosis and aspergillus and viruses like respiratory syncytial virus (rsv), influenza, rhinoviruses and coronaviruses, are not spread through the air. 11 while a large percentage of hais are spread through surface contact (such as hands) or by catheters, intravenous (iv) lines or surgical incisions, many nosocomial infections can also spread through the air. it was previously believed that most pathogenic bacteria could not survive as bioaerosols and spread significant distances to infect patients. however, many airborne pathogenic bacteria are viable but not culturable, and some experts have estimated that as little as 1% of viable bacteria are culturable by standard microbiological techniques. 11, 12 for example, heidelberg et al. reported that viable counts of serratia marcescens, klebsiella planticola and cytophaga allerginae in 4-hour-old bioaerosols were, respectively, 48, 73 and 66% of the original counts even though none of the bacteria was culturable on tryptic soy agar plates. 12 many pathogens present on sneezes evaporate in less than a second into small droplet nuclei of about 2 mm diameter. 13, 14 such small droplet nuclei can remain suspended for hours and travel long distances before settling. 14 a number of studies have reported airborne transmission of many pathogenic bacteria to humans including mrsa, coagulase-negative staphylococci, corynbacterium diphtheriae, neisseria meningitidis, bordetella pertussis, acinetobacter and pseudomonas. 15e20 a mouse study reported two strains of klebsiella pneumoniae which could infect and multiply in mouse lungs after airborne exposure. 21 therefore it may be concluded that while many nosocomial bacterial infections are spread by contact or by iv routes, the airborne route is also an important source of many hais. 11 pathogens from hospital water are another underappreciated and underdiagnosed source of hospital infection. 22, 23 over 30 published studies employing both epidemiology and molecular biology techniques [such as polymerase chain reaction (pcr) and dna probes] have confirmed that contaminated hospital water sources can cause nosocomial outbreaks from many pathogens including legionella, mycobacteria, pseudomonas, stenotrophomonas, serratia, acinetobacter, aeronomas and moulds such as fusarium, aspergillus and exophialia. 22,23 hospital water can also be contaminated with amoebae and viruses. 24 it is estimated that waterborne nosocomial pseudomonas infections kill 1400 annually in the usa. 22 legionella is found in many hospital water systems and can persist for years. viable legionella were found in the water systems of 14 of 20 (70%) us hospitals and 17 of 20 (85%) spanish hospitals. 25, 26 environmental pulsed-field gel electrophoresis studies have confirmed that specific legionella strains can persist for as long as 17 years in hospital water supplies. 27 viable pathogens can grow in many sources of hospital water including drinking water, hand-washing water, ice, dialysis water, shower water, water in storage tanks and distribution systems, water from decorative pools/fountains, and carpets, furniture, ventilation ducts and building materials that have become wet. contaminated environmental surfaces (such as bedside rails) are also an under-recognised source of hospital infections. 28 many surfaces in hospitals contain viable pathogens such as mrsa and vre. 28 in rooms of patients with diarrhoea, viable mrsa has been collected from 59% of the room surfaces and viable vre has been collected from 46% of room surfaces. 29,30 many strains of mrsa and vre can remain viable for several weeks to several months on dry surfaces. 31, 32 infections can also be spread to hospitalised patients via drugs, intravenous solutions, cleaning solutions or by foodstuffs. a review of 2250 hais obtained via contaminated substances reported that the most commonly involved items were disinfection materials (n ¼ 622 patients), heparin solutions (n ¼ 451), red blood cells, clotting factors and other blood products (n ¼ 333), albuterol inhalers (n ¼ 143), total parenteral nutrition (n ¼ 109), propofol (n ¼ 53), rantidine (n ¼ 50) and ultrasound gel (n ¼ 36). 33 the remaining part of the results section will concentrate on research on interventions to reduce hais. table i gives a summary of 48 nonpharmacological interventions that have either been proven to reduce nosocomial infections or some level of evidence suggests may be effective. hand washing, gloving, gowning and personal items frequent and adequate hand washing is the best way to prevent spread of most nosocomial infections. the extreme importance of hand washing has been known since at least 1847, when dr ignaz semmelweis discovered that washing hands before performing obstetric exams on pregnant women reduced childbirth-related infectious mortality from more than 10% to less than 1%. 34 however, rates of hand washing among healthcare providers usually range from only about 20 to 50% per hospital patient encounter, although some studies have reported hand-washing rates as high as 81%. 35e37 viable pathogens are often found on hands of healthcare providers. various studies have reported the following pathogens on the respective percentage of healthcare providers' hands: acinetobacter spp. 3e15%, clostridium difficile 14e59%, klebsiella spp. 17%, mrsa up to 16.9%, pseudomonas spp. 1.3e25%, rotavirus 19.5e78.6%, vre to 41% and yeasts (including candida) 23e81%. 38 most studies have reported that increases in hand-washing rates significantly reduce rates of hais including mrsa although a few studies have reported negative results. 37,39e45 alcohol-based hand-washing solutions are generally considered to be more effective than soap and water. compared with plain soap and water, some studies have reported significantly lower rates of nosocomial infections when alcohol-based solutions or chlorhexidine-or triclosan-based hand-washing agents are used. 36,43,46,47 an australian hospital study noted a hand hygiene programme that switched from soap to a chlorhexidine/isopropyl alcohol solution reduced mrsa bacteraemia by 57% (p ¼ 0.01) and reduced clinical isolates of lactamase-resistant e. coli and klebsiella by 90% (p < 0.001). 41 some studies have also reported that the economic savings that alcohol or chlorhexidine hand-washing solutions provide by reducing nosocomial infections will far more than pay for the cost of the hand-washing solutions. 36, 47 it is estimated that hand washing with plain soap for 30 s removes most soil and dirt, eliminates about 90% of transient hand flora but a low percentage of resident hand flora. 48 hand washing for 15 s with a soap containing chlorhexidine or triclosan removes most soil and dirt and about 99.9% of transient flora and about 50% of resident flora. hand rubbing for 15 s with an alcohol-based gel does not remove soil or dirt, but kills about 99.999% of transient flora and about 99% of resident flora. 48 many healthcare providers prefer using alcoholbased solutions instead of soap and water, and compliance rates are generally higher when alcohol-based hand-washing solutions are used. 49 use of alcohol-based cleaners saves time and these generally abrade and irritate the skin less than antiseptic soaps. 49 however, some people complain that alcohol-based cleaners dry out and crack their skin. 49 hospitals and healthcare providers may want to experiment with several alcohol or chlorhexidine-based hand cleaners. soap and water may still have to be used in cases when hands are visibly soiled. in that case, staff and visitors should wash hands carefully for at least 15 s with soap and water. 49 table ii lists six abridged 'golden rules' to improve hand hygiene compliance proposed by dr gunter kampf. 50 in 2002, the us centers for disease control and prevention (cdc) published new guidelines for hand hygiene. 51 major changes of these guidelines over the 1995 guidelines included use of waterless alcohol-based cleaners (unless hands are visibly soiled), prohibition of artificial fingernails and an institutional mandate to provide staff education and develop a multidisciplinary programme to monitor compliance. to measure effectiveness of these new cdc guidelines, an anonymous survey of 1359 staff in 40 hospitals was made after the new guidelines had been in force for a year. this survey found that mean hand-washing rates were only 56.6%, and 45% of the hospitals had no multidisciplinary programme to improve compliance. 51 it is not certain what type of glove provides the best protection for infection control. some studies have suggested that latex gloves are somewhat better in preventing penetration of water and virus than vinyl gloves. 52 however, about 3e16% of healthcare workers are sensitive to latex and sometimes experience severe respiratory reactions to it. if latex gloves are used in the healthcare setting, only the powder-free gloves should be used since these release much lower levels of latex allergens than the powdered latex gloves. nitrile gloves also have good barrier penetration but are more expensive and heavier than either latex or vinyl gloves. 52 gowns are often used in rooms of patients with infectious disease. data on gown use and nosocomial prevention are sparse. one study reported that use of disposable gowns in an intensive care unit (icu) was associated with a 54% reduction in vre (p < 0.01). 53 gown use in this study also produced an annual net benefit of $419,346 in the icu by averting an estimated 58 vre cases. 54 another study reported that use of gowns was associated with a modest and insignificant drop in mrsa cases. 55 rates of gown usage by healthcare providers and staff are generally mediocre, with one study reporting mean gown usage in rooms of patients with contact precautions was only 76% for healthcare providers and 65% for visitors. 56 shoe and head covers are often recommended for use in areas containing immunocompromised or surgical patients. although bacterial pathogens have been collected from shoes, research on the use of shoe covers and/or separate hospital shoes and spread of pathogens has been meagre. 57 one study reported that wearing gowns and shoe covers in bone marrow surgery did not significantly reduce patient infection risk (as measured by antibiotic therapy). 58 an experimental laboratory study involving sham surgery reported significantly lower levels of airborne bacteria when headgear was worn versus no headgear. 59 various studies have reported that nosocomial pathogens are present on many items of healthcare providers such as laboratory coats, stethoscopes, blood pressure cuffs, ekg electrodes, pens, finger rings, neck ties, artificial nails and ambulances. 60e68 to prevent spread of nosocomial infections, these items should be disinfected or cleaned regularly. disposable one-use electrodes even a small number of nosocomial infections prevented will outweigh the cost of effective hand hygiene products. 5. encourage senior staff to set a good example to motivate junior staff. 6. have adequate staff:patient ratios. are now available. 63 sometimes, pieces of equipment such as stethoscopes or blood pressure cuffs are dedicated to one patient only in order to limit spread of pathogens. proper cleaning techniques and proper cleaning chemicals can also significantly reduce hospital pathogen levels and risk of nosocomial infections. one study utilised a fluorescent marker solution to determine cleaning efficacy of 13 369 surfaces found in 1119 patient care rooms of 23 us hospitals. 69 terminal room cleaning after patient discharge was able to adequately clean only a mean of 49% of the standardised surfaces, including less than 30% for toilet hand holds, bedpan cleaners, room door knobs and bathroom light switches. carling et al. recommended that hospitals monitor performance of cleaning personnel and provide feedback and training as needed to optimise cleaning effectiveness. 69 several studies have reported that hospital cleaning personnel often receive little initial training and, after receiving instruction, often do a much better job of eliminating pathogens by their cleaning. 70, 71 an illinois prospective study reported that following a cleaning educational programme, average rates of cleaning icu surfaces rose from 48 to 87%. rates of vre infection were reduced by 64% [95% confidence interval (ci): 0.19e0.68]. 72 a british retrospective icu study reported that significantly higher rates of mrsa infection were associated with inadequate surface cleaning and nurse understaffing. 73 research on chemicals used to clean non-porous surfaces (such as floors, walls, tables etc.) in hospitals and their effects on reducing nosocomial infection has been sparse. 74 in a 2004 literature review, no randomised controlled trials and only four cohort studies could be identified. 74 three studies detected no significant differences between nosocomial infection rates when comparing surface cleaning with aldehydes, quaternary ammonium compounds, active oxygen cleaners or ortho-benzyl para-chlorophenol compared with plain detergent solutions. the fourth study reported that use of 1:10 hypochlorite (bleach) solution was associated with a significantly lower rate of c. difficile infection in bone marrow patients compared with cleaning with quaternary ammonium compounds. 75 another study of 17 rooms which housed vre-positive patients found that 16 (94%) of the rooms' surfaces contained viable vre before cleaning, but 0 (0%) had viable vre after thorough cleaning with a 10% bleach solution (p < 0.001). 71 hydrogen peroxide vapour may be used to decontaminate rooms containing pathogens. a british study compared manual cleaning of rooms (via a protocol compliant with uk standards) with a 5 h protocol using 40 min of 500 ppm hydrogen peroxide vapour to decontaminate rooms. 76 in 10 surgical ward rooms, 89% of 124 swab samples were positive for viable mrsa before manual cleaning, and 66% of 124 matched swabs were still positive for viable mrsa after cleaning. by comparison, in six other surgical rooms, viable mrsa was found on 72% of 85 swabs before hydrogen peroxide treatment, but on only 1% of 85 matched swabs following hydrogen peroxide treatment. during the hydrogen peroxide disinfection, hydrogen peroxide levels in adjacent rooms were no greater than 1 ppm at head height. 76 more study is needed on the safety and efficacy of this hydrogen peroxide vapour technology. research is currently underway to use copperoxide-impregnated textiles and paints in order to prevent spread of infections. 77 better nutrition can also play a critical role in reducing nosocomial infections. malnutrition is very common in hospitalised patients. a review of 110 published studies in acute care patients reported that malnutrition ranged from 13 to 78% of all hospitalised patients and 42e91% of hospitalised elderly. 78 malnutrition was measured by such parameters as weight, weight loss, body mass index, grip strength, respiratory function, nutritional intake and blood levels of albumin, and prealbumin. 78 many nutrients play a key role in maintaining immunity including protein, omega-3 fatty acids, vitamins a, b 6 , b 12 , c, d, and e; selenium, zinc, copper and iron. 79 most of these nutrients become depleted following acute illness. 79 malnutrition is a major risk factor for infection. a study of 630 hospitalised patients reported that the odds ratio risk of hais was 4.98 times as great (95% ci: 4.6e6.4) in severely malnourished patients compared with adequately nourished patients. 80 other studies have reported that malnourished elderly are significantly more likely to acquire nosocomial infections and are significantly more likely to acquire pneumonia compared with well-nourished elderly. 81, 82 better nutrition may play a major role in reducing nosocomial infection in acutely ill hospital patients who cannot eat by the regular oral route. in recent years, 'immunonutrition' enteral formulas containing larger quantities of antioxidant vitamins, zinc and other trace metals, omega-3 fatty acids and amino acids like glutamine have become more commonly used. meta-analysis has calculated that enteral immunonutrition in hospitalised patients is associated with a 46% lower risk of nosocomial pneumonia (11 studies, p ¼ 0.007), a 55% lower risk of bacteraemia (nine studies, p ¼ 0.0002), a 78% lower risk of abdominal abscesses (six studies, p ¼ 0.005), and a 34% lower risk of urinary tract infections (10 studies, p ¼ 0.05) compared with patients receiving standard enteral formula. 83 many hospitalised patients develop serious c. difficile infections after several antibiotic courses. probiotic bacteria and yeasts can be helpful in preventing or clearing infections by c. difficile and other bacteria. meta-analysis of 25 studies reported that supplemental saccharomyces boulardii, lactobacillus spp. or bifidobacterium spp. were associated with significantly lower levels of antibiotic-associated diarrhoea. 84 meta-analysis of six studies indicated that s. boulardii was effective in reducing the incidence of c. difficile diarrhoea. 84 yoghurt containing active lactobacillus spp. has been found to be effective in preventing or clearing infections caused by c. difficile and vre in hospitalised patients. 85, 86 housing patients in separate rooms, pathogen surveillance and 'search and destroy' strategies for nosocomial infections housing patients in separate rooms may reduce risk of hais. a quebec observational study of a 14bed icu measured rates of nosocomial infections during a 2.5-year period. 87 the incidence of nosocomial mrsa, pseudomonas and candida infections per 1000 patient-days were respectively 4.1, 3.9, 38.4 for patients housed in multiple patient icu rooms and 1.3, 0.7, 13.8 for patients housed in single rooms (p < 0.001 for all three comparisons). 87 however, another study conducted in two british icus reported that isolating patients had little effect on mrsa acquisition rates. 88 screening patients at hospital admission for common pathogens like s. aureus may be an effective way to prevent the pathogens from becoming established infections. a recent study estimated the health and economic impacts of preadmission s. aureus screening and subsequent decolonisation therapy for the 7.1 million us patients who undergo elective surgery annually. an s. aureus screening and decolonisation protocol for all elective surgical patients was projected to save 935 inpatient lives and save $231 million in net medical costs annually. 89 for some nosocomial infections such as mrsa, 'search and destroy' control strategies have been developed. 90 such search and destroy protocols involve a number of interventions including: (i) active surveillance which includes a nasal swab for mrsa cultures upon patient admission and every third day throughout hospitalisation. (ii) contact precautions including proper gloving, gowning and mask use. (iii) treatment of carriers with antibiotics and surface disinfectants. (iv) microbiological controls: starting 48 h after the end of treatment three control samples were taken at colonised sites. if mrsa was isolated, treatment was resumed. (v) isolation or cohorting: mrsa positive patients were placed in separate rooms or cohorted with other patients with mrsa infection. (vi) educational programme on infection controls for healthcare workers. such a search and destroy mrsa programme was found to reduce mrsa infection in a spanish icu from 3.5 to 1.7 cases per 1000 patient-days (p ¼ 0.024). 90 the netherlands has been addressing the mrsa problem since the 1980s with a programme of patient isolation, search and destroy protocols and restrictive antibiotic usage. 91, 92 by 2001, mrsa comprised less than 1% of clinical s. aureus specimens collected in netherlands hospitals, while mrsa comprised 28%, 33%, 19% and 50% of clinical s. aureus cultures respectively in belgium, france, germany and the usa. 92 molecular biology techniques such as pcr and gel electrophoresis techniques have been very useful in hospital surveillance and tracking of nosocomial infections. 93 such techniques can be used to test samples from patients, staff and environmental substrates. implementation of an enhanced infection control programme which included molecular typing to assess microbial clonality was associated with an 11% reduction of nosocomial infections in a large hospital (p ¼ 0.027). 94 this infection control programme was calculated to annually prevent 270 nosocomial infections and save us$2.2 million in net healthcare costs. inadequate nurse staffing may increase risk for nosocomial infections. 73 a us study of 15 846 patients in 51 icu units served by 1095 nurses measured rates of nosocomial infections and nurse staffing levels. 95 icu units with higher nurse staffing had significantly lower rates of centralline-associated infections, ventilator-associated pneumonia, ducibitus ulcers and 30 day mortality (p < 0.05 for all comparsions). 95 a 12-month study from a 1394-bed taiwanese hospital reported that higher nursing staff levels were associated with significantly lower levels of urinary tract infections (p < 0.001), respiratory infections (p ¼ 0.004) and pressure ulcers (p ¼ 0.031). 96 public reporting of hais may provide a good incentive for hospitals to reduce nosocomial infection rates. in the usa, since 2003, a number of states have passed laws mandating reporting of hai rates. 97 there is some concern that such reporting of infections may undercount the true nosocomial infection rates. 97 there is also concern about the need for proper adjustment of infection rates for factors such as age, chronic health problems and preadmission health of the patients received by specific hospitals. 97 many administrative problems have beset these mandating laws. for example, the state of illinois passed the 'hospital report card act' (sb 59) in 2003 mandating public reporting of several types of nosocomial infections. 98 however, by january 2008 none of the reporting systems had been implemented. 98 about 80e95% of hospital-acquired urinary tract infections originate from urinary catheters. 99 urinary catheters should be used only if necessary and should be removed as soon as practicable. 99 ,100 some studies have indicated that early removal of urinary catheters can reduce urinary tract infection rates by up to 40%. 101 about 15% of urinary hais have been linked to improper handwashing and poor aseptic techniques in cleaning the urinary meatus area and inserting and maintaining the urinary catheters. 100 however, studies have shown that vigorous twice-daily meatal cleaning does not seem to reduce urinary infection rates. 100 many studies and meta-analyses have reported that silver alloy/silver hydrogel-tipped urinary catheters significantly reduce urinary tract infections. a 1998 meta-analysis of eight published studies reported that use of silver-tipped urinary catheters was associated with a mean 41% reduction of urinary tract infections (95% ci: 0.42e0.84). 102 a more recent study reported that using silver-tipped catheters (both short-and long-term users) was associated with a 57% reduction in urinary tract infections. 103 none of the 50 bacteria and yeasts isolated from these silver-tipped catheters developed any resistance to silver. 103 these studies have also indicated that while silver-tipped urinary catheters cost more than standard catheters, the saving in nosocomial urinary infections far more than pays for the extra cost of the silver-tipped catheters. 102, 103 however, according to meta-analysis of urinary catheters inserted for less than 30 days, post-1995 studies have reported that silver-tipped catheters reduce urinary tract infections by a smaller margin than in pre-1995 studies. 104 the reason for this possible reduced effectiveness of silver-tipped catheters after 1995 is not known. the use of nitrofurazone-coated catheters was associated with a 32e 98% reduction in urinary tract infections in three recent published studies. 104 a number of interventions can significantly reduce the morbidity and mortality of central venous catheter (cvc)-related infections. 105 cvcs should be used only when necessary and should be removed as soon as practical, since longer catheterisation periods significantly increase risk for bloodstream infection. 106 three studies reported that the use of extensive barrier precautions (long-sleeved gown, sterile gloves, mask, cap and large sterile sheet drape) when inserting a central line was associated with a significantly lower rate of bloodstream infections compared with when only gloves and a small drape were used. 107 these studies also found that extensive barrier precautions were very cost-effective in terms of saving costs of nosocomial infections. 108 other studies have reported that subclavian central venous insertion is associated with significantly lower bloodstream infection rates compared with femoral insertion. 109 meta-analysis of eight studies reported that use of antiseptic chlorhexidine-containing solutions to prepare the catheter site was associated with a 51% lower risk of catheterrelated bloodstream infections compared with when iodine based solutions were used (95% ci: 0.28e0.88). 110 the institution of multiple interventions at the same time ('bundling') may be the best strategy to reduce cvc-related infections. 105 a huge study of catheter-related bloodstream infections was conducted in 103 icus and analysed 375 757 patientcatheter-days. 111 an education programme was conducted in icus that included hand washing, using extensive barrier precautions when inserting a cvc, cleaning skin with chlorhexidine, and avoiding the femoral site and the use of unnecessary catheters. catheter-related bloodstream infections were 7.7/1000 catheter-days at baseline to 1.4/1000 at 16e18 months follow-up (82% reduction, p < 0.002). another study reported that a multidimensional educational programme for central catheter insertion and maintenance reduced bloodstream infections from 10.8 to 3.7/ 1000 catheter-days (66% reduction, p < 0.0001) and produced a net saving of from $0.2 to 2.8 million in 18 months secondary to reduced bloodstream infection rates. 112 the use of coated cvcs can also significantly reduce the risk of nosocomial infections. 113 a twoyear study at a large michigan hospital reported that using chlorhexidine/silver sulfadiazene-coated catheters reduced bloodstream infections in hospitalised patients by 35% (p < 0.0003). 114 metaanalyses and many studies have reported that the use of cvcs coated with chlorhexidine/silver sulfadiazene significantly reduces rates of catheterrelated infections and significantly lowers hospital costs. 115 cost savings were estimated to be $196 for each chlorhexidine/silver sulfadiazene-coated catheter used. 116 use of new agents such as lysostaphin in catheters and catheter lock solutions may also reduce infection. 113 lysostaphin is an enzyme which effectively breaks up and kills staphylococci in biofilms on catheters. 117 haemodialysis patients are at high risk of many nosocomial infections including s. aureus, coagulasenegative staphylococci, many types of gramnegative bacteria and candida. 118 temporary catheters have the greatest risk of infection and should not be used any longer than necessary. a review of eight studies calculated that mean rates of bacterial infections in haemodialysis patients were about 6.3/1000 days when temporary catheters were used, 2.8/1000 days with cuffed temporary catheters, 0.4/1000 days with polytetrafluoroethylene grafts, and 0.14/1000 days when arteriovenous fistulas are used. 118 renal patients are at significant risk for hepatitis c (hcv) transmission from haemodialysis procedures. risk of hcv transmission can be significantly reduced by using separate haemodialysis machines and equipment for hcv þ and hcv à patients, proper gloving and other barrier precautions by healthcare workers, proper cleaning of machines and sending all tubing and dialysis units for either disposal or disinfection and reprocessing after each use. 119 a spanish study reported that hcv þ prevalence fell from 30.5% (121 patients) in 1993 to 6.8% (161 patients) (p < 0.05) in 2003 following the institution of universal precautions and increased cleaning along with the separation of hcv þ and hcv à patients. no serconversions were noted during this time in 335 hcv à haemodialysis patients following separation of hcv þ and hcv à haemodialysis. 119 preventing ventilator-associated pneumonia although prompt use of proper antibiotics is the cornerstone for treating ventilator-associated pneumonia (vap), there are many non-pharmacological interventions which can significantly reduce risk of vap incidence. longstanding methods of reducing risk of vap include: (i) avoiding tracheal intubation whenever possible and using noninvasive positive pressure ventilation instead; (ii) placing the patient in semi-erect position of 30e45 above horizontal reduces risk of aspiration-related vap; and (iii) using enteral feeding rather than parenteral feeding whenever possible. 120 recent meta-analysis has also indicated that the following interventions are associated with significantly lower levels of vap: (i) kinetic bed therapy (15 studies; rr: 0.38; 95% ci: 0.28e0.53); (ii) subglottic secretion drainage (five studies; rr: 0.51; 95% ci: 0.37e 0.71); (iii) heat and moisture exchangers vs heated humidifiers (eight studies; rr: 0.69; 95% ci: 0.51e 0.94); (iv) oral decontamination with chlorhexidine (seven studies; rr: 0.74; 95% ci: 0.56e0.96). 121 five studies employing multiple interventions were able to significantly reduce rates of vap by 31e57%. 121 one of these studies involved four hospitals and employed an intensive educational programme for icu nurses and respiratory therapists coupled with posters and fact sheets posted in the icu. 122 following these broad based interventions, vap rates fell from 8.75 to 4.74/1000 ventilator-days (46% reduction, p < 0.001). 122 about 2e5% of all surgical patients develop a significant infection at the wound site. 123 while antibiotics play a major role in preventing and treating surgical infections, many other factors are important in preventing surgical infections. higher rates of surgical infections are associated with operations of two or more hours, a contaminated or dirty procedure, or inadequate scrubbing procedures. 123 traditionally patients have been shaved at surgical sites, but it is now believed that clipping hair is better since shaving leaves small cuts in the skin. a review of three trials involving 3193 surgical patients reported that there were significantly more surgical site infections when patients were shaved versus clipped (rr: 2.02; 95% ci: 1.21e3.36). 124 cleaning surgical sites with antiseptics such as iodine compounds or chlorhexidine has long been recommended to reduce risk of surgical infection. however, meta-analysis of six studies found that bathing or swabbing sites with 4% chlorhexidine solutions was associated with only a marginal decline in surgical site infection rates compared with bathing with plain soap or placebo solutions (rr: 91%; 95% ci: 0.80e1.04). 125 warming the patient before or during surgery has also been shown to significantly reduce rates of surgical infection. 126 warming may reduce surgical infection rates by improving blood circulation and immune function in the surgical areas. an ultraclean air-filtered operating room coupled with use of whole-body ventilated exhaust suits by operating personnel was associated with a 60% drop in deep sepsis rates compared with standard operating room procedures (p < 0.001). 16 multiple interventions simultaneously may prove to be the most effective way to reduce surgical infections. institution of a comprehensive surgical infection control programme was associated with a 63% drop in surgical-related infections for coronary artery bypass graft patients (or: 0.37; 95% ci: 0.22e0.63). 127 this infection control programme included prospective surveillance and reporting, chlorhexidine showers, discontinuation of shaving, elimination of ice baths for cardioplegia solution, limitation of operating room traffic, reducing use of flash sterilisation and elimination of postoperative tap-water wound washing for four days. laparoscopic surgery should be done instead of open surgery whenever possible, since laparoscopic surgeries generally have significantly lower rates of infection, adhesions and other complications. many studies and meta-analyses have reported much lower infection rates when laparoscopic surgery is performed instead of open surgery for many types of abdominal procedures including perforated peptic ulcer surgery, cholecystectomy, splenectomy, lysis of small intestine adhesions causing obstruction, appendectomy, rectal cancer surgery and ventral hernia repair. 128e135 relatively few studies have been conducted involving sterilisation of surgical instruments and medical devices such as endoscopes. cleaning must also precede sterilisation or high-level disinfection. surgical and medical instruments may be sterilised or disinfected by a number of methods including autoclaving, ethylene oxide chambers, or solutions containing phenolics, aldehydes, quaternary ammonium compounds, hydrogen peroxide, peracetic acid or chlorine compounds. 136 all of these techniques have advantages and disadvantages. autoclaving provides excellent sterilisation but not all equipment can withstand the heat. ethylene oxide chambers provide excellent disinfection but they must be monitored for potential ethylene oxide gas leaks. disinfectant aldehydes such as glutaraldehye and ortho-phthaladehyde can cause respiratory, skin and eye irritation. peracetic acid systems provide good sterilisation but are relatively expensive and can only be used for immersible instruments. 136 preventing waterborne hospital infections a number of interventions have been proven effective in reducing rates of hospital waterborne infections. numerous studies have found that replacing tap water with sterile water for drinking, bathing and procedures can significantly reduce rates of many hospital infections including crytosporidium, legionella, aeromonas and stenotrophomonas. 22 sterile sponges can be used for bathing. boiling and water filtration in hospital water systems can also sterilise water, but these systems need to be monitored closely because many problems can develop which cause these systems to fail. 22 daily cleaning of patient shower areas with a detergent and phenolic compound has been shown to significantly decrease airborne levels of moulds including aspergillus. 137 heating water to more than 50 c has been shown to significantly reduce levels of legionella spp. in storage tanks and hospital water systems; however, water heating alone will not usually eliminate all legionella in a contaminated hospital water system. 138 some studies have found that the uv-light water treatment can greatly reduce levels of legionella in hospital water systems. 139 copperesilver-based ionisation systems can also significantly reduce waterborne concentrations of legionella, moulds and gram-negative bacteria such as p. aeruginosa and actinetobacter baumannii. 140e142 a spanish hospital saw legionella infection rates fall from 2.45 to 0.18 cases per 1000 discharges following installation of a copperesilver ionisation system (p < 0.001). 140 routine surveillance of hospital water supplies for legionella is highly recommended in cases of confirmed legionella infections; however, it is controversial as to whether such routine testing is needed in hospitals with no legionella infection history. 138, 143 all water leaks and water damage should be repaired and remediated within 24 h to prevent growth of pathogenic bacteria and moulds. 144 hospitals should avoid using indoor decorative fountains since they encourage legionella and the splashing water facilitates ready aerosolisation of the organism. hepa filtration is relatively inexpensive and probably should be used for all hospital rooms. various studies have found that the hepa filtration in hospitals can significantly reduce airborne levels and/or infection rates for several aerosolised pathogens. many studies have reported that the hepa filters in patient rooms can significantly reduce both airborne aspergillus concentrations and rates of human aspergillus infections. 145e147 meta-analysis of six non-randomised controlled trials reported that hepa filtration for neutropenic patients was associated with a significant drop in mortality due to mould infections (rr: 0.29; 95% ci: 0.15e0.54). 148 meta-analysis of six randomised controlled studies reported that hepa filtration for neutropenic patients was associated with only a marginal drop in overall mortality (rr: 0.86; 95% ci: 0.65e1.14). 148 use of portable hepa filters has been found to significantly reduce airborne levels of mrsa and p. aeruginosa in hospitals. 149, 150 a porcine study reported that hepa filtration was associated with significantly lower rates of porcine respiratory syndrome virus (prsv). 151 hepa air filtration has been shown to reduce airborne concentrations of droplet nuclei (which transport tuberculosis) by 90%. 152 recently, a new hospital air filtration system has been developed by airinspace technologies (montigny le bretonneux, france). this portable immunairä system forms a protective hood around the patient, filters air at 60 air changes per hour and uses a 'cold plasma' system to destroy microbes. early tests have indicated that such a system has a more than 99% single-pass efficiency in destroying bacteria, viruses and moulds such as aspergillus. 153 more study of this and other air filtration systems is needed. provision of adequate outdoor air ventilation rates is also essential to dilute out and control hospital pathogens. a study with army recruits reported significantly higher rates of acute respiratory disease when housed in poorly ventilated barracks compared with well-ventilated barracks. 154 the american society for heating, refrigerating and air conditioning engineers (ashrae) has proposed standards of at least four outdoor air changes per hour (ach) for hospital rooms, 15 outdoor ach for operating rooms and six outdoor ach for icus. 155, 156 hospitals undergoing construction or renovation have increased dangers for airborne and dustborne pathogens and may require additional outdoor ach as well as barrier protections. 155, 156 uv light machines in rooms or in ventilation systems can effectively kill mycobacteria, legionella and many viruses, but uv light is not effective in killing many species of bacteria and moulds. 155 special interventions for control of tuberculosis tuberculosis (tb) remains a serious health problem in both the developed and developing world. 157 recent cdc guidelines have recommended a number of administrative, engineering and personal protection measures to control tb spread in healthcare settings. 158 recommended administrative controls include tb testing for all patients at risk of tb, implementing a written tb control plan in the hospital and housing infected patients in separate rooms. all rooms housing tb patients should have at least 12 outdoor ach, have a negative pressure of at least 0.01 inch water, and the rooms of patients with actual or suspected tb should be checked visually with tests such as smoke tests. hepa air filters in patient rooms and uv irradiation in the ventilation systems or upper part of rooms is also strongly recommended to reduce airborne tb levels. hepa masks or other respiratory protection need to be worn by healthcare workers and visitors to rooms of infectious tb patients. proper cleaning and disinfecting of instruments used by tb patients are also essential. 158 controlled studies for individual interventions of tb control programmes are lacking. 157 however, risk of tb transmission can be greatly reduced when many infection control measures are applied simultaneously. a 1000-bed hospital in atlanta, georgia, used a variety of controls for tb including administrative (patient isolation, staff tb education programme, tb tests to staff every six months and hiring a nurse epidemiologist), negative-pressure tb rooms, and hepa masks by all healthcare workers in respiratory protection areas. 159 over a 28-month period, the number of tb exposure incidents fell from 4.4 to 0.6 per month (p < 0.001). the rate of tuberculin skin test conversions among healthcare workers also fell from 3.3% to 1.7% in this period (p < 0.001). 159 many non-pharmacological interventions have been shown to significantly reduce rates of hais, but are often overlooked in clinical practice. widely varied interventions such as proper hand washing, better nutrition, housing patients in separate rooms, sufficient numbers of nursing staff, coated urinary and cvcs, hepa air filters, copperesilver water ionisation and numerous interventions for ventilated and surgical patients have all been documented to significantly reduce risk of nosocomial morbidity and/or mortality. many of these studies have also indicated that these infection control interventions will more than pay for themselves in terms of reduced total medical costs. the hospital environment is a complicated ecosystem and many interventions are needed for optimal infection control. while many hospitals are using a number of these infection control strategies, relatively few hospitals are employing most of the broad range of infection control methods available today. multiple interventions ('bundling') often give better results than single interventions. 160 most bundling studies have used only two to five infection control interventions at the same time. 160 larger interventional studies should be undertaken which employ large numbers of infection control methods simultaneously. such multifaceted infection control protocols will probably result in larger declines in nosocomial infection rates than strategies employing only one to five interventions. however, it is difficult to sort out the efficacy of individual interventions when many interventions are simultaneously used. aboelela et al. have suggested that in studies with many interventions, groups of several interventions or bundles can be studied as one intervention. 160 current levels of multidrug-resistant bacteria will increase in the future as antibiotics are heavily used in both human and veterinary medicine and relatively few new antibiotics are being developed. multifactorial non-pharmacological infection control strategies will not only substantially reduce the numbers of nosocomial infections, but should also significantly reduce hospital antibiotic usage. lower overall antibiotic use will reduce risk of antibiotic-resistant organisms and should improve efficacy of antibiotics given to patients who do acquire nosocomial infections. multiple-intervention infection control strategies should significantly reduce mortality, morbidity and overall medical costs. there needs to be more support for improved hospital infection control on the part of patient advocacy groups, nursing, medical and public health associations, hospital administrators, health insurance companies, business and labour groups, the media and public officials. research and implementation of multifaceted hospital infection control strategies should clearly be one of the highest priority items facing healthcare in the early 21st century. estimating health-care associated infections and deaths in u.s. hospitals invasive methicillin resistant staphyloccocus aureus infections in the united states proportion of hospital deaths potentially attributable to nosocomial infections the costs of nosocomial infections epidemiology and economic evaluation of severe sepsis in france: age, severity, infection site, and place of acquisition (community, hospital or icu) as determinants of workload and cost clinical and economic consequences of ventilator associated pneumonia: a systemic review decreasing ventilator-associated pneumonia 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with mrsa impact of ring wearing on hand contamination and comparison of hand hygiene agents in a hospital neck ties as vectors for nosocomial infections artificial nails: are they putting patients at risk? a review of the research can methicillinresistant staphylococcus aureus be found in an ambulance fleet? identifying opportunities to enhance environmental cleaning in 23 acute care hospitals effect of a training program for hospital cleaning staff on prevention of hospital acquired infection reduction of clostridium difficile and vacomycin-resistant enterococcus contamination of environmental surfaces after an intervention to improve cleaning methods reduction in acquisition of vancomycin-resistant enterococcus after enforcement of routine environmental cleaning measures mrsa acquisition in an intensive care unit does disinfection of environmental surfaces influence nosocomial infection rates? a systematic review environmental control to reduce transmission of clostridium difficile tackling contamination of the hospital environment by methicillin resistant staphylococcus aureus (mrsa): a comparison between conventional terminal cleaning and hydrogen peroxide vapour decontamination copper-oxide impregnated textiles with potent biocidal activities malnutrition in acute care patients: a narrative review contribution of selected vitamins and trace elements to immune function malnutrition is an independent risk factor associated with nosocomial infections relations between undernutrition and nosocomial infection in elderly patients risk factors for nosocomial pneumonia in a geriatric hospital: a case-control, one-center study immunonutrition in the intensive care unit. a systemic review and consensus statement meta-analysis of probiotics for the prevention of antibiotic associated diarrhea and the treatment of clostridium difficile disease use of probiotic lactobacillus preparation to prevent diarrhoea associated with antibiotics: randomised double blind controlled trial probiotic treatment of vancomycin-resistant enterococci: a randomised controlled trial single rooms may help to prevent nosocomial bloodstream infections and cross-transmission of methicillin-resistant staphylococcus aureus in intensive care units isolation of patients in single rooms or cohorts to reduce spread of mrsa in intensive-care units: prospective two-centre study budget analysis of rapid screening for staphylococcus aureus colonization among patients undergoing elective surgery in us hospitals methicillin resistant staphylococcus aureus control in an intensive control unit: a 10 year analysis methicillin resistant staphylococcus aureus control in hospitals: the dutch experience low prevalence of methicillin-resistant staphylococcus aureus (mrsa) at hospital admission in the netherlands: the value of search and destroy and restrictive antibiotic use application of molecular techniques to the study of hospital infection medical and economic benefits of a comprehensive infection control program that includes routine determination of microbial clonality nurse working conditions and patient safety outcomes relationships between nurse staffing and patient outcomes coming soon: state reports on infection rates hospital safety reports past due: officials debate reasons for three studies delays decreasing urinary tract infection in a large academic community hospital preventing catheter-related bacteriuria. should we? can we? how? urosepsis in the critical care unit the efficacy of silver alloy-coated urinary catheters in preventing urinary tract infections: a meta-analysis effect of silvercoated urinary catheters: efficacy, cost-effectiveness, and antimicrobial resistance systematic review: antimicrobial urinary catheters to prevent catheterassociated urinary tract infection in hospitalized patients prevention of central venous catheter-related infections: what works other than impregnated or coated catheters? prevention and control of nosocomial infections using maximal sterile barriers to prevent central venous catheter-related infection: a systematic evidence-based review use of maximal sterile barriers during central venous catheter insertion: clinical and economic outcomes complications of fermoral and subclavian venous catheterization in critically ill patients: a randomized controlled trial chlorhexidine compared with povidone-iodine solution for vascular catheter-site care: a meta-analysis an intervention to decrease catheter-related bloodstream infections in the icu effect of an education program on decreasing catheter-related bloodstream infections in the surgical intensive care unit health care-associated infections: major issues in the early years of the 21st century are antiseptic-coated central venous catheters effective in a real-world setting? prevention of nosocomial bloodstream infections: effectiveness of antimicrobialimpregnated and heparin-bonded central venous catheters cost-effectiveness of 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acquired legionellosis: solutions for a preventable infection evaluation of ultraviolet light for disinfection of hospital water hospital acquired legionnaires disease in a university hospital: impact of the coppere silver ionization system impact of copper and silver ionization on fungal colonization of the water supply in health care centers: implications for immunocompromised patients in vitro efficacy of copper and silver ions in eradicating pseudomonas aeruginosa, stenophomonas maltophilia and acinetobacter baumannii: implications for on-site disinfection for hospital infection control surveillance of hospital water and primary prevention of nosocomial legionellosis: what is the evidence? cleaning and restoration certification (iicrc). iicrc standard and reference for professional mold restoration s520. 2715 east mill plain boulevard impact of air filtration on nosocomial aspergillus infections control of construction associated nosocomial aspergillosis in an antiquated hematology unit efficacy of high-efficiency particulate air filtration in preventing aspergillosis in immunocompromised patients with hematologic malignancies the influence of high-efficiency particulate air filtration on mortality and fungal infection among highly immunocompromised patients: a systematic review reduction in mrsa environmental contamination with a portable hepa-filtration unit airborne dissemination of epidemic pseudomonas aeruginosa in the nottingham cystic fibrosis population: a role for portable hepa filtration? abstract presented at the 2006 federation of infection societies meeting in further evaluation of alternative air-filtration systems for reducing the transmission of porcine reproductive and respiratory syndrome virus efficacy of portable filtration units in reducing aerosolized particles in the size range of myobacterium tuberculosis evaluation of a new mobile system for protecting immune-suppressed patients against airborne contamination building-associated risk of febrile acute respiratory diseases in army trainees control and management of hospital water quality ventilation for acceptable indoor air quality control and prevention of healthcareassociated tuberculosis: the role of respiratory isolation and personal respiratory protection guidelines for preventing the transmission of mycobacterium tuberculosis in health care settings preventing the nosocomial transmission of tuberculosis effectiveness of bundled behavioural interventions to control healthcareassociated infections: a systemic review of the literature acknowledgements i thank all of the infection control researchers who have published useful papers. none declared. none. key: cord-323668-evzzfu04 authors: yin, zhixin; guan, dawei; fan, qin; su, juan; zheng, wenling; ma, wenli; ke, changwen title: lncrna expression signatures in response to enterovirus 71 infection date: 2013-01-11 journal: biochemical and biophysical research communications doi: 10.1016/j.bbrc.2012.11.101 sha: doc_id: 323668 cord_uid: evzzfu04 abstract outbreaks of hand, foot, and mouth disease caused by enterovirus 71 (ev71) have become considerable threats to the health of infants and young children. to identify the cellular long noncoding rnas (lncrnas) involved in the host response to ev71 infection, we performed comprehensive lncrna and mrna profiling in ev71-infected rhabdomyosarcoma cells through microarray. we observed the differential expression of more than 4800 lncrnas during infection. further analysis showed 160 regulated enhancer-like lncrna and nearby mrna pairs, as well as 313 regulated rinn’s lncrna [m. guttman i. amit, m. garber, c. french, m.f. lin, d. feldser, m. huarte, o. zuk, b.w. carey, j.p. cassady, m.n. cabili, r. jaenisch, t.s. mikkelsen, t. jacks, n. hacohen, b.e. bernstein, m. kellis, a. regev, j.l. rinn, e.s. lander. chromatin signature reveals over a thousand highly conserved large non-coding rnas in mammals. nature 458 (2009) 223–227, a.m. khalil, m. guttman, m. huarte, m. garber, a. raj, d. rivea morales, k. thomas, a. presser, b.e. bernstein, a. van oudenaarden, a. regev, e.s. lander, j.l. rinn. many human large intergenic noncoding rnas associate with chromatin-modifying complexes and affect gene expression. proc. natl. acad. sci. usa 106 (2009) 11667–11672] and nearby mrna pairs. the results provided information for further research on the prevention and treatment of ev71 infection, as well as on distinguishing severe and mild ev71 cases. enterovirus 71 (ev71) is a typical positive-strand rna virus belonging to the picornaviridae family [1] . ev71 infection is a major cause of hand, foot, and mouth disease (hfmd) in infants and young children [2, 3] this infection can also cause neurological diseases such as aseptic meningitis, encephalitis, and acute flaccid paralysis, which can lead to permanent paralysis and even death [4, 5] . outbreaks of ev71 infection have been reported in many countries [6] . in recent years, ev71 infection has become a considerable threat to public health in china [7] , where the government has reported 1,619,706 cases of hfmd (with 509 deaths) in 2011 and 1,587,849 cases (with 463 deaths) from january to august 2012 (http://www.moh.gov.cn/publicfiles//business/htmlfiles/ mohjbyfkzj/s3578/list.htm). however, to date, no effective vaccine or therapy is available to prevent or treat this infection. in humans, cellular immunity is important in preventing the development of serious complications after ev71 infection [8, 9] . thus, understanding the cellular events after ev71 infection can facilitate the development of new strategies for preventing and treating this infection. in 2004, shih [10] reported that ev71 infection leads to increased levels of mrnas encoding chemokines, proteins involved in protein degradation, complement proteins, and proapoptotis proteins. the infection also results in the decreased expression of genes encoding proteins involved in host rna synthesis. in 2006, leong et al. [11] identified 152 down-regulated genes and 39 upregulated genes in rhabdomyosarcoma (rd) cells infected with ev71. in 2010, 64 micrornas were up-or down-regulated more than twofold in response to ev71 infection [12] . in the last decade, long noncoding rnas (lncrnas) have been shown to play important roles in gene expression regulation, dosage compensation, genomic imprinting, nuclear organization and compartmentalization, as well as nuclear-cytoplasmic trafficking [13] [14] [15] [16] [17] . recent studies have demonstrated the changes in host lncrna expression in response to virus infection. after infectious bursal disease virus and marek's disease virus infection in chicken, eight and two lncrnas are differently expressed, respectively [18] . in 2010, peng et al. [19] reported the differential expression of more than 500 lncrnas in mice after severe acute respiratory syndrome coronavirus (sars-cov) infection. other studies have shown that most lncrnas are similarly regulated in response to influenza virus infection, and that they have distinctive kinetic expression profiles in type i interferon receptor and stat1 knockout mice during sars-cov infection [19] . these findings suggest the widespread differential expression of lncrnas in response to 0006 virus infection and their involvement in regulating the host response, including innate immunity [19] . to determine which cellular lncrnas play roles in the host response to ev71 infection, we performed lncrna and mrna microarray analyses in mock-and ev71-infected rd cells. human rd cells were grown in eagle's minimum essential medium (mem; gibco) supplemented with 10% fetal bovine serum (fbs; gibco). when the cells had grown to 90% confluence in 25 cm 2 flasks, they were infected with ev71 (gdfs-3; isolated and identified from guangdong province, china in 2008) [20] at a multiplicity of infection of 100 50% tissue culture infectious doses (tcid50). after adsorption for 1 h at 37°c, the inoculum was removed and eagle's mem with 2% fbs was added. the culture was maintained at 37°c. at 24 h post-ev71 infection, the medium was removed, an appropriate volume of buffer rlt was added, and the total rna was extracted using an rneasy mini kit according to the manufacturer's protocol (qiagen). rna was also extracted from mockinfected cells. the quality and the concentration of the rna samples were monitored at absorbance ratios of a260/a280 and a260/230 using a nanodrop nd-1000 spectrophotometer and standard denaturing agarose gel electrophoresis. sample labeling and array hybridization were performed according to the agilent one-color microarray-based gene expression analysis protocol (agilent technology) with minor modifications. briefly, mrna was purified from total rna after the removal of rrna (mrna-only™ eukaryotic mrna isolation kit, epicentre). each sample was amplified and transcribed into fluorescent crna along the entire length of the transcripts without a 3 0 bias using the random priming method. the labeled crnas were purified by using an rneasy mini kit (qiagen). the concentration and specific activity of the labeled crnas (pmol cy3/lg crna) were measured using nanodrop nd-1000. about 1 lg of each labeled crna was fragmented by adding 5 ll of 10â blocking agent and 1 ll of 25â fragmentation buffer, followed by heating the mixture at 60°c for 30 min. finally, 25 ll of 2â ge hybridization buffer was added to dilute the labeled crna. about 50 ll of hybridization solution was dispensed into the gasket slide and assembled onto an arraystar human lncrna array v2.0 slide (arraystar, usa). the slides were incubated for 17 h at 65°c in an agilent hybridization oven. the hybridized arrays were washed, fixed, and scanned using an agilent dna microarray scanner (part number g2505c). agilent feature extraction software (version 11.0.1.1) was used to analyze the acquired array images. quantile normalization and subsequent data processing were performed using the genespring gx v12.0 software package (agilent technologies). after quantile normalization of the raw data, lncrnas and mrnas with ''present'' or ''marginal'' (''all targets value'') flags in mock-and ev71infected samples were subjected to further data analysis. differentially expressed lncrnas and mrnas between the two samples were identified through fold-change (greater than twofold) filtering. lncrnas with enhancer-like function were identified using the gencode annotation [21] of human genes [22] . the selection of lncrnas with enhancer-like function involved the exclusion of transcripts mapped to the exons and introns of annotated protein coding genes, as well as the natural antisense transcripts overlapping with the protein coding genes and all known transcripts. rinn's lncrnas were identified based on the studies of rinn [23, 24] . human homeobox transcription factors (hox) cluster lncrnas were also identified based on the study of rinn [25] . qpcr was performed to confirm the expression of lncrnas by microarray analysis. briefly, cdna was synthesized from total rna using a primescript rt reagent kit with a gdna eraser (taka-ra). primers for four lncrnas were designed and synthesized (table 1). then, qpcr was performed using a lightcycler 480 (roche applied science). the 10 ll pcr reactions included 1 ll of cdna product and 5 ll of sybr premix ex taq ii (takara). the reactions were incubated at 95°c for 1 min, followed by 40 cycles at 95°c for 5 s, 60°c for 10 s, and 72°c for 15 s. all reactions were run in triplicate. after reaction, the threshold cycle value (ct) data were determined using default threshold settings, and the mean ct was determined from the duplicate pcrs. human 18s rrna was used for normalization. the expression levels of lncrnas were measured in terms of ct, and then normalized to 18s using 2 àddct [26] . cytopathic effects were observed 24 h post-infection. total rna was extracted from mock-and ev71-infected cells 24 h postinfection. the od260/od280 ratios were approximately 2.1, and the od260/od230 ratios were more than 1.9, which suggested that the total rnas were sufficiently pure for the succeeding experiments. subsequently, mrna was purified, crna was prepared, and array hybridization was performed using arraystar human lncrna array v2.0. after quantile normalization of the raw data, the expression profiles of 22971 lncrnas and 18194 mrnas were obtained from mock-and ev71-infected cells (tables s1 and s2 ). the distributions of the log2 ratios of lncrnas and mrnas between ev71-and mock-infected samples were almost the same. fig. 1 shows the heat maps of the expression ratios (log2 scale) of lncr-nas and mrnas. to identify differentially expressed lncrnas and mrnas, we performed fold-change filtering between mock-and ev71-infected cells (fold change > 2.0). we found that 2990 lncrnas and 1718 mrnas were up-regulated, whereas 1876 lncrnas and 2552 mrnas were down-regulated in ev71-infected cells (tables s3 and s4 ). we performed qpcr assays to confirm the expression pattern of four differentially expressed lncrnas in rd cells. a general consistency between the qpcr and microarray analysis results was confirmed in four lncrnas (ap000688.29, ac002511.1, rp5-843l14.1, and rp4-620f22.3) in terms of regulation direction and significance. specifically, a 3.31-fold down-regulation (2.25-fold in microarray analysis) was observed in ap000688.29, 3.33-fold up-regulation (2.77-fold in microarray analysis) in ac002511.1, 2.29-fold up-regulation (2.40-fold in microarray analysis) in rp5-843l14.1, and 2.99-fold up-regulation (2.22-fold in microarray analysis) in rp4-620f22.3 (fig. 2) . rinn et al. [25] identified numerous transcripts from the four hox loci, which included 101 mrna, 75 introns, and 231 intergenic transcripts. these lncrnas, which were expressed in temporal and site-specific manners, possibly used the same enhancers as hox genes and may have the same global regulating functions as hox. in this paper, the profiling data of all probes targeting these 407 discrete transcripts are presented in table s5 . the data showed that 43 mrnas can be detected in rd cells, with seven of them being differentially expressed. then, 300 transcribed noncoding rnas (including introns and intergenic transcripts) were detected, with 64 of them being differentially expressed. using chromatin-state maps, rinn's studies [23, 24] have identified 3019 lncrnas with clear evolutionary conservation and association with distinct and diverse biological processes, such as cell proliferation, rna binding complexes, immune surveillance, embryonic stem cell pluripotency, neuronal processes, morphogenesis, gametogenesis, and muscle development. the profiling data of all probes for these lncrnas are provided in table s6 , which indicated that 477 from the detected 2200 lncrnas were differentially expressed. among them, 190 were down-regulated and 287 were up-regulated. further analysis resulted in 313 differentially expressed lncrnas and nearby coding gene pairs (distance < 300 kb) for each comparison between mock-and ev71-infected cells (table s7) . among the 163 pairs, lncrnas and nearby coding genes were regulated in the same direction (up or down), whereas 150 pairs were regulated in the opposite direction. in 2010, using the gencode annotation [21] of human genes, orom et al. [22] identified a set of lncrnas with enhancer-like function from human cell lines. the depletion of these lncrnas resulted in a concomitant decrease in the expression of neighboring genes. the profiling data of all probes for lncrnas with enhancerlike function are shown in table s8 ; 1025 enhancer-like lncrnas were detected and 252 of them were differentially expressed (fold change > 2). among these 252 lncrnas, 160 had differentially expressed nearby coding genes (distance < 300 kb) for each comparison, as shown in table s9 . in 70 of the 160 pairs, lncrnas and nearby coding genes were regulated in the same direction (up or down); in 90 of the 160 pairs, they were regulated in the opposite direction. inferring the putative functions of protein-coding genes located near lncrnas is an important approach to lncrna research [23, 27] . in this paper, we combined differentially expressed nearby mrna pairs of 313 differentially expressed rinn's lncrnas and 160 enhancer-like lncrnas, and abandoned the duplicated mrnas. then, we used the david functional annotation chart [28, 29] for functional enrichment analysis of the differentially regulated proteincoding gene and lncrna pairs. the most significant functional groups consisted of annotation terms of alternative splicing, splice variant, phosphoprotein, nucleus, cytoplasm, and acetylation (fig. 3) . we hypothesized that the lncrnas can modulate host responses through nearby protein-coding genes. continuous interactions between viruses and hosts during their co-evolution have shaped their immune system. consequently, viruses have manipulated host immune-control mechanisms to facilitate their propagation. previous studies on virus-host interactions and viral pathogenesis have largely focused on proteincoding genes. in the last decade, evidence of host-cellular microrna modulation of the expression of various viral genes has been reported. this modulation plays a pivotal role in the host-pathogen interaction network [30] . recent studies have shown that virus infection alters the expression profiles of host lncrnas. for example, eight lncrnas are differentially expressed in virus-infected birds [18] . in 2010, comprehensive deep sequencing showed that more than 500 lncrnas were differentially expressed in mice after sars-cov infection [19] . however, lncrnas in other virus infections are not well documented. in the present study, using arraystar microarray analysis, we identified the differentially expressed lncrnas in rd cells after ev71 infection, together with nearby differentially expressed mrna pairs. in a study on the molecular mechanisms of host response to ev71 infection, hsih et al. [11] found the up-regulation of mrnas encoding chemokines, complement proteins, proteins involved in protein degradation, and proapoptosis proteins. they also observed the down-regulation of several genes encoding proteins involved in host rna synthesis in ev71-infected sf268 cells. further investigations have shown that ev71 infection alters the transcription of genes encoding components of cytoskeleton, protein translation, and protein modification; cellular transport proteins; protein degradation mediators; cell death mediators; mitochondrial-related and metabolism proteins; as well as cellular receptors and signal transducers in rd cells. recent microrna profiling analysis in hep2 cells has identified 64 micrornas whose expression levels changed more than twofold in response to ev71 infection. their potential conserved target genes encode proteins with functions in neurological processes, immune responses, and cell death pathways. these proteins are known to be associated with the extreme virulence of ev71 [12] . in this study, lncrna and mrna expression analyses identified differentially expressed lncrna and mrna pairs. functional enrichment analysis further indicated that the mrnas were associated with alternative splicing, splice variant, phosphoprotein, nucleus, cytoplasm, and acetylation. these results and those of future works can expand the molecular mechanisms of the host response to ev71 infection. some lncrnas reportedly serve as enhancers and have positive effects on gene expression. evf-2 ncrna forms a complex with the homeodomain-containing protein dlx2 and causes transcriptional enhancement [31] . the binding of heat-shock rna-1 ncrna with heat-shock transcription factor 1 leads to the induction of heatshock proteins [32] . an isoform of ncrna steroid receptor rna activator is also co-activated with steroid receptor responsiveness [33] . recently, orom et al. [22] showed that noncoding rna-activating 1-7 enhance the expression of nearby genes. in our study, we identified 160 differentially expressed enhancer-like lncrna and mrna pairs, and 4l.5% (70/160) of these pairs were regulated in the same direction. we speculated that some of these lncrnas function as enhancers that activate nearby genes; however, further research is needed to prove this hypothesis. identifying the putative functions of nearby genes of lncrnas may aid in understanding the functional roles of lncrna [23, 27] . peng et al. [19] performed functional enrichment analysis on the nearby protein-coding genes of differentially expressed lncrnas in sars-cov infected mouse. they found that the most significant functional group consisted of annotation terms related to gene expression, including transcription regulation, nuclear and dnabinding transcription factor activity, as well as regulation of rna metabolic process. in this study, functional enrichment analysis of differentially expressed mrnas with differentially expressed nearby lncrna partners showed that they were functionally related with alternative splicing, splice variant, phosphoprotein, nucleus, cytoplasm, and acetylation. although the functions of lncrnas during virus infection have not been explored, we speculate that ev71 infection may change some lncrna expression levels that further regulate the expression of proteins related with alternative splicing and signal transduction. ev71 infection may be asymptomatic or may cause diarrhea, rashes, and hfmd. ev71 can also cause severe neurological disease [4, 5] . distinguishing mild and severe cases of ev71 infection in the early infection phase is very important in determining the appropriate treatment process. recent reports have shown that the expression levels of five micrornas significantly increase in coxsackievirus a16 (cav16)-infected patients compared with fig. 3 . functional enrichment analysis on differently regulated mrnas which were with differently expressed nearby lncrnas. the mrnas were from the 160 regulated enhancer-like lncrna and nearby mrna pairs, and 313 regulated rinn's lncrna and nearby mrna pairs. the functional enrichment analysis was performed by utilizing the david functional annotation chart [28, 29] . ev71-infected ones. the combination of three micrornas also shows a moderate ability to differentiate between cva16 and ev71. these data indicate that microrna expression profiles can serve as supplementary biomarkers for diagnosing and classifying enteroviral hfmd infections [34] . we suggest that further studies on the putative differential expression profiles of lncrnas in different ev71-infected cases can help distinguish mild and severe cases. in summary, we identified the lncrnas putatively involved in the host response to ev71 infection, which provided deeper insight into the mechanisms underlying ev71 infection. after determining the role of lncrnas in the regulation of host-ev71 interactions, the protection and treatment methods for ev71 infection can be improved, and severe cases can be distinguished from mild ones in the earlier phase. epidemics to eradication: the modern history of poliomyelitis enterovirus-associated encephalitis in the california encephalitis project acute encephalitis caused by intrafamilial transmission of enterovirus 71 in adult an overview of the evolution of enterovirus 71 and its clinical and public health significance appearance of mosaic enterovirus 71 in the 2008 outbreak of china the 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long noncoding rna expression in response to virus infection and altered innate immune signaling the complete sequence analyses of enterovirus 71 strain from the fatal case of the hand, foot and mouth disease during an epidemic in guangdong gencode: producing a reference annotation for encode long noncoding rnas with enhancer-like function in human cells chromatin signature reveals over a thousand highly conserved large non-coding rnas in mammals many human large intergenic noncoding rnas associate with chromatin-modifying complexes and affect gene expression functional demarcation of active and silent chromatin domains in human hox loci by noncoding rnas analyzing real-time pcr data by the comparative c(t) method genomic and transcriptional colocalization of protein-coding and long non-coding rna pairs in the developing brain systematic and integrative analysis of large gene lists using david bioinformatics resources bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists cellular versus viral micrornas in hostvirus interaction the evf-2 noncoding rna is transcribed from the dlx-5/6 ultraconserved region and functions as a dlx-2 transcriptional coactivator rna-mediated response to heat shock in mammalian cells a steroid receptor functions coactivator sraas an rna and is present in an src-1 complex serum microrna expression profile distinguishes enterovirus 71 and coxsackievirus 16 infections in patients with hand-foot-and-mouth disease supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbrc.2012.11.101. key: cord-339578-eg19rfvi authors: garcia-garcia, maria luz; calvo, cristina; ruiz, sara; pozo, francisco; del pozo, victoria; remedios, laura; exposito, nadia; tellez, ana; casas, inmaculada title: role of viral coinfections in asthma development date: 2017-12-05 journal: plos one doi: 10.1371/journal.pone.0189083 sha: doc_id: 339578 cord_uid: eg19rfvi background: viral respiratory infections, especially acute bronchiolitis, play a key role in the development of asthma in childhood. however, most studies have focused on respiratory syncytial virus or rhinovirus infections and none of them have compared the long-term evolution of single versus double or multiple viral infections. objective: our aim was to compare the frequency of asthma development at 6–8 years in children with previous admission for bronchiolitis associated with single versus double or multiple viral infection. patients & methods: a cross-sectional study was performed in 244 children currently aged 6–8 years, previously admitted due to bronchiolitis between september 2008 and december 2011. a structured clinical interview and the isaac questionnaire for asthma symptoms for 6-7-year-old children, were answered by parents by telephone. specimens of nasopharyngeal aspirate for virological study (polymerase chain reaction) and clinical data were prospectively taken during admission for bronchiolitis. results: median current age at follow-up was 7.3 years (iqr: 6.7–8.1). the rate of recurrent wheezing was 82.7% in the coinfection group and 69.7% in the single-infection group, p = 0.06. the number of wheezing-related admissions was twice as high in coinfections than in single infections, p = 0.004. regarding the isaac questionnaire, 30.8% of coinfections versus 15% of single infections, p = 0.01, presented “wheezing in the last 12 months”, data that strongly correlate with current prevalence of asthma. “dry cough at night” was also reported more frequently in coinfections than in single infections, p = 0.02. the strongest independent risk factors for asthma at 6–8 years of age were: age > 9 months at admission for bronchiolitis (or: 3.484; ci95%: 1.459–8.317, p:0.005), allergic rhinitis (or: 5.910; 95%ci: 2.622–13.318, p<0.001), and viral coinfection-bronchiolitis (or: 3.374; ci95%: 1.542–7.386, p:0.01). conclusions: asthma at 6–8 years is more frequent and severe in those children previously hospitalized with viral coinfection-bronchiolitis compared with those with single infection. allergic rhinitis and older age at admission seem also to be strong independent risk factors for asthma development in children previously hospitalised because of bronchiolitis. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 more frequently in coinfections than in single infections, p = 0.02. the strongest independent risk factors for asthma at 6-8 years of age were: age > 9 months at admission for bronchiolitis (or: 3.484; ci95%: 1.459-8.317, p:0.005), allergic rhinitis (or: 5.910; 95%ci: 2.622-13.318, p<0.001), and viral coinfection-bronchiolitis (or: 3.374; ci95%: 1.542-7.386, p:0.01). there is strong evidence that respiratory viruses play a key role in the development of asthma in children. classic studies by sigurs et al. showed that respiratory syncytial virus (rsv) bronchiolitis, severe enough to require hospitalization, is a risk factor for asthma at the age of 7, 13 and 18 [1, 2, 3] . ng asthma at 6 years being more than four-fold higher compared with hrvnegative cases [4, 5] . other studies have reported that early human metapneumovirus (hmpv) infection during infancy is also an independent risk factor for the development of preschool asthma [6] ]. however, most studies have focused on rsv or hrv infections and only one of thm has evaluated the long-term evolution of viral coinfections, although focused exclusively on human bocavirus (hbov) and hrv coinfections in 3 to 35 month-old children with their first or second wheezing episode [7] . our main objective was to compare the frequency of asthma and other respiratory symptoms, at 6-8 years of age, in children with previous admission with bronchiolitis associated with viral co-infection versus simple viral infection. this is a substudy of an ongoing prospective investigation of respiratory tract infections in children, approved by the medical ethics committee. inclusion criteria were children 6-8 years of current age, with a previous hospitalization with bronchiolitis at 0-24 months of age, between september 2008 and december 2011. during the hospital stay a physician filled out a study-questionnaire with the following variables: age, sex, month of admission, clinical diagnosis, history of prematurity, need for oxygen therapy-evaluated via transcutaneous oxygen saturation -, axillary temperature (! 38˚c), presence of infiltrates and/or atelectasis in chest x-rays, administration of antibiotic therapy, length of hospital stay, total white blood cell (wbc) count, c-reactive protein (crp) serum levels and blood culture results (for those cases where such tests were performed) and results of the virological study. parents were contacted by phone between october 2016 and january 2017, and were invited to take part in the study. a telephone interview based on a structured questionnaire was performed, to obtain information on wheezing episodes, related hospital admissions, physician-diagnosed atopic dermatitis, allergic rhinitis, food allergy, use of bronchodilators and maintenance medication for asthma. information on pet contacts, parental smoking habits, presence of allergy, eczema and asthma in first order family members (mother, father or siblings) that had been diagnosed by a medical doctor was also recorded. furthermore, the present investigation used the isaac questionnaire for asthma symptoms for 6-7-year-old children, previously validated and translated to spanish [8] , which was answered by parents over the phone. the researchers were blinded to the status of the child when the interviews were performed. informed consent was obtained from parents or legal guardians. exclusion criteria were refusal to participate. specimens consisted of nasopharyngeal aspirates (npa) that were taken from each patient at admission. each specimen was sent for virological investigation to the respiratory virus and influenza unit at the national microbiology center (isciii, madrid, spain). npas were processed within 24 hours after collection. upon receipt, three aliquots were prepared and stored at -70˚c. both the reception and the npa sample processing areas were separate from those defined as working areas. polymerase chain reactions (pcr) methods for detection of sixteen respiratory viruses. three reverse transcription (rt)-nested pcr assays were performed to detect a total of sixteen respiratory viruses. in these assays, the reverse transcription and first amplification round were carried out in a single tube using the qiagen onestep rt-pcr kit (qiagen). influenza a, b and c viruses were detected by using primer sets only to amplify influenza viruses in a multiplex pcr assay as previously described [9] . a second multiplex pcr was used to detect parainfluenza viruses 1 to 4 (piv), human coronaviruses (cov) 229e and oc43, enteroviruses (ev) and hrv [10] . presence of rsv a and b types, hmpv, hbov and adenoviruses (adv) were investigated by a third multiplex rt-nested pcr-brq method [11] (s1 table) . bronchiolitis. all the classic criteria, present in an initial episode of acute onset expiratory dyspnea with previous signs of viral respiratory infection-whether or not this was associated to respiratory distress or pneumonia-were applied in diagnosing bronchiolitis [12] . the term recurrent wheezing was used to imply more than one episode of wheezing verified by a physician. the children who answered affirmatively to question 2 of the isaac questionnaire, "wheezing in the last 12 months", being the one that in the validation studies has shown a better correlation with the current prevalence of asthma, were considered to have asthma [13] . allergic rhinitis. was defined as rhinitis appearing at least twice after exposure to a particular allergen and unrelated to infection. viral coinfection. was defined as the detection of more than one viral pathogen in the same sample. continuous variables were described using mean and standard deviation. categorical variables were described using absolute and relative frequencies. clinical characteristics of patients with simple infections were compared with those associated with coinfections. to compare qualitative variables, chi 2 test or fisher's exact test was used if there were 5 items of data in a cell. for quantitative variables, as all of them followed a normal distribution, the means were compared using the students´s t to compare two groups. a two-sided value of p < 0.05 was considered statistically significant. multivariate stepwise logistic regression analysis was used to calculate the adjusted odds ratios (or) with 95% confidence intervals for estimating the association between different factors and asthma. all analyses were performed using the statistical package for the social sciences (spss), version 21.0. of the 351 children previously admitted with bronchiolitis, with positive viral detection and current age between 6 and 8 years, 244 (52 coinfections and 192 single infections) could be located and agreed to participate in the study. clinical characteristics during admission for bronchiolitis are presented in table 1 . no significant clinical differences could be detected between both groups, coinfections and single infections, in terms of age at admission, gender, prematurity, fever, hypoxia or length of hospital stay. the most frequently identified viruses in single infections were: rsv (100, 52%), hrv (29, 15%), hmpv (15, 8%), adv (5, 2.6%) and hbov (3, 1.6%). the most frequent coinfections were rsv + hrv (15, 29%) and rsv + hbov (12, 23%). with respect to comparability between both groups, no differences were found between coinfections and single infections regarding allergic rhinitis, atopic dermatitis or food allergy ( table 2) . several possible hereditary and environmental factors for the development of recurrent wheezing or asthma were also evaluated. no differences were observed in the family history of asthma or atopy. nearly one-third of children were exposed to tobacco smoke, with similar percentages in both groups ( table 2) . median current age at follow-up contact was 7.3 years (iqr: 6.7-8.1), with a slight predominance of females (52.5%). the rate of recurrent wheezing was 82.7% in the coinfection group and 69.7% in the single-infection group, p = 0.06, needing rehospitalization for wheezing 21% and 29% respectively, p = 0.251. the number of wheezing-related admissions was twice as high in coinfections than in single infections, p = 0.004. a similar percentage of children were prescribed inhaled corticosteroids in both groups. however, montelukast was used more frequently in coinfections than in simple infections, p = 0.01 (table 3) . regarding the isaac questionnaire answers, nearly 70% had "ever wheezed", without significant differences between both groups. however, 30.8% of coinfections versus 15% of single infections, p = 0.01, presented "wheezing in the last 12 months", data that strongly correlate with current prevalence of asthma. "dry cough at night" was also reported more frequently in coinfections than in single infections, p = 0.02. the questions: "wheezing during exercise?" and "ever had asthma?" were affirmatively answered with similar frequency in both groups. (table 4 ). in the combined coinfection and single infection group, several possible hereditary and environmental risk factors for asthma at the age of 6-8 were evaluated using a univariate analysis ( table 5 ). the risk of asthma was increased in children previously hospitalized for bronchiolitis with viral coinfection (or = 2.498; 95% ci: 1.229-5.077), in those who were older at the table 5 with a p-value < 0.10 were entered in a stepwise logistic regression analysis to estimate which factors were independently related to the development of asthma at 6-8 years. age > 12 months at admission for bronchiolitis (or: 7.389; 95%ci: 2.683-20.352, p<0.001), age > 9 months at admission for bronchiolitis (or: 3.484; ci95%: 1.459-8.317, p:0.005), allergic rhinitis (or: 5.910; 95%ci: 2.622-13.318, p<0.001), and viral coinfection-bronchiolitis (or: 3.374; ci95%: 1.542-7.386, p:0.01) were the strongest independent risk factors for asthma at 6-8 years of age. our current study shows for the first time to our knowledge, that there is a strong and independent association between severe bronchiolitis associated with viral coinfection and the development of asthma at 6-8 years old. viral coinfections are usually detected in up to 30% of children with an acute respiratory tract infection [14] . our group, in previous studies, found viral coinfections in 22.8% of 318 infants less than 2 years old admitted with bronchiolitis [11] and in 35% of 2,993 children less than 14 years old admitted with lower respiratory tract infection [15] . our current results confirm that the most frequently identified viral coinfections are dual rsv-hrv and rsv-hbov infections. results from cohort studies evaluating the severity of acute respiratory viral coinfections are conflicting. our group previously found that clinical severity was associated mainly with rsv infections, either alone or in combination with other viruses [15] . however, asner et al. [16] in a recent systematic review and meta-analysis found no differences in clinical disease severity between viral coinfections and simple infections, although an increased risk of mortality was observed amongst preschool children with coinfections. they conclude that large, prospective, well designed studies with objective outcomes are needed to better understand the clinical significance of viral respiratory coinfections. regarding the role of early viral respiratory coinfections in the development of wheezing or asthma, so far, no study evaluating this possible association has been published. our results confirm that recurrent wheezing and asthma are very common in the first years of life after a severe bronchiolitis. but the likelihood of developing asthma was 2.5 times higher if bronchiolitis was associated with viral coinfection compared to single infections. in addition, the number of rehospitalizations for asthma was also significantly higher in coinfections than in single infections, suggesting that coinfections are associated not only with more frequency of asthma but also with greater severity. the mechanism by which respiratory infections are associated with subsequent asthma is not fully understood but the different inflammatory responses released after viral infections seem to play a relevant role. the induction of a prevalent th2-type response, with the production of il-4, il-5, and il-13, results in a less robust and less efficient reaction to the infection, clinically presenting as increased disease severity and risk for future recurrent wheezing [17, 18] . several reports showed that thymic stromal lymphopoietin (tslp) is a key initiator of allergic airway responses [19] and significant negative correlation has been described between tslp levels in induced sputum and fev-1 in asthmatic children [20] . tslp is secreted by rsv-infected airway epithelial cells (aecs) to promote the activation of dendritic cells (dcs) [21, 22] . the activated dcs can then induce a th2 cell polarization response in the lung, which could contribute the development of asthma in rsv-infected individuals, as well as induction of exacerbations in asthmatic rsv-infected patients. it has been suggested that the ability of rsv to induce tslp expression by aecs is responsible for the subsequent th2 aspects of the immune response [23] . other recent studies performed in young children with hrv infection, have also found higher levels of nasal tslp [24, 25] . on the other hand, various genetic studies in humans have identified both il-33 and its receptor st2, as being key regulators in the development of asthma [26] and to have a strong th2-promoting ability in animal models of asthma [27] . il-33 is responsible for the immunopathophysiological response observed following neonatal rsv infection in mice. its presence in nasal aspirates of human infants with severe rsv, together with the finding that by blocking the receptor of interleukin 33 in vivo [28] , th2 responses are greatly reduced, suggest it has a role in disease severity and asthma [29] . our findings, in a recent study conducted in hospitalized infants with bronchiolitis and in healthy controls, showed that nasal tslp and il-33 are detected more frequently in viral coinfections than in single infections. in addition, infants with dual rsv+hrv infection were 9 times more likely to have detectable nasal tslp and this association was independent of other factors such as age or illness severity [30] . it is worth noting that no patient with bronchiolitis but with negative viral detection had detectable levels of nasal tslp or il-33, suggesting that the release of these proteins may be mediated by respiratory viruses. it can be hypothesized that this stronger tslp response after viral coinfection bronchiolitis, could stimulate a vigorous production of th2-associated effector cytokines, such as il-4, il-5, il-13, as was reported in asthmatic adults by ying et al, that could be associated with higher frequency of wheezing and asthma development later on [31] . as far as we know, only lukkarinen et al [7] have compared the systemic th1-type, th2-type and proinflammatory cytokine profiles in young children with simple versus viral coinfection, being hrv and hbov1 the viruses involved. they included hospitalized children less than 35 months of age with their first or second wheezing episode. they observed that wheezing children with hrv had higher proinflammatory responses than did those with hbov1 or those with coinfection hrv+hbov1, suggesting that hbov1 may interfere with hrv-induced immune responses. furthermore, this immunological response was accompanied by the clinical finding that children with hrv+hbov1 wheeze tended to develop recurrent wheezing later and less often than did those with hrv wheeze. our previous and current results suggest that an interaction may also occur in rsv and hrv coinfections, but in an opposite way, towards a predominant th2 response that could increase the development of recurrent wheezing/asthma. the risk of asthma in our study, like in other cohort studies on hospitalized children with lower respiratory symptoms, was directly dependent on age [4, 32] : the adjusted or was 3.4 for asthma in children with bronchiolitis aged > 9 months and 7.3 for asthma in school age in bronchiolitis children aged > 12 months. however, lukkarinen et al [33] , in a 7-year followup found asthma inversely dependent on age, probably because they studied infants with wheezing only, vs. bronchiolitis, with or without wheezing, in other studies as in ours. this different inclusion criteria may bias the kind of patients included in the studies. rhinitis is a known risk factor for asthma in children [34] . our results suggest that rhinitis is an independent risk factor for asthma persistence in school-age children with previous severe bronchiolitis. lauhkonen et al [35] in a prospective follow-up study, in 102 children hospitalised for bronchiolitis, also found that current asthma was associated with prolonged rhinitis and a positive skin prick test at five to seven years. on the other hand, recent reports have demonstrated that the severity of rhinitis and asthma are closely related in children [36] . thus, as other authors have stated [37] , all these results highlight the convenience to assess nasal symptoms in infants and children with recurrent wheezing and asthma. in conclusion, asthma at the age of 6-8 is more frequent and severe in those children previously hospitalized with viral coinfection bronchiolitis compared with those with single infection. moreover, viral coinfection, allergic rhinitis and older age at admission seem also to be strong independent risk factors for asthma development in children previously hospitalised because of bronchiolitis. however, given the complexity of the immunological mechanisms involved, more studies are needed to confirm this association and better 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initiator of allergic airway inflammation in mice increased expression of thymic stromal lymphopoietin in induced sputum from asthmatic children tslp-activated dendritic cells induce an inflammatory t helper type 2 cell response through ox40 ligand tslp-activated dendritic cells induce human t follicular helper cell differentiation through ox40-ligand thymic stromal lymphopoietin is induced by respiratory syncytial virus-infected airway epithelial cells and promotes a type 2 response to infection rhinovirus infection in young children is associated with elevated airway tslp levels rhinovirus-induced airway cytokines and respiratory morbidity in severely premature children defining the contribution of snps identified in asthma gwas to clinical variables in asthmatic children il-33 is more potent than il-25 in provoking il-13-producing nuocytes (type 2 innate lymphoid cells) and airway contraction role of interleukin 33 in respiratory allergy and asthma respiratory syncytial virus disease is mediated by age-variable il-33 thymic stromal lymphopoietin, il-33, and periostin in hospitalized infants with viral bronchiolitis thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of th2-attracting chemokines and disease severity wheezing rhinovirus illnesses in early life predict asthma development in high-risk children prednisolone reduces recurrent wheezing after first rhinovirus wheeze: a 7-year follow-up allergic rhinitis as a predictor for wheezing onset in school-aged children following up infant bronchiolitis patients provided new evidence for and against the united airway disease hypothesis severity of rhinitis and wheezing is strongly associated in preschoolers: a population-based study preschool-age wheezing phenotypes and asthma persistence in adolescents key: cord-356188-rwf78stz authors: oshansky, christine m.; thomas, paul g. title: the human side of influenza date: 2012-07-01 journal: journal of leukocyte biology doi: 10.1189/jlb.1011506 sha: doc_id: 356188 cord_uid: rwf78stz a clear understanding of immunity in individuals infected with influenza virus is critical for the design of effective vaccination and treatment strategies. whereas myriad studies have teased apart innate and adaptive immune responses to influenza infection in murine models, much less is known about human immunity as a result of the ethical and technical constraints of human research. still, these murine studies have provided important insights into the critical correlates of protection and pathogenicity in human infection and helped direct the human studies that have been conducted. here, we examine and review the current literature on immunity in humans infected with influenza virus, noting evidence offered by select murine studies and suggesting directions in which future research is most warranted. influenza infection in humans can cause a wide range of disease symptoms, from asymptomatic to serious illness, characterized by sudden onset of fever, myalgia, and respiratory indications, including nonproductive cough, sore throat, and rhinitis. in addition, children infected with influenza commonly present with otitis media, nausea, and vomiting and more frequently, complain of rhinorrhea [1, 2] . historically, the pediatric and elderly populations are considered vulnerable during influenza epidemics [3] [4] [5] [6] , and although still debated, studies suggest that children have a unique role in the spread of influenza during epidemics [7] [8] [9] . children under 2 years old are more susceptible to secondary complications [10] , and infants with severe respiratory tract viral infection are often at greater danger for pulmonary dysfunction, including wheezing, allergy, and asthma later in life [11] [12] [13] . in contrast, elderly individuals are at higher risk for developing hemorrhagic bronchitis, pneumonia, and death when infected with influenza [14] , and this population generally has a less-robust immune response following influenza immunization. although rsv infections are the leading cause of hospitalizations of children in the united states, influenza accounts for the highest incidence of infection in children under 2 years of age [15] . influenza epidemics occur annually and can cause significant morbidity and mortality worldwide. during 1976 -2007, the cdc estimated that in the united states alone, annually, 8.5% (6309) of all "deaths with underlying pneumonia and influenza causes" (74, 363) were influenza-associated, and 2.1% (23, 607) of "deaths with underlying respiratory and circulatory causes" (1,132,319) were influenza-associated [16] . furthermore, individuals with underlying respiratory and cardiac disease or diabetes mellitis, infected with influenza, are often at higher risk for developing hemorrhagic bronchitis or pneumonia and associated comorbidities. in 2008, it was estimated that there were 28,000 -111,500 deaths attributable to influenza in children ͻ5 years of age globally, with developing countries carrying the largest burden [17] . in the united states, annual influenza epidemics account for an estimated $10 billion (12% of the total economic burden) in direct medical costs [18] and an estimated $44 -$163 million in hospitalization costs for children [19] . introduction of a swine-origin influenza into the human population in 2009 quickly disseminated globally, causing the first pandemic of the 21st century. this novel h1n1 virus, hereafter referred to as a(h1n1)pdm09 [20] , disproportionately affected children and young adults [21, 22] . observed mortality increased between four and 10 times in the pediatric population during the initial wave of a(h1n1)pdm09 infection in 2009 compared with previous influenza seasons [23] , whereas those individuals born prior to 1950 had pre-existing, cross-reactive antibodies against a(h1n1)pdm09 [24] . as a result, within the population of those with laboratory-confirmed a(h1n1)pdm09, 2% were older individuals (ͼ60 years), whereas 79% were ͻ30 years old. risk factors for severe a(h1n1)pdm09 infection are similar to those for seasonal influenza but also include obesity. the majority of the severe or fatal cases have accompanying chronic illnesses, such as cardiac disease, chronic respiratory diseases, and diabetes [25] . several studies suggest that pregnant and postpartum women are at increased risk for complications from infections with a(h1n1)pdm09 [26 -28] . furthermore, severity of a(h1n1)pdm09 infection was related to the length of time between symptom onset and the initiation of antiviral treatment with na inhibitors [29] . influenza infections are associated with secondary bacterial infections, or superinfections, most notably, streptococcus pneumoniae and staphylococcus aureus (reviewed in ref. [30] ). in children, the most common secondary bacterial infections lead to acute otitis media, which is found in 50% of patients with symptomatic influenza infection, with five to six cases observed annually for every 100 children ͻ2 years of age [10] . since the emergence of a(h1n1)pdm09, approximately one-third of fatal infections are associated with bacterial coinfection [31, 32] , but this number includes cases where respiratory support is needed, adding to the risk of secondary infections. in fatal cases of those infected by a(h1n1)pdm09, diffuse alveolar damage alone, diffuse alveolar damage associated with necrotizing bronchiolitis, and diffuse alveolar damage with hemorrhage were found, and in most of the cases where necrotizing bronchiolitis was found, the individuals had bacterial coinfections [33] . vaccination remains an effective and primary preventive strategy to avert influenza infection. the world health organization and cdc recommend that children and adults over the age of 6 months receive an influenza vaccination annually [34] . currently, there are three licensed seasonal vaccines administered in the united states: 1) a tiv, administered by i.m. injection; 2) a laiv, delivered intranasally; and 3) an i.d.-administered tiv preparation [35] . each includes three circulating strains of influenza grown in eggs, reflecting annual surveillance data that predict which strains (a/h1, a/h3, and b) are most likely to be circulating the following season in northern and southern hemispheres. the i.m. tiv is approved for use in children and adults over 6 months of age, whereas the newer i.d. tiv is approved for adults aged 18 -64 years. laiv is approved for use in healthy children and adults between 2 and 49 years of age. the current influenza vaccines work to induce memory recall responses, primarily via humoral immune responses against the ha and na surface glycoproteins, and ha inhibition antibody responses following immunization correlates with protection against infection [36, 37] . the main component standardized in influenza vaccines is the ha protein, and it is well-characterized in safety trials and challenge studies that the amount of ha within the vaccine correlates with level of protection elicited by immunization [38, 39] . beginning during the northern hemisphere's 2010 -2011 influenza season, a vaccine containing 60 g ha/vaccine strain, as opposed to 15 g in other preparations, was approved as an alternative tiv for elderly individuals (over age 65 years) [35] . following vaccination, ascs rapidly proliferate upon antigen exposure, and cell numbers peak 1 week postimmunization in healthy adults and children [40, 41] . igg and iga ascs decline to low levels by 4 -6 weeks postimmunization [42, 43] . this increase in the total numbers of ascs corre-sponds to nab levels postvaccination, peaking at 4 weeks in adults and children [43] . laiv elicits a strong serum and mucosal influenza-specific antibody response [44] , and studies in young children (6 -59 months of age) showed that those receiving laiv had significantly reduced incidence of influenza infection [45] . although limited data exist, influenza-specific ascs and nab titers are increased to a greater degree following tiv immunization compared with laiv vaccination [43, 46, 47] , but the role of neutralization of laiv occurring in antigen-experienced individuals is not well understood. influenza immunization is effective, and studies in healthy children ͻ15 years of age have shown tiv efficacies ranging from 31% to 90% (reviewed in ref. [48] ). since 2010, universal influenza vaccination is recommended, i.e., for all individuals at least 6 months of age, but traditional efforts remain focused on those individuals at greatest risk of serious disease: young children, elderly, individuals with pulmonary or cardiovascular disorders, and those who are immunocompromised or pregnant [35] . vaccination programs also elicit indirect benefits, including herd immunity, by which immunized individuals protect those who are immunocompromised or other nonvaccinated individuals [49, 50] . this effect was observed in a retrospective epidemiological study in japan and the united states examining mortality and influenza vaccination rates between 1949 and 1998 [51] . as children have long been considered to have an important role in the spread of influenza during epidemics [7] [8] [9] , japanese officials legislated compulsory vaccination of school-aged children in 1977, and as the policy changed to optional immunization in 1987, it became clear that mortality rates were inversely correlated with overall vaccination coverage. however, recent reviews of vaccine studies in various populations have concluded that overestimation of the benefits of vaccination is common, particularly within the elderly population [52, 53] , but studies that take into account selection biases do suggest that vaccination is associated with lower risk of hospitalization for pneumonia or influenza, as well as all-cause mortality [52, 54, 55] . furthermore, in october 2009, the united states commenced a national influenza h1n1/2009 vaccination campaign, and it is estimated that ϳ41.2% and 43% of the u.s. population received the 2009 -2010 and 2010 -2011 influenza vaccines, respectively [56] . following implementation of this campaign, the frequency of positive influenza cultures reported to the cdc declined quickly. immunization and herd immunity resulting from prior exposure of a(h1n1)pdm09 likely resulted in decreased cases of influenza during the 2010 -2011 influenza season. undoubtedly, influenza vaccines can be improved, particularly for the most vulnerable populations. continuous epidemiological surveillance of circulating influenza strains is required for keeping pace with antigenic drift and shift (reviewed in ref. [57] ). antigenic drift is an endless battle, whereby viral variants emerge as a result of point mutations within the viral genome, escaping antibody-mediated viral neutralization. whereas many mutations occur throughout the viral genome, the point mutations within the ha or na proteins can change antibody-binding domains, such that individuals no longer have adequate, protective b cell memory responses. in contrast, major qualitative changes in antigenicity as a result of antigenic shift occur as a result of genetic reassortment and replacement of one or more influenza gene segments. this, in turn, creates a new influenza virus subtype, to which little or no immunity may exist within the population. antigenic shift remains a chief concern, in that highly pathogenic influenza viruses could potentially gain efficient human-to-human transmission. as influenza viruses frequently undergo antigenic drift and less frequently, shift, a desirable vaccine candidate would be able to induce highly specific, cross-reactive antibodies and t cell responses. one strategy used in vaccine improvement includes the use of adjuvant to increase antigen uptake by apcs and enhance cytokine production. neither of the u.s.-licensed tivs nor laiv include adjuvant, but there are several licensed vaccines available outside of the united states that use oil and water emulsions for seasonal influenza as well as for a/h5n1 (reviewed in ref. [58] ). for instance, novartis vaccines and diagnostics (switzerland) manufactures fluad using mf59 adjuvant, and glaxosmithkline biologicals (belgium) produces pandemrix, which includes as03. fluad has been used in europe since 1997 and was recently approved for use in canada in adults 65 years and older. mf59 and as03 are both squalene-based with surfactants included to stabilize the emulsions. clinical trials of mf59-adjuvanted vaccines are well-tolerated and elicit higher antibody responses in adults [59 -61] and in children [62] [63] [64] compared with unadjuvanted vaccine preparations. the concept of a universal influenza vaccine, where one vaccine elicits protection against multiple influenza subtypes, is becoming a reality. the influenza m2 ion channel is expressed at high levels on the surface of influenza-infected cells and has a conserved ectodomain (m2e) that is 24 aa in length [65] . animal studies have demonstrated improved viral clearance and cross-protection upon lethal challenge in mice following passive m2 mab transfer or m2-immunized mice (reviewed in ref. [66] ). phase i clinical safety trials evaluating vax102, manufactured by vaxinnate, have reported increased m2e antibody responses [67] . creation of a universal influenza vaccine that targets conserved regions of the ha stem region is particularly appealing, and several groups have demonstrated encouraging results using broadly neutralizing mab to regions of ha that are cross-reactive between group 1 and group 2 influenza strains [68 -70] . structural analyses indicate that these antibodies bind to conserved pockets of the a helix of the ha stalk, located below the trimeric head region [71] [72] [73] . it is therefore within the realm of possibility to focus immunization strategies to these conserved regions in the ha and m2 proteins to elicit effective antibody, and presumably t cell, responses in the case of emergent influenza viruses. still, it remains a mystery as to why these conserved epitopes have not generated a high level of cross-protection in populations repeatedly immunized or infected with diverse viral strains. factors that limit influenza vaccine efficacy in humans include age, immunocompetence, and pre-existing antibody levels from previous infections or immunizations. whereas key lessons have been learned using animal models of influenza infection, studies done using human participants are invaluable for describing the unique and often complex characteristics of the human immune response to infection and immunization. here, we describe central findings obtained from human influenza challenge studies, natural infections, and ex vivo characterization of human immune cells ( table 1 ). figure 1 indicates where questions remain for the human immune response to infection. effective control and clearance of influenza infection can involve most components of the innate and adaptive immune responses. in mammals, the cellular innate immune system acts as a first-line defense consisting of multipotent hematopoietic stem cells that differentiate into nk cells, mast cells, eosinophils, and basophils, as well as macrophages, neutrophils, and dcs, collectively known as phagocytic cells. phagocytes function as apcs, engulfing and digesting opsonized pathogens and apoptotic cells. these cells essentially act as scavengers to uptake toxic metabolic byproducts and produce myriad inflammatory mediators, which ideally, result in the killing of viruses, bacteria, and parasites. however, abundant inflammation often has a role in creating immunopathology and can contribute to diverse inflammatory diseases, including asthma [123] [124] [125] . in the absence of protective immune memory, the innate response limits initial viral replication, while the adaptive immune response develops. influenza primarily infects epithelial cells lining the respiratory tract but can infect directly or be phagoctyosed by "professional" innate immune cells, dcs, and macrophages, which mature in response to viral stimuli or signals from other infected cells. after activation, mature dcs migrate to draining lns, where they have a major role in antigen presentation to naïve t cells. influenza infection of human monocyte-derived dcs can result in decreased dc maturation from ns1 suppression of type i ifn production [126] . seasonal h1n1 viruses, including a(h1n1)pdm09, elicit weak, proinflammatory cytokine gene expression in human dcs [127] . we know from murine models that influenza contains ligands that activate several prr signaling pathways, including tlr3 and retinoic acid-inducible gene i (rig-i), which recognize dsrna, and tlr7, which recognizes ssrna [128 -130] . type i (ifn-␣/␤) and type iii (ifn-1-3) ifns mediate expression of more than 300 isgs via the jak/stat pathway and are important in the induction of antiviral responses against influenza viruses [131] . stimulation of these pathways in dcs and macrophages leads to the induction of a potent antiviral response, including production of proinflammatory cytokines il-6 and ifn-␣ within the respiratory tract by day 2 following infection, paralleling the peak of viral replication, mucus production, and disease symptoms. tnf-␣ and il-8 levels peak between day 3 and 6 after infection as symptoms begin to subside [95] . antimicrobial peptides, such as collectins, defensins, and cathelicidins, are important innate effectors in the human defense against influenza infection. collectins, or collagenous lectins, are found primarily in mucosal secretions and include sp-a and sp-d; sp-d has been shown to inhibit ha activity by binding to n-linked, high-mannose oligosaccharides in a calcium-dependent manner, while enhancing activation of neutrophils in in vitro assays [132, 133] . in murine models, sp-a and sp-d have roles in promoting viral clearance from lungs and reducing inflammatory cytokines [134, 135] , but their role during human influenza infection is less well-defined. influenza virus impairs calcium metabolism and subsequent calcium deactivation in neutrophils [136] , and interestingly, sp-d can protect neutrophils from calcium deactivation if preincubated with virus, presumably by inhibiting ha activity [132] . in humans, the only cathelicidin, human cathelicidin 18, the active form of which is ll-37, is found in diverse cell types, including neutrophils, nk cells, monocytes, b cells, ␥␦ t cells, mast cells, as well as epithelial cells lining the lungs [137, 138] . like surfactants, ll-37 shows antiviral activity against influenza and can reduce viral titers in in vitro studies [139] . defensins have a key role in host defense and act primarily to disrupt lipid membranes leading to lysis and subsequent death of bacteria, fungi, and enveloped viruses [140] . there are two classes of defensins, ␣-defensin and ␤-defensin, which were first described in models of host defense against bacterial pathogens. both types are found in the respiratory and gastrointestinal tracts. the ␣-defensins in humans include hnp-1, -2, -3, and -4, all found in high concentrations within neutrophil granules and released within the lungs in response to proinflammatory signals during infection and inflammation [141] . in vitro, hnp-1 and hnp-2 act as chemokines for human monocytes [142] , and hnp-1, -2, and -3 can induce neutrophil internalization of influenza in vitro [143] . moreover, the ␣-defensins may have a role in directly inactivating influenza virus as a/wsn preincubated with hnp-1 showed decreased infection in vitro in a dosedependent manner [144] . hnp-1 can also inhibit influenza virus replication via modulation of cellular pathways, including the pkc pathway [145] . heterogeneous populations of monocytes comprise ϳ10% of blood leukocytes in humans (4% in the mouse), and their roles in controlling pathogens and inflammation can be characterized by functional cellular subsets. human blood monocytes can be divided into three populations based primarily on cell surface expression of cd14 (lps receptor) and cd16 (fc␥riii). the first subgroup consists of "classical monocytes", characterized by cd14 expression and lack of cd16 expression (cd14 ϩ cd16 ϫ ). the cd14 ϩ cd16 ϫ monocytes comprise 80 -90% of blood monocytes and resemble murine inflammatory ly6c ϩ (gr1 ϩ ) monocytes [146] . the second subgroup is defined by high expression of cd14 and cd16 (cd14 ϩ cd16 ϩ ) and release tnf following lps stimulation [147] . the third monocytic subgroup is characterized by low expression of cd14 and high expression of cd16 (cd14 dim cd16 ϩ ), and at least one study has shown increased numbers in the blood of sepsis patients [148] . cd14 dim blood monocytes function as efficient producers of proinflammatory cytokines in response to viruses and nucleic acids [149, 150] . few studies have examined the role of monocytes during influenza infection in humans, particularly regarding the specific subsets mentioned above, but comparison of ifn-␥ production from t cells cocultured with cd64 ϩ cd16 ϫ and cd64 ϫ cd16 ϩ monocytes [119, 120] cellular immunity class i hla presents peptides from internal and external viral proteins. [121, 122] showed that cd64 ϫ cd16 ϩ monocytes (equivalent to the cd14 ϩ cd16 ϩ monocytes) induced a greater response to influenza a antigen [151] . in vitro, human dcs and macrophages treated with type i or type iii ifn prior to influenza infection result in potent antiviral activity and decreased viral replication [127] . among dc populations, cd11c ϫ cd14 ϫ hla-dr ϩ cd123 ϩ pdcs were found to produce more ifn-␣ in response to a high dose of a/pr8/8/34 (h1n1) [152] . whereas increased numbers of total cd14 ϩ cells are observed in the blood of influenza-infected individuals, decreased blood myeloid dcs and pdcs were observed (defined in this study as hla-dr ϩ cd14 ϫ cd16 ϫ cd11c ϩ and hla-dr ϩ cd14 ϫ cd16 ϫ cd123 ϩ , respectively) [153] . however, increased numbers of pdcs were observed in nasal secretions compared with healthy controls within this cohort. the ns1 protein is a well-characterized antagonist of the host's antiviral response, acting primarily to circumvent ifn-␣/␤ responses (reviewed in ref. [154] ), and in vivo models show that its deletion results in virus attenuation and higher levels of ifn [155] . type i ifn antagonism can result in inhibition of adaptive immunity by interfering with dc maturation and thus, the ability of dcs to activate t cells [126] . the ns1 protein from influenza b virus inhibits isgylation, an important host antiviral effector mechanism, by interacting with the n-terminal domain of isg15 [156] . murine models have provided several clues regarding the actual role of monocytes during the innate immune response to influenza infection. in vivo, ccr2-dependent recruitment of inflammatory monocytes is critical for host defense and contributes to the innate immune response, as well as downstream adaptive responses [157] . during influenza infection, ccr2deficient mice have reduced monocyte and increased neutrophil recruitment, but, despite higher viral lung titers, in one model of infection these mice were protected from severe pneumonitis [158, 159] . in the lungs, ccr2-mediated recruitment of tnf and inos-producing dcs augments influenzaspecific t cell responses [160] . at least one murine study suggests that pregnant mice have increased infiltration of neutrophils (cd11b high ly6g high mhcii ϫ ) and macrophages (cd11b high ly6g ϫ mhcii high ) and elevated, proinflammatory cytokine levels, despite no change in viral titers in the lungs compared with nonpregnant animals [161] . in vitro, influenza infection of murine alveolar epithelial cells resulted in monocyte production of mcp-1 (ccl2, the ligand for ccr2) and rantes, and monocyte migration was dependent on ccr2 expression [162] . although some murine models suggest that neutrophils may have little role in mediating viral clearance [163] , type i interferon (ifn-␣/␤)-dependent generation of ly6c hi monocytes following influenza infection may be necessary to limit excess neutrophil infiltration, preventing tissue damage caused by uncontrolled inflammation [164] . in addition, the newly described innate lymphoid cells have been implicated in having a role in airway integrity following influenza infection [165] . thus, innate immune cells, particularly monocytes, have at least two roles during influenza infection: to boost the innate immune response by producing proinflammatory cytokines and to enhance influenza-specific t cell responses. however, the lack of clear correspondence between murine and human monocytic subsets has made the application of these data to the human situation difficult and highlights the need for specific studies focused on human innate immune responses. during rsv and influenza infection of infants, neutrophils are recruited to the upper and lower airways in high numbers [103, 104] , and fatal cases during the a(h1n1)pdm09 pandemic with necrotizing bronchiolitis had more neutrophils within the lung infiltrate [33] . the neutrophil chemoattractant il-8, produced by bronchial epithelial cells, is elevated early during experimental human influenza infection [95] and is increased in the serum of a/h5n1-infected individuals [96] . increased il-8 levels in bals of humans are associated with the infiltration of neutrophils and may have a role in neutrophil-mediated lung injury and development of acute respiratory distress syndrome [97] . ex vivo influenza infection of pb-mcs has been shown to induce apoptosis in neutrophils [166] and peripheral blood monocytes [167] . influenza infection of neutrophils resulted in increased hydrogen peroxide production, enhanced surface expression of fas antigen and fasl, and secretion of fasl into the supernatant [166] . interestingly, this study showed that influenza infection of neutrophils resulted in increased internalization of escherichia coli, suggesting a role in controlling bacterial secondary infections in humans. moreover, standard chemotaxis assays during experimental influenza infection [168 -170] or chemokine receptor expression during ex vivo influenza infection [105] demonstrated impaired chemotaxis of monocytes and granulocytes. still, although all of these studies suggest that neutrophils can be regulated and recruited during human influenza infection, the specific protective and pathological roles that they play in vivo remain unclear. nasopharygeal aspirates of infants infected with influenza have increased levels of proinflammatory cytokines such as il-6, tnf-␣, il-10, or ifn-␥ compared with rsv-or human metapneumovirus-infected infants [98 -100] , and mip-1␣ levels are higher in infants with more severe influenza infections characterized by hypoxic bronchiolitis [101] . variability between these studies is likely a result of seasonal differences observed year-to-year. individuals infected with a/h5n1 have high viral loads detected within respiratory secretions and peripheral hypercytokinemia [96, 102] . severe disease is associated with macrophage infiltration into the lungs, and chemoattractants of monocytes and macrophages, such as ifn-inducible protein 10 (ip-10), monokine induced by ifn-␥ (mig), and mcp-1, were found to be elevated in the serum of influenza-infected individuals and were highest in those infected with a/h5n1 [96] . therefore, peripheral (or local) hypercytokinemia likely contributes to enhanced immunopathogenesis during influenza disease. although there is no difference in the duration of viral shedding in children hospitalized for a/h1n1 or a/h3n2 infections compared with those infected with influenza b virus, children with influenza a exhibited different serum cytokine profiles. serum ifn-␥ levels were similar during acute-phase infection [171] , but levels were increased 3 weeks following influenza b infection compared with influenza a infection. furthermore, serum il-4 levels were significantly higher during the acute and recovery phases of influenza a infection. influenza strain differences were also observed in umbilical cord blood lymphocytes treated with uv-inactivated viruses. uv-inactivated influenza b virus induced less ifn-␥, il-4, or il-10 relative to uv-inactivated a/h1n1, a/h3n2, and a/h2n2 [172] . together, these data strongly suggest that innate cells and their associated responses have an important role in influenza disease manifestation, by providing protection and by contributing to pathological outcomes. the murine studies cited here clearly demonstrate that the local inflammatory environment can be dramatically different from the blood profile. the vast majority of experimental data in humans is restricted to blood leukocytes, with minimal emphasis placed on sites of infection. further, we lack good, functional analysis of these cells in humans, and most studies provide correlational, cross-sectional observations. as a result, there is a significant gap in our knowledge of innate immune cells in affected tissues. the history of respiratory virus challenge studies dates back to the 1918 influenza pandemic [173] , and early studies in humans infected with influenza identified the principal correlate of protection against influenza infection as strain-specific, virus-neutralizing antibodies directed against the ha. these studies (reviewed in ref. [80] ) suggested that the presence of serum antibody is associated with protection, as illness is less frequently observed in those people with pre-existing nab [74] . subsequent studies showed that protection is also correlated with the development of serum and mucosal anti-na or anti-ha igg and iga [37, 47, [75] [76] [77] [78] , but this protection can be incomplete, as individuals with detectable nab can still be infected [79, 174] . antibodies specific for the na have been shown to reduce disease severity by restricting virus release from infected cells and enhancing viral clearance (reviewed in ref. [175] ). in addition to ha and na, antibodies are produced to np, matrix m2 protein, and pb1-f2 [176] , although the significance of these responses remains unclear. of course, antigenic shift and antigenic drift can both thwart the efficacy of serological memory. the recent circulation of a(h1n1)pdm09 has provided a modern "experiment of nature", demonstrating the importance of pre-existing antibody from prior infection or vaccination [81] [82] [83] . even prior to 2009, it was well-recognized that heterosubtypic immunity has a large role in protection against antigenic variants of influenza [84, 85] , and vaccine studies support the idea that cross-protective, adaptive immune responses occur [86] . likewise, neutralizing polyclonal antibodies cross-reactive against h5 can also be produced in individuals vaccinated against seasonal influenza, as well as those naturally infected with a(h1n1)pdm09 [87, 88] . compared with seasonal influenza infections prior to 2009, ϳ31% of b cell epitopes are conserved in a(h1n1)pdm09, with 17% of those conserved within the surface viral proteins [177] . the ha of a(h1n1)pdm09 contains sequences homologous with that of a/h1n1 (1918) demonstrated by the a/h1n1 (1918) mab 2d1, which binds to the sa antigenic site of the ha [89] . furthermore, this particular antibody can neutralize ha activity in vitro, and mab-treated mice show reduced viral titers when challenged with a(h1n1)pdm09 [90] . most neutralizing antibodies bind to the exposed regions of the ha that surround the receptor-binding site and interfere with attachment to sialic acids on the cell surface. therefore, generation of crossprotective neutralizing antibodies is elusive, as the external viral proteins are most exposed to immune pressure, but universal immunization against conserved ha stalk and m2e domains is promising [71] [72] [73] . human pbmcs include at least three major subsets of dcs: cd141 ϩ mdcs, cd1c ϩ mdcs, and pdcs (reviewed in ref. [178] ). dcs process and present antigen to t cells and therefore have an important role in regulating the adaptive immune response. depending on the local cytokine milieu, mdcs can differentiate into macrophages or tissue-specific dcs (i.e., epithelial langerhans cells or interstitial dcs). as influenza primarily infects epithelial cells lining the respiratory tract, lung-resident dcs and macrophages are particularly important for efficient development of an adaptive immune response. once activated, mdcs function as apcs to activate t cells; on the other hand, pdcs are major producers of type i ifn upon tlr activation. in response to influenza infection in vitro, human dcs and macrophages undergo maturation, secreting type i ifn and inducing t cell proliferation and ifn-␥ production [179 -181] . cellular immune responses (cd8 ϩ ctls and cd4 ϩ th cells) are important for virus clearance in murine models of influenza infection, with cd4 ϩ and cd8 ϩ t cell-depleted mice displaying delayed viral clearance. the relevance of these observations remains somewhat controversial in humans. regard-less, as in mice, the human lung contains resident respiratory virus-specific cd8 ϩ memory t cells [182] , which can effectively kill virus-infected cells. human cd8 ϩ t cells recognize antigen in the context of surface hla molecules and generally recognize a broad range of influenza epitopes, including those from structural and nonstructural influenza proteins [121] . approximately 41% of cd4 ϩ and 69% of cd8 ϩ t cell epitopes are conserved in a(h1n1)pdm09 from the prior circulating seasonal h1n1 [177] . furthermore, cd8 ϩ intraepithelial t cells can mount fast and efficient recall responses upon viral infection [183] , and immunization studies in humans have shown that t cell responses peak within 1 week after symptom onset [174] . in terms of pandemic viruses, individuals infected with a(h1n1)pdm09 were shown to have rapid, virus-specific cd8 ϩ t cell recall responses early during infection, but this ctl response wanes over time [184] . likewise, vaccinated individuals, who were then naturally infected with a(h1n1)pdm09, had t cell responses increase by twofold early after infection [174] . memory cd4 ϩ or cd8 ϩ t cells isolated from pbmcs of healthy individuals are also cross-reactive against proteins of heterologous viruses, including those from a(h1n1)pdm09 and a/h5n1 [91] [92] [93] [94] , but immunocompromised patients do not have lasting antibody or cd4 ϩ /cd8 ϩ memory responses following natural a(h1n1)pdm09 infection [185] . furthermore, ctls, isolated from a(h1n1)pdm09-or a/h5n1-inexperienced individuals with a memory cd45ra med cd62 ligand lo ccr7 ϫ phenotype, will respond robustly to peptide pools and influenza-infected cells [34, 177, 186, 187] , suggesting that t cells may provide some level of protection against conserved epitopes [110] . although nonconserved regions of the virus may effectively escape recognition by t cells [188] , the majority of t cell responses is elicited against conserved, internal viral proteins, generating much interest in vaccine or therapeutic strategies that involve t cell-mediated immunity. a frequently neglected population of lymphocytes, ␥␦ t cells comprise 1-5% of t cells in the blood of humans, the majority of which are v␥9v␦2 t cells. these ␥␦ t cells are considered to be a major innate-like t cell subset (reviewed in ref. [189] ), and in vitro studies suggest that activated human v␥9v␦2 t cells may have a role in the antiviral response by killing influenza-infected, monocyte-derived macrophages and producing high levels of ifn-␥ [190, 191] . in particular, neonates are known to have compromised cellular immune responses and represent an important target population for influenza immunization. despite being such a critical target population, there are relatively few neonatal models of respiratory infections, and for ethical reasons, it is appropriately difficult to acquire data from human neonates. in the case of rsv infection, murine neonate models suggest that the age of primary viral infection can determine the immunopathology of a secondary viral infection [192] [193] [194] . during rsv infection, younger murine neonates (յ7 days) will develop an immune response characterized by increased eosinophil and t cell recruitment to the lungs, enhanced airway hyper-responsiveness, and higher il-13 levels upon reinfection 12 weeks later, whereas older mice (ն4 weeks) develop an immune response characterized by decreased t cell and eosinophil recruitment to the lungs upon reinfection [192, 193] . likewise, a neonatal mouse model of influenza a virus showed that early infection was associated with decreased cd8 ϩ t cell recruitment and function and enhanced airway inflammation [195] . these studies highlight an important concept, whereby infection too early results not in protective immunity but immunopathogenesis. consistent with an age-dependent defect in adaptive immunity, during rsv and influenza infection of infants, few cd4 ϩ or cd8 ϩ t cells or cd56 ϩ nk cells are found in the upper and lower airways [100, 103, 104] , in contrast to the observed infiltration of lymphocytes in adult a/h5n1 influenza [106] and sars cov infections [196, 197] . moreover, fatal cases of a(h1n1)pdm09 had increased numbers of cd8 ϩ t cells and granzyme b ϩ cells in their lungs [33] . this observation may represent a correlation with higher levels of viral antigen or may indicate a pathogenic role of exuberant cd8 ϩ t cell responses. on the other hand, the elderly have been shown to have reduced ctl activity compared with younger individuals, and this can have a negative impact on immunization efficacy [111] [112] [113] . however, prevaccination t cell responses do not correlate with postvaccination antibody titers [114] , and as the elderly also have decreased antibody responses following vaccination, t cell responses may provide a better correlate of vaccine efficacy in the elderly population [115] . interestingly, unstimulated memory t cells from older individuals are in a higher state of differentiation than those of younger individuals, and it was shown that fewer senescent, influenza-specific t cells, characterized as killer cell lectin-like receptor subfamily g member 1 (klrg1) hi cd57 hi , are associated with a more robust antibody response following tiv delivery [110] . the immunodominance hierarchy in hla-a*02 individuals is often reported as directed primarily against the conserved influenza a m1 58 -66 (gilgfvftl) [198, 199] . however, studies suggest that although this cd8 ϩ t cell response to m1 58 -66 is conserved in hla-a*02 individuals, it may not be strictly immunodominant, as greater responses were observed against internal np and polymerase pb1 subunit proteins in some individuals [122, 200] . in terms of hla presentation of influenza peptides, the hla-a*02:01-restricted m1 58 -66 peptide-specific ctl response is characterized by expression of v ␤ 17 tcr use [201] , and in hla-a*02:01 individuals, v ␤ 17 ϩ ctls were the dominant cells responding to in vitro influenza exposure. in contrast, cord blood from hla-a*02:01 infants, thus having no prior influenza exposure, was characterized by a m1 58 -66 -specific cd8 ϩ t cell response that was less dependent on the presence of the cd8 ϩ v ␤ 17 ϩ t cell population [109] . therefore, maturation of the ctl response upon subsequent influenza infections is likely related to effective clearance of viral infections and has important implications for therapeutic interventions. although children beyond infancy who are considered high-risk for influenza infection, i.e., children with underlying diseases, such as asthma, sickle cell disease, and those receiving solid organ transplantation, have sig-nificant increases in serum nab and t cell responses, children with systemic lupus erythematosus are unable to mount significant t cell responses following tiv immunization [108] . these findings suggest that the pathogenesis of respiratory viral infections in infants may be associated with the absence of immunological memory or a failure to develop an appropriate, well-regulated t cell response. undoubtedly, host genetics have a role in the outcome of many infectious diseases, including influenza. mouse models have provided some clues regarding the role of the host genetic component on the inflammatory response in the respiratory tract. briefly, these studies found that influenza-induced pathology is altered depending on the inbred mouse strain used for infection. the genetic background of the host also controls cumulative and maximal viral titer, underscoring the importance of limiting viral replication early during influenza infection [111, 202] . in humans, certain populations worldwide have been found to be more prone to severe disease during influenza infection. for instance, aboriginal peoples have more severe illness and increased mortality during the a/h1n1 pandemics of 1918 and 2009 compared with those of non-aboriginal descent [29, 116 -118] . it is important to note, however, that poor socioeconomic conditions and metabolic diseases, such as diabetes, are more common in indigenous populations [203] , and this may contribute to influenza-related disease. moreover, hla alleles are proposed to have some role during influenza infection, as differential ctl responses exist in individuals with varying hla alleles [204] . gene polymorphisms located in human chromosomes 1 and 17 were also determined to be associated with severe pneumonia during a(h1n1)pdm09 infection [120] . interestingly, two of these snps are mapped to genes fcgr2a and c1qbp. the protein encoded by fcgr2a is involved in phagocytosis and clearance of immune complexes and expressed on the surface of phagocytic cells. the protein encoded by c1qbp is a critical factor involved in complement activation. notably, presence of low-avidity antibodies and subsequent formation of immune complex-mediated complement activation in the lungs of patients with severe influenza infection are associated with severe disease following influenza infection [107] . recently, "systems biology" strategies have emerged to improve vaccine efficacy, measuring expression patterns to expose protective molecular signatures following immunization or natural infection [46, 205] . confirming in vitro mouse studies, which suggest that mbl can neutralize influenza [206, 207] , human polymorphisms identified within mbl2 are associated with poor antibody responses to influenza vaccination [119] . although mbl deficiency has been observed to influence the outcome in several respiratory diseases, including pneumococcus infection, tuberculosis, and sars cov (reviewed in ref. [208] ), it did not have an obvious role in a(h1n1)pdm09 infection of naïve individuals [209] . these studies highlight the involvement of host ge-netics in development of enhanced disease, although they likely represent a small fraction of the potential regulatory host genetic elements. studies of innate and adaptive immune responses to influenza virus infection have been restricted primarily to animal models of disease and inflammation. as our immunological tools become more sophisticated, more emphasis should be placed on human immunology as opposed to relying solely on animal infection models. moreover, the vast majority of experimental data in humans is restricted to blood leukocytes, with minimal emphasis placed on the innate immune response, particularly at sites of infection. as a result, there is limited scientific knowledge regarding the specific roles of immune cells in affected tissues. understanding the innate response in particular is critical to appreciate early 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diabetes prevalence among american indians and alaska natives and the overall population-united states preferential hla usage in the influenza virus-specific ctl response systems vaccinology: learning to compute the behavior of vaccine induced immunity complement-dependent neutralization of influenza virus by a serum mannose-binding lectin human mannan-binding lectin inhibits the infection of influenza a virus without complement mannose-binding lectin deficiency and respiratory tract infection no association between mannose-binding lectin deficiency and h1n1 2009 infection observed during the first season of this novel pandemic influenza virus the authors are funded in part by u.s. national institutes of health/national institute of allergy and infectious diseases contracts hhsn266200700005c (st. jude center of excellence for influenza research and surveillance) and hhsn272200800058c (systems influenza) and the american lebanese syrian associated charities (alsac). key: cord-318852-gouz6tth authors: lee, j.s.e.; goy, r.w.l.; sng, b.l.; lew, e. title: considerations and strategies in the organisation of obstetric anaesthesia care during the 2019 covid-19 outbreak in singapore date: 2020-04-20 journal: int j obstet anesth doi: 10.1016/j.ijoa.2020.04.003 sha: doc_id: 318852 cord_uid: gouz6tth • identifying ‘high-infection risk’ parturients is challenging in the covid-19 outbreak. • a multidisciplinary approach is required to provide obstetric anaesthesia services. • protocols for labour analgesia and caesarean delivery allow safe provision of care. • infection control resource management allows for the allocation of finite resources. • organisational changes are required to respond to the covid-19 outbreak. since the outbreak of the coronavirus disease in 2019 (covid-19), singapore has seen the emergence of 1309 cases with six mortalities (as of 5 april 2020). 1 the 'disease outbreak response system condition' (dorscon) orange alert was declared by our local health authorities in early february, signifying a severe disease of limited community spread. 2 the kk women's and children's hospital provides tertiary obstetric care for approximately 11 000 deliveries a year. even in a global pandemic, we need to maintain essential obstetric services at our institution. here we share our experience in the provision of an obstetric anaesthesia and analgesia service, through careful re-organisation and resource planning, aimed to prevent disease transmission amongst our patients and healthcare staff. a multidisciplinary task force comprising of obstetricians, obstetric anaesthesiologists, neonatologists, infectious disease physicians and operating theatre and delivery suite nursing managers was formed before the first covid-19 case was identified in singapore. there was an urgent need to review pre-existing institutional infection control protocols that were established during the outbreaks of sars and mers-cov, in keeping with emerging evidence from the current global outbreak. 3 as the epidemiologic and infectious characteristics of the virus were initially unknown, the task force reviewed and revised infection control protocols based on emerging information. specific challenges were, firstly, the problem associated with making evidence-based clinical decisions given the lack of published information. there was a need to establish communication channels with staff to facilitate frequent updates. good communication among medical staff, taskforce stakeholders, institutional senior leadership and health ministry officials is pivotal if ground staff are to obtain timely information, updates and clarifications in a rapidly-evolving situation. our major considerations included: 1. identification of the 'high infection-risk' parturient. 2. clinical management of the 'high infection-risk' parturient, presenting for labour epidural and delivery. 3. organisational changes to respond to the crisis. because of the rapidly changing risk criteria for covid-19 'cases', identifying the 'high infection-risk' parturient was a challenge. prompt identification was needed so that these women could be isolated and treated appropriately, minimising the infection risk to healthcare staff and other patients. this problem arose for several reasons. firstly, infected patients may be infectious during the incubation period but do not exhibit symptoms or signs of acute infection. 4 secondly, viral shedding can occur 8-37 days from the onset of symptoms in infected patients. 5 thirdly, patients may have nonspecific symptoms that are similar to the 'common flu'. 6 these characteristics limit the usefulness of temperature screening prior to admission for labour or caesarean delivery. screening for travel history may also be unreliable, not only because of under-reporting in some countries, but because as the infection spreads globally, local community transmission occurs. consequently, all parturients with an acute respiratory tract infection or a positive travel or contact history were considered of high infection-risk and isolated until they tested negative for the virus. it is in this context that local health authorities mandated that healthcare staff performing aerosol-generating procedures (agp) such as tracheal intubation and extubation, bag-mask ventilation, orogastric tube insertion and bronchoscopy, don full personal protective equipment (ppe) regardless of risk status. protective equipment includes a splash-resistant gown, double-gloves, goggles or face shield with national institute for occupational and safety health (niosh) n95 mask, with or without a powered air purifying respirator (papr). 7 management in the labour ward 'high infection-risk' parturients are placed in negative pressure ( -2.5 pa) rooms and are asked to wear a surgical mask. to minimise infection risk, no visitors are allowed. early labour epidural analgesia is encouraged to reduce the use of nitrous oxide/oxygen inhalation analgesia and also general anaesthesia with airway instrumentation for intrapartum caesarean delivery, both of which are associated with aerosolization of the virus. high-flow oxygen administration via face mask was identified as a factor causing hospital ward outbreaks during the sars epidemic and should be avoided. 8 however, nitrous oxide/oxygen inhalation, where appropriate, is provided to the parturient while awaiting epidural catheter insertion. an experienced anaesthesiologist, donned in ppe against possible aerosol generation from nitrous oxide/oxygen use, should perform the neuraxial procedure to improve the chances of success. goggle design may limit peripheral vision and visual acuity, hampering the ability to successfully perform the procedure to a greater degree in a novice anaesthesiologist. the decision whether to provide combined spinal-epidural analgesia or epidural analgesia is left to the discretion of the anaesthesiologist. when possible, we prefer to proceed with caesarean delivery in the 'high infection-risk' patient only after she has been de-isolated or has tested 'negative' for covid-19 from the first nasopharyngeal swab. however, should an emergency caesarean delivery be required, it is performed in a dedicated negative-pressure operating room with staff donning full ppe. should this type of room be in use, we proceed with surgery in another operating room, adopting the necessary precautionary measures when accommodating a patient with covid-19. 9 for cesarean delivery the anaesthetic technique of choice remains neuraxial anaesthesia as this minimises the risk of aerosolisation associated with airway intervention. 10 the authors also prefer a combined spinal-epidural technique if surgery is expected to be prolonged, in order to minimise the risk of a mid-procedure conversion to general anaesthesia. if the risk of a failed neuraxial anaesthetic is anticipated to be high or general anaesthesia is planned, the papr should be worn over the n95 mask from the outset. after rapid-sequence induction, intubation by an experienced anaesthesiologist is performed with a video-laryngoscope to maximise the chances of rapid successful intubation. face mask-ventilation is avoided when possible, and if rescue maskventilation is needed in the setting of failed intubation, small tidal volumes used until the cuff on the tracheal tube has been inflated. we advocate the use of disposable equipment (e.g. disposable laryngoscope blades) and consumables. an anaesthetic nurse runner is stationed in the anteroom should further drugs or equipment be required. a difficult airway cart is placed either in the operating room or anteroom. depending on the pathological lung changes present, patients with pneumonia may need sophisticated ventilators capable of delivering varied ventilatory settings and lung recruitment manoeuvres. after delivery, the neonate should be isolated as a precautionary measure. no skin-to-skin contact with the mother is allowed, to minimise infection risk. after surgery, patients are monitored in the operating room until ready for discharge to the postpartum ward, with the patient wearing a surgical mask en route. the operating room and the transportation route to the ward are disinfected. patients who need the postoperative intensive care unit (icu) for indications such as ventilatory support and/or management of obstetric-related complications should be transferred there directly, accompanied by healthcare staff donned in full ppe. all soiled consumables, used gloves, masks and gowns should be discarded in biohazard bags and sealed. we generally avoid activating a category 1 caesarean delivery in 'high infectionrisk' parturients by encouraging our obstetric colleagues to perform frequent surveillance of their fetal status and cardiotocography tracings, and to forewarn operating theatre staff of impending likely caesarean delivery. this allows the operating room team to prepare in advance, to allow expedient delivery having undertaked precautionary measures for staff protection. with collaborative teamwork and communication, we believe it is possible to achieve decision-to-delivery intervals of 30 min, even for category 1 cases. since the implementation of this workflow pattern in early february 2020, we have managed 11 'high infection-risk' parturients for caesarean delivery as of 25 mar 2020. although none eventually tested positive for the covid-19 virus, the surgeries were still performed in a negative pressure operating room with staff dressed in full ppe. two patients had general anaesthesia and were transported postoperatively to the icu while still intubated; spinal anaesthesia was initiated for eight patients, and one patient had extension of labour epidural analgesia. to increase the availability of isolation beds, all elective surgical procedures were postponed and only operations deemed essential or urgent allowed to continue. a prudent approach to the use of protective face masks is necessary to balance the need to conserve a finite resource against the overwhelming need to protect healthcare staff from the disease. while staff are advised to wear surgical masks in all clinical areas, only frontline staff who come into close contact with 'high infection-risk' patients (e.g. screeners and those who perform agps) need to wear n95 masks. all staff should undergo n95 mask fitting. guidance on the use of ppe was formulated and disseminated. all leave applications were rescinded and approved only on an 'as needs' basis, to ensure everyone is available to deal with the crisis at hand. to mitigate the risk of service disruption due to disease transmission among staff, we organised the department into modular teams segregated by physical location. bedside handovers are done via focused clinical summaries, with the staff donning the appropriate protection. as the sars-cov-2 virus is transmitted through close contact, good hand hygiene and social distancing are reinforced. advances in information technology are fully exploited, with department meetings and educational activities being conducted via video-conferencing platforms. we also use secure communication applications on mobile devices to build channels of communication among working teams for the rapid dissemination of information. all healthcare staff monitor and log their temperatures twice daily. a staff clinic manages staff with fever (>37.5°c) and/or respiratory symptoms and facilitates the prompt identification of institutional nests of outbreaks. with the implementation of modular teams and the consequent increase in overnight duties, we recognise that more staff may experience acute stress, fear of infection and burn-out. 11, 12 hence, we are working with hospital support groups to provide further support and feedback channels. in order to provide sustainable and safe obstetric anaesthesia during an infectious disease pandemic, a collaborative effort among anaesthesiologists, intensivists, obstetricians, neonatologists, nursing, infectious disease physicians and environmental services is required to minimise infection risks to both patients and healthcare workers. constant review of the criteria of the 'high infection-risk' patient, aligned to the evolving global situation, facilitates effective screening, isolation, and management. protocols and workflows for the management of such patients in labour and operative deliveries also need frequent revision to enhance efficiency while optimising the use of finite resources. lastly, establishing good communication channels among health officials, institutional leadership and ground staff is pivotal for the timely dissemination of updated information and obtaining feedback. updates on covid-19 (coronavirus disease 2019) local situation what do the different dorscon levels mean? available at a pneumonia outbreak associated with a new coronavirus of probable bat origin a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical features of patients infected with 2019 novel coronavirus in wuhan, china protecting health-care workers from subclinical coronavirus infection why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? what we do when a covid-19 patient needs an operation: operating room preparation and guidance expert recommendations for tracheal intubation in critically ill patients with novel coronavirus disease 2019 risk perception and impact of severe acute respiratory syndrome (sars) on work and personal lives of healthcare workers in singapore healthcare workers emotions, perceived stressors and coping strategies during a mers-cov outbreak highlights identifying 'high-infection risk' parturients is challenging in the covid-19 outbreak a multidisciplinary approach is required to provide obstetric anaesthesia services protocols for labour analgesia and caesarean delivery allow safe provision of care infection control resource management allows for the allocation of finite resources organisational changes are key: cord-324788-echu0zmf authors: aich, palok; potter, andrew a; griebel, philip j title: modern approaches to understanding stress and disease susceptibility: a review with special emphasis on respiratory disease date: 2009-07-30 journal: int j gen med doi: nan sha: doc_id: 324788 cord_uid: echu0zmf studies in animals and humans link both physical and psychological stress with an increased incidence and severity of respiratory infections. for this manuscript we define stress as the physiological responses an individual undergoes while adjusting to a continually changing environment. it is known that stressors of various types (psychological/physical) can alter the physiological levels of certain hormones, chemokines and cytokines. these alterations send information to the central nervous system to take necessary action which then sends messages to appropriate organs/tissues/cells to respond. these messages can either activate or suppress the immune system as needed and failure to compensate for this by the body can lead to serious health-related problems. little is known how stress affects disease susceptibility, yet understanding this mechanism is important for developing effective treatments, and for improving health and food quality. the current review focuses on (a) the effects of psychological stressors in humans and animals, (b) various methodologies employed to understand stress responses and their outcomes, and (c) the current status of the attempts to correlate stress and disease with respiratory disease as model system. the methodologies included in this review span traditional epidemiological, behavioral and immunological studies to current high throughput genomic, proteomic, metabolomic/metabonomic approaches. with the advent of various newer omics and bioinformatics methodologies we postulate that it will become feasible to understand the mechanisms through which stress can influence disease onset. although the literature in this area is limited because of the infancy of this research area, the objective of this review is to illustrate the power of new approaches to address complex biological questions. these new approaches will also aid in our understanding how these processes are related to the dynamics and kinetics of changes in expression of multiple genes at various levels. from the preceding discussion it is clear that stress can be defi ned in many different ways depending upon the objectives or perspective of the researcher. all these defi nitions, however, share a common component of adaptive physiological responses following challenges to homeostasis. the adaptive reactions to stressors may involve mobilization of a wide variety of physiological responses including the immune response. stress responses usually include physical perturbations that can encompass either the entire body or specifi c cellular compartments. considering the volume of work in various areas of stressors and their effects the main objective of the current review is to focus on one type of stress, which includes psychological stressors. for our purposes, stress can be defi ned as a psychologically perturbing condition occurring in response to adverse external infl uences capable of affecting physical health. transportation, fear (ie, fright and fl ight response), overcrowding and weaning in the form of social reorganization are a few of the important types of psychological stressors identifi ed in the literature. these stressors have been linked to many conditions including immune suppression, disease susceptibility, hypertension and reproductive dysfunctions. [4] [5] [6] stress is a major concern because it is ubiquitous, recurring in nature and has detrimental effects on health. in this review we will primarily deal with important psychological stressors and their infl uence on respiratory disease which is one of the most widely studied models. for many years, psychological stress has been shown to signifi cantly increase disease susceptibility. 6, 7 until 20 years ago, researchers investigating the psychological factors contributing to human disease focused primarily on coronary heart disease and cancer and neglected studies on infectious diseases. 8 however, interest in this area started to shift with the publication of evidence that psychological factors infl uenced immune function. 9 furthermore, there was an increasing awareness that stress and other psychological factors played a role in the onset and progression of acquired immunodefi ciency syndrome (aids). 10 these studies demonstrated a signifi cant role for psychological stressors in compromising immunity and interest in the effects of stressors in other diseases was initiated. considerable emphasis has been placed on respiratory diseases in understanding the onset and severity of the disease as a result of psychological stress. 11 increased risk of fatal bacterial respiratory infections following a primary viral infection has been observed in a wide variety of species. this phenomenon is called viral-bacterial synergy and was fi rst established following human infl uenza epidemics when a variety of secondary bacterial respiratory infections were associated with increased mortality. 12 studies have also linked a variety of psychological stressors with an increased incidence and severity of respiratory infections in humans 13, 14 and animals. 15, 16 it is known that respiratory disease has a huge economic impact in the areas of human health care, animal welfare and the food industry. 17, 18 to focus the review, we will restrict our discussion to research related to a comparative analysis of the effects of psychological stress on respiratory disease in humans and cattle. there are reports which have shown direct connections between stress and immune system function. 19 similarly, other studies have shown that social stressors could also increase the risk for upper respiratory infection. 20 a viral challenge study provides the strongest evidence for a link between stress and susceptibility to the common cold. 21 other studies have extended these results by considering a wider range of psychosocial factors. 22 the effects of stress on health are often mediated by a number of psychological factors. social support can often act as a buffer against the effects of stress as cohen and colleagues showed that social support reduced viral replication rate and increased mucociliary clearance of infection. 11, 13 in another report, they examined the effects of stress and social support in a routine study of upper respiratory tract illnesses. 23 under low levels of stress, high levels of social support were associated with a decreased risk of infection, whereas social support had no effect when levels of stress were high. a separate study was done to examine the associations between psychosocial factors (stress, social support, fl uctuations in mood) and viral exacerbations of asthma. the study involved naturally occurring illnesses rather than experimentally-induced infections but it maintained several important features of the methodology used by cohen and colleagues. 21, 24 for example, stress was measured at the start of the study by measuring the immune responses in terms of leukocytes present in peripheral blood in order to determine the extent to which stress could predict subsequent illness. in addition, effects of personality, 25 smoking status, and alcohol consumption 21 were also studied as possible predictors of susceptibility to respiratory viral infections. before considering the effects of psychosocial factors on respiratory virus-induced exacerbations of asthma, it is essential to have strong evidence that these viruses play a direct role in asthma. until recently, it appeared to be only a weak association between asthma and upper respiratory tract infection in adults. 26, 27 the absence of a stronger association in these epidemiological studies of adult asthmatics could, at least in part, have been due to diffi culties in isolating human rhinoviruses and coronaviruses. indeed, results from a study using enzyme-linked immunosorbent assays for antibodies to human coronavirus and semi-nested reverse transcriptase polymerase chain reactions for detections of rhinovirus suggested a stronger association between these viruses and asthma in adults. 28 in summary, this study confirmed that psychosocial factors and health-related behaviors were associated with increased susceptibility to colds, which then led to an exacerbation of asthma. this conclusion was made in the context of a study where both diseases (cold and asthma) were verifi ed using objective measures. the well established buffering effect of social support was observed in the high stress group and possible confounders such as demographics, health-related behaviors or personality could not account for this effect. alcohol consumption, personality and demographic factors were also shown to be important predictors of susceptibility. in contrast smoking was related to illness severity but not disease susceptibility. these results show that one must consider a range of psychosocial factors, personality traits, demographic factors, and health-related behaviors in studies of individual differences in susceptibility and severity of upper respiratory tract infections. while studies have correlated stress-related behavior with disease susceptibility, research has also been performed to determine the immunological basis of the relationship between stress and disease. exposure to viral agents that cause upper respiratory disease provokes illness in some individuals but not others. moreover, the severity of clinical symptoms among those who develop illness can vary substantially. evidence from prospective epidemiological studies 20, 23, 29 and from experimental viral-challenge studies 21, 30 show that individuals reporting greater psychological stress have both a higher incidence and a greater severity of illness. however, past attempts to identify the behavioral patterns and biological responses linking psychological stress with upper respiratory viral illness have been unsuccessful. 20, 21, 30 other studies reported that psychological stressors acutely activate the production of interleukin-6 (il-6), a pro-inflammatory cytokine. il-6 release in response to infection is thought to be mediated by glucocorticoids, thus providing a hypothetical pathway by which stressors (via the induction of glucocorticoid production) could control cytokine release. 31 in addition, il-6 triggers additional release of glucocorticoids, possibly exacerbating the stress response through positive feedback. at least one source of il-6 is epithelial cells as evidenced by their production of il-6 in vitro and in vivo when exposed to rhinovirus. 32 because a local increase in the concentration of this pro-infl ammatory cytokine precedes the development of acute signs and symptoms of illness, it has been implicated as a mediator in the pathway for symptom expression. in fact, il-6 concentrations in nasal secretions were associated with upper respiratory tract symptoms among persons infected with infl uenza a virus (a/texas and a/kawasaki) and rhinovirus (strain hanks' and type 39). 13 cohen and colleagues addressed the hypothesis that il-6 production in response to infl uenza a virus infection represents a viable pathway through which psychological stress infl uences the severity of illness. to achieve that goal, they measured levels of psychological stress in a group of adult subjects before experimentally infecting them with influenza a virus. self-reported respiratory symptoms, mucus weights, and local il-6 concentrations were then measured on the day before and for seven days after virus exposure. the data collected supported the conclusion that the level of psychological stress predicts the severity of illness and also the magnitude of the cytokine response. the data were then examined for evidence that il-6 mediated the association between stress and illness. these analyses suggested that most of the effects of psychological stress on clinical symptoms and mucus weights could be accounted for by changes in il-6. however, it is possible that il-6 itself is not the causal link but rather just a marker (covariate) for other pro-infl ammatory cytokines which were elevated during the course of experimental infection. for example, in another related study 33 reported that interferon α and il-6 levels (but not tumor necrosis factor [tnf], il-8, il-1, or il-2) increased early in the course of infection and both correlated with viral titers, increases in body temperature, mucus production and symptom scores. there is also an issue regarding the correlational nature of the mediational analysis. although consistent with the hypothesis that the association between stress and illness was mediated by il-6, the data do not permit causal inference. for example, this pattern of data is also consistent with increases in il-6 occurring in response to tissue damage associated with illness symptoms. even with these reservations, this was the fi rst aich et al study to provide evidence consistent with the hypothetical model that psychological stress infl uences upper respiratory infections through a biological pathway. 13 there are two parts to the stress response: sympatheticadrenal-medullary (sam) and the hypothalamic-pituitaryadrenal axis (hpa). the hpa is the core stress axis in mammals and together with the sam system co-ordinates response to the diverse range of stressors from psychological to physical. there is considerable interplay between both neuronal systems especially between the noradrenergic nucleus locus ceruleus which provides central regulation of the sam and the parvocellular neurones which regulate the hpa. the sam, by triggering catecholamine release, provides the acute stress response whilst the hpa governs longer term stress defence mechanisms. together those systems regulate energy utilisation and metabolic activity throughout the body. 34 the sam system produces the immediate shock response by acting on the hypothalamus, which activates the adrenal medulla and the sympathetic autonomic nervous system (ans) (figure 1 ). the sam produces the "fi ght-or-fl ight" response which increases alertness, blood fl ow to muscles, heart rate, blood pressure, respiration rate, etc. and might decrease activity in the digestive system. the hpa system regulates release of the hormone crf to activate the anterior pituitary and uses another hormone, adrenocorticotropic hormone (acth), to activate the adrenal cortex to release a group of hormones including cortisol ( figure 2 ). cortisol and other glucocorticoid hormones have various effects such as conservation of glucose for neural tissues, elevation or stabilization of blood glucose levels, mobilization of protein reserves, conservation of salts and water, suppression of wound healing and the immune system. according to seyle's general adaptation syndrome (gas) theory the general adaptation syndrome divides the stress response into three stages: stage 1: alarm reaction (sam and hpa activity increases and result in the "fi ghtor-fl ight" response); stage 2: resistance (hpa activity takes over, bodily resources are at maximum and if the stress is experienced for short term the body returns to normalcy); stage 3: exhaustion (with very prolonged stress, bodily systems are ineffective. sympathetic ans action reappears. adrenal cortex damage causes parasympathetic action, omics approaches in understanding stress and disease susceptibility eg, energy storage failure. the immune system collapses, and stress-related diseases increase). while it has been assumed that psychological (psychogenic) and physical (neurogenic) stressors are most closely aligned with depression, the suspicion has arisen that systemic stressors, including immune alterations, may also act in such a provocative capacity. 35 communication occurs between the immune, endocrine, autonomic and central nervous systems 36 such that psychological events that affect central neurochemical processes may affect immune activity. conversely, immune activation may affect hormonal processes and the activity of central neurotransmitters. thus, by virtue of the neurochemical effects imparted, immune activation may affect behavioral outputs and may even be related to behavioral pathology such as depressive illness. 37 the hypothesis that altered immune activation may occur as a result of various stressors emerged over time. the initial theory came from hans seyles' gas, which was derived from observation and experimentation on laboratory animals. using a variety of stressors (ie, pain, thermal extremes and starvation), seyle described a common nonspecifi c stress response pathway. 38 after initial perception of a stressor, the animal mounts an emergency alarm or fi ght-or-fl ight response. this catecholamine-driven reaction results in increased cardiovascular function and an overall increase in metabolism. if the stressor persists, the resistance phase or 'conservation withdrawal reaction' is initiated, which is a physiological coping reaction to the increased demands of maintaining homeostasis. chronic stress leads to the exhaustion phase and may lead to pathology. seyle's theory unifi ed the stress phenomena because it provided a common response pathway to all the varied stressors encountered. this pathway, the hpa axis, involves perception in the brain with release of hypothalamic corticotropin-releasing factor (crf) and vasopressin which stimulates the anterior pituitary to secrete acth ( figure 2 ). circulating acth causes the adrenal cortex to produce glucocorticoids (gc). glucocorticoids cause gluconeogenesis with conversion of lipid to glucose for the central nervous system (cns) and other functions. thus, gas allowed the identifi cation of stressors (and presumably the status of well being of the animal) by measurement of gc levels. it was an attractive theory because gc levels were relatively easy to measure. 2 unfortunately the gas theory has proven to be too simplistic. mason's experiments with rhesus monkeys aich et al exposed to different types of stressors revealed a disparity in neuroendocrine responses provided by specifi c stressors. 38 for instance, monkeys subjected to emotional stress had elevated serum gc levels but those subjected to a heat-stress regime failed to show gc elevation. in addition to gc, other neuroendocrine mediators were characteristically produced in response to specifi c stressors. 38 emerging information on the response to stressors suggests that there are at least four different avenues of neuroendocrine responses. these involve the autonomic nervous system, the hpa axis, neuropeptides, neurotransmitters and neuroimmunological peptides and receptors. 2 interactions between the immune and nervous systems play an important role in modulating host susceptibility and resistance to infl ammatory disease. neuroendocrine regulation of inflammatory and immune responses as well as onset of disease can occur at multiple levels: (a) systemically through the anti-infl ammatory action of gcs released via hpa axis stimulation, (b) regionally through production of gcs within the affected tissue as well as by sympathetic enervation of immune organs such as the thymus and (c) locally at sites of infl ammation. estrogens also play an important role in immune modulation and contribute to the approximately 2-to 10-fold higher incidence of autoimmune/infl ammatory diseases in females of all mammalian species. 39 during infl ammation, cytokines from the periphery activate the central nervous system through multiple routes. this results in stimulation of the hpa axis which in turn, through the immunosuppressive effects of the glucocorticoids, generally inhibits infl ammation. recent studies indicate that physiological levels of glucocorticoids are immunomodulatory rather than solely immunosuppressive causing a shift in patterns of cytokine production from a th1-to a th2-type pattern. interruptions of this loop at any level and through multiple mechanisms, whether genetic, or through surgical or pharmacological interventions, can render an infl ammatory resistant host susceptible to infl ammatory disease. over-activation of this axis, as occurs during stress, can also affect severity of infectious diseases through the immunosuppressive effects of the glucocorticoids. these interactions have been clearly demonstrated in many animal models using a variety of species and infectious agents. 39 the results from these models are also relevant to human infl ammatory, autoimmune and allergic illnesses including rheumatoid arthritis, systemic lupus erythematosus, sjogren's syndrome, allergic asthma and atopic skin disease. while many genes and environmental factors contribute to susceptibility and resistance to autoimmune/infl ammatory diseases, a full understanding of the molecular mechanisms by which a combination of neuropeptides, neurohormones and neurotransmitters can modulate immune responses is essential for effective design of future interventions. 39 it is clear from the previous discussion and review of previous research that the relationship between stress and disease susceptibility is very complex and intertwined with cascades of events. in order to understand or characterize stress-induced disease susceptibility one needs to identify various biological or functional pathways and interaction networking involved at any given time. to follow the cascade of molecular events happening over time one needs to employ methodologies which are holistic in nature and can provide global information related to the kinetics of multiple changes in gene expression and multiple biomolecular interactions. with the advent of various high-throughput genomic approaches it has become possible to explore complex biological processes and in one step obtain information about gene expression at the transcriptional (genomics) and translational (proteomics) level, as well as to identify metabolites arising from these responses (metabolomics/metabonomics). these methodologies together are often referred to as 'omics' approaches. although these methodologies are still in their infancy they have started showing promise in understanding systems' biology and various disease processes. work employing omics approaches to understand mechanism of stress and disease susceptibility is limited at this time. there are reports which describe the effects of oxidative stress on cellular apoptosis 40 but there are no reports on the effects of psychological stressor and disease susceptibility in animals using holistic methodologies such as various omics approaches. the literature in this area is very scarce because these methodologies are very new and few results are currently available. the main studies to correlate stress and disease susceptibility was done either in plant systems 41 or a correlation between oxidative stress and infectious disease. 42 the review by hirai and saito established, using liquid chromatogram based mass spectrometric proteomic and metabolomic and cdna microarray based transcriptomic analyses, that these omics studies can reveal the genomic networking involving several pathways related to oxidative stress response and key metabolic pathways. 41 bioinformatics and statistical approaches specifi c for detailed transcriptomics or cluster analyses of expressed genes have been developed and are useful for extracting information on various functional genomic responses. [59] [60] [61] it is important, however, to understand the translation of transcriptomic information into protein expression and modifi cations since it is the protein that acts as biomarkers for various disease processes or conditions. several proteomic tools have been developed. these include gel-based two-dimensional (2d) gel electrophoresis (ge), 47 2d fluorescence difference gel electrophoresis (2dige) 62 or gel-free multidimensional protein identifi cation technology (mudpit), 63,64 isotope-coded affinity tag (icat™), 65 surface-enhanced laser desorption-ionization time-of-flight (seldi-tof) ms 66,67 or isobaric tag for relative and absolute quantitation (itraq) 68 methodologies. while these technologies are being matured more appropriate biological questions can be addressed. in 2d-ge whether traditional or newer dige system, proteins are fi rst focused (fi rst dimension) in terms of their pi values followed by sds-page (second dimension). in contrast to traditional 2d-ge, dige system utilizes the fl uorescence labeling of protein samples and two-to-three different samples can then be run on a single gel which makes it superior to traditional 2d-ge in terms of eliminating gel to gel variation and comparative analysis. although 2d-ge is a very good way of separating complex protein mixtures, it is limited in terms of high-throughput analysis and sensitivity. because of low sensitivity it is mostly limited to the analysis of high abundant proteins of a system. efforts have been made to improve the sensitivity and analysis capacity of proteomics techniques. as a result, other methodologies such as 2d-hplc, mudpit, icat, and itraq have been developed and are gaining popularity. although various bioinformatic and data analyses approaches have been developed to analyze gel images and mass spectrometric based protein characterization a highthroughput methodology in this area has yet to be developed. functional genomic techniques of transcriptomics and proteomics and available bioinformatic and statistical analyses promise unparalleled global information during the analyses of complex biological responses. however, if these technologies are used in isolation, the large multivariate data sets produced are often diffi cult to interpret and have the potential to ignore key metabolic events to understand the true biology. high resolution 1 h nmr spectroscopy used in conjunction with pattern recognition provides one such tool for defi ning the dynamic phenotype of a cell, organ, or organism in terms of a metabolic phenotype. in a recent review the benefi ts of this metabonomics/metabolomics approach to problems in toxicology have been discussed. 69 one of the major benefi ts of this approach is its high-throughput nature and cost-effectiveness on a per sample basis. using such a method, the consortium for metabonomic toxicology (comet) is currently investigating approximately 150 model liver and kidney toxins. this investigation will allow the generation of expert systems where liver and kidney toxicity can be predicted for model drug compounds, providing a new research tool in the fi eld of drug metabolism. the review also included how metabonomics may be used to investigate co-responses with transcripts and proteins involved in metabolism and stress responses such as during drug-induced fatty liver disease. by using data integration to combine metabolite analysis and gene expression profi ling, key perturbed metabolic pathways can be identifi ed. detailed omics-based bioinformatics studies, to correlate stress-dependent disease susceptibility, have yet to analyze this complex biological response. a few preliminary studies have been reported in the literature. a recent study has shown the adverse effects of using mechanical ventilators for respiratory support. 70 this acute lung injury because of mechanical stretch can lead to high mortality and this study has utilized recent advances in bioinformatic-intense candidate gene searches to correlate the lung injury and gene expression profi les in a rodent model analyzed with microarrays. 70 the authors used 2,769 mouse/rat orthologous genes identifi ed on rg_u34a and mg_u74av2 arrays and the expression profi les were simultaneously analyzed by signifi cance analysis of microarrays. this combined ortholog and signifi cance analysis of microarrays approach identifi ed 41 up-and 7 downregulated ventilator-induced stress-related candidate genes. results were validated by comparable expression levels obtained by either real-time or relative rt-pcr for 15 randomly selected genes. k-mean clustering of 48 candidate genes clustered several well-known lung injury associated genes (il-6, plasminogen activator inhibitor type 1, ccl-2, cyclooxygenase-2) with a number of stressrelated genes (myc, cyr61, socs3). the only unannotated member of this cluster (n = 14) was riken_1300002f13 est, an ortholog of the stress-related gene33/mig-6 gene. the authors speculated that the ortholog-signifi cant analysis of microarray approach is a useful, time-and resourceeffi cient tool for identifi cation of candidate genes in a variety of complex disease models such as ventilator induced lung injury. 70 using microarray-based genomic approaches work has also been initiated to identify hypertension-related genes in rat model. 71 though it is a common belief that the stressors mentioned earlier can compromise host immune responses and enhance susceptibility to various diseases, very little work has been done in this area to understand the mechanism of stress dependent disease susceptibility in mammals. kelley in 1980 identifi ed eight stressors that typically occur in modern livestock production units: heat, cold, crowding, mixing, weaning, controlled-feeding, noise and restraint. all these stressors were shown to alter components of the immune system in animals and these changes in immune function may ultimately explain the physiological basis of diseaseenvironment interactions. 72 another study in 1993 showed that neurotransmitters and neuroendocrine hormones can modify the function of immune cells. conversely, cytokines produced by immune cells can alter brain homeostasis. 73 this connection is further manifested by experimental studies showing a relationship between stress and resistance to infection. human subjects under high stress were shown to be more susceptible to infection with common cold viruses. furthermore, a diversity of experimental animal models confi rmed that laboratory stressors such as forced exercise, avoidance learning, restraint, isolation and cold exposure made animals more susceptible to primary infection with a variety of viruses and bacteria. the cellular and molecular basis for the observed modulation of host resistance is not fully understood but involves altered functioning of both t-lymphocytes and cells of the hypothalamic-pituitary adrenal axis. also involved is the altered production of cytokines and hormones produced by the immune system and brain. 73 emau and colleagues in 1987 showed that the onset of immunodepression by stress or viral infection in the pathogenesis of bovine pneumonia permits super infection of the lungs with mannheimia haemolytica (formerly called pasteurella haemolytica) which results in exudative fi brinous pneumonia. 74 although these studies provide a preliminary basis of stress-induced bovine respiratory disease (brd), there was a lack of necessary information to determine the molecular basis of such dependence, which can be found by studying changes at the level of gene expression (transcriptional and proteomic) and metabolites. a recent study compared proteomics of epithelial lining fl uid from lungs of weaned and nonweaned cattle. 75 the study was done over a shorter period of time (36 h) with a combination of stressors (weaning, transportation) and important questions such as to determine specifi city and duration (stability) of the protein biomarkers are yet to be addressed. the group also studied the proteomic profi le of bronchoalveolar lung lavage fl uid following treating the calves with dexamethasone to determine the effects of glucocorticoids, which is elevated following induction of stress. 76, 77 more studies are however needed (a) to fi nd biomarkers associated with stressors and infection, (b) to determine the duration of the biomarkers so that these are sustainable enough to identify and predict stressed animals (which might be more susceptible to infection in real life situation such as in feedlot cattle). disease models have been developed to study the molecular mechanisms underlying the viral-bacterial synergy which results in fatal brd infections. [78] [79] [80] we have confi rmed with the infectious brd model that the stress of weaning 81 significantly enhances the viral-bacterial synergy leading to fatal bacterial respiratory infection. 82 the major stressors young omics approaches in understanding stress and disease susceptibility cattle experience include maternal separation or weaning, dietary changes, transportation, social reorganization and other environmental effects (figure 3 ). with this in mind, we focused our initial studies to understand the role of weaning on the clinical response to brd. fifteen suckling calves were removed from their mothers 24 h prior to viral infection (abruptly weaned/stressed, aw). a second group of 15 calves was weaned 2 wks prior to viral infection (pre-conditioned/ control, pc); 2 wks was chosen as an appropriate interval to eliminate psychological and physiological effects associated with breaking the maternal/nutritional bond and to adapt to the dietary change and social re-organization that follows weaning ( figure 4 ). all calves were transported to vaccine and infections disease organization vido, and aerosol challenged with bovine herpesvirus-1 (bhv-1) followed by m. haemolytica; this combined viral-bacterial infection induces fatal pneumonia in 50%-70% of calves. 83 our clinical analyses revealed a signifi cant difference in brd clinical disease when comparing freshly weaned calves (80% mortality) versus pre-conditioned calves (40% mortality). 84, 85 increased mortality associated with fresh weaning was characterized by a decrease in both survival time post-infection and, interestingly, decreased lung pathology which suggested a systemic reaction. contrary to expectations, transportation induced a signifi cant cortisolemia in pre-conditioned but not freshly weaned calves ( figure 5 ). transportation is known as an important stressor which increases blood cortisol level. [86] [87] [88] [89] however, the stressor combination of weaning and transportation may have different physiological markers than commonly expected. cortisol is a potent regulator of pro-infl ammatory cytokine transcription by acting as a negative regulator of nfκb activation, and can be used as an indicator of stress. 90 fatal bacterial respiratory infection in calves was usually associated with an accelerated decline in serum cortisol levels. therefore, we hypothesized that increased respiratory disease susceptibility was associated with enhanced pro-infl ammatory responses in freshly weaned calves. elevated body temperature and interferon-gamma (ifn-γ) levels in nasal secretions during bhv-1 infection of freshly weaned calves were similar to previous reports, 82, 91 supporting this hypothesis. these immune responses were signifi cantly increased despite no signifi cant difference in virus shedding. furthermore, increased il-10 expression, during bhv-1 infection of pre-conditioned calves, was consistent with reduced pro-infl ammatory responses. 82 we conducted bovine microarray analyses of rna isolated from blood mononuclear cells to determine if changes in gene expression correlated with either stress or the severity of brd infection; results support the conclusion that differential regulation of pro-infl ammatory responses is a major mechanism contributing to increased disease susceptibility. conserved responses included an enhanced potential to aich et al respond to pathogen-associated molecular patterns through increased expression of toll-like receptors (tlrs). tlr induction of innate immune responses is mediated primarily through activation of nuclear factor kappab (nfκb). we also observed differential expression of β-defensin 5 (a host defense peptide induced by tlr4 signaling) in freshly weaned calves, consistent with reduced cortisol levels. 92 in addition, when we compared expression of select innate immune genes (eg, tlr2, tlr4, ifn-γ and 2'5' o-adenylate synthetase) between day 4 (post bhv-1 infection) and day 0 (prior to bhv-1 infection) for aw-and pc-groups, results revealed a trend of increased expression of the select genes on day 4 irrespective of the stress situation (table 1 ). this trend of activation of select immune genes clearly revealed that following bhv-1 infection the innate immune response was enhanced. this observation contradicts the current hypothesis that the primary viral enhances the secondary bacterial infection by compromising the immune system. 78, 93 our studies revealed that the anti-infl ammatory gene il-10 was upregulated on day 4 in pc groups while β-defensin was activated on day 4 in aw groups (table 1) . il-10 which acts as an anti-infl ammatory gene should be activated on day 4 to control pro-infl ammatory responses as a result of bhv-1 infection in either aw or in both groups; instead it was only over expressed in control (pc) groups. on a similar token β-defensin on day 4 should also be observed in both groups to control bhv-1 infection. these results are particularly interesting to explore further to understand the detailed mechanism of stress-induced disease correlation. although advances have been made in understanding various disease processes, successful intervention often depends upon the stage at which disease is detected. thus, identifying markers for disease or its cause, as well as understanding the mechanism of disease onset and susceptibility, are very important. systems biology approaches such as omics (genomic, proteomic, metabonomic) and multivariate analysis to understand mechanisms and to identify biomarkers provide potential tools to address these questions. 69, [94] [95] [96] it is particularly important to employ a combination of molecular approaches such as omics to understand complex processes such as stress and its correlation with disease susceptibility. with such goals in mind, we identifi ed and characterized a group of protein, metabolite and elemental biomarkers using serum samples from bhv-1 infected as well as from stressed animals. the trend of each group of biomarkers distribution was analyzed to correlate with the groupings based on stress condition or infection. 53 multivariate analysis revealed distinct differential trends in the distribution profi le of proteins, metabolites and elements following a stress response both before and after primary viral infection. a group of acute phase proteins, metabolites and elements could be specifi cally linked to either a stress response (decreased serum amyloid a and cu, increased apolipoprotein ciii, amino acids, low-density lipoprotein [ldl], p and mo) or a primary viral respiratory infection (increased apolipoprotein a1, haptoglobin, glucose, amino acids, ldl and cu, decreased lipid and p). thus, combined omics analysis of serum samples revealed that multimethod analyses could be used to discriminate between the complex biological responses to stress and viral infection. there are reports which suggest that transport can be an important stressor which alters blood leukocyte populations and sets the stage for brd. 97, 98 currently we are conducting experiments in which transportation of the animals is eliminated, such that stressors related to a new environment are removed. it was documented that abrupt weaning (separation of suckling calves from their dams) causes a prolonged psychological stress response. 81 this stress response manifests as an increase in vocalization by both calves and cows and a significant increase in time spent walking by the calf. consequently, time spent eating and resting also decreases for abruptly weaned calves. statistically, these changes in behavior return to baseline values after four to fi ve days of weaning. as is evident from the review of the literature that traditionally effects of stress have been studied by attempting to understand behavioral patterns of the subject and cellular immune response of the host. it is, however, clear that amount of data to understand such a complex condition (such as stress and stress-induced diseases) is not adequate and enough. behavioral studies may not always be correlated to a particular stressor as there might have been other parameters affecting the observations. attempts have also been made to use cortisol as a unique biomarker for stress, but cortisol is diurnal hence cannot be a reliable marker. while it is important to understand cellular immunology to establish the mechanism of stress-induced disease susceptibility, however without identifying and establishing the immune markers specifi c for stressor it will not lead into any meaningful inference. systems biology approaches using various omics and bioinformatics methodologies can identify a group of biomarkers at various levels of host immune response, gene expression and metabolism. association of the identifi ed biomarkers and the patterns in changes in their expression level above or below a determined threshold level with specifi c stressor(s) could then be used to defi ne stress situation, identif ication of individuals susceptible to stressinduced disease. moreover, establishing a pattern in changes of biomarkers is more reliable than a single biomarker. duration of identifi ed biomarkers can further strengthen the methodology for applying in real life situation. the current review established the importance of psychological stress and its effects on infectious disease severity and susceptibility with emphasis on respiratory disease. as evident from the literature, initial work focused mainly on understanding the roles of various stressors on physiology and behavior. theories emerged as a consequence of these studies which implicated the hpa axis and the sympathetic nervous system (sns) in altered immunity. however, physiological responses studied in a laboratory may not necessarily be consistent with real life phenomena, especially when attempting to analyze heterogeneous populations such as cattle or humans. experiments with human subjects cannot always be properly controlled because of ethical concerns and therefore using appropriate animal models to mimic natural disease outcomes may be more informative. with the use of high-throughput omics approaches it should be possible to extract more detailed information out of these complex biological systems and understand the complex biology and mechanisms by which stress augments disease susceptibility. more studies are needed to understand the physiological differences between acute versus chronic stress as well as the combined effects of various stressors. it is becoming more apparent that stress not only makes an individual more susceptible to a disease but can also enhance disease severity. therefore, it is very important to understand the kinetics, specifi city and stability or duration of biomarkers associated with a specifi c stressor or combination of stressors before we can predict 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vaccination against experimental bovine herpesvirus-1 and pasteurella haemolytica respiratory tract infection: onset of protection effect of stress on viral-bacterial synergy in bovine respiratory disease: novel mechanisms to regulate infl ammation comparative approaches to the investigation of responses to stress and viral infection in cattle transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress acute phase proteins in cattle after exposure to complex stress effects of road transportation on lymphocyte subsets in calves transportation stress alters the circulating steroid environment and neutrophil gene expression in beef bulls hypercortisolemia in acute stroke is related to the infl ammatory response induction of interferon activity in nasal mucus by association of levamisole by systemic route -inactivated virus by local route effect of stress on viral-bacterial synergy in bovine respiratory disease: a review of potential mechanisms immune mechanisms of pathogenetic synergy in concurrent bovine pulmonary infection with haemophilus somnus and bovine respiratory syncytial virus proteomic characterization of the interstitial fl uid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery unlocking the mysteries of virus-host interactions: does functional genomics hold the key? a comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging in escherichia coli cells effect of transport stress on respiratory disease, serum antioxidant status, and serum concentrations of lipid peroxidation biomarkers in beef cattle effect of transportation on blood serum composition, disease incidence, and production traits in young calves. infl uence of the journey duration this project was funded by the saskatchewan agriculture and food rural revitalization (safrr), genome bc and genome prairie for the 'pathogenomics of innate immunity' research program and the ontario cattleman's association (oca). this manuscript is published with permission of the director of vaccine and infectious disease organization as article number 512. a.a.p. is the holder of an nserc senior industrial research chair. key: cord-315726-ltjurdrq authors: acheson, d.w.k. title: food and waterborne illnesses date: 2009-02-17 journal: encyclopedia of microbiology doi: 10.1016/b978-012373944-5.00183-8 sha: doc_id: 315726 cord_uid: ltjurdrq there are many different biological, chemical, or radiological agents that when added to food can result in many different types of illness. some may be rapidly fatal; others require long-term exposure to result in illness. some lead to short-term illness and others result in long-term complications. the universe of such agents and situations is enormous. this article summarizes some of the principal foodborne microbiological agents that clinicians and those involved with public health have to deal with. while the range of agents is broad and the list is long there are several basic mechanisms such as ingestion of preformed toxins or toxin production once a microbe is present in the intestine that facilitate sorting these agents into some logical framework. however, at the end of the day it is always key to think about ingested agents as a cause for illness, whether that illness be confined to the intestinal system or more systemic. in principle all foodborne illness is preventable and of the key messages to consumers and health care professionals is to know if you or your patient is at greater risk from foodborne illness. if one is dealing with an ‘at risk’ patient, it is important they be educated on what foods to avoid and what precautions to take to minimize the likelihood of acquiring a foodborne illness. while treating most foodborne illness is straightforward, prevention is clearly the path of choice. the topic under discussion is foodborne illness. while there are many causes of foodborne illness the focus of this text is on microbes. the text approaches the issues by discussing illness due to toxins preformed in foods and toxins made once the microbes have been ingested, illness due to other mechanisms that affect the gastrointestinal tract, and finally foodborne illness that has manifestations other than purely gastrointestinal. a wide variety of the common foodborne pathogens is discussed, with a brief description of what they are, the types of illness they cause, and the kinds of food most frequently associated with them along with some commentary with regard to treatment. food-and waterborne illness typically brings to mind the image of an individual who develops an acute gastrointestinal illness following exposure to contaminated food or water. however, the definition of illness that may be attributed to food or water is broad and encompasses exposure to toxins, carcinogens, metals, prions, allergens, and other factors, in addition to the classic infective pathogens. as reviewing each of these agents in detail is beyond the scope of this article, our focus will be on foodand waterborne infections only; an extensive list of foodborne pathogens is given in table 1 . this article discusses the current epidemiology of foodborne illness, provides an overview of the various toxins and organisms considered to be the more important foodborne agents, and discusses some preventative approaches that can be used to help ensure consumers stay safe with regard to the food they prepare and eat at home. the clinical symptoms, treatment, and long-term consequences of various foodborne infections are also briefly reviewed. foodborne illness typically consists of acute gastrointestinal upset with nausea, vomiting, diarrhea, and abdominal cramps. typically, symptoms resolve without the need for significant medical intervention and without long-term consequence. however, on occasion foodborne infection causes severe illness or death. unfortunately, in the early stages of illness, differentiating between a patient with an inconsequential infection and the patient who may develop life-threatening sequelae can be difficult. some systemic consequences of infection occur several days or weeks after the initial exposure. examples include the hemolytic uremic syndrome (hus) secondary to shiga toxin-producing escherichia coli (stec), the development of guillain-barré syndrome (gbs) after campylobacter infection, and the association of a number of enteric bacterial pathogens with reactive arthritis and postinfectious irritable bowel syndrome. the true burden of foodborne illnesses in the united states and in other parts of the world is largely unknown; however, the number of suspected deaths worldwide from foodborne pathogen exposure is staggering. several million children die each year worldwide from acute diarrheal disease and resulting dehydration, the majority of which is likely due to contaminated food or water. in the united states, until recently, we had very little data on the numbers and outcomes of foodborne infection. the development of the foodborne diseases active surveillance network (foodnet) in 1996 by the centers for disease control and prevention (cdc) has provided, for the first time, the opportunity to determine the epidemiology of foodborne disease in the us population. foodnet is the (eip) , and is a collaborative venture with eip program sites, the us department of agriculture (usda), and the food and drug administration (fda). foodnet performs populationbased active surveillance for confirmed cases of campylobacter, e. coli o157:h7, listeria, salmonella, shigella, vibrio, yersinia, and hus, as well as cryptosporidium and cyclospora infections. in 2006, surveillance occurred within a defined population of 44.9 million americans using information from clinical microbiology laboratories in ten states. foodnet monitors only confirmed cases of diarrheal infection, missing cases that never present to medical attention. however, through additional surveys, foodnet has the capacity to determine the frequency of diarrhea and the number of physician visits within the study population. utilizing foodnet and other data, the cdc provides our current best estimate of the true burden of foodborne infections in the united states. mead and his colleagues from the cdc estimate that there are 76 million illnesses, 325 000 hospitalizations, and 5000 deaths annually due to foodborne infections. this means that, on average, somewhere between one in three and one in four americans will have a foodborne infection each year. while these data provide an excellent estimate of disease prevalence in the united states, they also illustrate some major gaps in our knowledge of foodborne infections. specifically, determining attribution can be very difficult. for example, in the context of sporadic infections, the precise food that has caused the illness and the point at which the food was contaminated are usually unknown. or indeed whether the infection was acquired through person-to-person spread or by some other route is difficult to ascertain. also, in 62 million cases, or 82% of the estimated 76 million infections each year, no specific pathogen is identified. disease due to unidentified agents results in 265 000 hospitalizations and 3200 deaths, which begs the question as to whether these are due to known pathogens or foodborne infections yet to be discovered. our ignorance as to the cause of more than 80% of the estimated foodborne illness is a daunting problem. however, many new agents have been discovered and linked to foodborne disease in the last 30 years. table 2 offers a list of some recently described food-and waterborne pathogens, some are new pathogens and others are agents previously recognised but infrequently linked to illness or considered nonpathogenic. for example, campylobacter jejuni was once thought to be an unusual cause of bacteremia but is now known to be one of the most frequent bacterial causes of enteritis in the united states. in 2006, the most recent year for which preliminary foodnet data are available, the cdc confirmed 17 252 laboratory-confirmed cases of infections from the foodnet sites. incidence varied dramatically between the foodnet sites. for example, campylobacter affected 6.27 per 100 000 people in georgia and 26.82 per 100 000 in california. salmonella infections varied from 11.01 per 100 000 in oregon to 20.04 per 100 000 in georgia. though the explanation for these geographic differences is unknown, they seem to suggest true regional variation of foodborne pathogens. another trend observed in foodnet data was the preponderance of cases in the young and elderly. in 2006, foodnet identified 71 cases of hus in children aged below 18 years (rate: 0.68 per 100 000 children); 47 (66%) of these cases occurred in children aged below 5 years (rate: 1.63). across all age groups, clinical outcomes differed by pathogen. while the total number of listeria monocytogenes and e. coli o157:h7 infections were less than for some of the other pathogens, they were associated with much higher hospitalization rates and death rates than any of the other bacterial pathogens monitored (table 3) . table 3 reflects the lack of correlation between the propensity for an organism to cause disease and its propensity to result in the death of the patient. since foodnet began to operate in 1996, the accumulated data have also revealed a seasonal trend, with a spike in infection with the three major pathogens (salmonella, c. jejuni, and e. coli o157:h7) during the summer months (figure 1) . the summer predominance of bacterial foodborne infections is likely multifactorial. clearly, warmer weather allows for more rapid bacterial growth on food that is improperly refrigerated. consumer habits also change in the warmer months, with more picnics and barbecues, contributing to problems with keeping food refrigerated, increased risk of cross contamination, and so on. foodnet surveillance effectively monitors trends in the rates of infection over time and there have been a number of changes since foodnet surveillance began, with the estimated annual incidence of several infections changing significantly from baseline to 2006 (figure 2 decreased significantly (41% ci ¼ 34-48%). in contrast, significant increases in incidence compared with baseline occurred for salmonella enteritidis (28%, ci ¼ 4-57%), salmonella newport (42%, ci ¼ 7-87%), and salmonella. javiana (92%, ci ¼ 22-202%). the estimated incidence of salmonella heidelberg and salmonella montevideo did not change significantly compared with baseline. while foodnet produces excellent data on the epidemiology of foodborne illness overall, it has several important areas of weakness. it does not survey for many of the common foodborne pathogens, including viruses, which are thought to cause the vast majority of foodborne illness. similarly, it does not address the cause of illness in patients who do not have a stool sample sent for analysis: those who either do not seek medical care or do seek care but do not have a stool sample analyzed. in an adjunctive study reported by the cdc, 11% of 10 000 residents interviewed through random phone consultations reported an episode of diarrhea during the previous month. this translates to 1.4 episodes of diarrhea per person per year, which if multiplied roughly by the population of the united states, represents 375 million diarrheal cases per year. in this study, merely 8% of those with a diarrheal episode sought medical care, and of those, only 20% reported submitting a stool sample for culture. thus, our best data on the causes of acute gastrointestinal disturbance from foodnet surveillance are based on cultures of less than 2% of diarrheal episodes. nonetheless, despite the current limitations of our evaluation of foodborne illness, the endeavors of state, local, and federal authorities have been critical to improving our knowledge of disease frequency, pathogen epidemiology, and the establishment of control systems to limit food contamination. the knowledge gained from foodnet surveillance allows for targeted efforts to improve food safety and education. as noted previously, the diversity of foodborne pathogens listed in table 1 is far too extensive to be discussed completely within the scope of this article. in the following sections therefore, many of these microbial agents will be discussed briefly with a focus on typical modes of transmission, the foods they frequently contaminate, and the specific serious consequences that may ensue from infection. although foodborne agents cause disease by a wide variety of mechanisms, the mode of infection often falls into one of the following three categories: (1) ingestion of preformed toxins produced by bacteria in food prior to consumption; (2) infection with pathogens present in food which, following ingestion, produce toxins in the gastrointestinal tract; and (3) infection with organisms having various virulence factors that permit the microbes to be invasive, cause local damage, or create physiologic perturbations that result in clinical disease. of the three mechanisms, the preformed toxin is the most consistently transmitted via food. as each toxigenic organism requires a specific environment to stimulate toxin production, each has a predilection for certain types of food. as a result, different types of foods confer different risks for toxin ingestion. the major toxins of the common foodborne pathogens are discussed in detail in the next few sections and were reviewed by sears and kaper in 1996 . c. botulinum produces one of the best-known and deadly preformed toxins. the organism's natural habitat is soil, and therefore, its spores frequently contaminate fresh fruits and vegetables. commercial food sources have been occasionally implicated, but the majority of outbreaks have been traced to home-canned foods, especially vegetables, fruits, fish, and condiments. recent outbreaks have been attributed to chili, carrot juice, and home-prepared fermented tofu. generally the disease is rare, and in the united kingdom, only 62 cases have been recognized between 1922 and 2005. a recent report from the united kingdom provides a brief review of c. botulinum and foodborne botulism as well as descriptions of the six episodes (33 cases with three deaths) of this disease that occurred in the united kingdom between 1989 and 2005. to prevent botulism, the clostridium spores must be destroyed by heating food to a temperature of 120 c for 30 min, usually with the aid of a pressure cooker. in an anaerobic environment with a ph above 4.6, any surviving spores will germinate and produce their deadly toxins. there are seven antigenically distinct types of botulinum toxin, each of which is designated by a letter, a-g. types a, b, e, f, and g are associated with human disease, with type a accounting for about 25% of outbreaks and type b 8%. once ingested, the toxin is absorbed through the proximal small intestine and spreads via the bloodstream to the peripheral cholinergic nerve synapses where it irreversibly blocks acetylcholine release. a flaccid paralysis results, with cranial nerves affected first, followed by respiratory muscle paralysis and death if left untreated. the diagnosis of botulism is clinical and treatment should be initiated prior to confirmation with laboratory data, as the traditional mouse bioassay for toxin detection requires 4 days for final results. samples such as food, vomitus, serum, gastrointestinal washings, and feces are all reasonable specimens to test. newer pcr-and enzyme immunoassay-based detection methods are now being used. early in the course of disease, treatment may include emetics or gastric lavage to remove unabsorbed toxin. a trivalent (a, b, e) horse serum antitoxin decreases the progression and duration of paralysis, but it does not reverse existing paralysis. pentavalent and heptavalent antitoxins are also being investigated. human botulism immune globulin intravenous (big-iv) may also be beneficial. botulism carries a significant mortality rate, of up to 25%, with type a toxin. of those who survive the acute phase of illness, most recover completely. see later in the article for a discussion of infant botulism, which is a similar condition but not due to preformed toxin. a second well-known group of preformed toxins are those produced by s. aureus. s. aureus produces a variety of enterotoxins, defined by their antigenicity as enterotoxins a-h. staphylococcal enterotoxins a through g are responsible for 95% of staphylococcal food poisoning outbreaks. on rare occasions, other staphylococcal species, including coagulase negative staphylococci, have been found to produce similar enterotoxins. the toxins are small proteins with similar tertiary structures and biologic activity, including superantigen properties. ingestion of as little of 100-200 ng of toxin is considered sufficient to cause disease in humans. compared with botulinum toxin, staphyloccocal toxins are not inactivated by heating or boiling; nor are they susceptible to ph extremes, proteases, or radiation. as a result, once formed in food, these toxins are almost impossible to remove. the mechanism through which the toxin acts is not fully understood, but is suspected to be via stimulation of the autonomic nervous system and gut inflammation. as the toxin is not absorbed systemically, protective immunity is not induced following exposure. typically, patients become symptomatic between 1 and 7 h after ingestion of the food containing staphylococcal enterotoxin, with nausea (73-90%), vomiting (82%), and abdominal cramps (64-74%). diarrhea occurs in a large proportion of patients (41-88%), but fever is rare. treatment of affected individuals is supportive and symptoms usually abate within 2 days. there is no need to treat with antibiotics directed toward s. aureus. s. aureus is present in the mucous membranes and skin of most warm-blooded animals. food is most often contaminated with s. aureus through the fingers or nose of a food worker. the toxin is produced when contaminated food is stored at room temperature for a sufficient length of time to allow the organism to grow and produce toxin. the bacterial population must be greater than 10 5 organisms per gram of contaminated food before appreciable amounts of toxin will be produced to elicit illness. a number of different foods have been associated with staphylococcal food poisoning, including egg products, cooked meat products, poultry, tuna, mayonnaise, and particularly cream-filled desserts and cakes. this disease is more frequently associated with food from the home or a service establishment rather than commercially prepared food. it has also occasionally occurred in large outbreaks with thousands of affected individuals. a third example of preformed toxins are those of b. cereus, a gram-positive, spore forming aerobe that causes two distinct clinical syndromes: a short-incubation period emetic syndrome and a long-incubation period diarrheal syndrome. the organism is known to produce up to three enterotoxins. cereulide and the tripartite hemolysin bl have been identified specifically as emetic and diarrheal toxins, respectively. nonhemolytic enterotoxin, a homologue of hemolysin bl, has also been associated with the diarrheal syndrome. the toxins associated with the diarrheal illness are not preformed but produced by the organism during the vegetative growth phase in the small intestine. the emetic toxin, named cereulide, is thought to be an enzymatically synthesized peptide produced as the organism grows in food, especially starchy foods such as rice and pasta. like staphylococcal toxin, cereulide is resistant to heat, ph variation, and proteolysis, and is therefore rarely destroyed during food preparation. its exact pathogenic mechanism remains unknown, but it has been shown to stimulate the vagus afferent by binding to the 5-ht3 receptor. the emetic syndrome presents much like s. aureus-related foodborne disease, occurring 1-6 h after exposure and causing nausea and vomiting. fever is not characteristic of the illness and full recovery usually occurs. diagnosis can be made by finding the organism in the food or vomitus of the patient, or through detection of the emetic toxin through bioassays or the enterotoxins by commercial immunoassays. new approaches include the use of real-time pcr to detect cereulide-producing b. cereus genes in potentially contaminated food. derived from various types of food, a number of naturally occurring toxins may cause human foodborne illness. many are associated with consumption of seafood contaminated by algae. others are due to fungal contamination of food or inherent to certain fruits and vegetables. scombroid poisoning typically occurs after the ingestion of spoiled, dark-fleshed fish, especially tuna and mackerel. the clinical symptoms of poisoning, including flushing, headache, palpitations, dizziness, nausea, vomiting, and diarrhea, are attributable to excess levels of histamine present in temperature-abused fish. histamine is produced by bacterial metabolism of the amino acid histidine in fish muscle. bacterial replication and histamine production occur when fish is not frozen promptly after being caught or is stored at room temperature for several hours. symptoms of intoxication begin within minutes to several hours following ingestion. most resolve fully within hours, but, occasionally, bronchospasm or circulatory collapse may occur. the diagnosis is clinical, and treatment consists of antihistamines. elevated histamine levels in the contaminated fish or the patient's serum may be diagnostic, but few laboratories, other than regulatory laboratories, are equipped to undertake this analysis. ciguatera poisoning is due to the ingestion of neurotoxins from tropical and subtropical marine fin fish, including mackerel, groupers, barracudas, snappers, amberjack, and triggerfish. it affects 50 000 individuals yearly, mainly in the caribbean and south pacific islands. the toxin is produced in reef algae, the dinoflagellates (e.g., gambierdiscus toxicus). it spreads through the food chain via consumption of smaller organisms and fish by larger predators, accumulating at dangerous levels in the flesh of large fish. two groups of compounds are implicated in ciguatera fish poisoning: the lipid-soluble ciguatoxins, which activate nerve synapse sodium channels, and the water-soluble maitotoxin, which induces neurotransmitter release by binding to calcium channels. in humans, these toxins cause gastrointestinal symptoms 3-6 h after ingestion, including nausea, vomiting, and watery diarrhea. neurologic symptoms follow, with weakness, heat-cold temperature reversal, vertigo, ataxia, paresthesias, and dysathesias of the perioral region, palms, and soles. death and serious cardiovascular complications are uncommon. most symptoms resolve within a week, but neurologic symptoms can persist for months. the diagnosis of ciguatera is clinical; however, the toxin can be detected in fish using a mouse bioassay or newer enzyme immunoassays. five main types of shellfish poisoning have been described: paralytic, neurotoxic, diarrheic, amnestic, and azaspiracid. like ciguatera, illness is due to toxins generated by algae, usually dinoflagellates, which accumulate in the shellfish. the paralytic variant of shellfish poisoning is due to saxitoxin, an agent that blocks neuronal sodium channels and prevents propagation of the action potential. clinically, this results in a rapid-onset, life-threatening paralysis. brevitoxin, the agent responsible for neurotoxic shellfish poisoning, also binds sodium channels but does not cause paralysis; instead, it produces a clinical syndrome similar to but less severe than ciguatera. symptoms of nausea, vomiting, and paresthesias occur within hours of exposure and resolve completely within 3 days. diarrheic shellfish poisoning causes gastrointestinal disturbance with nausea, vomiting, and diarrhea. the toxin acts by increasing protein phosphorylation. amnestic shellfish poisoning, also known as toxic encephalopathic poisoning, causes outbreaks of disease in association with consumption of mussels. manifestations include nausea, vomiting, diarrhea, severe headache, and, occasionally, memory loss. the toxin domoic acid is a glutamate receptor agonist that causes excitatory cell death. diagnosis of human illness due to shellfish toxins is clinical based on symptom profile and prompt onset of symptoms after shellfish consumption. the exception to this is amnestic poisoning, which may not cause symptoms until 24-48 h after exposure. the toxins can be detected using either mouse bioassays or high-performance liquid chromatography (hplc), but this is done primarily for research purposes or in monitoring. owing to the serious consequence of shellfish poisoning, large-scale surveillance systems for contamination of shellfish populations have been implemented. tetrodotoxin is present in certain organs of the puffer fish and if ingested can cause rapid paralysis and death. symptoms may occur in as little as 20 min or after several hours. the illness progresses from gastrointestinal disturbance to almost total paralysis, cardiac arrhythmias, and death within 4-6 h after ingestion of the toxin. the diagnosis is clinical and based on history of exposure. mouse bioassays and hplc have been used to detect tetrodotoxin in food. aflatoxins are produced by certain strains of fungi (e.g., aspergillus flavus and aspergillus parasiticus) that grow in various types of food. most human exposure occurs through mold-contaminated corn or nuts, especially tree nuts (brazil nuts, pecans, pistachio nuts, and walnuts), peanuts, and other oilseeds. because mycotoxins can be produced prior to or after harvest, eliminating them from food is nearly impossible. aflatoxin b1 is the most common and toxic, but there are several types of toxins (b2, g1, and g2). they are potent mutagens and carcinogens, with b1 causing deoxyribonucleic acid (dna) damage in the p53 tumor suppressor gene. exposure to the aflatoxin predisposes the patient to hepatocellular carcinoma, especially in conjunction with chronic hepatitis b infection. with a high ingested dose of aflatoxin, a condition known as aflatoxicosis may occur, characterized by fever, jaundice, abdominal pain, and vomiting. aflatoxin exposure is common in asia and parts of africa but uncommon in the united states. the diagnosis is clinical, but assays to detect the toxins in food exist. serum and urine markers have also been developed to quantify exposure. vibrios currently, there are over 40 vibrio species, a group of gram-negative marine organisms, most of which are not human pathogens. the most common and severe human illness is caused by vibrio cholerae o1, the species responsible for seven cholera pandemics. the previous six were caused by the 'classic' biotype and the seventh pandemic, which began in 1961, was caused by the 'el tor' biotype. in the united states, cholera is mainly acquired through consumption of gulf coast seafood or through foreign travel. a clean water supply is critical to cholera prevention, as the organism is resistant to washing, refrigeration, and freezing of a wide variety of seafood and fresh produce. because stomach acidity does kill many of the organisms, more than 10 6 v. cholerae are usually required for infection; those with decreased gastric acidity may be infected with lower doses. the incubation period is usually 1-3 days, but may be as short as a few hours or as long as 5 days. infection causes voluminous watery diarrhea. hypotension and shock may result within the first 12 h of infection. the primary virulence factor is the cholera toxin, which targets an intestinal g-protein, producing cyclic adenosine monophosphate (camp). the increase in camp produces watery diarrhea by inhibiting intestinal sodium absorption and increasing chloride and bicarbonate secretion. the toxin is transmitted to the organism via a bacteriophage. indeed, in recent years a new pathogen, v. cholerae o139, evolved in the indian subcontinent. non-o1 strains were not previously associated with human epidemics, but this pathogen appears to have acquired the cholera toxin and other virulence factors through horizontal transmission and bacteriophage infection. vibrio parahaemolyticus also inhabits marine environments and is acquired principally through the ingestion of raw shellfish. this vibrio has been a major foodborne pathogen in japan, but is less common in the united states. in recent years there has been global dissemination of v. parahaemolyticus serotype o3-k6. infection is characterized by diarrhea, abdominal cramps, nausea, and vomiting, with fever and chills present in about 25% of cases. dysentery occurs in a minority of patients, more often in children than in adults. occasionally, wound infections and septicemia occur. symptoms may appear in as little as 4 h, but are typically present 12-24 h after exposure. disease is attributed to a 23 kda protein called thermostable direct hemolysin (tdh). the gastroenteritis is usually self-limiting. patients require fluids, and antibiotics may be useful if intestinal symptoms persist. vibrio vulnificus is another free-living estuarine organism that is frequently isolated from shellfish, most often acquired through raw oyster or clam consumption. it is the most common life-threatening vibrio infection in the united states. individuals with diabetes, immunosuppressive disorders, and liver disease including hemochromatosis and alcoholic liver disease are especially susceptible to infection. in these groups the case fatality ratio may exceed 50%. infection presents with fevers, chills, nausea, vomiting, and diarrhea. hypotension and sepsis ensue. large hemorrhagic bullae erupt and progress to necrotic ulcers. v. vulnificus is an encapsulated organism, thereby resistant to the bactericidal activity of normal human serum. the pathogenesis of v. vulnificus is not well understood but has been summarized recently by gulig and colleagues. the organisms are sensitive to the amount of transferrin-bound iron in the host, which may explain the increased susceptibility in patients with hemochromatosis. definitive diagnosis may be made from blood, stool, or wound cultures. due to the severity of infection, antibiotics should be initiated promptly. v. vulnificus is susceptible to many antimicrobials, including tetracycline, ciprofloxacin, trimethoprim-sulfamethoxazole, ampicillin, and chloramphenicol. clostridium perfringens is an anaerobic, spore-forming, gram-positive rod associated with two distinct types of foodborne disease. the species has been divided into five distinct types, a-e. type a causes the majority of human infections and is usually linked to the consumption of meat or poultry (typically high-protein foods) that have been stored between 15 and 60 c for more than 2 h. at this temperature, clostridial spores germinate and begin vegetative growth. at an infective dose of 10 5 vegetative cells, ingested clostridial spores transiently colonize portions of the intestine and produce enterotoxin. ingestion of preformed toxin or nongerminated spores will not usually result in disease. the enterotoxin (cpe) is a heat-labile 35 kda protein encoded by the cpe gene. c. perfringens types a, c, and d all carry this gene, but for unclear reasons only type a is frequently associated with foodborne disease. cpe functions by a complex mechanism, inserting itself into the host cell membrane and altering membrane permeability. clinically, diarrhea and severe abdominal cramps develop 6-14 h after exposure; vomiting and fever are less common. diagnosis is complicated by the presence of c. perfringens in the bowel microflora of many asymptomatic individuals. however, a number of tests are able to detect the enterotoxins in stool, including enzyme immunoassays or latex agglutination. c. perfringens type c causes the second distinct foodborne illness, mainly in developing countries. it causes a necrotizing enterocolitis seen in the context of malnutrition. the type c strains produce enterotoxin and type 'a' and 'b' toxins. the b toxin appears to be responsible for the cell necrosis associated with infection. as the b toxin is inactivated by intestinal proteases, illness occurs in patients in whom these enzymes are inadequate (e.g., in malnutrition) or in the presence of trypsin inhibitors found in undercooked pork or sweet potatoes. infant botulism results from the germination of ingested spores of botulinum toxin-producing clostridia that colonize the large intestine. the spores germinate within the intestine and produce botulinum toxin. of the various potential environment sources such as soil, dust, and foods, honey is the one dietary reservoir of c. botulinum spores that has definitively been linked to infant botulism by both laboratory and epidemiological studies. children aged 12 months are very susceptible to developing infant botulism. honey continues to be an important exposure source in young infants and cases continue to occur. jars of honey bear a label advising parents to not feed honey to children less than 12 months old. the two main e. coli species associated with foodborne illness are stec and enterotoxigenic e. coli (etec). the former are relative newcomers to the scene of foodborne pathogens. the first stec to be associated with disease in humans was e. coli o157:h7 following two outbreaks of hemorrhagic colitis in 1982. since then, at least 60 different serotypes of stec have been associated with clinical disease and have become recognized as the most common cause of hus. not all stec have been associated with human illness and the more virulent forms are often referred to as enterohemorrhagic e. coli (ehec) that are characterized by having the ability to attach and efface intestinal epithelium, produce shiga toxins (stx), and carry a specific plasmid. stec bacteria colonize the intestinal tracts of many mammalian species, particularly ruminants (cattle, sheep, and goats). most human illness is due to the ingestion of contaminated bovine products, but an increasing number of reports associate infection with fecally contaminated fresh produce (lettuce, alfalfa sprouts, unpasteurized apple cider, spinach) and water. one of the key virulence factors of stec is bacteriophage-encoded stx. the two main types are stx1 and stx2, but there are at least five subtypes of stx2 (stx2, 2c, 2d, 2e, and 2f). the infectious dose of some stec (e.g., o157:h7) is known to be very low, in the region of 10-100 organisms. symptoms typically develop 2-4 days after ingestion, but may occur in as little as 1 day or as long as 8 days. nonbloody or bloody diarrhea is the primary acute manifestation. treatment of stec and its major complications is currently largely supportive. controversy exits as to the role of antibiotics, with concern that treatment of pediatric patients with certain antimicrobials (e.g., fluoroquinolones and trimethoprim-sulfamethoxazole) may actually increase the likelihood of serious complications such as hus. several recent reviews relating to foodborne e. coli infections have been written, and the reader is referred to them for more details. a well-described example of a long-term consequence following infection with a foodborne and waterborne pathogen is the hus resulting from stec infection. in the united states, 1.5% of patients will require a renal transplant following hus. in up to 20% of patients with hus, the pancreas is also damaged, causing some patients to develop permanent diabetes mellitus. etec infection is a common cause of disease in developing countries, and is frequently associated with travelers' diarrhea. etec are transmitted through contaminated water and food and have caused a number of large outbreaks in the united states; however, their importance in sporadic disease is not known. incubation periods range from 12 h to 2 days, and typical symptoms are abdominal discomfort and watery, nonbloody diarrhea without fever. etec have two significant virulence characteristics: the ability to colonize the intestine and the capacity to produce enterotoxins. a variety of colonization factor antigens (cfa) and two different types of toxins, known as heatstable (st) and heat-labile (lt) toxins, have been found in etec. the st group consists of small peptides that effect intracellular concentrations of cyclic guanosine monophosphate (gmp). the lt toxins are structurally and functionally much like the cholera toxin. oral rehydration is the mainstay of treatment and is often life saving for infants. antibiotic therapy is not routinely required. salmonella salmonella are one of the most common causes of foodborne illness in humans. they can be divided into two broad categories: those that cause typhoid and those that do not. the typhoidal salmonella, such as s. typhi and s. paratyphi, colonize humans and are acquired through the consumption of food or water contaminated with human fecal material. the much larger group of nontyphoidal salmonella are found in the intestines of other mammals and, therefore, are transmitted through food or water that has been contaminated with fecal material from a wide variety of animals and poultry. more than 2300 serovars of salmonella are differentiated by their somatic (o) antigens and flagellar (h) antigens. in the united states, most typhoid is the result of food contamination by an asymptomatic chronic carrier, or from foreign travel. typhoid fever continues to be a global health problem, but is uncommon in the united states; only 60 outbreaks occurred between 1960 and 1999. in contrast, the number of cases of nontyphoidal salmonella increased steadily over the last four decades. s. enteritidis infection due to contamination of hen eggs is a particular problem, with an estimated contamination rate of 1 in 10 000 eggs. the bacteria penetrate intact eggs lying in fecal material or infect them transovarially before the shell is formed. other common sources of nontyphoidal salmonellosis are inadequately pasteurized milk, foods prepared with raw eggs, meat, poultry, and fecally contaminated fresh produce. the infectious dose of s. typhi is thought to be around 10 5 organisms. typhoid infection is characterized by high fevers, abdominal discomfort, and a rose-colored macular rash. the infective dose of nontyphoidal salmonella may vary from <100 to 10 6 depending on the host, the food vehicle, and the type of salmonella. these species tend to cause bloody or nonbloody diarrhea, fever, nausea, vomiting, and abdominal discomfort. in all types of salmonella the most critical virulence determinant is their ability to cross the intestinal epithelium and cause invasive disease. the most pressing problem regarding salmonella is the emergence of multidrug-resistant strains. for example, s. typhimurium phage type dt104 is resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. in europe quinolone-resistant strains of salmonella have been detected. campylobacter, which was not recognized as a foodborne pathogen until the mid 1970s, is now one of the most common bacterial foodborne infections diagnosed in the united states. campylobacter are gram-negative, spiral, microaerophilic organisms. two species of campylobacter, c. jejuni and c. coli, are responsible for the vast majority of human disease, with c. jejuni causing 90% of infections and c. coli near 10%. campylobacter fetus, campylobacter upsaliensis, campylobacter hyointestinalis, and campylobacter lari have occasionally been associated with gastroenteritis. in human studies, infectious doses as low as 100 organisms may result in disease, and one drop of chicken juice may contain 500 infectious organisms. campylobacter are more frequently associated with sporadic disease than with outbreaks, and person-to-person spread does not appear to be common. c. jejuni and c. coli are intestinal commensals in many animals and birds, including domestic pets. the main vehicle for human infection is poultry, but other raw meats, milk, and water have also been implicated. surface water can be contaminated with campylobacter and waterborne outbreaks have been reported. the pathogenicity of campylobacter depends on its motility; in vitro, nonmotile strains are not capable of invading intestinal epithelial cells. typical infection causes diffuse colonic inflammation with marked inflammatory cell infiltration of the lamina propria, which may be mistaken for inflammatory bowel disease. symptoms usually occur within 2-3 days after exposure, but may occur as quickly as 10 h or as late as 7 days. high fevers, headache, and myalgias may precede the onset of nausea, vomiting, and diarrhea. the diarrhea may be loose and watery or grossly bloody. abdominal cramps and pain may predominate. interestingly, the disease is sometimes biphasic, with an apparent settling of symptoms after 4-5 days followed by a recrudescence. local complications resulting from direct spread of the organisms from the gastrointestinal tract are rare and include cholecystitis, hepatitis, acute appendicitis, and pancreatitis. the case fatality rate is low, approximately 0.5 per 1000 infections. however, long-term complications may occur including gbs. gbs affects 1-2 persons per 100 000 in the united states each year, or less than 1 person per 1000 infected. a recent article by hughes and cornblath address this issue and they point out that about a quarter of patients with gbs have had a recent c. jejuni infection, and that axonal forms of the disease are especially common in these people. the pathogenesis of injury is molecular mimicry, in which the immunologic response to the core oligosaccharides of campylobacter lipopolysaccharide cross-react with a variety of neuronal glycosphingolipids. up to 20% of individuals affected by gbs require mechanical ventilation, and another 20% will have permanent neurologic deficits. the overall risk of developing gbs following campylobacter infection is considered to be 1 in 1000. the percent of cases of gbs linked to prior infection with campylobacter is estimated to be 30-40%. at least 11 campylobacter serotypes have been associated with gbs, but serotype o:19 is thought to be the most common association. the interval between infection and the development of gbs may be as short as 1 week or as long as 6. those with a rapid onset of gbs are suspected to have had prior exposure to the critical campylobacter serotypes and therefore primed for a rapid immune response. diagnosis of campylobacter is confirmed by stool culture. pcr and enzyme immunoassays are now available and may become useful for species-specific antigen detection. as with salmonella, a growing number of campylobacter are developing antimicrobial resistance. fluoroquinolones are generally very active against campylobacter when they are susceptible, and there was a period when it appeared that these would be the drugs of choice. however, the increasing problems with fluoroquinolone resistance now makes fluoroquinolones much less desirable and not a drug of choice in first-line therapy. in sweden, quinolone resistance in clinical isolates of c. jejuni increased more than 20fold in the early 1990s; macrolide resistance is also increasing. of the three members of the genus yersinia, y. enterocolitica and y. pseudotuberculosis are considered to be foodborne pathogens, whereas y. pestis is typically not. overall, yersinia cause less foodborne illness than salmonella or campylobacter, and the majority of isolates in food, environmental samples, and human stool are nonpathogenic species. y. enterocolitica is divided into biogroups, with more than 50 'o' antigens used to designate strains. most human disease is associated with serotypes o3, o5, o8, or o9. y. enterocolitica is an invasive organism. all pathogenic strains carry a plasmid pyv, coding for the virulence proteins yersinia outer proteins (yops) and adhesin a (yada), which block phagocytosis, opsonization, and complement activation; and yersinia enterotoxin (yst), invasin (inv), and attachment-invasion proteins (ail), which mediate invasion and serum resistance. a variety of tests, including pcr and dna hybridization, congo red absorption, salicin fermentation, and esculin hydrolysis, can be used to determine if a strain is pathogenic. y. enterocolitica infection results in mesenteric lymphadenitis, enteritis, and diarrhea. most infections are selflimited, but symptoms can be prolonged, lasting several weeks or longer. complications such as ulceration and intestinal perforation may occur. the classic long-term complication following yersiniosis is the development of reactive arthritis, occurring most commonly in patients who are hla-b27-positive. although antibiotic therapy is not routinely required, many antimicrobials are effective; ceftriaxone or fluoroquinolones are recommended for serious infection. yersinia infection is most frequently associated with raw or undercooked pork consumption. swine are the major reservoir of these organisms, though pathogenic human strains have been found in sheep, dogs, cats, and wild rodents. milk is a frequently reported source, and since y. enterocolitica can survive and indeed multiply in milk at 4 c, small numbers of organisms can become a significant health threat, even if the milk is refrigerated. six serotypes and four subtypes of y. pseudotuberculosis have been described, but serotype o1 is associated with about 80% of human disease. the clinical picture is similar to that of y. enterocolitica. l. monocytogenes is a pathogen of great concern because of the high mortality rate associated with infection. listeriosis is the major concern from exposure to l. monocytogenes and although rare and usually occurring only in high-risk populations (1800 cases per year estimated to occur in the united states) is associated with high morbidity and mortality rates, with a case fatality rate of over 20%. of the seven listeria species, only l. monocytogenes is a significant human pathogen. it is common in the environment, present in soil, water, on plants, and in the intestinal tracts of many animals. thirty-seven different types of mammals, at least 17 species of birds, and between 1 and 10% of humans are carriers of listeria. although the organism is readily killed by heat and cooking, the fact that it is ubiquitous makes recontamination a real risk. of particular concern is that the organism is able to grow and multiply at refrigerator temperatures in certain foods, so even minor contamination of a product may result in high levels of bacteria after extended storage. the infectious dose is not known, with some studies suggesting it may be as high as 10 9 organisms, and others suggesting that it may be as low as several hundred. the more critical determinant of listeria infection is likely individual susceptibility, with the elderly, pregnant women, the immunocompromised, and newborns having higher rates of infection and higher mortality rates. foods associated with listeriosis include unpasteurized milk, soft cheeses (e.g., feta, camembert, and brie), coleslaw, smoked seafood, luncheon meats, and hot dogs. human infection occurs sporadically and in outbreaks. infected individuals suffer a mild, transient gastroenteritis 2-3 days after contaminated food is consumed. most immunocompetent adults have no further symptoms. susceptible individuals may suffer, after a period of days, fevers and mylagias, septicemia, meningitis, or encephalitis. pregnant women have a 12-fold increased risk of infection, and transplacental transmission may cause spontaneous abortion, premature birth, neonatal sepsis, and meningitis. once the diagnosis is established, l. monocytogenes is readily treated by penicillins or aminoglycosides. l. monocytogenes has also been associated with febrile gastroenteritis and is linked with a variety of food items. generally, such episodes are self-limiting and do not lead to listeriosis. it is unclear how frequently l. monocytogenes causes enteritis since it is not an organism that is routinely looked for in this context. shigellae are unusual in that they are not present in fecal material from animals such as poultry, beef, and pork, and are therefore not transmitted in the same manner as nontyphoid salmonella, campylobacter, or e. coli. instead, these bacteria are highly host adapted, infecting only humans and some nonhuman primates. transmission occurs when a food product is contaminated by human fecal material. there are four different species of shigella (s. dysenteriae, s. flexneri, s. sonnei, and s. boydii) and all cause human disease. in the united states and other developed countries, most infection is due to s. sonnei, though s. flexneri is also common. one of the most striking features of shigellosis is the very small inoculum of organisms required to cause disease: as few as 10-100 of the most virulent genus, s. dysenteriae, are sufficient to cause dysentery in healthy adult volunteers. this low infectious dose permits person-to-person spread, with 20% of persons in a household becoming infected when an index case is identified in a family. given that these organisms are not typically present in food other than through human contamination -either directly during food preparation or indirectly from contamination with human fecal materialall shigellosis could be considered to be due to person-toperson spread. a variety of foods have been implicated in shigellosis including salads (potato, tuna, shrimp, macaroni, and chicken), raw vegetables, dairy products, poultry, and common-source water supplies. shigella often cause bloody diarrhea. some species carry stx and may cause hus like e. coli o157:h7. treatment with antibiotics shortens the duration of fever, diarrhea, and bacteremia, and reduces the risk of lethal complications. it also shortens the duration of pathogen excretion in stool, thereby limiting the spread of infection. a recent concern, however, is the increasing antibiotic resistance of shigella species. antibiotic resistance occurs quickly in shigella, attributed to horizontal transfer of resistance genes on integrons. multidrug-resistant isolates have been discovered in several developing countries. e. sakazakii is a motile, peritrichous, gram-negative rod that was previously referred to as a 'yellow-pigmented enterobacter cloacae'. e. sakazakii is a recently identified foodborne pathogen that has been implicated most frequently in causing illness in neonates and children from 3 days to 4 years of age. a recent review by bowen and braden of 46 cases indicated that e. sakazakii has a mortality rate of 40-80%. twelve infants had bacteremia, thirty-three had meningitis, and one had urinary tract infection. most newborns with e. sakazakii infections die within days of infection. death is usually attributed to sepsis, meningitis, or necrotizing enterocolitis. the case fatality rates vary from 40 to 80%. sources of e. sakazakii associated with infant infections have not been identified in many cases; however, epidemiological investigations have implicated rehydrated powdered infant formula as well as equipment and utensils used to prepare rehydrated formula in hospital settings. enteroinvasive e. coli (eiec) is not frequently recognized as a foodborne pathogen, but infection has been linked to water and other foods such as cheese. eiec causes morbidity and mortality in young children in developed countries, but is a more important pathogen in developing countries due to poor hygiene and sanitation. a number of prominent serogroups found to be eiec have been described, including o28, o112, o124, o136, o143, o144, o147, and o164. clinically, eiec produces disease similar to shigellosis, with watery diarrhea or dysentery. eiec should be considered in those subjects with dysentery and substantial fecal leukocytes, in whom other invasive organisms have been ruled out. enteropathogenic e. coli (epec), like shigella species, is transmitted mainly by the fecal-oral route from one infected individual to another. epec has no known animal reservoir and is transmitted via food and water once contaminated by an infected person. epec is a major cause of infantile diarrhea worldwide, but mostly affects the developing world. the organisms have caused major outbreaks in various developed countries, but their role in sporadic disease is unknown because we lack routine diagnostic testing for these bacteria. clinically, epec infection presents with a watery, nonbloody diarrhea. low-grade fever and vomiting are common. in the developing world, mortality rates may be high, especially among infants. enteroaggregative e. coli (eaec) get their name from the way in which they adhere to epithelial cells in culture, in a 'stacked brick' pattern. these bacteria have been associated with acute or persistent diarrhea among immunocompromised patients and in developing countries. currently, there is no known animal reservoir for eaec, and fecal-oral spread from one person to another is considered to be the usual route of transmission. as with epec, contamination of food and water from infected individuals is probably important. in hiv patients with persistent eaec-associated diarrhea, antibiotic treatment has resulted in clearing of the organisms and in improvement in symptoms, suggesting that these bacteria are true pathogens, but they may be more opportunistic than other foodborne bacteria. aeromonads are gram-negative, facultatively anaerobic, motile, oxidase-positive bacilli that have been associated with foodborne illness. they are present in soil, freshwater, and sewage, and can contaminate fresh produce, meat, and dairy products. the infection rate tends to peak during the summer months. of the various species, aeromonas hydrophila, aeromonas caviae, aeromonas veronii, and aeromonas jandaei are most frequently associated with acute enteritis and foodborne infections. all typically cause persistent watery diarrhea. patients often have abdominal pain and dysenteric-like symptoms can occur, but fecal leukocytes and red cells are usually absent from stool. nausea, vomiting, and fever may occur in up to 50% of patients. infection is usually self-limiting and full recovery occurs in most healthy individuals without antimicrobial therapy, often making the diagnosis of academic interest only. the exception may be the patient with persistent diarrhea in whom no other cause has been identified. a number of protozoa have been associated with consumption of contaminated food and water. according to a review by karanis and colleagues, at least 325 waterassociated outbreaks of parasitic protozoan disease have been reported. giardia lamblia and cryptosporidium parvum account for the majority of outbreaks (132, 40.6 and 165, 50.8%, respectively), entamoeba histolytica and cyclospora cayetanensis were the etiological agents in nine (2.8%) and six (1.8%) outbreaks, respectively, while toxoplasma gondii and isospora belli were responsible for three outbreaks each (0.9%) and blastocystis hominis for two outbreaks (0.6%). balantidium coli, the microsporidia, acanthamoeba, and naegleria fowleri were responsible for one outbreak each (0.3%). however, questions remain in the literature as to whether some of these less frequently seen agents are truly the cause of illness or simply 'detected at the time'. c. parvum is an apicomplexan protozoan parasite that causes diarrhea in both immunocompetent and immunocompromised individuals. its pathogenic potential in immunocompromised patients first became evident during the early acquired immunodeficiency syndrome (aids) epidemic. its ability to affect healthy individuals was confirmed in 1993, when more than 400 000 people in milwaukee developed cryptosporidiosis as a result of contaminated municipal drinking water. cryptosporidia are typically waterborne, but foodborne and person-toperson spread have occurred. the primary reservoirs are bovine and human. symptoms tend to occur 5 days after ingestion of the oocysts. once ingested, the oocysts release four sporozoites, which then attach to and invade intestinal epithelial cells, especially in the jejunum and ileum. as a result, infection may be missed by diagnostic evaluation such as endoscopy. the diagnosis is made by a modified acid-fast or kinyoun stain for oocysts in the stool, or using commercially available immunofluorescence assays. typically, cryptosporidiosis causes watery diarrhea, abdominal cramping, nausea, and vomiting. fever is infrequent. in the immunocompetent, infection is selflimiting and recovery is the rule after a week or two. immunocompromised hosts do not clear the infection, and malabsorption may become a significant and lifethreatening problem. unfortunately, there is no known treatment for c. parvum infection, and current methods of water purification are ineffective for removal of the organism from the public water supply. g. lamblia is probably the most common enteric protozoan worldwide. though it may not cause dramatic enteric disease and has few systemic complications, giardiasis can lead to profound malabsorption and misery. only g. lamblia is known to infect humans. like other enteric protozoa, it is transmitted via the fecal-oral route and is most commonly spread through contaminated water. disease is caused by ingestion of cysts, which excyst in the proximal small intestine and release trophozoites. the trophozoites divide by binary fission and attach intimately to the intestinal epithelium via a ventral disk. the infectious dose is as low 10-100 cysts. clinical symptoms vary greatly; infection may be asymptomatic, or at the other extreme, may result in substantial abdominal discomfort, chronic diarrhea, protein-losing enteropathy, and intestinal malabsorption. g. lamblia can be diagnosed by fecal microscopy looking for either cysts or trophozoites. currently, many laboratories use commercially available kits utilizing either fluorescence microscopy with specific antibodies or enzyme immunoassays. metronidazole is the drug of choice for treatment. e. histolytica is the second leading cause of parasitic death in the world, with more than 40 000 deaths annually. it is spread through fecal contamination of food and water or by person-to-person contact. amebic cysts are the infectious agent. they may survive for weeks in an appropriate environment. following ingestion, they pass unharmed through the stomach, travel to the small intestine, and excyst to form trophozoites. the trophozoites then colonize the large bowel and either multiply or encyst, depending on local conditions. the trophozoites invade the colonic epithelium, resulting in ulceration of the mucosa and amebic dysentery. they may also spread hematogenously to the portal circulation, causing parenchymal liver damage and amebic abscesses. the onset of symptoms in amebic dysentery may be gradual, initially presenting with mucoid stools and constitutional symptoms before progressing to bloody stools, abdominal pain, and fever. amebic abscesses may develop months to years after exposure. there are two types of entamoeba: e. histolytica is pathogenic while e. dispar is a commensal. microscopic examination of the stool has been the standard technique used to diagnose amebic dysentery, but this technique cannot distinguish between the two species. in the patient with classic symptoms of amebic dysentery, this distinction may not be important. however, elisa and stool pcr techniques are now commercially available and allow specific identification of e. histolytica. once the diagnosis is made, in the united states, metronidazole is the only drug available for treatment. in invasive amebic infections metronidazole should be followed by a luminal agent such as paromomycin or iodoquinol to eliminate bowel colonization by cysts. c. cayetanensis has caused a number of outbreaks in north america associated with consumption of imported raspberries in 1996-99. cyclospora has also been associated with basil and snow peas, undercooked meat and poultry, and contaminated drinking and swimming water. in immunocompetent patients, cyclospora infection results in self-limiting diarrhea with nausea, vomiting, and abdominal pain. in immunocompromised patients there can be a chronic cycle of diarrhea with anorexia, malaise, nausea, and abdominal discomfort followed by transient remissions. infection is diagnosed through detection of oocysts in stool by direct stool microscopy and oocyst autofluorescence. the infection can be treated successfully with trimethoprim-sulfamethoxazole. t. gondii is an intracellular pathogen that invades the human host from the gastrointestinal tract and causes symptomatic or asymptomatic toxoplasmosis. the vast majority of persons infected with t. gondii are asymptomatic. however, there is a risk of reactivating infection at a later time should the individual become immunocompromised. this is especially a concern in patients with aids. there is a greater risk of this when the cd4 lymphocyte count falls below 100 cells/ml. primary maternal infection during pregnancy can be transmitted to the fetus and can result in serious sequelae and there are an estimated 400-4000 cases of congenital toxoplasmosis in the united states each year. felines of all types are the only animals in which t. gondii can complete its reproductive cycle; thus cats are a major source of infection. with regard to foods, humans may become infected from consuming undercooked contaminated meat from an infected animal or from consuming food that has been contaminated in the environment with oocysts in the soil and then not cooked (e.g., fresh produce). according to cdc, viruses account for many more cases of foodborne infection than bacterial causes. viral syndromes range from simple gastroenteritis to life-threatening hepatitis. viruses contaminate both food and water, but they do not reproduce in these media; nor do they produce toxins. several viruses, such as the noroviruses, cause large outbreaks, while others are only associated with sporadic disease. the difficulty in diagnosing viral illness has precluded the acquisition of large amounts of epidemiologic data. however, the advent of rapid tests such as enzyme immunoassays is beginning to change this and will eventually lead to a better understanding of the epidemiology and disease burden caused by the various foodborne viral pathogens. noroviruses (genus norovirus, family caliciviridae) are a group of related, single-stranded rna, nonenveloped viruses that cause acute gastroenteritis in humans. norovirus is the official genus name for the group of viruses provisionally described as 'norwalk-like viruses' (nlv). noroviruses are the principal cause of epidemic, nonbacterial gastroenteritis in the united states. mead and colleagues estimate these viruses cause 23 million infections, 50 000 hospitalizations, and 300 deaths annually. norwalk virus (now called norovirus) was first described after a large outbreak in 1972. noroviruses have been associated with many large outbreaks in cruise ships, nursing homes, banquet halls, and other institutional settings. the primary source of infection is feces-contaminated drinking water, but the virus may also be spread through food that has been stored or washed in contaminated water or handled by an infected food service worker. noroviruses are highly contagious, with fewer than 100 viral particles sufficient to cause disease, and are resistant to freezing, heating, ph extremes, and disinfection. symptoms tend to occur 48 h after exposure and consist of vomiting and diarrhea. the diarrhea is watery without red cells, leukocytes, or mucus. the disease is usually selflimiting, resolving in 1-3 days without long-term sequelae. diagnosis can be made using transmission electron microscopy to find norovirus particles in stool, vomitus, or food. serologic testing, enzyme immunoassays, and pcr techniques also establish the diagnosis. the only treatment required is to prevent dehydration. handwashing will have a significant impact on the spread of the infection. a number of other viruses have also been associated with outbreaks of acute enteritis and are suspected to be spread through the fecal-oral route. table 1 includes a list of potential foodborne viruses: rotavirus, enteric adenovirus, saporo-like viruses, coronaviruses, toroviruses, reoviruses, and the smaller-sized viruses such as caliciviruses, astroviruses, parvoviruses, and picobirnaviruses. all cause a similar acute illness with a self-limiting noninflammatory, watery diarrhea. hepatitis a is an rna virus, belonging to the family picornaviridae, with a worldwide distribution. it is spread via the fecal-oral route through contaminated food and water, and person-to-person spread. in sporadic infections, up to 50-75% of susceptible household contacts of the affected individual are infected with hepatitis a. large outbreaks have been traced to a variety of foods including contaminated water, shellfish, milk, potato salad, and fresh fruits. one of the largest outbreaks in the united states was in 2003 from green onions. symptoms develop 30 days after exposure on average, with a range of 15-50 days. the lengthy incubation period complicates tracing the source of infection. during the incubation period and the first week of acute illness, hepatitis a virus can usually be detected in stool. therefore, there is a prolonged phase when an individual is asymptomatic, but may transmit the disease to others, a significant concern in relation to food workers and foodborne transmission. an inactivated viral vaccine was licensed in 1995 and the cdc and the american academy of pediatrics have been implementing an incremental hepatitis a immunization strategy for children since then. in endemic countries, childhood infection and immunity are almost universal; childhood disease tends to be asymptomatic. in the united states, disease typically occurs after foreign travel to an endemic region. it may present with fever, jaundice, fatigue, abdominal pain, nausea, and diarrhea. diagnosis of the acute infection may be established serologically and treatment is supportive. an immune globulin may also be used for pre-or postexposure prophylaxis. hepatitis e virus was first described in 1978 after an epidemic affecting 52 000 individuals occurred in kashmir, causing 1650 cases of fulminant hepatic failure and 1560 deaths. it is a small rna virus from the caliciviridae family usually transmitted through contaminated drinking water. hepatitis e is responsible for most of the epidemics of hepatitis in the developing world and is transmitted through contaminated water. it is the major etiological agent for acute hepatitis and acute liver failure in endemic regions. it causes severe liver disease among pregnant females and patients with chronic liver disease. person-to-person spread occurs rarely, with secondary attack rates of 0.7-2.2% in household contacts of infected individuals. foodborne spread has not yet been documented. hepatitis e is endemic to india, southeast and central asia, parts of africa, and mexico. it has an incubation period of 2-9 weeks, although most people develop symptoms around 40 days postexposure. clinically, the disease is similar to hepatitis a, with constitutional symptoms followed by jaundice. most patients recover, but mortality rates of up to 3% have been reported, with pregnant women at higher risk. the diagnosis is made serologically. hepatitis e vaccines remain experimental. preventing illness in the first place is clearly the most desirable approach when dealing with food safety and foodborne illness and there are many approaches to take with regard to prevention. prevention is particularly important when it comes to individuals who are young, elderly, or have compromised immune systems, and there are a number of steps that can be undertaken to minimize the potential risk. at the outset, it is important to recognize that certain groups are at much greater risk than others. this is well illustrated in the context of listeriosis in which the likelihood of developing illness varies in relation to a variety of underlying conditions ( table 4) . for example, there is a 2584 times greater risk of a transplant patient becoming sick from listeriosis as compared to an individual under the age of 65 with no underlying medical conditions. according to the council for agricultural science and technology (cast), a majority of foodborne illnesses can be attributed to improper food-handling behaviors (tables 5 and 6). leading causal behaviors are failure to (1) hold and cool foods appropriately, (2) practice proper personal hygiene, (3) prevent cross-contamination, (4) cook to proper internal temperatures, and (5) procure food from safe sources. information related to the proper handling of food can be found at www.foodsafety.gov. risk assessment of listeria monocytogenes in ready-to-eat foods. technical report (microbiological risk assessment series; no. 5), food and agriculture organization of the united nations and the world health organization, 2004. behaviors that 80% of a national panel of food safety experts (n ¼ 28) rated as being of special importance to pregnant women and/or infants and young children, with those rated as important to both groups presented first. table 6 consumer food-handling behaviors of special importance to elderly and immune compromised individuals elderly and immune compromised individuals avoid soft cheeses, cold smoked fish, and cold deli salads l. monocytogenes avoid hot dogs and lunchmeats that have not been reheated to steaming hot or 165 f store eggs and poultry in the refrigerator salmonella enteritidis avoid raw or partially cooked eggs, foods containing raw eggs. cook eggs until both the yolk and white are firm. use a thermometer to make sure that foods containing eggs are cooked to 71.1 c (160 f) cook shellfish until the shell opens and the flesh is fully cooked; cook fish until flesh is opaque and flakes easily with a fork nlv obtain shellfish from approved sources nlv; vibrio species avoid eating raw or undercooked seafood/shellfish (clams, oysters, scallops, and mussels). cook fish and shellfish until it is opaque; fish should flake easily with a fork. when eating out, order foods that have been thoroughly cooked and make sure they are served piping hot behaviors that 80% of a national panel of food safety experts (n ¼ 28) rated as being of special importance to the elderly and/or immunocompromised individuals, with those rated as important to both groups presented first. emerging infections; enteropathogenic infections; epidemiological concepts and historical examples; global burden of infectious diseases antimicrobial resistance in nontyphoidal salmonella campylobacter jejuni infections: update on emerging issues and trends infections of the gastrointestinal tract invasive enterobacter sakazakii disease in infants food poisoning exotoxins of staphyloccocus aureus guillain-barré syndrome escherichia coli o157:h7 and other shiga toxin producing e. coli strains waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt enterohaemorrhagic escherichia coli in human medicine food-related illness and death in the united states diarrheagenic escherichia coli pathogenesis and diagnosis of shiga toxin-producing escherichia coli infections salmonella typhimurium dt104: a virulent and drug resistant pathogen bacillus cereus food poisoning and its toxins enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion the enteric toxins of clostridium perfringens fish and shellfish poisoning key: cord-321584-4bu0lps0 authors: mitchell, brett g.; russo, philip l.; kiernan, martin; curryer, cassie title: nurses' and midwives’ cleaning knowledge, attitudes and practices: an australian study date: 2020-09-30 journal: infect dis health doi: 10.1016/j.idh.2020.09.002 sha: doc_id: 321584 cord_uid: 4bu0lps0 background: as frontline providers of care, nurses and midwives play a critical role in controlling infections such as covid-19, influenza, multi-drug resistant organisms and health care associated infections. improved cleaning can reduce the incidence of infection and is cost effective but relies on healthcare personnel to correctly apply cleaning measures. as nurses and midwives have the most contact with patients and as an important first step in improving compliance, this study sought to explore nurses' and midwives’ knowledge on the role of the environment in infection prevention and control and identify challenges in maintaining clean patient environments. methods: cross-sectional online survey of 96 nurses (rn/en) and midwives (rw) employed in clinical settings (e.g. hospital, aged care, medical centre, clinic) in australia. results: nurses and midwives broadly stated that they understood the importance of cleaning. however, cleaning responsibilities varied and there was confusion regarding the application of different disinfectants when cleaning after patients with a suspected or diagnosed infection post-discharge. most would not be confident being placed in a room where a previous patient had a diagnosed infection such as multi-drug resistant organism. conclusion: greater organisational support and improving applied knowledge about infection control procedures is needed. this includes correct use of disinfectants, which disinfectant to use for various situations, and cleaning effectively following discharge of a patient with known infection. the cleanliness of shared medical equipment may also pose current risk due to lack of cleaning. as frontline providers of care, nurses and midwives play a vital role in prevention and control of infections such as covid19, influenza, multi-drug resistant organisms (mdros) and health care associated infections (hcais) more broadly. however, nurses' and midwives' compliance with infection control policies can vary between settings and individual workers [1e3] . subjective indicators such as visible dirt, personal appearance and whether a patient had been identified as being infectious, can inform nurses' decision-making regarding even basic standard precautions such as handwashing [1,3e6] . this reliance on personal judgement rather than consistent application of clinical standards for infection prevention and control could potentially lead to crosscontamination and subsequently, increase rates of infection. experience, organisational structure (including staffing ratios and training), individual knowledge, and personal accountability may also impact on compliance with optimal infection control practice and governance [7e11] . beyond individual factors, the hospital environment has been shown to be a contributing factor in the spread of hcais and mdros [12, 13] . moreover, pathogens can survive for days or months on surfaces that have not been cleaned, posing an ongoing risk for transmission [14] . consequently, there is a higher risk of a patient acquiring a pathogen from the previous room occupant [15, 16] . improved cleaning can reduce the incidence of hcais and is cost effective [12, 17] , but relies on healthcare personnel to correctly and consistently apply cleaning measures. nurses and midwives have the most contact with patients across healthcare settings. therefore, they have a critical role in infection prevention and control. as an important first step in improving compliance and precursor to further work, this study sought to explore: 1. what are enrolled nurses, registered nurses and midwives' knowledge on the role of the environment in the infection prevention control, and 2. what are the barriers and challenges for nurses and midwives to maintaining a clean patient environment? this paper reports findings from a cross-sectional, online survey of nurses and midwives employed in clinical roles. registered nurses (rn), enrolled nurses (en) and registered midwives (rm) who are currently employed in clinical settings in australia. participants were recruited via advertisements placed in written and electronic materials published by professional associations (such as the australian college of nursing and the australian nurses and midwives association), via workplace emails and newsletters, and through social media (facebook, twitter) targeting nurses and midwives. the advertisements provided broad information about the survey, and included an online link to the study information, electronic consent form, and non-identifiable survey. ten $20 gift cards were randomly allocated as a participation incentive. participants who were not registered nurses, midwives or enrolled nurses were excluded from the study, in addition to those currently unemployed or not working in clinical roles (e.g. hospital, residential aged care facility, medical centre, or clinic). the survey was open for responses between 1st december 2019 and 13th march 2020; at which time the survey was closed due to dwindling response rates. interested participants accessed the online link as provided in the study invitation. screening questions were used to exclude individuals who did not meet criteria for eligibility. eligible participants then completed an online consent form before gaining access to the survey. descriptive and exploratory analysis of survey results was performed. qualitative (free-text) responses to open-ended questions were collated and each response read individually. qualitative analysis (constant comparison, frequency counts/ranking) was used to identify and group responses into common themes. overview 132 participants accessed the online survey. of these, 28 were subsequently excluded from the survey (n z 19 not currently working in a clinical setting; n z 6 not rn, en or rm; and n z 3 did not provide consent). of the 104 eligible participants, 96 consented to participate and commenced the survey, representing our sample size. 79 participants completed the full survey. the use of ip address cross referenced against demographic information suggested there were no repeat responses from the same individual. participant demographic data is presented in table 1 . there was representation across all age groups, with diversity in the highest qualification obtained, the length of time at their current employer and the jurisdiction in which they worked. most participants worked in a hospital setting. importance of cleaning participants were asked to nominate the most important reasons for cleaning the environment in healthcare settings. seventy-four (94%) participants indicated that the main reason for cleaning was to reduce the risk of infection transmission. healthcare accreditation was found to be the least important reason for cleaning (n z 35, 44%). we asked participants to indicate who was responsible for cleaning four items, two frequently touched items (bed rails and nurse call bells) and two items of shared medical equipment (iv pole and iv pump). the majority of participants indicated that nursing/midwifery staff were responsible for cleaning the iv pole (73%, n z 58) and pump (79%, n z 62). there was less certainty about who was responsible for cleaning bed rails and nurse call bells. fortypercent (40%, n z 32) indicated it was a nursing/midwifery responsibility. ten percent of participants did not know who was responsible for cleaning shared medical equipment (iv pole and pump). participants were asked to nominate what method or product they would use to clean in various situations. results are presented in table 2 . using a likert scale, participants were asked to indicate how much they agreed with four statements relating to the use and application of disinfectant products in clinical settings (fig. 1) . while the effectiveness on patient safety was well understood, there was less certainty about disinfectants and their use. participants were shown four visual representations of how to clean a surface, using different directional movements such as circular, up and down, one-directional or sshaped (serpentine). of those that answered, 61% (n z 48) correctly identified the best way to clean a surface (i.e., answer z c, serpentine). regarding cleaning of shared medical equipment such as a blood pressure cuff, a small number reported 'probably don't clean' (14%, n z 11) this equipment. the majority (81%, n z 64) reported using wipes to clean shared medical equipment (supplementary material, table s1 ). participants were shown pictures of three patient hospital rooms (fig. 2) . room a showed a patient lying present in the bed with various equipment. room b appeared to be empty, with the bed looking slightly rumpled. room c showed a patient lying in bed and was less cluttered in appearance than room a. participants were then asked to nominate which room presented the lowest risk of infection (a, b, c, or 'don't know'). the majority chose room a, a cluttered room occupied by a patient. using a free text option, participants were asked what one item/piece of equipment they thought posed the greatest risk of infection transmission from the environment. the most common responses were hospital furniture the themes from participants around barriers to cleaning effectively were a lack of information and training, resources (cleaning products and equipment), lack of dedicated cleaning staff, and organisational structures. the free-text survey comments (supplementary material, sq1-2) stressed the need for more readily accessible information including simple wall charts with information about which product to use and where and improved labelling on wipes and cleaning agents. more education was needed about which products were recommended for patients presenting with infections such as c. difficile or multi-drug resistant organism (mdro). product useability was important, with single-use disinfectant wipes preferred, especially where staff experienced competing time pressures. a lack of policies and guidelines to inform infection control practices and lack of clear role definitions and staff accountability were also identified. in contrast to most comments, seven participants perceived that 'nothing' impacted their ability to clean equipment between patients, i.e., cleaning always occurred even when staff were pressed for time. most participants reported having received information about cleaning importance, correct product usage and availability. twenty-three percent (n z 18) had received information within last 3 months, 19% (n z 15) in the previous 3e12 months and 15% (n z 12) reported having received information in the last 1e3 years. however, 32% (n z 25) either 'do not recall' or have 'never' received any information about the importance of cleaning, product availability in their organisation, nor how to correctly apply products for infection control purposes. the majority of training received (25%, n z 20) was provided by an infection control team (table s2) . additionally, using a likert scale, participants were asked to indicate level of agreement with four statements regarding cleaning effectiveness (table s3) . despite the majority indicating confidence in their cleaning ability (usually 46%, n z 36; always 23%, n z 18), most did not feel comfortable being admitted to a room where the previous patient had a multi-drug resistant organism (never 42%, n z 33; only sometimes 34%, n z 18). it is well accepted that the clinical environment plays a role in the transmission of infections such as multi-drug resistant organisms (mdros) and healthcare associated infections (hcais) [2,7,8,12,14,15,17e20] . ineffective cleaning practices by nursing and midwifery staff may also contribute to a high pathogen-load being present within hospital settings [15, 19, 21] . as an important first step to improving environmental hygiene, this study found that nurses and midwives broadly stated that they understood the importance of cleaning, albeit, there is variation in cleaning responsibilities. moreover, cleaning of shared medical equipment may pose current risk in terms of lack of cleaning. in keeping with aiken et al. [22] , this study found that nursing and midwifery staff play a key role in cleaning duties as part of their working role. however, our findings suggest there was ambiguity about who was responsible for cleaning patient areas or certain items (such as iv pumps). there was also less certainty regarding how or when to use disinfectants and about the effectiveness of disinfectants on different groups of micro-organisms. these findings could be a result of any number of factors, including appropriateness of the product, lack of product information or of education. the implications of inappropriate product use may result in ineffective cleaning, thus increasing the risk of hcais. there are also health and safety implications for disinfectant use. in terms of the process of cleaning, 39% (n z 31) of participants did not identify the correct way to clean (wipe) a surface (i.e. s-shaped or serpentine). therefore, this finding, coupled with a lack of understanding about product (disinfectant) choice, will result in less effective cleaning and increase transmission risks. as pathogens can survive on uncleaned or inappropriately cleaned surfaces for long periods of time, it is vital that shared medical equipment is consistently and correctly cleaned to reduce the risk of hcais. genomic analyses by lee et al. [23] of vre transmission pathways within an intensive care unit identified the key role shared medical equipment has in icu. factors for suboptimal cleaning of shared equipment may include insufficient stocks of equipment to allow for cleaning and rotation between patients, lack of product at the point of use and perceived lack of time [1, 7, 19] . understanding reasons for this are important and we will be following this up in future work. survey participants called for more easily accessible information about the different types of cleaning products and what they were used for, greater accessibility to products, greater clarity around cleaning roles and who was responsible for maintaining particular items or patient areas, as well as increased accountability on the behalf of staff and hospital management. factors that may influence a decision for cleaning to take a secondary role include the perception of infection risk from the environment versus other competing patient care requirements, as well as understanding cleaning responsibilities. when we asked participants to identify which room posed the highest infection risk, the majority of participants chose the most cluttered room. this indicates good understanding among respondents that cluttered environments can hamper cleaning. however, the correct answer was 'don't know', i.e., while this room may reflect challenges undertaking cleaning, it does not necessarily relate to risk. pathogens are invisible to the naked eye and any of the rooms may pose an infection risk [15] . factors influencing risk would include the type of infection or pathogen from an unknown but colonised patient, as well as the effectiveness of cleaning. none of the provided images illustrated this. the subjectivity of choosing a room which is cluttered is consistent with other work, which found that compliance with even basic infection protocols such as handwashing and wearing gloves was individually and subjectively based [3e5] . variations in product use and cleaning practices [21, 24, 25] , information transfer and communication pathways [26e28], and organisational culture [20] , can all influence cleaning outcomes. improving staff knowledge around product use, communication, training, audit and utilising an implementation framework have been shown to improve cleaning outcomes, reduce risks for patients and are costeffective [12, 17, 29] . in our study, most participants (68%, n z 54) indicated that they had received information about the importance of cleaning, the types of products available in their organisation and product application. however, 11% (n z 9) had last received that information more than 3 years prior and a further 32% (n z 25) did not recall or had never received any cleaning information. another key theme emerging from survey comments was the lack of simple information about particular cleaning products. participants called for easy instructions to support correct product usage and application. these findings suggest the need for improved and structured education of nurses and midwives around cleaning on a regular basis, as well as improved communication. education could be provided in any number of ways, including from nurse educators, online platforms or from representatives from industry supplying products and equipment. of course, nurses are only one professional group in healthcare. shared medical equipment is also used by medical and allied health. potentially the same issues exist in these professional groups regarding knowledge of cleaning and responsibilities around who cleans equipment they use. this study is limited by the use of a cross-sectional study design and the accuracy of self-report responses provided. the vast majority of surveys were undertaken prior to the covid-19 pandemic taking hold in australia, so biases associated with this are expected to be limited. the sample size, while not large is a further limitation. nonetheless, this study provides a useful snapshot of nurses' and midwives' knowledge of infection control and cleaning processes, something that to our knowledge has not been undertaken before. we identified gaps in training and knowledge, as well as unclear responsibilities for cleaning certain objects. these findings can be used to inform workforce education and planning and hospital cleaning policies. similarly, the findings lay the foundation for future research exploring solutions to try and improve the cleaning of shared medical equipment. greater organisational support, clear policies detailing cleaning responsibility, and improving the applied knowledge and personal efficacy of nurses and midwives regarding infection prevention and control is needed. this includes the correct use of disinfectants, which disinfectant to use for various situations, and how to clean effectively following discharge of a patient with a suspected or known infection. the cleanliness of shared medical equipment may also pose current risk due to lack of cleaning. ethics approval for this study was granted by [blinded for review]. factors influencing nurses' compliance with standard precautions in order to avoid occupational exposure to microorganisms: a focus group study comprehensive systematic review of healthcare workers' perceptions of risk and use of coping strategies towards emerging respiratory infectious diseases why healthcare workers don't wash their hands: a behavioral explanation infection prevention and control: who is the judge, you or the guidelines? dirt and disgust as key drivers in nurses' infection control behaviours: an interpretative, qualitative study infection prevention as "a show": a qualitative study of nurses' infection prevention behaviours evaluation of infection prevention and control policies, procedures, and practices: an ethnographic study hospital staffing and health careeassociated infections: a systematic review of the literature resourcing hospital infection prevention and control units in australia: a discussion paper staff perceptions of the sources and control of meticillin-resistant staphylococcus aureus exploring the context for effective clinical governance in infection control an environmental cleaning bundle and health-careassociated infections in hospitals (reach): a multicentre, randomised trial beware biofilm! dry biofilms containing bacterial pathogens on multiple healthcare surfaces; a multi-centre study the role of environmental cleaning in the control of hospital-acquired infection risk of organism acquisition from prior room occupants: a systematic review and meta-analysis prior room occupancy increases risk of methicillin-resistant staphylococcus aureus acquisition cost-effectiveness of an environmental cleaning bundle for reducing healthcare-associated infections where does infection control fit into a hospital management structure? why do nurses miss infection control activities? a qualitative study hospital organisation, management, and structure for prevention of health-care-associated infection: a systematic review and expert consensus variation in hospital cleaning practice and process in australian hospitals: a structured mapping exercise nurses' reports on hospital care in five countries defining the role of the environment in the emergence and persistence of vana vancomycin-resistant enterococcus (vre) in an intensive care unit: a molecular epidemiological study cleaning the grey zones of hospitals: a prospective, crossover, interventional study variations in hospital daily cleaning practices translating infection control guidelines into practice: implementation process within a health care institution communication interventions to improve adherence to infection control precautions: a randomised crossover trial exploring performance obstacles of intensive care nurses effectiveness of a structured, framework-based approach to implementation: the researching effective approaches to cleaning in hospitals (reach) trial thank you to the participants in this survey. supplementary data to this article can be found online at https://doi.org/10.1016/j.idh.2020.09.002. bm, mk and pr designed the project. bm is the chief investigator for the project. bm and cc drafted the paper. all authors provided critical input into the paper. all authors approved the manuscript. two of the authors (bm and plr) are editorial board members with the journal. all authors were blinded to this submission in the journal's electronic editorial management system and none of the authors played any editorial role in handling this paper whatsoever. one author (mk) has paid employment from a company which sells cleaning products, as well as university appointments. no company played no any role in the design, analysis, interpretation or presentation of this paper. in kind support is provided by the higher education institutions with which the chief investigators are affiliated. plr is supported by national health and medical research council early career fellowships app1156312. not commissioned; externally peer reviewed. key: cord-328287-3qgzulgj authors: moni, mohammad ali; liò, pietro title: network-based analysis of comorbidities risk during an infection: sars and hiv case studies date: 2014-10-24 journal: bmc bioinformatics doi: 10.1186/1471-2105-15-333 sha: doc_id: 328287 cord_uid: 3qgzulgj background: infections are often associated to comorbidity that increases the risk of medical conditions which can lead to further morbidity and mortality. sars is a threat which is similar to mers virus, but the comorbidity is the key aspect to underline their different impacts. one uk doctor says "i’d rather have hiv than diabetes" as life expectancy among diabetes patients is lower than that of hiv. however, hiv has a comorbidity impact on the diabetes. results: we present a quantitative framework to compare and explore comorbidity between diseases. by using neighbourhood based benchmark and topological methods, we have built comorbidity relationships network based on the omim and our identified significant genes. then based on the gene expression, ppi and signalling pathways data, we investigate the comorbidity association of these 2 infective pathologies with other 7 diseases (heart failure, kidney disorder, breast cancer, neurodegenerative disorders, bone diseases, type 1 and type 2 diabetes). phenotypic association is measured by calculating both the relative risk as the quantified measures of comorbidity tendency of two disease pairs and the ϕ-correlation to measure the robustness of the comorbidity associations. the differential gene expression profiling strongly suggests that the response of sars affected patients seems to be mainly an innate inflammatory response and statistically dysregulates a large number of genes, pathways and ppis subnetworks in different pathologies such as chronic heart failure (21 genes), breast cancer (16 genes) and bone diseases (11 genes). hiv-1 induces comorbidities relationship with many other diseases, particularly strong correlation with the neurological, cancer, metabolic and immunological diseases. similar comorbidities risk is observed from the clinical information. moreover, sars and hiv infections dysregulate 4 genes (anxa3, gns, hist1h1c, rasa3) and 3 genes (hba1, tfrc, ghitm) respectively that affect the ageing process. it is notable that hiv and sars similarly dysregulated 11 genes and 3 pathways. only 4 significantly dysregulated genes are common between sars-cov and mers-cov, including nfkbia that is a key regulator of immune responsiveness implicated in susceptibility to infectious and inflammatory diseases. conclusions: our method presents a ripe opportunity to use data-driven approaches for advancing our current knowledge on disease mechanism and predicting disease comorbidities in a quantitative way. electronic supplementary material: the online version of this article (doi:10.1186/1471-2105-15-333) contains supplementary material, which is available to authorized users. the term "comorbidity" refers to the coexistence of multiple diseases or disorders in relation to a primary disease or disorder in an individual [1] . a comorbidity relationship between two diseases exists whenever they appear simultaneously in a patient more than chance alone [2] . it represents the co-occurrence of diseases or presence http://www.biomedcentral.com/1471-2105/ 15/333 to chronic obstructive pulmonary disease (copd) [6, 7] , obesity [8] , mental disorders [9] , immune-related diseases [10] , cancer [11] etc. comorbidity can be attributed to the disease connections on the molecular level, such as dysregulated genes, ppis (protein-protein interactions), and metabolic pathways as potential causes of comorbidity [1, 3, 12, 13] . from a genetic perspective, a pair of diseases is connected because they have both been associated with the same dysregulated genes [14, 15] , whereas from a proteomics perspective phenotypically similar diseases are related via biological modules such as ppis or molecular pathways [16, 17] . population-based disease association is important in conjunction with molecular and genetic data to uncover the molecular origins of diseases and disease comorbidities. patient medical records contain important clarification regarding the co-occurrences of diseases affecting the same patient [2] . during the last few years, several researchers have been conducted the disease comorbidity analysis to understand the origins of many diseases [1, 12, 18] . goh, cusick, valle, childs, vidal, barabasi et al. and feldman, rzhetsky, vitkup et al. built networks of gene-disease associations by connecting diseases that have been associated with the same genes [14, 15] , whereas lee, park, kay, christakis, oltvai and barabási et al. constructed a network in which two diseases are linked if metabolic reactions are associated between them [13] . disease association studies from proteomic point of view have been studied by rual, venkatesan, hao, hirozane-kishikawa, dricot, li, berriz, gibbons, dreze, ayivi-guedehoussou et al. and stelzl, worm, lalowski, haenig, brembeck, goehler, stroedicke, zenkner, schoenherr, koeppen et al. [19, 20] . rzhetsky, wajngurt, park and zheng et al. inferred the comorbidity links between 161 disorders from the disease history of 1.5 million patients [12] . however, all of these efforts have focused on the role of a single molecular or phenotypic measure to capture disease-disease relationships. in our work we have used disease-gene associations, ppis, molecular pathways and clinical information to obtain statistically significant associations and comorbidity risks among diseases. inflammation is a hallmark of many serious human infectious diseases associated to a wide variety of infections, such as hiv-1 [21] . uk doctor max pemberton says "i'd rather have hiv than diabetes" as life expectancy among diabetes patients is lower than that of hiv [22] . however, hiv has a comorbidity impact on the diabetes. also the flu can cause complications, including bacterial pneumonia, or the worsening of chronic health problems. asthma is the most common comorbidity in patients hospitalized for swine influenza (h1n1) infection [23] . dengue can cause myocardial impairment, arrhythmias and, occasionally, fulminant myocarditis [24] . chronic medical conditions, such as heart disease, lung disease, diabetes, renal disease, rheumatologic disease, dementia, and stroke are risk factors for influenza complications [25] . common chronic infections such as periodontitis or infection with helicobacter pylori may also increase stroke risk [26] . moreover, the severity of pneumonia in patients coinfected with influenza virus and bacteria is significantly higher than in those infected with bacteria alone. the incidence of flu is higher in children and younger adults than in older individuals, but influenzaassociated morbidity and mortality increase with age, especially for individuals with underlying medical conditions such as chronic cardiovascular diseases [27] . during the ageing process the immune system becomes compromised and it causes an increasing inflammation [28] . in particular, chronic inflammation (inflammageing) and metabolic function are strongly affected by the ageing process [29] . the ageing of populations is leading to an unprecedented increase different diseases like cancer and fatalities. it is reported that 80% of the elderly population has three or more chronic conditions [30] . on the other hand, respiratory viruses are an emerging threat to global health security and have led to worldwide epidemics with substantial morbidity and mortality [31] . coronaviruses (covs) cause respiratory and enteric diseases in human and other animals that induce fatal respiratory, gastrointestinal and neurological disease. severe acute respiratory syndrome (sars) is an epidemic human disease, is caused by a coronavirus (cov), called sarsassociated coronavirus (sars-cov) [32] . sars patients may present with a spectrum of disease severity ranging from flu-like symptoms and viral pneumonia to acute respiratory distress syndrome and death [33] . most of the deaths were attributed to complications related to sepsis, ards and multiorgan failure, which occurred commonly in the elderly for comorbidities [34] . age and comorbidity (e.g. diabetes mellitus, heart disease) were consistently found to be significant independent predictors of various adverse outcomes in sars [35] . children with sars have better prognosis than adults [34] . advanced age and comorbidities were significantly associated with increased risk of sars-cov related death, due to acute respiratory distress syndrome [35] . mild degree of anaemia is common in the sars infected patients and patients who have recovered from sars show symptoms of psychological trauma [34] . another novel coronavirus mers-cov, which is a new threat for public health, has similar clinical characteristics to sars-cov, but the comorbidity is the key aspect to underline their different impacts [36, 37] . mers-cov causes respiratory infections of varying severity and sometimes fatal infections in humans including kidney failure and severe acute pneumonia [38] . despite sharing some clinical similarities with sars (eg, fever, http://www.biomedcentral.com/1471-2105/15/333 cough, incubation period), there are also some important differences such as the rapid progression to respiratory failure, which we have studied on comorbidities point of view. infection with the human immunodeficiency virus-1 (hiv) and the resulting acquired immune deficiency syndrome (aids) affects cellular immune regulation [39] . hiv infection severely impacts on the immune system causing phenotypic changes in peripheral cells and dysregulates the innate immune system [40] . significant number of hiv-1 infected patients exhibits osteopenia and osteoporosis, leading to higher incidence to develop weak and fragile bones during the course of disease [41] . hiv has also been associated with an increased risk of developing both diabetes and cardiovascular disease [42] . infection with hiv weakens the immune system and reduces the body's ability to fight infections that may lead to cancer [43, 44] . people infected with human immunodeficiency virus (hiv) have a higher risk of some types of cancer (kaposi sarcoma, non-hodgkin lymphoma, cervical cancer, anal, liver, lung cancer, and hodgkin lymphoma) than uninfected people [45] . many people infected with hiv are also infected with other viruses that cause certain cancers [46, 47] . hiv infection even when controlled by highly active antiretroviral therapy (haart) is being linked to chronic inflammation [48] . people with hiv-1 infection appear to have a markedly higher rate of chronic kidney disease than the general public [49] . it is because some of the risk factors associated with hiv-1 acquisition are the same as those that lead to kidney disease because of the virus itself and some therapies (e.g. haart therapy). antiretroviral therapy for hiv may increase the risk of developing metabolic syndrome (abdominal obesity, hyperglycaemia, dyslipidaemia and hypertension) and thus predispose to type 2 diabetes and cardiovascular disease. many of the biologic factors thought to be causally associated with inflammation in hiv disease are also thought to be causally associated with the inflammation of ageing [50] . infections (acute and chronic conditions) are often associated to comorbidity that increases the risk of medical conditions which can lead to further morbidity and mortality. comorbidities related to flu have been recently investigated [51] . comorbidities for tuberculosis have also been studied recently [52, 53] . to understand the overall mechanism we have studied the comorbidity associations of sars and hiv infections. both hiv and sars are emerging infectious diseases in the modern world; each of these diseases has caused global societal and economic impact related to unexpected illnesses and deaths [54] . sras is a significant public health threat and hiv is a long term chronic infection. since these two infections are associated with high mortality rates and there are no clinically approved antiviral treatments or vaccines available for either of these infections, we have selected these two infections for our study. centred on the sars and hiv-1 infections we have investigated highly heterogeneous disease comorbidity networks using the disease-gene associations, ppi subnetwork, molecular pathways and clinical information. we have presented a systematic and quantitative approach to discover human disease comorbidities using different sources of available mrna expression, protein-protein interactions, signalling pathways, disease-gene associations, disease-disease associations and disease-drug associations data. it has been shown that sars coronavirus infects and replicates in a wide variety of host cells in susceptible animals and human beings [55, 56] . to understand the host response to this pathogen, we analysed the gene expression patterns of sars infected patients, compared to normal subjects using oligonucleotide microarrays from the ncbi geo (http://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse1739) [55] . we analysed the microarray gene expression data of over 8,700 genes from the pbmcs of 10 sars patients, and compared with healthy control samples. we found that 274 genes (p < 0.01, > 1.5 fold change) were differentially expressed as compared to healthy controls in which 120 genes were significantly up regulated and 154 genes were significantly down regulated (see additional file 1: table s1 ). on the other hand, monocytes are the key immune responsive cells whose function is adversely impacted by hiv-1. hiv-1 infection radically alters the monocyte phenotype, which is reflected in an hiv-1 induced gene expression analysis. monocyte gene expression microarray data were collected for control and hiv patients from the ncbi geo (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=gse18464) [57] . to find out the significant dysregulated genes during the hiv-1 infection, we have performed global gene expression analysis. we found that 186 genes (p < 0.01, > 1.5 fold change) were differentially expressed as compared to healthy controls in which 71 genes were up regulated and 115 genes were down regulated (see additional file 2: table s2 ). considering the significantly dysregulated genes of sars (274 genes) and hiv-1 (186 genes) infections, and gene-disease associations information, we have constructed two gene-disease associations networks (gdn), which are used to explore the shared genetic associations and disease comorbidity. starting from the bipartite graph we generated biologically relevant network projections and constructed multi-relational gene-disease network in which nodes are diseases or genes, and edges indicate association between gene and disease. this bipartite http://www.biomedcentral.com/1471-2105/15/333 graph consists of two disjoint sets of nodes, where one set corresponds to all known genetic disorders and the other set corresponds to all of our identified significant genes for sars and hiv-1 infections. the list of disorders, disease genes, and associations between them were obtained from the online mendelian inheritance in man (omim) [58] , a compendium of human disease genes and phenotypes (see details in the methods section). we classified each disorder into one of 21 disorder categories based on the physiological system affected as introduced in goh, cusick, valle, childs, vidal, barabasi et al. [14] . in the gdn, nodes represent diseases class or genes, and two disorders are connected to each other if they share at least one gene in which mutations are associated with both diseases groups (figures 1 and 2 ). the number of interlinked genes between sars infection and other diseases indicates that immunological, hematological, neurological, metabolic and dermatological diseases categories are strongly associated with the sars infection (see figure 1 and additional file 3: table s3 ). few genes are also shared between more than 2 categories of diseases i.e those disease groups are also associated through at least that genes. for an instance, the gene atm shared among sars infection, cancer and immunological diseases. therefore, cancer and immunological diseases are also interrelated through the gene atm. among all these disease classes immunological diseases class is tightly correlated with the sars infection due to the highest number of genes (12 genes) shared between them. on the other hand, the number of associated genes between hiv infection and other diseases indicates that neurological, metabolic, cancer and hematological diseases categories are strongly correlated with the hiv infection (see figure 2 and additional file 4: figure 1 the gene-disease association network centred on the sars infection is constructed based on the different categories of diseases that are connected and showed comorbidities with the sars infection through the different genes. red colour represents different categories of disorders and green colour represents different genes that are common with the other categories of disorders. the size of a disease node is proportional to the number of dysregulated genes shared between the infections/disorder groups. a link is placed between a disorder and a disease gene if mutations in that gene lead to the specific disorder. http://www.biomedcentral.com/1471-2105/15/333 figure 2 the gene-disease association network centred on the hiv infection is constructed based on the different categories of diseases that are connected and showed comorbidities with the hiv-1 infection through the different genes. red colour represents different categories of disorders and green colour represents different genes that are common with the other categories of disorders. the size of a disease node is proportional to the number of dysregulated genes shared between the infections/disorder groups. a link is placed between a disorder and a disease gene if mutations in that gene lead to the specific disorder. table s4 ). few hiv dysregulated genes are also shared between more than 2 categories of diseases such as the gene tgfb1 is shared among hiv infection, cancer and skeletal diseases. it is notable that 11 significant genes (4 upregulated and 7 downregulated) are similarly dysregulated in the both sars and hiv infections. to observe the association of sars and hiv infections with other 7 important diseases (chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, type 1 and type 2 diabetes), we have collected mrna microarray raw data associated with each disease from the gene expression omnibus (http://www.ncbi.nlm.nih.gov/geo/) accession numbers are gse9006, gse9128, gse15072, gse7158, gse8977 and gse7621 [59] . after several steps of statistical analysis we have selected the most significant over and under expressed genes for each infection and disease. we also performed cross compare analysis to find the common significant genes between each disease and sars/ hiv-1 infection. we observed that sars infection shares 21, 12, 16, 5, 11, 11, 11 and 13 genes corresponding to the chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, hiv-1 infection, type 1 and type 2 diabetes. on the other hand, hiv-1 infection shares 11, 10, 17, 9, 7, 11, 9 and 7 genes corresponding to the chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, sars infection, type 1 and type 2 diabetes. then we built disease-disease relationships network for sars and hiv-1 infection with other diseases (see figures 3 (a) and (b) and additional file 5: table s5 and additional file 6: table s6 ). since genes do not function alone and they coordinate their activities in the form of complexes or molecular pathways. therefore two diseases are potentially inter-correlated to each other if they share at least one commonly associated pathway. for this http://www.biomedcentral.com/1471-2105/15/333 reason we have used reactome pathway database [60] and selected the pathways related to these 7 diseases as well as sars and hiv-1 infections. we have observed that diseases and infections shared pathways between them as shown in figures 3 (a) and (b) and additional file 5: table s5 and additional file 6: table s6 . dysregulation in a protein subnetwork may yield the dysfunction of multiple protein subnetworks. therefore, multiple diseases may be caused by the malfunction of a protein complex. so, two diseases are potentially related to each other if they share one or more commonly associated protein subnetwork. to identify the association between diseases based on the ppi subnetwork, we used significantly associated disease protein pairs data from the hprd data base [61] . to find statistically significant associations among diseases, we built disease networks centred on the sars and hiv infections in which two diseases are comorbid if there exists one or more protein subnetwork that are associated with both diseases. the disease similarity network and the protein-protein interaction network are integrated systematically and comprehensively in a simple and compact manner to formulate the disease comorbidity for the sars and hiv-1 infections as shown in figures 4 and 5. we showed that sars and hiv infections shared ppi subnetworks with the other 7 diseases or infections similar to the gene-disease and pathway-disease associations as shown in figures 4 and 5 . based on the gene expression, protein-protein interaction and molecular pathways data, we have found that both sars and hiv-1 infections have a strong association with other 8 diseases or infections (chronic heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, hiv/sars infection, type 1 and type 2 diabetes). these diseases and infections are also strongly correlated among them. we present the correlation strength and distance between a pair of these diseases and infections in figure 6 . we show that some diseases (such as kidney disorders, breast cancer, osteoporosis and heart failure) are more associated with the sars infection (see figure 6 ). kidney disorder is also tightly connected with the hiv-1 infection. the probability of occurring comorbidities between the more tightly connected diseases is more than that of others. it is notable that the patient medical records contain important evidence regarding the co-occurrences of diseases affecting the same patient. so, we constructed a phenotypic disease comorbidity network using 32 million medical records of 13039018 patients data from medpar and analysed its structural properties to better understand the connections among diseases and infections. nodes are unique diseases and edges indicate co-morbidity of the diseases. we included edges between disease pairs for which the co-occurrence is significantly greater than the random expectation based on population prevalence of the diseases. as pointed out in [2] , the relative risk (rr ij ) overestimates relations involving rare infections and diseases, and underestimates relationships between very common disorders or infections. on the other hand, φ-correlation underestimates comorbidity between rare and frequent diseases, and discriminates associations between disorders of similar appearances. thus, we built a network by selecting only the statistically significant network edges having rr ij ≥ 20 and φ ij ≥ 0.06. figure 7 summarises the set of all comorbidity associations among all diseases expressed in the study population by constructing a phenotypic disease network (pdn). in the pdn, nodes are disease phenotypes identified by unique icd-9-cm (the international classification of diseases) disease codes, and links connect phenotypes that show significant comorbidity according to the relative risk rr ij ≥ 20 and the correlation φ ij ≥ 0.06. our phenotypic disease network consists of 336 unique diseases nodes and 1018 co-morbidity relationships. sars-associated coronavirus icd-9-cm diagnosis code is 079.82, which is under the group of "viral and chlamydial infection in conditions classified elsewhere and of unspecified site" and icd-9-cm diagnosis code 079. moreover, the icd-9-cm code 480.3 is for the pneumonia due to sars associated coronavirus. so we have considered both icd-9-cm codes 079.82 and 480.3 for our phenotypic sars comorbidity study. in our 3 digit code data we have considered 079 and for 5 digit code data we have considered 480.3. considering the relative risk rr ij ≥ 10 between the disease group 079 and other disorder categories, we have constructed the pdn as shown in figure 8 (a), and considering the relative risk rr ij ≥ 20 between the disease group 480.3 and other disorder categories, we have constructed the pdn as shown in figure 8 (b). we presented only the most significant relative risk associations (see additional file 7: table s7 and additional file 8: table s8 ). the icd-9-cm diagnosis code for the human immunodeficiency virus (hiv) infection is 042 to 044, which is under the group of "infectious and parasitic diseases" and icd-9-cm code (001-139). so we have considered both 3 digit and 5 digit icd-9-cm codes for our phenotypic comorbidity studies related to hiv infection. considering the relative risk rr ij ≥ 20 between the disease group 042 and other disorder categories, we have constructed the pdn as shown in figure 9 (a) and considering the relative risk rr ij ≥ 100 and φ-correlation φ ij ≥ 0.06 between the disease groups under the sub categories of 042 and other disorder categories, we have constructed the pdn as shown in figure 9 (b). only the most significant relative risk association is represented (see additional file 9: table s9 and additional file 10: table s10 ). to observe the trend of phenotypic relative risk corresponding to the number of shared genes between 2 http://www.biomedcentral.com/1471-2105/15/333 diseases, we have computed the number of shared genes between two diseases and their corresponding phenotypic relative risk of the occurrence of comorbidities as shown in figure 10 . we observed that with increasing number of shared biomarker genes between 2 diseases, the phenotypic relative risk is also increased. we may predict existing diseases of a patient and the prospective disease comorbidities through the identification of highly up and down dysregulated genes. so based on the available data we could predict the disease comorbidities and the level of the comorbidities using the regression model as figure 10 . it is notable that ageing is also a "disease", not a natural process, for which age-related diseases increase exponentially with chronological time. so, to understand the impact of ageing on the disease comorbidities for sars and hiv infections we have considered the ageing data from the genage database (http://genomics.senescence. info/genes/human.html) [62, 63] . after cross comparing http://www.biomedcentral.com/1471-2105/15/333 human lung epithelial cells are likely among the first targets to encounter invading severe acute respiratory syndrome-associated coronavirus (sars-cov) [32] . thus, a comprehensive evaluation of the complex epithelial signalling to sars-cov is crucial to better understand sars pathogenesis. since both of the sars-cov and mers-cov infections cause severe lung pathology we compare and contrast the genes expression level of sars-cov infection and mers-cov infection. to compare between sars-cov and mers-cov infections, and the affect on the disease comorbidities, we have performed the time series microarray data analysis for the both types of infections on lung compared to controls. we have considered gene expression microarray data from the ncbi geo (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=gse45042) [64] . from the analysis of sars-cov vs mock-infected controls (treated the same way except without the virus) we have found 215 genes are highly significant and from the analysis of mers-cov vs mock we have found 234 gens are highly significant (see details in the additional file 11: table s11 and additional file 12: table s12 ). interestingly, only 4 genes (nfkbia, egr1, ddit31 and ifit2) are common between these two infections (see figure 13 ). however, only 2 genes (nfkbia and egr1) play an important role and differentially expressed among the both infections in lung and also in sars infected pbmcs. then from the hierarchical cluster analysis of the differentially expressed genes of the lung infection by sars-cov and mers-cov, we observed distinct groups of genes that were significantly changed over time (see additional file 13: figure s1 and additional file 14: figure s2 , and additional file 11: table s11 and additional file 12: table s12 ). the log fold changes of the common 4 genes (nfkbia, egr1, ddit31 and ifit2) expression level for the infection of mers-cov and sars-cov are presented in the figures 14 and 15 . we observed that the log fold changes of nfkbia genes expression level is sharply upregulated in the both types of infections corresponding to time point. so nfkbia is an important bio-marker for the both mers-cov and sars-cov infections. it is also observed that the inflammatory genes nfkbia is a highly over expressed in the both pbmcs and lung cells for the infection of sars and also for the infection of mers in the lung cells (see figure 16 ). indeed, the immune system plays a pivotal role in the outbreak of the inflammatory state. so in case of sars infection, the nfkbia gene plays an important role for the disease comorbidities. on the other hand, similar diseases share common genes and could be treated by the same drugs [17] , which may allow us to make predictions for new uses of existing drugs. for an instance, the anti-diabetic drug metformin plays a major protective effect against cancer development and increases significantly higher survival rate of the cancer patients [65] . the finding is that the earlier the metformin regimen was initiated, the greater the http://www.biomedcentral.com/1471-2105/15/333 preventive benefit for the cancer patient. there is an evidence that the antiviral medication, ribavirin, does not work in case of sars infection [66] . to this end, we used connectivity map (cmap), which is a database of more than 1,400 drug transcriptional signatures in several cell lines [67] . this database allows to identify of molecules that induce similar or opposite transcriptional changes relative to the query signature, based on their connectivity enrichment scores. as a query signature we used our 274 highly dysregulated genes for the sars infection. we generated the connectivity score value ranges between +1 and -1, where a highly positive score indicates that the drug induces changes similar to those induced by viral infection, while a highly negative score indicates that the drug reverses the expression of the sars signature. based on the connectivity score we have selected most potential positive and negative regulators of sars viral response (see details in the additional file 15: table s13 ). potential negative regulators indicate that drugs reverse the sars signature gene expression. among the negative potential regulator, the drug molecule tetracycline, zalcitabine, gibberellic acid, prestwick-642 and sulfaquinoxaline are more potential for the mcf7 cell line and vorinostat for the hl60 cell line. based on the data demonstrate the efficacy of different drug against sars virus can be predicted effective drug treatment for the emergent viruses. furthermore, immunomodulatory drugs that reduce the excessive host inflammatory response to respiratory viruses have therapeutic benefit to reduce the sars infection as well as disease comorbidities. we presented and analysed multi-relational disease comorbidity relationships of sars and hiv-1 infections with other diseases or infections based on the associations of genetics, proteomics, molecular signalling pathways and phenotypic disorders. the combination of molecular biology, genetics and clinical medicine has greatly facilitated understanding of how different diseases relate to each other. based on the combined genetics, ppis, pathways and clinical data, our disease networks can disclose potentially novel disease relationships that have not been captured by previous individual studies. the underlying hypothesis behind this line of research is that once we catalogue all disease-related genes, ppi complex and signalling pathways, if we do not consider environmental changes, we will be able to predict the susceptibility of each individual to future diseases using various molecular biomarkers and it will help us to enter an era of predictive medicine. our results indicate that such a combination of molecular and population-level data could help to build novel hypotheses about disease mechanisms. furthermore, if two or more diseases have associated comorbidity, the occurrence of one of them in a patient may increase the likelihood of developing the other diseases. we have also studied the differences between mers-cov and sars-cov in the host response. this enables rapid assessment of viral properties and the ability to anticipate possible differences in human clinical responses to mers-cov and sars-cov and their impact on comorbidities with respect to the general comorbidities conditions. we used this information to predict potential effective drugs against sars-cov, a method that could be more generally used to identify candidate therapeutics in future disease outbreaks. these investigation approach may also help to generate hypotheses and make rapid advancements in characterising the new viruses. we also found that patients' response of sars appears to be mainly an innate inflammatory response using nfk-bia, rather than any specific immune response against a viral infection such as hiv. however, hiv infection and highly active antiretroviral therapy (haart) also increase the immune reconstitution inflammatory syndrome (iris) and inflammation through the nf-κb pathways [68] . moreover we have studied before about the impact of hiv infection on bone diseases and infection (e. g. osteoporosis and osteomyelitis). we observed that genes (e.g. rankl) and pathways (e.g. nf-κb) that are dysregulated by hiv infection also impact on the bone remodelling and bone related diseases. it is also recognised that inflammation plays a role in cancer aetiology, and various studies have found that inflammation may causes iris, obesity and tumour-promoting effects [69] . moreover, inflammation is an important concomitant cause of many major age-associated pathologies such as cancer, neurodegeneration and diabetes [70] . our study provides important evidence to associate diseases with the ageing process at the system level and helps to understand more about the comorbidities of the complex diseases. the ageing process itself is accompanied by a chronic low-grade inflammation, which is termed "inflammageing". the combination of metabolic-driven and age-driven inflammatory pathways plays a pivotal role in disease progression. this observation suggests that inflammageing and meta-inflammation can share stimuli and pathogenic mechanisms for comorbidities. we suppose that what is happening for the comorbidities we investigate is similar to what found for prions [71, 72] . similar to most infectious agents, prion causes inflammatory responses by activating innate immunity through glial cells in the brain. the complete transcriptome of the prion brain at 10 different time points is observed during the 22-week period [71, 72] . at the beginning of the disease, both normal and diseased mouse networks were the same. although the disease started in the most unique network of prion accumulation and replication it is progressed to the other networks. based on this approach we may propose a pathway model for comorbidities how hubs genes dysregulate several other pathways to influence comorbidities. the number of dysregulated pathways could be proportional to the amount of dysregulation of hub genes. our pathway model may states that the hubs that are over turned on, may direct the signal to the different pathways creating comorbidities as shown in figure 17 . for the infection, one of the pathways related to the inflammation starts dysregulation. with increasing time, both confidence level of inflammation and the number of dysregulated pathways are increased. moreover, with the increasing of inflammation the number of diseases for the comorbidities may increase. so initially infection dysregulates one signalling pathway of any cells and that causes other pathways may be dysrupted. in this way disrupting pathways increase more diseases in the same patient and make multimorbidity. disease genes play a central role in the human interactomes. overlapping component genes serve as bridges across the relatively independent functional modules or pathways. so perturbation in one pathway, such as the nf-κb signalling pathway, could be propagated throughout the other relevant pathways. we found sars and hiv-1 infections share 11 significantly dysregulated genes as well as molecular pathways. both sars and hiv-1 viruses may infect and find an already existing comorbidity or generate a new comorbidity through the perturbation of the infected pathways. furthermore, it may provide us an opportunity to investigate the role of other http://www.biomedcentral.com/1471-2105/15/333 genes from the same pathway in the disease space. therefore, pathways could be used to represent the underlying biology of diseases and make prediction of disease comorbidities. in most of the cases, the correlativeness among genes, pathways and diseases are many-to-many, e.g. a disease is associated to many different genes and pathways; and a pathway is associated to many different diseases. this study suggests that a single pathway can be involved in many diseases whereas a disease may have dysregulation in many biological processes. hence, if a drug is already available to treat a disease through modulating the activity of a pathway, then it could potentially be used to treat other diseases that are strongly linked with the same pathway. on the other hand, when a disease shows dysregulation in multiple pathways, a pathwayguided combined drug may be employed in the treatment. moreover, the protein subnetwork-based approach to diseases may aid in drug discovery, in fact it can potentially be used to treat other diseases that are linked to the same protein complex. thus, our findings not only potentially help us to understand how different diseases are related based on their underlying molecular mechanisms but also provide insights into the design of novel, protein complex-guided therapeutic interventions for diseases. extending the concept of subclassifying patient cohorts to the single patient level refers to as personalised medicine. during the last few years, acceptance level of the personalised medicine is sharply increased as it has been apparent that standard treatment approaches are rarely efficient across the entire patient population. advances in highthroughput molecular assay technologies in the fields of genomics, proteomics and other "omics" is increasing the diagnostic and therapeutic strategies for personalised treatment. as a result, declining per-sample cost has given rise to numerous public repositories of biomolecular data. in particular, the availability of these data sets for many different diseases presents a ripe opportunity to use data-driven approaches to advance our current knowledge of disease relationships in a systematic way. the identified disease patterns can then be further investigated with regards to their diagnostic utility or help in predicting novel therapeutic targets. medicine will focus on each individual patient. it will become intrinsically proactive and will increasingly focus on wellness rather than disease. proactive and personalised medicine will bring fundamental changes to healthcare, taking carefully http://www.biomedcentral.com/1471-2105/15/333 targeted preventative or therapeutic action at the earliest indications of risk or disease comorbidities. we are entering into the genomic era of medicine, where a patient's genetic/genomic data is becoming important for clinical decision making, including disease risk assessment, disease diagnosis and subtyping, drug therapy and dose selection, risk assessment for adverse drug reaction, and family planning [73] . today multi-scale and complex biomedical data are gathered and analysed to uncover combinations of predictive disease profiles. our genome, as well as multiple proteomes, multiple transcriptomes, multiple metabolomes, and other personalised data sets obtained at different points in our lives, will be readily available at affordable prices for each individual. in the near future, clinicians will have to consider genetic/genomic implications to patient care throughout their clinical workflow, including electronic prescribing of medications. therefore, for the implementation of the personalised medicine system, a model could be developed that will take individual genetic data. dysregulated biomarker genes will be identified from this genetic data and disease will be identified from the gene-disease association database. based on the information of the existing disease, the model will predict disease comorbidities using the disease-disease associations database. this will provide us to detect many diseases at the earliest detectable phase, even weeks, months, or maybe years before the symptoms appear and it will afford crucial insights into optimizing of our wellness. thus, personalised medicine will give fundamental new insights into disease mechanisms, and hence will open new opportunities for diagnosis, therapy and prevention from the disease comorbidities. in this study, we have considered all available categories of omics and phenotypic data to quantify the sars and hiv-1 infections centred comorbidity associations. we have shown that the phenotype disease network (pdn) has a heterogeneous structure where some diseases are highly connected while others are hardly connected at all. our findings showed that disease progression can be represented and studied using network methods, offering the potential to enhance our understanding of the origin and evolution of human diseases. detecting comorbidity in a large population is of clinical interest due to the fact that it may reveal new information useful for cause of diseases as well as for new treatment strategies. this study demonstrates the value of an integrated approach in revealing disease relationships and new opportunities for therapeutic applications. so we can say that this kind of approach will be helpful for making evidence-based recommendations about disease comorbidities. moreover, considering environmental factors (such as physiological stress, diet), ethnic group and gender discriminations are important factors in the comorbidity analysis. our network approach could be extended as a comorbidity map by integrating diet, exercise and other factors as in [74] . the gene-disease associations data used in this study were collected from the online mendelian inheritance in man (omim) database (http://www.ncbi.nlm.nih.gov/ omim/). this omim database is the best-curated repository of all known disease genes and their associated http://www.biomedcentral.com/1471-2105/15/333 figure 17 progressive temporal activation of pathways. a schematic view of networks becoming disease comorbidities increased for the perturbation of the pathways dysregulation advances with time. the red circles indicate increased levels of dysregulated gene expression relative to control and the red linked indicate dysregulated pathways that have been increased from infection as compared with normal control. the green indicated transcripts that are the same in control and infection condition. the four panels represent the network with time intervals of the infection progression. with time the inflammation confidence level is increased which is indicated by confidence interval. disorders [75, 76] . genotype-phenotype relationships, as summarised in the omim database, contained more than 5000 human disease-genes associations involving 1500 diseases and 3000 disease associated genes. each entry of the omim is composed of four fields, the name of the disorder, the associated gene symbols, its corresponding omim id, and the chromosomal location. we selected the entries with the "(3)" tag, for which there is strong evidence that at least one mutation is cause of the disorder. omim initially focused on monogenic disorders but in recent years has expanded to include complex traits and the associated genetic mutations that confer susceptibility to these common disorders [58] . subsequently we classified each disorder into 21 primary disorder classes based on the physiological system affected as introduced in goh, cusick, valle, childs, vidal and barabasi et al. [14] . disorders having distinct multiple clinical features are assigned to the "multiple" class. this classification scheme reflects the phenotypic similarities among diseases in the same class and has been successfully used in the recent studies of systematic disease analysis [77] . the gene expression data used in this study was obtained from the ncbi gene expression omnibus (geo) (http://www.ncbi.nlm.nih.gov/geo/) [59] . we have considered 10 different data sets for our analysis (accession numbers are gse1739, gse45042, gse17400, gse9006, gse9128, gse15072, gse7158, gse8977, gse18464 and gse7621) [32, 55, 57, 64, [78] [79] [80] [81] [82] . these data sets contain data from the patients of different age and sex. after several rounds of filtering, normalization and statistical analysis, we had microarrays representing sars, mers, hiv-1 infections and 7 other human diseases (heart failure, kidney disorders, breast cancer, parkinson, osteoporosis, type 1 and type 2 diabetes). http://www.biomedcentral.com/1471-2105/15/333 the protein-protein interaction (ppi) data for human was obtained from the human protein reference database (hprd) [61] . hprd contains the maximum number of ppi data among all publicly available literature-derived databases for human ppi [83] . we have used the reactome knowledge base of human biological pathways database for our pathways association analysis [60] . for the cross compare analysis between the sars and hiv infections, and ageing process we have download ageing data from the human ageing genomic resources (http://genomics.senescence.info/ download.html) [62, 63] . they have collected human ageing genes after an extensive review of the literature. these genes are commonly dysregulated during the ageing process. to test the validity of the proposed disease associations, we examined the disease co-occurrence information at the population level. we obtained statistically significant pairwise comorbidity associations reconstructed from over 32 million medical records in the us medicare claims database recorded in the icd-9-cm format (http://www. icd9data.com), which are frequently used for epidemiological and demographic studies and collected from [2] . we used medpar records from 1990 to 1993, where the dates and reasons for all hospitalizations were reported in icd-9-cm format and it contains the diagnoses of 13039018 elderly patients. each record consists of the date of visit, a primary diagnosis and up to 9 secondary diagnosis. all diagnoses are specified by icd9 codes of up to 5 digits. the first three digits specify the main disease category while the last two are used to give additional information about the disease. in total, the icd-9-cm classification consists of 657 different categories at the 3 digit level and 16,459 categories at the 5 digit level [2] . to determine whether some existing drug compounds can reverse the sars infection signature, we used the publicly available connectivity map (cmap) database [67] . cmap provides associations among genes, chemicals and disease or infection conditions. it is a collection of genome-wide transcriptional data from cultured human cells treated with 1,400 different compounds. the method of global gene expression analysis using oligonucleotide microarrays has proven to be a sensitive method to develop and refine the molecular determinants of human disorders [55] . using this technology, we compared the gene expression profiles of sars, hiv and other diseases. to avoid the problems of comparing microarray data of different platforms and experimental systems, we normalized the gene expression data in each microarray sample (disease state or control) using the z-score transformation (z ij ) for each disease gene expression matrix using z ij = where sd is the standard deviation, g ij represents the expression value of gene i in sample j. this transformation allows for the direct comparison of gene expression values across various microarray samples and diseases. to combined more than one data series or experiments for a given disease, we employed a linear regression approach to obtain a combined t-test statistic between two conditions. data were log 2 -transformed and we calculated expression level for each gene using a linear regression model : where y i is the gene expression value and x i is a disease state (disease or control). the coefficients β 0 and β 1 are the parameters of this model and were estimated by least squares. the t-test statistic, when estimating the value of β 1 , is the same as the standard t-test statistic between disease and control states. time series microarray gene expression data analysis was divide into two steps: pre-processing and identification of statistically significant points by t-test, anova and regression analysis to find differently expressed gene profiles in different time points. in the first step, we preprocessed the experimental data using different statistical methods and finally followed by post less normalization, recommended by the golden spike project [84] . in the second step, we have used a most suitable method "masigpro" (microarray significant profiles) to identify differentially expressed genes in time-course microarray experiments, which is a two step regression method successfully applied on more than one groups of time-series [85, 86] . this two steps regression strategy is used to find genes with significant temporal expression changes and significant differences between experimental groups. this procedure first adjusts this global model by the leastsquared technique to identify differentially expressed genes and selects significant genes applying false discovery rate control procedures. then stepwise regression is applied as a variable selection strategy to study differences between experimental groups and statistically significant profiles. after finding differentially gene expression profiles among the group of experiments, the next step is to cluster them according to their profile similarities. the hierarchical clustering and the median gene expression profiles of clusters are performed according to the "masigpro" package in r [85] . student's unpaired t-test was performed to identify genes that were differentially expressed in patients over normal samples and significant genes were selected. a threshold of at least 1.5 fold change and a p value for the t-tests of less than 0.01 were chosen. in addition, a two-way anova with bonferroni's post-hoc test was used to establish statistical significance between groups (< 0.01). pathways and functional categories were considered as over-represented when fisher's exact test p value was < 0.01. for presenting the signalling and http://www.biomedcentral.com/1471-2105/15/333 interaction pathways of the different significant genes, we used cytoscape for data integration and network visualization [87, 88] and reactome functional interaction (fi) cytoscape plugin for knowledge base of human biological pathways and network processes [60] . for the gene disease association, we have considered the neighbourhood based benchmark and topological methods, which are better suited to our networks [89] . in this case, topological refers to methods that rely only on the structure of the network to draw conclusions. we construct a gdn from gene-disease associations where the node in the network can be either a disease or a gene. this network can also be regarded as a bipartite graph. diseases are connected when the diseases share at least one significant dysregulated genes. let a particular set of human diseases d and a set of human genes g, genedisease associations attempt to find whether gene g ∈ g is associated with disease d ∈ d. if g i and g j , the sets of significant up and down dysregulated genes associated with diseases i and j respectively, then the number of shared dysregulated genes (n g ij ) associated with both diseases i and j is as follows: the co-occurrence refers to the number of shared genes in the gdn. the common neighbours is the based on the jaccard coefficient method, where the edge prediction score for the node pair is as: where e is the set of all edges. the number of shared pathways and protein subnetwork that links between diseases i and j are calculated using the equation 1 and the link prediction score is measured using the equation 2. to estimate the correlation starting from disease cooccurrence, we need to quantify the strength of disease association for comorbidities by dipicting a distance between two diseases. for the analysis of the phenotypic data, we used the relative risk (rr ij ) as the quantified measures of comorbidity tendency of two disease pairs and checked φ-correlation (φ ij ) to measure the robustness of the comorbidity associations. the rr ij is observing in a pair of diseases i and j affecting the same patient. when two diseases co-occur more frequently than expected by chance, we will get rr ij > 1 and φ ij > 0. however, rr ij and φ ij are not independent of each other and each carries unique biases that are complementary [1, 2] . so, we used both measures of comorbidity to ensure the robustness of our investigations. the rr ij allows us to quantify the co-occurrence of disease pairs compared with the random expectation which is calculated as: (3) where n is the total number of patients in the population, p i is the incidences/prevalences of disease i, p j is the incidence of disease j and c ij is the number of patients that have been diagnosed with both diseases i and j. for rr ij >= 1 comorbidity is larger than expected by chance and for rr ij < 1 comorbidity is smaller than expected by chance. to calculate the significance of the relative risk rr ij , we used the katz, baptista, azen and pike et al. method to estimate confidence intervals [90] . according to their estimation, the 99% confidence interval for the rr ij between two diseases i and j is calculated by: lower bounds of the confidence interval (lb) = rr ij * exp(−2.56 * σ ij ) and upper bounds of the confidence interval (ub) = rr ij * exp(2.56 * σ ij ), where σ ij is given by: disease pairs within the 99% confidence interval are only considered if the lb value is larger than 1 when rr ij is larger than 1, or if the ub value is smaller than 1 when rr ij is smaller than 1. relative risk measure is intrinsically biased towards overestimation of relationships between rare diseases and underestimates the co-morbidity of more frequent diseases [2] . this bias can be reduced by introduction of a φ-correlation measure. we can quantify the strength of comorbidities by calculating the correlation coefficient associated with a pair of diseases i and j as: where c ij is the number of patients affected by both diseases, n is the total number of patients in the studied population, and p i and p j are the morbidity or incidence of the i th and j th diseases respectively. the φ-correlation is the pearson's correlation for the variables which only take 0 or 1 values [91] . for φ ij > 0 comorbidity is larger than expected by chance and for φ ij < 0 comorbidity is smaller than expected by chance. we can determine the significance of φ = 0 by performing a t-test. this consists of calculating t according to the formula: t = φ √ n−2 √ 1−φ 2 , where n is the number of observations used to calculate φ. to predict the comorbidities considering the primary or index disease we have calculated the conditional relative risk (conditional rr ij ) as follows: for all possible disease pairs i and j, for the cases that one index disease (i) is present (k = true) or absent (k = false). (p = 0.01). we have weighted the edges using a mutual information metric which quantifies how much greater the edge relationship is with respect to co-occurrence. the mutual information weight between two diseases i and j is defined as w ij = c ij p i + p j − c ij (6) where c ij is the observed co-occurrence and p i and p j are the morbidity or prevalence of the i th and j th diseases respectively. to compare between sars-cov and mers-cov, a gene set enrichment analysis was undertaken using gsea [92] . to find out the correlation (similarities) and distance (dissimilarities) among the diseases from the integrated analysis of multidimensional data (gene expression and protein protein interaction), we have applied euclidian distance measurement and metric multi-dimensional scaling (mds) using majorization [93] . mds is a set of methods for discovering hidden structures in multidimensional data. based on a proximity matrix derived from variables measured on objects as input entity, these distances are mapped on a lower dimensional spatial representation. optimization problem is used to find mapping in target dimension of the data based on given pairwise proximity information while minimize the objective function. the particular objective function (or loss function) we used in this work is a sum of squares, commonly called stress. we used majorization to minimize stress and this mds solving strategy is known as smacof (scaling by majorizing a complicated function). stress majorization is an optimization strategy used in multidimensional scaling (mds) where, for a set of nm-dimensional data items, a configuration x of n points in r(<< m)-dimensional space is sought that minimizes the stress function σ (x). here r is 2 that means the (r × n) matrix x lists points in 2-dimensional euclidean space. we have applied the cost function σ to measures the squared differences between ideal (m-dimensional) distances and actual distances in r-dimensional space as follows: x 1 of dimension n 1 × p as the individual's or judge's configuration, and x 2 of dimension n 2 × p as the object's configuration matrix. the least squares metric multidimensional scaling or mds problem is the minimization of σ and over all m × p configurations x. here w ij are given non-negative weights and d ij are given non-negative dissimilarities. the d ij (x) are the euclidean distances between rows i and j of x. thus d ij (x1, x2) = p s=1 x 1is − x 2js 2 (8) where w ij ≥ 0 is a weight for the measurement between a pair of points (i, j), d ij (x) is the euclidean distance between i and j, and δ ij is the ideal distance between the points (their separation) in the m-dimensional data space. note that w ij is used to specify a degree of confidence in the similarity between points (e.g. 0 can be specified if there is no information for a particular pair). a configuration x which minimizes σ (x) gives a plot in which points that are close together correspond to points that are also close together in the original m-dimensional data space. programming scripts are freely available at www.cl.cam. ac.uk/~mam211/comor/. http://www.biomedcentral.com/1471-2105/15/333 information regarding each of the clusters and genes is described in additional file 12: table s12 . additional file 15: table s13 . connectivity map results of predicted drugs per instance (for each drug and cells line) to reverse sars-cov for early and sustained signature (drugs with negative enrichment scores). the impact of cellular networks on disease comorbidity a dynamic network approach for the study of human phenotypes comor: a software for disease comorbidity risk assessment comorbidity of cardiovascular disease, diabetes and chronic kidney disease in australia. canberra: australian institute of health and welfare mortality after incident cancer in people with and without type 2 diabetes impact of metformin on survival comorbidities in chronic obstructive pulmonary disease comorbidities of chronic 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and phenotype networks obtaining confidence intervals for the risk ratio in cohort studies applied multiple regression/correlation analysis for the behavioral sciences. routledge gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles a majorization algorithm for solving mds submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank fp7-health-f5-2012 for providing financial support, under grant agreement n 305280 (mimomics). additional file 1: table s1 . highly statistical significantly differential expressed genes between sars and control group in pbmcs. table s2 . highly statistical significantly differential expressed genes between hiv and control group. table s7 . phenotypic disease association for sars infection based on the icd9 codes at the 3-digit category level. only statistically significant links with high relative risk rr ij are considered. additional file 8: table s8 . phenotypic disease association for sars infection based on the icd9 codes at the 5-digit category level. only statistically significant links with high relative risk rr ij are considered.additional file 9: table s9 . phenotypic disease association for hiv infection based on the icd9 codes at the 3-digit category level. only statistically significant links with high relative risk rr ij are considered. additional file 10: table s10 . phenotypic disease association for hiv infection based on the icd9 codes at the 5-digit category level. only statistically significant links with high relative risk rr ij are considered.additional file 11: table s11 . highly statistical significant differentially expressed genes between sars-cov and reference group (mock) in lung epithelial cells.additional file 12: table s12 . highly statistical significant differentially expressed genes between mers-cov and reference group (mock) in lung epithelial cells.additional file 13: figure s1 . median expression profile of sars-cov vs mock using hierarchical clustering (ward method, pearson correlation) of 215 statistical significantly differential expressed genes (p < 0.001). the information regarding each of the clusters and genes is described in additional file 11: table s11 .additional file 14: figure s2 . median expression profile of mers-cov vs mock using hierarchical clustering (ward method, pearson correlation) of 234 statistical significantly differential expressed genes (p < 0.001). the the authors declare that they have no competing interests.authors' contributions mam and pl designed and mam implemented the analysis of the paper. mam and pl wrote the manuscript. both authors contributed to and approved the manuscript. key: cord-355872-z6vsjmxn authors: colón-lópez, daisy d.; stefan, christopher p.; koehler, jeffrey w. title: emerging viral infections date: 2019-08-15 journal: genomic and precision medicine doi: 10.1016/b978-0-12-801496-7.00010-1 sha: doc_id: 355872 cord_uid: z6vsjmxn the emergence of viral infections is driven by multiple factors including changes in human behavior, population growth, reservoir host distribution, viral diversity and environmental changes. effective surveillance methods, diagnostic assays and containment measures are pivotal to preventing widespread outbreak of a new viral infection. however, the limited understanding of some emerging viruses poses numerous challenges for effective intervention. in this chapter we discuss various genomics-based methods and strategies to overcome these inherent challenges of emerging and re-emerging viral infections with a focus on current viral threats. we also provide an outlook on the use of genomic tools in personalized medicine and potential solutions to current and foreseeable challenges. the mastomys natalensis rat, are increased in west africa during the dry seasons when the rats are more likely to be inside individuals' houses [21] . fluctuations in climate resulting in increasing crop yields, and the associated rat population, followed by a severe drought can lead to increased cases of lassa fever. furthermore, climactic changes are altering vector-borne infectious disease risks due to expanded vector ranges. for example, there is an increased risk of lyme disease in canada due to tick range expansion [22] , and climate change is expanding the mosquito range, and the associated infectious disease risk, of aedes albopictus in the united states [23] . rna viruses represent a significant source of viral outbreaks due partly to the higher mutation rate and error prone nature of rna replication itself [24] . newly introduced mutations into a viral genome can confer new pathogenic characteristics such as enabling transmission to new species or increased virulence. for instance, mutations within the severe acute respiratory syndrome coronavirus (sars-cov) genome allowed transmission from bats to humans [24, 25] . the better understanding about the viral agents responsible of causing outbreaks in humans and the underlying factors driving emergence, the better the chances of preparedness and effective intervention for prevention of a new global epidemic ( figs. 1 and 2) . this chapter provides an overview of the genomics and precision medicine methodologies relevant to the topic of emerging and re-emerging viral threats. the chapter is divided up into two main sections: viral genomics and host genomics, and include examples of their applications in pathogen detection, population susceptibility, diagnostics, and outbreak response. the seminal investigations on the transmission of yellow fever virus by walter reed and colleagues in 1901 [27, 28] ushered in an era of discovery and classification of a wide variety of viral pathogens including smallpox virus, poliovirus, and measles virus. technological advancements including viral cell culture, plaque assays, enzyme-linked immunosorbent assays, and monoclonal antibodies pushed human virology forward. a second wave of viral discovery and characterization is currently ongoing, harvesting the power of next-generation sequencing to identify unknown viruses in acute febrile illness patients [29] [30] [31] . increasingly, efforts are underway to identify the next emerging pathogen by sequencing and identifying the viruses circulating at the human/animal/pathogen interface [32, 33] . while detection of viruses in wildlife does not predict transmission, identifying mutations or gene acquisitions through surveillance and continued discovery will provide valuable insight in the future. a major hindrance for precision medicine toward emerging zoonotic pathogens is the limited global characterization of potential threats. taxonomic placement of viruses is problematic due to the lack of universal genes, such as the bacterial 16s rna gene, limiting classification to viruses with defined biological characteristics [34] . in fact a mere 5000 total species are currently classified by the international committee on taxonomy of viruses (ictv) which pales in comparison to the estimated 320,000 viral species infecting mammals alone [35] . metagenomic sequencing has highlighted this significant gap in classified vs unknown viromes, and while biological characterization will not also always be defined, knowledge can be gained from genomic sequences including evolutionary relationships and genome structure [34] . continuing to gather viral genomic information from environmental, plant, and animal samples will continue to fill gaps in our knowledge base and further prepare us for detecting and predicting emerging pathogens. metagenomic sequencing has been used for the discovery of previously unclassified human pathogens. in 2008 lujo virus was discovered in south africa sequence diversity of lassa virus complicates molecular diagnostic tool development. a phylogenetic tree (a) was generated using and alignment of full length and near full length lassa virus s segment nucleic acid sequences available in genbank. diversity of lassa virus is linked to geographic location as shown here for viruses sequenced from nigeria, sierra leone, guinea, mali, and togo. selected sequences from each country [starred in (a)] were aligned in (b). the forward and reverse primers (red arrows) and the probe (green arrow) from a previously published lassa virus josiah real-time rt-pcr assay [26] are indicated. the primers and probe are exact matches to the lassa virus josiah sequence; however, contain mismatches within other lassa virus isolates, including ones found in sierra leone. [31] . five patients with undiagnosed hemorrhagic fever were discovered after air transfer of a critically ill index case. the disease resulted in a case fatality rate of 80%. unbiased pyrosequencing of rna extracts from human samples identified approximately 50% of an arenavirus genome which was thereafter gap filled using primers designed off the sequenced genome. phylogenetic analysis confirmed the identification of a novel old world arenavirus [31] . similarly, deep sequencing identified bas-congo virus, of a novel rhabdovirus, in three human cases in the democratic republic of congo [30] . this virus was associated with high fever and rapid death and was found to have only 34% amino-acid identity with other rhabdoviruses. the discovery and characterization of these viruses has allowed novel diagnostics to be developed, including real-time pcr, leading to a better preparedness for future outbreaks. sequencing human samples from outbreaks has resulted in the discovery of several viruses; however, 70% of emerging human pathogens result from wildlife [36] . analysis of samples from non-human hosts including arthropods, bats, rodents, and domestic animals offers potential insight into the transmission, evolution, and treatment of these pathogens [37] . deep sequencing on more than 220 invertebrate species resulted in the discovery of 1445 phylogenetically distinct viromes filling in phylogenetic and evolutionary gaps [38] . characterization of bacterial and viral relationships in mosquito arthropods demonstrated a symbiotic relationship between the bacterium and host, limiting dengue virus infection and potentially revealing new antiviral strategies [39, 40] . the discovery of middle-east respiratory syndrome-coronavirus (mers-cov) in camels demonstrated these animals as a potential reservoir and host for transmission [41, 42] . while detection of viruses in wildlife does not predict transmission, identifying mutations or gene acquisitions through surveillance and continued discovery will provide valuable insight in the future. in addition to pathogen discovery, metagenomic sequencing is increasingly used to monitor viral genomic changes as an outbreak is occurring [43] [44] [45] [46] . the ebola virus outbreak in west africa resulted in 26,648 cases and 11,017 documented deaths, and genomic sequencing was applied in near real-time to provide information to aid in containing the outbreak [44, 45] . sequencing results early in the outbreak provided valuable insight into origination and transmission routes demonstrating the outbreak started from a single introduction in guinea in december 2013 and was sustained by human to human transmissions [2] . sequencing provided molecular evidence that ebola virus was transmissible by sexual intercourse leading to changes in cdc recommendations for survivors [45, 47] and establishing programs to support national testing of semen and other body fluids in male survivors [48] . rapid outbreak sequencing allowed for the identification of transmission chains in sporadic clusters following the outbreak [46] , further adding insight to end the epidemic. sequencing data from sierra leone early in the outbreak also found increased ebola virus diversity that could have an impact on sequence-based therapeutics, vaccines, and diagnostic assays being fielded at the time [44] . having such rapid sequencing information during an outbreak would inform responders about the potential efficacy of diagnostics and sequence-based countermeasures, either reassuring decision makers or informing them of the need to modify strategies based on the findings. while sequencing has found a niche in the discovery and epidemiologic tracing of emerging infections, its roll for diagnostics is still in its infancy. emerging pathogens often occur in austere environments or countries with limited infrastructure not conducive to sequencing technologies. sequencing still has significant hurdles in decreasing the technical and mechanical requirements for routine clinical use. however, next-generation sequencing offers limitless potential due the necessity for little to no prior knowledge in sample composition. newer technologies such as nanopore sequencing are looking to minimize these hurdles by creating smaller foot-prints and near real-time sequence analysis with minimal sample preparation [49] ; however, difficult paths to their acceptance beyond laboratory derived tests to full regulatory approval remain. knowledge of viral genomic sequences in designing rapid point of care molecular diagnostics such as pcr is invaluable. the last decade has seen a significant increase in the use of pcr as a primary diagnostic due to its speed, sensitivity, and cost. however, due to the lack of available genomic sequences and high genetic variation within certain viral species, finding a conserved target can be difficult. the advent of multiplex pcr has allowed more targets to be captured in a single assay, lowering costs and increasing throughput. for example, this technique has been used to subtype influenza a and b viruses [50] . other diagnostic devices such as the biofire filmarray can run multiple pcrs at once on a single device allowing the user to select between different panels [51] [52] [53] [54] [55] . whether singleplex or multiplex, pcr remains the most rapid and sensitive method for the detection of viral genomes directly from clinical matrices. coordinating with detection, viral genome sequences are increasingly being used to predict and guide antiviral therapy. during the ebola virus outbreak, sequence analysis of the viral genome over time demonstrated changes which could make the pathogen resistant to therapeutics such as sirnas, phosphorodiamidate morpholino oligomers (pmos), and antibodies [56] . the discovery of the crispr/cas9 system and its ability to destroy dsdna has brought to light its potential in mutating essential sites or removing proviral dna from infected cells during and hiv infection crispr [57] . similarly studies involving herpesviruses, large dsdna viruses, have demonstrated the ability to destroy latent herpesvirus from infected cells [58, 59] . these methods require prior knowledge of the viral genome promising a future where antiviral therapies are targeted specifically toward an individual's own specific infection. emerging and re-emerging viral diseases pose unique challenges to the use of omics and precision medicine tools. how does one predict when and where a pathogen will jump a species barrier or emerge into a new population that could rapidly spread into new geographic regions? to further complicate the scenario, a new human viral infection may not present with obvious signs of an infection. such a virus may get introduced and transmitted across populations while remaining asymptomatic or unrecognized for long periods of times, like the hepatitis c virus (hcv) and hiv [10] , for instance. viruses rely on their small-sized genomes to encode enough information to hijack the host's cellular machinery for target-cell recognition and entry, genome replication, protein synthesis, viral participle assembly, and propagation. evolutionarily, hosts evolved conserved mechanisms to generate an immune response to detect and limit an infection and to prevent re-infection in the future. the early innate immune response functions to slow the infection once the immune system recognizes there is an infection (extensively reviewed in [60] ) and guides the subsequent adaptive immune response to the pathogen. successful control of these immunological processes depend on tight control of the host's immunological and inflammatory pathways by regulating gene transcription. these processes result in measurable changes in the relative expression levels of coding and non-coding rna and protein, even when an infection is asymptomatic [61] [62] [63] [64] [65] or difficult to diagnose [66] . the development of low-cost and less-time consuming genomics had facilitated the study of pathway-specific transcript alterations during viral infections. consequently, various transcriptomic-based assays have been developed to study, characterize, and identify signs of infection using transcript signatures. for example, zaas and colleagues identified a unique immunological gene expression profile capable of differentiating viral from bacterial respiratory infections in humans [64, 65] , laying to foundations for appropriate antibiotic use without direct pathogen identification. traditionally, diagnosing viral infections is made based on clinical symptoms, pcr and serological testing, and virus isolation. while the world of omics is expanding with new and improved platforms, host transcriptome-based assays are becoming more feasible and widely available. those of more relevance to this chapter are targeted and agnostic next-generation sequencing (ngs) platforms for genome-wide association studies (gwas) and transcriptomics as well as microarray-based assays for amplification-independent profiling of transcripts. gwas have direct applications to precision medicine whereas host-based transcriptomic assays have shown to have multiple applications for clinical diagnosis, and pathogen identification and characterization. these technologies were not specifically developed for emerging or re-emerging viruses but can be leveraged for these purposed based on their applications. gwas may provide insights into the genetic factors driving population susceptibility to viral infections [67, 68] . this information can be utilized to identify populations within geographic regions at higher or lower risk of being infected with a new pathogen. higher-risk populations may include those with increased direct exposure to animal reservoirs of zoonotic pathogens or their arthropod vectors. other population subgroups without defined topographical relationships can be determined as well. a few examples of these are demographic, age and, gender groups that can be classified based on genetic polymorphisms and other susceptibility markers. in this setting, gwas can provide guidance in outbreak preparedness and intervention. the information provided by gwas-based studies is not focused on the directed response to an infection but on the information already stored within the host's genome. on the other hand, transcriptomic approaches identifies the interactions between the pathogen and the host's genome by evaluating transcript levels, typically mrnas. the most commonly used transcriptomic methods are rna-seq [69, 70] and microarrays [65, 66] . rna-seq is generally suitable for unbiased transcriptomic profiling and provides better understanding of global transcriptomic changes. this agnostic method is appropriate for identifying changes in the human transcriptome as a result of an emerging viral infection to show specific mechanisms of immune response evasion and other effects in the host's biology at the transcriptomic level. conversely, targeted-approaches depend on previous knowledge of host-transcriptomics. studies with human cohorts and animal models have developed gene signatures that discriminate between viral and bacterial respiratory infections [61] [62] [63] [64] [65] . other groups have developed sepsis classifiers to guide treatment options in clinical settings [71, 72] . a goal of developing these disease classifiers is to use them to predict disease outcome prior to the onset of symptoms and to assist in decision making when a symptomatic disease with an unknown etiology is suspected. effective and accurate discernment between a viral and a bacterial infection can make the difference in administering the appropriate therapeutics and potentially saving lives. when a specific viral species can't be identified, a gene classifier may be useful to determine potential disease outcomes such as lethality. as technology continues to advance, the linkages of genomics and precision medicine with emerging infectious diseases will continue to strengthen. improvements with sequencing accuracy and speed are pushing pathogen-specific genomics to near real-time [73, 74] , allowing rapid access to critical information within a timeframe that can impact an outbreak response [44, 45] . confidence in the results from sequencing is leading to clinically actionable diagnostic patient testing [29] , and it is reasonable to foresee rapid, pathogen agnostic diagnostic assays routinely utilized in the clinic. population growth, expansion into rural geographic regions, and rapid travel has greatly expanded the pathogen/host/environment interactome, putting a greater number of people at risk for formerly exotic infectious diseases such as ebola virus or even the next unknown, soon-to-emerge pathogen. applying genomics and the host transcriptome to generalize the response to a viral or bacterial pathogen would allow for more appropriate antibiotic use without requiring direct pathogen detection. besides the benefits to antibiotic stewardship in an era where antimicrobial resistance is expanding rapidly, such an application can greatly improve clinical care in the event of an emerging, highly virulent unknown unknown organism. opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the u.s. army. report of an international commission. ebola haemorrhagic fever in zaire emergence of zaire ebola virus disease in guinea zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria zika virus, a cause of fever in central java, indonesia zika virus outbreak on yap island, federated states of micronesia zika virus, french polynesia, south pacific zika virus outbreak, bahia, brazil zika virus spreads to new areas-region of the americas chimpanzee reservoirs of pandemic and nonpandemic hiv-1 hiv epidemiology. the early spread and epidemic ignition of 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public health practitioners emerging virus diseases: can we ever expect the unexpected? sars-cov and emergent coronaviruses: viral determinants of interspecies transmission comprehensive panel of real-time taqman polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and new world hantaviruses yellow fever: 100 years of discovery the etiology of yellow fever: an additional note actionable diagnosis of neuroleptospirosis by next-generation sequencing a novel rhabdovirus associated with acute hemorrhagic fever in central africa genetic detection and characterization of lujo virus, a new hemorrhagic fever-associated arenavirus from southern africa author correction: the discovery of bombali virus adds further support for bats as hosts of ebolaviruses a metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in neoromicia bats within south africa consensus statement: virus taxonomy in the age of 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rapid outbreak sequencing of ebola virus in sierra leone identifies transmission chains linked to sporadic cases possible sexual transmission of ebola virus-liberia implementation of a national semen testing and counseling program for male ebola survivors-liberia the sequence of sequencers: the history of sequencing dna multiplex pcr for typing and subtyping influenza and respiratory syncytial viruses evaluation of the biofire filmarray respiratory panel and the genmark esensor respiratory viral panel on lower respiratory tract specimens preliminary evaluation of biofire filmarray((r)) gastrointestinal panel for the detection of noroviruses and other enteric viruses from wastewater and shellfish application of biofire filmarray blood culture identification panel for rapid identification of the causative agents of ventilator-associated pneumonia cost justification of the bio-fire filmarray meningitis/encephalitis panel versus standard of care for diagnosing meningitis in a community hospital clinical evaluation of the biofire filmarray((r)) biothreat-e test for the diagnosis of ebola virus disease in guinea evaluation of the potential impact of ebola virus genomic drift on the efficacy of sequence-based candidate therapeutics antiviral goes viral: harnessing crispr/cas9 to combat viruses in humans rna-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection crispr/cas9-mediated genome editing of herpesviruses limits productive and latent infections antiviral actions of interferons differential evolution of peripheral cytokine levels in symptomatic and asymptomatic responses to experimental influenza virus challenge a genomic signature of influenza infection shows potential for presymptomatic detection, guiding early therapy, and monitoring clinical responses a host transcriptional signature for presymptomatic detection of infection in humans exposed to influenza h1n1 or h3n2 a host-based rt-pcr gene expression signature to identify acute respiratory viral infection gene expression signatures diagnose influenza and other symptomatic respiratory viral infections in humans detection of tuberculosis in hiv-infected and -uninfected african adults using whole blood rna expression signatures: a case-control study genome-wide association study identifies two risk loci for tuberculosis in han chinese apobec3g variants and protection against hiv-1 infection in burkina faso a conserved transcriptional response to intranasal ebola virus exposure in nonhuman primates prior to onset of fever comparison of transcriptomic platforms for analysis of whole blood from ebola-infected cynomolgus macaques a community approach to mortality prediction in sepsis via gene expression analysis robust classification of bacterial and viral infections via integrated host gene expression diagnostics nanopore sequencing as a rapidly deployable ebola outbreak tool minion as part of a biomedical rapidly deployable laboratory key: cord-319933-yp9ofhi8 authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: chapter 38 animal models of human viral diseases date: 2013-12-31 journal: animal models for the study of human disease doi: 10.1016/b978-0-12-415894-8.00038-5 sha: doc_id: 319933 cord_uid: yp9ofhi8 abstract as the threat of exposure to emerging and reemerging viruses within a naive population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. by using animal models in this endeavor, the response to viruses can be studied in a more natural context to identify novel drug targets, and assess the efficacy and safety of new products. this is especially true in the advent of the food and drug administration's animal rule. although no one animal model is able to recapitulate all the aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to be considered before an animal study are the route of challenge, species of animals, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand the disease progression, pathogenesis, and immunologic responses in humans. furthermore, to test vaccines and medical countermeasures, well-developed animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease. a good animal model of viral infection should allow many parameters of infection to be assayed, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations, and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for initially testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for the pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most experiments performed in small animal models when possible, and nhps only used to fill in remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen-free (spf), transgenic, and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables larger or more frequent blood samples to be collected, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains; thus, greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic animals are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically the closest species to humans; thus, disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns of experimentation on nhps, along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure that the least amount of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, route of exposure and the dose should be as close as possible to the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are also often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on the viruses for each family that are the greatest concern for public health worldwide. poliovirus (pv) is an enterovirus in the picornavirus family and causes poliomyelitis. 1 humans are the only natural host for the virus, but a number of nhp species are also susceptible. all three serotypes of pv cause paralytic disease, but it is relatively rare with only 1-2% of infected individuals ultimately developing paralysis. humans typically acquire and transmit the virus by the oral-fecal route, although transmission by aerosol droplets may also be possible. 2 the virus replicates in the oropharyngeal and intestinal mucosa, made possible by the resistance of pv to stomach acids. 3 cd155 expression in peyer's patches and m cells suggest that these cell types may be important during initiation of infection. 4 replication at extraneural sites 38 . in vivo models of viral diseases affecting humans precedes invasion into the central nervous systems (cnss), when it occurs. two effective vaccines, the salk killed polio vaccine delivered by the intramuscular route and the sabin live attenuated polio vaccine delivered orally, have been used very successfully to eliminate the disease from most parts of the world. 5 the world health organization has led a long and hard-fought global polio eradication campaign, with much success, but full eradication has not yet been achieved. since 2003, between 1000 and 2000 cases of pv infection are reported worldwide each year. 6 thus, animal models are also needed to test new vaccine approaches that could be used toward eradication of polio in the areas where it still persists. additionally, the recent focus of work with pv animal models has been fraught with urgency, as to gain understanding of pv pathogenesis before the eradication effort is complete and work with this virus ceases. animal models for the study of pv consist of nhp models and mouse models. mice are susceptible to certain adapted pv strains: p2/lansing, p1/lsb, and a variant of p3/leon. mice infected intracerebrally with p2/lansing develop disease with some clinical and histopathological features resembling that of humans. 7 wild-type mice are not susceptible to wildtype pv; however, the discovery of the pv receptor (cd155) in 1989 led to the use of hcd155 transgenic mice as a model of pv infection. 8 these mice are not susceptible to pv by the oral route and must be exposed intranasally or by intramuscular infection to induce paralytic disease. 9 interestingly, hcd155 mice that have a disruption in the interferon (ifn)-a/b receptor gene are susceptible to oral infection. 10 this finding has given rise to speculation that an intact ifn-a/b response may be responsible for limiting infection in the majority of individuals exposed to pv. thus, mouse models have proven to be very useful in gaining a better understanding of pv disease and pathogenesis. rhesus macaques are not susceptible to pv by the oral route, but they have been used extensively to study vaccine formulations for safety and immunogenicity, for monitoring neurovirulence of the live attenuated sabin vaccine, and in the past for typing pv strains. 11 bonnet monkeys are also susceptible to oral inoculation of pv, which results in the gastrointestinal shedding of virus for several weeks, with paralysis occurring in only a small proportion of animals. consistent paralytic disease can be induced in bonnet monkeys (macaca radiata) through exposure to pv by infection into the right ulnar nerve (at the elbow), resulting in limb paralysis that resembles human paralytic poliomyelitis both clinically and pathologically. 12 as such, bonnet monkeys can be used to study pv distribution and pathology and the induction of paralytic poliomyelitis or provocation paralysis. 13 hepatitis a virus causes jaundice, which is a public health problem worldwide. the incubation period lasts from 15 to 45 days with an average of 28 days. transmission between humans occurs by the oral-fecal route, person-to-person contact, or ingestion of contaminated food and water. 14 hepatitis a virus causes an acute and self-limited infection of the liver with a spectrum of signs and symptoms ranging from subclinical disease, to jaundice, fulminant hepatitis, and in some cases death. 15, 16 the disease can be divided into four clinical phases: (1) incubation period, during which the patient is asymptomatic but virus replicates and possibly transmits to others. (2) prodromal period, which might last from a few days to a week with patients generally experiencing anorexia, fever (<103 f), fatigue, malaise, myalgia, nausea, and vomiting. (3) icteric phase, in which increased bilirubin causes characteristic dark brownish colored urine. this sign is followed by pale stool and yellowish discoloration of the mucous membranes, conjunctiva, sclera, and skin. most patients develop an enlarged liver, and approximately 5-15% of the patients have splenomegaly. (4) convalescent period, with resolution of the disease and recovery of the patient. rarely, during the icteric phase, extensive necrosis of the liver occurs. these patients show a sudden increase in body temperature, marked abdominal pain, vomiting, jaundice, and the development of hepatic encephalopathy associated with coma and seizures, all signs of fulminant hepatitis. death occurs in 70-90% of patients with fulminant hepatitis. 16 experiments showed that hepatitis a causes disease only in humans, chimpanzees, several species of south american marmosets, stump-tailed monkeys, and owl monkeys via the oral or intravenous (iv) routes. [17] [18] [19] [20] it is known that cynomolgus macaques are infected with hepatitis a virus in the wild. 21 amado et al. used cynomolgus macaques (macaca fascicularis) for experimental hepatitis a infections. 17 the animals did not exhibit clinical signs of disease, but viral shedding was observed in saliva and stool as early as 6 h postinoculation (pi) and 7 days pi, respectively. although mildto-moderate hepatic pathology was observed in all macaques, seroconversion and mildly increased alanine aminotransferase (alt), an enzyme associated with liver function, were observed in some of them. because this study had a very small group of animals (four macaques), the data should not be considered as conclusive, and more studies are needed to better define the cynomolgus macaque model. although hepatitis a virus is transmitted by the oralfecal route, studies in chimpanzees and tamarins showed that the iv route was much more infectious than oral route was. there was no correlation between dose and development of clinical disease for either species or experimental routes, and similar to cynomolgus macaques, none of these species showed clinical signs of disease. 20 inoculation of common marmosets (callithrix jacchus) with hepatitis a virus did not produce clinical signs of disease as seen in other nhp models. 22, 23 liver enzyme levels increased on day 14 pi, and monkeys had measurable antihepatitis a antibodies by day 32 pi. an experimental study with cell culture-adapted hepatitis avirus in guinea pigs challenged by oral or intraperitoneal routes did not result in clinical disease, increase in liver enzymes, or seroconversion. 24 viral load was detected in stool and serum between days 14 and 52 and 21 and 49 days, respectively. liver pathology showed mild hepatitis. furthermore, histopathology indicated that virus replicated in extrahepatic tissues such as spleen, regional lymph nodes, and intestinal tract. in summary, none of the animal models for hepatitis a infection is suitable for studying pathogenesis of the virus because all clinical and most of the laboratory parameters remain within normal range or only slightly increased after the infection. one possibility is to test the safety of vaccines against hepatitis a virus in those models with demonstrable viral shedding. noroviruses, of which norwalk is the prototypic member, are responsible for up to 85% of reported food-borne gastroenteritis cases. in developing countries, this virus is responsible for approximately 200,000 deaths annually. 25 a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants. 26, 27 symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation normally is for 1-3 days, with symptoms enduring for 2-3 days. 28 viral shedding is indicative of immunocompromised status within an individual with the elderly and young having a prolonged state of shedding. 29 transmission occurs predominately through the oral-fecal route with contaminated food and water being the major vector. 30 a major hindrance to basic research into this pathogen is the lack of a cell culture system. therefore, animal models are used not only to determine the efficacy of novel drugs and vaccines but also for understanding the pathogenesis of the virus. therapeutic intervention consists of rehydration therapy and antiemetic medication. 31 no vaccine is available, and development of one is expected to be challenging given that immunity is short lived after infection. 32 nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus can be monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity are measured exclusively by assay of viral shedding. 33 it was determined that more virus was needed to create an infection when challenging by the oral route than when challenging by the iv route. chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route. although none of the animals developed disease symptoms, viral shedding within the feces was observed within 2-5 days postinfection and lasted anywhere from 17 days to 6 weeks. viremia never occurred, and no histopathological changes were detected. the amount and duration of viral shedding were in line with what is observed upon human infection. 34 a recently identified calicivirus of rhesus origin, named tulane virus, was used as a surrogate model of infection. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever 2 days postinfection. viral shedding was achieved for 8 days. the immune system produced antibodies that dropped in concentration within 38 days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease. 35 a murine norovirus has been identified and is closely related to human norwalk virus. however, clinically, the viruses present a different disease. the murine norovirus does not induce diarrhea nor vomiting and can develop a persistent infection in contrast to human disease. [36] [37] [38] porcine enteric caliciviruses can induce diarrheal disease in young pigs and an asymptomatic infection in adults. 39 gnotobiotic pigs can successfully be infected with a passaged clinical noroviruses isolate orally. diarrheal disease developed in 74% of the animals, and 44% were able to shed virus in their stool. no major histopathological changes or viral persistence was noted. 40 calves are naturally infected with bovine noroviruses. experimentally challenging calves with an oral inoculation of a bovine isolate resulted in diarrheal disease [14] [15] [16] the link between equine cases and the human disease was confirmed in 1938 by observing 30 cases of fatal encephalitis in children living in the same area as the equine cases. during this outbreak, eeev was isolated from the cnss of these children as well as from pigeons and pheasants. 44 eeev primarily affects areas near salty marshes and can cause localized outbreaks of disease in the summer. the enzootic cycles are maintained in moist environments such as coastal areas, shaded marshy salt swamps in north america (na), and moist forests in central america and south america (sa). 45 birds are the primary reservoir, and the virus is transmitted via mosquitoes. furthermore, forest-dwelling rodents, bats, and marsupials frequently become infected and may provide an additional reservoir in central america and sa. despite known natural hosts, the transmission cycles in these animals are not well characterized. 44 reptiles and amphibians have also been reported to become infected by eeev. eeev pathogenesis and disease have been studied in several laboratory animals. as a natural host, birds do not generally develop encephalitis except pheasants or emus, in which eeev causes encephalitis with 50-70% mortality. 46 young chickens show signs of extensive myocarditis in early experimental infection and heart failure rather than encephalitis is the cause of death. 47 besides the heart, other organs such as pancreas and kidney show multifocal necrosis. additionally, lymphocytopenia has been observed in the thymus and spleen in birds. 45 eeev causes neuronal damage in newborn mice, and the disease progresses rapidly, resulting in death. 48 similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, whereas inoculation via the subcutaneous route causes a pantropic infection eventually resulting in encephalitis. 49, 50 guinea pigs and hamsters have also been used as animal models for eeev studies. 51, 52 guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed and resulted in brain lesions in these animals. 52 subcutaneous inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis and death. in addition, parenchyma necroses were observed in the liver and lymphoid organs. 51 intradermal, intramuscular, or iv inoculations of eeev in nhps cause disease but does not always result in symptoms of the nervous system. intracerebral infection of eeev results in nervous system disease and fatality in monkeys. 53 the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in monkeys when they are infected by the peripheral route. 54 intranasal and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but less drastic than those caused by intracerebral injections. 54 the aerosol route of infection also progresses to uniformly lethal disease in cynomolgus macaques. 55 in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed, and nhps became moribund and were euthanized between days 5 and 9 days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals. 56 similar clinical signs and pathology were observed when common marmosets were infected with eeev by the intranasal route. 57 both aerosol and intranasal nhp models had similar disease progression and pathology as those seen in human disease. a common marmoset model was used for comparison studies of sa and na strains of eeev. 57 previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected intranasally with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized 4-5 days after the challenge. although sa strain-infected animals developed viremia, they remained healthy and survived the challenge. epizootics of viral encephalitis in horses were previously described in argentina. more than 25,000 horses died from western equine encephalitis virus (weev) in the central plains of the united states in 1912. 58 weev was first isolated from the brains of horses during the outbreak in the san joaquin valley of california in 1930. although it was suspected, the first diagnosis of weev as a cause of human encephalitis occurred in 1938, when the virus was recovered from the brain of a child with fatal encephalitis. 44 in horses, the signs of disease are fever, loss of coordination, drowsiness, and anorexia, leading to prostration, coma, and death in about 40% of affected animals. 59 weev also infects other species of birds and often causes fatal disease in sparrows. weev infection occurs throughout western na and sporadically in sa as it circulates between its mosquito vector and wild birds. 44 chickens and other domestic birds, pheasants, rodents, rabbits, ungulates, tortoises, and snakes are natural reservoirs of weev. 60,61 weev has caused epidemics of encephalitis in humans, horses, and emus, but the fatality rate is lower than that for eeev. 62 predominately young children and those older than 50 years demonstrate the clinical symptoms of the disease. 63 severe disease, seizures, fatal encephalitis, and significant sequelae are more likely to occur in infants and young children. 64, 65 typically, the disease progresses asymptomatically with seroprevalence in humans being fairly common in endemic areas. species used to develop animal models for weev are mice, hamsters, guinea pigs, and ponies. studies with ponies resulted in viremia in 100% of the animals 1-5 days pi. fever was observed in 7 of 11 animals, and six exhibited signs of encephalitis. 44 after subcutaneous inoculation with weev, suckling mice started to show signs of disease by 24 h and died within 48 h. 66 in suckling mice, the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on days 4-5 postinfection. mice were severely ill by day 8 and appeared hunched and dehydrated. death occurred between days 7 and 14, and both brain and mesodermal tissues such as heart, lungs, liver, and kidney were involved. 66, 67 intracerebral and intranasal routes of infection resulted in a fatal disease that was highly dependent on dose, while intradermal and subcutaneous inoculations caused only 50% fatality in mice regardless of the amount of virus. 49 studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases. 68 cns involvement was evident with intracerebral, intraperitoneal, and intradermal inoculations. 68 weev is highly infectious to guinea pigs. 69 intraperitoneal inoculation of weev is fatal in guinea pigs regardless of virus inoculum, with the animals exhibiting signs of illness on days 3-4, followed by death on days 5-9 (nalca, unpublished results). very limited studies have been performed with nhps. the intranasal route of infection causes severe, lethal encephalitis in rhesus macaques. 54 reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model would be useful for testing of vaccines and therapeutics against weev. 70 venezuelan equine encephalitis virus (veev) is maintained in nature in a cycle between small rodents and mosquitoes. 45 the spread of epizootic strains of the virus to equines leads to high viremia followed by a lethal encephalitis, and tangential spread to humans. veev can easily be spread by the aerosol route making it a considerable danger for laboratory exposure. in humans, veev infection causes a sudden onset of malaise, fever, chills, headache, and sore throat. 45, 71, 72 symptoms persist for 4-6 days, followed by a 2-to 3-week period of generalized weakness. encephalitis occurs in a small percentage of adults ( 0.5%); however, the rate in children may be as high as 4%. neurologic symptoms range from nuchal rigidity, ataxia, and convulsions to the more severe cases exhibiting coma and paralysis. the overall mortality rate in humans is <1%. 45 laboratory animals such as mice, guinea pigs, and nhps exhibit different pathologic responses when infected with veev. the lymphatic system is a general target in all animals infected with cns involvement variable between different animal species. the disease caused by veev progresses very rapidly without showing signs of cns disease in guinea pigs and hamsters. mortality is typically observed within 2-4 days after infection and fatality is not dose dependent. 44 veev infection lasts longer in mice, which develop signs of nervous system disease in 5-6 days and death 1-2 days later. lethal dose in mice changes depending on the age of mice and the route of exposure. 56 in contrast to guinea pigs and hamsters, the time of the death in mice is dose dependent. mortality is observed generally within 2-4 days after infection and fatality is not dose dependent. subcutaneous/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans. 73 virus begins to replicate in the draining lymph nodes at 4 h pi. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis. 74, 75 aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure do. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death. 56, 74, 75 intranasal challenge of c3h/hen mice with high dose veev caused high morbidity and mortality. 76 viral titers in brain peaked on day 4 postchallenge and stayed high until animals died on day 9-10 postchallenge. protein cytokine array done on brains of infected mice showed elevated interleukin (il)-1a, il-1b, il-6, il-12, monocyte chemoattractant protein-1 (mcp-1), ifng, mip-1a, and regulated and normal t-cell expressed and secreted levels. this model was used successfully to test antivirals against veev. 77 xi. viral disease veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at 12-72 h after infection and lasted <12 h. secondary fever generally began on day 5 and lasted 3-4 days. 78 veev-infected nhps exhibited mild symptoms such as anorexia, irritability, diarrhea, and tremors. leucopenia was common in animals exhibiting fever. 79 supporting the leucopenia, observed microscopic changes in lymphatic tissues such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection. 78 the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day 6 postinfection during the secondary febrile episode. cynomolgus macaques develop similar clinical signs including fever, viremia, lymphopenia, and encephalitis upon aerosol exposure to veev. 80 chikungunya virus is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics mainly within africa and southeast asia. 45 the virus is transmitted by aedes mosquitoes. given the widespread endemicity of aedes mosquitoes, chikungunya virus has the potential to spread to previously unaffected areas. this is typified by the emergence of disease for the first time in 2005 in the islands of the southwest indian ocean, including the french la reunion island, and the appearance in central italy in 2007. 81, 82 the incubation period after a mosquito bite is 2-5 days followed by a self-limiting acute phase that lasts 3-4 days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported. 83 individuals typically develop a stooped posture due to the pain. for approximately 12% of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions, including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of 3 and 18 years have an increased risk of developing neurological manifestations. 84 there is no effective vaccine or antiviral. wild-type c57bl/6 adult mice are not permissive to chikungunya virus infection by intradermal inoculation. however, it was demonstrated that neonatal mice were susceptible, and severity was dependent upon age at infection. six-day-old mice developed paralysis by day 6, and all died by day 12, whereas 50% of nine-day-old mice were able to recover from infection. by 12 days, mice were no longer permissive to disease. infected mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications. 85, 86 an adult mouse model has been developed by injection of the ventral side of the footpad of c57bl/6j mice. viremia lasted 4-5 days accompanied by foot swelling and noted inflammation of the musculoskeletal tissue. 87, 88 adult ifna/br knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within 3 days. this model was useful in identifying the viral cellular tropism for fibroblasts. 85 imprinting control region (icr) cd1 mice can also be used as a disease model. neonatal mice subcutaneously inoculated with a passaged clinical isolate of chikungunya virus developed lethargy, loss of balance, and difficulty in walking. mortality was low, 17% and 8% for newborn cd1 and icr mice, respectively. the remaining mice fully recovered within 6 weeks after infection. 86 a drawback of both the ifna/ br and cd1 mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised. 89 long-tailed macaques challenged with a clinical isolate of the virus developed a similar clinical disease to humans. initially, the monkeys developed high viremia with fever and rash. after this period, viremia resolved and virus could be detected in lymphoid, liver, meninges, joint, and muscle tissue. the last stage mimicked the chronic phase in which virus could be detected up to two months after infection, although no arthralgia was noted. 90 dengue virus is transmitted via the mosquito vectors aedes aegypti and aedes albopictus. 91 given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to dengue virus. this results in approximately 50 million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia. 92 it is estimated that there are 20,000 deaths each year caused by dengue hemorrhagic fever (dhf). 93 there are four serotypes of dengue virus, numbered 1-4, which are capable of causing a wide spectrum of disease that ranges from asymptomatic to severe with the development of dhf. 94 incubation can range from 3 to 14 days, with the average being 4-7 days. the virus targets dendritic cells and macrophages after a mosquito bite. 95 typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure. 94 thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis. 96 severe disease has a higher propensity to occur upon secondary infection with a different dengue virus serotype. 97 this is hypothesized to occur due to antibodydependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in developing an animal model, it is important to note that mosquitoes typically deposit 10 4 -10 6 pfu, and is therefore the optimal range to be used during challenge. a comprehensive review of the literature regarding animal models of dengue infection was recently published by zompi et al. 98 several laboratory mouse strains including a/j, balb/c, and c57bl/6 are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis. [99] [100] [101] a higher dose of an adapted dengue virus strain induced dhf symptoms in both balb/c and c57bl/6. 102, 103 this model can also yield asymptomatic infections. a mouse-adapted (ma) strain of dengue virus 2 introduced into ag129 mice developed vascular leak syndrome similar to the severe disease seen in humans. 104 passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased, and animals died due to vascular leak syndrome. 105 another ma strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability. 103 intracranial injection of suckling mice with dengue virus leads to death and has been used to test the efficacy of therapeutics. 106 scid mice engrafted with human tumor cells develop paralysis upon infection, and are thus not useful for pathogenesis studies. 107,108 df symptoms developed after infection in nod/scid/il2rgko mice engrafted with cd34 ã¾ human progenitor cells. 109 rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and laboratory-adapted strain of dengue virus 2. 110 a passaged clinical isolate of dengue virus type 3 was recently used to create a model in immunocompetent adult mice. interperitoneal injection in c57bl/6j and balb/c caused lethality by day 6-7 postinfection in a dose-dependent manner. the first indication of infection was weight loss beginning on day 4 followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, aspartate aminotransferase (ast) and alt, were also observed. viremia was established by day 5. this model mimicked the characteristic symptoms observed in human dhf/dss cases. 111 a novel model was developed that used infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of dengue virus type 2. infected mosquitoes then fed upon the mouse footpad to allow for the transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the amount of mosquitoes it was exposed to, with four to five being optimal. detectable viral rna was in line with what is observed during human infection. severe thrombocytopenia developed on day 14. this model is intriguing given that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus. 112 nhp models have used a subcutaneous inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection. 113 the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes. 114 throughout these preliminary studies, no clinical disease was detected. to circumvent this, a higher dose of dengue virus was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day 3 and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed. 115 further development would allow this model to be used for testing of novel therapeutics and vaccines. although primates do not develop disease upon infection with dengue, their immune system does produce antibodies similar to those observed during the course of human infection. this has been advantageous in studying ade. sequential infection led to a crossreactive antibody response, which has been demonstrated in both humans and mice. 116 this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was 3-to 100-fold higher than was previously reported; however, no clinical signs were apparent. 117 the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. japanese encephalitis virus ( jev) is a leading cause of childhood viral encephalitis in southern and eastern asia and is a problem among military personnel and travelers to these regions. it was first isolated from the brain of a patient who died from encephalitis in japan in 1935. 118 culex mosquitoes, which breed in rice fields, transmit the virus from birds or mammals (mostly domestic pigs) to humans. the disease symptoms range from a mild febrile illness to acute meningomyeloencephalitis. after an asymptomatic incubation period of 1-2 weeks, patients show signs of fever, headache, stupor, and generalized motor seizures, especially in children. the virus causes encephalitis by invading and destroying the cortical neurons. the fatality rate ranges from 10% to 50%, and most survivors have neurological and psychiatric sequelae. 119, 120 jev virus causes fatality in infant mice by all routes of inoculation. differences in pathogenesis and outcome are seen when the virus is given by intraperitoneal inoculation. 121 these differences depend on the amount of virus and the specific viral strains used. the biphasic viral multiplication after peripheral inoculation is observed in mice tissues. primary virus replication occurs in the peripheral tissues and the secondary replication phase in the brain. 122 hamsters are another small animal species that are used as an animal model for jev. fatality was observed in hamsters inoculated intracerebrally or intranasally, while peripheral inoculation caused asymptomatic viremia. studies with rabbits and guinea pigs showed that all routes of inoculation of jev produce asymptomatic infection. 123 serial sampling studies with 12-day-old wistar rats inoculated intracerebrally with jev indicated that jev causes the overproduction of free radicals by neurons and apoptosis of neuronal cells. 124 following a study in 2010 by the same group, showed that although cytokines tumor necrosis factor (tnf)-a, ifn-g, il-4, il-6, il-10, and chemokine mcp-1 increased gradually and peaked on days 10 pi with jevin rats, the levels eventually declined, and there was no correlation with the levels of cytokines and chemokines and neuronal damage. 125 intracerebral inoculation of jev causes severe histopathological changes in brain hemispheres of rhesus monkeys. symptoms such as weakness, tremors, and convulsions began to appear on days 6-10, with indicative signs of encephalomyelitis occurring on days 8-12 postinfection for most of the animals followed by death occurring on days 8-12 postinfection for most of the animals followed by death. 126 although intranasal inoculation of jev results in fatality in both rhesus and cynomolgus monkeys, peripheral inoculation causes asymptomatic viremia in these species. 123, 127 west nile virus west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in 1937. 128 after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early 1950s 129, 130 and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia. 131 the virus first reached the western hemisphere in the summer of 1999, during an outbreak involving humans, horses, and birds in the new york city metropolitan area. 132, 133 since 1999, the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from 3 to 6 days. 134 the mortality rate after neuroinvasive disease ranges from 4% to 11%. 131, [135] [136] [137] the most severe complications are commonly seen in the elderly, with reported case fatality rates from 4% to 11%. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. although many early laboratory studies of wn encephalitis were performed in nhps, mice, rat, hamster, horse, pig, dog, and cat models were used to study the disease. [138] [139] [140] [141] [142] [143] [144] inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e. asymptomatic, fever, encephalitis). 141 thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subcutaneous inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia. 145, 146 wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and intraperitoneal routes, resulting in encephalitis and 100% mortality. intradermal route pathogenesis studies indicated that langerhans dentritic cells are the initial viral replication sites in the skin. 147, 148 the infected langerhans cells then migrate to lymph nodes, and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases. [149] [150] [151] [152] tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared normal during the first 5 days, became lethargic at approximately day 6, and developed neurologic symptoms by days 7-10. 143 many of the severely affected animals died 7-14 days after infection. viremia was detected in the hamsters within 24 h after infection and persisted for 5-6 days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the above-mentioned monkey experiments by pogodina et al., persistent wnv infection was found in the brains of hamsters. the etiologic agent of severe acute respiratory syndrome (sars), sars-coronavirus (cov), emerged in 2002 as it spread throughout 32 countries in a period of 6 months, infecting >8000 people and causing nearly 800 deaths. 153, 154 the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to 6 days and can be detected in stool, suggesting other modes of transmission are also possible. 155 although other members of the family usually cause mild illness, sars-cov infection has a 10% case fatality with the majority of cases in people over the age of 15 years. 156, 157 after an incubation period of 2-10 days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough. 158 in some sars, cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within 2 weeks and coincides with the induction of high levels of neutralizing antibodies. 159 in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses. 160 infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection. virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues. 161 sars-cov can replicate in many species, including dogs, cats, pigs, mice, rats, ferrets, foxes, and nhps. 162 chinese palm civets, raccoon dogs, and bats are possible natural hosts. no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (w10%), viral replication, and pathology. 163 in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models use mice, hamsters, ferrets, and nhps (table 38 .1). mouse models of sars-cov typically are inoculated by the intranasal route under light anesthesia. young, 6-to 8-week old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinates, with a peak on day 2 and clearance by day 5 postexposure. there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis). intranasal sars-cov infection of c57bl/6 (b6) also yield reduced weight gain and viral replication in the lungs, with a peak on day 3 and clearance by day 9. 171 in contrast, balb/c mice 13-14 interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. 129s mice and b6 mice show outcomes to sars-covinfection similar to those observed for balb/c mice but have lower titers and less prolonged disease. one problem is that it is more difficult to obtain large numbers of mice older than 1 year. a number of immunocompromised knockout mouse models of intranasal sars-cov infection have also been developed. 129svev mice infected with sars-cov by the intranasal route develop bronchiolitis, with peribronchiolar inflammatory infiltrates, and interstitial inflammation in adjacent alveolar septae. 172 viral replication and disease in these mice resolve by day 14 postexposure beige, cd1ã�/ã�, and rag1ã�/ã� mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs. signal transducer and activator of transcription-1 (stat1) ko mice infected intranasally with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths. the stat1 ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. syrian golden hamsters (strain lvg) are also susceptible to intranasal exposure of sars-cov. after the administration of 10 3 tcid 50 (tissue culture infective dose), along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis. there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. some mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred. 163 damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge. 167 clinical disease has been observed in several studies. sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day 4 postexposure as well as mild alveolar damage in 5-10% of the lungs, occasionally accompanied by severe pathology within the lungs. 173 with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or intratracheal routes generally results in a very mild infection, which resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness, but others have rash, lethargy, and respiratory signs and pathology. 170 rhesus have little to no disease and only have mild findings upon histopathological analysis. agms infected with sars-cov have no overt clinical signs, but dad and pneumonitis have been documented. viral replication has been detected for up to 10 days in the lungs of agms; however, the infection resolves and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were thought to be involved in the sars-covoutbreak. intratracheal and intranasal inoculation of civets with sars-covresults in lethargy, decreased aggressiveness fever, diarrhea, and conjunctivitis. 174 leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. common marmosets have also been shown to be susceptible to sars-cov infection. 175 vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine have used live-attenuated, killed, dna and viral-vectored vaccine strategies. 176 an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease. 177 as such, immune mice had th2-type immunopathology upon sars-cov challenge. 178 severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported. 179 additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance. 180 mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. ma sars and human ace2 transgenic mice are available. 181 all mammals experimentally or naturally exposed to rabies virus have been found to be susceptible. this highly neurotropic virus is a member of the lyssavirus genus and is transmitted from the bite of an infected animal to humans. 182 the virus is able to replicate within the muscle cells at the site of the bite, and then travel to the cns. once reaching the cns by retrograde axonal transport, the virus replicates within neurons creating inflammation and necrosis. the virus subsequently spreads throughout the body via peripheral nerves. 183 a typical incubation period is 30-90 days, and is highly dependent upon the location of the bite. proximity to the brain is a major factor for the onset of symptoms. the prodromal stage lasts from 2 to 10 days and is when the virus initially invades the cns. flu-like symptoms are the norm in conjunction with pain and inflammation at the site of the bite. subsequently, there are two forms of disease that can develop. in 80% of cases, an individual develops the encephalitic or furious form. this form is marked by hyperexcitability, autonomic dysfunction, and hydrophobia. the paralytic, or dumb form, is characterized by ascending paralysis. ultimately, both forms result in death days after the onset of symptoms. once the symptoms develop, there is no proven effective therapy. in the developing world, death is caused by the lack of access to medical care including postexposure prophylaxis. in na, fatal cases result because of late diagnosis. 184 syrian hamsters have been challenged with rabies virus intracerebrally, intraperitoneally, intradermally, and intranasally. all animals died as a result of the exposure, although intracerebral and intranasal inoculation led to only the furious form depicted by extreme irritability, spasms, excessive salivation, and cries. the virus used had been isolated from an infected dog brain and passaged in swiss albino mice. animals inoculated by intracerebral injection develop disease within 4-6 days, whereas all other routes of entry develop disease within 6-12 days. 185 this model has been used to study and test novel vaccine candidates. 186 mice have been extensively studied as an animal model for rabies. it was shown that swiss albino mice intracerebrally injected with a virus isolated from a dog developed only the paralytic form of disease 6 days after the initial challenge. balb/c mice are universally susceptible to intracerebral injection of rabies virus within 9 days. disease symptoms include paralysis, cachexia, and bristling appearing 1-3 days before death. 187 a more natural route of infection via peripheral injection into masseter muscles was tested on icr mice. these mice developed neurological signs including limb paralysis, and all died within 6-12 days. 188 icr mice have been instrumental in analyzing novel vaccines and correlates of protection. 189 this line of mice was also used to assess the value of ketamine treatment to induce coma during rabies infection. 190 another mouse line used is the p75 neurotrophin receptor-deficient mouse. this mouse developed a fatal encephalitis when inoculated intracerebrally with the challenge virus standard. 191 bax-deficient mice have also been used to determine the role of apoptotic cell death in the brain during the course of infection. 192 a viral isolate from silver-haired bats can also be used in the mouse model. this strain is advantageous given that it is responsible for the majority of deaths in north na. 193 early death phenomenon is typified by a decrease to time of death in a subset of individuals and animals that have been vaccinated and subsequently exposed to rabies. 194 this trend has been demonstrated experimentally in swiss outbred mice and primates. 195, 196 cynomolgus and rhesus were both infected with passaged rabies virus to create an nhp model. a high titer of virus was needed to induce disease, but exposure was found to not be universally fatal. the animals that survived beyond 4 weeks within the experiment did not develop clinical disease nor succumb to infection. primates that did develop disease refused food and had progressively less activity until death. this lasted from 24 h up to 4 days, with all animals with symptoms dying within 2 weeks. 196 bats have been experimentally challenged with rabies. vampire bats, desmodus rotundus, intramuscularly injected with a bat viral isolate displayed clinical signs including paralysis in half of the population of the study animals. of those who did develop disease, the duration was 2 days, and incubation period ranged from 7 to 30 days. regardless of disease manifestation, 89% of challenged animals died. 197 skunks can be challenged intramuscularly or intranasally with either challenge virus strain or a skunk viral isolate. interestingly, the challenge virus strain more readily produced the paralytic form, whereas the street form of rabies developed into the furious form. however, the challenge strain virus resulted in a shorter incubation period of 7-8 days in comparison to 12-14 days seen with the street virus. 198 filoviridae consists of two well-established genera, ebola virus and marburg virus (marv) and a newly discovered group, cuevavirus 199 (table 38 200 two other ebola viruses are known; ta㯠forest (tafv; previously named cote (circumflex over the 'o') d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. the disease in humans is characterized by aberrant innate immunity and a number of clinical symptoms such as fever, nausea, vomiting, arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, anorexia, and numerous others. 201 approximately 10% of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.). 199 natural transmission in an epidemic is thought to be through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, intramuscular, intraperitoneal, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures. 202 because filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type 1 ifn response. 203 however, wild-type inbred mice are susceptible to filovirus that has been ma by serial passage. 204 balb/c mice, which are the strain of choice for intraperitoneal inoculation of ma-ebov, are not susceptible by the aerosol route. 205 for aerosol infection of immunocompetent mice, a panel of bxd (balb/ c ã� dba) recombinant inbred strains were screened, and one strain, bxd34, was shown to be particularly susceptible to airborne ma-ebov, with 100% lethality to low or high doses (w100 or 1000 pfu). these mice developed weight loss of >15% and succumbed to infection between days 7 and 8 postexposure. the aerosol infection model uses a whole-body exposure chamber to expose mice aged 6-8 weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately 1.6 mm and a geometric standard deviation of approximately 2.0 for 10 min. another approach uses immunodeficient mouse strains such as scid, stat1 ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by intraperitoneal or aerosol routes. 206 mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection. liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps. although most mouse studies have used ma-ebov or ebov, an intraperitoneal ma marv model is also available. 207 ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds. 208 hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models. 209 an intraperitoneal ma-ebov infection model has been developed in syrian hamsters. 210 this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, uses male 5-to 6-week-old syrian hamsters that are infected with 100 ld50 of ma-ebov. virus is present in tissues and blood collected on day 4, and all animals succumbed to the disease by day 6. detailed accounts of this model have been presented at international scientific meetings by ebihara and feldmann et al. but have not been reported in a scientific journal at the time of writing this chapter. 211 guinea pig models of filovirus infection have been developed for intraperitoneal and aerosol routes using guinea pig-adapted ebov (gp-ebov) and marv (gp-marv). 212, 213 guinea pigs models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent (hourly) intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. hartley guinea pigs exposed to aerosolized gp-marv or gp-ebov become moribund at times comparable to that of nhps, generally succumbing to the infection between 7 and 12 days postexposure. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses (100 or 1000 pfu target doses) but lethality drops with low (<1000 pfu) presented doses of airborne gp-marv, and more protracted disease is seen in some animals. 213 weight loss of between 15% and 25% is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day 5, occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day 6 postexposure. blood chemistries (i.e. alt, ast, alkaline phosphatase (alkp), and blood urea nitrogen) indicating problems with liver and kidney function are also altered late in the disease course. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans. 214 old world primates have been primarily used for the development of intraperitoneal, intramuscular, and aerosol models of filovirus infection. uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, in agms and marmosets. [215] [216] [217] [218] [219] low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. prominent features of the infections are onset of fever by day 5 postexposure, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. clinical disease parameters may have a slightly delayed onset in aerosol models. petichial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species. dyspnea late in infection is a prominent feature of disease after aerosol exposure. a number of pronounced pathology findings include multifocal necrosis and fibrin lesions, particularly within the liver and the spleen. lymphocytolysis and lymphoid depletion are also observed. multilead, surgically implanted telemetry devices are useful in the continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection. the most recently developed telemetry devices can aid in plethysomography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate. 220 outbreaks caused by nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra outbreaks have yet to be reported outside of australia. 221, 222 hendra was the first member of the genus to be identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from human to human or bat to human. nipah has the distinction of being able to be transmitted by humans, although the exact route is unknown. 223 the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized to be needed for successful transmission. 224 both viruses have a tropism for the neurological and respiratory tract. hendra virus incubation period is 7-17 days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting. 225 disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure. 226 nipah virus has an incubation period of 4 days to 2 weeks. 227 much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within 24-48 h. 228 survivors of infection typically make a full recovery; however, 22% suffer permanent sequelae, including persistent convulsions. 229 at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because observed human cases are all at terminal stages. the best small animal representative is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died xi. viral disease 38 . in vivo models of viral diseases affecting humans within 4-17 days postinfection. the progression of disease and timeline are highly dependent on dose and route of infection. intranasal inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas intraperitoneal resulted in tremors and paralysis within 24 h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, while the brain was the most affected organ. this model has been used for vaccination and passive protection studies. [230] [231] [232] the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge. 233, 234 inoculation with hendra virus via the subcutaneous route leads to a generalized vascular disease with 20% mortality. clinical signs were apparent 7-16 days postinfection with death occurring within 2 days of cns involvement. higher inocula have been associated with the development of encephalitis lesions. intradermal and intranasal injections do not lead to disease, although the animals are able to seroconvert upon challenge. inoculum source does not affect clinical progression. nipah virus challenge only develops disease upon intraperitoneal injection and results in weight loss and transient fever for 5-7 days. virus was shed through urine and found to be present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder. 235 ferrets display the same clinical disease as seen in the hamster model and human cases. 236, 237 upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within 6-9 days including fever, coughing, and dyspnea. lesions do develop in the ferrets' brains, but to a lesser degree than seen in humans. cats have also been used as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is 4-8 days and leads to respiratory and neurological symptoms. 238, 239 this model has proven to be useful in a vaccine challenge model. squirrel and agms are representative of the nhp models. within the squirrel monkeys, nipah virus is introduced by either the intranasal or iv route and subsequently leads to clinical signs similar to that in humans, although intranasal challenge results in milder disease. upon challenge, only 50% animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in the lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge. 240 agms have been found to be a very consistent model of both viruses. intratracheal inoculation of the viruses results in 100% mortality, and death within 8.5 and 9-12 days postinfection for hendra and nipah, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis. 241, 242 the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue and nipah virus can be located in urine. 243 pigs have been investigated as a model as they develop a respiratory disease upon infection with both nipah and hendra. [244] [245] [246] oral inoculation does not produce a clinical disease, but subcutaneous injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as 4 days postinfection. nipah can also be transmitted between pigs. nipah was able to induce neurological symptoms in 20% of the pigs, even though virus was present in all neurological tissues regardless of symptoms. 247 within the pig model, it seemed that nipah had a greater tropism for the respiratory tract, while hendra for the neurological system. horses also are able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within 2 days. 248, 249 animals died within 4 days postexposure and were found to have interstitial pneumonia with necrosis of alveoli. 250, 251 virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but found to be nonpermissive to infection. 232, 252 suckling balb/c mice succumb to infection if the virus is inoculated intracranially. 253 embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within 4-5 days postinfection. 254 respiratory syncytial virus is responsible for lower respiratory tract infections of 33 million children under the age of 5 years, which in turn results in three million hospitalizations and approximately 200,000 deaths. 255 within the united states, hospital costs alone amount to >600 million dollars. 256 outbreaks are common in the winter. 257 the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and further spreads to the lower respiratory tract. incubation for the virus is 2-8 days. respiratory syncytial virus is highly virulent leading to very few asymptomatic infections. 258 disease manifestations are highly dependent upon the age of the individual. primary infections in neonates produce nonspecific symptoms including the overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting the virus at this age results in an increased chance of developing childhood asthma. 259 young children develop recurrent wheezing, whereas adults exacerbate previous respiratory conditions. 260 common clinical symptoms are runny nose, sneezing, and coughing accompanied with fever. mortality rates in hospitalized children are 1-3% with the greatest burden of disease seen in 3-4-month-olds. 261 there is no vaccine available, and ribavirin usage is not recommended for routine treatment. 262 animal models were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated respiratory syncytial virus vaccine (fi-rsv). this vaccineinduced severe respiratory illness in infants who received the vaccine and were subsequently infected with live virus. 263 mice can be used to model disease, although a very high intranasal inoculation is needed to achieve clinical symptoms. 264, 265 strain choice is crucial to reproducing a physiological relevant response. 266 age does not affect primary disease manifestations. 267 however, it does play a role in later sequelae showing increased airway hyperreactivity. 268 primary infection produces increased breathing with airway obstruction. 264, 269 virus was detected as early as day 3 and reached maximum titer at day 6 postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. cotton rats are useful given that it is a small animal disease model. the virus is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after intranasal inoculation. 270, 271 it has been reported that viral replication is 50-to 1000-fold greater in the rat model than in the mouse model. 272 the rats develop mild-to-moderate bronchiolitis or pneumonia. 273 although age does not seem to factor in clinical outcome, it has been reported that older rats tend to take longer to achieve viral clearance. viral loads peak by the fifth day, dropping to below the levels of detection by 8. the histopathology of the lungs seems to be similar to that in humans after infection. 274 this model has limited usage in modeling the human immune response to infection as challenge with the virus creates a th2 response, whereas humans tend to skew toward th1. [275] [276] [277] fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally via intranasal inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is thought to be useful in studying mucosal immunity during infection. 278 chimpanzees are permissive to replication and clinical symptoms of respiratory syncytial virus including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms nor high levels of viral replication. 279 bonnet monkeys were also tested and found to develop an inflammatory response by day 7 with viral rna detected in both bronchial and alveolar cells. 280 the chimpanzee model has proven to be useful for vaccine studies. 281, 282 sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine respiratory syncytial virus. 283 lambs were also found to be susceptible to human respiratory syncytial infection. 284, 285 when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after 6 days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within 14 days. during the course of disease, viral replication peaked at 6 days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features between them and humans. 286, 287 the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; 16 ha and 9 na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within 10 days. 288 the rate of infection is estimated at 10% in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately 40,000 people in the united states die each year from seasonal influenza. 289 thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing. 290 influenza virus replicates in the upper and lower airways, peaking at approximately 48 h postexposure. infection can be more severe in infants and in children under the age of 22 years, people over the age of 65 years, or immunocompromised individuals in whom viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis. 291 pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyrogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza itself, accounting for approximately 27% of all influenza-associated fatalities. 292 death, often due to ards can occur as early as 2 days after the onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation. 288 the h5n1 avian strain of influenza has lethality rates of approximately 60% (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus, the potential for global spread is a major concern. 293 the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. 294 lethality rate can vary with the virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, superinfection and sepsis, severe with ards, and neurologic manifestations. 290 also, models can use seasonal or avian strains and models have been developed to study transmission, important for understanding the potential for more lethal strains such as h5n1 for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines. 295 inoculation is by the intranasal route, using approximately 60 ml of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with an mmad of <5 mm. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c57bl/6j mice. males and females have equivalent disease, but influenza is generally more infectious in younger 2-to 4-week-old (8-10 g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. mice lack the mxa gene, which is an important part of the human innate immune response to influenza infection. the mouse homolog to mxa, mx1, is defective in most inbred mouse strains. 296 weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs. for more virulent strains, mice may require euthanasia as early as 48 h postexposure, but most mortality occurs from 5 to 12 days postexposure accompanied by decreases in rectal temperature. 297 pulse oximeter readings and measurement of blood gases of oxygen saturation are also used to determine the impact of influenza infection on respiratory function. 298 virus can be isolated from bronchial lavage fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild-tomoderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha-1-acidglycoprotein and lung weight are also frequently present. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa 2,3 predominates over sa 2,6 which is why h1, h2, and h3 subtypes usually need to be adapted to mice and h5n1, h2, h6, and h7 viruses do not require adaptation. 299 to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. however, h5n1 and the 1918 pandemic influenza virus can cause lethal infection without adaptation. 300 h5n1 infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia. 301 as in humans, the virulence of h5n1 is attributable to damage caused by an overactive host immune response. additionally, mice infected with the 1918 h1n1 influenza produces severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia. 302 in superinfection models, a sublethal dose of influenza is given to mice followed 7 days later by intranasal inoculation of a sublethal dose of a bacterial strain such as s. pneumoniae or s. pyrogenes. 303 morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to 2 weeks. at least one transmission model has also been developed in mice. with h2n2 influenza, transmission rates of up to 60% among cage mates can be achieved after infection by the aerosol route and cocaging after 24 h. 304 domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans. [305] [306] [307] ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa 2,6 and sa 2,3) within the airways similar to that of humans. 308 influenza was first isolated from ferrets infected intranasally with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments. 309 when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by intranasal. inoculation of 0.25-0.5 ml containing approximately 10 4 -10 6 egg id50 dropwise to each nostril. influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts 3-5 days for mildly to moderately virulent strains. 310 ferrets are more susceptible to influenza a than to influenza b strains and are also susceptible to avian influenza h5n1 strains without adaptation. 311 virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirror that seen in humans. nonadapted h1n1, h2n2, and h3n2 have mild-to-moderate virulence in ferrets. strains of low virulence have predominant replication in the nasal turbinates. clinical signs and other disease indicators are similar to that of humans with a mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia. 312 replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract ferrets correlates with severe disease and lethal outcome. h5n1-infected ferrets develop severe lethargy, greater ifn response, transient lymphopenia, and replication in respiratory tract, brain, and other organs. 313 old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models, which are deficient for h5n1 vaccine studies because there is a lack of correlation with hemagglutination inhibition. 314 of old world primates, cynomolgus macaque (m. fascicularis) are most frequently used for studies of vaccines and antiviral drug therapies. 315, 316 h5n1 and h1n1 1918 infections of cynos are very similar to those in humans. 317 cynos develop fever and ards upon intranasal inoculation of h5n1 with necrotizing bronchial interstitial pneumonia 318 nhps are challenged by multiple routes (ocular, nasal, and tracheal) simultaneously 1 ã� 10 6 pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h5n1 results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the 1918 h1n1 caused a very high mortality rate due to an aberrant immune response and ards and had >50% lethality (humans only had a 1-3% lethality). ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains such as h3n2. 299 influenzainfected rhesus macaques represent a mild disease model pathogenesis for vaccine and therapeutic efficacy studies. 319 other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates such as squirrel and cebus monkeys. 320 rats (f344 and sd) inoculated with rat-adapted h3n2 developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes. 321 additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets. 322 cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies. 323, 324 cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted. 325, 326 nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at 24 h postexposure. virus was cleared from the lung by day 3 and from the nares by day 66, but animals had bronchial and alveolar damage, and pneumonia for up to 3 weeks. there is also a s. aureus superinfection model in cotton rats. 327 coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic pig influenza models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h5n1. 328, 329 although pigs infected with influenza may have fever, anorexia, and respiratory signs such as dyspnea and cough, mortality is rare. 330 size and space requirements make this animal difficult to work with, although the development of minipig (ellegaard gottingen) models may provide an easier-to-use alternative. incubation period, animals exhibit signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers. 331 mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans. 332,333 after 2-6 days of incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever. [334] [335] [336] depending on the severity of the disease when the symptoms start, 10-20% of the hospitalized patients might die in 3-6 days or 12-17 days after the disease onset. 334 hepatic failure, renal failure or disseminated intravascular coagulation (dic), and encephalitis are demonstrated within patients during postmortem examination. mice are one of the most susceptible animal species to rvfv infection. subcutaneous or intraperitoneal routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice. 337, 338 mice start to exhibit signs of decreased activity and ruffled fur by day 2-3 postexposure. immediately after these signs are observed, they become lethargic and generally die 3-6 days postexposure. ocular diseases or hemorrhagic form of the disease has not been observed in mice models so far. 334 increased viremia and tissue tropism were reported in mice with 338 increased liver enzymes and lymphopenia observed in sick mice. rats and gerbils are also susceptible to rvfv infection. rats' susceptibility is dependent on the rat strain used for the challenge model. there was also noted an age dependence in susceptibility of rats. although wistar-furth and brown norway strains and young rats are highly susceptible to rvfv infection, fisher 344, buffalo and lewis strains, and old rats demonstrated resistance to infection. 339, 340 similar pathologic changes such as liver damage and encephalopathy were observed in both rats and mice. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils. 341 similar to the rat model, the susceptibility of gerbils was also dependent on age. so far, studies showed that rvfv does not cause uniform lethality in an nhp model. intraperitoneal, intranasal, iv, and aerosol routes have been used to develop the nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort. 342 monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to those seen in humans such as pathological changes in liver and hemorrhagic disease. there was no ocular involvement in this model. recently, smith et al. compared iv, intranasal, and subcutaneous routes of infection in common marmosets and rhesus macaques. 343 marmosets were more susceptible to rvfv infection than were rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subcutaneous routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of intranasally challenged marmosets. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, seems to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans. 344 incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts 1-9 days. during the prehemorrhagic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients. 345, 346 over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. [347] [348] [349] [350] until recently, the only animal that manifests disease is the newborn mouse. infant mice infected with cchfv intraperitoneally caused fatality around day 8 postinfection. 351 pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day 6) and the brain (day 7). the recent studies using knockout adult mice were successful to develop a lethal small animal model for cchfv infection. 352, 353 bente et al. infected stat1 knockout mice by the intraperitoneal route. in this model, after the signs of fever, leucopenia, thrombocytopenia, viremia, elevated liver enzymes, and proinflammatory cytokines, mice were moribund and succumbed to disease in 3-5 days of postexposure. the second model was developed by using ifn-alpha/beta (ifna/b) receptor knockout mice. 353 similar observations were made in this model as in the stat1 knockout mouse model. the animals were moribund and died 2-4 days after exposure with high viremia levels in the liver and spleen. other laboratory animals, including nhps, show little or no signs of infection or disease when infected with cchfv. 348 butenko et al. used agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day 4 postinfection, the animals did not exhibit signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. in 1975, fagbami et al. infected two patas monkeys (cercopithecus erythrocebuserythrocebus) and one guinea baboon (papio papio) with cchfv. 347 although all three animals had low level viremia between days 1 and 5 after inoculation, only the baboon serum had neutralizing antibody activity on day 137 postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia, 354 and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least 3 months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes. 355 shepherd et al. infected 11 species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. 349 although scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (tatera brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and m. coucha), and syrian hamsters were negative. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed the onset of viremia earlier than those infected by the subcutaneous or intraperitoneal routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather by rodents. 356 rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by the inhalation of infected rodent saliva, feces, and urine. 357 human infections can normally be traced to a rural setting with activities such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention. 358 the viruses have a tropism for endothelial cells within the microvasculature of the lungs. 359 there are two distinct clinical diseases that infection can yield; hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses. 360 hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul. 358 incubation lasts two to three weeks and presents as flulike in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops, which can further progress to shock in approximately 15% patients. the overall mortality rate is 7%. infection with dobrava and hantaan viruses are typically linked to the development of severe disease. hps was first diagnosed in 1993 within the southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus. 361 this virus is still the leading cause within na of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay. 362, 363 the first report of hps in maine was recently documented. 361 andes virus was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans. 364 the fulminant disease is more lethal than that observed for hfrs with a mortality rate of 40%. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation. 365 incubation typically occurs 14-17 days after exposure. 366 unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within 48 h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted. 367 vaccine development has been hampered by the vast diversity of hantaviruses and the limited number of outbreaks. 368 syrian golden hamsters are the most widely used small animal models for hantavirus infection. hamsters inoculated intramuscularly with a passaged andes viral strain died within 11 days postinfection. clinical signs did not appear until 24 h before death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged sin nombre isolate. no hamsters developed any symptoms during the course of observation. although an antibody response to the virus that was not dose dependent was determined via an enzymelinked immunosorbent assay. hamsters infected with andes virus were found to have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day 8 and lasted up to 12 days postinfection. infection of hamsters with andes virus yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver. 369 lethal disease can be induced in newborn mice but does not recapitulate the clinical symptoms observed in human disease. 370 adult mice exposed to hantaan virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease. 371, 372 knockout mice lacing ifn-a/b were found to be highly susceptible to hantaan virus infection. 373 in a study looking at a panel of laboratory strains of mice, c57bl/6 mice were found to be most susceptible to a passaged hantaan viral strain injected intraperitoneally. animals progressed to neurological manifestation including paralyses and convulsions and succumbed to infection within 24-36 h postinfection. clinical disease was markedly different than that observed in human cases. 372 nhps have been challenged with new world hantaviruses; however, no clinical signs were reported. 374, 375 cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease. 376, 377 challenge with andes virus to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within 5 days postinfection. aerosol exposure led to 4 of 6 monkeys and 8 of 11 iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all these viruses share common clinical manifestations. 378 lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april. 379 this virus is responsible for 100,000-500,000 infections per year, leading to approximately 5,000 deaths. 380 outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases sprung up in germany, the netherlands, the united kingdom, and the united states due to transmission to travelers on commercial airlines. 381 transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex. 379 humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease can occur in 20% of individuals. the incubation period is from 5 to 21 days, and the initial onset is characterized by flulike illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease, which is permanent for a third of those affected. the overall fatality is about 1%; however, of those admitted to the hospital, it is between 15% and 25%. there is no approved vaccine, and besides supportive measures, ribavirin is effective only if started within 7 days. 382, 383 the primary animal model used to study lassa fever is the rhesus macaque. 384 aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus more closely mirrored the disease course and histopathology observed in human infection. 385 iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt. 386 disease was dose-dependent with iv, intramuscular, and subcutaneous inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be used, and mimic a more natural route of infection. 387 within this model, the nhp becomes viremic after 4-6 days. clinical manifestations were present by day 7, and death typically occurred within 10-14 days. 388, 389 intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns. 390 this pathogenicity is seen in select cases of human lassa fever. 391, 392 a marmoset model has recently been defined using a subcutaneous injection of lassa fever virus. virus was initially detected by day 8 and viremia achieved by day 14. liver enzymes were elevated, and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases. 393 mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is completely dependent upon the major histocompatibility complex (mhc) background and age of animal along with the route of inoculation. 394 guinea pig inbred strain 13 was found to be highly susceptible to lassa virus infection. the outbreed hartley strain was less susceptible, and thus, strain 13 has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus. 395 infection with pichinde virus that has been passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death. 396, 397 the guinea pig is an excellent model given that it not only results in similar disease pattern as humans but also the viral distribution is similar along with the histopathology and immune response. 398, 399 infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig model. the virus was injected intraperitoneally resulting in the animals becoming lethargic and anorexic within 6-7 days. virus was first detected at 3 days and reached maximum titers within 5 days. neurological symptoms began to appear at the same time, and all the animals died by day 9. pneumonitis, pulmonary hemorrhage, and edema were also present. 400 these results were recapitulated with a nonadapted pichinde virus. [401] [402] [403] globally, diarrheal disease is the leading cause of death with rotavirus being one of the main etiological agents responsible. according to the world health organization, rotavirus alone is responsible for a third of all hospitalization related to diarrhea and 500,000-600,000 deaths per year. 404 the virus is very stable due to its three-layer capsid, which allows it to be transmitted via the oral-fecal route, depositing itself in the small intestine. rotavirus is highly contagious, and only 10 viruses are needed to cause symptomatic disease. 405 the host determinant with the greatest influence on clinical outcome is age. neonates typically are asymptomatic, which is suggested to be due to the existence of maternal antibodies. hence, the most susceptible age group is 3 months to 2 years, coinciding with a drop in these protective antibodies. 406 within this age range, children will develop noninflammatory diarrhea. virus replicates in the intestinal villus enterocytes resulting in their destruction and malabsorption of needed electrolytes and nutrients. symptoms of disease include watery, nonbloody diarrhea with vomiting, fever, and potentially dehydration that lasts up to a week. 407 there is a short episode of viremia during the course of infection. 408 mice can be used as both an infection and disease model depending upon age at challenge. mice <14 days old develop disease, whereas older mice are able to clear the infection before the onset of symptoms. 409 this halts the study of active vaccination against disease in the infection model. in the adult mouse model, the course of the infection is monitored via viral shedding within the stool. 410 infant mice, specifically balb/c, receiving an oral inoculation of a clinical strain of virus developed diarrhea within 24 h postinfection, and 95% of those exposed developed symptoms within 72 h postinfection. symptoms lasted from 2 to 4 days with no mortality. viral shedding was at its peak at 24 h and lasted up to 5 days. there were noted histopathological changes within the small intestine localized to the villi that was reversible. 407 within the adult mouse model, oral inoculation of a mouse rotavirus strain showed viral shedding by 3 days lasting up until 6 days postinfection. 410 these mouse models have been used to study correlates of protection and therapeutic efficacy including gastro-gard ã� . 409, 411, 412 rats can also be used as disease models depending upon the strain of rat. 413, 414 suckling fischer 344 rats were exposed to a simian strain of rotavirus orally. the rats were susceptible to diarrheal disease till they were 8 days old with age determining the length of viral shedding. 415 rats have mainly been used to study the correct formulation for oral rehydration. these rodents are large enough to perform in situ intestinal perfusions. within these studies, 8-day-old rats were infected with a rat strain of rotavirus by orogastric intubation. within 24 h postinfection, the rats developed diarrhea, at which point the small intestine was perfused to compare differing solutions of oral rehydration. 416 gnotobiotic pigs are also used given that they can be infected with both porcine and human strains. 417 they are susceptible to developing clinical disease from human strains up to 6 weeks of age. they allow for the analysis of the primary immune response to the virus given that they do not receive transplacental maternal antibodies and are immune competent at birth. 418 another advantage of this model is that the gastrointestinal physiology and mucosal immune system closely resemble that of humans. 419 this model has been useful in studying correlates of protection. gnotobiotic and colostrum-deprived calves have also been used as an experimental model of rotavirus infection. they are able to develop diarrhea and shed live virus. 420 gnotobiotic lambs can also develop clinical disease upon oral inoculation with clinical strains. 421 infant baboons, agms, and rhesus macaques have all proven to be infection models with severity measure by viral shedding. 422, 423 retroviridae human immunodeficiency virus type 1 the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects 0.6% of the world's population. a greater proportion of infections and deaths occur in sub-saharan africa. worldwide, there are approximately 1.8 million deaths per year with >260,000 being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv-1 and hiv-2, with hiv-2 having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv-1. 424 hiv targets t-helper cells (cd4ã¾), macrophages, and dendritic cells. 425 acute infection occurs 2-4 weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd8ã¾ t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd4ã¾ t-cells decline to <200 cells per microliter; thus, cell-mediated immunity becomes impaired, and the person is more susceptible to opportunistic infections and certain cancers. humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of intact and functional human innate and adaptive immune responses. 426 the scidhu hiv infection model has proven to be useful, particularly in screening antivirals and therapeutics. 427 a number of different humanized mouse models have been developed for the study of hiv, including rag1ã�/ã�gcã�/ã�, rag2ã�/ ã�gcã�/ã�, nod/scidgcã�/ã� (hnog), nod/ scidgcã�/ã� (hnsg), nod/scid blt, and nod/ scidgcã�/ã� (hnsg) blt. cd34ã¾ human stem cells derived from the umbilical cord blood or fetal liver are used for humanization. 428 hiv-1 infection by intraperitoneal injection can be successful with as little as 5% peripheral blood engraftment. 429 vaginal and rectal transmission models have been developed in blt scidhu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv-1 viremia occurs within approximately 7 days pi. 430 in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans. 431 importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hiv-infected humanized mice, and at least some mechanisms of pathogenesis that occur in hiv-infected humans also occur in the hiv-infected humanized mouse models. 432 the advantage of these models is that these mice are susceptible to hiv infection, and thus, the impact of drugs on the intended viral targets can be tested. one caveat is that although mice have a "common mucosal immune system," humans do not, due to the differences in the distribution of addressins. 433 thus, murine mucosal immune responses to hiv do not reflect those of humans. there are a number of important nhp models for human hiv infection. simian immunodeficiency virus (siv) infection of macaques is widely considered to be the best platform for modeling hiv infection of humans. importantly, nhps have similar, pharmacokinetics, metabolism, mucosal t-cell homing receptors, and vascular addressins to those of humans. thus, although the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through the use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero, or through breast milk. [434] [435] [436] there are also macaque models to study the emergence and clinical implications of hiv drug resistance. 437 these models most routinely use rhesus macaques (macaca mulatta), cynomolgus macaques (macaca fasicularis), and pigtailed macaque (macaca nemestrina). animals of all ages are used, depending on the needs of the study. for instance, the use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently used. studies are performed in bsl-2 animal laboratories, and nhps must be of simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied. 438 challenges may be through a single high dose. iv infection of rhesus macaques with 100 tcid 50 of the highly pathogenic siv/deltab670 induces aids in most macaques within 5-17 months (mean of 11 months). 439 peak viremia occurs around week 4. aids in such models is often defined as cd4ã¾ t-cells that have dropped to <50% of the baseline values. alternatively, repeated low-dose challenges are often used, depending on the requirements of the model. 440, 441 because nhps infected with hiv do not develop an infection with a clinical disease course similar to that in humans, siv or siv/hiv-1 laboratory-engineered chimeric viruses (simian-human immunodeficiency virus or shiv) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease, which progresses to aids and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv-1 and is more closely related to hiv-2. however, the polymerase region of siv is 60% homologous to that of hiv-1, and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors; thus, hiv-1 rt is usually put into siv for such studies. 442 sivmac239 is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt or integrase inhibition. 443 nhps infected with sivmac239 have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd4ã¾ t-cell depletion. additionally, sivmac239 uses the cxcr5 chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry. 444 nhps infected with shiv strains may not develop aids, but these models are useful in testing vaccine efficacy. for example, rt-shivs and env-shivs are useful for the testing and evaluation of drugs that may target the envelope or rt, respectively. 442 one disadvantage of the highly virulent env-shiv (shiv-89.6 p) is that it uses the cxcr4 coreceptor. of note, env-shivs that do use the cxcr5 coreceptor are less virulent; viremia develops and then resolves without further disease progression. 445 simian-tropic (st) hiv-1 contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for three months, followed by clearance. 446 a number of routes are used for siv or shiv infection of nhps, with iv inoculation being the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than does the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) one month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase the susceptibility to infection by atraumatic vaginal instillation. 447 upon vaginal instillation of 500 tcid50 of shiv-162p3, peak viremia was seen around 12 days postexposure with >10 7 copies per milliliter and dropping thereafter to a constant level of 10 4 rna copies per milliliter at 60 days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac251. 448 an example of an intrarectal model used juvenile (2year-old) pigtailed macaques, challenged intrarectally with 10 4 tcid 50s of siv mne027 to study the pathogenesis related to the virulence factor, vpx. 449 here, viremia peaked at approximately 10 days with >10 8 copies per milliliter. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistently high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of highly active antiretroviral therapy on virus and t-cells in the male genital tract, adult (3-to 4-year old) male cynomolgus macaques were intravenously inoculated with 50 aid50s of sivmac251, and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization. 450 pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course. 451 importantly, mother-to-infant transmission models have also been developed. 452 pregnant female pigtailed macaques were infected during the second trimester with 100 mid 50 shiv-sf162p3 by the iv route. four of nine infants were infected; one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission and the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and terminal-repeat retrotransposon in miniature (trim) alleles), differences between challenge strains, and challenge routes. 453 nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the longterm clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade. 454 unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study that used an adenovirus-based vaccine approach. 455 vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv is more difficult to protect than mucosal and is used as a "worst-case scenario." however, efficacy at one mucosal route is usually comparable to that at other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect. 456 there are >100 human papillomaviruses (hpvs), with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately a third of these are transmitted sexually. of these, virulent subtypes such as hpv-16, hpv-18, hpv-31, hpv-33, and hpv-45 place individuals at high risk for cervical and other cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist that use animal papillomaviruses in their natural host, or a very closely related species. 457, 458 these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines. 459 wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which are a very closely related species. 460 in this model, papillomas can range from cutaneous squamous cell carcinomas on the one end of the spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) cause florid warty lesions in the mucosa of the oral cavity within 4-8 weeks postexposure in experimental settings. 461 the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv-6 and hpv-11 make this a useful model. 462 these lesions typically spontaneously regress 4-8 weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged 10 weeks to 2 years, with no history of copv, are typically used for these studies. infection is achieved by the application of a 10 ml droplet of virus extract to multiple 0.5 cm 2 scarified areas within the mucosa of the upper lip of anesthetized beagles. 463 bovine papillomavirus (bpv) has a wider host range than do most papillomaviruses, infecting the fibroblasts cells of numerous ungulates. 458 bpv-4 infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques, rhesus papillomavirus, is very similar to hpv-16 and is associated with the development of cervical cancer. 464 mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, but they are often used to look at immunogenicity of vaccines. 465, 466 herpesviridae please see chapter 25. monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including squirrels. 467, 468 mpxv was discovered during the pox-like disease outbreak among laboratory java macaques in denmark in 1958. no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until 1970 in zaire (the present drc) with the continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of 2003, a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and the western hemisphere. among 72 reported cases, 37 human cases were laboratory confirmed during an outbreak. 469, 470 it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-toperson spread occurs by large respiratory droplets or direct contact. 471 most of the clinical features of human monkeypox are very similar to those of ordinary smallpox. 472 after a 7-to 21-day incubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash. 471, 473 typical maculopapular rash follows the prodromal period generally lasting 1-3 days. the average size of the skin lesions is 0.5-1 cm, and the progress of lesions follows the order macules through papules, vesicles, pustules, umblication then scab and desquamation and lasts typically 2-4 weeks. fatality rate is 10% among the unvaccinated population and death generally occurs during the second week of the disease. 469, 471 mpxv is highly pathogenic for a variety of laboratory animals, and so far, many animal models have been developed by using different species and different routes of exposure (table 38. 3). because of the unavailability of variola virus to develop animal models and resulting disease manifestations in humans that are similar, mpxv is one of the pox viruses that are used very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat1-deficient c57bl/6 mouse, prairie dogs, african dormice, ground squirrels are highly susceptible to mpxv by different exposure routes. [474] [475] [476] [477] [478] [479] [480] [481] cast/eij mice, one of the 38 inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose-dependent mortality after intranasal exposure to mpxv. studies with the intraperitoneal route of challenge indicated a higher susceptibility to mpxv with an almost 50-fold less ld50 when compared to the intranasal route. 474 scid-balb/c mice were also susceptible to the intraperitoneal challenge route, and the disease resulted in mortality day 9 postinfection. 476 similarly c57bl/6 stat1 ã�/ã� mice were infected intranasally with mpxv, and the infection resulted in weight loss and mortality 10 days postexposure. mice models mentioned here are very promising for screening of therapeutics against pox viruses, but testing in additional models will be required for advanced development. high doses of mpxv by intraperitoneal or intranasal route caused 100% mortality in 6 days postexposure and 8 days postexposure, respectively, in ground squirrels. 480 the disease progressed very quickly, and most of the animals were lethargic and moribund by day 5 postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv in ground squirrels by subcutaneous route resulted in systemic disease and the mortality in 6-11 days postexposure. the disease resembles hemorrhagic smallpox with nose bleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. because in the us outbreak the virus was transmitted by infected prairie dogs, this animal model has recently been studied much further and used to test therapeutics and vaccines compared to other small animal models. 475, 481, 491, 492 studies using intranasal, intraperitoneal, and intradermal routes of exposure showed that mpxv was highly infectious to prairie dogs. by using the west african mpxv strain, the intraperitoneal route caused a more severe disease and 100% mortality than challenge by the intranasal route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to the intraperitoneal route, the intranasal route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in intraperitoneally infected animals. 481 recent studies by hutson et al. used intranasal and intradermal infections with west african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and generalized pox lesions. 475 therefore, this model can be used for testing therapeutics and vaccines against pox viruses. the african dormouse is susceptible to mpxv by the foodpad injection route or intranasal route. 478 mice exhibited decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of 200 and 2000 pfu provided 100% mortality with a mean time to death of 8 days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and hemorrhage in lungs were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. considering the limited availability of ground squirrels and african dormice, lack of reagents to these species, and resemblance to hemorrhagic smallpox disease, these models are not very attractive for further characterization and vaccine and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal models for mpxv. 482, 483, 485, 489, 490 during our studies by using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day 6 postexposure. 482 complete blood count and clinical chemistries showed abnormalities similar to those of human monkeypox cases with leukocytosis and thrombocytopenia. 493 whole-blood and throat swabs had viral loads peak around day 10, and in survivors, gradually decrease until day 28 postexposure. because doses of 4 ã� 10 4 , 1 ã� 10 5 , or 1 ã� 10 6 pfu resulted in lethality for 70% of the animals, whereas a dose of 4 ã� 10 5 pfu resulted in 85% lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, before animals can develop pox lesions, they succumbed to disease. with the low challenge dose, pox lesions were observed, but they were few in comparison to the iv model. mpxv causes dose-dependent disease in nhps when given by the iv route. 490 studies showed that with 1ã� 10 7 pfu iv, challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an intratracheal infection model deposits virus into the trachea, delivering directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation by skipping the upper respiratory system. fibrinonecrotic bronchopneumonia was described in animals that received 10 7 pfu of mpxv intratracheally. 489 although a similar challenge dose of intratracheal mpxv infection resulted in a similar viremia in nhps than with the aerosol route of infection, the timing of the first peak was delayed by 5 days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately 100-fold lower. this suggests that local replication is more prominent after aerosol delivery compared to that after intratracheal delivery. an intrabronchial route of exposure resulted in pneumonia in nhps. 490 delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and the intratracheal route of infection models, the number of pox lesions was much less than in the iv route of the infection model. a major downside of the iv, intratracheal and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus into the blood stream or into the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human variola virus (varv) infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is decided to be used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b is one of the most common infections worldwide with >400 million people chronically infected and 316,000 cases per year of liver cancer due to infection. 494 the virus can naturally infect both humans and chimpanzees. 495 hepatitis b is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture. 496 the age at which one is infected dictates the risk of developing chronic disease. 497 acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms last for a few weeks before resolving. 498 after this acute phase, life time immunity is achieved. 499 of those infected, <5% will develop the chronic form of disease. chronicity is the most serious outcome of disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is 100 times more likely to develop in a chronically infected individual than in a noncarrier. 500 the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepadna virus suggest that x protein may be responsible. 501 many individuals are asymptomatic until complications emerge related to chronic carriage. chimpanzees have a unique strain that circulates within the population. 502, 503 it was found that 3-6% of all wild-caught animals from africa are positive for hepatitis b antigen. 504 natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease. 505 to date, the use of chimpanzees provides the only reliable method to ensure that plasma vaccines are free from infectious particles. 506 this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially attuned to these studies given that their immune response to infection directly mirrors humans. 507 other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hepatitis b, none develop hepatic lesions or liver damage as noted by monitoring of liver enzymes. 508 mice are not permissible to infection, and thus, numerous transgenic and humanized lines that express hepatitis b proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease. 495 recently, the entire genome of hepatitis b was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection. 509 hepatitis b can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses. 510 the woodchuck hepatitis virus was found to induce hepatocellular carcinoma. 511 within a population, 65-75% of all neonatal woodchucks are susceptible to chronic infection. 512 a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within 3 years of the initial infection in woodchucks, whereas human infection takes much longer. 513 the acute infection strongly resembles what occurs during the course of disease in humans. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage. 514 challenge with virus in neonates leads to a chronic infection, while adults only develop the acute phase of disease. 515 a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. it was found to develop an acute hepatitis with a productive infection. 516 hepatitis d is dependent upon hepatitis b to undergo replication and successful infection in its human host. 517 there are two modes of infection possible between the viruses: coinfection in which a person is simultaneously infected or superinfection in which a chronic carrier of hepatitis b is subsequently infected with hepatitis d. 518 coinfection leads to a similar disease as seen with hepatitis b alone; however, superinfection can result in chronic hepatitis d infection and severe liver damage. 519 both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d. 520 a recently published report demonstrated the use of a humanized chimeric mouse to study the interactions between the two viruses and drug testing. 521 the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus be a low passage clinical isolate; thus, animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that which occurs in natural disease. to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the above characteristics cannot always be satisfied, however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for licensure to satisfy the food and fda's "animal rule." this rule applies to situations in which vaccines and therapeutics cannot safely or ethically be tested in humans; thus, licensure will come only after preclinical tests are performed in animal models. many fields in virology are moving toward standardized models that can be used across institutions to test vaccines and therapeutics. a current example of such an effort is within the filovirus community, where animal models, euthanasia criteria, assays, and virus strains are in the process of being standardized. the hope is that these efforts will enable results of efficacy tests on medical 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ferret model by an intranasal virosome-formulated influenza subunit vaccine local innate immune responses and influenza virus transmission and virulence in ferrets regional t-and b-cell responses in influenza-infected ferrets human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals live, attenuated influenza virus (laiv) vehicles are strong inducers of immunity toward influenza b virus [comparative study the ferret: an animal model to study influenza virus pathogenesis of avian influenza a (h5n1) viruses in ferrets severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction neuropathology of h5n1 virus infection in ferrets evaluation of three strains of influenza a virus in humans and in owl, cebus, and squirrel monkeys a computationally optimized hemagglutinin virus-like particle vaccine elicits broadly reactive antibodies that protect nonhuman primates from h5n1 infection evaluation of intravenous zanamivir against experimental influenza a (h5n1) virus infection in cynomolgus macaques aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus pathology of human influenza a (h5n1) virus infection in cynomolgus macaques (macaca fascicularis integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics integration of clinical data, pathology, and cdna microarrays in influenza virus-infected pigtailed macaques (macaca nemestrina kinetic profile of influenza virus infection in three rat strains the guinea pig as a transmission model for human influenza viruses influenza-induced tachypnea is prevented in immune cotton rats, but cannot be treated with an anti-inflammatory steroid or a neuraminidase inhibitor the antiviral potential of interferon-induced cotton rat mx proteins against orthomyxovirus (influenza), rhabdovirus, and bunyavirus the cotton rat as a model to study influenza pathogenesis and immunity the cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis co-infection of the cotton rat (sigmodon hispidus) with staphylococcus aureus and influenza a virus results in synergistic disease pathogenicity of a highly pathogenic avian influenza virus, a/chicken/yamaguchi/7/04 (h5n1) in different species of birds and mammals domestic pigs have low susceptibility to h5n1 highly pathogenic avian influenza viruses [comparative study research support animal models in influenza vaccine testing rift valley fever: an uninvited zoonosis in the arabian peninsula prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the 1993 outbreak in egypt rift valley fever the occurrence of human cases in johannesburg the pathogenesis of rift valley fever epidemic rift valley fever in egypt: observations of the spectrum of human illness crc handbook series in zoonoses rift valley fever virus in mice. i. general features of the infection the pathogenesis of rift valley fever virus in the mouse model the susceptibility of rats to rift valley fever in relation to age inbred rat strains mimic the disparate human response to rift valley fever virus infection the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis experimental rift valley fever in rhesus macaques development of a novel nonhuman primate model for rift valley fever crimean-congo hemorrhagic fever: a global perspective crimean-congo hemorrhagic fever the clinical pathology of crimean-congo hemorrhagic fever experimental congo virus (ib-an7620) infection in primates crimean congo hemorrhagic fever: a global perspective viremia and antibody response of small african and laboratory animals to crimean-congo hemorrhagic fever virus infection a comparative study of the crimean hemorrhagic faverdcongo group of viruses [comparative study ribavirin efficacy in an in vivo model of crimean-congo hemorrhagic fever virus (cchf) infection pathogenesis and immune response of crimean-congo hemorrhagic fever virus in a stat-1 knockout mouse model crimean-congo hemorrhagic fever virus infection is lethal for adult type i interferon receptorknockout mice possibility of extracting hyperimmune gammaglobulin against chf from donkey blood sera study of susceptibility to crimean hemorrhagic fever (chf) virus in european and long-eared hedgehogs. tezisy konf vop med virus field's virology epidemiological studies of hemorrhagic fever with renal syndrome: analysis of risk factors and mode of transmission a short review hantavirus pulmonary syndrome. pathogenesis of an emerging infectious disease field's virology centers for disease control & prevention. outbreak of acute illness-southwestern united states genetic diversity, distribution, and serological features of hantavirus infection in five countries in south america first reported cases of hantavirus pulmonary syndrome in canada [case reports an unusual hantavirus outbreak in southern argentina: person-to-person transmission? hantavirus pulmonary syndrome study group for patagonia hantavirus pulmonary syndrome: the new american hemorrhagic fever the incubation period of hantavirus pulmonary syndrome cardiopulmonary manifestations of hantavirus pulmonary syndrome hantavirus pulmonary syndrome a lethal disease model for hantavirus pulmonary syndrome pathogenesis of hantaan virus infection in suckling mice: clinical, virologic, and serologic observations infection of hantaan virus strain aa57 leading to pulmonary disease in laboratory mice hantaan virus infection causes an acute neurological disease that is fatal in adult laboratory mice functional role of type i and type ii interferons in antiviral defense andes virus dna vaccine elicits a broadly cross-reactive neutralizing antibody response in nonhuman primates andes virus infection of cynomolgus macaques wild-type puumala hantavirus infection induces cytokines, 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phylogenetic relationships with human and other primate genotypes antibody to hepatitis-associated antigen. frequency and pattern of response as detected by radioimmunoprecipitation hepatitis and blood transfusion perspectives on hepatitis b studies with chimpanzees hla a2 restricted cytotoxic t lymphocyte responses to multiple hepatitis b surface antigen epitopes during hepatitis b virus infection primates in the study of hepatitis viruses transfer of hbv genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice asymmetric replication of duck hepatitis b virus dna in liver cells: free minus-strand dna a virus similar to human hepatitis b virus associated with hepatitis and hepatoma in woodchucks effects of age and viral determinants on chronicity as an outcome of experimental woodchuck hepatitis virus infection hepadnavirusinduced liver cancer in woodchucks animal models of hepadnavirus-associated hepatocellular carcinoma hepatitis b viruses and hepatocellular carcinoma hepatitis b virus replication in primary macaque hepatocytes: crossing the species barrier toward a new small primate model animal models of hepatitis delta virus infection and disease experimental hepatitis delta virus infection in the chimpanzee expression of the hepatitis delta virus large and small antigens in transgenic mice experimental hepatitis delta virus infection in the animal model humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation key: cord-336663-fawcn6em authors: liu, chunyan; xiao, yan; zhang, jing; ren, lili; li, jianguo; xie, zhengde; xu, baoping; yang, yan; qian, suyun; wang, jianwei; shen, kunling title: adenovirus infection in children with acute lower respiratory tract infections in beijing, china, 2007 to 2012 date: 2015-10-01 journal: bmc infect dis doi: 10.1186/s12879-015-1126-2 sha: doc_id: 336663 cord_uid: fawcn6em background: human adenoviruses (hadv) play a significant role in pediatric respiratory tract infections. to date, over 60 types of hadv have been identified. here, hadv types are characterized in children in the beijing area with acute lower respiratory tract infections (alrtis) and the clinical features and laboratory findings of hospitalized hadv-infected cases are described. methods: respiratory specimens were collected from pediatric patients with alrtis in the emergency department or from those admitted to beijing children’s hospital between march 2007 and december 2012. infections with common respiratory viruses were determined by pcr or rt-pcr. hadv positive samples were further typed by pcr and sequencing. results: among 3356 patients with alrtis, 194 (5.8 %) were found to have hadv infection. hadv infection was primarily confined to children (88.35 %) less than 5 years of age. a total of 11 different types of hadv were detected throughout the study period, with hadv-b7 (49.0 %) and hadv-b3 (26.3 %) as the most prevalent types, followed by hadv-c2 (7.7 %) and hadvc1 (4.6 %). newly emerging and re-emergent types or variants, hadv-b55 (n = 5), hadv-c57 (n = 3), and hadv-b14p1 (n = 1), were identified. results also included the reported first case of co-infection with hadv-c2 and hadv-c57. clinical entities of patients with single hadv infection (n = 49) were similar to those with mixed hadv/respiratory syncytial virus (rsv) infections (n = 41). patients with hadv-b7 infection had longer duration of fever and higher serum levels of muscle enzymes than hadv-b3-infected patients. conclusions: during the study period, hadv-b7 and hadv-b3 were the predominant types identified in pediatric alrtis. hadv-b7 infection tends to have more severe clinical consequences. the presence of newly emerging types or variants and co-infection with different types of hadv highlights the need for constant and close surveillance of hadv infection. acute lower respiratory tract infections (alrtis) are the leading cause of pediatric morbidity and mortality worldwide, particularly in developing countries. in infants and young children, alrtis are most frequently caused by respiratory viruses. one such virus, human adenovirus (hadv), plays a significant role in pediatric respiratory tract infections, accounting for 2-5 % of the overall respiratory illnesses and 4-10 % of the pneumonias [1, 2] . although most cases are mild and indistinguishable from other viral causes, alrtis caused by hadv can be severe, or even fatal, and are associated with the highest risk of long term respiratory sequelae [3] . thus, hadvassociated alrtis are of particular interest to both clinicians and researchers. hadv are responsible for a wide spectrum of clinical diseases, including respiratory illness (both upper and lower respiratory tract), pharyngoconjunctival fever, conjunctivitis, cystitis, gastroenteritis, and neurologic and venereal disease [4] . hadv were first isolated in 1953 as respiratory pathogens [5, 6] . to date, over 60 types of hadv have been identified and classified into seven species (a to g) [7] [8] [9] [10] [11] [12] [13] [14] . cases of severe infection, outbreaks in closed populations, and even epidemic outbreaks have been associated with the newly emerging or re-emergent types or variants [15] [16] [17] . interestingly, different types of hadv display various tissue tropisms that correlate with different clinical manifestations of infection. hadv infections of the respiratory tract are predominantly caused by hadv-b (including subspecies b1 and b2), hadv-c, or hadv-e. the predominant types vary among different countries and regions and they change over time because transmission of novel strains between countries or across continents may occur [18] . type identification is critical to epidemiological surveillance, detection of new strains, and understanding of hadv pathogenesis. however, because most clinical laboratories do not type the isolates, there is little published information about epidemiologic and clinical features of hadv infections by type in children with alrtis. to identify hadv types and species in children with alrtis in beijing area and to characterize clinical features and laboratory findings of hospitalized hadvinfected cases, respiratory specimens were collected from hospital-admitted pediatric patients with alrtis and typed hadv positive samples using pcr and sequencing. the study protocol was approved by the ethical review committee of beijing children's hospital. individual written informed consent was obtained from the parents or guardians of all participants. from march 2007 to december 2012, pediatric patients with alrtis who presented in emergency department or were admitted to respiratory department or intensive care unit, beijing children's hospital, were recruited for the study. the study site hospital is a tertiary comprehensive pediatric hospital with over 900 beds and more than twenty clinical departments. alrtis were defined as the presence of signs and symptoms of respiratory tract infection (i.e., fever, coughing, rhinorrhea, oropharyngeal hyperemia, swelling of tonsils), and lower respiratory signs (tachypnea, dyspnea, retractions, or wheezing/rales upon auscultation). the patients were diagnosed with bronchitis, bronchiolitis or pneumonia. chest x-rays were taken for all patients and the criteria for diagnosing pneumonia are the presence of lung infiltrates indicated by chest radiography. nasopharyngeal aspirate or throat swab specimens were collected in virus transport media from each patient. no repeated samples were collected from any patient. all samples were stored at −80°c prior to use. total nucleic acids (dna and rna) were extracted from 200 μl nasopharyngeal aspirate or throat swab specimens using the nuclisens easymag™ automated extraction system (biomérieux, marcy l'etoile, france) according to the manufacturer's instructions and eluted in 60 μl elution buffer. the presence of common respiratory viral agents, including parainfluenza virus (piv) type 1-4, influenza virus (ifv), respiratory syncytial virus (rsv), human rhinovirus (hrv), enterovirus (ev), human coronavirus (hcov 229e, nl63, hku1, and oc43), human metapneumovirus (hmpv), human bocavirus (hbov), and hadv was determined by multiplex rt-pcr, single rt-pcr, or pcr assays as previously described [19, 20] . blank virus transport media here served as a negative control for nucleic acid extraction and pcr. hadv positive samples were further amplified using a nested pcr procedure that targeted hyper variable regions 1-6 of the hexon gene as described by lu and erdman [21] . expected amplicons ranged from 688 bp to 821 bp (secondary amplification) in length. sequencing was performed in both directions using the amplification primers. sequences were proof read and assembled using seqman software v7.1.0 (dnastar inc., wi, u.s.). for assignment of molecular identity and identification of the closest match, sequence alignment was performed using the basic local alignment search tool (blast) against ncbi genbank database (http://www.ncbi.nlm.nih.gov). clinical data were retrospectively recorded by careful analysis of patient medical files in beijing children's hospital, using a predefined microsoft excel spreadsheet. patients' demographic, clinical, and radiologic findings were collected. continuous variables were summarized as means ± standard deviations (sd) or medians. for categorical variables, percentages of patients in each category were calculated. differences between groups were assessed using pearson's chi square test or fisher's exact test for categorical variables and the one way anova, independent-samples t test, mann-whitney u test, and kruskal-wallis test for continuous variables. all analyses were performed using spss software, version 19.0 (ibm corporation, ny, u.s.). all tests were calculated in a two-tailed manner and a p value of <0.05 was considered statistically significant. from march 2007 through december 2012, a total of 3356 patients with alrtis (2766 with pneumonia, 309 with bronchitis and 281 with bronchiolitis) were enrolled in this study. the mean age of study participants was 3.87 ± 4.03 years (median 1 year; age range, 0.5 month to 17 years and 17 months). there were 2085 male participants with a male-to-female ratio of 1.64:1. at least one respiratory virus was detected in nasopharyngeal aspirate or throat swab specimens of 2322 (69.2 %) enrolled participants. rsv (33.4 %) was the most commonly detected viral pathogen, followed by hrv (26.6 %) and piv (13.6 %). one hundred and ninety-four patients (5.8 %, 194/3356) were found to have hadv infection, representing 8.7 % (194/2232) of patients with positive respiratory samples. male paticipants were more likely to be infected with hadv (135 boys and 59 girls, male to female ratio = 2.3:1). the mean age of infection was 2.13 ± 2.68 years (median, 1 year; age range, 1 month to 15 years). most of hadv-infected cases (88.35 %) were under 5 years of age and the highest percentage of hadv infections (42.47 %) occurred in infants (age group 0-< 1 year), followed by the age group 1-< 2 years (27.84 %). additionally, one or more other respiratory viruses were detected in 69.6 % (n = 135) of 194 hadv-infected participants. dual viral infection was identified in 75 cases, triple infection in 45 cases, quadruple in 13 and quintuple in 2. rsv (n = 56) was the most frequently co-detected virus, followed by hrv (n = 53) and piv (n = 42). hbov (n = 24), hcov (n = 15), ifv (n = 8), ev (n = 6), and hmpv (n = 5) were also found to be co-infected with hadv. one hundred and ninety-four hadv-positive specimens were all successfully typed by hexon gene amplifying and sequencing. throughout the study period, four species (a, b, c, e) of hadv, including 11 different types were identified. additionally, hadv-b7 (n = 95; 49.0 %) and hadv-b3 (n = 51; 26.3 %), which belong to species b, were the most prevalent hadv types, accounting for 75.3 % of all hadv-associated infections. hadv-c2 (7.7 %), hadv-c1 (4.6 %), hadv-c5 (3.6 %), hadv-b55 (2.6 %), hadv-c6 (1.5 %), hadv-c57 (1.5 %), hadv-a31 (1.0 %), hadv-b14 (0.5 %), and hadv-e4 (0.5 %) were also detected. interestingly, sequencing results from one specimen showed superimposed peaks in the chromatograms. to confirm the possibility of multiple hadv strains in that sample, pcr products were cloned and sequenced further. distinct hexon genes of different types (hadv-c2 and hadv-c57) were verified. hadv detection rate varied through the years, ranging from 2.55 % in 2007 to 9.15 % in 2010 (fig. 1) . additionally, although hadv was detected throughout the year, cases commonly peaked in winter and spring season (fig. 2) . furthermore, different types of hadv did not remain constant across the whole study period (fig. 2) . specifically, hadv-c1, −c2, −b3, and -b7 were detected throughout the study; hadv-c5 in all years except 2007; hadv-c6 and hadv-c57 in years 2008, 2009, among the 194 hadv-positive cases, 150 hospitalized cases were included in the clinical analysis, and 44 cases from the emergency department for which the details of the medical records were not available were excluded. pneumonia (n = 142, 94.7 %) was the most common clinical diagnosis, followed by bronchitis (n = 7) and bronchiolitis (n = 1). additionally, almost all hospitalized hadv-infected patients presented with fever (144/150, 96.0%) and coughing (149/150, 99.3 %) ( table 1 ). the mean peak body temperature was 39.53 ± 0.67°c (n = 144, range 37.3 − 41.4°c) and febrile seizures were noted in two febrile patients. in addition to respiratory symptoms, diarrhea, vomiting, skin rash, and conjunctivitis were noted in 21.3 %, 9.3 %, 10.0 % and 4.7 % of the patients respectively. twenty-two patients (14.7 %) had underlying diseases, which included congenital heart disease (8 patients), airway anomaly (malacia, stenosis, 11 patients), bronchopulmonary dysplasia (1 patient), asthma (2 patients), or primary immunodeficiency (1 patient). seventeen patients (11.3 %) required admission to the intensive care unit and 38 patients (11.3 %) received mechanical ventilation including both noninvasive (n = 31) and invasive (n = 7) modes. analysis revealed that the mean value of white blood cell (wbc) count was 10.09 ± 4.61 × 10 9 /l ( table 2) . leukocytosis (wbc > 12.0 × 10 9 /l) was observed in 37 (24.7 %) patients. eighty-one patients (54.0 %) had elevated serum c-reaction protein (crp). given rsv was the virus most frequently co-detected with hadv, differences among patients with single hadv infection (n = 49) and those with hadv/rsv coinfections, including both dual infections (n = 18) and multiple infections (hadv/rsv with one or more other respiratory viruses, n = 23), were assessed ( table 1 ). the mean age of patients with multiple infections (1.20 ± 0.81) and dual infections (1.53 ± 2.41) was significantly younger than those with single hadv infection (3.68 ± 3.64) (p = 0.001). however clinical characteristics and laboratory findings showed no significant differences among different groups. because hadv-b7 and hadv-b3 were the most predominant type among patients with hadv infection, the clinical entities of patients with single hadv-b7 infection (n = 30) and those with single hadv-b3 infection (n = 15) were also compared to exclude the possible effect of other respiratory virus infection (table 2) . patients with single hadv-b7 infection showed longer duration of fever (22.07 ± 21.52 vs 9.73 ± 7.31, p = 0.038) than patients with hadv-b3 alone. immunoglobulin was more frequently used in single hadv-b7 infected patients than in single hadv-b3 group (p = 0.038). patients with hadv-b7 alone also tend to require longer hospital stay (18.50 ± 12.87 vs 12.33 ± 4.95, p = 0.082) than those with single hadv-b3 infection, although no significant difference was found. biochemical tests demonstrated aspartate aminotransferase (ast), alanine aminotransferase (alt), lactate dehydrogenase (ldh) and hydroxybutyrate dehydrogenase (hbdh) levels were significantly higher in the single hadv-b7 infected group. two patients died in-hospital. both of them required icu admission and died of multiple organ failure. one was a 17 month-old boy with multiple underlying conditions of complex congenital heart disease and tracheobronchial malformation. the other was a previously healthy 18 month-old boy. analysis indicated that both fatal patients were infected with hadv-b7 but no other respiratory viruses. hadv is a significant causative agent of respiratory tract illnesses in both children and adults. here, the molecular showed that the hadv infection rate in the current study population was 5.8 %, which was consistent with previous reports from china and other countries [22] [23] [24] . results showed that most patients with hadv infection were younger than 5 years (88.35 %), which is similar to numbers reported in previous studies [1, 22, 23, [25] [26] [27] . this may because the immune systems of young children are not well developed, which leaves them prone to more severe hadv disease. this may also suggest that schoolage children are exposed to the most common endemic types of hadv early in life, thereby establishing a protective immunity resulting only in mild clinical symptoms, such that upper respiratory tract infection does not require care in an emergency department or hospital in this age group. over a period of 6 years, 11 different types of hadv belonging to 4 species (hadv-a, b, c, e) were identified in respiratory specimens from children with alrtis. hadv-7 and hadv-3 of species b comprised the most prevalent types and presented throughout the duration of the study. although these results were consistent with previous reports from korea and argentina [22, 28, 29] , investigations from croatia, peru, canada, france showed that species c predominated [1, 25, 26, 30] . this difference in type prevalence may be attributed to difference in regions, year of study, and population recruited. notably, some newly emerging or re-emergent types or variants were here identified, although only in rare cases. five patients were found to have hadv-b55 (formerly named hadv-11a), which is an uncommon re-emergent type that once caused an outbreak of respiratory tract infection in a senior high school in shanxi province, china in 2006, including one fatal case [15] . subsequently, hadv-b55 has been associated with several outbreaks of respiratory disease in other provinces in china [31] . an emerging variant, hadv-b14p1 (formerly known as 14a), was also found. recently, hadv-b14p1 has been associated with several large outbreaks of acute respiratory infection, which included severe and even fatal cases in the united states and europe [16, 32] . additionally, in 2011, an outbreak of febrile respiratory illness that affected 43 students in gansu province, china was reported to be caused by hadv-b14p1 [33] . one hadv-b14 infected patient who presented with bronchopneumonia and required hospitalization in april 2010 was identified. by further sequencing the fiber gene (data not shown), this strain was confirmed to be hadv-b14p1 because it contained a unique characteristic 6-nuleotid deletion in fiber knob region as reported by kajon et al. [32] . last, this is the first report of detection of hadv-c57 in respiratory samples collected from pediatric patients with alrtis and the first of co-detection of hadv-c57 with hadvc-2. hadv-c57 (formerly designated strain 16700) was first isolated from the feces of a healthy child as part of an acute flaccid paralysis surveillance program. computational genomic and bioinformatic analysis showed hadv-c57 to be a recombinant virus with fiber gene nearly identical to hadv-c6 and a unique hexon distinct from all viruses in species hadv-c [34, 35] . out of the three hadv-c57-infected cases identified here, one was a previously healthy 9month-old male who presented with bronchopneumonia and conjunctivitis requiring hospitalization. because only a small number of hadv-c57 positive cases were found here and all were co-infected with other respiratory viruses, the pathogenic role of hadv-c57 in respiratory infections will require further investigation. hadv type is traditionally determined by virus isolation and subsequently serum neutralization tests, in which antibodies raised against specific type are used to suppress cytopathic effects in tissue culture assays. by nature of its design, this test can only reveal the dominant type. by applying pcr-based identification targeting hexon or fiber genes, co-infections with multiple hadv types (types from same or different species) have been reported in both immunocompromised and immunocompetent patients [28, 29, [36] [37] [38] . in current study, results showed that one specimen contained both hadv-c2 and hadv-c57 by cloned sequencing the pcr products. these were amplified directly from respiratory samples using universal primers of hexon gene. this co-infected phenomenon was confirmed using the fiber gene sequencing results with type-specific primers (data not shown). the specimen was collected from a previously healthy 2.7-year old boy, presenting with fever, coughing and seizure at emergency department on december 10, 2009. co-infection of different hadv types has never been reported in any previous studies of mainland china. the clinical implications of such co-infection remain unclear, and its role in hadv pathogenesis and evolution will require further study. consistent with the report from guangzhou, southern china [24] , results here showed that 69.6% of hadvinfected participants were co-infected with one or more other respiratory tract viruses and that rsv was the most frequently co-detected virus. however, no significant differences in clinical characteristics and laboratory findings were found between patients with single hadv infection and those co-infected with rsv except that coinfections were more frequently observed in younger children. similarly, a study from peru also did found no higher prevalence of any clinical manifestations in coinfected patients than in those infected with hadv alone [26] . the results of another report from chile showed the clinical severity to be the same in patients with single hadv infection and those with mixed rsv-hadv infections [39] . these data demonstrate that, as more sensitive molecular methods become more frequently used to identify pathogens, co-detection of different viruses in the same specimen may also become more common. however, the clinical role of such coinfections will still require independent investigations. both hadv-b7 and hadv-b3 may cause severe or even fatal pneumonia in even immunocompetent children. several previous studies showed that patients infected with hadv-b7 tend to have higher case-fatality rates than those with hadv-b3 [40, 41] . two fatal cases were recorded during the study period, and both of these patients were infected with hadv-b7 alone. analysis revealed that patients with hadv-b7 infection had longer duration of fever and higher serum levels of muscle enzymes than hadv-b3-infected patients. patients with hadv-b7 infection also tended to require longer hospital stays although no significant difference was found. these differences have excluded the possible interference by any other co-infected respiratory viruses since this work only evaluated the patients with hadv infection alone. such results may suggest that hadv-b7 infection tended to cause more extrapulmonary tissue damage (such as liver and heart) and may have more severe clinical consequence. this is a cross-sectional study. only one respiratory sample was collected from each patient and no viral load analysis was performed. although hadv is a pathogen that for long has been known to cause respiratory tract infection, asymptomatic carriage of the virus may persist for weeks [18] . the detection of hadv in nasopharyngeal aspirate or throat swab with the use of a pcr assay could represent convalescent-phase shedding, so detection may not suggest the current infection. in summary, a total of 11 different types of hadv were identified in children with alrtis and hadv-b7 and hadv-b3 were the most predominant types. clinical entities of patients with single hadv infection were similar to those with mixed hadv/rsv infections. hadv-b7 infection tends to have more severe clinical consequences. the presence of newly emerging types or variants and co-infection with different types of hadv highlights the need for constant and close surveillance of adenovirus infection. ck, median (iqr) (u/l) epidemiology of severe pediatric adenovirus lower respiratory tract infections in manitoba, canada community-acquired pneumonia requiring hospitalization among u.s. children long term sequelae from childhood pneumonia; systematic review and meta-analysis diagnostic procedures for viral, richettsial and chlamydial infections isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture recovery of new agent from patients with acute respiratory illness a case of urethritis caused by human adenovirus type 56 isolation and characterization of a novel recombinant human 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hospital 2008-2010: clinical and virological features an outbreak of acute respiratory disease in china caused by human adenovirus type b55 in a physical training facility molecular epidemiology and brief history of emerging adenovirus 14-associated respiratory disease in the united states outbreak of febrile respiratory illness associated with human adenovirus type 14p1 in gansu province, china. influenza other respir viruses computational analysis of two species c human adenoviruses provides evidence of a novel virus evidence of frequent recombination among human adenoviruses pcr analysis of egyptian respiratory adenovirus isolates, including identification of species, serotypes, and coinfections highincidence of human adenoviral co-infections in taiwan molecular typing of clinical adenovirus specimens by an algorithm which permits detection of adenovirus coinfections and intermediate adenovirus strains adenovirus and respiratory syncytial virus-adenovirus mixed acute lower respiratory infections in chilean infants genome type analysis of adenovirus types 3 and 7 isolated during successive outbreaks of lower respiratory tract infections in children adenovirus serotype 3 and 7 infection with acute respiratory failure in children in taiwan we would like to thank all participating physicians and nurses of beijing children's hospital for their assistance and collaboration in the sample and clinical data collection. this study was supported by grants from the national major s & t research projects for the control and prevention of major infectious diseases in china (2012zx10004-206) and national science and technology supported projects (2013bai09b11). the authors report no conflicts of interests.authors' contributions cl analyzed data, performed statistical analysis, drafted and reviewed manuscript. yx and jz carried out the molecular studies. lr participated in study design and coordination and helped to review the manuscript. jl designed the primers of sequencing typing. zx analyzed data and reviewed manuscript. bx, yy and sq collected samples and data. jw and ks conceived of the study. all authors read and approved of the final manuscript. availability of data and materials not applicable submit your next manuscript to biomed central and take full advantage of: key: cord-346539-kxnrf5g5 authors: riggioni, carmen; comberiati, pasquale; giovannini, mattia; agache, ioana; akdis, mübeccel; alves‐correia, magna; antó, josep m.; arcolaci, alessandra; kursat azkur, ahmet; azkur, dilek; beken, burcin; boccabella, cristina; bousquet, jean; breiteneder, heimo; carvalho, daniela; de las vecillas, leticia; diamant, zuzana; eguiluz‐gracia, ibon; eiwegger, thomas; eyerich, stefanie; fokkens, wytske; gao, ya‐dong; hannachi, farah; johnston, sebastian l.; jutel, marek; karavelia, aspasia; klimek, ludger; moya, beatriz; nadeau, kari; o'hehir, robyn; o'mahony, liam; pfaar, oliver; sanak, marek; schwarze, jürgen; sokolowska, milena; torres, maría j.; van de veen, willem; van zelm, menno c.; wang, de yun; zhang, luo; jiménez‐saiz, rodrigo; akdis, cezmi a. title: a compendium answering 150 questions on covid‐19 and sars‐cov‐2 date: 2020-06-14 journal: allergy doi: 10.1111/all.14449 sha: doc_id: 346539 cord_uid: kxnrf5g5 in december 2019, china reported the first cases of the coronavirus disease 2019 (covid‐19). this disease, caused by the severe acute respiratory syndrome‐related coronavirus 2 (sars‐cov‐2), has developed into a pandemic. to date it has resulted in ~6.5 million confirmed cases and caused almost 400,000 related deaths worldwide. unequivocally, the covid‐19 pandemic is the gravest health and socio‐economic crisis of our time. in this context, numerous questions have emerged in demand of basic scientific information and evidence‐based medical advice on sars‐cov‐2 and covid‐19. although the majority of the patients show a very mild, self‐limiting viral respiratory disease, many clinical manifestations in severe patients are unique to covid‐19, such as severe lymphopenia and eosinopenia, extensive pneumonia, a “cytokine storm” leading to acute respiratory distress syndrome, endothelitis, thrombo‐embolic complications and multiorgan failure. the epidemiologic features of covid‐19 are distinctive and have changed throughout the pandemic. vaccine and drug development studies and clinical trials are rapidly growing at an unprecedented speed. however, basic and clinical research on covid‐19‐related topics should be based on more coordinated high‐quality studies. this paper answers pressing questions, formulated by young clinicians and scientists, on sars‐cov‐2, covid‐19 and allergy, focusing on the following topics: virology, immunology, diagnosis, management of patients with allergic disease and asthma, treatment, clinical trials, drug discovery, vaccine development and epidemiology. over 140 questions were answered by experts in the field providing a comprehensive and practical overview of covid‐19 and allergic disease. the first cases of the coronavirus disease 2019 (covid19) , caused by the novel severe acute respiratory syndrome-related coronavirus 2 (sars-cov-2), were reported in china in december 2019 1 and rapidly led to pandemic. currently, ~6.8 million confirmed cases of covid-19 and near 400,00 covid-19-related deaths have been reported globally. 2 these numbers, which are still rising, likely underestimate the cumulative incidence of covid-19 due to several factors; these include limitations of current diagnostic tests, the extent of population testing and reporting, and the type and timing of community mitigation strategies adopted by each country, among others. 3 covid-19 shows a complex clinical profile with many different presentations. like in many other viral infections, subclinical, mild, moderate, or severe cases (10-20% of patients require hospitalization and 2-4% intensive care unit, icu) presenting with or without pneumonia are observed. asymptomatic cases are common but, to date, there is a lack of epidemiological surveys that provide a clear percentage of asymptomatic cases. 4, 5 the covid-19 pandemic is the world's gravest public health crisis of the 21st century, and there is an urgent need for reliable and updated scientific and clinical information. covid-19 is a zoonosis to cd147 (known as basigin or extracellular matrix metalloproteinase inducer), which is expressed in human airway and kidney epithelium, as well as in innate cells and lymphocytes, 14 and to tmprss4, which is highly expressed in intestinal epithelial cells. 15 in addition, antibody-dependent enhancement of sars-cov-2 cell entry may also contribute to infection as reported for sars-cov. 16 sars-cov-2 may use receptors that have been reported for other coronaviruses, such as cd26, aminopeptidase n and glutamyl aminopeptidase for cell invasion. 13, 17, 18 among these, cd26 (encoded by dpp4) has emerged as a putative receptor for sars-cov-2 because structural analyses predict that the spike protein of sars-cov-2 binds to cd26. 19 this receptor has been shown to be expressed in the human epithelium and immune cells. 14 there is limited evidence about covid-19-associated polymorphisms. ace might be one of the candidate genes that influences pneumonia progression in sars. it is conceivable that the d allele influences the renin-angiotensin system via elevation of serum or local ace levels, which may damage the endothelium or epithelium of the lungs. 20 the variance in covid-19 prevalence and mortality cannot be explained by an ace insertion or deletion polymorphism alone, or one polymorphism of any single gene. however, polymorphisms in genes of toll-like receptors, inflammasome, intracellular molecular sensors, interferons (ifns) 21 and interleukins (ils) may contribute. structural proteins of sars-cov-2 virions, such as the spike glycoprotein, envelope, membrane and nucleocapsid, are the main immunogenic molecules (figure 1) . 22 ,23 sars-cov-2 adaptive responses develop mainly to the spike protein, and immunodominant t and b cell epitopes have been reported. 24 intracellularly, the viral rna replicase complex, and non-structural and translated proteins, activate innate immune pathways. this leads to an ifn type i response, nf-kb activation in epithelial cells, as well as activation of nlrp3 and other inflammasomes, in macrophages and dendritic cells. 23 this article is protected by copyright. all rights reserved the spike protein of sars-cov-2 has a receptor-binding domain that binds ace2 with higher affinity than sars-cov. 8 in addition, the sars-cov-2 spike protein harbors a polybasic furin cleavage site (prrar) with an insertion of 4 amino acid residues, which is distinct from that found in sars-cov and other sars-like viruses. this allows effective cleavage by furin and other proteases and determines viral infectivity and host range. 6 the severe lymphopenia observed in covid-19 25 is similar to that reported in hiv infection and acquired immune deficiency syndrome. the latter is characterized by cd4+ t cell lymphopenia, whereas covid-19 causes general lymphopenia. however, severe lymphopenia development in covid-19 happens in weeks, whereas hiv-induced lymphopenia takes years. 26 hiv and sars-cov-2 are both rna viruses and share some similarities in their replication pathways; hence certain rna replication drugs may work in both diseases (figure 1) . 27 there are 2 strains of sars-cov-2 that are clinically relevant. genome analysis of sars-cov-2 from human samples shows high rates of mutation and deletion in several viral genes, including the spike-glycoprotein gene. 28 covid-19 treatments, including future vaccination against sars-cov-2, may drive the genetic evolution of the virus affecting virulence and pathogenicity. for example, a report on a 382-nt deletion in orf8 (figure 1 ) of sars-cov2 isolated from patients in singapore implied that mutations may arise as a result of human adaptation and could be associated with attenuation. 29 nevertheless, the emergence of a sars-cov-3 is possible as long as there is close contact between humans and living animals that harbor coronaviruses. data from 96 covid-19 patients in china show sars-cov-2 detection in respiratory samples for a median of 18 days (13-29 days) . in this study, sputum and saliva were not analyzed separately. viral shedding was significantly longer in patients with severe disease, with a median of 21 days (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) days), compared to mild disease, 14 days (10-21 days). furthermore, glucocorticoids treatment this article is protected by copyright. all rights reserved longer than 10 days significantly extended the duration of sars-cov-2 shedding. 30 viral load differed significantly by sample type, with respiratory samples showing the highest, followed by stool samples, and serum samples showing the lowest (figure 2) . 30 another study of 78 patients with covid-19 (33 asymptomatic vs 42 symptomatic) has estimated that the duration of viral shedding from nasopharynx swabs was 8 days (3-12 days) for asymptomatic vs 19 days (16-24 days) for symptomatic patients. 31 the viral load range from 1.34 × 10 11 copies per ml to 7.52 × 10 5 in sputum of patients who died or survived, respectively. 32 tmprss2 and tmprss4 promote sars-cov-2 infection of ace-expressing human enterocytes 15 causing diarrhea in adults and children. 33,34 sars-cov-2 has been detected in stool samples by reverse transcription polymerase chain reaction (rt-pcr) (figure 2) . the median duration of the virus in stool samples (22 days, interquartile range 17-31 days) was significantly longer than in respiratory samples (18 days, 13-29 days) . 30 however, sars-cov-2 released into the intestinal lumen was inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of patients with covid-19. therefore, the intestine is a potential site of sars-cov-2 replication, which may contribute to local and systemic illness and overall disease progression but unlikely to contribute to the spreading of covid19. 15 section 2: immunology of covid-19 from previous sars studies, it is known that the median seroconversion time for detectable igg was 17 days after infection. 35 detectable levels of sars-specific igg and neutralizing antibodies persisted for up to 720 days. this suggests that there is antibody-mediated protection from sars-cov recurrent infection for up to 2 years. 36 there are inconsistent reports on the humoral response to sars-cov-2. one study with 285 covid-19 patients reported that sars-cov-2 virus-specific igg and igm peaked 17-19 days and 20-22 days after symptom onset, respectively. 37 on the other hand, another study of 26 hospitalized covid-19 patients showed that seroconversion could take up accepted article to 50 days. 38 these discrepancies may be related to the time of sars-cov-2 diagnosis or the clinical characteristics of each cohort and warrant additional studies. systemic iga responses may play a relevant role in the pathogenesis of covid-19. 39 mucosal iga likely exerts a protective role by preventing sars-cov-2 adherence to epithelial cells. circulatory iga may also contribute to sars-cov-2 neutralization. in addition, iga has the ability to either promote inflammation, through the formation of immune complexes, or to dampen it via fc-mediated inhibitory itam-signaling. 40, 41 a seroconversion study in covid-19 patients has found and association between disease severity and sars-cov-2-specific iga levels. these were significantly higher than sars-cov-2-specific-igm and -igg levels in critically ill covid-19 patients. 39 whether this association, previously unseen in sars-cov infection, 42 is due to a protective or detrimental role of iga in covid-19 remains to be elucidated. preliminary findings indicate that asymptomatic and mild cases of covid-19 can generate detectable levels of sars-cov-2-specific antibodies in serum. however, seroconversion is observed less frequently in asymptomatic compared to mild or severe cases, and many asymptomatic cases yield undetectable sars-cov-2-specific antibody responses. 37, [43] [44] [45] so far, no robust data are available on the qualitative differences in humoral responses between asymptomatic and symptomatic covid-19 patients. it is not clear which molecular mechanisms underlie the milder symptoms of covid-19 in children as compared to adults. children may mount a sars-cov-2 antibody response characterized by more efficient production of the so-called natural antibodies, which arise from activated igm+ memory b cells. 46 these cells, which are more prevalent in children than in adults, presumably produce broadly neutralizing antibodies early during the infection. this article is protected by copyright. all rights reserved b cell receptor-sequencing has been conducted in the blood of covid-19 patients. naive b cells exhibited little clonal expansion, whereas cd27+cd38+ memory b cells showed the highest expansion levels among diverse b cell subsets. covid-19 patients significantly expanded specific bcell receptor clones compared to those in the healthy controls. these findings suggest that b cells experience unique clonal variable, diversity, and joining gene segment rearrangements upon sars-cov-2 infection. 47 the lifespan and functionality of these b cells remain to be elucidated. the term "herd immunity" refers to the generation of population immunity that protects a region, or country, from infection. 48 the number of confirmed covid-19 cases has reached approximately 6.5 million. 2 the world population is estimated to be 7.8 billion. to ascertain the extent of herd immunity, it is pivotal to define the prevalence of sars-cov-2-exposed humans. it is thought that 67% is the minimum percentage of symptomatic or asymptomatic covid-19 population required for herd immunity. 48 that is to say that worldwide herd immunity may occur when ⁓5 billion humans have a protective immune response against sars-cov-2. to date, there are no reliable data, particularly on the number of asymptomatic individuals that show seroconversion, to determine the degree of herd immunity. 49 il-4 is pleiotropic and could theoretically cause negative effects on immune responses. however, based on phase ii and iii studies with dupilumab (an il-4rα-specific monoclonal antibody that blocks il-4 and il-13 signaling) in the context of atopic dermatitis, chronic rhinosinusitis with nasal polyps and asthma, no increased risk of infections to viral or bacterial pathogens have been documented. 50 furthermore, dupilumab had no impact on responses to non-live vaccines. 51 this article is protected by copyright. all rights reserved allergic airway disease patients appear to be underrepresented among covid-19 patients. 52-54 this could be partly attributed to the low ace2 expression detected in allergic patients, with or without concomitant asthma. 55 furthermore, allergen challenge, which induces t helper (th)-2 inflammation, 56 has been shown to reduce ace2 expression in a murine model of asthma, and ace2 expression was inversely associated with type 2 biomarkers (il-13, ige, exhaled nitric oxide fraction). 57 these results are in line with previous work showing that decreased ace expression in the airway epithelium of asthmatic subjects was associated with eosinophilic inflammation. 58 on the other hand, the analysis of nasal airway transcriptome data from 695 children identified that tmprss2 is highly upregulated by type 2 inflammation through the action of il-13. therefore, the reduced ace2 expression seen in asthmatic patients may be compensated by an increase in tmprss2 production. 59 eosinopenia has been reported in ⁓50-70% of severe covid-19 patients. a minority of covid-19 patients present with eosinophilic inflammation. 25,60 the th1/th2 cytokine balance may play a role, particularly as it pertains to il-5, which promotes eosinophilopoiesis and eosinophil survival and activation. eosinophilic inflammation suggests the dominance of type 2 inflammation, which may play a protective role against sars-cov-2. on the other hand, it may be the result of a hypersensitivity reaction to drugs used to treat covid-19. 61-63 anti-il-5 treatment, which induces eosinophil deficiency, results in a higher viral load in influenza and rhinovirus infection. this might be due to the ability of eosinophils to bind and inactivate the influenza a virus and respiratory syncytial virus (rsv). 64 a similar role seems possible in sars-cov-2 infection, where type-2 asthma patients potentially benefit from antiviral eosinophil responses. on the other hand, covid-19 post-mortems did not show lung eosinophilia 62 , which argues against its local protective role in sars-cov-2 infection, although it is important to control for glucocorticoid-driven eosinophil reduction in these studies. 61 this article is protected by copyright. all rights reserved eosinopenia is commonly reported in severe covid-19. 65, 66 the underlying mechanisms are largely unknown and most likely multifactorial. a number of possible explanations have been proposed: decreased eosinophilopoiesis; defective eosinophil egression from the bone marrow; and eosinophil apoptosis induced by type 1 ifn released during the acute infection. 61 also, increased eosinophil migration and retention within inflamed tissues has been described, 67 but disputed for the aforementioned reasons. 62 there is no evidence for an enhanced susceptibility of patients on anti-il-5/il-5r treatment to develop viral infections. observational studies in covid-19 patients reported elevated eosinophil counts with a favorable outcome, whereas eosinopenia was observed in more severe cases. 25, 68 neither was there proof of causation nor evidence for enhanced tissue presence in lungs of covid-19 patients. 69 there is neither evidence for a protective effect of these biologicals nor a negative effect regarding sars-cov-2 infection. importantly, maintaining proper asthma control is imperative and so is to follow up on severe asthmatics during the covid-19 pandemic, for example via telemedicine. 50 more than 1 billion people worldwide are infected with helminths, with those living in resource-poor tropical areas being disproportionately affected. helminth co-infection has been shown to influence the severity of viral infection in mice. for example, murid herpesvirus 4 respiratory infection, prior infection with schistosoma mansoni, reduced disease severity. 70 however, immune responses to pulmonary coronaviruses and murid herpesvirus 4 are different and therefore the impact of helminth co-infection is yet to be determined. this is particularly important as the pandemic is now spreading through the helminth-endemic regions of the word. 71 this article is protected by copyright. all rights reserved sars-cov-2 infects human t cells via cd147-binding. 72 t cells are severely affected by sars-cov-2, which reduces t cell counts nearly 2 times below the reference limit. this effect is more pronounced in critically ill covid-19 patients. 60, 73, 74 in addition to the reduction in t cell numbers, a recent study found that cd4+ and cd8+ t cells as well as natural killer cells displayed reduced antiviral cytokine production in covid-19 patients. a reduced cytotoxic potential was identified in covid-19 patients, particularly in those that required icu, and was associated with high il-6 serum levels. 75 circulating sars-cov-2−specific cd8+ and cd4+ t cells have been reported in ∼70% and 100% of covid-19 convalescent patients, respectively. 76 cd4+ t cell responses to the spike protein were robust and correlated with sars-cov-2-specific-igg and -iga titers. the m, spike and n proteins each accounted for 11-27% of the total cd4+ response, with additional responses commonly targeting nsp3, nsp4, orf3a and orf8, among others. for cd8+ t cells, spike and m proteins were recognized, with at least 8 sars-cov-2 orfs targeted. interestingly, sars-cov-2−reactive cd4+ t cells were detected in ∼40-60% of unexposed individuals, which indicate cross-reactive t cell recognition between circulating 'common cold' coronaviruses and sars-cov-2. 76 three sars-recovered individuals, 9-and 11-years post-infection, were analyzed for t-cell responses against 550 sars-cov peptides that may share homology with mers-cov. sarsspecific memory t cells persisted at 9 and 11 years post-sars infection in the absence of antigen exposure. 77 based on these data, it is likely that specific sars-cov-2 epitopes elicit a persistent t cell response, which may also confer protection against other 'common cold' coronaviruses. 76 however, long-term studies on the natural history of sars-cov-2 infection are pending. different mechanisms have been proposed for lymphopenia: 1) t cell exhaustion. the expression of programmed cell death-1 marker (also known as pd-1), which is associated with t-cell exhaustion, was higher in t cells from covid-19 patients than in healthy controls; the expression of pd-1 and tim-3 (another exhaustion marker) increased as covid-19 progressed. 78 2) activation of p53 signaling in lymphocytes, which suggests a role for apoptosis for in lymphopenia. 3 this article is protected by copyright. all rights reserved pyroptosis, which induces lymphopenia and may be proinflammatory. 79 cov-2, which may also cause a cytopathic effect on infected t cells. 5) other mechanisms of lymphopenia that remain to be studied are bone marrow suppression during cytokine storm syndrome (css; see below) and sequestration in the lungs during extensive bilateral pneumonia. 60 lymphopenia can be used as an early predictor of severity and clinical outcome. a significant reduction in lymphocyte counts was common in severe and critically ill covid-19 patients. a continuing or gradual decrease of lymphocyte counts was indicative of poor prognosis and usually required icu admission ( table 1) 60 . in agreement with this, a number of studies have identified lymphopenia as an independent risk factor for mortality in covid-19. 25,80 in covid-19 patients, decreases were observed in total lymphocytes, cd4+ and cd8+ t cells, b cells and natural killer cells. t cell and natural killer cell counts were below normal levels, while b cell counts were at the low end of the normal range. a reduction in specific subsets of lymphocytes, such as cd16+cd56+ natural killer cells and regulatory t cells, was reported in severe covid-19 patients. 60 css is associated with a wide variety of diseases, both infectious and noninfectious. it is a complex cascade of multicellular activation events that leads to an excessive or uncontrolled release of proinflammatory cytokines. css-associated inflammation begins at a local site and spreads throughout the body via the systemic circulation and can cause multi-organ failure and hyperferritinemia. 60,81 css encompasses the activation of large numbers of blood cells, including b cells, natural killer cells, macrophages, dendritic cells, neutrophils, monocytes, resident tissue cells and epithelial and endothelial cells. their activation cause a massive release of pro-inflammatory cytokines, which this article is protected by copyright. all rights reserved drives pathology. 82 the cells involved in css during covid-19 have not been fully determined yet. were the most important cell types releasing a large amount of proinflammatory cytokines. 60, 83 which cytokines are most elevated during css? multiple proinflammatory cytokines and inflammasome activation may contribute to css pathogenesis. 60 elevated serum ferritin, il-6, il-1β, ifn-γ , cxcl10 (known as ip-10) and ccl2 immunosuppression is a double-edged sword in viral infections. 86 this article is protected by copyright. all rights reserved primary immunodeficient patients are a high-risk group in the current pandemic, but to date it is unknown if a particular immunodeficiency poses a higher risk of severe disease. international primary immunodeficiency monitoring is being carried out and few cases have been documented. patients at higher risk are those with complications resulting from their primary immunodeficiency and strict follow-up must be done in those cases. a consensus has been established that baseline chronic treatment should be continued in those patients if they are asymptomatic or mildly symptomatic. furthermore, recommendations regarding primary immunodeficient patients adhere to individual national guidelines emphasizing social distancing and strict hygiene measures. systematic testing of primary immunodeficient patients is not advised, however recommendations may change as the pandemic evolves. 89 there are no longitudinal studies analyzing t regulatory cells in covid19 systemic dysregulation of metabolism, such as that seen in obesity and diabetes is a risk factor of sars-cov-1 and sars-cov-2 infection and of covid-19 severity 69 . these diseases lead to chronic systemic inflammation, upregulation of sars-cov-2 receptors in the lungs and the periphery, and they disturb the glucose and lipid metabolism of tissues and immune cells. 14, 91, 92 ards is an acute life-threatening inflammation of the lung due to infection, trauma, or inflammatory conditions. excessive inflammation leads to alveolar damage and increased permeability of endothelial and epithelial cells. this results in protein-rich fluid accumulation in the interstitium and the air space, which causes impaired gas exchange and hypoxemia. reactive oxygen species, leukocyte proteases, chemokines, and cytokines also contribute to lung injury. the barrier impairment of the lung microvascular barrier is central to the pathogenesis of ards. 93 in covid-19 patients, ards is more common in the elderly, those with multiple comorbidities, and those with continuing or gradually progressing neutrophilia and lymphopenia, and a higher level of creactive protein, lactate dehydrogenase, d-dimer and procalcitonin. 60, 88 there are at least 2 clinical phenotypes of ards: 1) near normal pulmonary compliance with isolated viral pneumonia; 2) decreased pulmonary compliance. 95, 96 what specific therapies can be suggested for ards? different treatments were suggested for ards. corticosteroid treatment is generally not recommended, although widely used in critically ill patients. convalescent plasma (cp) was administered to a small number of patients and was associated with virus clearance and clinical improvement ( table 2) . low tidal mechanical ventilation, positive end-expiratory pressure, prone positioning ventilation, and fluid management guidelines were associated with improved outcomes. extracorporeal membrane oxygenation could be used according to the inclusion and exclusion criteria of the eolia trial. other potential therapies such as mesenchymal stem cell therapy and cytokine inhibitors are still in trials and without definite results. 60,97 bcg is a live attenuated vaccine that was developed against tuberculosis at the beginning of the 20th century. bcg vaccination induces metabolic and epigenetic modifications by enhancing trained immunity (innate immunity to subsequent infections). 98 it was hypothesized that general bcg vaccination policies adopted by different countries might have impacted the transmission patterns and/or covid-19-associated morbidity and mortality. 99 the mechanisms underlying kawasaki disease -a generalized vasculitis, in young children, of unknown, potentially post-viral etiology-are poorly understood. the rare covid-19-associated inflammatory syndrome also features vasculitic changes, affects older children too and is often only associated with positive sars-cov-2 serology, but not viral shedding. its mechanisms need to be elucidated and may include post-infectious, antibody and immune-complex mediated pathology. in adults, there are occasional cases of covid-19-associated cutaneous vasculitis, possibly a localized manifestation of the disease that leads to severe generalized vasculitis in some children. [104] [105] [106] interestingly kawasaki-like disease was not reported in chinese cases and the first months of european cases. the season of the disease and environmental factors should be considered. the chinese epidemic was mainly from january to march whereas the usa epidemic started in mid-march and is still ongoing. initial results of acute phase reactants such as c-reactive protein, alanine transaminase, lactate dehydrogenase, d-dimer, procalcitonin, serum ferritin and il-6 on admission were used to evaluate the severity and predict the mortality. however, dynamic changes of these variables will be more precise in predicting the recovery or progression of covid-19. continuing or progressively increasing levels of c-reactive protein, procalcitonin, d-dimer and lactate dehydrogenase were shown to be associated with a high risk of death in severe covid-19 patients. 25,60,107 patients with acute respiratory illness (i.e., fever and at least one sign/symptom of respiratory disease such as cough or shortness of breath) and a history of contact with a confirmed or probable covid-19 case during the 14 days before symptom onset. patients with any acute respiratory illness in the context of a pandemic should have sars-cov-2 infection in their differential diagnosis. special attention should be given to patients with sudden onset of anosmia, loss of taste, gastrointestinal symptoms or skin lesions without respiratory symptoms who also have epidemiological links. 5,25,108 smell loss is now a well-established diagnostic symptom of covid-19 and can be present in otherwise asymptomatic patients, making it a useful tool in initial diagnosis. 109 this has resulted in anosmia to be included in the list of symptoms used in early screening tools for possible covid-19 in many international bodies. 109 rapidly progressive respiratory failure and sepsis, elevated serum proinflammatory cytokine levels, elevated acute phase reactants (e.g. c-reactive protein), cell-free-hemoglobin-leukopenia and markers of disseminated intravascular coagulation. 110 rt-pcr to generate cdna from sars-cov-2 rna extracted from respiratory samples, followed by quantitative pcr (figure 2 ). 111 common gene targets for sars-cov-2 include the envelope, nucleocapsid, spike, rna-dependent rna polymerase, and orf1 genes. it is recommended to include in the analysis, at least, 2 target genes. 112 nasopharyngeal and oropharyngeal (throat) swabs are the primary specimens for sars-cov-2 rt-pcr testing. lower respiratory tract specimens (i.e. sputum, endotracheal aspirate or bronchoalveolar lavage) may have higher viral loads and be more likely to yield positive tests ( figure 2 ). however, these locations carry a high risk of aerosolization and therefore should be reserved for severe patients with a negative test on an upper respiratory tract specimen and high suspicion for lower respiratory tract sars-cov-2 infection. 113, 114 serology is useful to determine prior exposure to sars-cov-2 within a given period of time (the length of time following infection that one remains positive is unknown) (figure 2) . detection of this article is protected by copyright. all rights reserved antibodies specific to the receptor binding domain of the spike protein indicates neutralization capacity, hence informing better about the development of protective immunity. 37, 111, 115 the antibody response occurs later than initiation of symptoms as well as of the detection of viral rna by rt-pcr in respiratory tract specimens, which usually peaks within the first week of symptom onset (figure 2) . although antibodies to sars-cov-2 have been detected as early as the first week after symptom onset, igm, iga and igg seroconversion commonly occurs between the 2 nd and 3 rd week of clinical illness onset. thereafter, igm starts to decline, reaching low levels by week 5 and almost disappears by week 7, while iga and igg persist beyond this period. 37, 39, 111, 116 the main approaches include nucleic acid amplification on respiratory samples using mobile devices (rt-pcr or isothermal nucleic acid amplification) and viral antigens or host antibodies (viral protein fragments) detection using immunoassays. 117 however, individual tests need validation in large populations before use and their sensitivity, specificity, positive and negative predictive values have to be accurately ascertained. otherwise, they may lead to covid-19 under or over diagnosis, thus undermining the public health efforts to control the disease. 118 a high rate of false negatives with antigen point-of-care assays may be due to the fact that the majority of patients produce antibodies against sars-cov-2 only after the second week after of infection ( figure 2 ). 119 furthermore, an effective antibody response is connected with several determinants, comprising severity of the disease, age and nutritional status of the patient, medications administered and concomitant infections. 118 nucleic acid amplification using rt-pcr directly targeting the virus is not affected by the above-mentioned limitations. 120 this article is protected by copyright. all rights reserved positive by the fifth and final test. 121 patients with an initial positive sars-cov-2 result had an increased risk of progressing to severe cases. altogether, these findings underscore how the timing of the immune response influences rt-pcr tests for sars-cov-2, and the importance of combining rt-pcr and seroconversion data for covid-19 diagnosis. the decision to discontinue home isolation/quarantine should be adapted to specific groups of patients based on factors such as symptom severity, healthcare systems´ capacity, laboratory diagnostic resources and local epidemic status. patients with suspected or confirmed symptomatic covid-19 can discontinue self-isolation/quarantine if all the following 4 conditions are met: a) resolution of fever (without the use of fever-reducing medications) for at least 3 days; b) clinical improvement in respiratory symptoms (e.g., cough, shortness of breath) for at least 3 days; c) at least 8 days have passed since the onset of symptoms for mild cases or at least 14 days for severe cases and immunocompromised patients; d) 2 negative rt-pcr tests from respiratory specimens taken 24 hours apart. if there is limited or no testing capacity, the combined symptom/test-based strategy should be reserved to hospitalized covid-19 cases and healthcare workers, whereas for mild or asymptomatic covid-19 cases (suspected or confirmed) the symptom-based strategy (condition a) and b) and c)) without lab testing is considered acceptable to end the self-isolation period. 122 pandemic strategies for risk minimization should be elaborated, harmonized and followed as such in allergy clinics, centers and practices. 123 in the eaaci/aria position paper by pfaar et al. 124 experts in the field have developed practical recommendations for optimizing allergic patients 'care whilst ensuring the safety of all health care professionals (figure 3) . general guidance from national health authorities should be strictly followed (i.e., world health organization, who; european centre for disease prevention). in-person consultations should be minimized to the lowest necessary level and triaged by telemedicine whenever possible (figure 4 ). 125 special attention should be paid to dataprotection in adherence to national data-security and -protection laws. non-delayable diagnostic and therapeutic measures should strictly follow reasonable preventive measures. several specific considerations regarding diagnostic and therapeutic measures are important in different allergic diseases ( figure 5) . moreover, socio-psychological aspects play a fundamental role in the care of allergic patients during the current pandemic and should be especially recognized and followed. stress caused by isolation and stigmatization due to allergic symptoms may amplify the development of allergic symptoms. 126 virtual doctor consultations have been regarded as an alternative to on-site clinical encounters and are increasing during the covid-19 pandemic. 124 initially, pre-visit telephonic communication is helpful to screen for patients with potential sars-cov-2 infection. 127 the epidemiological history should be investigated to determine if patients have fever or respiratory symptoms. in addition, previsit specific triage improves the efficiency of the patient's visit, thus reducing the length of stay in the hospital. to reduce face-to-face meetings, physicians can train some patients to self-treat at home based on the diagnosis obtained through a telephone consultation (figure 4) . a strict screening protocol is needed to identify sars-cov-2 infected patients (figure 4) . ideally, only sars-cov-2 negative patients (diagnosed via rt-pcr and/or rapid test) should come to the clinic. in places where systematic testing is unavailable, at least, normal temperature and negative epidemiological history should be mandatory to proceed to the outpatient departments. patients with a body temperature higher than 37.3ºc should have additional screening examinations, including routine blood tests, chest computed tomography scanning and even throat swabs for sars-cov-2 rt-pcr testing. 128 the indication and urgency of the tests for diagnosis should be considered. contraindications for skin, provocation and lung function tests can be explained beforehand to the patient, which helps to accepted article avoid unnecessary in-person consultations. 124 any test generating aerosol particles should be avoided because it is considered high risk (figure 4) . personal protective equipment (ppe) must be used when collecting biological samples. biological samples collected on-site from suspected or confirmed covid-19 patients (e.g. antibody assays, rna isolation, flow cytometry) should be processed following bsl-2 practices. during and after the covid-19 pandemic, the usage of bsl-2 facilities is mandatory for all newly arriving patient samples to prevent spreading the disease. research procedures involving sars-cov-2 isolation or culture should be conducted in a bsl-3 facility. 124,129 patients with common allergic diseases do not develop distinct symptoms or severe outcomes. allergic children show a mild course similar to non-allergic children 5 . in a recent study of 182 hospitalized children, 43 of them were reported with allergies. allergic rhinitis was the most prevalent allergic disease (83.7%), followed by drug allergy, atopic dermatitis, food allergy and asthma. in this study, allergic children showed a reduced increase in acute phase reactants, procalcitonin, d-dimer and aspartate aminotransferase levels compared to all patients. there were no deaths in allergic children in that study. 130 clinical history is very helpful to identify seasonality-and exposure-related symptoms driving the diagnosis of pollen-induced allergic rhinitis. an atopy test (in vivo or in vitro) reinforces the diagnosis. however, covid-19 can be superimposed on allergic rhinitis symptoms. 124 symptoms such as fever, fatigue and sudden loss of smell, are suggestive of covid-19 and should be closely monitored. pandemic? this article is protected by copyright. all rights reserved n95 facial masks have been proven useful in reducing allergen exposure by blocking pollen access to nose and mouth. on the other hand, surgical masks do not protect against inhalation of small airborne contaminants and are not designed to seal tightly against the user´s face, hence the contaminated air can pass through the gaps. 131 there are no conclusive data on the impact of allergic rhinitis on covid-19 susceptibility 132 . a recent study with 24 allergic rhinitis patients demonstrated a reduction of ace2 expression in nasal brush samples following an allergen challenge. 55 also, this study reported lower ace2-expression in the epithelium of asthmatic patients. on the other hand, tmprss2 is highly upregulated by type 2 inflammation through the action of il-13. 59 therefore, further studies are necessary to determine if allergic rhinitis patients have an altered risk of sars-cov-2 infection as compared to non-allergic individuals. although limited, the available evidence suggests that, compared to non-allergic individuals, allergic there is no scientific evidence that treatments for allergic rhinitis either increase susceptibility to sars-cov-2 infection or the severity of covid-19. therefore, allergen avoidance measures, nasal saline douches, and background controller therapies recommended by current guidelines for allergic rhinitis, such as nasal corticosteroids or second-generation h1-blockers, should be continued as prescribed, both in non-infected and covid-19 diagnosed patients. 124 the loss of smell in chronic rhinosinusitis is caused by type-2 inflammation of the olfactory epithelium. 135 in covid-19, the exact mechanism of potential olfactory neuropathy is still unclear. 136 however, a study found that sustentacular cells of the olfactory epithelium express ace2 and tmprss2, which enable sars-cov-2 entry and may subsequently impair the sense of smell. 137 a considerable percentage of covid-19 patients experience loss of smell as an early sign of the disease. 109 in many patients smell recovers in 1-2 weeks and there is no indication that intranasal corticosteroid treatment has a positive impact on the recovery. 138 on the other hand, there is no evidence suggesting that this treatment has a negative impact on symptomatology and/or accepted article development of covid-19. consequently, it is recommended to continue regular intranasal corticosteroid treatment for chronic rhinosinusitis (figure 5) . 124 an asthma exacerbation is difficult to differentiate from covid-19 ards or pneumonia by the patient, especially if it is triggered by rhinovirus, or other common respiratory viruses, because both conditions have dry cough and dyspnea. the british thoracic society advises patients with asthma experiencing fever, fatigue and loss of taste or smell to alert their physician as these are indicative of covid-19. 144 the distinction can be made by the physician based on the presence of wheeze, which is generally (but not always) absent in covid-19 pneumonia, as well as high-resolution chest tomography and viral diagnostic tests. 124 patients with controlled asthma are not at higher risk of severe infection than the general population. 53,54 ace2 expression was shown to be decreased in patients with allergic asthma 55 and in those receiving inhaled corticosteroids. 145 on the other hand, ace2 expression in asthmatic patients was increased in african-americans, in males and associated with diabetes, 55 and type 2 inflammation in children is associated with increased expression of tmprss2. 59 it is clear though that uncontrolled asthma is a risk factor of severe covid-19, thus all efforts should be focused on treating asthma by regular use of controller medication, including inhaled corticosteroids and biologicals. 53,146,147 there is no evidence available that patients on inhaled corticosteroids are at higher risk of covid-19 infection or of more severe symptoms than the general population. it is strongly advised by international scientific societies that patients continue with their routine control medication including inhaled corticosteroids during the pandemic (figure 5) . 132,148 this article is protected by copyright. all rights reserved recent evidence indicates that inhaled corticosteroid treatment reduces the expression of viral membrane receptors used to infect the human airways in a dose-dependent manner. 145 on the other hand, the immune suppression exerted by corticosteroids may impair anti-viral responses. 141 however, there are no clinical studies investigating the effect of inhaled corticosteroid on sars-cov-2 infection rates. spirometry is essential for the diagnosis of new asthma cases as stated by the global initiative for asthma guidelines. therefore, it should be conducted, but under special conditions (negative pressure chamber, etc.) and only in areas with low sars-cov-2 infection incidence. healthcare providers performing lung function testing need to wear maximum ppe (filtering face-piece particles 2 or 3 face mask, goggles, or disposable face shield covering the front and sides of the face, clean gloves, and clean isolation gowns), and the spirometer devices should be properly disinfected between patients (figure 3) . 149 an alternative, less precise, is monitoring morning and evening peak expiratory flow variability over a week. 150, 151 the global initiative for asthma guidelines state that routine spirometry should be avoided, especially in high-risk areas of covid-19 transmission. if spirometry needs to be performed, maximum ppe should be used (figures 3 and 4) . 148 the treatment of asthmatic patients can be monitored using personal devices measuring forced expiratory volume and peak expiratory flow. many of these devices are equipped with remote transmission functions and thus are amenable for the telemedicine management of patients. 152 pandemic? this article is protected by copyright. all rights reserved there is no evidence suggesting that the current approach to treat asthmatic patients during an exacerbation should change during the covid-19 pandemic. moreover, there is no proof that a short course of systemic corticosteroids impacts the evolution of covid-19. thus, oral corticosteroids should be given as usual for the treatment of an asthma exacerbation ( figure 5) . 144, 148 in the few cases in which patients are treated with long-term oral corticosteroids in addition to their high dose inhaled corticosteroids this should be continued in the lowest dose possible to prevent exacerbations. 148 the cause of the asthma exacerbation should be studied thoroughly to rule out potential exacerbations due to viral infections. 89 the preferred treatment is a pressurized metered-dose inhaler with a spacer. each patient should have an individual spacer, and this should not be shared at home. the use of nebulizers should be avoided when possible because they increase the risk of disseminating viral particles, which could affect other patients and healthcare personnel. 148 anti-ige treatment with omalizumab (or other biologics indicated for asthma) should be continued in non-infected patients. self-administration devices at home, whenever this option is available, are preferred, to minimize face-to-face contact in the clinic. in infected patients, omalizumab administration should be delayed until complete clinical recovery and viral clearance is achieved ( figure 5) . 50,153 endotype is associated with severe asthma in obese patients, are obese asthmatic patients more likely to develop severe covid-19? obesity, as part of the metabolic syndrome, increases the risk of severe covid-19. this is due to the pre-existent systemic low-grade inflammation and increased expression of sars-cov-2 entry receptors (ace2, tmprss2 and cd147). 154, 155 obese patients tend to have worse asthma control, increased hospitalizations and suboptimal response to standard controller therapy. thus, both difficult-to-control asthma and underlying metabolic syndrome are risk factors for severe covid-19. this article is protected by copyright. all rights reserved the il-6/th17 endotype encountered in late-onset obese asthma might be an additional risk factor. 156,157 the dermatological manifestations of covid-19 range from an un-specific macular erythematous rash, urticarial lesions, chickenpox-like vesicles and acro-ischemic lesions. 158, 159 they can result from local inflammation due to circulating immune complexes or from systemic manifestations leading to vasculitis and thrombosis. 160 these patients are also at increased risk of drug hypersensitivity lesions ( figure 6 ). 161 there is no evidence that patients with barrier defects such as atopic eczema have a higher risk for sars-cov-2 infection or skin complications during covid-19. however, patients with atopic dermatitis are often on systemic immunosuppressants and should be monitored closely. optimal topical treatment regime should also be encouraged in all patients. 162 hand hygiene procedures are pivotal to prevent self-infection and virus spreading. however, extensive water contact enhances dry skin, disturbs the commensal microbiota and leads to barrier disruption in healthy individuals. moreover, it exacerbates diseases with an intrinsic barrier defect such as atopic dermatitis. 163, 164 effective skin-care after hand hygiene is therefore essential to prevent barrier disruption and sensitization events. here, emollients containing hyaluronic acid, vitamin e, ceramide or urea are recommended. 165 dupilumab is approved for the treatment of moderate-to-severe atopic dermatitis. first data from italy on dupilumab-treated non-infected in high epidemic areas, and current evidence from dupilumab trials, suggest no negative effect of dupilumab regarding viral infections 166 with reports on a reduced number of herpes simplex superinfections and less bacterial superinfections. [167] [168] [169] accepted article this article is protected by copyright. all rights reserved the current eaaci statement on the usage of biologicals in the context of covid-19 advices no change of therapy in non-infected individuals and to withhold/delay the application of biologicals for a minimum of two weeks or the resolution of the disease in case of sars-cov-2 infection (figure 5) . 50 this is based on expert opinion in the light of missing data and may be adapted if more information becomes available. acro-ischemic lesions on toes and fingers have been identified in a subgroup of covid-19 patients. 25, 170 the data available are scarce and it is unclear if preventive or active anticoagulation should be initiated. however, acro-ischemic lesions could predate other sars-cov-2 symptoms in children and young adults. covid-19-induced skin lesions can be related to thrombovascular events (i.e. petechiae, acroischemia, dry gangrene) or to typical viral infections (i.e. erythematous rash, urticaria, maculopapular exanthema). 161 drug hypersensitivity has to be considered as a differential diagnosis, mainly in the second group, being a distinction difficult during the acute phase. diagnosis relies mostly on clinical observations. in that regard, an accurate chronology of the reaction and the drug exposure timeline is very informative 63 . laboratory and histopathological findings may also help. immunomodulatory drugs (including azithromycin), hydroxychloroquine/chloroquine and ifns, are the ones most frequently involved in hypersensitivity reactions. most reactions are non-immediate and further studies are required to clarify whether this increased frequency is caused by the drug immunogenicity or simply derives from a greater consumption as compared to other treatments. 161 this article is protected by copyright. all rights reserved drug provocation tests are not recommended because reactions can occur during the tests, including the generation and spreading of virus-containing aerosols. however, they may be considered after careful risk-benefit assessment in cases of urgent need, such as chemotherapy in cancer patients, perioperative drugs and radiocontrast media in subjects needing urgent procedures, and antibiotics if no effective alternative drug is available. 124 most ait products authorized for use in europe indicate that ait should be discontinued in case of infection; the same principle will apply to the covid-19 pandemic. patients on subcutaneous or sublingual ait, who are diagnosed with covid-19, those suspected of sars-cov-2 infection or symptomatic patients with a positive contact to sars-cov-2 individuals, ait should be interrupted until the patient has recovered. in patients not infected or who have recovered from the infection, ait could be continued ( table 3) . these recommendations are conditional and could change as clinical data evolve. 124 ,134 ait should continue in non-infected patients or those recovered from covid-19 ( figure 5 ). this is especially important in patients with life-threatening conditions such as venom allergy. it is possible to extend the intervals between vaccines during subcutaneous ait, as done for inhalant allergens, to minimize visits to the allergy clinic. if venom ait was stopped due to sars-cov-2 infection, it is unclear when it should be re-initiated because data from convalescent patients is scarce. 134 in patients diagnosed with covid-19 or cases with suspected sars-cov-2 infection, oral immunotherapy dosing should continue as indicated in the dosing plan and in coordination with the treating physician. oral immunotherapy can be continued in non-infected patients and those who have recovered from covid-19 ( figure 5 ). in areas with high level of sars-cov-2 community transmission, visits to the allergy clinic for oral immunotherapy up-dosing should be postponed. 124, 134 accepted article this article is protected by copyright. all rights reserved these patients are generally on symptomatic treatment. they need to look out for symptoms suggesting hypoxia or pneumonia, such as shortness of breath, deep shallow breathing, chest pains or persistent tachycardia. special attention needs to be given to those with risk factors for disease progression, such as patients older than 65 years, cardiac or pulmonary comorbidities and immunosuppression. 171, 172 prophylactic low molecular weight heparin, or heparin, has been recommended by the who in severe to critically ill covid-19 patients. 141 however, the international society on thrombosis and haemostasis recommended that all hospitalized covid-19 patients, not just those in icu, should receive prophylactic low molecular weight heparin in the absence of contraindications. 173 during the sars outbreak in 2003, corticosteroids did not change the course of the viral infection and delayed viral clearance. 174 on the other hand, a retrospective study on sars patients in hong kong suggested a better survival rate in patients treated with prednisolone for milder pneumonia or methylprednisolone in more severe cases. 175 recently, chinese experts stated that, in covid-19 patients, systemic corticosteroids should be considered on individual indications in a low-to-moderate dose and for no longer than a week. 176 the national institutes of health in their covid-19 treatment guidelines advises against the use of systemic corticosteroids in non-critically ill patients. 177 there are over 170 clinical trials on covid-19 treatment registered now in the international databases and very few have been completed. currently promoted pharmacological treatments are, at the most, based on anecdotic data collected in small numbers of covid-19 patients. these studies did not satisfy evidence-based medicine criteria, but caught general attention through news media, for example hydroxychloroquine (see below). tocilizumab is a humanized monoclonal antibody specific for il-6r, and it is approved for the treatment of rheumatoid arthritis. a positive response to tocilizumab points towards an imbalanced innate immune response in severe covid-19. luo et al. 178 reported that of the 15 patients treated with tocilizumab, 7 of them critically ill, 11 of the patients recovered within a week. prompt resolution of symptoms and encouraging results have also been reported in uncontrolled or retrospective trials. [179] [180] [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] these zoonotic beta-coronaviruses share structural and genomic similarities that are useful to patients? this article is protected by copyright. all rights reserved fang et al. 192 suggested that there is ace2 overexpression upon treatment with ace inhibitors, thiazolidinediones and ibuprofen. there were concerns pertaining to the use of nonsteroidal antiinflammatory drugs in covid-19 patients. the european medicines agency clarified that no scientific evidence established a link between ibuprofen, or other nonsteroidal anti-inflammatory drugs, and a risk to worsen covid19. 193 section 7: clinical trials and drug discovery in covid-19 adaptations for clinical trials during the pandemic must include all concerned parties such as there are drugs that interfere with ace2 and tmprss2, which are molecules used by the virus to enter the cell. 9, 195 for example, camostat mesylate is a clinically proven serine protease inhibitor with affinity for tmprss2. it has shown activity against sars-cov-2 in human lung calu-3 cells. 9 several drugs that target virus internalization are being investigated, including chloroquine phosphate and hydroxychloroquine, which have shown limited efficacy in humans and raised concerns due to side effects (see below). 196 drugs designed to inhibit the viral replication machinery may be effective against sars-cov-2. for example, remdesivir inhibits viral rna polymerases, which prevents sars-cov-2 replication (see below). it is uncertain whether lopinavir-boosted ritonavir and other antiretrovirals improve clinical outcomes or prophylaxis among patients at high risk of sars-cov-2 infection. 200 additional potential candidates include other broad-spectrum antiviral drugs such as arbidol and favipiravir and phytochemicals with anti-viral activity such as resveratrol ( figure. 1) . 197 in a cohort of severe covid-19 patients, compassionate-use of remdesivir showed clinical improvement in 68% of patients (36 out of 53). 201 of note, a double-blind, randomized, placebocontrolled trial of intravenous remdesivir was conducted in 1,063 adults hospitalized with covid-19 with evidence of lower respiratory tract involvement; remdesivir was superior to placebo in shortening the time to recovery in adults hospitalized with covid-19 and evidence of lower respiratory tract infection. 202 furthermore, in a study of 5 hiv-positive hospitalized patients with severe covid-19, three of them were given lopinavir-boosted ritonavir and 2 darunavir-boosted cobicistat for 14 days. four patients recovered and 1 remained hospitalized. 203 meplazumab is a cd147-specific humanized monoclonal antibody that has been shown to prevent sars-cov-2 infection of fibroblasts (veroe6 cells). 72 currently, there is insufficient evidence to draw any conclusions on the benefits of meplazumab for the therapy of covid-19 patients. in an observational chinese study, adults hospitalized with covid-19 pneumonia (n=17) who were treated with an intravenous infusion of meplazumab as an add-on therapy showed a higher recovery rate compared to controls (n=11). 198 however, these results should be interpreted with caution because they were generated in a non-randomized, non-stratified study, with a small sample size. large-scale studies are needed to assess the effectiveness and safety profile of meplazumab as a potential therapy for covid-19. cp therapy for covid-19 treatment has yielded promising results. for example, in a trial of 10 severe covid-19 patients, 205 cp therapy was well tolerated and improved the clinical outcomes. the viral load was undetectable after cp transfusion in 7 patients who had viremia. no severe adverse effects were observed. other clinical trials have shown the beneficial effect of cp therapy in covid-19 patients and ongoing clinical trials will provide additional data on its efficacy, safety and optimal timing for treatment ( table 2) . in this regard, it is unclear whether in patients with a high viral load, such as severely ill patients, cp therapy may drive tissue pathology through immune complexes or complement activation. baricitinib, fedratinib, and ruxolitinib are potent and selective jak-stat signaling inhibitors approved for indications such as rheumatoid arthritis and myelofibrosis. these drugs are powerful antiinflammatory medications that may reduce the systemic levels of cytokines associated with covid-19. 206 indeed, in a pilot study of 12 covid-19 patients, baricitinib limited the css and was beneficial for the patients. 207 the use of jak inhibitors has been associated with a higher risk of opportunistic viral infections, such as herpes zoster, which suggests that the reduced inflammation caused by jak inhibitors may limit, to some extent, anti-viral responses. 208 this article is protected by copyright. all rights reserved ivermectin (avermectin b1a and avermectin b1b) is an anti-parasitic drug that has shown broadspectrum anti-viral activity in vitro. in sars-cov-2-infected fibroblasts (vero-hslam cells), a single addition of ivermectin at 2 h post-infection reduced viral rna ~5000-fold at 48 hours. 209 however, plasma concentrations of total and unbound ivermectin did not reach the ic50 determined in vitro, even at a 10-times higher dose than approved by the food and drug administration (usa). 210 consequently, the likelihood of a successful clinical trial using ivermectin is low. in an observational study of 1,446 covid-19 patients, 811 received hydroxychloroquine treatment, which did not change the risk of intubation or death. 211 furthermore, in a brazilian randomized control study evaluating 2 different doses of chloroquine in covid-19 patients with severe respiratory symptoms, mortality was 2.5 times higher in the high-dose chloroquine arm. 212 moreover, pre-published results from us veterans health administration hospitals did not support any advantages of hydroxychloroquine administered alone or with azithromycin. 213 in addition, the results of a clinical study conducted in 821 individuals showed that hydroxychloroquine did not prevent illness compatible with covid-19 or confirmed infection when used as postexposure prophylaxis within 4 days after exposure. 214 however, because of the retraction of two main papers on hydroxychloroquine treatment for covid-19 patients, this area requires further attention by the european medicines agency and the food and drug administration. mesenchymal stem cells may exert antiviral mechanisms in the context of sars-cov-2 infection. the basal ifn-stimulated gene expression of mesenchymal stem cells is high, which enhances their responsiveness to ifn signaling, potentially inducing broad viral resistance. mesenchymal stem cell therapy may potentiate the low ifn-i and -iii levels and moderate ifn-stimulated gene response reported in sars-cov-2-infected ferrets and covid-19 patients. 215 it is is being used in some centers but its efficacy in covid-19 has not been proven. data available are mainly experimental with few records in humans and no reports on its efficacy in randomized clinical trials. 216 common anti-hypertensive drugs inhibit ace, but not ace2. importantly, ace2 opposes ace actions and lowers blood pressure by converting angiotensin-ii (a vasoconstrictor peptide) into its metabolites-angiotensin (1-7) (vasodilators). 217 other common related antihypertensive drugs are angiotensin-2 receptors blockers, which block at-1, a receptor for angiotensin-ii, through which it exerts its vasoconstrictor effect. however, at-1 is not known to be used by sars-cov-2 to infect cells. it was shown in animal models that ace inhibitors might increase ace2 expression, thus increasing susceptibility to infection. it has not been proven in humans but it raised the concerns during the covid-19 pandemic. 217 based on the data available to date, antihypertensive treatment with these medications should be continued. 218 at the moment, the animal model that resembles more closely human covid-19 is the rhesus macaque, whose ace2 receptor is identical to that in humans. this model recently showed that sars-cov-2 reinfection was hampered due to infection-acquired immunity and demonstrated the therapeutic effect of remdesivir in covid-19 prior use in human clinical trials. 219, 220 the murine ace2 receptor is different from humans, hence humanized murine models with recombinant human ace2 are necessary. 221 previous vaccine research for sars/mers facilitates rapid translation. 222 in the who vaccine single-domain antibodies have been investigated as potential therapeutics for influenza, rsv and hiv in addition to coronaviruses. sars-cov-2 mainly targets the respiratory tract, hence the development of vaccines directed to the respiratory epithelia and lung parenchyma using a nebulizer has been considered to maximize bioavailability and function. 229 although active research against respiratory viruses has focused on aerosolized plasmid dna vaccines, other forms of vaccine administration are currently further advanced in clinical trials. 222 veterinary medicine commonly uses aerosolized coronavirus vaccines for chicken farms. 230 a novel vaccine platform requires careful evaluation and should ideally include toxicological studies in valid animal models. early progress towards sars vaccines has facilitated a "running start" but standards of care and safety must be maintained. acceleration rather than omission of clinical trials is key. preliminary data from oxford university is anticipated by mid-2020. 222 of note, a doseescalation, single-center, open-label, non-randomized, phase 1 was conducted in 108 healthy individuals that received an ad5 vectored covid-19 vaccine. the vaccine was tolerable and this article is protected by copyright. all rights reserved immunogenic at 28 days post-vaccination. sars-cov-2-specific antibodies peaked at day 28 postvaccination and specific t-cell responses were detected from day 14 post-vaccination. 231 an important aspect is that covid-19-associated mortality is very high, almost unavoidable when the pandemic control fails. this is due to rapid community spread, high community virus, especially in the elderly and co-morbid, but also in younger non-comorbid persons, including healthcare workers, young adults and children. the covid-19 pandemic also seems to be characterized by a significant level of asymptomatic spread. [232] [233] [234] the iceberg of covid-19: are there asymptomatic cases below the surface? the the differences are almost entirely due to the timing and effectiveness of public health interventions. countries that failed to control did too little, too late, and allowed sars-cov-2 to rip through their population, with catastrophic outcomes. those that intervened early effectively stopped the disease transmission. 235 this article is protected by copyright. all rights reserved it is difficult to determine as it varies greatly from country to country, depending on how well countries control their epidemics with widespread testing, case isolation and vigorous contact tracing, testing and isolation if positive. in countries that do this well, the r0 can be very low indeed. in countries that fail to control the spread of the virus, the r0 is high but unknown as sars-cov-2 spreads untested and therefore undetected. it has been estimated to be ~2.2. 236 sars-cov-2 transmits more readily than either sars-cov or mers-cov. the r0 of sars-cov-2 is controversial but if left unchecked it is likely to be greater than 3-4. however, the r0 number cannot be precisely defined as no country has left it to spread completely unchecked. in any case, even when preventative measures are taken, the r0 of sars-cov-2 is higher than that of sars-cov (1.7-1.9) and mers-cov (<1). 237 there is a considerable frequency of very mild covid-19 patients as well as asymptomatic sars-cov-2-infected people. this makes transmission control more challenging than either sars-cov or mers-cov, where illness is frequently more severe. children are at low risk of severe covid-19 outcomes. 238, 239 most patients in pediatric age with sars-cov2 infection presented with no or mild clinical manifestations, including fever, fatigue and dry cough. they were typically managed with supportive treatments only and they had generally a favorable prognosis with a recovery within 2 weeks. [240] [241] [242] young children also frequently carry other respiratory viruses, which potentially limit sars-cov-2 infection, as reported for other viral infections. 243 differences between children and adults in the regulation of ace2 expression may also play a role. 46 ace2 mrna expression was high in type i and ii alveolar epithelial cells, in nasal and oral mucosa and nasopharynx, in smooth muscle cells and endothelium of vessels from the stomach, small intestine, colon, and in the kidney of human adults (mean age 52±22). 244 interestingly, a recent study demonstrated age-dependent ace2 gene expression in the nasal epithelium, which was lowest in younger children and increased with age. 245 in addition, cd147, cd26 and their molecular interaction proteins seem to be differently expressed in peripheral blood mononuclear cells and t cells in children in comparison with adults. 14 many children remain asymptomatic, even when they have radiologic pneumonia detected on screening. 238 given that children are effective transmitters of other respiratory viruses, 246 it is expected that they will be just as good at transmitting sars-cov-2. bats are likely the natural reservoir of sarsseverity (see questions below). data on ethnicity and covid-19 are scarce and further research on ethnicity and covid-19 outcomes is needed. 250 however, the data available show a disproportionate number of covid-19 deaths in black, asian and minority ethnic backgrounds. in fact, one third of uk icu admissions are reportedly from them. 251 in the usa, african americans had more covid-19 diagnoses and deaths, after adjusting for age, poverty, comorbidities, and epidemic duration. these disparities are also seen in the hispanic and asian communities. 252 pregnant women may be at a higher risk of poorer covid-19 outcomes because they have deficient ifn-α and ifn-λ responses to viral infections. 253 however, reported pregnancy outcomes in covid-19 are reassuring as they appear similar to non-pregnant adult females. 254 this article is protected by copyright. all rights reserved testing treatments is problematic because pregnant women are excluded from most trials. 255 it is known that azithromycin doubles innate ifn production from virus-infected lung cells. 256 it is safe for all trimesters of pregnancy 257 and has been shown effective in high-quality clinical trials of virusinduced lung disease. 258, 259 given that the human ace2 protein is encoded on the x chromosome, this may be relevant for malefemale differences in outcomes. particularly in males with rare ace2 coding variants as they will express those variants in all ace2-expressing cells compared to a mosaic pattern of expression in females. 260 males may also have differences in certain innate antiviral responses compared to female counterparts. 261 there is reasonably robust data of covid-19 deaths in hospitals because most people who die in hospital are tested. deaths outside hospitals are likely underestimated as people are dying in care homes where mortality approaches ~40%, 267 and may die without being tested and diagnosed. it is difficult to determine prevalence as testing practices vary so much from country to country. seroprevalence studies will help to collect these data. covid-19 was introduced rapidly to many industrialized countries as a result of air travel. 268 most of europe and the usa probably did not react in a timely and efficient manner, resulting in the rapid spread and subsequent high mortality rates. in light of the devastating situation in many european countries and the usa, less industrialized countries had a little more time to better prepare to control the pandemic. 269 an important factor for prevalence studies is the percentage of the population that has undergone a diagnostic test, which seems to be at lower levels in developing countries. this article is protected by copyright. all rights reserved respiratory viruses spread less readily in summer than in winter for reasons that are not well understood. dry air and higher temperatures are slowing down the spread of respiratory viruses. absence of school attendance, more time outdoors, greater household ventilation, warmer temperatures facilitating virus inactivation and higher vitamin d levels are all likely to play a part. although social distancing measures are implemented, the summer weather should play a role in hampering the spread of covid-19. however, based on the analogy of previous influenza pandemic, it is unlikely that summer, on its own, could stop transmission of sars-cov-2. [270] [271] [272] it largely depends on the sars-cov-2 seroprevalence developed in each country, which is still unknown. countries that have had widespread transmission may be hit by a second wave, but presumably with less severe consequences. countries that effectively controlled the pandemic are at a higher risk of second wave of covid-19 if those effective controls are relaxed due to the limited viral transmission and lack of active immunization. sars-cov-2 has spread worldwide in humans, causing mild or no disease in many cases. it will continue circulating similar to other human coronaviruses (229e, hku1, nl63, oc43), and it may well become an endemic, seasonal virus. 273 the main route of sars-cov-2 transmission is via respiratory droplets and aerosols. [274] [275] [276] avoidance of high virus loads, acquired through aerosol and droplet transmission, is paramount to prevent severe outcomes. consequently, social distancing, masks and hand sanitation are undoubtedly effective because they prevent the droplet and surface contact-associated initial high virus load and the increased risk of severe disease. [277] [278] [279] what is the evidence supporting social distancing and face mask to prevent sars-cov-2 infection? this article is protected by copyright. all rights reserved a systematic review and meta-analysis has found that transmission of viruses was lower with physical distancing of 1 m or more, compared with a distance of less than 1 (or 0.18) and protection was increased as distance was lengthened. in addition, face mask use could result in a large reduction in risk of infection (or 0.15), with stronger associations with n95 or similar respirators compared with disposable surgical masks or similar. eye protection also was associated with less infection (or 0.22). 280 therefore, the covid-19 pandemic can be controlled if social distancing is combined with widespread testing, case isolation, vigorous contact tracing and personal protection. indeed, severe and critical illness among chinese healthcare workers before january 10 th was 45%, a time when personal protection equipment and infectious control measures were likely not implemented. after february 1 st , when personal protection measures were in place, the percentage of severe and critically ill chinese healthcare workers dropped to 8.7%. 281 sars-cov-2 remained viable in aerosols for 3 h with a ~10-fold reduction in infectious titre. 282 sars-cov-2 was more stable on plastic and stainless steel than on copper and cardboard; viable virus was detected up to 3 days after application to plastic and 2 days to stainless steel, on each surface the virus titer was reduced nearly ~100-fold. 282 importantly, sunlight exposure inactivated 98% of infectious sars-cov-2 every 6.8 minutes in simulated saliva and every 14.3 minutes in culture media. this study suggests that persistence, and subsequently exposure risk, may vary significantly between indoor and outdoor environments. 272 therefore, it is convenient to minimize contact with surfaces touched by others (even before sars-cov-2 existed), particularly at indoor environments, for example when using public transportation. in 248 covi̇d-19 patients, the estimated median time from symptom onset to viral clearance in the nasal swabs was 11 days, while in asymptomatic cases it was 2 days. 283 in patients that recovered, the median duration of viral shedding was ⁓20 days, while in non-survivors it was detected until death. the longest duration of viral shedding in survivors was 37 days. 110 accepted article and found that a slow viral clearance is associated with an increased risk of high disease severity with a 1% mortality rate. 285 the individual variation in the transmission of an infection is described by a factor called "dispersion factor or k". the lower "k" value, the more transmission comes from a small proportion of individuals acting like superspreaders. superspreading clusters have been observed in past coronavirus outbreaks (sars/mers), where a small number of infected individuals was responsible for a large proportion of secondary transmissions, with an estimated "k" of about 0.16 for sars and 0.25 for mers. 286 it is unclear whether superspreading clusters have contributed to the covid-19 outbreak. a simulation of early outbreak trajectories estimated that "k" for covid-19 is higher than for sars and mers. 286 however, in a recent preprint study, the estimate of "k" for sars-cov-2 was around 0.1, suggesting that around 10% of infected patients may have been responsible for 80% of secondary transmissions. 287 individual variation in infectiousness is difficult to measure, as it is mostly empirical, but the identification of any sars-cov-2 superspreading will be of primary importance for pandemic control. the designation of covid-19-dedicated wards and personnel within hospitals is useful to limit nosocomial sars-cov-2 infections. 124 it also allows other non-covid-19 conditions to be treated using routine healthcare resources more safely and effectively. maintaining such separation requires intensive sars-cov-2 testing in view of the high asymptomatic infection rate. 288 community-based strategies are effective at controlling the transmission of sars-cov-2. australia, hong kong, japan, singapore, south korea, and new zealand have all controlled effectively. their cumulative covid-19 mortality is >100-fold less than that in belgium, france, italy, spain and the uk, countries which have had difficulties to adequately control the pandemic. 147, 289, 290 it is important to implement measures to contain the spread of the virus, such as developing models to predict sars-cov-2-related mortality. 291 closing live animal markets is likely to reduce the risk of future viral outbreaks although this is not a practical way to prevent viral outbreaks for multiple reasons including social and economic. lifestyle factors that may influence sars-cov-2 infection susceptibility and covid-19 severity include smoking, stress, diet and alcohol intake, among others. for example, smoking has been shown to increase the susceptibility to respiratory tract infections and its severity, 294 and it is a risk factor for severe covid-19. 295 moreover, alcohol consumption may impair anti-viral immunity; 296 in vitro studies with human monocytes have shown that both acute and prolonged alcohol exposures inhibit type i ifn induction upon toll-like receptor-8 and -4 stimulation 297 . dietary habits may also play a role as obese patients have been shown to have a higher risk of developing severe covid-19. 298 furthermore, there are bioactive food compounds with antiviral activity, such as resveratrol, 299 although the amount of them obtained through the diet is unlikely to play a relevant role in covidit is known that respiratory virus infection causes perturbations in the gut microbiota and that germfree mice are more susceptible to viral infections, which intimates a role for the microbiota in covid19 . 300 however, the impact of the commensal microbiota on sars-cov-2 infection susceptibility and covid-19 severity is unknown. 301 an essential step is identifying the bacterial species interacting with sars-cov-2. this is rather challenging given the large number of bacterial species in the lung and respiratory tract, 302 and especially in the gut. however, a number of lung metagenomic studies have reported an abundance of prevotella in the lung of sars-cov-2 infected patients 303 . while in accepted article silico analysis have revealed that prevotella proteins may promote viral infection 304 , prospective studies are necessary to ascertain if this is a consequence of the infection or a risk factor for it. it is well-established that epithelial barrier defects and/or damage favor the development of th2 immunity. 305, 306 increased hygiene, in general, as well as overexposure to epithelial barrier opening molecules, such as detergents, can promote the onset of allergic disease. 307 to date, there is no evidence linking the covid-19 protective measures (gloves, hand-sanitizers, etc.) with increased allergy prevalence. in this regard, multifactorial epidemiological studies are needed. these studies should consider the impact on allergic diseases of virus-specific type 1 responses and psychosocial and environmental changes caused by the pandemic and efforts to contain it. although there has been a significant change in pollution parameters, unfortunately this reduction in pollution is transient and consequently unlikely to be significant. the exposome-related allergy and asthma risk is multifactorial. it includes climate change, biodiversity, the microbiome and nutrition among others, which have not changed during the pandemic. 308 in addition, although pollution levels have dropped, climate change still occurs at an accelerated pace. lifestyle changes during the lockdown 309 , weight gain and increased exposure to indoor allergens and pollutants may even increase the incidence of allergic diseases in the long-run. with the rapid spread of covid-19 at a pandemic scale, we are overwhelmed and drowned with a wealth of information. a global fight to contain the pandemic has started in which we need international solidarity and prompt sharing of accurate scientific information. we strongly support the this article is protected by copyright. all rights reserved continue scit or slit: non-infected individuals this article is protected by copyright. all rights reserved hypersensitivity reactions to drugs may occur more often during the pandemic due to the increased use of drugs and drug interactions, which can result in morbilliform rash, erythroderma, early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia effect of changing case definitions for covid-19 on the epidemic curve and transmission parameters in mainland china: a modelling study presumed asymptomatic carrier transmission of covid-19 eleven faces of coronavirus disease 2019 the proximal origin of sars-cov-2 phylogenetic network analysis of sars-cov-2 genomes structural 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plasma for covid-19 patients in wuhan effect of convalescent plasma therapy on viral shedding and survival in covid-19 patients treatment of covid-19 patients with convalescent plasma the authors thank the european academy of allergy and clinical immunology this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord-318944-13zk6cco authors: bizzoca, maria eleonora; campisi, giuseppina; lo muzio, lorenzo title: covid-19 pandemic: what changes for dentists and oral medicine experts? a narrative review and novel approaches to infection containment date: 2020-05-27 journal: int j environ res public health doi: 10.3390/ijerph17113793 sha: doc_id: 318944 cord_uid: 13zk6cco the authors performed a narrative review on severe acute respiratory syndromecoronavirus-2 ( sars-cov-2) and all infectious agents with the primary endpoints to illustrate the most accepted models of safety protocols in dentistry and oral medicine, and to propose an easy view of the problem and a comparison (prevs post-covid19) for the most common dental procedures. the outcome is forecast to help dentists to individuate for a given procedure the differences in terms of safety protocols to avoid infectious contagion (by sars-cov-2 and others dangerous agents). an investigation was performed on the online databases pubmed and scopus using a combination of free words and medical subject headings (mesh) terms: “dentist” or “oral health” and “covid-19” or “sars-cov-2” or “coronavirus-19”. after a brief excursus on all infectious agents transmittable at the dental chair, the authors described all the personal protective equipment (ppe) actually on the market and their indications, and on the basis of the literature, they compared (before and after covid-19 onset) the correct safety procedures for each dental practice studied, underlining the danger of underestimating, in general, dental cross-infections. the authors have highlighted the importance of knowing exactly the risk of infections in the dental practice, and to modulate correctly the use of ppe, in order to invest adequate financial resources and to avoid exposing both the dental team and patients to preventable risks. the era of corona-virus-disease-19 is an important historical period from various points of view, from the world health to the huge cascade of socio-economic implications. everyday habits have been turned upside down, and the way of life of people all over the globe, engaged in all activities, especially in the health sector, will be involved in this necessary change. dentists, being in close contact with the patient's droplets and aerosols generated, have to revise the operating protocols to protect the team and the patients from the risk of infectious diseases. unfortunately, the pandemic covid-19 will not stop immediately and everyone will have to face each other very long working and social recovery times of the population. in this time, a large part of the population will avoid dental treatment other than those imposed by pain or urgency, both due to money issues and, principally, for a psychological reason: it will not be easy to overcome the fear of infection. for many, the dental practice is a source of possible infections, considering that the first person at risk is the dentist himself. the scenario in dental practices is very complex and several problems can arise which are dangerous for the dental practice [1] [2] [3] [4] [5] [6] [7] [8] [9] . for an infection to emerge, it is necessary that an adequate number of specific microorganisms can infect a person or groups. the classic contamination paths clearly incorporate all the dental unit (team and patient): body fluids in direct contact with the wound site during operation, injuries of the skin and the mucosa with sharp objects, body fluids and contaminated material contact with eyes, aerosols arising during the operation with air produced by turbine and ultrasonic devices, contamination via droplet, and surgical smoke formed during electro-cautery or laser applications [10, 11] . the first problem raised with respect to covid-19, is related to the easy spread of viral agents in the air during dental procedures [12] [13] [14] [15] . hence, aerosol is the most aggressive source of covid-19 as well as other viral infections, placing dentists and their collaborators at the first line of the exposure to risk scale within the context of healthy personnel [10, 16] . the second problem is related to the persistence of the biological agent in operating rooms. the aerosol produced by high rotation instruments and ultrasound could remain for several hours in the air and on the surfaces [17, 18] . although it can save the operator, if well protected, during the therapeutic acts, it means that the air will be contaminated, thus presenting a risk for operators after removing the ppe (personal protective equipment) and for the next patients. this covid-19 pandemic has shown that several people can be positive and spread the viral agents around without any symptoms or signs of biological agents. so, the dental team, a part performing the double triage [1] , should consider each patient as sars-cov-2 positive until proven otherwise and use protective equipment in order to preserve their own health and the health of all patients as attending the dental office. for these reasons, it is necessary to use rigid and precise operating protocols capable of classifying dental procedures based on risks for the team as well as for the patients. this study was born from the awareness of a necessary change in decision making processes. it involves a rereading of relevant literature in order to build protocols addressed to dentists, to assess and modulate the risks of contagion in the dental practice. moreover, it proposes, on the basis of information from literature, a classification of dental procedures based on the risk of contagion of infectious agents, showing what will change for the dentist and the oral medicine expert. an investigation was performed on the online databases pubmed and scopus using a combination of free words and mesh terms: "dentist" and "covid-19" or "sars-cov-2" or "coronavirus-19", and "oral health" and "covid-19" or "sars-cov-2" or "coronavirus-19". only studies fulfilling the following inclusion criteria were considered eligible for inclusion in this study: (i) performed on human subjects, (ii) written in the english language, and (iii) published in 2019-2020. the manuscript titles list was highlighted to exclude irrelevant publications and search errors. the final selection was performed by reading the full texts of the papers in order to approve each study's eligibility based on sars-cov-2 and other infective agents involved in dentistry. data selection and revision was performed by two independent reviewers (meb, university of foggia and gc, university of palermo). they singularly analysed the papers, and in agreement, included 142 papers in this narrative review. the authors, in consideration of the importance of the emerging topic, decided to include also guidelines, online documents, reviews, experts' opinions, renouncing the prisma-related design of regular systematic reviews ( figure 1 ). after a brief excursus on all possible infectious agents, the authors described, on the basis of the literature selected, all personal protective equipment (ppe) actually on the market and their indications. then, they compared (before vs after covid-19 era) the correct safety procedures for each dental practice selected, underlining the danger of underestimating, in general, dental cross infections, if focused only on the newest sars-cov-2. results are summarised in tables 1-8. different classes of bacteria, viruses, and fungi can cause human infections. three factors are important for the transmission of these infectious agents: an infectious agent, a receptive subject and a transmission mode. the pathogens involved in infections during health care mainly derive from staff, from patients (and possible careers), but also from inanimate environmental sources ( figure 2 ). these human sources can: 1) have active infections, 2) be asymptomatic or in an incubation period, or 3) be colonized transiently or chronically with pathogen microorganisms. the infection is the consequence of the contact between a contagious agent and a potential host. moreover, the same characteristics of the host can influence the onset and the severity of the infectious disease. however, several other factors can modify the virulence and behavior of infectious disease such as the number of infectious agents, the transmission way and the pathogenicity [19] . predictors of the disease evolution in a specific subject could be: immune status at exposition time, age, comorbidity, and virulence of the agent [20] . there are two main ways of infective transmission, namely vertical (from mother to fetus: transplacental, during vaginal birth or breast feeding) and horizontal (sexual and non-sexual). in a dental setting, infectious agents are transmitted in the horizontal, non-sexual route [21] . in the non-sexual horizontal transmission, direct or indirect contact (e.g., herpes simplex virus, respiratory syncytial virus, s. aureus), droplets (e.g., influenza virus, b. pertussis) or airways (e.g., m. tuberculosis) are possible routes. other viruses can be transmitted by the blood (e.g., hepatitis b and c viruses and hiv) via percutaneous or mucous membrane exposure [3, 4, 14, 22] . in synthesis, the three main routes of the transmission are [23] : contact transmission: contact transmission can be through direct contact and indirect contact. during direct contact transmission, pathogens are transmitted from an infected person to another subject without an intermediate object or person (for example, mucous membrane or breaks contact blood or other blood-containing body fluids infected, or contact hsv lesion without gloves) [3, 4, 14, 15, 18, 22, 24, 25] . during indirect contact transmission, pathogens are transmitted to the host through objects or human body carrying those pathogens [17, 18, 22, [26] [27] [28] [29] [30] . moreover, all the personal protective equipment (ppe), such as uniforms or isolation gowns, can be contaminated by infectious agents during the treatment of a patient colonized or infected. • droplet transmission: some infectious agents can reach the host through the direct and indirect contact routes or through droplets [3, 15, [31] [32] [33] . droplets can carry infectious pathogens travelling for short distances directly from the respiratory tract of the infectious subjects to host reaching susceptible mucosal surfaces [3, 15, [31] [32] [33] . respiratory droplets are produced during coughs, sneezes, or talks [34] or by airway health procedures. the nasal mucosa, conjunctivae, and mouth are good portals for respiratory viruses [35] . to date, the maximum distance that a droplet can reach is not known, even if pathogens transmitted by a droplet do not run across long distances [19] . the size of droplets has traditionally been defined as being >5 µm [19] . several types of droplets, including those with diameters of 30 µm or greater, can remain suspended in the air [36] . the sizes of the droplets can determine the maximum distance reached: largest droplets, between 60 and 100 microns, totally evaporate before spontaneously falling 2 m away [37] . for respiratory exhalation flows, the critical factor is the exhalation air velocity: these droplets are carried more than 6 m away by exhaled air at a velocity of 50 m/s (sneezing), more than 2 m away at a velocity of 10 m/s (coughing), and less than 1 m away at a velocity of 1 m/s (breathing) [37] . airborne transmission: this means of transmission consists of dissemination of airborne droplet or small particles containing infectious pathogens that remain infective over time and distance (e.g., spores of aspergillus spp., and m. tuberculosis) [31, 33, [38] [39] [40] [41] [42] . several infectious agents can involve dentist and his team [10] (table 1) . • sars-cov-2 determines covid-19 (coronavirus disease 2019), an infectious disease characterized by several important systemic problems such as coronavirus associated pneumonia. the principal symptoms are fever, cough, and breathing difficulties; the most patients have mild symptoms, some progress to severe pneumonia [43] . the diagnosis is performed with the identification of the virus in swabs of patient throat and nose. covid-19 can involve the respiratory tract determining a mild or highly acute respiratory syndrome due to the production of pro-inflammatory cytokines, such as interleukin (il)-1beta and il-6 [44] . one mechanism that can make the coronavirus lethal is the induction of interstitial pneumonia linked to an over-production of il-6 [44, 45] . based on this principle, several researchers have started to use an anti-arthritis drug, tocilizumab, for its anti-il-6 action [46] [47] [48] [49] . herpes simplex virus (hsv) can determine a primary infection with minor, ulcerative lymphadenopathy gingivostomatitis [50] and a recurrent infection with cold sores. herpetic whitlow, an hsv infection of the fingers is usually caused by direct contact of the same fingers with infected saliva or a herpetic lesion [51] [52] [53] . skin, mucosal lesions, and secretions such as saliva can determine the transmission [54, 55] . lesions are usually characterized by vesicles and sequent crusting. acyclovir can be used for the treatment of the diseases. it is sufficient to wear gloves in order to avoid the herpetic whitlow when the clinician treats patients with hsv lesions [10, 56] . • varicella zoster virus (vzv) can determine chickenpox (primary disease), usually in children, and shingles, which is very painful (secondary disease), for the reactivation of a virus residing in sensory ganglia during the latency period [57] [58] [59] . chickenpox disease is highly contagious and spreads via-airborne routes [60] [61] [62] . the virus can infect nonimmune dental team via inhalation of aerosols from a patient incubating the disease. masks and gloves can be not sufficient for complete protection of the healthcare workers [10] . [77] . it is present in saliva and could infect susceptible subjects via direct contact or aerosols [78] . human t-lymphotropic virus is involved in adult t cell leukaemia and spastic paraparesis. this virus can be transmitted through blood [79] [80] [81] [82] and, in a dental setting, it can infect via sharp instruments injuries [10] . hepatitis b virus (hbv) causes acute hepatitis and it is an important risk-agent for the health care staff [83, 84] . the possible ways of transmission are sexual intercourse, through blood, contaminated material injuries, and perinatal way [85] [86] [87] [88] . so, all operators of the dental team should be vaccinated [10, 89] . hepatitis c virus (hcv) causes "non-a" and "non-b" hepatitis and it is transmitted like hbv [85] [86] [87] 90] . the primary infection is often asymptomatic and the most of infected subjects become carriers of the virus with risk of development of chronic liver disease that could evolve in hepatocellular carcinoma [10] . human immunodeficiency virus (hiv) infects the immune system of susceptible subjects, t-helper cells particularly. it can be transmitted like hbv (sexual intercourse, blood borne and perinatal ways) [91, 92] . moreover, this infection have oral manifestations that can help in diagnosis: e.g., oral candidiasis, oral hairy leukoplakia, oral necrotising ulcerative gingivitis and oral kaposi's sarcoma [10, [93] [94] [95] [96] . • cytomegalovirus (cmv) is part of the herpes virus family and can cause diseases with several manifestations [97] . mumps virus is part of the paramyxoviridae group. this pathogen often affects the parotid glands, and the consequently characteristic symptom is swelling of these salivary glands [98] . moreover, this virus can cause inflammation of the ovaries, testis, pancreas or meninges with several complications. after the introduction of the vaccine against measles, mumps, and rubella (mmr), mumps incidence has decreased, even if several mumps cases have recently been reported [99] . • mycobacterium tuberculosis causes tuberculosis and is a bacterium transmitted by inhalation, ingestion and inoculation. the main symptoms are cervical lymphadenitis and lung infections. in order to prevent infection, the dental team should be adequately vaccinated and wear ppe [100] [101] [102] [103] [104] [105] . this bacterium is resistant to chemicals and, for this reason, sterilization and disinfection protocols must be rigorously performed [10] . • legionella spp. is a gram-negative bacterium that causes legionellosis and generally it resides in water tanks. legionellosis occurs with pneumonia, sometimes lethal in older people. since this pathogen lives in water, it can be easily transmitted during dental procedures through aerosols from incorrectly disinfected water circuits [106, 107] . in fact, water circuits that remain unused for long periods of time should be checked regularly to prevent legionella bacteria from residing [106, 107] . • treponema pallidum causes syphilis and dental team must wear gloves in order to adequate protect themselves [10, 108] . meningococcal spp. are gram-negative bacteria. they are located on the nasopharyngeal mucosa and their presence is generally asymptomatic. the bacterium is easily transmitted, especially during adolescence, when people get together. as already mentioned, colonization of the nasopharynx is common, and while the resulting disease is rare, at times, it can cause death or permanent disability [109, 110] . staphylococcus aureus is an important agent involved in nosocomial infections. this bacterium causes a wide range of diseases that can be mild or life-threatening (e.g., bacteraemia, pneumonia, and surgical site infection [111] ). in addition, s. aureus can easily have antimicrobial resistance. this bacterium principally resides on the epithelium of the anterior nares in human beings [112] . group a streptococcus (gas) is a gram-positive, beta-haemolytic bacterium. this pathogen is responsible for several diseases in human beings, such as acute pharyngitis, impetigo and cellulitis. it can also cause serious invasive diseases such as necrotizing fasciitis and toxic shock syndrome (tss) [113] [114] [115] . the bacterium mainly resides in human nose, throat and on skin and it is often transmitted without symptoms [116] [117] [118] . obviously, asymptomatic subjects are less contagious than the symptomatic carriers of this bacterium. gas is transmitted through respiratory droplets spread in the air, for example during coughing, sneezing and nasal secretions [117, 118] . in addition, this bacterium can spread through close interpersonal contact during a kiss, using the same dishes and sharing the same cigarette. streptococci mutans mainly colonize dental surfaces after tooth eruption and is associated to the development of caries [119] . this bacterium may be transmitted horizontally between children during the initial phases of the s. mutans colonization in nursery environments [120] . there is scientific evidence of vertical transmission of s. mutans from mother to child [121] . • some periodontal bacteria (e.g., a. actinomycetemcomitans, p. gingivalis) are considered person-to-person transmitted, but it is still unclear if transmission is governed only by domestic pathways, without definitive implications for the dental office. vertical transmission of a. actinomycetemcomitans is between 30% and 60%, while that of p. gingivalis is rarely observed. horizontal transmission ranges from 14% to 60% for a. actinomycetemcomitans and between 30% and 75% for p. gingivalis [122] . certainly, by understanding the spread mechanisms of these bacteria, it would also be possible to prevent a number of systemic diseases [123] . the dental team must adapt several precautions to avoid these infections; an adequate training and information of the personnel is mandatory in order to control infections in the dental office. the individual protection methods include a series of enforcement with the aim to reduce the risks of contamination, unfortunately without being able to eliminate them. the basic principle of infection control is to approach to each patient as if he was an infected patient (by one of the main microbes listed above) and to correctly carry out the protection methods [124] . adequate personal protective equipment (ppe) must be selected based on a risk assessment and the procedure to be performed. the precautions for infection control require wearing gloves, aprons, as well as eye and mouth protection (goggles and mask, such as medical masks and filtering face piece or fpp) for each procedure involving direct contact with the patient body fluids. whenever possible "single use" or "disposable" equipment should be used [10] (table 2) . if the necessary precautions are not taken, it is inevitable that operators can become infected through contact of the mucous membranes with blood, saliva, and aerosols from a potentially infective patient [10] . in healthcare setting, masks are used in order to: protect personnel from contact with patient infectious material; 2. protect patients from infectious agents carried by healthcare workers; 3. limit the potential spread of infectious respiratory aerosol between patients [19] . masks can be worn with goggles in order to protect mouth, nose and eyes, or with a face shield to provide more complete face protection. we must distinguish masks from particle respirators that are used to prevent inhalation of small particles which may contain infectious agents transmitted through the respiratory tract. the mouth, nose, and eyes are sensitive portals to the entry of infective pathogens, such as skin cuts. medical masks: • could be flat or pleated (some are like cups) and fixed to the head with straps or elastic bands; • does not offer complete protection against small particle aerosols (droplet nuclei) and should not be used during contact with patients with diseases caused by airborne pathogens; • they are not designed to isolate the face and therefore cannot prevent inhalation by the health personnel wearing them; • they must be replaced if wet or dirty. there are no standards that evaluate the efficiency of the medical mask filter. aorn (association of peri-operative registered nurses) recommends that medical (surgical) masks filter at least 0.3 µ particles or have a bacterial filtration efficiency of 90%-95% [126] . surgical masks (sm) are used to prevent that large particles (such as droplets, sprays or splashes), containing pathogens, could reach nose and mouth [127] . although their purpose is to protect patients from healthcare professionals (and healthcare team from patients) by minimizing exposure to saliva and respiratory secretions, they do not create a seal against the skin of the face and therefore are not indicated to protect people from airborne infectious diseases. masks are available in several shapes (modeled and unprinted), dimensions, filtration efficiency and attachment method (ribbons, elastic through the ear). masks are disposable and must be changed for each patient. instead, during the treatment of patients with respiratory infections, particulate respiratory masks must be worn. particulate respirators (with filtering percentage) in use in various countries include: [126] . ffp2 european respirators are comparable to n95, and they are indicated for prevention of infectious airborne diseases. however, ffp3 respirators offer the highest level of protection against infectious agents and are the only ffp class accepted by the health and safety executive (hse) as regards the protection in the healthcare environment in the united kingdom [126] . the powered air purifying respirator is also considered a standard part of ppe in certain situations, including aerosol generation procedures in high risk environments. in the event of a pandemic infection, any aerosol generation procedure on infected patients should only be carried out with an ffp3 respirator. non-urgent procedures should be postponed until the infection resolves. in the us, the national institute for occupational safety and health (niosh) defined the following particulate filter categories in 2011, in title 42 code of federal regulations, section 84 (table 3) . there are several models of ffp2 and ffp3 respirators, both with valves and without valves. however, this is not a filter but a valve that regulates the flow of air at the outlet and therefore makes it easier to exhale. therefore, these masks are designed to be able to filter very well the air that comes in the mouth, nose, and lungs of those who wear them. instead, these masks are not designed specifically to prevent the wearer from infecting someone else with their own breathing. in practice, if a mask has a valve, it can let out particles, even if it manages to block almost all the inlet ones. and therefore, a healthy person can use it effectively so as not to get infected. for a sick person or one who could be contagious, however, using it could infect others by letting germs pass from their breath outwards. it is important to say that there is no specific test that has been done to verify the possibility that the virus spreads from an infected person passing through a mask equipped with a valve [128] . surgical masks, on the other hand, are similar in both directions. they have been designed to prevent healthcare workers and surgeons in particular from infecting their own breath with patients, who may have open wounds on the operating table, but also work to protect the healthcare staff themselves against a potentially contagious person. their effectiveness, however, is much lower also because they do not prevent the breath from spreading and allow a lot of air to pass through and to the mouth and nose [128] . the choice of individual eye protection devices (such as goggles or face mask) varies according to the exposure circumstances, other ppe worn. and the need for personal vision [10] . in order to protect the eyes, eyeglasses and contact lenses are not considered suitable [129] . eye protection must be effective but at the same time comfortable and allow sufficient peripheral vision. there are different measures that improve the comfort of the glasses, for example anti-fog coating, different sizes, the possibility of wearing them on prescription glasses. although they provide adequate eye protection, glasses do not protect from splash or spray the other parts of the face. disposable or sterilizable face shields can be used in alternative to glasses. face shield protects the other areas of the face besides the eyes (glasses only protect the eyes). the face shields that extend from the chin to the forehead offer better protection of the face and eyes from spray and splashes [83] . the removal of a facemask, goggles, and mask can be safely performed after removing dirty gloves and after performing hand hygiene. gowns and coveralls are additional personal protective equipment in the health sector [83] . operator hygiene, including wearing appropriate clothing and ppe, has a dual purpose: on the one hand, to defend the operator himself in an environment where the infectious risk is high, and on the other hand to prevent the operator from becoming responsible transmission of infections. to increase the protective function of the uniform or to carry out those procedures in which high contamination is expected, additional disposable clothing can be worn [83] . these clothes can be ppe certified for biological risk and for this recognition must comply with the requirements of the technical standards, namely european standards are en 14126 and iso 16604 (dpi) and en 24920 (dm). the material constituent is mainly tnt (texture not texture), which is suitable for "disposable" use in this specific area. to offer greater protection of the part front of the body, the most exposed to risk, it is required that such lab coats have standard features within the heterogeneity of the models, for example: back closure, covered or heat-sealed seams, long sleeves with cuffs tight and high collar. obviously, for these devices, comfort and practicality are also required, so the operator must be able to move freely and perceive good perspiration [83] . different types of gowns and overalls are available with varying levels of protection. the level of protection depends on various factors including the type of tissue, the shape and size of microorganisms, the characteristics of the conveyor, and various external factors [130] . in high-risk environments, it is recommended to use waterproof and fluid-resistant gowns or overalls. during minor oral surgery, surgical gowns must be worn with tight cuffs that must be inserted under the gloves. fabric work uniforms must be washed daily on a hot 60 • c cycle. fabric uniforms are not considered ppe since the material they are made of is absorbent and therefore offer little protection against infectious pathogens. during all dental procedures, it is impossible to avoid contact of the hands with blood and saliva [10] . that is why all operators must wear protective gloves before performing any type of procedure on patients [10] . gloves must be changed with each patient and at every contact with contaminated surfaces to prevent cross-infection [10] . not only the dentist, but also other dental team members must wear gloves during dental procedures [10, 131] . gloves used in dental clinic can be distinguished basically in two categories: those for purely use clinical and those for instrumentation reordering procedures and of the operational area. when cleaning dental appliances and instruments, more durable gloves should be worn than normal non-sterile gloves to prevent injury [10] . regarding clinical gloves, a clear distinction must be made between them procedures that require invasive action on the patient, or however at clear biological risk, and the procedures that do not require them, or in any case present a negligible biological risk for the operator. the two types of gloves resulting from this distinction are found in the words "inspection gloves" and "surgical gloves" one commonly used nomenclature [83] . both disposable products, from a macroscopic point of view usually have some obvious differences: • surgical gloves in general always distinguish the right side from the left, they are long enough to be worn over the cuffs of the gowns and always packaged in sterile pairs, • the inspection glove is usually an ambidextrous device, shorter and thinner than the previous one and rarely sterile [132] . in general, clinical gloves are made of latex, nitrile or vinyl. latex and nitrile have proven to be more resistant than and therefore are generally preferred. gloves contain powder to make them easier to wear, but which can cause skin irritation [10] . powder-free gloves exist on the market and they should be used when such reactions occur [10] . some people may experience allergies and contact dermatitis due to latex [10] . latex-free gloves for allergy sufferers are also available [10] . also, the weather of use is an absolutely relevant parameter in terms of protection. the use of the glove, especially if in latex, involves development not perceived of microperforations which become particularly significant from a numerical point of view after 60 min and which induce an increase in biological risk [133] . the simultaneous use of two pairs of gloves considerably reduces the passage of blood through microperforations [134] . there are no significant reductions in manual skills and the sensitivity of the operator wearing the double glove [132] . it was confirmed that the formation of microperforations can be also induced by washing gloves with soap, chlorhexidine, or alcohol. moreover, particular attention should be paid also while waiting for the total drying of the alcoholic substances applied on the hands, which has also proven to be potentially harmful to the integrity of the device, before wearing gloves [132] . other personal protective equipment include the disposable cap (headgear) and shoe covers. a disposable cap device is recommended for clear hygienic reasons, such as containment operator contamination and prevention of dispersion of dandruff in the environment, and even more generic protective functions for the worker, such as: interlocking with subsequent tearing of hair and possibly scalp from a part of moving and/or rotating organs, the burning of the hair due to flames or incandescent bodies, and hair fouling due to various agents, including powders and drops of blood-salivary material [83] . dentist personal hygiene is an absolute necessity for infection prevention [23] . the image that the doctor presents of himself and his study is related to the trust that the patient will show towards the doctor and the treatment itself, in an era in which there is increasing information and awareness of the risk. specific notes of hygiene include: • hair, if a doctor hair can touch the patient or dental equipment, should be attached to the back of the head or a surgical cap should be worn [23] ; • facial hair should be covered with a mask or shield [23] ; • jewels should be removed from the hands, arms, or facial area during the patient treatment [23] ; • nails should be kept clean and short to prevent the perforation of the gloves and the accumulation of debris [23] ; • full forearm and hand washing are mandatory before and after treatment [23] . it is very important to maintain an excellent level of hand hygiene in protection techniques that affects all members of the dental team [10] . "hand hygiene" includes several procedures that remove or kill microorganism on the hands [83] : • during handwashing, water and soap should be used in order to generate lather that is distributed on all surface of the hands and after rinsed off; • hand antisepsis, to physically remove microorganisms by antimicrobial soap or to kill microorganisms with an alcohol-based hand rub; • surgical hand rub procedure that kills transient organisms and reduces resident flora for the duration of a surgical procedure with antimicrobial soap or an alcohol-based hand rub [135] . there are different types of soap: • plain soap, that have no antimicrobial properties and works physically removing dirt ad microorganism; • alcohol-based hand rub, used without water, kills microorganism but does not remove soil or organic material physically; antimicrobial soap kills microorganism and removes physically soil and organic material [135] . in 1975 and in 1985, the cdc published a guideline on how to wash the hands, stating that the hands should be washed with antimicrobial soaps before and after procedures performed on patients [10] . the use of gloves is not an alternative to hand washing [10] . hand washing is different if it is a routine procedure or a surgical procedure: in the first case, normal or antibacterial soaps are sufficient [89] . alcohol-containing agents are preferable [10] . cold water must be of choice when washing hands because the repeatedly use of hot water can cause dermatitis [10] . it is recommended to wash hands using liquid soap for a minimum duration of 60 s. it is very important to reduce the number of microorganisms before each surgical procedure; that is why applying antibacterial soaps and acts a detailed cleaning followed by liquids containing alcohol is recommended [10] . despite the fact that the antibacterial effects of alcohol containing cleansers arise quickly, such antiseptics including compounds of triclosan, quaternary ammonium, chlorhexidine, and octenidine must be included [10] . before surgical hand washing, rings, watches, and other accessories must be taken off and no nail polishes or other artificial must be present [11, 89] . the use of disposable paper towels is preferable for drying hands. after every procedure and after taking off the gloves, it is highly recommended to wash hands once again with regular soaps. if soap and water are not readily available, it can be used an alcohol-based hand sanitizer that contains at least 60% alcohol [10] . • must consider all sharp objects contaminated with the patient blood and saliva as potentially infectious; • do not hood the used needles in order to avoid an accidental injection [83] ; • put all used sharp objects in suitable puncture resistant bins [83] . it is necessary to clean all instruments with detergent and water before sterilization [10] . during washing, it is advisable to avoid splashes of water a wear gloves and face protection. the instruments that penetrate the tissues must be sterilized in an autoclave [83] . it is advisable to heat sterilize items that touch the mucosa or to at least disinfect them, for example, with the immersion in a 2% glutaraldehyde solution in a closed bid, naturally following the instructions of the producer [83] . anything that cannot be autoclaved must be disinfected. the handpieces should be able to drain the water for two minutes at the start of the day. not autoclavable handpieces can be disinfected using viricidal agent. after sterilization, all instruments must be kept safely in order to avoid recontamination for a maximum of 30 days, 60 days if closed in double bags [83] . sterilization completely kills all vital agents and spores too. the classic sterilization procedure expects the use autoclave, with cycles at 121 • c for 15-30 min, or at 134 • c for 3-4 min [23, 83] . it is necessary to thoroughly wash and dry all items before sterilizing them as dirt and water can interfere with sterilization [83] . steam sterilization cannot be used for all facilities and a possible alternative can be the use of chemical sterilization using ethylene oxide gas, formaldehyde gas, hydrogen peroxide gas, liquid peracetic acid, or ozone [83] . the disinfection processes do not destroy the bacterial load, rather reducing it to acceptable levels. commonly used disinfectants are described below (table 4 ). the action of cleaning and disinfection can be manual or automatized. for example, it is possible to use ultrasonic baths in order to clean complex, articulated, or notched stainless-steel instruments such as cutters. the washer-disinfectors provide a high temperature passage (generally 90 • c for one minute), which drastically reduces the microbial contamination of the items. the final rinse must be carried out with high quality water (table 5 ). it is necessary to have always a perfect protection of operative room with disinfected surfaces [10] . there are two ways to make a surface aseptic [23] : • clean and disinfect contaminated surfaces [23] and • prevent surfaces from being contaminated by using surface covers [23] . a combination of both can also be used [23] . the following chemicals are suitable for surface and equipment asepsis: • chlorine, e.g., sodium hypochlorite • phenolic compounds • water-based, water with ortho-phenylphenol, tertiary amylphenol, or o-benzyl-p-chlorophenol • alcohol-based ethyl or isopropyl alcohol with ortho-phenylphenol or tertiary amylphenol • iodophor-butoxy polypropoxy polyethoxy ethanol iodine complex [23] . in the literature there are still little information on 2019-ncov. similar genetic features between 2019-ncov and sars-cov indicate that covid-19 could be susceptible to disinfectants such as 0.1% sodium hypochlorite, 0.5% hydrogen peroxide, 62%-71% ethanol, and phenolic and quaternary ammonium compounds [4] . it is important to pay attention to the duration of use, dilution rate, and especially the expiration time following the preparation of the solution [4] . a recent paper pointed out that surface disinfection could be performed with 0.1% sodium hypochlorite or 62%-71% ethanol for one minute in order to eliminate sas-cov-2 [139] . after each treatment, work surfaces should be adequately cleaned and decontaminated with ethyl alcohol (70%). if blood or pus is visible on a surface, it is necessary to clean and disinfect that surface with sodium hypochlorite (0.5%). it is necessary to wear protective gloves and care taken to minimize direct skin, mucosal or eye contact with these disinfectants. in addition to disinfection with chemicals, a ultraviolet-c (uv-c) irradiation lamp can be used [140] . the uv light system for disinfection has several advantages, including: does not require room ventilation, does not leave residues after use and have a wide action spectrum in a very short time [140] . the uv-c lamp must be activated only when the room is empty, without staff and without patient. in the literature, there are no cases of damage to the materials present in the room; despite this, the acrylic material can be degraded if subjected to repeated exposure to uv-c light and for this reason it is recommended to cover it during disinfection with uv-c [141] . ultraviolet light has a wavelength between 10 and 400 nm, while ultraviolet-c (uv-c) light has a wavelength between 100 and 280 nm, and the greatest germicidal power is obtained with a wavelength of 265 nm [142] . the germicidal effect of uv-c light causes cell damage thus blocking cell replication [141] . in descending order of inactivation by uv-c light, there are bacteria, viruses, fungi, and spores [143] . uv-c rays can be generated by low pressure mercury lamps and pulsed xenon lamps which emit high intensity pulsed light with a higher germicidal action [141] . uv-c rays are equipped with high energy which decreases exponentially with the increase of distance from the light source: objects or surfaces closer to the uv-c source will have a greater exposure and therefore will have to be disinfected for less time than distant objects [142] . depending on the nature of the object affected by uv-c light, it can block the light rays or allow itself to be passed through allowing the irradiation of the objects placed behind it. for example, the organic material completely absorbs the uv-c light and blocks its diffusion. for this reason, the surfaces must be manually cleaned to remove the organic substances before decontamination with ultraviolet light [142] . the extent of inactivation of the microorganisms is directly proportional to the uv-c dose received and this, in turn, is the result of the intensity and duration of exposure [142] . therefore, according to the data in the literature, the use of uv-c rays for disinfection has proven effective in reducing the overall bacterial count and significantly more effective than just manual disinfection on surfaces [141] . in addition, to encourage the exchange of air, it is recommended to ventilate the rooms between one patient and another. if it is not possible to allow the exchange of natural air (at least 20-30 min), forced ventilation systems with high efficiency particulate air (hepa) filters must be used, paying attention to the periodic replacement of the filters. recommendations for environmental infection prevention and control in dental settings [136] : • establish a protocol for cleaning and disinfection of surfaces and environments of which health personnel must be informed; • cover with disposable films all the surfaces that are touched during the procedures (for example switches, it equipment) and change these protections between each patient; • surfaces that are not protected by a barrier should be cleaned and disinfected with a disinfectant after each patient; • use a medium level disinfectant (i.e., tuberculocidal indication) if a surface is visibly contaminated with blood; • for each disinfectant, follow the manufacturer's instructions (e.g., quantity, dilution, contact time, safe use, disposal) [136] (table 6 ). if proper maintenance is not carried out, microbial pathogens (e.g., pseudomonas or legionella spp.) can multiply in duwls. these organisms grow in the biofilm on the internal surfaces of the tubes, where they cannot be attacked with chemicals. to prevent the formation of this biofilm, the systems should be drained at the end of each day [144] . in dental unit water lines (duwl), water must flow and they must be washed regularly: it is recommended to rinse for two minutes at the beginning and end of each day and for 20-30 s between patients [144] . different agents for disinfection of duwl are available. all handpieces and ultrasonic meters must be equipped with backstop valves and must undergo periodic maintenance and inspection. the filters used in the duwl must be checked periodically or, if they are disposable, they must be changed daily. recommendations for dental unit water quality in dental settings: • use water compliant with environmental protection agency (epa) standards for drinking water (i.e., ≤ 500 cfu/ml of heterotrophic water bacteria), • follow the recommendations for water quality monitoring given by the manufacturer of the unit or waterline treatment product, • use sterile water or sterile saline for the irrigation during surgical procedures [136] . any waste containing human or animal tissue, blood or other body fluids, drugs, swabs, dressings or other infective material is defined as "clinical waste" and it must be separated from non-clinical waste [144] . used disposable syringes, needles, or other pointed instruments must be disposed of in a special rigid container, in order to avoid injury to operators and operators in charge of waste disposal. the waste must be kept in a dedicated area before it is collected, away from public access, and excessive accumulation of waste must be avoided [4, 144] . the whole dental team must be vaccinated against hepatitis b in order to increase personal protection [83] . individuals who have already been vaccinated should monitor their levels of immunity against hbv over time and make booster shots [145] . all dental health care professionals should also receive the following other vaccinations: flu, mumps (live-virus), measles (live-virus), rubella (live-virus), and varicella-zoster (live-virus) [10] . in addition, the rubella vaccine is strongly recommended especially for women who have pregnancy uncertainty [131] . the influenza vaccine is very useful for dental health professionals as they are at risk for respiratory droplets infections by working in close proximity to the patients [10] . when the covid-19 vaccine is ready, healthcare professionals should take it. as additional infection prevention and health care worker measures, rapid tests can be used in dental practices to diagnose covid-19 before each treatment. this is because, as mentioned above, a patient without symptoms is not necessarily a healthy patient. from all these data, it is evident that the dentist and his team need to use rigid and precise operating protocols in order to avoid infectious contagion [23] . several authors proposed some right procedures in the operative dentistry [2] [3] [4] 10, 23, 83, 139, [146] [147] [148] [149] . for this reason, we reassume them in a precise operative protocol organized for all the patients and characterized by some defined steps: prevention of infections must be a priority in any healthcare setting and therefore also in any dental clinic. to do this, staff training and information, adequate management of resources, and use of well-defined operating protocols is necessary. adequate management of the protection for operators (and therefore also for patients) begins with the roles of the secretariat. in order to better organize the workflow, the secretariat must provide a telephone triage. it would be advisable to phone each patient to make sure he is healthy on the day of the appointment. patients with acute symptoms of any infectious disease should be referred at the time of symptom resolution. the medical history of patients may not reveal asymptomatic infectious disease of which they are affected. this means the operator must adopt the same infection control rules for all patients, as if they were all infective. in addition, the secretariat must organize appointments in order to avoid crowding in the waiting room. it would be advisable for the patient to present himself alone, without companions (only minors, the elderly and patients with psycho-physical conditions can be accompanied). in some urgent and non-deferrable cases, it is necessary to treat the patient despite being in the acute phase of infection with any virus. examples of urgent treatments are: pulpitis, tooth fracture, and avulsion [2] . in these cases, the operator must implement the maximum individual protection measures. in the waiting room all material (e.g., magazines, newspapers, information posters) that can represent a source of contamination must be eliminated so that the room is easy to disinfect. patients are requested to go to the appointment without any superfluous objects. at the entrance of the dental structure, the patient must wear shoe covers, disinfect the hands with hydroalcoholic solution according to the following indications, affix any jacket on a special hanger and disinfect the hands again with hydroalcoholic solution. if there are several patients in the waiting room, they must be at least two meters away from each other. the correct hand disinfection procedure with hydroalcoholic solution is as follows: a) apply a squirt of sanitizer in the palm of hand, b) rub hands palm against each other, c) rub the back of each hands with the palm of the other hand, d) rub palms together with your finger interlaced, e) rub the back of fingers with the opposite palms, f) rotate thumbs in the other hand, g) do a circle on palm with finger clasped, h) once dry, hands are safe. the same procedure is performed for washing hands with soap and water. the operators must be adequately dressed in the correct ppe. healthcare professionals will need to remove any jewel before starting dressing procedures. all the necessary ppe must already be positioned clearly visible and intact, in a room that will be distinct from the one where the undressing phase will take place. in both areas, hydroalcoholic solution and/or items necessary for washing hands with soap and water should be available. in the dressing room there must be trays for the collection and subsequent disinfection of the non-disposable ppe and special containers for the collection of waste where to dispose of the disposable ppe. a dressing and undressing procedure is described below, imagining that the dentist has to operate under a high risk of infection. dressing and undressing procedures must be particularly considered. dressing procedure: a) eliminate jewels and personal items from the pockets of the uniform; b) long hair must be tied and inserted into a cap not mandatory for single use (no tufts of hair must come out of the cap); c) wear shoe covers; d) perform social hand washing or disinfection with antiseptic gel; e) wear the first pair of gloves of the right size; f) wear the water repellent gown by tying it on the back without double knots (first the upper part and then the lower part, the latter must be tied on the front) being careful not to leave parts of the uniform exposed; g) wear the mask (ffp2-ffp3) which must adhere well to both the nose and the mind; h) put on the disposable water-repellent cap and be tied under the chin, the excess ribbons must be inserted inside the gown; i) wear glasses/protective screen; j) wear a second pair of gloves for direct patient assistance. these gloves must cover the cuffs of the disposable gown. undressing procedure: a) remove the second pair of (dirty) gloves being careful not to contaminate the underlying gloves; b) gloves still worn with a hydroalcoholic solution are disinfected and a new pair of gloves is worn on them; c) the face shield is removed: if it is disposable it should be trashed, and if it is not disposable, it should be placed in a container with disinfectant; d) the second pair of gloves is removed without contaminating the underlying gloves; e) the gloves are rubbed with hydroalcoholic solution and a new pair of gloves is worn; f) disposable gown removal starting from the top, then the bottom, rolling it up to touch the inside, clean; g) throw disposable shirts and second pair of gloves; h) the gloves are rubbed with hydroalcoholic solution and a new pair of gloves is worn; i) remove the water-repellent cap; j) the gloves are rubbed with hydroalcoholic solution and a new pair of gloves is worn; k) remove mask taking it by the elastics with the head bent forward and down; l) both the first pair and the second pair of gloves are removed; m) hands are disinfected with hydroalcoholic solution. before entering the surgical room, the patient must be dressed in a disposable gown and headgear worn in order to avoid any contagion on clothing and hair. 7. before dental session patient should rinse and gargle with a specific mouthwash. chlorhexidine is commonly used for pre-procedural oral rinses in dental offices, but its capacity of 2019-ncov destruction has not yet been demonstrated [4] . instead, pre-procedural oral rinses with oxidizing such as 1% hydrogen peroxide or 0.2% povidone-iodine are recommended [4] . so, the pre-procedural use of mouthwash, especially in cases of inability to use a rubber dam, can significantly reduce the microbial load of oral cavity fluids [3] . in fact, even if oral rinses seem to "limit" the viral load, virus can spread through the complete respiratory tract and it is not scientifically possible to guarantee that this reduction is constant during the operative manoeuvre (e.g., cough, sneezing, runny nose). then the following pre-operative procedure is recommended to the patient: a) 1% hydrogen peroxide 15" gargle followed by 30" rinse, b) do not rinse with water at the end of the rinse and continue with chlorhexidine 0.20% 60" rinse with final gargle of 15" [146] . at the end of the procedure, the patient must be appropriately undressed, and have another oral rinse performed before washing hands and face thoroughly. 8. after every patient, carefully clean all surfaces, starting from the least contaminated to the most potentially infected, taking care not to overlook the handles of the doors and the various drawers, worktops and all the devices used during the treatment and which are not disposable or autoclavable. cover switches, mice, computer keyboards, and anything else that may be more difficult to clean with disposable film. the worktops must be free from anything that is not strictly necessary to perform the service. an accurate disinfection of the surfaces includes a preventive cleaning of the same in order to eliminate the soil which otherwise would not allow the disinfectant to inactivate the microorganisms [29] . in the same way, if you want to use disinfectant wipes, you must use one to cleanse and after another to disinfect. as regards spray disinfectants, the percentage of dilution and the time of application vary from product to product: you must follow the instructions provided by the company. moreover, alcohol-based disinfectants (75%), 0.5% hydrogen peroxide, 0.1% sodium hypochlorite are recommended to be left to act on the surfaces for 1 min. disinfect the circuits of the treatment center at each patient change. between patients, the tubing of high-volume aspirators and saliva ejectors should be regularly flushed with water and disinfectant such as 0.1% sodium hypochlorite. always air the rooms after each patient (at least 20-30 min) or use germicidal lamps. clean floors with bleach at least two times a day. 9. during every procedure minimize the use of an air/water syringe: dry the site with cotton rollers when possible; use suction at maximum power (it might be an idea to use autoclavable plastic suction cannulas that have a greater suction capacity than normal disposable pvc cannulas) or use two saliva ejectors; in the case of exposed carious dentine, try to remove it as manually as possible using excavators; be sure to first mount the rubber dam, disinfect the crown with pellets soaked in 75% alcohol and recommend with the second operator to position the aspirator as correctly as possible to avoid excessive spraying and/or splashing; do not use air-polishing; avoid intraoral x-rays as they stimulate salivation, coughing and/or vomiting; prefer exams like opt (orthopantomography) or cbct (cone beam computed tomography). in case of extractions, it is preferable to use resorbable sutures to seal the post-extraction site. in the case of patients who are definitely positive for any infectious agent or on which there are greater possibilities of positivity highlighted by the medical history, it is necessary to plan their treatment at the end of the day. do not touch patient card and pens with dirty gloves. it is good practice to cough or sneeze into the elbow. the operator must avoid touching his eyes, nose and mouth with dirty gloves or hands. 10. isolation with rubber dam [4] . isolating the oral cavity with the use of rubber dams greatly reduces (about 70%) the spread of respiratory droplets and aerosols containing saliva or blood coming from the patient and aimed to the operator area of action [4] . after positioning the dam, the operator must provide an efficient high-volume intraoral aspiration in order to prevent the spread of aerosol and spray as much as possible [148] . if rubber dams cannot be used for any reason, the operator should prefer to use manual tools such as hand scalers [4] . 11. anti-retraction handpiece [4] . during the covid-19 pandemic, operators should avoid using dental mechanical handpieces that do not have an anti-retraction function [4] . mechanical handpieces with the anti-retraction system have valves (anti-retraction) that are very important in order to prevent the spread and dispersion of droplets and aerosol [148, 149] . 12. all instruments which have been used for the treatment of a patient or which have only been touched by operators during a session and which cannot be sterilized according to standard protocols, must be disinfected (e.g., immersed in a container with phenol) [23] . this tools bagged in disinfection solution must remain in solution for about 10 min [23] . some materials, such as polysulphide, polyvinylsiloxane, impression compound, and zoe impressing materials, after being in the patient mouth, are rinsed with water and immersed in a 5.25% sodium hypochlorite solution for about 10 min [23] . the alginate or polyether impressions are also rinsed with water, sprayed with a 5.25% sodium hypochlorite solution and placed in a container for about 10 min [23] . wax, resin centric relation records, and zoe are rinsed with water and sprayed with a 5.25% sodium hypochlorite solution and placed in a plastic bag for about 10 min [23] . provisional restorations and complete dentures removed from the patient mouth are immersed in a 5.25% sodium hypochlorite solution for 10 min [23] . otherwise, removable partial prostheses with metal bases are treated with 2% glutaraldehyde solution and placed in a plastic bag for 10 min [23] . a novel and useful indication is that of classifying each common dental procedure according to the likelihood of a contagion by one or more infective agents (via saliva, blood, droplets or aerosol) for the team and for the patient (under the cure or the subsequent), nevertheless its type and intrinsic operative difficulty (table 7) . according to this paradigm, all dental procedures involving the use of the air-water syringe and/or rotating/ultrasound/piezo tools are able to produce high levels of aerosols and droplets and for this reason the dentist must consider them dangerous for himself, the dental team, and the subsequent patients. meanwhile, procedures, even if refined (e.g., soft tissues biopsy for oral cancer suspicion) but characterized by a low/absent production of aerosol and droplets, must be considered not particularly threatening. for all these considerations, the dental team must reconsider its operative protocols and modulate the ppe use according to level of risk of common dental procedures of generating droplets or aerosols. table 8 presents the use of different ppes for each common dental procedure in pre-covid vs post-covid era. it is definitively clear that the use of air-water syringe and/or rotating/ultrasound/piezo tools able to produce high levels of aerosols and droplets need the use of the safest ppe in order to reduce/eliminate viral or other infectious agent diffusion within the dental setting. table 8 . proposal of modulation of personal protective equipment (ppe) according to level of risk or common dental procedures both in pre-covid and post-covid era (bold style means the introduction of the new ppe due the transition from a risk category to a higher one). in the face of the covid-19 pandemic, new biosafety measures are necessary to reduce contagion. dentistry is a profession that works directly with the oral cavity and is therefore very exposed to this virus or other infectious agents. because of this, some measures need to be taken to minimize contagion. in fact, dentists can play an important role in stopping the transmission chain, assuming correct procedures in order to reduce the viral agent diffusion, or in promoting undesirable infectious disease diffusion, if operating in adherence to adequate safety protocols. dental-care professionals must be fully aware of 2019-ncov and other viral agent spreading modalities, how to identify patients with active infections and, most importantly, to prioritize self and patient protection. 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efficacy of rubber dam isolation in reducing atmospheric bacterial contamination severe acute respiratory syndrome and dentistry approaches to the management of patients in oral and maxillofacial surgery during covid-19 pandemic interim guidance for management of emergency and urgent dental care; ada: niagara falls plan estratégico de acción para el periodo posterior a la crisis creada por el covid-19 key: cord-352433-sts48u9i authors: galanti, marta; shaman, jeffrey title: direct observation of repeated infections with endemic coronaviruses date: 2020-07-07 journal: j infect dis doi: 10.1093/infdis/jiaa392 sha: doc_id: 352433 cord_uid: sts48u9i background: although the mechanisms of adaptive immunity to pandemic severe acute respiratory syndrome coronavirus 2 (sars-cov-2) are still unknown, the immune response to the widespread endemic coronaviruses hku1, 229e, nl63, and oc43 provide a useful reference for understanding repeat infection risk. methods: here we used data from proactive sampling carried out in new york city from fall 2016 to spring 2018. we combined weekly nasal swab collection with self-reports of respiratory symptoms from 191 participants to investigate the profile of recurring infections with endemic coronaviruses. results: during the study, 12 individuals tested positive multiple times for the same coronavirus. we found no significant difference between the probability of testing positive at least once and the probability of a recurrence for the betacoronaviruses hku1 and oc43 at 34 weeks after enrollment/first infection. we also found no significant association between repeat infections and symptom severity, but found strong association between symptom severity and belonging to the same family. conclusions: this study provides evidence that reinfections with the same endemic coronavirus are not atypical in a time window shorter than 1 year and that the genetic basis of innate immune response may be a greater determinant of infection severity than immune memory acquired after a previous infection. the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) appears to have emerged in humans in the hubei province of china during november 2019 [1] . human-to-human transmission was confirmed in early january, and since then the virus has rapidly spread to all continents except antarctica. the outbreak was declared a pandemic by the world health organization on 11 march 2020. as of 4 july 2020, it has spread to >180 countries, with 10 922 324 confirmed cases and 523 011 deaths reported [2] . symptoms associated with sars-cov-2 vary from none to extremely severe, with elder adults and people with underlying medical conditions more at risk for developing severe and potentially fatal disease [3] . at present, there is no vaccine or approved antiviral treatment for sars-cov-2, and therapies rely principally on symptom management. many institutions across the world are working to develop a sars-cov-2 vaccine, and clinical trials with some vaccine candidates have already begun [4] . as the pandemic progresses, infecting millions of people across the world, a key question is whether individuals upon recovery are prone to repeat infection. there have been reports of individuals again testing positive by polymerase chain reaction (pcr) weeks after recovering from a sars-cov-2 infection. however, in korea, as reported by the korean centers for disease control and prevention, viable sars-cov-2 was not isolated in cell culture of respiratory samples from potentially reinfected individuals [5] ; thus, these subsequent positive results may have been due to inactive genetic material detected by molecular testing. a recent animal challenge study showed evidence of (at least) short-term protection against reinfections in rhesus macaques experimentally reinfected 4 weeks after first infection [6] . the immune response following reexposure to a virus depends highly on the pathogen and on host-pathogen interactions. some viruses such as measles elicit lifelong immunity; others, like influenza, do not. moreover, when a reinfection is prone to occur, there can be differences in symptom severity. reinfection with the same virus may be less symptomatic, as shown for subsequent influenza infections among children [7] . however, reinfection can also result in a more severe disease via antibody-dependent enhancement, a phenomenon in which antibodies raised against a virus bind with but are unable to neutralize a different strain of the same virus [8] two main processes appear to be responsible for the shortlived immunity engendered against some pathogens: (1) waning of antibodies and memory cells in the host system; and (2) antigenic drift of the pathogen that enables escape from the immunity built against previous strains. reinfections with the respiratory viruses have been reported in previous studies, in which individuals were infected in 2 sequential challenges with the same h1n1 virus [7, 9] . studies focusing on respiratory syncytial virus have provided evidence of subsequent reinfection with very similar strains or with the same strain in <1 year [10, 11] . serological studies have documented subsequent infections with endemic coronaviruses [12] . sequential rhinovirus infections have also been reported in a number of studies; however, this finding could be due to the multitude (>150) of antigenically distinct types of rhinovirus in circulation [13] . to contextualize the issue of protective immunity to sars-cov-2, we here present findings from a recent proactive sampling project carried out in new york city that documented rates of infection and reinfection among individuals shedding seasonal cov (types: hku1, 229e, nl63, and oc43). the results are discussed and analyzed in the broader context of coronavirus infections. data are derived from sampling performed between october 2016 and april 2018 as part of the virome project, a proactive sampling of respiratory virus infection rates, associated symptom self-reports, and rates of seeking clinical care. we enrolled 214 healthy individuals from multiple locations in the manhattan borough of new york city. cohort composition is described in [14] and includes children attending 2 daycares, along with their siblings and parents; teenagers and teachers from a high school; adults working at 2 emergency departments (a pediatric and an adult hospital); and adults working at a university medical center. the cohort was obtained using convenience sampling, and all participants were <65 years of age. while the study period spanned 19 months from october 2016 to april 2018, some individuals enrolled for a single cold and flu season (october-april) and others for the entire study period. participants (or their guardians, if minors) provided informed consent after reading a detailed description of the study (columbia university medical center institutional review board aaaq4358). nasopharyngeal samples were collected by study coordinators once a week irrespective of participant symptoms. samples were screened using the genmark esensor respiratory viral panel (rvp) system for 18 different respiratory viruses, including coronavirus 229e, nl63, oc43, and hku1. sample collection and extraction followed the same protocol as shown in [15] . in addition, participants completed daily self-reports rating 9 respiratory illness-related symptoms (fever, chills, muscle pain, watery eyes, runny nose, sneezing, sore throat, cough, chest pain), each of which was recorded on a likert scale (0 = none, 1 = mild, 2 = moderate, 3 = severe); see supplementary text 1, supplementary tables 1 and 2, and galanti et al [14] for further survey details. for this analysis, only the 191 participants who contributed at least 6 separate pairs of nasopharyngeal samples in the same season were included. we defined an infection (or viral) episode as a group of consecutive weekly specimens from a given individual that were positive for the same virus (allowing for a 1-week gap to account for false negatives and temporary low shedding). we classified all infection episodes as symptomatic or asymptomatic according to individual symptom scores in the days surrounding the date of the first positive swab of an episode. we considered multiple criteria for discriminating between symptomatic and asymptomatic episodes, as a standard definition for symptomatic infection does not exist in the literature. table 1 reports the 5 symptomatic thresholds used; all of the symptom scores are described in reference to a -3 to +7-day window around the date of the initial positive swab for each infection episode. the daily symptom score is defined as the sum of the 9 individual symptoms (range, 0-27) on a given day. total symptom score is the daily symptom score summed over the -3 to +7-day window. see supplementary text 1 for details and examples on how symptom scores were calculated (supplementary tables 1 and 2) per the definitions in table 1 . we applied standard methods of survival analysis to our longitudinal records of infections to estimate (1) the probability of infection with each endemic coronavirus type; and (2) the probability of being reinfected with the same coronavirus type following a previous documented infection. more specifically, the probability of infection and reinfection by time t, i (t),was estimated as: is the standard kaplan-meier estimator and time t is measured in either weeks from enrollment in the cohort, for the first analysis, or weeks from the previous documented infection (with a specific coronavirus type), for the second analysis. here d i are the participants testing positive exactly i weeks after enrollment (after first infection) and n i are the participants who are still enrolled i weeks after enrollment (after first infection). the denominator n i corrects for participants withdrawing from the study at different times by right censoring. the kaplan-meier estimators are compared statistically using the log-rank test. we used fisher exact test to analyze the difference between symptoms developed during subsequent infections and analysis of variance (anova) comparison to test differences in symptom scores reported by different family clusters. we restricted the last analysis to the family clusters within the cohort that presented at least 3 coronavirus infections during the study. among all participants enrolled, 86 individuals tested positive at least once during the study for any coronavirus infection. fortyeight individuals tested positive at least once for oc43, 31 tested positive for 229e, 15 tested positive for nl63, and 28 tested positive for hku1. figure 1 shows a kaplan-meier plot estimating the probability of becoming infected with each coronavirus within x weeks following enrollment (see supplementary table 3 for the number of individuals infected and censored at each time point). oc43 was the most widely diffused virus; the probability of testing positive following 80 weeks in the study was 0.47. in contrast, nl63 was the least frequently isolated coronavirus type; the probability of testing positive after 80 weeks was 0.17. among the study participants, 12 individuals tested positive multiple times during the study for the same coronavirus: 9 tested positive multiple times for oc43, 2 tested positive twice for hku1, 1 tested positive twice for 229e, and none tested positive multiple times for nl63. among the 9 participants with multiple oc43 infections, 3 individuals experienced 3 separate infection episodes, and the other 6 experienced 2 separate episodes. the median time between reinfection events was 37 weeks. the shortest time for a reoccurrence of infection was 4 weeks (oc43), and the longest was 48 weeks (oc43). among the 12 individuals testing positive multiple times for the same coronavirus, 9 were children aged between 1 and 9 years at enrollment, and 3 were adults aged between 25 and 34 years (see supplementary table 4 and supplementary figure 1 for characteristics and timelines of the repeated infections). figure 2 shows a kaplan-meier plot estimating the probability of becoming reinfected with the same betacoronavirus (oc43 and hku1) within x weeks after a previously documented infection (see supplementary table 5 for the number of individuals infected and censored at each time point). a comparison between the data shown in figure 2 and figure 1 found no significant differences between the probability of testing positive at least once and the probability of a recurrence for both hku1 and oc43 at 34 weeks after enrollment/first infection. to control for false-positive pcr results, we tested the sensitivity of the findings to different choices of the positivity threshold used in rvp testing (see supplementary text 2 and supplementary figures 2-5) . the probability of reinfection with betacoronaviruses at >38 weeks after prior infection was robust across different thresholds, whereas short-term reinfection signals could be an artifact due to pcr amplification. this shifted threshold also yields a statistically significant difference between the probability of testing positive at least once and the probability of a recurrence after first infection until week 43 (p = .04). there was no significant difference in the likelihood of experiencing symptomatic infection between the first and subsequent infection episodes by any of the 5 definitions provided in table 1 ; that is, severity of symptoms neither increased nor decreased significantly upon second infection. in particular, all of the individuals who were completely asymptomatic during the first recorded occurrence did not report any symptoms during subsequent infection(s) with the same coronavirus type. however, there was a significant association between severity of symptoms associated with any coronavirus infection and belonging to the same family cluster (p < .0001, 1-way anova). figure 3 shows the total symptom score associated with any coronavirus infection for infections grouped by family cluster. as the sars-cov-2 pandemic spreads to millions of individuals worldwide, it is extremely important to understand the mechanisms of protective immunity elicited by infection. until direct observations of adaptive immune response to sars-cov-2 become available, analyses of protective immunity elicited by other coronaviruses may offer useful insights. several studies in the last 4 decades have shown that infections with the 4 endemic coronaviruses 229e, oc43, nl63, and hku are common in the general population [12, 16] . infection with these viruses generally produces mild and even asymptomatic infection [17] . serological studies have shown that >90% of the population presents a baseline level of antibodies against these endemic coronaviruses, with first seroconversion occurring at a young age [16, 18] . shortly after infection, baseline antibody titers increase sharply; this response has been demonstrated for both natural and experimentally induced infections [12, 19, 20] . antibody titers start increasing roughly 1 week following infection, reach a peak after about 2 weeks [20] , and by 4 months to 1 year have returned to baseline levels [12, 20] . a challenge study [20] showed that the likelihood of developing an infection after inoculation correlated with participants' concentration of antibodies at enrollment. moreover, a positive correlation has been shown between antibody rise after infection, severity of clinical manifestation, and viral shedding [19] , with milder cases linked to less substantial postinfection antibody rises. instances of natural reinfections with the same virus type have been documented previously [12] in which repeated infections with oc43 and 229e were recorded by serological testing. subsequent infections were separated by at least 8 months, though study participants were tested every 4 months. participants in a separate challenge study were inoculated with coronavirus 229e and then rechallenged with the same virus after 1 year [20] . in most cases, reinfection occurred, though it presented with decreased symptom severity and shortened duration of shedding. the adaptive immune response to coronavirus is mainly directed toward the most variable part of the virus, a region that is not conserved across types; consequently, cross-reactive protection between different types does not appear to be an important factor [21, 22] . in addition, the effects of antigenic drift on reinfection have not been elucidated [23] , and more studies are warranted to understand whether repeat infections are ascribable to rapid virus evolution rather than a decline in antibody titers. the mild pathogenicity of seasonal coronavirus infection (with immune response often localized to the upper respiratory trait) is also often regarded as the reason for short-lived immunity. coronavirus infections, and the adaptive immunity acquired toward them, have also been studied in animals. in a study on porcine respiratory coronavirus, which causes subclinical infections in pigs, antibody titers waned approximately 1 year after experimental infection [24] . in contrast, an experimental study on murine coronavirus, which produces severe, systemic infections in mice, has shown an interplay between virus-specific antibodies and t cells, that upon survival in the host lead to lifelong protection against reinfection [25] . similarly, a longer immunity profile has been hypothesized for severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) due to their increased severity and to the systemic response that infection induces [21] . specific antibodies were detectable for at least 2 years in sars and mers survivors [26, 27] . although longitudinal studies on sars survivors have not detected specific sars immunoglobulin g antibody persistence 5 years after infection, they have found that specific memory t cells persist in the peripheral blood of recovered sars patients, and at higher levels in patients who experienced severe disease [28] . whether the presence of these memory t cells would be enough to induce a fast, protective response upon reinfection with sars has not been assessed. our study confirms that seasonal coronaviruses are widespread in the general population, with infections directly documented for a large fraction of the participants in our study. the methods for our analysis are based on the hypothesis that infection probabilities are comparable among participants enrolled at different times in the study. however, the seasonality of endemic coronaviruses, which are mostly absent during the summer months, and the relative magnitude across years of seasonal coronavirus epidemics are limitations. in the united states, the prevalence of oc43 during the 2016-2017 season was much higher than during the 2017-2018 season, whereas the opposite trend was observed for hku1 [29] . moreover, our estimates of infection and reinfection probabilities must be considered as a lower bound, due to the occurrence of weekly swabs missed by the participants and due to the design of the study itself, which may have missed infections of short duration in between consecutive weekly tests. nevertheless, this study confirms that reinfections with the same coronavirus type occur in a time window shorter than 1 year, and finds no significant association between repeat infections and symptom severity. instead, it suggests the effect of possible genetic determinants of innate immune response, as individuals asymptomatic during first infection did not experience symptoms during subsequent infections, and members of the same families reported similar symptom severity. genetic variations associated with immune responses have been associated with increased severity and exacerbation of symptoms due to respiratory infections [30, 31] . . total symptom score associated with infections by any coronavirus type. the score is calculated as the sum of daily symptom scores for the -3/+7 day window around the test date, as indicated for definition 3 in table 1 . each point represents an infection event, and each cluster represents a family group. each family group 1 to 9 is composed of a parent and 1-4 children. for each box, the red line indicates the median, and the bottom and top edges of the blue box are the 25th and 75th percentiles. the dashed lines extend to the most extreme data points that are not outliers, whereas the outliers are indicated by the red "+" symbol. we recognize that the self-reporting of symptoms is an important limitation in this analysis and that parents reported symptoms for their dependents, which possibly introduced bias. moreover, the majority of the repeated coronavirus infections were found in children, a cohort more vulnerable to infection because of their immature immune system [32] , and 26% of the episodes in the repeated infections were coinfections with other respiratory viruses (see supplementary table 2 ). another potential limitation of our study is the high sensitivity of pcr tests, which can amplify very small amounts of genetic material, possibly not ascribable to active infections. however, the occurrence of repeated infections separated by at least 38 weeks was corroborated by repeating the analysis with different positivity thresholds for the rvp. still, without virus sequencing, we cannot exclude the possibility that subsequent positives are the resurgence of the same infection rather than new infections, especially for infections reoccurring within a short time window. additional analyses involving viral sequencing and serological testing would be necessary to confirm repeated infections and to help disentangle the effects of antigenic drift and antibody waning. more studies analyzing the genetic basis of individual response to coronavirus infections are also warranted. even though endemic coronaviruses are very rarely associated with severe disease, their widespread diffusion, together with the fact that oc43 and hku1 belong to the same betacoronavirus genus as sars-cov-2, offers important opportunities for investigation. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. disclaimer. the funders had no role in study design, data collection and analysis, preparation of the manuscript, or decision to submit the manuscript for publication. financial support. this work was supported by the defense advanced research projects agency (contract number w911nf-16-2-0035). potential conflicts of interest. j. s. and columbia university disclose partial ownership of sk analytics. j. s. also discloses consulting for bni. m. g. reports no potential conflicts of interest. both authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. the proximal origin of sars-cov-2 centers for disease control and prevention. coronavirus disease 2019: symptoms. 2020 national institutes of health. nih clinical trials of investigational vaccine for covid-19 begins reinfection could not occur in sars-cov-2 infected rhesus macaques. biorxiv influenza a: infection and reinfection antibody-dependent enhancement of virus infection and disease influenza a reinfection in sequential human challenge: implications for protective immunity and "universal" vaccine development immunity to and frequency of reinfection with respiratory syncytial virus molecular analysis of respiratory syncytial virus reinfections in infants from coastal kenya rises in titers of antibody to human coronaviruses oc43 and 229e in seattle families during 1975-1979 prolonged shedding of rhinovirus and re-infection in adults with respiratory tract illness longitudinal active sampling for respiratory viral infections across age groups asymptomatic summertime shedding of respiratory viruses repeated endemic coronavirus infection â�¢ jid 2020:xx (xx xxxx first infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood rates of asymptomatic respiratory virus infection across age groups development of a nucleocapsid-based human coronavirus immunoassay and estimates of individuals exposed to coronavirus in a u.s. metropolitan population enzymelinked immunosorbent assay for detection of antibody in volunteers experimentally infected with human coronavirus strain 229 e the time course of the immune response to experimental coronavirus infection of man middle east respiratory syndrome vaccines antibody to virus components in volunteers experimentally infected with human coronavirus 229e group viruses viral infections of humans neutralizing antibody decay and lack of contact transmission after inoculation of 3-and 4-day-old piglets with porcine respiratory coronavirus effective clearance of mouse hepatitis virus from the central nervous system requires both cd4+ and cd8+ t cells longitudinal profile of antibodies against sars-coronavirus in sars patients and their clinical significance persistence of antibodies against middle east respiratory syndrome coronavirus lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study national respiratory and enteric virus surveillance system genetic susceptibility to respiratory syncytial virus bronchiolitis is predominantly associated with innate immune genes genetic associations with viral respiratory illnesses and asthma control in children evolution of the immune system in humans from infancy to old age key: cord-337284-joq1aqn6 authors: barrera‐lópez, pedro; pérez‐riveros, erika d.; moreno‐montoya, josé; ballesteros, silvia marcela; valencia, sergio a.; de la hoz‐valle, josé a. title: co‐infection of other respiratory pathogens and hiv in covid‐19 patients: is there a pattern? date: 2020-07-24 journal: j med virol doi: 10.1002/jmv.26331 sha: doc_id: 337284 cord_uid: joq1aqn6 the pandemic caused by sars‐cov‐2 has led to the elaboration of multiple studies to increase knowledge and understanding, hence, having the ability to accomplish an adequate and timely diagnosis and give an optimal treatment according to the patient's condition. the clinical manifestations of covid‐19 pose a series of challenges both in understanding and delimiting the disease secondary to the sars‐cov‐2 infection. this is due to the fact that the main axis of this disease is the endothelial compromise and the production of a "cytokine storm", triggering multiple organ failure and death. given that a complete understanding of its pathophysiology and clinical behavior has not yet been achieved, we wondered if co‐infection with other respiratory viruses modifies its performance and outcomes described so far. a literature search was performed, obtaining 68 articles, of which 25 were analyzed. the analysis showed us that there is a high variety both in the types of associated infections and in the clinical behavior of patients and their outcomes. therefore, we consider that the search for other infections should be performed exhaustively, especially in those cases that may be susceptible to treatment such as influenza a, hiv, or bacterial infections. as well as optimize the analysis of these cases and establish if there are characteristics that allow establishing the possibility of carrying an additional infection to that of sarscov2 and the implications for the management and prognosis of the patient. this article is protected by copyright. all rights reserved. the pandemic caused by sars-cov-2 has led to the elaboration of multiple studies to increase its knowledge and understanding, hence, having the ability to accomplish an adequate and timely diagnosis and give an optimal treatment according to the patient's condition. this disease has increased exponentially to more than 13.4 million confirmed cases globally, with a current mortality rate of 4.3% (582,547 deaths) 1 . the clinical manifestations of covid-19 pose a series of challenges both in understanding and delimiting the disease secondary to the sars-cov-2 infection. these manifestations have shown an important evolution, initiating with a respiratory tract infection that in its most severe form could progress to an acute respiratory distress syndrome or reach a multisystemic compromise. this is due to the fact that the main axis of this disease is the endothelial compromise and the production of a "cytokine storm", triggering multiple organ failure and death 2 . as a result of these changes in the comprehension of the disease and given that a complete understanding of its pathophysiology and clinical behavior has not yet been achieved, we wonder if co-infection with other respiratory viruses modifies its performance and outcomes described so far. to elucidate this query, a literature research was performed through the "pubmed" platform focused on co-infection reports (the terms "covid-19" "sars-cov-2" and "co-infection" were included with the boléan "or" operators for the first 2 terms and "and" for the last one in all the fields). in every scenario, the research was restricted to english and spanish written articles, published until june 1, 2020. we obtained 65 articles, of which 43 were excluded after reading its titles and abstracts, given the lack of focus on co-infections or an inadequate result description. the remaining 22 articles were chosen for the analysis, in addition to 3 studies of co-infection with hiv, according to the recommendation of experts [3] [4] [5] (figure 1 ). the analysis of these articles shows us that there is a high variety both in the types of associated infections and in the clinical behavior of patients and their outcomes. most of the reports focused on co-infection with respiratory pathogens, however, we found reports of unusual concomitant infections, such as periorbital cellulitis. the general analysis, that includes a total of 300 patients, revealed that the most frequent pathogen associated with co-infection was influenza a 6-8 (mentioned in 8 of the 19 articles focused on co-infection with respiratory pathogens), possibly following a seasonal pattern; these studies showed a slight predominance of the male sex, without preference for any age group. a single patient was diagnosed with cmv by serology 9 , and multiple bacterial infections including legionella were described, but these were mostly related to health care in seriously ill patients 10 . the most common radiological finding was "ground glass", nonetheless, it is not exclusive; the findings range from disseminated interstitial involvement to welldefined consolidations without following a pattern associated with co-infections. only one study related co-infection with an increase in the severity of the disease, but in general, there is no evidence of clinical findings or a particular prognosis in these patients. cases of severity and mortality are related to variables such as age, presence of comorbidities, lymphopenia, and elevation of d-dimer, and proinflammatory biomarkers (pcr, procalcitonin, or il-6), which are covid-19 risk factors, regardless of a co-infection. in addition, 6 of the studies included analyzed hiv patients (for a total of 63 reported cases). even in patients with adequate cd4 count, undetectable viral load and receiving antiretroviral management, the reported mortality is once again related to age and comorbidities such as hypertension, dyslipidemia, obesity, and diabetes mellitus. it is noteworthy that 4 of these patients reported a delayed serological response for igm and igg (beyond day 20), regardless of their immunity state; as well as a varied radiological finding between consolidation and peripheral compromise with a predominance of the "ground glass" pattern 11,12 . (table 1) the previous analysis does not show a pattern of clinical behavior or characteristic outcomes between sars-cov-2 infection and other pathogens. therefore, we consider that the search for other infections should be performed exhaustively, especially in those cases that may be susceptible to treatment such as influenza a (oseltamivir), hiv (antiretroviral therapy), or bacterial infections. we also suggest that reports of co-infections should be divided according to their origin, for instance, if it is a co-infection with a respiratory virus at the beginning of the disease, or if it is an opportunistic bacterium associated with health care; all with a detailed description of the evolution of the patient, changes in laboratories and at radiological level. the foregoing, to optimize the analysis of these cases and establish if there are characteristics that allow establishing the possibility of carrying an additional infection to that of sars-cov-2 and the implications for the management and prognosis of the patient. the authors declare that they have no competing interests. the authors did not participate in any open calls or received any financial support from their institution or any other entity during the development of the study. therefore, they did not have any receipt of any research funding, nor are currently receiving any research funding, reason why there are no competing interests. table table 1 . synthesized data of covid-19 and co-infection articles. coronavirus covid-19 global cases by the the pathogenesis and treatment of the `cytokine storm' in covid-19 covid-19 in patients with hiv: clinical case series incidence and severity of covid-19 in hiv-positive persons receiving antiretroviral therapy: a cohort study hiv-infected individuals: a single-centre, prospective cohort. lancet hiv co-infection with sars-cov-2 and influenza a virus in patient with pneumonia co-infection with influenza a and covid-19 co-infection with covid-19 and influenza a virus in two died patients with acute respiratory syndrome, bojnurd, iran a case of coinfection with sars-cov-2 and cytomegalovirus in the era of covid-19 sars-cov-2 and legionella coinfection in a person returning from a nile cruise not applicable infection . key: cord-329263-o5e2go23 authors: kaplan, nasser m.; dove, winifred; abd‐eldayem, sawsan a.; abu‐zeid, ahmad f.; shamoon, hiyam e.; hart, c. anthony title: molecular epidemiology and disease severity of respiratory syncytial virus in relation to other potential pathogens in children hospitalized with acute respiratory infection in jordan date: 2007-11-26 journal: j med virol doi: 10.1002/jmv.21067 sha: doc_id: 329263 cord_uid: o5e2go23 human respiratory syncytial virus (hrsv) is the major viral cause of acute lower respiratory tract infections in children. few data about the molecular epidemiology of respiratory syncytial virus in developing countries, such as jordan, are available. the frequency and severity of infections caused by hrsv were assessed in hospitalized jordanian children <5 years of age compared with other potential etiological agents. overall a potential pathogen was detected in 78% (254/326) of the children. hrsv was detected in 43% (140/326) of the nasopharyngeal aspirates. hrsv was found more frequently during the winter (january/february), being less frequent or negligible by spring (march/april). analysis of 135 hrsv‐positive strains using restriction fragment length polymorphism showed that 94 (70%) belonged to subgroup a, and 41 (30%) to subgroup b. there were also two cases of mixed genotypic infection. only four of the six previously described n genotypes were detected with np4 predominating. there were no associations between subgroup or n‐genogroup and disease severity. hrsv was significantly associated with more severe acute respiratory infection and the median age of children with hrsv was lower than for those without. next in order of frequency were adenovirus (116/312: 37%), human bocavirus (57/312: 18%), rhinovirus (36/325: 11%), chlamydia spp. (14/312: 4.5%), human metapneumovirus (8/326: 2.5%), human coronavirus nl63 (4/325: 1.2%), and influenza a virus (2/323: 0.6%). influenza b; parainfluenza viruses 1–4, human coronavirus hku1 and mycoplasma pneumoniae were not detected. j. med. virol. 80:168–174, 2008. © 2007 wiley‐liss, inc. acute respiratory infection is the major cause of death in children <5 years of age, and occurs predominantly in developing countries [bryce et al., 2005] . human respiratory syncytial virus (hrsv) is the leading viral cause of acute respiratory infection in infants and young children in terms of prevalence and effect [shay et al., 2001] . hrsv causes substantial annual winter epidemics in temperate climates, representing a major cause of pediatric hospitalizations and a serious economic burden [viegas et al., 2004] . hrsv can cause reinfections throughout the child's life and it can infect infants in the presence of maternal antibodies. this could be due to either an inadequate immune response or to the extensive genetic variability of the virus. although antigenic variation is not essential for reinfection, growing evidence suggests that it may contribute to reinfections by immune evasion [viegas and mistchenko, 2005] . it has also been reported that hrsvspecific t-cell responses do not provide protection against reinfection, and reinfection does not boost hrsv-specific t-cell proliferation [bont et al., 2002] . hrsv is an enveloped rna virus that is classified within the pneumovirus genus as a member of the family paramyxoviridae [collins et al., 2001] . the virus has a non-segmented, single-stranded, negative-sense genome. its genome encodes 10 proteins, including two major surface glycoproteins (g and f), two matrix proteins (m 1 and m 2 ), a small hydrophobic protein (sh), and three nucleocapsid associated proteins (n, p, and l) [collins et al., 1984] . the two antigenic glycoproteins g and f are responsible for host cell attachment and viral entry by membrane fusion, respectively [palomo et al., 2000] . hrsv is divided into two major antigenic subgroups, a and b, on the basis of reactivity with monoclonal antibodies against the major structural glycoproteins g and f [mufson et al., 1985] . however, these two subgroups can be further subdivided into genotypes by restriction analysis and nucleotide sequence variability [sullender et al., 1993; peret et al., 1998] . the n (nucleoprotein) gene is relatively well conserved between virus isolates, but the g gene shows much greater variability. the variation in these genes has been used to define hrsv typing schemes as n gene differences resulting in the np1-np6 genotypes. the purpose of the present study was to examine the molecular epidemiology of hrsv in jordan. we also compared the disease severity of hrsv subgroups a and b and their associated genotypes in hospitalized jordanian children set in the context of other potential respiratory pathogens. this prospective cross-sectional study was conducted over six consecutive months from december 2003 to may 2004. children younger than 5 years of age with acute respiratory infection admitted to the pediatric wards of king hussein medical centre and queen alia hospital, amman, jordan were enrolled in the study irrespective of the severity of their illness. king hussein medical centre, a tertiary referral hospital, and queen alia hospital, a district general hospital, provide hospital pediatric care for amman, the capital city of jordan, and its surroundings. the study was approved by the medical research ethical committee of the king hussein medical centre, amman, jordan and signed informed consent was obtained from each of the children's parents or legal guardians for participation in the study. the clinical diagnosis of acute respiratory infection and assessment of its severity was made by using the world health organization standard protocol for acute respiratory infection based on the presence of cough, tachypnoea, chest indrawing, and wheezing for <7 days duration [pio, 2003] . severe disease was defined as present in children with a respiratory rate >60/min and chest indrawing. oxygen saturation (po 2 ) was measured by using pulse oximetry (nellcor puritan bennett npb-195, uk) and a po 2 85% used as the cut-off for giving supplementary oxygen. a standardized questionnaire containing clinical, socio-demographic, therapeutic, and outcome data was completed for each patient. nasopharyngeal aspirates were collected by instilling 1 ml sterile phosphate-buffered saline through a sterile nasopharyngeal mucous extractor. the aspirates were frozen at à808c until analyzed in the department of medical microbiology, university of liverpool, uk. the genome of hrsv was detected in nasopharyngeal aspirates by reverse transcription-polymerase chain reaction (rt-pcr) [greensill et al., 2003] . hrsvpositive strains were classified into subgroup a and b by restriction fragment length polymorphism analysis [cane and pringle, 1992] . total rna and dna were extracted separately from nasopharyngeal aspirates by using the commercial rneasy and qiaamp dna mini kits (qiagen, crawley, west sussex, uk) according to manufacturer's instructions. chain reaction for hrsv the primers: n1 (5 0 -gga aca agt tgt tga ggt tta tga ata tgc-3 0 ) and n2 (5 0 -ctt ctg ctg tca agt cta gta cac tgt agt-3 0 ) were used to amplify the nucleocapsid (n) gene between nucleotides 858 and 1,135 giving a 278-bp product [cane and pringle, 1991 ]. an aliquot of 10 ml of the extracted viral rna serving as a template for cdna synthesis was added to 40 ml of pcr mixture. the final 50 ml mastermix reaction contained 1â pcr buffer, 3 mm mgcl 2 , 5 mm dithiothreitol (dtt), 0.4 mm deoxyribonucleotide triphosphates (dntps), 0.4 mm of primers mixture containing equal volumes of the two primers at a concentration of 20 mm each, 20 units (u) of rnase inhibitor (rnasin), 25 u of murine leukemia virus reverse-transcriptase, and 2.5 u of amplitaq gold dna polymerase (roche diagnostics ltd., burgess hill, uk). the rt-pcr was performed in a perkin elmer gene-amp 2400 thermal cycler (norwalk, ct) according to the following program: 30 min reverse transcription cycle at 508c, followed by 5 min reverse transcriptase inactivation and dna polymerase activation cycle at 948c, then forty pcr cycles (1-min denaturation at 948c, 1 min of primer annealing at 558c, and 1 min of primer extension at 728c), and a final extension cycle of 728c for 10 min. ten microliter volumes of each amplified dna pcr product were separated by electrophoresis on a 2% (wt/ vol) agarose-tris-borate-ethylene diamine tetracetic acid gel, stained with ethidium bromide, and visualized under ultraviolet light (syngene gel documentation and analysis system, ingenius, cambridge, uk). analysis of hrsv aliquots of 2 ml from hrsv positive amplified dna were separately digested directly and without prior purification by a set of five restriction endonuclease enzymes namely hindiii, psti, bglii, rsai, haeiii (roche). the restriction patterns were analyzed by electrophoresis on a 2% (wt/vol) agarose-tris-borateethylene diamine tetracetic acid gel, stained with ethidium bromide, and visualized under ultraviolet light. hrsv amplicons were typed into six nucleoprotein (np) genotypes on the basis of their restriction endonuclease digestion profiles [cane and pringle, 1992] . hrsv subgroup a included np2, np4, and np5, while subgroup b included np1, np3, and np6 genotypes. detection of other respiratory pathogens was performed according to previously published protocols. rt-pcr was used for the detection of human metapneumovirus (hmpv) [greensill et al., 2003] , influenza a and b, and parainfluenza virus 1-4 [templeton et al., 2004] , human rhinovirus (hrv) and human coronaviruses nl63 and hku1 [choi et al., 2006; sloots et al., 2006] . pcr was used to detect human bocavirus (hbov) [allander et al., 2005] , adenoviruses, chlamydia spp., and mycoplasma pneumoniae [couroucli et al., 2000] . epi info version 3.3.2 (centers for disease control, atlanta, ga) was used for statistical analysis by applying chi-square and student's t-tests. p values <0.05 were considered statistically significant. a total of 326 children (188, 58% male) with a median age of 5 months were recruited, and 326 nasopharyngeal aspirates were collected and analyzed, however the volume of some nasopharyngeal aspirates was inadequate for both dna and rna extractions. of the 326 children studied 15 (5%) had some underlying condition which might have contributed to disease severity. of these 13 had cardiopulmonary disease, predominantly congenital heart disease (but none had bronchopulmonary dysplasia), one was premature and one had immunodeficiency. in all 8 (53%) of those with an underlying condition had severe disease. a total of 72/326 (22%) patients had no pathogens detected by pcr (table i) , but 254 (78%) had at least one potential respiratory pathogen detected which consisted of 140/ 326 (43%) hrsv, 116/312 (37%) adenoviruses, 57/312 (18%) hbov, 36/325 (11%) rhinovirus, 14/312 (4.5%) chlamydia spp., 8/326 (2.5%) hmpv, 4/325 (1.2%) human coronavirus nl63, and 2/323 (0.6%) influenza a virus. overall 106/326 (33%) infants had mixed infections (table ii) and of these 83 were infected with 2 potential pathogens, 22 with 3, and 1 with 4 potential pathogens. a total of 67 hrsv co-infections (48% of all hrsv infections) were detected; 30 with adenoviruses, 10 with hbov, 10 with adenovirus and hbov, 8 with rhinoviruses, 3 with chlamydia spp., 1 with rhinovirus and hbov, 1 with chlamydia spp. and adenovirus, 1 with chlamydia spp. and hbov, 1 with chlamydia spp. and rhinovirus, 1 with adenovirus and rhinovirus, and 1 with chlamydia spp., rhinovirus and hbov. no hrsv/hmpv co-infections were detected. influenza b, parainfluenza viruses 1-4, human coronavirus hku1 and m. pneumoniae were not detected in this study. restriction fragment length polymorphism analysis was performed for 98% (137/140) of hrsv-positive strains; three weakly positive strains were not subjected to further subgrouping analysis. hrsv subgroup a was detected in 70% (94/135) and subgroup b in 30% (41/135) of the samples. there were two cases of mixed genotypic infections, one np2/np4 (a/a) and one np4/np3 (a/b). genotyping analysis of hrsv strains showed that 63 (47%) were np4, 31 (23%) np2, 26 (19%) np1, and 15 (11%) np3. each of the four genotypes co-circulated during the january-march period of the study (table iii) . all the children in the study had lower respiratory tract infections mainly bronchiolitis and bronchopneumonia and routine cultures of their blood and respiratory secretions had detected no potential bacterial or fungal pathogens. although there were no deaths, three children were admitted to the intensive care unit, one infected only with hrsv subgroup b (np1), one infected only with hbov, and one who had congenital heart disease with no pathogens detected. a total of 139 (43%) children had severe, and 187 (57%) had mildmoderate acute respiratory infection. significantly (p < 0.0005) more hrsv-infected children had severe disease (100/140; 71%) compared to those uninfected with hrsv (39/186; 21%). the median age of hrsvinfected patients was 4.8 months (range 1-48 months) and 82 (59%) were male, compared with a median age of 6 months and 104 (56%) male patients in the hrsvnegative patients (p < 0.05). severe hrsv infections occurred in all age groups, however it was significantly more likely to occur in those under 6 months of age (p < 0.01). thus 62 (62%) in the 0-6 months age group, 30 (30%) in the 7-12 months group, and 8 (8%) in those older than 1 year had severe disease. hrsv subgroup a and b were associated with 66 and 33 cases of severe disease, respectively (p ¼ 0.2). there was no significant difference between the individual hrsv genotypes as potential causes of severe disease. in the 100 patients with severe acute respiratory infection in whom hrsv was detected, it was the sole pathogen detected in 53 (53%) patients, however in the remaining 47 cases, it was found as a mixed infection with adenovirus (20 patients), hbov and adenovirus (9 patients), hbov (8 patients), chlamydia spp. (3 patients), rhinovirus (2 patients), hbov and rhinovirus (1 patient), chlamydia spp. and hbov (1 patient), chlamydia spp. and adenovirus (1 patient), adenovirus and rhinovirus (1 patient), and chlamydia spp., hbov and rhinovirus (1 patient). there was no significant difference (p > 0.5) in the prevalence of severe disease between those where hrsv was the sole pathogen (53/ 73:73%) and those with hrsv and another potential pathogen (47/67:70%). the median age of children with severe acute respiratory infection was 4 months for those infected only with hrsv, and 6 months for those co-infected with hrsv and other potential respiratory pathogens (p < 0.05). in the 40 patients with mildmoderate acute respiratory infection in whom hrsv was detected, it was the only pathogen in 20 (50%) patients, however in the remaining 20 cases, it was found as a mixed infection with adenovirus (10 patients), rhinovirus (6 patients), hbov (2 patients), adenovirus and hbov (1 patient), and chlamydia spp. and rhinovirus (1 patient). the median age of children with mild-moderate acute respiratory infection was 4 months for those infected only with hrsv, and 9.5 months for those co-infected with hrsv and other potential respiratory pathogens (p ¼ 0.05). of the 36 children infected with rhinovirus 24 (67%) had mild/moderate and 12 (33%) severe disease. the median age of those infected with rhinovirus was 4.5 months not significantly different from that of those infected with hrsv. although 12 of the children infected with rhinovirus had severe disease, only one of these severe infections occurred in the absence of coinfection with any of the other eleven potential respiratory pathogens. of the 24 children with mild/moderate disease 17 (71%) were co-infected with other pathogens. there was a similar prevalence of rhinovirus infection in january-april. this study has confirmed that hrsv is the most frequent cause of severe acute respiratory infection in young children in jordan. the fact that a large proportion of hrsv infections (71%) were associated with more severe disease is not unexpected because only hospitalized patients were involved in this study. however significantly more hospitalized infants with severe respiratory disease than mild/moderate disease due to hrsv were detected compared to infection with the other potential pathogens. worldwide, two subgroups of hrsv circulate independently within human populations, with group a being the more prevalent [peret et al., 2000] . in a 3-year study conducted in the usa [walsh et al., 1997] , group b viruses predominated for 2 years, while group a predominated in the third year. information on the relative frequencies of subgroups a and b in the middle east and africa is scarce. in the present study, subgroup a was predominant (70% vs. 30%), as previously shown in jordan [bdour, 2001] , and yemen [al-sonboli et al., 2005] . this predominance of subgroup a hrsv is the most common pattern worldwide [cane, 2001] . only four of the six previously described n genotypes [fletcher et al., 1997] were found among the hrsv strains. all four genotypes were identified during the january-march period of the study, with genotype np4 predominating (table ii) . however all four genotypes co-circulated at the same time. some authors have concluded that infection with group a hrsv is associated with more severe disease [peret et al., 2000] but others have found no such association [fletcher et al., 1997; walsh et al., 1997 ]. in the present study hrsv subgroup a infection was not associated with more severe disease than infection with subgroup b (table iii) . the medical staffs were unaware of group and genotype results and thus potential bias leading to a differential misclassification of the disease severity is unlikely. although the study extended through only 6 months of the year, there was a demonstrable peak of acute respiratory infection due to hrsv during january and february, as shown in a previous study in jordan [al-toum et al., 2006] . hrsv was less frequent or almost absent in march-may. hrsv-infected children were significantly younger than hrsv-negative children, and severe hrsv infections most frequently (90%) affected children younger than 12 months of age in agreement with a previous report from jordan [meqdam and nasrallah, 2000] . although it has been shown previously that children infected with group a hrsv were significantly older than were those infected with group b hrsv [hall et al., 1990] , there was no significant difference in the ages of these groups (p ¼ 0.2) in the present study. there were no significant differences between the different hrsv genotypes as potential causes of severe disease, so it was difficult to establish a relationship between genotypes and disease severity. overall 102 (31.2%) of the children had more than one potential pathogen detected in their nasopharyngeal aspirates. this is perhaps not surprising as the respiratory viral seasons often co-incide [choi et al., 2006] , and increasing the range of potential pathogens sought (in this study 10 viral and 2 bacterial pathogens) will reveal more co-infections. in 68 children the copathogens were hrsv with one or more other pathogen. however in no case was a co-infection with hrsv and hmpv encountered. such co-infection has previously been linked with more severe acute respiratory infection in some [greensill et al., 2003; semple et al., 2005; foulongne et al., 2006] but not all studies [maggi et al., 2003; wilkesmann et al., 2006] . hmpv was less frequently detected (2.5% of cases) than in some other hospital based studies [al-sonboli et al., 2005; semple et al., 2005] . however the peak prevalence of hmpv infections does seem to vary year by year and the hrsv and hmpv seasons are not always concurrent [serafino et al., 2004; choi et al., 2006] . after adenovirus the second most common hrsv copathogen was the newly described hbov. the finding of hbov in jordanian children has already been reported [kaplan et al., 2006] . it was infrequently found as sole pathogen which suggested that further studies were needed to assess its role as a respiratory pathogen. however recent report from edinburgh, uk has demonstrated that it is an important respiratory pathogen in children and not found in children without acute respiratory infection [manning et al., 2006] . adenoviruses were detected in 116 of the 312 children (37%), a prevalence that is significantly higher than those reported recently from germany (12.9%), brazil (6%), and india (1.5%) [grondahl et al., 1999; maitreyi et al., 2000; straliotto et al., 2002] . however only the german study used rt-pcr. the latter two employed immunofluoresence and viral culture, respectively. in 75 (65%) of the cases of adenovirus infection it was found together with another potential pathogen. the german study showed that in 5% of the cases when an agent was detected there was more than one potential pathogen and in most cases this was adenovirus with another pathogen [grondahl et al., 1999] . it is unclear why we had such a high detection rate of adenovirus infection and it warrants further study. rhinoviruses were the fourth commonest respiratory pathogen detected (36:11%). this prevalence is double that (5.8%) described for lower respiratory tract infections in korean children [choi et al., 2006] but somewhat lower than that (44%) found recently in australia . rhinovirus was present as a copathogen in 24 (67%) of jordanian children whereas in australia in almost one-third of the rhinovirus infections a co-pathogen was detected . hrvs are well-recognized causes of upper respiratory tract infection. however in the australian study it was detected in almost half the samples from patients with lower respiratory tract infection , again somewhere higher than the 11% prevalence in jordanian children, hayden [2004] has recently reviewed the role of rhinoviruses in lower respiratory tract infection and found that they were responsible for between 12% and 24% of such infections in children. in one study it was present as a co-pathogen in almost 50% of infections [papadopoulos et al., 2002] . better therapies and prevention strategies are needed to decrease the burden of acute respiratory infection particularly that due to hrsv. thus further molecular epidemiological studies over longer periods of time are warranted to better determine the role of the different hrsv genotypes in the epidemiology and the severity of disease and their inter-relationship with other respiratory pathogens. this could inform better therapeutic approaches and vaccine development. cloning of a human parvovirus by molecular screening of respiratory tract samples respiratory syncytial virus and human metapneumovirus in children with acute respiratory infections in yemen epidemiology and clinical characteristics of respiratory syncytial virus infections in jordan frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses and bocavirus during acute respiratory tract infections respiratory syncytial virus subgroup a in hospitalized children in zarqa natural reinfection with respiratory syncytial virus does not boost virus-specific t-cell immunity who estimates of the causes of death in children molecular epidemiology of respiratory syncytial virus respiratory syncytial virus heterogeneity during an epidemic: analysis by limited nucleotide sequencing (sh gene) and restriction mapping (n gene) molecular epidemiology of respiratory syncytial virus: rapid identification of subgroup a lineages the association of newly 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syncytial virus in a community circulation patterns of group a and b human respiratory syncytial virus genotypes in 5 communities in north america standard case management of pneumonia in children in developing countries: the cornerstone of the acute respiratory infection programme dual infection in infants by human metapneumovirus and human respiratory syncytial virus is associated with severe bronchiolitis respiratory syncytial virus and metapneumovirus in children over two seasons with a high incidence of respiratory infections in brazil bronchiolitis-associated mortality and estimates of respiratory syncytial virus-associated deaths among us children, 1979-1997 evidence of human coronavirus hku1 and human bocavirus in australian children viral etiology of acute respiratory infections among children in porto alegre, rs, brazil analysis of respiratory syncytial virus genetic variability with amplified cdnas rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2,3 and 4 molecular epidemiology of human respiratory syncytial virus subgroup a over a six-year period (1999-2004) in argentina respiratory viruses seasonality in children under five years of age in severity of respiratory syncytial virus infection is related to virus strain human metapneumovirus infections cause similar symptoms and clinical severity as respiratory syncytial virus infections key: cord-328196-fpk9elm8 authors: sykes, jane e. title: immunodeficiencies caused by infectious diseases date: 2010-05-13 journal: vet clin north am small anim pract doi: 10.1016/j.cvsm.2010.01.006 sha: doc_id: 328196 cord_uid: fpk9elm8 immunodeficiencies caused by infectious agents may result from disruption of normal host barriers or dysregulation of cellular immunity, the latter serving to promote survival of the infectious agent through immune evasion. such infections may be followed by opportunistic infections with a variety of other microorganisms. classic infectious causes of immunodeficiency in companion animals are the immunodeficiency retroviruses, including feline immunodeficiency virus and feline leukemia virus. other important causes include canine distemper virus; canine parvovirus 2; feline infectious peritonitis virus; rickettsial organisms that infect leukocytes; leishmania; and fungal pathogens, such as cryptococcus. considerable research effort has been invested in understanding the mechanisms of pathogen-induced immunosuppression, with the hope that effective therapies may be developed that reverse the immunodeficiencies developed and in turn assist the host to clear persistent or life-threatening infectious diseases. jane e. sykes, bvsc(hons), phd the classic example of immunodeficiency caused by an infectious agent is the acquired immunodeficiency syndrome, caused by human immunodeficiency virus (hiv). similarly, the best known pathogens of companion animals causing immunodeficiencies are the feline retroviruses feline immunodeficiency virus (fiv) and feline leukemia virus (felv). however, several other pathogens are capable of disrupting normal immune function. many infectious agents disrupt host barriers to infection. this may result from the inflammatory response to a pathogen or direct damage by the microbe itself. examples include disruption of the gastrointestinal mucosal barrier by canine parvovirus, destruction of nasal turbinates by aspergillus fumigatus in canine sinonasal aspergillosis, or paralysis of the respiratory cilia by bordetella bronchiseptica. anaplasma phagocytophilum disables neutrophil function, ensuring its survival within a cell normally charged with antimicrobial substances. viruses, such as canine distemper virus, cause lymphopenia; the outcome of infection depends on the balance between viral destruction of the immune system and the ability of the remaining immune defenses to eliminate the virus. disruption of immune function by infectious agents may serve to promote the infectious agent's survival through host immune evasion. immunosuppression having the greatest impact clinically often occurs as a result of infection with organisms that are able to persist within the host. ideally, a pathogen is able to adapt such that it can coexist with the host, without causing death of the host or severe illness, in a way that maximizes the pathogen's transmission efficiency. the types of opportunistic infections that occur in patients that are immune compromised as a result of an underlying immunosuppressive infection depend upon the mechanisms of immunosuppression. impairment of normal host barrier function or the function of granulocytes is generally associated with a broad spectrum of bacterial infections and sometimes infection with opportunistic fungi, such as aspergillus spp impairment of cell-mediated immunity (cmi) results in infections with opportunistic pathogens, such as nocardia spp, mycobacterium spp, toxoplasma gondii, and a variety of fungal pathogens. reactivation of dormant pathogens, such as feline herpesvirus, may also occur with depression of cmi. the purpose of this article is to highlight some of the mechanisms by which persistent infectious microorganisms cause acquired immunodeficiency in companion animal species, and the consequences of the resulting disturbance in immune function. canine distemper virus (cdv) causes canine distemper, a common disease of dogs worldwide that is associated with a high degree of morbidity and mortality. the virus also infects several other species, including foxes, raccoons, skunks, ferrets, and free-ranging and captive felids. disease in dogs is most prevalent in regions where vaccination of young dogs against the disease is either not performed or is poorly timed, and epidemics continue to occur in shelter environments in developed countries. 1 canine distemper virus is a morbillivirus related to measles virus and has been used to study the pathogenesis of measles virus infection. morbilliviruses are enveloped rna viruses that survive poorly in the environment. based on genetic variation within the viral hemagglutinin (h) gene, a multitude of different strains of cdv exist that vary in their geographic distribution, cell tropism, and virulence. although cdv infects a variety of different cell types, including epithelial, mesenchymal, neuroendocrine, and hematopoietic cells, the marked tropism of cdv for immune cells is critical in respect to its ability to cause immunosuppression. viral components involved in cdv-induced immunodeficiency include the viral hemagglutinin; the v protein (a nonstructural phosphoprotein); and the nucleocapsid (n) protein. dogs are generally exposed to cdv through contact with infected oronasal secretions. the virus initially infects monocytes within lymphoid tissue in the upper respiratory tract and tonsils and is subsequently disseminated via the lymphatics and blood to the entire reticuloendothelial system. direct viral destruction of a significant proportion of the lymphocyte population, and especially cd41 t cells, occurs within the blood, tonsils, thymus, spleen, lymph nodes, bone marrow, mucosa-associated lymphoid tissue, and the hepatic kupffer cells. [1] [2] [3] this viral destruction is associated with an initial lymphopenia and transient fever that occurs a few days after infection. subsequently, there is a second stage of cell-associated viremia, after which cdv infects cells of the lower respiratory; gastrointestinal tract; central nervous system; urinary tract; and red and white blood cells, including additional lymphoid cells. elimination of cdv by the host depends on humoral and cmi. 1, 4 because the virus is lymphocytolytic, the outcome of infection depends on the rate at which the host is able to remove the virus before the virus has sufficient time to cause severe immune system injury. dogs mounting a partial immune response may undergo recovery from acute illness but fail to eliminate the virus completely, leading to a spectrum of more chronic disease manifestations that often involve the uvea, lymphoid organs, footpads, and especially the cns. opportunistic infections may also have the chance to develop in these dogs. dogs with canine distemper may develop profound lymphopenia and leucopenia. lymphopenia results from generalized depletion of t and b cells in a variety of tissues ( fig. 1) . cd41 t cells are preferentially depleted during the acute phase, which is followed by cd81 cell depletion. 5, 6 necrosis of hematopoietic cells within the bone marrow may result in leucopenia. 7 infection of ferrets has been used as a model of cdv-induced immunosuppression. 8 cdv infection of ferrets leads to dramatic reduction in cell-mediated immune function with markedly depressed lymphocyte proliferative activity, and to some extent delayed type hypersensitivity responses. the virus enters lymphocytes following binding of the viral h gene to the primary receptor for the virus, signaling lymphocyte activation molecule (cd150, slam). the expression of slam appears to be upregulated in response to cdv infection. 9 slam is also expressed on antigen-presenting cells, such as dendritic cells and activated monocytes, and infection of these cells, which may predominate in the chronic phase of infection, has been hypothesized to be associated with impaired antigen presentation. 1, 6 infection of dendritic cells within the thymus may lead to impaired maturation and selection of t cells, with subsequent release of immature cd5-t cells, including cells that may have the potential for autoreactivity. 6 lymphocyte apoptosis also occurs independent of viral infection in canine distemper, although the mechanisms have not yet been elucidated. 10 the presence of the viral v protein is essential to permit rapid replication of cdv in t cells and critical in cdv-mediated immunosuppression. this protein almost completely antagonizes alpha interferon, tnf-alpha, il-6, gamma-interferon, and il-2 in the acute phase of infection. 3 suppression of the cytokine response is associated with severe immunosuppression and a fatal outcome in ferrets. finally, the n protein of morbilliviruses may interfere with the immune response through the binding of the cd32 (fc-gamma) receptor on b cells, resulting in impaired differentiation of b cells into plasma cells. 11 binding of this receptor on dendritic cells 12 is associated with impairment of antigen presentation by dendritic cells and resulting disruption of t cell function. the most common secondary infections in canine distemper are secondary bacterial infections that contribute to bronchopneumonia. bordetella bronchiseptica is also a common co-pathogen in dogs with distemper. dogs may be diagnosed with bordetellosis in the early stages of distemper, the underlying cdv infection being overlooked. other opportunistic infections that have been identified in dogs with distemper include toxoplasmosis, 13 salmonellosis, 14 nocardiosis, 15, 16 and generalized demodicosis (sykes and colleagues, unpublished observations, 2006) . in one study from brazil, canine distemper was the most common underlying immunosuppressive disease predisposing to nocardiosis in dogs. 16 infection with pneumocystis carinii was associated with cdv infection in a mink, 17 and concurrent neosporosis and canine distemper was reported in a raccoon. 18 although parvoviruses do not cause chronic, persistent infections in dogs and cats, parvoviral replication creates the perfect storm for development of acute and severe opportunistic bacterial infections. the combination of leukopenia, disruption of the gastrointestinal barrier, and the immature immune system of the young animals that are most susceptible to these viruses is associated with the common development of sepsis, which is frequently the cause of death. canine parvovirus 2 (cpv-2) and feline panleukopenia virus (fpv) are small, nonenveloped dna viruses. since its emergence in 1978, cpv has subsequently mutated to cpv-2a; cpv-2b; and in the last decade, cpv-2c, which was first documented in italy and has subsequently spread to dogs on every continent, with the exception of australia. the cpv-2c strain appears to be particularly virulent and there has been some debate regarding the ability of current vaccines to protect against it and the ability of commercially available snap elisa tests to detect the virus. 19 cpv and fpv have tropism for rapidly dividing cells. as such, they exert an effect on the host that resembles the outcome of treatment with a chemotherapeutic drug. the virus binds and enters cells using the transferrin receptor. 20 cells preferentially involved are the crypt cells of the gastrointestinal tract, bone marrow, and lymphoid tissue. leukopenia results from sequestration of neutrophils within damaged gastrointestinal tissue and is compounded by destruction of white cell precursors within the bone marrow. damage to the gastrointestinal barrier can result in translocation of enteric bacteria. in the face of the massive immunosuppression that ensues as a result of virus-induced neutropenia and lymphopenia, the host fails to contain bacterial replication and bacteremia and sepsis ensue. treatment of secondary infections with broad-spectrum parenteral antimicrobial drugs is critical to permit recovery of dogs and cats from parvoviral infection. bacterial causes of sepsis reported in infected animals include escherichia coli, salmonella spp, and clostridium difficile. giardia infection also exacerbates illness. 21 immunosuppression may also contribute to replication of other co-infecting enteric viruses, such as enteric coronavirus, which in turn exacerbate the damage to the gastrointestinal mucosa. similarly, cpv-induced immunosuppression potentiates the development of postvaccinal canine distemper encephalitis. 22 the importance of secondary infections in the pathogenesis of parvovirus infections is highlighted by the fact that experimental infection of germfree cats is not associated with development of clinical illness, despite the associated reduction in white cell count. 23 feline leukemia virus and feline immunodeficiency virus are common causes of viralinduced immunodeficiency in cats, although the underlying mechanisms by which they exert immunodeficiency are still incompletely understood. subtypes of felv and fiv are defined based on variations in the env gene sequence, which also influences their pathogenicity. there are four different subtypes of the gamma retrovirus felv: felv-a, felv-b, felv-c, and felv-t. each subtype uses a different receptor to enter cells ( table 1) . [24] [25] [26] [27] all sykes cats infected with felv-b, felv-c, and felv-t are co-infected with felv-a, with felv-a being the only type that is transmitted between animals. the other subtypes arise through recombination or point mutation within felv-a during the course of infection and influence the clinical expression of disease (see table 1 ). felv-t, a t-cell tropic variant, is unique amongst gamma retroviruses in that it requires two host proteins to enter and infect cells. 27 as a result of its t-cell tropism, felv-t infection may be particularly associated with immunodeficiency in cats. transmission of felv-a primarily occurs through prolonged, close contact with salivary secretions, although other routes of transmission, including through biting, can also occur. after an initial phase of viremia, felv replicates within rapidly dividing lymphoid, myeloid, and epithelial cells, such as those lining the intestinal crypts. 28 as with distemper, when cellular destruction exceeds the ability of the host's immune system to suppress viral replication, persistent viremia and progressive felv-related disease results. clinical outcomes of felv infection include tumor development, especially lymphoma or leukemia; non-regenerative anemia; marrow failure, which in turn can result from myelophthisis, myelodysplasia, or myelofibrosis; neurologic manifestations, such as anisocoria; reproductive failure; gastrointestinal disease; and immunodeficiency. the development of opportunistic infections may result from marrow failure or cell-mediated immunodeficiency. the immunosuppressive properties of felv have been linked at least in part to the transmembrane viral envelope peptide, immunodeficiencies caused by infectious diseases p15e. 29 this viral protein inhibits t-and b-cell function, inhibits cytotoxic lymphocyte responses, alters monocyte morphology and distribution, and has been associated with impaired cytokine production and responsiveness. [30] [31] [32] kittens persistently infected with felv have impaired t-cell, and to a lesser extent, b-cell function. [33] [34] [35] [36] infected cats may develop lymphopenia, thymic atrophy, and depletion of lymphocytes within lymph node paracortical zones. cd41 t-cell malfunction may contribute to a decreased humoral and cellular immune response in affected cats. 37, 38 the response to vaccination may also be impaired. neutrophil function is also impaired in cats that are felv-infected. [39] [40] [41] opportunistic infections documented in cats that are felv-infected include bacterial infections of the upper and lower urinary tract, hemoplasmosis, respiratory tract infections, feline infectious peritonitis (fip), and chronic stomatitis, although there is little evidence in the literature to support an increased prevalence of these infections in cats with felv as opposed to cats not infected with felv. some infections, such as cryptococcosis, appear to occur with the same frequency in cats that are felv positive as in cats that are felv negative, but may be more severe and refractory to therapy (fig. 2) . 42 fiv is a lentivirus that is primarily transmitted between cats by biting. fiv invades cells via the primary receptor cd134, which is expressed on feline cd41 t lymphocytes; b lymphocytes; activated macrophages 43, 44 ; and the secondary receptor cxcr4, a chemokine receptor. the mechanisms of immunosuppression in fiv infection are complex, and despite more than 20 years of research on the subject, not completely understood. paradoxically, immune suppression and immune hyperactivation have been documented in infected cats. a comprehensive review of the subject is beyond the scope of this article but has been recently published elsewhere. 45 central to fiv-induced immunosuppression is a progressive reduction in cd41 tcell numbers. the number of cd41 t cells in peripheral blood declines shortly after infection, owing to initial viral replication within target activated cd41 t cells and macrophages. after this acute phase of infection, numbers of cd41 t cells rebound and viremia is suppressed (fig. 3) . neutropenia can also occur during this phase 46 and it has been suggested that this may result from neutrophil apoptosis. 47 cd41/cd251 t regulator cells have recently been shown to be infected and activated during acute infection. when activated, these cells inhibit proliferation and induce apoptosis of other activated cd41 or cd81 t cells, which may also contribute to persistence of fiv and further immunosuppression. 45, 48, 49 evidence also points to altered dendritic cell function during acute fiv infection. 50, 51 the impairment of t-cell function in acute fiv infection has been suggested to result from cytokine dysregulation, immunologic anergy, and increased apoptosis. 45 in turn, this is associated with an inability to mount a primary immune response to opportunistic pathogens. a prolonged asymptomatic period follows, sometimes lasting years or even the lifetime of the cat, which is associated with a gradual decline in cd41 t-cell numbers; a reduction in the cd41/cd81 ratio; generalized lymphoid depletion; and in some cats, hyperglobulinemia, which results from b-cell hyperactivation. in addition to a decline in cell numbers, although activated, paradoxically, t cells develop a reduced ability to respond to antigenic stimulation. altered lymphocyte expression of cell surface molecules, including cd4, cytokine receptors and major histocompatibility complex (mhc) ii antigens, and continued alteration of dendritic cell function, also contribute to immunosuppression. dysregulation of cytokine production occurs. cats chronically infected with fiv fail to produce il-2, il-6, and il-12 in response to t gondii infection, instead producing elevated levels of the antiinflammatory cytokine il-10. 45, 52 ultimately these changes lead to opportunistic infections, most commonly bacterial infections of the mouth; chronic bacterial skin infections; persistent viral upper respiratory tract infections; mycobacterial infections; hemoplasmosis; toxoplasmosis; and parasitic infections, such as demodicosis and severe flea burdens. the immune response. 53 the mechanism of t cell depletion is not clear, because the virus does not infect lymphocytes, only monocytes and macrophages. infection of antigen-presenting cells, specifically dendritic cells, by the virus has been hypothesized to cause t-cell apoptosis. 53 despite the profound t-cell deficiency that accompanies fip, opportunistic infections are rarely reported, perhaps partly as a result of the rapidly fatal clinical course of disease. perhaps the best examples of bacterial infections causing immunodeficiency are those of the tick-borne pathogens ehrlichia canis and anaplasma phagocytophilum, which are described later in this article. bartonella spp. and hemotropic mycoplasmas (hemoplasmas) may also be capable of inducing chronic immunodeficiencies. human infection with bartonella bacilliformis infection may be immunosuppressive and many patients have succumbed with secondary bacterial infections, especially salmonellosis. 54 impaired leukocyte function, cyclic cd81 lymphopenia, and diminished expression of adhesion molecules and mhc class ii molecules by cd81 and b lymphocytes, respectively, were documented in one study of bartonella vinsonii subspecies berkhoffii-infected dogs. 55 hemoplasma-induced immunosuppression is not a new phenomenon and has been recognized as a problem in experiments involving chronically infected laboratory rodents and in sheep chronically infected with mycoplasma ovis. 56, 57 the clinical importance of immunosuppression induced by bartonella spp and hemoplasmas in cats and dogs requires further investigation. ehrlichia canis is a gram negative intracellular bacteria that causes canine monocytic ehrlichiosis (cme), arguably the most important infectious disease of dogs exposed to ticks worldwide. the organism is transmitted by the brown dog tick, rhipicephalus sanguineus. the organism infects monocytes, in which it forms morulae. in the united states, disease is diagnosed most frequently in dogs living in the southeastern and southwestern states, but because of chronic, subclinical infection, dogs can be transported to non-endemic regions and subsequently develop disease. different strains of e canis exist but the degree by which these vary in virulence is poorly characterized. the course of cme has been divided into acute, subclinical, and chronic phases, although in naturally infected dogs, these phases are often not readily distinguishable. clinical signs of acute disease include depression, inappetence, fever, and weight loss. ocular and nasal discharges, edema, hemorrhages, and neurologic signs may also occur. the organism replicates in reticuloendothelial cells with generalized lymphadenopathy and splenomegaly, and transient cytopenias, especially thrombocytopenia, may occur. after the acute phase, which may last up to 6 weeks, a subclinical phase may develop that lasts months to years. during this phase, the organism appears to evade host immune responses through antigenic variation. ultimately, a small percentage of these infected dogs develop chronic cme. chronic cme is characterized by signs that include lethargy, inappetence, fever, weight loss, bleeding tendencies, pallor, lymphadenopathy, splenomegaly, dyspnea, anterior uveitis, polyuria/polydipsia, muscle wasting, polyarthritis, and edema. dogs with severe chronic ehrlichiosis may develop marrow failure, with aplastic pancytopenia. severe disease may also be associated with a protein-losing nephropathy and development of neurologic signs. some dogs have bone marrow plasmacytosis and peripheral granular lymphocytosis. hyperglobulinemia is a frequent finding on the serum chemistry profile and usually results from a polyclonal gammopathy, although monoclonal gammopathies have also been reported. 58 high antibody titers to e canis, occasionally exceeding 1:1,000,000, are also common. the chronic phase may also be associated with development of secondary opportunistic infections. the precise underlying mechanism of the immunodeficiency that develops and how it relates to successful persistence of e canis has not been elucidated. not all dogs that develop chronic infections are pancytopenic, so leukopenia alone does not explain the predisposition for opportunistic infection. furthermore, the types of infections reported, such as viral papillomatosis; generalized demodicosis; protozoal infections, such as neosporosis and opportunistic mycoses, suggest a defect develops in cmi (fig. 4) . 59 e canis infection has also been suggested to predispose dogs to development of canine leishmaniasis. 60 infection of a canine cell line with e canis resulted in suppression of mhc class ii expression. 61 in one study, acute experimental infection with e canis was not associated with measurable suppression of cmi or humoral immune responses. 62 alterations in immune responses during chronic infection require further evaluation. like e canis, anaplasma phagocytophilum is an obligate, tick-transmitted intracellular bacteria that forms morulae within leukocytes. in contrast to e canis which infects monocytes, a phagocytophilum infects granulocytes, primarily the neutrophil, 63 and causes granulocytic anaplasmosis, a disease of humans, dogs, horses, ruminants, and occasionally cats (fig. 5) . the vector ticks are generally those belonging to the ixodes persulcatus complex, primarily i scapularis and i pacificus in the united states, and i ricinus in europe. numerous small wild mammals, deer, and possibly birds, act as reservoir hosts for the organism. several genetic variants have been identified and there is increasing evidence of strain variation in host specificity and pathogenicity. immunosuppression resulting from a phagocytophilum infection results primarily from impairment of neutrophil function by the bacteria. after inoculation into the host, a phagocytophilum attaches to sialylated ligands on the surface of neutrophils, after which it enters neutrophils via caveolae-mediated endocytosis, bypassing phagolysosomal pathways. a phagocytophilum then actively disables neutrophil bactericidal functions, in particular neutrophil superoxide production, thus promoting its own survival. 64, 65 a phagocytophilum also reduces neutrophil mobility and phagocytosis, 66 and reduces endothelial adherence and transmigration of neutrophils. 67 by inhibiting neutrophil apoptosis, the organism is able to survive in a well-differentiated cell that normally has a very short lifespan. the impairment of neutrophil function and leukopenia that develop as a result of a phagocytophilum infection is occasionally associated with development of opportunistic infections in some humans and animals with granulocytic anaplasmosis. the best example of this is tick pyemia, which is a debilitating lameness and paralysis that develops in infected lambs in europe, most commonly as a result of disseminated staphylococcus aureus or pasteurella spp infection. infection with a phagocytophilum may influence the outcome of infection with borrelia burgdorferi, which can be co-transmitted by ixodes ticks, possibly as a result of impaired neutrophil function. 68 leishmaniasis, caused by the protozoal parasite leishmania infantum, is a chronic progressive disease transmitted by the sand fly. the mechanisms of immunosuppression induced by this organism are perhaps the best studied amongst protozoal parasites. the disease is most common in the mediterranean basin and south america. the organism causes a systemic disease in dogs characterized by lymphadenopathy, crusting skin lesions, weight loss, anemia, ocular lesions, polyarthritis, and proteinlosing nephropathy. the infection is often associated with other infections, especially ehrlichiosis and babesiosis, and occasionally with neoplastic disease, especially hematopoietic tumors. 69 leishmania infantum invades mononuclear phagocytes, evading the phagolysosome, and survives within them through inhibition of the respiratory burst, inhibition of macrophage function and apoptosis, and impairment of antigen presentation through inhibition of mhc class i and mhc class ii molecule expression. the protozoan also appears to impair macrophage and neutrophil chemotaxis, and interferes with il-12 transcription. 70 the leishmania spp surface protein gp63 is a key protein that mediates entry and survival within macrophages. it also allows the organism to resist complement and was recently shown to bind to and suppress the activity of nk cells. 71 several fungal pathogens are capable of causing immunosuppression, including aspergillus spp, candida spp, and cryptococcus spp. cryptococcus neoformans and cryptococcus gattii are highly immunosuppressive fungal pathogens, although co-infections with other pathogens are rarely documented. cryptococcal organisms possess several potent virulence factors that are capable of suppressing or orchestrating the immune response in favor of fungal growth and persistence. the cryptococcal capsular polysaccharide, glucuronoxylomannan, has attracted the most attention in this regard (fig. 6) . it effectively inhibits phagocytosis and interferes with migration of leukocytes from the bloodstream into tissues by causing them to shed selectin. it can also deplete complement and directly inhibits t-cell responses. 72, 73 there is a shift from a th1 to a th2 immune response, the th1 response being normally required for organism clearance. the cryptococcal urease enzyme was shown to promote accumulation of immature dendritic cells within the lung, and an associated shift in the immune response to a non-protective th2-cytokine dominated response. 71 this review highlights the mechanisms of immunosuppression in just a small subset of the huge variety of infectious agents that are capable of inducing immunosuppression to promote their own survival within the host. the degree of immunosuppression and the mechanisms by which immunodeficiency develops are highly variable and complex. pathogen surface molecules and cellular receptor tropisms play an important role in determining the initial immune cells infected. because of the cascading mechanisms involved in normal immune cell recruitment, cytokine and antibody production, pathogens frequently disrupt the function of immune cells that do not undergo direct infection. considerable research effort has been invested in understanding the mechanisms of pathogen-induced immunosuppression, with the hope that effective therapies may be developed that reverse the immunodeficiencies developed and in turn assist the host to clear persistent or life-threatening infectious diseases. pathogenesis and immunopathology of systemic and nervous canine distemper tropism illuminated: lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system receptor (slam [cd150]) recognition and the v protein sustain swift lymphocyte-based invasion of mucosal tissue and lymphatic organs by morbillivirus lymphocyte-mediated immune cytotoxicity in dogs infected with virulent canine distemper virus immunohistochemical analysis of the lymphoid organs of dogs naturally infected with canine distemper virus phenotypical characterization of t and b cell areas in lymphoid tissues of dogs with spontaneous distemper 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ehrlichia subversion of host innate responses mechanism of evasion of neutrophil killing by anaplasma phagocytophilum defective phagocytosis in anaplasma phagocytophilum infected neutrophils diminished adhesion of anaplasma phagocytophilum-infected neutrophils to endothelial cells is associated with reduced expression of leukocyte surface selectin anaplasma phagocytophilum-infected neutrophils enhance transmigration of borrelia burgdorferi across the human blood brain barrier in vitro extranodal gammadelta-t-cell lymphoma in a dog with leishmaniasis how protozoan parasites evade the host immune response leishmania surface protein gp63 binds directly to human natural killer cells and inhibits proliferation direct inhibition of t-cell responses by the cryptococcus capsular polysaccharide glucuronoxylomannan cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective t2 immune response within the lung the authors thank dr ellen e. sparger for her review of the retroviral section of this article. key: cord-305457-t7qw1oy2 authors: zhang, youhong; enden, giora; wei, wei; zhou, feng; chen, jie; merchuk, jose c. title: baculovirus transit through insect cell membranes: a mechanistic approach date: 2020-09-21 journal: chem eng sci doi: 10.1016/j.ces.2020.115727 sha: doc_id: 305457 cord_uid: t7qw1oy2 baculovirus systems are used for various purposes, but the kinetics of the infection process is not fully understood yet. we investigated the dynamics of virion movement from a medium toward the interior of insect cells and established a mechanistic model that shows an excellent fit to experimental results. it also makes possible a description of the viral dynamics on the cell surface. a novel measurement method was used to distinguish between infected cells that carry virions on their surfaces, cells that carry virions in their interior, and those carrying virions both inside and on their surface. the maximum number of virions carried by a cell: 55 viruses/cell, and the time required for viral internalization, 0.8 [formula: see text] , are reported. this information is particularly useful for assessing the infection efficacy and the required number of virions needed to infect a given cell population. although our model specifically concerns the infection process of sf9 insect cells by baculovirus, it describes general features of viral infection. some of the model features may eventually be applicable in the studies towards palliation of the covid-19 outbreak. the baculovirus (bv) expression vector system (bevs) is of great interest for the production of recombinant proteins in a wide range of fields-from basic science research to the development of biomedical applications and the production of bio-insecticides [1] [2] [3] [4] . the production of recombinant proteins in bevs is greatly affected by the characteristics and kinetics of the viral infection process-including, for instance, the multiplicity of infection (moi), time of infection (toi), cell cycle, cell line selection, and culture state [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] , but the early events of viral infection, such as the attachment of virions to their receptors and the kinetics of their entry into the cell, are still not entirely understood. this gap hampers the development of efficient and specific genetically engineered proteins and gene therapy vectors based on bva goal that has lately gained much interest [1] [2] [3] [4] 8] . since baculovirus was used for efficient gene transfer to mammalian cells in the 1990s [16] [17] , several effective baculovirus-based gene therapy vectors had been created and tested, demonstrating favorable therapeutic efficacy in laboratory and preclinical phase studies [18] [19] [20] [21] . although the entry processes and the mechanisms involving bvs infection of insect cells or transduction of mammalian cells remain poorly understood, it has been shown that a similar mechanism may mediate gp64 binding to permissive host cells [22] [23] [24] and that bvs enter insect and mammalian cells through a clathrin-mediated endocytosis [25] [26] . thus, comprehensively characterizing the kinetics underlying the early steps of bvs infection of insect cells is crucial both for improving the production of recombinant proteins in insect cells and for designing therapy vectors and enhancing gene transduction efficacy in therapeutic studies. mathematical modeling and simulations may serve as effective predictive tools in pursuing this goal [8, [14] [15] 27] , as they allow dynamical simulation of the process and estimating variables that are hard to measure directly, such as the rate of virion internalization after its attachment to the cell. quantitative models were also employed to simulate the infection dynamics of infectious diseases. padmanabhan and dixit (2011) [28] constructed a kinetic model of hepatitis c virus (hcv) which accounted explicitly for the dependence of hcv entry target cells on cd81 expression. iwami et al. (2012) [29] optimized a mathematical model to describe the kinetics of the simian/human immunodeficiency virus (shiv) infection which improved the understanding of shiv and human immunodeficiency virus type-1 pathogenesis. such models, and the virtual experiments that they facilitate, are useful for interpreting the mechanistic aspects of the viral infection process and may save time, manpower, and physical resources in predicting their outcomes. the main purpose of the current study was to construct such a model, focusing on the early stages of a high-moi bv infection of insect cells [14, 30] , based on and tested against experimental results. our model embodies an interdisciplinary integration of microbiology, biology, engineering and mathematics and sets a basic mechanistic approach to the early stages of the infection process. it disregards the effect of non-infective viral mutants which might enter the cell without infecting it. it also does not address the effectiveness of infection, which may vary depending on the elapsed storage time, mainly due to viral aggregation within the viral stocks [31, 32] . as we used the same storage time in all experiments, particle aggregation did not affect the measured results. consequently, we disregarded storage time in our model. based on detailed and accurate measurements, our model enhances our understanding of the early bv infection process and can be potentially extended to describe the early stages of other infection processes, such as gene delivery by viruses in mammalian cells. it also provides a basis for modelling the production of foreign proteins. the present model is a first step towards the longterm goal of establishing a more elaborated model of the production process, aimed at saving time, manpower, and physical resources. the insect cell line used in this study was sf9 (cctcc-gdc0008 note that the units of moi used here are the ratio of viral particles to cells, as counted by a flow cytometer (becton dickinson, san jose, tx), while, traditionally, it is measured as plaque-forming units (pfus) per cell or as tcid50/cell. : the ratio of fcm counts to end-point dilution assay titers ranges from 1.0 to 9.4, with an average of 3.7 and a standard deviation of 2.4 [31] . thus, the mois of 100, 200 and 400 bvs/cell used in present experiments were equivalent to 25, 50 and 100 tcid50/cell. an improved method of fcm was used to quantify baculovirus titer, as described previously [31] [32] [33] . prior to the fcm analysis, in the final 1-ml phosphate-buffered saline (pbs, ph 8.0) solution, virion samples were fixed with 0.1% (w/v) paraformaldehyde for 30 min at 4 °c and then stained with a specific nucleic acid dye, sybr green i, at a commercially 6 available concentration (1× 10 −4 ) for 10 min at 80 °c. yellow-green fluorescence microspheres (1 μm in diameter, molecular probes, eugene, or) were added (as an internal reference to the bv) to the control sample [34] . all samples were analyzed using a facs calibur flow cytometer (becton dickinson, san jose, tx) and the threshold was set on green fluorescence (fl1-h) to eliminate the background interference, as seen in fig. 1 . bv particles were congregated in r1 and bead particles were congregated in r2. comparing the number of virion particles with yellow-green fluorescent microspheres, the bv titer was determined by fcm using cellquest software (becton dickinson, san jose, tx). to avoid the coincidence of viral particles (i.e., two or more particles simultaneously being within the sensing zone), the samples were diluted such that the total event rate was kept below 500 events/s. cells were counted with a hemocytometer using trypan blue (0.5% w/v) exclusion to distinguish dead from live cells. to experimentally monitor the early stages of the infection process and detect infection in cells to which virions attach, we directly tracked gp64-the major envelope protein of the bv, which plays a crucial role in the early stages of infection. gp64 is essential for the attachment of virions to cells in a culture and for their transport between cells in infected tissues [35] , as it is involved in the binding of the virions to host cell receptors [36] and acts to locally reduce the membrane ph, which triggers membrane fusion. once the virus has entered the cell by endocytosis, gp64 is necessary for stripping the nucleocapsid from its capsid proteins and for releasing the virion into the cytosol [37] [38] [39] . to label gp64 specifically and thereby detect only infected cells, we used the monoclonal antibody acv1, which binds to gp64 but does not reduce its ability to attach to cells [25, [40] [41] [42] [43] . to differentiate between virions that are attached to the cell surface from those that have already entered it, we either permeabilized the cells or left them intact (see methods) assuming that acv1 labeled bvs attaches to the cell surface in both permeabilized and nonto count the number of non-permeabilized infected cells, the samples were stained with 5 μl acv1-pe without adding the permeabilization reagents. after a 30 min incubation at 4 °c in the dark, the cell samples were washed again. finally, to obtain the optimal fluorescence signal, the cell samples were examined by fcm within 30 min. the infected and uninfected cells were detected using the scatter plot with forward scattering (fsc-h) versus side scattering (ssc-h), and then the ratio of infected to total cells was analyzed by the bd cellquest software. control cell samples were treated using the same steps, but without adding acv1-pe (data not shown). we began by establishing the capacity of sf9 cells to carry autographa californica multicapsid nucleopolyhedrovirus (acmnpv)an important reference for moi optimization. we detected the virion titer by flow cytometry (fcm), using the fluorescent dye sybr green i to stain the nucleic acids of the virions [31] [32] [33] [34] . the sf9 cells were infected with acmnpv at mois of 114, 219, or 421 bvs/cell, and the obtained virion and cell profiles are shown in table 1 experimental data on free viruses in the medium and the infected cell concentrations for each moi. the maximum number of virions that can attach to a cell, n, is defined here as: where it is assumed that moi ≥ . here is the final measured concentration of virions in the medium, is the final measured concentration of infected cells 9 h post-infection (ℎ ), as shown in fig. 3 and in the initial stage of the study, we characterized the kinetics of non-infected sf9 cells, which were grown under the same conditions as those used in the infection experiments (see methods in the attachments), but without bv infection. it was found that the simple first-order growth rate fits satisfactorily the collected data. the use of more sophisticated kinetic forms is therefore unnecessary the kinetic expression for the growth of non-infected cells is: the solution of this expression is: where is the cell concentration, is time, and is the growth rate factor. regression of expression (3), based on all experimental data points in a 60ℎ experiment, yielded = 0.022[ℎ −1 ] . this kinetic rate constant was used in the mathematical model described below. the value is lower than data reported previously for these cells, but it should be kept in mind that anyway the growth rate of the cells is sensibly lower that the infection rate. this consideration is reinforced further by the success of the assumption of constant cell concentration rate presented in 3.5. the dynamics of the early stages of the infection process in sf9 cells are shown in fig. 4 for mois = 114, 219, and 421. the percentage of infected cells, as evaluated without permeabilization, , increased until 4 ℎ , reached a maximum of 79%, and then slowly decreased to 65% at 9 ℎ . this maximum will be explained below in terms of the balance between virus attachment and virus internalization. in contrast, the percentage of the infected cells, as evaluated when permeabilizing reagents were used in the analysis, , increased monotonically, reached a maximum of 97%, and then, either remained unchanged, or moderately increased to a plateau. this effect was observed with only minor differences between the three examined mois (although higher mois resulted in a slightly higher ), suggesting that almost all cells were infected by or before approximately 6 ℎ . to model the dynamics of the early infection process, we used the following variables: x the infection process is viewed here as two serial steps: the first is the virion-cell encounter and its attachment to the cell surface, as defined in eqs. (6) and (9); the second step consists of the internalization of the virion into the cell, assumed here to last a certain time, , as reflected in eqs. (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) . in addition, we assume that (1) the latter bears the simplifying assumption that after the first virion attachment to each untreated cell, it will appear as "infected" until all its n attachment sites are used and all its surface virions internalized. one of the simplest ways to depict macroscopically the mechanism of virion attachment to cells is to assume that the rate of attachment is proportional to both the number of free virions and the number of unused attachment sites on the cell. denote the initial virion concentration in the medium as 0 and the number of virions removed from a unit volume of the medium (via attachment to cells) as ( ) . the concentration of free virions in the medium is therefore, = 0 − . assuming that the rate of virus attachments is proportional both to free virus concentration and to the concentration of attachment sites, the differential equation describing the adsorption rate of virions can be written as: where ( ) = ( ) + ( ) is the total cell concentration hours after toi, and equation (6) the material balance on the infected cells in the system yields: at toi, (0) = 0 ( 1 0 ) a balance on the pool of viable cells will include a cell growth term, already shown in eq. (1) to be a first-order process with a kinetic constant , and a second term for viable cell depletion by infection: at toi, (0) = (0) = 0 where 0 for each moi is shown in table 1 . in this case, cells containing only internalized virions cannot be distinguished from uninfected cells, since acv1 conjugated with a fluorescent pe cannot enter into nonpermeabilized cells. assuming that the internalization time, , is identical for all virions, we can calculate ( ), i.e., the number of virions (per unit volume of the medium) attached to cell surfaces (and, therefore, detectable) at any given time. this value is equal to ( ) for < , and is equal to the difference between the number of virions that were attached to the cells between ( − ) and for ≤ , since virions that arrived earlier than ( − ) have already been internalized and cannot be detected. therefore, denoting by ( ) the number of internalized viruses at time per unit volume of the medium, we obtain: the rate of change of ( ), ( ), at time , is: and, following, eq. (6): the rate of viral internalization, ( ), at a time , is: where ⌊ ⌋ denotes the "whole value function" (also called the "floor function"), which returns the largest integer that is smaller than or equals to x (rounded down). it follows that, for any ≥ , a crude approximation to ( ) is made here by assuming that the concentration of darkened cells is proportional to ( − ) for ≥ . noting that, for < , ( ) = ( ), we obtain: the solution of this model, represented in equations (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) , was fitted to the experimental data and the kinetic parameters k and , , and were determined. the optimization was accomplished using the matlab and polymath codes. the optimized parameters are shown in table 2 . eqs. (6) (7) (8) (9) (10) (11) (12) (13) were solved numerically using matlab and the parameters were evaluated with respect to the measured data in the time interval 0 ≤ ≤ 3ℎ. table 3 for moi = 114. with the acmnpv-sf9 system shown in table 2 . this indicates that different baculovirus may have different internalization half-time value. since the internalization time for each specific virus-host system is expected to be a characteristic constant, it seems that viral vectors with shorter internalization times would have some advantages in gene therapy, since they enter host cells more rapidly while the cells are robust. therefore, the estimation of the internalization time for any potential virus-host combinations may be very important. the very low value of the constant indicates that the actual accumulation rate of dark cells ⁄ is substantially lower than the floor function shown in eq. (19) , and yet follows a similar pattern, given the quality of the fit. based on the results shown above, a crude approximation to ⁄ can be made assuming that the generation rate of darkened cells is proportional to : as an example, fig. 7a demonstrates the typical dynamics of the system variables ( ), the low growth factor, = 0.022, suggests that the cell concentration, may be treated as nearly constant at the range 0 ≤ ≤ 3ℎ. this observation is further supported by the experimental behavior of the cell concentration shown in fig. 6 . in this section, we neglect the changes in and consider it to be a constant equal to 0 . in this case, is set to zero and eqs. (6) (7) (8) (9) (10) (11) (12) can be integrated analytically to yield a closed-form solution for , , , , and . (21) where = 0 ; = ( 0 − 0 ), and is equivalent to in eq. (5). the closed-form solutions of , , and are shown in the appendix. next, we used the closed-form solution with the parameters shown in table 3 note that the asymptotic value of ( ), is independent of the kinetic constant . in fact, this is the total number of attachment sites on the cells, as might be expected. as defined above, ( ) is the number of virions per unit volume that have been captured by cells between 0 and . it reaches maximum when all cells are saturated by n virions. n is a constant, specific to each virus-host system the approximated solution, based on a constant value of the total cell concentration, appears to be successful for our set of initial conditions. the solution of eq. (6) can be replaced by the closed-form expression given above (eq. (21)) without noticeable changes in the predictability capacity of the model. in the present multi-disciplinary research, we propose a mathematical model of the early the baculovirus expression vector system: a commercial manufacturing platform for viral vaccines and gene therapy vectors baculovirus as versatile vectors for protein expression in insect and mammalian cells engineering the transposition-based baculovirus expression vector system for higher efficiency protein production from insect cells improved pfastbac™ donor plasmid vectors for higher protein production using the bac-to-bac® baculovirus expression vector system optimization of egfp expression using a modified baculovirus expression system physiological and environmental factors affecting the growth of insect cells and infection with baculovirus application of on-line our measurements to detect actions points to improve baculovirus-insect cell cultures in bioreactors modeling the population dynamics of 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envelope variants for improved delivery to human airway epithelial cells gene therapy for treatment of chronic hyperammonemia in a rat model of hepatic encephalopathy a novel coronavirus from patients with pneumonia in china emergence of a novel coronavirus disease (covid-19) and the importance of diagnostic testing: why partnership between clinical lab oratories, public health agencies, and industry is essential to control the outbreak *the maximum number of virions that sf9 cells can carry: 55 viruses/cell, is reported. *cells that carry virions on their surface, in their interior, or both are distinguished *analytical mathematical solution renders satisfactory results this research was financially supported by the national natural science foundation of china (20876120 and 20910102037) and the nature science foundation of hubei, china (2013cfa101). we thank professor qi yipeng of wuhan university, china, for kindly providing wt acmnpv. the authors declare no competing interests, financial or other. key: cord-318061-xe8lljz0 authors: overgaauw, paul a.m.; vinke, claudia m.; van hagen, marjan a.e.; lipman, len j.a. title: a one health perspective on the human–companion animal relationship with emphasis on zoonotic aspects date: 2020-05-27 journal: int j environ res public health doi: 10.3390/ijerph17113789 sha: doc_id: 318061 cord_uid: xe8lljz0 over time the human–animal bond has been changed. for instance, the role of pets has changed from work animals (protecting houses, catching mice) to animals with a social function, giving companionship. pets can be important for the physical and mental health of their owners but may also transmit zoonotic infections. the one health initiative is a worldwide strategy for expanding collaborations in all aspects of health care for humans, animals, and the environment. however, in one health communications the role of particularly dogs and cats is often underestimated. objective: evaluation of positive and negative one health issues of the human–companion animal relationship with a focus on zoonotic aspects of cats and dogs in industrialized countries. method: literature review. results: pets undoubtedly have a positive effect on human health, while owners are increasing aware of pet’s health and welfare. the changing attitude of humans with regard to pets and their environment can also lead to negative effects such as changes in feeding practices, extreme breeding, and behavioral problems, and anthropozoonoses. for the human, there may be a higher risk of the transmission of zoonotic infections due to trends such as sleeping with pets, allowing pets to lick the face or wounds, bite accidents, keeping exotic animals, the importation of rescue dogs, and soil contact. conclusions: one health issues need frequently re-evaluated as the close human–animal relationship with pet animals can totally differ compared to decennia ago. because of the changed human–companion animal bond, recommendations regarding responsible pet-ownership, including normal hygienic practices, responsible breeding, feeding, housing, and mental and physical challenges conforming the biology of the animal are required. education can be performed by vets and physicians as part of the one health concept. the one health initiative or concept is a worldwide strategy that recognizes that public health is connected with animal health and the environment. it concerns multidisciplinary collaboration between physicians, veterinarians, environmental scientists, public health professionals, wildlife experts, and many others [1, 2] . with a multisectoral and transdisciplinary approach, public health threats can be better monitored and controlled. the resulting synergism enhances the knowledge of how diseases, known as zoonotic diseases, can be shared between animals and people with the goal of this article is based on an existing post-graduate course for veterinarians, vet technicians, family doctors, midwifes, and specialists such as pediatricians which explores healthy human-animal relationships. the existing evidence-based knowledge contained in this course has been actualized by performing a literature search to add new relevant publications. a literature search was conducted through 2 march 2020, using the national library of medicine's pubmed for the terms "one health" and "companion animals"; "pet ownership"; "households" and "pets"; "dogs" or "cats" or "pets" and "mental" or "physical health" or "children"; "animal assisted therapy"; "dogs" or "cats" and "nutritional problems" or "overweight" or "obesity" or "homemade" or "raw meat diets"; "dogs" or "cats" and "behavior problems" or "aggression" or "fear" or "anxiety" or "abnormal repetitive behavior"; "dogs" or "cats" and "breeding" or "genetic problems"; "dogs" or "cats" and "zooanthroponoses"; "pets" and "anthropomorphism"; "dogs" or "cats" or "exotic animals" or "rescue dogs" or "soil" and zoonoses. for some topics the internet was accessed and used as reference if additional information was not available as scientific publication. the authors selected articles that described pivotal and novel insights in the different topics. all searches were carried out without filters. the titles of all found articles were screened for relevance to the topic, and appropriate titles were assessed and selected based on their abstracts. if a selected article was a review, it was read and relevant citations were used to find primary literature on the subject. additional studies were found using the bibliographies of selected articles. occasionally, reviews were directly used as sources, mostly to convey background information that is not in the core focus of this article. original articles in english and different national languages (dutch, german, french, spanish, if available) were included. specific searches were made for citations dated after the year 2000 to ensure more recent literature on the topic had not been missed. a pet or companion animal is an animal that lives in or around the house and is fed and cared for by humans. until the 1960s, pets were mainly kept as utility animals, for example as draft dogs or watch dogs or for pest control when it comes to cats. due to major changes that have taken place in society since the second world war, such as increased leisure time and prosperity, but also individualization of humans, animals are nowadays kept as pets and are regarded by many owners as valued family members, e.g., over 90% in the united kingdom [13] . pet ownership is still increasing in many industrialized countries and these animals are more often considered a member of the family [5] . even in china, a country where pets were banned in urban areas until 1992, pet ownership has grown quite rapidly in the major cities. the rate of pet ownership of all usa households increased from 57.6% to 59% in 2018. dogs continue to dominate in popularity among american households. approximately 38% of households nationwide owned a dog, bringing the population of pet dogs to nearly 77 million, while 25% of households owned cats, with a total population of 58 million [14] . in 2018, an estimated 80 million european households owned at least one pet animal; 24% of households owned dogs and 25% owned cats [15] . there are 85 million pet dogs and 104 million pet cats in europe which is a 15% increase of dogs within 8 years (74 million dogs in 2010) and a 22% increase of cats (85 million in 2010) [16] . pet cats in europe thereby are more popular than dogs. an explanation of the higher popularity of cats may be the number of single-person households in the eu that rose on average by 2.0% per annum between 2006 and 2016 to 32.5%, and the growth of two-family households grew by 1% to 31% in this period [17] . also, dual-earner families are widespread as a result of quite a steep growth in female employment over the past two decades [18] . when animals live with humans, they too benefit from human interaction. over the past decades, animal welfare has evolved to recognize that animals are sentient beings capable of experiencing positive and negative emotions. the social and ethical dimensions of animal welfare, which are concerned with how human society morally regards and treats non-human animals, are also increasingly being recognized [19] . in dutch law, the intrinsic value of kept animals is expressly incorporated and used as a guiding ethical principle that forms the basis of any further legislation. the intrinsic value is thereby defined that animals are sentient beings that can feel pain and discomfort. therefore, they should be kept free of stress, pain, disease, hunger, thirst, and should be able to show natural behavior known as the five freedoms of r. brambell 1965 [20] . in industrialized countries animal keepers, their owners, are legally required to provide such circumstances and can be prosecuted if they infringe the law. of course, in the field there is animal abuse, negative animal welfare conditions, and animal diseases. however, in general, caretaking for companion animals is nowadays performed at a high level. new insights into animal behavior has had its influence on the general public. for example, owners are aware or are told by vets and pet shops that rabbits should not be kept alone but at least in pairs due to their need for social contact [21] . there are various reasons to keep pets, such as love, warmth, and companionship. companion animals have an important emotional value, and promote the socialization of the lonely elderly because they facilitate additional contact with people. pets form a goal in life, reduce stress, and ensure that the owner keeps physically active. around 95% of dog owners and 93% of cat owners expressed that owning their pet makes them happy and 44% of owners selected this as one of the reasons they got their pet in the first place [22] . however, the function of companion animals consists of more than just providing a socializing being. studies show other benefits of having a pet, such as the positive effect on individuals' mental and physiological health status. most research addressing the health benefits of pet ownership show reductions in distress and anxiety, decreases in loneliness and depression, and increases in physical condition [23] . the positive benefits to human health from interacting with animals, focusing on the companion animal, have also be described with the term "zooeyia" [24] . in fact, 63% of owners agreed that having a pet makes them physically healthier, with dog owners more likely to agree, most probably because dog owners exercise more (85% agreeing, compared to 41% of cat owners). besides, 84% of owners agreed that having a pet makes them mentally healthier. expressed reasons are the non-judgmental nature of their pets, their playfulness, or physical contact [22] . another demonstrated positive influence is the blood pressure and heart rate lowering effects that occurs when stroking a friendly-looking dog or even being in the presence of a friendly animal, while it is not necessary to own a pet to obtain these stress-moderating benefits [23] . many studies have demonstrated the association between pet ownership and cardiovascular health and dog owners appear to have a significantly greater chance of survival after a heart attack compared to people without pets [25] [26] [27] . pets can therefore play an important role in reducing absenteeism and visits to family doctors or the hospital [28] . it has been estimated that pet ownership saved australia $1 billion in 1994 [29] , while it may reduce the use of the national health service (nhs) in the uk to the value of £2.45 billion per year [30] . dogs also play an increasing role as co-therapist or as supporter for people with psychological or physical disabilities. the benefits of these animal-assisted activities are improved mood, decreased physiological distress, depression, dementia, and loneliness [31, 32] . examples include resident or visiting dogs in prisons, nursing homes [33] , mental institutions, and hospitals where they can reduce patient anxiety in a hospital emergency department [34] , reduce pain perceptions in children after surgery [35] , or calm young patients at a pediatric dental clinic [36] . since dogs have extremely sensitive noses, they are used for several purposes such as tracking, bomb detection, and search and rescue. in recent years, canine olfaction has also been more recognized as a diagnostic tool for identifying pre-clinical disease status, such as diabetes (ketones), different forms of cancer, and infections from biological media samples [37] . animal-assisted therapies can act as co-therapies to facilitate psychotherapy or to provide specific types of therapeutic interventions such as improving motor skills or behavior [23] . such interventions were effective in improving the state of children or adults with or at risk of developing mental disorders such as attention deficit hyperactivity disorder (adhd), post-traumatic stress disorder (ptsd), or autism spectrum disorder (asd) [38] [39] [40] , and for the treatment of ptsd in military veterans [41, 42] . assistance or service animals are trained to perform tasks for the benefit of individuals who have disabilities such as hearing loss, physical disabilities, emotional disabilities, seizure disorders, or diabetes [23] . finally, a wide range of emotional health benefits from childhood pet ownership has been identified, particularly for those suffering from low self-esteem and loneliness. there is evidence of an association between pet ownership and educational and cognitive benefits, increased social competence, social networks, social interaction, and social play behavior [43, 44] . significantly less absenteeism from school through sickness among children who live with pets has also been reported [13] . having a dog or cat in the house during the first year of life may protect against childhood asthma and allergy [45, 46] . it can therefore be concluded that companion animals contribute significantly toward the public health, but also increasingly, the health of individually challenged persons through animal-assisted interventions [47] . providing companion animals with feed by humans, has been considered an advantage for companion animals in their relationship with humans. however, the feeding practices can also have a negative impact on companion animals [48, 49] . obesity in cats and dogs is a disease which is rapidly increasing with significant and lifelong implications for animal welfare. although no universally accepted definition of canine and feline obesity exists, the american veterinary medical association defined obesity being more than 30% above the ideal weight of an animal. overweight is defined as 10%-20% above the ideal weight. using body condition scores, it has been estimated that in the united states, 54% of dogs and 59% of cats are obese or overweight. a study in the united kingdom reported 65% of adult dogs and 37% of juvenile dogs as being obese or overweight [50, 51] . overweight dogs are more likely to be diagnosed with, e.g., urinary tract diseases. obese and overweight dogs are at risk developing orthopedic disorders and hypothyroidism [52, 53] . obese cats are at higher risk for developing urinary tract disease, diabetes mellitus, and neoplasia [54, 55] . other diet-related problems in companion animals can be caused by the changed feeding behavior of humans, e.g., by providing companion animals with bone and raw feed (barf) or vegan diets. risks for companion animals associated with barf or vegan diets are the presence of microbial hazards, insufficient nutrition, and in raw meat diets the presence of risk materials like thyroid tissue. through contact with their animals, it is possible that risks could even develop for owners. there could be an increased risk of human salmonellosis because of the presence of salmonella spp. in the diet which can spread to humans through diet leftovers or by contact with animal feces. recently a review was published on the risks of barf feeding [56] . the authors concluded that the data for the nutritional, medical, and public health risks of raw feeding are fragmentary, but they are increasingly forming a compelling body of formal scientific evidence. publications were found reporting the presence of escherichia coli o157, salmonella typhimurium, campylobacter spp., and antibiotic resistant bacteria in the feed. nutritional problems, such as calcium/phosphorous imbalances and specific vitamin deficiencies [57] are also reported. moreover, homemade diets are inherently susceptible to nutritional imbalances and deficiencies [58] . awareness about climate change, public health and animal welfare has incited a major change in dietary choices among many individuals. the number of vegans in the world keeps growing, even quadrupling from 150,000 to 600,000 individuals between 2014 and 2019 in affluent countries such as the uk [59] . the popularity of veganism goes beyond the scope of the human diet, as more people are interested in the possibility of feeding their companion animal a vegan diet than ever before. to create animal-free complete cat food requires replacing nutrients in animal-based materials with plant-based materials. different sources are used such as corn, rice, peas, soy, potato, and different oils and seeds. any further nutrients that are missing from plant-based materials, such as taurine and carnitine, are replaced with synthetically produced versions [60, 61] . feeding trials using vegan animal food are either not performed due to testing costs or kept private due to the highly competitive vegan pet food market [62] . additionally, they reported testing 24 vegetarian diets for cats and dogs and found that one was lacking protein and six did not meet all amino acid concentration requirements. vegan animal food may not contain meat, but it does contain grains, soy, and corn. plant-based products, such as grains, can be a source of health problems because of the presence of mycotoxins, for example [63] . warm, humid storage conditions can lead to the formation of mycotoxins such as aflatoxins, produced by the fungi aspergillus flavus and aspergillus parasiticus. many regular animal feeds also contain plant-based products, therefore the negative impact of feeding vegan diets to companion animals, especially obligate carnivores such as the cat or the ferret, seems therefore more related to diet insufficiency than to microbial health risks. additionally, addressing behavioral problems, the "free" provision of food might fulfil the consumptive part of feeding behavior of our companion animals but does not fulfil the appetitive phase. especially this phase of feeding patterns can have consequences for the companion animals' mental health and may result in behavioral problems if appetitive physical and mental challenges remain chronically absent in the human-animal relationship. in the human-companion animal bond, pets may develop abnormal behavior, including excessive aggression, fear and anxiety, or even abnormal repetitive behavior. abnormal repetitive behavior (arbs) were first noticed in zoo-, shelter-, and laboratory animals: all animals housed under stimulus-poor conditions and with limited space. however, companion animals can develop arbs as well, if the individual's adaptive capacity is exceeded due to, e.g., a lack of social contact, physical exercise, mental challenges, and in uncontrollable and unpredictable environments (e.g., separation, mistreatment, or inadequate application of cages). arbs can either be classified in stereotypies or compulsive disorders [64] with stereotypies generally defined as unvarying repetitive behavior patterns with no obvious goal or function [65] . the terminology of compulsive disorders is preferably chosen for repetitive behavior patterns that are goal-directed and show variability in the repetitive (motor) patterns [66, 67] . under chronic conditions without possibilities to adapt (cope), companion animals may develop stereotypies or compulsive disorders like, e.g., tail chasing, polyphagia, compulsively self-directed licking and/or biting the coat [68] , or feather pecking in parrots [69] . self-directed patterns can result in serious degrees of alopecia, lick granuloma, or even self-inflicted injuries (auto-mutilation) with a risk of infection. two main reasons underlie the development of arbs in our companion animals. first of all, a lot of companion animals are social species eager for social contact. in the human-companion animal bond, the need for social contact with either conspecifics and/or humans [70, 71] can remain unfulfilled if owners work from nine to five, five days a week with the pet staying alone at home on a daily basis. on the other hand, a cat which is originally a solitary hunter with a complex dynamic social structure may start overgrooming or house soiling in the presence of another cat in the territory. such situations might occur in multi-cat households, in the presence of neighboring cats, and in in-stable grouped housing conditions in shelters [72, 73] . secondly, most companion animals are species that are eager for mental and physical challenges on a daily basis. the lack of foraging opportunities, the appetitive phase of feeding behavior [74] might be another reason for the possible development of arbs in companion animals. foraging is often regarded as a high priority behavior [75, 76] , i.e., an internally motivated behavioral pattern that should be performed, or otherwise may induce a state of chronic stress, which may result in behavioral pathology like arbs as described in many other animal species [77] [78] [79] . foraging patterns may include walking, running, jumping, nose pushing, digging, and overseeing the area, all active patterns that imply the daily need for physical exercise and mental challenges in most of our companion animals. nonetheless, our pets mostly, if not always, get their food for free with minimal foraging challenges, except for going out 3-5 times a day. for some individuals (and especially some dog breeds, e.g., malinois, border collies, and pit bull terriers) [80, 81] , situations and contexts with limited challenges can make them more vulnerable to the development of arbs. as well as arbs, other problematic behavior may develop in our companion animals, and the prevalence of some of this behavior is even higher than that of arbs, for example excessive interspecific and/or intraspecific aggression, fear, and anxiety. at what moment, and which type of problem behavior may develop, depends on the intermingled factors of, e.g., genetics, early life experiences (maternal-child bonding, weaning, socialization [82] ), daily environment, and multiple factors in and around the human-companion animal bond. the history of breeding animals goes back to a time when humans and animals shared each other's habitat. dogs originally have been selectively bred to support human needs, such as hunting, herding, obedience, guarding, rescuing, and for companionship. this artificial selection has generated a large number of dog breeds, displaying a large variation of behavior, size, head shape, coat color, and coat texture [83, 84] . unfortunately, in the last 100 years, intensive selection for extreme looks and a narrow gene pool of many breeds has interfered in the genetic make-up of dogs, leading to unfavorable anatomy (extreme large, or extreme "teacup" small), and genetic predisposition to numerous health, welfare, and behavioral problems [85] . over 700 inherited disorders and traits have already been described in the domestic dog [86] . one type of dog with a distinct dysmorphology is the brachycephalic dog. brachycephalic dogs are characterized by a large head and round face due to a shortened muzzle, a high and protruding forehead, and widely spaced large eyes. these facial features fit the concept of baby schema ("kindchenschema") proposed by konrad lorenz [87] . infantile (cute) faces are biologically relevant stimuli for rapidly and unconsciously capturing attention and eliciting positive or affectionate behavior, including the willingness to care [88] . the appeal of brachycephalic animals has led to specimens that are the so-called "over-typed" dogs and cats with a too short nose, excessively protruding eyes, too straight angulations, etc. breeding animals with this type of severe skull and muzzle abnormalities leads to physical and physiological hardship and limits their natural behavior [89, 90] . this violates their integrity and is a big risk for their welfare. selectively breeding animals in order to express specific traits does not only alter existing animals, but also creates new ones, turning animals into an instrument for human use [91] . the bambino sphynx cat is an example of so called "mutant breeding", where breeders deliberately stack in two steps the recessive inheriting mutations, which leads to hairlessness in sphynx cats, on the dominant inheriting (lethal) mutation responsible for the shortened legs of the munchkin cat. the lack of hair in combination with short legs interferes with the normal physiology of the cat with regard to the manner of movement, thermoregulation, and skin health. one may argue that artificial selection in exchange for money, status, or aesthetic reasons violates the animal's dignity and integrity [92] . conclusively, artificial selection for excessive traits can have direct consequences for individual health and welfare, may obstruct and prevent a pet from fulfilling its behavioral needs, and conflicts with the current moral way of thinking on animal dignity and integrity. pet animals are not only perceived in 80%-90% as family members or partners but are almost treated like humans. in one study, up to 62% of owners agreed with the statement "my dog is more important to me than any human being" [93] . this kind of behavior is the result of the attribution of human cognitive processes and emotional states to animals, such as feelings of happiness, love, or guilt. people believe that animals have awareness, thoughts, and feelings. this behavior is called anthropomorphism, personification, or humanization and can also be applied to plants, gods, or objects. anthropomorphism appears to be caused by the perceived similarity between humans and animals and the extent to which people have developed an affectionate bond with their dogs and cats [94] . human empathy provides the basis for the attribution of empathy to other animals, as well as attributions of the communicative ability of other animals [95] . anthropomorphistic behavior can be harmless, such as talking to pets, which many owners do and one of the reasons for this may be the unique ability of humans to recognize facial expressions. talking to pets is also found to be linked to social intelligence [94] . however, it can lead to animal welfare problems when the feelings of owners no longer match the needs and the intrinsic values of their animal. examples of this are designer dog clothes, animal perfumes, and jewelry, thought the use of protective coats in colder climates for small, short-haired breeds, e.g., chihuahuas, is considered useful. the large number of obese pets can also be partly attributed to anthropomorphism. studies from north america, europe, and australia to determine what proportion of animals, mainly dogs, are overweight or obese reported prevalences of between 22%-44% [96] . in 2018, an estimated 60% of cats and 56% of dogs in the usa were overweight or obese [50] . when the owner takes a treat with coffee, it is believed that the dog should also get it. even chocolate treats are given, when these are potentially fatal for dogs and cats. this also applies to a good meal that is shared with the pet. dog owners who did not consider obesity to be a disease, maybe because the facial features of their pets fit the concept of baby schema [87] , were more likely to have obese dogs [97] . an often-unrecognized risk for pets is reverse zoonotic disease transmission, the so-called zooanthroponosis. a review on this subject reported 56 articles dealing with human to animal disease transmission [98] . most of the articles dealt with bacterial pathogens but also viral, parasitical, and fungal pathogens were studied in these publications. animals reported to have been infected or inoculated with human diseases included wildlife, livestock, companion animals, and other animals or animals not explicitly mentioned. the majority of the studies focused on human to wildlife transmission with an emphasis on mycobacterium spp. for companion animals, mrsa-infection was especially reported but m. tuberculosis, influenza a, and candida albicans were also discussed. for all groups of animals, microsporum spp. and trichophyton spp. were identified as infectious agents originating from humans [99] . recent publications report a different kind of zooanthroponosis: the transmission of high-risk, multidrug-resistant pathogens from humans to animals [100] . a major issue mentioned is the transmission of high-risk clones of extended-spectrum beta-lactamase (esbl) producing bacteria including escherichia coli, enterobacter cloacae, and klebsiella pneumonia [100, 101] . the transmission of carbapenem-resistant ndm-5 producing e. coli from previously hospitalized humans to dogs has also been suggested [102] . transmission of hospital acquired antibiotic resistant bacteria from human patients to their pets has been confirmed, such as the vim-2 producing pseudomonas aeruginosa st233 strain in brazil [103] . this increased transmission of high-risk multidrug-resistant pathogens from humans to animals was related to the closer relationships between humans and companion animals. some authors doubt the generalized pet-effect on human mental and physical health because of conflicting results that are prevalent in this area of science and the lack of publication of negative results [13, [104] [105] [106] . the majority of research evidence was also considered inconclusive due to methodological limitations such as reliance on self-reports, small sample sizes that may not be representative of the general population, homogeneous populations, varying research designs, narrow range of outcome variables that were examined, and the use of cross-sectional designs that do not consider long-term health outcomes [107] [108] [109] . other studies found for example that pet ownership was associated with a higher incidence of heart attacks and readmissions in heart attack patients instead of a lower incidence [110] or that pet owners had higher diastolic blood pressure than those without pets [111] . müllersdorf (2010) showed that pet owners had better general health but suffered more from mental problems such as anxiety, insomnia, and depression, than those who did not own pets [112] . other studies failed to support earlier findings that pet ownership is associated with a reduced use of general practitioner services [113] or psychological or physical benefits on health for community dwelling older people [114] . negative effects of pet ownership include dog and cat bites or scratches, the spreading of disease (zoonoses), and fall injuries, caused by falling or tripping over dogs and cats [115] . allergic reactions may be a consequence of animal contact and affect 15%-30% of individuals (often genetically) predisposed [116] . allergies relating to more uncommon pets such as fish, birds, and amphibians seem to be increasing in prevalence [117] . other studies prove that pet ownership in early life did not appear to either increase or reduce the risk of asthma or allergic rhinitis symptoms in children aged 6-10 years. therefore, advice to avoid or to specifically acquire pets for primary prevention of asthma or allergic rhinitis in children should not be given [118] . there are also less-positive effects that pets can have on health. more excessive forms of anthropomorphism became clear in our study for the presence of zoonotic parasites in healthy dogs and cats. fifty percent of owners allow pets to lick their faces. sixty percent of the pets visit the bedroom; 45-60% (dogs-cats) are allowed on the bed, and 18-30% (dogs-cats) sleep with their owner in bed. six percent of pets always sleep in the bedroom. of the cats, 45% are allowed to jump onto the kitchen sink [119] . this means that in addition to the detected zoonotic parasites (the hazard), there was a significant potential exposure to these pathogens. in addition to parasites, other pathogens such as bacteria, viruses, and fungi can also be transmitted by animals by direct contact through biting, licking, scratching, sneezing or coughing, handling pets or their body fluids or secretions and by indirect contact through contaminated bedding, food, water, or bites from an arthropod vector [120] . not every individual will develop symptoms after being infected with a zoonosis. this is the result of various factors such as the causative pet species, housing, the degree of contact and contamination, the ability of a micro-organism to cause disease in humans and animals, but especially due to the degree of immunity of the recipient. in order to assess the risk of disease transmission from pets it is important that the nature and frequency of contacts between pets and their owners or other people are evaluated [120] . we traditionally know that young children (age < 5 years), the elderly (age ≥ 65 years), patients with an impaired immunity, and pregnant women that carry a fragile fetus are at more than average risk of becoming ill after an infection. moreover, they may have more severe disease, have symptoms for a longer duration, or develop more severe complications compared to other patients. young children (notably those aged 3-5 years) and some people with developmental disabilities often have suboptimal hygiene practices or higher risk contact with animals which further increases risk [121] . in children, hand-to-mouth behavior is part of their natural development and they mouth their fingers and other objects. in a meta-analysis, the average indoor hand-to-mouth frequency ranged from 6.7 to 28.0 contacts/hour and the average outdoor frequency ranged from 2.9 to 14.5 contacts/hour. the lowest value was attributed to the 6-to 11-year-olds and the highest to the 3-to < 6-month-olds [122] . fifteen percent of dog owners and 8% of cat owners always wash their hands after contact with the animals [119] . in addition, due to improved healthcare in recent decades, the group of immunocompromised patients has increased sharply. this includes, for example, patients with diabetes, post-splenectomy, after placement of implants and patients being treated with chemotherapy or immunosuppressants. the risk groups are also referred to as yopis (young, old, pregnant, and immune suppressed). patient surveys and epidemiological studies suggest that the occurrence of pet-associated zoonotic disease is low overall [121] . many of these pathogens are not reportable and presumably underdiagnosed or not recognized by family doctors due to the general, mostly flu-like, symptoms. therefore, any reported frequency of such infections is likely underestimated. to get a better picture of zoonotic risks, a risk analysis is required where a risk score can be calculated using exposure, contagiousness of the infection, and its consequences in the human. in addition, the disease burden of an infection for the population can be calculated and expressed in disability adjusted life years (dalys). this quantifies the health loss based on two components: the life years lost due to premature death and secondly the proportional loss of quality of life as a result of the disease. there is a trend towards closer physical contact between owners and their pets or their environment which poses an increased risk of transmission of zoonotic pathogens. these trends (a general direction in which something is developing or changing) will be explained. our publication [119] showed that a high percentage of pets were allowed in the bedroom and in bed, with 6% always sleeping in bed with their owner. is that a problem? already from a hygienic point of view, it is not advisable to sleep with animals or take them into bed. they do not pay attention to where they are walking outside and do not wipe their feet after arriving home. dogs like to roll in carcasses and both dogs and cats regularly lick the anus and thereafter the fur. in a pilot study with 28 healthy dogs and 22 healthy cats that slept with their owners, we tested 68% of the dogs (19) and 32% of the cats (7) positive for enterobacteriaceae on the fur or footpads. fleas and flea larvae were found on 14% of pets [123] . as a result of our publication, chomel investigated in 2011 whether transmission of infections by sleeping with pets and licking the face could be found in literature. he reported that also in the usa, france and the uk, a relatively large number of pets slept in bed with their owner (14%-33% of dogs and 45%-60% of cats) [124] . similar results of sleeping with pets were also reported from canada (26% of pets slept with children), the czech republic (45%), and qatar (63.3%) [125] [126] [127] . chomel found bacterial infections such as yersinia pestis (plague), bartonella henselae (cat scratch disease), methicillin-resistant staphylococcus aureus, and sometimes fatal bite wound infections such as capnocytophaga canimorsus and pasteurella multocida. furthermore, parasite infections such as cheyletiella spp. were reported. feline cowpox is a rare viral infection, but it can be transferred to the human after direct contact. both animals and humans reveal local exanthema on arms and legs or on the face. in most cases the disease is self-limiting, but immunosuppressed patients can develop a lethal systemic disease resembling smallpox [128] . it can be concluded that, although uncommon with healthy pets, the risk of transmission of zoonotic agents by close contact between pets and their owners through bed sharing is real and has even been documented for life threatening infections such as plague [129, 130] . although pets do not transmit arthropod-borne diseases to people (e.g., lyme borreliosis, ehrlichiosis, anaplasmosis), they do bring zoonotic disease vectors such as ticks and fleas, in close proximity to people, e.g., when they are sleeping with their animals [121] . while fleas are considered a vector of bartonella henselae (the causative agent of cat scratch disease) tickborne diseases are reported as increasing as ticks expand their ranges [131, 132] . with an estimated 65,000 cases a year, lyme borreliosis is responsible for the largest disease burden of any vector-borne disease in the european union [133] . another increased risk associated with close contact with fur is when it is contaminated with zoonotic parasite eggs. especially with echinococcus multilocularis (fox tapeworm) or e. granulosus (hydatid worm, or dog tapeworm). these eggs are immediately infective and may cause serious health problems in the human, many years post infection [134, 135] . despite a low prevalence of infectious (embryonated) eggs of toxocara spp. on dog's fur, the potential zoonotic risk should not be disregarded [136] . the same risk is applicable for the persistence of sporulated toxoplasma gondii oocysts in dogs' fur [137] . in relation to this, it is noteworthy that many publications report striking increases of ringworm, a common zoonotic fungal skin infection in mainly children caused by microsporum spp., trichophyton spp. or arthroderma spp., where the presence of pets is always mentioned [94] . however, nowhere has it been suggested that close contact with infected pets in bed increases the infection risk [138, 139] . rodents or rabbits are mainly infected with t. mentagrophytes, while m. canis is primarily found in dogs and cats. infection occurs by direct or indirect contact with infected hair, scales, or materials. infected animals may be asymptomatic carriers without clinical signs. examples are 16% m. canis carriage in a study of european cats [140] , 27% in suspected brazilian cats [141] , and the isolation of t. mentagrophytes dermatophytes from 4% of clinically healthy rabbits and 17% of guinea pigs in dutch pet shops [142] . licking the face of humans by mainly dogs is an expression of their naturally submissive, positive social behavior. the owner is recognized by the dog as the dominant superior in the ranking. in a pack of dogs, submissive dogs lick their dominant counterparts at the corners of the mouth from a typical submissive attitude [143, 144] . owners apparently allow this as a token of affection from their pet. such behavior is more common in young animals and has been considered as attention-seeking or care-soliciting gestures. it indicates to the owner the strength of the social bond between dogs and people [120] . licking or nudging of veterans by service dogs may help take their mind off any negative thoughts, emotions, or memories that they might be experiencing [145] . on the internet, many images can be found of mainly dogs, but also cats and even rats licking their owner's face. various studies show that around 40%-50% of owners allow this [119, 120] . the question is whether this is harmful due to the potential transmission of infections. the review by chomel (2011) shows in the literature that infections, especially pasteurella spp. and capnocytophaga canimorsus, were reported to have transmitted to humans by dogs, cats, kittens, and rabbits [124] . pasteurella multocida meningitis has been reported in 36 infants where 87% had been exposed directly or indirectly to the oropharyngeal secretions of household dogs or cats through licking or sniffing [146] . zoonotic transmission via this route is also assumed for various other pathogens such as gastric helicobacter spp. [147] , periodontal pathogens [148] , and bartonella henselae the etiological agent in cat scratch disease. the b. henselae bacteria may cause ocular complications, including parinaud oculoglandular syndrome, a severe eye infection. the route of infection is unknown, although direct conjunctival inoculation, most likely with infected flea feces, seems to be most plausible [149] . knowing, however, that b. henselae is present in up to 40% of cat saliva [150] , it is more plausible that salivary fluid could be rubbed directly into the eye from the skin after been licked by a cat. there are several anecdotal reports of infections in mainly young children that were transmitted by being licked. one recent example is an 8-month-old baby that presented with fever and preseptal cellulitis with purulent discharge. the causative agent was surprisingly corynebacterium bovis, a bacterium that is normally found in bovine mastitis. it became clear that the dog was frequently allowed to lick the baby's face and was fed on raw meat [151] . there is an ineradicable belief among a large part of the public that the licking of human wounds by dogs can disinfect them and that the saliva thereby has healing properties [152] . in addition, it is regularly reported that a dog's tongue is believed to be sterile. this is of course not the case and the oral flora of a dog comprises hundreds of species (including pathogenic) bacteria, fungi, and viruses [153, 154] . various wound healing saliva components have indeed been demonstrated in human and animal studies [155, 156] . the bactericidal effects of male and female dog saliva facilitate the hygienic function of maternal licking of the mammary and anogenital areas by protecting newborns from fatal coliform enteritis caused by e. coli and neonatal septicemia caused by streptococcus canis. however, the saliva is only slightly, and non-significantly, bactericidal against wound bacteria such as coagulase positive staphylococci and pseudomonas aeruginosa [157] . capnocytophaga canimorsus and pasteurella multocida are common commensals in the oral cavity of dogs, cats, and other species [158, 159] . transmission has been reported after the licking of mucous membranes or open wounds [160] [161] [162] . in patients at high risk, severe wound infections, sepsis, disseminated intravascular coagulation, or death can occur. patients with immunodeficiency, splenectomy, or alcohol dependence are at a particularly increased risk of infection with c. canimorsus [159] . even immunocompetent persons who have been licked by a dog can develop fatal sepsis [163] . a further negative effect of companion animal ownership is, of course, accidents inflicted on humans by these animals. these accidents can involve tripping over a cat or being dragged by an enthusiastic dog, but in literature, most evidence points towards biting and scratching incidents. dog biting and cat scratching incidents can cause physical health problems both at the time of infliction but also afterwards by triggering trauma-related secondary infections. dog biting incident reports are numerous, and numbers vary from country to country. in the uk, 18.7 dog bites per 1000 population per year were reported [164] , while a commission in the netherlands reported in 2008, 150,000 bite accidents per year in a population of 16 million (9 events per 1000 inhabitants) [165] . children are especially vulnerable to dog bites. the majority of dog bites occurred in children 5 years of age or younger (68.0%) and almost all (89.8%) of the dogs were known to the children [166] . recently, a systematic review has been published that analyzed more than 26,000 bites from the literature of the past 30 years about the risk of bites relative to specific breeds of dogs, combining bite incidence with bite severity [167] . the analysis by breed revealed that pit bulls were responsible for the highest percentage of reported bites across all studies (22.5%), followed by mixed breed (21.2%), and german shepherds (17.8%). dog bite incidents can result in medical treatment, hospitalization and even death. in the netherlands it was calculated that from the 150,000 dog bite victims per year, around 50,000 seek medical attention and 230 are hospitalized [165] . between 3% and 30% of dog bites become infected and complications become more severe when infection occurs. more than 100 species of bacteria have been isolated from bacterial infections of dog bites, suggesting that most oral flora of dogs have the potential to be pathogenic [168] . the top 3 pathogens found are pasteurella, staphylococcus, and streptococcus. in literature, specific attention is given to wound infections caused by capnocytophaga canimorsus, because this bacterium is seen as the relatively deadliest pathogen. it was suggested that only 2% of dog bite wounds contained capnocytophaga spp. [154] while others reported infection percentages of 4.2% [169] . wound infection with this bacterium can lead to severe complications like septicaemia, meningitis, osteomyelitis, peritonitis, endocarditis, pneumonia, purulent arthritis, and disseminated intravascular coagulation. c. canimorsus septicaemia has been associated with 30% mortality. the true number of c. canimorsus infections is probably largely underestimated due to the fastidious growth of the organism. however, infected dog bites in predisposed persons should be taken seriously especially after splenectomy [170] . cat bite incidents occur less frequently. in only 5%-10% of reported bite incidents in australia, cats are to blame. the long incisor teeth inflict less severe superficial wounds but because of the penetrating effect, joint and tendon infections more easily occur. in a review it is reported that 28%-80% of cat bites become infected mostly by pasteurella multocida [169] . the bacterium bartonella henselae can also be transmitted by cats through biting incidents but the transmission of this bacterium is much more related to a cat scratch accident. even less frequently reported animal biting incidents are those inflicted by rodents (2% of cases in australia). these bites have an infection rate of approximately 10%. this could result in rat bite fever in humans, an infection with streptobacillus monoliformis or spirillum minus, characterized by the triad of fever, rash, and arthritis [169] . cat scratch disease (csd) was first described in a french boy in 1931 and is a common, often self-limited, disease that usually presents as tender lymphadenopathy caused by bartonella henselae [171] . the cat is considered the primary reservoir for this bacterium, with infected fleas and ticks serving as vectors and humans and dogs as accidental hosts. vector transmission of this bacterium occurs via two primary routes: inoculation of bartonella contaminated arthropod feces via animal scratches, most often cat scratches, or by self-inflicted contamination of wounds induced by the host scratching arthropod bites [172] . immunocompromised human hosts (kidney transplant patients or patients with hiv) are especially susceptible to infection. in these individuals, the disease may be present as a more disseminated form with hepatosplenomegaly, meningoencephalitis, or angiomatosis [173] . an increasing number of pocket pets and exotic pets are kept by humans. numbers of ornamental birds in europe are estimated as 50 million, fish tanks 15.5 million (300 million ornamental fish), small mammals 27 million, and reptiles 8 million [15] . these animal species can also be a source of many zoonotic diseases, especially in young children and immunocompromised individuals. most cases of these conditions are not serious, and deaths are very rare but some of these diseases can be life threatening, such as rabies, rat bite fever infections, and plague [174] . however, there is also a trend to keep more unusual exotic animals, legally or illegally. these are "wild" animals that are kept in the home such as bats, foxes, skunks, raccoons, meerkats, prairie dogs, kinkajous, sloths, monkeys, apes, prosimians (mammals), parrots, mynah birds, finches (birds), crocodiles, turtles, tortoises, lizards, snakes (reptiles), frogs, toads, newts, salamanders (amphibians), fish, eels, rays (fish), crabs, crayfish, snails, insects, spiders, and millipedes (invertebrates) [175] . even fruit bats are kept as pets [176] and it is known that bats harbor a higher proportion of zoonoses than all other mammalian orders, including rabies-like viruses that are highly pathogenic for people. [177] . another example of a zoonosis is monkeypox following the importation of prairie dogs in the usa [178] . most reptiles and amphibians such as turtles, lizards, and frogs carry salmonella bacteria in their gut, in most cases without visible signs of infection. the infection may cause symptoms of sickness, diarrhea and fever in humans [179] . zoonotic transmission of salmonella infections causes an estimated 11% of salmonellosis annually in the united states. in cases involving pet turtles, almost half (45%) of infections occurred in children younger than 5 years [180] . salmonella infections are often transferred by feeder rodents [181] and outbreaks highlight the importance of improving public awareness and education in countries who receive imported reptiles [182] . it is advised to exclude reptiles, amphibians, rodents, exotic species, baby poultry, and raw animal-based pet food items from the households of patients at high risk. in lower risk households, an understanding of the risk of salmonellosis and other pet-associated zoonoses and preventive hygiene measures is needed. the pet trade in general, with its high turnover and diversity of available species, creates a reservoir for pathogens originating from all over the globe. reptiles account for approximately 10% of live animal shipments imported to the united states [182] . importation of live reptiles and amphibians for commercial purposes is for a great part unregulated at eu level except cites and customs regulations [176] . in a risk assessment study, the five pathogens with the highest public health risk caused by the import of exotic animals were salmonella spp., crimean-congo hemorrhagic fever virus, west nile virus, yersinia pestis, and arenaviruses. the risk via legally imported animals was considered low, but substantial for illegally imported animals due to the unknown health status of the animals [183] . avoiding exposure to exotic pet pathogens in the home is difficult and best achieved by not keeping them in the first place. otherwise, it is advised to always wash the hands immediately and thoroughly after contact with exotic pets and after handling raw (including frozen or defrosted) mice, rats and chicks. children should be supervised so that they do not put their mouths close to or kiss exotic animals. reptiles and other animals should be kept out of rooms in which food is prepared and eaten. the number of abandoned and homeless dogs and cats in europe is estimated to be over 100 million. countries with more than 1 million abandoned animals are italy, romania, russia, and ukraine [184] . there is an increasing trend to rescue and import dogs from countries with stray animal problems, in europe often from southern or eastern europe and in the us from puerto rico, the dominican republic, mexico, the middle east, turkey, china, and korea [185] . many charities and independent groups are involved to rescue dogs and seek adoption in more animal-friendly countries [186] . new owners primarily choose to adopt from abroad based on a desire for a particular dog they had seen advertised and on concern for its situation, while some were motivated by previously having been refused dogs from local rescues [187] . in the usa 85% of all household dogs were neutered and today there are no longer enough dogs being born in the usa annually to replace the approximately 8 million dogs that die each year. developing countries have hundreds of millions of street dogs available for export, for example egypt has an estimated 15 million, india, 30 million, and afghanistan, 100 million [188] . in 2006, more than a decade ago, the centers for disease control (cdc) estimated annual dog imports at around 287,000. today the number of imported dogs is estimated to be more than a million a year [189] . imported dogs are reintroducing diseases and parasites that were previously eliminated in the usa [190] . in the usa new and lethal strains of distemper and canine influenza as a result of imported rescue dogs were reported as well as canine brucellosis, rabies, and vector-borne diseases like ehrlichiosis, heartworm, babesiosis, and leishmaniasis [188, 191] . in many southern european countries, there is also a risk of exposure to diseases not encountered in the northern, importing, countries. animals could be infected with anaplasma phagocytophilum, babesia canis, brucella canis, borrelia burgdorferi, dirofilaria immitis (heartworm), dirofilaria repens (subcutaneous worm), echinococcus multilocularis (fox tapeworm), echinococcus granulosus (hydatid or dog tapeworm), ehrlichia canis, hepatozoon canis, leishmania infantum, linguatula serrata (tongue worm), onchocerca lupi, rabies, rickettsia conorii, strongyloides stercoralis, and thelazia callipeda. except b. canis, e. canis, and h. canis, the infections are zoonotic and regularly reported [192] [193] [194] [195] [196] [197] [198] [199] [200] [201] [202] . most of these are transmitted by ticks, sand flies, or mosquitoes that are non-endemic in the receiving countries, but a reservoir of infections has been created and the risk is that vectors will become present as result of climate change. [203] . animals that are infected with rabies or echinococcus spp. may infect people directly. the importation of dogs from endemic, predominantly mediterranean, regions to northern europe, as well as travelling with dogs to these regions carries a significant risk of acquiring an infection. pet owners are therefore advised not to travel with dogs and to seek the advice of their veterinarian prior to importing a dog from an endemic area or travelling to such areas [193] . for this reason, esccap developed maps on their website featuring european countries and regions with advice on endemic parasites, diseases and recommended treatments when travelling with dogs [204] . a three-year european union funded project entitled callisto (companion animal multisectorial interprofessional and interdisciplinary strategic think tank on zoonoses), has investigated zoonotic infectious diseases transmitted between companion animals and humans and food producing animals [176] . the committee advised that special attention should be given to stray cats and dogs. stray dogs, in particular, may pose serious health and welfare problems for humans and animals [81] , including the transmission of zoonotic diseases such as rabies. consideration should be given to controlling companion animal movement between areas of the eu endemic for particular zoonoses and areas that are not currently endemic for that disease. reliable figures are not available, probably because of the fact that many voluntary organizations are responsible for saving and transporting these rescue animals. such data are essential in order to be able to quantify the actual risks of zoonotic diseases attributable to companion animals and to develop sustainable interventions to prevent transmission to humans and livestock [176] . as long as there are no official guidelines, to prevent the spread of (zoonotic) diseases to new countries, the time, expense, disease risk, and the implications of adopting a dog from abroad should all be carefully considered before importation. as an alternative, the conditions for native dogs could be improved by supporting local charities to organize neutering campaigns and rehoming programs, to build local animal shelters and to improve attitudes towards dogs and their living conditions [205] . cats and dogs harbor the enteric nematodes toxocara canis and toxocara cati, and cats are the final host for the protozoal parasite toxoplasma gondii. these parasites can be transmitted to humans because they have an oral-fecal transmission cycle. humans can be infected by ingestion of infective toxocara spp. eggs or toxoplasma oocysts from contaminated soil (gardens, sandpits, and playgrounds) [206, 207] . a recent meta-analysis of data from published records indicates that public places are often heavily contaminated with a pooled global prevalence of toxocara eggs of 21% [208] . both parasites are considered by cdc as part of five neglected parasitic infections. these infections are considered neglected because relatively little attention has been devoted to their surveillance, prevention, and/or treatment. the diseases that they cause have been targeted as priorities for public health action based on the number of people infected, the severity of the illnesses and the ability to prevent and treat them [209] . tens of millions of people worldwide are estimated to be exposed to, or infected with, toxocara spp. and recent findings suggest that the effect of toxocarosis on human health is increasing in some countries. almost one fifth (19%; 1.4 billion individuals) of the world's human population is seropositive to toxocara. the highest seroprevalence rates were found in africa (mean: 37.7%) and the lowest in the eastern mediterranean region (mean: 8.2%) [210] . toxocara larvae migrate into the body of the human to several organ systems with a preference for the central nervous system (brain, eyes). human toxocarosis can manifest itself as syndromes known as visceral larva migrans, ocular larva migrans, neurotoxocarosis, and covert or common toxocarosis [211] . asthma is one of the most common chronic respiratory diseases worldwide, with a negative impact on the quality of life and socio-economic status of patients. two decades ago, the first evidence was published that suggested that toxocara infection is a neglected risk factor for childhood asthma [212] . the finding that children infected with toxocara spp. are more likely to have asthma compared to non-infected children was recently confirmed in a systematic review and meta-analysis [213] . cognitive or developmental delays in children or young adults who become infected is of particular concern. toxocarosis appears to be associated with decreased cognitive function [214, 215] . the annual toxoplasma oocyst burden measured in community surveys has been reported as up to 434 oocysts per square foot (4670 per square meter) and is greater in areas with loose soil, that cats like to use to cover their feces in gardens, children's play areas, and especially sandboxes, also called sandpits and sand piles [207] . because a single oocyst can possibly cause infection, this oocyst burden represents a major potential public health problem. an estimated one third of the world's population harbor anti-toxoplasma antibodies. due to keeping pigs indoors, more education and awareness, the prevalence of the disease in the usa and europe declined by 50% over the last decades [216] . during an acute invasion of toxoplasma parasites there is mild to major tissue damage without clinical symptoms (latent toxoplasmosis). the most important form is congenital toxoplasmosis when a woman receives her first exposure to toxoplasma during pregnancy. in early pregnancy, this can lead to abortion or to malformations that are not compatible with life shortly after birth. congenital infections may also be characterized by mental retardation and ocular defects. acquired infection after birth may result in clinical symptoms such as lymphadenitis, fever, and malaise and probably leads to a clinically symptomatic disease state more frequently than the congenital condition, with an estimated incidence of 30% of all ocular toxoplasmosis cases [216] . playing in a sandbox is also found to be a predominant risk factor for s. typhimurium salmonellosis in children aged 4-12 years [217] . this can be the result of fecal contamination of the sand by dogs and cats that have been fed raw meat (see section 5.1.1.). young children are especially at risk as they put their hands or other objects in their mouths every 2-3 min [208] . it has also been reported that children ingest a median of 40 mg of soil per day and that one child consumed 5-8 g of soil per day on average [218] . it is therefore advisable not to let children play in public places or on playgrounds with loose sand, but only in sandboxes that can be covered. furthermore, washing hands after playing outside is important and fingernails must be trimmed to prevent sand being left behind. in this context, a strange trend can be observed as young children play in mud baths on 29 june "as a way to connect and celebrate the natural joys of playing in the mud". this international mud day originated in 2008 and was initiated by an australian pedagogue who had observed this phenomenon during a visit to nepal [219] . if the origin of the soil needed for producing the mud is unknown, there is of course an infection risk for the above discussed parasites. to prevent the transmission of zoonotic diseases from pets, risk analysis is of great value. this starts with an assessment of the potential zoonoses in an area, depending on the endemicity (the hazard, h). hazard characterization also includes prevalence in animals (the reservoir), virulence for man, transmission routes, and survival of the agent in the environment. the second step is exposure assessment (e). who is exposed to the potential hazard and for how long or how often? how much of the potential pathogen is needed to become a health risk? this inevitably is directly related to human behavior in relation to their pets. the third step is to assess the impact of getting infected (i). how serious is the disease, what is the chance of complications, and what economic consequences may be expected (e.g., labor hours lost)? each of the parameters can be ranked in classes from negligible to the most serious possibility. ranking is based on literature data, own observations (measuring), or experts' opinions. the final risk assessment can be achieved by multiplying the outcome of hazard characterization, exposure assessment and impact (h × e × i = a number). the outcome can be compared with other zoonotic agents and a ranking of significance can be made [220] . there is one important parameter to reduce the risk of contracting a zoonotic infection that can directly be influenced, which is exposure. recommendations are particularly targeted to households with very young children, the elderly, pregnant women, or immunocompromised patients. they are based on reducing exposure to hazards and involve four categories of advice (table 1) . table 1 . recommendations to prevent the transmission of zoonotic pathogens from pets [121, 221] . • washing the hands thoroughly -after animal contact, at least before eating and drinking and before preparing food or drinks -after handling raw pet food -after handling pet habitats or equipment -after cleaning up feces -after removing soiled clothes or shoes reflecting on the changed human-companion animal bond, it can be concluded that pets undoubtedly have a positive effect on human health. conversely, the human-pet bond seen nowadays is facing many challenges, putting pet welfare under more pressure due to issues such as anthropomorphism, which mainly results in obesity, breeding on extreme appearance rather than health, behavioral problems connected to unfulfilled species specific mental and physical needs, and the provision of inadequate food because owners mistakenly think they feed more naturally. with regard to the negative effects of pets this article attempts to give an impression of increasing trends in the human-companion relationship that can be observed in society, which appears to increase the risk of transmission of infection between pets and humans. it is mainly a consequence of the increasing contacts between humans and pets and with pathogens secreted by animals in the shared environment. more than 6 out of every 10 known infectious diseases in people can be spread from animals, and 3 out of every 4 new or emerging infectious diseases in people come from animals [222] . the recent pandemic of the covid19 coronavirus (sars-cov-2), that may be originated from bats, is a good example of a recent emerging zoonotic infectious disease. a few cats and dogs have tested positive but are not considered as a source of infection for people. the proportion of zoonotic human disease that is attributable to pets is largely unknown. reports about the frequency of such infections are likely underestimated [120] ; however, the risk of infection is relatively small for many zoonoses and the severity of the disease is often limited. a person's age and health status may affect their immune system, and thereby increase his or her chances of getting a disease from animals. pregnant women should avoid contact with pet rodents, reptiles, cat feces, and raw meat to prevent infection of the unborn child, abortion, or birth defects. if symptoms occur in immunocompetent, non-pregnant persons between 5 and 64 years of age, these are mainly of a general nature such as diarrhea or flu-like symptoms. physicians do not regularly ask about the presence of pets or pet contact, nor do they discuss the risks of zoonotic diseases with patients, regardless of the patient's immune status, which means that many cases of zoonotic diseases go undiagnosed. the general public and people at high risk of pet-associated disease are not aware of the risks associated with high risk pet practices or recommendations to reduce them. since unfamiliarity with hazards reinforces fear, communication plays an important role in this. veterinarians play a key role in education regarding risk reduction by giving advice about responsible pet ownership and the required preventive hygiene. healthcare providers such as family doctors, school doctors, and pediatricians can also provide information about safe pet ownership. physicians should ask as part of the medical history about eventual contact with pets, particularly with patients at high risk [120] . the "one health" initiative aims to reduce this professional gap between vets and physicians [2] . when giving recommendations to prevent zoonotic transmission, one of the often-made remarks is that people nowadays are already too hygienic. it is assumed that there is a protective influence of postnatal infection and that it might be lost in the presence of modern hygiene. this belief is based on the hygiene hypothesis that was formulated in 1989 [223] . it was observed that hay fever in young adults was inversely related to the number of siblings in their family. this hypothesis focused exclusively on allergic conditions as a result of the modern way of life and the assumption that modern hygiene was reducing contact with bacteria. another view is that some chronic inflammatory disorders have increased over the last decades as a result of decreased frequency of infections due to pathogenic organisms [224] . another related theory is the "old friends" mechanism that is based on the positive influence of gut parasites, non-pathogenic environmental bacteria (saprophytes, pseudocommensals), and gut commensals or microbiota. however, decreased exposure to these microorganisms is not the only reason for the increasing frequency of allergies and chronic inflammatory disorders in industrialized countries. nowadays there is more information about the immunological roles of skin, oral mucosa, and gut microbiota as well as helminths and the influence these have on the immune system [225] . gut flora may be modified due to diet, obesity, hygiene, antibiotics, but also to psychological stress, vitamin d deficiency, and pollution [225] [226] [227] . pollution also has a significant effect on the development of several respiratory problems and diseases. not only due to outdoor pollution such as fine dust, harmful solids, liquids, or gases [228] , but also due to indoor molds as result of insufficient ventilation in energy efficient homes [229] . finally, there is a clear increase in the allergen production of house dust mites and pollen leading to more exposure and sensitization in susceptible individuals [230] . all together it must be clear that there is much more known about other causes of increasing allergies worldwide than simply excessive cleanliness as suggested in the hygiene hypothesis. regarding field infections with helminths such as trichuris trichiura in early life, these are associated with a reduced prevalence of allergies later in life and infants of helminth-infected mothers have a reduced prevalence of eczema. hookworm infections in developing countries are associated with a reduced prevalence of asthma [231] . the rate of eczema in such countries was found to be about five times higher in infants whose mothers had never had helminths compared with persistent helminth-infected mothers [232, 233] . helminths are nowadays even used under controlled conditions to stimulate immunity. examples are trichuris suis therapy for crohn's disease and necator americanus larvae to treat crohn's disease and other autoimmune disorders [234] . there are no clinically apparent childhood infections found to be associated with protection from allergic disorders [235] . it can even result in an opposite effect in the case of toxocara infection by children after soil contact. a recent meta-analysis showed that children infected with toxocara spp. are more likely to have asthma compared to non-infected children [213] . this parasite and asthma both have elevated immunoglobulin e (ige) levels and eosinophilia in common. that means that precautions should be taken in children to prevent soil contact not only to prevent toxocara infection, but also to prevent acquired ocular toxoplasmosis. there is no need, therefore, to stimulate the contraction of pathogenic bacteria or helminths to achieve a healthy gut microbiota and to reduce allergic conditions. recommendations based on the hygiene hypothesis should preferably be based on results from controlled studies to prevent unintentional negative effects. it is both humans and companion animals who experience negative effects of a changed human-companion animal bond. the education given by vets to their clients should therefore also focus on preventing these negative health and behavioral effects. for instance, by giving science-based advice on feeding practices. in general, regulating authorities should encourage the development of enforcement criteria for breeding dogs and cats to reduce health and welfare risks. pets undoubtedly have a positive effect on human health and well-being, while owners are increasingly aware of pet health, welfare, and well-being. anthropomorphism, also resulting in behavioral problems and breeding on appearance rather than health, and trends such as keeping exotic animals and importing rescue dogs may result in an increased risk of contracting zoonotic infections. recommendations regarding responsible pet ownership, including normal hygienic practices, responsible breeding, feeding, housing, and mental and physical challenges conform the biology of the animal, are key in preventing such negative aspects of the human-animal bond. there is no need to stimulate unhygienic practices following the hygiene hypothesis. education can be performed by vets and physicians as 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friends' hypothesis metabolic and metagenomic outcomes from early-life pulsed antibiotic treatment current understanding of the human microbiome environmental and health impacts of air pollution: a review. front. public health 2020, 8, 14 abridged version of the awmf guideline for the medical clinical diagnostics of indoor mould exposure house dust mite allergy under changing environments interactions between helminth parasites and allergy the impact of helminths on the response to immunization and on the incidence of infection and disease in childhood in uganda: design of a randomized, double-blind, placebo-controlled, factorial trial of deworming interventions delivered in pregnancy and early childhood immunologic profiles of persons recruited for a randomized, placebo-controlled clinical trial of hookworm infection an update on the use of helminths to treat crohn's and other autoimmune diseases infections presenting for clinical care in early life and later risk of hay fever in two uk birth cohorts this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord-316513-dbzj101e authors: sen-crowe, brendon; mckenney, mark; elkbuli, adel title: utilizing technology as a method of contact tracing and surveillance to minimize the risk of contracting covid-19 infection date: 2020-07-04 journal: am j emerg med doi: 10.1016/j.ajem.2020.07.003 sha: doc_id: 316513 cord_uid: dbzj101e nan j o u r n a l p r e -p r o o f currently, there are two major forms of testing in the u.s.: testing for sars-cov-2 rna and serologic testing. 1, 2 however, only testing those experiencing symptoms is not a practical way of obtaining a true picture of the infection status of the nation, as the majority of infected individuals do not exhibit symptoms. 3 healthcare personnel who are in close contact with ill patients may be asymptomatic themselves and unknowingly transmit the infection to others. a call for new methods of testing and surveillance on a large scale will be important if we hope to control the spread of sars-cov-2 infections. fortunately, advancements in technology allow diagnostics and surveillance to be readily available. one innovative approach that has gained popularity is the use of a smart ring. on example is the oura ring (ooura health ltd.'s, oulu, finland) which can detect physiologic changes and alert the possibility of infection. understanding changes in vital signs such as body temperature, respiratory and heart rate may enable early detection of signs of infection. for example, one study at west virginia univeristy, rockefeller neuroscience institute predicted symptoms 24 hours prior to onset based on physiologic changes detected by the oura ring, and aim to achieve a 3-day forecast in the future. 4 implementation of preventative measures before symptoms are apparent when there may be a risk of viral shedding may translate into a much larger benefit than we anticipate and has the potential to reduce infection in other healthcare workers and patients. in addition, early detection and contact tracing has the potential to conserve hospital resources that have become scarce throughout the pandemic. for example, there has been a shortage of ventilators. 5 areas that have been flagged as high-risk due to surveillance can be supplied with additional hospital resources to match their case load. hence, targeted distribution of hospital resources to these areas can make the difference for a hospital to adequately treat those in critical condition without the need for triaging treatment between patients. testing for covid-19 cdc diagnostic test for covid-19 transcript for the cdc telebriefing update on covid-19 wvu rockefeller neuroscience institute and oura health unveil study to predict the outbreak of covid-19 in healthcare professionals ventilator stockpiling and availability in the us. johns hopkins center for health security australian government -department of health key: cord-325613-oamw57gx authors: zhong, peipei; zhang, hailin; chen, xiaofang; lv, fangfang title: clinical characteristics of the lower respiratory tract infection caused by a single infection or coinfection of the human parainfluenza virus in children date: 2019-05-29 journal: j med virol doi: 10.1002/jmv.25499 sha: doc_id: 325613 cord_uid: oamw57gx background: human parainfluenza virus (hpiv), usually combined with other pathogens, causes lower respiratory tract infection (lrti) in children. however, clinical characteristics of hpiv coinfection with other pathogens were unclear. this study aimed to investigate the viral and atypical bacterial etiology of lrti in children and compare the clinical characteristics of hpiv single infection with those of coinfection. methods: this study included 1335 patients, aged between 1 to 71 months, diagnosed with lrti in yuying children's hospital, zhejiang, china, from december 2013 to june 2015. nasopharyngeal secretions were collected, and respiratory pathogens were detected using multiplex polymerase chain reaction. the clinical data of patients were collected and analyzed. results: at least 1 pathogen was detected in 1181/1335 (88.5%) patients. the pathogens identified most frequently were respiratory syncytial virus, human rhinovirus, hpiv, adenovirus, and human metapneumovirus. the coinfection rate was 24.8%. hpiv coinfection with other viruses was more associated with running nose, shortness of breath, and oxygen support compared with hpiv single infection. moreover, hpiv coinfection with atypical bacteria was more related to running nose, moist rales, and longer hospital duration compared with hpiv single infection, and also to longer hospital duration compared with coinfection with other viruses. conclusions: this study demonstrated that viral infections were highly associated with lrti and the rate of coinfection was high. hpiv single infection was milder than coinfection with other viruses. moreover, hpiv coinfection with atypical bacteria was more serious than hpiv single infection and coinfection with other viruses. commercialized multiplex pcr kits have been developed, allowing the simultaneous detection of a large panel of viruses and atypical bacteria in clinical practice. 3 several studies used this molecular diagnostic tool to show that viral infections accounted for a large proportion of lrtis. in addition, mixed infections were identified frequently. [4] [5] [6] several studies discussed the clinical characteristics of coinfection compared with single virus infection, but most of them analyzed on a general basis or focused solely on respiratory syncytial virus (rsv). [5] [6] [7] as one of the most common pathogens of lrti, human parainfluenza virus (hpiv) is found worldwide, especially in children aged less than 5 years. 8 hpiv can cause severe respiratory infection and accounts for 9 to 30% inpatient children admitted due to acute respiratory infection. 9 an increasing number of recent studies focused on hpiv, especially the epidemiology and presentation of four types of hpiv. 10 however, a few studies discussed coinfections with hpiv. therefore, this study aimed to investigate the viral and hpiv-positive patients were divided into three groups based on the test results: hpiv single infection, coinfection with other viruses, and coinfection with atypical bacteria. the diagnosis of lrti for each patient was performed by at least two attending physicians based on zhu futang practice of pediatrics, 8th edition. 11 it was defined according to the clinical symptoms, including severe cough, fever, tachypnea, wheezing, and respiratory distress signs such as nasal flaring, retraction, cyanosis, and abnormal auscultatory findings (wheezing and crackles), or radiologic evidence indicative of an lrti. clinical syndromes of bronchitis, bronchiolitis, and pneumonia were included in the lrti category. bronchitis was diagnosed based on the clinical manifestations including severe cough with or without fever, symmetrical breath sounds without permanent rales on auscultation, and increased bronchovascular shadows in chest x-ray examination. bronchiolitis was recognized in patients aged <24 months with lower respiratory symptoms of wheezing, tachypnea, and signs of respiratory distress such as nasal flaring, intercostal/subcostal retractions, and central cyanosis. the diagnosis of pneumonia was established based on clinical findings, including fever, tachypnea, and respiratory distress, with the presence of focal or diffuse crackles, decreased vesicular sounds, and radiographic findings such as patchy and macular shadows and/or atelectasis, and/or air bronchograms. the pertussis-like cough was defined based on clinical signs including spasmodic cough, inspiratory whoop, and posttussive vomiting. data included demographic information, subjective symptoms, physical examination findings, hospital course and management, radiographic findings, and laboratory results. the data were analyzed using spss (version 17.0; spss, inc., il). they were expressed as mean, standard deviation, median, quartile, frequency, and percentage. continuous variables with a normal distribution were compared using analysis of variance, whereas other variables were compared using the mann-whitney u test. the categorical data were evaluated using the χ 2 and fisherʼs exact tests. a p value less than .05 was considered statistically significant (two-tailed). the study was submitted to the local ethics committee for approval. oral information was given together with a paper explaining the content of the study. a consent form was signed by a parent or legal guardian before the inclusion of each patient in the study. the flow of the study is depicted in figure 1 table 1 ). the total coinfection rate was 24.8%. hcov showed the highest coinfection rate of 65.0%, followed by infb (63.9%), hbov (59.3%), adv (56.5%), and hrv (51.7%). table 3 ). the clinical characteristics of hpiv-positive patients were compared (table 4 ). the most common diagnosis was pneumonia, followed by bronchiolitis and bronchitis. a few patients (5.4-12.5%) in each group had a pertussis-like cough. hpiv coinfection with other viruses was more associated with running nose and shortness of breath (χ 2 = 5.235; p = 0.022; χ 2 = 7.87; p = 0.005), and more patients needed oxygen support (χ 2 = 6.539; p = 0.011) compared with hpiv single infection. neutrophil percentage was higher in coinfection with viruses than in hpiv single infection (χ 2 = 5.744; p = 0.017). moreover, hpiv coinfection with atypical bacteria was more related to running nose (χ 2 = 6.511; p = 0.011), moist rales (χ 2 = 5.167; p = 0.023), and longer hospital duration (χ 2 = 5.904; p = 0.015) compared with hpiv single infection, and also to longer hospital duration compared with coinfection with other viruses (χ 2 = 4.847; p = 0.028). this study revealed a high coinfection detection rate of 24.8%. the previously reported rate was 18 to 65% in patients with ari. 4, 5, 17, 19 it appears that coinfections are related to the prolonged period of viral persistence in the mucosa of the respiratory tract. 20 the large difference in the coinfection rate is probably due to the age and severity of patients enrolled. infants and toddlers have an extremely high rate of virus coinfection compared with older children and adults. 19 singleton et al 21 coinfection with virus took the major part, of which hrv, rsv, and adv were the most frequently detected agents. the fastest growing virus hrv was most commonly found in combination with hpiv, possibly due to the same age and seasonal distribution and the specific characteristics of the two viruses. 18, 22 children aged less than 3 years had a higher positive rate of hpiv compared with children older than 3 years, indicating that young children are vulnerable to respiratory infection. [15] [16] [17] only eight patients were coinfected with hpiv and atypical bacteria in the present study. one hypothesis to explain the relative paucity of the codetections is that infections with these pathogens exhibit different age and seasonal distributions. several studies focused on the association between the severity of illness and coinfection, but no consensus has been reached. some studies showed that viral coinfection did not increase severity, 17, 23 some studies indicated that virus single infection increased the risk of severe situations, 24 laboratory examination pct＞0.5 ng/ml 6 (9.7) 3 (6.7) 0 (0) the unit for peak and bottom of wbc was 10 9 /l; the normal value was 4-12 × 10 9 /l. d the unit for crp was mg/l; the normal value was 0-8 mg/l. e leukocytosis was defined as wbc more than 12 × 10 9 /l. f crp increase was defined as crp more than 8 mg/l. g leukopenia was defined as wbc less than 4 × 10 9 /l. the quantitative data were expressed as median(quartile). the count data were expressed as numbers (%) of each group. global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the global burden of disease study xtag rvp assay: analytical and clinical performance comparative evaluation of six commercialized multiplex pcr kits for the diagnosis of respiratory infections epidemiology and microbiological investigations of community-acquired pneumonia in children admitted at the emergency department of a university hospital etiology and clinical characteristics of single and multiple respiratory virus infections diagnosed in croatian children in two respiratory seasons viral coinfection in childhood respiratory tract infections the burden of single virus and viral coinfections on severe lower respiratory tract infections among preterm infants: a prospective birth cohort study in brazil human parainfluenza virus infection in severe acute respiratory infection cases in beijing, 2014-2016: a molecular epidemiological study. influenza other respir viruses the role of human parainfluenza virus infections in the immunopathology of the respiratory tract epidemiology and clinical presentation of the four human parainfluenza virus types zhu futang practice of pediatrics an advanced fragment analysisbased individualized subtype classification of pediatric acute lymphoblastic leukemia impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses. influenza other respir viruses evaluation of epidemiological and clinical features of influenza and other respiratory viruses clinical evaluation of viral acute respiratory tract infections in children presenting to the emergency department of a tertiary referral hospital in the netherlands prevalence of respiratory viruses among children hospitalized from respiratory infections in shenzhen, china virological and clinical characterizations of respiratory infections in hospitalized children epidemiology and clinical characteristics of acute respiratory tract infections among hospitalized infants and young children in severe lower respiratory tract infection in infants and toddlers from a non-affluent population: viral etiology and co-detection as risk factors human bocaviruses: possible etiologic role in respiratory infection viral respiratory infections in hospitalized and community control children in alaska coinfections of the respiratory tract: viral competition for resources respiratory viral coinfection and disease severity in children: a systematic review and metaanalysis multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children. influenza other respir viruses the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis single, dual and multiple respiratory virus infections andrisk of hospitalization and mortality viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection interference between the influenza viruses: i. the effect of active virus upon the multiplication of influenza viruses in the chick embryo in vitro and ex vivo analyses of coinfections with swine influenza and porcine reproductive and respiratory syndrome viruses modeling inoculum dose dependent patterns of acute virus infections clinical and epidemiological characteristics of human parainfluenza virus infections of children in southern taiwan epidemiology and clinical presentation of parainfluenza type 4 in children: a 3-year comparative study to parainfluenza types 1-3 human parainfluenza virus types 1-4 in hospitalized children with acute lower respiratory infections in china etiologic spectrum and occurrence of coinfections in children hospitalized with community-acquired pneumonia clinical manifestations in infants and children with mycoplasma pneumoniae infection clinical characteristics of the lower respiratory tract infection caused by a single infection or coinfection of the human parainfluenza virus in children the authors thank the colleagues at the department of childrenʼs hospital, for their support during the study. zhejiang province (2015c37026). the authors declare that there are no conflict of interest. http://orcid.org/0000-0003-0531-4917 key: cord-326961-ti6mrzxf authors: aly, mariam mohsen; elchaghaby, marwa aly title: impact of novel coronavirus disease (covid-19) on egyptian dentists’ fear and dental practice (a cross-sectional survey) date: 2020-10-12 journal: bdj open doi: 10.1038/s41405-020-00047-0 sha: doc_id: 326961 cord_uid: ti6mrzxf objectives: this study aimed to evaluate the fear of infection among egyptian dentists practicing during the current coronavirus disease 2019 (covid-19) pandemic and to explore the dentist’s knowledge about guidelines to fight the virus and to assess various modifications in dental practice. methods: an online survey was submitted to dental professionals. data were collected through a validated questionnaire consisting of 23 closed-ended questions. the gathered data were statistically analyzed. results: an overall 216 dentists completed the survey. a total of 200 (92.6%) dental professionals were afraid of becoming infected with covid-19 while 196 (90.7%) became anxious to treat patients showing suspicious symptoms. the majority of the participants were aware of the mode of transmission of covid-19 and a lot of them were updated with the current disease control and prevention (cdc) or world health organization (who) guidelines for cross-infection control. conclusions: covid-19 pandemic has a significant impact on dental professionals. in december 2019, patients with pneumonia of unknown cause were detected in the city of wuhan and within a couple of weeks, it reached other parts of the world, which generated a major public health crisis worldwide and imposed a challenge to healthcare systems across the six continents. 1, 2 the world health organization (who) named the chinese outbreak covid-19 and declared it to be a public health emergency of international concern posing a high risk to countries with vulnerable health systems. 3, 4 the spread of coronavirus (covid-19) has posed significant challenges for dentistry and medicine, and dental and medical schools, in all affected countries. 5 the reaction speed and response type to this disease have been very variable around the world according to different healthcare systems, economies, and political views. 6 due to the nature of dental settings, the risk of cross-infection may be huge between dental practitioners and patients through direct transmission by a sneeze, cough, or droplet inhalation, or contact transmission such as ocular contact or through mucous membranes of the eyes and nose and saliva. 3 for dental clinics and hospitals in regions and countries that are potentially influenced by covid-19, firm, and effective infection control protocols are crucially required. this is a result of the unique attributes of dental procedures where an enormous number of droplets and aerosols could be generated, the usual protective measures in daily clinical practice are not effective enough to counter the spread of covid-19, especially when patients are in the incubation period and are unaware that they are infected or prefer to conceal their infection. 7 different practical guidelines were recommended for dental professionals by the centers for disease control and prevention (cdc), the american dental association (ada), and the who to control the spread of covid-19 and like other contagious infections, these recommendations include personal protective equipment, hand washing, detailed patient evaluation, rubber dam isolation, anti-retraction handpiece, mouth rinsing before dental procedures, and disinfection of the clinic. 8 in a pandemic, fear raises anxiety and stress levels in healthy persons and escalates the symptoms. the number of persons whose mental health is affected tends to be greater than the number of persons affected by infection. 9 fear and anxiety are powerful emotions that may be associated with the overwhelming reports on the pandemic by social, electronic, and print media, the absence of wellstructured scientific evidence and knowledge about covid-19, time-consuming screening and diagnostic methods, insufficient personal protection equipment, unclear treatment, and immunization. 5 mild anxiety is natural and fosters preventive and safeguarding behavior. although the ada has published preventive guidelines, the majority of dentists are still reluctant and fearful of treating patients in such a situation. 10 before effective approaches to reinforce dental professionals can be developed, it is crucial to recognize their specific sources of anxiety and fear. focusing on addressing those concerns, rather than teaching general approaches to reduce stresses, should be the main focus of support efforts. 11 this study aimed to evaluate the fear of infection among egyptian dentists practicing during the current covid-19 pandemic and to explore the dentist's knowledge about guidelines to fight the virus and to assess various modifications in dental practice. the present study was a cross-sectional study of a random sample of egyptian dental professionals. the strobe guidelines were used to ensure the reporting of this observational study. data were collected via an online survey link and received a response through an online survey submission. any egyptian dentist having a bachelor of dentistry or equivalent degree was invited to participate while dental students were not included in this survey. trial registration this study has been registered with clinicaltrials.gov under the title: fear and practice modification among dentists during covid-19 pandemic with an identifier: nct04383626. to assess the prevalence of fear of becoming infected, the sample size was calculated (http://www. nss. gov. au/ nss/ home.nsf/ pages/ sample+ size+ calculator) using the following assumptions: confidence level = 95% ci = 5%, the total number of dentists with an estimated percentage of fear of becoming infected = 87% according to ahmed et al. 10 the number was increased by 25% to allow for nonresponse. the required sample size was 216. this online survey tried to evaluate: data sources and measurement data were gathered using an english language self-administrated questionnaire, which developed based on a similar study by ahmed et al. 10 after taking their permission. on the first page of the questionnaire, the participants were briefed about the purpose of the study and the voluntary and confidential nature of their participation. informed consent was obtained from each participant in the form of answering a question (yes/ no) before proceeding with answering the questionnaire. the questionnaire design consisted of 23 close-ended, multiplechoice questions (yes/no/unaware or yes/no/don't know) in three different sections. the first section of the questionnaire with four questions was related to the demographic data of the participants. the second section with 7 questions focused on the dentists' fear of becoming infected with covid-19 and the third section with 12 questions was designed to gather information regarding their knowledge about guidelines to fight the virus and to assess various modifications in dental practice. to limit selection bias, all dental professionals who participated in this study were randomly selected and invited to respond to a self-administered anonymous questionnaire. to limit information bias, all participants were offered the same explanation regarding the nature and the aim of the study. statistical methods collected data were analyzed by ibm-spss (version 22.0) software package for microsoft windows. descriptive statistics were calculated as frequencies and percentages. a total of 216 dental professionals completed the questionnaire. most of them were general dental practitioners 113 (52.3%), below the age of 40 years with no sex predilection. approximately about 146 (67.6%) of dentists enrolled were working in private clinics as illustrated in table 1 . fear of dental personnel during the covid-19 outbreak was assessed using the questionnaire as shown in table 2 information regarding the knowledge of dental personnel about guidelines to fight the virus and various modifications in dental practice were also obtained using the questionnaire as illustrated in table 3 . most of the respondents 203 (94.0%) were aware of the mode of transmission of covid-19 but only 156 (72.2%) were updated with the current cdc or who guidelines for cross-infection control. unfortunately, only 55 (25.5%) of dental personnel took the patient's body temperature, 103 (47.7%) requested a patient's travel history before performing any dental treatment, and only 154 (71.3%) deferred dental treatment of patients showing suspicious symptoms. regarding the use of personal protection, 180 (83.3%) of dentists believed that the surgical mask wasn't enough to prevent cross-infection of covid-19, and 175 (81.0%) believed that n-95 mask should be routinely worn. although the majority of dentists 183 (84.7%) followed the universal precautions of infection control routinely, only 53 (24.5%) of the respondents reported the use of rubber dam isolation and 104 (48.1%) reported the use of high-volume suction. one hundred and ninety-nine (92.1%) of participants practiced washing hands with soap and water or sanitizer before and after treatment of patients, while 134 (62.0%) of participants were aware of the proper authority to contact if they came across a patient with a suspected covid-19 infection. this cross-sectional study assessed the fear of infection between egyptian dentists practicing during the present covid-19 pandemic and to explore their knowledge about guidelines to fight the virus and various modifications in dental practice through an online survey. data were gathered using a questionnaire based on the previous study of ahmed et al. 10 that was validated through intra-class correlation with a strong relation of 0.74. the online survey method is considered a useful tool for collecting data quickly with many benefits including saving time and money, ease of use, the capability to prohibit errors when entering and editing data, and rapid transmission of survey results. 12 in a pandemic, fear, anxiety, and stress levels increase. correspondingly, the rate of distress among healthcare staff is higher compared with the general population, because they are more at risk for infection. 9, 13 this is a result of the unique attributes of dental procedures including the proximity to the patients, the enormous number of droplets and aerosols generated during dental procedures, 1 as the main route for transmission of coronavirus is through droplets and aerosols, the likelihood of dental professionals becoming infected and further spreading the virus is elevated. 10 as a considerable viral load was consistently detected in the saliva of patients diagnosed with the infection by up to 91.7%, 1 a high percentage of dentists reported their fear of becoming infected with covid-19 from a patient or a co-worker in this study. as usual protective measures in daily clinical practice are not effective enough to counter the spread of covid-19, especially when patients are in the incubation period and are unaware that they are infected or prefer to conceal their infection, 7 the majority of dentists stated being anxious when providing treatment to a patient who is coughing or showing suspicious symptoms. these findings triggered a large number of participated dentists to close their dental practice until the number of covid-19 cases starts declining. while dental professionals often accept the elevated risk of infection, as part of their chosen profession, they usually show concern about transmitting the infection to their families, especially if family members are elderly, immunocompromised, or possess a chronic medical condition. almost all participants in the study feared for their families as they battle covid-19, which can be linked to the fact that about 3000 healthcare providers in china became infected and transmitted the infection to family members. despite the admission that transmission occurs mostly via symptomatic individuals, there are reports of asymptomatic individuals who transmitted the disease to multiple family members. 14, 15 these results were in accordance with the finding of ahmed et al., 10 who reported that a large number of dentists fear becoming infected by their patients or co-workers and are also fearful of providing treatment to any patient reporting suspicious symptoms. the majority of the respondents were aware of the mode of transmission of covid-19 and many of them were updated with the current cdc or who guidelines for cross-infection control. however, a great number of participants reported not using rubber dam isolation or high-volume suction for every patient in their practice. concerning the mode of transmission of covid-19, the utilization of rubber dams can dramatically diminish the production of saliva-and blood-contaminated aerosol or spatter, especially when dental ultrasonic devices and high-speed handpieces are used. also, it could significantly decrease airborne particles in the~3-foot diameter of the operational area by 70%. the use of high-volume suction is also considered a crucial means to limit aerosols evacuation during dental treatment and should be used for most of the patients. considering the benefits, there is no excuse for not using a rubber dam during dental procedures, especially while using rotary instruments. 16, 17 many of the participants considered that the surgical mask wasn't enough to counter the cross-infection of covid-19 and that the n-95 mask should be routinely worn in dental practice during the current outbreak. it can be explained as surgical masks protect the oral and the nasal mucous membranes from droplet spatter, but they do not provide complete protection against the inhalation of airborne infectious agents unlike n-95 masks. 5, 16 hand hygiene was performed by most of the dentists. current evidence indicates that the covid-19 virus is transmitted through respiratory droplets or contact. contact transmission appeared directly when a contaminated hand contacted the mouth, the nose, or the eye; the virus can also be transmitted indirectly from one surface to another by contaminated hands. therefore, hand hygiene is very important to prevent the spread of the covid-19 virus. 18, 19 conversations with front-line caregivers may help reduce anxiety. the focus should be on supportive communication, clear guidance when recommendations exist attempts to minimize misinformation, and efforts to reduce anxiety. transparent and thoughtful communication could contribute to trust and a sense of control. ensuring that workers feel they get adequate rest, can tend to critical personal needs and are supported both as healthcare professionals and as individuals will help maintain individual and team performance over the long run. 14, 18 limitation of the study this research was subjected to some limitations: • time constraints: the rapid and extensive nature of the new coronavirus may intensify the respondent's responses and feelings. also, a change in their behavior might occur. • internet access: this study was conducted online so it was limited to participants who had internet access. conclusions covid-19 pandemic has a significant impact on dental professionals. this is reflected in the high percentage of dental professionals experiencing fear and anxiety of getting infected while providing dental care. some dental professionals often accepted the elevated risk of infection as part of their chosen profession and introduced several modifications in their dental practices in compliance with the recommended guidelines. while others want to close down their dental practice for a period of time until the number of covid-19 cases starts declining. the majority of dental professionals were knowledgeable about the mode of transmission of covid-19 and many of them were updated with the current cdc or who guidelines for cross-infection control. covid-19 pandemic and role of human saliva as a testing biofluid in point-of-care technology an epidemiological study on covid-19: a rapidly spreading disease human saliva: non-invasive fluid for detecting novel coronavirus (2019-ncov) world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) common misconceptions regarding covid-19 among health care professionals: an online global cross-sectional survey dentistry and coronavirus (covid-19) -moral decision-making coronavirus disease 2019 (covid-19): emerging and future challenges for dental and oral medicine dentists' awareness, perception, and attitude regarding covid-19 and infection control: cross-sectional study among jordanian dentists pandemic fear" and covid-19: mental health burden and strategies fear and practice modifications among dentists to combat novel coronavirus disease (covid-19) outbreak understanding and addressing sources of anxiety among health care professionals during the covid-19 pandemic online survey software as a data collection tool for medical education: a case study on lesson plan assessment covid-19 and anxiety: a review of psychological impacts of infectious disease outbreaks supporting the health care workforce during the covid-19 global epidemic presumed asymptomatic carrier transmission of covid-19 interim infection prevention and control recommendations for healthcare personnel during the coronavirus disease 2019 (covid-19) pandemic possible aerosol transmission of covid-19 and special precautions in dentistry knowledge and attitude of dental practitioners related to disinfection during the covid-19 pandemic world health organization. recommendations to member states to improve hand hygiene practices to help prevent the transmission of the covid-19 virus: interim guidance which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder this study was self-funded. competing interests: the authors declare no competing interests.ethics approval: approval for conducting this study was obtained from the ethics committee of the scientific research, faculty of dentistry, cairo university. dentists were briefed about the general objectives of the study, along with the voluntary nature of participation and informed consent was attained from each participant. the questionnaires were completed anonymously to ensure the confidentiality of the information provided.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-333041-69n2wwn3 authors: pal, anandita; gowdy, kymberly m.; oestreich, kenneth j.; beck, melinda; shaikh, saame raza title: obesity-driven deficiencies of specialized pro-resolving mediators may drive adverse outcomes during sars-cov-2 infection date: 2020-08-11 journal: front immunol doi: 10.3389/fimmu.2020.01997 sha: doc_id: 333041 cord_uid: 69n2wwn3 obesity is a major independent risk factor for increased morbidity and mortality upon infection with severe acute respiratory syndrome coronavirus (sars-cov-2), which is responsible for the current coronavirus disease pandemic (covid-19). therefore, there is a critical need to identify underlying metabolic factors associated with obesity that could be contributing toward increased susceptibility to sars-cov-2 in this vulnerable population. here, we focus on the critical role of potent endogenous lipid metabolites known as specialized pro-resolving mediators (spms) that are synthesized from polyunsaturated fatty acids. spms are generated during the transition of inflammation to resolution and have a vital role in directing damaged tissues to homeostasis; furthermore, spms display anti-viral activity in the context of influenza infection without being immunosuppressive. we cover evidence from rodent and human studies to show that obesity, and its co-morbidities, induce a signature of spm deficiency across immunometabolic tissues. we further discuss how the effects of obesity upon sars-cov-2 infection are likely exacerbated with environmental exposures that promote chronic pulmonary inflammation and augment spm deficits. finally, we highlight potential approaches to overcome the loss of spms using dietary and pharmacological interventions. collectively, this mini-review underscores the need for mechanistic studies on how spm deficiencies driven by obesity and environmental exposures may exacerbate the response to sars-cov-2. obesity is an independent risk factor for increased morbidity and mortality upon infection with the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) responsible for the current covid-19 pandemic. several studies underscore the notion that obesity, in addition to a range of other co-morbidities and dietary factors, may increase the risk for sars-cov-2 (1-10). as an example, in a study from mexico, the odds of having covid-19 among obese patients with a bmi > 30 kg/m 2 was 61% higher than that of control non-obese patients (1) . generally, amongst patients with symptoms, those with severe or critical conditions had much higher bmi and prevalence of obesity than the normal population or covid-19 negative patients (2) (3) (4) (5) (6) (7) (8) (9) (10) . one study used the uk biobank data (n = 285,817) to show that obesity almost doubled the risk of infection, adjusted for age, sex, ethnicity and socioeconomic status (9) . thus, it is clear that obesity results in a higher risk of increased severity of infection with sars-cov-2. these findings mirror influenza infection, as obesity also independently increases risk for influenza severity and death (11) . the high rate of obesity worldwide (e.g., in the u.s. over 40% of the adult population is obese) combined with the enhanced morbidity and mortality in obese individuals from infection with sars-cov-2 represents a public health emergency. therefore, there is a critical need to identify the underlying factors by which obese patients are at high risk of infection and complications with sars-cov-2. in this mini-review, we focus on a unique aspect of fatty acid metabolism that may provide a link between obesity and immune dysregulation to sars-cov-2 infection. these significant insights could evoke new areas of investigation at a mechanistic level and ultimately therapeutic strategies for this vulnerable population. a wide range of metabolic factors contribute toward impaired innate and adaptive immunity in obesity. here, we discuss the role of fatty acid-derived metabolites belonging to the specialized pro-resolving mediator (spm) family. these potent lipid autacoids known as resolvins, protectins, maresins, and lipoxins are synthesized during the transition of inflammation to resolution and are critical for turning damaged tissue to homeostasis (12) . spms are predominately synthesized from the n-3 polyunsaturated fatty acids (pufa) known as eicosapentaenoic (epa) and docosahexaenoic (dha) acids ( figure 1a) . some spms are also synthesized from arachidonic acid, an n-6 pufa ( figure 1b) . for further details on these metabolites and their immunoresolvants properties, we refer the reader to elegant reviews from serhan et al. (12, 13) . there is strong literature to support a role for spms in improving outcomes upon bacterial, parasitic, and viral infections (14, 15) . to exemplify, the dha-derived spm known as protectin dx (pdx), an isomer of protectin d1 (pd1), enhanced mouse survival upon lethal h5n1 infection including under conditions where antiviral drugs failed to confer protection (16, 17) . mechanistically, pdx inhibited viral replication by targeting the nuclear export machinery for viral transcripts. pdx specifically blocked viral transcripts from being transported to nxf1, an mrna transporter. furthermore, pulmonary pdx levels were lowered upon influenza infection and were dependent on 12/15-lipoxygenase activity. these effects were unique to pdx as other pufa-derived metabolites did not confer any improvement in survival. another study suggested that metabolites of the dha-derived spm family have utility as adjuvants for influenza vaccination. the spm precursor 17-hydroxydocosahexaenoic acid (17-hdha) increased antibody levels and improved survival upon ph1n1 influenza vaccination and infection in lean mice by promoting b cell differentiation toward the formation of cd138 + long-lived antibody secreting cells (18) . at a molecular level, this was driven by 17-hdha upregulating the expression of key transcription factors including blimp-1, the master regulator of b cell differentiation toward antibody secreting plasma cells. similarly, administration of dietary dha ethyl esters, the parent compound of dha-derived spms, also boost antibody levels of obese mice (19, 20) . dha improved antibody levels upon influenza infection by increasing the concentration of 14hydroxydocosahexaenoic acid (14-hdha), which in turn drove the formation of long-lived cd138 + antibody secreting cells (19) . therefore, these studies suggest that spms have a role in controlling influenza infection through differing mechanisms including improving aspects of humoral immunity. furthermore, there is also in vitro evidence that the n-6 pufa-derived spm known as lipoxin b4 can stimulate antigen-specific igg production from memory b cells in subjects that were vaccinated for influenza (21) . in this case, lipoxin b4 upregulated the expression of blimp-1 and xbp1 to increase the abundance of memory b cells. the effects of spms are not just limited to influenza virus. for instance, aspirin-triggered resolvin d1 is reported to have anti-inflammatory effects on murine ocular inflammation driven by infection with herpes simplex virus (22) . in addition, aspirin triggered resolvin d1 can clear mouse bacterial infections such as pulmonary pneumonia, which can lower the need for antibiotics (23, 24) . the cellular targets of spms in the context of viral infection and obesity are emerging. there is strong evidence for the role of spms in controlling chronic inflammation in obesity by targeting monocyte and macrophage polarization (25) . this is particularly relevant for covid-19 as adipose tissue presumably expresses high levels of the human angiotensin converting enzyme (ace2), the receptor for sars-cov-2. ace2 expression levels are likely higher in adipose tissue of the obese compared to the lungs, suggesting that adipose tissue may be a major target for sars-cov-2 (26). as described above, there is strong evidence on how spms drive b cell differentiation toward long-lived antibody secreting cells. however, it is unclear how spms influence other aspects of humoral immunity to promote antibody production. for instance, the abundance of t follicular helper cells, which are required to promote b cell activation and germinal center formation, is lowered in obesity (27) . it remains unclear if spms could be targeting the abundance of these cells to improve germinal center formation and function. in addition, obesity impairs pulmonary outcomes upon influenza infection, including lung inflammation characterized by dysregulated memory cd8 + t cell metabolism (28) . given evidence to show that spms can control t cell differentiation and function, there is a need to figure 1 | metabolic pathways by which specialized pro-resolving mediators (spms) are synthesized from polyunsaturated fatty acids (pufa). (a) epa and dha are long-chain n-3 pufas that serve as precursors for the biosynthesis of spms of the resolvin, protectin, and maresin families through the use of differing enzymes. epa and dha can be synthesized from the essential short-chain n-3 pufa known as alpha-linolenic acid. (b) the biosynthesis of lipoxins from the n-6 pufa arachidonic acid. arachidonic acid can be synthesized from the essential n-6 pufa linoleic acid. key enzymes for fatty acid elongation and desaturation in addition to spm biosynthesis are indicated for the n-3 and n-6 pufa pathways. for simplicity, the biosynthesis of all spm intermediates is not shown for the n-3 and n-6 pathways. understand the mechanisms by which spms may control the abundance and function of pulmonary t cell populations (29) . there is evidence that obesity generally drives a unique signature of spm deficiency (19, (30) (31) (32) (33) (34) (35) (36) (37) . table 1 summarizes the results of these studies. to exemplify, obese mice compared to lean controls display a rapid reduction in dha-derived spm precursors and spms in white adipose tissue within 4 days of consuming a high fat diet (37) . others have also reported a reduction of not only dha-derived spms but also metabolites from the epa pathway upon long term consumption of obesogenic diets in white adipose tissue and liver, which are central in driving complications of obesity (30, 32, 34, 42) . as described below, these deficiencies can be overcome through dietary administration of epa-or dha-enriched marine oils. on the contrary, one study demonstrated that in a model of liver steatosis, select spms were elevated, which may be due to an attempt to lower chronic inflammation (38) . however, in this study, the liver content of epa and dha, the parent fatty acids of spms, were lower in obese mice relative to lean controls. spm deficiencies are not just limited to adipose tissue and liver. when mice were fed a western diet, there was a significant loss of pdx in the spleen, which was reversed upon administration of dha ethyl esters in the diet (19) . a significant reduction of 14-hdha, 17-hdha, and pdx was also reported in mice consuming a high fat diet with a modest effect on 14-hdha in the bone marrow (33) . the effects were evident in male but not female obese c57bl/6j mice, suggesting sex differences c57bl/6j mice spleen and bone marrow 14-hdha, 17-hdha and pdx were lower in obese male but not female mice. 14-hdha was lowered in the bone marrow of obese male but not female mice humans with the metabolic syndrome and weight loss neutrophils metabolic syndrome patients who lost weight in a weight loss program had a 2-fold increase in rve1 compared to those participants who were in the weight maintenance group and did not lose weight (41) in spm deficiencies. in support of this notion, it is known that synthesis of dha is higher in women than men (43) . the notion of sex-differences in spm metabolism is also consistent with a human study that showed females were protected from endothelial impairments driven by inflammation due to elevated levels of spms compared to males (44) . the sex-differences are intriguing, as data on covid-19 prevalence shows that males are disproportionally at higher risk for becoming infected than females across all ages (45) . studies with human samples have validated murine studies by demonstrating that obese humans compared to lean controls display deficiencies of key spm precursors in circulation. a major finding was that leukocytes isolated from obese patients had reduced levels of 17-hdha and an unbalanced formation of dha-derived resolvins along with an increased production of the potent chemokine leukotriene b 4 (31) . this study found impaired activity of 15-lipoxygenase, a key enzyme required for spm biosynthesis to be the cause of the deficiency. interestingly, the impairment was not due to reduced cellular uptake of dha, consistent with rodent studies that show no impairment in dha levels (33) . furthermore, when leukocytes were treated in vitro with 17-hdha, the biosynthesis of downstream metabolites was rescued, demonstrating 15-lipoxygenase to be a potential therapeutic target for improving circulating levels of spms (31) . the observations on spm deficiencies with obesity are generally consistent with models of type 2 diabetes, a major comorbidity of obesity ( table 1) . for instance, in wounds of db/db mice, select spms were lowered relative to littermate controls (39) . in another study, 17-hdha and pd1 were decreased in white adipose tissue of db/db mice, consistent with studies using diet-induced obese mice, although 18-hepe levels were elevated compared to controls (37) . in type 2 diabetic subjects, circulating maresin 1 (mar1) levels were decreased compared to controls; furthermore, mar1 was further decreased in those type 2 diabetics with foot ulcers (40) . mar1 is of significance given its role in regulating murine insulin sensitivity and adipose tissue inflammation in models of genetic and diet-induced obesity (46). finally, a recent study showed weight loss elevated rve1 levels in human subjects with metabolic syndrome (41) , suggesting that the effects of obesity on spms could be potentially reversed through weight loss ( table 1) . recent studies have noted that individuals living in areas with higher levels of ambient air pollution are at a higher mortality risk from covid-19 (47, 48) . this was also noted with previous sars pandemics (49) . obese individuals are uniquely susceptible to environmental exposures and it is currently unknown whether there is a higher rate of mortality from covid-19 in obese patients that live in areas with increased air pollution. epidemiological studies have indicated an association between obesity and air pollution (50, 51) . studies of obese humans and animal models have demonstrated a greater decrement in pulmonary function after exposure to the criteria air pollutant ozone (o 3 ), enhanced production of proinflammatory cytokines, and markers of oxidative stress (52, 53) . it is currently unclear why obese individuals are more susceptible to the health effects of environmental exposures. however, experimental data have noted that obese mice and humans exposed to air pollutants have increased pulmonary and systemic tnfα, il-17, markers of lung injury, and airspace neutrophilia (54) . in addition to increased inflammation, acute exposure to o 3 significantly reduces pulmonary and systemic dha-derived spm precursors and spms (55) . treatment of mice with 17-hdha, 14-hdha, and pdx significantly decreased o 3induced pulmonary inflammation (55) . this suppression of spm production was also noted in a murine model of nanotoxicity wherein obese mice exposed to nanoparticles had a significant suppression in pulmonary expression of 5-lipoxygenase and 12/15-lipoxygenase and the production of epa-and dhaderived spms (56) . taken together, these data suggest that the susceptibility of obese individuals to environmental lung diseases may drive an altered pulmonary immune response and a state of spm deficiency that increases the morbidity and mortality to respiratory infections, including covid-19. given that spm deficiencies in obesity are potentially contributing toward poor outcomes upon sars-cov-2 infection, administration of spms may be beneficial (57) . this hypothesis assumes that spms would target key mechanisms by which sars-cov-2 drives an uncontrolled and dysregulated pulmonary response. sars-cov-2 can drive a cytokine storm, which may be a potential target for intervention as spms are known to have dual anti-inflammatory and pro-resolving properties including restricting excessive immune cell infiltration (12, 58) . for instance, tnf-α, il-6, il-1β, il-8, il-12, monocyte chemoattractant protein 1 (mcp1), interferon-gamma inducible protein (ip10) and macrophage inflammatory protein 1a (mip1a) have been implicated in driving complications associated with sars-cov-2 (59) . furthermore, uncontrolled infiltration of immune cells into the lungs, due to excessive reactive oxygen species and secretion of proteases promote pulmonary destruction and thereby lower blood oxygen upon sars-cov-2 infection (60). thus, spms or their parent compounds may have utility in improving pulmonary cytokine production and recruitment of pulmonary immune cells upon infection. in support of this notion, in a mouse model of infection with non-typeable haemophilus influenzae, the aspirin triggered rvd1 decreased the concentration of pulmonary tnfα and il-6 in addition to driving the clearance of macrophages (61) . there are several approaches that could increase levels of spms. one is through dietary intervention in which the parent compounds of spms, notably epa and dha, can be delivered as either over-the-counter supplements or as prescription supplements such as lovaza, vascepa, and epanova. it is important to note that over-the-counter formulations of these fatty acids are not the same as prescriptions due to differences in dose, purity, and composition of the fatty acids. nevertheless, a recent study showed that an spm precursor containing marine oil strongly upregulated spms of the epa and dha series within hours of administration accompanied by enhanced neutrophil and monocyte phagocytosis of bacteria (62) . however, a major limitation of this approach is that dietary epa and dha may not be as potent as direct intervention with spms (12) . a more directed approach is to deliver spms rather than the parent compounds although the mode of delivery remains to be established. one recent study showed that spms were delivered using nanoparticles in a model of intestinal wound healing, which led to activation of pro-repair pathways in the colonic mucosa (63) . furthermore, changes in dietary patterns may be another viable option. the western diet is associated with impaired pulmonary outcomes and a shift toward a mediterranean diet may prevent a deficiency of spms (64) . an additional consideration is the potential role of n-6 pufas on outcomes related to sars-cov-2 infection. n-6 pufas are highly abundant in the western diet and there is some suggestion that select n-6 pufas such as linoleic acid could be driving spm deficiencies due to competition between the n-6 and n-3 fatty acids for specific enzymes that control spm biosynthesis (65, 66) . this is particularly important to consider given that parenteral nutrition in a hospital setting is enriched in n-6 pufa-enriched oils (67) . thus, increasing n-3 pufa levels alone may not be enough to increase downstream spms in the obese but could require changes in the intake of n-6 pufas. of course, n-6 pufas themselves are also critical for synthesis of spms such as lipoxins (12) . thus, additional studies on the complex relationship between dietary n-6 and n-3 pufas with downstream spm biosynthesis, particularly in the context of viral infection are essential. overall, there is no current evidence to support changes in dietary pufa intake for improving outcomes upon sars-cov-2 infection, but is an important area of investigation at the pre-clinical and clinical level. finally, our understanding of the mechanisms by which sars-cov-2 exerts its effects are just emerging (60), although how the virus impairs outcomes in obese individuals currently remains unknown. there is no evidence for a role for spms in controlling the host's response upon sars-cov-2 infection. therefore, there is a critical need to evaluate and understand the kinetics of spm biosynthesis in human and animal models of obesity during sars-cov-2 infection using mass spectrometrybased lipidomics. supporting experiments with gain and loss of function approaches in animal models are also required to establish that spm deficiencies in obesity exacerbate the response to the infection. it is also important to consider the host genetic profile (34) , which could be a major consideration in developing dietary or pharmacological approaches to overcoming spm deficiencies and improving outcomes to sars-cov-2 for the obese. in summary, spms are key players in inflammation resolution and the infectious response. deficiencies in spms, driven by obesity, its 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increase peripheral blood specialized pro-resolving mediators concentrations and reprogram host immune responses: a randomized double-blind placebo-controlled study resolvin e1 is a pro-repair molecule that promotes intestinal epithelial wound healing an official american thoracic society workshop report: obesity and metabolism. an emerging frontier in lung health and disease interactive effects of maternal and weaning high linoleic acid intake on hepatic lipid metabolism, oxylipins profile and hepatic steatosis in offspring linoleic acid: a nutritional quandary parenteral nutrition and lipids the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 pal, gowdy, oestreich, beck and shaikh. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-333853-p2kbjwpy authors: smee, donald f.; wong, min-hui; russell, andrew; ennis, jane; turner, jeffrey d. title: therapy and long-term prophylaxis of vaccinia virus respiratory infections in mice with an adenovirus-vectored interferon alpha (mdef201) date: 2011-10-13 journal: plos one doi: 10.1371/journal.pone.0026330 sha: doc_id: 333853 cord_uid: p2kbjwpy an adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mdef201) was evaluated for efficacy against lethal vaccinia virus (wr strain) respiratory infections in mice. mdef201 was administered as a single intranasal treatment either prophylactically or therapeutically at doses of 10(6) to 10(8) plaque forming units/mouse. when the prophylactic treatment was given at 56 days prior to infection, it protected 90% of animals from death (100% protection for treatments given between 1–49 days pre-infection), with minimal weight loss occurring during infection. surviving animals re-challenged with virus 22 days after the primary infection were protected from death, indicating that mdef201 did not compromise the immune response against the initial infection. post-exposure therapy was given between 6–24 h after vaccinia virus exposure and protection was afforded by a 10(8) dose of mdef201 given at 24 h, whereas a 10(7) dose was effective up to 12 h. comparisons were made of the ability of mdef201, given either 28 or 1 day prior to infection, to inhibit tissue virus titers and lung infection parameters. lung, liver, and spleen virus titers were inhibited to nearly the same extent by either treatment, as were lung weights and lung hemorrhage scores (indicators of pneumonitis). lung virus titers were significantly (>100-fold) lower than in the placebo group, and the other infection parameters in mdef201 treated mice were nearly at baseline. in contrast, viral titers and lung infection parameters were high in the placebo group on day 5 of the infection. these results demonstrate the long-acting prophylactic and treatment capacity of mdef201 to combat vaccinia virus infections. the threat of using orthopoxviruses, variola and monkeypox, as bioterror weapons has led to increases in vaccination particularly in military personnel as well as the investigation of countermeasures (antiviral treatments) for such infections [1] . a number of compounds have been identified that exhibit direct antiviral activity against these and related poxviruses in animal models [2, 3] . from these investigations, three compounds stand out as being clinical candidates, cidofovir [4] , cmx001 (an orally active prodrug of cidofovir) [5] , and st-246 [6] . cidofovir, cmx-001, and st-246 have all been used in emergency settings to treat vaccination complications [7, 8] . immune system stimulation via exogenous recombinant interferon (rifn) is effective for treating vaccinia virus respiratory infections in mice [9] . single daily doses of rifna or rifnc rescued mice from lethality when administered 1-2 days after virus challenge and reduced lung virus titers. both vaccinia keratitis and vaccinia-induced skin lesions were treated with rifn (topically and injected respectively) in monkey models with success [10, 11] . vaccinia and other poxviruses have evolved comprehensive mechanisms to antagonize the interferon system [12] , that include blocking ifn gene induction, disrupting extracellular and intracellular signaling and slowing ifn induced gene (pkr and 2959 oas) activation. treatment with exogenous rifn only compensates for host ifn gene suppression and swamping the ifn binding protein so it must begin early after infection before the virus suppresses activation of the antiviral state. interferon has a short half-life of three hours [13] , which requires frequent treatment with large bolus doses resulting in commensurate adverse effects often leading to patient-initiated cessation of treatment [14, 15] . pegylated rifn has improved the in vivo half-life to allow for weekly injections, but results in a reduced activity of the protein and an increased cost. a needle-free, single dose drug capable of achieving steady-state method of delivering interferon would maximize the therapeutic benefits of ifn, while minimizing the bolus-induced toxicity. to this end, a replication-deficient adenovirus 5 (ad5) vector containing the human consensus interferon alpha gene, referred to as def201, has been developed. intranasal administration of def201 allows for the ad5 vector to infect respiratory cells and drives constitutive expression of the ifn transgene and secretion of fully glycosylated ifn. ad5-vectored mouse interferon (mdef201) resulted in sustained ifn levels [16] , that completely protected mice from a lethal western equine encephalitis virus infection when given intramuscularly at 10 7 plaque forming units (pfu)/ mouse up to 7 days prior to virus challenge [16] . against venezuelan equine encephalitis virus infection, mdef201 prevented death when administered intramuscularly at 10 7 pfu/mouse 24 h prior to infection but not when given 6 h after infection [17] . intranasally-administered mdef201 was also used to treat mice infected intranasally with sars virus (resulting in a lethal respiratory infection) [18] . it was 100% protective when administered prophylactically at 10 6 pfu/ mouse up to 14 days pre-virus exposure, with similar protection afforded by a 10 8 dose administered therapeutically at 12 h after infection [18] . adenoviral vectored human consensus ifn (def201), was recently used intranasally to treat hamsters infected intraperitoneally with yellow fever virus (resulting in a lethal hepatic infection) [19] . protection from death was observed for single administrations of def201 at 10 7.5 pfu/animal given 7 days before to 2 days after virus challenge, which demonstrated both prophylaxis and therapy in this model. def201 treatment reduced yellow fever virus titers in liver and spleen, and resulted in decreases in serum alanine transaminase levels. herein, mdef201 was evaluated for prophylaxis and therapy of vaccinia virus (wr strain) respiratory infections in mice. in this model the virus infection spreads systemically to include other tissues besides the lungs, particularly the liver and spleen [20] . increases in lung weight and lung hemorrhage (indicative of pneumonitis) are hallmarks of vaccinia infection, as is severe body weight loss. the results show that a single intranasal dose of mdef201 produced a remarkably prolonged 8-week prophylactic effect, and was also active therapeutically. the experiments were conducted in accordance with protocol 552 approved by the institutional animal care and use committee of utah state university. the work was done in the aaalac-accredited laboratory animal research center of utah state university in accordance with the national institutes of health guide for the care and use of laboratory animals (revision; 2010). female balb/c mice (charles river labs, wilmington, ma) weighing approximately 13-15 g at the time of first treatment were used. after a 48-hour quarantine period the animals were randomly assigned to cages. all work with these animals was performed in the biosafety level 2 area of the aaalacaccredited laboratory animal research center at utah state university. the adenovirus vectored mouse interferon alpha, subtype 5 construct mdef201 was prepared at the robert fitzhenry vector laboratory (mcmaster university, hamilton, canada). the methods of preparation have been described previously [16] . briefly, the mouse interferon alpha gene (subtype 5) was cloned into a replication deficient ad-5 vector (deletions of e1 and e3 gene deletions), amplified in 293 cells and purified by cesium chloride gradient centrifugation. stock solutions of mdef201 were stored at 280uc and were thawed on ice and diluted in saline to the appropriate dose immediately prior to a single administration. gilead sciences (foster city, ca) provided the positive control compound cidofovir. vaccinia virus (wr strain) was purchased from the american type culture collection (atcc, manassas, va). the virus was propagated in african green monkey kidney (ma-104) cells (from ma bioproducts, walkersville, md) for use in these studies. mice were anesthetized with ketamine/xylazine (50/5 mg/kg) by intraperitoneal (i.p.) injection for intranasal treatments and intranasal infection. the animals were initially infected intranasally with approximately 1-2610 5 pfu of vaccinia virus in a 50-ml volume. mdef201 was administered intranasally (50-ml) either prior to or after vaccinia virus exposure, according to the experimental schedule. placebo-treated mice were given saline. in order to maintain consistency since intranasal treatment alters the environment for infection, all mice were given intranasal saline when the mdef201 treatments were given 1 day before or after virus infection, or else additional placebo groups were used to accommodate delivery of intranasal liquid. all animals were weighed every other day. in some instances a normal control group (untreated, uninfected) of 10 mice was maintained throughout the study. for certain studies, the normal control group was infected with virus at the end of the initial 21-day antiviral study to serve as naïve, infected controls for the re-infection. treated mice surviving the primary infection were re-infected on day 22 with approximately 5610 5 pfu units of vaccinia virus in a 50-ml volume. this dose was larger than for the primary infection to account for the increased age of the mice, and older mice are less susceptible to vaccinia infection. the age-matched normal control (naïve) animals were infected for the first time with the same virus challenge. survival and body weights in these groups were determined over a 21-day period. separate animals were maintained for determination of tissue virus titers, lung hemorrhage scores, and lung weights. groups of 5 infected mice per day were sacrificed for removal of tissues. lungs were given a hemorrhage score (color change from pink to plum which occurs regionally in the lung rather than gradually over the entire lung) ranging from 0 to 4 (entire lung affected). lungs, spleens, livers, and snouts were weighed prior to homogenization which releases infectious virus for titration. homogenization of soft tissues was done in 1 ml of cell culture medium using a stomacher. snouts were ground in 1 ml of medium using sterilized mortars and pestles. samples were serially diluted in 10-fold increments and plaque titrated in 12-well microplates of vero cells. plaques were stained at three days with 0.2% crystal violet in 10% buffered formalin, then the plaques were counted with the aid of a light box. plaque numbers were converted to pfu per gram of infected tissue. initially, survivor numbers were compared by multiple group chi square analysis. when statistical significance was found, survivor numbers were evaluated using the two-tailed fisher exact test. differences in the mean day of death, tissue virus titers, lung weights, and lung hemorrhage scores were statistically analyzed using the two-tailed mann-whitney u-test. analyses were performed using the instath computer software program (graphpad software, san diego, ca), comparing treated and placebo groups. the short-term prophylactic activity of mdef201 administered at various intranasal doses one day prior to virus challenge is presented in table 1 . mdef201 was uniformly, 90-100% protective from a lethal vaccinia challenge at doses of 10 5 to 10 7 pfu/animal. this protective effect can be attributed to the mifn transgene because the empty adenovirus vector was not protective and produced similar complete mortality as the placebo control. cidofovir (positive control compound) was also 100% protective at 100 mg/kg/day when administered by intraperitoneal route starting 1 day post-infection. table 1 also shows the effects of treatment with mdef201 and cidofovir on lung infection parameters. a dose-responsive effect on lung virus titer was seen with mdef201 treatments, with the highest dose causing nearly an 800-fold reduction in lung virus titer. mdef201 doses of 10 5 and 10 6 pfu/mouse reduced virus titers 8-and 125-fold, respectively. all three mdef201 treatments significantly reduced lung weights and lung hemorrhage scores. in contrast, cidofovir treatment caused a only a 40-fold reduction in virus titer, and protected lungs from virus-induced pathology. lung virus titers and lung weights were also intermediate between the 10 5 and 10 6 mdef201 dosage groups. neither the control, nor adenovirus empty vector inhibited lung infection parameters. to measure the window of prophylactic activity, 10 7 pfu/mouse of mdef201 was administered at various times prior to virus infection ( table 2 ). in the first experiment a single dose of mdef201 was 100% protective when administered between 28 days and 1 day pre-virus exposure. mean body weights during the course of the primary infection are presented in figure 1a . all of the treatments with mdef201 largely protected the animals against weight loss. a slight drop in weight occurred on day 1 following intranasal exposure to virus, but the mice were back to the initial starting weight by day 3. seven placebo-treated animals survived the infection, differing from that of table 1 where no animals survived. this likely occurred because the mice at the time of infection were 28 days older, and susceptibility of the animals to infection diminishes with age. severe weight loss in the placebo group persisted until day 11 of the infection. after that time, the seven animals that survived the infection began to recover body weight. the surviving mice from experiment 1 were re-infected with a 5-fold higher challenge of vaccinia virus than was originally given, and all of these animals survived the re-challenge ( table 2) . infection of age-matched naïve mice resulted in expected 100% mortality. weight loss occurred in all mdef201 treated groups, but was much less severe than in naïve, infected mice ( figure 1b) . rebound from weight loss during the re-infection was fastest in the placebo group. this group contained the most seriously afflicted animals during the primary infection, thus would be expected to have a more robust immune response than treated animals that did not get as sick. based upon the successful extended prophylaxis up to 28 days pre-infection, a second experiment ( table 2 ) was initiated using starting times as early as 56 days before virus challenge. mdef201 was 90% protective when administered at -56 days, and 100% protective when given at all shorter time points (,49 days). because the virus challenge dose was higher than that given in experiment 1 (see footnote ''b'' in table 2 ), the result was that all placebo-treated animals in experiment 2 died from the primary infection. minimal weight loss occurred in groups treated prophylactically with mdef201 ( figure 1c ). decreases in body weight in mdef201 groups were primarily seen on days 5-9 of the infection, with the nadir being day 7. the surviving mice that were treated with mdef201 in the second experiment (table 2) were re-infected with a 2-fold higher challenge of vaccinia virus, and all of these animals survived. infection of age-matched naïve mice resulted in 100% mortality. weight loss occurred in all mdef201 treated groups, but was minimal and occurred on days 3-5 ( figure 1d ). this is in contrast to the rapid weight decline in naïve, infected mice. body weights during the re-infection were similar for most mdef201 groups. slightly less rapid recovery in body weight over 21 days was seen in the mdef201 group treated prophylactically at -56 days compared to the other treated groups. viral titers in various tissues were determined from mice treated with mdef201 either at 28 days or 1 day prior to infection. lung virus titers in mdef201 treated groups were at least 100-fold less than in placebo treated mice (figure 2a ). liver and spleen virus titers were below or near the level of detection in animals treated with mdef201, but increased over time in placebos ( figures 2b and 2d) . snout virus titers in mdef201 treated groups were significantly less than placebos on day 3 but not on day 5 ( figure 2f ). in comparing virus titers in the -28 day mdef201 group to that of the -1 day mdef201 group, the amounts of detectable virus were nearly equivalent in lung, liver and spleen. differences were observed in snout virus titers between these two groups, but due to variability the differences were not statistically significant. lung weights and lung hemorrhage scores were determined in infected mice in parallel with the determination of viral titers. lung weights in mdef201 treated animals were near normal, whereas on day 5 the placebos exhibited a large increase in lung weight ( figure 2c) . similarly, lung scores were near normal in the two groups treated with mdef201, but were severe on day 5 in the placebo group ( figure 2e ). the extent of inhibition of lung intranasal treatments with mdef201 (10 7 pfu/mouse) were given one time only on the indicated day prior to virus exposure. in experiment 1 the primary intranasal challenge was 1610 5 pfu of virus. since this challenge dose failed to cause 100% mortality (as mice age their susceptibility to infection wanes), the primary virus challenge dose was increased to 2.5610 5 pfu of virus for experiment 2. reinfection of both sets of mice was performed intranasally with 5610 5 pfu of virus. the data accompany those of table 2 . thus, the initial number of mice per group for each figure corresponds to the total number per group in the table. doi:10.1371/journal.pone.0026330.g001 disease in the -28 days mdef201 group was comparable to that seen in the -1 day mdef201 group. the results from these experiments indicate that mdef201 has an extremely long-acting prophylactic activity against vaccinia virus infections in mice. only in animals treated 56 days prior to infection was there a hint of waning activity, since one animal died from the infection in that group. mdef201 at two different doses was administered after infection to combat a vaccinia virus infection. preliminary studies with low dose mdef201 (10 5 or 10 6 pfu/mouse), given at 6, 12, or 24 h after infection provided no protection from the lethal infection (data not shown). however, higher doses of mdef201 (10 8 pfu/mouse) were 100% protective when administered either at 6, 12, or 24 h after infection (table 3) . a 10 7 pfu/mouse dose was 80-90% protective when given at 6 and 12 hours, but only 30% protective when administered at 24 hours. survival time in the lower dosage group receiving treatment at 24 hours was significantly increased relative to the placebo control. cidofovir was 100% protective when administered at 100 mg/kg/day for two days starting at 24 h. body weight changes for treatments with 100% survival during the course of the infection are shown in figure 3 (10 8 mdef201 and cidofovir). weight loss in mdef201 treated mice increased with longer delay to commencement of treatment, with all animals regaining their pre-challenge weight by day 18 of the experiment. given the weight loss in the +24h group, further delays in treatment would likely result in some mortality. mice treated daily with cidofovir starting 24h post-vaccinia challenge lost less weight. in these studies, a single intranasal dose of mdef201 was found to exhibit a potent prophylactic activity that endured for at least 56 days. an endpoint in the duration of the protection could not be projected because treatment at -56 days resulted in only one death intranasal treatments with mdef201 (10 7 pfu/mouse) were given one time only on the indicated day prior to intranasal virus exposure (2.5610 5 pfu). there were no survivors in the placebo group on day 7 for data determinations. this experiment was conducted concurrently with the second experiment in table 2 . each data point represents the mean for five animals per group. doi:10.1371/journal.pone.0026330.g002 and caused minimal weight loss during the infection. indeed, weight loss during infection for the -56 day treatment group was equivalent to the -1 day treatment group. investigation of other parameters of infection (tissue virus titers, lung weights, and lung hemorrhage scores) revealed that the inhibition of these parameters with a -28 day mdef201 treatment was quite comparable to that of a -1 day treatment. prophylaxis starting a day before infection could effectively be achieved with doses of 10 5 through 10 7 pfu/mouse. extended prophylaxis up to 56 days preinfection was only investigated at the 10 7 dose. other investigators have shown that def201 in both mouse and human constructs are active prophylactically [16, 17, 18, 19] . kumaki et al. showed 100% protection against sars infection in mice by a 10 6 dose given only at -14 days (other time points were not assessed) [17] . thus, it is possible that prophylaxis earlier than 14 days would be protective against sars infection. against yellow fever virus infections in hamsters, def201 was effective when given 7 days prior to infection but not at -21 days [19] . this breadth of viral efficacy indicates the potential of def201 to function as a truly broad-spectrum antiviral. remarkably, mdef201 was effective in mice against vaccinia virus infection when given nearly two months prior to virus exposure. this indicates that ad5-vectored ifn induced a longterm antiviral state that was completely protective with a single dose. this extended prophylaxis appears to exist in the absence of measurable serum ifn levels, since wu et al. [15] were only able to detect interferon alpha protein in mice treated intramuscularly with mdef201 at 24 and 48 h, but not at 7 days (times in between were not investigated). this study raises a number of important questions relative to the long acting protective effect of mdef201 against vaccinia virus infection in mice. questions such as how much interferon is produced, how long is it produced, and what cells are responsible for producing it are being asked and are currently under investigation. it has already been shown that interferon is successfully produced by the vector when administered intramuscularly to mice and the protein is detectable in the serum [16] . from that work we also know that the levels of interferon rise very quickly (within 3-5 h), are transiently high, and come down within several days. we can infer that the interferon protein will be delivered successfully to the lung and nostrils (and have since confirmed this in a separate study, unpublished). given that the protection lasts longer than the previously measured levels of interferon protein persist, we assume that the interferon is activating an immunological cascade that then protects the animal from infection by inducing an antiviral state. thorough investigation of induced genes, length of induction, and localization of induction are planned, but are beyond the scope of this study. the choice of intranasal route of administration is important, as this route has been demonstrated to bypass pre-existing immunity to the adenovirus vector [21] . intranasal delivery is also less invasive and easier to administer to large numbers of patients in the event of a mass infection due to a natural outbreak or intentional release. adenovirus was selected to be the vector for interferon due to its rapid infectivity, breadth of human safety data, and facility of intranasal administration. mdef201, administered by intranasal route, has already been shown to be effective in the treatment of sars coronavirus respiratory infections in mice [18] . this is the first description of adenoviral vectored interferon alpha being used, by any route, to prevent or treat vaccinia virus infections. a limited therapeutic window of time exists for treating vaccinia virus infections with interferon-based drugs like mdef201, and to achieve treatment higher doses of mdef201 are needed than for prophylaxis. indeed, low dose mdef201 (10 5 -10 6 pfu/mouse) that were effective prophylactically were not effective when administered even 6 h after infection. higher doses (10 8 pfu/ mouse) were required for full protection from lethality when given at +24 h, however considerable body weight loss resulted due to infection (figure 3 ). the single 10 8 dose administered at 24 h was not as effective as daily doses of 100 mg/kg of cidofovir that acts directly on vaccinia to inhibit replication. this may be due in part to the lag time between administration of mdef201 and the production of therapeutic levels of ifn within 6 h [15] . secondly, in situ produced ifn must overcome a high level of ifn system down regulation caused by the established vaccinia infection [12] . it has been demonstrated by other investigators that both mdef201 and def201 are effective as a treatment only when given within 1-2 days after virus exposure [16, 17, 18, 19] . how well ad-vectored ifn protects infected animals will depend upon each virus' virulence including the number and expression of different ifn system antagonists. however, the treatment efficacy of def201 in these unrelated viruses indicates real potential as a broad spectrum antiviral, and the extrapolation of these treatment windows into the clinical scenario may be significant. in these studies antiviral activity depended on the amount of mdef201 administered, which was the case in other published studies with unrelated viruses [16, 17, 18, 19] . the production of a steady-state level of interferon may bypass the need for bolus dosing associated with traditional interferon treatment, and thus reduce toxic side effects. nevertheless, safety of def201 is a consideration for eventual human use, and safety and toxicology studies are ongoing. it will need to be determined how long the ifn-induced antiviral state persists and its effects on treated individuals. this is the first study demonstrating that surviving mice treated with mdef201 are protected against re-infection with the same virus. this is not surprising, as mdef201 treatment did not completely prevent vaccinia virus production in the lungs and snouts of the animals (figures 2a and 2f) , thus driving an acquired immune response. the veracity of that immune response in mdef201 treated animals most likely was weaker than it would be in more severely infected mice, based upon weight comparisons to surviving placebos ( figure 1b) . assuming an adequate safety profile, one can envision the use of def201 as a means of quickly providing significant protection to first responders and medical chain personnel confronted with a deliberate release of variola or monkeypox virus into the environment. even if infected, def201 treated individuals could still acquire an immunity to the virus, as demonstrated by the present studies, thus rendering them 'vaccinated' against future exposure. the intranasal method of delivery of def201 facilitates rapid prophylaxis in people entering a suspected poxvirus environment. moreover, def201 has the potential to treat a large number of unrelated viruses simultaneously, for which there are no current treatments. pathogenesis and potential antiviral therapy of complications of smallpox vaccination a review of compounds exhibiting antiorthopoxvirus activity in animal models progress in the discovery of compounds inhibiting orthopoxviruses in animal models countermeasures to the bioterrorist threat of smallpox development of cmx001 for the treatment of poxvirus infections st-246 antiviral efficacy in a nonhuman primate monkeypox model: determination of the minimal effective dose and human dose justification severe eczema vaccinatum in a household contact of a smallpox vaccinee progressive vaccinia in a military smallpox vaccinee -united states prevention of lethal respiratory vaccinia infections in mice with interferon-alpha and interferon-gamma effect of human leukocyte interferon on vaccinia and herpes virusinfected cell cultures and monkey corneas prevention of vaccinia lesions in rhesus monkeys by human leucocyte and fibroblast interferon the interferon system and vaccinia virus evasion mechanisms pharmacokinetics of interferon alpha-2b in healthy volunteers pharmacokinetic properties of a 40 kda branched polyethylene glycol-modified form of consensus interferonalpha (peg-cifn) in rhesus monkeys phase i trial of consensus interferon in patients with metastatic renal cell carcinoma: toxicity and immunological effects pre-and post-exposure protection against western equine encephalitis virus after single inoculation with adenovirus vector expressing interferon alpha alpha interferon as an adenovirus-vectored vaccine adjuvant and antiviral in venezuelan equine encephalitis virus infection singledose intranasal administration with mdef201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal sars-cov balb/c mouse model treatment of yellow fever virus with an adenovirus-vectored interferon, def201, in a hamster model effects of cidofovir on the pathogenesis of a lethal vaccinia virus respiratory infection in mice nasal delivery of an adenovirus-based vaccine bypasses pre-existing immunity to the vaccine carrier and improves the immune response in mice key: cord-315730-fzgxuak7 authors: penman, sophie l.; kiy, robyn t.; jensen, rebecca l.; beoku‐betts, christopher; alfirevic, ana; back, david; khoo, saye h.; owen, andrew; pirmohamed, munir; park, b. kevin; meng, xiaoli; goldring, christopher e.; chadwick, amy e. title: safety perspectives on presently considered drugs for the treatment of covid‐19 date: 2020-07-17 journal: br j pharmacol doi: 10.1111/bph.15204 sha: doc_id: 315730 cord_uid: fzgxuak7 intense effort is underway to evaluate potential therapeutic agents for the treatment of covid‐19. in order to respond quickly to the crisis, the repurposing of existing drugs is the primary pharmacological strategy. despite the urgent clinical need for these therapies, it is imperative to consider potential safety issues. this is important due to the harm‐benefit ratios that may be encountered when treating covid‐19, which can depend on the stage of the disease, when therapy is administered and underlying clinical factors in individual patients. treatments are currently being trialled for a range of scenarios from prophylaxis (where benefit must greatly exceed risk) to severe life‐threatening disease (where a degree of potential risk may be tolerated if it is exceeded by the potential benefit). in this perspective, we have reviewed some of the most widely‐researched repurposed agents in order to identify potential safety considerations using existing information in the context of covid‐19. taleb-gassabi, & dayer, 2017) . a standard dose of lopinavir-ritonavir is 400 mg/100 mg twice a day for hiv-1 treatment, and this has also been used for sars-cov-2 treatment . the most frequent reported aes for lopinavir-ritonavir treatment are gastrointestinal disturbances including diarrhoea, nausea and vomiting (chandwani & shuter, 2008) . dose-related diarrhoea have been reported in up to 25 % of patients and are thought to occur through a number of mechanisms including decreased proliferation of intestinal epithelial cells, disruption of intestinal barrier function, inducing endoplasmic reticulum stress and activating the unfolded protein response (x. wu, li, peng, & zhou, 2014) . diarrhoea is also a symptom in some covid-19 patients and so lopinavir-ritonavir has the potential to exacerbate this. pancreatitis has been reported in a small number of patients following lopinavir-ritonavir treatment although this was more frequent in those with a pre-existing history of pancreatitis (chandwani & shuter, 2008; oldfield & plosker, 2006) . additionally, patients with underlying liver diseases should have regular monitoring of hepatic function (palacios et al., 2006) . caution should be exerted for those patients taking concomitant medication as lopinavir-ritonavir inhibits p-glycoprotein (p-gp) and cytochrome p450 (cyp) -3a4, which therefore may alter the pk of other compounds (l. zhang, zhang, & huang, 2009) . a covid-19 drug interaction website has been developed by the liverpool drug interaction group which details ddis with lopinavir-ritonavir and a number of drugs, which in some cases can lead to potentially serious and/or life-threatening reactions (group, 2020; abbvie inc., 2016) . since the sars-cov-2 outbreak, 79 clinical trials have been registered (up to 8 th july 2020) to test lopinavir-ritonavir as a potential treatment for sars-cov-2 with variable outcomes in terms of efficacy. in one trial of 199 patients with confirmed sars-cov-2, 13 patients on the lopinavir-ritonavir arm were withdrawn due to aes . in a different trial, patients who were administered lopinavir-ritonavir (200 mg/ 50 mg) also experienced gastrointestinal aes (y. li et al., 2020) . chloroquine and its derivative, hydroxychloroquine, are widely used as inexpensive and safe antimalarial drugs. in particular, the established good tolerability of chloroquine/hydroxychloroquine has made them safe to use even in pregnancy (villegas et al., 2007) . in addition to anti-malarial activity, both drugs have immunomodulating effects and are used for the treatment of autoimmune diseases including systemic and discoid lupus erythematosus, psoriatic arthritis, and rheumatoid arthritis. chloroquine/hydroxychloroquine concentrate extensively in acidic vesicles including the endosomes, golgi vesicles, and the lysosomes (ohkuma & poole, 1981) . this leads to lysosomal membrane permeabilisation or dysfunction of several enzymes including acid hydrolases and palmitoyl-protein thioesterase 1 (rebecca et al., 2019; savarino, boelaert, cassone, majori, & cauda, 2003; schrezenmeier & dorner, 2020) . although the precise mechanisms of the anti-viral effects are not fully understood, it has been proposed that chloroquine/hydroxychloroquine can prevent virus infection (pre-infection) by interfering with the glycosylation of cellular receptors and impair viral replication by increasing endosomal ph (post-infection) (savarino et al., 2003; savarino et al., 2004; vincent et al., 2005) . owing to their efficacy against viruses (mostly demonstrated in vitro) including influenza, hiv, coronavirus oc43, and sars-cov, a large number of clinical trials (>230) have been registered worldwide using chloroquine/hydroxychloroquine alone, or in combination with other drugs (e.g. azithromycin) for the treatment of covid-19. despite promising in vitro antiviral results for hydroxychloroquine/chloroquine, there is no convincing evidence of efficacy at present (gao, tian, & yang, 2020; gautret, lagier, parola, hoang, meddeb, mailhe, et al., 2020; gautret, lagier, parola, hoang, meddeb, sevestre, et al., 2020; magagnoli, 2020; mathian et al., 2020; million et al., 2020; tang, 2020; yao et al., 2020) . a post-exposure prophylaxis randomised controlled trial of 821 participants failed to show any benefit of hydroxychloroquine (n=414) compared with placebo (n=407) (boulware et al., 2020) . at the time of writing, the recovery trial (clinical trial identifier nct04381936) which is the largest randomised control trial so far conducted for the treatment of covid, has stopped recruiting to the hydroxychloroquine arm (1542 patients compared with 3132 on standard care) because of no beneficial effect either in terms of mortality or hospital stay (p. . there are still many other trials on-going testing the efficacy of hydroxychloroquine for either prophylaxis or treatment. both chloroquine and hydroxychloroquine have been in clinical use for many years for rheumatoid diseases, and thus their safety profile is well established. dose-dependent retinal toxicity has long been recognized as the major ae with long-term use of chloroquine/hydroxychloroquine (marmor et al., 2011) . besides retinal toxicity, gastrointestinal, liver and renal toxicity have also been reported (giner galvan, oltra, rueda, esteban, & redon, 2007; michaelides, stover, francis, & weleber, 2011; mittal, zhang, feng, & werth, 2018) . as both drugs are mainly metabolised in the liver and excreted by renal clearance, their use in patients with liver or renal impairment may worsen the function of these organs. for chloroquine treatment, prescribing information recommends the full dose at all degrees of renal impairment but suggests that monitoring of renal function may be useful . for hydroxychloroquine, reductions in dosage are advised for patients with impaired renal function, as well as those taking concomitant medications with known risks of kidney damage (concordia pharmaceuticals inc, 2017) . this article is protected by copyright. all rights reserved. a serious ae associated with chloroquine/hydroxychloroquine is cardiotoxicity, which can take many forms including cardiomyopathy in rare instances. prolonged treatment or high dosage of chloroquine/hydroxychloroquine has been shown to increase of the risk of qt interval prolongation, polymorphic ventricular tachycardia, and sudden cardiac death (chatre, roubille, vernhet, jorgensen, & pers, 2018) . a large epidemiological analysis in patients with rheumatoid arthritis has recently shown that 30-day cardiovascular mortality was increased by more than 2-fold when hydroxychloroquine was combined with azithromycin. the lethal ventricular arrhythmias are primarily due to inhibition of a potassium channel (the inward rectifier kir2.1 channel) and may occur at low µm concentrations (ic50=8.7 m) (rodriguez-menchaca et al., 2008) . while therapeutic doses of chloroquine typically result in plasma concentrations of 2-5 µm, much higher concentrations in the heart are expected based on a 400-fold increase observed in rat pk studies (mcchesney, banks, & fabian, 1967; walker, dawodu, adeyokunnu, salako, & alvan, 1983) . both drugs act on various potassium channels including the inward rectifier currents (kir2.1 and kir6.2) and rapid delayed rectifier currents (kv11.1/herg) (ponce-balbuena et al., 2012; rodriguez-menchaca et al., 2008; sánchez-chapula, navarro-polanco, culberson, chen, & sanguinetti, 2002) . the binding of chloroquine to the inward rectifier kir2.1 channel can be stabilized by negatively charged and aromatic amino acids (rodriguez-menchaca et al., 2008) . to a lesser extent, chloroquine also blocks the rapid delayed rectifier ikr, possibly through cation-π and π-stacking interactions with tyrosine 652 and phenylalanine 656 in the s6 domain of herg (sánchez-chapula et al., 2002) . the effect of inhibition of these potassium channels on the heart rate appears to be complex. however, blocking the herg channel has proven to be the most common mechanisms by which drugs cause qt interval prolongation (traebert & dumotier, 2005) . the binding of chloroquine/hydroxychloroquine to proteins is also stereoselective, but whether one of the chloroquine/hydroxychloroquine enantiomers has a stronger interaction with the kir2.1 channel is not known. caution is needed when hydroxychloroquine is used in combination with other drugs (including azithromycin), which increase the qt interval because of a pharmacodynamic synergistic interaction. given the comorbidities in many patients with covid-19, especially those with underlying cardiovascular disease, and the fact that covid-19 itself is associated with cardiac manifestations, this may increase the risk of cardiotoxicity associated with the use of chloroquine/hydroxychloroquine. indeed, excessive qtc prolongation was observed in 36 % of patients as reported by bessiere at al. and greater qtc prolongation was also seen in patients taking the combination of hydroxychloroquine and azithromycin than those taking hydroxychloroquine alone, highlighting the importance of pharmacodynamic interactions (bessiere et al., 2020; mercuro et al., 2020) . furthermore, a phase iib trial in brazil showed that a higher dose of chloroquine (600 mg twice daily) in patients hospitalised with covid-19 had a higher fatality rate (30 %) compared with 15 % in the lower dose (450 mg twice daily) group (borba et al., 2020) . qtc interval prolongation >500 msec was observed in 19 % of the high dose group compared with 11 % of the low dose group. the us prophylaxis randomised control trial however did not show any increase in cardiovascular aes (boulware et al., 2020) . we await the publication of the recovery trial to determine whether there was an excess of cardiovascular events. however, it is important to note that despite the size of the recovery trial (n = 1542 patients), it may still be under-powered to identify an excess number of cardiovascular events when compared with standard of care. remdesivir is an investigational compound that was developed for the treatment of ebola (mullard, 2018; tchesnokov, feng, porter, & gotte, 2019) . remdesivir is a monophosphoramidate prodrug and acts as a broad-spectrum antiviral that can be incorporated into viral rna (agostini et al., 2018; sheahan et al., 2020; warren et al., 2016) . many anti-virals are proving to be ineffective against covid-19 due to the presence of a proofreading exoribonuclease (exon) specific to coronaviruses, encoded in non-structural protein 14 (nsp14) (agostini et al., 2018) . remdesivir is able to evade this viral proofreading, meaning its incorporation into viral rna results in the inhibition of rna-dependent rna polymerases (rdrps), thereby preventing subsequent viral replication (warren et al., 2016) . furthermore, arshad et al. suggest that the maximum serum concentration (cmax) of remdesivir is sufficient to inhibit 90 % of sars-cov-2 replication, a parameter which is suspected to be of vital importance in the treatment of covid-19 (arshad et al., 2020) . remdesivir is administered intravenously, with single doses ranging between 3 to 225 mg being well patients receiving remdesivir via the uk early access to medicines scheme (eams) is similar to that which was evaluated for ebola treatment: a loading dose of 200 mg on day 1, followed by 100 mg daily for 5 -10 days depending on symptom severity (medicines and healthcare products regulatory agency, 2020b). as such, it is likely that many of the aes observed in the ebola study will translate to covid-19 patients treated with remdesivir. this article is protected by copyright. all rights reserved. mild to moderate alt and aspartate transaminase (ast) elevations were observed in several ebola patients during the multiple-dose study, thus reflecting observations made in human hepatocytes in vitro (clinical trials.gov, 2019; world health organisation, 2018) . this is likely to be due to the high cell permeability of hepatocytes, in combination with the effective intracellular metabolism of remdesivir to its active form within the liver (world health organisation, 2018) . emerging data has suggested that sars-cov-2 may target ace2 on hepatocytes leading to liver injury as evidenced by a significant increase in alt and bilirubin in severe cases of covid-19 (guan et al., 2020) . therefore, it is likely that differentiating between covid-19-induced transaminase elevations and those induced by remdesivir presents challenges (bangash, patel, & parekh, 2020; c. zhang, shi, & wang, 2020) . however, a recent study found that only 4.1 % of covid-19 patients receiving remdesivir treatment suffered serious (grade 3 or 4) transaminase elevations, with there being no significant difference between the remdesivir-and placebo-treated groups (beigel et al., 2020) . this data implies that remdesivir is relatively well-tolerated in sars-cov-2-positive patients. regardless, as advised by the drug manufacturer, daily liver function tests are essential in any patients receiving remdesivir, with suggested discontinuation of the drug in patients whose alt levels reach ≥ 5 times the upper limit of normal (uln) (gilead, 2020) . adhering to these guidelines is of particular importance in patients with pre-existing liver disease, or in those taking other medications which can also induce transient alt and ast elevation (world health organisation, 2018) . the reported differences between preclinical and clinical data regarding the safety of remdesivir highlight the inadequacies of preclinical models in some contexts. for example, with regards to covid-19, a concerning element of theoretical toxicity is that which affects the respiratory system. a study using mice models of middle east respiratory syndrome coronavirus (mers-cov) found remdesivir improved pulmonary pathology in infected mice and rhesus monkeys, and no respiratory toxicity was observed (gilead, 2020; sheahan et al., 2020) . in contrast, a respiratory safety study in rats showed that remdesivir had no impact on tidal volume or minute volume, but did increase respiratory rate, which returned to baseline by 24 hours post-dose (world health organisation, 2018). clearly, increased respiratory rate is a manifestation of covid-19, and there would be problems in assessing causality if remdesivir was also likely to cause of respiratory problems in a clinical setting. fortunately, a recent double-blind, randomized, placebo-controlled trial showed there to be no significant differences in adverse respiratory events between the remdesivir-treated and control arms (beigel et al., 2020) . in addition to this, preclinical safety studies performed in rats and cynomolgus monkeys suggested that the kidney was the target organ for remdesivir-induced toxicity (gilead, 2020) . this was a significant concern before the initial covid-19 clinical trials, as it is known that sars-cov-2 can cause acute kidney failure in severe cases (ronco, reis, & husain-syed, 2020) . however, this has not this article is protected by copyright. all rights reserved. been reflected in covid-19 clinical trials, where the presence of biomarkers indicative of renal injury have not differed in patients treated with remdesivir compared to those on placebo (beigel et al., 2020; gilead, 2020) . however, due to the inclusion of the solubility enhancer sulfobutylether βcyclodextrin sodium (sbecd) within remdesivir formulations, remdesivir is contraindicated in patients with severe renal impairment (egfr < 30 ml/min) (european medicines agency, 2020). finally, remdesivir is not exempt from ddis. co-administration of remdesivir with several antibiotics including rifampicin is contraindicated, which could cause problems for any patients being treated concomitantly for tuberculosis (group, 2020) . this occurs because of enzyme induction which reduces systemic exposure to remdesivir. a similar interaction has also been seen with enzyme-inducing anticonvulsants, including carbamazepine, phenytoin, and phenobarbital (group, 2020) , where reduction in remdesivir exposure may lead to inadequate treatment of covid-19. favipiravir is another broad-spectrum anti-viral prodrug which undergoes intracellular phosphoribosylation to produce its active form, favipiravir-ribofuranosyl-5′-triphosphate (favipiravir-rtp) (yousuke furuta, komeno, & nakamura, 2017) . it is thought that this anti-viral primarily acts by inducing lethal mutagenesis of rna viruses, although it also selectively and potently inhibits viral rdrp by acting as a pseudo purine nucleotide (dawes et al., 2018; sangawa et al., 2013) . favipiravir is currently licensed in japan for the treatment of novel and re-emerging influenza (yousuke y. furuta et al., 2002) . its extensive spectrum of activity against various rna virus polymerases led to favipiravir being cited as a potentially 'crucial pandemic tool', even before the outbreak of the novel coronavirus, covid-19 (adalja & inglesby, 2019) . the pk of favipiravir was initially characterised in healthy japanese volunteers (madelain et al., 2016) . a cmax of 51.5 µg/ml was found to occur 2 hours post-administration, but plasma concentrations decreased rapidly due to the relatively short half-life of favipiravir (between 2 and 5.5 hours) (madelain et al., 2016) . however, both cmax and half-life increase slightly after multiple doses and it has been suggested that favipiravir is capable of reaching a cmax in humans sufficient to inhibit 90 % of sars-cov-2 replication, thus establishing it as an important compound in the ongoing search for covid-19 therapies (arshad et al., 2020) . marked differences in cmax have been observed between japanese and american patients with cmax values in japanese subjects being on average 13.26 µg/ml greater than those in american subjects (pmda, 2014) . this highlights the need for relevant covid-19 clinical trials to include a diverse range of subjects so that factors such as weight and ethnicity can be considered to optimise dose. the bioavailability of favipiravir is high at 97.6 % and only 54 % of the drug is plasma protein-bound, suggesting high tissue penetration would be likely (madelain et al., 2016; pmda, 2014) . in vivo work in mice showed that the half-life of favipiravir in the lungs is double that of favipiravir in plasma, indicating slower elimination from the lungs (pmda, 2014) . this is thought to be of high importance in covid-19, where viral load is particularly high in the lungs. for influenza treatment in adults, 1600 mg favipiravir is given twice on day 1 of treatment, followed by 600 mg twice daily from days 2 to 5 (pmda, 2014). however, the dosing period has been extended in ongoing covid-19 clinical trials: up to 10 days in chictr2000029996 and 14 days in chictr2000029548 (guan et al., 2020) . it is therefore essential that all pk parameters are monitored in these trials as differences, including increased cmax and decreased clearance, are expected during this prolonged dosing regimen which may impact upon safety. favipiravir has been linked to teratogenicity and embryotoxicity, and is therefore contraindicated in pregnancy (yousuke furuta et al., 2013) . overall, favipiravir is generally thought to have a good safety profile (asrani, devarbhavi, eaton, & kamath, 2019; group, 2020; nhs, 2019) . this is likely to be due to the fact that unlike other antiviral drugs such as ribavirin, favipiravir does not appear to disrupt non-viral rna or dna synthesis. however, very little is known about the long-term safety of favipiravir, as in previous clinical trials patient follow-up has been as little as 5 days . this is perhaps less of a concern in covid-19 as treatment is time-limited. drug-drug interactions have been reported with favipiravir. for example, coadministration with favipiravir can increase exposure to paracetamol by around 15 %, which may be a concern for patients with pre-existing liver disease as paracetamol is the leading cause of acute drug-induced liver injury (dili) in the uk and usa (asrani et al., 2019; group, 2020) . favipiravir can also increase patient exposure to many contraceptives, including progesterone-only pills, combined pills, and several contraceptive implants, which may cause discomfort, prolonged vaginal bleeding, and nausea (group, 2020; nhs, 2019) . whether the increased exposure to oestrogens caused by concomitant treatment with favipiravir can enhance the risk of thrombosis is not known but should be monitored, given the overwhelming evidence that covid-19 increases the risk of blood clots (atallah, mallah, & almahmeed, 2020; di micco et al., 2020; spiezia et al.) . interestingly , large clots are most common in patients under the age of 50; almost 25 % of women aged between 15 -49 in the usa currently use either oral or long-acting contraceptives, and thus represent a particular risk group (hurley, 2020; prevention, 2019). sars-cov-2 virus is capable of eliciting an immune reaction in the infected individual. laboratory examinations have revealed that inflammatory factors such as interleukin (il)-6, il-1, il-10 and tumour necrosis factor-α (tnfα) are upregulated during infection and can instigate an inflammatory response in the lower airways leading to lung injury in some instances guo et al., 2020) . additionally, in patients with severe symptoms of covid-19, there may be activation of a cytokine storm, which can cause significant tissue damage (mehta, mcauley, et al., 2020; shi et al., 2020) . a smaller proportion of patients can progress to a hyper-inflammatory state which in covid-19 has been suggested to resemble secondary haemophagocytic lymphohistiocytosis (shlh), a rare syndrome characterised by uncontrollable fever, cytopenia, raised ferritin levels and acute respiratory distress (seguin, galicier, boutboul, lemiale, & azoulay, 2016) . interleukin and tnf-α levels show the greatest increase in those who require admission to the intensive care unit (icu), suggesting that the cytokine storm is instrumental in severe covid-19 cases (huang et al., 2020) . therefore, there has been a logical progression towards the use of immunosuppressive agents as potential therapies to alleviate inflammation and hyperinflammation associated with covid-19 (mehta, mcauley, et al., 2020) . dexamethasone is a glucocorticoid that can be administered both orally and intravenously. it acts as a glucocorticoid receptor agonist and is over 20 times more potent than endogenous cortisol, thus resulting in dose-dependent suppression of pro-inflammatory genes through a number of pathways in common with other steroids (papich, 2016; whelan & apfel, 2013; yasir & sonthalia, 2019) . low doses of glucocorticoids have an anti-inflammatory effect while higher doses are immunosuppressive (buttgereit et al., 2002) . dexamethasone can be used for inflammatory diseases such as rheumatoid arthritis (crohn's & colitis foundation, 2018; freeman, 2008) , but is recommended for short-term treatment (spanning from one to 21 days) because of the major adverse effects which can occur with long-term treatment. one of the commonest uses of dexamethasone is for reducing cerebral oedema. as of 8 th july 2020, dexamethasone was undergoing evaluation in 17 clinical trials. on 16 th june, the results of the dexamethasone arm of the recovery trial were announced. the trial results, which are available in preprint form, showed that 2104 patients had received either oral or intravenous lowdose (6 mg) dexamethasone daily for ten days (peter horby et al., 2020) . when compared to 4321 control patients receiving usual care only, it was shown that dexamethasone reduced deaths by one third in sars-cov-2 positive patients requiring ventilation, and by one fifth in patients receiving this article is protected by copyright. all rights reserved. oxygen. no benefit was observed for patients with milder covid-19 symptoms who did not require respiratory support (peter horby et al., 2020) . recent work has found that tissue inflammation and organ dysfunction seen in fatal cases of covid-19 are not consistent with sars-cov-2 distribution in tissues and cells (dorward et al., 2020) . tissuespecific tolerance to the virus may therefore important, and suggests that fatalities arising from covid-19 may be mainly due to host-mediated immune response rather than pathogen-mediated end-organ inflammation. this is consistent with the dexamethasone result in the recovery trial. dexamethasone has a bioavailability of 70-78 % and is 77 % protein bound in plasma (spoorenberg et al., 2014) . it is 6-hydroxylated by hepatic cyp3a4 to 6α-and 6β-hydroxy-dexamethasone, and can also be reversibly metabolised to 11-dehydroxymethasone and back to dexamethasone by renal corticosteroid 11-beta-hydrogenase isozyme 1 (diederich et al., 1998; diederich, hanke, oelkers, & bähr, 1997; tomlinson, maggs, park, & back, 1997) . unlike many glucocorticoids which are predominantly excreted in urine, only about 10% of dexamethasone is excreted in urine (dexcel pharma technologies ltd.). glucocorticoids are generally safe drugs when given at low doses and for short periods of time (< 3 weeks), with the risk of adverse events increasing with dose and therapy duration (yasir & sonthalia, 2019 ). short-term use of dexamethasone can result in increased appetite, mood changes, and insomnia, but most of the adverse reactions are self-limiting (nhs, 2020). dexamethasone can lead to b and t cell depletion, and hence lymphopenia (marinella, 2020) , which interestingly is also found in up to 80 % of patients with covid-19 (liu, blet, smyth, & li, 2020) . however, despite this, the recovery trial was able to show a mortality benefit in the most severely affected covid-19 patients. a critical issue may be the dose that is administered -in recovery, 6 mg/day was administered over 10 days, which is a relatively low dose. a recent systematic review and meta-analysis of corticosteroid treatment in patients with coronavirus infection suggested that corticosteroids were associated with higher rates of bacterial infections, longer time spent in hospital and higher rates of mortality (z. yang et al., 2020) . however, most of the studies analysed in this meta-analysis were retrospective observational studies, generally of poor quality and did not analyse the effects according to steroid dose. other studies which have used low-to-moderate-dose corticosteroids as treatment for diseases such as viral and bacterial pneumonia reflect the results of the recovery trial, with low dose corticosteroids resulting in decreased mortality and morbidity in patients with severe pneumonia (h. li et al., 2017; stern et al., 2017) . in these studies, low-to-moderate dose corticosteroids (40-50mg prednisolone, which equates to 6-7.5 mg dexamethasone) were given to patients for between 7 and 10 days (stern et al., 2017) (national institute for health and care excellence, 2020b). in keeping with the known adverse effects of corticosteroids, the systematic review showed that hyperglycaemia was significantly more frequent in the corticosteroid-treated group (stern et al., 2017) . dexamethasone can be involved in both pharmacokinetic and pharmacodynamic interactions. combining it with other immunosuppressants may increase the risk of serious infection (national institute for health and care excellence, 2020c). co-treatment with ibuprofen or other nsaids increases the risk of gastrointestinal bleeding (national institute for health and care excellence, 2020c), while its gluconeogenic effects can lead to hyperglycaemia, which in diabetic patients can lead to increased insulin doses being required (consilient health ltd., 2020). dexamethasone is a cyp3a4 inducer, and may therefore interact with remdesivir, a cyp3a4 substrate, potentially reducing its plasma exposure. although clinicians should be aware of this interaction, the risk is small given that both drugs are indicated for 10 days or less. both tocilizumab and sarilumab are humanised anti-il-6 receptor monoclonal antibodies used for the treatment of moderate -severe rheumatoid arthritis, whereas siltuximab is a chimeric, human-mouse anti-il-6 receptor monoclonal antibody used for treatment of multicentric castleman's disease (mcd) (deisseroth et al., 2015 ; national institute for health and care excellence, 2020e). due to their long half-life, il-6 inhibitors do not need to be taken daily; however, given that they are currently indicated for chronic diseases, patients receive il-6 inhibitor treatments for life or until treatment failure (janssen biotech inc; roche pharma; sanofi-aventis). clinical trials to assess the efficacy and safety of tocilizumab, sarilumab and siltuximab for the treatment of the inflammatory phase of covid-19 are ongoing. whilst the exact dosing regimens vary between trials, covid-19 patients will be receiving a single or short course intravenous infusion or subcutaneous injection of the il-6 inhibitor (clinical trial identifiers nct04317092, nct04315298, nct04327388, nct04330638, nct04322188). due to their similarity, it is not surprising that tocilizumab, sarilumab and siltuximab have comparable safety profiles. thus far, evidence from clinical trials in patients with rheumatoid arthritis and mcd or post-marketing have revealed that il-6 inhibitors are generally well-tolerated. participants were enrolled on these trials for a minimum of 6 months and in some cases up to 24 months. individuals with diabetes, a history of recurrent infection, age ≥ 65 and corticosteroid use have been shown to be at an increased risk of developing a more serious infection following il-6 inhibitor use (jones et al., this article is protected by copyright. all rights reserved. 2010). whilst adverse reactions were typically seen following chronic il-6 inhibitor treatment, the potential for covid-19 patients to develop an adverse drug reaction (adr) following a single or small number of doses should not be ignored. the most common infections reported in patients receiving anti-il6 therapy include skin infections, respiratory infections, urinary tract infections and in some cases, opportunistic infections ranging from tuberculosis to herpes (emery et al., 2008; smolen et al., 2008) . liver injury has also been reported with a liver biopsy from a female patient who had taken tocilizumab for a month revealing focal this article is protected by copyright. all rights reserved. necrosis of hepatocytes with steatosis and early fibrosis (mahamid et al., 2011) . covid-19 also has effects on the liver, and again causality assessment may be difficult (guan et al., 2020) . the prescribing instructions for tocilizumab and sarilumab indicate that liver function tests are required every 4 -8 weeks following treatment commencement and then every 3 months thereafter (roche pharma; sanofi-aventis). if liver enzymes are 1 -3 x uln, the dose of tocilizumab and sarilumab can be reduced until alt or ast have normalised and then treatment resumed at the therapeutic dose. where laboratory findings are > 3 -5 x uln, treatment with il-6 inhibitors must be paused and then recommendations for 1 -3 x uln followed. if elevations persist or are > 5 x uln, tocilizumab and sarilumab treatment must be discontinued immediately (roche pharma; sanofi-aventis). whilst sarilumab and siltuximab are associated with abnormalities in liver function tests, they are typically short-lived and asymptomatic (livertox, 2016 (livertox, , 2017b . pre-existing liver disease can worsen symptoms of dili, and in some cases increase susceptibility (david & hamilton, 2010) . tocilizumab, sarilumab and siltuximab are expected to undergo metabolism via catabolic pathways and not cyp450 processes (mccarty & robinson, 2018) . therefore, due to the lack of hepatic metabolism, it is assumed that the pk of the il-6 inhibitors will not be altered in patients with preexisting liver disease (abou-auda & sakr, 2010). however, tocilizumab, sarilumab and siltuximab have been shown to restore and improve cyp levels (janssen biotech inc, 2019; roche pharma, 2013; sanofi-aventis, 2017). this is of particular importance as cyp levels may remain elevated following treatment discontinuation due to the long half-life of the compounds. therefore, this may be a consideration for further evaluation for any dosing adjustment requirements if patients are taking medication that are metabolised by cyp enzymes. anakinra is a 17 kd, recombinant human il-1 receptor antagonist that blocks the activity of proinflammatory cytokines il-1α and il-1β (cawthorne et al., 2011; dinarello, simon, & van der meer, 2012) . anakinra is primarily used in combination with methotrexate for reducing the symptoms and slowing the progression of joint damage in rheumatoid arthritis (national institute for health and care excellence, 2020a). it is also used for rare inflammatory conditions such as cryopyrin-associated periodic syndromes and still's disease (national institute for health and care excellence, 2020a). it is administered via subcutaneous injection and is supplied as a single-use, pre-filled syringe containing 100 mg/0.67 ml (swedish orphan biovitrum ltd, 2007) . rheumatoid arthritis patients and those with still's disease and a body weight > 50 kg must be administered 100 mg anakinra, while patients with still's disease with a body weight < 50 kg should have weight-based dosing starting at 1 -2 mg/kg this article is protected by copyright. all rights reserved. (swedish orphan biovitrum ltd, 2007). the recommended starting dose for patients with cryopyrinassociated periodic syndromes is 1 -2 mg/kg. if tolerated, the dose can be increased to 3 -4 mg/kg to a maximum of 8 mg/kg (swedish orphan biovitrum ltd, 2007) . anakinra has a short terminal half-life of approximately 4 -6 hours and so must be administered daily, preferably at the same time each day (amgen inc., 2001) . anakinra is currently not licensed for intravenous administration or treatment of shlh but its use is endorsed by clinicians, where intravenous infusion, as opposed to subcutaneous injection, can achieve quicker and greater maximal plasma concentrations (carter, tattersall, & ramanan, 2018; la rosée et al., 2019; mehta, cron, hartwell, manson, & tattersall, 2020) . thus far, 21 clinical trials have been registered to assess the use of anakinra in patients with severe covid-19. additionally, two recent studies have reported positive outcomes with anakinra in covid-19 induced acute respiratory distress syndrome (cavalli et al., 2020; clinical trials.gov, 2020c; huet et al., 2020) . participants were dosed 100 mg twice daily subcutaneously for 72 hours followed by 100 mg daily for 7 days in addition to standard of care (huet et al., 2020) . this retrospective study found that anakinra reduced rates of mortality and the need for mechanical ventilation in icu patients (huet et al., 2020) . anakinra was administered either subcutaneously or intravenously in the covid-19 biobank study (huet et al., 2020) . participants received subcutaneous injections at a dose of 100 mg twice daily or via slow intravenous infusion at 10 mg/kg per day until there was a 75 % reduction in serum c-reactive protein levels and sustained respiratory improvements (cavalli et al., 2020) . whilst no safety concerns emerged with anakinra administered subcutaneously, it was discontinued due to a lack of clinical improvement and limited reduction in c-reactive protein (cavalli et al., 2020) . by contrast, intravenous anakinra was well-tolerated and improved clinical outcomes. notably, 72 % of patients had improved respiratory function in comparison to 50 % within the standard treatment group (cavalli et al., 2020) . in both studies, cases of alt ≥ 3 x uln were observed in both the anakinra and the standard treatment arms. four cases of bacteraemia following intravenous anakinra were reported in the covid-19 biobank study, but there were no cases of bacterial infection in the ana-covid study (cavalli et al., 2020; huet et al., 2020) . whilst both studies are encouraging, they should be considered proof-of-concept trials and larger randomised trials are still needed (cavalli et al., 2020; huet et al., 2020) . subcutaneous administration of anakinra is associated with injection site reactions (kaiser et al., 2012) . in a review of five rheumatoid arthritis clinical trials, 71 % of participants receiving anakinra therapy reported injection site reactions in comparison to 28 % of participants on placebo (mertens & singh, 2009) . injection site reactions can range from immediate to delayed. in immediate cases, the reaction manifests as a burning sensation whereas delayed reactions present as a rash, pruritus or swelling (kaiser et al., 2012) . anakinra has also been reported to lead to infection, neutropenia, this article is protected by copyright. all rights reserved. thrombocytopenia, headache, and blood cholesterol increase when administered subcutaneously (swedish orphan biovitrum ltd, 2007) . injection site reactions that arise immediately can be eased by placing an ice pack on the injection site before and after anakinra administration and delayed reactions can be treated with topical corticosteroids or anti-histamines (kaiser et al., 2012) . increases in serious infection rate are common following anakinra use and frequently include upper respiratory infections, sinusitis, urinary tract infection and bronchitis (bresnihan et al., 1998; cohen et al., 2002; r. m. fleischmann et al., 2003) . whilst rare, cases of opportunistic infection have been reported in anakinra monotherapy or in those receiving anakinra in combination with immunosuppressive agents (salvana & salata, 2009; swedish orphan biovitrum ltd, 2007) . neutrophil counts must be monitored during the first six months of anakinra treatment and quarterly henceforth (swedish orphan biovitrum ltd, 2007) . in patients where the anc is < 1.5 x 10 9 /l, treatment must be discontinued immediately (swedish orphan biovitrum ltd, 2007) . the higher doses being used in covid-19 trials and the potential for a greater cmax due to intravenous administration potentially raise additional safety concerns. however, earlier detection of aes should be possible since the duration of treatment will be shorter than that used in rheumatoid arthritis, coupled with the fact that patients will already be hospitalised. anakinra is catabolised and eliminated via glomerular filtration (swedish orphan biovitrum ltd, 2007; b.-b. yang, baughman, & sullivan, 2003) . caution should be exercised and dose-adjustments may be required in moderate to severe renal impairment (swedish orphan biovitrum ltd, 2007; b.-b. yang et al., 2003) . during general infections and inflammatory diseases, cyp enzymes are primarily downregulated (mallick, taneja, moorthy, & ghose, 2017) . similar to il-6 inhibitors, it may be possible that anakinra treatment restores cyp levels in infected patients (swedish orphan biovitrum ltd, 2007) . therefore, caution should be exerted in covid-19 patients receiving concomitant medications with a narrow therapeutic window drug. mild interactions can occur between anakinra and warfarin, clopidogrel, clozapine and phenytoin (group, 2020) . baricitinib is an oral disease-modifying anti-rheumatic drug (dmard), traditionally used in the treatment of moderate to severe active rheumatoid arthritis (al-salama & scott, 2018). by acting as an atp-competitive kinase inhibitor, baricitinib can selectively and potently inhibit janus kinases (jaks) -1 and -2 in a reversible manner. jaks are essential in the transduction of intracellular signals for various cytokines involved in the inflammatory and immune responses, and so by inhibiting these kinases, baricitinib is able to relieve symptoms of rheumatoid arthritis for many patients (fridman et al., 2010) . as described previously, a common characteristic of covid-19, much like another beta-coronavirus disease sars, is a profuse inflammatory response (huang et al., 2020; stebbing et al., 2020) . increased levels of pro-inflammatory cytokines, such as interferon (ifn) -γ and il-1β, have been observed in confirmed covid-19 cases (huang et al., 2020; mehta, mcauley, et al., 2020; russell et al., 2020) . furthermore, the levels of some specific cytokines appear to be related to disease severity; patients requiring admission to intensive care units show increased levels of tnfα and monocyte chemoattractant protein 1 (mcp1). the rationale behind repurposing baricitinib as a treatment for covid-19 is centred on this potential for severely ill patients to present with a cytokine storm (mehta, mcauley, et al., 2020; russell et al., 2020) . by dampening the inflammatory response, it is postulated that baricitinib will be able to relieve covid-19 symptoms. data modelled using artificial intelligence techniques suggests baricitinib may work by inhibiting virus entry into cells via an endocytic regulator known to be involved in coronavirus internalisation, ap2-associated protein kinase 1 (aak1) (burkard et al., 2014; richardson et al., 2020) . baricitinib, as well as being capable of jak1 and jak2 inhibition, is a high-affinity inhibitor of aak1 . patients tend to tolerate baricitinib well, and it has a relatively good safety profile (keystone et al., 2015) . however, as with tocilizumab and sarilumab treatment, a very common (≥ 1/10) ae observed in patients taking baricitinib, but not in the placebo arm, is upper respiratory tract infection, which may be related to its ability to suppress the immune system (eli lilly, 2017) . patients taking baricitinib have the potential to develop respiratory tract infections which may make it difficult to distinguish whether any deterioration is due to covid-19 or a secondary infection. other opportunistic infections including herpes zoster and urinary tract infections were also more common in the treated arm compared to placebo, and dose reduction is recommended for patients with a history of chronic infections (eli lilly, 2017; josef s. smolen et al., 2018) . secondary infections are not uncommon in severe covid-19 patients and so the use of a drug that may make patients increasingly prone to infections will depend on the harm-benefit ratio for severe cases of covid-19 (world health organisation, 2020a). baricitinib is currently still being trialled in patients with covid-19 with a therapeutic dose of 2 -4 mg once daily which is the same as the recommended dosage for the treatment of rheumatoid arthritis (cantini et al., 2020; richardson et al., 2020) . there have been a small number of reports from patients taking this recommended dosage for the treatment of rheumatoid arthritis presenting with deep vein thrombosis (dvt), which was severe in some of these cases (taylor et al., 2019) . this is a cause for this article is protected by copyright. all rights reserved. concern as there are increasing reports of covid-19 patients, especially those who are critically ill and in the icu, with thrombotic complications including pulmonary embolism and other venous and arterial thrombotic events (klok et al., 2020; middeldorp et al.) . as baricitinib has been reported to cause dvt, there is the potential for disease-drug interactions with covid-19 patients taking baricitinib potentially more likely to develop thrombotic complications. in order to mitigate this risk, alternative jak inhibitors, which have a lower risk of thrombotic events, such as ruxolitinib, may be considered in the context of covid-19 (alvarez-larran et al., 2018) . however, unlike baricitinib, ruxolitinib is primarily metabolised by cyp3a4 (l. p. h. yang & keating, 2012) . this means that prescribing ruxolitinib instead of baricitinib may increase the risk of cyp3a4-related ddis (ogu & maxa, 2000) . baricitinib is not predicted to be involved in any problematic ddis. coadministration with both cyp3a inhibitors (fluconazole) and inducers (rifampicin) failed to result in any clinically relevant changes to baricitinib exposure (eli lilly, 2017). emerging reports have revealed that patients with covid-19 experience renal impairment, which could be attributed ace2 receptor expression on kidney endothelial cells (varga et al., 2020) . baricitinib should not be given to patients with renal impairment as the majority of the drug is cleared through the kidneys, and monitoring of renal function will be important to prevent aes related to over-exposure to baricitinib in those with deteriorating renal function (eli lilly, 2017) . type 1 ifns are a group of cytokines produced during viral infection. notably, ifn-β-1a has a leading role in activating genes involved in immunomodulation, suppressing the inflammatory response and anti-viral effects (sallard, lescure, yazdanpanah, mentre, & peiffer-smadja, 2020) . whilst a variety of type 1 ifns exist, in vitro evidence has shown that ifn-β-1a and ifn-β-1b are the most potent in the inhibition of sars-cov and mers-cov (chan et al., 2013; hensley et al., 2004) . within the lungs, ifnβ-1 has been shown to upregulate levels of the enzyme cluster of differentiation 73 (cd73), which inhibits vascular leakage, increases the secretion of anti-inflammatory adenosine and preserves pulmonary endothelial barrier function (kiss et al., 2007; sallard et al., 2020) . however, in vivo research has revealed that timing of administration of ifn-β-1 is imperative for positive effects. when administered shortly after mers-cov infection, ifn-β-1 protected mice from lethal infection, whereas delayed administration failed to effectively inhibit viral replication or pro-inflammatory cytokines, leading to fatal pneumonia (channappanavar et al., 2019) . interestingly, in vitro evidence has revealed this article is protected by copyright. all rights reserved. that sars-cov-2 is more sensitive to ifn-β-1 treatment than mers-cov and sars-cov, and thus supports the tenet that treatment with ifn-β-1 may be beneficial for covid-19 patients (lokugamage, schindewolf, & menachery, 2020; sheahan et al., 2020; thiel & weber, 2008) . it is assumed that treatment of covid-19 patients with ifn-β-1 will strengthen the host immune response and prevent the worsening of severe respiratory tract manifestations. ifn-β-1 therapy has been used for the long-term management of multiple sclerosis (ms) and has been associated with a number of aes. when administered subcutaneously in ms patients, the most common aes were flu-like symptoms, injection site reactions, worsening of ms symptoms, menstrual disorders, mood alterations and laboratory abnormalities (walther & hohlfeld, 1999) . the most common laboratory abnormalities were neutropenia, leukopenia, lymphopenia and raised aminotransferases (walther & hohlfeld, 1999) . a genome-wide association study of patients with ifnβ induced liver injury showed that rs2205986 which has been linked to differential expression of interferon regulatory factor (irf)-6 is a predisposing factor (kowalec et al., 2018) . this may be related to the fact that irf6 leads to apoptosis in the presence of ifn-β. depression is a common ae reported in patients receiving subcutaneous ifn-β-1 therapy, and thus caution is needed when administering to those with a previous or current history of depressive disorder (biogen) . whilst rare, careful monitoring of clinical manifestations such as new onset hypertension, thrombocytopenia, impaired renal function and fever are required in order to identify cases of thrombotic microangiopathy (tma) (biogen) . tma is rare and has been reported at different time points of ifn-β-1 therapy (biogen; nishio et al., 2016; yam, fok, mclean, butler, & kempster, 2018) . laboratory findings of a decreased platelet count, increased serum lactate dehydrogenase (ldh) and red blood cell fragmentation are suggestive of tma (biogen) . if diagnosed, patients must discontinue ifn-β-1 therapy and will require plasma exchange (biogen) . sng001 is an inhaled form of ifn-β-1a produced by synairgen. the company have tested the efficacy and safety of the drug for the prevention and treatment of symptoms associated with respiratory viral infection in asthma and chronic obstructive pulmonary disease (copd) (synairgen plc, 2018). a randomised, placebo-controlled phase 2 trial is currently ongoing to assess the safety and efficacy of inhaled sng001 for the treatment of patients with covid-19 (nct04385095). data from the asthma trials have revealed that when administered via inhalation, high levels of ifn-β-1a are achieved within the lungs with lower levels within the circulation leading to improvements in lung function, antiviral responses and better asthma control (djukanović et al., 2014) . inhaled sng001 seems to have a good safety profile; 5 patients within the sng001 arm reported cardiac palpitations whereas no cases were reported in the placebo arm, but symptoms were mild and not considered clinically significant (djukanović et al., 2014) . this article is protected by copyright. all rights reserved. a clinical trial has been undertaken in hospitalised covid-19 patients where the triple combination of ifn-β, lopinavir-ritonavir and ribavirin was compared to lopinavir-ritonavir and ribavirin (hung et al., 2020; shalhoub, 2020) . patients in the triple combination therapy arm achieved negative tests results faster than those in the control arm, with improved patient symptoms, decreased viral shedding and decreased overall length of stay in the hospital compared to those in the control group (hung et al., 2020) . aes reported in both groups included nausea and diarrhoea. however, due to polypharmacy in this trial, it was difficult to determine the effect of ifn-β on sars-cov-2 alone. ifn-β has reported ddis with other covid-19 therapies including chloroquine and hyrdroxychloroquine, and with anakinra, sarilumab and tocilizumab (group, 2020) . ddis have also been reported with metamizole (analgesic), linezolid (antibacterial), clozapine (antipsychotic), zidovudine (hiv antiretroviral therapy) and some immunosuppressants (adalimumab, azathioprine and pirfenidone) (group, 2020) . reviewing the safety of potential covid-19 treatments (table 1) is complex due to the fast-moving pace of research in this field. for example, chloroquine and hydroxychloroquine with or without an accompanying macrolide antibiotic, have consistently been at the forefront of covid-19 research efforts since the outbreak began. however, the astonishing developments over a week or so have led to retraction of a highly publicised paper, and results from a post-exposure prophylaxis trial and a treatment trial (recovery), both of which have shown no beneficial effect of hydroxychloroquine (boulware et al., 2020; mehra, ruschitzka, & patel, 2020) . this highlights that the rapid rate of discoveries surrounding covid-19 therapies generates the need to update this perspective frequently, in order to ensure that the safety of any newly repositioned therapies, novel developmental compounds, or new therapeutic combinations are investigated. for example, the potential use of heparin in novel forms, including nebulised therapy (clinical trial identifier nct04397510), as an antiviral agent is currently the subject of several investigational trials. in addition, the potential utility of nitazoxanide is currently the subject of several clinical trials (clinical trials.gov, 2020a; pepperrell, pilkington, owen, wang, & hill, 2020; rajoli et al., 2020) . it is clearly essential that the harm:benefit ratio of any pharmaceuticals being considered for use in the treatment of covid-19 are thoroughly considered. this ratio changes dependent upon the disease stage and is correlated to potential mortality. for example, a higher risk may be accepted for patients in the later stage of severe disease than the same therapeutic agent administered in mild disease. this difference in harm-benefit analysis becomes even more striking when considering the use of such agents to prevent infection. as is the case for many highly contagious viruses, prevention by prophylaxis would be incredibly valuable. some of the agents described in this review, including chloroquine and ritonavir have been suggested as potential prophylactic agents, but to date, data on efficacy have been disappointing (rathi, ish, kalantri, & kalantri, 2020; spinelli, ceccarelli, di franco, & conti, 2020) . clearly, treatment duration for prophylaxis is expected to be longer than for treatment of covid-19, and this may further alter the harm-benefit ratio, reinforcing the need for safety considerations at the outset of any clinical trials. similarly, the evaluation of therapy risk also applies to long-term recovery. as the current pandemic progresses, it is becoming apparent that being discharged from hospital does not necessarily mean that patients are free from covid-19 symptoms. large numbers of patients who have survived severe sars-cov-2 infection may have incurred long-term health problems, including some permanent loss of lung and kidney function (foundation, 2020; su et al., 2020; summers, 2020) . consequently, it is probable that long-term therapies will be required for many patients to maintain, or ideally restore, normal physiological organ function. it is vital that therapies which will be used to treat patients during their long-term recovery are also undergoing evaluation for their safety, particularly as many of these agents may need to be administered over much longer periods of time than initial covid-19 treatments. the identification and characterisation of biomarkers of disease and safety will be invaluable in the further development and deployment of therapies for covid-19. disease biomarkers, for example of lung injury or the hyperinflammatory reponse, may allow the stratification of therapy in order to select the agent best suited to the stage of disease. moreover, biomarkers should be considered to monitor patient safety in cases of known aes. for example, the manufacturer's guidelines for remdesivir recommend daily liver function tests due to the risk of transaminase elevations (gilead, 2020) . these tests are essential, particularly with regards to covid-19 where increased alt levels are reported to be common amongst hospitalised patients (bangash et al., 2020; l. zhang et al., 2009 ). looking to the future, improvements in the specificity, predictivity and reliability of drug-induced organ damage, through academic-industry partnerships such as the biomarker qualification program in the critical path institute in the us, and the european innovative medicines initiative consortium transbioline, will help improve clinical assessment of covid-19 drug safety issues. continued enhancements in the speed, predictivity, and human translation of safety assessment for toxicity of anti-viral compounds is clearly warranted, and this may include animal models of sars-cov-2 as well as in vitro models, in order to assess efficacy alongside safety. such a full understanding for individual therapies will indicate the combinations that can have the potential to provide the best this article is protected by copyright. all rights reserved. synergy for benefit, while forewarning of the potential for increased risk/harm through pharmacokinetic or toxicodynamic interaction. although outside the scope of this review, a vaccine for covid-19 remains the greatest hope to end the pandemic and protect the population. as of 8 th july 2020, according to who there are 21 vaccines in clinical trial stages and 139 in preclinical stages of evaluation (world health organisation, 2020b). currently, potential vaccines are only just beginning to be tested for efficacy in humans in early phase studies, and therefore safety data will begin to emerge as larger numbers of individuals are administered the vaccine. safety data regarding preliminary vaccinations against sars and mers are limited, but the available information may be useful during the development of covid-19 vaccines due to the similarities between the coronavirus strains (padron-regalado, 2020). one safety concern relevant to coronaviruses is the potential for the induction of antibody-dependent enhancement (ade), a phenomenon which was observed in cats vaccinated against feline infectious peritonitis coronavirus, and has also been seen in patients vaccinated against zika virus and dengue virus (khandia et al., 2018; padron-regalado, 2020; vennema et al., 1990) . ade can occur when nonneutralising antibodies bind to virus particles and increase their uptake into host cells, instead of rendering them non-infectious (padron-regalado, 2020; tirado & yoon, 2003) . this caused concern in initial sars vaccine development, but can reportedly be avoided by using truncated versions of the viral s glycoproteins (he et al., 2004) . acknowledging safety concerns such as this, as well as the ways they can be attenuated, may be paramount in the timely development of a vaccine against covid-19. in conclusion, although expanding extremely rapidly, the field of therapies to treat covid-19 remains in its infancy. safety will continue to play a major role in therapeutic success, as apparent with recent reports of increased cardiac toxicity associated with the use of chloroquine/hydroxychloroquine in the treatment of covid-19, despite its long history of use as an antimalarial. above all, this perspective has exemplified the need to view safety concerns in the context of the individual and specific phase of disease in order to formulate a comprehensive harm-benefit balance. importantly, an awareness of potential safety concerns will support the development of the next stage of therapy targeting prophylaxis and recovery post-covid infection. it is imperative that safety scientists look to rise to the challenge of covid-19 by utilising their expertise in mechanistic understanding, biomarker development and toxicokinetic modelling in order to support the development of covid-19 therapies that can be used effectively and safely. aa, bkp, cbb, ceg, rlj, rtk, shk, slp and xm declare that that they have no conflicts of interest. ao declares no direct conflict of interest but is director and cso for tandem nano ltd and a co-inventor of patents relating to drug delivery of infectious disease medicines. aec reports no direct conflict of interest but receives research funding for the support of sp and rlj from servier pharmaceuticals and astrazeneca, these are unrelated to the published work. aec receives additional unrelated research funding from janssen pharmaceuticals. ao has received consultancy and /or research funding from viiv healthcare, merck, astrazeneca, gilead, and janssen unrelated to the current paper. db received educational grants and/or consultancy from abbvie, novartis, merck, gilead and viiv healthcare outside the submitted work. mp receives research funding from various organisations including the mrc, nihr, eu commission and health education england. he has also received partnership funding for the following: mrc clinical pharmacology training scheme (co-funded by mrc and roche, ucb, eli lilly and novartis); and a phd studentship jointly funded by epsrc and astra zeneca. he has also unrestricted educational grant support for the uk pharmacogenetics and stratified medicine network from bristol-myers squibb and ucb. none of the funding received is related to the current paper. figure 1 : overview of the mechanisms of action of the repurposed drugs undergoing clinical trials for the treatment of covid-19 that will be reviewed in this perspective. compounds in red represent those that are viral entry inhibitors, compounds in green represent disruptors of cellular viral processing, compounds in blue are modulators of the hyperinflammatory phase of infection and compounds in yellow stimulate host immunomodulatory and anti-viral activity. abbreviations: ace2, angiotensin converting enzyme 2; il-6, interleukin-6; il-1, interleukin-1; jak, janus kinase; rdrp, rna-dependent rna polymerases; stat, signal transducer and activator of transcription proteins; p, phosphate; nf-κb, nuclear factor kappalight-chain-enhancer of activated b cells; ifn-β, interferon-beta; isre, interferon stimulated response element. tocilizumab: a new anti-rheumatic drug broad-spectrum antiviral agents: a crucial pandemic tool coronavirus susceptibility to the antiviral 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coronavirus infection: a systematic review and meta-analysis in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) liver injury in covid-19: management and challenges scientific and regulatory perspectives on metabolizing enzyme-transporter interplay and its role in drug interactions: challenges in predicting drug interactions single-cell rna expression profiling of ace2, the putative receptor of wuhan 2019-ncov. biorxiv covid-19 and the cardiovascular system key: cord-324775-3x5os79m authors: crowe, j.e. title: human respiratory viruses date: 2008-07-30 journal: encyclopedia of virology doi: 10.1016/b978-012374410-4.00488-x sha: doc_id: 324775 cord_uid: 3x5os79m viruses are the leading causes of acute lower respiratory-tract infection in infancy. respiratory syncytial virus (rsv) is the most common pathogen, with hmpv, piv-3, influenza viruses, and rhinoviruses accounting for the majority of the remainder of acute viral respiratory infections. humans generally do not develop lifelong immunity to reinfection with these viruses; rather, specific immunity protects against severe and lower respiratory-tract disease. respiratory virus infections of humans are the most common and frequent infections of man. hundreds of different viruses can be considered respiratory viruses. viruses that enter through the respiratory tract include viruses that replicate and cause disease that is restricted to the respiratory epithelium, and other viruses that enter through the mucosa but also spread by viremia causing systemic disease. an example of the latter is measles virus. sars coronavirus is another. in general, viruses that do not cause viremia are capable of reinfecting the same host multiple times throughout life. in contrast, infections with systemic viruses induce lifelong immunity. probably, the high rate of reinfection of mucosally restricted viruses reflects the difficulty and metabolic cost of maintaining a high level of immunity at the vast surface area of the mucosa. virus-specific iga levels are maintained at high levels generally only for several weeks or months after infection. the anatomy and the cell types of the respiratory tract dictate to a large degree the type of disease observed during respiratory virus infection. the demarcation between the upper and lower respiratory tracts is the vocal cords. the structures of the upper respiratory tract, which are all interconnected, include the nasopharynx, the larynx, the eustachian tube and middle ear space, and the sinuses. significant collections of lymphoid tissue reside in the upper respiratory tract, the tonsils and the adenoids. the lower respiratory tract structures include the trachea, bronchi, bronchioles, alveoli, and lung tissue. the cell types that line the respiratory tract are complex, and exhibit different susceptibilities to virus infection. the predominant cell types are ciliated and nonciliated epithelial cells, goblet cells, and clara cells. smooth muscle cells are prominent features of the airways down to the level of the bronchioles, and the lung possesses type i and ii pneumocytes. the disease syndromes that are associated with respiratory viruses generally follow the anatomy of the respiratory tract. different viruses appear to have tropisms for different cells or regions of the respiratory tract; therefore, there are particular associations of viruses with clinical syndromes. the clinical diagnoses for infections with disease manifestations in the respiratory tract are rhinitis and the common cold, sinusitis, otitis media, conjunctivitis, pharyngitis, laryngitis, tracheitis, acute bronchitis, bronchiolitis, pneumonia, and exacerbations of reactive airway disease or asthma. clinical syndromes with more systemic illness due to respiratory viruses include the influenza syndrome, measles, severe acute respiratory syndrome (sars), and hantavirus pulmonary syndrome (hps). the principal causes of acute viral respiratory infections in children became apparent through large epidemiologic studies conducted soon after cell culture techniques became available. the landmark studies of association of viruses with clinical syndromes were performed in the 1960s and 1970s. recent studies have increased our understanding of the causes of viral respiratory infection in infants, especially because of the advent of molecular tests such as the polymerase chain reaction (pcr), which is more sensitive than cell culture. respiratory syncytial virus (rsv), parainfluenza viruses (pivs), adenoviruses, and influenza viruses were identified initially as the most common causes of serious lower respiratory tract disease in infants and children. more recently, human metapneumovirus (hmpv) was identified as a major cause of serious illness. in the last 10 years, a number of additional viruses have been associated with respiratory illness, as discussed below. however, still, infectious agents are not identified in 30-50% of clinical illnesses in large surveillance studies, even using sensitive diagnostic techniques such as viral culture on multiple cell lines, antigen detection assays, and rt-pcr based methods. it is not known if these illnesses are due to identified pathogens that are simply not detected due to low titers of virus in patient samples or if there are novel agents that are yet to be identified. reactivation of latent viruses, such as the herpesviruses hsv and cmv, and adenoviruses occurs in immunocompromised humans, particularly subjects with late-stage hiv infection, those with organ transplantation, and patients with leukopenia and neutropenia caused by chemotherapy. cmv is the most frequently recovered virus from diagnostic procedures such as bronchoalveolar lavage in immunosuppressed patients with pneumonia. these patients also suffer more frequent and more severe disease including mortality with common respiratory viruses, including rsv, hmpv, piv, influenza viruses, rhinoviruses, and adenoviruses. nosocomial transmission including large unit outbreaks is not uncommon, and can result in high frequency of transmission. picornaviridae a wide variety of picornaviruses cause respiratory disease, including rhinoviruses, the enteroviruses a to d including coxsackieviruses a/b, echoviruses, non-polio enteroviruses, and parechoviruses 1-3. enterovirus infections occur most commonly in the summer months in temperate areas, which differs from the season of many of the other most common respiratory viruses such as paramyxoviruses and influenza virus. rhinovirus infections occur year-round. rhinovirus is a genus of the family picornaviridae of viruses. rhinoviruses are the most common viral infective agents in humans, and a causative agent of the common cold. there are over 105 serologic virus types that cause cold symptoms, and rhinoviruses are responsible for approximately half of all cases of the common cold. rhinoviruses have single-stranded positive-sense rna genomes. the viral particles are icosahedral in structure, and they are nonenveloped. rhinovirus-induced common colds may be complicated in children by otitis media and in adults by sinusitis. most adults, in fact, have radiographic evidence of sinusitis during the common cold, which resolves without therapy. therefore the primary disease is probably best termed rhinosinusitis. rhinovirus infection is associated with exacerbations of reactive airway disease in children and asthma in adults. it is not clear whether rhinovirus is restricted to the upper respiratory tract and induces inflammatory responses that affect the lower respiratory tract indirectly, or whether the viruses spread to the lower respiratory tract. in the past, it was thought that these viruses did not often replicate or cause disease in the lower respiratory tract. however, recent studies discern strong epidemiological associations of rvs with wheezing and asthma exacerbations, including episodes severe enough to require hospitalization. likely, rhinoviruses can infect the lower airways to some degree, inducing a local inflammatory response. another possibility is that significant local infection of the upper respiratory tract might induce regional elaboration of mediators that causes lower airways disease. association of rhinovirus infection with lower respiratory tract illness is difficult to study because cell diagnosis by cell culture is not sensitive. rt-pcr diagnostic tests are difficult to interpret because they are often positive for prolonged periods of time and even asymptomatic individuals may have a positive test. comprehensive serologies to confirm infection are difficult because of the large number of serotypes. nevertheless, most experts believe rhinoviruses are a common cause of lower respiratory tract illness. these viruses cause oral lesions and often are associated in children with a disease syndrome termed 'hand-footand-mouth disease'. the pharyngitis associated with this infection often is marked by the very characteristic findings of herpangina, a clinical syndrome of ulcers or small vesicles on the palate and often involving the tonsillar fossa associated with the symptoms of fever, difficulty swallowing, and throat pain. outbreaks commonly occur in young children, in the summer. non-polio enteroviruses are common and distributed worldwide. although infection often is asymptomatic, these viruses cause outbreaks of clinical respiratory disease, sometimes with fatal consequences. studies have associated particular types with clinical syndromes, as enterovirus 68 with wheezing and enterovirus 71 with pneumonia. the term 'echo' in the name of the virus is an acronym for enteric cytopathic human orphan, although this may be an archaic notion since most echoviruses are associated with human diseases, most commonly in children. there are at least 33 echovirus serotypes. echoviruses can be isolated from many children with upper respiratory tract infections during the summer months. echovirus 11 has been associated with laryngotracheitis or croup. epidemiology studies also have associated echoviruses with epidemic pleurodynia, an acute illness characterized by sharp chest pain and fever. these viruses have been assigned a new genus of the family picornaviridae because of distinctive laboratorybased molecular properties. the most common member of the genus parechovirus, human parechovirus 1 (formerly echovirus 22) is a frequent human pathogen. the genus also includes the closely related virus, human parechovirus 2 (formerly echovirus 23). human parechoviruses usually cause mild respiratory or gastrointestinal illness. most infections occur in young children. there is a high seroprevalence for parechoviruses 1 and 2 in adults, and a few clear descriptions of neonatal cases of severe disease. respiratory syncytial virus rsv is a single-stranded negative-sense nonsegmented rna genome virus of the family paramyxoviridae, genus pneumovirus. it is one of the most infectious viruses of humans and infects infants at a very young age, often in the first weeks or months of life. it is the most common viral cause of severe lower respiratory tract illness in children and one of the most important causes of hospitalization in infants and children throughout the world. there is one serotype, but circulating viruses exhibit an antigenic dimorphism such that there are two antigenic subgroups designated a and b. reciprocal cross-neutralization studies using human sera showed that the antigenic groups are about 25% related. reinfection is common and can be caused by viruses of the same subgroup. yearly, epidemics of disease often peak between january and march in temperate regions. rsv infection causes mild upper respiratory tract infection in most infants and young children, but results in hospitalization in 0.5-1% of infants. most children have been infected by the age of 2 years. there is an association of rsv infection early in life and subsequent asthma, although a causal relationship is controversial. most hospitalized infants are otherwise healthy, but some groups are considered high risk for severe disease such as premature infants especially those with chronic lung disease and infants born with congenital heart disease. immunocompromised patients of any age are at risk of severe disease. these viruses constitute a group of four distinct serotypes (types 1-4) of single-stranded rnaviruses belonging to the family paramyxoviridae. when considered as a group, they are the second most common cause of lower respiratory tract infection in young children. piv3 is the most common cause of severe disease. repeated infection throughout life is common. first infections are more commonly associated with lower respiratory tract disease, especially croup, while subsequent infections typically are limited to the upper respiratory tract. pivs are detected using cell culture with hemadsorption or immunofluorescent microscopy, and rt-pcr. in 2001, investigators in the netherlands described a new human respiratory virus, hmpv. evidence of near universal seroconversion was found in the general population by 5 years of age, suggesting ubiquitous infection in early childhood. this virus, a member of the genus pneumovirus with rsv, differs from rsv in that it lacks the ns1 and ns2 nonstructural genes that counteract host interferons and it possesses a slightly different gene order. studies of the role of hmpv in pediatric lower respiratory tracts infection (lri) in otherwise healthy children in the united states, using a prospectively collected 25-year database and sample archive representing about 2000 children, revealed that nearly 12% of lri in children was associated with a positive hmpv test. this and similar studies suggested that the virus is one of the major respiratory pathogens of early childhood. the clinical features of hmpv lri were similar to those of other paramyxoviruses, most often resulting in cough, coryza, and a syndrome of bronchiolitis or croup. interestingly, hmpv seemed to be clinically intermediate between rsv and piv in that it tended to cause bronchiolitis with similar frequency to rsv but more frequently than piv, while causing croup less often than the latter. studies in subjects with conditions predisposing to increased risk of respiratory illness suggest that hmpv plays a significant role in exacerbations of asthma in children and adults, lri in immunocompromised subjects, and in the frail and elderly. measles virus, a paramyxovirus of the genus morbillivirus causes infection with systemic disease, also known as rubeola. the virus is spread both by direct contact/fomite transmission and by aerosol transmission, and therefore is one of the most highly contagious infections of man. the classical symptoms of measles include 3 or more days of fever that is often quite high and a clinical constellation of symptoms termed 'the three cs': cough, coryza, and conjunctivitis. a characteristic disseminated maculopapular rash appears soon after onset of fever. transient mucosal lesions in the mouth of a characteristic appearance (koplik's spots) are considered diagnostic when identified by an experienced clinician. the virus causes a number of systemic effects and can be complicated by severe pneumonia, especially when primary infection occurs in an unvaccinated adult or immunocompromised person of any age. mortality in developing countries is high, especially when infection occurs in the setting of malnutrition. these emerging pathogens that are grouped in their own new genus henipaviruses may not be respiratory pathogens in a conventional sense, but they are paramyxoviruses that probably infect humans by the respiratory route. nipah virus is a newly recognized zoonotic virus, named after the location in malaysia where it was first identified in 1999. it has caused disease in humans with contact with infectious animals. hendra virus (formerly called equine morbillivirus) is another closely related zoonotic paramyxovirus that was first isolated in australia in 1994. the viruses have caused only a few localized outbreaks, but their wide host range and ability to cause high mortality raise concerns for the future. the natural host of these viruses is thought to be a certain species of fruit bats present in australia and the pacific. pigs may be an intermediate host for transmission to humans in nipah infection, and horses in the case of hendra. although the mode of transmission from animals to humans is not defined, it is likely that inoculation of infected materials onto the respiratory tract plays a role. the clinical presentation usually appears to be an influenza-like syndrome, progressing to encephalitis, may include respiratory illness, and causes death in about half of identified cases. influenza is a single-stranded segmented negative-sense rna genome virus of the family orthomyxoviridae. there are three types of influenza viruses: influenza virus a, influenza virus b, and influenza virus c. influenza a and c infect multiple species, while influenza b infects humans almost exclusively. the type a viruses are the most virulent human pathogens among the three influenza types, and cause the most severe disease. the influenza a virus can be subdivided into different subtypes based on the antibody response to these viruses. the subtypes that have been confirmed in humans in seasonal influenza, ordered by the number of known human pandemic deaths, are: h1n1 which caused the 1918 pandemic, and h2n2 which caused the 1957 pandemic of avian influenza that originated in china, h3n2 which caused the pandemic of 1968. currently, h3n2, h1n1, and b viruses cause annual seasonal epidemics. in addition, h5n1 virus infection of humans occurred during an epizootic of h5n1 influenza in hong kong's poultry population in 1997. the disease affected animals of many species and exhibited a high rate of mortality in humans. the virus is spreading throughout asia, carried by wild birds. human-to-human transmission does not occur efficiently at this time; however, there is widespread current concern about the potential for an h5n1 pandemic if the virus acquired transmissibility among humans. the h7n7 avian virus also has unusual zoonotic potential. in 2003 this virus caused an outbreak in humans in the netherlands associated with an outbreak in commercial poultry on several farms. one death occurred and 89 people were confirmed to have h7n7 influenza virus infection. h1n2 virus appears to endemic in pigs and humans. h9n2, h7n2, h7n3, and h10n7 human infections have been reported. influenza b virus is almost exclusively a human pathogen, and is less common than influenza a. it mutates less rapidly than influenza a, and there is only one influenza b subtype. in humans, common symptoms of influenza infection and syndrome are fever, sore throat, myalgias, headache, cough, and fatigue. in more serious cases, influenza causes pneumonia, which can be fatal, particularly in young children and the elderly. influenza pneumonia has an unusually high rate of complication by bacterial superinfection with staphylococcal and streptococcal bacterial pneumonia occurring in as many as 10% of cases in some clinical series. viruses of the family adenoviridae infect both humans and animals. adenoviruses were first isolated in human lymphoid tissues from surgically removed adenoids, hence the name of the virus. in fact, some serotypes establish persistent asymptomatic infections in tonsil and adenoid tissues, and virus shedding can occur for months or years. these double-stranded dna viruses are less than 100 nm in size, and have nonenveloped icosahedral morphology. the large dsdna genome is linear and nonsegmented. there are six major human adenovirus species (designated a through f) that can be placed into 51 immunologically distinct serotypes. human respiratory tract infections are mainly caused by the b and c species. adenovirus infections can occur throughout the year. sporadic outbreaks occur with many of the serotypes, while others appear to be endemic in particular locations. respiratory illnesses include mild disease such as the common cold and lower respiratory tract illness, including croup, bronchiolitis, and pneumonia. conjunctivitis is associated with infection by species b and d. there is a particular constellation of symptoms called 'pharyngoconjunctival fever' which is very frequently associated with acute adenovirus infection. in contrast, gastroenteritis has been associated most frequently with the serotype 40 and 41 virus of species f. immunocompromised subjects are highly susceptible to severe disease during infection with respiratory adenoviruses. the syndrome of acute respiratory disease (ard), especially common during stressful or crowded living conditions, was first recognized among military recruits during world war ii and continues to be a problem for the military following suspension of vaccination. ard is most often associated with adenovirus types 4 and 7. members of the genus coronavirus also contribute to respiratory illness including severe disease. there are dozens of coronaviruses that affect animals. until recently, only two representative strains of human coronaviruses were known to cause disease, human coronavirus 229e (hcov-229e) and hcov-oc43. a recent outbreak of sars-associated coronavirus (sars-cov) showed that animal coronaviruses have the potential to cross species to humans with devastating effects. there has been one major epidemic to date, between november 2002 and july 2003, with over 8000 cases of the disease, and mortality rates approaching 10%. sars-cov causes a systemic illness with a respiratory route of entry. sars is a unique form of viral pneumonia. in contrast to most other viral pneumonias, upper respiratory symptoms are usually absent in sars, although cough and dyspnea occur in most patients. typically, patients present with a nonspecific illness manifesting fever, myalgia, malaise, and chills or rigors; watery diarrhea may occur as well. recently, investigators reported the identification of a fourth human coronavirus, hcov-nl63, a new group 1 coronavirus. evidence is emerging that hcov-nl63 is a common respiratory pathogen of humans, causing both upper and lower respiratory tract illness. human coronavirus (hcov) hku1 was first described in january 2005 following detection in a patient with pneumonia. several cases of respiratory illness have been associated with the virus, but the infrequent identification suggests to date that this putative group 2 coronavirus causes a low incidence of illness. several herpes viruses cause upper respiratory infections, especially infection of the oral cavity. herpes simplex pharyngitis is associated with characteristic clinical findings, such as acute ulcerative stomatitis and ulcerative pharyngitis. herpes simplex virus 1 (hsv-1) and herpes simplex virus 2 (hsv-2), also called human herpesvirus 1 (hhv-1) and human herpesvirus 2 (hhv-2), respectively, cause oral lesions, although over 90% of oral infections are caused by hsv-1. primary oral disease can be severe, especially in young children, who sometimes are admitted for rehydration therapy due to poor oral intake. a significant proportion of individuals suffer recurrences of symptomatic disease consisting of vesicles on the lips. epstein-barr virus (ebv) mononucleosis syndrome is often marked by acute or subacute exudative pharyngitis; in some cases, the swelling of the tonsils in ebv pharyngitis is so severe that airway occlusion appears imminent. most of the viruses of the family herpesviridae can cause severe disease in immunocompromised patients (especially hematopoietic stem cell transplant patients), including cytomegalovirus (cmv), ebv, varicella-zoster virus, herpesvirus 6, herpesvirus 7, and herpesvirus 8. a new virus was identified recently in respiratory samples from children with lower respiratory tract disease in sweden. sequence analysis of the viral genome revealed that the virus is highly related to canine minute virus and bovine parvovirus and is a member of the genus bocavirus, subfamily parvovirinae, family parvoviridae. this virus was tentatively named human bocavirus (hbov). hbov has been identified as the sole agent in a limited number of respiratory samples from children hospitalized with respiratory tract disease. it remains to be seen whether the virus is causative of or merely associated with disease in these preliminary studies. over 400 cases of hps have been reported in the united states. the disease was first recognized during an outbreak in 1993. about a third of recognized cases end in death. the four corners area outbreak is well known; however, cases now have been reported in 30 states. patients with hps usually present with a febrile illness beginning with symptoms of a flu-like illness. physical examination is not specific, often only with findings of fever, and increased heart and respiratory rates. in addition to the respiratory symptoms, abdominal pain and fever are common. diagnosis is often delayed until a severe illness occurs requiring mechanical ventilation. rotaviruses are dsrna enteric viruses that are the most common cause of severe viral infectious gastroenteritis in children. clinical series suggest that some children with gastroenteritis suffer upper respiratory symptoms during the prodrome of disease manifestation, and virus can be recovered from respiratory secretions. some reports suggest that rotavirus infection is associated with lower respiratory tract illness, although this association is unclear. these dsrna viruses (named using an acronym for respiratory enteric orphan virus) are not clearly associated with respiratory disease, but seroconversion rate is high in the first few years of life, and they probably cause minor or subclinical illness. pharyngitis occurs with primary hiv infection and may be associated with mucosal erosions and lymphadenopathy. polyomaviruses are small dsdna genome nonenveloped icosahedral viruses that may be oncogenic. there are two polyomaviruses known to infect humans, jc and bk viruses. eighty percent or more of adult us subjects are seropositive for these viruses. jc virus can infect the respiratory system, kidneys, or brain. bk virus infection causes a mild respiratory infection or pneumonia and can involve the kidneys of immunosuppressed transplant patients. given the overlap in the winter season of these viruses in temperate areas, it is not surprising that co-infections with two or more viruses occur. in general, when careful studies using cell culture techniques were used for virus isolation, more than one virus was isolated from respiratory secretions of otherwise healthy subjects with acute respiratory illness in about 5-10% of cases in adults and 10-15% in children. there is little evidence that more severe disease occurs during co-infections, although there is insufficient evidence on this point to be definitive. the incidence of two molecular diagnostic tests being positive (generally rt-pcr, for these rna viruses) is expected to be higher than that of culture, because molecular tests can remain positive for an extended period of time after virus shedding has ended. respiratory viruses generally have two main modes of transmission, large particle aerosols of respiratory droplets transmitted directly from person-to-person by coughing or sneezing, or by fomites. fomite transmission occurs indirectly when infected respiratory droplets are deposited on hands or on inanimate objects and surfaces with subsequent transfer of secretions to a susceptible subject's nose or conjunctiva. most respiratory viruses, unlike measles virus or varicella zoster virus, do not spread by small particle aerosols across rooms or down halls. therefore, contact and droplet precautions are sufficient to prevent transmission in most settings; handwashing is critical in healthcare settings during the winter season. ribavirin is a nucleoside antimetabolite pro-drug that is activated by kinases in the cell, resulting in a 5 0 triphosphate nucleotide form that inhibits rna replication. the drug was licensed in an aerosol form in the us in 1986 for treatment of children with severe rsv lower respiratory tract infection. the efficacy of aerosolized ribavirin therapy remains uncertain despite a number of clinical trials. most centers use it infrequently, if ever, in otherwise healthy infants with severe rsv disease. intravenous ribavirin has been used for adenovirus, hantavirus, measles virus, piv, and influenza virus infections, although a good risk/benefit profile has not been established clearly for any of these uses. a humanized mouse monoclonal antibody directed to the f protein of rsv, 'palivizumab', is licensed for prevention of rsv hospitalization in high-risk infants. it is efficacious in half or more of high-risk subjects. a more potent second-generation antibody is being studied in clinical trials. experimental treatment of both immunocompetent and immunocompromised rsv-infected subjects has been reported but the efficacy of this approach is not established. there are four licensed drugs in the us for treatment or prophylaxis of influenza. 'amantadine' and 'rimantadine' are two of the drugs that interfere with the ion channel activity caused by the viral m2 protein of influenza a viruses, which is needed for viral particle uncoating following endocytosis. the other two drugs, 'oseltamivir' and 'zanamivir', are neuraminidase inhibitors that act on both influenza a and b viruses by serving as transition state analogs of the viral neuraminidase that is needed to release newly budded virion progeny from the surface of infected cells. the cell surface normally is coated heavily with the viral receptor sialic acid. resistance to the ion channel inhibitors arises rapidly during prophylaxis or treatment, and in 2006 resistance levels became so common in circulating viruses that the cdc no longer recommends use of these drugs. 'interferon-a' has been shown to protect against rhinovirus infections when used intranasally. this biological drug causes some side effects, such as nasal bleeding, and resistance to the drug developed during experimental use, so the molecule is no longer being developed for this purpose. 'pleconaril' has been tested for treatment of rhinovirus infection, as it is an oral drug with good bioavailability for treating infections caused by picornaviruses. this drug acts by binding to a hydrophobic pocket in the vp1 protein and stabilizing the protein capsid, preventing release of viral rna into the cell. the drug reduced mucus secretions and other symptoms and is being further examined. 'acyclovir' and related compounds are guanine analog antiviral drugs used in treatment of herpes virus infections. hsv stomatitis in immunocompromised patients is treated with 'famciclovir', or 'valacyclovir', and immunocompetent subjects with severe oral disease compromising oral intake are sometimes treated. these compounds have also been used prophylactically to prevent recurrences of outbreaks, with mixed results. intravenous acyclovir is effective in hsv or varicella zoster virus pneumonia in immunocompromised subjects. 'ganciclovir' with human immunoglobulin may reduce the mortality associated with cmv pneumonia in hematopoietic stem cell transplant recipients and has been used as monotherapy in other patient groups. 'cidofivir' is a nucleotide analog with activity against a large number of viruses, including adenoviruses. intravenous cidofovir has been effective in the management of severe adenoviral infection in immunocompromised patients but may cause serious nephrotoxicity. there are licensed vaccines for influenza viruses. in the us, both a trivalent (h3n2, h1n1, and b) inactivated intramuscular vaccine and a live attenuated trivalent vaccine for intranasal administration is available. the efficacy of these vaccines is good when the vaccine strains chosen are highly related antigenically to the epidemic strain. antigenic drift caused by point mutations in the ha and na molecules leads to antigenic divergence, requiring new vaccines to be made each year. the influenza genome is segmented, which allows reassortment of two viruses to occur during co-infection, which sometimes leads to a major antigenic shift resulting in a pandemic. pandemics occur every 20-30 years on average. there is current concern about the potential for an h5n1 pandemic, and experimental vaccines are being tested for this virus. to date, h5n1 vaccines have been poorly immunogenic compared to comparable seasonal influenza vaccines. vaccines were developed for adenovirus serotypes 4 and 7, and these were approved for preventing epidemic respiratory illness among military recruits. essentially, these were unmodified viruses given by the enteric route in capsules, instead of the respiratory route, which is the natural route of infection leading to disease. inoculation by the altered route resulted in an immunizing asymptomatic infection. all us military recruits were vaccinated against adenovirus from 1971 to 1999 with near complete prevention of the disease in this population, but the sole manufacturer of the vaccine halted production in 1996 and supplies ran out 3 years later. since 1999, adenovirus infection has reemerged as a significant problem in the military with approximately 10% of all recruits suffering illness due to adenovirus infection during basic training; some deaths have occurred. live attenuated vaccine candidates are under development and being tested in phase i and ii clinical trials for rsv and the pivs. mutant strains with reduced pathogenicity were isolated in the laboratory, tested, and sequenced. now, vaccine candidates are being optimized by combining mutations from separate biologically derived viruses into single strains using recombinant techniques for generating rnaviruses from cdna copies, a process called reverse genetics. subunit vaccines have been developed for rsv, but there are safety concerns about their use in young infants because formalin inactivated vaccine induced a more severe disease response to infection in the 1960s. there are no vaccines against rhinoviruses as there is little or no cross-protection between serotypes, and it is not feasible to develop a vaccine for over 100 serotypes. efforts to develop coronavirus vaccines are in the preclinical stage. viruses are the leading causes of acute lower respiratory tract infection in infancy. rsv is the most common pathogen, with hmpv, piv-3, influenza viruses, and rhinoviruses accounting for the majority of the remainder of acute viral respiratory infections. humans generally do not develop lifelong immunity to reinfection with these viruses; rather, specific immunity protects against severe and lower respiratory tract disease. risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level viral infections in relation to age, atrophy, and season of admission among children hospitalized for wheezing parainfluenza viruses what have we learned from the tucson children's respiratory study? epidemiology of respiratory syncytial virus infection in washington, dc. part ii. infection and disease with respect to age, immunologic status, race, and sex characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season fields virology human t-cell leukemia viruses: general features m yoshida, university of tokyo, chiba, japan 2008 elsevier ltd. all rights reserved.glossary px region htlv sequence between the env and 3 0 ltr, encoding tax, rex and other small regulatory proteins. rex trans-modulator of viral rna splicing and transport. tax pleiotropic regulator activating viral and cellular replication interacting with cellular transcription factors, tumor suppressor proteins, and cell cycle checkpoints. human t-cell leukemia virus 1 (htlv-1) is the first established tumorigenic retrovirus of humans; exogenous to humans this virus is classified as the species human t-cell leukemia virus, in deltaretroviridae, within the family retroviridae. htlv-1 infection is associated with leukemia and neural disease, adult t-cell leukemia (atl) and htlv-1-associated myelopathy/tropical spastic parapasis (ham/tsp), respectively. the genomic structure of the virus with genes for nonstructural proteins established a distinct viral genus that includes bovine leukemia virus. htlv-1 has no oncogene, but nevertheless transforms t cells rather efficiently and is identified as the etiologic agent of atl. htlv-1 has unique regulatory proteins, tax and rex, and tax has been identified as a critical molecule not only in regulation of viral replication but also in induction of atl. after long and enormous efforts to identify a retrovirus in human tumors, htlv was described in t-cell lines as a convincing human retrovirus. the first report of the virus (htlv) was from a patient with mycosis (mf) in the us, and another (adult t-cell leukemia virus (atlv)) was from a patient with atl in japan. subsequently, the mf case was characterized as atl and the two isolates were established to be the same following a comparison of their genomes.a prototypical retroviral genome contains the gag, pol, and env genes encoding the virion proteins including core proteins, reverse transcriptase, and surface glycoprotein, respectively. acute leukemia viruses generally have an oncogene acquired from cellular genes that substitutes a part of the gag, pol, and env sequences. in contrast to these genomes, htlv has additional genes in an extra px region between env and the 3 0 ltr (ltr -long terminal repeat). this unique genomic structure classified htlv as a member of a distinct genus of the retroviridae, which includes htlv-1, and-2, bovine leukemia virus (blv), and simian t-cell leukemia viruses (stlv-1, -2, and -3). htlv-2 was isolated from a patient with hairy t-cell leukemia and its genome similarity to the type 1 is about 60%.stlvs have been isolated from various species of old world nonhuman primates, including the japanese macaque, african green monkey, pig-tailed macaque, gorilla, and chimpanzee. their genomes share 90-95% homology.blv infects and replicates in b cells of cows and sheep and induces b-cell lymphoma. key: cord-321132-xdpb3ukt authors: lhomme, sebastien; garrouste, cyril; kamar, nassim; saune, karine; abravanel, florence; mansuy, jean-michel; dubois, martine; rostaing, lionel; izopet, jacques title: influence of polyproline region and macro domain genetic heterogeneity on hev persistence in immunocompromised patients date: 2014-01-15 journal: j infect dis doi: 10.1093/infdis/jit438 sha: doc_id: 321132 cord_uid: xdpb3ukt hepatitis e virus (hev) can chronically infect immunocompromised patients. the polyproline region (ppr) and the macro domain of orf1 protein may modulate virus production and/or the host immune response. we investigated the association between the genetic heterogeneity of hev quasispecies in orf1 and the outcome of infection in solid-organ transplant patients. both sequence entropy and genetic distances during the hepatitis e acute phase were higher in patients whose infection became chronic than in those who cleared the virus. hence, great quasispecies heterogeneity in the regions encoding the ppr and the macro domain may facilitate hev persistence. hepatitis e virus (hev) can chronically infect immunocompromised patients. the polyproline region (ppr) and the macro domain of orf1 protein may modulate virus production and/or the host immune response. we investigated the association between the genetic heterogeneity of hev quasispecies in orf1 and the outcome of infection in solidorgan transplant patients. both sequence entropy and genetic distances during the hepatitis e acute phase were higher in patients whose infection became chronic than in those who cleared the virus. hence, great quasispecies heterogeneity in the regions encoding the ppr and the macro domain may facilitate hev persistence. keywords. hepatitis e; chronic infection; orf1; polyproline region; macro domain. hepatitis e virus (hev) is a major cause of enterically transmitted non-a, non-b hepatitis. it is also responsible for large outbreaks of waterborne acute hepatitis in tropical and subtropical countries as well as sporadic infections worldwide. hepatitis e is a zoonotic disease in industrialized countries, with pigs, wild boar, and deer being the major animal reservoirs of hev [1] . hev, genus hepevirus, family hepeviridae, is an unenveloped, single-stranded, positive-sense rna virus. like all rna viruses, hev exists as a mixture of closely related variants defining a quasispecies. the approximately 7.2 kb genome of hev has a coding region consisting of 3 open reading frames (orfs). orf2 encodes the capsid protein; orf3 encodes a small protein implicated in virus egress [2] , whereas orf1 encodes a nonstructural protein that contains several putative functional domains. these include at least 4 enzyme activities: methyltransferase, cystein protease, helicase, and rna-dependent rna polymerase [2] . homologies with other plant and animal positive strand rna viruses have been used to identify other domains: the y domain, the polyproline region (ppr), and the macro domain. the ppr is genetically very diverse and could correspond to an intrinsically disordered region involved in virus adaptation [3] . in addition, the ppr does not seem to be required for hev replication in vitro or in vivo [4] . nonstructural virus genes that are not essential for replication are usually involved in modulating host immune responses [5] . the genomes of several virus families, including all members of coronaviridae, rubella virus, and alphaviruses (togaviridae), and hev, have macro domains. the macro domain of the mouse hepatitis virus (mhv) influences the pathogenicity of the virus [6] . hev can lead to chronic hepatitis in solid-organ transplant (sot) patients [7] . but our knowledge of the factors associated with the development of chronic infection in sot patients exposed to hev is far from complete. our working hypothesis was that the genetic heterogeneity of the ppr or the macro domain play a role in the outcome of hev infection in immunocompromised patients, as the ppr could modulate the host immune response and the macro domain could influence virus pathogenicity. we therefore analysed the characteristics of hev quasispecies at the acute phase of hepatitis e in 2 groups of sot patients, one whose infection became chronic and the other who cleared the virus. we studied 14 sot patients who became acutely infected with hepatitis e between january 2004 and june 2009. the infection was diagnosed by the detecting hev rna using the real time polymerase chain reaction (pcr) and immunoglobulin m/immunoglobulin g anti-hev antibodies by a commercial enzyme-linked immunosorbent assay [8] . each patient was assigned to one of 2 groups according to the outcome of the infection. the first group (group i; 8 patients) had chronic infections, defined by persistently elevated liver enzyme activity and serum that was positive for hev rna for more than 6 months after diagnosis. the second group (group ii; 6 patients) had resolving infections. serum samples were collected from all patients at the acute phase of their infection and stored at −80°c. each patient underwent an exhaustive clinical and laboratory examination, including the immunosuppressive drugs used, the hepatic enzyme activities, and white blood cell count. the serum concentration of hev rna was measured by real-time pcr [9] . virus genotype was determined by sequencing a 189nucleotide fragment within the orf2 gene. the sequences were compared to reference hev strains (genbank) as described elsewhere [10] . nucleic acids were extracted from serum samples and analysed by 1-step rt-pcr with the sense primer hevorf1-s1 and anti-sense primer hevorf1-a1 using the super script iii enzyme (invitrogen, cergy-pontoise, france). the sequences of the primers are listed in supplementary table 1 . the sequence amplified included the ppr (nucleotide [nt] 2137-2340) and the macro domain (nt 2341-2829), using the genome 1b l08816 as a reference. the pcr products were purified using qiaprep (qiagen, courtaboeuf, france) miniprep kits and stored at −20°c. in total, 10 ng of the amplified sequence were directly ligated into 1 µl of pcr 4 vector (kit topo ta cloning; invitrogen) containing a gene conferring resistance to ampicilline. recombinant plasmids were used to transform escherichia coli-competent cells, and transformants were grown on ampicillin-coated plates at 37°c for 18 hours. the complementary dna (cdna) clones (20 from each patient sample) were analysed by pcr with the primers hevorf1-s1 and hevorf1-a1 (supplementary table 1 ). the pcr products of these amplifications were purified (quick-spin columns; qiagen) and sequenced using the fluorescent dye terminator method for big dyeterminator cycle sequencing (applied biosystems, paris, france) with the primers hevorf1-s1, hevorf1-s2, hevorf1-s3, hevorf1-a1 and hevorf1-a2 (supplementary table 1 ) on an applied biosystems abi 3130 xl analyzer. sequences were aligned using clustal x and compared with those of hev strains obtained from genbank. we quantified the complexity of the hev quasispecies by calculating the shannon entropy: s = −σ i ( p i ln p i ), where pi is the frequency of each sequence in the viral quasispecies. the normalized entropies for both nucleotides and amino-acids, sn, were calculated using sn = s/ln n, where n is the total number of sequences analysed. we quantified diversity as the mean genetic distance calculated for all pairs of nucleotide sequences using mega 4.0. the numbers of synonymous (ks) substitutions per synonymous site and nonsynonymous (ka) substitutions per nonsynonymous site were calculated with the jukes-cantor correction for multiple substitutions using mega 4.0. the ka/ks ratio is an indicator of the positive (>1) or negative (<1) selection pressure on a quasispecies [11] . proportions were compared by fisher exact test. quantitative variables were compared with the nonparametric mann-whitney test. correlations between complexities or diversities of quasispecies were estimated by calculating spearman rank correlation coefficient. a p-value below .05 was considered to be statistically significant. the sequences have been deposited in the genbank database under accession numbers kc911858 to kc912137. the clinical and biological characteristics of the patients are summarized in supplementary table 2 . there was no significant difference between patients with chronic infections and those with resolving infections in terms of gender, age, or immunosuppressant treatment. the alt activities of individuals with a chronic infection tended to be lower. patients whose infection became chronic had lower total, cd3, cd4, and cd8 lymphocyte counts, but the differences were not significant. the serum hev rna concentrations at the acute phase of the 2 groups of patients were similar. they were all infected with hev genotype 3f strains, except one whose infection was genotype 3c. we compared the sequence heterogeneity of 2 regions of orf1 in patients with chronic hev infection and those with resolving infections. the mean nucleotide sequence entropy (nt complexity) of the ppr was higher in group i [7] . the viral determinants associated with the persistence of such an infection are poorly documented. we therefore investigated the influence of the genetic heterogeneity of hev quasispecies on the outcome of infection, focusing on the ppr and the macro domain. both the complexity and diversity of the ppr and the macro domain were higher in viral population of the patients whose infection became chronic than in those who cleared the virus. it has been shown that the quasispecies heterogeneity in the orf2 region encoding the capsid protein during the acute phase of infection is associated with the development of a chronic hev infection [12] . our data support the finding that great genetic heterogeneity of the quasispecies in patients whose infection become chronic seems to favor the appearance of variants that can persist, as reported for hcv infections [13] . although the diversity of the region preceding the ppr was higher in patients with chronic infection, nt and aa complexities were not different in the 2 groups (data not shown), suggesting that the higher heterogeneity in chronic patients was not a general effect seen across the entire region studied. this great genetic heterogeneity could also reflect an inadequate control of the viral replication, but no correlation was found between quasispecies heterogeneity and viral concentrations. ppr appears to be dispensable for in vitro hev replication, but it is required for in vivo infection, suggesting that it is involved in infecting cells with innate immunity [4] . although the role of the macro domain in the replication of hev is unknown, it does not seem to be essential for the replication of coronavirus in cell culture [14] . it was recently suggested that genes that are dispensable for virus replication are involved in modulating the host immune response, like down-regulating interleukin 1β (il-1β) or tumor necrosis factor α (tnf-α) secretion [5] . the macro domain is also involved in the inflammation caused by the mouse hepatitis virus (mhv), and the substitution of a strictly conserved amino-acid residue is responsible for reducing the secretion of inflammatory cytokines [6] . the great quasispecies heterogeneity in patients whose infection became chronic may include some variants that reduce inflammatory cytokine production, which could facilitate hev persistence. this could explain the lower serum concentrations of interleukin 1 (il-1) receptor antagonist and tnf-α found in patients whose infection became chronic [12] . we find that the complexities and diversities of the ppr and the macro domain are correlated. these 2 regions may well have evolved together as the protein encoded by orf1 does not seem to be cleaved [15] . we also studied the correlations between the orf1 and orf2 as this study and our previous one on orf2 diversity [12] were carried out on the same patients (except for 3). we also find that the complexity and diversity of the regions in orf1 and orf2 that were studied are correlated (data not shown), suggesting that they, too, have evolved together. finally, we find no differences in the ka/ks ratios of the studied regions, even though the ka/ks ratios of the ppr seemed to be higher in patients who cleared the virus. alternatively, the ka/ks ratio may not allow us to infer differences in selection pressure. in both regions, ka/ks ratios indicate negative selection, probably due to structural or functional constraints. a limitation of our study is the small number of patients in each group and an implication of immunological factors in the evolution toward chronicity cannot be excluded. we conclude that the high genetic heterogeneity of the ppr and the macro domain at the acute phase of an hev infection is associated with persistence of the virus. this association may be due to the appearance of mutants able to modulate the host immune response. further investigation is now needed to confirm this association. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org/). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. molecular virology of hepatitis e virus the hepatitis e virus polyproline region is involved in viral adaptation deletions of the hypervariable region (hvr) in open reading frame 1 of hepatitis e virus do not abolish virus infectivity: evidence for attenuation of hvr deletion mutants in vivo immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response mouse hepatitis virus liver pathology is dependent on adp-ribose-1′′-phosphatase, a viral function conserved in the alpha-like supergroup hepatitis e virus and chronic hepatitis in organ-transplant recipients characteristics of autochthonous hepatitis e virus infection in solid-organ transplant recipients in france genotype 3 diversity and quantification of hepatitis e virus rna hepatitis e virus genotype 3 diversity statistical methods for detecting molecular adaptation hev quasispecies and the outcome of acute hepatitis e in solid-organ transplant patients the outcome of acute hepatitis c predicted by the evolution of the viral quasispecies adp-ribose-1′′-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture early secretory pathway localization and lack of processing for hepatitis e virus replication protein porf1 financial support. this work was supported by institut national de la santé et de la recherche médicale u1043.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-356040-qdpkidn8 authors: ghazawi, feras m.; lim, megan; dutz, jan p.; kirchhof, mark g. title: infection risk of dermatologic therapeutics during the covid‐19 pandemic: an evidence‐based recalibration date: 2020-07-03 journal: int j dermatol doi: 10.1111/ijd.15028 sha: doc_id: 356040 cord_uid: qdpkidn8 recommendations were made recently to limit or stop the use of oral and systemic immunotherapies for skin diseases due to potential risks to the patients during the current severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) covid‐19 pandemic. herein, we attempt to identify potentially safe immunotherapies that may be used in the treatment of cutaneous diseases during the current covid‐19 pandemic. we performed a literature review to approximate the risk of sars‐cov‐2 infection, including available data on the roles of relevant cytokines, cell subsets, and their mediators in eliciting an optimal immune response against respiratory viruses in murine gene deletion models and humans with congenital deficiencies were reviewed for viral infections risk and if possible coronaviruses specifically. furthermore, reported risk of infections of biologic and non‐biologic therapeutics for skin diseases from clinical trials and drug data registries were evaluated. many of the immunotherapies used in dermatology have data to support their safe use during the covid‐19 pandemic including the biologics that target ige, il‐4/13, tnf‐α, il‐17, il‐12, and il‐23. furthermore, we provide evidence to show that oral immunosuppressive medications such as methotrexate and cyclosporine do not significantly increase the risk to patients. most biologic and conventional immunotherapies, based on doses and indications in dermatology, do not appear to increase risk of viral susceptibility and are most likely safe for use during the covid‐19 pandemic. the limitation of this study is availability of data on covid‐19. the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), also named 2019 novel coronavirus disease covid-19, is the causative agent of the ongoing pandemic. 1 and mip-1a. 5, 6 this is consistent with the reported elevation of proinflammatory cytokines in sars 7 and mers infections. 8 the massive inflammatory cell infiltration and elevated proinflammatory cytokine/chemokine responses result in acute lung injury and ards. 4, 9, 10 part 2: infectious risks associated with biologics: evaluating cytokine knockout data and reviewing data from randomized controlled trials (rcts) and biologic treatment registries infecting tnf-a à/à , tnf receptor 1 (r1) à/à , and tnfr2 à/à mice with mouse hepatitis virus-3 (mhv-3, belongs to the coronavirus family) revealed that a deficiency of either tnf-a or tnfr1 decreased morbidity and mortality (table 1) . 11 tnf receptors 1/2 knock-out mice infected with sars-cov were protected from infection-related morbidity. 12 collectively, tnf-a promotes the deleterious effects of coronavirus infection presumably through excessive inflammation. from clinical trials ( the b-lymphocyte antigen cd20 is highly expressed on b cells starting at the pre-b-cell stage and on mature b cells, and it is downregulated during terminal differentiation into plasma cells. while the precise function of cd20 is not fully elucidated, igm expression in immature and mature b cells from cd20-deficient mice was markedly reduced compared to wildtype. 16 furthermore, reduced humoral immunity to adeno-associated viral antigens was demonstrated in cd20-deficient mice. 17 a patient who lacked cd20 expression due to homozygous mutations reported intermittent respiratory infections, associated with persistent hypogammaglobulinemia and strong reductions in circulating memory b cells. 18 no significant differences in urti, nasopharyngitis, bronchitis, cough, and sinusitis between rituximab (anti-cd20) 19 and placebo were demonstrated in a double-blind rct for rheumatoid arthritis (ra). 20 however, in a prospective, open-label rct, it was noted that lung infections/ pneumonia were higher in the rituximab treatment arm by more than twofold (11% vs. 5% in control, no confidence intervals were presented). 21 the role of cd20 + cells in presenting antigen to t cells and in generation of antibodies to protect from new infections remains unclear. the il-12/il-23 common pathway plays a key role in the induction of inflammation in adaptive immune responses, where il-12 induces a th1 immune response with a downstream induction of cytokines such as tnf, interferon (ifn)-c, and il-23 promotes a th17 immune response through the induction of inflammatory cytokines such as il-17 and il-22. 22 mice defective in both il-12/23 (p40 à/à ) and il-12 alone (p35 à/à ) were infected with a murine coronavirus (mhv). 23 il-12 and il-12/23 knockout mice had similar survival to wild-type animals. 23 therefore, il-12 does not seem to contribute to antiviral function or survival. mice deficient in il-23 alone (p19 à/à ) were infected with murine coronavirus, and viral control was similar to wild-type mice, demonstrating that il-23 does not significantly confer protection from infection. 24 this was also demonstrated thorough neutralization of mice using anti-il-23p19specific and anti-il-12/23p40 antibodies, followed by infection of mice with mhv. 25 in the absence of il-12/23 signaling, specific antiviral t-cell response was intact. 25 clinical trials using il-12/23 or il-23 inhibitors demonstrated no significant increase in respiratory adverse events (table 2) . furthermore, the psolar study reported that ustekinumab had no increased risk of serious infections. 13 of note, a recent case study reported covid-19 in a patient during il-23 inhibitor (guselkumab) treatment for psoriasis, and the patient had a good outcome. 26 il-17 is a proinflammatory cytokine with important roles in tcell activation and neutrophil mobilization and activation. 27 il-17 expression is induced during influenza infection as part of the th1 immune response that contributes to viral clearance. 28 however, a growing body of evidence suggests that il-17 is also associated with promotion of viral infections and tissue pathology. this is thought to occur through direct suppression including tnf-a, il-1b, and il-6. 32 in humans, chronic mucocutaneous candidiasis has been attributed to the disruption of th1 and th17 pathways. this was illustrated in patients with identified mutations in il-17ra and stat1 genes. 33 these patients have no increased risk of viral infections. 34 clinical trials using il-17 inhibitors demonstrated no significant increase in respiratory adverse events (table 2) . a recent case report reported a patient receiving therapy with an il-17 inhibitor (ixekizumab) who was completely asymptomatic but tested positive for covid-19. 35 ity. 38 the use of anakinra in clinical trials was associated with a slightly higher frequency of serious infectious episodes, primarily pneumonia (2.1% vs. 0.4%, comparative risk 5.25), than the placebo group. 39 it appears that normal il-1 expression/ function is required to mount an optimal antiviral immune response. il-4 is a key regulator in humoral and th2 adaptive immunity. mouse models demonstrated that the constitutive overexpression of il-4 prior to rsv infection delayed viral clearance, increased the density of the lymphocytic infiltrate in the lungs, and diminished induction of primary cytotoxic t lymphocyte responses. 40 conversely, il-4 à/à mice cleared rsv readily after primary infection, with minimal pathology. 40 cyclosporine is a calcineurin inhibitor that blocks il-2 signaling and t-cell proliferation. 52, 53 the most common infectious side effects from cyclosporine were flu-like symptoms seen in 15% of patients enrolled in an rct for chronic idiopathic urticaria. 54 psoriasis registries examining cyclosporine reported infection trials that combined mmf with corticosteroids had significantly higher rates of infections, up to 59%. 62 mmf is reported to increase patients' susceptibility to viral infections, 63 and an increase in nasopharyngitis and urtis was noted comparing prednisone plus mmf to prednisone monotherapy in pemphigus vulgaris. 62 of note, mmf was used to treat eight patients with mers with a 100% survival rate; however, when analyzing the severity of illness and treatment, mmf was given to less severely ill patients. 64 azathioprine azathioprine inhibits purine synthesis and downregulates b-cell and t-cell function. 65, 66 documented types of infection with use of azathioprine include lower respiratory tract infections (lrti) and urti, which had rates of 5% and 5-20%, respectively. 67, 68 thirty-six percent of patients in one study had infections of moderate intensity. 69 there were no registries evaluating the prevalence of infections during azathioprine therapy for dermatologic uses. one systematic review evaluating the off-label use of azathioprine found mild infections reported in 0.36% of patients and severe infections in only 0.30% of patients 70 (table 3 ). the use of methotrexate (mtx), 71 a folic acid antagonist that inhibits nucleotide synthesis, 72 infection were similar to the placebo group. 76 a review of infectious risks in rheumatoid arthritis (ra) patients indicated that although mtx has previously been implicated not only with increased risk of infection but also increased severity, the evidence was not clear. 77 the review concluded that mtx appears to be associated with minimal, if any, increased infection risk in the ra population. 77 hydroxychloroquine is an antimalarial medication that inhibits lysosomal functions and interferes with a myriad of immune pathways. 78 its exact mechanism in many dermatologic processes has never been fully elucidated. hydroxychloroquine has been shown to have a favorable side effect profile in terms of infection risk in many clinical trials. 79, 80 it is currently under investigation in numerous phase 2 clinical trials as treatment for covid-19 as it may inhibit viral fusion to the host cell and inhibit viral assembly and release. 81 apremilast is a phosphodiesterase 4 (pde4) inhibitor, 82 with side effects including nasopharyngitis and urti. 83 the incidence of urti in the apremilast-treated groups is comparable to placebo ranging from 4.8 to 26.0% and 4.4 to 14.0%, with higher rates being accounted for from one study examining apremilast in palmoplantar psoriasis (table 3 ). overall, rates of infection were not increased in patients treated with apremilast. [84] [85] [86] [87] [88] [89] a recent case was reported of a patient with erythrodermic psoriasis, with contraindication to most treatments due to a recurrent brain oligodendroglioma who had psoriasis thalidomide thalidomide, 91 an immunomodulatory drug with a range of activity that is not fully characterized, 92 is effective for various refractory dermatoses, but its side effect profile is unfavorable, and risks of teratogenicity and neuropathy often preclude its use. 91 table 3 highlights four rcts where there was no increased risk of infection in thalidomide compared to placebo. prolonged use of oral corticosteroids is generally avoided due to side effects. 93 none of the following studies reported infection as an adverse reaction. 94 immunosuppression is not a comorbidity that is commonly reported in covid-19 patients despite it commonly being referred to as a risk factor. 104 the limited data do not suggest increased risk of severe complications compared to the general population. lei et al. 105 reported two heart transplant patients in china who survived covid-19 infections. two reported renal transplant patients who contracted covid-19 and succumbed to the illness had similar clinical courses compared to non-transplant patients. 106 transplant recipients may practice more stringent physical distancing practices compared to the general population, resulting in falsely low numbers. the literature surrounding sars and transplant recipients is sparse. risk factors for severe sars included hypertension, diabetes, coronary heart disease, hepatitis, and pregnancy with a mortality rate with ≥1 risk factor compared to none of 54.5% vs. 7.5%; p < 0.01. 107 there is no evidence that suggests transplant recipients had poorer outcome in the sars epidemic. a retrospective cohort study of a mers outbreak in korea revealed that the number of affected immunosuppressed patients was low and did not identify any transplant patients. 108 immunosuppression was not identified as a poor prognostic factor in mers infection. 109 immunomodulatory regimens have revolutionized the treatment of dermatological diseases. with the current covid-19 pandemic, it is imperative to examine the evidence and conduct a risk-benefit analysis for each patient. there may be patients who require more or less treatment, for instance some patients with existing comorbidities may require a more conservative figure 1 a pictorial representation of covid-19 risk assessment of dermatologic treatments where green represents "safe" and red represents "higher risk" approach. 110 the greatest risk of infections in biologics appear to occur with cd20 inhibition (fig. 1) . for non-biologic immunotherapies, the greatest risk of infection appears to occur with the use of high doses of oral corticosteroids. a slight increased infection risk is seen with cyclosporine, although cyclosporine has been shown to inhibit coronavirus replication and did not increase susceptibility in 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spontaneous urticaria similar efficacy with omalizumab in chronic idiopathic/spontaneous urticaria despite different background therapy subcutaneous immunoglobulin for maintenance treatment in chronic inflammatory demyelinating polyneuropathy (path): a randomised, double-blind, placebo-controlled, phase 3 trial a randomized doubleblind trial of intravenous immunoglobulin for bullous pemphigoid a randomized doubleblind trial of intravenous immunoglobulin for pemphigus inhibition of neutrophil responses by cyclosporin a. an insight into molecular mechanisms product monograph randomized double-blind study of cyclosporin in chronic 'idiopathic' urticaria long term safety of nine systemic medications for psoriasis: a cohort study using the biobadaderm registry drug safety of systemic treatments for psoriasis: results from the german psoriasis registry psobest infections in moderate to severe psoriasis patients treated with biological drugs compared to classic systemic drugs: findings from the biobadaderm registry suppression of coronavirus replication by cyclophilin inhibitors cyclosporin a inhibits the replication of diverse coronaviruses preferential suppression of lymphocyte proliferation by mycophenolic acid and predicted long-term effects of mycophenolate mofetil in transplantation mycophenolate mofetil treating pemphigus vulgaris with prednisone and mycophenolate mofetil: a multicenter, randomized, placebo-controlled trial risk of herpes zoster infection in patients with pemphigus on mycophenolate mofetil treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia cd28-dependent rac1 activation is the molecular target of azathioprine in primary human cd4+ t lymphocytes product monograph azathioprine in severe adult atopic dermatitis: a double-blind, placebo-controlled, crossover trial azathioprine dosed by thiopurine methyltransferase activity for 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multicenter, double-blind, randomized, parallel-group trial hydroxychloroquine in systemic lupus erythematosus: results of a french multicentre controlled trial (plus study) in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) apremilast in psoriasis and beyond: big hopes on a small molecule otezla â (apremilast) efficacy and safety of apremilast in systemic-and biologic-naive patients with moderate plaque psoriasis: 52-week results of unveil safety and efficacy of apremilast through 104 weeks in patients with moderate to severe psoriasis who continued on apremilast or switched from etanercept treatment: findings from the liberate study apremilast for the treatment of moderate-to-severe palmoplantar psoriasis: results from a double-blind, placebo-controlled, randomized study apremilast, an oral phosphodiesterase 4 (pde4) inhibitor, in patients with moderate to severe plaque psoriasis: results of a phase iii, randomized, controlled trial (efficacy and safety trial evaluating the effects of apremilast in psoriasis [esteem] 1) efficacy and safety of apremilast, an oral phosphodiesterase 4 inhibitor, in patients with moderate-to-severe plaque psoriasis over 52 weeks: a phase iii, randomized controlled trial (esteem 2) apremilast for moderate hidradenitis suppurativa: results of a randomized controlled trial covid-19 pulmonary infection in erythrodermic psoriatic patient with oligodendroglioma: safety and compatibility of apremilast with critical intensive care management thalidomid â (thalidomide) binding of thalidomide to alpha1-acid glycoprotein may be involved in its inhibition of tumor necrosis factor alpha production product monograph the role of systemic steroids and phototherapy in the treatment of stable vitiligo: a randomized controlled trial placebo-controlled oral pulse prednisolone therapy in alopecia areata a comparison of the efficacy, relapse rate and side effects among three modalities of systemic corticosteroid therapy for alopecia areata a randomized comparative study of oral corticosteroid minipulse and lowdose oral methotrexate in the treatment of unstable vitiligo adverse events of low-to medium-dose oral glucocorticoids in inflammatory diseases: a meta-analysis the international society of dermatology risk of infectious complications in patients taking glucocorticosteroids but not tnf antagonists, are associated with adverse covid-19 outcomes in patients with inflammatory bowel diseases: results from an international registry recovery trial: the uk covid-19 study resetting expectations for clinical trials the natural history of influenza infection in the severely immunocompromised vs nonimmunocompromised hosts coronaviruses and immunosuppressed patients. the facts during the third epidemic prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and meta-analysis first cases of covid-19 from china covid-19 in kidney transplant recipients prognostic factors for severe acute respiratory syndrome: a clinical analysis of 165 cases host susceptibility to mers-cov infection, a retrospective cohort study of the 2015 korean mers outbreak middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission available at interleukin-1 is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection adalimumab therapy for moderate to severe psoriasis: a randomized, controlled phase iii trial certolizumab pegol for the treatment of chronic plaque psoriasis: results through 48 weeks from 2 phase 3, multicenter, randomized, doubleblinded secukinumab in plaque psoriasis-results of two phase 3 trials a global phase iii randomized controlled trial of etanercept in psoriasis: safety, efficacy, and effect of dose reduction infliximab induction and maintenance 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for the treatment of bullous pemphigoid efficacy and safety of mycophenolate mofetil vs. methotrexate for the treatment of chronic plaque psoriasis evaluation of mycophenolate mofetil as a steroid-sparing agent in pemphigus: a randomized, prospective study mycophenolate mofetil (cellcept) for psoriasis: a two-center, prospective, open-label clinical trial a randomized, investigator-masked, double-blind, placebo-controlled trial on thalidomide in severe cutaneous sarcoidosis comparative efficacy of thalidomide and prednisolone in the treatment of moderate to severe erythema nodosum leprosum: a randomized study effect of thalidomide on clinical remission in children and adolescents with refractory crohn disease: a randomized clinical trial thalidomide in the treatment of the mucocutaneous lesions of the behcet syndrome. a randomized, double-blind, placebo-controlled trial cyclosporin in the treatment of patients with atopic eczema -a systematic review and meta-analysis long-term efficacy and safety of azathioprine in ulcerative colitis effectiveness of methotrexate in moderate to severe psoriasis patients: realworld registry data from the swiss dermatology network for targeted therapies (sdntt) real-world data on the efficacy and safety of apremilast in patients with moderate-to-severe plaque psoriasis risk of serious infections, cutaneous bacterial infections, and granulomatous infections in patients with psoriasis treated with anti-tumor necrosis factor agents versus classic therapies: prospective meta-analysis of psonet registries the international society of dermatology the authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. jpd is a senior scientist at bc children's hospital research institute. key: cord-303054-s1clwunc authors: velly, lionel; gayat, etienne; jong, audrey de; quintard, hervé; weiss, emmanuel; cuvillon, philippe; audibert, gerard; amour, julien; beaussier, marc; biais, matthieu; bloc, sébastien; bonnet, marie pierre; bouzat, pierre; brezac, gilles; dahyot-fizelier, claire; dahmani, souhayl; de queiroz, mathilde; maria, sophie di; ecoffey, claude; futier, emmanuel; geeraerts, thomas; jaber, haithem; heyer, laurent; hoteit, rim; joannes-boyau, olivier; kern, delphine; langeron, olivier; lasocki, sigismond; launey, yoan; saché, frederic le; lukaszewicz, anne claire; maurice-szamburski, axel; mayeur, nicolas; michel, fabrice; minville, vincent; mirek, sébastien; montravers, philippe; morau, estelle; muller, laurent; muret, jane; nouette-gaulain, karine; orban, jean christophe; orliaguet, gilles; perrigault, pierre françois; plantet, florence; pottecher, julien; quesnel, christophe; reubrecht, vanessa; rozec, bertrand; tavernier, benoit; veber, benoit; veyckmans, francis; charbonneau, hélène; constant, isabelle; frasca, denis; fischer, marc-olivier; huraux, catherine; blet, alice; garnier, marc title: guidelines: anaesthesia in the context of covid-19 pandemic date: 2020-06-05 journal: anaesth crit care pain med doi: 10.1016/j.accpm.2020.05.012 sha: doc_id: 303054 cord_uid: s1clwunc abstract objectives: the world is currently facing an unprecedented healthcare crisis caused by covid-19 pandemic. the objective of these guidelines is to produce a framework to facilitate the partial and gradual resumption of intervention activity in the context of the covid-19 pandemic. methods: the group has endeavoured to produce a minimum number of recommendations to highlight the strengths to be retained in the 7 predefined areas: (1) protection of staff and patients; (2) benefit/risk and patient information; (3) pre-operative assessment and decision on intervention; (4) modalities of the pre-anaesthesia consultation; (5) specificity of anaesthesia and analgesia; (6) dedicated circuits and (7) containment exit type of interventions. results: the sfar guideline panel provides 51 statements on anaesthesia management in the context of covid-19 pandemic. after one round of discussion and various amendments, a strong agreement was reached for 100% of the recommendations and algorithms. conclusion: we present suggestions for how the risk of transmission by and to anaesthetists can be minimised and how personal protective equipment policies relate to covid-19 pandemic context the outbreak of covid-19 (sars-cov-2) has been spreading globally outside the first chinese outbreak since january 2020 and the world health organization (who) declared a pandemic situation on march 11, 2020 . the epidemic situation has led to a drastic reduction in hospital activities. the evolution of the pandemic allows us to resume some of these activities. beyond this resumption, the persistence of the virus defines a new situation that will have to be taken into account for the care of patients in the coming months. the size and type of activities that will resume depend on many factors outside the organisation of care within our establishments. these factors include the availability of personal protective equipment, anaesthesia/critical care drugs, and critical care beds. finally, it seems important to point out that the epidemic situation is fluctuating not only in time but also in space, so it will be necessary to modulate the recommendations according to the region of exercise and the incidence of covid-19 cases. we need to organise access to this care by meeting a dual imperative: 1) providing access to quality care for patients whose procedures cannot (or can no longer) be postponed, and 2) limiting the risk of contamination of these patients and healthcare professionals. the choice of specific measures to be implemented for the management of a patient in this context will be guided by the risk associated with the patient and the risk associated with the procedure. the persons at risk of serious forms of covid-19 are:  people aged 70 years and over (although people aged 50 to 70 years should be monitored more closely);  people with a history of cardiovascular disease: complicated high blood pressure, history of stroke or coronary artery disease, heart surgery, nyha stage iii or iv heart failure;  insulin-dependent diabetics who are unbalanced or have secondary complications;  people with chronic respiratory disease that may decompensate for a viral infection;  people with morbid obesity (body mass index > 30 kg/m 2 ).  concerning the risk related to surgery, two situations have been identified:  surgery with a high risk of contamination of caregivers by aerosolisation of sar-cov-2 (intervention with opening or exposure of the airways: lung resection surgery, ent surgery, neurosurgery of the base of the skull, rigid bronchoscopy);  major surgery, with a high risk of postoperative critical care stay, where the perioperative respiratory risk inherent to surgery and anaesthesia is likely to be increased by sar-cov-2 infection or even porting. the objective of these guidelines is to produce a framework to facilitate the partial and gradual resumption of intervention activity in the context of the covid-19 pandemic. the group has endeavoured to produce a minimum number of recommendations to highlight the strengths to be retained in the 7 predefined areas. the basic rules of universal good medical practice in perioperative medicine were considered to be known and were therefore excluded from the recommendations. the recommendations made concern 7 fields: to the drafting of the recommendations to adopt a format of expert opinion. the recommendations were then drafted using the terminology "experts suggest doing" or "experts suggest not doing". proposed recommendations were presented and discussed one by one. the aim was not to necessarily arrive at a single, convergent expert opinion on all the proposals, but to identify points of agreement and points of divergence or indecision. each recommendation was then evaluated by each of the experts and subjected to an individual rating using a scale ranging from 1 (complete disagreement) to 9 (complete agreement). the collective rating was based on a grade grid methodology. in order to validate a recommendation, at least 70 per cent of the experts had to express a favourable opinion, while less than 20 per cent expressed an unfavourable opinion. in the absence of validation of one or more recommendations, the recommendation(s) was/were reformulated and submitted again for scoring with the aim of reaching consensus. the experts' synthesis work resulted in 51 recommendations. after one round of scoring, a strong agreement was reached for 100% of the recommendations and algorithms. in order to protect them during this pandemic, strict safety measures should be implemented. these measures should be carried out all throughout the patient's healthcare pathway: preanaesthetic assessment, operating theatres, recovery rooms, intermediate care units and critical care units. these safety measures will be implemented directly by providing healthcare professionals with adequate ppe, but also indirectly by supplying patients with the right equipment. administrative measures (patient information, preoperative laboratory testing, check-up modalities, anaesthesia modalities, dedicated healthcare pathways, patient and surgery selection), which also help protecting staff members, will be detailed in the following/other chapters. staff members should apply strict social and physical distancing measures when not caring for patients (team rounds, discussions about patients, hand-offs, breaks, meals...): they must keep at least 1 to 2 meters apart from one another, especially during times when wearing a mask is not possible. using alcohol-based hand sanitiser and put on a surgical mask type ii/iir when entering a hospital. this also applies to kids for whom fitted masks should be provided. page 11 of 57 j o u r n a l p r e -p r o o f 11 alcohol-based hand sanitiser before and after every contact with the patient or his surroundings, in addition to wearing a surgical mask type ii or iir and eye protection (goggles) during any clinical examination which requires the patient to take off his mask.  setting up a safety distance in addition to specific physical distancing devices (like temporary plexiglass barriers, interphones…) for those whose work position requires them to be in physical proximity to other people. these devices should be cleaned frequently, following the same cleaning procedures that are used on other surfaces;  removing magazines, documents and other commonly used objects from waiting rooms and common areas, including children's toys;  regularly cleaning surfaces (counters, computers, phones...) and equipment (blood pressure cuffs, pulse oximeter, stethoscopes…) after each patient. during this covid-19 pandemic, every patient could potentially be contaminated and should therefore protect other patients and hospital staff by applying alcohol-based hand gel and wearing a surgical mask type ii or iir. [1] [2] [3] by blocking large droplets, surgical masks protect staff members from droplet and contact transmission. 4 surgical masks can provide protection for healthcare professionals against droplet transmission within a one-meter radius of the patient. four rcts compared the efficiency of n95 or ffp2 masks and surgical masks in healthcare workers performing non aerosol-generating procedures. 5-8 a meta-analysis including these studies reported no significant difference in the occurrence of viral respiratory infections (rc 1,06; 95% ic 0, 25) between the 2 types of mask. 9 only one study specifically evaluated coronaviruses and reported no significant difference between the 2 types of masks in non-aerosol generating procedures. 6 1.3. operating theatre 12 r1.3.1 -experts suggest that healthcare professionals involved in airway management (intubation, extubation, supraglottic airway insertion and/or removal…), or those who could be brought to do so in some given situations, wear a fit tested respirator mask (respirator n95 or ffp2 standard, or equivalent) in addition to a disposable face shield or at least, in the absence of the latter, safety goggles, regardless of the patient's covid-19 status (table 1) there is a great risk of becoming infected during airway management. therefore, strict safety measures should be applied during aerosol-generating procedures such as bag mask ventilation, endotracheal intubation, open/endotracheal suctioning and extubation. the use of a respirator ffp (filtering face piece mask) type 2 is recommended by the french society of hospital hygiene (sf2h) and the french-speaking society of infectious disease for all healthcare professionals manipulating the airway. 10 respirators are tight fitting masks, designed to create a facial seal that protect the person wearing them from droplets and airborne particles inhalation. however, wearing this type of mask can bring more discomfort than wearing a surgical mask (overheating, page 13 of 57 j o u r n a l p r e -p r o o f 13 respiratory resistance...). they have the advantage of blocking at least 94% of aerosol particles (total inward leaking < 8%) and are more effective than surgical masks type ii/iir in blocking < 5 µm particles. 11 nonetheless, a poorly fitted n95 or ffp2 respirator does not protect more than a surgical mask. a leak test must be performed systematically. furthermore, a beard (even a stubble one) reduces the mask's adherence to the face and thus decreases its global efficiency. in case of n95 or ffp2 respirators shortage, some experts suggested using n99 or ffp3 respirators which block at least 99% of aerosol particles (total inward leaking < 2%). however, the problem with these respirators in that the air is most often exhaled through an expiratory valve without being filtered. they do not filter the wearer's exhalation, only the inhale. this one-way protection puts others around the wearer at risk, in a situation like covid-19. covid-19 can also be transmitted by aerosol contact with conjunctiva 12 and lead to a respiratory infection. 13 the fact that unprotected eyes increase the risk of transmission has been demonstrated with coronaviruses. 14 face shields provide a barrier against high velocity aerosol particles and are commonly used as alternatives to safety goggles as they provide greater face protection. 15 using a droplets simulator loaded with influenza viruses (mean droplet diameter: 3.4 µm) and a breathing simulator, it was demonstrated/shown that the use of a face shield reduces the risk of aerosol inhalation by 70%. 16 when spraying fluorescent dye (particle diameter = 5 µm) from a distance of 50 cm towards a mannequin head equipped with an n95 respirator and a face shield, no contamination was noted in either nostrils nor eyes nor mouth folds. the same researchers found that using safety goggles in combination with an n95 respirator did not prevent some eye contamination. 17 face shields also contribute to sparing n95 or ffp2 respirators by limiting their contamination with aerosol projections. n95 or ffp2 respirators can be used for up to 8 hours. during the pandemic period, and a minimal distance of 7-8 meters if an extubation is performed in the recovery room. whenever possible, in order to spare n95 or ffp2 respirators and to protect staff members and other patients, extubation should be performed in the operating theatre by the person who performed the intubation. if this is not possible, the same precautions should be taken in the recovery room for staff protection. in the latest world health organization (who) recommendations for covid-19, health care personnel and other staff are advised to maintain a one-meter distance away from a person showing symptoms of disease. 19 the centre for disease control and prevention recommends a two-meters separation. 20 however, these distances are based on estimates of range that have not considered the possible presence of a high-momentum cloud carrying the droplets long distances recent work has shown that exhalations, sneezes and coughs emit turbulent multiphase flows that can contain pathogen-bearing droplets of mucosalivary fluid. 21 when sneezing or coughing, these droplets/gas clouds can travel in the air for up to 7 to 8 meters. 22 this new understanding of respiratory emissions dynamics has implications on social distancing strategies during the covid-19 pandemic. similarly, swabs taken from air exhaust outlets in covid+ patients' rooms were found to contain rna fragments, suggesting that small virus-laden droplets may be displaced by airflows. 23 however, in this study, no viral culture was done to demonstrate virus viability. for these reasons, extubation should remain exceptional in the recovery room, and giving out surgical masks type ii/iir to patients after their extubation is essential.  administration of nebulised treatment by a device other than vibrating membrane nebulisers. r1.5.3 -when the patient's covid-19 status is unknown, experts suggest using a closed suction system for tracheal suctioning. if this system is unavailable, it is necessary to interrupt the patient's ventilation during suctioning, ideally with the help of a second operator. respiratory droplets are the main source of contamination in healthcare professionals. 2 during aerosol-generating procedures, there is a consensus on the efficiency of n95 or ffp2 respirators (see questions 1.3) and the wear of protective gear such as a fluid resistant long-sleeved gown or a combination of a conventional gown and a plastic apron. 10, 24 the number of asymptomatic patients carrying the virus is high 25 , which is why caregivers should systematically use protection during high-risk procedures. 10,24,25 1.6. paediatric particularities r1.6.1 -experts suggest allowing only one parent to be present during kids' preanaesthetic assessment. and gloves, when performing any procedure with a high transmission risk, particularly when examining the oral cavity. r1.6.3 -experts suggest wearing an n95 or ffp2 respirators, a head cap, a gown with an apron, gloves and a face shield or, failing that, protective goggles, when performing airway procedure in children who are awake in the recovery room, regardless of their covid status. during this covid-19 pandemic, applying enhanced safety measures for the paediatric population is justified due to the existence of a significant proportion of possibly asymptomatic covid+ children (up to 16% depending on the series) and the likely difficulty in complying with social distancing and safety measures (difficulty of continuous wearing of the surgical mask) by children. [26] [27] [28] these findings imply that anaesthesia staff should wear a surgical mask type ii/iir, protective goggles (or a face shield) and gloves when performing any procedure with a high risk of transmission, and particularly when examining the oral cavity during anaesthesia consultation. r2.1 -in asymptomatic patients, during a covid-19 pandemic, experts suggest evaluating the benefit/risk ratio of the intervention according to criteria related to the patient, the pathology and the procedure ( table 2) . the circulation of sars-cov-2 in the population and the existence of asymptomatic carriers affect the risk-benefit ratio of performing a planned surgical procedure during the covid-19 pandemic and require rigorous evaluation. this consideration must integrate three types of criteria related to the patient, the pathology and the procedure. the data in the literature, although heterogeneous and with a low level of evidence, identify several patientrelated risk factors for serious forms of covid-19 potentially associated with an increase in postoperative complications: asa class, obesity, age (> 65 years, < 1 year), underlying respiratory (asthma, copd, cystic fibrosis) or cardiovascular (hypertension, coronary artery disease and chronic heart failure) pathology, obstructive sleep apnoea syndrome, diabetes, and immunosuppression. 29, 30 this increase in perioperative risk is, however, offset by the potential deleterious effect of cancelling or postponing the procedure on the patient. 31 the loss of chance in the absence of intervention must be estimated and the effectiveness and availability of therapeutic alternatives (curative or waiting) explored. finally, two types of factors related to the surgical procedure must be considered: resource utilisation and the risk of transmission of cov-2-sars to the healthcare team. surgical time and expected length of stay provide an indication of the staff and hospital resources required. for each intervention, the foreseeable use of postoperative management in a critical care area must be anticipated in order to adapt surgical activity to the supply available at the time. transfusion needs must also be assessed due to the difficulties of public access to blood donation collection points. the number of personnel required must be taken into account as it increases the risk of contamination of the health care team due to the impossibility of complying with the recommendations for intraoperative distancing. finally, the risk related to the type of anaesthesia and the type of surgery must be evaluated. upper airway management has been identified as a high-risk event for potential transmission of the aerosolised airway secretion virus that persists several minutes after the procedure. 32, 33 the same risk is observed for upper aerodigestive tract and thoracic procedures. finally, the risk related to the surgical site must take into account the probability of postoperative mechanical ventilation, the consequences of which could be aggravated in the context of an infection, or even portage, with sars-cov-2. during the preanaesthetic consultation, detailed information must be provided to the patient and/or his/her legal representative about the perioperative strategy decided regarding his specific situation in the context of covid-19 pandemic. the message must be clear, objective and based on the currently available data, while trying to be reassuring for the patient and/or his legal representative. this message must be given orally during the consultation but also disseminated through a document (established and validated by each structure), which can be given to the patient and/or his legal representative during the preoperative consultation (surgical or preanaesthetic). this information must appear in the medical record. in the appendix, based on current data, we propose examples of model documents (appendix 1, 2 and 3). in the event of cancellation or postponement of the intervention, it is essential to keep in touch with the patient, mostly through the surgical teams, and to reassess the possible alternatives and the feasibility of the procedure according to the evolution of the circumstances. if the decision of postponement or cancellation of the surgery is taken by the patient, it must be recorded in the medical record. the use of a standardised questionnaire increases the completeness of the symptom collection and the reproducibility of the medical examination. it is an appropriate tool for collecting accurate information from a large number of subjects. the data collected are easily quantifiable and traceable. the essential qualities of such a questionnaire are acceptability, reliability and validity. the questions must be formulated to be understood by the largest number of patients, without ambiguity, and be based on validated items. because of the wide variety of symptoms attributable to the sars-cov-2, the questionnaire should be designed to look for the most frequent symptoms (fever, dry cough, etc.) and/or the most evocative ones (anosmia, ageusia, etc.), without however declining all the unusual symptoms that have been reported in the literature. an example of a standardised questionnaire distinguishing between major and minor symptoms is proposed for adults in the appendix #4 and for children in the appendix #5. cov-2 infection at the minimum during the preanaesthetic consultation/teleconsultation and during the preanaesthetic visit. whenever possible, searching symptoms during a phone call with the patient or his legal representative 48-72 hours before the intervention is also recommended to avoid a last-minute postponement of surgery. assessment of specific perioperative risk during the covid-19 pandemic requires, as in the usual situation, the joint consideration of the surgical, patient and anaesthetic risks. in addition, searching usual and/or evocative symptoms of sars-cov-2 infection is an important time of the preanaesthetic consultation in the current pandemic context and during the first months following the easing of the lockdown. the presence of major (i.e., very frequent or relatively characteristic) and/or minor (i.e. more inconsistent and/or less specific) symptoms allows to orient the preoperative covid-19 status assessment, and then to estimate the benefit/risk balance of maintaining or postponing the surgery, taking into account the risk of contamination of health personnel and others patients within the care structure. 34 the integration of these different risks must be collectively weighed against the potential consequences of postponing or cancelling a scheduled intervention. 31 this search for symptoms compatible with a sars-cov-2 infection must take place at the time of the preanaesthetic consultation in order to discuss the postponement of the intervention, if possible, and to anticipate the protective measures that should be applied for the health personnel, and the care circuit that should be used. the questionnaire can be completed by the patient himself, by a nurse just before the consultation or by the anaesthesiologist during the consultation. then, it must be explained that the patient must immediately contact the anaesthesia team, without waiting for admission to the hospital, in case one or more symptoms compatible with a sars-cov-2 infection appear between the preanaesthetic consultation and the day of the intervention. it will also be necessary to explain the importance of the strictest compliance with protective measures, particularly hand-washing and wearing systematically a face mask outside home, between the preanaesthetic consultation and the day of the intervention. if the local organisation allows it, a contact with the patient 48 to 72 hours prior to its admission to the hospital, to ensure that no symptoms have appeared, can also be planned. this timeframe can be adapted locally, the objective of this contact being to have a pcr performed and its results available before coming to the hospital for surgery if the patient has become symptomatic since the preanaesthetic consultation. however, taking into account that the delay between the preanaesthetic consultation and the intervention may correspond to the incubation period of the disease, and that spontaneous reporting by the patient of the onset of symptoms since the consultation will not be systematic nor exhaustive, the search for these same symptoms must be systematically renewed during the "physical" preanaesthetic visit the day before or on the day of surgery. fever, although non-specific, is a very common symptom of symptomatic sars-cov-2 infections, present in 75% to 95% of cases. [35] [36] [37] [38] the presence of fever is a major symptom and an important warning sign that should raise the suspicion of a possible sars-cov-2 infection during the current pandemic. however, since the sensation of fever is highly imperfectly correlated with the temperature objectively measured, 39 it is suggested that patient's temperature should be measured during the preanaesthetic consultation. in addition, antipyretic drug intake should also be systematically collected at the same time as the temperature measurement because acetaminophen (or even nsaids when taken as self-medication by the patient) can normalise the patient's temperature. as the delay between the pre-anaesthetic consultation and the intervention may correspond to the incubation period of the disease, an objective measurement of the patient's temperature must be renewed during the preanaesthetic visit the day before or on the day of the intervention. (figures 1 and 2) for the preoperative covid-19 status assessment and perioperative strategy before scheduled or emergency surgery. these 2 algorithms are the result of a work that tried to take into account a maximum number of clinical situations in a maximum number of structures, while trying to keep it simple. if local provisions, linked to access to diagnostic tests, to the typology of patients, to the prevalence of the virus in the geographical area concerned, or to an agreement between the different specialties at the local level, have led to propose a local algorithm different from those proposed, we suggest that the local algorithm may take precedence over those proposed here. if the patient presents with signs compatible with a sars-cov-2 infection but that the pcr is negative, the evocative paraclinical signs are absent, the ct-scan shows no signs of sars-cov-2 viral pneumonia, and the serology performed after at least 7-10 days of symptoms is negative, a differential diagnosis is then the most likely, and the intervention will be postponed until this other pathology has recovered. in a completely asymptomatic patient, a distinction should be made between: 1) surgeries with opening or exposure of the airways (ent surgery, thoracic surgery, oral surgery, surgery of the base of the skull, rigid bronchoscopy, etc.) for which there is a significant risk of aerosolisation for the operating theatre staff, motivating the realisation of a pcr even in an asymptomatic patient as long as the virus is circulating in the population; and 2) surgeries for which a sars-cov-2 infection could have serious postoperative consequences, thus motivating pcr testing. these surgeries can probably be summed up as "major" surgeries (open-heart surgery, major abdominal or pelvic surgery, organ transplantation, etc.), particularly due to their frequent respiratory impact, since the risk of synergy between sars-cov-2 and perioperative lung injury is not known. to date, this preoperative screening for covid-19 indicated by the type of surgery is based on pcr and there is no indication to perform a thoracic ct scan in this context. in these two situations, the pcr will ideally be performed in the 24 hours preceding the intervention, at most 48 hours, in order to have an idea of the viral carriage as close as possible to the high-risk procedure while taking into account the time required to obtain the results in each structure in order to have them available before the intervention. finally, non-major surgeries in an asymptomatic patient can be performed in a conventional non-covid-19 circuit. 46 if possible, it is suggested that the close contacts of these patients (such as the immediate neighbours in the postoperative recovery room) should be traced to facilitate contact tracing if the patient develops symptoms consistent with sars-cov-2 infection in the days following surgery. it should be noted that if the presence of antibodies in the plasma of a convalescent patient 7 to 10 days after the onset of symptoms has been reported, the positivity of the serology is sometimes later (up to several weeks). in addition, the antibody titre and their neutralising character against sars-cov-2 may vary depending on the patient. [47] [48] [49] [50] [51] [52] furthermore, diagnostic performances vary greatly depending on the type of kit used in the laboratory. finally, the neutralising character of the detected antibodies depends on the viral antigens against which the detected antibodies are directed. [47] [48] [49] [50] [51] [52] consequently, the only place of serology in the diagnostic strategy to date is in addition to a chest ct-scan and a new pcr sample if the first pcr in a symptomatic patient is negative and the symptoms have been evolving for at least 7 to 10 days. new data may change its place in the diagnostic algorithm in the future, especially if it allows the formal detection of patients who are genuinely cured and protected against re-infection, so that surgery can be performed without risk for the patient and staff. by definition non-deferrable, the surgery has to take place. however, pcr sampling should be performed in symptomatic or mildly symptomatic patients who have had close contact with a covid-19 patient within the last 15 days, or who themselves have risk factors for severe forms of covid-19 or are operated from surgery with postoperative respiratory risk. surgery is performed without waiting for the results. in the case of major surgery, a postoperative surveillance in the intensive care unit (potentially already justified by the complexity of the surgery and/or the patient's comorbidities) may be considered, especially in a symptomatic patient, as a risk of synergy between perioperative lung injury and infection/carry of sars-cov-2 cannot be excluded at this time. an outpatient procedure, the experts suggest that the covid-19 status should be sought, at a minimum by using the standardised questionnaire (paediatric version, appendix 5) at the call on d-1. if the interview proves positive, the procedure is rescheduled at least 15 days later. if the questioning does not appear to be interpretable, the child will, depending on the degree of urgency of the procedure, either be rescheduled or hospitalised with a pcr screening test. severe forms of covid-19 are uncommon in children compared to adults, with an estimated incidence of resuscitation of 0.6% of symptomatic forms. 53 clinical manifestations are generally limited to a mild form with fever, myalgia, dry (or productive) cough, runny nose and digestive disorders (nausea, vomiting, diarrhoea, abdominal pain) in 54% of cases. [53] [54] [55] finally, more specific to covid-19 is the presence of anosmia and/or ageusia without nasal obstruction, which are strongly suggestive of this pathology. 1, 2 the presence of skin signs such as pseudo frostbite or urticarial elements are also signs suggestive of covid-19 in children and adolescents. in all cases, the majority of reported paediatric cases are familial in origin and a history of covid-19 in the family environment should be considered a risk factor for this disease in children, even if the child is asymptomatic. 56, 57 radiological signs are identical to those in adults but are inconsistently found (43% of cases on average) and therefore do not contribute much to the diagnosis in this population. 56, 57 the same limitation applies to pulmonary ultrasonography given the lack of studies in the paediatric population. 58 biologically, the published series show lymphopenia or hyperlymphocytosis associated with increased crp. 56 it is important to note that recent studies conducted on cohorts of individuals on an epidemiological basis tend to show that for one person expressing the disease, 7 people are asymptomatic, which reflects the limitations of the clinic to screen all potentially contaminating patients (prepublication study 1) [9-10]. taking into account these elements and the asymptomatic or paucisymptomatic nature of the disease, the problem of the preoperative assessment in paediatrics is above all that of diagnosing this pathology in children, given the risks incurred by caregivers (representing between 3 and 15% of covid-19 infections) [6] , but also that of nosocomial contamination of other patients given the particularly high number of reproductions of this condition (between 2 and 3.5). 56, 57 in the same vein, ambulatory surgery should in theory be favoured in order to avoid cases of nosocomial contamination. it is therefore proposed to perform a pcr test for the virus for each paediatric patient before surgery. in the context of the emergency department, pcr is carried out on admission of the child, but surgery can be performed before the results are obtained. r4.1.1 -during covid-19 crisis, the experts suggest that telemedicine is an alternative to face-to-face consultation and must be used to reduce patient in-visit. the current outbreak of covid-19 has placed a heavy burden on global medical systems, particularly with regard to the preoperative assessment of patients for surgery. for all elective surgeries in france and in many countries for major surgery, preoperative physical assessment by physicians had become a standard of care. the current crisis has reduced this possibility because patients should not be exposed to potentially contagious structures. in for patients, prior agreement to carry out a telemedicine evaluation is a mandatory step. it is advisable to send beforehand a guide to prepare the teleconsultation (including: connection modalities, health questionnaire on current treatments, information documents...) to facilitate the smooth running of the consultation. if necessary, a person close to the patient or an interpreter may, if present during the tlc, assist the doctor in carrying data of the clinical examination within the limits of his or her competence. not all patients desire remote evaluation, and the exact reasons for this have not been elucidated. patient selection is an important step for virtual preoperative evaluation. for example, patients in whom arranging travel is complicated underwent successful telemedicine preoperative evaluation before oral and maxillofacial surgery with no complications, highlighting this patient population as one in whom remote evaluation may be beneficial. the use of telemedicine preoperative evaluation has been studied in a variety of patient populations. all types of surgery can be performed with telemedicine evaluation but major surgery (cardiac, vascular, thoracic, etc.) and patients with many comorbities or treatment are obstacles to the development of this technique. similarly, patients must be able to connect to a platform and know how to use the software. failure to undergo a preoperative anaesthesia evaluation may contribute to day of surgery cancellation, which has a negative financial impact on both patients and hospitals. up to 25% of day of surgery cancellations are due to inadequate preoperative workup, and it is well established that preoperative clinics reduce risk of such cancellations and delays. with telemedicine, we found a 1.3% last minute cancellation rate, consistent with the international average, in patients who underwent telehealth evaluation as opposed to an in-person visit, thus suggesting an equivalent performance between the 2 evaluation options. teleconsultation is carried out using tools that guarantee the security of patient data. it is carried out in conditions that must guarantee : authentication of the healthcare professionals involved in the procedure; identification of the patient; access by healthcare professionals to the patient's medical data required to perform the procedure; access by the patient to the patient's medical data required to perform the procedure. informed consent is an important factor in surgery and telemedicine itself is no different. the evaluation of the practices is advised to optimise these new modalities. as stated in the introduction, in the context of the covid-19 pandemic, the resumption of surgical activity is subject to several major limitations: the strain on the supply of certain anaesthesia drugs, the change in hospitalisation capacities, the risk of contamination of healthcare providers and patients and the application, throughout the patient's journey, of the "distancing" principle. in addition, some peculiarities of covid-19 patients (risk of drug interactions, worsening of the condition, etc.) are to be taken into account. these limitations lead us to propose an adaptation of anaesthesia procedures. favour strategies that reduce the exposure of health professionals to a risk of contamination while maintaining optimal safety conditions for the patient is one of the most important objectives. when safety conditions are met (especially for postoperative follow-up), outpatient management should probably be prioritised. r5.1.2 -experts suggest giving priority whenever possible to regional anaesthesia. regional analgesia and infiltration techniques should also be considered. tensions on drug stocks and even shortages of drugs such as propofol, midazolam, atracurium, cisatracurium or rocuronium require the choice of anaesthesia protocol that spares these drugs, which are otherwise subject to quotas. to do so, the experts propose several principles: -prefer regional anaesthesia (ra) for anaesthesia and analgesia, rather than general anaesthesia. in the context of -peripheral and topical local anaesthesia allow postoperative follow-up directly in the room or in a dedicated space, without going through the recovery room in accordance with regulations. this facilitates compliance with distancing measures specific to the current epidemic context. 65 in children, since ra techniques are regularly associated with general anaesthesia or sedation, they do not make it possible to bypass the recovery room. -when ga is required, inhaled anaesthesia should probably be preferred in this context to intravenous targetcontrolled anaesthesia. -monitoring of the depth of anaesthesia when possible, and of curarisation may be required in order to best adapt drug dosages. 66 these recommendations apply to both elective and emergency care. in conjunction with the institution's pharmacy, it is important to monitor local stock trends. epidemics" published by the srlf-sfar and to the "airway management principle" sheet, which are also applicable in the operating theatre. during the covid-19 pandemic period, the intubation of a covid+ patient in the operating theatre is based on the same rules as those issued in critical care units, due to the risk of spraying of the virus during this risky procedure. in order to minimise the risk of aerosolisation and contamination of personnel, it is necessary to: -limit the number of staff present in the operating theatre -avoid ventilating the patient with a face mask during the preoxygenation phase. -stop oxygen before removing the bag valve mask. -intubate the patient by the most experienced senior using a video laryngoscope -connect the ventilator after inflating the intubation tube balloon. highly suspected patients. patient. if general anaesthesia is required, the patient's clinical condition and covid-19 status should be considered in the airway management strategy. -if the patient is covid+ or highly suspected: the procedure described by sfar 46 should be followed with rapid sequence induction and intubation. special attention should be paid to tracheal extubation with the same barrier precautions as for intubation. this applies to patients under emergency management when the covid-19 status is unknown. special attention should also be paid to hand hygiene. -if the patient is non-covid or asymptomatic, there is no need to modify usual procedures because of the covid-19 pandemic. routine airway management is recommended. if intubation is chosen, conventional induction is recommended according to standard recommendations, with adaptation of the induction sequence according to haemodynamic conditions, drug contraindications, and compliance with fasting conditions and the patient's age. the frequency of anaphylaxis related to atracurium has been estimated to be 1/22451 administrations. the frequency of anaphylaxis due to fast-acting myorelaxant is about 10 times higher (succinylcholine: 1/2080 and j o u r n a l p r e -p r o o f 27 rocuronium: 1/2499). 67 the severe over-risk of allergy to the patient linked to a rapid sequence induction does not seem to be justified by the sole risk of sars-cov-2 contamination of the caregivers, this risk being low when protective measures are well respected (cf. item 1). readers are invited to refer to "guidelines on muscle relaxants and reversal in anaesthesia". 66 in a non-covid patient, spontaneous ventilation anaesthesia or the use of supraglottic devices such as laryngeal masks is possible. we insist on the importance during the preoperative checklist to share with the operating theatre staff, in addition to the usual information, the covid status of the patient which will determine his perioperative circuit and the strategy adopted by the anaesthesia team for airway management. cov-2 is available online from the university of liverpool. 68 a summary is provided below for drugs frequently used in the perioperative period ( table 3) . the hydroxychloroquine has multiple cardiac adverse events, including significant qt prolongation. combinations with other drugs that prolong the qt interval, frequently used in the perioperative period such as halogenated drugs, droperidol, ondansetron, or hypothermia related to surgery and anaesthesia may increase the risk of developing a serious arrhythmia, such as ventricular fibrillation. the combination of hydroxychloroquine and azithromycin, proposed by some, carries a risk of additive/synergistic qt interval prolongation. ecg monitoring is essential. in addition, the combination of lopinavir/ritonavir carries a risk of overdosage with amide type local anaesthetics (lidocaine, levobupivacaine, bupivacaine, prilocaine, mepivacaine, ropivacaine), ketamine, midazolam, sufentanil, oxycodone or tramadol due to ritonavir-related cytochrome p3a inhibition, but also to underdosage of propofol and morphine due to increased biotransformation of products metabolised by cytochrome p2c9 and p2c19 or by glucuronidation. remdesivir, tocilizumab, and interferon beta do not show significant interactions with drugs normally used perioperatively, nor do they have cardiac effects. nsaids may be associated with worsening of symptoms during respiratory viruses, with an increased risk of empyema. 70 despite recent alerts, there is no scientific evidence to date linking nsaid use to the aggravation of sars-cov-2 infection. a precautionary principle applies. 71 thus, in a patient with an established or strongly suspected sars-cov-2 infection, the prescription of nsaids will be avoided. however, in asymptomatic patients, there appears to be no contraindication to their use if their benefit is established. 72, 73 discontinuation of corticosteroids is not recommended in patients on long-term therapy. 70 steroid treatment of patients with covid-19 is controversial and is not currently recommended. 74 the single intraoperative injection of dexamethasone, at the usual recommended doses, does not appear to present an over-risk in the asymptomatic patient. anaesthesia is indicated, experts suggest that rapid sequence anaesthesia be performed regardless of the patient's covid-19 status. in the context of covid-19 pandemic, obstetric patients present two particularities. first, unlike scheduled surgical activities, obstetrical activity in essence cannot be postponed and therefore remained at its usual level at the peak of the pandemic. the organisation of care had to be adapted, with the establishment of specific care channels for women infected with sars-cov-2 or suspected of being infected, not only to optimise the care of these women, but also to avoid the contamination of other pregnant women and of caregivers working in maternity wards. these covid-positive or suspected covid-positive/non-covid channels are logically maintained as long as the pandemic persists. the resumption of surgical activity during the covid-19 outbreak exposes no-covid-19 patients and healthcare workers to contamination. the following expert proposals should be discussed within each institution in a collegial manner (extended executive board, operating theatre committee, healthcare infection control practices advisory committee) and lead to protocols that take into account the specific characteristics of each institution (architectural constraints, recruitment) and the local incidence of covid-19 infection. appropriate signage has to be applied throughout the specific covid-19 pathway. in the context of non-covid patients management in the operating theatre, the aim of this guideline was to avoid both the occurrence of nosocomial sars-cov-2 infection 87 and the contamination of caregivers by asymptomatic patients 88 . for any planned surgical procedure, the risk/benefit balance must be discussed in a multidisciplinary manner, given the probably high postoperative morbidity and mortality in this epidemic context. 88 management of "non-covid" patients must be considered in a specific pathway. 89 this pathway covers the entire patient's hospitalisation day: from the anaesthesia consultation to discharge from the hospital after surgery, following the guidelines for protection (chapter 1). suggest that for both adults and children, priority should be given to outpatient treatment and enhanced recovery after surgery as much as possible. in the context of covid-19 outbreak, outpatient management should be considered and preferred to conventional hospitalisation when feasible. outpatient management reduces the length of stay, thereby reduces the risk of patient exposure and the risk of contamination in case of asymptomatic infection. 90 outpatient management of surgical emergencies should be considered whenever possible. 91 outpatient pathways for resumption of activity during the pandemic period need to consider several points: 1/ the planning and convocation schedules should be staggered to avoid waiting times and gathering of patient; 2/ the use of single or isolated rooms should be preferred to wait or exit lounges; 3/ limit admissions in the postoperative recovery room must be applied as much as possible, in particular after performing locoregional anaesthesia. depending on the local outpatient surgery units, this recommendation may limit the number of patients treated. finally, waiting areas for companions should be arranged in order to respect the safe distances. 91, 92 the number of companions should be limited to one person per patient (adult or child). in case of conventional hospitalisation, enhanced recovery after surgery should be preferred as far as possible in order to reduce, once again, the length of stay. in the same way, hospitalisation on the day of surgery should be considered if the healthcare institution ensures that there is no risk of infected patient by the covid-19 (for example by a phone call the day before hospitalisation). the rapidly changing covid-19 pandemic situation requires a periodic review of the measures taken and an analysis of the clinical, social and economic context derived from each decision. the resumption of surgical activity will be gradual and spread over time. the objective is to summarise, as a priority and progressively, those activities that prove decisive in limiting the loss of chance for patients awaiting cancer or non-cancer surgery. 93 the gradual deployment of surgical activity in a controlled number of operating theatres will make it possible to achieve efficiency in open operating theatres and facilitate compliance with reinforced hygiene rules to ensure the safety and protection of patients and caregivers. experts suggest that public and private facilities agree to propose a common approach to the provision of care adapted to the population and regional conditions of the covid-19 pandemic. the pace of rescheduling elective surgery in children and adults will vary according to geographical location, epidemiological pressure, and the possibility of redeploying staff from critical care to operating theatres. elements to be evaluated for the resumption of surgical activity are the following:  timing of resumption: there should be a sustained reduction in the rate of new covid-19 cases in the geographical area concerned for at least 14 days before the resumption of elective surgery. 94  any resumption must be authorised by the relevant regional and national health authorities.  facilities are able to safely treat all patients requiring hospitalisation without the need for a crisis care organisation.  the facility has an appropriate number of critical and non-critical non-covid and covid+ beds, ppe, ventilators, drugs, blood products and all necessary medical and surgical equipment. the facility has a number of trained and educated staff appropriate to the planned surgical procedures, the patient population and the facility resources. health care staff fatigue and the impact of stress must be considered in order to perform planned procedures without compromising patient safety or staff safety and well-being. 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joint task force of the chinese society of anesthesiology and the chinese association of anesthesiologists clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of covid-19 infection temporal dynamics in viral shedding and transmissibility of covid-19 preparing for a covid-19 pandemic: a review of operating room outbreak response measures in a large tertiary hospital in singapore the covid-19: role of ambulatory surgery facilities in this global pandemic statement from the ambulatory surgery center association regarding elective surgery and covid-19 at guidelines for ambulatory surgery centers for the care of surgically necessary/time-sensitive orthopaedic cases during the covid-19 pandemic approaching surgical triage during the covid-19 pandemic at <https://journals american college of surgeons, american society of anesthesiologists: joint statement: roadmap for resuming elective surgery after covid-19 we asked the experts: covid-19 outbreak: is there still a place for scheduled surgery? "reflection from pathophysiological data the authors want to thank all the reviewers from others medical specialties who brought their expertise on several hand disinfection with a hydro-alcoholic based hand gel, a surgical mask type ii/iir.patients must wear a surgical mask type ii/iir when leaving their unit and heading for the or.coded covid-19 dedicated routes should be followed.surgical mask type ii/iir. discuss 1) a chest ct-scan, and/or 2) a control of the pcr on a new sample, and/or 3) a serology* key: cord-321756-a7eh4dkb authors: kwofie, theophilus b; anane, yaw a; nkrumah, bernard; annan, augustina; nguah, samuel b; owusu, michael title: respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana date: 2012-04-10 journal: virol j doi: 10.1186/1743-422x-9-78 sha: doc_id: 321756 cord_uid: a7eh4dkb background: acute respiratory tract infections are one of the major causes of morbidity and mortality among young children in developing countries. information on the viral aetiology of acute respiratory infections in developing countries is very limited. the study was done to identify viruses associated with acute lower respiratory tract infection among children less than 5 years. method: nasopharyngeal samples and blood cultures were collected from children less than 5 years who have been hospitalized for acute lower respiratory tract infection. viruses and bacteria were identified using reverse transcriptase real-time polymerase chain reaction and conventional biochemical techniques. results: out of 128 patients recruited, 33(25.88%%, 95%ci: 18.5% to 34.2%) were positive for one or more viruses. respiratory syncytial virus (rsv) was detected in 18(14.1%, 95%ci: 8.5% to 21.3%) patients followed by adenoviruses (adv) in 13(10.2%, 95%ci: 5.5% to 16.7%), parainfluenza (piv type: 1, 2, 3) in 4(3.1%, 95%ci: 0.9% to 7.8%) and influenza b viruses in 1(0.8%, 95%ci: 0.0 to 4.3). concomitant viral and bacterial co-infection occurred in two patients. there were no detectable significant differences in the clinical signs, symptoms and severity for the various pathogens isolated. a total of 61.1% (22/36) of positive viruses were detected during the rainy season and respiratory syncytial virus was the most predominant. conclusion: the study has demonstrated an important burden of respiratory viruses as major causes of childhood acute respiratory infection in a tertiary health institution in ghana. the data addresses a need for more studies on viral associated respiratory tract infection. acute respiratory infections (ari) are one of the major causes of morbidity and mortality in young children throughout the world especially in developing countries [1, 2] . data from who estimated the burden of ari at 94,037,000 disability-adjusted life years (dalys) and 3.9 million deaths in 2001 [3] . similar report from a metaanalysis study demonstrates that throughout the world 1.9 million (95% ci 1.6-2.2 million) children died from ari in 2000, 70% of them in africa and southeast asia [2] . a further systematic analysis also estimated 1.575 million (uncertainty range: 1.046 million -1.874 million) deaths of children worldwide in 2008 as due to ari [4] . majority of acute lower respiratory tract infections (alrti) in developed countries have been reported to be often due to viral pathogens of which most common are rsv, piv, influenza viruses, adv, human coronaviruses and bocaviruses [5] [6] [7] . on the contrary, information on these viruses in developing countries is limited probably due to paucity of modern diagnostic molecular techniques. these infections are therefore treated unsuccessfully with antibiotics based on suspicion of bacterial causes [8] . apart from the public health concern of nosocomial infections that are associated with viral respiratory infections [9, 10] , significant costs derived from long duration of hospitalization and several healthcare visits could also aggravate the poor socio-economic status and increased child mortality in developing countries including ghana. lessons from the outbreak of the severe acute respiratory syndrome (sars) epidemics which resulted in the death of 776 individuals [11] and the recent emergence of a novel swine flu pandemic emphasize the risk posed by respiratory viral infections in humans [12] . this study was done to determine the burden of respiratory viruses among children hospitalized at the komfo anokye teaching hospital for acute lower respiratory illness using the real time polymerase chain reaction (rt-pcr). this was a hospital based cross-sectional study of children less or equal to five years and hospitalized for acute lower respiratory tract illness. the study was performed at the children's ward of the komfo anokye teaching hospital (kath), ghana from january to december 2008. kath is approximately a thousand bed tertiary medical facility located in ashanti region, kumasi, the confluence of the transportation network in the central part of ghana. its position makes it the most accessible tertiary and referral medical facility in ghana attending to a population of over 4.4 million in the ashanti region and beyond [13] . to have a year round incidence of the various viral agents, recruitment was done throughout the year 2008. screening and recruitment were started at the beginning of every week till two or three patients were recruited. after a maximum of three patients have been obtained it was suspended till the beginning of the next week. this cycle was repeated till the maximum of 11 patients were recruited for each month. at the recruitment station all patients arriving at the unit were screened but only those less than five completed years were recruited. patients recruited into the study should have features of severe pneumonia or very severe pneumonia as defined by the who-imci (world health organisation integrated management of childhood illness) protocol [14, 15] . severe pneumonia was said to be present if the child has a history of cough and/ or difficult breathing of less than 3 weeks duration, with lower chest wall recession. severe pneumonia in addition to cyanosis and/or inability to feed or drink was classified as very severe pneumonia. a child was recruited into the study only after the guardian or parents had consented after the objectives of the study had been explained in english or the local dialect. a standardized case record form was then used to record the history of the illness as well as the presenting clinical features of pneumonia. five (5 ml) of blood sample was then taken into 25 ml of brain heart infusion broth (bhib) and quickly sent to the bacteriological laboratory for culture. nasopharyngeal specimens were also taken using the nasopharyngeal flocked swab (copan, italy). the swab was gently inserted up the nostril towards the pharynx until resistance was felt and then rotated 3 times to obtain epithelial cells. it was then withdrawn and put into 2.5 ml phosphate buffered saline (x1). the samples were transported on ice to the laboratory within few hours of collection. they were then vortexed, transferred into 1.5 ml eppendorf tubes (eppendorf, germany) and kept at -80°c for polymerase chain reaction (pcr). both samples were taken by experienced prior trained nurses of the children ward. children were excluded if both samples could not be obtained for any reason. viral nucleic acids were extracted from samples using qiaamp viral mini kit (qiagen, germany) according to the manufacturers' instruction [16] . rt-pcr amplification was performed for the detection of adv, rsv, influenza a (flu a), influenza b (flu b), parainfluenza 1(piv 1), parainfluenza 2 (piv 2) and parainfluenza 3 (piv 3) using the light cycler version 1.5 (roche, germany). primers highly specific to each of the viruses were used ( table 1 ). the reaction mixture for rna viruses was made up of 5 μl of rna extract, 1 μl deoxynucleotide (dntp) triphosphate (10 mm) (qiagen), 1 μl bovine serum albumin (bsa)(1 mg/ml)(qiagen), 12.5 μl qiagen one step rt-pcr buffer x5 (containing 12.5 mm mgcl 2 ) (qiagen), 1 μl qiagen one step enzyme mix, 1 μl each of sense and antisense primer(10 μm each), 0.5 μl probe and 2.0 μl of rnase free water. dna virus reaction mixture was made up of 5 μl pcr buffer (10x), 1 μl of dntp mix (10 mm each), 1 μl of mgcl 2 (50 mm), 0.5 μl of hot star taq dna polymerase, 0.5 μl of probe, 1 μl of bsa (1 mg/ml) and 1 μl each of sense and antisense primers. the pcr conditions for piv, rsv and influenza viruses consisted of an initial reverse transcription at 20 minutes for 50°c, taq dna polymerase activation at 95°c followed by forty five (45) cycles of denaturation at 15 seconds for 95°c and annealing at 15 seconds for 58°c. the initial reverse transcription step was omitted in the case of adv. for each batch of tests that were run, rnase free water (qiagen, germany) was used as negative control and in vitro transcribed gene of the various viruses was used to determine the detection limit of the assay as positive control. the in vitro transcripts were prepared in our collaborative institution (bonn institute of virology, bonn, germany) and transported to ghana via cooling chain. the in vitro transcripts were prepared by amplifying the genes of interest of the viruses with specific set of primers. the products were ligated to the pcr2.1 vector using the topo ta cloning kit (invitogen corp, carlsbad, ca) and transformed into competent e.coli cells. after overnight incubation, desirable colonies were selected and purified using qiamp spin-miniprep kit (qiagen, germany). pcr was performed on the purified product and rna transcripts were then synthesized from the lowest dilution (with clear band) using ambion megascripts t3 kit (invitogen corp, calsbad, ca). the final products were purified using rneasy mini kit (qiagen, germany) and the concentrations of the transcripts were determined using a spectrophotometer (themoscientific, u.s.a). the copy numbers of the rna transcripts were determined following the method of fronhoffs [21] . ten-fold dilutions of the rna transcripts were prepared in carrier rna-rnase free water (310 μg of carrier rna in 310 ml of rnase free water) and tested. the detection limit was determined as the last dilution after which all other replicates gave negative results. the detection limits were respiratory syncytial virus (rsv): 20 copies/μl, piv 1: 1 -78 copies/μl, piv 2: 400 copies/μl, piv 3: 30 copies/μl, influenza a: 120 copies/μl and influenza b: 480 copies/μl. to determine the specificities, our assays were tested on other different viruses and they turn out negative. bacterial isolates were identified using conventional biochemical methods including urease and indole production, citrate utilization, hydrogen sulphide, gas production and fermentation of sugars. the biochemical media used included simon's citrate medium, urea and triple sugar iron agar (tsi). coagulase tests were performed for all staphylococci organisms. data obtained was double entered into a spreadsheet database prepared with microsoft ® excel. it was then compared and cleaned for abnormal wrongful entries. statistical analysis was done using stata se statistical software version 11.2(texas, usa) after the data had been imported. categorical variables such as age groups and their association with respiratory agents were analyzed using the fischer's exact test. continuous variables were expressed as medians with their inter-quartile ranges. a non-parametric k-sample test on the equality of medians was used to evaluate the differences in the medians of the various subgroups of the continuous variables. for all analysis done, a p-value of less than 0.05 was considered statistically significant. the study protocol was approved by the six (20.0%) out of 30 children less than six months of age were found to be positive for viral infection ( table 2 ). the highest number of children infected were between 6 months and 2 years, the proportions infected for the various age groups were however not significantly different. clinical presentation of patients were compared to general pathogens identified (table 3 ) and the individual viral pathogens (table 4) . generally, there were no detectable significant differences in the clinical presentation as well as the gender and duration of illness of patients for the various pathogens isolated. the study also investigated the seasonal variation of viruses. rsv infections were detected throughout the year however the peak infection rate occurred during the minor rainy seasons (october) (figure 1 ). adenovirus infections on the other hand had two main peaks occurring in the month of april and october. viral agents play an important role in acute lower respiratory infections and may herald the onset of pneumonia caused by secondary bacterial infections [22] . although information on the causes of respiratory illness in tropical countries is very scanty, available data indicates about one -third of the cases of respiratory tract infections are due to viruses [23] . the present study identified viruses in 25.8% of patients hospitalized for alrti with rsv being the most predominant. the overall prevalence is comparable to previous studies done in other developing countries [24] and the predominance of rsv is in accordance with the assertion that this virus is the single most frequent lower respiratory tract pathogen in infants and young children worldwide [25] [26] [27] . adenoviruses, the second highest viruses detected in this study have been reported to be responsible for 5-10% of lower respiratory tract infections with the highest rate occurring in younger children [28] [29] [30] [31] . our study similarly recorded a 10.2% detection rate of adenoviruses however there was no statistical difference in the age specific prevalence. this could possibly be due to the few number of younger children (less than one year) enrolled in this study. this study generally recorded higher cases of tachypnoea, chest recessions and very severe pneumonia in rsv compared to adenovirus infected patients but the differences were not statistically significant. although similar clinical presentations have been found to be associated with rsv [32, 33] , our small sample size could account for our inability to detect these differences. among patients who were poorly fed, those without viral or bacterial infections were found to be higher (p = 0.03). poor feeding and malnutrition has generally been associated with less risk for respiratory viral infection in developing countries [34] [35] [36] however the possibility of increased mortality could occur especially when bacterial infections are involved. further case controlled studies are therefore needed in developing countries to investigate this observation there have been fewer studies of piv infections in developing countries and most of them do not differentiate the subtypes of the viruses. our study recorded a 3.1% prevalence of piv infections with the predominant type being piv-3. similar hospital based studies have been reported in other developing countries [37] [38] [39] [40] . piv infections have been strongly associated with croup [41, 42] . the present study recorded no case of croup among piv patients. viral associated bacteremia was detected in two patients (1.7%). low rate of secondary bacterial infections has generally been reported in developing countries with the isolation rate varying between 2 and 10% [43] [44] [45] . studies in some developed countries however reported the converse [46, 47] probably due to the higher sensitivity of the bacteria identification techniques used. the blood culture identification system used in this study may be limited in the detection of bacteria associated with lower respiratory tract infection. perhaps the detection rate could have increased if the present study had identified bacterial pathogens from nasopharyngeal aspirates or washings as reported by hishinki et al. [47] . the isolation of staphylococcus aureus in an rsv positive patient in the present study was similar to studies reported by berman et al., [48] and cherian et al., [43] . in their case, the bacterium was associated with fatality in the children. the present study observed that all subjects enrolled in this study were treated empirically with antibiotics in accordance with the paediatric emergency treatment protocol for managing severe and very severe pneumonia. similar occurrences have been reported where patients were treated unsuccessfully with multiple antibiotics [8] . policy makers may therefore consider reviewing the clinical algorithm for patient management as the economic cost both to the health service and the patient's household derived from the use of antibiotics could be enormous. the period of this study experienced major rainfalls in the months of may to july and minor rainfalls in the month of september to october. the month of october recorded the highest detection of viral infections (57.6%; 19/33) with the commonest being rsv. this phenomenon has been reported in other developing countries [43, 49] . the occurrence of rsv infections in the rainy and cold season could be due to overcrowding as a result of populations staying more to their homes. more studies are however needed to define the seasonality of respiratory viruses in tropical countries. our study had some limitations which include underestimation of the overall prevalence of respiratory viruses since we did not test for coronaviruses, human metapneumovirus, bocaviruses and rhinoviruses which have also been reported in hospitalized patients. also the negative results recorded for influenza a and piv 2 and the low prevalence of influenza b in our samples could be due to the low sensitivity of the assay for these viruses. the limit of detections of influenza a, influenza b and piv 2 are 120 copies/μl, 480 copies/μl and 400 copies/μl respectively as such viral copies below these limits of detections could be missed by our assay. this study has demonstrated that respiratory viruses are associated with a considerable number of hospital admissions in ghana. hospitalized children presenting with symptoms of alrti may not necessarily need treatment with antibiotics but rather antiviral drug options could be explored. the importance of further cross-sectional and longitudinal studies of viral-associated respiratory infections among out-patients and healthy populations cannot be over emphasized. this will contribute immensely to overcoming the paucity of data on the importance of respiratory associated viruses to the burden of disease and the morbidity and mortality of under five children. acute respiratory infections are the leading cause of death in children in developing countries estimates of worldwide distribution of child deaths from acute respiratory infections world health organisation: burden of disease in dalys by sex and mortality stratum in who regions, estimates for 2001. the world health report global, regional, and national causes of child mortality in 2008: a systematic analysis progress in the surveillance of respiratory syncytial virus (rsv) in europe development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children risk of secondary bacterial infection in infants hospitalized with respiratory syncytial viral infection the clinical spectrum of respiratory syncytial virus disease in the gambia an outbreak of acute respiratory disease in trinidad associated with para-influenza viruses newly discovered coronavirus as the primary cause of severe acute respiratory syndrome emergence of a novel swine-origin influenza a (h1n1) virus in humans komfo anokye teaching hospital, centre of excellence integrated approach to child health in developing countries integrated management of childhood illness by outpatient health workers: technical basis and overview. the who working group on guidelines for integrated management of the sick child qiagen: protocol: purification of viral rna (spin protocol) a method for the rapid construction of crna standard curves in quantitative real-time reverse transcription polymerase chain reaction epidemiology of acute respiratory infections in children of developing countries. review of infectious diseases viral respiratory infections and their role as public health problem in tropical countries (review) respiratory syncytial virus infection in tropical and developing countries update: respiratory syncytial virus activity-united states, 1996-97 season epidemiology and aetiology of acute bronchiolitis in hong kong infants viral etiology of acute lower respiratory tract infections in hospitalized young children in northern taiwan respiratory adenoviral infections in children: a study of hospitalized cases in southern taiwan in 2001-2002 frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real time pcr assay adenovirus pneumonia in infants and factors for developing bronchiolitis obliterans: a 5-year follow-up epidemiology and clinical presentation of respiratory syncytial virus infection in a rural area of southern mozambique clinical presentation and severity of viral community-acquired pneumonia in young nepalese children the etiology of pneumonia in malnourished and well-nourished gambian children respiratory syncytial virus infections in malnourished children viruses associated with acute lower respiratory tract infections in children from the eastern highlands of papua new guinea (1983-1985) epidemiology of acute respiratory tract infections among young children in kenya. review of infectious diseases viral etiology and epidemiology of acute respiratory infections in children in etiology of acute lower respiratory tract infections in gambian children: i. acute lower respiratory tract infections in infants presenting at the hospital viral etiology of respiratory infections in children under 5 years old living in tropical rural areas of senegal: the evira project respiratory syncytial virus and parainfluenza virus parainfluenza viruses bronchiolitis in tropical south india etiology of acute respiratory infections in children in tropical southern india. review of infectious diseases diagnosis of childhood pneumonia in the tropics association of invasive pneumococcal disease with season, atmospheric conditions, air pollution, and the isolation of respiratory viruses incidence of bacterial coinfection with respiratory syncytial virus bronchopulmonary infection in pediatric inpatients acute lower respiratory tract illnesses in cali, colombia: a two-year ambulatory study seasonal variation in respiratory syncytial virus infections in children in evaluation of quantitative and typespecific real-time rt-pcr assays for detection of respiratory syncytial virus in respiratory specimens from children pring-akerblom p: rapid and quantitative detection of human adenovirus dna by real-time pcr comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children simultaneous detection of influenza viruses a and b using real-time quantitative pcr respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana the study was partly supported by the komfo anokye teaching hospital (ghana) and the kumasi center for collaborative research in tropical medicine (ghana). we will like to thank all directorate of child health doctors, nurses and patients at the komfo anokye teaching hospital. we also thank the bonn institute of tropical medicine (germany) for supporting us with laboratory materials. authors' contributions yaa, aa and bn co-worked on the data collection and contributed to laboratory analysis of the samples. sbn performed statistical analysis of the data and contributed to writing of the manuscript. tbk planned, initiated the study and contributed to writing of the manuscript. ow carried out the laboratory analysis of the samples and contributed to writing of the manuscript and interpretation of the data. all authors have read and approved the manuscript. the authors declare that they have no competing interests. key: cord-323311-xl2fv0qx authors: kahn, r. e.; morozov, i.; feldmann, h.; richt, j. a. title: 6th international conference on emerging zoonoses date: 2012-09-07 journal: zoonoses public health doi: 10.1111/j.1863-2378.2012.01539.x sha: doc_id: 323311 cord_uid: xl2fv0qx the 6th international conference on emerging zoonoses, held at cancun, mexico, 24–27 february 2011, offered 84 participants from 18 countries, a snapshot of current research in numerous zoonoses caused by viruses, bacteria or prions. co‐chaired by professors heinz feldmann and jürgen richt, the conference explored 10 topics: (i) the ecology of emerging zoonotic diseases; (ii) the role of wildlife in emerging zoonoses; (iii) cross‐species transmission of zoonotic pathogens; (iv) emerging and neglected influenza viruses; (v) haemorrhagic fever viruses; (vi) emerging bacterial diseases; (vii) outbreak responses to zoonotic diseases; (viii) food‐borne zoonotic diseases; (ix) prion diseases; and (x) modelling and prediction of emergence of zoonoses. human medicine, veterinary medicine and environmental challenges are viewed as a unity, which must be considered under the umbrella of ‘one health’. several presentations attempted to integrate the insights gained from field data with mathematical models in the search for effective control measures of specific zoonoses. the overriding objective of the research presentations was to create, improve and use the tools essential to address the risk of contagions in a globalized society. in seeking to fulfil this objective, a three‐step approach has often been applied: (i) use cultured cells, model and natural animal hosts and human clinical models to study infection; (ii) combine traditional histopathological and biochemical approaches with functional genomics, proteomics and computational biology; and (iii) obtain signatures of virulence and insights into mechanisms of host defense response, immune evasion and pathogenesis. this meeting review summarizes 39 of the conference presentations and mentions briefly the 16 articles in this special supplement, most of which were presented at the conference in earlier versions. the full affiliations of all presenters and many colleagues have been included to facilitate further inquiries from readers. addition to the summaries later of six presentations on this topic, this special supplement includes an article, monitoring of west nile virus infections in germany by dr. u. ziegler et al. which identified west nile virus (wnv) antibodies in migratory birds, but not in resident birds, in domestic poultry or in local horse populations throughout germany. the wnv antibody-positive species were found in birds that migrate to tropical africa or southern europe; however, wnv-specific rna could not be found in any of the samples. the conference opened with a presentation from professor m. a. diuk-wasser and her colleagues j. simpson and c. m. fosom-o'keefe (all yale school of public health, new haven, ct, usa) and g. molei, p. m. armstrong, and t. g. andreadis (center for vector biology and zoonotic species at the connecticut agricultural experiment station, new haven, ct, usa), ecology of west nile virus in the north-eastern united states. professor diuk-wasser began by noting that west nile virus (wnv) was introduced into new york city in 1999 by unknown means and was now considered endemic throughout the usa, with 29,700 human cases and 1,180 deaths in the usa since 1999. it had been hypothesized that increased biodiversity leads to a decreased risk of exposure to zoonotic pathogens (keesing et al., 2006) . at issue is whether this 'dilution effect' or 'zooprophylaxis' for vector-borne pathogens applies only when vectors are generalist feeders, because the link between host diversity and pathogen transmission might break down when vectors exhibit host preferences. in the north-eastern united states, wnv perpetuates in an enzootic transmission cycle involving culex spp. mosquitoes and virus-competent avian hosts. previous studies had detected that a large proportion of c. pipiens and c. restuans bloodmeals were derived from american robins (turdus migratorius), suggesting a key role for this bird species in the wnv transmission cycle (kilpatrick et al., 2006; molaei et al., 2006) . the new haven-based research team tested for preferential feeding by conducting equal choice experiments (robins versus other bird species) (simpson et al., 2009) and by comparing the proportion of culex spp. bloodmeals acquired from robins to the proportion of robins in the local bird community. both methods indicated preferential feeding for robins. they were also able to identify robin communal roosts as amplification foci in greater new haven (diuk-wasser et al., 2010) . then, through field-informed mathematical modelling, they determined that host preferences were indeed key drivers of wnv transmission and that landscape attributes (such as urbanization) in combination with mosquito abundance and a measure of host community competence were the strongest predictors of pathogen prevalence (simpson et al., 2011) . thus, it was clear that pathogen prevalence and human risk of infection were best predicted by assessing the relative pathogen competence and attractiveness to vectors of all species in the host community, rather than using simple measures of biodiversity. in the next presentation, interactions among multiple tick-borne pathogens in a natural reservoir host, professor fish and his colleagues j. brown, m. fitzpatrick, s. usmani-brown, p. cislo and p. krause (yale school of public health, new haven, ct, usa) stressed that species interactions within a parasite community drive infection risk in a wildlife population (telfer et al., 2010) . at least five tick-borne pathogens are known to be transmitted by ixodes scapularis, the principal vector of lyme disease in the united states: (i) borrelia burgdorferi, an agent of lyme disease; (ii) anaplasma phagocytophilum, an agent of human anaplasmosis; (iii) babesia microti, an agent of human babesiosis; (iv) borrelia miyamotoi, an agent of relapsing fever; and (v) the powassan encephalitis virus. two or more of these pathogens can be transmitted either simultaneously by a single tick or sequentially by successive tick-bites, resulting in 240 different permutations of mixed-infection studies. in the context of pathogen prevalence of ixodes scapularis nymphs, borrelia burgdorferi, has been found in 19.8% of samples from the north-east and mid-western united states, while babesia miroti has been found in 14.7% of samples from block island, rhode island. professor fish explained that several types of co-infections have been explored in an experimental system employing laboratory colonies of i. scapularis ticks and peromyscus leucopus white-footed mice, a natural reservoir host for these pathogens. outcomes of mixed infections in mice have been measured by r o , the fitness parameter and basic reproductive rate which indicates the number of secondary tick infections resulting from a primary infection (levin and fish, 2004) . the observed outcomes of dual mixed infections have been variable with both positive and negative effects on r o , while interactions have been mutual, unidirectional or null. these diverse pathogen interactions play an important role in determining the infection prevalence of host-seeking nymphs in nature, and consequently, in the risk of infection for humans. professor h. henttonen (finnish forest research institute, vantaa, finland) and his team h. leirs, e. r. kallio, k. tersago and l. voutilainen in collaboration with university of antwerp, belgium; university of liverpool, united kingdom; and the universities of helsinki and jyväskylä, finland, studied biome specific rodent dynamics and hanta epidemiologies in europe. their research sought to understand the main biomes and forest coverage in europe, the european hanta viruses and their carriers, and the biome specific dynamics of hanta virus carriers and the biome specific transmission dynamics and epidemiologies. within the bunyaviridae family of viruses, hantaviruses infect rodents (and insectivores) and cause haemorrhagic fever with renal syndrome (hfrs) in humans in the old world and hantavirus cardiopulmonary syndrome (hcps) in the new world. in a large european union project, eden (emerging diseases in a changing european environment, 2011), rodent-borne (robo) viral infections have been studied, along with tick-borne pathogens, leishmaniasis, west nile virus, malaria and rift valley fever. the most important aim of professor henttonen and his colleagues was to clarify the differences in boreal (northern) and temperate europe in the human epidemiology of nephropathia epidemica, by far the most common hantaviral disease in europe, caused by puumala hantavirus (puuv). the population dynamics of the host species, the bank vole, differ greatly in various parts of europe, driven by predation in the north and masting events in the temperate zone. consequently, the causes of rodent fluctuations are different. in addition, the role of landscape patterns (homogenous taiga vs. fragmented temperate forests) in rodent/virus dispersal is significant, as well as local environmental conditions (e.g. temperature and moisture), which affect virus survival outside the host. for example, in room temperature, puuv remains infectious for at least 2 weeks outside the host, and possibly for much longer in cold temperatures and in moist conditions. these research findings are essential for human risk evaluation with regard to both long-term and seasonal occurrence of puuv in the environment. in spite of chronic infection of bank voles and the excretion of puuv in their faeces, urine and saliva, the shedding period is limited, which has significant implications for seasonal transmission dynamics in rodents. thus, within the same host/virus system, biomespecific puuv epidemiologies occur (kallio et al., 2009; tersago et al., 2009) , thereby highlighting the need for geographically comparative studies in europe (metla, 2012) . professor v. sambri and his team, p. gaibani, f. cavrini, a. m. pierro, m. p. landini and g. rossini (all regional centre for microbiological emergencies [crrem] , unit of clinical microbiology, st orsola-malpighi university hospital, bologna, italy) investigated usutu: a novel human pathogenic mosquito-borne flavivirus. this virus belongs to the japanese encephalitis serogroup within the mosquito-borne cluster of the genus flavivirus in the family flaviviridae. first isolated from mosquitoes of the genus culex in south africa in 1959, the usutu virus (usuv) has since been isolated from mosquitoes, rodents and birds throughout sub-saharan africa and europe. the virus is thought to be maintained in nature in a mosquito-bird transmission cycle in areas with a minimum of at least ten hot days >30°c, but no mammalian reservoir has yet been identified. professor sambri pointed out that it was not until september 2009 that usuv was found in the liver of a patient who underwent an orthotropic liver transplant (gaibani et al., 2010) . further study of the plasma and genome sequencing analysis confirmed the presence of usuv viremia. then usuv was detected in the livers of an additional four patients from the same area suffering from acute meningo-encephalitis during 2008/2009. both serological assay and molecular assay have been used as new tools for the diagnosis of usuv infection. thus, it is now clear that usuv is a new emerging flavivirus pathogenic for humans. further studies are required to discover both the geographical distribution of this virus and the mechanisms by which humans acquire the virus. since this conference presentation, there has been increased awareness of the seriousness of usuv (vázquez et al., 2011) . according to the world health organisation (who) and unicef, 1.5 million children under the age of five die from diarrhoea annually (unicef/who, 2009 astroviruses cause infections within the small intestine and are associated with at least 10% of all sporadic cases and >25% of all hospitalized cases. these rapidly evolving, nonenveloped, single-stranded rna viruses can be transmitted directly from infected individuals and animals, and indirectly through contaminated food and water. professor schultz-cherry's laboratory was the first to demonstrate that astroviruses induce diarrhoea by a novel mechanism: they possess an enterotoxin that disrupts intestinal epithelial barrier function independent of cellular damage or an inflammatory response (koci et al., 2003) . this occurs within 24 h post-infection because of reorganization of the tight junction protein occludin and the actin cytoskeleton (moser et al., 2007) . in essence, within a complex pathogenic process, astroviruses cause diarrhoea by increasing intestinal barrier permeability. this is the first evidence showing that a viral coat protein is an enterotoxin. of great interest, the toxin can act independently of species barriers. given the increasing isolation of astroviruses from diverse species, there is increasing evidence that toxicogenic astroviruses could be associated with zoonotic disease. professor m. g. katze (department of microbiology and washington national primate research center, university of washington, seattle, wa, usa) set out a unifying approach to molecular biology in his presentation, systems and computational biology: emerging tools for exploring emerging viruses. he emphasized that modern day virologists and immunologists must do better in their search to understand how a virus kills and how effective vaccines can be developed, especially because traditional virology has yielded surprisingly little information about why some virus strains cause severe diseases while others remain innocuous. he pointed out that the case fatality rate for the 1918 influenza pandemic was about 2.5% and that particular h1n1 virus may have infected as much as one-third of the world's population. issues arise not only in understanding a virus, but also in understanding how hosts respond. for example, the 1918 virus infection resulted in very high expression of inflammatory, antiviral and immune cell genes very early in host infection (kash et al., 2006) . significant progress in overcoming existing and emerging viruses depends on biologists, mathematicians and computer specialists working together within a systems biology paradigm. such research begins with either in vitro studies of virus replication on cell lines or primary cell cultures, moving to nonhuman primate models of virus infection. then samples from the experiments are investigated at multiple time points and conditions; and high throughput data are then examined by data processing to prepare systematic evaluations of different host responses. data integration involving data analysis and modelling of key genes and pathways is then possible, followed by iterative processing of host perturbations and the use of viral mutants to discover specific applications to translational research. such a systems biology approach requires not only continuing experiments with virusinfected experimental systems but also significant efforts to maintain the hardware and software of an extensive laboratory computational infrastructure. it is this computing infrastructure, which permits the laboratory to go quickly from samples to pathway visualization, as the data analysis workflow moves from microarray images to gene expression data to pathway models. the mission of this virolab is to develop steadily over the years to come a virtual laboratory to confront the viruses involved in 14 infectious diseases -influenza, ebola, marburg, hepatitis c, sars-cov, vaccinia, herpes simplex, west nile, hiv-1, siv, measles, lassa, chikungunya and dengue fever. the three key characteristics of this integrated approach to so many infectious diseases are as follows: (i) to use cell culture, primary cells, nonhuman primate and human clinical models to study viral infection; (ii) to combine traditional histopathological, virological and biochemical approaches with functional genomics, proteomics and computational biology (haagmans et al., 2009); and (iii) to obtain signatures of virulence and insights into mechanisms of host defense response, viral evasion and pathogenesis (casadevaill et al., 2011) . for example, with the study of all respiratory viral diseases, a unifying hypothesis is that highly pathogenic respiratory viruses use both unique and common strategies to remodel the host cell to enhance virus replication, regulate disease severity and promote virus transmission (chang et al., 2011) . a highly significant new tool for studying these emerging viruses is next generation sequencing (ngs) which has already 'changed the way we think about scientific approaches in basic, applied and clinical research' to such an extent that 'the potential of ngs is akin to the early days of polymerase chain reaction (pcr), with one's imagination being the primary limitation to its use' (peng et al., 2011) . already, a good understanding of the 'timing' and extent of immune (innate)-mediated injury after virus infection has been achieved. furthermore, molecular 'disease' signatures associated with different pathogens in multiple animal species have been described at micro-rna, mrna, protein level, metabolite and lipid levels. such successful modelling of molecular events has made possible verifiable prediction about key nodes and bottlenecks, enabling the identification of novel host cell drug targets (diamond et al., 2010) . the translational impact of this research, in professor katze's view, will be immense, revealing a completely new and expanded host defense repertoire consisting of non-annotated noncoding rnas. despite all of these achievements, four crucial questions remain unanswered: (i) is systems biology too complicated and too expensive to become the pre-eminent approach in virology and immunology? (ii) are mathematicians and computer scientists up to the challenges? (iii) how will new technologies like next generation sequencing impact virus systems biology research, especially in the context of rna sequencing? (iv) how can new principal investigators best be identified and appointed? (virolab, 2012) . it has long been recognized that the emergence of any zoonoses is a complex process involving 'ecological interactions at the individual, species, community and global scale' (childs et al., 2007, p.2) . this topic began with a presentation from professor a. a. aguirre that focused on the ecological framework in which any zoonotic disease should be considered. the role of bats as an important reservoir host for many dangerous zoonotic pathogens was then considered in some detail (cf. daniels et al., 2007; field et al., 2007; gonzalez et al., 2007; wang and eaton, 2007) . professor a. a. aguirre (department of environmental science and policy, george mason university and executive director, smithsonian-mason global conservation studies program, front royal, virginia, usa) presented emerging zoonotic diseases of wildlife: developing global capacity for prediction and prevention. he began by explaining that conservation medicine and more recently ecohealth have emphasized the need to bridge disciplines, thereby linking human health, animal health and ecosystem health under the paradigm that 'health connects all species in the planet' (aguirre et al., 2002) . in his view, the recent convergence of global problems such as climate change, biodiversity loss, habitat fragmentation, globalization, infectious disease emergence and ecological health demands integrative approaches breaching disciplinary boundaries. the international union for conservation of nature (iucn) maintains a red list of threatened species -an important initiative in view of the 869 animal extinctions that have already occurred, of which 3.7% were caused by disease (smith et al., 2006) . professor aguirre noted that the u.s. agency for international development (usaid) has been a major leader in the global response to the emergence and spread of highly pathogenic avian influenza (hpai). since mid-2005, it has programmed approximately $500 million to build capacities in more than 50 countries for monitoring the spread of hpai among wild bird populations, domestic poultry, and humans, and to mount a rapid and effective containment of the virus when it is found. recent analyses indicate that these efforts have contributed to significant downturns in reported poultry outbreaks and human infections and a dramatic reduction in the number of countries affected. furthermore, the usaid bureau for global health, office of health, infectious disease and nutrition (gh/hidn) recently funded two cooperative agreements, predict and respond, under its avian and pandemic influenza and zoonotic disease program to continue and expand this work. the goal of predict is to establish a global early warning system for zoonotic disease emergence that is capable of detecting, tracking and predicting the emergence of new infectious diseases in high-risk wildlife (e.g. bats, rodents and nonhuman primates) that could pose a major threat to human health. the goal of respond is to improve the capacity of countries in high-risk areas to respond to outbreaks of emergent zoonotic diseases that pose a serious threat to human health. the geographical scope of this expanded effort is directed to zoonotic 'hotspots' of wildlife and domestic animal origins (jones et al., 2008) . predict includes a program of smart (strategic, measurable, adaptive, responsive and targeted) surveillance that focuses on preventing the 'spilling over' from wildlife to humans or to halt these diseases rapidly after that spillover by understanding what factors induce emergence and rapidly identifying ways of prevention, control, and mitigation. the overall aim of smart is to promote an integrated, global approach to emerging zoonoses. this integration requires commitment from a broad coalition of partners and stakeholders including government agencies, universities and non-governmental organizations, collaborating for specific purposes and to generate in the future new international structures able to respond to these emerging zoonoses. with 1.5 billion animals being imported into the united states each year, as well as an extensive international trade in illegal animal exports ) and some 75% of emerging zoonoses worldwide having wildlife origins, professor aguirre stressed that ecohealth has become a necessity, not an optional policy goal. dr. g. a. marsh and his colleague dr. l.-f. wang (australian animal health laboratory [aahl], geelong, victoria, australia) began their presentation, bats: a mixed bag of new and emerging viruses, by pointing out that the ''old'' bat viruses were represented by many zoonotic pathogens, including rabies virus, yellow fever virus, st louis and japanese encephalitis viruses, and west nile virus. now bats have been identified as natural reservoirs for a number of new and emerging viruses -ebola virus, marburg virus, hendra virus and sars-like coronaviruses. there are some 1000 different bat species; and they often roost in high-density colonies of over one million flying mammals, which have, in a very real sense, been travelling for millions of years, exposing themselves to many pathogens; therefore, the resulting complexity is not surprising. key research questions include (i) why do bats seem to be able to co-exist with a great diversity of viruses without showing disease signs? (ii) what triggers the spillover of bat viruses into other animals? (iii) do bats control viral infection differently from other mammals? attempts to isolate viruses from bats have generally been unsuccessful. therefore, in an effort to improve the success rate for virus isolation, dr. marsh and his team have recently developed primary cell culture lines from numerous different species of bats (crameri et al., 2009) . the use of these bat cell lines, in combination with improved sampling techniques, has lead to recent isolation of hendra virus from a number of bat urine samples collected in several locations across queensland, australia, including those associated with human and horse virus spillover events (smith et al., 2011) . furthermore, this henipavirus surveillance program has led to the isolation of a number of novel viruses from two different virus families, whose zoonotic potential is not yet known. in an attempt to understand virus/host interactions, as well as to provide insight into the key factors involved in future spillover events, aahl has launched a number of international collaborative projects in south-east asia and ghana, west africa. c. kohl (sonntag et al., 2009) . the phylogenetic analysis of the genome sequence of bat adv-2 demonstrated a close relationship to canine adenovirus 1 and 2 (cadv-1 and cadv-2) (kohl et al., 2012) . the very similar genome organization supported the hypothesis of a shared ancient ancestor. interestingly, both cadvs are presenting untypical pathological features within the family adenoviridae. these adenoviruses were found to have an unusually broad host range and are causing a rather higher pathogenicity in a variety of carnivore hosts. the untypical pathological features might be understood as signs of a missing adaptation host and could provide a model to study ancient inter-species transmission events. this section of the conference addressed cross-species transmission of selected pathogens. in addition to the summaries below of three presentations on this topic, this special supplement includes an article, epidemiological survey of tryanosoma cruzi infection in domestic owned cats from the tropical southeast of mexico by dr. m. jiménz-coello et al. setting out how a significant public health problem in mexico has been caused by the crossspecies transmission of american trypanosomiasis (at) from triatomine bugs to domestic cats, representing a potential risk to humans. speaking on behalf of an extensive team of collaborators from a number of institutions -c. osborne, p. cryan, t. j. o'shea, l. m. oko, c. ndaluka, c. h. calisher, a. berglund, m. l. klavetter, r. a. bowen and k. v. homes -dr. s. r. dominguez (section on pediatric diseases, the children's hospital, university of colorado school of medicine, aurora, co, usa) began by noting that the first pandemic of the twenty-first century, the deadly sars virus, had its natural reservoir in bats. in his presentation, alphacoronaviruses in new world bats: prevalence, persistence, phylogeny and potential for interaction with humans, he suggested that bat coronaviruses (covs) may well be the ancestors of all group 1 and 2 covs. today bats had become a primary species encountered by humans in terms of potential exposure to significant disease agents. their research was tackling three important unanswered questions: (i) what is the prevalence and diversity of bat covs in new world bats? (ii) do bat covs persist in bat populations and/or individual bats? (iii) what are the potential interactions of infected bats with the human population? a 3-year study (osborne et al., 2011) had collected clinical and environmental samples from bats at 16 rural sites and 5 urban sites throughout colorado, as well as bat carcasses obtained from various counties throughout the state from the colorado department of public health and environment. of the 1,002 faecal or anal swab samples, 75, that is, 7%, were positive for cov rna. the highest prevalence of the virus was in juvenile bats; although rates of prevalence varied from year to year, late spring was the time when the virus peaked. although bat covs persisted within bat populations and their roosts, individually tagged cov-infected bats cleared their infections within 6 weeks without apparent illness. new world bats of the same species in geographically distinct locations and over the course of several years harbour similar covs, and some new world bat covs may be able to infect bats of different genera. strikingly, bats, which had known or potential contact with humans, had a high prevalence of 10-20% of cov infection. it is clear that significant opportunities exist for zoonotic transmission of coronaviruses from bats to humans and vice versa, especially as more than 95 viruses have already been isolated from or detected in bat tissues. noting that many mammalian and avian species in addition to bats are susceptible to coronavirus infection, receptor proteins that include ace2, apn and cea-cam1. the recent emergence of sars coronaviruses from civets, bats and/or other reservoir species into humans depended upon a few amino acid substitutions in the receptor-binding domain (rbd) of s from the animal viruses that allowed them to recognize human ace2 instead of, or in addition to, receptors of their natural hosts (li, 2008) . alphacoronaviruses of pigs, cats, dogs and human coronovirus 229e use apn receptors of the host species, and all four viruses recognize feline apn (tusell et al., 2007) . in contrast, for human alphacoronavirus nl63, the receptor-binding motif (rbm) with its three loops in the rbd binds specifically to human ace2. in the rbds of the cat virus, fipv, professor holmes and her research team predicted three loops structurally similar to the nl63 rbms, and they constructed chimeric fipv rbds containing one, two or three rbms from nl63. receptor-binding assays using enzyme-linked immunoassays (elisa), flow cytometry and co-immunoprecipitation identified three loops (rbms) in fipv rbd that are required for binding to feline apn. furthermore, substitution of only a few key amino acid residues within the rbms of fipv altered apn specificity and viral host range. thus, the emergence of alphacoronaviruses into new host species can occur when spontaneous mutations arise in the rbms that permit binding to variants of the apn receptor protein expressed by different host species. considering the interaction between human and swine h1n1 viruses since 1900, professor h. d. klenk (institute of virology, philipps university, marburg, germany) presented the mechanisms of pathogenicity and host adaptation of influenza viruses in the light of the new h1n1 pandemic. he explained that there was now a clear scientific consensus that wild aquatic birds are the natural hosts for a large variety of influenza a viruses. occasionally these viruses are transmitted from this reservoir to other species, such as chickens, pigs and humans, leading to devastating outbreaks in domestic poultry and the possibility of human influenza pandemics. by the end of february 2010, there had been 15,921 deaths, with the world health organization later confirming cases in 171 countries and territories, with deaths in at least 139 countries and territories before the spread of the h1n1 virus diminished. however, professor klenk set out the evidence to support his view that the pathogenic and pandemic potential of this new h1n1 virus is not yet exhausted. the host range and pathogenicity of any virus are polygenic traits depend on the interaction of different viral proteins with specific host factors. it has long been known that proteolytic activation and receptor specificity of the hemagglutinin (ha) are important determinants for pathogenicity and interspecies transmission, respectively. there is now considerable evidence that ha mutations altering receptor specificity and cell tropism of the 2009 pandemic influenza a virus (h1n1v) are linked to the d222g amino acid substitution and are associated with a particularly severe outcome of infection (liu et al., 2010) . it should be remembered that the viral polymerase has to enter the nucleus of the infected cell to promote replication and transcription of the viral genome. adaptive mutations in polymerase subunits of avian viruses improve binding to importin alpha, a component of the nuclear pore complex in mammalian cells. as a result, nuclear transport of these proteins and efficiency of replication are enhanced. thus, the interaction of the viral polymerase with the nuclear import machinery is an important determinant of host range. some of the structural features typical for avian viruses have been preserved in the polymerase of the 2009 pandemic influenza a virus (h1n1v) suggesting that this virus has the potential to further adapt to humans. recent studies have shown that the ns1 protein, another important determinant of pathogenicity and host range, is sumoylated and that this modification enhances virus growth. interestingly, ns1 of h1n1v is not sumoylated (xu et al., 2011) . taken together, these observations support the view that the pathogenic and pandemic potential of the new virus is not yet exhausted. furthermore, because of the firm evidence of ha polymorphism in position 222, mutants and other mutations with altered receptor specificity will have to be closely monitored. in the subsequent discussion, it was noted that when a virus becomes highly pathogenic, this might block its spread if additional hosts are not readily available. furthermore, the role of co-infection with bacterial inflection was highly relevant in the 1918-1919 influenza pandemic and might well be relevant in a future pandemic. there have been at least three influenza pandemics every century since 1700, with some evidence of earlier epidemics and pandemics after 1500. in the cambridge world history of human disease, a. w. crosby (1993; p. 810) has noted that although the black death and world wars i and ii killed higher percentages of the populations at risk, the 1918-1919 influenza pandemic was possibly 'in terms of absolute numbers, the greatest single demographic shock that the human species has ever received'. the summaries below of seven presentations on this topic highlight the diversity of influenza viruses in north america (cf. nelson et al., 2011) , while other relevant research has been published with respect to swine influenza viruses (sivs) in europe (kyriakis et al., 2011) . considerable research has now been carried out into how the highly pathogenic h5n1 avian influenza virus spreads from wild birds and ducks to chickens and other species, including humans (rabinowitz et al., 2010; ma et al., 2008) . the studies of how influenza viruses can be genetically altered to become more transmissible have become a matter of much controversy palese and wang, 2012) . in addition, to the summaries below, this special supplement includes an article, lessons from emergence of a/goose/guangdong/1996-like h5n1 highly pathogenic avian influenza viruses and recent influenza surveillance efforts in southern china, in which dr. x.-f. wan has considered the emergence and ecology of influenza a viruses in southern china, especially the highly pathogenic h5n1 virus. backed by an extensive team of collaborators, professor a. d. m. e. osterhaus (head, department of virology, erasmus medical centre, rotterdam, the netherlands) began his presentation, emerging and neglected influenza viruses, by explaining the complex aetiology of the influenza a, b and c viruses. while humans can serve as host species for all three viruses, influenza a can also be present in other mammals and avian species, influenza b in seals and influenza c in pigs. the severity of the disease is relatively high with influenza a, moderate with influenza b and low with influenza c, with the prevalence in humans high with both influenza a and b viruses, but lower with influenza c. furthermore, a clear distinction needs to be made between seasonal influenza, avian influenza and pandemic influenza. there are two different mechanisms of host adaptation -sequential mutations and genome reassortment. most recently, the new h1n1 swine flu pandemic outbreak of 2009 drew attention to the speed with which an influenza virus could move around the world. however, the fact that this particular virus was not as virulent as first anticipated proved crucial in confronting the virus, even though it spreads rapidly among humans, unlike the much more virulent h5n1 avian flu virus, from which more than 300 people have died from more than 500 verified cases from 2003 to 2011 (world health organization (who), 2012). although clinical evidence of h5n1 avian influenza appears predominantly in diving ducks, a number of dabbling duck species -mallard, teal, wigeon and gadwall -appear to spread h5n1, generally acquired from wild birds, without showing major signs of disease. the likelihood of a major pandemic linked to h5n1 has not decreased in the last 5 years, even though publicity has certainly decreased. furthermore, professor osterhaus pointed out that the recent h1n1 pandemic influenza outbreak indicated that the scientific community was wrong in its earlier belief that 'a pandemic strain could only arise from a subtype that had not previously been widely disseminated in humans [because] the h1n1 virus has shown that human varieties characterized by different hemagglutinin (ha) molecules may follow separate lines of evolution and may generate potentially pandemic strains within an existing human ha subtype. hence, it is essential to develop methods for estimating how many antigenically different subtypes may reside within each ha type' (cf. rappuoli et al., 2009) . in the light of the continuing prevalence of many subtypes of influenza, there is a critical need for improved monitoring, especially in asia and africa, as part of a move from a reactive to a proactive approach, with greater research into the possibility of developing a universal vaccine. although there are increasing opportunities for virus infections to emerge and spread rapidly in our global society, new tools are being provided by research in molecular biology, epidemiology, genomics and bioinformatics. already early warning systems based on state of the art virus detection techniques, as well as targeted intervention strategies based on data about the mutual virus-host interaction have been instrumental in dealing with numerous viral threats, including sars and avian influenza. the extensive research of the department of virology at erasmus medical centre in rotterdam was highlighted by a further presentation, influenza pneumonia: the role of the alveolar macrophage, given by dr. d. van riel. highly pathogenic avian influenza (hpai) h5n1 virus causes severe, often fatal, pneumonia in humans. the pathogenesis of hpai h5n1 virus is not completely understood, although the alveolar macrophage (am) is thought to play an important role. the am resides in the pulmonary alveolus, the primary site of hpai h5n1 virus replication in humans. it had been shown previously that hpai h5n1 virus attaches abundantly to these am (van riel et al., 2006) . the aim of this study was to determine the response of primary human am to hpai h5n1 virus, seasonal h3n2 virus or pandemic h1n1 virus, and to compare these responses with that of macrophages cultured from monocytes. hpaiv h5n1 infection of am compared with that of macrophages cultured from monocytes resulted in a lower percentage of infected cells (up to 25% versus up to 84%), lower virus production and lower tnf-alpha induction. infection of am with h3n2 or h1n1 virus resulted in even lower percentages of infected cells (up to 7%) than with hpai h5n1 virus, while virus production and tnf-alpha induction were comparable. in conclusion, this study revealed that macrophages cultured from monocytes are not a good model to study the interaction between am and influenza viruses. furthermore, the interaction between hpai h5n1 virus and am could contribute to the pathogenicity of this virus in humans, because of the relatively high percentage of infected cells rather than virus production or an excessive tnf-alpha induction (van riel et al., 2011 2). one virus of each pair was wild type, while the other carried the h274y na mutation conferring resistance to na inhibitor oseltamivir. within each pair, the wild-type and oseltamivir-resistant virus caused disease of equal severity in ferrets and replicated to comparable virus titers in the upper respiratory tract. then, to assess the fitness of drug-resistant h5n1 influenza viruses, the research team considered virus-virus interactions within the host by co-inoculating ferrets with mixtures of the oseltamivirsensitive and oseltamivir-resistant h5n1 viruses in varying ratios (e.g. 100/0; 80/20; 50/50; 20/80; 0/100). using this novel approach, they demonstrated that the proportion of a/vietnam/1203/2004-h274y clones tended to increase, while the proportion of a/turkey/15/2006-h274y clones tended to decrease. their findings suggest that the h274y na mutation can affect the fitness of two h5n1 viruses differently and is dependent on background na sequence. dr. govorkova pointed out that antigenic and genetic diversity, virulence, the degree of na functional loss of h5n1 virus and differences in host immune response can also contribute to such differences. therefore, the risk of emergence of drug-resistant influenza viruses with uncompromised fitness should be monitored closely and considered carefully in pandemic planning. in a collaboration with c. corzo, k. juleen and m. gramer they initiated an active surveillance program in healthy pigs in multiple sites in 2009, during a period coincident with the emergence of the h1n1 pandemic in humans. their study, active surveillance for influenza viruses in north america, presented an analysis from 12 months of data which indicated that similar viruses can be detected in both active and passive surveillance schemes and that there has been an explosion of diversity in swine influenza viruses (siv) in the united states. not only were a number of pandemic h1n1 infections in swine detected, but a number of pandemic/endemic swine virus reassortants were found, albeit from healthy animals (ducatez et al., 2011) . virologically, the pattern of disease surveillance grounded in the activities of state diagnostic laboratories collecting information from diseased animals is representative; however, epidemiologically this data from diseased animals is not representative. reverse zoonoses have had a huge impact on siv in the united states (vincent et al., 2008) , and the pandemic virus is now endemic. however, in considering whether any particular reassortment causes alarm, it must be acknowledged that there is not yet a good model of risk, so h3, like h1, is going to be found in pigs for some time to come, but the consequences of this diversity in siv are not yet clear. the extensive collaboration now taking place in the study of swine influenza was evident in the presentation vessel pendulum began by explaining the three elements of how swine could be considered as a mixing vessel for influenza a viruses as formally proposed by scholtissek et al. (1985) : (i) swine are susceptible to infection with influenza a viruses from avian and human viruses; (ii) the avian viruses can adapt within the pig, producing novel reassortants; and (iii) these reassortants can then be shed and are infectious to man. the goal of this presentation was to test the first part of the mixing vessel hypothesis, concerned with the susceptibility of swine to avian and human influenza viruses, making use of both mixing vessel studies in pigs and genetic markers to investigate adaptation. dr. lager noted that the emergence of the h5n1 highly pathogenic avian influenza virus that can transmit from avian species directly to man, and the presumption that the 1918 h1n1 influenza jumped from birds to man has expanded our understanding of the swine mixing vessel hypothesis as a potential, but not exclusive, source of human pandemic viruses (taubenberger et al., 2005) . moreover, the emergence of the 2009 pandemic h1n1 virus has re-emphasised swine as a potential source of pandemic virus. in this study, all of the challenge viruses (avian h5, h7, h9) induced a similar effect in pigs; challenge viruses did replicate in pigs; the infections were subclinical with mild pneumonias; most infections resulted in seroconversion; and none of them transmitted to contact controls. this series of studies suggests pigs could be easily infected with avian viruses; however, an adaptation step is needed to generate fit viruses that transmit among swine. parallel studies are currently underway testing the susceptibility of pigs to human seasonal influenza viruses. future studies using reverse genetics could investigate potential genetic markers for adaptation of avian viruses to swine which may provide insight into the interspecies transmission of influenza viruses. a in this study, an attempt was made to recreate the 2009 pandemic virus by co-infecting cells (in vitro) or a group of pigs (in vivo) with eurasian (sp04) and north american triple reassortant (ks07) sivs (ma et al., 2010a) . infected pigs were co-housed with two groups of sentinel animals to investigate virus maintenance and transmission. the origin of each gene segment of viruses was determined, which were isolated from supernatants collected from co-infected cells or nasal swabs and bronchioalveolar fluid samples collected from infected and sentinel animals. different reassortant viruses were identified from co-infected cell lines; however, no virus with the genotype of ph1n1 was found. less reassortant viruses were found in the lungs of co-infected pigs in contrast to those in co-infected cells. interestingly, only the intact ks07 was detected from nasal swabs from the second group of sentinel pigs. these results demonstrated that multiple reassortant events can occur within the lower respiratory tract of the pig; however, only a specific gene constellation is able to be shed from the upper respiratory tract. however, in this study, it was not possible to generate the ph1n1 constellation using co-infection with the techniques described above and previously (ma et al., 2010b) . in . she began by reflecting on the ability of swine to act as a reservoir for many influenza viruses, becoming infected with low mortality, regardless of influenza virus strain. the objective of the study was to further understand the porcine response to influenza and to compare this response to other animals infected with the same virus. to accomplish this objective, they used statistical and functional analysis of global gene expression to compare host transcriptional response during acute infection by a contemporary h1n1 pandemic influenza virus (a/california/04/2009) in swine, non-human primates and mice. using their data, they compared and contrasted the biological pathways most significantly associated with gene expression changes during acute infection across these species. their goal was to leverage data collected in their previous studies (ma et al., 2011; safronetz et al., 2011) to better understand influenza virus pathogenesis through a cross-species analysis that considered three crucial questions: (i) which genes change over the course of acute infection? (ii) what are the top functions altered during infection? (iii) how does functional response compare across the three species? despite challenges to data integration and interpretation, including the differences in transcript representation and annotation on the microarrays for the different species, the researchers found notable differences in response to influenza in the lungs of the three species. although similar functional groups of genes changed with infection in all three species, the nature of that response was species specific. swine exhibited an elevated transcriptional response that tapered by resolution of influenza. mice exhibited a decrease in many acute phase and immune response genes quickly followed by a steady increase in expression. host response in macaques was most pronounced and maintained over time. in considering the transcription of immune-related genes in swine, mice and nonhuman primates, they found that although the number of immune-related genes changing in each species was similar, the precise genes changing were very different, with only 14 immune response genes commonly differentially expressed across all three species. this suggested that the nature of immune response within each species may be quite different. in response to the perennial question after any scientific experiment, ''where do we go from here?'' they offered four ideas: (i) time series analysis could reveal unique response kinetics across species, thereby leading to targeted analysis; (ii) data integration across multiple data types, including transciptomics, proteomics, mirna and ngs could generate a more complex, multidimensional view of response; (iii) as annotation of the different species-specific genomes improves, this information could be integrated into future analyses, making a better understanding of the biological responses to infection possible; and (iv) the gathering of this additional information could empower more precise analysis on what makes each species uniquely susceptible or resistant to influenza. in the firm view of these particular six researchers, studies such as this are necessary for a deeper understanding of influenza pathogenesis and demonstrate the utility of systems biology in the study of emerging viruses. three relevant articles on this topic have been published below, highlighting the global dimensions of both infection and treatment, no matter where the virus first emerges. the need for geographical comparative studies of the emerging hantavirus, puumala hantavirus (puuv), has already been indicated by professor henttonen and his team in their presentation summarized earlier in the opening topic of this meeting review. in a further investigation into the same hantavirus, dr. eckerle and her colleagues have presented an article within this special supplement entitled atypical severe puumala hantavirus infection and virus sequence analysis of the patient and regional reservoir host. in this article, they focus on the difficulties in the diagnosis of and treatment for a single patient and performed virus sequence analysis showing regional clustering in reservoir and host. in their more wide-ranging conference presentation, they investigated cytokine expression in a cohort of patients hospitalized with acute severe hantavirus infection during an epidemic in germany in 2010 (cf. faber et al., 2010) . elevated proinflammatory cytokines during the early phase of disease compared to healthy controls and increase in immunosuppressive tgf-b from early to later phase of disease supported the hypothesis of an immune-mediated pathogenesis of puumala hantavirus (sadeghi et al., 2011) . this finding indicates that the immune status of the host for old-world hantaviruses plays an important role, not only the virus itself. in a further article published in this special supplement, how ebola virus counters the interferon system, a. kühl and s. pöhlmann have reviewed which components of the innate immune system could be effective against the zoonotic transmission of ebola virus (ebov) to humans, which results in severe haemorrhagic fever and high case-fatality rates. their focus is on how the interferon (ifn) system, as a key innate defense against viral infections, is targeted by distinct ebov proteins, and on how specific effector molecules of the ifn system could form a potent barrier against the spread of ebov in humans. finally, in lassa fever in west africa: evidence for an expanded region of endemicity, dr. n. sogoba and his colleagues h. feldmann and d. safronetz have stressed the importance of increased surveillance for lassa virus across west africa. the seven presentations summarized below cover a number of haemorrhagic fever viruses. for example, an important example of a highly contagious and life-threatening haemorrhagic fever virus is crimean-congo haemorrhagic fever virus (cchfv), caused by a tick-borne virus of the bunyaviridae family (elliott, 1990) , first recognized in the crimea in 1944, with an identical virus isolated in the congo in 1956; the incidence and geographical spread of this disease with its high human fatality rate have increased significantly in the past 10 years. however, the causes of this increase are not yet clear (maltezou and papa, 2011) . in the light of the need to develop new therapies and effective, safe vaccines, the next seven research presentations could prove to be of considerable significance, not only for cchfv, but also for the hendra, nipah, lujo and ebola viruses. although these viruses have certain common features in their causes and consequences, each haemorrhagic fever virus needs to be carefully studied as a distinct entity. dr (peyrefitte et al., 2010) . moreover, it has already been shown that cchfv causes liver damage in infected patients and in the animal model (bereczky et al., 2010) . the research objectives were to consider: (i) how does cchfv affect hepatocarcinoma cell lines? (ii) is cchfv able to enter and replicate into these cell lines? (iii) does cchfv modulate the in vitro cellular response? to better understand the cchfv pathogenesis in liver cells, they analysed in vitro the host response induced after cchfv infection in huh7 (unable to produce ifn-beta) and hep-g2 (capable of producing ifn-beta) cell lines. they noticed that while in huh7, cchfv infection elicited at day 3 a cytopathogenic effect, no visible effect was seen in cchfv-infected hepg2. this intriguing feature led them to analyse the viral parameters expecting a differential cellular response. both cell lines were shown to be permissive to cchfv and with a high viral yield as monitored by plaque titration assay, genomic and antigenomic strand quantification. these cchfv-infected hepatocarcinoma cell lines induced only il-8 secretion. in addition, a pro-apoptotic effect was observed in huh7 but not in hepg2. interestingly, no type-i ifn was detected for hep-g2 during the kinetic study, suggesting a strong inhibition of ifn secretion. they concluded that cchfv does enter and replicate in hepatocytes and that hepatocytes could be involved in cchf pathogenesis associated with antigen presenting cells for cchfv dissemination. while cchfv did not induce ifn-beta secretion in hepatocyte cell lines, cchfv did induce the secretion of il-1 in hepatocyte cell lines. furthermore, cchfv induced a higher secretion of il-8 in the apoptotic huh 7 cell line than in the nonapoptotic hep-g2 cell line. thus, this research indicated that il-8 production and apoptosis seemed to be markers of cchfv pathogenesis in hepatocyte cell lines. professor t. w. geisbert (university of texas, medical branch, galveston, tx, usa) presented an evaluation of countermeasures against hendra and nipah viruses in nonhuman primate models. he pointed out that the henipaviruses, hendra virus (hev) and nipah virus (niv) are enigmatic emerging pathogens that can cause severe and often fatal neurologic and/or respiratory disease in both animals and humans. guinea pigs, hamsters, ferrets and cats have been evaluated as animal models of human hev infection. a research team led by professor geisbert recently evaluated african green monkeys as a nonhuman primate model for henipavirus infection and discovered that they are the first consistent and highly susceptible nonhuman primate models of hev and niv infection rockx et al., 2010) . the severe respiratory pathology, neurological disease and generalized vasculitis manifested in both hev-and nivinfected african green monkeys provides an accurate reflection of what is observed in henipavirus-infected humans. these nonhuman primate models were then employed to evaluate several post-exposure treatments including ribavirin (which did not work) and a human anti-henipavirus monoclonal antibody (which was successful). dr the research was motivated by the awareness that neutralizing antibodies are probably the major effectors against this viral infection. the rationale of using rv vectors for the development of a niv vaccine was fourfold: (i) rv-vectored vaccines are not pathogenic regardless of the route of administration or the immune status of the host; (ii) rv-based vaccines are very efficacious even after a single immunization by the oral route; (iii) rv-based vaccines have the ability to target macrophages and dendritic cells, to induce th1 t-cell response and are capable of inducing long-lasting immunity; and (iv) postexposure prophylaxis using recombinant rv vaccines is very effective, even when the cns is already infected (faber et al., 2009a,b) . the niv g gene was inserted into the non-pathogenic rv vectors spbaangas or spbaangas-gas, resulting in spbaangas-ng or the double gas variant spbaan-gas-ng-gas, respectively. further research led to four significant conclusions: (i) there are no detectable amounts of niv g present in recombinant nivg-rv particles; (ii) the presence of an niv g gene does not increase, but rather decreases the pathogenicity of the recombinant viruses; (iii) priming with nivg-rv triggers a strong niv g-specific memory response, which correlates inversely with vaccine concentration used for the priming; and (iv) a single immunization with nivg-rv is probably sufficient to protect against a niv challenge infection. arenaviruses are rodent-borne bisegmented ambisense rna viruses, which include lassa fever virus, lymphocytic choriomeningitis (lcm) and tacaribe the index case for this acute febrile illness virus was a travel agent living on a farm during 2008 in lusaka, zambia, who infected a local cleaner, as well as a paramedic and a nurse in johannesburg, south africa, all of whom died, with the paramedic infecting a further nurse who was treated with ribavirin and survived . the name of the virus originated from the first two letters of the two key cities, lusaka and johannesburg. four of the five infected persons died of haemorrhagic fever-like symptoms paweska et al., 2009 ). viral genome sequencing revealed that this virus differed from other arenaviruses by at least 36% and is highly pathogenic, with a case fatality rate (cfr) of 80% paweska et al., 2009) . in view of the uniqueness and high virulence of lujo virus (ljv), the research team developed a reverse genetics system to study the molecular characteristics of this novel arenavirus. this system will facilitate studies of ljv biology, development of antiviral screening assays and pathogenesis studies in animal models. t. cutts (national microbiology laboratory, public health agency of canada, canadian science centre for human and animal health, winnipeg, manitoba, canada) with his colleagues s. theriault (chief, applied biosafety research program, same centre) and g. kobinger (chief, special pathogens program, same centre) presented cytofixô inactivation of veroe6 cells infected with zaire ebola virus (zebov) both in vitro and in vivo. first, it was pointed out that removing infected tissues from high-containment laboratories requires implementation of a number of different decontamination techniques to render the organism inert and is subject to flexibility according to the laws of the country in which the laboratory is located. according to the canadian biosafety guidelines 4th edition, an organism may be removed from containment once it has been rendered inert, but no procedure is in place to validate these biosafety guidelines, and it is up to the individuals to implement the relevant guidelines (public health agency of canada, 2004, p. 28. chap. 3.1.4). methods such as gamma irradiation, formalin fixation, acetone and methanol permeation, plus the use of various other chemical agents, are common practices to preserve cellular tissue or blood components and to inactivate organisms (elliott et al., 1982; mitchell and mccormick, 1984; preuss et al., 1997; villinger et al., 1999; sanchez et al., 2007) . such methods still raise questions as to their effectiveness or their redundancy. furthermore, these inactivation steps can lead to the alteration of the target organism possibly affecting the qualitative and quantitative results. the focus of the applied biosafety research program was to evaluate and develop technologies and procedures relevant to biocontainment in the context of the laboratory, as well as to prevent unintentional and intentional release of dangerous organisms into the environment. using the commercial product, cytofix/cytoperm tm from bd biosciences, this research sought to inactivate vero e6 cells which had been infected with the deadly zaire ebola virus (zebov). the aim of the research was to determine the effectiveness and duration of cytofix/ cytoperm for fixing the cellular material infected with zebov. the veroe6 cells were infected with the wildtype zebov and a mouse adapted zebov(mazebov) and assayed after a 5-min and 20-min exposure to cyto-fixô followed by neutralization. samples of blood from a non-human primate infected with zebov were drawn at 7 dpi and assayed for effectiveness in the same manner as the in vitro studies with cytofixô. in addition, vero e6 cells infected with mazebov were treated in the same manner and injected into balb/c mice to compose the in vivo studies. cytoxicity and neutralization assays were used to determine the effect (if any) the treatment had on both the virus and the health of host cells. results of the tissue culture tcid50 assay showed that a 5-min exposure to cytofixô inactivated a large portion of the cells containing infectious virions, while after a 20-min exposure, no detectable levels of virus were observed. blood samples from the non-human primates showed similar results to the cell culture assay having no detectable virus from infected cells after 20 min of exposure. in vivo studies with mice showed that both a 5min and 20-min exposure time to cytofixô had a 100% survival rate after 28 days post-infection, while the positive controls succumbed after 4 to7 dpi. because laboratories differ in their preferences of technique, the time of inactivation also varies. what this research demonstrated was the effectiveness of a quick procedure of 20 min for inactivating viruses within cells infected with zebov, thereby rendering organisms safe to remove from containment. has not yet been linked with disease in humans, the presence of antibodies against rebov in people working closely with infected macaques and swine indicates that humans can be infected with this virus (miller et al., 1990; miranda et al., 1991; barrette et al., 2009 ). however, research has been hampered by the fact that the only available disease model for rebov to date has been cynomolgus macaques. seeking new rebov disease models, the research team assessed various rodent models -the balb/c mouse, hartley guinea pig, syrian hamster and stat1 )/) mouse that lacked the signal transducer and activator of transcription 1 (durbin et al., 1996) . although virus replication occurred in guinea pigs and hamsters, progression to disease was only observed upon inoculation of stat1 )/) mice. despite certain drawbacks set out in the journal article, the stat1 )/) mouse can be used to investigate the determinants of differences in pathogenicity in various rebov strains, as well as to assess vaccination and antiviral therapies (miller et al., 1990; miranda et al., 1991; durbin et al., 1996; barrette et al., 2009; de wit et al., 2011) . the unity of human, animal and ecosystem health outlined by professor aguirre, as well as the interactions among multiple tick-borne pathogens in a natural reservoir host set out by professor fish and his research team, both summarized in topic 1 above, highlight the necessity of cross-disciplinary collaboration in studying zoonotic bacterial diseases (daszak et al., 2007, pp. 470-471) . such collaboration is especially important in studying tick-borne infectious disease, which emerged so extensively in the united states during the last three decades of the twentieth century (paddock and yabsley, 2007, p. 290) . now, in an article published in this special supplement, beyond lyme: etiology of tick-borne human disease with emphasis on the southeastern united states, drs. stomdahl and hickling have explained that tick distributions are in flux, especially in the south-eastern united states, requiring health providers to think 'beyond lyme' to identify the specific tick species that bite humans and the different pathogens these ticks carry. in an international context, drs. wood and artsob have set out the increasing importance of travel-associated rickettsioses in their article, spotted fever group rickettsiae: a brief review and a canadian perspective. in a third article published in this special supplement, drs. verma and stevenson present an article on epidemiology of leptospirosis with its one million cases worldwide. in leptospiral uveitis -there's more to it than meets the eye! they hypothesize in detail about how the eye inflammation uveitis is triggered and stress the impact that 'understanding how this bacterium is able to induce this inflammatory process will be a key to the better management and prevention of the disease'. this continuum of basic research leading to understanding a disease and then to managing that disease and finally to preventing it offers a pattern of scientific discovery that is relevant to many other emerging zoonotic diseases. opening his presentation, the foodborne pathogen campylobacter jejuni exploits mammalian host cell receptors and signaling pathways, professor konkel noted that the per cent of c. jejuni isolates that are resistant to antibiotics is continuing to increase and that c. jejuni infections are frequently associated with serious sequelae, including guillain-barré syndrome. it is well understood that infection with c. jejuni is often a consequence of eating foods contaminated with undercooked poultry. however, c. jejuni pathogenesis is a highly complex process that is dependent on many factors including motility, adherence, cell invasion, protein secretion, intracellular survival and toxin production. acute illness, characterized by the presence of blood and leucocytes in stool samples, is specifically associated with c. jejuni invasion of intestinal epithelial cells. dissecting bacteria-host cell interactions are critical to understanding the infection caused by c. jejuni. previous work has shown that maximal invasion of host cells by c. jejuni is dependent on synthesis of the c. jejuni cadf and flpa fibronectin (fn) binding proteins and requires the secreted campylobacter invasion antigens [cia(s)] (larson et al., 2008) . to test the hypothesis that maximal cell invasion requires specific signalling events, binding and internalization assays were performed in the presence of numerous inhibitors of cell signaling pathways. the research team found that c. jejuni cell invasion utilizes components of focal complexes (fcs), as invasion is significantly inhibited by wortmannin (an inhibitor of pi-3 kinase) and pp2 (a c-src inhibitor). they further demonstrated that a wild-type strain of c. jejuni results in the activation of the rho gtpase rac1. these observations are consistent with the proposal that c. jejuni binding to host cell-associated fn and secretion of the cia proteins trigger integrin receptor activation, which in turn promotes intracellular signalling and actin cytoskeletal rearrangement. on the basis of these data, they concluded that c. jejuni utilizes a novel mechanism to promote host cell invasion. the research findings professor konkel presented were recently published in cellular microbiology (eucker and konkel, 2012) . simple, fast and specific tests for pathogen identification are essential for epidemiological investigation of numerous diseases. within the field of immunodiagnostics, a quantitative determination of either antibody or antigen by antigen-antibody interaction can be made by lateral flow tests (also known as a dipstick or rapid tests). dr. e. baranova and her colleagues p. solov'ev, n. kolosova and s. biketov (all state research center for applied microbiology, obolensk, russia) began the presentation, development of lateral flow tests for the fast identification of zoonotic disease agents, by pointing out that lateral flow (lf) tests can be used in the field, as a diagnostic tool that produces results that can be read visually by the naked eye within 20 min after sample application. the creation of an algorithm for the development of an appropriate lf test to identify biopathogens requires the development of a target antigen, obtaining specific antibodies (biketov et al., 2010) and then creating a lf-test formulation to be trial tested. the target antigens must have the ability to induce species-specific antibodies, as well as be characterized by surface localization with multiple epitope presentation on the surface. the antibodies need to have a specificity and sensitivity sufficient for application in the lf detection format, as well as the capacity to be preserved after labelling with gold particles and after immobilization on a surface. over a period of 22 months, the research team developed and tested in the field lf tests for the detection of bacillus anthracis, which causes anthrax, yersinia pestis, which causes bubonic plague, and francisella tularensis, which is the causative agent of tularaemia (or rabbit fever). all three of these lf tests have now been made available as commercial products and are being used throughout russia for the rapid identification of these dangerous pathogens. drs. j. d. trujillo and p. l. nara (center for advanced host defences, immunobiotics and translational comparative medicine, iowa state university, ames, iowa, usa) have developed and validated a new approach to the diagnosis of infectious agents. dr. trujillo explained that they are employing novel polymerase chain reaction (pcr)-based methods for the detection and differentiation of current and emergent mycoplasma species relevant to human and animal medicine and biodefense. their presentation, titled novel sybr ò real-time pcr assay for detection and differentiation of mycoplasma species in biological samples from various hosts, began by explaining the relevance of mycoplasma species, which are endemic, strict or opportunistic pathogens in human and animal medicine. moreover, mycoplasma species are important re-emerging pathogens and foreign animal diseases. importantly, mycoplasma species are difficult to culture or are un-culturable, and thus are difficult to impossible to detect by conventional diagnostic methods. moreover, current pcr methods have limited breath of species detection and differentiation, requiring the use of species-specific assays that are costly and time-consuming. their goal was to develop a pilot mycoplasma genus diagnostic assay to validate the novel application of high-resolution melt (hrm) methodology for rapid, sensitive and cost-effective detection and differentiation of various pathogenic mycoplasma species. dr. trujillo presented the validation and utilization of sybr ò green dye in real-time pcr (qpcr) mycoplasma detection and differentiation assay (panmyco qpcr). this pcr assay utilizes primers specific for this genus (modified from s. c. baird et al., 1999) . this pcr assay results in the generation of small dna fragments of various base pair lengths called pcr amplicons. each amplicon has a melt temperature (tm) that is determined following qpcr. sequence of amplicon representative of the mycoplasma species present defines the melt temperature (tm) and allows for the use of amplicons tm in species identification with limited resolution and excellent sensitivity. the panmyco qpcr assay has similar sensitivity to a conventional nested pcr assay for mycoplasma bovis with a linear detection range of one colony forming unit (trujillo et al., 2009 ). additional work presented described increasing species resolution of this assay, by defining unique melt profiles for each mycoplasma species amplicon utilizing precision melt software from biorad, ca, usa to perform hrm analysis. greater than 30 different species of mycoplasma found in bovine, caprine, ovine, avian and porcine hosts have been characterized with the panmyco qpcr and hrm analysis. occasionally, this testing has resulted in the detection of multiple species in a single sample or discovery of novel or emergent mycoplasma species. this data analysis method allows for the sensitive detection and rapid differentiation of numerous mycoplasma species in many different hosts. dr. trujillo concluded that this novel real-time pcr assay can detect and potentially differentiate all known mycoplasma species. moreover, this presentation demonstrated the novel use of genus-specific sybr green pcr and hrm analysis for the detection, differentiation and discovery of medically important pathogens. several additional translational research projects have been launched to demonstrate the importance and utility of the pan myco qpcr assay in the context of infectious disease surveillance. one translational research project focuses on validation of this novel molecular methodology for field detection assays. there is increasing awareness of the need for improved laboratory investigation, risk assessment, contingency planning and simulation exercises to respond effectively to zoonotic diseases (lipkin, 2008; westergaard, 2008a and 2008b; escorcia et al., 2012) . in view of the need to research into and respond to so many emerging zoonoses, it is relevant to note the fourfold classification of emerging zoonoses proposed earlier by silvio pitlik: type 1: from wild animals to humans (hanta); type 1+: from wild animals to humans, with further human-to-human transmission (aids); type 2: from wild animals to domestic animals to humans (avian flu); and type 2+: from wild animals to domestic animals to humans, with further human-to-human transmission (sars) (kahn et al., 2009: p. 410) . confronting outbreaks of these emerging zoonoses is often possible with an imaginative combination of laboratory investigation and extensive fieldwork (borchert et al., 2011; robinson, 2011) . three distinctive articles appear below on outbreak responses to zoonotic diseases, highlighting the importance of linking together basic research, practical action and an integrated one health-oriented approach. in a. grolla and nine co-authors from eight different institutions in five different countries have explained how two mobile laboratories were set up and capable of running within <24 h of arrival, providing safe, accurate, rapid and reliable diagnostic services as the ebola zaire outbreak began in the democratic republic of the congo. finally, in emerging and exotic zoonotic disease preparedness and response in the united states: coordination of the animal health component, dr. r. l. levings has set out the integrated approach of emergency management and diagnostics, veterinary services, animal and plant health inspection service, united states department of agriculture in the prevention of, the preparedness for, the response to and the recovery from a zoonotic disease outbreak. in all three of these areas -basic research, practical action and an integrated one health-oriented approach -much has been achieved in recent years, but much also remains to be achieved as soon as possible. even when those diseases are not transmitted to humans, there are substantive challenges, as highlighted in the next case study by woods on combating brucellosis in cattle in zimbabwe. in a practical, problem-oriented presentation, dr. p. s. a. woods (veterinary public health section, faculty of veterinary science, university of pretoria, onderstepoort, south africa and university of reading) with r. s. beardsley (pharmaceutical health services research, school of pharmacy, university of maryland, baltimore, maryland, usa) and n. m. taylor (veterinary epidemiology and economics unit, school of agriculture, policy and development, university of reading, reading, united kingdom) asked can we increase farmers' perception of their brucellosis susceptibility to improve adoption of preventive behaviors amongst small-scale dairy farmers in zimbabwe? she explained the background to the problem, presented a model that was used to develop a strategy to confront the disease and then set out the results and recommendations of the research team. brucellosis is an extremely infectious bacterium that causes abortion in cows, different syndromes in other animal species and malaria-like undulant fever, arthritis, depression and epididymitis in people. however, it had been controlled in zimbabwe until 2001 when financial constraints forced the government veterinary services to curtail disease surveillance and discontinue free vaccinations. small-scale farmers did not seek vaccination from other sources, partly because they were unaware of the necessity of vaccination, and also at that time brucellosis was absent from small-scale farming areas. however, uncontrolled cattle movements from 2000 to 2009 linked to invasions of large-scale farms resulted in dispersal of possibly brucella-positive cattle and movement of the disease into small-scale herds. the result was that brucellosis became a potential problem in these herds and now presents a serious zoonotic threat. preventing brucellosis requires movement control to stop brucella-positive cattle entering an area, as well as live vaccine for female calves. although there is no human-to-human spread of the disease, it is essential that people do not handle new-born calves or abortions from brucella-positive cows, nor drink unpasteurized milk from brucella-positive cows (arimi et al., 2005) . in essence, reducing the risk of brucellosis requires that farmers adopt appropriate preventive behaviours, with these control efforts and changes in behaviour being communitydirected in order to be sustainable. it was this stress upon community direction that formed the basis for funding by the wellcome trust to investigate the hypothesis that the level of a farmer's knowledge about brucellosis would influence subsequent preventive behaviour. the approach, based partly on the 'health belief model' (rosenstock et al., 1988) was grounded in the expectation that each small-scale farmer would make health behaviour choices according to individual perceptions about the disease and personal beliefs about their abilities and the costs required to change the risks of their cattle and families acquiring the disease. in this project, the independent variable was the level of an individual farmer's knowledge about brucellosis, while the dependent variables were two key preventive behaviours -decreasing cattle disease by calfhood vaccination and preventing zoonotic disease by milk pasteurization. the research was carried out in partnership with a national network of small-scale dairy cooperatives with all activities conducted with existing local personnel. the aim was to tailor the educational program to the initial knowledge or awareness of each community of farmers, recognizing the considerable difference in knowledge levels between-and within communities. local teams, not outsiders, developed appropriate educational materials, targeting those with the lowest levels of knowledge. completed survey questionnaires indicated a significant relationship between the initial level of farmers' knowledge about brucellosis and their calf brucellosis vaccination practices. the range of brucellosis knowledge among some 210 small-scale farmers in southern zimbabwe was considerable, with 38% of farmers being unaware of the disease, 12% having limited knowledge and 50% having good knowledge. however, even amongst those farmers with a relatively high level of knowledge, 78% of farmers had not vaccinated their calves at the time of the survey. furthermore, there was a disappointingly low uptake of milk boiling despite a significant increase in knowledge about raw milk as a mode of infection for humans. although the information sessions did increase farmers' awareness of the dangers of zoonotic brucellosis, an exaggerated perception of the effectiveness of calf vaccination decreased the likelihood of safe milk practices. this outcome indicated the importance of reaching the women who were responsible for milk and food preparation. ongoing research is investigating whether increasing the role of nurses and environmental health technicians to emphasize human infection and to reach different family members, within a research paradigm which combined veterinary and human medicine, would increase the uptake of milk hygiene practices. there is increasing awareness of the need to balance transparency with carefully designed information disclosure strategies in the face of sudden outbreaks of foodborne diseases (national research council, 2011; taylor, 2011) . both consumers and producers must be rapidly informed of any significant dangers with specific food products; however, considerable misinformation can be spread if laboratory results are incomplete or inconclusive (palm et al., 2012) . recent experience with e. coli-infected sprouts in germany and listeria-infected cantaloupes the united states has highlighted the difficulties in identifying the original source of a disease outbreak, as well as the swiftness with which an unexpected food-borne disease can cause sickness and death (armour, 2011; blaser, 2011; buchholz et al., 2011; frank et al., 2011) . it should be noted that that there was no easily identified zoonotic link in either of these two food-borne diseases derived from bacteria, which killed 29 people in the united states and 50 throughout europe during 2011; however, as professor c. kastner points out later, a significant number of these food-borne diseases do have a zoonotic origin (parker et al., 2011) . two articles linked to this topic are published in this special supplement. first, there is emerging antimicrobial resistance in commensal e. coli with public health relevance by dr. a. käsbohrer and her colleagues. their aim was to assess the prevalence of and trends in antimicrobial resistance through active monitoring programs along the food production chains for poultry, pigs and cattle, as well as to collect isolates for resistance testing and then select certain isolates for further phenotypic and genotypic characterization. the research team found alarming rates of resistance to antimicrobials in zoonotic bacteria and commensals, as set out in their article, which could compromise the effective treatment for human infections. this work provides a basis on which to improve both risk assessment and risk mitigation strategies in the face of the increasing antimicrobial resistance to zoonotic bacteria and parasitic organisms within both humans and animals. second, in american trypanosomiasis infection in fattening pigs from the south-east of mexico, m. jiménz-coello and her colleagues have investigated the extent to which the protozoa trypanosoma cruzi (t. cruzi) is presenting in fattening pigs in yucatan, mexico, threatening parasitic infections in animals destined for human consumption. tackling the question of how to refine national and international strategies to combat food-borne zoonotic diseases, professor c. kastner (food science institute, kansas state university, manhattan, kansas, usa) considered the public health and economic impact of foodborne zoonotic diseases. he began by noting that each year in the united states, according to statistics from the centers for disease control, 48 million people become sick from food-borne diseases, 128,000 are hospitalized and 3,000 die. a significant portion of these diseases have a zoonotic origin, with extensive product recalls and domestic as well as international trade disruptions (fung et al., 2001) . therefore, more than 20 years ago, the us department of agriculture established a food safety consortium (2011) which focuses on food-borne zoonotic diseases involving beef in kansas, pork in iowa and poultry in arkansas. the continuing aim of that consortium is to develop long-term control strategies that identify the critical control points and control technologies, as well as short-term strategies to address incidental contamination, whether accidental or intentional. the us livestock industry in general and kansas in particular are vulnerable to food-borne zoonotic diseases. for example, in kansas, sources of contamination include feed, feedlots (which vary in size from 1,000 to 150,000 head per lot) and packing plants (which vary in size from 3,000 to more than 5,000 head per day per plant). beef processing points where mixing of different ingredients occurs are the most critical points for both incidental and intentional contamination. in the light of these challenges, a biosafety level 3 research facility, the biosecurity research institute (bri) (2011) has been built on the kansas state university campus, to evaluate strategies to detect and control food-borne zoonotic diseases from production through processing. furthermore, in minneapolis, minnesota, ncfpd (national center for food production defense, 2011) has been operational since 2004 as a department of homeland security center of excellence. ncfpd has adopted a systems approach whose goals include to: (i) ensure significant improvements in supply chain security, preparedness and resiliency; (ii) develop rapid and accurate methods to detect incidents of contamination and to identify the specific agent(s) involved; (iii) apply strategies to reduce the risk of food-borne illness because of intentional contamination in the food supply chain and to develop the tools to facilitate recovery from contamination incidents; (iv) deliver appropriate and credible risk communication messages to the public; and (v) develop and deliver highquality education and training programs to develop a cadre of professionals equipped to deal with future threats to the food system. these research centers are essential to minimize the threat of food-borne zoonotic diseases. t. cutts (national microbiology laboratory, public health agency of canada, canadian science centre for human and animal health, winnipeg, manitoba, canada) presented comparative inactivation studies of listeria monocytogenes at room and refrigeration temperatures on behalf of a research team that included b. carruthers, c.-l. cross, s. theriault (chief, applied biosafety research program, same center) and himself. listeria monocytogenes, a non-sporulating, gram-positive bacillus, is found chiefly in ruminants, but can affect all species and causes listeriosis, an infrequent but serious illness that affects the central nervous system of humans and domestic animals (bortolussi, 2008; chan and weidmann, 2009 ). listeriosis can be acquired from the consumption of contaminated foods and has an incubation period ranging from 2 to 70 days (bortolussi, 2008; chan and weidmann, 2009 ). because of this variable incubation period and the fact that listeriosis leads to a mortality rate of 20-30%, the applied biosafety research program at the national microbiology laboratory of the public health agency of canada considered the significance of proper decontamination of listeria in food processing environments (chan and weidmann, 2009 ). the importance of this work is indicated by the fact that somewhere from 1 to 10% of ready-to-eat foods are thought to be contaminated with listeria (public health agency of canada, 2004 and . recently, listeria monocytogenes has gained notoriety because of its ability to grow at the low temperatures, high salt and low ph used in food processing plants (bortolussi, 2008) . therefore, a study was undertaken to determine the bactericidal efficacy of various liquid disinfectants and the effect that low temperatures have on the ability of these disinfectants to inactivate l. monocytogenes at conditions found in food processing plants. at both room and refrigeration temperature (4°), ethanol, javex, su393 and peracetic acid (paa) products outperformed all others. surprisingly, there was no significant variation in performance at room temperature compared with refrigeration temperature. however, as some organisms undergo changes during a temperature shift, it is crucial to test each disinfectant at the temperature at which it will be employed. bleach was found to be effective but is toxic, corrosive and residue forming, while the paa and ethanol compounds do not form residues and are not corrosive. as a result of these studies, major canadian food-processing plants have changed their decontamination procedures and are no longer using quaternary ammonium compounds (quats), which were previously used extensively. positive relations have been built up between companies and laboratories, leading to more relevant laboratory studies and industrial applications (public health agency of canada, 2012). a prion (proteinaceous infectious particle) has been defined as a 'malformed version of a normal cellular protein that apparently ''replicates'' by recruiting normal proteins to adopt its form, [thus becoming] capable of infecting other cells of the same, or a different organism' (prusiner, 2003; thain and hickman, 2004, p. 573) . although two nobel prizes in medicine have been awarded for prion research, to carleton gajusek in 1966 and to stanley prusiner in 1997, the precise nature of the infectious agent remains unclear to such an extent that controversy continues about whether a prion is solely protein (brooks, 2011, pp. 75-100) . whatever the cause, prion diseases are fatal chronic neurological diseases that affect the brains and nervous systems of many mammals, including humans (imran and mahmood, 2011) . prions can be detected in tissues by a number of research techniques, including infective bioassay, animal inoculation, western blot and immunochemistry. it is clear that prions can cause spongiform encephalopathies within both humans and animals (e.g. creutzfeldt-jakob disease, kuru, scrapie, transmissible mink encephalopathy, feline spongiform encephalopathy and bovine spongiform encephalopathy) (blood et al., 2007 (blood et al., , p. 1456 . summaries of the three presentations below offer further insights into the nature of prion diseases. in prion diseases, professor j. j. badiola and dr. c. akin (university of zaragoza, zaragoza, spain) focused on the 1986 outbreak of bovine spongiform encephalopathy (bse) ('mad cow disease') in the united kingdom, which led to a better understanding of the epidemiology and molecular characteristics of the disease. epidemic bse affected mainly the united kingdom, with a total of 184,615 positive animals compared to 5,765 in all other member states of the european union (oie, 2012). control and eradication of transmissible spongiform encephalopathies (tses) became a priority, not only in europe, but throughout the world. in 2000, a reinforcement of the passive surveillance program and the establishment of an active one were established by the european commission for all the european union member states (european commission, 2001) . passive surveillance, focused on animals with clinical signs of the disease, and active surveillance was carried out in the following target groups: healthy slaughtered, fallen stock, emergency slaughtered and animals culled under bse eradication. apart from these measures, specific risk materials (e.g. tonsils, intestines, spleen, spinal cord and skull, including the brain and eyes) were defined and prohibited from being included in the human food chain. moreover, a banning of all meat and bone meal for animal feed was established (european commission, 2009) . the result of these powerful eradication measures has been a rapidly decreasing number of new bse cases, with less than 50 cases detected worldwide in 2010, 45 of which were in the european union (oie, 2012) . the impressive containment of bse in the united kingdom from 35,090 reported cases in 1993 to 11 in 2010 is testimony to the determination with which scientists, politicians, civil servants and farmers have worked together to bring the disease under control. professor c. i. lasmézas (dept of infectology, the scripps research institute, scripps, florida, usa) began her presentation, zoonotic potential of new animal prion diseases: assessment in non-human primates, by noting that the first demonstration of the transmissibility of a prion disease to non-human primates (nhps) was made in 1966 by carleton gajdusek when he transmitted kuru to chimpanzees. since then, animal and human prion diseases have been transmitted to a range of nhps. cynomolgus macaques have shown the highest selectivity with regard to the prion strain by which they can be infected and therefore seem to be the species of choice to assess the risk that any given animal prion strain can be pathogenic for humans (lasmézas et al., 1996) . prions were thought to be very difficult to transmit from one species to another; however, the experience of studying scrapie highlights the difficulties inherent in studying prion diseases in the laboratory. scrapie had been transmitted orally to other ruminants (goats) but only intracerebral inoculations had successfully transmitted scrapie to monkey, mouse or mink. however, the oral transmission of bovine spongiform encephalopathy (bse) to domestic cats in 1990 forced a revision of this earlier belief. transmissions of bse have now occurred orally to sheep, goat, monkey, mink, cheetah, puma, cat and mouse. intracerebral transmission of bse has also occurred to pig. furthermore, intraspecies oral transmission of bse has taken place within numerous speciesmonkey, mink, sheep, goat, cow, hamster and mouse. vcjd (variant creutzfeldt-jakob disease) is a new human disease, which was caused by eating ruminant-derived food products contaminated with bse. vcjd poses a public health problem because of the absence of preclinical diagnostic test, the long incubation periods of prion diseases in humans (possibly extending up to 50 years) and the transmissibility of the disease by blood transfusion. the research team at the french commissariat a l' energie atomique (cea) demonstrated that bovine spongiform encephalopathy (bse) was transmissible to macaques within 3 years with a 100% infection rate and caused a disease indistinguishable from the human variant of creutzfeldt-jakob disease (lasmézas et al., 1996) . this provided a model to study carefully the peripheral pathogenesis of vcjd, the oral infectious dose of bse and evaluate the risk of human-to-human transmission of vcjd by blood transfusion (herzog et al., 2004) . further, the research team used the macaque model to assess the zoonotic potential of emerging forms of bse called l-or h-type. the l-type bse presents with higher pathogenicity to macaques than classical bse (comoy et al., 2008) . therefore, continued precautionary measures remain necessary to protect the human food chain. experiments are ongoing at the national institute of allergy and infectious disease, hamilton, montana, to assess the risk linked to chronic wasting disease that is spreading throughout the usa. the closing acknowledgements of professor lasmézas to 35 other researchers indicated both the complexity and importance of continuing work in prion diseases. furthermore, since the cancun meeting further important research has been published (hamir et al., 2011) . infectivity distribution studies of animals infected with bse prions animals are a matter of considerable importance in seeking to elucidate the route of infectious prions from the gut to the central nervous system (cns) open questions about this lethal journey from the gut to the brain, including where in the gut the disease begins, the initial steps of the neuronal bse pathogenesis, the ascension of bse prions to the brain, the haematogenous spread and the centrifugal contamination of the periphery (buschmann and groschup, 2005; hoffmann et al., 2007) . the scale of the research task was indicated by the fact that 1,400 samples were collected per animal autopsy, leading to some 200,000 frozen samples collected and archived at the friedrich-loeffler-institut. tissue samples were collected from the gut, the central and autonomous nervous system (ans) of the challenged bovines and then examined for the presence of pathological prion proteins (prp sc ). there was some variation among different animals. however, a distinct accumulation of prp sc was observed in the distal ileum, confined to follicles and/or the enteric nervous system, in almost all animals . bse prions were found in the sympathetic nervous system starting from 16 months post-inoculation (mpi) on as well as in the parasympathetic nervous system from 20 mpi on (kaatz et al., 2012) . a clear dissociation of prion infectivity and detectable prp sc deposition was obvious in tongue (balkema. the earliest presence of infectivity in the brainstem was detected at 24 mpi, while prp sc -accumulation was detected first after 28 mpi. in summary, these results deciphered for the first time the centripetal spread of bse prions along the ans to the cns starting already half way during the incubation period. bse prions spread in cattle from the gut to the brain along the sympathetic, parasympathetic and spinal cord routes, possibly in that order of importance. spinal cord involvement may even not be necessary at all, but bse infectivity in the form of prp sc spills over into the periphery already in the pre-clinical phase. the modelling and prediction of emerging zoonoses is a fast-growing field of considerable complexity. of the five papers relevant to this topic, two have been published in full below in this special supplement. dr. g. zanella and her colleagues consider modelling transmission of bovine tuberculosis in red deer and wild boar in normandy, france. their mathematical model of the mycobacterium bovis infection within and between species takes into account the transmission of m. bovis through infected offal -the viscera of animals killed by hunters and left behind. when an animal was hunted in the brotonne forest in normandy prior to 2002, it was eviscerated in situ and only the carcass taken away, with the raw viscera left behind. since 2002, offal disposal has been required in brotonne forest; however, the regulation has not always been observed by hunters (unpublished correspondence with g. zanella, 16-17 december, 2011) an important benefit of mathematical modelling is that it permits consideration of all the elements involved in disease transmission within a population, thereby complementing field data, as well as testing the effects of control measures. thus, the direct transmission of the m. bovis infection within the red deer and wild boar populations can be distinguished from indirect transmission through contaminated offal. the model indicates that offal destruction is the key factor in infection control for both red deer and wild boar. the authors conclude that, in principle, the structure of this model is relevant to the situations where dead animals play an important role in disease transmission between two or more species. in a further article published in this special supplement, constructing ecological networks: a tool to infer risk of transmission and dispersal of leishmaniasis, dr. c. gonzález-salazar and professor c. stephens set out the role of ecological networks as a powerful tool for understanding and visualizing inter-species ecological and evolutionary interactions. taking the example of leishmaniasis in mexico, they show that such networks can be used not only to understand potential ecological interactions between species involved in the transmission of the disease, but also to identify the potential role of the environment in disease transmission and dispersal. strikingly, they show how potential interactions can be inferred from geographical data, rather than by direct observation. their findings have led to the prediction of additional reservoirs in mexico of many new species, including bats and squirrels. the resulting model can be used to understand and map potential transmission risk, as well as construct risk scenarios for the dispersal of leishmaniasis from one geographical region to another. such a risk assessment tool for leishmaniasis will be especially useful in the light of the bill and melinda gates foundation decision in january 2012 to join with 13 major pharmaceutical companies and the world health organization in targeting leishmaniasis as one of the neglected tropical diseases to receive improved drugs, diagnostics, vector control strategies and vaccines (bill & melinda gates foundation, 2012; boseley, 2012) . however, the possibility of new reservoirs suggests it is hard to imagine that leishmaniasis can be completely eradicated. nevertheless, it is increasingly clear that leishmaniasis has a disturbing capacity to jump from species to species, so efforts to control the disease must be given a high priority (unpublished correspondence with c. stephens, february 1, 2012; cf. flanagan et al., 2011) . it is difficult to model and predict the distribution and impact of a new emerging virus. for example, the emergence in november 2011 in europe of a midge-borne virus member of the bunyaviridae family, named schmallenberg virus after the location in germany where it was first detected, has caused serious birth defects in lambs, goats and cattle (ecdc, 2011) . scientists, farmers, veterinarians, public health officials and consumers are all confronted with the uncertainty inherent in facing a new animal pathogen (farmers weekly, 2012) . appropriately, at the same time as this new virus has emerged, the animal health and veterinary laboratories agency (ahvla) has set up a new independent advisory group to evaluate veterinary surveillance in england and wales, although their original intent was in part to consider funding reductions (trickett, 2012) . modelling risk factors for zoonotic influenza infections is challenging because the infections are often rare; the laboratory assays are often difficult and imprecise, and the most definitive studies require intensive resources. this was the view of professor g. c. gray (emerging pathogens institute and college of public health and health professions, university of florida, gainesville, florida, usa) in his presentation, modeling risk factors for zoonotic influenza infections in man: challenges and strategies for success. in particular, serologic detections of these infections in humans may be confounded by crossreacting antibody, waning antibody from the infection of interest, inaccurate matching of the enzootic pathogen and the laboratory strain, laboratory errors and weakly powered statistical comparisons. the underlying question which professor gray and his research team is tackling is: which human, animal and environmental factors predict disease? these three factors can be viewed as a venn diagram with its intricate interactions. like understanding cardiovascular disease, how a person acquires a zoonotic influenza infection is a complex process, and predictive laboratory assays are imprecise. for example, with avian influenza viruses (especially h5n1, popularly known as 'bird flu'), poultry veterinarians, turkey workers, hunters and people without indoor plumbing may be at increased risk of aiv infection but infections are rare. subclinical or mild infections do occur; and occasionally aiv causes severe disease in persons exposed to sick birds. although aiv transmission from human-to-human seems rare, further cohort studies and more sensitive serological assays are needed. a basic scientist often tests hypotheses by: (i) carefully setting up an experimental setting; (ii) isolating confounding factors; and (iii) looking for statistically significant associations with an outcome. such a process is not possible for a number of emerging disease problems such as human infections with swine influenza virus (siv). experimental studies are not possible. hence, epidemiologists must perform observational studies of people most likely to be infected with siv and by looking at possible risk factor associations, infer causality. one must first determine settings where the prevalence of siv in expected to be high and then study those workers. for example, sivs are often endemic in large-scale modern production facilities. risk factors for sow-herd siv seropositivity involve pig density, whether there is an external source of breeding pigs, the total animals on the site and the closeness of barns. similarly, risks factors for finisherherd siv positivity must be considered -the number of siv-positive sows, size of herd, pig farm density and farrow-to-finish type of farm (poljak et al., 2008) . however, siv surveillance in pigs is largely passive and voluntary, so recognizing which pig workers to study is a challenge. detection of siv infections in man often requires a sentinel event (e.g. human illness with pig exposure or sick pigs). as pigs do not always have clinical signs of novel virus infection and often there is no compensation system to protect pig farmers, the pork industry is reluctant to permit the study of their workers for siv infection (gray and baker, 2011) . therefore, these observational studies are currently very difficult. professor gray concluded by pointing out that although there are numerous challenges in conducting epidemiological studies for zoonotic influenza, there are six substantive ways to control confounding variables: (i) design every study carefully; (ii) use non-animal-exposed controls; (iii) employ validated laboratory assay using zoonotic influenza strains; (iv) use multivariate modeling to examine cross-reacting serologic responses due to human viruses and vaccines; (v) consider proportional odds modeling; and (vi) consider employing a second unique serologic test (see gpl, 2012) . with the support of 26 co-authors from 21 different institutions, dr. k. j. linthicum (united states department of agriculture, agricultural research service, center for medical, agricultural & veterinary entomology, gainesville, fl, usa) presented two case studies about forecasting emerging vector-borne diseases. dr. linthicum began by pointing out that global climate variability, often linked to el niño conditions, can be used to forecast emerging vector-borne disease spread in local areas (linthicum et al., 1999) . these forecasts are possible because specific pathogens, their vectors and hosts are sensitive to temperature, moisture and other ambient environmental conditions. with consistent and reliable satellite observations, global sea temperatures, climate and vegetation can be observed. first, temperature plays a major role in its impact on aides aegypti mosquitoes transmitting dengue haemorrhagic fever virus in southeast asia (linthicum et al., 2008) and possibly also on how ae. aegypti transmits chikungunya virus in africa and asia , as well as on how anopheles species mosquitoes transmit p. vivax malaria in the republic of korea. vectorial competence is dependent upon the extrinsic incubation (ei) period in the mosquito vector. the ei represents the time from ingestion of the virus while feeding on a viremic host to the virus arriving in the salivary glands. the shorter the ei period, which occurs during higher ambient temperatures, the greater the vectorial competence (garrettjones and shidrawi, 1969) . if data are available for a specific local area on the daily humanbiting rate (ha) of the mosquitoes, the daily rate of blood feeding (a) and the length of the ei cycle (n), it is possible to calculate vectorial capacity (rattanarithikul et al., 1996) . second, accurate measurements and understanding of how exceptionally heavy rainfall and flooding affects aides and culex mosquitoes and the introduction of virusinfected mosquitoes into susceptible vertebrate hosts enables forecasts to be made about when and where rift valley fever (rvf) will develop in sub-saharan africa and middle east (anyamba et al., 2009) . outbreaks of rift valley fever are known to follow periods of widespread and heavy rainfall associated with the development of a strong inter-tropical convergence zone over eastern africa (davies et al., 1985) . during periods of elevated transmission, there is a significantly increased risk of globalization of these and other arboviruses; however, the forecasting methods described provide 2.5-5 months early warning before an outbreak and provide ample time for disease mitigation before the first cases appear (anyamba et al., 2010) . furthermore, the emergence and expansion of a number of disease vectors (e.g. mosquitoes, mice, locust) often follow the trajectory of the green flush of vegetation in semiarid lands. the ability to predict periods of elevated risk enables better prevention, containment or exclusion strategies to be drawn up to limit globalization of emerging pathogens. thus, it has been possible for the food & agricultural organization (fao) to create a system of alerts -the emergency prevention system for transboundary animal and plant pests and diseases (empress, 2012) . subsequent to dr lithicum's presentation, significant further work has been done to provide a genome-scale overview of gene expression in the malaria-transmitting mosquito anopheles gambiae (maccallum et al., 2011) , as well as to expand the vectorbase website with regularly updated genome information on two other mosquito species, aedes aegypti and culex quinquefasciatus, and numerous other organisms, including the tick species ixodes scapularis (lawson et al., 2009; niaid, 2012) . the ultimate aim of this research is to create a database that will facilitate a systems-level view of gene expression for many different organisms. reflecting on the numerous types of statistical analysis that are used to estimate confidence intervals for proportions in scientific studies, dr. s. guillossou and his colleagues professors h. m. scott and j. a. richt (dept. of diagnostic medicine and pathobiology, college of veterinary medicine, kansas state university, manhattan, kansas, usa) utilized the final presentation of the conference, estimates of low prevalences and diagnostic test estimates: what confidence do we really have? to illustrate the differences, limits and sometimes chaotic behaviour of different statistical approaches. dr. guillossou pointed out that there were more than 15 different methods for determining a 95% confidence interval of a proportion. he stressed that it is always important to report the method of statistical analysis being utilized. in his view, the agresti-coull interval approach presents a satisfactory compromise between computational requirements and coverage probability (newcombe, 1998; brown et al., 2001) . ideally, the effects of coverage probability should be estimated and the most appropriate method chosen before reporting the findings or using proportions as inputs in any epidemiological study. what did this 6th international conference on emerging zoonoses achieve? there was the opportunity to meet old friends and make new friends, to share one's academic work and to reflect on what lies ahead with emerging zoonoses. it is now clear that human medicine, veterinary medicine and environmental challenges are a unity which must be considered under the umbrella of 'one health' (one health initiative, 2012) . viruses are continuing to jump from animals to people with unexpected consequences, because the evolution of any virus is impossible to predict. even the recent relatively mild swine flu virus infected 10% of the human population and killed some 100,000 people globally -far less than would have been the case if the virus had mutated to a more deadly form, as might easily have happened. the reality is, as professor nathan wolfe, professor in human biology at stanford university, has commented: 'as a species, we're not that focused on the things that have the most potential to be devastating to us as a global population, such as viruses. unless people take these things seriously, we're going to look back and say we had all the tools necessary to try to address these risks, and we basically ignored them because they weren't dramatic like a car accident or a hurricane' (geddes, 2011; kahn, 2011; wolfe, 2011) . this conference, many others and the 7th international conference on emerging zoonoses to be held in 2014 in berlin, are aimed at creating, improving and using the tools essential to address the risks of viral contagions in a global society. none. the organizing committee of the conference wishes to acknowledge the excellent services of the conference organizers, target conferences of tel aviv, israel, and the welcome financial contributions of medimmune, boehringer ingelheim vetmedica gmbh, prionics ag, center of excellence for emerging and zoonotic animal diseases (ceezad) and national center for foreign animal and zoonotic disease defense (fazd), as well as the poster prize donated by wiley-blackwell. we are also grateful to the wiley-blackwell staff who have contributed so significantly to this special supplement, especially rachel robinson and peter tubman, as well as to dr. klaus osterrieder for his helpful comments and to the presenters who have approved or improved every summary in this meeting review. this material is based upon work supported by the u.s. department of homeland security under grant award number 2010-st-ag0001. the views and conclusions contained in this supplement are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the u.s. department of homeland security. additional funding has been provided by the kansas bioscience authority. conservation medicine: ecological health in practice prediction of a rift valley fever outbreak 2010: prediction, assessment of the rift valley fever activity in east and southern africa 2006-2008 and possible vector control strategies climate teleconnections and recent patterns of human and animal disease outbreaks risk of infection with brucella abortus and escherichia coli o157:h7 associated with marketing of unpasteurized milk in kenya fallout from listeria outbreak hits walmart: retail detection and identification of mycoplasma from bovine mastitis infections using a nested polymerase chain reaction bse infectivity in the absence of detectable prp(sc) accumulation in the tongue and nasal mucosa of terminally diseased cattle discovery of swine as a host for the reston ebolavirus 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transmission to macaques vectorbase: a data resource for invetebrate vector genomics inference between agents of lyme disease and human granulocytic ehrlichiosis in a natural reservoir host structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections climate and satellite indicators to forecast rift valley fever epidemics in kenya vector-borne diseases -understanding the environmental, human health, and ecological considerations, workshop summary pathogen discovery altered receptor specificity and cell tropism of d222g hemagglutinin mutants isolated from fatal cases of pandemic a (h1n1) 2009 influenza virus the pig as a mixing vessel for influenza viruses: human and veterinary implications pandemic h1n1 virus causes disease causes upregulation of genes related to inflammatory and immune response, cell death, and lipid metabolism in pigs identification and characterization of a highly virulent triple 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confidence intervals for the single proportion: comparison of seven methods geographical distribution of countries that reported bse confirmed cases since 1989 alphacoronaviruses in new world bats: prevalence, persistence, phylogeny, and potential for interaction with humans ecological havoc, the rise of the white-tailed deer, and the emergence of amblyomma amricanum-associated zoonoses in the united states 2012: h5n1 influenza: facts and fear molecular epidemiology of human pathogens: how to translate breakthroughs into public health practice development of an algorithm for assessing the risk to food safety posed by a new animal disease and investigation teams, and members of the outbreak control integrative deep sequencing of the mouse lung transcriptome reveals differential expression of diverse classes of small rnas in response to respiratory virus infection differential activation profiles of crimean-congo hemorrhagic fever virus-and dugbe virus-infected antigen-presenting cells 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real-time pcr detection and differentiation assay for mycoplasma species in biological samples mutational analysis of aminopeptidase n, a receptor for several group 1 coronaviruses, identifies key determinants of viral host range 2009: diarrhoea: why children are still dying and what can be done usutu virus -potential risk of human disease in europe markedly elevated levels of interferon (ifn)-c, ifn-a, interleukin (il)-2, il-10, and tumor necrosis factor-a associated with fatal ebola virus infection swine influenza viruses: a north american perspective bats, civets and the emergence of sars a foot and mouth disease simulation exercise involving the five nordic countries contingency planning: preparation of contingency plans what is the optimal therapy for patients with h5n1 influenza assessment of rodents as animal models for reston ebolavirus writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h5n1) virus modification of non-structural protein 1 of influenza a virus by sum01 key: cord-355171-oi3ezlsl authors: macintyre, c. r.; seale, h.; yang, p.; zhang, y.; shi, w.; almatroudi, a.; moa, a.; wang, x.; li, x.; pang, x.; wang, q. title: quantifying the risk of respiratory infection in healthcare workers performing high-risk procedures date: 2013-12-05 journal: epidemiol infect doi: 10.1017/s095026881300304x sha: doc_id: 355171 cord_uid: oi3ezlsl this study determined the risk of respiratory infection associated with high-risk procedures (hrps) performed by healthcare workers (hcws) in high-risk settings. we prospectively studied 481 hospital hcws in china, documented risk factors for infection, including performing hrps, measured new infections, and analysed whether hrps predicted infection. infection outcomes were clinical respiratory infection (cri), laboratory-confirmed viral or bacterial infection, and an influenza infection. about 12% (56/481) of the study participants performed at least one hrp, the most common being airway suctioning (7·7%, 37/481). hcws who performed a hrp were at significantly higher risk of developing cri and laboratory-confirmed infection [adjusted relative risk 2·9, 95% confidence interval (ci) 1·42–5·87 and 2·9, 95% ci 1·37–6·22, respectively]. performing a hrp resulted in a threefold increase in the risk of respiratory infections. this is the first time the risk has been prospectively quantified in hcws, providing data to inform occupational health and safety policies. healthcare workers (hcws) are at increased risk of healthcare-associated infections due to the front-line nature of their work. transmission of highly infectious diseases from infected patient to other patients and hcws occurs constantly in hospitals and healthcare centres and has been well documented [1] [2] [3] . although hcws are aware of infection control measures, low levels of compliance with standard precautions by this group are frequent [4, 5] . hcws are less willing to adhere to infection-control practices when they work for extended hours [6] , with probable reasons for low compliance being insufficient time, scarcity of equipment, lack of knowledge and low perception of risk [5] . the three principal routes of transmission of respiratory pathogens are contact transmission (direct and indirect), droplet transmission, and airborne transmission. for any pathogen, more than one transmission route may occur, but many pathogens are known to be transmitted by one predominant mode. in droplet transmission, pathogens or droplets which are larger than 5 μm, such as influenza virus and bordetella pertussis are transmitted from an infected patient to hcws through breathing, talking, coughing, sneezing, as well as through performing high-risk procedures (hrps) [2, 7, 8] . however, influenza virus has also been documented to be transmitted by the airborne route, which results in infectious particles being present in the air for longer periods of time [9] [10] [11] [12] . respiratory infectious diseases, even those with limited airborne transmission, are more likely to be transmitted from patients to hcws during hrps such as suctioning and intubation which generate respiratory aerosols [13] . many studies suggest that both invasive and non-invasive procedures are likely to increase the probability of hcws being infected [13, 14] . some studies have reported that non-invasive positive pressure ventilation (nppv) can be a risk of severe acute respiratory syndrome (sars) transmission to hcws [15] [16] [17] . cardiopulmonary resuscitation, manual ventilation, bronchoscopy and suctioning have also been documented to increase the risk of hcws being infected with sars and tuberculosis (tb) [18] [19] [20] [21] , while tracheal intubation has been significantly associated with risk of sars transmission to hcws [17] . while it has been well documented that tb and sars can be transmitted to hcws during aerosol-generating procedures, there are some data suggesting h1n1 can transmit via such procedures [13] . seasonal influenza also causes outbreaks in healthcare settings [22] . hcws are one of the most vulnerable groups likely to be infected with influenza infection in acute-care facilities due to the high exposure rates in such settings [23] . an attack rate of nosocomial influenza could reach 11-59% in hcws in a healthcare environment [24] . as such, hcws are a priority group for preventive strategies such as influenza vaccination [25, 26] . although various guidelines and policies for infection control measures are implemented in healthcare settings worldwide, the risk of transmission of infectious diseases while performing hrps has not been well quantified. this study aims to describe the range of exposure to hrps in hcws and to quantify the risk of respiratory infections occurring in hcws who perform hrps. we prospectively studied 481 hospital hcws from wards including emergency and respiratory wards from nine hospitals in beijing, china over a 5-week period from 1 december 2008 to 15 january 2009. these 481 subjects were a control group in a larger study [27] . the hospital wards were selected as high-risk settings in which repeated and multiple exposures to respiratory infections are expected. participants were hospital hcws aged 518 years and who were provided with written information about the study. staff who agreed to participate provided informed consent and a copy of the information sheet with the participants' initials was retained as documentation [27] . the study protocol was approved by the institutional review board and human research ethics committee of the beijing ministry for health. participants were asked to record on a daily basis whether they had performed one of the following: provision of nebulizer medications, suctioning, intubation, aerosol-generating procedures and chest physiotherapy. the following information was also collected: number of hours worked, estimated number of daily contacts with patients, number of daily contacts with influenza-like illness (ili) patients, and hand-washing adherence. the use of personal protective equipment such as gloves, gowns, eye shields, foot/hair covers was documented by study participants in a daily selfreport diary, and details of clinical and demographics were also recorded. all study participants were followed up for a period of 31 days and monitored daily for the onset of respiratory symptoms. if any symptom developed, combined nasal and throat swabs (double rayon-tipped, plastic-shafted swab) were taken and tested for respiratory viral or bacterial infection (fig. 1) . the nose and throat swabs were tested at the laboratories of the beijing centres for disease control and prevention. viral dna⁄rna was extracted from 300μl of each respiratory specimen using the viral gene-spin ™ kit (intron biotechnology inc., korea) according to the manufacturer's instructions [27] . we tested nose and throat swabs for the following: adenoviruses, human metapneumovirus (hmp), coronaviruses 229e/nl63 and oc43/hku1, parainfluenza viruses 1, 2 and 3, influenza viruses a and b, respiratory syncytial virus (rsv) a and b, and rhinovirus a/b by nucleic acid testing using a commercial multiplex polymerase chain reaction (pcr), with the seeplex® rv12 detection kit (seegen inc., korea). details of laboratory methods have been described in a previous publication [27] . we also tested for bacterial colonization. a multiplex pcr (seegen inc.) was used to detect streptococcus pneumoniae, mycoplasma pneumoniae, b. pertussis, legionella spp, chlamydophilia and haemophilus influenza type b. after preheating at 95°c for 15 min, 40 amplification cycles were performed under the following conditions in a thermal cycler (geneamp pcr system 9700, applied biosystems, usa): 94°c for 30 s, 60°c for 1·5 min, and 72°c for 1·5 min. amplification was completed at the final extension step at 72°c for 10 min. the multiplex pcr products were visualized by electrophoresis on an ethidium bromide-stained 2% agarose gel. the controls represent hcws in their usual working conditions, without any interventions. this study is a post-hoc analysis of data collected during the primary trial on hrps in the control arm. the prospective data collection and measurement of clinical endpoints in a group of hcws working under usual conditions afforded the opportunity to measure the association of incident infection with hrps. the primary outcomes of the study were: clinical respiratory infection (cri)presence of two or more respiratory symptoms or one respiratory symptom (e.g. cough, runny nose, shortness of breath, sore throat) and one systemic symptom (e.g. fever, lethargy, chills); laboratory-confirmed viral infection (influenza a and b, parainfluenza, rsv, coronavirus, hmp virus, adenovirus, rhinovirus); laboratoryconfirmed viral or bacterial infection (and of the above viruses or a bacterial infectionpertussis, hib, pneumococcus, mycoplasma, legionella); and influenza a or b (categorized as 'influenza' if either strain were present). the outcomes were tested against predictor variables such as age, education, category of hcw, influenza vaccination uptake, and performance of hand washing and hrps. the total number of hrps performed over the study period was calculated. a binary variable defining whether or not hcws performed any hrps during the study period was created and analysed with other predictor variables against incident infection during the study period. poisson regression was used for the analysis of the outcomes, using egret software (cytel, usa). a p value of 40·05 was considered significant in the analysis. a total of 481 hcws were recruited into the study. demographic characteristics of study participants are described in table 1 . of these, 369 (76·7%) were females; and 52% (252/481) of the participants were aged 535 years. the breakdown of participants by area was: respiratory ward 75 (16%); emergency department 72 (15%); respiratory clinic 16 (3·3%); paediatric department 15 (3·1%); infection fever clinic 6 (1·2%); and other wards 297 (62%). of the 481 hcws, 236 (49%) were doctors and 245 (51%) were nurses and others. during the study period, the uptake of influenza vaccine in hcws was low in both 2007 and 2008 (19·3% and 18·1%, respectively). fifty-six (11·6%) out of 481 hcws performed at least one hrp during the study, with the most common activity being airway suctioning (66%, 37/56). figure 2 shows the number of days on which a hcw reported performing a hrp. thirty-four (61%) out of 56 hcws, reported performing a hrp more than once during the study period. the aggregated number of days a hrp was performed was 264. in addition, hcws on the respiratory ward (33%) were more likely to perform hrps than those in the emergency department (16·7%). nurses and other hcws (16%, 39/245) were significantly more likely than doctors (7%, 17/236) to perform hrps (p < 0·01). the weekly incidence of cri was 18/1000 hcws, for viral or bacterial infection, 16/1000; for any viral infection, 6/1000; and for influenza, 2·5/1000. hcws who performed hrps had a significantly higher risk of cri [relative risk (rr) 2·5, 95% (ci) 1·3-6·5, p < 0·01) and laboratory-confirmed viral or bacterial infection (rr 2·6, 95% ci 1·4-5, p < 0·01) than those who did not perform hrps (table 2) . by poisson regression analysis, adjusting for other variables, only hrps determined the risk of an infection outcome, as shown in tables 3-5. the relative risk for cri in hcws who performed a hrp was 2·9 (95% ci 1·42-5·87, p < 0·01) ( table 3 ). the rr for a laboratory-confirmed pathogen (viral or bacterial) in symptomatic hcws was 2·9 (95% ci 1·37-6·22, p = 0·01), in those who performed a hrp (table 4 ). for the outcome of any respiratory viral pathogen, the rr was 3·3 (95% ci 1·01-11·02, p = 0·05) ( table 5 ). hand washing, influenza vaccination and use of surgical or cloth face masks did not affect the risk of infection outcomes. we also tested for other variables, e.g. number of hours worked, number of patients the hcw was in contact with during the study period, and number of contacts with patients with ili; however, none of these had a significant association with infection outcomes. there were no significant association between laboratory-confirmed influenza and hrps (data not shown); but the numbers of influenza-positive cases were low (six cases) in the study. we examined the association between hrp and the risk of respiratory infection in hcws. our findings demonstrated that hcws who perform a hrp have a greater risk of respiratory infections than those who did not perform a hrp. this is consistent with observational findings of other studies [15, 16, 18, 21, 28] ; however, we have been able to quantify the magnitude of this risk in our study as a threefold increase in risk. many factors influence the nosocomial spread of infectious diseases, and hcws are the initial point of contact with patients in both acute and long-term healthcare settings. our findings suggest that targeted interventions and policies are warranted to offer greater protection to hcws who perform hrps. this has occupational health and safety implications for hcws routinely engaged in hrps. more than 10% of hcws performed a hrp during a 1-month period, and the majority of those performed more than one hrp. this suggests that interventions to reduce transmission of respiratory infections may be more efficient if targeted to hcws performing hrps. there may be certain settings such as emergency wards, intensive-care units and respiratory wards where hrps are more commonly performed, making these important targets for interventions. there are some limitations to our study. our study was conducted in china, so the results, particularly around frequency of performing hrps, may not be generalizable to different hcw populations in other contexts. there are variations in infection control practices from hospital to hospital, even within china. however, the quantification of risk for hcws who perform hrps has implications for hcws everywhere. the fact that this was a control group in a larger trial is a strength, rather than a limitation, in that this group had rigorous follow-up and documentation of incident infection as well as risk factors (including hrps). this provides more robust data than, for example, an observational study such as a case-control study, because it was prospective and measured infection in a group that went about usual practice. to date, there is much policy debate and direction about hrps, but no data whatsoever to inform the actual risk associated with hrps. despite the limitations of the analysis, we believe that the data we present in this paper are a useful addition to current knowledge. hcws are at higher risks of contracting respiratory infections, and are subject to generic guidelines around infection control. these include hand hygiene before and after the patient care; wearing of personal protective equipment such as gowns, goggles, gloves, n95 respirators or surgical masks; presence of minimum number of hcws when performing a procedure in a single room; and in addition, it is recommended that such procedures should be performed in a sterilized room [13, 29, 30] . we have shown in two large randomized controlled trials that the risk of respiratory infection in hcws can be reduced with the use of n95 respirators [27, 31] . we also show that in highrisk wards, targeted use in situations of self-identified risk, such as when performing hrps or barrier nursing a patient, is less effective than continuous use of a respirator in that ward while on shift [31] . this suggests that hcws are unable to identify all situations of risk when left to decide whether or not they should wear a respirator. this is the first time the risk of hcws performing hrps has been prospectively quantified, and this finding has important occupational health and safety implications for hcws, particularly in settings such as emergency and respiratory wards where hrps are frequently performed. the traditional approach to hospital infection control has not consistently categorized staff in terms of whether they perform hrps in order to apply guidelines. we found that the majority (89%) of hcws do not perform hrps. this proportion may vary in different country, hospital and ward settings; our study suggests that categorizing hcws by whether or not they perform hrps in their work may serve as a useful classification in order to tailor guidelines appropriately or increase the attention to adherence with existing guidelines. the minority of hcws performing hrps should receive optimal respiratory protection, and high-risk wards should have guidelines in place to minimize the risk to hcws. transmission of bacterial infections to healthcare workers during intubation and respiratory care of patients with severe 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control measures for 2009 h1n1 influenza in healthcare settings, including protection of healthcare personnel should noninvasive ventilation be considered a high-risk procedure during an epidemic? transmission of severe acute respiratory syndrome during intubation and mechanical ventilation illness in intensive care staff after brief exposure to severe acute respiratory syndrome aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review the transmission of tuberculosis in confined spaces: an analytical review of alternative epidemiological models possible sars coronavirus transmission during cardiopulmonary resuscitation a practical approach to airway management in patients with sars occupational tuberculous infections among pulmonary physicians in training influenza in the acute hospital setting requiring influenza vaccination for health care workers: seven truths we must accept vaccination against classical influenza in health-care workers: self-protection and patient protection acip recommendations: introduction and biology of influenza, 2010-11 influenza prevention & control recommendations world health organisation a cluster randomized clinical trial comparing fit-tested and non-fit-tested n95 respirators to medical masks to prevent respiratory virus infection in health care workers which preventive measures might protect health care workers from sars? infection prevention and control during health care for confirmed, probable, or suspected cases of pandemic (h1n1) 2009 virus infection and influenza-like illnesses epidemic and pandemic prone acute respiratory diseases -infection prevention and control in health care a randomised clinical trial of three options for n95 respirators and medical masks in health workers dr seale is in receipt of an nhmrc australian-based public health training fellowship (1 012 631) . professor macintyre receives funding from influenza vaccine manufacturers gsk and csl biotherapies for investigator-driven research. dr seale has received funding from sanofi pasteur, gsk and csl biotherapies for investigator-driven research and for conference presentations. key: cord-334027-xhfmio7k authors: fagre, anna c.; kading, rebekah c. title: can bats serve as reservoirs for arboviruses? date: 2019-03-03 journal: viruses doi: 10.3390/v11030215 sha: doc_id: 334027 cord_uid: xhfmio7k bats are known to harbor and transmit many emerging and re-emerging viruses, many of which are extremely pathogenic in humans but do not cause overt pathology in their bat reservoir hosts: henipaviruses (nipah and hendra), filoviruses (ebola and marburg), and coronaviruses (sars-cov and mers-cov). direct transmission cycles are often implicated in these outbreaks, with virus shed in bat feces, urine, and saliva. an additional mode of virus transmission between bats and humans requiring further exploration is the spread of disease via arthropod vectors. despite the shared ecological niches that bats fill with many hematophagous arthropods (e.g., mosquitoes, ticks, biting midges, etc.) known to play a role in the transmission of medically important arboviruses, knowledge surrounding the potential for bats to act as reservoirs for arboviruses is limited. to this end, a comprehensive literature review was undertaken examining the current understanding and potential for bats to act as reservoirs for viruses transmitted by blood-feeding arthropods. serosurveillance and viral isolation from either free-ranging or captive bats are described in relation to four arboviral groups (bunyavirales, flaviviridae, reoviridae, togaviridae). further, ecological associations between bats and hematophagous viral vectors are characterized (e.g., bat bloodmeals in mosquitoes, ingestion of mosquitoes by bats, etc). lastly, knowledge gaps related to hematophagous ectoparasites (bat bugs and bed bugs (cimicidae) and bat flies (nycteribiidae and streblidae)), in addition to future directions for characterization of bat-vector-virus relationships are described. bats and the viruses they harbor have been of interest to the scientific community due to the unique association with some high consequence human pathogens in the absence of overt pathology. virologic and serologic reports in the literature demonstrate the exposure of bats worldwide to arboviruses (arthropod-borne viruses) of medical and veterinary importance [1] . however, the epidemiological significance of these observations is unclear as to whether or not bats are contributing to the circulation of arboviruses. historically, a zoonotic virus reservoir has been considered a vertebrate species which develops a persistent infection in the absence of pathology or loss of function, while maintaining the ability to shed the virus (e.g., urine, feces, saliva) [2] [3] [4] . haydon et al. extended this definition of a reservoir to include epidemiologically-connected populations or environments in which the pathogen can be permanently maintained and from which infection is transmitted to the defined target population. the significance of the relative pathogenicity of the infectious agent to the purported reservoir host has been debated [5] . in the case of bats as a reservoir species, rigorous field and experimental evidence now exist to solidify the role of the egyptian rousette bat (rousettus aegyptiacus) as the reservoir for marburg virus [6] [7] [8] . considering arboviruses, additional criteria must be met in order to consider a particular vertebrate species a reservoir. reviewed by kuno et al., these criteria include the periodic isolation of the infectious agent from the vertebrate species in the absence of seasonal vector activity, and the coincidence of transmission with vector activity [9] . further, the vertebrate reservoir must also develop viremia sufficient to allow the hematophagous arthropod to acquire an infectious bloodmeal [10] in order for vector-borne transmission to occur. bats have long been suspected as reservoirs for arboviruses [11] , but experimental data that would support a role of bats as reservoir hosts for certain arboviruses remain difficult to collect. here we synthesize what information is currently known regarding the exposure history and permissiveness of bats to arbovirus infections, and identify knowledge gaps regarding their designation as arbovirus reservoirs. the order bunyavirales is divided into eight families, four of which pose threats to public health and veterinary medicine-families nairoviridae, peribunyaviridae, phenuiviridae, and hantaviridae [12] . while bats have been demonstrated to host hantaviruses, these viruses do not rely on an arthropod in their transmission cycle and thus will not be discussed [13] . viruses in order bunyavirales that have been experimentally examined in bats or described in field studies are descried in table 1 . members of the genus orthonairovirus of medical and veterinary significance include crimean congo hemorrhagic fever virus (cchfv) and nairobi sheep disease virus (nsdv) [12] . cchfv is transmitted by ticks in genera rhipicephalus and hyalomma [14] . while neither live virus nor nucleic acid of cchfv has been detected from bats, serologic evidence suggests past infection of populations of bats across a diverse geographic range [15] [16] [17] . further, bats are often parasitized by both soft and hard ticks, which occupy a diverse range of ecological niches in endemic countries [18] [19] [20] . a 2016 seroprevalance study by müller and colleagues examining 16 african bat species (n = 1, 135) found that the prevalence of antibodies against cchfv was much higher in cave-dwelling bats (3.6%-42.9%, depending on species) than foliage-living bats (0.6%-7.1%) [15] . they also screened 1,067 serum samples by rt-pcr, but all were negative for cchfv nucleic acid [15] . experimental studies to assess the ability of bats to support replication of cchfv have not been published. members of the genus orthobunyavirus include many viruses of importance to human and veterinary medicine, including bunyamwera virus, california encephalitis virus, jamestown canyon virus, kaeng khoi virus, and la crosse encephalitis virus [12] , but limited evidence exists regarding the exposure or potential involvement of bats in the circulation of viruses in this family. kaeng khoi virus (kkv) has been isolated from cimicid bugs (order: hemiptera, family: cimicidae) (stricticimex parvus and cimex insuetus) and from suckling wrinkle-lipped bats (tadarida plicata ) in caves in thailand, but was not isolated from soft ticks tested in the same area (ornithodorus hermsi) [21] . additionally, kkv has been implicated in the case of several mine workers who reported illness and were discovered to have seroconverted [22] , demonstrating spillover of this virus to humans in association with the cave environment, and suggesting that cimicids may play a role in vectoring virus between bat and human hosts. to date, no experimental data have been generated to address this hypothesis. spence and colleagues attempted to experimentally infect jamaican fruit bats (artibeus jamaicensis) via intramuscular injection with nepuyo virus (group c serogroup), yet no infectious virus was subsequently recovered from the bats [23] . this is interesting considering two strains of nepuyo virus were isolated from jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (artibeus literatus) in honduras, and protective sera were found in jamaican fruit bats in trinidad. [24, 25] . bats of undetermined species were involved in a large serosurvey in brazil that examined antibodies in wildlife against the gamboa serogroup orthobunyaviruses, though none were found to be positive [26] . seven and twelve species of trinidadian bats were examined for antibodies by hi against caraparu (group c serogroup) and maguari (bunyamwera serogroup) viruses, respectively, and were all found to be negative [25] . viruses in the genus phlebovirus (family phenuiviridae) of importance to human and animal health include rift valley fever virus (rvfv) and severe fever with thrombocytopenia syndrome virus (sftsv) [12] . bats of the species miniopterus schreibersii (n = 1) and eptesicus capensis (n = 2) were experimentally infected with rvfv and the m. schreibersii bat's urine and liver tested positive for antigen [27] . a recent study by balkema-buschmann and colleagues experimentally infected egyptian rousette bats (rousettus aegyptiacus) with vaccine strain mp-12 and recovered infectious virus from spleen and liver of some animals [28] . oelofsen & van der ryst (1999) examined 350 samples from 150 field-caught bats in africa, yet none were positive for antigen by use of elisa [27] . kading et al (2018) detected neutralizing antibodies against rvfv in egyptian rousette bats and little epauletted fruit bats (epomophorus labiatus) in uganda, a country that has recently experienced human cases of rvfv [29, 30] . whether or not bats serve as a reservoir of rvfv during interepidemic periods remains to be determined. bangui virus (bgiv) is an unclassified bunyavirus and was isolated from an unidentified bat in the central african republic (car) [31] . mojuí dos campos virus (mdcv) is another ungrouped bunyavirus isolated from an unidentified bat species [32, 33] . the family flaviviridae includes many high-consequence emerging arboviruses, including zika virus (zikav), yellow fever virus (yfv), and dengue virus (denv). flaviviruses associated with bats that do not appear to utilize an arthropod vector ("no-known vector flaviviruses") have been reviewed elsewhere [56] . viruses in family flaviviridae that have been experimentally examined in bats or described in field studies are descried in table 2 . interestingly, despite denv isolations from artibeus spp. bats in the wild, experimental infections of great fruit-eating bats (a. intermedius) with denv-2 and jamaican fruit bats with denv serotypes 1 and 4 resulted in low levels of viremia, low rates of seroconversion, and lack of detection of viral rna in the organs via rt-pcr, indicating that bats may not act as a suitable reservoir host [57] [58] [59] . experimental infection of the indian flying fox (pteropus giganteus) resulted in no viremia or clinical signs, but intracerebral inoculation of little brown bats (myotis lucifugus) resulted in irritability, paralysis, and death [60, 61] . denv nucleic acid and anti-denv antibodies have been detected in mexican bats on the gulf and pacific coast, and nucleic acid has been detected in the liver and/or sera of wild-caught bats in french guiana [62, 63] . anti-denv antibodies have been detected in multiple bat species in uganda [29] . however, a survey in central and southern mexico analyzing 240 individuals representing 19 bat species by rt-pcr resulted in no detection of viral nucleic acid [64] . a 2017 study by vicente-santos and colleagues examined 12 bat species from costa rica and found a cumulative seroprevalence of 21.2% (51/241) by prnt and a prevalence of 8.8% (28/318) in organs tested by rt-pcr [65] . no infectious virus was isolated and viral loads were considered too low for the bats to function as amplifying hosts. rather, vicente-santos and colleagues surmised a spillover event from humans to bats, with bats functioning as a dead-end host [65] . the serum of jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (a. literatus) from grenada (n = 50) were also tested for antibodies against denv 1, 2, 3, and 4, and none were seropositive [66] . while field evidence supports the exposure of bats to denv in multiple geographic areas, experimental infections conducted to date are consistent in that bats are not likely to support denv replication and circulation to levels high enough to infect blood-feeding mosquitoes. multiple studies conducted experimental infections of insectivorous bats with japanese encephalitis virus (jbev) and found that bats were susceptible to infection with this virus. three species of bats (big brown bats (eptesicus fuscus), little brown bats (myotis lucifigus) and eastern pipistrelles (pipistrellus subflavus)) were inoculated with jbev in the laboratory and maintained infection while held under simulated hibernation conditions. bats infected prior to hibernation were viremic upon arousing from hibernation, with circulating virus detectable as long as 112 days after the initial infection [67] . big brown bats also demonstrated recurrent viremia in the absence of clinical signs in a subsequent study [68] . importantly, researchers demonstrated a mosquito-bat-mosquito transmission cycle and postulated this may be an overwintering mechanism for jbev since mosquitoes did successfully transmit jbev to bats at low temperatures [67] . eastern pipistrelles also became infected with jbev after consumption of infected mosquitoes, demonstrating that bats could be infected orally as well as through a mosquito bite [67] . no demonstrable pathologic effects noted during infection of three bat species [big brown bats (eptesicus fuscus), little brown bats (myotis lucifigus) and mexican free-tailed bats (tadarida brasiliensie mexicana) with various strains of jbev or st. louis encephalitis virus (slev) [69] . no pathology nor viremia was appreciated when pipistrelles (pipistrellus abramus) were infected with jbev [70] . while experimental data demonstrated that some bat species can sustain jbev infections and support mosquito-borne transmission of this virus, the epidemiological significance of these observations in the field remains unclear. jbev has been isolated from wild-caught bats in taiwan (miniopterus fuliginosus and hipposideros armiger terasensis [32, 71] , china (rousettus leschenaultia and murina aurata [72, 73] , japan (miniopterus schreibersi fuliginosus and rhinolophus cornutus cornutus [74] . antibodies against jbev have been detected in wild-caught bats in indonesia (unspecified species) [75] , china (rousettus leschenaultia, cynopterus sphinx, taphozous melanopogon, miniopterus schreibersii, pipistrellus abramus, rhinolophus macrotis and miniopterus fuliginosus [76, 77] , australia (pteropus scapulatus and pteropus gouldi) [78] , taiwan (unspecified species) [79] , india (pteropus giganteus, hipposideros pomona, hipposideros speoris, hipposideros bicolor, hipposideros cineraceus, megaderma lyra, cynopterus sphynx, and rhinolophus rouxi) [80] [81] [82] , and japan (miniopterus schreibersi fuliginosus, rhinolophus ferrum equinum nippon, vespertilio superans, myotis macrodactylus, rhinolophus cornutus cornutus, pipistrellus abramus, myotis mystacinus, plecotus auritus sacrimontis, and murina leucogaster hilgendorfi) [83] . multiple isolations of jbev from locations where the virus is endemic, in addition to the fact that genetic characterization of isolates has supported their similarity to strains identified from human and mosquito isolates, support the role of bats in ongoing circulation of jbev [84] . another medically-important flavivirus with both field-obtained information and in vivo experimental inoculation is slev. a 1983 study by herbold and colleagues demonstrated that 9% of wild-caught eptesicus fuscus and myotis lucifugus (n = 390) in ohio possessed neutralizing antibodies to slev [85] . other serosurveillance efforts in north america and grenada focused on detection of slev in free-ranging bat populations have resulted in largely negative findings [66, 86] . following experimental infection, viremia and transplacental transmission (albeit infrequent) was appreciated in mexican free-tailed bats (tadarida brasiliensis) [69, 87] . the viremia in these bats reached 4 log units, likely too low a titer to facilitate transmission to a blood-feeding mosquito [10] . upon inoculation, little brown bats (myotis lucifugus) appear to be resistant or only slightly susceptible to slev [69] . herbold and colleagues (1983) demonstrated that inoculation of eptesicus fuscus with slev results in infection and virus was maintained throughout hibernation (70 days), with viremia developing four days following arousal (105 days post-infection) [85] . low levels of viremia upon experimental inoculation in conjunction with low seroprevalence data indicate this virus likely does not utilize bats as a reservoir host in nature. to date, biosurveillance testing of bats in central america for wnv have turned up negative results. grenadian artibeus jamaicensis and artibeus literatus (n = 50) bats were negative for wnv neutralizing antibodies by prnt [66] , 14 trinidadian bat species (n = 384) were negative by elisa for wnv antibodies [88] , and 16 mexican bat species (n = 146) tested for wnv rna by rt-pcr were negative [89] . in north america, results have been negative or indicative of low levels of circulation in bat populations tested. tissues from 312 field-collected bats representing seven species in illinois tested by rt-pcr were all negative for wnv, and the same study reported one big brown bat (eptesicus fuscus) with neutralizing antibodies (n = 97) [90] . a field survey taking place in new jersey and new york reported one big brown bat and one northern long-eared bat (myotis septentrionalis) with neutralizing antibodies to wnv (n = 83) [86] . in another field study, only two of 149 free-tailed bats (tadarida brasiliensis) possessed neutralizing antibodies against wnv [91] . in uganda, kading et al. (2018) detected neutralizing antibodies to wnv in 2/8 african straw-colored flying foxes (eidolon helvum), and 3/44 little epauletted fruit bats (epomophorus labiatus) [29] . simpson and o'sullivan (1968) demonstrated experimental inoculation of african straw-colored flying foxes did not result in viremia though two of three bats developed neutralizing antibody. in the same study, two of three egyptian rousette bats were infected but only trace viremia was detected and seroconversion was not appreciated [43] . experimental inoculation of free-tailed bats (tadarida brasiliensis) did not result in viremia, and infection of big brown bats resulted in low titers (10-180 pfu/ml) [91] , not capable of supporting transmission to feeding mosquitoes [10] . attempts to experimentally infect vampire bats (desmodus rotundus) and black mastiff bats (molossus rufus) by mosquito bite (aedes aegypti) were unsuccessful [11] . experimental inoculation of multiple bat species (eumops perotis, carollia perspicillata, phyllostomus hastatus and bats in the genus mollosus) were similarly unsuccessful [92] . still, kading et al. detected a significant neutralizing antibody titer against yfv in one egyptian rousette bat in uganda in 2012, indicating bats are exposed to this virus in nature [29] . uganda has experienced outbreaks of yfv in recent years [93] . while multiple african bat species (eidolon helvum, rousettus aegyptiacus, and rousettus angolensis) demonstrated viremia following inoculation with zikav, mops condylurus did not become viremic, although did contain low virus titers in the kidney [43, 44] . experimentally-infected little brown bats were susceptible to the zikav by the intraperitoneal, intradermal, intracerebral and intrarectal routes of exposure, but not susceptible intranasally [94] . however, it is unclear how zikav could circulate in bat populations. kading et al. (2018) did not detect neutralizing antibodies to zikav among 292 ugandan bats screened. flavivirus infections of bats with an emphasis on the potential role in zika virus ecology has been reviewed elsewhere [95] . flavivirus serology has been historically challenging due to the cross-reactivity of viral epitopes to circulating antibodies [96] . therefore, the results of serologic surveillance studies must be interpreted cautiously [29, 97] . further, multiple methods exist for antibody detection (e.g., hi, prnt, elisa), and the biological significance of neutralizing vs. non-neutralizing antibodies must be taken into account. in 2010, the serum of 140 mexican bats from three species (glossophaga soricina, artibeus jamaicensis, and artibeus literatus) was assayed by prnt using wnv, slev, and denv 1-4, and 26 were positive for flavivirus-specific antibodies (19%). none of the titers exceeded 80, and all samples were also negative when tested for flavivirus nucleic acid by rt-pcr [97] . in a 2015 serosurvey, eight bats (2.6%) displayed non-specific hemagglutination-inhibition (hi) results indicating cross-reactivity or antibodies against an undetermined flavivirus [88] . kading and colleagues performed a serosurveillance study in ugandan bats and identified 13.6% (85/626) had non-specific flavivirus antibodies by plaque reduction neutralization assay (chaerephon pumilus, hipposideros ruber, mops condylurus, nycteris macrotus, eidolon helvum, epomophorus minor, and rousettus aegyptiacus) [29] . still, results generally supported the widespread exposure of bats in uganda to flaviviruses [29] . in 2018, sotomayor-bonilla and colleagues reported that liver and spleen samples from 12 mexican bat species tested negative using pan-flavivirus ns5 primers [98] . a recent study in brazil suggested a lack of arboviral circulation in bat populations, as 103 individuals from 9 species were tested for molecular and serologic evidence of alphavirus and flavivirus infection and all were negative [99] . results of experimental infection of egyptian rousette bats with wnv and of angolan free-tailed bats (mops condylurus) with ntaya virus resulted in very low levels of viremia, while infection of african straw-colored fruit bats with ntaya virus resulted in neither pathology nor detectable viremia [43] . few studies have examined the presence of viruses in genus coltivirus in bat populations, and to date, a single isolation has been made (table 3 ) [127] . a 1984 study by chastel and colleagues failed to detect antibodies to eyach virus (reoviridae, colorado tick fever group) in the serum of two field-caught bats [128] . to date, five orbiviruses have been isolated from wild-caught bats and serologic evidence exists for exposure of australian and south american bats to orbiviruses (table 3) . while no evidence of human exposure exists for these bat-associated orbiviruses, bukakata (bukv) and fomede (fomv) appear to be strains of the chobar gorge species [129] . cgv was isolated from ornithodoros species ticks in nepal, and serum from nearby humans and ruminants possessed anti-cgv antibodies, indicating past exposure [130] . further investigation is warranted to determine the true vector-host association of these viruses and their zoonotic potential. viruses in family reoviridae that have been experimentally examined in bats or described in field studies are descried in table 3 . viruses in genus alphavirus (family togaviridae) that have been experimentally examined in bats or described in field studies are descried in table 4 . enzootic circulation of chikv is understood to occur among non-human primates and forest-dwelling mosquitoes [142] , but other vertebrates including rodents, bats, reptiles and amphibians have been shown to support chikv replication [143, 144] . the range of peak viremia developed by big brown bats was relatively low, but within the range of infectivity to blood feeding mosquitoes [10, 143] . when indian flying foxes (pteropus giganteus) and big brown bats were experimentally infected with chikv, bats developed viremia but no clinical signs of disease, indicating they could play a role in the natural transmission of this virus [60, 143] . experimental infection of african straw-colored flying foxes did not result in viremia or seroconversion to chikv, supporting a separate study which reported lack of viremia in experimentally infected egyptian rousette bats and african straw-colored flying foxes [43, 44] . in 2015, the serum of 42 wild-caught grenadian bats (genus artibeus) were subjected to prnt and 15 (36%) were found to possess neutralizing antibody to chikv [66] . chikv has been circulating in central and south america since 2013 [145] . whether or not bats are contributing to the natural circulation of chikv in endemic areas or areas of introduction remains to be determined. serological evidence exists supporting exposure of bats to encephalitic alphaviruses in the field, and experimental data demonstrate the susceptibility of bats to infection with alphaviruses including veev. four mexican bat species were examined for molecular evidence of infection with venezuelan equine encephalitis virus (veev), western equine encephalitis virus (weev), and eastern equine encephalitis virus (eeev). no individual bats were positive for weev or eeev, but 3% (5/150) representing all four species were positive for veev [89] . field-caught jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (artibeus literatus) were negative by prnt for eeev and weev antibodies, but 2.6% (1/38) had neutralizing antibodies to veev [66] . similarly, the serum of 384 bats representing 14 species was subjected to elisa, and 2.9% (11/384) contained veev-specific antibodies. elisa and hi assays for eeev and weev antibodies, respectively, were all negative [88] . four species of wild-caught bats from the northeastern united states were tested for neutralizing antibody against eeev and weev. samples were negative for antibodies against weev, but 1.3% of the 128 bats tested did possess eeev-neutralizing antibody [47] . bats of the genera myotis and eptesicus were experimentally infected with eeev, and developed viremia but failed to develop neutralizing antibodies. infection of big brown bats by bite of culiseta melanura and aedes aegypti mosquitoes was successful. more non-hibernating than hibernating bats were seropositive for eeev [146] . in a recent serosurveillance study, 2/432 bats were seropositive by plaque reduction neutralization assay to babanki virus (bbkv) and 9/626 egyptian rousette bats had non-specific alphavirus antibodies (table 4 ) [29] . multiple isolates of bbkv were obtained from cx. perfuscus mosquitoes collected from multiple locations in uganda during this same sampling period as when bats were sampled [147] . mosquito blood meals from bats comprised 7.5% of the total blood meals identified from the species cx. perfuscus [148] . it is unclear whether bats contribute to the transmission cycle of bbkv or are merely incidentally exposed through mosquito bites ten pteropus poliocephalus bats were experimentally infected with ross river virus, and five developed low (log 10 2.2 tcid 50 /100 µl) detectable and short-lived (2 days) viremia. still, 2% of the colonized mosquitoes (aedes vigilax) that fed on the bats between days 1-4 post-infection became infected [148] . kading et al. (2014) modeled that for viremias <log 10 2.0/ml, the probability of a mosquito becoming infected was around 0.1 or less given the low circulating titer and the volumetric constraints of a small mosquito blood meal; therefore, at least 10 mosquitoes would need to feed on an animal with a low viremia in order for one mosquito to ingest virus [10] . in the case of rrv, published data demonstrate that infection of mosquitoes fed on rrv-viremic bats is still possible despite a viremia and low titer [149] . therefore, if bat and mosquito populations are in high numbers, 50% of bats develop a detectable viremia, and 2% of mosquitoes become infected, mosquito-borne transmission could take place even though experimentally-determined efficiencies are low [149] . antibodies against mayaro virus were detected by hi in 37 trinidadian bat species tested [25] . a number of hematophagous arthropods feed on bats, including bat flies (genera nycteribiidae and streblidae), bat bugs and bed bugs (family cimicidae), and ticks (families argasidae and ixodidae) [18, 20, 21, [167] [168] [169] [170] [171] [172] . viruses of medical and veterinary significance have also been isolated from these arthropods [21, [173] [174] [175] . however, the contribution that these ectoparasites play in the circulation of medically important viruses among bats and other hosts is unclear and necessitates further investigation. kading and schountz (2016) reviewed instances in the literature where mosquito blood meals have been identified as originating from bats [95] . information on primary mosquito vectors feeding on bats is very limited. tiawsirisup et al. 2012 collected mosquitoes from five genera inside a bat cave in thailand to investigate sylvatic circulation of jbev. while these collections included arbovirus vectors cx. quinquefasciatus and cx. tritaenhiorhynchus, the only blood-fed mosquitoes collected from the cave were cx. quinquefasciatus, at least 20 of which had fed on leschnault's rousette (rousettus leschenaulti) bats [176] . culiseta morsitans theobald mosquitoes (vector of eastern equine encephalitis virus) were found to have fed on eastern pipistrelle bats (pipistrellus subflavus), but these blood meals comprised only 1% of the total blood meals identified from this mosquito species [177] . no information was found on any blood meals from bats being detected in aedes (stegomyia) species, but it is unclear how much investigation has been done in this area. sixteen of 20 field-collected ae. funereus mosquitoes (vector of rrv) had fed on pteropus alecto bats [149] . in africa, mosquito species in which bat blood meals have been identified and are known to be associated with a number of medically-important arboviruses include: coquillettidia (cq.) fuscopennata (theobald) (yfv, sindbis, chikungunya viruses), culex (cx.) perfuscus edwards (wnv, oropouche, sindbis, wesselsbron, usutu, babanki viruses), cx. (cx.) neavei theobald (wnv, babanki, spondweni, sindbis, koutango viruses), and cx. (cx.) decens group (wnv, chikungunya, babanki viruses [148, 178] . while mosquitoes in the subgenus cx. (cx.) are recognized as primary vectors of wnv, sindbis, babanki, and usutu viruses, only for babanki virus has additional field data been collected so far that support a potential role for bats in virus circulation (discussed above) [29] . bat flies (order: diptera; families: nycteribiidae, streblidae) are highly host-specific obligate hematophagus ectoparasites of bats [179, 180] . a novel fusogenic orthoreovirus, tentatively named mahlapitsi virus (mahlv), was discovered in bat flies (eucampsipoda africana) associated with egyptian rousette bats [181] . a novel orthobunyavirus, tentatively named wolkberg virus (wbv), was isolated from the same species of bat flies in a similar geographic region [182] . dengue virus rna was detected by rt-pcr in bat flies (strebla wiedemanni, trichobius parasiticus) associated with common vampire bats (desmodus rotundus) [100] . bartonella spp. bacteria have also been found infecting both fruit bats and the bat flies parasitizing them in madagascar, providing a documented example of a pathogen-vector-bat association involving bat flies [183] . more research in this area is needed to elucidate the role of bat ectoparasites in spreading pathogens among individual bats in a roost. in addition to parasitizing humans by infesting their dwellings, bed bugs and other arthropods in family cimicidae are found in close association with bat populations [170] . some cimicids are known to play a role in alphavirus transmission. cliff swallow bugs (oeciacus vicarious) transmit fort morgan virus and buggy creek virus [173, 174, 184, 185] among passerine birds. tonate virus, which is closely related to veev, and also causes fatal encephalitis in man, has also been isolated from swallow bugs in the 1970's [186] . as previously described, bat bugs (cimex insuetus and strcticimex parvus) host the orthobunyavirus kkv which likely causes disease in humans [21, 22] , but this also remains a largely understudied research area. ticks and mites are known to parasitize bats and they both fill similar ecological niches. many studies have identified viruses in bats that cluster phylogenetically with other arboviruses transmitted by ticks. other isolations are indicative of bats playing a role in viruses isolated from either hard-bodied or soft-bodied ticks. for example, estero real virus was first isolated from ticks (ornithodoros tadaridae) obtained from a palm tree colonized by cuban bats [187] . due to the close ecological association between egyptian rousette bats (r. aegyptiacus) and soft-bodied ticks in caves, the argasid tick ornithodoros faini was screened for marburgvirus rna but all pools were found to be negative [19] . to date, evidence does not support the role of arthropods in the transmission of filoviruses in nature. bats are also known to host mite species in families macronyssidae, dermanyssidae, and spinturnicidae, though the significance of these arthropod's role in virus transmission between bats and other vertebrates is largely unknown [188] [189] [190] . species in macronyssidae and dermanyssidae have also been demonstrated to efficiently transmit arboviruses (further reviewed in [191] ). a recent study explored viruses of potential arthropod-origin in the fruit bat hypsignathus monstrosus and identified five viruses: one dicistrovirus (family dicistroviridae, order picornavirales), one nodavirus (family nodaviridae), and two tombus-like viruses. they also detected a related tombus-like virus isolated from fig wasps and primitive crane flies co-habitating with bats, suggesting the ability of arthropods to host "bat-associated" viruses [192] . tombus viruses typically infect plants, but tombus-like viruses infect a wide range of hosts, including arthropods and marine invertebrates [193] [194] [195] . ingestion of arthropods by fruit bats has been documented [196, 197] , and could be a potential mechanism explaining the association between viromes of arthropods and frugivorous bat species. with regard to experimental infections, one must take into account the method of inoculation (e.g., intracerebral, subcutaneous, intraperitoneal, intramuscular), the inoculating titer, as well as whether or not vector bites were associated with the infection. in analyzing serologic results, one must take into account whether the assay is detecting neutralizing or non-neutralizing antibodies, and consider the dynamics of antibody decay which may impact detection sensitivity [198] . arthropod saliva has been shown to potentiate arbovirus infection, affecting not only the magnitude of peak viremia but also the viral loads in certain organs [199] . the inoculating viral strain used is also important, as it should align geographically with where the species under study resides to truly characterize their potential as a reservoir species. komar et al. (2003) presented detailed methods for estimating the reservoir competence of vertebrate species for mosquito-borne viruses [200] , including calculating a reservoir competence index based on the susceptibility to infection, mean daily infectiousness, and the duration of infectiousness. this reservoir competence index is interpreted as the relative number of infectious vectors that derive from a particular vertebrate species [200] , and would be easily transferrable to evaluating the reservoir competence of bats. further, bats are very long-lived (up to 25 years) compared to other small mammals, potentially increasing the chances of exposure and subsequent transmission to other vertebrates or invertebrate vectors [1] . cell culture also offers a viable starting point for initial assessments of viral replication in a particular host species, particularly when in vivo studies are not feasible. to date, many cell lines derived from the organs of bats have been established [201] . importantly, the intent of this review is not to vilify bats, but to analyze the existing literature and its support for bats as arboviral reservoirs, in addition to identifying areas for future study. bats provide vital ecosystem services, such as arthropod suppression, pollination, and seed dispersal [202] . past depopulation efforts in response to viral outbreaks resulted in increased viral spillover, and are not a viable means of disease control and have even resulted in higher virus infection rates in the bat species when colonies repopulated from neighboring roosts [203] . further work should encompass directed field surveillance complemented by both in vitro and in vivo approaches, with field surveillance efforts focused on optimization of non-lethal and non-invasive sampling [204] [205] [206] [207] . when possible, longitudinal sampling of identifiable animals through use of marking or tagging allows for monitoring of seroconversion and viremia persistence, providing a chance to better characterize transmission periodicity and seasonal changes in viremia profiles. to truly elucidate the role of bats as reservoirs for arboviruses, field surveillance studies documenting natural infection and transmission dynamics among vector and vertebrate species must be supplemented with experimental infections to characterize viremia profiles and infectiousness to vectors, virus-induced pathology, and immune kinetics following infection. with bats, these tasks are not trivial, and carry significant challenges in both the field and the lab. these challenges are evidenced by the few arboviruses for which there are substantial field and laboratory data involving the infection of bats. while many studies have presented serologic data indicative of past exposure of free-ranging bats to arboviruses of medical and veterinary importance, these studies should be followed up with laboratory assessments of reservoir host competence to shed light on the true epidemiological significance of the field data. further, the detection of viral nucleic acid in free-ranging bats does not necessarily implicate the species as an arboviral reservoir. rather, recovery and isolation of live virus at biologically relevant titers, and demonstrating the persistence of the pathogen in nature among connected populations of a potential reservoir species is more definitive. unfortunately, few established bat colonies exist for use in vivo viral pathogenesis studies, limiting the achievability of these studies. to fulfill the vertebrate reservoir host paradigm, in vivo infections must support findings in the field. the isolation of marburg virus from egyptian rousette bats in uganda in addition to experimental infections demonstrating viremia and shedding in the absence of overt pathology support the role of this bat species as the reservoir for marburg virus [6, 7, 208] . for arboviruses, the combination of field work and in vivo pathogenesis studies are lacking. still, several examples have emerged from this review that point to bats as potentially competent amplifying hosts for arboviruses. kaeng khoi virus was isolated from both bats and cimicid bugs in thailand, and was also shown to be the causative agent behind sick mine workers [21] . some bat species did develop a viremia at a level that would be infectious to mosquitoes when inoculated experimentally with chikv, and bats have been found exposed to chikv during field investigations [66, 143] . experimental evidence supporting transmission of rrv from pteropus poliocephalus to recipient mosquitoes in addition to identification of rrv-seropositive bats trapped in australia and indonesia highlights the need for further investigation into the role of bats in the ecology of this disease [75, 149, 157] . a mosquito-bat-mosquito transmission cycle was established in the lab for jbev [67] . serological evidence from the field documenting bat exposure to jbev, the sustained viremia in bats during hibernation, and demonstration of mosquito transmission from and to bats in the laboratory collectively demonstrate the capability of bats to function as reservoir hosts for this virus. circumstantial evidence from field-sampled mosquitoes and bats supports the cycling of babanki virus among bats and mosquitoes in uganda [29] , but experimental data are still lacking. for rvfv, bats evaluated in the lab have supported virus replication, and multiple populations of bats in the field have been found with neutralizing antibodies or natural infection with rvfv [1, 28, 29] . additional studies are warranted on these and other viruses for which additional field or experimental data are needed to support the role of bats as a possible reservoir. author contributions: a.c.f. and r.c.k. designed the study. a.c.f. and r.c.k. analyzed the data. a.c.f. and r.c.k. prepared the manuscript. funding: this research received no external funding. the authors declare no conflict of interest. bats: 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capture-mark-recapture estimation of population size based on a single sampling session optimizing viral discovery in bats optimizing non-invasive sampling of an infectious bat virus experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera key: cord-345472-qrddwebe authors: sebina, ismail; phipps, simon title: the contribution of neutrophils to the pathogenesis of rsv bronchiolitis date: 2020-07-27 journal: viruses doi: 10.3390/v12080808 sha: doc_id: 345472 cord_uid: qrddwebe acute viral bronchiolitis causes significant mortality in the developing world, is the number one cause of infant hospitalisation in the developed world, and is associated with the later development of chronic lung diseases such as asthma. a vaccine against respiratory syncytial virus (rsv), the leading cause of viral bronchiolitis in infancy, remains elusive, and hence new therapeutic modalities are needed to limit disease severity. however, much remains unknown about the underlying pathogenic mechanisms. neutrophilic inflammation is the predominant phenotype observed in infants with both mild and severe disease, however, a clear understanding of the beneficial and deleterious effects of neutrophils is lacking. in this review, we describe the multifaceted roles of neutrophils in host defence and antiviral immunity, consider their contribution to bronchiolitis pathogenesis, and discuss whether new approaches that target neutrophil effector functions will be suitable for treating severe rsv bronchiolitis. respiratory syncytial virus (rsv) is the leading viral cause of lower respiratory infections (lri) among young infants [1, 2] . in 2015, an estimated 33 million cases and 120,000 deaths due to rsv infection were reported worldwide [2] . the estimated cost of treating severe cases of rsv-induced bronchiolitis is over $300 million per year [3] , and an fda-approved vaccine against rsv remains elusive [4, 5] . a greater understanding of the molecular mechanisms underlying the immunopathogenesis of rsv-bronchiolitis may reveal novel pathways that can be targeted for improving treatment therapies for severe rsv patients. severe viral bronchiolitis in infancy is a major independent risk factor for the development of chronic asthma in later life [6] . therefore, ameliorating severe bronchiolitis in infancy may also act as a preventative strategy for later asthma in susceptible individuals. the prevailing view is that severe bronchiolitis results from excessive inflammation and consequent immunopathology, rather than virus-induced cytology. neutrophils are the dominant inflammatory cell in the airways of paediatric patients with rsv bronchiolitis [7] [8] [9] , accounting for up to 80% of the cellular infiltrate at peak symptomatology [8] , and yet the precise contribution of neutrophils to antiviral immunity remains ill-defined. in the context of anti-bacterial or anti-fungal immunity, neutrophils mediate protection through the production of antimicrobial peptides, respiratory oxygen species (ros), cytokines and chemokines, and the formation of neutrophil extracellular traps (nets). new information from omics-based technologies and advances in multi-parameter flow cytometry are shedding new light on neutrophil diversity and function, suggesting a greater complexity to a cell that is often unfavourably viewed as a blunt tool on account of its spectacular prowess at microbial killing and short life-span. here, we review the multifaceted roles of the neutrophil in host defence, antiviral immunity, and immunopathogenesis in the context of viral lris. we find that neutrophils make an important contribution to innate antiviral immunity and help to optimise the induction of an effective adaptive immune response. however, as a consequence of their ability to initiate and amplify the magnitude of an inflammatory reaction, it is clear that a failure to adequately regulate this response can lead to excessive collateral damage and a loss of tissue function, leading to significant morbidity from an rsv infection. we consider a number of novel approaches that might be employed to either harness or suppress the actions of neutrophils to promote antiviral immunity and/or ameliorate the immunopathology associated with severe rsv infection. an estimated four billion neutrophils are produced in the bone marrow every hour [10, 11] , and accordingly, neutrophils are by far the most abundant leukocyte in blood, representing approximately 60% of the total white blood cell count. as well as circulating in large numbers, neutrophils enter and surveil tissues in the steady state [11] . neutrophils are one of the host's first defensive measures against a sterile or microbial insult. neutrophils employ three major defensive mechanisms: phagocytosis, degranulation (secreting an array of anti-microbial peptides), and netosis [12, 13] . during phagocytosis, neutrophils utilise a combination of surface-expressed opsonic receptors, intracellular signalling cascades, and cytoskeletal rearrangements to engulf and internalise microbes [14, 15] . neutrophils contain cytoplasmic granules, which store antimicrobial molecules and facilitate cross-talk with other immune cells [16] . these granules (summarised in table 1 ) are subdivided into primary or azurophilic granules (containing, e.g., myeloperoxidase, cd63), secondary or specific granules (containing, e.g., lipocalin-2 and lactoferrin), and tertiary or gelatinase granules (containing, e.g., cathelicidins and neutrophil collagenase) [16] . this classification is based on their production during granulopoiesis and expression of distinct protein markers [16] . as a consequence of phagocytosis, the internalised microbe is trapped in 'bag-like' structures known as phagosomes, which fuse with neutrophil granules for the safe destruction of microbes [14] . upon fusion, antimicrobial molecules (summarised in table 1 ) are released into the phagosome, whereupon they kill the trapped microbe [16, 17] . table 1 . neutrophil granules and their anti-microbial properties. neutrophils can also eliminate microbes by releasing extracellular dna traps composed of decondensed nuclear chromatin, histones, and protein granules, in a process termed netosis [28, 29] . classical activation of netosis depends on nadph oxidase-mediated production of ros [30] , which promotes chromatin decondensation via histone deamination or citrullination. this process is aided by the downstream activation of the protein-arginine deiminase type 4 (pad4), a nuclear enzyme that citrullinates arginine residues, converting amine groups to ketones [31] [32] [33] . ros production can also activate azurophilic granules to release myeloperoxidase (mpo) and neutrophil elastase (ne). if these enzymes translocate to the nucleus, they can disrupt chromatin packaging [15, 17] , and promote the release of chromatin, which then interacts with other granular and cytosolic proteins to form 'nets' [30] . these nets are important for immunity against fungi and some bacterial species [22, 32, 34] . for example, people who are unable to produce nets develop chronic granulomatous disease and are highly susceptible to invasive aspergillosis [23, 35] . similarly, individuals lacking mpo are susceptible to recurrent fungal infections [23] . in pad4-deficient mice, neutrophil killing of shigella flexneri or group a streptococcus is diminished [36] . collectively, these studies demonstrate that netosis is an important weapon in the host's defensive armoury. however, if left unchecked, net-induced responses may also mediate severe pathology. excessive netosis can damage the epithelium in pulmonary aspergillosis [37] , damage the endothelium in transfusion-related acute lung diseases [38] , and exacerbate rhinovirus infection-induced allergic asthma [39] . therapeutic approaches to block netosis are now being assessed for the treatment of infectious diseases (including bacterial sepsis), autoimmune diseases (including systemic lupus erythematosus [40] , rheumatoid arthritis [41] ) and chronic lung disorders (such as cystic fibrosis [42] ). neutrophils are a vital component of the innate immune system, with key roles in pathogen recognition, the killing of invading pathogens, the presentation of antigens to t-cells, the recruitment of other inflammatory cells, and the production of cytokines [18, 43] . for sensing invading pathogens, neutrophils employ a vast array of pattern recognition receptors (prrs), including toll-like receptors (tlrs), c-type lectin receptors (e.g., dectin-1) and cytoplasmic sensors of ribonucleic acids (rig-i and mda5) [18, 44, 45] . neutrophils also express nucleotide-binding oligomerisation domain (nod)-like receptors, important for inflammasome formation [46] . the sensing of pathogens through these prrs activates the effector functions of neutrophils, including ros production, net formation, and degranulation (as highlighted in section 2). activated neutrophils may also influence the quality of innate and adaptive immune responses by affecting the trafficking and function of other innate cells, such as dendritic cells (dcs) [47] [48] [49] [50] [51] [52] [53] . for example, the neutrophil-derived chemokine ccl3 supports the rapid recruitment of dcs to the inoculation site during leishmania major infection [54] . dc function can also be affected: in the context of an infection with toxoplasma gondii, neutrophil-depletion attenuated interleukin (il)-12 and tnf production by splenic dcs [55] . neutrophils also affect the recruitment of other cells too, which they accomplish via the secretion of pro-inflammatory mediators such as danger-associated molecular patterns (damps; e.g., high mobility group box 1 (hmgb1), double stranded dna, and s100 complex proteins), pro-inflammatory cytokines (e.g., il-1α, il-1β, il-6, il-17, and tnf) and chemokines (e.g., cxcl1, cxcl2, cxcl8, ccl2, and ccl3) [12, 56, 57] . neutrophils can also initiate adaptive immune responses by directly presenting antigens themselves, or by acting as accessory cells, to support t-cell responses. for instance, human neutrophils upregulate mhc-ii and co-stimulatory molecule (cd40 and cd80) expression and can present antigens to cd4 + t-cells following phagocytosis [58] . neutrophil acquisition of these antigen-presenting properties (mhc-ii, cd40, and cd80 expression) is associated with increased activation and proliferation of cd4 + t-cells in response to tetanus toxoid. neutrophils can also direct t-cell recruitment to the site of infection. for example, in response to influenza virus infection, lung-infiltrating neutrophils were found to deposit a long-lasting chemoattracting trail (expressing the chemokine cxcl12) in the lung to guide antigen-specific cd8 + t-cells into specific niches [59] . in the absence of neutrophils, influenza-specific cd8 + t-cells were lower in the lung, leading to increased viral load and delayed viral clearance. neutrophils can also influence cd4 + t cell helper (th) responses, in particular, th17 immune responses [50, 51, 60] . in a mouse model of allergic asthma, neutrophil cytoplasts (enucleated cell bodies) augmented dc-mediated th17 responses in the lymph nodes, which subsequently increased asthma-like pathology in the lung [51] . collectively, these studies demonstrate that neutrophils are important participants in innate immunity and contribute to effective adaptive immune responses. the vast majority of human respiratory viruses, including rsv, rhinovirus, influenza, coronavirus, adenovirus, and parainfluenza virus, can cause bronchiolitis [61]. however, rsv-induced bronchiolitis is the leading cause of hospitalisation and death among infants within the first two years of life [1, 62] . rsv is highly contagious and persists outside of the host for almost six hours [63] . this prolonged survival facilitates its spread to susceptible individuals, mainly via inoculation of open mucous membranes lining the eyes and buccal cavity. upon inoculation, rsv infects the nasopharyngeal epithelium of the upper respiratory tract, replicates in epithelial cells, and then spreads to the lrt via the bronchiolar epithelium [9, 64] . this occurs within 1-3 days post infection, with peak infectivity occurring at 5-7 days post-inoculation [3, 65] . during this period, rsv triggers extensive inflammation (characterised by increased neutrophilia and levels of inflammatory cytokines), mucus hypersecretion, and oedema in the airways [1, 9, 66] . in severe cases, increased mucus production and deposition of cellular debris can occlude the bronchiole lumen, contributing to bronchiolar obstruction, air trapping, and lobar collapse [9] . patients with severe disease experience dyspnea, wheezing, and cough, the latter often persisting for three or more weeks. mild cases of bronchiolitis are manageable in outpatient departments. however, severe disease can be life threatening, necessitating urgent mechanical ventilation and admission to intensive care units for some patients. individuals with severe rsv-induced bronchiolitis during infancy are more likely to develop impaired lung function, recurrent wheezing, and asthma in adulthood [67] . whether neutrophils play a beneficial or detrimental role during rsv bronchiolitis remains difficult to ascertain clinically due to difficulties in sampling young infants. however, the predominance of neutrophils in the airway wall and alveoli of lung autopsy samples from fatal cases of rsv-related lri [9, 68] or in the airways of paediatric patients (~80% of the total cell infiltrate) with severe rsv-induced bronchiolitis [8] would suggest that they are not innocent bystanders. we postulate that neutrophils promote lung pathophysiology during rsv bronchiolitis through the production of pro-inflammatory cytokines and the releasing of cytotoxic molecules (illustrated in figure 1 ). these products, released through distinct cellular processes (e.g., netosis, degranulation) contribute to mucus hyperproduction, airway epithelial cell (aec) death and sloughing, and oedema ( figure 1 ). in rsv-infected mice, the depletion of neutrophils decreases ne, mpo, and mmp9 levels in the airways [69] . depletion of neutrophils has also been shown to lower tnfα levels and attenuate airway mucin production in the lungs of rsv infected mice [70] . in addition, in vivo antagonism of cxcr2 (a chemokine receptor important for neutrophil recruitment into the lung) reduces mucus production in the airways of rsv-infected mice [71] . in vitro, co-culture of rsv-infected aecs with neutrophils increases aec damage compared with rsv-infected aecs cultured without neutrophils [72] . collectively, these findings suggest that neutrophils can contribute to pathogenesis; however, a limitation of modelling rsv in mice is that the virus replicates poorly, and hence this is not a particularly tractable system to assess the beneficial properties of neutrophils, such as their antiviral activities. (1), degranulation (2), respiratory oxygen species (ros) production (3), and the release of neutrophil extracellular traps (netosis) (4) are associated with increased lung inflammation, systemic fever, mucus hypersecretion, airway obstruction, and epithelial cell death. together, these factors contribute to increased lung damage during severe rsv bronchiolitis. infantile severe viral bronchiolitis remains a major risk factor for persistent wheezing and chronic asthma in childhood [73, 74] . therefore, excessive neutrophilic inflammation in infants with severe rsv bronchiolitis may contribute to the onset of type 2 inflammation and cause long-term defects in lung development that predispose susceptible infants to the development of asthma in later childhood. thus, regulating excessive neutrophilic responses during severe rsv-bronchiolitis might protect against the loss of lung function, curtail the development of allergic sensitisation, and reduce the prevalence of asthma. in light of this, it is important to understand (1) the molecular events governing neutrophil recruitment to the lung, (2) the type of neutrophils recruited to the lung, (3) the molecular signals that control optimal neutrophil activation and function, and (4) whether specific neutrophil subtypes can be targeted therapeutically to improve the management of severe rsv bronchiolitis. we discuss these questions in the sections below. the earliest stages of neutrophil recruitment are initiated by damps (table 2) , including hmgb1, dna, and histones, which are released by damaged or dying cells [75] . indeed, hmgb1 is elevated in the nasopharynx of rsv-infected children compared with those infected with other viruses [76] . animal models of rsv-bronchiolitis have improved our knowledge on the mechanisms of disease development and progression (reviewed extensively in [77] [78] [79] ); however, as figure 1 . potential roles of neutrophils in the pathophysiology of severe respiratory syncytial virus (rsv) bronchiolitis. excessive neutrophil-derived inflammatory cytokine production (1), degranulation (2), respiratory oxygen species (ros) production (3), and the release of neutrophil extracellular traps (netosis) (4) are associated with increased lung inflammation, systemic fever, mucus hypersecretion, airway obstruction, and epithelial cell death. together, these factors contribute to increased lung damage during severe rsv bronchiolitis. infantile severe viral bronchiolitis remains a major risk factor for persistent wheezing and chronic asthma in childhood [73, 74] . therefore, excessive neutrophilic inflammation in infants with severe rsv bronchiolitis may contribute to the onset of type 2 inflammation and cause long-term defects in lung development that predispose susceptible infants to the development of asthma in later childhood. thus, regulating excessive neutrophilic responses during severe rsv-bronchiolitis might protect against the loss of lung function, curtail the development of allergic sensitisation, and reduce the prevalence of asthma. in light of this, it is important to understand (1) the molecular events governing neutrophil recruitment to the lung, (2) the type of neutrophils recruited to the lung, (3) the molecular signals that control optimal neutrophil activation and function, and (4) whether specific neutrophil subtypes can be targeted therapeutically to improve the management of severe rsv bronchiolitis. we discuss these questions in the sections below. the earliest stages of neutrophil recruitment are initiated by damps (table 2) , including hmgb1, dna, and histones, which are released by damaged or dying cells [75] . indeed, hmgb1 is elevated in the nasopharynx of rsv-infected children compared with those infected with other viruses [76] . animal models of rsv-bronchiolitis have improved our knowledge on the mechanisms of disease development and progression (reviewed extensively in [77] [78] [79] ); however, as pneumoviruses are host-specific, a major limitation with using mice to study human rsv stems from its inability to replicate efficiently in this setting. to better understand the pathogenic processes that underlie viral bronchiolitis, we and others have preferred to inoculate mice with pneumonia virus of mice (pvm), which is orthologous to human rsv [9, 80] . because pvm is a natural pathogen in rodents, a low inoculum dose (e.g., 10 pfu) can be employed, and this allows for greater insights into host-pathogen interactions over time as the virus replicates in the airway epithelium. importantly, inoculation with a low dose of pvm can induce the more severe pathologies of infant bronchiolitis, such as alveolar epithelial cell apoptosis, bronchial epithelial necrosis, multifocal acute alveolitis, intra-alveolar oedema, haemorrhage, and increased neutrophilia [76, 78, 81] . these pathological features are similar to those observed in autopsy samples obtained from fatal cases of rsv bronchiolitis [9] . although there are limitations with the use of pvm (highlighted in [78] ), it is proving a useful model to understand the cellular and molecular processes that underlie pathogenesis. unlike wild-type (wt) control mice, plasmacytoid dendritic cell (pdc)-depleted, toll-like receptor (tlr)7-deficient, or interferon regulatory factor (irf)7-deficient neonatal mice develop severe pathology, characterised by increased neutrophilia and lung inflammation in response to acute pvm infection [80] [81] [82] . in the absence of the tlr7-irf7 signalling pathway, massive amounts of preformed airway epithelial cell hmgb1 is released into the extracellular environment, increasing the level of neutrophilic inflammation and amplifying tissue pathology, most notably tissue oedema, epithelial sloughing, and cell death [76, 80, 82, 83] . the cell death occurs through programmed necroptosis and further increases the levels of hmgb1, perpetuating the inflammatory response [76] . rsv infection of primary human airway epithelial cells also induces necroptosis-dependent hmgb1 release [76] . intriguingly, therapeutic inhibition of necroptosis during severe viral bronchiolitis protected mice against the subsequent development of experimental asthma in later-life [76] , indicating that severe bronchiolitis is causally associated with subsequent asthma. examination of the infant bronchiolitis revealed that pharmacological inhibition of necroptosis or immunoneutralisation of hmgb1 decreases the expansion of il-13-producing type-2 innate lymphoid cells (ilc2s), and prevents alterations to the airway wall such as thickening of the airway smooth muscle (asm) layer [76, 83] . in vitro, asm cells cultured with il-33/il-2-activated ilc2s isolated from the lungs of pvm-infected mice undergo increased cell division, elucidating a cellular and molecular pathway by which excessive inflammation and immunopathology promotes both type 2 inflammation and an aberrant repair response that alters the architecture of the developing lung [83] . therefore, therapeutic targeting of the hmgb1 signalling axis may act as a novel asthma preventative by dampening ilc2-mediated type-2 inflammation and associated asm remodelling. low circulating pdc numbers in infancy are associated with acute lris [84] . interestingly, the temporal depletion of pdc in neonatal mice predisposes to severe pvm bronchiolitis due to a lack of immunoregulation by regulatory t-cells [80] . the adoptive transfer of splenic regulatory t-cells to infected pdc-depleted neonatal mice decreased hmgb1 and il-6 levels, epithelial sloughing, and lung neutrophil infiltration. despite the lack of effect on viral load, the attenuated inflammatory response improved the clinical score. additionally, the adoptive transfer of regulatory t-cells in early life was sufficient to protect against the later development of experimental asthma [80] . these data suggest that therapies that enhance immunoregulation will both ameliorate the severity of bronchiolitis and break its nexus with the development of asthma. damps can indirectly promote neutrophilic inflammation by inducing the production of neutrophil-active chemoattractants (table 2) , such as il-8 [85] and leukotriene b4 (ltb4) [86] . il-8 levels correlate with neutrophil numbers in the airways and are elevated in nasal washes of rsv-infected children [68] . moreover, genetic polymorphisms in the il-8-encoding gene that increase il-8 production are associated with increased severity of rsv bronchiolitis [87] , and pharmacological inhibition of il-8 in humans reduces neutrophilia and improves clinical outcomes [88] . levels of the eicosanoid ltb4 are elevated in endotracheal aspirates of infants with rsv bronchiolitis compared with healthy controls [89] , and are associated with increased neutrophilic responses [90] . treatment of rsv-infected mice with the leukotriene inhibitor zileuton reduces cellular inflammation (including neutrophils) in the lung, prevents rsv-induced weight loss and decreases airway pathology compared with untreated control mice [91] . mice lacking ccl3 or its receptor ccr1 display significantly reduced numbers of airway neutrophils in response to infection with pvm [92, 93] . moreover, the blockade of ccl3/ccr1 in combination with antiviral therapy improves the survival of mice infected with a lethal pvm dose [94] . damps can also promote γδ-t cells [95] and cd4 + th17 cells [96] to produce il-17a [96] [97] [98] , which is elevated in nasal aspirates from rsv-infected infants in comparison with uninfected controls [99] . il-17a augments neutrophil recruitment into the lung by stimulating the production of il-8 and leukotrienes by microvascular endothelial and lung epithelial cells [100, 101] . in addition, il-17 activates p38 mapk-induced expression of endothelial adhesion markers (e-selectin, vcam-1, and icam-1) on lung endothelial cells, promoting trans-endothelial migration of neutrophils. lack of il-17 in rsv-infected mice reduces neutrophil numbers in the lung, limits mucus production, and improves the cytotoxic t-cell (ctl) responses against rsv [99] . however, the mechanism by which il-17 deficiency enhances ctl responses to rsv remains unknown. nonetheless, inhibiting il-17 signalling may prevent neutrophil-induced inflammation and pathology during rsv infection. induce secretion of pro-inflammatory cytokines, drive ilc2 responses, induce necroptosis and aec death [76, 83] neutrophils have traditionally been viewed as a homogenous cell population. however, it is now appreciated that diversity exists at the molecular, phenotypic, and functional level [11, 13, 112, 113] . this heterogeneity is a consequence of differences in proliferative capacity, maturation status, transcriptional and epigenetic properties, and environmental cues in tissues [11] . in one study that employed fucci-474 reporter mice to identify cells at different stages of the cell cycle, subsequent mass cytometry and transcriptomic analysis revealed the existence of three distinct neutrophil subsets in the bone marrow [114] . these subsets consisted of a highly proliferative precursor that differentiated into two non-proliferating subsets, one immature and one mature. the precursor neutrophils were ly6gand cxcr2-negative, but expressed high levels of ckit and cxcr4. the immature subset displayed low levels of surface ly6g and cxcr2 and was positive for cxcr4. the mature subset displayed higher levels of ly6g, cxcr2, and cd101 expression and lacked surface cxcr4. immature and mature neutrophil subsets were subsequently identified in human blood, with immature neutrophils associating with increased tumour burden [114] , suggesting that neutrophil heterogeneity influences disease outcomes. importantly, different neutrophil subsets have been identified in circulation and in the lung during acute rsv-bronchiolitis [115, 116] . flow cytometric analysis revealed four distinct neutrophil subsets according to their differential expression of cd16 and cd62l [115] . these included a mature subset (defined as cd16 hi cd62l hi ), an immature one (cd16 lo cd62l hi ), a suppressive subset (cd16 hi cd62l lo ), and a progenitor subset (cd16 lo cd62l lo ). the relative frequency of these subsets changed over the course of infection, suggesting that they may play different roles, although functional differences between these subsets remain to be shown. in a separate study in rsv-infected patients, geerdink and colleagues identified that neutrophils in balf, compared to those in blood, displayed heightened expression of the inhibitory immune receptor leukocyte-associated immunoglobulin-like receptor-1(lair-1) [117] . flow cytometry analysis also revealed increased sirp-α, siglec-9, cd11b, and reduced cd62l and cd31 expression on activated lair-1-expressing neutrophils. agonistic ligation of lair-1 was shown to limit net formation by balf neutrophils in vitro. of note, rsv-infected lair-1-deficient mice present with greater neutrophilic inflammation upon inoculation with rsv [118] , supporting the notion that agonism of lair-1 is a logical strategy to ameliorate the severity of viral bronchiolitis. in a separate study performed in infants with an rsv infection, the neutrophils in the nasopharyngeal aspirates were shown to exhibit lower cd62l and cd31 (pecam-1) and higher cd54 (icam-i), cd11b, and cd18 expression compared to those in peripheral blood [116] . cd18 and icam-1 expression on neutrophils are also increased in rsv-infected infants compared with uninfected control infants [119] . in a more recent study, the addition of physiological concentrations of neutrophils to human rsv-infected nasal cultures in vitro increased epithelial cell damage, characterised by lower ciliary activity, cilium loss, less tight junction expression, and greater detachment of epithelial cells, compared to cultures with rsv-infected epithelial cells alone [120] . high-throughput analysis of lung-infiltrating neutrophils using the latest transcriptomic and high-dimensional flow cytometry is now needed to better identify different neutrophil subsets and associate these with beneficial and deleterious outcomes. such knowledge, together with an understanding of the molecular signals controlling neutrophil recruitment and function, is likely to reveal novel prognostic biomarkers and targets for therapeutic intervention. neutropenic individuals are highly susceptible to bacterial and fungal infections. the evidence with regard to viral infections is less clear [18] , suggesting that neutrophils are not critical participants. however, neutrophils do appear to be required for optimal host immunity against an rsv infection. to accomplish this, neutrophils detect virus-associated molecular patterns through pattern recognition receptors (such as tlrs), produce an array of antimicrobial products, and assist the adaptive immune responses. for example, tlr4 expression on neutrophils is important for generating optimal immunity against rsv infection [121, 122] . the expression of tlr4 is lower on blood and airway neutrophils of infants with severe rsv infection compared with uninfected controls [122] . in humans, two tlr4 gene mutations are associated with an increased risk of developing severe rsv bronchiolitis [123] . of note, the beneficial or detrimental roles of tlr-signalling in neutrophils during respiratory viral infections may be context-dependent. for instance, tlr4-signalling may be detrimental in rsv infection but protective against influenza. therapeutic tlr4 antagonism lowers tnf-α, il-1α, ptgs2, and cxcl1 gene expression and improves survival of mice infected with a lethal dose of influenza a [124] . some neutrophil anti-microbial products may be exploited for boosting anti-viral immunity during rsv infection. for instance, inflammatory neutrophils produce cathelicidins; cationic proteins important for host defence. intriguingly, in infants hospitalised with viral bronchiolitis, those with the lowest levels of ll-37 were more likely to be infected with rsv, suggesting that ll-37 is beneficial [125] . human neutrophils stimulated in vitro with rsv virions secrete ll-37, inhibiting the formation of new viral particles and reducing the apoptosis of infected epithelial cells [126, 127] . neutrophil-derived ll-37 may support the production of interferons, which are required for viral control. in mice, treatment with an exogenous ll-37 peptide protected against body weight loss, increased interferon-lambda production, and lowered rsv load [128] . neutrophils may also influence the quality of adaptive immune responses that develop during rsv infection. for example, neutrophils may aid in the rapid recruitment of virus-specific cd8 + t-cells. in infants with rsv infection, a systemic influx of bone marrow-derived neutrophil precursors in blood precedes robust cd8 + t-cell activation and a reduction in severe disease symptoms [129] . however, the precise mechanisms through which neutrophils influence cd8 + t-cells remain unclear. collectively, these studies suggest that neutrophils can act beneficially in mediating immune protection against rsv infection. however, perturbation of optimal neutrophil function during infection may amplify their effector responses and thus contribute to the pathology that occurs in severe rsv patients. excessive neutrophilia is associated with increased tissue damage during severe rsv bronchiolitis [130] . in part, this is attributed to molecular dysregulation of neutrophil-effector pathways, including ros production, netosis, degranulation, and over-secretion of proteolytic enzymes [101, 111, [131] [132] [133] [134] , (highlighted in figure 1 ). excessive ros production activates indiscriminate oxidative stress in the lung, contributing to lung damage [108] [109] [110] . indeed, markers of oxidative stress are elevated in the lungs and blood of patients with severe rsv [108] . in vivo treatment of mice with antioxidants significantly reduces rsv-induced inflammation and pathology [108] . ne levels are heightened in nasal aspirates and serum from paediatric patients with acute rsv infection compared with uninfected controls [68, 106] , and nets are readily detected in balf samples obtained from patients with an rsv infection [111] . in another study that sampled patients with rsv bronchiolitis, blood neutrophils showed little net formation, whereas neutrophils from the airways underwent netosis [117] . intriguingly, in an ex vivo model, this process was diminished, with an agonistic antibody directed against lair-1, which was shown to be elevated on airway neutrophils but not on circulating neutrophils [117] . in vitro stimulation of human neutrophils with rsv virions induces ros-dependent netosis via pad-4 citrullination and downstream activation of the pi3k/akt, erk, and p38 mapk pathways [131] . the deposition of net products in the culture was shown to trap rsv virions in dna lattices coated with ne and mpo, implicating a beneficial antiviral effect of nets [131] . however, in calves with a severe bovine rsv infection, the widespread airway obstruction is caused by luminal plugs composed of mucins, cellular debris, and nets [111] . as dna-rich mucus is associated with airway obstruction [111, 121] , excessive net release likely contributes to pathogenesis, although this has yet to be demonstrated clinically or in an experimental animal model of bronchiolitis. findings from other experimental systems such as allergic asthma have demonstrated that nets can exacerbate immunopathology [39] . virus-associated exacerbations of asthma are attenuated following inhibition of netosis, either through blockade of ne or degradation of nets with dnase [39] . mechanistically, netosis inhibition reduced type 2-mediated allergic inflammation and lowered airway hyper-reactivity [39] . clearly, as with most inflammatory processes, netosis is a double-edged sword. a greater understanding of netosis in the context of acute lris is needed; however, on balance it appears that limiting netosis in severe rsv bronchiolitis would be advantageous. neutrophil-derived toxic granules and proteolytic enzymes also contribute to the pathogenesis of rsv bronchiolitis. rsv increases the expression of matrix metalloproteinase-9 (mmp-9), which augments the influx of neutrophils to the site of infection [107] . elevated levels of mmp-9 are detected in the respiratory secretions of rsv-infected paediatric patients and are associated with increased risk of requiring mechanical ventilation [133] [134] [135] . hence, mmp-9 activity is considered a useful predictor of disease severity in children with rsv-induced respiratory failure [133] . genetic mmp-9 deficiency in mice decreases viral burden and lung inflammation [134] , although it is still unclear whether neutrophil-derived mmp-9 plays a dominant role in the mmp-9-induced pathology during rsv infection. specific ablation of mmp-9 in neutrophils using cre-recombinase-loxp systems would help address this question. in summary, severe neutrophilia and excessive production of powerful antimicrobial mediators appears to cause significant collateral damage, resulting in airway obstruction and the clinical symptoms that characterise severe rsv bronchiolitis. pharmacological tools to inhibit the excessive neutrophilic response in the lung are likely to ameliorate the severity of rsv bronchiolitis. to achieve this goal, therapeutic strategies should seek to modify the expression of cytokines and/or chemokines, which regulate neutrophil trafficking/recruitment into the lung, and neutrophil activation and function in response to infection (as proposed in figure 2 ). macrolide antibiotics, in particular azithromycin, which is efficacious against il-8 and neutrophil-induced inflammation, decrease the severity of symptoms during viral bronchiolitis [88, 136, 137] . a randomised trial in infants hospitalised with rsv bronchiolitis demonstrated that, compared with the placebo-control group, a 14-day treatment with azithromycin significantly decreased wheezing episodes, time to recovery, and overall respiratory morbidity over the subsequent year in comparison [88] . notably, this was associated with lower il-8 levels in nasal lavage fluid, suggesting that neutrophil numbers were attenuated, although this was not specifically assessed [88] . preclinical studies in mice have also demonstrated that azithromycin decreases virus-induced neutrophil accumulation in the lung by abrogating the expression of neutrophil inflammatory mediators such as cxcl1 [136] . in a follow-up study to the clinical trial, azithromycin treatment modified the composition of the upper airway microbiome, reducing the abundance of moraxella [138] . this phenotype was associated with lower odds of developing recurrent wheezing episodes over the next 12 months [138] . ongoing phase ii clinical trials are evaluating whether the addition of azithromycin to routine bronchiolitis care in hospitalised infants reduces the frequency of recurrent wheeze episodes during preschool years (clinicaltrials.gov identifier: nct01486758). viruses 2020, 12, x for peer review 10 of 20 would help address this question. in summary, severe neutrophilia and excessive production of powerful antimicrobial mediators appears to cause significant collateral damage, resulting in airway obstruction and the clinical symptoms that characterise severe rsv bronchiolitis. pharmacological tools to inhibit the excessive neutrophilic response in the lung are likely to ameliorate the severity of rsv bronchiolitis. to achieve this goal, therapeutic strategies should seek to modify the expression of cytokines and/or chemokines, which regulate neutrophil trafficking/recruitment into the lung, and neutrophil activation and function in response to infection (as proposed in figure 2 ). macrolide antibiotics, in particular azithromycin, which is efficacious against il-8 and neutrophil-induced inflammation, decrease the severity of symptoms during viral bronchiolitis [88, 136, 137] . a randomised trial in infants hospitalised with rsv bronchiolitis demonstrated that, compared with the placebo-control group, a 14-day treatment with azithromycin significantly decreased wheezing episodes, time to recovery, and overall respiratory morbidity over the subsequent year in comparison [88] . notably, this was associated with lower il-8 levels in nasal lavage fluid, suggesting that neutrophil numbers were attenuated, although this was not specifically assessed [88] . preclinical studies in mice have also demonstrated that azithromycin decreases virusinduced neutrophil accumulation in the lung by abrogating the expression of neutrophil inflammatory mediators such as cxcl1 [136] . in a follow-up study to the clinical trial, azithromycin treatment modified the composition of the upper airway microbiome, reducing the abundance of moraxella [138] . this phenotype was associated with lower odds of developing recurrent wheezing episodes over the next 12 months [138] . ongoing phase ii clinical trials are evaluating whether the addition of azithromycin to routine bronchiolitis care in hospitalised infants reduces the frequency of recurrent wheeze episodes during preschool years (clinicaltrials.gov identifier: nct01486758). therapeutic inhibition strategies may aim to: (1) block excessive neutrophil granulopoiesis in the bone marrow (e.g., neutralising g-csf production and function); (2) antagonise chemokine-mediated neutrophil activation and chemotaxis to the lung microenvironment (e.g., using cxcr2 small molecule inhibitors); (3) (4) (5) (6) regulate neutrophil function in the lung (e.g., blocking the production of antimicrobial products such as mmps (3), ros (4), inflammatory cytokines (5) , and deposition of nets (6)). therapeutic anti-viral products (7) may also limit rsv-induced recruitment of neutrophils in the lung during infection. targeting chemokine receptors, for instance cxcr2, which is important for neutrophil migration, may also be a therapeutic option (figure 2 ). danirixin, a selective and reversible antagonist of cxcr2, decreases the activation and transmigration of neutrophils to the lungs of rats treated with aerosolised lipopolysaccharide [139] . a clinical trial to evaluate the efficacy of danirixin in rsv-infected infants <2 years of age (clinicaltrials.gov identifier: nct02201303) is ongoing. pharmacological inhibition of mmp activity has also been employed as a therapeutic strategy for treating chronic lung diseases [25, 140, 141] . for example, marimastat, a broad-spectrum mmp inhibitor, reduces bronchial hyperresponsiveness to inhaled allergens in atopic asthmatic individuals [140] . similarly, mice treated with another broad-spectrum mmp inhibitor, r-94138, also exhibited reduced airway inflammation, characterised by reduced eosinophils and lymphocytes recruited to the lung airways during experimental allergic asthma [141] . as mmp-9 levels are significantly elevated in rsv-infected children [133] [134] [135] , selective inhibition of mmp-9 activity may be of significant benefit for the treatment of rsv bronchiolitis (figure 2 ). inhibition of netosis may also be a therapeutic option. strategies to achieve this might involve the targeting of neutrophil granules (including elastases or mpo), neutrophil-activating pattern recognition receptors (e.g., tlrs) or enzymes (nadph-oxidase and pad4). the ne inhibitor azd9668 improves lung function in patients with bronchiectasis [142] ; however, it is yet to be tested for efficacy in infants with a severe rsv infection. similarly, selective inhibition of mpo with pf-1355, which attenuates tissue injury in experimental immune complex-mediated alveolitis [143] , may offer a therapeutic benefit against severe rsv bronchiolitis ( figure 2 ). the detection of pathogen-associated molecular patterns by pattern recognition receptors on neutrophils can induce netosis. therefore, pharmacological disruption of these interactions may attenuate netosis. for instance, tak-242, a small molecule tlr4 inhibitor [144] that reduces inflammation, may also be used to regulate netosis. considering that the formation of nets is largely dependent on the activity of nadph oxidase or pad4, the inhibition of either enzyme is likely to decrease the magnitude of neutrophil-associated tissue damage. genetic deletion of pad4 or pad4 inhibition by cl-amidine or bb-cl-amidine improves clinical outcomes in several different mouse models of inflammatory disease [40, 145, 146] , although whether pad4 inhibitors are beneficial against a severe rsv infection remains unknown. the modulation of neutrophil numbers or function is evidently a promising target to ameliorate the severity of rsv bronchiolitis. however, several feasibility issues remain, particularly in relation to safety and specificity. selective targeting of neutrophils is challenging due to their close relationship to other myeloid lineages, such as monocytes and macrophages. this may alter tissue homeostasis and immune defence against other intracellular pathogens such as mycobacterium tuberculosis or listeria monocytogenes. moreover, since neutrophils are important for robust antimicrobial host defence, their therapeutic inhibition may predispose the host to other infectious agents or effect colonisation by commensals, which can become pathobionts if left unchecked. to overcome this, one strategy would be to target specific neutrophil subsets, and thus ablate the pathogenic population without compromising the beneficial effect of neutrophils in mediating antimicrobial host defence. new insights are needed to identify approaches that can support this possibility. most therapeutic approaches in the pipeline have focused on inhibiting excessive neutrophil function in treating disease pathology. however, an alternative approach is to modify neutrophil function to boost host immunity against pathogenic microbes, particularly in the elderly. for instance, the cholesterol-lowering medication simvastatin has been associated with improved neutrophil function and immunity against bacterial infection in humans [147] . in this study, adding simvastatin to the normal treatment regimen significantly reduced the severity of bacterial pneumonia and sepsis in patients older than 62 years [147] . mechanistically, simvastatin acted by augmenting the migratory accuracy of neutrophils into tissues, albeit with reduced netosis and ne release, which promoted optimal non-inflammatory bacterial control. therefore, pharmacological approaches to rejuvenate neutrophils in the elderly might be beneficial in boosting immunity against rsv. there remains a pressing need to develop new treatments to reduce the significant morbidity and mortality associated with severe rsv bronchiolitis. neutrophils contribute to host immunity against rsv; however, they are 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respiratory syncytial virus bronchiolitis: upper airway microbiome alterations and subsequent recurrent wheeze danirixin: a reversible and selective antagonist of the cxc chemokine receptor 2 the effect of marimastat, a metalloprotease inhibitor, on allergen-induced asthmatic hyper-reactivity inhibition of matrix metalloproteinases prevents allergen-induced airway inflammation in a murine model of asthma phase ii study of a neutrophil elastase inhibitor (azd9668) in patients with bronchiectasis pf-1355, a mechanism-based myeloperoxidase inhibitor, prevents immune complex vasculitis and anti-glomerular basement membrane glomerulonephritis tak-242, a small-molecule inhibitor of toll-like receptor 4 signalling, unveils similarities and differences in lipopolysaccharide-and lipid-induced inflammation and insulin resistance in muscle cells neutrophil-mediated ifn activation in the bone marrow alters b cell development in human and murine systemic lupus erythematosus locally instructed cxcr4(hi) neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps simvastatin improves neutrophil function and clinical outcomes in pneumonia. a pilot randomized controlled clinical trial this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-339852-9rq7zzqs authors: theamboonlers, apiradee; samransamruajkit, rujipat; thongme, chittima; amonsin, alongkorn; chongsrisawat, voranush; poovorawan, yong title: human coronavirus infection among children with acute lower respiratory tract infection in thailand date: 2006-11-30 journal: intervirology doi: 10.1159/000097392 sha: doc_id: 339852 cord_uid: 9rq7zzqs objective: this study was performed to further identify the previously uncharacterized human coronavirus 229e (hcov-229e) and human coronavirus oc43 (hcov-oc43) in thailand by using the rt-pcr technique. in addition, we performed this study in order to delineate the prevalence, the potential clinical impacts and evaluation of the genetic characterization of this pathogen in young children who presented with acute lower respiratory tract infections (alri). methods: we obtained nasopharyngeal secretions (nps) from 226 children <5 years of age who were either attending the outpatient department or hospitalized with alri from march 2002 to july 2003. all clinical, laboratory, rt-pcr, direct sequencing and phylogenetic analysis data were collected and analyzed. results: of the 226 nps samples from infants and young children presented with alri, 8 (3.54%) were positive for hcov-229e, 2 (0.88%) were positive for hcov-oc43, and 1 (0.44%) had co-infection. the following clinical presentations were noted: fever (100%), rhinitis (44%), acute bronchiolitis (44%), viral pneumonia (33%), viral pneumonia triggering asthma exacerbation (11%) as well as viral pneumonia causing bpd exacerbation (11%). all positive samples were subjected to direct sequencing. the amino acid sequences had 82-99% similarity to previous sequences stored in the genbank database. conclusion: the molecular technique we applied to detect human coronavirus appears justified as a valuable diagnostic approach to elucidate the prevalence, cause and clinical implications of alri among infants and young children. sionally a more severe lower respiratory tract illness in this patient group [2, 3] . due to the diffi culty in distinguishing between respiratory pathogens clinically and by laboratory-based analysis, studies on the impact of respiratory infections are still quite limited. despite improved sensitivity of diagnostic techniques, the causes of a signifi cant part of lower respiratory tract infections have as yet eluded identifi cation [4] . the development of new diagnostic techniques like rt-pcr has facilitated identification of coronavirus infection in recent years [5] . hospitalized infants with acute bronchiolitis and pneumonia have previously been shown to harbor coronavirus as one of a minor contributory factor [6] . coronavirus belongs to the coronaviridae family and contains a single molecule of linear, single-stranded rna of positive polarity, ranging from 27.6 to 31.3 kb (100-150 nm) in size, a nucleocapsid protein (n) and a lipid envelope with three major membrane proteins. coronaviruses are subdivided into three groups, human corona viruses 229e (hcov-229e) and human coronavirus oc43 (hcov-oc43) have been recognized as causes of upper respiratory tract disease including common cold and also are group 1 and 2 coronaviruses, respectively [7] . recently, another group (hcov, hcov-nl63) has been reported in the netherlands [6] . based on the structure and function of the polymerase gene, there is a valid region for phylogenetic comparisons as well as for developing a consensus polymerase chain reaction assay applicable to fi nd the differentiation of novel coronaviruses such as sars [8, 9] . this might prove very useful as these novel coronaviruses have been tentatively identifi ed by electron microscopy in association with a variety of human and animal diseases, although further characterization and definite identifi cation of these agents as coronaviruses has been extremely diffi cult and insensitive. rt-pcr is currently a rapid and sensitive method for the diagnosis of hcov [5] . coronaviruses are mostly associated with respiratory, enteric, hepatic and central nervous system diseases. nevertheless, other organs such as kidney, heart, and eye can also be infected [7] . coronavirus comprises a large family of viruses infecting a broad range of vertebrates, from mammalian to avian species. in humans and fowl, coronaviruses primarily lead to upper respiratory tract infections, whereas porcine and bovine coronaviruses cause enteric infections that result in high economic loss [7] . as for the antigenic composition of coronaviruses, research of the human strains in particular has hardly advanced because the studies have been limited to cell cul-ture or serology [10] [11] [12] and the apparently antigenic cross-reactivity between asra-cov and hcov-229e and hcov-oc43 [13] . thus, hcovs are very diffi cult to detect and the prevalence and epidemiological data are scanty, especially in asian countries. one recent report from hong kong showed that hcov infections were also found in 4.4% (hcov-nl63 1.5%, hcov-oc43 0.3%, hcov-229e 2.6%) of the children studied [14] . for isolation of suspected cases, contact tracing is only available for controlling the infection. a rapid and accurate diagnosis test is very important. since the antibody response takes (such as sars coronavirus) [14] 10-28 days after onset [19] , detection of viral rna by rt-pcr is likely to be the best option for early detection [6] . in this study we applied molecular biology techniques to identify hcov in nasopharyngeal secretions (nps) for the study on the prevalence of molecular characterization and clinical correlation of coronavirus infections in hospitalized infants and young children with acute lower respiratory tract infection (alri). the study protocol was approved by the ethics committee of the faculty of medicine, chulalongkorn university. parents were informed as to the study objective and their written consents were obtained before specimen collection. we collected np samples of 131 male and 95 female children at a mean age of 1.3 years (ranging from 24 days to 15 years) with alri attending either the outpatient clinic or admitted to king chulalongkorn memorial hospital between march 2002 and july 2003. all samples were kept at -70 ° c until further examination. patients' demographic data, clinical presentations, duration of hospitalization, basic laboratory data including complete blood count, chest radiographs and treatment were recorded for analysis. np specimens were centrifuged and the supernatant was analyzed for coronavirus rna by rt-pcr. briefl y, rna was extracted from 50 l of nps applying the guanidinium-isothiocyanate method as described elsewhere [15] and subsequently reverse transcribed into cdna using primers mf3 for hcov-oc43 and pcon4.2 for hcov-229e. the resulting cdna was amplifi ed by pcr using primer mf1 as a sense primer, and again mf3 as an antisense primer for hcov-oc43 [16] . for a replicase gene, of hcov-229e, pcon3 was used as an outer sense primer, pcon4.2 as an outer antisense primer, pcon1 as an inner sense primer, and pcon2.2 as an inner antisense primer base on the sequence accession no. x69721. the details of the primers are shown in table 1 . the amplifi cation reaction was performed in a thermocycler (9600 perkinelmer cetus, norwalk, conn., usa) applying the following conditions: initial denaturation at 95 ° c for 3 min, followed by 30 cycles each of 1 min at 95 ° c for denaturation, 1 min at 60 ° c for primer annealing, and 1 min at 72 ° c for extension and concluded by a fi nal ex-tension step at 72 ° c for 10 min. after electrophoresis in a 2% agarose gel stained with ethidium bromide on preparation, the expected 217-bp product for hcov-229e and the 334-bp product for hcov-oc43 were visualized on a uv transilluminator (gel doc 1000, bio-rad, calif., usa). all the specimens were positive for ␤ -actin which served as an internal control. for further sequencing, the pcr product bands of interest were purifi ed from residual agarose using the perfect prep gel cleanup kit (eppendorf, westbury, n.y., usa) according to the manufacturer's specifi cations and subsequently subjected to 2% agarose gel electrophoresis in order to ascertain their purity. to determine the concentration of the amplifi ed dna, we measured every sample's absorption at 260 nm in a uv spectrophotometer (shimadzu uv 160 a, tokyo, japan). the concentration was calculated according to the formula 1 od 260 = 50 g doublestranded dna. between 10 and 30 ng/ l (3-6 l) of each dna sample were subjected to cycle sequencing using 4 l of dye terminator, 2 l of dna sequencing buffer (big dye terminator v.3.1 cycle sequencing ready reaction, foster city, calif., usa) and 3.2 pmol of specifi c primer in a fi nal reaction volume of 20 l in a thermocycler (9600 perkinelmer cetus). this amplifi cation round was performed according to the manufacturer's specifi cations, using primers pcon1 and pcon2.2 for hcov-229e and mf1 and mf3 for hcov-oc43 to amplify the particular dna strand of interest for further sequencing. the results were analyzed by the sequence navigator program and submitted to the genbank database (ay751810-19, ay754762). the nucleotide sequences were also compared with the sequences published previously in genbank, applying the blast program (www.ncbi.nlm.nih.gov/blast). in order to determine the relationship between various coronaviruses isolated from the patients, we generated unrooted phylogenetic trees of nucleotide sequences comprising the polymerase gene region for hcov-229e and the m gene for hcov-oc43. ten nucleotide sequences from patients and references were multiple aligned to construct a phylogenetic tree using the clustalw package run-ning under bioedit version 5.0.9 (www.mbio.ncsu.edu/bioedit). phylogenetic trees were made with the dnaml software of the phylip version 3.6 (http://evolution.genetics.washington.edu/ phylip.html) by using 1,000 bootstraps. the obtained coronavirus sequences were aligned with those of known species stored in gen-bank and subjected to phylogenetic analysis. all isolates from this cluster grouped on the same node of the phylogenetic tree were assumed to be identical. the clinical data were analyzed and were expressed as percentage where applicable. in the present study, virus diagnosis was performed by virus identifi cation from nps obtained from the 226 infants and young children with alri attending our hospital. for hcov rt-pcr analysis, we used primer sets specifi c for the known hcov-oc43 and hcov-229e ( table 1 ). eight of 226 (3.54%) proved positive for hcov-229e and 2 of 226 (0.88%) for hcov-oc43, 1 (0.44%) showed co-infection. out of the 226 specimens, we found hcov infection in 9 patients. the clinical diagnoses of these patients with lower respiratory tract infection included acute bronchiolitis in 4 (44%), viral pneumonia in 3 (33%), viral pneumonia triggering asthma exacerbation in 2 (22%), and viral pneumonia causing bpd exacerbation in 1 (11%). their common clinical presentations were fever (100%), upper respiratory tract infection (44%), and severe cough (33%). wheezing was found in 4 (44%) and crepitations were more common in 6 (66%). their mean age was 17 8 14.9 months. the mean hospitalization required was at 6 8 2.24 days, signifi cantly more than that experienced by infants and children infected with rsv. there were 6 infants (66%) co-infected with rsv. all were treated with a bronchodilator yet only 4 (44%) responded to therapy. nobody required intensive care unit admission. all were subjected to chest radiographs, which yielded abnormal results. most chest radiographs showed perihilar infi ltrates and hyperaeration. three (33%) had patchy infi ltrates simulating bacterial infection, although 5 (55%) had been given antibiotics and all required oxygen supplement for at least 1 day after admission. a summary of the clinical data is shown in table 2 . we determined the cdna sequences of hcov-229e and hcov-oc43 by direct sequencing of the rt-pcr products and submitted them to genbank. the two samples positive for hcov-oc43, cu60th (ay751817) and cu82th (ay751818), showed 99% relative similarity to the reference strain ay382775. the following 8 samples were positive for hcov-229e: cu60th (ay751815), cu65th (ay751814), cu71th (ay754762), cu109th (ay751812), cu110th (ay751816), cu122th (ay751811), cu126th (ay751813) and cu129th (ay751810) exhibiting 82% similarity to the reference strains x69721 and ay518894 (hcov-nl). the accession numbers of references for both hcov-229e and hcov-oc43 to perform the phylogenetic analysis were: ay518894 (hcov-nl), x69721 (hcov) and af304460 (hcov-229e); af353511 (porcine epidemic area virus, pedv); aj271965 (transmissible gastroenhcov-oc43 (ay751817-18) sequences obtained in our study were aligned with those of known genotype stored in genbank and subjected to phylogenetic analysis, shown along with the hcov-229e and hcov-oc43 in different geographical areas in fi gures 1 (hcov-229e) and 2 (hcov-oc43). alris like acute bronchiolitis and viral pneumonia are major causes of morbidity in infants and young children ! 5 years of age. the majority of infections are caused by four groups of respiratory virus: rsv, parainfl uenza viruses, infl uenza viruses and adenoviruses. in the past, virus detection was markedly limited due to technical obstacles and diffi culties in virus isolation. however, owing to advances in molecular diagnostics in recent years, we are able to identify newly and deadly emerging respiratory pathogens such as sars which is also caused by coronavirus infection. thus, we realized the necessity to further explore these potential respiratory pathogens in more detail. coronaviruses have been divided into three groups or serotypes, based on their distinct genome organization and phylogenetic analyses [6, 7] . group 1 comprises hcov-229e, porcine enteric gastroenteritis virus, tgev, pedv and respiratory (prcov) coronavirus, canine coronavirus and feline coronavirus; group 2 consists of hcov-oc43, bovine coronavirus, turkey coronavirus, murine coronaviruses, porcine hemagglutinating encephalomyelitis virus, rat coronavirus, and group 3 comprises avian coronavirus including infectious bronchitis virus, turkey coronavirus, rabbit coronavirus and the sars coronavirus as a distinct entity located on a different node on the phylogenetic tree. in this study, we have identifi ed human coronavirus hcov-oc43 and hcov-229e infection in infants and young children presenting with alri. we found the human coronavirus-229e being predominant in our population. only one sample contained both. hcov-229e and hcov-oc43 have previously been proven responsible for infecting people of all age groups and causing severe lower respiratory tract infection primarily in frail patients such as young children and elderly individuals [17] [18] [19] . human coronavirus hcov causes approximately 10-20% of common colds and 6% of infl uenza-like illnesses in all age groups [20] . the clinical features associated with coronavirus infection appear to be similar to those observed with other respiratory viruses, such as rsv, parainfl uenza virus and human metapneumovirus. symptoms of upper respiratory tract infection such as rhinitis, coryza, and of lower respiratory tract infection such as tachypnea, wheezing and chest wall retraction were frequently observed in our patients. wheezing was observed in 44% compared to 9% reported from hong kong [14] . this may be explained by the difference in host response. hypoxemia was present without exception. thus, this may indicate yet another potentially severe effect of this virus infection and future impact on healthcare providers. additionally, chest hyperinfl ation, which refl ects small to medium airway obstruction caused by airway infl ammation, has proven a relatively common fi nding in our population ( 1 50%). co-infection with rsv and other pathogens has been previously described. interestingly, we found 6 (66%) patients co-infected with rsv ( table 1 ) . hence, rsv and human coronavirus might share a similar seasonal pattern. nevertheless, confi rmation of the underlying mechanism of co-infection will require further longitudinal studies conducted over several bronchiolitis seasons. the role of viral respiratory tract infections in triggering reactive airway exacerbation, especially in asthma patients, has been the subject of much research interest. rsv, parainfl uenza and rhinoviruses have previously been recognized as a primary trigger of reactive airway exacerbation. our data indicate that human coronavirus may also be important in this regard, although its role in the pathogenesis of wheezing still warrants further study. more importantly, the protection from this virus may reduce the cost of hospitalization and reduce the morbidity of these children. the hcov-229e sequences obtained from our samples showed 82% similarity to previously published data. hence, the availability of the hcov, hcov-nl and our genome sequences might contribute to the development of a variety of diagnostic assays to determine both prevalence and clinical impact of hcov infections in humans. moreover, further studies conducted on the remaining genes would be required in order to arrive at more specifi c diagnoses and elucidate the related clinical implications. acute respiratory infections: a review an outbreak of coronavirus oc43 respiratory infection in normandy, france coronavirus-related nosocomial viral respiratory infections in a neonatal and paediatric intensive care unit: a prospective study impact of respiratory virus infections on persons with chronic underlying conditions evaluation of nested polymerase chain methods for the detection of human coronaviruses 229e and oc43 osterhaus adme: a previously undescribed coronavirus associated with respiratory disease in humans coronavirus derived expression systems identifi cation of a novel coronavirus in patients with severe acute respiratory syndrome anderson lj; sars working group: a novel coronavirus associated with severe acute respiratory syndrome development and application of an enzyme immunoassay for coronavirus oc43 antibody in acute respiratory illness antigenic relationships amongst coronaviruses purifi cation and biophysical properties of human coronavirus 229e antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229e and oc43 human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong human metapneumovirus infection in thai children direct diagnosis of human respiratory coronaviruses 229e and oc43 by the polymerase chain reaction rhinovirus and coronavirus infection-associated hospitalizations among older adults coronavirus infection in acute lower respiratory tract disease of infants respiratory viral infections in the elderly surveillance of community-acquired viral infections due to respiratory viruses in rhône-alpes (france) during winter 1994 to 1995 we are grateful to the thailand research fund, senior research scholar and center of excellence in viral hepatitis fund, chulalongkorn university. also, we would like to express our gratitude to the entire staff of the center of excellence in viral hepatitis, chulalongkorn university, for their efforts in contributing to the present study. we would also like to thank venerable dr. mettanando bhikkhu of the foundation of king rama ix the great and ms. petra hirsch for reviewing the manuscript. intervirology 2007;50:71-77 key: cord-331413-fejho1of authors: nakayama, eiichi; hasegawa, keiko; morozumi, miyuki; kobayashi, reiko; chiba, naoko; ubukata, kimiko; iitsuka, taketoshi; tajima, takeshi; sunakawa, keisuke title: rapid optimization of antimicrobial chemotherapy given to pediatric patients with community-acquired pneumonia using pcr techniques with serology and standard culture date: 2007-12-31 journal: journal of infection and chemotherapy doi: 10.1007/s10156-007-0535-6 sha: doc_id: 331413 cord_uid: fejho1of abstract children (n =117; mean age 2.4 ± 2.9 years) were diagnosed as having community-acquired pneumonia (cap) using clinical symptoms, chest x-rays, and hematological data. the causative pathogen was determined using real-time polymerase chain reaction (pcr) (6 bacteria), multiple reverse transcription-pcr (mpcr; 11 viruses), bacterial culture, and serology. the initial chemotherapy was evaluated based on the pathogens identified using pcr. we found 27 viral cases (23.1%), 25 bacterial cases (21.4%), 45 mixed infections with virus and bacteria (38.5%), 10 mycoplasma pneumoniae (8.5%), 7 mixed infections with m. pneumoniae and another pathogen (6.0%), 1 chlamydophila pneumoniae (0.9%), and 2 unknown pathogens (1.7%). streptococcus pneumoniae and haemophilus influenzae accounted for 58 (49.5%) and 27 (23.0%) of the cases, respectively. the median values (50%) of the white blood cell count (wbc) and c-reactive protein (crp) using the box-and-whisker and plot method, respectively, were 11.7 × 103 mm−3 and 1.4mg/dl in viral infections, 15.6 × 103 mm−3 and 4.8mg/dl in mixed infections with virus and bacteria, 17.8 × 103 mm−3 and 6.3mg/dl in bacterial infections, 6.7 × 103 mm−3 and 1.4mg/dl in m. pneumoniae infections, and 21.5 × 103 mm−3 and 6.4mg/dl in mixed infections with m. pneumoniae and other bacterial infections. sulbactam/ampicillin (n =61), carbapenems (n =12), and ceftriaxone (n =7) were selected for the patients suspected of having bacterial infections alone or mixed infections with bacterial and viruses in accordance with our criteria defined tentatively. for those with m. pneumoniae and c. pneumoniae infections, azithromycin or minocycline was initially used. treatments averaged 3–5 days. the empirical chemotherapy was improper in 9.4% of cases in relation to the etiologic agents finally identified. we conclude that rapid and comprehensive identification using pcr can provide optimal antimicrobial chemotherapy for cap patients. community-acquired pneumonia (cap) is one of the most common infections occurring in children. [1] [2] [3] cap is caused by multiple etiologic agents including viruses, streptococcus pneumoniae, haemophilus infl uenzae, mycoplasma pneumoniae, chlamydophila pneumoniae, and other agents. [4] [5] [6] the rates of these causative microorganisms are quite different depending on many factors including the detection methods, seasonal epidemics, and the antibiotics predominantly used. [7] [8] [9] in japan, antimicrobial chemotherapy for patients with cap is begun empirically based on (1) chest x-rays, (2) clinical fi ndings including respiratory status, (3) age, and (4) laboratory tests such as white blood cell count (wbc) and c-reactive protein concentration (crp). recently, the guidelines to optimize empirical chemotherapy for cap patients were published. 10 however, we believe the goal of chemotherapy is to select the most appropriate antibiotic for every patient within a short time after admission, based on laboratory results and immediately determining the causative agent. recently, the detection of viruses and bacteria using polymerase chain reaction (pcr) in addition to serological diagnosis has determined etiologic agents with high precision. [11] [12] [13] [14] [15] [16] in children with cap, the determination of the causative pathogen is diffi cult because of the diffi culty in collecting direct clinical samples from alveoli, unlike in adults. because the pathogens must be identifi ed using indirect nasopharyngeal samples that have low invasiveness, the physician has to determine whether the isolated microorganism is the etiologic agent or not. therefore, a system to estimate the causative pathogens using obtainable clinical samples on the day of hospitalization is needed to quickly select the appropriate chemotherapy. our aim here was to use a multiplex pcr (mpcr) for viruses in parallel with bacterial detection using real-time pcr 17 in nasopharyngeal samples that were obtained from patients with pediatric cap. conventional bacterial cultures using the same samples and serological diagnosis with paired sera from the patient were performed to verify the results of the pcr. the clinical fi ndings and laboratory test results from the patient were compared for every causative pathogen, and the appropriateness of the antimicrobial agents selected empirically according to the clinical fi ndings was evaluated. pediatric patients with cap (male: n = 59; female: n = 58) were admitted to the pediatric department of hakujikai memorial hospital, tokyo, from may 2004 to april 2005. the criteria for hospitalization were: (1) the presence of pulmonary infi ltrates found in the chest x-ray, (2) acute respiratory symptoms (e.g., tachypnea), and (3) deterioration of the general clinical state. we excluded patients requiring intensive therapy including artifi cial ventilation, those with chronic respiratory disease, those with congenital heart disease, those hospitalized with the same disease within a 1-month period, and patients with congenital or acquired immunosuppressive conditions. identifi cation of the causative pathogens after informed consent obtained from the child's parents or guardians, blood samples were taken to determine wbc, crp, and serum antibody titers for several pathogens. nasopharyngeal samples were also collected to determine the causative pathogens. each sample was used for: (1) real-time pcr screening for six bacterial pathogens, (2) multiple-reverse transcription pcr (mpcr) screening for 11 viral pathogens, and (3) conventional bacterial culture. these techniques were performed immediately after collection of the samples at the laboratory of molecular epidemiology for infectious agents, kitasato institute for life sciences. streptococcus pneumoniae, haemophilus infl uenzae, mycoplasma pneumoniae, chlamydophila pneumoniae, streptococcus pyogenes, and legionella pneumophila were identifi ed within 1.5 h after using the real-time pcr with the rti kit 17 (takara bio, kyoto, japan) and stratagene mx3000p (stratagene, la jolla, ca, usa). the sensitivity and specifi city were high at 95% and 98%, respectively, as compared with standard culture as previously described. 17, 18 for the identifi cation of viruses, an mpcr kit (maximbio, san francisco, ca, usa) was used according to the manufacturer's instructions. the mpcr kit identifi es seven viruses: respiratory syncytial virus (rsv), adenovirus (adeno), infl uenza virus a (flua), infl uenza virus b (flub), and parainfl uenza virus-1, -2, and -3 (piv-1, piv-2, piv-3). in addition, four primer sets to identify rhinovirus (rhino), 19 human metapneumovirus (hmpv), 20 human bocavirus (hbov), 21 and coronavirus 19 were prepared and the pcrs were performed using the same conditions as used for the mpcr kit. mpcr required 5 h. bacterial culture was performed according to the manual of clinical microbiology. 22 serotyping of s. pneumoniae was performed using antiserum purchased from statens serum institut (denmark). antibody titers against m. pneumonia, c. pneumonia, rsv, adeno, flua and flub, and piv-1, -2, and -3 were determined in paired sera from the acute and convalescent phase using the complement fi xation (cf) test, hemagglutination inhibition (hi) test, or enzyme-linked immunosorbent assay (elisa). when a signifi cant rise in antibody titer was noted in the convalescent phase, the corresponding microorganism was considered to be the causative pathogen. a fourfold rise in titer for m. pneumoniae and adeno; for rsv using the cf assay; and for piv-1, -2, and -3, and flua and flub using the hi assay were used as indicators. chlamydophila pneumonia was diagnosed using elisa and the identifi ed patient had an index value (id) of 1.35 for igg in the paired sera. the decision to begin antimicrobial chemotherapy was based on four conditions described previously; 23 clinical course, chest x-ray fi ndings, age, and the laboratory fi ndings. for clinical observations we used: (1) the presence or absence and timing of fever and respiratory symptoms such as tachypnea, wheezing, and retractive breathing; (2) presence or absence of nasal discharge and its properties; and (3) recurrent fever during the period of recovery of common cold-like symptoms. the diagnosis of tachypnea used world health organization (who) criteria. 24 chest x-rays were divided into typical pneumonia (segmental or bronchial pneumonia), atypical pneumonia (ground-glass appear-ance, skip lesion, pleurisy, and segmental), and viral pneumonia (bronchial pneumonia and interstitial shadow). with regard to age, patients aged 5 years or older were usually thought to have m. pneumoniae infection and those aged 4 years or younger to have viral or mixed viral and bacterial infections. bacterial infection and mixed infection were suspected in patients aged 4 years or younger with a wbc count of 13 × 10 3 mm −3 or greater, or a crp value of 3.8 mg/dl or greater. these values may be low in some cases in the early stage after onset and the wbc may readily fl uctuate for various reasons in infants. however, ultimately the estimation of infection to be bacterial, viral, mycoplasmal, or mixed was determined using a composite of the clinical course, chest x-rays, age, and the laboratory data. of the four parenteral antibiotics of ampicillin-sulbactam (sbt/abpc), panipenem (papm), meropenem (mepm), and ceftriaxone (ctrx), a single agent was selected for the patients suspected of having a bacterial infection alone or a viral-bacterial mixed infection from four conditions described above. the respective doses were as follows: sbt/abpc 100-120 mg kg −1 day −1 (q.i.d.), papm and mepm 60 mg kg −1 day −1 (t.i.d.), and ctrx 40-60 mg kg −1 day −1 (b.i.d.). the antibiotics were administered for 3 days after defervescence (37.5°c). the febrile period varied considerably in patients with viral and bacterial mixed infections, so the antibiotic was withdrawn 1-2 days after defervescence where we judged the antibiotic had been effective against the bacterial pathogen. for patients suspected of having m. pneumoniae or c. pneumoniae infection from the four conditions, azithromy-cin (azm) or minocycline (mino) was used. we did not use antimicrobial agents for patients where viral infection alone was strongly suggested from the four conditions described above. criteria for etiological classifi cation table 1 shows tentative criteria for the etiological classifi cation in cap patients. seven categories are as follows: (1) bacterial, (2) mixed infection of viral and bacterial, (3) viral, (4) mycoplasmal, (5) mixed infection of mycoplasmal (chlamydial) and bacterial, (6) mixed infection of mycoplasmal (chlamydial) and viral, and (7) unknown that could not identify any etiological agent. statistical analysis of the difference in clinical fi ndings in relation to the causative pathogens was performed using the fisher's exact test. wbc and crp values on the day of admission were analyzed using the box-and-whisker plot method. the lower hinge, median, and upper hinge of the box corresponded to the 25%, 50%, and 75% percentiles, respectively; half of the cases were included in the box. the dotted line in each box is 1.5 times the quartile deviation. one hundred and seventeen cap cases were 59 male and 58 female patients during may 2004 to april 2005. the as described previously, the decision to admit was determined using chest x-rays (i.e., segmental pneumonia, bronchial pneumonia, atypical pneumonia, or interstitial shadows), acute respiratory symptoms, and deterioration in their general state. fifty-eight cases (49.6%) had previously received oral antimicrobial agents within the 2 weeks prior to their hospitalization. viral pathogens mpcr with 11 viruses were correlated with serological test results ( table 2 ). all pcr positive cases for rsv, adeno, flua, flub, piv-1, and piv-3 showed signifi cantly high antibody titers to the corresponding virus. the specifi city of pcr for these agents was 100%; however, the sensitivity of the pcr was 63.6%-100% for viral infections alone and only 21.1%-75.0% for mixed infections. simultaneous infections with two viruses with adeno and rsv or hmpv and piv-3 were identifi ed in two patients each. the cumulative positive cases determined by serology and pcr were 23 rsv (19.7%) cases, 23 piv1-3 (19.7%), 13 adeno (11.1%), 9 flua (7.7%), 9 flub (7.7%), 6 rhino (5.1%), and 3 hmpv (2.6%) cases. no cases having corona and hbov infection were identifi ed. the bacteria suspected to be the causative pathogens was determined by standard culture and real-time pcr for six pathogens: streptococcus pneumoniae, haemophilus infl uenzae, mycoplasma pneumoniae, chlamydophila pneumoniae, streptococcus pyogenes, and legionella pneumophila (table 3) in the patients suspected of having an infection caused by s. pneumoniae and h. infl uenzae, the real-time pcr results were all positive with early threshold cycles of 15-25 that indicated more than 1000 cfu per sample. direct bacterial count showed more than 10 4 cfu per sample using standard culture and was found 83.3% and 84.6% of the time, respectively, for s. pneumoniae and h. infl uenzae. seventeen cases identifi ed as m. pneumoniae infection showed single (ten cases; 8.5%) and mixed (seven cases; 6.0%) infections with other microorganisms. fourteen (82.3%) of these cases were identifi ed rapidly by real-time pcr. the rate of individual pathogens in the patients distributed by age is shown in fig. 2 . viral infection and mixed infection with viruses and bacteria were the most frequent among children 6 months to 3 years of age, bacterial infection alone occurred in those aged 1-6 years, and m. pneumoniae occurred in those aged 3 years or older. clinical characteristics according to etiologic agents table 4 the following clinical fi ndings on admission were classifi ed according to the respective criteria: (1) chest x-ray fi ndings, (2) with/without asthma, (3) respiration rate, (4) wbc, and (5) crp. they were analyzed statistically using the fisher's exact test. as expected, signifi cant differences were noted in chest x-rays among the fi ve groups with signs of interstitial pneumonia being frequently found in patients with viral infection, segmental pneumonia in those with bacterial infections, bronchial or segmental pneumonia in those with mixed infections, and with atypical pneumonia in patients having m. pneumoniae infections. viral infection and viral and bacterial mixed infections were more frequent in children with asthma compared with other infections. with regard to respiratory rate, tachypnea was not observed in m. pneumoniae infections. wbc and crp differed signifi cantly between viral and bacterial infection, and between bacterial or mixed and m. pneumoniae groups. wbc and crp values compared with causative pathogens wbc and crp values of 112 patients on the day of admission were plotted according to the respective causative pathogens (figs. 3 and 4) . patients with an unclear causative pathogen (n = 2), c. pneumoniae (n = 1), and mixed infections with m. pneumoniae and virus (n = 2) were excluded. the data were analyzed using the box-and-whisker plot method where each box encompasses 50% of the cases. as shown in fig. 3 , the median wbc values in the patients with viral, viral and bacterial, bacterial, m. pneumoniae, and m. pneumoniae and bacterial infections were 11.7 × 10 3 , 15.6 × 10 3 , 17.8 × 10 3 , 6.7 × 10 3 , and 21.5 × 10 3 mm −3 , respectively. in patients with defi ned viral and bacterial mixed infections, the 50% box of wbc values was located between those of the viral and bacterial cases. as shown in fig. 4 the relation between the empirically selected antibiotic for 117 cases and the causative pathogens is shown in table 5 . the decision to use antimicrobials and the selective criterion for the initial treatment were as outlined in patients and methods. antibiotics were used in 97 patients (82.9%), namely sbt/abpc in 61 (52.1%); ctrx in 8 (6.8%); a carbapenem, either mepm or papm, in 12 (10.3%); azm in 10 (8.5%); mino in 3 (2.6%); and sbt/abpc and azm in 2 (1.7%) patients. no antibiotic was used in 20 (17.0%) patients. the initially selected antibiotic was retrospectively considered inappropriate in 9 patients with viral infection (adeno, 4; rsv, 3; infa, 1; piv-3, 1), 1 with m. pneumonia and viral mixed infection, and 1 patient with an undetermined causative pathogen. out of the 117 cases, antimicrobials were inappropriately used in 11 (9.4%) cases. clinically, defervescence was achieved within 24 h after admission in all the patients with bacterial infections. the average duration of antimicrobial treatment was 3-5 days. the duration of antibiotic therapy was also 4 days in four patients in whom s. pneumoniae or h. infl uenzae was isolated from blood cultures taken at admission. after the termination of treatment, no patient experienced a relapse or was refractory to treatment. however, we experienced four cases having recurrent fever whose hospitalization was slightly prolonged, but this was not due to relapse of pneumonia and all of them recovered spontaneously without further antimicrobial use. identifi cation of the causative pathogens in children with cap is not always easy because sputum or bronchoalveolar lavage (bal) samples cannot be obtained as routinely performed in adults. to diagnose children, the causative pathogen is suspected from the history, chest x-ray, and blood examination data while taking into account the patient's age, and then the antibiotic is selected empirically. recently in japan, 10 the guidelines for the treatment and management of respiratory tract infection in pediatric patients have been proposed to promote optimal chemotherapy, and similar guidelines are found in other countries. 25, 26 however, the incidence rate of pathogenic microorganisms in pediatric cap in other countries likely differs due to many factors such as the health insurance system, vaccination programs, kinds of antibiotics predominantly used, and population density. there are published studies of the use of pcr methods to determine the pathogens in cap, [11] [12] [13] [14] [15] [16] and the simultaneous detection of both bacteria and viruses is expected to enhance the accuracy of cap diagnosis. here our aim was to identify bacteria and viruses using pcr within a short time frame and to evaluate the pcr results in relation to clinical fi ndings. as previously described, 17 dna/rna samples were extracted from cliniin the near future, it is anticipated we will also be able to accomplish viral identifi cation with real-time pcr. we used nasopharyngeal samples as a source to identify the causative pathogens. these materials are appropriate to identify viruses, mycoplasma pneumoniae, and chlamydophila pneumoniae, but this sample type is questionable for streptococcus pneumoniae and haemophilus infl uenzae because they can be present in normal individuals. a positive result for s. pneumoniae and h. infl uenzae by real-time pcr should be carefully considered as to whether it indicates their causal relation with the infection or not where the physician should consider the chest x-rays, clinical symptoms, clinical laboratory fi ndings such as wbc and crp, bacterial amounts, and infl ammation fi ndings of leucocytes in nasopharyngeal samples. the rate of viruses and bacteria identifi ed in this study as the causative pathogens was similar to the data reported by michelow et al. 12 and juvèn et al., 5 although the rate of h. infl uenzae differed. this was presumably because hib vaccination has not been approved in japan 27 where there are cases of pneumonia due to hib and nontypable h. infl uenzae. three cap cases with hib having positive blood cultures and positive real-time pcr were found among our 117 cases. chlamydophila pneumoniae was detected in only one patient possibly because there were few children older than 6 years in this study. as for the relation between the diagnosis of cap and blood examination test, although some studies have only concluded that crp and wbc can provide useful informa-tion for pneumococcal pneumonia, [28] [29] [30] [31] these values are used by japanese pediatricians as useful references in addition to routine chest x-ray to diagnose pneumonia. in clinical practice, we have observed these values do not fl uctuate for about half a day after the onset of bacterial infection. in addition, in the cases of adenovirus infection, and virus or m. pneumoniae infection in school-age children, the values of wbc and crp are relatively high. the physician must be mindful of these kinds of exceptional cases; however, as shown in our data, signifi cant differences in these values are found and are correlated to the causative pathogens. at present, we believe the time from onset to presentation in hospital is relatively uniform in japan as compared with other countries because of our universal health insurance system. in this study, the antimicrobial agent that was empirically selected was found to have been inappropriately administered to 9.4% of the 117 cases. using our techniques, we found the treatment for most cases was completed within 3-5 days. in general, the dosing period recommended in the guidelines is 7-10 days; however, we consider this is somewhat long because the antibiotics acted against the causative bacteria within a shorter period. of the 58 strains of s. pneumoniae, 25 were penicillin resistant streptococcus pneumonia (prsp), and none of the patients experienced a relapse after treatment with sbt/abpc and a carbapenem antibiotic (data not shown). viral and bacterial mixed infections were identifi ed in 40.2% of the cases in addition to viral infection in 23.1%. thus, involvement with the viral-related cases was 74 (63.2%) cases in total. furthermore, cases relating to virus infections may be present, which could not be demonstrated when 5 days or more had elapsed after onset. timing of acquiring the clinical samples is also very important to demonstrate the causative viruses. in the future, we expect comprehensive and rapid identifi cation of the causative pathogens outlined here will become routine in clinical practice. community-acquired pneumonia etiology and treatment of pneumonia diagnosis and management of pneumonia in children community-acquired pneumonia in children etiology of community acquired pneumonia in 254 hospitalized children management of community-acquired pediatric pneumonia in an era of increasing antibiotic resistance and conjugate vaccines mycoplasma pneumoniae and chlamidia pneumoniae in pediatric community-acquired pneumonia: comparative effi cacy and safety of clarithromycin vs erythromycin ethylsuccinate etiology of childhood pneumonia: serologic results of a prospective, population-based study rationalised prescribing for community-acquired pneumonia: a closed loop audit practice guidelines for respiratory tract infections in childhood etiology and treatment of community-acquired pneumonia in ambulatory children epidemiology and clinical characteristics of communityacquired pneumonia in hospitalized children rapid identifi cation of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-pcr: feasibility study national disease burden of respiratory viruses detected in children by polymerase chain reaction epidemiological investigation of nine respiratory pathogens in hospitalized children in germany using multiplex reverse-transcriptase polymerase chain reaction population-based incidence of severe pneumonia in children in kiel simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time pcr with pathogen-specifi c molecular beacon probes assessment of real-time pcr for diagnosis of mycoplasma pneumoniae pneumonia in pediatric patients simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays human metapneumovirus infection in japanese children detection of human bocavirus in japanese children with lower respiratory tract infections manual of clinical microbiology etiology and clinical study of community-aquired pneumonia in 157 hospitalized children world health organization british thoracic society of standards of care committee. bts guidelines for the management of community acquired pneumonia in childhood a practical guide for the diagnosis and treatment of pediatric pneumonia high prevalence of type b β-lactamase-non-producing ampicillin-resistant haemophilus infl uenzae in meningitis: the situation in japan where hib vaccine has not been introduced differential diagnosis of viral, mycoplasmal and bacteraemic pneumococcal pneumonias on admission to hospital white blood cells, c-reactive protein and erythrocyte sedimentation rate in pneumococcal pneumonia in children c-reactive protein in viral and bacterial respiratory infection in children bacteremic pneumococcal pneumonia in children the authors are grateful to akiko ono and matsumoto masato of meiji seika kaisha for their assistance. key: cord-349647-cfjrwt44 authors: girkin, jason; maltby, steven; singanayagam, aran; bartlett, nathan; mallia, patrick title: chapter 8 in vivo experimental models of infection and disease date: 2019-12-31 journal: rhinovirus infections doi: 10.1016/b978-0-12-816417-4.00008-1 sha: doc_id: 349647 cord_uid: cfjrwt44 abstract human and animal models continue to play a crucial role in research to understand host immunity to rhinovirus (rv) and identify disease mechanisms. human models have provided direct evidence that rv infection is capable of exacerbating chronic respiratory diseases and identified immunological processes that correlate with clinical disease outcomes. mice are the most commonly used nonhuman experimental rv infection model. although semipermissive, under defined experimental conditions sufficient replication occurs to induce host immune responses that recapitulate immunity and disease during human infection. the capacity to use genetically modified mouse strains and drug interventions has shown the mouse model to be an invaluable research tool defining causal relationships between host immunity and disease and supporting development of new treatments. used in combination the insights achieved from human and animal experimental infection models provide complementary insights into rv biology and yield novel therapeutic options to reduce the burden of rv-induced disease. that deliberately expose humans to an infectious agent are required, when there are more than enough naturally acquired infections to yield sufficient subjects for studies. despite the ubiquity of viral colds there are a number of factors that make studies of naturally acquired infections problematic. although rvs are the commonest etiological agents of viral colds there are several other viral causes (and nonviral causes of upper airway symptoms), and the clinical syndromes caused by different virus types are indistinguishable. 2, 3 other factors contributing to variability in naturally acquired infections include different routes of inoculation (eye, nasopharynx, direct contact, aerosol, etc.), different inoculation doses, variability in the perception of symptoms leading to differences in time to presentation and host factors (immune status, smoking, age, etc.) that influence viral pathogenicity. further, the heterogeneous nature of naturally occurring infections requires that large patient numbers are needed to identify statistically significant effects of treatment. therefore human experimental infection studies are an attractive proposition as they allow for a known etiological agent to be administered at a standard dose, route of inoculation, and time point to a selected group of recipients with similar characteristics (e.g., age, smoking history, health/disease, and antibody status). detailed follow up can be carried out in a controlled clinical setting with sample collection at defined time points in relation to the time of infection. as the clinical syndrome induced by rv challenge in young healthy volunteers is benign and self-limiting, experimental rv infection in this group is relatively uncontroversial. perhaps the only risk to subjects was the possibility of additional infectious agents present in the inoculum and good manufacturing practice (gmp)-prepared stocks are now required by regulators for experimental infection studies in humans to contravene this risk. 4 studies in healthy volunteers have been central to establishing the key aspects of the biology of rv infection including routes of acquisition of infection, 5à8 clinical symptoms, 8, 9 inflammatory and immune responses, 10 involvement of the lower airway, 10, 11 and correlates of immune protection of rv infection. 12 virus challenge studies have also been used in healthy volunteers to evaluate a vast array of potential treatments. however, none of these studies have led to the licensing of a single treatment for the common cold. 13à51 licensing approval was sought for an antiviral drug, pleconaril, for the treatment of rv infection. 21 approval was denied by the food and drug administration as the adverse effects outweighed the benefits in healthy subjects with self-limiting colds. the lack of approval of any antiviral treatments casts doubt on whether continued investment in viral challenge studies is justified. however, the recognition that rv infection is associated with more severe clinical manifestations in people with chronic lung diseases such as asthma and copd provided a new impetus to research and a new direction to human experimental infection studies. up until the early 1990s the prevailing view was that rv infection resulted in a self-limiting, mild upper respiratory tract syndrome only. there were occasional reports of rv infection associated with more severe clinical illness such as pneumonia 52 but both scientific and pharmacological research tended to be focused on other respiratory viruses such as influenza and respiratory syncytial virus, as these were considered to be more serious human pathogens. colds had long been associated with asthma exacerbations but early studies investigating virus infection in asthma and copd exacerbations reported low detection rates. 53à55 the consensus was that asthma exacerbations were predominantly triggered by allergen exposure and copd exacerbations by acute bacterial infection. the development of highly sensitive and specific molecular diagnostic techniques using polymerase chain reaction (pcr) technology led to a revolution in viral diagnostics and a reevaluation of the role of respiratory viruses in a range of clinical syndromes. this was particularly pertinent to human rvs, which are either difficult or impossible to culture (e.g., rv-c strains) and due to the large number of serotypes diagnostic serology testing is not feasible. pcr-based diagnostic tests have a much greater sensitivity for the detection of rvs and studies using pcr revealed that the range of clinical illness associated with rv infection was much broader than previously recognized and included more severe disease syndromes such as pneumonia, 56 bronchiolitis, 57 acute rhinosinusitis, 58 and influenza-like illness. 59 in addition rvs could be detected in most asthma exacerbations, 60 and in a substantial proportion of copd exacerbations. 61 asthma is estimated to affect 360 million people worldwide and copd affects 174.5 million people and was the cause of 3.2 million deaths in 2015. 62 much of the enormous morbidity, mortality, and healthcare costs associated with asthma and copd are related to acute exacerbations. therefore the recognition that rvs are a major cause of asthma and copd exacerbations stimulated new interest in their biology and treatment. as part of this research investigators considered whether experimental infection studies in humans could be extended from healthy volunteers to patients with asthma and copd. respiratory viruses can be detected in up to 80% of asthma exacerbations in children and 60%à80% of exacerbations in adults, 60,63à65 with rvs the commonest virus detected. the recognition of the role of rvs in asthma exacerbations stimulated research into their biology in an attempt to develop treatments for virus-induced exacerbations. this research included investigating whether experimental rv infection could be used in people with asthma in the same way it had been used in healthy individuals. the first experimental rv challenge of subjects with asthma was carried out in 1985 at dalhousie university, canada. of the 21 volunteers inoculated, 19 became infected but only 4 had $ 10% decrease in forced expiratory volume in 1 second (fev 1 ) and an increase in airway hyperreactivity (ahr). the authors felt that these findings suggested "that other viral pathogens may play a more important role in precipitating asthma attacks." 66 it is unclear why this study failed to induce features of asthma exacerbations but it would be almost another decade before further experimental rv infection studies in people with asthma were attempted. experimental infection studies in allergic (nonasthmatic) subjects suggested that rv infection could induce changes in lower airway physiology similar to that seen in asthma. 67, 68 in 1994 an experimental infection study from the university of southampton, united kingdom included a small group of people with allergic asthma and reported that upper respiratory symptoms were more severe in atopic subjects but did not report on lower airway symptoms or physiology. 69 concurrently a study using pcr to detect viruses in naturally acquired asthma exacerbations strongly supported a role for rv. 60 subsequent studies were carried out by research groups in the united kingdom, 70 the netherlands, 71à74 and the united states 75, 76 in volunteers with mild, intermittent asthma. these studies demonstrated that rv infection induced airway obstruction, 77, 78 increased ahr, 70à73,79 and airway inflammation 70,72,79à84 and rv could be detected in the lower airways, 75,85 thereby supporting a causative role for rvs in asthma exacerbations. having established that respiratory viruses are a trigger for asthma exacerbations, research focused on investigating why rvs cause a benign, self-limiting illness in healthy subjects but result in more severe manifestations in people with asthma. a study of naturally acquired infections in cohabiting couples discordant for asthma suggested that people with asthma were not more susceptible to virus infection but had more lower respiratory tract symptoms. 86 airway inflammation during naturally acquired infections was greater in people with asthma compared with those without asthma but the number of subjects in this study was small and the viruses detected were different between the two groups. 87 viral challenge studies are ideally suited to addressing this research question as people without asthma matched for characteristics such as age and gender can be infected simultaneously. most of the earlier infection studies did not include a control group of healthy volunteers and therefore could not address the question as to whether host responses to infection differ in people with asthma. studies that did include nonasthmatic controls produced somewhat inconsistent results with one study reporting no differences in lower airway inflammatory cells, 70 another reporting increased nasal inflammatory mediators in asthma 88 and discrepant results regarding virus-induced respiratory symptoms. 88, 89 these divergent results were likely related to differences in sampling methods and timing, antibody status of subjects, and choice of healthy controls (e.g., atopic vs nonatopic). the first study to show clear differences between subjects with and without asthma in their responses to rv infection was published in 2008. 90 in this study, rv challenge induced more respiratory symptoms, greater lung function impairment, increased bronchial hyperreactivity, and eosinophilic and neutrophilic lower airway inflammation in asthmatic compared with normal subjects with direct correlations between loss of lung function and the degree of neutrophilic, eosinophilic inflammation, and nasal viral load. 90 in addition, the study provided insights into potential mechanisms of differential responses to viral infection in people with asthma. despite being infected with the same dose of virus, postinoculation virus loads tended to be higher in the asthmatic subjects compared with the healthy controls, suggesting that antiviral immunity may be impaired in people with asthma with subsequent failure to control viral replication. virologic and clinical outcomes were related to deficient interferon (ifn)-γ and interleukin (il)-10 responses and to augmented t-helper type 2 (t h 2) responses (il-4, il-5, and il-13), indicating that excessive t h 2 or impaired t h 1 (or il-10) immunity may be important mechanisms. when infected with rv in vitro, alveolar macrophages and bronchial epithelial cells from subjects with asthma demonstrated deficient production of ifnβ and ifnλ, and this was related to the severity of virus-induced asthma exacerbations. 91 other reports subsequently confirmed that ifn production is deficient in asthma, 92, 93 but this finding has not been replicated in all studies. 94à96 it may be that this phenomenon only occurs in a subset of people with asthma, or that it is seen in more severe or poorly controlled asthma. such patients were not initially included in challenge studies as these were limited to mild, wellcontrolled asthma not requiring inhaled corticosteroids. in 2014 rv challenge was shown to be safe in a small group of people with wellcontrolled asthma requiring long-term use of inhaled corticosteroids. 97 a larger study confirmed this and reported significantly more upper and lower respiratory symptoms, greater reduction in peak expiratory flow and fev 1 , increased viral loads, increased bronchoalveolar lavage (bal) eosinophils, and increased nasal il-4, il-5, and il-13 in subjects with moderate asthma using inhaled corticosteroids. 98 this study also identified novel mediators of virus-induced asthma exacerbations including il-33, 98 il-25, 99 and il-18. 100 poor asthma control was associated with more severe virus-induced exacerbations, greater t h 2 inflammation and higher virus load. 101 therefore responses to virus infection may differ depending on asthma severity and control, which may account for some of the discrepant results in earlier experimental infection studies. these successful viral challenge studies should pave the way for further studies in subjects with moderate asthma that should reveal new insights into the pathogenesis of exacerbations that may not have been obtained from studies in mild asthma. the evidence that emerged from research, including experimental infection studies, that asthma is associated with deficient ifn responses led to the development of inhaled ifnβ as a treatment for asthma exacerbations. a clinical trial of inhaled ifnβ reported that treatment can reduce the severity of virus-induced exacerbations in a subgroup of patients assessed with more severe asthma. 102 the development of inhaled ifn as a novel asthma treatment is a clear demonstration of the potential of experimental rv infection studies to contribute to the discovery of new treatments for virus-induced asthma exacerbations. the global initiative for obstructive lung disease defines copd as "a common preventable and treatable disease, characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases. exacerbations and comorbidities contribute to the overall severity in individual patients." 103 acute exacerbations of copd are the major drivers of morbidity, mortality, and healthcare costs in copd and prevention of exacerbations a major unmet need. 104 acute bacterial infection was believed to be the main cause of copd exacerbations and this is reflected in the widespread use of antibiotics in copd exacerbations. 105, 106 although copd exacerbations are preceded by upper respiratory symptoms in up to two-thirds of cases, virus detection rates in the pre-pcr era were low. 53, 107 as with asthma, the role of viruses in copd exacerbations was reexamined using pcr-based detection methods. although detection rates of respiratory viruses in copd exacerbations are more variable than in asthma, respiratory viruses can be detected in 50%à64% of copd exacerbations, with rvs the predominant virus type detected. 108à111 despite this emerging evidence implicating respiratory viruses in a significant proportion of copd exacerbations, both scientific and clinical research continued to focus on bacterial infection. as evidence of this, it was almost two decades after the first experimental rv infection study was carried out in asthma that a similar study in copd was attempted. despite the excellent safety record of experimental infection studies in asthma, caution was warranted in repeating these studies in copd as there are major differences between these two populations. copd patients are older, current or ex-smokers, and have impaired lung function with irreversible airflow obstruction. all these factors have the potential to result in a more severe response to experimental rv challenge, compared with the younger, nonsmoking patients with relatively normal baseline lung function recruited to the asthma infection studies. the first experimental infection study in copd was a small pilot study published in 2006 that established the safety of rv infection in four patients with moderate airflow obstruction (fev 1 50%à80% predicted) and not using regular inhaled therapy. 112 the subjects developed symptoms consistent with a copd exacerbation following rv inoculation, together with objective markers of exacerbation with falls in lung function and increases in upper airway inflammatory markers. all the subjects recovered completely without treatment and no adverse events were reported. subsequently the same research group carried out two larger studies of experimental rv infection in subjects with copd and non-copd control subjects. 113, 114 these studies replicated the findings of the pilot study, wherein rv infection manifested in cold symptoms, lower respiratory symptoms consistent with exacerbations of copd, including airway inflammation and worsened airflow obstruction. 113, 114 these studies provided important causal evidence linking virus infection to copd exacerbations. studies from naturally acquired infection were supportive of this link but do not provide definitive evidence as pcr evaluation detects viral nucleic acid and therefore does not prove the presence of live virus and samples are only collected after exacerbation onset. respiratory virus nucleic acid can also be detected in copd patients with stable disease, although is usually elevated during copd exacerbations. 115 in experimental infection models, rv was present in airway samples prior to exacerbation onset, virus load increased in parallel with the increase in symptoms, airflow obstruction and inflammation, and clearance of virus was associated with exacerbation resolution. 113, 114 strong correlations were observed between virus load and airway neutrophil numbers, neutrophil elastase, il-8, il-6, and tumor necrosis factor-alpha (tnfα), granulocyte macrophage colony stimulating factor, most of which of also correlated with levels of epigenetic regulator, histone deacetylase 2 and these inflammatory responses were greater in patients with copd. in these studies, rv infection is the sole experimental agent responsible for increased inflammatory markers in patients with copd, providing strong evidence that rv infection directly causes exacerbations in copd patients. another advantage of virus challenge studies over naturally acquired infections is the ability to carry out detailed and repeated lower airway sampling during the course of the exacerbations, including the use of bronchoscopy. this has provided a wealth of mechanistic data regarding the pathogenesis of virus-induced exacerbations including the presence of inflammatory mediators, 113, 114 inflammatory cells, 113, 116, 117 oxidative and nitrosative stress, 114 and impaired antiviral ifn responses. 113 a novel observation that emerged from these studies was that secondary bacterial infections occurred in 60% of experimental virus-induced exacerbations, 118 whereas coinfection was rarely reported in naturally acquired exacerbations. 119 analysis of the respiratory microbiome following experimental rv infection suggest that secondary infection occurs due to an outgrowth of previously present airway bacteria. 120 potential mechanisms of secondary bacterial infection include reduced antimicrobial peptides 118 and increased glucose in the airways. 121 a subsequent study of naturally acquired exacerbations sampling at multiple time points during exacerbations confirmed the validity of this observation. 122 therefore although the numbers of copd subjects recruited to virus challenge studies to date is small, rv infection appears to be safe in this population and replicates the features of naturally acquired infection. further studies including larger numbers of patients are needed to validate these findings and further investigate the mechanisms of virus-induced exacerbations in copd. since the first studies in healthy volunteers, experimental rv infection has been extended to patients with asthma and copd and has contributed enormously to expanding our understanding of the biology of rv infection and how it affects patients with chronic airway diseases. a summary of the key findings from human experimental infection studies in people with asthma and copd is provided in table 8 .1. these studies have tended to have a narrow focus on rv infection and host immune responses. it is clear from both in vivo and in vitro studies that there are interactions between respiratory virus infections and other factors that exacerbate asthma and copd such as bacteria, 127 allergens, 128 and air pollution. 129 these factors have been somewhat neglected in viral challenge studies and are a promising field of future research that is starting to be addressed. 124, 130 as mentioned previously, the use of the viral challenge model in asthma is much further advanced compared with copd. one study identified ifn-deficiency in copd but this has not been replicated. with the development of inhaled ifn as a therapy option for asthma, the role of ifn in copd requires urgent further investigation. other areas of future research include the effects of virus infection on novel pathways such as lipidomics 131 and metabolomics 121 in asthma and copd. respiratory viruses have also been identified as triggers of exacerbations in other airway diseases such as cystic fibrosis 132, 133 and bronchiectasis 134, 135 and there is evidence of impaired antiviral immunity in these diseases. 136 experimental infection studies may help to define the role of virus infection in these patient populations. 124 11 aa exposed to allergen 1 placebo, 10 aa exposed to rv, 9 aa exposed to rv and allergen perhaps the most promising use of the virus challenge model will be to accelerate the process of drug development. 137, 138 virus challenge studies have been used to evaluate the effects of existing asthma therapies on virus-induced exacerbations. 123, 125 recently the first study using viral challenge to evaluate a novel, unlicensed drug in asthma was also published. 126 although these studies had negative results they demonstrate the potential of the viral challenge model in drug development. the key to successful drug development is the identification of clinically relevant mechanisms of rv infection or immunopathology that can be experimentally manipulated for therapeutic benefit. this is where human experimental rv infection is complemented by work in animal models. there are a number of options for modeling human rv infection in animal models and they provide the ability to investigate specific disease components and mechanisms that would be otherwise impossible in humans. human experimental infection models have the advantage of identifying disease correlates, but the degree of experimental manipulation possible is extremely limited and the safety and effectiveness of interventions must first be evaluated in animals. animal models have proven useful for mechanistic studies across a range of diseases. models of rv infection have been reported in several animal species, including mouse, cotton rat, and nonhuman primates. each of these experimental systems provides its own advantages and disadvantages and a substantial contribution to the knowledge base of the biology of rv infection. animal models provide a range of benefits to complement human experimental approaches. 139 experimental animals can be readily manipulated to induce consistent disease outcomes, such as the induction of allergic airway disease (aad) to model asthma or cigarette smokeàinduced copd. animal models have less variability than human populations providing more consistent experimental outcomes and statistical power in intervention studies. experimental environment, exposures (e.g., previous infection history), endpoints, and interindividual variability can be controlled. further, a broad array of tools are available to characterize disease outcomes, including reagents, genetically modified animal strains, experimental protocols, and assessment techniques (e.g., lung function testing). sample tissues can be easily isolated at experimental endpoints, which are difficult or impossible to sample in humans (e.g., lung tissues, draining lymph nodes, bone marrow). further, animal models serve as a valuable preclinical system for the assessment of novel interventions, to provide proof-of-principle safety and efficacy findings prior to exposure of healthy human volunteers. mice in particular have been extensively used to model disease, including virus infection and exacerbations of asthma and copd. 138à140 as a result, a wealth of tools and techniques are available to study immune mechanisms and pathophysiology in mice. well-characterized protocols and reagents support the induction of disease states and allow detailed characterization of immune responses (e.g., fluorescently tagged monoclonal antibodies to quantify immune cell subsets). a range of transgenic and knockout mouse strains are available that allow for the careful dissection of relevant disease mechanisms. further, reagents are available to assess the effects of novel interventions on disease outcomes (e.g., blocking antibodies and various forms of innate immunity activators; see chapter 9: emerging therapeutic approaches). the expansion of mouse rv infection models has paralleled our understanding of rv biology in humans. 141 initial approaches focused on understanding the effects of rv infection in isolation, identifying the mechanisms and cell types mediating lung pathology. increasingly complex experimental models are now being used to characterize long-term effects of infection on airway function and the effects of rv infection on preexisting airway disease (e.g., asthma exacerbations). 138à140 one of the biggest developments in mouse rv infection models was the protocol for isolation of highly purified, concentrated (high titers) of rv from henrietta lacks (hela) immortalized human epithelial cell lines and later, the intracellular adhesion molecule 1 (icam-1) transfected rhabdomyosarcoma cell lines. 142, 143 prior to this, clarified infected hela cell lysates were used for in vitro experiments. 144 some investigators also used infected hela cell lysates in mouse models. 145 efforts to improve the quality and validity of the model made use of a partial purification protocol to generate viral stocks. 146 for mouse models of rv infection or exacerbations, the refined, high-titer, rv purification protocol is the gold standard. a major barrier that hindered the early development of mouse models was the species specificity of rv infection. "major-group" rv strains, which make up approximately 90% of all rv strains, enter the cell through binding of icam-1. 147,148 rv binding to icam-1 is limited to human and chimpanzee and does not occur in other species, including mouse. 149 as a result, major-group rv strains cannot infect mouse cells and fails to replicate or induce pathology in mouse models. early attempts to develop rv mouse models failed to detect sufficient viral replication to induce disease. 150 a major advance enabling the development of mouse rv infection models was the initial recognition that the minor-group rv-a1, which use the host cell receptor low-density lipoprotein receptor, can infect the mouse epithelial cell line la-4. 151 this recognition suggested that minorgroup rv viruses (e.g., rv-a1) may be useful to model infections in mice in vivo. indeed, inoculation of wild-type balb/c mice with rv-a1 induced lung pathology, mucus production, and inflammatory cytokine production. 152 notably, rv infection was also sufficient to induce exacerbations of preexisting asthma in sensitized and challenged mice (as discussed in more detail below). 152 an alternate approach was also sought to allow modeling of majorgroup rv infection in mice. the same study by tuthill et al. demonstrated that transfection of la-4 cells with a chimeric icam-1 receptor containing the human extracellular receptor domains allowed infection and replication by the major-group virus rv-a16. 151 this finding provided the basis for developing a transgenic mouse strain expressing a chimeric mouseàhuman icam-1 receptor. chimeric receptor expression in hu-icam tg mice is sufficient to support in vivo infection with rv-a16, resulting in airway inflammation, mucus production, viral replication, and inflammatory cytokine production. 152 of note, the hu-icam tg mouse was generated by random insertion of a chimeric transgene and little is known about the transgene insertion site within the genome. use of this transgenic strain requires additional experimental considerations (e.g., genotyping and use of heterozygous animals in experiments), which has limited its broad utility. additional variations have also been reported in the literature, which aim to broaden the available mouse models. genetic rv-a1 variants have been generated by serial passage through mouse embryonic fibroblasts in vitro and lung epithelial cells in vivo, which exhibit increased growth in mouse cells. 153 inoculation of balb/c mice with the rv-a1/ m2m7 variant [8 3 10 6 plaque forming units (pfu)] allowed for recovery of virus from mouse lung after 24 hours, when mice were also pretreated with intranasal hydrochlorous acid to increase epithelial permeability. 153 successful use of mouse models also depended on the development of streamlined rv isolation protocols that have allowed consistent and rapid isolation of virus stocks, to limit variability between experiments. the current gold-standard protocols for rv isolation, rv-a1 infection of wild-type balb/c mice, and the infection of transgenic mice expressing chimeric mouseàhuman icam-1 receptor with rv-a16 are published and readily available. 141 a range of studies have assessed the effects of primary rv infection in mice, contributing to our understanding of the mechanisms underlying disease pathogenesis. studies have used similar protocols, typically performing intranasal inoculation of b10 6 à10 8 tcid 50 (tissue culture infective dose in 50% of culture, a titration of infection units of pathogens that do not form plaques in culture) rv-a1 and assessing responses over 1 week following infection in balb/c or c57bl.6 mice. in the initial publication by bartlett et al., intranasal inoculation of wildtype balb/c mice with 5 3 10 6 tcid 50 rv-a1 induced a range of disease features similar to human disease. 152 rv infection induced airway inflammation characterized by increased neutrophil numbers at 24 and 48 hours postinfection and increased lymphocyte numbers persisting for 1-week postinfection. 152 tissue pathology was characterized by perivascular and peribronchial inflammation, increased mucus production, and elevated inflammatory cytokine production (including mip-2, kc, mip-3α, ip-10, rantes, itac, il-6, il-1β, ifnα/β/λ/γ). 152 furthermore, rv infection resulted in the development of an rv-specific adaptive antibody response by 7 days postinfection. 152 further studies have provided insights into the effects of rv infection alone in mice. following inoculation of wild-type c57bl/6 mice with 1 3 10 8 tcid 50 rv-a1, detectable rv positive-and negative-strand rna were recovered from the lung, indicative of active viral replication and viral rna was detectable up to 7 days postinfection. 145 the study also noticed a small increase in airway neutrophils and lymphocytes in the presence of uv-inactivated rv-a1, although uv-inactivated rv-a1 (and major-group virus rv-39) failed to induce inflammatory cytokine production. 145 rv-a1 infection also increased airway responsiveness to methacholine challenge at days 1 and 4 postinfection. 145 inoculation with rv-a1 or uv-inactivated rv-a1 induced pi3k activation in airway epithelial cells and pretreatment with the pi3k inhibitor ly294002 in vivo dampened neutrophilic inflammation and inflammatory cytokine production (kc, mip-2, mip-1α, ifnγ). 145 inoculation of c57bl/6 mice with rv-a1 (5 3 10 7 tcid 50 ) leads to discontinuous expression of zonula occludens-1, suggesting that infection disrupts airway epithelial barrier function. 154 rv infection also stimulated il-15 production and release into the airways, which is dependent on type i ifn production and stimulates activation of natural killer (nk) and cd8 1 t cell responses. 155 treatment with an il-15àil-15ra complex increased expression of il-15, il-15rα, ifnγ, and cxcl9 and stimulated increased nk, cd8 1 , and cd4 1 t cell recruitment and activation. 155 ccl7 and irf-7 are the most upregulated lung transcripts following rv-a1 infection. 156 blocking ccl7 or irf-7 function reduced lung neutrophil and macrophage accumulation and ifn responses and blocking ccl7 also reduced ahr. 156 this publication also delineated ahr from inflammatory infiltrates showing instead a relationship between classical proinflammatory transcription factors (nfκb) and ahr. as alluded to previously, the use of knockout mice in particular has provided insights into a number of mechanisms regulating rv-induced pathology. key roles for neutrophils and the neutrophil chemokine receptor cxcr2 in mediating rv-induced pathology were identified. inoculation of cxcr2 2/2 mice with rv-a1 (4.5 3 10 6 tcid 50 ) resulted in reduced airway neutrophil numbers, reduced inflammatory cytokine production (tnfα, mip-2, kc), decreased mucus production, and decreased cholinergic responsiveness, with no alteration in viral load, compared with wild-type control animals. 157 further, antibody depletion of neutrophils and infection of tnfr 2/2 mice also reduced ahr, compared with control animals. 157 these findings provide evidence for a role of neutrophilic inflammation, potentially via tnfα production, on downstream pathology following rv infection. roles for pattern recognition molecules have been demonstrated for mda5, toll-like receptor 3 (tlr3) and tlr7. infection of mda5 2/2 mice resulted in delayed type i ifn (ifnα/β) and suppressed type iii ifn expression, with a slight early increase in viral load in the lung. 158 in contrast, inoculation of tlr3 2/2 mice resulted in normal ifn responses and no difference in viral yield. 158 both mda5 2/2 and tlr3 2/2 mice had reduced neutrophil numbers, inflammatory cytokine production (cxcl1, cxcl2, ccl2, cxcl10) and airways responsiveness, compared with wild-type controls. 158 differing roles for nfκb signaling pathways in rv-induced inflammation and type i inf responses in antiviral immunity have been demonstrated. disruption of nfκb signaling in p65 1/2 mice resulted in reduced neutrophil numbers and inflammatory cytokine production (cxcl1, cxcl5, cxcl2), while ifn production and viral loads are unaltered. 159 in contrast, ifnar1 2/2 mice have unaltered neutrophilic inflammation, a persistent increase in lymphocyte numbers and cytokines ccl5, cxcl10, and cxcl11, with reduced ifnα production and increased viral load. 159 a pathogenic role for the proinflammatory molecule muc18 has also been demonstrated, with increased expression of antiviral genes (mx1, ip-10), reduced neutrophil inflammation and viral load in muc18 2/2 mice following rv-a1 inoculation (1 3 10 7 pfu). 160 studies using tbet 2/2 mice (a key regulator of t h 1 cell differentiation) have also demonstrated the key role for t h 1-polarized t cells in the response to rv infection. tbet 2/2 mice developed a t h 2/ t h 17-polarized immune response to rv infection (5 3 10 6 tcid 50 ) with increased il-13 and il-17a production, deficient nk cell responses, and decreased neutralizing antibody development. 161 cd4 1 t cells contributed to increased airway eosinophil numbers and mucus production following rv infection in tbet 2/2 mice. 161 studies using tslp receptor-deficient mice (tslpr 2/2 ) demonstrated that rv-a1 infection interferes with tolerance to an inhaled allergen, via a mechanism requiring tslp, il-33, and activation of ox40l on lung dendritic cells. 162 after observing increased levels of the tnf super family member protein, tnfsf10 (trail or cd253) production over a time course of rv-a1 infection in mice, girkin et al. compared rv-a1 infection in tnfsf10 2/2 mice to wild-type balb/c mice and observed an almost complete ablation of inflammatory responses to rv-a1. 163 following rv infection, peribronchiolar inflammation and lung histopathology were reduced in tnfsf10 2/2 mice; neutrophil and lymphocytes in bal remained at baseline; and cd4 1 t cells, cd8 1 t cells, nks, plasmacytoid dendritic cells (pdcs), and myeloid dendritic cells were all reduced in flow cytometry of total lung cells. 163 tnfsf10 2/2 mice were protected from rv-induced ahr, and failed to develop rv-induced exacerbations of allergic airways disease. 163 an interesting proviral effect of trail was also identified whereby tnfsf10 2/2 mice had reduced viral load and anti-trail antibodies reduced viral load (whereas recombinant trail administration increased viral load) in beas2b cells infected with rv-a1 in vitro. 163 this effect on viral load was independent of ifn responses and may be associated with an unidentified role of apoptosis in rv replication, which remains to be explored. some studies have assessed the effect of primary rv infection on clinically relevant sequelae, including secondary bacterial infection and the effects of premature birth on infection. exposure of epithelial cells in culture to rv-a1 resulted in increased bacterial attachment and translocation through an epithelial monolayer (nontypeable haemophilus influenzae (nthi), pseudomonas aeruginosa, staphylococcus aureus), 154 suggesting a potential mechanism underlying secondary bacterial infections following viral infection. a subsequent study demonstrated that primary inoculation with rv-a1 (5 3 10 6 tcid 50 ) delayed the clearance of nthi in vivo, associated with suppressed chemokine production (kc, mip-2) and neutrophilic inflammation through a tlr2-mediated mechanism. 164 the model has also been used to assess immune alterations relevant to premature birth and bronchopulmonary dysplasia, risk factors for viral-induced exacerbations. exposure of neonatal mice to hyperoxia (75% oxygen) in early life increased inflammatory cytokine expression (il-12, ifnγ, tnfα, ccl2, ccl3, ccl4) and suppressed early ifn responses following rv-a1 infection (9 3 10 6 pfu) at 14 days of age. 165 one study has also assessed the effect of rv infection timing on subsequent development of aad. inoculation of 7-day-old mice with rv-a1 and subsequent induction of house dust mite (hdm)-induced allergic airways disease had additive effects with increased neutrophilia and ahr in female mice, although rv inoculation had no additional impact in male mice. 166 these studies extend the use of rv infection in mice to new areas, including mechanisms of early life infection susceptibility, to mechanisms of secondary bacterial infection/compromised antimicrobial immunity and experimental exploration of clinical risk factors associated with increased likelihood to develop virus-induced exacerbations of respiratory diseases. mouse models are valuable tools for the preclinical testing of novel treatments. several studies have used the mouse rv infection model to assess intervention strategies, including vaccine development and drug treatment. primary inoculation of balb/c with rv-a1 (1 3 10 6 tcid 50 ) rapidly induced circulating rv-specific igg antibody production within 4 days, which binds capsid protein vp1 and those antibodies were crossreactive to another minor strain rv (rv-29). 167 repeated rv infections were necessary to induce rv-specific iga responses and neutralizing antibodies, but administration of cpg or subcutaneous immunization with freund's adjuvant promoted neutralizing antibody development and may inform potential vaccine strategies. 167 pretreatment with the plant flavanol quercetin before and during rv-a1 infection effectively reduced viral replication, inflammatory cytokine production (kc, mip-2, tnfα, ccl2, ifnα, and ifnλ2), and ahr. 168 treatment with the cancer therapeutic gemcitabine (20,20-difluorodeoxycytidine) reduced rv load, inflammatory cytokine levels (tnfα, il-1β), and reduced lung lymphocyte numbers. 169 treatment with corticosteroid therapy (fluticasone proprionate) suppressed ifn responses to rv and reduced airway inflammation, leading to increased mucus production and reduced antimicrobial responses. 170 effects on viral load, mucin production, and antibacterial response could be reversed by administration of recombinant ifnβ. 170 despite promising findings in mouse models, quercetin has not entered clinical trials for the treatment of rv infection, likely due to a previous randomized community clinical trial in 2010 that showed little benefit of quercetin supplementation on upper respiratory infections. 171 these findings may highlight the limitation of mouse models, which (while valuable) do not always fully recapitulate human disease mechanisms. animal models to study rv-mediated exacerbations of airway disease have also been developed. these models combine experimental rv infection with models of airways disease, including asthma, copd, and chronic rhinosinusitis. models of asthma typically consist of administration of a sensitizing agent [e.g., ovalbumin (ova) or hdm] and subsequent challenge in the airways to induce an eosinophilic, allergic airways disease. copd is typically induced by prolonged and repeated exposure of mice to cigarette smoke or treatment with elastase. after airways disease is established, mice are then inoculated with rv to induce disease exacerbations. these models are explained and expanded upon in the following sections. researchers have also used double-stranded rna (dsrna) administration as a surrogate for virus infection to exacerbate preexisting disease (reviewed in refs. [139, 140] ). however, these approaches fail to model the complexity of virus infection and are beyond the scope of the current book chapter. many studies have characterized the effects of rv infection on preexisting asthma and have provided insights into the immune cell types involved, key molecules, and responses to potential therapies. in the initial report of an rv (rv-a1) exacerbation model, ova-sensitized and challenged balb/c mice were inoculated with rv-a1 during the allergen challenge phase. 152 the combination of virus and allergen challenge increased airway neutrophil, eosinophil, and lymphocyte numbers; increased cytokine production (il-4, il-13, and ifnγ), increased ahr; and increased mucus gene expression. 152 subsequent studies have identified key functional roles for macrophages, gamma-delta (γδ) t cells, dendritic cell subsets, and neutrophils in rv-induced immunopathology. in a similar model, rv-a1 inoculation into ova-sensitized/challenged mice increased macrophage lung infiltration and eotaxin-1/ccl11 expression. 172 eotaxin was expressed by pulmonary macrophages in the lung after combined virus infection and allergen challenge. 172 further, macrophage depletion or antieotaxin treatment reduced rv-induced airway eosinophilia and ahr. 172 macrophage activation state also modulates the response to rv infection in allergen sensitized/challenged mice and shapes the resulting pattern of inflammation. rv infection in asthma exacerbation models induced an il-13expressing macrophage population, with m2 polarized phenotype. 173 depletion of il-13-producing cells in cd11b-dtr mice or ccr2 2/2 mice reduced airway inflammation and ahr. 173 interestingly, while rv infection of ova-treated wild-type mice contributes to mixed neutrophilic and eosinophilic airway inflammation and m2 macrophage phenotype, il-4r 2/2 mice exhibit neutrophil inflammation alone and increased m2 polarization of pulmonary macrophages but still have exacerbated airway responses. 174 γδt cells dampen exacerbation responses. γδt cells are increased in rv-induced asthma exacerbation models and blocking responses with anti-γδtcr antibody worsened exacerbations with increased ahr, and increased numbers of t h 2 cells and eosinophils, with no effect on virus load. 175 pdcs were recruited to the lung during rv-induced inflammation and subsequently promoted t h 2 responses in the lung draining lymph nodes, in a process mediated by il-25. 176 depletion of pdcs with an antibody or treatment with anti-il-25 reduced eosinophil numbers, decreased lung pathology, reduced cytokine production (il-5, il-13), and reduced ahr. 176 functional roles for neutrophils, and neutrophil extracellular traps (nets), have also been provided in rv-induced asthma exacerbation models. chronic low-dose hdm exposure and rv infection have additive effects on neutrophilia and induce ahr. 177 a more recent study demonstrated that rv infection in an hdm-mediated asthma model results in double-stranded dna release into the airways and administration of genomic dna alone was sufficient to mimic characteristic components of rv-induced exacerbations. 178 further, blocking neutrophil elastase or degrading nets by applying dnase into the airways reduced eosinophil and lymphocyte numbers, tissue pathology, and cytokine production (il-5, il-13). 178 a number of studies have highlighted the functional roles of specific molecules in rv-induced mouse exacerbation models, as potential therapeutic targets to validate in patient populations. in addition to roles during rv infection alone highlighted above, mda5 and tlr3 are also involved in rv-induced exacerbations. mda5 2/2 and tlr3 2/2 mice have decreased inflammatory responses and ahr, while mda5 2/2 also had decreased ifn responses (ifnβ/λ2/λ3). 158 midline 1 (a e3 ubiquitin ligase) is upregulated in an hdm-induced model, and short interfering rnaàmediated inhibition prior to rv inoculation reduced neutrophil numbers and mucus production production. 179 the monocyte chemotactic protein ccl2 is produced by epithelial cells and macrophages following rv-induced exacerbation and administration of an anti-ccl2 antibody reduced eosinophil numbers and ahr. 180 foxa3-overexpressing transgenic mice produce excess mucus in their airways and rv infection increased foxa3 expression. 181 in foxa3-deficient mice (foxa3 2/2 ), rv clearance is increased, with increased ifnβ activation. 181 il-25 expression is also increased in rv-induced exacerbations and blocking the il-25 receptor reduced type 2 cytokine production (il-4, il-5, il-13, il-25, il-33, tslp), mucus production, and numbers of eosinophils, neutrophils, t cells, and innate lymphoid type 2 cells. 99 combining dsrna administration with rv-a1 inoculation worsened preexisting allergic airways disease. repeated dsrna administration after ova sensitization/ challenge resulted in neutrophilic lung inflammation and tissue pathology and combined dsrna and rv-a1 inoculation increased expression of tslp, tnfα, and ifnλ in the lung. 182 key roles for pattern recognition receptors have also been demonstrated in rv asthma exacerbation models. hdm-allergic tlr7 2/2 mice had a decreased antiviral response, with reduced ifn release (ifnα/β/λ1/λ2/λ3) and increased virus replication, associated with increased eosinophil and lymphocyte numbers, increased il-5 and ccl11, and ahr. 183 administration of ifn or transfer of wild-type tlr7-competent pdcs could restore antiviral responses and reduce disease exacerbation. 183 ova-allergic tlr2 2/2 mice also had reduced macrophage, neutrophil, and eosinophil numbers and suppressed ahr after rv inoculation. 146 bone marrow transfer experiments demonstrated that tlr2 2/2 bone marrow could protect from exacerbations, while transfer of wild-type bone marrow restored responses in tlr2 2/2 mice. 146 transfer of wild-type macrophages into tlr2 2/2 mice could also restore exacerbations. 146 as previously mentioned, a role for trail has also been demonstrated, with hdm-allergic trail-deficient mice (tnfsf10 2/2 ) protected from rv-induced ahr and induction of airway inflammation. 163 rv-induced asthma exacerbation models have also been used to assess the responses to existing therapies and as preclinical models for novel therapies. treatment of hdm-allergic mice with the long acting beta-2 agonist salmeterol reduced ahr and eosinophil numbers during rv exacerbation, and limited chemokine levels (ccl11, ccl20, cxcl2) through modulation of pp2a. 184 the findings of this study were focused on pp2a as a novel therapeutic target rather than promoting salmeterol monotherapy (which was associated with adverse events and tolerance to β2-agonists with chronic salmeterol use 185 ). an approach to block majorgroup rv virus infection was assessed through administration of antihuman icam-1 antibody, which prevented entry of rv-a16 and rv-14 and reduced neutrophil and lymphocyte numbers, cytokine production (il-4, il-5, il-6, ccl1, ccl11), mucus production, and virus load in human transgenic mice in an ova-allergic model. 186 treatment with a nontoxic anthraquinone derivate reduced rv-induced ahr, neutrophil, and eosinophil airway inflammation; inflammatory cytokine production; and mucus hypersecretion while also boosting type 1 ifn response and reducing viral yields, with associated decreased akt, hif-1α, and vegf production. 187 treatment with an antiinflammatory vap-1/ssao inhibitor, pxs-4728a, or the macrolide antibiotic azithromycin also reduced neutrophil numbers and pxs-4728a reduced ahr following rv-a1 inoculation in hdm-allergic mice. 188 8.11.2 mouse chronic obstructive pulmonary disease exacerbation models rv also plays an important role in virus-induced exacerbations of copd. several studies have assessed the effects of rv inoculation in animal models of copd. exposure to elastase and lipopolysaccharide (lps) once per week for 4 weeks induces features of copd, including airway inflammation, goblet cell metaplasia, and altered lung function. 189 addition of rv-a1 led to persistence of viral rna ( . 14 days postinfection), deficient ifn responses (ifnα/β/γ) and increased ahr, lung volume, cytokine production (tnfα, il-5, il-13), and mucus production, compared with elastase/lps administration alone. 189 a subsequent study attempting to replicate the elastase/lps model of copd found that a single elastase treatment followed by rv-a1 inoculation was enough to increase airway neutrophil and lymphocyte numbers, increased inflammatory cytokine production (tnfα, cxcl10, ccl5), mucus hypersecretion, and ahr. 190 in the same elastase-induced model, fluticasone proprionate treatment reduced ifn responses, increased viral load, suppressed airway immune cell numbers (lymphocytes and neutrophils), suppressed inflammatory cytokines (il-6, tnfα), and increased mucus production, following rv-a1 exacerbation. 170 the differences in the experimental approaches required to elicit an rv-induced exacerbation in these different studies is likely due to the quality of virus inoculum used by the different investigators, which as explained previously, is influenced by purification approach. the first study used only crude virus-infected cell lysates, whereas the later study employed a highly purified virus inoculum. several studies have also reported on rv infection in a cigarette smo-keàinduced copd model. in an initial study, 8 weeks of cigarette smoke exposure resulted in increased viral persistence, neutrophilia, and increased mucus production following rv infection. 191 subsequent studies demonstrated that goblet cell gene expression was reduced following treatment with a gamma-secretase inhibitor (gsk l685,458) to limit notch activation. 192 further, supplementation of feed with quercetin reduced rvinduced lung inflammation (including neutrophilia), goblet cell metaplasia, and ahr. 193 to our knowledge, only one study has assessed the effect of rv infection in a chronic sinusitis model. a mouse model of chronic allergic rhinosinusitis was induced by 5 weeks of repetitive nasal ova challenges. 194 increased rv-a1 yields were reported in the nasal tissue of mice with rhinosinusitis, although inflammatory cytokine production and histopathology were unaffected. 194 this study served to illustrate the range of additional diseases where rv infection has been shown to be relevant in human populations where animal models are available for future research (e.g., cystic fibrosis). a limited number of studies reported the use of rv infection in other animals, namely cotton rats and nonhuman primates. we note that historical studies assessing "rv" infection in other animal species are referring to genetically distinct viral genera and should not be confused with human rv (e.g., equine rv and bovine rv). for example, while human rv and equine rv were both originally assigned to the rhinovirus genus, they have been reclassified into enterovirus and apthovirus, respectively. equine rv has subsequently been renamed "equine rhinitis virus." the cotton rat (sigmodon hispidus) is a recognized model for human respiratory infection, particularly for respiratory syncytial virus, as well as adenoviruses, parainfluenza virus, measles, and human metapneumovirus (reviewed in ref. [195] ). to date, two studies have reported on rv infection in cotton rats, providing evidence that cotton rats are partially permissive to rv major-group infection. intranasal inoculation of rv-a16 (10 7 pfu) into cotton rats induced lower respiratory histopathology, increased mucus production, and induction of inf-activated genes. 196 immunization with live rv-a16-induced high levels of circulating antibodies and protected from subsequent infection, while prophylactic transfer of anti-rv-a16 serum also protected from disease. 196 further, this protection was transferred effectively from mother to newborn, limiting viral yields in subsequently infected progeny. 196 in a later study, the same group provided evidence that infection with rv-b14 (10 6 pfu) induced similar disease pathology. furthermore, immunization with rv-b14 provided protection from subsequent infection with either rv-b14 (an rv-b group virus) or rv-a16 (an rv-a group virus), demonstrating some degree of crossreactivity to very different major-group viruses. 197 chimpanzees and gibbons are the only nonhuman primates that support rv infection, although rv infection has also been reported in the vervet monkey cells, with consistent infection requiring high dose exposure. 198 initial rv infection studies in chimpanzees were reported in 1968, using rv-b14 and rv-a43 199 and in gibbons in 1969. 200 subsequent studies in chimpanzees and gibbons assessed the antiviral effects of drug treatments on rv infection, using bis-benzimidazole and triazinoindole, respectively. 201, 202 administration of soluble truncated form of human icam-1 can prevent subsequent infection in chimpanzees. 203 however, it has been noted that neither chimpanzees nor gibbons develop "cold" symptoms following rv infection and the high costs and logistics of these studies has limited further progress. chimpanzees are an endangered species and require considerable resources and facilities for research. current chimpanzee research is limited to the united states and gabon. however, the national institutes of health in the united states have indicated that they are seeking to eliminate the use of chimpanzees in research. all but one species of gibbon are endangered. thus clinical research using nonhuman primates in the future to characterize rv infection are likely to be limited or nonexistent. as rv does not normally infect rodents, an attenuated mengovirus infection model has been proposed as an alternative option to model rv infection. mengovirus also belongs to the picornaviridae family and normally causes systemic infection in rodents. using an attenuated mengovirus, intranasal inoculation of 10 7 pfu into rats increased airway neutrophil and lymphocyte numbers, induced lung tissue pathology, and increased expression of cxcl1 and ccl2. 204 a subsequent report using a genetically attenuated mengovirus vmc(0) in mice also induced lower respiratory tract infection with increased lung neutrophil and lymphocyte numbers, expression of cxcl1, cxcl2, cxcl5, il-17a, infs, and chemokines cxcl10 and ccl2. 205 other respiratory virus infections are associated with acute exacerbations of asthma and copd, including respiratory syncytial virus, influenza, human coronavirus, human parainfluenza virus, human metapneumoviruses, and adenoviruses. 206 animal models for these infections have largely been limited by the specificity of viruses to humans. it is unclear to what extent the mechanisms causing pathology differ between different viruses (or between strains of the same virus). a detailed discussion of the disease processes induced by each of these different virus infections is beyond the scope of this chapter. a detailed analysis of the relevant disease mechanisms in each infection setting is necessary to inform our understanding of disease exacerbations and ideally to identify common mechanisms between viruses that can be targeted for therapy. no animal model can completely recapitulate naturally occurring human rv infection. while animal models provide important insights into disease mechanisms, it is important to also recognize their limitations. there are recognized limitations of mice as models of human respiratory disease. 207 these include differences in response/symptoms between other species and humans. there are differences in respiratory tract architecture in human, nonhuman primate, and mouse airways. they range from dichotomas (each airway splits into two), trichotomas (airways split into threes), or monopodial branching (central airway continues while subordinate airways branch out) with differences in airflow inhomogeneities covered in detail by miller et al. 208 there are also differences in mucus production processes in mouse compared with human airways. the short lifespans of laboratory animals do not capture the life-course of human disease, mice do not naturally develop asthma or copd, and most current models of asthma represent eosinophilic, allergic patterns of disease. it remains unclear to what extent the current models and pathophysiology truly reflect human disease (particularly considering recognized heterogeneity of the human population). rv has evolved for efficient replication in the human respiratory tract. due to the decreased efficiency of rv entry into nonhuman epithelial cells (and likely differences in the nuances of cellular machinery required for replication), a high amount of viral load is required to elicit a biological response to rv in laboratory animals (e.g., 10 6 tcid 50 in mouse vs 5à10 tcid 50 in experimental human infection models). human rv strains also demonstrate limited viral replication and different replication kinetic between mouse and man. these differences highlight the importance of confirming findings in relevant patient cohorts/samples and the utility of using human experimental models in parallel with animal models. this point is not purely for academic consideration. more so, it is important to take into consideration clinical trial design and outcomes. for example, mouse models highlighted the key relevance of il-5 in asthma pathology through use of knockout mice 209 and antibody blockade. 210 however, the initial randomized control trial assessing anti-il-5 therapy (mepolizumab) in a broad asthma population failed to demonstrate any clinical effect. 211 it was not until subsequent trials limited recruitment to patients with demonstrable eosinophilic asthma (a patient subset that is more closely modeled by the experimental mouse system) that clinical improvements were observed. 212, 213 8.14.2 future directions for animal models in a similar way to experimental human infection models, there has been a narrow focus in animal models. in mice, focus has largely been on rv infection alone with a growing body of literature assessing asthma exacerbations. while difficult to model in mice, rv-induced copd exacerbations models are emerging through use of elastase administration and cigarette smokeàinduced copd. a summary of key findings from mouse models of rv-induced exacerbations of airway disease is presented in table 8 .2. limited studies have reported on rv effects or potential interventions in these models. as with human experimental infections, animal infection models may also be relevant to an expanding array of diseases in the future (e.g., cf, bronchiectasis). to date, there has been limited assessment across different rv strains in both animal and human studies. the primary focus of rv models has been on rv-a1 in mice, or rv-a16 and rv-a39 in human, possibly due to the availability of these strains and the ease of growing these strains in cell culture. in particular (due to its relatively recent discovery), rv-c infection has yet to be assessed in animal models. there has so far been difficulty in generating sufficient quantities of rv-c for research purposes (particularly at infectious titers required for mouse models). with the recent establishment of a suitable cell line (e8 hela cells) that supports rv-c replication this gap in the literature will likely be rectified. 214 189 elastase/lps 1 rv-a1 nil deficient antiviral immunity increased inflammation exaggerated ahr increased mucin expression as is the case for the majority of human virusàmouse infection models, the mouse is semipermissive to rv infection and as such a high-titer inoculum is required to induce prolonged replication and robust, reproducible host immune responses. a mouse-adapted rv strain (rv-1bm) has been generated by serial passage in mouse epithelial cells (la-4 cells) though this mouse-adapted virus has only been characterized in vitro with primary mouse tracheal epithelial cells 215 and has not yet been tested in vivo. the clinical translation of novel therapies identified in animal models for the treatment of rv infection in humans is yet to come to fruition. however, there are multiple molecules currently in the drug development pipeline, ranging from virus-targeting molecules, drugs targeting host factors of the viral replication cycle, and biologics such as innate immune stimulators and cytokine blocking monoclonal antibodies, all of which are elaborated on in chapter 9, emerging therapeutic approaches. there are significant opportunities for further research in both human and nonhuman models, including assessment of infections in various unexplored disease backgrounds that are exacerbated by rv infection (e.g., cystic fibrosis) and expansion of studies using newly identified rv strains (e.g., rv-c strains). human and mouse rv experimental infection models effectively complement each other and have contributed immensely to our understanding the mechanisms shaping rv-induced pathology. human experimental rv challenge studies have shed light on the biology of rv infection and the mechanisms associated with rv-induced exacerbations of chronic respiratory diseases. mouse models of rv infection in particular are readily manipulatable to identify cause and effect between specific molecules and disease outcomes for preclinical testing. an excellent example of how human and mouse models complement each other is the growing understanding of the disease mechanisms during rvinduced asthma exacerbations. human experimental infection revealed a potential role for induced type 2 immunity following rv infection in individuals with asthma. 90, 98 subsequent mouse model studies have demonstrated a causal role for rv-induced type 2 immune effector molecules in exacerbations, 99, 152, 176, 179 allowing preclinical assessment of the efficacy and safety of novel therapies. findings from these studies have not yet resulted in the development of approved therapies for rv infections, cold symptoms, or exacerbations of respiratory diseases associated with rv infection. however, the wealth of knowledge derived from experimental rv infections has broadened our understanding and identified many potential therapeutic 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exacerbations of asthma con: mice are not a good model of human airway disease lower respiratory tract structure of laboratory animals and humans: dosimetry implications interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model interleukin-5 and eosinophils induce airway damage and bronchial hyperreactivity during allergic airway inflammation in balb/ c mice effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response mepolizumab for prednisonedependent asthma with sputum eosinophilia mepolizumab for severe eosinophilic asthma (dream): a multicentre, double-blind, placebo-controlled trial mutations in vp1 and 3a proteins improve binding and replication of rhinovirus c15 in hela-e8 cells temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells key: cord-312438-zr9zx7pv authors: hoo, regina; nakimuli, annettee; vento-tormo, roser title: innate immune mechanisms to protect against infection at the human decidual-placental interface date: 2020-09-10 journal: front immunol doi: 10.3389/fimmu.2020.02070 sha: doc_id: 312438 cord_uid: zr9zx7pv during pregnancy, the placenta forms the anatomical barrier between the mother and developing fetus. infectious agents can potentially breach the placental barrier resulting in pathogenic transmission from mother to fetus. innate immune responses, orchestrated by maternal and fetal cells at the decidual-placental interface, are the first line of defense to avoid vertical transmission. here, we outline the anatomy of the human placenta and uterine lining, the decidua, and discuss the potential capacity of pathogen pattern recognition and other host defense strategies present in the innate immune cells at the placental-decidual interface. we consider major congenital infections that access the placenta from hematogenous or decidual route. finally, we highlight the challenges in studying human placental responses to pathogens and vertical transmission using current experimental models and identify gaps in knowledge that need to be addressed. we further propose novel experimental strategies to address such limitations. the human placenta is the temporary extra-embryonic organ that is present only during pregnancy and is the anatomical boundary between the mother and fetus. it has a range of functions including transport of nutrients and gases, and hormonal production (1) . the placenta forms a physical, selective barrier between the maternal and fetal circulations, preventing transfer of pathogens. the uterine mucosal lining, the endometrium, is transformed into the decidua during early pregnancy (2) . a range of innate immune mechanisms can respond to pathogens in both the decidua and the placenta (3, 4) . the maternal-fetal interface is a protective barrier against pathogens, but some pathogens can transfer from the mother to fetus by different routes and cause fetal infection (3, 4) . vertical transmission during pregnancy can occur on distinct boundaries between the mother and the fetus: (i) the intervillous space (ivs), where placental villi is in direct contact with the maternal blood, (ii) the implantation site or decidua basalis, where maternal cells are in direct contact with the invading fetal trophoblast, and (iii) the fetal membranes, which are in direct contact with the uterine cavity (5) . defense mechanisms in the cervix, such as the production of mucus and antimicrobial peptides (amp), limit ascending infection from pathogens present in the lower genital tract, that otherwise may access the uterine cavity (6) . however, some pathogens can escape antimicrobial strategies at the cervix and ascend to the uterus, where they can bypass the fetal membranes and lead to the inflammation of the membranes-also known as chorioamnionitis-and infection of the amniotic fluid (7, 8) . pathologic and immune features of chorioamnionitis and intra-amniotic infection are generally associated with bacterial invasion and inflammation [refer to (8, 9) for a comprehensive review on these mechanisms]. here, we focus on infections and innate immune mechanisms at the uterine-placental interfacecases (i) and (ii) (figure 1) . infections at the uterine-placental interface are commonly associated with viruses, parasites and few bacteria ( table 1) . viral pathogens such as human cytomegalovirus (hcmv), zika (zikv), and rubella virus are the most common vertically transmitted pathogens through the decidual-placental interface ( table 1 ) (26, 27) . non-viral pathogens, such as toxoplasma gondii and listeria monocytogenes, can cross the placental barrier via cell-to-cell transmission ( table 1 ) (28, 29) . fetal infection can result in various forms of congenital anomalies in humans ( table 1) . understanding the pathogenic mechanisms used by infectious agents is central to preventing vertical transmission and controlling infection during pregnancy. how the innate immune cells and mechanisms in the placenta and the uterus recognize and respond to protect both the fetus and mother remains controversial due to technical and ethical constraints. however, there are several different models currently used to interrogate the uterine-placental interface in pregnancy. firstly, mice are frequently used as a pregnancy model for infection. although the murine models have provided important insights into the pathogenesis of various infection agents in the context of pregnancy, there are still limitations with this approach. the anatomy of placentation, length of gestation, and use of inbred strains, make extrapolation to humans problematic (30, 31) . secondly, a range of human trophoblast and choriocarcinoma cell lines are used as in vitro models for infection with pathogens. in contrast to the first trimester trophoblast in vivo, these cell lines do not recapitulate normal human trophoblast characteristics such as expression of the human leukocyte antigen (hla) class i and methylation of elf5 (32, 33) . thirdly, human primary placental explants are frequently used. the syncytium dies rapidly in these cultures and it is virtually impossible to standardize the types of villi sampled (30) . therefore, these in vitro experimental factors should be taken into careful consideration when interpreting studies of infection of trophoblast. in this review, we cover the innate immune features of the decidual-placental interface throughout gestation. we identify the gaps in knowledge and highlight the limitations of current studies and experimental models. finally, we discuss novel experimental strategies for understanding how infection affects pregnancy in humans. the trophoblasts of the placenta are the barrier between fetal and maternal tissues. they are derived from the trophectoderm, the outer layer of the blastocyst that forms an inner mononuclear layer with an outer primary syncytium following implantation (34) . the trophoblast in contact with the maternal cells can be: (i) syncytiotrophoblast (sct), a single layer multinucleated, syncytial layer formed by fusion of the underlying villous cytotrophoblast (vct), and (ii) extravillous trophoblast (evt), that invade from the cytotrophoblast shell and anchoring villi into the transformed maternal endometrium, the decidua (2) . the function of evt is to transform the uterine spiral arteries so that maternal blood is delivered to the intervillous space at low pressure. the arteries are surrounded by interstitial evt that destroys the smooth muscle cells of the arterial media, known as "fibrinoid" change (35, 36) . subsequently, endovascular evt (eevt) moves down the spiral arteries from the placentadecidua boundary (35) . these eevt form a plug of cells, limiting surges of arterial blood from damaging the delicate villi. evt invasion transforms the arteries to support optimal regulation of blood flow into the placenta during fetal development (36) . the plugs dissipate between 8 and 10 weeks of gestation when the full hemochorial circulation is established (37) . maternal blood then flows into the ivs, and establishes direct contact with the sct allowing for proper nutrient and gas exchange between the mother and the fetus. hofbauer (hb) cells are fetal macrophages of the human placenta (38) . hb cells can be detected in the placental villous stroma as early as 3 weeks post-conception and are present throughout pregnancy (1, 39) . they are likely to have a variety of functions including control of villous remodeling and differentiation, hormonal secretion, and trophoblast turnover (1, 40) . several lines of evidence have led to the postulation that hb cells may have a role in infection during pregnancy. hb cells with zikv viral particles detected intracellularly have been shown (41, 42). human immunodeficiency virus 1 (hiv-1) has also been detected in hb cells from first trimester infected placenta (43) . whether the hb cells can serve as a reservoir or limit virus replication is still unknown. isolated hb cells from healthy term placenta show elevation of pro-inflammatory cytokines such as il-6, mcp-1, ip-10, and ifn-α upon in vitro infection with zikv (44) . hb cells from the first trimester placenta are also permissive for zikv infection and replication (23) . however, this must be interpreted with caution because in vitro culture of hb cells do not entirely recapitulate the complexity of villous stromal microenvironment, such as presence of hormone and growth factors, all of which will influence the function and activity of hb cells (45) . the sct is the barrier between maternal blood and the placental core as it separates the ivs from the underlying fetal villous stroma. blood-borne pathogens such as viruses and parasites can potentially be transmitted through the sct barrier (figure 1) . how can pathogens cross the sct barrier and the vct to infect the villous stroma? although the sct is an efficient barrier due to its stiff, highly dense actin cytoskeleton network and continuous membrane (46), the syncytium undergoes continuous breaks or gaps and dynamic repair processes (47) . breaks in the syncytium could potentially lead to transmission of pathogens into the underlying vct. our recent work showed that a novel population of maternal macrophages (m3) is associated with the sct in early pregnancy and might be involved in repairing the breaks in the syncytium (10). it is intriguing that m3 macrophages infected with intracellular pathogens could possibly gain access to the underlying vct via the syncytial breaks (figure 2) . only a few viral entry receptors on the sct are described. notably, the sct lacks expression of zikv entry receptors, axl, and tyro3 (48) and the hcmv entry co-receptor integrin α/β (49) . this is further supported by the transcriptomic expression of viral receptors in placental cells (10, 50, 51). expression of surface receptors commonly used by zikv such as axl and hcmv such as nrp2 and pdgfra are lowly expressed by the sct (50) . in addition, there is minimal co-expression of ace2, the receptor gene for human severe acute respiratory syndrome coronavirus 2 (sars-cov-2), and tmprss2, the viral spike protein serine protease gene (50, 52) . in line with this, there is no conclusive and direct evidence of vertical transmission of sars-cov-2 in a placenta from a healthy individual. there are some reports showing sars-cov-2 is predominantly localized at the sct of the second trimester placenta (53, 54) and can lead to severe inflammatory infiltrate in the ivs (55) . however, these findings are presented in a very small number of patients with severe disease or pre-existing pregnancy complications (54, 55) . alternative transplacental mechanisms have been postulated at the syncytial barrier. neonatal fc receptor (fcrn) is expressed on the apical surface of the sct and functions to selectively figure 2 | toll-like receptors and potential inflammatory response at the sct-blood interface. predominant tlrs found in the human placenta from early and term pregnancies. tlr2 and tlr4 are expressed in human placenta sct, vct, and in hb cells. infiltration of infected maternal blood, infected immune cells, or release of pathogenic determinant such lipopolysaccharide (lps), peptidoglycan, or parasite materials such as hemozoin or gpi (glycosylphosphatidylinositol) into the ivs will activate tlr-mediated signaling, leading to the production of a wide range of cytokines and chemokines. severe infection is characterized by massive immune cell infiltration including monocytes and neutrophils from systemic circulation and overproduction of inflammatory cytokines upon tlr activation. this may lead to sct inflammation and damage. sct also secretes antimicrobial peptides as innate immune mechanisms. figure is created by biorender.com. frontiers in immunology | www.frontiersin.org transport maternal igg (56) . fcrn could be exploited by certain viruses to enter the placenta including zikv, hiv-1, and hcmv (19, 57, 58) . transferrin receptor 1 (tfr1) is expressed on the apical end of the sct, and functions as the primary iron transporter into the basal side of the sct to provide sufficient iron stores into fetal circulation (59) . tfr1 has been associated with viral entry into a broad host cell range, including hepatitis c virus (60, 61) suggesting a possible mechanism of viral transport across the sct barrier. some pathogens, although unable to cross the sct barrier, can still adhere to the syncytium and cause further pathology. for instance, plasmodium falciparum infected red blood cells can bind with high affinity to chondroitin sulfate a expressed on the sct, resulting in local inflammation, syncytial breaks, and damage (62) (63) (64) . although the sct is an effective barrier to most pathogens, local inflammation, tissue damage, and fcrn or tfr1-mediated viral entry at the sct can potentially allow pathogen to breach the syncytial barrier, giving opportunity for transmission from maternal blood into placental villi (figure 2 ). during the first trimester of pregnancy, fetal evt invades deeply into the uterus. the decidua basalis, the region located at the implantation site, is populated at this time by a distinctive subset of innate lymphocytes, decidual natural killer cells (dnk), which constitute up to 70% of leukocytes. we have identified three major populations of dnk by single-cell rna-sequencing with unique phenotypes and functions in early pregnancy (10). in addition, there are populations of decidual macrophages (dms) (∼20%), conventional dendritic cells (dcs) and small proportions of t cells (∼10-15%), whereas b cells, plasma cells, mast cells, and granulocytes are virtually absent (10) (figure 1) . the proportion of immune cells will vary throughout pregnancy, with an increase in the proportion of t cells at term (51) . systemic infections will reach all organs including the decidua. whether pathogens can also access the decidua via the cervix is still unclear. chlamydia trachomatis, a common sexuallytransmitted intracellular bacteria, was detected in glandular epithelial cells and unidentified decidual cells in decidual biopsies (11) . this suggests the possibility of infections ascending and spreading from cell-to-cell from the lower genital tract into endometrial glands and vascular endothelium. the decidua basalis is in close contact with fetal cells and the maternal vasculature (figure 1) . first trimester dms and decidual stromal cells are susceptible to zikv infection and replication ex vivo (23) . hence, infection could possibly spread from infected maternal immune and non-immune cells at the decidua, into uninfected vct in the columns of the anchoring villi, and finally into the fetal compartment. however, this is likely to be limited to certain microorganisms which are capable of cell-to-cell spread, have an intracellular host niche, and are able to escape host innate defense mechanisms (table 1) . hcmv, the most common cause of congenital infection, is mostly reported to infect from the decidua (11, 65) . women with primary hcmv infection and first pregnancy are more likely to transmit the virus to their fetus, compared to multiparous women with previous infection and demonstrable antibodies (66) (67) (68) . low affinity maternal antibodies against hcmv correlate with higher viral loads detected in the decidua, whereas patients with intermediate to high neutralizing antibodies have minimal viral replication (65) , suggesting that maternal immunity against hcmv reduces risk of vertical transmission. hcmv protein was also detected in a range of cells within the decidua including endothelial, decidual stromal cells, dcs and macrophages (11, 65) , suggesting that that infected maternal leukocytes could initiate transmission through contact and infection of endothelial cells that line decidual blood vessels. despite the evidence of decidual infection, the mechanism of vertical transmission for hcmv is still in debate. dnks have been proposed to play a protective role against hcmv infection through several mechanisms including modulation of their cytotoxic effector function (69) and the interactions between the killer-cell immunoglobulin receptors (kirs) expressed by dnk and hla molecules expressed in the infected cells (70, 71) . activating kir2ds1 by dnks has been demonstrated to be more cytolytic against hla-c2 hcmv-infected maternal decidual stromal cells (70) . similar cytotoxic response was also observed when peripheral blood nk cells expressing kir2ds1 were exposed to hcmv-infected fibroblasts (71) . hence, this implies that in the decidua, dnks are capable of eliminating harmful infection depending on the combination of kir/hla interactions between dnk and infected cells. dnks are also able to control hiv-1 infection in vitro through production of ifn-γ (72) . the role of dnk in controlling viral infection may protect against potential risk of vertical transmission from the decidua. pattern recognition receptors (prr) are encoded in the germ-line and recognize specific, conserved pathogen-associated molecular patterns (pamps). these include gram-negative bacteria lipopolysaccharide (lps), gram-positive bacteria lipoteichoic acids, lipoprotein, dna, rna, glucans, and peptidoglycans (73, 74). pathogen recognition is not only an essential component of the innate immune response against infection, but also plays an important role in bridging the innate and adaptive systems by toll-like receptors (tlr) activation of antigen presenting cells by up-regulation of major histocompatibility complex (mhc) and co-stimulatory molecules (75) . tlrs, the most studied family of prr, are type i transmembrane proteins with large extracellular domains containing leucine-rich repeats that are expressed at the cell surface or intracellularly (76) . each tlr recognizes distinct pamps, leading to the activation of the transcription factor nf-κb and/or the interferon-regulatory factor (irf) family, and the production of a wide range of cytokines and chemokines, including type i ifns (76, 77) expression of tlrs is dynamic and changes in response to different pathogens and cytokines (74) . tlr2 (which recognizes bacterial proteoglycan) and tlr4 (which recognizes bacterial lps) are the most well-studied, with immunohistochemical evidence of expression in healthy primary sct at term (78) (79) (80) . in contrast, in the first trimester, tlr2 and tlr4 proteins are expressed in vct and evt, but minimally in sct (81, 82) (figure 3) . there is therefore variation in tlr2 and tlr4 expression in the different trophoblast lineages across pregnancy. why and how such dynamic regulation of tlr expression occurs during gestation requires further investigation in a broader range of human placental samples (different donors, gestation stages, genetic background, sampling regions). it is likely that alteration in cytokines profiles in the microenvironment as pregnancy progresses (83) may result in the variation in the expression of tlrs in the placenta. current evidence is only limited to in vitro tlr2/4 stimulation studies using placental explants and primary first trimester trophoblast cells, which drives the expression of figure 3 | toll-like receptors and potential inflammatory response at the decidua. predominant tlrs found in the human placenta from early and term pregnancies. tlr2 and tlr4 are expressed in evt. dm and dnk also express a wide range of tlr families, where stimulation of tlr agonists lead to the production of a variety of cytokines and chemokines. infiltration of infected cells and release of pamps in the decidua, which will activate tlr-mediated signaling. overproduction of inflammatory cytokines at the decidua may lead to local inflammation. figure is created by biorender.com. pro-inflammatory cytokines il-6, il-8, tnf-α, and ifn-γ (78, 80, 81) . tlr2 and tlr4 proteins are expressed in hb cells, confirmed by co-expression of cd68 in healthy term placentas (78) . in early pregnancy, our findings indicate that only tlr4 but not tlr2 transcripts are expressed in steady-state hb cells (10) (figure 4) . enhancement of il-6 and il-8 secretion upon stimulation of isolated first trimester hb cells with tlr4 agonist, lps (84), does suggest a role for tlrs on hb cells in bacterial recognition and placental inflammation during early pregnancy. hb cells are postulated to have a role in viral replication (41, 42), however evidence on the expression and function of viral nucleic acid sensing receptors tlr3, tlr7, tlr8, and tlr9 in hb cells is lacking. our findings show that tlr7, which recognizes viral single-strand rna (ssrna) (85) is expressed in steady-state hb cells (figure 4) (10) . other tlrs have also been shown to be expressed in decidua cells. dms and dnks isolated from first trimester pregnancies show steady state level expression of tlr1-9 transcripts and respond to a broad range of pamps, including heat-killed bacteria, microbial membranes, and nucleic acids (86) . stimulating primary dms with these pamps produces high levels of tnf-α, il-1β, il-6, il-8, il-12, il-10, and il-1ra, whereas dnks secrete il-6, il-8, and ifn-γ (86) . this study suggests that, in addition to the physiological roles of dms and dnks in accommodating the uterus for placentation, dms and dnks may play a role in pathogen recognition and antimicrobial response via activation of tlr signaling (figure 3) . the extent to which subsets of dms or dnks population (10) are critical for tlr-mediated response at the decidua is currently unknown. in malaria endemic populations, single nucleotide polymorphisms (snps) within the tlr4 coding and tlr9 promoter regions are associated with variation in disease severity and parasitemia control (87, 88) . in the case of pregnancy malaria, primiparous infected mothers with common tlr4 and tlr9 polymorphic variants are correlated with severe complications such as low birth weight and maternal anemia (89) . this highlights the importance of studies involving large cohorts of individuals which include genotyping from pregnant mothers living in malaria endemic regions (see section on "challenges and future perspective"). animal models have also been used to study the functional role of tlr signaling, particularly for pathogens that are intracellular at some stage of their life cycle ( table 1) . tlr4 and tlr9 are strongly activated by malaria parasite pamps such as glycosylphosphatidylinositol (gpi), dna, and hemozoin (90, 91) (figure 2) . in a mouse model of placental malaria, tlr4, and myd88 signaling activation resulted in placental expression of pro-inflammatory markers, such as il-6 and tnf-α (92, 93) . these studies also demonstrated that malaria parasite infection and inflammation in the mouse placenta lead to reduced fetus growth rate and disorganization of the vascular space in the placenta (92, 93) . however, tlr-mediated inflammation and pathology in the human placenta upon malaria infection is unknown and remains to be further investigated. studies of congenital toxoplasmosis are also currently limited to animal models. tlr2 and tlr4 are associated with recognition of t. gondii's infection in mice (94) . engagement of the t. gondii ligand by tlr2 and tlr4 at the sct-blood or in the evt-decidua compartments is plausible, although there is still no direct evidence for such host-parasite interaction in humans. tlr11 has a role in controlling t. gondii infection in mice (95, 96) , however in humans tlr11 is a pseudogene and is not expressed (97) . cytosolic prrs play an important role in fighting against viral infection by eliciting host type i interferons (ifn) antiviral response through recognition of single and double stranded rna (ssrna and dsrna) (98, 99) . examples of prrs are the cytosolic retinoic acid-inducible gene-i-like (rig-i) and the melanoma differentiation-associated protein 5 (mda5) receptors, both expressed in the sct and vct of term placenta (100) . in the human placenta, there is limited information on the function of rig-1 and mda5, but they may play a crucial role in recognizing a variety of rna viruses, including zikv and dengue virus (101) . the nucleotide binding oligomerization domain-like receptors (nod-like receptors; nlr) recognizes intracellular pathogen products which have entered into the host cytoplasmic compartment (74) . both nod-1 and nod-2 receptors, which are known to detect intracellular bacterial peptidoglycans (102) , are expressed in the sct in the first trimester and term placentas (103, 104) . the nlr pyrin-containing 1 and 3 proteins (nlrp1 and nlrp3) form the major inflammasome complexes, which contribute to activation of inflammatory caspases and pathogen clearance (105, 106) . activation of nlrp3 and aim2 inflammasomes, together with high expression of il-1r, il-1β, and caspase-1 was recently shown in the placental tissue of mothers infected with p. falciparum with significant pathology (107) . in a murine model of intra-amniotic inflammation induced by bacterial lps, tissue sections from the decidua basalis region expressed high levels of nlrp3, but negligible caspase-1 activation suggesting a possible non-canonical activation of the nlrp3 inflammasome (108) . our analysis shows that decidual dm1 expresses high levels of nlrp3 transcript at steady state compared to other cell types (10) (figure 4) , thus dm1 may play a role in nlrp3-mediated pathogen recognition during early pregnancy. amp secreted by epithelial and immune cells are small peptides that bind and destroy most groups of pathogens-bacteria, yeasts, fungi, and viruses (109) . in addition to direct killing of pathogens, amps can rapidly modulate innate host immune responses by recruiting myeloid cells and lymphocytes to the site of infection and mediating activation of tlr (110, 111) . the human placenta expresses high levels of β-defensins, a family of broad spectrum antimicrobial peptides which participate in direct bactericidal and anti-viral activity (112) . specific subtypes of β-defensins (hbd-1, 2, and 3) are expressed in scts (112) , suggesting these amps can target potentially bacterial or viral infection from the maternal blood. recognition of pamps by prrs during infection leads to production of pro-inflammatory cytokines that can aid in clearing the pathogen (74) . studies on the direct role of proinflammatory cytokines on the placenta in the case of infection is limited. inflammatory mediators can directly influence infection outcome and fetal development, but they can also cause damage to the placenta if produced in excess (113). amongst the proinflammatory cytokines associated with uterine-placental infection during pregnancy, the antiviral ifn are the most well-characterized. ifns are secreted by a variety of cell types as the first line of defense against viral infection (114) . type i ifns, including ifn-α and ifn-β, are potent antiviral cytokines. ifn-α and ifn-β bind to the ifnar1/2 receptor and lead to expression of ifn stimulated genes (isgs), which control virus infection through a variety of mechanisms (114) . loss of ifnar in the placenta leads to vertical transmission and fetal mortality in murine herpesvirus-68 (mhv68) infected mice (115) . in the mouse model of zikv infection, type i ifn-mediated signaling is essential for the control of viral replication in the placenta, but can also lead to significant placental pathology and fetal mortality (116, 117) . the mechanism of type i ifn-mediated placental pathology has been recently elucidated. ifn-induced transmembrane (ifitm) protein, which normally blocks viral entry into host cells, impairs syncytin-mediated fusion of vct to form sct, leading to aberrant placental development (118) . type ii ifn, ifnγ, predominantly produced by nk and cd4+ t cells is crucial in controlling parasitic infection, such as t. gondii in mice (94, 119) . however, elevated levels of ifnγ in response to t. gondii infection can lead to pathological effects during pregnancy including fetal demise (119, 120) . severe placental pathology and fetal death have also been associated with elevation of ifnγ during pregnancy in a murine model of malaria (121). hence, proper regulation of type i and ii ifn-mediated signaling at the uterine-placental interface is crucial in limiting pathogen replication, whilst preserving a balanced environment for normal placental development (122) . type iii ifn, ifnλ, are constitutively secreted by the human sct, which presumably confers antiviral effects against zikv infection (123) (124) (125) . tryptophan metabolism by ido indoleamine 2,3-dioxygenase (ido) is a host intracellular enzyme which metabolizes the amino acid tryptophan (126) . ido has been associated with maternal immunoregulation during pregnancy (127) . it also plays a key role in the control of bacterial and viral replication, through limiting the bioavailability of tryptophan (128) . ido also inhibits the replication of several parasitic pathogens including t. gondii in human fibroblasts (129) and leishmania spp in human macrophages (130) . mouse infection with l. monocytogenes showed that ido is elevated in an ifn-γ-dependent manner in stromal cells of the metrial gland and decidua basalis; a crucial process to resolve bacterial infection in the mouse placenta (131) . our findings also show ido1 expression is enriched in epithelial glandular and dc1 cell type in the first trimester decidua (10) (figure 4) . the presence of ido in decidua suggests that the enzyme might have a central role in limiting parasitic, viral, and bacterial replication, thus preventing their spread to the fetus. research on how the human placenta safeguards itself against infections is challenging due to obvious logistical and ethical issues in obtaining tissue from early in gestation (box 1). although animal experimental models have provided important insights relating to the immune responses to pathogenic infection, major differences between human and animal placentas must be considered (30, 31) . likewise, differences between strains of pathogens adapted for mice compared with human clinical isolates should be taken into account as this may lead to variation in pathogenesis and cellular response. one such example is the use of mouse cmv, which is unable to cross the mouse placental barrier, unlike the hmvc counterpart which can be transmitted transplacentally in humans (132) . therefore, all data obtained from studies of infection in pregnant animals needs careful interpretation and consideration prior to translation to clinical infection in humans. inherent properties of trophoblast cell lines, primary cultures or explants vary between donors, and are likely to be confounded by the area of the placenta that is sampled and as well as stage of gestation (133) . for instance, villous placental explants will vary depending on the types of villi sampled and the presence of box 1 | perspective of vertical transmission and innate immune function during pregnancy and infection. a variety of maternal infections can lead to vertical transmission ( table 1) . the exact mechanisms these pathogens use to escape host defense and cross the placental barrier into the fetal compartment are not entirely known. experimental models that recapitulate infection of the human placenta and thus vertical transmission are challenging to set up. more data and representative experimental models are needed to answer these questions: (i) how do different pathogens escape or modulate the maternal-fetal host innate immune barrier (ii) why do some pathogens lead to congenital infection but not others? studying infected human placentas will be essential in understanding this but access to these samples is difficult especially in low and middle-income countries (lmic) where maternal infection is particularly prevalent (who, maternal mortality index 2019). despite evidence of expression in primary placental tissue, functional studies on important innate immune features such as tlrs, amps, rig-i, mda5, nlrs, and ido during infection and pregnancy are lacking. understanding how different cell types at the uterine-placental interface (hb cells, dnks, and dms) respond to pathogen challenge is essential, but remains under-researched. a critical obstacle is to also extrapolate the protective and pathological mechanisms of cytokines from mouse to human infection. therefore, systematic comparison of the innate immune effector mechanisms across gestation, in the placenta and decidua from natural human infection vs. healthy pregnancy, will provide a more accurate representation in clinical settings. attached decidual tissue (133) . caution is therefore needed when interpreting data using these experimental models. to overcome such limitations, population-based cohort studies of women with infection during pregnancy with extensive tissue sampling should be performed. these need to include and focus on lmic where infection is still a major cause of maternal and fetal mortality and morbidity. cohort studies and epidemiological surveillance on maternal infections can offer significant insights into disease pathogenesis and accelerate clinical interventions (134) . collaborations between clinicians and researchers for population-based cohort collection and sample processing will be instrumental to achieving this goal. biological samples such as blood or placenta collected from controls and infected pregnant individuals could be stored and cryopreserved retrospectively. to capture the overall heterogeneity of infected and non-infected placenta samples, sampling, and biobanking criteria of different regions of placenta should be considered (135) . protocols are now available to use frozen tissue processed for single-cell/nuclei and spatial genomics (136, 137) . hence, application of single-cell "omics" on infected vs. healthy human placental and decidual samples will enable us to evaluate cellular heterogeneity in response to infection. the capacity to detect transcripts specific to host or pathogen mrna from the same tissue using in situ nucleic acid hybridization methods will provide direct quantification of infection burden and identification of potential target host cells within the same tissue (138) . recent advances in spatial transcriptomics methods have also allowed gene expression signatures to be quantified and resolved from individual tissue sections (139) . combination of these emerging technologies with new methods to integrate single-cell and spatial data computationally (140) will provide an unbiased approach to characterize and profile the transcriptome of individual cells in situ from the placenta and decidua in response to infections. we anticipate that high-throughput datasets generated from cohort sampling studies will unravel novel cell states and tissue spatial localization associated with placental infections and inflammation. this will also allow us to better characterize not only the innate immune response or makers of infection, but also other adaptive immune states in the human placenta (box 1). the use of in vitro models will also further define host responses to infection. the recent generation of human trophoblast stem cells (htscs) (141) and three-dimensional (3d) trophoblast organoids (142, 143) offer a great opportunity to study infections in early pregnancy where the access to first trimester placental samples is a concern. more importantly, the htscs and trophoblast organoids fulfill the criteria characteristic of human first trimester trophoblast in vivo (32) . both htscs and trophoblast organoids can differentiate in vitro into sct and evt with appropriate media (142, 143) allowing infection experiments on both the major trophoblast subpopulations present at the two major sites of contact between maternal and fetal cells. sequencing of both host and pathogen transcriptomes from infected trophoblast at single-cell resolution will also advance our understanding on host-pathogen interactions in placentas (144, 145) . further refinement of the trophoblast organoid and htscs culture system is needed to address key biological questions unanswered by current models. these include studying the effect of infection on cellular crosstalk between trophoblast and other primary placental cells such as hb cells, or decidual cells in culture, such as dnk or decidual stromal cells. adaptation of crispr/cas9 genome editing technology for the trophoblast organoids or htscs will offer novel insights into essential host genes required for vertical transmission and placental defense mechanisms in humans. major maternal and fetal complications as a result of infection are still a concern, especially in lmic with highest prevalence reported in countries of sub-saharan africa (who, maternal mortality index 2019). profound limitations on current study models and ethical regulations on studying human placenta have significantly delayed the development of therapies and vaccines for maternal-fetal infection. how vertical transmission occurs and how the uterine-placental innate immune system reacts to infection remain as major unresolved questions. revolutionary advances in single-cell genomics, imaging, computational, and stem cell biology methods are currently underway to study the molecular and cellular mechanisms of human diseases. therefore, it is now an exciting time to apply these transformative technologies to comprehensively address fundamental questions on host-pathogen interaction at the human uterine-placental interface. rh, an, and rv-t wrote and edited the manuscript. all authors contributed to the article and approved the submitted version. rh and rv-t were 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situ rna analysis platform for formalin-fixed, paraffin-embedded tissues visualization and analysis of gene expression in tissue sections by spatial transcriptomics spatial mapping of cell types by integration of transcriptomics data derivation of human trophoblast stem cells trophoblast organoids as a model for maternalfetal interactions during human placentation self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta scdual-seq: mapping the gene regulatory program of salmonella infection by host and pathogen single-cell rna-sequencing we would like to thank ashely moffett for the useful discussions and critical review of the manuscript. we are also very grateful to sarah aldridge, loren gibson, damiana alvarez, carlos talavera-lopez, and anna arutyunyan for their insightful comments and corrections. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 hoo, nakimuli and vento-tormo. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-322380-udjoghr6 authors: nash, anthony a.; dalziel, robert g.; fitzgerald, j. ross title: early stages of infection after pathogen entry date: 2015-02-06 journal: mims' pathogenesis of infectious disease doi: 10.1016/b978-0-12-397188-3.00003-2 sha: doc_id: 322380 cord_uid: udjoghr6 in this chapter, we describe the early stages of the pathogenic cycle involving entry into the host. for example, some bacteria may enter into epithelial cells, proliferate and then spread at the epithelial surface before being transmitted to another host. some pathogens then migrate past the epithelial layer to deeper tissues causing invasive infections. examples of some of the major bacterial, viral and fungal pathogens affecting the epithelial surfaces in the body are provided. the body produces a strong inflammatory immune response to pathogens at the epithelial surface. an introduction to the components of the inflammatory response including the phagocytic cells and the lymphatic system is provided. in addition, the nutritional requirements for invading bacteria including the importance of iron is discussed. molecular weight proteins, coded for by the cell, and formed in response to infection with nearly all viruses (see chapter 9). the interferon formed by the infected cell is released and can act on neighbouring or distant cells, protecting them from infection. freshly formed virus particles from the first infected epithelial cell enter the fluids bathing the epithelial surface and are borne away to initiate fresh foci of infection at more distant sites. interferons too can reach these sites, and as more and more interferon is formed on the epithelial sheet, more and more cells are protected, so that the infectious process is slowed and finally halted. other antiviral factors probably play a part, and the adaptive immune response itself comes into action in the final stages. interferons are produced a few hours after infection of the first epithelial cell at a site where they are needed and without the delay characteristic of the immune response. the immune response provides resistance to subsequent re-infection, but it does not appear to be of primary importance in recovery from respiratory infections of this type. the spread of infection is very rapid on epithelial surfaces that are covered with a layer of liquid because of the ease with which the microorganism in the fluid film encounters cells and is disseminated over the surface. this is true for the respiratory infections mentioned above, and also for infections of intestinal epithelium, such as those caused by the human diarrhoea viruses, e.g. rotavirus. the argument does not apply, however, to local infections of the skin. in this case, where the microorganism is not carried across the epithelial surface in a liquid film to establish fresh foci of infection, the whole process takes a this bacterium commonly infects the stratum corneum and causes erythrasma, a scaly condition of the axilla, groyne and between toes. much longer time. papilloma (wart) viruses, for instance, cause infection in a discrete focus of epidermal cells; indeed a wart consists of a clone of cells produced by the division of a single initially infected cell. the inevitably slow evolution of single virus-rich lesions should mean that immune responses have the opportunity to respond to and limit the infection. papillomaviruses, however, escape the attention of the immune system. in the basal layer of the epidermis, adjacent to the antibodies and immune cells that arrive from dermal blood vessels, the virus infection is incomplete; in this layer of the epidermis, only a subset of virus genes are transcribed, no virus structural proteins are produced and no virus dna replication occurs, therefore no virus particles are produced. some of the proteins which are produced are involved in driving the cell into proliferation or differentiation, others act to downregulate the ifn system and mhc presentation. the infected basal cell is therefore not recognised and not a target for the immune response. as the cells move further away from these immune forces, approaching the epidermal surface and becoming keratinised, more and more virus is produced for liberation to the exterior. neither antibodies nor immune cells are present on this dry surface to influence virus multiplication and shedding. the respiratory viruses described above have a hit and run type of infection of epithelial cells, and are very successful parasites. a number of other viruses, including measles and chickenpox, infect inconspicuously via the respiratory tract, then spread systemically through the body and only emerge again to cause widespread respiratory infection and shedding to the exterior after a prolonged incubation period. the limitation of rhinoviruses and human coronaviruses to the surface of the upper respiratory tract is at least partly determined by their optimum growth temperature. many of them replicate successfully at 33 c, the temperature of nasal epithelium, but not very well at the general body temperature, 37 c. thus, they do not spread systemically nor to the lung. it is also likely that a lack of expression of the virus receptor involved in entry of the virus to the cell plays a major role in determining spread or lack thereof. viruses of the influenza and parainfluenza groups can infect the lung as well as the nasal mucosa, but they are generally limited to the epithelial surfaces. the limitation is not absolute. occasionally in adults and more often in infants, influenza and parainfluenza viruses spread to infect the heart, striated muscle or the central nervous system. the spread of infection from epithelial surfaces is also controlled by the site of virus maturation from cells. influenza and parainfluenza viruses are liberated (by budding) only from the free (external) surface of epithelial cells, as is appropriate for infection limited to surface epithelium. a similar restriction in the topography of budding is seen with rabies virus in the infected salivary gland. however, vesicular stomatitis virus is released only from the basal surface of the epithelial cell, from whence it can spread to subepithelial tissues and then through the body; topographical restriction in the site of virus release from epithelial cells reflects the polarisation of function in these cells, which in turn depends on the maintenance of tight junctions between them. many bacterial infections are largely confined to epithelial surfaces (table 3 .1). this is a feature, for instance, in diphtheria and streptococcal infections of the throat, gonococcal infections of the conjunctiva or urethra and most salmonella infections of the intestine. to a large extent this is because host antibacterial forces, to be described at a later stage, do not permit further invasion of tissues. under most circumstances, these bacteria are not able to overcome the host defences, but gonococci and streptococci, at least, often spread locally through tissues and occasionally systemically through the body. for example, group a streptococcus occasionally causes necrotizing fasciitis or 'flesh-eating disease'. gonococci cause a patchy infection of the columnar epithelium of the male urethra, reaching subepithelial tissues 3à4 days after infection; the yellow discharge consists of desquamated epithelial cells, inflammatory exudate, leucocytes and gonococci. subepithelial spread probably takes the infection to other parts of the urethra and to local glands. most gram-negative bacteria have only a very limited ability to invade a given host. in man, e. coli, proteus spp. and pseudomonas aeruginosa are only capable of invasion when defences are impaired or when bacteria are inadvertently introduced into a suitable site in the body (see chapter 2). they cause systemic infection in debilitated, malnourished, or immunosuppressed patients; they produce sepsis in the uterus after abortion, and when they are introduced into the body by intravascular devices or catheters. certain gram-negative bacteria penetrate the intestinal epithelium but get no further, as in shigella dysentery and salmonellosis. one or two highly specialised gram-negative bacteria penetrate intestinal epithelium, enter lymphatics and spread systemically through the body to cause enteric or typhoid fever (salmonella typhi and paratyphi). a few bacteria show a temperature restriction similar to that described above for rhinoviruses, which prevents anything more than local spread. for instance, the lesions in leprosy (mycobacterium leprae) are confined to cooler parts of the body (skin, superficial nerves, nasal mucosa, testicles, etc.). other mycobacteria (mycobacterium ulcerans and m. marinum) occur in water and enter human skin through superficial abrasions, especially in warm countries, and cause chronic skin ulcers. these bacteria, which also infect fish, have an optimum growth temperature of 30à33 c and remain restricted to the skin. fungi of the dermatophyte group (ringworm, athlete's foot 1 ) infect skin, nails and hair, but are restricted to the dead keratinised layers of epithelium. fungal antigens are absorbed from the site of infection and immune (including allergic) responses are generated, which at least partly account for the failure to invade deeper tissues. some of the important microorganisms that regularly establish systemic infections after traversing epithelial surfaces are listed in table 3 .2. there is one important distinction between intracellular and extracellular microorganisms. if an obligate intracellular microbe is to spread systemically from the body surface, it must first enter the blood or lymph. this means gaining access to the lumen of a subepithelial lymphatic or blood vessel, either as a free microorganism, or alternatively after entering a mobile cell (leucocyte) that will carry it to other parts of the body. the microorganism cannot replicate until it reaches a susceptible cell, and the absence or shortage of such cells except at the body surface would prevent or seriously hinder its spread through 1 fungi causing this condition flourish in a moist environment, and athlete's foot is restricted to those who encase their feet in shoes. however, those who do not wear shoes (e.g. tropical africa) are vulnerable to other fungi that enter skin at sites of injury and cause deeper lesions called mycetomas. the body. thus, rotaviruses and rhinoviruses replicate at the epithelial surface but cannot infect leucocytes, and in any case would be unlikely to find susceptible cells elsewhere in the body if they entered blood or lymphatic vessels. certain viruses (yellow fever, poliovirus) spread through the body to reach susceptible target organs (liver, central nervous system) after free virus particles have entered vessels below the skin or intestinal epithelium. measles virus and tubercle bacilli infect leucocytes, which carry them through the body to organs such as the liver, spleen, skin and lung. a remarkable example of an intracellular bacterium which can manipulate host cells to assist in dissemination is m. leprae. m. leprae targets schwann cells which are their primary niche and re-programmes them so that they return to a stem cell-like state. the properties of these cells include plasticity and migration and this facilitates the dissemination of m. leprae within those cells by differentiation and via macrophage release. these findings demonstrate how at least one intracellular bacteria can hijack host cell programming in order to promote bacterial spread within the host. if, on the other hand, the microbe is able to replicate outside cells and does not have to find a susceptible cell, it can in principle multiply locally, in the blood and lymph, and in whatever part of the body it gets to. extracellular replication itself, however, conveys a serious disadvantage, because the microorganism is exposed to all the antimicrobial forces that the body can summon up. indeed, bacteria and other microorganisms that are capable of extracellular replication generally advertise their presence by releasing a variety of products into surrounding fluids, many of which cause inflammation and thus bring antibacterial agents such as immunoglobulins, complement and leucocytes to the site of the infection. lymphatics are also dilated and carry the infecting organisms to lymph nodes for further exposure to antibacterial and immune forces. intracellular microorganisms in contrast, although exposed to the infected cell's own defence mechanisms, are directly exposed to the general bodily defences only during transit from one infected cell to another. however, if the infected cell is recognised as such by the immune defences, it can be destroyed (see chapters 6 and 9). a number of bacteria and protozoa, such as mycobacterium tuberculosis, legionella pneumophila, brucella abortus or leishmania donovani, carry out much of their multiplication in macrophages that have ingested them. although they are not obligate intracellular parasites, this shifts the hostàmicrobe battlefield into the cell. the battle is then waged in the infected macrophage, whose antimicrobial powers (see chapter 4) and participation in immune defences (see chapters 6 and 9) become of critical importance. the infected host has a variety of defences that operate without delay, before the immune response comes into action (table 3 .3). these 'early' defences are referred to in this and in subsequent chapters, and they are the type of defences that mattered before the immune system had evolved. many microbes have strategies for interfering with these defences. for example, cells infected with a virus can commit suicide before the virus has completed its growth cycle in the cell. this is called apoptosis and occurs after reovirus, hiv, and other infections. the fact that viruses (e.g. adenoviruses) have developed mechanisms for inhibiting apoptosis indicates that it plays an important part in defence. apoptosis occurs also in bacterial infections. when uropathogenic strains of e. coli infect bladder epithelium, the host responds by apoptosis of the infected cells. the actual value of this response is not clear. after traversing the epithelial cell layer, a microorganism encounters the basement membrane. the basement membrane acts as a filter and can to some extent hold up the infection, but its functional integrity is soon broken by inflammation or epithelial cell damage. the invading microorganism has now reached the subepithelial tissues (figure 3 .1), and here it is exposed to three important host defence systems. these are (i) the tissue fluids, (ii) the lymphatic system leading to the lymph nodes and (iii) phagocytic cells. these three host defence mechanisms are of supreme importance and come into play whatever part of the body is infected, whether the nasal mucosa, meninges, urethra, cardiac muscle or liver lobule. each depends for its action on the inflammatory response, because this response brings the phagocytes and serum factors to the site of infection and promotes drainage from the site by the lymphatic system. therefore a short account of the inflammatory response will be given, and after this, each of the three antimicrobial factors will be considered separately. table 3 .2 shows some of the important microorganisms that regularly spread through the body in spite of these antimicrobial factors. the capillary blood vessels supplying a tissue bring oxygen and low molecular weight materials to the cells, taking away carbon dioxide and metabolic or secretory products. there is also a constant passage of plasma proteins and leucocytes from capillaries into normal tissues, and these are returned to the blood via the lymphatic system after entering lymphatic capillaries. indeed, their presence in tissues is inferred from their presence in lymphatics draining these tissues. the cells are nearly all t lymphocytes, which leave blood capillaries by actively passing through endothelial cells. after moving about and performing any necessary tasks in the tissues, the lymphocytes penetrate lymphatic capillaries and thus enter the lymph. the lymph, with its content of proteins and cells, then passes through the local lymph nodes and generally at least one more lymph node before entering the thoracic lymph duct and being discharged into the great veins in the thorax or abdomen. blood lymphocytes also enter lymph nodes directly and in larger numbers through post-capillary venules. the constant movement of lymphocytes from blood to tissues or lymph nodes, and back via lymphatics to the blood again, is called lymphocyte recirculation. circulating lymphocytes are mostly t cells, and in the course of their continued entries into tissues and lymph nodes they have regular opportunities to encounter any microbial antigens that may be present. there is in fact a regular monitoring of tissues by t lymphocytes, and this is referred to as immune surveillance. the various plasma proteins occur in the tissues in much the same proportion as in plasma, the actual concentrations depending on the structure of the capillary bed. as determined by concentrations in local lymphatics, the leaky sinusoids of the liver let through 80à90% of the plasma proteins into liver tissue, the less leaky capillaries of the intestine admit 40à60% into intestinal tissues and capillaries of skeletal muscle with their continuous lining only 10à30% (figure 3.2) . thus, immunoglobulins, complement components, etc. occur regularly in normal tissues, but in lesser concentrations than in blood. there is some discrimination against very large molecules because the largest immunoglobulins (igm) do not leave the blood vessels and are not detectable in afferent lymph. there is a prompt and vigorous change in the microcirculation when tissues are damaged or infected. capillaries and post-capillary vessels are dilated, gaps appear between endothelial cells, and the permeability of these vessels increases, allowing leakage from the blood of a protein-rich fluid. increased amounts of immunoglobulins, complement components and other proteins are then present in tissues, and fibrinogen, for instance, may be converted into fibrin so that a diffuse network of fibrils is laid down. circulating leucocytes (especially neutrophils and monocytes) adhere to endothelial cells, and this is followed by active passage (diapedesis) of leucocytes between endothelial cells and out into tissues. the affected part now shows the four cardinal signs of inflammation, being red and warm (vasodilation), swollen (vasodilation, cell and fluid exudate) and often painful (distension of tissues, presence of pain mediators). lymphatic capillaries also become dilated, taking up the inflammatory fluids and carrying them to local lymph nodes. there is a greatly increased turnover of plasma components in the inflamed tissue. initially, the predominant cell is the neutrophil, a reflection of the situation in the blood, but neutrophils only live for a day or two in tissues, and as the acute inflammatory state subsides mononuclear cells become more prominent, especially macrophages, which phagocytose dead neutrophils and tissue debris. the initial stages of the inflammatory response tend to be consistent, whatever the nature of the tissue insult, and this is partly because the changes are caused by the same mediators of acute inflammation. these include histamine (released from mast cells lying close to blood vessels), kinins (polypeptides derived from precursors in plasma; see glossary) and products of complement activation by the alternative pathway (c3a and c5a). some of the kinins are highly active and kallidin, for instance, a decapeptide formed from kallidinogen (an α 2 globulin) is about 15 times more active (on a molar basis) than histamine in causing inflammation. most bacteria form inflammatory materials during their growth in tissues, but these are not very potent compared with the activation of c3 and other molecules by carbohydrates (e.g. polysaccharides) present on bacterial surfaces (see figure 6 .6). macrophages, when they are stimulated, release a variety of inflammatory mediators and, in addition, immune-mediated inflammation results from interaction of microbial antigen with antibody (via c3a and c5a) or reaction of antigen with ige antibody on mast cells. the final mediators include molecules such as tnf (tumour necrosis factor), icam-1 (intercellular adhesion molecule-1) and elam-1 (endothelial cell leucocyte adhesion molecule-1). inflammatory responses, like other powerful tissue responses, must be controlled and terminated, and the mediators of inflammation not only have a variety of inhibitors but are also inactivated locally (e.g. kinins inactivated by kininases). at a later stage, prostaglandins (a family of 20-carbon fatty acid molecules) and leukotrienes (a group of biologically active lipids) come into play. 2 they are produced from leucocytes, endothelial cells and platelets, and they both mediate and control the response. if inflammation is due to infection with one of the pyogenic bacteria and the infection continues, then the continued supply of inflammatory and chemotactic products from the multiplying bacteria maintains vasodilation and the flow of neutrophils to the affected area. there is an increase in the number of circulating neutrophils, because of an increase in the rate of release from the bone marrow. the bone marrow holds a vast reserve supply with 20 times as many neutrophils as are present in the blood. if the tissue demand continues, the rate of production in the bone marrow is increased, and circulating neutrophils may remain elevated in persistent bacterial infections such as subacute bacterial endocarditis. neutrophil production in the bone marrow is regulated by certain colonystimulating factors, and it is a serious matter if something goes wrong and the marrow supplies are exhausted. a fall in circulating neutrophils (neutropenia) during a bacterial infection is of ominous significance. 2 the terminology becomes complicated. eicosanoids are produced by metabolism of arachidonic acid and include leukotrienes, prostaglandins, thromboxanes and lipoxins. viruses produce inflammatory products in tissues in the form of necrotic host cell materials or antigenàantibody complexes, but these are less potent than bacterial products, and the acute inflammatory response is of shorter duration, neutrophils being replaced by mononuclear cells. mononuclear infiltrates are also favoured in virus infections because the infected tissues themselves are often one of the sites for the immune response, with mononuclear infiltration and cell division. after extravasation from blood vessels, leucocytes would not automatically move to the exact site of infection. neutrophils show random movement in tissues and also a directional movement (chemotaxis) in response to chemical gradients produced by chemotactic substances. monocytes show little or no random movement, but they too respond to similar chemotactic substances. chemotactic substances such as leukotrienes, c3a and c5a are formed during the inflammatory response itself. also, many bacteria, such as staphylococcus aureus or s. typhi, form chemotactic substances, and thus automatically betray their presence and attract phagocytic cells. it would obviously be an advantage to an infectious agent if no inflammatory or chemotactic products were formed, but for most large microorganisms (bacteria, fungi, protozoa) these products seem an almost inevitable result of microbial growth and metabolism. however, some pathogens have evolved ways of interfering with the chemotactic process by producing substances that block chemotactic receptors. the early stages of the inflammatory response in particular are known to have an important protective effect against microorganisms. in experimental staphylococcal skin infections, for instance, if the early inflammatory response is inhibited by adrenalin, and the early delivery of plasma factors and leucocytes to the site of infection thus reduced, bacteria multiply more rapidly and produce a more severe lesion. perhaps it is not surprising that many bacterial pathogens can suppress the early inflammatory response. for example the chemotactic inhibitory protein of s. aureus (chips) bind to the c5a and fmp receptors on macrophages, blocking the recognition of c5a and formylated peptides. if inflammation becomes more severe or widespread, it is generally modulated by increased output of corticosteroid hormones, but at the same time it is backed up by a general metabolic response in the body. this is called the acute phase response. the liver releases about 30 different proteins, including c-reactive protein and serum amyloid protein, which undergo 1000-fold increases in concentration, as well as mannose-binding protein, haptoglobulins (α 2 -glycoproteins), protease inhibitors and fibrinogen. the exact function of these acute phase proteins is not clear, but they are protective; they fix complement, opsonize and inhibit bacterial proteases. their presence is associated with an increased erythrocyte sedimentation rate. the patient may develop headache, muscle pains, fever and anaemia, with decreased iron and zinc and increased copper and ceruloplasmin in the serum. proteins in muscle are broken down, partly to provide energy required during fever and fasting, and partly to provide amino acids needed by proliferating cells and for the synthesis of immunoglobulins and acute phase proteins. many of the features of the acute phase response appear to be due to the action of interleukin-1 (see glossary), and also il-6 and tnf released from macrophages and lymphocytes. it is a complex response, which on the whole would be expected to serve useful purposes, although some less obviously beneficial 'side effects' may be unavoidable. tissue fluids normally contain variable amounts of plasma proteins, including igg antibodies as discussed above. in the absence of specific antibodies and complement, tissue fluids make a good culture medium for most bacteria, but bacterial multiplication almost inevitably causes some inflammation. powerful inflammatory events are set in motion when molecules on the bacterial surface (e.g. endotoxin) activate the alternative complement pathway. larger amounts of igg as well as activated complement components, will then be present in tissue fluids. at a later stage, secretory products from phagocytes (lysosomal enzymes, oxygen radicals, lactoferrin, etc.) will also be present, and finally tissue breakdown products and additional antimicrobial substances liberated from dead platelets, neutrophils and macrophages. a complex network of lymphatics lies below the epithelium at body surfaces. after reaching subepithelial tissues, foreign particles of all kinds, including microorganisms, rapidly enter lymphatic capillaries after uptake by or passage between lymphatic endothelial cells. there is a particularly rich superficial plexus of lymphatics in the skin and in the intestinal wall. microorganisms scratched or injected into the skin inevitably enter lymphatics almost immediately. the intestinal lymphatics not only take up microorganisms that have breached the epithelial surface but also have an important role in the uptake of fat in the form of chylomicrons. microorganisms in peripheral lymphatics are rapidly moved (within minutes) to the local lymph nodes strategically placed to deal with the flow of lymph before it returns to the blood. the rate of flow of lymph is greatly increased during inflammation, when there is increased exudation of fluid from local blood vessels and the lymphatics are dilated. microorganisms carried to the node in the lymph are exposed to the macrophages lining the marginal sinus (figure 3 .3), and these cells take up particles of all types from the lymph and thus filter it. the efficiency of filtration depends on the nature of the particles, on the physiological state of the macrophages, and also on the particle concentration and flow rate, the efficiency falling off at high particle concentrations or high flow rates (see chapter 5). all infecting microorganisms are handled in the same way and delivered via lymphatics to the local lymph node. when there has already been microbial multiplication at the site of initial infection, very large numbers may be delivered to the node. the efficiency of the node as a defence post depends on its ability to contain and destroy microorganisms rather than allow them to replicate further in the node and spread to the rest of the body. the antimicrobial forces are the macrophages of the node, the neutrophils and serum factors accumulating during inflammation, and the immune response which is initiated in the node. under normal circumstances, as the first trickle of microorganisms reaches the node, the most important event is the encounter with macrophages in the marginal sinus. microorganisms escaping phagocytosis by these cells enter the intermediate sinuses where they run the gauntlet of a further set of macrophages before leaving the node. if there is an inflammatory reaction in the node, a substantial migration of neutrophils into the sinuses greatly increases the phagocytic forces and thus the filtering efficiency. there is usually a further node to be traversed before the lymph is discharged into the venous system. as well as functioning as filters, the lymph nodes, of course, are sites where the immune response comes into play. soon after infection, as inflammatory products of microbial growth arrive in the node, there is some swelling and inflammation. the microbial antigens, some of which are already associated with antigen-presenting cells encountered at the body surface, generate an immune response, and there is further swelling of the node as cells divide and additional lymphoid cells are recruited into the node from the blood. the ability of viruses and other intracellular microorganisms to bypass the defences of the node and spread to the bloodstream is discussed in chapter 5. specialised phagocytic cells are divided into two main types: the macrophages, scattered through all the major compartments of the body (see chapter 4) and the circulating neutrophils. the phagocytic cells to which microbes are exposed in the subepithelial tissues are the local macrophages (histiocytes) and also the cells arriving from the small blood vessels during inflammation. these comprise the blood monocytes which become macrophages after extravasation, and the neutrophils. from the time of the russian zoologist, elie metchnikoff, who described phagocytosis in 1883, the importance of the phagocyte in defence against disease organisms has been accepted, and children have learnt of the white blood cells that act both as scavengers and policemen, removing debris, foreign particles and microorganisms. because of the central importance of the phagocytic defence mechanisms, the subject will receive a chapter to itself. in addition to being able to resist host defence mechanisms, a pathogenic organism à be it an obligate intracellular, facultative intracellular or extracellular pathogen à must also overcome the problem of obtaining essential nutrients if it is to be successful. two examples illustrate this point. iron is essential for bacterial growth but most iron is sequestered in the host and the concentration of free iron in body fluids is too low (ca. 10 218 m in serum) to support growth. in the host, iron is bound by both intracellular (ferritin, haemosiderin and haem) and extracellular (transferrin in serum and milk, and lactoferrin in milk), fe-binding proteins with high association constants for iron. in order to overcome this problem, bacteria have evolved numerous ways of acquiring iron from the host in concentrations high enough for growth. for example, listeria monocytogenes produces a soluble reductant which removes iron from transferrin. a common strategy by many bacterial pathogens is the synthesis of low molecular weight compounds called siderophores, which have an extraordinary affinity for iron. at the same time the bacteria express outer membrane proteins that act as receptors for the fe-siderophore complexes so that fe is taken up into the cell. e. coli has been extensively studied, and three classes of siderophore have been recognised; ferrichrome, the hydroxymates and aerobactin. salmonella and shigella spp. and p. aeruginosa produce more than one siderophore. salmonella is also known to synthesise receptors for siderophores other than its own, which could be advantageous when present with other organisms, particularly in the competitive environment of the gut. the mycobacterial siderophores (mycobactins) are lipid soluble and membrane associated, and exochelins are water soluble, extracellular and are the more important of the two. another common bacterial strategy for dealing with the fe shortage involves the synthesis of new outer membrane proteins which interact directly with the host's own fe-binding proteins. this is the method used by neisseria meningitidus and n. gonorrhoeae which are able to scavenge iron directly from the host. perhaps the most dramatic example of fe uptake and storage is exhibited by yersinia. yersinia pestis is the agent of bubonic plague and has been responsible for devastating epidemics throughout human history. this pathogen persists in wild rodent populations in many parts of the world except australia, and is transmitted to humans by the bites of fleas. the blockage of the proventriculae of fleas by y. pestis forces infected fleas to bite and subsequently regurgitate the infected blood meal into the bite wound of a new host. the ensuing bacteraemia in rodents completes the rodentàfleaàrodent cycle essential for y. pestis spread. however the ecology, pathogenicity and host range of yersinia pseudotuberculosis (the predicted ancestor of y. pestis) and yersinia enterocolitica are quite different from y. pestis. these are orally transmitted from contaminated food or water. as described in chapter 2, they invade peyer's patches and disseminate to mesenteric lymph nodes (where they multiply extracellularly) and occasionally beyond causing septicemic plaguelike infections; normally these infections are self-limiting. despite their disease-causing differences, the three yersinia species do have some common pathogenic strategies, in particular the mechanism(s) for acquiring iron. yersinia have two important sets of pathogenicity genes: the 70 kb virulence plasmid which has genes encoding proteins involved in inhibition of phagocytosis to which we will return in chapter 4, and chromosomal genes encoding virulence factors including an 'invasin' (involved in interaction with peyer's patches) and the pgm (pigmentation) locus. virulent y. pestis strains accumulate huge quantities of exogenous haemin to form pigmented colonies on haemin agar (hence the alternative acronym for the locus, hms). contiguous with the hms is the ybt gene cluster responsible for the synthesis of the siderophore yersiniabactin. in y. pseudotuberculosis, hms and ybt are not contiguous, and in y. enterocolitica, hms is absent. inactivation of hms renders y. pestis avirulent and unable to develop blockages in fleas, whereas inactivation of ybt in y. enterocolitica hugely reduces virulence for laboratory animals. in fact, the ybt gene cluster has been designated the 'high pathogenicity island' (hpi) because biotype ib strains of y. pestis, y. tuberculosis and y. enterocolitica (new world strains) all possess ybt biosynthetic genes and kill mice with very low infectious doses. in contrast, biotypes 2à5 (old world strains) are much less virulent for mice and do not possess ybt genes. accordingly, the capacity to acquire high concentrations of iron for metabolism is associated with a high virulence phenotype. other nutrients in short supply in the mammalian host are aromatic amino acids like tryptophan. interestingly, the expression of the trp operon (which comprises the genes responsible for the synthesis of tryptophan) is controlled by fe as well as by tryptophan levels. a functional aromatic biosynthetic pathway is absolutely vital for growth in vivo since aromatic amino acids are generated via chorismic acid. the latter is a branch point at which the biosynthesis of p-amino benzoic acid (paba) also begins. paba is required as a precursor of folic acid; it is as a competitor for paba that the sulphonamide drugs work. by introducing lesions in one or more genes in this system (aroa and arod), it has been possible to attenuate strains of pathogenic bacteria such as s. typhimurium, e. coli, and aeromonas salmonicida, such that their initial invasive properties are unaltered but their ability to grow in vivo is severely restricted. in some cases, such crippled constructs can be used as vaccines to protect against subsequent infection or manipulated to carry genes encoding heterologous antigens which may offer immune protection. plague in the genomic area apoptosis; an innate immune response to virus infection reprogramming adult schwann cells to stem cell-like cells by leprosy bacilli promotes dissemination of infection cd1d, a sentinel molecule bridging innate and adaptive immunity, is downregulated by the human papillomavirus (hpv) e5 protein: a possible mechanism for immune evasion by hpv the struggle for iron à a metal at the hostàpathogen interface yersiniabactin iron uptake: mechanisms and role in yersinia pestis pathogenesis the high-pathogenicity island of yersiniae the acute phase response during parasitic infection human papillomavirus 16-encoded e7 protein inhibits ifn-gammamediated mhc class i antigen presentation and ctl-induced lysis by blocking irf-1 expression in mouse keratinocytes key: cord-306266-8qdrshz3 authors: scully, crispian title: respiratory medicine date: 2014-06-25 journal: scully's medical problems in dentistry doi: 10.1016/b978-0-7020-5401-3.00015-1 sha: doc_id: 306266 cord_uid: 8qdrshz3 ●. upper respiratory infections are commonplace, especially in young people, and are often contagious; ●. lower respiratory infections are often contagious and some are potentially fatal; ●. asthma is common and may be life-threatening; ●. chronic obstructive pulmonary disease is common and disabling; ●. tuberculosis worldwide is an important infection, affecting people with hiv/aids or malnutrition particularly; ●. lung cancer is common and usually has a poor prognosis. • upper respiratory infections are commonplace, especially in young people, and are often contagious the respiratory tract consists of the upper respiratory tract (urtnose, paranasal sinuses, pharynx and larynx; discussed in ch. 14) and the lower respiratory tract (lrt): the respiratory airways (trachea, bronchi and bronchioles) and lungs (respiratory bronchioles, alveolar ducts, alveolar sacs and alveoli), discussed in this chapter. protective mechanisms in the respiratory tracts include a mucociliary lining. particles or pathogens are trapped in the mucus and driven by ciliary action (the ciliary elevator) to the pharynx. mucociliary trans port declines with age but any effect on clinical infection has not been proved. lymphoid tissues of the waldeyer ring (adenoids, palatine and lingual tonsils) are important in developing an immune response to pathogens. however, the best respiratory defence mechanism is the cough reflex, the components of which include cough receptors, affer ent nerves, the cough centre, and efferent nerves and effector muscles. impairment of any of these -as may be seen in older patients or those with conditions associated with lowered consciousness (e.g. sedative use and neurological disease) -can weaken protection. dysphagia or impaired oesophageal motility may exacerbate the tendency to aspi rate foreign material. the alveolar defence mechanisms include mac rophages, immunocytes, surfactant, phospholipids, immunoglobulin g (igg), ige, secretory iga, complement components and factor b; many immune defects manifest with recurrent respiratory infections. lung function is vital to gas exchange -the blood absorbs oxygen and releases carbon dioxide. normal gas exchange requires adequate alveolar ventilation, normal ventilation/blood flow relationships and adequate alveolar-capillary membrane surface area. breathing (ven tilation) depends on respiratory drive, which reacts to the respiratory load. this process requires work and results in gas exchange. oxygen is transported in combination with haemoglobin in erythro cytes and a small amount dissolved in plasma. the oxyhaemoglobin dissociation curve is sigmoidal; once the oxygen saturation falls below 95%, the amount of o 2 transported to the tissues and brain falls rapidly. high temperatures, acidosis, raised co 2 and raised 2,3 diphosphoglycerate (2,3dpg) levels encourage oxygen offloading, whereas fetal haemoglobin and carboxyhaemoglobin have the con trary effect. chronic hypoxaemia (e.g. at high altitudes) stimulates release of erythropoietin from the kidneys, with a rise in red cell pro duction, and raised 2,3dpg. athletes have abused erythropoietin to gain competitive advantage (ch. 33). the most common lrt disorders are asthma and chronic obstructive pulmonary disease (copd). respiratory disorders are common, and are often caused or aggravated by tobacco smoking. they may significantly affect general anaesthesia (ga) and conscious sedation (cs), since they are often a contraindica tion to use of benzodiazepines, opioids, ga agents and other respira tory depressants. impaired gas exchange leads to laboured breathing and can cause significant incapacity. features include cough, sputum production, wheeze, dyspnoea, chest pain, cyanosis, fingerclubbing ( fig. 15.1) , use of accessory muscles of respiration with indrawing of the intercostal spaces (hyperinflation), and abnormalities in chest shape, movements, respiratory rate and breath sounds. cough may be a feature of any respiratory problem but, if chronic, may herald serious disease -for example, copd, cancer or infec tion such as tuberculosis. mucoid or mucopurulent sputum is often a feature ( fig. 15 .2); purulent sputum indicates acute bronchitis, bronchiectasis or lung abscess. blood (haemoptysis) or bloodstained sputum, though common in acute infections (especially in preexisting copd), bronchiectasis and pulmonary embolism, may herald an even more serious condition -for example, possibly one due to carcinoma or tuberculosis. wheezing is caused by airways obstruction and is a typical sign of asthma or copd. breathlessness (dyspnoea) is distress ing, and may be caused by respiratory or cardiovascular disease, or by anaemia, and is particularly ominous if it persists at rest. excessive resistive load, such as in asthma, copd and cystic fibro sis, impairs airflow. elastic load increases because of, for example, interstitial fibrosis, muscle paralysis and obesity. diagnosis of respiratory disorders is from the clinical features sup ported by imaging (especially chest radiography). spiral computed tomography (ct) can now scan the lungs in a quick 20-30second breathhold and therefore, instead of producing a stack of individual ct slices, which may be misaligned due to patient movement or breathing in between slices, provides highresolution three dimensional images. respiratory function tests can measure individual components of the respiratory process. spirometry is the basic screening test for assess ing mechanical load problems, the quantification involving determi nation of the vital capacity (vc) -slow vital capacity (svc) and/or forced vital capacity (fvc) -and the speed of maximal expiratory flow (mef; fig. 15 .3). in health, about 75% of a normalsized vc is expelled in 1 second (fev 1 ). the peak flow meter, which measures the peak expiratory flow rate (pefr; the earliest portion of forced expiration), is a simple measure of airflow obstruction, when the fev 1 is a much smaller fraction of the vc. in lung restriction, the diminished vc can be mostly expelled in about 1 second. serial meas urements (e.g. in asthma) provide valuable information about disease progress. the reversibility of airways obstruction is usually assessed by spirometry before and after use of a bronchodilator agent. arterial blood gas analysis yields considerable information about gas exchange efficiency. arterial hypoxaemia in adults is defined as pao 2 below 10.7 kpa breathing room air, although it is not usually treated as clinically important unless below 8 kpa, when oxygen saturation will be 90% or less (table 15 .1). arterial carbon dioxide tension (paco 2 ) is used as an inversely pro portional index of 'effective' alveolar ventilation. hence, a high paco 2 is taken to indicate poor alveolar ventilation. alveolar hypoventila tion (raised paco 2 ) with a normal ph probably represents a primary ventilatory change present long enough for renal mechanisms to compensate, as in chronic ventilatory failure. ventilation/blood flow relationships are most simply assessed by considering the size of the difference between the amounts of oxygen and carbon dioxide in the blood and in the air; the differences are small if the lungs are work ing efficiently. disparity between ventilation/blood flow ratios results in abnormally wide differences -and then alveolar-arterial po 2 and arterial-alveolar pco 2 gradients will be abnormal. alveolar capillary surface area is assessed by measuring the uptake of carbon monoxide -usually abnormal in diffuse interstitial inflam matory and fibrotic processes and in emphysema. assessing bronchial reactivity and the exercise response can help evaluate breathlessness. simple exercise testing provides information about overall fitness and the appropriateness of cardiorespiratory responses. radionuclide lung scanning, blood gas analysis and sputum cytology or culture are sometimes needed in addition. management can include oxygen administration by mask or nasal cannula (figs 15.4 and 15.5) . lrt disorders can cause significant incapacity and are often a con traindication to ga, and even to cs. asthma is common, affecting 2-5% of the overall population; it is on the increase, particularly in childhood, with a frequency of up to 20% in some highincome countries. asthma usually begins in childhood or early adult life; about half the patients with asthma develop it before age 10 years. bronchial hyperreactivity causes reversible airway obstruction from smooth muscle constriction (bronchospasm), mucosal oedema and mucus hypersecretion. there are two main types, extrinsic (allergic) and intrinsic asthma (table 15 .2). extrinsic (allergic) asthma, the main childhood type, may be pre cipitated by allergens in animal dander, feathers or hair, drugs (e.g. nonsteroidal antiinflammatory drugs [nsaids] and some antibiot ics), food (e.g. eggs, fish, fruit, milk, nuts), house dust (mite allergens) or moulds. patients frequently have or develop other allergic diseases, such as eczema, hay fever and drug sensitivities. extrinsic asthma is associated with ige overproduction on allergen exposure, and release of mast cell mediators (histamine, leukotrienes, prostaglandins, bradykinin and platelet activating factor), which cause bronchospasm and oedema. about 75% of asthmatic children lose their asthma or improve by adulthood. intrinsic asthma is usually of adult onset and not aller gic, but appears rather to be related to mast cell instability and airway hyperresponsivity. triggers include emotional stress, gastro oesophageal reflux or vagally mediated responses. either type of asthma can be triggered by: infections (especially viral, mycoplasmal or fungal); irritating fumes (e.g. traffic or cigarette smoke); exercise (possibly due to cold air); weather changes; emotional stress; foods (e.g. nuts, shellfish, strawberries or milk) or additives (such as tartrazine); and drugs (e.g. aspirin and other nsaids, beta blockers and angiotensinconverting enzyme inhibitors [aceis]). in wellcontrolled patients with asthma, clinical features may be absent. during an asthmatic episode, symptoms may include dysp noea, cough and paroxysmal expiratory wheeziness with laboured expiration. the frequency and severity of attacks vary widely between individuals (table 15 .3). patients may become distressed, anxious and tachycardic, have reduced chest expansion and be using accessory respiratory muscles to increase their ventilatory effort. nasal polyps are common, especially in aspirinsensitive asthmatics. children with asthma initially suffer from repeated 'colds' with cough, malaise and fever, often at night. asthma is typically diagnosed when the patient has more than one of the following -wheeze, cough, difficulty breathing and chest tightness -particularly if these are frequent and recurrent; are worse at night and in the early morning; occur in response to, or are worse after, exercise or other triggers, such as exposure to pets, cold or damp air, or with emotions or laughter; or occur without an association with colds. there is often: ■ a personal history of atopic disorder ■ a family history of atopic disorder and/or asthma ■ widespread wheeze, heard on chest auscultation ■ a history of improvement in symptoms or lung function in response to adequate therapy. a prolonged asthmatic attack, which is refractory to treatment, may lead to lifethreatening status asthmaticus (persisting for more than 24 hours). failure of the patient to complete a sentence, indrawing of the intercostal muscles, a rapid pulse, a silent chest and signs of exhaustion are suggestive of impending respiratory arrest. diagnosis of asthma is from the clinical history and presentation, based on recognizing a characteristic pattern of episodic symptoms in the absence of an alternative explanation. investigations include a chest radiograph (to exclude other diagnoses, such as a pneumo thorax), spirometry (serial pefr), skin tests and blood examination (usually eosinophilia, raised total ige and specific ige antibody concentrations, which may help identify allergens). occasionally, a histamine or methacholine challenge is used if the diagnosis is unclear. in children with an intermediate probability of asthma, who can perform spirometry and have evidence of airways obstruction, assess the change in fev 1 or pefr in response to an inhaled bronchodilator (reversibility) and/or the response to a trial of treatment for a speci fied period; if there is significant reversibility, or if a treatment trial is beneficial, a diagnosis of asthma is probable. management includes patient education, smoking cessation advice, avoidance of identifiable irritants and allergens, and use of drugs. home use of peak flow meters allows patients to monitor progress and detect any deterioration that may require urgent modification of treatment. treatment should be based on the amount by which peak flow is reduced (a pefr diary should be kept). drugs used for asthma management (table 15 .4) include oxygen, shortacting β 2 agonists (sabas; such as salbutamol), corticosteroids, leukotriene receptor antagonists and omalizumab (a recombinant humanized monoclonal antiige antibody that reduces the antigen specific ige). inhaled longacting β 2 agonists (labas) may be needed ( fig. 15.6 ). deaths from asthma are usually a result of failure to recognize dete rioration or reluctance to use corticosteroids. other factors that have been studied include: ■ air pollution -there is an association between air pollution and aggravation of existing asthma ■ allergen avoidance -there is no consistent evidence of benefit ■ breast-feeding -there is evidence of a protective effect in relation to early asthma ■ electrolytes -there is no consistent evidence of benefit ■ fish oils and fatty acid -there is no consistent evidence of benefit ■ house dust mites -measures to reduce the numbers of house dust mites do not affect asthma severity ■ immunotherapy -allergenspecific immunotherapy is beneficial in allergic asthma ■ microbial exposure -there is insufficient evidence to indicate that the use of probiotics in pregnancy reduces the incidence of childhood asthma ■ modified milk formulae -there is no consistent evidence of benefit pets -there are no controlled trials on the benefits of removing pets from the home ■ tobacco -exposure to cigarette smoke adversely affects quality of life, lung function, need for rescue medications and longterm control with inhaled steroids. there is an association between maternal smoking and an increased risk of infant wheeze ■ weight reduction -there is an association between increasing body mass index and symptoms of asthma. elective dental care should be deferred in severe asthmatics until they are in a better phase; this can be advised by the patient's general practitioner. asthmatic patients should be asked to bring their usual medica tion with them when coming for dental treatment. local anaesthe sia (la) is best used; occasional patients may react to the sulphites present as preservatives in vasoconstrictorcontaining la, so it may be better, where possible, to avoid solutions containing vasoconstric tor. adrenaline (epinephrine) may theoretically enhance the risk of arrhythmias with betaagonists and is contraindicated in patients using theophylline, as it may precipitate arrhythmias. relative analgesia with nitrous oxide and oxygen is preferable to intravenous sedation and gives more immediate control. sedatives in general are better avoided as, in an acute asthmatic attack, even ben zodiazepines can precipitate respiratory failure. ga is best avoided, as it may be complicated by hypoxia and hyper capnia, which can cause pulmonary oedema even if cardiac function is normal, and cardiac failure if there is cardiac disease. the risk of post operative lung collapse or pneumothorax is also increased. halothane or, better, enflurane, isoflurane, desflurane and sevoflurane are the preferred anaesthetics, but ketamine may be useful in children. allergy to penicillin may be more frequent in asthmatics. drugs to be avoided, since they may precipitate an asthmatic attack (see later), include those listed in box 15.1. acute asthmatic attacks may also occasionally be precipitated by anxiety; it is important to attempt to lessen fear of dental treatment by gentle handling and reassurance. even routine dental treatment can trigger a clinically significant decline in lung function in approximately 15% of asthmatics. acute asthmatic attacks are usually selflimiting or respond to the patient's usual medication, such as a betaagonist inhaler, but status asthmaticus is a potentially fatal emergency (ch. 1). there may be complications caused by the antiasthmatic drugs (table 15 .5). gastrooesophageal reflux is not uncommon, with occasional tooth erosion. periodontal inflammation is greater in asthmatics than in those without respiratory disease. persons using steroid inhalers may develop oropharyngeal candidosis or, occasionally, angina bullosa haemorrhagica. guidelines on the management of asthma may be found at: http://www.sign.ac.uk/guidelines/fulltext/101/index.html, http:// www.nice.org.uk/guidance/qualitystandards/indevelopment/asthma. jsp and http://www.britthoracic.org.uk/portals/0/guidelines/ asthmaguidelines/qrg101%202011.pdf (all accessed 30 september 2013). churg-strauss syndrome (css) is a rare, potentially fatal, systemic vasculitis similar to polyarteritis nodosa (pan), characterized by severe asthmalike attacks with peripheral eosinophilia, and intravas cular and extravascular granuloma formation with eosinophil infiltra tion and skin lesions in 70%. cardiopulmonary involvement is the main cause of death. css is diagnosed if at least 4 of the 6 criteria listed in box 15.2 are positive. the 5year survival of untreated css is 25%. combination treatment with cyclophosphamide and prednisolone (prednisone) provides a 5year survival of 50%. management problems relating to patients with css may include res piratory impairment and corticosteroid treatment (ch. 6). chronic obstructive pulmonary disease (copd; chronic obstructive airways disease, coad) is a common, chronic, slowly progressive, irre versible disease (most frequently a combination of chronic bronchitis and emphysema), characterized by breathlessness and wheeze (airways obstruction), cough and sputum. chronic bronchitis is defined as the excessive production of mucus and persistent cough with sputum production, daily for more than 3 months in a year over more than 2 consecutive years. it leads to production of excessive, viscous mucus, which is ineffectively cleared from the airway, obstructs and stag nates, and becomes infected, usually with streptococcus pneumoniae, moraxella catarrhalis and haemophilus influenzae. patchy areas of alveolar collapse can result. emphysema is dilatation of air spaces dis tal to the terminal bronchioles with destruction of alveoli, reducing the alveolar surface area available for respiratory exchange. copd is now the preferred term for conditions with airflow obstruction because of a combination of airway and parenchymal damage; patients were previ ously diagnosed as having chronic bronchitis or emphysema. copd is characterized by airflow obstruction -defined as an fev 1 / fvc ratio reduced to less than 0.7. if fev 1 is 80% or more, a diagno sis of copd should only be made if there are respiratory symptoms (e.g. dyspnoea or cough). the airflow obstruction is not fully revers ible, does not change significantly over months, and is usually progres sive in the long term. the most important causes of copd include cigarette smoking, environmental pollution, dusts, chemicals or occupational exposures to various substances. exposure to smoke from home cooking or heating fuels may contribute. deficiency of the antiproteolytic enzyme alpha1antitrypsin is a rare cause of emphysema. there is often significant airflow obstruction before the person is aware of it and so copd typically remains undiagnosed until patients are in their fifties. differentiation from asthma is important (table 15 .6). a diagnosis of copd should be considered in patients over the age of 35 who have a risk factor (e.g. smoking) and exertional breath lessness, chronic cough, regular sputum production, frequent winter 'bronchitis' or wheeze. clinical judgment is based on history, physical examination, confirmation of airflow obstruction using spirometry (postbronchodilator spirometry) and assessment of the severity of dyspnoea (tables 15.7 and 15.8). copd is characterized by breathlessness and wheeze (airways obstruction), cough and an early morning mucoid sputum production. to investigate symptoms that seem disproportionate to spirometric impairment progressive dyspnoea, low oxygen saturation, carbon dioxide accumu lation (hypercapnia) and metabolic acidosis mean that patients may ultimately become dyspnoeic at rest ('respiratory cripples'), especially when recumbent (orthopnoea), and eventually develop respiratory failure, pulmonary hypertension, right ventricular hypertrophy and rightsided heart failure (cor pulmonale). two clinical patterns of copd are recognized: ■ 'pink puffers' -patients with emphysema who manage to maintain normal blood gases by hyperventilation, and are always breathless but not cyanosed; rather they are pink from vasodilatation ■ 'blue bloaters' -patients with chronic bronchitis who lose their co 2 drive, fail to maintain adequate ventilation and become both hypercapnic and hypoxic with central cyanosis, cor pulmonale and oedema (for these patients, the respiratory drive is from the low po 2 and thus oxygen administration is contraindicated) (table 15 .9). the diagnosis of copd is based upon clinical history and presen tation. investigations include a chest radiograph (which may show hyperinflated lung fields with loss of vascular markings); arterial blood gases (which should be measured if pulse oximetry shows oxygen satu ration less than 92%); spirometry; and lung function tests. fev1 is reduced in all cases (fev 1 of less than 40% signifies severe copd) and the flow-volume curve shows a typical pattern, with reduced flow rates at mid and lowerlung volumes. a ratio of fev 1 :fvc of less than 70% confirms airways obstruction. patients with copd and their family should be educated about the disease, and about required lifestyle changes and medication. nondrug therapy includes: stopping smoking (nicotine replacement therapy or bupropion may help); exercise by pulmonary rehabilitationof proven benefit; weight loss (improves exercise tolerance); and vaccination (pneumococcal and influenza vaccines). drug therapy includes shortacting bronchodilators (anticholinergic drugs [ipra tropium bromide]) and β 2 agonists (salbutamol) to treat the reversible component of airway disease; corticosteroids (inhaled or systemic); and antibiotics (amoxicillin, trimethoprim or tetracycline). mucolytics, such as carbocisteine, reduce acute exacerbations by almost onethird. longterm oxygen therapy (ltot) reduces mortality. people with stable copd who remain breathless or have exacerba tions, despite using shortacting bronchodilators, should be offered the following as maintenance therapy: ■ if fev 1 is 50% of predicted or more: use either a longacting β 2 agonist (laba) or longacting muscarinic antagonist (lama). ■ if fev 1 is less than 50% predicted: either a laba with an inhaled corticosteroid (ics) in a combination inhaler, or a lama. offer a lama in addition to a laba plus ics to people with copd who remain breathless or have exacerbations, despite taking laba plus ics, irrespective of their fev 1 . provide pulmonary rehabilitation for all who need it; noninvasive ventilation (niv) is the treatment of choice for persistent hyper capnic ventilatory failure during exacerbations not responding to medical therapy. the frequency of exacerbations should be reduced by appropriate use of inhaled corticosteroids and bronchodilators, and vaccinations. bronchodilators (shortacting β 2 agonists [saba] and shortacting muscarinic antagonists [sama]) should be the initial empirical treat ment for the relief of breathlessness and exercise limitation. ics have potential adverse effects (including nonfatal pneumonia) in people with copd. offer a oncedaily lama in preference to fourtimes daily sama to people with stable copd who remain breathless or have exacerbations, despite using shortacting bronchodilators as required, and in whom a decision has been made to commence regular maintenance bronchodilator therapy with a muscarinic antagonist (see above). most patients -whatever their age -are able to acquire and main tain an adequate inhaler technique. bronchodilators are usually best administered using a handheld inhaler device (including a spacer device if appropriate). patients with distressing or disabling dyspnoea, despite maximal therapy using inhalers, should be considered for nebulizer therapy. they should be offered a choice between a face mask and a mouth piece to administer their nebulized therapy, unless the drug specifically requires a mouthpiece (for example, anticholinergic drugs). some patients with advanced copd may require maintenance oral corticosteroids when these cannot be withdrawn following an exacer bation. these individuals should be monitored for the development of osteoporosis and given appropriate prophylaxis. theophylline should only be used after a trial of saba and laba, and only to those who are unable to use inhaled therapy, as there is a need to monitor plasma levels and interactions. the dose of theo phylline prescribed should be reduced at the time of an exacerbation if macrolide or fluoroquinolone antibiotics (or other drugs known to interact) are given. there is insufficient evidence to recommend prophylactic antibiotic therapy in the management of stable copd. mucolytic drug therapy should be considered in patients with a chronic cough productive of sputum. if patients remain symptomatic on monotherapy, their treatment should be intensified by combining therapies from different drug classes, such as: ■ β 2 agonist and theophylline ■ anticholinergic and theophylline. inappropriate oxygen therapy in people with copd may depress respiration. ltot is indicated in patients with copd who have a pao 2 of less than 7.3 kpa when sta ble, or a pao 2 greater than 7.3 kpa and less than 8 kpa when stable, and one of: secondary polycythaemia, nocturnal hypoxaemia (oxygen saturation of arterial blood [sao 2 ] of less than 90% for more than 30% of the time), peripheral oedema or pulmonary hypertension. to reap the benefits of ltot, patients should breathe supplemental oxygen for at least 15 hours per day. to ensure that all those eligible for ltot are identified, pulse oximetry should be available in all health care settings. the assessment of patients for ltot should comprise the measurement of arterial blood gases on two occasions at least 3 weeks apart in patients who have a confident diagnosis of copd, who are receiving optimum medical management and whose copd is stable. patients should be warned about the risks of fire and explosion and told not to smoke when using oxygen. ambulatory oxygen therapy should be considered in patients on ltot who wish to continue oxygen therapy outside the home, and who have exercise desaturation, are shown to have an improvement in exercise capacity and/or dyspnoea with oxy gen, and are motivated to use oxygen. adequately treated patients with chronic hypercapnic respiratory failure who have required assisted ventilation during an exacerbation, or who are hypercapnic or acidotic on ltot, should be referred to a specialist centre for consideration of longterm niv. advanced emphysema is occasionally treated with sur gery -excision of large acquired bullae or, rarely, lung transplantation. patients with copd who need dental care can be classified as follows: ■ patients at low risk -experience dyspnoea on effort but have normal blood gas levels. these patients can receive all dental treatment with minor modifications. ■ patients at moderate risk -experience dyspnoea on effort, are chronically treated with bronchodilators or recently with corticosteroids, and pao 2 lowered. a medical consultation is advised to determine the level of control of the disease before any dental treatment. ■ patients at high risk -have symptomatic copd that may be end stage and poorly responsive to treatment. with these patients, a medical consultation is essential before any dental treatment is carried out. patients with copd are best treated in an upright position at midmorning or early afternoon, since they may become increasingly dyspnoeic if laid supine. it may be difficult to use a rubber dam, as some patients are mouthbreathers and not able to tolerate the additional obstruction. la is preferred for dental treatment, but bilateral mandibular or palatal injections should be avoided. patients with copd should be given relative analgesia only if absolutely necessary, and only in hospital after full preoperative assessment. cs with diazepam and midazolam should not be used, as benzodiazepines are respiratory depressants. patients should be given ga only if absolutely necessary, and intravenous barbiturates are contraindicated. secretions reduce airway patency and, if lightly anaesthetized, the patient may cough and contaminate other areas of the lung. postoperative respiratory complications are more prevalent in patients with preexisting lung diseases, especially after prolonged operations and if there has been no preoperative preparation. the most important single factor in preoperative care is cessation of smok ing for at least 1 week preoperatively. respiratory infections must also be eradicated; sputum should first be sent for culture and sensitivity, but antimicrobials such as amoxicillin should be started without await ing results. the medical management of copd should be optimized prior to surgery. the ultimate clinical decision about whether or not to proceed with surgery should rest with a consultant anaesthetist and consultant surgeon, taking account of comorbidities, functional status of the patient and necessity for the surgery. composite assessment tools, such as the american society of anesthesiologists (asa) scoring system, and not just lung function, are the best criteria for the assessment of patients with copd before surgery. those taking corticosteroids should be treated with appropri ate precautions (ch. 6). interactions of theophylline with other drugs, such as adrenaline (epinephrine), erythromycin, clindamycin, azithro mycin, clarithromycin or ciprofloxacin, may result in dangerously high levels of theophylline. ipratropium can cause dry mouth. guidelines for the management of copd may be found at: http:// publications.nice.org.uk/chronicobstructivepulmonarydisease cg101 (accessed 30 september 2013). respiratory viruses usually spread by touch or airborne transmission and the very small particles (2-0.2 micrometres) can avoid the upper respiratory tract defences and the mucociliary elevator to reach the lung alveoli. a range of viruses can cause lower respiratory tract infections (lrtis ; table 15 .10). some viruses (e.g. influenza and respiratory syncytial) can spread from the upper to the lower respira tory tract via infection of the respiratory epithelium and can lead to bacterial superinfection and pneumonitis (pneumonia). mycoplasmal (atypical) pneumonia and tuberculosis (tb) may be direct infections. epidemics of a potentially fatal severe acute respiratory syndrome (sars) have been caused by a coronavirus that originated in china and spread worldwide; h5n1 bird influenza also arose as an epidemic; and a similar epidemic, but of swine influenza (h1n1), emanated from mexico (see later). bacterial infections, such as pneumonia or lung abscess, can also result from material aspirated into the lungs, and are usually unilat eral. those who aspirate more than others have, as a result, more frequent lrti and this is seen in alcohol and other drug abusers, as well as comatose patients. exogenous penetration and contamination of the lung can result from trauma (e.g. a stab wound or road traffic accident) or surgery. entamoeba histolytica can occasionally cause pneumonia -by direct extension from an amoebic liver abscess (table 15 .11). patients with endocarditis, or septic pelvic or jugular thrombo phlebitis, may experience lrti acquired haematogenously and then it is often bilateral. immunocompromised persons (e.g. those with human immunode ficiency virus/acquired immune deficiency syndrome [hiv/aids] and transplant recipients) and people with bronchiectasis or cystic fibrosis are also susceptible to respiratory infections by a range of opportun istic microbes. pneumocystis jiroveci (p. carinii), for example, is a com mon cause of potentially fatal pneumonia in immunocompromised patients -especially those with hiv/aids (chs 20 and 21). clinical features of lrti vary according to the part of the respiratory tract mainly affected: ■ bronchiolitis causes rapid respiration, wheezing, fever and dyspnoea -but is restricted mainly to infants. ■ bronchitis causes cough, wheezing and sometimes dyspnoea. ■ pneumonia causes cough, fever, rapid respiration, breathlessness, chest pain, dyspnoea and shivering. antimicrobial therapy is indicated, particularly for pneumonia. antivirals have not been highly effective. oxygen may be needed. pneumococcal vaccine is indicated for older people. the majority of lrtis are severe illnesses, and are contraindications to all but emergency dental treatment. ga is hazardous and absolutely contraindicated. dental treatment should be deferred until recovery, or be limited to pain relief. influenza is mainly a communitybased infection transmitted in house holds and communities. healthcareassociated influenza infections can arise in any healthcare setting, most commonly when influenza is also circulating in the community. influenza is a contagious disease caused by influenza virus types a, b or c. type a has two main subtypes (h1n1 and h3n2); it causes most of the widespread influenza epidemics and can occasionally be fatal. type b viruses generally cause regional outbreaks of moderate severity, and type c viruses are of minor significance. a person can spread influenza starting 1 day before they feel sick and for another 3-7 days after symptoms start. influenza can be pre vented or ameliorated by vaccination each autumn; this is especially indicated for older people and those with cardiorespiratory disease. influenza attacks virtually the whole respiratory tract; symptoms appear suddenly after 1-4 days and include fever, sore throat, nasal congestion, headache, tiredness, dry cough and muscle pains (myalgia). most people recover in 1-2 weeks but infection can be lifethreatening, mainly because primary influenzal viral pneumonia can lead to sec ondary bacterial pneumonia or can exacerbate underlying conditions (e.g. pulmonary or cardiac disease). the old and very young, and those with chronic disorders, are more likely to suffer complications, such as pneumonia, bronchitis, sinusitis or otitis media. influenza has also been followed by depression, encephalopathy, myocarditis, myositis, pericarditis, reye syndrome and transverse myelitis. rest, maintenance of fluid intake, analgesics, antipyretics, and avoid ance of alcohol and tobacco help relieve symptoms. aspirin must never be given to children under the age of 16 years who have 'flulike symptoms, and particularly fever, as this can cause reye syndrome. zanamivir (an antiviral that works against influenza types a and b) can shorten the symptoms by approximately 1 day, if treatment is started during the first 2 days of illness. other antiviral drugs include amantadine, oseltamivir and rimantadine; they may be helpful but their use is restricted mainly to immunocompromised persons, since they can cause adverse effects. influenza can be a severe contagious illness so all but emergency den tal treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. influenza type a subtype h5n1 can cause an illness known as 'avian influenza' or 'bird 'flu' in birds, humans and many other animal spe cies. hpai a(h5n1) -'highly pathogenic avian influenza virus of type a of subtype h5n1' -is the causative agent and is enzootic in many bird populations, especially in southeast asia. it has spread globally and resulted in the deaths of over 100 people and the slaughter of mil lions of chickens. a vaccine that could provide protection (prepandrix) has been cleared for use in the european union. h5n7 is a more recent emergent infection, similar in many respects. swine influenza is common in pigs in the midwestern united states, mexico, canada, south america, europe (including the uk, sweden and italy), kenya, china, taiwan, japan and other parts of eastern asia. transmission of swine influenza virus from pigs to humans is not com mon, but can produce symptoms similar to those of influenza. a 2009 outbreak in humans ('swine 'flu') was due to an apparently new strain of h1n1 arising from a reassortment produced from strains of human, avian and swine viruses. it can pass from human to human. antiviral agents such as oseltamivir may help. vaccines are now available. an outbreak of a lifethreatening febrile respiratory infection appeared in 2003, originating from guangdong, china, and was named severe acute respiratory syndrome (sars). caused by a newly recognized coronavirus (sarsassociated coronavirus, sarscov), sars spread via close contact to many countries across the world. according to the world health organization, 8437 people worldwide became sick with sars during the course of the first recognized outbreak and 813 died. the incubation period of 2-7 days is followed by a high fever (above 38.0°c), malaise, headache and myalgia. some people also experience mild upper respiratory symptoms and, after 2-7 days, lower respiratory signs -a dry cough and dyspnoea, potentially progressing to hypox aemia. sars can cause a pneumonia with a mortality approaching 10%, particularly in older or immunocompromised people. artificial ventilation has been needed in 10-20% of cases. antiviral agents, such as oseltamivir or ribavirin, may help. inactivated vaccines, virally and bacterially vectored vaccines, recombinant protein and dna vaccines, as well as attenuated vaccines, are under development. sars is a severe illness, and all but emergency dental treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. for all contact with suspect sars patients, careful hand hygiene is important, including handwashing with soap and water; if hands are not visibly soiled, alcoholbased handrubs may be used as an alternative to handwashing. if a suspected sars patient is admitted to hospital, infection control personnel should be notified immediately. infection control measures (www.cdc.gov/ncidod/hip/iso lat/isolat.htm; accessed 30 september 2013) should include standard precautions (e.g. hand hygiene): healthcare personnel should wear eye protection for all patient contact; contact precautions (e.g. gown and gloves for contact with the patient or their environment); and airborne precautions (e.g. an isolation room with negative pressure relative to the surrounding area and use of an n95 filtering disposable respirator for persons entering the room). pneumonia is classed as 'primary' if it occurs in a previously healthy individual, and is usually lobar; it is called 'secondary' if it follows some other disorder, such as previous viral respiratory infections, aspir ation of foreign material, lung disease (bronchiectasis or carcinoma), depressed immunity (e.g. alcoholism or immunosuppression), or aspir ation of oral bacteria ( pneumonia causes cough, fever, rapid respiration, breathlessness, chest pain, dyspnoea and shivering. complications can include lung abscess or empyema (pus in pleural cavity). it is important to avoid alcohol and tobacco, but use analgesics and antipyretics to relieve the symptoms. broadspectrum antimicrobi als given promptly and empirically usually include a macrolide (azithromycin, clarithromycin or erythromycin), quinolone (moxiflox acin, gatifloxacin or levofloxacin), or doxycycline for outpatients. for in patients, cefuroxime or ceftriaxone plus a macrolide is used. prophylaxis includes immunization against influenza and pneumococci. pneumonia is a severe illness and all but emergency dental treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. ventilatorassociated pneumonia (vap) is discussed later. legionellosis is a bacterial respiratory infection caused by one of the family legionellaceae, gramnegative aerobic bacilli, ubiquitous in water and soil but particularly preferring warm aquatic environments. the term legionnaire's disease was coined as a result of an outbreak of the previously unrecognized respiratory disease in an american legion meeting in philadelphia in 1976, but it is now recognized worldwide, many infections being contracted during travel abroad, particularly to spain, turkey and some other mediterranean areas. legionella bacteria can be found in natural freshwater environments, usually in insufficient numbers to cause disease. legionella grow best in warm water, as in hot tubs, cooling towers, hot water tanks, large plumbing systems, or the airconditioning systems of large buildings. though there are over 30 legionellaceae, most infections are caused by legionella pneumophila. disease is contracted by inhalation of contaminated mist or vapour, mainly (approximately 46%) through aerosolization of infected water in airconditioning systems, hotwater systems, humidifiers, nebulizers, showers and spa pools. outbreaks have mostly been linked to aerosol sources in the community, cruise ships and hotels, with the most likely sources being whirlpool spas, air conditioning units in large buildings, potable (drinking) water systems, and water used for bathing. risk factors include: ■ exposure to: recent travel with an overnight stay outside of the home (outbreaks of travelassociated legionellosis are infrequently identified but more than 20% of cases are thought to be associated with recent travel) whirlpool spas recent repairs or maintenance work on domestic plumbing ■ systemic illhealth: alcohol use chronic kidney disease diabetes immune defects liver disease malignancy smoking. illness mainly affects males over 45, smokers, heavy drinkers, older people and the immunocompromised. also vulnerable are travellers, especially middleaged and older tourists, and conference or business groups, possibly because of tiredness or age. many young people have been exposed to infection and become seropositive, but remained healthy. there is no evidence of persontoperson transmission of legionellosis. legionellosis manifests as one of two clinical syndromes (table 15 .14). legionnaire's disease is typically a lobular type of pneumonia, which can be fatal but is fortunately rare; infection can range from discrete patches of inflammation and consolidation to involvement of whole lobes. pontiac fever is milder and usually subsides rapidly, often with out treatment. people who should be tested for legionnaire's disease include those with pneumonia in the following groups: because legionella is commonly found in the environment, clinical isolates are necessary to interpret the findings of an environmental investigation. diagnosis can be by rapid urine molecular testing for l. pneumophila antigen, and culture of respiratory secretions on selective media. sensitivity and specificity of the diagnostic tests are shown in table 15 .15. pontiac fever is a selflimited illness; most cases recover within 1 week and few benefit from antibiotic treatment. overall mortality in legionnaire's disease may be as high as 10%, and over 25% in older people and up to 80% in the immunocompromised. erythromycin is standard treatment; cephalosporin is an alternative. legionella species are present in roughly twothirds of potable water samples collected from domestic and institutional taps and drinking fountains, and from a similar percentage of dental units, but water from these dental units often has higher bacterial concentrations (ch. 31). there are reports of legionella infections in dental unit water lines, and antibodies and occasionally frank infection demonstrated in dental staff; at least one patient appears to have contracted and died from infection emanating from a dental practice. prevention is crucial, involving (ch. general aspects tuberculosis (tb) , an infection caused by mycobacteria, affects approxi mately onethird of the world's population (1.5 billion people); it is a major global health problem, some 2 million people dying from it annu ally. tb disproportionately affects the poorest persons in both high income and developing countries. in highincome countries, most human tb arises from mycobacterium tuberculosis, transmitted from person to person through the air. tb usually affects the lungs initially (pulmonary tb) but can also involve brain, kidneys, spine and other parts. from victorian times to about the second world war, mycobacterium bovis infection from infected cows' milk (bovine or btb) was a major cause of morbidity and mortality; it was clinically and pathologically indistin guishable from infection caused by m. tuberculosis. cattletesting and a slaughter programme became compulsory in 1950 and, by the 1980s, the incidence of tb in cattle had been substantially reduced. tuberculosis from m. bovis in cows' milk was virtually eliminated in highincome countries by the tuberculin testing of cattle and pasteurization of milk. in the developing world, many cattle still have tb, and btb is still seen. btb has also increased in highincome countries over the last two dec ades and an infection rate of up to 38% in badgers -and transmission to cattle -may explain this. tb is not spread by touch or by drinking glasses, dishes, sheets or clothing. it is usually transmitted by infected sputum, typically from close contacts such as family members, but is unlikely to be transmit ted between normal social contacts. tb can present an occupational risk to healthcare professionals, including dental staff. one outbreak of drugresistant tb in new york involved at least 357 patients, most of whom contracted tb in one of 11 hospitals; nearly 90% of the patients were also hivpositive, and most were young males of hispanic or african heritage. tb has been transmitted between pas sengers during longhaul airline flights. the risk of transmitting tb though air circulation is now low because the highefficiency particu late air (hepa) filters on newer commercial aircraft are of the same type as those used in hospital respiratory isolation rooms; indeed, the number of times air is cleaned each hour exceeds the recommendation for hospital isolation rooms. subsaharan africa has the highest rates of active tb per capita, driven primarily by the hiv epidemic. the absolute number of cases is highest in asia, with india and china having the great est burden of disease globally. in the usa and most western european countries, the majority of cases occur in foreignborn residents and recent immigrants from countries in which tubercu losis is endemic. immunocompromised people -such as diabetics and severely immuno deficient patients, like those with hiv/aids (about 30% of south africans with hiv/aids also have tb) -and patients in prisons or institutions are at risk. tb also mainly affects medically neglected persons, such as vagrants, alcoholics, intravenous drug abusers or older homeless people. the main groups at increased risk for infection therefore include people who are resourcepoor or immunoincompetent, especially: tb in developing countries is particularly widespread and is increasing, the highest rises in incidence being in southeast asia, subsaharan africa and eastern europe. in highincome countries, the incidence is also rising, probably because of worsening social deprivation, homelessness, immigration, hiv infection and intra venous drug abuse. it is now as common in london as in the devel oping world, and is seen especially in immigrants, such as those from the indian subcontinent, africa and south asia. this increase appears to be a result of the development of tb disease in individu als who may have been infected for some time and of new infections acquired in the uk, or as a result of travel to other countries where tb is common. london accounted for the highest proportion of cases in the uk in 2011 (39%), followed by the west midlands region (11%); 74% of these were born outside the uk and mainly originated from south asia and subsaharan africa. in 2011, there was a rise in the number of tb cases compared to 2010, as well as an increase in drug resistance. more information on tb, including statistics, can be found at: http:// www.hpa.org.uk/publications/infectiousdiseases/tuberculosis/ and http://www.tbfacts.org/tbstatistics.html (both accessed 30 september 2013). initial infection with tb is usually subclinical. about 10% of those infected develop overt disease; of these, half will manifest within 5 years (primary tb), while the remainder will develop postprimary disease. inhaled mycobacteria may cause subpleural lesions (primary lesion) and lesions in the regional lymph nodes (primary complex). body defences usually localize the mycobacteria, though these remain viable; infected persons are not obviously ill and are unlikely to know they are infected (latent ; table 15 .16). latent tb infection (ltbi) usu ally becomes active only after many years, if body defences become weakened (box 15.3). however, active tb can develop shortly after mycobacteria enter the body, if body defences are impaired such as in ageing, drug or alcohol abuse, or hiv/aids. also, in massive infec tions, acute active tb can result, typically causing a chronic productive cough, haemoptysis, weight loss, night sweats and fever. erythema nodosum may be associated. extrapulmonary tb is less common; it may appear as glandular involvement in the neck or elsewhere, and is less infectious than pulmonary tb. lymph node tb may lead to lymphadenopathy, caseation of the nodes and pressure symptoms -for example, on the bronchi. postprimary tb follows reactivation of an old primary pulmonary lesion and results in features ranging from a chronic fibrotic lesion to fulminating tuberculous pneumonia. the pulmonary lesions may extend and lead to a pleural effusion. reactivation or progression of primary tb may also result in widespread haematogenous dissemina tion of mycobacteria -'miliary tb'. multiple lesions may involve the central nervous system, bones, joints, and cardiovascular, gastrointes tinal and genitourinary systems. clinical presentation in tb is thus variable, depending on the extent of spread and the organs involved. as it frequently passes unrecog nized for so long, the mortality is high. similar illnesses to tb may also be caused by atypical (nontuber culous) mycobacteria, such as m. avium complex (mac; see below). the diagnosis of tb is suggested by the history and confirmed by physical examination, a massively raised erythrocyte sedimentation rate (esr), positive tuberculin skin tests (tsts; mantoux or heaf test for a delayed hypersensitivity reaction to protein from m. tuberculosis [purified protein derivative; ppd]) and chest imaging. hypersensitivity develops with 2-8 weeks of infection and can be detected by conversion of the tst from negative to positive, but tsts are neither 100% sensi tive nor specific. a positive mantoux reaction indicates previous immu nization (bcg; bacille calmette-guérin -live attenuated m. bovis) or current infection -not necessarily disease. chest radiography may show scarring and hilar lymphadenopathy. computed tomography (ct) may show areas of calcification or highlight a tuberculous abscess. smears and culture of sputum, blood, laryngeal swabs, bronchoalveolar lavage, gastric aspirates or pleural fluid may be tested for mycobacteria. polymerase chain reaction (pcr) techniques have greatly acceler ated the diagnosis and speciation, though ziehl-neelsen, auramine or rhodamine microbial stains are still used. the mycobacteria growth indicator tube (mgit) system gives results as early as 3-14 days. blood assay for m. tuberculosis (bamt) may be positive by interferongamma release assay (igra). some 15% of people over 65 years have a positive igra. the igra can be used in place of (but not in addition to) tst. igras measure the immune reactivity to m. tuberculosis. white blood cells from most persons that have been infected with m. tuberculosis will release interferongamma (ifnγ) when mixed with m. tuberculosis antigens. a positive test result sug gests that m. tuberculosis infection is likely; a negative result suggests that infection is unlikely. latent infection (ltbi) can be diagnosed with either a tuberculin skin test or an igra (more specific). igra gives a result within 24 hours and should be used biological therapy is given, such as for rheumatoid arthritis or inflammatory bowel disease. prior bcg vacci nation does not cause a falsepositive igra test result. more informa tion on the igra is available at: http://www.cdc.gov/tb/publications/ factsheets/testing/igra.htm (accessed 30 september 2013). active tb is diagnosed by sputum microscopy and culture in liquid medium with subsequent drugsusceptibility testing. nucleic acid people who should be tested for tb include those who have symp toms, those who have had close daytoday contact with active tb disease (family member, friend or coworker), those who have hiv infection or aids, those with lowered immunity, those who are required to for employment or school, and those about to be treated with biological agents. the top priority of tb control programmes is to identify and give complete treatment to all patients with active disease. tb is a notifi able disease and contact tracing is an important aspect of limiting spread. treatment with antibiotics is indicated for people who are sick with tb, those infected but not sick, and those who are close contacts of infectious tb cases. treatment for 'symptomatic sputumpositive' patients, which should be instituted as soon as possible, is combination chemotherapy, usually isoniazid plus rifampicin plus pyrazinamide or ethambutol for 2 months, with continuation of daily isoniazid and rifampicin for a further 4 months. treatment for 'asymptomatic' patients who are believed to have been infected by contacts, but are not unwell, includes isoniazid for 6 months or isoniazid and rifampicin for 3 months. rifapentine is a longacting rifampicin used once weekly. fluoroquinolones (moxifloxacin) may also act against tb. there may be resistance to one or more than one antibiotic. currently, given the potential risk of drugresistant tb being present, treatment is usually started with isoniazid, rifampicin, pyrazinamide and ethambutol (or a quinolone such as gatifloxacin or moxifloxacin) for 2 months, then isoniazid and rifampicin for 4 months. all antituberculous drugs (table 15 .17) have potentially serious adverse effects and require careful monitoring. if patient compliance is considered to be poor, directly observed therapy (dot), where drugs are dispensed by and taken in the presence of a healthcare profes sional, may be indicated. new drugs are on the horizon. immunization using bcg is advocated for schoolchildren, highrisk individuals and healthcare professionals -although its efficacy has been questioned. new vaccines are in development. chemoprophylaxis with isoniazid and rifampicin is indicated in a number of situations (box 15.4) . tb can become resistant to the drugs used to treat it particularly when the drugs are misused or mismanaged. this may occur, for example, when: in some developing countries, approximately 10% of cases are multi ple antibioticresistant; this is termed multidrugresistant tuberculosis (mdrtb); in the uk, only a small minority currently fall into this category but the number of cases is increasing. mdrtb is defined as resistance to rifampicin and isoniazid; it may be atypical in presenta tion and the infection disseminates. more than 4% of people with tb worldwide have mdrtb, and eastern europe has a high prevalence. mdrtb is seen mainly in people with hiv/aids and in hiv/aids and in africans. bedaquiline, is a new antitubercular agent the first active agent against tuberculosis to be registered since 1963. extensively drugresistant tuberculosis (xdrtb) is a rare type of mdrtb, not only resistant to isoniazid and rifampin, but also to any fluoroquinolone and at least one of three injectable secondline drugs (i.e. amikacin, kanamycin, or capreomycin). xdrtb is of special concern for immunocompromised people (e.g. with hiv/aids), who are more likely to develop tb, and have a higher risk of death if they do develop it. xdrtb is most often encountered in people from eastern europe, russia and africa. it has been transmitted in healthcare facilities and is now seen worldwide. it is essentially untreatable, though capreomycin has been used effectively to treat mdrtb in hivpositive individuals. totally drugresistant tb was reported initially in 2007-2009 in india, iran and italy; it is spreading, despite denials, and is most disquieting. chronic ulcers, usually on the tongue dorsum, are the main oral manifestation of tb. they result from coughing of infected sputum from pulmonary tb, including in hivinfected persons with tb, but are rare and such cases (usually middleaged males) may result from neglect of symptoms or default from treatment. occasionally, the diagnosis is made from biopsy of an ulcer after granulomas are seen microscopically. acidfast bacilli are rarely seen in oral biopsies, even with the help of special stains, so unfixed material should also be sent for culture if possible. tuberculous cervical lymphadenopathy is the next most common form of the infection and is particularly com mon among those from south asia. most tb lymphadenitis is pain less, with several enlarged, matted nodes, but systemic symptoms are present only in a minority and only about 15% have pulmonary mani festations on radiography (fig. 15.7) . diagnosis relies on tuberculin testing, which can be positive in both tuberculous and non tuberculous mycobacterial cervical lymphadenitis. any person with lymphadenop athy and recent conversion from a negative to positive tuberculin test should be suspected of having mycobacterial infection, and this should prompt biopsy (e.g. fineneedle aspiration biopsy) for culture or histo logical confirmation. pcr will improve diagnosis, as culture must wait 4-8 weeks for a result. oral complications of antitubercular therapy are rare, but rifabutin and rifampicin can cause red saliva. pulmonary tb is of high infectivity, as shown by cases of tuber culous infection of extraction sockets and cervical lymphadenitis in 15 patients treated by an infected member of staff at a dental clinic. dental staff who themselves were hivpositive, working in a dental clinic for hivinfected persons in new york, have died from tb con tracted occupationally. transmission of mdrtb between two dental workers may have occurred in an hiv dental clinic. infection control is thus important, so staff with tb are usually precluded from their occupation until treated. management of a patient with tb depends upon the level of poten tial infectivity (table 15.18) . patients with open pulmonary tb are con tagious, and dental treatment is thus best deferred until the infection has been treated. treatment with appropriate drugs for 2 weeks drasti cally reduces the infectivity of patients with pulmonary tb. if patients with open pulmonary tb must be given dental treatment, special pre cautions should be used to prevent the release of mycobacteria into the air, to remove any that are present and to stop their inhalation by other persons. reduction of splatter and aerosols, by minimizing cough ing and avoiding ultrasonic instruments, and use of a rubber dam, are important. improved ventilation, ultraviolet germicidal light, new masks and personal respirators, and other personal protective devices, such as hepa filters, are indicated ( fig. 15.8) . mycobacteria are very resistant to disinfectants, so that heat sterilization must be used. la is safe and satisfactory. relative analgesia is contraindicated because of the risk of contamination of the apparatus. ga is also contraindicated for dental treatment because of the risk of contamina tion of the anaesthetic apparatus and because of impaired pulmonary function. aminoglycosides, such as streptomycin, enhance the activity of some neuromuscular blocking drugs and in large doses may alone cause a myasthenic syndrome. possible drug interactions are shown in table 15 .19. other factors, such as alcoholism or intravenous drug use (ch. 34), hepatitis (ch. 9) or hiv disease (ch. 20), may also influence dental management. mycobacteria other than tuberculosis (mott) are widely distributed in water, soil, animals and humans, and rarely cause disease. severe mott infections have been seen, however, in individuals predisposed because of defects in the interleukin12 (il12) and interferongamma (ifngamma) pathways. mycobacterium abscessus, a bacterium found in water, soil and dust, has been known to contaminate medications and products, including medical devices. healthcareassociated m. abscessus can cause a vari ety of infections, usually of the skin, but it can also cause lung infec tions in persons with various chronic lung diseases and is increasingly recognized as an opportunistic pathogen in cystic fibrosis (cf) patients persontoperson transmission of atypical mycobacteria is not important in acquisition of infection, except for skin infections. on rare occasions, mott skin infections have followed tattooing with contaminated tattoo inks. many people become infected with and har bour mott in their respiratory secretions without any symptoms or evidence of disease. individuals with respiratory disease from mott do not readily infect others and, therefore, do not need to be isolated. mott are generally not infectious to others. infection with m. abscessus is usually caused by injections of con taminated substances or by invasive medical procedures employing contaminated equipment or material. infection can also occur after accidental injury where the wound is contaminated by soil. there is very little risk of transmission from person to person. mac complex, m. scrofulaceum and m. kansasii are possible causes of tuberculous cervical lymphadenitis. mac may also infect the lungs (similar to tb), skin or lymph nodes. lung disease is also caused occasionally by m. kansasii, mainly in middleaged and older persons with underlying chronic lung conditions. m. fortuitum and m. chelonae may cause skin and wound infections and abscesses, frequently associated with trauma or surgery. m. marinum may cause 'swimming pool granuloma', a nodular lesion that may ulcerate, usually on an extremity. m. ulcerans may produce chronic ulcerative skin lesions, usually of an extremity. m. abscessus skin infections present with swollen and/ or painful areas that are usually red, warm and tender to the touch, and which can also develop into boils or pustules. other features of m. abscessus infection are fever, chills, muscle aches and malaise. cervical lymphadenitis due to mac, m. scrofulaceum and m. kansasii may affect otherwise healthy young children, most commonly pre school females who have unilateral cervical lymphadenopathy, typically in the submandibular or jugulodigastric nodes, and they may form a 'cold abscess'. mott is the usual cause in children under 12 years but tb is more common in older patients. absence of fever or tuber culosis, a positive tuberculin test and failed response to conventional antimicrobials are highly suggestive of mott, but definitive diagnosis is by smear, culture or pcr of biopsy material obtained by fineneedle aspiration or removal of nodes. treatment is based on results of laboratory testing, which should identify the appropriate antibiotic. preventive treatment of close contacts of persons with disease caused by mott is not needed. most mott are resistant to standard antitubercular medication and, though it is possible that clarithromycin or clofazimine may have some effect, excision of affected nodes is the usual recommended therapy. water from dental units may contain mott species; mycobacterial proliferation in biofilms may explain the extent of this contamination (ch. 31). aspiration syndromes are conditions in which foreign substances are inhaled into the lungs and which can have consequences ranging from asphyxia to infection and lung abscess. dental restorations or frag ments of teeth, plaque, gastric contents and other materials may be aspirated, especially if material enters the pharynx, and particularly if the cough reflex is impaired for any reason. most commonly, aspiration syndromes involve oral or gastric contents associated with gastrooesophageal reflux disease (gord), swallowing dysfunction (ch. 7), neurological disorders and structural abnormalities, such as a pharyngeal pouch. cricopharyngeal dys function involves cricopharyngeal muscle spasm or achalasia of the superior oesophageal sphincter, and can be seen in infants who have a normal sucking reflex but have incoordination during swallowing, pos sibly secondary to delayed development or cerebral palsy. anatomical disorders, such as cleft palate, pharyngeal pouch, oesophageal atresia, tracheooesophageal fistula, duodenal obstruction or malrotation, and motility disorders, such as achalasia, may have an aspiration risk. infirm older patients are also at risk of aspiration, especially if they are bedbound or have neurological disorders. isolated superior laryngeal nerve damage, vocal cord paralysis, cerebral palsy, muscular dystrophy and riley-day syndrome (familial dysautonomia) are all associated with increased risk of aspiration. ventilatorassociated pneumonia (vap), as defined by the centers for disease control and prevention (cdc), is present when the chest radiograph shows new or progressive infiltrate, consolidation, cavitation or pleural effusion in conjunction with either new onset of purulent sputum or change in character of sputum, and an organism isolated from blood, or the isolation of an aetiological agent from a specimen obtained via suction aspiration through an endotracheal or tracheostomy tube. the major route for acquiring endemic vap is oropharyngeal colo nization by endogenous flora or by exogenously acquired pathogens from intensive care units. vap is the most commonly reported health careacquired infection in patients receiving mechanical ventilation, with prevalence rates consistently in the 10-20% range. mortality rates in vap are at least double those in patients without vap, ranging from 24% to 85% when the infection is caused by a multidrugresistant gramnegative pathogen. the healthcare infection control practices advisory committee of the cdc has developed guidelines for the prevention of vap. these include strategies aimed at preventing aspiration of contaminated oral or gastric material (e.g. raising the head of the bed and draining subglottic secretions), and interventions to alter bacterial coloniza tion of stomach (e.g. stress ulcer prophylaxis and selective digestive decontamination) and mouth. oral hygiene, suctioning and the provi sion of moisture to lips and oral mucosa, plus toothbrushing, may be important in prevention of vap. there are also strategies for manag ing ventilator circuits (e.g. replacement of ventilator circuits, use of closed rather than open suction, and use of heat moisture exchange as opposed to heated circuit technology). lung abscess is a localized infection leading to cavitation and necro sis. while some cases result from aspiration of foreign material, most develop from pneumonia caused by infection with staph. aureus or klebsiella pneumoniae. bronchial obstruction by carcinoma is another important cause. symptoms resemble those of suppurative pneumonia. there is a risk of infection spreading locally or leading, via septicaemia, to a brain abscess. diagnosis rests mainly on the chest radiograph, which may sometimes show cavitation or a fluid level. antimicrobial chemotherapy, postural drainage and relief by bronchoscopy of any obstruction are indicated. a wellrecognized cause of lung abscess is inhalation of a tooth or fragment, a restoration or rarely, an endodontic instrument. when undertaking endodontics or cementing restorations, such as inlays or crowns, a rubber dam or other protective device should always be used to avoid the danger of inhalation. lung abscesses may also result from aspiration of oral bacteria, particularly anaerobes, especially in infirm older patients or those who are intubated. the other main dangers in dentistry are with ga, particularly if an inadequate throat pack has been used. patients who inhale tooth frag ments or dental instruments must have chest radiographs (lateral and posteroanterior) and, if necessary, bronchoscopy. loeffler syndrome appears to be an allergic reaction, usually to the parasitic worm ascaris lumbricoides, or drugs such as sulphonamides. it manifests with pulmonary infiltrates (and abnormal chest radio graph) and eosinophilia (eosinophilic pneumonia). the disease usually clears spontaneously. sarcoidosis, so named because skin lesions resembled a sarcoma, is a multisystem granulomatous disorder, seen most commonly in young adult females in northern europe, especially in people of african heritage. the aetiology is unclear but propionibacterium acnes and p. granulosum have been implicated and associations have been reported with exposure to inorganic particles, insecticides, moulds and occupations such as firefighting and metalworking. serum sam ples contain antibodies directed against mycobacterium tuberculosis antigens. sarcoidosis is associated with hladrb1 and dqb1, and a butyrophilinlike 2 (btnl2) gene on chromosome 6. thelper 1 (th1) cells release il2 and ifnγ, and augment macrophage tumour necrosis factor alpha (tnfα) release. cd25 regulatory t cells cause a limited impairment of cellmediated immune responses (partial anergy) but no obvious special susceptibility to infection. sarcoidosis affects the thorax in 90%, but has protean manifestations and can involve virtually any tissue (table 15 .20). sarcoid most typi cally causes löfgren syndrome (fever, bilateral hilar lymphadenopathy, arthralgia and erythema nodosum, especially around the ankles; figs 15.9 and 15.10). other common presentations may include pulmonary infiltration and impaired respiratory efficiency, with cough and dyspnoea in severe cases, or acute uveitis, which can progress to blindness. susceptibility to lymphomas has been suggested but not confirmed. because of its vague and protean manifestations, sarcoidosis is under diagnosed. in the presence of suggestive clinical features, helpful investigations include: chest radiography (enlarged hilar lymph nodes); raised serum angiotensinconverting enzyme (sace ; table 15 .21) in acute disease (this is insensitive, nonspecific and a poor guide to therapy); positive gallium67 citrate or gadolinium or positron emis sion tomography (pet) scans; labial salivary gland or transbronchial biopsy (for histological evidence of noncaseating epithelioid cell granulomas) -except in löfgren syndrome, which is a classical clini cal diagnosis. 18 fdeoxyglucose pet is helpful in identifying sites for biopsy. nonspecific findings may include mild anaemia, leukopenia, eosinophilia, hypergammaglobulinaemia, raised esr and low serum albumin. hypercalcaemia is common because of extrarenal produc tion of active vitamin d and can result in renal damage. alkaline phosphatase, 5'nucleotidase, lysozyme and adenosine deaminase levels are raised in hepatic sarcoidosis. evidence of impaired delayed hypersensitivity reactions to some antigens may be useful. kveim skin tests are not now used. half the patients with sarcoidosis remit within 3 years and about 66% remit by 10 years. patients with only minor symptoms usually need no treatment but corticosteroids, sometimes with azathioprine, methotrexate, tetracyclines, hydroxychloroquine, infliximab or etaner cept, are given if there is active organ disease (ocular disease, progres sive lung disease, hypercalcaemia or cerebral involvement). biopsy of the minor salivary glands frequently shows noncaseating granulomas and association with other features of sarcoidosis, par ticularly hilar lymphadenopathy. this is an important diagnostic find ing that may obviate more invasive procedures. sarcoidosis can involve any of the oral tissues but has a predilection for salivary glands. asymptomatic swelling of the parotid glands or cervical nodes, and less frequently the lips, may accompany systemic disease. superficial or deepseated red submucosal nodules may develop intraorally and on the lips. nontender, wellcircumscribed, brownishred or violaceous nodules with superficial ulceration have also been reported. the oral and lip lesions may occasionally precede systemic involvement. there is enlargement of the major salivary glands in about 6% of cases; some have xerostomia, and the association of salivary and lacri mal gland enlargement with fever and uveitis is known as uveoparotid fever (heerfordt syndrome). salivary swelling may also be seen with out other features of heerfordt syndrome. the salivary gland swellings usually resolve on treatment of sarcoidosis but this may take up to 3 years. facial palsy and other cranial neuropathies may be seen. there is also an association with sjögren syndrome, when ssa and ssb serum autoantibodies are found. rarely there is an association of thyroiditis with addison disease, sjögren syndrome and sarcoidosis (tass syndrome). there is a group of patients who have histologi cal features of sarcoid in one or more sites in the mouth, such as the gingivae, but no systemic manifestations. a few of these patients may ultimately develop other more or less systematized disease but the majority probably have isolated lesions. such cases, where no exog enous cause for the granulomatous reaction can be found, are regarded as having 'sarcoidlike' reactions (orofacial granulomatosis) and treat ment is unnecessary. however, patients should be kept under observa tion for as long as possible. management of patients with systemic sarcoidosis may include con sideration of respiratory impairment, uveitis and visual impairment, renal disease, jaundice or corticosteroid treatment. la is safe and satisfactory. cs is contraindicated if there is any res piratory impairment. ga should only be given in hospital. lung cancer is the most common cancer in highincome countries in males and most frequently affects adult urban cigarettesmokers. bronchogenic carcinoma accounts for 95% of all primary lung cancer and has also become increasingly common in women (because of increased tobacco use), to the extent that the mortality rate for the two sexes has become almost equal. metastases from cancers elsewhere are also frequently found in the lungs. recurrent cough, haemoptysis, dyspnoea, chest pain and recurrent chest infections are the predominant features. local infiltration may cause pleural effusion, lesions of the cervical sympathetic chain (horner syndrome), brachial neuritis, recurrent laryngeal nerve palsy or obstruction of the superior vena cava with facial cyanosis and oedema (superior vena cava syndrome). there are many nonmetastatic extrapulmonary effects of bron chogenic (or other) carcinomas -for example, weight loss, anorexia, fingerclubbing, neuromyopathies, thromboses (thrombophlebitis migrans), muscle weakness, various skin manifestations and ectopic hormone production (of antidiuretic hormone, adrenocorticotropic hormone, parathyroid hormone and thyroidstimulating hormone). metastases from bronchogenic cancer are common and typically form in the brain (which may manifest with headache, epilepsy, hemi plegia or visual disturbances), liver (hepatomegaly, jaundice or ascites) or bone (pain, swelling or pathological fracture). the diagnosis is based on history and physical examination, supported by radiography, ct and magnetic resonance imaging (mri), sputum cytology, bronchoscopy and biopsy. spiral ct appears to detect tumours at an early stage. the overall 5year survival rate is only 8%. radiotherapy is the most common treatment. only some 25% of patients are suitable for surgery but, even then, the 5year survival is only about 25%. chemotherapy has been disappointing, except in smallcell carcinomas. dental treatment under la should be uncomplicated. cs should preferably be avoided. ga is a matter for specialist management in hospital, as patients often have impaired respiratory function, espe cially after lobectomy or pneumonectomy. this, along with any muscle weakness (myasthenic syndrome, eaton-lambert syndrome) that can make the patient unduly sensitive to the action of muscle relaxants, makes ga hazardous. oral cancer may be associated with lung cancer, and vice versa, or develop at a later stage (ch. 22). such synchronous or metachronous primary tumours must always be ruled out. metastases can occasionally affect the orofacial region and cause enlargement of the lower cervical lymph nodes, epulislike softtissue swellings or labial hypoaesthesia or paraesthesia in the jaw. soft palate pigmentation is a rare early oral manifestation. lung cancer is a fairly common cause of death in dental techni cians, but it is unknown whether this is due to smoking alone or to dust inhalation. cystic fibrosis (cf) is one of the most common fatal hereditary dis orders. inherited as an autosomal recessive trait, with an incidence of about 1 in 2000 births, it is the most common inherited error of metabolism and is seen mainly in people of european descent. the gene responsible is on chromosome 7q. cf is caused by defects in the cystic fibrosis transmembrane conductance regulator (cftr), a protein that appears to be part of a cyclic adenosine monophosphate (camp)regulated chloride channel, regulating cl − and na + transport across epithelial membranes, and ion channels and intracellular fluid flow in sweat, digestive and mucus glands. the basic defect in cf is abnormal chloride ion transport across the cell membrane of nearly all exocrine glands. the blockage of salt and water movement into and out of cells results in the cells that line the lungs, pancreas and other organs producing abnormally thick, sticky mucus that can obstruct the airways and various glands, especially in the respiratory tract and pancreas. involved glands (lungs, pancreas, intestinal glands, intrahepatic bile ducts, gallbladder, submaxillary and sweat glands) may become obstructed by this viscid or solid eosino philic material. recurrent respiratory infections result in a persistent productive cough and bronchiectasis, with the lungs becoming infected with a variety of organisms including staph. aureus, haemophilus influenzae, pseudomonas aeruginosa, strep. pneumoniae, burkholderia cepacia, and sometimes mycoses or mycobacteria. mycobacterium abscessus is a nontuberculous mycobacterium increasingly recognized as an opportunistic pathogen in cf patients. viral infections, such as mea sles, can have severe sequelae. pancreatic duct obstruction leads to pancreatic insufficiency, with malabsorption and bulky, frequent, foulsmelling, fatty stools. gallstones, diabetes, cirrhosis and pancreatitis may result. sinusitis is very common. growth is frequently stunted. the mutations can also cause con genital bilateral absence of the vas deferens, so fertility is impaired in most males with cf. in women, fertility may be impaired by viscid cervical secretions, but many women have carried pregnancies to term. most patients have a high concentration of sodium in their sweat (also reflected in the saliva); a sweat test showing sodium and chloride values of more than 60 mmol/l is considered positive, between 40 and 60 mmol/l equivocal, and less than 40 mmol/l negative. physiotherapy and postural drainage are crucially important. clearance of sputum is helped by water aerosols and bronchodila tors (terbutaline or salbutamol), but mucolytics such as carbocisteine, methyl cysteine and dornase alfa are of questionable effectiveness. treatment with ivacaftor, a cftr potentiator, improves chloride transport through the ion channel. amoxicillin and flucloxacillin are effective prophylactic antimicrobi als and may be given by aerosol. vaccination against measles, whoop ing cough and influenza is important. a low fat intake, adequate vitamins and oral pancreatic enzyme replacement (pancreatin) are also necessary. doublelung or heart-lung transplantation may eventually become necessary. sinusitis is very common; most cf patients have recurrent sinusitis and nasal polyps. the major salivary glands may enlarge and hyposali vation sometimes occurs. the lowfat, highcarbohydrate diet and dry mouth may predispose to caries. enamel hypoplasia and black stain may be seen, and both dental development and eruption are delayed. tetracycline staining of the teeth was common but should rarely be seen now. pancreatin may cause oral ulceration if held in the mouth. la is satisfactory but cs is usually contraindicated because of poor respiratory function. ga is contraindicated if respiratory function is poor. lung disease, such as bronchiectasis, liver disease and diabetes, may complicate treatment. bronchiectasis is dilatation and distortion of the bronchi. causes include: ■ congenital defects, which should be considered in all patients include cystic fibrosis, kartagener syndrome, alpha1antitrypsin deficiency, collagen defects (e.g. marfan syndrome) there is no identifiable underlying cause in about 50% of adults and 25% of children. the damaged and dilated bronchi lose their ciliated epithelium and therefore mucus tends to pool, causing recurrent lrtis, typically with strep. pneumoniae, haemophilus influenzae or pseudomonas aeruginosa. overproduction of sputum, which is purulent during exacerbations, a cough (especially during exercise or when lying down) and finger clubbing are typical features, with recurrent episodes of bronchitis, pneumonia and pleurisy. haemoptysis is not uncommon. in advanced bronchiectasis, chest pain, dyspnoea, cyanosis and respiratory failure may develop. complications may include cerebral abscess and amyloid disease. chest radiography and pulmonary function tests are required. high resolution ct (hrct) is useful. postural drainage is important. antimicrobials, such as amoxicillin, cephalosporins or ciprofloxacin, are given for acute exacerbations and for longterm maintenance treatment. ga should be avoided where possible and is contraindicated in acute phases. workers exposed to airborne particles may develop pulmonary disease (pneumoconiosis), which ranges from benign (e.g. siderosis) to malig nant, as in mesothelioma from asbestosis (see appendix 15.1), but any pneumoconiosis can cause significant incapacity. ga may be contraindicated; the physician should be contacted before treatment. berylliosis may be a hazard in some dental technical laboratories, when lung cancer is more frequent. respiratory complications following surgical operations under ga include segmental or lobar pulmonary collapse and infection. they are more common after abdominal surgery or if there is preexistent respiratory disease or smoking (see also ch. 3), and can be signifi cantly reduced by smoking cessation, preoperative physiotherapy and bronchodilators, such as salbutamol. if postoperative pulmonary infection develops, sputum should be sent for culture, and physiotherapy and antibiotics should be given. the common microbial causes are strep. pneumoniae and haemophilus influenza; in this case, suitable antibiotics include amoxicillin and erythromycin. hospital infections may include other microorganisms, such as mrsa, klebsiella, pseudomonas and other gramnegative bacteria. inhalation (aspiration) of gastric contents can cause pulmonary oedema and may be fatal (mendelson syndrome); it is most likely if a ga is given to a patient who has a stomach that is not empty, has a hiatus hernia or is in the last trimester of pregnancy. prevention is by ensuring the stomach is empty preoperatively; if it is not, an anaes thetist should pass an endotracheal tube. antacids or an h2receptor blocker, such as cimetidine or ranitidine, may be given by mouth pre operatively to lower gastric acidity. if gastric contents are aspirated, the pharynx and larynx must be carefully sucked out. systemic corticosteroids have been recommended but probably do not reduce the mortality. respiratory distress in premature infants may be caused by immaturity of surfactantproducing cells, when the alveoli fail to expand fully; this necessitates endotracheal intubation for many weeks. it may, in turn, result in midface hypoplasia, palatal grooving or clefting, or defects in the primary dentition. the same oral effects may be seen with prolonged use of orogastric feeding tubes. the degree to which subsequent growth corrects these deformations is currently unknown, though the palatal grooves typically regress by the age of 2 years. using soft endotracheal tubes does not obviate this problem and, at present, the best means of avoiding palatal grooving appears to be the use of an intraoral acrylic plate to stabilize the tube and protect the palate. acute respiratory distress syndrome (ards) is a sequel to several types of pulmonary injury and some infections, including those with oral viridans streptococci. patients with endstage pulmonary disease are considered for potential transplantation, usually using a lung from a braindead organ donor. a combination of ciclosporin, azathioprine and glucocorticoids is usu ally given for lifelong immunosuppression to prevent a tcell, alloim mune rejection response. inhaled nitric oxide modulates pulmonary vascular tone via smooth muscle relaxation and can improve ventilation/perfusion matching and oxygenation in diseased lungs. early graft failure following lung transplantation has been described by various investi gators as reimplantation oedema, reperfusion oedema, primary graft failure or allograft dysfunction. pathologically, this entity is diffuse alveolar damage. see also chapter 35. a meticulous presurgery oral assessment is required and dental treatment must be undertaken with particular attention to establishing optimal oral hygiene and eradicating sources of potential infection. dental treatment should be completed before surgery. for 6 months after surgery, elective dental care is best deferred. if surgical treat ment is needed during that period, antibiotic prophylaxis is probably warranted. cardiopulmonary transplantation (heart and lung transplantation) is the simultaneous surgical replacement of the heart and lungs in patients with endstage cardiac and pulmonary disease, with organs from a cadaveric donor. all transplant recipients require lifelong immunosuppression to pre vent a tcell, alloimmune rejection response. see also chapter 35. a meticulous presurgery oral assessment is required and dental treatment must be undertaken with particular attention to establishing optimal oral hygiene and eradicating sources of potential infection. dental treatment should be completed before surgery. for 6 months after surgery, elective dental care is best deferred. if surgical treat ment is needed during that period, antibiotic prophylaxis is probably warranted. national institutes of health: national institute of allergy and infectious diseases healthcare infection control practices advisory committee guideline for the prevention of healthcare associated pneumonia nosocomial pneumonia: state of the science extensively drugresistant tuberculosis as a cause of death in patients coinfected with tuberculosis and hiv in a rural area of south africa a review of the possible role of oral and dental colonization on the occurrence of health careassociated pneumonia: underappreciated risk and a call for interventions reducing ventilatorassociated pneumonia through advanced oraldental care: a 48month study apic infection control and applied epidemiology: principles and practice sepp. ventilatorassociated pneumonia and oral care: a successful quality improvement project guidelines for preventing the transmission of mycobacterium tuberculosis in healthcare settings a randomized trial of dental brushing for preventing ventilatorassociated pneumonia pneumonia associated with a dental unit waterline the pathogenesis of ventilatorassociated pneumonia: its relevance to developing effective strategies for prevention aspects of human disease 32 chronic obstructive pulmonary disease (copd) aspects of human disease 31 in vitro antibacterial activities of oral care products against ventilatorassociated pneumonia pathogens the impact of a simple, lowcost oral care protocol on ventilatorassociated pneumonia rates in a surgical intensive care unit intermittent suction of oral secretions before each positional change may reduce ventilatorassociated pneumonia: a pilot study current trends and newer concepts on diagnosis, management and prevention of respiratory tract infections key: cord-347000-zxytdb0b authors: foweraker, juliet title: recent advances in the microbiology of respiratory tract infection in cystic fibrosis date: 2009-01-20 journal: br med bull doi: 10.1093/bmb/ldn050 sha: doc_id: 347000 cord_uid: zxytdb0b introduction: infection is a major cause of morbidity and mortality in patients with cystic fibrosis (cf). research on cf infection has highlighted differences from other respiratory infections—both in the range and the nature of the organisms—especially in chronic infection. this is a rapidly advancing field of microbiology and is bringing insights into the complexity and adaptations of bacteria causing chronic infection in the respiratory tract. areas of agreement and controversy: the epidemiology of some infections in cf has changed, with reduction in spread of burkholderia cenocepacia following patient segregation. conversely, epidemic strains of pseudomonas aeruginosa have emerged, which spread between patients; previously, most p. aeruginosa strains were patient-specific. studies on hypermutators, quorum sensing, biofilm growth and the development of molecular identification have shed light on pathogenicity, microbial adaptation to the host and complexity of infection in cf. non-tuberculous mycobacteria are emerging pathogens in cf; however, there is much to learn about pathogenicity and treatment of these infections. species of aerobic and anaerobic bacteria, more commonly encountered in the upper tract, are found in significant numbers in cf sputum. the significance of this is however under debate. finally, although the clinical relevance of conventional antibiotic susceptibility testing for chronic cf pathogens has been questioned, there are no clear alternatives. emerging areas for developing research: much has been learnt about pathogenicity, evolution of cf pathogens and development of antibiotic resistance. the need is to focus on clinical relevance of these observations to improve diagnosis, prevention and treatment of cf infection. cystic fibrosis (cf) is an inherited disorder due to a range of mutations in the cf gene affecting the camp-regulated chloride channel (cf transmembrane regulator or cftr). in the lungs, this leads to dehydration of the peri-ciliary layer such that the mucociliary escalator cannot perform its function of clearing particles (including microorganisms) from the airways. patients develop acute and later chronic respiratory infection leading to a cycle of further damage and less ability to clear infection. the range of micro-organisms causing infection differs from those in patients without cf with the commonest pathogens being staphylococcus aureus and pseudomonas aeruginosa. 1, 2 antibiotics are used to clear early infection, treat acute exacerbations of chronic infection and reduce the frequency of exacerbations (by use of long-term systemic or inhaled drugs). these treatments have had a major impact on the quality and survival of patients with cf. there are still however questions about the best way to diagnose infection and the use of antibiotics in patients who will need repeated courses during their lifetime. bacteria in chronic infection have different characteristics from those causing acute infection, including atypical phenotypes leading to difficulty in identifying some species in the laboratory. antibiotic resistance-both from infection with innately resistant bacteria and also the development of multi-resistant strainsis an increasing challenge to cf. although infection remains the main cause of morbidity and mortality, 3 recent advances in understanding the role of different species and modes of pathogenicity hold much promise for improved treatments. this review will focus on recent advances in cf microbiology, look at their relevance to the diagnosis and management of infection and highlight areas that need further development. specific antibiotic treatment strategies or practical guidelines for the diagnosis of infection will not be discussed here but are well reviewed elsewhere. 4, 5 staphylococcus aureus staphylococcus aureus is the most frequent pathogen in young infants with cf, but an uncommon cause of lower respiratory tract infection in patients without cf. it has been found in broncho-alveolar lavage (bal) fluid in 30% of cf children of average age of 3 months 6 and often persists despite treatment. 7 the use of anti-staphylococcal antibiotics has been clinically effective but the role of long-term prophylaxis for s. aureus is uncertain. 8 the small-colony variant (scv) phenotype of s. aureus is common in cf patients. 7 these bacteria have adaptations including antibiotic resistance and the ability to survive inside host cells, which may contribute to persistence in the cf airways. the colonies are very small, non-pigmented and are slow growing, so may be missed during routine culture, but the use of certain media may make them easier to recognize. the slow growth makes identification and susceptibility testing in automated systems unreliable. longitudinal studies have shown a significantly higher rate of genome alterations in persisting clones of s. aureus in cf sputum than those in the nose of healthy individuals, presumably in response to the selective pressures of the host response and repeated use of antibiotics in cf. 9 potential mechanisms for genome alteration are bacteriophage mobilization and hypermutability. hypermutator bacteria have mutations in dna repair or error avoidance genes such that the spontaneous mutation rate is raised. mutator strains of s. aureus have been identified in cf, some due to inactivation of a dna mismatch repair gene muts. 10 another mechanism that may help the long-term survival of s. aureus in the cf lung is the ability to form biofilms. this may be important for infection of medical devices and prostheses such as artificial heart valves and orthopaedic implants. the role of biofilms in cf is however unclear and needs further investigation. the increase in methicillin-resistant s. aureus (mrsa) in the general population has been a cause of concern in many countries. this is not because these organisms are necessarily more pathogenic, rather than treatment options are limited. the mechanism of resistance to methicillin confers resistance to all b-lactams ( penicillins and cephalosporins) and many strains of mrsa have also acquired resistance to unrelated antibiotics, such as quinolones and aminoglycosides. major attempts are being made to control the spread of resistant s. aureus, 11 with some success in the uk. however, the prevalence of mrsa in cf patients is increasing 12 and vancomycin-resistant s. aureus, although very rare, are being seen in non-cf patients. treatment alternatives to the glycopeptides are now available, but experience with them in cf is limited. several clearance regimens have been proposed, some surprisingly successful (up to 94%). 13 non-encapsulate and non-type b capsulate haemophilus influenzae are more common in infants and young children with cf than in older patients. there is little information on the epidemiology of long-term infection with h. influenzae in cf. a recent study showed that infections in cystic fibrosis infection was a dynamic process with most patients sequentially infected with different strains. repeated isolation of the same clone was only seen in 5 of 27 cf patients. 14 there was an increase in antibiotic resistance over time, with 37% of patients infected with h. influenzae resistant to two or more antibiotic classes. resistance included decreased susceptibility to ciprofloxacin, which is rarely seen outside cf. one-third of patients had hypermutable strains. in another cf study, a hypermutable h. influenzae was associated with mutation in muts. 15 over 11 months, this organism gradually became resistant to the antibiotics used to treat the patient. persistence of h. influenzae may be linked to ability to form biofilms. this was shown in otitis media in non-cf patients. structures consistent with biofilms have now been observed in the bal of asymptomatic cf patients. clinical concentrations of azithromycin have been shown to inhibit biofilm formation and reduce established biofilms of h. influenzae in vitro without affecting bacterial growth. 16 although streptococcus pneumoniae is a common respiratory tract pathogen, it is unusual in cf. rates of colonization range from 3 to 5% in different series. a recent study showed multi-resistance and possible hypermutators in isolates from cf. 17 streptococcus pneumoniae from cf respiratory samples were better at producing biofilms in vitro than blood culture isolates from non-cf patients, 18 which suggests that these organisms have the ability to persist in the cf lung. it is still however unclear whether s. pneumoniae acts as a harmless commensal in cf or has a role in acute exacerbation or facilitating infection with other pathogens. pseudomonas aeruginosa is the most common pathogen cultured from cf sputum. it may be seen early in infancy and is often cultured intermittently during childhood. eventually, chronic infection develops and this leads to a faster decline in lung function. the age at which infection becomes chronic has increased with better treatment of cf. this is particularly due to the practice of intense treatment with antipseudomonal antibiotics when p. aeruginosa is first cultured, which can delay the onset of chronic infection and increase life expectancy in cf. the patient may be clinically stable when chronically infected with p. aeruginosa but has repeated episodes of acute exacerbation with increased breathlessness, sputum production and feeling unwell. these exacerbations are also marked by a reduction in lung function. exacerbations are generally treated with a combination of systemic antibiotics. 5 inhaled antibiotics, most commonly colistin or tobramycin, can be used to help clear early infection. they can be effective as long-term prophylaxis, reducing frequency of acute exacerbation once infection is chronic. more recently, long-term azithromycin prophylaxis has been used in cf. this followed experience with treating diffuse pan-bronchiolitis, a condition described in japan that also leads to chronic infection with p. aeruginosa. the benefit of long-term oral azithromycin has been shown in cf, 19 with fewer exacerbations, better lung function and a reduction in inflammatory markers. macrolide antibiotics may act because of their anti-inflammatory activity or by modifying the pathogenicity of p. aeruginosa (see below). early epidemiological studies showed that cross-infection with p. aeruginosa was rare and that cf patients were infected with their own unique strains presumably acquired from the environment. some siblings did have the same strain but it was unclear if this was due to cross-infection or acquisition from a common source. in contrast, more recent studies revealed the spread of the so-called epidemic strains, some of which were multi-resistant. this would not necessarily be a particular concern except that some, for example, the liverpool epidemic strain (les), may be more pathogenic and can superinfect patients already colonized with other strains of p. aeruginosa. infected patients had more exacerbations, time in hospital and courses of antibiotics. 20 furthermore, when patients were first infected with multiresistant epidemic p. aeruginosa early aggressive treatment often failed. 21 this led to a policy in the uk of segregating patients with epidemic strains. the les genome was recently sequenced and work is progressing to determine the basis of transmissibility and enhanced pathogenicity. most strains of les are hyper-producers of pyocyanin. this is a putative pathogenicity factor with effects including reduced beating of respiratory epithelium cilia and induction neutrophil apoptosis, and is regulated by quorum sensing (qs) (lasa). 22 qs systems have been extensively studied in p. aeruginosa. molecular signals allow communication between bacteria. 23 they are secreted and at a certain concentration can trigger expression of genes. in p. aeruginosa, these include those that code for pathogenicity factors, biofilm formation and motility. it is thought that 5% of genes in p. aeruginosa are regulated by qs and this mechanism allows precise regulation of genes in response to environmental stimuli and cell density. some plants naturally form antagonists to qs molecules (acylated homoserine lactones) as a defence mechanism. qs is therefore a potential target for new antimicrobial therapies. in the laboratory, bacteria isolated from chronically infected patients differ from the usual p. aeruginosa cultured from acute infection. clonal bacteria can form colonies with different appearance referred to as colonial morphotypes. the classical cf isolate is mucoid, due to hyperproduction of alginate. other morphotypes include bacteria that look like coliforms, non-pigmented forms and slow-growing scvs. 24 these latter are hydrophobic, have reduced swimming motility and form good biofilms in vitro. they may be missed in the routine laboratory because they can take more than 48 h to appear. phenotypic variation is not restricted to colonial morphology but includes type iii secretion, auxotrophy, lipopolysaccharide (lps) modification (smooth to rough) and both resistance and hypersusceptibility to antibiotics. variability in antibiotic susceptibility in a population of bacteria from a cf sputum includes differences between bacteria of the same clone and morphotype. as a result, susceptibility testing is poorly reproducible as the result may depend on which bacteria are picked for testing. 25 the role of antimicrobial susceptibility testing for helping the clinician who chose the antibiotics to treat acute exacerbations in cf is controversial. there are anecdotal reports of poor correlation between antimicrobial susceptibility and treatment of acute exacerbation. methods to improve the detection of resistant sub-populations have been described. 25, 26 however, the only big study to correlate antibiotic resistance to clinical response showed that lung function (forced expiratory volume in one second) improved in patients in spite of treatment with antibiotics to which their p. aeruginosa were resistant in vitro. 27 antibiotics seldom if ever eradicate chronic infection with p. aeruginosa from the cf lung. even the resolution of symptoms and signs of an exacerbation may not be matched with a significant reduction in bacterial load. it may be therefore that antibiotics are effective without actually inhibiting or killing the bacteria-the so-called sub-minimum inhibitory concentration (mic) effect. this may also explain the successful treatment of infection with antibiotics, even though the bacteria are resistant. for example, at sub-mic concentration, ceftazidime can inhibit attachment to cells and ciprofloxacin can reduce alginate production. 28 although p. aeruginosa is intrinsically resistant, azithromycin may reduce pathogenicity by inhibition of qs and biofilm formation. pseudomonas aeruginosa is innately resistant to many antibiotics and acquired resistance is increasing. 29 synergy testing has been advocated as a way to find combinations of antibiotics effective against multiresistant p. aeruginosa. time-kill curves, chequerboard methods and the multiple combination bactericidal test (mcbt) are the most commonly used methods and synergy can be shown in vitro. different results can however be obtained with different methods and there is no accepted reference standard. 30 there are few studies that attempt to correlate the results of synergy testing with the clinical outcome. one randomized prospective study compared the mcbt with traditional single antibiotic susceptibility testing for determining treatment of acute exacerbation with a range of multi-resistant species in cf including p. aeruginosa, stenotrophomonas maltophilia, achromobacter xylosoxidans and burkholderia cepacia complex (bcc). this showed no better outcome when antibiotics were chosen on the basis of the result of mcbt. 31 the role of current methods of synergy testing is controversial, but there is a definite need for clinically validated in vitro tests to guide treatment of multi-and pan-resistant bacteria at acute exacerbation and when patients receive lung transplants. the rapid development of antibiotic-resistant p. aeruginosa may be partly due to the presence of hypermutator strains. these bacteria have defects in their ability to correct mistakes in dna replication because of a range of mutations, including in the mismatch repair genes such as muts. strong mutators may have a mutation rate as high as 1â 10 25 to 10 24 , compared with the normal rates of 1â 10 28 to 10 27 . one study showed hypermutators in 37% of cf patients with chronic infection with p. aeruginosa. 32 this was the highest prevalence described in a natural system. since then hypermutators have been shown in other chronic lung diseases. 33 hypermutator strains were found amongst isolates of p. aeruginosa early in infection suggesting that they may have an advantage in initiating infection as well as in chronic infection, however they were infrequent. 34 it was questioned whether mutator strains would be at a disadvantage because of the high rate of damaging spontaneous mutations. however, their ability to rapidly adapt to a changing environment becomes an advantage in an ecological niche repeatedly exposed to antibiotics. in the large population of bacteria in the cf lung, an organism with a mutation for antibiotic resistance may be present even before the antibiotic is given. it has been practice in cf to give two anti-pseudomonal antibiotics to reduce the emergence of resistant strains. the recent observation of hypermutators in cf shows the potential importance of that recommendation. in common with other bacteria seen in cf, some p. aeruginosa form biofilms in vitro and can be observed in biofilm-like aggregates in expectorated sputum. biofilms are communities of bacteria within an acellular matrix, usually attached to a surface-in cf, the respiratory epithelium. in the patient, biofilms may also contain other species of bacteria and host-derived products. bacteria in biofilms are difficult to eradicate as they can resist the host immune system and are more antibiotic resistant by a variety of mechanisms. antibiotics may bind to the extracellular matrix or have reduced activity in areas of poor oxygenation or low ph. also the bacteria in a biofilm live in a gradient of oxygen, nutrients and metabolites. they are therefore diverse, having adapted to the different micro-environments. the growth phase (actively dividing or dormant) may affect their susceptibility to some antibiotics. 35 the variability in antibiotic susceptibility seen in the population of bacteria cultured from a single sputum under the same in vitro conditions may be because bacteria are present in biofilm fragments in the sputum. these organisms may have adapted to a range of different active concentrations of antibiotic encountered in different levels of the biofilm. concern that laboratory methods developed to measure the antibiotic susceptibility of planktonic bacteria causing acute infections may not be relevant to cf has led to development of methods to test antibiotic susceptibility in a biofilm. evaluation of one method to determine the optimum antibiotics to clear chronic infection is in progress. 36 a recent study using whole genome analysis showed an accumulation of mutations in 68 genes of p. aeruginosa over 8 years in a patient with cf. 37 this evolutionary divergence was thought to favour adaptation. early and late isolates of p. aeruginosa from 29 patients were compared to look at these genes. one of the commonest mutations was in las r, the principle qs regulator in p. aeruginosa. this interferes with many pathogenicity factors including biofilm formation. this is difficult to reconcile with the proposed importance of biofilms in persistence of infection. other genes commonly mutated after chronic colonization are also involved in pathogenicity e.g. exo a, the regulator of type iii secretion and antibiotic resistance (genes coding for components of multi-drug efflux pumps). the picture that may be emerging is therefore of an organism with a great ability to adapt to its environment that becomes less pathogenic with time, presumably in itself a device for long-term survival in its host. a recent study has shown that antibiotic-resistant genes are not over-represented in hypermutators and that hypermutability may play an important more general role in genome evolution and adaptation in the cf lung. 38 pseudomonas aeruginosa and burkholderia spp. (see below) in cf have a range of strategies to enable them to persist in the cf lung, including constantly adapting to a changing environment. 3 not only are these bacteria difficult to eradicate, but also the phenotypic diversity observed in the laboratory makes the design and validation of new tests of antibiotic susceptibility very difficult. future research is needed to understand the mechanisms of pathogenicity and persistence, treatment to prevent or clear infection and the development of appropriate laboratory test of susceptibility relevant to clinical outcome. isolates of the species b. cepacia ( previously pseudomonas cepacia) have been shown to consist of phenotypically similar but genotypically distinct genomovars. 39 subsequently, the number of genomovars has increased and all have achieved species status. presently, the so-called bcc comprises at least 15 species. 40 they are natural soil bacteria, found in the rhizosphere and can be plant symbionts or pathogens. while they can be opportunist pathogens affecting patients who are severely immuno-suppressed or following contamination of fluids or medical devices, they only cause significant primary infection in patients with cf or chronic granulomatous disease (cgd). while patients with cgd have a specific immune defect in the oxidative burst pathway, the reason for the association with cf is still unknown. patients with non-cf bronchiectasis do not get infected with bcc. these are complex organisms with a range of pathogenicity factors, many regulated by qs systems, similar to p. aeruginosa. 41 burkholderia spp. are resistant to colistin and often multi-or even panresistant. some combinations of antibiotics are effective in vitro but there is little evidence to correlate the results with the clinical response to treatment. 31 the importance of bcc in cf was first recognized following outbreaks in the 1980s. 42 by the 1990s, the et12 strain of burkholderia cenocepacia was the most common in the uk and canada. this was highly transmissible and pathogenic and led to many deaths due to a rapidly progressing pneumonia sometimes with bacteraemia, referred to as the 'cepacia syndrome'. bcc survives well in natural and healthcare environments and some strains most notably the et12 lineage of b. cenocepacia easily spread between patients. controls to reduce cross-infection including segregating patients have been the main reason that infection and mortality has declined significantly. in the uk, the most common species seen is burkholderia multivorans and most new infections are caused by unrelated clones, suggesting an environmental source rather than person-to-person spread. infection with bcc is more common in older cf patients but can occur at any time in the patient's life. cepacia syndrome can occur when the organism is first acquired or unexpectedly many years after initial infection. it is most frequently due to b. cenocepacia; however, both b. multivorans and burkholderia dolosa can also cause the 'cepacia syndrome'. 43 of the potential pathogenicity factors (lps, specific cytotoxins and the ability to form biofilms) many are controlled by qs. in addition, bcc are able to survive in cultured human epithelial cells and macrophages. severity of disease may be strain rather than species-specific; however, it may also be dependent on patient factors and interactions with other pathogens. of note, neither the presence of cable pili nor the presence of b. cepacia epidemic strain marker is now considered as a reliable indicator of virulence or transmissibility. 42 chronic infection may develop with acute exacerbations and a gradual decline in lung function. while transmissible species can replace other burkholderia spp. causing chronic infection, exacerbations are not associated with acquisition of a new species or strain. 44 factors that lead to more severe disease with certain species or strains are still unclear. the relevance of other members of the complex is unclear. prompt and accurate laboratory identification of bcc is very important because of the implications for the patient's care and the need to prevent cross-infection. commercial identification systems are not reliable but the use of selective culture media and molecular identification methods have greatly improved detection. the epidemiology of b. pseudomallei is totally different from bcc. this is a well-recognized human pathogen that can cause melioidosis with severe pneumonia in individuals who are immuno-competent and do not have the cf gene defect. there are reports of infection in cf patients living in or travelling to endemic areas. 45 not all infections lead to significant disease. the bacteria may however persist in the cf lung with the risk of later reactivation. other gram-negative bacilli (e.g. achromobacter xylosoxidans, stenotrophomonas maltophilia, pandoraea apista) some gram-negative bacilli are either more prevalent in cf now (s. maltophilia) or have been recognized more recently due to improvements in laboratory identification (a. xylosoxidans, pandoraea apista). these bacteria may have been selected by prolonged use of antipseudomonal antibiotics with the increasing life expectancy of cf patients. recent studies of s. maltophilia support earlier observations that this organism may be of limited clinical significance in cf. 46 despite increasing culture from cf patients with more severe disease, it is not associated with pulmonary deterioration. it is intrinsically resistant to meropenem and can acquire resistance to many other antibiotics. while co-trimoxazole is the treatment of choice in symptomatic infection, the best management of co-trimoxazole-resistant infections is uncertain and in vitro susceptibility testing of other agents with current methods is not reliable enough to be used to determine treatment. like stenotrophomonas, achromobacter ( previously alcaligenes) xylosoxidans may chronically colonize the cf lung but has not been shown to lead to significant deterioration in lung function, but more data are needed. 47 pandoraea apista has only recently been identified in cf sputum. as it is intrinsically resistant to colistin, it may also be mis-identified as a burkholderia sp. more information on the clinical relevance of this organism is needed, but cross-infection with clinical deterioration has been described in six patients in a danish cf centre. 48 a wide range of other gram-negative bacteria can be isolated from cf sputum including species of the genera ralstonia, chryseobacterium, comamonas, moraxella, acinetobacter, bordetella, inquilinus and members of the family enterobacteriaceae. 49 there are insufficient data to comment on the clinical relevance of these bacteria and they may be harmless colonizers. it is important however that they are correctly identified. this is so that any association with significant disease may be recognized but also because some may be mistaken for known pathogens. uk reference laboratory reports suggest that around 10% bacteria referred as members of the bcc were initially mis-identified. a similar mis-identification rate occurs between a. xylosoxidans and atypical multi-resistant forms of p. aeruginosa. a recent paper highlights the difficulty identifying cf bacteria with atypical phenotypes using traditional methods (colony morphology and biochemical identification) and the authors advocate the use of nucleic acid-based identification methods such as species-specific pcr or ribosomal rna gene sequencing. 50 non-tuberculous mycobacteria (ntm) are being increasingly recognized as a cause of infection in cf. this may be partly because more clinicians are sending samples for ntm culture and laboratories are getting better at isolating ntm. however, there is also thought to be a genuine increase in the prevalence of lung infection with ntm as patients with cf survive longer. automated liquid systems allow more rapid culture of ntm and may be more sensitive than the traditional use of lowenstein jensen agar slopes. the high number of p. aeruginosa in cf sputum can however cause problems with decontamination and make ntm culture difficult. 51 ntm are ubiquitous in the environment and can be found in dust, food and drinking water. cf patients are therefore repeatedly exposed to ntm and it is not surprising that their sputum can be infected. infection in cf is restricted to the lungs without the disseminated ntm disease found in patients with aids. in a study of 21 usa cf centres, 128 (13%) of 986 cf patients over 10 years old had ntm cultured from at least one of three sputa over 12 months. 52 of these 25 (2.5%) met the ats criteria for ntm disease, although the relevance of these criteria for patients with cf has not been assessed. infection with ntm was more common in older patients and was associated with better lung function and co-infection with s. aureus rather than p. aeruginosa. there appears to be a geographical influence on the overall prevalence; this ranged from 24% in new orleans to 7% in boston. it is uncertain however whether these differences relate to different climate or vegetation or reflect the antibiotic and steroid usage in those cf clinics. members of the mycobacterium avium complex were most frequently isolated (72% of patients), with m. avium, the predominant species. almost all patients had unique strains with no evidence of cross-infection and 10% had mixed species. mycobacterium abscessus only accounted for 16% of ntm cultured in the us study but has been increasingly reported in cf patients in europe. 53 this seems to be associated with a more severe infection, and research on pathogenicity is needed. m. abscessus colonies can either be rough or smooth depending on the expression of glycopeptidolipid (gpl). it has been proposed that the smooth form (with gpl) forms biofilms in vitro and is less pathogenic-favouring colonization whereas the rough form produces little gpl, infects human monocytes and causes persistent invasive infection in a mouse model. 54, 55 a similar relationship between colonial morphology and virulence has been proposed for m. avium and m. kansasii. there have however been no studies to test the clinical relevance of these observations. in common with other bacteria that may persist in biofilms, m. abscessus appears less susceptible to antibiotics when grown in biofilms than in planktonic cultures. 56 the clinical relevance of ntm can be difficult to assess in cf as these patients usually have other pathogens in their sputum. in addition, ntm of different species may be cultured on different occasions such that it can be difficult to distinguish repeated acquisition and loss from the environment from established infection. all isolates therefore need to be identified rather than assuming that repeat positive cultures of mycobacterium spp. indicate persistent infection. difficulties with identification and changes in taxonomy mean that previous infection attributed to m. fortuitum complex or with m. chelonae may have been due to m. abscessus. the role of in vitro susceptibility testing of m. abscessus is in question as correlation with clinical response is poor. only susceptibility testing to the macrolides (clarithromycin, azithromycin) is recommended for m. avium complex. 57 areas that need more work include reliable culture and identification of ntm, clinically relevant tests to measure antibiotic susceptibility and more understanding of pathogenicity mechanisms, in particular, for m. abscessus. building on the ats guidelines, more specific definitions for cf to distinguish transient carriage, colonization and clinical infection are needed for trials of treatment. various species of aspergillus, most commonly aspergillus fumigatus, can be cultured from cf sputum. the fungal spores are common in the air and water, and are usually derived from rotting vegetation. aspergillus may contaminate sputum containers or even laboratory cultures. in cf, it can cause allergic broncho-pulmonary aspergillosis (abpa). diagnostic criteria for abpa in cf have been published 58 and patients are treated with steroids and antifungals. aspergillomas have also been seen in cf but are rare. a recent paper has described a bronchitis in six patients with infection with aspergillus without the features of abpa who responded to antifungal therapy. 59 scedosporium apiospermum has also been associated with the abpa-like picture in cf. 60 candida spp. are commonly cultured from cf sputum. this is not unexpected given that much antibiotic treatment in cf is broad spectrum and can lead to an overgrowth of candida spp. on mucosal surfaces. some cultures are therefore due to oral contamination of sputum but colonization of sputum is also possible. there is no current evidence of a significant role for candida spp. in lower airway infection. the possible role of normal oral flora in cystic fibrosis sputum bacteria usually labelled as normal flora in the mouth and upper airways are often grown from sputum and even from samples taken at bronchoscopy. these were thought to be due to contamination with saliva, however these bacteria may be present in such large numbers that it is more likely that sputum was seeded during small-volume aspiration and multiplication takes place in the sputum. 61 two main lines of investigation have focused on these bacteria. careful culture techniques have shown the large number and diversity of anaerobes that can be cultured from cf sputum. 62 the use of 16s rrna gene profiling by terminal restriction fragment length polymorphisms directly from cf sputum has shown a great diversity of anaerobic and aerobic species. 63 what is the significance of these observations? it may be that these flora are just bystanders (commensals). alternately, some may actually be pathogenic and have been missed because they are present in a mixture with an assumed pathogen such as p. aeruginosa. this could even explain why cf patients may improve when treated with antibiotics to which the conventional pathogen is resistant in vitro. 27 another explanation is that members of the 'normal' flora may interact with the conventional pathogen. in one study, a viridantype streptococcus was shown to enhance the pathogenicity of p. aeruginosa in an agar bead lung infection model in rats. 61 this viridans-type streptococcus and a coagulase-negative staphylococcus, both isolated from cf sputum, also upregulated p. aeruginosa genes. these included those coding for pathogenicity factors such as elastase (lasb). respiratory viral infections are common in children, but there have been relatively few well-controlled studies on the specific role of respiratory viruses in cf. 64 this is partly because the techniques for identifying viruses by culture or serology have been too insensitive. respiratory viruses are thought to be a trigger or co-factor in acute infective exacerbations of copd 65 and it has been proposed that they may play a role in developing chronic infection with bacterial pathogens. early danish data showed that cf patients were more likely to acquire their first p. aeruginosa or develop chronic pseudomonal infection between october and march-the peak season for respiratory viruses. this finding was consistent over 25 years in spite of major changes in treatment of bacterial infection. the rapid technical advances, for examples, with the use of micro-arrays, should allow more in-depth studies of the role of viral infections in cf. 66 influenza a can have a significant impact in patients with cf. anti-viral drugs are recommended in the uk for patients with chronic lung disease including cf, both for treatment and prevention after exposure. annual vaccination is also recommended for patients with cf. the microbiology of cf is complex. a pattern of acute, intermittent and eventually chronic infection is seen. both familiar pathogens and bacteria not normally seen in the respiratory tract are encountered. the relevance of new insights into the pathogenicity and epidemiology of old pathogens in cf is being assessed and the significance of other micro-organisms, previously not recognized in cf, is being investigated. although there have been exciting advances in basic science, we still need to understand what triggers exacerbations, how to test bacteria from cf in the laboratory for the most accurate and clinically relevant results, and what are the best options for prevention and treatment of infection. lesson learned from the complex interactions between bacteria and adaptations to chronic infection seen in the cf lung hold promise for better management of infection in cf. these may also help develop strategies to improve the outcome of other chronic infections, both in the lung and elsewhere. microbiology of sputum from patients at cystic fibrosis centers in the united states microbiology of lung infection in cystic fibrosis persistent and aggressive bacteria in the lungs of cystic fibrosis children report of the uk cystic 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characterization of unusual bacteria isolated from respiratory secretions of cystic fibrosis patients and description of inquilinus limosus gen. nov., sp. nov superiority of molecular techniques for identification of gram-negative, oxidase-positive rods, including morphologically nontypical pseudomonas aeruginosa, from patients with cystic fibrosis atypical mycobacterial infection in patients with cystic fibrosis: update on clinical microbiology methods nontuberculous mycobacteria. i: multicenter prevalence study in cystic fibrosis age-related prevalence and distribution of nontuberculous mycobacterial species among patients with cystic fibrosis hypervirulence of a rough variant of the mycobacterium abscessus type strain spontaneous reversion of mycobacterium abscessus from a smooth to a rough morphotype is associated with reduced expression of glycopeptidolipid and reacquisition of an invasive phenotype differential antibiotic susceptibility of mycobacterium abscessus variants in 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rhinovirus diversity key: cord-324880-s1oqkqef authors: xu, lili; gao, hengmiao; zeng, jiansheng; liu, jun; lu, cong; guan, xiaolei; qian, suyun; xie, zhengde title: a fatal case associated with respiratory syncytial virus infection in a young child date: 2018-05-11 journal: bmc infect dis doi: 10.1186/s12879-018-3123-8 sha: doc_id: 324880 cord_uid: s1oqkqef background: respiratory syncytial virus (rsv) is the most common viral cause of pediatric bronchiolitis and pneumonia worldwide. risk factors for high mortality and prolonged morbidity after rsv infection include premature birth, bronchopulmonary dysplasia, congenital heart disease, and down syndrome. however, some previously healthy, full-term children who are infected with rsv also require hospitalization and even experience severe sequelae or death. case presentation: in this report, we present the case of an rsv-associated death of a child who was born at full-term and developed normally up to the age of 2 years old. cardiopulmonary arrest occurred within 3 days after the onset of symptoms, which included cough and high fever. complete brain edema was prominent, and encephalopathy was developing. viral antigen detection and microbiome analyses of oral swab and nasopharyngeal aspirate specimens verified an rsv infection, while bacterial culture of blood specimens yielded negative results. the rsv strain detected in this patient was subtyped as rsvb9, and no mutation was found in the six antigenic sites for targeted drugs or vaccines. conclusions: the patient had a severe infection associated with rsv, which was very likely the cause of her central nervous system infection and acute neurological complications. respiratory syncytial virus (rsv) is the major cause of lower respiratory tract illness in children. for most children, an initial rsv infection normally occurred within the first 2 years of life. in infants less than 1 year of age and with lower respiratory infection, up to 80% are due to rsv [1] . in most cases, the virus is not fatal. the most severe infections and well-defined high-risk groups, including infants with a history of premature birth, and those with chronic lung disease, congenital heart disease, cystic fibrosis and immunodeficiency [2] . for those high-risk infants, palivizumab, a humanized monoclonal antibody which has produced favorable results to date, is strongly recommended to be administered prophylactically [3] . however, most children with rsv infection were previously healthy, and it is often difficult to predict deterioration of rsv infection [2, 4] . rsv-related encephalitis with acute encephalopathic symptoms such as seizure, severe sequelae and even death following rsv infection in children without underlying disease has sporadically been reported [5] . rsv-related encephalitis usually develops within 1 to 2 days after the onset of clinical symptoms, such as high fever, cough, and fatigue. however, the mechanism underlying the rapid progression of related encephalitis remains unclear. central nervous system (cns) infection, coinfection with bacteria, and dysfunction of the host immune system may be possible causes. next-generation sequencing (ngs) accompanied with metagenomic analysis can be used as a nonselective method for pathogen discovery and is increasingly applied as a diagnostic tool to investigate the causes underlying unexplained encephalitis in patients. two key features of this methodology are: (1) it makes no assumptions about the type of pathogen and has the potential to detect nucleotides from all species, and (2) the causative pathogen may not necessarily be the most abundant signal in the ngs results and may sometimes be present as only a low-level signal compared to all other signals associated with commensal pathogens. it can, however, provide unbiased and sensitive identification. in this report, we present the case of a 2-year-old girl who was not born prematurely and had no underlying disease whose sudden death may have been related to an rsv infection identified by conventional methods and metagenomic analyses. the patient in this fatal case was a 2-year-old girl who was born full-term and had developed normally. she had no medical history of asthma or pneumonia and no familial history of immunodeficiency. she had no brothers or sisters. the patient was admitted to the hospital due to 3 days of fever and 15 min of respiratory and cardiac arrest. the symptoms started on 10 november (3 days before admission), with a fever (up to 39.8°c) but no chills, rash or convulsions. ibuprofen was given orally. the body temperature decreased for 4 to 5 h but then climbed to 40.3°c. shortness of breath accompanied the fever, and the body temperature did not decrease obviously after the oral administration of ibuprofen. wheezing caused by the retention of phlegm in the throat and a single paroxysmal cough occurred. the patient occasionally coughed up a small amount of yellow phlegm and had a slightly runny nose. she had no asthma, breathing difficulty or hemoptysis; she had lethargy and a poor appetite but no vomiting or diarrhea. on 11 november (2 days before admission), the patient still had a high fever, and her body temperature fluctuated around approximately 40°c. the patient's body temperature did not obviously decrease after she was given oral ibuprofen and acetaminophen alternately. acute infection was considered from then on. the patient received an intravenous infusion of 0.14 g zithromax and 120 mg rographolide as well as aerosol inhalation of 2 mg budesonide, but her fever persisted, and her body temperature rose to a peak of 40°c. on 12 november (1 day before admission), the patient still had a persistent fever and wheezing due to the retention of phlegm in her throat. she had shortness of breath, a light cough, and a drooping spirit, accompanied by a rash on the torso and limbs. her appetite was slightly improved, and she had no vomiting or convulsions. on 13 november, the patient had sudden respiratory and cardiac arrest 3 h before admission. she was immediately and continuously treated with cardiopulmonary resuscitation by physicians and the intravenous injection of adrenaline (4 times). she was treated with trachea cannula and mechanical ventilation, and her heart beat recovered approximately 15 min later, but the patient remained in a deep comatose state with no spontaneous breathing. then, the patient was transferred to our hospital and immediately underwent electrocardiogram (ecg) monitoring. bloody fluid was visible in the indwelling gastrointestinal decompression tube, and the blood-gas analysis showed metabolic acidosis. the patient was treated with sodium bicarbonate to correct the acidosis. she was diagnosed with an acute cns infection and brain hernia. after cardiopulmonary resuscitation, she was admitted to the pediatric intensive care unit (picu). the head ct scan showed extensive brain swelling, decreased brain parenchymal density, narrowed cerebral ventricles and cisterns. these findings prompted a diagnosis of extensive brain edema and hernia. the chest posteroanterior radiograph showed fuzzy, coarse bilateral lung markings, visible small patchy shadows in the right inferior lung and a clear pulmonary hilus. these findings were diagnosed as pneumonia. routine blood examination results suggested the presence of a bacterial infection; thus, the patient was treated with vancomycin and meropenem to control an infection. after that, immunoglobulin (1 g/kg) was administered for immune support. the patient was still in a deep coma state, and light reflexes of both pupils were absent. the patient's spontaneous breathing was weak and irregular, and she had no response to painful stimulation. compared with earlier, her rash was reduced, and the pulmonary lesions shown on the chest posteroanterior radiograph were slightly absorbed. immunoglobulin (1 g/ kg) was continuously administered to neutralize pathogens. on 15 november (3 days after admission), transcranial doppler ultrasound assessment showed that the patient's anterior and posterior cerebral circulation corresponded to the diagnostic criteria for brain death. on 17 november (5 days after admission), various organ functions failed, and the patient could not tolerate a spontaneous breathing test. her guardian chose to quit treatment, and the patient died. viral antigen detection based on both an immunofluorescence assay and the luminex xtag respiratory viral panel assay was positive for rsv in the patient's nasopharyngeal aspirates (which were collected on 14 nov, the 5th day of disease onset and the 2nd day of admission) and negative for adenovirus, influenza a and b viruses, parainfluenza virus 1-4, human metapneumovirus, enteroviruses and rhinoviruses, human coronavirus hku1, 229e, nl63 and oc43, and human bocavirus. because the patient's guardian refused to consent to lumbar puncture, cerebrospinal fluid (csf) was not available for the detection of cns infection. the blood biochemistry results are summarized in table 1 . the amounts of red blood cells (rbcs), hemoglobin, and platelets continuously decreased after the onset of symptoms. extremely high levels of the percentage of t lymphocytes was 46.6%, of which helper t cells and suppressor t cells accounted for 29.6 and 13.2%, respectively. the ratio of cd4/cd8 was 2.2. the proportions of b lymphocytes and nk cells were 45. 6 and 3%, respectively. all these immunological indexes indicated dysfunction of the patient's immune system. oral swab, nasopharyngeal aspirate, and serum specimens collected on 14 nov (the 5th day of disease onset and the 2nd day of admission) were subjected to multiplex metagenomic analyses using an ngs platform. the nucleic acid library was constructed as previously described [6] . the amplified nucleic acid libraries were then analyzed using an illumina hiseq 4000 sequencer for a single read of 126 bp. the raw sequence reads were filtered using previously described criteria [7] to obtain valid sequences. when bacteriophages, plant-origin sequences resulting from food debris in the oral cavity, and contamination from the reagents used in the sample processing step (murine leukemia virus (mlv), for example) were excluded, only human rsv (based on the ncbi taxonomy database) was identified, with at least one specific sequence from the oral swab (2137 reads) and the nasopharyngeal aspirate (146 reads). no virus-related sequences were detected in the serum specimen. meanwhile, large numbers of sequence reads related to bacteria, including streptococcus mitis, streptococcus parasanguinis, streptococcus pneumoniae, streptococcus salivarius, streptococcus infantis, streptococcus suis, neisseria meningitidis, neisseria gonorrhoeae, haemophilus sputorum, haemophilus parainfluenzae, enterococcus cecorum, and other conditioned pathogenic bacteria were also detected in the oral swab, nasopharyngeal aspirate, or/and serum specimens (fig. 1) . however, although the metagenomic analysis showed sequence reads assigned to kingdom bacteria, the bacterial culture of the blood specimens yielded negative results. based on the random distribution of reads of the rsv virus genome, the complete length of the genome was obtained using ngs methods and gap amplification. this rsv strain was subtyped as rsvb; it was found to cluster in the ba genotype and had the signature 60-bp duplication in the g gene. the newly identified virus was named rsvb/bch-y/2016, and the full genome sequence was deposited in genbank under accession number ky924878. the phylogenetic analysis was conducted with representative sequences from nearly all rsvb subgroups (ba1-10, gb1-4, sab1-4, uru1-2) from genbank; rsvb/bch-y/2016 belonged to ba9 subgroup (fig. 2) . the nucleotide homology comparisons revealed that the g gene of this strain was most closely related (share 98.82% homology) to strain rsvb/gz/13-730, which was isolated from a child in guangzhou, china, in 2013. for the six most important antigenic sites (ø, i, ii, iv, v, vi) in the fusion protein for drug or vaccine (such as palivizumab) targeting [8, 9] , no mutation was found in rsvb/bch-y/2016. rsv infection generally causes symptoms such as fever, cough and wheezing. while respiratory failure may occur in severe cases, the fatality rate is not high in clinical cases. the patient in this case study was infected with rsv, and the disease progressed rapidly, with extensive cerebral edema and hernia. there are prior reports of neurological complications of rsv infection, which mainly include central apnea, seizures, and encephalopathy [10] . apnea is a common occurrence in rsv-infected patients younger than 2 months of age and is a frequent indication for intubation [11, 12] . the mechanism of rsv-related apnea is unknown, but a significantly reinforced laryngeal chemoreflex or an immature respiratory center in infancy has been proposed [11, 13] . evidence of an encephalitic pathology caused by rsv infection is sparse. the presence of rsv in csf, detected using polymerase chain reaction, was reported in a single case of a 4-month-old boy with febrile seizure [14] . the pathogenesis of rsvrelated encephalitis is not yet fully understood. however, it has been hypothesized that rsv may enter the cns through the hematogenous/blood-brain barrier route or through invasion, causing the release of several humoral neurotoxic cytokine mediators [15] . the diseases that we considered in the course of treatment included the following: (1) severe hand, foot and mouth disease (usually caused by enterovirus, type 71): this disease has a sudden onset in most cases, and symptoms include fever, rashes and herpes appearing on the oral mucosa, hands, and feet. this disease progresses quickly in a small number of cases, especially in children younger than 3 years old. at 1-5 days after onset, complications such as meningitis, encephalitis, encephalomyelitis, pulmonary edema and circulatory disorders occur. a very small number of infections worsen and lead to death. however, symptoms such as herpes and pulmonary edema were not observed in this patient, and both viral pathogen analyses showed negative results for enteroviruses, so this speculation was not supported. (2) high pathogenic influenza virus infection: the typical symptoms include fever, cough, sore throat, headache, body pain and fatigue. some patients progress quickly to severe pneumonia, acute respiratory distress syndrome (ards), shock and acute necrotic brain injury. however, this child underwent repeated influenza a virus antigen detection, and all results were negative; thus, this speculation was not supported. (3) fulminant myocarditis: the initial symptoms primarily include fever, cough, fatigue and vomiting. the disease develops rapidly and can lead to sudden congestive heart failure, cardiogenic shock, and adam-strokes syndrome. ecg sometimes demonstrates st-t changes, myocardial infarction, and arrhythmia. the cardiac ultrasound may show left ventricular enlargement. however, no abnormalities were observed in the ecg or heart ultrasound analyses of our patient; thus, this speculation was not supported. because the patient's guardian refused to consent to lumbar puncture, a csf specimen was not available for further validation. we could not be sure whether the neuroinvasion of rsv did occur in this case because rsv has only rarely been identified in cns specimens [12] . this finding is somewhat similar to that of influenza encephalitis. britton et al. have described cases of influenza-associated neurological disease without testing for influenza viruses in csf specimens [16] . whether the influenza virus invades the cns or not is still controversial. fujimoto et al. reported that influenza virus rna was detected in the csf of 71.4% (5/7) of patients who developed influenza-associated acute encephalopathy/encephalitis [17] . however, in other reports, only a small number of patients were positive for viral rna in the csf and brain, and there was a lack of inflammation in the brain tissue of fatal cases [18] [19] [20] [21] . in the reported cases, no influenza rna was detectable in the csf. however, this finding does not exclude influenza as a cause of the encephalitis because the viral rna may have been cleared by the time the csf was taken. the fact that the rna is no longer detectable does not fig. 1 heatmap based on the read numbers of pathogens derived from the oral swab, nasopharyngeal aspirate, and serum specimens. the clinical specimens are listed in the bottom row, and the pathogen names are presented in the left column. the boxes colored from blue to red represent the metagenomic sequencing reads observed (the reads varied between 2 0 and 2 15 ) exclude an earlier cns insult by the virus causing the current symptoms. for the case reported here, although direct evidence of rsv infection in the cns was not available, the clinical symptoms together with the laboratory findings and metagenomic analysis results suggested that the patient may have had severe sepsis that potentially resulting from an rsv infection with a high probability of cns infection and acute neurological complications. combining ngs with metagenomic analysis provides an important clinical tool to identify unexpected or novel pathogens in patients. the advantage of this methodology is maximized when the causative pathogen presents a low-level signal compared to all other signals representing environmental and commensal pathogens. improving the optimization and implementation of protocols suitable for clinical samples will no doubt improve microbial diagnosis in clinical practice. our findings, in conjunction with previously reported cases emphasize the need for more awareness of the neurological complications of rsv infection. its clinical manifestations may include seizures, encephalopathy, and focal neurological findings. early recognition of neurological complications of rsv infection is important for initiating effective treatment to reduce mortality and long-term morbidity. the datasets supporting the conclusions of this article are included within the article. authors' contributions llx performed the lab analysis and drafted the manuscript. hmg, jsz, jl and cl collected clinical data and monitored the patient. xlg performed the pathogen detection. zdx and syq participated in the study design and coordinated the drafting of the manuscript. all the authors read and approved the final manuscript. fig. 2 phylogenetic analysis based on the complete g gene sequence of the rsv detected in this patient and other representative sequences from nearly all rsvb subgroups (ba1-10, gb1-4, sab1-4, uru1-2) from genbank. the nucleotide phylogenetic tree was constructed using the neighbor-joining method with nucleotide p distances and 1000 bootstrap replicates in the molecular evolutionary genetics analysis program (mega, version 4.0, usa) rsv infections: state of the art the burden of respiratory syncytial virus infection in young children updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection predicting deterioration in previously healthy infants hospitalized with respiratory syncytial virus infection examination of neurological prognostic markers in patients with respiratory syncytial virus-associated encephalopathy metagenomic analysis of viral genetic diversity in respiratory samples from children with severe acute respiratory infection in china deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases structure of rsv fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody neutralizing epitopes on the respiratory syncytial virus fusion glycoprotein extrapulmonary manifestations of severe respiratory syncytial virus infection-a systematic review risk factors for respiratory syncytial virus associated apnoea neurological complications of respiratory syncytial virus infection: case series and review of literature reflex apnoea response and inflammatory mediators in infants with respiratory tract infection detection of subgroup b respiratory syncytial virus in the cerebrospinal fluid of a patient with respiratory syncytial virus pneumonia respiratory syncytial virus-related encephalitis: magnetic resonance imaging findings with diffusion-weighted study the spectrum and burden of influenza-associated neurological disease in children: combined encephalitis and influenza sentinel site surveillance from australia pcr on cerebrospinal fluid to show influenzaassociated acute encephalopathy or encephalitis influenza-associated acute encephalopathy in japanese children in 1994-2002 neurologic complications associated with novel influenza a (h1n1) virus infection in children encephalitis and encephalopathy associated with an influenza epidemic in japan detection of influenza virus rna by reverse transcription-pcr and proinflammatory cytokines in influenza-virus-associated encephalopathy this study was approved by the ethical review committee of beijing children's hospital. written informed consent for the further analysis that followed treatment of the patient and the sequencing that followed the routine care was obtained on the patient's behalf from her parents. written informed consent provided by the patient's parents was obtained on the patient's behalf for the publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal. the authors declare that they have no competing interests. key: cord-315304-pge45105 authors: kotton, c.n.; kuehnert, m.j.; fishman, j.a. title: organ transplantation, risks date: 2015-03-06 journal: reference module in biomedical sciences doi: 10.1016/b978-0-12-801238-3.02629-5 sha: doc_id: 315304 cord_uid: pge45105 viruses are among the most common causes of opportunistic infection after solid organ and hematopoietic stem cell transplantation (sot and hsct). viral infection is associated with both direct (invasive disease) and indirect (immune modulation) effects affecting susceptibility to other infections and promoting allograft rejection. the transplantation recipient is susceptible to a broad array of viral pathogens. some may be transmitted with the allograft as donor-derived infections, while others result from exposure, either soon after the transplant or from more distant exposures when infection is latent and reactivates in the setting of immune suppression. simultaneous infections with multiple viral or viral and nonviral pathogens are common. the risk for viral infection is a function of the intensity of exposure and virulence of the specific virus, the intensity of immune suppression used to prevent graft rejection or graft-versus-host disease, underlying immune deficits, and factors affecting host susceptibility. studies of viral latency, reactivation, and of the cellular effects of viral infection will provide clues for future strategies in prevention and treatment of viral infections. this article covers specific issues relating to viral infection in sot and hsct; additional details regarding these viral infections are also found elsewhere in this text. transaminases, coagulopathy, graft dysfunction, and either fever or leukocytosis within several weeks after transplantation. given the rarity of such infection diagnosed in normal hosts, there may be a predilection for this in transplant recipients. transplant-transmitted infection is rare and might be difficult to recognize, but physicians should consider the possibility, particularly when unexplained neurologic complications occur. these investigations underscore the challenge in detecting and diagnosing infections that occur in recipients of organs or tissues from a common donor. the potential for disease transmission from donor source may not be considered in recipient evaluation. in these investigations, the ability to connect illnesses to a common organ donor was facilitated by the fact that multiple recipients were hospitalized at the same facility. as organ and tissue transplantation becomes more common, the potential risks of disease transmission may also increase. because of improved diagnostic assays, donor-transmitted infections are increasingly recognized, although often times, the impact of infection is not well understood. the advent of polymerase chain reaction (pcr) and other genetic material-based tests have allowed for detection of active viremia, in contrast to serologic tests, which reflect past acquisition of infection. this is important not only for recipient diagnosis, but also for recognition of donor infection retrospectively; donor diagnosis is only possible if appropriate specimens are stored postmortem. recognition of viral infection transmitted through transplantation is increasing, likely both due to increased recognition of unexpected symptoms consistent with transmission, and due to increasingly sophisticated diagnostic testing in both the recipient and the donor. investigation of potential donor-transmitted infection requires rapid communication among physicians in transplant centers, organ procurement organizations, and public health authorities. an immediate system for tracking and disseminating pertinent patient data to evaluate donor-derived infection and associated adverse event outcomes is needed. until such a system can be established, clinicians should report unexpected outcomes or unexplained illness in transplant recipients to their local organ and tissue procurement organizations. given the frequency of latent viral infection, notably among herpesviruses, reactivation of latent infection provides a major source of infection after transplantation. the specific virus, the tissue infected, stimuli for activation, and the nature of the host immune response impact the nature of viral latency. some viruses are metabolically inactive when latent, while others continue to replicate at low levels that may be determined by the effectiveness of the host's immune response. multiple factors contribute to viral reactivation after transplantation, including graft rejection and therapy, immune suppression (especially reduction of t-cell mediated, cytotoxic immunity), inflammation, and tissue injury. numerous cellular pathways are involved in the control of viral replication and are activated after transplantation, such as nuclear factor kb, ikb, and jak-stat (the janus family of protein tyrosine kinases (jaks) and signal transducers and activators of transcription (stat) proteins). antirejection therapy can also result in a significant release of pro-inflammatory cytokines which may increase viral replication. in general, reactivation of viruses, especially late after transplantation, should suggest new immune defects (e.g., cancer) or relative over-immunosuppression. latency and reactivation has been best studied in the herpesviruses, which establish lifelong, latent infection after primary infection. in general, latency is considered to be the absence of viral replication, with viral genomes present in the cell without replication or spread. studies of other viruses, such as friend virus in mice, suggest that protective antiviral immunity is an active process mediated by 'leaky' (low-level) viral replication. the existence of true latency, as opposed to low-level replication, remains controversial. herpesviruses make 'latency' proteins that both control viral persistence within the target cell and influence other cellular processes. the latent state is characterized by low levels or the absence of detectable viral antigens, minimal transcription of productive or lytic cycle genes, and expression of the latency-associated viral transcripts. viral latency may be occasionally interrupted, leading to reactivation and spread of infectious virus with or without recurrent disease. ebv establishes latency in b lymphocytes in association with expression of a limited set of viral genes. immune control of hsv infection and replication occurs at the level of skin or mucosa during initial or recurrent infection and in the dorsal root ganglion, where latency and reactivation are controlled by immune mechanisms mediated by interferons, myeloid and plasmacytoid dendritic cells, cd4(+) and cd8(+) t cells, and other cytokines. despite similarities, the molecular details and mechanisms of latency and reactivation vary considerably among the herpesviruses. mechanisms responsible for maintenance of latency are unclear. reactivation of cmv has been extensively studied. cmv viral genomes can be found in cd14+ monocytes and cd34+ progenitor cells, although the primary reservoir for latent cmv and the mechanisms by which latency is maintained are unknown. allogeneic immune responses and fever (via tumor necrosis factor-a (tnf-a)) have been shown in vitro to increase both cmv promoter activity and viral replication. immune suppression is not essential for the reactivation of latent cmv, but serves to perpetuate such infections once activated. subclinical activation of cmv is common and increasing diagnosed by sensitive molecular assays. for other viruses such as bk polyomavirus, specific types of tissue damage such as warm ischemia and reperfusion injury may precipitate viral activation; they have been linked to an inflammatory state in grafts (via activation of tnf-a, nuclear factor kappa b (nf-kb), neutrophil infiltration, and nitric oxide synthesis), tubular-cell injury, and enhanced expression of cell-surface molecules, all of which may contribute to viral activation. thus, immune injury, inflammatory cytokines, and ischemia-reperfusion injury stimulate viral replication and change expression of virus-specific cell-surface receptors. the hosts' direct pathway antiviral cellular immune response within allografts is less effective due to mismatched major histocompatibility antigens between the organ donor and host with dependence on indirect pathways of antigen presentation. these factors may render the allograft more susceptible to viral infection. common reactivation infections after transplantation include cmv, hbv, hcv, hiv, hsv-1 and hsv-2, hpv, and vzv (as zoster). other less clinically common viral infections related to reactivation include the polyoma viruses bk and jc, human herpes virus 6 (hhv-6), human herpes virus 7 (hhv-7), and hhv-8. reactivation of one virus may lead to reactivation of others; multiple studies have shown that infection with hhv-6 and/or hhv-7 are risk factors for cmv disease and cmv infection may trigger hhv-6 and hhv-7 reactivation. while some reactivation infections routinely cause significant clinical disease, such as cmv, hsv, and vzv, others may cause more variable illness. hhv-6, for example, reactivates with immunosuppression, with clinically significant infection after hsct. by contrast, the role of both hhv-6 and hhv-7 in sot recipients is less well defined; while reactivation is common, clinical disease is generally not evident. based on epidemiologic exposures, new infections from the environment are commonly acquired after transplantation. the respiratory viruses are the most common new infections after transplantation, including rsv, influenza, parainfluenza, and adenovirus. newer respiratory pathogens (metapneumovirus and sars coronavirus) also cause major infections in immunocompromised hosts. gastrointestinal viruses such as rotavirus or norwalk virus are common and can cause significant diarrhea and dehydration; diarrheal syndromes may alter absorbance and metabolism of calcineurin inhibitors (e.g., cyclosporine and tacrolimus), with unexpectedly elevated levels of tacrolimus. nonimmune patients can acquire primary ebv, cmv, vzv, parvovirus b19, and other infections in the post-transplantation period. in the absence of previous immunity and with the attenuation of immunity due to the immunosuppressive regimen, new infections are often more severe and prolonged than in the general population. for example, parvovirus b19 infection is often more persistent and relapsing in transplantation patients, occasionally complicated by the unusual findings of hepatitis, myocarditis, pneumonitis, glomerulopathy, arthritis, or transplantation graft dysfunction. the effects of viral infection are conceptualized as 'direct' and 'indirect' (see table 2 ). this classification serves to separate the tissueinvasive viral infection (cellular and tissue injury) from effects mediated by inflammatory responses (e.g., cytokines) or by alterations in host immune responses. syndromes such as fever and neutropenia (e.g., with cmv infection) or invasive disease resulting in pneumonia, enteritis, meningitis, and encephalitis are considered direct effects. indirect effects of viral infections are generally thought to be immunomodulatory responses to viral infections mediated by cytokines, chemokines, and/or growth factors. the impact of these effects is diverse and includes systemic immune suppression predisposing to other opportunistic infections (notably with cmv or hcv infections). in addition, viral infection may alter the expression of cell-surface antigens (e.g., major histocompatibility antigens) provoking graft rejection and/or cause disregulated cellular proliferation (contributing to atherogenesis in cardiac allografts, obliterative bronchiolitis in lung transplantation, or to oncogenesis). increased viral replication and persistence may contribute to allograft injury (fibrosis) or chronic rejection. infection with one virus may stimulate replication of other viruses in a form of viral 'cross talk'. as was noted above, infection with hhv-6 and/or hhv-7 serve as risk factors for cmv disease and vice versa. the direct and indirect effects of hhv-6 reactivation can be significant: hhv-6 infection is associated with high levels of il-6 and tnf-a, and in pediatric renal or bone marrow transplantation, hhv-6 reactivation is strongly associated with acute rejection. co-infection with hcv and cmv predicts an accelerated course for hepatitis. co-infection with cmv and ebv increases the risk for post-transplantation lymphoproliferative disorders (ptld) by 12-20-fold. a more theoretical concern is that t-cell responses against viral infections are thought to produce cross-reacting immune responses against graft antigens, possibly via 'alternative recognition' within the t-cell receptor. this cross-reactivity is termed 'heterologous immunity' and may provoke abrogation of graft tolerance. the hhv family has eight members affecting humans, all of which can cause significant disease in transplantation recipients. the risk of many of these infections is reduced by the use of valganciclovir or acyclovir after transplantation. hsv-1 and à 2 (hhv-1 and à2) usually cause oral and genital ulceration, although it may occur in more unexpected areas as well. recurrent disease can be problematic for some patients and warrants consideration of secondary prophylaxis with antiviral therapy. vzv (hhv-3) is common with an incidence of herpes zoster among 869 patients after sot of 8.6% (liver 5.7%, renal 7.4%, lung 15.1%, and heart 16. . ptld constitutes a spectrum of disease, which is often responsive to reduced immunosuppression in previously immune hosts. ebv-seronegative individuals with primary infection after transplantation are at increased risk for ebv-mediated ptld. the clinical presentation of cmv (hhv-5) can range from a 'cmv syndrome' including fever, malaise, leukopenia, to a 'flu-like' illness with myalgias and fatigue, to a more significant end-organ disease with pneumonitis, colitis, encephalitis, hepatitis, or chorioretinitis. cmv is the single most important pathogen in transplantation recipients due to direct and indirect effects (see above). hhv-6 commonly reactivates after transplantation, especially after hsct where it is associated with hepatitis, pneumonitis, cmv reactivation, bone marrow suppression, and encephalitis. hhv6 causes less symptomatic clinical infection after sot, although the indirect effects of reactivation have not been studied. hhv-7 commonly reactivates after transplantation. the clinical symptoms caused by hhv-7 are uncertain, although neurological symptoms seem to be significant, especially in children. hhv-8 causes kaposi's sarcoma and is seen in sot recipients at a rate 500-1000 higher than the general population, with a prevalence of 0.5-5% depending on the patient's (and donor's) country of origin. hepatitis b and c are among the most common indications for liver transplantation, and can complicate other transplantations as well. hepatitis c is currently the most common indication for liver transplantation, accounting for 40-45% of cases in recent times. recurrent post-transplantation hepatitis c infection poses a conundrum between treating the hepatitis c and reducing immunosuppression without precipitating rejection. given the risk of precipitating graft dysfunction, hepatitis c treatment with interferon and ribavirin was often deferred in extra-hepatic transplant recipients. novel therapies for hepatitis c should reduce the risk of treatment complications after transplant. for hepatitis b, the goal is complete viral suppression before and after transplantation, using antiviral agents and sometimes hepatitis b immunoglobulin. respiratory viruses are the most common community-acquired infections in transplantation recipients. given the increased rates of pneumonia and bacterial and fungal superinfection, prevention (vaccination, avoidance of sick individuals) is essential. diagnosis of respiratory viruses within a few hours via enzyme-linked immunosorbent assay (elisa) or immunofluorescent staining is available in many medical centers. viral cultures are time consuming and expensive. respiratory syncytial virus and parainfluenza are the most common community-acquired respiratory viruses, followed by influenza and adenovirus. antiviral medications (rimantidine, amantidine, or oseltamivir) may prevent or reduce the severity of illness. the use of ribavirin or rsv table 2 direct and indirect effects of cmv infection fever and neutropenia syndrome (leukopenia, fever, myalgia, fatigue, thrombocytopenia, hepatitis, nephritis) increases risk of ptld gastrointestinal invasion with colitis, gastritis, ulcers, bleeding, or perforation increases risk of cardiovascular events hepatitis increases risk of immunosenescence, mortality pancreatitis chorioretinitis increases risk of new-onset diabetes mellitus after transplantation immune globulin in adults to prevent rsv infection is not well studied. ribavirin is sometimes used for documented rsv infections of the lower respiratory tract in transplant recipients. metapneumovirus and severe acute respiratory syndrome-associated coronavirus (sars-cov) are emerging pathogens in transplantation patients. the clinical spectrum of disease from metapneumovirus ranges from symptomatic (even fatal) to asymptomatic cases. some groups have suggested a possible correlation with graft rejection in lung transplant recipients. diagnosis is often made using molecular assays; the full impact of this infection in transplantation is yet to be realized. sars, caused by a zoonotic coronaviruses, is a highly contagious and rapidly progressive form of viral pneumonia, which spread from asia to many parts of the world in early 2003. a number of transplantation patients were infected, some of whom died. the impact of sars and resulting infection control issues was significant for both active organ transplantation (i.e., concerns about transmitting donorderived infections) as well as routine follow-up care for transplantation patients, some of who deferred healthcare visits. gastrointestinal viruses such as rotavirus or norwalk virus may cause significant diarrhea and dehydration; prolonged infections for months have been seen in transplant recipients. enteroviral infections in the summer months in the northern hemispheres are common and can have a more complicated and prolonged course in renal transplantation recipients. bk virus is associated with a range of clinical syndromes in immunocompromised hosts: viruria and viremia, ureteral ulceration and stenosis, and hemorrhagic cystitis. the majority of patients with bk virus infections are asymptomatic. infection by jc polyomavirus has been observed in renal allograft recipients as both nephropathy (in association with bk virus or alone) and/or progressive multifocal encephalopathy (pml). jcv establishes renal latency but receptors are present in multiple tissues including the brain. infection of the central nervous system generally presents with focal neurologic deficits or seizures and may progress to death following extensive demyelination. human papilloma virus (hpv) infections can cause significant disease in renal transplantation recipients, including oral, skin, genital, and rectal lesions ranging from warts and dysplasia to malignancy (especially squamous cell carcinoma). the recent arrival of a vaccine for genital hpv infections may help reduce these infections. in transplantation recipients, parvovirus b19 infection can cause erythropoietin-resistant anemia, pancytopenia, myocarditis, or pneumonitis. direct renal involvement with glomerulopathy and allograft dysfunction has been reported in renal transplantation recipients. clinical and virologic responses to treatment with intravenous immunoglobulin are usually excellent. several zoonotic viruses have caused major illness and death in the transplantation setting, including wnv, rabies, and lymphocytic choriomeningitis virus (lcmv). all have been recently reported as donor-derived infections related to sot, with clinically subtle infections in the donor and often deadly infections in the recipients. wnv is more morbid after sot; the risk of meningoencephalitis in a sot patient infected with wnv has been estimated to be 40%, compared with <1% in normal hosts. donors in endemic areas should be screened for wnv, as the prevalence can be high. aside from donor-derived infections, rabies and lcmv have not been reported. although hiv-infected patients are living longer and dying less often from complications related to acquired immunodeficiency syndrome (aids), they are experiencing significant morbidity and mortality related to end-stage liver and renal disease. preliminary studies suggest that both patient and graft survival are similar in hiv-negative and hiv-positive kidney and liver transplantation recipients. however, hcv infection appears to be accelerated even in controlled hiv infection. ongoing multicenter trials of transplantation in hiv infections are continuing. drug interactions between the immunosuppressive regimen and antiretroviral drugs necessitate careful monitoring. rapid and sensitive molecular biology-based assays for many of the common viruses after transplantation have replaced, for the most part, serologic testing and in vitro cultures for the diagnosis of infection. serologic assays are generally less sensitive in transplant patients, as humoral immune responses may be delayed or absent. in one series, 29% of patients with parvovirus b19 infection as shown by pcr assay had a negative igm assay. quantitative molecular tests allow the optimization and individualization of antiviral therapies for prevention and treatment of infection. this advance is most significant in the management of cmv, ebv, hepatitis b, and hepatitis c viruses, where quantitative assays (such as viral loads or antigenemia tests) guide antiviral therapy. nonquantitative (i.e., qualitative) assays are less useful in management as they do not assess responses to therapy and cannot differentiate primary infection, from reactivation or reinfection. for example, chromosomal integration of latent hhv-6 dna (which happens in 1-3% of immunocompetent subjects) leads to high levels of viral dna, whether or not the infection is active; one group concludes that any diagnosis of hhv-6 encephalitis should not be made without first excluding chromosomal hhv-6 integration by measuring dna load in csf, serum and/or whole blood. latent infections due to ebv and cmv may be qualitatively positive by pcr, confirming the need for quantitative assays. blood tests may not always accurately reflect the level of end-organ diseases; thus, it may be useful to test specific affected tissues as well the blood. in this regard, histologic evaluation of tissues using pathogen-specific immunohistochemistry may augment systemic assays. patients with cmv colitis, for example, may have negative molecular or antigenemia blood assays for cmv. in addition, patients may shed cmv in secretions without true infection, limiting the diagnostic capacity of a positive culture. bk polyomavirus may be detectable in the urine (either by cytology, looking for the classic decoy cells, or by pcr) before it is detectable in the blood, providing a window of opportunity for reducing immunosuppression possibly in advance of invasive disease. adenovirus may be detectable in local infections, such as cystitis, with negative blood assays. the treatment of viral infections in the renal transplantation recipient includes: the reduction of immunosuppression, antiviral therapy, diagnosis and treatment of co-infections (such as cmv, ebv, hhv-6, or à7), and use of adjunctive therapies such as immunoglobulins or colony stimulating factors. the overall level of immunosuppression has a major impact on both the risk of reactivation of latent infection and the ability to clear such an infection. reducing the immunosuppressive regimen during active viral infection can be a major contribution toward clearing infection, although it presents a risk of graft rejection. as protective cytotoxic immunity to viruses is generally t-cell (cd8 +) mediated, an initial reduction of antimetabolites (if neutropenic) and calcineurin inhibitors merits consideration. in contrast, a reduction of the steroid dose during the acute phase of a febrile illness may cause acute adrenal insufficiency. when available, antiviral therapies (such as acyclovir, ganciclovir, ribavirin, lamivudine, and oseltamivir) are often essential. the toxicity of some agents (such as cidofovir, foscarnet, and ribavirin) may complicate management, notably in the face of reduced renal function in patients receiving calcineurin inhibitors as part of the immunosuppressive regimen. the duration of therapy is often longer in transplant patients than in normal hosts, and often reflects the ability to reduce the overall level of immunosuppression, that is. less immunosuppression may result in a shorter treatment time. the increased information provided by molecular diagnostics may allow for more directed treatment regimens. antiviral therapy is often used for prophylaxis in the post-transplantation period, especially for individuals at risk for primary infection. in general, acyclovir and related agents are used to prevent hsv and vzv and ganciclovir or valganciclovir to prevent cmv as well as hsv and vzv. the relative advantages of universal prophylaxis (i.e., the use of antiviral medication in all susceptible patients for a period after transplantation) or monitoring with preemptive (early) therapy remain to be established. meta-analyses suggest that universal prophylaxis with antiviral medications in sot recipients reduces cmv disease and cmv-associated opportunistic infections, graft rejection, and mortality; their use is recommended for high risk individuals (cmv-positive recipients and in cmv-negative recipients of cmv-positive organs). some studies indicate that universal prophylaxis and preemptive therapy are effective in reducing the incidence of cmv disease. in the hsct setting, where the risks of bone marrow toxicity from valganciclovir are higher, many programs choose to use preemptive monitoring and therapy for cmv. various adjunctive therapies have been helpful in treating and preventing viral infections. immunoglobulins (i.e., intravenous immune globulins (ivig), cmv, and hbv hyper-immune globulins (both prepared from plasma preselected for high titer antibodies to cmv and hbv, respectively), as well as monoclonal antibodies such as those for rsv) have been helpful in preventing and treating viral infections, likely due to both direct and indirect immunomodulatory properties. a significant percent of patients have post-transplantation hypogammaglobulinemia, which has been linked to increased mortality and may benefit from globulin repletion. this may be most apparent in the setting of active infections, as well as prophylaxis. immunostimulatory agents, such as interferon-alpha used to treat hepatitis c, can be helpful at treating the viral infection but may perturb the relationship between the graft and the host, precipitating rejection. reversal of neutropenia can be done using colony stimulating factors such as g-csf, which is generally well tolerated in sold organ transplantation patients. vaccines should be given to patients as early as possible in the course of organ failure and well in advance of transplantation to optimize immune responses. in general, a response to vaccination is more likely to occur when given pre-transplantation rather than after transplantation. nonlive viral vaccines such as hepatitis b, hepatitis a, injectable influenza, rabies, hpv, injectable polio may be given both pre-and post-transplantation (see table 3 for classifications). live viral vaccines such as attenuated influenza for protection against varicella in nonimmune subjects, as well as zostavax, for protection against zoster), yellow fever, oral polio, and vaccinia (smallpox) should generally be avoided after sot and in patients who are chronically immunosuppressed (such as those with gvhd); some may be given after hsct. consideration of future travel or trips home should also be considered in the pre-transplantation period a prospective survey of human herpesvirus-6 primary infection in solid organ transplant recipients ast infectious diseases community of practice (2013) vaccination in solid organ transplantation parvovirus b19 infection after transplantation: a review of 98 cases transmission of lymphocytic choriomeningitis virus by organ transplantation infection in solid-organ transplant recipients prospective study of polyomavirus type bk replication and nephropathy in renal-transplant recipients zoonoses in solid-organ and hematopoietic stem cell transplant recipients cmv: prevention, diagnosis and therapy viral infection in the renal transplant recipient prevention of infection in adult travelers after solid organ transplantation updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation a seroprevalence study of west nile virus infection in solid organ transplant recipients idsa clinical practice guideline for vaccination of the immunocompromised host notes from the field: a cluster of lymphocytic choriomeningitis virus infections transmitted through organ transplantation -iowa hhv-6 dna level in csf due to primary infection differs from that in chromosomal viral integration and has implications for the diagnosis of encephalitis key: cord-318282-ocgfgx9r authors: boyce, john m; cookson, barry; christiansen, keryn; hori, satoshi; vuopio-varkila, jaana; kocagöz, sesin; öztop, a yasemin; vandenbroucke-grauls, christina mje; harbarth, stephan; pittet, didier title: meticillin-resistant staphylococcus aureus date: 2005-10-31 journal: the lancet infectious diseases doi: 10.1016/s1473-3099(05)70243-7 sha: doc_id: 318282 cord_uid: ocgfgx9r nan the epidemiology and the clinical management of mrsa infections have continued to evolve in recent years in the usa. the prevalence of mrsa has increased progressively since the early 1980s, and by 2002 mrsa accounted for nearly 60% of nosocomial s aureus infections acquired in intensive-care units. 1 mrsa strains have been reported to account for 30-62% of nosocomial s aureus bloodstream infections and 42-60% of s aureus surgical-site infections. [2] [3] [4] [5] [6] [7] in some facilities, the proportion of health-care-associated s aureus infections caused by mrsa is even higher. vancomycin-intermediate s aureus (visa) strains, which have vancomycin minimum inhibitory concentrations (mics) of 8-16 mg/ml, have continued to cause occasional health-care-associated infections, although transmission within health-care facilities of such strains has seldom been documented. 8 at least three well-documented isolates of vancomycin-resistant s aureus (vrsa) that have vancomycin mics of 32 mg/ml or greater have been recovered from patients in the usa. 9 although it is possible that the relatively high frequency of co-colonisation by vancomycinresistant enterococci (vre) and mrsa among patients in the usa may create more opportunities for in-vivo transfer of the vana gene complex from vre into mrsa strains, it is not known if this has affected the frequency of vrsa in the usa. by contrast with the uncommon occurrence of visa and vrsa, mrsa strains that are still classified as susceptible to vancomycin (ie, mics of 1-4 mg/ml) have caused an increasing number of health-careassociated infections that respond poorly to vancomycin therapy. 8, 10, 11 the poor response to vancomycin therapy in such cases appears to be associated with several strain characteristics. patients infected with mrsa strains with vancomycin mics of 1-4 mg/ml have responded to vancomycin therapy less frequently than patients with infections caused by strains with mics of less than 0·5 mg/ml. 10 in some cases, poor clinical responses may have been due to strains with hetergenous resistance to vancomycin-so-called hetero-visa. 6 fowler and colleagues 14 found that mrsa bloodstream infections that persisted for more than 7 days despite appropriate vancomycin therapy were significantly more likely than short-duration mrsa infections to have been caused by strains that exhibited (1) higher rates of survival in vitro after exposure to thrombin-induced platelet microbicidal protein (p=0·005), (2) defective delta-lysin production (suggestive of loss of accessory gene regulator [agr] function; p=0·057) and (3) agr type ii (p=0·037). in two prospective randomised trials of treatment of ventilator-associated pneumonia (vap), vancomycin therapy was significantly less effective than treatment with linezolid (p=0·001) among a subset of patients with vap caused by mrsa. 12 unfortunately, the microbiological characteristics of mrsa from these pneumonia studies were not described in detail. the above trends suggest that although vancomycin remains the drug of choice for most serious health-careassociated mrsa infections, clinicians in the usa may need to consider the use of other agents-eg, linezolid, daptomycin, or tigecycline-in some clinical situations based on the characteristics of the infecting strain and the body site affected. another major change in the epidemiology of staphylococcal infections in the usa is the rapid emergence of community-acquired mrsa strains since the late 1990s. [13] [14] [15] [16] although such infections have been more common among population groups such as young children, native american and pacific islander communities, prisoners, military personnel, men who have sex with men, intravenous drug users, and individuals involved in amateur or professional competitive sports, spread within the general community is likely occurring. 13, [15] [16] [17] a relatively small number of unique uk mrsa was first described in england in the early 1960s, just months after meticillin had been introduced into clinical practice. mrsa has waxed, waned, and waxed again over the following decades. the international union of microbiology and who's staphylococcal centre is based in the laboratory of healthcare associated infection of the health protection agency (hpa), and was the first to identify and number epidemic mrsa strains (emrsa 1-17), plot their spread, and identify the existence of other epidemic mrsa strains in europe as part of the harmony network project. 25 community mrsa are evident in england but have not yet emerged to be the problem encountered in some other countries. in the early 1990s, about 2% of s aureus bacteraemias were due to mrsa; the mean figure is now about 45%, although there is a large range, with some hospitals encountering the organism infrequently. current english data show that the occurrence of meticillinsensitive strains has also increased, so merely focusing on the percentage of total s aureus bacteraemias can be misleading. ever since the organism was first described, hospitals have continued to vary in mrsa occurrence within and between different cities and in different wards within the same hospital. the reasons are complex and may include aspects of the reporting system; a comparison of reporting methods is underway. we know that the current prevalent uk epidemic mrsa (emrsa strains 15 and 16) differ genetically from their predecessors and also contain more or different toxins. however, we still do not understand why certain mrsa strains have this epidemic potential. when such strains spread to other countries they seem to exploit the more stressed or less "infection control compliant" hospitals or wards. mrsa are opportunistic pathogens colonising more patients than they infect. the patient case mix has also changed. mrsa has always preferably colonised and infected elderly people. improved nutrition and medical advances has meant that elderly patients are now admitted to "high-risk" units-eg, cardiovascular and orthopaedic wards with periods of care on intensive care units. such units can then "carousel" forum strains appear to be responsible for many of the community-acquired mrsa infections occurring in the usa. 17 most of such strains contain the mobile genetic element staphylococcal cassette chromosome mec (sccmec) type iv, which is relatively uncommon among health-care-associated mrsa strains in the usa. 18 emergence of community-acquired mrsa has also required that clinicians alter their approach to empiric treatment of community-acquired skin and soft tissue infections. physicians have been encouraged to routinely culture skin lesions to determine if community-acquired mrsa is the cause, and in geographic areas where community-acquired mrsa are relatively common, empirical therapy of such infections with clindamycin, trimethoprim-sulfamethoxazole (co-trimoxazole), doxycycline or minocycline, or linezolid is becoming common. if empiric clindamycin is administered, testing community-acquired mrsa isolates for inducible clindamycin resistance by using the "d-test" is recommended. 19 by contrast with the netherlands and some other parts of northern europe, no standard set of measures has been adopted in all health-care facilities in the usa for controlling transmission of mrsa. measures recommended by the society for healthcare epidemiology of america for controlling health-care-associated mrsa include the use of screening cultures to detect colonised patients, placing patients in private rooms or cohorting patients, wearing gloves for room entry, gowns for substantial contact with patients or their environment, and hand hygiene before and after patient contact. 20 however, there is considerable debate among experts in the usa about whether or not the use of screening cultures to detect patients at high risk of mrsa is necessary or practical in all health-care settings. given the considerable body of evidence that screening cultures, when combined with contact precautions, are beneficial and cost-effective, [20] [21] [22] it is disconcerting that a recent survey of infectious disease consultants found that only 50% of 463 respondents favoured the use of screening cultures to detect multidrug-resistant pathogens, and only 30% worked in facilities where screening cultures were routinely done. 23 the costs of surveillance cultures, potential logistic problems that may result from an increased number of patients requiring isolation, and lack of controlled trials demonstrating the efficacy of screening cultures are often cited as concerns by those who do not use screening cultures. further studies are necessary to establish the relative efficacy of control measures such as screening cultures, cohort nursing, increased staffing levels, and improved hand hygiene adherence rates in controlling transmission of mrsa in health-care facilities. 24 at present, there is no consensus regarding which measures are most appropriate for reducing transmission of community-acquired mrsa in community settings and for preventing the spread of these strains in health-care facilities. in the past year i have served as a consultant to dial corporation and woodward laboratories, who produce hand hygiene products, including alcohol-based hand rubs and soaps. i am currently a consultant to gojo industries. infectious diseases section, hospital of saint raphael, new haven, ct, usa forum mrsa-affected patients to other parts of the hospital, spreading the organisms elsewhere. there is also mrsa spread between orthopaedic wards and residential or nursing homes, which now care for more severely ill patients. another change has been the increasing involvement of paediatric populations; this is being studied specifically. it is interesting that obstetrics and paediatrics had encountered mrsa infrequently in previous decades in the uk. perhaps the development of mixed specialty wards has facilitated its introduction into this population? implementation of effective measures is being deleteriously affected by other changing environmental factors. reduced hospital bed numbers have resulted in increased interward transfers of patients, which have been shown to increase the risk of mrsa acquisition. decreased lengths of stay have resulted in patients being discharged before they present with mrsa infections. we have thus reduced the effectiveness of our alert organism laboratory surveillance systems. our data also show that inter-city transfers of patients has encouraged national mrsa spread. bed occupancy in many hospitals is over 90% and in some parts over 100%. some uk infectioncontrol teams have problems closing mrsa-affected wards because of the pressures on waiting lists. however, the relation between bed occupancy and mrsa rates is not clear-cut. 26 insufficient numbers of skilled staff further compounds the problem. studies have shown that compliance with infection-control practices decreases as workloads increase, resulting in more mrsa crossinfection. these practices include mrsa screening, effective hand hygiene, wound and intravascular/urinary device care, and appropriate isolation measures. when we did an intra-uk comparison of hospitals in 1995, those that were better able to control mrsa outbreaks had fewer delays in identifying mrsa patients (28·6% vs 50%), fewer inter-hospital transferred patients (9·4% vs 29·4%), and no problems with mupirocin resistance (0% vs 14·7%) compared with those less able to control the problem. a recent english mrsa systematic review 27 concluded that there were many problems with the quality of the reviewed papers. absence of evidence should not be taken to mean that there is an absence of effect. the authors emphasised that the current guidelines for mrsa control should be followed until evidence emerges that other approaches are effective. prescribing of certain antibiotics probably contributes to the acquisition of mrsa, although the evidence for this is not clear-cut. the authors indicated the ways in which better studies could be designed to inform the most cost-effective means to produce sustained improvements in compliance. mrsa control guidelines were first written in 1985 and were similar to the "search and destroy" approach used in non-endemic countries. in 1998 we revised them in the light of the endemic problems encountered in many english hospitals. 28 there has been much debate as to how to best control endemic mrsa and other writers in this forum have outlined many of these issues. our analyses of the current situation will further inform the most effective mrsa control measures. new guidelines have been written and a consultation on them is underway. several interventions will be required. the department of health and the hpa in consultation with many other bodies and health-care workers are introducing a suite of initiatives with various performance indicators and outcome measures that should enable improvements in infection control. however, these initiatives will require substantial funding. multidisciplinary involvement will be crucial, as well as ownership of infection control by all healthcare workers and sharing of good practice. there are clearly many issues that need to be discussed with policy makers; bed managers also need to work closely with infection-control teams. in some parts of england mrsa-free wards are being created, elsewhere mrsafree or fast-track orthopaedic hospitals are being assessed. i believe that effective control and real reductions in mrsa will be possible. screening strategies are critical-eg, planned screening and eradication of mrsa before admissions, monitoring of acquisitions on high-risk units and among patients who have been in hospital for prolonged periods (ie, more than a week), and rotating screening programmes on wards to provide feedback data to inform interventions such as hand hygiene improvement campaigns. a good example of this is the cleanyourhands campaign, which uses a multifaceted approach including the use of alcohol handrubs and patient empowerment. rapid mrsa detection systems are being examined for costeffectiveness. many systems have been used to isolate patients-eg, an open ward, side rooms, four-bedded cohorting, or an isolation ward. whatever strategy is used should be validated by screening strategies. modelling studies have further informed isolation strategies. 27 the hpa and others have done much to ensure that the news media are well informed about mrsa and have provided leaflets to educate the public. however, we will also need to do more to look at infection-control standards in nursing homes and to plan the eradication of mrsa from patients better after discharge. epidemic mrsa has a long history in australia. the bacterium was first isolated in 1968 in sydney and spread rapidly through teaching hospitals over the next 10 years, such that by the end of the 1980s the prevalence was 10-25% in hospitals in the cities on the eastern side of australia. annual surveillance studies by the australian group on antimicrobial resistance have allowed us to track the evolution of both epidemic and community mrsa for the past 18 years. the current prevalence (2003) for multidrug-resistant epidemic mrsa varies for the state capital cities, and is approximately 35% in sydney, 22% in melbourne, 16% in brisbane, 14% in adelaide, and 3% in perth. recent epidemiological typing studies have enabled the identification of these epidemic strains as predominantly st239-mrsa-iii (uk emrsa 1, 4, 11, vienna, portuguese/brazilian clones) with two variants, one in melbourne and adelaide and the other in sydney, canberra, and brisbane. active screening and infection-control policies initiated in the early 1980s following a single hospital outbreak have prevented the establishment of epidemic mrsa in any western australian hospital. since 1997, increasing numbers of st22-mrsa-iv (uk emrsa 15) have been detected clinically, on screening, and in the annual surveillance studies. this non-multidrug-resistant clone is the major epidemic strain in western australia, where it comprises 21% of all mrsa isolates and 80% of all epidemic strains. a similar trend is occurring on the eastern side of australia, with sydney having the highest prevalence of this strain (15% of all mrsa). nurses coming from the uk are an important source of these isolates, which are often detected on pre-employment screening. this clone has also become established in many long-term care facilities. in addition to nosocomial epidemic mrsa we have been documenting the evolution of true community-acquired mrsa strains in australia over the past 20 years, such that now we have at least 23 clones that have evolved independently around the country. it is somewhat ironic that western australia, the state with the lowest rate of noscomial epidemic mrsa, should now have the highest rate of communityacquired mrsa (75% of all mrsa isolates). these strains, although frequent in admitted patients, rarely seem to be transmitted to others in the hospital. when identified, patients colonised or infected with community-acquired mrsa are managed with enhanced precautions (single room if possible, attention to hand hygiene, contact precautions, and extra cleaning during admission and on discharge). to date, we have not had any major hospital outbreak with community-acquired mrsa, despite an increasing prevalence in the community. my experience with mrsa has been as a western australian, where we have successfully used a "search and destroy" policy for at least 20 years. it is state policy that any patient who has been hospitalised outside the state in the preceding year be isolated and screened for mrsa before joining the general hospital population. in addition, staff who have worked clinically outside western australia are also screened pre-employment. this screening has led to my colleagues in sydney, melbourne, and brisbane calling us "fortress wa". we have had a number of outbreaks over the years, particularly in my own hospital, which has a lack of single rooms, but swift screening of contact patients and staff, isolation of colonised patients, attention to hand hygiene, and enhanced ward cleaning has terminated the outbreaks rapidly. we also routinely screen all patients in high-risk areas (intensive care, burns, vascular, and spinal units) on admission, discharge, and after prolonged stay. decolonisation is initiated for persistently colonised staff and patients likely to have frequent readmissions. in general, topical hexachlorophene or triclosan together with mupirocin nasal ointment is used. however, if oral carriage is detected or the topical regimen fails, systemic therapy with two agents is given. i cannot speak for my colleagues on the other side of australia who have lived with endemic nosocomial mrsa for many years, but i believe that there are a variety of practices mostly aimed at containment rather than eradication. in brisbane, where the prevalence has been very high, rates have dropped substantially with the move to new hospital accommodation and intensification of infection-control procedures. it is perhaps easiest for those of us who do not have endemic nosocomial mrsa. we have a very defined end point-no endemic mrsa-that we wish to achieve. for those with endemic mrsa, realistic end points for any containment programme must be established and measures instigated to ensure the attainment of those outcomes. containment or eradication is important-we are all aware of the emergence of vrsa carrying the vana gene complex. the coexistence of mrsa and vre provides the environment for gene transfer. policies for the control of both these organisms are therefore of paramount importance. community-acquired mrsa presents a new challenge. fortunately in western australia, most of the community strains are panton-valentine leucocidin (pvl) negative. the infections seen are therefore of similar severity to those caused by susceptible s aureus. however, there are disturbing data indicating that our most frequent community-acquired mrsa clone can, and has, acquired pvl on several occasions. we are currently contemplating what community strategies should be implemented, in particular whether we should follow the example of denmark where a search and destroy policy has been used within the community. it would not be feasible for us to do this with all our community-acquired mrsa isolates but it may be possible for those with pvl. this toxin is carried on a prophage and is therefore potentially mobile. if we can prevent wide dissemination of the pvl genes we could perhaps limit the associated disease severity. the australian public is very aware of "the golden staph" tag by which the press has labelled mrsa. there is episodic press coverage, both print and television, of personal stories and individual hospital problems. however, understanding of antimicrobial resistance of any kind is minimal within the community. providing and disseminating relevant, understandable information that will change community and prescriber attitudes is a challenge that should be met by our profession, regulatory authorities, and politicians. i declare that i have no conflicts of interest. head of the department of microbiology and infectious diseases and associate professor of the school of biomedical sciences, curtin university, perth, australia japan japan has one of the highest prevalences of mrsa in the world. among s aureus bloodstream isolates in 2001, nearly 70% were meticillin resistant. to control mrsa in hospitals, it is essential to identify potential mrsa patients immediately after admission. since microbiological testing usually takes 3-4 days, the identification of those who are at high risk for mrsa carriage is required to decide whether proper precautions should be taken for each new patient. no mechanism to identify patients within hospitals currently exists in japan-there is no general database that includes previous medical and laboratory results of patients that can be shared by clinicians between different hospitals. in addition, people can attend any hospital they choose-from a large teaching hospital to a general hospital-without an appointment. without mrsa screening on admission, this situation makes it difficult to know whether the patient has already been colonised or infected with mrsa in the past. ignorance about mrsa is a huge problem among the public and media in japan. although there is some interest in the subject, the risks and safety issues surrounding mrsa are generally not that well known. mrsa carriage is seen as a "stigma" for patients and because colonisation with mrsa is not usually differentiated from an infection, the media are quick to generate a scandal and attach blame to hospitals and doctors when mrsa is reported. hence, most hospitals tend to refuse mrsa-positive patients to avoid potentially damaging repurcussions for the hospital. moreover, this situation can lead to clinicians hesitating to identify mrsa carriage of patients who are being transferred to another hospital. in many countries it is strongly recommended that clinicians at the transferring hospital should notify mrsa carriage status of the patient to the infection control team at the receiving hospital before transfer. however, this procedure has not been followed stringently in japan. an mrsa flagging system to highlight previously identified patients within the same hospital or upon readmission is expected to become available soon to most clinicians. 29 this flagging system needs to be computerised and added on to the individual database in each hospital. however, it is still difficult to share microbiological results through a single medical computer network system. each hospital needs to develop mrsa flagging software for this purpose. although japanese hospitals are extensively computerised, the main purpose of the hospital computer system is for ordering and accounting for medical procedures. the data sorting function from previous laboratory results is usually optional. we have recently developed a small computer programme on our hospital network to indicate an "alert spot" on the individual patient record window. if a patient has a previous history of being mrsa positive, a redcoloured alert spot automatically appears on the computer display. this alert system helps us to recognise whether the patient is a potential or current mrsa carrier without checking any previous microbiological results, which is usually a tedious and time-consuming procedure for busy clinicians and hospital officers. this function will be introduced to a new hospital computer system in the future. ideally, these data will become indispensable information for inter-hospital transfers via a shared database that should be set up in the future. an alternate route of importing mrsa into hospitals is from the community. 30 only a few studies of mrsa prevalence in the community have been published from japan. one study reported that 35 out of 818 (4·3%) healthy children aged 3-5 years old carried mrsa in their anterior nares. 31 this prevalence of community mrsa among children is high, and deserves further prevention efforts. mrsa surveillance is currently mandatory in large general and university hospitals but not in middle to small-sized hospitals that are the most common types of hospital in japan. for example, geriatric hospitals with less than 200 beds are usually one of the most important reservoirs of mrsa. mrsa-positive patients are sent to us from these types of institutions without information of mrsa carriage. therefore, it is important that mrsa surveillance should be made mandatory by legislation. another less common strain of mrsa is visa, which was first reported in japan. 32 although the hetero-visa strain (a possible precursor of visa) was identified in 9·3% of mrsa isolates in seven university hospitals, 33 a new visa strain has not been identified in japan so far, because therapeutic drug monitoring has become widely available from both "in-house" and commercial laboratories since 2000. controlling the use of vancomycin means there is less chance for the emergence of vancomycin-resistant strains. educational awareness of mrsa should be provided to all health-care workers as well as the general public, and should be supported by the government or relevant associations and academic societies. mrsa carriage should be notified properly when the patient is transferred from one hospital to the next. senior lecturer, department of infection control science, juntendo university, graduate school of medicine, tokyo, japan finland is a country with low mrsa incidence. the incidence figures are based on mandatory reporting by clinical microbiological laboratories of each new mrsa isolation since 1995 and analysis of mrsa strains at the national reference laboratory at the national public health institute. in recent years, however, we have seen a worrying increase in the number of mrsa cases. the number of reported cases has increased from 120 in 1997 to 1460 in 2004 (from 2·3 to 28·04 cases per 100 000) in a population of 5·2 million. for many years mrsa remained below 1% among invasive cases, but during 2004 we saw the first signs that the figures were worsening substantially. at the same time, the coverage and efficacy of mrsa reporting has remained stable, thus we assume that we are facing a true change. strict mrsa prevention measures are taken following each newly identified mrsa case, regardless of whether it is symptomless carriage or infection. in response to three large mrsa epidemics in southern finland, national mrsa prevention consensus guidelines were adopted in 1995. the updated version of these guidelines was published in 2004. 34 the new guidelines cover both acute care hospitals and long-term health-care facilities, including homes for elderly people. the main principles are promotion of hand disinfection, identification of mrsa risk patients (carriers and contacts of known mrsa cases), rapid microbiological identification of mrsa, contact isolation of mrsa cases, and treatment of mrsa infections. emphasis is placed on trying to target preventive measures at the very first steps of possible mrsa transmission. according to the guidelines, every patient who has been treated in a hospital outside of finland during the previous year and is transferred to a finnish hospital is screened for mrsa colonisation at the time of hospital admission. patient records of each mrsa case are labelled accordingly, and an "mrsa alert" is also added to electronic patient records in many institutions. these actions have proven to be effective in controlling several rather large hospital mrsa outbreaks in the 1990s. some of the international mrsa clones have also repeatedly entered our country via patients or health-care workers who have come from abroad, but they have caused few secondary cases or outbreaks. during the past couple of years, the mrsa situation has changed in finland. in 1997-99, one-fifth of our cases showed no contact with the health-care system, and were thus considered community-acquired mrsa. 35 usually, these mrsa strains possess only beta-lactam resistance, carry the sccmec type iv genes, and cause problems in younger age groups. however, the proportion of elderly people with mrsa has increased steadily. in 2004, over 50% of cases were found in individuals aged 75 years or older. in 2002, over half of finnish mrsa cases were reported in long-term care facilities, and outbreaks are forum occurring in these facilities. 36 these two settingscommunity acquisition and long-term care facilitiescreate new and demanding challenges to our mrsa prevention policy. the finnish health-care system is currently struggling with increasing output demands and decreasing funding. the nursing staff is often overwhelmed with work, and patient wards are crowded and often lack single-bed isolation rooms, creating problems for infection control. in long-term care facilities, we are witnessing the lack of trained personnel and problems in finding functional placement options for mrsa carriers. there has been a considerable amount of debate about the ethical right to isolate mrsa carriers in the nursing home setting. it is difficult to find a solid answer that would hold for every case. in our decisions about mrsa-positive people we try to respect two basic principles: the patient's right for proper medical treatment and the individual's right to live a normal life. the actions taken now and over the next few years are crucial in determining how the trend of mrsa in finland progresses. at present, we still have the chance to prevent the situation from becoming much worse. in 2005, the finnish government launched a fund for the health districts to update their resources on emerging hospital infection issues, including mrsa prevention. sweden, denmark, norway, iceland, and finland have also started a joint initiative, provoked by the scandinavian society for antimicrobial chemotherapy, to try to find ways to keep mrsa levels below 1% among s aureus isolates in each country. at the very least, this cooperative effort will mean that information on the mrsa situation is exchanged, attempts are made to improve mrsa laboratory diagnostics, common reasons for why mrsa is emerging sought, and attempts made to increase awareness among the public and health-care workers. in countries where mrsa case levels have remained low, the only right and ethical decision is to try to fight hard to keep the situation from becoming worse. this battle requires new investments now, but it will save money and resources in the future. chief physician, hospital bacteria laboratory, department of bacterial and inflammatory diseases, national public health institute, 00300 helsinki, finland turkey mrsa emerged in turkey in the 1980s as a major clinical problem in hospitals, and to date has continued to be one of the most problematic nosocomial pathogens. the extent of resistance varies nationally, regionally, and even institutionally. in addition to meticillin, these strains are also resistant to routinely used antimicrobials, and thus infections caused by such isolates cause serious treatment difficulties. 37 unfortunately for turkey, accurate and recent population-based national surveillance for communityacquired and hospital-acquired drug-resistant microorganisms does not exist. however, data from sporadic reports of the number of cases suggest an urgent need for surveillance. available reports provide a fragmented and incomplete picture to guide our understanding of the problem. the most recent and only national resistance screening programme data supported by the turkish scientific and technological research council is from 1993. in this study, 1826 clinical isolates both from outpatient and inpatient clinics (including surveillance isolates) from 29 different hospitals were collected and screened. overall, mrsa resistance varied from 7% to 55% at different centres. 38 this range of resistance may have been due to variations in patient population, hospital care practices, and infection control activities. other factors-eg, size, locality, and type of hospital-were also contributing factors to the wide disparity in resistance. among the identified isolates, 178 nosocomial strains were randomly selected for evaluation of the mechanisms of meticillin resistance, and the presence of meca was found to be the most frequent type (94%). 38 when various other studies from different centres were evaluated up to 1995, it was noted that up to 40% of strains in some places were mrsa. in a further evaluation extending to 1999, this figure rose to 70%. 39 more recently, data from the past 5 years from individual centres across turkey state the percentage of mrsa strains is 65·5%. [40] [41] [42] [43] when tested, all strains were reported to be vancomycin and teicoplanin susceptible but potential emergence of resistance to these drugs should be kept in mind, since this has prompted the overuse of glycopeptides in empirical and even prophylactic therapy. 44, 45 evaluation of reports showed no correlation between mrsa rates and sex, profession, hospital department, or carriage. it was difficult to evaluate the age distribution and age group at risk. ideally at each hospital multidrugresistant strains should be regularly identified. clonal typing methods should be used to identify the relations between these strains, and to see which strains predominate in the country. we evaluated our hospital's main nosocomial mrsa clone and tried to track the sources of the clonal types in different clinics by pulsed field gel electrophoresis. one major type was identified among their strains. this single clone was compared with another turkish university hospital's clone and several main international clones including the iberian and brazilian clone, and it was found to be different from them. 46 predominance of this clone or any other clone is not known. this type of evaluation needs to be done by most hospitals. with enough funding a national netherlands yes, they do not like to see us coming: physicians, nurses, and hospital administrators may shrink away when the banner of mrsa is raised by the hospital infection-control practitioner-it always means expensive and timeconsuming isolation, may mean temporary removal of staff, or the partial or complete locking of wards that have to stop admissions. yet the incidence of mrsa in the netherlands remains one of the lowest of europe. in 2003, mrsa was isolated from 1601 people, one-fifth of whom were health-care workers, which means that approximately 1280 patients newly acquired mrsa in the course of a year. to put this number into perspective, the netherlands has 16 million inhabitants, 124 hospitals, and approximately 9 million patient-days of stay in hospital. a timely implementation of a national policy of stringent control measures, and a long-standing tradition in parsimonious antibiotic use surely contributed to keeping mrsa at bay. as in many other countries, after a short appearance in the late 1960s, mrsa entered the netherlands in the early to mid-1980s. three large hospitals in rotterdam, amsterdam, and utrecht experienced outbreaks that were eventually controlled by strict infection-control measures that included isolation of patients, screening of patients and hospital staff members, and closure of wards. both in amsterdam and in utrecht, mrsa was introduced by a patient who had been transferred from a hospital abroad. these early experiences prompted the dutch working party on infection prevention (wip) to formulate national mrsa guidelines. this working group is funded by the ministry of health, and its task is to develop guidelines for infection prevention in hospitals, nursing homes and institutions for the mentally handicapped, and dental care and homecare. the guidelines issued by the wip are considered professional standards and are used as such by the dutch public health inspector; this undoubtedly contributes to the adherence to the guidelines by nearly all health institutions. the mrsa guidelines are based on three main principles. first, patients with mrsa are always isolated in single rooms, whether they have an active mrsa infection or not-carriers are also considered potential sources of transmission. isolation for being a carrier raises anxiety in the patient and their relatives. however, 30% of adults are staphylococcal carriers, and they develop an infection only occasionally, so, in itself, carriage of a staphylococcus that has additional resistance is not a source of worry for the individual patient. second, patients suspected of potential carriage are always placed in isolation; potential carriage is considered in all patients transferred from hospitals abroad to dutch hospitals, in patients from dutch hospitals, or from nursing homes with an actual problem of mrsa, and in patients who have been nursed in the same room as a patient in whom mrsa is detected unexpectedly. this isolation may come forum epidemiological surveillance group should be established. 47 data emerging from various centres indicate that in turkey-like many other countries-both infections and the rate of mrsa is gradually increasing. the main factors that contribute to this organism's resistance are misuse and overuse of antimicrobials, which include constant pressure on the physician to prescribe antimicrobials when they are not indicated, the patient's failure to finish a prescribed antibiotic regimen, and also the availability of antimicrobials without a prescription. we believe that control of such factors, alongside implementing good infection-control procedures in turkey, will result in decreasing rates of mrsa. studies from other countries have shown that surveillance activities-eg, setting up a national/regional system to collect and evaluate data from all centres and updating data on antibiotic resistance patterns that will form the basis for prevention guidelines of nosocomial infections-decrease infection rates by as much as 20%. the negative impact of nosocomial infections on our health-care system is not well documented. data obtained from limited number of studies are not suitable for comparison with each other and neither are data from other countries. 48 thus, a national nosocomial infection control project (nosoline), supported by the turkish hospital infections society, has recently started at the hacettepe university hospital, ankara. the project's aim is to develop a regular, standardised surveillance system, allowing electronic data transfer and online announcement of the results to the member centres. to achieve its goals, the problems of gaining financial support and recruiting qualified personnel for data evaluation and processing need to be solved, and additionally all major hospitals will need access to the system. 49 despite the absence of extensive data, reports of recent percentages of up to 70% of nosocomial mrsa infections confirm the urgent need for an effective antimicrobial usage policy for our country. to prevent resistant isolates spreading within and between hospitals, proper infection control procedures should be enforced, which currently most hospitals lack. in time we hope to prevent the uncontrolled availability of antimicrobials, increase awareness of such problems to physicians and the community, and have more available funds to support programmes in hospitals. we declare that we have no conflicts of interest. as a shock to people who become ill abroad, are repatriated, and arrive with a sigh of relief in a hospital in the netherlands, only to be faced with strict isolation measures, where even the family members have to wear gowns and masks upon entering the patient's room. an often-heard reaction is "am i a leper?", and it may be taxing to the attending physician to explain the reasoning behind the precautionary measure. third, contacts of patients with mrsa-both other patients and hospital staff-are screened for mrsa carriage and treated with mupirocin nasal ointment if found positive. this screening can prove trying for staff, because they are not allowed to return to work unless negative. fortunately, mrsa is usually quickly lost in normal community life or upon treatment with mupirocin nasal ointment. on occasion, a staff member proves to be a "stubborn" carrier, and several courses of mupirocin and the use of disinfectant soap are needed to clear the carriage. this upsets personal life and the working schedules of staff colleagues, who have to fill in for their colleague during the period he or she is not allowed to work. fortunately, untreatable carriage is very unusual, but may lead to the necessity to change job. the wip guidelines for mrsa are also published in english and can be found at http:// www.wip.nl. this very active searching policy implies that many of the mrsa strains that are identified in dutch hospitals are not actually causing infections, but are merely colonisers at the moment that they are detected. indeed, of the 1601 strains isolated in the past year, one-fifth were from health-care workers, who very seldom have an active infection when they are screened; the remainder were from patients who may have had an active infection or may just have been colonised. in the netherlands, mrsa occurs mainly in isolated cases or in smaller outbreaks, and it is not yet endemic in any hospital. because of this, it is possible to institute strict control measures whenever needed, although every time they cause great upheaval in the implicated ward, which has to isolate patients and screen patients and staff. the decreased therapeutic options, the nightmare of impending vancomycin resistance, and the higher numbers of therapeutic failures that accompany infections with mrsa provide a firm ground for the dutch policy. it is therefore strongly supported by the inspectorate of health care and by all microbiologists, infectious disease specialists, and infection-control officers involved. there is the odd dissenting voice of a surgeon or intensive-care physician who points to his colleagues in the uk or the usa where mrsa is rampant-"medical life is possible over there isn't it?" part of the unease of physicians is the result of misunderstandings and some "urban myths"-more appropriately "hospital myths"about mrsa. for instance, surgeons may think that they cannot operate on a patient who carries mrsa. nothing could be further from the truth. yes, elective surgery might be postponed, but all necessary surgery should go on as planned, with extra precautions. again, it is too often forgotten that 30% of all adults are staphylococcus carriers, and that the carriage in itself does no harm. up to now there have been neither legal nor ethical qualms about the dutch "search, isolate, and destroy" strategy, as in the end, all sections of medical life in the netherlands-physicians, nurses, and administratorssee the value of prevention. after careful explanation, even if some measures may tax the ingenuity of administrators and staff, the experience is almost invariably one of excellent cooperation, and most people involved enjoy the process of working for the greater good. although the exact burden of disease caused by mrsa remains largely unknown, most experts would agree that mrsa infections represent an important clinical and public-health problem. we hardly need to call readers' attention to the thousands of articles published over the past three decades about epidemiological and microbiological aspects of mrsa. yet uncertainty remains about the best approach to prevent and control this worldwide plague. in this issue of the lancet infectious diseases, several authors offer insight into mrsa control approaches used in different areas of the world. countries like finland, denmark, and the netherlands have managed to keep mrsa at a low level using surveillance cultures of patients and personnel, strictly enforced contact precautions, and judicious use of broad-spectrum antibiotics. unfortu-nately, these countries are facing now the paradoxical situation that transmission of community-acquired mrsa may jeopardise their well-established strategies to control nosocomial mrsa. denmark, for instance, has seen a substantial increase in mrsa since 2003, due to the epidemic spread of genetically distinct communityacquired mrsa strains. 50 conversely, many middleincome countries (eg, turkey, argentina) and some highincome countries (eg, italy, greece, uk, usa) that were not able to install stringent counter-measures now have hyper-endemic mrsa and are obliged to concentrate available resources to prevent mrsa infections in highrisk populations-eg, dialysis, transplant, or critically ill patients. a few countries with endemic mrsa (eg, australia, france, belgium) have managed to stabilise or even decrease mrsa prevalence in confined geographic areas. on the other hand, several asian countries (eg, china, south korea, japan) have more or less ignored this public-health problem for a long time, resulting in some of the highest mrsa incidence rates worldwide. in these countries, financial incentives for physicians' antibiotic prescribing linked to the pharmaceutical reimbursement system have strongly influenced antibiotic overuse and increased antibiotic selection pressure on mrsa; an issue that has only recently been adequately addressed at the policy level. why do mrsa rates vary so much across countries? differences are caused largely by uneven control and isolation measures, hand hygiene practices, antibiotic prescribing behaviours, and allocation of resources. 51 cultural and economic factors pervade all aspects of mrsa control, which can only be fully successful if strict measures and policies are installed at an early stage of mrsa dissemination, sufficiently supported by financial and staff resources. especially at the early phase of a nationwide mrsa epidemic, the full clinical impact of mrsa may not be visible, leading to misconceptions among clinicians and policy makers that mrsa may not be a threat to patient safety. do we have any hope for the future? as mrsa surveillance systems and control strategies improve in quality and become more coherent among different countries, international pressure may start to be applied to induce change in countries where infection-control policies are lax or non-existent. the situation with mrsa might become comparable to that observed for other infectious problems such as severe acute respiratory syndrome and mad cow disease-economic and political pressure may contribute to compliance and uniformity in control measures and to allocation of resources to improve patient safety. 52 yet stringent mrsa control worldwide will remain difficult to implement and will require intensive surveillance efforts and substantial resources. to achieve this goal may be possible, as shown by several examples where successful action against mrsa has been endorsed by strong policy support. adequate hand hygiene decreases the transmission of mrsa, although the practice is difficult to enforce, because of psychological, practical, and organisational barriers. promoting hand hygiene to improve patient safety and decrease health-care-associated infections worldwide constitutes a core component of the first global patient safety challenge ("clean care is safer care") of the who world alliance for patient safety launched in 2004. if successful, clean care is safer care will certainly have a positive impact on mrsa transmission and other antibiotic-resistant infections. low mrsa prevalence in a country is good news in that preventive measures are more likely to succeed than if endemic mrsa levels are already present. unfortunately, for key questions regarding the most cost-effective control of endemic mrsa, we have only weak or contradicting evidence. several well-conducted studies from france, germany, the uk, and the usa have recently illustrated this dilemma. [53] [54] [55] [56] [57] whatever the final outcome of this ongoing debate, health authorities and policy makers are well-advised to put effort and money into their mrsa control efforts. mrsa is everybody's business, not only that of hospital epidemiologists and a few opinion leaders. national nosocomial infections surveillance (nnis) system report, data summary from predicting methicillin resistance and the effect of inadequate empiric therapy on survival in patients with staphylococcus aureus bacteremia occurrence and antimicrobial resistance pattern comparisons among bloodstream infection isolates from the sentry antimicrobial surveillance program outcomes analysis of delayed antibiotic treatment for hospital-acquired staphylococcus aureus bacteremia the impact of methicillin resistance in staphylococcus aureus bacteremia on patient outcomes: mortality, length of stay, and hospital charges adverse clinical and economic outcomes attributable to methicillin resistance among patients 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infections successful long-term program for controlling methicillin-resistant staphylococcus aureus in intensive care units we declare that we have no conflicts of interest. infection control program, department of internal medicine, university of geneva hospitals, switzerland key: cord-340028-6oicmeam authors: zhavoronkov, alex title: geroprotective and senoremediative strategies to reduce the comorbidity, infection rates, severity, and lethality in gerophilic and gerolavic infections date: 2020-03-31 journal: aging (albany ny) doi: 10.18632/aging.102988 sha: doc_id: 340028 cord_uid: 6oicmeam the recently identified sars-cov-2 betacoronavirus responsible for the covid-19 pandemic has uncovered the age-associated vulnerability in the burden of disease and put aging research in the spotlight. the limited data available indicates that covid-19 should be referred to as a gerolavic (from greek, géros “old man” and epilavís, “harmful”) infection because the infection rates, severity, and lethality are substantially higher in the population aged 60 and older. this is primarily due to comorbidity but may be partially due to immunosenescence, decreased immune function in the elderly, and general loss of function, fitness, and increased frailty associated with aging. immunosenescence is a major factor affecting vaccination response, as well as the severity and lethality of infectious diseases. while vaccination reduces infection rates, and therapeutic interventions reduce the severity and lethality of infections, these interventions have limitations. previous studies showed that postulated geroprotectors, such as sirolimus (rapamycin) and its close derivative rapalog everolimus (rad001), decreased infection rates in a small sample of elderly patients. this article presents a review of the limited literature available on geroprotective and senoremediative interventions that may be investigated to decrease the disease burden of gerolavic infections. this article also highlights a need for rigorous clinical validation of deep aging clocks as surrogate markers of biological age. these could be used to assess the need for, and efficacy of, geroprotective and senoremediative interventions and provide better protection for elderly populations from gerolavic infections. this article does not represent medical advice and the medications described are not yet licensed or recommended as immune system boosters, as they have not undergone clinical evaluation for this purpose. aging is a complex, multifactorial process [1] that leads to loss of function and is the primary risk factor for major human pathologies including cancer, diabetes, cardiovascular disorders, and neurodegenerative diseases [1, 2] . although there is still much debate in the scientific community, proposals have been made to classify aging as a disease in order to develop therapeutic strategies to prevent or delay the onset of age-related illnessess [3] [4] [5] . increasing frailty with age leads to an increased risk of many diseases. these diseases are commonly referred to as age-related [6] . many pathogens are more infectious and prevalent in the elderly, [7] [8] [9] [10] and may be referred to as gerophilic (from greek, géros "old man" and philia, "love"). some infections, including covid-19, are not exclusively gerophilic, as younger people may also become infected. however, these individuals have mild symptoms or remain asymptomatic, while the elderly experience substantially more severe symptoms and lethality. the term gerolavic (from greek, géros "old man", and epilavís, "harmful") may more appropriately the online resources our world in data [16] and the lancet's global burden of disease [17] provide deep visual insight into the global burden of disease by cause and demographics. in 2017, there were 56 million deaths globally; over two-thirds of these (76%) were in people over 50 years of age. according to the online period life tables put out by the us social security administration [18] , the annual chance of death in 2015 (the probability of dying within one year) for a person over 80 was 5.2%, increasing to 14.8% by the age 89. according to estimates by the us centers for disease control [19] , approximately 5,945,690 individuals older than 65 had symptomatic influenza during the 2017-2018 season, resulting in 3,329,586 medical visits, 540,517 hospitalizations, and 50,903 deaths. hence, the death rate for those hospitalized with influenza was 9.4% for patients over 65. however, many of the covid-19 patients over 65 years have one or more comorbities [20] , and it is often difficult february 11, 2020 . the figures are adopted and generated from [12] (http://weekly.chinacdc.cn/en/article/id/e53946e2-c6c4-41e9-9a9b-fea8db1a8f51). to attribute the cause of death exclusively to the gerolavic coronavirus. currently, there are no accurate statistics linking smoking status, lifestyle, and behavior to the severity and lethality of covid-19. despite this, it is possible that these factors, as well as frailty and comorbidities, play a substantial role. gerolavic diseases such as covid-19 may not significantly increase the yearly death rates for each individual age group; however, these diseases substantially accelerate death from multiple conditions, and compress the process to less than two weeks. comorbidity is also the likely cause of the substantial differences in death rates among different countries due to differences in patient demographics, levels of preparedness, when the epidemic began locally, and reporting [13] . only the data available from china, where the epidemic has subsided, and the diamond princess cruise ship were used for this study. one of the possible causes of the age-associated increases in covid-19 infection rate, severity, and lethality is immunosenescence. immunosenescence is a well-known age-related process contributing to the global burden of disease [21] . it is among the major factors underlying the difference between younger and older populations in the response rate to vaccinations and the virulence of infectious diseases [22] [23] [24] . among the factors contributing to immunosenescence is the chronic involution of the thymus gland with increased age. indeed, the infection rates of covid-19, separated by age, are correlated with involution of the thymus [12] . the thymus gland is most active early in life, reaching maximum size within the first year. its activity then declines with age until an individual reaches 40 to 50, after which there are negligible traces of the thymus remaining, replaced by fibrotic tissue [25] . as a result of thymic involution, the number of naïve t cells exiting the thymus decreases significantly, with substantial declines in older age [26] . besides thymic involution, there are many other factors driving immunosenescence and the increase in multimorbidity that occurs during aging [22, 27, 28] . figure 2 illustrates the hypothesised reciprocal relationship between immunosenescence and infectious disease acquisition. in this model, age-associated immunosenescence leads to a reduced ability to resist infection, while infection produces biological damage and loss of homeostasis. this ultimately contributes to accelerated aging and the development of age-related diseases, and further accelerates immunosenescence. in support of this model, infections and other age-related diseases are among the main causes of death in the developed world and in developing countries. due to the gerolavic nature of covid-19, the classical preventative measures and treatment strategies used for targeting infectious diseases may not be as effective, and there is a need for alternative geroprotective and senoremediative strategies. there are multiple clinical trials in progress using established medical interventions to treat covid-19, with the number of studies rapidly increasing [23] . for a list of promising sars-cov-2/covid-19 targets and treatment approaches, please refer to the global health drug discovery institute's portal dedicated to covid-19 [29] . here we compare the expected benefit of treatments for elderly populations (60 years and older) that are currently in development, including standard preventative strategies such as vaccines and antivirals targeting sars-cov-2, and the potential added benefit of speculative geroprotective strategies such as rapalogs, nad+ boosters, senolytics, and stem cell treatment. these additional measures may be used in isolation or as adjuvant therapies to reduce infection risk, symptom severity, or improve vaccine efficacy. vaccine development is one of the most successful approaches for combating viral diseases globally, and is often regarded as one of the greatest advances in biomedical science and integrated healthcare. currently, there are around 60 active clinical trials related to a sars-cov-2 vaccine, most of them taking place in china [30] . broadly speaking, the success of a vaccine partly depends on the similarity of the vaccine strain with the viral pathogenic strain in question. in addition, an individual's immune response must be sufficiently strong to mount a reaction to the vaccine that can later confer protection against the pathogen, should exposure occur. our current strategy for targeting annual influenza viral outbreaks focuses on effective vaccination based on predictions of strain variants. people >60 years of age with chronic medical conditions, such as type 2 diabetes or cardiovascular disease, direct immunosuppression from hiv, posttransplant or biologic treatment, pregnant individuals, or those with bmi>40, are believed to be at higher risk for influenza infection due to a weakened immune response [31] . similarly, vaccines do not provide complete protection in older populations due to agerelated declines in immune function and accumulation of multi-morbidities. outbreaks can occur in elderly nursing homes even when vaccination rates reach 80-98% uptake [32] . thus, even when a successful vaccine for sars-cov-2 becomes available, a geroprotective agent might be used in combination with the vaccine to boost the immune response. currently in most countries, the influenza vaccine formulation is determined 6-9 months before the expected outbreak season and the strains are based on the precedent season's viruses. as a result, vaccine efficacy is expected to differ from season to season. thus, an ongoing additive geroprotective therapy is of high importance [33, 34] and is applicable beyond the current pandemic. while vaccines may be the best preventative strategy for reducing the infection rates, severity, and lethality of covid-19, the rates to vaccines in the elderly will likely be lower [35] and vaccine potentiation strategies [36] may be explored and evaluated in clinical trials. while chemoprophylaxis is not routinely indicated and is not considered a replacement for vaccination, using influenza as an example, prophylactic treatment prior to symptom onset in high-risk groups or after close contact exposure to the virus is an alternative preventative strategy against viral disease [31] . for influenza, the neuraminidase inhibitors oseltamivir and zanamivir are occasionally given prophylactically to high-risk individuals in long-term care facilities during outbreaks [37] . nevertheless, there is currently no definitive benefit proven for antiviral treatment outside of these specific circumstances, as it comes at a cost and may be associated with side effects; for example, zanamivir can induce bronchospasms in patients with chronic respiratory disease and asthma. pharmacotherapy for individuals with infection remains the cornerstone of clinical practice. the success of antiviral treatment is condition-specific, ranging from new, direct-acting antiviral drugs that offer a potential cure for hepatitis c [38] ; to the highly active antiretroviral drugs that enable hiv positive individuals the prospect of a healthy life expectancy while on treatment; to antiviral drugs for herpes simplex types 1 and 2 that lead to symptom alleviation but do not eradicate the latent infection; to antivirals for seasonal influenza that are believed to reduce symptom duration, and reduce complications and transmission risk. other anti-influenza medications licensed for treatment, aside from oseltamivir and zanamivir, consist of an intravenous neuraminidase inhibitor, peravamir, and a novel oral inhibitor of cap-dependent endonuclease, baloxavir. neuraminidase inhibitors are effective against both influenza a and b, while an additional class of antivirals that are no longer recommended for treatment of influenza due to reduced efficacy, neurological side effects, and widespread resistance, adamantanes (m2 inhibitors, amantadine and rimantadine), are only active against influenza a [31] . although many patients with influenza exhibit minimal clinical improvement upon treatment with these medications, they are currently recommended for treatment of all hospitalised patients, even prior to laboratory confirmation of influenza infection. evidence shows that the greatest benefit is seen when these drugs are administered 24-30 hours prior to symptom onset, in which case they reduce symptom duration by 0.5-3 days and reduce transmission risk [39] [40] [41] [42] . according to the recent covid-19 treatment guidelines in china [43] , symptomatic treatment for covid-19 patients is recommended for mild cases and consists of aging rest, isolation, adequate hydration, analgesia, and antipyretic medication. moderate and severe cases (mostly hospitalized) require additional measures, such as careful fluid balance, intravenous antibiotics for superinfections, oxygen supplementation, non-invasive ventilation with or without positive pulmonary pressure, and in some cases intubation and mechanical ventilation. although the projected global infection rates are variable, we share a common concern that outside of china there may be an insufficient number of beds for hospitalization and ventilation units if the disease spread does not slow down. even asymptomatic covid-19 infections can induce lung fibrosis, which may lead to reduced function of the respiratory system. further, severe cases are often complicated by bacterial infections and pneumonia, leading to fibrosis. therefore, covid-19 rehabilitation may include antifibrotic compounds, anti-copd, and regenerative medicine therapies. there are multiple interventions proposed in the academic literature to remedy age-associated increases in infection rates, severity, and lethality for a variety of infections. for example, regular increased physical activity has been proposed to reduce immunosenescence [44] . fahy et al. [45] and horvath [46] have suggested that a combination of the potentially geroprotective compound metformin, recombinant human growth hormone (rhgh), and dehydroepiandrosterone (dhea) may reverse biological age, as measured using the methylation aging clock, and immunosenescence [45] . geroprotectors were previously proposed to enhance human radioresistance in extreme conditions [47] . while there is no clinical evidence yet suggesting age reversal or improved immune function in the elderly, efforts are being made to identify new geroprotectors using human data and artificial intelligence [48] [49] [50] . further, the use of natural compounds that mimic the effects of known geroprotectors is generally recognized as safe [51] . however, attempts have been made to develop criteria for the evaluation of geroprotectors for clinical validation. there are multiple strategies proposed to restore immune function in the elderly [52] , and multiple databases of geroprotectors exist [53, 54] . however, to date the only known geroprotectors backed by promising clinical evidence of improved immune response to viral infection in the elderly, although still limited by a lack of large clinical trials, are sirolimus (rapamycin) and everolimus. these may be used as single agents in combination with other treatments (figure 3 ). sirolimus (rapamycin) is a well-known geroprotector, known to effectively increase lifespan and slow aging in many species, including yeast [55, 56] , drosophila [57, 58] , c. elegans [59] , and mice [60] [61] [62] [63] [64] . it also delays age-related diseases in humans [65] [66] [67] [68] , and blagosklonny proposed rapamycin for the prevention of multiple age-related diseases in humans [69] [70] [71] [72] . sirolimus and rapalogs are commonly used as immunosuppressants. rapalogs, the derivatives and mimetics of rapamycin, target critical factors in the rapamycin (tor) pathway. everolimus (rad001), another close structural derivative of sirolimus developed by novartis, acts as an immunosuppressant; but like sirolimus, it has many other properties beyond immunosuppression [73] . paradoxically, these compounds also exert immunostimulatory effects, such as boosting t cell responses in reaction to pathogen infection and vaccination [74] . nevertheless, this would not be the first case of a physiological paradox in clinical medicine. the administration of beta-blockers to heart failure patients at first seemed contradictory, as these compounds slow down an already failing heart, but proved to provide the most benefit for the treatment of heart failure patients. likewise, hormonal treatment of hormone-dependent cancers, such as testosteronedependent prostate cancer, seems incongruous. however, administration of a synthetic version of gonadotropin-releasing hormone (gnrh) in a different dosing regime from the cyclical secretion that occurs physiologically, which normally indirectly increases testosterone levels, actually reduces hormone levels. therefore, it might be possible that a drug that is known to be an immunosuppressant might in a different dosing regimen prove to be an immunostimulant. however, extremely cautious clinical validation is required as this treatment might carry significant risks; indeed, there is some indication that morbidity from coronavirus infections occurs from secondary overactive immune responses [75, 76] . in addition to rapamycin, other agents that inhibit mtor, such as torin1, torin2, azd8055, pp242, ku-006379 and gsk1059615, may act similarly to rapamycin in low-doses and may have a geroprotective effect [77] [78] [79] . substantial pre-clinical validation would be required to apply these compounds to specific age-associated diseases and to explore clinical applications of these compounds in human clinical trials. multiple clinical observations suggested that patients with cytomegalovirus (cmv) disease who were treated with rapamycin demonstrated better outcomes and were better able to control cmv viremia than patients treated with standard calcineurin inhibitor-based immunosuppression following transplantation [74, 80] . in 2009, two seminal studies of sirolimus demonstrated the immunostimulatory effects of rapamycin on the cd8+ memory t cell response following pathogen infection [74, 80] . later studies also showed that monkeys treated with sirolimus exhibited increased recall responses and enhanced differentiation of memory t cells following vaccination with modified vaccinia ankara [81] . additional clinical studies by mannick et al. [82, 83] demonstrated the immunostimulatory role of rapalogs in the elderly using the novartis rapalog everolimus (rad001), a close structural analog of sirolimus (rapamycin). administration of everolimus ameliorated immunosenescence in healthy elderly volunteers and enhanced the response to the influenza vaccine by around 20% at doses that were well tolerated [82] . further studies demonstrated enhanced immune function and reduced infection in elderly patients receiving tolerable doses of everolimus. mannick et al. also conducted a phase 2a randomized, placebo-controlled clinical trial which demonstrated that a low-dose combination of dactolisib (bez235) and everolimus in an elderly population was safe and associated with a significant (p=0.001) decrease in the rate of reported infections [83] . mannick and colleagues further conducted a phase 2a randomized, placebo-controlled clinical trial that demonstrated that a low-dose combination of dactolisib (bez235), a pi3k inhibitor [84] and catalytic mtor inhibitor, and everolimus in an elderly population was safe and associated with a significant (p=0.001) decrease in the rate of reported infections [83] . a follow-up trial of dactolisib alone (bez235 rebranded as rtb101) for prevention of respiratory tract infections in the elderly did not meet the primary endpoint and further trials were withdrawn [85] . in prior studies, everolimus (rad001) was used as a standalone agent or in combination with dactolisib, which may explain the phase 3 failure of bez235/rtb101. there are over 95 phase 3 and phase 4 studies for these agents [86] , and they are generally well tolerated even in high doses. even though it may not be commercially viable due to the patent expirations, clinical trials should be conducted to evaluate the effectiveness of these agents for protection against sars-cov-2 (covid-19) and other gerophilic and gerolavic infections. metformin is a drug approved to treat type 2 diabetes but appears to target a number of aging-related mechanisms, including decreasing igf-1 levels, inhibiting mtor, and inhibiting mitochondrial complex 1. metformin is currently in the first large-scale human aging clinical trial of aging, the targeting aging with metformin (tame) study, which is investigating its effect on time to a new occurrence of a composite outcome that includes cardiovascular events, cancer, dementia, and mortality [87] . metformin would likely still be contraindicated in elderly patients with advanced chronic kidney disease and egfr<15. a reduced dose would potentially be required for egfr<30 due to a risk of lactic acidosis. the effects on gerophilic and gerolavic infections should be carefully examined in the context of the tame study, and other clinical trials involving metformin. nicotine adenine dinucleotide (nad) is a cofactor of multiple fundamental enzymes. it is involved in metabolic regulation through the krebs (citric acid) cycle, oxidative phosphorylation, and cellular signaling, as well as cellular senescence and dna repair through the poly-adp-ribose polymerases (parps), sirtuins, and cd38. nad levels decrease with aging, and benefits of nad supplementation have been reported in multiple animal studies. although no proof of a similar effect in humans has been shown, several clinical trials are in progress [88] [89] [90] [91] . supplementation with nicotinamide riboside (nr) in one human study produced an improvement in exercise capacity in a population with a mean age of 71 [92] . this compound was also shown to reduce blood pressure in hypertensive patients [93] . nicotinic acid is another nad precursor that is converted in the body to nad by the enzymes naprt, nmnat, and nads. large-scale trials of nicotinic acid for cardiovascular disease [94, 95] showed some efficacy, but produced adverse side effects, such as headache, skin flushing, and dizziness [96] . nad acts at a cellular level and it is still unclear whether oral or intravenous supplementation with nad donors, such as nr and nicotinic acid, will increase nad levels and exert a clinical benefit in humans. however, covid-19 patients may benefit tremendously from these compounds, as sars-cov-2infected patients have increased levels of cd38+, and nad has been shown to enhance dna repair via parp pathways [97] . caution should be exercised when conducting any clinical trials for nad boosters against gerophilic and gerolavic infections, as the underlying biology of nas metabolism and viral infections is still poorly understood. recent studies in humans demonstrate that nr supplementation reduces the levels of circulating inflammatory cytokines [98] , while nicotinamide mononucleotide (nmn) may reduce the expression of these cytokines [99] . other studies implicate nad in increased cytokine production [100] and the nad+consuming enzyme cd38 in increased inflammation [101] . additional immunological studies of nad boosters must be performed before clinical trials may be conducted. however, considering the large consumer base of nr and nmn supplements, it may be possible to conduct metastudies on influenza and sars-cov-2 infectivity, severity, and lethality. senolytics are drugs that are postulated to selectively destroy senescent cells, which accumulate with aging and exhibit senescence-associated secretory phenotype (sasp), through senolysis, apoptosis, immunosurveillance, or other mechanisms of action [102] . sasp is now hypothesised to lead to nad depletion and thus initiate or perpetuate an increase in sterile chronic inflammation. many drug classes, ranging from fibrates to cardiac glycosides, have been reported to have senolytic properties in animal models [103] . however, recent promising human data have been reported with the tyrosine kinase inhibitor dasatinib in combination with the plant flavonol quercetin in a trial by the mayo clinic [104] ; flavonoid polyphenols have also proven beneficial. in addition, pre-clinical and clinical data suggest that flavonoids may be used for prophylaxis in upper respiratory tract infections [105] . although senolytic drugs would have a scientifically plausible role in biological age reversal and thus reduction of mortality from gerolavic viruses like sars-cov-2, it has not been shown that these classes of drugs would protect against infection or could be used as adjuncts to vaccination. in addition, there remains the risk that senolytics would not be sufficiently specific to discriminate between deleterious senescent cells and quiescent (dormant) cells, which might still differentiate into the mature cell types of a given tissue, and could thus deplete beneficial protective stem cell reserves. it has been shown in multiple studies that calorie restriction leads to increased lifespan and improved cardiometabolic markers, even when initiated in middle age [106] . caloric restriction should be considered as a preventive measure on a long-term basis and is indicated for younger individuals. some elderly patients already have frailty syndromes and evident sarcopenia/ osteopenia, which limits the suitability of intermittent caloric restriction. nevertheless, the aging benefits of time-restricted feeding and intermittent fasting go beyond simple caloric restriction due to the production of ketones. ketones are active signaling molecules that play a major role in the ppar, sirtuin, nad and cd38 pathways, encourage autophagy (the removal of damaged cellular materials), modulate the immune response, and have been explored in clinical trials as an adjuvant therapy for cancer treatments [107] . within 8-12 hours of food restriction, ketones are believed to rise to 0.2 to 0.5mm and continue to increase within the first 48 hours to 1 to 2mm [108] . under fasting conditions the major body ketone in the plasma, beta-hydroxybutyrate (bhb), increases. bhb is believed to confer the major metabolic benefit of fasting and is in development as an independent therapeutic supplement. an age-related decrease of thymic function consequently reduces the levels of specific t cell subsets [109] . foxp3+ regulatory t (treg) cells are critical in homeostasis of the immune system and are believed to start declining in numbers at around 50-60 years of age; this remains one of the fundamental drivers of immunosenescence. there are two known origins for treg cells: thymus-derived treg cells and peripherally-derived treg (ptreg) cells. thus, inducing a peripheral treg response in older individuals might be a feasible strategy for increasing treg cell levels until we have more plausible options for thymic rejuvenation. foxp3 transcription factor (tf) is the most important regulator of tregs and ageassociated immunosenescence. foxp3 tf expression is regulated by chemical modification by sirtuin (sirt) and histone deacetylases, in particular sirt1 and hdac9 [110, 111] . interestingly, nad is essential for sirtuin action. therefore, it is plausible that nad and nad-related compounds such as nr and nmn, which are under investigation as therapeutic interventions that increase serum and cellular nad levels, also act via sirt1 along the foxp3 and treg axis, and play a role in immunosenescence and "inflammaging". a brief summary of the conventional and geroprotective and senoremediative strategies for patients 60 or older is provided in table 1 . while there are decades of clinical evidence supporting the use of rapalogs, such as sirolimus, everolimus, and metformin, substantial meta-analysis and additional clinical trials must be conducted to understand the population-level and individual effects of these drugs taken as single agents and in combination in the context of gerolavic diseases. in this paper i propose conducting clinical trials on these known geroprotectors as a preventative measure before patients are exposed to disease (figure 4 ). in the case of covid-19 as the number of cases worldwide increases, meta-analysis of infection rates, severity, and lethality should be performed rapidly to evaluate the effects of geroprotectors, with particular focus on rapamycin. since covid-19 engages the immune system to damage the lungs, it may be entirely plausible that the immunomodulatory properties of rapamycin may go beyond prevention and may provide an effective treatment option. however, this hypothesis must be validated using meta-analysis before being proposed for a clinical trial. as covid-19 causes substantial lung damage, antifibrotics, senolytics and other geroprotectors may be explored in clinical trials to assist in patient recovery to prevent a reduction in respiratory function. since senescence varies among individuals, a person's chronological age is not as important as their biological age. for several years, scientists have sought accurate aging biomarkers that may predict an individuals' biological age and, independently of immunosenescence, their risk of morbidity and mortality. these biomarkers, or "clocks", could then be used to test for the effectiveness of proposed geroprotective treatments and as surrogate markers in anti-aging clinical trials. while there are no reliable aging clocks to evaluate immunosenescence and inflamaging [112] , these biomarkers may be rapidly developed using historical data. at present, age clocks trained on clinical blood tests [113] , transcriptomic [114] and proteomic data [115] , methylation clocks [46, 116] , microbiomic clocks [117] and other clocks have been described. recent advances in artificial intelligence have enabled the development of multimodal multi-omics age-predictors, able to learn complex non-linear patterns and extract the most important features [113, 118] . none of these currently have robust clinical validation and cannot yet serve as companion biomarkers for geroprotective and antiaging interventions intended to ameliorate the population-level effects of infectious diseases during flu seasons and pandemics. we call for rigorous clinical validation and further development of biological aging clocks that could, in the future, allow us to measure the effectiveness of the numerous speculative geroprotective and senoremediative interventions described herein. covid-19 is a gerolavic infection. efficacy of the vaccine will likely be significantly reduced due to immunosenescence and multimorbidity. possible immunogenicity and mild viral prodrome symptoms as a result of vaccination. antibodies for covid-19 antibodies of serum of recovered individuals. antibodies targeting specific sars-cov-2 proteins. successfully trialed in other viral diseases including ebola. reduction in disease severity and lethality in exposed individuals. risk of systemic immune reactions and certain blood borne infections. selective small molecule inhibitors targeting sars-cov-2 proteins such as 3c-like protease. multiple examples from influenza. neuraminidase and endonuclease inhibitors. reduction in disease duration, severity, and lethality in exposed individuals. mild side effects such as nausea, vomiting, diarrhea, etc. non-steroidal antiinflammatory drugs (nsaids), antibacterials, pain management. multiple clinical trials, common use. reduction in severity of disease. mild side effects such as nausea, vomiting, diarrhea, etc. [24] . theoretically, treatments found to be effective against sars and mers are the most promising starting points for treatments likely to be effective against sars-cov-2. the sars outbreak of 2002 was rapidly contained, and no new cases have been reported since 2004 [119] . since the scale of the outbreak did not provide any commercial benefit for the pharmaceutical industry to develop effective drugs for sars, much of the discovery efforts stopped after the epidemic. when the news of sars-cov-2/covid-19 emerged in early january 2020, it was difficult to justify a business case for small biotechnology companies to allocate resources to the effort. by january 28 th , however, insilico medicine allocated resources to generate and test small molecules against the sars-cov-2 3c-like protease [120, 121] . as the scale of the current covid-19 pandemic remains uncertain, it is still difficult to justify allocating scarce company resources to full-scale drug discovery and drug development programs, which may cost tens or even hundreds of millions of dollars [122] . multiple biotechnology companies are in the same situation and will not be able to proceed without substantial backing from government agencies, nonprofit organizations, or bigger pharmaceutical companies. however, given the gerophilic and gerolavic nature of covid-19, strategies targeting age-associated pathologies and immunosenescence, which could decrease the comorbidity, infection rates, severity, and lethality of the disease, will remain commercially-viable even when the pandemic subsides. in addition, respiratory infections are now the third leading cause of death in the world, following cardiac disease and stroke [123] , further justifying the need for these interventions. considering the gerolavic nature of covid-19, where the majority of the seriously affected population is older than 60, classical prevention and treatment strategies may not be effective. given the severity and lethality of the pandemic, even healthcare systems in developed countries will find it challenging to cope with the increased disease burden and hospital needs. conventional approaches to prevention such as vaccines are much needed, but even these do not offer complete protection in the elderly due to multi-morbidity and agerelated immune declines. therefore, interventions that enable immunocompromised elderly to mount an immune response to newly developed vaccines are necessary to help eradicate the disease and reduce the associated mortality. to avoid substantial loss of life and quality of life, primarily among the elderly and vulnerable populations, governments and healthcare systems should investigate preventative and intervention strategies stemming from recent advances in aging research. as discussed in this paper, small clinical studies have shown that several geroprotective and senoremediative interventions, such as treatment with aging sirolimus and rapalogs, can induce immunopotentiation, increase resistance to infection, and reduce disease severity in the elderly, without severe side effects. serendipitously, during the revision of this article, another group utilizing computational approaches proposed using melatonin and sirolimus (rapamycin) in combination to treat the covid-19 infection outside the context of geroprotection [124] . many of these predicted geroprotectors are available as supplements; however, no meta-analysis or metaclinical trials have been performed at scale to evaluate their effectiveness. the covid-19 pandemic highlights the paucity of clinical trials on the effects of dietary supplements and drugs on aging and immunosenescence. the existence of pseudoscience and anecdotal promotion in the supplement industry does not mean that protective compounds do not exist. dietary supplement vendors and pharmaceutical companies need to actively engage in preclinical and clinical research to evaluate the effectiveness of the currently available products on immunosenescence and aging. this paper is not intended to encourage the use of rapalogs or other potential geroprotectors during the covid-19 pandemic. it may be possible that some of the potential geroprotectors described in this paper are harmful to the elderly after infection, and may actually increase disease severity and lethality. however, it may be possible to conduct clinical trials on the efficacy of geroprotectors previously tested in human clinical trials in treating covid-19 and other gerophilic and gerolavic infections. to combat the growing covid-19 pandemic, researchers have united globally to tackle a disease that is impacting lives and healthcare systems around the world. after carefully analysing preliminary data, we suggest that covid-19 has a gerophilic and gerolavic profile, being more infectious and more severe in the elderly. in this paper, we review the current literature on speculative aging reversal treatments, such as experimental geroprotective strategies using everolimus (rad001) and sirolimus (rapamycin). we summarize the current possible interventions and identify the lack of clinical evidence to support their immediate use with the aim of encouraging further, more rigorous reviews of geroprotective compounds such as rapalogs, metformin, senolytics, and conventional and investigational nad+ boosters. we also suggest that further clinical studies should be carefully designed and adequately powered to determine if these interventions might provide clinical benefit as adjuncts to vaccines and antiviral treatments by acting as immune response potentiators. lastly, as with many other diseases, covid-19 is more common and severe in elderly populations, and we thus invite further research and clinical validation in the field of biological aging clocks. these markers could potentially be used in the future to measure and analyze immunosenescence and the efficacy of interventions claimed to slow down or reverse age-related immune decline. this perspective is of a highly speculative nature presented during the time of a global covid-19 pandemic. it is intended for a professional audience to stimulate ideas and aid the global efforts of the scientific community to develop effective new treatments for this disease. this article does not represent medical advice or recommendations to patients. there is no clinical evidence to support the use of the treatments described in this article for this indication and the authors do not advise anyone to self-administer these drugs as covid-19 prevention or treatment. furthermore, this perspective is based on the limited data from the first weeks of the covid-19 outbreak. the demographic distribution of the infected and diseased may change and differ in different countries with different social customs and different ethnicities. the media should exercise caution and seek expert medical advice for interpretation when referring to this article to avoid misinterpretation or unsafe messages being delivered to the community amidst exceptional coverage of this disease in the media at present. the author would like to thank dr. quentin vahaelen of insilico medicine, dr. evelyne bischof of the jiaotong and shanghai university of medicine and the university of basel, and mr. dara vakili of imperial college london for advice and valuable contributions. the author would like to thank dr. richard faragher for the valuable suggestions and comments on the new term "gerolavic" to describe the infections harmful to the elderly, which did not previously exist; and rachel stewart for edits, formatting, and reference management. no funding has been provided for this work. 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2019-ncov potential covid-2019 3c-like protease inhibitors designed using generative deep learning approaches how to improve r&d productivity: the pharmaceutical industry's grand challenge acute respiratory infections are world's third leading cause of death network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 key: cord-335871-zieuc7vk authors: brazee, patricia l.; sznajder, jacob i. title: targeting the linear ubiquitin assembly complex to modulate the host response and improve influenza a virus induced lung injury date: 2020-05-13 journal: arch bronconeumol doi: 10.1016/j.arbres.2020.04.019 sha: doc_id: 335871 cord_uid: zieuc7vk abstract influenza virus infection is characterized by symptoms ranging from mild congestion and body aches to severe pulmonary edema and respiratory failure. while the majority of those exposed have minor symptoms and recover with little morbidity, an estimated 500,000 people succumb to iav-related complications each year worldwide. in these severe cases, an exaggerated inflammatory response, known as “cytokine storm”, occurs which results in damage to the respiratory epithelial barrier and development of acute respiratory distress syndrome (ards). data from retrospective human studies as well as experimental animal models of influenza virus infection highlight the fine line between an excessive and an inadequate immune response, where the host response must balance viral clearance with exuberant inflammation. current pharmacological modulators of inflammation, including corticosteroids and statins, have not been successful in improving outcomes during influenza virus infection. we have reported that the amplitude of the inflammatory response is regulated by linear ubiquitin assembly complex (lubac) activity and that dampening of lubac activity is protective during severe influenza virus infection. therapeutic modulation of lubac activity may be crucial to improve outcomes during severe influenza virus infection, as it functions as a molecular rheostat of the host response. here we review the evidence for modulating inflammation to ameliorate influenza virus infection-induced lung injury, data on current anti-inflammatory strategies, and potential new avenues to target viral inflammation and improve outcomes. tratamiento dirigido al complejo de ensamblaje de cadenas lineales de ubiquitina para modular la respuesta del huésped y mejorar el daño pulmonar inducido por el virus de la gripe a la infección por el virus de la gripe se caracteriza por síntomas que van desde la congestión leve y los dolores corporales hasta el edema pulmonar grave y la insuficiencia respiratoria. aunque que la mayoría de las personas expuestas presentan síntomas leves y se recuperan con poca morbilidad, se estima que cada año 500 000 personas en todo el mundo fallecen por las complicaciones relacionadas con esta infección. en estos casos graves, se produce una respuesta inflamatoria exagerada, conocida como "tormenta de citoquinas", que causa daños en la barrera epitelial respiratoria y el desarrollo del síndrome de distrés respiratorio agudo (sdra). los datos de estudios retrospectivos en humanos, así como de modelos animales experimentales de infección por el virus de la gripe, resaltan la delgada línea que existe entre una respuesta inmune excesiva y una inadecuada, cuando la respuesta del huésped debe mantener el equilibrio entre el aclaramiento viral y la introduction seasonal influenza a viral infection affects a significant proportion of the population worldwide, with an estimated 500,000 people succumbing to iav-related complications each year. while most patients infected with influenza a virus (iav) recover without major sequelae, severe viral pneumonia is one of the most common causes of acute respiratory distress syndrome (ards) [1] [2] [3] [4] . clinically, ards presents with bilateral pulmonary infiltrates, hypoxemia, pulmonary edema and widespread lung inflammation that lead to high mortality rates due respiratory and to multiple organ failure [1, [4] [5] [6] . ards patients can be sub-grouped based on the severity of the inflammatory response, where patients with hyper-inflammation have worse clinical outcomes, spending more days on mechanical ventilation, experiencing increased incidence of organ failure and a higher mortality rate compared to hypo-inflammatory ards patients [7] . impairment of gas exchange in iav-induced ards, in large part, is due to damage to the respiratory epithelial barrier and edema accumulation [1, [4] [5] [6] [7] [8] . during iav infection, an exaggerated inflammatory response, known as "cytokine storm", can occur leading to the development of hyper-inflammatory ards, increasing iav-induced morbidity and mortality ( figure 1 ) [4] . post-mortem studies of lungs from iav-infected patients show extensive diffuse alveolar damage characterized by edema, cellular infiltration, thickening of alveolar walls, and necrosis [9] . interestingly, a study of critically ill patients showed no differences in pulmonary viral load between those who died and those who recovered, while mortality directly correlated with exuberant inflammation, further supporting a maladaptive host response as the major driver of iav-induced lung injury [10] [11] [12] [13] [14] . similar observations are being reported in patients with severe coronavirus disease (covid19) , where severe lung damage is associated with increased pro-inflammatory cytokines and respiratory failure from ards is the leading cause of mortality [15, 16] . with no virus-specific treatment options currently validated, therapies which target the inflammatory response are currently being considered for patients with severe covid-19 [17, 18] . however, as it has been reported for severe iav infections, current antiinflammatory drugs have pleiotropic effects and may lack the specificity needed to carefully calibrate the host response. respiratory epithelial cells, as primary targets for iav infection and replication, initiate inflammatory signaling [19] [20] [21] [22] . in response to respiratory epithelial derived cytokines, innate immune cells, such as neutrophils, monocyte-derived inflammatory macrophages, and natural killer (nk) cells are recruited to the airspace [23] . together with tissue resident alveolar macrophages, recruited innate immune cells are critical for control of viral replication through both lysis and clearance of virus-infected cells [23] [24] [25] [26] . however, in addition to controlling viral spread, innate immune cells contribute to the overproduction of pro-inflammatory cytokines that enhance iav-induced lung injury ( figure 2 ). the pulmonary immune response must be carefully balanced, simultaneously promoting viral clearance and limiting excessive inflammation to maintain proper lung function. findings from animal models of iav infection have shown that modulation of the host immune response is associated with reduced lung injury and improved survival [11, 14, 27, 28] . blockade of specific immune cell subsets has been shown to improve outcomes in mouse models of severe iav infection. for example, genetic deletion of the chemokine receptor ccr2 inhibited the recruitment of monocyte-derived inflammatory macrophages during iav infection and resulted in reduced lung injury with improved survival. however, loss of this myeloid cell population resulted in a delay in viral clearance [29, 30] . moreover, adoptive transfer of nk cells from iav-infected lungs, as compared to nk cell from naïve lungs, resulted in increased mortality of influenzainfected mice [31] , suggesting that inflammation-dependent activation, rather than recruitment, drives the observed pathology. taken together data from these animal models suggest that the determinant of influenza severity be orchestrated by respiratory epithelial cells. the linear ubiquitin assembly complex (lubac) is a multi-protein e3 ubiquitin ligase complex composed of two stabilizing proteins, the heme-oxidized iron responsive element binding protein 2 ubiquitin ligase-1l (hoil-1l) and shank-associated rh domain-interacting protein (sharpin), and a catalytic component, hoil-1-interacting protein (hoip) [32] [33] [34] (figure 3 ). the proteins within the heteromeric complex contain multiple domains for interactions within the complex, ubiquitin binding, as well as catalytic activity [32] [33] [34] [35] [36] . lubac is an essential regulator of nf-κb activation and has been shown to act as a molecular rheostat, regulating the amplitude of the epithelial-driven inflammatory response dung iav infection [35, 36] . the respiratory epithelium actively participates in the first line of defense against pathogens by orchestrating host innate immunity [23, [37] [38] [39] . as iav replicates within the respiratory epithelium cells, the cytosolic pattern recognition receptor (prr), rig-i, is activated and initiates formation of a signaling platform to which lubac is recruited. lubac covalently attaches met-1 linked linear ubiquitin chains to the nf-κb essential modulator (nemo), a component of the inhibitor of nf-κb (iκb) kinase (ikk) complex along with ikkα and ikkβ [40, 41] . due to the high affinity of nemo's ubiquitin binding domain for linear chains, linear ubiquitination of nemo facilitates the recruitment of additional ikk complexes, which results in the efficient trans-autophosphorylation and activation of proximal ikkβ followed by the phosphorylation and degradation of iκbα and robust nf-kb activation ( figure 3 ) [34, 35, 40, 42, 43] . recent reports show that destabilization of respiratory epithelial lubac, via loss of the non-catalytic component hoil-1l, dampens the host response during severe influenza and promotes survival with reduced lung injury as well as reduced viral titers. however, when lubac activity is abolished through deletion of hoip, the alveolar epithelia driven inflammatory response is inhibited and mortality is increased. these findings highlight the fine line between an excessive and an inadequate immune response and suggest that therapeutic modulation of lubac activity may be crucial, as it functions as a rheostat regulating the amplitude of the host response to iav infection. lubac covalently attaches linear ubiquitin chains to nemo, which facilitates the recruitment of additional ikk complexes. stably docked ikk complexes result in the efficient transautophosphorylation and activation of proximal ikkα/β, followed by the phosphorylation and degradation of iκbα. nf-κb translocates to the nucleus to stimulate transcription of inflammatory genes. current anti-influenza strategies are limited to yearly vaccination or administration of antiviral drugs, however, short therapeutic windows, viral mutation, and resistance to current therapies limit their effectiveness. despite available vaccination and anti-viral drugs, the most recent pandemic in 2009 resulted in an estimated 151,700 -575,400 deaths in its first year of circulation worldwide [44] . the pandemic strain contained a novel assortment of viral genes not previously identified in animal or human populations. from its first detection in april 2009, it was only 3 months until resistance to anti-viral drugs was reported, and it took an additional 3 months before the first vaccine offering protection from the pandemic strain was administered [45] . in addition to emerging pandemic strains, between 291,000 and 646,000 people worldwide die from seasonal influenza-related respiratory illnesses each year [46] . novel mutations and reassortments of the virus will inevitably lead to the next iav pandemic; therefore, the use and development of therapeutics that target conserved host pathways, rather than the virus itself, hold promise to curtail the impact of viral infection. moreover, a heterogeneous response to iav with the same virulence exists within the population, suggesting that host factors play a crucial role regulating the host response and determining the severity of lung injury [2, 3, 47] . additionally, experimental evidence from human studies and animal models of severe iav show that viral titers do not always correlate with severity of disease, but rather ards induced "cytokine storm" is the major driver of morbidity and mortality [4, 28, 38, 48] . severe iav infection is associated with inflammatory cytokines in humans and mice. due to their pleiotropic and redundant effects, targeting of individual cytokines may not be a suitable approach to reduce pathology during iav infection. instead, dampening of the immune response may be more effective, as was the case with lubac destabilization noted above [36] . fda-approved anti-inflammatory drugs, including corticosteroids and statins, have been proposed for the treatment of "cytokine storm" associated with severe iav infection [49] . moreover, current data regarding their efficacy is limited to mouse models and retrospective patient observations [49, 50] . corticosteroids have been shown to be effective in limiting the inflammation in in some lung pathologies [49] [50] [51] . however, observational studies of the impact of corticosteroid treatment of iav-infected patients suggest against their use; with administration associated with higher incidence of hospital-acquired pneumonia, longer duration of mechanical ventilation, and increased mortality [50] . similarly, clinical evidence does not support corticosteroid treatment for covid-19 lung injury [52] . statins are another class of drugs recognized for their ability to dampen inflammation [49] . while experimental evidence from mouse models using statins during iav infection have been inconclusive regarding their benefit [49, 53, 54] , retrospective analysis of patient data suggests an association between statin treatment and lower iav mortality rates [55] . these examples highlight the need for new avenues of drug discovery and validations, as no currently available immune modulators have convincingly demonstrated their ability to improve outcomes during influenza infection. lubac represents a potential new target for limiting the pathological inflammation that occurs during iav infection. several chemical inhibitors as well as peptides that bind hoip have been used to inhibit lubac activity in cell culture [56] [57] [58] [59] and in vitro assays [60, 61] and support the specific targetablility of lubac (figure 4) . currently, lubac inhibitors fall into two categories: those that target the catalytic activity of hoip (ie. bay117082, gliotoxin, hoipin) or those that disrupt the interaction between lubac components to destabilize the complex (ie. stapled peptides). bay117082, a small molecule commonly used as an inhibitor of nf-κb activation. it has been observed that treatment of raw 264.7 macrophages with bay117082 prevented il-1 stimulated formation of linear ubiquitin chains. further investigation revealed that bay117082 irreversibly inhibits lubac through a chemical reaction with cysteine residues in the active site of hoip, the catalytic unit of lubac [58] . while bay117082 represents a potent inhibitor of lubac activity, it targets multiple components of the ubiquitin system, including inhibition of e2 ubiquitin conjugating enzymes, and possible proteasome inhibition [58] . as such, use of bay117082 is not suitable for the study of lubac-dependent physiological functions or therapeutic targeting of lubac activity in disease. gliotoxin, a fungal metabolite, was identified in a highthroughput screening for lubac inhibitors using a time-resolved fret-based screening system. while gliotoxin is known to have multiple cellular targets, it is able to inhibit lubac activity and downstream activation of nf-κb at 10x lower concentrations [61] . gliotoxin's strong, irreversible binding to the catalytic site of hoip makes it a selective inhibitor of lubac activity [61] . interestingly, the potency of gliotoxin has been shown to vary between cell types, with myeloid and lymphoid cells being more sensitive to gliotoxin-mediated nf-κb inhibition than epithelial cells [61, 62] . however, this irreversible inhibition may quench the inflammatory response and increase susceptibility to secondary infections. hoipins are synthetic small molecules that reversibly inhibit lubac though targeting of hoip activity, displaying both lubac specificity as well as low cytotoxicity. several derivatives have been made with varying degrees of efficacy (hoipin-1-8), with hoipin-8 showing significantly enhanced ability to prevent lubac-mediated nf-κb activation in response to tnf-α without cytotoxicity compared to the other derivatives in vitro [57] . conversely, stapled α-helical peptides developed based on specific lubac structures disrupt interactions necessary for stable complex formation [56, 63] . stapled peptides are a class of synthetic macrocycles where the secondary α-helix structure is stabilized by the introduction of a hydrophobic bridge or "staple" that rigidifies specific areas to inhibit protein:protein interactions [56] . stapled peptides based on the hoip ubiquitin binding domain have been shown to successfully inhibit lubac activity in vitro by disrupting its interaction with hoil-1l and destabilizing the overall complex [56, 63] . while several inhibitors of lubac have been developed and shown promise in vitro, no data is available detailing their efficacy in vivo. further investigation in to these compounds which target lubac stability to modulate the degree of lubac activity is warranted as they may be therapeutically beneficial for the treatment of hyper-inflammatory response during viral infection, where a graded host response is necessary. within the past 150 years, iav has been the causative agent of at least five pandemics (1889, 1918, 1957, 1968, 2009) [47, 64] . in addition to iav, novel viral threats, such as the coronavirus outbreaks of 2003, 2015 and 2019, quickly spread worldwide before virus-specific vaccines or pharmacological options could be developed. thus, therapies that target conserved host pathways may provide a novel universal treatment strategies, regardless of viral sequence. findings from animal models of iav infection have shown that inhibition of the exuberant host immune response is associated with reduced lung injury and improved survival [11, 14, 27, 28] . however, current fda-approved anti-inflammatory drugs, such as corticosteroids and statins, have failed to show benefit during severe iav infection [49, 50] . beyond viral infections, the amplitude of the inflammatory response has been shown to be a critical determinant in outcome during bacterial sepsis in community acquired pneumonia, a common complication post iav infection [51, 65] . analysis of a cohort of patients showed that reductions in the inflammatory response during bacterial pnumonia, both due to host inability to mount a response or the administration of anti-inflammatory steroids, lead to increased mortality [51, 65] . these clinical observations highlight the need to balance the inflammatory response during viral infection, not only to improve lung injury during the primary viral infection, but also to prevent poor outcomes to secondary infections. in addition to the seasonal threat of influenza, we must also be cautious in the regulation of inflammation in treatment of the ongoing covid-19 pandemic. while a subgroup of patients with severe covid-19 develop 'cytokine storm' [15, 16, 18] , it must be remembered that current anti-inflammatory drugs have pleiotropic effects and lack the specificity needed to carefully calibrate the host response. as such, newly developed pharmacologics such as those that target lubac, a molecular rheostat of inflammatory signaling, have the potential to fine tune inflammation and moderate the host response. further investigation of compounds which modulate lubac activity is warranted, as they may be 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ring-between-rings--keeping the safety on loaded guns sharpin forms a linear ubiquitin ligase complex regulating nf-kappab activity and apoptosis lubac, a novel ubiquitin ligase for linear ubiquitination, is crucial for inflammation and immune responses. microbes infect role of linear ubiquitination in health and disease linear ubiquitin assembly complex regulates lung epithelial-driven responses during influenza infection the airway epithelium: soldier in the fight against respiratory viruses alveolar epithelial cells: master regulators of lung homeostasis alveolar edema must be cleared for the acute respiratory distress syndrome patient to survive recruitment of the linear ubiquitin chain assembly complex stabilizes the tnf-r1 signaling complex and is required for tnf-mediated gene induction analysis of nuclear factor-kappab (nf-kappab) essential modulator (nemo) binding to linear and lysine-linked ubiquitin chains and its role in the activation of nf-kappab involvement of linear polyubiquitylation of nemo in nf-kappab activation the e3 ligase hoip specifies linear ubiquitin chain assembly through its ring-ibr-ring domain and the unique ldd extension estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study h1n1 pandemic timeline estimates of global seasonal influenza-associated respiratory mortality: a modelling study hospitalized patients with 2009 h1n1 influenza in the united states influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions immunomodulatory therapy for severe influenza corticosteroids for severe influenza pneumonia: a critical appraisal transcriptomic signatures in sepsis and a differential response to steroids. from the vanish randomized trial clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury effect of statin treatments on highly pathogenic avian influenza h5n1, seasonal and h1n1pdm09 virus infections in balb/c mice the effect of rosuvastatin in a murine model of influenza a infection association between use of statins and mortality among patients hospitalized with laboratory-confirmed influenza virus infections: a multistate study biophysical and biological evaluation of optimized stapled peptide inhibitors of the linear ubiquitin chain assembly complex (lubac) small-molecule inhibitors of linear ubiquitin chain assembly complex (lubac), hoipins, suppress nf-kappab signaling the anti-inflammatory drug bay 11-7082 suppresses the myd88-dependent signalling network by targeting the ubiquitin system essential role of the linear ubiquitin chain assembly complex in lymphoma revealed by rare germline polymorphisms fragment-based covalent ligand screening enables rapid discovery of inhibitors for the rbr e3 ubiquitin ligase hoip gliotoxin suppresses nf-kappab activation by selectively inhibiting linear ubiquitin chain assembly complex (lubac) in vitro and in vivo effects of gliotoxin, a fungal metabolite: efficacy against dextran sodium sulfate-induced colitis in rats cooperative domain formation by homologous motifs in hoil-1l and sharpin plays a crucial role in lubac stabilization the pathology of influenza virus infections genomic landscape of the individual host response and outcomes in sepsis: a prospective cohort study key: cord-346836-6jyv0q5e authors: ikegami, tetsuro; makino, shinji title: the pathogenesis of rift valley fever date: 2011-05-06 journal: viruses doi: 10.3390/v3050493 sha: doc_id: 346836 cord_uid: 6jyv0q5e rift valley fever (rvf) is an emerging zoonotic disease distributed in sub-saharan african countries and the arabian peninsula. the disease is caused by the rift valley fever virus (rvfv) of the family bunyaviridae and the genus phlebovirus. the virus is transmitted by mosquitoes, and virus replication in domestic ruminant results in high rates of mortality and abortion. rvfv infection in humans usually causes a self-limiting, acute and febrile illness; however, a small number of cases progress to neurological disorders, partial or complete blindness, hemorrhagic fever, or thrombosis. this review describes the pathology of rvf in human patients and several animal models, and summarizes the role of viral virulence factors and host factors that affect rvfv pathogenesis. rift valley fever (rvf), a mosquito-borne zoonotic disease among humans and ruminants, is caused by rift valley fever virus (rvfv) belonging to family bunyaviridae, genus phlebovirus [1, 2] . rvf is endemic to sub-saharan african countries and has caused major outbreaks in several countries including kenya, tanzania, somalia, south africa, madagascar, egypt, sudan, mauritania, senegal, saudi arabia, and yemen [3] . pregnant ruminants infected with rvfv typically are subject to highrate abortions, fetal malformation, and subclinical-to-fatal febrile illness, while newborn lambs usually die by acute hepatitis [4] [5] [6] . rvfv infection in humans primarily causes a self-limiting febrile illness; however, some patients develop hemorrhagic fever, neurological disorders, or blindness after the febrile period [5, 7, 8] . in endemic area, floodwater aedes mosquitoes, such as ae.mcintoshi or ae.vexans, serve as vectors, and the virus could be transmitted into offspring transovarially [9, 10] . heavy rainfall or flooding of river banks due to construction of dams increases the number of permanent fresh water species of mosquitoes such as culex pipens, which play a role in amplifying rvfv among mosquitoes, ruminants and humans [10] [11] [12] [13] [14] [15] . an outbreak of rvf in developed countries, e.g., the u.s. or europe, could force a curtailing of livestock movement to prevent rvfv spread, causing massive economic loss, and a substantial degree of panic in our society, because the body fluids of infected animals contain infectious rvfv [16, 17] , and mosquitoes such as culex spp. aedes spp. or anopheles spp. might further spread rvfv into other mosquitoes, humans and animals [18] [19] [20] . effective vaccines and antiviral drugs are necessary for the containment of outbreaks and treatment of rvf patients, respectively. however, neither safe and effective vaccines nor efficient treatment is available. a correct understanding of rvf pathogenesis is essential for the development of effective vaccines and antiviral drugs against rvf. in this review, we will describe clinical and pathological findings of rvf in humans and animals and discuss viral and host factors that affect rvf pathogenesis. most rvf patients suffer from a self-limiting, febrile illness. however, some patients develop neurological disorders, vision loss, hemorrhagic fever, or thrombosis as shown in figure 1 . in the 1930s-40s, many rvfv laboratory infections occurred due to a lack of appropriate biosafety procedures [21] [22] [23] [24] [25] . however, the patients in most of these and later outbreaks suffered from self-limiting and nonfatal illness [4, 6, 22, 23, [25] [26] [27] [28] . typically, the incubation period for rvf is 4 to 6 days. symptoms start abruptly with severe chills, malaise, dizziness, weakness, severe headache, nausea and/or sensation of fullness over the liver region [6, 22, 23] . these symptoms are followed by an elevated body temperature (38.8 °c to 39.5 °c); decreased blood pressure; pain in the back, shoulders, neck or legs; rigor; shivering; flushed face; red eye with sore; constipation; insomnia and/or photophobia. occasionally, other symptoms are seen which include epistaxis, abdominal pain, lack of gustatory discrimination, vomiting and/or diarrhea [4, 6, 22, 25, 26, 28] . some lessening of symptoms can be observed on the 3rd day, and the body temperature often decreases to a normal level by the 4th day after the onset of symptoms. however, within 1 to 3 days after the recovery of body temperature, some patients again experience a temporal recurrence of high fever with a severe headache for a few days [6, 25, 26] . moreover, patients may have a long-lasting high fever for as much as 10 days [6] . after body temperature becomes normal, some patients may develop a massive coronary thrombosis [26] , persistent aching of legs for two weeks [4, 22] , or persistent abdominal discomfort for weeks [4] . the palpable enlargement of the liver and spleen is not common. in the convalescence phase, patients often experience weakness, malaise, a tendency to sweat, frequent headaches, pain on motion of the eye, and a sense of imbalance. virus has been demonstrated in the blood during the febrile period (3-4 days) , whereas neutralizing antibody also starts appearing around the 4th day of the onset of symptoms [6, 22, 24, 25, 27] . maar et al. described a case of encephalitis in a rvf patient [29] . the patient exhibited symptoms of sudden fever, rigor, and retro-orbital headache for two days. he had fever again at the 22nd day after the onset of illness and experienced neck rigidity lasting for five days from the 25th day. subsequently, he was sometimes confused and otherwise mentally affected, and experienced temporal vision loss without detectable retinopathy. he also exhibited convulsive attacks, hyperflexia and fever until the 50th day. his serum contained anti-rvfv hemagglutination (hai) antibodies of 1:160 at the 25th day and 1:640 at the 40th day, while his cerebrospinal fluid (csf) contained 1:2 of hai antibody at the 28th day and 1:64 at the 50th day. the csf also contained an increased number of white blood cells consisting mainly of lymphocytes at the 28th day, indicative of the possible occurrence of viral meningitis or meningoencephalitis. the patient recovered after treatment with amantadine, rifampicin, and dexamethasone for two weeks, although the effect of therapy could not be evaluated precisely. another case with encephalitis and retinitis was described by alrajhi et al. [30] . the patient had a fever, ataxic gait, and bilateral retinal hemorrhage. she could not count fingers, and the csf contained many leukocytes, including lymphocytes. her consciousness level was decreased. she was discharged on day 30 of the illness to her home, at which time she was awake, blind, quadreparetic, and incontinent. moreover, her neurologic conditions did not improve for the next year. an additional report described a patient who had persistent hemiparesis for four months after the onset of illness [31] , and another paper reported 12 rvf patients, who developed neurological signs and symptoms, including meningeal irritation, confusion, stupor and coma, hypersalivation, teeth-grinding, visual hallucinations, locked-in syndrome, and choreiform movement of upper limbs [32] ; in these patients, the histopathological lesions in brains were characterized by focal necroses associated with an infiltration of round cells, mostly lymphocytes and macrophages, and perivascular cuffing [32] . some patients suffer from maculopathy or retinopathy. patients noticed the loss of central vision or blurred eye occurring at various times after infection; e.g., from immediately after the disease onset to several weeks or months later. one or both eyes could be affected [33] [34] [35] , and the affected eyes had macular edema with exudates containing a white mass covering the macular area with or without retinal hemorrhage, vasculitis, infarction or vitreous haze [34] [35] [36] [37] [38] [39] . in addition, retinal detachment [35, 36] , uveitis [38, 39] , or arterial occlusion [35, 36, [39] [40] [41] was reported in some patients. in many cases, a complete recovery of vision does not occur, and chorioretinal scarring can remain in macular and paramacular areas, in spite of the resorption of exudates [34, 35, [37] [38] [39] [40] [41] , while some patients show partial improvement in vision after several months of rvfv infection [34, 36, 38, 40 ]. fatal rvf cases often involve hemorrhagic manifestations but the time to death varies among cases [42] [43] [44] . most typically, the illness starts suddenly, and the patients experience fever, rigor, nausea, vomiting, headache, injected conjunctives, drowsiness, and/or body pains. the patients may also have such symptoms as macular rash over the entire trunk, ecchymoses on the arms, limbs, and/or eyelids, bleeding from the gums and/or gastrointestinal mucosal membrane, low blood pressure, hematemesis, melena, diarrhea, throat pain, pneumonitis, jaundice, and/or hepatosplenomegaly [42, 43] . typically, elevation of alanine aminotransferase (alt), aspartate aminotransferase (ast), lactate dehydrogenase (ldh), and reduction of platelet count and hemoglobin are seen in these patients [42, 45] . in many cases, death occurs in 3 to 6 days after patients become symptomatic; however, in some cases, death occurs in 12 to 17 days after the onset of symptoms. postmortem examination shows diffuse necrosis of hepatocytes which more greatly affect the centrilobular area than the portal area, which may indicate association with acute hepatic injury in this type of pathogenesis [42, 44] . it should be noted that some patients who do not exhibit jaundice or hemorrhage, die from renal failure or disseminated intravascular coagulation (dic) accompanied by an elevation of alt, ast, ldh or d-dimer, or a decrease in platelet count [46, 47] . a group of rvf patients who died from typical hemorrhagic fever also had encephalitis in addition to hepatic and gastro-intestinal necroses [32] , which demonstrates the neuroinvasiveness of rvfv in hemorrhagic patients. another type of fatal case of rvfv infection was described by schwentker et al. [21] . they reported that the temperature of the patient fell to normal on the 4th day after the onset of symptoms, whereas two papular areas of several centimeter in diameter were found on the patient's thigh and leg on the 5th day and remained until day 8. after temporal recovery by the 12th day, the patient experienced phlebitis of the popliteal vein, which was followed by infarcts in the lungs on the 20th, 26th and 34th days at multiple locations; these eventually caused a fatal embolus in the pulmonary vessels on the 45th day of illness. the liver of the patient was normal, did not contain infectious rvfv, and no typical rvf lesions were confirmed at the postmortem histopathological examination. however, there was a large thrombus in the inferior vena cava, a part of which might have detached and caused an embolus in the pulmonary artery. the neutralizing antibody showed up on the 6th day of illness, and its titer increased toward the 12th day. in a retrospective study in egypt, no increases in the total number of abortions were seen during an rvf outbreak, and the serological conversion rate of aborted women before and after outbreak was 31.1% and 27.5%, respectively [48] . a report describing a potential vertical infection of rvfv concerned a pregnant woman, who experienced fever, headache, dizziness and generalized muscle ache four days before delivery during the rvf outbreak in saudi arabia in 2000 and developed igg specific to rvfv [49] . her newborn baby presented with an anti-rvfv igm antibody, as well as alt/ast elevation, jaundice, extension of the activated partial thromboplastin time (aptt: test for the deficiency of intrinsic pathway factors) and the prothrombin time (pt: test for the deficiency of extrinsic pathway factors), and died on the 6th day after birth [49] . although it is unknown whether the newborn baby died from rvf, it is possible that a vertical transmission in utero might have occurred in this case. the clinical symptoms of rvf vary among patients. the determinant of host susceptibility to induce hemorrhagic fever in humans has not been characterized. it is also unknown how rvfv causes diseases such as neurological disorders, vision loss or thrombosis in the presence of protective antibodies. several animal models have been used to understand the pathology of rvf, and the advantages and disadvantages of different animal models are summarized in table 1 . we discuss the pathological findings in various different animal models in the next chapter. mice are one of the most susceptible animal species to rvfv infection [6, 8] , and rvf pathology in infected mice mimics the pathological findings in newborn lambs [6] . most of the mice infected with wild-type (wt) rvfv zh548 or zh501 strains die in 3 to 5 days [50] [51] [52] , whereas they die faster by infection with other wt isolates [6, 53] . infected mice show ruffed fur with decreased activity in 2 to 3 days, and then become more lethargic while lying with their back legs wide apart [6, 52] . the symptom is often followed by death within one hour [6] . occasionally, mice survive this stage, yet have hind limb paralysis at days 8-9 post infection (p.i.) and die from encephalitis [52] . the rectal temperature of infected mice is often normal or decreased to below normal [54] . also, the clotting time of blood derived from rvfv-infected mice is significantly extended, and it clots normally by mixing with normal sera, a finding that may indicate the shortage of coagulation factors is important for the extension of clotting time [54] . the liver is the major target organ of rvfv, while the enlargement of liver is not common [6, 52] . liver lesions are characterized by fulminant hepatitis, with coagulative necroses leaving the portal space intact [50, 52, 55, 56] . depletion of glycogen in hepatocytes is also common at an early stage [6, 51, 55] . infected hepatocytes in mice contain eosinophilic intranuclear inclusion bodies [6, 52] , which are not reactive to the feulgen reaction, a nucleic acid stain [57] . the intranuclear inclusion bodies in rvfv-infected cultured cells were visualized by an indirect immunofluorescent assay with antisera against rvfv and found to have a filamentary shape [58] . inclusion bodies are formed by the nss protein [59] , a viral nonstructural protein, and the 10-to-17 amino acids at the carboxyl terminus of nss are responsible for the formation of the filamentous structures via self-association [60] . viral antigens start accumulating in hepatocytes at day 2, and their abundance increases extensively at day 3 p.i. [52] . the infected hepatocytes are stained by using a terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay, indicating that rvfv replication induces apoptosis in hepatocytes [52] . mice that survived the early hepatitis phase often are able to regenerate hepatocytes [52, 61] . although swollen endothelial cells can be observed in the liver [6] , antigens are not easily detectable in endothelial cells or kuppfer cells, which indicate that these cells are not the primary targets of rvfv [52, 55] . in addition to hepatocytes, viral antigens have been detected in, odontogenic and gingival epithelium; lipocytes; pituicytes; olfactory neurons and multiple types of neurons in the brain; mononuclear phagocytes; cardiac myofibers; and in perineural, periosteal, adrenocortical, endosteal, perivascular, bone marrow stromal, fibroblastic reticular, and vascular smooth muscle cells, as well as in cells morphologically consistent with dendritic, pancreatic islet, and adrenal medullary cells; however, no viral antigens were reported in any ocular structure, including the retina [52] . apoptosis of lymphocytes were found in the thymus, spleen, lymph nodes and mucosa-associated lymphoid tissues [52] . congestion and hemorrhage are common to the liver, spleen, lymph nodes, large intestine, kidneys and brain [6, 52] , but are uncommon in the jejunum [54] . in some mice that survived acute viral hepatitis, a sharp decrease in viral antigens occurred at 8 days p.i., and no virus could be detected in the sera, liver, lung, pancreas, large intestine and ovaries [52] . in the late stage of infection, however, lethal meningoencephalitis characterized by neuronal necrosis, microhemorrhages, and perivascular cuffs occurs in mice that survived the acute hepatitis [52] . the susceptibility of rats to rvfv differs among rat strains [62] . peters et al. demonstrated that 10-to-15-week-old inbred rats from u.s. breeders exhibited three different responses to subcutaneous (s.c.) rvfv inoculation [63, 64] . wistar-furth (wf) and brown norway strains were highly susceptible to rvfv and died within four days p.i. by liver necrosis, while the fisher 344, buffalo, da and lewis strains were largely resistant to rvfv infection [64] . aci and maax strains proved to be moderately susceptible and showed ascending paralysis; lesions were mainly in the brain and spinal cord and characterized as mild-to-severe necrotizing encephalitis and encephalomyelitis with focal necrosis with neutrophilic infiltrate and perivascular cuffing primarily with lymphocytes. in the aci and maax strains, viruses were undetectable in the liver and blood, whereas 5-to-6 log pfu/g of viruses could be detected from brain tissue, even in the presence of neutralizing antibody in the serum. the intracranial injection of rvfv uniformly caused encephalitis in these rats, including the resistant lewis strain. the immunosuppression of the resistant lewis rats by treating animals with cyclophosphamide 1 day prior to s.c. rvfv infection resulted in death at around 5-to-7 days p.i. with increased viral titers in the serum, liver, spleen, brain, kidneys and adrenal gland, although the virus titers in these organs in the lewis rat were still lower than those in the corresponding organs of the wf strain [65] . these data suggest that the lewis rat encodes a gene(s) important for the resistant phenotype. interestingly, wf (wf/mol) and lewis rats (lewis/mol) obtained from a european breeding colony are resistant and susceptible to rvfv, respectively; hence, these rats showed the opposite susceptibilities to rvfv infection to those of the same strains from u.s. breeders. furthermore, both wf and lewis rats obtained from another european breeder were resistant to rvfv infection; taken together, these findings indicate the possible genetic variability of inbred rats among different breeders [66] . cross-breeding experiments culminated in findings that indicated the resistance of wf/mol rat was segregated as a single mendelian dominant locus [66] . findlay et al. also showed that the albino rat of the glaxo strain had an age-dependent susceptibility to rvfv via the i.p. route infection [62] ; rats younger than 15 days died in 2 to 4 days with extensive liver necrosis, whereas 26-day-old rats survived rvfv infection [62] . the syrian hamster is one of the most susceptible rodents to rvfv. death occurs in 2 to 3 days p.i. after intraperitoneal inoculation with massive liver necrosis [6, 67] . the pathological changes are similar to those seen in mice [6] . administration of low titers of neutralizing antibodies protects hamsters from fatal liver necrosis, yet infected hamsters die from encephalitis by day 11 [67] . the gerbil, meriones unguiculatus, represents a unique rvfv animal model, which produces fatal encephalitis with minimal liver involvement after infection of non-neuroadapted wt rvfv. the gerbil has proven moderately susceptible to rvfv, and the survival rate of 10-week-old gerbils after s.c. inoculation is reported to range from 50 to 100%, dependent on the strain and inoculation dose [68] . death was reported to occur around 1 to 3 weeks in a dose-independent manner; s.c. inoculation of 10 7 , 10 5 , 10 3 , 10 1 pfu of zh501 resulted in 90%, 100%, 60% and 60% survival of outbred tum:(mon) gerbils, respectively, and 50%, 90%, 70% and 70% survival of inbred mon/tum gerbils, respectively [68] . the infected gerbils exhibited hind-limb paralysis, generalized weakness and wasting. gerbils also showed an age-dependent resistance to rvfv infection. most of the 3-to 5-week-old tum:(mon) gerbils died after 10 7 pfu s.c. inoculation of zh501 from encephalitis, whereas 90% of 7-week-old gerbils were able to survive the infection. after s.c. inoculation, rvfv replicated temporally in the livers of both 4-week-old and 10-week-old gerbils on days 1 and 2 (~10 3 pfu/g), while subsequent efficient virus replication in the brain occurred in 4-week-old gerbil (from day 4 to day 7 up to 10 7 pfu/g), but not in 10-week-old gerbil (temporal increase up to 10 2 at day 7) [68] . intracerebral wt rvfv inoculation of 50 pfu into 10-week-old gerbils resulted in an efficient viral replication (~10 7 pfu/g) in the brain and the mean time to death was six days, which is not statistically different from that of 4-week-old gerbils, which may indicate the presence of host factors influencing the neuroinvasiveness in an age-dependent manner [68] . histopathologically, minimal multifocal necroses of hepatocytes are seen at days 1 or 2 after the s.c. inoculation of wt rvfv, while focal necrotizing encephalitis with neuronal necrosis, a neutrophilic infiltrate, and perivascular cuffing are seen in the brain at later time points [68] . mild, necrotizing encephalitis without detectable infectious rvfv could be observed even in clinically normal rvf-infected gerbils [68] . rhesus macaques are moderately susceptible to rvfv infection [6] . after i.p or intranasal inoculations, body temperatures of infected macaques increased to 39-40 °c at 1 to 4 days p.i. and the febrile period lasted for 24 to 120 h, whereas some infected animals did not show any febrile reactions [6] . peters et al. first described hemorrhagic fever-like illness in rhesus macaques that were experimentally infected with a wt rvfv zh501 strain [69] . three out of fifteen rhesus macaques intravenously inoculated with rvfv zh501 became ill; two became moribund and were euthanized on days 7 and 3, and one recovered from illness, whereas the others showed temporal viremia, the maximum viral titer of which occurred around day 2, and were clinically normal. all three monkeys exhibited lassitude, weakness, the cessation of food intake, petechiae, ecchymoses and bleeding from nares, gums or venipuncture sites. clear extensions of aptt, slight extension of pt, and a decrease in the number of platelets were observed in the two dead monkeys, possibly indicating a deficiency of coagulation factors and platelets. histopathologically, the dead monkeys showed moderate focal or midzonal coagulative necrosis of the liver involving approximately 1/3 to 2/3 of hepatocytes, necrosis in the ventricular myocardium, fibrin thrombi in the glomeruli and small intertubular vessels of renal medulla in the kidneys, and mild depletion of lymphocytes from white pulp and the deposition of eosinophilic amorphous fibrin-like material in red pulp cords in the spleen [69] . morrill et al. reported that after intravenous inoculation of 1 × 10 5 pfu zh501 strain into 17 rhesus macaques, three developed signs of hemorrhagic fever, seven were clinically ill but survived, and the other seven survived without clinical signs [70] . serum interferon (ifn)- was detected from 6-24 h p.i. and from 24-30 h p.i. in the surviving monkeys and in those dying, respectively, and the delayed ifn response was preceded by viremia in two of the three lethally-infected monkeys [70] . no surviving monkeys developed signs of encephalitis or retinal complications in follow-up observations at two months to two years [70] . morrill et al. also demonstrated that the administration of recombinant leukocyte a ifn (5 × 10 5 u, i.m) at 6 h after rvfv intravenous inoculation reduced the peak viremia titer by 100-times and cleared viruses by 48 h p.i. [71] . these studies suggested the importance of ifn- in limiting viral replication. findlay et al. reported that three species of african monkey, i.e., the green guenon (cercopithecus callitrichus), the sooty mangabey (cercocebus fuliginosus) and the patas guenon (erythrocebus patas), did not exhibit any febrile reaction after inoculation of rvfv, whereas virus was detected in the blood [72] . in contrast, four species of south american monkeys, two brown capuchin monkeys (cebus fatuellus and cebus chrysopus) and two common marmosets (callithrix jacchus and callithrix penicillata) exhibited febrile reactions for 1 to 2 days upon rvfv infection, which may indicate that south american monkeys are more susceptible to rvfv infection than african monkeys [72] . davies et al. reported that rvfv-infected baboons (papio anubis) had viremia for 3 to 4 days without developing significant clinical signs [73] . daubney et al. originally reported an outbreak of rvf in a herd of sheep in kenya in 1930, which was characterized as a high rate of abortion in pregnant ewes and high mortality of newborn lambs [4] . a later study by easterday et al. described that the mortality of adult sheep following experimental rvfv infection was approximately 20% [74] . typically, sheep with more than one week old were relatively resistant to rvfv infection, yet did exhibit fever (39 to 40 °c), viremia, diarrhea, nasal discharge, and decreased activity [74, 75] . nine-to ten-week-old young adult sheep (ripollesa breed) that were subcutaneously inoculated with rvfv had corneal and choroidal edema with inflammatory infiltrate, which could be associated with drainage failure or inadequate corneal dehydration after transient viremia [76] . on the other hand, 7-to 11-month-old yansaka sheep subcutaneously inoculated with rvfv died during the viremic febrile phase and displayed symptoms of epistaxis (2 days p.i.~), severe and bloody diarrhea, conjunctival hemorrhage, widespread petechiae and ecchymoses in hairless areas, pulmonary edema/hemorrhage, and thrombi formation in the blood vessels of the heart, kidneys and brain. rvfv-infected west african dwarf or the ouda breed did not exhibit such rapid hemorrhagic symptoms and rather exhibited marked coagulative hepatic necrosis, and brain lesions, including mild gliosis, neural degeneration, neurophagia, and satellitosis [77] . interestingly, yansaka, west african dwarf and ouda also had increased prothrombin time, which may have indicated that hemorrhage was induced by a combination of vascular endothelial damage and an inability to clot blood in response to the damage [77] . the inconsistency of symptoms and mortality in adult sheep described in these publications suggest the divergence of host genetic background, even within the same breed of sheep, affects susceptibility to rvfv infection. several studies examined the effects of rvfv vaccine candidates for protecting pregnant ewes from wt rvfv infection. however, insufficient immunogenicity of inactivated vaccines or residual virulence of live-attenuated vaccines induced fetal malformations. pregnant ewes were untreated (n = 8) or vaccinated (n = 50) once with formalin-inactivated rvfv and then challenged with wt rvfv zh501 at 45 days post vaccination [78] . abortion occurred in both unvaccinated and vaccinated ewes, between 6 to 18 days p.i., and 50% of the ewes aborted their fetuses. one out of 50 vaccinated ewes and one out of eight unvaccinated ewes died. these data may indicate that the inactivated rvfv vaccine induced an insufficient immunity for sheep. necropsy at 19 days p.i. revealed that 100% of the ewes had either aborted or dead fetuses, while the dead or aborted lambs showed extensive liver necrosis typical of rvf [78] . coetzer et al. reported that immunization of pregnant ewes with a live-attenuated smithburn vaccine strain at 42 to 74 days of pregnancy could cause a teratogenic effect in the fetus, including arthrogryposis, hydranencephaly, or mineralization of brain with or without hydrop amnii, and two out of six lambs from the nine vaccinated ewes showed such effects [79] . hunter et al. described that pregnant ewes inoculated with live-attenuated mp-12 vaccine strain at 28 to 56 days of gestation either miscarried or produced lambs showing teratogenic effects (11 out of 75 lambs from 50 vaccinated ewes), such as cerebellar hypoplasia, spinal hypoplasia, hydranencephaly, prognathia inferior, brachygnathia inferior, arthrogryposis, scoiliosis, lordosis, kyphosis, or dormed head [80] . the abortion and teratogenous effects did not occur when pregnant ewes were vaccinated with mp-12 at the third trimester of pregnancy, i.e., at 90-110 days of gestation [81, 82] . on the other hand, pregnant ewes vaccinated at 15 days of gestation with a rvfv clone 13 (c13) strain, which has a 69% in-frame deletion of nss [83] , did not cause abortion or fetal malformations and were protective against wt rvfv challenge [84] . these data may imply the involvement of mp-12 nss in the abortion of ewes and the teratogenic effects in lambs. rvfv infection causes an acute and fatal disease in newborn lambs [5] . rvfv-infected newborn lambs usually exhibit obvious illness, including elevated body temperature (40 to 41 °c), loss of appetite, decreased activity, and prostration, about 12 to 18 h prior to death [85] . the mortality rate in rvfv-infected newborn lambs is 95 to 100% [5] . studies of experimental infection of 1-4 day-old lambs with rvfv via s.c. resulted in necrosis of isolated hepatocytes (12-18 h p.i.), focal coagulative necrosis of hepatocytes (24-33 h p.i.), and extensive hepatocyte necrosis (48-51 h p.i.) with a progressive increase in viral antigens, whereas no viral antigens could be detected in the endothelial or kupffer cells in the liver, suggesting that hepatocytes are the primary target of rvfv [86] . the necrosis is predominantly centrilobular or midzonal, and yet there is no definite distribution pattern in liver necrosis [5, 87] . some infected lambs also exhibited necrosis in the villi at the distal jejunum and ileum and depletion of lymphocytes in the spleen, whereas the brain and eyes had no lesions [87] . overall, the liver pathology of newborn lambs resembles that of mice or hamsters, which are extremely susceptible to rvfv. however, the rvfv neurovirulence in lambs is less prominent when compared with that in rodents. rvfv also causes diseases in other animals including goats, cattle, camels, dogs, cats, and ferrets, but does not cause any symptomatic diseases in rabbits, guinea pigs, birds, horses, pigs and other animals, as reviewed in detail previously [4] [5] [6] 8, 63, [88] [89] [90] [91] [92] [93] . as of the present, the mechanism of species-specific susceptibility to rvfv infection is unknown. rvfv infection shows unique pathogenesis in each animal model. because viral replication and host antiviral responses most probably contribute to viral pathogenecity, an understanding of rvf pathogenesis requires identification and characterization of the viral virulence factors and host antiviral factors. the next chapter describes the viral determinant of virulence. the rvfv genome is comprised of three rna segments named the s-, m-and l-segments ( figure 2 ) [1, 2] . the s-segment encodes n and nss genes in an ambisense manner, the m-segment. nsm (nsm2), 78 kd (nsm1), gn and gc genes, and the l-segment, the rna-dependent rna polymerase (l) gene [10] . rvfv virions bind to an unidentified cellular receptor, and enter the cells in a ph-dependent manner [94] , probably through a clathrin-mediated endocytic pathway, as described for another phlebovirus [95] . after viral uncoating, viral ribonucleocapsid (rnp) composed of viral genomic rna segments and n protein [96] is released into the cytoplasm, and the viral polymerase, which probably is attached to the rnp exerts primary transcription to synthesize viral mrna [97] . both n mrna and nss mrna are transcribed during primary transcription as early as 40 min after infection from an efficiently packaged, viral-sense (negative-sense) s-segment and anti-viral-sense (positive-sense) s-segment, respectively; the packaging mechanism of rvfv rnp and the presence of specific cis-signals in the genomic rna are not known [97] . viral rna replication starts around 1 to 2 h after infection [97] and an increase in the amount of viral genomic rna results in increases in viral mrnas and proteins. the rnp is packaged into viral virions probably by its interaction with the cytoplasmic domains of gn/gc at the golgi apparatus, as reported for other bunyaviruses [98, 99] . the three different rna segments could be co-packaged in a coordinated manner, in which the co-packaging of m and s-segments could support the packaging of the l-segment [100] . the rvfv virion surface is highly symmetric t = 12 icosahedral lattice [101] , which is formed by a shell of 122 glycoprotein capsomers most probably composed of 720 gn-gc heterodimers [102] . the rvfv m-segment encodes 78 kd, nsm, gn and gc proteins in m mrna (figure 2 ). those proteins are synthesized from a single open reading frame of m mrna at different augs present at the 5' region of m mrna by leaky scanning of ribosomes, while the n-terminus of gn and gc is co-translationally cleaved by host proteins [103] [104] [105] . nsm is synthesized from the 2nd aug, and the c-terminus is generated by cleavage at the n-terminus of gn, while the 78 kd protein is synthesized from the 1st aug, and the c-terminus is identical to that of gn. among seven viral proteins, nss and nsm are nonstructural proteins which are not incorporated into virions [59, 104] . the 78 kd protein has not been studied in detail, while an "80 kd" protein induced by rvfv infection (most probably corresponding to the current 78 kd protein) is known to be incorporated into virions [106] , which may indicate that the 78 kd protein is a structural protein. nss, 78 kd protein and nsm are dispensable for viral replication in cell cultures [83, [107] [108] [109] . infection of 12-week-old female wf rats with a recombinant rvfv zh501 strain lacking both 78 kd protein and nsm induced acute fatal hepatic disease, causing the deaths of some infected rats around four days p.i., or a delayed fatal neurologic disease, resulting in death of some of infected animals around 13 days p.i. (mortality rate: 50 to 70%), while wt zh501-infected rats developed acute hepatitis and 100% died. these data suggest that nsm is not essential for virulence and lethality [110] . on the other hand, a recombinant rvfv mp-12 lacking the expression of both 78 kd and nsm induced more extensive apoptosis than did mp-12 in cultured cells and the expression of nsm significantly inhibited the cleavage of caspase-8 and -9 induced by staurosporine [111] , demonstrating that nsm protein suppresses apoptosis. thus, nsm probably suppresses apoptosis in infected hosts and affects viral pathogenicity. nss is known to be a major virulence factor of rvfv; a rvfv c13 strain, which has a 69% in-frame deletion of nss [83] , and is completely attenuated in mice or sheep [51, 84] . further studies showed that nss inhibits the synthesis of ifn- mrna under the conditions that transcription factors, such as ifn regulatory factor 3 (irf-3), nf-b and activator protein (ap)-1, are activated in wt rvfv zh548-infected cells [112] . it was found that nss can bind and sequester the p44 subunit of tfiih, an essential transcription factor for rna polymerase i and ii; and hence nss was reported to prevent the assembly process of the tfiih complex, resulting in the suppression of host mrna transcription [113] . le may et al. also described studies demonstrating that a region of nss, corresponding to amino acid 210 to 230, specifically binds to sin3a-associated protein (sap30) and forms a complex that represses the histone acetylation required for the transcriptional activation of ifn- promoter; this repression worked even after the binding of irf-3 to the ifn- promoter [114] ; hence, recombinant zh548 carrying a deletion at amino acid 210 to 230 of nss cannot suppress the ifn- mrna synthesis. it should be noted that c13 nss bound to sap30, yet it did not suppress ifn- expression [114] . although the inability of c13 nss to suppress ifn- expression may have been due to its poor accumulation in infected cells, further studies are required to know how the binding of nss to sap30 can lead to the suppression of ifn- mrna synthesis. it is also uncertain whether the general host transcriptional suppression is required for the inhibition of ifn- mrna synthesis. because nss induces host general transcription suppression [113] , rvfv might have the ability to replicate under host transcription suppression. actinomycin d (actd) is a general inhibitor of host rna synthesis. viral titers of several cytoplasmic rna viruses including arenaviruses [115] [116] [117] , measles virus [118] , sindbis virus [119] , rubella virus [120] , polio virus [121, 122] , coronaviruses [123, 124] , and lactic dehydrogenase elevating virus [125] could be reduced in the presence of actd, while the viral rna synthesis is often unaffected [115, 118, 123] . we found that expression of nss protein is essential for the rvfv mp-12 strain to actively synthesize viral proteins and produce high titers of infectious viruses in the presence of actd [126] ; cells infected with recombinant rvfv mp-12 lacking nss failed to synthesize viral proteins; and while cells infected with mp-12 lacking nss accumulated phosphorylated eif2. the latter made cellular translation initiation inactive through the activation of dsrna-dependent protein kinase (pkr) at around 8 h p.i. [126] , resulting in the suppression of viral protein synthesis. further studies revealed that mp-12 nss promoted the degradation of pkr through the proteasome pathway and prevented an accumulation of phosphorylated eif2, thereby securing efficient viral protein synthesis under host transcription shut-off induced by actd [126] . habjan et al. reported that wt rvfv nss also induced pkr degradation and demonstrated that an rvfv c13 strain carrying biologically inactive nss induced fatal hepatic disease in c57bl/6 mice lacking pkr [127] ; these mice are competent for inducing type-i ifns in response to viral rna replication or poly (i):poly (c) [128] . pkr is one of several ifn-stimulated genes (isgs) and plays an important role in inhibiting viral replication in vivo; other rna viruses, including vesicular stomatitis virus (vsv) and influenza virus, also replicate more efficiently in mice lacking pkr than in those with an intact pkr [129] . in addition to pkr, ifn-induced mxa proteins is also known to inhibit rvfv replication [130] . although the genetic diversity of rvfv strains is relatively low (approximately 5% in primary sequences [131] ), the susceptibilities to rat ifn-/ differed among rvfv strains. most of the sub-saharan rvfv strains are very sensitive to rat ifn-/ (ed 50 : 0.3-0.7 units), whereas egyptian strains, including zh501 and zh548, and a zimbabwean isolate (2269/74) are relatively resistant to rat ifn-/ (ed 50 : 50-200 units) [132] . all of those rvfv isolates show a similar sensitivity to human ifn- (ed 50 : 70-880 units). in summary, rvfv nss induces the shut-down of host transcription, including transcription of both type-i ifn and isgs mrnas, to prevent antiviral responses. nss also induces the degradation of pkr to prevent eif2-mediated host and viral translational shut-off and promote an efficient viral protein synthesis. although nss is a major virulence factor to escape host innate immune responses, the virulence of rvfv could be controlled in a polygenic manner. the rvfv mp-12 strain is a highly attenuated strain derived from wt rvfv zh548 [133] and encodes a functional nss gene. mutations in the m-and l-segments are major determinants of mp-12 attenuation [134, 135] . in contrast, the c13 strain encodes an s-segment lacking a functional nss gene, while the m-and l-segments of c13 strain are still virulent phenotypes [51, 83] . mice lacking ifn-ar (ifn-ar -/mice) are susceptible to both mp-12 and c13, while the viral replication kinetics of mp-12 and c13 differ in those mice; the highest titer of viremia was reached within 28 h p.i. and around 48 h p.i. in c13-infected mice and in mp-12-infected mice, respectively [51] . thus, there is a possibility that m-and l-segments of c13 may facilitate a rapid replication of c13 in these mice, reaching the highest virus titer substantially earlier compared to mp-12-infected mice. recently, we found that wt rvfv zh501 virus stock contains two major viral populations, rzh501-m847-a (glu at aa.123 of gn protein) and rzh501-m847-g (gly at the corresponding site); the difference in the amino acid is mapped within one of the neutralizing epitopes in gn protein [61, 136] . although it is not known how the two different populations have emerged in the zh501 virus stock, which was amplified once in the mouse brain and passaged twice in frhl cells and twice in vero e6 cells, the rzh501-m847g replicated less efficiently than rzh501-m847a in infected mice, whereas both of them replicated efficiently in tissue cultures such as mrc-5, veroe6, j774.1, and nih3t3 cells. our study showed that one amino acid change in the gn can substantially alter replication and pathogenesis of zh501 in vivo, and yet the mechanism of the gn mutation in the pathogenesis remains unknown. clearly further studies will be needed for understanding the roles of viral proteins in rvfv pathogenesis. rvfv encodes several virulence factors, and the major virulence factor nss plays an important role in evading host innate immune responses. in the next chapter, we discuss how humans or host animals develop protective immune responses against highly virulent wt rvfv. adequate immunity against rvfv can attenuate the virulence of rvfv in animals and vaccination against rvfv can save animals from lethal rvfv challenge [84, 133, [137] [138] [139] [140] . the passive transfer of neutralizing antibodies is sufficient for protection from lethal rvf [67, [141] [142] [143] [144] , whereas the role of the cellular immune response for the protection is not sufficiently evaluated. yet, mandell et al. demonstrated that mice immunized with virus-like particles containing n have a higher survival rate (survival: 9/16) than those not containing n after lethal rvfv challenges (survival: 3/16). because anti-n protein antibody is unlikely to neutralize rvfv, these data may point to the involvement of cellular immune responses against n protein for rvfv protection [145] . alternatively, antiviral immune responses might be induced by forming a complex between anti-n antibody and n proteins released from dead cells or infected cells in vivo [146] . if n proteins are present in cell surface as reported in influenza virus-infected cells [147, 148] , complement-mediated cell lysis could be another mechanism to support the elimination of infected cells [146] . aerosol exposure is one of the most likely routes for both laboratory infections and bioterrorism attack. immunization of rats by s.c. inoculation of a formalin-inactivated rvfv vaccine (tsi-gsd-200) partially protected lethal rvfv aerosol exposure at 187 days post immunization (survival: 72/105: 69%), whereas 11 out of 72 surviving rats developed encephalitis without clinical signs at 27-28 days post-challenge, which was revealed during necropsy [149] . another study showed that three vaccinated rats challenged with rvfv aerosol developed uveitis, although no histopathological analysis was presented [141] . mice are highly susceptible to wt rvfv aerosol exposure (ld 50 : 1 to 2 pfu), which may cause lethal hepatitis, but not pneumonia [150] . a study in mice vaccinated at several different routes with formalin-inactivated rvfv vaccine (ndbr-103) [149] and challenged with rvfv subcutaneously showed that the immunization route affected survival rates; s.c. immunization, intraperitoneal (i.p.) immunization, and intraduodenal (i.d.) immunization resulted in survival rates of 97.5%, 100% and less than 20%, respectively. another study reported that fewer than 20% of mice immunized via s.c. or i.d. and about 50% of mice immunized via i.p. survived after aerosol route challenge of rvfv in the 2-week observation period [151] . findings from this study using aerosol rvfv challenge indicated that the mucosal immunity elicited by i.d. immunization successfully lowered the occurrence of olfactory bulb encephalitis, but failed to prevent necrotic hepatitis, and immunity induced by s.c. or i.d. did not prevent hepatitis, olfactory bulb encephalitis and multifocal encephalitis, while i.p. immunization completely prevented the occurrence of hepatitis, but not multifocal encephalitis [151] . these reports seem to indicate that immunization with inactivated vaccines via s.c. cannot prevent rvfv-induced diseases after aerosol challenge. it will be important to establish a reliable countermeasure against bioterrorism by use of rvfv through further detailed characterization of rvfv replication in various organs after aerosol challenge and examination of the efficacy of immunization of current live-attenuated rvfv vaccine candidates, such as mp-12 or c13, for preventing rvf-induced diseased after rvfv aerosol challenge. exposure of animals to aerosol containing rvfv probably results in initial rvfv infection in lung epithelial cells, such as type i alveolar epithelial cells. both infection and release of rvfv occur in polarized epithelial cells, such as caco-2 cells (human colorectal adenocarcinoma cells), at apical and basolateral membranes [152] . infection by punta toro virus (ptv), which belongs to the phlebovirus genus and causes a lethal necrotic hepatitis in hamsters [153, 154] and mice [155] , results in virus being released into the basolateral membrane [156] , which may contribute to systemic viral spread in infected animals. as described in section 2.3, several studies point to the possibility that unidentified host genetic factors influence the susceptibility of inbred rat species to rvfv. the primary rat hepatocytes derived from resistant american lewis rats or wf/mol rats are less permissive to rvfv infection than those derived from susceptible american wf rat or lewis/mol rats [66, 157] , whereas rvfv replicates efficiently in primary cortical glial cells derived from wf/mol rats [66] or spontaneously transformed cell lines generated from embryonic thymus cells of either resistant lew rats or susceptible wf rats [132] , suggesting that hepatocytes in those resistant inbred rats are under the control of a host factor(s) that restricts efficient rvfv replication. peritoneal macrophages (pm) derived from resistant lewis rat or susceptible wf rat were treated with 1.0 or 10 u of ifn, which resulted in a 150-or 3300-fold reduction of zh501 replication in lewis pm, and a 4-or 250-fold reduction in wf pm, respectively, possibly indicative of the role of ifn to increase resistance in lewis rats [158] . on the other hand, primary hepatocytes derived from susceptible lewis/mol rats and resistant wf/mol rats are resistant to rvfv after treatment of the cells with rat type-i ifn, suggesting the resistance induced by type-i ifn is not necessarily important for the host specific resistance seen in wf/mol rats [66] . a recent study showed that mbt/pas mice, but not balb/cbyi mice, are highly susceptible to rvfv zh548 infection [159] . experiments using microarray and quantitative real-time pcr showed that rvfv zh548 replication in mouse embryonic fibroblast (mef) cells derived from susceptible mbt/pas mice induced higher levels of ifnb1 and ifna4 mrnas than did those derived from resistant balb/cbyj mice, while mef cells derived from mbt/pas mice failed to induce several isgs, including the ifn regulatory factor 7 (irf7) mrna, the 2'-5' oligoadenylate synthetase-like 2 (oasl2) mrna, and the ifn-induced 17 kda protein (isg15) mrna [159] . the knockout of isg15 or oasl2 mrna expressions increased viral replication in mef cells derived from resistant balb/cbyj mice, leading the authors to suggest that mbt/pas mice have a defect in their ifn responses which controls rvfv spread [159] . it is of interest to know whether the mice lacking isg15 or oasl2 genes are susceptible to rvfv. it is unknown whether the susceptibility of inbred rat to rvfv is due to a deficit in some isgs. the host factors determining the host susceptibility to ptv infection have been explored; ptv infection in mice mimics rvfv-induced lethal hepatitis. c57bl/6j mice show an age-dependent susceptibility to ptv infection; the mice gradually become resistant to ptv from 5 to 7 weeks, and they are resistant at eight weeks of age [155] . the 8-week-old c57bl/6j mice show a delayed viremia, when compared to 4-week-old mice, and the peak viremia titers in the 8-week-old mice are 5-to 10-times lower than those in the 4-week-old mice [155] . likewise, ptv replication was lower in primary cultured hepatocytes, kupffer cells and peripheral blood monocytes isolated from 8-week-old c57bl/6 mice than in those cells obtained from 3-week-old c57bl/6 mice [160, 161] . adding stress to the 8-week-old mice by daily handling and observation increased their susceptibility to ptv replication [162] . the role of toll-like receptor (tlr) 3, which is a pathogen recognition receptor recognizing double-stranded rna, in the susceptibility of the mice to ptv was studied by using 8-week-old tlr3 -/mice of the c57bl/6 background [162] . the wt mice infected with ptv had a 100% mortality and high levels of il-6 induction (yet no remarkable increase in tnf-), whereas mice lacking tlr3 infected with ptv had increased survival rates, slightly earlier reduction of serum virus titers, and decreased levels of serum il-6 [162] . on the other hand, il-6 -/mice were more susceptible to ptv infection and supported higher levels of viremia than did wt mice, which possibly means that il-6 is indispensable for protective immunity. a similar attenuation of virulence has been also reported for west nile virus infection in tlr3 -/mice [163] . the authors hypothesized that over-production of il-6 in wt mice would be detrimental to the outcome of ptv infection [162] , pointing out that the balance of cytokines might alter the pathogenesis of ptv. stat-1 is a key molecule in the ifn signaling pathway [164] . it was reported that ptv replicated substantially better in the brains of stat-1 -/mice than in wt mice. also ptv titers in sera, spleens and livers of the stat-1 -/mice were high, which may emphasize the importance of ifn responses for limiting viral replication in the liver, spleen and brain [165] . since the first recognition of rvf in an outbreak in 1930, more than 80 years has passed. although there has been good progress in characterizing clinical, pathological, and virological features of rvfv infection, rvfv still causes outbreaks in african countries or the arabian peninsula [3, 10] . the development of effective, safe, highly immunogenic and economic vaccines for animals and humans will prevent rvf in the endemic countries [137] . there are several important questions to address in controlling rvfv and in further understanding rvf pathogenesis. some of them include determining the: (1) mechanism that triggers hemorrhagic fever, (2) route of viral entry to the brain, (3) mechanism of prolonged diseases in rvf patients in the presence of neutralizing antibodies, and (4) significance of vaccination to prevent rvf after aerosol exposure. addressing these questions will have a substantial impact on understanding of rvf pathogenesis and development of anti-rvfv reagents. fields virology rift valley fever virus enzootic hepatitis or rift valley fever: an undescribed virus disease of sheep cattle and man from east rift valley fever the virus of rift valley fever or enzootic hepatitis rift valley fever rift valley fever-a review rift valley fever virus (family bunyaviridae, genus phlebovirus). isolations from diptera collected during an inter-epizootic period in kenya rift valley fever virus (bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention rift valley fever surveillance in the lower senegal river basin: update 10 years after the epidemic risk factors for severe rift valley fever infection in kenya rift valley fever during rainy seasons, madagascar climate and satellite indicators to forecast rift valley fever epidemics in kenya prediction of a rift valley fever outbreak prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the 1993 outbreak in egypt prevalence of rift valley fever immunoglobulin g antibody in various occupational groups before the 2007 outbreak in tanzania. vector borne zoonotic dis replication and dissemination of rift valley fever virus in culex pipiens vector potential of selected north american mosquito species for rift valley fever virus vector competence of a houston, texas strain of aedes albopictus for rift valley fever virus report of a fatal laboratory infection complicated by thrombophlebitis laboratory infections with the virus of rift valley fever am rift valley fever : a report of three cases of laboratory infection and the experimental transmission of the disease to ferrets human infection with rift valley fever virus and immunity twelve years after single attack rift valley fever; accidental infections among laboratory workers rift valley fever; i. the occurrence of human cases in johannesburg rift valley fever in south africa. 2. the occurrence of human cases in the orange free state, the north-western cape province, the western and southern transvaal. b. field and laboratory investigation rift valley fever in south africa: 2. the occurrence of human cases in the orange free state, the north-western cape province, the western and southern transvaal. a epidemiological and clinical findings rift valley fever encephalitis. a description of a case rift valley fever encephalitis clinical studies on rift valley fever. part 2: ophthalmologic and central nervous system complications rift valley fever affecting humans in south africa: a clinicopathological study rift valley fever ocular manifestations: observations during the 1977 epidemic in egypt epidemic maculopathy rift valley fever retinitis macular changes in rift valley fever rift valley fever in man, complicated by retinal changes and loss of vision ocular disease resulting from infection with rift valley fever virus ocular complications of rift valley fever outbreak in saudi arabia ocular complications of rift valley fever ocular manifestations of rift valley fever fatal rift valley fever of man in rhodesia clinico-pathological picture in five human cases died with rift valley fever rift valley fever virus infections in egypt: pathological and virological findings in man epidemic rift valley fever in saudi arabia: a clinical study of severe illness in humans rift valley fever hepatitis complicated by disseminated intravascular coagulation and hepatorenal syndrome acute renal failure associated with the rift valley fever: a single center study rift valley fever as a possible cause of human abortions vertical transmission of fatal rift valley fever in a newborn the s segment of rift valley fever phlebovirus (bunyaviridae) carries determinants for attenuation and virulence in mice genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein nss the pathogenesis of rift valley fever virus in the mouse model rift valley fever virus in mice. iii. further quantitative features of the infective process rift valley fever virus in mice. i. general features of the infection rift valley fever virus hepatitis: light and electron microscopic studies in the mouse pathogenicity of different strains of rift valley fever virus in swiss albino mice the feulgen reaction 75 years on demonstration of nuclear immunofluorescence in rift valley fever infected cells identification of a major non-structural protein in the nuclei of rift valley fever virus-infected cells the carboxy-terminal acidic domain of rift valley fever virus nss protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein rapid accumulation of virulent rift valley fever virus in mice from an attenuated virus carrying a single nucleotide substitution in the m rna the susceptibility of rats to rift valley fever in relation to age pathogenesis of rift valley fever inbred rat strains mimic the disparate human response to rift valley fever virus infection pathogenesis of rift valley fever virus (rvfv) in inbred rats resistance to rift valley fever virus in rattus norvegicus: genetic variability within certain 'inbred' strains active and passive immunization against rift valley fever virus infection in syrian hamsters the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis experimental rift valley fever in rhesus macaques pathogenesis of rift valley fever in rhesus monkeys: role of interferon response prevention of rift valley fever in rhesus monkeys with interferon-alpha the infectivity of rift valley fever for monkeys the pathogenicity of rift valley fever virus for the baboon experimental rift valley fever in lambs and sheeps clinical, virological and serological response of the west african dwarf sheep to experimental infection with different strains of rift valley fever virus experimental infection of young adult european breed sheep with rift valley fever virus field isolates. vector borne zoonotic dis experimental infection of three nigerian breeds of sheep with the zinga strain of the rift valley fever virus abortion in vaccinated sheep and cattle after challenge with rift valley fever virus hydrops amnii in sheep associated with hydranencephaly and arthrogryposis with wesselsbron disease and rift valley fever viruses as aetiological agents teratogenicity of a mutagenised rift valley fever virus (mvp 12) in sheep further evaluation of a mutagen-attenuated rift valley fever vaccine in sheep pathogenicity and immunogenicity of a mutagen-attenuated rift valley fever virus immunogen in pregnant ewes characterization of clone 13, a naturally attenuated avirulent isolate of rift valley fever virus, which is altered in the small segment evaluation of the efficacy and safety of the rift valley fever clone 13 vaccine in sheep the pathogenesis of rift valley fever in lambs distribution of viral antigen in tissues of new-born lambs infected with rift valley fever virus the pathology of rift valley fever. i. lesions occurring in natural cases in new-born lambs the clinical aspects of rift valley fever virus in household pets. i. susceptibility of the dog the clinical aspects of rift valley fever virus in household pets. ii. susceptibility of the cat susceptibility of dogs and cats to rift valley fever by inhalation or ingestion of virus pigs and rift valley fever rift valley fever in camels rift valley fever development and characterization of a rift valley fever virus cell-cell fusion assay using alphavirus replicon vectors helenius, a. entry of bunyaviruses into mammalian cells structure of the rift valley fever virus nucleocapsid protein reveals another architecture for rna encapsidation rift valley fever virus nss mrna is transcribed from an incoming anti-viral-sense s rna segment role of the cytoplasmic tail domains of bunyamwera orthobunyavirus glycoproteins gn and gc in virus assembly and morphogenesis the glycoprotein cytoplasmic tail of uukuniemi virus (bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging mechanism of tripartite rna genome packaging in rift valley fever virus threedimensional organization of rift valley fever virus revealed by cryoelectron tomography electron cryo-microscopy and singleparticle averaging of rift valley fever virus: evidence for gn-gc glycoprotein heterodimers rift valley fever virus m segment: phlebovirus expression strategy and protein glycosylation rift valley fever virus m segment: use of recombinant vaccinia viruses to study phlebovirus gene expression synthesis, proteolytic processing and complex formation of nterminally nested precursor proteins of the rift valley fever virus glycoproteins protein synthesis in rift valley fever virus-infected cells nsm and 78-kilodalton proteins of rift valley fever virus are nonessential for viral replication in cell culture the nsm proteins of rift valley fever virus are dispensable for maturation, replication and infection rescue of infectious rift valley fever virus entirely from cdna, analysis of virus lacking the nss gene, and expression of a foreign gene rift valley fever virus lacking nsm proteins retains high virulence in vivo and may provide a model of human delayed onset neurologic disease nsm protein of rift valley fever virus suppresses virus-induced apoptosis nss protein of rift valley fever virus blocks interferon production by inhibiting host gene transcription tfiih transcription factor, a target for the rift valley hemorrhagic fever virus a sap30 complex inhibits ifn-beta expression in rift valley fever virus infected cells replication and physical parameters important for preparing purified junin virus inhibition of pichinde virus replication by actinomycin d inhibition of lymphocytic choriomeningitis virus replication by actinomycin d and 6-azauridine the effects of actinomycin d on rna synthesis in measles virus-infected cells requirement for host transcription in the replication of sindbis virus rubella virus replication: effect of interferons and actinomycin d the effect of rifampicin, actinomycin d and mitomycin c on poliovirus and foot-and-mouth disease virus replication the inhibition by actinomycin d of poliovirus multiplication hep 2 cells inhibition of coronavirus 229e replication by actinomycin d differential in vitro inhibition of feline enteric coronavirus and feline infectious peritonitis virus by actinomycin d inhibition of replication of lactic dehydrogenase virus by actinomycin rift valley fever virus nss protein promotes post-transcriptional downregulation of protein kinase pkr and inhibits eif2alpha phosphorylation nss protein of rift valley fever virus induces the specific degradation of the double-stranded rna-dependent protein kinase deficient signaling in mice devoid of double-stranded rna-dependent protein kinase essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection inhibition of bunyaviruses, phleboviruses, and hantaviruses by human mxa protein complete genome analysis of 33 ecologically and biologically diverse rift valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry viral determinants of virulence for rift valley fever (rvf) in rats mutagen-directed attenuation of rift valley fever virus as a method for vaccine development rna polymerase i-mediated expression of viral rna for the rescue of infectious virulent and avirulent rift valley fever viruses reassortant rvfv between mp-12 and zh501 by reverese genetics. the university of texas medical branch use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the g2 glycoprotein of rift valley fever virus rift valley fever vaccines immunization against rift valley fever virus. studies onthe immunogenicity of lyophilized formalin-inactivated vaccine the development of a formalin-killed rift valley fever virus vaccine for use in man rift valley fever; the neurotropic adaptation of the virus and the experimental use of this modified virus as a vaccine efficacy of a rift valley fever virus vaccine against an aerosol infection in rats baculovirus expression of the m genome segment of rift valley fever virus and examination of antigenic and immunogenic properties of the expressed proteins evaluation of a formalin-inactivated rift valley fever vaccine in sheep long term existence of rift valley fever virus in immune mice flick, r. a replication-incompetent rift valley fever vaccine: chimeric virus-like particles protect mice and rats against lethal challenge contributions of antinucleoprotein igg to heterosubtypic immunity against influenza virus early presence of ribonucleoprotein antigen on surface of influenza virus-infected cells expression of influenza a virus internal antigens on the surface of infected p815 cells rift valley fever vaccine for humans respiratory infectivity of a recently isolated egyptian strain of rift valley fever virus mucosal priming alters pathogenesis of rift valley fever bidirectional infection and release of rift valley fever virus in polarized epithelial cells pathogenesis of a phleboviral infection (punta toro virus) in golden syrian hamsters induction of severe disease in hamsters by two sandfly fever group viruses, punta toro and gabek forest (phlebovirus, bunyaviridae), similar to that caused by rift valley fever virus punta toro virus infection of c57bl/6j mice: a model for phlebovirusinduced disease assembly and polarized release of punta toro virus and effects of brefeldin a immunoelectron microscopy of rift valley fever viral morphogenesis in primary rat hepatocytes the effects of aging in vitro and interferon on the resistance of rat macrophages to rift valley fever virus (rvfv) a new mouse model reveals a critical role for host innate immunity in resistance to rift valley fever role of hepatocytes and kupffer cells in agedependent murine hepatitis caused by a phlebovirus, punta toro effect of macrophage source and activation on susceptibility in an age-dependent model of murine hepatitis caused by a phlebovirus tlr3 deletion limits mortality and disease severity due to phlebovirus infection toll-like receptor 3 mediates west nile virus entry into the brain causing lethal encephalitis fagard, r. stat1 and pathogens, not a friendly relationship punta toro virus (bunyaviridae, phlebovirus) infection in mice: strain differences in pathogenesis and host interferon response the authors (ti and sm) were supported by grant number 5 u54 ai057156 through the western regional center of excellence. ti was also supported by r01ai08764301 from the national institute of allergy and infectious diseases and internal funding from the sealy center for vaccine development at the university of texas medical branch. sm was also supported by a grant from department of homeland security. key: cord-332516-eaqpiq1o authors: joseph, carol; togawa, yu; shindo, nahoko title: bacterial and viral infections associated with influenza date: 2013-08-27 journal: influenza and other respiratory viruses doi: 10.1111/irv.12089 sha: doc_id: 332516 cord_uid: eaqpiq1o influenza‐associated bacterial and viral infections are responsible for high levels of morbidity and death during pandemic and seasonal influenza episodes. a review was undertaken to assess and evaluate the incidence, epidemiology, aetiology, clinical importance and impact of bacterial and viral co‐infection and secondary infection associated with influenza. a review was carried out of published articles covering bacterial and viral infections associated with pandemic and seasonal influenza between 1918 and 2009 (and published through december 2011) to include both pulmonary and extra‐pulmonary infections. while pneumococcal infection remains the predominant cause of bacterial pneumonia, the review highlights the importance of other co‐ and secondary bacterial and viral infections associated with influenza, and the emergence of newly identified dual infections associated with the 2009 h1n1 pandemic strain. severe influenza‐associated pneumonia is often bacterial and will necessitate antibiotic treatment. in addition to the well‐known bacterial causes, less common bacteria such as legionella pneumophila may also be associated with influenza when new influenza strains emerge. this review should provide clinicians with an overview of the range of bacterial and viral co‐ or secondary infections that could present with influenza illness. bacterial secondary infections or co-infections associated with cases of influenza are a leading cause of severe morbidity and mortality, especially among high-risk groups such as the elderly and young children. vaccines and antiviral and antibiotic therapies are now readily available to clinicians for control and prevention of primary and secondary bacterial infections. thus, information on the overall range, incidence and severity of influenza co-infections and secondary infections associated with different influenza strains, aetiological agents, different age groups and their underlying risk conditions is very important contextually for clinicians and public health specialists involved in implementing policy and treatment regimes for this disease spectrum. bacterial infection may be concurrent with influenza viral infection, and the resulting co-infection can lead to an enhanced pneumonic illness or may occur shortly after influenza virus has been largely cleared from the lungs, when the host appears to be more susceptible to bacterial infection. 1, 2 morbidity and mortality are recognised to be greater in cases of influenza-associated bacterial infection compared with bacterial pneumonia without influenza infection 3 with all age groups affected by this synergistic process. the annual increase in influenza activity during winter months is usually accompanied by an increase in cases of community-acquired pneumonia (cap). the most com-mon causes of cap are streptococcus pneumoniae (s pneumoniae), staphylococcus aureus (s aureus) and haemophilus influenzae (h influenzae). s pneumoniae is the most frequently isolated pathogen associated with influenza 4 , although deaths, especially in children are also associated with s aureus infection, as highlighted by the recent emergence of community-acquired methicillin-resistant staphylococcus aureus (mrsa). 5 the objective of this review is to assess and evaluate the incidence, epidemiology, aetiology, clinical importance and impact of co-infection and secondary infection associated with influenza. as there are few review articles on this topic, it also aims to integrate some newly published reports. the main focus of this review is pulmonary infection while some less common extra-pulmonary complications as shown by case reports are also included. information in this review should supplement the information available to clinicians on antimicrobial treatment therapies, including antibiotic sensitivity information, local guidelines and local antimicrobial susceptibility data as well as local availability of medicines. bacterial outcomes have been extensively studied in both influenza pandemic and epidemic periods and latterly within the context of available viral and bacterial vaccines that protect against primary and secondary infection. in addition to prevention strategies, treatment of influenza complications has also become available through antiviral and antibiotic therapies. the goal of combined therapies for prevention and treatment must surely be a reduction in the proportion of bacterial infections associated with influenza. however, the wider use of antibiotics for treatment of bacterial influenza needs to be considered alongside the corresponding requirement for appropriate use of antibiotics, in order to reduce the increasing burden of antibiotic resistance of bacterial strains implicated in co-infection with influenza. the mechanisms by which co-infection and secondary infection take place are complex. reports from past influenza pandemics show an extremely high frequency of lung colonisation by bacterial species that are commonly found in the nasopharynx. most evidence suggests that virusinduced changes in the respiratory tract prime the upper airway and lung for subsequent bacterial infection. secondary bacterial infections are facilitated by virus-induced cytopathology and resulting immunological impairment, which may be caused in part by the overproduction of inflammatory cytokines. 6 modification of the immune response either by diminishing the ability of the host to clear bacteria or by amplification of the inflammatory cascade is likely to contribute to the severity of the resulting infection. 7 animal studies using murine models have shown that influenza predisposes to bacterial pneumonia. 6, [8] [9] [10] lag times of 7-21 days have been calculated in studies for onset of bacterial infection following seasonal influenza 11 although much shorter times from onset to death have been recorded in pandemic periods. [12] [13] [14] influenza a is the dominant strain associated with co/ secondary bacterial infection with evidence that specific n2 seasonal subtypes cause more severe infection than other subtypes. 15 influenza b, although generally regarded as having less impact on morbidity and mortality in healthy persons can also cause severe secondary bacterial infection during seasonal influenza episodes, especially within younger age groups. 5, 16, 17 methods we searched the national library of medicine through pubmed using the search terms: ('influenza' and 'secondary infection'), ('influenza' and 'pneumonia') and ('influenza' and 'co-infection'). we also searched the references of the identified articles for additional articles. we included studies on both influenza type a and b, and also seasonal and pandemic influenza. the search was limited to studies of disease in humans that were published in english from 1918 to the end of 2011. we only selected studies in which pathogens were identified. we then reviewed abstracts and titles and selected studies that were relevant to the topic of interest. bacterial co-infectiona bacterial pneumonia occurring simultaneously with onset of influenza virus illness secondary bacterial infectiona bacterial pneumonia occurring after influenza illness onset or clearance of influenza virus viral co-infection -influenza virus and one or more other respiratory viruses detected simultaneously by microbiological examination of respiratory samples. the three influenza pandemics of the 20th century (1918 h1n1, 1957 h2n2 and 1968 h3n2) are all associated with secondary bacterial pneumonia. 18 this pandemic has been extensively researched, mainly due to its global impact and estimated 40-50 million deaths in an era of unknown virological cause and absence of antibiotic therapy, in order to understand its aetiological and epidemiological features. british and french army camps in 1916/ 17 were the initial setting for major outbreaks of purulent bronchitis associated with haemophilus influenzae, pneumococcus, streptococcus and staphylococcus. over a period of 2 months in 1918 at 37 large american army camps, secondary bacterial infection occurred in approximately 17% of those diagnosed with influenza of which approximately 35% were fatal. 4 oxford describes these army camp outbreaks as progenitors of the ensuing h1n1 pandemic of 1918. 19 overall, it has been estimated that in the us military, the mean influenza attack rate during the course of the 1918/ 1919 pandemic was 23%, the mean percentage of influenza cases that developed pneumonias was 16% and the mean percentage of pneumonia cases that were fatal was 34%. 4 recent re-analyses of post-mortem lung cultures from 1918 showed evidence of bacterial infection in >90% of the specimens. 18, 20 experts now support the sequential infection hypothesis and believe that bacteria were secondary invaders to pulmonary tissues weakened by the influenza virus. they suggest that the scale and range of bacterial invaders was random, and in the case of large group outbreaks, depended on the occurrence of particular bacteria in the respiratory tract of persons at the time of infection and on their occurrence in contacts. the fatal outcome of influenza pneumonia was therefore determined partly by virally depressed local and general pulmonary resistance and partly by the virulence and nature of the invading bacteria. 12 brundage explains the high transmission rates in military camps and other crowded settings as due to 'cloud adults'affected persons who increased the aerosolisation of colonising strains of bacteria to other susceptible persons. military personnel were deemed to be highly susceptible because of their closed community style living and their physically weakened state. in american civilian populations, attack rates and deaths were similar among younger adults and overall were approximately 28% for influenza with 30% of associated pneumonias being fatal. 21 studies also show that, in all age groups, deaths were strongly correlated with pneumonia cases than with influenza clinical cases alone. children had the highest rates of clinical influenza infection, whereas young adults had the highest influenza pneumonia rates and associated fatality rates. 20, 22, 23 1957 h2n2 pandemic the asian influenza pandemic was similarly characterised by waves of influenza followed by an increase in hospitalisations and deaths from pneumonia. one us study showed these were associated with s pneumoniae, h influenzae and s aureus 24 , while a dutch study of 158 asian influenza deaths documented that s aureus and pneumococci were recovered from 59% and 15% of lung cultures, respectively. 25 a british study of 140 hospitalised cases of pneumonia over a two-month period in 1957, a high proportion of whom also had evidence of confirmed influenza a infection, showed that s aureus was isolated from 27% of the cases and pneumococci and h influenzae from 15% and 4%, respectively. 26 mortality was 47% in the staphylococcal group compared with 16% in the nonstaphylococcal group: eight of the 18 staphylococcal deaths were in persons with no previous disease while seven were in cases with chronic chest disease. in the 1968 h3n2 hong kong pandemic, a three-fold increase in the incidence of staphylococcal pneumonia was found in one hospital study compared with the number of pneumonic cases in the previous year. of 128 patients with pneumonia during the pandemic influenza period, 26% were proven staphylococcal pneumonia cases and a high correlation between pneumonia and influenza infection was documented. 27 in england and wales, the national experience of the hong kong influenza in 1968/69 reported that mortality was substantially lower than in previous influenza winters. 28 however, excess respiratory deaths were recorded in the second wave of the pandemic in 1969/70 and increased by approximately 55% and circulatory system deaths by 4%. deaths in the elderly increased by 10%, in those aged 40-60 years by 8% and in younger adults by 4%. 29 in the united states, significant excess pneumonia-influenza mortality occurred in all nine geographical areas of the country in the first wave in 1968/69 and followed influenza activity by several weeks. 30 the first pandemic of the 21st century in 2009 is still being researched but so far has shown a similar pattern to previous pandemics with a high proportion of cases and deaths occurring in younger age groups compared with nonpandemic influenza seasons. a study of the first 47 deaths in new york city showed 13 (28%) had evidence of invasive bacterial co-infection. s pneumoniae was most commonly identified [8 patients (17%)], followed by s pyogenes [3 patients (6%)]. one paediatric case had post-mortem evidence of both bacteria. 13 a multi-centre review of 77 deaths in another us study between may and august 2009 found evidence of concurrent bacterial infection in specimens from 22 (29%) of the 77 patients, including 10 caused by s pneumoniae. 31 an analysis of 631 patients admitted to hospital over the five-month pandemic period in 2009 in the uk with confirmed pandemic influenza infection reported that 102 cases had radiological evidence of pneumonia and that mortality in cases with radiographic pneumonia was significantly higher than in cases without (p = 0á0008). four cases of pneumonia (4%) had positive bacteriological findings, three of whom diedtwo children with methicillin-resistant staphylococcus aureus (mrsa) and one adult with s pneumoniae in sputum. one adult had s aureus bacteraemia and survived. 32 in a study of 68 autopsy reports from a total of 457 pandemic influenza deaths in the uk, 28 (41%) reported that bacterial secondary infection was the significant complication; pneumococcus was the most common agent identified (25%). 33 in argentina, nasopharyngeal swab samples from 199 cases of confirmed h1n1 pandemic infection were tested for 33 additional microbial agents using masstag pcr methods. at least one additional agent of potential pathogenic importance was detected in 152 samples, including s pneumoniae (41%); h influenzae (68á4%); s aureus (23%); and methicillinresistant s aureus (mrsa, 4%). other viruses such as rsv, and influenza b were found in 20 samples and other bacterial pathogens in five. the presence of s pneumoniae was strongly correlated with severe disease and was present in 56á4% of severe cases versus 25% of mild cases (p = 0á0004). in subjects 6-55 years of age, the adjusted odds ratio (or) of severe disease in the presence of s pneumoniae was highly significant (p = 0á0001). this study demonstrated that the presence of s pneumoniae in nasopharyngeal swab samples could predict severe disease outcome, the risk being more acute in persons aged between 6 and 55 years. in this lowrisk age group, severity of disease could be predicted with 90á97% accuracy via a multivariable logistic regression model. 34 in the united states, those aged 5-19 years influenza and co-secondary infections ª 2013 blackwell publishing ltd experienced overall the largest relative increase in pneumococcal hospitalisations during the 2009 pandemic influenza period compared with seasonal baseline estimates for this age group and mirrored both temporal and geographical influenza activity across the country. 35 no national relative increase occurred in persons aged <5 years or aged 65 years or more, suggesting that the pandemic influenza virus was the likely cause of the increase in the younger age group. a prospective, observational, multicenter study conducted in 148 spanish intensive care units (icu) and with 645 patients, all of whom had confirmed h1n1 pandemic influenza infection showed that co-infection occurred in 113 (17á5%) of patients. s pneumoniae was identified as the most prevalent bacteria (54á8%). co-infection was associated with increased icu mortality (26á2% versus 15á5%), but cox regression analysis adjusted by potential confounders did not confirm a significant association between co-infection and icu mortality. 36 a study of 100 fatal cases of h1n1 influenza in the united states showed 26% overall were due to bacterial co-infection, mainly caused by s. pneumoniae. 37 similarly, a study of paediatric h1n1-associated mortality in the united states demonstrated that 28% of fatal cases had evidence of co-infection 38 while a study in england of 70 h1n1 paediatric deaths confirmed bacterial co-infection in 20% of the cases. 14 a french study measured levels of procalcitonin (pct)a recognised marker of bacterial infection, in patients with h1n1 influenza pneumonia admitted to hospital and was able to conclude that levels of 0á8 lg/l or more discriminated well between isolated viral and mixed bacterial and viral pneumonia. 39 this information together with clinical judgement may help to identify patients for whom antibiotic therapy may be inappropriate. the overall conclusion to date from epidemiological and clinical studies of the 2009 h1n1 pandemic is that worldwide incidence was low and infection was mostly mild. the fact that much of the 2009 pandemic occurred outside the regular season for pneumococcal disease in temperate regions may help to explain the lack of a marked increase in risk of pneumococcal infection at this time. 40 death rates although low, were more prevalent in younger age groups than the elderly and excess deaths were recorded in children by one international european study 41 and in england where the childhood mortality rate was six per million population compared with an estimate of two per million population for seasonal influenza among children aged <14 years. 14 as in the pandemics of the 20th century, s pneumoniae, h influenzae and s aureus were the main bacterial infections associated with severe infection or death in this pandemic. measures to prevent and treat their adverse impact on pandemic influenza cases in the future should now be incorporated into pandemic plans. 42 the literature on co-infections with other viruses during pandemic periods is sparse in comparison with that available for influenza-associated bacterial infections. there are no reports documenting solely viral co-infections during the 1957 and 1968 pandemics, possibly because these infections were not sufficiently severe to merit hospitalisation and/or enhanced microbiological investigation. the 2009 pandemic first appeared in some countries during their normal seasonal activity. as a result, national virological surveillance schemes were able to demonstrate the emergence of the new h1n1 strain against a background and subsequent decline of circulating seasonal h1n1 and h3n2 strains. in new zealand, 13 cases of pandemic h1n1 cases co-infected with seasonal h1n1 were detected, all with mild disease. 43 in argentina, 20 of 199 persons investigated with pandemic disease were co-infected with another respiratory virus including rsv (a or b), rhinovirus and coronavirus. seven of these cases were classed as having severe disease (hospitalisation or death with no other risk factors related to underlying disease) and 13, mild disease (ambulatory cases). 34 a us study of 173 cases of pandemic h1n1 found co-infection with other viruses in 20 cases (11á6%), rhinovirus being the most common agent. 44 in england and wales, the health protection agency's national virological surveillance scheme for community cases of influenza 45 detected 14 (3%) specimens where cases had evidence of pandemic h1n1 infection together with rsv, human metapneumovirus (hmpv), rhinovirus or parainfluenza virus (personal communication j field). seasonal influenza activity provides more opportunities for monitoring the changing epidemiology and microbiological features of influenza-related co/secondary infection but essentially mirrors that of findings in recent pandemics. maxwell first noted that bacterial pneumonia could occur during interepidemic periods when sporadic cases of influenza were investigated. 46 mccullers shows that from 1968 to 1999 excess deaths directly attributed to pneumonia and influenza (p&i deaths) from selected us cities data were more commonly associated with influenza a(h3n2) rather than h1n1 or influenza b infections. 7 meningococcal infections were observed to increase in the presence of both influenza a 47,48 and influenza b 49 , but no causal relationship was identified in a later study. 50 in sweden, pneumococcal infections were calculated to increase by 12-20% per influenza season over a ten-year study period with a lag time of 1-3 weeks for pneumococcal disease following peaks in influenza incidence. 11 in canada, a recent observational study showed that the seasonality and time lag of pneumococcal disease was only partially related to influenza seasonality. other factors such as reduced temperatures and daylight hours were important for the regular appearance of pneumococcal disease each winter. however, the study did find that influenza increased the risk of pneumococcal disease through enhancing pneumococcal invasion in colonised individuals, but had minimal impact on the transmission dynamics of pneumococcal infection. 40 transmission studies using animal models show increased incidence and severity of bacterial pneumonia after influenza infection is pneumococcal strain dependent. different strains may increase the duration of pneumococcal carriage and enhance the bacterial pneumonia. 10 in another study using infant mice, influenza virus was shown to be essential for pneumococcal transmission in a co-housed group although other indirect effects by which the virus altered the immune response of the mice were also considered to be important reasons in the dynamics and synergism between influenza and pneumococcal infection. 9 the introduction of a sevenvalent pneumococcal conjugate vaccine (pcv7) for infants has been shown in the united states to lead to a reduction in pneumococcal infections in vaccinated children and in adults through herd immunity effects. 51 a significant fall in influenza-associated pneumonia hospitalisations was observed among vaccinated children and unvaccinated adults; the vaccine acted to prevent the secondary pneumococcal pneumonia that followed influenza infection. 51 a nine-valent pneumococcal conjugate vaccine (pnccv) in south africa was shown to prevent 31% of pneumonias associated with any of seven respiratory viruses in hospitalised children. the study concluded that a significant proportion of viral pneumonia is due to bacterial co-infection and is preventable by a bacterial vaccine. 52 a study of influenza-related paediatric deaths in the united states over the 2003/04 influenza season found 24 of 102 deaths to have a bacterial cause, mainly s aureus. 53 other us studies of that season also noted a rise in reports of community-acquired s aureus infections in children and young adults, a significant proportion being mrsa infections. 54 similar findings were obtained for the 2006/07 influenza season in the united states 55 and by kallen who reported 51 cases of influenza-related s aureus, 37 of which were mrsa infections in young adults. where outcome of illness was known, deaths occurred in 24 of 47 cases. 56 finelli notes that the proportion of influenza-related s aureus paediatric deaths increased fivefold between 2004 and 2007. 5 although less common than influenza a-associated mortality, deaths do result from influenza b infection, and awareness is growing of the role influenza b co-infection may play in severity of influenza-related illness. a review of influenza surveillance data in the united states from 2004 to 2007 revealed that anywhere from 23 to 38% of the annually reported paediatric deaths attributable to influenza were from influenza b, and many of the fatal cases had evidence of bacterial co-infection. 5 england during the seasonal outbreak of influenza in 2010/ 2011 included four cases of influenza b infection, of which three were fatal. 17 case reports of three healthy women with no known risk factors and severe co-infections of s. pyogenes in two and s. pneumonia in the third that required intensive resuscitation measures recently in switzerland 16 , further underscore the potential impact bacterial co-infection with influenza b can have on morbidity and mortality. multi-viral co-infections have mainly been identified in paediatric studies. between two and six viral co-infections were reported per child in china, 57 a mix of influenza a h1 and h3 with influenza b in japanese children 58 and coinfection with influenza and human metapneumovirus (hmpv) in two successive winters 2002-2004, also in japan. 59 a one-year study in peru found 5á5% of virological surveillance samples to be positive for influenza plus additional respiratory viruses 60 while a similar finding of 3% was obtained from the community-based virological surveillance data in england and wales for the 2010/11 winter season (personal communication j field). none of these reports suggested that the cases had severe disease. the 2009 pandemic is the first pandemic where virological and microbiological tests for the disease have been conducted intensively at a global level. as a consequence, a wide range of less common pathogen pairings were encountered. in south africa, co-infection with hiv or active tuberculosis was a common finding among the investigated early fatal cases, signifying that these conditions could be associated with increased mortality risk. 61 from a tb endemic area, it has been reported that an immunocompromised cancer patient was co-infected with both tb and pandemic h1n1a rare finding. 62 in mexico, a study of 126 hiv patients with respiratory symptoms found that in the 30 patients co-infected with pandemic h1n1 virus, illness opportunistic infections were more severe and involved longer hospital stays (p = 0á0013), higher hospitalisation rates (p < 0á0001) and increased deaths (p = 0á026). deaths were also associated with delayed administration of oseltamivir (p = 0á0022). 63 in the united states, hopkins et al. reported six cases of bacterial tracheitis (bt) that were isolated in conjunction with influenza a (h1n1). no previous h1n1 cases have presented as bt in the literature to date. 64 six cases of bacterial co-infection due to legionella pneumophila were reported from italy; the authors commenting on the fact that bacterial co-infections associated with the influenza influenza and co-secondary infections ª 2013 blackwell publishing ltd a h1n1 pandemic have not been well described yet because of lack of data. 65 the first case of panton-valentine leukocidin (pvl) necrotising pneumonia due to influenza a (h1n1) and community-acquired methicillin-resistant staphylococcus aureus was reported from spain, 66 the authors recommend that other clinicians become aware of this coinfection. similarly, co-infection with dengue virus and pandemic influenza h1n1 is likely to be a feature in tropical countries where seasonal patterns of these two viruses were contemporaneous, as documented in a case report from puerto rico. 67 during seasonal influenza activity in 2006, influenza a h3n2 infection was associated with a fatal paediatric case of campylobacter jejuni infection in malaysia. 68 co-infection with campylobacter spp. has not been previously described together with influenza virus. this review has documented the adverse impact of co/ secondary bacterial infection with influenza infection, and the higher rates of severe morbidity or mortality that subsequently occur in all age groups. streptococcus pneumoniae continues to be the dominant pathogen involved in this synergistic process 7 followed mainly by staphylococcus aureus 5 and haemophilus influenzae. 12, 18 legionella pneumophila a less common bacterium was also found to be associated with influenza infection in the 2009 h1n1 pandemic. 65 pneumonia remains the single commonest cause of death in children <5 years. 69 clinicians and public health officials now have several means by which influenza-associated pneumonias can be prevented or ameliorated. control and prevention, through viral and bacterial vaccines, and prophylaxis and treatment, through the application of antiviral and antibiotic therapies all contribute to reducing the global burden of these infections. all of these measures however have limitations which impede their effectiveness. antibiotic resistance has been recognised as a growing concern for many years and during the 2009 pandemic, an increasing number of oseltamivir-resistant influenza strains were detected, particularly among immunocompromised patients with influenza infection. 70, 71 other threats to combating influenza epidemics or pandemics in the future might include timely production and administration of effective vaccines and logistical issues associated with stockpiling and distribution of antiviral and antibiotic drugs. 42, 72 most vaccine effectiveness studies select high-risk groups within which to show enhanced protective effects and typically present data on levels of protection against hospital admissions or mortality associated with influenza rather than prevention of secondary infections as a clinical end point. 73 pneumococcal vaccine effectiveness studies also focus on prevention of invasive pneumococcal disease and may or may not include information on influenza vaccine status as a confounding variable. 74 some studies have focused on the protective effect of dual influenza and pneumococcal vaccinations in the elderly. in sweden, influenza and pneumococcal polysaccharide vaccines together reduced hospital admissions for influenza, pneumonia and invasive pneumococcal disease by 32, 22 and 54%, respectively. overall mortality was also reduced by 27%. 75 a study of prior influenza vaccination in relation to its effect on severity and mortality in patients with cap during seasonal influenza periods showed that prevention of the predisposing viral illness reduced the risk for more severe secondary pneumonia. however, outside the influenza season, no significant influence of influenza vaccination status on cap severity was found. 76 most of the subjects in this study were elderly and therefore in the risk group for influenza vaccination. increasing the uptake of influenza vaccination in younger age groups could contribute to the overall prevention of influenza-attributable pneumococcal disease either directly or through protection from influenza via herd effects on other individuals. 40 the 2009 pandemic provided the first opportunity for significant international use of antivirals to prevent the spread of influenza and to treat infected individuals. given prophylactically, antivirals aid prevention of infection either in the absence of vaccine protection or within high-risk groups and may also prevent transmission within closed communities or households. when used for treatment, observational studies have confirmed that oseltamivir given within 48 hours of symptom onset (the recommended time for maximum effectiveness) improves survival in patients with severe influenza and may reduce secondary bacterial infection. 77 of 70 paediatric deaths related to the 2009 pandemic and investigated in one study, 45 (64%) had received antiviral therapy but only seven (10%) within 48 hours of onset. 14 21% of the deaths in this cohort occurred in healthy children. the authors recommend that vaccination of children should be extended to include nonrisk groups and that antiviral treatment should be given as early as possible after symptom onset. 14 antibiotic treatment should be guided by information on the likely bacterial pathogens associated with the influenza virus circulating in the community. recommendations should also defer to local cap management practice because inappropriate use of antibiotics increases antibiotic resistance. if invasive bacterial infection is suspected, early antiviral treatment and appropriate use of antibiotics should be administered. aggressive use of antimicrobial therapy early in the course of infection may reduce severe morbidity and mortality of influenza-associated bacterial infection. 6 the articles in this review suggest that influenza-related bacterial infections overall may account for up to 30% of cap cases, mainly in the elderly. 42 in the developing world, this percentage is much higher, but children are the main sufferers, and pneumonic infections are the leading cause of death in children aged <5 years. many of these deaths are preventable through immunisation, treatment and access to health care. the recent initiative of the global alliance on vaccines and immunisation, which supports global coverage of conjugate hib and pneumococcal vaccines in childhood programmes, estimates that the incidence of severe pneumonia and associated mortality in children in developing countries may be reduced by 50% through this programme. 69 however, other inequities are likely to persist between developed and developing countries with reference to access to adult respiratory vaccines, the ability to stockpile antivirals and antibiotics as part of pandemic planning and remain a major obstacle to global-improved public health goals. antibiotic usage to combat bacterial infection is recognised as adversely contributing to changes in sensitivity and resistance of these drugs with evidence that communityacquired mrsa infections are leading to high levels of morbidity and mortality in individuals with influenza, especially children. 5 if influenza vaccine coverage was extended to all children and low-risk adults aged <65 years, protection against influenza infection would be enjoyed by a larger proportion of the population, overall attack rates should fall, there should be an associated reduction in the incidence of secondary bacterial infections, a subsequent fall in the number of antibiotic prescriptions for these infections and therefore a slowing down in the rise of antibiotic resistance. obviously, this simplistic approach overlooks the vast organisation of economic and human resources needed to achieve these outcomes, but nevertheless, they remain a public health goal. interventions through vaccination have been shown to be cost saving and of cost benefit in influenza epidemic and pandemic settings, 78 but again, depend on staying one step ahead of the viruses and bacteria they seek to eradicate. the co-infections with more than one strain or subtype of influenza virus reported in this review have not yet provided any evidence of re-assortment threats or the emergence of new influenza strains. in 2001/02, a re-assortment between h1n1 and h3n2 produced a small number of cases of h1n2 infection in some countries, but circulation of the new strain was not sustained the following winter and did not confer more severe levels of illness compared with other subtypes. however, drifted strains occur on a regular basis and give rise to high levels of primary and secondary infection, particularly when there is a mismatch to strains contained within the seasonal vaccine. in conclusion, ample evidence exists to show that severe bacterial infection can be a consequence of influenza infection, both in pandemic and seasonal episodes. planning for future pandemics must therefore give equal emphasis to prevention and treatment of both these conditions, partic-ularly in high-risk groups such as the very young and the elderly. influenza infection leads to increased susceptibility to subsequent bacterial superinfection by impairing nk cell responses in the lung respiratory viral infection predisposing for bacterial disease: a concise review disease severity in patients with simultaneous influenza and bacterial pneumonia interactions between influenza and bacterial respiratory pathogens: implications for pandemic preparedness influenza-associated pediatric mortality in the united states: increase of staphylococcus aureus coinfection how do viral infections predispose patients to bacterial infections? insights into the interaction between influenza virus and pneumococcus influenza virus coinfection with bordetella bronchiseptica enhances bacterial colonization and host responses exacerbating pulmonary lesions influenza a virus facilitates streptococcus pneumoniae transmission and disease influenza enhances susceptibility to natural acquisition of and disease due to streptococcus pneumoniae in ferrets occurrence of invasive pneumococcal disease and number of excess cases due to influenza deaths from bacterial pneumonia during 1918-19 influenza pandemic for the new york city department of health and mental hygiene 2009 h1n1 influenza investigation team. fatalities associated with the 2009 h1n1 influenza a virus paediatric mortality related to pandemic influenza a h1n1 infection in england: an observational populationbased study influenza virus neuraminidase contributes to secondary bacterial pneumonia coinfection of influenza b and streptococci causing severe pneumonia and septic shock in healthy women group a streptococcal infections during the seasonal influenza influenza and co-secondary infections ª 2013 blackwell publishing ltd outbreak 2010/11 in south east england predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness world war i may have allowed the emergence of "spanish" influenza bacterial pathogens and death during the 1918 influenza pandemic the epidemiology of influenza statistics of influenza morbidity with special reference to certain factors in case incidence and case-fatality the incidence of epidemic influenza pulmonary infections complicating asian influenza bacteriology and histopathology of the respiratory tract and lungs in fatal asian influenza importance of staphylococcus aureus in pneumonia in the 1957 epidemic of influenza a bacterial pneumonia during the hong kong influenza epidemic of 1968-1969 national experience with hong kong influenza in the united kingdom excess deaths attributable to influenza in england and wales: age at death and certified cause national influenza experience in the usa bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza a (h1n1) -united states risk factors for hospitalisation and poor outcome with pandemic a/h1n1 influenza: united kingdom first wave predictive clinicopathological features derived from systematic autopsy examination of patients who died with a/h1n1 influenza infection in the uk 2009-10 pandemic streptococcus pneumoniae coinfection is correlated with the severity of h1n1 pandemic influenza impact of the 2009 influenza pandemic on pneumococcal pneumonia hospitalizations in the united states h1n1 semicyuc working group. community-acquired respiratory coinfection in critically ill patients with pandemic 2009 influenza a(h1n1) virus pandemic influenza a (h1h1) pathology and pathogenesis of 100 fatal cases in the united states deaths among children-united states can procalcitonin help identify associated bacterial infection in patients with severe influenza pneumonia? a multicentre study invasive bacterial diseases network, fisman d. evaluation of coseasonality of influenza and invasive pneumococcal disease: results from prospective surveillance higher all-cause mortality in children during autumn 2009 compared with the three previous years: pooled results from eight european countries bacterial pneumonia and pandemic influenza planning pandemic (h1n1) 2009 and seasonal influenza a (h1n1) co-infection rate and influence of respiratory virus coinfection on pandemic (h1n1) influenza disease. infectious diseases society of america 48th annual meeting, vancouver. oral abstract session hpa -health protection agency homepage -protecting people, preventing harm, preparing for threats the relation of influenza virus and bacteria in the etiology of pneumonia influenza a and meningococcal disease influenza a and meningococcal disease bacterial meningitis after influenza invasive pneumococcal and meningococcal disease: association with influenza virus and respiratory syncytial virus activity? impact of pneumococcal conjugate vaccination of infants on pneumonia and influenza hospitalization and mortality in all age groups in the united states a role for streptococcus pneumoniae in virus-associated pneumonia influenza special investigations team influenza-associated deaths among children in the united states severe community-acquired pneumonia due to staphylococcus aureus, 2003-04 influenza season severe methicillinresistant staphylococcus aureus community-acquired pneumonia associated with influenza-louisiana and georgia staphylococcus aureus community-acquired pneumonia during the 2006 to 2007 influenza season multipathogen infections in hospitalized children with acute respiratory infections eleven cases of co-infection with influenza type a and type b suspected by use of a rapid diagnostic kit and confirmed by rt-pcr and virus isolation prevalence of human metapneumovirus and influenza virus infections among japanese children during two successive winters the peru influenza working group changes in the viral distribution pattern after the appearance of the novel influenza a h1n1 (ph1n1) virus in influenzalike illness patients in peru interim report on pandemic h1n1 influenza virus infection in south africa coinfection with mycobacterium tuberculosis and pandemic h1n1 influenza a virus in a patient with lung cancer severe 2009 pandemic influenza a (h1n1) infection and increased mortality in patients with late and advanced hiv disease h1n1 influenza a presenting as bacterial tracheitis pandemic influenza and pneumonia due to legionella pneumophila: a frequently underestimated coinfection necrotising pneumonia due to influenza a (h1n1) and community-acquired methicillin-resistant staphylococcus aureus clone usa 300: successful management of the first documented paediatric case co-infection with dengue virus and pandemic (h1n1) fatal influenza a (h3n2) and campylobacter jejuni coinfection what are the implications for childhood pneumonia of successfully introducing hib and pneumococcal vaccines in developing countries oseltamivir resistance in adult oncology and hematology patients infected with pandemic (h1n1) 2009 virus detection of oseltamivir sensitive/resistant strains of pandemic influenza a virus (h1n1) from patients admitted to hospitals in thailand bacterial pneumonias during an influenza pandemic: how will we allocate antibiotics? contribution of vaccine-induced immunity toward either the ha or the na component of influenza viruses limits secondary bacterial complications epivac study group. effectiveness of the 23-valent polysaccharide pneumococcal vaccine against invasive pneumococcal disease in people 60 years or older effects of a largescale intervention with influenza and 23-valent pneumococcal vaccines in elderly people: a 1-year follow-up influenza vaccination is associated with reduced severity of community-acquired pneumonia early versus late oseltamivir treatment in severely ill patients with 2009 pandemic influenza a (h1n1): speed is life public health and economic impact of vaccination with 7-valent pneumococcal vaccine (pcv7) in the context of the annual influenza epidemic and a severe influenza pandemic the authors have no competing interests. key: cord-301225-h178zpb3 authors: gautret, philippe; parola, philippe; wilson, mary elizabeth title: fever in returned travelers date: 2018-11-26 journal: travel medicine doi: 10.1016/b978-0-323-54696-6.00056-2 sha: doc_id: 301225 cord_uid: h178zpb3 predominant causes of fever vary by different geographic areas of exposure. malaria is the most common overall cause of systemic febrile illness in travelers returning from tropical areas; dengue is the most common cause in travelers to some regions. the approach to a febrile patient must consider travel and exposure history, incubation period, mode of exposure, and impact of pretravel vaccination. initial symptoms of self-limited and life-threatening infections may be similar; focal signs and symptoms can help to limit the differential diagnosis. routine laboratory results can provide clues to the final diagnosis. while fever may be the manifestation of a self-limited infection, it can also presage an infection that could be rapidly progressive and lethal. international travel expands the list of infections that must be considered but does not eliminate common, cosmopolitan infections. initial attention should focus most urgently on infections that are treatable, transmissible, and that may cause serious sequelae or death. 1 the characteristics of the places visited and the recency of travel will affect the urgency and extent of the initial workup. the recent emergence of the middle east respiratory syndrome coronavirus (mers-cov) in the arabian peninsula and of ebola in west africa and the recent epidemics of chikungunya and zika virus diseases underline the necessity of being aware of the possible implication of emerging pathogens in imported fever. 2 this chapter will focus on identifying the cause of fever in a returned traveler. the reader should refer to other sources for the specifics of therapy. fever in the absence of other prominent findings has been reported in 2%-3% of european and american travelers to developing countries. among 784 american travelers who traveled for 3 months or less to developing countries, 3% reported fever unassociated with other illness. 3 these results are similar to those reported by steffen et al. 4 in which 152 of 7886 (almost 2%) of swiss travelers with short-term travel to developing countries reported "high fever over several days" on questionnaires completed several months after return. of those with fever, 39% reported fever only while abroad, 37% had fever while abroad and at home, and 24% had fever at home only. analysis of the geosentinel surveillance network database found that 28% of ill returned travelers seeking care at a geosentinel clinic had fever as a chief reason for seeking care. 5 among patients with travel-related hospitalization, febrile illnesses predominated, accounting for 77% of admissions in a study from israel. 6 findings from eight studies, each with at least 100 cases, that examined causes of fever after tropical travel are shown in table 56 .1. [5] [6] [7] [8] [9] [10] [11] [12] [13] the geographic region of exposure helps to explain the marked differences in the relative likelihood of various diagnoses, as has been shown in a study by freedman et al. 14 malaria was the most common diagnosis among those requiring hospitalization for fever in most recently published series. in a geosentinel study including 3655 cases of potentially life-threatening tropical diseases, 91% of which had fever as a symptom, 77% were caused by malaria 15 ; in the study by bottieau and colleagues 9 falciparum malaria was the only tropical disease that was fatal (n = 5). in a geosentinel study 17% of febrile illnesses were caused by infections that are preventable with vaccines or specific chemoprophylaxis (e.g., falciparum malaria). 14 common cosmopolitan infections were found in 34% of returned febrile travelers in the bottieau study. infections, such as respiratory tract infections, hepatitis, diarrheal illness, urinary tract infections, and pharyngitis, with a broad or worldwide distribution, account for more than half of fevers in some series, 8 and the cause of fever remained undefined in about one-quarter of cases. 5, 9, 10 while, overall, malaria is the most common specific infection causing systemic febrile illness, dengue fever, mononucleosis, rickettsial infections, and enteric fever are also important infections. their relative rank varies by geographic location, with top three diagnoses being falciparum malaria, rickettsial infections, and dengue after travel to sub-saharan africa; dengue, falciparum, and vivax malaria after travel to southeast asia; enteric fever, dengue, and vivax malaria after travel to south central asia; and dengue, vivax malaria, and enteric fever after travel to latin america and caribbean. 16 leptospirosis is likely underrecognized because of difficulty in confirming the diagnosis in many laboratories. the major increase in chikungunya virus infections in indian ocean islands, asia, and now the americas has been reflected in an increase in cases in travelers to those regions (and even local spread of infection introduced by travelers in europe). 17 incubation time is a valuable tool in evaluating a febrile patient. knowledge of the incubation periods can allow one to exclude infections that are not biologically plausible. for example, dengue fever typically has an incubation of 3-14 days. thus fever that begins >2 weeks after return from thailand is not likely to be related to dengue fever. remote travel is sometimes relevant, but most severe, acute life-threatening infections result from exposures that have occurred within the past 3 months. important treatable infections that may occur >3 months after return include malaria, amebic liver abscess, and visceral leishmaniasis. in the study by o'brien et al. 11 analyzing patients hospitalized with fever after travel, 96% were seen within 6 months of return from travel; in the study by bottieau et al. 9 of patients referred for fever after tropical travel, fever occurred during travel or within 1 month of return home in 78%. although the initial focus should be on travel within the past 3-6 months, the history should extend to include exposures a year or more earlier, if the initial investigation is unrevealing. more than a third of malaria-infected travelers in a study from israel and the united states had illness that developed >2 months after return from endemic areas. 19 onset of illness >6 months after return occurred in 2.3% of malaria patients reported to the cdc in 2009. 20 table 56 .2 lists many of the infections seen in travelers by time of onset of symptoms relative to the exposure and the initial clinical presentation. in assessing potential incubation period one must take into account the duration of the trip (and points of potential exposure during travel) and time since return. the fever pattern and clinical findings for many infections are similar. relevant exposures can also occur in transit (e.g., on an airplane flight or cruise ship). 18 during the workup the clinician should keep in mind that fever after exotic travel may reflect infection with a common, cosmopolitan pathogen acquired during travel or after return home. at the same time it should be noted that unfamiliar infections can be acquired in industrialized countries (such as plague, rocky mountain spotted fever, tularemia, lyme disease, hantavirus pulmonary syndrome in north america, and visceral leishmaniasis, hemorrhagic fever with renal syndrome and other hantaviral infections, and tickborne encephalitis in europe). a detailed review of the clinical course, supplemented by the physical examination and laboratory data, will help to determine more likely causes and also to identify any infections that might require urgent interventions, hence expedited diagnostic studies. the process involved in the evaluation can be summarized in the following questions: the course of medical care, patients may become colonized or infected with bacteria that are extremely resistant to usual antibiotic therapy, as has recently been reported with the new delhi metallo-β-lactamase resistance mechanism, 22 or they may have other hospital-acquired infections. the history should include a review of pretravel vaccines, including dates of vaccination, types of vaccines received, and number of doses for multidose vaccines. vaccines vary greatly in efficacy, and knowledge of vaccine status can influence the probability that certain infections will be present. for example, hepatitis a and yellow fever vaccines have high efficacy and only rare instances of infection have been reported in vaccinated travelers. in contrast, the typhoid fever vaccines (oral and parenteral) give incomplete protection. 23 the protective efficacy with the available typhoid vaccines was estimated to be 60%-72% in field trials in endemic regions. 24 many febrile infections are associated with focal signs or symptoms, which may help to limit the differential diagnosis. undifferentiated fever can be more challenging. the following sections discuss common infections that can be acquired by a single bite of an infective arthropod, ingestion of contaminated food or beverages, swimming in contaminated water, or from direct contact with an infected person or animal are most often seen in short-term travelers. casual sexual contact with new partners is common in travelers (5%-50% among short-term travelers) and inquiry about sexual exposures should be included as part of the history of an ill traveler. a canadian study found that 15% of travelers reported sex with a new partner, or potential exposure to blood and body fluids through injections, dental work, tattoos, or other skin-perforating procedures during international travel. 21 thus history is important to review even in returned travelers who are not acutely ill. in many instances travelers will be unaware of exposures. for example, patients with mosquito-borne and tickborne infections may not recall any bites. in contrast, patients who have had freshwater exposure (such as swimming, wading, bathing, or rafting) that places them at risk for schistosomiasis will typically recall the exposure with focused questioning, though they may have been unaware that the exposure carried any risk for infection. the provider should also inquire about medical care during travel. travel for the purpose of seeking medical care (medical tourism) has expanded; travelers may undergo extensive surgery including cardiac surgery and organ transplantation overseas. in was 4.2% in persons with prior dengue infection who became infected with a new serotype. 31 diagnosis is usually confirmed by serologic tests; viral isolation or detection of viral ribonucleic acid (rna) by polymerase chain reaction (pcr) is available in some laboratories. because specific igm antibodies take several days to develop (usually present by day 5 of illness), serologic diagnosis may not be possible in the early febrile period. 32 igg antibody response can be difficult to interpret because of extensive cross reactions with other flaviviruses (e.g., yellow fever, japanese encephalitis, west nile, zika). in recent years, several diagnostic methods, including real-time pcr (rt-pcr) and ns1 antigen detection, have been proposed to optimize the early diagnosis of denv in travelers. 32 however, it is likely that only a minority of cases that occur in travelers are documented. a recent prospective study of dutch travelers found that seroconversion to denv occurred in 1.2% (incidence was 14.6 per 1000 person-months). 28 in the geosentinel database, confirmed or probable dengue fever was the most common specific diagnosis in patients with febrile systemic illness who had traveled to tropical and subtropical areas in the caribbean, south america, south central and southeast asia. 14 in 2009, dengue was the second most frequent cause of fever among 6392 patients with travel-associated health complaints seen in geosentinel european sites (no dengue hemorrhagic fever/dengue shock syndrome), a significant increase over 2008. 33 chikungunya. chikungunya (chik) fever is a tropical arboviral disease responsible for acute polyarthralgia, which can last for weeks to months. after half a century of focal outbreaks in africa and asia, the disease has emerged or reemerged in many parts of the world in the past decade, and has unexpectedly spread, with large outbreaks in africa, around the indian ocean, in the americas, and rare autochthonous transmission in temperate areas. it has now become an important global public health problem, with several ongoing outbreaks occurring worldwide. 34 since the beginning of this outbreak, several million cases of chikungunya virus disease have occurred in autochthonous populations and in travelers who were diagnosed after they returned home from epidemic areas. chik virus, usually transmitted by a. aegypti mosquitoes, has now been repeatedly associated with a new vector, a. albopictus (the "asian tiger mosquito"), which has spread into tropical and subtropical areas previously occupied predominantly by a. aegypti. 35 introduction into europe and spread has been described. 36 zika. zika viral infection is a tropical arboviral disease usually transmitted by a. aegypti and a. albopictus mosquitoes and responsible for acute fever. zika virus has spread rapidly throughout latin america and the caribbean since its initial identification in the americas in brazil in 2015. although infections are asymptomatic or relatively mild in approximately 80% of persons, severe complications have been described, including guillain-barré syndrome, myelitis, and encephalitis; miscarriage, prematurity, and multiple fetal neurologic and developmental abnormalities. 37 common symptoms in a recent series of travel-associated cases included exanthema (88%), fever (76%), and arthralgia (72%). 38 given the clear evidence of different methods of sexual transmission of zika virus, infected travelers should be counseled on the need for and duration of barrier contraception to limit onward sexual transmission of the virus. rickettsial infections are widely distributed in developed and developing countries and often named for a geographic region where they are found, though names can mislead. rickettsial diseases are increasingly being recognized among international travelers. a recent study of ≈7000 returnees with fever as a chief reason to clinical presentations, with focus on more common diseases causing each. other chapters provide more detailed discussions of diarrhea, skin diseases, and respiratory diseases. always look for malaria. malaria remains the most important infection to consider in anyone with fever after visiting or living in a malarious area. in nonimmune travelers falciparum malaria can be fatal if not diagnosed and treated urgently. although most patients with malaria will report fever, as many as 40% or more may not have fever at the time of initial medical evaluation. 25 risk of malaria varies greatly from one endemic region to another, but in general risk is highest in parts of sub-saharan africa; most severe and fatal cases in travelers follow exposure in this region. tests to look for malaria should be done urgently (same day) and repeated in 8-24 hours if the initial blood smears are negative. in recent years rapid diagnostic tests for malaria have become valuable tools for the diagnosis of malaria in both endemic and nonendemic areas. 26 prompt evaluation is most critical in persons who have visited areas with falciparum malaria in recent weeks. in the united states in 2009, 81% of reported patients with acute falciparum malaria had onset of symptoms within a month of return to the country; another 15% had onset of illness before arriving in the country. 20 use of chemoprophylaxis may ameliorate symptoms or delay onset. no chemoprophylactic agent is 100% effective, so malaria tests should be done even in persons who report taking chemoprophylaxis. many antimicrobials (e.g., tmp-smx, azithromycin, doxycycline, clindamycin) have some activity against plasmodia. taking these drugs for reasons unrelated to malaria may delay the onset of symptoms of malaria or modify the clinical course. although fever and headache are commonly reported in malaria, gastrointestinal (gi) and pulmonary symptoms may be prominent and may misdirect the initial attention toward other infections. thrombocytopenia and absence of leukocytosis are common laboratory findings. a prospective study of 335 travelers and migrants with suspected malaria found white blood cell (wbc) count <10,000 cells/l, platelet count <150, 000/µl, hemoglobin <12 g/dl, and eosinophils <5% to be associated with malaria parasitemia. 27 dengue. dengue, a mosquito-transmitted flavivirus that exists in four serotypes, is the most common arbovirus in the world. it is increasing in incidence in endemic areas and is an increasingly common cause of fever in returned travelers. 28, 29 dengue is found in tropical and subtropical regions throughout the world. among travelers, dengue is seen most often in visitors to southeast asia and latin america (including the caribbean) and infrequently in travelers to africa, though infections may be underrecognized. 30 because humans are the main reservoir for the dengue virus (denv), which is transmitted primarily by the aedes aegypti mosquito that inhabits urban areas and lives in close association with humans, travelers visiting only urban areas can become infected. symptoms of dengue, also known as breakbone fever, typically begin 4-7 days (range 3-14 days) after exposure. common findings are fever, frontal headache, and myalgia. approximately 50% of patients have skin findings, which can be a diffuse erythema or a maculopapular or petechial eruption. intense itching may be present toward the end of the febrile period. leukopenia, thrombocytopenia, and elevated transaminases are common laboratory findings. the most serious forms of infection-dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss)-in many studies have been observed more often in persons who have a second dengue infection with a different serotype. in a well-characterized outbreak in cuba, 98.5% of dhf/dss cases were in persons with a prior dengue infection. the attack rate of dhf/dss suspicion of intestinal perforation or other complication. diagnosis should be confirmed by recovery of salmonella typhi (or s. paratyphi) from blood or stool. 44 culture of bone marrow aspirate may have a higher yield than blood or feces but is generally not favored by clinicians and patients. serologic tests lack sensitivity and specificity. increasing resistance of s. typhi to many antimicrobials makes it important to isolate the organism and to do sensitivity testing. the emergence of multidrug resistance and decreased ciprofloxacin susceptibility in salmonella enterica serovar typhi in south asia have rendered older drugs, including ampicillin, chloramphenicol, trimethoprim sulfamethoxazole, ciprofloxacin, and ofloxacin, ineffective or suboptimal for typhoid fever. 45 multiple studies have identified the indian subcontinent as a destination with relatively high risk for enteric fever in travelers, especially those visiting friends and relatives (vfrs). 46 the efficacy of typhoid vaccines in published studies varies widely depending on the type of vaccine, number of doses, and population studied. as noted, the efficacy of commonly used vaccines may be 60%-70%. 24 the important observation for clinicians evaluating returned travelers is that typhoid fever remains a concern (albeit lower) in persons who have received a typhoid vaccine. infections with s. paratyphi may be relatively more common as a cause of typhoid fever in vaccinated populations because vaccine protects mainly against s. typhi. 44 notably, the course of s. paratyphi a was not found to be milder than that of s. typhi infection. 46 leptospirosis. although leptospirosis has a broad geographic distribution, infections in humans are more common in tropical and subtropical regions. recreational activities of travelers, including whitewater rafting in costa rica and other sports involving water exposures, have been associated with sporadic cases and large outbreaks. 47 among 158 competitive swimmers in the eco-challenge in malaysia in 2000, 44% met the case definition for acute leptospirosis. 48 although clinical manifestations may be protean, common findings include fever, myalgia, and headache. among 353 cases reported from hawaii, 39% had jaundice and 28% conjunctival suffusion. 49 other findings such as meningitis, rash, uveitis, pulmonary hemorrhage, oliguric renal failure, and refractory shock may be present. a summary of 72 sporadic leptospirosis cases in travelers from europe and israel shows that the majority were reported from southeast asia, were male (84%), the disease was associated with water activities in 91%, and 90% were hospitalized with no mortality. 50 multiple different serovars exist, and clinical presentation and severity vary with infecting serovar. in israeli travelers 55% had severe leptospirosis, usually associated with ictero-hemorrhagic serogroup. 51 owing to lack of sensitive and specific diagnostic tests to confirm infection early in the course in most institutions, early empiric therapy is recommended for suspected infection, especially if severe. agents used include doxycycline (and other tetracyclines), penicillins, and ceftriaxone. follows exposure to fresh water infested with cercariae that penetrate intact skin. the disease, seen primarily in nonimmunes, manifests 3-8 weeks after exposure. clinical manifestations include high fever, myalgia, lethargy, and intermittent urticaria. 52 dry cough and dyspnea, sometimes with pulmonary infiltrates, are noted in the majority of patients. 53 eosinophilia, often high grade, is usually present. in one outbreak involving 12 travelers the median duration of fever was 12 days (range 4-46 days) and 10 of 12 had eosinophilia during the first 10 weeks of infection. 52 in most cases the disease is acquired in africa (not only sub-saharan); however, in the last decade an seek medical care suggested that 2% of imported fevers are caused by rickettsioses and that 20% of these patients are hospitalized. 5 most infections are acquired in sub-saharan africa, where spotted fever group (sfg) rickettsioses are second only to malaria as the most commonly diagnosed diseases in returnees with systemic febrile illness. 14 rickettsia rickettsii, the cause of rocky mountain spotted fever in the united states, is found throughout the americas from canada to brazil. rickettsial infections such as african tick-bite fever (r. africae), mediterranean spotted fever (r. conorii), and murine or endemic typhus (r. typhi) are important treatable infections in travelers. 39 many additional rickettsioses have emerged throughout the world. these are being increasingly recognized in travelers, probably reflecting increased travel to high-risk areas, such as southern africa, and increased awareness among clinicians. 40, 41 diagnosis is usually confirmed with serologic tests or molecular tools such as pcr-based assay on skin biopsies or after eschar swabbing. clinical presentations of the rickettsial infections are varied, depending on the species. most rickettsial infections are transmitted by arthropods, such as ticks and mites, and an eschar may mark the inoculation site. eschars are often small (<1 cm in diameter), asymptomatic, and may be overlooked. in south african tick-bite fever eschars are often multiple (>50% of cases). rashes may be present, but many rickettsial infections (even among the sfg) are spotless. r. australis, r. africae, and rickettsialpox can cause a vesicular rash that may be mistaken for varicella, monkeypox, or even smallpox. high fever, headache, and normal or low wbc cell count and thrombocytopenia are characteristic. lymphadenopathy may be present. infections may be confused with dengue fever. rickettsiae multiply in and damage endothelial cells and cause disseminated vascular lesions. without treatment, the illness may persist for 2-3 weeks. response to tetracyclines is generally prompt. patients with suspected rickettsial infections should be treated empirically while awaiting laboratory confirmation. other tickborne infections, human monocytic ehrlichiosis, and human granulocytic ehrlichiosis (granulocytotropic anaplasmosis), 42 are most commonly diagnosed in the united states but are also found in europe, africa, and probably asia. clinical findings include prominent fever and headache. these infections may also be associated with leukopenia and thrombocytopenia, and respond to treatment with tetracyclines. when epidemiologic and clinical aspects of rickettsial diseases were investigated in 280 international travelers reported to the geosentinel surveillance network during 1996-2008, 231 (82.5%) had spotted fever (sfg) rickettsiosis, 16 (5.7%) scrub typhus, 11 (3.9%) q fever, 10 (3.6%) typhus group (tg) rickettsiosis, 7 (2.5%) bartonellosis, 4 (1.4%) indeterminable sfg/tg rickettsiosis, and 1 (0.4%) human granulocytic anaplasmosis; 197 (87.6%) of sfg rickettsiosis cases were acquired in sub-saharan africa and were associated with higher age, male gender, travel to southern africa, late summer season travel, and travel for tourism. 43 enteric fever. enteric fever (typhoid and paratyphoid fever) is another infection that causes fever and headache and can be associated with an unremarkable physical examination, though a faint rash (rose spots) may appear at the end of the first week of illness. laboratory findings include a normal or low wbc count, thrombocytopenia, and elevation (usually modest) of liver enzymes. gi symptoms such as diarrhea, constipation, and vague abdominal discomfort may be present, as well as dry cough. in contrast to the abrupt onset of fevers in dengue and rickettsial infections, the onset of typhoid fever may be insidious. leukocytosis in a patient with typhoid fever should raise countries and was responsible for an outbreak of eosinophilic meningoencephalitis in travelers to jamaica in 2000. 58 african trypanosomiasis (sleeping sickness), transmitted by an infective tsetse fly, initially causes a nonspecific febrile illness. a chancre marks the site of the bite. if untreated, trypanosomes can infect the cns and cause lethargy. several cases have been seen in travelers after exposures, especially in tanzania and kenya. patients with malaria, typhoid fever, and rickettsial infections often have severe headache, but cerebrospinal fluid (csf) is typically unremarkable in these infections. cerebral malaria causes altered mental status and can progress to seizures and coma. mefloquine taken for malaria chemoprophylaxis has rarely been associated with seizures and other neuropsychiatric side effects, but fever typically is absent. neuroschistosomiasis can be seen in travelers, but fever usually is not present at the time of the focal neurologic changes, caused by tissue reaction to ectopic schistosome egg deposition in the nervous system. sexually transmitted infections such as hiv and syphilis, whether acquired at home or during travel, can involve the cns. lyme and ehrlichiosis are other treatable infections that can cause prominent neurologic findings. other treatable infections that are unfamiliar to clinicians in many geographic areas include q fever, relapsing fever, brucellosis, bartonellosis, anthrax, and plague. diagnoses to be considered in patients with persistent or relapsing fevers include nonfalciparum malaria, typhoid fever, tuberculosis, brucellosis, cytomegalovirus (cmv), toxoplasmosis, louseborne relapsing fever (borrelia recurrentis), melioidosis (burkholderia pseudomallei), q fever (coxiella burnetii), visceral leishmaniasis, histoplasmosis (and other fungal infections), african trypanosomiasis, and infections that may be unrelated to exposures during travel, such as endocarditis. for fever with prominent respiratory symptoms, please refer to references 59-64 and chapter 59. results of routine laboratory findings may provide clues to the diagnosis in the febrile traveler. an elevated wbc count may suggest a bacterial infection, but a number of bacterial infections, such as uncomplicated typhoid fever, brucellosis, and rickettsial infections, are associated with a normal or low wbc count. in the past hepatitis a virus was the most common cause of hepatitis after travel to developing regions. with the wide use of the hepatitis a vaccine, acute hepatitis a now is seen primarily in persons who failed to receive vaccine (or immunoglobulin) before travel. hepatitis b remains a risk for unvaccinated persons. hepatitis e, transmitted via fecally contaminated water or food, clinically resembles acute hepatitis a. cases have been reported in travelers. 65 mortality may be 20% or higher in women infected during the third trimester of pregnancy. many common as well as unusual systemic infections cause fever and elevation of liver enzymes. among those that may be a concern, depending on geographic exposures, are yellow fever, dengue and other hemorrhagic fevers, typhoid fever, leptospirosis, rickettsial infections, toxoplasmosis, q fever, syphilis, psittacosis, and brucellosis. transaminases are often elevated in these infections. parasites that directly invade the liver and bile ducts (e.g., amebic liver abscess and liver flukes) often cause right upper quadrant pain, tender liver, and elevated alkaline phosphatase. drugs and toxins (sometimes found in herbal drugs or nutritional supplements) can damage the liver, so a careful review of these agents should be part of the history. important focus was documented in laos with infection due to s. mekongi. 54 amebic liver abscess. an amebic abscess can cause fever and chills that develop over days to weeks. although focal findings may not be prominent, 85%-90% of patients will report abdominal discomfort and about 70%-80% will have right upper quadrant tenderness on examination. 55 extension of infection to the diaphragmatic surface of the liver may lead to cough, pleuritic or shoulder pain, and right basilar abnormalities on chest x-ray, which may initially suggest a pulmonary process. the abscess can be seen on ultrasound and serology for entamoeba histolytica is usually positive. several infections in addition to exotic infections such as ebola and marburg can cause fever and hemorrhage in travelers and many are treatable. ebola and marburg are transmitted mostly through direct contact with patient body fluids and are rarely seen in international travelers. during the recent ebola epidemic in west africa, falciparum malaria was the most frequent cause of fever in travelers to the affected area. 56 leptospirosis, meningococcemia, and other bacterial infections can cause hemorrhage. rickettsial infections can produce a petechial rash or purpura, and severe malaria may be associated with disseminated intravascular coagulation. many viral infections, in addition to dengue, can cause hemorrhage. most are arthropod-borne (especially mosquito or tick) or have rodent reservoir hosts. among those reported in travelers are dengue fever (dhf), yellow fever, lassa fever, crimean congo hemorrhagic fever, rift valley fever, hemorrhagic fever with renal syndrome (and other hantavirus-associated infections), kyasanur forest disease, omsk hemorrhagic fever, and several viruses in south america (junin, machupo, guanarito, sabia). lassa fever responds to ribavirin therapy if started early. several of the viruses can be transmitted during medical care, so it is important to institute barrier isolation in a private room pending a specific diagnosis. identification of viral agents causing hemorrhage may require the assistance of staff working in special laboratories, such as one available at cdc. (assistance is available through the special pathogens branch, division of viral and rickettsial diseases, cdc, atlanta, ga 404-639-1511 and other specialized laboratories.) even when specific treatment is not available, good supportive care can save lives. neurologic findings in the febrile patient indicate the need for prompt workup. high fever alone or in combination with metabolic alternations precipitated by systemic infections can cause changes in the mental status in the absence of cns invasion. one must consider common, cosmopolitan bacterial, viral, and fungal infections that cause fever and cns changes. additional considerations in travelers include japanese encephalitis, rabies, west nile, polio, tickborne encephalitis, and a number of other geographically focal viral infections, such as nipah virus. outbreaks of meningococcal infections (meningococcemia and meningitis) have been associated with the annual hajj pilgrimage to mecca in saudi arabia. beginning in 2000, for the first time ever, infection with neisseria meningitidis serogroup w-135 caused outbreaks of meningococcal disease in pilgrims and subsequently in their contacts in multiple countries. pilgrims vaccinated with the quadrivalent meningococcal vaccine (serogroups a, c, w-135, and y) can still carry n. meningitidis in the nasopharynx. dengue fever can cause neurologic findings that mimic japanese encephalitis. in a study in vietnam, dengue-associated encephalopathy was found in 0.5% of 5400 children admitted with dhf. 57 meningitis may be present in leptospirosis. the parasite angiostrongylus cantonensis causes sporadic infection in many prompt diagnosis and urgent treatment may be necessary to save the patient's life. fig. 56 .1 provides an algorithm for the approach to a febrile patient following travel. useful algorithms based on expert opinion and review of published literature are also available. 67, 68 during the evaluation and treatment, the clinician should also keep in mind the public health impact. familiar infections (e.g., salmonellosis, campylobacteriosis, gonorrhea) may be caused by multidrug-resistant organisms. it is especially important to recognize the potential for multidrug resistance in infections, such as typhoid fever, that can be lethal. absence of response to what should be appropriate treatment should lead the clinician to consider drug resistance, the possibility of the wrong diagnosis, or the presence of two infections. particularly in patients with acute undifferentiated fever, rickettsial diseases must be considered, and patients with severe disease may be treated empirically. use of empirical doxycycline treatment for patients with fever of unknown origin should be discussed, especially when empirical treatment with β-lactams has failed or, in severe cases, in association with β-lactams. 69 a number of case reports document the simultaneous presence of malaria and typhoid fever, amebic liver abscess and hepatitis a, and other dual infections. 70, 71 conclusion it should be reminded that some febrile illnesses in travelers are still associated with high mortality and should be rapidly suspected and treated. place of exposure and local epidemiology are key elements in the diagnosis process. knowledge of the epidemiology of infections in a given geographic area is valuable, but detailed, up-to-date information about a specific location may be unavailable. electronic databases are a useful source of current information about disease outbreaks and alerts about antimicrobial resistance patterns. eosinophilia is sometimes an incidental finding on laboratory testing. when it is found in a person who has visited or lived in tropical, developing countries, it is a clue that should suggest several specific parasitic infections. 66 for further details see chapter 58. a careful, complete physical examination should be carried out, looking with special care for rashes or skin lesions, lymphadenopathy, retinal or conjunctival changes, enlargement of liver or spleen, genital lesions, and neurologic findings. the initial laboratory evaluation in a febrile patient with a history of tropical exposures should generally include all or most of the following: • complete blood count with a differential and estimate of platelets • liver enzymes • blood cultures • malaria and dengue rapid diagnostic tests • urinalysis and urine culture • chest radiograph if malaria is suspected, it is essential not only to request the appropriate tests for malaria but also to make certain that tests are done expeditiously and by knowledgeable persons. in patients with diarrhea or gi symptoms (or if enteric fever is suspected), stool culture should be requested. in a patient with persisting fever a repeat physical examination will sometimes identify new findings (e.g., new rash, splenomegaly) that can provide useful clues to the diagnosis. table 56 .3 lists tests used to diagnose common infections in febrile returned travelers. the process of travel may lead to medical problems. the immobility associated with travel may predispose to deep vein thrombosis; sinusitis may flare up during or after air travel, related to changes in pressure during ascent and descent. noninfectious disease causes of fever, such as drug fever, and pulmonary emboli, should also be considered if initial studies do not confirm the presence of an infection. in the study by bottieau et al. 9 noninfectious causes accounted for 2.2% of the fevers. spectrum of disease and relation to place of exposure among ill returned travelers geosentinel surveillance network. acute and potentially life-threatening tropical diseases in western travelers-a geosentinel multicenter study geosentinel surveillance network. geosentinel surveillance of illness in returned travelers infection with chikungunya virus in italy: an outbreak in a temperate region transmission of the severe acute respiratory syndrome on aircraft delayed onset of malariaimplications for chemoprophylaxis in travelers malaria surveillance-united states blood and body fluid exposure as a health risk for international travelers emergence of a new antibiotic resistance mechanism in india, pakistan, and the uk: a molecular, biological, and epidemiological study the effect of oral and parenteral typhoid vaccination on the rate of infection with salmonella typhi and salmonella paratyphi a among foreigners in nepal comparison of enteric-coated capsules and liquid formulation of ty21a typhoid vaccine in a randomised controlled field trial illness after international travel approach to fever in the returning traveler health problems in a large cohort of americans traveling to developing countries health problems after travel to developing countries fever in returned travelers: results from the geosentinel surveillance network epidemiology of travel-related hospitalization fever in travelers returning from malaria-endemic areas: don't look for malaria only fever in returned travelers: a prospective review of hospital admissions for a 2 1 2 year period etiology and outcome of fever after a stay in the tropics fever as the presenting complaint of travelers returning from the tropics fever in returned travelers: review of hospital admissions for a 3-year period prospective observational study of fever in hospitalized returning travelers and migrants from tropical areas fever in travelers returning from tropical areas: prospective observational study of 613 cases hospitalised in marseilles assessment of the clinical presentation and treatment of 353 cases of laboratory-confirmed leptospirosis in hawaii travel-associated zoonotic bacterial diseases travel-related leptospirosis in israel: a nationwide study outbreak of schistosomiasis among travelers returning from mali, west africa pulmonary manifestations of early schistosome infection among nonimmune travelers travel-related schistosomiasis acquired in laos amebic liver abscess geosentinel surveillance network. differential diagnosis of illness in travelers arriving from sierra leone prospective case-control study of encephalopathy in children with dengue hemorrhagic fever an outbreak of eosinophilic meningitis caused by angiostrongylus cantonensis in travelers returning from the caribbean cruise ships: high-risk passengers and the global spread of new influenza viruses influenza virus infection in travelers to tropical and subtropical countries outbreak of coccidioidomycosis in washington state residents returning from mexico update: outbreak of acute febrile respiratory illness among college students fungal infections among returning travelers an outbreak of acute pulmonary histoplasmosis in members of a trekking trip in martinique, french west indies running like water-the omnipresence of hepatitis e diagnostic significance of blood eosinophilia in returning travelers practice guidelines for evaluation of fever in returning travelers and migrants approach to fever in the returning traveler rickettsioses as causes of cns infection in southeast asia concurrent falciparum malaria and salmonella in travelers: report of two cases simultaneous amoebic liver abscess and hepatitis a infection difficulties in the prevention, diagnosis, and treatment of imported malaria evaluation of rapid diagnostic tests for malaria in swedish travelers clinical and laboratory predictors of imported malaria in an outpatient setting: an aid to medical decision making in returning travelers with fever travel-related dengue virus infection dengue in travelers dengue virus infection in africa epidemiologic studies on dengue in santiago de cuba, 1997 early diagnosis of dengue in travelers: comparison of a novel real-time rt-pcr, ns1 antigen detection and serology travel-related imported infections in europe chikungunya: its history in africa and asia and its spread to new regions in 2013-2014 chikungunya virus infection chikungunya virus, southeastern france zika virus geosentinel surveillance network. travel-associated zika virus disease acquired in the americas through february 2016: a geosentinel analysis tick-borne rickettsioses around the world: emerging diseases challenging old concepts african tick bite fever in travelers to rural sub-equatorial africa rickettsia africae, a tick-borne pathogen in travelers to sub-saharan africa human ehrlichioses multicenter geosentinel analysis of rickettsial diseases in international travelers clinical importance of salmonella paratyphi a infection to enteric fever in nepal treatment of typhoid fever in the 21st century: promises and shortcomings typhoid and paratyphoid fever in travelers outbreak of leptospirosis among white-water rafters-costa rica update: outbreak of acute febrile illness among athletes participating in eco key: cord-303966-z6u3d2ec authors: shears, p. title: poverty and infection in the developing world: healthcare-related infections and infection control in the tropics date: 2007-10-22 journal: j hosp infect doi: 10.1016/j.jhin.2007.08.016 sha: doc_id: 303966 cord_uid: z6u3d2ec in many hospitals serving the poorest communities of africa and other parts of the developing world, infection control activities are limited by poor infrastructure, overcrowding, inadequate hygiene and water supply, poorly functioning laboratory services and a shortage of trained staff. hospital transmission of communicable diseases, a high prevalence of human immunodeficiency virus and multidrug-resistant tuberculosis, lack of resources for isolation and disinfection, and widespread antimicrobial resistance create major risks for healthcare-related infections. few data exist on the prevalence or impact of these infections in such environments. there is a need for interventions to reduce the burden of healthcare-related infections in the tropics and to set up effective surveillance programmes to determine their impact. both the global (g8) international development summit of 2005 and the united nations millennium development goals (mdgs) have committed major resources to alleviating poverty and poor health in the developing world over the next decade. targeting resources specifically to infection control in low-resource settings must be a part of this effort, if the wider aims of the mdgs to improve healthcare are to be achieved. summary in many hospitals serving the poorest communities of africa and other parts of the developing world, infection control activities are limited by poor infrastructure, overcrowding, inadequate hygiene and water supply, poorly functioning laboratory services and a shortage of trained staff. hospital transmission of communicable diseases, a high prevalence of human immunodeficiency virus and multidrug-resistant tuberculosis, lack of resources for isolation and disinfection, and widespread antimicrobial resistance create major risks for healthcare-related infections. few data exist on the prevalence or impact of these infections in such environments. there is a need for interventions to reduce the burden of healthcare-related infections in the tropics and to set up effective surveillance programmes to determine their impact. both the global (g8) international development summit of 2005 and the united nations millennium development goals (mdgs) have committed major resources to alleviating poverty and poor health in the developing world over the next decade. targeting resources specifically to infection control in lowresource settings must be a part of this effort, if the wider aims of the mdgs to improve healthcare are to be achieved. ª 2007 the hospital infection society. published by elsevier ltd. all rights reserved. 'the widening gap between the developed countries and the poorest communities of the developing world has become a central issue of our time.' this is not a quote from the global (g8) international development summit of 2005, or from international awareness events such as the live aid and live 8 concerts e it is the opening sentence of the pearson report of 1969, a commission set up by the world bank to investigate why, after a decade of 'development', little impact had been achieved for the poorest communities of the tropics. 1, 2 in 1984, the alma ata conference of the world health organization (who) stated as its aim 'health for all by 2000'. a visit to a village affected by human immunodeficiency virus (hiv) and malaria in tropical africa, to a shanty town in south asia with inadequate water and sanitation ( figure 1 ), or to a displaced community in southern somalia suffering from cholera, dysentery and rift valley fever, suggest that these aims are yet to be fulfilled. many medical journals are currently devoting part of their current issues to the themes of poverty and infection in the developing world, in recognition of the commitments made by the g8 summit and the united nations (un) millenium development goals (mdgs) to improve maternal healthcare, reduce childhood mortality and the impact of human immunodeficiency virus (hiv)/ acquired immunodeficiency syndrome (aids), malaria and other communicable diseases. 3 if g8 and the un mdgs are to be catalysts for change, it will be necessary to look beyond international conferences and mission statements and to concentrate on specific objectives that can be implemented in resource-poor settings. from the viewpoint of hospital infection control, the concern is how hospital and healthcarerelated infections affect poorer communities of the developing world, and what can be done to begin to make a contribution to reducing the associated morbidity and mortality. a visit to a sub-saharan african country, where i had been looking at laboratory services and hospital infection control, left two particular memories. the first is the overwhelming disadvantages faced by health workers surrounded by hiv/aids, multidrug-resistant tuberculosis, overcrowding and lack of resources for the most basic of hygiene activities. the second is the impossibility of knowing the magnitude of hospital-related infections and optimum treatment strategies without effective laboratory services. these comments would certainly have been equally appropriate 10 or 15 years ago. might 2007 be a turning point? with the current emphasis of governments and un agencies to focus again on poverty and health, there is an opportunity to move healthcare-related infections, and the support required in terms of laboratory development, education, hospital infrastructure, and appropriate professional training, into a focused agenda, rather than the 'cinderella' position that has until now been the case. the ebola virus outbreak in southern sudan in 1976 was a vivid reminder of the potential magnitude of hospital-acquired infection in tropical africa. 4 on 6 august 1976, a student from nzara presented to the local district hospital at maridi in equatoria province, southern sudan, suffering from a severe febrile disease. he died a week later. over the next week, a nurse, a cleaner, and a hospital messenger became unwell, and then further medical and auxiliary staff. by the time a ministry of health/who team arrived in maridi approximately six weeks after the first case, one of the two doctors in the hospital had been infected and died, all six of the medical assistants had been infected and five had died, and twenty student nurses had died. further spread and deaths occurred until the impact of basic infection control strategies, involving gloves, gowns and masks for healthcare workers, and hygienic measures in dealing with body fluids and the deceased, brought the outbreak to an end. while the maridi ebola outbreak was an extreme case, it emphasised that the priorities for healthcare-associated infection control in the tropics, particularly away from tertiary or university hospitals in capital cities, cannot be a simple translation of expertise from the west. while limited systematic data exist on the overall patterns and prevalence of infection, experience from the field, and a review of available literature, indicate that the following are the major groups of infection control areas: since the 1976 outbreaks in sudan and zaire (now democratic republic of the congo, drc), nosocomial transmission of ebola virus has been reported in drc (kikwit), uganda, and probable cases in gabon and sudan. nosocomial transmission of other viral haemorrhagic fevers has included lassa fever in nigeria, and marburg haemorrhagic fever in angola. 5e8 prevention of transmission is difficult in resource-limited settings, with no total protective clothing or isolation facilities available. specific guidelines for control in district hospitals have been drawn up by who, and these cover issues including patient isolation, locally produced protective clothing, waste disposal, disinfectants, and community education. 9 early diagnosis of the first cases, often in the community or peripheral health centres, is the mainstay of ensuring that preventive measures are initiated in a timely fashion and that transmission is kept to a minimum. if severe acute respiratory syndrome, pandemic influenza or h5n1 avian flu were to become pathogens in these underresourced tropical areas, the pattern of the 1976 ebola outbreaks could be repeated. in any hospital infection control programme in much of africa, and large parts of asia and latin america, prevention of the spread of hiv, between patients, from patients to health workers, and from health workers to patients, is a priority. largely due to the un and who global aids programme, there is considerable literature and guidance available. 10 at the level of the individual, poorly resourced hospital with limited reuseable equipment and disinfectants, hiv is a major concern for all health workers and local health education and support programmes have been shown to be effective. 11 with the rise in hiv, there has been an increasing prevalence of tuberculosis, both in hiv-affected patients but also in the wider community, and in many areas an increasing prevalence of multidrug-resistant tuberculosis (mdrtb). this poses a risk for nosocomial transmission between patients, but particularly for health workers who will have direct contact, often with undiagnosed cases, and with less than adequate protective clothing. 12 there are likely to be few rural hospitals in africa with a supply of pfr95 masks. the strict barrier precautions described by uk and centers for disease control and prevention (cdc) guidelines are unlikely to be practicable and workers dealing with these issues daily have developed more appropriate strategies. 13 nosocomial spread of other communicable disease where hospital hygiene is limited, and wards are crowded, the risk of transmission of communicable disease is high. the source may be an inadequately isolated index case, relatives who are staying in the hospital to provide food and general care for patients, or contaminated food or water in the hospital. although from observation such transmission must occur, few such episodes have been recorded. nosocomial outbreaks of cholera have been published from tanzania and mozambique and these reports suggest that outbreaks of other faecal-oral infections, particularly shigellosis, typhoid and hepatitis a and e, must also occur. 14, 15 other reported hospital communicable disease outbreaks have included measles and non-typhoidal salmonellae. 16, 17 for communicable diseases in the tropics, the boundary between hospital and community infection is blurred. among relatives camping within the hospital compound, there may be a case of undiagnosed tuberculosis? is the infected person part of the community or part of the hospital? this distinction between public health and healthcare-related infection is particularly complex in the setting of refugee camps. 18, 19 direct hospital acquired infections studies of surgical and other hospital-related infections have been published from several countries in africa, including kenya, nigeria, tanzania, ethiopia and burkina faso. 20e24 these studies show a similar pattern of pathogens that are seen globally; staphylococci, enterobacteriaceae and pseudomonas spp., with high levels of antimicrobial resistance and a high and somewhat arbitrary use of antimicrobials. the studies are necessarily selective e only those hospitals in which there is a functioning microbiology laboratory, and which have sufficient staff, can undertake such work. in addition, with limited laboratory facilities, only the more easily culturable bacteria may be isolated and the results may give only a partial picture of the true prevalence and antimicrobial resistance patterns of pathogens. in situations where more sophisticated microbiological studies have been undertaken in two particular non-western environments e the tsunami area of south east asia and repatriated combatants from afghanistan e unusual and multiply resistant isolates have been found. 25, 26 occurrence of multi-resistant bacterial pathogens a common theme in all published studies of hospital infections in the tropics is the high prevalence of antimicrobial-resistant bacterial pathogens. this raises one of the major areas of concern. in terms of hygiene facilities, crowding and laboratory support, many hospitals in the tropics are like those of the 'pre-penicillin era', yet within them are the multi-resistant bacteria of the twenty-first century. data in many areas are limited or non-existent, but, where surveys have been done, meticillin-resistant staphylococcus aureus and extended-spectrum b-lactamase-producing and other multi-resistant gram-negative bacteria have been shown to be widely disseminated. 27e31 the problem is compounded as antimicrobials, including quinolones and third-generation cephalosporins, are available without prescription, many areas have no laboratory facilities to provide accurate susceptibility data, and, despite several past attempts, there are no functioning international resistance surveillance programmes. 32, 33 hospital infrastructure and facilities the 'catalogue of disadvantage' for a rural district hospital in africa demonstrates the commitment of local staff in providing even the most basic services (figure 2 ). the catalogue includes: poor building infrastructure, inadequate water supply, electricity for perhaps only a few hours a day, overcrowded wards with patients lying on the floor as well as on beds, lack of resources for cleaning the environment, beds or equipment, often an absence of soap, and remoteness from regional centres or the capital city. maridi, the hospital of the 1976 ebola outbreak in southern sudan, is 1800 km from khartoum, with a road link not possible for three months in the rainy season. this situation is compounded by civil strife in the democratic republic of congo, southern somalia or darfur, for example, where hospitals may have influxes of patients with both infected injuries and communicable diseases. 34, 35 as patients with communicable diseases are admitted from crowded refugee camps, they may become the index cases for further spread within the hospital if infection control facilities are overstretched (figure 3 ). there is no director of infection prevention and control in an african rural district hospital. nor is there usually a dedicated infection control nurse, almost certainly not a clinical microbiologist, and rarely clinicians with sufficient time to work with the laboratory staff. there have been developments in training personnel specifically to work in infection control in larger hospitals, those supported in some way by outside agencies, and in teaching hospitals in larger cities. most published work on training for infection control has been from south africa, with an understandable emphasis on hiv transmission, but many hospitals will have developed local, basic policies. 36 the requirements to improve hospital infection control in the tropics have been considered in a number of valuable papers. 37e39 the key areas that have been identified include the following: e setting up national and regional infection control networks. many of these will only occur when there are the resources for major economic change and that is why it is necessary to consider poverty reduction, institutional as well as individual, rather than to focus on improving infection control as simply a 'health' or 'medical' problem. whether g8 or the mdgs for 2015 can achieve this poverty reduction remains to be seen. 40 past experience of the pearson commission and health for all by 2000 suggests that targets alone, without addressing the fundamental causes of poverty or ill health, need to be handled with care. in the mean time, what can be done to assist the improvement of infection control in resource-poor settings? local health workers themselves are the most likely to achieve progress. the resources for hiv infection control that are part of the global aids programme represent one means for local improvement. the main who strategy to improve infection control internationally is the global patient safety challenge. 41 this has involved many meetings and recommendations, but whether these will have a significant impact in resource-poor, tropical settings has yet to be seen. however, the initiative may give support to policy-makers in giving a priority to infection control. certainly, links that have been established between hospitals in the uk and individual hospitals in africa can provide muchneeded resources, can share expertise and provide training opportunities. the existence of the internet has in many ways revolutionised the ability of even remote hospitals to acquire information, to download who or cdc guidelines, to communicate with distant colleagues, and in some situations to be part of a telemedicine network. such initiatives include the raft network in francophone africa (réseau en afrique francophone pour la télémédicine) and the who health initiative for access to research information (hinari) programme. 42, 43 if the aim of an internetlinked computer in every village in africa or asia or latin america, or at least in hospitals in those areas is to be achieved, there are practical issues of connectivity and band width that have to be faced. providing laptop computers without adequate connectivity and support will not be productive. 44 efficient internet links will enable very real possibilities of support between hospitals in the uk and rural tropical hospitals. the absence of effective laboratory services at district hospital level is a major impediment to the long-term control of hospital-associated infections. unless the causative pathogens can be identified, and where appropriate the antimicrobial susceptibilities determined, there is a considerable risk that multiply resistant bacteria will become the dominant pathogens, with few antimicrobials available for treatment. within the mdgs, no specific mention is made of infection control or laboratories; without these, we cannot achieve the three specific health goals of: (i) improving maternal health, (ii) reducing child mortality and (iii) tackling aids, malaria and other communicable diseases. healthcare-associated infections have been near the top of the political agenda in the uk, resulting in increased funding and commitment for change. for the developing world the same priority is required if the aims of g8 and the mdgs are to be approached. the g8 and global health: what now? what next? partners in development: report of the commission on international development achieving the millennium development goals report of a who/international study team ebola outbreak in kikwit, democratic republic of the congo: discovery and control measures outbreak of ebola hemorrhagic fever uganda review of cases of lassa fever in nigeria: the high price of poor medical practice lessons from nosocomial haemorrhagic fever outbreaks centers for disease control and prevention and world health organisation. infection control for viral haemorrhagic fevers in the african health care setting the joint united nations programme on hiv/aids. project update self reported infection control practices and perceptions of hiv/aids risk amongst emergency department nurses in botswana tuberculosis infection among health care workers in kampala, uganda practical and affordable measures for the protection of health care workers from tuberculosis in low income countries hospital outbreaks of cholera transmitted through close person to person contact a hospital outbreak of cholera in maputo nosocomial outbreaks e a potential threat to the elimination of measles? nosocomial outbreak of neonatal salmonella enteritidis in a rural hospital in northern tanzania epidemiological assessment of health and nutrition of ethiopian refugees in emergency camps in sudan communicable diseases in complex emergencies: impact and challenges nosocomial infections at kenyatta national hospital intensive care unit in aerobic bacterial nosocomial infections in paediatric surgical patients at a tertiary health institution in lagos risk factors for surgical site infections in a tanzanian hospital: a challenge for the traditional national nosocomial infections surveillance system index wound infection in tikur anbessa hospital surgical department survey of nosocomial infection prevalence on the surgery department of the central national hospital of ouagadougou rare bacteria species found in wounds of tsunami patients. gram negative bacteria from patients seeking medical advice in stockholm after the tsunami catastrophe multidrug resistant acinetobacter extremity infections in soldiers first report of mrsa from hospitalised patients in sudan staphylococcus aureus bacteraemia in the dakar fann university hospital nasal carriage of meticillin resistant staphylococcus aureus among health care personnel in abidjan (cote d'ivoire) extended spectrum beta lactamases among gram negative bacteria of nosocomial origin from an intensive care unit of a tertiary health facility in tanzania occurrence of extended spectrum beta lactamase enzymes in clinical isolates of klebsiella species from harar region, eastern ethiopia surveillance of antimicrobial resistance at a tertiary hospital in tanzania surveillance of antimicrobial resistance: the whonet programme burden of injury during the complex emergency in northern uganda the disease profile of poverty: morbidity and mortality in northern uganda in the context of war, population displacement and hiv-aids south african health service must strengthen infection control measures infection control in africa south of the sahara nosocomial infections in developing countries: cost effective control and prevention risk of nosocomial infection in tropical africa countdown to 2015: will the millenium development goal for child survival be met? clean care is safer care'; the global patient safety challenge the raft network: 5 years of distance continuing medical education and teleconsultations over the internet in french speaking africa who's health internetwork access to research initiative (hinari) access to electronic health knowledge in five african countries: a descriptive study none declared. none. key: cord-350715-x92g6bnk authors: zheng, yutong; yan, meitian; wang, lan; luan, liang; liu, jing; tian, xiao; wan, nan title: analysis of the application value of serum antibody detection for staging of covid‐19 infection date: 2020-07-23 journal: j med virol doi: 10.1002/jmv.26330 sha: doc_id: 350715 cord_uid: x92g6bnk coronavirus disease 2019 (covid‐19) has now spread all over the world. the national health commission of the people's republic of china reported 78,439 cured and discharged cases, 4634 deaths, 83,462 confirmed cases and 760,818 close contacts as of june 25, 2020. joint detection of nucleic acids and antibodies has become an important laboratory diagnostic for covid‐19 patients. disease progression and infection stage can be established based on the biological characteristics of these tests. however, there have been few studies of the different infection stages of covid‐19. we conducted a retrospective analysis to explore the clinical characteristics of covid‐19 patients at different infection stages and to characterize the characteristics of specific serum antibodies at each stage. these data will provide a theoretical basis for clinical diagnosis and treatment. this article is protected by copyright. all rights reserved. patients. in this study we explored the clinical value of specific serum antibody detection in covid-19 patients. nucleic acid and specific antibody detection sars-cov-2 nucleic acids in nasopharyngeal swab samples were detected using a model 7500 pcr gene amplification instrument (abi, foster city, ca, usa). a cycle threshold value of > 40 was considered negative and a value of <40 was considered positive. fasting venous blood (2-5 ml) was collected and centrifuged. the serum was separated and stored at -20°c. specific serum igm and igg antibodies were quantitatively detected using an axceed 260 magnetic particle-based chemiluminescence immunoanalyzer (bioscience, tianjin, china). a chemiluminescence signal cutoff value (s/co) of <1 was considered negative and >1 was considered positive. this article is protected by copyright. all rights reserved. statistical methods spss statistics software version 25.0 (ibm, armonk, ny, usa) was used for all statistical analyses. count data were expressed as frequency (%) and the chi-square test was used to assess differences between groups. continuous data with normal distributions were expressed as x ±s, and differences between two groups or among multiple groups were assessed using the student's t test and analysis of variance, respectively. non-normally distributed variables were expressed as medians and interquartile ranges and differences between groups were assessed using the mann-whitney u test. graphpad prism 7.0( graphpad software,san diego,ca)was used to produce all figures. a total of 723 covid-19 patients were enrolled, comprising 290 male patients and 433 female patients with an average age of 61.30±14.55 years. moderate cases made up the largest number of patients, accounting for 72.20% of the total. mild cases were rarest, accounting for only 1.11% of the total, while severe and critical cases accounted for 23.24% and 3.46% of the total, respectively. according to the biological characteristics of nucleic acids and specific serum igm and igg antibodies, the 723 covid-19 cases were classified into infection stages ( table 1) . analyses of the characteristics of different periods were performed ( table 2 ). patients who were nucleic acid negative at admission could be divided into two categories:① the nucleic this article is protected by copyright. all rights reserved. acid test had a positive record, but was negative more than twice before admission, and remained negative many times after admission; or ② nucleic acid test results were negative since the onset of the disease, with diagnosis made based on lung imaging, symptoms, epidemiological history and serum antibody results. because most patients had a long disease course when they were admitted to hospital, there were more cases in the active, middle/late and convalescent stages of infection (p<0.001). figure 1a ). this article is protected by copyright. all rights reserved. on january 20, 2020, the national health commission decided to include covid-19 pneumonia in statutory infectious disease category b and to take preventive and control measures for category a infectious diseases 5 . on january 30, the who called the epidemic "an emergency of international concern". sars-cov-2 is a novel enveloped β coronavirus with an rna genome. in the early stages of the epidemic, nucleic acid detection was taken as direct evidence of infection. however, the accuracy of nucleic acid detection was affected by the quality of detection kits, sample collection methods, operator ability, rna stability, patient condition and concurrent drug use 6 this article is protected by copyright. all rights reserved. in addition, we found that levels of igg were persistently high after the active stage of infection, indicating that disease progression coincided with emergence of these antibodies. we found that levels of igg in patients with different clinical severity were higher than those of igm. this finding may be related to the high affinity and easy detection of igg antibodies and the longer course of disease of the patients included in the study following their admission to hospital. however, levels of serum specific igm tables table 1. combined analysis of nucleic acid and serum antibody testing. ① the nucleic acid test had a positive record, but was negative more than twice before admission, and remained negative many times after admission.② the nucleic acid test result was negative since the onset of the disease. table 2 . analysis of covid-19 infection stages. combined detection of two detection methods to determine the stage of infection nucleic acid+ igm-igg-①window period ②igm begins to appear but is still below the detection limit in the early stage of infection. this article is protected by copyright. all rights reserved. igm+igg-the early stage of infection. the middle and late stage of infection (the result of early antibody test of the patient is unknown, the possibility of recurrent infection can not be ruled out). the active stage of infection, but at this time the body has developed a certain immune capacity. igm-igg-it may be in the infection recovery stage where the nucleic acid turns negative, the igm antibody disappears and the igg antibody begins to appear but is still below the detection limit. igm-igg+ previous infection. the convalescent stage of infection, igm decreased but still above the detection limit. nucleic acid-② igm-igg-this type of patient was diagnosed by pulmonary ct, symptoms and epidemiological history on admission. (1) the possible "window period" of false negative nucleic acid. (2) the convalescent stage in which the nucleic acid turned negative, the igm antibody disappeared and the igg antibody began to appear but was still below the detection limit. igm+igg-may be in the acute stage of infection, consider the possibility of false negative nucleic acid. consider the possible active stage of infection with false negative nucleic acid. ① the nucleic acid test had a positive record, but was negative more than twice before admission, and remained negative many times after admission. ② the nucleic acid test result was negative since the onset of the disease. as of 24:00 on june 25 th, the latest situation of covid-19 epidemic situation there are 9211083 confirmed cases by novel coronavirus outside china consensus of 2019-ncov nucleic acid testing experts on the issuance of novel coronavirus's diagnosis and treatment plan for pneumonia (seventh edition for trial implementation):health office medical care administration file〔2020〕184 [s].beijing:national health commission of the people's republic of national health commission of the people's republic of china.pneumonia infected by novel coronavirus is included in the management of legal infectious diseases cause analysis and countermeasures of false negative results of novel this article is protected by copyright. all rights reserved. accepted article coronavirus nucleic acid test false positive analysis of chemiluminescence microparticle immunoassay in hiv detection ping'an zhang.the diagnostic value of joint detection of serum igm and igg antibodies to 2019-ncov in 2019-ncov infection research progress on mechanism of antibodydependent enhancement is covid-19 receiving ade from other coronaviruses? immunoregulation with mtor inhibitors to prevent covid-19 severity: a novel intervention strategy beyond vaccines and specific antiviral medicines dengue fever, covid-19 (sars-cov-2), and antibody-dependent enhancement (ade): a perspective is covid-19 receiving ade from other coronaviruses susceptibility of the elderly to sars-cov-2 infection: ace-2 overexpression, shedding, and antibody-dependent enhancement (ade) similarities and evolutionary relationships of covid-19 and related viruses. preprints identification of epitopes on sars-cov nucleocapsid protein that induce the cross-or specific-reactivity among sars-cov, hcov-oc43 and hcov-229e effectiveness of convalescent plasma therapy in severe covid-19 patients proc. natl. acad. sci first infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood this study was supported by research grants from the natural science the authors declare that there are no conflict of interests. yz and nw contributed to data analysis, graphics and writing the paper. yz, nw, my, lw and ll contributed to data collection and graphics development. jl and xt contributed to editing the paper. no date are available.this article is protected by copyright. all rights reserved. key: cord-344093-3bniy5b5 authors: peteranderl, christin; herold, susanne title: the impact of the interferon/tnf-related apoptosis-inducing ligand signaling axis on disease progression in respiratory viral infection and beyond date: 2017-03-22 journal: front immunol doi: 10.3389/fimmu.2017.00313 sha: doc_id: 344093 cord_uid: 3bniy5b5 interferons (ifns) are well described to be rapidly induced upon pathogen-associated pattern recognition. after binding to their respective ifn receptors and activation of the cellular jak/signal transducer and activator of transcription signaling cascade, they stimulate the transcription of a plethora of ifn-stimulated genes (isgs) in infected as well as bystander cells such as the non-infected epithelium and cells of the immune system. isgs may directly act on the invading pathogen or can either positively or negatively regulate the innate and adaptive immune response. however, ifns and isgs do not only play a key role in the limitation of pathogen spread but have also been recently found to provoke an unbalanced, overshooting inflammatory response causing tissue injury and hampering repair processes. a prominent regulator of disease outcome, especially in—but not limited to—respiratory viral infection, is the ifn-dependent mediator trail (tnf-related apoptosis-inducing ligand) produced by several cell types including immune cells such as macrophages or t cells. first described as an apoptosis-inducing agent in transformed cells, it is now also well established to rapidly evoke cellular stress pathways in epithelial cells, finally leading to caspase-dependent or -independent cell death. hereby, pathogen spread is limited; however in some cases, also the surrounding tissue is severely harmed, thus augmenting disease severity. interestingly, the lack of a strictly controlled and well balanced ifn/trail signaling response has not only been implicated in viral infection but might furthermore be an important determinant of disease progression in bacterial superinfections and in chronic respiratory illness. conclusively, the ifn/trail signaling axis is subjected to a complex modulation and might be exploited for the evaluation of new therapeutic concepts aiming at attenuation of tissue injury. interferons (ifns) are well described to be rapidly induced upon pathogen-associated pattern recognition. after binding to their respective ifn receptors and activation of the cellular jak/signal transducer and activator of transcription signaling cascade, they stimulate the transcription of a plethora of ifn-stimulated genes (isgs) in infected as well as bystander cells such as the non-infected epithelium and cells of the immune system. isgs may directly act on the invading pathogen or can either positively or negatively regulate the innate and adaptive immune response. however, ifns and isgs do not only play a key role in the limitation of pathogen spread but have also been recently found to provoke an unbalanced, overshooting inflammatory response causing tissue injury and hampering repair processes. a prominent regulator of disease outcome, especially in-but not limited to-respiratory viral infection, is the ifn-dependent mediator trail (tnf-related apoptosis-inducing ligand) produced by several cell types including immune cells such as macrophages or t cells. first described as an apoptosis-inducing agent in transformed cells, it is now also well established to rapidly evoke cellular stress pathways in epithelial cells, finally leading to caspase-dependent or -independent cell death. hereby, pathogen spread is limited; however in some cases, also the surrounding tissue is severely harmed, thus augmenting disease severity. interestingly, the lack of a strictly controlled and well balanced ifn/trail signaling response has not only been implicated in viral infection but might furthermore be an important determinant of disease progression in bacterial superinfections and in chronic respiratory illness. conclusively, the ifn/ trail signaling axis is subjected to a complex modulation and might be exploited for the evaluation of new therapeutic concepts aiming at attenuation of tissue injury. (73) cell death induction, e.g., bcl-2-associated x protein, caspase-8, fas-associated protein with death domain, fas ligand, and tnf-related apoptosis-inducing ligand (trail) dsrna, polyi:c (4, 110) iav (4, 5, 10, 115) sendai virus (110) trail virus control by apoptosis induction in infected cells iav (6, 170, 171) tissue injury by apoptosis of both infected and non-infected alveolar epithelial cells, lung macrophages iav (5, 7, 10) rsv (137) necrosis of fibroblasts, dendritic cells, and epithelial cells iav (146, 147, 168) increased cellular infiltration cov (175) decreased expression of na,k-atpase, impaired epithelial fluid reabsorption iav (11) introduction in 1957, isaacs and lindenmann (1) first recognized the potential of a soluble and probably cell-derived factor to combat influenza virus infection and named this factor interferon [(ifn) from latin interferre, to interfere]. since then, three subgroups of ifns have been defined, primarily by their differential receptor usage. while the groups of type i ifn and type iii ifn comprise largely agents directly limiting pathogen spread by improving cellular counter measurements, ifn-γ, the sole type ii ifn, has been mainly implicated in the modulation of innate and also adaptive immune responses (2, 3) . accordingly, type i and iii ifns are key signaling molecules in viral control, and lack of both signaling pathways results in increased viral loads and disease severity. still, there is accumulating evidence that not only lack of an antiviral response but that also an unbalanced overshooting activation of ifns contributes to an exaggerated inflammatory reaction, tissue injury, reduced proliferative capacity, and thus enhanced disease severity ( table 1) . especially in viral infections, this effect has not only been tracked down to ifn signaling in general but specifically to the exaggerated production of key effector ifn-stimulated genes (isgs) (4) . a prominent example is the tnf-related apoptosis-inducing ligand (trail) that displays an ambivalent role in viral infection (5-7) ( table 1) . whereas first identified as factor produced by immune cells in non-respiratory infection (8, 9) , trail is now especially well studied in influenza a virus (iav) infection, where it is released in high amounts from bone marrow-derived macrophages upon pathogenassociated molecular patterns (pamps) recognition and type i ifn production (10) . macrophage-released soluble trail, but also membrane-bound cell-associated trail, acts via distinct receptors on infected but also on non-infected, neighboring cells. in viral infection, its preliminary role is to drive infected cells into apoptosis to limit virus spread. however, studies performed within the last decade demonstrate that trail's antiviral activity seems to be outweighed by the functional and structural damage it induces not only in infected but also in bystander cells such as uninfected cells of the alveolar epithelium (10, 11) . this process is not only relevant in promoting viral disease progression but has further implications in bacterial superinfection and probably also in chronic diseases. the recognition of the ambivalent role of ifn-driven signaling in vivo is a first important step to better understand disease progression and to envision novel treatment options for primary viral respiratory infection targeting distinct host-derived signaling mediators such as trail. it is a commonly accepted concept that-as janeway (12) already proposed in 1989-immune activation toward invading pathogens is mounted upon recognition of pamps. pamps are evolutionary conserved biomolecules such as proteins, lipids, nitrogen bases, sugars, and complexed biomolecules such as lipoglycans that are essential to the survival of a given pathogen (13) . pamps are recognized by distinct pattern recognition receptors (prrs) that are germ-line encoded and-similar to pamps-usually show a high evolutionary conservation. the first recognized and probably most intensely studied family of prrs are the toll-like receptors [tlrs; reviewed in mogensen (14) ; leifer and medvedev (15) ]. in viral infection, both host cell membrane-localized tlrs (tlr2, tlr4, detecting viral envelope proteins) and endosomal tlrs (tlr3, tlr7, tlr8, and tlr9 , nucleic acid sensors) initiate signal transduction cascades leading to ifn production (figure 1 ). tlr activation results in either myeloid differentiation factor 88 (myd88) or tir-domaincontaining adaptor protein-inducing ifn-β (trif) recruitment that both trigger various downstream signaling events, eventually leading to ifn regulatory factor (irf)3, irf7, and nfκb nuclear translocation as well as map kinase and activator protein 1 (ap-1) activation (16, 17) . similar to endosomal tlrs, the cytosolic retinoic acidinducible gene (rig)-i-like receptors (rlrs) are specialized to recognize viral nucleic acid contents and are central prrs relevant to mount an antiviral response, providing resistance to most rna (e.g., orthomyxoviruses) and some dna (e.g., reoviruses) viruses [reviewed in ref. (18, 19) ]. both melanoma differentiation-associated gene 5 (mda-5) and rig-i recognize dsrna, 5′-triphosphate rna, or the synthetic analog to dsrna, polyi:c (20, 21) . both drive the dimerization of the mitochondriaassociated adaptor protein ifn-β promoter stimulation 1 (ips-1) (also named mavs, visa, cardif). a subsequently activated cascade including tradd (tnf receptor type 1-associated death domain protein), traf3 (tnf receptor-associated factor 3), and tank (traf family member-associated nf-κb activator) induces the phosphorylation of irf3 and irf7, resulting in type i ifn production (22) . the third rlr, lgp2, so far has primarily been implicated to regulate rig-i or mda-5 as a cofactor; however, a recent study by stone et al. (23) demonstrated a novel, non-redundant, and independent role of lgp2 in west nile virus infection. another class of prr, the nucleotide oligomerization domain (nod)-like receptors (nlrs), has mainly been implicated in bacterial recognition (24) , still several nlrs are activated as well upon virus infection. especially, nlrp3 is known to recognize rna of different viruses including hepatitis c virus, measles virus, influenza, and vesicular stomatitis virus (vsv) (25) (26) (27) (28) , resulting in inflammasome formation and caspase-1-dependent activation of il-1β and il-18 (29) (30) (31) . in addition, virus infections are sensed also by a structurally diverse group of viral rna and dna sensors residing in the cytoplasm. these include the cyclic gmp-amp synthase that synthesizes the second messenger cgamp. cgamp in turn activates stimulator of ifn genes (sting), tank-binding kinase 1, and irf3, triggering ifn production (32) (33) (34) . moreover, sting itself acts as a prr and has been implicated in dna virus recognition including hsv, adenovirus, vaccinia virus, and papilloma virus and in sensing of retroviral rna-dna hybrids (35) and rna viruses after being activated by rig-i (36, 37) . another cytosolic nucleic acid sensor, pkr, is well known for its phosphorylation of eukaryotic initiation factor 2 α (eif2α) in response to viral dsrna. phosphorylation of eif2α results in its deactivation, host translational shut-off, and the limitation of viral replication. of note, the pkr-eif2α-driven inhibition of protein synthesis can contribute to an ips-1-dependent ifn-β induction (38) . furthermore, pkr has been implicated in efficient type i ifn activation by tlr3 in response to dsrna (39) and can mediate-at least partially-activities of irf1 (40) . in addition to type i ifns, also type iii ifns exert antiviral activity and are widely expressed after viral recognition, being produced by most cell types including epithelial, endothelial, fibroblast, and polymorphonuclear cells [reviewed in ref. (41, 42) ]. like type i ifns, type iii ifns are induced in viral infection by the prr rig-i as well as tlr3 and tlr9 and rely on the activation of the same transcriptional activators, including irf3, irf7, and nfκb. these observations initially led to the conclusion that type i and type iii ifn comprised two completely redundant systems to induce isgs in response to pamp recognition. however, more recent data suggest distinct selection mechanisms for either type i or type iii ifn expression. as such, ips-1 specifically induces ifn-λ, but not type i ifn, when located at the peroxisomal membrane instead of the mitochondrial membrane in response to rig-i activation by reovirus, sendai virus, or dengue virus challenge (43) . interestingly, type iii ifn induction is largely independent toward ap-1 translocation, which facilitates an instantaneous induction of ifn-λ after viral recognition, highlighting it as an important immediate factor driving innate microbial defense mechanisms. release of ifns upon pathogen recognition is a highly conserved mechanism-found from teleost fish to insects and mammals-to prepare the surrounding cells as well as the host defense against the invading threat (44, 45). whereas often high-level ifn production relies on specialized sentinel cells such as macrophages or dendritic cells (dcs), mostly all cells of the multicellular organisms are able to respond to at least one type of ifn by expression of respective receptors. receptor binding then induces a signal transduction cascade relying on the janus kinase (jak) and signal transducer and activator of transcription (stat), which results in efficient transcription of a plethora of different isgs in infected as well as bystander cells (46, 47) . ifns engage a classical canonical signal transduction cascade employing jak/stat molecules after binding to their respective receptors. herein, type i ifns ligate to their common heterodimeric receptor consisting of the ifn-α receptor (ifnar)1 and ifnar2 subunits, whereas type iii ifns act via a interleukin-10 receptor 2 (il-10r2)/ifn-λ receptor 1 (ifnlr1) heterodimer that to date has been reported to be restricted in its expression to epithelial cells (48) . in type i ifn signaling, ifnar engagement leads to the activation of the receptor-associated protein tyrosine kinases jak1 and tyrosine kinase 2 followed by the recruitment or repositioning of already associated but elsewise latent cytoplasmatic transcription factors stat1 and stat2. consequently, stat1/stat2 are phosphorylated on conserved tyrosine residues, they disassemble, undergo conformational changes enabling their heterodimerization as well as the exposure of a nuclear localization sequence. subsequently, the stat1/stat2 heterodimer translocates into the nucleus where it interacts with the irf9 to form the trimeric ifn-stimulated factor 3 (isgf3). isgf3 binds cognate dna sequences, the ifn-stimulated response elements (isre), finally leading to isg induction. also type iii ifn interaction with the il-10r2/ifnlr1 receptor complex triggers stat1/stat2 heterodimerization, nuclear translocation, and isgf3 assembly (49) . interferon signaling results in the induction of isgs evoking different cellular responses against viral infection, both in infected as well as in non-infected cells, including direct antiviral, immune-modulatory, or cell death-inducing effects to enable an immediate and robust response to a pathogen challenge. many isgs directly interfere with viral replication on an intracellular level. well-studied examples of antiviral isgs comprise ifn-induced transmembrane proteins (ifitms) effective in iav, west nile virus, and dengue virus infection (50, 51) , the myxovirus resistance protein a (mxa) that interferes with vsv mrna production and binds the iav nucleocapsid to prevent nuclear translocation of viral genetic material (52) (53) (54) , the 2-5-oligoadenylate synthase (oas), which activates rnase l triggering viral rna degradation, or the prr pkr, which besides activating the ifn response has a major impact on viral protein translation by inhibiting the eif2α (55) . more recently identified isgs include the plasminogen activator inhibitor 1 that blocks iav infection by inhibiting glycoprotein cleavage executed by extracellular airway proteases (56) or the antiviral isg20 that limits iav viral replication via its exonuclease activity most likely by interfering with the viral np (57) . accordingly, ifn pretreatment usually results in the establishment of an antiviral state that limits viral replication and spread from the start of infection and thus favors milder disease outcomes. ifn-α pretreatment has been demonstrated to limit viral spreading of seasonal iav strains and thus decrease morbidity and mortality in mice, guinea pigs as well as ferrets (58) (59) (60) . as shown by a study by tumpey et al. (61) , this effect can be attributed to the early induction of antiviral isgs including mxa. importantly, type i ifn pretreatment also dampens early replication of highly pathogenic avian influenza in ferrets (58) . also in respiratory syncytial virus (rsv) infection, treatment with recombinant ifn-α results in significantly decreased lung viral titers, alveolar inflammatory cell accumulation, and clinical disease in rsv-infected mice (62) . in addition, respiratory infections caused by emerging coronaviruses (cov) can be ameliorated by type i ifn pretreatment strategies. in an in vivo macaque model (macaca fascicularis) of severe acute respiratory syndrome (sars)-cov infection, it could be demonstrated that pretreatment with pegylated ifn-α significantly diminished cov replication and excretion and resulted in reduced pulmonary damage (63) . macaques also serve as a preclinical model for middle east respiratory syndrome (mers)-cov, and similar to sars-cov, ifn-α in combination therapy with ribavirin reduces viral replication and severe histopathological changes (64) . in line, genetic alteration leading to an enhanced type i ifn signaling has been demonstrated to limit iav-induced disease outcomes, as a recent study by xing et al. (65) reported that deletion of trim29, a negative regulator of nemo, which leads to nfκb induction and therefore enhanced type i ifn production, is protective in vivo in iav-infected mice. conversely, the genetic depletion of ifn signaling in ifn receptor-deficient mice can result in a lack of viral control, resulting in enhanced viral titers in different viral infections including rsv or iav (66, 67) . still, it must be noted that this effect is often mild in ifnar-or ifnlr-deficient animals, which is probably related to a certain redundancy between type i and type iii ifn signaling in limiting viral spreading in epithelial cells (68) . in contrast, ifnar/ ifnlr-double knockout or stat1 knockout animals that are deficient in both type i and type iii ifn signal transduction succumb more readily to infection due to excessive viral replication (69) (70) (71) . vice versa, mutations in key isgs such as ifitm3 are associated with increased iav disease severity in mice and humans (72) . however, ifn pretreatment and genetic loss-of-function approaches generally are not relevant to human respiratory virus-induced hospitalizations, where patients already present with ongoing respiratory infection and inflammation, and preclinical studies underline that type i ifn signaling in an already inflamed organ is rather detrimental and enhances tissue injury, and lack of type i ifn in vivo may even ameliorate disease outcome. accordingly, in cases where the antiviral defense was not compromised (e.g., in animals with efficient type iii ifn signaling) ifnar-deficient mice infected with sendai virus or iav were reported to be more resistant to infection-induced morbidity and mortality (73, 74) . similarly, in sendai virus in vivo infection, wetzel et al. (75) showed that increased ifn-β levels in the lung homogenate correlates to increased morbidity and mortality, and also for sars-cov, a recent study demonstrates that high type i ifn induction in an already ongoing viral infection contributes to mortality in sars-cov-infected mice (76) . also for iav infection, type i ifn application after infection has been proven to drive disease severity (74) . of note, the detrimental effects of type i ifns were especially pronounced in mice lacking central antiviral factors, namely the ifit protein in sendai virus infection and mxa in iav. interestingly, beilharz et al. (77) demonstrated that application of low doses of ifn-α reduces viral load, which to a certain degree led to attenuated disease progression, whereas high dose application of type i ifn contributed to morbidity (77) . in line, high expression levels of isgs have been shown to correlate to worse outcomes in ards patients (78) . this observation corresponds to reports stating that the ifn threshold needed to induce antiviral isgs-showing a beneficial effect in acute respiratory viral infection-is by at least 10-fold lower than the ifn dose necessary to trigger isgs that show immunomodulatory, death-inducing, or anti-proliferative effects and thus can contribute to disease progression (79) (80) (81) (82) . altogether, these data demonstrate that ifns may significantly contribute to unbalanced inflammation and tissue injury during respiratory viral infection depending on expression levels and duration of ifn-related signaling events. to date, the underlying mechanisms leading to the ifndependent enhanced disease progression are not fully understood but often result from a dysregulated ifn signaling response. one mode of action of ifn and ifn-stimulated isgs is to stimulate negative feedback loops on ifn signaling. for example, suppression of jak1 or stat1 via specific phosphatases, expression of suppressor of cytokine signaling (socs)1 and socs3, or ubiquitination and endocytosis of the ifn receptors (83) (84) (85) (86) desensitize cells to ifn signaling and allow recovery and the return to homeostasis after microbial challenge. as demonstrated by bhattacharya et al. (87) , the lack of ifnar downregulation and thus the failure to initiate ifn-desensitization contributes to increased inflammatory signaling, extensive lung injury and, importantly, also impaired tissue regeneration (87) . moreover, ifns are immunomodulatory and shape the specific responses of cells of the immune system, which has been implied to influence disease progression both positively and negatively. in a recent study, type i ifns have been associated in the regulation of innate lymphoid immune cells (ilc)2 in iav infection, where they-in concert with ifn-γ and il-27-promote an ilc2-dependent restriction of immunopathology (88) . moreover, type i ifns play an important role in stimulating the immune response driven by dcs; they stimulate the expression of mhc molecules as well as the co-stimulatory ligands cd80 and cd86 and thus activate t cell responses (89, 90) . additionally, ligand-driven activation of ifnar enhances the proliferation of cd8 positive t cells, especially early in infection. however, late in infection, type i ifns were also implied in decreasing t cell expansion upon sars-cov and arenavirus infection (76, 91) , which might potentially be related to the above described desensitization upon prolonged ifn signaling and might be detrimental if initiated too early in infection. in line, pinto et al. (92) reported an impairment of t cell responses upon type ifn induction in west nile virus infection. in b cells, the lack of ifnar has been demonstrated to result in enhanced release of neutralizing antibodies in iav infection (93) implying a repressive role for type i ifn in b cell antibody production. however, immunization studies by le bon et al. (94) reported the necessity of ifnar on b cells for efficient igm and igg production, underlining the need for further studies to understand the detailed effects of ifn-dosage and timing adaptive immunity activity upon respiratory viral infection. type i ifns additionally induce the production of high levels of pro-inflammatory cytokines that have been closely linked to worsened outcomes of acute respiratory viral infection. especially in iav, disease severity and disease progression are linked with an overshooting, ifn-driven inflammatory response, in which further exogenous supplementation with type i ifn in fact correlates with increased morbidity and mortality (74, 95) . in non-human primates, iav infection with a highly pathogenic h5n1 isolate evokes a strong induction of type i ifn, resulting in severe lung injury by a necrotizing bronchiolitis and alveolotis (96) . ifn levels in turn have been demonstrated to cause elevated pro-inflammatory cytokine levels after in vivo iav infection and additionally, in human alveolar macrophages, the release of pro-inflammatory cytokines (e.g., mcp-1) are preceded by a robust type i ifn response (97) . importantly, also in human infection with h5n1, levels of pro-inflammatory cytokines are strongly elevated in bronchoalveolar lavage fluid, and cytokine levels have been associated with organ damage and worsened disease outcomes (98, 99) . still, it should be noted that due to strain differences in virus-elicited prr activation and, importantly, ifn antagonism by the iav non-structural (ns)1 protein, ifn levels and disease severity do not always directly correlate; actually, the extent to which ns1 can suppress the ifn response relates to prolonged viremia and thus can also be a determinant of virus pathogenicity both in human bronchial epithelial cells and in an in vivo model of iav infection (100, 101) . alongside iav, also in rsv infection the induction of high levels of pro-inflammatory cytokines has been directly related to type ifn, as rsv-infected but ifnar-deficient mice presented with significantly diminished pro-inflammatory cytokine release, which translated into an attenuated disease course (67) . also in sars-cov, the late phase type i ifn induction relates to accumulation of inflammatory macrophage populations and elevated lung cytokine levels (76) . in addition to antiviral, immunomodulatory, and pro-inflammatory isgs, ifn signaling results in the transcription and translation of cell death-inducing isgs. in the context of viral infection, these factors provide a mode to block viral spreading and reinfection by killing those infected cells, in which the internal activation of antiviral isgs is not sufficient to restrict viral replication. thus, the infected cell is sacrificed to prevent the release of infectious progeny virions to limit viral spreading. however, especially in the lung, the disruption of the alveolar epithelial barrier by cell death of infected cells, but importantly also non-infected bystander cells induced by factors such as trail, significantly contributes to worsened disease outcomes. controlled cell death or apoptosis can be induced by intrinsic and extrinsic signals. the intrinsic apoptosis pathway is initiated by diverse intracellular stimuli that influence the expression and activation of b cell lymphoma (bcl)-2 family proteins that govern the permeabilization status of the outer mitochondrial membrane. once cytochrome c is released from the mitochondria, it binds to the intracellular adaptor protein, apoptotic peptidase activating factor 1, forming the so-called apoptosome that in turn recruits pro-caspase-9 (102). caspases (cysteine-aspartic proteases) exert their action by cleaving other proteins and substrates. herein, initiator caspases such as caspase-8 and caspase-9 target other downstream caspases, whereas effector caspases, including caspase-3, -6, and -7, directly cause apoptosis by cleaving and thus inactivating or disassembling a vast array of cellular integral proteins and complexes (103) . the extrinsic apoptosis pathway relies on an extracellular signal exerted by ligands of the tumor necrosis factor (tnf) receptor (tnfr) superfamily, including trail, tnf-α, and fas ligand (fasl) (104) . their ligation to their respective cell surface-expressed death receptors (dr) leads via the signal transmission by fas-associated protein with death domain (fadd) to the activation of the initiator caspases-8 or -10, finally stimulating effector caspases including caspase-3 (105) . to date, several type i and type iii ifn-induced, proapoptotic factors have been identified (106) . both caspase-4 and caspase-8 have been shown to be upregulated upon type i ifn signaling (4, 107); caspase-8 enhances the fadd-driven extrinsic apoptosis pathway, whereas the less-studied caspase-4 may promote pro-il-1β cleavage and inflammasome-driven cell death (pyroptosis) in macrophages (108, 109) . chattopadhyay et al. (110) demonstrated that sendai virus infection and polyi:c treatment resulted in bcl-2-associated x protein (bax) activation and apoptosis induction via one of the key transcription factors of ifn genes, irf3. in addition irf5 was reported to enhance trail-dependent extrinsic apoptosis by nuclear translocation resulting in the translation of to date undefined factors that increase cell death upstream of caspase-8 activation (111) . furthermore, both rlrs, rig-i and mda-5, trigger the proteins puma and noxa that induce bcl and the ifn/trail signaling axis frontiers in immunology | www.frontiersin.org march 2017 | volume 8 | article 313 thus activate the intrinsic mitochondrial apoptotic cascade (112) . also, pkr influences a cell's susceptibility to apoptotic signals, as it was demonstrated to sensitize to the fadd/caspase-8 apoptosis pathway upon type i ifn signaling after challenge with iav or dsrna (4) and the oas-rnasel system has been suggested to contribute to ifn-α-related cell death induction, but the exact mechanisms remain to be elucidated (113) . finally, also two classical initiators of the extrinsic apoptosis cascade are induced as isgs. both fasl and its receptor fas are upregulated on mrna levels by ifn-α (114) , and fasl was reported to be induced by type i ifn in iav infection in the murine lung in vivo (115) . also, the proapoptotic factor trail (or tnfsf10, apo2l) is induced by ifn-mediated and isgf3-executed transcriptional activation, as has been shown by sato et al. (116) , who revealed the presence of the isre sequence within the trail promoter region (116) . in iav infection, trail is released in high amounts from infected alveolar macrophages depending on a pkr-and ifn-β-driven autocrine signaling loop. binding of ifn-β to macrophageexpressed ifnar activates a jak/stat-dependent release of trail, which then acts through its receptor dr5 on the alveolar epithelial cells (5, 10) . however, certain prerequisites may decrease the ability of a cell to undergo apoptosis, including a shortage in pro-caspase-8 availability, expression of cellular fadd-like il-1β-converting enzyme-inhibitory proteins (c-flips) that block fadd-driven caspase activation, inactivation, or degradation of fadd itself, or expression of cyld, which acts as a receptor-interacting serine/threonine-protein (rip)1 kinase de-ubiquitinase and thus stabilizes rip1. however, in these cases ifn signaling can still promote a caspase-independent, programmed inflammatory cell death by activating the necroptosis pathway (117, 118) . necroptosis is induced by a complex formation by rip1 and rip3 kinases that activate both poly-adp-ribose (par) polymerase 1 (parp-1) and/or mixed lineage kinase domain-like (mlkl), leading to atp depletion, calpain activation, par polymer accumulation or cell membrane permeabilization, and release of damage-associated molecular patterns, respectively [reviewed in ref. (119, 120) ]. both a type i ifn-dependent jak/stat-driven activation of pkr as well as signaling by the prr dai (dnadependent activator of irfs) initiates necroptosis via rip1/rip3 activation, respectively (117, 121) . importantly, the activation of proapoptotic and pro-necroptotic pathways in respiratory infection can result in a structural disruption of the airway and the alveolar epithelial barrier, which is a major hallmark of respiratory disease and its progression to the acute respiratory distress syndrome (122, 123) . in virusinduced lung injury, especially expression of trail, which can initiate both apoptosis as well as necroptosis has been correlated with more severe outcomes. as described earlier, trail belongs to the superfamily of tnf ligands and has been reported to be inducible by both type i and type iii ifns. trail has been found to be present in various cells of the immune system, among them natural killer (nk) cells, t cells, nk t cells, dc subsets such as ifn-γ-producing killer dcs and macrophages, and can be displayed in large amounts on the cell surface or be shed upon ifn-and/or pro-inflammatory cytokine signaling (124) (125) (126) . in addition to cells of the immune system, fibroblasts have been shown to produce trail after ifnγ treatment or viral challenge. also, club cells and the alveolar epithelium have been reported to produce trail (127) (128) (129) (130) . similar to other ligands of the tnf superfamily, trail is a homotrimeric type ii transmembrane protein with a conserved c-terminal extracellular domain that mediates receptor binding and can be cleaved by metalloproteinases to generate a soluble mediator (131) . however, trail can induce cell death also in its membrane-bound form, that is, similar to trail expression levels and trail shedding, upregulated by type i ifn (126) . direct cell-to-cell trail-dr interactions have been demonstrated to play a role in macrophage, nk as well as cd4 + t cell-mediated induction of cellular death (132, 133) . in humans, five different binding partners for trail are present: the membrane-bound dr4 (trail-r1) and dr5 (trail-r2) that both induce a proapoptotic signaling cascade, the membrane-bound anti-apoptotic decoy receptors (dcr)1 and dcr2, and the soluble interaction partner osteoprotegerin (134) . in the murine system, only dr5 has been identified to ligate to trail (135) . in the human respiratory compartment, both dr4 and dr5 have been demonstrated to be present under steady-state conditions (136, 137) . however, upon viral infection, cell-sensitivity to trail-induced apoptosis is enhanced, which has been attributed to increased trail receptor expression especially on infected cells, as dr levels are markedly increased in iav-, adenovirus-, and paramyxovirus-infected cells in contrast to non-infected bystander cells (10, 138, 139) . of note, studies on the dependency of dr upregulation upon type i ifn signaling after iav infection have yielded conflicting results in different strains of mice (10, 74) , highlighting the complex interplay of ifn-induced cascades in a host-and tissue-specific context, whereas the exact virus-and host-specific mechanisms for dr regulation remain less well defined. moreover, previous assumptions that also dcr expression would correlate with cell-sensitivity to trail-induced cell death could not be experimentally verified (125) . tumor necrosis factor-related apoptosis-inducing ligand ligation to the proapoptotic receptors dr4 or dr5 triggers a trimerization of the receptors. subsequently, depending on additional stimuli, presence or absence of adaptor molecules or inhibitory proteins, different signaling pathways can be activated (figure 2) . in the classical trail-dependent extrinsic apoptosis induction, the proteins rip, tradd, and fadd are subsequently recruited to the dr cytoplasmic domain upon trail ligation (140, 141) . these factors and the proapoptotic drs all share a cytoplasmic death domain (dd), which is lacking or truncated and thus inactive in the dcr. the dd plays a central role in the concerted formation of the death-inducing signaling complex (disc). disc formation exposes a second functional domain of fadd, the death effector domain that is directly able to recruit pro-caspase-8 figure 2 | trail/dr5-mediated cellular signaling pathways. in presence of rip1, tradd, and fadd, trail ligation to dr5 results in apoptosis induction, which is initiated by recruitment of the pro-caspase-8 or -10 to fadd. these in turn activate the effector caspases-3 and -7, which leads to dna fragmentation and apoptosis induction. in addition, tradd can trigger a traf2-and jnk-dependent activation of bax and subsequent release of mitochondrial cytochrome c, inducing the pro-caspase-9 activation. in the presence of cyld, c-flip or absence of sufficient amounts of fadd or pro-caspase-8, trail ligation to dr triggers the interaction of rip1 and rip3 kinase, which in turn cause cell death via induction of mlkl and/or parp-1. in the presence of ciaps, fadd is not recruited to dr5 upon trail ligation, and tak1 is activated by tradd/traf2 interactions. tak1 induces nemo followed by iκb degradation and nfκb activation, as well as mkk and jnk activation leading to ap-1 nuclear translocation; both events promote the production of cytoprotective factors such as xiap, ciaps, and c-flip. additionally, tak1 triggers ampk activation and thus mtorc inhibition, which results in enhanced autophagic activity. abbreviations: ap-1, activator protein 1; tnf, tumor necrosis factor; trail, tnf-related apoptosis-inducing ligand; dr5, death receptor 5; rip1, receptor-interacting serine/threonine-protein kinase 1; tradd, tnf receptor type 1-associated death protein; fadd, fas-associated protein with death domain; traf, tnf receptor-associated protein; jnk, janus kinase; bax, bcl-2-associated x protein; c-flip, cellular fadd-like il-1β-converting enzyme-inhibitory proteins; rip, receptor-interacting serine/threonine-protein; mlkl, mixed lineage kinase domain-like; parp-1, poly-adp-ribose (par) polymerase 1; ciap, cytoprotective factors including inhibition of the autophagic machinery; xiap, x-linked inhibitor of apoptosis protein; ampk, amp-activated protein kinase; mtorc, mammalian target of rapamycin complex; traf2, tnf receptor-associated protein 2; mkk, mitogen-activated protein kinase. and pro-caspase-10. how exactly disc formation induces caspase activation is still under debate. the most probable scenarios include either an autocatalytic cleavage of caspase-pro-domains enabled by the spatial proximity between pro-caspases (generated by their recruitment to disc), by pro-caspase dimerization, or by pro-caspase conformational stabilization (125) . removal of the pro-domain of caspase-8 and caspase-10 results in the activation of the effector caspases-3 and -7, which cleave dna fragmentation factor 45 and lead to apoptosis (142, 143) . moreover, trailbinding to dr4 and dr5 can induce the jnk either via caspase-8 or recruitment of tnf receptor-associated protein 2 (traf2) to the disc complex, which results in the activation of the intrinsic apoptotic cascade by bax-dependent mitochondrial cytochrome c release (144) . in addition, trail signaling is also able to induce necroptosis by both activating the rip1/rip3 kinase downstream effectors parp-1 and mlkl, contributing to epithelial cell death and tissue injury (145) (146) (147) . it has become apparent in recent years that trail signaling is closely linked to induction of autophagy, a process generally associated with the blockade of apoptosis and necrosis. indeed, autophagy has been reported to improve cellular survival in cell stress by catabolic removal of cytoplasmic long-lived proteins and damaged organelles. it also contributes to viral clearance and the transfer of viral material to endosomal-/lysosomallocated tlr7 or mhc class ii compartments for the activation of adaptive immunity (148). several studies outline that trail ligation to dr5 can result in a traf2-dependent activation of tak1 (map3k7) that has been attributed a central role in trail-induced autophagy activation (149) . tak1 modulates the ikk-dependent translocation of nfκb, and it also induces jnk activation via mitogen-activated protein kinase. both events lead to expression of autophagy-related factors including inhibition of the autophagic machinery (ciap)1, ciap2, x-linked inhibitor of apoptosis protein, and c-flip (150, 151) . especially, c-flip has been associated with desensitization of cells to trail-induced apoptosis, favoring autophagy-related cascades (152) . another study revealed that upon trail signaling the amp-activated protein kinase (ampk) is activated. ampk in turn inhibits the mammalian target of rapamycin complex 1 that itself is an inhibitor of autophagy, thus the activation of the autophagic machinery is promoted (153) . the decision if trail signaling results rather in necroptotic or apoptotic cell death or in activation of autophagy seems to be dependent on the presence of ciaps that promote rip kinase ubiquitination and degradation (146) , but also on the balance between active caspases and autophagic proteins such as beclin-1 (154, 155) . this suggests a scenario where autophagy is activated as cell protective mechanism until cell stress-as executed by enhanced trail signaling or additional viral infection-increases over a threshold to favor cell death induction. accordingly, as trail signaling is not restricted to infected cells, excessive cell death activation might be limited by autophagy induction in non-infected bystander cells. however, autophagy is not only related to cell survival but can also positively affect apoptosis and induce-even if the exact mechanisms are still under debate-autosis, the autophagy-related cell death, another mode of trail to trigger cell death (156, 157) . of note, autophagy activation needs to be placed into its virus-specific context, as some viruses, including dengue virus, poliovirus, and coxsackie b virus (158) , can exploit autophagic pathways for their own replication and thus promote apoptosis and tissue injury. as discussed above, trail is a potent activator of cell death. however, its signaling outcomes can differ largely depending on its delivered form (e.g., membrane-bound versus soluble), the availability of drs on the target cell membrane, alternate intracellular pathways that might be activated and finally the pathogen itself, as it might exploit trail-induced pathways for its own survival and replication. in acute respiratory infection, trail signaling is often part of an ifn-driven overshooting inflammatory reaction that promotes unspecific tissue injury and thus disease severity by increasing functional and structural changes in infected but also non-infected cells, as will be outlined below. the release and effects of trail have been especially well studied in iav infection in the last decade. earlier studies reported that within 3 days after infection, bronchial, bronchiolar, and alveolar epithelial cells undergo apoptosis (159) . this early induction of cell death is mainly attributed to direct apoptosis induction by the virus itself, as iav actively promotes apoptosis for efficient viral replication (160) . herein, the viral ns1 and pb-f2 proteins not only play a crucial role (161, 162) but also the viral m2 protein has been implicated in this process as it inhibits autophagy in infected cells (163) . in addition, our own data revealed that later in iav in vivo infection, the recruitment of bone marrowderived macrophages via the cc chemokine receptor type 2 (ccr2)-cc-chemokine ligand 2 (ccl2) axis significantly contributes to alveolar cell apoptosis and structural damage of the alveolar epithelium (5) . studies by wurzer et al. (164) had previously demonstrated that iav promotes the production of proapoptotic factors in an auto-and paracrine fashion via nfκb transcriptional activation by iav (164) . subsequently, brincks et al. (6) elucidated that human peripheral blood mononuclear cell treated with iav released trail and that increased trail levels correlated with type i as well as ii ifn induction. additionally, trail sensitivity was increased in influenza virusinfected cells. in line, our investigations could elucidate that iav triggers a pkr-dependent translocation of nfκb that results the production of type i ifns. these in turn induce, via ligation to the ifnar receptor complex, expression and shedding of trail by bone marrow-derived macrophages (10) . in addition, davidson et al. (74) demonstrated that type i ifn application to iavinfected mice increased morbidity and lung injury, which could be attributed to both dr5 and trail upregulation inducing epithelial cell apoptosis. importantly, högner et al. also reported that the iav strain used in these studies, a/pr8 (h1n1), which is highly pathogenic for mice, induced an approximately 800-fold induction in macrophage trail expression, whereas the lower pathogenic virus a/x-31 (h3n2) only stimulated trail by a factor of eight. of note, the relation between trail induction and iav strain-specific pathogenicity also translates to the highly pathogenic avian h5n1 iav, causing severe pneumonia in mice as well as in humans (165, 166) . moreover, human infection with both the highly pathogenic h5n1 as well as the pandemic 1918 h1n1 iav strains are characterized by a massive influx of mononuclear phagocytes into the alveoli, which is correlated with extensive alveolar epithelial cell apoptosis (97, 167) . additionally, macrophages gained from bronchoalveolar lavages of patients presenting with ards caused by the pandemic h1n1/2009 virus strain showed high surface expression and release of trail (10) . another recent report demonstrates that in highly pathogenic avian influenza, in addition to macrophages also the alveolar epithelium might be involved in causing elevated levels of trail in the alveolar space (130) . besides its role in apoptosis, trail signaling upon iav infection has also been implicated in the induction of necroptosis in fibroblasts, dcs, and lung epithelial cells (146, 147, 168) . rodrigue-gervais et al. (146) demonstrated that lack of cipa2 promotes rip3 kinase-mediated necroptosis in response to trail-but also the proapoptotic factor faslreleased from hematopoietic cells. this contributed to severe lung epithelial degeneration and increased mortality, even though viral control was not compromised. nogusa et al. (147) further elucidated that iav-induced necroptosis depends on rip3 kinase activation of mlkl, and that rip3 kinase deficiency, similar to ciap2-deficiency, increased iav-susceptibility in vivo. in iav infection, as mentioned earlier, dr5 expression is elevated on infected alveolar epithelial cells, but not in noninfected cells in vivo, which might impact on trail susceptibility to apoptosis induction (10) . however, both infected as well as neighboring bystander cells were found to be targeted for apoptosis induction by macrophage-released trail. nonetheless, we could recently show that specifically in noninfected cells within the iav-infected lung, trail severely compromises the function of the ion channel na,k-atpase, which was mediated via induction of the stress kinase ampk (11) , thereby potentially revealing a cross-link to trailinduced autophagic cell stress pathways in bystander cells both in vitro and in vivo. the trail-induced and ampk-mediated downregulation of the na,k-atpase, a major driver of vertical ion and fluid transport from the alveolar airspace toward the interstitium, resulted in a reduced capacity of iav-infected mice to clear excessive fluid from the alveoli. thus, trail signaling contributes to intensive edema formation, a hallmark of disease in virus-induced ards (123) . notably, this effect of trail on na,k-atpase expression was induced independently of cell death pathways elicited by caspases, as treatment of cells and mice with a specific caspase-3 inhibitor diminished apoptosis in alveolar epithelial cells but still allowed for the reduction of the na,k-atpase (11) . conclusively, treatment of iav-infected mice with neutralizing antibodies directed against trail or the abrogation of recruitment of trail + bone marrow-derived macrophages inhibited apoptosis of both non-infected and bystander cells. thus, lung leakage due to loss of alveolar barrier function was reduced, whereas alveolar fluid clearance capacity was enhanced, resulting in reduced edema, improved survival, and outcome upon iav challenge in vivo. however, trail has also been shown to be upregulated on nk, dc, and on cd4 + and cd8 + t cells after iav infection (169) . studies by brincks et al. demonstrated that especially cd8 + t involved in cytotoxic t cell responses toward iav and drive iav-infected cells into apoptosis via trail, thus contributing to efficient virus clearance (6, 170) . in addition, both fasl and trail are involved in dc-mediated ctl activation and cytotoxicity against iav-infected cells (6, 171) . furthermore, studies showed delayed viral clearance upon neutralizing anti-trail antibody administration (169, 172) . our data, however, demonstrate that the transfer of trail-deficient bone marrow into irradiated wild-type mice, resulting in loss of trail production by bone marrow-derived macrophages upon iav infection, does not impact on the capacity to fully clear viral particles from the lung at day 7 after infection, suggesting that other compensatory mechanisms are recruited to guarantee viral clearance (10) . taken together, in iav infection, trail acts both as an important mediator of infected cell killing but particularly as a detrimental factor contributing to tissue injury and impaired inflammation resolution when released in excessive amounts by recruited immune cells. respiratory syncytial virus is an important cause of respiratory tract infections especially in children worldwide. generally, there seem to be virus-elicited anti-apoptotic mechanisms active in the lung epithelium, as rsv-infected primary human airway cells show a minimal cytopathic effect (173) . however, several cell lines including small airway cells, primary tracheal-bronchial cells, and a549 and hep-2 showed increased expression of trail and its ligands dr4 and dr5 in an in vitro rsv infection model (174) . moreover, soluble trail released from leukocytes was elevated in the bronchoalveolar lavage fluid of patients with rsv-associated respiratory failure, suggesting that similar to iav, trail contributes to rsv-induced epithelial injury and disease progression (137) . also in cov respiratory tract infection, trail levels, but less so fasl, have been reported to be markedly elevated (175, 176) . for sars-cov that presents with a severe damage to both the upper and lower respiratory tract (177) , especially dcs respond with a strong induction of trail production, which was suggested to correlate to increased cellular lung infiltrations present in sars-cov patients (175) . interestingly, sars-cov infection drives cells into apoptosis by a pkr-driven but eif2α-independent pathway (178) , which might-similarly as seen in iav infection-suggest a pkr-induced and autocrine/paracrine executed activation of apoptosis. also mers-cov, which causes pneumonia and respiratory failure, has been demonstrated to induce profound cell death within 24 h of infection, irrespective of viral titers produced by the infected cells. however, type i ifn expression is strongly reduced in mers-cov in comparison to seasonal human cov in in vitro infection models, including human monocyte-derived macrophages, calu-3, and human lung fibroblasts (179, 180) , which might also dampen downstream trail induction. therefore, the exact mechanism by which mers-cov promotes cell death remains to be investigated. recurrently, viral infections of the respiratory tract are followed by outgrowth of colonizing gram-positive bacteria that aggravates the course of illness. this is well documented for iav, where "super" infections with streptococcus pneumoniae and staphylococcus aureus are the most frequent and increase viral pneumonia-associated morbidity and mortality (181) . during the 1918 iav pandemic, bacterial pneumonia was evident in most cases (182) and also during the recent 2009 h1n1 pandemic, coinfections were a relevant factor for severe disease in a young patient population without comorbidities (183) . interestingly, virus-induced elevation of the type i ifn response levels might promote secondary bacterial outgrowth by several mechanisms [reviewed in ref. (184) ]. in line, it has been repeatedly demonstrated that lack of type i ifn signaling results in better bacterial clearance and increased survival rates in iav-and s. pneumoniae-superinfected mice (185) (186) (187) . herein, ifn-induced apoptosis induction as well as depletion or impaired recruitment of lymphocyte subsets necessary for bacterial control play a critical role (188, 189) . bacterial clearance from the lung has been reported to rely on sufficient phagocyte generation, recruitment, and survival. type i ifn has been demonstrated to cause apoptosis in bone marrow-derived granulocytes, affecting the numbers of recruited neutrophils (189) , but also to impair expression of the cytokines cxcl1 (or kc) and cxcl2 (or mip-2), thus inhibiting neutrophil recruitment to the lungs with severe effects on survival of superinfected mice (185) . a recent report by schliehe et al. (190) elucidated the mechanistic background for impaired cxcl1 expression and secretion and demonstrated that type i ifns activate the histone methyltransferase setdb2, which in turn represses the cxcl1 promoter and thus impairs neutrophil recruitment and bacterial clearance. moreover, type i ifn production decreases ccl2 production, thus inhibiting macrophage recruitment, which as well has been reported to have detrimental effects on bacterial clearance and disease progression in bacterial superinfection after viral insult in vivo (186) . in addition, type i ifns also impair γδ t cell function and il-17 release, which was shown to increase susceptibility to s. pneumoniae superinfection after iav challenge (187) . also in s. aureus pneumonia, a robust type i ifn response is correlated to excessive morbidity and tissue injury (191) . in a model of polyi:c, s. aureus (methicillinresistant strain, mrsa) superinfection, polyi:c treatment prior to bacterial infection enhanced type i ifn levels and decreased bacterial clearance and survival (192) . furthermore, shepardson et al. (193) demonstrated that late type i ifn induction rendered mice more susceptible to secondary bacterial pneumonia in a model of iav-mrsa superinfection. only limited data are available on a direct role of trail in respiratory disease progression due to bacterial superinfections. in a model of iav-haemophilus influenza infection, neither deficiency for cc chemokine receptor type 2, inhibiting bone marrow-derived macrophage recruitment, nor deficiency of fas or tnfr1 impacted outcome (194) . yet, during s. pneumoniae single infection, early cell death of macrophages is thought to limit an exuberant inflammatory reaction and accordingly, a study by steinwede et al. (195) revealed that neutrophil-derived trail limits tissue injury by inducing cell death in dr5-epressing lung macrophages in bacterial mono-infection (195) . in contrast, in the iav-s. pneumoniae superinfection mouse model, iavinduced trail has a detrimental effect on overall mortality (7), as trail-induced epithelial injury enhanced bacterial outgrowth of s. pneumoniae-administered at day 5 after iav infection-markedly. importantly, administration of anti-trail neutralizing antibodies enhanced bacterial control by the host organism. thus, the activation of ifn/trail-mediated signaling in viral infection has detrimental implication for outcome of secondary bacterial infection following viral insult, rendering the ifn/trail signaling axis an interesting therapeutic target not only in respiratory viral infections but also in complicating bacterial superinfection. an increasing number of reports connect progression of chronic respiratory disease to acute respiratory virus infection or proapoptotic signaling events. in fact, trail has been reported to be a critical determinant for promoting the development of chronic lung disease in early life (196) ; targeting trail by genetic deletion or neutralizing antibody application in early-life respiratory infections ameliorated infection-induced histopathology, inflammation, as well as emphysema-like alveolar enlargement and lung function. furthermore, trail was also shown to play a role in the development of allergy and asthma. trail is not only elevated in the sputum of asthmatic patients but has also been reported to be highly expressed in an experimental mouse model of asthma, where it induces ccl20 secretion by bronchial epithelial cells, thus promoting th2 cell responses and airway hyperreactivity (197) . in copd, acute exacerbations driven by viral and bacterial infection are a major factor increasing both mortality and morbidity, and both influenza and s. pneumoniae have been identified among the most common causes of copd exacerbations (198) . indeed, primary bronchial epithelial cells isolated from subjects with copd show an impaired production of type i ifn (199) , which has been implied in the enhanced susceptibility of copd patients to respiratory infections; however, even in absence of high ifn induction, both an abnormally elevated loss of alveolar epithelial cells due to apoptosis as well as elevated trail and dr5 levels were reported (200) , implying a possible link between viral/bacterial induction of trail and acute exacerbations in copd. trail induction has also been directly linked to cigarette-smoke exposure, a common cause of copd, and trail deficiency resulted in decreased pulmonary inflammation and emphysema-like alveolar enlargement in vivo (201) . moreover, increased levels of both trail and dr5 were associated to impaired lung function and increased systemic inflammation in human copd patients (202) . while alveolar epithelial cell death is closely connected to idiopathic pulmonary fibrosis (ipf), trail and its receptors dr4 and dr5 in aec were shown to be upregulated in ipf lungs (129) . also, in pulmonary arterial hypertension virus infection is considered to be a possible risk factor (203) , and pulmonary hypertension has been reported to be a side effect of prolonged treatment with type i ifn (204, 205) . in line, trail has been closely linked to disease progression in pulmonary hypertension. trail has been found to be increased within pulmonary vascular lesions of patients with pulmonary hypertension (206) and also in a mouse model of hypoxia-induced pulmonary hypertension, levels of soluble trail correlated with right ventricular systolic pressure, right ventricular hypertrophy, and pathologic alterations (33, 34) . importantly, neutralizing antibody-treatment against trail showed positive effects on survival while reducing pulmonary vascular remodeling (207) . notably, the extent to which infection-induced trail release causes or exacerbates chronic lung disease or in how far trail production in chronic lung diseases affects susceptibility to respiratory viral and complicating bacterial infection remains to be elucidated. respiratory viral infections are major causative agents for lung injury and ards; however, in many cases antivirals are not sufficient to limit disease (208). besides the fact that most viruses are subject to strong selective pressures that favor quickly evolving, drug-resistant virus variants, recent advances in understanding the processes that contribute to tissue injury and ards highlight a crucial role of immune-related, ifn-driven events. therefore, novel therapeutic strategies often aim to improve the outcome of severe respiratory infection by modulating host cell responses; however, to date, clinical trials trying to improve severe viral infections or ards outcomes by targeting host pathways have not resulted in approval of new drugs (122) . of note, for establishment of such therapies it has to be considered that the timing and intensity of induction and amplification as well as of dampening and termination of the ifn-driven immune response needs to precisely match the pathogen-and organ-specific requirements of a given infection. a non-controlled regulation of these processes may lead to either an unrestricted pathogen spreading or, on the other extreme, to an overshooting inflammatory response, including the increased production of pro-inflammatory and proapoptotic mediators, elevated levels of recruited immune cells, and/or aberrant repair processes. notably, both too low and too high levels of ifn-induced effects facilitate disease progression with a possible increase of fatal outcomes in ards patients (78) . accordingly, preclinical in vivo studies of ifn-directed therapies yielded seemingly adverse results, depending on the context, timing, and dosage of ifn modulation. however, in multiple settings of acute respiratory viral infection, studies demonstrate that an exaggerated signaling derived from type i ifn in an already inflamed tissue contributes to worsened outcomes, and importantly, might favor secondary bacterial superinfection [e.g., ref. (75, 76, 209) ]. interestingly, davidson et al. (209) demonstrated that type iii ifn release upon influenza challenge-in contrast to type i ifn induction-does not trigger an unbalanced inflammatory response that critically contributes to respiratory disease progression in vivo, highlighting it as a possible therapeutic option in iav-induced lung injury. most likely, this effect derives from the lack of the ifn-λr1/il-10r2 receptor complex, but presence of ifnar, on immune cells, including bone marrowderived macrophages. nonetheless, other reports identify ifn-λ as a driver of macrophage polarization to an inflammatory m1 phenotype (41) that has been attributed to further promote an overshooting inflammatory response, highlighting the need for further studies of type iii ifn biology in pathogen-associated disease progression. as generally ifn-directed therapeutic approaches target various downstream signaling events that might both act beneficially as well as detrimentally on viral replication and pathogenesis, a further approach is to address specific isgs that primarily show detrimental effects on disease progression. as outlined above, trail or its downstream signaling events might comprise a suitable target for adjunct therapies in addition to antivirals. accordingly, our own data in a preclinical mouse model of iav infection demonstrate a clear benefit of the systemic application of neutralizing antibodies against trail at days 3 and 5 postinfection for lung injury, morbidity, and mortality (10, 11) . targeting trail as a major determinant of disease severity in respiratory viral infections including iav, but also rsv and cov, may yield therapeutic approaches that are superior to ifn-directed strategies, as they seemingly do not bear the risk of compromising host defense. yet, it should be thoroughly excluded that blocking trail-induced cell death of infected cells will not lead to an overwhelming viral spreading, especially as reports on viral loads upon trail inhibition in preclinical models of iav are controversial (6, 10, 170) . accordingly, additional studies are needed to understand how and to which extent virus-infected cells can be killed or viral spreading can be controlled by other means in the absence of trail. moreover, targeting pathways and signaling hubs downstream of trail/drs, such as ampk (11), in a well-timed and lung compartment-specific way, may open new therapeutic avenues but requires more detailed preclinical studies on efficacies and side effects. a valid approach might be the use of a combination therapy of such a treatment together with a classical antiviral drug therapy limiting viral replication; however, exact dosage, timing, kinetics, and application routes remain to be defined. cp and sh performed bibliographic research and drafted the manuscript. this work was supported by the german research foundation (sfb-tr84 b2, sfb1021 c05, kfo309 p2/p8, exc147), by the german center for lung research (dzl), and by the german center for infection research (dzif). 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critical link between trail and ccl20 for the activation of th2 cells and the expression of allergic airway disease viral and bacterial etiologies in acute exacerbation of copd detected using a multiplex real-time polymerase chain reaction impaired antiviral stress granule and ifn-β enhanceosome formation enhances susceptibility to influenza infection in chronic obstructive pulmonary disease epithelium alveolar epithelial and endothelial cell apoptosis in emphysema: what we know and what we need to know a pathogenic role for tumor necrosis factor-related apoptosis-inducing ligand in chronic obstructive pulmonary disease increased serum trail and dr5 levels correlated with lung function and inflammation in stable copd patients viral infection and pulmonary hypertension: is there an association? irreversible pulmonary hypertension associated with the use of interferon alpha for chronic hepatitis c interferoninduced pulmonary hypertension the role of the osteoprotegerin/tumor necrosis factor related apoptosis-inducing ligand axis in the pathogenesis of pulmonary arterial hypertension inhibition of tumor necrosis factor-related apoptosis-inducing ligand (trail) reverses experimental pulmonary hypertension luks am. ventilatory strategies and supportive care in acute respiratory distress syndrome ifnλ is a potent anti-influenza therapeutic without the inflammatory side effects of ifnα treatment the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-353787-24c98ug8 authors: jackson, j. a. title: immunology in wild nonmodel rodents: an ecological context for studies of health and disease date: 2015-04-27 journal: parasite immunol doi: 10.1111/pim.12180 sha: doc_id: 353787 cord_uid: 24c98ug8 transcriptomic methods are set to revolutionize the study of the immune system in naturally occurring nonmodel organisms. with this in mind, the present article focuses on ways in which the use of ‘nonmodel’ rodents (not the familiar laboratory species) can advance studies into the classical, but ever relevant, epidemiologic triad of immune defence, infectious disease and environment. for example, naturally occurring rodents are an interesting system in which to study the environmental stimuli that drive the development and homeostasis of the immune system and, by extension, to identify where these stimuli are altered in anthropogenic environments leading to the formation of immunopathological phenotypes. measurement of immune expression may help define individual heterogeneity in infectious disease susceptibility and transmission and facilitate our understanding of infection dynamics and risk in the natural environment; furthermore, it may provide a means of surveillance that can filter individuals carrying previously unknown acute infections of potential ecological or zoonotic importance. finally, the study of immunology in wild animals may reveal interactions within the immune system and between immunity and other organismal traits that are not observable under restricted laboratory conditions. potentiating much of this is the possibility of combining gene expression profiles with analytical tools derived from ecology and systems biology to reverse engineer interaction networks between immune responses, other organismal traits and the environment (including symbiont exposures), revealing regulatory architecture. such holistic studies promise to link ecology, epidemiology and immunology in natural systems in a unified approach that can illuminate important problems relevant to human health and animal welfare and production. recent technological advances in the de novo sequencing and analysis of nucleic acids are revolutionizing the measurement of gene expression in nonmodel organisms, with promising applications in the study of the immune system (1) . although other phenotypic measurements of immunity remain relevant and useful, albeit limited in scope or technically difficult to apply in nonmodel organisms (2) , these advances mean that studying the immunology of such organisms in the natural environment has become easier and can take on a genomewide perspective embodied, for example in techniques such as rnaseq. this can, in turn, be accompanied by powerful analytical approaches derived from systems biology (3) and statistical methodologies applied in ecology. when these elements are combined with the monitoring of natural fluctuation or experimental perturbation, it opens up the possibility of 'reverse engineering' the regulatory architecture of the immune system and its interaction with other organismal traits and with natural environmental pressures (3) . such approaches, using natural systems, complement the strengths and weaknesses of modern immunology (4) . here, the great strengths are derived from the very refined use of inbred and genetically manipulated mice under controlled conditions that negate environmental variation. this is very successful for unpicking the structure of molecular pathways and workings of cellular populations, but relevance for natural environmental variation disappears where genetically unrepresentative individuals are studied under homogenous laboratory conditions and in the absence of a natural flora and fauna of symbionts (4, 5) . (here symbiont is defined as any organism involved in an intimate association with the host, including parasitic, commensal and mutualistic associations.) the present review will be concerned with how this 'blind spot' in modern immunology can be addressed by a focus on natural populations. it will scan the horizon for unique ways in which studies of nonmodel rodents can contribute to our wider understanding of the biology of the immune system and the way it interacts with the environment to determine health. additionally, it will consider how immunological measurement, interpreted in the light of paradigms from laboratory mouse immunology, can define individual variation relevant to ecological and epidemiological studies of infectious disease in the natural environment (1, 6) . rather than produce an exhaustive list of possible interests, though, this review will concentrate on three broad reasons to study immunology in naturally occurring vertebrate hosts, reasons that seem particularly exciting because they could have major practical implications for human health and the welfare and productivity of domesticated animals. each of these themes will be considered in turn and then the reasons why nonmodel rodents (species excluding mus musculus, m. domesticus and rattus norvegicus) may be particularly useful models. finally, selected case studies will be discussed that have begun to approach some of the issues raised. whilst the focus in these case studies is on the measurement of expression in selected candidate genes, they also illustrate the potential for future work using broader transcriptomic approaches, such as rnaseq. much of the ill-health experienced in modern human populations is not caused directly by infectious agents but linked to aberrant inflammation (7) . thus, conditions including cancer (8) , diabetes (9) , asthma and allergies (10) and various neurological (11, 12) and psychiatric disorders (13) have, in part, immunological aetiologies. trends in these immune-based conditions have been, broadly, upward in 'westernized' human populations, the short time scale indicating the involvement of an environmental variable or variables (14) associated with 'modern' anthropogenic environments. in addition, the effects of environmental factors on individuals are likely to be modulated by genetic variation inherited from wild ancestral populations (15, 16) amplifying individual variability in propensity to disease. a major challenge facing biomedical science, then, is to pinpoint the environmental causes for this variance in health. in particular, given the observed epidemiological trends in immunologically based disease, the questions might be asked: what are the immunological changes that occur in the transition from natural to more anthropogenic environments, and what triggers these changes? several causal mechanisms have been considered, but to parasite immunologists one particularly influential and intuitive (but unproven) line of thought is that increased dysregulation of the immune system is ultimately caused by the host's co-adaptation to stimuli from co-evolved symbionts. these symbionts might include macroparasites and other agents of chronic infection that tend to be lost in anthropogenic environments (17) . this idea is embodied in what has been termed the 'hygiene' (5) or 'old friends' (13) hypothesis. surprisingly, modern immunology is not well placed to take up the challenge of identifying real-world environmental drivers of the immune phenotype. this is because the remarkable laboratory models that have been established for revealing the molecular details of immunological pathways are unsuited to studying how these pathways interact with complex environments under natural conditions. it has often been noted that the genetics of laboratory mice does not reflect the natural situation (4). typically, laboratory lineages are partly or completely inbred and often generated in a haphazard way that makes the range of allelic variation fixed in their genomes unrepresentative of natural variation (18) . in the case of the fully inbred (isogenic) lines, their genomewide homozygosity is itself highly unnatural. whilst these disadvantages would perhaps be overcome (19) by a range of carefully generated wild-derived inbred and outbred lines (20) , a much less tractable limitation lies in the inability to recreate natural environmental influences in the laboratory (5) . thus, wild animals experience a range of complex symbiont exposures and environmental stressors that cannot be sufficiently replicated in captivity (5) . as such, wild mammals (and especially wild nonmodel rodents, due to some of the advantages discussed below) would seem a natural starting point to approach the problem of identifying environmental stimuli that drive immunological development and homeostasis in the wild. as previously considered in more detail by friberg et al. (5) , progress could be made either through in situ studies in natural populations tracking the effects of environmental variables using manipulative experimental or observational approaches (see for example, the wood mouse case study below), or through transplantation of naturally occurring lineages to (and monitoring of the changes occurring in) experimentally manipulated anthropogenic environments. not all humans in modern environments develop immunologically based diseases (even though increasing numbers do), and those that succumb often have identifiable genetic predispositions. as noted above, causative environmental factors likely exert their effects upon a background of significant immunogenetic variability inherited from wild ancestral populations. the subject area of wild rodents as models for this immunogenetic variability was reviewed in detail by turner and paterson (15) and will only be considered here sufficiently to provide a general overview relevant to the present article. briefly, a parallel challenge to the one of identifying environmental factors driving immunopathological phenotypes in anthropogenic environments described above, then, is the one of revealing genetic variation that places individuals at risk (15) . in other words, finding genetic variation that natural selection has shaped in a way that, although adaptive in some natural settings, has the potential to be maladaptive in anthropogenic environments. such variation results from balancing selection, or from directional selection or genetic drift that has not proceeded to fixation, in ancestral populations. here, selective agents (for example, naturally occurring pathogens) that were present in the past, or neutral processes, may have driven into wild populations alleles that are broadly deleterious in novel anthropogenic environments. where potentially deleterious genes have been fixed, though, only variation due to environmental factors (see section above) or the wider genetic background is important. immunogenetic polymorphism driven by balancing or directional selection is thought to cause at least some of the variation underlying immunopathological conditions in modern humans (21) (22) (23) (24) (25) (26) and could, equally, be responsible for a great deal of natural variation in wild animals. although it is increasingly recognized that adaptive evolution has structured the polymorphism in genes controlling the vertebrate immune system (26) (27) (28) , our knowledge of the dynamics of this selection is still rudimentary. crucially, the types of genes and pathways that tend to undergo balancing selection, or frequent intermittent directional selection, in the natural environment are poorly known. also, the nature of the selection involved and the phenotypic manifestations of the polymorphisms are not well understood. studies of wild populations (29) (30) (31) (32) (33) are pivotal in this regard, as the wild is the only place in which natural selection, and the phenotypes upon which it operates, can be measured. furthermore, given the conservation of the mammalian immune system, it seems likely that similar genes and pathways may be the target of pathogen-mediated balancing selection across taxa. future studies into the causes of immunogenetic variation (and the characterization of the associated phenotypes) in natural populations are thus likely to feed insights into the biomedical and veterinary fields through focussing attention on the types of gene predisposed to drive immunopathology due to ancestral adaptive evolution. another goal of immunological studies in wildlife is to provide an improved understanding of the dynamics of infection in natural populations (6, 34) . and rodents are of particular interest in this regard, given their ecological importance and role as reservoirs for many zoonotic infectious agents (35, 36) . in general, the dynamics of zoonotic and other ecologically important infectious diseases are likely to be influenced by heterogeneities amongst individual hosts (37) , which will affect disease severity and transmission. as the immune system is the host's defence against infectious agents, variation within it is likely to generate much of this heterogeneity (32, 38) . the measurement of immunological variation in real ecological contexts may, then, allow us to better define individual heterogeneity, and moreover, to address its environmental causes and epidemiological consequences (1, 39) . this could help anticipate infectious disease risks in the environment. for example, such information may help to identify variations in immune defence that occur in time [e.g. seasonal (40) (41) (42) ] or space [e.g. habitat-specific or along invasion fronts (43, 44) ] and that alter susceptibility to infectious agents; or it may help identify species (45, 46) , or subsets within populations (47) , whose immunological profiles indicate an increased likelihood to serve as permissive reservoir hosts for certain pathogens. for example, studies on variability in the expression of tnfa and mx2 genes in bank voles (myodes glareolus) have suggested the possibility of environmentally driven landscape-level patterns that may affect the epidemiology of zoonotic puumala hantavirus (47). as the most numerous group of mammals (~1500 species) the rodents represent, through sheer weight of numbers and their wide distributions, very significant potential reservoirs for emerging zoonotic infections. much emphasis has recently been placed on emerging and re-emerging infectious diseases from wildlife reservoirs [for example, the one health initiative (48) ] and the importance of undertaking pro-active monitoring programmes. in addition to infection risk for humans and domesticated animals, it is likely that epidemic and endemic infectious disease may also contribute significantly to the dynamics of wildlife themselves and indirectly to higher level ecosystem functioning. discovery of infectious agents of ecological importance, or that present a risk of zoonotic emergence is, however, limited by the fact that diagnostic techniques are specific to individual pathogens, or groups of pathogens. whilst next-generation sequencing (ngs) approaches are becoming available that allow very broad nonspecific surveys for pathogen sequences, these are costly and many individuals would have to be processed were a population to be sampled randomly. given this, the detection of potential emergent infectious agents in wildlife could, in some cases, be facilitated by the monitoring of immunological expression (49) . this would narrowly focus attention on individuals with aberrant immunological profiles that might be indicative of infection states. for example, these individuals could then be selected for further ngs studies (50, 51) in order to detect pathogen-specific sequences correlating with the aberrant immune expression profiles. whilst endemic reservoirs may be highly co-adapted with their pathogens and be relatively asymptomatic (52) (perhaps without anomalous immune expression profiles), it is sometimes the case that infectious agents emerging from wildlife reservoirs may do so via an intermediate 'amplifier' host that does succumb to significant disease. such hosts may shed more infective particles into the environment than the endemic reservoir and also show signs of acute immune responses. for example, putative amplifier hosts may have been involved in the emergence of the sars coronavirus. here, the ultimate natural reservoir appears to include horseshoe bats (rhinolophus spp.), but the infection was likely transmitted to human hosts indirectly via other wild mammals, including the palm civet (paguma larvata) (53), which is highly susceptible to the virus and sheds high titres of infective particles (54) . in scenarios where the aim is to identify emergent disease risks before any infectious agent is specifically identified, immune expression studies may provide a way to filter potential diseased amplifier individuals from natural populations and focus attention on these for further study. it is also likely that the pattern of immune expression may be indicative of the type of pathogen involved [as is increasingly being exploited in medical diagnostics (55) ] and that this may help target subsequent efforts at identification. studies in wild nonmodel rodents may also yield unexpected general insights into the fundamental biology of the mammalian immune system. thus, the laboratory model of mouse immunity cannot properly address many aspects of environmental variation seen in nature, and studies in humans are also limited in this way and by what tissues may be sampled or experimental approaches undertaken. on the other hand, immune responses in wild animals, once they can be interpreted using post-genomic and systems biology approaches, may reveal functional pathways and interaction networks that were not previously understood, precisely because they relate to stressful environmental conditions that cannot ethically be replicated in laboratory or domestic animals, or in humans. an example where a focus on natural populations may feed-back insights into general immunology is given in the section below dealing with immunodynamics in field vole populations (56) . included in the fundamental biology of the immune system might be the costs of immunity embodied in classical ecological immunology (16, 57) . this revolves around the concept of trade-offs: that immune responses impose a penalty in the form of competition for energy allocation to, or functional interference with, other life-history traits. understanding these costs, and the interaction of immunity with other organismal traits, is an active and exciting area of research (16) that may generate insights relevant to human health and biomedical science. there is also relevance to agriculture, where production traits reflecting reproductive or growth parameters might interact with immunity in ways that are not yet fully appreciated. whilst many advances have been made in the field relating to costs of immunity (16) , further studies in natural populations using the type of holistic, genomewide approach possible with transcriptomic methods hold the promise of further advances. why not just well-studied laboratory or farmed species? some studies may necessarily focus on particular species or local faunas because of specific concerns about infectious disease risks. where more general questions are to be answered, though, an obvious starting point for studies of immune function in natural systems would be to use the wild counterparts of standard laboratory models or other well-studied domesticated species. in particular, house mice (covered elsewhere in this special issue) would seem an obvious choice given their status as a central model in immunology. this would allow the use of robust measurements based on pre-existing antibody reagents (see next section) and would also allow interpretation of immunological patterns in the context of a very detailed organism-specific knowledge base. looking beyond the small number of laboratory and domesticated species, though, several considerations make it advisable to additionally consider other naturally occurring species. firstly, as they occur in the 'wild' today, rats and mice may have patchy population structures that are narrowly focussed on unnatural anthropogenic environments and are the result of a long history of anthropogenically linked dispersal. in the case of the mouse, for example, dispersal is believed to have occurred from the fertile crescent in the near east (58) across most of the globe following the expansion of human civilizations (59) . this history of co-habitation and dispersal means that house mice may harbour symbiont assemblages restricted by lineage sorting (extinction) during dispersal/colonization events and, as invasive organisms, they may have acquired new infections in their relatively recent evolutionary history. in response to selective forces acting during dispersal and gain/loss of infectious threats, such invasive species may also have undergone rapid genetically based changes in immune function (43, 60) . these considerations (lack of a natural symbiont flora/fauna, an unusual evolutionary history of dispersal and frequent occupation of anthropogenic habitats) make rats and mice weaker natural models to assess 'hygiene hypothesis' or 'old friends' ideas. here, where the hypothesis is that immunopathological phenotypes in anthropogenic environments arise from a lack of stimuli from co-evolved symbionts, models are needed where the host co-exists in its natural setting with a complete assemblage of co-evolved symbionts. in contrast to the house mouse and norway rat, common naturally occurring murine and microtine rodent species (e.g. apodemus, myodes and microtus spp. in europe), whose ecology has been well studied, often occur in relatively extensive, evenly distributed and persistent populations. these are likelier to have been stably linked in the long term with natural or quasi-natural habitats and with a diverse, co-adapted symbiont assemblage. transcriptomic studies are facilitated by a previously annotated genome, and this may affect the choice of study species. increasing genomic information is becoming available for several well-studied taxa, including the european species apodemus sylvaticus, microtus agrestis and m. glareolus, and annotated genomes have been assembled for peromyscus maniculatus and microtus ochrogaster in north america. however, transcriptomic studies can be carried out through de novo assembly without a pre-existing species-specific genome (61) and allow the exploitation of almost any species for ecological immunology studies. naturally occurring rodents recently used for ecological immunological studies in other parts of the world include, for example, organisms as diverse as the capybara (hydrochoerus hydrochaeris) (62, 63) or the pallid atlantic forest rat (delomys sublineatus) (64) in south america. in general, naturally occurring rodents represent unparalleled models for work in ecological immunology and immunogenetics. whilst their close relation to the laboratory mouse allows interpretation in terms of modern mechanistic immunology, their high population densities, amenability to longitudinal sampling and experimental manipulation in the field, short generation times, relatively spatially static populations and, ultimately, their adaptability to the laboratory environment, make these organisms uniquely tractable study systems. given the diversity and abundance of rodents, it is likely that most researchers will have, at close hand, naturally occurring rodent systems that may serve as useful ecological models or that are practically relevant as reservoirs of transmissible infectious disease. finally, it should not be forgotten that there is very likely to be value, for its own sake, in carrying out studies across a wide diversity of host systems (16) . this will be useful in revealing the generalities in immune function across species and will also provide insights from the specialized adaptations that individual species use to meet specific sets of circumstances. there is a strong emphasis in modern immunology on the use of antibody reagents against immunological biomolecules for robust molecular phenotypic measurements. gene expression measurements at the mrna level, in comparison, are generally considered a more problematical approach that is 'resorted to' if necessary, particularly in the case of analyses of individual genes by real-time pcr (qpcr). this is based primarily on the fact that the expression of bioactive protein may not always track upstream mrna concentrations (65) . complex kinetics in the pathway between mrna and protein, and the stability of the proteins themselves, can prevent a simple relationship. often there is poor concordance of matched transcriptomic and proteomic data sets across a range of organisms (66) . such a lack of gene-by-gene correlation, though, is much less relevant than the information content that transcriptomic profiles carry in relation to the biological status of the individual. this information content is attested to by the increasing use of transcriptomic approaches in modern biology. more specifically, early indications in wild rodents (1, 47, 56, 64) suggest that gene expression measurements, especially if interpreted with the complexity of post-transcriptional dynamics in mind, do contain much useful information. practical issues restrict the usefulness of antibody-based methods in nonmodel rodents. due to structural variability in many immune molecules (especially canonical cytokines and cell surface markers), the transferability of commercially available off-the-shelf reagents amongst rodents, even within the subfamily murinae, is limited. whilst some commercial assays and reagents may indeed be found to cross-react amongst rodent species (for example, many commercial antibodies targeting immunoglobulins), this approach may involve trialling dozens of others unsuccessfully. this has been the experience of researchers targeting cytokines in the cricetid, peromyscus maniculatus, in north america (67), or even the murine, apodemus sylvaticus, in western europe (49, 68) . thus, developing a broad panel of assays may require the de novo production of monoclonal and/or polyclonal antibodies, which is technically feasible but is costly and time-consuming (4-6), especially given that two separate antibodies may be required per target (if setting up a microplate elisa, for example). a promising and more economical strategy will be to explore further the potential of established and emerging nucleic acids measurement (4, 6, 69) . this approach can focus on panels of candidate genes, selected on the basis of the existing knowledge base for mouse immunological pathways. even more powerfully, rnaseq (70, 71) , because it encompasses the whole transcriptome (10s of thousands of genes, depending on sequencing coverage), largely removes concerns associated with measuring single genes as it allows a focus on whole pathways whose concerted variation is much more likely to reflect the phenotype. this is a technique that, although very expensive per sample, can be used in an unbiased way to identify informative and reliable marker genes that can then be measured by cheaper technologies in larger sample sizes. as an approach directed at the entire transcriptome, it is likely to supersede oligonucleotide microarray methods, due to general technical superiority (72) and, particularly in the case of studies in nonmodel species, the requirement of microarrays for prior sequence information. targeting of single genes (or small panels of genes) may be carried out by qpcr (quantitative real-time pcr, often abbreviated as rt-pcr) or other emerging technologies that measure nucleic acids more directly, for example digital pcr (73) or new developments of microarray-like systems (74) . the latter technologies are likely to be technically more robust than qpcr, which although still invaluable, suffers from sensitivity to variable reaction kinetics amongst sam-ples; however, they are currently more expensive, and in the case of digital pcr less applicable to high-throughput applications. finally, the point should be made that, whatever molecular measurement approach taken (protein or rnabased), this should ultimately be cross-referenced to functional measures of immunity (such as susceptibility to pathogens). in natural populations, this cross-referencing can be achieved either by observational epidemiological studies, or, more powerfully (and with greater difficulty), by manipulative experimental infectious challenges. the advent of genomewide transcriptomic (and other high throughput) measurements arguably allows rapid progress in the study of immunity in natural systems through exploratory, data-led approaches that might loosely be covered by the term 'reverse engineering' (75) . biologists often attempt to explain natural systems using a forwardengineering-like philosophy, where a high level model or concept is used to direct the interpretation of data. although this approach (in some form or other) is likely to remain indispensible and also corresponds to many biologists idea of the basic scientific method, it is vulnerable to arbitrary choice of starting model and is not necessarily the exclusive, or most direct, route to understand complex systems (especially when starting from a low knowledge base). in contrast, in reverse engineering, large sets of responses (as generated by, for example, transcriptomic or multiplexed protein measurements) can be correlated with environmental and organismal variables of interest across perturbations (either natural or experimental), allowing interaction networks to be inferred (3). this can help establish how molecular pathways interact with each other and the environment to generate the observed phenotype. a reverse-engineering-like philosophy has, in part, featured in the examples dealt with below, and although these work with limited panels of measurements, they illustrate the potential for future studies using broader transcriptomic approaches. the immune system has co-evolved with commensal microbes to the extent that it requires cues from these organisms to programme its normal development (76, 77) . moreover, as a result of this, immunopathological phenotypes are to be expected where microbial exposures occur that are outside the parameters within which natural selection has operated during evolutionary history (77, 78) . this developmental interaction between microbiota and immune system is, in part, channelled by toll-like receptors (tlrs) of the innate immune system (76, 79, 80) . the specificities of these receptors (81) and the inflammatory programmes they recruit are essentially directed against microbes. however, recent studies in wild rodents, which will briefly be reviewed here, suggest that natural exposures to macroparasites modify systemic tlr-mediated responses to bacterial molecular patterns. macroparasites, given their widespread occurrence in natural vertebrate populations, may thus be part of an extended co-evolved interaction network, involving commensal microbes and innate antimicrobial responses, which can drive the development and homeostasis of the immune system. cotgrave forest, nottinghamshire a wood mouse population at cotgrave forest, nottinghamshire, was monitored (49, 68, 82) over time, with a series of cross-sectional samples between 2006 and 2008. a range of ex vivo tlr-mediated responses (to defined tlr agonists) were measured in cultured splenocytes from subsets of animals. due to a pattern of positive covariation amongst tlr-mediated tumour necrosis factor alpha (tnf-a) protein responses (tlrs 2, 3-5, 7, 9) measured in the early part of the sample series, and the especially strong associations of tlr2-mediated tnf-a production with measures of macroparasite infection, this last response in particular was chosen to be measured in all samples. focussing on tlr2-mediated response across the whole study period, this was found to be associated with certain macroparasites, especially the gastrointestinal heligmosomatid nematode, heligmosomoides polygyrus, and the blood-sucking ectoparasitic louse, polyplax serrata. the magnitude and direction of the associations, though, changed across the study period. the changing associations corresponded to different environmental conditions following a perturbation (population crash) in the mouse population during the middle part of the study. early in the study, the abundances of mice and macroparasites were high, but following the mouse population crash, the later part of the study was characterized by lower macroparasite abundance. corresponding changes occurred in the association between macroparasites (p. serrata and h. polygyrus) and tlr2-mediated tnf-a responses, with a strong negative coefficient before the perturbation (at high infection levels) and a positive one subsequently (at lower infection levels). complementary laboratory experiments using the mouse-heligmosomoides bakeri model supported a causal effect of nematode infection upon tlr-mediated responses. this model is relevant because h. bakeri is a very close relative (59, 83) of the h. polygyrus occurring in wood mice. previously na€ ıve inbred mice exposed to single (cba, balb/c, c57bl/6, swr) and trickle h. bakeri infections (balb/c, c57bl/6), typically up-regulated tlr responses signalling through myeloid differentiation primary response gene 88 (myd88) at some point during the infection course, usually coinciding with peak standing worm burdens. this is consistent with a permissive effect of myd88 signalling on gastrointestinal nematode infection demonstrated through experiments with myd88 à/à and interleukin one receptor one (il1r1) à/à mice (84, 85) . it is interesting, though, that in the trickle infection experiments, resistance developed in both balb/c and c57bl/6. this contrasts with more permissive infection phenotypes previously achieved under similar experimental regimens, where the susceptible strain c57bl/6 supports chronic infection and can continue accumulating worms to the point of lethality (86) . furthermore, in wild wood mice there is a linear accumulation of h. polygyrus with age and no indication of acquired immunity in the form of abundance as a decelerating function of age indicators (82) . it may also be significant that the laboratory experiments, in initially na€ ıve animals, produced results consistent with the positive abundance -tlr response association seen in the wood mouse population during times of low parasite abundance, but were not consistent with the negative association seen at times of high parasite abundance. the latter negative association (and perhaps the permissive infection phenotypes noted above) could be accounted for by the well-known immunosuppressive effects of chronic heligmosomatid infections. these effects would likely involve adaptive regulatory t-cell responses that could feedback negatively onto innate immune responses (87) . taken together, all of this information indicates that macroparasite infection exposures may have significant and context-dependent effects on the innate responses that mediate interactions of the immune system with microbes. the mechanism for this remains to be determined, but as discussed by friberg et al. (5) , the possibilities include effects on tlr signalling by worms that are direct, via secreted immunomodulators, or indirect, via feedback from adaptive immune responses. other indirect effects could be mediated by altered exposure to microbes or innate damage signals resulting from the activities of macroparasites at their site of infection. there is some reason to believe that one or both of these last mechanisms might be important. thus, heligmosomatid excretory-secretory products (88) , and the regulatory (87) and th2 (89) immune responses that these parasites typically trigger, generally reduce tlr-mediated signalling. in the laboratory experiments, though, heligmosomatid infections actually increased tlr responses (perhaps consistent with activation by tlr ligands, which are primarily microbialor damage-associated patterns). this re-emphasizes the possibility that co-infections with macroparasites contribute to the network of interactions between commensal microbes and the immune system. moreover, due to the epidemiological association of ectoparasitic lice with systemic responses in the cotgrave forest study, it seems that antiparasite responses at peripheral sites beyond the gut (78) may also be involved. the existence of such an extended interaction network is highly relevant to our understanding of how microbiotal exposures programme immunity. because macroparasites are often absent in anthropogenic environments (5) , this may contribute to the disruption of co-evolved interactions (cf. the hygiene hypothesis). thus, studies in a wild system have pointed towards the need for further work to establish the role of natural macroparasite communities in the formation of microbiotal assemblages and the contribution of this to health. background evolutionary fitness in the natural environment is not measureable in the laboratory, and so studies of hostpathogen community dynamics in the wild are essential to fully understand immune responses in their wider context as components of antipathogen strategies that maximize fitness. studies in wild field voles, briefly reviewed below, have aimed to identify distributional infection patterns associated with different antipathogen strategies in natural populations and to link these to expression signatures in immune-relevant genes. such gene expression markers can then be used to track the life-history correlates of different putative strategies and may also give insights into the immunological mechanisms involved. when considering adaptations to infection exposures, two strategies are available to a host and these may often be deployed together, although there may be some emphasis on one relative to the other. these strategies are resistance, where the host prevents infection (denies access to the pathogen), and tolerance, where the host allows access to the pathogen whilst actively mitigating the negative effects of infection. identifying patterns of tolerance and resistance in natural populations is problematical but can be approached using a general framework like that summarized in jackson et al. (56) . in cross-sectional samples ('snapshot' destructive samples of individuals), tolerance in identifiable groups of animals may be measured as the regression slopes (reaction norms) of fitness measures (for example, body condition) against infection load. here there is the problem that, in individuals from natural populations, the infection dose and the time course of infection are not standardized. reaction norms, though, can also be supplemented by consideration of the phase curves (temporal trajectories of fitness in relation to infection load) of infection courses in longitudinally sampled individual animals. these allow a known infection load to be related to fitness measurements at a later time point. in the study briefly reviewed below, both approaches were used, with initial identification of a tolerance-like pattern in cross-sectional samples, which was then corroborated and extended by focussed analyses in longitudinal samples. immune gene expression profiles in field voles at kielder forest, northumberland gene expression measurements are increasingly being used in studies of infectious disease in nonmodel rodent systems (1, 6, 49, 64, (90) (91) (92) . in work carried out in the well-studied kielder voles (m. agrestis) system (39), measurements of a panel of candidate genes (representing different immunological pathways) were taken in sets of cross-sectional and longitudinal samples from individual voles at two sites in each of two seasons (2008) (2009) (2010) . this design aimed to capitalize on the respective strengths of the two sampling modes: the greater range and precision of measurement in destructively sampled 'snapshot' cross-sectional samples (where more tissues can be interrogated and manipulated in the laboratory), also the stronger inference of causality in time series data from repeat-sampled individually marked animals (longitudinal samples). in cross-sectional samples, expression profiles were measured in ex vivo stimulatory assays of cultured splenocytes (with stimulants including tlr2 and tlr7 agonists and mitogen), whilst in longitudinal samples, constitutive expression was recorded in peripheral blood. in addition, a range of infection and condition measures were recorded in the sample animals. early analyses in a partial cross-sectional data set for immune gene expression (2008) (2009) , and without considering pathogen data, suggested the value of the measurement approach through the existence of significant variation of expression in relation to season, life-history stage and individual condition (1) . further analyses, on the full data set, searched for patterns of resistance or tolerance to pathogens. initially focussing on the more detailed cross-sectional data, and using body and organ condition (weight adjusted for standard length) as a fitness measure, it was possible to recognize a predominant pattern indicative of tolerance to macroparasite infection in mature males (where macroparasites accumulated with age indicators and were associated with increasing body condition) and a pattern indicative of resistance in immature males (where macroparasite abundances were decelerating functions of age indicators and not associated with body condition) (56) . although unexpected, the positive association of body condition with macroparasite infection in mature males was in the context of negative changes in other life-history components and not inconsistent with a negative overall impact of infection exposure on fitness. high expression of the transcription factor gata-binding factor 3 (gata3) in mitogen-stimulated splenocytes was found to mark both tolerant animals (amongst mature males) and resistant animals (amongst immature males) in the cross-sectional set. furthermore, analyses of timelagged associations in mature males in the longitudinal data suggested that macroparasite infections triggered gata3 responses [as might be expected in laboratory models (93) (94) (95) ], which in turn gave rise to increases in body condition. this corroborated the cross-sectional analyses and supported a hypothesis that gata3 activity stimulated by macroparasites is part of a complex of (tolerance) responses leading to the readjustment of body condition in mature males. constitutive gata3 expression in peripheral blood also correlated with survival in longitudinally monitored animals, with high relative expression of gata3 predicting poorer survival in younger animals but having a progressively more positive effect on survivorship with increasing age. in this observational field study, the existence of confounding processes that might produce the cross-sectional patterns attributed to tolerance should be considered. indeed, the multifaceted nature of the kielder study, with cross-sectional and longitudinal components providing a range of measurements in different population strata, increases the opportunities for comparing predictions to data. two main confounding processes might be relevant in terms of their potential to generate tolerance-like patterns. one of these is differential mortality (dm): where, amongst animals heavily infected with macroparasites, those in poor condition die more quickly, biasing the average condition of the survivors upwards. another possibility is correlated risk (cr): where parasite load may be linked to good condition because hosts in good condition forage more or range more and encounter more parasite infective stages as a result. the dm and cr scenarios were poorer explanations for the observed patterns from a number of perspectives. under cr, increased condition would precede increased acquisition of parasites and the gata3 responses they trigger; but in temporal series for individual mature males, these two sets of events, in reality, occurred in the reversed sequence. this observed sequence and the direction of association also contradict the prediction of dm of a negative effect of macroparasite infection (and the gata3 responses triggered) on subsequent condition (which follows if infection is a major cause of mortality)in reality there was a positive association. perhaps most importantly, the dm and cr scenarios do not explain the age-specific changes in the relationship between gata3 expression and survival in longitudinal data, or in the relationship between gata3, condition and macroparasite infection in the cross-sectional data. thus, if dm were true, gata3 expression in peripheral blood would be expected to show a consistent negative association with individual survival, especially in classes of animal with an apparent gata3associated tolerance pattern. however, in reality gata3 expression decreases survival in smaller males (where a tolerance pattern is not seen) but tends to increase survival in larger animals (which do show the gata3-associated tolerance pattern). furthermore, if cr were true, better conditioned animals would generally have higher parasite exposure and higher gata3 expression; but in reality, this is only seen in mature males and not in immature males. it might also be argued that a special case of cr, which could explain the latter age-specific pattern, is that better conditioned mature males range more, or have more social contacts, due to increased breeding activity. even in this case, though, the wide-ranging males would be expected to have better testis condition (if undergoing increased behaviours associated with breeding); but in our study, gata3 expression was negatively correlated with testis condition. thus, the details of the study were consistent with a hypothesis of elevated gata3 expression mediating resistance in immature males and tolerance in mature males; there were major inconsistencies with alternative dm and cr interpretations. gata3 is a master transcription factor involved in the differentiation and development of th2 (t-helper type 2 cell) cells (96) and would be expected to mark th2 activity in the splenocyte cultures studied. the seemingly dual aspect of gata3 (involved in tolerance and resistance) is not biologically implausible, given that th2 responses have long been associated with resistance to macroparasite infections in laboratory models but are also linked to wound-healing mechanisms that might be involved in tolerance (97) . it would seem possible, however, that gata3expressing th2 cells might drive different downstream effector responses in resistance and tolerance, and this is one aspect that is worthy of further study. whilst regulatory components of the immune system have previously been considered as possible mediators of tolerance (98) , through their ability to dampen effector responses, this was not supported in the kielder study. thus, the antiinflammatory cytokines interleukin (il) 10 and transforming growth factor beta one (tgf-b1) and the transcription factor forkhead box p3 (foxp3), whose expression characterizes many regulatory t-cell subsets, were measured but were not associated with tolerance patterns. gata3 expression in tolerant males was associated with a complex of life-history readjustments likely to impact fitness (represented by schematic phase curves in figure 1 ). apart from the increase in body condition, there was a decrease in a fecundity indicator (testis condition) and increasing survival as animals aged. possibly, then, the tolerance strategy increases fitness via improvements in residual reproductive value (potential for future reproduction) during macroparasite infection. this is epidemiologically significant, as the transmission of infectious agents may become focussed through tolerant subsets of the population with consequences for the population dynamics, life histories and co-evolutionary dynamics of the interacting organisms. but there are also insights that can be fed back into laboratory immunology. the results raise the possibility that th2 cell activity may show ontogenetic changes, sometimes mediating resistance and at other times mediating tolerance. this dichotomy could be relevant to the lab-oratory as, typically, laboratory experiments are carried out in restricted life-history stages under restricted environmental conditions, and this may have skewed our view of what responses are likely to occur in systems outside of the laboratory. there is also a relevance to vaccination strategies in real-world situations, which may need to encompass the possibility that, under some conditions and in some subsets of a population, similar immunogenic stimuli may result in resistance or tolerance responses (that might not be protective in terms of preventing infection, but could have some benefit in terms of ameliorating disease). this may be especially significant, given that most vaccine adjuvants used in practical contexts are th2-inducing aluminium salts (alums). although much interesting work has been done in nonmodel rodents using narrow focusses on individual immune responses, a renewed effort addressing the immune system (and its interaction with wider organismal traits and the environment) in a more holistic way seems likely to pay dividends. the pivotal technological means to do this are now accessible, in the form of nextgeneration sequencing analyses of the transcriptome. early indications from studies using qpcr panels of candidate genes suggest that gene expression measurements in natural populations do convey interpretable information about individual status, especially where combined with defined stimulatory treatments of cultured cell populations. associations occur with season, life-history stage and body condition. furthermore, there are strong indications that infection pressures are key drivers of many aspects of expression in the immune system in nature and that, as a result of these pressures, wild mammals can be confidently predicted to adopt phenotypes very different to those seen in laboratory rodents. all of this supports the utility of immune gene expression measurements for ecologically and epidemiologically motivated studies; it also confirms the interest for our basic understanding of the immune system: where the diverse combinations of environmental conditions seen in the wild may reveal interaction networks that remain hidden under controlled laboratory conditions. finally, studying the diversity of immune function in natural and anthropogenic environments (and, ultimately, further dissection of this variation under experimental conditions) will help us resolve the environmental stimuli (and genotype 9 environment interactions) that affect the formation of immunopathological phenotypes such as those responsible for so much variation in human health and animal welfare. δ body condition δ survival probability δ testis condition large mature male immature male gata3 blood gata3 mit-s m gata3 mit-s m figure 1 life-history responses in male field voles (microtus agrestis) associated with gata3 expression triggered by 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essential role for tlr4 and myd88 in the development of chronic intestinal nematode infection genetic variation in resistance to repeated infections with heligmosomoides polygyrus bakeri, in inbred mouse strains selected for the mouse genome project cd4 + cd25 + regulatory t cells control innate immune reactivity after injury impairment of dendritic cell function by excretory-secretory products: a potential mechanism for nematode-induced immunosuppression th2 cytokines down-regulate tlr expression and function in human intestinal epithelial cells tnf-alpha expression and promoter sequences reflect the balance of tolerance/ resistance to puumala hantavirus infection in european bank vole populations heligmosomoides polygyrus infection is associated with lower mhc class ii gene expression in apodemus flavicollis: indication for immune suppression? expression profiling of lymph node cells from deer mice infected with andes virus saag-4 is a novel mosquito salivary protein that programmes host cd4(+) t cells to express il-4 blood feeding by the rocky mountain spotted fever vector, dermacentor andersoni, induces interleukin-4 expression by cognate antigen responding cd4(+) t cells the immune response to parasitic helminths: insights from murine models gata3 and the t-cell lineage: essential functions before and after t-helper-2-cell differentiation evolution of th2 immunity: a rapid repair response to tissue destructive pathogens decomposing health: tolerance and resistance to parasites in animals acknowledgements i gratefully acknowledge the colleagues with whom i have worked on nonmodel rodent systems and whose ideas have influenced my own thinking, also acknowledged is funding from the leverhulme trust (rpg-301) and nerc (ne/l013517/1). key: cord-318063-bainw3d6 authors: haque, mainul; sartelli, massimo; mckimm, judy; abu bakar, muhamad title: health care-associated infections – an overview date: 2018-11-15 journal: infect drug resist doi: 10.2147/idr.s177247 sha: doc_id: 318063 cord_uid: bainw3d6 health care-associated infections (hcais) are infections that occur while receiving health care, developed in a hospital or other health care facility that first appear 48 hours or more after hospital admission, or within 30 days after having received health care. multiple studies indicate that the common types of adverse events affecting hospitalized patients are adverse drug events, hcais, and surgical complications. the us center for disease control and prevention identifies that nearly 1.7 million hospitalized patients annually acquire hcais while being treated for other health issues and that more than 98,000 patients (one in 17) die due to these. several studies suggest that simple infection-control procedures such as cleaning hands with an alcohol-based hand rub can help prevent hcais and save lives, reduce morbidity, and minimize health care costs. routine educational interventions for health care professionals can help change their hand-washing practices to prevent the spread of infection. in support of this, the who has produced guidelines to promote hand-washing practices among member countries. health care-associated infections (hcais) are those infections that patients acquire while receiving health care. 1 the term hcais initially referred to those infections linked with admission to an acute-care hospital (earlier called nosocomial infections), but the term now includes infections developed in various settings where patients obtain health care (eg, long-term care, family medicine clinics, home care, and ambulatory care). hcais are infections that first appear 48 hours or more after hospitalization or within 30 days after having received health care. 2 multiple studies indicate that the most common types of adverse events affecting hospitalized patients are adverse drug events, hcais, and surgical complications. [3] [4] [5] [6] [7] the us center for disease control and prevention identifies that nearly 1.7 million hospitalized patients annually acquire hcais while being treated for other health issues and that more than 98,000 of these patients (one in 17) die due to hcais. 8 the agency for health care research and quality reported that hcais are the most common complications of hospital care and one of the top 10 leading causes of death in the usa. 9 out of every 100 hospitalized patients, seven patients in advanced countries and ten patients in emerging countries acquire an hcai. 10 other studies conducted in high-income countries found that 5%-15% of the hospitalized patients acquire hcais which can affect from 9% to 37% of those admitted to intensive care units (icus). 11, 12 multiple research studies report that in europe hospital-wide prevalence rates of hcais range from 4.6% to 9.3%. [13] [14] [15] [16] [17] [18] [19] [20] [21] the who reports however that hcais usually receive public attention only when there are epidemics. 22 hcais also have impact on critically ill patients with around 0.5 million episodes of hcais being diagnosed every year in icus alone. 7, 14, 23 icu patients are often in a very critically ill, immuno-compromised status which increases their susceptibility to hcais. 24, 25 brief history there has been long-standing awareness that the practice of medicine can do harm as well as good. for example, hippocrates, the father of modern medicine, stated more than 2,500 years ago that "i will use treatments for the benefit of the ill in accordance with my ability and my judgment, but from what is to their harm and injustice i will keep them." 26 it was also recognized (eg, by semmelweis discussing puerperal fever) many years ago that coming into hospitals (in particular) can be dangerous. 27 in this century, the idea that medicine could cause harm, including death is described as "unintended physical injury resulting from or contributed to by medical care, including … [its] absence … that requires additional monitoring, treatment or hospitalization, or … results in death." 28, 29 offering another perspective, an american natural sciences writer noted that hcais are now killing around 100,000 people, many more than hiv/aids, cancer, or road traffic accidents. 30 the hungarian obstetrician professor (dr) ignaz phillip semmelweis is largely considered as the medical doctor who realized that health care providers could communicate disease. his work identified the mode of communication and spread of puerperal sepsis while working at the maternity hospital in vienna. in 1847, he observed higher rates of maternal mortality among patients treated by obstetricians and medical students than among those cared for by midwives. at that time, he also found that a pathologist had died of sepsis after wounding himself with a scalpel while carrying out an autopsy on a patient with puerperal sepsis. the pathologist's illness mirrored that of women with puerperal sepsis, and semmelweis wrote that both a scalpel and a physicians' contaminated hands could transmit organisms to mothers during labor. he introduced chlorinated lime hand washing to the obstetric hospital staff, resulting in large improvements in maternal mortality rates. 31 however, semmelweis' theories were dismissed by most of the medical establishment because of a lack of appropriate statistical analysis of the data. nevertheless, after koch's postulates were published in 1890, the germ theory of disease and semmelweis' theory of transmission of disease from doctor to patient were found to be valid. semmelweis was therefore the first to describe an hcai and provide an intervention to avert its spread through hand hygiene. 32 a survey conducted in 183 us hospitals with 11,282 patients reported that 4% of patients had at least one hcai with the most common microorganism being clostridium difficile. most infections were surgical site infections (ssis), pneumonia, and gastrointestinal infections. 33 a study 2 years earlier by the same group found that 6% (51) of patients had suffered from hcais with the top 75.8% acquiring ssis, urinary tract infections (utis), pneumonia, and bloodstream infections. staphylococcus aureus was the most frequently detected microorganism. 34 the group conducted a comparative study between 2011 and 2015 and found a statistically significant (p<0.05) reduction of hcais in ssis, utis, and central line infections, probably due to a national initiative. 35 hcais are also problematic elsewhere in the world. for example, a study in singapore reported 11.9% (646) patients with hcais, primarily undetermined clinical sepsis, and pneumonia caused mainly by s. aureus and pseudomonas aeruginosa. 36 this study also reported that the acinetobacter species and p. aeruginosa were extremely resistant to carbapenem. 36 a recent european study found that 2,609,911 new patients were identified as having hcais annually in the european union and european economic area. 37 this study revealed that for every 20 patients hospitalized, at least one acquired an hcai which was preventable. 37 klebsiella pneumoniae and the acinetobacter species were exceedingly resistant to multiple antimicrobials, and the lack of new antimicrobials increases the huge burden in europe. 37 in greece, the hcai prevalence rate was 9.1%. the frequent types of hcais were lower respiratory tract infections (lrtis), bloodstream infections, utis, ssis, and systemic infections. 38 one systematic review and meta-analysis regarding hcais in southeast asian countries (brunei, myanmar, cambodia, east timor, indonesia, laos, malaysia, the philippines, singapore, thailand, and vietnam) found an overall prevalence rate of 9.1% with the most common microorganisms being p. aeruginosa, the klebsiella species, and acinetobacter baumannii. 39 a study conducted in eight university hospitals of iran (ranging from 60 to 700 beds) reported an overall hcai frequency of 9.4%, the most common hcais were bloodstream infections, ssis, utis, and pneumonia. 40 19-4.28) . being admitted to an icu is not in itself a self-determining hcai risk factor. the or for all hcais of acquiring an infection was 3.24 (95% ci 2. 34-4.47) in patients with hospital stays longer than 8 days. 33 seventy-one percentage (71%) of the studied patients received antimicrobials, but 9.4% had at least one evidence of infection. 33 another study revealed that the average number of microbes ranged from on (9.67×1011), working surfaces (1.64×1012), door handles (1.71×1012), and highest in taps (2.08×1012). 41 the highest number (23) of pathogens were isolated from door handles, and the peak variance of pathogens were on hospital floors (7). among those microbes, those that were disease-producing were 46.14%, 53.86% were nonpathogenic, the most common was s. aureus at 14.42% and 45.2% of the total bacterial isolates comprised bacillus subtilis. a study conducted in ghana reported that gentamicin was the most effective antibiotic (100%) on both gram-positive and gram-negative organisms, but of the 12 antibiotics tested (ampicillin, cefuroxime, cotrimoxazole, cefotaxime, tetracycline, amikacin, gentamicin, chloramphenicol, cefixime, cloxacillin, and erythromycin), six were resistant to either gram-positive or gram-negative organisms. 41 most of the hcais in the us are triggered by the eskape group, comprising the antimicrobial-resistant gram-negative microorganisms (k. pneumoniae, a. baumannii, p. aeruginosa, and enterobacter spp.) and the grampositive species, enterococcus faecium and s. aureus. [42] [43] [44] multiple studies report that gram-negative organisms are responsible for 10%, 45 20%-40%, 46 of hcais and that antimicrobial resistance places a significant burden on the global health care system, particularly in low resource countries. 47, 48 this problem is exacerbated as research and development into new antimicrobials targeting gram-negative organisms has rapidly decreased in recent years. 48 among the newer aminoglycosides, plazomicin has been found to be active against the extended-spectrum betalactamase (esbl) generating strains of enterobacter spp., escherichia coli, and k. pneumoniae 49 and more effective in laboratory experiments against a. baumannii than gentamicin, tobramycin, and amikacin. 50 plazomicin has a better safety profile than other drugs, with no report of damage to the cochlea, auditory nerve, vestibular, and renal system in healthy volunteers, even with high and multiple doses. 51 another study found that, in a comparison between hcais due to methicillin-sensitive s. aureus and methicillin-resistant s. aureus (mrsa), isolates were statistically significantly (p<0.005) more resistant to ciprofloxacin, clindamycin, trimethoprim/sulfamethoxazole, erythromycin, gentamicin, and tetracycline. 52 hospital waste, especially contaminated surgical waste, often acts as a reservoir for pathogenic virulent microorganisms, and it suggested that 20%-25% of the waste produced by health care outlets is considered to have high potential to cause hcais, it therefore needs appropriate handling and disposal. 53 45, 55 some of these gram-negative microorganisms have a much higher rate (20%-40%) of resistance than others 45 with the organisms isolated from device-associated hcais having the highest antimicrobial resistance phenotypes. 56 in the latter study, although similar to the percentage resistance for most phenotypes was that in an earlier research study, 45 an upsurge in the scale of the resistance fractions against e. coli pathogens was observed, especially with fluoroquinolones. 56 acinetobacter, burkholderia spp. and pseudomonas spp. isolates were 100% were 92% resistant to cephalosporins respectively. burkholderia spp. was again totally resistant to fluoroquinolones and acinetobacter spp. and pseudomonas spp. were 94.2% and 95.8% resistant, respectively. the same study reported that 86.4% acinetobacter spp. and 62.5% pseudomonas spp. showed a high resistance to carbapenems, the preferred drug regime in icus. carbapenems were found more effective against burkholderia spp. with 20% resistance. 57 in another study, enterobacteriaceae community were found to be completely resistant to third-generation cephalosporins. 58 over 80% of the klebsiella spp. community were resistant to ciprofloxacin, gentamicin, piperacillin, tazobactam, and imipenem showing 48.6% resistance. e. coli was equally resistant although carbapenems were effective in almost haque et al 80% cases. although citrobacter spp.-related hcais are a relatively minor proportion, they also show resistance toward cephalosporins, fluoroquinolones, and aminoglycosides. 58 another study reported that although the acinetobacter spp. were 76.99%-92.01%, resistant to most antimicrobials, only 30% of acinetobacter spp. isolated were susceptible. 59 it can be seen therefore that the causative pathogenic microorganisms differ from country to country as does patterns of resistance. alongside infections due to cross-contamination between patients and health workers, patients being susceptible to common infections due to diminished immune responses, and infections at surgery sites (ssis), many hcais are due to implants and prostheses. these include central line-associated bloodstream infections (clabsis), catheter-associated utis, and ventilator-associated pneumonia (vap). 57, 60, 61 clabsis clabsis substantially increase morbidity, mortality, and health care costs, and great attention has been paid to addressing these. 62, 63 as a consequence, in 2009, 25,000 fewer clabsis occurred in the icus of us hospitals than in 2001, a 58% reduction, with about 6,000 lives saved and estimated financial savings of us$414 million in potential excess health care costs, although the costs of reducing such infections is very high. 64 it is estimated that it costs ~$1.8 billion between 2001 and 2009 to save an additional 27,000 lives. 64 despite this investment, a considerable number of clabsis still occur, especially in outpatient hemodialysis centers and inpatient wards. 64 another study also reported the link between clabsis and considerable morbidity and mortality, although there is a wide variation in reported infection rates (from 20% to 62.5%) in emerging economies. 65 a study conducted in taiwan reported the occurrence of clabsis as 3.93 per 1,000 central-catheter days. 66 the most common causative pathogens were gram-negative (39.2%), gram-positive (33.2%), and candida spp. microorganisms (27.6%). 66 in this study, patients developed clabsis 8 days from the time of insertion of the central line catheter. 66 multivariate analysis showed that a higher pitt bacteremia score (or 1.41; 95% cl=1.18-1.68) and the prolonged interval between the onset of clabsis and catheter removal (or 1.10; 95% ci=1.02-1.20) were associated with higher death rates. 66 another similar study identified prolonged catheter in situ, pediatric icu stay, and intravenous nutrition were significant prognosticators of peripherally inserted central catheter-related clabsis among hospitalized children. 67 ssis ssis (formerly termed "wound infections") are still one of the most common adverse events that occur in hospitalized patients undergoing surgery or in outpatient surgical measures, regardless of the advances in preventive procedures. 68 ssi is the most common complication in postoperative surgical patients, associated with significant morbidity, high death rates, and financial stress on national budgets and individual patients. [69] [70] [71] ssis are defined as infections arising up to 30-90 days after surgery in patients receiving an organ, group of cells, or device and affecting both the incisional site and deeper tissues around the surgery location. 72, 73 the type of surgery determines the proportion of ssis. between 2% and 36% of patients may develop ssis, with the highest risk for orthopedic followed by cardiac and intraabdominal surgery. 14, 72, 74, 75 the length of hospital stay for patients with ssis increases from 4 to 32 days as compared with patients with no post-surgical infections. [76] [77] [78] approximately 25% of patients with ssis develop severe sepsis and shock and are moved to an icu. 65 ssis cause statistically significant morbidity, mortality, and financial burdens for individuals and for communities. [69] [70] [71] 78 hcais are common following cardiac surgery, with a reported incidence rate of between 5.0% and 21.7%, 79, 80 often accompanied with multiple organ failure and prolonged hospital stays, leading to increased mortality rates. 79, 80 the three most common locations for hcais after cardiac surgery are lungs, central venous catheters, and surgical sites. 69 ssis followed by cardiac surgery classically present with localized cellulitis (erythema, warmth, and tenderness), purulent discharge, sternal instability, chest pain, and systemic upset with deep infections. [81] [82] [83] ssis are devastating for orthopedic patients as it is very difficult to rid the bones and joints of the infection. 83 one saudi arabian study reported an incidence of ssis in orthopedic patients of 2.55% (79 of 3,096 patients) with the most common pathogens being staphylococcus species including mrsa (29.11%); acinetobacter species (21.5%); pseudomonas species (18.9%), and enterococcus species (17.7%). 84 surgical wound contamination potentials, patients' clinical conditions, type of surgery, and length of surgery were variables statistically significantly associated with ssis and should be viewed as risk factors. 85 the movement and number of staff and the structural features of the operating theater also affect the incidence of ssis. 85, 86 one study found that 73.33% cases of ssis following orthopedic surgery were culture positive, and a total of 35 bacterial strains were isolated, among which 65.72% were grampositive isolates and 34.28% were gram-negative bacteria. 87 infection and drug resistance 2018:11 submit your manuscript | www.dovepress.com health care-associated infections and prevention strategy about 68.6% of all bacterial isolates were resistant to cefuroxime used in the management of orthopedic ssis. this study also found that diabetes mellitus, smoking, operations lasting more than 3 hours, the absence of antibiotic prophylaxis, and a history of previous surgery were positive risk factors associated with a significant upsurge in ssis. 87 ssis comprise at least 14%-22.2% of all hcais for abdominal surgery [88] [89] [90] and often lead to extended hospitalization and higher antimicrobial costs. 71 the microorganisms generally involved in such ssis include s. aureus, coagulasenegative staphylococci and enterococcus spp., and e. coli. 71 s. aureus has been known to be a major cause of hcais for over 100 years. 91 when first introduced, nearly all strains were susceptible to penicillin, but since its wide and often irrational use, s. aureus started to become resistant by producing β-lactamase enzyme. 91 by 1960, 95% hospital variants of s. aureus were resistant. 91, 92 to help combat resistance, several new penicillins were developed to resist staphylococcal β-lactamase, such as methicillin, oxacillin, cloxacillin, and flucloxacillin. 91 however, within 1 year of methicillin being marketed in 1960, the first mrsa strain of s. aureus was reported in england. 93 the mrsa strain represents 50% of hcais in the us and europe and causes infections that are very difficult to manage because of their potential resistance to multiple antimicrobials. [94] [95] [96] in one study, the incidence of ssis was after gastrectomy in 11.3%, after colorectal surgery in 15.5%, after hepatectomy in 11.3%, and after pancreaticoduodenectomy in 36.9%. 97 while the incidence of ssis was higher in the absorbable stitching material than the silk group for all surgical procedures, the difference was not statistically significant. 97 a japanese study on abdominal surgery reported an overall ssi rate of 14.4%. the ssi rates in the suture-less, vicryl, and silk groups were 4.8%, 14.8%, and 16.4%, 88 respectively, again with no statistically significant differences between the groups. in colorectal surgery, the ssi rate in the polyglactin 910 (absorbable, synthetic, usually braided suture; vicryl tm ) group was 13.9%, which was statistically significantly lower than that of the silk group (22.4%; p=0.034). the incidence of deeper ssis in the vicryl group, including deep incisional ssis (issis) and organ/space ssis (osis), was statistically significantly lower than that in the silk group (p=0.04). 88 the ssi rates did not differ among the suture types overall in gastric surgery or in appendectomy. 98 a us study of pediatric patients found that while this was only 2.5% of the caseload, colorectal surgery contributed to 7.1% of the ssis. 98 the ssi rates of all types of colorectal surgery were 5.9% (issis: 3.2%; osis: 2.7%) with the uppermost being total abdominal colectomy (11.4%) trailed by partial colectomy (8.3%) and colostomy closure (5.0%). 98 inflammatory bowel diseases caused the topmost health problems in a comparison of all colorectal diagnosed diseases (24.9%; issis: 22%; osis: 28.6%). hirschsprung's disease (14.2%; issis: 15.4%; osis: 12.8%) and anorectal malformations (12.4%; issis: 17.6%; osis: 6.4%) were the next major group in colorectal diseases. 98 finally, a study utilizing univariate analysis defined 13 statistically significantly variables related to ssis. those were patients aged over 60 years, lower functional status, diabetes mellitus, congestive heart failure, immunocompromising disease, anticancer medications, immunosuppressive agents, impaired immune system, open cholecystectomy, laparotomy, an american society of anesthesiologists score above 2, drain insertion, and dirty wound. 99 using multivariate regression analysis, this study also found that immunosuppressive agents (or =2. 5 internationally, utis are the most common hcais and one of the top ranking microbial infections, representing around 40% of hcais, with significant consequences for morbidity and mortality and substantial financial implications. 14, 99, 100 although cautis are typically benign, some patients have potentially pathogenic virulent bacteria but are asymptomatic, and these patients were associated with a three-times higher mortality than in non-bacteriuric patients. 101, 102 multivariate analysis indicates the risk factors for cautis including prolonging the duration of the catheter, female sex, older age, diabetes mellitus, the absence of systemic antibiotics, catheter insertion outside the operating room, and a breach in the closed system of catheter drainage. 101, 103 the rate of cau-tis has been estimated to be about 5% per day, regardless of the duration of the indwelling catheter, with e. coli being the main infecting pathogenic microorganism, although a wide spectrum of other microorganisms were identified, including eukaryotic fungus. 104, 105 the repetitive inappropriate administration of antimicrobials often leads to greater bacterial resistance. cautis habitually lead to biofilm formation on both the extraluminal and intraluminal portal catheter surface, largely from extraluminal microorganisms. [106] [107] [108] the biofilm defends microbes from both antimicrobials and host defense mechanisms. 109 haque et al inserted and cleaned, in patients with long-term indwelling catheters, fever from cautis is common with a frequency fluctuating from one per 100 to one per 1,000 catheter days. 105 patients in institutional care with long-term indwelling catheters have a greater risk for the presence of pathogenic microorganisms and other urinary tract diseases than those without catheters. 105 one meta-analysis found that cautis were linked with statistically significantly higher death rates (or =1.99; 95% ci =1.72-2.31; p<0.00001; i 2 =54%; eight studies; 62,063 patients) and days in the icu (weighted mean difference of +12 days; 95% ci =9-15; p<0.00001; i 2 =96%; seven studies; 13,011 patients) and hospital (mean difference +21 days; 95% ci =11-32; p<0.0001; i 2 =98%; five studies; 10,183 patients). 110 an australian health care-associated urinary tract infection (hcauti) non-concurrent cohort study carried out for 4 consecutive years found that patients had an extra 4 days (95% ci =3.1-5.0 days) of hospitalization. 111 this study further reported that the infection rate was statistically significantly minimized utilizing a cox regression model (hr =0.78; 95% ci =0.73-0.83) when patients were released from the hospital. 111 hcautis very rarely cause death (hr =0.71; 95%ci =0.66-0.75), especially in large hospitals when compared to other health care institutes, even when compared with age and sex (hr =0.74; 95% ci =0.69-0.78), although elderly patients more often died (hr =1.40; 95% ci =1.38-1.43). 111 vap the death risk for patients in the icu is not only because of their original illness but often because of hcais. 2, 54, 112 pneumonia is the second commonest hcai in icus, affecting more than one-quarter of patients. 113, 114 around 86% of hcais are associated with motorized automatic ventilation and vap. 113 between 9% and 27% of patients with assisted ventilation develop this kind of pneumonia, and vap has been identified internationally as a potential major cause of death. 114 the average critical time to develop vap following endotracheal intubation and mechanical ventilation was 2-3 days. 115 patients usually develop a fever, altered bronchial sounds, white blood cell counts reduced, changes in sputum, and causative organisms are often identified. [116] [117] [118] [119] [120] [121] a us study found a range of vap of between 1.2 and 8.5 per 1,000 ventilator days 122 although an international group reported a much higher occurrence of vap of 13.6/1,000 ventilator days. 123 in asian countries, a different picture of 3.5-46 infections/1,000 ventilator days emerges, 124 with a very high incidence rate in india of 40.1 per 1,000 ventilator days. 125 the initial 5 days of mechanical ventilation is the most critical time for the development of vap, with a mean duration of 3.3 days between intubation and the development of vap. [119] [120] [121] [122] [123] [124] [125] [126] another recent indian study reported that non-fermentative gram-negative bacilli 127 were the predominant organisms, followed by pseudomonas and klebsiella genus. in this study, s. aureus reduced in prevalence from 50% to 34.9% between 2011 and 2013, but between 2012 and 2013 vancomycin-resistant enterococci increased from 4.3% to 8.3%, while methicillin resistance among s. aureus exceeded 50% in 2013. in addition, an upwavard trend in resistance by pseudomonas genus was observed for piperacillin-tazobactam, amikacin, and imipenem. the incidence of non-fermenters' resistance continued to be very high except for amikacin and imipenem (33.1%) and polymyxin-b (2.4%). 127 a study at chonnam national university hospital in south korea of the transtracheal aspirates or bronchoalveolar lavage of patients suffering from vap found that s. aureus (44%) was the most frequently detected causative microorganism followed by a. baumannii (30%), p. aeruginosa (12%), stenotrophomonas maltophilia (7%), k. pneumoniae (6%), and serratia marcescens (2%). 128 in addition, s. aureus was found as mrsa and 69% of acinetobacter baumannii were imipenem-resistant. 128 no statistically significant variance was observed in the imipenem-resistant a. baumannii 128 between the earlier and late vap-related study groups (73% [8/11] vs 67% [14/21] , p=1.000). 128 in this study, 67% of k. pneumoniae was esbl-positive. 128 vap was frequently linked with substantially increased morbidity, including prolonged icu and hospitalization, and higher ventilator days and health care costs. 129 in the uk and the republic of ireland, a european study of hcais connected with respiratory infection found a prevalence rate of 7.59%. among these hcais, 15.7% were pneumonia, and 7% were lower respiratory tract infections other than pneumonia (lrtiop). 130 around 21% of patients in both the groups were having artificial ventilation, which was much higher when compared to the rest of the patients with hcais. mrsa was the principal invading microorganism for both pneumonia and lrtiop. although the patients with lrtiop suffered more from c. difficileinduced diarrhea than pneumonia, this was not statistically significant. 130 a recent chinese study reported that 14.94% (895) of inpatients acquired a lrti which prolonged their hospital stay and increased the costs per individual case by us$2,853.93. 131 another study revealed that 9.6% of patients developed hcais, of which respiratory tract infections were the highest at 65.8%. 132 the most frequently identified respiratory pathogen was gram-negative acinetobacter species (40.4%), and among these 21% were mdr. 132 submit your manuscript | www.dovepress.com health care-associated infections and prevention strategy a significant number of patients develop pneumonia after surgery which includes both hospital-acquired pneumonia (pneumonia developing 48-72 hours after admission) and (as discussed above) vap (pneumonia developing 48-72 hours after endotracheal intubation). 133 postoperative pneumonia has been described as one of the leading consequences of all types of surgery with a high incidence of morbidity and mortality. 134 it increases hospital stays on an average of 7-9 days and increases health care costs from us$12,000 to us$40,000. 114, 135, 136 hcais hcais are a major safety concern for both health care providers and patients. they continue to escalate at an alarming rate, especially in emerging economies, with infection rates 3-20 times higher than in high-income countries. 1, 2, 137 hcais increase morbidity, mortality, length of hospital stays, and costs; 138-140 therefore, more research and changes in practice are needed to ensure hospital safety and prevent hcais. 32, [141] [142] [143] the annual costs for hcais alone in the usa are between us$28 and us$45 billion, but with even this amount of spending, 90,000 lives are still lost per year: hcais are among the top five killers in the usa. 14, [144] [145] [146] [147] the who advocates that effective hand hygiene is the single most important practice to prevent and control hcais, which form colonies with mdr microbes. 1, 2, 148, 149 several studies report that a simple and straightforward process, taking only a few seconds to clean hands with an alcohol-based hand rub helps prevent hcais and save lives, reduce morbidity, and minimize health care costs. 150, 151 however, factors such as the availability of alcohol-based hand rubs and up-to-date knowledge of the importance of hand washing hinder good practice in hand hygiene. for example, an australian observational study of community nurses highlighted poor practices of hand hygiene in comparison with a standard protocol. 152 the who promotes and advocates that all health care workers (hcws) must wash their hands before touching a patient, before clean/aseptic procedures, after body fluid exposure/risk, after touching a patient, and after touching patient surroundings. 153 the center for disease control and prevention has developed a comprehensive plan and guidelines for the prevention of hcais which covers basic infection prevention and control (ipc); antibiotic resistance; device-and procedure-associated infections; disease/ organism-specific infections; and guidance for health workers working in specific settings. 154 this guidance, like that of the who and the uk royal college of nursing (rcn) also emphasizes the importance of hand washing. [153] [154] [155] the rcn also promotes and advocates that all health care profes-sionals must receive compulsory "infection control training as part of their induction and on an ongoing annual basis. it is particularly important that knowledge and skills are continually updated." 155 multiple research studies indicate that policy changes and the adoption of novel multifactorial, multimodal, multidisciplinary strategies offer the greatest possibility of success in terms of hand hygiene improvement and the reduction of hcais. [156] [157] [158] [159] [160] [161] [162] [163] [164] [165] [166] [167] instigating best practice in health care stems "from a response to factors that are outside a purely scientific understanding of infection and not simply understood as a deficit in knowledge." 168, 169 good practice for infection prevention among hcws can be ensured through compliance to ipc guidelines. 168 specific individuals acting as "change champions" can act as arbitrators or negotiators, contributing to changing behaviors and implementing best practice to ensure patient safety. [168] [169] [170] [171] this calls for educational interventions that reflect the philosophies, principles, and community understanding of dirt and infection. 169 an educational intervention involving 4,345 health professionals in three public hospitals in the usa successfully improved hand hygiene immensely with the use of alcohol hand rub. nurses, physicians, and allied hcws improved from 14% to 34%, 4.3% to 51%, and 12% to 44%, respectively. 172 other studies also highlight how behavior change around hand washing can result from educational interventions. 149, 151, 172 health professionals must protect themselves with barriers for example, gloves, gowns, face masks, protective eyewear, and face shields, 173 to decrease the work-related transmission of microorganisms. regular use of personal protective equipment (ppe) 173 devices protects both the professional and the patient from potentially infectious body fluids. 173 nevertheless, the use of ppe does not confirm 100% protection, 174 for example, needlestick injury can breach ppe, and, in many occasions, issues might go unrecognized which might cause a dangerous health hazard including hepatitis b or hiv. 175 respiratory microorganisms, for example, influenza virus, bordetella pertussis, haemophilus influenzae, neisseria meningitidis, and mycoplasma pneumoniae, severe acute respiratory syndrome-associated coronavirus, group a streptococcus, adenovirus and rhinovirus, and tubercular bacilli 176 are easily dispersed through droplets (particles ≤5 µm in size) in closed health care settings and often cause endemics and epidemics. 176 [178] [179] [180] [181] [182] meticulous cleaning of hospital surfaces is therefore vital to maintain standards and reduce the risk of hcais. 183 several studies conclude that ultraviolet devices and hydrogen peroxide vapor technologies successfully eradicate potentially dangerous hospital microorganisms adhering to the surfaces in ward or patient rooms. [183] [184] [185] [186] furthermore, hydrogen peroxide vapor efficiently sterilizes and sanitizes all clinical areas where potentially dangerous microbial mdr microorganisms and spores were suspected to be present. 187 in the early to mid-19th centuries in both europe and usa, thousands of young women died from puerperal sepsis and fever, the diseases rampant in the charity maternity clinics of the time 188 and, due to the efforts of (among others) dr ignaz phillip semmelweis and dr oliver wendell holmes, the fight against puerperal fever was won and it was confirmed that hcais were transmitted via the hands of hcws. [188] [189] [190] [191] [192] despite the development of many hi-tech methods, hand washing with soap and water or alcohol rub is still the most important means of maintaining personal hygiene and preventing hcais. 192 however, due to the rise of antibioticresistant bacteria and a reluctance of some hcws to implement best practice infection control, hcais remain one of the biggest causes of death in most countries. therefore, it is essential that strategic, policy, and education initiatives continue to focus on managing and controlling such (predominantly needless) infections. the topic of hcais is a very broad issue, and it has therefore not been possible to cover all aspects of hcais in one paper; hence, we have been selective in selecting key aspects of the current debate. patient safety and quality: an evidence-based handbook for nurses healthcare -associated infections: a public health problem incidence of adverse events and negligence in hospitalized patients: results of the 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health care. who guidelines on hand hygiene in health care: first global patient safety challenge clean care is safer care. geneva: who hand hygiene: back to the basics of infection control a short history of midwifery. philadelphia; london: w. b. saunders company the authors are grateful to dr zakirul islam, associate professor and head of the department, pharmacology and therapeutics, eastern medical college, comilla, bangladesh for his cooperation in converting the video abstract from a powerpoint file to video format. the authors report no conflicts of interest in this work. infection and drug resistance is an international, peer-reviewed openaccess journal that focuses on the optimal treatment of infection (bacterial, fungal and viral) and the development and institution of preventive strategies to minimize the development and spread of resistance. the journal is specifically concerned with the epidemiology of antibiotic resistance and the mechanisms of resistance development and diffusion in both hospitals and the community. the manuscript management system is completely online and includes a very quick and fair peerreview system, which is all easy to use. visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors. health care-associated infections and prevention strategy key: cord-317499-mxt7stat authors: saraya, takeshi; kurai, daisuke; ishii, haruyuki; ito, anri; sasaki, yoshiko; niwa, shoichi; kiyota, naoko; tsukagoshi, hiroyuki; kozawa, kunihisa; goto, hajime; takizawa, hajime title: epidemiology of virus-induced asthma exacerbations: with special reference to the role of human rhinovirus date: 2014-05-26 journal: front microbiol doi: 10.3389/fmicb.2014.00226 sha: doc_id: 317499 cord_uid: mxt7stat viral respiratory infections may be associated with the virus-induced asthma in adults as well as children. particularly, human rhinovirus is strongly suggested a major candidate for the associations of the virus-induced asthma. thus, in this review, we reviewed and focused on the epidemiology, pathophysiology, and treatment of virus-induced asthma with special reference on human rhinovirus. furthermore, we added our preliminary data regarding the clinical and virological findings in the present review. more than 200 different types of viruses, such as human rhinovirus (hrv), human metapneumovirus (hmpv), respiratory syncytial virus (rsv), and human parainfluenza virus (hpiv), are known to cause acute respiratory illness (ari; tsukagoshi et al., 2013) . we recently reported the issue of "virus-induced exacerbation in asthma and chronic obstructive pulmonary disease" (kurai et al., 2013a) , however, among these causative viruses, hrv is now recognized to have a major impact on asthma pathogenesis (fujitsuka et al., 2011) . from this perspective, we reviewed the literature regarding the epidemiology of hrv-induced asthma in adults, together with preliminary epidemiological data obtained at our institution. hrv belongs to the genus enterovirus and family picornaviridae (turner and couch, 2007) . hrv possesses a single strand positive-sense rna (ssrna) genome of approximately 7.2 kb. the viral capsid is composed of four viral proteins (vp1-4) which are assembled into 60 protomers, resulting in a small icosahedral structure with a diameter of about 28-30 nm (turner and couch, 2007) . genetically, hrv is classified into three species; hrv-a, -b, and -c (simmonds et al., 2010) . furthermore, these species of hrv have more than 150 genotypes (andries et al., 1990; arakawa et al., 2012; kiyota et al., 2013 kiyota et al., , 2014 . molecular epidemiological studies suggest that the dominant species are hrv-a and -c, while hrv-b is relatively rarely detected (arakawa et al., 2012; kiyota et al., 2013) . in particular, the vp1 and vp2 proteins have variations in their amino acid sequences, accounting for the large number of viral serotypes (turner and couch, 2007) . the host receptor for hrv in respiratory epithelial cells is the intracellular adhesion molecule 1 (icam-1, cd54) for the 84 major hrv serotypes (hrv-a and -b), or low-density lipoprotein receptor (ldlr) for the other minor hrv serotypes. the receptor for hrv-c is not yet known. it has been suggested that the optimal temperature for replication of hrv is relatively cool (33-35 • c), which would limit infections to the upper airway; however, large or medium sized airways lower in the respiratory tract are now also considered cool enough for hrv replication, in spite of the higher temperature of the lung parenchyma (37 • c; mcfadden et al., 1985) . therefore, hrv is potentially a causative agent of more severe ari such as bronchiolitis and pneumonia (turner and couch, 2007; watanabe et al., 2010; smuts et al., 2011; arakawa et al., 2012) , and may be associated with virus-induced asthma linsuwanon et al., 2009; fujitsuka et al., 2011; smuts et al., 2011) . hrv might therefore be involved in various aris and additional respiratory complications (kiyota et al., 2013) . lieberman et al. (2009) reported that the detection of any virus include hrv, the sensitivity rates for nasopharyngeal swab (73.3%) was superior than that of oropharyngeal swab (54.2%), respectively. the common cold is the third most common primary diagnosis in office visits (hsiao et al., 2010) , and this disease is generally selflimiting, usually lasting up to 10 days (fashner et al., 2012) . among the general population, hrv infection causes common colds at a frequency of 25-53% (makela et al., 1998; van gageldonk-lafeber et al., 2005) . tyrrell et al. (1993) reported that intranasal www.frontiersin.org inoculation with either hrv serotypes 2, 9, and 14, coronavirus type 229e, or rsv in healthy volunteers induced patterns of symptom development which were not substantially different from each other. however, individual signs or symptoms occurred earliest in hrv infections, then in coronavirus, and lastly in rsv, appearing up to 5 days after inoculation, which demonstrated the long incubation periods of rsv in volunteers (tyrrell et al., 1993) . hrv has been implicated in patients with acute otitis media, exacerbation of chronic obstructive pulmonary disease, common cold, and lower respiratory tract infections in neonates, the elderly and immunocompromised. arruda et al. (1997) researching the frequency and natural history of hrv infections in adults during autumn, demonstrated that the first symptom noticed most often was sore throat (40%) in hrv culture-or pcr-positive patients, and stuffy nose in hrv-negative patients (27%), using nasal wash specimens. respiratory symptoms typically develop after 1-2 days after inoculation in studies, and uncomplicated hrv infections usually peak 2-4 days after inoculation. the median duration of hrv colds is 1 week, but up to 25% last more than 2 weeks (gwaltney et al., 1967; rotbart and hayden, 2000) . it should be noted that in illness caused by hrv, viral shedding occurs naturally for up to 21 days, but predominantly over a 3-4 days period. hrv-a type16 (hrv-16), a major group virus commonly used for experimental human infection, and hrv-a type1 (hrv-1), which has been used in animal models of hrv infection, are closely related. grunberg et al. (1999) reported that experimental hrv-16 infection via nasal inhalation leads to a transient decrease of fev 1.0 in patients with asthma, and this decreased lung function was correlated with enhanced cold symptoms and / or airway hyperresponsiveness. contoli et al. (2006) demonstrated that type iii interferon (ifn-λ) production levels in ex vivo cell cultures derived from bronchial epithelial cells (becs) and macrophages obtained from asthmatic patients, were lower than in those derived from healthy controls. furthermore, deficient interferon-λ production was correlated with hrv viral load, severity of clinical symptoms and fev 1.0 . message et al. (2008) demonstrated that the severity of intranasally inoculated hrv-induced clinical illness in asthmatic subjects was correlated to virus load and lower airway virus-induced inflammation. on the other hand, demore et al. (2009) reported that no difference in clinical symptoms, and patterns of viral shedding, was noted between subjects with persistent allergic asthma and healthy subjects after experimental infection with hrv. these different results after experimental hrv infection in individual studies in asthmatic patients and healthy subjects might be dependent on the severity of the asthma of those subjects who enrolled in the studies. indeed, in several reports, neither defective ifn induction by hrv, nor increased hrv replication was observed in primary human becs derived from subjects with well controlled asthma (lopez-souza et al., 2009; bochkov et al., 2010; sykes et al., 2014) . a few animal models for rhinovirus infection have been showed because a major group of hrv (i.e., hrv-16) did not bind mouse icam-1. only a minor group of hrv (i.e., hrv-1b) infected the mouse. in this regard, bartlett et al. (2008) generated a transgenic balb/c mouse expressing a mouse-human icam-1 chimeric receptor for hrv-16 infection. this study also showed asthma exacerbation model by intraperitoneally sensitized with ovalbumin with aluminum hydroxide followed by intranasal inoculation of hrv-1b or uv-inactivated hrv-1b. although data regarding virus respiratory infections (vris) as precipitators of asthma attacks in adults are less clear, nicholson et al. (1993) reported that vris are as commonly linked to exacerbations in adults as they are in children (johnston et al., 1996; fujitsuka et al., 2011) . this study showed that viruses were detected in 44% of clinical exacerbative episodes with a decrease in peak expiratory flow rate (pefr) of 50 ml/minute or more, and the most commonly identified virus was hrv, followed by coronaviruses and parainfluenza viruses (nicholson et al., 1993) . thus, the virus most commonly detected in asthma exacerbations appears to be hrv. although hrv is well known as the most frequent cause of the common cold, the implications of hrv infection vary according to respiratory diseases. table 1 shows the frequency of hrv infection in various adult respiratory diseases such as exacerbation of asthma (nicholson et al., 1993; atmar et al., 1998; tan et al., 2003) , common cold (makela et al., 1998; van gageldonk-lafeber et al., 2005) , exacerbation of copd (seemungal et al., 2001; rohde et al., 2003; tan et al., 2003; beckham et al., 2005; papi et al., 2006; hutchinson et al., 2007; ko et al., 2007; mcmanus et al., 2008; kherad et al., 2010; dimopoulos et al., 2012; perotin et al., 2013) , community acquired pneumonia (jennings et al., 2008; johnstone et al., 2008; johansson et al., 2010; lieberman et al., 2010; fry et al., 2011; wootton et al., 2011; luchsinger et al., 2013; takahashi et al., 2013; huijskens et al., 2014) , exacerbation of idiopathic pulmonary fibrosis (wootton et al., 2011) , and asymptomatic infection (fry et al., 2011) . the risk of exacerbations of asthma in adults is elevated after children return to school, and around december 25th (the christmas holiday in westernized countries), and this is likely to be due to social interactions with children at these times. prospective monitoring studies using reverse transcription polymerase chain reaction (rt-pcr) indicate that as many as 85% of acute asthma exacerbations in children, and about 60% in adults, were associated with the presence of upper respiratory tract (urt) infections. corne et al. (2002) found that the detection rates of hrv in asthmatic (10.1%) and healthy participants (8.5%) were similar, but the lrt symptoms were significantly more severe and longer lasting in the asthmatic group than in the healthy group based on one definition of urt and lrt symptoms ( table 2 ; johnston et al., 1995) . there is no common antigen across all strains of hrvs; therefore, no reliable diagnostic method for hrv infection has been established using hrv antigens or hrv-specific antibody. although viral culture is the conventional method for hrv detection, culture methods are not practical in clinical settings for the detection of hrv, because of its slow growing character and requirement for specific culture conditions. furthermore, the diagnostic capability of molecular amplification techniques frontiers in microbiology | virology exacerbation of asthma 26-36 nicholson et al. (1993) , tan et al. (2003) , atmar et al. (1998) common cold 25-53 makela et al. (1998) cited and adapted from johnston et al. (1995) . such as nucleic acid sequence-based amplification and rt-pcr is superior to those of culture methods (loens et al., 2006) . experimental hrv infections have been shown to lead to a longlasting excessive airway narrowing in volunteer subjects with asthma (cheung et al., 1995; grunberg et al., 1999) . of note, rhinovirus, unlike influenza and other viruses, causes minimal cytotoxicity (fraenkel et al., 1995) , and the amount of epithelial damage does not correlate with the severity of the symptoms. hrv infection can cause additive or synergistic effects in exacerbation of asthma via the influx of additional inflammatory cells in the airways with preexisted inflammation, resulting in airway cholinergic hyperresponsiveness (nagarkar et al., 2010) , as an allergic response. the effects of hrv infection such as enhanced contractility of airway smooth muscle (asm) cell and impaired relaxation to cholinergic or β-adrenaergic agonists are attributed solely to binding of the virus to its host receptor icam-1 on the asm cell surface. this proasthmatic-like effect was recognized even in the situation of complete inhibition of viral replication in vitro, but not in the setting of pretreatment of asm with neutralizing antibody directed against for icam-1 (grunstein et al., 2001) . thus, the hrv attachment to icam-1 itself can affects the contractility of asm cells in the absence of any cytopathic effects, and chun et al. (2013) reported that a 549 cells infected with hrv in vitro produced a higher value of il-8 and rantes than those of rsv or adenovirus. in addition, only the combination of hrv with der f1 (house dust mites antigen) acted synergistically to induce il-8 production. these findings are the reason why the hrv can be a major pathogen for acute exacerbation of asthma. we present a schema for pathogenesis in hrv associated asthma exacerbations (figure 1) , which requires the following steps, (1) hrv attachment to airway epithelial cells, (2) an innate immune response which leads to epithelial damage, (3) infection-related airway remodeling. when rt-pcr is used to either supplement or replace conventional culture techniques, viruses have been found in approximately one half to three quarters of adults experiencing an acute wheezing episode (jackson and johnston, 2010) , and the majority (59%) of viruses identified were hrvs (nicholson et al., 1993) . however, the evidence is weak, and mechanisms are poorly understood. initially, hrv-a and -b attach to airway epithelial cells via icam-1 or ldlr (kennedy et al., 2012) . the receptor or receptors for the recently identified group hrv-c have yet to be clarified. hrv-infected becs secrete a wide range of cytokines and chemokines such as il-1, il-6, ccl5/rantes (regulated on activation, normal t cell expressed and secreted), cxcl8/il-8, gm-csf, and cxcl10/interferon-inducible protein 10 (ip-10; jackson and johnston, 2010; proud, 2011) , which induce neutrophilic, lymphocytic, and eosinophilic inflammation together with airway hyperresponsiveness and airway remodeling (wark et al., 2002; proud, 2011) . clearance of viral pathogens begins with interferon secretion, and the underproduction of these factors has been postulated to lead to viral-induced exacerbations. there are three types of interferons, based on the receptors they bind: type i (ifn-α/β), type ii (ifn-γ), www.frontiersin.org and type iii (ifn-λ). hrv infection induced epithelial expression of mrna for both type i and type iii ifns, and it has been suggested that impaired epithelial production of ifn-β and ifnλ in asthmatic subjects may contribute to viral exacerbations of asthma (wark et al., 2005; contoli et al., 2006) . contoli et al. (2006) showed significant inverse correlations between ex vivo production of ifn-λ and severity of symptoms, bronchoalveolar lavage viral load and airway inflammation, and a strong positive correlation with reductions in lung function during in vivo infection. genome-wide association studies showed that single nucleotide polymorphisms involve in various diseases. interferon-λ polymorphisms may effect on the incidence of hrv infection (russell et al., 2014) . message et al. (2008) reported virus load in asthmatic subjects as being related to increased lower airway inflammation, and in turn increased lower airway inflammation being related to increased symptoms, reductions in lung function, and increases in bronchial hyperreactivity. these data suggest a causal role for hrv infection in the pathogenesis of asthma exacerbations. investigating virus-allergen interactions, durrani et al. (2012) demonstrated that another mechanism that increased expression and cross-linking of the high-affinity ige receptor, fcεri, on plasmacytoid dendritic cells is associated with reduced hrv-induced ifn-α and ifn-λ1 secretion, and allergic asthmatic children have significantly reduced hrv-induced ifn-α and ifn-λ1 production after cross-linking of fcεri. type 2, or inducible, nitric oxide synthase (inos) is the major nos isoform found in epithelial cells and can generate substantial amounts of nitric oxide (no). the no molecules both inhibit the replication of hrv in airway epithelial cells, and suppresses hrv-induced cytokine production (proud, 2005) . although the measurement of fractional no concentration in exhaled breath (feno) may be used to support the diagnosis of asthma (dweik et al., 2011) , however, increasing of feno seems to be not always correlated with viral load during the period of hrv infection (sanders et al., 2004) . other factors such as allergy, allergen exposure, tobacco smoke, particulates, ozone, stress, and infections such as sinusitis commonly contribute to exacerbations of asthma. grainge et al. (2011) reported that repeated bronchoconstriction in asthma promotes airway remodeling, and there is now clear evidence that airway remodeling begins in early childhood, and can be present even before clinical diagnosis of asthma is established (pohunek et al., 2005) . increasing evidence regarding hrv-induced wheezing or exacerbation of asthma raises the possibility that hrv infections could contribute to the initiation and subsequent progression of airway remodeling, which involves multiple factors such as increased epithelial release of mucin5ac (mu5ac), activin a, amphiregulin, matrix metalloproteinase 9 (mmp9), epidermal growth factor (egf), fibroblast growth factor (fgf), and vascular endothelial growth factor (vegf). hrv infection upregulates production of muc5ac from epithelial cells, which leads to airflow obstruction in asthma (hewson et al., 2010) . activin a is a member of the tgf-β superfamily and amphiregulin, a member of the egf family, alters repair processes (leigh et al., 2008) . both activin a and amphiregulin have been linked to subepithelial basement membrane thickening in asthma. mmp9 appears to have important roles in asthma exacerbation and airway remodeling (sampsonas et al., 2007) . expression of vegf and its receptors is increased in asthmatic subjects, and vegf is the major proangiogenic activator in asthmatic airways (feltis et al., 2006; simcock et al., 2007) . kuga et al. (2000) reported that 61.5% of adult asthmatic patients with common cold suffered an asthma attack, and common cold was significantly associated with acute exacerbations of asthma. they also stated that hrv infection might be important as the virus was detected by rt-pcr in throat gargles (kuga et al., 2000) . virus-induced exacerbation of asthma is a critical issue for the general physician. however, among asthmatic patients with exacerbative status, distinguishing between those patients which have vris, and those who do not, is difficult. furthermore, epidemiological data regarding adult asthma exacerbations have been sparsely reported. to investigate the prevalence of vri in exacerbations of adult asthma in both hospitalized or not-hospitalized patients, characterization of clinical and radiological findings was performed. a prospective observational cohort study was conducted at kyorin university hospital, tokyo, japan from august 2012 to august 2013 (kurai et al., 2013b) . all patients with respiratory symptoms associated with exacerbation of asthma were included, and samples were collected by nasopharyngeal or oropharyngeal swab, and subjected to a pcr method to detect common respiratory viruses. the 44 patients who were enrolled consisted of hospitalized (n = 15) or not-hospitalized patients (n = 29; table 3 ). in these two groups, the subject's backgrounds were similar for age, sex, smoking rates, and duration of illness, however, the measured value of spo 2 was significantly lower in hospitalized patients (87 ± 2.3%) than in non-hospitalized patients (96.2 ± 0.7%). the incidence of vri was significantly higher in the former group (46.7%, n = 7) than in the latter group (6.9%, n = 2; p = 0.002). in the latter group, influenza virus alone was detected in both patients. furthermore, all hospitalized patients (100%, n = 15) had wheezing or severe exacerbation based on the ats (american thoracic society)/ers (european respiratory society) statement (reddel et al., 2009) , whereas, among nonhospitalized patients, only nine patients (31%) were considered as having a severe exacerbation (p < 0.001), and 10 patients (38.4%) had wheezing (p < 0.001). these findings suggested that virus infection was certainly associated with the hypoxemia and / or wheezing which resulted in a severe or serious asthma attack, based on the japanese guidelines (ohta et al., 2011) or the ats/ers statement (reddel et al., 2009) . previous studies using ohta et al. (2011) , † † defined by reddel et al. (2009) . ** p < 0.01, *** p < 0.001. all data are presented as (mean ± sd). pcr-based viral diagnostics found that viral respiratory infections were detected in up to 85% of exacerbations of asthma in children and about 50% of exacerbations in adults (nicholson et al., 1993; johnston et al., 1995) , which is similar to our results. serum inflammatory or allergic markers are not different between the hospitalized and non-hospitalized patients ( table 3) . in hospitalized patients, the viruses identified were hrv (n = 5), hmpv (n = 1), and rsv (n = 1). at the time of admission, the virus-positive group (n = 7) had significant lower values of spo 2 (81.4 ± 3.9%) than those of the virus-negative group (n = 8, spo 2: 91.8 ± 1.3%, p < 0.007), and for the patients whose data are available, the frequency of hypercapnea (paco 2 45 torr) was significantly higher in the virus positive group (66.7%, n = 4) than in the virus negative group (0%; p = 0.014; table 4 ). the mechanisms for hypercapnea in virus infected individuals have not been elucidated. however, cheung et al. (1995) reported that hrv infection causes long lasting excessive airway narrowing in response to methacholine in asthmatic subjects. we speculated that smooth muscle might have a role in exaggerated airway narrowing in virus positive asthmatic patients, as described by king et al. (1999) . interestingly, the incidence of ground glass opacities (ggo) on high resolution computed tomography seemed to be higher for virus-positive hospitalized patients than for virus-negative patients, but it did not reach statistical significance. for example, figure 2a shows a patchy ggo with thickening of interlobular septa in a 28-year-old woman who was admitted during an asthma attack induced by hrv-a. figure 2b also shows ggo in a 62-year-old man with an asthma attack caused by hrv-c infection. these ggo in both patients could only be detected in hrct, not in chest x-ray. these results suggested that hrv was the major cause of virusinduced asthma, and was possibly involved in lower airway or lung parenchyma features, appearing as ggo. viral infection significantly exaggerated the respiratory status (low spo 2 and hypercapnea) when compared to that of virus-negative asthma exacerbative patients at the time of admission. indeed, in recent years, hrv has been recognized as a common cause of hospital admission, both as an agent of bronchopneumonia and through exacerbation of chronic pulmonary conditions, even in the elderly over 65 years of age (pierangeli et al., 2011) . curiously, after initiation of treatment with intravenous steroid, both the virus-positive and -negative groups had no significant difference in duration of respiratory failure, wheezing, days in hospital, and even in the time required for steroid treatment. no established treatment for prevention of hrv-induced asthma is available, and we describe the exploratory interventions as follows. inhaled corticosteroid (ics) is the main drug for regular asthma therapy. ics treatment improved airway hyperresponsiveness in asthmatic patients experimentally challenged with hrv, however, ics treatment did not reduce accumulation of inflammatory cells, except for eosinophils in bronchial epithelium (grunberg et al., 2001) . double-stranded rna (dsrna), a viral product and a ligand for the toll-like receptor-3 (tlr3), upregulates the expression of inflammatory chemokines in airway epithelial cells. matsukura et al. (2013) reported that treatment of beas-2b cells with fluticasone propionate significantly and dose-dependently inhibited dsrna-induced expression of ccl5, cxcl8, and cxcl10 protein and mrna. to confirm the effect on ssrna, such as that of hrv, would need further studies. leukotriene receptor antagonist was prescribed in asthmatic patients with or without ics. montelukast treatment did not improve asthma control or cold symptom scores when hrv were experimentally inoculated into mild asthmatics, or healthy subjects (kloepfer et al., 2011) . it is uncertain whether leukotriene receptor antagonist treatment is effective in the reduction of asthma symptoms associated with hrv infection. zambrano et al. (2003) reported that high serum ige levels in mildly asthmatic children with experimental hrv infection may be associated with enhanced lower respiratory symptoms and elevation of inflammatory markers, such as nasal eosinophil cationic protein and expired nitric oxide, than those of healthy subjects and/or low ige asthmatic patients. the prevalence of asthma was closely associated with the serum ige levels standardized for age and sex (burrows et al., 1989) , and airway hyperresponsiveness appears to be closely linked to the allergic diathesis, as reflected by the serum total ige level (sears et al., 1991) . omalizumab, an anti-ige monoclonal antibody, was indicated in inadequately controlled moderate-to-severe persistent allergic asthma patients who were treated with high dose ics. durrani et al. (2012) showed that the ige receptor fcεri is inversely associated with ifn-α and ifn-λ1 secretion when plasmacytoid dendritic cells derived from allergic asthmatic children were challenged with hrv. omalizumab downregulates fcεri expression on dendritic cells (prussin et al., 2003) , which may reduce exacerbation of asthma associated with increased production of ifns, through fcεri. no drugs are clinically used in hrv infection, although several drugs have been tried for treatment and prevention of hrv infection. these drugs are summarized in a review (jacobs et al., 2013) . ifns had a potential protective role in viral induced asthma (cakebread et al., 2011; gaajetaan et al., 2013) . becker et al. (2013) showed that exogenous ifn-α, ifn-β, ifn-λ1, and ifn-λ2 inhibited hrv replication in becs from healthy donors. macrolides are known to possess anti-inflammatory and immunomodulatory actions extending beyond their antibacterial activity in pulmonary inflammatory disorders (takizawa et al., 1995; min and jang, 2012) . erythromycin inhibits hrv infection by reducing icam-1 expression on the surface of human tracheal epithelial cells, and modulates inflammation by suppressing the production of proinflammatory cytokines (suzuki et al., 2002) . yamaya et al. (2014) reported that the mucolytic drug ambroxol hydrochloride, antibiotic drug of levofloxacin (yamaya et al., 2012b) , and bronchodilators (tiotropium, tulobuterol, and procaterol) for asthma or copd (yamaya et al., 2011 (yamaya et al., , 2012a (yamaya et al., , 2013 may have a beneficial effect in hrv infection, by inhibiting hrv replication and partly reducing icam-1 expression and acidic endosome production, via the inhibition of nf-kappab activation (yamaya, 2012) . we reviewed the previous reports regarding hrv-induced asthma exacerbations, together with our results from an institutional prospective study. hrv is a major pathogen for asthma exacerbations, and certainly associated with more serious clinical conditions such as hypoxemia or hypercapnea in hospitalized patients. further accumulation of evidence of virus-induced asthma for multidisciplinary assessment would be helpful for physicians in recognizing the condition or understanding the pathogenic mechanisms. two groups 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copd exacerbations the mechanics of exaggerated airway narrowing in asthma: the role of smooth muscle genetic analysis of human rhinovirus species a to c detected in patients with acute respiratory infection in kumamoto prefecture genetic analysis of the vp4/vp2 coding region in human rhinovirus species c in patients with acute respiratory infection in japan effects of montelukast on patients with asthma after experimental inoculation with human rhinovirus 16 viral etiology of acute exacerbations of copd in hong kong the correlation between the exacerbation of bronchial asthma and picornavirus (human rhino virus) infection in throat gargles by rt-pcr virusinduced exacerbations in asthma and copd respiratory viral infection in admitted adult patients human rhinovirus infection enhances airway epithelial cell production of growth factors involved in airway remodeling identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling respiratory viruses in adults with community-acquired pneumonia high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season in vitro susceptibility to rhinovirus infection is greater for bronchial than for nasal airway epithelial cells in human subjects community-acquired pneumonia in chile: the clinical relevance in the detection of viruses and atypical bacteria viruses and bacteria in the etiology of the common cold basic research on virus-induced asthma exacerbation: inhibition of inflammatory chemokine expression by fluticasone propionate thermal mapping of the airways in humans respiratory viral infection in exacerbations of copd rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and 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species and their clinical presentation among different risk groups of non-hospitalized patients viral infection in acute exacerbation of idiopathic pulmonary fibrosis virus infection-induced bronchial asthma exacerbation inhibitory effects of tiotropium on rhinovirus infection in human airway epithelial cells levofloxacin inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells procaterol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells tulobuterol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells ambroxol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells experimental rhinovirus challenges in adults with mild asthma: response to infection in relation to ige the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-326328-9w2p3xla authors: jenkins, ian a.; saunders, michael title: infections of the airway date: 2009-06-25 journal: paediatr anaesth doi: 10.1111/j.1460-9592.2009.02999.x sha: doc_id: 326328 cord_uid: 9w2p3xla infections of the airway in children may present to the anesthetist as an emergency in several locations: the emergency department, the operating department or on intensive care. in all of these locations, relevant and up to date knowledge of presentations, diagnoses, potential complications and clinical management will help the anesthetist and the surgical team, not only with the performance of their interventions, but also in buying time before these are undertaken, avoiding complications and altering the eventual outcome for the child. diseases such as epiglottitis and diphtheria may show diminished incidence but they have not gone away and their clinical features and essential management remain unchanged. paradoxically, perhaps, some conditions such as lemierre’s syndrome appear to be making a comeback. in these instances, clinicians need to be alert to these less common conditions, not only in regard to the disease itself but also to potentially serious complications. this article describes those infections of the airway that are most likely to present to the anesthetist, their attendant complications and recommendations for treatment. given that both viral and bacterial infections may produce disorders of similar presentation and sometimes may do this concurrently, it is more logical to examine these disorders by the site and nature of their condition rather than by their infective origin. in a 6-year series of head and neck infections in children, it was noted that 49% affected the peritonsillar space, 22% the retropharyngeal and 2% para-pharyngeal (1) . tonsillitis, peritonsillar abscess, retro-or parapharyngeal abscesses may compromise the airway and so these will be described in turn, together with rarer but important conditions and their complications. bacterial tonsillitis & peritonsillar abscess (quinsy). bacterial tonsillitis can cause airway compromise without extension into surrounding tissue (figure 1 ), although this is unusual and more likely in mononucleosis (see below). the lingual tonsil has also been implicated in airway compromise (2) . the typical flora associated with tonsillitis comprises aerobes; streptococcus pyogenes, staphylococcus aureus and pneumococci, and anaerobes; fusobacterium spp., prevotella spp., porphyromonas spp. and actinomyces spp. aerobes predominate in the acute primary infection, whereas anaerobes are associated with abscess formation or extension across tissues that form deeper infections through fascial planes (3) . penicillin resistance in beta-lactamase forming organisms is common and third generation cephalosporins or amoxicillin-clavulanate should be used, in part, because they allow growth of 'nonpathogenic interfering bacteria' which compete with the pathogens (3) . where the infection has extended outside of the tonsil itself, antibiotics effective against anaerobes should be used; metronidazole, carbapenems or beta-lactamase resistant penicillins (e.g. amoxicillin-clavulanate, piperacillin-tazobactam). if the patient is toxic, then clindamycin or linezolid should be added for their ability to prevent bacterial exotoxin release (4) . there is some controversy whether surgical removal of tonsils that have caused airway compromise should be undertaken early because of possible excessive bleeding (5) . when pus has formed, surgical drainage is usually indicated to prevent spontaneous rupture and the serious risks of aspiration, extension into the mediastinum or laterally causing erosion of blood vessels (6) . infectious mononucleosis (im) can cause compromise of the airway ( figure 2 ) and this has been reported in as many as 25-60% of children presenting with im (7) (8) (9) . although most authors since the 1960s have advocated the use of glucocorticoids to avert the need for surgical intervention, several series note that, despite steroids, 40-88% of patients with airway obstruction required tonsillectomy (7, 9) . in these reports, no cases of excessive hemorrhage were noted. the use of glucocorticoids does reduce duration of fever, pharyngitis and abnormal hematological findings and does not appear to be associated with a predilection for development of peritonsillar abscess (10). the bacterial organisms involved in these infections are identical to those producing peri-tonsillar abscesses and can be considered together from the point of view of their microbiology and clinical management as 'deep neck infections' (11, 12) . the retropharyngeal space contains loose connective tissue and lymph nodes that drain the nasopharynx, paranasal sinuses, middle ear, teeth and adjacent bones. retropharyngeal abscesses are more common in young children and this may be because lymph tissue in this area involutes and atrophies in older patients (13) . most studies give a male to female ratio of nearly 2 : 1 (11, 14) and a median age of 32.5-36 months (15, 16) . the incidence of this condition appears to be markedly on the rise (15) . in a recent series covering 11 years (1995-2006) and 162 children with retropharyngeal abscesses, page (11) noted a linearly rising incidence over the study period. computed tomography was performed in 94% and had an accuracy of 68%. the principal symptoms were fever, sore throat, torticollis and neck pain. there was obstruction of the airway in only 8%. the commonest clinical signs were lymphadenopathy, local tenderness and limited range of movement. however, more specific findings were somewhat less common, e.g. pharyngeal bulge in 23% (figure 3 ), tonsillar deviation in 12% and drooling in 10%. at drainage, 80% of cases showed cloudy fluid or frank pus. a single organism was grown in 25% and polymicrobial in 79%; organisms include group a b-hemolytic streptococci, streptococci, staph. aureus, of which 30% were methicillin-resistant, moraxella, haemophilus and other mixed oropharyngeal flora. features associated with surgical drainage were symptoms for 2 days or more, prior administration of antibiotics and fluid on computed tomography scan with a cross-sectional area of >2 cm 2 . there was a trend indicating pharyngeal bulge as a factor but not significant (p = 0.051). some authors disagree with the primacy of surgery as the treatment of choice (16, 17) particularly where clindamycin was used in all cases and combined with cefuroxime in most (17) . airway compromise may be more frequent in those below 1-year-old group (18) .the anesthetic management of airways obstructed by such abscesses can be challenging. the airway must be secured without rupturing the abscess and soiling the airway with pus. inflammation may also have extended to adjacent tissues and the glottis may be hard to visualize and even to locate (19) . where there is stridor a cautious inhalational induction with an otolaryngologist present is appropriate, either in the or or in the icu. the use of a cuffed endotracheal tube will be useful to prevent soiling of the lungs from either spontaneous rupture or surgical drainage of the abscess. the use of cuffed tubes, even in young children, has been shown to be safe (20) (21) (22) . administration of glucocorticoids does not feature in the large series quoted here and there is no evidence of benefit (11, 16) . more than two-thirds of deep neck abscess contain beta-lactamase producing organisms and most abscesses contain anaerobes (6) . antibiotic treatment must take these features into account. complications of these abscesses are descending mediastinitis and lemierre's syndrome. mediastinitis following retropharyngeal abscess (see figure 4 ) may be more common in younger patents and those with methicillin-resistant staph. aureus (mrsa) infections (15) . mediastinitis has been associated with a high mortality ranging from 16.5 to 50% and the place of surgery to drain the areas affected a subject of debate ct with contrast -retropharyngeal abscess with descending mediastinitis: a, abscess; b, nasogastric tube. but should probably depend on response to maximal antibiotic therapy and monitoring of both clinical infective markers and radiological appearances (23). this is a relatively rare but possibly increasingly seen complication of pharyngo-tonsillar infections (24, 25) ; 'the forgotten disease' (24) . in 1936, lemierre described a condition of 'anaerobic postanginal septicaemias' associated with 'bacillus funduliformis' -now known as fusobacterium necrophorum -and 'b. symbiophiles' (26). this paper described an evolving condition beginning with suppuration at the local site, followed by thrombophlebitis with septic emboli a later feature. no specificity was attributed to f. necrophorum but rather a recognition that the picture may be caused by normally saprophytic anaerobes, possibly working in synergy (26) , and indeed its growth may be promoted by coexisting aerobes (27) . the septicemic phase typically occurs 4-5 days after the onset of a sore throat or tonsillitis and is characterized by a rise in temperature and rigors, whereas the original pharyngeal ⁄ tonsillar condition may have improved (28) . the infection has local effects, classically causing thrombosis in the ipsilateral internal jugular vein (ijv), and more distant spread with suppuration most commonly affecting the lungs, but also causing septic arthritis and osteomyelitis, meningitis and liver, renal and skin abscesses ('necrobacillosis') (28). lemierre's syndrome is normally associated with previously healthy adolescents and young adults. mortality in the preantibiotic era was 90% and still runs at 4-12% depending on promptness of detection (29) and where fulminant, cavitating suppuration can still be seen (30) . however, the full syndrome has been reported in a 3-year old child (31) . additionally, ijv thrombosis has been reported in a 5-month old with mastoiditis infected with f. necrophorum (32) and in a 3-month-old female infant with a retropharyngeal abscess infected with mrsa (33) . examples of both thrombosis of the ijvs and thrombosis in the pulmonary artery are shown in figures 5 and 6 in a patient presenting with a retropharyngeal abscess complicated by descending mediastinitis (seen above in figure 4) where streptococcus milleri was the organism isolated from blood and abscess. the incidence of this condition is rising. whether this is due to better forms of detecting the anaerobic bacteria involved is doubtful, as this is a condition with a distinct clinical presentation (25, 26, 30) . another theory is that the move away from treating pharyngitis and tonsillitis with antibiotics in primary care has allowed bacterial infections to cause the conditions in which these anaerobic saprophytes can cause opportunistic, but disastrous, infections (25, 27, 28) . additionally, there may be a genetic explanation why some individuals develop a serious infection from a normally harmless saprophyte (34). ct with contrast -retropharyngeal abscess with thrombophlebitis: a, right internal jugular vein with intraluminal thrombosis (also seen on left); b, internal carotid artery; c, trachea with endotracheal tube; d, nasogastric tube; e, descending retropharyngeal abscess; f, replogle tube placed into abscess cavity. ct scan with contrast -thrombus sitting within branch of left pulmonary artery (same patient as figure 5 ). treatment should be intravenous to start with, using high-dose penicillin with metronidazole or monotherapy with clindamycin. in penicillin allergy, clindamycin should be used because f. necrophorum can show resistance to macrolides. a total duration of 2-6 weeks is recommended as viable bacteria can be found in necrotic abscess formations for some weeks (27) . the clinical case for or against anticoagulation is not clear; however, in cases of propagating thrombosis or embolization, heparin administration is advisable (27) . septic presentations in young people, even when these seem to affect distal sites such as the lungs or the liver should be accompanied by examination of the head, neck and throat to exclude a rare but classic disease (27, 30) . initially described in 1836, this is a diffuse infection of the submandibular and sublingual spaces. symptoms of severe pain, fever, malaise and dysphagia occur with swelling that can be large enough to cause airway compromise. normally associated with dental caries, sickle cell disease, immunodeficiency and trauma, it can occur de novo in children (35, 36) . in one series from india, the proportion of children was 24% (35) . it has been described in a 4-month-old infant (37) . bacterial isolates are variable; staphylococci, a-haemolytic streptococci and anaerobes such as bacteroides, peptococci and peptostreptococci (36) . antibiotic treatment covering these organisms should include metronidazole, penicillin, flucloxacillin or penicillin-betalactam inhibitor combinations: ticarcillin-clavulanate, amoxicillin-clavulanate or piperacillin-tazobactam. where penicillin allergy occurs, clindamycin is effective (36) . reduction of edema with glucocorticoids has been proposed (38) but there are no controlled studies to support this. surgical drainage is sometimes indicated where there is accumulation of pus (35) . recurrent respiratory papillomatosis (rrp) is caused by the human papilloma virus (hpv), usually types 6 and 11; the same types seen in more than 90% of genital condylomata and has a prevalence varyingly reported as 3-5 per 100 000 population (39, 40) . it usually affects the larynx and typical lesions are shown in figure 7 . the squamouscolumnar junction is the most frequently affected area (39) but this can extend distally to the larger airways in as much as 29% of patients and intrapulmonary in 7% (41) . the obstruction is of slow onset and the presenting symptom usually hoarseness but if the diagnosis is delayed respiratory obstruction may supervene (39) , especially with concomitant acute respiratory infection (40) . a more aggressive course is seen in the younger the age of first presentation and in those infected with type 11 (40) . possibly, 25% of all childbearing women worldwide harbor hpv in the genital tract. although cesarean section possibly reduces transmission, it is thought that transmission may occur in utero (42) . the papillomata are treated with laser (co 2 or ktp) or endoscopic micro-debridement. surgical access to these lesions is most often achieved using a suspension laryngoscope and anesthesia achieved with either spontaneous ventilation with insufflation of gases into the hypopharynx or by jet ventilation. the latter can be used either supra-or infra-glottically by rigid cannulae fixed to the laryngoscope (43) . in either case, an antisialogogue is administered and topical lidocaine sprayed onto the larynx (max. 6 mgaekg )1 ) (44, 45) . disadvantages of jet ventilation, particularly supra-glottically, are the potential dispersion of papillomatous matter distally and the need for muscle relaxation and, if used sub-glottically, one must ensure adequate escape of gases to prevent barotrauma. however, contamination of the field and the or with vapors are then avoided (46) . advantages of spontaneous ventilation are the preservation of the patient's respiratory drive, a potential safety factor, and the relative containment of debrided material. additionally, total intravenous anesthesia can reduce or supplant the need for vapors (44) (45) (46) . tracheostomy is generally avoided as this is associated with increased distal spread (41) . attention has been increasingly paid to 'adjuvant therapies' such as: interferon -given subcutaneously for 6 months, ribavirin and acyclovir. more recently intra-lesional injections of cidofovir have become the most common adjuvant in resistant cases and it has been given systemically for intrapulmonary lesions with some success. however, there is an association with carcinogenicity in animals and so it needs to be used selectively. recently, a quadrivalent vaccine has been developed against types 6, 11, 16 and 18, the major causes of rrp, and it is hoped that this will reduce the exposure of infants to this vertically transmissible disease (40) . historically, epiglottitis has been associated primarily with haemophilus influenzae infections, typically occurring in children aged 3 months to 5 years, with a peak incidence between 1 and 3 years, and characterized by a rapid onset of fever, drooling and stridor (47) . haemophilus influenzae is a gramnegative coccobacillus that affects only humans. serious infections are usually caused by the capsulated forms, serotypes a to f. however, type b (hib) was responsible for approximately 85% of invasive disease in children prior to immunization against this type (48) and epiglottitis accounted for about 12% of hib infections (49) . for those nations who undertook widespread immunization in the late 1980s and early 1990s, the incidence of hib-related infections dropped dramatically. the adult form seems different; a slower onset of symptoms where dysphagia and sore throat precede stridor (47), a more diffuse anatomic involvement, justifying the term supraglottitis (50) , and half the need for airway intervention (47) . in the uk, there was a resurgence in 2003 with over 260 cases of hib infections, causing a booster program to be launched (51) . the uk vaccine is now thought to be only 57% effective (52) . this may be due to its acellular composition. where whole cell vaccines are utilized, effectiveness rates of 95-100% have been demonstrated in california and the gambia (53) . in the postvaccination era, epiglottis is more likely to be an infection with a 'vaccination breakthrough' type b, or infection with any of the other organisms associated with this condition; h. parainfluenzae, group a streptococci, pneumococci or staphylococci (54) (55) (56) . in immuno-compromised patients, this can also include candida spp., herpes simplex type 1, varicella zoster and parainfluenza. vaccination failure is thought to be due either insufficient antibody level (57, 58) or to a defect in immunological priming and a decrease in avidity of anticapsular antibodies (59) . the child will be pyrexial and possibly toxic, dysphagic, possibly drooling, anxious and will often adopt a characteristic posture, sitting forward with the head extended; the so-called sniffing position (60) . this situation requires skilled airway management with inhalational induction, the presence of an otolaryngologist prepared to undertake emergency tracheostomy (58) and administration of broad spectrum antibiotics (54, 55, 61) . practice varies considerably regarding imaging prior to securing the airway. either computed tomography or lateral neck radiographs (looking for the 'thumb sign') are still frequently undertaken in some centers; 84% of cases in one us series (55) . stridor is a late feature and imaging should not delay securing the airway (45, 50) and consequently is very often not performed at all, where the priority is seen to be clinical diagnosis and the securing of the airway with confirmation of diagnosis at laryngoscopy (58) . the child can be re-intubated nasally and then managed on the pediatric intensive care unit (picu). practice varies between keeping the child sedated and possibly ventilated, or allowing the child to wake and breath spontaneously. if the child is awake, some form of physical restraint is frequently used to prevent accidental extubation and a heat and moisture exchanger attached to the endotracheal tube to prevent drying of secretions. the choice of antibiotics will vary depending on local flora but should always include an agent with beta-lactamase resistance; a third generation cephalosporin or a penicillin-derived drug combined with a beta-lactamase inhibitor (55, 58, 62) . some authors recommend administration of steroids (50,55) but this practice is not universal and does not alter outcomes with respect to intubation, duration of intubation, icu stay or hospitalization (47) . in a more recent series from denmark, the use of steroids was associated with longer hospital stay. this may have been because those receiving steroids were more severely compromised patients but, again, no evidence of benefit was demonstrated (63) . extubation is usually possible after 48 h (45). inhalational anesthesia can be employed so that the airway can be reassessed and extubation then performed under controlled conditions. this is a fairly common childhood disease characterized by a distinctive barky, hoarse cough progressing to stridor. this is initially associated with inspiratory subcostal recession and then, as the condition worsens, sternal recession and expiratory stridor. these symptoms are often preceded by a nonspecific upper respiratory tract infection for 12-48 h. denny et al. carried out an 11-year study showing that croup occurs between 6 months and 3 years of age, with a peak incidence during the second year. the parainfluenza viruses accounted for 74.2% of all isolates with 65% parainfluenza type1 (64) . respiratory syncytial virus (rsv), influenza viruses a and b, and mycoplasma pneumoniae were the only other agents isolated in appreciable numbers. rsv caused croup in children less than 5 years of age whereas the influenza viruses and m. pneumoniae were significant causes of croup only in children more than 5-6 years old (64) . an increasing variety of viruses are now associated with croup, undoubtedly because of improving methods of detection. in 2004, human metapneumovirus was demonstrated 20% of previously virusnegative samples, a fifth of these had presented with croup (65) . coronavirus was first described in german children in 2005 (66) and bocavirus recently in korea, with less predominance of parainfluenza 1 (67) . parainfluenza virus has specific effects on the ion transport of respiratory epithelium, which causes more airway secretions and exacerbates the effects of tissue edema (68) . mortality is relatively rare and an extensive review of the literature estimates overall mortality at 1 in 30 000 cases (60) . although the diagnosis is usually clear, the differential should include foreign body aspiration, epiglottitis, bacterial tracheitis, tonsillitis and peri-tonsillar and retropharyngeal abscesses, tracheobronchiomalacia or vascular rings. mediastinal masses can also present with 'croup' in all age groups (69) . on a worldwide basis, laryngeal diphtheria still occurs and also presents at any age (see below). the presentation is usually with acute onset of a barky, coarse cough, hoarseness and respiratory distress with stridor ( figure 8 ). pyrexia is usually present but the child should not drool nor appear toxic. laboratory and radiologic tests are not needed to confirm the diagnosis. indeed, such unnecessary disturbance may make the situation worse. only where the diagnosis remains uncertain in the absence of clinical features either for or against the diagnosis of croup should imaging take place. simple antero-posterior and lateral neck and chest radiographs may then indicate the presence of one of the differential diagnoses. it is clearly safer for the radiography to be performed in the ed, or or icu, rather than the child travel to the radiology department for such investigations (60) . the management consists of minimal disturbance, making a clinical assessment of the degree of severity and administering glucocorticoids, nebulized epinephrine, antipyretics. the use of humidified gases, although a traditional therapy, has been shown not to have any benefit on outcome (70) . most studies employ the 'westley score' of assessing the severity of the condition (71) but this has not found its way into general clinical practice because of reported inter-observer variance (60) . a meta-analysis of studies showed that treatment with glucocorticoids was associated with nearly a fivefold reduction on the rate of intubation (72) . if intubated, the duration of intubation and the rate of reintubation are reduced (73), although a meta-analysis was repeated in 1999, selecting only for randomized controlled studies, which found that while there was general benefit on administering glucocorticoids, no effect on intubation rates was demonstrated (74) . although the inhaled route is of benefit, the best routes to give glucocorticoids are intramuscular or oral (75) . it appears dexamethasone at 150 lgaekg )1 is superior to prednisolone 1 mgaekg )1 (76) . it has been observed that the duration of the anti-inflammatory effect of one dose of dexamethasone is 2-4 days and the natural history of croup last approximately 72 h, thus making subsequent doses superfluous (60) . regarding the exact dose of dexamethasone required, two recent studies suggest that 150 lgaekg )1 is as effective as 600 lgaekg )1 (77, 78) . should the clinical condition be severe on presentation or deteriorate despite steroids, value of nebulized epinephrine has long been established (71, 79, 80) . these effects have a duration of less than 2 h but there does not appear to be a 'rebound' in deterioration (71) . although the first studies were done using racemic epinephrine, the more commonly available 'l l-epinephrine' is just as effective (81) . a systematic review of dosage concluded that use of 3-5 ml of 'neat' 1 : 1000 l l-epinephrine was safe and effective (82) . paradoxically perhaps, the only report of ventricular tachycardia and a myocardial infarction in an otherwise healthy 11-year old with normal echocardiography and coronary anatomy was associated with the use of diluted racemic epinephrine (83) . should the above measures fail, then consideration can be given to the administration of a heliumoxygen mixture ('heliox'), which is examined in a separate section. otherwise, the child should be intubated in a controlled manner with the most skilled anesthetic practitioner available. the usual provisos apply: a full range of airway instrumentation and the presence of an otolaryngologist prepared to undertake tracheostomy should intubation fail. preservation of spontaneous ventilation till the airway is secured is still recommended, especially when there may be co-existing problems such as underlying tracheal stenosis or compression (69, 84) and best achieved with nonirritant inhalational vapors such as sevoflurane and halothane. typical appearances of the larynx on intubation are shown in figure 9 . endotracheal tubes of at least one whole size smaller than predicted by age should be available and so it is unrealistic to expect a leak round the endotracheal tube once the airway is secured. the croup -diffuse acute laryngotracheitis. child should be cared for in the intensive care unit and the size of the tube may well dictate whether the child should be managed spontaneously ventilating (and possibly awake) or remaining sedated and ventilated. the tube is left in place until there is a clear leak indicating resolution of the edema. if the clinical course extends beyond 48 h further dosing of steroids should be considered (60, 73) . normally this condition resolves within a week. if this does not occur then other diagnoses, mentioned above, should be considered. some clinicians prefer to extubate with the child re-anesthetized but spontaneously ventilating to assess the adequacy of the airway. if there is any suspicion of bacterial secondary infection, then treatment for bacterial tracheitis should be commenced (see appropriate section below). from a world viewpoint, diphtheria is still very much with us. it is caused by cornyebacterium diphtheriae and cornyebacterium ulcerans. it is a condition commencing with a croup-like illness, cough and sore throat, but often progressing to death through sepsis (disseminated intravascular coagulation and renal failure), suffocation by the 'pseudomembranes' of serocellular exudate forming in the respiratory tract, and direct effects of the powerful exotoxin that has affinity for neural endings (paralysis), cardiac muscle (heart block and myocardial failure) and the adrenal glands (hypotension with endocrine failure) (85) . electrocardiogram abnormalities can occur in the first week; the overall incidence of cardiomyopathy is 10-20% and this has a mortality of about 50% (86) . immunization was commenced in the uk in 1942 and the incidence of deaths plummeted, so that by 1952 it was extremely rare (87) . the last child deaths from diphtheria infection in the uk were in 1994, in an immigrant child infected in pakistan, and then in may 2008, in a child that was not immunized and had moved from europe to the uk in late 2007. swabs from family and hospital contacts proved negative (88) . sporadic cases occur in countries with developed immunization programs as a result of immigration (89) . unless there is a high index of suspicion, it is a diagnosis easily missed. even if correct management is instituted early, there is an appreciable mortality of 8-10% (86, 89) . presence of pseudomembranes and 'bull neck' are predictive of development of cardiomyopathy (86) . a breakdown in primary immunization and booster programs led to the major outbreak in former soviet states 1990-1995, where nearly 50 000 cases occurred (90) . in a large series of 154 children in 1 year in vietnam, treatment consisted of specific antitoxin, high-dose penicillin (or erythromycin if allergic) and intravenous hydrocortisone for severe neck edema. if edema or laryngeal pseudomembranes were causing obstruction, tracheotomy was performed (86). it is possible that this condition is becoming relatively more common. over a 10-year period in vermont, usa, of the airway infections causing picu admissions, tracheitis was the most common. whereas only 17% of viral croup admissions were intubated, 83% of those with tracheitis were. only 6% of picu admissions in this period had epiglottitis (91) . immunizations against haemophilus type b and the efficacy of glucocorticoids in viral croup may have changed the spectrum of picu admissions. the clinical presentation often follows a prodrome of viral upper respiratory infection, but with sudden progression of cough, stridor and pyrexia with a toxic element (92) (see figure 8 ). the endoscopic features are erythema, edema and thick purulent secretions (91) -see figure 10 ; sometimes these have the appearance of pseudomembranes (93) . the microbiology is mixed; predominantly staph. aureus -the most frequent according to most authors (91, 93, 94) , streptococcus pneumoniae, lancefield group a streptococci, moraxella catarrhalis and haemophilus spp. (92, 93) . the presence of moraxella was associated with a higher intubation rate (93) . these bacterial infections may occur in the presence of influenza virus a or b (91) but the presence of viruses did not relate to the intubation rate (93) . the therapy consists primarily of antibiotics; these should cover the likely flora and so include third generation cephalosporins with anti-staphylococcal agents, e.g. cefotaxime and flucloxacillin. glucocorticoids are not thought to have any beneficial effect on the course of the disease, which may develop into full blown sepsis with pneumonia and acute respiratory distress syndrome (91, 94) . salamone reported a 10-year series of 94 cases and described a subset of patients that have exudative pseudomembranes that required repeat debridement by bronchoscope and termed this 'exudative tracheitis' (93) . with increasing respiratory distress intubation is necessary. if the diagnosis is in doubt then inhalation induction should be considered and the presence of an otolaryngologist is recommended especially where tenacious secretions may require immediate bronchoscopy and bronchial toilet (93) . respiratory difficulties observed with obstructed airways may not be due simply the proximal obstruction itself. increasing respiratory effort is capable of quite large decreases in intrapleural pressure. this will increase the pressure gradient across the pulmonary capillary and override the starling equilibrium. the resulting transudation of fluid will cause acute pulmonary edema (95) . hypoxia itself will cause failure of the integrity of the alveolar-capillary membrane and exacerbate the transudative leak. the result is decreased pulmonary compliance which then, in turn, exacerbates the child's dyspnea and respiratory compromise (95, 96) . two gas physics equations provide the rationale for substituting helium for the nitrogen in air where there is airway compromise. the poiseuille equation applies to laminar flow: (dp is pressure gradient, r is radius, g is viscosity, l is length of airway). here, helium has nothing to offer; its viscosity (188 micropoises) approximates to those of nitrogen and oxygen (167 and 192, respectively). however, with a narrowed airway segment, as in many of the conditions referred to above, turbulent flow is more likely to occur. to maintain flow across a restricted cross-sectional area, gas velocity must increase. in these circumstances, turbulent flow is associated with a 'reynolds number' greater than 2000 (no units) as given by the following: (q is density and v average velocity). here, helium has a density 0.18 gael )1 , whereas nitrogen and oxygen densities are 1.25 and 1.43, respectively. a helium-oxygen mixture of 79 : 21 has a density of 0.43 gael )1 ; this means that, for a given velocity, the reynolds number will drop by three times and the propensity for turbulent flow drops accordingly (97) . 'heliox' refers to mixtures of helium and oxygen. potentially this could be in any ratio as found in the bespoke mixtures utilized in sub-aqua diving. however, medically, it is commonly provided as 79 : 21%. this can be added to an oxygen blender and the output monitored, thus providing a variable fio 2 as needed (97, 98) . in patients that are already hypoxic, the introduction of heliox should be done incrementally. as the viscosity of the inspired gases drops, any benefit or deterioration in oxgenation should be monitored carefully. when used in croup, heliox has been shown to procure improvements in croup scores similar to nebulized epinephrine (99) . reviews of the available evidence to support the use of heliox in this situation have demonstrated a need for further studies in this area bacterial tracheitis: erythema and mucosal edema with copious purulent secretions. (97, 98, 100) . helium has high thermal conductivity and so care needs to be taken with respect to humidification and avoidance of patient hypothermia (101, 102) and it should not be used in nebulizers as these depend on the generation of turbulence to deliver the drug (102) . acute infectious upper airway obstructions in children infectious mononucleosis complicated by lingual tonsillitis current management of upper respiratory tract and head and neck infections impact of antibiotics on expression of virulence-associated exotoxin genes in methicillin-sensitive and methicillin-resistant staphylococcus aureus early adenotonsillectomy for relief of acute upper airway obstruction due to acute tonsillitis in children microbiology and management of peritonsillar, retropharyngeal, and parapharyngeal abscesses the management of severe infectious mononucleosis tonsillitis and upper airway obstruction airway obstruction in children with infectious mononucleosis acute tonsillectomy in the management of infectious mononucleosis corticosteroids and peritonsillar abscess formation in infectious mononucleosis clinical features and treatment of retropharyngeal abscess in children surgical management of retropharyngeal space infections in children respiratory emergencies in children pediatric retropharyngeal abscesses: a national perspective pediatric mediastinitis as a complication of methicillin-resistant staphylococcus aureus retropharyngeal abscess retropharyngeal abscess in children: clinical presentation, utility of imaging, and current management intravenous antibiotic therapy for deep neck abscesses defined by computed tomography retropharyngeal abscess anesthetic management of a wooden parapharyngeal foreign body abscess the use of cuffed versus uncuffed endotracheal tubes in pediatric intensive care absence or presence of a leak around tracheal tube may not affect postoperative croup in children the future of the cuffed endotracheal tube descending necrotizing 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influenzae type b conjugate vaccines paediatric epiglottitis: the influence of the haemophilus influenzae b vaccine, a tenyear review in the sheffield region epiglottitis in the hemophilus influenzae type b vaccine era: changing trends antibody concentration and clinical protection after conjugate vaccination in the united kingdom paediatric acute epiglottitis: not a disappearing entity haemophilus influenzae type b vaccine failure in children is associated with inadequate production of high-quality antibody acute epiglottitis, sevoflurane and hib vaccination haemophilus influenzae type b epiglottitis as a cause of acute upper airways obstruction in children acute epiglottitis: epidemiology, clinical presentation, management and outcome croup: an 11-year study in a pediatric practice human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children croup is associated with the novel coronavirus nl63 the association of newly identified respiratory 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experience with ippb in the treatment of acute laryngotracheobronchitis inhalation of racemic adrenaline in the treatment of mild and moderately severe croup. clinical symptom score and oxygen saturation measurements for evaluation of treatment effects prospective randomized double-blind study comparing l-l-epinephrine and racemic epinephrine aerosols in the treatment of laryngotracheitis (croup) the safety of nebulization with 3 to 5 ml of adrenaline (1 : 1000) in children: an evidence based review pediatric myocardial infarction after racemic epinephrine administration acute infective airway obstruction associated with subglottic stenosis coryneform bacteria, listeria and erysipelothrix clinical features and predictors of diphtheritic cardiomyopathy in vietnamese children death of a child infected with toxigenic cornyebacterium diphtheriae in london unexpected combination of acute croup and myocarditis: case report current situation and control strategies for resurgence of diphtheria in newly independent states of the former soviet union changing epidemiology of life-threatening upper airway infections: the reemergence of bacterial tracheitis tracheitis in pediatric patients bacterial tracheitis reexamined: is there a less severe manifestation? systemic complications associated with bacterial tracheitis negative pressure pulmonary edema in children -pathogenesis and clinical management pulmonary edema associated with croup and epiglottitis heliox administration in the pediatric intensive care unit: an evidence-based review use of heliox in children a randomized comparison of helium-oxygen mixture (heliox) and racemic epinephrine for the treatment of moderate to severe croup heliox in croup heliox questions heliox questions the authors would like to acknowledge the help given by dr rob hawkes, paediatric radiologist, royal hospital for children, bristol, regarding the authors have declared no conflicts of interest. none. key: cord-339328-wizu3arz authors: jain, sanjay k. title: the promise of molecular imaging in the study and treatment of infectious diseases date: 2017-02-02 journal: mol imaging biol doi: 10.1007/s11307-017-1055-0 sha: doc_id: 339328 cord_uid: wizu3arz infectious diseases are a major threat to humanity, and it is imperative that we develop imaging tools that aid in their study, facilitate diagnosis, and guide treatment. the alarming rise of highly virulent and multi-drug-resistant pathogens, their rapid spread leading to frequent global pandemics, fears of bioterrorism, and continued life-threatening nosocomial infections in hospitals remain as major challenges to health care in the usa and worldwide. early diagnosis and rapid monitoring are essential for appropriate management and control of infections. tomographic molecular imaging enables rapid, noninvasive visualization, localization, and monitoring of molecular processes deep within the body and offers several advantages over traditional tools used for the study of infectious diseases. noninvasive, longitudinal assessments could streamline animal studies, allow unique insights into disease pathogenesis, and expedite clinical translation of new therapeutics. since molecular imaging is already in common use in the clinic, it could also become a valuable tool for clinical studies, for patient care, for public health, and for enabling precision medicine for infectious diseases. humans in the modern era are increasingly prone to virulent infections. selection and dissemination of bsuper bugs^by overuse (misuse) of antimicrobials, global travel and the rapid spread of infections, and the increasing use of medical procedures-implants, catheters, immunosuppressive/cancer therapies-are leading to life-threatening infections that are a growing threat to humanity [1] [2] [3] [4] . in 2011, about 722,000 americans developed hospital-acquired infections, resulting in 75,000 deaths in the usa. bacteria, notably from the enterobacteriaceae family (26.8 %) , clostridium difficile (12.1 %), staphylococcus aureus (10.7 %), and pseudomonas aeruginosa (7.1 %), accounted for the majority of these infections [5, 6] . similarly, viral infections such as influenza cause millions of new cases and several thousand deaths annually in the usa [7] . the burden of hospital-acquired and other infections is substantially higher globally. for example, mycobacterium tuberculosis, the causative agent for tuberculosis (tb), was responsible for 10.4 and 1.8 million new cases and deaths in 2015 alone [8] . the alarming rise of multi-drug-resistant (mdr) and extensively drugresistant (xdr) strains, as well as hiv co-infection, continues to fuel this epidemic [8] [9] [10] . global travel and rapid spread of infections-swine flu, sars-cov, ebola, and zika virus-have led to several recent global pandemics [11, 12] . finally, several infectious pathogens, e.g., yersinia pestis (causes plague) from the enterobacteriaceae family, are also recognized as biothreat agents [13] . these processes in vivo is a key to controlling infections. therefore, the world molecular imaging society has made a commitment for advancing imaging tools for infectious diseases and inflammation by creating the imaging of infection interest group (ioi-ig). this interest group is tasked with the mission to globally advance the development of new imaging technologies for infectious diseases and the translation of effective imaging tools to provide unique insights into disease mechanisms, enable drug and vaccine development, guide treatments, and benefit patients (fig. 1 ). as part of these efforts, the ioi-ig organized several sessions at the 2016 world molecular imaging congress (wmic). this included a 1-day workshop focused on imaging of infection and inflammation, which brought together individuals from multiple scientific disciplines and highlighted the latest developments in the field. in addition, the workshop also had a panel discussion on the challenges in funding research, with panelists from the us national institutes of health. a similar workshop is planned at the 2017 wmic (philadelphia, pa) and will focus on leveraging current resources, promoting collaborations, clinical translation, and finding strategies to overcome the challenges in funding research in this emerging field. rapid and accurate diagnosis and localization of infections are essential for effective early interventions and appropriate management. however, traditional diagnostic tools, namely microscopy, microbiology, and molecular techniques, are dependent upon sampling suspected sites of infection, often blindly, and then performing assays that can be time consuming. while major advances are being made in the application of molecular techniques such as nucleic acid amplification (naa), deep sequencing, and matrix-assisted laser desorption/ionization, they are all dependent on the availability of a relevant clinical sample. easily obtained clinical samples such as blood or urine may lack relevance and not contribute to a specific diagnosis, especially with deep-seated infections, and invasive biopsies are often required. biopsies can be costly, risky (anesthesia, dangers of surgery), and prone to incorrect sampling (limited to the tip of the biopsy needle) as well as lead to the introduction of artifacts or contamination. molecular imaging techniques detect crucial biological and biochemical changes at the sites of infection, often at the earliest phase of the disease, thereby allowing the clinician to promptly identify not only the infective or inflammatory focus but also the class of the causative pathogen and establish the best therapeutic approach for patients. tomographic imaging can evaluate disease processes deep within the body, noninvasively and rapidly. moreover, the ability of imaging to conduct noninvasive, longitudinal assessments in the same individual is a fundamental advantage over current traditional tools. other major advantages of imaging are its ability to provide a holistic, three-dimensional assessment of the whole organ or body, less likely to be limited by sampling errors and co-relating well with the overall disease process (table 1) . selected recent examples highlighting the unique role of imaging in infectious diseases are briefly described in this overview. optical imaging methods have been used extensively to study disease pathogenesis, largely in small laboratory animals. for example, real-time analysis of mycobacterium marinum infection in the transparent zebrafish has provided new insights into the temporal kinetics of host-pathogen interactions [14] . similarly, immune responses to central nervous system (cns) viral infections were visualized in real time using intravital twophoton laser scanning microscopy [15] . while highly sensitive, optical imaging is limited by the depth of the signal and is therefore well suited for the study of laboratory animal models but has limited clinical utility. the limited depth of penetration of signal is not a problem using nuclear medicine tools. for example, 2-deoxy-2-[ 18 f]fluoro-d-glucose ([ 18 f]fdg) positron emission tomography (pet) and computed tomography (ct) were used to monitor the spatial evolution of individual pulmonary lesions in a cohort of infected mice that develops tb lesions akin to humans [16] [17] [18] and study the temporal evolution of reactivation pulmonary tb (relapse) [19] . it was noted that the fate of individual pulmonary lesions in the same animal varied substantially. more interestingly, several lesions also arose de novo within regions with no prior lesions suggesting that dormant bacteria may also reside outside tb lesions. similar variability of tb lesion within the same host was also reported in non-human primates [20] . molecular imaging can therefore provide unique insights into disease pathogenesis that are not possible with traditional tools. other applications include assessing hideouts of infections, defining the diversity of the microbial populations (microbiome), and providing end points to assess antimicrobial or vaccine efficacy or predict stable cure. current antimicrobial dosing regimens are based on achievable serum concentrations. however, a growing number of studies support the importance of monitoring drug concentration in table 1 . evaluate disease processes deep within the body, noninvasively, and relatively rapidly longitudinal assessments in the same individual-fundamental advantage over traditional tools provide holistic, three-dimensional assessment of the whole organ or body representative of the overall disease (versus tip of a biopsy needle) and therefore less prone to sampling error imaging in infectious diseases role setting overall goal(s) pathogenesis preclinical unique insights into disease pathogenesis, e.g., assessing hideouts of infections, defining the diversity of the microbial populations (microbiome) studying multi-compartment antimicrobial pharmacokinetics expedite bench-to-bedside translation of new therapeutics, e.g., surrogate end points to assess antimicrobial or vaccine efficacy or predict stable cure clinical trials unique insights into disease pathogenesis-noninvasive visualization of processes deep inside the body phase 0 studies to determine compartment-specific antimicrobial penetration/binding (sites of infection, necrotic/fibrotic lesions, privileged sites-cns) to inform appropriate dosing of novel drugs; determine accumulation at non-target sites to assess potential toxicities; current us food and drug administration (fda) guidelines require tissue drug distribution studies at the infected sites patient settings enabling precision medicine by providing unique insights into disease pathogenesis, antimicrobial pharmacokinetics, etc. clinical trials and patient settings rapidly and specifically distinguish an infectious process from other diseases (malignancy, sterile inflammatory processes, etc.) determine the site (e.g., extension/metastasis to other organs or privileged sites) and extent of disease provide information on the class of the infectious pathogen, which could help in targeted empiric antimicrobial treatments monitoring and prognostication preclinical noninvasive longitudinal assessments, especially in studies utilizing larger, more expensive animal species; serial assessments in the same animal could significantly reduce sample size, inter-animal variability (outbreed animals), and therefore cost of the studies clinical trials early end points for treatment trials to assess activity of treatments and to predict stable cure assessing host-directed treatments for infections enable adaptive designs patient settings rapidly detect treatment failures due to drug-resistant organisms or other reasons rapidly monitor treatment responses in patients with drug-resistant organisms and individualize treatments early end points for duration of treatment and predict stable cure enabling precision medicine public health rapid determination of the infectious risk of a patient to the population based on response to treatment and extent and location of disease rapid diagnosis and monitoring of biothreat agents infected tissues, rather than serum alone. inadequate drug concentrations in target tissues can lead to treatment failure and selection of drug-resistant organisms. additionally, altered metabolism in diseased states may lead to organ toxicities. vancomycin, a widely used antimicrobial to treat drug-resistant, gram-positive bacteria, highlights these limitations. studies performed in response to treatment failures noted in patients with standard doses of vancomycin revealed that it penetrates poorly into infected tissues, with levels only one sixth of plasma [21] . conversely, high levels of vancomycin can cause nephrotoxicity. based on these data, new recommendations have been developed for vancomycin [22] , which, unfortunately, had been inappropriately under-dosed for decades. noninvasive bioimaging can be used to assess the distribution of drugs into multiple compartments of interest, simultaneously. for example, given the importance of rifampin for successful cure, potential for shortening the duration of tb treatments, and the limited availability of in situ data [23] , dynamic pet imaging was performed over 60 min after injection of 11 c-rifampin in live, m. tuberculosis-infected mice [24] . 11 c-rifampin rapidly distributed and quickly localized to the liver. areas under the concentration-time curve for the first 60 min (auc 0-60 ) in infected and uninfected mice were uniformly low in the brain (10 to 20 % of blood values). however, lower concentrations were noted in necrotic lung tissues of infected mice than in healthy lungs, demonstrating the need for higher dosing than those determined by serum levels alone. an ideal imaging agent for diagnosing infections needs to be both sensitive and specific. while current, noninvasive techniques, such as ct, magnetic resonance imaging (mri), and ultrasound, can rapidly detect anatomic pathology, they are non-specific and cannot reliably differentiate infectious lesions from non-infectious processes such as cancer or autoimmune diseases. similarly, highly sensitive, current nuclear imaging tools such as [ 111 in]oxine-tagged white blood cell, single-photon emission computed tomography (spect), and [ 18 f]fdg pet, which are increasingly being used for infections, are non-specific [25] . finally, all these imaging tools are dependent upon host responses, which could be altered in immunosuppressed states (e.g., cancer, aids). radiolabeled antibiotics or antimicrobial peptides have been previously tested as pathogen-specific imaging tracers [26, 27] . however, radiolabeled antibiotics have demonstrated variable specificity and an inability to reliably differentiate infection from sterile inflammation [28] . radiolabeled peptides lack mechanistic binding or uptake, but recent pet tracers may show promise [29] . tracers targeting pathogen-specific metabolic processes have also been evaluated in preclinical studies [30, 31] . [ 124 i]fiau, a nucleoside analog substrate for thymidine kinase, was developed as a γ-herpesvirus and bacteria-specific tracer [32] but was later found to lack specificity, presumably due to host mitochondrial metabolism pet could specifically detect enterobacteriaceae in vivo in various different sites including the brain, and the pet signal was not affected by immunosuppressive cancer treatments [36] . f-18 analogs of trehalose have also been developed as potential imaging agents for mycobacteria [37] , while aspergillus fumigatus has been imaged in vivo using ga-68-radiolabeled siderophores [38] . investigators have also utilized radiolabeled antibodies to image a. fumigatus and yersinia in murine models [39, 40] . similarly, santangelo et al. developed a technique to image simian immunodeficiency virus (siv) replication using a cu-64-labeled siv gp120-specific antibody and were able to detect virus-specific pet signals in treated and untreated monkeys [41] . other approaches have utilized tc-99m-labeled oligomers to specifically target the ribosomal rna in the pathogen [42, 43] . finally, using endogenous cest contrast, liu et al. were able to image bacteria-specific mri signals from c. novyi-nt-infected tumors in mice [44] . noninvasive imaging, including non-specific agents, can be used to monitor disease, and serial imaging has been used to assess antimicrobial treatments in animals and humans [9, [45] [46] [47] . for example, phase iii tb trials entail treating hundreds of patients ≥6 months and monitoring for ≥1 year thereafter for relapse [48] . as m. tuberculosis can infect any part of the lung (or extra-pulmonary sites), traditional tools such as sputum culture or naa (e.g., xpert® mtb/rif) do not always correlate with the overall disease burden. recently, chen et al. reported that [ 18 f]fdg pet and ct imaging were superior to traditional (sputum) microbiology for monitoring response to treatments in adults with mdr-tb [49] . in this study, quantitative changes in lesion volumes on ct imaging were predictive of treatment responses. moreover, quantitative changes in [ 18 f]fdg pet not only correlated with treatment responses but were also predictive of long-term treatment outcomes. in another more recent study, given the absence of clinical or microbiological markers of disease, low-radiation exposure pulmonary ct imaging was successfully used to monitor treatment response in a 2-year-old child with xdr-tb and guide an individualized drug regimen [9] . imaging agents with higher specificity for tb-associated inflammation have been shown to be promising in animal models [45] . pathogen-specific imaging could be an even better predictor of treatment responses [36] . for example, both 18 f-labeled maltohexaose and [ 18 f]fds pet could rapidly identify therapeutic failures associated with drug-resistant infections [34, 36] . given that several infectious agents are easily transmissible (e.g., via aerosol), imaging could also help with rapid determination of the infectious risk of a patient to the population. this could be especially relevant for highly drug-resistant organisms or biothreat agents, where rapid diagnosis and therapeutic monitoring would be critical to prevent spread to others in the community. a large majority of infectious pathogens can be handled using standard precautions. however, animals infected with pathogens designated as biosafety level 3/4 (bsl-3/4) require appropriate containment. in addition, work with biothreat pathogens requires regulatory approvals, as well as highly trained personnel [50] . to achieve containment, several groups have installed imaging equipment inside the bsl-3/4 barrier (e.g., niaid, university of pittsburg). while advantageous, maintaining expensive equipment within these barriers can be complicated. therefore, other groups have utilized animal isolation approaches achieved by isolating infected-animals inside sealed, biocontainment devices that can be transported to the imaging equipment housed in standard environments [46, 51, 52] or polycarbonate plastic tubes extending from the biocontainment space to the imaging equipment [53] . a tail vein catheter system was also used for on-table drug delivery to infected animals while sealed inside the biocontainment device [24, 54] . animal containment allows for easier maintenance of the equipment, which can also be shared with other investigators that do not work within the bsl-3/4 barrier. unlike traditional microbiological or molecular techniques, imaging can provide key spatial information to dictate treatment, enabling detection and therapeutic monitoring of infections in patients with deep-seated infections for whom traditional clinical samples (e.g., blood, urine) would be insensitive and high risk/ impractical (e.g., biopsy for brain infection) or where rapid assessment of therapeutic effect is needed. in fact, in our clinical practice at johns hopkins hospital, oncology patients with fever and neutropenia or patients with fever of unknown origin (fuo) routinely undergo whole body imaging to look for foci of fig. 2 . relative risk of infections with treatment-and radiation-induced cancer. the risk of mortality due to infections (tb as an example) compared to the risk of radiation-induced cancer due to commonly used imaging techniques. the risk of mortality for mdr-and xdr-tb patients on treatment is similar to that due to cancers. even drug-susceptible tb patients on treatment have significantly higher risk of mortality than radiation-induced cancers with optimized imaging. the risk of radiation-induced cancer and mortality has been theoretically estimated based on acute radiation exposure (100 msv), and actual risks are likely to be much lower for smaller amounts of radiation. infection. however, current imaging modalities are not specific, and biopsies are often non-diagnostic, leading to uncertainty and overuse (or misuse) of broad-spectrum antimicrobials. ct and other nuclear medicine techniques are often perceived to deliver high levels of radiation. however, recent technological developments have significantly lowered radiation exposure and also allowed rapid scans that avoids the need for sedation in children. for example, the effective dose for each chest ct in the child treated for xdr-tb was 0.4-0.7 msv [9] . this is equivalent to 3 months of natural background radiation, a single screening mammography, or four trans-atlantic airplane round trips [55] [56] [57] . moreover, no sedation was required for this child as scan times were short (3 s). we calculated the risks related to infections, with tb as an example, and compared them to the various imaging techniques used currently (fig. 2) . the risk of mortality for patients with mdr-and xdr-tb on treatment is similar to that due to cancers [8, 52, 58] . moreover, even patients with drugsusceptible tb on treatment have several orders of magnitude higher risk of mortality than the theoretical risk due to radiationinduced cancers [59, 60] . development of nuclear imaging tracers with short half-life isotopes and rapid elimination from the body could further limit radiation exposure. while no studies should be performed without an excellent rationale and clinical indication, we need to be pragmatic about the (minimal) risks of imaging, especially when dealing with infections due to mdr organisms. given sub-pharmacological dosing, there are several approaches for translating pet tracers to humans [61] , allowing more rapid translation of new imaging tools to the clinic. some pet tracers discussed above are currently being tested in humans. recently, a study in healthy volunteers demonstrated that 18 f-fds was safe, well tolerated, and rapidly cleared, following a single, intravenous dose [62] , suggesting significant potential for use in humans. during the past decade, developing countries, especially the brics nations (brazil, russia, india, china, south africa), have witnessed significant increases in the installation and use of advanced imaging. for example, new delhi (india) alone has 9100 clinical mri scanners (several with 3 t strength) [63, 64] . moreover, ct, mri, and pet/ct costs are substantially lower,~$50-$100 for ct and mri and $300 for pet per scan at private (for-profit centers) in developing nations such as india than in the usa [53] . mobile pet scanners and 68 ga pet agents that can be generated without the need of a cyclotron hold great promise for use in remote areas [29] . china has already overtaken the usa to become the largest economy in purchasing power parity, with india expected to become the second largest economy in purchasing power parity by 2050 [65] . infections are rampant in the developing world, and given the considerable number of people living in big cities, advanced imaging has the potential to become a powerful routine clinical tool for the early diagnosis and monitoring of infectious diseases in developing countries. acknowledgements. i would like to thank m. mahesh (chief physicist-johns hopkins hospital) for reviewing the data on the radiation risks related to imaging techniques. funding this review was funded by the national institutes of health (nih) director's transformative research award r01-eb020539 (s.k.j.) and the r01-hl131829 (s.k.j.) as well as nih director's new innovator award dp2-od006492 (s.k.j.). the funders had no role in review design, decision to publish, or preparation of the manuscript. antibiotic resistance: the last resort mortality following bacteraemic infection caused by extended spectrum beta-lactamase (esbl) producing e. coli compared to non-esbl producing e. coli escherichia coli harboring mcr-1 and blactx-m on a novel incf plasmid: first report of mcr-1 in the united states emergence of plasmidmediated colistin resistance mechanism mcr-1 in animals and human beings in china: a microbiological and molecular biological study multistate pointprevalence survey 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mortality in patients with drug-susceptible pulmonary tuberculosis radiation doses for pediatric nuclear medicine studies: comparing the north american consensus guidelines and the pediatric dosage card of the european association of nuclear medicine inds for pet molecular imaging probes-approach by an academic institution biodistribution and radiation dosimetry of the enterobacteriaceae-specific imaging probe [ 18 f]fluorodeoxysorbitol determined by pet/ct in healthy human volunteers commentary-radiology in india: the next decade radiology in india: trends in medical imaging technology 2050 the author declares that he has no conflict of interest. key: cord-354720-fu19u2b0 authors: white-dzuro, gabrielle; gibson, lauren e.; zazzeron, luca; white-dzuro, colin; sullivan, zachary; diiorio, daren a.; low, sarah a.; chang, marvin g.; bittner, edward a. title: multisystem effects of covid-19: a concise review for practitioners date: 2020-11-04 journal: postgraduate medicine doi: 10.1080/00325481.2020.1823094 sha: doc_id: 354720 cord_uid: fu19u2b0 while covid-19 has primarily been characterized by the respiratory impact of viral pneumonia, it affects every organ system and carries a high consequent risk of death in critically ill patients. higher sequential organ failure assessment (sofa) scores have been associated with increased mortality in patients critically ill patients with covid-19. it is important that clinicians managing critically ill covid-19 patients be aware of the multisystem impact of the disease so that care can be focused on the prevention of end-organ injuries to potentially improve clinical outcomes. we review the multisystem complications of covid-19 and associated treatment strategies to improve the care of critically ill covid-19 patients. coronavirus disease 2019 (covid19) , the disease caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first described in wuhan, china in december 2019 and rapidly evolved into a worldwide pandemic [1] . of patients affected with the virus, approximately 5% to 14% will become critically ill [2] [3] [4] . while covid-19 generally begins as a respiratory tract infection, it can have damaging effects on every organ system. when the virus does spread systemically, the result is often multisystem critical illness associated with a high risk of death. higher sequential organ failure assessment (sofa) scores have been associated with increased mortality in critically ill patients with covid-19 [5] . it is important that clinicians managing these critically ill patients be aware of the multisystem impact of the disease so that care can be focused on the prevention of end-organ injuries to potentially improve clinical outcomes. here, we review the multisystem complications of covid-19 and treatment strategies to improve the care of critically ill covid-19 patients. the multisystem manifestations of covid-19 (figures 1-3) result from a combination of the direct effects of the viral infection and the indirect effects of the body's significant inflammatory response to the virus. post mortem findings in covid-19 patients show viral elements within endothelial cells, an accumulation of inflammatory cells, and cellular apoptosis in multiple organs [6] . these widespread effects largely reflect the virus' ability to use the angiotensinconverting enzyme 2 (ace2) receptors to gain entry into endothelial cells [7] . ace2 breaks down angiotensin ii, a pro-inflammatory factor in the lung, and viral inhibition of this enzyme may also be a contributing factor to the lung injury and multiorgan dysfunction that result from sars-cov-2 infection [8] . the indirect effects of the virus result from the host's response to the viral infection, and are associated with a cytokine storm characterized by very high circulating levels of pro-inflammatory cytokines, including tumor necrosis factor (tnf)-α, interleukins, granulocyte-colony stimulating factor, and chemokines [9] . this hyper-inflammatory response combined with hypercoagulability [10] can lead to venous thromboembolism, further increasing the risk of multisystem failure and mortality. the severity of covid-19-related respiratory disease varies significantly, from mild symptoms requiring minimal oxygen support with nasal cannula to acute hypoxemic respiratory failure requiring intubation and mechanical ventilation. for about 80% of the patients, the disease will be mild and mostly restricted to the upper airways. the remaining 20% of the patients will develop pulmonary infiltrates as the virus reaches the alveoli, where it preferentially infects the peripheral and subpleural units [4] . in an earlier phase of the disease, patients often show preserved lung mechanics with normal compliance, despite having severely impaired oxygenation and increased minute ventilation [11] . there have been a number of phenotypes that have been proposed to characterize covid-19 pneumonia (i.e. 'l' vs 'h' phenotype), however, these phenotypes are controversial [12] . on ct imaging, ground-glass opacities are seen in up to 98% of the patients, which commonly suggests interstitial, rather than alveolar, edema. these characteristics have been categorized as 'type l' and include a low lung elastance, low recruitability, and a poor response to positive end-expiratory pressure (peep). in some patients, the disease progresses into a clinical scenario that resembles typical acute respiratory distress syndrome (ards), which may be due to the development of ventilator-induced lung injury, worsening of covid-19 disease, or bacterial superinfection [13] . these patients are found to have high lung elastance, extensive consolidations on ct imaging, and significant response to higher peep. these lung findings are categorized as 'type h.' standard lung-protective ventilation should be used for all stages of the disease, despite the wide spectrum of clinical characteristics. there has been significant debate regarding the optimal form of respiratory support for non-intubated patients with increasing oxygen requirements and dyspnea. early intubation has been encouraged to reduce the risk of virus aerosolization and self-induced lung injury. however, early intubation may result in unnecessary intubation of patients who would have otherwise improved with less invasive support [14] . other strategies that may improve ventilation in these patients include prone positioning and nitric oxide, which is being tested both as an antiviral agent and for its benefits as a pulmonary vasodilator. while prone positioning in awake spontaneously breathing patients is generally well tolerated and has been shown to improve oxygenation and reduce respiratory rate, the effects on clinical outcomes is unclear [15] . the use of noninvasive ventilation and high flow nasal cannula in covid-19 patients is controversial given the potential risk for aerosolization and exposure to health care workers, and can be minimized when delivered in a negative pressure environment. in-hospital airway management is challenging and we have previously described a practical, stepwise protocol for safe in-hospital airway management in covid-19 patients [16] . covid-19 has profound effects on the hematologic system. lymphopenia is a characteristic laboratory finding in covid-19 patients and has prognostic potential in determining severe cases [17] . throughout the disease course, serial monitoring of lymphocyte count dynamics and inflammatory indices such as lactate dehydrogenase, c-reactive protein, and interleukin-6, may allow identification of patients with poor prognosis and trigger timely intervention. other biomarkers, including high serum procalcitonin and ferritin, have emerged as poor prognostic factors [5] . patients with covid-19 infection present a complicated clinical paradigm as current research has suggested that they are at risk of both impaired hemostasis as well as thrombotic events. elevated fibrinogen and d-dimer levels are the most common coagulopathy seen in hospitalized covid-19 patients. while patients with severe covid-19 infection commonly meet the clinical criteria for disseminated intravascular coagulopathy (dic), their thrombocytopenia is generally mild, and microangiopathy is often not present. however, given that some patients can develop fulminant dic with consumption of coagulation factors, platelet count, pt/aptt, d-dimer, and fibrinogen should be monitored closely. while abnormal coagulation parameters are common in patients with covid-19 infection, covid-19 infection seems to be rarely associated with bleeding. correction of the coagulopathy should only be pursued in patients with active bleeding or those requiring an invasive procedure. patients with covid-19 are also at an increased risk of venous thromboembolic (vte) disease. vte disease is likely due to the combined effects of systemic inflammation, abnormal coagulation, multiorgan dysfunction, and critical illness [18] . multiple studies have shown the correlation between elevated d-dimer and disease severity [5, 19, 20] . clinically, d-dimer levels have been followed in patients with covid-19 infection to determine not only disease severity and clinical progression but also risk of thromboembolic events. while elevated serum d-dimer levels have been associated with increased mortality in covid-19 patients, there is no current evidence supporting the use of d-dimer levels to guide anticoagulation. pharmacologic vte prophylaxis is generally recommended in all hospitalized covid-19 patients with no specific contraindication. a prospective cohort study that examined the rate of thromboembolic events in 184 icu patients all receiving standard dosing of vte prophylaxis and found a 31% incidence of thromboembolic events. given this finding, the authors suggest increased dosing of prophylaxis medications [21] . the current evidence is limited on the optimal dosing of vte prophylaxis medication in critically ill patients with covid-19 and studies to evaluate therapeutic anticoagulation in critically ill patients with covid-19 are currently ongoing (nct04359277). furthermore, post-hospital discharge vte prophylaxis should be considered in these patients on a case-by-case basis. assessment for vte is challenging in these patients, and imaging should only be pursued when clinical examination and condition support the laboratory studies. the decision for treatment with anticoagulation should be made based on imaging findings when possible. a review by flaczyk et al. compares published guidelines for management of covid-19 associated coagulopathy in critically ill patients and provides a framework for patient management [22] . include direct viral damage of nervous tissue, injury resulting from the excessive inflammatory response, unintended host immune response effects after the acute infection (e.g., guillain-barré syndrome as reported in a case series of four patients [24] ), and injury resulting from the effects of systemic illness. most covid-related neurologic complications in critically ill patients fall into this latter category, and manifest as encephalopathy, delirium, and critical illness myopathy. a variety of neurological manifestations have been reported, including headache, encephalitis, stroke, seizures, hyposmia, and hypogeusia [25] . the prevalence of hyposmia and hypogeusia suggests direct viral infection of the olfactory nerve [23] and sars-cov 2 has been detected in human neuronal cells on postmortem analysis [26, 27] . several studies have also reported meningoencephalitis manifested by headache, fever, altered mental status, and signs of meningeal irritation as a presenting symptom of covid-19 that may be secondary to the indirect inflammatory effects of the virus [28] [29] [30] [31] [32] . a case of covid-associated acute necrotizing hemorrhagic encephalitis within the temporal lobes and thalami has also been reported [30] . sars-cov-2 has been detected in the csf of a patient with encephalitis [29] . there have been several cases of young patients presenting with large-vessel strokes [33] and 5.7% of a hospitalized cohort in wuhan had an acute stroke (80% ischemic, 20% hemorrhagic) [23] . given the prevalence of hypercoagulability, neurologic thromboembolic events should be strongly considered in a patient with a fluctuating neurologic examination [34] . careful clinical assessment in conjunction with imaging and cerebrospinal fluid examination may be essential for diagnosing covidrelated neurological disorders. a significant proportion of patients receive high amounts of sedation to ensure comfort and facilitate ventilator synchrony [35] . neuromuscular blockade has also been utilized when the effects of sedation are inadequate. spontaneous awakening trials are vital to alert clinicians to neurologic changes and enable them to take appropriate action. this neurologic assessment is especially important before initiating therapeutic anticoagulation, given the significant morbidity in patients with unidentified intracranial hemorrhage. a growing body of literature is reporting high rates of acute encephalopathy and agitated delirium in critically ill covid-19 patients [36] [37] [38] [39] . delirium management strategies should be routinely employed, given the prevalence of the disorder in covid-19 patients. between 5% and 25% of the hospitalized covid-19 patients will have evidence of myocardial involvement [40, 41] , and preexisting cardiovascular disease has been linked to more severe infections [42] . cardiovascular complications include infarction, myocarditis, heart failure, and dysrhythmias. there are multiple proposed etiologies for adverse cardiovascular outcomes, including an increased metabolic demand, a hyperinflammatory state, and increased procoagulant activity [42] . there is also evidence that the virus may cause direct damage to the heart via ace2 receptors located within the cardiac tissue. angiotensin ii is converted to angiotensin (1-7) by ace2, and the downregulation of ace2 among covid-19 patients has been correlated with increased viral load [43] . since angiotensin (1-7) has anti-inflammatory and anti-fibrotic effects that counter the pro-inflammatory effects of angiotensin ii, there has been concern that patients taking angiotensin-converting enzyme inhibitors or angiotensin receptor blockers might be more susceptible to viral infection and propagation [44, 45] . however, the reduction in angiotensin ii activity and upregulation of the anti-inflammatory ace2 effects from these drugs may be beneficial in covid-19 infection [46] . many use the lack of evidence of harm in covid-19 to support the continuation of such medications [45] , given the risk of heart failure exacerbation with their withdrawal [47] . elevated troponins have been found in many patients with covid-19, which may be secondary to increased cardiac physiologic demand, hypoxia, and/or direct myocardial injury. myocarditis has also been identified on autopsy of some patients with covid-19 [48, 49] and presents with variable clinical severity in covid-19 patients. fulminant viral myocarditis [48, 50] or diffuse lymphocytic infiltrates characteristic of viral myocarditis [43, 51] are uncommon findings in covid-19 patients. the patients who have developed covid-19 myocarditis are typically younger and healthier, often without underlying cardiovascular disease. myocarditis may result from a cytokine storm in patients who mount a vigorous immune response to the virus. differentiating myocarditis from acute coronary syndrome (acs) can be challenging [52] . serum troponin values will be elevated in both conditions, and electrocardiogram (ecg) in patients with myocarditis can demonstrate a range of findings, in some cases mimicking acs. echocardiographic evaluation is more likely to show global dysfunction with myocarditis, while a focal wall motion abnormality is more suggestive of acs. ecg and echocardiographic abnormalities are markers of severity in covid-19 patients, and are correlated with worse outcomes; moreover, troponin elevations in severe covid-19 patients have been directly associated with an increased risk of mortality. diagnostic confirmation of covid-19 myocarditis via biopsy is unlikely to change management and therefore is not generally recommended. instead, it may be reasonable to empirically treat covid-19 patients with suspected myocarditis with a course of corticosteroids. given the hyperinflammatory and hypercoagulable state, covid-19 patients may be at increased risk for acute myocardial infarction. patients should be directed toward appropriate standard of care therapy [53] while also avoiding unnecessary and costly procedures that can both lead to morbidity and increase the risk of infectious exposure to staff. approximately a quarter of patients presenting with covid-19 will develop acute heart failure, with the majority lacking a prior diagnosis of hypertension or cardiovascular disease. it is unclear if heart failure in these patients is a new cardiomyopathy or an exacerbation of an undiagnosed condition. given the high prevalence of cardiac dysfunction and concern for causing pulmonary edema, fluids should be administered judiciously in covid-19 patients. a diagnosis of stress cardiomyopathy should be considered in any critically ill covid-19 patient with an abrupt decline in cardiac function. focused echocardiography can be performed at the bedside to allow for rapid evaluation while minimizing exposure to additional staff. typical echocardiographic findings include impaired systolic function with global hypokinesis or regional wall motion abnormalities that are not limited to a coronary distribution [54] . in stress cardiomyopathy, systolic dysfunction is typically transient, with most patients recovering within days to weeks. however, due to ongoing infection and respiratory compromise, the prognosis for covid-19 patients who develop stress cardiomyopathy is often poor. management is mostly supportive, and the use of mechanical circulatory support should be considered for patients in whom myocardial recovery is likely. a 'pediatric multi-system inflammatory syndrome temporally associated with covid-19' (pim-ts) syndrome has been described in children, which shares certain features of toxic shock syndrome and atypical kawasaki disease and has been linked to a number of deaths and rapid decompensation in children [55] . common features include rash, fever, gastrointestinal symptoms, and severe cardiac dysfunction from myocarditis and cardiogenic shock. repeat echocardiography, cardiac catheterization, supportive treatment, steroids, and ivig may be required. gastrointestinal (gi) manifestations of covid-19 are common and include diarrhea, vomiting, and abdominal pain. the sars-cov-2 rna can be readily detected in stool specimens, even where respiratory tests are negative [56] . sars-cov-2 has a tropism for the gi tract, with the viral ace2 receptor found on gastrointestinal epithelial cells. critically ill patients with covid-19 are at high risk of hepatobiliary, hypomotility, and ischemic gi complications [57] , with small vessel thrombosis and viral enteroneuropathy as likely etiologic factors. consequently, serial abdominal exams with limited sedation are required when there is clinical suspicion of intraabdominal pathology. liver injury occurs in 15% to 78% of covid-19 patients, with the most common finding being abnormal transaminase levels [58, 59] . most liver injuries are mild and transient, but more severe liver damage can occur and is more likely in patients with severe covid-19 infection. mechanisms of liver injury may include direct viral infection of hepatocytes, immune-related injury, and drug hepatotoxicity. in patients with transaminitis, medications with potential hepatotoxicity, including acetaminophen, statins, and hydroxychloroquine, should be adjusted or discontinued. acute kidney injury (aki) is a common complication of covid-19 infection, occurring in 0.5-15% of hospitalized covid-19 patients [5] , and up to 23% of critically ill patients [60, 61] . the median onset of aki from hospitalization ranges from 7 [62] to 15 days [5] , and it has been identified as an independent risk factor for morbidity and mortality [62] . aki can occur through several proposed mechanisms, including acute tubular necrosis induced by sepsis, fluid restriction, rhabdomyolysis, or hypoxia. furthermore, intrinsic tissue injury by direct viral invasion of the renal tubular cells, interstitum, or glomeruli has also been proposed. varying degrees of acute tubular necrosis, lymphocytic infiltration, and viral rna have been found on postmortem examination of covid-19 patients, suggesting the direct invasion of kidney tubules [63] . management of aki in covid-19 patients must account for classical and viral-specific risk factors of renal damage. in a recent study, approximately 30-40% of critically ill patients with aki required renal replacement therapy (rrt) [34, 61] . the preferred modality is continuous rrt or sustained lowefficiency dialysis, although the american society of nephrology notes that whatever is available in these resourcescarce times will suffice. if the aki is secondary to cytokine storm, some have questioned whether using high-volume hemofiltration would be preferred to clear inflammatory molecules, but current evidence shows no difference in outcome compared to standard volume filtration [18] . while the effects of covid-19 on the endocrine system remain largely unknown, given the expression of ace2 on the majority of endocrine glands, dysfunction of these systems should be considered in critically ill patients. the ace2 receptor is expressed throughout the pancreas and dysfunction of both the exocrine and endocrine systems is seen in patients with covid-19 infection. while pancreatitis is uncommon, elevated lipase or amylase have been seen in up to 17% of the patients with severe disease [64] . similar to other infections, patients with diabetes mellitus (dm) are more at risk for covid-19 than the general population and are more likely to have a severe course due to compromised innate immunity and downregulated ace2 levels [18] . covid-19 patients with dm have higher serum levels of inflammatory biomarkers and are more susceptible to cytokine storm [18] . furthermore, infection with sars-cov-1 was shown to cause new-onset diabetes in approximately 8% of the patients during hospitalization that may not be classic type 1 or type 2 diabetes but a new type of diabetes [65, 66] . in some patients, hyperglycemia persisted for 3 years after recovery from the virus. while a similar effect has not yet been reported in covid-19, close monitoring of blood glucose during the acute and convalescent phase of the illness is indicated. obesity is associated with severe covid-19, which may be explained by ace2 expression in adipose tissue. furthermore, visceral and subcutaneous adipose tissue produces proinflammatory cytokines that are found in greater abundance in obese patients with covid-19 as compared to non-obese patients [67] . this may predispose obese patients with covid 19 to an exaggerated cytokine response in the presence of sars-cov-2, manifesting as more severe disease and ards [68] . hypothalamic and pituitary tissues express ace2 and given the evidence of viral injury of nervous tissue, it is reasonable to assume that sars-cov-2 may affect the hypothalamus-pituitary as well, either directly or via immune-mediated hypophysitis [69] . accordingly, central hypocortisolism should be suspected in critically ill covid-19 patients with unexplained hypotension or shock. diabetes insipidus is common in patients with pituitaryhypothalamic disorders and, in conjunction with insensible water loss from fever and the conservative fluid management strategy employed in critically ill covid patients, could result in hypovolemia and hypernatremia. impairment of the host's cortisol stress response was a primary immunologic strategy utilized by sars-cov-1 for facilitating viral spread [70] . the similarities between the two viral strains suggest that sars-cov-2 may employ the same strategy. therefore, patients with severe covid-19 may be more prone to develop critical illness-related corticosteroid insufficiency. the absence of lymphopenia in patients with covid-19 could be used as a marker of hypocortisolism (absolute or relative) and clinicians may want to adopt a low threshold for initiating glucocorticoid therapy in the presence of shock. data regarding the impact of covid-19 on thyroid function are limited. a case report suggests that covid-19 may lead to subacute thyroiditis in some patients, which is suspected to have viral or postviral origin [71] . recent guidelines advise patients with underlying hypothyroidism or hyperthyroidism to continue prescribed medications since uncontrolled thyroid disease may increase the risk for viral infection and complications [72] . a variety of cutaneous manifestations such as erythematous rash, generalized urticaria, and chickenpox-like lesions have been associated with covid-19, and can present even before the onset of symptoms and with an incidence of up to 36% of the patients depending on the cutaneous manifestation [73, 74] . interestingly, there was no association between cutaneous findings and disease severity [74] . urticarial eruptions typically preceded additional symptoms of covid-19 infection, were noted to occur even in the absence of fevers, and tended to be consistent on histology with a viral exanthem [53, 75, 76] . acrocyanosis and limb ischemia have been described in a cohort of critically ill patients with elevated d-dimer, fibrinogen, and fibrinogen degradation products [77] . in this small cohort, 57% of the patients developed dic. livedo reticularis, a cutaneous manifestation commonly associated with dic, was seen in two patients with only mild-moderate disease [78] . the authors hypothesize that these findings may represent microthrombosis of cutaneous vasculature, as similar pathophysiology has been noted in other organ systems in covid-19 patients. chilblains-like lesions, which are erythematous areas on the feet, described colloquially as 'covid toes,' may represent endothelitis secondary to systemic covid19 . in a small cohort of patients with severe covid-19 and purpuric skin rash, biopsy demonstrated a thrombogenic vasculopathy of both affected and normal-appearing skin as well as localization of sars-cov-2 spike glycoprotein causing complement activation [79] . these findings are consistent with a catastrophic microvascular injury mediated by systemic viral spread and associated with a procoagulant state. current evidence does not suggest that dermatologic findings are associated 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of muscle relaxants and corticosteroids paresis acquired in the intensive care unit: a prospective multicenter study gwd, leg, lz, cwd, zs, dad, sal, mgc, and eab wrote and reviewed the article.peer reviewers on this manuscript have no relevant financial or other relationships to disclose. the authors declare to have no competing interests. gwd reports no conflict of interest. leg reports no conflict of interest. lz reports no conflict of interest. cwd reports no conflict of interest. zs reports no conflict of interest. dad reports no conflict of interest. sal reports no conflict of interest. mgc reports no conflict of interest. eab reports no conflict of interest. key: cord-353786-284qn075 authors: chen, zhi-min; fu, jun-fen; shu, qiang; chen, ying-hu; hua, chun-zhen; li, fu-bang; lin, ru; tang, lan-fang; wang, tian-lin; wang, wei; wang, ying-shuo; xu, wei-ze; yang, zi-hao; ye, sheng; yuan, tian-ming; zhang, chen-mei; zhang, yuan-yuan title: diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus date: 2020-02-05 journal: world j pediatr doi: 10.1007/s12519-020-00345-5 sha: doc_id: 353786 cord_uid: 284qn075 since december 2019, an epidemic caused by novel coronavirus (2019-ncov) infection has occurred unexpectedly in china. as of 8 pm, 31 january 2020, more than 20 pediatric cases have been reported in china. of these cases, ten patients were identified in zhejiang province, with an age of onset ranging from 112 days to 17 years. following the latest national recommendations for diagnosis and treatment of pneumonia caused by 2019-ncov (the 4th edition) and current status of clinical practice in zhejiang province, recommendations for the diagnosis and treatment of respiratory infection caused by 2019-ncov for children were drafted by the national clinical research center for child health, the national children’s regional medical center, children’s hospital, zhejiang university school of medicine to further standardize the protocol for diagnosis and treatment of respiratory infection in children caused by 2019-ncov. since december 2019, an epidemic caused by novel coronavirus (2019-ncov) infection has occurred unexpectedly in china. at present, 2019-ncov infection has been a legal class b infectious disease of the law of the people's republic of china on the prevention and treatment of infectious diseases, and managed as a class a infectious disease for infection prevention and control practices. as of 8 pm, 31 january, more than 20 pediatric cases have been reported in china. of these cases, ten were identified in zhejiang province, with an age of onset ranging from 112 days to 17 years. following latest national recommendations for diagnosis and treatment of respiratory infections caused by 2019-ncov (the 4th edition) and current status of clinical practice in zhejiang province, recommendations for the diagnosis and treatment of respiratory infection caused by 2019-ncov for children were drafted out by the national clinical research center for child health, national children's regional medical center, children's hospital, zhejiang university school of medicine to further standardize the protocol of respiratory infection in children caused by 2019-ncov. etiology 2019-ncov is a novel human coronavirus in addition to coronavirus 229e, nl63, oc43, hku1, middle east respiratory syndrome-related coronavirus (mersr-cov) and severe acute respiratory syndrome-related coronavirus (sars-cov). 2019-ncov is enveloped single-stranded plus stranded rna virus with a diameter of 60-140 nm, spherical or elliptical in shape and pleomorphic [1] . it has been reported that the consistency of whole genome-wide nucleotide sequences of 2019-ncov with sars-like coronavirus in bats (bat-sl-covzc45) ranges from 86.9 [1] to 89% [2] . the nucleotide sequence of spines protein on the envelope of the virus is also highly consistent with that of bat-sl-covzc45 (84%) and sars-cov (78%). the physicochemical property of 2019-ncov has not been clarified clearly yet. it is thought that 2019-ncov is sensitive to ultraviolet radiation and heating. for example, according to researches on sars-cov and mers-cov, the virus can be inactivated by heating at 56 °c for 30 minutes and by using lipid solvents such as 75% ethanol, chlorinecontaining disinfectant, peroxyacetic acid and chloroform, but not by chlorhexidine, according to the researches on sars-cov and mers-cov. the main sources of the infection are patients infected by 2019-ncov with or without clinical symptoms [2] . in addition, patients in the incubation period may also have potency to transmit the virus based on the case evidence. the novel virus is spread through respiratory droplets when patients cough, talk loudly or sneeze. close contact is also a source of transmission (e.g., contact with the mouth, nose or eye conjunctiva through contaminated hand). whether transmission can occur through mother-infant vertically or breast milk has not been established yet. there are general susceptibilities in all groups, with the elderly and people with basic diseases more likely to become severe cases. children may have mild clinical symptoms after infection [3] . the incubation period of 2019-ncov infections ranges from 2 to 14 days, though most often rangs from 3 to 7 days [3] . at the onset of the disease, infected children mainly present with fever, fatigue and cough, which may be accompanied by nasal congestion, runny nose, expectoration, diarrhea, headache, etc. most of the children had low to moderate fever, even no fever. dyspnea, cyanosis and other symptoms can occur as the condition progresses usually after 1 week of the disease, accompanied by systemic toxic symptoms, such as malaise or restlessness, poor feeding, bad appetite and less activity. the condition of some children may progress rapidly and may develop into respiratory failure that cannot be corrected by conventional oxygen (nasal catheter, mask) within 1-3 days. in these severe cases, even septic shock, metabolic acidosis and irreversible bleeding and coagulation dysfunction may occur. rapid respiration rate and moist rales on auscultation usually indicate pneumonia. the criteria for rapid respiratory rate are as follows: ≥ 60 times/min for less than 2 months old; ≥ 50 times/min for 2-12 months old, ≥ 40 times/min for 1-5 years old, ≥ 30 times/min for > 5 years old (after ruling out the effects of fever and crying). with the aggravation of the disease, respiratory distress, nasal flaring, suprasternal, intercostal and subcostal retractions, grunting and cyanosis may occur. maternal hypoxemia caused by severe infection can lead to intrauterine asphyxia, premature delivery and other risks. neonates, especially preterm infants, who are more likely to present with insidious and non-specific symptoms need more close observation. according to the current conditions of the reported cases, the children mainly belong to family cluster cases. most of them have good prognosis, and in mild cases recover 1-2 weeks after disease onset. no deaths in children have been reported up to now. routine blood test white blood cell count is usually normal or reduced, with decreased lymphocyte count; progressive lymphocytopenia in severe cases. c-reactive protein (crp) normal or increased. procalcitonin (pct) normal in most cases. the level of pct > 0.5 ng/ml indicates the co-infection with bacteria. others elevation of liver enzymes, muscle enzymes and myoglobin, and increased level of d-dimer might be seen in severe cases. nucleic acid testing is the main method of laboratory diagnosis. 2019-ncov nucleic acid can be detected by rt-pcr or by viral gene sequencing of throat swabs, sputum, stool or blood samples. other methods 2019-ncov particles can be isolated from human respiratory epithelial cells through virus culture [4, 5] , but this experiment cannot be carried out in general laboratories. virus antigen or serological antibody testing kits are not available at present. chest x-ray examination in the early stage of pneumonia cases, chest images show multiple small patchy shadows and interstitial changes [4] , remarkable in the lung periphery [2] . severe cases can further develop to bilateral multiple ground-glass opacity, infiltrating shadows, and pulmonary consolidation, with infrequent pleural effusion. chest ct scan pulmonary lesions are shown more clearly by ct, including ground-glass opacity and segmental consolidation in bilateral lungs, especially in the lung periphery. in children with severe infection, multiple lobar lesions may be present in both lungs. patients should be suspected of 2019-ncov infection who if they meet any one of the criteria in the epidemiological history and any two of the criteria in clinical manifestations. city and neighboring areas, or other areas with persistent local transmission within 14 days prior to disease onset. 2. children with a history of contacting patients with fever or respiratory symptoms who have a travel or residence history in wuhan city and neighboring areas, or in other areas with persistent local transmission within 14 days prior to disease onset. 3. children with a history of contacting confirmed or suspected cases infected with 2019-ncov within 14 days prior to disease onset. 4. children who are related with a cluster outbreak: in addition to this patient, there are other patients with fever or respiratory symptoms, including suspected or confirmed cases infected with 2019-ncov. 5. newborns delivered by suspected or confirmed 2019-ncov-infected mothers. 1. fever, fatigue, dry cough; some pediatric patients may have no fever. 2. patients with the above-mentioned chest imaging findings (refer to the section of imaging features); 3. in the early phase of the disease, white blood cell counts are normal or decreased, or with decreased lymphocyte count. suspected cases who meet any one of the following criteria: 1. throat swab, sputum, stool, or blood samples tested positive for 2019-ncov nucleic acid using rt-pcr; 2. genetic sequencing of throat swab, sputum, stool, or blood samples being highly homologous with the known 2019-ncov; 3. 2019-ncov granules being isolated by culture from throat swab, sputum, stool, or blood samples. it is necessary to strengthen the awareness of the early identification of the disease. in clinic, screening was conducted mainly according to the epidemiological history and fever or respiratory symptoms, and pathogen examination should be carried out on time. furthermore, effective isolation measures and appropriate treatment need to be provided promptly. even if the common respiratory pathogen tests are positive, children who have a history of close contact with 2019-ncov-infected cases also are recommended for timely 2019-ncov pathogen testing. this type of patient includes those with asymptomatic infection, upper respiratory infection (uri) and mild pneumonia. symptoms include fever, cough, sore throat, fatigue, headache or myalgia. some patients show pneumonia signs on chest imaging. these patients do not have any of the severe or critical symptoms and complications described below. disease progresses to meet any of the following conditions [6]: 1. significantly increased respiration rate: rr ≥ 70/min (≤ 1 year), rr ≥ 50/min (> 1 year). 2. hypoxia: spo 2 ≤ 93% (< 90% in premature infants) or nasal flaring, suprasternal, intercostal and subcostal retractions, grunting and cyanosis, apnea, etc. 3. b l o o d g a s a n a ly s i s : p a o 2 < 6 0 m m h g , paco 2 > 50 mmhg. 4. consciousness disorders: restlessness, lethargy, coma, convulsion, etc. 5. poor feeding, bad appetite, and even dehydration. 6. other manifestations: coagulation disorders (prolonged prothrombin time and elevated level of d-dimer), myocardial damage (increased level of myocardial enzyme, electrocardiogram st-t changes, cardiomegaly and cardiac insufficiency in severe cases), gastrointestinal dysfunction, raised level of liver enzyme and rhabdomyolysis. disease progresses rapidly along with organ failure with any of the following conditions: 1. respiratory failure which requires mechanical ventilation patients present with acute respiratory distress syndrome (ards) and are featured by refractory hypoxemia, which cannot be alleviated by conventional oxygen therapy, such as nasal catheter or mask oxygen supplement. 2. septic shock in addition to severe pulmonary infection, 2019-ncov can cause damage and dysfunction of other organs. when dysfunction of extrapulmonary system such as circulation, blood and digestive system occurs, the possibility of sepsis and septic shock should be considered and the mortality rate increases significantly. 3. accompanied by other organ failure that needs icu monitoring and treatment. viruses such as sars virus, influenza virus, parainfluenza virus, adenovirus, respiratory syncytial virus and metapneumovirus can cause viral respiratory infections. patients commonly present with fever, cough and dyspnea, and interstitial pneumonia in some cases. most of the cases have normal or decreased white blood cell counts, while severely infected children show reduced level of lymphocyte count. similar to 2019-ncov, all of these viruses can transmit through respiratory tract or direct contact, which is characterized by clustering onset. epidemiological exposure history plays an important role in differential diagnosis, which is mainly confirmed by laboratory examinations. patients with bacterial pneumonia mostly display high fever and toxic appearance. in the early stage of the disease, cough is not obvious but moist rale can be heard. chest image may show small patchy shadows, or segmental or even lobar consolidation. blood routine examination shows increased white blood cell count with neutrophil predominating, as well as elevated crp. noninvasive pneumonia is usually accompanied by obvious cough. antibiotics are effective. blood or deep sputum culture is helpful for diagnosis of bacterial pneumonia. mycoplasmal pneumonia may occur in any season and in schools and child-care institutions with small epidemic. patients are predominantly school-age children, but the number of younger children is increasing. they usually start with high fever and cough. chest images may reveal manifestations including reticular shadows and small patchy or large consolidation. blood routine examination shows that the white blood cell count is normal or increased and crp is slightly elevated. nucleic acid detection of airway secretion and serum mycoplasma-specific igm determination are helpful for differential diagnosis. it should be noted that patients with 2019-ncov infection can have co-infection or superimposed infection with other viruses or bacteria simultaneously. the four principles of "early identification", "early isolation", "early diagnosis", and "early treatment" should be emphasized. medical isolation is supposed to be carried out once the suspected cases are identified. the confirmed cases should be admitted to the designated hospitals. avoid using broad-spectrum antibiotics and corticosteroids. antibiotics, corticosteroids, bronchoalveolar lavage, mechanical ventilation, and other more invasive intervention, such as blood purification and extracorporeal membrane oxygenation (emco) should be applied cautiously, based on cost-benefit evaluation. monitoring patient's conditions closely and adjusting the therapeutic protocols timely through multidisciplinary cooperation are of great significance. suspected case should be isolated in a single room, while confirmed cases can be arranged in the same room. during the course of treatment, pay close attention to the changes in children's conditions, regularly monitor vital signs, spo 2 , etc., and identify the severe and critical cases as early as possible. the general strategies include bed rest and supportive treatments; ensuring sufficient calorie and water intake; maintaining water electrolyte balance and homeostasis, and strengthening psychotherapy for elder children when necessary. there are no effective antiviral drugs for children at present. interferon-α2b nebulization can be applied, and the recommended usage is as follows: irrational use of antibiotics should be avoided. bacteriological monitoring should be strengthened. if there is evidence of secondary bacterial infection, appropriate antibiotics should be used timely. corticosteroids should be avoided in common type of infection. however, it can be considered in the following situations: intravenous methylprednisolone (1-2 mg/kg/day) is recommended for 3-5 days, but not for long-term use [3, 5, [9] [10] [11] . intravenous immunoglobulin can be used in severe cases when indicated, but its efficacy needs further evaluation. the recommended dose is 1.0 g/kg/day for 2 days, or 400 mg/kg/ day for 5 days [6, 9, 12, 13] . bal is not suitable for most patients, and there is an increased risk of cross-infection. the indications should be strictly controlled. bal can be considered if patients have obvious symptoms of airway obstruction, refractory massive atelectasis indicated by imaging, a significant increase in peak pressure during ventilator therapy, decrease of tidal volume, or poor oxygenation which cannot be reversed by conservative treatments. in case of circulation dysfunction, vasoactive drugs should be used to improve microcirculation on the basis of adequate liquid support [3] . patients with acute renal injury should be given continuous blood purification on time. in the meantime, paying attention to the brain function monitoring if neccessary. if intracranial hypertension and convulsion occurs in children, it is necessary to reduce the intracranial pressure and control convulsion timely [6, 12, 13] . in the case of respiratory distress that occurs despite nasal catheter or mask oxygenation, heated humidified high-flow nasal cannula (hhhfnc), non-invasive ventilation such as continuous positive airway pressure (cpap), or non-invasive high-frequency ventilation can be used. if improvement is not possible, mechanical ventilation with endotracheal intubation and a protective lung ventilation strategy should be adopted. continuous blood purification should be considered in cases of multiple organ failure (especially acute kidney injury), or capacity overload and life-threatening imbalance of water, electrolyte, and acid-base. the therapeutic model may include continuous veno-venous hemofiltration (cvvh), continuous veno-venous hemodiafiltration (cvvhdf), or heterozygosis. if combined with liver failure, plasma exchange is feasible. coronavirus investigating, and research team. a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster national recommendations for diagnosis and treatment of pneumonia caused by 2019-ncov (the 4th edition). national health commission and national administrative office of chinese tradition medicine /42945 63ed3 5b432 09b31 739bd 0785e 67/files /7a930 91112 67475 a99d4 30696 2c8bf 78.pdf. access 29 clinical features of patients infected with 2019 novel coronavirus in wuhan another decade, another coronavirus diangosis and treatment guideline of community acquired pneumonia in children role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial expert consensus on the diagnosis and treatment of viral pneumonia in children corticosteroid therapy for critically ill patients with middle east respiratory syndrome clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected the editorial board, chinese journal of pediatrics. diagnosis and treatment guideline of community acquired pneumonia in children (2013 the first part) guidelines for the diagnosis and treatment of adenovirus pneumonia in children (2019 edition) pediatric branch, chinese medical association thoracic vascular anesthesiology society, extracorporeal life support committee, pediatric surgery branch, chinese medical association. consensus of extracorporeal membrane oxygenation support for acute fulminant myocarditis in children publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ecmo therapy should be considered when mechanical ventilation, blood purification, and other means are ineffective, and cardiopulmonary failure occurs which is difficult to correct.1. ecmo indications [14] . 2. pao 2 /fio 2 < 50 mmhg or oxygen index (oi) > 40 for more than 6 h, or severe respiratory acidosis (ph < 7.15). 3. high mean airway pressure (map) during mechanical ventilation, or severe air leakage and other severe complications. 4. circulation function cannot be improved with conventional treatment, or large amounts of vasoactive drugs are required to maintain basal blood pressure, or lactic acid levels continuously rise.contraindication [14] ecmo should be contraindicated or used with caution if the duration of mechanical ventilation is more than 2 weeks, or if serious brain failure or bleeding tendency occurs. children with body temperature back to normal for at least 3 days, significant improvement in respiratory symptoms,and completion of two consecutive negative tests of respiratory pathogenic nucleic acid (sampling interval of at least 1 day) can be discharged. if necessary, home isolation for 14 days is suggested after discharge. special vehicles should be used to transfer infected patients. strict protection for staff of transportation and disinfection for the vehicle are of vital importance. enforcing close to the lines of notification of the project for transportation of pneumonia cases infected with novel coronavirus (trial version) published by the national health commission of people's republic of china is required. medical personnel are supposed to have good personal protection, hand hygiene, ward management, environmental ventilation, object surface cleaning and disinfection, medical waste management and other hospital infection control work, based on the standard prevention protocol, to minimize nosocomial infection. infectious diseases department, and isolation ward: equipped with medical overalls, caps, disposable isolation clothing, surgical masks, and goggles or face shield for daily medical activities and ward rounds; fit with goggles or face shield is stressed when collecting respiratory samples; using latex gloves in addition when contacting with blood, body fluid, secreta, or excreta; wearing surgical masks, goggles or face shield, latex gloves, medical protective clothing (disposable impermeable isolation clothing can be added), and respiratory hood when necessary, to prevent aerosol or splash during operations of endotracheal intubation, bronchoscopy, airway nursing, and sputum suction. 4. medical personnel should wear and remove personal protective equipment in strict accordance with the onoff procedure, rather than leaving ward with the contaminated equipment, so as to avoid cross-contamination in different zones. 5. patients and their accompanying family members are required to wear surgical masks. 1. channels for medical personnel and patients in the isolation ward should be separated and equipped with medical personnel channel buffer zone. 2. wearing gloves cannot replace hand hygiene. 3. perform strict visiting regulations for pediatric patients admitted in isolation, and ask visitors to have personal protection according to relevant regulations when necessary. 4. optimize medical procedures to reduce the frequency of medical personnel's contact with patients. 5. attention should be paid to the strict elimination and disinfection of the patient's secretions and excretions.author contributions all authors drafted part of the manuscript and approved the final version.funding none. ethical approval none. all authors declared no conflict of interest related to this paper. key: cord-322899-uxvlagt3 authors: gorji, ali; ghadiri, maryam khaleghi title: the potential roles of micronutrient deficiency and immune system dysfunction in covid-19 pandemic date: 2020-11-06 journal: nutrition doi: 10.1016/j.nut.2020.111047 sha: doc_id: 322899 cord_uid: uxvlagt3 preliminary studies indicate that a robust immune response across different cell types is crucial in the recovery from covid-19. an enormous number of investigations point to the vital importance of various micronutrients in the interactions between the host immune system and viruses, including covid-19. there are complex and multifaceted links between micronutrient status, the host immune response, and the virulence of pathogenic viruses. micronutrients play a critical role in the coordinated recruitment of innate and adaptive immune responses to viral infections, particularly in the regulation of pro-and anti-inflammatory host responses. furthermore, inadequate amounts of micronutrients not only weaken the immune system in combating viral infections, but also contribute to the emergence of more virulent strains via alterations of the genetic make-up of the viral genome. this study aimed to evaluate the evidence which suggests the contribution of micronutrients in the spread as well as the morbidity and mortality of covid-19. both the presence of micronutrient deficiencies among infected subjects and the effect of micronutrient supplementation on the immune responses and overall outcome of the disease could be of great interest to weigh the use of micronutrients in the prevention and treatment of covid-19 infection. these investigations could be of great value in dealing with future viral epidemics. coronaviruses (cov) are a large group of rna viruses that primarily target the human respiratory system and can lead to a wide range of illnesses from the common cold to severe respiratory syndromes. in the last two decades, outbreaks of cov-related infections, including the severe acute respiratory syndrome (sars)-cov and the middle east respiratory syndrome (mers)-cov, led to great public health problems and concerns. [1] a new coronavirus termed covid-19 is currently associated with an increasing number and rate of morbidities and fatalities. the genetic analysis of the covid-19 exhibited more than 50% sequence identity to mers-cov and 80% to sars-cov. [2] the innate immune system represents the first line of defense against viruses, which can inhibit virus replication, improve virus clearance, promote tissue repair, and activate a prolonged adaptive immune response against the viruses. [3] viruses, such as cov, could affect the function of the immune system in different manners, such as dysregulation of the macrophage antiviral response, induction of excessive cytokine-mediated immune system responses, and the activation of complement and coagulation cascades, which may result in enhanced infectivity and worse outcomes. [4] since there is currently no effective drug or vaccine, boosting the immune system could be a reasonable option to combat covid-19. a functional immune system is a prerequisite for the host's ability to prevent or limit viral infections. it is well-known that the nutrition of the host may influence the immune system and its susceptibility to viral infection. numerous studies pointed to the increase in either susceptibility to or severity of various viral infections in the nutritionally deficient subjects. [5] in addition to the host's response, various micronutrients can have a significant influence on disease severity via the modulation of viral pathogenesis, such as mutations in the viral genome. [6] on the contrary, a viral pathogen in the micronutrient deficient population could replicate to a new, more pathogenic strain. [7] the objective of this review was to provide a collection of evidence points to the key role of various micronutrients on the interactions between the host immune system and viruses, particularly coronaviruses. furthermore, we describe the evidence that may support the contribution of micronutrient deficiency and immune system dysfunction to the viral outbreaks, including covid-19. references for this review were identified through searches of pubmed for articles published from january 1961 to april 2020, by use of the terms "coronavirus", "immune system", "micronutrients", "vitamin", and "covid-19". relevant articles were identified through searches in google scholar and springer online archives collection. articles resulting from these searches and relevant references cited in those articles were reviewed. articles indicate a strong link between viral infections; particularly cov infection, with micronutrients and the immune system were selected. articles published in english and spanish were included. the recruitment of various immune cells, including antibody-secreting cells and follicular helper t cells as well as activated cd4 + and cd8 + t cells, along with igm and igg covid-19-binding antibodies have been reported in a patient with non-severe covid-19. [8] in the initiation stage of severe covid-19, increased amounts of pro-inflammatory cytokines and chemokines, like interleukin-2 (il-2), il-6, granulocyte-colony stimulating factor, interferon (ifn) γ-induced protein 10, monocyte chemoattractant protein-1, vascular endothelial growth factor, macrophage inflammatory protein-1α, and tumor necrosis factor-α (tnf-α), as well as lymphopenia have been detected in the serum of some patients. however, infection with covid-19 in its critical stage exacerbates the secretion of t-helper-2 (th2) cytokines, such as il-4, il-1ra, and il-10, which suppress the inflammatory response [9, 10] . the preliminary data indicate the ability of the immune system to recognize covid-19 and initiate an effective immune response across different cell types leading to successful recovery from the infection in cases with mild-to-moderate symptoms. the majority of subjects with covid-19 only have mild-to-moderate symptoms. however, the cytokine storm aggravates the infection severity and worsens prognosis. [9, 10] poor outcomes with enormously high values of pro-inflammatory cytokines have been also reported in patients infected with mers-cov and sars-cov. [11] in addition, it has been suggested that a significantly elevated amount of platelets in patients with covid-19, which is associated with a longer average hospitalization and poor prognosis, may be stimulated by the higher inflammatory cytokine levels. [12] baseline total lymphocytes were significantly higher in survivors than non-survivors. in survivors, lymphopenia improved during hospitalization, whereas severe reduction of lymphocytes continued to decrease until death in non-survivors. the values of serum ferritin and il-6 were markedly greater in non-survivors than survivors. [13] due to the supporting role of micronutrients on the host immune responses to viral infections (table 1) , it is not surprising if micronutrient deficiency would be associated with a weakened immune system and a higher risk for the occurrence and severity of viral infections. zinc homeostasis is essential for sustaining proper immune function. [14] zinc plays an important role in host-virus interactions due to its effect on nucleic acid synthesis and repair, apoptosis, inflammation, and redox homeostasis. [15] zinc baseline level is a crucial factor that can affect antiviral immunity, especially in zinc-deficient populations. [16] zinc deficiency is associated with impaired immune responses and leads to a higher risk of respiratory viral infections, particularly in elderly subjects. [17] zinc is involved in the modulation of the pro-inflammatory response by targeting nuclear factor kappa b (nf-κb). zinc deficiency enhances the production of pro-inflammatory cytokines, such as il-1β, il-6, and tnf-α, and reduces the lytic activity of natural killer (nk) cells. furthermore, zinc deficiency leads to a decrease in antibody production via the alteration of the function and number of various immune cells. [14] the zinc-finger domain is found in various proteins encoded by the genome of different cov, such as sars-cov [18] , and plays a key role in viral replication and transcription. [19] a specific mutation within the zinc-finger domain of cov caused reduced antiviral response. [20] disruption of the zinc-binding function of cov-229e nonstructural protein-13 (nsp13) or deletion of the entire zinc-binding domain affects both transcription and replication of cov. [21] furthermore, it has been shown that the zinc-binding domain may start to unfold during the first transition of sars-cov and lead to a reduction in pathogen virulence. selenium deficiency not only weakens the host immune system against viral infections but also leads to viral genome mutations from benign variants to highly pathogenic viruses. [4] inadequate antioxidant protection against various mutating rna viruses, including sars-cov, has been observed in subjects with blood selenium concentrations of less than 1 μm/l. iodide modulates the transcriptional immune signature of human peripheral blood immune cells and induces greater cytokine and chemokine secretion, such as il-6, il-8, and il-10. [36] iodide is found in the salivary glands, nasal mucosa, and lung secretions. [37] the sodium-iodine symporter, a plasma membrane glycoprotein that mediates active iodide transport in different tissues, contributes to the oxidation of iodide in the lungs, which improves the antiviral respiratory defense system. [38] the oral intake of potassium iodide copper is an essential nutrient for the development and maintenance of the human immune system. copper is crucial for the generation and response of il-2 to adaptive immune cells, the production of antibodies, maintaining intracellular antioxidant balance, and self-protection of immune cells. [41, 42] copper deficiency can lead to increased viral virulence, decreased il-2 level and t-cell proliferation, and reduced phagocytic ability. [43] copper exhibits a potent antiviral property, possibly via binding electron donor groups on viral proteins or nucleic acids. [44] copper antiviral effects may also due to the regulatory roles of copper on certain enzymes, which are critical for the function of various types of immune cells. [42, 43] in addition, activated macrophages accumulate copper within the phagosome to inactivate the pathogens. this event plays an important role in the control of pulmonary infections. [45] intravenous copper administration results in a greater copper concentration in the lung [46] , promotes the anti-inflammatory forkhead box p3-positive t-cells. [59] vitamin a deficiency leads to the reduced weight of the thymus, decreased lymphocyte proliferation, impaired tcell-mediated response, and enhanced pathogen binding to respiratory epithelial tissues. [60] vitamin a inhibits viral replication, promotes the immune response, and decreases morbidity and mortality of some viral infections. [61] the beneficial effects of vitamin a on morbidity and mortality of some viral infections, such as measles and hiv, could be due to increased antibody production and lymphocyte proliferation as well as enhanced t-cell lymphopoiesis. [62] clinical investigations and in vitro studies have indicated that vitamin a is the main regulator of mucosal immunity and could affect immune responses to mucosal infections. levels as well as an enhancement of cd4 + /cd8 + ratio and tnf-α value. [41] there are several experimental and clinical studies indicating the antiviral effects of b vitamins. patients with hiv suffered from a high prevalence of vitamin b1 deficiency. vitamin b1 affects hiv infection via non-genomic mechanisms, which may lead to beneficial effects in patients with hiv. [72] vitamin b2 alone or in combination with uv light has a potent antiviral effect on a wide range of viruses, such as mers-cov. [73] deficiencies in vitamins b6, b9, and b12 render people more susceptible to viral respiratory infections, such as influenza. [74] it has been suggested that a vitamin a-vitamin b6 conjugate analogue can exert an antiviral effect by regulating transcription and/or replication of various rna viruses, including coronavirus. [75] vitamin c a wide range of studies points to the importance of vitamin c in immune host responses to viral infections. vitamin c promotes the production, function, and migration of immune cells, and enhances serum values of antibodies and complement proteins. [76] vitamin c also supports the differentiation and proliferation of lymphocyte and enhances apoptosis, chemotaxis, and ifn production. [77] clinical trials and experimental studies suggested that vitamin c inhibits the pro-inflammatory cytokines, like tnf and il-6, and increases the proinflammatory cytokines, such as tnf, il-6, and il-1β. [78] vitamin c exerts an antiviral immune response against the influenza virus via the enhancement of ifn-il-1α/β production. [79] vitamin c enhances the resistance of broiler chicks [80] and chick embryo tracheal organ cultures [81] to infections induced by an avian coronavirus. vitamin c reduced the cytokine levels (tnf-α and il-1β) in an animal model of acute respiratory distress syndrome (ards); suggesting its beneficial effect for the treatment of similar inflammatory disorders. [82] indeed, the intravenous administration of vitamin c in patients suffering from sepsis and ards significantly reduced the mortality rate. [83] several investigations have suggested that vitamin c in high dosages has direct virucidal effects. [84] several clinical trials have shown a significantly lower incidence of rti in vitamin c-treated subjects. [85] on the contrary, vitamin c deficiency enhances the risk of respiratory infections, particularly in the elderly. vitamin d deficiency was associated with higher illness severity, multiple organ dysfunctions, and mortality in critically ill subjects, particularly with sepsis and pneumonia. vitamin e supports the integrity of epithelial membranes and increases il-2 production, nk cell activity, t-cell-mediated functions, and lymphocyte proliferation. furthermore, vitamin e initiates t-cell activation, promotes th1 proliferation, and inhibits th2 response. [101] vitamin e supplementation causes a higher il-2 and ifn-γ production with a lower lung virus titer in animals with the influenza virus. [102] vitamin e deficiency markedly increases the viral pathogenicity and heart damage in mice infected with coxsackieviruses-b3. [103] administration of vitamin e increased lymphocyte proliferation as well as il-2 and ifnγ production in healthy subjects and aged mice after influenza infections. [102] a modest level of vitamin e supplementation regulates the cellular free radical-antioxidant balance, enhances the antibody response, and activates the immune cells of broilers vaccinated with the infectious bronchitis virus. [104] h1n1-infected mice have shown positive associations between anti-inflammatory cytokine il-10 level and vitamin e metabolism. [105] vitamin e and selenium exhibit strong control over viral replication and mutation. in a nutritional deficiency condition of these micronutrients, rna viruses are able to convert to more virulent strains. [106] vitamin e deficient mice failed to exhibit an appropriate immune response to hsv-1 infection. [107] a significant increase in lung and serum vitamin e levels has been observed a few days after infection with the influenza virus in mice. [108] critically ill patients who were admitted to an icu with ards have shown a significant reduction of vitamin e plasma level. [109] the use of vitamins e and c in critically ill patients reduced the incidence of ards and pneumonia and shortened icu length of stay. [110] the most vulnerable groups to the severe-critical complications of covid-19 are the elderly above the age of 60 and subjects with chronic diseases, including hypertension, diabetes, and cardiovascular or respiratory diseases. [111, 112] although only 36% of patients infected with covid-19 in italy were over the age of 70, more than 80% of deaths occurred in people of this age. [113] furthermore, older adults are more susceptible to severe covid-19 at admission. [114] immune function in older adults can be modified by nutritional and pharmacological interventions. [115] aging causes alterations in every component of the immune system, which leads to increased morbidity and mortality following infectious diseases. [116, 117] the altered function of the immune system in the elderly can be promoted via the manipulation of cytokine production, changes of metabolic pathways in immune cells, and immune-system rejuvenation aimed at reactivating the generation of new lymphocytes. [118] micronutrient interventions have shown a promising impact in targeting the immune system impairments observed in the elderly and improve the infection-related morbidity and mortality. [119, 120] micronutrient deficiencies affect approximately two billion people worldwide and contribute essentially to the global burden of several diseases. [121] for instance, zinc deficiency affects approximately 30% of the world population with ranging from 4% to 73% across different countries and implicated in about 16% of lower rti. [122] micronutrient deficiencies decrease the ability to resist infections and are common causes of immunodeficiency in the developing countries. [123] although micronutrient deficiencies are one of the major public health challenges in developing countries, about 30% of the population in industrialized societies are also affected. [124] silent epidemics of micronutrient deficiencies could be due to insufficient intake and/or sufficient intakes in association with impaired absorption owing to infection, inflammation, or chronic diseases. [123, 125] approximately 35% of populations who are above the age of 50 in europe, usa, and canada have an obvious deficiency of one or more essential micronutrients. [126] in addition to an insufficient intake of micronutrients, older people lose their ability to produce endogenous antioxidants. [127] italy, spain, and france have experienced the highest covid-19 death toll in europe and the elderly in these countries have shown the highest prevalence of vitamin d deficiency compared with many other european countries. [128, 129] approximately 60% of people who died from covid-19 in italy were living in the lombardy region. during the cold seasons, up to 60-90% of the population in this region shows deficient/insufficient values of vitamin d. [130] the lombardy region, the most air polluted area of italy, has a high rate of hospitalizations and respiratory illnesses. [131] air pollution associated with increased ozone values absorbs uvb radiation and leads to vitamin d deficiency. [132] the overall prevalence of low vitamin d status is above 40% in the us population. [133] several clinical studies indicate the vital role of micronutrients in the prevention and treatment of viral infections. [134, 135] micronutrient deficiencies, including zinc and vitamins b2, b6, b12, c, and d, were reported to be common in the ecuadorian elderly, which weakened their immune system and placed them at greater risk of viral rti. [74] administration of zinc and vitamin a significantly decreased the incidence of pneumonia in children with pneumonia [136] , and oral zinc supplementation could shorten the duration of symptoms of respiratory infection. [137] food and agriculture organization of the united nations has reported that nutrition and antiviral drugs are equal in the treatment of hiv infection and regular intake of micronutrients is crucial for promoting the immune response and maintaining good health for both infected and uninfected individuals. [138] early administration of vitamin a reduced the mortality rate of patients with ebola virus disease during the western african outbreak. [139] supplementation of micronutrients in elderly subjects enhanced the number of t-cells and lymphocytes, improved lymphocyte response to mitogen, increased il-2 levels and nk cell activity, promoted the response to the influenza virus vaccine, and reduce the duration of viral diseases. [41, 140] some commonly used drugs, such as antibiotics, can lead to various micronutrient depletions, such as iron and vitamins a, b, and d. [141] a combination of micronutrient supplementation in older adults may decrease antibiotic usage and causes a higher post-vaccination immune response. [126] interestingly, some countries with higher morbidity and mortality of covid-19, such as italy and spain, have a greater consumption of antibiotics compared to other european countries. [142, 143] mice treated with antibiotics are unable to stimulate cytokine release in the lung and augment protective t-cell responses following influenza infection. [144] infectious disease outbreaks could indeed be the result of infection by a virus whose virulence has altered as a result of replicating in a nutritionally deficient host so that a non-virulent virus becomes a pathogen due to changes in its genome. in addition, available data strongly suggest that the association of unpredictable occurrence of novel viral pathogens combined with decreased host immunity and micronutrient deficiency poses a twofold threat to human health in the near future. further investigating the role of micronutrients and their substitution on immune system activity, therefore, may present a highly cost-efficient and uncomplicated measure with promising long-term benefits on future viral outbreaks. the development of novel vaccinations and drugs targeting pathogens that cause currently relevant diseases is often an expensive and risky process associated with a narrow spectrum efficacy due to their selective applications. furthermore, the use of novel vaccines and drugs is usually restricted to the global population due to their high costs. a decade ago, the copenhagen consensus project on hunger and malnutrition concluded that efforts to provide micronutrients for the global population generate higher returns than any other public health measure. 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weight gain the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord-321949-s1qu3odd authors: anderson, evan j; weber, stephen g title: rotavirus infection in adults date: 2004-01-28 journal: lancet infect dis doi: 10.1016/s1473-3099(04)00928-4 sha: doc_id: 321949 cord_uid: s1qu3odd rotavirus has been recognised for 30 years as the most common cause of infectious gastroenteritis in infants and young children. by contrast, the role of rotavirus as a pathogen in adults has long been underappreciated. spread by faecal-oral transmission, rotavirus infection in adults typically manifests with nausea, malaise, headache, abdominal cramping, diarrhoea, and fever. infection can also be symptomless. rotavirus infection in immuno-compromised adults can have a variable course from symptomless to severe and sustained infection. common epidemiological settings for rotavirus infection among adults include endemic disease, epidemic outbreak, travel-related infection, and disease resulting from child-to-adult transmission. limited diagnostic and therapeutic alternatives are available for adults with suspected rotavirus infection. because symptoms are generally self-limiting, supportive care is the rule. clinicians caring for adults with gastroenteritis should consider rotavirus in the differential diagnosis. in this review we intend to familiarise clinicians who primarily provide care for adult patients with the salient features of rotavirus pathophysiology, clinical presentation, epidemiology, treatment, and prevention. infective gastroenteritis causes substantial morbidity and mortality worldwide. although various bacterial species have long been associated with gastrointestinal disease, 1 specific viral causes of these infections were not delineated until the early 1970s. however, with the discovery of norwalk virus in 1972 2 and rotavirus in 1973, 3 the causative agents for most non-bacterial gastroenteritis infections were identified. almost immediately, the spectrum of viruses causing gastrointestinal infection in adults was recognised as differing from that in children. among children younger than 2 years, nearly half of all cases of diarrhoea requiring admission to hospital can be attributed to rotavirus infection. 4 by contrast, among adults most non-bacterial outbreaks of gastroenteritis can be linked to the norwalk-like viruses. 5 the important part played by viral pathogens besides the norwalk-like viruses in adults with gastroenteritis is not yet fully appreciated. specifically, the contribution of pathogens that typically affect children is not recognised by most clinicians who take care of adults. such is the case for adult infections caused by the common paediatric pathogen rotavirus. here we review important features of rotavirus microbiology and pathophysiology, along with relevant clinical and epidemiological features of rotavirus infection. in 1973, bishop and colleagues 3 described unique viral particles obtained from the duodenal mucosa of children with gastroenteritis. viruses with similar morphological appearance had been seen in 1963 in the intestinal tissue of mice with diarrhoea. 6 under the electron microscope, the 70 nm diameter viral particles first described in these reports had a wheel-like appearance, prompting the name rotavirus, from the latin rota (figure). 7 rotavirus is a non-enveloped virus now classified within the reoviridae family. 11 segments of doublestranded rna reside within the core. the rna encodes six viral proteins (vp) that make up the viral capsid, and six non-structural proteins (nsp). the core is surrounded by an inner capsid, composed mostly of vp6, the primary group antigen, 1, 8 and includes the epitope detected by most common diagnostic assays. other structural proteins also seem to confer some degree of group specificity. 9 the outer capsid is primarily composed of vp4 and vp7. 10 vp4 contributes the spoke-like projections to the wheel-shaped appearance of rotavirus. this vp is cleaved by trypsin in vitro to yield vp5* and vp8*, which appear to play an important part in cellular attachment. 9 the inner and outer capsids give the viral particle the double-layered icosahedral structure visualised on negative-stain electron microscopy. 10 seven distinct groups of rotavirus (named a to g) have been shown to infect various animal species. of these, only groups a, b, and c have been reported as human pathogens. 9 group a is the primary pathogen worldwide and is the group detected by commercially available assays. additional subgroups and serotypes can be identified by further characterisation of vp4, vp6, and vp7 antigens. 10 group b seems to be limited to causing epidemic infection in asia and the indian subcontinent, whereas group c rotavirus causes endemic infections that frequently go unrecognised. rotavirus spreads from person to person, mainly by faecaloral transmission. although rotavirus has been detected in urine and upper-respiratory samples, 11, 12 these body fluids are not believed to be commonly associated with transmission. after ingestion, rotavirus particles are carried to the small intestine where they enter mature enterocytes 13 through either direct entry or calciumdependent endocytosis. 14 after cytolytic replication in the mature enterocytes of the small intestine, new rotavirus particles can infect distal portions of the small intestine or be excreted in the faeces. more than 10 10 -10 11 viral particles per gram of faeces are excreted by children during infection. 8, 15 the amount of rotavirus excreted by adults might be more variable. in at least one study shedding was 10-100-fold lower in travellers' diarrhoea. 16 symptom-free adults can shed rotavirus in quantities so low as to be undetectable by most routine assays. 17 the mechanism by which rotavirus induces diarrhoea is poorly understood. few investigations have incorporated the study of human mucosal samples. the reports that are available describe various findings: villous shortening, flattening, and atrophy, denudation of microvilli, mitochondrial swelling, distension of the endoplasmic reticulum, depressed disaccharidase concentrations, and infiltration of mononuclear cells. 14, 18, 19 additional hypotheses about the pathophysiology of rotavirus gastroenteritis have been generated from animal studies. in one review the diminished ability of the intestinal epithelium to absorb fluid and nutrients, 14 stimulation of the enteric nervous system, 20 and local villous ischaemia and shortening resulting in impaired nutrient absorption were noted. a murine model of rotavirus infection suggests that rotavirus nsp4 acts as an enterotoxin, potentially by increasing calcium-dependent signalling of chloride secretion. 21, 22 the diarrhoea induced by rotavirus is unlikely to be completely explained by any one process, rather that several mechanisms contribute simultaneously. 14 these mechanisms are summarised in panel 1. 3, 13, 14, [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] resolution of rotavirus gastroenteritis depends greatly on the immunological response of the host. in a normal host, rotaviral antigens are transported to peyer's patches, undergo processing by b cells, macrophages, or dendritic cells and are presented to helper t cells. this cascade culminates in stimulation of rotavirus-specific b cell and cytotoxic t-lymphocyte-precursor expansion. 29 bernstein and colleagues 30 noted that stool rotavirus iga concentrations peaked 14-17 days after infection and persisted for longer than 1 year, but at declining concentrations. the researchers suggested that serum rotavirus iga is a more consistent marker of rotavirus immunity than other antibody measurements. 30 however, rotavirus-specific iga is frequently undetectable in duodenal fluid or faeces in the first week of infection, although symptoms might resolve within that time. 29 this pattern suggests a mechanism independent of humoral immunity. offit 29 notes that infected mature villous epithelial cells are steadily replaced by less-mature enterocytes, which may be less susceptible to rotavirus invasion. increased peristalsis improves clearance of viral particles and the non-specific activity of interferons can prevent vp translation. 29 de bouissieu 31 has reported that interferon ␣ concentrations correlate with a trend towards shorter duration of diarrhoea among patients who have rotavirus infection. although many physicians presume that rotavirus infection will confer lifelong immunity, multiple investigations show that re-infection can occur. bishop and colleagues 32 noted that infection with rotavirus during the neonatal period did not protect against developing rotavirus infection during the first 3 years of life but did lessen the severity of such infections. in a prospective study of 200 mexican infants followed up from birth, valazquez and colleagues 33 noted that by age 2 years 96% of infants had experienced a primary rotavirus infection. during the same period, nearly 70% of the infants experienced a second infection. more than 10% of the children studied had five or more rotavirus infections during the first 2 years of life. yolken and colleagues 34 had previously noted that by age reduced absorptive surface denudation of microvilli; shortening, flattening, and atrophy of villi; invasion of villi by rotavirus causing ischaemia and shortening 3, 18, 19 functionally impaired absorption depressed disaccharidase concentrations; impaired co-transport of glucose and sodium; decreased sodium-potassium atpase activity impairing electrochemical gradient 3, 18, 19, [23] [24] [25] cellular damage impairing absorption mitochondrial swelling; distension of endoplasmic reticulum; mononuclear cell infiltration 3, 13, 18, 19 enterotoxigenic effects of rotavirus protein nsp4 induces increased intracellular calcium concentrations; in murine models, acts like a toxin to induce diarrhoea 21, 22 stimulation of enteric nervous system stimulation of intestinal secretion of fluid and electrolytes; stimulation of intestinal motility resulting in decreased intestinal transit time 14, 20 altered epithelial permeability increased paracellular permeability by weakening tight junctions between cells [26] [27] [28] 2 years, more than 85% of children had antibodies to two different rotavirus serotypes. therefore, although nearly all adults have antibodies to rotavirus, 35 they might still be susceptible to infection. rotavirus can elude host defences and induce repeat infection through several mechanisms. there are multiple groups, subgroups, and serotypes of rotavirus. initial antibody response to infection is serotype specific, with limited production of cross-reactive antibodies. 36 subsequent rotavirus infections increase antibodies that cross-react with multiple serotypes. 36 additionally, certain elements of the rotavirus-specific immune response are short-lived. rotavirus-specific secretory iga is sometimes not detectable in faeces as early as 1 year after infection. 37 elias 38 reported that rotavirus fluorescent antibody titres peaked in children at age 1-3 years but subsequently fell to almost undetectable concentrations in individuals older than 70 years. 38 in a review of multiple studies, jiang and colleagues 36 an appreciation of the typical presentation of rotavirus infection in children is critical to understanding the spectrum of disease among adults. primary infection with rotavirus typically occurs in infants between ages 6 months and 2 years, although infection in neonatal intensive-care units and severe infection in infants younger than 6 months are well documented. 51, 52 in all age-groups, the classic presentation of rotaviral infection is fever and vomiting for 2-3 days, followed by non-bloody diarrhoea. 53 the diarrhoea may be profuse, and 10-20 bowel movements per day are common. when examined, the stool from affected patients is generally devoid of faecal leucocytes. especially when associated with vomiting, the diarrhoea of rotavirus infection can precipitate severe and even life-threatening dehydration. infants with repeat rotavirus infections are generally less-severely affected than those with primary disease. 33 among adults, rotavirus infection has been associated with a wide spectrum of disease severity and manifestations. as such, it is difficult to provide a concise description of a typical clinical presentation. nevertheless, prospective studies of voluntary rotavirus ingestion have provided some insight, although the participants in these studies were primarily young healthy adults. data from several such trials are summarised in the table. 35, [46] [47] [48] 54 for these volunteers, illness most frequently began 2-6 days after ingestion and continued for 1-4 days. about two-thirds of study participants had an antibody response, with more than half of all participants eventually shedding rotavirus. symptoms were less common than evidence of infection, but most frequently included diarrhoea, fever, headache, malaise, nausea, or cramping. one participant passed 11 stools in 1 day. 35 among the volunteers, rotavirus particles were detectable in the stool from the start of symptom onset and persisted for more than 10 days in some. three studies document rotavirus readministration to volunteers, noting that symptoms and antibody response were much less common. 35, 47, 54 in several reports of rotavirus outbreaks among adults similar symptoms have been described. in a prospective study of 98 families with neonates followed up from shortly after birth, only 17 of 43 adults who had serological evidence of rotavirus infection were symptomless. 55 14 had diarrhoea and 11 had abdominal cramping. none had symptoms that necessitated medical care or absence from work. in a separate study, 14 parents of children with rotavirus gastroenteritis developed serological evidence of infection, but only three had diarrhoea. 56 grimwood and colleagues 57 found, in a study of children with rotavirus in 28 families, that 18 of 54 adult family members exposed to rotavirus developed evidence of infection, and all but four were symptomatic. in a study of college students during a rotavirus outbreak, of the 83 individuals who met the criteria for rotavirus infection, 93% had diarrhoea, 90% abdominal pain or discomfort, 83% loss of appetite, 81% nausea, and more than 50% had fatigue, vomiting, headache, chills, subjective or low-grade fever, or myalgia. 58 patients with underlying immunodeficiency are at risk of sustained symptoms and rotavirus dissemination, a phenomenon already recognised among children. this pattern was first described in 1980 when two of four children with underlying primary immunodeficiency who had rotavirus infection developed chronic diarrhoea that at least temporarily responded to administration of human milk containing a high titre of rotavirus antibodies. 59 a geriatric patient with impaired naturalkiller-cell activity and impaired cellular and humoral immunity had rotavirus shedding for at least 35 days. 60 gilger and colleagues 61 noted that in four children who had various immune deficits and chronic diarrhoea from rotavirus, rotavirus was identified in the liver and kidney. whether the involvement of liver or kidney was important is unclear. natural infection quantity of cross-reactive neutralising antibody responses against multiple serotypes, 40-42 of virus-specific secretory iga at intestinal mucosa surface, 37, 43 and of virus-specific iga and igg in serum 41, 44 challenge studies serum serotype specific neutralising antibody; 35, 45 intestinal neutralising antibody; 46,47 serum rotavirus igg, but not necessarily with large doses; [46] [47] [48] and raised prechallenge titres of antibody to specific epitope of rotavirus vp7 49 vaccine-induced serum antibodies might be important in protection from natural infection, but interpretation is complicated because of differences in methods of existing studies; 36 and heterotypic antibody response to rotavirus vaccination might be protective against usual infecting serotypes 36, 50 other investigators have assessed the course of rotavirus infection in patients with malignant disease. a wide spectrum of clinical manifestations and severity of illness have been reported. bolivar 62 screened 90 adults who had various solid tumours and leukaemia, with and without diarrhoea. he noted that two patients had rotavirus infection, both of whom had undergone bone-marrow transplantation and had developed graft-versus-host disease. diarrhoea lasted for 10 days before resolution. in three subsequent studies in bone-marrow-transplant patients results varied. yolken and colleagues 63 prospectively assessed patients undergoing bone-marrow transplantation for infectious gastroenteritis and found that rotavirus occurred less frequently than clostridium difficile or adenovirus, but still occurred in nine of 78 patients. in one patient, adenovirus occurred concomitantly with rotavirus infection. of the eight remaining patients, all developed vomiting, seven had abdominal cramps, six had respiratory illness (infiltrates on chest radiography with appropriate clinical signs and symptoms) and four had diarrhoea. five of the eight rotavirus-infected patients eventually died. troussard and colleagues 64 also prospectively assessed patients undergoing bone-marrow transplantation and noted rotavirus infection in eight of 94 patients. adenovirus occurred concomitantly in two patients. seven of the patients had isolates positive for rotavirus in the winter months, with acute onset, vomiting, and diarrhoea. in a later study of the same topic, rotavirus was noted in four of 60 adult asymptomatic stem-cell-transplant patients followed up prospectively. 65 no patient with gastroenteritis had rotavirus isolated. rotavirus infections in adult patients infected with hiv-1 frequently present as a chronic diarrhoea with sustained viral shedding in stools. albrecht and colleagues, 66 between 1987 and 1991, detailed a retrospective assessment of 106 samples from 66 patients infected with hiv-1 who had otherwise unexplained diarrhoea. 35 samples from patients without diarrhoea served as controls. 13 samples from nine case patients were positive for rotavirus; two of these samples were rotavirus recurrences 6 months after the initial episode. no symptom-free patients had rotavirus infection. rotavirus was associated with diarrhoea of 2-8 weeks' duration in all patients and with abdominal cramping in eight patients. thomas and colleagues 67 looked prospectively at 862 samples from 377 uk hiv-1-positive patients with diarrhoea. rotavirus was third in frequency to adenovirus and coronavirus, occurring in 11 (2·4%) samples. the median cd4 count of patients with rotavirus infection was nine. hrdy 68 described five typical settings for rotavirus infections in adults. we propose modification of these classifications to the following: endemic disease, epidemic outbreaks, travelrelated gastroenteritis, and infections transmitted from children to adults. although substantial overlap exists between the groups, our classifications clarify separate risk factors and clinical features. rotavirus infection in children is seasonal, with peak incidence in winter months in temperate climates. iturriza-gomara and colleagues 69 noted that, in the uk between 1995 and 1998, notable numbers of infections began in december or january, peaking in march or april and falling to almost zero by july. in the usa, kapikian and colleagues 56 found that rotavirus cause 59% of diarrhoea cases necessitating admission to hospital in children between november and april, but could not be linked to cases from may to october. in several studies findings suggest that adult disease is not as season-specific as childhood disease. cox ffu=focus forming unit. nr=data not recorded in original paper and taken as not having occurred in calculation of summary percentages. *data included when >50% infectious dose ingested (>9 ffu). †15 of 38 patients had mild illness (including one patient with no antibody response or shedding). ‡one of four volunteers experienced illness but no specific symptoms were recorded. §summary percentages after rotavirus ingestion calculated only from data from studies in which full clinical syndrome of illness described. all percentages have been rounded to the nearest whole percentage point. adult serum in routine hospital samples are present throughout the year. igm concentrations increased with older age, and antibodies reached 20% in march and fell to 10-11% during the summer months. cox and medley attributed the high rates to igm persistence, igm crossreactivity, or possibly to non-seasonal high infection rates in adults. other researchers have also found that rates of adult disease do not mirror the winter seasonality of infection in paediatric patients. 71 these studies suggest that endemic disease in adults may not arise solely from unrecognised transmission of rotavirus from children to adults. the contribution of rotavirus as a cause of endemic gastrointestinal disease varies according to geographic distribution and characteristics of patients. in a small prospective study in the uk, rotavirus caused 4·1% of acute diarrhoea in adults admitted to hospital. 72 similarly, 3% of acute diarrhoea in switzerland, 73 3% of infectious diarrhoea pathogens in a swedish clinic for infectious diseases, 74 5% of adults with gastroenteritis requiring admission in thailand, 75 2-4% of adults older than 15 years with gastroenteritis presenting to their family physician in the netherlands, 76 and nearly 4% of individuals older than 45 years in michigan 77 were due to rotavirus. in studies in other geographic areas even higher rates of infection have been seen. in japan, nakajima and colleagues 71 reported that group a rotavirus had a role in 14% of patients with diarrhoea. pryor and colleagues 78 noted that rotavirus was second only to campylobacter spp as a cause of diarrhoea among australian adults, accounting for 17% of all cases. in indonesia, 42% of patients presenting with diarrhoea had rotavirus-positive stools compared with 11% of control samples. 79 in a study of mexican adults, 63% of patients presenting with acute gastroenteritis during winter months were positive for rotavirus. 80 even these results might underestimate the true prevalence of endemic rotavirus infection. group c rotavirus is not routinely detected by commercial assays but it does contribute to endemic rotavirus infection worldwide. in a study in the uk, 43% of patients were seropositive for group c rotavirus. 81 among adults, clusters of rotavirus infections most frequently occur in communities that are otherwise sheltered from more routine exposure to rotavirus-infected children. 82 one of the largest outbreaks involved nearly 3500 people in 1964, in an isolated area of micronesia. 83 since then, other outbreaks have occurred among closed communities, including a finnish military base, 84 an israeli kibbutz, 85 and an isolated south american indian community. 86 outbreaks of rotavirus infection have also occurred in long-term health-care facilities, particularly those with close living quarters; compromised host immunity and multiple comorbid disorders might help facilitate the spread of infection. 87-91 cubitt and colleagues 88 described an epidemic of rotavirus among staff and patients in an extended-stay geriatric hospital, in which 15 of 39 residents developed symptoms and seven had confirmed rotavirus infection. halvorsrud and orstavik 92 described an outbreak of 92 cases of acute gastroenteritis among nursing-home patients with identification of rotavirus by comparing acute and convalescent antibody titres. rotavirus has been suggested as the causal pathogen in 5% of diarrhoea outbreaks in a study of institutions caring for elderly residents. 93 among adults, rotavirus outbreaks are not confined to geriatric populations. group a rotavirus was associated with an outbreak of gastroenteritis among college students in the district of colombia. 58 rotavirus also caused a waterborne outbreak of gastroenteritis in 1981 in eagle-vail and avon, co, usa in which severity of symptoms correlated with the amount of tap water consumed. 94 finally, griffin and colleagues 95 screened 263 outbreaks of gastroenteritis in the usa between 1998 and 2000 and found that rotavirus was implicated in three outbreaks. uniquely affecting asia, group b rotavirus has been associated with outbreaks affecting large numbers of adults in broad geographic distributions of china and india. 96, 97 rotavirus has been implicated as an important contributor to travellers' diarrhoea among adults, especially among those visiting central america and the caribbean. in a study of travellers returning from jamaica, rotavirus was identified in 9% of individuals with diarrhoea, making the virus second only to enterotoxigenic escherichia coli as a cause. 98 in two studies of us students travelling in mexico, electron microscopy identified rotavirus in about 25% of patients who had diarrhoea, compared with 3% and 15%, respectively, of symptom-free patients. 99, 100 in a third study, a substantial rise of antibodies to rotavirus was seen in 17% of two student groups travelling to mexico. 101 by contrast, only 5-6% seroconverted to norwalk virus. 101 ryder and colleagues 102 found rotavirus in 26% of panamanian travellers to mexico who had diarrhoea. sheridan and colleagues 103 similarly found that 36% of us peace corps volunteers and 30% of panamanian travellers visiting mexico had at least a four-fold increase in rotavirus antibody titres. adult travellers with rotavirus shed 10-100 times less rotavirus than do paediatric patients. 99, 100 although rotavirus can be linked to adult gastroenteritis in each of the other settings, adults who are in contact with children are at particularly high risk of infection. transmission of rotavirus within families from children to parents seems to be a common event. wenman and colleagues 55 showed prospectively that rotavirus infection occurred in 36 of 102 adults caring for children with rotavirus infection. by contrast, only four of 86 adults whose children had no documented rotavirus infection became infected. 55 grimwood and colleagues 57 confirmed this finding in a report that a third of adult family members in new zealand developed evidence of rotavirus infection. the same phenomenon has been seen among parents of more severely ill children. kim and colleagues 104 found evidence of rotavirus infection in 55% of adult contacts of children who were admitted to hospital with rotavirus, compared with 17% of adult contacts whose children were not infected. more casual contact might also be sufficient to facilitate rotavirus transmission from children to adults. rodriguez and colleagues 105 reported that nine of 12 adults experienced illness after exposure to children infected with rotavirus in a playgroup. although substantial evidence is lacking, child-to-adult transmission of rotavirus is accepted to occur with some frequency on paediatric wards. many paediatric nurses, medical students, and house officers experience symptoms of gastroenteritis during the winter months when most paediatric rotavirus infections are encountered. von bonsdorff and colleagues 106 described paediatric nurses at several different locations with acute gastroenteritis caused by rotavirus. among seven hospital staff that developed diarrhoea after direct contact with children with diarrhoea staying in hospital, a rise in antibody titres was detected in three. 104 interestingly, in the same study, six of 45 medical students reported gastroenteritis. three of the students had rotavirus particles present on electron microscopy and were noted to be more ill than the parents of the children who were infected. all had diarrhoea for 3-6 days and two of the three had low-grade fever and vomiting. another case report supports transmission of rotavirus from children to hospital caretakers. 107 electron microscopy, which permits visualisation of the pathognomonic wheel-like appearance, was initially used for diagnostic purposes, but elisa or eia have become more commonly used. commercial assays are reliable, convenient, and inexpensive, but require at least 10 4 -10 7 virions to generate a positive result. 108, 109 the false-positive rate of commercial assays is 3-5%. 69 one of the biggest limitations of most commercial assays is that they do not detect non-group-a rotavirus. 68, 110 other more sensitive and newer methods are being used in research. one such method is pcr, which is up to 1000 times more sensitive than immunoassays. 109 in one study using pcr, 30% of otherwise healthy children shed virus for 25-57 days after symptoms developed. 111 although stool cultures are routinely tested for bacterial pathogens, the low frequency of detecting a positive result calls the usefulness of this practice into question. rotavirus infection can occur in a similar number of patients to some bacterial pathogens. sending a sample of rotavirus antigen for testing by elisa or eia could potentially cut costs if by doing so either hospital stay or procedures could be avoided. such a cost-benefit analysis in adult patients has not been published. one limitation is that adults might shed less rotavirus in faeces than do children, further hampering diagnosis. 100 we suggest that obtaining rotavirus antigen testing for patients admitted to hospital with risk factors for rotavirus infection will be cost effective if additional inpatient studies can be avoided. determination of rotavirus infection may also be beneficial if infectious patients can be isolated to prevent nosocomial spread. a positive rotavirus antigen test might also allow physicians to avoid prescribing antibiotics for travel-related rotavirus infections. treatment of rotavirus infections is primarily directed at symptom relief and restoration of normal physiological function. oral rehydration should be attempted initially. in most developing countries, oral rehydration salt solutions are used extensively in children. most adults can be managed by encouraging them to drink fluids. an additional intervention that has been used is administration of lactobacillus spp bacteria to shorten the duration of diarrhoea. 112, 113 although seldom used in children, codeine, loperamide, and diphenoxylate can help with symptom relief and control of the volume of diarrhoea. 114 bismuth salicylate, in a placebo-controlled double-blinded trial, was efficacious in treating the symptoms of rotavirus diarrhoea. 115 trial use of bismuth salicylate can be considered in adults when other coexistent infectious causes have been ruled out. if symptoms cannot be controlled and the patient becomes dehydrated, administration of intravenous fluids and hospital admission might be necessary. rarely, extraordinary measures have been attempted to help resolve rotavirus infections. for example, human breastmilk has been provided to immunodeficient infected children to help resolve chronic diarrhoea. 59 this option, however, is not practical in adults. several groups report oral administration of human serum immunoglobulins possessing antirotavirus activity to bind free rotavirus antigen. guarino and colleagues 116 noted a mean duration of diarrhoea of 76 h in children who received one oral dose of 300 mg/kg human serum immunoglobulin, compared with 131 h in children who did not. in a study involving three immunocompromised children with chronic rotavirus diarrhoea, oral administration of human serum immunoglobulins (igg 150 mg/kg) cleared rotavirus antigen in all three, but rotavirus antigen recurred in two. 117 among adults, oral immunoglobulin administration of 5-6 g daily for 5 days to bone-marrow-transplant recipients has been successful. 118 prevention of rotavirus infection can be facilitated by avoiding exposures and faecal-oral spread. contact with sick children and potentially contaminated food and water should be avoided. since 43% of rotavirus virions placed on human fingers survive for 60 min, thorough hand washing is critical in prevention. 119 contact isolation for patients diagnosed with rotavirus infection is necessary, generally for the duration of hospital stay, because of sustained faecal shedding of low concentrations of virus. 120 gloves, gowns, isolation, and rigorous hand washing should be used in the care of individuals infected with rotavirus. 121 sattar and colleagues 122 reported that rotavirus survives best in low humidity on non-porous surfaces at room temperature or cooler. phenolic disinfectants do not inactivate rotavirus; instead hypochlorite or sodium dichloroisocyanurate tablets with a free chlorine concentration of at least 20 000 parts per million are recommended. 110 a 70% ethanol solution is also effective in inactivation of rotavirus and can help to prevent environmental spread. 120 rotavirus infection in adults has been successfully prevented by use of a commercially available review disinfectant spray on rotavirus contaminated fomites under experimental conditions. 123 given the substantial disease-related morbidity and mortality associated with rotavirus, the development of an effective vaccine is a priority. although multiple vaccines were under development, the tetravalent rhesus-human reassortant rotavirus vaccine (rrv-tv) seemed to produce the best results. the rrv-tv vaccine prevented about half of rotavirus infections, but was much more effective in preventing severe disease. 124, 125 shortly after the vaccine was approved, the vaccine adverse-event monitoring system noted by mid-1999 an excess of cases of intussusception among recently vaccinated infants, eventually prompting the vaccine to be withdrawn. two sharply differing perspectives on the risk and benefits of the rrv-tv vaccine and the advisory committee on immunization practices' confirmation of its decision to withdraw its recommendation for the vaccine have been put forward. 126, 127 other vaccines are under development. vaccines have been primarily developed to attempt to decrease the severity of rotavirus infections in children. although vaccines seem to be fairly safe in adults from the vaccine trials, we are unaware of any plan to consider vaccination in adult patients. vaccination could theoretically be used in adult patients considering travel to central america or the caribbean or among immunocompromised patients to prevent or lessen the severity of rotavirus diarrhoea. despite recognition as an important cause of gastroenteritis in children, rotavirus's role in adult gastroenteritis is underappreciated. immunity to rotavirus is incomplete and most people have multiple infections over their lifetime. adults with rotavirus can be asymptomatic, but the most common symptoms are nausea, malaise, headache, abdominal cramping, diarrhoea, and fever. adults at particular risk of rotavirus infection are travellers, adults exposed to infected children, and immunocompromised people. group a rotavirus, the most common human pathogen, can be diagnosed with many different commercial assays, but all have limited sensitivity. rotavirus testing might be beneficial in certain clinical settings if detecting rotavirus would change management of patients or prevent nosocomial spread. treatment is primarily symptomatic. rotavirus should be considered in the differential diagnosis of adult infectious gastroenteritis. we have no conflicts of interest. we identified sources for this review by searches of medline with use of the key words "rotavirus infection" and "adult." the search strategy and selection criteria included all english and human studies from the year 1970 until the present. we further reviewed the abstracts for relevance before inclusion. review of the references of the papers retrieved by the initial search allowed for additional studies to be identified and considered for inclusion. visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis viral gastroenteritis molecular epidemiology of "norwalk-like viruses" in outbreaks of gastroenteritis in the united states epizootic diarrhea of infant mice: identification of the etiologic agent relationship between viruses from acute gastroenteritis of children and newborn calves rotavirus infections: guidelines for treatment and prevention rotaviruses and their replication fields' virology nonenteric sources of rotavirus in acute diarrhea rotavirus infection of the oropharynx and respiratory tract in young children infantile enteritis viruses: morphogenesis and morphology pathogenesis of rotavirus diarrhea rotaviruses of man and animals human rotavirus in an adult population with travelers' diarrhea and its relationship to the location of food consumption excretion of serotype g1 rotavirus strains by asymptomatic staff: a possible source of nosocomial infection duodenal mucosal damage in 31 infants with gastroenteritis structural and functional abnormalities of the small intestine in infants and young children with rotavirus enteritis role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein diarrhea induction by rotavirus nsp4 in the homologous mouse model system rotavirus infection impairs intestinal brush-border membrane na(+)-solute cotransport activities in young rabbits d-glucose transport in piglet jejunal brush-border membranes: insights from a disease model absence of a campmediated antiabsorptive effect in an undifferentiated jejunal epithelium rotavirus alters paracellular permeability and energy metabolism in caco-2 cells intestinal permeability assessed with polyethylene glycols in children with diarrhea due to rotavirus and common bacterial pathogens in a developing community rotavirus-induced structural and functional alterations in tight junctions of polarized intestinal caco-2 cell monolayers host factors associated with protection against rotavirus disease: the skies are clearing induction and persistence of local rotavirus antibodies in relation to serum antibodies rotavirus induces alpha-interferon release in children with gastroenteritis clinical immunity after neonatal rotavirus infection: a prospective longitudinal study in young children rotavirus infections in infants as protection against subsequent infections epidemiology of human rotavirus types 1 and 2 as studied by enzyme-linked immunosorbent assay oral administration of human rotavirus to volunteers: induction of illness and correlates of resistance the role of serum antibodies in the protection against rotavirus disease: an overview role of coproantibody in clinical protection of children during reinfection with rotavirus distribution and titres of rotavirus antibodies in different age groups mechanisms of protection against rotavirus in humans and mice protective effect of naturally acquired homotypic and heterotypic rotavirus antibodies anti-rotavirus g type-specific and isotype-specific antibodies in children with natural rotavirus infections protection against rotavirus disease after natural rotavirus infection: us rotavirus vaccine efficacy group fecal antibody responses to symptomatic and asymptomatic rotavirus infections serum antibody as a marker of protection against natural rotavirus infection and disease studies in volunteers with human rotaviruses effects of antibody to rotavirus on protection of adults challenged with a human rotavirus protection of adults rechallenged with a human rotavirus human rotavirus studies in volunteers: determination of infectious dose and serological response to infection identification of vp7 epitopes associated with protection against human rotavirus illness or shedding in volunteers homotypic and heterotypic epitope-specific antibody responses in adult and infant rotavirus vaccinees: implications for vaccine development clinical manifestations of rotavirus infection in the neonatal intensive care unit rotavirus gastroenteritis in infants aged 0-6 months in melbourne, australia: implications for vaccination comparison of serum and mucosal antibody responses following severe acute rotavirus gastroenteritis in young children orbivirus acute gastroenteritis of infancy rotavirus infection in adults. results of a prospective family study human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children spread of rotavirus within families: a community based study foodborne outbreak of group a rotavirus gastroenteritis among college students: district of columbia chronic rotavirus infection in immunodeficiency prolonged shedding of rotavirus in a geriatric inpatient extraintestinal rotavirus infections in children with immunodeficiency rotavirus screening in adult cancer patients infectious gastroenteritis in bone-marrow-transplant recipients virus recovery from stools of patients undergoing bone marrow transplantation infectious gastroenteritis: an uncommon cause of diarrhoea in adult allogeneic and autologous stem cell transplant recipients rotavirus antigen detection in patients with hiv infection and diarrhea enteric viral infections as a cause of diarrhoea in the acquired immunodeficiency syndrome epidemiology of rotaviral infection in adults molecular epidemiology of human group a rotavirus infections in the united kingdom between 1995 and 1998 serological survey of antigroup a rotavirus igm in uk adults winter seasonality and rotavirus diarrhoea in adults aetiology of acute diarrhoea in adults etiology of acute infectious diarrhea in a highly industrialized area of switzerland enteropathogens in adult patients with diarrhea and healthy control subjects: a 1-year prospective study in a swedish clinic for infectious diseases rotavirus as a cause of severe gastroenteritis in adults etiology of gastroenteritis in sentinel general practices in the netherlands the tecumseh study. xv: rotavirus infection and pathogenicity acute diarrhoea in adults: a prospective study enteropathogens associated with acute diarrhea in community and hospital patients in jakarta, indonesia acute gastroenteritis associated with rotavirus in adults seroepidemiology of human group c rotavirus in the uk rotavirus infections in adults in association with acute gastroenteritis gastroenteritis due to rotavirus in an isolated pacific island group: an epidemic of 3,439 cases rotavirus epidemic in adults two sequential outbreaks of rotavirus gastroenteritis: evidence for symptomatic and asymptomatic reinfections an outbreak of rotavirus diarrhea among a nonimmune, isolated south american indian community rotavirus infection in a geriatric population an outbreak of rotavirus infection in a long-stay ward of a geriatric hospital epidemic of viral gastroenteritis in an elderly community an outbreak of rotavirus infection in a geriatric hospital outbreaks of astrovirus type 1 and rotavirus gastroenteritis in a geriatric inpatient population an epidemic of rotavirusassociated gastroenteritis in a nursing home for the elderly outbreaks of infectious intestinal disease in residential institutions in england and wales 1992-1994 a community waterborne gastroenteritis outbreak: evidence for rotavirus as the agent outbreaks of adult gastroenteritis traced to a single genotype of rotavirus investigation of an outbreak of adult diarrhea rotavirus in china emergence of adult diarrhoea rotavirus in calcutta, india epidemiology, etiology, and impact of traveler's diarrhea in jamaica rotavirus in travelers' diarrhea: study of an adult student population in mexico human rotavirus in an adult population with travelers' diarrhea and its relationship to the location of food consumption norwalk virus and rotavirus in travellers' diarrhoea in mexico travelers' diarrhea in panamanian tourists in mexico traveler's diarrhea associated with rotavirus infection: analysis of virus-specific immunoglobulin classes human reovirus-like agent infection: occurrence in adult contacts of pediatric patients with gastroenteritis common exposure outbreak of gastroenteritis due to type 2 rotavirus with high secondary attack rate within families rotavirus associated with acute gastroenteritis in adults rotavirus-associated gastroenteritis in two adults probably caused by virus reinfection viral infections of the gastrointestinal tract improved detection of rotavirus shedding by polymerase chain reaction control of outbreaks of viral diarrhoea in hospitals: a practical approach extended excretion of rotavirus after severe diarrhoea in young children oral bacterial therapy reduces the duration of symptoms and of viral excretion in children with mild diarrhea bacteriotherapy with lactobacillus reuteri in rotavirus gastroenteritis rotavirus infections: guidelines for treatment and prevention bismuth subsalicylate in the treatment of acute diarrhea in children: a clinical study oral immunoglobulins for treatment of acute rotaviral gastroenteritis oral administration of human serum immunoglobulin in immunodeficient patients with viral gastroenteritis: a pharmacokinetic and functional analysis severe rotavirus-associated diarrhoea following bone marrow transplantation: treatment with oral immunoglobulin rotavirus survival on human hands and transfer of infectious virus to animate and nonporous inanimate surfaces red book: 2003 report of the committee on infectious diseases infection control for hospitalized children institutional outbreaks of rotavirus diarrhoea: potential role of fomites and environmental surfaces as vehicles for virus transmission prevention of surface-to-human transmission of rotaviruses by treatment with disinfectant spray randomised placebo-controlled trial of rhesushuman reassortant rotavirus vaccine for prevention of severe rotavirus gastroenteritis efficacy of the rhesus rotavirus-based quadrivalent vaccine in infants and young children in venezuela reappraisal of the association of intussusception with the licensed live rotavirus vaccine challenges initial conclusions the first rotavirus vaccine and intussusception: epidemiological studies and policy decisions for personal use. only reproduce with permission from the lancet. a 73-year-old woman presented with a 2-year history of persistent ulcerous nasal injury that had progressed slowly and without fever, respiratory, or any general symptoms, until the upper lip was affected. a painless ulcero-vegetating injury was seen but the patient was otherwise normal; chest radiographs did not show abnormalities. further examination showed soft tissue attenuation and an ulcered mass lesion in the nasal cavity (figure). the definitive diagnosis was made by isolating mycobacterium tuberculosis from tissue removed during biopsy and antituberculous therapy was started. the lesion had resolved at completion of treatment.tuberculosis of head and neck occurs infrequently and involvement of the nose is rare. granulomatous lesions within the nasal cavity may represent either local disease or a manifestation of a systemic disorder and the differential diagnosis must include tuberculosis. although almost forgotten in industrialised countries, this unusual form of tuberculosis can appear mainly in females and the elderly. it is not thought to be contagious, or to produce noticeable symptoms or physical signs. key: cord-341548-gazsszs6 authors: buscho, r. o.; saxtan, d.; shultz, p. s.; finch, e.; mufson, m. a. title: infections with viruses and mycoplasma pneumoniae during exacerbations of chronic bronchitis date: 1978-04-17 journal: j infect dis doi: 10.1093/infdis/137.4.377 sha: doc_id: 341548 cord_uid: gazsszs6 the association of viral and mycoplasma pneumoniae infections with acute exacerbations of chronic bronchitis was studied by serologic or isolation techniques in 46 adult men during the five years from 1964 through 1968. serologic evidence of viral or m. pneumoniae infection was detected in 25% of 166 episodes of exacerbation and 14% of 138 remission periods (p = 0.02). influenza a virus, parainfluenza virus type 3, and coronavirus oc43 predominated; infections with other viruses were infrequent. infection with m. pneumoniae was detected serologically in four patients, but this organism was never isolated from sputum specimens. rhinoviruses were isolated from frozen-stored sputum specimens in 2.7% of the episodes of exacerbation and from 0.55% of the remission intervals (p not significant). these data suggest that although exacerbations of chronic bronchitis may be accompanied by viral and m. pneumoniae infections, patients with chronic bronchitis also acquire such infections without a worsening of their respiratory status. most of the microorganisms that infect the respiratory tract can be recovered from individuals undergoing exacerbations of chronic bronchitis [1, 2] . however, the role of viral and mycoplasmal infections in exacerbations of chronic bronchitis has not been fully documented. moreover, the observations regarding infections in chronic bronchitics in a designated patient population during any specified period may not be comparable to those in other populations. because of the high prevalence and morbidity of chronic bronchitis among patients of veterans administration hospitals, we have conducted surveillance of one of these groups to assess the impact of respiratory tract infections on the natural course of this disease and to investigate further the occurrence and relative importance of viruses and mycoplasmas in exacerbations [3] . evidence of viral and mycoplasma pneumoniae infections (obtained mainly by serologic testing and to a lesser extent by isolation of organisms) was correlated with the pattern of clinical disease in patients with chronic bronchitis. design of study. forty-six male adults with chronic bronchitis, outpatients at the veterans administration hospital, hines, ill., constituted the study group. these patients all fulfilled the criteria for the diagnosis of chronic bronchitis, defined as a chronic or recurrent increase in the volume of mucoid or mucopurulent bronchial secretions sufficient to cause expectoration, during at least three months of each of the two years prior to entry into the study group. their clinical and microbiological status was studied longitudinally during the five years from 1964 through 1968. monthly or in alternate months and during periods of exacerbation, each patient reported to the outpatient clinic for examination. at each visit a symptom questionnaire was completed by the examiner, a physical examination was performed, pulmonary function tests were done, a sputum specimen was collected for isolation of virus and mycoplasma, and a serum specimen was obtained for serologic evaluation. the questionnaires and definitions of remissions and exacerbations were modeled after the guidelines reported by the medical research council [4] . exacerbations were defined as an increase in cough or sputum production since the patient's previous visit. symptoms of upper airway infection were not included among these criteria. an exacerbation was considered satisfactorily tested for viral and m. pneumoniae infections when serum pairs were obtained before and after this episode and a sputum specimen was collect-edior mycoplasmal and viral isolation. one hundred sixty-six (62%) of 270 exacerbations were tested in this manner. for comparison, 138 intervals when patients did not experience an exacerbation or were otherwise in remission were similarly tested. population characteristics. all patients were veterans who attended the pulmonary outpatient clinic on a regular basis. they visited the clinic for monthly follow-up an average of 10.3 months per year and made an average of 11.0 total visits per year. persons who were habitual poor attenders were not included in the study population. the ages of the 46 patients at the beginning of the study period ranged from 31 to 79 years; the distribution of age was as follows: 30-39 years, two patients; 40-49 years, 13; 50-59 years, nine; 60-69 years, 13; and 70-79 years, nine. the median age decade was 50-59 years. all patients were or had been cigarette smokers. the range of pack-years (one pack per man per year) was five to 125 (median, 49 years). pulmonary function tests on entry into the study disclosed a marked degree of obstructive pulmonary disease. the mean values of forced vital capacity (fvc) , forced expiratory volume (fev 1)' and the ratio fev 1/fvc were 2.45 liters, 1.31 liters, and 52%. expressed as a percentage of normal, the fev 1 for study patients ranged from 10% to 80%, but two-thirds of the group had values of 40%. similarly, the fev 1/fvc ratio ranged .from 30% to 80% of normal, and two-thirds of the group had values of~50%. procedures for isolation of virus. sputum specimens for viral isolation were stored at -70 c for five to eight years. when ready for use, speci-mens were rapidly thawed, and 0.4 ml was suspended in 2.0 ml of chilled veal infusion broth treated with 500 units of penicillin rrnl, soo p,g of streptomycin rml, and 0.02 mg of amphotericin b /ml of broth for 1 hr at 4 c. a volume of 0.2 ml of the treated sputum suspension was inoculated into each of two roller-tube cultures of human diploid fibroblasts (vvi-38 strain) for recovery of picornaviruses, adenoviruses, herpesviruses, and coronavirus strain 229e. because of the long period of storage of the sputum specimens, no attempt was made to isolate myxoviruses. wi-38 roller-tube cultures were obtained from flow laboratories, rockville, md. maintenance medium for these cell cultures consisted of eagle's minimal essential medium supplemented with 2% inactivated fetal calf serum; the complete medium was adjusted to ph 7.0 with 7.sc;o nahc0 3 [s] . cultures were incubated on a rotating drum at 33 c for at least 21 days and often for as long as 28 days. subpassages of negative cultures were not made. cell cultures were examined three times a week for cpe, and the maintenance medium was changed at these times. viral isolates \.vere identified by procedures previously described [5] . mycoplasma. a fresh agar medium consisting of pleuropneumonia-like organism agar (difco, detroit, mich.), 20% horse serum, 10% yeast extract, penicillin g (2,000 unitsj ml). amphotericin b (5.6 jlg/ml), and thallium acetate (0.05%) was used for the isolation of all mycoplasmas. duplicate plates were inoculated with sputum. one plate was incubated aerobically and the other anerobically at 37 c for four weeks. mycoplasma isolates were identified by growth inhibition procedures using specific hyperirnmune antiserum [6] . included in the test were antisera to m. pneumoniae, mycoplasma orale) mycoplasma hominis, mycoplasma [errnentans, mycoplasma arthriditis, and mycoplasma salioarium. approximately one-half of the cultures were passaged on agar a second time from an area selected adjacent to the zone of inhibition in an attempt to identify mixed cultures of mycoplasmas. none were detected. serologic procedures. serial serum specimens were tested by cf for antibodies to influenza a and b viruses, respiratory syncytial virus (rsv), parainfluenza virus types 1, 2, and 3, adenoviruses, and coronavirus types 229e and oc43 and for m. pneumoniae. microtiter cf tests containing 1.7-1.8 units of complement were employed after fixation overnight at 4 c. four units of each viral and mycoplasmal antigen were used. serologic evidence of infection was defined as a fourfold or greater rise in antibody titer between the acuteand convalescent-phase sera. occurrence of acute respiratory illness in patients with chronic bronchitis. the total number of exacerbations experienced by each patient during their participation in this study ranged from one to 14 (mean, 5.9). the number of exacerbations of anyone patient usually was directly proportional to the number of years of participation in the surveillance program. the mean number of years of patient participation was 3.7. only one patient reported no exacerbations during the four-year period of the study. exacerbations of chronic bronchi tis tended to occur more often in the winter months than in the summer months, although they occurred frequently in all months of the year (table 1) . serologic evidence -of viral and m. pneumoniae infections. infections with respiratory virus or m. pneumoniae were detected at least once in 40 of the 46 patients studied. the mean num-379 ber of infections was 1.9 (range, 1-6). although patients included in the study for a longer time experienced somewhat more virus and m. pneumoniae infections, the relationship was not linear, a finding suggesting that individual differences in rates of infection could not be attributed exclusively to the duration of participation in the study. fourfold or greater rises in titer of antibody to virus or m. pneumoniae were detected in 50 (30.1%) of 166 exacerbations of chronic bronchitis in the 46 adult patients studied (table 2) . by comparison, viral and m. pneumoniae infections were detected in 38 (27.5%) of 138 remissions. these rates were not significantly different and suggest that patients with chronic bronchitis can undergo infections with these agents apparently without an acute worsening of their respiratory status. when rises in titers of antibody to multiple agents which occurred simultaneously during a single episode were excluded from this analysis, the distribution of single-agent infections could be assessed (table 3) . infections defined as a rise in titer of antibody to a single agent (a virus or m. pneumoniae) were detected in 41 (24.7%) of 166 exacerbations of chronic bronchitis and only 19 (13.8%) of 138 remissions, a difference which was significant (p = 0.02). the frequency of individual viral and m. pneumoniae infections was analyzed using only these single-agent antibody titer rises (table 4) . fortyone (82%) of 50 rises in antibody titer during exacerbation were single-agent antibody titer rises, and during remission 19 (50%) of the 38 rises were single-agent antibody titer rises. the higher incidence of multiple-agent antibody titer rises in remission intervals may be a reflection of the study design. more attention was paid to specimen collection during exacerbations with the result that the average intervals defined by available serum pairs were 1.5 months for exacerbation and 3.2 months for remission. myxovirus and coronavirus infections predominated (table 4) . influenza a virus, parainfluenza virus type 3, and coronavirus oc43 accounted for nearly two-thirds of all infections. in each instance these infections occurred more often in exacerbation than in remission, a finding that suggests their association with the occurrence of exacerbations of chronic bronchitis; however, the difference was significant only for influenza a virus and coronavirus oc43 (p < 0.05). all other viral infections occurred as often in exacerbation as in remission. m. pneumoniae infection alone occurred in 2.4~o of exacerbations but not in any remission. fifteen additional rises in titer of antibody to m. pneumoniae occurred simultaneously with respiratory virus infections. these rises were equally distributed between periods of exacerbation and remission. viral infections among patients with chronic bronchitis occurred as defined outbreaks ( figure 1) in this study, viral and m. pneumoniae infections were common in patients during exacerbation, but such infections occurred also in these patients without worsening of their respiratory status. we reported our findings in two ways: as rises in titer of antibody to a single agent and as rises in titer of antibody to two or more agents within the same interval. rises in titers of antibody to multiple agents predominated in remission intervals, which tended to be longer than exacerbation intervals. this difference may have accounted for some variation in the antibody titer which was not indicative of specific viral infection. antibody titer rises to multiple agents also may reflect heterotypic responses and overestimate the number of infections. by analyzing antibody titer rises to single agents in this report, we estimated the importance of infection with each specific virus, but the approach precluded assessment of the role of dual infection with two viruses or a virus and m. pneumoniae in exacerbations. rises in titer of antibody to m. pneumoniae, however, were detected most often in association with a titer rise to one or more respiratory viruses. the possibility that m. pneumoniae can function as a secondary or synergistic pathogen in respiratory tract infections cannot be excluded and requires further investigation. that viruses may playa role in exacerbations of chronic bronchitis is suggested by the finding that one-fourth of exacerbations were associated with viral infections. this rate was twice that of viral infection in remission. infections with influenza a virus and coronavirus oc43 occurred significantly more often in exacerbations than in remissions. coronaviruses have been recognized recently as etiologic agents of respiratory illness in children and adults [8] [9] [10] . gump and coworkers also reported association of these agents with exacerbations of chronic bronchitis [11] . they found that four of seven infections with coronavirus oc43 and two of seven infections with coronavirus 229e were associated with an exacerbation. evidence of this relationship has accumulated very slowly because of the requirement for recovery of strain oc43 in human fetal trachea organ cul-tures, a difficult procedure. infection with coronavirus 229e was not detected either by isola tion or rise in antibody titer in the patients in our study. the finding in this study of rhinovirus infections in only 2.7% of exacerbations is considerably lower than the 23% recovery rate for rhinoviruses in exacerbations reported by eadie [14, 15] . carilli et al., however, failed to isolate these agents [16] . in his controlled study, stenhouse found that rhinovirus infection was not more common in' subjects with bronchitis than in the control population, but that rhinovirus infection was more likely to be associated with the development of acute lower respiratory tract symptoms [14j. the extended period of frozen storage of specimens before tissue culture inoculation may have contributed to our low recovery rate of rhinoviruses. sommerville first detected rsv infection in 50% of 82 exacerbations [17j. carilli et al. [16j and mcnamara et al. [13j also found rsv associated with a significant number of exacerbations, 17% and 12%, respectively. unlike these investigators, we detected rsv infection by serologic procedures in only one remission interval of only one of our patients. the paucity of m. pneumoniae infections detected in exacerbations in this study agrees with the results of another recent report [18] . in a number of prospective studies, m. pneumoniae infections have been associated with 8.7% and 9.5% of exacerbations [12, 13j and with no exacerbations [19] . one consistent finding, however, has been the failure to recover the organism from sputum, even from patients with rising antibody titers. these results question the significance of serologic responses alone only in this infection and should be further investigated. m. saliuarium seems ubiquitous in samples of expectorated sputum as well as in specimens obtained during bronchoscopy [2oj . our findings are similar to those reported in a number of other studies on viral and m. pneumoniae infections in exacerbations of chronic bronchitis (table 5) . in various studies the rates of infection in exacerbations ranged widely (4%-64%). these data have been interpreted as constituting strong, but not conclusive, evidence for an etiologic role of viruses or m. pneumoniae in the pathogenesis of acute exacerbations of chronic bronchitis. however, the available data cannot be easily compared because different sam-piing procedures and methods were employed. in addition, individual authors often did not specify the number of remission intervals examined but reported only infections detected during exacerbations and compared patients with chronic bronchitis with healthy (control) populations. furthermore, the definition of an exacerbation is a subjective judgment, and different interpretations of the criteria will affect the percentage of association with infection. two groups of investigators have derived a more striking correlation of infection with exacerbation by interpreting their findings in a timeweighted fashion. thus gump et al. found that the incidence of infection was 32% per patient week of exacerbation but only 0.9% per patient week spent in remission [11j. similarly, lamy et al. compared patient months spent in exacerbation and remission with an incidence of infection of 52% and 3.7%, respectively [24] . our data did not permit a time-weighted analysis since the duration of exacerbation was not recorded. when the duration of exacerbation was estimated using the interval between the collection of two samples of serum bracketing an exacerbation, we tested 257 months of exacerbation and 442 months of remission. the rate of viral infection was 15.4% per exacerbation month and 4.3% per remission month. this trend is similar to that in the data of gump et al. [11] . a special problem in interpretation is posed by the detection of infection in patients with chronic bronchitis without an associated acute compromise in their respiratory status. in our study approximately one-third of all viral and m. pneumoniae infections detected belonged in this category. whether subclinical infections contribute to continuing clinical deterioration remains unexplored, and long-term epidemiologic and clinical surveillance of patients with chronic bronchitis will be required to answer this question. role of infection in the cause and course of chronic bronchitis and emphysema role of infection in chronic bronchitis virus and mycoplasma infections in exacerbations of chronic bronchitis definition and classification of chronic bronchitis for clinical and epidemiological purposes. a report to the medical research council the role of viruses, mycoplasmas and bacteria in acute pneumonia in civilian adults mycoplasma species identification based upon growth inhibition by specific antisera elementary statistics with applications in medicine coronavirus infections in military recruits: three-year study with coronovirus strains oc43 and 229e the tecumseh study of respi-383 frequency of and relationship between ou tbreaks of coronavirus infection coronavirus infection in acute lower respiratory tract disease of infants role of infection in chronic bronchitis virological studies in chronic bronchitis viral and mycoplasma pneumoniae infections in exacerbations of chronic lung disease rhinovirus infection in acute exacerbation of chronic bronchitis: a controlled prospective study viral antibody levels and clinical status in acute exacerbations of chronic bronchitis: a controlled prospective study a virologic study of chronic bronchitis respiratory syncytial virus in acute exacerbations of chronic bronchitis mycoplasma infections in patients with chronic obstructive pulmonary disease pinkerton, 1. infective agents and chronic bronchitis a search for mycoplasma infections in patients with chronic bronchitis infection with influenza and parainfluenza viruses in chronic bronchitis persistence of viral antibodies in patients with chronic bronchitis pilot study of factors associated with exacerbations in chronic bronchitis debacker-willame, e. respiratory viral infections in hospital patients with chronic bronchitis: observations during periods of exacerbation and quiescence key: cord-309274-2npxrrhr authors: lee, m.k.; chiu, c.s.; chow, v.c.; lam, r.k.; lai, r.w. title: prevalence of hospital infection and antibiotic use at a university medical center in hong kong date: 2007-02-02 journal: j hosp infect doi: 10.1016/j.jhin.2006.12.013 sha: doc_id: 309274 cord_uid: 2npxrrhr hospital infection prevalence surveys were performed in our 1400-bed university medical centre in hong kong from 1985 to 1988. we investigated the rates of four major hospital-acquired infections (hais) (pneumonia, symptomatic urinary tract infection, surgical site infection and laboratory-confirmed bloodstream infection) in order to identify current distribution and any changes after 15 years. a one-day point prevalence study was performed on 7 september 2005. all inpatients were surveyed for hais, community-acquired infections (cais), risk factors, pathogenic isolates and antibiotics prescribed. infections were diagnosed according to centers for disease control and prevention (cdc) criteria. in total, 1021 patients were surveyed; of these, 41 had 42 hais (4% prevalence) and 389 (38%) were receiving antibiotics. the commonest hai was pneumonia (1.4%) followed by bloodstream infection (0.9%) and symptomatic urinary tract infection (0.8%). the prevalence of postoperative surgical site infection was 5.6%. the nosocomial prevalence rate was highest in the intensive care unit, followed by the pediatric and neonatal intensive care units, children's cancer centre/bone marrow transplant unit and orthopaedics with traumatology. meticillin-resistant staphylococcus aureus and pseudomonas aeruginosa were the commonest pathogens. the rates are significantly lower than previously and reflect the increased resources for infection control made available following the outbreak of severe acute respiratory syndrome (sars). hospital-acquired infections (hais) are common causes of morbidity and mortality among hospitalized patients. surveillance for nosocomial infections (nis) is an important component of an effective infection control programme. 1 the national nosocomial infections surveillance (nnis) system coupled with feedback and interventions successfully reduced the infection rate among participating hospitals. 2 unfortunately such prospective surveillance is costly and labour intensive. prevalence surveys are relatively inexpensive and easy to perform, although rates obtained are higher than comparable incidence rates and may be statistically less stable. 3 despite their limitations they provide a useful estimate of hai. french et al. conducted serial point prevalence surveys in our hospital 15 years ago. these proved to be a practical and useful way of documenting the trend of hais and assessing the impact of an infection control programme. 4 conducting a prevalence survey also raises awareness among healthcare professionals of infection risks in hospital settings. 5, 6 the objectives of this study were to determine the rate of nosocomial infection and the use of antibiotics. knowledge of the current situation and major changes in distribution of nosocomial infections should help to prioritize resource allocation, allow a more effective infection control policy and enable a targeted prospective surveillance programme. the prince of wales hospital (pwh) is a 1400-bed teaching hospital in hong kong. our laboratory is accredited by the national association of testing authorities (nata) and offers a wide range of bacteriology and virology services. the study was performed on 7 september, 2005. all inpatients at 09:00 as documented by the hospital computer system were included except those who were temporarily under observation in the accident and emergency department. cdc definitions for nosocomial infections were used. hai was defined as occurrence of infection after hospital admission, without evidence that the infection was present or incubating (48 h) on admission. 7 four major infections were surveyed: pneumonia, symptomatic urinary tract infection (suti), surgical site infection (ssi) and laboratory-confirmed bloodstream infection (lcbsi). infections of more than one site in the same patient were counted as separate infections. only active infections (i.e. those undergoing antimicrobial treatment, symptomatic or considered ongoing by medical staff) were included. demographic information, admission diagnosis, use of medical device and antibiotic were recorded by the ward nursing staff who had attended briefing sessions on each ward on the point prevalence survey with instructions on correct filling of a data collection form. other relevant data were recorded by our investigators (infection control nurses and medical microbiologists: total ¼ 10) on a separate standardized data collection form. they visited each ward and examined the clinical records, microbiology results and drug charts. a return visit was made if patient or medical record was not available. completed data collection forms were checked on the same day by the survey coordinator to avoid absence of data. microbiological data were collected up to two weeks after the study period. statistical analysis was conducted using spss software (spss, inc., chicago, il, usa). p < 0.05 was considered statistically significant. the results were fed back to the hospital infection control committee and individual departments after the survey. in all, 1021 patients were surveyed and their distribution by specialty is shown in table i . ages ranged from 1 day to 102 years. the mean and median age was 46.9 and 52 years respectively; 52% were male and 48% were female. hospitalization ranged from 1 to 886 days. most patients (42.4%) were admitted for two days or less, nearly 80% admitted for 10 days or less. the mean and median durations of hospitalization were 12.5 and four days respectively. in 607 patients, 801 devices were present. peripheral intravenous catheter (n ¼ 442), urinary catheter (n ¼ 269) and central venous catheter (n ¼ 61) were the most common. of these, 427 patients had one device, 166 had two and 14 patients had three. twenty patients were receiving mechanical ventilation. forty-four hais were diagnosed in 43 patients. two patients had acquired the infections in another hospital and were excluded, leaving 42 hais in 41 patients giving a 4% hai rate. the most common hai was pneumonia (1.4%), followed by ssi (1.1%), lcbsi (0.9%) and finally suti (0.8%) ( table i) . there were two cases (18%) of ventilator-associated pneumonia (vap) among the 11 cases of hospitalacquired pneumonias (hap). fifty-seven percent of hospital-acquired urinary tract infections were catheter related. the icu had the highest prevalence rate of nosocomial infection (26.7%), followed by the pediatric/neonatal icus, children's cancer centre/ bone marrow transplant unit (ccc/bmt) and orthopaedics and traumatology (table i) . the prevalence of ssi among those patients who had undergone operation was 5.6% (table i) . approximately 11% of the surgical patients underwent operations classified as 'contaminated' or 'dirty' were diagnosed with ssi. four percent and 2% of those who had 'clean' and 'cleanecontaminated' operations had ssis (table i) . patients with nosocomial infection had a median hospital stay of 19 days compared to 4 days for non-infected patients. duration of hospital stay, operation, invasive ventilation and intravascular device were independent risk factors for hai (p < 0.05) (table ii) . sixty-six patients were diagnosed with cai. the prevalence rate was 6.5%. together with hai (41 patients with hai and two other patients with hai from other hospitals), the overall prevalence rate of infection in hospital was 10.7%. among 41 patients with hai, 97.6% had relevant microbiology and serological tests performed and 77.5% had positive cultures. the commonest organisms isolated were pseudomonas aeruginosa and meticillin-resistant staphylococcus aureus. a total of 389 patients (38%) were receiving antibiotics; 213 (20.9%) received one antibiotic, 132 (12.9%) received two and 31 (3%) received three or more. the five most commonly prescribed antibiotics were amoxicillin-clavulanate, metronidazole, cefuroxime, cloxacillin and gentamicin (table iii) . the commonest antibiotic combinations were cefuroxime þ metronidazole (n ¼ 20), gentamicin þ penicillin (n ¼ 13), ampicillin þ cloxacillin (n ¼ 11), ampicillin þ cefuroxime þ metronidazole (n ¼ 7) and gentamicin þ vancomycin (n ¼ 6). though some of our findings are similar to those in other parts of the world, they are useful for local healthcare workers. the last survey in our hospital was performed over 15 years ago and some interesting changes in nosocomial infection rate and antibiotic use were found. differences in methodologies and patient population do not allow stringent comparison of prevalence between different surveys and meaningful comparison of infection rates requires adjustment for intrinsic and extrinsic risk factors. the impact of case mix on infection rates was well illustrated with the example of hospital size in the study by sax's group. 8 detailed information on patient demographics, duration of hospital stay and invasive devices/antibiotic use has been illustrated for reference in this single hospital study. the cdc definition of infections was adopted. although it was not used in the previous surveys, it is now one of the most widely used and objective standards. all our investigators were qualified medical and nursing staff with relevant infection control training. inter-observer bias was minimized by ensuring that the investigators agreed on the interpretation of the diagnostic criterion before the survey. however, gastmeier et al. showed that despite the use of specific cdc criteria, there could still be subjectivity in the diagnosis of nosocomial infections. 9 the investigator effect was not known in the current survey. only four major infections were studied. the prevalence of hai was 4%. in point prevalence surveys using the cdc diagnostic criteria and involving the four major sites of infection (suti, lrti, ssi and bsi) conducted in france, italy, norway and lebanon, the prevalence of hai ranged from 3.5 to 6.8%. 5,10e13 the inclusion of all sites of infection tends to increase the overall prevalence rate by 20e30% as compared with studies that included only major sites. 6, 14 thus, if corrected, the hai prevalence rate estimated for all sites would be 5e6%. this rate was much lower than those found in 1985e1988 in pwh (7.3%), 15 as well as in other countries (6.5e13.95%). 6,14,16e19 apart from the overall rate, rate reductions were found in hap (from 1.8 to 1.4%), ssi (from 1.5 to 1.1%), and uti (from 2.6 to 0.8%) without making case-mix adjustments 15 (figure 1 ). this probably relates to improved infection control following the sars crisis in hong kong, as evidenced by increased resources and heightened awareness among healthcare workers (hcws). our hospital had one infection control nurse per 250 beds after 2003 as compared to the previous ratio of 1:700. infection control programmes, such as 'alert organism' reporting, biannual hand hygiene audit, regular infection control educational/training to hcws and ssi surveillance have since been implemented. heightened infection control awareness was demonstrated by the high compliance rate in hand hygiene audit among hcw. the low prevalence may also relate to a short duration of hospital stay and rapid transferal to convalescence hospitals after initial diagnosis and stabilization. the prevalence of nosocomial pneumonia, lcbsi and ssi was similar to that reported in european surveys. the prevalence of nosocomial lcbsi was relatively high, possibly due to a high overall blood culture rate and simple application of the diagnostic criteria. the rate of nosocomial urinary tract infection (uti) was lower than the 1.5e4.5% reported from other surveys. 5,10e12,16e17 the greatest problem with defining suti is that if the urine is not analyzed, uti cannot be diagnosed. secondly, the definition did not take into account urinary catheters, as symptoms of uti other than fever cannot be assessed in catheterized patient. 20 the frequency of suti therefore depended upon: (i) frequency of catheter use: 26% of the patients had an indwelling urinary catheter; (ii) urine analysis protocol: in our hospital, urine is only analysed when the signs and symptoms indicate a uti or when a catheterized patient develops a fever. symptoms of uti in patients with a serious major disease of other organs may have been ignored; and (iii) physician's diagnosis: data collection based on doctor's diagnosis may not have been sensitive enough to record all utis. furthermore asymptomatic bacteriuria was not evaluated. these factors may account for the underestimation of rate of uti compared with other studies. the distribution of nosocomial infection among different specialties was similar to published european studies. the prevalence of nosocomial infection was greatest in icu at 26.7%, which compared with 35.5% 15 years ago, 15 and was similar to that of other studies. 5, 6, 14, 18 the special pediatric unit ranked second for nosocomial infection. hai, not limited to ssi, has been shown to be twice as common in postoperative patients. 6, 11 thirty-eight percent of patients received at least one antimicrobial agent, more than in other studies (16.6e44%), but only 10% had an infection according to the cdc criteria. 14e18,21 indications for antibiotic prescription were reviewed retrospectively in order to investigate this discrepancy (table iii) . prophylactic use (6.3%) and treatment for infections that were not being investigated in the current survey (50%) may have accounted for antibiotic use in 37% of patients. the remaining 35% may have represented treatment of infections not fulfiling the cdc criteria or inappropriate antibiotic prescription. recent surveys showed that suboptimal antimicrobial prescription is frequent in our hospital. 22 though appropriateness of antibiotic prescription was not the aim of current study, unnecessary antibiotic combinations were noted (table iii) . broad-spectrum antibiotics, e.g. imipenem, meropenem and cefepime, accounted for about 20% of the antibiotics being prescribed on the day of survey. audit of 'big-gun' antibiotics in local university hospitals revealed that 20e25% of the prescriptions were not justified or suboptimal. 22, 23 the upcoming antibiotic stewardship programme targeting this group of drugs should help to optimize use of antimicrobials in the hospital. 23 in summary, provision of better resources appears to have had a definite effect in reducing hais. the icu, special pediatric and surgical/ orthopaedic units warrant targeted surveillance of nosocomial infection followed by a relevant infection control programme. another target would be improvement in nosocomial bloodstream infection by promoting proper intravenous catheter care. repeated prevalence surveys are useful for monitoring trends in rates of both hai and effectiveness of such intervention strategies. 4 the efficacy of infection surveillance and control programs in preventing nosocomial infections in us hospitals feeding back surveillance data to prevent hospital acquired infections prevalence, incidence and duration repeated prevalence surveys for monitoring effectiveness of hospital infection control prevalence of nosocomial infections in hospitals in norway the french prevalence survey study group. prevalence of nosocomial infections in france: results of nationwide survey in 1996 national nosocomial infection study site definition manual inter-hospital differences in nosocomial infection rates: importance of casemix adjustment experience with two validation methods in a prevalence survey on nosocomial infections prevalence of nosocomial infections in representative german hospitals prevalence of nosocomial infections in italy: result from the lombardy survey in 2000 national prevalence survey on hospital infection in norway a one-day prevalence survey of hospital acquired infections in lebanon prevalence of nosocomial infections in spain: epine study 1990e1997 prevalence of hospital infection at the prince of wales hospital a three-year survey of nosocomial and community-acquired infections, antibiotic treatment and re-hospitalization in a norwegian health region hospital-acquired infections in italy: a region wide prevalence study prevalence of hospital-acquired infection in a lithuanian hospital prevalence of nosocomial infection and antibiotic use at a university medical center in malaysia surveillance in infection control: are we making progress? prevalence of hospitalacquired infection in a tunisian hospital antibiotic guidelines and optimization programme optimising antimicrobial prescription in hospitals by introducing an antimicrobial stewardship programme in hong kong: consensus statement we are grateful to the nursing staff of the pwh, and especially to our microbiologists and icns, who assisted in the surveys. key: cord-344084-z4t2wkgk authors: ellwanger, joel henrique; kulmann-leal, bruna; kaminski, valéria de lima; rodrigues, andressa gonçalves; de souza bragatte, marcelo alves; chies, josé artur bogo title: beyond hiv infection: neglected and varied impacts of ccr5 and ccr5δ32 on viral diseases date: 2020-05-30 journal: virus res doi: 10.1016/j.virusres.2020.198040 sha: doc_id: 344084 cord_uid: z4t2wkgk the interactions between chemokine receptors and their ligands may affect susceptibility to infectious diseases as well as their clinical manifestations. these interactions mediate both the traffic of inflammatory cells and virus-associated immune responses. in the context of viral infections, the human c-c chemokine receptor type 5 (ccr5) receives great attention from the scientific community due to its role as an hiv-1 co-receptor. the genetic variant ccr5δ32 (32 base-pair deletion in ccr5 gene) impairs ccr5 expression on the cell surface and is associated with protection against hiv infection in homozygous individuals. also, the genetic variant ccr5δ32 modifies the ccr5-mediated inflammatory responses in various conditions, such as inflammatory and infectious diseases. ccr5 antagonists mimic, at least in part, the natural effects of the ccr5δ32 in humans, which explains the growing interest in the potential benefits of using ccr5 modulators for the treatment of different diseases. nevertheless, beyond hiv infection, understanding the effects of the ccr5δ32 variant in multiple viral infections is essential to shed light on the potential effects of the ccr5 modulators from a broader perspective. in this context, this review discusses the involvement of ccr5 and the effects of the ccr5δ32 in human infections caused by the following pathogens: west nile virus, influenza virus, human papillomavirus, hepatitis b virus, hepatitis c virus, poliovirus, dengue virus, human cytomegalovirus, crimean-congo hemorrhagic fever virus, enterovirus, japanese encephalitis virus, and hantavirus. subsequently, this review addresses the impacts of ccr5 gene editing and ccr5 modulation on health and viral diseases. also, this article connects recent findings regarding extracellular vesicles (e.g., exosomes), viruses, and ccr5. neglected and emerging topics in “ccr5 research” are briefly described, with focus on rocio virus, zika virus, epstein-barr virus, and rhinovirus. finally, the potential influence of ccr5 on the immune responses to coronaviruses is discussed. inflammatory cells play a crucial role in protecting the host from viral infections. leukocyte migration is a fundamental step of the inflammatory response to viruses, a process regulated by the interaction between chemokines and their receptors. therefore, dysregulations in the chemokine-mediated inflammatory process may contribute to viral pathogenesis (glass et al., 2003) . the c-c chemokine receptor type 5 (ccr5) interacts primarily with the chemokines ccl3 (mip-1α), ccl4 (mip-1β), and ccl5 j o u r n a l p r e -p r o o f (rantes) , which act as ccr5 agonists by stimulating cell migration and mediating inflammatory responses. on the other hand, the chemokine mcp-3/ccl7 is the main ccr5 antagonist ligand (blanpain et al., 1999; zlotnik and yoshie, 2000; glass et al., 2003; alkhatib, 2009 ). in addition to regulating the migration of non-specific leukocytes during inflammatory responses, ccr5 controls the action of specific cell types, including natural killer (nk) cells (khan et al., 2006; weiss et al., 2011) and regulatory t (treg) cells (wysocki et al., 2005; tan et al., 2009; dobaczewski et al., 2010) . ccr5 is also expressed by tissue-resident memory t cells. these ccr5 + cells support barrier immunity (davis et al., 2019) . the ccr5 is a g-protein-coupled receptor (gpcr), containing seven transmembrane α-helices, three extracellular loops, and three intracellular loops (tan et al., 2013) . figure 1 shows the structure of ccr5, highlighting its transmembrane domains and extra and intracellular loops. the specificity of interaction between ccr5 and chemokines is mediated by the second extracellular loop (samson et al., 1997) . helices 2 and 3 have a fundamental role in chemokine-induced ccr5 activation (govaerts et al., 2003) . the steps required from ligand binding culminating in cell migration encompass a series of intracellular interactions, including the g-protein heterotrimer and downstream effectors (lacalle et al., 2017) . after stimulation by chemokines or natural reactive antibodies and subsequent triggering of chemotaxis, ccr5 is phosphorylated and internalized in the cytoplasm (signoret et al., 2000; venuti et al., 2015; venuti et al., 2016; lacalle et al., 2017; venuti et al., 2017; venuti et al., 2018) . the number of available receptors on the cell surface is related to the rate of internalization and recycling of ccr5, which affects the activation of ccr5 and consequent signaling of specific pathways that culminate in chemotaxis processes (mueller et al., 2002) . of note, intracellular pools of ccr5 can be detected in the cells. these pools are probably formed by internalized or immature/precursor forms of ccr5 molecules (mirzabekov et al., 1999; kohlmeier et al., 2008; achour et al., 2009; guglielmi et al., 2011; shirvani et al., 2011) that can be rapidly expressed on the cell surface in response to viral stimuli and inflammatory responses (kohlmeier et al., 2008) . in other words, ccr5 molecules are recycled by cells. specifically, ccr5 recycling can be mediated by degradation followed by de novo synthesis (in response to stimulation by natural antibodies) or occur in the classic short-term system without de novo synthesis (in response to stimulation by ccl5, for example) (venuti et al., 2015; venuti et al., 2016) . the traffic of ccr5 between the plasma membrane and the intracellular medium is mediated by different molecules, including clathrins, β-arrestin 2, and extracellular signal-regulated kinase (erk) 1 (venuti et al., 2015; venuti et al., 2016; venuti et al., 2018) . also, intracellular cd4 regulates the expression of ccr5 on the cell surface (achour et al., 2009 ). the human ccr5 protein (352 residues) is encoded by the ccr5 gene [chromosome 3 (3p.21.31)], which is very polymorphic (blanpain et al., 2000; hoover, 2018) . among polymorphisms of the ccr5, the ccr5δ32 (rs333) has been intensively studied in different human populations. the frequency of the ccr5δ32 is quite variable. in general, the δ32 allele frequency is high in european-derived populations (for example, 16% in norway and 11% in germany) and low or absent in african and asian populations j o u r n a l p r e -p r o o f (solloch et al., 2017) . however, although the δ32 allele is more frequent in european populations, there are exceptions due to migratory events. for example, the frequency of the δ32 allele is high in south africa (13%) and chile (12%) (solloch et al., 2017) . also, the frequency of the δ32 allele can be quite variable within the same country. in brazil, the frequency of the allele in the general population is around 4-5% (silva-carvalho et al., 2016; solloch et al., 2017) . in the southern region of the country, the frequency can reach up to 9% due to the past migration of european populations to this region (boldt et al., 2009; pena et al., 2011; schauren et al., 2013) . figure 2 summarizes basic aspects of ccr5 and shows the frequency of the δ32 allele in various countries. the ccr5δ32 is the most studied genetic variant of the ccr5 gene because of its strong protective effect against hiv infection (considering susceptibility to ccr5-tropic strains). hiv entry into cd4 + t cells is mediated by the interaction of the virus with cd4 and with a co-receptor, usually ccr5. the ccr5δ32 variant is a 32 base-pair deletion in the ccr5 coding region, which causes a frameshift, resulting in a truncated protein that is not directed to the cell surface. ccr5δ32 in heterozygosis promotes a decrease in the expression of functional ccr5 on the cell surface compared to ccr5 wild-type cells. therefore, individuals with heterozygous genotype for ccr5δ32, if infected with hiv, have a small protection against disease progression due to the reduced expression of ccr5 on the surface of cd4 + t cells (reduced hiv-ccr5 interaction). in ccr5δ32 homozygous cells, no ccr5 is expressed in the plasmatic membrane. therefore, homozygous individuals for this polymorphism (δ32/δ32) show virtually total protection against hiv type 1 infection, since no ccr5 expression is verified on cell surface (no hiv-ccr5 interaction at cell surface is possible) (deng et al., 1996; dragic et al., 1996; huang et al., 1996; samson et al., 1996; wu et al., 1997; proudfoot, 2002; venkatesan et al., 2002; picton et al., 2012) . figure 3 illustrates the phenotypic effects of ccr5δ32 in human cells. the main results involving the triad "ccr5, hiv, and ccr5δ32" were published in 1996 in nature, cell and science papers by different groups (parmentier, 2015) . since then, the research involving ccr5 has explored the role of the ccr5 protein and ccr5δ32 polymorphism in different diseases, as well as the therapeutic potentials of ccr5 blockade. currently, the physical interaction of ccr5 with hiv is known in detail (shaik et al., 2019) ryst, 2015; latinovic et al., 2019) . also, a recent study has shown that molecules that inhibit ccr5 trafficking to the plasma membrane also have a therapeutic potential against hiv infection (boncompain et al., 2019) . research involving ccr5 has also brought other important advances in combating hiv infection. of note, there are already two cases of sustained remission of hiv infection following stem-cell transplantation using ccr5δ32 homozygous donor, the "berlin patient" (hütter et al., 2009) and the "london patient" (gupta et al., 2019; gupta et al., 2020) . articles that evaluated the involvement of ccr5 or ccr5δ32 in the infections caused by the mentioned viruses were analyzed. subsequently, the google scholar (https://scholar.google.com.br/) was consulted to detect relevant papers that were not indexed in pubmed, using the terms "ccr5" in association with the name of each of the viruses covered in the review. the authors of this review tried to include the largest number of original articles that addressed the topic covered in this work, intending to write a broad and complete article. however, some papers were not included because it was not possible to obtain clear conclusions from the studies. the reference lists of the consulted articles were also used to complement the selection of articles for this review. as previously mentioned in the introduction, a discussion addressing the involvement of ccr5 in tbev infection was not included in this work. articles addressing the involvement of ccr5 and ccr5δ32 in hiv infection were included only to present to the reader some basic and historical aspects related to such topics, cited mainly in the introduction section and figures, but not included as a major section. considering the importance of the coronavirus disease 19 (covid-19) pandemic, a section addressing the potential influence of ccr5 on the immune responses to coronaviruses was included in the article. review articles were also selected in pubmed and google scholar for writing the sections and paragraphs that address the basic aspects of viruses, exosomes, and diseases (e.g., epidemiological, molecular, clinical aspects). exceptionally, review articles with outstanding discussions regarding the role of ccr5 in viral infections were also cited in this work. regarding tables, it is important to note that the data available in the "population" columns are limited and often represent only general characteristics of the evaluated population. in many studies, a population can be composed of individuals from various ethnic groups. this limitation must be taken into account when evaluating the results of the studies cited in the tables. finally, also in "population" columns, "information not available" was used when this information was not clearly described in the cited article. west nile virus (wnv) is a neurotropic positive-sense single-stranded rna flavivirus endemic in various parts of the world. wnv transmission to humans occurs through the bite of infected mosquitoes, especially species of the culex genus (suthar et al., 2013) . although different animals can participate in the wnv transmission cycle, birds are the classic amplifier hosts. humans, horses and other mammals are deadend hosts (kramer et al., 2008; cdc, 2018) . among humans, blood transfusion, organ transplantation, and breast milk can also transmit the virus. vertical transmission may also occur. however, compared to transmission by mosquito bites, these routes of transmission are rare (kramer et al., 2008) . about 25% of wnv-infected individuals develop west nile fever, a clinical condition with variable symptoms and severity. in less than 1% of the infected individuals, wnv invades the central nervous system (cns), causing neurological manifestations (neuroinvasive disease), including meningitis, encephalitis, and acute flaccid paralysis. of note, west nile neuroinvasive disease shows a 10% to 20% fatality rate. severe illness is associated with older age and other factors, including genetic traces (petersen et al., 2013; sejvar, 2016) . wnv infection is considered the leading cause of arboviral encephalitis in the world (ciota, 2017) . the treatment of wnv infection is supportive (petersen et al., 2013) . it is known that the ccr5 protein interferes in the clinical course of wnv infection (glass et al., 2005; diamond, 2009; michlmayr and lim, 2014) , but the effects of ccr5δ32 on the susceptibility to this infection and disease progression are different. according to lim et al. (2010) and danial-farran et al. (2015) , ccr5δ32 has no important effect on susceptibility to wnv infection. in accordance, loeb et al. (2011) found no association between ccr5δ32 and wnv infection. conversely, there is robust populationbased data showing a strong association between the ccr5δ32 homozygous genotype and increased risk of developing symptomatic wnv infection lim et al., 2008; lim et al., 2010) . also, bigham et al. (2011) showed that the ccr5δ32 variant was associated with symptomatic wnv infection when the dominant model of inheritance was considered in the analysis. a recent meta-analysis confirmed the role of the ccr5 gene in wnv infection, specifically the association between the ccr5δ32 with severe disease (cahill et al., 2018) . table i summarizes the results of the studies that evaluated the ccr5δ32 genetic variant in the context of human wnv infection. in agreement with studies showing that ccr5δ32 homozygous genotype is a risk factor for symptomatic wnv infection in humans, ccr5-/-wnv-infected mice showed a reduced capacity of viral control, increased disease severity, impaired leukocyte trafficking towards the brain, and high mortality rates than ccr5 wild-type mice. these findings reinforce that ccr5 is a key molecule in the immune response against wnv (glass et al., 2005; durrant et al., 2015) . taking together, these pieces of evidence robustly support the role of ccr5 as a protective molecule on wnv pathogenesis. specifically, ccr5+ leukocytes play a fundamental role in combating wnv in the brain (glass et al., 2005; lim et al., 2006; michlmayr and lim, 2014; durrant et al., 2015) . in this sense, ccr5 plays a specific and non-redundant role in controlling wnv infection (lim and murphy, 2011; durrant et al., 2015; ellwanger et al., 2020a) . based on the data mentioned above, the lack of ccr5 expression linked to ccr5δ32 homozygosis is an important risk factor for increased severity of wnv-associated disease. the opposite effects of the ccr5δ32 genetic variant on both hiv and wnv infections are summarized in figure 5 . hereupon, individuals homozygous for ccr5δ32 and living in endemic areas of the wnv should take additional care to prevent wnv infection (e.g., use of repellents, mosquito nets). also, the use of ccr5 blockers to treat hiv infection may have a negative impact on populations living in wnv-endemic areas. to avoid this negative impact, hiv-infected individuals who live in such areas and who use ccr5 blockers must apply robust measures against mosquito bites lim et al., 2006; lim and murphy, 2011). j o u r n a l p r e -p r o o f influenza infection affects humans seasonally, causing recurrent epidemics and even pandemics in some years. in humans, the infection is caused basically by influenza a and influenza b, both enveloped negative-sense single-stranded rna viruses belonging to orthomyxoviridae family (krammer et al., 2018; petrova and russell, 2018) . influenza a is a zoonotic disease, and influenza b circulates primarily in humans. influenza infection affects mainly the respiratory tract, which can cause mild to severe disease depending on viral and host characteristics. secondary bacterial infection may also occur (krammer et al., 2018) . human co-infection with multiple influenza types is an important neglected problem (gregianini et al., 2019) . influenza is a prevalent infection worldwide, and new vaccines are produced annually, based on strains circulating each year in the northern and southern hemispheres (krammer et al., 2018; petrova and russell, 2018) . antivirals can be used in the treatment of influenza infection (krammer et al., 2018) . investments in new vaccines, antiviral drugs, and surveillance systems are needed to reduce the global burden associated with influenza infection (petrova and russell, 2018) . the severity of influenza infection is related to the intensity of proinflammatory responses and the predominant profile of cytokine production by the host . a body of evidence has shown that both ccl5 and ccr5 participate in the modulation of the immune response to influenza virus infection (matsukura et al., 1998; tyner et al., 2005; sládková and kostolanský, 2006; kohlmeier et al., 2008; oslund and baumgarth, 2011) . of note, ccr5 mediates the recruitment of nk cells to the lungs in influenza a infection (carlin et al., 2018) and participates in neutrophil action in the lungs during influenza pneumonia (rudd et al., 2019) . although flow cytometry data did not indicate significant changes regarding ccr5 expression on the surface of human monocytes after experimental influenza a infection (salentin et al., 2003) , various studies have shown that, in mice, the lack of ccr5 expression is associated with a higher risk of death by influenza infection (dawson et al., 2000; tyner et al., 2005; fadel et al., 2008; tavares et al., 2020) . based on these findings, it was postulated that pharmacological ccr5 blockade may have some undesirable effect on the immune response against the influenza virus in humans (fadel et al., 2008) . importantly, more research on this aspect is needed since this data suggests that, in humans, the ccr5 absence due to the ccr5δ32 polymorphism could affect pathogenesis and the lethality rate of influenza infection. falcon et al. (2015) evaluated the frequency of the ccr5δ32 in pandemic h1n1-infected spanish individuals and revealed an association between the polymorphism with fatal outcome. the ccr5δ32 was also associated with increased disease severity in other studies (keynan et al., 2010; rodriguez et al., 2013) . however, these results should be interpreted with caution since both studies were based on very small sample sizes (keynan et al., 2010; rodriguez et al., 2013) . importantly, other studies addressing humans reported no association between ccr5δ32 and severity of influenza infection (sironi et al., 2014; maestri et al., 2015; matos et al., 2019) . taking together, the body of evidence suggests that ccr5δ32 has little j o u r n a l p r e -p r o o f influence on severity of influenza infection (table 2) . results described by falcon et al. (2015) seem to be specific to that studied population, composed of individuals from 13 regions of spain. human papillomavirus (hpv) is a double-stranded dna virus belonging to the papillomavirus family. hpv is transmitted by direct contact (for example, through sexual intercourse). the hpv infection is quite common worldwide, and is usually controlled by the immune system. viral clearance occurs in most cases within 1-2 years after infection. however, if the infection is not controlled, hpv can cause noncancerous mucosal lesions or different types of malignant lesions such as anal cancer, penile cancer, vulvar cancer, head cancer, neck cancer, and especially cervical cancer. hpv is an oncogenic pathogen because it inhibits the activity of p53 and prb (retinoblastoma protein) tumor suppressor molecules through the action of e6 and e7 viral proteins, respectively. these proteins also exhibit other oncogenic mechanisms. worldwide, between 5-10% of all cancers in women are due to hpv infection (schiffman et al., 2016; sanjosé et al., 2018) . there are several hpv genotypes (>200), and those most associated with cancer are: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and probably 68 (schiffman et al., 2016) . of note, the genotype 16 (hpv16) has a prominent role in cancer development. vaccination is one of the most effective ways to prevent hpv infection, having important positive impacts on multiple aspects of population health. hpv vaccine protects the vaccinated individual from infection per se and from hpv-related cancer (schiffman et al., 2016; sanjosé et al., 2018) . the expression of ccr5 is increased in cervical cancer tissues (sales et al., 2014; che et al., 2016) . also, in vitro growth, proliferation, and invasive capacity of cervical cancer cells can be inhibited through ccr5 downregulation (che et al., 2016) . these findings suggest the involvement of ccr5 in the development of cervical lesions. in a study performed with indian individuals (singh et al., 2008) , the genotype and allele frequencies of ccr5δ32 were not different between cervical cancer patients and controls. however, when the patients were stratified by cancer stage (stages 1b to 4), the ccr5 heterozygous genotype was associated with stage 1b of cervical cancer. the hpv positivity rate among the evaluated patients was not described (singh et al., 2008) . the relationship between ccr5δ32, hpv infection, and cervical lesions is addressed in other studies (table 3 ). ccr5δ32 homozygous genotype was associated with increased susceptibility to hpv infection in a study performed with swedish individuals (zheng et al., 2006) . mangieri et al. (2019) investigated the potential influence of ccr5δ32 on susceptibility to hpv infection and cervical lesions in brazilian women. however, the genetic variant was not significantly associated with susceptibility to hpv infection (considering allele frequency, codominant model and dominant model) or hpv-associated lesions (mangieri et al., 2019) . in accordance, previous studies performed with brazilian women did not find an association between ccr5δ32 and susceptibility to hpv infection (suzuki et al., 2008) or between the polymorphism and hpv-related cervical lesions (suzuki et al., 2008; santos et al., 2016) . lastly, ccr5δ32 j o u r n a l p r e -p r o o f did not affect susceptibility to hpv infection in lithuanian individuals with laryngeal cancer (stumbrytė-kaminskienė et al., 2020) . in conclusion, although tissue analysis and evidence obtained in vitro suggest that the ccr5 is potentially involved in the pathogenesis of hpv, most studies point to a lack of involvement of ccr5δ32 in susceptibility to hpv infection or hpv-associated diseases. the the ccr5 and its ligands regulate the action of t cells and other leukocytes in the liver. thus, ccr5 regulates liver inflammation and participates in the local immune response against viruses (ajuebor et al., 2006; sanchooli et al., 2014) . in mice models of hepatitis, ccr5 deficiency was associated with increased liver inflammation, tissue injury, and liver failure (ajuebor et al., 2005; moreno et al., 2005; stevens et al., 2019) . deficiency of ccr5 expression is generally associated with reduced migration of inflammatory cells, which would translate into less inflammation. this reasoning is correct and applies to different situations and tissues (braunersreuther et al., 2007; muntinghe et al., 2009; kaminski et al., 2019a) . however, ccr5 is an immunoregulatory molecule (doodes et al., 2009; dobaczewski et al., 2010; christmann et al., 2011; hütter et al., 2011) and therefore its deficiency can also cause deregulation in the action of various immune cell types (e.g., nk and treg cells), increasing the inflammatory status in some tissues. in humans, multiple evidence has shown the involvement of ccr5 (protein and gene) in distinct aspects of hbv infection (ahn et al., 2006; trehanpati et al., 2009; ahmadabadi et al., 2013; yang et al., 2018) . interestingly, ccr5δ32 and other host genetic factors can affect the immunogenicity of the hbv vaccine (ganczak et al., 2017; ellwanger and chies, 2019b) . considering these aspects, the influence of the ccr5δ32 on susceptibility/resistance to hbv and disease severity is quite plausible. several studies investigated the role of the ccr5δ32 polymorphism in hbv infection. some of them found no association between those variables (arababadi et al., 2010; khorramdelazad et al., 2013; safari et al., 2017; zhang et al., 2018; moudi et al., 2019) . in other studies, the absence of ccr5δ32 allele in the sample, due to ethnic features of the evaluated population, precluded the analysis concerning the potential impact of this genetic variant on hbv infection (ahn et al., 2006; li et al., 2011) . some authors have reported significant influences of ccr5δ32 on hbv infection. suneetha et al. (2006) reported an association between the ccr5δ32 heterozygous genotype and chronic hbv infection. in contrast, thio et al. (2007) found an association between the ccr5δ32 allele and infection recovery in a study that analyzed individuals with persistent hbv infection and individuals who recovered from the infection. subsequently, thio et al. (2008) associated the hbv infection recovery with an epistatic effect between the ccr5δ32 and the rantes-403a promoter polymorphisms. finally, abdolmohammadi et al. (2016) found a protective effect of the ccr5δ32 genetic variant against hbv infection, once the δ32 allele was more frequent in controls compared to hbv-infected individuals. recently, we investigated the frequency of ccr5δ32 in hbv mono-infected and hbv/hiv coinfected brazilian individuals (ellwanger et al., 2020b) . a control group and hiv mono-infected individuals were also evaluated in our study. a total of 1113 individuals were studied, which represents the largest study involving ccr5δ32 and hbv infection to date (see table 4 for comparisons with other studies). we found a significant protective effect of ccr5δ32 on hbv/hiv co-infection, a result probably due to the partial protective effect of ccr5δ32 against hiv infection since no important impact of ccr5δ32 on susceptibility to hbv mono-infection was observed (ellwanger et al., 2020b) . the hepatitis c virus (hcv) was formally described in 1989 (choo et al., 1989) and, currently, hcv infection is one of the most important infectious diseases in terms of global public health burden. hcv is an enveloped single-stranded positive-sense rna virus, has seven genotypes, and belongs to the flaviviridae family, hepacivirus genus (pietschmann and brown, 2019) . it is estimated that 71 million individuals are chronically infected by hcv worldwide (viganò et al., 2019) . similar to the hbv infection, hcv-infected individuals can eliminate the virus naturally or develop chronic infection, which occurs in 55-85% of the cases. chronic infection can cause liver inflammation, cirrhosis, and hepatocarcinoma (lingala and ghany, 2015) . in addition to liver damage, hcv causes a series of immune-mediated extrahepatic manifestations, including rheumatologic, dermatologic, ophthalmologic, renal, pulmonary, neuropsychiatric, cardiovascular, and hematologic manifestations, especially mixed cryoglobulinemia (romano et al., 2018) . hcv therapy using direct-acting antivirals (daas) shows cure rates over 95% (pietschmann and brown, 2019) . early treatment of infected patients decreases death rates from hcv-associated liver disease, reduces disease transmission, and alleviates extrahepatic health problems. focusing the efforts on hcv treatment is extremely important because there is no effective hcv vaccine (viganò et al., 2019) . hcv seropositivity is an important risk factor for hiv infection (zwolińska et al., 2013) . like hiv, hcv is primarily transmitted through blood transfusion and sexual intercourse, and hcv/hiv co-infection is a major problem worldwide. depression of the immune system due to uncontrolled hiv infection may contribute to hcv progression (schlabe and rockstroh, 2018) . both susceptibility to hcv infection and j o u r n a l p r e -p r o o f disease progression are affected by viral and environmental factors and physio-metabolic, immune, and genetic components of the host (ellwanger et al., 2018a) . chemokines and chemokine receptors participate in the recruitment and activity of inflammatory cells in the liver, acting on anti-hcv immune responses and ultimately modifying the rate of inflammation and other histological manifestations observed during infection. based on this rationale, the ccr5 molecule was postulated as having an impact on hcv-induced liver injury, susceptibility to hcv infection, and modulation of the possibilities of viral clearance. the downregulation of ccr5 due to ccr5δ32 may interfere in these processes (ahlenstiel et al., 2004; coenen and nattermann, 2010) . in agreement, several polymorphisms in other immune system genes [especially human leukocyte antigen (hla), mannosebinding lectin (mbl), toll-like receptor (tlr), interleukins (il), and interferon (ifn) gene families] indeed modify both susceptibility to hcv infection and disease progression (ellwanger et al., 2018a) . especially the focus of this review, clinical response to hcv therapy is influenced by ccr5 gene polymorphisms (konishi et al., 2004; omran et al., 2013) . also, there is evidence showing that ccr5 haplotypes can affect susceptibility to hcv infection (huik et al., 2013) . a recent in vitro study suggested that ccr5 blockage could have a beneficial effect on the treatment of hcv infection since ccr5 antagonists (maraviroc and cenicriviroc) inhibit hcv replication (blackard et al., 2019) . the use of ccr5 antagonists in humans is safe (fätkenheuer et al., 2010; gulick et al., 2014; giaquinto et al., 2018) and these drugs have the potential to treat a number of diseases in which ccr5 is involved, including hcv-associated liver disease. although the use of ccr5 antagonists on hcv monoinfection is not yet approved, it can be useful specifically for co-infected hiv/hcv patients, where ccr5 blocking (maraviroc) is already recommended (haïm-boukobza et al., 2013; blackard et al., 2019) . however, the detailed patterns of ccr5 expression in different tissues and at various points in the clinical course of hcv infection are still poorly understood. according to a recent study, the expression of ccr5 in cd8+ t cells is increased in the liver of chronic hcv-infected patients (pirozyan et al., 2019) , but other studies have found mixed results regarding ccr5 expression on t cells in the context of hcv infection (lichterfeld et al., 2002; vincent et al., 2005; larrubia et al., 2007; zahran et al., 2020) . therefore, considering that the role of ccr5 in hcv infection is still uncertain, the potential use of ccr5 blockers to treat hcv mono-infection should be cautious. the influence of the ccr5δ32 variant on hcv infection susceptibility was investigated by several studies. woitas et al. (2002) found a significantly higher frequency of the ccr5δ32 homozygous genotype in hcv-infected individuals compared to controls, hiv-infected and hcv/hiv co-infected individuals, suggesting the ccr5δ32 as a risk factor for hcv infection. in agreement, the δ32 allele was a significant risk factor for infection when the authors compared the hcv-infected group to both controls and hivinfected individuals. moreover, the ccr5δ32 homozygous genotype was associated with increased hcv loads. in their study, it was observed an important deviation from the hardy-weinberg equilibrium in data from hcv-infected individuals; and a high portion of the individuals included in the study was hemophiliac j o u r n a l p r e -p r o o f (woitas et al., 2002) . hemophiliac individuals were at high risk of exposure to hcv and hiv until the mid-1980s (promrat et al., 2003; zhang et al., 2003) . considering that the ccr5δ32 homozygous genotype provides protection against hiv infection, a high frequency of this genotype in an hcv-infected group may be due to hiv resistance, but not to hcv, among individuals highly exposed to both viruses. due to those and other reasons, the results of woitas et al. (2002) were criticized by different authors (klein, 2003; mangia et al., 2003; poljak et al., 2003; promrat et al., 2003; zhang et al., 2003) . in this sense, no influence of ccr5δ32 on susceptibility to hcv infection were reported in studies performed with various populations (glas et al., 2003; mangia et al., 2003; poljak et al., 2003; promrat et al., 2003; zhang et al., 2003; ruiz-ferrer et al., 2004; wald et al., 2004; wasmuth et al., 2004; thoelen et al., 2005; goyal et al., 2006) . reinforcing the observations of those different studies, our group found no association between the ccr5δ32 and susceptibility to hcv infection or hcv/hiv co-infection in a study that evaluated a large number of brazilian individuals (ellwanger et al., 2018b) . bineshian et al. (2018) did not detect the ccr5δ32 allele in any iranian hcv-infected individual and controls included in their study, preventing any conclusion in terms of susceptibility in that population. finally, it is important to mention that in addition to the study by woitas et al. (2002) , a study performed in 2013 also found an association between the ccr5δ32 homozygous genotype with chronic hcv infection in europeans, but the authors of the study mentioned that specific factors regarding selection bias (e.g., co-exposure to hiv) may have influenced their results (suppiah et al., 2013) . taken together, the above-mentioned results called attention for the importance of performing genetic variant studies in different populations, exposed to different social and environmental factors and presenting distinct ethnic backgrounds. considering multiple clinical and histological parameters, two main different results were obtained when the ccr5δ32 genetic variant was evaluated in the context of hcv-related diseases: reduced liver inflammation in δ32 allele carriers (hellier et al., 2003; wald et al., 2004; goulding et al., 2005) ; and no association between the ccr5δ32 and clinical variables (glas et al., 2003; mangia et al., 2003; promrat et al., 2003; goyal et al., 2006; mascheretti et al., 2004; ruiz-ferrer et al., 2004; morard et al., 2014; ellwanger et al., 2018b) . in the context of persistence/resolution of hcv infection and viral control, in the one hand, studies described association of the ccr5δ32 allele with reduced rates of spontaneous viral clearance (nattermann et al., 2011; morard et al, 2014) , higher viral load (yilmaz et al., 2014) , and reduced anti-hcv immune response (ahlenstiel et al., 2009 ). on the other hand, studies have reported association between the ccr5δ32 variant and increased rates of spontaneous viral clearance (goulding et al., 2005; el-moamly et al., 2013) . no significant effect of the ccr5δ32 on viral clearance was reported by other authors (mascheretti et al., 2004) . the potential impact of the ccr5δ32 polymorphism on response to hcv therapy was also evaluated by some authors. ahlenstiel et al. (2003) found an association between the ccr5δ32 allele and reduced response rates to interferon-α monotherapy, but the polymorphism did not affect the response to the combined interferon/ribavirin therapy. this finding shows that the use of more robust therapeutic regimens j o u r n a l p r e -p r o o f compensates the undesirable effects of ccr5δ32 on hcv therapy with interferon-α monotherapy. of note, the effect of ccr5δ32 may be negligible in the context of modern hcv therapies (ahlenstiel et al., 2003) . other studies did not report significant effects of the polymorphism on response to hcv therapy (glas et al., 2003; promrat et al., 2003; goyal et al., 2006; mascheretti et al., 2004; suppiah et al., 2013; morard et al., 2014) . again, it is important to mention that the ethnic distribution of the ccr5δ32 allele could interfere with study results. konishi et al. (2004) , for example, did not detect the ccr5δ32 allele in any japanese individual included in their study focused on host genetic factors involved in the response to interferon therapy. lastly, the polymorphism also does not appear to be associated with any specific hcv genotype (glas et al., 2003; wasmuth et al., 2004; goyal et al., 2006) . ahlenstiel et al. (2004) highlighted that the impact of ccr5 on hcv infection was controversial. in 2020, many controversies remain, although some points are better defined. table 5 summarizes the main findings of the studies that evaluated ccr5δ32 on hcv infection. although some studies indicate an influence of the polymorphism on susceptibility to infection, most studies indicate that the δ32 allele has little (or no) influence on hcv susceptibility. the impact of the ccr5δ32 on hcv-related liver disease is quite variable and context-dependent. finally, available data suggest some benefit of ccr5 antagonists for the treatment of hcv mono-infection. however, these data are still limited and further studies evaluating this topic are needed. poliovirus (pv) is a single-stranded rna enterovirus of the family picornaviridae. there are three types of pv: wild pv type 1 (wpv1), type 2 (wpv2), and type 3 (wpv3). the pv replicates in the tonsils and intestinal tract. in few infection cases (~1%), the virus invades the cns and can cause poliomyelitis resulting in paralysis. poliomyelitis is a condition characterized by inflammation of the gray matter of the spinal cord and muscle paralysis unleashed by pv replication in motor neurons (racaniello, 2006; kew and pallansch, 2018; keohane et al., 2020) . polioencephalitis can also occur and is characterized by the pv outbreak occurred in that country (table 6 ). the authors found no statistically significant effect of the ccr5δ32 on pv infection; only a trend of association between the δ32 allele and increased risk of pv infection was observed (rosenberg et al., 2013) . however, this study had a very small sample size (only seven cases of severe pv infection were evaluated) and therefore the results were not conclusive. in addition, due to the declining number of pv infection cases in the world, the effect of ccr5δ32 will be increasingly difficult to be assessed in population-based studies. dengue virus (denv) is a single-stranded rna virus that belongs to the flaviviridae family, flavivirus genus. there are four denv serotypes, being all transmitted to humans by aedes mosquitoes (aedes aegypti and aedes albopictus) (guzman et al., 2016) . denv infection is a global health problem with huge impacts on public health systems, especially in tropical countries (bhatt et al., 2013) , being considered the most common arbovirosis in the world (stanaway et al., 2016) . globally, the incidence of symptomatic denv infection is within the range of 50 to 100 million cases per year, resulting in ~10,000 deaths each year (stanaway et al., 2016) . clinically, dengue illness is divided into three basic phases: acute febrile phase, critical phase, and recovery (convalescent) phase. dengue disease occurs with/without warning signs or severe dengue. warning signs (suggestive signs or symptoms of important fluid loss, capillary leakage, and shock, such as severe abdominal pain and mucosal bleeding; observed at the end of the febrile phase) allow the rapid identification of patients who need more clinical attention and supportive therapy, in an attempt to avoid severe dengue. when severe disease occurs, this condition can lead to serious organ involvement, shock, and hemorrhage, among other signals and symptoms (guzman et al., 2016) . infection with a denv serotype triggers long-term immunity to that specific serotype (homotypic denv). immunity to heterotypic denv also occurs, but it is transitory. therefore, an individual can have dengue disease more than once. severe dengue occurs more frequently in recurrent infection with a different viral serotype (murphy and whitehead, 2011; st john and rathore, 2019 ). an immune response mediates denv clearance and the resolution of dengue diseases, but it is also involved in the disease pathogenesis (murphy and whitehead, 2011) . some evidence suggests the participation of ccl5/ccr5 axis in the protection against denv (sierra et al., 2014) , as well as in the pathogenesis of dengue disease (islam et al., 2019) . indeed, denv infection is associated with increased frequency of human ccr5+ t cells (de-oliveira-pinto et al., 2012; badolato-corrêa et al., 2018) . in a study performed by marques et al. (2015) , lower viral replication was found in macrophages treated with ccr5 blockers. in the same study, ccr5-/-mice were protected from denv infection. these findings suggest that ccr5δ32 could be a protective factor against denv infection. however, xavier-carvalho et al. (2013) found no statistical difference in the frequency of ccr5δ32 polymorphism between brazilian children with j o u r n a l p r e -p r o o f severe denv infection and healthy controls. in the same direction, no effect of ccr5δ32 on susceptibility to denv infection was found in a small sample-size study performed with individuals from western australia (brestovac et al., 2014) and recent data suggested no important involvement of ccr5 gene or ccr5 polymorphisms in denv infection (cahill et al., 2018; ornelas et al., 2019) . finally, the ccr5δ32 allele was not identified in a small group of indian denv-infected individuals (islam et al., 2019) . table 6 shows some details of the studies involving ccr5δ32 and denv infection. taking together, the data mentioned above indicate that the effects of ccr5 on denv infection are very different between humans and rodents. however, it should be noted that the approach of each mentioned study is quite particular, and we cannot exclude some potential effects of ccr5 and ccr5δ32 on denv infection in humans. cmv is also linked to cancer development, once several cmv proteins (e.g., pul122, pul123, pus28, pul83, pul111a) activate pro-oncogenic pathways, including angiogenesis, escape of immune control and tumor suppressors, tumoral inflammation, invasion and metastasis, genome instability, and increased cell survival and proliferation (herbein, 2018) . poor socioeconomic condition favors cmv infection. antibodies indicating past cmv infection are found in ~60% of adults from high-income countries. in low-income countries, the rate of past infections can reach 100% (griffiths et al., 2015) . cmv can manipulate the immune system producing virokines (virus-encoded cytokine/chemokine homologs) and viroceptors (virus-encoded cytokine/chemokine receptor homologs), molecules that enable the virus to evade host immune defenses. such molecules can also facilitate viral replication (lucas et al., 2001; froberg, 2004; vomaske et al., 2012) . importantly, cmv-encoded proteins can interact with ccr5, (johnson et al., 2015) . however, mixed results were reported regarding the effect of cmv on ccr5 expression since there is evidence indicating that cmv infection may reduce ccr5 expression in various cell types (lecointe at al., 2002; varani et al., 2005) . interestingly, these mixed results may not be contradictory. cmv-infected cells may indeed exhibit decreased ccr5 expression, limiting hiv infection in these cells. however, cmv-infected cells release cmv-associated soluble factors that increase ccr5 expression in non-infected bystander cells, then facilitating hiv replication in such cells and, consequently, contributing to hiv pathogenesis (king et al., 2006) . there is evidence showing that variants in the ccr5 gene can influence multiple aspects of cmv infection (loeffler et al., 2006; sezgin et al., 2011) . for example, the ccr5 promoter polymorphism rs1800023 affects cmv replication (bravo et al., 2014; corrales et al., 2015) . in a study evaluating children, kasztelewicz et al. (2017) found no influence of ccr5δ32 on susceptibility to congenital cmv infection, severity of congenital cmv disease, or cmv-related sensorineural hearing loss at birth. as an individual genetic factor, ccr5δ32 was not statistically associated with the progression of cmv retinitis, a condition that cmv can cause in immunocompromised individuals (sezgin et al., 2011) (table 6 ). bunyaviridae. cchfv circulates in africa, europe, middle east, and asia countries, and can infect a variety of domestic animals and wild species, but without causing symptomatic illness. humans are accidental hosts of cchfv, for which the virus is transmitted mainly by tick-bites (especially ticks of the genus hyalomma), although other routes of transmission also exist, such as exposure to blood of infected animals. most cchfv-infected individuals have no symptoms or have mild nonspecific febrile syndrome. however, in some individuals, the infection can cause the crimean-congo hemorrhagic fever, a severe disease characterized by fever, myalgia, hemorrhage, among other manifestations. neurological complications can also occur, being the spectrum and intensity of the disease quite variable (ergönül, 2006; bente et al., 2013; garrison et al., 2019) . hemorrhagic fever (ergönül, 2006; saksida et al., 2010; bente et al., 2013; garrison et al., 2019) . in a study performed by arasli et al. (2015) , expression of the ccr5 ligands ccl2, ccl3, and ccl4 was increased in cchfv-infected adults compared to controls. considering these same chemokines in cchfv-infected children, only ccl4 was significantly increased compared to pediatric controls (arasli et al., 2015) . engin et al. (2009) evaluated the ccr5δ32 in 15 turkish cchfv-infected individuals and observed the wild-type homozygous genotype in all cases. in a subsequent study evaluating the turkish population, rustemoglu et al. (2017) found a protective effect of ccr5δ32 heterozygous genotype and δ32 allele on cchfv infection, since the genotype and allele frequencies were higher in controls than in cchfv-infected individuals. conversely, the wild-type genotype (normal ccr5 expression) was prevalent among infected j o u r n a l p r e -p r o o f individuals. these findings suggest that ccr5 contributes to susceptibility to cchfv infection and that ccr5 down-regulation due to ccr5δ32 results in some protection against the infection. however, further studies are needed to explain the mechanisms by which ccr5 participates in cchfv infection. in the same study, the ccr5δ32 was not significantly associated with disease severity, clinical parameters, or mortality rate (rustemoglu et al., 2017) . together, these findings indicate that the effect of ccr5δ32 is given specifically on resistance against cchfv infection, without affecting the pathogenesis/outcome of crimean-congo hemorrhagic fever. the genus enterovirus is composed of non-enveloped, positive-stranded rna viruses, belonging to the picornaviridae family. enteroviruses (ev) can infect the gastrointestinal tract, cns, and other organs, including heart (tapparel et al., 2013) . cardiomyopathy is a common consequence of ev infection in the heart. ev infection is associated with myocardial inflammation (myocarditis) and other damages to heart tissues (badorff et al., 2000; cooper, 2009; sagar et al., 2012; tapparel et al., 2013; weintraub et al., 2017) . damage heart tissues. viral persistence in individuals with enteroviral cardiomyopathy is associated with an increased mortality rates (kühl et al., 2005; lassner et al., 2018) . some studies suggest that ccr5 influences different aspects of the pathogenesis of viruses belonging to the picornaviridae family, including encephalomyocarditis virus (christmann et al., 2011; shaheen et al., 2015) , coxsackievirus b3 (valaperti et al., 2013) , and rhinovirus (muehling et al., 2017) , once ccr5 participates in the regulation of the host immune response during infection by these viruses. considering the effects of the genetic variant ccr5δ32 on ccr5 expression and ccr5-related immune responses, it is possible that ccr5δ32 also shows some impact on ev-related diseases. in a german study that evaluated patients with enteroviral (chronic/inflammatory) cardiomyopathy (lassner et al., 2018) , the ccr5δ32 was strongly associated with spontaneous viral clearance and better clinical outcome (reduced mortality rate) ( table 6 ). these findings indicate a critical involvement of the ccr5 molecule in the pathogenesis of ev cardiomyopathy. it was suggested that the ccr5δ32 genotyping could be used to assist in the prediction of the clinical progression of enteroviral cardiomyopathy: the δ32 allele as a predictor of a better prognosis, without the need of antiviral interferon-β (ifn-β) therapy; and the wild-type genotype as a predictor of a worse prognosis and immediate need of antiviral ifn-β therapy (lassner et al., 2018) . the clinical use of ifn-β is effective to eliminate the virus, avoid irreversible cardiac injury, and reduce mortality rates, but it is also associated to numerous adverse effects (kühl et al., 2012; lassner et al., 2018) . considering the prognostic value of the ccr5δ32 on the clinical course of enteroviral cardiomyopathy, it is necessary to evaluate the relationship between the ccr5δ32 and the disease in different human populations, mainly through genetic association studies. if this association is confirmed in other populations, the ccr5δ32 genotyping will be a broad-spectrum clinical tool, enhancing and driving the treatment of enteroviral cardiomyopathy. jev is the etiological agent of most cases of viral encephalitis in many countries, reaching ~30% mortality rate (van den hurk et al., 2009; tiwari et al., 2012; le flohic et al., 2013) . some evidence obtained in laboratory conditions (in vitro analysis and murine models) showed that jev infection induces the up-regulation of ccr5 gene (gupta and roa, 2011; pereek et al., 2014; zhang et al., 2019) . also, increased infiltration of ccr5 + cd8 + t cells was observed in the brains of jev-infected mice (zhang et al., 2019) . in this context, using a mouse model of japanese encephalitis, larena et al. (2012) showed that ccr5 protects the host against jev infection in the cns, being essential for disease recovery. ccr5-deficient mice showed higher viral loads in the brain and spinal cord as well as increased mortality rate, as compared to control mice (larena et al., 2012) . also using a mouse model of japanese encephalitis, kim et al. (2016) demonstrated that ccr5 controls the infiltration of cd4 + foxp3 + t regulatory cells (treg) in the cns, contributing to protection against japanese encephalitis. the infiltration and action of inflammatory cells in the brain are important to limit neuroinvasive infections, processes that are regulated by multiple factors, especially cytokines, chemokines and their receptors, including ccr5. however, the uncontrolled action of inflammatory cells can cause damage to the cns. of note, cd4 + foxp3 + treg cells regulate the immune responses, avoiding undesirable or excessive action of inflammatory cells (bardina and lim, 2012; veiga-parga et al., 2013; simonetta and bourgeois, 2013; campbell, 2015; kim et al., 2016) . according to these pieces of evidence, ccr5-mediated action of treg cells is critical for the control of japanese encephalitis. however, the role of ccr5 in jev infection may be more complex than it appears at first. liu et al. (2018) demonstrated that the use of the ccr5 antagonist maraviroc reduces the jev-induced inflammation in mice brain, increasing survival rate. this result suggests that potential deleterious effects of ccr5-mediated inflammation in the brain may override the effects of ccr5-mediated action of treg cells and, therefore, the use of ccr5 antagonists to treat japanese encephalitis may be beneficial. based on these complex and contradictory results, it can be concluded that ccr5 indeed has an important influence on japanese encephalitis, but it is not yet possible to state its specific roles, once they are varied and appear to be context-dependent. also, the results obtained in animal models may not fully represent the disease course in humans. indeed, some evidences suggest the involvement of ccr5 in the pathogenesis of japanese encephalitis in humans. in indian individuals with mild cases of japanese encephalitis, a significant dowregulation of the ccr5 gene was observed as compared to healthy controls (chowdhury and khan, 2019) . however, the ccr5δ32 does not have a relevant influence on the disease. deval et al. (2019) investigated the effect of various genetic variants, including ccr5δ32, on the development of japanese encephalitis in individuals from north india (table 6 ). no statistically significant difference was found when the ccr5δ32 (as an individual factor) was compared between cases and controls (deval et al., 2019) . considering that both studies mentioned above were performed in the indian population, studies evaluating the potential effects of ccr5 and ccr5δ32 on japanese encephalitis in other populations are necessary. puumala virus (puv) infection is a zoonosis endemic in europe. puv is an enveloped singlestranded rna virus, belonging to the bunyaviridae family, genus hantaviridae. most puv infections are mild or subclinical. in some infected individuals, puv is responsible for the development of nephropathia epidemica, a milder form of hemorrhagic fever with renal syndrome, a condition typically caused by other hantaviruses (settergren, 2000; mustonen et al., 2013; lebecque and dupont, 2019 ). an in vitro study found increased ccr5 gene expression in primary monocytes infected by hantaviruses (hantaan virus, sin nombre virus and andes virus) (markotić et al., 2007) . in this sense, host genetic factors and the immune responses affect different aspects of puv infection and progression of nephropathia epidemica (mustonen et al., 2013) , although the role of ccr5 in this disease is little known. (table 6 ). of note, in their study, the authors stated hantavirus infection as the cause of nephropathia epidemica, not specifying the puv detection. no statistical difference was observed in ccr5δ32 genotype frequencies between cases and controls, indicating that ccr5δ32 does not influenced the susceptibility to hantavirus infection in the studied population. considering only nephropathia epidemica cases, the study revealed an association between the wild-type homozygous genotype (functional ccr5) and increased severity of the disease. conversely, the heterozygous genotype was considered a protective factor against increased disease severity ( in 2019, the multiple roles of ccr5 in physiological and pathological conditions gained the attention of the scientific community and lay public due to ccr5 gene editing in human embryos, the "crispr babies", performed by a chinese biophysicist. the researcher claimed to have used crispr gene editing technology to edit the ccr5 of germline cells to mimic the effects of ccr5δ32, aiming to generate babies resistant to hiv infection. the ethical, safety, and legal aspects related to this procedure have caused an intense and broad discussion in the media and scientific literature (cohen, 2019; daley et al., 2019; greely, 2019; rosenbaum, 2019) , and this case culminated in a three years prison sentence for the biophysicist responsible for the procedure (cyranoski, 2020). also, the consequences of the ccr5 absence have brought many concerns to light, once the ccr5 protein participates in tissue development processes, control of immune responses, and several other physiological processes. considering these concerns, our group and others described some undesirable effects associated to natural ccr5 absence (due to the ccr5δ32 homozygous genotype) and ccr5 editing (ellwanger et al., 2019; wang and yang, 2019; xie et al., 2019) . besides gene editing techniques, the expression of ccr5 can be artificially modulated through the use of pharmacological antagonists (e.g. maraviroc), chemokine ligands, and monoclonal antibodies (fantuzzi et al., 2019) . the use of ccr5 antagonists is well established for the treatment of hiv infection and is currently being tested for the treatment of many other diseases, such as cancer (pervaiz et al., 2019; huang et al., 2020a) , stroke (joy et al., 2019), and cocaine addiction-related disorders (hall et al., 2020; nayak et al., 2020) . taking together, it is increasingly clear that the specific inhibition of ccr5 expression through different techniques is gaining pace in different clinical contexts. the pharmacological ccr5 blockade has many benefits in different clinical situations, particularly in the treatment of hiv infection. also, the potential of gene editing (especially in somatic cells) for the treatment of genetic diseases is very promising. however, considering viral infections, two aspects must be considered, as follows: i) the non-redundant characteristics of the ccr5 should be taken into consideration when studying the ccr5 protein and the effects of ccr5δ32 on viral infections: the traditional concepts of redundancy and robustness of the chemokine system consider that the absence of a specific chemokine or chemokine receptor is adequately compensated by other molecules. although these concepts are generally correct for some chemokines/receptors and for some physiological conditions, the lack of ccr5 expression may affect the protection against some specific infectious diseases once the ccr5 absence is not perfectly compensated for by other receptors (ellwanger et al., 2020a) . the nonredundancy of ccr5 is relevant especially for infections by neuroinvasive flaviviruses (bradina and lim, 2012) . for example, the absence of ccr5 due to ccr5δ32 is considered deleterious in wnv infection lim et al., 2008) and in some aspects of tbev infection (ellwanger and chies, 2019a) . the non-redundant role of ccr5 is also likely in jev infection (larena et al., 2012) . it is possible that those individuals using ccr5 antagonists (e.g., for the treatment of hiv infection) and living in areas endemic for neuroinvasive viruses, especially wnv and tbev, may be at increased risk of j o u r n a l p r e -p r o o f developing viral encephalitis-related problems. although, it is still necessary that studies consistently evaluate this assumption, since the available evidence does not support risks for the use of ccr5 antagonists in endemic areas of flaviviruses (martin-blondel et al., 2016) . as mentioned in the topic addressing wnv in this review, it is prudent to recommend to individuals using ccr5 antagonists to adopt measures to minimize the risk of neuroinvasive infections, such as the use of mosquito repellents and mosquito nets (considering the risk of wnv infection), and to limit their exposure to tick-infested areas (considering the risk of tbev infection). concerns regarding the use of ccr5 antagonists in areas of jev circulation have also been raised by other authors (kim et al., 2016; larena et al., 2012) . if the connection between the use of ccr5 antagonists and increased risk of neuroinvasive infections is confirmed in methodologically wellcontrolled studies, these recommendations should be considered of essential importance for users of ccr5 antagonists. "extracellular vesicles" (evs) is a general term used to designate different membranous vesicles released by various cell types. evs include microparticles, microvesicles, nanovesicles, nanoparticles, ectosomes, exosomes, exovesicles, exosome-like vesicles, oncosomes, among other vesicle types. evs act in the transport of different molecules (e.g. micrornas, mrnas, proteins) between cells and tissues in a regulated and precise manner. evs promote the maintenance of physiological processes, also participating in the pathogenesis of various diseases, such as cancer and inflammatory diseases (colombo et al., 2014; théry et al., 2018) . besides, the regulation of multiple aspects of the immune system is strongly influenced by evs (o'neill and quah, 2008; colombo et al., 2014; ellwanger et al., 2016) . the multiple roles of evs in viral infections began to be studied more intensively in recent years, representing an emerging topic in the field of infectious diseases. currently, the relationship between evs and viral infections has already been explored (to a greater or lesser extent) in the context of the following . evs participate in immune evasion processes, ultimately allowing viruses to bypass host defenses (kaminski et al., 2019b) . for example, hiv is likely to usurp the exosome biogenesis pathway in such a way that enhances its infectivity, while increasing its evading strategies from the host immune defenses (ellwanger et al., 2017a) . regarding tbev, exosomes were indicated as important participants in both viral infection and pathogenesis; in this case, tick-derived exosomes facilitate tbev transmission to the host. also, exosomes derived from tbev-infected host neurons can facilitate the spread of the virus in the cns . exosomes have shown multi-dimensional roles in denv life cycle. they can modulate negatively or positively denv pathogenesis, where they are suggested as instrumental for dengue j o u r n a l p r e -p r o o f hemorrhagic fever development in reason of the transportation of specific micrornas (mishra et al., 2019) . since hcv can be found inside exosomes (liu et al., 2014) , it is not suprising that blood-derived exosomes from hcv-infected patients can transmit the virus to other cells (bukong et al., 2014) . these findings suggest that exosomes and other evs assist in the transport of hcv particles/components between cells, ultimately facilitating viral infection. of note, evs can transport multiple viral components or molecules from virus-infected cells that end up facilitating the spread of the virus in the host. on the other hand, evs can act in favor of the host, transporting molecules that assist host defenses in the elimination or control of infections (kaminski et al., 2019b) . evs can transport a range of cytokines and chemokines, such as il-1, il-2, ifn-α, ifn-β, ccl2, ccl3, ccl4, and cxcl10 (chen et al., 2011; konadu et al., 2015; hosseinkhani et al., 2018; kodidela et al., 2018; gao et al., 2019a; gao et al., 2019b; aiello et al., 2020; chiantore et al., 2020) . besides, evs and microparticles also transport chemokine receptors (shen et al., 2016; liang et al., 2018) , including ccr5 tokarz et al., 2019) . interestingly, evs from ccr5 wild type cells can deliver ccr5 molecules to ccr5 δ32/δ32 cells, making them susceptible to hiv infection . a similar phenomenon has been reported involving microparticles, hiv, and cxcr4 (rozmyslowicz et al., 2003) . ccr5 expression is also influenced by the release of evs, specifically microparticles (renovato-martins et al., 2017) . therefore, evs have the potential to attribute greater complexity to ccr5 roles in viral infections. it is possible that the presence of ccr5 in cells is not only dependent on mechanisms of membrane expression and internalization of the receptor. it can also be dependent on ccr5 delivery mediated by evs. however, the actual impact of evs on ccr5-mediated immune response in infections remains to be determined and deserves further investigation. finally, a truncated ccr5 soluble form (tsimanis et al., 2005) , as well as soluble cxcr4, have also been reported in the plasma of humans (malvoisin et al., 2011) . of note, the truncated ccr5 soluble form has ~22 kda (half the size of the ccr5 found on cell membranes) and is truncated at the end of the second extracellular loop (the third extracellular loop and the subsequent three transmembrane regions are absent) (tsimanis et al., 2005) . however, the existence of cell-free soluble ccr5 is still controversial and does not represent a consensus in the scientific community. such doubts occur mainly in consideration of the structure of the receptor, which is highly characteristic of a cell membrane-associated molecule. we believe that evscontaining ccr5 can explain potential (misleading) detections of soluble ccr5. the importance of circulating evs-containing ccr5 on viral infections represents an additional and interesting open question to be investigated. chávez et al. (2013) (ellwanger et al., 2017b) . specifically, using a mouse model of rocv-associated encephalitis, ccr5-deficient mice showed increased survival rate and reduced levels of brain inflammation compared to mice expressing ccr5, indicating the participation of ccr5 in rocv-associated encephalitis (chávez et al., 2013) . although other studies discussed in this review have shown an important involvement of ccr5 in infection by flaviviruses [especially as an important molecule for the resolution of neuroinfection, in opposition to results of chávez et al., (2013) ], the role of the ccr5 in rocv infection represents a neglected topic. this knowledge gap should be addressed in further studies since the reemergence of rocv in brazil is a concern in terms of public health (figueiredo, 2007; ellwanger et al., 2017b) . although no other rocv outbreaks have been reported in brazil after the 1980s, there is evidence of rocv circulation in animals (casseb et al., 2014; pauvolid-correa et al., 2014; silva et al., 2014) . ccr5 expression on t cells. of note, zikv is another mosquito-borne flavivirus that recently reemerged in many countries, affecting brazil in particular. zikv infection is associated with microcephaly and other human development problems (baud et al., 2017) . zachova et al. (2019) showed that epstein-barr virus (ebv)-infected b cells have increased ccr5 expression compared to ebv-non-infected cells. also, recent evidence points to a pivotal role of the ccr5 in the attenuation of rhinovirus-associated inflammation, by controlling the activity of cd4 + foxp3 + treg cells (hossain et al., 2020) . altered levels of cytokines and chemokines are associated with several aspects of zikv (barros et al., 2018; naveca et al., 2018; khaiboullina et al., 2019; lima et al., 2019) , ebv (piovan et al., 2005; ehlin-henriksson et al., 2009; cohen et al., 2017) , and rhinovirus infections (rajan et al., 2014; shelfoon et al., 2016; hansel et al., 2017) . however, the potential role of the chemokine receptor ccr5 in the pathogenesis of zikv, ebv, and rhinovirus represents open questions in the field of ccr5 research. coronaviruses are positive-sense rna viruses that belong to the coronaviridae family (li et al., 2020) . coronaviruses infect a wide range of species, including humans. until 2019, humans have faced two major outbreaks of high pathogenic coronaviruses, the severe acute respiratory syndrome coronavirus (sars-cov) outbreak and the middle east respiratory syndrome coronavirus (mers-cov) outbreak (fung and liu, 2019) . in 2019, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in wuhan (hubei province, china) and rapidly spread to more than 180 countries/regions (interactive dashboard of global covid-19 cases -johns hopkins university: https://arcg.is/0fhmtx ; dong et al., 2020) . of note, sars-cov-2 infection causes the "coronavirus disease 2019" and therefore is also called "covid-19". considering the ongoing sars-cov-2 pandemic and the clinical variability observed in the affected individuals, ranging from mild to severe respiratory disease j o u r n a l p r e -p r o o f 2020b), the following question arose: does ccr5 have any clinically relevant influence on sars-cov-2 or other coronaviruses infections? recent data indicated that sars-cov-2 binds to the ace2 receptor (andersen et al., 2020) , using this receptor for entry into human cells. therefore, it is unlikely that ccr5 or ccr5δ32 have some significant effect on the susceptibility or resistance to sars-cov-2 infection. however, ccr5 may have some impact on the clinical course of sars-cov-2 infection. the pattern and intensity of the human immune responses regulate the clinical progression and even the outcome of infections by coronaviruses (li et al., 2020) , including sars-cov-2 (qin et al., 2020; shi et al., 2020) . sars-cov-infected mice showed increased expression of ccr5 mrna in lungs along with the production of ccl2, ccl3 and ccl5 chemokines, the major ccr5 natural agonists (chen et al., 2010) . intracranial infection of mice with mouse hepatitis virus (mhv, a murine coronavirus) induces an increased ccr5 expression, which contributed to mhv-induced demyelination through ccr5-mediated macrophage recruitment to the cns (glass et al., 2001) . subsequent studies using mhv-infected mice showed that ccr5 participates in the regulation of cd4 + and cd8 + t cell activities against the virus in the cns (glass and lane, 2003a; glass and lane, 2003b) . also, sars-cov-infected human monocyte-derived dendritic cells showed increased ccr5 expression in vitro (law et al., 2009) . taking together, the findings mentioned above suggest that ccr5 could have some influence on the clinical course of sars-cov-2 infection. however, future studies addressing humans are needed to clarify this suggestion. in this sense, we emphasize that so far, there is no evidence showing a clear involvement of ccr5 in sars-cov-2 human infection. the ccr5δ32 is a highly penetrating polymorphism, and exerts a robust phenotypic effect on ccr5. however, the expression of ccr5 is regulated by other polymorphisms in addition to ccr5δ32 (mehlotra, 2019) . also, the ccr5 expression is influenced by non-coding genetic elements. the effect of the genetic backround of the host can also be exacerbated or diminished depending on the environmental conditions to which the individual is exposed (ellwanger et al., 2018c; kulkarni et al., 2019) . taking together, these factors help to explain why many of the effects exerted by ccr5 on a given disease are specific to a certain population, ethnic group, or individual. research involving ccr5 and hiv began in the mid-1990s. since then, many achievements in the field of ccr5/hiv research have been made, such as the identification of ccr5δ32 as a strong protective factor against hiv infection and the development of ccr5 antagonists for the treatment of hiv infection. currently, these drugs are being tested for the treatment of other inflammatory and infectious diseases. in this context, the use of ccr5 antagonists has raised some concerns, since the blockade of ccr5 can affect j o u r n a l p r e -p r o o f or even weaken the host defenses against certain infections, especially neuroinvasive infections by flaviviruses. however, to date, there is no strong evidence indicating that the use of ccr5 antagonists has increased the susceptibility of individuals to problematic flaviviruses infections. the frequency of ccr5δ32 is quite varied among human populations, and therefore the effects of the allele in a particular population may not apply to another population. moreover, the effects of ccr5 and ccr5δ32 are disease-specific. for instance, the ccr5δ32 does not significantly affect susceptibility to wnv infection. however, the effect of the ccr5 and ccr5δ32 on wnv disease progression is very robust. some animal-derived findings suggest the involvement of the ccr5 in the pathogenesis of denv infection. however, the effects of ccr5 on denv infection are very different between humans and rodents. of note, ccr5δ32 does not significantly affect susceptibility to denv infection or disease progression. moreover, the effect of ccr5δ32 on hbv-related disease is population-specific. interestingly, cmv release virokines, which are molecules that can manipulate the host immune response and affect the ccr5 function. the examples cited above highlight the varied impacts of ccr5 and ccr5δ32 on viral infections. the few studies involving cchfv, ev, and hantavirus infection have indicated important effects of ccr5δ32 on these conditions. based on these findings, further studies should investigate the role of ccr5δ32 and ccr5 protein in such infections, considering populations with distinct genetic backgrounds. some evidence suggested the participation of ccr5 in infections by zikv, ebv, and rhinovirus. also, a mouse model of rocv-associated encephalitis suggested an important role for ccr5 in host immune responses against the virus. however, the roles of ccr5 and ccr5δ32 in infections by zikv, ebv, rhinovirus, and rocv are still poorly understood and need to be investigated in future studies. the role of evs in the transport of ccr5 between cells indicates that the expression of ccr5 on the cell surface may also depend on the release of evs containing ccr5. also, the transport of host molecules and viruses through evs adds complexity to the topics covered in this review and should be taken into account in future studies that investigate the role of ccr5 in viral infections. gene editing technologies have the potential to be used to treat different diseases, mainly when applied to somatic cells. however, ccr5 gene editing in human embryos presents a number of ethical problems. besides, the absence of ccr5 can have deleterious effects in certain conditions, such as increased susceptibility to symptomatic wnv infection. finally, this article showed that the participation of ccr5 in different viral infections is complex and varied and, therefore, cannot be generalized. this article also pointed out neglected gaps in knowledge involving ccr5 that should be addressed in future studies. the authors declare no conflicts of interest. ccr5 polymorphism as 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mimicry by hiv exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral rna and proteins to the vertebrate neuronal cells chemokines: a new classification system and their role in immunity protective effect of ccr5-δ32 against hiv infection by the heterosexual mode of transmission in a polish population american 395 symptomatic wnv+ individuals; 145 symptomatic but noninfected individuals; 1318 controls (blood donors)the ccr5δ32 homozygous genotype was strongly associated with increased risk of symptomatic wnv infection glass et al. (2006) american 224 symptomatic wnv+ individuals; 1318 controls (blood donors)corroborating data from glass et al. (2006) , the ccr5δ32 homozygous genotype was strongly associated with increased risk of symptomatic wnv infection lim et al. (2008) american 634 wnv+ individuals; 422 non-infected individuals ccr5δ32 homozygous genotype was associated with clinical symptoms of wnv infection; no association between ccr5δ32 and susceptibility to wnv infection lim et al. (2010) american, canadian 385 symptomatic wnv+ individuals; 328 asymptomatic wnv+ individuals; 1318 controls [blood donors from glass et al. (2006)] no association of ccr5δ32 and wnv infection considering symptomatic and asymptomatic wnv+ individuals; considering a dominant model of inheritance of ccr5δ32 and using controls from glass et al. (2006) , the ccr5δ32 was associated with symptomatic wnv infection and infection risk bigham et al. (2011) key: cord-317198-mean7sj9 authors: giamberardin, heloisa i.g.; homsani, sheila; bricks, lucia f.; pacheco, ana p.o.; guedes, matilde; debur, maria c.; raboni, sonia m. title: clinical and epidemiological features of respiratory virus infections in preschool children over two consecutive influenza seasons in southern brazil date: 2016-02-09 journal: j med virol doi: 10.1002/jmv.24477 sha: doc_id: 317198 cord_uid: mean7sj9 this study reports the results of a systematic screening for respiratory viruses in pediatric outpatients from an emergency department (ed) in southern brazil during two consecutive influenza seasons. children eligible for enrollment in this study were aged 24–59 months and presented with acute respiratory symptoms and fever. naso‐ and oropharyngeal swabs were collected and multiplex reverse transcription pcr (rt‐pcr) was performed to identify the respiratory viruses involved. in total, 492 children were included in this study: 248 in 2010 and 244 in 2011. in 2010, 136 samples (55%) were found to be positive for at least one virus and the most frequently detected viruses were human rhinovirus (hrv) (18%), adenovirus (adv) (13%), and human coronavirus (cov) (5%). in 2011, 158 samples (65%) were found to be positive for at least one virus, and the most frequently detected were hrv (29%), adv (12%), and enterovirus (9%). further, the presence of asthma (or, 3.17; 95% ci, 1.86–5.46) was independently associated with hrv infection, whereas fever was associated with adv (or, 3.86; 95% ci, 1.31–16.52) and influenza infections (or, 3.74; 95% ci, 1.26–16.06). ten patients (2%) were diagnosed with pneumonia, and six of these tested positive for viral infection (4 hrv, 1 rsv, and 1 adv). thus, this study identified the most common respiratory viruses found in preschool children in the study region and demonstrated their high frequency, highlighting the need for improved data collection, and case management in order to stimulate preventive measures against these infections. j. med. virol. 88:1325–1333, 2016. © 2016 wiley periodicals, inc. viral acute respiratory infections (aris) in pediatric outpatients represent a significant burden on emergency departments (eds) and the patients' families, mainly during influenza seasons, being associated with around 20% of all deaths in pre-school children worldwide, with 90% of these deaths due to pneumonia. [izurieta et al., 2000; fiore et al., 2009; bezerra et al., 2011; gessner et al., 2011; munywoki et al., 2011] . however, information about the etiology of viral aris in preschool children, particularly in outpatients, is limited, especially in latin america. community respiratory viruses (crvs) are frequently found in this population and could be involved in severe infections in young children and patients with comorbid conditions [ampofo et al., 2006; ampofo et al., 2008; bender et al., 2009] . therefore, understanding the dynamics of their circulation among various age groups is essential, not only to help pediatricians in their diagnosis but also for developing preventive measures against these infections. curitiba city in southern brazil has a temperate climate with low temperatures and high humidity in winter. previous studies have shown a seasonal pattern of prevalence of respiratory viruses in curitiba, with an increased incidence in winter. during this period, influenza and respiratory syncytial viruses are most frequently associated with increased respiratory disease morbidity and lead to an extensive respiratory disease burden [tsuchiya et al., 2005; coelho et al, 2007; debur et al., 2010; raboni et al., 2011] . however, the majority of these studies have been conducted in hospitalized patients or outpatients screened under an influenza surveillance program. although reports that a high number of outpatients consistently present with aris, few of them have evaluated the pathogens involved in aris in this group of patients [pavia, 2013; adams et al., 2015] . this study reports, the results of a laboratory-based surveillance for respiratory viruses in preschool children who were treated in the ed of a pediatric referral hospital during two consecutive influenza seasons. a cross-sectional study was carried out from july to november in both 2010 and 2011. children considered eligible for this study were outpatients who were aged 24-59 months and attended the ed of pequeno pr ıncipe hospital (pph), curitiba, brazil; these children had been diagnosed with acute upper or lower respiratory infections or asthma-related conditions. patients whose parents signed the consent form, completed the form (demographic, clinical, and influenza vaccine data), and met all of the following inclusion criteria were enrolled in the study: (i) aris with or without fever (>100˚f/38˚c); (ii) that occurred <7 days before the ed visit; and (iii) for which samples had been obtained. each patient was included only once. patients exhibiting symptoms that began >7 days before the ed visit, patients who were administered oseltamivir during the 3 days (6 doses) prior to enrollment, and those from whom samples could not be obtained were excluded from the study. detailed demographic information was obtained for all children enrolled by using medical charts and brief interviews. in addition, clinical data, outcomes, comorbidities, and the time spent in school or a day-care center were assessed for each child. aris comprise a large and heterogeneous group of diseases such as the following: flu syndrome; common cold; pharyngitis; laryngitis; tracheitis; asthma and wheezing crisis; bronchiolitis; and pneumonia of bacterial, viral, and other etiologies. this study was approved by the institutional review board (irb: #820-10), and written informed consent was obtained from the individuals (parents, tutor, or relatives) responsible for the patients. the pequeno pr ıncipe hospital is the largest pediatric referral hospital of curitiba and attends children referred from various health units in the city and metropolitan region for tertiary and quaternary medical care. it also has a large number of specialized clinics and emergency rooms for primary and secondary medical care, the study was conducted with these group of patients. samples from naso-and oropharyngeal mucosa were collected with swabs and transferred to virological transport media (tryptose phosphate buffer enriched with gelatin) and shipped at 4˚c to the virology laboratory. crvs were detected using multiplex rt-pcr. the viral genome was extracted using the viral gene-spin tm kit (intron biotechnology inc., korea) according to the manufacturer's instructions. the multiplex rt-pcr technology used for the samples from 2010 enables simultaneous detection of 12 viruses (seeplex 1 rv 12 ace detection, seegene, korea): human adenovirus (adv); human coronavirus (cov) types 229e/nl63 and oc43/hku1; human metapneumovirus (mpv); parainfluenza virus types 1, 2, and 3 (piv-1, piv-2, and piv-3); influenza a (flua) and influenza b (flub) viruses; respiratory syncytial virus types a and b (rsv-a, rsv-b); and human rhinovirus types a and b (hrv a/b). the multiplex rt-pcr technology used for the 2011 samples enables simultaneous detection of 15 viruses (seeplex 1 rv 15 ace detection, seegene, korea): the 12 viruses of the seeplex 1 rv 12 ace kit plus human enterovirus (ev), human bocavirus (bov), and parainfluenza virus type 4 (piv-4). subtyping of influenza a viruses was performed by using real time rt-pcr (qrt-pcr) according to the cdc protocol [who, 2010] . sample size estimation was based on a 95% bilateral confidence interval for the expected proportion of children with infection caused by influenza virus. the expected proportion was assumed to range from 0.07 to 0.40, the number of subjects included was 250, and the maximum confidence interval precision (range) was chosen to be 0.12. data were compiled using jmp version 5.2.1 (sas institute inc., cary, nc) and analyzed using the r package version 3.0.1 (r core team; 2014). descriptive statistics were used to describe the general characteristics of the study population. quantitative variables with normal and non-normal distributions are presented as means ae standard deviation and medians with interquartile ranges (iqrs, 25th-75th percentiles), respectively, whereas qualitative variables have been expressed as numbers and percentages with 95% confidence intervals (cis). chi-square or fisher's test were used for comparison of qualitative variables. the student's t-test or the mann-whitney u-test was applied to compare quantitative variables, as appropriate. five viruses (hrv, adv, piv, flu, and cov) were selected for a binomial analysis to assess the impact of viruses on the health of the children. the presence or absence of the virus was compared with different variables (demographic, clinical features, and host characteristics). the selection of these viruses was based on either the high frequency of infections or previously reported severity of infections caused by them. only cases with mono-detection were included in this evaluation. variables with an associated p-value < 0.2 in the univariate analysis were subjected to multivariate logistic regression to identify independent predictors of these viruses infection. odds ratios (ors) and 95% cis were calculated. all p-values were based on two-tailed comparisons, and the level of significance was set at p < 0.05. initially, 505 patients were enrolled, of these 13 were later excluded because: (i) inappropriate sample collection (01); (ii) date of onset of symptoms !7 days (06); (iii) medical record was not found (01); (iv) not meeting ili acute condition (03); and (v) age >59 months (02). a total of 492 children between the ages of 24 and 59 months were included in the study during two influenza seasons (248 in 2010 and 244 in 2011). the mean age of the children was 39.1 (ae10.2) months, and 265 (54%) were male individuals. further, 376 (77%) of the 492 children attended school or daycare centers, of which 218 (58%) attended school or day care on a fulltime basis. comorbid conditions were present in 149 (30%) children, with asthma/bronchitis (n ¼ 129, 26%, as evidenced by reporting of previous episodes of wheezing with the use of bronchodilator medication) being most frequently reported (table i) . fever, cough, and pharyngeal erythema were the most common clinical manifestations. chest radiography was performed for 44 (9%) patients, and perihilar infiltrates, and pulmonary consolidation were found in 27/44 (61%) and 10/44 (23%) cases, respectively. six of the 10 patients (2%) with pulmonary consolidation tested positive for multiple viruses (4 hrv, 1 rsv, and 1 adv). nine (2%) patients required non-invasive respiratory support, and antibiotics were prescribed for 34 (7%) children. nasopharyngitis and influenza-like infection were diagnosed in 71% of the cases (table ii) . clinical complications occurred in eight (1.6%) patients: one had sinusitis, two presented with otitis media, one had tracheobronchitis, and one had pneumonia. of the 248 children evaluated in 2010, 136 (55%) samples were positive for at least one respiratory virus. hrv a/b (n ¼ 46, 18%), adv (n ¼ 33, 13%), and cov 229/nl 63 (n ¼ 13, 5%) were detected most frequently. viral co-infections occurred in 18 (7%) patients, mainly with the association of hrv a/b and (2) adv (n ¼ 5; 2%). in 2011, 157 out of 244 (65%) samples were positive for at least one respiratory virus. further, hrv a/b (n ¼ 71, 29%) and adv (n ¼ 29, 12%) were detected most frequently, followed by enterovirus (n ¼ 21, 9%). viral co-infections occurred in 26 (11%) patients and mainly involved an association of hrv a/b and adv (n ¼ 5, 2%), enterovirus and hrv a/b (n ¼ 5, 2%), or hrv a/b and parainfluenza 3 (n ¼ 5, 2%) (table iii, fig. 1 ). the distribution of the frequency of the viruses according to the age group showed that most of them were detected in children aged between 24 and 36 months, except for influenza a and human coronaviruses. notably, in both years, there was a higher rate of detection of influenza type b (5%; 25/492) than of type a (3.6%; 18/492). while 17 of the 18 samples containing influenza a were of subtype h3n2, one was of an undetermined subtype (0.4%), and none of them belonged to subtype h1n1pdm09. there was a fluctuation in the most frequently detected respiratory viruses according to the epidemiological week during the study, with a higher frequency of detection of influenza b in the first 6 weeks of the study, followed by influenza a h3n2. the proportion of influenza virus infection in the population studied was between 5% and 12% (95% ci, 0.05-0.12). further, a significant difference in the coverage rate of influenza vaccination was observed between 2010 and 2011 (88%; 95% ci, 85.9-93.6 vs. 24%; 95% ci, 19.2-30). over 90% of immunized patients received the monovalent h1n1pdm influenza vaccine in 2010, whereas in 2011, most of the immunized children received the trivalent vaccine (table iv) . binomial analysis of the five selected viruses showed that the presence of underlying asthma/bronchospasm was significantly associated with hrv infection (or, 3.17; 95% ci, 1.86-5.46), while fever and myalgia was less likely to be associated with hrv. on the other hand, the presence of fever was significantly associated with adv and influenza infections (or, 3.86; 95% ci, 1.3-16.5 and or, 3.74; 95% ci, 1.26-16.0, respectively), whereas the presence of underlying conditions did not increase the chance of acquiring these infections. none of the evaluated variables showed significant correlation with the presence of parainfluenza virus and coronavirus infections (table v) . this study showed crv circulation in a population of preschool children in post-pandemic years. human rhinovirus and adenovirus were the main viruses found in the 2 years following the pandemic. the profile of detected viruses was different from that previous reported, and viruses such as rsv were less frequent, while adenoviruses, which are generally associated with severe infections in hospitalized patients, were more frequently detected. this is probably due to differences in age groups, because previous studies with pediatric outpatients under 2 years old showed a pathogen profile similar to that of hospitalized children [milstone et al., 2012; hara et al, 2014; adams et al., 2015] . s cis cek et al. [2015] studied respiratory viruses in adult and pediatric patients in a 12-year follow-up study. similar to our results, in pediatric patients, they found that hrv was detected more frequently in outpatients, and rsv, in inpatients; however, the distribution of the detected viruses was not stratified according to age group. moreover, diagnosis was based on antigen detection and virus isolation, which are less sensitive than the method used in this study. likewise, zimmerman et al. [2015] re-] reported a higher frequency of rsv and influenza a viruses in outpatients, although they were between 6 months and 17 years of age, unlike the group assessed in this study. when comparing the two periods studied, we observed a higher prevalence of viral coinfection in 2011, due to increased virus detection owing to the use of rv 15 multiplex pcr. however, in both years, the coinfection rates were lower than those in various previous reports, wherein they ranged from 31% to 51% [lepiller et al., 2013; luchsinger et al., 2014; turunen et al., 2014] . though, most of these studies were carried out in hospitalized children who generally are younger and presented with their first episodes of viral respiratory infection. the frequency of infection caused by coronaviruses, which are usually associated with common cold, was 9.5%, a rate higher than that previously reported [pilger et al., 2011; hara and takao, 2015] . the molecular method used did not allow genotype differentiation, but in general, patients infected with coronavirus showed favorable outcomes, without reports of any complications. several factors contributed to the low influenza a h1n1pdm09 circulation in 2010 and 2011. first, vaccination coverage rates were high owing to the public immunization campaigns in 2010 and 2011: vaccination coverage in the studied age group in the city of curitiba was almost 100% [brazilian health ministry, 2011] . in addition, because of the magnitude of the 2009 pandemic in southern brazil, the population can be considered sensitized during the study period. second, the early use of oseltamivir might be a contributing factor. third, a high rate of influenza a h1n1pdm09 infection was observed in 2010 [libster et al., 2010] , which might have led to a protective effect in many children during the subsequent season. no gender-specific differences were found in the prevalence of crv infections, which were generally of low severity. the incidence of crvs was higher in children attending school and/or day-care centers; however, there was no significant difference in the amount of time spent in these places in the 2 years. similar to previous reports, in both seasons, the most prevalent underlying conditions associated with crv infection were asthma and wheezing crisis [tregoning and schwarze, 2010; turunen et al., 2014] . x-rays were performed in only 9% of the patients, and perihilar infiltrates were the most common radiologic findings, although none of the patients needed mechanical ventilation or intensive care treatment. one of the main challenges in the care of patients with aris is to determine clinical and demographic factors that can help in the etiological diagnosis of this infection. for this reason, we selected five rvs that are often detected in aris and used logistic regression to evaluate correlations between their prevalence and various parameters. an association was observed between hrv and asthma, and between the presence of fever and hadv and influenza infections. similarly, previous reports have linked hrv to atopic characteristics, and the susceptibility to hrv-induced wheezing has been recognized as an important risk factor for childhood asthma [tregoning and schwarze, 2010; mackay et al., 2013; turunen et al., 2014] . the strongest association was observed between fever and adenovirus and influenza infections, suggesting that screening for influenza by rapid tests would be recommended. as previously reported, children maintain the chain of transmission of influenza since they shed viruses for a more prolonged time and in higher concentrations; thus, control of these infections must be based on disease control in pediatric patients [ploin et al., 2007; d'onise and raupach, 2008; fiore et al., 2009] . this study had some limitations: (i) the difference in the number of viruses detected by the multiplex rt-pcr kits used in the 2 years may have influenced the detected virus profile, although no significant difference was found in the positivity rates; and (ii) the predefined sampling period, made it impossible to assess the seasonality of the infections, although this information is already available for the studied region. despite the advances in the establishment of a wide network of information on the circulation of influenza viruses, little is known about vaccination coverage as well as the impact of crvs in preschool children. our findings provide novel insights into the epidemiology and clinical impact of respiratory viruses on pediatric health and highlight the need for implementation of surveillance programs for respiratory viral infections, seeking to stimulate vaccination and to guide preventive measures to protect this population. bold values, statistical differences. comparison of human metapneumovirus, respiratory syncytial virus and rhinovirus respiratory tract infections in young children admitted to hospital seasonal invasive pneumococcal disease in 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viruses in pediatric outpatients with acute respiratory illness by real-time pcr using nasopharyngeal flocked swabs what is the role of respiratory viruses in community acquired pneumonia; what is the best therapy for influenza and other viral causes of cap? detection of human bocavirus and human metapneumovirus by real-time pcr from patients with respiratory symptoms in southern brazil influenza burden in febrile infants and young children in a pediatric emergency department laboratory diagnosis, epidemiology, and clinical outcomes of pandemic influenza a and community respiratory viral infections in southern brazil respiratory viral infections in infants: causes, clinical, symptoms, virology, and immunology viral respiratory infection in curitiba, southern brazil the first wheezing episode: respiratory virus etiology, atopic characteristics, and illness severity who. cdc protocol of real time rtpcr for influenza a (h1n1) viral infections in outpatients with medically attended acute respiratory illness during the 2012-2013 influenza season key: cord-314600-x8mmuf3y authors: biagi, carlotta; rocca, alessandro; poletti, giulia; fabi, marianna; lanari, marcello title: rhinovirus infection in children with acute bronchiolitis and its impact on recurrent wheezing and asthma development date: 2020-10-21 journal: microorganisms doi: 10.3390/microorganisms8101620 sha: doc_id: 314600 cord_uid: x8mmuf3y acute bronchiolitis represents the leading cause of hospitalization in infants. together with a respiratory syncytial virus, rhinovirus (rv) is one of the most common pathogens associated with bronchiolitis, and its genetic diversity (>150 types) makes the recurrence of rv infections each year quite typical. the frequency of rv infection and co-infection with other viruses and its impact on the clinical course of bronchiolitis have been studied by several authors with controversial results. some studies demonstrate that multiple virus infections result in more severe clinical presentation and a higher risk of complications, whereas other studies suggest no influence on clinical course. moreover, rv bronchiolitis has been reported to potentially contribute to the development of long-term sequelae, such as recurrent wheezing and asthma, in the pediatric population. in the present review, we summarize the most recent findings of the role of rv infection in children with acute bronchiolitis, its impact on subsequent asthma development, and the implication in clinical practice. rhinovirus (rv) is a non-enveloped single-stranded rna virus belonging to the enterovirus genus in the picornaviridae family. it is a highly contagious and ubiquitous virus. its transmission generally occurs through direct exposure to respiratory droplets/micro-droplets, even though it can also take place via contaminated surfaces, including direct person-to-person contact [1] . in temperate climates (i.e., many areas of the usa and europe), rv is responsible for annual outbreaks in the period from early fall to the end of spring [2] .rv has three different subgroups-a, b, and c-which consist of 80, 30, and 56 types, respectively [3] . the genetic diversity of rv (>150 types) makes the recurrence of rv infections each year quite typical and the development of an effective vaccine very difficult [4] . rv represents the main responsible agent of "common colds", mostly characterized by rhinorrhea, sore throat, cough, and diffuse malaise. rv can also cause many other upper and lower respiratory tract infections, such as otitis media, croup, pneumonia, and acute bronchiolitis [5] [6] [7] . acute bronchiolitis is the most frequent lower respiratory tract infection in children, especially in preterm infants, and represents the leading cause of hospitalization in infants, accounting for 18% of all pediatric hospital admissions in the united states [7, 8] . various definitions of bronchiolitis have been proposed [7, 9, 10] . bronchiolitis is defined by the american academy of pediatrics (aap) as a constellation of signs and symptoms, including a viral upper respiratory tract prodrome, followed by increased respiratory effort and wheezing in children under the age of two [9] .in europe, by contrast, the term bronchiolitis is generally referred to as a first episode of acute lower airway infection in infants younger than one year [7] . clinically, it is characterized by few days of rhinorrhea, fever, and cough, which precede the signs of lower respiratory distress associated with wheeze and/or crackles on chest auscultation. most children with bronchiolitis have an uneventful course. hospitalization is required in about 3% of cases, and admission to a pediatric intensive care unit (picu) in approximately 2-6% of the hospitalized cases [11] . according to the national institute for health and clinical excellence (nice) guidelines, indicators for hospital admission are respiratory rate over 60 breaths/minute, marked chest wall retractions, apnea, an oxygen saturation (spo2) lower than 92%, central cyanosis, poor oral fluid intake, inability or indifference to eating due to breathlessness [10] . even if the diagnosis of bronchiolitis is mainly based on history and clinical findings, and the treatment is primarily supportive, the identification of the causative organism should improve the understanding of the disease and open avenues for precision medicine. during the last 20 years, the methodologies for virus detection-immunofluorescence assay, but also molecular investigations, such as polymerase chain reaction-have improved, increasing the knowledge of the viral agents responsible for acute bronchiolitis. these techniques have led to the identification of the main responsible pathogen, respiratory syncytial virus (rsv), accounting for 70-80% of bronchiolitis, followed by rv [12] . approximately 20-40% of children with bronchiolitis seem to be infected by rv [13] . other viruses, such as adenovirus, influenza, parainfluenza, metapneumovirus, human bocavirus, and human coronavirus, are less frequently implicated. up to 30% of hospitalized infants with bronchiolitis have multiple respiratory virus co-infections [14] . some viruses might be detected because of colonization, prolonged viral-shedding post-infection, or incubation before clinical infection. indeed, respiratory viruses have been found in up to 40% of asymptomatic children. according to this finding, the interpretation of multiple coexisting viruses in symptomatic subjects should be interpreted with caution. the frequency of rv infection and co-infection with other viruses and its impact on the clinical course of bronchiolitis have been studied by several authors with conflicting evidence. moreover, rv bronchiolitis has been reported to potentially contribute to the development of long-term sequelae, such as recurrent wheezing and asthma, in childhood. the objective of this review is to provide an overview of the role of rv infection in children with acute bronchiolitis and to depict its potential impact on the clinical course of the illness and the subsequent development of asthma in the pediatric population. many studies have investigated whether the severity of acute bronchiolitis-mainly measured by clinical score indexes (csis), oxygen requirement, ventilatory support, pediatric intensive care unit (picu) admission, and length of hospital stay (los)-is associated with specific viral infections or co-infections, with controversial results. the results of the most important studies analyzing the correlation between the pathogen involved (rv vs. other respiratory viruses) and the severity of bronchiolitis are summarized in table 1 .only the studies published in the last 20 years and enrolling more than 100 children are considered and described below. several studies have investigated the relationship between specific viral pathogens and bronchiolitis csi based on different symptoms and signs, mainly heart and respiratory rate, clinical signs of respiratory distress, the difficulty in feeding, and oxygen saturation [15, 19, 20, 23, 24, 34] . as shown in table 1 , the majority of the studies reported that rv is associated with a milder csi compared to other viral agents [19, 20, 24] . in the single-center prospective study of midulla and colleagues, enrolling 182 infants hospitalized with bronchiolitis, rv appeared to be associated with a lower csi value at hospital admission when compared to other viral agents, firstly rsv (p = 0.05) [19] . similarly, miller and colleagues assessed the relationship between the viral pathogen and the bronchiolitis severity in 455 hospitalized infants over 4 years (2004) (2005) (2006) (2007) (2008) . they found that rv was related to a lower csi when compared to other viral pathogens (p < 0.001) [20] . a similar result emerged when the same authors specifically compared rv and rsv bronchiolitis, concluding that rv was once again associated with lower csi (p = 0.013) [24] . on the contrary, the group of papadopoulos found that rv was associated with the risk of a higher csi at hospital admission when compared to other viral groups in 118 children with bronchiolitis, both at the univariate analysis (p = 0.004) and multivariate analysis (p = 0.022) [15] . corresponding results emerged from another single-center retrospective study analyzing data of 180 infants with bronchiolitis. however, in this study, rv infection was associated with a more severe csi than the other viral types only at the univariate analysis (p = 0.041), while no significant differences emerged at the multivariate logistic regression analysis [23] . the more recent research focusing on this matter was the study retrospectively conducted by petrarca et al. they enrolled 486 patients during 12 consecutive epidemic seasons without finding significant differences in csi between rv and rsv-bronchiolitis [34] . the great heterogeneity of the reported results can be justified, at least in part, by the adoption of different csis that do not always consider the same parameters and whose point's range significantly differ from each other. moreover, many of these scores have not been formally validated, limiting their role in the assessment of the severity of bronchiolitis. therefore, many authors have considered other more objective criteria for the evaluation of bronchiolitis severity, mainly represented by oxygen therapy, ventilatory support, los, and picu admission. regarding oxygen therapy, rv infection seems to be associated with lower oxygen requirement and duration. in a cohort of 455 infants with acute bronchiolitis described by miller et al., rv infection was associated with a lower frequency of supplemental oxygen requirement than infants with other viral pathogens (p < 0.001) [20] . moreover, marguet and colleagues found that rvinfection decreased the risk of oxygen requirement compared to rsv-infection (or 0.29, 95% ci 0.09-0.90 at the logistic regression) in a prospective multicenter study enrolling 209 infants [17] . nevertheless, no significant differences emerged in the more recent and larger (486 cases), albeit retrospective, research of petrarca et al. [34] . similarly, no differences among viral pathogens emerged concerning ventilatory support, intended as continuous positive airway pressure (cpap) and/or intubation requirement during the hospitalization [22, 29, 37] . these data resulted from the multicenter airway research collaboration (marc), a program of the emergency medicine network, which was responsible for the enrollment of the largest cohorts of hospitalized infants with bronchiolitis, prospectively studied in multiple sites in the usa. in 2012, mansbach et al. analyzed data obtained from the enrollment of 2207 patients during three winter seasons. they found a viral pathogen in 2068 of cases and compared single-rv to single-rsv infection, as well as co-infections, without discovering significant differences in terms of cpap/intubation requirement [22] . in more recent years, a similar study was performed, also in finland. in 2015, jarttiet al. collected data from both the enrolled american and finnish cohorts, amounting to 2615 children <2 years of age, of whom 694 documented rv infection. to the best of our knowledge, this study was the only one to investigate the relationship between rv viral load and severity of the disease, and no significant differences emerged in cpap or intubation requirement [29] . similarly, hasegawa et al. compared enrolled american (1016 patients, of which 197 were rv-infected) and finnish (408 patients, of which 109 were rv-infected) cohorts of infants. here, they specifically identified also the rv-a, rv-b, and rv-c types responsible for the collected bronchiolitis (47%, 6%, and 47% in the american group and 23%, 3%, and 74% in the finnish group, respectively). no significant differences in terms of cpap and/or intubation requirement emerged between the three different rv types in both analyzed cohorts [37] . finally, also in a prospective multicenter study focusing on 363 hospitalized infants with moderate-severe bronchiolitis, no significant differences appeared between rv infection-even when they considered viral types-and other single virus-infections regarding either oxygen requirement, ventilation need, or nasogastric feeding [31] . furthermore, many studies have analyzed the hospital los as an index of bronchiolitis severity. only one study has reported a longer los in rv bronchiolitis compared to rsv bronchiolitis: paul and colleagues enrolled a cohort of 319 hospitalized children for acute bronchiolitis, focusing on single rv or single rsv infections (65 cases for the former, 162 cases for the latter). analyzing the mean los as clinical outcome emerged a greater los in the rv group compared with the rsv group (p = 0.032) [32] . more studies have observed instead a correlation between rv infection and a lower los when this virus is compared to other viral pathogens, especially rsv [17, 19, 26, 36] . in the prospective single-center study conducted in 2010 (182 infants), midulla et al. found that rv infection had a lower mean los than rsv-infection (p = 0.05) [19] . jartti and colleagues came to comparable results on a cohort of 408 children comparing rv infections to rsv infections in terms of los shorter or longer than three days (p = 0.03) [26] . similarly, marguet et al. studied 209 infants considering the duration of los > 5 days as an outcome, finding that rv infection decreased the risk of prolonged los compared to other viral infections, including rsv-bronchiolitis (or 0.11, 95% ci 0.03-0.37) [17] . finally, in 2019, bergroth and colleagues analyzed once again a finnish cohort obtained from the marc protocol study, specifically focusing on the three rv types. they demonstrated a correlation between rv-a and rv-c (but not rv-b) infection and a lower los when compared to rsv-infection (p = 0.003). this result appeared limited by the very low frequency of detected rv-b infection (3%) [36] . on the contrary, many other studies have not found significant differences in terms of los between rv bronchiolitis and other viral group-infection [18, 20, 25, 28, 30, 31, 33, 34] . analyzing the rate of admission to picu in bronchiolitis, bergroth et al. found that none of the 101 rv-infected patients of their cohort needed intensive care compared to 9% of the 145 rsv-bronchiolitis (p = 0.02) [36] . nevertheless, other studies have reported no significant differences in terms of admission to picu between the different types of viral infections [27, 34, 35] . in 2018, praznik and colleagues conducted a retrospective single-center study analyzing the site management of 761 children with bronchiolitis (138 treated as outpatients, 599 treated in the standard hospital setting, 24 admitted to picu). besides, in this study, no differences regarding the causative viruses were found [35] . moreover, data from the previously cited largest american and finnish cohorts studies in the context of marc's databases-focusing on genomic rv load and rv types-did not find significant differences in terms of picu admission [29, 37] . when taken all into account, these findings seem to support the milder severity course of rv bronchiolitis in comparison with the illness of other viral pathogens, especially rsv. however, it is important to consider the heterogeneity of the reported studies-from the study design (retrospective vs. prospective, case-control. or cross-sectional) to the cohort's inclusion criteria (such as age, <1 year vs. <2 years)-which may limit the relevance of these results. in particular, the age could represent an important factor, influencing rv and rsv severity. indeed, rsv dominates in young infants, whereas the prevalence of rv bronchiolitis increases steadily with age [18] [19] [20] 24, 26, 31, 36] . regarding the studies enrolling children under the age of two, three of them have conducted a parallel analysis using a stricter definition for bronchiolitis (age <12 months) [26, 29, 36] . no differences are seen in this subset of patients, with the exception of the study of jartti and colleagues [26] . in this study, rv etiology was associated with shorter hospital los compared with rsv in all children with bronchiolitis, but this finding lost its statistical significance in the subset aged <12 months. according to the studies reported in table 1 , the frequency of viral co-infections in bronchiolitis varies from 11% to 67% [30, 31] . specifically, the reported frequency of rv + rsv co-infection varies from 2.6% to 62.5% [15, 36] , while the frequencies of rv + non-rsv viral pathogens co-infections range from 5% to 20% [22, 29] . although it may be intuitive to think that multiple viral bronchiolitis tends to be more severe than single viral illness, only the retrospective study of richard and colleagues in 2008 found data supporting this hypothesis. in this study, children with viral co-infection (44 patients, 13 of which with rv + rsv bronchiolitis) were associated with a higher risk of admission to picu at the multivariate analysis (p = 0.02). moreover, the study observed that los tended to be prolonged in co-infections, but no statistically significant differences were found [16] . surprisingly, in a prospective single-center study enrolling 142 hospitalized children with bronchiolitis, co-infections were more frequently associated with mild (no hypoxia or feeding problems) and moderate (oxygen requirement, feeding problems) illness than to severe disease (mechanical ventilation requirement) (p = 0.003). in this study, infants with sole rsv infection had the most severe diseases. as suggested by the authors, the young age of these infants might have represented a bias in the results since a young age is a well-known risk factor for severe rsv-bronchiolitis, more important than multiple virus infections [21] . mansbachet al. described data on 2207 children with bronchiolitis, of which 658 were with multiple infections. they compared rv single infection with rsv single infection, rv + rsv co-infection, rv + non-rsv co-infection, and rsv + non-rv co-infection. this study did not find any significant differences in terms of ventilatory support (intended as cpap and/or intubation requirement) and admission to picu but reported the association between rv + rsv co-infection and longer los (p = 0.04). regarding this peculiar result, the authors suggested that it could be determined by the specific inflammatory property of rsv, which tends to reduce interferon (ifn)-î³ response during infection, possibly allowing an enhancement of rv replication. another suggested hypothesis is that rsv-infected endothelial cells increase the intercellular adhesion molecule-1 (icam-1) expression, the major receptor for rv, setting the stage for a more severe rv infection [22] . however, many other studies that have compared rv bronchiolitis with rsv + rv co-infection or other co-infections have not found any statistically significant difference in terms of csi [34] , respiratory support [31, 34] , admission to picu [27, 34] , and los [18, 25, 30, 34] . the great heterogeneity of these results may be due to the differences between the described researches, such as study design, sample size, and considered outcomes, but also may be partially related to the fact that rv is a very ubiquitous virus. its prevalence among asymptomatic children has been reported up to 40% [38] , and its detection in nasal specimens, together with other viruses during acute bronchiolitis, may lead to giving rv a causative role that it possibly does not always have [39] . factors that may play a role in the clinical impact of bronchiolitis are the viral load and the type of rv responsible for the infection. to the best of our knowledge, only two studies have reported up to now data about the correlation between rv viral load and bronchiolitis severity, without finding any statistically significant result [29, 31] . surely, future studies on this topic could add new information about this relevant matter. regarding the different rv types, few studies have focused on their clinical impact, probably because of the recent introduction of molecular detection methods. infection with rv-c has been associated with more-severe lower respiratory tract illness in the pediatric population compared to rv-a [40, 41] ; however, this finding has not been consistent across studies [38, 42, 43] . regarding acute bronchiolitis, rv-c seems clinically similar to rv-a and rv-b [28, 31, 37] . in 2014, selvaggiet al. genotyped 40 rv-infected patients, discovering a frequency of 45% of rv-a subtype and 55% of rv-c type [28] . skjerven and colleagues in 2016 similarly reported a higher prevalence of rv-c type in their 122 rv-infected patients (71.3% vs. 28.7% of rv-a/b types) [31] . in both studies, no significant differences in terms of hospital los and the level of supportive care emerged among the viral types. similarly, hasegawa et al. genotyped both the american and finnish cohorts without discovering significant differences in terms of los, picu admission, and cpap/intubation requirement between the three different rv types [37] . bergroth and colleagues genotyped their 101 rv patients and specifically compared them with rsv-bronchiolitis. they found that rv-a and rv-c infections (but not rv-b infection) were associated with lower los than rsv-infection (p = 0.003) [36] . nevertheless, the extremely low frequency of detected rv-b type (only three cases) prevents to draw conclusions about the role of this viral species on this matter. some viral agents, such as rsv, have been associated with the development of asthma in children [44] . in literature, rv bronchiolitis appears more clearly to potentially contribute to the development of recurrent wheezing and asthma in childhood. the results of the most important studies analyzing the correlation between rv infection and preschool wheezing or asthma are summarized in table 2 and described below. only researches with clinical outcomes are taken into account. supposing the role of viral pathogens in asthma development, in 2011, koponenet al. followed-up a cohort of 205 infants hospitalized for bronchiolitis at <6 months of age. here, 81% of these patients received a control/telephone interview at 5-6 years of age, focusing on the presence of current asthma during the preceding year [45] . current asthma was defined in case of a continuous maintenance medication for asthma, doctor-diagnosed wheezing, or prolonged (at least 4 weeks) cough apart from infection. the viral etiology of bronchiolitis was demonstrated in 97% of cases: rsv 70.5%, rv 12.7%, others 16.9%. at the adjusted multivariate analysis, non-rsv bronchiolitis resulted as an independent risk factor for preschool asthma (p = 0.01). although this study did not find a direct link between rv and asthma, the strong correlation between previous non-rsv bronchiolitis and the risk to the development of asthma led to focus the attention on rv, the second viral pathogen responsible for bronchiolitis [45] . in 2014, midullaet al. published data on a cohort of 313 infants hospitalized for bronchiolitis who underwent yearly follow-up up to three years from the time of discharge. they examined the correlation between viral pathogens (rsv in 73.9% of cases, rv in 14.9% of cases, other viral agents in 21.6% of cases) and the recurrence of wheezing. they observed that rv was the only virus associated with recurrent wheezing (defined as â�¥2 episodes in a year for three years) at multivariate analysis (p = 0.03). moreover, they confirmed the lack of association between rsv bronchiolitis and wheezing [46] . on the contrary, the study of amat and colleagues [47] on a cohort of 154 hospitalized infants for bronchiolitis did not find any difference between the kind of respiratory virus (rsv in 76% of cases, rv in 28.6%, other viruses in 70.1% of cases) and the subsequent development of recurrent bronchial obstruction (intended as â�¥3 respiratory symptoms documented in â�¥2 times). however, the same study demonstrated that rv + rsv co-infected bronchiolitis was associated with the risk of sensitization to aeroallergens at three years at the multivariate analysis (p = 0.02) [47] . once again, the cohorts enrolled in the context of marc's studies have given results also in terms of the correlation between rv bronchiolitis and the development of asthma [36, 49] . in 2019, bergroth and colleagues analyzed the use of asthma control medication four years after hospitalization for bronchiolitis in 349 children. they found that rv infection compared to rsv infection was associated with higher use of asthma control medication in the last year (p < 0.001). moreover, they genotyped rv, finding rv-a type in 24% of rv cases, rv-b in 3% of cases, and rv-c in 73%. at the multivariable analysis, rv-c was specifically associated with a higher risk for the use of asthma control medication four years after severe bronchiolitis (p < 0.001), more than rv-a type (p = 0.03). the authors supposed that specific rv-c risk genes, such as cadherin-related family members 3, could predispose to the development of asthma after infection with this rv type [36] . in 2020, mansbach and colleagues analyzed the american marc's cohort of 673 hospitalized infants for bronchiolitis, attending two years of follow-up after discharge. collecting nasopharyngeal aspiration three weeks after the discharge, they focused their attention on the hypothesis that a delayed viral clearance or a sequential infection may be related to the presence of recurrent wheezing by the age of three. recurrent wheezing was defined as having â�¥2 corticosteroid-requiring exacerbations in 6 months or â�¥4 wheezing episodes in a year that last â�¥1 day and affect sleep. they found that infants with rv bronchiolitis at hospitalization, followed by a new rv infection, had the highest risk of recurrent wheezing (p = 0.01). this result was limited by the small number of considered patients (eight infants); however, the authors suggested that it could indicate a specific role by subsequent several rv genotype infections in the genesis of wheezing [49] . finally, hunderi and colleagues conducted a prospective multicenter study enrolling 294 hospitalized children with moderate-severe bronchiolitis [48] . recurrent wheeze was defined as â�¥3 episodes of wheezing, including the bronchiolitis at the enrollment. the authors found that recurrent wheezing at the age of two years was not significantly associated with rv or rsv infection, with the rv type or with the viral load during acute bronchiolitis. these results contrasted with previous studies and did not support the hypothesis that rv infection in susceptible infants may predispose to the development of wheezing [48] . however, a longer follow-up period could have provided further insight into the role of rv infection in early sensitization and asthma development. unlike other respiratory viruses, such as rsv, rv does not have a cytopathic effect and is not able to cause direct airway epithelial cell destruction. however, it alters the epithelial barrier's function, dissociating the zonula occludens-1 from tight junction complex through the release of reactive oxygen species during viral replication [50] . this alteration can allow the absorption of higher amounts of aeroallergens, strictly related to the development of wheezing and asthma [39] . moreover, different rv types use several specific vehicles to enter into airway epithelium: rv-a and -b types use icam-1 or the low-density lipoprotein receptor (ldl-r), while recently, the cadherin-related family member 3 has been identified as a receptor for rv-c [36, 39] . these different ways of access may partially justify the recurrent finding that rv-a and rv-c are more often associated with wheezing in asthma exacerbations [39] . another peculiar aspect concerns the genetic variations at locus 17q21, which are related to the risk of asthma. indeed, early-life respiratory wheezing illnesses are a stronger risk factor for asthma in children with 17q21 locus risk variants than in children without it, and this is more evident for episodes triggered by rv than rsv [36, 51] . some immunologic factors of the innate and adaptive immune response result typical of rv infection and may contribute to the development of wheezing or asthma. recent studies have observed that a th-2-mediated immune response is more frequent after rv infection. key factors in this immunologic pattern are interleukin (il)-4, il-5, il-10, il-13, il-25, il-33, and thymic stromal lymphopoietin (tslp). all these mediators are now well known for taking part in the remodeling of the airway's activation and subsequent wheezing development [39] . moreover, there are shreds of evidence about the fact that through th2 cells activation, previous rv infection results are associated with the development of the so-called "indicators of atopy": eosinophilia and allergen specific ige [52] . rv bronchiolitis has been found statistically associated with eosinophilia, especially with a blood eosinophil count >400/mm 3 [34, 46] , more frequently than non-rv bronchiolitis [19, 28, 30] . moreover, as mentioned earlier, amat and colleagues in 2018 found a correlation between co-infected rv + rsv bronchiolitis and the increased risk of sensitization to aeroallergens at three years at multivariate analysis [47] . the other peculiar rv-related immunological aspects are represented by the low ifn responses, especially ifn-î³. low ifn responses in early life increase the likelihood of respiratory illnesses, including those associated with wheezing. moreover, studies on airway epithelial cells cultured from patients with asthma have observed a diminished production of ifn-î², ifn-î³, and ifn-î», which facilitates rv replication during infection. reduced ifn-î³ responses in infancy are also observed in "atopic children", and this fact may contribute to explain why atopy is a risk factor for virus-induced wheezing [52] . other studies have also identified differences in ifn-î» 1-3 levels in infants with rv or rsv bronchiolitis that may explain different clinical courses [28] . however, exposure to rv does not lead to asthma in all children, suggesting that personal risk factors (genetic, allergy, and antiviral immunity) and environmental exposure (farm, urban, microbes, and nutrition) also play a role. it remains to be elucidated whether rv bronchiolitis contributes to asthma development or is a marker of asthma susceptibility. in this sense, rv may be a revealing factor for those with early airway inflammation (i.e., epithelial barrier dysfunction, th2 polarized inflammation), low ifn responses (i.e., impaired viral defense), and/or genetic variations (i.e., virus-specific risk genes, single nucleotide polymorphisms), acting as a clinically useful risk marker of asthma [53] . several studies have investigated the link between rv bronchiolitis and short-and long-term outcomes, such as the future risk of subsequent asthma. regarding the studies focused on the course of bronchiolitis, many of them have supported that rv is associated with milder disease severity in comparison with other viral pathogens, especially rsv. on the contrary, in the majority of cases, the comparison between sole rv bronchiolitis and multiple viral co-infections does not suggest any significant difference concerning the severity of illness. however, not all research studies are consistent with these results. moreover, the great heterogeneity of the investigations-from study design to analyzed outcome (csi, ventilatory support, picu admission, and los)-implies a certain obstacle to any interpretation of the literature data. comprehensively, the available data are insufficient to draw conclusions in this regard. thus, to date, the detection of the responsible virus pathogen does not seem to significantly impact the prognosis of bronchiolitis. future studies should focus on rv types and viral load, which may play a role in the clinical course of bronchiolitis. on the contrary, regarding long-term clinical outcomes, many studies accordingly have reported a strong association between rv bronchiolitis and the development of preschool wheezing and asthma. if confirmed in future trials, these findings may challenge the notion that the viral etiology of bronchiolitis does not affect clinical outcomes and support further research to guide therapeutic strategies to prevent the development of asthma. author contributions: c.b. and a.r. contributed to the manuscript's concept. c.b., a.r., g.p., and m.f. contributed to the update of the literature review and to the writing and drafting of the article. m.l. revised the entire manuscript. all authors agree to be personally accountable for the author's own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and documented in the literature. all authors have read and agreed to the published version of the manuscript. the abcs of rhinoviruses, wheezing, and asthma clinical and molecular epidemiology of human rhinovirus c in children and adults in hong kong reveals a possible distinct human rhinovirus c subgroup human rhinoviruses rhinovirus-from bench to bedside uncommon (ly considered) manifestations of infection with rhinovirus, agent of the common cold viral bronchiolitis in children risk factors for bronchiolitis hospitalization during the first year of life in a multicenter italian birth cohort clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis nice clinical guideline: bronchiolitis in children: table 1 trends in bronchiolitis hospitalizations in the united states respiratory syncytial virus: the influence of serotype and genotype variability on clinical course of infection role of respiratory 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cord-314390-q36ye9ff authors: kang, gagandeep title: viral diarrhea date: 2016-10-24 journal: international encyclopedia of public health doi: 10.1016/b978-0-12-803678-5.00486-0 sha: doc_id: 314390 cord_uid: q36ye9ff viral gastroenteritis is among the most common illnesses affecting humans and has greatest impact at the extremes of age. the spectrum of disease can range from asymptomatic infections to severe disease with dehydration. in contrast to bacterial pathogens, enteric viruses cannot multiply outside their host; hence, the original inoculum into the common source determines infectivity. prevention of contamination of food and water control primary cases, whereas careful nursing and handwashing prevent secondary cases. effective vaccines are available and widely used to prevent rotaviral gastroenteritis, but vaccines for other causes of viral gastroenteritis are not yet available. acute gastroenteritis is among the most common illnesses affecting humans and has greatest impact at the extremes of age, severely affecting children and the elderly. the spectrum of disease can range from asymptomatic infections to severe disease with dehydration, which can be fatal. diarrheal disease continues to be a major cause of mortality in young children, particularly in developing countries. prior to 1972, the etiology of most episodes of gastroenteritis was unknown, and cases were attributed to a multitude of causes, including teething, weaning, diet, old age, drugs and malnutrition, as well as infections. intensive investigation of enteric infections in the past three decades has resulted in the discovery of many new viral agents filling in the 'diagnostic gap' in diarrheal disease. with the identification of the norwalk virus, rotavirus, astroviruses, enteric adenoviruses and other caliciviruses in the 1970s and subsequently, it has become increasingly clear that viruses cause a significant proportion of the enteric illnesses that did not earlier have a defined etiology. with improvements in sanitation and hygiene, and better standards of living, the proportion of diarrheal disease attributed to bacteria has decreased, resulting in an increase in the proportion of cases associated with viral infections. developments of new assays to identify viruses have also resulted in the ability to identify the viral etiology of episodes and epidemics of gastroenteritis (desselberger and gray, 2003) . approximately, 5 billion episodes of diarrhea occur worldwide annually, with virtually all children infected with the most common agents by the age of 3 years. widespread use of oral rehydration therapy in the past two decades has resulted in a significant decrease in mortality due to diarrhea, by about 70% to approximately 1.4 million annual deaths, occurring mainly in developing countries (lozano et al., 2012) . infections with gastroenteritis viruses differ from bacterial enteric infections in that they affect children in both developing and developed countries, suggesting that they may also be transmitted by means unrelated to contaminated food or water (cheng et al., 2005) . although feco-oral spread is the major route of transmission for all enteric viruses, transmission through contact, fomites and a respiratory route is likely based on epidemiology and the recovery of these viruses from inanimate objects during outbreaks. the four distinct patterns of viral gastroenteritis, endemic childhood diarrhea, outbreaks in closed communities, other food or waterborne outbreaks among wider communities and viral gastroenteritis in immunocompromised patients, reflect the differences in the pathogens, transmission and host response (glass et al., 2001) . these have a direct bearing on strategies for prevention and control ( table 1) . the highest rates of viral gastroenteritis occur between 3 and 24 months of age. protection in early infancy is believed to be mediated by maternal antibodies, followed by acquisition of protective immunity through repeated exposure in early childhood. this pattern is seen with all viral enteropathogens. however, for the noroviruses and astroviruses, immunity is not long-lasting, suggesting waning of immunity or lack of cross-protection between different viral strains. childhood diarrhea is best exemplified by the group a rotaviruses, but a similar pattern of infection and illness is seen with enteric adenoviruses, astroviruses and sapoviruses. these agents infect children during the first few years of life, with first infections being symptomatic and protecting against subsequent disease. disease is caused by a limited number of specific serotypes and incidence decreases with increasing age. group a rotaviruses are the main cause of severe diarrhea in children under 5 years of age, and resulted in an estimated 450 000 deaths in 2008 (tate et al., 2012) . prior to vaccine introduction, reports from europe, australia, and usa indicate that rotavirus was responsible for 20-60% of cases of gastroenteritis requiring hospitalization. in other parts of the world, approximately 40-45% of gastroenteritis requiring admission is due to rotavirus. of the 'non-group a' rotaviruses, group b rotavirus has been identified in epidemic outbreaks of severe diarrhea in adults in china and in symptomatic infections in children. outbreaks of diarrhea due to group c rotavirus have been identified in asia, europe, south, and north america, but are not common. human noroviruses are associated mainly with milder cases of gastroenteritis in children, causing >20% of diarrheal disease in children in the community. after introduction of rotavirus vaccines, noroviruses have become the most common cause of gastroenteritis in children (koo et al., 2013) . enteric adenoviruses cause 10% of diarrheal disease in reports from developed countries and have a variable incidence of 2-30% depending on the region in developing countries. astroviruses were found in approximately 1% of cases, when electron microscopy was employed for detection, but with the availability of a commercial enzyme immunoassay and molecular techniques, the percentage of detection has increased in hospital and community settings to 5-10%, and several new types of astroviruses have recently been described. outbreaks in closed or semi-closed communities such as old-age homes, cruise ships and hospitals are mainly due to noroviruses. norovirus infections are a significant cause of outbreaks in adults in nursing homes and residential care facilities and can lead to an increased need for hospital care and increased mortality. nosocomial outbreaks occurring in hospitals have required the closure of wards in order to control infections. outbreaks due to noroviruses and due to mixed viral infections have been reported in military personnel and on cruise ships. attack rates as high as 30% have been observed among cruise ship passengers, and repeated outbreaks have continued even after cleaning and disinfection protocols were instituted on successive voyages (ahmed et al., 2014) . in addition to infections in adult patients, viral agents of gastroenteritis are an important cause of nosocomial infection in pediatric units. between 20% and 50% of cases of gastroenteritis caused by rotavirus in hospitals are considered to be of nosocomial origin, and nosocomial viral enteric infections have been documented in up to 6% of children admitted for >72 h in both developed and developing countries. infections in older individuals are usually due to noroviruses, although adenovirus infections have been documented. asymptomatic patients who excrete the virus and the relative resistance of these viruses to normal disinfectants may explain the prevalence and transmission of nosocomial infection. the microbiological contamination of food and water is a significant global problem. it is estimated that there are approximately 1.5 billion cases and over 3 million deaths worldwide annually (duizer and koopmans, 2004) . the microorganisms associated with about 50% of the foodborne disease outbreaks still go unrecognized, particularly those occurring in developing countries. the apparent failure to confirm a viral etiology in such outbreaks has been due largely to the lack of available tests, unavailability of food or water specimens and the failure to report outbreaks of mild gastrointestinal disease. all of these factors have resulted in a drastic underestimate of the true scope and importance of food-or waterborne viral infection. the most common types of food-and waterborne viral disease are infectious hepatitis due to hepatitis a virus and acute viral gastroenteritis associated with the human noroviruses. noroviruses, transmitted by the fecal-oral and the aerosol route, are the most common cause of outbreaks of nonbacterial gastroenteritis in industrialized countries, and emerging data indicates that they are also prevalent in developing countries. many outbreaks can be associated with the consumption of primarily or secondarily contaminated foods. shellfish and fruit implicated in outbreaks have been shown to be contaminated at the site where these foods are harvested or produced, whereas other foods, such as salads, cold foods, and sandwiches, have caused outbreaks after being contaminated by food handlers at the site of food preparation. shellfish, in particular, oysters and clams, which are raw or insufficiently cooked, are associated with noroviral outbreaks, frequently occurring because these shellfish filter contaminated seawater to feed and hence result in a concentration of virus. foodborne outbreaks due to rotaviruses, parvoviruses, and astroviruses are also occasionally reported. water is also a common source of outbreaks and may include water from municipal supplies, wells, recreational lakes, swimming pools, and ice machines. rotaviruses, noroviruses and some adenoviruses are important causes of waterborne disease outbreaks. post-recovery and secondary transmission is a particular concern in infections due to these agents (kapikian, 1994) . the main viral causes of severe gastroenteritis in immunosuppressed patients are cytomegalovirus (cmv) and epstein-barr virus (ebv), which mainly affect patients with aids and transplant recipients. cmv is a frequent pathogen in diarrhea associated with aids with cd4 counts <100 cells mm à3 . other viruses that produce hiv-associated gastroenteritis include astrovirus, picobirnavirus, calicivirus and adenovirus. there is evidence of gastroenteritis due to astrovirus and adenovirus in both child and adult bone marrow transplant recipients. noroviruses are now being increasingly recognized as a cause of chronic diarrhea in patients undergoing transplants. toroviruses have been found in association with diarrhea in immunocompromised children (krones and hogenauer, 2012) . criteria to define a virus as an etiologic agent of gastroenteritis include (1) the identification of the virus more frequently in subjects with diarrhea than in controls, (2) the demonstration of an immune response to the specific virus, and (3) demonstration that the beginning and end of the illness correspond to the onset and termination of virus shedding, respectively (glass et al., 2001) . so far, these include human caliciviruses, which are noroviruses and sapoviruses, rotaviruses, astroviruses and the enteric adenoviruses. in immunocompromised patients, gastrointestinal cmv and ebv infections also cause significant morbidity. coronaviruses, including the sars coronavirus, toroviruses, the aichivirus, certain non-polio enteroviruses and picobirnaviruses have also been found to be associated with diarrhea. similarly, in conditions such as hiv, it has been difficult to obtain definitive data on the role of enteric viruses in the causation of symptoms (thomas et al., 1999) . the study of rotaviruses, enteric adenoviruses, and astroviruses has been facilitated greatly by the ability to propagate these viruses in cell culture, which has allowed the production of reagents for use in diagnostic studies, a better understanding of factors correlated with immunity to infection, and the elucidation of each virus's life cycle. although human noroviruses have defied numerous attempts to propagate them in cell culture to date, the ability to culture murine norovirus has informed pathogenesis and the use of molecular tools has increased our ability to diagnose and study norovirus infections ( figure 1 ). rotaviruses are double stranded rna viruses comprising a genus within the family reoviridae. the mature virus particles are triple layered, approximately 70 nm in diameter, and possess icosahedral symmetry. the rotavirus genome consists of 11 segments of double-stranded rna that code for 6 structural viral proteins and 6 non-structural proteins. of the non-structural proteins, nsp4 is of particular interest, since it has enterotoxin-like activity and can induce diarrhea in mice. the classification of rotavirus into 7 different groups (a-g) is based on the antigenic specificity of the vp6 capsid proteins. of the 7 groups, only groups a, b and c are known to infect humans. severe, life-threatening disease in children worldwide is caused predominantly by group a rotaviruses. variability in the genes encoding the two outer capsid proteins vp7 and vp4 form the basis of the current strain typing of group a rotaviruses into g and p genotypes respectively. all known g serotypes correspond with genotypes; more p genotypes than serotypes have been identified. at least 15 g genotypes and >30 p genotypes have been identified to date. the strains most commonly reported include g1p , and more recently g12 strains in combination with different p types. unusual g and p types have been reported from different parts of the world. the temporal changes in circulating strains and the rapid evolution of rotaviruses by a variety of mechanisms provide challenges in epidemiological studies. these mechanisms of evolution include genetic drift, wherein accumulation of point mutations generates genetic lineages leading to the emergence of antibody escape mutants, and genetic shift through gene reassortment during dual infection of a single cell. hence methods of virus typing need to be regularly monitored and updated to identify emerging novel strains of epidemiological importance (parashar et al., 2013) . rotaviruses induce a clinical illness characterized by vomiting, diarrhea, abdominal discomfort, fever, and dehydration (or a combination of some of these symptoms) that occurs primarily in infants and young children and may lead to hospitalization for rehydration therapy. fever and vomiting frequently precede the onset of diarrhea. milder gastroenteric illnesses that do not require hospitalization are common. the highest attack rate is usually among infants and young children 6-24 months old. neonatal infections are largely asymptomatic. deaths from rotavirus gastroenteritis may occur from dehydration and electrolyte imbalance. the severity of diarrhea is measured by the vesikari score that includes duration and severity of diarrhea and vomiting, associated fever and degree of dehydration. in older children and adults, rotavirus gastroenteritis occurs infrequently, although subclinical infections are common. rotaviruses also induce chronic symptomatic diarrhea in immunodeficient children. rotavirus infections can be severe and sometimes fatal in individuals of any age who are immunosuppressed for bone marrow transplantation. rotavirus infections have also been associated with necrotizing enterocolitis and hemorrhagic gastroenteritis in neonates in special-care units. rotavirus antigenemia has been described early in infection in children requiring hospitalization, and rna has been extracted from serum of antigenemic children and csf of children with seizures, but the clinical significance of these findings require further investigation. rotaviruses infect the mature enterocytes on the tips of small intestinal villi, leading to villous atrophy with secondary hyperplasia of the crypts. it has been proposed that cellular damage is secondary to villus ischemia. the mechanism that induces the production of diarrhea is not well understood, although it appears to be mediated by the relative decrease of villous epithelium absorption in relation to the secretory capacity of the crypt cells, as well as the action of nsp4, the viral enterotoxin that has been shown to cause secretory diarrhea in rodents. there is a loss of intestinal permeability to macromolecules such as lactose, secondary to a decrease in disaccharidase in the intestine. the enteric nervous system is stimulated by this virus, leading to intestinal water and electrolyte secretion (parashar et al., 2013) . recent studies indicate that lack of specific carbohydrates in the enteric mucosa may influence the ability of rotaviruses to infect and cause disease (lependu et al., 2014) , but the immunologic mechanisms responsible for protection against infection by rotavirus are still not well known. older children and adults usually have asymptomatic or mild infection unless an overwhelming infectious dose is delivered. several studies have shown that local intestinal immunity produced in response to infection protects against subsequent severe episodes of diarrhea. it was believed that the antibody response in primary infection was homotypic with subsequent infections producing a broadening of the immune response, but the basis of strain-specific immunity is less clear now that it has been shown that two doses of a monovalent vaccine can present infection with multiple strains as well as a multivalent vaccine (parashar et al., 2013) . laboratory procedures for diagnosis of rotavirus include electron microscopy (em), passive latex agglutination assays (la), electropherotyping using polyacylamide gel electrophoresis (page), enzyme-linked immunosorbent assays (elisa) and reverse transcriptionpolymerase chain reaction (rt-pcr). in recent years, elisa has become the method of choice for screening. early studies on strain surveillance identified rotavirus serotypes using neutralization assays. monoclonal antibodies to specific serotypes were used. new methods have greatly improved data on circulating rotavirus strains and include multiplex rt-pcr based genotyping based on vp7 and vp4 genes, nucleotide sequencing for specific genes and whole genome sequencing. the name calicivirus is derived from the latin calyx, meaning cup or goblet, and refers to the cup-shaped depressions visible by electron microscopy. these cup-like depressions are more prominent in some strains, particularly the sapoviruses, leading to the characteristic star of david appearance from which caliciviruses get their name. caliciviruses (family caliciviridae) are a group of nonenveloped, icosahedral viruses with a single-stranded, positive sense rna genome. the genome is 7.5-7.7 kb in length and has three open reading frames (orfs). noroviruses can be genetically classified into six different genogroups (gi-gvi) which can be further divided into different genetic groups or genotypes. genogroups i, ii and iv infect humans, while genogroup iii is associated with bovine infections and genogroup v has been isolated in mice. noroviruses were initially named after the places where the outbreaks occurred, but a numeric classification system based upon numbering genogroups with roman numerals and genotypes with numbers is now used. for example the genogroup ii norovirus, lordsdale virus is a member of genotype 4, and therefore classified as a gii.4 norovirus. gii.4 viruses account for the majority of adult outbreaks of gastroenteritis and often sweep across the globe, with the sydney 2012 virus the most recent gii.4 to cause multi-country outbreaks (ramani et al., 2014) . sapoviruses, previously called the classical caliciviruses based on their morphology, have a similar system of strain designation as noroviruses and have 8 human genotypes in 5 genogroups, although up to 14 genogroups have been proposed with the inclusion of sapoviruses from other hosts (scheuer et al., 2013) . the incubation period for caliciviral infections is short, about 24-48 h, and the mean duration of illness is 12-60 h. nausea is prominent, with vomiting, non-bloody diarrhea, and abdominal cramps occurring in most cases. all age groups experience these symptoms, but diarrhea is relatively more prevalent among adults, whereas a higher proportion of children experience vomiting. from 25-50% of affected persons also report headache, fever, chills, and myalgias. adults have died during illness caused by noroviruses, presumably from electrolyte imbalance. late sequelae have not been reported, but the elderly often report persistence of constitutional symptoms for up to several weeks. routes of transmission that have been documented include water, food (particularly shellfish and salads), aerosol, fomites, and person-to-person contact. infectivity can last for as long as 4 days after resolution of symptoms. presymptomatic shedding has been suspected on epidemiologic grounds but not proven in volunteer studies. human noroviruses have not been grown in culture, making studies of pathogenetic mechanisms difficult. in studies carried out on volunteers, infection by calicivirus produces an expansion of the villi of the proximal small intestine. the epithelial cells remain intact with a shortening of the microvilli. the mechanism by which diarrhea is produced is unknown. in volunteer studies, infection by the norwalk virus induces a specific igg, iga and igm serum antibody response, even in persons with pre-existing antibodies. after norovirus infection, immunity appears to last for a few months but there is little or no evidence of long-term protection. volunteer studies conducted in the 1970s also suggested that some people were resistant to norwalk virus challenge. human blood group antigens (hbgas) have been shown to serve as cell attachment factors for noroviruses and thus determine susceptibility to certain human noroviruses. reduced infection and illness is observed in persons with serum antibodies that block strain-specific binding to hbgas hbga expression on cell surfaces is affected by the abo, secretor and lewis genotypes of an individual. in general, because gii.4 viruses can bind a larger range of hbgas in comparison to other genotypes, they have a larger susceptible population pool for infection (ramani et al., 2014) . data on sapovirus infections and immune responses is not yet available. electron microscopy was initially used for identification of these viruses, but is insensitive compared with molecular detection assays. currently, rt-pcr assays are the most common approach for establishing a diagnosis of norovirus infection. virus-specific primers are used to amplify conserved regions of the genome, usually in the polymerase or capsid genes. no single primer pair can detect all norovirus or sapovirus strains because of the high sequence diversity, but in most geographic regions, more than 90% of currently circulating strains can be detected using separate primer pairs for genogroup i and ii noroviruses and sapoviruses. real-time pcr assays have greater sensitivity and are increasingly used. antigen-detection elisa assays for noroviruses have been established in the last decade, but the first assays had a very narrow reactivity. more broadly reactive assays have been developed using monoclonal antibodies that recognize cross-reactive epitopes or multiple monoclonal antibodies, but not widely used. serologic assays also have been developed to detect immune responses to infecting norovirus strains, but are used more in epidemiological studies than for diagnosis in individual patients. human astrovirus is the prototype of the astroviridae, a family of non-enveloped positive sense rna viruses, measuring 38-41 nm. by direct electron microscopy, astroviruses recovered from stool display a distinctive surface star-like appearance. the genome of astrovirus consists of positive-sense, single stranded rna, 6.8 kb in length, organized in 3 orfs. all serotypes have at least three capsid proteins, p1, p2 and p3, with the p2 protein carrying the group-reactive epitopes and the p3 protein specifying serotype. astroviruses are classified into serotypes based on the reactivity of the capsid proteins with polyclonal sera and monoclonal antibodies. astroviruses can also be classified into genotypes on the basis of the nucleotide sequence of a 348-bp region of the orf2, and there is a good correlation with the serotypes. there are eight established genotypes. phylogenetic analyses have shown that it is common to find multiple astrovirus strains circulating in one region during a given period of time, and that there are also variations in the prevalent type with time, suggesting either a genetic shift or an introduction of new strains. serotype 1 is predominant in most studies, followed by 2, 3, 4 and 5. serotypes 6, 7 and 8 are rarely detected. new astroviruses have been described recently, based on sequencing of samples from children with diarrhea and controls. clinically, these viruses cause similar symptoms to caliciviruses. like rotaviruses, astrovirus infections occur through the year with peaks in the winter months. infections have been shown to occur mainly in childhood. other studies showed that most of the cases of infection were detected in children less than 5 years of age with the majority of the children being under 1 year of age. outbreaks of astrovirus infection involving children and elderly patients have been described and prolonged excretion documented in immunosuppressed, immunodeficient and aids patients. significantly higher seroprevalence rates of astrovirus have been reported in adults exposed to contaminated water compared with a control group. the pathogenesis of the disease induced by astrovirus has not been established, although it has been suggested that viral replication occurs in intestinal tissue. in animal studies, atrophy of the intestinal villi is observed, as well as inflammatory infiltrates in the lamina propria leading to osmotic diarrhea. symptomatic astrovirus infection occurs mainly in small children and the elderly, which suggests both an acquisition of antibodies with increasing exposure and a reduction in antibodies with advancing age. studies in adult volunteers indicate that people with detectable levels of antibodies do not develop the illness, although epidemiological observations suggest that human astrovirus infections may not induce heterotypic immunity, as an episode of astrovirus diarrhea is not associated with a reduced incidence of a subsequent episode. electron microscopy is an insensitive technique, because a high concentration of viral particles is required for detection and the typical five or six pointed star morphology is seen in less than 10% of particles. enzyme immunoassays have been developed including streptavidin-biotin assays for increased sensitivity of detection, and are used in most diagnostic laboratories. for epidemiological research, recently astrovirus-specific rt-pcr has been the screening method of choice. while some investigators have used highly sensitive primers targeted to conserved genomic regions coding for the nonstructural proteins and untranslated regions, others prefer to use primers from the capsid coding region, which can be less sensitive but provide typing information (guix et al., 2005) . all adenovirus particles are non-enveloped, 60-90 nm diameter, with icosahedral symmetry easily visible in the electron microscope by negative staining and are composed of 252 capsomers: 240 hexons and 12 pentons bearing fibers at the vertices of an icosahedron. the genome is linear, non-segmented, double-stranded dna of 30-38 kbp. based on their immunologic properties, oncogenicity in rodents, genome and morphology, adenoviruses are classified into six subgroups a-f with 51 serotypes. serotypes predominantly associated with human infections include h-40, h-41, which belong to subgenus f and occasionally h-31 in subgenus a. adenoviruses are widely recognized causes of respiratory, ocular, and genitourinary infections. however, serotypes 40 and 41 (previously called fastidious enteric adenoviruses) primarily affect the gut, contributing to 5-20% of hospitalizations for childhood diarrhea in developed countries. enteric adenoviruses have also been identified in pediatric gastroenteritis in developing countries. peak incidence is among children less than 2 years of age, but older children and adults may be infected, with or without symptoms (glass et al., 2001) . incubation is between 3 days and 10 days, with illness lasting greater than or equal to 1 week, longer than for other enteric viral pathogens. diarrhea is more prominent than vomiting or fever, and respiratory symptoms are often present. the lesions produced by serotypes 40 and 41 in the enterocytes lead to atrophy of the villi and compensatory hyperplasia in the crypts, with subsequent malabsorption and loss of fluids. a neutralizing antibody response made in response to infection results in control of disease and protection from reinfection with the same serotype. asymptomatic virus excretion can continue for prolonged periods even after an antibody response is documented in acute infection. although adenoviruses can be grown in culture, little data is available on the pathogenetic mechanisms of these agents of viral gastroenteritis. traditionally, adenoviruses have been detected and typed by electron microscopy, virus culture and neutralization assays. these assays are time-consuming and more rapid serological assays including immunofluorescence, enzyme immunoassays and latex agglutination have been developed. the rapid assays are useful in the diagnostic laboratory, but do not generally distinguish between serotypes. pcr-based techniques are more sensitive and relatively rapid, but have been shown to give discrepant results when compared to serotyping by neutralization. torovirus is a genus within the coronaviridae family, and toroviruses are known causes of diarrhea among cattle. these viruses have an envelope of 100-140 nm, with a helicoidal capsid and a single-stranded positive sense rna genome. torovirus was detected for the first time in human gastroenteritis in 1984. they are associated with persistent and acute diarrhea in children, and may represent an important cause of nosocomial diarrhea. coronaviruses are well-established causes of diarrhea in animals and respiratory disease in humans. these viruses are between 60 and 220 nm, with helicoidal symmetry and a spiculated envelope that gives them the appearance of a crown and a genome with positive sense single stranded rna. they have been identified in the stool of persons with gastroenteritis (usually children less than 2 years of age), but human controls have been found to shed them with higher frequency, raising doubt about their etiologic role in human diarrhea. diarrhea also occurs as part of the syndrome seen with the sars coronavirus and is likely to play a role in transmission. picobirnaviruses are small viruses, without an envelope, 30-40 nm in diameter, with an icosahedral capsid and a genome made up of two or three segments of bicatenary rna. reports from brazil documented human cases of diarrhea caused by picobirnavirus, which had been thought to be a cause of diarrhea only in animals. the importance of this pathogen is unknown, but it has been found in association with hiv and cryptosporidium infected individuals. aichivirus, in the genus kobuvirus, was first recognized in 1989 in oyster-associated non-bacterial gastroenteritis in man. aichivirus appears to morphologically resemble astroviruses when examined by electron microscopy. recently aichivirus was isolated from pakistani children and from japanese travelers with gastroenteritis returning from tours of southeast asian countries. pestivirus antigen was found in 23% of children less than 2 years of age with gastroenteritis of unknown etiology compared with 3% of controls in a study on an american indian reservation, but further studies on the role of this agent in gastroenteritis have been lacking. parvovirus-like particles have been identified by electron microscopy in stool specimens of both well and ill persons in britain. the relationship of these particles to disease is unclear, but they have been associated with shellfish-related outbreaks of gastroenteritis. enteroviruses cause a wide spectrum of disease, in which gastroenteritis plays a minor role. although the entry of polio, coxsackie, echo, or other enteroviruses through the gut may cause incidental mild diarrheal symptoms, the spread of the virus through the bloodstream to other organs (e.g., central nervous system, heart, pleura, pancreatic islets) produces major disease manifestations. although reports have linked some enteroviruses to illnesses in which diarrhea was the sole symptom, an outbreak or case of gastroenteritis should not be attributed to an enterovirus merely because it was isolated in the stool of an affected person, but attempts should be made to evaluate the amount, duration of shedding and relation to symptoms and antibody response to establish a causal relationship. treatment of viral gastroenteritis is symptomatic, and its aim is to prevent or treat the dehydration secondary to the disease. dehydration is assessed using blood pressure, pulse, heart rate, skin turgor, fontanelle depression, mucous membranes, eyes, extremities, mental status and activity, urine output and thirst. assessment of dehydration, particularly in children in community studies or by field workers relies on lethargy, restlessness, appearance of eyes, skin turgor and feeding/thirst. fluid and metabolic imbalances must be assessed and corrected. the most important factor predicting adverse outcome of viral gastroenteritis is delay in fluid and electrolyte therapy. clinically significant dehydration can occur within 6 h of onset of illness, especially during primary infection in children. malnutrition, malignancy, and immunodeficient states predispose to a more severe episode of illness or unremitting diarrhea that can persist until the underlying condition is corrected. oral rehydration therapy is recommended for preventing and treating early dehydration and continued replacement therapy for ongoing losses. intravenous therapy is required in severe dehydration, shock and decreased consciousness. in children, age-appropriate diet should be continued during oral rehydration and following intravenous rehydration. anti-emetics, anti-diarrheal agents and antibiotics should not be given to children, although anti-emetics and anti-diarrheals may be used in adults. studies have shown that antirotavirus immunoglobulin as bovine hyperimmune colostrum or human milk mat decrease the frequency and duration of rotavirus diarrhea. probiotics such as lactobacillus casei gg and saccharomyces boulardii reduce the frequency and duration of diarrhea (guandalini, 2006) . racecadotril, an enkephalinase inhibitor has been shown in some studies to be useful in treating rotaviral diarrhea in children. zinc supplements have been suggested to reduce severity and duration of illness. preventive measures can limit the number of episodes of viral gastroenteritis both within the home and in institutions. diaper-changing areas should be separate from food preparation areas. diapers should be disposed of directly in the changing area and should be placed in closed bags. hands should be washed after contact with soiled diapers and clothing. interruption of transmission of the infection is extremely important, especially in hospitals and centers which care for small children. therefore, it is necessary to reinforce hygiene measures, such as handwashing and clean all surfaces with suitable disinfectants. since viruses do not replicate outside a host, decreasing the potential inoculum is key to preventing further infection of susceptible hosts. further preventive measures in child care facilities and hospitals include isolation and cohorting of ill children. asymptomatic infections probably also play an important role in the spread of infection. studies with vaccines against group a rotavirus began in 1982. the first vaccine developed was the tetravalent human-rhesus reassortant vaccine, which induces protection against the four main rotavirus serotypes, g1-g4. efficacy studies showed a reduction in the appearance of severe gastroenteritis caused by rotavirus in vaccinated children, and the vaccine was approved in the usa in 1998. however, the detection of an increase in the risk of intussusception after vaccination led to its suspension. in 2006, two new licensed vaccines became commercially available. these were rotateqô, a live oral attenuated pentavalent vaccine from merck which is recommended by the cdc advisory committee on immunization practices to be given at 2, 4 and 6 months with the last dose administered no later than 32 weeks, and rotarixô from glaxosmithkline, a human strain derived monovalent live oral vaccine given in two doses at 2 and 4 months. both vaccines are licensed and used in over one hundred countries and the number continues to grow. the vaccines have lower efficacy in low and middle-income countries than in more industrialized countries. nonetheless, use of the vaccine has shown a dramatic effect on diarrheal hospitalizations in countries where the vaccines are widely used and a reduction in all cause diarrheal mortality in middle-income countries. a lower level of risk of intussusception of about one case in 20 000-50 000 vaccines has been identified in several countries with both vaccines, but overall, the benefits and lives saved by the vaccine are much greater than the risk and sequelae of intussusception (parashar et al., 2013) . owing to the high cost of these vaccines, vaccine manufacturers in developing countries have initiated the process of formulating and testing vaccines based on local strains which will address the issue of cost. dna based, subunit protein based and virus like particle vaccines may also provide an alternate method of prevention in the future. studies on virus-like particle based vaccines against norwalk virus were initiated with capsid proteins expressed in plants inducing an antibody response in experimental models. more recently, a monovalent genogroup i norovirus virus-like particle (vlp) vaccine was evaluated in secretor positive individuals and showed proof-of-concept efficacy. studies on a bivalent formulation including gii.4 vlps, given intramuscularly are now underway (ramani et al., 2014) . in summary, viral gastroenteritis is a major cause of morbidity in developed countries and mortality in developing countries. hygiene is the first preventive step in viral gastroenteritis and rehydration is the key to management of clinical illness. the range of organisms which can cause gastroenteritis is immense and vaccines will be important in reducing the impact of childhood gastroenteritis. global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis infectious diarrhea in developed and developing countries viral gastroenteritis foodborne viruses: an emerging problem gastroenteritis viruses: an overview probiotics for children: use in diarrhea human astrovirus diagnosis and typing: current and future prospects viral infections of the gastrointestinal tract noroviruses: the most common pediatric viral enteric pathogen at a large university hospital after introduction of rotavirus vaccination diarrhea in the immunocompromised patient host-pathogen co-evolution and glycan interactions global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study diagnosis, management, and prevention of rotavirus gastroenteritis in children epidemiology of human noroviruses and updates on vaccine development prevalence of porcine noroviruses, molecular characterization of emerging porcine sapoviruses from finisher swine in the united states, and unified classification scheme for sapoviruses estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis enteric viral infections as a cause of diarrhoea in the acquired immunodeficiency syndrome key: cord-332747-u46xryoo authors: mingorance, lidia; castro, victoria; ávila-pérez, ginés; calvo, gema; rodriguez, maría josefa; carrascosa, josé l.; pérez-del-pulgar, sofía; forns, xavier; gastaminza, pablo title: host phosphatidic acid phosphatase lipin1 is rate limiting for functional hepatitis c virus replicase complex formation date: 2018-09-18 journal: plos pathog doi: 10.1371/journal.ppat.1007284 sha: doc_id: 332747 cord_uid: u46xryoo hepatitis c virus (hcv) infection constitutes a significant health burden worldwide, because it is a major etiologic agent of chronic liver disease, cirrhosis and hepatocellular carcinoma. hcv replication cycle is closely tied to lipid metabolism and infection by this virus causes profound changes in host lipid homeostasis. we focused our attention on a phosphatidate phosphate (pap) enzyme family (the lipin family), which mediate the conversion of phosphatidate to diacylglycerol in the cytoplasm, playing a key role in triglyceride biosynthesis and in phospholipid homeostasis. lipins may also translocate to the nucleus to act as transcriptional regulators of genes involved in lipid metabolism. the best-characterized member of this family is lipin1, which cooperates with lipin2 to maintain glycerophospholipid homeostasis in the liver. lipin1-deficient cell lines were generated by rnai to study the role of this protein in different steps of hcv replication cycle. using surrogate models that recapitulate different aspects of hcv infection, we concluded that lipin1 is rate limiting for the generation of functional replicase complexes, in a step downstream primary translation that leads to early hcv rna replication. infection studies in lipin1-deficient cells overexpressing wild type or phosphatase-defective lipin1 proteins suggest that lipin1 phosphatase activity is required to support hcv infection. finally, ultrastructural and biochemical analyses in replication-independent models suggest that lipin1 may facilitate the generation of the membranous compartment that contains functional hcv replicase complexes. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 millions of humans are chronically infected by hepatitis c virus (hcv) worldwide [1] . chronic hcv infection is a major biomedical problem as it causes liver inflammation and fibrosis, which can lead to severe liver disease, such as cirrhosis and hepatocellular carcinoma [2, 3] . there is no vaccine against hcv and, although blood-screening tests and other prophylactic measures have reduced the dissemination of this pathogen, a number of newly acquired infections still occur associated with risk behavior or with unknown origin [4, 5] . however, chronic hcv infection can be successfully eradicated from chronically infected individuals through specific direct-acting antiviral (daa) combination therapies, virtually in all treated patients [6] . since these specific treatments have only been in place recently, there are no sufficient clinical data on the long-term benefit of these treatments in relieving the severity of advanced liver disease [7, 8] . hcv is a hepacivirus (flaviviridae) with a positive sense, single-strand rna genome that encodes a single open reading frame (orf) flanked by untranslated regions (utr), which are essential for viral polyprotein translation and viral genome replication. hcv orf is co-and post-translationally processed by cellular and viral proteases to produce ten major proteins. these have been functionally classified in a replication module, that includes the minimal viral components of the rna replicase (ns3, ns4a, ns4b, ns5a and ns5b) and an assembly module, which comprises the major structural components of enveloped hcv virions, the capsid protein (core) and the envelope glycoprotein complex formed by e1 and e2 heterodimers; as well as the polypeptides p7 and ns2, which are not structural components of virions but contribute to infectious particle assembly in a concerted action with the viral replicase [9] . hcv utilizes key aspects of cellular lipid metabolism for essentially every aspect of the virus replication cycle and strongly interferes with host cell lipid homeostasis [10] [11] [12] . in fact, chronic hcv patients display high rates of liver steatosis, severity of which inversely correlates with serum liver derived-lipoprotein [13] . thus, although host immune response remains a major component in hcv pathogenesis, direct interference of hcv infection with hepatocyte lipid metabolism may contribute to overall disease progression [13] . identified a limiting role of lipin1 pap activity in the generation of hcv replicase complexes. defective replicase assembly leads to strong inhibition of hcv propagation in lipin1-deficient cells but not that of another (+) strand rna virus, indicating a specific role for lipin1 in hcv infection. given the relevance of lipin1 for lipid metabolism and its steatogenic potential, we set out to independently verify data from published differential transcriptomic profile studies that suggested that hcv infection may alter lpin1 mrna abundance in cell culture [40, 41] . a specific and statistically significant lpin1 mrna induction (fig 1a) was observed in huh-7 cells after single cycle infection experiments [multiplicity of infection (moi) 10] with a cell cultureadapted genotype 2a hcv (d183) variant [42] at the peak of the infection (48 and 72 hours post-infection) and as compared with mock-infected cells (fig 1b) . lpin1 mrna induction was prevented when infected cells were treated with 1μm sofosbuvir (fig 1c) , an hcv rna polymerase inhibitor [43] that reduced viral rna accumulation by more than two orders of magnitude (fig 1d) , indicating that active hcv replication is required to induce lpin1 mrna accumulation. lpin1 mrna has been shown to be upregulated by mechanisms that involve induction of reactive oxygen species (ros), as treatment of the cells with ros-scavenger molecule n-acetylcysteine (nac) is capable of preventing lpin1 mrna induction under glucose deprivation [44] or during h 2 o 2 treatment [45] . given that transcriptional activation of a subset of lipogenic genes during hcv infection is also prevented by addition of antioxidants [46] , we sought to determine if lpin1 mrna induction by hcv infection is mediated by ros production and therefore dampened by nac treatment. lpin1 mrna induction was prevented in the presence of the antioxidant (fig 1e) , despite comparable hcv rna accumulation in mock-treated and nac-treated hcv-infected cells (fig 1f) , suggesting that virus replicationinduced ros production is required to induce lpin1 mrna accumulation. western-blot analysis confirmed that the observed transcriptional change leads to a correlative protein accumulation (fig 1g and 1h) ; reinforcing the notion that acute hcv infection alters lipin1 expression. in order to determine if lipin1 subcellular localization was altered during hcv infection, we performed confocal microscopy studies in control and hcv-infected huh-7 cells. lipin1 staining was observed as cytoplasmic punctated structures both in control and hcv-infected cells. to study if lipin1 signal colocalized with viral antigens, we performed double staining with antibodies against lipin1 and double-stranded rna (dsrna) or replicase subunits ns3 and ns5a. none of the viral antigens strictly colocalized with lipin1 (pearson´s<0.5) (s1a fig). however, mander´s coefficients indicate that the majority of lipin1 overlapped with a small fraction of ns3 and ns5a (s1c fig). in contrast to lipin1, lipin2 signal did not overlap with that of viral proteins ns3 and ns5a (s1b fig). our results indicate that no major lipin1 rearrangements are observed after hcv infection and that only a minor fraction of ns proteins colocalize with lipin1. to study if lipin1 plays any role in hcv infection, lipin1-deficient cells were generated by transducing human hepatoma (huh-7) cells with lentiviral vectors expressing shrnas targeting lpin1 mrna or a control vector expressing an irrelevant shrna. lipin1 expression silencing was verified by western-blot, typically 7 days post-transduction (fig 2a) . a partial (50%; shlpin1-1) and a more profound (>95%; shlpin1-2) reduction in lipin1 accumulation was observed after transduction with specific shrnas as compared with the control (fig 2b) . . rnas from mock-infected cells and hcv infected. impact of sofosbuvir (1μm; daa) (c, d) or n-acetylcysteine (10mm; nac) treatment (e, f) on hcv rna accumulation and lpin1 mrna levels. data are shown as average and standard deviation of two independent infection experiments performed in triplicate (n = 6). g-protein samples of infected and control cells collected at 48 hours post-infection were subjected to western-blot analysis to determine lipin1 and ns3 levels, using beta-actin as loading control h-quantitation of the relative lipin1 protein levels in mock and hcv-infected cells (n = 3). statistical significance was determined using student´s t-test ( ã p<0.05; ãã p<0.01). https://doi.org/10.1371/journal.ppat.1007284.g001 huh-7 cells were transduced with lentiviral vectors expressing control or lpin1-specific shrnas. seven days after transduction samples of the cells were collected for western-blot analysis using antibodies against lipin1 and lipin2 and actin as loading control. parallel cultures were subjected to an mtt assay to determine their viability as described in the methods section. (a) representative western-blot showing cellular lipin1 and lipin2 protein expression levels and a loading control (actin). (b) average expression values for lipin1 and lipin2 as determined by western-blot and cell viability as determined by an mtt assay. data are shown as average and sd of six independent transduction experiments (n = 6). (c) huh-7 cells were transduced with lentiviral vectors expressing control or lpin1-specific shrnas. at day 3 post-transduction, cells were infected at moi 0.01 with hcv d183 virus. samples of cell supernatants were collected at days 3 and 5 post-infection to determine the extracellular infectivity titer. average and sd of the infectivity titers at day 3 and 5 of two independent infections performed in triplicate (n = 6). (d) huh-7.5 cells were transduced with lentiviral vectors expressing control or lpin1-specific shrnas. silenced cells were infected 7 days after transduction at moi 0.05 with genotype 1a hcv (tncc) in the presence or absence of 2made (10μm; shcontrol+daa). intracellular hcv rna was determined by rt-qpcr 72 hours post-infection. data are shown as average and sd of three experiments performed in triplicate (n = 9). statistical significance was determined using student´s t-test ( ã p<0.05; ãã p<0.01). as expected, lipin1 and lipin2 expression is inversely correlated in lipin1 shrna-expressing cells (fig 2b) . the viability of lipin-deficient cells, as determined by mtt assay [47] was comparable to that of control cells (fig 2b) . these results illustrate that lipin1 shrna-expressing cells respond homeostatically to functionally compensate partial (shlpin1-1) and more pronounced (shlpin1-2) loss of lipin1 (fig 2b) . control and lipin1-deficient cells were infected with a genotype 2a d183 at moi 0.01 to study viral spread by determining extracellular infectivity titers at different times post infection. fig 2c shows limited propagation of this virus in lipin1-deficient cells, with a statistically significant reduction of up to two (shlpin1-1) and three orders of magnitude (shlpin1-2) in viral titer between the control and lipin1-deficient cell lines at day 5 post-infection. these results indicate that lipin1 is required for efficient hcv propagation and that homeostatic lipin2 accumulation (fig 2b) is not sufficient to support efficient hcv infection. since important differences in the interaction of different hcv genotypes with cellular lipid metabolism have previously been described [48] , we set out to determine if the observations made with a jfh1-derived virus (genotype 2a) were extensive to other hcv genotypes. we performed low multiplicity infections (moi 0.05) with genotype 1a tncc virus strain in lipin1-deficient huh-7.5 cell lines, which are susceptible to infection by this recombinant virus [49] . first, we verified that huh-7.5.1 also display a significant reduction in genotype 2a infection efficiency when lipin1 is silenced (s2b fig) . in order to determine tncc infection efficiency, intracellular hcv rna accumulation was determined 72 hours post-inoculation in control and lipin1-deficient huh-7.5 cells. viral rna detected under these conditions reflects the ability of genotype 1a to infect and replicate viral rna in the different cell lines, as treatment of control cells with an hcv polymerase inhibitor 2´-c-methyladenosine (2made; 10 μm) [50] reduced viral rna content by two orders of magnitude ( fig 2d) . lipin1 silencing consistently and significantly reduced tncc replication by approximately 3-fold both in shlpin1-1 and shlpin1-2 cell lines (fig 2d) , indicating that lipin1 is also limiting for genotype 1a hcv infection. to determine the specificity of these observations, identical lipin1-deficient and control huh-7 cultures were inoculated at moi 0.01 with a human alpha-coronavirus cov-229e bearing a gfp reporter gene (hcov-229e-gfp) [51] . inoculation of control and lipin1-deficient cells with this virus resulted in comparable progeny virus production, as determined by infectivity titration in cell supernatants 48 hours post-infection (s3 fig) . these results suggest that lipin1 is not rate limiting for cov-229e-gfp infection and that lipin1 expression is particularly limiting for hcv. to determine which aspects of the hcv replication cycle are limited by lipin1 silencing, single cycle infection experiments were conducted by inoculating control and lipin1-deficient cell cultures at moi 10 with genotype 2a d183 virus. infection efficiency was measured by titration of progeny virus infectivity present in the supernatant of infected cells and intracellular hcv rna accumulation at 48 and 72 hours post-infection. infection of lipin1-deficient cells resulted in a significant reduction of progeny infectious virus production in shlpin1-1 and shlpin1-2 cells as compared with the titers observed in the supernatants of control cells ( fig 3a) , reinforcing the notion that lipin1 silencing interferes with hcv infection. reduced virus production is likely due to parallel reduction of intracellular hcv rna levels observed in lipin1-deficient cells as compared with the control cell line (fig 3b) . this reduction was observed at all time points, except for that at 5 hours, indicating that the size of the inoculum and initial virus adsorption is comparable among the different cell lines (fig 3b) . thus, lipin1 silencing suppresses hcv infection by interfering with a step of the hcv lifecycle preceding intracellular hcv rna accumulation. next, we set out to determine if lipin1 silencing has any impact on persistent hcv infections to verify if lipin1 is also limiting for late aspects of the virus lifecycle. persistently infected cells continuously replicate viral rna, express viral antigens and secrete infectious virions. thus, it is a valuable system to measure steady-state hcv rna replication as well as infectious particle assembly and secretion. persistently infected cultures were transduced with the lentiviral vectors described above to produce persistently infected, lipin1-deficient cells (s4 fig) . shown as genome equivalents per microgram of total rna ge/μg). panels c, d-persistently infected cultures were generated by inoculation with jfh-1 virus at moi 0.01. once cultures reached >95% of hcv-positive cells, they were transduced with lentiviral vectors expressing control, hcv rna-targeting or lpin1-specific shrnas. at day 7 post-transduction, cells were split and samples of the cells and supernatants were collected 24 hours later to determine infectious virus production rate by infectivity titration hcv (c) and rna levels by rt-qpcr (d). all data are shown as mean and sd of 3 independent experiments performed in triplicate (n = 9). statistical significance was determined using student´s t-test ( ã p<0.05; ãã p<0.01). analysis of extracellular infectivity titers revealed that infectivity titers in lipin1-deficient and control cells were comparable, with the exception of a marginal reduction in shlpin1-2 expressing cells, indicating that lipin1 silencing does not strongly interfere with infectious virus production ( fig 3c) . intracellular hcv rna levels in lipin1-deficient cells were comparable to that of the control cells (fig 3d) , indicating that lipin1 expression is not rate limiting for hcv rna replication once infection has been established. taken together, the results shown above indicate that lipin1 is only limiting at early steps of hcv infection leading to viral rna accumulation. as an independent verification of the hypothesis that lipin1 is limiting for early aspects of hcv infection, we used a single cycle surrogate infection model based on the production of hcv virions bearing a defective reporter genome encapsidated by trans-complementation (hcv tcp ). hcv tcp are capable of producing abortive single-cycle infections, efficiency of which is proportional to the luciferase activity found in the target cells [52] . as expected from the results shown in fig 3, infection of lipin1-deficient cells with hcv tcp resulted in a 75% reduction in the reporter luciferase activity in shlpin1-1 cells and 90% in shlpin1-2 determined at 48 hours post-infection, proportionally to the degree of silencing in these cells ( fig 4a and 4b ). these results underscore the role that lipin1 plays at early steps of hcv infection and indicate that either viral entry or a step leading to efficient hcv rna replication is impaired in these cells. lpin-1 mrna is alternatively spliced in human liver to produce isoforms α and β, which may differ in catalytic activity, subcellular localization and gene expression regulation [53] . to determine the relative contribution of these isoforms to hcv infection, isoform-specific shrnas were generated and used to transduce huh-7 cells, as described above. lpin1α-specific shrna (shlpin1-3) reduced total lipin1 protein by 70%, while lpin1β-specific shrna (shlpin1-4) reduced total lipin1 expression by only 30% (fig 4a) . infection of control and lipin1 isoform-deficient cells with hcv tcp revealed that lipin1α silencing resulted in a strong (85%) reduction in hcv infection while lipin1β silencing resulted in a milder (55%) but significant reduction in hcv infection efficiency ( fig 4b) . these results indicate that both isoforms alpha and beta are limiting for hcv infection and suggest that the total amount of lipin1 present in the cell determines hcv infection efficiency. taken together, the results obtained with four different shrnas indicate that total lipin1 expression levels strongly correlate with hcv tcp infection efficiency (fig 4c) , underscoring a role for this host protein in early aspects of hcv infection. reduced hcv rna accumulation in a single cycle infection (fig 3) as well as reduced hcv tcp infection (fig 4) may be due to a defect in entry of incoming virions. hcv e1e2-pseudotyped retroviral vectors bearing a luciferase gene (hcv pp ) were used to measure viral entry because they constitute a sound model to study viral adsorption, receptor-mediated internalization and e1e2-mediated fusion in endosomes [54] . to assess the specificity of these observations, parallel cultures were inoculated with vsv-g pseudotyped retroviral vectors (vsv pp ). control and lipin1-deficient cell lines were infected with hcv pp (genotype 2a; jfh-1 strain) and vsvpp. as a positive control of inhibition of hcv entry, we used hydroxyzine (5 μm), which efficiently blocks hcv infection by interfering with viral entry [55] . as expected, hydroxyzine selectively inhibited hcv pp infection, as shown by reduced luciferase levels 48 hours postinoculation only in hcv pp -infected cells ( fig 5a) . interestingly, lipin1-deficient cells (shlpin1-1 and shlpin1-2) were fully susceptible to hcv pp and vsv pp infection, as comparable luciferase activity levels were found in all cell lines 48 hours post-inoculation ( fig 5a) . these results indicate that lipin1 is not rate limiting for receptor binding, particle internalization or e1e2-mediated endosomal fusion, which are steps recapitulated in this model [54] . based on the data presented thus far, we hypothesized that lipin1 is limiting for a step in the hcv lifecycle downstream of hcv entry, leading to hcv rna accumulation. to verify the hypothesis that lipin1 silencing causes strong reduction in initial hcv rna accumulation by interfering with a step downstream of viral entry, we bypassed this step of the viral replication cycle by transfecting a subgenomic hcv rna replicon bearing a reporter luciferase gene into control and lipin1-deficient cells. first, we evaluated hcv-ires driven primary translation of incoming genomes by transfection of a replication-deficient mutant replicon that bears an inactivation mutation in the catalytic site of ns5b rna polymerase [56] . luciferase activity measured at 5 hours post-transfection was not reduced in any of the cell lines, indicating that transfection efficiency and hcv ires-dependent primary translation was not significantly affected by lipin1 silencing (fig 5b) . a significant increase in renilla luciferase activity was observed in shlpin1-1 cells both when transfecting a replicon ( fig 5b) or a plasmid expressing renilla luciferase under a minimal rna polymerase ii promoter (s5a fig), suggesting that the increase in luciferase activity observed in these cells is not related with hcv. hcv rna replication was evaluated by measuring accumulation of a reporter luciferase gene at 5 and 48 hours post-transfection of a replication competent hcv subgenomic replicon. under these experimental conditions luciferase accumulation at 5 hours also represents hcv ires-driven primary translation of the input rna, while reporter luciferase activity at 48 hours depends on effective hcv rna replication. in contrast to primary translation, hcv rna replication inferred by luciferase activity at 48 hours was strongly reduced in lipin1-deficient cells (90% in shlpin1-1 and 99% in shlpin1-2 cells) (fig 5c) , indicating that initiation of hcv replication is dependent on normal lipin1 expression. this reduction was not due to a non-specific defect in luciferase expression, as co-transfection of a plasmid expressing renilla luciferase lead to comparable luciferase accumulation in all cell lines, discarding the possibility that death of transfected cells or other spurious effects are responsible for the reduced luciferase activity accumulation (s5a fig) . similar experiments were conducted in atg4b-deficient cells, as this host factor was shown to be limiting for primary hcv translation [57] . our studies confirmed that, while partial atg4b silencing (s5b fig overall, these data indicate that hcv rna replication is not initiated efficiently in lipin1-deficient cells and that blockade occurs at a step downstream translation of incoming hcv genomes. in order to determine if any of the known functions ascribed to lipin1 is required for hcv infection, we tested the ability to restore hcv infection susceptibility of silencing-resistant wild-type (wt) or mutant lipin1 versions bearing a mutation in the catalytic site responsible for its phosphatase activity (dxdxt) and a mutant in the lxxil motif, which is inactive both for transcriptional activation as well as for phosphatase activity [31] . control and lipin1-deficient cells were transfected with wt lipin1 beta cdna as well as with dxdxt and lxxil mutants. comparable overexpression levels of the wt and mutant proteins was obtained in each cell line, although overexpressed lipin1 levels were consistently higher in lipin1-deficient cells (fig 6a) . cells were subsequently inoculated with hcv d183 at moi 10 and relative infection efficiency was calculated by determining extracellular infectivity titers 48 hours post-infection. overexpression of wt lipin1 did not significantly alter susceptibility to hcv infection in control cells, although we observed a small but consistent reduction in extracellular infectivity titers when overexpressing wt and mutant lipin1 constructs (s6a fig using relative infection efficiency as readout of this set of experiments, we could clearly observe a statistically significant increase (2-fold) in the relative infection susceptibility in cells overexpressing wt lipin1 cdna as compared with mock-transfected cells or cells expressing similar or higher levels of the mutants (fig 6a) , suggesting that wt lipin1 modestly, though significantly, rescues hcv infection while phosphatase (dxdxt) and transcriptional coactivation (lxxil) mutants do not ( fig 6b) . these results suggest that lipin1 transcriptional coactivation capacity is not sufficient to support hcv infection while lipin1 phosphatase activity is essential. however, given that lxxil mutant is deficient both in transcriptional co-activation and pap activity [31], we cannot determine if transcriptional co-activation by lipin1 is also required to support hcv infection. the data described above suggest a role for lipin1 phosphatase activity in a step of hcv replication cycle between translation of the viral genome and formation of functional replicase complexes. data regarding primary translation where inferred from a surrogate model of translation based on a reporter luciferase gene ( fig 5b) . to address if indeed viral polyprotein is properly processed and inserted into detergent-resistant microdomains to form the characteristic membranous ultrastructures bearing the viral replicase, we used a replication-independent surrogate model of polyprotein expression. this system is based on a vector encoding the portion of the viral polyprotein corresponding to the replicase (ns3-ns5b) under the transcriptional control of the t7 polymerase and the translational control of encephalomyocarditis virus (emcv) ires (ptm-ns3/5b) [58] . replication-independent polyprotein overexpression control and silenced cells were inoculated with hcv e1e2 (hcv pp ) or vsv-g-pseudotyped retroviral vectors (vsv pp ) 7 days post-transduction. control cells treated with hydroxyzine (5μm) were used as positive inhibition control. luciferase activity was determined in the different cell lines at 48 hours post-inoculation. hdx, hydroxyzine pamoate (5μm). panels b, c-control and lipin1-deficient cells were transfected with a replication-deficient mutant (b) or replication competent subgenomic hcv replicon bearing a luciferase gene (c). luciferase activity was determined in the different cell lines at 5 hours post-transfection for both replicons and 48 hours post-transfection for the replication-competent replicon rna. data are expressed as average and sd of three independent experiments performed in triplicate (n = 9). statistical significance was determined using student´s t-test ( ã p<0.05; ãã p<0.01). https://doi.org/10.1371/journal.ppat.1007284.g005 systems enable assessment of polyprotein processing as well as studying the formation of virus-derived membranous structures [16, 59] . control and lipin1-deficient cells were infected with a recombinant vaccinia virus expressing t7 rna polymerase (vact7) and subsequently transfected with the plasmid ptm-ns3/5b to enable viral replicase expression. sixteen hours post-transfection, cells were processed for lipin1 is required for hcv replicase formation western-blot using anti-ns3. accumulation of ns3 is comparable in control and both lipin1deficient cells, underscoring the notion that lipin1 is not limiting for polyprotein translation and processing (fig 7a) . similarly, ns3 and ns5a expression and subcellular distribution was similar in all cell lines (fig 7b) . these results suggest that there are no major differences in accumulation of viral proteins in lipin1-deficient cells and that a step downstream is affected in these cells. transmission electron microscopy (tem) of ultrathin cell sections of cells expressing hcv replicase components shows the expected accumulation of a mixture of characteristic doublemembrane vesicles (dmv) as well as multiple membrane vesicles (mmv) (fig 7d, 7e and 7f) that were not found in mock-transfected cells (fig 7c) , as reported in previous studies using similar systems [16] . individual vesicle diameter displays heterogeneous size distribution in which the predominant population is distributed between 125-150 nm (median 150 nm; average ± sd: 167 ± 81 nm, n = 279) with larger vesicles being less predominant (fig 7g and s7 fig) . this size distribution is compatible with that observed similar replication-deficient systems and during hcv infection. treatment of these cells with 100 nm daclatasvir (dctv), resulted in a strong reduction in the number of vesicles per section area (s7b fig) , without significantly altering the size distribution of the remaining vesicles (fig 7g) , as reported by berger et al. [59] . similarly, the diameter distribution of the vesicles found in lipin1-deficient cells was comparable to that in control cells ( fig 7g) . however, hcv-induced structures were significantly less abundant 16 hours post-transfection in lipin1-deficient cells than in controls cells (s7c fig) . this reduced abundance is illustrated by a significant reduction in the fraction of cells displaying vesicular structures in lipin1-deficient cell cultures (fig 7h) despite comparable transfection efficiency and viral protein expression levels, indicating that lipin1 may be required in a critical step leading to formation of the hcv-induced vesicular compartment. to validate the tem results independently, we set out to establish a biochemical assay to evaluate replication-independent replicase complex formation. one of the characteristics of the hcv replicase complexes is that they are located in detergent-resistant membranes (drm) [19, 20] . in this sense, ns proteins are associated with replicase complexes that co-sediment with drm markers such as caveolin-2 or sigma-1 receptor (sigmar1) in low-density fractions in isopycnic gradient ultracentrifugation experiments [19, 21] . control and lipin1deficient cells were infected with vact7 and subsequently transfected with limiting doses of the plasmid ptm-ns3/5b [16] . parallel samples were treated with dctv (100 nm). sixteen hours post-transfection, cell lysates were generated and subjected to equilibrium ultracentrifugation in 10-40% sucrose gradients. gradient fractions were collected and subjected to western-blot analysis to determine the impact of lipin1 silencing on ns3, sigmar1 and actin sedimentation profiles. fig 8a shows how, as previously shown for replicon and jfh1-infected cells, ns3 can be detected in drm fractions (fractions 3-5), as determined by the presence of sigmar1 in those fractions (fractions 3-5; s8a fig), although in this experimental system, unlike during viral infection [21] , most ns3 co-sediments together with solubilized proteins, as shown for actin (fractions 9-12; s8a fig) . drm-associated ns3 is reduced in dctv-treated and lipin1-deficient cells, while total ns3 expression remains unchanged (fig 8a) . four independent experiments were performed in which relative-drm associated ns3, normalized to that found in solubilized fractions, was calculated for each experimental condition (fig 8b) . lipin1-deficient cells display a consistent and statistically significant reduction in the drmassociated, but not total ns3 abundance (fig 8a and 8b; shlpin1-1 and shlpin1-2) , similar to that observed in control cells in the presence of dctv (figs 8a and 8b; shcontrol+dctv) , consistent with the tem data (figs 7 and s7 ) and the notion that ns3 in drm fractions may reflect the abundance of replicase complexes formed in these cells. this reduction is not due to an overall reduction in cellular drm abundance in lipin1-deficient cells, as lipin1-deficient cells display similar sigmar1 distribution pattern as the control cells (s8 fig). overall, tem data and drm floatation assays strongly suggest that lipin1 is rate limiting for the generation of replicase complexes from fully processed polyprotein subunits. hepatitis c virus replication cycle is tightly linked to host cell lipid metabolism and interference with cellular lipid homeostasis contributes to viral pathogenesis [3] . one of the most evident consequences of this interference is the high prevalence of liver steatosis among chronically infected patients [13, 60] . this clinical manifestation of the infection has been linked to, among others, chronic er stress, mitochondrial dysfunction and metabolite depletion induced by hcv infection, which result in the activation of persistent homeostatic adaptation of the cellular lipid metabolism to permit cell survival, at the cost of pathogenic metabolic alterations [11, [61] [62] [63] [64] . among the different regulatory networks that have been shown to be stimulated during hcv infection, pparα [61] , pgc-1α [65] , hif-1 [66] and srebp [46, 67] have also been shown to regulate transcription of lpin1 mrna [31, 45, 68, 69] . thus, it is likely that stimulation of one or several of these regulatory networks by hcv infection results in the lpin1 mrna transcriptional activation observed in this ( fig 1a) and other studies [40, 41] . importantly, prevention of lpin1 mrna accumulation with nac ( fig 1e) did not significantly interfere with hcv rna replication (fig 1f) , suggesting that enhanced lpin1 mrna accumulation is not required for efficient hcv infection. we favor the hypothesis that ros induced by hcv protein accumulation actively participates in lpin1 induction, as treatment with the antioxidant nac prevented lpin1 mrna accumulation, similar to what has been shown for other srebp-regulated genes during hcv infection [46] . accumulation of lpin1 mrna during hcv infection results in concomitant protein accumulation (fig 1g and 1h) . however, post-translational mechanisms such as phosphorylation, acetylation or sumoylation regulate lipin1 protein stability, membrane association as well as subcellular localization thus influencing the activity of lipin1 as pa-phosphatase and as transcriptional coactivator [70] . hence, it is difficult to predict the implications of lipin1 protein accumulation during hcv infection. thus, future studies on the interference of hcv infection with cellular lipin1 functions will be required to determine its role in hcv-related pathogenesis, particularly in its contribution to steatosis. lipin1 is required for hcv replicase formation the data presented in this study provide evidence that lipin1 is rate limiting for hcv infection at an early step of the infection leading to formation of membranous hcv replicase complexes, downstream of viral polyprotein expression and processing. we provide evidence for reduced accumulation of viral rna during single cycle infection experiments (fig 2d) , that is reminiscent of a faulty initiation of viral replication, as suggested by reduced replication of a transfected subgenomic replicon in lipin1-deficient cells (fig 5d) . data obtained in replication-independent polyprotein expression models suggest that generation of the membranous compartment that contains functional replicase complexes is severely limited in lipin1-deficient cells, as suggested by a significant reduction of the fraction of cells where these structures could be visualized by tem (figs 7 and s7 ). this hypothesis is further supported by a significant reduction in drm-associated ns proteins in lipin1-deficient cells (fig 8) , which may reflect limitations in the association of viral replicase subunits with cholesterol and sphingolipid-rich membranes in lipin1-deficient cells [19, 20, 71] . four different shrnas targeting lpin1 mrna decrease susceptibility to hcv infection proportionally to their ability to reduce total lipin1 protein accumulation (fig 4) . these results, together with the cdna rescue experiments (fig 6) , strongly reduce the possibility of observing rnai-associated off-target phenomena. interestingly, homeostatic accumulation of lipin2 protein in lipin1-deficient cells (fig 2a and 2b) is not sufficient to compensate for lipin1 loss to support efficient hcv infection. the notion that, despite being capable of mutually compensating basic liver functions [36, 37], lipin1 and lipin2 play non-redundant functions in the liver has previously been proposed [36, 72, 73] . lipin1 is tightly regulated at many different levels and its activity accommodates pap activity in response to different physiological situations such as fasting and insulin signaling [70] . compelling evidence indicates that, while lipin1 and lipin2 cooperate to maintain liver lipid homeostasis, the two proteins differ in many aspects. for instance, lipin1 is transcriptionally induced by pgc-1α and it is also an inducible amplifier of this transcriptional network [31], whereas lipin2 is not [36] . lipin1 is sumoylated and sumoylation regulates its nuclear localization and function, whereas lipin2 sumoylation could not be demonstrated, despite the presence of a canonical sumoylation motif in its primary sequence [74] . lipin1 enzymatic activity is blocked by mammalian target of rapamycin (mtor)-dependent phosphorylation in response to different metabolic stimuli [30, 75], whereas lipin2 is constitutively active even when phosphorylated [76] . thus, lipin2 is considered more as a constitutive phosphatidic acid phosphatase with lower specific activity than lipin1 [76] . in addition to these differential regulatory networks, it has been shown that in vitro pap activity of purified lipin1 and lipin2 is differentially influenced by the composition of the substrates (liposomes and lipid-detergent micelles) as well as the ph at which the assay is performed [76] . this differential lipid substrate recognition may be reminiscent of the different preferential association with membranes of different subcellular compartments (s1 fig) [73, 76] . these differences suggest that, while lipin1 and lipin2 may share some common features, they are not functionally interchangeable, particularly not in the case of hcv infection [70] . our data support the notion that lipin1 silencing has a strong impact on hcv infection without affecting basic cellular functions (fig 2b) or significantly interfering with infection by an unrelated virus (s3 fig). despite great efforts and different overexpression systems, functional rescue of lipin1 functions by wt lipin1 cdna overexpression in lipin1-deficient cells only lead to a small but consistent rescue of virus infection efficiency, which was only observed when overexpressing wt lipin1 (figs 6 and s6) . given the multiple transcriptional, post-transcriptional and post-translational regulation levels existing for lipin1 expression, it is conceivable that only a fraction of the overexpressed lipin1 is fully competent to sustain hcv infection. moreover, high overexpression levels could only be achieved in lipin1-deficient cells (fig 6a) , underscoring the notion that intracellular lipin1 levels are tightly regulated by the host. nevertheless, overexpression of a mutant lipin1 lacking phosphatase activity (dxdxt) or a mutant inactive as transcriptional coactivator (lxxil) were not capable of enhancing hcv infection in lipin1-deficient cells as compared with the wt lipin1 ( fig 6b) . these data reveal that lipin1 phosphatase activity is required for lipin1 to support hcv infection and suggest that, while transcriptional co-activation by lipin1 may be important, this function is not sufficient to support hcv replication. lipins are important enzymes in the main pathway for de novo phospholipid biosynthesis by providing dag derived from the glycerol-3-phosphate pathway to produce pc and pe through the kennedy pathway [70] . production of membranous replicase compartments likely requires de novo synthesis of pc and/or pe, which are major components of biological membranes that depend on dag biosynthesis [29] . in fact, local pc biosynthesis is required for efficient replication of (+) rna viruses and certain pc species accumulate in hcv-infected cells [26, 28] . although increased lipin2 accumulation may be sufficient to compensate lipin1 silencing at the whole-cell level [37], it is possible that acute, local demand of de novo synthesized phospholipids is required at defined suborganellar compartments during early steps of hcv infection and that lipin1-deficiency shortens or alters the availability of different membrane components, demand that may not be satisfied by lipin2, given the differential regulation [76] and subcellular localization of these two proteins (s2 fig). remarkably, deletion of the yeast lipin homologs pah1/smp2 (s. cerevisae) or ned1 (s. pombe) gene, results in deregulated proliferation of the er and nuclear envelope membranes [77, 78] , with concomitant enhancement in (+) rna virus replication [79, 80] . the membranous alterations and elevation of total phospholipid content observed in pah1-deficient yeast have been ascribed to transcriptional activation of pah1-independent alternative phospholipid biosynthetic programs due to pa accumulation [77, 80] . our data in mammalian cells are more compatible with a shortage of phospholipid production, which may be at the basis of the reduced abundance viral membranous structure (figs 7 and 8) . this opposite outcomes of infection may derive from the fact that yeast use mainly pa (lipin1 pap substrate) as a precursor for pc biosynthesis, while mammals mainly use dag (lipin1 pap product) as precursor for pc biosynthesis through the kennedy pathway [77, [80] [81] [82] . moreover, in contrast to yeast and lower eukaryotes, which express only one lipin gene, three different lipin genes coordinate glycerolipid homeostasis in mammals [72] . thus, interfering with expression of one of the members of the family may not be sufficient to observe the same effects observed when deleting pah1, as transcriptional and posttranslational homeostatic compensations are in place in mammals, particularly between lipin1 and lipin2 in liver tissue [36, 37] . in this sense, deletion of either lipin1 or lipin2 in mouse models results in a relatively balanced liver phospholipid content while simultaneous deletion of both lipins is embryonically lethal [37] . accordingly, only minor alterations of er membranes [83] and no significant alterations in total pc levels [36] have been reported in lipin1-deficient mouse liver. given the fact that hcov-229e is fully capable of replicating in these cells (s3 fig), it is unlikely that a general disruption of de novo phospholipid biosynthesis occurs in lipin1-deficient cells, particularly since hcov-229e infection also induces profound er membrane rearrangements required for replication [84] , some of which are structurally similar to dmvs observed during hcv infection [85] . thus, we favor the hypothesis that a subcellular pool of glycerophospholipids is managed by lipin1 in huh-7 cells and that lipin1 silencing perturbs local levels of pa and dag, limiting local availability of precursors of structural components of virus-induced membranes. alternatively, lipin1 deficiency may alter local amounts of important signaling molecules, in particular, that of its substrate (pa) or its product (dag). deregulation of the local pa and dag pools may cause important alterations for the host cell, as both metabolites are potent chemical messengers that regulate different aspects of cellular homeostasis [86] [87] [88] . regarding pa conversion into dag by lipins, it has been shown that pah1 (yeast lipin1 homolog) phosphatase activity is critical for transforming local pools of pa into dag at the er membrane to facilitate membrane fusion events mediated by snare complexes [89, 90] . mammalian lipin1 phosphatase activity is also critical for transforming local pools of pa that accumulate at the surface of mitochondria to promote mitochondrial fission [91] or at the surface of endolysosomes to facilitate autophagy [92] . thus, lipin1 and probably other members of the lipin family modulate different aspects of intracellular membrane signaling. given that the function of host factors known to be involved in functional hcv replicase biogenesis, like vapa, vapb and osbp [11] are indirectly regulated by local pa/dag pools [93] , it is tempting to propose that lipin1 silencing interferes with the function of one or several of these, or other yet uncharacterized cellular factors. in contrast to what has been reported for other host factors required for hcv replicase complex formation [58] , we did not find evidence of lipin1 protein relocalization during hcv infection (s1 fig). thus, determining the precise mechanism by which lipin1 regulates hcv replicase formation is challenging, as association of lipin1 with different cell membranes is transient and highly regulated by posttranslational modifications [70] . moreover, some of the known lipin1 cellular functions may be compensated by other lipins, particularly lipin2 in the liver. nevertheless, our data clearly indicate that lipin1 participates at early stages of hcv replication and that the aforementioned homeostatic compensations by other lipins in regards to cellular metabolism may constitute an advantage when considering lipin1 as a host target for anti-hcv therapy. hcv antiviral compounds 2´-c-methyladenosine (2made), sofosbuvir and daclatasvir were obtained from boc sciences (ny, usa), selleckchem (texas, usa) and medchem express (new jersey, usa) respectively and dissolved in dmso to obtain 10mm stock solutions. nacetylcysteine (nac) and puromycin were obtained from sigma-aldrich (missouri, usa), dissolved in water to a final concentration of 0.5 m and 50 mg/ml respectively. hydroxyzine pamoate was purchased from sigma-aldrich (missouri, usa) and dissolved in dmso to a final 10 mm concentration. human hepatoma huh-7 and derived subclones huh-7.5, huh-7.5.1 (clone 2) have been described [94] [95] [96] and were kindly provided by dr. chisari (tsri-la jolla, ca). hek-293t cells [97] were kindly provided by dr. ortin (cnb-madrid, spain). cell cultures were maintained subconfluent in dulbecco´s modified eagle´s medium (gibco) supplemented with 10 mm hepes (gibco), 100u/ml penicillin/streptomycin (gibco), 100μm non-essential amino acids (gibco) and 10% fetal bovine serum (sigma-aldrich). genotype 2a (jfh-1 strain), d183 adapted virus (d183) and genotype 1a (tncc) virus have been described elsewhere [42, 49, 56] . tncc plasmid was kindly provided by dr. jens bukh (huidovre hospital; copenhagen, denmark). recombinant viruses were generated by electroporation of an in vitro transcribed rna as described previously [42] , using clone 2 cells (jfh-1 and d183) virus and huh-7.5 cells for tncc. supernatants containing the infectious virus were used to inoculate naive huh-7.5.1 clone 2 cells at low multiplicity of infection (moi 0.01 ffu/cell) to prepare working virus stocks. for tncc infections, supernatants of electroporated cells were directly used. lentivirus production. lentiviral vectors shrna expression were produced by co-transfection of plasmids pmdl-rre, pmd2g and prsv-rev (a gift from didier trono-addgene plasmids #12251; #12253; #12259), together with the genomic plasmid (mission plko-puro; sigma-aldrich) into hek-293t cells [21] . selected shrna target sequences are: cgagagaa agtggttgacata (shlpin1-1); (cctcagacagaaatgcagttt) shlpin1-2; cact cccagtccttccggttc (shlpin1-3); cctgttccatccttcggaaag (shlpin1-4). plasmids encoding a atg4b shrna (atg4b800) [57] , non-targeting shrna (shcontrol) as well as an shrna targeting hcv ires (shhcv)were previously described [21] . a lentiviral vector plasmid encoding lipin1-alpha cdna was purchased from genecopeia (cat nbr: ex-t7124-lv153). to generate lipin1 beta isoform, exon 7 sequence was inserted using neb-q5 mutagenesis kit (neb). similarly, silent mutations were introduced in shlpin1-2 target sequence to obtain wt lipin1beta protein expression from a cdna resistant to silencing. mutations in the conserved motifs dxdxt (didgt>eidgt) and lxxil (lghil>lghff) were also introduced in lipin1beta cdna using neb-q5 kit, based on previously described mutations [31, 98] . the sequence of the entire lpin1 cdnas was determined by sanger sequencing before use, to assess the introduction of only the desired mutations. coronavirus 229e-gfp. recombinant human coronavirus 229e-gfp [51] was kindly provided by dr. volker thiel (university of bern, switzerland). virus was propagated by infection (moi 0.01) in huh-7 cells. supernatants were collected at different time points and titrated by end-point dilution and immunofluorescence microscopy. lentiviral vectors expressing control and lpin1-specific shrnas were used to inoculate huh-7 cells. twenty-four hours later, cells were subjected to selection with 2.5μg/ml of puromycin to assess the lowest lentivirus dose capable of conferring puromycin resistance to 100% of the cell population. selected cell populations were subsequently cultured in the presence of puromycin until lpin1 silencing was ascertained by western-blot using anti-lipin1 antibodies, typically at day 6-7 post lentiviral transduction, time at which all experiments were performed in the absence of puromycin. before execution of all the experiments shown in this study, lipin1 expression was assessed by western-blot. cell viability was determined by a thiazolyl blue tetrazolium blue (mtt) formazan formation assay [47] . control and lipin1-deficient cell lines (5. 10 4 cells/well) were plated onto 12-well plates and were inoculated with d183 virus at a moi 10 ffu/cell. samples of the cells and supernatants were collected 24, 48 and 72 hours post-infection. for multiple cycle infection experiments (moi 0.01), samples of the supernatants were collected at day 3, 5 and 7 post-inoculation. cells were split 1:3 in the multiple cycle infection experiments at days 3 and 5 to maintain the cultures subconfluent. extracellular infectivity titers were determined by endpoint dilution and infection foci counting as previously described [99] . intracellular hcv rna was determined by reverse transcription and quantitative pcr (rt-qpcr) as previously described [99] . total protein samples were prepared in laemmli buffer and separated using polyacrylamide denaturing gel electrophoresis (sds-page). proteins were subsequently transferred onto pvdf membranes and incubated with 5% milk (lipins) or 3% bsa in pbs-0.25% tween20 for one hour at room temperature (rt). primary antibodies against lipin1 (clone b-12; santa cruz), lipin2 (h-160; santa cruz), ns3 (clone 2e3; biofront), beta-actin (ab8226; abcam) and tubulin (clone aa2; sigma-aldrich) were diluted in pbs-0.25% tween20 and incubated for 1 hour (four hours for lipins) at rt. membranes were subsequently washed for 20 minutes with pbs-0.25% tween20 three times. horseradish peroxidase-conjugated secondary antibodies were incubated for 1 hour at room temperature in 5% milk-pbs-0.25% tween20 and subsequently washed three times for 20 minutes at room temperature. protein bands were detected using enhanced chemoluminescence reactions and exposure to photographic films. specific bands were quantitated using the imagej software [100] on non-saturated, scanned films. confocal microscopy was performed with a leica tcs sp8 laser scanning system (leica microsystems). images of 1024 × 1024 pixels at eight bit gray scale depth were acquired sequentially every 0.13-0.3 μm through a 63x/1.40 n.a. immersion oil lens, employing las af v 2.6.0 software (leica microsystems). colocalization indexes were calculated using jacop plugin for image j [101] from a minimum of 10 regions of interest (roi). images were processed using imagej, were medians of 1 pixel were obtained for the different channels, only for illustration, not for analysis. color levels, brightness and contrast were manipulated for illustration using technical and biological controls as reference. total rna extraction was performed using the gtc extraction method [102] . purified rna (10-500 ng) was subjected to rt-qpcr using random hexamers and a reverse transcription kit (applied biosystems). quantitative pcr was performed using 2x reaction buffer from (applied biosystems) and specific oligonucleotides as previously described [99, 103] . standard curves were prepared by serial dilution of a known copy number of the corresponding amplicon cloned in a plasmid vector. control and lipin1-deficient huh-7.5 cells were inoculated with tncc virus (moi 0.05). due to the relatively low propagation levels of the tncc virus in this experimental setup, parallel cultures were infected and treated with 2´-c-methyladenosine (2made; 10μm) to determine the levels of non-replicative, background hcv rna. cells were incubated for 72 hours at 37˚c, time after which samples of the cells were collected to determine intracellular hcv rna levels by rt-qpcr. to establish persistently infected cell cultures huh-7 cells were inoculated at moi 0.01 with jfh-1 hcv strain as previously described [24] . cell cultures were maintained subconfluent for two weeks, time after which infection rates reach nearly 100% of the cells, as assessed by immunofluorescence microscopy. at this point cells were split and transduced with the corresponding lentiviral vectors in order to generate lipin1-deficient cell cultures as well as control cell lines. once silencing had been verified by western-blot, typically at day 6-8 post-transduction, cells were split and samples of the cells and supernatants were collected 24 hours later to determine intracellular hcv rna levels by rt-qpcr and extracellular infectivity titers by end-point dilution and immunofluorescence microscopy. infectious, spread-deficient hcv particles produced by trans-complementation (hcv tcp ) have previously been described [52] . briefly, huh-7.5.1 clone 2 cells expressing core-e1 and e2-ns2 regions from jfh-1 by lentiviral transduction, were electroporated with a jfh-1 subgenomic dicistronic replicon bearing a firefly luciferase gene with reagents kindly provided by dr. ralf bartenschlager (u. of heidelberg). supernatants containing hcv tcp were collected 36, 48 and 72 hours post-electroporation, pooled and assayed for viral infectivity. hcv tcp infection efficiency was determined by inoculating naïve huh-7 cells with the electroporation supernatants and measuring luciferase activity 48 hours post-infection using a commercially available kit (promega). retroviral particle production pseudotyped with different viral envelopes has previously been described [54, 55] with the materials kindly provided by dr. f. l. cosset (inserm, lyon). control and lipin-deficient cell lines were inoculated with hcv pp and vsv pp and incubated for 48 hours, time at which total cell lysates were assayed for luciferase activity using a commercially available kit (promega). a selective hcv entry inhibitor, hydroxyzine pamoate (hdx) from sigma-aldrich (missouri, usa), was used as positive control of inhibition [55] . a plasmid containing the sequence corresponding to a subgenomic jfh-1 replicon bearing a firefly luciferase reporter gene was kindly provided by dr. ralf bartenschlager (u. of heidelberg) [52] . after digestion with the restriction enzyme mlui, the linearized plasmid was transcribed in vitro using a commercial kit (megascript t7; ambion-paisley, uk). the resulting products were digested with dnase and precipitated with licl. pelleted rna was washed with 75% and 100% ethanol, and resuspended in nuclease-free water. in vitro transcribed rna was transfected into the different cell lines together with a plasmid expressing renilla luciferase under a minimal promoter (prl-null; clontech-california, usa) using lipofectamine 2000 and the manufacturer´s recommendations (life technologies-california, usa). firefly and renilla luciferase activities were measured in the sample using a commercial kit (dual luciferase assay system; promega-wisconsin, usa) at different times post-transfection. lipin1-deficient cells were generated by lentiviral transduction of shlpin1-2 shrna. at day 3 post-transduction, control and lipin1-deficient cell populations (5 x 10 4 cells/m24 well) were transfected in suspension using lipofectamine 2000 with plasmids (800 ng/m12 well) expressing wt lipin1beta isoform shlpin1_2-resistant cdna or dxdxt or lxxil motif mutants [31] . transfected cell cultures were incubated for 48 hours and subsequently inoculated at moi 10 with d183 virus. infection efficiency was determined by measuring extracellular infectivity titers 48 hours post-infection. parallel cultures were used to determine relative wt and mutant protein expression efficiency by western-blot. infectivity titers were measured as described above. the relative impact of cdna expression was estimated by determining the ratio between the infectivity found in lipin1-deficient cells and the control cells transfected with the same plasmid. in order to average experiments with different raw infection efficiency, all the experiments were referenced to the ratio in the mock-transfected cells. control and lipin1-deficient huh-7 cells were inoculated with cov-229e (moi 0.01) for 2 hours at 37˚c. cells were washed twice with warm pbs and replenished with dmem-10% fcs. extracellular infectivity titers were determined 48 hours post-infection by end-point dilution and fluorescence microscopy in huh-7 cells. huh-7 cells were inoculated at moi 10 with a recombinant vaccinia virus expressing the t7 phage rna polymerase (vact7) [104] . two hours later, cells were transfected with the plasmid ptm-ns3/5b [16, 58] (kindly provided by dr. lohmann; u. of heidelberg) and lipofectamine 2000 (thermofisher-massachussets, usa) following the manufacturer´s recommendations in terms of total dna per well (typically 4μg per 35mm dishes with 7.5 x 10 5 cells/well) and 50% of the recommended lipofectamine:dna ratio. transfected cells were cultured in the presence of the dna replication inhibitor cytosine β-d-arabinofuranoside (arac; sigma-aldrich) for 16 hours to prevent vact7 replication [105] . when indicated, media was also supplemented with 100nm daclatasvir (dctv). total cell extracts were used to determine viral protein accumulation by western-blot using anti-ns3 antibody (clone 2e3; biofront) and β-actin (abcam; ab8226) as loading control. for ultrastructural electron microscopy studies, control and lipin1-deficient cells expressing ns3-5b polyprotein (see above) were cultured on glass coverslips and fixed in situ after polyprotein expression with a mixture of 2% paraformaldehyde (taab) and 2.5% glutaraldehyde (taab) (1h at room temperature), post-fixed with 1% osmium tetroxide in pbs (45 min), treated with 1% aqueous uranyl acetate (45 min), dehydrated with increasing quantities of ethanol and embedded in epoxy resin 812 (taab). ultrathin, 70-nm-thick sections were cut in parallel to the monolayer, transferred to formvar-coated em buttonhole grids and stained with aqueous uranyl acetate (10 min) and lead citrate (3 min). sections were visualized on a jeol jem 1200 exii electron microscope (operating at 100 kv). quantitation of hcv-induced structures was performed as follows. to quantitate the differences in total vesicle abundance, tem sections were visually inspected under the microscope for the presence/absence of vesicular structures. the number of positive cells and total number of cells were inserted in a 2 x 2 contingency table to determine the statistical significance of the differences between control and lipin1-deficient cells using two-tailed fisher´s exact test or two-tailed chi square test. in addition, we determined the frequency of hcvinduced vesicles in dctv-treated control cells by dividing the number of structures per inspected area and calculating the average and sd of the frequencies found in the different images. the diameters of individual vesicles were determined manually using size-calibrated images and image j software. lipin1-deficient and control cells (7.5 x 10 5 cells) were infected with vact7 virus (moi 10) and transfected with limiting doses of ptm-ns3/5b plasmid (typically 800 ng/ well), as higher plasmid doses may difficult observing the reported differences. cells were lysed by adding 250 μl of tne (50mm tris-hcl ph 7.5, nacl 150 mm and edta 2 mm) buffer containing 0.5% triton x-114 and protease inhibitors (complete; roche-basel, sw). lysates were incubated for 30 minutes on ice before clearing them by 10-minute centrifugation at 12,000 r.p.m. clear supernatants were mixed 1:1 with 60% sucrose tne solution. this mixture was applied on top of a 40% sucrose-tne cushion and was overlaid with 20% and 10% sucrose-tne until completing the discontinuous gradient. gradients were centrifuged for 16 hours at 120,000 x g. fourteen fractions were collected from the top and analyzed by sds-page and western-blot using antibodies against ns3 (clone 2e3; biofront), sigmar1 (s-18; santa-cruz), caveolin-2 (epitomics; 3643-1) and beta actin as described previously [21] . ns3 signal was quantitated using imagej software and the fraction of drm-associated ns3 was determined as the ratio of ns3 signal in fractions 3, 4 and 5 to the total ns3 signal in the gradient. global epidemiology and burden of hcv infection and hcv-related disease immunobiology and pathogenesis of viral hepatitis liver injury and disease pathogenesis in chronic hepatitis c from non-a, non-b hepatitis to hepatitis c virus cure reversion of disease manifestations after hcv eradication risk for hepatocellular carcinoma after hepatitis c virus antiviral therapy with direct-acting antivirals: case closed? the molecular and structural basis of advanced antiviral therapy 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to generate tgn to cell surface transport carriers. cold spring harbor perspectives in biology growth of human hepatoma cells lines with differentiated functions in chemically defined medium highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication interferon modulation of cellular micrornas as an antiviral mechanism production of high-titer helper-free retroviruses by transient transfection the cellular functions of the yeast lipin homolog pah1p are dependent on its phosphatidate phosphatase activity robust hepatitis c virus infection in vitro nih image to imagej: 25 years of image analysis a guided tour into subcellular colocalization analysis in light microscopy single-step method of rna isolation by acid guanidinium thiocyanate-phenol-chloroform extraction expression of the splicing factor gene sfrs10 is reduced in human obesity and contributes to enhanced lipogenesis cytoplasmic expression system based on constitutive synthesis of bacteriophage t7 rna polymerase in mammalian cells transient expression of the vaccinia virus dna polymerase is an intrinsic feature of the early phase of infection and is unlinked to dna replication and late gene expression we are grateful to drs. bartenschlager key: cord-334369-xgw7o5gd authors: innes, elisabeth a.; chalmers, rachel m.; wells, beth; pawlowic, mattie c. title: a one health approach to tackle cryptosporidiosis date: 2020-01-23 journal: trends parasitol doi: 10.1016/j.pt.2019.12.016 sha: doc_id: 334369 cord_uid: xgw7o5gd cryptosporidiosis is a significant diarrhoeal disease in both people and animals across the world and is caused by several species of the protozoan parasite cryptosporidium. recent research has highlighted the longer-term consequences of the disease for malnourished children, involving growth stunting and cognitive deficits, and significant growth and production losses for livestock. there are no vaccines currently available to prevent the disease and few treatment options in either humans or animals, which has been a significant limiting factor in disease control to date. a one health approach to tackle zoonotic cryptosporidiosis looking at new advances in veterinary, public, and environmental health research may offer several advantages and new options to help control the disease. infection occurs through oral ingestion of the oocyst stage of the parasite from contaminated faeces, food, drink, and pasture (for grazing animals), and following ingestion, the sporozoites are released from the oocysts and invade and undergo asexual development in the epithelial cells of the gastrointestinal tract of the host. this is followed by a sexual phase of development resulting in the production of potentially genetically diverse oocysts that are shed fully infective in the faeces. oocysts may also 'hatch' before they are shed from the host, causing reinfection and exponential increases in parasite burden, leading to chronic infection, particularly in immunocompromised hosts [4] . cryptosporidium parasites have a remarkable capacity to reproduce in the host. the rapid multiplication of the parasite in the gut cells causes tissue damage and destruction of the intestinal epithelial cells with stunting of the villi, reducing the absorptive surface of the gut, leading to malnutrition, dehydration, and diarrhoea [5] . during acute infection, and for a week or so following clinical recovery, the infected host will shed very large numbers of infectious oocysts in faeces [6] . work with neonatal calves has shown that one calf can shed around 100 million infectious oocysts in faeces, which is a massive source of environmental contamination and poses an infection risk for other vulnerable hosts [7] . cryptosporidium oocysts are microscopic (most species are 4-6 μm in diameter) and have a tough waxy wall composed of lipids and glycoproteins, which enables the parasite to survive a wide range of conditions, including temperatures from -22°c to 60°c. this wall protects the parasite against many common disinfectants, which makes cryptosporidium hard to control on the farm, in drinking water, swimming pools, and in fresh produce [8, 9] . not all cryptosporidium spp. in livestock are of public health significance, but the species of most zoonotic importance for human infection is c. parvum [1] , which will be the focus for this review. livestock, in particular young calves, are very vulnerable to cryptosporidiosis and a recent modelling study estimated the global load of cryptosporidium parasites in livestock manure to be in the region of 3.2 × 10 23 oocysts per year, with cattle being the predominant source [10] . therefore, improving our understanding of environmental transmission routes of zoonotic cryptosporidium and oocyst survival is important in assessing and mitigating against disease risk and is essential for a one health approach to tackle human and animal cryptosporidiosis. currently, there are no effective vaccines to prevent cryptosporidiosis in humans or in livestock and there are few safe and effective therapeutic options available. cryptosporidiosis is a disease that is well placed for a collaborative interdisciplinary approach to tackle it effectively and previous authors have advocated the benefits of adopting a one health approach [11] . this review will discuss progress made in this area, with greater emphasis on the benefits and opportunities from tackling the disease in livestock, and look at recent advances in scientific technologies that may lead to much needed prevention and control strategies. human infection is usually of the small bowel via the faecal-oral route, although there is growing evidence for respiratory involvement in some populations [12] . gastrointestinal symptoms, glossary calcium-dependant protein kinase 1 (cdpk1): the calcium-dependant protein kinase family are expanded in apicomplexan parasites and are plantlike and do not occur in animal cells, which makes them interesting targets for drug development. crispr/cas9: a technology that enables genome editing by removing, adding, or altering sections of the dna sequence. disability adjusted life years: a measure of overall disease burden, expressed as the number of years lost to ill health, disability, or early death. finished water: water that has passed through a water treatment plant. gnotobiotic: an animal derived by aseptic caesarean section and reared in isolator conditions. gp60: a 60-kda glycoprotein gene of cryptosporidium used as a target for molecular typing studies. hyperimmune colostrum: produced by immunisation of cows during pregnancy, resulting in high levels of specific antibodies in the colostrum, the first milk produced after birth. mesophilic: an organism that grows best in moderate temperature (20°c-45°c). multilocus fragment typing (mlft): multilocus fragment typing uses tandem repeat units within the genome, often called micro-or minisatellites, which are amplified by pcr and the amplicons visualised by gel electrophoresis. oocyst: environmentally resistant transmissive life cycle stage of cryptosporidium spp. organoids: self-organised threedimensional tissue culture organ models derived from stem cells that show realistic micro-anatomy. pk/pd: pharmacokinetics (pk) describes the drug concentration in body fluids and pharmacodynamics (pd) describes the observed effect of the drug. potable: water that is safe to drink or to use in food preparation. rapid gravity filtration system: type of filter used in water purification. thermophilic: an organism that grows best in high temperatures (41-122°c) . zoonotic: pertaining to a zoonosis, a disease that can be transmitted from animals to people. cryptosporidiosis is a significant diarrhoeal disease in both people and animals worldwide. young children and people with compromised immune systems are very vulnerable to disease and the importance of cryptosporidium in human health was first recognised during the aids epidemic in the 1980s [75] . from 2007 to 2017 the global prevalence of cryptosporidium in hiv/aids patients was 10.9% [76] . a large-scale epidemiology study involving 22 500 children in sub-saharan africa and south east asia found that cryptosporidium was a major cause of severe diarrhoea in very young children and was the only gastrointestinal pathogen presenting a significant risk of death [15] . cryptosporidium is also recognised as an important food-borne pathogen [8] , being responsible for more than 8 million cases of food-borne illness annually [77] . burden of disease studies, focussed on acute illness, estimate 4.2 million disability adjusted life years lost in children under 5 years [64] , with longer term sequelae, including growth faltering and cognitive defects. disease impact studies in high-income countries found longer term impacts following acute infection involving persistent abdominal pain, myalgia/arthralgia and fatigue [78] , irritable bowel syndrome [79] , and a recent study has shown a strong association between cryptosporidium infection and human colon cancer [80] . cryptosporidiosis is a major diarrhoeal disease in neonatal calves and other animals. in the cattle industry, production losses associated with the disease include death of the calf, costs incurred in the diagnosis, treatment and supportive therapies employed, and the extra costs of feed and husbandry for the animals to reach market weight [81] . cattle infected with cryptosporidium and monitored from birth to 210 days showed a correlation with infection and a lower live weight gain along with poorer production performance [82] . long-term production impacts on growth and carcase weights following acute cryptosporidiosis in lambs was also reported on australian sheep farms [83] . outbreaks of cryptosporidiosis associated with contaminated water supplies can result in significant economic and health impacts. the large waterborne outbreak in wisconsin affected 403 000 people and was estimated to cost usd 96.2 million [84] . in sweden it was estimated that 50 000 sick leave days were attributed following a waterborne cryptosporidiosis outbreak where the attack rate was 45% of 60 000 residents [85] . detection of cryptosporidium oocysts in public water supplies often results in condemnation of supplies, public notices to boil water, and provision of bottled water. in a recent waterborne outbreak in ireland a boil water notice was put on for 158 days, affecting over 120 432 people and costing around eur 19 million [86] . including profuse watery diarrhoea, abdominal pain, and nausea, typically start between 2 and 10 days after ingestion of oocysts and arise from malabsorption and inflammation [13] . some populations are especially vulnerable to cryptosporidium, and the parasite was included in the world health organization's neglected diseases initiative in 2004 [14] . this was in recognition of the links to poverty, the disproportionate impact on young, malnourished children with subsequent growth and development impairment [15, 16] , and the severe impact on immunocompromised people. long-term health consequences are also now recognised in people (box 1). globally, cryptosporidiosis affects a large number of animal species, including the main livestock animals such as: cattle, sheep, goats, pigs, rabbits, horses, donkeys, water buffalo, camels, and poultry [17] and zoonotic cryptosporidium spp. have also been reported in wildlife species, including rabbits [18] , deer [19] , wild mammals [20] , and fish [21] . infection with c. parvum is one of the most important causes of diarrhoeal disease in neonatal (b6 weeks old) calves in many countries worldwide [22] . the parasite is the most frequently diagnosed cause of neonatal calf diarrhoea in the uk i and c. parvum also causes significant disease in lambs and goats [23] . sheep and goat production is an important agricultural industry in europe, particularly in less favoured areas for agriculture, where they are an important source of meat and dairy products. it is estimated that 98% of the goat population is in developing countries, mainly in africa and asia, where they are an important source of meat, milk, fibre, and fertilizer and help to improve the livelihoods of the world's poorest communities [24] . children often play an important role in tending grazing livestock in many countries and, due to their close proximity to the animals, are very likely to come into contact with cryptosporidium parasites. recent data from china has also shown the presence of c. parvum in yaks, which may pose a public health risk [25] . the parasite causes disease in both dairy and beef herds and the prevalence of infection can be high in young calves, particularly in intensively reared management systems, with various clinical outcomes being reported, ranging from death of the calf to growth retardation and some animals with no obvious clinical signs [26] . the disease can also cause long-term production impacts in livestock (box 1). the minimal infectious dose for neonatal calves is very low, with 17 oocysts causing infection and a correlation of a dose-dependent relationship between oocyst intake and clinical signs [27] . clinical signs in acutely infected calves include yellow watery faeces, reluctance to feed, dehydration, and in severe cases can result in death [22] . the gut takes a few weeks to recover from infection and regain its ability to effectively absorb nutrients [5] . oocyst shedding in the faeces of infected calves can be observed from day 4 postinfection, lasting for a further 10 days to 2 weeks and shedding can occur after diarrhoea has ceased. therefore, asymptomatic animals may also pose a risk of infection to other animals and to people. this is particularly pertinent in managing risk of zoonotic transmission of cryptosporidium at open farms and petting zoos [28] . transmission pathways for zoonotic cryptosporidium oocysts figure 1 (key figure) illustrates some of the main sources and transmission pathways for zoonotic cryptosporidium parasites and further information about transmission sources to people are shown in box 2. environmental contamination with cryptosporidium oocysts is widespread globally and it has been suggested that cryptosporidium poses the biggest pathogen threat to the water industry [29] . of 199 documented outbreaks of human parasitic protozoal disease due to water contamination in the period 2004-2010, cryptosporidium spp. accounted for the majority of outbreaks [30] . how the oocysts reach water courses is not clear, as fate and transfer of oocysts from host faecal material through the environment to water courses is not well understood. most scientists and water industry experts consider this mainly to be due to overland flow, particularly in periods of high rainfall or around intense rainfall events [31] . the effect of climate change (which is predicted to increase winter precipitation and summer extreme precipitation events) on pathogen loading to surface waters was recently explored, but predicted to have limited effect on the risk to public health, using models for catchment pathogen loads [32] . as livestock pastures frequently surround reservoirs ultimately destined for human drinking water, this can cause problems for water providers. the true incidence of human cryptosporidiosis is not known, although the population at most risk is clearly children under the age of five and the immunocompromised. diagnosis requires laboratory confirmation, usually through testing a stool sample [33] . stool samples will not necessarily be tested for cryptosporidium unless the patient falls into a selected group (e.g., young children; hiv-aids; recent foreign travel) or a request is made. traditionally, stained microscopy is used to observe oocysts, and infection rates among (presumably immunocompetent) diarrhoea patients of 6.1% and 2.1% in developing and developed areas, respectively, have been reported, contrasting with 24% and 14% among hiv positive diarrhoea patients [34] . differences in the sensitivity, specificity, and the positive predictive value of the diagnostic tests can impact burden of disease estimates; in the global enteric multicentre study, an improved elisa was used and revealed the significant contribution of cryptosporidium to childhood morbidity and mortality [16] . differences in access to health care, sample submission, and diagnostic practice contribute to the ascertainment of cryptosporidiosis and, along with variation in reporting requirements, make interpretation of surveillance data within and between countries difficult [35] . in 2016, there were 3.8 confirmed cases per 100 000 population reported in the eu/eea by 21/32 countries [36] . one study in the uk that measured under-ascertainment found that for every case reported to national surveillance there were an estimated 8.2 undiagnosed cases in the community [37] . diagnosis of cryptosporidium infection in livestock is traditionally done by the direct examination of faecal samples using microscopy and modified ziehl-neelsen or auramine phenol staining. people may become infected directly by ingestion of infective oocysts through the faecal-oral route. this may be occupational [87] , through contact with infected animals [88] , or indirectly through the consumption of contaminated drinking water [89] or food [8] . proportional source attribution data are sparse for cryptosporidium. one study in canada attributed source to sporadic cryptosporidiosis cases from exposure data and found that water was the most commonly reported likely source of infection (48% of cases), followed by contact with livestock (21%), person-to-person contact (15%), food-borne transmission (8%), and contact with pets (8%) [90] . more recent expert elicitation generally concurs with this order [91] . general social and economic zoonotic risk factors have also been identified for human cryptosporidiosis. c. parvum was more common in areas with lower human population densities, where there are a higher ratio of the number of farms to human inhabitants, or a higher ratio of the number of private water supplies to human inhabitants and in areas with high ruminant livestock density [92] . in england and wales, c. parvum was linked to living in an area with a high estimate of cryptosporidium applied to land from manure [93] . additionally, through the identification of the infecting species and subtypes, the variable transmission and epidemiology of cryptosporidiosis becomes clearer and interventions can be directly applied, whether these are broadly hygiene and sanitation measures on farms, in homes, and institutions, or in potable or recreational water and the food chain. although most c. parvum subtypes are zoonotic, a subspecies that is human-adapted has been proposed as c. parvum anthroponosum [94] . geographical analysis indicates that the human-adapted species (c. hominis, some c. parvum subtypes) are relatively more prevalent in resource-poor countries, whereas zoonotic c. parvum dominates in north america, europe, australia, and parts of the middle east [95] . transmission varies seasonally; in most european countries cryptosporidiosis cases are mainly reported in late summer and autumn (august-october) [35] , with some countries also having a smaller peak in spring. there is good evidence from outbreaks and molecular analytical epidemiology that the spring peak is mostly attributable to c. parvum and may be related to lambing and calving, while the summer/autumn peak is mainly c. hominis and is associated with swimming pools and foreign travel [96] . immune chromatography lateral flow assays are used to give a rapid diagnostic and are widely used for point-of-care testing on farms. molecular assays are essential to give further information on parasite species and genotype [38] . different cryptosporidium species can be detected using multiplex pcr assays [39] or by direct sequencing but this is not done as a routine diagnostic test. molecular-based diagnostics are widely used in commercial labs, often using real time pcr (that is helpful to detect low concentrations of oocysts) and are increasingly used in human diagnostics. diagnostic epidemiology studies on a whole catchment scale are essential when considering riskbased water safety assessments [40] . such studies are scarce but have shown links between livestock and wildlife c. parvum infection with water contamination. human infection through contamination of the public water supply has resulted in outbreaks and hospitalisation of consumers. in one study in scotland, where this had occurred, further investigation showed that livestock and wild red and roe deer were contributing to catchment parasite loading and water contamination [19] . another catchment study in cumbria, uk, concluded that the distribution of cryptosporidium species in surface waters, livestock, and wildlife were also linked [40] . gene sequencing and pcr-restriction fragment length polymorphism analysis showed the presence of the same c. parvum strain in water and faecal samples collected from neonatal calves on dairy farms in parana, brazil, highlighting the risk of zoonotic strains contaminating water supplies used for human and animal consumption [41] . a recent study in australia showed the presence of zoonotic cryptosporidium spp. in livestock and wild mammals in water catchments [20] . in addition, c. parvum has been detected in edible marine fish in european waters, indicating the extent of environmental contamination with this zoonotic pathogen [21] . application of genotyping tools assignment to species level is useful in determining zoonotic potential of the parasite, but to determine transmission dynamics and source of infection, more discriminatory power is required [42] . genotyping within c. parvum has mainly been based on a single locus through sequencing of a polymorphic region of the gp60 gene, which has a putative role in virulence. whilst this is a useful library typing tool, it does not provide adequate differentiation for local or regional epidemiological questions, such as outbreak investigations. a literature review of multilocus genotyping schemes for c. parvum showed that these are usually based on single nucleotide polymorphisms or on micro/minisatellite regions [43] . in multilocus fragment typing (mlft), repeat units within the genome (micro/minisatellites) are amplified and either sequenced or analysed for size, reflecting the length polymorphisms due to variable numbers of repeat motifs, which forms the basis for genotyping. alleles at different loci, or markers, are combined to give a multilocus genotype. robinson and chalmers [43] favour fragment sizing for surveillance and outbreak investigations, due to the potential to provide rapid, cost-effective results that are discriminatory enough to address source attribution. work in bovine-derived c. parvum assessed an mlft typing tool and demonstrated good discriminatory ability compared with gp60 sequencing alone [44] . it would be desirable for a one health approach to develop an integrated genotyping approach that could be used by both veterinary and public health researchers, as advocated in a recent eu cost action ii . the markers that mlft is based on are single copy genes and as such this approach has limitations for environmental samples where there is likely to be a low quantity of c. parvum dna present. this presents a problem for whole-catchment approaches, where mlft could be a useful tool in determining c. parvum transmission dynamics and innovative application of amplification technologies could be used to overcome such barriers. there are currently few options to help prevent and treat cryptosporidiosis in humans with no vaccines and only one fda approved drug, nitazoxanide, available. nitazoxanide has efficacy in immunocompetent people but is not effective in severely immunocompromised individuals [45] . education about routes of transmission and hand washing are practical preventative environmental public health measures to help reduce disease incidence [46] . due to the lack of vaccines for cryptosporidiosis in livestock, prevention and control of disease currently involves supporting the resilience of calves in the first few weeks of life. this will include making sure they receive adequate quantities of good quality colostrum in the first few hours of life [47] and housing in clean, dry, and warm pens with raised feeding and water troughs to minimise exposure to cryptosporidium parasites in the environment. deep and clean straw bedding will also help minimise contact with contaminated faeces and regular cleaning out of calf pens along with steam cleaning, as oocysts are inactivated at temperatures above 60°c [48] . disinfection with recommended products can help to reduce build-up of contaminated faeces on the farm, where disinfectants containing hydrogen peroxide are the most effective [22] . a recent study has also suggested that disinfection of calf pens with hydrated lime may help to reduce onset and severity of cryptosporidiosis in calves [49] . it is advisable to house calves in similar age groups, as the younger calves are more susceptible to disease and may be vulnerable to infection if they are moved into an environment contaminated by older calves. calves affected by cryptosporidiosis should be housed separately until at least 1 week after diarrhoea stops, as the animal may still be shedding oocysts. calves can be given supportive rehydration therapy and kept warm, clean, and dry. there are currently two licenced treatment products for use in calves; one is halofuginone lactate [50] , which is used as a preventative treatment and is given within 24 hours of the onset of clinical signs and continued for 7 days. the drug acts to reduce shedding of the parasite and to reduce the severity and duration of diarrhoea, although it can be toxic in dehydrated animals so it needs to be used appropriately. paromomycin [51] is recently available in uk under veterinary prescription and can be applied for 7 days, following a diagnosis of cryptosporidium infection, and has been shown to reduce oocyst shedding and diarrhoea, although there is also a risk of toxicity with this drug. treatment of manure to reduce viability of cryptosporidium a recent study used spatially explicit process-based modelling to estimate the global cryptosporidium loads in livestock manure and estimated this to be in the region of 3.2 × 10 23 oocysts per year, with cattle being the predominant source [10] . as manure is applied to land, oocysts can be transported via run-off into surface waters and may be a source of infection to both animals and people. asia has the highest oocyst load from livestock manure, followed by africa, south america, and europe [10] . the effective management of manure and slurry on farms to reduce viability of cryptosporidium oocysts will have an impact on disease risk in the wider environment and in particular for water catchments [52] . such on-farm practices include the proper composting of manure, as heat (n60°c) will inactivate the oocysts; slurry storage, as ammonia and low ph will help to inactivate oocysts; and treatment with mesophilic and thermophilic anaerobic digestion to significantly reduce oocyst viability [10] . fencing of livestock away from streams and water courses and use of vegetated and riparian buffer strips can help to slow down the transfer of cryptosporidium oocysts from livestock faecal matter into water courses [52] . applying measures to prevent and control cryptosporidiosis in livestock will have significant benefits for livestock health and welfare and will increase the efficiency of production, bringing economic benefits to livestock farmers in many regions of the world. in addition, applying methods on farm to minimise the environmental contamination with faeces containing infective cryptosporidium oocysts will also help to minimise risk to other animals and to people through protection of the environment and water catchments (figure 2 ). there has been considerable recent investment in cryptosporidium drug discovery and there are now several classes of compounds of lead, late lead, and preclinical status. these include bumped kinase inhibitors that target cryptosporidium calcium-dependant protein kinase 1 (cdpk1) and are effective in several in vivo models of cryptosporidiosis, including gnotobiotic piglets and calf models, which showed a reduction in oocyst shedding and clinical signs [53, 54] . compounds that target trna synthetases, enzymes that charge trnas with their cognate amino acid for use in protein synthesis, have also proven effective against cryptosporidium [55] . several groups carried out screens of compounds with activity on related parasites and identified new anti-cryptosporidial compounds [56] . these compounds progressed from in vitro screens, to small animal models, and to neonatal calf studies. because c. parvum infects both humans and cattle, new medicines for treating human patients may also be effective for controlling livestock cryptosporidiosis. a major question in drug discovery is a requirement for gastrointestinal versus systemic exposure. because cryptosporidium are usually located in the brush border epithelium of the gastrointestinal tract, systemic exposure may not be required to cure the infection [57] . current drug efforts are working to better understand the pk/pd distribution required to cure cryptosporidiosis. modelling this for both human and cattle will be important in developing effective treatments. vaccines for livestock? there are currently no vaccines available to prevent cryptosporidiosis in farm livestock and as the disease mainly occurs in very young calves it might be difficult to generate protective immunity to the parasite quickly enough through active vaccination. an alternative strategy such as that currently used to vaccinate dams against other pathogens causing diarrhoeal disease (e.g., rotavirus, coronavirus, and escherichia coli) may also work for cryptosporidium by generating specific immunity in the dam, which is passed to the calf through the colostrum, thus helping the calf to resist the disease during these crucial early weeks of life [58] . promising results using a passive immunity approach have been achieved through immunisation of dams with recombinant p23 and cp15 proteins to generate hyperimmune colostrum containing specific antibodies and other immune factors that can help protect neonatal calves against disease caused by cryptosporidium infection [59] . the protective immune mechanisms in calves against cryptosporidium parasites are not well understood and are likely to involve both humoral and cell-mediated immune responses [22, 58] . antibody responses against several dominant cryptosporidium antigens, such as gp15, cp15, and cp23 that are thought to play a role in parasite attachment and invasion of host cells, may be important as vaccine candidates [60] . both animals and humans develop protective immunity after exposure to and recovery from cryptosporidium infections. understanding what drives immunity will aid development of much needed vaccines. recent work by sateriale et al. [61] developed a new mouse model to dissect immunological responses to cryptosporidium. they isolated a mouse-specific cryptosporidium tyzzeri strain that infects the small intestine of immunologically competent mice, closely mimicking human infection. this model is genetically tractable for both the host and parasite, and promises to provide mechanistic understanding of protective immunological responses that can drive rational vaccine design. an effective vaccine that would minimise disease in livestock and reduce shedding of oocysts in faeces would have a significant impact on livestock health and welfare and also would reduce the contamination of the farm environment and wider catchment areas with infective oocysts [58] . a large outbreak of cryptosporidiosis that occurred in scotland in 2000 was associated with drinking water supplied from loch katrine and the catchment area surrounding the loch had a large sheep and cattle population. livestock were removed from the area by the water authorities as a precaution and a rapid gravity filtration system was introduced, which successfully reduced the cryptosporidium oocysts from the finished water [62] . an important example of health authorities working together with farmers to help protect water catchments is one of the largest water supply systems in the usa, providing over 9 million people in the new york city area with 4.5 billion litres daily from a single source of unfiltered water. farmers in the catskills area had intensified livestock production, leading to increased pollution from faeces and manure getting into the water shed [63] . the new york city authorities worked with the farmers to set up a whole farm planning system to customise pollution control on each farm to maximise effectiveness and minimise cost iii . by continuing to work effectively with the farmers, the new york city authorities were able to protect the environment in the water catchment area and avoid the multi-billion dollar cost of filtering the water supply, illustrating very well how a managed environment will produce good quality water. adopting a one health approach offers several opportunities to help tackle cryptosporidiosis, in particular, disease caused by c. parvum and other zoonotic species that affect both animals and people. the impact of the disease is far reaching and recent studies have highlighted the longterm consequences of clinical disease in both humans and livestock animals (box 1). the true burden of disease in both public [64] and veterinary health is unknown, as many incidences of infection and disease go undiagnosed or unreported. with livestock farming moving towards increasingly efficient and sustainable production to minimise impact on the environment, there is much to be gained from tackling cryptosporidiosis. as livestock are also a major source of infectious and long-lived oocysts that can infect other animals, wildlife, and people [22] , measures to reduce oocyst shedding and methods of storing and treating farm manure [10] to prevent oocyst contamination of the wider environment are important mitigation strategies. new diagnostic targets rapid point-of-care detection is needed to enable quick and effective intervention and treatment. in addition, an integrated genotyping approach, which could be used by both veterinary and public health researchers [44] , would help in disease surveillance identifying sources of infection and routes of transmission, examine evolving epidemiology patterns, and aid in the analysis of parasite virulence. sensitive detection and genotyping methods are required when looking at environmental samples that may contain very few oocysts and a test to confirm oocyst viability would be highly desirable for analysis of infection risk. a recent eu research project involving research organisations and industrial partners across europe looked at emerging technologies for early detection of waterborne pathogens, including cryptosporidium, to enable improved water safety and public health (aquavalens, 2017 iv) . collaboration and cooperation between countries on surveillance strategies and techniques would be of great benefit to enable sharing of information and disease epidemiology data. protective immunity and prospects for vaccination developing vaccines for use in livestock to prevent disease and reduce oocyst shedding would be a key priority for a one health approach. this intervention would not only benefit animal health and welfare, it would also have additional benefits to reduce parasite contamination of the environment through reduced oocyst shedding and would therefore also benefit public health [58] . the most feasible approach would be to target cattle where the parasite is highly prevalent in the first instance, as they are the most significant contributors to contaminated manure globally [10] . there are currently no human vaccines available and this would be a very desirable target, particularly for high-risk groups such as young children in developing countries and immunocomprised individuals, where infection with the parasite can have very severe clinical consequences. the feasibility of a vaccination approach has been supported by studies showing the development of a degree of resistance to the parasite following repeated exposure [65] . in addition, sero-epidemiology studies in scotland looked at the prevalence of antibodies against cryptosporidium sporozoite antigens in populations before and after the introduction of enhanced filtration to remove cryptosporidium oocysts for the water supply and found a significant reduction in seroprevalence, indicating a potential reduction in immune resilience to the trends in parasitology parasite [66] . several promising candidate antigens have been identified, along with knowledge of likely protective immune responses and different vaccine delivery strategies to target mucosal immune responses, recently reviewed [60] . of interest is the level of cross-protective immunity between c. hominis and c. parvum, as this will be an important consideration for vaccine design. a serological study was conducted in children in bangladesh and found that although most children were infected with c. hominis, they also had cross-reactive antibodies to the c. parvum antigen cp23 [67] . a further study looked at cross-protective immunity between the two species using an in vivo gnotobiotic pig model and showed than prior infection with c. hominis protected against a further c. hominis challenge and gave partial protection against a c. parvum challenge [68] , showing that there is a degree of cross-immunity between the two cryptosporidium species. the gnotobiotic piglet model shows similar clinical signs of diarrhoea as seen in human infection following challenge with c. hominis and is therefore a relevant clinical model with which to test out new therapies and vaccines [69] . neonatal cattle [27] and sheep [70] are also relevant clinical models for c. parvum in particular, to improve understanding of host-parasite interactions, to test out novel therapeutic agents, and to provide sources of cryptosporidium oocysts for research and diagnostic purposes. recently, there has been some exciting progress on the in vitro culture of cryptosporidium parasites using hollow-fibre technology [71] and the demonstration of the complete life cycle of c. parvum when cultured in human and mouse intestinal organoids [72, 73] . heo et al. microinjected cryptosporidium into the lumen of three-dimensional human-derived organoids [72] , while wilke et al. infected monolayers of mouse-derived organoids cultured at an air-liquid interface in transwells [73] . both culture systems support long-term growth (n20 days) and produce viable, infectious oocysts. additionally, wilke et al. generated transgenic parasites in vitro in organoid co-culture and used these parasites to perform a genetic cross [73] . these advances will help to reduce the numbers of animals used in research and for production of oocysts. important advances have also been made in the genetic engineering of cryptosporidium parasites [74] . use of crispr/cas9 in cryptosporidium has enabled the generation of reporter strains to advance drug discovery. manipulation of genes of interest allows us to interrogate the function of genes directly in the parasite for the first time. further tool development, including protein tagging and conditional knockdown, will improve our understanding of the parasite biology and host pathogen interactions [74] . these new technologies are very exciting, as both host and parasite are accessible and tractable for fundamental research, and provide more relevant models for drug and vaccine discovery. similarly, other hosts and species of cryptosporidium may be amenable to culture and laboratory exploration in the future. this would be especially impactful for c. hominis, which lacks an in vitro system and relies on a resource intensive gnotobiotic piglet animal model. along with increasing awareness and understanding of the impact of cryptosporidiosis in people and animals, there is a renewed impetus to tackle cryptosporidiosis. understanding key transmission routes and main sources of infection can also help to develop mitigating strategies to prevent direct infection and to protect against indirect transmission through water, food, and the environment. cryptosporidium really lends itself to a one health approach and with the recent technological advances and increased awareness of the long-term impacts for people, animals, and our natural environment, the opportunity exists to work collaboratively across the different sectors to tackle this global disease (see outstanding questions). a key target would be the development of a outstanding questions would a vaccine relying on passive transfer of immunity be effective in neonatal calves to reduce incidence of cryptosporidiosis and shedding of oocysts into the environment? would an integrated genotyping approach in veterinary and public health help with source tracking, epidemiology, and surveillance? what treatments would be effective to reduce viability of cryptosporidium oocysts in manure? will some of the new anti-cryptosporidial drug compounds identified be effective in both people and livestock? will the application of crispr/cas9 in cryptosporidium enable the manipulation of genes of interest to facilitate the direct interrogation of the function of specific genes? trends in parasitology vaccine that could be used to help prevent disease and oocyst shedding in neonatal calves, as this would not only improve the health and welfare of the animals, it would also reduce environmental contamination as these animals are a main reservoir of c. parvum oocysts. due to the very young age of the calves when they first encounter the parasite, a vaccination approach involving passive transfer of immunity in colostrum is most likely to be successful. applying an integrated genotyping approach that may be used in both veterinary and public health would aid in source tracking and surveillance and inform epidemiology studies. treatment of livestock and human faecal waste to reduce viability of cryptosporidium oocysts would help to minimise contamination of the environment with infectious parasites and protect human and animal health. there have been some very exciting new advances in research into cryptosporidium parasites, involving the development and application of relevant clinical in vivo models of disease, new in vitro systems, and the ability to conduct genetic manipulation studies. these will greatly advance our knowledge of host-parasite interactions and help facilitate the testing of new therapeutic compounds and vaccine targets. by combining our knowledge and expertise from the veterinary, public, and environmental health areas, along with the new advances in biology, genetics, and host-pathogen 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england and wales we would like to thank hazel simm at moredun research institute for graphic design. key: cord-302379-jh6jxwyn authors: jevon, phil; abdelrahman, ahmed; pigadas, nick title: management of odontogenic infections and sepsis: an update date: 2020-09-25 journal: br dent j doi: 10.1038/s41415-020-2114-5 sha: doc_id: 302379 cord_uid: jh6jxwyn the management of odontogenic infections has improved over recent decades, but further improvements are still required. the ongoing education of gdps and their dental teams on this issue continues to be important, especially during the current covid-19 pandemic, where remote triage poses additional difficulties and challenges. odontogenic infections can lead to sepsis, a potentially life-threatening condition caused by the body's immune system responding in an abnormal way. this can lead to tissue damage, organ failure and death. a patient with non-odontogenic-related infection could also present with sepsis at a dental practice. early recognition and prompt management of sepsis improves outcomes. gdps and their dental teams should be trained in the recognition and management of sepsis. age-specific sepsis decision support tools have been developed by the uk sepsis trust to help dental staff recognise and manage patients with suspected sepsis. the aim of this article is to provide an update on the management of odontogenic infections and sepsis. although the management of odontogenic infections has improved over recent decades, further improvements are needed and the ongoing education of gdps and their dental teams on this issue is essential. in addition, the covid-19 pandemic has imposed new difficulties and challenges; for example, telephone triage and prescription of antibiotics, and it is important to be up-to-date with current guidelines. 1, 2 odontogenic infections can lead to sepsis, 3,4 a potentially life-threatening condition caused by the body's immune system responding abnormally. this can lead to tissue damage, organ failure and death. 5 a patient with nonodontogenic-related infection could also present with sepsis at a dental practice. early recognition and prompt effective treatment of sepsis improves outcomes. 5 the dental team should be trained in the principles of the management of sepsis. 6 agespecific sepsis decision support tools have been developed by the uk sepsis trust 7 to assist the dental team to recognise and manage patients with suspected sepsis. the aim of this article is to provide an update on the management of odontogenic infections and sepsis in the dental practice. the morbidity and mortality rate of odontogenic infections has dropped significantly over the past 70 years. 8, 9, 10 this dramatic drop is undoubtedly linked to the discovery of antibiotics, the improvement of the general population health standards, and a better understanding of appropriate medical and surgical management of these cases. further improvements are needed and ongoing education of the dental team on this issue is very important. odontogenic infections pass through three key stages: 11 • stage 1: 1-3 days; soft and mildly tender swelling • stage 2: 2-5 days; hard, red and severely sore swelling • stage 3: 5-7 days; abscess formation. there is a strong belief that once the abscess has formed, surgical drainage is mandatory to achieve resolution. 12 medical management has a role in selected cases. 13 seven principles have been proposed to achieve the best outcome in managing odontogenic infections: 11 1. establish the severity of the infection 2. assess host defences 3. elect the setting of care 4. surgical intervention 5. medical support 6. antibiotic therapy 7. frequently evaluate the patient. a careful history and thorough clinical examinations are essential to determine the severity of any infection. history-taking will highlight factors like immune system competence and the level of systemic reserves to fight infections. a physical examination can identify clinical observations outside normal limits. several clinical and haematological parameters have been used as prognostic indicators for the severity of the infection. c-reactive protein (crp), fever and anatomical locations have been investigated for the assessment of the extent of odontogenic infections and presumed duration of hospital stay. 14 additional factors must be considered to establish the infection severity: • anatomical location • airway compromise. there are a number of potential spaces between the musculoskeletal head and neck structures and the regional fasciae and organs better known as fascial spaces. a summary of these spaces and their level of risk 11, 12 is found in table 1 . in healthy and systemically well patients without trismus, infections of low-risk spaces can be initially treated in a primary care dental practice, while infections spreading to higher risk spaces should be managed more aggressively and may need to be treated in a secondary care centre. ludwig's angina was described by karl friedrich wilhelm von ludwig in 1836 as a rapidly and frequently fatal progressive gangrenous cellulitis and ooedema of the soft tissues of the neck and floor of the mouth. 15 thankfully, mortality rates have reduced significantly with the introduction of antibiotics, improved oral and dental hygiene, and timely surgical intervention. 15 the majority of ludwig's angina infections are odontogenic; 16 peritonsillar or parapharyngeal abscesses, mandibular fractures, oral lacerations/piercing and submandibular sialoadenitis are other recognised causes. 15 a compromised airway is synonymous with ludwig's angina and the initial assessment of a patient with ludwig's angina should follow the familiar 'airway, breathing, circulation, disability, exposure' (abcde) approach. signs of a compromised airway in these patients could include noisy (gurgling) breathing with drooling saliva, stridor, dyspnoea, tachypnoea, tachycardia, dysphagia and trismus. the initial immediate management usually includes positioning the patient in an upright position and administering oxygen 15 litres/minute, 15 while colleagues call 999 for an ambulance. a healthy immune system is essential to the maintenance of host defence against infection. multiple medical conditions can affect it. 17 box 1 summarises the common factors that can cause immune system compromise. the concept of 'physiologic reserve' represents a significant driver of outcome in patients fighting infection. this can be defined as the capability of an organ to carry out its activity under stress. 18 age is an essential factor that is inversely related to the physiologic reserve; that is, decreased respiratory, cardiovascular and metabolic reserve. 19 elect the setting of care an uncomplicated localised dental abscess in a healthy young person, who does not show signs and symptoms of a worsening immune response, can be safely treated in a dental practice. early and adequate intervention is essential in order to prevent avoidable deterioration with invasion of adjusted anatomical spaces and symptoms of sepsis ( fig. 1) . similarly, severe neck infection in an immunocompromised elderly person warrants treatment in a secondary care setting. the clinical decision to choose the setting of care is not always straightforward though, prompting the need for clear secondary care referral criteria. although there are no agreed national guidelines on when to admit to a secondary care setting, criteria for hospital admission have been proposed 2,20 (box 2). a careful history, thorough clinical examinations and a high index of suspicion will enable the gdp to diagnose and appropriately manage patients presenting with odontogenic sepsis. early surgical intervention has been advocated to improve the clinical outcome of odontogenic infection. the dramatic improvement in the outcome of sever odontogenic infection is directly linked to the immediate establishment of a safe airway, followed by early surgical intervention. once the airway has been deemed patent and not at risk of being compromised, in either a hospital setting or dental practice, the principles of management are very similar. thorough knowledge of head and neck anatomy will enable the surgeon to access the abscess cavities using incisions in safe places without damaging any vital structures like blood vessels or nerves. most of the odontogenic infections can be drained through intraoral access. five principles must be followed: 11 21 although abscess formation takes place between the fifth and seventh days, early elimination of the infection source and surgical intervention will decompress the involved anatomical spaces. 14 relying on antibiotics only in relieving dental infection is likely to be less effective and can cause antimicrobial resistance. 22 two of the challenges to performing adequate drainage of any odontogenic infection in dental practice are: 23 1. achieving adequate local anaesthesia 2. risk of spreading the infection to other anatomical spaces. the ability to deliver safe, adequate local anaesthesia is essential for any dental procedure. the mechanism of action of the local anaesthetic solution depends on the tissue ph. in the presence of infection, tissue ph becomes more acidic, which slows down the degree of ionisation, resulting in less optimal or failed anaesthesia. 23 to overcome this problem, the injection of the anaesthetic solution at a distance from the inflammatory site is required (nerve blocks). it will also avoid infection spread to different tissue spaces. although surgical drainage is the classic approach to most of the odontogenic infection, medical support has a critical role in controlling the disease. 24 adequate hydration, nutrition and control of fever are essential to optimise the medical care for patients presenting with odontogenic infections. stabilisation of any underlying systemic disease (for example, uncontrolled diabetes) is extremely important. 24 box 2 criteria for referral to secondary care 2, 20 • difficulty in swallowing and dehydration first-line antibiotics for dental abscess in dental practices (adults and children more than 12 years) odontogenic infections are multi-microbial with a combination of facultative and anaerobes species. facultative streptococcus viridans group are commensal gram-positive bacteria and include s. anginosus, s. intermedius and s. constellatus. these organisms are abundant in the mouth and most frequently associated with orofacial cellulitis and abscess. after a few days, the anaerobes (prevotella and porphyromonas) predominate. the majority of the facultative streptococci that cause odontogenic infections are sensitive to penicillin. 25 approximately a quarter of strains of prevotella and porphyromonas are penicillin-resistant. 26 the scottish dental clinical effectiveness programme (sdcep) has published evidencebased guidance on antibiotic prescription in dental practice. penicillin-based antibiotics remain the first line for the treatment of odontogenic infections. metronidazole is effective against anaerobic bacteria. 1, 27, 28 the antibiotic doses recommended in the sdcep's guidance are based on the doses recommended by the british national formulary (bnf) 13 (table 2 ). in secondary care settings, the antibiotics are prescribed in accordance with the local hospital antimicrobial therapy. consultation with the on-call microbiologist is a common practice for severe cases and cases which are not responding to first-line treatment. the last principle, but as vital as the previous ones, is the periodic re-evaluation of these patients. in outpatient settings, the recommended follow-up is after two days. 29 forty-eight hours will allow the drainage to cease and the immune system to overcome the initial insult from the infection. if no improvement or deterioration of symptoms is noted, further escalation in care must be provided. the review interval, however, depends on the clinical course of the infection. a patient with a rapidly developing swelling and mild temperature may need review within 24 hours, but a patient with a chronic abscess and no systemic symptoms will need to be reviewed at the end of the antibiotic treatment. causes of treatment failure include: • failure to remove the source of infection • underlying systemic disease; for example, uncontrolled diabetes • antibiotic-related factors -patient non-compliance, drug not reaching site secondary to inadequate drainage, wrong antibiotic choice or incorrect dose. in hospital settings, more frequent evaluations are essential as the disease is expected to be more aggressive. the covid-19 pandemic has dramatically changed dental practice since march 2020. guidance on the management of acute dental problems is available. 2, 30 this is likely to change as the situation evolves. advice, analgesia and antimicrobials (when indicated) should form the basis of primary care dental triage when using remote consultation (telephone call or video call). 1 while assessing the patient, covid-19 status should be established and documented, as this will determine how the patient's care will be managed should referral to an urgent dental care centre or secondary care be required. patients should be advised that dental treatment options are currently severely restricted and that they should call back in 48-72 hours if their symptoms have not resolved. 2 the sdcep's flowchart (fig. 2) helps the remote management of patients by guiding the gdp to categorise the patient into one of three management groups. 2 the sdcep has also recently updated their drugs for the management of dental problems worryingly, there has been a rise in anecdotal reports of antibiotics apparently being overprescribed for dental pain since the outbreak of covid-19. 31 this pandemic has demonstrated the havoc a pathogen can unleash when we have no protection against it. inappropriate use of antibiotics increases the likelihood that resistant bacteria will evolve 31 and it is essential that gdps remain guardians against antimicrobial resistance. 1,32 antibiotics should only be prescribed if it is likely that the patient has a bacterial infection, and the principles of prescribing and follow-up (as detailed earlier) should be followed. it is estimated that 234,000 patients develop sepsis in the uk every year, 5 with 70% of sepsis cases originating in the primary care setting. annually, there are approximately 44,000 deaths from sepsis in the uk 5 and six million deaths worldwide. 33 although deaths from sepsis due to odontogenic infection are very rare, they have been reported. 34 the incidence of sepsis is on the increase, possibly due to: 35 • a growing elderly population • an increased use of invasive surgery • an increased incidence of bacterial resistance • an increased number of immunocompromised patients. a localised infection which progresses into an uncontrolled systemic response is usually the cause of sepsis. progression to acute physiological deterioration with the risk of multiple organ failure and death can be swift. • pneumonia: 50% • urinary tract: 20% • abdomen: 15% • skin, soft tissue, bone and joint: 10% • endocarditis: 1% • device-related infection: 1% • meningitis: 1% • others: 2%. in normal circumstances, the body's immune system will prevent or fight infection (bacteria, viruses, fungi). however, the immune system can sometimes go into overdrive, resulting in vital organs and other tissues being targeted. this can result from any injury or infection in the body. although a wide variety of different microorganisms (for example, streptococcus, e. coli, mrsa or clostridium difficile) can cause sepsis, it is usually caused by common bacteria that don't normally make patients ill. 36 any infection can lead to sepsis (box 3), though pneumonia (commonly referred to as chest sepsis) is the cause in half of the cases. 5 the national institute for health and care excellence (nice) 6 the uk sepsis trust has developed age-specific sepsis decision support tools to assist the dental team to assess both adult and paediatric patients who may have sepsis. 7 utilisation of these sepsis decision tools will help determine if red flag sepsis (see below) is present, prompting appropriate timely action. the prompt transfer of patients presenting with orofacial infections suspected of sepsis to an acute hospital setting for early treatment should ultimately improve sepsis survival rates. 37 the care quality commission (cqc) 38 endorses these sepsis decision tools and, ideally, all three should be readily available in the dental practice. the 'gdp sepsis decision support tool for primary dental care' (fig. 3) should be applied to all adults and young people aged 12 years and over with fever (or recent fever), symptoms presenting with a source of orofacial/dental infection (including post-operative infection) or have clinical observations outside normal limits. 7 it details what to look out for if the patient has presumed infection and, in particular, what constitutes red flag sepsis. red flag sepsis is a definition from the uk sepsis trust which lists a set of easyto-assess clinical parameters, the presence of one of which in the context of infection identifies sepsis with a high risk of death and a requirement for urgent treatment (fig. 3) . 7 if red flag sepsis is present: there are two paediatric sepsis decision tools, one for children aged 5-11 years (fig. 4) and one for children <5 years (fig. 5) . these should be used in children who have a suspected source of orofacial/dental infection (including post-operative infection) or have clinical observations outside the normal range. 7 the paediatric sepsis decision tools take into account paediatric considerations, including differences in paediatric physiology. covid-19 infection can cause sepsis on its own. 5 unfortunately, the sepsis signs and symptoms for a number of initial conditions can be very similar. this stresses the importance for dental teams to be familiar with sepsis and the decision tools described here for safe management of such patients. in addition, evidence suggests that, for a period of time following sepsis, patients may be vulnerable and develop further infections including covid-19; therefore, they have an increased risk of readmission with infective complications (including sepsis). 5 the nice advises that patients with suspected sepsis are assessed following a structured set of observations to stratify the risk of acute illness or death. 6 the royal college of physicians' national early warning score (news) 2 is widely used by the ambulance service and in hospitals, and reliably detects deterioration in adults, triggering review, treatment and escalation of care, particularly sepsis. 41 although news2 hasn't yet been validated for use in primary care, nhs england is encouraging its widespread use in this sector. 42 the cqc has created a webpage titled 'dental mythbuster 25: sepsis' on its website, 38 providing helpful information relating to the management of sepsis in the dental practice, including online links to professional guidelines (nice and uk sepsis trust) as well as what to expect from the cqc, relating to sepsis, when they review dental practices to determine whether they are safe and well-led. when reviewing dental practices, the cqc will ask dental staff what systems and processes are in place to manage a patient with a bacterial infection, including procedures for follow-up and referral for specialist care when necessary. this will include treating patients who: • are not responding to conventional oral antibiotic treatment • cannot have their infection drained at an initial appointment. the cqc will also ask what advice is given to patients, including when they should seek emergency advice or treatment, if symptoms worsen or when the dental surgery is closed. odontogenic infections can lead to sepsis, which can result in tissue damage, organ failure and death. this article has outlined the management of odontogenic infections, including the latest covid-19 guidelines. drugs for the management of dental problems during covid-19 pandemic management of acute dental problems during covid-19 pandemic severe odontogenic infections with septic progress -a constant and increasing challenge: a retrospective analysis medical emergencies: sepsis in primary dental care professional resources sepsis: recognition, diagnosis and early management nice guideline uk sepsis trust. clinical tools. 2020. available online at changing trends in deep neck abscess. a retrospective study of 110 patients deep neck abscesseschanging trends life-threatening oro-facial infections peterson's principles of oral and maxillofacial surgery deep space neck infection: principles of surgical management prescribing in dental practice increasing frequency and severity of odontogenic infection requiring hospital admission and surgical management ludwig's angina ludwig's angina management of head and neck infections in the immunocompromised patient organ reserve, excess metabolic capacity, and aging influence of aging and environment on presentation of infection in older adults criteria for admission of odontogenic infections at high risk of deep neck space infection irrigating drains for severe odontogenic infections do not improve outcome antibiotic selection in head and neck infections pharmacology of local anaesthetics used in oral surgery is conservative treatment of deep neck space infections appropriate? evaluation of bacterial spectrum of orofacial infections and their antibiotic susceptibility severe odontogenic infections. part 2. prospective outcomes study management of acute dental problems: guidance for healthcare professionals sdcep. drug prescribing for dentistry: dental clinical guidance deep neck infection covid-19 guidance and standard operating procedure when to prescribe antibiotics royal college of surgeons, faculty of general dental practice (uk) & bda. open letter on prescribing antibiotics during covid-19 assessment of global incidence and mortality of hospital-treated sepsis. current estimates and limitations death from overwhelming odontogenic sepsis: a case report sepsis guidance implementation advice for adults sepsis decision support tool for primary dental care dental mythbuster 25: sepsis resuscitation council uk. the abcde approach basic guide to medical emergencies in the dental practice: second edition news) 2: standardising the assessment of acute-illness severity in the nhs national early warning score (news) the recognition and management of sepsis in the dental practice has also been discussed, including the age-specific sepsis decision support tools developed by the uk sepsis trust. key: cord-347039-eap592i7 authors: lee, seung-hwan; dimock, ken; gray, douglas a; beauchemin, nicole; holmes, kathryn v.; belouchi, majid; realson, john; vidal, silvia m. title: maneuvering for advantage: the genetics of mouse susceptibility to virus infection date: 2003-08-31 journal: trends in genetics doi: 10.1016/s0168-9525(03)00172-0 sha: doc_id: 347039 cord_uid: eap592i7 abstract genetic studies of host susceptibility to infection contribute to our understanding of an organism's response to pathogens at the immunological, cellular, and molecular levels. in this review we describe how the study of host genetics in mouse models has helped our understanding of host defense mechanisms against viral infection, and how this knowledge can be extended to human infections. we focus especially on the innate mechanisms that function as the host's first line of defense against infection. we also discuss the main issues that confront this field, as well as its future. genetic studies of host susceptibility to infection contribute to our understanding of an organism's response to pathogens at the immunological, cellular, and molecular levels. in this review we describe how the study of host genetics in mouse models has helped our understanding of host defense mechanisms against viral infection, and how this knowledge can be extended to human infections. we focus especially on the innate mechanisms that function as the host's first line of defense against infection. we also discuss the main issues that confront this field, as well as its future. viral infections in humans are notable for the diversity of host responses, rates of progression, and disease outcomes. a large body of evidence indicates that these differential responses depend not only on viral factors but also on inherited components affecting host susceptibility. significant advances in our understanding of the host response to infection in humans and other animal species have recently been achieved through the use of mouse models of infection. in laboratory mice, years of inbreeding have fixed deleterious alleles, which can be detected as susceptibility phenotypes. two major breakthroughs advanced this field. one was our ability to manipulate the mouse genome using transgenic and gene knockout technologies. this 'reverse genetics' approach allows direct testing of specific genes for their role in host resistance or susceptibility to infection. the other was our ability to clone genes responsible for natural variation in susceptibility to infection among inbred strains of mice, through molecular genetics. this 'forward genetics' approach can uncover novel mechanisms of host defense that are crucial to effective and protective responses to infection. to date more than 30 mouse loci (table 1 ) and many fewer human genes ( table 2 ) have been associated with the outcome of virus infection [1, 2] . each locus controls infection by a single virus family or strain. this is probably related to the vast diversity of virus life cycles and the likelihood that the product of each host resistance gene interacts with unique molecules encoded by individual viruses (fig. 1) . in this review we illustrate the contribution of mouse genetics to our understanding of mechanisms of host resistance to virus infection. these mechanisms might manifest themselves as: (1) barriers to infection at the host cell membrane; (2) intracellular host responses to infection; or (3) recognition or destruction of infected cells. viruses used as examples are described in table 3 . barriers to infection at the host cell membrane the first step in the life cycle of a virus is attachment to a receptor on the cell surface and delivery of the viral genome into the interior. usually the virus takes advantage of a host molecule with an unrelated function, such as an adhesion molecule or complement regulator, for its own benefit. receptors are recognized as important determinants of virus host range and tissue tropism; and some host resistance/susceptibility loci encode molecules that are expressed on the cell surface. for example, sjl mice are 10 000 times more resistant to a lethal dose of mouse hepatitis virus (mhv) -a murine coronavirus -than c57bl/6 or balb/c mice [3] . resistance is due to allelic variation in the gene encoding carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1) [4] . susceptible strains carry the ceacam1a allele, which encodes the principal mhv receptor, cea-cam1a. resistant strains, such as sjl, are homozygous for the ceacam1b allele, which encodes a 27-amino-acid substitution in the n-terminal virus-binding domain of ceacam1a [4] . an immunoreceptor tyrosine-based inhibitory motif (itim) (see glossary) is present in the cytoplasmic domain of ceacam1, also suggesting a potential immunomodulatory function for this molecule during mhv infection. although no role for human ceacam1a has been described during viral infection of humans [5] , a possible immunomodulatory function is consistent with the observation of a ceacam1-mediated suppression of cd4 þ t-cell activation during infection by the pathogenic bacteria neisseria gonorrhoeae, neisseria meningitidis and haemophilus influenzae. these human pathogens bind to domain 1 of ceacam1, very close to the site recognized by mhv [6] . another example of natural host resistance is the restriction of ecotropic murine leukemia virus (mulv) infection by the mouse fv4 gene. binding of retroviral env glycoproteins to cellular receptors triggers fusion of viral and cellular membranes, and allows the virus nucleocapsid to enter the cell. the fv4 (or akvr) locus is a defective endogenous ecotropic provirus that encodes an env protein, which also has a fusion defect [7, 8] . it has been proposed that the env protein encoded by fv4 blocks mulv infection by interacting with the cellular receptor and restricting the amount of receptor available for exogenous retrovirus attachment (box 1) [9] . in humans, resistance to human immunodeficiency virus (hiv) is also mediated by a barrier at the cell surface. cd4 is the primary or 'attachment' receptor for hiv, but cd4 is necessary but not sufficient for the productive entry of hiv into target cells. the identification of ccr5 as a coreceptor for hiv prompted genetic screening of individuals that escape hiv disease despite high-risk behavior [10] . individuals carrying a homozygous 32 bp deletion in the coding sequence of ccr5 (ccr5d32) are extremely resistant to the 'r5'strain of hiv because the deletion results in a frame shift and generates a nonfunctional receptor [10] . the ccr5d32 mutation is found predominantly in the caucasian population and is absent in africans, american indians and east asians [11] . it has been speculated that this distribution is consistent with resistance to an agent that predates hivand caused enormous mortality. one candidate is yersinia pestis, the causative agent of bubonic plague, although other pathogens targeting the macrophage/monocyte lineage cannot be excluded [12] . delivery of a viral genome to the interior of an infected host cell does not guarantee that an infection will be glossary advanced intercross lines : mouse lines generated by producing an f2 generation between two inbred strains and then intercrossing in each subsequent generation, but avoiding sibling matings. this provides increasing probability of tightly linked genes recombining. congenic strain : a mouse strain that has been bred to be identical to an inbred strain, except for a selected differential chromosomal segment. congenic strains are derived by backcrossing to a parental inbred strain for at least ten generations while selecting for heterozygosity at a particular locus. consomic strain : a variation on a congenic strain in which recombinants between two inbred strains are backcrossed to produce a strain that carries a single chromosome from one strain on the genetic background of the other. epistasis : the interaction between alleles at different loci that allows an allele at one locus to alter the effects of alleles at a different locus. immunoreceptor tyrosine-based activation motif (itam) : a cytoplasmic tyrosine-containing motif that is the site of tyrosine phosphorylation. these motifs are associated with tyrosine kinases and other phosphotyrosinebinding proteins involved in cellular activation. immunoreceptor tyrosine-based inhibitory motif (itim) : a cytoplasmic tyrosine-containing motif. in contrast to itams, phosphorylation of the tyrosine residues within itims recruits the src-homology-2-domain-containing tyrosine phosphatase shp-1 and/or shp-2, transducing a negative signal that inhibits cellular activation. interferons (ifns) : a group of immunoregulatory proteins that are produced by cells infected with a virus and have the ability to inhibit viral growth. ifns are classified as type i (ifn-a/b), which have antiviral properties, and as type ii (ifn-g), which is known as immune interferon. despite the use of different receptors, certain ifn-a/b and ifn-g functions are shared because the signal transduction pathways activated through these receptors partly overlap. murine leukemia virus (mulv) : there are four subgroups of naturally occurring mulvs based on receptor usage on mouse cells: ecotropic mulvs, which are able to infect only mouse cells utilizing the cationic amino acid transporter as a receptor; amphotropic mulvs, which are able to infect mouse cells as well as those of other species (including human) by binding to an inorganic phosphate transporter protein; polytropic mulvs, which can infect cells from mouse as well as nonmurine species using yet another cellular receptor protein, thought to be a g-protein-coupled receptor; and xenotropic mulvs, which use receptors from cells of most species except mice. quantitative trait loci (qtls) : the locations of genes that affect a trait that is measured on a quantitative (linear) scale, such as body weight or blood pressure in animals, as identified by statistical analysis. these traits are typically affected by more than one gene, and also by the environment. recombinant congenic strain : a variation on recombinant inbred strains in which the initial outcross is followed by several generations of backcrossing before inbreeding. retroviral interference : the phenomenon by which prior infection of cells with a retrovirus confers strong resistance to infection of the same cells by a retrovirus that utilizes the same receptor; however, cells remain susceptible to viruses that use a different receptor. this process results from masking or downregulation of the receptor due to interaction with the glycoprotein of the established virus. an important element of resistance to retroviral infection is provided by endogenous retroviral elements. some retroviral elements in the mammalian genome are transcriptionally active but carry deletions and point mutations that render them unable to replicate. some of their gene products, such as the fv4 and fv1 proteins, interfere with infection by their exogenous relatives thus providing a selective advantage to their hosts. for example, fv4 encodes an env protein that interferes with ecotropic murine leukemia virus (mulv) infection (see main text) at the level of viral entry. examples of this phenomenon can be found in mice, chickens and cats, suggesting that env-mediated retroviral interference is commonly used for limiting virus spread [76] . gene therapy based on the principle of receptor interference has demonstrated that introduction of fv4-envtransduced bone marrow cells into a thymectomized host confers resistance to friend leukemia virus-induced leukemogenesis [77] . these findings suggest that a similar approach could be used as a therapeutic strategy to inhibit infections by other retroviruses in vivo, including immunodeficiency virus. for example, transfer of a gene encoding a normally processed but fusion-defective retroviral env protein into susceptible cells would interfere with viral entry and potentially reduce the infectiousness of viruses emerging from the cell. the fv1 locus [78, 79] encodes a retrovirus capsid-like protein [80] that restricts mulv infection at a post-entry stage, before integration of the provirus [81] . because all retroviruses replicate in very similar ways, it is conceivable that similar restriction mechanisms could be found in humans. interestingly, although an fv1 ortholog was not detected in nonmurine species, a mechanism of preintegration interference, resistance factor 1 (ref1), has been demonstrated in human cell lines [82] . at the time of writing, the ref1 gene has not been cloned, but there is speculation that, like fv1, ref1 might consist of endogenous retroviral sequences. a recent study suggests the existence of a ref1-like restriction in humans and nonhuman primates that determines lentivirus tropism [83] . consistent with this proposal, the target for this restriction is within the capsid protein of hiv [83] . an example of an alternative mechanism of resistance to retroviral infection is provided by fv2, which determines the progression of friend virus complex-induced erythroleukemia. fv2 is identical to ron, which encodes stk, a member of the met family of receptor tyrosine kinases. susceptible mice express a positive-acting truncated version of stk lacking most of the extracellular domain, which is associated with proliferation of sffv-infected erythroblasts [84] . successful, because potent defense mechanisms act at the intracellular level. one of the initial cellular responses to viral infection is the production of type i interferons (ifns). ifns, in turn, stimulate the expression of several gene products, including the double-stranded rna-activated protein kinase (pkr) and rnase l, which leads to the establishment of an antiviral state in cells, characterized by a general inhibition of protein synthesis. the ifn-induced mx1 protein is one of the best-studied determinants of innate immune responses to viral infection. the inbred mouse strain a2g is resistant to doses of mouse-adapted influenza virus (an orthomyxovirus) that are lethal for other inbred strains [13] . resistance in these mice is determined by a single dominant locus, mx1, whose gene product, mx1, rapidly accumulates in the nuclei of cells following influenza virus infection, and blocks virus spread [14] . susceptible mice produce no functional mx protein due to either a nonsense mutation (cba/j) or gene deletion (balb/c) [15] . the mx1 product belongs to the dynamin superfamily of large gtpases conserved in all vertebrates. in mice there are actually two mx gene products, mx1 and mx2. mx1 protein blocks transcription of influenza virus, probably via interaction with the pb2 subunit of the influenza virus polymerase [16] . by contrast, the mx2 protein is cytoplasmic and has been shown to inhibit viruses that replicate in the cytoplasm, such as vesicular stomatitis virus (vsv) and members of the family bunyaviridae [14] . mx proteins inhibit virus replication by interfering with the transport of viral nucleocapsids, and by sorting them to locations where they become unavailable for the generation of new virus particles [14] , possibly to promyelocytic leukemia protein nuclear bodies (pml nbs) [17] , which are known to be a site of proteasome-mediated degradation. humans also possess mx genes, mxa and mxb, but only the mxa gene product has antiviral properties. however, in contrast to its mouse mx1 ortholog, the mxa gtpase accumulates in the cytoplasm of ifn-treated cells, where it inhibits not only the replication of orthomyxoviruses, vsv and bunyaviruses but also the replication of other rna viruses such as measles virus, coxsackievirus b4 and semliki forest virus [14] . a mechanism of resistance to flaviviruses, which also appears to be ifn-mediated, was recently revealed by the cloning of flv, which confers resistance (flv r ) or susceptibility (flv s ) to flavivirus infection. virus titers in the brains of resistant mice infected with murray valley encephalitis virus, yellow fever virus or west nile virus are orders of magnitude lower than in susceptible animals. viral yields in tissue cultured from resistant or susceptible animals are also dramatically different, indicating that the flv gene product acts intracellularly on flavivirus replication [18] . genetic mapping localized flv1 to chromosome 5 [19] and, more recently, it was demonstrated that flv susceptibility is associated with a nonsense mutation in a member of the 2 0 ,5 0 -oligoadenylate synthetase (oas) family, oas1b [20, 21] . oas proteins are induced by ifns and play a central role in producing the antiviral state by binding double-stranded rna and catalyzing the synthesis of 2 0 ,5 0 -oligoadenylates (2-5a), which, in turn, interact with and activate rnase l to degrade singlestranded viral and cellular rnas. whereas oas1b genes from resistant mice encode full-length proteins, those from susceptible mice encode proteins truncated at the c-terminus that might not form active synthetases. in contrast to the single oas1 gene found in humans, there are eight closely linked oas1 genes in the murine genome [20, 22] , but only oas1b is associated with resistance to flaviviruses. in addition to resistance imparted by barriers to virus entry or by intracellular antiviral defense mechanisms, cytotoxic cells can control the level of viral replication in an animal (box 2). host recognition of virus-infected cells is mediated by two players: natural killer (nk) cells and cytotoxic t cells. herpesviruses avoid recognition and activation of the adaptive immune system by downregulating major [24] . strains of the c57bl background carry a dominant resistance allele, cmv1 r , that restricts viral replication in target organs, whereas other mouse strains carry a recessive susceptibility allele, cmv1 s , that allows rapid proliferation of the virus [25] . cmv1 r encodes an activating nk-cell receptor, ly49h [26 -28] , which is absent in susceptible strains. mouse strains express different repertoires of ly49 molecules, which are part of a large family of polymorphic receptors expressed on overlapping populations of nk cells. upon binding of mhc class i ligands, different ly49 molecules deliver either activating or inhibitory signals that modulate nk-cell function [29] . recent studies have revealed that ly49h recognizes mcmv-infected cells via direct interaction with the viral antigen, m157, which shares structural homology with mhc class i molecules and is expressed on the surface of infected cells [30, 31] . m157 also binds to an inhibitory receptor, ly49i, expressed in susceptible strain 129 mice, suggesting that m157 could have evolved as a mechanism to escape nk-cell killing by targeting inhibitory receptors [30] . in resistant mice, ly49h might have evolved as a countermeasure against virus-encoded mhc class i homologs, providing an overriding activating signal to the nk cell, and promoting the elimination of mcmv-infected cells (fig. 2a) . considering the prevalence of human cytomegalovirus (hcmv) in the human population, we expect that there are likely to be hcmv-specific activating receptors present on human leukocytes that might function in a manner analogous to ly49h. the mhc (box 2) is a set of genes, present in all vertebrates, with immunological and nonimmunological functions. mhc class i genes play a crucial role in in vertebrates, host defense against virus infection is mediated by innate and adaptive immunity. innate immunity constitutes the first line of defense, providing a rapid response through germ-line encoded proteins that pre-exist or are induced within hours of infection. adaptive immunity is a slower, yet highly specific response mediated by b cells and t cells that confers effective and long-lasting protection against infection, and is characterized by immunological memory. major histocompatibility complex (mhc) molecules are highly polymorphic glycoproteins encoded by mhc class i and ii genes, which are mainly involved in the presentation of peptide antigens to t cells. mhc class i molecules bind peptides derived from proteins synthesized in the cytosol, such as viral proteins, and present them to cytotoxic cd8 þ t cells. by contrast, mhc class ii molecules are loaded with peptides derived from exogenous antigens engulfed within intracellular vesicles, for presentation to cd4 þ t cells. mhc class i molecules also play an important role in modulating the cytotoxic activity of natural killer (nk) cells. nk cells are a component of the innate system, so named because of their propensity to kill certain neoplastic and virus-infected cells without prior sensitization. the cytotoxic activity of nk cells is regulated by signals elicited by inhibitory receptors containing immunoreceptor tyrosine-based inhibitory motifs (itims), or stimulatory receptors associated with immunoreceptor tyrosine-based activation motifs (itam)-bearing adaptor molecules. inhibitory receptors interact with mhc class i molecules and prevent cell killing of healthy cells by autologous nk cells. in situations where mhc class i is downregulated, such as during tumorigenesis or viral infection, reduced inhibitory signals result in nk-cell-mediated lysis [85] . activating nk-cell receptors recognize stress-induced or pathogenencoded mhc class i-like proteins and stimulate killing of cells expressing these molecules [85] . review trends in genetics vol. 19 no.8 august 2003 combating viral infection in mice (fig. 3a) . congenic mice with intra-mhc recombinations provide an important tool for dissecting the contribution of mhc class i subregions to virus infection. for example, comparisons of t-cell responses in h2 congenic mice that differ in their recovery from friend leukemia virus infection were used to localize friend leukemia virus resistance loci rfv1 and rfv2. rfv1 mapped to the h2d subregion and determined t-cell activation. rfv2 mapped to the ia subregion and determined unresponsiveness of t cells in a proliferation assay [35] . resistance to acute lethal infection of mcmv is also controlled by genes linked to h2, with the k haplotype being more resistant than b or d. resistance was associated with genes of subregions k/ia and d. interestingly in this model, transfection of macrophages with sequences encoding mhc molecules such as h2-k d , d d or k b renders these cells, which are major reservoirs for mcmv, sensitive to mcmv infection [37] , consistent with a role for mhc molecules as mcmv receptors. in humans, association analysis between mhc and specific viral infections has also suggested a differential role of specific alleles in susceptibility to hiv, hepatitis b virus (hbv) and hepatitis c virus (hcv) (fig. 3b) . for example, hla-drb1*1302 is associated with resistance to chronic hbv infection in both african and european populations [42, 43] whereas hla-b*35-cw*04 is associated with the rapid progression of aids in caucasian populations [38] . although polymorphisms in the classical mhc class i and class ii genes are known to be related to antigen presentation, it is important to note that there are other genes within the mhc class iii region, such as components of the complement cascade (c2, c4), cytokines -tumor necrosis factor (tnf)-a and -b -and proteins involved in antigen processing (lmp2, tap1), which also have an important function in immunity [51] . therefore, disease resistance or susceptibility that maps to the mhc must be interpreted with caution, to differentiate the role of antigen presentation from the other roles of mhcassociated genes. although advances in genomic analysis have paralleled the discovery of host susceptibility traits that are determined by single alleles, most differences in host susceptibility to viral infection are the result of interactions among different genes with multiple alleles. the study of infectious disease under complex genetic control in humans is complicated by a variety of factors including genetic heterogeneity, low penetrance and environmental factors. the tools currently available to the mouse geneticist make the identification and isolation of disease susceptibility genes much more attainable. an example is the response to theiler's murine encephalomyelitis virus (tmev), a rodent model for multiple sclerosis [52] . in genetically susceptible mice, certain tmev strains cause persistent infection, inflammation, and a chronic demyelinating disease. one of the loci determining theiler's murine encephalomyelitis virus persistence (tmevp1), maps to the mhc region and corresponds to the h2d gene [52] . mice transgenic for the h2d b allele acquired resistance to persistent virus infection, suggesting that the gene effect was at the level of peptide presentation by h2d b [53] . using the amount of viral rna in the spinal cords of persistently infected mice as a phenotype, and a genetic dissection strategy based on genome scans, a second locus, tmevp2, was localized near the type ii interferon (ifn-g) gene on distal chromosome 10 [54] . analysis of a panel of congenic mice that inherited different portions of mouse chromosome 10 revealed that tmevp2 was actually two distinct loci, now termed tmevp2 and tmevp3, and excluded the ifn-g gene from the candidate region [55] . recently, a strong candidate for tmevp3, named tmevpg1, has been identified as a noncoding rna expressed in immune cells infiltrating the central nervous system of resistant mice, where an inverse relationship with ifn-g mrna levels has been observed [56] , suggesting a role for this rna in regulating ifn-g synthesis. consistent with this observation, the same reciprocal pattern of rna expression was observed for tmevpg1, the human counterpart, and ifn-g in human nk cells and t cells. several complementary avenues promise significant acceleration in the identification of genes that determine resistance or susceptibility in complex models of virus infection. the well-defined sets of recombinant congenic strains (rcs) [57] , consomic (chromosome substitution) strains [58] and advanced intercrossed lines [59] that are now available can be used to map a locus of interest and to characterize the specific phenotypic component of disease susceptibility under genetic control. also, traditional genetic mapping techniques are now complemented by high-throughput methods for studying gene function and regulation. for example, high-density oligonucleotide arrays [60] or cdna arrays [61] allow for gene expression monitoring on a genome-wide scale and offer an opportunity to establish functional links between genotype and phenotype. a strategy that combines congenic mapping with microarray expression profiling would aid in the identification of novel susceptibility genes and biochemical pathways not previously known to be involved in disease etiology. furthermore, the availability of the human and mouse genome sequences represents the ultimate breakthrough in quantitative trait loci (qtl) studies by placing the identification of candidate genes within a defined genetic interval only a (computer) mouse click away. one of the current limitations of using inbred strains of mice to study susceptibility to viral infection is the limited extent of genetic variation, mainly due to a small number of original progenitor strains [62] . therefore, although these resources are valuable, they do not permit the identification of all possible resistance or susceptibility loci. wild-derived inbred strains of mice are an important source of additional resistance alleles [63] that is just beginning to be exploited, as demonstrated by the characterization of flv. chemical mutagenesis, a method that has been successfully applied to the study of other model organisms, from bacteria to drosophila, is another promising strategy for creating novel susceptibility alleles in the mouse. the alkylating agent n-ethyl-n-nitrosourea (enu) introduces point mutations and creates random variation throughout the genome [64] . this is highly advantageous for defining host susceptibility phenotypes because it represents an unbiased method for identifying previously unrecognized physiological pathways because no assumptions are made about the relevant genes. enu mutagenesis yields models of simple inheritance that are readily accessible to analysis by positional cloning [64] . the effort to understand the genetic basis of susceptibility to viral disease is driven by three considerations: (1) the increased public awareness of the toll imposed by viruses on the host; (2) the increase in susceptible human populations because of longer life expectancy, frequently accompanied by chronic illness, and the consequences of advances in medical technology, including immunosuppressive therapies for organ transplantation or treatment of malignancy; and (3) the need to develop new therapies for infections caused by multidrug-resistant human killer-cell immunoglobulin-type receptor (kir) is considered to be a functional homolog of mouse ly49. an epistatic interaction between kir3ds1 and hla-b delays progression to aids, suggesting that hla-b behaves as a ligand for kir3ds1 [32] . the peptide presented on hla-b that is responsible for this interaction remains to be identified. because kir3ds1 receptor is also associated with the adaptor molecule dap12, the intracellular signaling cascade leading to cellular activation and killing of virus-infected cells seems to be similar to that triggered by mouse ly49h. by using inhibitory small molecules, represent promising approaches to antiviral therapy. therapies based on the principle of receptor interference for viruses other than retroviruses (box 1) could be explored. type i ifn induces an intracellular antiviral response against many different rna and dna viruses. mouse genetics has provided a starting point for dissecting intracellular mechanisms of host defense, and has revealed that several ifn-inducible gene products have inhibitory effects on a much more restricted number of viruses than expected. the precise nature (sequence or structural) of determinants recognized by effector proteins such as mx or oas1b remain to be characterized. the identification of additional effector molecules and viral targets must continue to be an important area of active research, particularly in view of the existence of orthologous human genes. mouse genetics has also demonstrated that recognition and destruction of virus-infected cells by nk cells is mediated by specific interactions between activating nkcell receptors and viral target molecules. viruses pose a particular problem for specific recognition because all the components of the virus are synthesized and assembled in the host cell. important questions to be answered include: (1) do other activating nk-cell receptors have specialized functions in virus recognition? (2) is the mhc class i fold a recognition-associated structure? (3) what is the extent of the repertoire of target molecules for nk-cell-activating receptors? in addition, studies of mouse nk-cell receptors, such as the ly49 family, have provided a conceptual framework for understanding human nk-cell biology. because no functional human ly49 counterpart has been identified, prime candidates for the recognition of viral pathogens by human nk cells are members of the killercell immunoglobulin-like receptor (kir) family [65] . although the ligands of activating kir receptors remain to be identified, it has been proposed that activating kir molecules have evolved to recognize infectious agents (fig. 2b) [32] . it is suspected that several common and/or chronic human diseases under complex genetic control are triggered by a viral infection. this idea is supported by experimental evidence derived from mouse models for initiation and exacerbation of atherosclerosis following mcmv infection [66] , for diabetes [67] or dilated cardiomyopathy [68] following coxsackievirus infection, and for multiple sclerosis-like disease following tmev [33] or mhv [69] infection. identifying genes that control susceptibility to acute and chronic viral infection in these models is a crucial step in understanding the development of these complex pathologies. comparisons between mouse and human mechanisms of host resistance or susceptibility to viral infection have increased our awareness not only of their differences but also, more importantly, of their similarities. in the future, the use of comparative genomic approaches in animal model systems will provide more comprehensive knowledge of the impact of viruses on human health. forward genetics of infectious diseases: immunological impact genetics of susceptibility to human infectious disease resistance to fatal central nervous system disease by mouse hepatitis virus strain jhm. i. genetic analysis specificity of coronavirus/ receptor interactions human aminopeptidase n is a receptor for human coronavirus 229e crystal structure of murine sceacam1a[1,4]: a coronavirus receptor in the cea family molecular characterization of the akvr-1 restriction gene: a defective endogenous retrovirus-borne gene identical to fv-4r fv-4 resistance gene: a truncated endogenous murine leukemia virus with ecotropic interference properties fv-4: identification of the defect in env and the mechanism of resistance to ecotropic murine leukemia virus chemokine receptors as hiv-1 coreceptors: roles in viral entry, tropism, and disease global distribution of the ccr5 gene 32-basepair deletion dating the origin of the ccr5-delta32 aids-resistance allele by the coalescence of haplotypes resistance of mice to mouse adapted influenza a virus interferon-induced mx proteins: dynamin-like gtpases with antiviral activity influenza virus-susceptible mice carry mx genes with a large deletion or a nonsense mutation function of the mouse mx1 protein is inhibited by overexpression of the pb2 protein of influenza virus interferon-induced antiviral mx1 gtpase is associated with components of the sumo-1 system and promyelocytic leukemia protein nuclear bodies genetically determined resistance to infection with group b arboviruses. ii. increased production of interfering particles in cell cultures from resistant mice development and characterization of new flavivirus-resistant mouse strains bearing flv(r)-like and flv(mr) alleles from wild or wild-derived mice positional cloning of the murine flavivirus resistance gene a nonsense mutation in the gene encoding 2 0 -5 0 -oligoadenylate synthetase/l1 isoform is associated with west nile virus susceptibility in laboratory mice gene structure of the murine 2 0 -5 0 -oligoadenylate synthetase family selective rejection of h-2-deficient lymphoma variants suggests alternative immune defence strategy inhibition of natural killer cells by a cytomegalovirus mhc class i homologue in vivo cmv-1, a genetic locus that controls murine cytomegalovirus replication in the spleen susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the c-type lectin superfamily vital involvement of a natural killer cell activation receptor in resistance to viral infection murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to ly49h the ever-expanding ly49 gene family: repertoire and signaling direct recognition of cytomegalovirus by activating and inhibitory nk cell receptors recognition of a virus-encoded ligand by a natural killer cell activation receptor epistatic interaction between kir3ds1 and hla-b delays the progression to aids genetics of susceptibility to theiler's virus infection serial backcross analysis of genetic resistance to mousepox, using marker loci for rmp-2 and rmp-3 h-2d control of recovery from friend virus leukemia: h-2d region influences the kinetics of the t lymphocyte response to friend virus genetic control of sensitivity to moloney leukemia virus in mice. iii. the three h-2 linked rmv genes are immune response genes controlling the antiviral antibody response are mhc proteins cellular receptors for cmv? hla and hiv-1: heterozygote advantage and b*35-cw*04 disadvantage influence of combinations of human major histocompatibility complex genes on the course of hiv-1 infection hla alleles determine human t-lymphotropic virus-i (htlv-i) proviral load and the risk of htlv-i-associated myelopathy a tumor necrosis factor-alpha (tnf-alpha) promoter polymorphism is associated with chronic hepatitis b infection hla-drb1*1301 and *1302 protect against chronic hepatitis b association between an mhc class ii allele and clearance of hepatitis b virus in the gambia class ii hla alleles and hepatitis b virus persistence in african americans influence of mhc class ii genotype on outcome of infection with hepatitis c virus. the hencore group. hepatitis c european network for cooperative research association between mhc class ii alleles and clearance of circulating hepatitis c virus. members of the trent hepatitis c virus study group genes of the major histocompatibility complex class ii influence the outcome of hepatitis c virus infection association between hla class ii genotype and spontaneous clearance of hepatitis c viraemia influence of hla haplotypes on the clinical courses of individuals infected with hepatitis c virus hla drb1 and dqb1 alleles and haplotypes influencing the progression of hepatitis c complete sequencing and gene map of a human major histocompatibility complex (mhc) the infection of mouse by theiler's virus: from genetics to immunology fvb mice transgenic for the h-2db gene become resistant to persistent infection by theiler's virus mapping loci influencing the persistence of theiler's virus in the murine central nervous system two loci, tmevp2 and tmevp3, located on the telomeric region of chromosome 10, control the persistence of theiler's virus in the central nervous system of mice tmevpg1, a candidate gene for the control of theiler's virus persistence, could be implicated in the regulation of gamma interferon recombinant congenic strains derived from a/j and c57bl/6j: a tool for genetic dissection of complex traits analysing complex genetic traits with chromosome substitution strains simultaneous detection and fine mapping of quantitative trait loci in mice using heterogeneous stocks high density synthetic oligonucleotide arrays quantitative monitoring of gene expression patterns with a complementary dna microarray the mosaic structure of variation in the laboratory mouse genome wild mice: an ever-increasing contribution to a popular mammalian model enu mutagenesis: analyzing gene function in mice divergent and convergent evolution of nk-cell receptors does cytomegalovirus play a role in atherosclerosis coxsackieviruses and diabetes the transition from viral to autoimmune myocarditis mouse hepatitis virus infection of the central nervous system: chemokine-mediated regulation of host defense and disease susceptibility to hiv infection and progression of aids in relation to variant alleles of mannose-binding lectin genetic restriction of hiv-1 pathogenesis to aids by promoter alleles of il10 mutation of gene of mannose-binding protein associated with chronic hepatitis b viral infection tuberculosis and chronic hepatitis b virus infection in africans and variation in the vitamin d receptor gene host response to ebv infection in x-linked lymphoproliferative disease results from mutations in an sh2-domain encoding gene the x-linked lymphoproliferative-disease gene product sap regulates signals induced through the co-receptor slam endogenous retroviruses and the evolution of resistance to retroviral infection a gene therapy model for retrovirus-induced disease with a viral env gene: expression-dependent resistance in immunosuppressed hosts susceptibility to two strains of friend leukemia virus in mice inheritance of susceptibility to friend mouse leukemia virus. 11. spleen foci method applied to test the susceptibility of crossbred progeny between a sensitive and a resistant strain positional cloning of the mouse retrovirus restriction gene fv1 physical mapping of the fv-1 tropism host range determinant of balb/c murine leukemia viruses a conserved mechanism of retrovirus restriction in mammals cellular inhibitors with fv1-like activity restrict human and simian immunodeficiency virus tropism fv2 encodes a truncated form of the stk receptor tyrosine kinase natural killer cells, viruses and cancer we are grateful to christine di donato for critical reading of the manuscript. our laboratories are supported by grants from the canadian institutes of health research (cihr) and natural sciences and engineering research council of canada (nserc). seung-hwan lee is supported by a cihr doctoral scholarship and silvia m. vidal is a junior scientist of cihr. key: cord-331673-xv1tcugl authors: reina, giacomo; peng, shiyuan; jacquemin, lucas; andrade, andrés felipe; bianco, alberto title: hard nanomaterials in time of viral pandemics date: 2020-07-15 journal: acs nano doi: 10.1021/acsnano.0c04117 sha: doc_id: 331673 cord_uid: xv1tcugl [image: see text] the sars-cov-2 pandemic has spread worldwide during 2020, setting up an uncertain start of this decade. the measures to contain infection taken by many governments have been extremely severe by imposing home lockdown and industrial production shutdown, making this the biggest crisis since the second world war. additionally, the continuous colonization of wild natural lands may touch unknown virus reservoirs, causing the spread of epidemics. apart from sars-cov-2, the recent history has seen the spread of several viral pandemics such as h2n2 and h3n3 flu, hiv, and sars, while mers and ebola viruses are considered still in a prepandemic phase. hard nanomaterials (hnms) have been recently used as antimicrobial agents, potentially being next-generation drugs to fight viral infections. hnms can block infection at early (disinfection, entrance inhibition) and middle (inside the host cells) stages and are also able to mitigate the immune response. this review is focused on the application of hnms as antiviral agents. in particular, mechanisms of actions, biological outputs, and limitations for each hnm will be systematically presented and analyzed from a material chemistry point-of-view. the antiviral activity will be discussed in the context of the different pandemic viruses. we acknowledge that hnm antiviral research is still at its early stage, however, we believe that this field will rapidly blossom in the next period. t he current emergence caused by sars-cov-2 is dramatically changing the everyday life of all of us. due to the high globalization, new viruses can spread all over the world much faster than ever, infecting the communities worldwide. the current technologies and measurements are able to sensibly slow down the infection spread. however, their cost is tremendously high, impacting the healthcare systems and causing the shutdown of industries and the lockdown of the population. the impact of sars-cov-2 on the worldwide economy is estimated to be a 2−3% decline, making this the biggest crisis since the world wars. 1 a possible vaccine is hopefully expected to come in 1−2 years, originating a buffer period pretty much uncertain for many people. vaccination is the only way known to accelerate the flock immunity without causing further death by this pandemic. contemporary history has seen the spread of other viral pandemics such as h2n2 flu (1956−1958) , h3n3 flu (1968), hiv (peak reached between 2005 and 2012), sars (2009), while mers (2012 to now) and ebola (1975 to now) viruses are in a prepandemic phase. the continuous colonization of wild nature lands may touch unknown virus reservoirs causing the spread of contagious epidemics. due to these facts, there is a clear urgency in the development of viral treatments to avoid the risk of new pandemics. 2 in particular, the possibility to have smart antiviral tools able to efficiently disinfect surfaces, block the viral spreading, enhance the survival of infected people, and boost immunization are highly desirable. in particular, more investments in the next years are expected in the antiviral research. hard nanomaterials (hnms) have been extensively studied for many types of applications including drug delivery, bioimaging, and biosensing. 3−5 the use of nanomaterials in biomedical research is highly developed, reaching in some cases clinical approval. 6 despite that, nanomaterials have been mainly developed for cancer therapy, while scarce attention has been spent on their application in viral infections. 7 the continuous virology research has more and more increased into viral replication machinery, allowing the preparation and rationalization of more sophisticated vaccine formulations and viral inhibitors. the use of hnms may be one of the keys to provide more effective biomedical agents with a wide spectrum of activity in viral pandemics. 8 an increasing number of reports describe how hnms can be successfully applied to block viral spread. hnms can be used at different stages of viral infection: blocking viral entry, hampering interaction with infected host cells, and modulating immune responses. due to their core composition (e.g., metal oxides, noble metals), hnms can have an important antiviral activity inactivating some specific proteins of the capsid or dysregulating radical homeostasis in the virus particles. additionally, surface functionalization can sensibly increase hnm antiviral activity, enabling the mimicking of host cells or enhancing the targeting efficiency. 9 in this review, we will critically analyze different strategies for the application of hnms as antiviral agents. the rational and the synthetic strategies will be highlighted and correlated to different relevant examples. particular attention will be paid on the mechanisms of antiviral action and on hnm applicability and efficacy. for the sake of clarity, this review is divided into three parts, namely: blocking viral entry, antiviral activity in host cells, and stimulation of immune system. we have focused on the different stages of infection. in the section dedicated to blocking viral entry, the application of hnms for surface disinfection and inactivation of the virus prior to interaction with host cells will be described. subsequently, the interaction of hnms after internalization into host cells will be addressed, stressing their antiviral delivery features and their activity in viral replication blockage, leading to host cell survival. then, activation of immune response induced by hnms, triggering the innate and the adaptive (e.g., nanovaccines) immunity, will be presented. the different adopted strategies will be correlated to the nanomaterial core (e.g., composition, size, shape) and surface (e.g., chemistry, surface charge) properties. finally, limits (e.g., unknown long-term toxicity), advantages (e.g., high and wide spectrum virucidal activity), and perspectives will be discussed with particular attention to the applications in viral pandemics (e.g., hiv, sars, and influenza viruses). this review is addressed to material and biomaterials scientists who are interested in antiviral research. we acknowledge that application of hnms as antiviral agents is still in the early stages; however, we believe that the research on this topic is going to grow soon. thus, with this contribution we genuinely hope to inspire researchers in the preparation of smart and efficient hnm antiviral agents. the last decades have been characterized by increasing investigation on viruses. in particular, their surface charge, protein composition, and host cell entry mechanism have been elucidated, allowing to formulate the first-generation wide spectrum antivirals. blocking the viral entry is one of the most common known antimicrobial procedure to stop infections at the early stage. in this context, antiviral materials have been used for surface disinfection or for epidemic limitation in humans and animals. due to their high surface to volume ratio, composition, and tunable surface chemistry, hnms are now more and more studied as powerful agents in blocking viral entry. as for other biological interactions, the attachment and entry of viruses into host cells are mediated by multivalent interactions between the surface of the virus and cell surface receptors. 10 nanomaterials can display multivalency that makes them able to compete with the host cells on virus attachment, limiting their infectivity. the mechanism of antiviral actions relies on the inactivation of the capsid proteins. as a matter of fact, hnms have been used for: (1) blocking target proteins for viral entry, (2) capsid protein oxidation, (3) mimicking cell surface, and (4) mechanical rupture of viruses ( figure 1 ). these strategies target proteins and mechanisms of entry common in most of the viruses, thus allowing the preparation of wide spectrum antiviral agents. in this section, the application of different hnms as powerful inhibitors of viral entry will be discussed. noble nanoparticles. noble nanoparticles (nps), made of gold and silver, are attractive as antiviral agents for their surface functionalization versatility and their capacity to cleave disulfide bonds. their use in disinfection has been extensively studied for different types of viruses. the morphology and the size of the nps play a crucial role in their ability to efficiently interact with the capsids and in their toxicity for the organism. these nanomaterials are characterized by a very large specific surface area (inversely proportional to the particle diameter). as the particle size becomes smaller and smaller, the percentage of surface atoms increases, creating many unsaturated bonds due to lack of neighboring atoms. as a consequence, agnps and aunps have unstable atoms with high surface energy. this kind of structure provides a lot of contact adsorption sites and reaction points for further modifications. these chemical features allow to easily combine surface np atoms with other atoms through chemical bonds. besides the composition of the metal core, several studies have pointed out the importance of the control of the surface chemistry. the surface groups can: (1) stabilize nps in the biological media, (2) insert targeting agents, and (3) enhance the circulation time inside the body. antiviral efficiency can be also enhanced by the multivalency effect, where highly branched ligands are used to locally augment the local concentration of the targeting molecules. in this section the main strategies and results for silver (agnps) and gold (aunps) nanoparticles in blocking viral entry will be critically discussed. silver nanoparticles. many studies have shown that naked agnps have a good effect on the control and prevention of a variety of viral diseases (table 1) . however, the antiviral mechanism of nanosilver is still unclear. the antiviral action is associated with the following mechanisms: nanosilver can prevent the virus from entering the host cells and inhibit the virus from binding to the cell receptor, thereby stopping the virus from infecting the targeted cells. agnps may be able to bind the viral surface protein and inhibit the interaction between the virus and the cell membrane receptors (figure 2 , left). however, it has been also reported that agnps can inactivate the virus through denaturation of surface proteins containing cysteine and methionine residues present on the viral capsid, in a similar way reported for bacteria. for example, agnps smaller than 10 nm were shown to interact with the sulfur-bearing residues of gp120 glycoprotein knobs distributed on the lipid membrane of hiv-1 virus, preventing the virus from binding to cd4 receptor site on the host cells, thus inhibiting the viral infection. 11 by means of a viral adsorption assay, it was shown that the agnp mechanism of anti-hiv action is based on the inhibition of the initial stages of the hiv-1 cycle. to demonstrate that the antiviral effect of agnps is due to the particle structure rather than to silver ions present in solution, the antiviral activity of silver sulfadiazine (agsd) and silver nitrate (known antibacterial silver salts) was evaluated. both salts showed a much lower therapeutic index than agnps in vitro, indicating that silver ions themselves are less efficient. 12 these results point out that the antiviral efficacy is not only related to the dose of ag + ions present in solution but is also regulated by different other parameters (e.g., size, charge, and surface functionalization) associated with the nanosize dimension. for instance, in the case of herpesviridae and paramyxoviridae viruses (both enveloped viruses with embedded viral-encoded glycoproteins), agnps can effectively reduce their infectivity, by blocking the interaction between the viral particles and the host cells with an antiviral activity strictly dependent on the size and ζ potential of the agnps. as a general observation, it was reported that smaller nanoparticles have better antiviral effect. this effect was associated with the increase of the surface area, where smaller-sized agnps could bind more efficiently to the viral particles exerting a higher antiviral activity. 13 another study reported the impairment of peste des petits ruminants virus (pprv) replication after incubating infectious viral particles with agnps, which did not exhibit any virucidal effect even up to 900 μg/ml. this result suggested that the anti-pprv activity of the agnps is due to the inhibitory effect 13 alternatively, nanosilver can be combined with viral nucleic acids to change the capsid structure, affect the replication of viral genetic material, and make the virus inactive. for example, tem analyses have shown that nps can cause a change of the structure of the ad3 virus from a hexahedral shape to an irregular shape, destroying its fibers and capsid proteins, leading to inhibition of the virus from binding to the host cells and destroying the dna structure, preventing adenoviral infection. 15 nanosilver can also bind directly to the doublestranded dna of hepatitis b virus to inhibit its replication. 16 in other studies, it has been demonstrated that silver ions released from nanosilver can directly damage the viruses. based in this property, an interesting application has been proposed. agnps were used as a coating on polyurethane condoms, (hsv) . the hypothesized mechanism is that silver ions are transferred directly from oxidized nps to biological targets, such as viral membrane proteins gp120 and gp41. in addition, a small amount of silver ion is also released from the coated contraceptives to improve the antiviral level. 17 although the studies on naked agnps to reduce viral infectivity have shown their potential as broad-spectrum antiviral agents, the understanding of the specific antiviral action mechanism still needs to be elucidated in depth. many studies have shown that the antiviral performance of naked agnps is related to their size, and smaller nanoparticles have better antiviral activities. 16 in addition to particle size, the antiviral action of agnp morphology has also attracted interest to fight against coronavirus. agnps and two types of silver nanowires were able to significantly cause an inhibitory effect on coronavirus transmissible gastroenteritis (tgev)-induced host cell infection and tgev replication. the mechanism is likely based on a direct interaction of agnps with tgev surface proteins (e.g., tgev glycoproteins) to inhibit the beginning of viral infection. it is possible that agnps and ag nanowires alter the structure of some surface proteins of tgev and then inhibit their recognition and adhesion to the cellular receptor papn. 18 although the potential of agnps as antiviral agents has been commonly recognized, unfortunately, their wide biological applications are limited by the risks of self-aggregation and environmental pollution. silver ions can be released from the surface of agnps and potentially pollute the environment, and their agglomeration into bulkier particles or fibers may change their biological characteristics, diminishing the antiviral effect. in several cases, it has been reported that naked agnps may affect human health. 25 therefore, research and development of agnps whose surface is modified or stabilized by protecting molecular layers is an urgent need to overcome these problems ( table 2) . poly(n-vinyl-2-pyrrolidone) (pvp) is the most commonly used stabilizer of agnps. the pvp-coated agnps are able to inhibit the activities of hiv-1, herpes simplex 2 virus (hsv-2), and respiratory syncytial virus (rsv). 11, 26, 27 but compared to foamy carbon, small-sized pvp and bsacoated agnps showed poor antiviral activity to the hiv-1 virus. 11 for rsv, pvp-coated agnps have a specific binding capacity to the viral surface, evidencing a regular spatial arrangement and a clear interaction with g-protein. 26 in addition, to improve the stability of agnps, their surface modification with antiviral drugs was proved to reduce the drug resistance caused by the drugs administered alone. tannic acid-modified agnps showed good antiviral effects on hsv-2 infection in vitro and in vivo. the viral infection was inhibited only when these nps directly interacted with hsv-2 virions. indeed, the pretreatment of host cells with such agnps did inhibit the entry of hsv-2. due to the high affinity of tannins to proteins and sugars, tannic acid can bind glycoproteins on the surface of viruses to make them inert, impairing glycoprotein function and preventing viruses from attaching and entering host cells. 28 the surface modification can also exert a synergistic antiviral effect. agnps decorated with polyphosphonium-oligochitosan (pqpoc) exhibited moderate to excellent antiviral activity against hav, nov, and coxb 4 . in addition, agnps could interact with the virion glycoproteins and prevent viral attachment and penetration. pqpoc can also serve as an effective virus inhibitor by blocking the interaction of the targeted virus with the host through the electrostatic interaction between the cationic polymers and the negatively charged binding sites of the virus. 29 surface-modified agnps can also prevent viral infection by competitive adsorption on host cells. the process of infection of cells by herpes simplex virus type 1 (hsv-1) involves the interaction between viral envelope glycoproteins and heparan sulfate (hs) on cell surface. therefore, researchers designed agnps capped with mercaptoethanesulfonate (ag-mes) to compete with the cellular hs through the sulfonate end groups, thereby blocking the virus from entering the cells. 30 a few years ago, it was shown that curcumin could prevent the replication and the budding of rsv, 31 but the disadvantage of poor solubility and low bioavailability limited its clinical application. 32 curcumin was used as a reducing and capping agent to prepare stable curcumin agnps (cagnps) under physiological conditions. cagnps could reduce cytopathic effects induced by rsv and showed efficient antiviral activity against infection by directly inactivating the virus prior to entry into the host cells. its antiviral effect was higher than curcumin alone or unmodified agnps ( figure 3 ). 33 alternatively, zhu et al. prepared agnps surface-modified with oseltamivir, amantadine, and zanamivir (ag@otv, 34 ag@am, 35 and ag@znv 36 ), by chemical methods. the results showed that these nanoparticles can directly interact with the virions, resulting in viral function damages. overall different studies have reported the capacity of agnps to block viral entry. however, there is not a concerted antiviral mechanism, but their activity differs from case to case, based on viral particle adsorption, capsid structure alteration, or surface protein denaturation. for agnps, the antiviral activity can be associated with different parameters including size, shape, surface charge, and functionalization but also to the topical release of silver ions able to disturb the viral cycle replication. as described before, bare agnps can be used as disinfectant agents, however their use in biological media is acs nano www.acsnano.org review limited by their low colloidal stability and potential cytotoxicity. surface functionalization can alleviate cytotoxicity, but it can also mask the nanoparticle surface, reducing their affinity for viral particles, thus reducing agnp antiviral activity. for these reasons, agnps at the moment could find application mainly for surface disinfection and for topical administration. further studies are needed to prepare safer agnp formulations for systemic administration. in particular, the clarification of the antiviral mechanisms and the use of surface functional groups able to stabilize agnps in biological fluids without affecting their prominent antiviral activity are probably the most important challenges to tackle. gold nanoparticles. compared to agnps, aunps exhibit reduced toxicity on healthy cells, making them more attractive for in vivo and clinical applications. 38 indeed, aunps have been successfully tested as inhibitors of viral entry into the host cells. aunps interact with hemagglutinin (ha), where au is able to oxidize the disulfide bond of this glycoprotein causing its inactivation, thus impeding the membrane fusion of the virus with host cells. targeting ha has emerged as an alternative strategy to the actual therapies (e.g., matrix protein 2 and neuramidase), especially to pandemic viruses that show an accelerated mutation speed of their surface proteins, hence a resistance to conventional treatments increasing their infectivity and mortality. 38 this strategy has been applied to influenza (e.g., h1n1, hcv) and herpes viruses. 39−44 the activity of aunps is proportional to the surface area exposed. as a consequence, the size and the morphology of these metal nps play a substantial role in their antiviral activity. recently, kim et al. have reported that porous aunps are able to inhibit influenza a infection more efficiently than nonporous aunps. 39 this effect has been associated with the higher surface area of the porous material that favors their interaction with capsids and thus increases their antiviral activity ( figure 4 ). besides the per se antiviral activity, aunp surface modifications have been developed in order to enhance their overall therapeutic benefits. the engineering of tailored aunps with selected ligands has allowed the preparation of efficient antiviral nanoagents. the target ligands can be introduced directly during the particle synthesis via ligand exchange reactions or ligand modifications. for instance, direct reduction of gold ions in the presence of gallic acid produced homogeneous aunps able to sensibly reduce herpes simplex virus infection in vitro. 40 compared to free ligand nps, functionalized aunps benefit from the multivalency effect and higher circulation times, decreasing the needed therapeutic concentrations. 39 functionalized aunps can present organic groups that mimic host cell surfaces or other specific molecular patterns that selectively target the virus. normally, negative charges are used to mimic cell surfaces and favor the interaction between the particles and the capsid. in particular, sulfonates and organic sulfates have been used for their capacity to attract the virus via capsid protein interaction and block the ha activity. 41 aunps functionalized with sulfonates showed an increasing inhibition of influenza a compared to the nanoparticles capped with succinic acid. 42 this study also demonstrated that there is not a correlation between the negative charge and the antiviral activity, but instead the inhibition depends mainly on the organic groups used. thiolcapped aunps also displayed powerful inactivation of bovine viral diarrhea virus in vitro. 43 multivalency has been exploited in more complex systems using dendrons as capping agents. this strategy allows to generate higher concentrations of the target ligand in close proximity to the aunps and to increase the binding efficiency of the nanoparticles to the capsid. the driving force of the antiviral efficiency relies on the concentration of the targeting agent onto the particles. sulfonated dendrons were grafted to aunps via a sulfide bond and tested for hiv inhibition. 44 the results showed that acs nano www.acsnano.org review the decorated aunps exerted a higher affinity to the virus. additionally, comparing aunps functionalized with different generation dendrons, those with a third generation displayed the highest inhibition performance with an ic 50 below 0.1 μmol/ml, thus making them attractive for in vivo translation. it is worth noting that the inhibition efficiency is strictly dependent on the available sulfonate groups present on the surface of the nps, making crucial a thorough characterization of the material. 44 the size of the aunps clearly plays an important role in the concentration of targeting ligands exposed per particle. 45 indeed, too big nps have a limited surface area, while too small would not allow an efficient grafting of the dendrons due to steric hindrance. for instance, it has been shown that dendron-functionalized aunps showed a size-dependent antiviral activity for influenza virus, where 14 nm particles exhibited a higher efficiency than 2 nm aunps. this has been associated with the low functionalization grade of the small nanoparticles and to the inappropriate spatial distribution of the interacting ligand/receptor pairs. the development of viral proteomics has profoundly transformed the antiviral and disinfection strategies. in particular, small molecules and peptides able to target and block the viral biochemical machinery have been developed. however, despite these efforts into the drug design, many of these molecules suffer from poor biological effect, low concentration in the diseased areas, and undesired side effects. in this context, aunps have been coupled to biologically inactive small molecules to create biologically active multivalent aunp therapeutics. a bright example has been reported by bowman et al., where the authors functionalized aunps with sdc-1721, a small membrane fusion inhibitor of hiv. 46 the results demonstrated that, while pure sdc-1721 has low activity, functionalized aunps are able to inhibit hiv replication at μm concentrations. similar results have been reported using targeting peptides. in particular, it was evidenced that the functionalized aunps can sensibly reduce the ic 50 up to 2 orders of magnitude compared to pure peptides. 47 preliminary results in vivo confirmed the biosafety of the aunps. 48 nanoparticles generating reactive oxygen species. one of the main advantages of using nps compared to oxidized metals relies on the slow release of ions and clusters from these particles, leading to an enhancement of the antiviral activity. additionally, the use of metal nps containing cu or fe in ionic form catalyzes the generation of radicals via fenton and fenton-like reactions oxidizing the capsid proteins and consequently blocking the viral infection at early stage. for instance, copper ions (derived from sulfates or iodide salts) have been widely used as antiviral agents because of their activity on several kinds of enveloped and non-enveloped viruses including influenza virus, 49−51 herpes simplex virus 52−54 and hepatitis a virus. 55 their mechanism of action relies on the formation of cu + ions (from soluble salts or nanoparticles) that generate hydroxyl radicals. 56 the use of metallic copper nanostructures in the form of particles or sheets has shown only a moderate efficiency due to the low concentration and low release of cu + . 56 for these reasons, cu + salts, where the copper ions are readily present in their active monocationic form, have been favored. in particular, cui nanoparticles (stable at room temperature) have been extensively studied for deactivation of feline calicivirus 56 and h1n1 pandemic influenza virus. 57 however, the use of copper salts at high concentrations can irreversibly alter reactive oxygen species (ros) homeostasis of healthy cells, provoking a general toxicity for the organism, limiting their applications to disinfection. 56 nanostructured cuprous and cupric oxides have been also extensively employed as antiviral agents for in vitro applications. for instance, cuprous oxide nanoparticles (cuonps) were successfully employed against hepatitis c. 58 in particular, it was found that these nps exerted a favorable antiviral activity with no cytotoxic effects. cuonps target the binding and entry step of viral infection to hepatic cells ( figure 5 ). similar results were reported on the use of cuonps against hsv-1, however without any profound investigation on the antiviral mechanism. 59 alternatively, zinc salts have been successfully used as antimicrobial agents from research up to clinical trials for viral warts. 60, 61 more recently, zno nanoparticles (znonps) were developed for the treatment of hsv-2. znonps were prepared with a tetrapod morphology. 62 the results showed that they can mimic cell surface interacting with the hs present on the viral capsid. additionally, these particles have been used for photocatalysis showing to efficiently destroy the viral proteins upon uv irradiation. 62 besides all these interesting examples, in vivo applications are still needed to validate this therapeutic modality. due to the generation of high levels of ros, the toxicity of copper nanoparticles has been widely debated. the antiviral activity of copper nanoparticles is generally associated with the release of cu + ions in solution, thus the leakage of cytotoxic cationic species can be modulated by surface functionalization before in vitro and in vivo applications. on the other side, the use of nanomaterials generating ros can find applications in textile and surface coating. the general broad virucidal efficiency of copper oxide nanoparticles shown for h1n1 pandemic influenza 57 should be tested on sars-cov-2 and might be used for improving mask protection efficiency. carbon nanomaterials. due to their diversity, versatility, and tunable surface chemistry, carbon nanomaterials have been attractive for several types of applications. in particular, the past decade has seen a tremendous raise in the preparation of performant carbon-based nanomaterials in the antiviral field. fullerene and its derivatives are the most studied carbon nanomaterials for their virucidal activity. due to the lack of solubility of pristine fullerene, functionalization strategies have been developed to prepare water-soluble drugs. investigations in the biomedical field evidenced the membranotropic capacity of fullerene derivatives. 63 by modulating shape and functions, fullerene derivatives have been shown to possess antiviral properties through inhibition of viral entry and blockage of viral replication. from these results, the attention has been directed also to other carbon nanomaterials. in particular, functional carbon dots (cds) and graphene oxide (go) have been investigated for their ability to block viral entry into host cells. glycofullerenes. the emerging of mortal viruses, like ebola or zika, and the lack of suitable treatments led the academic and the industrial communities to look for alternative therapeutic routes. most of these pathogens are rna enveloped viruses, and they share common infection mechanisms that can be targeted for the preparation of wide small spacer between core c 60 and surrounding fullerenes. b large spacer between core c 60 and surrounding fullerenes. www.acsnano.org review spectrum antivirals. the external surface of the envelope of these viruses is covered by glycans that tightly interact with lectin receptors on host cells. 64 this strong interaction allows the attachment of the virions to the cells, followed by internalization and infection. blocking lectin receptors is a general strategy used to stop viral infection at an early stage. fullerenes have been widely investigated as antiviral molecules, drug carriers, or tissue scaffolds. 65 fullerene applications have been recently extended to the design of mannosylated derivatives to block the entry of viral particles into host cells. mannose, due to the high affinity with lectin receptors, competes with the virus in the interaction with the host cells. for example, one of the targets is the inhibition of viral particles through the interaction of mannose with the dendritic cell-specific icam-grabbing non-integrin (dc-sign). dc-sign receptors mediate the interactions between dcs and t cells. 66, 67 to exploit these characteristics, mannose was combined with fullerene in the design of the so-called glycofullerenes to study their capacity to inhibit ebola, dengue, and other pathogens. for this purpose, different glycofullerenes were synthesized by changing the number of mannose units (from 12 to 36) and the spacers between the fullerene moieties and by varying steric hindrance in order to obtain a library of molecules. 66 the synthetic route is composed of three steps based on "click chemistry": (1) assembly of glycodendrons by cu(i)-catalyzed azide−alkyne cycloaddition (cuaac), (2) synthesis of alkynesubstituted bingel-hirsch hexakis-adducts, and (3) the coupling between the last two products again by cuaac. to increase the number of mannose moieties up to 36, the glycodendron core was changed from malonate to trialkynyl pentaerythritol. in order to compare the different derivatives, in vitro studies were performed. jurkat cells (lymphocyte t cd4 immortalized cells) expressing dc-sign were used to prove the inhibition capacity of the glycofullerenes on viral infection of ebola ( figure 6 , route a). the study revealed an ic 50 in the μm range for the 12 mannose fullerene, a lower efficiency with the 36 mannose fullerene with a short spacer (peg, with 2 ethylene oxide units), while a nanomolar ic 50 was achieved with 36 mannose fullerenes with a longer spacer (peg, with 3 ethylene oxide units) ( table 3 ). this first proofof-concept study was then expanded, aiming to obtain a better antiviral activity by increasing the valence and inserting longer and flexible spacers. 68 based on these studies, another class of multivalent fullerene dendrimers was then designed. a fast and controlled synthetic route was developed to achieve giant globular multivalent fullerenes, containing hundreds of functional groups. the first study was performed with tridecafullerenes containing 120 mannoses. 69 the molecular structure is composed of 12 hexakis c 60 surrounding a c 60 core ( figure 6 , route b). compared to the previous study, an ic 50 3 orders of magnitude lower was measured on the inhibition of ebola virus (table 3) . 66, 68 in order to present more carbohydrates at the periphery of the dendrimer, a trialkynyl pentaerythritol derivative allowed to afford a tridecafullerene with 360 carbohydrates. in this case, the molecule was synthesized with a c 60 tridecafullerene bearing α(1,2)mannobioside. 70 the use of this disaccharide was already investigated, showing an increase of affinity with dc-sign receptors by a factor of 3−4. 71 the synthetic strategy exploited also the use of strainpromoted copper-free cycloaddition of azides to alkynes (spaac) for the coupling of the core fullerene to the surrounding fullerenes. spaac allows an easier purification avoiding the removal of cytotoxic copper ions. the inhibition performance of this molecule was studied in vitro with viral pseudoparticles of dengue and zika. the comparison was made between 360 and 120 disaccharides tridecafullerenes and 36 disaccharide monofullerene. the results highlighted a picomolar ic 50 inhibition on both zika and dengue models for the 360 disaccharide glycofullerene ( table 3) . the ability to inhibit other types of viruses allows the use of glycofullerenes as broad spectrum antiviral drugs. moreover, the negligible toxicity to other cells proved the biocompatibility of these molecules. following an alternative strategy, a supramolecular assembly of monodisperse glycofullerenes, leading to the formation of micelles, was achieved and tested. 72 these micelles present a uniform and spherical shape ( figure 6 , route c). the aggregation synthetic route is faster compared to a controlled synthesis of giant glycofullerenes, but it might suffer from low reproducibility and batch-to-batch differences between each formulation. this self-assembled c 60 functionalized with 6 or 12 mannoses exposes a large amount of carbohydrate at the surface, leading to an inhibition of ebola virus in the nanomolar range (ic 50 of 424 nm for six mannoses and 196 nm for 12 mannoses, respectively, table 3 ). further in vitro studies evidenced again a good biocompatibility of these glycofullerenes. while there are no in vivo studies yet, these promising results enlarge the panel of molecules in the fight against new emerging viruses. functionalized fullerenes have been used for their ability to compete with viral particles through lectin receptors in host cells. there has been a tremendous advancement in the functionalization of fullerenes leading to the preparation of derivatives with a high amount of mannose, capable to enhance the multivalency effect and thus to increase the therapeutic outcome. first, glycofullerenes do not have an intrinsic virucidal activity. they can reduce the infectivity, but they are not able to completely inactivate the virus. second, the mechanism of action of glycofullerenes relies on their interaction with host cells and not with viral particles. thus, for a therapeutic application, they should be injected at different time points, ensuring that the local concentration is therapeutically relevant to prevent the virus from invading the host cells. in addition, glycofullerenes can be internalized into host cells losing their viral "shield" activity. on the other hand, the well-developed surface chemistry of glycofullerenes can be used for other key receptors involved in viral entry. for instance, in the case of the current sars-cov-2 pandemic, a similar click chemistry strategy can be used to anchor ligands recognized by human lung ace2 receptors and so inhibiting viral entry. 73 other carbon nanomaterials. alongside fullerenes, other carbon nanomaterials (nms) have been scrutinized for their ability to block viral entry. cds and go are the most known and studied carbon nms with marked antiviral properties. cds are zero-dimensional carbon nanoparticles. they are generally produced via hydrothermal decomposition of carbon containing "low-cost" precursors. the use of cds in the biomedical field has been encouraged by their easy preparation, low toxicity, fluorescence properties, and easy surface functionalization. pristine cds have shown moderate viral blocking activity for hiv infection in vitro. 74 this has been associated with the surface of the material rich in carboxylic and hydroxyl groups prone to form noncovalent acs nano www.acsnano.org review interaction with viral membranes. moreover, due to the complexity of the biological systems these nonspecific interactions could not be so effective in vivo, likely reducing the antiviral efficacy. therapeutic targeting molecules can be grafted onto a cd surface to enhance their antiviral activity. in this context, the design of multifunctional cd platforms can be obtained through two different strategies. the first consists in a single-step reaction that foresees the insertion of the therapeutic molecule directly into the step of preparation. target molecules are decomposed with the other precursors, generating the desired functional cds. this protocol is fast and efficient, however the drug loading as well as its activity are hard to estimate. indeed, the hydrothermal treatment can alter the chemical structure of the active molecule, thus vanishing its therapeutic effect. for these reasons, the reaction conditions must be carefully controlled. 75 the second method is a twostep reaction and implies the postfunctionalization via amide formation on the surface of the cds rich in carboxylic groups. this strategy offers a better chemical control, but the yield and the drug loading may not be quantitative and high, respectively. different functionalized cds were prepared to hamper host cell viral entry. for instance, benzoxazine (a low water-soluble antiviral agent) was incorporated into the cd structure during their preparation (figure 7) . the as-prepared cds showed a broad spectrum viral blocking capacity in vitro for enveloped (e.g., japanese encephalitis virus, dengue virus, and zika virus) and non-enveloped viruses (e.g., porcine parvovirus and adenovirus-associated virus). 76 these positive results were explained by the efficient binding and deactivation induced by the multivalent effect of the cds to the viral particles amino-functionalized cds were also tested for the treatment of human norovirus. in this study, cds were functionalized with 2,2′-(ethylenedioxy)bis(ethylamine) (eda) and 3ethoxypropylamine (epa) via amide bond formation. 77 these nms exerted a good viral blockage. in particular, epafunctionalized cds were able to inhibit 100% of viral infection at concentration of 2 μg/ml, while in the case of cds prepared with the other amines, 80% of inhibition was reported. these effects have been associated with the higher positive charge of cd-eda compared to cd-epa. another surface group used for viral targeting is boronic acid (ba), which can bind glycosylated surfaces forming boronic esters. this strategy was successfully adopted to treat hiv where the boronic groups, linked to different nanoparticles (e.g., silica nanoparticles and nanodiamonds) can target gp120 receptors on the viral envelope inhibiting the infection. 78 another recent study proposed the use of cd functionalized with phenylboronic acid for prevention of hiv infection. 74 the functional materials showed good inhibition properties compared to nonfunctionalized cds by preventing the binding to the target cell in vitro. overall, the use of cds for stopping host cell viral entrance has shown good results in vitro. however, there is a lack of proofs in vivo limiting their applications to surface disinfection or masks. in addition, most of the in vitro studies foresee first the contact of the cds with the viral particles and then their incubation with host cells. deeper investigations should be performed adding the nms at other time points (for instance in infected cells) to understand if the antiviral activity is maintained. in addition, cds have been successfully used for photodynamic therapy (generating radicals upon light irradiation) in cancer treatment. the same approach may be used to combat viral infections, where the antiviral activity induced by the surface modification can be sensibly enhanced by ros generation under irradiation. graphene materials, and in particular go and reduced go (rgo), have been used for different biomedical applications including drug delivery, biosensing, and tissue engineering. 5 go platforms have shown also interesting antimicrobial activity. 79 regarding viral infection, go was used to block the virus entrance in host cells. go and rgo can be considered as two-dimensional materials that contain hydrophilic and hydrophobic domains allowing to adsorb many biological molecules including nucleic acids and proteins. go showed low interaction with viruses, however its surface functionalization with target molecules can sensibly enhance its affinity for the viral particles. additionally, go can be used as photothermal agent (generation of heat by nir irradiation) or photodynamic therapy (using visible light irradiation) inactivating the capsids by local thermal shock or by radical formation during irradiation, respectively. the use of phototherapies may significantly augment the antiviral properties of the materials. however, we must keep in mind that these therapeutic modalities can be applied only to disinfection, since the radical/heat production may be harmful for the healthy tissues in vivo. photodynamic therapy has been successfully exploited using bacteriophage ms2 as a model virus. 80 in this study, go was functionalized with an aptamer recognized by the viral surface. the results showed that this functionalization is able to enhance the binding efficiency of the ms2 capsids onto the go surface compared to nonfunctionalized go. subsequently, irradiation in the visible light was able to disinfect the solution, while nonfunctionalized go showed much less activity due to the lack of adsorption. despite these interesting results, this pioneer work remains at an early research stage since the use of high light dose (300 w for 10−140 min) and the lack of material recovery and reuse make its application for surface disinfection difficult. go can be also used as a platform to link antiviral agents. encouraging results were reported using go with hypericin for the treatment of a recently appeared duck reovirus. 81 more recently, deokar et al. reported an original rgo-based multifunctional platform for hsv-1 treatment. 82 in this work, the authors functionalized the material with organic sulfate groups and iron oxide magnetic nanoparticles (fenps). the rgo functionalized with the sulfate is able to mimic the host cell surface and to bind hsv-1. subsequently, the viral particles captured onto the rgo-fenp surface can be concentrated via magnetic precipitation and destroyed via photothermal therapy. this approach is highly efficient for disinfection with low energy (1.6 w/cm 2 for 7 min) and cost effectiveness. hs is a common entry receptor in various types of viruses (e.g., herpes viruses, human papillomavirus, dengue virus). 83 the use of organic sulfate-functionalized graphene sheets mimicking hs, like go and rgo, has been already explored. however, it is worth noting that these nms are prone to strongly adsorb proteins in culture environments (coronation), likely inhibiting their antiviral efficacy. 84 high loadings of sulfate groups were introduced onto rgo using polyglycerol sulfate. 85, 86 this approach has been used for inhibition of orthopoxvirus, pseudorabies virus, and african swine fever virus in vitro. 85, 86 graphene has also been used as antiviral material. polysulfates and fatty amines were grafted onto graphene surface via triazine chemistry for the treatment of herpes simplex virus. 87 this strategy promotes the synergy between the electrostatic and hydrophobic interactions, showing incredibly high inhibition efficacy. overall, graphene materials have shown a good capacity to block host cell viral entry. disinfection with graphene family materials is also promising, offering the possibility to couple high viral binding with phototreatments. regarding the go and rgo activity in cellular environments, different parameters must be considered such as protein coronation, blood circulation time, and activity in vivo. so far, the use of sulfonic groups introduced via diazonium salt decomposition has been largely privileged. we take this opportunity to encourage future studies using other targeting groups (e.g., boronic acids) and grafting methods (e.g., epoxide ring opening or hydroxyl esterification reactions). mechanical disruption of the capsid. the most direct way to suppress viruses and stop the spreading of viral infection is to inactivate them before the attachment to the host cells, by binding to the acceptor proteins. 88 one of the most conserved targets of viral attachment ligands is the heparan sulfate proteoglycan (hspg), previously mentioned. hspgs are expressed on the surface of almost all eukaryotic cell types, and many viruses like hiv-1, hsv, human papilloma virus (hpv) exploit hspgs as the target of their viral attachment ligands. bearing in mind this behavior, different studies have used hspg-mimicking materials to target this type of virus−cell interaction and to achieve broad spectrum efficacy. for example, in one key study, aunps were functionalized with mercaptoethanesulfonate (mes) based on its mimicry of hs (au-mes nps). au-mes nps were shown to interfere with viral attachment, viral entry, and cell-to-cell spreading. 89 the importance of the polyvalent interactions with the virus makes these nps a good candidate for antiviral therapy. however, au-mes nps presented virustatic activity, meaning that upon dilution of the nps, the virus recovers its infectivity due to the reversibility of the cell-virion interaction. this problem was solved by stellacci and colleagues who developed nps coated with mercapto-1-undecanesulfonate (mus) ligands (au-mus nps). the long aliphatic and flexible linkers provide stronger associations with the viral particles compared to au-mes nps, leading to local distortions and eventually inducing a global deformation and breaking of the capsid that inactivates its contagion irreversibly (figure 8 ). 90 these au-mus nps were tested against different hspgdependent viruses, showing a high viricidal activity over hsv, hpv, and rsv ( figure 9a) . furthermore, the activity of the au-mus nps was studied in vivo using mice infected with rsv, indicating that the material can prevent pulmonary dissemination of the infection and showing potential use as medically relevant virucidal drugs to fight viral infections. more recently, the concept was further extended to cyclodextrins modified with mercapto-1-undecanesulfonate, proposing this system as a broad spectrum virucidal macromolecule. 91 besides au-nps, a similar mechanism of disruption was studied with other materials like graphene or go. in a study, in which the toxicity of graphene was evaluated theoretically, it was also shown that graphene nanosheets can interrupt the hydrophobic protein−protein interaction, which is essential to biological functions. 92 this feature was attributed to the hydrophobic nature of graphene. thus, it seems energetically favorable for graphene to slide between the interface of two proteins in contact, due to hydrophobic interactions. in another study inspired by this behavior, the authors performed molecular dynamics simulations of graphene nanosheets in the proximity of the surface of the ebola viral matrix protein vp40 showing that the nanosheets can break the hydrophobic interactions in vp40, a key protein for the replication and stability of ebola virus (figure 8 ). 93 these findings suggest that graphene nanosheets might have potential antiviral activity against ebola; however, there is a lack of experimental evidence that corroborates this mechanism of disruption. on the other hand, go was tested experimentally against pseudorabies virus and porcine epidemic diarrhea virus (pedv), showing significant decrease in the infectivity. 94 it was found that the negatively charged surface of go is important for the adsorption of the virus, whose surface is positively charged, and that go could directly interact with the viral particles and destroy their structures due to the sharp edges of the material (figures 8 and 9b) . the mechanical disruption of the capsid is a peculiar antiviral mechanism associated with some nms. in particular, the use of specific sulfonates able to mimic heparan sulfate can be also used to target sars-cov-2 infection. 95 blocking the viral entry, via liquid/surface disinfection or once in the body, is a powerful strategy to hamper early stage viral contagions. on the other hand, when infections have already spread and reached middle and late stages, alternative pharmacological strategies are required. the study of the viral pathogenesis and machinery inside host cells has allowed the preparation of different drugs. due to the present pandemic, such antiviral drugs are now of top interest in the scientific and medical communities. so far, few antiviral drugs are clinically available, and their mechanisms of action consist on the inhibition of reverse transcriptase (hiv, hepatitis), dna inhibition of polymerase (herpes, hiv), inhibition of protease (hiv), blockage of ion channels (influenza), and inhibition of neuramidase (hiv, influenza, hepatitis). however, these drugs suffer from moderate to severe side effects. additionally, rapid mutations in the viral machinery make them resistant to the treatments, making the control and the stop of the infection challenging. the use of hnms in drug delivery has shown several advantages. first, hnms can increase drug solubility and its circulation time. additionally, they can be functionalized with targeting molecules able to direct the drug to the desired organs and so avoiding side effects and reducing the dosage. more recently, new antiviral mechanisms were discovered. in particular, it was found that different types of hnms are able to change the ros homeostasis in infected host cells, stopping the viral replication and preserving the cell survival. in this section both drug delivery materials and hnms with ros modulation properties will be critically presented ( figure 10) . nanomaterials for drug delivery. different hnms have been widely tested for drug delivery applications. the advantages of this strategy are several including enhance drug solubility and the possibility of targeting and multivalent effects. as a potential broad spectral antiviral agent, agnps can prevent the virus from adsorbing to the host cell in the early stage of infection and thus show strong antiviral activity. after the cells are infected by the virus, there are ways to inhibit cell apoptosis (figure 2, right) . for example, lv et al. studied the anti-tgev activity of agnps in swine testicle cells and explored the possible mechanism of agnp inhibition of tgev infection-induced apoptosis. 18 the results showed that these agnps are able to decrease cell apoptosis through the activation of p38/mitochondria-caspase-3 signaling. 18 although the use of agnps has shown good antiviral action to further improve the therapeutic effects and reduce both side effects and drug resistance, the way of binding drugs or genes and other therapeutic agents to agnps has received particular attention. it has been observed that agnps inhibit the activities of neuraminidase and hemagglutinin, preventing the h1n1 influenza virus from attaching to host cells. at the same time, the potential molecular mechanisms revealed that caspase-3-mediated apoptosis was inhibited by ros generation. agnps modified with polyethylenimine (pei) can bind sirna. ag@pei@sirna exhibited superior abilities for enhanced cellular uptake and blocking ev71 virus infection acs nano www.acsnano.org review and significantly decreased the apoptotic cell population, which prevented the spread of ev71 virus. 37 in addition to drugs and sirna, neutralizing antibodies in combination with agnps were developed. the results demonstrated that there is an additive effect between the antibody and agnps when combined against cell-associated hiv-1 infection in vitro. 96 the membranotropic properties of fullerenes were widely exploited. for example, pristine c 60 , after accumulation at the cell membrane, can translocate into the cytoplasm by crossing the membrane through multiple energy-dependent pathways despite its hydrophobic character. 97 fullerenes can be made water dispersible using surfactants, sonication or first dissolving them in appropriate organic solvents (dmso). the use of fullerenes as inhibitors of viruses started in 1993 with a study focused on hiv infection. 98 this work revealed the interaction between c 60 derivatives and hiv protease through molecular modeling and experimental verification. hiv protease (hiv-pr) is involved in the mechanism of replication in the maturation of hiv virion, cleaving newly synthesized proteins. the active site of hiv protease has a cavity of 10 å closed to the diameter of c 60 cage. 99 molecular docking and experiments on hiv-pr catalytic activity revealed a blockage of the active site of the enzyme by van der waals interactions leading to an antiviral effect. the following studies were based on a structure−activity relationship between functionalized fullerenes and hiv-pr with the aim to increase the antiviral activity. the introduction of pyrrolidinium salts onto c 60 was tested against the activity of hiv-1 strain. 100 in a second study, c 60 bearing two ammonium groups was applied against the activity of hiv-1 and hiv-2 strains. 101 the results showed the importance of having two moieties in a precise position on the fullerene cage and the influence of the charge of the different salts sensibly increasing its antiviral activity. further investigations using different functional groups (e.g., amino acid derivatives) were explored. c 60 functionalized with aminobutyric acid and aminocaproic acid was able to inhibit hiv viral replication at subnanomolar concentrations. 63 in another work, anionic and cationic pyrrolidinium salts and amino acid functionalized fullerene derivatives were used for the inhibition of hiv reverse transcriptase (hiv-rt). fullerene compounds were compared to nevirapine, an available drug against hiv-rt, revealing a better inhibition compared to the pure drug. 102 the antiviral property of carboxylated fullerenes was confirmed by another study. 103 results obtained in vitro on cem cell line showed low toxicity and a submicromolar ec 50 against hiv-1 and hiv-2 strain viral replication. several mechanisms of hiv protease inhibitors have been hypothesized and simulated. some studies investigated the action of fullerene derivatives on viral replication cycle and the virus maturation ( figure 11 ). 98, 104 for the latter, it was suggested an impairment due to a strong interaction between fullerene and the immature capsid. 105 other rna viruses share ways of viral replication similar to hiv. 106 c 60 functionalized with an amino acid derivative was investigated against hepatitis c rna polymerase (hcv-rp). 102 this essential enzyme for viral replication was inhibited in a submicromolar range, similarly to benzo-1,2,4thiadiazine, a potent specific inhibitor of hcv-rp. another publication highlighted the effect of a c 70 poly(carboxylic acid) derivative on different strains of influenza virus (e.g., a, h1n1, h3n2, and b). 107 the inhibition was comparable to tamiflu, rimantadine, ribavirin, and amantadine, but the mechanism of action remains still unclear. these data emphasize that fullerenes c 60 or c 70 can be potent universal antiviral drugs for rna enveloped viruses. however, clear elucidations of the mechanisms of action and in vivo studies are still a missing point. due to their high surface/weight ratio and capacity to pass through cell membranes, carbon nanotubes (cnts) have been extensively explored for drug and gene delivery applications. 108 cnts have been also successfully used for the delivery of antiviral agents. compared to pristine materials, oxidized cnts (ox-cnts) showed an inhibitory activity for hiv viruses per se. 109 this effect has been associated with the oxygenated groups that increase the hydrophilicity and the colloidal stability of the material. the antiviral efficiency has been correlated to the ox-cnt interaction with host cells. 109 however, it is not clear how and what kind of mechanism blocks the viral machinery in host cells. different anchoring strategies have been explored to link antiviral drugs onto cnt surface. for instance, ox-cnts were covalently linked to 2amino-3-nitro-1-(3,5-dimethylbenzyl)-aniline (chi360) and n-(2-aminophenyl-3-nitro-)-3,5-dimethylbenzenesulfonamide (chi415), two active non-nucleoside reverse transcriptase inhibitors for hiv treatment. 109 following the covalent conjugation, only a moderate antiviral effect was observed compared to pure drugs, indicating that most probably the nm cell trafficking played a key role on the virucidal activity. 109 in another study, ox-cnts functionalized with cyclodextrin were used for the delivery of acyclovir (a prodrug inhibitor of the viral dna polymerases) for the treatment of hsv-1. preliminary results showed that when acyclovir was delivered via the nanotubes, the viral antireplicative effect was higher than the free drug. 110 more recently, a similar approach was applied to herpes virus using cyclodextrin and pei-functionalized cnts for co-delivery of cidofovir and plasmid dna. however, the antiviral effect of the materials was not explained, and the transfection effect was not satisfactory. 111 functionalized cnts have been also used for delivery of ribavirin in vivo using grass carp as an animal model for the study of grass carp reovirus. ox-cnts were first functionalized via amidation with bsa, and then ribavirin was covalently bound to the protein via esterification (figure 12) . 112 in vivo tests demonstrated that, when ribovirin was shuttled by ox-cnts, the antiviral efficiency was significantly increased without any evident toxicity and no significant changes in ros-generating enzymatic activities. the use of cnts in drug delivery is however still controversial. 113 graphene-based materials have been limitedly studied as antiviral drug delivery carriers. only a few examples can be found in the literature. graphene quantum dots (gqds) were used for drug delivery of chi360 and chi415 and tested in vitro against hiv similarly to cnts. 109 both the prepared gqd-chi360 and gqd-chi415 showed a high antiviral activity once into host cells, with low toxicity. go has been also used for the delivery of dnazyme into hepatic cells allowing to block the hepatitis c infection. this specific dna single strand is able to recognize the viral mrna and to silence its expression. 114 overall, carbon nms proved to be interesting carriers for antiviral drugs. however, several questions need to be answered before their safe application as antiviral materials. different reports have demonstrated that pristine cnts display relevant toxicity for healthy cells, but by oxidation of the tubes, the side effects can be sensibly reduced. 109 in addition, more investigations should be performed on cnt toxicity. these nanocarriers indeed are not "innocent delivery agents", but they play a key role in drug internalization pathway and in host cell machinery that might be averse to the expected therapeutic effects. 109 so far, cnt application in biological systems has been studied for 20 years. however, due to their possible toxicity, their real application in clinics seems to be steeper and difficult to achieve. 112 materials tuning reactive oxygen species. ros homeostasis in infected cells has been studied for both rna and dna viruses. for instance, it was shown that infection of mice with influenza a decreased the concentration of lung glutathione and the antioxidant vitamin c, providing evidence that the viral infection was associated with oxidative stress in vitro as well as in vivo. 115 similarly, in hiv infection, induced oxidative stress in host t cells and high concentrations of antioxidants are able to slow down the cell-to-cell viral spreading. 115 the increase of ros concentration is a common process in most of the viral infections. however, the mechanism of radical generation is different from case to case. several proofs suggest that modulating ros homeostasis in infected cells can slow down or block the infection. 116 hnms have been shown to be powerful allies against viral infections. in particular, metal or metal oxide nms, once internalized, can regulate the radical production into infected host cells. in this scenario, the nms can work following two different mechanisms: (1) enhancing radical activity or (2) quenching ros inside cell compartments. in the first case, metal oxide nps are able to convert superoxide ions into more reactive hydroxyl radical species via fenton or fenton-like reactions. the excess of superoxide ions is able to oxidize the viral proteins and the genetic material and therefore efficiently block the infection. this approach has been reported using zno nps. 117, 118 in these studies, zno nps have been successfully applied for the treatment of h1n1 influenza virus and herpes simplex virus. the preliminary results showed that pegylated zno particles were able to efficiently reduce the viral infection with ic 50 similar to acyclovir. more importantly, toxicity of zno was modulated by functionalization with peg, which allowed a higher colloidal stability and a more controlled release of zn 2+ ions to catalyze ros formation. indeed, ros (e.g., superoxide and hydroxyl radicals) produced by the nps should be highly reactive and should not only damage the exogenous biological molecules but also attack different cell compartments. overproduction of ros may reduce virus spread but also induce cell death. interestingly, this approach is applicable only at the early infection stage (after 1 h incubation of the host cells with a virus), but it loses its activity at later time points. these 117, 118 this specific mechanism of action restricts zno nps to an application only at the early stage of infections. however, the same approach with other metals able to induce fenton or fenton-like reactions (e.g., fe, cu, and mn) has not been reported yet. the choice of proper capping agents may allow to control the metal ion release and thus tune the ros-mediated antiviral activity. we would like to take an advantage here to suggest the growth of the antiviral research in this direction. another successful approach relies on the reduction of ros concentration in host cells. ros scavenging is able to alleviate the toxicity of the infection enhancing cell viability, giving time to start its endogenous antiviral mechanisms. so, this approach may both block infection and ensure host cell survival. in this context, selenium nps (senps) have been extensively studied for their antiviral activity. the mechanism of action of these nps relies on the quenching of the radicals into host cells due to the infection, stopping the mitochondria depolarization and the consequent apoptotic cascade. 119 additionally, senps can also adsorb onto the viral capsid sensibly reducing their infectivity. senps can be prepared via classical mixing of selenium salt precursors in the presence of a reducing agent. more recently, senps have been instead biosynthesized from actinobacteria showing good stability and capacity to inhibit dengue virus in vitro. 120 moreover, senps were used to carry different antiviral drugs including zanamivir, 121 oseltamivir, 122 amantadine, 123 and ribavirin. 119 their functionalization with the desired drug can be easily achieved adding the molecule during their synthesis through the se ion controlled reduction. these nms have been applied for the treatment of h1n1 virus. notably, senps with ribavirin (administered via intranasal absorption every 24 h for 3 days) showed that infected mice had much less alveolar collapse and perivascular and peribronchiolar edema, compared to the group challenged with the virus (figure 13 ). 119 due to their efficacy and low toxicity, senps can be considered a useful material for the treatment of other viral diseases including sars-cov-2. as a matter of fact, oxidative stress as well as chronic inflammation may contribute to the aggravation of the covid-19 symptoms and to the general spread of the infection. 124 the use of senps could eventually alleviate the toxicity of infected patients, giving time for the immune system to react against the contagion. however, the lack of preclinical studies on senps, together the scarce knowledge of their biosafety and long-term toxicity, still remain the main challenges to tackle for clinical translation. the vast majority of the studies show that human survival to viral attack is based on the stimulation and response of our immune system. when a virus enters and starts infecting tissues, the body reacts and triggers a strong immune response to overcome the pathogen invasion and spread. normally, two different immunological reactions take place. the oxidative stress induced by infection causes the activation of the inflammasome through upregulation of pro-inflammatory cytokines such as il-1β, pro-il-18, and nlrp3. 122 excessive upregulation of this mechanism leads to cell damage and eventually to pyroptosis activated by caspases. 122 it was also shown that generation of radicals is able to depolarize mitochondria, hence affecting host cell respiration and inducing ros-mediated apoptosis at the late stage of infection. 123 besides, activation of pro-inflammatory cytokines can alert the immune system blocking the infection (the innate acs nano www.acsnano.org review immune response). 122 the second immune response is specific (the adaptive immune response). immune cells are trained to attack the virus (cellular response), while specific antibodies are produced by b cells (humoral response). in the last years, hnms were proved to tune the immune responses, demonstrating to be a possible alternative against viral infection. in this section the most relevant strategies for the activation of innate and adaptive immune responses using nms will be described ( figure 14) . innate immune response. innate immune response is the first response that takes place in the presence of any type of infection. during this early stage, interferon stimulating genes (isgs) are upregulated in the infected cells. 125 this selfdefense mechanism slows down the viral replication and alerts sentinel immune cells that start producing proinflammatory cytokines and trigger inflammation. 125 however, many viruses are able to escape this complex mechanism, retarding the immune response and spreading the infection. the interaction of hnms with the immune system has been more and more studied. in the case of antiviral hnms, many examples can be found in the literature where the nms not only slow down the infection but also tune the innate immune response. certain hmns can display an intrinsic immune stimulation. we have already mentioned above that in the early stage of infection, agnps mainly prevent the virus from entering the host cell through the interaction with the external capsid, but in vitro cellular experiments lack to understand the complex interaction with primary immune cells. recent studies have shown that agnps can potentially induce the expression of genes involved in innate and adaptive immunity-associated pathways, which are known to play crucial role in immune regulation. for example, toll-like receptor 7 can be upregulated by agnps after 24 h, by recognizing the singlestranded rna of the viruses and regulating the antiviral immune response. 126 another study showed that in rsvinfected mice treated with agnps, the particles reduced the production of pro-inflammatory tnf-α and il-6 cytokines, but potentiated the anti-rsv activity of neutrophils in an experimental mouse model. 127 however, activation of agnps in the reduction of rsv has been noted only when the nm was intranasally inoculated together with the virus, and no results have been reported on the use of agnps administrated on infected mice. in the case of influenza virus infection of lung epithelial cells, it was found that agnps targeted infected lung epithelial cells and reduced viral replication, by preventing autophagy. however, the blockage of the autophagic flux by agnps does not inhibit viral replication in already infected cells. therefore, agnps are more suitable as viral preventive agents due to their pro-inflammatory response rather than drugs. 128 more recently, agnps were combined with graphene materials and exploited as antiviral material for the treatment of porcine reproductive and respiratory syndrome virus (prrsv). 129 go-agnps were able to clump the virus diminishing its fusion with cell membrane. additionally, once go-agnps were internalized in host cells, they stimulated the isgs that blocked viral budding and its diffusion to other cells in vitro (figure 15 ). 129 similarly, cds used for the treatment of rsv and prrsv were able to activate the innate immune response via an upregulation of isgs in vitro. 130 gold nanorods were applied to boost the innate immune response against rsv in vivo. 131 interestingly, it was shown that these nanorods (when intranasally administered with rsv) were not only able to activate the isgs but also to tune the production of pro-and anti-inflammatory cytokines, resulting in the blocking of the infection with a reduced pulmonary inflammation. 131 as drug carriers, hnms can also affect the internalization pathways, modulate drug efficacy, and the immune cell responses. for instance, it was found that the isoprinosine immunomodulatory antiviral drug displays a much higher antiviral efficacy in vivo when delivered with cnts than as a pure drug (in acs nano www.acsnano.org review zebrafish larvae food administration), probably due to a better cellular uptake and the anti-inflammatory properties of the nanotubes. 132 fullerenes also exhibit immunomodulation properties through the release of cytokines by bovine alveolar macrophages. 133 a study explored the influence of functionalization of c 60 with molecules presenting different surface charges like hydroxyl groups and amino acids. 134 the study concluded that negative charges upregulated tnf-α up-secretion in raw 264.7 macrophages, but highly positively or negatively charged surfaces increased cell toxicity. the upregulation of the cytokine tnf-α is a proof that fullerene can promote cellular immune response. despite these preliminary results, the actual mechanisms of interaction between hnms and the immune system are still at early stage of understanding. we must consider that the immune response needs to be proportional to the infection grade. if on one side nanosized immuno-boosters can alert more efficiently the sentinel cells, the use of hnms that trigger an exaggerated immune response can promote excessive inflammation, damaging healthy cells and promoting uncontrolled side effects. in the particular context of sars-cov-2, the use of agnps, fullerenes, or other pro-inflammatory hnms at the middle and late infection stage may cause an aggravation of the symptoms due to already diffused inflammation. in particular, most of the studies showed the ability to reduce infection when hnms were first incubated with the pathogenic virus, thus limiting their potential use in the early stage infection. 124 the formulation of hnms able to both alert the immune system (e.g., upregulating cytokine) and control lung inflammation (e.g., ros scavenger) even after the early stage infection may be a possible strategy for treatments against sars viruses. adaptive immune response. adaptive immune response is the specific response that the immune system exerts against pathogens. this mechanism is particularly active toward viral infections where the immune system produces specialized lymphocytes (to fight the virus), called memory b cells (to be effective in case of new infections) and antibodies (corresponding to the humoral response). 135 the stimulation of adaptive responses in case of specific infections can be induced artificially through the introduction of attenuated pathogens, stimulating the production of specific antibodies. this is the principle of vaccination, which is the most common procedure for immunization of large areas of population against many kinds of lethal viruses. 135 besides, the use of viral proteins as antigens in the vaccine formulation leads to neutralizing antibodies, but, due to the low immunogenicity of isolated proteins, does not always stimulate sufficiently the immune system to reach total protection. more recently, nanotechnology has been applied to develop more efficient vaccines (e.g., nanovaccines). the use of nanostructures with a size similar to virus (virus-like nanoparticles) sensibly enhances the response helping to reach immunity. 135 hnms can adsorb viral particles and present them to the immune system. 136, 137 this method of vaccination has been successfully applied in vivo for the challenge of herpes simplex 2 virus. hsv-2 starts its spreading in vaginal tissues and then diffuses to the neurons causing death in mice. zno nps (teardrop morphology), after vaginal inoculation with hsv-2, are able not only to prevent viral cell adhesion but also to expose viral antigens to t cells and dcs, leading to immunization. the preclinical trials acs nano www.acsnano.org review against hsv-2 showed a survival to infection higher than 90%. this approach highlights the possibility to couple cell mimicking nms to other co-adjuvants for the formulation of large spectrum nanovaccines ( figure 16 ). 136, 137 hnms have been also applied to the delivery of antigens, exposing them to the immune system. fullerenes were found as suitable carriers for the delivery of drugs or nucleic acids. 138 functionalized fullerene can also self-assemble into virus-sized nps. 139 investigated as vaccines in cancer immune therapy, 140 polyhydroxy fullerenes (called fullerenols) display interesting properties for antiviral therapy, based on their capacity to selfassemble into virus-like particles (vlps) and so to enhance the immunogenicity of the antigens. 141 the great advantage of this strategy relies on the easy encapsulation process during the self-assembly making fullerenols versatile for the formulation of different kinds of vaccines. these vlps were investigated against hiv-1 141 and hepatitis c viruses. 142 compared to conventional protein-or peptide-based vaccines intended to induce antigen-specific adaptive immune responses, dna vaccines are more stable, cost-effective, easy to manufacture, and safe in handling. 143 however, dna vaccines have the disadvantage of being poorly immunogenic. 144 fullerenol vlps allow to avoid the use of other adjuvants. in the case of a vaccine against hiv-1, fullerenol vlps penetrated easily into the cells resulting in an enhancement of dna transfection. this was proved in a study using fullerenol encapsulating dna encoding the hiv-1 envelope protein gp145 ( figure 17 ). 141 in vitro assays were performed in human embryonic kidney cells line (hek293) showing good transfection ability. following various immunization routes (e.g., activation of toll-like receptor signaling or effector memory t cell immune response), fullerenol vlps can induce an innate and a cellular immunity. a similar study was performed for hepatitis c using the hcv recombinant protein as antigen, 142 confirming the potential efficacy of using fullerenols as antiviral vaccines. nevertheless, these results require further mechanistic investigations. indeed, in vitro studies also evidenced a suppressive effect of acquired immune response of c 60 pyrrolidine tris-acid and fullerenol c 60 (oh) 36 . 145 the fullerenol had a dose-dependent effect on t cell receptormediated activation and antibody production by b cells under anti-cd40/il-4 stimulation. however, the molecular mechanism is still unknown. other hnms including aunps (two subcutaneous injections in guinea pigs) and nanodiamonds (three subcutaneous injections in balb/c mice) were explored as carriers of viral proteins for immunization of swine transmissible gastroenteritis virus and h7n9 influenza, respectively, with good preliminary results. 146, 147 in these cases, the vaccine formulation relies on the adsorption of the viral antigen onto the surface of the nanoparticles. however, an effective vaccination depends on several factors. first of all, the size of the nps plays a key role on the immune system. for instance, size-dependent vaccination efficacy has been reported in mice immunization against the foot-and-mouth disease virus (intraperitoneal and subcutaneous injection, every 7 days for 7 weeks) using aunps as antigen carriers. in this study, the most effective activity to stimulate the immune system was exerted by particles with a diameter in the range of 8 nm. 148 both smaller or bigger particles evidenced a drop-off of the immunization effect. this aspect cannot be ascribed to the antigen concentration, but must be associated only to the acs nano www.acsnano.org review nanoparticle size; however, the mechanism of interaction remains unknown. the selected antigen plays also a crucial role in the preparation of wide spectrum vaccines. for instance, in the case of influenza, two major membrane glycoproteins, hemagglutinin and neuraminidase, are generally used as antigens. however, the antibodies produced by this vaccination strategy are selective to the dominant epitope which has a low effectiveness or is totally ineffective against other epitopes or other kinds of influenza viruses. m2 (a viral protein responsible for the budding and scission of the influenza virus) is commonly expressed in different types of influenza viruses with a high rate of conservation but with low antigenicity. it has been shown that aunps functionalized with m2e protein have a high immunization capacity in comparison to the antigen alone. mice immunized with aunps (two intranasal injections), and then challenged, showed a survival rate higher than 90% to california-h1n1pdm, victoria-h3n2, and vietnam-h5n1 infections. 149 this strategy shows that hnms can be used to boost the immune response of low immunogenic molecules, providing a wide spectrum vaccination potential. unfortunately, it has not been determined if the budding process in sars-cov-2 is mediated by viral proteins or via the host cell's endosomal sorting complex, thus more research on the sars-cov-2 viral machinery is highly desirable. all these approaches are based on the capacity of the nanoparticles to adsorb the antigens and expose them to the immune system in the appropriate conformation to produce the neutralizing and protective antibodies. however, the adsorption is not an easy process to control. for instance, aunps have been ineffective in the immunization against sars-cov, when s viral proteins were used as antigens. 150 in particular, the immunization with the protein alone was more efficient than when it was adsorbed onto the aunp surface. this failure was associated with a conformational change or denaturation of the antigen, which did not lead to the production of the specific antibody. 150 covalent chemistry strategies offer a higher control on the antigen quantification with higher reproducibility and possibility to bind different groups onto the surface of hnms. for example, calcium phosphate nanoparticles (capnps) were successfully covalently functionalized with hen egg lysozyme as model antigen showing an immunization 100 times higher in vivo compared to antigens alone using (one subdermal injection in mice). 151 a similar approach was used with iron oxide nps using mannose (to target dcs) and hepatitis b antigen showing good immunological activity in vitro (two subdermal injections in mice at 14 days distance). 152 more recently, other types of vaccination strategies have been applied using hnms. a smart example has been reported using multifunctional capnps on herpes virus. in this study, capnps have been covalently functionalized with alum/mpl as the adjuvant and two peptides as antigens selected via reverse vaccination. the nm stimulated the immune system generating highly efficient antibodies able to block cell-to-cell infection of herpes virus in vivo (three intramuscular injections in mice every 14 days), increasing the survival rate of immunized mice to 100% against the controls (20% survival, figure 18 ). 153 the recent history has shown the spread of different viral pandemics such as h1n1 flu, hiv, and sars. nowadays, the sars-cov-2 pandemic global lockdown has profoundly changed the daily life of most humans, causing uncertainty in short and middle time perspectives. in this context, the scientific community has responded to protect the population by studying new vaccines and disinfection methods to be applied in the near future. despite this tough work, a sars-cov-2 vaccination will hopefully be available in 1−2 years, making this epidemic transient period gloomy with increased instability. the study of more effective vaccines and the production of a wide range antiviral agents is nowadays an extremely hot topic. hnms comprise a family of materials that share a nanostructured hard core and a tunable surface chemistry. in this contribution, we have methodically reviewed different hnms for antiviral properties. hnms can have antiviral properties per se, blocking the viral replication and diffusion, or their antiviral properties can be tailored, playing with surface chemistry. hnms can be used to block viral entry and arrest infection at the early stage. the mechanisms rely on different actions including breaking of capsid disulfide bonds (e.g., noble metal nanoparticles), capsid oxidation (e.g., cuonps), mimicking of cell surface (e.g., carbon nms), or mechanical disruption (e.g., aunps or graphene). surface functionalization additionally confers a higher specificity and pharmacological activity toward the targeted virus. in particular, the high local concentration of ligands on functionalized hnm surface imparts a high multivalent effect, enhancing the viral trapping efficiency of nms. interestingly, hnms that show an intrinsic antiviral activity can further enhance their antiviral efficacy via surface functionalization. some antiviral hnms are good photosensitizers (e.g., cuonps) or exert photothermal activity (e.g., carbon nms), thus their antiviral activity can be trigged by light stimulation. more importantly, most of the settled strategies target common viral entry mechanisms and can be adopted to fight a wide viral spectrum. hnms have been also applied as antiviral agents by their interaction with host cells. hnms are able to block viral replication machinery in host cells (e.g., cnts and fullerenes), inhibiting endogenous enzyme activity. additionally, hnms can be explored for the delivery of antiviral molecules, showing a better antiviral activity and reducing side effects in vitro and in vivo. some hnms can also regulate the ros homeostasis of host cells, reduce apoptosis, and enhance host cell survival during the infection. finally, we have reviewed the role of hnms on immunity. in particular, hnms can stimulate the innate immune response, mainly inducing overexpression of interferon and cytokines. this effect can alert sentinel cells and generally warn the immune system of the infection. hnms can be also used for the activation of the adaptive immune response, foreseeing vaccination. due to the similar size of a virus, functionalized hnms with antiviral molecules (virus-like particles) can enhance their immunogenicity. certainly a huge effort should be done for translation of the research into clinics. indeed, hnm applications as antivirals are still in the early phase of research. several challenges still need to be tackled before their safe use. at the current stage, some hnms have been approved only for surface disinfection. for instance, cunps have been used in filters for the preparation of highly efficient broad spectrum antiviral masks. 50 some hnms have been already clinically approved. for example, fenps were approved for imaging and as a drug to treat iron deficiency anemia in adult patients with chronic kidney disease, while aunps are in clinical trials for the treatment of prostate cancer (photothermal therapy). 154, 155 however, at the moment no clinical trials are running for the use of hnms as antiviral agents. in fact, there are still several concerns on the applications of hnms in drug formulations. compared to molecules, where mainly concentration and exposure routes are concerned, solving hnm toxicity issues is much more complicated. composition, size, shape, and surface functionalization must be considered to respond to the requirement for safety regulations. additionally, interaction on hnms with the immune system must be better elucidated. in particular, the activation of the immune system and complement activation-related pseudoallergy must be taken into account. 156 for instance, ferumoxytol (a fenp-based drug) has been reported to generate severe anaphylactic reactions in humans, 18 of which were fatal. 156 this is due to the possible interaction of hnms with mast cells, provoking their degranulation/activation and release of histamine even at the first exposure (pseudoallergic-mediated hypersensitivity). besides, the mechanism of activation of these cells is still unknown, although it was found that it depends on the hnm composition, size, and surface chemistry and on the corona formation. 156 on the other hand, it has been demonstrated that hnms can travel to the draining lymph nodes, targeting resident dendritic cells and macrophages. therefore, they are able to interact with antigen presenting cells to stimulate innate and adaptive immune responses. 156 we believe that a joint venture of different chemists, materials scientists, virologists, toxicologists, and medical doctors can push forward the preparation and safe application of hnms in this field, hoping to prevent and eventually block the rise of new viral pandemics. alberto bianco − cnrs, immunology, immunopathology and therapeutic chemistry, upr 3572, university of strasbourg isis, 67000 strasbourg, france; orcid.org/0000-0002-1090-296x; email: a.bianco@ibmc-cnrs.unistra.fr giacomo reina − cnrs, immunology, immunopathology and therapeutic chemistry, upr 3572, university of strasbourg isis, 67000 strasbourg, france; orcid.org/0000-0002-8147-3162; email: g.reina@ibmc-cnrs.unistra.fr hard nanomaterial, nonpolymeric organic or inorganic materials with sizes comprised between 1 and 100 nm; antiviral, medical term used for any agent or drug altering virus integrity or process involved in viral infection disease; viral infection, process by which viruses invade the body through multiple pathways and multiply in susceptible host cells; viral pathogenesis, approach in biomedical research to understand the process by which a viral infection leads to disease, including mechanism of infection into the host (e.g., viral entry, viral replication) and factors that affect this mechanism (e.g., virus susceptibility to host defenses); membranotropism, the ability of an organism or an agent to interact with biological barriers; immunogenicity, capacity of an exogenous substance or material to trigger an immune response in humans and other animals or to induce a humoral and/or cellular immune responses; multifunctional platform, material modified with different functionalities to achieve a variety of combined treatments aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 recent advancements in two-dimensional nanomaterials for drug delivery metal-based nanomaterials in biomedical applications: antimicrobial activity and cytotoxicity aspects promises, facts and challenges for graphene in biomedical applications mediated tumor targeting using ultrasmall-hybrid nanoparticles: from animal to human with theranostic aguix nanoparticles. theranostics 2020 nanotherapeutic anti-influenza solutions: current knowledge and future challenges nanoparticles: alternatives against drug-resistant pathogenic microbes an overview of functional nanoparticles as novel emerging antiviral therapeutic agents high 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b-cell targeting vaccines: tailoring of humoral immune responses by functionalization with different tlr-ligands hbs antigen and mannose loading on the surface of iron oxide nanoparticles in order to immuno-targeting: fabrication, characterization, cellular and humoral immunoassay induction of herpes simplex virus type 1 cell-to-cell spread inhibiting antibodies by a calcium phosphate nanoparticle-based vaccine progress in nanomedicine: approved and investigational nanodrugs gold nanoshell-localized photothermal ablation of prostate tumors in a clinical pilot device study engineered nanomaterials and type i allergic hypersensitivity reactions shiyuan peng − cnrs, immunology, immunopathology and therapeutic chemistry, upr 3572, university of strasbourg isis, 67000 strasbourg, france lucas jacquemin − cnrs, immunology, immunopathology and therapeutic chemistry, upr 3572, university of strasbourg isis, 67000 strasbourg, france andreś felipe andrade − cnrs, immunology, immunopathology and therapeutic chemistry, upr 3572, university of strasbourg isis, 67000 strasbourg, france complete contact information is available at: https://pubs.acs.org/10.1021/acsnano.0c04117 the authors declare no competing financial interest. the authors gratefully acknowledge the financial support from the eu graphene flagship project (no. 881603). this work was partly supported the agence nationale de la recherche (anr) through the labex project chemistry of complex systems (anr-10-labx-0026_csc). we wish to acknowledge the centre national de la recherche scientifique (cnrs) and the international center for frontier research in chemistry (icfrc). s.p. is indebted to the chinese scholarship council for supporting her ph.d. internship as a visiting ph.d. student. a.f.a. wish to thanks the eur csc graduate school (strasbourg, france) for supporting his master studies. key: cord-302247-moor7dfc authors: richards, james; rodan, ilona title: feline vaccination guidelines date: 2001-05-31 journal: veterinary clinics of north america: small animal practice doi: 10.1016/s0195-5616(01)50602-6 sha: doc_id: 302247 cord_uid: moor7dfc the 1998 report of the american association of feline practitioners and academy of feline medicine advisory panel on feline vaccines was developed to help veterinary practitioners formulate vaccination protocols for cats. the current panel report updates information, addresses questions, and speaks to concerns raised by the 1998 report. in addition it reviews vaccine licensing, labeling, and liability issues and suggests ways to successfully incorporate vaccination protocol changes into a private practice setting. patients are healthy before vaccination. because vaccination alone does not completely protect animals from infection and disease, environmental conditions should be addressed and exposure to infectious agents should be minimized. the overall objectives of vaccination are to vaccinate the largest possible number of individuals in the population at risk, vaccinate each individual no more frequently than necessary, and vaccinate only against infectious agents to which individuals have a realistic risk of exposure and subsequent development of disease. kittens younger than 16 weeks of age are generally more susceptible to infection than are adult cats and typically develop more severe disease. they represent the principal target population for vaccination. 40 maternal antibody interference is the most common reason why some animals are not immunized after vaccination, and it is also the reason why a series of vaccinations is necessary for kittens younger than 12 weeks of age. 20 vaccination needs of adult cats should be assessed at least once yearly and, if necessary, modified on the basis of an assessment of their risk. it is recommended that administration sites for parenteral vaccine be chosen in accordance with the guidelines established by the american association of feline practitioners and adopted by the vaccine-associated feline sarcoma task force (table 1) . 46 use of multiple-dose vials is discouraged, because inadequate mixing may result in unequal distribution of antigen and adjuvant, possibly resulting in decreased efficacy or an increased likelihood of adverse events; iatrogenic contamination is an additional risk. the panel discourages the use of polyvalent vaccines other than those containing combinations of feline panleukopenia virus, feline herpesvirus-1 (fhv-1), and feline calicivirus (fev), exclusively. this opinion is based on the belief that as the number of antigens in a vaccine increases, so too does the probability of associated adverse events. additionally, use of polyvalent vaccines may force practitioners to administer vaccine antigens not needed by the patient. feline panleukopenia is caused by feline parvovirus (fpv). the virus remains infectious for months to years in the environment and is primarily spread via the fecal-oral route. fomites (e.g., cages, food bowls, litter boxes, health care workers) play an important role in the transmission of the organism. clinical signs of infection include lethargy, an-orexia, vomiting, diarrhea, fever, and profound panleukopenia; mortality is highest in young susceptible cats. 12 in utero infection with fpv is a common cause of cerebellar hypoplasia. 12 vaccination against fpv is highly recommended for all cats. immunity to 'feline panleukopenia is primarily through antibody response to natural infection, vaccination, or passive transfer of maternal antibodies from queen to kittens. maternal antibody may interfere with immunization when antibody titers are high during the neonatal period. maternal antibody titers generally wane sufficiently to allow immunization by 12 weeks of age. 47 immunity conferred by feline panleukopenia vaccines is considered to be excellent, and most vaccinated animals are completely protected from infection and clinical disease. serologic and challenge exposure data indicate that a parenteral fpv vaccine induces immunity that is sustained for at least 7 years. 4s , 49 after the initial series of vaccinations and revaccination 1 year later, cats should be vaccinated no more frequently than once every 3 years. modified-live virus (mlv) vaccines and adjuvanted inactivated virus vaccines for parenteral administration as well as an mlv vaccine for topical (intranasal) administration are available and effective. experimental studies have shown that intranasal administration of canine parvovirus-2 vaccines to puppies is less effective than parenteral administration in overcoming maternal antibody interference (ronald schultz, phd, personal communication, 2000). the most likely reasons are that fewer virus particles reach lymphoid tissue when the product is given intranasally compared with parenteral administration and viral replication in lymphoid tissue is required for immunization with mlv parvovirus vaccines. although studies have not been performed in cats, the same phenomenon may occur in this species. caution is appropriate when contemplating the use of intranasal fpv vaccines for primary immunization of kittens, especially those residing in environments where exposure to fpv is likely. it has been found recently that some cats with panleukopenia-like disease were infected with canine parvovirus-2b. studies show that fpv vaccines provide excellent protection not only from fpv but also from canine parvovirus-2b; thus, canine parvovirus infection should not be a concern for cats immunized as a result of vaccination with fpv vaccines. 39 serious adverse events associated with fpv vaccines are rare. tumor formation at the site of a topically administered vaccine has not been reported. vaccination of pregnant queens with modified-live fpv vaccines may possibly result in neurologic disease in developing fetuses 52 ; the same concern applies to kittens vaccinated at less than 4 weeks of age. the use of mlv vaccines should be avoided in pregnant queens and kittens less than 1 month of age. 52 iimost often, the product approved for use annually is given for initial vaccination followed 1 year later and every 3 years after that by administration of the product approved for use every 3 years; however, vaccination interval must comply with local and state statutes. ilfelv testing is recommended before vaccination; infected cats do not derive any benefit from vaccination. *'this product is not the same as the b. bronchiseptica vaccine approved for use in dogs; the product approved for use in dogs should not be used in cats. mlv = modified-live virus; felv = feline leukemia virus; sc = subcutaneously. feline viral rhinotracheitis caused by fhv-1 and fcv infection account for up to 90%. of all cases of infectious upper respiratory tract disease in cats. 17 both viruses are shed in ocular, nasal, and pharyngeal secretions of infected cats.42 organisms are transmitted from cat to cat directly through sneezed macrodroplets or indirectly via contaminated fomitesy the disease is self-limiting; however, infected cats may develop chronic oculonasal disease. latent infection is lifelong for cats infected with fhv-1; reactivation can occur during periods of stress or after corticosteroid administration. some cats infected with fcv become persistently infected and shed virus for prolonged periods (months to years). although rarely serious in adult cats, disease caused by these viruses may be severe, and· sometimes fatal, in kittens. lameness and chronic oral inflammatory syndromes have been linked to calicivirus infection and vaccination with modified-live calicivirus vaccines. 3 ,7, 10, 11, 45, 57 risk of exposure to either fhv-1 or fcv is high, because both organisms are widespread tn the feline population. vaccination against fhv-1 and fcv is highly recommended for all cats. immunity is through humoral and cell-mediated immune responses to natural infection or vaccination or through passive transfer of maternal antibodies from queen to kittens. maternal antibody may interfere with induction of a systemic immune response; however, by the time that kittens are 12 weeks of age, maternal antibody titers wane sufficiently to allow parenteral immunization. topically administered (intranasal, conjunctival) vaccines are capable of inducing a local immune response in the face of high maternal antibody titers.27 serologic and challenge exposure data indicate that parenteral fhv-1 and fcv vaccines induce protection that lasts at least 3 years. 48 ,49 after the initial series of vaccinations and revaccination 1 year later, cats should be vaccinated once every 3 years. regardless of the route of administration, fhv-1 and fcv vaccines induce only relative but not complete protection. at best, these vaccines induce an immune response that lessens the severity of disease; vaccinates are not immune to infection nor are they protected from all signs of disease. 2o currently available fcv vaccines probably do not induce protection from all isolates of the virus. 9 mlv and inactivated virus vaccines for parenteral administration and mlv vaccines for topical (intranasal and conjunctival) administration are available. if a susceptible cat is born into or is entering an environment in which viral upper respiratory tract disease is endemic (e.g., some catteries, boarding facilities, shelters), the use of a topical product may be advantageous. administration of such products to kittens as young as 10 to 14 days of age could be considered in these situations; however, products that also contain modified-live fpv antigens should not be administered to kittens younger than 4 weeks of age. 62 adverse events associated with vaccination against fhv-l and fev include mild transient fever, sneezing, conjunctivitis, oculonasal discharge, lameness, and, for parenteral products, pain at the injection site. 9 ,11 sneezing, conjunctivitis, oculonasal discharge, and ulceration of the nasal philtrum are believed to occur more frequently with vaccines licensed for topical use. tumor formation at the site of a topically administered vaccine has not been reported. rabies is transmitted mainly through bite wounds of infected mammals. more cats than dogs develop rabies in the united states 29 ; although they are relatively resistant to rabies, both species serve as potential sources of infection for human beings. 21 ,29 treatment is ineffective in cats that develop clinical signs and should not be attempted because of the high potential for zoonotic infection. 21 all instances of suspected or known rabies virus infection must be reported to local health department officials. proper precautions and quarantine procedures as outlined by local regulations and described in the compendium of animal rabies prevention and control, 2000 26 should be followed. although vaccine-associated sarcomas have been reported to develop in association with administration of a variety of vaccines, current data suggest that they are more frequently associated with administration of feline leukemia virus (felv) vaccines and adjuvanted rabies virus vaccines. 28 inflammatory reactions are commonly observed at sites where adjuvanted rabies virus vaccines have been administered, and concern has arisen regarding the possible association between these reactions and vaccine-associated sarcomas. 35 with the exception of a recently approved canarypox virus-vectored recombinant feline rabies virus vaccine (pure-vax feline rabies vaccine; merial limited, iselin, nj), all rabies virus vaccines currently on the market contain adjuvants. in rats, inflammation induced by the recombinant product seems to be minimal,34 but whether the use of this vaccine is associated with a reduced likelihood of vaccineassociated sarcoma formation in cats is not yet known. the recombinant product is currently licensed only for annual administration. rabies virus vaccination is highly recommended for all cats and is required by law in some states and municipalities. manufacturers are required by the us department of agriculture to establish, by means of experimental challenge exposure studies, the minimum duration of immunity for the rabies virus vaccines that they sell, and products approved for use every year or every 3 years are available. statutes governing the administration of rabies virus vaccines vary considerably throughout the united states; veterinarians should comply with the legal requirements of their area. felv infects domestic cats throughout the world. transmission is through transfer of virus in the saliva or nasal secretions resulting from prolonged intimate contact (e.g., mutual grooming), biting, or sharing of food and water utensils. the virus may also be transmitted by transfusion of blood from an infected cat, in utero, or through the milk. 33 exposure to virus persisting in the environment on fomites or in aerosolized secretions is not an efficient means of viral transmission. clinical signs of felv infection are primarily related to neoplasia, anemia, and diseases resulting from immunosuppression. kittens are the most susceptible to infection; resistance increases with maturity. experimental data demonstrate that kittens younger than 16 weeks of age are most susceptible to infection, with cats older than this being relatively resistant. 23 cats at greatest risk include outdoor cats (free-roaming pets, stray cats, and feral cats). also at risk are cats residing in open multiple-cat environments, cats living with felv-infected cats, and cats residing in households with unknown felv status. the decision to vaccinate an individual cat against felv infection should be based on the cat's age and its risk of exposure. vaccination against felv is recommended for cats at risk of exposure (i.e., cats not restricted to a closed, felv-negative, indoor environment), especially those younger than 4 months of age. vaccination is not recommended for cats with minimal to no risk of exposure, especially those older than 4 months of age. the ability of a particular vaccine brand to induce an immune response sufficient to resist persistent viremia varies from study to study.s3 because protection is not induced in all vaccinates, preventing exposure to infected cats remains the single best way to prevent felv infection. vaccination against felv does not diminish the importance of testing cats to identify those that are viremic. it is of critical importance that viremic cats not be in contact with other cats, especially those younger than 4 months of age. as a result, the felv infection status of all cats should be determined.13 adverse events associated with vaccination against felv include local swelling or pain, transient lethargy or fever, and postvaccination granuloma formation. although vaccine-associated sarcomas have been reported to develop in association with administration of other vaccines, current data suggest that they are more frequently associated with administration of felv vaccines and adjuvanted rabies virus vaccines. 28 if vaccination is deemed appropriate, annual revaccination is recommended. cats should be tested for felv infection before initial vaccination and when there is a possibility that they have been exposed to felv since they were vaccinated. the enzyme-linked immu-nosorbent assay is the preferred screening test; the indirect immunofluorescent assay is the preferred confirmatory test.13 individuals confirmed to be infected with felv need not receive felv vaccines, but they should be segregated from uninfected cats. chlamydia psittaci is a bacterial pathogen of the conjunctiva and respiratory tract of cats. transmission is through direct cat-to-cat contact; fomite transmission is less likely, because the organism is unstable in the environment. serous conjunctivitis, which may initially affect only one eye, is the most common clinical sign. sneezing or nasal discharge may develop, but if it does develop, it is usually mild. clinical signs are usually evident 5 to 10 days after infection and resolve with appropriate antimicrobial treatmenu 8 isolation rates have been reported to range from a£proximately 1% for cats without signs of respiratory tract disease to approximately 14% for cats with concurrent upper respiratory tract disease. 56 the highest rates of infection are reported for cats between 5 weeks and 9 months of age. 61 immunity conferred by c. psittaci vaccines is similar to that conferred by fhv-1 and fcv vaccines in that vaccinates are protected from severe clinical disease but not from infection. 20 the frequency of adverse systemic events associated with c. psittaci vaccines is higher than that associated with other commonly used vaccines; reactions include lethargy, depression, anorexia, lameness, and fever 7 to 21 days after vaccination. 55 because signs of disease associated with c. psittaci infection are comparatively mild and respond favorably to treatment, and because adverse events associated with the use of c. psittaci vaccines are of greater concern than adverse events associated with the use of many other products, routine vaccination against c. psittaci infection is not recommended. vaccination may be considered for cats in multiple-cat environments, where infections associated with clinical disease have been confirmed. if vaccination is deemed appropriate, annual revaccination is recommended. feline coronaviruses (fcovs) vary considerably in pathogenic potential and have historically been grouped into two biotypes: feline enteric coronaviruses, which typically cause subclinical to mild enteric infections, and feline infectious peritonitis (fip) viruses, which cause fip. currently, pip viruses are believed to be generated as mutant variants in feline enteric coronavirus-infected cats. 58 ,59 fcovs are widespread in feline populations worldwide, with seropositivity rates highest in crowded multiple-cat environmentsy transmission of the virus is mainly via the fecal-oral route. in environments in which fcov infection is endemic (e.g., most multiple-cat environments), 35% to 70% of cats are shedding fcovs in the stool at any given time. 16, 22 most infected cats remain healthy, although a few (usually between 1% and 5%) ultimately develop fip. affected cats rarely survive regardless of treatment. 43 kittens are most often affected with fip, but the disease reportedly can develop in cats of all ages. a genetic predisposition has been suggested, with higher disease incidence in certain lines. 15, 43 considerable controversy surrounds the ability of the currently available fip vaccine (primucell-fip; pfizer animal health, exton, pa) to prevent disease. some studies demonstrate protection from disease i9 ,24; others show little benefit from vaccination. 36 ,51 antibody-dependent enhancement of disease in vaccinates has been demonstrated in experimental challenge exposuj'e studies,50 but it is uncertain whether antibody-dependent enhancement occurs in a natural setting. discrepancies between study results are probably attributable to differences in test methodology (e.g., strain and dose of challenge virus, genetic predisposition of the test animals). protection from disease has not been demonstrated in animals vaccinated when younger than 16 weeks of age. most kittens born and reared in environments in which fco v infection is endemic are infected before reaching this age. l , 22 in these instances, vaccination of infected cats has not proven beneficial. at this time, there is no evidence that the vaccine induces clinically relevant protection, and its use is not recommended. canis infection is not recommended. at the time of this writing, the product has not been independently evaluated for efficacy. based on studies conducted by the manufacturer, it is reasonable to consider vaccination as adjunctive treatment for individual infected cats 4 months of age or older to hasten resolution of clinical signs. if the vaccine induces an immune response that accelerates lesion resolution, the number of infectious fungal spores produced by vaccinates may be reduced as well; thus, it is reasonable to consider vaccination as one component of a comprehensive treatment program in multiple-cat environments in which m. canis infection is endemic. nonetheless, the ability of this product to hasten elimination of endemic infections from such environments has not been evaluated. the revaccination interval is not stipulated on the label. major adverse events reportedly associated with the use of this product are pain, temporary hair loss, and formation of sterile abscesses or granulomas at the vaccine site. 38 bordetella bronchiseptica is a small, aerobic, gram-negative coccobacillus long recognized as a respiratory tract pathogen of several species of animals. the natural route of transmission in cats is believed to be via the aerosol or intranasal route. 8 experimental challenge exposure studies have shown that b. bronchiseptica can act as a primary pathogen in cats; inoculation of specific-pathogen-free kittens results in self-limiting disease characterized by variable degrees of fever, nasal or ocular discharge, sneezing, induced or spontaneous coughing, pulmonary rales, and submandibular lymphadenopathy.8 bronchopneumonia associated with naturally occurring b. bronchiseptica infection has been reported in kittens and adult cats. 60 other factors, including nutritional status, overcrowding, coinfection with other agents such as fev or fhv-1, and suboptimal hygiene, may influence the outcome of exposure. 41 .s 4 seroprevalence surveys suggest that exposure to the organism is common, with infection rates varying from population to population. the highest rates of seropositivity (often over 80%) are found among cats in rescue shelters and multiple-cat households, especially when there is a history of respiratory tract disease. the lowest rates are found among cats in households with few cats and no history of respiratory tract disease. 4 • 37 similarly, isolation rates vary. b. bronchiseptica was isolated from the oropharynx in 19 of 614 (3.1%) asymptomatic cats and from the distal trachea in 6 of 614 (1%) asymptomatic cats from shelters in lousiana. 2s in a recent survey of 740 cats in the united kingdom, none of the household cats were found to be infected, but 9% of cats from breeding colonies and 19% of cats from rescue shelters were found to be carrying the organism. s in the same survey, 9% of healthy cats and 14% of cats with respiratory tract disease tested positive for the organism. an additional finding was a strong positive association between oropharyngeal isolation of b. bronchiseptica and residence in households containing dogs with a recent history of respiratory tract disease. definitive diagnosis of disease associated with b. bronchiseptica infection may be difficult, in part, because signs of infection often mimic those associated with fhv-l or fev infection. isolation of b. bronchiseptica from a cat with respiratory tract disease is supportive of the diagnosis, but carriage of the organism in asymptomatic cats precludes establishing a direct cause-and-effect relation. resolution of disease with appropriately chosen antimicrobial medication might suggest a causative role for b. bronchiseptica, but the self-limiting nature of many cases of viral upper respiratory tract disease prevents attributing disease resolution solely to antimicrobial treatment. a vaccine (protex-bb; intervet) to prevent disease caused by infection with b. bronchiseptica has recently been licensed. the product contains a live reduced-virulence culture of b. bronchiseptica and is licensed for administration via the intranasal route in cats 4 weeks of age and older. efficacy of the vaccine has not been independently evaluated; however, in studies conducted by the manufacturer to gain vaccine licensure, vaccinated 4-week-old specific-pathogen-free cats experienced less severe signs of disease than did unvaccinated controls when exposed to challenge 3 weeks after vaccination. similar results were obtained when 8-week-old kittens were exposed to challenge 72 hours after vaccination. as of this writing, studies to evaluate the duration of protection induced by the vaccine have not been completed and the revaccination interval is not yet stipulated on the label. routine use of this vaccine is not recommended. it is reasonable to consider vaccinating cats entering or residing in multiple-cat environments (e.g., shelters, catteries, boarding facilities) where disease associated with b. bronchiseptica infection has been confirmed. the ability of the product to reduce the prevalence of infection or the severity of disease in such environments has not been evaluated, however. infection of cats with the protozoan giardia lamblia is associated with acute or chronic gastrointestinal disease ranging in severity from subclinical to severe. 32 , 64 because infected cats shed cysts intermittently, diagnosis of g. lamblia infection is often cumbersome and usually requires multiple fecal examinations. several methods of diagnosis are available, including examination of a fecal smear, the zinc sulfate centrifugation method, and use of an enzyme-linked immunosorbent assay to test feces. 64 there are currently no approved treatment methods for cats, and although treatment commonly controls signs of disease, it is uncertain that it clears infection. 63 treatment effectiveness is highly variable, and resistant organisms are commonly encountered. 31 ,63 g. lamblia is transmitted via the fecal-oral route; cysts may be ingested from contaminated water, from direct cat-to-cat transmission especially in crowded environments (e.g., through mutual grooming), from exposure to contaminated litter boxes, and from consuming prey. 3d, 31 giardiasis is a recognized zoonotic disease, but the role of cats in transmission of the organism is not well established. 2 , 6, 64 a vaccine has recently been licensed by the us department of agriculture (fel-o-vax giardia; fort dodge animal health) as an aid in the prevention of disease associated with c. lamblia infection and reduction in the severity of shedding of cysts. this vaccine is composed of quantified, homo gena ted, and chemically inactivated g. lamblia trophozoites, and it contains an adjuvant commonly found in other feline products from the manufacturer but different from the adjuvant in the manufacturer's canine product. the vaccine is approved for use in cats 8 weeks of age and older. at the time of this writing, the vaccine has not been independently evaluated for efficacy, but in studies conducted by the manufacturer to gain vaccine licensure, vaccinates had a statistically significant reduction in severity of clinical signs (diarrhea), duration of cyst shedding, and prevalence of infection (percentage of cats with trophozoites at the end of the trial) compared with control animals. protection was demonstrated to persist for at least 1 year after vaccination. routine use of this vaccine is not recommended, but because vaccinates had less severe clinical disease and shed cysts for a shorter time, it is reasonable to consider vaccination as part of a comprehensive control program in environments where exposure to g. lamblia is clinically significant. when parasite exposure is ongoing, revaccination at annual intervals is recommended. some vaccinates may shed cysts subsequent to c. lamblia exposure; thus, proper hygiene and sanitation practices should be implemented even with vaccinated cats. the ability of this product to aid in hastening elimination of endemic infection from multiple-cat environments has not been evaluated. a study of naturally occurring feline coronavirus infections in kittens enteric protozoal infections detection of feline calicivirus antigens in the joints of infected cats prevalence of antibodies against bordetella bronchiseptica in cats with a history of respiratory disease prevalence and risk factors for feline bordetella bronchiseptica infection lameness in kittens after vaccination studies on natural transmission of bordetella bronchiseptica in cats problems with respiratory virus vaccination in cats acute arthritis of cats associated with feline calicivirus infection investigation of vaccine reactions and breakdowns after feline calicivirus vaccination comments on cerebellar ataxia and its congenital transmission in cats by feline panleukopenia virus american association of feline practitioners and the academy of feline medicine recommendations for feline leukemia virus testing and recommendations for feline immunodeficiency virus testing report of the american association of feline practitioners and academi'of feline medicine advisory panel on feline vaccines the inheritance of susceptibility to feline infectious peritonitis in purebred catteries patterns of feline coronavirus infection and fecal shedding from cats in multiple-cat environments viral upper respiratory infection in cats infectious diseases of the respiratory tract protection against feline infectious peritonitis by intranasal inoculation of temperature-sensitive fipv vaccine immunoprophylaxis and immunotherapy infectious diseases of the dog and cat fecal shedding of feline coronavirus in adult cats and kittens in an abyssinian cattery feline leukemia virus infection: agerelated variation in response of cats to experimental infection independent evaluation of a modified-live feline infectious peritonitis virus-vaccine under experimental conditions (louisiana experience) isolation and characterization of bordetella bronchiseptica from cats in southern louisiana compendium of animal rabies prevention and control vaccination against feline viral rhinotracheitis in kittens with maternally derived feline viral rhinotracheitis antibodies epidemiologic evidence for a causal relation between vaccination and fibrosarcoma tumorigenesis in cats rabies surveillance in the united states during 1997 protozoal and miscellaneous infections giardia: diagnosis and treatment giardiasis in dogs and cats felv and non-neoplastic felv-related disease local postvaccinal reactions of a recombinant rabies vaccine the potential role of inflammation in the development of postvaccinal sarcomas in cats independent evaluation of a modified-live fipv vaccine under experimental conditions (university of liverpool experience) seroprevalence and isolation rate of bordetella bronchiseptica in cats in the uk dermatophytosis: advances in therapy and control in august jr canine parvovirus (cpv-2b) infection in cats basic and clinical immunology feline husbandry: diseases and management in the multi-cat environment feline calicivirus infection an overview of feline enteric coronavirus and infectious peritonitis virus-infections feline panleukopenia and other enteric viral diseases acute and chronic faucitis of domestic cats. a feline calicivirus-induced disease feline sarcoma task-force meets diseases of the cat: medicine and surgery. philadelphia, wb saunders duration of immunity in cats vaccinated with an inactivated feline panleukopenia, herpesvirus, and calicivirus vaccine long-term immunity in cats vaccinated with an inactivated trivalent vaccine antibody-dependent enhancement of feline infectious peritonitis virus infection independent evaluation of a modified-live fipv vaccine under experimental conditions (cornell experience) hydranencephaly and cerebellar hypoplasia in two kittens attributed to intrauterine parvovirus infection feline leukemia virus-a review of immunity and vaccination bordetella bronchiseptica infection in the cat reaction rate in cats vaccinated with a new controlled-titer feline panleukopenia-rhinotracheitis-calicivirus-chlamydia psittaci vaccine prevalence of feline chlamydia psittaci and feline herpesvirus 1 in cats with upper respiratory tract disease chronic oral infections of cats and their relationship to persistent oral carriage of feline calici-, immunodeficiency, or leukemia viruses a comparison of the genomes of fecvs and fipvs and what they tell us about the relationships between feline coronaviruses and their evolution feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses bordetella bronchiseptica infections in cats prevalence of chlamydia psittaci in different cat populations in britain other feline viral diseases consultations in feline internal medicine key: cord-303741-1ou0cy5k authors: stafstrom, carl e.; jantzie, lauren l. title: covid-19: neurological considerations in neonates and children date: 2020-09-10 journal: children (basel) doi: 10.3390/children7090133 sha: doc_id: 303741 cord_uid: 1ou0cy5k the ongoing worldwide pandemic of the novel human coronavirus sars-cov-2 and the ensuing disease, covid-19, has presented enormous and unprecedented challenges for all medical specialists. however, to date, children, especially neonates, have been relatively spared from the devastating consequences of this infection. neurologic involvement is being increasingly recognized among adults with covid-19, who can develop sensory deficits in smell and taste, delirium, encephalopathy, headaches, strokes, and peripheral nervous system disorders. among neonates and children, covid-19-associated neurological manifestations have been relatively rare, yet reports involving neurologic dysfunction in this age range are increasing. as discussed in this review, pediatric neurologists and other pediatric specialists should be alert to potential neurological involvement by this virus, which might have neuroinvasive capability and carry long-term neuropsychiatric and medical consequences. severe and at times fatal symptoms caused by the novel human coronavirus, severe acute respiratory syndrome (sars)-cov-2, and the associated coronavirus disease 2019 (covid19) , are ravaging the world. while symptoms of covid-19 are primarily pulmonary (fever, dry cough, fatigue, pneumonia), it is becoming increasingly recognized that multiple organ systems can be affected, including the brain, with neurological involvement affecting up to~36% of patients [1] [2] [3] [4] [5] . information gained from studies of related coronaviruses in recent epidemics of severe acute respiratory syndrome (sars, 2002) and middle east respiratory syndrome (mers, 2012) suggests that all three coronaviruses might have neurologic consequences [6, 7] , though the relative severity and frequency of neurologic involvement caused by coronaviruses varies and thus the extent to which sars and mers epidemics inform our understanding of covid-19 remains unclear [5] . nevertheless, the possibility has been raised that sars-cov-2 could invade the brain and cause neurological disease [2, 8] . while appealing conceptually, data supporting the idea that the sars-cov-2 virus can infect the peripheral and central nervous systems (pns, cns) are limited, as discussed below. table 1 lists definitions of relevant terms that are often used in the literature. neurotropic viruses vary in their invasiveness, virulence, and propensity to cause inflammation [9] . the purpose of this review is twofold: (1) to discuss the available data about covid-19 infections in neonates and children, and (2) to provide a perspective about potential neurologic involvement in neonates and children with covid-19 infections, in view of neurobiological development. a few points need clarification up front. first, data about the virus and its effects are accumulating rapidly and our understanding of its consequences will evolve over time. second, much of the literature about covid-19 currently exists as case reports or small series; obviously, the impact of such publications is limited, and greater understanding will emerge only as large, rigorous studies are published. third, we must be mindful that a positive test for sars-cov-2 in a patient with a neurological symptom does not necessarily imply that the virus caused the symptom. the covid-19 epidemic has escalated rapidly and spread widely across the globe, with cases continuing to accrue at an alarming rate. the first case was reported from wuhan, china in mid-december 2019 and three months later, in march 2020, the world health organization declared covid-19 a pandemic. as of this writing (late august, 2020), more than 23 million cases of covid19 have been documented worldwide (with many more mildly symptomatic cases likely not reported), with over 800,000 deaths, and more than 175,000 deaths in the united states alone (www.cdc.gov, accessed 24 august 2020). documentation of the numerous clinical presentations, manifestations, and disease course have proliferated in the medical literature. a pubmed search (23 july 2020) using the keyword covid-19 revealed an astounding number of published reports already (34, 310) , over the course of only a few months. of those citations, only 501 reports (1.5%) also included the keyword neonate, attesting to the low published incidence in newborns. when searching pubmed with the key terms covid-19, neonate and neurological or brain, fewer than 10 articles emerged. therefore, at least so far, covid-19 does not seem to be affecting neonates very often from a neurological point of view; pubmed counts probably underestimate the occurrence of neurological involvement in children, being biased toward areas of the world with greater medical resources. these observations do not preclude the potential for neonatal brain involvement in covid-19 nor exclude the possibility of long-term medical, neurodevelopmental, and psychosocial consequences of the disease. indeed, as time goes on, a wider spectrum of neurologic manifestations will likely emerge with as yet undetermined long-term sequelae. as the covid-19 pandemic continues, certain trends are becoming evident. first, while the number of cases and deaths continue to rise, the disease does not affect infants and children nearly as frequently as adults [9, 10] . to date, approximately 2-5% of cases of covid-19 involve children, who appear to be less severely affected than adults, mainly with pulmonary symptoms [11] [12] [13] . second, disease severity in children who develop covid-19 is usually milder than in adults, and children with severe disease often have an underlying co-morbidity such as immunosuppression [10, 14] . indeed, there is accumulating evidence that adults with covid-19 infection manifest with multiple organ system involvement, including the cns and pns, and that older and sicker individuals carry a higher risk for neurologic problems [1, 4, 15] . these age-dependent differences in disease expression and severity have clear implications for healthcare professionals who deal with the pediatric population because children remain at risk for incurring and spreading the virus, yet many remain asymptomatic. several hypotheses have been posited as to why children are less affected by covid-19, including age-related differences in immune responses [16] , a neutralizing antibody response due to prior exposure to coronaviruses [17] , lower prevalence of co-morbidities in children, and age-specific differences in sars-cov-2 receptor function [18] , neurovirulence, intrinsic biological protective mechanisms, and other host factors [19] . none of these hypotheses is supported compellingly by extant data at present. while the number of affected neonates and children remains small, pediatric practitioners cannot become complacent about the potential for neurologic involvement in covid-19. furthermore, the recently described multisystem inflammatory syndrome-children (mis-c), raises the specter that covid-19 or its after-effects also target children (see section 4) [20, 21] . as data from china, europe and other areas affected early by the covid-19 pandemic are reported, some patterns are emerging regarding pregnant women and neonates. first, it is clear that vertical transmission covid-19 from a pregnant mother to her fetus occurs quite rarely. more than a dozen publications attest to this observation, together encompassing over 100 patients (table 2 lists a few relevant publications, selected from the largest available series and omitting small series and single case reports). none of these reports documents unequivocal vertical transmission. in many of the babies, onset of symptoms occurred in the neonatal period but not immediately at birth, so the exact timing of infection remains uncertain. overall, the data does not support robust transplacental transfer of sars-cov-2, but recent case reports are providing proof-of-principle that the virus can be transmitted intrauterine from infected mother to fetus [22, 23] . an especially apropos case demonstrated maternal viremia, placental infection shown by immunohistochemistry, and high placental viral load with subsequent neonatal viremia, implying transplacental transfer of sars-cov-2 from pregnant mother to fetus [24] ; this newborn presented with neurological symptoms as discussed in section 3. of all the infants reported during the first month of life, most had documented exposure to affected family members [13, 32] which emphasizes the importance of controlling horizontal virus transmission from affected family members to the neonate [33] . while this trend may need revision [27, 34] , so far, it is encouraging that most covid-19-positive pregnant women do not transmit the disease to their unborn children. similarly, there is no evidence that covid-19-positive pregnant women incur covid-19 or develop more severe disease than similar-aged women who are not pregnant. however, the impact of chronic inflammation caused by maternal viral infection on fetal development, pregnancy outcomes and long-term neurodevelopment is unknown. similarly, the timing of maternal viral infection with respect to major milestones of in utero neurodevelopment (i.e., second trimester vs. late third trimester) is an unknown and critical consideration for further research and long-term neurodevelopmental followup. there is legitimate concern about the impact of acute and chronic stress during the pandemic (i.e., worry about the medical complications of sars-cov-2 infection, family disruption, job loss, economic pressures, educational uncertainties, food availability, etc.) on the pregnant patient and developing fetus [35] [36] [37] [38] . furthermore, it is reassuring that there is no definitive evidence that the virus is present or can be transmitted in the breast milk of covid-19-positive women [28, 39, 40] . the current american academy of pediatrics recommendation is that covid-19-positive mothers can breast feed directly while wearing a mask or feed expressed breast milk, using appropriate breast and hand hygiene [41] . extensive guidelines are available regarding principles of management of pregnant women with covid-19 and their newborns [40] [41] [42] . again, all of these observations are preliminary and subject to modification over time. although covid-19 primarily affects the pulmonary system, it is a multisystem infection (e.g., gastrointestinal tract, kidneys, liver, heart) and involvement of the pns and cns are increasingly recognized [43] [44] [45] [46] . data on neurological signs and symptoms are limited but increasing, with a wide spectrum of acute and chronic manifestations becoming apparent [47] . in a series of 214 hospitalized adults with covid-19, 88 of whom had "severe" infections, 36.4% of the entire group was reported to manifest some neurologic involvement, including alteration of consciousness, encephalopathy, headache, cerebrovascular disease, and skeletal muscle injury (myalgia, weakness) [1] . ischemic strokes, many affecting young adults with large vessel occlusions, have garnered considerable concern that the etiology may be a prothrombotic state caused by virus-induced inflammation of the vascular epithelium [48, 49] . many of the young adult stroke victims had other vascular risk factors such as diabetes or hypertension, which emphasizes the importance of comorbidities with systemic inflammatory conditions in disease manifestations and severity. stroke has not been reported in children with covid-19 [50] . the occurrence of encephalitis remains controversial, as virus has not been recovered from cerebrospinal fluid (csf) [51] and overall, a surprisingly small number of covid-19 patients develop classic encephalitic symptoms. autopsy studies are beginning to be published. the wide spectrum of postmortem findings include mostly secondary changes to the cns such as hypoxemia and ischemia, rarely localized perivascular and interstitial neuroglial activation with neuronal loss and axonal degeneration [52, 53] , and no other major cns abnormalities [54] . no pediatric autopsy cases have reported neuropathological involvement. clearly, more data are needed before this issue can be clarified. in the chinese series [1] , only 2 out of 214 patients had seizures (1%), which is not greater than the general population, so it is uncertain whether infected patients are at higher risk. the few patients with seizures are reported mainly in case reports [55, 56] . the lack of frequent seizures is rather curious, especially if encephalopathy is indeed a frequent complication of covid-19; this data may be related to sampling bias rather than actual non-occurrence. a few reports of seizures have appeared in adults using electroencephalography (eeg) [57, 58] , but a more concerted effort to evaluate the brain electrophysiology of children with covid-19 would be informative. the examples of seizures in children with covid-19 described in section 3.6 appear to be largely anecdotal. many additional cases are necessary to conclude whether there is increased seizure susceptibility in the pediatric population. other cns symptoms include headache, dizziness and delirium, all of which can occur as a nonspecific consequence of systemic infection or inflammation of the respiratory tract as well as via a cns mechanism. although headaches are reported frequently, the pain often appears to be nonspecific or associated with inflammation or migraine exacerbation rather than meningeal irritation [59] . the most commonly reported symptoms related to the pns are decreased taste (hypogeusia or ageusia) and smell (hyposmia or anosmia) [60, 61] . a neural mechanism is suspected for hyposmia in covid-19, because decreased smell is often the first symptom experienced and occurs in mild disease in the absence of significant local inflammation or mucosal congestion that are typical of the more benign coronavirus or non-coronavirus nasal infections [62] . a few adolescents with covid-19 have been reported with decreased taste or smell [63] ; these symptoms appear to be very uncommon in children but deserve a more concerted ascertainment effort. several cases of guillain-barré syndrome (gbs) have been reported in adults with covid-19, raising the possibility of post-infectious autoimmune responses against the pns [64, 65] . two case reports of children with gbs who developed covid-19 symptoms about 3 weeks later confirms that gbs can occur with covid-19, though this association remains quite rare given the widespread prevalence of covid-19 [66, 67] . finally, the possibility of central demyelination has been raised, e.g., in the form of multiple sclerosis (ms), among patients with covid-19 [68] ; this concern is relevant in that various disease-modifying agents used to treat ms could theoretically exacerbate ms symptoms [69] . fortunately, there is no evidence that covid-19 triggers central demyelinating disease in children [50] . the lack of unequivocal reports of sars-cov-2 being recovered from the csf of individuals affected with presumed neurological involvement nor in brain tissue from the limited number of autopsied cases strengthens the possibility that the virus does not often directly cause the symptoms but rather, that the neurological sequelae are secondary to hypoxia, cytokine involvement, or some other non-direct mechanism (see section 6). it is appropriately concerning that chronic neurologic diseases such as epilepsy, amyotrophic lateral sclerosis, multiple sclerosis, etc., might be exacerbated during concurrent covid-19 infection or that covid-19 may unmask preexisting cns pathology that might have been unrecognized or asymptomatic. as just discussed, neurologic involvement in children, and in particular neonates, with covid-19 appears to be scarce but may be under reported [70] . a few selected examples of case reports of neurologic involvement in neonates and children are presented in table 3 ; such case reports have limited generalizability, and many lack sufficient details to ascribe causality between sars-cov-2 and neurologic symptoms. most children were assumed to have contracted covid-19 from a family member and some children had concurrent infection with other viruses, confounding any argument for causality. importantly, csf was negative for sars-cov-2 in all children on whom spinal fluid was obtained. all children recovered within a few days or weeks, contrasting with the severe and prolonged courses in many adults. available evidence does not allow distinction between a direct effect of sars-cov-2 causing neurologic dysfunction, versus the symptoms instead being secondary to an over activated immune response (see section 5) . in summary, case reports of neurological involvement in babies and children are rare but accumulating, and the recovery of most infants with early neurologic symptoms implicates some virus-or host-related factors that minimize massive neurological devastation. this newly recognized kawasaki syndrome-like hyperinflammatory disorder presents with acute hypotension and cardiogenic shock and is proliferating across the globe. it is likely a post-infectious syndrome or inflammatory reaction following asymptomatic or mildly symptomatic covid-19 [76] . children can develop toxic shock-like symptoms, hypoxia-ischemia, and significant end organ damage to the heart, kidneys, and other organs. while definitive data is not available, there is concern that the inflammation that hallmarks mis-c may have adverse consequences on the developing brain. while no consistent neurologic picture has emerged, several mis-c patients have had cns involvement as part of their course. in a small series of 6 children with mis-c, 4 patients had neurologic symptoms, including headache, altered mental status, and aseptic meningitis [77] . headache was the most common symptom in a series of 58 children with mis-c associated with sars-cov-2, affecting 26% of patients [78] . a series 21 children from france notes that 57% of patients were "irritable" and another 29% had "other neurological features", though these were not specified [79] . in a larger survey of 186 children, 5-11% had neurologic involvement, depending on age, including encephalitis, seizures or mental status alteration, but details are not provided [80] . finally, 4 of 27 children with covid-19 associated mis-c developed new neurologic symptoms including encephalopathy, headache, weakness, ataxia, and dysarthria [81] ; two patients had lumbar punctures and csf was negative for sars-cov-2 in both. three of the four patients had an eeg; each showed diffuse slowing. brain mri scans of all four children showed abnormal signal intensities of the splenium of the corpus callosum (a finding seen in previous cases of encephalopathy and thought to indicate inflammation-induced focal myelin edema [81, 82] . a recent systematic review of eight studies notes neurological symptoms in~25-50% of children with mis-c, depending upon inclusion criteria [83] . a putative impact on the immature cns and developing immune system, including neural-immune maturation, cannot be overlooked, and the long-term neurologic impact of both covid-19 and mis-c on the developing brain need urgent elucidation. sars-cov-2 is a highly contagious and pathogenic rna virus that is transmitted via droplet, contact, or aerosol routes. the virus gains entry into epithelia of the pulmonary system (upper or lower respiratory tracts), digestive tract, or nasopharynx. the virus is composed of single-stranded rna enveloped by surface proteins (s, e, m, n). the spike (s) glycoprotein serves as the attachment site onto angiotensin converting enzyme type 2 (ace-2) receptors on epithelial membranes. the normal function of ace-2 receptors is to provide protection against pulmonary and endothelial injury [84] . sars and sars-cov-2 share~79% genome sequence identity and both viruses infect epithelium by the binding of spike proteins to ace-2 receptors. while most prevalent on airway and pulmonary epithelium [85] , ace-2 receptors are also reportedly present to a lesser degree on neurons and perhaps glia [2] . binding of the s protein to ace-2 receptors leads to proteolytic cleavage of s by the transmembrane protease tmprss2 [5] . viral rna then enters the epithelial cell and replicates rapidly, translating viral proteins and inducing a host immune response. this immune response can be adaptive, attacking and inactivating the virus. by contrast, the immune response can be maladaptive and induce a massive immune reaction, accompanied by a hyper-inflammatory response hallmarked by excessive cytokine secretion and signal transduction (cytokine storm), and robust cellular immune activation and recruitment [86] . the large-scale cytokine storm consists of a massive release of pro-inflammatory humoral agents such as interleukin-6 (il-6), interferon gamma (ifn-y), mcp-1/ccl2 (monocyte chemoattractant protein 1/chemokine ligand 2), il-1, il-12, il-8, tnfα (tumor necrosis factor alpha), and cxcl 10 (c-x-c motif chemokine ligand 10) that exacerbate the underlying pathophysiology [87, 88] . this cytokine release subsequently feeds forward an overactive and dysregulated cellular immune response defined by macrophage, monocyte, neutrophil, and t-cell hyperactivation and recruitment [89] . the impact of this systemic cytokine storm on neurodevelopment is under investigation in preclinical models [90] and should be the focus of future prospective studies. subsequently, the replicated viruses exit the cell, leading to further infection. it is unknown why children, and neonates in particular, seem to be relatively resistant to covid-19 and its severe symptoms, including neurological manifestations. the cytokine response to coronavirus infection appears to be less robust in young children although the recognition of mis-c may suggest host-dependent genetic susceptibility to enhanced cytokine and/or inflammatory responses [91] , but other mechanisms are also plausible [19] . it remains controversial whether ace-2 inhibitors would provide symptomatic relief or prevent the covid-19 disease, and evidence for the effectiveness of these agents in children and neonates is not yet available [92] . the cellular and molecular basis of sars-cov-2 neurotropism, neuroinvasiveness, and neurovirulence are poorly understood [9] : does the virus get into the brain and if so how, and what does it do in the cns once there (e.g., infects neural cells? causes disease?). neurological involvement in covid-19 might be associated with at least four potential mechanisms: 1. a direct neurotropic or neuroinvasive effect of sars-cov-2 (e.g., anosmia, encephalopathy), 2. a secondary effect of the systemic inflammatory responses triggered by the viral infection (e.g., encephalopathy), 3. a secondary effect associated with the vascular and prothrombotic effect of the viral infection on the cns or pns vasculature (e.g., strokes, necrotizing leukoencephalopathy), 4. an immune-mediated para-infectious or post-infectious autoimmune effect in response to the viral infection (e.g., gbs, acute disseminated encephalomyelitis). figure 1 summarizes, in schematic fashion, some hypothetical possibilities about how the virus may infect the brain directly, whether the neurological symptoms and signs may be related to systemic or hyperactivation of immune responses, or both [87] . it is important to consider the mechanisms associated with neurological manifestations of covid-19, with an aim toward developing therapeutic options. the possibilities of direct neurotropism and hyper-responsiveness to immune activation (cytokine storm) are considered separately below, though these mechanisms might work synergistically. the sars-cov-2 virus attaches to olfactory epithelium using the ace-2 receptor. after cell entry, the virus replicates and induces a massive immune response leading to excessive cytokine release, comprising a maladaptive immune response. theoretically, virus particles may reach the cns retrogradely via cranial nerve pathways: v from corneal epithelium or oropharyngeal cutaneous sensory receptors; i via the cribiform plate, infecting olfactory sensory neurons; vii and ix from tongue chemoreceptors; x via pulmonary mechanoreceptors. once reaching cns nuclei including brainstem and cortex, a variety of neurologic signs and symptoms are possible. however, it must be noted that the virus has not been recovered from csf or brain tissue, making all of these pathways hypothetical at this point. abbreviations: np, nasopharynx; gi, gastrointestinal; ace-2, angiotensin converting enzyme type 2 receptor; pns, peripheral nervous system; cns, central nervous system; ich, intracranial hemorrhage; gbs, guillain-barre syndrome; bbb, blood-brain barrier. definitive demonstration of direct viral invasion would require a positive csf reverse transcriptase-polymerase chain reaction (rt-pcr) for sars-cov-2, recovery of infective virus from the csf as demonstrated by viral cultures or "plaque assay" [93] , intrathecal synthesis of antibodies to sars-cov-2, or autopsy-demonstrated sars-cov-2 antigen or rna in brain tissue [5] . current published evidence meeting these strict criteria is minimal. while it is plausible that the virus infects the brain through one of the anatomical pathways discussed below, the lack of viral recovery from the cns gives pause to that notion. neuroinvasion has been demonstrated for the related sars and mers viruses [94] , but sars-cov-2 has not been recovered from the csf or brain tissue. animal models of sars and mers have shown that the virus can enter through epithelium of the nasopharynx and travel retrogradely to the cns [95, 96] . interestingly, wild type mice are not vulnerable to infection and disease by human coronaviruses, but transgenic mice with human ace-2 receptors do develop respiratory and neurological symptoms when infected [95, 97] . in such transgenic mice, intranasal exposure to sars or mers leads to brain infection. one of the proposed portals of entry is via olfactory sensory neurons, crossing the cribiform plate into the olfactory bulb, with subsequent retrograde travel along the olfactory nerve (cranial nerve i) to the brainstem, thalamus, and basal ganglia, all areas that are connected to the olfactory cortex. please note that it has yet to be proven that sars-cov-2 infects olfactory sensory neurons. emerging animal models may clarify whether sars-cov-2 is similarly neuroinvasive as sars and whether this isage dependent the sars-cov-2 virus attaches to olfactory epithelium using the ace-2 receptor. after cell entry, the virus replicates and induces a massive immune response leading to excessive cytokine release, comprising a maladaptive immune response. theoretically, virus particles may reach the cns retrogradely via cranial nerve pathways: v from corneal epithelium or oropharyngeal cutaneous sensory receptors; i via the cribiform plate, infecting olfactory sensory neurons; vii and ix from tongue chemoreceptors; x via pulmonary mechanoreceptors. once reaching cns nuclei including brainstem and cortex, a variety of neurologic signs and symptoms are possible. however, it must be noted that the virus has not been recovered from csf or brain tissue, making all of these pathways hypothetical at this point. abbreviations: np, nasopharynx; gi, gastrointestinal; ace-2, angiotensin converting enzyme type 2 receptor; pns, peripheral nervous system; cns, central nervous system; ich, intracranial hemorrhage; gbs, guillain-barre syndrome; bbb, blood-brain barrier. definitive demonstration of direct viral invasion would require a positive csf reverse transcriptase-polymerase chain reaction (rt-pcr) for sars-cov-2, recovery of infective virus from the csf as demonstrated by viral cultures or "plaque assay" [93] , intrathecal synthesis of antibodies to sars-cov-2, or autopsy-demonstrated sars-cov-2 antigen or rna in brain tissue [5] . current published evidence meeting these strict criteria is minimal. while it is plausible that the virus infects the brain through one of the anatomical pathways discussed below, the lack of viral recovery from the cns gives pause to that notion. neuroinvasion has been demonstrated for the related sars and mers viruses [94] , but sars-cov-2 has not been recovered from the csf or brain tissue. animal models of sars and mers have shown that the virus can enter through epithelium of the nasopharynx and travel retrogradely to the cns [95, 96] . interestingly, wild type mice are not vulnerable to infection and disease by human coronaviruses, but transgenic mice with human ace-2 receptors do develop respiratory and neurological symptoms when infected [95, 97] . in such transgenic mice, intranasal exposure to sars or mers leads to brain infection. one of the proposed portals of entry is via olfactory sensory neurons, crossing the cribiform plate into the olfactory bulb, with subsequent retrograde travel along the olfactory nerve (cranial nerve i) to the brainstem, thalamus, and basal ganglia, all areas that are connected to the olfactory cortex. please note that it has yet to be proven that sars-cov-2 infects olfactory sensory neurons. emerging animal models may clarify whether sars-cov-2 is similarly neuroinvasive as sars and whether this isage dependent [97, 98] . however, since mice are not naturally susceptible to the clinical and immunopathological manifestations of coronaviruses affecting humans, translational studies of pathogenic mechanisms and vaccine development become complicated. extensive efforts to modify mice with transgenic approaches have begun to provide informative models. as mentioned, the olfactory epithelium has been touted as a potential site of viral entry into the brain, and hence explain hyposmia [99] .detailed genetic and immunohistochemical examinations of cell types of the olfactory system reveal that ace-2 and tmprss2 are present on olfactory epithelial cells (especially supporting or "sustenacular" cells) but not on olfactory sensory neurons themselves [54, 85, 100] . moreover, there is some evidence of virus-induced cell death in other coronavirus infections but not yet for sars-cov-2 [84, 101] . likewise, the virus might enter via the sensory system of the tongue that mediates taste, with transmission via cranial nerves vii, ix, and x to the nucleus tractus solitarius, thalamus and eventually, brain. finally, trigeminal nociceptors via cranial nerve v from either the corneal epithelium or buccal epithelium could theoretically reach the cns. these potential pathways could explain the symptoms of hypogeusia and altered vision. however, sars-cov-2 has not been recovered from the brain. transynaptic transport from lower respiratory tract mechano-and chemoreceptors to the brainstem medullary cardiorespiratory centers has been proposed as a hypothetical mechanism that could exacerbate brainstem dysfunction and perhaps even worsen respiratory effort [102] ; however, this hypothesis lacks objective validation and remains controversial. other potential routes for virus to enter the cns are through the bloodstream (hematogenous) or via disruption of the blood brain barrier (bbb). from the systemic circulation, the virus might travel to the cerebral circulation where it can damage capillary epithelium and access the brain. interestingly, there is scant evidence that sars-cov-2 produces a significant or sustained viremia [103] . the bbb is essential for transport of molecules into the brain and exclusion of pathogens and overall maintenance of cerebral homeostasis [104] . the bbb is a dynamic structure, consisting of several cell types and proteins, each with its own maturational profile-astrocyte foot processes, pericytes, tight junction proteins, and extracellular matrix, providing structural and functional support. virus attachment to ace-2 receptors at the bbb might facilitate trafficking of the virus into the cns, facilitating endothelial damage and edema [89] . notably, while the bbb is structurally complete at birth and is sufficiently functional in the neonate to provide protection against many pathogens, its full physiological maturation may take several months [105] . in the context of covid-19 infection, the bbb may be dysfunctional, disrupted either by inflammatory response or the virus itself, allowing transmission of the virus or activated immune cells from the circulation into the cns [8, 84] . the release of inflammatory cytokines by activated glia and neural mast cells exacerbate the inflammation [89] . similarly, flow of the virus through lymphatic channels of the interstitial space of the brain could breach the blood-csf barrier and permit virus entry [106] . to date, there is no evidence for the presence of the virus in pathological specimens of the pns or cns, in part due to the dearth of comprehensive autopsies [52] [53] [54] . obviously, patient care has focused on critical pulmonary and life-support measures so neuropathologically-focused autopsy studies have been uncommon. animal studies of covid-19 will be crucial to complement information gained from prior studies of the other coronaviruses. such animal models will provide more information about mechanisms of virus entry into the nervous system and how the virus affects neural function, neural-immune maturation and neurodevelopment, as well as the critical and yet unanswered question of long-term neurological sequelae of covid-19 [107] . that is, if there is predilection of the virus for certain neural structures or chronic neuroinflammation, long-term consequences may arise in various neural functions such as learning, memory, cognition, seizure predisposition, and other functions. all of this is speculative at present. another essential question, alluded to above, is whether cns disease contributes to the respiratory failure seen in covid-19 patients. ongoing or severe hypoxia can exacerbate ongoing symptoms in other organs. in particular, cns respiratory control centers in the brain stem, nearby the vagus nerve, has been speculated to play a role in respiratory failure [102, 108] . at present, there is some reason for guarded optimism for young patients within the devastating covid-19 pandemic. children, particularly neonates, are less likely to become infected and develop severe symptoms, and their propensity to spread the virus is controversial [109] . there is at best slight evidence for vertical transmission of sars-cov-2 or covid-19 disease from pregnant mother to fetus; rather, neonates are more likely incur the disease by exposure to affected individuals postnatally, and breast milk transmission has not been shown (table 4) . a variety of practical guidelines have been developed for the care of pregnant women who have or are suspected to have covid-19 positivity. analogous guidelines for the care of adult covid-19 patients with neurologic problems are also available and need to be developed for children [110] . table 4 . summary of covid-19 infections in children. severe infection caused by the novel coronavirus, sars-cov-2, has predominant pulmonary involvement but can also affect multiple other organ systems, including the cns and pns. symptoms are less frequent and usually less severe in children and particularly in neonates. vertical transmission of sars-cov-2 from pregnant mother to fetus is rare but anecdotal case reports support this possibility. most cases of covid-19 in early life are due to exposures to infected patients (horizontal transmission). there is no reported transmission of sars-cov-2 via breast milk. regarding neurologic involvement in covid-19, there are plausible mechanisms by which the virus can gain entry into the cns and subsequently incur acute neurologic symptoms, either directly or through immune dysfunction ( table 5 ). the occurrence of long-term medical and neuropsychiatric sequelae is unknown. children can be resilient and yet remain vulnerable to coping with the challenges of covid-19 in the context of other acute and chronic diseases. youngsters may not understand the need for social distancing, prolonged quarantine, and other preventative measures, and it is anticipated that stress-related post-traumatic symptoms will develop in some young people, whether or not they actually acquire symptoms. in children with comorbid chronic conditions and developmental disabilities, the challenges are even more profound. therefore, neuropsychological surveillance and studies of the long-lasting effects of this pandemic on neurodevelopment are critical [111] . finally, the emergence of the hyper-inflammatory multisystem syndrome (mis-c) supplants any conclusion that covid-19 is benign or negligible in the pediatric age range. therefore, it behooves neurologists and other pediatric specialists who deal with neonates and young children to be aware of the potential neurologic involvement of this novel, potentially devastating virus. future animal models should evaluate the impact of sars-cov-2 on maternal infection, inflammatory signal transduction through the maternal-placental-fetal axis, and brain development. the importance of large-scale immunization should a vaccine become available, cannot be over emphasized as should the role of systemic inflammation, neuroinflammation, and neural-immune interactions in novel pathophysiology and symptomology. additionally, mechanism-specific targeted therapies could emerge from basic science studies of sars-cov-2 infection. table 5 . neurological involvement in covid-19. acute neurological involvement in adults with covid-19 can include decrease taste/smell, headache, confusion, peripheral nerve dysfunction, strokes, and encephalopathy. neurological involvement of covid-19 in neonates and children is still quite rare but recent case reports warrant vigilant surveillance. neurological involvement of covid-19 in neonates and children is still quite rare but recent case reports warrant vigilant surveillance. sars-cov-2 has 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for and against vertical transmission for severe acute respiratory syndrome coronavirus 2 development, maintenance and disruption of the blood-brain barrier review: the blood-brain barrier; protecting the developing fetal brain sars-cov2 entry and spread in the lymphatic drainage system of the brain in vitro and animal models for sars-cov-2 research is the collapse of the respiratory center in the brain responsible for respiratory breakdown in covid-19 patients covid-19 super-spreaders: definitional quandaries and implications child neurology society; in collaboration with the neurocritical care society ethics committee aan position statement neurotropic mechanisms in covid-19 and their potential influence on neuropsychological outcomes in children this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license research in the laboratory of stafstrom is supported by the mathias koch memorial fund, the sandra and malcolm berman foundation, and the paine foundation. research in the laboratory of jantzie is supported by the national institutes of health (1r01hl139492) and the department of defense (w81xwh-18-1-0167) . we thank carlos pardo-villamizar for his insightful comments and critique of the manuscript. the authors declare no conflict of interest. key: cord-352222-zq9o66i4 authors: rajatonirina, soatiana; razanajatovo, norosoa harline; ratsima, elisoa hariniana; orelle, arnaud; ratovoson, rila; andrianirina, zo zafitsara; andriatahina, todisoa; ramparany, lovasoa; herindrainy, perlinot; randrianirina, frédérique; heraud, jean-michel; richard, vincent title: outcome risk factors during respiratory infections in a paediatric ward in antananarivo, madagascar 2010–2012 date: 2013-09-12 journal: plos one doi: 10.1371/journal.pone.0072839 sha: doc_id: 352222 cord_uid: zq9o66i4 background: acute respiratory infections are a leading cause of infectious disease-related morbidity, hospitalisation and mortality among children worldwide, and particularly in developing countries. in these low-income countries, most patients with acute respiratory infection (ari), whether it is mild or severe, are still treated empirically. the aim of the study was to evaluate the risk factors associated with the evolution and outcome of respiratory illnesses in patients aged under 5 years old. materials and methods: we conducted a prospective study in a paediatric ward in antananarivo from november 2010 to july 2012 including patients under 5 years old suffering from respiratory infections. we collected demographic, socio-economic, clinical and epidemiological data, and samples for laboratory analysis. deaths, rapid progression to respiratory distress during hospitalisation, and hospitalisation for more than 10 days were considered as severe outcomes. we used multivariate analysis to study the effects of co-infections. results: from november 2010 to july 2012, a total of 290 patients were enrolled. co-infection was found in 192 patients (70%). co-infections were more frequent in children under 36 months, with a significant difference for the 19–24 month-old group (or: 8.0). sixty-nine percent (230/290) of the patients recovered fully and without any severe outcome during hospitalisation; the outcome was scored as severe for 60 children and nine patients (3%) died. risk factors significantly associated with worsening evolution during hospitalisation (severe outcome) were admission at age under 6 months (or = 5.3), comorbidity (or = 4.6) and low household income (or = 4.1). conclusion: co-mordidity, low-income and age under 6 months increase the risk of severe outcome for children infected by numerous respiratory pathogens. these results highlight the need for implementation of targeted public health policy to reduce the contribution of respiratory diseases to childhood morbidity and mortality in low income countries. respiratory infections are a major cause of infectious diseaserelated morbidity, hospitalisation, and mortality among children under 5 years old worldwide, and particularly in developing countries [1] . these diseases are responsible for 4 million deaths worldwide every year [4] : they are thus among the leading causes of death and the most common infectious cause of death [2] . although many episodes of respiratory infection are mild, 7 to 13 percent are severe enough to warrant hospitalisation [3] . these diseases are responsible for at least 6% of the world's disability and death [1] . it is estimate that there are 33.7 million cases of respiratory infection annually among african children and longitudinal studies suggest that the overall mortality rate among children in developing countries is between 6.6% and 14.1% [5] . despite respiratory infections being a public health priority, data on their epidemiology in africa are sparse. diagnostic methods to identify the pathogen rapidly are not available in lowincomes settings, so most patient with respiratory infections are treated empirically with antibiotics based on local epidemiology [6] . administering appropriate therapy at the appropriate time would improve outcomes. these diseases also have an economic impact, as they lead to substantial consumption of antimicrobial agents in both community and hospital settings. furthermore, the high frequency of respiratory infections and the excessive use of antimicrobial agents are major contributors to the development of antibiotic-resistant pathogens. knowing the local epidemiology of respiratory infections is essential if appropriate empiric therapy is to be prescribed. evaluation of mixed infections and the relative importance of each potential pathogen may also contribute to improving the understanding of the pathogenesis of these infections [7, 8] . our study aimed to evaluate the risk factors associated with the evolution and outcome of respiratory illnesses in patients aged under 5 years old hospitalised in one of the four main public hospitals in antananarivo. the study was conducted at the ''centre hospitalier de soavinandriana'' (cenhosoa) in antananarivo, the capital city of madagascar, which is located in the central highlands. antananarivo consists of administrative, commercial, industrial and residential areas, with patches of agricultural land, mostly rice fields. the city is divided into six administrative districts. in madagascar, hib vaccine was introduced in 2008, and pneumococcia vaccine in 2012 cenhosoa is a national referral hospital in the third district of the urban municipality of antananarivo; hospitalization and care at this hospital is paid for by the patients. it is located near the institut pasteur of madagascar (ipm). the paediatric and neonatology ward of cenhosoa has 54 beds, and the annual number of children hospitalized for respiratory infection was estimated to be 150. a prospective, hospital-based, study was conducted at cen-hosoa from november 2010 to july 2012. the case definition and eligibility criteria used during this study are described in figure 1 . demographic, socio-economic, clinical and epidemiological data were recorded in case report forms (crfs). paediatricians completed the crfs which were then validated by a clinical research officer. instruction sheets were provided to clinical research officers to ensure accurate data collection. the data collected was managed by a senior medical epidemiologist. on admission, trained personnel using standard operating procedures had collected nasal and throat swabs, and sputum sample by aspirating airway secretion from the hypopharynx through the nose or tracheal aspirates. nasopharyngeal secretions, tracheal aspirates and sputum were obtained before start of antibiotic therapy. specimens were transported within 30 minutes of sampling to the ipm for tests of respiratory pathogens. hiv serostatus was not determined; the prevalence of hiv in madagascar is low, estimated to be around 1 per 1000 [7] . the study was approved by the national ethics committee of the ministry of health of madagascar. written informed consent was obtained from parents or legal guardians of children enrolled in the study. consenting to be enrolled in the study required consent to provide samples for diagnosis of influenza and other respiratory pathogens and to fill out a case report forms (crfs). refused consent and hospitalization within the two weeks before consultation were reasons for exclusion from the study. virological analyses were performed by national influenza centre (nic). a panel of multiplex real-time rt-pcr was used for the detection of viral pathogens in nasopharyngeal swabs. primers and probes were obtained from the authors of reported studies or were developed at the nic [8] . viral rna and dna were extracted from 140 ml of each specimen using the qiaamp viral rna mini kit and qiaampminelute virus kit (qiagen, courtaboeuf, france) according to the manufacturer's protocols. an abi 7500 fast real-time pcr system (applied biosystems) was used for amplifications. each sample was subjected to five different amplification reactions, each containing primers and probes specific for two or three viruses such that the following 12 respiratory viruses were targeted as previously described: parainfluenza virus 1-3 (piv), human coronaviruses (hcov) -oc43, -229e, -nl63 and hku1, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), human rhinovirus (hrv), adenovirus (adv) and bocavirus (bov). as part of our routine influenza surveillance, screening for influenza viruses is performed by mdck cell inoculation and realtime rt-pcr according to the cdc protocols [9] . bacteriological analyses were performed by the medical analysis laboratory of ipm gram-stained sputum preparations were examined for polynuclear neutrophils (pn) and epithelial cells. the areas of maximal purulence were examined and graded as follows: class 1: more than 25 cells and fewer than 10 pn, class 2: more than 25 cells and 10-25 pn, class 3: more than 25 cells and more than 25 pn, class 4: 10-25 cells and more than 25 pn and class 5: fewer than 10 cells and more than 25 pn. only the classes 4 and 5 present a high positive predictive value. if the laboratory determined that the sample was of oral origin it had been rejected. so, classes 1 to 3 had been considered as rejection criteria. quantitative sputum cultures were performed on each specimen by a previously described method [10] : sputum specimens were homogenized with an equal volume of digest-eurh on a vortex mixer; 10 ml aliquots of the homogenate were serially diluted in saline and 10 ml aliquots of the dilutions were plated with a sterile inoculator on blood columbia agar with nalidixic acid and colistin (anc), chocolate polyvitex (pvx) agar, and drigalski agar. plates were incubated under 5% co 2 at 35uc for 24 to 48 hours. colonies growing on plates inoculated with suitable dilutions were counted to enumerate each bacterial species present. the threshold of significance was set at $10 7 /ml (for one or two species only). isolates were identified by standard laboratory methods. mono-infection was defined as the presence of only one pathogen as determined by viral and bacterial analysis. patients for which more than one pathogen was detected were considered to have co-infections. the outcome was scored as severe if the patient died, or presented a wrong clinical evolution during hospitalisation (complication) or the hospitalization was longer than 10 days (3 rd quartile). analyses were performed with r software [11] . the nonparametric wilcoxon test was used to compare means. qualitative variables were compared with the fisher exact test. statistical differences were considered significant for p-values,0.05. multivariate backward step-by-step logistic regressions were performed to take into account confounders, bias and interactions involving pathogens linked to the dependent variable. all variables with a pvalue,0.20 were included in the initial model. five age groups were defined for the analysis: [0-5] months, [6] [7] [8] [9] [10] [11] [12] months, [13] [14] [15] [16] [17] [18] months, [19] [20] [21] [22] [23] [24] months, [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] months, .36 months. from november 2010 to july 2012, 290 children were recruited from the 301 hospitalized patients eligible. eleven eligible patients refused to participate in the study, and they did not differ from included patients for age, sex or diagnosis on admission. the baseline demographic and clinical characteristics of the patients are shown in table 1 . the sex ratio (male/female) was 1.3. the mean of age was 1.1 years [95% ci: 1.0 to 1.2], the age range was from 1 day to 5 years, and 250 (86%) patients were 2 years old or younger. the diagnoses on admission (table s1 ) were significantly different between the age groups: bronchiolitis was found more frequently among patients under 6 months old (55%, p,0.01); ltri and pneumonia were more frequent among patients aged over 12 months (respectively, 68% and 85%,p,0.01). forty-nine (17%) patients had at least one underlying disease, with asthma being the most frequent (5%). some of the children had underlying conditions, particularly atopy (52; 18%) and passive exposure to tobacco smoke (113; 39%). seventy-one (24%) had been hospitalized during the previous 12 months. patients aged under 18 months had significantly more prior hospitalisations (p = 0.01). the difference between sexes was not statistically significant. around 45% (131/290) of patients had been treated with antibiotics before hospitalisation (table s2 ). significantly more patients under than over 12 months old had received antibiotic treatment before hospitalisation (p,0.01). the difference between sex was not statistically significant. the frequency of bacterial detection was lower in the group with prior antibiotic treatment (49% versus 65%, p = 0.01). no such relationship was found for viral results. accurate information about immunisation was available on vaccination cards for 82 (26%) children. eighty-seven percent of children had completed vaccination against bcg, 77% had received one haemophilus influenzae type b vaccination. no child was vaccinated against streptococcus pneumoniae and only two were vaccinated against influenza. the average of duration of hospitalisation was 7.9 days [95% ci: 7.1 to 8.6], range 1 to 79 days. the median duration of hospitalisation was 7 days, and 61% of patients were hospitalised for more than 7 days. the duration of hospitalisation for living patients was 8.0 [7.2-8.7 ] days, and for fatal cases was 3.9 [1.4-9.2]. there were no significant differences between sex and age groups. discharge information was available for all cases: 79% (230/ 290) of patients fully recovered without any complication during their hospital stay; the condition of 60 children worsened during hospitalisation, and nine patients (3%) died. all deaths were of children under 18 months of age, and six (67%) were of children less than 6 months old. for the patients who died, streptococcus pneumoniae (56%, 5/9) was the pathogen most frequently detected, followed rsv (33%, 3/9) and influenza type a virus (22% 2/9). for multivariate analysis of factors possibly associated with coinfection, sex, age group, number of rooms in the home and antibiotic treatment before hospitalisation were included in the initial model. the multivariate analysis indicated that children under 36 months of age were more often infected by more than one pathogen; the difference was statistically significant for the 19-24 months old group (adjusted or = 8.0) ( table 1) . previous antibiotic treatment before hospitalization was a significantly protective factor against co-infection (or = 0.5). in univariate analysis, influenza a, rhinovirus, streptococcus pneumoniae, and haemophilus influenza type b were significantly associated with co-infections. haemophilus influenzae and s. pneumoniae were most often associated with co-infection (adjusted or = 20.5 and 11.1, respectively) ( table 1) . univariate analysis showed that a severe outcome was significantly more frequent for patients with comorbidity (or = 3.9), including prematurity or congenital disease (such as heart disease or cerebral malformation). also, two of the three patients presenting with malnutrition died. hospitalization during the 12 months prior to admission was also associated with a severe outcome (or = 2.1). a low household income, of less than 455 us$ (less than 182 us$ or = 4.4 and between 182 and 455 us$ or = 3.5) was associated with severe outcomes. for multivariate analysis, age group, monthly household income, admission diagnosis, comorbidities, previous hospitalisation, and infection with influenza virus type a, rsv and rhinovirus were included in the initial model. multivariate analysis (table 3 ) adjusted for these variables showed that age below 6 months (or = 5.3), living in a household with low income (or = 4.1), and the presence of comorbidities (or = 4.6) were significantly aggravating factors. infection with an influenza type a virus was a significantly protective factor against a severe outcome (or = 0.4 [95% ci = 0.2-0.9]). in the paediatric ward of cenhosoa, most children under 36 months, hospitalised for respiratory infections, were co-infected, and the most frequently encountered pathogens were h. influenza, s. pneumonia, influenza a and rhinoviruses. during hospitalisation, the evolution of respiratory infections was worse for the youngest children and for those presenting comorbidities and living in lowincome household. over all, viral infections made a large contribution to the burden of infectious respiratory disease. viral pathogens were found in 85% of the patients and bacterial pathogens in 57%, although for 45% of the patients had been treated with antibiotics before hospitalisation, and this presumably affected the observed prevalence of bacterial infections. our findings for viruses were similar to those of other studies. for example, the detection rate for viral pathogens was 72% in a study in vietnam [12] , 50% in a study in mozambique [13] , both focusing on hospitalised children. these results are consistent with those for the paediatric ward in cenhosoa. our findings are also consistent with another report from madagascar indicating that rsv (30.5%) and by influenza a (22.6%) are the most frequent viral causes of community respiratory infection [8] . few studies in developing countries have addressed the role of bacterial co-infection in severe respiratory illness. most focus on the viral aetiology because molecular diagnosis tools are available for these pathogens allowing to identify the infection in 80%-95% of cases [14, 15] . in developing countries, laboratory methods to identify bacterial pathogens are more difficult to implement, because of the lack of appropriate facilities and material in hospital laboratories and then, long transit times are prejudicial for stability of specimens. in our study the transit time was reduced because of the proximity of the cenhosoa and the ipm. unfortunately, our findings for the rate of bacterial infection may be artificially low due to prior antibiotic use. in our study, streptococcus pneumoniae was the most common bacterial pathogen as in other studies of hospitalised patients with acute respiratory illness, and haemophilus influenzae type b was the next most frequent [16] [17] [18] [19] . primary infection by a respiratory virus increases the risk of secondary bacterial pneumonia, and viral and bacterial coinfection is common in young children with pneumonia in developing countries (about 20-30% of episodes) [20, 21] . evidence of probable mixed viral-bacterial infection has been recorded in up to 45% of cases of community acquired pneumonia in children [22] [23] [24] [25] . a large proportion of our patients had coinfection (70%; 192/273) and the [0-6] months age group had the highest risk. these results are consistent with a study of patients with community respiratory infection in madagascar [8] . viral and bacterial co-infection was found in 57% of our patients; the most frequent combination was streptococcus pneumoniae with one of several respiratory viruses (88%). data from gambia and nigeria indicate that mixed bacterial and viral infections may occur in 8 -40% of cases of childhood pneumonia [26, 27] . recent data from south africa indicate that in at least 31% of cases, viral-associated pneumonia may be due to concurrent infection with streptococcus pneumoniae in the absence of vaccination with pneumococcal conjugate vaccines [28] . appropriate management of respiratory infections can have a significant impact on child mortality and it is important to target priority groups of children most at risk of dying: these groups include very young infants, children infected with hiv and malnourished children [29] . in our study, the outcome was severe for all of the children who presented malnutrition and 67% (2/3) died. chisti et al show that the combination of pneumonia with severe or moderate malnutrition is associated with a high risk of death: the rr ranges from 2.9 to 121.2 for severe malnutrition, and from 2.5 to 15.1 for moderate malnutrition. for moderate malnutrition, rr = 1.2 to 36.5. several other studies also lead to the same results [30] [31] [32] [33] . ballard et al. report that malnutrition and level of parental education both have an impact on the incidence of respiratory infections [34] . pneumonia is the leading causes of childhood mortality, and is responsible for approximately 21% of deaths of african children under five years of age each year. however, little information is available about the causes of childhood pneumonia in developing countries. in our study, only 3% of the patients died; this value is low. note that the study population consists of patients from families with a socioeconomic level that is above the average for the general population in madagascar. the patients who died were more frequently co-infected and under 6 months old. these results are consistent with other studies on this topic in developing countries [35, 36] . however, study design may have an impact on the mortality rate observed: indeed all fees for hospitalisation were paid by the study to allow follow-up to be optimised. the study identified living in a low-income household as a risk factor for severe outcome. this is important for patient management during hospitalisation and also highlights the consequences of financial barriers to access to vaccination against influenza and pneumococci. these vaccinations were not included in the panel of vaccinations in expanded programme of immunisation (epi) during this period in madagascar. they were only available in private centres and were expensive, and therefore inaccessible for much of the population. only two of the patients included in this study had received vaccination against influenza and none had received vaccination against pneumococci according to their vaccination cards. our statistical analysis identified influenza a infection as a protective factor against a fatal outcome; however, it is well-known that influenza a may have an impact on high mortality during major epidemics [37] [38] [39] . the co-circulation of influenza a virus, rsv and the high frequency of streptococcus pneumoniae in co-infections in children aged less than 5 years raised issues about the indirect impact of influenza vaccination on respiratory infections. in particular, the benefits of establishing a system of vaccination against infectious agents for which a vaccine is available should be considered to provide access to a larger proportion of the population. in conclusion, we advocate to further research in developing countries to document in more detail the impact of viral and bacterial co-infections on the prognosis of severe respiratory illness. as numerous infections found in our study could be avoided by immunisation, we recommend that appropriate vaccination must be rapidly available for all the malagasy children. this would reduce mortality among the youngest children, as this age group currently suffers the greatest burden of morbidity and mortality associated with respiratory infections. table s1 diagnosis at admission according with age groups. (docx) estimates of world-wide distribution of child deaths from acute respiratory infections community-acquired pneumonia epidemiology and etiology of childhood pneumonia study of community acquired pneumonia aetiology (scapa) in adults admitted to hospital: implications for management guidelines community-acquired pneumonia in children british thoracic society guidelines for the management of community acquired pneumonia in childhood bayesian mapping of pulmonary tuberculosis in antananarivo viral etiology of influenza-like illnesses in antananarivo who | cdc protocol of realtime rtpcr for influenza a (h1n1) (n.d.). who. available remic référentiel en microbiologie médicale r: a language and environment for statistical computing. r foundation for statistical computing viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city viral acute respiratory infections among infants visited in a rural hospital of southern mozambique intranasal fluticasone propionate does not prevent acute otitis media during viral upper respiratory infection in children human bocavirus and acute wheezing in children epidemiology and etiology of childhood pneumonia etiology of community-acquired pneumonia in hospitalized patients in northern israel etiology of severe pneumonia in children in developing countries the bacterial etiology of community-acquired pneumonia in korea: a nationwide prospective multicenter study etiology of acute lower respiratory tract infections in gambian children: ii. acute lower respiratory tract infection in children ages one to nine years presenting at the hospital diagnoses of acute lower respiratory tract infections in children in rawalpindi and islamabad induced sputum in the diagnosis of childhood community-acquired pneumonia etiology of community-acquired pneumonia in 254 hospitalized children etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections malnutrition as an underlying cause of childhood deaths associated with infectious diseases in developing countries etiology of acute lower respiratory tract infections in gambian children: ii. acute lower respiratory tract infection in children ages one to nine years presenting at the hospital the etiology of pneumonia in malnourished and well-nourished gambian children a trial of a 9-valent pneumococcal conjugate vaccine in children with and those without hiv infection challenges to improving case management of childhood pneumonia at health facilities in resource-limited settings multihospital surveillance of pneumonia burden among children aged ,5 years hospitalized for pneumonia in bangladesh bacterial aetiology and outcome in children with severe pneumonia in uganda etiologic agents and outcome determinants of community-acquired pneumonia in urban children: a hospital-based study improvements in nutritional management as a determinant of reduced mortality from community-acquired lower respiratory tract infection in hospitalized children from rural central africa the effects of malnutrition, parental literacy and household crowding on acute lower respiratory infections in young kenyan children bacterial etiology of serious infections in young infants in developing countries: results of a multicenter study. the who young infants study group neonatal pneumonia in developing countries excess mortality associated with the 2009 a(h1n1)v influenza pandemic in antananarivo this study would not have been possible without the excellent support from all the clinicians (dr emma rasoanirina, dr roland randriamirado, dr harimboahangy rakotoarison, dr lucien radozahana, dr rani razafindrakoto and dr domohina rakotovao), and all the nurses of the paediatric ward of the cenhosoa, antananarivo. we are deeply indebted to all staff who contribute, every day, to the hospital surveillance programme. we also acknowledge the tremendous work of all technicians of the virology unit and cbc.disclaimer: the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the ministry of health. conceived and designed the experiments: sr. performed the experiments: sr jmh vr. analyzed the data: sr vr nhr. contributed reagents/ materials/analysis tools: nhr ao ehr lr rr ph fr zza ta. wrote the paper: sr nhr ehr vr. key: cord-320548-oigyut2k authors: zumla, alimuddin; memish, ziad a; maeurer, markus; bates, matthew; mwaba, peter; al-tawfiq, jaffar a; denning, david w; hayden, frederick g; hui, david s title: emerging novel and antimicrobial-resistant respiratory tract infections: new drug development and therapeutic options date: 2014-09-01 journal: lancet infect dis doi: 10.1016/s1473-3099(14)70828-x sha: doc_id: 320548 cord_uid: oigyut2k the emergence and spread of antimicrobial-resistant bacterial, viral, and fungal pathogens for which diminishing treatment options are available is of major global concern. new viral respiratory tract infections with epidemic potential, such as severe acute respiratory syndrome, swine-origin influenza a h1n1, and middle east respiratory syndrome coronavirus infection, require development of new antiviral agents. the substantial rise in the global numbers of patients with respiratory tract infections caused by pan-antibiotic-resistant gram-positive and gram-negative bacteria, multidrug-resistant mycobacterium tuberculosis, and multiazole-resistant fungi has focused attention on investments into development of new drugs and treatment regimens. successful treatment outcomes for patients with respiratory tract infections across all health-care settings will necessitate rapid, precise diagnosis and more effective and pathogen-specific therapies. this series paper describes the development and use of new antimicrobial agents and immune-based and host-directed therapies for a range of conventional and emerging viral, bacterial, and fungal causes of respiratory tract infections. the emergence of diffi cult-to-treat known and novel bacterial, viral, and fungal respiratory tract pathogens with epidemic potential is of major global concern. treatment options are limited by increasing antimicrobialdrug resistance. however, new viral infections causing severe respiratory tract disease with pandemic potential have focused global attention. 1 a substantial rise in the number of patients with multidrug-resistant pulmonary tuberculosis 2 and pan-drug-resistant bacteria 3 has been noted. increasing use of immunosuppressive agents, broad-spectrum antibiotics, and anticancer agents, coupled with resistance to azoles, has led to an increase in the number of invasive pulmonary fungal infections 4 with resultant high morbidity and mortality. successful treatment outcomes for patients with respiratory tract infections across all health-care settings require appropriate, eff ective, and pathogen-specifi c drug or alternative treatments. we describe a range of conventional and emerging viral, bacterial, and fungal causes of respiratory tract infections for which new antimicrobial drugs and immune-based and host-directed therapies are being developed and studied. the outbreak of severe acute respiratory syndrome coronavirus (sars-cov), 5 re-emergence of avian infl uenza a h5n1, 6 global circulation of oseltamivirresistant seasonal infl uenza a h1n1, 7 and subsequent emergence of the pandemic infl uenza a h1n1 strain pdm09 virus (which continues to circulate), 8 have shown the potential limitations of current antiviral treatments for severe respiratory viral infections. epidemic waves of avian infl uenza a h7n9, 9 sporadic cases of avian infl uenza a h10n8, 10 the ongoing outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection, and the burden of common respiratory viruses 11 -such as seasonal infl uenza, respiratory syncytial virus, rhinoviruses, and adenoviruses-show that the development of more eff ective therapies to reduce morbidity and mortality is urgently needed. research is focused on the repurposing of available antiviral drugs for generic or specifi c use and for two classes of antiviral drugs are approved for the prevention and treatment of infl uenza in most countries: m2 inhibitors (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir, peramivir, zanamivir, and laninamivir; table 1). in general, antiviral treatment is indicated as early as possible for any patient with confi rmed or suspected infl uenza who has severe, complicated, or progressive illness or is admitted to hospital, and in outpatients at higher risk of infl uenza complications. 12, 13 time to treatment after onset of symptoms, illness severity, and extent of viral replication are key variables with respect to response. starting of treatment should not be delayed for diagnostic testing. m2 inhibitors-also known as adamantanes-are ineff ective against infl uenza b viruses and recently circulating infl uenza a h3n2 and 2009 pandemic infl uenza a h1n1 viruses, which are resistant because of an s31n mutation in the m2 ion channel. 12 however, a proportion of avian infl uenza a h5n1 strains will be susceptible, 14 and the combined use of an adamantane and a neuraminidase inhibitor improves antiviral activity for susceptible isolates. 15 two neuraminidase inhibitors are approved for use in most countries: oseltamivir and zanamivir. laninamivir is approved for use in japan only, and peramivir in china, japan, and south korea. several observational studies have shown that when adults admitted to hospital with severe infl uenza are given oseltamivir, mortality falls and clinical outcomes improve, especially when treatment is initiated within 2 days of the onset of symptoms (but positive eff ects are noted when it is begun as late as 4-5 days after onset). 12, 13, 16, 17 oseltamivir reduces mortality in infl uenza a h5n1 infection when given before the onset of respiratory failure, 18 and might be benefi cial when started as late as 6-8 days after symptom onset. 19 in patients admitted to hospital with severe infl uenza a h7n9 infection, reduction of viral load after treatment with oseltamivir correlated with improved outcome, whereas the emergence of virus resistant to neuraminidase inhibitors that harbours an arg292lys substitution is associated with poor outcomes and poor response to oseltamivir and peramivir. 20 the standard duration of oseltamivir treatment is 5 days; longer treatment is recommended for critically ill patients with respiratory failure, who often have prolonged viral replication in the lower respiratory tract despite treatment. 13 whether increased doses provide greater antiviral eff ects in such patients is under investigation. a randomised controlled trial 21 of patients in hospital (76% of whom were children) showed no virological or clinical advantages when a double dose of oseltamivir was given rather than a standard dose. no additional benefi t was noted with high-dose oseltamivir in adults admitted with infl uenza a, although a faster virological response was noted in those with infl uenza b. 22 however, in a randomised controlled trial 23 of 18 critically ill patients with 2009 pandemic infl uenza a h1n1, a triple-dose oseltamivir regimen was associated with signifi cantly higher proportions of viral clearance at 5 days than was standard therapy (78% vs 11%; p=0·015). 23 studies of intravenous neuraminidase inhibitors that are underway should provide further data on the value of high-dose therapy. zanamivir and laninamivir have generally similar profi les of susceptibility. for example, the his275tyr mutation confers high-level resistance to oseltamivir carboxylate and reduced susceptibility to peramivir in n1containing viruses but does not substantially diminish susceptibility to zanamivir and laninamivir. 30 inhaled zanamivir has not been studied in detail in severely ill patients or those admitted to hospital, in whom eff ective delivery to sites of viral replication and tolerability could be an issue. by contrast, intravenous zanamivir has been used widely on a compassionate basis since the 2009 h1n1 pandemic, particularly for late treatment of critically ill adults with 2009 pandemic infl uenza a h1n1 virus infection and those with suspected or proven oseltamivir resistance. 31 one trial 32 has shown no drug-related trends in safety measures, and a subset of 93 patients positive at baseline for infl uenza showed a median decrease in nasopharyngeal viral rna load of 1·42 log 10 copies per ml after 2 days of treatment. a phase 3 trial in patients who have been admitted to hospital is underway (nct01014988). a phase 2 randomised controlled trial of inhaled laninamivir in uncomplicated infl uenza failed to show superiority in illness alleviation (primary endpoint) compared with placebo. the trial, involving 639 patients, tested 40 mg and 80 mg doses of the inhaled drug. the median time to alleviate fl u symptoms was 102·3 h for the 40 mg dose and 103·2 h for the 80 mg dose, compared with 104·1 h for the placebo (nct01793883). das181 has host-directed receptor-destroying action, which is inhibitory for parainfl uenza and infl uenza viruses, including those resistant to amino adamantanes and neuraminidase inhibitors. 15 when delivered topically, it is eff ective in animal models of lethal infl uenza caused by the h5n1 and h7n9 viruses, including the neuraminidase-inhibitor-resistant arg292lys -containing variant. 35 in a phase 2 randomised controlled trial, 36 inhaled das181 reduced pharyngeal viral replication in uncomplicated infl uenza but did not reduce nasal viral loads or improve clinical outcomes. case reports 37 suggest that inhaled or nebulised das181 might be eff ective in immunocompromised hosts with severe parainfl uenza lung disease. favipiravir (t-705; 6-fl uoro-3-hydroxy-2-pyrazinecarboxamide) is active against infl uenza a, b, and c viruses, including strains resistant to approved antivirals, and a broad range of other rna viruses when given at series somewhat higher concentrations. 38 combinations of favipiravir and neuraminidase inhibitors have additive and synergistic eff ects in preclinical models, 39 but clinical trials have been restricted to uncomplicated infl uenza so far. these clinical trials (combination amantadine, ribavirin, and oseltamivir vs oseltamivir mono therapy [nct01227969], nitazoxanide vs oseltamivir vs combination vs placebo [nct01610245], favipiravir vs placebo randomised controlled trial in outpatients [nct02008344, nct2026349]), which have not been published, suggest that favipiravir has antiviral eff ects similar to those of oseltamivir. 40 a randomised controlled trial 41 showed that favipiravir shortened the time to alleviation of infl uenza symptoms by about 15 hours compared with placebo, and further studies are underway. nitazoxanide is an oral antiparasitic drug with immunomodulatory eff ects, including upregulation of interferon and various interferon-inducible genes and a specifi c infl uenza-inhibitory eff ect related to blockade of haemagglutinin maturation. 42 nitazoxanide inhibits infl uenza replication in vitro 43 and in a phase 2 randomised controlled trial 44 had signifi cant antiviral eff ects (1·0 log 10 reduction in nasal viral loads) and resulted in a signifi cantly faster time to alleviation of illness (roughly 20 h diff erence in medians from placebo) in uncomplicated infl uenza. 44 a placebocontrolled randomised trial of nitazoxanide versus oseltamivir-and the com bination thereof-in uncomplicated infl uenza and a hospital-based study of its use in severe respiratory illness are in progress (nct01610245). non-randomly assigned studies and case reports suggest that convalescent plasma with neutralising antibodies is a useful add-on therapy for patients with sars and severe infl uenza pneumonia, including that caused by infl uenza a h5n1. 45 a recently published systematic review of available sars and infl uenza treatment studies employing convalescent plasma or serum found a signifi cant overall mortality benefi t. 46 a prospective observational study 47 showed lower crude mortality and faster nasopharyngeal viral clearance in plasma-treated patients who were admitted with severe 2009 pandemic infl uenza a h1n1 infection, whereas in a randomised controlled trial 48 a reduction in mortality was reported in severe illness when hyperimmune globulin was given within 5 days of the onset of symptoms (table 2) . heterosubtypic haemagglutinin stem-neutralising antibodies, which are highly eff ective in animals, 49 are entering clinical evaluation in human beings. the combination of antivirals with diff erent mechanisms of actions (eg, a neuraminidase inhibitor with a polymerase inhibitor such as favipiravir, 38 a broad-spectrum antihaemagglutinin-neutralising antibody, 49 (20) oral rimantadine and nebulised saline (21) post-hoc analysis showed faster cough resolution but no signifi cant diff erences in the proportion of patients shedding virus by treatment day 3 (57% zanamivir plus rimantadine, 67% placebo plus rimantadine), or in the durations of hospitalisation and supplemental oxygen use underpowered because of low enrolment oseltamivir and corticosteroids (19) more rapid improvement in partial pressure of oxygen, fraction of inspired oxygen, and sequential organ failure assessment scores; shorter ventilator use (median 7 days vs 15 days, p=0·03); and faster viral clearance in the sirolimus than in the control group rct=randomised controlled trial. 51 in a retrospective study 53 of critically ill adults, mortality rates did not diff er between those who received a triple combination of antiviral drugs and those receiving oseltamivir only, and a randomised controlled trial sponsored by the national institute of allergy and infectious diseases in higher-risk outpatients is underway (nct01227969). host-directed therapies aim to reduce the damaging consequences of the host immune response to the pathogen. combinations of antivirals with host-directed therapies such as the immunomodulator sirolimus, an mtor inhibitor that blocks host pathways needed for viral replication (table 2) , 54 might also enhance antiviral activity. other host-directed therapies inhibiting cellular targets needed for effi cient viral replication (eg, the raf-mek-erk mitogenic kinase cascade and the ikk-nf-κb module) might provide future options for clinical testing. 15 the role of adjunctive immunomodulatory therapies in severe infl uenza and other respiratory viral infections remains uncertain. several observational studies show that systemic corticosteroids given for 2009 pandemic infl uenza a h1n1-associated viral pneumonia increased the risk of mortality and morbidity (eg, secondary infections), especially when there was a delay in initiation, or absence of, eff ective antiviral therapy. 45 their use might delay viral clearance and increase the risk of the emergence of resistance 20 and fungal infections. 45 other potential adjunctive therapies for infl uenza include intravenous immunoglobulin, n-acetylcysteine, statins, macrolides, peroxisome proliferator-activated receptor agonists, celecoxib, mesalazine, plasmapheresis, and haemo perfusion. 45 chloroquine was eff ective against infl uenza a h5n1 infection in one animal model 55 but was ineff ective in other animal models and one human randomised controlled trial. 56, 57 interferons mers-cov infection can cause severe respiratory disease, and has higher mortality in those with medical comorbidities. although empirical treatment with a range of antivirals has been tried for severe respiratory tract infections caused by mers-cov and sars-cov, no regimens have been rigorously assessed in clinical trials (panel). 58,59 mers-cov elicits attenuated innate immune responses with delayed proinfl ammatory cytokine induction in cell culture and in vivo. 60, 61 it is also readily inhibited by type 1 interferons (interferon alfa and especially interferon beta), suggesting a potential therapeutic use for interferons. early pegylated interferon alfa therapy was eff ective in a sars primate model, and treatment with interferon-alfa-consensus-1 plus systemic corticosteroids was associated with improved oxygen saturation and more rapid resolution of radiographic lung opacities than were systemic corticosteroids alone in an uncontrolled study of patients with sars patients. 62 further studies of interferons in mers-cov seem warranted. ribavirin was used extensively in patients with sars without any benefi cial eff ects and was complicated by haemolytic anaemia and metabolic disturbances in many cases. 58, 59 a combination of interferon alfa 2b and ribavirin reduced lung injury and moderately decreased viral replication (<1·0 log 10 reduction in lung titres) when given to rhesus macaques within 8 h of inoculation with mers-cov. 63 the treatment combination was given to several severely ill patients with mers, but the infections proved fatal, probably because of late administration in the advanced stage of the disease. 64, 65 ribavirin has in-vitro inhibitory eff ects against mers-cov. 66 the use of protease inhibitors with lopinavir and ritonavir as initial therapy in sars was associated with signifi cantly less death (2·3% vs 15·6%, p<0·05) and intubation (0% vs 11·0%, p<0·05) than was use of ribavirin alone in a matched historical cohort (n=44 for lopinavir and ritonavir as intial treatment vs n=634 for the matched historical cohort). 68 however, one study reported that nelfi navir and lopinavir have high 50% eff ective inhibitory concentrations (ec 50 ) against mers-cov in vitro, 66 whereas another found inhibition with lopinavir at clinically achievable concentrations. 69 several drugs have shown inhibitory eff ects against mers-cov in cell cultures, including interferons, ciclosporin, and mycophenolic acid. 66, 67, 69 mycophenolic acid was inhibitory at clinically achievable concentrations, and the combination of mycophenolic acid and interferon β1b lowered the ec 50 of each drug by one-to-three times. 66 dipeptidyl peptidase 4 (dpp4), also known as cd26, is a functional receptor for mers-cov, and an anti-cd26 polyclonal antibody showed in-vitro inhibitory eff ects on mers-cov. 70 by contrast, inhibitors of the enzymatic action of dpp4 (eg, gliptins) did not inhibit viral replication. timely administration of neutralising antibodies could have a high likelihood of therapeutic success. 46 treatment with convalescent plasma (from patients who have recovered from sars-cov infection) containing high levels of neutralising antibody within 2 weeks of illness onset resulted in a higher proportion of discharges at day 22 than did treatment more than 14 days after onset (58% vs 16%, p<0.001). 71 75 showed worse outcomes when systemic corticosteroids were given in sars. consequently, their use should be avoided unless a carefully controlled prospective study is done to test their eff ectiveness when combined with an antiviral. several observational studies have shown that systemic corticosteroids given for 2009 pandemic infl uenza a h1n1-asssociated viral pneumonia or acute respiratory distress syndrome increased the risk of mortality and morbidity (eg, secondary bacterial or fungal infections), especially if there is delay or lack of eff ective antiviral therapy. 45 use of systemic corticosteroids has probably contributed to delayed viral clearance and emergence of antiviral resistance in patients with severe infl uenza a h7n9 infection requiring extracorporeal membrane oxy genation. 20 infl uenza increases the risk of invasive aspergillosis, especially among immunocompromised patients, and this is often a silent infection in the early stages, 76 so direct surveillance with aspergillus antigen and pcr testing on respiratory secretions is advisable. patients treated for fungal infections will have to undergo antifungal therapeutic drug monitoring. 77 data are insuffi cient to support routine use of any of the immune therapies. better animal data and careful systematic clinical studies, including serial virological measurements of priority treatments such as convalescent plasma and interferons (and randomised controlled trials if case numbers are suffi cient), are needed. currently, clinical management of patients with severe respiratory tract infections due to mers-cov largely relies on meticulous intensive care supportive treatment and prevention of complications. research done in patients with haemopoietic stem-cell transplants shows that adoptive transfer of antigenspecifi c t cells can restore protective immunity and prevent or reverse disease due to opportunist viral infections such as cytomegalovirus. 78 in transplant recipients, transfer of donor-derived t cells can result in resolution of infection through expansion of virusspecifi c t cells, with associated clinical improvement. 79 transfer of donor t cells is associated with the risk of severe acute graft-versus-host disease, and thus most t-cell therapies have been done in patients who have low lymphocyte counts. lymphopenia enables only a very low number of t cells to be transferred, which then proliferate in lymphopenic hosts, most likely as a result of the interleukins 7 and 15 if the patient does not receive immunosuppressive treatment during t-cell therapy. 80 t-cell therapy targeting cytomegalovirus strains resistant to drug treatment is clinically relevant in lung transplant recipients. 81 t-cell expansion requires time to induce clinical regression of viral infection. several other approaches might be applicable in situations that necessitate fast clinical action-eg, use of synthetic mhc antigens loaded with the relevant peptide from the pathogen of interest (so-called tetramer or multimer mhc-peptide complexes), which engage pathogenspecifi c lymphocytes expressing the pathogen-specifi c t-cell receptors. pathogen-specifi c t cells can be isolated through use of soluble mhc-peptide complexes, and can immediately be transferred into patients for salvage treatments for viral infections. 82 t-cell expansion can also be achieved with several stimuli targeting several infectious pathogens. 83 expansion of t cells targeting several antigens of cytomegalovirus, epstein-barr virus, and adenovirus provides broad antiviral specifi city after stem-cell transplantation. 84 an alternative approach to series become independent of ex-vivo expansion of t cells is the identifi cation of t-cell receptors that would recognise viral infected cells that could be transferred into recipient eff ector cells. 85 t cells can also be engineered to produce an antiviral rna that would block viral infection. 86 synthetic antisense molecules, such as phosphorodiamidate morpholino oligomers, are structurally similar to rna but the phosphorodiester linkage is replaced with a neutral phosphorodiamidate linkage and the ribose ring with a six-membered morpho lino ring. 87 they change gene expression by inhibiting translation, disrupting rna secondary structure, and interfering with pre-mrna splicing. 88 the usefulness of phosphorodiamidate morpho lino oligomers coupled to argininerich cell-penetrating peptides has been repeatedly demonstrated against bacterial pathogens 89 and could be a viable option for any microbial gene of interest. specifi c biological therapy for infectious pathogens targets not only drug-resistant pathogens but also their immune evasion mechanisms. 90 an antibody directed against cd19 (a b-cell marker) fused to a t-cell signalling molecule can be expressed in t cells and could kill target cells once they encounter their nominal target antigen. such cd19 chimeric-antigen-receptor cells are used to remove epstein-barr-virus-positive lymphoma cells in the case of post-transplantation proliferative diseases. 91 similar approaches can be used for the eff ective removal of pathogen-infected cells when very specifi c antibodies exist and if target molecules are expressed on infected cells only. 92 the frequency and spectrum of resistance to antibiotics in specifi c bacterial pathogens that cause respiratory tract infections continues to increase worryingly. multidrugresistant streptococcus pneumoniae-with resistance to three or more antibiotics-was initially noted in 1977 in south africa 93 and subsequently in many other countries, with alarming rates of 30-50% of s pneumoniae that are multidrug resistant in the usa and spain. [94] [95] [96] the european antimicrobial resistance surveillance system showed that 22·2% of s pneumoniae were intermediate penicillin susceptible, 10·9% were penicillin resistant, and 21·1% were resistant to erythromycin. 97 concerns about multidrug-resistant and pan-antibioticresistant gram-negative bacteria 98, 99 are focused on klebsiella pneumoniae, enterobacter spp (production of extended spectrum β lactamase, klebsiella pneumoniae carba penemase, ndm1, and ampc), acinetobacter baumannii, and pseudomonas aeruginosa. in one survey of us health centres, 78% of gram-negative bacteria were resistant to all antibiotics except colistin (to which 62% of acinetobacter spp, 59% of pseudomonas spp, and 52% of enterobacter spp were resistant). 98 therapeutic options to treat these infections are limited. 100, 101 carbapenems are recommended for organisms that produce extended-spectrum β lactamases. 101 in a metaanalysis, [102] [103] [104] [105] [106] doripenem was more eff ective for p aeruginosa infections than were comparators in a modifi ed intention-to-treat analyses. polymyxin b and colistin are concentration-dependent bactericidal agents that bind to bacterial cell membranes and have reliable activity against acinetobacter spp. novel β-lactamase inhibitors 107 and antibiotic com bination therapies 108 might provide stopgap measures for fulfi lling clinical need. antibiotic development pipelines remain thin, 109, 110 and global attention is focused on increasing awareness for investments into the development of new antibacterial agents 111 and other antibacterial innovations, coupled to raising global awareness for more prudent use of available drugs. 112 in 2012, an estimated 1·3 million people died worldwide from tuberculosis, 170 000 of whom had multidrugresistant disease. 113 multidrug-resistant tuberculosis, which is caused by mycobacterium tuberculosis bacilli resistant to at least isoniazid and rifampicin, is now widespread globally, with an estimated half a million cases in 2012. 2 extensively drug-resistant tuberculosisresistance to rifampicin, isoniazid, any fl uoroquinolone, and at least one of the three injectable second-line drugs, amikacin, kanamycin, and capreomycin-has been reported in 92 countries. 113 who recommends use of second-line drugs for 18-24 months or longer for extensively drug-resistant or multidrug-resistant disease. 114, 115 treatment success rates are low in both individualised and standard regimens and new drugs and regimens are needed. in the past 5 years, a promising pipeline of new drugs for the treatment of multidrug-resistant and extensively drug-resistant tuberculosis has emerged. 115 progress has been made by repurposing drugs that are already available, including re-engineering existing antibacterial compounds and redesigning scaff olds, leading to discovery of new compounds. 116, 117 two new drugs, delamanid (opc-67683) and bedaquiline (tmc207 or r207910), have been approved by regulatory authorities. these new drugs are combined with older drugs to treat multidrug-resistant disease. 118, 119 host-directed adjunct therapies several approaches to rational development of adjunct immune-based therapies for multidrug-resistant tuberculosis have been developed. 120, 121 non-steroidal antiinfl ammatory drugs can reduce m tuberculosis load and series alleviate lung disease 122 in mice. 123 effl ux pump inhibitors such as verapamil and reserpine reduce macrophageinduced drug tolerance, and thus could be used as adjunct host-directed therapies. 124, 125 phosphodiesterase inhibitors such as cilostazol and sildenafi l improve mycobacterial clearance and decrease time to sterilisation by reducing tissue infl ammation. 126 a range of adjunct immunotherapy approaches implicating cytokines or their inhibitors and other biological immunomodulatory compounds are being assessed as means to limit damage from infl ammatory responses against m tuberculosis. various cytokine regimens, including interferon c or interleukin 2, have been assessed, with variable eff ect. 127, 128 the antiinfl ammatory eff ects of macrolide antibiotics need to be further studied. 129 whole genome sequencing might allow for rapid determination of resistance patterns of m tuberculosis strains, enabling tailored treatment regimens. other immunomodulatory strategies include restoration of eff ective antipathogen-directed immunoresponses-and consequent decreasing of damaging host responses in lung tissues-in multidrug-resistant tuberculosis with infusions of the patient's own bonemarrow-derived stromal cells. a phase 1 trial showed that the procedure is safe, 130 and phase 2 trials are planned to assess the eff ects of mesenchymal stromal cell adjunct therapy on clinical and microbiological outcomes. invasive fungal respiratory tract infections are increasingly reported worldwide (table 3) . 131, 132 the two most common pulmonary fungal pathogens are aspergillus fumigatus and pneumocystis jirovecii. they increasingly represent primary causes of morbidity and mortality in critically ill patients across europe, africa, and asia as a result of more people living with hiv, increased use of immunomodulatory drugs in patients with cancer, transplantations, and use of broad-spectrum antibiotics. some patients with relapsed or micro biologically unconfi rmed multidrug-resistant tuber culosis have alternative diagnoses, including chronic pulmonary aspergillosis, and more com prehensive searches for alternative fungal diagnoses in smear and culture negative cases should be done in patients with multidrug-resistant disease. 133 aspergillus is the most important fungal cause of invasive pulmonary disease, and a fumigatus is the cause in more than 75% of cases. voriconazole is the most eff ective treatment for invasive aspergillosis but resistance has been noted on all continents except south america. 134, 135 widespread use of the azoles as fungicides in agriculture has led to the environmental development of pan-azole resistance. 136 resistance can also emerge during treatment, typically to itraconazole, and is possibly linked to a combination of low blood concentrations of the drug and high fungal loads. [137] [138] [139] modelling suggests that more than 6·5 million people have severe asthma with fungal sensitisations, as much as 50% of adults with asthma who attend secondary care have fungal sensitisation, and an estimated 4·8 million adults have allergic bronchopulmonary aspergillosis. 140, 141 people with asthma who are sensitised to a fumigatus have a much higher rate of bronchiectasis than do those who are unsensitised. reclassifi cation of aspergillosis in adults with cystic fi brosis by aspergillus serology (ige and igg) and both pcr and antigen on sputum showed three distinct classes of aspergillosis. 18% had allergic bronchopulmonary disease, 15% had aspergillus sensitisation, and 30% had aspergillus bronchitis; the remaining patients had no disease. long-term oral antifungal therapy is benefi cial for 60-80% of patients with asthma, but is of unproven benefi t in cystic fi brosis. 142 resistance in a fumigatus has been reported throughout europe in roughly 4% of samples from patients with cystic fi brosis. 143, 144 a new fungus causing disseminated infections in patients with aids was identifi ed in 2009. 145 molecular identifi cation on the basis of its1 and its2 sequencing showed that all isolates of this new species were tightly clustered and were most similar to emmonsia pasteuriana and emmonsia parva, and slightly more distantly related to histoplasma capsulatum. clinical features of infection included fever, loss of weight, anaemia, skin lesions akin to those in disseminated histoplasmosis, and a chest radiograph similar to that noted in pulmonary tuberculosis. the fungus was cultured from skin and blood, but not sputum or csf. signifi cant clinical responses were noted when patients were given intravenous amphotericin b followed by itraconazole. 145 a large combination study 146 series did not reach its primary endpoint of reduced mortality, although patients with positive galactomannan seemed to benefi t most. guidelines for management of invasive aspergillosis still favour voriconazole over all other treatments and combination therapy is not usually recommended. a tablet formulation of posaconazole, which is more bioavailable than the oral suspension, is available and can be given once a day, 147 and the us food and drug administration has approved an intravenous suspension of the drug. the only new drug to be approved is isavuconazole, a broad-spectrum azole, which will be available in intravenous and oral forms (application for approval was submitted in july, 2014). itraconazole seems safe in the fi rst trimester of pregnancy, whereas fl uconazole increases the risk of fallot's tetralogy by a factor of three to one in 1000. 148 drivers for the development of new antifungal drugs include inadequate response rates, the absence of oral preparations of echinocandins, drug interactions, important drug toxic eff ects (especially amphotericin b and voriconazole), and triazole and echinocandin resistance. several drugs are being repurposed for use as antifungals, and new drugs are under development (table 4) . [149] [150] [151] [152] [153] [154] [155] sertraline, which is used for depression, has synergistic activity with fl uconazole in a murine model of cryptococcal infection. 156 calcineurin and targets of rapamycin inhibitors have antifungal activity, which is synergsitic with that of azoles. 157 hsp90 inhibitors initially developed for cancer treatment can improve fl uconazole activity in vitro and in animals. 158 enoxacin, a fl uoroquinolone antibiotic, shows activity in a murine candidiasis model. 159 although azoles are important for the treatment of invasive pulmonary aspergillosis, the degree of immunosuppression and other immunological factors have a role in treatment outcomes. antifungal immune responses could be improved by adaptive transfer of pathogen-specifi c t cells directed against invasive and pulmonary fungal infections, particularly infections with candida, aspergillus, and mucormycetes, especially after allogeneic stem-cell transplantation. t-cell responses are mhc class i restricted (for cd8positive t cells) or mhc class ii restricted (for cd4positive t cells), and thus an eff ective t-cell response needs to match the genetic background of the patient. t-cell transfer was developed on the basis of the promising fi nding that transfer of pathogen-specifi c t-cell clones induces clinically signifi cant responses. 160, 161 several approaches have been used to obtain these pathogen-specifi c t cells. anti-pathogenspecifi c t cells can be expanded ex vivo under appropriate conditions (usually with the help of recombinant cytokines, synthetic peptides, or cellular components representing the pathogen). responder t cells are identifi ed by interferon-γ production, removed via an interferon-capture assay, and transferred into the patient. this approach requires time for expansion of t cells (either the patient's own or those of an mhc-matched donor). this protocol enabled the expansion of aspergillus spp, candida spp, with the terms "respiratory tract", "pneumonia", "infections", "bacteria", "virus", "fungus", and "mycobacteria". we also combined these terms with the words "antibiotic", "antibiotic resistance", "treatment", "drugs", "drug development", "drug pipeline", "antibiotic development", "host-directed", "therapy", "adjunct therapy", "steroids", and "immunotherapy". we complemented the search with publications from who, the us centers for disease control and prevention, http://clinicaltrials. gov, and google scholar. we also reviewed studies cited by articles identifi ed by this search. and mucor spp-reactive t cells defi ned by interferon-γ production. upon re-encounter with the nominal target antigen, the t cells proliferated and increased the antifungal reactivity of phagocytes. 162 new and antimicrobial-resistant species of bacteria, viruses, and fungi continue to emerge because of the remarkable genetic and adaptable plasticity of the microbiota. 163 respiratory tract infections are among the top two causes of death globally. 164, 165 microorganisms do not respect international boundaries, and ease of travel and airborne spread make them a threat to global health security. the increasing frequency of antibiotic resistance and limited therapeutic options emphasise the urgent need for more international cooperation to tackle new emerging microbial threats and multidrug-resistant microbes. development of new therapeutic options needs to be coupled to international regulations on the use and prescription of antimicrobial drugs. dsh and az coordinated the writing of this series paper and wrote the draft outline, and subsequent and fi nal drafts. all authors contributed relevant text and tables on their expert sections or sections and contributed to fi nalising the paper. fgh has served as non-paid consultant for multiple companies engaged in marketing and/or clinical development of antivirals for respiratory viral infections including several whose therapeutics are discussed in this review (adamas, biocryst, gsk, genentech, janssen, roche, romark, toyama/medivector, visterra). dwd holds founder shares in f2g, a university of manchester spin-out company. he acts as a consultant to trinity group, t2 biosystems, glaxosmithkline, sigma tau, oxon epidemiology, and has consulted for merck and astellas and he has been paid to give talks on behalf of astellas, gilead, and pfi zer. all other authors declare no confl icts of interest. interspecies transmission and emergence of novel viruses: lessons from bats and birds clinical features and rapid viral diagnosis of human disease associated with avian infl uenza a h5n1 virus history of swine infl uenza viruses in asia writing committee of the world health organization consultation on northern hemisphere infl uenza vaccine composition for 2013-2014. who recommendations for the viruses used in the 2013-2014 northern hemisphere infl uenza vaccine: epidemiology, antigenic and genetic characteristics of infl uenza a(h1n1)pdm09, a(h3n2) and b 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candida species and aspergillus species isolates activity of mgcd290, a hos2 histone deacetylase inhibitor, in combination with azole antifungals against opportunistic fungal pathogens modeling nikkomycin z dosing and pharmacology in murine pulmonary coccidioidomycosis preparatory to phase 2 clinical trials in vitro and in vivo antifungal activities of t-2307, a novel arylamidine advancing antifungal r&d. www.f2g.com/f2g-ltd-completes-30-million-fi nancing-round-to-fund-pre-clinical-and-clinical-developmentof-novel-anti-fungal-compounds/ (accessed may the antidepressant sertraline provides a promising therapeutic option for neurotropic cryptococcal infections signaling cascades as drug targets in model and pathogenic fungi progress and prospects for targeting hsp90 to treat fungal infections inhibition of candida albicans virulence factors by novel levofl oxacin derivatives restoration of viral immunity in immunodefi cient humans by the adoptive transfer of t cell clones adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specifi c t-cell lines clinical-scale generation of multi-specifi c anti-fungal t cells targeting candida, aspergillus and mucormycetes towards an ecology of the lung: new conceptual models of pulmonary microbiology and pneumonia pathogenesis deaths due to respiratory tract infections in africa: a review of autopsy studies global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study key: cord-304088-xkg0ylz8 authors: zhu, han; rhee, june-wha; cheng, paul; waliany, sarah; chang, amy; witteles, ronald m.; maecker, holden; davis, mark m.; nguyen, patricia k.; wu, sean m. title: cardiovascular complications in patients with covid-19: consequences of viral toxicities and host immune response date: 2020-04-21 journal: curr cardiol rep doi: 10.1007/s11886-020-01292-3 sha: doc_id: 304088 cord_uid: xkg0ylz8 purpose of review: coronavirus disease of 2019 (covid-19) is a cause of significant morbidity and mortality worldwide. while cardiac injury has been demonstrated in critically ill covid-19 patients, the mechanism of injury remains unclear. here, we review our current knowledge of the biology of sars-cov-2 and the potential mechanisms of myocardial injury due to viral toxicities and host immune responses. recent findings: a number of studies have reported an epidemiological association between history of cardiac disease and worsened outcome during covid infection. development of new onset myocardial injury during covid-19 also increases mortality. while limited data exist, potential mechanisms of cardiac injury include direct viral entry through the angiotensin-converting enzyme 2 (ace2) receptor and toxicity in host cells, hypoxia-related myocyte injury, and immune-mediated cytokine release syndrome. potential treatments for reducing viral infection and excessive immune responses are also discussed. summary: covid patients with cardiac disease history or acquire new cardiac injury are at an increased risk for in-hospital morbidity and mortality. more studies are needed to address the mechanism of cardiotoxicity and the treatments that can minimize permanent damage to the cardiovascular system. coronavirus disease of 2019 , caused by infection from severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has spread across the world as a serious pandemic [1, 2] . sars-cov-2, an enveloped virus with non-segmented, single-stranded, positive-sense rna genome [3] , is a member of the coronaviridae (cov) family which causes a predominantly respiratory illness with a wide range of clinical severity, ranging from asymptomatic or mildly symptomatic (fever, cough, dyspnea, myalgias, fatigue, and diarrhea) in a large proportion of patients to severe acute respiratory distress syndrome (ards) and fatal multi-organ failure [1, 4-6, 7••] . the disease has a case-fatality rate that ranges from less than 0.5% to more than 7% (average,~3.8%) [8] , with an infectivity greater than that of influenza [9] . its high transmissibility and relatively high rate of causing serious complications has led covid-19 to become a serious public health threat worldwide. among various physiological consequences of severe covid-19, cardiovascular complications have emerged as some of the most significant and life threatening. covid-19 may present with respiratory failure from pneumonia and ards, with or without distributive ± cardiogenic shock [10, 11, 12•] , and severe cardiac injury manifesting as markedly elevated troponin and heart failure [12•, 13-14] . cardiac injury has also been associated with increased mortality [15••] . in a cohort study of 416 patients with confirmed covid-19, elevated troponin was present in 19.7% of patients during hospitalization and was found to be an independent risk factor for in-hospital mortality [15••] . the increased incidence of cardiac injury among those with severe systemic inflammatory response syndromes (sirs) and shock in the setting of covid-19 also highlights an important relationship between the immune response to the virus and the cardiovascular system. in addition, a high prevalence of pre-existing cardio-metabolic disease has been noted among those with severe covid-19 [16, 17] , and those with pre-existing cardiovascular conditions suffer increased mortality during covid-19 infection [18] . in particular, the reported case fatality rates for covid-19 are 10.5% in patients with cardiovascular disease, 7.3% in patients with diabetes, and 6.0% in those with hypertension, higher than the case-fatality rate of 3-4% observed world-wide for patients without these co-morbidities [7••] . last but not least, the increased frequency of adverse cardiovascular events following the resolution of covid-19, similar to other viral infections such as influenza [19] , may also play a role in worsening the mortality of patients with covid-19. thus, understanding the relationship between the viral-host immune response and the cardiovascular system will be critically important in our care and management of patients with covid-19 going forward. in order to better understand the biology of viral immune response and how it impacts the heart, we explore here the basic biological mechanisms underlying viral entry into the host cells and the subsequent immune response. coronaviruses are enveloped viruses with a single-strand, positive-sense rna genome approximately 26-32 kilobases in size, which is the largest known genome for an rna virus. six coronaviruses (covs) are known to infect humans: 229e, oc43, sars-cov, nl63, hku1, and mers-cov [3] . in humans, cov infections primarily involve the upper respiratory tract and gi tract [3] . studies have demonstrated that sars-cov-2, as well as other corona viruses, requires the angiotensin-converting enzyme 2 (ace2) for cellular entry [20] . ace2 is a type i integral membrane protein that serves an important role in cardiorenal homeostasis. it is also highly expressed in lung alveolar cells, providing the main entry site for virus into human hosts [21] . it is plausible that the high expression of ace2 in the lung, gut, heart, and kidneys may facilitate direct damage by the virus throughout the course of infection. one key protein on the virus-the spike protein (s)-facilitates viral entry into the target cells by the binding of its surface unit, s1, to the ace2 receptor on the host cell [21] [22] [23] , followed by cleavage by hostcell protease tmprss2 [24] . other important sars-cov-2 components include the hemagglutinin-esterase protein, the membrane (m) protein, the nucleocapsid protein, the small envelope protein, the internal protein, and group-specific proteins, which could become targets for vaccines in the future [25] . of note, sars-cov-2 also contains an rna-dependent rna polymerase which is the target of the anti-viral agent remdesivir, currently being studied randomized clinical trials for use against covid-19 disease [26] . the host immune response to viral entry is also important to discuss, as pathogenesis in the later stages of sars-cov and sars-cov-2 infection results not only from direct viral toxicity but also from immune dysregulation and hyperactivity [27, 28] . progress in this field, however, has been hindered by the failure to replicate in mice, ferrats, or non-human primates the lethal human immune response in ards with the original sars-cov strain [29, 30] . this has led to the development of mouse-or rat-adapted strains of sars-cov that have been able to replicate the extensive and often lethal pulmonary disease [31] . the majority of studies addressing the immune response to respiratory viral infections involve mice infected with a variety of natural and mouse-adapted pathogens. the process of respiratory viral invasion into the body begins with infection of the airway epithelial cells and the activation of lung-resident dentritic cells (rdcs) via acquisition of the invading pathogen or antigens from infected epithelial cells. these rdcs then become activated, process antigen and migrate to the draining (mediastinal and cervical) lymph nodes (dln). naïve circulating t cells in the dlns then recognize antigens presented on the dcs in the form of mhc/peptide complexes. in combination with additional costimatory signals, t cells then become activated, proliferate, and migrate to the infected site [32, 33] . upon arrival to the site of infection, t cells produce and release antiviral cytokines including interferon (ifn)-γ, tumor necrosis factor (tnf)-α, and interleukin (il)-2; chemokines including cxc chemokine ligand (cxcl)-9, 10, and, 11, and cytotoxic molecules such as perforin and granzyme b [34] . ifn-γ and other effector cytokines directly inhibit viral replication and enhance antigen presentation, while the chemokines released by activated t cells recruit more innate and adaptive cells to combat the pathogen. granzyme b and other cytotoxic molecules also directly kill infected cells to eliminate the pathogen [35] [36] [37] [38] . recent data from china on sars-cov-2 [28, 39] as well as prior data from sars-cov [40] demonstrate a rapid reduction of t lymphocytes (both cd4+ and cd8+) in the peripheral blood of infected patients [27, 41, 42] . this is in direct contrast to the proliferative lymphocyte responses seen with other viral infections such as epstein-barr virus (ebv), human immunodeficiency virus (hiv)-1, or cytomegalovirus (cmv), but similar to what happens during other acute viral infections, such as influenza. the loss of lymphocytes precedes even the abnormal radiographic changes on chest xray [43•] . despite reduction in t lymphocyte counts, peripheral blood analysis on a patient infected with sars-cov-2 demonstrated increased markers of t lymphocyte activation [44] , as evidenced by high proportions of hla-dr and cd38 double-positive fractions [28] . additionally, there was an increased percentage of highly proinflammatory ccr6+ th17 among cd4+ t cells, and an increased concentration of cytotoxic granules in cd8+ t cells (31.6% perforin positive, 64.2% granulysin positive, and 30.5% granulysin/perforin double-positive) [28] . interestingly, one group found that production of ifn-γ by cd4+, t cells but not cd8+ t cells or nk cells tended to be lower in severe cases compared with moderate cases [41] . cd4+ t cells, in particular, are felt to be especially important in the host-immune defense against sars-cov infections [45] . in addition, disturbances in t regulatory cells (tregs) were noted in severe cases-with a significantly lower proportion of c45ra+ naïve tregs (ntregs) and a slightly higher proportion of their memory counterparts cd45ro+ memory tregs (mtregs) [41] . on recovery, there is a rapid and significant restoration of cd3+, cd4+, and cd8+ t cells along with b cell and nk cell counts 2-3 months after onset of disease [40] . memory cd4+ t cells returned to normal 1 year after onset, whereas other cell counts including total t lymphocytes, cd3+, cd4+ and naïve cd4+ t cells were still lower than healthy controls [43•] . the mechanism of lymphocytopenia in peripheral blood is unclear but thought to be due to sequestration, with release of sequestered cells upon recovery [21] . taken together, these changes in lymphocyte populations suggest dramatic dysregulation, evidence of t cell "exhaustion" and shifts in the adaptive immune response to sars-cov and sars-cov-2 infections [42] . in addition to changes in lymphocyte populations, changes in innate immunity likely also contribute to viral pathogenesis, particularly as seen in severe lung and systemic inflammation secondary to cytokine storm. in ards, increased levels of cytokines such as tnf-α, ifn inducible protein 10 (ip10), il-6, and il-8 are thought to contribute to tissue destruction and poor outcomes [46] , attributed to hyperactivation of macrophage/monocyte lineage cells. sars-cov-2-infected patients have high levels of il-1 beta, ifn-γ, ip-10, and monocyte chemoattractant protein-1 (mcp-1), which probably leads to activated t-helper-1 cell response [1] . compared with patients who did not require icu admission, those requiring icu admission had higher conentrations of granulocyte colony-stimulating factors (gcsf), ip-10, mcp-1, macrophage inflammatory protein-1 α (mip-1-α), and tnf-α, suggesting that cytokine storm might affect disease severity [47] . additionally, increased levels of type i ifn and a dysregulated ifn-stimulated gene (isg) response have been seen in patients with severe sars [48, 49] . last but not least, sars-specific igg antibodies are produced in the late acute stage (about 2 weeks from symptom onset), gradually increase throughout the course of the disease and are felt to be associated with disease outcome [50] . the development of anti-sars-cov-2 antibodies is highly relevant for both protecting against viral replication/expansion in the infected host as well as providing a source of anti-sars-cov-2 convalescent plasma to treat patients with severe disease, although supporting data for efficacy is currently lacking. this is being tested in covid-19 positive patients under an expanded access program by the fda [51] . myocardial injury, manifesting as elevated serum troponin levels, has been described in many patients infected with covid-19, and mortality has been associated with increase in troponin levels > 99th percentile of the upper limit of normal and with electrocardiographic and echocardiographic abnormalities [12•, 18, 52] . in addition, reports of the rarer manifestation of fulminant myocarditis with markedly elevated troponin levels have been reported [12•, 13, 14] . there are several thoughts on the mechanism of injury, including direct myocardial injury by the virus through ace2 entry, hypoxia-induced myocardial injury, microvascular damage and endothelial shedding, and cytokine/ inflammation-mediated damage [15••] . direct viral toxicity on cardiomyocytes has occurred in the setting of other viral infections such as coxsackievirus-induced myocarditis. in this case, the entry of the coxsackievirus is through the coxsackievirus and adenovirus receptor (car) and through release of protease 2a coded by coxsackievirus particles, disrupting the dystrophin cytoskeleton complex [53] [54] [55] . in the case of coronavirus, as mentioned above, the spike (s) protein of coronaviruses facilitates viral entry into target cells. entry depends on binding of the surface unit, s1, of the s protein to ace2, allowing the virus to attach to the surface of the target cell. in addition, entry requires s protein priming by cellular proteases, which entails s protein cleavage at the s1/s2 and s2' site and allows fusion of viral and cellular membranes, a process driven by the s2 subunit [23] . it was found that sars-2-s shares 76% amino acid identity with sars-s, and both engage ace2 and employ the cellular serine protease tmprss2 for s protein priming for host cell entry [22] . interestingly, injection of sars-cov spike protein into mice worsened acute lung failure in vivo, and was attenuated by blockade of the renin-angiotensin pathway [21] . also of note, tmprss2 is highly expressed in the lung and kidneys, but is present in only low to moderate levels in the heart and blood vessels, suggesting other mechanisms of injury for the latter organ systems [56] . lastly, the amount of viral load in sars-cov-2 infection correlates with disease severity, with higher viral loads on presentation correlating with worse disease outcomes [57] . this study highlights the potential importance of direct viral toxicity in the pathogenesis of covid-19 infections. in addition to direct damage caused by the virus, there has also been speculation of an ischemic effect, either in the form of demand ischemia from lung pathology or direct toxicity by the virus on the macro-or microvascular level. it has been suggested that, because ace2 is expressed on the endothelium, it may induce endothelial shedding and dysfunction contributing to vascular damage, local inflammation, and production of procoagulant factors predisposing to thrombosis, similar to the increase in myocardial infarctions observed after influenza infections [19, 58] . in addition to endothelial inflammation and dysfunction, an increased incidence of abnormal coagulation parameters and of disseminated intravascular coagulation (dic) has been noted in patients with sars-cov-2 infection [59, 60] , contributing to risk of thrombosis and ischemic events that could damage the myocardium. early reports of fulminant myocarditis have alerted the medical and scientific communities that myocardial inflammation may play a role in cardiac injury during viral infection [12•, 13, 14] . however, the exact mechanism of this is currently unclear [12•, 13] , as acute lymphocyte infiltrates were not noted in the myocardium of a sars-cov-2-infected ards patient autopsy [39] , where only a few mononuclear inflammatory cells were seen. currently, there is great interest in obtaining the pathological specimens from patients presenting with markedly elevated troponin and fulminant myocarditis in order to evaluate for lymphocyte-induced myocardial injury in sars-cov-2 infection. consistent with this, no myocardialspecific epitopes have thus far been identified in the setting of sars-cov or sars-cov-2 infection. several hla-a*02:01-specific t cells recognizing sars-cov epitopes have been identified in the peripheral blood mononuclear cells (pbmc) of sars-recovered individuals, including immunogenic epitopes localized to spike (s) and nucleocapsid (n) protein of sars-cov [61, 62] . of note, mri-verified acute myocarditis has been reported in association with mers-cov [63] , although the exact mechanism by which it occurs is unknown. although no evidence of direct lymphocytic infiltration of the myocardium exists, the dysregulation of t cells can likely contribute to the cytokine storm and multi-organ damage in the setting of coronavirus infection. a recent retrospective, multi-center study of 150 patients confirmed that inflammatory markers, including elevated ferritin (mean 1297.6 ng/ml in non-survivors vs 614.0 ng/ml in survivors, p < 0.001) and il-6 (p < 0.0001) were associated with more severe covid-19 infection, suggesting that systemic inflammation may be a significant driver of multi-organ damage [18, 64] . a separate group has also reported that the serum cytokines il-2r, il-6, il-10, and tnf-α are increased in patients with severe disease [41] . this systemic release of cytokines, characterized by increased il-2, il-6, il-10, gcsf, ifn-γ, mcp-1, mip-1-α, and tnf-α, likely contributes to cardiac injury in a situation analogous to cardiotoxicity in the setting of chimeric antigen receptor (car)-t cell therapy. a prior study demonstrated that cardiac injury and cardiovascular events in the form of elevated troponin and left ventricular systolic dysfunction are common post-car-t; in a cohort of 137 patients with post-car-t cytokine release syndrome (crs), 21% had elevated troponin and 12% developed cardiovascular events including cardiac arrest, decompensated heart failure, and arrhythmias [65] . notably in this study, a shorter time from crs onset to the administration of the il-6 inhibitor tocilizumab was associated with a lower rate of cardiovascular events [65] . of note, tociluzumab may have some benefit in covid-19 infection, suggesting a common mechanism of injury in the two settings [66] . the exact mechanism by which cytokines/chemokines damage the myocardium is unknown, but cardiomyocyte and endothelial cell death in the presence of inflammatory cytokines such as tnf-α has been well documented in the literature [67, 68] . age and gender differences in covid-19 infection rates have raised interest in possible differences in age-and genderdependent immune responses to viral exposure. children account for the minority of laboratory-confirmed cases of covid-19 in china and appear less susceptible to severe disease [69] . although the functions of both innate and adaptive immune immunity declines with aging, this does not typically start until late adulthood, and thus would not fully explain the decreased severity of disease in children compared with even young adults. [70] [71] [72] . the effect of age on the immune system can be demonstrated by the low protective titers among 50% of adults older than 65 who receive the influenza vaccine [73] . additional information about the differential response of sars-covand sars-cov-2 with aging comes from animal models; in comparison with sars-cov-ma15-infected young c57bl/6 mice, infection of aged mice (12 months) is associated with severe reduction in the number of virus-specific cd8+ t cells in the lungs [66] . in addition to the effect of aging, gender is also thought to play a role in outcomes in sars-cov-2 infection. one study demonstrated a higher incidence of sars-cov-2 infection in older adult males compared with females [74] . sex differences in immune response have been noted in literature, although the reasons are not clear. males experience greater severity and prevalence of bacterial, viral, fungal and parasitic infections than females, who also mount a more robust response to antigenic challenges including infection and vaccination [75] [76] [77] [78] . we (davis et al) have used machine learning approaches to identify a cluster of genes involved in lipid biosynthesis, previously shown to be upregulated by testosterone, which correlates with poor virus-neutralizing activity in men [79] . interestingly, the stronger immune response in females is thought to explain why females are more prone to immunemediated pathologies including autoimmune disease and cytokine storm [78] . however, in the case of covid-19, there is no data to currently support a female-predominance toward cytokine storm; if anything, males are prone to more severe disease and mortality [74] . thus, it would be important to gather more data and larger number cohort studies on the current sars-cov-2 pandemic to study further delineate whether there is a gender-dependent risk for cytokine storm and subsequent cardiac injury in covid-19 infection. various therapies have emerged to treat the various aspects of viral pathogenesis and subsequent immune response, and an understanding of which aspect of disease pathogenesis they target may aide the clinician in knowing when to use them. treatments which target steps early in the infection process ("respiratory phase" in fig. 1 ) are meant to suppress viral replication and aid the host immune response to fight the virus. when the immune response becomes hyperactive ("sepsis/shock" phase in fig. 1 ) with cytokine storm, immunomodulators that target various harmful inflammatory cytokines may help mitigate end-organ toxicity. unfortunately, there are currently no fda-approved therapies for sars-cov-2, and no substantial randomized trial data to support any therapy thus far. however, several promising therapies are actively being test in patients including the recently announced multicenter, randomized solidarity trial (remdesivir, hydroxychloroquine) sponsored by the world health organization [51] . anti-microbial agents which target the early (pre-systemic) phase of infection include remdesivir, hydroxychloroquine, and azithromycin. remdesivir is a drug which targets the rna polymerase, suppresses viral replication, and has strong in vitro evidence of efficacy against sars-cov-2 [26] . although randomized trial data are still pending [51] , there have been anecdotal reports of improvement after compassionate use of remdesivir [80] . lopinavir-ritonavir is another rna polymerase inhibitor which showed initial promise, but a recently published study from beijing showed no significant difference in the treatment versus control group, suggesting that the effect, if present, was insufficient to cause a significant clinical response [81] . chloroquine has traditionally been used as an anti-malaria drug, but has shown reasonable efficacy against sars-cov-2 in vitro [26] . a single arm study of 20 covid-19-positive patients treated in france demonstrated some efficacy for the combination of azithromycin and hydroxychloroquine, a safer variation of chloroquine, in sars-cov-2 [80] . another small non-randomized study showed similar results [82] however, these were small, non-randomized studies in whose definition of efficacy was based on viral clearance rather than mortality benefit. a subsequent pilot randomized trial in shanghai comparing hydroxychloroquine and placebo has shown no difference in virologic clearance [83] . azithromycin is an antimicrobial agent generally used for antibacterial purposes; however, it also has been shown to inhibit zika virus tropism in the human brain and has been suggested as an adjunct for treatment of intracellular microbes in the past [80, 81] . since both chloroquine/ hydroxychloroquine and azithromycin lead to corrected qt (qtc) prolongation, their use either alone or in combination should be monitored for this potential cardiotoxic side effect [84] . the second group of therapies is immunomodulators, used to target immune hyperactivity and the cytokine storm that occurs in the later stages of covid-19 infection [64] . monoclonal antibodies have shown some promise in early studies. the il-6 inhibitors tocilizumab and sarilumab have shown early benefit and are being studied in ongoing randomized trials [51] . other therapies including meplazumab (anti-cd147), eculizumab (targets complement), adalimumab (tnf-α inhibitor), and ivig (saturation of fc receptors on macrophages, suppression of chemokines/cytokines) are also intended toward reduction of deleterious immune effects [51] . interestingly, immune stimulatory agents aimed at enhancing beneficial aspects of the immune response are also being tested-including ifn-β1a, pd-1 inhibitors, and donor convalescent plasma [51] . there are conflicting data to support the use of glucocorticoids in covid-19 related ards [85] , and even potential evidence of harm in the form of decreased viral clearance in sars and mers [86] and increased mortality in sars-cov2 [87] . without evidence of acute lymphocytic myocarditis in cardiovascular injury, we do not currently advocate for the routine administration of glucocorticoids for elevated troponin in covid-19 patients. randomized trials on both glucocorticoids and ivig in severe cases of covid-19 are ongoing [51] . sars-cov-2 has become a worldwide health threat, with the numbers of infected patients growing rapidly. an increased incidence of cardiac injury has been observed among those with severe infection. the mechanism of cardiac injury is unclear but likely involves a combination of direct viral damage and immune-mediated damage by inflammatory cytokines/chemokines and cytotoxic immune cell response in the later stages of infection. the host immune response and contributors to cytokine storm in sars-cov-2 infection are complex. significant depletion and dysregulation of t lymphocytes may contribute to immune dysregulation and hyperactivity. cardiac damage in the setting of cytokine storm may be analogous to that seen in cardiotoxicity from car-t. treatments for covid-19 are bimodal, with one group of treatments targeted toward early infection and viral replication, and another group targeted toward immune modulation in the later, systemic inflammatory phase of infection. with the advent of single cell immune phenotyping technologies, it will be important to perform comprehensive immune surveys of infected patients to better understand systemic perturbations with the infection and downstream cardiovascular effects. more data are needed to help guide us toward definitive treatment and protection of the cardiovascular system in covid-19 infection. 1 hypothesis of sars-cov-2 pathogenesis and immune response in cardiovascular injury. spike protein (sars-2-s) on the virus is activated by cellular serine protease tmprss2 highly expressed in lung, renal, and gastrointestinal cells and engages with angiotensinconverting enzyme 2 (ace2) that is highly expressed on respiratory epithelial cells for entry into the host cell. early infection is characterized by viral replication and direct damage by the virus to host cells, via ace2/tmprss2-mediated cell entry. as infection progresses, pro-inflammatory signals upregulate inflammatory cytokine production by cells in the adaptive and innate immune system, leading to cytokine storm and multi-organ damage clinical features of patients infected with 2019 novel coronavirus in wuhan a novel coronavirus outbreak of global health concern epidemiology, genetic recombination, and pathogenesis of coronaviruses clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china articles clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study full spectrum of covid-19 severity still being depicted characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention an interactive web-based dashboard to track covid-19 in real time estimation of the reproductive number of novel coronavirus (covid-19) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis cardiovascular considerations for patients, health care workers, and health systems during the coronavirus disease 2019 (covid-19) pandemic characteristics and outcomes of 21 critically ill patients with covid-19 in washington state this paper is of importance because it describes an example of fulminant myocarditis which can occur in association with covid-19 infection sars-cov-2: a potential novel etiology of fulminant myocarditis first case of covid-19 infection with fulminant myocarditis complication: case report and insights 2020;1-8. this paper is of major importance because it is the first large retrospective study demonstrating cardiac injury as an independent predictor of mortality in covid-19 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prolongation, torsade de pointes, and regulatory issues: a narrative review based on the study of case reports risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease systemic corticosteroid therapy may delay viral clearance in patients with middle east respiratory syndrome coronavirus infection clinical features of 69 cases with coronavirus disease publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-312795-0e4esl2o authors: puig-domingo, m.; marazuela, m.; giustina, a. title: covid-19 and endocrine diseases. a statement from the european society of endocrinology date: 2020-04-11 journal: endocrine doi: 10.1007/s12020-020-02294-5 sha: doc_id: 312795 cord_uid: 0e4esl2o nan coronavirus disease 2019 (covid-19) outbreak requires that endocrinologists from all over europe move on, even more, to the first line of care of our patients, also in collaboration with other physicians such as those in internal medicine and emergency units. this will preserve the health status and prevent the adverse covid-19-related outcomes in people affected by different endocrine diseases. people with diabetes in particular are among those in high-risk categories who can have serious illness if they get the virus, according to the data published so far from the chinese researchers, but other endocrine diseases such as obesity, malnutrition, and adrenal insufficiency may also be impacted by covid-19. therefore, since the responsibilities of endocrinologists worldwide due to the current covid-19 outbreak are not minor we have been appointed by the european society of endocrinology (ese) to write the current statement in order to support the ese members and the whole endocrine community in this critical situation. in addition, endocrinologists, as any other healthcare worker under the current covid-19 outbreak, will need to self-protect from this viral disease, which is demonstrating to have a very high disseminating and devastating capacity. we urge health authorities to provide adequate protection to the whole workforce of health professionals and to consistently test for covid-19 the exposed personnel. a decrease in the number of healthcare professionals available for active medical practice in case they contract the disease as it is happening in certain countries, is itself, a threat for the healthcare system and the well-being of our patients. the virus seems to have spread from infected animals and human-to-human transmission is now more than evident, with a high suspicion that non-symptomatic individuals act as the major vectors. it spreads like any other respiratory infectious disease, through contaminated airdroplets that come out of the mouth of infected persons when talking, coughing, or sneezing. the virus can survive in the environment from a few hours to a few days, depending on surfaces and environmental conditions, and touching affected surfaces. the mouth, nose, and ocular mucosa appears to be the major way of transmission. general symptoms are relatively nonspecific and similar to other common viral infections targeting the respiratory system, and include fever, cough, myalgia, and shortness of breath. the clinical spectrum of the virus ranges from mild disease with nonspecific signs and symptoms of acute respiratory illness, to severe pneumonia with respiratory failure and septic shock. possibly, an overreaction of the immune system leading to an autoimmune aggression of the lungs could be involved in the most severe cases of acute distress respiratory syndrome. there have also been reports of asymptomatic infection and research in this matter is currently ongoing worldwide to elucidate the real prevalence of the disease and the true relative mortality ratio. increased risk of morbidity and mortality in patients with diabetes regarding covid-19 infection older adults and those with serious chronic medical conditions like heart disease, lung disease, and diabetes are at the highest risk for complications from covid-19 infection. chronic hyperglycemia negatively affects immune function and increases the risk of morbidity and mortality due to any infection and is associated to organic complications. this is also the case for covid-19 infection [1] . during the influenza a (h1n1) pandemic, the presence of diabetes tripled the risk of hospitalization and quadrupled the risk of icu admission once hospitalized. among covid-19 mortality cases in wuhan, china, major associated comorbidities included hypertension (53.8%), diabetes (42.3%), previous heart disease (19.2%), and cerebral infarction (15.4%) [2] . in addition, as for seasonal influenza, new data regarding covid-19 indicate that the infection potentiates myocardial damage and identifies underlying heart disorders as a new risk factor for severe complications and worsening of prognosis [3] . among the confirmed covid-19 cases in china by feb 11, 2020, the overall mortality reported is 2.3% [4] . this data refers mostly to hospitalized patients [4, 5] . among persons with no underlying medical conditions, the reported mortality in china is 0.9%. there is a lack of data regarding the number of nonsymptomatic cases, as in most countries universal microbiological screening has not been performed. it is presumed that the prevalence of the infection is probably high or very high in the community, thus leading to an overestimation of the prevalence of case fatality. however, mortality is strongly increased with the presence of comorbid diseases, including previous cardiovascular disease (10.5%), diabetes (7.3%), chronic respiratory disease, hypertension, and cancer, each at 6%. among 60-year-old people and older, mortality has been reported to be 14.8% in those >80 years, 8% for those between 70 and 79 years and 3.6% in the group of 60-69 years. compared with non-icu patients, critically ill patients are older (median age 66 vs. 51 years) and have more previous comorbidities (72% vs. 37%) [6] . worldwide mortality rates may vary by region, but this information is not yet consistently available and comparable, as public health policies applied and health registers used in every region of the world are not homogeneous. social distancing as well as home confinement of the whole population are now widely adopted in many countries in europe and worldwide as measures hopefully effective in contrast to the spread of infection. we recommend that due to the increased dangers of developing covid-19, persons with diabetes should strictly adhere to these preventive measures and adopt them also within their homes in order to avoid being in contact with their relatives. therefore, under these circumstances, it is recommended that people with diabetes try to plan ahead of time what to do in case they get ill. it is important to maintain a good glycemic control, because it might help reduce the risk of infection itself and may also modulate the severity of the clinical expression of the disease. contact with healthcare providers, such as endocrinologists in the case of type 1 diabetes, and including also internal medicine specialists and general practitioners for type 2 diabetes patients may be advisable. however, routine appointments in person are not recommended for people with diabetes, as they should avoid crowds (waiting rooms). therefore, we recommend phone calls, video calls, and emails as the main way for patients to keep in touch with their healthcare provider team, in order to guarantee an optimal control of the disease. moreover, it is advised to ensure adequate stock of medications and supplies for monitoring blood glucose during the period of home confinement. people with diabetes who are infected with covid-19 may experience a deterioration of glycemic control during the illness, like in any other infectious episodes. implementation of "sick day rules" is therefore mandatory to overcome potential diabetes decompensation. contacting the healthcare provider team by telephone, email, or videoconference is also mandatory in case of possible symptoms of covid-19 infection in order to seek advice concerning the measures to avoid risk of deterioration of diabetes control or the possibility to be referred to another specialist (pneumologist or infectious disease doctor) or in the emergency services of the referral hospital to avoid the most serious systemic complication of the viral infection itself. obesity there is a general lack of data regarding the impact of covid -19 in people suffering from obesity. however, as for what is currently being the experience in some hospitals in spain, cases of young people in which severe obesity is present may evolve toward destructive alveolitis with respiratory failure and death (puig-domingo m, personal experience). there is no current explanation for this clinical presentation, although it is well known that severe obesity is associated to sleep-apnea syndrome, as well as to surfactant dysfunction, which may contribute to a worse scenario in the case of covid-19 infection. also, deterioration of glycemic control is associated with an impairment of ventilatory function and thus may contribute to a worse prognosis in these patients. in addition, type 2 diabetes and obesity may concur in a given patient, which typically is also frequently accompanied by an age >65. in summary, these patients may be at a higher risk of impaired outcomes in the case of covid-19 infection. regarding undernourished subjects, covid-19 infection is associated to a high risk of malnutrition development, mostly related to increased requirements and the presence of a severe acute inflammatory status. these patients show also a hyporexic state, thus contributing to a negative nutritional balance. estimated nutritional requirements are 25-30 kcal/kg of weight and 1.5 g protein/kg/day [7] . a nutrient dense diet is recommended in hospitalized cases including high protein supplements (2-3 intakes per day) containing at least 18 g of protein per intake. adequate supplementation of vitamin d is recommended particularly in areas with large known prevalence of hypovitaminosis d and due to the decreased sun exposure [8, 9] . if nutritional requirements are not met, complementary or complete enteral feeding may be required, and in case that enteral feeding may not be possible due to inadequate gastrointestinal tolerance, the patient should be put on parenteral nutrition. covid-19 patients' outcome is expected to improve with nutritional support [10] . adrenal insufficiency is a chronic condition of lack of cortisol production. live-long replacement treatment aiming to mimic physiologic plasma cortisol concentrations is not easy for these patients. based on current data, there is no evidence that patients with adrenal insufficiency are at increased risk of contracting covid-19. however, it is known that patients with addison's disease (primary adrenal insufficiency) and congenital adrenal hyperplasia have a slightly increased overall risk of catching infections. moreover, primary adrenal insufficiency is associated to an impaired natural immunity function with a defective action of neutrophils and natural killer cells [11] . this may explain, in part, this slightly increased rate of infectious diseases in these patients, as well as an overall increased mortality. this latter could also be accounted by an insufficient compensatory increase of the hydrocortisone dosage at the time of the beginning of an episode of infection. for all these reasons, patients with adrenal insufficiency may be at higher risk of medical complications and eventually at increased mortality risk in the case of covid-19 infection. so far, there are no reported data on the outcomes of covid-19 infection in adrenal insufficient subjects. in the case of suspicion of covid-19, a prompt modification of the replacement treatment as indicated for the "sick days" should be established when minor symptoms appear. this means in the first instance to at least double the usual doses of glucocorticoid replacement, to avoid adrenal crisis. in addition, patients are also recommended to have sufficient stock at home of steroid pills and injections in order to maintain the social confinement that is required in most of the countries for impeding the covid-19 outbreak spread. if a person with endocrine and metabolic diseases has fever with cough or trouble breathing and may have been exposed to covid-19 (if living in or visited a country affected in the 14 days before getting sick, or if having been around a person who may have had the virus), a call to the physician or nurse for advice should be made. some countries have set up covid-19 phone lines for the public. the personnel in charge of these phone lines will prioritize arrangements, if needed, regarding what should be the next step in the healthcare protocol. if the person is advised to go to the hospital, it is recommended to put on a face mask. in countries with explosive outbreak, most of the people have already bought a face mask by their own initiative. fluid samples taken from the nose or throat will be used for microbiologic diagnosis. there is currently no specific treatment for covid-19, but since the majority of cases are mild, only a limited amount of people will require hospitalization for supportive care. however, in most of the countries in which the outbreak has been declared and recognized, particularly in china, the northern regions of italy, iran, and spain, the situation has been very challenging and the requirement of hospitalization has led national health systems to the limit of their capacities [12] . what to do in case of confinement at home? individuals and families affected or suspected to be affected by covid-19 that stay at home should follow proper measures for infection prevention and control. management should focus on prevention of transmission to others and monitoring for clinical deterioration, which may prompt hospitalization. affected persons should be placed in a wellventilated single room, while household members should stay in a different room or, if that is not possible, maintain a distance of at least one meter from the person affected (e.g., sleep in a separate bed) and perform hand hygiene (washing hands with soap and water) after any type of contact with the affected person or their immediate environment. when washing hands, it is preferable to use disposable paper towels to dry them. if these are not available, clean cloth towels should be used and replaced when wet. to contain respiratory secretions, a medical mask should be provided to the person affected and worn as much as possible. individuals who cannot tolerate a medical mask should use rigorous respiratory hygiene-i.e., the mouth and nose should be covered with a disposable paper tissue when coughing or sneezing. caregivers should also wear a tightly fitted medical mask that cover their mouth and nose when in the same room is present the person affected. an ese "decalog" for endocrinologists in the covid-19 pandemic adequately protect yourself and ask for covid-19 testing if exposed avoid unnecessary routine appointments in person put in place online/email/phone consultation services. 4. closely monitor glycemic control in patients with diabetes recommend to persons with diabetes a strict adherence to general preventive measures counsel persons with diabetes about specific measures related to their disease management (sick day rules) in case of infection by covid-19 counsel persons with diabetes particularly if aged over 65 and obese about referrals for management in case of suspected infection by covid-19 avoid undernourishment with dietary or adjunctive measures if clinically indicated closely monitor clinical conditions of patients with adrenal insufficiency adapt increased replacement treatment if clinically indicated in patients with adrenal insufficiency infections in patients with diabetes mellitus: a review of pathogenesis characteristics of and public health responses to the coronavirus disease 2019 outbreak in china clinical considerations for patients with diabetes in times of covid-19 epidemic characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention interim clinical guidance for management of patients with confirmed coronavirus disease (covid-19) clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan espen expert statements and practical guidance for nutritional management of individuals with sars-cov-2 infection controversies in vitamin d: summary statement from an international conference skeletal and extraskeletal actions of vitamin d: current evidence and outstanding questions management of disease-related malnutrition in hospitalized patients with covid-19. statement of the nutrition section primary adrenal insufficiency is associated with impaired natural killer cell function: a potential link to increased mortality for the zhongnan hospital of wuhan university novel coronavirus management and research team, evidence-based medicine chapter of china international exchange and promotive association for medical and health care (cpam), a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) conflict of interest the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-323112-e78zpa9c authors: waterer, grant; wunderink, richard title: respiratory infections: a current and future threat date: 2009-07-16 journal: respirology doi: 10.1111/j.1440-1843.2009.01554.x sha: doc_id: 323112 cord_uid: e78zpa9c despite all the medical progress in the last 50 years pulmonary infections continue to exact and extremely high human and economic cost. this review will focus on the human, pathogen and environmental factors that contribute to the continued global burden or respiratory diseases with a particular focus on areas where we might hope to see some progress in the coming decades. despite all the advances in medical science in the past four decades, lower respiratory tract infections remain the fourth most common cause of death in middle to high income countries and the leading cause of death in low income countries. 1 mortality aside, respiratory tract infections have an enormous economic cost. the annual global cost is unknown, but in the usa the direct cost of community and nosocomial lower respiratory tract infections was estimated at us$15 billion in 1985. 2 by 2001 the economic burden of non-influenza viral respiratory tract infection alone had increased to us$40 billion in the usa. 3 including direct and indirect costs (such as loss of work time), the cost of influenza alone in the usa is currently estimated at us$87 billion per year, 4 although total indirect costs have been estimated by others at over us$100 billion annually. 5 clearly, the global cost of respiratory infection must be in the trillions (us$) per year. in this final article in the pulmonary infectious diseases series we will discuss why pulmonary infections remain a major health problem and are likely to continue to be so well into the foreseeable future. the first and most obvious reason why pulmonary infections remain a problem is that potential pathogens abound in our everyday environment. while some pathogens are dependant on human-to-human spread, there are numerous examples of pathogens that normally invade their host directly from an environment source. typical examples include legionella spp., non-tuberculous mycobacteria and a plethora of fungi and molds, including cryptococcus, histoplasma, aspergillus and coccidiomycosis, just to name a few. while our immune systems have adapted in response to environmental threats, these pathogens will always remain a threat, particularly in subjects whose immune response becomes compromised by age or disease. human disease may also result from pulmonary pathogens crossing directly from animals to humans. among the well-recognized respiratory zoonoses are chlamydia psittaci (psittacosis), coxiella burnetti (q-fever), fracisella tularensis (tularaemia) and of course viral infections such as influenza and hanta. while many of these infections can be avoided by strict hygiene protocols, wherever there is close proximity between humans and animals, such as in large parts of the developing world, some inter species transmission inevitable. the largest risk in the environment, however, comes from other humans, as the vast majority of pulmonary infections are acquired from other infected individuals. this is particularly true of viral infections. the more crowded the human environment, the faster the spread of respiratory pathogens through the population. during the severe acute respiratory syndrome (sars) outbreak, extensive public health campaigns aimed at reducing spread of respiratory droplets by good cough hygiene, the avoidance of work, school or day care during acute infections, and a massive uptake in mask wearing in the general populace had a dramatic effect on the rates of all viral respiratory infections in hong kong. 6 as relaxing these efforts led to a return to normal rates of viral transmission, much more can clearly be done to reduce community spread of pulmonary pathogens, especially during pan/epidemics. nosocomial transmission of pulmonary pathogens is also a major environmental problem. hospital outpatient clinics are a well-documented source of transmission of multi-resistant bacteria between patients with cystic fibrosis 7 and bronchiectasis. 8 intensive care units, particularly those with long-term ventilated patients, are also a frequent source of recurrent cross infection with multi-resistant bacteria. 9 environmental pools of organisms within hospitals themselves have also been commonly associated with outbreaks of nosocomial pneumonia with pathogens, such as legionella, 10,11 aspergillus 12,13 and mucormycosis. 14 while it is clear that strict infection control can reduce nosocomial infection rates, 15 the practical necessity of pooling vulnerable hosts together combined with the inevitable ageing of health-care facilities will ensure that nosocomial outbreaks continue to be a problem. not only is the average age increasing, but the number of very elderly people in whom senescencerelated immune compromise is common has dramatically increased over the past few decades in most western countries. a very large number of age-related immune deficits have been described. key changes increasing the risk of pulmonary infections include the production of lower affinity antibodies, reduced phagocytic ability and reduced responsiveness of naïve cd4-positive t cells. 16 what is also important is that the pro-inflammatory response becomes progressively less regulated in the elderly, 17 often termed 'inflamm-ageing'. 18 the excess pro-inflammatory cytokine response is almost certainly a significant factor in the increased the risk of septic shock, ards and multi-organ failure in elderly patients with pulmonary sepsis. 19 further complicating the fact that we have more vulnerable aged people in our communities is that we cluster them together. the close contact between elderly individuals in nursing homes and other agedcare residences frequently leads to rapid spread of new pathogens throughout the facility, as many case series have documented. [20] [21] [22] [23] [24] [25] while this close clustering is to some extent an economic necessity, much more study is required into how to limit the spread of respiratory pathogens in aged-care facilities, particularly in epidemic circumstances. age aside, advances in medical care have also meant a vastly expanded pool of people with chronic organ failure living in the community. cardiac failure, chronic renal failure and diabetes in particular are all associated with a significantly greater risk of death from pneumonia. 26 unfortunately, it also appears that immunization, at least against influenza, has substantially reduced efficacy in these vulnerable groups. [27] [28] [29] adding to increasing age and increasing numbers of people with chronic organ failure in our communities is the additional factor of deliberate immunosuppression. in recent years the marked increase in tumour necrosis factor antagonists and monoclonal antibodies targeting specific lymphoid populations in patients with inflammatory arthritis (and especially rheumatoid disease) has significantly over taken patients on immunosuppressant therapy after solid organ transplantation as the major cause of iatrogenic immunosuppression. an increased risk of tuberculosis in particular has been a major problem with tnf antagonist therapy. 30 at the milder end of iatrogenic immunosuppression is recent evidence that inhaled corticosteroids, commonly used in asthma and copd, probably increase the incidence of pneumonia. 31 however, whether this statistical increase in pneumonia is clinically important remains unclear as total mortality is not increased and total hospitalizations are lower, suggesting that the small increase in risk of pneumonia is more than compensated for by other beneficial effects. austrian and gold demonstrated that it takes time, possibly days, for antibiotics to alter the natural course of pneumonia. 32 more recent papers demonstrating potential benefits of combination antibiotic therapy in pneumococcal pneumonia show a similar delay between the onset of antibiotic therapy and an identifiable benefit, 33, 34 and a review of all deaths from community-acquired pneumonia in young adults in the uk also found that many presented too late to benefit from any available therapy. 35 patients delay presentation to medical care when they have pneumonia for many different reasons. in some cases it is possible that the onset of disease is so swift that they are unable to seek help, especially if they live alone. however, many complex psychological and practical factors, including financial ability to access health-care, factor into the decision to delay medical treatment. 36 the lack of potential for new antibiotics to alter early mortality requires new approaches to be developed. drotecogen alpha does appear to reduce some of the organ damage in patients with pneumonia and sepsis but the survival benefit is modest. 37 as discussed in the review on streptococcus pneumoniae, 38 reducing the virulence of invading pathogens is one promising line of research. in nosocomial pneumonia, and especially ventilator-associated pneumonia, delayed recognition and hence delayed initiation of therapy is also associated with increased mortality. 39, 40 due to the frequent lack of significant inflammatory response and often the very non-specific nature of patient symptoms (if any), diagnosis of nosocomial pneumonia remains a clinical challenge. all recent guidelines have called for significant research in new diagnostic methods; [41] [42] [43] however, this remains a significant unmet need. human pulmonary pathogens are so well adapted to their human hosts that many cause little or no disease in animals. some pathogens have also developed substantial adaptations to evade our immune responses to them. structural change to the cell wall of mycobacterium tuberculosis enabling it to resist digestion after phagocytosis is one well-characterized adaptation. an example of the extent to which human pathogens can adapt is the production by some viruses of an il-10 like protein that can directly downregulate immune response. 44 as well as adapting to our innate immune response, pathogens also modify their genome in direct response to our attempts to reduce their virulence. the multitude of mechanisms by which bacteria have become antibiotic resistant is well documented, and due to these adaptations we now have problems with a wide range of pulmonary pathogens such as panresistant pseudomonas aeruginosa 45 and extensively drug-resistant m. tuberculosis. 46 even in the community setting drug-resistant pathogens, such as methicillin-resistant staphylococcus aureus, are beginning to become a significant concern as a cause of pneumonia in some regions. 47 viruses can also adapt to anti-viral agents, for example neuraminidase inhibitor-resistant influenza. 48 while not responding to any external pressure, the constant modification of viral genomes also ensures a steady supply of pathogens. antigenic drift and antigenic shift continue to keep influenza near the top of the list of pulmonary pathogens. new pathogens, like the coronavirus responsible for sars, 49 are also certain to continue to emerge, much as hiv did in the 1980s. finally, if new threats do not arise naturally, there is always the unfortunate possibility that humans will deliberately introduce them. 50 given the capacity for pathogens to adapt, it seems likely that regardless of what antimicrobials we develop, the development of resistance is inevitable. however, even if antibiotic resistance does not develop, clearing out one pathogen simply creates space for another to move in. bronchiectasis and cystic fibrosis are classic examples of a procession of bacteria occupying vacated niches. non-tuberculous mycobacteria, which have increased significantly in the past few decades as problematic pulmonary pathogens, 51 are another example of bacteria finding new niches. the emerging data on serogroup replacement in pneumococci in response to pneumococcal vaccination 52 are further evidence that the efficacy of any strategy we develop to reduce bacterial infections is likely to reduce over time as bacteria adapt to the niche available. human pathogens have evolved with us and are well adapted to overcome our innate immune responses. when pressure has been applied, either through antibiotics, antivirals or vaccination, pathogens have either shown the capacity to adapt to them or new pathogens have occupied the vacated niche. as we continue to increase the population of vulnerable hosts, pulmonary infections will remain a major health problem for the foreseeable future. new antibiotics and antivirals may help with specific threats, but will not address most of the fundamental problems. new therapeutic and diagnostic approaches coupled with clinical vigilance, strict infection control and solid public health measures are the hopes for reducing the burden of pulmonary infectious disease over the coming decades. world health organization. top ten causes of death economic costs of respiratory tract infections in the united states the economic burden of non-influenza-related viral respiratory tract infection in the united states the annual impact of seasonal influenza in the us: measuring disease burden and costs economic burden of respiratory infections in an employed population respiratory infections during sars outbreak environmental contamination with an epidemic strain of pseudomonas aeruginosa in a liverpool cystic fibrosis centre, and study of its survival on dry surfaces pseudomonas cross-infection from cystic fibrosis patients to non-cystic fibrosis patients: implications for inpatient care of respiratory patients acquisition and cross-transmission of staphylococcus aureus in european intensive care units prospective 3-year surveillance for nosocomial and environmental legionella pneumophila: implications for infection control nosocomial legionnaires' disease: lessons from a four-year prospective study construction-related nosocomial infections in patients in health care facilities. decreasing the risk of aspergillus, legionella and other infections impact of air filtration on nosocomial aspergillus infections. unique risk of bone marrow transplant recipients nosocomial pulmonary mucormycosis with fatal massive hemoptysis the international nosocomial infection control consortium (inicc): goals and objectives, description of surveillance methods, and operational activities immunosenescence: emerging challenges for an ageing population innate immunity and inflammation in ageing: a key for understanding age-related diseases inflammageing and lifelong antigenic load as major determinants of ageing rate and longevity early mortality in patients with community-acquired pneumonia: causes and risk factors respiratory syncytial virus outbreak in a longterm care facility detected using reverse transcriptase polymerase chain reaction: an argument for real-time detection methods outbreak of chlamydia pneumoniae infection in a japanese nursing home two outbreaks of severe respiratory disease in nursing homes associated with rhinovirus a preventable outbreak of pneumococcal pneumonia among unvaccinated nursing home residents in new jersey during an outbreak of multidrug-resistant pneumococcal pneumonia and bacteremia among unvaccinated nursing home residents sequential outbreak of influenza a and b in a nursing home: efficacy of vaccine and amantadine prognosis and outcomes of patients with community-acquired pneumonia. a meta-analysis t cell responses are better correlates of vaccine protection in the elderly response to influenza vaccination in community and in nursing home residing elderly: relation to clinical factors the comparison of antibody response to influenza vaccination in continuous ambulatory peritoneal dialysis, hemodialysis and renal transplantation patients. scand role of cytokines in rheumatoid arthritis: an education in pathophysiology and therapeutics long-term use of inhaled corticosteroids and the risk of pneumonia in chronic obstructive pulmonary disease: a meta-analysis pneumococcal bacteremia with special reference to bacteremic pneumococcal pneumonia monotherapy may be suboptimal for severe bacteremic pneumococcal pneumonia combination antibiotic therapy lowers mortality among severely ill patients with pneumococcal bacteremia a national confidential enquiry into community acquired pneumonia deaths in young adults in england and wales. british thoracic society research committee and public health laboratory service i really should've gone to the doctor': older adults and family caregivers describe their experiences with community-acquired pneumonia severe community-acquired pneumonia as a cause of severe sepsis: data from the prowess study new insights into pneumococcal disease appropriateness and delay to initiate therapy in ventilator-associated pneumonia clinical importance of delays in the initiation of appropriate antibiotic treatment for ventilator-associated pneumonia guidelines for the management of hospital-acquired pneumonia in the uk: report of the working party on hospital-acquired pneumonia of the british society for antimicrobial chemotherapy clinical practice guidelines for hospitalacquired pneumonia and ventilator-associated pneumonia in adults. can guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia viral interleukin 10 (il-10), the human herpes virus 4 cellular il-10 homologue, induces local anergy to allogeneic and syngeneic tumors pan-drug-resistant pseudomonas aeruginosa causing nosocomial infection at a university hospital in taiwan management of multidrug-resistant tuberculosis: update staphylococcus aureus community-acquired pneumonia during the 2006 to 2007 influenza season emergence of resistance to oseltamivir among influenza a(h1n1) viruses in europe identification of a novel coronavirus in patients with severe acute respiratory syndrome bioterrorism for the respiratory physician when and how to treat pulmonary nontuberculous mycobacterial diseases serotype replacement and multiple resistance in streptococcus pneumoniae after the introduction of the conjugate pneumococcal vaccine key: cord-345381-9cckppk2 authors: klimek, ludger; pfaar, oliver; worm, margitta; eiwegger, thomas; hagemann, jan; ollert, markus; untersmayr, eva; hoffmann-sommergruber, karin; vultaggio, alessandra; agache, ioana; bavbek, sevim; bossios, apostolos; casper, ingrid; chan, susan; chatzipetrou, alexia; vogelberg, christian; firinu, davide; kauppi, paula; kolios, antonios; kothari, akash; matucci, andrea; palomares, oscar; szépfalusi, zsolt; pohl, wolfgang; hötzenecker, wolfram; rosenkranz, alexander r.; bergmann, karl-christian; bieber, thomas; buhl, roland; buters, jeroen; darsow, ulf; keil, thomas; kleine-tebbe, jörg; lau, susanne; maurer, marcus; merk, hans; mösges, ralph; saloga, joachim; staubach, petra; jappe, uta; rabe, klaus f.; rabe, uta; vogelmeier, claus; biedermann, tilo; jung, kirsten; schlenter, wolfgang; ring, johannes; chaker, adam; wehrmann, wolfgang; becker, sven; freudelsperger, laura; mülleneisen, norbert; nemat, katja; czech, wolfgang; wrede, holger; brehler, randolf; fuchs, thomas; tomazic, peter-valentin; aberer, werner; fink-wagner, antje-henriette; horak, fritz; wöhrl, stefan; niederberger-leppin, verena; pali-schöll, isabella; pohl, wolfgang; roller-wirnsberger, regina; spranger, otto; valenta, rudolf; akdis, mübecell; matricardi, paolo m.; spertini, françois; khaltaev, nicolai; michel, jean-pierre; nicod, larent; schmid-grendelmeier, peter; idzko, marco; hamelmann, eckard; jakob, thilo; werfel, thomas; wagenmann, martin; taube, christian; jensen-jarolim, erika; korn, stephanie; hentges, francois; schwarze, jürgen; o´mahony, liam; knol, edward f.; del giacco, stefano; chivato pérez, tomás; bousquet, jean; bedbrook, anna; zuberbier, torsten; akdis, cezmi; jutel, marek title: use of biologicals in allergic and type-2 inflammatory diseases during the current covid-19 pandemic: position paper of ärzteverband deutscher allergologen (aeda)(a), deutsche gesellschaft für allergologie und klinische immunologie (dgaki)(b), gesellschaft für pädiatrische allergologie und umweltmedizin (gpa)(c), österreichische gesellschaft für allergologie und immunologie (ögai)(d), luxemburgische gesellschaft für allergologie und immunologie (lgai)(e), österreichische gesellschaft für pneumologie (ögp)(f) in co-operation with the german, austrian, and swiss aria groups(g), and the european academy of allergy and clinical immunology (eaaci)(h) date: 2020-09-07 journal: allergol select doi: 10.5414/alx02166e sha: doc_id: 345381 cord_uid: 9cckppk2 background: since the beginning of the covid-19 pandemic, the treatment of patients with allergic and atopy-associated diseases has faced major challenges. recommendations for “social distancing” and the fear of patients becoming infected during a visit to a medical facility have led to a drastic decrease in personal doctor-patient contacts. this affects both acute care and treatment of the chronically ill. the immune response after sars-cov-2 infection is so far only insufficiently understood and could be altered in a favorable or unfavorable way by therapy with monoclonal antibodies. there is currently no evidence for an increased risk of a severe covid-19 course in allergic patients. many patients are under ongoing therapy with biologicals that inhibit type 2 immune responses via various mechanisms. there is uncertainty about possible immunological interactions and potential risks of these biologicals in the case of an infection with sars-cov-2. materials and methods: a selective literature search was carried out in pubmed, livivo, and the internet to cover the past 10 years (may 2010 – april 2020). additionally, the current german-language publications were analyzed. based on these data, the present position paper provides recommendations for the biological treatment of patients with allergic and atopy-associated diseases during the covid-19 pandemic. results: in order to maintain in-office consultation services, a safe treatment environment must be created that is adapted to the pandemic situation. to date, there is a lack of reliable study data on the care for patients with complex respiratory, atopic, and allergic diseases in times of an imminent infection risk from sars-cov-2. type-2-dominant immune reactions, as they are frequently seen in allergic patients, could influence various phases of covid-19, e.g., by slowing down the immune reactions. theoretically, this could have an unfavorable effect in the early phase of a sars-cov-2 infection, but also a positive effect during a cytokine storm in the later phase of severe courses. however, since there is currently no evidence for this, all data from patients treated with a biological directed against type 2 immune reactions who develop covid-19 should be collected in registries, and their disease courses documented in order to be able to provide experience-based instructions in the future. conclusion: the use of biologicals for the treatment of bronchial asthma, atopic dermatitis, chronic rhinosinusitis with nasal polyps, and spontaneous urticaria should be continued as usual in patients without suspected infection or proven sars-cov-2 infection. if available, it is recommended to prefer a formulation for self-application and to offer telemedical monitoring. treatment should aim at the best possible control of difficult-to-control allergic and atopic diseases using adequate rescue and add-on therapy and should avoid the need for systemic glucocorticosteroids. if sars-cov-2 infection is proven or reasonably suspected, the therapy should be determined by weighing the benefits and risks individually for the patient in question, and the patient should be involved in the decision-making. it should be kept in mind that the potential effects of biologicals on the immune response in covid-19 are currently not known. telemedical offers are particularly desirable for the acute consultation needs of suitable patients. course in allergic patients. many patients are under ongoing therapy with biologicals that inhibit type 2 immune responses via various mechanisms. there is uncertainty about possible immunological interactions and potential risks of these biologicals in the case of an infection with sars-cov-2. materials and methods: a selective literature search was carried out in pubmed, livivo, and the internet to cover the past 10 years (may 2010 -april 2020). additionally, the current german-language publications were analyzed. based on these data, the present position paper provides recommendations for the biological treatment of patients with allergic and atopy-associated diseases during the covid-19 pandemic. results: in order to maintain in-office consultation services, a safe treatment environment must be created that is adapted to the pandemic situation. to date, there is a lack of reliable study data on the care for patients with complex respiratory, atopic, and allergic diseases in times of an imminent infection risk from sars-cov-2. type-2-dominant immune reactions, as they are frequently seen in allergic patients, could influence various phases of covid-19, e.g., by slowing down the immune reactions. theoretically, this could have an unfavorable effect in the early phase of a sars-cov-2 infection, but also a positive effect during a cytokine storm in the later phase of severe courses. however, since there is currently no evidence for this, all data from patients treated with a biological directed against type 2 immune reactions who develop co-vid-19 should be collected in registries, and their disease courses documented in order to be able to provide experience-based instructions in the future. conclusion: the use of biologicals for the treatment of bronchial asthma, atopic dermatitis, chronic rhinosinusitis with nasal polyps, and spontane-ous urticaria should be continued as usual in patients without suspected infection or proven sars-cov-2 infection. if available, it is recommended to prefer a formulation for self-application and to offer telemedical monitoring. treatment should aim at the best possible control of difficult-to-control allergic and atopic diseases using adequate rescue and add-on therapy and should avoid the need for systemic glucocorticosteroids. if sars-cov-2 infection is proven or reasonably suspected, the therapy should be determined by weighing the benefits and risks individually for the patient in question, and the patient should be involved in the decision-making. it should be kept in mind that the potential effects of biologicals on the immune response in covid-19 are currently not known. telemedical offers are particularly desirable for the acute consultation needs of suitable patients. the clinical symptoms of infection with the novel coronavirus (severe acute respiratory coronavirus 2; sars-cov-2) became known as the "coronavirus disease 2019 (covid-19)" on february 11, 2020 [1] . the international committee on taxonomy of viruses (ictv) called this novel human pathogenic virus sars-cov-2 [1] . the global spread of the sars-cov-2 pandemic and patients with severe covid-19 courses pose a major challenge to healthcare systems worldwide. the coronavirus that caused the severe acute respiratory syndrome (sars-cov) in 2002/2003 has approximately an 80% nucleotide sequence identity with sars-cov-2 [1] . sars-cov-2 is a betacoronavirus of the subgenus sarbecovirus, subfamily orthocoronavirinae, and the 7 th member of the coronaviridae family that can infect humans. it can be isolated from human samples obtained from respiratory secretions, nasal and pharyngeal swabs and isolated on cell cultures [1, 2, 3] . it is covered by a lipid membrane that can be disrupted by detergents and is different from the middle east respiratory syndrome-related coronavirus (mers-cov), from sars-cov, and from the coronaviruses responsible for the common cold (229e, oc43, nl63, and hku1) [1] . abbreviations. angiotensin-converting enzyme 2 the incubation period after an infection with sars-cov-2 can be of up to 14 days, during which the infected person can be asymptomatic but nevertheless transmit the virus. in a high number of patients, the infection leads to symptoms of the upper and lower airways, and, less frequently, also of other organ systems (nervous system, gastrointestinal tract, kidneys, blood vessels). in the worst case scenario, multi-organ failure and respiratory failure can result, as has been described for other coronavirus infections (sars-cov-1, mers-cov) [4, 5, 6] . in more severe cases, infection with sars-cov-2 can lead to pneumonia, severe acute respiratory syndrome, renal failure, and death [4, 7, 8, 9, 10] . higher age and comorbidities such as chronic airway diseases (particularly copd), diabetes mellitus, cardiovascular diseases, obesity, and immune deficiency of various origins have been described as risk factors of a severe course [4, 7, 8, 9] . the need for intensive care treatment and invasive ventilation is associated with high mortality. we will present clinical and immunological aspects that have to be considered with regard to the covid-19 pandemic in patients treated with biological therapy against ige and mediators of type 2 inflammation (table 1) . the characteristics of the immune response after infection with sars-cov-2 are still insufficiently understood. while various forms of the course of covid-19 and the infection with the virus have been described, it is still unclear which immunological background influences the course of the disease. this is also true for the role of the innate table 1 . recommendations on treatment with biologicals during the covid-19 pandemic in patients with asthma, atopic dermatitis, urticaria, or crswnp. recommendations for biological treatment in non-infected patients during the covid-19 pandemic or in patients who have recovered from covid-19 infection termination of biological treatment is not generally necessary; and biologicals should be continued as scheduled, particularly in severe cases, based on an individual risk-benefit analysis. in mild-to-moderate covid-19 courses, or when sars-cov-2 infection is suspected, biologicals can be continued in the indications discussed here if a patient-based risk-benefit analysis supports the decision and the patient consents after having been informed about the limited availability of data. prolongation of the injection interval can be considered (as indicated in the summary of product characteristics) to limit the necessary physician-patient contacts to a minimum. in severe covid-19 courses, prolongation of the injection interval (as indicated in the summary of product characteristics) or treatment interruption should be considered in the indications discussed here. the risk of the possible requirement of systemic glucocorticosteroids must be considered. biological treatment can be continued during the current covid-19 pandemic in asymptomatic patients with negative pcr tests, in patients without known exposure or contact with sars-cov-2-positive people, and in patients who have completed an adequate quarantine period. in a quarantine situation, telemedical support might be feasible, in particular with the aim of continuing the basic therapy with topical steroids, inhaled bronchodilators, antihistamines, etc. in accordance with the relevant guideline recommendations or with the aim to expand those therapies according to the patient's needs. biological therapy in patients without evidence of sars-cov-2 infection can be started for approved indications during the current covid-19 pandemic, self-administration of biologicals should generally be preferred; this is made easier if user-friendly pen systems for self-application are available. adequate patient training is required. practices and allergy centers must be prepared for the current covid-19 pandemic by following the recommendations of the who and of national and regional authorities. these recommendations should be continuously updated and adapted to new scientific findings and recommendations made by authorities. crswnp = chronic rhinosinusitis with nasal polyps. and adaptive immune system with regard to sars-cov-2 infection. while natural killer (nk) cells traditionally play an important role in the early phase of viral infections, cd8 + t helper cells come into action in the subsequent phase [11] . early antibody secretion and production in the mucosa-associated lymphatic tissue initially include antigenspecific igm, iga, and, later, igg antibodies, and are essential for immune response [12, 13, 14] . macrophages are activated and secrete inflammatory cytokines, with type 1 interferons (type 1 ifn) playing the most prominent role. in infections with other coronaviruses (e.g., sars-cov-1), type 1 ifn is responsible for the adequate initiation of the immune reaction, and patients with delayed or insufficient ifn production have a more severe disease course [6] . the activation of apoptosis or pyroptosis in epithelial cells serves as an antiviral defense, but excessive immune reactions can also contribute to local tissue damage through synergistic effects [15] . an excessive production of pro-inflammatory cytokines has already been observed in sars-cov-1, mers-cov, and recently also in sars-cov-2 infections, and has been described as a cytokine storm [4, 5] . natural igm, and probably also mannose-binding lectin (mbl), are believed to be the first line of defense against sars-cov-2 [16] . these antibodies and mbl recognize glycans and are abundant in children and young adults. however, they decrease dramatically with age and are over 50 times lower at the age of ≥ 60 than at the age of 20 -30 years [16] . as they are part of the innate immunity, they are the only antibodies able to recognize sars-cov-2 before the adaptive immune response is initiated [16] . if the virus enters the lungs early enough, it can replicate in an unhindered manner, as no or only little resistance exists. the resulting inflammation with a massive activation of local mediators (complement and coagulation cascades interleukin-6 (il-6), cytokine storm) can cause damages that lead to complications or, in some cases, even to death [16] . furthermore, the ability of sars-cov-2 viruses to penetrate the cell via receptors such as ace2 and tmprrss2 could explain the different severities of covid-19 in different patient groups [17] . extensive damage to the lungs leads to rapid clinical deterioration and usually to the need for intensive care treatment, which can be observed typically 7 -14 days after infection. the risk of kidney, liver, and/or other organ damage as well as of consumption coagulopathy is significantly increased. affected patients usually have highly increased interleukin (il)-1 β, il-6, il-8, and tnf-α levels ( figure 1 ) [18] . the therapeutic blockade of one or more of these cytokines has been discussed as a potential future therapy option for severely affected patients in whom il-6 can be massively increased [19] . il-6 plays a central role in the cytokine storm, and tocilizumab has already been used as a biological with anti-il-6 effects in covid-19 [20, 21] . approved indications for anti-il-6 or anti-il-6r antibodies (such as tocilizumab, sarilumab) currently include, for example, rheumatoid arthritis, juvenile rheumatoid arthritis, and castleman disease. the immune reactions of type 1 and type 3 described here are contained by other cytokines, such as il-10 and tgf-β, and type 2 inflammation could possibly counteract the cytokine storm. increased levels of eosinophilic granulocytes, as one of the key cells of type 2 inflammation, have been ascribed a protective effect in severe viral infections, although the mechanism of action has not yet been identified [22] . on the other hand, low blood eosinophil counts could simply reflect the severity of the infection. the interaction of sars-cov-2 with its receptor on the cells of the respiratory system, the membrane-bound angiotensin-converting enzyme 2 (ace2), which is responsible for the entry of the virus into the host cell, is far better investigated [17] . therefore, the reduced expression of ace2 in the airway epithelial cells of patients with allergic asthma is being discussed as a potentially protective factor against sars-cov-2 infection [23] . it can be assumed that only the interaction of the individual cytokine responses leads to an adequate and effective immune response in coronavirus infections. however, imbalances between type 1, type 2, and type 3 reactions might have a significant negative or positive impact on the course of the viral infection ( figure 1 ). figure 1 . sequence of immunological events in sars-cov-2 infection: if sars-cov-2 infects cells via the surface receptors ace2 and tmprss2, this leads to active replication and release of the virus, the cells decay through pyroptosis. damps are released that are recognized by neighboring epithelia, endothelia, and alveolar macrophages and trigger the release of pro-inflammatory cytokines such as il-6, ip-10, mip1α, mip1β, and mcp1. this attracts monocytes, macrophages, and t cells, which, when ifn-γ is added, activate another inflammatory, self-reinforcing cascade. in defective immune responses (left), this can lead to accumulation of immune cells and overproduction of pro-inflammatory cytokines, which then damage the lung and may lead to a cytokine storm with multi-organ failure. additionally, non-neutralizing antibodies produced by b cells may enhance the infection and lead to further organ damage. in a healthy immune response (right), the initial inflammation attracts virus-specific t cells to the infection site where they can eliminate the infected cells before the virus spreads further. neutralizing antibodies selectively block the virus, alveolar macrophages recognize and phagocytize affected cells and neutralized viruses. altogether, these processes clear the viral infection with minimal damage of respiratory tissue and lead to recovery. reproduced from tay et al. [65] . ace2 = angiotensin-converting enzyme 2; ade = antibodydependent enhancement; damp = damage-associated molecular pattern; g-csf = granulocyte colonystimulating factor; ifn = interferon; il = interleukin; mip1α = macrophage inflammatory protein 1α; sars-cov-2 = severe acute respiratory syndrome coronavirus 2; tnf = tumor necrosis factor. so far, there is insufficient evidence to indicate which risk factors cause a severe course of covid-19. a history of lung disease has been considered a potential risk factor for developing covid-19 and possibly also for a severe course. bronchial asthma, which is the most important allergic indication for biologicals targeting ige and type 2 inflammation, is possibly one of these diseases. however, since many patients with pulmonary disease also have other comorbidities, some of the suspected factors could turn out to be confounders once further studies are carried out. thus, it remains unclear whether patients with type-2-associated bronchial asthma without any other possible risk factors should be considered high risk for a severe covid-19 course. the currently available data rather contradict this. there is only limited data on covid-19 in connection with a type-2-associated disease, and its prognostic value is very limited. the currently available studies do not indicate an increased risk for patients with allergies, asthma, or other atopy-associated diseases ( table 2) [8, 24, 25, 26, 27, 28, 29] . for example, in wuhan and italy, the percentage of seriously ill or deceased covid-19 patients with known bronchial asthma was far below the prevalence of asthma in these places [18] . it also remains unclear why in many patients not only lymphopenia but also eosinopenia was detected at the time of admission [8] . neither decreased nor increased eosinophil levels have so far been clearly associated with certain clinical courses of sars-cov-2 infection. in recent years, several biologicals have been approved in europe that block ige antibodies or the interleukins il-4, il-5, and il-13, which are relevant in type 2 inflammation, or their receptors [30, 31, 32, 33, 34, 35, 36, 37, 38, 39] . omalizumab has been approved for the treatment of severe allergic bronchial asthma in adults and in children older than 6 years with proven sensitization against a perennial airborne allergen and reduced lung function. another indication is antihistamine-resistant chronic spontaneous urticaria in adults and in adolescents older than 12 years. mepolizumab, benralizumab, and reslizumab are report of 3 patients taking oral glucocorticosteroids because of breathing difficulties due to covid-19 and known asthma who were hospitalized 1 week later with acute respiratory insufficiency wang et al. [27] (wuhan, china) 2 of 69 studied patients had asthma zhang et al. [8] (wuhan, china) study of 140 patients of whom 2 had chronic urticaria, 1 had asthma, and 10 had unclear adverse drug reactions grasselli et al. [29] (lombardy, italy) study including 1,591, of whom 205 had a history of: bronchial asthma, anemia, inflammatory bowel disease, chronic respiratory insufficiency, endocrine disorders, chronic pancreatitis, diseases of the connective and supporting tissue, organ transplantation, epilepsy, neurological disease (reported as "other" in the study) dreher et al. [28] (aachen, germany) result: covid-19 patients with a history of respiratory disease develop ards more frequently (58 vs. 42%; 14 vs. 11 patients; of these, 4 vs. 2 patients with asthma; n = 50) ards = acute respiratory distress syndrome. il-5 or il-5 receptor blockers, available for adults and, partially, also for children. dupilumab has been approved for the treatment of: (i) atopic dermatitis in adolescents older than 12 years, (ii) severe type-2-dominant asthma in adults and (iii) chronic rhinosinusitis with nasal polyps (crswnp) in adults. table 2 shows the situation of approval in germany, austria, luxembourg, and switzerland. self-administration by the patient is listed separately as it significantly facilitates care for suitable patients during the sars-cov-2 pandemic. viral infections of the upper and lower airways have been associated with the development and exacerbation of allergic disease [40, 41, 42] . infection and the persistence of virus particles in the mucosa could inhibit the efficacy of the local innate immune system and promote type 2 inflammation. the blocking of type 2 inflammation by therapeutic antibodies against ige, il-5, or the il-5 or il-4/-13 receptors has so far not been suspected to increase the risk of viral infections. however, il-4 has a dual role in viral infections due to two different haplotypes in the il-4 gene. it can promote infections with the herpes virus and the norovirus [43] as well as with the ebola virus, which is related to the coronavirus [44] . on the other hand, il-4 can also inhibit viral infections by promoting innate immunity [45, 46, 47] . thus, more evidence from clinical observations is necessary to be able to provide clear recommendations with regard to covid-19. table 4 gives an overview of the frequency of viral infections occurring as adverse events in trials on these monoclonal antibodies. there have been reports on the lower incidence of viral infections under anti-ige treatment with omalizumab, since this therapy may increase the functionality and the production of ifn-α by plasmacytoid dendritic cells (pdc). this leads to an enhanced antiviral defense and to a reduction of virus-induced asthma exacerbations [40, 48] . also, for type 2 blockade with anti-il-5 antibodies (mepolizumab, reslizumab) or anti-il-5 receptor antibodies (benralizumab), the risk of respiratory viral infections in the active-agent study groups was equal to or lower than the risk in the placebo groups (table 4 ). it has not yet been clarified whether the blockade of type 2 inflammation or of ige influences the risk of developing covid-19 or its course. in the case of a cytokine storm, possible negative effects induced by blocking the type 2 immune response situation are conceivable; but these effects require further investigation. the first reports show that the disease course is not worse in covid-19 patients with eosinophilic diseases under biological therapy [24, 49] . however, further study results should be awaited, especially considering the fact that sars-cov-2 changes rapidly due to mutations [50] . meta-analyses by agache et al. [51, 52, 53] have shown a slightly increased rate (low to medium risk of association) of adverse events when anti-il-5/5r, anti-il-4/13r, and anti-ige are used in severe asthma, independently of covid-19. thus, there is no clear recommendation regarding the decision-making to continue or temporarily interrupt a biological therapy during an infection with sars-cov-2. treatment interruption could entail the risk of suboptimal control of the allergic disease or, in the case of exacerbations, the need for systemic glucocorticosteroids, for which an increased risk of a possibly more severe covid-19 course has been described [54] . recommendations for the management of allergic/atopyassociated diseases under anti-type-2 therapy during the covid-19 pandemic (table 1) to ensure an appropriate, high-quality, and accurate care for patients on anti-type-2 treatment with underlying atopic-eosinophilic or allergic disease, antibody therapy should be continued and remain unchanged during the ongoing pandemic when there is no evidence of sars-cov-2 infection. to cope with the current shortfalls in hospitals and the more difficult hygiene conditions, telephone or telemedical follow-up should be considered in suitable patients when technical and medical requirements allow for it. for this purpose, comprehensive patient training with regard to documentation of the disease activity and, where applicable, to self-administration of the medication is desirable. this is facilitated by the partial availability of user-friendly pen systems for self-application. in general, in countries with low infection numbers and a consequent relaxation of covid-19-associated restrictions, there is no contraindication for starting biological therapy in patients without evidence of a current sars-cov-2 infection. according to the current state of knowledge, biological therapy for the indications discussed here can be continued in mild to moderate cases of sars-cov2 infection/ covid-19 disease, if an individual consideration of risks and benefits supports this decision. the risks and benefits have to be assessed by a specialist, and it is recommended to in-form the patient about the fact that only limited data are available. in severe courses of covid-19, prolongation of the dosing interval or treatment interruption should be considered. when doing so, the risk of the potential requirement of treatment with systemic glucocorticoids should also be taken into account. in a quarantine situation, a telemedical approach might be feasible, in particular with the aim of continuing or expanding the basic therapy with topical steroids, inhaled bronchodilators, antihistamines, etc. in accordance with the relevant guideline recommendations [36, 37, 54, 55, 56, 57, 58] . if hospitalization due to the exacerbation of asthma-or type-2-associated diseases becomes necessary, current guidelines on diagnosis and treatment must be followed. sinus surgery for crswnp should, if possible, be delayed in patients with suspected or confirmed covid-19 disease. in the case of urgently indicated systemic therapy for severe atopic dermatitis, consideration should be given to therapy with either biologicals, classic immunosuppressants, or systemic glucocorticosteroids, although systemic glucocorticosteroids are not recommended due to their broad immunosuppressive effect (see above). for cyclosporin a (cya) as an approved therapeutic option for atopic dermatitis, in vitro studies have suggested antiviral effects [60] . t-celldirected immunosuppression performed after organ transplantation (cya, tacrolimus) is being discussed as a possible protective factor against serious clinical complications of sas-cov-2 infection [61] , as well as the use of cya in covid-19 [62, 63] . however, reliable clinical data have not yet been published. possible metabolic interactions between cya and lopinavir/ritonavir (cyp3 inhibitors) have to be taken into account. severe covid-19 courses have been reported in two patients with atopic dermatitis treated with dupilumab [64] . the currently available data suggest that the risk of developing a severe course of covid-19 is probably not increased in patients with allergies and atopy-associated diseases. however, there is a lack of study results including subgroup analyses on seriously ill atopy patients and their treatment. the effects of ige or type 2 inflammation blocking on sars-cov-2 infection have not yet been clarified. in cases of a mild to moderate covid-19 course, we advise to continue biological therapy for the indications mentioned here, if the patient-based assessment of the benefits and risks supports this approach and if the patient agrees after adequate information about the limited availability of data. in severe courses of covid-19, prolongation of the dosing interval or treatment interruption should be considered for the indications discussed here. this assessment should be patient-based and should consider the risk of the possible requirement of systemic glucocorticosteroids. in all other patients, in whom neither a suspected nor a proven sars-cov-2 infection is present, the use of biologicals for the treatment of bronchial asthma, atopic dermatitis, crswnp, and spontaneous urticaria can be continued unchanged or can be re-started in the current sars-cov-2 pandemic. the use of telemedicine for treatment support and patient education is recommended and can facilitate the continuation of biological administration by self-injection. s. korn received speaker and personal adviser's fees from astrazeneca, gsk, novartis, and sanofi outside the submitted work. u. darsow was a lecturer, principal investigator, and consultant for alk abello, bencard, and novartis pharma outside the submitted work. o. palomares received research grants and/ or personal fees from allergy therapeutics, amgen, astrazeneca, diater, glaxosmithkline, s.a., inmunotek s.l., novartis, sanofi-genzyme, and stallergenes outside the submitted work; he was a member of advisory boards of novartis and sanofi-genzyme. t. biedermann was a consultant to or received personal lecture fees or research grants from alk-abelló, celgene-bms, lilly deutschland gmbh, mylan, novartis, phadia-thermo fisher, sanofi-genzyme, regeneron. r. valenta received research grants from viravaxx, vienna, austria, and from hvd life sciences, vienna, austria, and acts as a consultant for viravaxx. r. buhl received personal lecture and/or consultant fees from astrazeneca, boehringer ingelheim, chiesi, cipla, novartis, roche, sanofi, and teva as well as research support for universitätsmedizin mainz from boehringer ingelheim, glaxosmithkline, novartis, and roche, outside the submitted work. r. brehler received personal fees for lectures and/or consultancy and/or clinical studies from alk, allergopharma, almirall, astra zeneca, bencard, gesellschaft zur förderung der dermatologischen forschung und fortbildung e.v., gesellschaft für information und organisation mbh, gsk, dr. pfleger, hal, leti, merck, novartis, oto-rhino-laryngologischer verein, pierre fabre, pohl boskamp, stallergenes, thermo-fischer, biotech tools, genentech, circassia. a. bossios reports personal consultant and/or lecture fees from novartis, astrazeneca, gsk, and teva outside the submitted work. j. bousquet reports personal fees from chiesi, cipla, hikma, menarini, mundipharma, mylan, novartis, sanofi-aventis, takeda, teva, uriach, kyomed-innov, and purina outside the submitted work. j. schwarze received personal fees from mylan, f2f events; support from industry for educational acivities of the scottish allergy and respiratory academy as well as the children and young people's allergy network scotland outside the submitted work; support from industry for eaaci; j. schwarze is eaaci secretary general 2019 -2021. j. hagemann received speaker fees from novartis pharma. m. wagenmann received personal consultant and/or speaker fees from alk-abelló, allergopharma, 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cell line hep3b il-4 enhances interferon production by virus-infected human mast cells preseasonal treatment with either omalizumab or an inhaled corticosteroid boost to prevent fall asthma exacerbations no evidence of increased risk for covid-19 infection in patients treated with dupilumab for atopic dermatitis in a high-epidemic area -bergamo sars-cov-2 immunogenicity at the crossroads. allergy. 2020. epub ahead of print efficacy and safety of treatment with dupilumab for severe asthma: a systematic review of the eaaci guidelines-recommendations on the use of biologicals in severe asthma efficacy and safety of treatment with biologicals (benralizumab, dupilumab, mepolizumab, omalizumab and reslizumab) for severe eosinophilic asthma. a systematic review for the eaaci guidelines -recommendations on the use of biologicals in severe asthma stellungnahme zur anwendung von glukokortikosteroiden bei entzündlichen erkrankungen der oberen atemwege (u. a. allergische rhinitis/chronische rhinosinusitis) während der aktuellen covid-19-pandemie -empfehlungen des ärzteverbandes deutscher allergologen (aeda), des deutschen berufsverbandes der hno-ärzte (bvhno) und der ags klinische immunologie eaaci guidelines on allergen immunotherapy: house dust mite-driven allergic asthma guideline for the diagnosis and treatment of asthma -guideline of the german respiratory society and the german atemwegsliga in cooperation with the paediatric eaaci guidelines on allergen immunotherapy: hymenoptera venom allergy cyclophilins and cyclophilin inhibitors in nidovirus replication immunosuppression drug-related and clinical manifestation of coronavirus disease 2019: a therapeutical hypothesis epub ahead of print cyclosporine therapy during the covid-19 pandemic is not a reason for concern role of cyclophilin a during coronavirus replication and the antiviral activities of its inhibitors safety of dupilumab in severe atopic dermatitis and infection of covid-19: two case reports the trinity of covid-19: immunity, inflammation and intervention considerations on biologicals for patients with allergic disease in times of the covid-19 pandemic: an eaaci statement praxis für kinderpenumologie/allergologie am kinderzentrum dresden (kid) praxis und klinik für dermatologie/allergologie am schwarzwald-baar klinikum germany 60 klinische abteilung für allgemeine hno 74 division of allergy and immunology 82 klinik für dermatologie, allergologie und venerologie medizinische hochschule hannover, 83 hno-klinik key: cord-310205-j57x9ke6 authors: alcaide, maria l.; bisno, alan l. title: pharyngitis and epiglottitis date: 2007-06-08 journal: infect dis clin north am doi: 10.1016/j.idc.2007.03.001 sha: doc_id: 310205 cord_uid: j57x9ke6 acute pharyngitis is one of the most common illnesses for which patients visit primary care physicians. most cases are of viral origin, and with few exceptions these illnesses are both benign and self-limited. the most important bacterial cause is the beta-hemolytic group a streptococcus. there are other uncommon or rare types of pharyngitis. for some of these treatment is required or available, and some may be life threatening. among those discussed in this article are diphtheria, gonorrhea, hiv infection, peritonsillar abscess, and epiglottitis. sore throat accounts for 1% to 2% of all patient visits to office-based primary care practitioners, hospital outpatient departments, and emergency departments in the united states [1] . data from the national medical care survey indicate approximately 7.3 million annual visits for children [2] and 6.7 such visits for adults [3] . thus, familiarity with the principles of diagnosis and management of this disorder is essential for primary care physicians seeing patients of all ages. the hallmarks of acute pharyngitis are sore throat of varying degrees of clinical severity, pharyngeal erythema, and fever. most cases of pharyngitis are of viral origin, and with few exceptions these illnesses are both benign and self-limited. a large proportion of cases of pharyngitis is associated with rhinovirus [4] and coronavirus colds or with influenza. the most important cause of bacterial pharyngitis is the beta-hemolytic group a streptococcus (streptococcus pyogenes, gas). there are other uncommon or rare types of pharyngitis, and for some of these treatment is also required or available. the recognized microbial causes of pharyngitis are listed in table 1 , which shows the syndromes of respiratory illness caused by the various agents [5] [6] [7] [8] [9] [10] . it still is not possible to determine the cause in a sizable proportion of cases. a major task of the primary care physician is to identify those patients with acute pharyngitis who require specific antimicrobial therapy and to avoid unnecessary and potentially deleterious treatment in the great majority who suffer from a benign, self-limited, usually viral infection. in most cases this distinction can be made easily by attention to the epidemiologic setting, history, and physical findings augmented by performance of a few simple and readily available laboratory studies. the results of epidemiologic investigations are influenced by the season of the year, the age of the population, the severity of illness, and the diagnostic methods used to detect cases. most cases of pharyngitis occur during the colder months of the year, during the respiratory disease season. viral agents such as rhinoviruses tend to have annual periods of peak prevalence in the fall and spring; coronaviruses have been found most often in the winter. influenza appears in epidemics, which in the united states usually occur between december and april. in military recruits, adenoviruses cause the syndrome of acute respiratory disease during the colder months. in civilians, acute respiratory disease occurs in the winter, and epidemics of pharyngoconjunctival fever occur in the summer. streptococcal pharyngitis occurs during the respiratory disease season, with peak rates of infection in winter and early spring. spread among family members in the home is a prominent feature of the epidemiologic behavior of most of these agents, with children being the major reservoir of infection [11] . as shown in table 1 , a number of bacteria may cause acute pharyngitis, but most of these are rare or unusual causes of the syndrome. moreover, the benefit of antimicrobial therapy for some of these agents (ie, arcanobacterium haemolyticum, non-group a beta hemolytic streptococci) is unclear. thus, gas is the only commonly occurring cause of sore throat for which antimicrobial therapy is definitely indicated. gas is estimated to be the cause of 15% to 30% of cases of acute tonsillopharyngitis occurring during the cooler months of the year in schoolaged children [12] and of approximately 10% of cases in adults [13, 14] , but there is considerable variability from study to study [15] . nationally, however, approximately 53% of children [2] and 73% of adults [3] who have sore throat receive antimicrobial agents, and a substantial proportion receives antimicrobial agents not recommended as treatments of choice for gas pharyngitis. the following discussion provides recommendations by which such unnecessary and/or inappropriate therapy may be minimized. the characteristic clinical findings are summarized in table 2 . the presence of marked odynophagia, exudative tonsillopharyngitis ( fig. 1) , anterior cervical adenitis, fever, and leukocytosis is highly suggestive of gas pharyngitis. none of the signs and symptoms listed in the table is specific for ''strep throat,'' however. moreover, patients vary widely in the severity of their symptoms. many cases are milder and nonexudative. only approximately half of children presenting with sore throats and positive throat cultures have tonsillar or pharyngeal exudates [12] . patients who have had a tonsillectomy may have milder symptoms. children less than 3 years of age may have coryza and crusting of the nares; exudative pharyngitis caused by gas is rare in this age group. on the other hand, the presence of cough, coryza (in children older than 3 years), hoarseness, diarrhea, conjunctivitis, and/or anterior stomatitis is highly indicative of viral rather than streptococcal infection. scarlet fever results from infection with a streptococcal strain that elaborates streptococcal pyrogenic exotoxins (erythrogenic toxins) to which the patient is not immune. although this disease usually is associated with pharyngeal infections, it may follow streptococcal infections at other sites such as wound infections or puerperal sepsis. nowadays, the clinical syndrome is similar in most respects to that associated with nontoxigenic strains, save for the scarlatinal rash. the latter must be differentiated from those of viral exanthems, drug eruptions, staphylococcal and streptococcal toxic shock syndrome, and kawasaki disease. when confronted with a patient who has acute pharyngitis, the clinician must decide whether the likelihood of gas infection is high enough to warrant a confirmatory diagnostic test. patients lacking the suggestive clinical and epidemiologic findings or manifesting signs and symptoms indicative of viral pharyngitis (see table 2 ) need not be tested or treated with antimicrobial agents. attempts have been made to incorporate clinical and epidemiologic features of acute pharyngitis into scoring systems that attempt to predict the probability that a particular illness is caused by gas [14, [16] [17] [18] [19] . these clinical scoring systems are helpful in identifying patients at such low risk of streptococcal infection that further testing is usually unnecessary. selective use of diagnostic studies for gas will increase the proportion of positive test results and also the percentage of patients with positive tests who are truly infected rather than merely streptococcal carriers. the signs and symptoms of streptococcal and nonstreptococcal pharyngitis overlap too broadly, however, to allow the requisite diagnostic precision on clinical grounds alone. although some have suggested otherwise [1, 20] , empiric antimicrobial treatment based on clinical and epidemiologic grounds alone has been found suboptimal in cost effectiveness [21] and by prospective analyses [18] . therefore, if the clinician is unable to rule out strep throat on clinical and epidemiologic grounds, further testing is required (see later discussion) [22] . a properly performed and interpreted throat culture is the gold standard for the diagnosis of gas pharyngitis. it has a sensitivity of 90% or higher, as judged by studies employing duplicate throat cultures. obtaining definitive results of the throat culture takes 18 to 48 hours. in the minority of patients who are severely ill or toxic at presentation and in whom clinical and epidemiologic evidence leads to a high index of suspicion, oral antimicrobial therapy may be initiated while awaiting the results of the throat culture. if oral therapy is prescribed, the throat culture serves as a guide to the necessity of completion of a full antimicrobial course or, alternatively, of recalling the patient for an injection of penicillin g benzathine. early initiation of antimicrobial therapy results in faster resolution of the signs and symptoms, but two facts should be kept in mind. first, gas pharyngitis usually is a self-limited disease; fever and constitutional symptoms are markedly diminished within 3 or 4 days after onset even without antimicrobial therapy. thus, antimicrobial therapy initiated within the first 48 hours of onset will hasten symptomatic improvement only modestly. second, it has been shown that therapy can be postponed safely up to 9 days after the onset of symptoms and still prevent the occurrence of the major nonsuppurative sequela, acute rheumatic fever [23] . the issues alluded to in the previous discussion may be obviated in part by the use of rapid antigen detection tests (radt), which can confirm the presence of gas carbohydrate antigen on a throat swab in a matter of minutes. currently available commercial test kits yield results that are highly specific for the presence of gas. thus, a positive radt can be considered equivalent to a positive throat culture, and therapy may be initiated without further microbiologic confirmation. unfortunately, the sensitivity of most of these tests ranges between 70% and 90% when compared with the blood agar plate culture [24] . for this reason it is necessary to back up negative radts with a conventional throat culture. one possible exception to the need for such backup relates to adults, in whom the prevalence of gas pharyngitis is relatively low and the risk of a first attack of acute rheumatic fever in north america is minimal [25, 26] . a positive radt or throat culture does not differentiate between the presence of acute streptococcal infection and chronic gas carriage [27] . in the appropriate clinical setting, however, a positive test should be considered as confirmatory of strep throat. follow-up throat cultures are not indicated routinely for asymptomatic patients who have received a complete course of therapy for gas pharyngitis, because most such patients are streptococcal carriers. exceptions include patients who have a history of rheumatic fever, patients who develop acute pharyngitis during outbreaks of either acute rheumatic fever or poststreptococcal acute glomerulonephritis, and outbreaks of gas pharyngitis in closed or semiclosed communities. follow-up throat cultures also may be indicated when ''ping-pong'' spread of gas has been occurring within a family. treatment of gas pharyngitis is recommended to prevent acute rheumatic fever, prevent suppurative complications [28] , shorten the clinical course (although only modestly) [28] , and reduce transmission of the infection in family and school units. there is no definitive evidence that such therapy can prevent acute glomerulonephritis. a number of antibiotics have been shown to be effective in therapy of gas pharyngitis. these include penicillin and its congeners (such as ampicillin and amoxicillin), numerous cephalosporins, macrolides, and clindamycin. penicillin, however, remains the treatment of choice because of its proven efficacy, safety, narrow spectrum, and low cost. amoxicillin often is used in place of oral penicillin v in young children; the efficacy appears equal, and this choice is related primarily to superior palatability of the suspension. erythromycin is a suitable alternative for patients allergic to penicillin, although increases in gas resistance to this agent have been reported in certain localized areas of the united states. first-generation cephalosporins also are acceptable for penicillin-allergic patients who do not manifest immediate-type hypersensitivity to beta-lactam antibiotics. there is debate regarding the relative efficacy of cephalosporins vis-a-vis penicillin [29] in eradicating gas from the pharynx and about the utility of shorter courses of certain antimicrobial agents in treating strep throat. for a further discussion of this topic, the reader is referred to the infectious diseases society of america (idsa) practice guideline [25] . penicillin, however, remains the preferred therapy according to guidelines published by the american heart association [30] , american academy of pediatrics [31] , and idsa (table 3 ) [25] . the idsa guideline also offers guidance in management of patients who have recurrent episodes of culture-or radtpositive acute gas pharyngitis. preliminary investigations have demonstrated that once-daily amoxicillin therapy is effective in the treatment of group a beta hemolytic streptococcus pharyngitis [32] [33] [34] . in the most careful of these studies, feder and colleagues [32] randomly assigned 152 children to receive amoxicillin, 750 mg once daily, or penicillin v, 250 mg three times daily. compliance was monitored by urine antimicrobial activity, and serotyping was performed to distinguish treatment failures from new acquisitions. the two regimens a although shorter courses of azithromycin and some cephalosporins have been reported to be effective for treating group a streptococcal upper respiratory tract infections, evidence is not sufficient to recommend these shorter courses for routine therapy at this time. b amoxicillin often is used in place of oral penicillin v for young children; efficacy seems to be equal. the choice is related primarily to acceptance of the taste of the suspension. c for patients who weigh ! 27 kg. d dose should be determined on basis of the benzathine component. for example, mixtures of 9 â 10 5 u of benzathine penicillin g and 3 â 10 5 u of procaine penicillin g contain less benzathine penicillin g than is recommended for treatment of adolescents or adults. e available as stearate, ethyl succinate, estolate, or base. cholestatic hepatitis, rarely, may occur in patients, primarily adults, receiving erythromycin estolate; the incidence is greater among pregnant women, who should not receive this formulation. f these agents should not be used to treat patients with immediate-type hypersensitivity to beta-lactam antibiotics. were equally effective in eradicating gas from the pharynx. if additional investigations confirm these results, once-daily amoxicillin therapy, because of its low cost and relatively narrow spectrum, could become an alternative regimen for the treatment of group a beta-hemolytic streptococcal pharyngitis. suppurative complications of gas pharyngitis include peritonsillar infection, retropharyngeal abscess, cervical lymphadenitis, mastoiditis, lemierre syndrome, sinusitis, and otitis media. such complications have become relatively rare since the advent of effective chemotherapy. peritonsillar infection may take the form of cellulitis or abscess (quinsy) and is the most common form of deep oropharyngeal infection. although peritonsillar abscesses may occur as complications of gas pharyngitis, the abscesses themselves frequently contain a variety of other oral flora including anaerobes, with or without gas [35] [36] [37] . they occur more frequently in adolescents and adults than in young children. pharyngeal pain is usually severe, and dysphagia is common. on examination, there is inflammation and swelling of the peritonsillar area with medial displacement of the tonsil, and patients speak with a ''hot potato'' voice. trismus may be present. peritonsillar cellulitis may be treated with antibiotics alone, but abscesses require drainage by direct aspiration or by incision and drainage. given the polymicrobial nature of these infections, parenteral antibiotics such as clindamycin, penicillin with metronidazole, or ampicillin-sulbactam are frequently employed. their superiority over penicillin has not been demonstrated definitively, however. lemierre syndrome (postanginal septicemia) is caused by an acute oropharyngeal infection with secondary septic thrombophlebitis of the internal jugular vein and, often, bacteremic spread to lungs or elsewhere [38] . the most common causative organism is fusobacterium necrophorum. the clinical findings include acute presentation with fever, swelling and tenderness at the angle of the jaw, and neck rigidity. dysphagia and dysphonia can occur. the peritonsillar area is inflamed only early in the disease. emergency medical treatment with intravenous antibiotics such as clindamycin, penicillin with metronidazole or ampicillin-sulbactam is usually sufficient, but at times ligation of the internal jugular vein is required. acute rheumatic fever and poststreptococcal acute glomerulonephritis are the delayed nonsuppurative sequelae of gas pharyngitis. although the former occurs only after gas upper respiratory infection, the latter may occur after infection of the throat or skin (streptococcal pyoderma). discussion of these entities is beyond the scope of this article. non-group a beta-hemolytic streptococci are classified biochemically by species. for clinical purposes, however, they usually are identified by their expression of the lancefield cell-wall antigens. certain of these strains clearly are capable of causing acute pharyngitis when ingested in high inocula. streptococci of serogroup g have been linked to common-source outbreaks of pharyngitis, usually related to a food product. one unusual species of group c organisms, streptococcus equi subspecies zooepidemicus, has given rise to common-source epidemics of pharyngitis, usually caused by consumption of unpasteurized dairy products. several of these outbreaks have been associated with poststreptococcal acute glomerulonephritis [39] . groups c and g streptococci are common commensals of the human pharynx. they form large colonies similar to those of gas and belong to the species streptococcus dysgalactiae subspecies equisimilis. physicians who do not back up negative radts with throat cultures will not identify these organisms. the extent to which they cause sporadically occurring episodes of true acute pharyngitis is unclear, but several careful studies suggest that group c streptococci may cause episodes of pharyngitis that mimic gas infection [40, 41] . moreover, a community-wide outbreak of group g streptococcal pharyngitis occurred in connecticut during the winter of 1986-1987 [42] . because of the difficulty in differentiating benign colonization from true infection with groups c or g streptococci, the benefit, if any, of antimicrobial therapy in sporadically occurring cases is unknown. should therapy be elected, agents listed in table 3 would be appropriate, but probably for a shorter duration, because non-group a streptococci never have been shown to cause acute rheumatic fever. arcanobacterium haemolyticum. arcanobacterium (formerly corynebacterium) haemolyticum is a gram-positive bacillus that is a relatively uncommon cause of acute pharyngitis and tonsillitis. symptomatic infection with this organism closely mimics acute streptococcal pharyngitis. rarely, a haemolyticum produces a membranous pharyngitis that can be confused with diphtheria. two features of pharyngeal infection with this organism are notable. it has a predilection for adolescents and young adults, and it frequently provokes a generalized rash that may resemble that of scarlet fever [43] [44] [45] . the organism is detected more readily on human-than sheep-blood agar plates and thus may not be identified in the routine throat culture. thus, the clinician should suspect a haemolyticum infection in a teenager or young adult who has an acute pharyngitis and scarlatiniform rash but a negative culture or radt for gas. should antimicrobial therapy of a patient who has a haemolyticum pharyngitis be elected, a macrolide or, alternatively, a betalactam antimicrobial agent may be prescribed. neisseria gonorrhoeae. neisseria gonorrhoeae is an uncommon, sexually transmitted cause of acute pharyngitis seen in persons who practice receptive oral sex. the risk of acquisition by this means is highest in men who have sex with men (msm). rates of pharyngeal gonorrhea in msm have been reported as being as high as 15%. a recent study in san francisco found n gonorrhoeae in the pharynx of 5.5% of msm and concluded that the pharynx was the most common site of infection in this population [46] . because of this high prevalence, the united states centers for disease control and prevention (cdc) guidelines for sexually transmitted diseases recommend a yearly pharyngeal test for n gonorrhoeae in msm who receive oral intercourse [47] . although the presence of gonorrhea in the pharynx is common, the occurrence of symptomatic pharyngitis is rare. in the abovementioned san francisco study, there was no association between the presence of n gonorrhoeae and pharyngeal symptoms. when symptomatic oral infection does occur, pharyngitis and tonsillitis are the most common manifestations. sore throat with an erythematous pharynx, bilateral tonsillar enlargement, at times with grayish-yellowish exudate, and occasionally with cervical lymphadenopathy have been reported [48] . gingivitis and glossitis also have been described. the diagnosis should be confirmed by culture on thayer-martin medium. currently, ceftriaxone (125 mg in a singular intramuscular dose) is the only cdc recommended therapy for uncomplicated pharyngeal gonorrhea. concomitant therapy for chlamydia is recommended if this infection has not been ruled out. corynebacterium diphtheriae. pharyngeal diphtheria is caused by corynebacterium diphtheriae. humans are the only reservoir of the organism. asymptomatic carriers account for 3% to 5% of the population in endemic areas, and transmission occurs through respiratory secretions. although diphtheria has become extremely rare in the united states and other developed countries with effective childhood immunization programs, there are reasons for the primary care physician and infectious disease specialist to be familiar with this disease. first, there have been a few indigenous cases in the united states in the last 10 years in unimmunized or underimmunized individuals in the lower socioeconomic groups [49] . second, a large proportion of adults in north america and western europe lack protective serum levels of antitoxic immunity and are at risk for acquiring the infection when traveling to endemic areas. in 2003, a fatal case occurred in a pennsylvania resident who traveled to haiti to assist in building a church [50] . epidemics of diphtheria involving thousands of cases occurred in the 1990s among residents of the newly independent countries of the former soviet union. third, early diagnosis and treatment are important predictors of ultimate prognosis. respiratory diphtheria is typically caused by toxin-producing (''toxigenic'') strains of c diphtheriae and rarely by toxigenic strains of corynebacterium ulcerans. the main pathogenic factor is an exotoxin capable of producing a severe local reaction of the respiratory mucosa with the formation of a dense necrotic coagulum and pseudomembranes. these pseudomembranes can lead to airway obstruction resulting in suffocation and death. the toxin is also responsible for severe cardiac and neurologic complications [51] . the incubation period of 2 to 4 days is followed by malaise, sore throat, and low-grade fever. the most notable physical finding is the grayish-brown diphtheritic pseudomembrane that may involve one or both tonsils or may extend widely to involve the nares, uvula, soft palate, pharynx, larynx, and tracheobronchial tree. it is firmly adherent to the mucosa, and its removal provokes bleeding. in severe cases, there is swelling of the soft tissues of the neck (''bull neck''), cervical adenopathy, profound malaise, prostration, and stridor. cardiac complications manifested as myocarditis with cardiac dysfunction occur in 10% to 25% of cases, usually when the pharyngeal manifestations are improving. neurologic complications can occur also and are related directly to the severity of the primary infection, the immunization history, and the time between the onset of symptoms and institution of treatment. the diagnosis of diphtheria requires a high index of suspicion and specific laboratory techniques. diagnosis should be suspected on epidemiologic grounds and in the presence of pharyngitis with pseudomembranes, especially if extending to the uvula and soft palate and bleeding when dislodged. plating on loeffler's or tindale's selective media can identify black colonies with metachromatic granules, but definitive diagnosis requires demonstration of toxin production by immunoprecipitation, polymerase chain reaction (pcr), or immunochromatography. because successful treatment is inversely related to the duration of the disease, therapy should be started once the diagnosis seems likely on clinical grounds and while awaiting laboratory confirmation. treatment of diphtheria includes diphtheria antitoxin and antibiotics. equine diphtheria antitoxin is only available through the national immunization program of the cdc. the therapeutic dose and mode of administration are recommended by the american academy of pediatrics according to the duration and extension of the disease [52] . antibiotics are effective in decreasing local infection, decreasing toxin production, and decreasing spread. intramuscular penicillin g, switched to oral penicillin v once the patient is able to swallow, and erythromycin are the antibiotics of choice. respiratory diphtheria (in contrast to cutaneous diphtheria) does not induce protective immunity, so diphtheria toxoid should be administered to patients during convalescence. prevention of transmission is crucial and is accomplished by strict isolation. close contacts should be cultured and started on prophylactic antibiotics while awaiting culture results; if not fully immunized, they should receive diptheria toxoid. both penicillin and erythromycin are efficacious in eradicating the carrier state, but erythromycin has been shown to be superior in some reports. c ulcerans is an animal pathogen that causes bovine mastitis but can be transmitted to humans through the consumption of raw milk. it can produce diphtheritic toxin and a clinical disease undistinguishable from c diphtheriae. mycoplasma pneumoniae and chlamydophila pneumoniae are known causes of lower respiratory tract infections; they also can be found in the throats of patients who have symptomatic pharyngitis and of asymptomatic carriers. although these agents probably cause some cases of acute pharyngitis, either as primary pathogens or copathogens, the frequency with which this occurs is still unclear. the pharyngeal manifestations that have been described include erythema, tonsillar enlargement, and, less often, exudate with cervical lymphadenopathy. in a recent italian study, 133 children who had acute tonsillopharyngitis were tested for m pneumoniae and c pneumoniae with acute-and convalescent-phase titers and pcr on nasopharyngeal aspirates. thirty-six of the children (27%) had serologically confirmed acute m pneumoniae infection, and 10 (7.5%) had serologically confirmed c pneumoniae infection. five of the latter also had a positive nasopharyngeal pcr for this organism [53] . the children were assigned randomly to receive azithromycin plus symptomatic treatment or symptomatic treatment alone and were followed for 6 months. in the short term, there was no difference in the outcomes of children with or without atypical infection. a significantly decreased rate of recurrent upper and lower respiratory infections occurred in patients with atypical infections who were randomized to azithromycin, however. these results require confirmation. infectious mononucleosis clinical manifestations. infectious mononucleosis (im) or ''glandular fever'' is caused by the epstein-barr virus (ebv). the virus is present in the oropharyngeal secretions of patients who have im and is spread by personto-person contact. infection with ebv is frequent in childhood but usually is asymptomatic. clinical manifestations are more common when the infection is acquired in adolescence or young adulthood. thus, most cases of im occur between ages 15 and 24 years. symptoms develop after an incubation period of 4 to 7 weeks. following a 2-to 5-day prodromal period of chills, sweats, feverishness, and malaise, the disease presents with the classic triad of severe sore throat accompanied by fever as high as 38 c to 40 c and lymphadenopathy. pharyngitis with associated tonsillitis occurs in 70% to 90% of the patients. tonsillar exudates are present in approximately one third of cases, and palatal petechiae may also be present lymphadenopathy is bilateral, particularly posterior cervical, but can involve axillary and inguinal areas. about 10% of patients have a rash of variable morphology, but administration of ampicillin or amoxicillin provokes a pruritic maculopapular eruption in 90% of patients. hepatomegaly is present in 10% to 15% of patients who have im, and splenomegaly occurs in almost half of the patients. this classic clinical presentation of im occurs in most of the children and young adults. older adults may not exhibit pharyngitis or lymphadenopathy, and disease can be manifested only with fever and more prominent hepatic abnormalities (typhoidal presentation) [54] . ebv infection can be complicated by a variety of neurologic and oncologic conditions that are beyond the scope of this article. the hematologic findings include leukocytosis with 60% to 70% lymphocytosis and thrombocytopenia that usually is mild but occasionally may be severe. the lymphocytosis usually is found at presentation and peaks 2 to 3 weeks after onset of the disease. the presence of more than 10% atypical lymphocytes in peripheral blood is one of the characteristic features of im and supports the diagnosis. the differential diagnosis at initial presentation includes gas pharyngitis, other respiratory viral infections (see table 1 ), cytomegalovirus infection, and, if suggested by epidemiologic history, the acute retroviral syndrome (see later discussion). rarely, entities such as toxoplasmosis, hepatitis a, human herpesvirus 6, and rubella must be considered. in most cases, the diagnosis is readily confirmed if suspected. initial diagnostic studies should include throat culture or radt for gas and a serologic test for the presence of heterophile antibodies. the latter are antibodies directed against antigens in erythrocytes from different animal species. they are present in approximately 90% of affected adolescents and adults within the first 1 to 3 weeks of illness and may persist for up to 1 year. spot and slide tests that use horse or purified bovine erythrocytes and allow rapid screening are commercially available. when combined with a compatible clinical presentation, a positive rapid test for heterophile antibodies can be considered diagnostic of im. false-negative tests can occur in up to 10% of patients, however, especially in children and older adults and in the early stages of the disease. approximately 10% of patients who have a classic mononucleosis syndrome have negative tests for heterophile antibodies. in such patients, ebv-specific antibodies should be assayed. the most useful of these for general clinical purposes is the igm antibody to viral capsid antigen. this test is present at clinical presentation and persists for 4 to 8 weeks. antibody to epstein-barr nuclear antigen first appears 3 to 4 weeks after onset and persists for life [55] . many patients who have heterophile-negative im are found by the aforementioned tests to be infected with ebv. in the majority of the remainder, serologic studies confirm infection with cytomegalovirus, which can produce a syndrome closely mimicking that induced by ebv. im is predominantly a self-limited disease, and studies have failed to detect any benefit of using antiviral agents. most symptoms resolve within 3 weeks of onset. physical activity is tailored to patient tolerance. because of the risk of splenic rupture, contact sports and heavy lifting should be avoided until the spleen returns to normal size, usually in approximately 3 to 4 weeks. the use of corticosteroids has been studied in clinical trials, but no clear benefit has been demonstrated [56, 57] . steroids, however, may be useful in the management of severe complications such as airway obstruction, hemolytic anemia, severe thrombocytopenia, and aplastic anemia. within days to weeks after initial infection with hiv type 1, 50% to 90% of patients develop a constellation of symptoms known as the ''acute retroviral syndrome.'' fever, sore throat, lymphadenopathy, maculopapular rash, myalgia, arthralgias, and mucocutaneous ulcerations are the landmarks of the syndrome [58] [59] [60] [61] . a nonexudative pharyngitis is present in 50% to 70% of patients. other oropharyngeal findings include ulcers and thrush. oral ulcers, which occur in 10% to 20% of the patients, appear in the first days of the illness and last for approximately 1 week. their distribution is usually in the inner lips and in the floor of the mouth, but tonsils, soft palate, and uvula also can be involved. fever occurs in almost 100% of patients who have acute retroviral syndrome, and it is usually high. lymphadenopathy occurs in 40% to 70% of the patients, is nontender, usually develops after 1 week of illness, and involves cervical, axillary, and inguinal regions. skin rash, which occurs in 40% to 50% of patients, usually is maculopapular, disseminated, and almost invariably involves the neck and upper trunk. the rash usually spares the distal extremities, although palms and soles can be affected. the clinical findings of fever, pharyngitis, and lymphadenopathy may simulate im. atypical lymphocytosis, although infrequent, also could lead to a misdiagnosis of mononucleosis. acute retroviral syndrome can be differentiated from mononucleosis, however, by its more acute onset, the absence of exudate or prominent tonsillar hypertrophy, the frequent occurrence of rash (rare in mononucleosis except after treatment with ampicillin or amoxicillin), and the presence of oral ulcerations (table 4 ) [62] . it is important for the clinician to recognize that the elisa commonly used to diagnose hiv-1 infection are negative in the first 3 to 4 weeks after infection and therefore are not useful in this setting. tests for p24 antigen or, preferably, quantitative assays for plasma hiv rna by branched chain dna or pcr should be performed. the viral load can be anticipated to be very high during this acute phase of infection. an hiv antibody test always should be performed later in time to confirm the diagnosis. treatment of acute retroviral syndrome with highly active antiretroviral medication has been controversial, current recommendations consider the use of antiretroviral medications in the setting of acute hiv infection to be optional [63] . potential benefits of antiretroviral therapy in acute hiv would be to decrease the severity of acute disease and to decrease viral replication. studies have demonstrated improvement of laboratory markers of disease progression when highly active antiretroviral therapy is used in acute hiv infection [64, 65] . these results would suggest a decrease in progression and transmission of the disease. treating acute hiv infection, however, also can have potential risks, including known side effects to medications, possible development of resistance, and adverse effects on quality of life. if the patient and clinician elect to start antiretroviral medications, the goal should be suppression of viral replication. whereas most respiratory viruses can cause symptoms indistinguishable from the common cold or acute pharyngitis, some viruses may produce more distinctive clinical syndromes. adenovirus. adenovirus is a common cause of viral pharyngitis. it is manifested clinically as an upper respiratory infection with fever, cough, rhinorrhea, and sore throat, usually more pronounced than in the common cold. the pharynx is erythematous and frequently may have exudates that mimic streptococcal pharyngitis [66] . a distinctive syndrome associated with adenovirus infection in children is pharyngoconjunctival fever. the disease occurs in outbreaks and is characterized by conjunctivitis, pharyngitis, rhinitis, cervical adenitis, and high fever. although adenoviral infections commonly occur in winter months, pharyngoconjuntival fever has been implicated in outbreaks in summer camps [67] and associated with contaminated swimming pools and ponds. several types of adenovirus also have been implicated in outbreaks of influenza-like illnesses with sore throat, rhinorrhea, and tracheobronchitis, known as acute respiratory disease of army recruits [68, 69] . these infections are self limited, and symptomatic treatment alone is recommended. coxsackie virus. most enteroviral infections occur in the summer and fall and present as febrile illnesses with sore throat, cough, or coryza. distinctive manifestations of enteroviral infection are herpangina and hand-footand-mouth disease. herpangina most often is caused by coxsackie a and is most frequent in infants or young children. it usually presents acutely with fever, sore throat, odynophagia, diffuse pharyngeal erythema, and a vesicular enanthem. headache and vomiting can be preceding symptoms. the oral lesions consist of 1-to 2-mm gray-white papulovesicles that progress to ulcers on an erythematous base and may be present on the soft palate, uvula, and anterior tonsillar pillars [70] . they are moderately painful and usually number less than a dozen. hand-foot-and-mouth disease also is caused by coxsackie a. it is characterized by a febrile vesicular stomatitis with associated exanthema. the oral findings include small, painful vesicles in the buccal mucosa and tongue that can coalesce and form ulcerative bullae. the lesions are similar to those seen in herpangina, but there is an associated peripheral rash involving hands and feet that can extend proximally. these coxsackie diseases are self limited, and treatment is symptomatic. herpes simplex virus. several studies have documented primary human herpes simplex (hsv) type 1 infection as a cause of pharyngitis in college students [71, 72] . hsv2 occasionally can cause a similar illness as a consequence of oral-genital contact [73] . this form of pharyngitis represents primary infection in immunocompetent patients but can occur as a reactivation of latent virus in immunocompromised hosts. although the characteristic presentation consists of small vesicles and ulcerations in the posterior pharynx and tonsils, hsv also may produce pharyngeal erythema and exudates at times indistinguishable from strep throat. lesions may extend to the palate, gingiva, tongue, lip, and face. general symptoms include fever, malaise, inability to eat, and cervical lymphadenopathy. treatment of hsv oral infection with antiherpetic medication is efficacious in reducing the duration of signs and symptoms as well as viral shedding. acyclovir, valcyclovir, and famciclovir are all useful in treating hsv infection. acetaminophen or nonsteroidal anti-inflammatory drugs are effective analgesics and antipyretics. treatment should be started as soon as symptoms develop and is useful in both viral and bacterial diseases. oral hydration and gargles with salt water may help alleviate the pharyngeal complaints. oral cough suppressants, decongestants, and antihistamines are helpful, depending on the symptoms present. lozenges containing local anesthetics are widely available over the counter and seem to provide temporary relief of sore throat [74, 75] . several authors have reported that adjuvant therapy with dexamethasone decreases the duration of throat pain in patients who have severe odynophagia [76] [77] [78] . the regimens used and the magnitude of the effect varied among the studies. no adverse effects have been reported, but duration of follow-up often has been limited. one randomized, double-blind, placebo-controlled trial of adjuvant dexamethasone therapy in children found efficacy only in patients who had radt-positive pharyngitis, and the benefit was judged to be only ''of marginal clinical importance'' [79] . the authors do not recommend use of corticosteroids in the therapy of gas pharyngitis. acute epiglottitis or supraglottitis is an inflammatory process of the epiglottis and adjacent structures that can lead to life-threatening acute respiratory obstruction. in the past, epiglottitis occurred most frequently in children between 2 and 4 years of age and was associated mainly with haemophilus influenzae type b (hib) infection. since the initiation of the childhood vaccination programs, epiglottitis caused by this organism is much less common. nevertheless, there are still cases of hib epiglottitis in both immunized and nonimmunized children, and the possibility of an infection with this pathogen cannot be excluded completely in vaccinated patients [80, 81] . bacteria associated with epiglottitis nowadays include streptococcus pneumoniae, staphylococcus aureus, and beta hemolytic streptococci. multiple agents including other bacteria, viruses, and fungi have been implicated in rare cases. the typical presentation in children includes fever, irritability, sore throat, and rapidly progressive stridor with respiratory distress. the affected child adopts a forward-leaning position, drooling oral secretions while trying to breathe. adults usually present with sore throat and a milder disease, although airway compromise can occur also. physical examination of patients suspected of having epiglottitis requires careful inspection of the oropharyngeal and suprapharyngeal area. the diagnosis requires direct visualization of an erythematous and swollen epiglottis under laryngoscopy. because of the risk of airway obstruction, this procedure should be performed in children only when skilled personnel and equipment to secure the airway are available [82] . once the airway has been secured, culture of the surface of the epiglottis along with blood cultures should be obtained to guide antibiotic therapy. management focuses on two important aspects: close monitoring of the airway with intubation if necessary and treatment with intravenous antibiotics. because of the aforementioned possibility of failure of vaccination, antibiotics should be directed against hib in every patient regardless of immunization status. cefotaxime, ceftriaxone, or ampicillin/sulbactam are appropriate choices. steroids are used commonly in the management of acute epiglottitis although no randomized trial has been done to support this practice. when a case of hib epiglottitis is diagnosed, the american academy of pediatrics recommends that postexposure prophylaxis with rifampin be given to household contacts when there is at least one child in the household younger than 4 years of age, a child in the household younger than 12 months of age who has not received the primary series of hib vaccine, or an immunosuppressed child, regardless of that child's hib immunization status [83] . acute pharyngitis is an extremely common disorder that usually runs a benign course. in almost all cases, the primary care physician must discriminate between a viral sore throat, which requires only symptomatic management, and gas pharyngitis, which requires specific antimicrobial therapy. this distinction is important so that gas pharyngitis can be treated appropriately to minimize the risk of suppurative and nonsuppurative complications. equally important is minimizing unnecessary and potentially deleterious overtreatment of viral infections with antimicrobial agents. this article has outlined the epidemiologic, clinical, and laboratory findings that assist in decision making. the clinician also must be alert to the occurrence of rare but serious upper respiratory infections that may be life threatening and require special forms of therapy (eg, diphtheria, parapharyngeal suppurative processes, acute epiglottitis). principles of appropriate antibiotic use of acute pharyngitis in adults antibiotic treatment of children with sore throat antibiotic treatment of adults with sore throat by community primary care physicians: a national survey clinical significance and pathogenesis of viral respiratory infections a collaborative study of the aetiology of acute respiratory infection in britain 1961-4. a report of the medical research council working party on acute respiratory virus infections virology of middle ear virologic studies of acute respiratory disease in young adults. iv. virus isolations during four years of surveillance acute respiratory illness in an american community. the tecumseh study acute pharyngitis and tonsillitis in university of wisconsin students group a streptococci, mycoplasmas, and viruses associated with acute pharyngitis principles and practice of infectious diseases diagnosis of streptococcal pharyngitis: differentiation of active infection from the carrier state in the symptomatic child a clinical score to reduce unnecessary antibiotic use in patients with sore throat the prediction of streptococcal pharyngitis in adults group a streptococcal pharyngitis in adults the diagnosis of strep throat in adults in the emergency room a streptococcal score card revisited empirical validation of guidelines for the management of pharyngitis in children and adults streptococcal pharyngitis: evaluation of clinical syndromes in diagnosis principles of appropriate antibiotic use for acute pharyngitis in adults: background a cost-effectiveness analysis of diagnosis and management of adults with pharyngitis diagnosing strep throat in the adult patient: do clinical criteria really suffice? the role of streptococcus in the pathogenesis of rheumatic fever rapid diagnosis of pharyngitis caused by group a streptococci practice guidelines for the diagnosis and management of group a streptococcal pharyngitis management of acute pharyngitis in adults: reliability of rapid streptococcal tests and clinical findings treatment failures and carriers: perception or problems? antibiotics for sore throat are cephalosporins superior to penicillin for treatment of acute streptococcal pharyngitis? treatment of acute streptococcal pharyngitis and prevention of rheumatic fever: a statement for health professionals. committee on rheumatic fever, endocarditis, and kawasaki disease of the council on cardiovascular disease in the young, the american heart association committee on infectious diseases. group a streptococcal infections once-daily therapy for streptococcal pharyngitis with amoxicillin randomized, single-blinded comparative study of the efficacy of amoxicillin (40 mg/kg/day) versus standard-dose penicillin v in the treatment of group a streptococcal pharyngitis in children treatment of streptococcal pharyngitis with amoxycillin once a day microbiology and management of peritonsillar, retropharyngeal, and parapharyngeal abscesses peritonsillitis: abscess of cellulitis? the microbiology of peritonsillar sepsis the evolution of lemierre syndrome: report of 2 cases and review of the literature group c and group g streptococcal infections: epidemiologic and clinical aspects epidemiologic evidence for lancefield group c beta-hemolytic streptococci as a cause of exudative pharyngitis in college students clinical and microbiological evidence for endemic pharyngitis among adults due to group c streptococci community-wide outbreak of group g streptococcal pharyngitis arcanobacterium haemolyticum in children with presumed streptococcal pharyngotonsillitis or scarlet fever incidence and pathogenicity of arcanobacterium haemolyticum during a 2-year study in ottawa corynebacterium hemolyticum as a cause of pharyngitis and scarlatiniform rash in young adults prevalence and incidence of pharyngeal gonorrhea in a longitudinal sample of men who have sex with men: the explore study sexually transmitted diseases treatment guidelines gonococcal tonsillar infection-a case report and literature review status report on the childhood immunization initiative: reported cases of selected vaccine-preventable diseases-united states fatal respiratory diphtheria in a u.s. traveler to haiti-pennsylvania principles and practice of infectious diseases red book: 2006 report of the committee on infectious diseases. elk grove (il): american academy of pediatrics acute tonsillopharyngitis associated with atypical bacterial infection in children: natural history and impact of macrolide therapy infectious mononucleosis in middle age epstein-barr virus (infectious mononucleosis) epstein-barr virus infections: biology, pathogenesis, and management steroids for symptom control in infectious mononucleosis acute human immunodeficiency virus type 1 infection acute aids retrovirus infection. definition of a clinical illness associated with seroconversion primary hiv infection clinical presentations and virologic characteristics of primary human immunodeficiency virus type-1 infection in a university hospital in taiwan clinical picture of hiv infection presenting as a glandular-fever-like illness is antiretroviral treatment of primary hiv infection clinically justified on the basis of current evidence? highly active antiretroviral treatment initiated early in the course of symptomatic primary hiv-1 infection: results of the anrs 053 trial effect of combination antiretroviral therapy on t-cell immunity in acute human immunodeficiency virus type 1 infection clinical presentation and characteristics of pharyngeal adenovirus infections outbreak of pharyngoconjunctival fever at a summer camp-north carolina large, persistent epidemic of adenovirus type 4-associated acute respiratory disease in u.s. army trainees large epidemic of adenovirus type 4 infection among military trainees: epidemiological, clinical, and laboratory studies viral exanthems pharyngitis associated with herpes simplex virus in college students acute respiratory disease of university students with special reference to the etiologic role of herpesvirus hominis acute pharyngotonsillitis caused by herpesvirus type 2 efficacy and tolerability of ambroxol hydrochloride lozenges in sore throat. randomised, double-blind, placebo-controlled trials regarding the local anaesthetic properties demonstration of dose response of flurbiprofen lozenges with the sore throat pain model efficacy of single-dose dexamethasone as adjuvant therapy for acute pharyngitis effectiveness of oral dexamethasone in the treatment of moderate to severe pharyngitis in children a pilot study of 1 versus 3 days of dexamethasone as add-on therapy in children with streptococcal pharyngitis oral dexamethasone for the treatment of pain in children with acute pharyngitis: a randomized, double-blind, placebo-controlled trial epiglottitis and haemophilus influenzae immunization: the pittsburgh experience-a five-year review paediatric acute epiglottitis: not a disappearing entity airway infectious disease emergencies red book: 2006 report of the committee on infectious diseases key: cord-317028-f3bpwm5j authors: olmsted, russell n. title: prevention by design: construction and renovation of health care facilities for patient safety and infection prevention date: 2016-08-09 journal: infect dis clin north am doi: 10.1016/j.idc.2016.04.005 sha: doc_id: 317028 cord_uid: f3bpwm5j the built environment supports the safe care of patients in health care facilities. infection preventionists and health care epidemiologists have expertise in prevention and control of health care–associated infections (hais) and assist with designing and constructing facilities to prevent hais. however, design elements are often missing from initial concepts. in addition, there is a large body of evidence that implicates construction and renovation as being associated with clusters of hais, many of which are life threatening for select patient populations. this article summarizes known risks and prevention strategies within a framework for patient safety. the built environment encompasses a broad range of physical design elements, including spaces for care of patients, support services, electronics, and major technical equipment; building systems that provide air and water; and surfaces and finishes. this spectrum of spaces and surfaces collectively is referred as the environment of care (eoc). in general, these are less frequently a source of microorganisms causing health care-associated infection (hai) compared with other sources, such as the patient's endogenous microflora, especially when an invasive device is present, or a surgical procedure. 2 carriage of microbes on hands of health care personnel (hcp) also is a more likely mechanism of exposure to potential pathogens. even so, the proportional contribution of the eoc as a reservoir of pathogens is estimated at 20%. 3 over the past several years there have been several studies showing that the eoc is a significant source of multidrug-resistant organisms (mdros), clostridium difficile, and norovirus. 4 in addition, investigation of the role of the eoc has found that admission to a patient room previously occupied by a patient with an mdro or c difficile is a risk factor for their acquisition by the next occupant. 5 specific pathogens can suggest an environmental source; for example, from demolition of drywall or gaps in maintenance of key mechanical systems, which include aspergillus spp, fusarium spp, rhizopus spp, bacillus cereus, legionella spp, a wide range of gram-negative bacteria, and nontuberculous mycobacteria. 2 when hais are caused by opportunistic pathogens it is important to apply key principles such as chain of transmission and the following criteria to determine whether reservoirs are present in the environment and to help guide implementation of mitigation strategies, if applicable. 1. the organism can survive after inoculation onto the fomite. 2 2. the organism can be cultured from in-use fomites. 3 . the organism can proliferate in or on the fomite. 4 . some measure of acquisition of infection cannot be explained by other recognized modes of transmission. 5. retrospective case-control studies show an association between exposure to the fomite and infection. 6. prospective case-control studies may be possible when more than 1 similar type of fomite is in use. 7. prospective studies allocating exposure to the fomite to a subset of patients show an association between exposure and infection. 8. decontamination of the fomite results in the elimination of infection transmission. annual spend on construction or renovation of health care facilities is approximately $40 billion. 6 because cost of construction per square meter ranges from $4300 to $12,920, there has been some modulation in the build of larger inpatient rooms. 7 a large proportion of current construction projects therefore involve a shift toward olmsted construction of outpatient facilities. a recent survey of providers found that the types of outpatient projects include ambulatory surgery centers (48%), freestanding imaging (23%), health system-branded clinics in retail space (23%), health system-branded general medicine and family care in the community (53%), immediate care facilities (49%), medical office buildings (60%), and telehealth (23%). 7 this finding reflects the general direction toward home-based and ambulatory-based care delivery with an emphasis on population health and value-based purchasing. disturbance of the eoc, especially from construction, renovation, or remediation, can result in exposure of patients and personnel to microorganisms present in air, water, or on surfaces. other maintenance activities, such as repair and remediation work (eg, installing wiring for new information systems, removing old sinks, and repairing elevator shafts) can also disrupt and release contaminants. aging equipment, deferred maintenance, and natural disasters provide additional mechanisms for the entry of environmental pathogens into high-risk patient-care areas. to mitigate contamination of patient-care areas several infection preventionists developed the use of infection control risk assessment (icra). 8 icra is a process that begins during planning and design of construction and renovation to ensure that elements of infection prevention are incorporated into the project. it includes strategies such as physical barriers to contain and confine dust, soil, and contaminants (eg, fungal spores) that may be released into the air during demolition, once construction begins. use of an icra has been incorporated into design standards as well as healthcare infection control practices advisory committee guidelines that address construction and renovation. although the percentage of hais directly related to construction is unknown, the morbidity, mortality, and costs of mitigation of these preventable infections are considerable. the mechanism of exposure of patients to airborne pathogens during construction is often from disturbance of building materials or surfaces that have been contaminated; for example, intrusion of water onto drywall substrate where fungal spores are present. demolition of these substrates releases bursts of spores into the air and, if not contained and removed, can result in exposure of occupants. vonberg and gastmeier 9 reviewed outbreaks of infection caused by aspergillus spp and found that almost half were associated with construction or renovation in hospitals. in addition, they identified that the infective dose of invasive pulmonary aspergillosis in immunocompromised patients can be as low as 1 colony forming unit/m 3 . this finding highlights the critical need for isolation and containment of construction activities from other occupied spaces. patient populations at increased risk of fungal infection and that are exposed to contaminated airborne spores include those undergoing hematopoietic stem cell or solid organ transplant, undergoing chemotherapy for conditions such as leukemia, in receipt of high-dose steroid therapy, the critically ill, neonates, those undergoing cardiac surgery, and those with chronic lung disease. kanamori and colleagues 10 recently reevaluated disease outbreaks from airborne fungal spores associated with construction. they found it encouraging that since 2010 there have been fewer reports of outbreaks. although not necessarily a direct causal relationship, this points to the efficacy of icra in mitigating transmission and protecting patients, personnel, and visitors in facilities that have ongoing construction or renovation. their search of the literature found 28 construction-associated outbreaks between 1976 and 2014, aspergillus spp being the most common pathogen, and a predominance of pulmonary infection with attributable mortality approaching 60%. the spectrum of microorganisms present in water is broad and includes gramnegative bacteria (eg, legionellae and pseudomonas spp), nontuberculous mycobacteria, protozoa, and fungi. 2 potable water provided by municipal water authorities must meet federal standards for drinking water and these are enforced by the us environmental protection agency. 11 water supplied to the health care facility is then distributed through an extensive network of plumbing to fixtures such as handwashing stations, ice machines, medical equipment (eg, automated endoscope reprocessors), and utility systems. this distribution network readily supports development of biofilm, and the microbial contaminants embedded in this matrix of extracellular polymeric substances, mainly composed of exopolysaccharides, proteins, and nucleic acids, protects microorganisms from disinfectants that are otherwise effective against planktonic forms. 12 stagnant water in this network, often from renovation of areas in the facility that has resulted in redundant lengths of pipework that are left in place and capped, also enhances development of biofilm. in addition, disruption of water utility systems during construction or renovation can disrupt biofilm and release contaminants into the water delivery network, posing a possible risk to patients, including those far away from the work area. a recent, extensive review of waterborne disease outbreaks found that the more susceptible patient populations, such as the critically ill, neonates, transplant recipients, surgical patients, and those with hematological disease, are often the sentinel signal of a new cluster. 13 of late the types of devices and architectural features that were a source of infections are growing in complexity. this article discusses these outbreak investigations and emphasizes the need to be vigilant for their detection and mitigation. .waterborne healthcare-associated outbreaks and infections continue to occur and were mostly associated with well-recognized water reservoirs as previously described. moreover, recent studies document electronic faucets (p. aeruginosa, legionella, m. mucogenicum), decorative water wall fountains (legionella), and heater-cooler devices used for cardiac surgery (m. chimaera) as water reservoirs. 13 there are some landmark investigations of waterborne disease outbreaks worth highlighting because these investigations have informed guidelines for construction and renovation in the united states. hand hygiene is the foundation of infection prevention and control. the 2 primary methods hcp use to clean their hands are alcohol-based hand rub (abhr) or soap and water and a handwashing station. hota and colleagues 14 reported an outbreak of pseudomonas aeruginosa infections that, ironically, centered on handwashing stations in intensive care units (icus) and transplant units. the outbreak unit featured single-occupancy patient rooms with convenient access to a handwashing station near the entrance in each for use by hcp. biofilm within the drains of these were identified as the source and the sink design included a shallow basin with the faucet spout directly over the drain. this arrangement resulted in splashes of water contaminated olmsted with p aeruginosa out of the drain contaminating surfaces near the sink, including a medication preparation area and the patient bed. interventions to mitigate contamination from sinks included a physical barrier between the sink and adjacent countertops, offset of the faucet spout so it did not discharge directly into the drain, and lower water pressure. the lessons from this outbreak have been incorporated into guidelines published by the facility guidelines institute (fgi). 15 key design features in current fgi guidelines include: basins that reduce the risk of splashing and are made of porcelain, stainless steel, or other solid surface material basin size of no less than 929.08 cm 2 (144 square inches) with 22.86 cm 2 (9 square inches) width or length sealed to prevent water intrusion into supporting cabinet, wall, and countertop discharge of water from faucet spout is at least 25.4 cm (10 inches) above the bottom of the basin and avoids dropping directly into the drain water pressure in station fixture is regulated allows controls for sink fixture to be wrist blade, single lever, or sensor activated water feature: not allowed decorative water features have been a popular element of design. however, there have been 2 recent outbreaks of legionnaires' disease associated with these. the first involved 2 patients with extended hospital stay preparing for stem cell transplant for treatment of leukemia. 16 investigation identified a decorative water fountain as the source; of note, testing of water identified a diverse range of other microorganisms in addition to legionella pneumophila serogroup 1. this fountain had been turned off for several months and the investigators commented that stagnation in the water circulation conduits likely promoted development of biofilm. haupt and colleagues 17 investigated a cluster of legionnaires' disease associated with a decorative water wall that was installed in a public corridor near the main entrance lobby of a community hospital. eight cases were detected and the only common risk factor was visiting the hospital where this feature was in use; most patients simply walked through the lobby past the water wall. l pneumophila serogroup 1 was detected from the water in this fountain despite adherence with the manufacturer's instructions for cleaning and maintenance. this incident and other evidence has led the fgi to state that, "unsealed, open water features are not permitted." 15 the microbiome of the inpatient room has undergone renewed appreciation following a considerable body of evidence that finds significant risk of acquisition of pathogens such as mdros or c difficile related to infection or colonization in the room's prior occupant, for as long as 3 weeks. 5 however, this contamination can be removed with attention and focus on thorough cleaning and disinfection of surfaces in rooms that are touched with high frequency, combined with real-time feedback. 18, 19 the evidence that mdros can persist in the environment for a prolonged time, as described earlier, in combination with the observed efficiency of cross-transmission of these in multibed rooms, has led to a preference for single-patient rooms. this design also enhances safety related to a variety of other potential harms, supports patient privacy, and lessens disruption from ambient noise. 20 by contrast, the lack of spatial separation between patients in multibed rooms or wards has been associated with increased risk of respiratory viral infections and bacterial infection when patient with similar devices are in the same room. 21, 22 other investigators identified a temporal association between fewer bloodstream infections, detection of mdros, and prevalence of antibiotic resistance with redesign of patient-care units from open wards to single-patient rooms. 23 studies of cross-transmission of microorganisms in health care facilities have identified that surfaces nearer to patients are more likely to be contaminated. further, patients with acute infection, especially when symptoms result in contamination of the immediate environment with body fluids containing the pathogen (eg, diarrhea), result in higher microbial burden on environmental surfaces. 5 this finding is particularly true of norovirus, for which studies find that person-to-person transmission depends on close or direct contact as well as short-range aerosol exposures. also, experience with control of outbreaks of norovirus highlight the need to clean and disinfect frequently touched surfaces (eg, patient and staff bathrooms, utility rooms, tables, chairs, commodes, computer keyboards and mice, and items in close proximity to symptomatic patients). 24 the fgi guidelines serve as a foundational resource for the design of health care facilities. 15 they are used as a basis for regulation and a national standard in 42 states as well as being cited by the joint commission, the department of housing and urban development, and the indian health service as normative, national standards. the guidelines are consensus-based and developed by the health guidelines revision committee and are updated every 4 years. the 2014 guidelines include 2 separate standards, one for hospitals and outpatients and the other for residential health, care, and support facilities. importantly, they provide minimum design standards; not necessarily parameters that involve daily operations of facilities. many of the elements discussed later are addressed in these guidelines and readers are referred to this resource for more details. icra is the core framework of design and construction/renovation. it is a component of the overall safety risk assessment called for in the fgi 2014 guidelines. icra calls for design recommendations and infection control risk mitigation recommendations (icrmr) that are applied to the construction project being planned. key aspects that icra needs to address include: design elements that support infection prevention and control proactive planning for mitigating sources of infection both within and external to the construction project that will be affected identify potential risk for transmission of airborne and waterborne pathogens during construction, renovation, and commissioning develop icrmrs to mitigate identified risks (see appendix a for a stepwise approach to developing icrmrs) there is evidence that an effective icra process can prevent hais. 25 fig. 1 provides examples of effective containment methods. heating, ventilation, and air conditioning heating, ventilation, and air conditioning (hvac) is a building system that is designed to provide comfort, support aseptic procedures, remove contaminants from air, and deliver an acceptable indoor air quality. fgi 2014 includes the american society of heating, refrigerating, and air-conditioning engineers (ashrae) 170 standard for design of hvac for health care facilities. this standard provides a wide range of parameters for hvac systems that supply patient-care, procedural (eg, surgery suite), and support areas. parameters included in ashrae 170 include air changes per hour, design temperature and relative humidity ranges, and pressure relationships to adjacent areas. 26 universal or acuity-adaptable and single-occupancy patient-care rooms the fgi commissioned a systematic review of available evidence on the value of singlepatient rooms 20 that found suggestive, albeit low-quality, evidence that this prevents infection and improves overall patient safety and experience of care. the addition of adaptability of these based on the patient's need also is worth considering. additional elements for adult icus have been described elsewhere and support this need for flexibility to accommodate changes in care practices and advances in technology. 27 planning for airborne-infection isolation rooms (aiirs) should be based on the local epidemiology and risk assessment for the prototype airborne disease, tuberculosis. other diseases in which aiirs are used include chickenpox and measles, and experience with these can also help inform on location and number of aiirs for a facility planning team as part of icra. there are also procedures that increase the risk of transmission if performed on someone with active pulmonary tuberculosis (eg, bronchoscopy), and these need to be considered when identifying the optimal number of aiirs. more recently, emerging and remerging diseases, such as ebola and middle east respiratory syndrome coronavirus, have highlighted the need to plan and respond as appropriate, from frontline facilities that receive patients to regional and national emergency preparedness and response. details of regional facilities designed for definitive treatment of patients with infections such as ebola are described elsewhere. 28 protective environment room a protective environment (pe) room is designed to provide a filtered supply of highefficiency particulate air (hepa) to rooms used to care for patients who are severely immunocompromised (eg, solid organ transplant patients or allogeneic neutropenic patients). these rooms need to be designed to ensure that rooms are well sealed by maintaining ceilings that are smooth and free of fissures, open joints, and crevices; sealing walls above and below the ceiling; and, once occupied, to monitor for leakage. additional details are available elsewhere. 2 recommendations for quality processes to ensure protection of immunocompromised patients during construction have been published. 29 design features were identified earlier in the review of water as a reservoir. reliable, readily available access to devices and products to use for hand hygiene is the foundation of infection prevention and control. the fgi guidelines include both handwashing stations and proactive planning for placement of dispensers of abhr that are readily visible to hcp. the move to single-patient rooms has resulted in most rooms having an attached bathroom/shower for use by the patient. for critically ill patients, the ability to use the bathroom is less likely; the exception might be a cardiac icu. swing-out or folddown fixtures (swivette toilets) should be avoided, because they are prone to mechanical problems and leakage, are difficult to use (especially for the acutely ill), and may not be rated for the bariatric patient population. alternatives for the icu population to manage human waste include body fluid disposal systems or plumbed, bedpan flushing/disinfection devices. if planned, it is important for these to be convenient for hcp because there are aesthetic and safety barriers to transport of human waste over long distance. importantly, fixtures used for disposal of human waste should be limited to that purpose and not used for other activities, such as hand hygiene. the renewed attention on the inanimate environment as a source of pathogens has stimulated interest in strategies that can support infection prevention. there are several antimicrobial treatments that have been applied to inanimate surfaces; nonporous and soft surfaces such as textiles and privacy curtains. the types of antimicrobial treatments include photoreactive substances that release antimicrobials when exposed to natural sunlight, heavy metals like copper and silver, organosilane, triclosan, and quaternary ammonium compound. there is suggestive evidence that many of these can reduce the concentration of microorganisms on environmental surfaces. 30 direct evidence that these treatments reduce the incidence of hais in which transmission from the inanimate eoc is involved is lacking. this lack of evidence reflects the complex environment in an acute care facility given the myriad of sources by which personnel can contaminate their hands (eg, shared equipment) so treating several surfaces in a room may still not be more effective than processes and olmsted real-time feedback to personnel who clean and disinfect the built environment. guidance is available for some of the surfaces and finishes being planned for health care facilities. highlights of these include 31 : 1. nonupholstered surfaces should be capable of being easily cleaned; minimize surface joints and seams. 2. upholstered surfaces used in patient-care areas should be impervious (nonporous); untreated (non-high performance) woven fabrics should not be used. upholstered surfaces should be durable and resist tearing, peeling, cracking, or splitting; damaged surfaces are more difficult to clean effectively. upholstered furniture in patient-care areas should be covered with fabrics that are fluid-resistant, nonporous, and can withstand cleaning with hospital-grade disinfectants. cdc guidelines have identified that, ".compared to hard-surface flooring, carpeting is harder to keep clean, especially after spills of blood and body substances. it is also harder to push equipment with wheels (eg, wheelchairs, carts, and gurneys) on carpeting." 2 there are several recommendations on carpeting in these guidelines but a key one is to avoid the use of carpeting in high-traffic zones in patient-care areas or where spills are likely (eg, burn therapy units, operating rooms, laboratories, and icus). walls should be cleanable and able to withstand repeated exposure to chemical surface disinfectants. ceilings in areas needing special hvac requirements, such as aiir, operating room, pe, and so forth, are an important aspect of ensuring that the room envelope is sealed to maintain desired pressurization and contain contaminants. for operating rooms, fgi 2014 guidelines call for a monolithic ceiling as a strategy to facilitate effective envelope seal. the fgi convened a futures summit to assist with development of upcoming editions of their guidelines. this summit identified the following trends and needs going forward, and awareness of these is important for infection preventionists and health care epidemiologists in applying relevant infection prevention strategies 32 : more health care provided at home more access to medical care in the community more specialized diagnosis and treatment facilities hospitals provide only for the sickest or those with most complicated needs navigators and health coaches provide assistance to patients, providers, and/or payers increased use of technology for health care monitoring and communication continued government involvement in regulating health care health care will increasingly be provided in outpatient facilities and residential care settings of numerous types. acute care facilities will see slower growth and be focused on providing care to higher-acuity patients with more complex treatment and care needs. as a society, the united states needs to encourage development of high-value, high-engagement models of care; how the design of health care facilities can relate to this goal should be considered. a 4-year cycle for document development is not optimal for responding to the rapidly changing health care landscape. documents focused on fundamental design requirements are important but do not address complex health care delivery needs. fgi also needs to facilitate development of best practice and alternative concept guidance for health care design. scientific studies that inform design of the eoc are challenging. even so, zimring and colleagues 33 called for the use of evidence-based design (ebd) to drive design and construction of health care facilities. the steps in the process of ebd include: 1. framing of goals and models; most recently the emphasis is on patient-centered care 2. incorporation of health care facility guidelines; for example, fgi 3. planning and design 4. operations: daily care delivery issues that may require variation from design parameters; for example, reducing the temperature in the operating room to address comfort of surgeons and perioperative personnel other resources are available to improve the effectiveness of the planning and design. these resources include a comprehensive safety risk assessment tool sponsored by the agency for healthcare research and quality and fgi. 34 elements of this tool include icra, patient handling, medication safety, mitigating risk of patient falls, and security. a manual for incorporating infection prevention into construction projects is also available. 35 infection prevention and control is an essential component of the built environment. when absent or when there are disruptions, risk of exposure of patients and disease outbreaks often result. however, there are well-established, evidence-based guidelines to assist infection preventionists and health care epidemiologists with identifying strategies for prevention in collaboration with the multiple disciplines involved in construction and renovation (eic 2003 2 , fgi 2014 15 ). the icra remains the keystone of designing in prevention at the inception of a project through the completion and commissioning phases. future trends in care delivery in the united states are going to have a significant impact on construction and renovation of health care facilities; however, involvement and subject matter expertise provided by infection preventionists/health care epidemiologists will remain a core component into the future. step 1: using the following table, identify the type of construction project activity (types a-d) step 2: using the following table, identify the patient risk groups that will be affected. if more than 1 risk group will be affected, select the higher risk group: type a inspection and noninvasive activities includes but is not limited to: removal of ceiling tiles for visual inspection only; eg, limited to 1 tile per 4.6 m 2 (50 square feet) painting (but not sanding) wallcovering, electrical trim work, minor plumbing, and activities that do not generate dust or require cutting of walls or access to ceilings other than for visual inspection type b small-scale, short-duration activities that create minimal dust includes but is not limited to: installation of telephone and computer cabling access to chase spaces cutting of walls or ceiling where dust migration can be controlled type c work that generates a moderate to high level of dust or requires demolition or removal of any fixed building components or assemblies includes but is not limited to: sanding of walls for painting or wall covering removal of floorcoverings, ceiling tiles, and casework new wall construction minor duct work or electrical work above ceilings major cabling activities any activity that cannot be completed within a single work shift type d major demolition and construction projects includes but is not limited to: activities that require consecutive work shifts requires heavy demolition or removal of a complete cabling system new construction step 10: do plans allow for an adequate number of isolation/negative airflow rooms? step 11: do the plans allow for the required number and type of handwashing sinks? step 12: do the infection prevention and control staff agree with the minimum number of sinks for this project? verify against fgi design and construction guidelines for types and area. step 13: do the infection prevention and control staff agree with the plans relative to clean and soiled utility rooms? step 14: plan to discuss containment issues with the project team; eg, traffic flow, housekeeping, debris removal (how and when). the begum's fortune (extraordinary voyages #18) guidelines for environmental infection control in health-care facilities: recommendations of cdc and the healthcare infection control practices advisory committee (hicpac) epidemiology and control of nosocomial infections in adult intensive care units understanding and preventing transmission of healthcareassociated pathogens due to the contaminated hospital environment evidence that contaminated surfaces contribute to the transmission of hospital pathogens and an overview of strategies to address contaminated surfaces in hospital settings construction at $1,140.8 billion annual rate healthcare facilities management magazine. chicago: american hospital association apic state-of-the-art report: the role of infection control during construction in health care facilities nosocomial aspergillosis in outbreak settings review of fungal outbreaks and infection prevention in healthcare settings during construction and renovation environmental protection agency (epa). 2016 u.s. drinking water contaminantsstandards and regulations pseudomonas aeruginosa in hospital water systems: biofilms, guidelines, and practicalities healthcare outbreaks associated with a water reservoir and infection prevention strategies outbreak of multidrug-resistant pseudomonas aeruginosa colonization and infection secondary to imperfect intensive care unit room design facility guidelines institute, published by the american society for healthcare engineering a cluster of cases of nosocomial legionnaires disease linked to a contaminated hospital decorative water fountain. infect control an outbreak of legionnaires disease associated with a decorative water wall fountain in a hospital environmental cleaning intervention and risk of acquiring multidrug-resistant organisms from prior room occupants maintain the gain: program to sustain performance improvement in environmental cleaning the use of single patient rooms versus multiple occupancy rooms in acute care environments why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? transmission of urinary bacterial strains between patients with indwelling catheters -nursing in the same room and in separate rooms compared impact of conversion from an open ward design paediatric intensive care unit environment to all isolated rooms environment on incidence of bloodstream infections and antibiotic resistance in southern israel guideline for the prevention and control of norovirus gastroenteritis outbreaks in healthcare settings effect of building construction on aspergillus concentrations in a hospital refrigerating and air-conditioning engineers (ash-rae). ansi/ashrae/ashe standard 170-2013. ventilation of health care facilities guidelines for intensive care unit design planning and response to ebola virus disease: an integrated approach consensus guidelines for implementation of quality processes to prevent invasive fungal disease and enhanced surveillance measures during hospital building works self-disinfecting and microbiocide-impregnated surfaces and fabrics: what potential in interrupting the spread of healthcare-associated infection? health care furniture design -guidelines for cleanability (bifma hcf 8.1-2014) the future of health care as predicted using scenario planning evidence-based design of healthcare facilities: opportunities for research and practice in infection prevention safety risk assessment for healthcare facility environments step 3: match the: patient risk group (low, medium, high, highest) with the planned: construction project type (a, b, c, d) on the following matrix, to find the: class of precautions (i, ii, iii, or iv) or level of infection control activities required. class i to iv or color infection control matrix for class of precautions: construction project by patient risk step 4: identify the areas surrounding the project area, assessing potential impact. step 5: identify specific site of activity; for example, patient rooms, medication room, and so forth.step 6: identify issues related to ventilation, plumbing, electrical (in terms of the occurrence of probable outages).step 7: identify containment measures, using prior assessment. what types of barriers (eg, solids wall barriers)? will hepa filtration be required? (note: renovation/construction area will be isolated from the occupied areas during construction and will be negative with respect to surrounding areas).step 8: consider potential risk of water damage. is there a risk from the compromising of structural integrity (eg, wall, ceiling, roof)?step 9: work hours. can or will the work be done during non-patient-care hours? key: cord-324635-27q3nxte authors: bouza, emilio; brenes, francisco josé; domingo, javier díez; bouza, josé maría eiros; gonzález, josé; gracia, diego; gonzález, ricardo juárez; muñoz, patricia; torregrossa, roberto petidier; casado, josé manuel ribera; cordero, primitivo ramos; rovira, eduardo rodríguez; torralba, maría eva sáez; rexach, josé antonio serra; garcía, javier tovar; bravo, carlos verdejo; palomo, esteban title: the situation of infection in the elderly in spain: a multidisciplinary opinion document date: 2020-09-08 journal: rev esp quimioter doi: 10.37201/req/057.2020 sha: doc_id: 324635 cord_uid: 27q3nxte infection in the elderly is a huge issue whose treatment usually has partial and specific approaches. it is, moreover, one of the areas where intervention can have the most success in improving the quality of life of older patients. in an attempt to give the widest possible focus to this issue, the health sciences foundation has convened experts from different areas to produce this position paper on infection in the elderly, so as to compare the opinions of expert doctors and nurses, pharmacists, journalists, representatives of elderly associations and concluding with the ethical aspects raised by the issue. the format is that of discussion of a series of pre-formulated questions that were discussed by all those present. we begin by discussing the concept of the elderly, the reasons for their predisposition to infection, the most frequent infections and their causes, and the workload and economic burden they place on society. we also considered whether we had the data to estimate the proportion of these infections that could be reduced by specific programmes, including vaccination programmes. in this context, the limited presence of this issue in the media, the position of scientific societies and patient associations on the issue and the ethical aspects raised by all this were discussed. authors for their corrections and amendments. the final document has been reviewed by all the authors. we will now review the questions posed, the arguments made and the conclusion reached for each one. what do we mean when we talk about the elderly? how many are there in spain? how many will there be in the near future? presentation: the who publishes reports on ageing and health, or old age and its consequences, on a regular basis, at least since the 60's. cited here are a few more. we reproduce a paragraph in full [1, 2] "today, for the first time in history, most people can aspire to live beyond the age of 60. in low and middle-income countries, this is largely due to the significant reduction in mortality in the early stages of life, especially during childbirth and infancy, and in mortality from infectious diseases. in high-income countries, the sustained increase in life expectancy today is mainly due to the decline in mortality among older people". the report focuses on a redefinition of healthy ageing based on the notion of functional capacity: the combination of the individual's intrinsic capacity, relevant environmental characteristics and the interactions between the individual and these characteristics. in spain, according to data from the national institute of statistics [3] , 18 .7% of the population is currently over 65. that's about 8.7 million people. if we focus on those over 85, they currently account for 6% of the total population (about 2.8 million). forecasts for 2031 put the number of people over 65 years old at 12 million (26.2% of the population) and those over 85 at 3.9 million (8.5% of the population). thus, between 1960 and 2031, the number of people over 65 will have increased by a factor of 5 (from 2.5 to 12 million), and the percentage will have increased by a factor of 3 (from 8.2 to 26.2 %), while the number of people over 80 will have increased by a factor of 10 (from 370,000 to 3.9 million) and the percentage will have increased by a factor of 6 (from 1.2 to 8.5 per %). once the figures have been established, it is necessary to clarify that, according to the dictionary of the royal spanish academy of language (drael), "old" is "that person of age, commonly one who has turned 70". however, age is a purely theoretical value to distinguish a person as "old" or "elderly". taking the age of 65 as the threshold for the onset of old age dates back to the late 19th century, when less than 10% of those born reached that age. today, more than 90% of people reach the age of 65, so this age limit is shifting towards older ages. nowadays the concept of "old" is more related to "function" than to age. thus, the drael defines health as "that state in which the organic being normally exercises all its functions". therefore, one of the most relevant aspects in considering a person "old" is that they need help to carry out the activities of daily life (bathing, dressing, feeding, moving, etc.). we can find totally independent people in their 80's and others with a high degree of dependency in their 60's. formato es el de la discusión de una serie de preguntas preformuladas que fueron discutidas entre todos los presentes. empezamos discutiendo el concepto de "anciano", las razones de la predisposición a la infección, las infecciones más frecuentes y sus causas, y la carga laboral y económica que suponen para la sociedad. también preguntamos si teníamos datos para estimar la proporción de estas infecciones que podrían ser reducidas por programas específicos, incluyendo programas de vacunación. en este contexto, se discutió la baja presencia de este problema en los medios de comunicación, la posición de las asociaciones científicas y de pacientes sobre el problema y los aspectos éticos que todo esto plantea. the ageing of the population in more developed societies is an incontrovertible fact. in the face of the indisputable success in achieving a longer life for a large proportion of the population, questions arise as to the viability of social protection systems. by 2030, over 25% of the population will be classed as elderly and their quality of life will depend, to a large extent, on avoiding preventable diseases such as infectious diseases. it is a well-known fact that the elderly constitutes a risk group for distinct types of infectious diseases, whose diagnosis and treatment are hindered by several factors. around this fundamental fact, however, we find a lack of answers to simple questions about the size of the problem, its epidemiology, the capacity of the social response to it and the need to plan useful preventive measures to minimise risk and reduce costs. for this reason, the health sciences foundation, which has prevention as one of its main objectives, has organised a discussion and opinion meeting on the infectious diseases situation in the elderly in spain, aiming to answer a series of questions accepted by all the participants. greater difficulty in eliminating secretions. in the digestive tract it is common to find diverticuli in the mucosa that act as microorganism reservoirs. also, losses in secretory function with a tendency to gastric achlorhydria, but, above all, motor function which at oesophageal level, can favour aspiration phenomena. in the urogenital system there are usually alterations arising from pregnancy, childbirth, previous surgeries and local manipulations that make the free flow of urine difficult. in this vein, it is worth adding the frequency of subjecting the elderly to diagnostic or therapeutic examinations that may favour infections. in addition to the deterioration of mechanical barriers, there are losses in non-specific defence mechanisms. these include limitation to increase blood flow and vascular permeability at the infection entry points. the ability to mobilise polymorphonuclear leukocytes rapidly and the agility of phagocyte function is also impaired. chemotactic capacity decreases from the age of 70, as does the capacity for the intracellular destruction of microorganisms. ageing is associated with a chronic, progressive, nonspecific, low-level pro-inflammatory state, for which the english literature has coined the term "inflammageing", which favours an environment conducive to infection and further limits the possibilities of an effective response to it. the deterioration of adaptive immunity ("immunosenescence") associated with the ageing process has been known for years and affects both innate and acquired immunity [4] [5] [6] . immunosenescence includes qualitative losses in t-lymphocyte subpopulations with decreased activity of cd-4 helpers, cytotoxic cd-8s and a limitation in generating t-cell growth factor. ageing determines a tendency to invert the cd4/cd8 t-cell ratio. the number of dendritic cells decreases with age and the response of nk cells to stimulating cytokines is limited. it also increases the activity of cd-8 suppressors. b-lymphocytes are limited in their ability to produce antibodies and to respond to external antigens. furthermore, there is an increase in the production of autoantibodies and circulating immune complexes. a third group of factors that add to the microorganisms and the individual are environmental and social factors, such as hygiene neglect, poverty, isolation and a sedentary lifestyle. the fact of living in nursing homes and the increase in hospitalisations favours an insufficiently quantified environmental exposure [7] . there are multiple factors that explain the higher incidence of infections in the elderly. the clearest are those that have to do with alterations of the defensive barrier mechanisms. immunosenescence is a complex concept involving various alterations in the immunity of the elderly. what are the main clinical syndromes of infection in the elderly? the frequency and even the aetiology of infections af-therefore, the "elderly" is an enormously heterogeneous group in aspects such as the prevalence of chronic diseases (ischaemic heart disease, hypertension, diabetes, copd, etc), the need for consumption of drugs and the existence or nonexistence of physical, mental (dementia, depression) and social (loneliness, isolation, poverty) problems. conclusion: -the definition of elderly is artificial and refers to any person over a certain age (which can be set at 65, 70 or older) who has serious limitations in the exercise of their physical, mental or social functions. -in our society, currently, almost 20% of the population would meet a definition of elderly based exclusively on the criterion of age, but it is estimated that, with this criterion, the percentage in spain will be greater than 25% by the year 2031. the changes that take place throughout the ageing process favour the existence of infections. the simplest explanation is that with age the numerator of the aggression/defence equation increases (greater arrival of microorganisms that are also more virulent) and the denominator decreases (less defence capacity on the part of the organism). we can therefore divide the causes of the elderly person's predisposition to infection into those that depend on the microorganisms and those that depend on the host's defence mechanisms. there is no evidence that the microbiota of the elderly is quantitatively different from that of younger populations, nor necessarily more aggressive. however, it is an incontestable fact that previous infections, antimicrobial treatments, the greater ease of microorganism acquisition and living in proximity to other elderly people, can predispose the elderly to colonization and subsequent infection by multi-resistant microorganisms, with the presence of "superinfections", with a worse response to antimicrobials and increased resistance to them. in terms of host defence mechanisms, there are many factors that make the elderly more labile. mechanical barriers, for example, are the first element of defence, but they deteriorate progressively throughout the ageing process, facilitating the entry of microorganisms. the skin and mucous membranes experience physiological losses and often also those resulting from local or systemic diseases. the most important changes are: thinning, with loss of epithelial and mucosal cells, worse hydration and vascularization, loss of elasticity, decrease in mucous gland secretions of antimicrobial peptides, worse healing, loss of cellular macrophages in the skin (langerhans cells) and immobility with increased local pressure in certain areas. in the respiratory system, there is a decrease in the number of cilia and a slowing down of their activity, a reduction of alveolar macrophages, a decrease of the cough reflex and pend on their situation. in independent elderly people, the most common infections are respiratory conditions caused by viruses or bacteria prevalent in the community, urinary tract infections and intra-abdominal infections. in contrast, in institutionalised elderly people, bladder catheter-related utis, aspiration pneumonias, skin and soft tissue infections, and infections of the gastrointestinal tract predominate. which microorganisms are most common? how does the problem of multi-resistance impact on the elderly? presentation: it is important to remember that infections in the elderly may be caused by a greater variety of microorganisms than in the younger population, so it is essential to obtain samples for culture before administering empirical antimicrobial treatment [8] . thus, for example, while the vast majority of utis in young patients are caused by e. coli, in the elderly their relative importance is less. in the case of pneumonia, there is a higher incidence of gram-negative bacilli (gnb) and as far as meningitis is concerned, they are rarely of viral aetiology, while we must consider gnb and listeria monocytogenes. in a spanish study, including 333 elderly patients (mean age 81.6 years), with utis, the most frequently isolated microorganisms were e. coli, (67%), enterococcus faecalis (15%), klebsiella pneumoniae (10%) and pseudomonas aeruginosa (9%). in up to 8% of cases, more than one microorganism was isolated in the urine. the frequency of bacteraemia was higher with e. coli and lower with e. faecalis and p. aeruginosa and bacteraemia was not associated with a worse prognosis [22] . the frequency of multi-resistance increases with age and comorbidity. in this spanish study, the proportion of extended-spectrum beta-lactamase (esbl) producing e. coli and k. pneumoniae isolates was 20.1% and 36.3%, respectively. in the previously mentioned study of patients attending the emergency department, the elderly accumulated more risk factors for multi-resistance (p < 0.001) and suffered from septic syndrome more frequently (p < 0.001) [16] . there are few studies that analyse the overall aetiology of respiratory infections in older patients, and most work focuses on describing specific populations or groups of pathogens. the aetiological affiliation rate of respiratory infections in the elderly is very low (<30%), and this is due, among other things, to the difficulty many patients have in producing sputum and to the high frequency of empirical treatment [21] . if we analyse the aetiology of cap, the most frequent pathogen is s. pneumoniae (20-80%), followed by h. influenzae (3-39%), respiratory viruses (3-30%), legionella spp.(1-17%) and gnb (3-14%) . it is also necessary to remember the importance of viral pathogens in this population, since the prescription rate of unnecessary antimicrobials is very high in them (46% of the elderly with viral symptoms) [23] . in a study conducted in china, in 6 sentinel hospitals, it was observed that 31.64% of elderly patients with respiratory infection had a viral aetiology (41.8% among extra-hospital infections and 25.7% among fecting the elderly vary depending on the clinical environment (home, nursing home, hospital) and the functional status of the patient. in older, independent and healthy people, respiratory conditions caused by viruses or bacteria prevalent in the community, urinary tract infections (utis), whether catheter-related or not, and intra-abdominal infections (cholecystitis, diverticulitis) are common. in contrast, in institutionalised elderly people, utis related to the bladder catheter, aspiration pneumonia, skin and soft tissue infections and those of the gastro-intestinal tract (git) predominate. in hospitalised elderly people we have to consider nosocomial pneumonia, intravascular catheter associated infections and c. difficile infections as the most prevalent [8] [9] [10] [11] [12] [13] [14] . there is limited data analysing the comparative overall frequency of the different syndromes. in elderly people living in nursing homes, utis (at least 30-40% of healthcare-associated infections), respiratory infections, skin and soft tissue infections and those of the git predominate [15] . in a recent spanish multicentre descriptive study, conducted in 49 emergency departments, 11,399 patients were included, of whom 4,255 (37.3%) were at least 65 years old. compared to younger adults, older patients (mean 78.8 years) had respiratory, urinary and intra-abdominal infections more often, while there was no difference in the frequency of other syndromes [16] . these data are confirmed in chinese studies that analyse elderly patients attending emergency departments and also show a significantly higher incidence of respiratory and urinary infections [17, 18] . in the case of utis, the relative prevalence is influenced by the gender of the patient. thus, for long-term care facility (ltcf) residents and in hospitalised elderly people, uti is the number one cause of infection and is the second most common in older women living in the community [19] . the incidence in men ranges from 0.05/person year (1/20) in men aged 65-74 and reaches 0.08 (1/12) in men over 85. in women, the incidence of uti increases with menopause (0.07 per person/ year: 1/14), increasing to 0.13 per person-year (1/7.5) after age 85 [20] . in indwelling catheter-wearing patients, the incidence of utis is 3.2 cases per 1,000 catheter days, compared to only 0.57 per 1,000 days for all residents (x18). urinary tract bacteraemia was 3-39 times more common in patients with permanent urinary catheterization [20] and uti is also the most frequent cause of community-acquired bacteraemia in the elderly (40-57%). with respect to respiratory infections, the annual incidence of community acquired pneumonia (cap) ranges from 8-18.2 episodes per 1,000 people over 65 years of age and represents 30-40% of hospitalisations in this age group [21] . in japan, 96% of deaths from pneumonia occur in patients over 65 years of age. the risk of cap is 4 times higher in those over 65 compared to those under 45 and 10.8 times higher in those over 85 compared to adults aged 50-64. viral infections are also common in this age range, as we will see later. the most prevalent infections in the elderly de-is estimated at between 4 and 5 episodes per 1,000 days of stay in the residence [33, 34] . the figures rise to 11 for those with some kind of prosthetic material [35] . we have several european halt studies (healthcare-associated infections and antimicrobial use in long term care facilities), with participation from 24 countries, including spain, with a prevalence of infection of 4.7% and 5% at two different times [36] [37] [38] . a french multi-centre study, conducted in 578 nursing homes with 445,000 beds, shows an infection prevalence of 11.23% [39] . the first data on infection in nursing homes in spain come from the epinger study, conducted in community health centres in catalonia, which reported a prevalence of 6.5%, although it should be pointed out that in catalonia the concept of the community health centre would include medium-long term patients, while in the rest of the spanish autonomous communities this concept would be limited to nursing homes [40] . in another study, conducted by san sebastian's fundación matía, an infection prevalence between 6.44% and 4.80% was reported [41] . data derived from the vincat study in catalonia show a prevalence of healthcare-associated infection in long-term care centres of 10.2%, with a great diversity, depending on the type of care unit (subacute 22.3%, palliative 18.7%, convalescent 11.7%, long stay 8.1%) [42] . home is the most recommendable place for the healthy elderly to live, and even for the elderly patient, with healthcare falling to primary care professionals, although sometimes with the collaboration of some hospital resources. the ministry of health, social services and equality has for the first time published the results of the primary care clinical database (bdcap), a tool that allows for a more precise and systematized knowledge of the main health problems in spain dealt with by the doctors on the healthcare frontline. thanks to this register, a detailed picture of the health problems of the spanish population is available from primary care [43] . in this database, infections appear among those over 64 years old with an elevated frequency of 634.1 cases a year per 1,000 people (569.9‰ men and 682.7‰ women). the most frequent correspond to the respiratory system (317 cases/1000 persons/year), followed by urinary tract infections with (84.4 cases/1000 persons/year) and clear female predominance. finally, nosocomial infections are those that occur in hospitalized patients and are present more than 48 hours after admission. they are acquired by transmission from the environment, from other patients or from healthcare personnel. they are considered to be the most preventable cause of serious adverse events in hospitalised patients [44] . in general, these infections are related to invasive diagnostic or therapeutic procedures (urethral catheterization, surgical procedure, vascular catheter, invasive mechanical ventilation), all of which have in common the disruption of the host's own defences by a device or an incision, allowing the invasion of nosocomial infections) [24] . the most common cause was influenza (14% of all patients studied). rsv is also a significant pathogen in this population [25, 26] . the most important cause of git infection in the elderly is clostridioides difficile. c. difficile (c-diff) infection is currently the most prevalent nosocomial infection, affecting in more than 70% of the episodes patients over 65 years of age [27] . moreover, it is in this population that c-diff causes the highest morbidity and mortality, with an increase in c-diff-related mortality from 5.7 to 23.7 deaths per million population per year from 1999 to 2004 [28] in patients with an average age of 84 years having been described in the usa. it is interesting to note the safety of using the same therapeutic options in elderly patients, including faecal microbiota transplantation [29, 30] . the microorganisms causing infection in the elderly are qualitatively the same as in the population of other age groups, although there are quantitative variations. where do they get these infections? what proportion are acquired in nursing homes? at home? in hospital? in addition to the hospital and home environment, the elderly can acquire infections elsewhere, and in particular in other care units. this is the reason why, almost 20 years ago (2002), the term "health care-associated infection" began to be used, which is not only limited to hospitalized patients, but also extends the concept to patients in contact with the health system (home care of patients with high comorbidity and complexity; day care centres; major outpatient surgery units; outpatient dialysis centres; community health centres for chronic or convalescent patients). to a great extent, it is in nursing homes where patients with more comorbidities, polypharmacy consumption, a high degree of dependency and a high prevalence of invasive devices (bladder catheter, nasogastric tube, percutaneous gastrostomy) will be treated. in addition, the environment can facilitate the transmission of microorganisms between residents and healthcare personnel, as well as between residents. for all these reasons and the excessive or inappropriate use of broad-spectrum antibiotics, either empirically or prophylactically, multi-drug-resistant (mdr) infections can be generated. implementation of effective preventive measures in this population is very difficult to organise. in the united states of america, it is estimated that approximately 1.5 million people live in nursing homes and suffer between 1.6 and 3 million episodes of infection annually [31] . the prevalence of infections in these residences is estimated at 10% of the residents [32] and the incidence of new infections infectious diseases are the second cause of such admissions (16.2%), only surpassed by cardiovascular diseases (28.6%). pneumonia and sepsis are the most common infections causing admission in this population [48] . the elderly population also has longer hospital stays (5.5 days for those over ≥65) than those between 45 and 64 (5.0 days) and those between 15 and 45 (3.7 days) [49] . the elderly are treated by virtually every unit in a hospital but it is worth mentioning that those over 65 years of age represent 40% of those admitted to intensive care units [50] . the other group of interest is that of specialised geriatric units, not available in all hospitals, which have been shown to improve the functional status of patients and reduce the number of discharges to long-term care homes [51] . in a study by saliba et al., conducted in israel [52] , out of a total of 81,077 hospital admissions in the elderly between 2001 and 2010, the proportion of admissions due to infectious diseases rose from 16.9% in 2001 to 19.3% in 2010. globally, the most frequent infections causing admission were: those of the lower respiratory tract (lrt) (41.0%), followed by the utis (21.4%), upper respiratory tract (10.2%) and hepatobiliary (9.8%). in spain we do not have precise answers to the questions asked. the proportion of serious infections in the elderly requiring hospitalisation depends on several factors: type of infection, severity of infection and other factors such as the degree of frailty of the elderly, their place of residence and their ability to receive care at home. the environment and the resources available also influence the hospitalisation decision. however, in our environment, most serious infections in the elderly will require hospitalisation for at least a few hours. in spain, serious infections in the elderly can be treated by different professionals depending on the type and severity of the infection, and the environment in which it occurs. a high percentage are treated by "generalists" hospital specialists, or geriatricians. where infectious disease specialists are available they are of course involved in their management, either in beds in their own departments or as consultants. they can also be treated by specialists of the affected organ such as orthopaedic surgeons in the case of infections of prosthetic material, or vascular surgeons in the case of infections of vascular ulcers. and if, in the end, hospital admission is not decided, the patient is cared for by the primary care team. as an example, we have collated the urinary tract infections treated at the hospital general universitario gregorio marañón between 2015 and 2018. when uti is the main diagnosis that motivates admission (about 700 cases a year) about 90% of cases are cared in the medical departments. when it comes to secondary diagnosis (about 2,000 cases per year), the internal medicine and geriatrics departments take care of about 75% of the cases. preventive programmes, such as flu vaccination programmes, reduce the need for hospitalisation for respiratory infections by nearly 30%, both inside and outside spain [53] [54] [55] . microorganisms that are part of the patient's usual microbiota (endogenous microbiota), or selected by the selective antibiotic pressure (secondary endogenous microbiota), or by one found in the hospital environment (exogenous microbiota). to understand the main epidemiological data on hospital infections, the epine study (estudio de prevalencia de las infecciones nosocomiales en españa (study on the prevalence of nosocomial infections in spain)) was developed. this is a multi-centre system for monitoring nosocomial infections, based on the production of an annual prevalence study, which has been conducted since 1990 in a large group of hospitals in spain and was promoted by the spanish society of preventive medicine, public health and hygiene. its methodology guarantees a homogeneous and systematic collection of information, which allows us to understand the prevalence of healthcare-associated infections (hais) at a national level, by autonomous regions and hospitals. since 2012, every 5 years the epine study has been produced jointly with the european study (in 2012 and 2017) under the coordination of the ecdc [45] . based on the latest data published, in november 2017 (313 hospitals and 61,673 patients), a prevalence of nosocomial infection in patients over 65 years of age of 6.07% (infections acquired during the current admission), 7.45% (infection acquired during the current or previous admission) and 8 .76% (the total, including the centre's own or imported) has been reported. it should also be noted that this register shows that in 22% of patients over 65 years of age admitted for an infection, the infection had been acquired in the community (patient's home). the home, nursing homes and community health centres, healthcare centres other than hospitals and the hospital itself are often the places where the elderly acquire infections. the studies reviewed allow us to estimate a prevalence of infection of between 4 and 10% in nursing homes in spain, depending on their complexity, and between 6 and 9% in hospitalised elderly people. in primary care and in the residential environment, there is no homogeneous epidemiological record of this problem. what proportion of severe infections in the elderly require hospitalisation? by whom are they treated? in the united states of america, patients over 65 years of age account for almost 40% of total adult admissions and the cost of these hospitalisations represents nearly 50% of the total cost for hospitalisation, although those over 65 years of age account for less than 20% of the total adult population [46, 47] . those over 65 years of age are admitted to hospital three times more often than those between 45 and 65 years of age, and those aged 85 or over account for 9.2% of all hospital discharges, although they represent only 1.8% of the population as a whole. moreover, in our opinion, in these departments, emergency assessment should not be focused only on the isolated episode for which the patient consults, but the particulars of the elderly person, their functional, mental and social situation should be taken into account. this is a huge workload for the ed. finally, we should bear in mind that the training of ed physicians on these issues is limited [63] as a direct consequence of the self-training of current professionals, which is not always complete, and the lack of a regulated medical specialty in the ed. in spain, between 15 and 25% of emergency department visits occur in the elderly. elderly people come in 14.5% of the time for infections and one third of the infections seen in the emergency departments occur in the elderly. the population over 65 years of age who attend the emergency department often have multiple pathologies and clinical manifestations of infection that may be atypical. in the spanish national health service, emergency activity accounts for a total of 47.2 million consultations per year, of which 26.5 million are attended to in primary care (pc) (outpatient or home), with an average attendance of 0.6 people/ year [64] one-third of emergency consultations in pc are related to infections [65] . in the older patient, infections are more frequent and serious, associated with greater morbidity and mortality [65] [66] [67] . among the elderly, the rate of infection reaches 634.1 cases per thousand people per year. the most frequent correspond to the respiratory system (317 cases per thousand), particularly those of the upper respiratory tract, followed by acute bronchitis and bronchiolitis and pneumonia [65, [67] [68] [69] . in second place are utis, mainly affecting women (114.8 cases per thousand compared to 44.2 per thousand for men) [67]. these are followed by skin and soft tissue infections [69] . most of these cases are dealt with in primary care and only those more serious situations and of uncertain diagnosis are referred. in 75%-80% of cases, cap is diagnosed in pc [65, 70] and streptococcus pneumoniae is the cause of two-thirds of these cases. invasive forms of pneumococcal disease (ipd) are less common, occur in patients with certain risk factors and have high mortality rates [70] . the vast majority of vaccination programmes in spain are carried out in primary care, but the vaccination schedule for older people is neither complete nor promoted as it should be. what is the workload represented by elderly patients in hospital emergency departments? the number of visits to hospital emergency departments (ed) has been increasing progressively for decades. this increase is greater in the elderly, whose population accounts for 15-25% of all visits to the hospital [56] . the incidence and impact of infection in the ed is estimated quite reliably. in spain it is 14.3%, 21% in the usa and around 30-40% in countries such as nicaragua and mexico [57] . the elderly are characterised by a higher probability of atypical presentation of diseases, of suffering from multiple diseases and of consuming many drugs. with regard to emergency care, this implies a more complex clinical evaluation, which translates into a greater request for additional tests and consultations with other specialists, longer stays in the ed (extended periods under observation and in ssus), as well as a greater probability of admission, discharge with undetected or untreated problems and return visits to the ed [58] . all this entails a high risk of adverse episodes [58] and a significant impact on healthcare pressure, resulting in a negative effect on ed saturation [59, 60] . likewise, the prevalence of the frail elderly in the community varies according to the diagnostic criteria. in a study conducted on elderly people admitted to the observation room of an ed in a spanish tertiary hospital, it was verified that only one of them did not have any fragility criteria and on admission almost half of them suffered significant dependence [61] . the detection of the high-risk or fragile patient is fundamental for these departments, for decision-making and in particular for discharge directly from the emergency department. we could highlight that in the recent work of the in-fur-semes group, in a study conducted in 49 spanish eds, 31.7% of infections occurred in patients over 70 years old. of these, 36% were urinary and 51.2% were lower respiratory. in conclusion, when compared with a similar study, conducted twelve years earlier, an increase in the prevalence of infections is observed, with an older patient profile, comorbidity, risk factors for mdr microorganisms and septic syndrome [62] . the latter almost always presents itself as an acute confusional syndrome, which implies a complex differential diagnosis. to what extent do you think that infection in the elderly is preventable? what proportion could be avoided with proper vaccination? in an article published by umscheid et al. [79] , not specifically addressing to the elderly field, it is estimated that 65%-70% of cases of catheter-related bacteraemia or catheter-associated urinary tract infection and 55% of pneumonias from mechanical ventilation or skin and soft tissue infections could be prevented in the hospital environment using the methodology currently available. an infection control programme for older patients includes methods for surveillance and recording of infections, recording and management of multi-resistant microorganisms, outbreak contingency plans, isolation policy and standard precautions, hand hygiene programmes, ongoing education of employees, resident health plans, audits and plans for reporting incidents to health authorities [80] . this set of resources is not available to most of the world's elderly. a group of experts, gathered in a delphi study on infection prevention measures in patients admitted to institutions for the elderly, agreed on 302 recommendations [81] but unfortunately the level of evidence on the effectiveness of each of them is very limited. data on the reduction of different infections by different measures are extremely scattered and limited. some examples are the reduction by 53% of periprosthetic infections with antibiotic prophylaxis [82] , a 60% reduction in episodes of influenza with the physical separation of the young and the elderly, [83] or a 48% reduction in episodes of pneumococcal pneumonia with the 23-valent vaccine [84] . makris et al. [85] conducted a study to test the effect of an infection control programme in 8 institutions for the elderly in the united states of america. they divided the centres into test centres (4) and control centres (4) and studied the incidence of infections in both groups before and after the programme was introduced. in the year prior to the intervention, test sites experienced 743 infections (incidence density rate, 6.33) and control sites 614 infections (incidence density rate, 3.39). in the intervention year, the test centres reported 621 infections, a decrease of 122 infections (incidence density rate, 4.15), while in the control centres, the number of infections increased slightly to 626 (incidence density rate, 3.15). the greatest reduction in infections at the testing centres was in upper respiratory tract infections (p = 0.06). the intervention programme consisted mainly of implementing environmental cleanliness, hand washing programmes and educational talks. therefore, and speculatively, we dare to estimate that a the infection rate in the elderly exceeds 500 episodes per 1,000 sick people per year. primary care handles the vast majority of these episodes and refers only the most serious cases. primary care is responsible for the vaccination programme for elderly people who attend to request it. the vaccination schedule for older people is neither comprehensive nor proactively promoted. what does infection in the elderly entail in terms of days of hospitalisation, financial expenditure and death? to approximate data/figures for variables such as "days of hospitalisation, economic expenditure and death" in a field as broad as "infection in the elderly" is enormously complicated. it must be taken into account that the infectious pathology is very varied and that it can affect people with different locations (community, community health centre or the hospital itself) and conditions. for example, with reference to nursing homes, lim et al. estimate 4 episodes of infection for every 1,000 cumulative days spent in the home in a small group in australia [71] , while much more extensive north american data report 12% of nursing home residents having an infection at the time of the study [32] . this leads to estimates of between 1.64 and 3.83 million episodes of infection per year [31] with annual costs of no less than us$ 1 billion, prior to 2000. in a study conducted in brazil, the cost of an infection in the elderly requiring admission is estimated at 28,714 brazilian reals (€ 6,305). patients are admitted for a median of 24 days compared to a median of 9 days for elderly people admitted for non-infectious causes [72] . of that cost, only 5% is attributable to the purchase of antibiotics. there is a greater volume of data for community-acquired pneumonia (cap) [73] [74] [75] [76] . the cost of cap varies greatly depending on where the treatment takes place. a spanish study [77] found a cost of only € 196 in the case of an outpatient, compared to €1,153 for pneumonia requiring hospitalisation. the costs were higher for subjects ≥65 years. mortality increases significantly in the older patient (25%) with respect to the general population (10%). it is worth noting a publication in spain with a sample of 2,049 subjects, where mortality due to pneumonia is more clearly related to the age group than to the aetiological agent [78] . we have not found precise data calculating overall clearly no one disputes the usefulness of ongoing education in many aspects of life and particularly in the reduction of nosocomial infections. that said, the literature review on the impact of educational programmes on nosocomial infection is irregular, fragmented and often difficult to assess. published studies generally include education as part of intervention programmes in which other measures are included, making it difficult to assess the role of education in isolation. it is also common to talk about the success or failure of an educational programme without detailing what the programme is, what content it has, how it has been implemented and how many people have accessed it. to complicate matters, in the case of the elderly, we have at least three different areas: home, nursing homes and institutions for the elderly and hospitals. in the first, the educational scope is very general and imprecise and is based on the public health and vaccination campaigns that are usually received not only by the elderly population but by the population in general. in the hospital field, we must assume that the literature produced on the impact of educational measures in the different syndromic entities generally includes the elderly population, but does not specifically differentiate it. most of the limited existing information, which we can consider specific to older people, is that generated in nursing homes and institutions that implement these programmes. a study conducted in the usa on 2,514 randomly selected nursing homes [91] asked the homes for information on 34 points related to infection control programmes. most of those responsible for control programmes, when they responded, claimed to have not only that responsibility but others as well (54%) and also to have no specific training in infection prevention (61%). there was great variability in practices carried out in each residence and 36% acknowledged having received an official citation for deficiencies in such control. those residences cited for deficiencies had a statistically lower proportion of staff trained in infection control. this is therefore an area with clear opportunities for improvement. in a systematic review on non-pharmacological infection prevention in long-term care facilities, only 24 papers were selected, the majority of which were randomised studies (67%) and the most common reason was prevention of pneumonia (66%). 54% showed favourable results for the interventions, but the studies had many potential biases [92] [93] [94] [95] [96] [97] [98] [99] . from these studies the 5 main quality markers in infection control in a nursing home were deduced, namely: percentage of long-term patients with pressure ulcers, urinary tract infection, bladder catheter, and vaccinated against influenza and pneumococcal infection. high quality infection control programme in nursing homes could reduce infection rates by up to 50%. but even if we estimate much lower figures, the impact on morbidity, mortality and the economy of such programmes would be enormous and would certainly outweigh their implementation costs. with reference to the second part of the question, the possibility of reducing the problem with vaccines, the data are again scattered and studied for different vaccines individually. in addition, information on the elderly must often be inferred from data on the general population. we refer readers to a recent review on the subject [86] . below is some data on the impact of vaccines of particular interest to the older population. gross et al. [87] in a meta-analysis of 20 cohort studies estimate the effectiveness of influenza vaccination at 56% in preventing respiratory infections, 53% in preventing pneumonia, 50% in preventing hospitalisations and 68% in preventing deaths. in the case of zoster, the vaccine's efficacy is estimated at more than 90% with minimal adverse effects [88] different pneumococcal vaccines have different impacts on the incidence of invasive pneumococcal disease (ipd) infection. a systematic review shows reductions in ipd incidence ranging from 61% as a combined effect of the use of pcv7, pcv10 and pcv13 in those over 65 in canada [89] to a 21% reduction as an effect of the use of pcv7 and pcv13 in israel [90] . with these data it is possible to imagine the added protection that adequate vaccine coverage would provide. an estimated 50,000 americans die each year from vaccine-preventable diseases, and 99% of those who die are adults [86] . increased provision of medical care in large care homes (e.g. those with more than 200-250 beds) could reduce the referral of many elderly residents to hospital emergency services. this provision of medical care would not necessarily be very complex and would cover both simple diagnostic material and the possibility of establishing and carrying out pharmacological therapeutic courses at the centre itself, the prescription of which in most cases still requires medical staff from outside the centre. it would be a way to reduce costs, lessen the burden on the elderly and reduce the overload on hospital emergency departments. it is impossible to give a precise answer to the questions asked, but it seems reasonable to assume that with appropriate prevention programmes, acquired infections in institutionalised elderly people could be reduced by up to 50%. strict adherence to a vaccination programme for the elderly would have an enormous impact on reducing suffering, death and economic waste. what data exist on the effectiveness of educational measures on the incidence of infection in the elderly? ties specifically dedicated to infection. by way of an example, in spain, this occurs among specialists in microbiology and infectious diseases and intensive care specialists. 9.-specifically promote research aimed at preventing infection in elderly patients. 10 .-introduce much more active involvement of patient associations in their management structures. what we say about societies primarily dedicated to the elderly, can be similarly assumed and applied to societies primarily dedicated to infectious diseases and microbiology. the role of the scientific societies dedicated to geriatrics and infectious diseases is to promote alliances in the common field of infection, in aspects of care, teaching and research. they need to look less to the interests of their members and be more proactive in promoting the interests of the patients they serve and incorporate patient associations more into their structures. capacity, understood as the possibility or potential for influence, is qualified by two variables. firstly, for offering free and truthful scientific information at the service of the community. and secondly, for facilitating the adoption of the best possible political decisions with consistency and realism. the rapprochement between professionals in the scientific and political fields must be adjusted to the interest of citizens, who can act as the third pillar in a transparent relationship model and as guarantor of equity befitting a democratic system of government [110] . while scientific experts advise and inform, it is the responsibility of politicians to make decisions and promote efficient measures to the benefit of the population. a complementary characteristic inherent to the scientific task is to exercise a dissemination action of the activity itself, in understandable terms and through accessible and reliable systems [111] . the configuration of platforms within scientific societies and the growing number of independent agencies advising political power represent a reality that aims to bring the contributions of science closer to the systems of governance [112] . in our country, the main function of the congress of deputies is legislative, which entails the approval of laws. the constitution recognises the legislative initiative of the government, the congress of deputies, the senate, the assemblies of the autonomous communities and the people's legislative initiative on the proposal of no less than 500,000 citizens, subject to the provisions of an organic law. these bills are known in spain as law projects when presented by the government and propositions in other cases. they are always submitted to the congress of deputies, except for the propositions of the senate which have to be considered scientific societies are professional associations that bring together generally specific groups (doctors, nurses, technicians, etc.) that essentially seek to defend the professional interests of their members. until now, it has not been common for groups of patients affected by different diseases under the thematic umbrella of each society to participate in them. in spain their impact and political credit is variable. among the most important objectives of most of these societies are such issues as training programmes for professionals, aspects related to the health education of the population in their particular field of competence, research grants, the preparation -sometimes in collaboration with societies of another related specialty -of specific diagnostic and therapeutic protocols, publications and congresses focussed on these topics, and a wide range of other activities, including health policy recommendations to the corresponding administrations that have a direct bearing on the issues discussed here. membership of societies is also not uniform, and often it is the more "senior" components of the profession that are most highly represented in them. their role, in our opinion, is to continue to improve the teaching, care and research produced in the societies' chosen fields in favour of patients, exercising ever greater mediation between the demands of patients and healthcare administration [100] . all societies must go far beyond issuing guidelines and therapeutic recommendations [81, [101] [102] [103] [104] [105] [106] [107] [108] [109] . in our view, scientific societies dealing with diseases of the elderly should promote, in the field of infectious diseases, among others, the following topics: 1.-encourage a proportionate share of its members to subspecialise in infectious diseases. 2.-coordinate and direct multidisciplinary teams specifically dedicated to the infection in the elderly and its prevention. 3.-participate more actively in specific programmes to reduce infections in the elderly, both at the nursing home level and at home and in hospital. 4.-implement vaccination campaigns in the elderly, taking particular advantage of admission to long-stay centres or hospital as opportunities to vaccinate. 5.-to design and disseminate educational projects on infection prevention practices for the elderly in their different environments. 6.-put pressure on health authorities to carry out a large national programme to reduce infection in older people. 7.-to include in the training programme of residents in geriatrics, a rotation in infectious diseases and microbiology as an essential part of the curriculum. 8.-create scientific and professional alliances with socie-to offer a unique system to access scientific information allowing the recovery of different types of documents such as: journals, books, images, theses, and conference proceedings. revista española de geriatría y gerontología (the spanish journal of geriatrics and gerontology) is the publication channel of the society of the same name, a publication founded in 1966 and the doyenne of the specialty in the spanish language [119] . medes is an initiative of the fundación lilly and its database, open and free, contains bibliographical references published since 2001 in a selection of 98 spanish journals covering 50 subjects in medicine, pharmacy and nursing, published in spanish, with 100,000 articles [120] . finally, pubmed is the widely implemented search engine, with free access to the medline database of citations and abstracts of biomedical research articles, offered by the united states national library of medicine and integrating 5,255 worldwide journals since 1966 [121] . the search was conducted with a double strategy: free text and controlled text using "mesh". in the first strategy, a free text search was conducted in the "science direct" and "clinical key" databases with the term 'infection in geriatrics' resulting in 508 and 1,191 findings respectively. the primo search engine (castilla y león online library) returned a total of 190 results for the same term. secondly, and also in free text, with the term 'infection in the elderly', we proceeded to consult revista española de geriatría (the spanish journal of geriatrics), which generated 195 results and medes (medicine in spanish) with 84 results. the second strategy of controlled text was conducted in the pubmed database, returning the following findings: <infection and geriatrics>: 997 results; <infection and aged> (people from 65 to 79 years old): 122.698 results and <infection and aged or aged, 80 and over>: 122.698 results (identical to the previous one). its development over the last decade has been progressive (from figures close to 4,000 in the 2009-2010 biennium, to over 5,000 from 2014 to 2017), excluding the year 2018 from the assessment. we have adopted their classification into thematic areas [122] and the twelve in which 95% of the results were concentrated are: sepsis and bacteraemia, pneumonia, urinary tract infections, central nervous system infections, endocarditis, prosthetic infections, skin infections, gastrointestinal infection, hiv infection, fever of unknown origin, multi-resistance and vaccinations. the scientific output on infections in the elderly, calculated by different databases, has been increasing in the last decade. how do the problems of the elderly impact on the mainstream media? how should the media contribute to the reduction of infection in the elderly? the impact of the problems of the elderly in the media is in the senate, which will later submit them to congress [113] . non-legislative bills, motions and proposals for resolutions are acts of a similar nature that seek the adoption of a non-legislative resolution by congress, by which congress expresses its position on a given subject or issue, or addresses the government urging it to act in a particular direction. the health and social services commission of the congress in the xii legislature offers access on its website to the 226 initiatives processed since its constitution in september 2016 until its dissolution in march 2019, representing an average of 75 per year [114] . of these, those referring to the field of infectious pathology as a whole do not exceed 3%. of particular relevance in the field of infectious pathology have been those relating to the national plan for the elimination of hepatitis c and antibiotic resistance. governance designates the effectiveness, quality and good orientation of state intervention, which provides the state with a good part of its legitimacy in what is sometimes defined as a "new way of governing". above all, it is used in economic, social and institutional operational terms [115] . an inherent aspect of the exercise of policy is the performance of "authority", which is equally composed of legitimacy (right to exercise), personal prestige (moral strength, leadership, honesty, knowledge, efficiency) and power (ability to administer and lead). it is precisely in the "personal prestige" where their synergy with the scientist (also covered by knowledge, honesty and leadership) should be the lever for the improvement of the society they both serve. initiatives on proposals or projects with reference to infection issues represent less than 3% of the total. of particular relevance in recent years have been those relating to the national plan for the elimination of hepatitis c and antibiotic resistance. in order to respond to the scientific output on infection in geriatrics, we will proceed to describe the data sources, the search methodology and the findings, in a way deliberately guided by the recommendations of professionals in our workplace libraries. sciencedirect [116] is a digital platform that has provided subscription access to a large research database, hosting more than 14 million publications from 3,800 academic journals and 35,000 e-books since 1997. clinical key [117], owned by "elsevier clinical solutions", has an intelligent search system, establishing the connection of medical terms with related content. it accesses a collection of resources of clinical guides, algorithms and patient files from fisterra, the database of monographs of medicines marketed in spain, the treaties of the medical surgical encyclopaedia, and books and journals in spanish from the cited publisher. primo is the discovery/search tool used by the castilla y león healthcare online library [118] as a small demonstration of this paradox -the contrast between the rising presence of the elderly in society and their lukewarm representation in the media-, we offer a chart with a comparison of publications on the websites of three generalist newspapers, "el país", "el mundo" and "abc", between the years 2016-2018, with the search for "elderly" and "infection" as key words. a total of 96 news items are recorded that mention the subject studied ( figure 1 ). this is little news, and in most cases linked to events and to the elderly as a risk group. this sample would require further media analysis to ratify this tendency in the treatment of the problems of the elderly and the infections they suffer, but it serves as the tip of the iceberg of relegation, insensitivity and atrophy in news treatment. since the onset of the economic crisis in 2008, the number of dedicated journalists specialising in social and health issues has been substantially reduced in order to divert manpower and resources mainly to political and economic content. if, in this situation, health, science and social issues have been scaled down and cut back in the operation of the media, the elderly, as journalistic content, have been pushed to the very margins of the newsrooms with complete normality; with no agenda, no specialists, no briefings, no planning, no contextualization; to see themselves as mere circumstantial, inconsequential, occasional content, with a light, sometimes frivolous treatment, lacking depth and sensitivity; building a narrative of topics, irrelevance and disconnection from their value and presence in society. this media portrayal of the elderly is in contrast to the ageing of the population, where reliable and accurate statistics limited, deficient, incomplete, unfocused, out of context, stereotyped and with a not particularly constructive, realistic or objective bias. the elderly are invisible in the media and when they appear, the content relating to them is characterised by simplification, victimhood, dramatization and superficiality. the image that the media convey of old age is linked to inactivity, unproductiveness, seniority, illness, dependence and deterioration. old age and its problems, circumstances, needs and contributions, as a social agent and subject, are not among the priorities and themes of general media planning. other groups, sectors, actors or social issues such as immigration, feminism, equality, children, domestic violence, ngos and their services, new technologies and their advantages, effects and risks, harassment in all its forms, health and sanitation, or scientific advances have much more visibility, relevance, monitoring, currency and presence in the media. the problems related to a stage of life that we can place at around 80 years provoke a disinterest and sidelining in the information and journalism that only is unblocked in the face of news related to events, diseases, negative or sensationalist facts or anecdotes, offering a fixed, unmoving and old-fashioned image of a sector of the population that, nevertheless, is increasing due to the increase in life expectancy. in a world where the 21st century grants youth and technology all the plaudits as to what is interesting and important, whether in the press, television, radio, websites or social networks, ageing and old age, as a concept, social and population sector, and newsworthy subject, are moved to a second or third tier on the podium of current affairs and information. citations regarding "infections" in the "elderly" in 3 major general journals of spain formation on the elderly and very elderly has been strengthened, is to promote health and healthcare information in relation to this sector of the population. in this context, the media would be in a position to treat and report, with much higher presence and representation criteria than at present, on the infections of the elderly within the framework of their health and well-being. it is very difficult to reach this third step without the two previous actions, since the handling of a health problem such as infection in the elderly by the media requires a commitment and responsibility in several phases that is part of a comprehensive strategy to provide a journalistic treatment of their problems on a par with their representation and contribution to society. it is necessary to present older people and the elderly removed from the clichés and stereotypes that link them directly and almost solely to the events, the deterioration of their health, family dependence or the hindrance or burden of their role and function in society. it is necessary to offer complete and balanced information in which tasks such as interest in culture, modernity, the future, technology or travel; their capacity to lea in civil society, family, business or education; their initiative in domestic and community tasks; their political or social contributions; or their skills in the practice of sport are inherent. in short, to show their vitality, enthusiasm, enterprise, activity, determination, solidarity or collaboration, beyond their problems or difficulties, which must also be reflected and analysed. it should not be forgotten that though the generation of elderly people now over 75/80 years old may have a more traditional, reserved and passive profile in certain cases -by no means in all-, the new generation of elderly people forecast for 2050, where their number will rise greatly, will experience a huge change with regard to the distorted image of the elderly today. the information that the general media dedicates to the problems of the elderly is minimal, distorted and biased. it is full of clichés and stereotypes that link them directly and almost exclusively to events, the deterioration of their health, family dependency or the hindrance or burden of their role and function in society. information on infections in this population group is even more scarce. presentation: the answer is yes, without a doubt [95, [127] [128] [129] [130] [131] [132] [133] [134] [135] [136] [137] [138] . the reasons are detailed below: studies conducted following scientific evidence criteria in recent years show that pharmaceutical care and the intervention of the pharmacist improve the overall quality of patient care, while the who itself states that point to a doubling of the number of older people by 2050. the data show that in 1960 in spain, there were 6.7 million children under 10 years and 0.4 million people over 80 years, in 2015 the number of children under 10 had fallen to 4.6 million, and over 80 risen to 2.7 million; by 2050 the trend becomes even more acute, with under 10 years predicted at 3.8 million, and over 80, 6.2 million [123, 124] . globally, in the century from 1950 to 2050, total population will triple; the population over 60 will grow by a factor of 10; and the population over 80 by a factor of 28, this last group going from 14 million in 1950 to 386 million in 2050. if information concerning and affecting the elderly continues to be ignored, marginalised and simplified in the media, they will neglect and fail in their mission of gathering information, analysis, data and opinions from a sector of the population with enormous influence on the life and events of a country. without rigorous, truthful, balanced, comprehensive and complete information on the phenomenon of old age, the view and expression of reality will be distorted, fragmented and fractured. to help reduce infection in the elderly, the media must take several steps beforehand and activate new information strategies and actions [125, 126] . review and reformulation of the contents for current events, relevance and interest agendas. the first step is to place general social and health issues on the same level of importance as national or international political, economic or sports information, with the consequent allocation of space, dedication and resources. enhancement of content for the elderly in social and health information. within the social and health content, the news of the old and elderly must be equated in relevance, dedication, selection, monitoring and treatment to other issues related to this journalistic field, with emphasis on the quantity and quality of the information, from the rigour, planning and contextualization to gather studies and data, human stories, opinions, difficulties and needs, social influence, contributions, and challenges in this sector of the population. the aim is to offer a complete, balanced, objective and true vision of their reality, their contributions, their heterogeneity, their variety, their complexity, their evolution and their demands and needs. the problems arising from the increase in age, health, coexistence and economic situation, as well as cultural, sociological, family and psychological aspects, must be approached with an informational style and treatment where ageing is considered from the standpoint of normality in life, with its ups and downs, and not as a hindrance, obstacle or inappropriate or unsustainable expense. the social and cultural role of the elderly, their knowledge and experience, their skills and abilities, should be valued as useful and enriching elements to society. promotion of health and sanitation information in the elderly. the next step for the media, once the general in-ed following scientific evidence criteria in recent years show that pharmaceutical care and the intervention of the pharmacist improve the overall quality of patient care, while the who itself states that pharmacists "contribute decisively to the rational use of medicines". what is the administration doing and what can it do to reduce these problems? from an educational point of view? from the legislative-regulatory point of view? in order to reduce these problems, the state administration must, among other things, launch: 1. prevention strategies and measures to control the transmission of the infection. 2.-vaccination programmes in the elderly. 3.-training and information programmes for health professionals, particularly in the area of rational use of antimicrobials and promotion of the use of appropriate definitions [140, 141] pharmacists "contribute decisively to the rational use of medicines". the decision on how to treat a given infection correctly with the most appropriate antimicrobial requires detailed knowledge of microbiological, clinical and pharmacological issues, but the causes of an optimal result go beyond this and extend to the so-called non-pharmacological basis, among which the behaviours of doctors, patients and pharmacists, as well as the relationships between them, play a fundamental role. the pharmacist is one of the apices of the so-called "human factor triangle" (made up of doctor-patient-pharmacist), a mirror image of the famous "davis triangle" (antimicrobial-microorganism-host). currently, pharmaceutical care aims to obtain the maximum clinical benefit from medicines and to achieve the lowest possible risk in the use of those medicines, which entails the identification, resolution and prevention of medication-related problems (mrp): adverse drug reactions (adr), drug-drug interactions (ddi), deficiencies in physician prescription, errors in the use of medication by the patient and breaking the vicious circle so frequent in the use of antimicrobials formed by self-medication -noncompliance -storage. pharmaceutical care is a process, which includes different stages: active dispensation (supply, delivery, dispatch >>> assistance, help, care), educational advice (health advice in response to a consultation/problem or instruction on the acquisition of a medicine) and pharmacotherapeutic follow-up (documentation and registration of the activity). as far as the hospital pharmacist is concerned, it must be said that they not only participate actively in the rational use of antimicrobials from their role as an active member of the pharmacy commission and the antimicrobial committee, but also get involved on a daily basis in the prudent and correct application of antimicrobial therapy, in order to obtain the most beneficial result from the clinical point of view and the most efficient from the pharmaco-economic point of view. this implies that: the appropriate antimicrobial has been prescribed in accordance with a correct diagnosis and the special characteristics of the elderly patient, it is dispensed under the proper conditions, administered at the indicated doses, at the intervals and for the period intended, it is used with the lowest possible cost, in such a way as to prevent or minimise the development of bacterial resistance and it achieves the desired therapeutic objective. in short, both the community and the hospital pharmacist as first-level health agents play a central role in the field of therapeutic adherence and rational use of antimicrobials, proposing their use in terms of quality of treatment and considering antimicrobials not only by virtue of the active ingredient contained in the corresponding pharmaceutical specialty, but also in terms of useful information ("software"). furthermore, both must take into account that antibiotics and vaccines are the paradigm of societal treatment and the treatment or non-treatment of an individual can affect the community [139] . conclusion: the answer is yes, without a doubt. studies conduct-another precaution is the sanitation of the space in which the elderly person stays so as to make it a healthy environment, including daily cleaning of surfaces, objects and utensils, ventilation, illumination preferably with natural light, and appropriate environmental temperature and humidity [156] . the tendency to unbalanced diets, malnutrition and low fluid intake increases susceptibility to infection. it is essential to promote healthy lifestyles and to provide structured plans for eating, drinking and exercise adapted to individual needs taking preferences and health problems into account [157] [158] [159] [160] [161] [162] . another strategy is the vaccination of the elderly and carers, adjusted for age, particular situation and the approved schedule in each autonomous community [163] . although infectious diseases in the elderly do not always have obvious signs and symptoms, the caregiver detects changes in their baseline situation that may lead to a suspicion of the presence of an infectious process, so education should be provided on how to proceed in the light of this suspicion and what to do when it is confirmed. finally, it is necessary to emphasise the effective management of treatment (dose, administration and side effects) and periodically monitor therapeutic adherence, avoiding self-medication, in order to achieve the optimal effects of non-pharmacological and pharmacological measures, so as to enable prevention, delay deterioration, recover or maintain health [164] . nurses develop interventions for prevention, monitoring and therapeutic adherence control, participating in the care plan for infection in the elderly. the implementation of many of the health promotion and care plans and regulations is the direct responsibility of the nursing profession. how do senior citizens' associations deal with this problem? the issue of health is a priority for the elderly and infection in particular is one of the most frequent causes of morbidity and mortality in the elderly, as has already been mentioned. elderly associations have traditionally focused on chronic rather than acute diseases and therefore have a huge role to play in this area. it is the mission of the elderly associations to encourage and promote the residence of the elderly in a family and social environment that is agreeable to them. it is well known that an older person who lives comfortably at home with family members has less risk of acquiring infections than one who lives alone. in the case of the elderly institutionalised in residences, the elderly associations have the mission to ensure the quality tions for the prevention and control of healthcare associated infections (hais). some examples of the above are programmes such as: "antibiotics: take them seriously" (2017); the "world antibiotic awareness week" (2018); the "european antibiotic awareness day" (2018). a national plan against antimicrobial resistance (pran) run by the spanish agency of medicines and health products (aemps) is essential [142, [143] [144] [145] [146] [147] . the administration has a constitutional mandate to promote health, which is of particular concern to groups as vulnerable as the elderly. among the measures to be implemented, those of an educational nature are especially necessary, both for patients and for their caregivers and healthcare personnel. from a legislative-regulatory point of view, we cannot forget that spain has one of the best health systems in the world. what is the role of nursing in managing and reducing infection in the elderly? how does the training of the caregiver affect this? nurses develop preventive interventions, participate in the monitoring, control, therapeutic adherence and care plan when the infection is established. these competencies are developed inside and outside of healthcare institutions. in the home setting, the focus is on education and providing support for safe practices [148] [149] [150] [151] . professionals, caregivers and elderly people have to distinguish modes of transmission, identify risk factors and susceptible people who may become reservoirs or constitute a vehicle of contagion and understand basic protective and barrier measures. the simplest, most effective and universal procedure is hand hygiene. the world health organization identifies five key times for washing: before and after contact with the person, before performing a clean/septic task, after the risk of exposure to body fluids, and after contact with the patient's environment [152] [153] [154] . when hygiene guidelines are given, it is worth noting other times: before, during and after handling or preparing food, before eating, before giving medication, before and after treating a wound or handling clinical devices, after using the bathroom and after handling used clothing, whether personal, bath or bedding, diapers or waste. after washing, it is important to dry the hands. personal hygiene and topical hydration are other prevention strategies. the skin constitutes a natural protective barrier and is particularly labile in the elderly. its daily care guarantees its integrity and protects it from external assault. this includes body hygiene and protective measures aimed at moisture control and injury prevention. some studies highlight the importance of oral hygiene in relation to respiratory diseases [155] . the great social esteem that existed in ancient cultures for the elder of the group or tribe is well known. he was not only the oldest person but also the biological father, the political leader and, in many cases, the religious authority. and, as anthropologists have pointed out more than once, the "hard disk" of the community, aware of past events of which the younger generations are not, thereby bringing the social group together and giving it its own identity. hence, the elders were not only respected but highly valued and even revered. it is enough to open the books of the bible, for example, to find testimonies of this. its pages over and over again reverential respect for the elder, applying such venerable terms as "patriarch". the bible attributes an extraordinary longevity to the first patriarchs (gen 5; 11,10-26), and even to the later patriarchs, like abraham (gen 17,1.17; 18,12) and moses (dt 31,1; 34,7), and to the prophets, it is difficult to represent them as young people. respect leads the bible authors to attribute centuries-long lives to them. longevity is a sign of their wisdom. the so-called wisdom literature bears good witness to this veneration for the elderly. in the book of ecclesiasticus we read: in your youth you did not gather. how will you find anything in your old age? how appropriate is sound judgment in the grey-haired, the contrast between the ancient civilization of israel and the archaic greek culture, as presented in the homeric poems, is surprising. it is difficult to imagine ulysses, hector or achilles as elders, even though in those poems there are also venerable subjects such as menelaus, agamemnon and priam. the contrast between agamemnon and achilles is particularly significant, for the poet paints the former as an ambitious and selfish man, with an excessive ego who confronts achilles, his best warrior, again and again. heroes, those beings that the greeks considered perfect and semi-divine, are by necessity young and in the fullness of their life force. in greek statuary of these institutions, that they are equipped with the appropriate medical, nursing and social services and that a systematic accreditation of these services is achieved. ideally, these centres should have very significant prevention measures in place and should work closely, on the one hand, with the primary care physicians responsible for the patients, and on the other hand, with the reference hospitals to which the patients have to be transferred at some point. elderly associations must continue to work to improve the care of the elderly in emergency departments, not only from a technical point of view, but also by ensuring the agility of the assessment and dignified conditions for the elderly in these departments. finally, the elderly who are hospitalised are patients who require very rapid mobilization, avoidance of exposure to multi-resistant microorganisms and the fastest possible transfer back to where they came from. elderly associations promote the provision of geriatric beds and services in all hospitals, where structures and organisations are set up specifically to serve the needs of elderly patients with a comprehensive idea of their care. as we have mentioned, prevention is better than cure, and in that sense, the elderly associations can play an important role in emphasizing to the authorities, to the groups of affected people and to healthcare personnel the importance of promoting vaccination campaigns [86] in short, associations for the elderly, whether they are focused on health or not, can play a very positive role that is often overlooked when it comes to improving health. they could work, if possible, promoting and propagating vaccination campaigns. they could also contribute more than they do to other forms of health education, from those oriented towards nutrition or physical activity, to those focused on fighting toxic habits or reporting abuse. all this is of general interest, as well as directly and indirectly affecting the field of infectious pathology. following the recommendations of the expert consensus on frailty in the elderly, active ageing and drug screening in polymedicated patients are important in preventing infections in these patients. elderly associations must play a major role in demanding quality care policies for elderly patients, both in the fields of prevention and treatment. target areas for intervention are the home environment, the outpatient system, nursing homes, hospital emergency departments and hospital care. patient associations can contribute more than they do to other forms of health education, from those oriented towards nutrition or physical activity, to those focusing on combating toxic habits or reporting abuse. what ethical aspects would you highlight in all these problems? modern systems of work organisation have made "efficiency" a major objective of the culture of the second stage of life. there is no doubt that in spain, for example, efficiency has increased three or fourfold in the last half century. and here is the origin of the problem. what do you do when you are no longer "efficient", at least in the way the economy defines efficiency? efficiency is a value that belongs to the category of socalled "instrumental values", "reference values" or "technical values". they are so called as they have no value in themselves, but only in reference to something else or another value. let's think, for example, of a drug. there is no doubt that it has value, at least financially. its most valuable asset is to relieve a symptom or cure a disease. if it wasn't good enough, we'd say "it's not good enough", and we wouldn't pay for it. this means that the value of the drug is in reference to something other than itself, such as well-being, health, life, etc. this happens to all technical instruments. if we were to find a more effective or less expensive drug, there is no doubt that we would choose it, because this is what efficiency is about: the cost/benefit ratio. efficiency is the unit of measurement for instrumental values. the problem is that not everything is instrumental. if they are always in the service of others, it means that these others must stand on their own, otherwise we fall into an infinite regression. these are called "intrinsic values" or "fundamental values". they are the most important in life. they are essential values, values that have worth in their own right, without reference to others. think, for example, of dignity. or many others, such as health, life, beauty, well-being, justice, solidarity, etc. these are all intrinsic values. without them, life is meaningless [165] . furthermore, they have the characteristic of not being measured in monetary units, nor is efficiency a criterion. "health is priceless" it has always been said; "true love is neither bought nor sold"; "only the foolish confuses value and price" said antonio machado. and the list could go on [166] . we can now understand the importance of promoting a culture of old age. during our working life there is no doubt that the fundamental criterion must be efficiency, and therefore economy. but that is, at the same time, the least human part of life. the day is not far off when that part of our existence can be transferred to the robots. and the problem arises: what will we humans do then? will we have anything to do? older people have a fundamental mission in our society, and that is to take charge of promoting intrinsic values and passing them on to younger generations. it's not all about economics. it's not all about efficiency. there are other values, which moreover are the most important, the most human. conclusion: promoting a new culture of the elderly should lead us to avoid not only the discrimination that has occurred throughout western culture, and particularly in recent centuries, but also to give impetus to the promotion of intrinsic values, the most humane, the most important in the lives of individuals and societies. this is the very im-it is impossible to see the decrepitude of the elderly person represented. the poet menander coined a sentence that soon became famous and that plautus translated into latin: quem di diligunt, adulescens moritur, "those loved by the gods die young" (bacchides, 816-817). perfection is in youth, and old age is almost embarrassing. aristotle says that "disease is an acquired old age, old age a natural disease" (gen. an. 784 b . it was important to remember this about the attitude of our culture, the western one, towards the elderly. they've never been held in high esteem. moreover, we can be seen that this esteem has been decreasing over time. this is demonstrated by the words we use to refer to this age group. "viejo" (old) comes from the latin vetus, the opposite of novus, both of which are terms that were designating things, not people. for people, the correct terms were senex and its opposite iuvenis. from senex comes our word "senescence", only used in a very limited sense today. cicero wrote a dialogue de senectute, using the correct term in his language. though, in the various spanish editions that exist, the translation is invariably sobre la vejez. (on old age). old age is not only an improper term, but also a derogatory one. no one sees it that way anymore, because they don't know about this process. but the transition from one term to another is an evident sign of the devaluation that the figure of the elder has undergone in western culture, even though it was originally already much lower than that of other cultures. if we add to this the spectacular increase in life expectancy at birth in the last century, it turns out that this devalued period, which until the beginning of the 20th century was almost anecdotal in the life of western society (it should be remembered that life expectancy at birth in spain had been stable at 25-30 years from the neolithic revolution to the end of the 19th century), has become a period of no lesser and sometimes greater duration than the active life of a person. so much so that human life today can very well be divided into three 30-year periods, the first of which is devoted to vocational training, the second to production, and the third.. it is not very clear to what, among other things, because the training we were given in the first 30 years was aimed at being productive in the second phase, but we were never educated for the "third age". the third and final phase of life, which today has an average duration of 30 years, is a continuous source of problems. it is, at least, in the economic order, as the present pension system seems difficult to maintain, and will be impossible in the near future. but, as important as this is, that's not the biggest problem. the most serious issue is that we have condemned the elderly to being a "passive class", whom inserso (the institute for the elderly and social services) has to ferry from one place to another in order to at least distract them. there is talk of discrimination and abuse of the elderly. in my opinion, the greatest discrimination is this, the fact that the elderly have been deprived of their own role in society; or, 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infections in long-term care facilities: revisiting the mcgeer criteria comunidad de madrid. prevención y control de las enfermedades transmisibles en atención primaria. gráficas monterreina s a plan nacional frente a resistencia a antibióticos plan nacional frente a la resistencia a los antibióticos (pran) comunidad de madrid. guía de uso de antimicrobianos en adultos con tratamiento ambulatorio programa marco para el control de las resistencias a los antimicrobianos en la comunidad de madrid (resiste) resolución nº 20/2017, de 14 de diciembre de 2017, por la que se ordenan determinados aspectos del ejercicio profesional enfermero en el ámbito de la prevención y control de infecciones infection prevention and control in households nursing challenges and implications guía de aplicación de la estrategia mundial de la oms para la mejora de la higiene de las manos y del modelo "los cinco momentos de la higiene de manos medidas de prevención de la trasmisión de organismos entre pacientes hospitalizados. higiene de manos the hygienic efficacy of different hand-drying methods: a review of the evidence the authors declare that they have no conflict of interest. this publication has been funded by glaxosmithkline. key: cord-320145-582kmoyo authors: cardinal, r. n.; meiser-stedman, c. e.; christmas, d. m.; price, a. c.; denman, c.; underwood, b. r.; shanquan, c.; banerjee, s. n.; white, s. r.; su, l.; ford, t. j.; chamberlain, s. r.; walsh, c. m. title: simulating a community mental health service during the covid-19 pandemic: effects of clinician-clinician encounters, clinician-patient-family encounters, symptom-triggered protective behaviour, and household clustering date: 2020-05-03 journal: nan doi: 10.1101/2020.04.27.20081505 sha: doc_id: 320145 cord_uid: 582kmoyo background. face-to-face healthcare, including psychiatric provision, must continue despite reduced interpersonal contact during the covid-19 (sars-cov-2 coronavirus) pandemic. community-based services might use domiciliary visits, consultations in healthcare settings, or remote consultations. services might also alter direct contact between clinicians. aims. we examined the effects of appointment types and clinician-clinician encounters upon infection rates. methods. we modelled a covid-19-like disease in a hypothetical community healthcare team, their patients, and patients' household contacts (family). in one condition, clinicians met patients and briefly met family (e.g. home visit or collateral history). in another, patients attended alone (e.g. clinic visit), segregated from each other. in another, face-to-face contact was eliminated (e.g. videoconferencing). we also varied clinician-clinician contact; baseline and ongoing "external" infection rates; whether overt symptoms reduced transmission risk behaviourally (e.g. via personal protective equipment, ppe); and household clustering. results. service organization had minimal effects on whole-population infection under our assumptions but materially affected clinician infection. appointment type and inter-clinician contact had greater effects at low external infection rates and without a behavioural symptom response. clustering magnified the effect of appointment type. we discuss infection control and other factors affecting appointment choice and team organization. conclusions. distancing between clinicians can have significant effects on team infection. loss of clinicians to infection likely has an adverse impact on care, not modelled here. appointments must account for clinical necessity as well as infection control. interventions to reduce transmission risk can synergize, arguing for maximal distancing and behavioural measures (e.g. ppe) consistent with safe care. many countries have reduced interpersonal contact to control infection during the covid-19 pandemic. the uk implemented interpersonal distancing on 16 march 2020 and "lockdown" on 23 march; its national health service (nhs) has reduced non-critical work and moved towards videoconferencing and telephone assessments where possible (1) . however, some face-to-face consultations remain necessary, including for urgent mental or physical health needs. for community-based health services -including physical care teams, community mental health teams (cmhts), mental health crisis teams, and other urgent care servicesthis raises the important question of how to structure patient contacts and clinical teams to minimize infection. covid-19 is a respiratory infection transmitted directly by airborne aerosols/droplets from an infectious person and indirectly via contaminated objects (fomites) (2) . asymptomatic people may be infectious (2, 3) . airborne transmission is reduced by asymptomaticity (4), physical distance, time, and personal protective equipment (ppe) (2) , and increased by symptoms such as coughing and aerosol-generating procedures (2) . fomite transmission is reduced by handwashing, cleaning/disinfection, and time, which cause viral inactivation (2, 5) . should community services bring patients to a central base (e.g. clinic) or visit patients at home? as of 8 april 2020, uk guidance advised ppe only for contact with patients having suspected or confirmed covid-19, rather than for all patients (6) . primary care and outpatient settings should segregate covid-19 and other patients in time or space, and allocate staff to one group or the other where possible (2) . if we assume that clinic settings have appropriate infection control procedures in place, such as physical separation and cleaning, then a key difference between this and home visits is exposure of the clinician to the home environment. this additional exposure includes other household members and fomites. does this pose a risk of increased transmission to the clinician and wider community? is this influenced by different models of service provision? other questions relate to clinician-clinician encounters, and the impact of clinical activity on disease transmission to patients and families. to what extent do clinician-clinician and clinician-patient encounters affect spread? this may be important not only for the infection of clinicians, with implications for healthcare capacity, but because clinicians may be in contact with more people than most in a time of "social distancing" and thus might have the potential to infect a relatively large number of patients -one aspect of concern about "super-spreading" (7) . clinicians may also work with patients particularly vulnerable to covid-19. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 3, 2020. . https://doi.org/10.1101/2020.04.27.20081505 doi: medrxiv preprint epidemiologists have modelled covid-19 spread across populations, and social measures to reduce spread given limited critical care capacity (4) . however, we were unable to find assessments of the impact of community consultation strategy or clinician-to-clinician contacts on disease spread (via pubmed to 29 march 2020 or via enquiries on 24 march to the nhs sustainability and transformation partnership for cambridgeshire). we therefore simulated a population of clinicians, patients, and patients' families via agent-based modelling (8) , seeding the population with an infectious disease with the approximate characteristics of covid-19. we examined spread under different conditions of interpersonal contacts, representing alternative ways of organizing community health services for necessary appointments. we report these simulations and make suggestions for infection control strategies applicable to such services. we took a broad approach because there are insufficient high-quality data to infer reliable parameters for many aspects of a complex model. uncertainties include: the infectivity profile over time; airborne transmission rates by contact type and time; the risks of fomite (surface) transmission in different contexts; many details of the network of patient and household community contacts (including community mixing in rural and urban areas, the use of public versus private transport, and the mix of patient residence between private homes and care facilities); the proportion of people in patients' households who must continue to work; the consequences of antigen and antibody testing; the likelihood of serious morbidity or death following infection; etc. therefore, we used a standard infectious disease model and applied simple "servicelevel" manipulations, including appointment type (varying the degree to which clinicians met patients and their family directly) and whether clinicians met each other. we tested the robustness of these effects by varying other inputs to summarize a wide range of unknown factors, such as the baseline and ongoing infection rates, and a form of inter-patient association or clustering. furthermore, clinical care has competing objectives: to deliver optimal healthcare, to safeguard patients and staff from infection, to ensure the service is robust to staff shortages, to provide continuity of care, etc. an action that improves infection control may have other adverse effects. we did not model a mixed objective function explicitly. instead, we focus on infection rates (including clinician infection rates) and discuss other potential effects of the service-level manipulations examined. fixed patient and disease-process parameters. we used a "susceptible-exposed-infectious-recovered" (seir) model (9) , modified to distinguish the symptomatic and infectious periods (figure 1a) , modelling individuals stochastically (appropriate for small populations). everyone began "susceptible". people were randomly assigned to become symptomatic (if infected) with probability p=0.67, based on influenza (10) and close to the 69% (confidence interval 46%-92%) estimated for sars-cov-2 (11) . otherwise, infection cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 3, 2020. . https://doi.org/10.1101/2020.04.27.20081505 doi: medrxiv preprint would lead to asymptomatic infectiousness. people became infectious 4.6 days after infection (4) . they remained infectious for 7 days, an uncertain value based on (12, 13) ; we used a simple model without temporal variation in infectivity during that time. if symptoms developed, they began 5.1 days after infection (4, 14, 15) and lasted 7 days (12) . recovery precluded re-infection (also uncertain). the transmission risk when a susceptible person was exposed to a symptomatic infectious individual was assumed to be p=0.2 for 24 hours' exposure. we reduced this proportionally for shorter periods of exposure: a process with a constant rate of infection (or half-life τ for remaining uninfected), e.g. pinfection(t) = 1 -0.5 t/τ , is approximately linear in this range. this value is uncertain: the basic reproduction number (r0) estimated for covid-19 (16) (17) (18) implicitly incorporates the duration of infectiousness, the interpersonal contact rate, and the transmission risk per contact (itself depending on contact duration and transmission risk per unit time) (19) . an r0 value of 2-3 (16) would be roughly equivalent to p=0.2 per 24h exposure if, for example, an infectious person infected 0.35 people/day for 7 days (2.5 people in total), e.g. via contact with susceptible others for 40 person-hours (e.g. 10 people, 4h each) per day. asymptomatic people were 50% as infectious biologically as their symptomatic counterparts (4, 10) . these values may be biologically inaccurate, and we did not implement realistic intersubject variability in these parameters. more sophisticated models exist (20) . however, the absolute rate of infection was not our chief concern (as above); rather, we focused on the effect of service arrangements (described below). relative differences due to service organization were assumed to be ordinally independent of the absolute transmission risk or other biological parameters. fixed population parameters. we simulated encounters between clinicians, patients, and patients' household contacts ( figure 1b) . a team of 20 clinicians was simulated, representing a multidisciplinary team (mdt). each clinician saw 5 new patients per day, without follow-up appointments. every clinician-patient interaction lasted 1h, based on a common uk "new patient" appointment slot in psychiatry. patients were assumed to come from independent households. patients could live with others (referred to here as family); the number of family members per patient was drawn from a poisson distribution with λ=1.37, from the mean uk household size of 2.37 (21) . everyone in a household was assumed to interact daily and every household contact was assumed to last 8h/day. clinicians' households were not simulated, for simplicity (though the likely effect of adding clinician households would be to increase clinician infection rates slightly). people were assumed to follow household social isolation, i.e. with the exception of clinician encounters, there were no explicit household-household contacts. in addition, "external" infection was simulated (see below). if a clinician became symptomatic, they were assumed to cease work for the duration of their symptoms and not meet others in the model during that time. their appointments were reassigned to other clinicians (who cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 3, 2020. . https://doi.org/10.1101/2020.04.27.20081505 doi: medrxiv preprint therefore saw more patients, without limit). symptomatic patients and family continued to interact with each other, and symptomatic patients were assumed still to require clinical care. we simulated a population of mean size 14,240 (20 clinicians, 6,000 patients, and on average 8,220 family members) for 60 consecutive days (1-60, with no allowance for weekends). days were simulated discretely; i.e. contacts were simulated (effectively) simultaneously for a given day, without regard to time of day. manipulations. the following aspects were varied, in all possible combinations. variable ranges were chosen to represent plausible extremes. • appointment type ("at"). in the "patient only" (po) condition, clinicians interacted only with the patient. this was intended to represent patients coming alone to a clinic, not interacting with others, with physical distancing and decontamination of clinic environments. in the "family contact" (fc) condition, clinicians also interacted for 0.2h (12 minutes) with each family member of the patient. this was intended to represent a home visit, but might also represent family accompanying the patient to a clinic and being present for a collateral history. in the "remote visit" (rv, e.g. videoconferencing) condition, no direct clinician-patient or clinician-family contact occurred. • clinician-clinician meetings ("cm"). in the "clinician meeting" condition, all clinicians met up for 1h per day (e.g. for a handover or mdt meeting). in the "no clinician meeting" condition, clinicians did not interact with each other in person (e.g. used videoconferencing instead). • baseline infection rate ("bl"). on day 0, the day before interpersonal contact simulation began, either 1% or 5% of the population were infected at random, reflecting approximate confidence interval (ci) extremes for the uk, 28 march 2020 (22) . clinicians and others had an equal chance of initial infection. • external infection ("ex"). every person had a 0%, 0.5%, 1%, or 2% chance each day of becoming infected from external sources (e.g. in supermarkets, on public transport, etc.). the external infection rate is not constant in an epidemic, being affected by prevalence and the rate of external contacts; the upper figure of 2% was chosen to represent a very high value (e.g. if 10% of "external" people were infectious and each modelled person had 24 person-hours of "external" contact per day at transmission risk p=0.2 per person-day exposure as above). • protective behavioural effect of symptoms upon transmission risk ("sx"). being symptomatic has biological consequences (modelled above) but also social consequences. for example, overt illness may increase physical distancing or cause clinicians to use ppe, reducing infection risks. we chose values to represent plausible extremes of any such effect. in one condition, being symptomatic had an effect to reduce transmission risk to 10% of what it would otherwise have been ("behavioural symptom effect present"); the relative risk of infection with h7n7 avian influenza a is approximately 9% when using a respirator (23) . alternatively, the transmission risk was unmodified ("behavioural symptom effect absent"). if present, this effect applied to all interpersonal contacts page 5 of 18 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 3, 2020. . (which may be unrealistic in that family members are unlikely to use ppe with each other; thus, any such behavioural effects may be smaller than those modelled). simulation. we simulated each condition 2000 times, using python (https://www.python.org/). we used r (https://www.r-project.org/) to analyse the total number of (a) people and (b) clinicians infected by the end of the virtual experiment. we used a generalized linear model (glm) with a poisson distribution and a log link function, then analysis of deviance with type iii sums of squares and α=0.05. however, the statistical power of a simulation is arbitrary (being determined by the effect size and the number of runs); thus, we present cis and do not report exact p values, reporting instead "p<α" for 10 -3 ≤p<0.05; "p≪α" for 10 -5 ≤p<10 -3 ; "p⋘α" for p<10 -5 . a significant interaction term implies non-additive effects of factors on the linear predictor, but because the predictors were on a log scale with respect to the dependent variable, interactions imply non-multiplicative effects of experimental factors on the number of people infected. not all patients live in small households: some live in much larger shared living facilities, such as care homes. clustering might be used as a route out of "lockdown". does such patient clustering amplify any infection-transmitting effect of clinicians, who are unusually mobile people during times of "lockdown" and may travel between clusters? we examined whether the effects of appointment type and clinician meetings depended on the degree of patient clustering in such "hubs", which we modelled as large households with multiple patients and their virtual "families" (or, similarly, care staff). we implemented this simply, by aggregating virtual households: thus, n=1 patients plus their family (household) per cluster as in experiment 1, or n=2 households per cluster, etc. for simplicity, all co-residents were assumed to interact with each other, as patients and family would in a single-patient household, and clinicians were assumed to have "family-level" contact with everyone else in the cluster during fc-type visits. the selection of patients requiring a visit on any one day was random; those patients were then assigned cyclically to available (not sick) clinicians. we varied the number of patients per household ("nph", 1-10, as a discrete predictor), at, and cm. we held all other parameters constant at values suggested by experiment 1 to give high power to detect such effects (no behavioural symptom effect; bl 1%; ex 0%). to examine the basis of the synergy between interventions observed in experiments 1-2, we ran deterministic plain seir models using the epidynamics r package. this form of model represents seir state changes via differential equations governed by rate parameters. it does not consider the timing, symptom, or interpersonal contact structure used in our agent-based model. we varied the proportion page 6 of 18 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 3, 2020. . https://doi.org/10.1101/2020.04.27.20081505 doi: medrxiv preprint exposed at time t=0 (1% or 5%, cf. experiment 1) and the transmission rate β. constants were: tfinal=1000 (for asymptote); birth/death rate μ=0; exposed-to-infectious rate σ=1/5; recovery rate γ=1/7. we examined the cumulative proportion infected (1 -susceptible). whole-population infection rates. in experiment 1, whole-population infection was dominated by baseline and external infection rates (with infection spreading primarily via intra-household contacts), plus the behavioural response to symptoms (all p⋘α), with only very small contributions from the appointment type and clinician-clinician meetings (figure 2a) . that is, neither appointment type nor clinician meetings had any appreciable effect on the total number of people infected. appointment type and clinician meetings had effects (e.g. at×cm×bl×ex×sx interaction, p<α), but these effects were very small (the overall difference in the proportion infected was 0.01 percentage points between fc and rv conditions; figure 2a) . the beneficial effects of symptom-induced protective behaviour were proportionally greater in conditions with lower external infection rates (bl×ex×sx, p⋘α; figure 2a) . effects of appointment type on clinician infection. in contrast, appointment type had more substantial effects on clinician infection rates (figure 2b) , which depended upon external infection rates and the behavioural symptom effect (at, p⋘α; at×ex×sx, p⋘α). as expected from the number of contacts involved, infection rates were consistently ranked "family contact" (e.g. home visit) > "patient only" (e.g. clinic) > "remote" for all conditions. however, these effects were lessened at high external infection rates. we figure 2b) . there was a strong effect of clinician meetings, more pronounced at low levels of external infection (figure 2b ; cm, p⋘α; cm×ex, p⋘α). we calculated the effect's magnitude as "new infections without clinician meetings ÷ new infections with clinician meetings" ("new infections" defined as before). the effect of eliminating clinician meetings was much larger with external infection at 0% (rv 0%, po 36%, fc 38%) than at 2% (rv 94%, po 95%, fc 95%), and numerically larger with a behavioural symptom effect (e.g. for 0% ex and po appointments, eliminating clinician meetings cut new infections to 25% [of that with meetings] when there was a behavioural symptom effect, but only to 47% without that behavioural effect). . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 3, 2020. effects of symptom-related behaviour on clinician infection. symptom-triggered behaviour had substantial effects on clinician infection rates (figure 2a,b ; sx, p⋘α; at×ex×sx, p⋘α), as it did for wholepopulation rates. the beneficial effects of symptom-triggered behaviour were proportionally greater with lower external infection rates, for higher-risk appointment types, and without clinician meetings. impact of patient clustering. figure 2c ,d shows experiment 2's results. predictably, greater clustering increased infection rates (whole-population and clinicians, nph, p⋘α). the effects of appointment type and clinician meetings on whole-population infection (at×cm×nph, p⋘α) were very small. the effects of appointment type on clinician infection were substantially magnified by greater clustering (at×nph, p⋘α). synergy between service manipulations. many of the effects of the modelled variables upon clinician infections were synergistic. preventing physical clinician meetings had a greater proportional effect when external infection rates were lower, and with a behavioural protective response to symptoms (as above). moving to lower-contact appointment types had a greater proportional effect when external infection rates were lower. not all manipulations were synergistic (e.g. appointment types had a greater proportional effect without a behavioural response to symptoms, but both manipulations were nonetheless helpful). in a simple deterministic seir model, linear changes in transmission rate had nonlinear effects on the cumulative infection rate (figure 2e) , which is consistent with the synergies observed in the full model. we modelled a hypothetical community clinical team, under different baseline and external infection rates. the fictional team organized its patient assessments in controlled environments in which only clinicianpatient contacts occurred (e.g. managed clinics), visits in which some clinician-family contacts occurred (e.g. home visits or family present for a collateral history), or remote assessments in which the clinician and patient did not meet physically (e.g. videoconferencing). clinicians met daily in person, or refrained from doing so. under our assumptions, these service arrangements had only a very small impact on infection rates across the population studied (clinicians, patients, and family together; figure 2a,c) , but some had a substantial effect on clinician infection rates (figures 2b,d) . clinicians may sometimes be a scarce resource, so higher clinician infection rates may have wider adverse effects on population health through lack of clinician availability. behavioural measures to reduce transmission in response to overt symptoms also had substantial effects on infection rates, despite a period of "silent" infectiousness and some infectious people never exhibiting symptoms. in our model, the infection risk to clinicians of appointment type directly reflected the degree of contacts with patients and family. this was expected, but we have quantified the relative importance of structural service manipulations. eliminating daily face-to-face clinician-clinician meetings also had a noticeable effect on clinician infection rates, which was most pronounced, proportionally, with the lowest-risk patient page 8 of 18 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 3, 2020. . https://doi.org/10.1101/2020.04.27.20081505 doi: medrxiv preprint encounter types. combined risk reduction methods interacted with each other, sometimes having more than a multiplicative effect, and were disproportionately more effective than each alone. all these effects lessened with increasing rates of infection from outside the modelled population. patient clustering increased wholepopulation infection and magnified the effect of appointment type on clinician infection, even for "patientonly" appointments. in our model, it was possible for multiple clinicians to visit a cluster; restricting which clinicians visit which clusters may be another important factor to consider. clustering or "hub" effects may also occur in other ways not modelled here -such as patients being in receipt of multiple services from one or several provider organizations -and would similarly serve to increase transmission further. changes in "lockdown" practices may affect external infection rates or clustering, requiring services to adapt to changing public health policy. multiple small improvements in infection control can have a total effect greater than the sum of its parts (figure 2e) . these results emphasize and quantify an obvious point that minimizing contact with additional people, such as household contacts of a patient, contributes to infection control. family contacts might occur during a home visit, but also if family accompany patients to a clinic. videoconferencing or other remote assessment obviously provides the best infection control of the methods modelled here. however, there are trade-offs between infection control and clinical care for different appointment types, which must be judged by individual teams and clinicians. in particular, the choice between home visits and clinic assessments is complex and goes beyond the "family contact" aspects modelled here ( table 1) . other infection control differences include exposure to others during transport (likely favouring home visits by clinicians), and the risk of fomite transmission in either environment (hard to quantify, but potentially less predictable and greater for home visits). relevant clinical differences go beyond infection control ( table 1) . a striking result was the degree to which clinician-clinician meetings affected clinician infection rates, in some cases synergistically with other infection control measures. current uk guidelines (2) include patient segregation, and segregation of primary care staff for covid-19 and other patients, but do not currently recommend the segregation of all clinical staff, or all staff, from each other as far as is possible. reducing contact between staff may be practical. dividing a clinical team into subteams may provide partial benefits ( table 2) . fomite transmission is also a risk to clinical teams: precautions available to clinical teams against fomite transmission overlap with but have some distinct elements from those for airborne transmission ( table 2) . the biological parameters we took for covid-19 spread were estimates from the published literature and in some cases are subject to high uncertainty; likewise our estimates of initial and ongoing infection rates. disease parameters were constant across subjects, other than symptomaticity given infection, which was stochastic. a paucity of contacts outside the household is highly atypical but corresponds to current uk page 9 of 18 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 3, 2020. . https://doi.org/10.1101/2020.04.27.20081505 doi: medrxiv preprint policy, if not necessarily universal practice. the behavioural effect of symptoms upon transmission risk was modelled in the same way for household contacts as for clinician-patient contacts, which is unrealistic in that clinicians are more likely to have access to ppe and rules mandating its use. clinicians' households were not modelled and would tend to increase clinician infection rates (particularly if clinicians share a household). we also modelled a series of one-off patient assessments; many patients, of course, are seen repeatedly by their clinical teams, or see many different healthcare teams routinely. however, all these aspects were constant across conditions. more important are limitations relating to differences between conditions. in the "family contact" (e.g. home visit) condition, we made assumptions about the duration of contact with family members, including that this plausibly encompassed the degree of surface as well as airborne transmission, and we assumed clinicians were not fomite vectors for direct transfer of virus between homes. in the "patient only" (e.g. clinic) condition, we assumed full segregation and cleaning between patients, with no inter-patient virus transfer. in the "no clinicians meeting" condition, we assumed a lack of fomite transmission between clinicians. any of these may be unrealistic. fomite transmission in either situation would likely increase infection rates and alter the impact of appointment type upon infection. we make our source code open for others to test different assumptions. staff segregation, as well as appointment type and additional protective measures when meeting overtly symptomatic people, may have important effects on covid-19 transmission in community clinical teams. infection control manipulations can synergize with each other, suggesting that maximal implementation of such measures should be adopted to the degree possible. appointment types must nevertheless meet the clinical need as well as infection control guidelines. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 3, 2020. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 3, 2020. . where possible. ► remote assessment where possible. ► "clean to dirty" progression as above. page 15 of 18 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 3, 2020. . figure 1 . overview of simulation methods. (a) individual people followed a "susceptible-exposedinfectious-recovered" model, with full immunity after recovery. whether an infected person developed symptoms or not was probabilistic (see methods) and if they developed symptoms, this occurred slightly after the infectious period began. (b) interaction between people (see methods). solid lines show fixed interactions; dashed lines show interactions that were varied. patients interacted with their household (family) members and family members interacted with each other. family sizes varied. in different conditions, clinicians did or did not interact directly with each other. clinicians interacted with their patients, except in the "remote visit" condition. in the "family contact" (e.g. home visit) condition, clinicians also interacted briefly with family (not all such interactions drawn). the initial level of infection and the rate of ongoing "external" infection (red arrows) were varied. additional manipulations, not shown here, included whether symptoms altered infectiousness in a behavioural, protective way (in contrast to an ever-present biological exacerbating way) and, in experiment 2, whether patient households were grouped together in larger clusters with mutual interaction. dotted line, 5% initially exposed). this effect underlies the synergy between measures that can be taken to reduce the transmission of infection: small changes in transmission risk sometimes cause dramatic changes in total infection. [rv, remote visits; po, patient only; fc, family contact. logarithmic scale. error bars/ribbons are 95% cis. "behav. sx effect" refers to the behavioural effect upon transmission when an infected person shows symptoms; either this reduces the transmission risk to 10% of its former value, e.g. via enhanced physical distancing or ppe, or has no effect.] page 16 of 18 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 3, 2020. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 3, 2020. clinicians meet each other next steps on nhs response to covid-19 covid-19: guidance for infection prevention and control in healthcare settings presumed asymptomatic carrier transmission of covid-19 impact of non-pharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents public health wales, health protection scotland, public health agency northern ireland, nhs. recommended ppe for primary, outpatient, community and social care by setting, nhs and independent sector the epidemiology of the outbreak of severe acute respiratory syndrome (sars) in hong kong--what we do know and what we don't agent-based modeling: methods and techniques for simulating human systems the mathematics of infectious diseases modeling targeted layered containment of an influenza pandemic in the united states estimation of the asymptomatic ratio of novel coronavirus infections (covid-19) self-isolation of you or someone you live with has symptoms viral dynamics in mild and severe cases of covid-19 early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application the reproductive number of covid-19 is higher compared to sars coronavirus estimation of the reproductive number of novel coronavirus (covid-19) and the probable outbreak size on the diamond princess cruise ship: a datadriven analysis preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak complexity of the basic reproduction number (r0) quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing dataset: families and households estimating the number of infections and the impact of non-pharmaceutical interventions on covid-19 in 11 european countries effectiveness of personal protective equipment and oseltamivir prophylaxis during avian influenza a (h7n7) epidemic, the netherlands perioperative covid-19 defense: an evidence-based approach for optimization of infection control and operating room management managing infection risks when handling the deceased: guidance for the mortuary, post-mortem room and funeral premises, and during exhumation (hsg283) [internet]. the stationery office key: cord-351319-ylg93l9q authors: evers, dorothea; van der bom, johanna g.; tijmensen, janneke; middelburg, rutger a.; de haas, masja; zalpuri, saurabh; de vooght, karen m. k.; van de kerkhof, daan; visser, otto; péquériaux, nathalie c. v.; hudig, francisca; zwaginga, jaap jan title: red cell alloimmunisation in patients with different types of infections date: 2016-08-18 journal: br j haematol doi: 10.1111/bjh.14307 sha: doc_id: 351319 cord_uid: ylg93l9q red cell alloantigen exposure can cause alloantibody‐associated morbidity. murine models have suggested that inflammation modulates red cell alloimmunisation. this study quantifies alloimmunisation risks during infectious episodes in humans. we performed a multicentre case–control study within a source population of patients receiving their first and subsequent red cell transfusions during an 8‐year follow‐up period. patients developing a first transfusion‐induced red cell alloantibody (n = 505) were each compared with two similarly exposed, but non‐alloimmunised controls (n = 1010) during a 5‐week ‘alloimmunisation risk period’ using multivariate logistic regression analysis. transfusions during ‘severe’ bacterial (tissue‐invasive) infections were associated with increased risks of alloantibody development [adjusted relative risk (rr) 1·34, 95% confidence interval (95% ci) 0·97–1·85], especially when these infections were accompanied with long‐standing fever (rr 3·06, 95% ci 1·57–5·96). disseminated viral disorders demonstrated a trend towards increased risks (rr 2·41, 95% ci 0·89–6·53), in apparent contrast to a possible protection associated with gram‐negative bacteraemia (rr 0·58, 95% ci 0·13–1·14). ‘simple’ bacterial infections, gram‐positive bacteraemia, fungal infections, maximum c‐reactive protein values and leucocytosis were not associated with red cell alloimmunisation. these findings are consistent with murine models. confirmatory research is needed before patients likely to develop alloantibodies may be identified based on their infectious conditions at time of transfusion. probably modulate alloimmunisation. identification of such factors might enable allocating extensively matched blood principally to high-risk patients. experimentally induced inflammation has consistently been demonstrated as a major determinant of red cell alloimmunisation in mice (hendrickson et al, 2006 (hendrickson et al, , 2007 hendrickson, 2008; smith et al, 2012) . in line with this, proinflammatory conditions related to sickle cell disease as well as febrile reactions to donor platelets were shown to enhance alloimmunisation in humans (yazer et al, 2009; fasano et al, 2015) . apart from one case report (hata et al, 2013) to the best of our knowledge, the influence of infection-associated inflammation on red cell alloimmunisation in humans has not been reported. in this nested case-control study, we quantified relative alloimmunisation risks for patients receiving red cell units during an infectious episode, according to the type of infection, its intensity, and the patient's inflammatory response to it. we performed a nested case-control study within a source population of previously non-transfused and non-alloimmunised patients in three university hospitals and three reference hospitals in the netherlands. using this design, we compared patients who developed red cell alloantibodies following transfusion with non-alloimmunised controls on the basis of supposed causal attributes, including various types of infections. details on the source population, including its eligibility criteria, and our case-control study design have been previously published (zalpuri et al, 2012a; evers et al, 2016) . to summarize, patients were eligible if they received their first red cell transfusion during the study period in one of the participating hospitals, provided this transfusion was preceded by a negative antibody screen and followed by an antibody screen, hereby permitting evaluation of alloantibody development. the study period per hospital depended on electronic availability of the necessary data between 1 january 2005 and 31 december 2013 (for details, see data s1). all red cell units were prepared by buffy-coat depletion of whole blood donations, subsequently filtered through a leucocyte depletion filter, and stored in saline, adenine, glucose, and mannitol (sagm) for a maximum of 35 days (cbo, 2011) . a patient was defined as a case upon developing a first, transfusion-induced red cell alloantibody directed against one of the following antigens: c, c, e, e, k, c w , fy a , fy b , jk a , jk b , lu a , lu b , m, n, s or s. anti-d immunised patients were not taken into consideration because we were unable to discriminate whether anti-d was caused by unmatched transfusions, or (mainly regarding fertile women) was due to recent anti-d administration in the context of a d-positive pregnancy or transfusion. patients who formed antibodies, yet either lacked exposure to a (documented or assumed) antigen-positive red cell unit or expressed the antigen themselves (i.e. auto-immunised patients) were deemed ineligible. in addition, alloimmunised patients were excluded if their first-time alloantibody positive screen occurred within 7 days of the first mismatched transfusion, as these were more likely to represent boosting of earlier primary immunisations. by consulting the nationwide alloimmunisation registry (http://www.sanquin.nl/producten-diensten/diagnostiek/trix), we additionally excluded patients previously diagnosed with alloimmunisation in other hospitals. considering the abovementioned criteria, we specifically aimed to exclude previously alloimmunised patients, including pregnancy-induced immunisations in women. finally, haemoglobinopathy patients and infants below 6 months of age were not included. each eligible case was matched to two randomly selected non-alloimmunised control patients based on the hospital and on the (lifetime) number of red cell transfusions received at the time of alloimmunisation. this 'incidencedensity sampling strategy' ensured that controls were exposed to at least the same amount of transfusions as their matched cases and thus formed a representative sample of the source population (rothman, 2007) . for all cases, we assumed that the last antigen-mismatched transfusion (the 'nth' or implicated transfusion) preceding the first positive screen most likely elicited alloimmunisation. if this last mismatched transfusion could not be identified due to incomplete typing of donor units, we assumed the last nontested unit preceding the first positive screen by at least 7 days to have elicited alloimmunisation. an 'alloimmunisation risk period' was then constructed, stretching from 30 days before up to 7 days after this implicated nth transfusion. a similar risk period around the nth transfusion was determined for the matched controls. the implicated transfusion and its alloimmunisation risk period are illustrated in fig 1. for all cases and controls, we recorded various clinical conditions during the alloimmunisation risk period. the study protocol was approved by the ethical review board at the leiden university medical centre in leiden and by the local board of each participating centre. patients in the netherlands are routinely screened for red cell alloantibodies at a maximum of 72 h prior to red cell transfusion. according to the dutch transfusion guideline (cbo, 2011), commercially available 3-cell screening panels are required to be homozygous positive for d, c, c, e, e, k, fy a , fy b , jk a , jk b , m, s and s. the presence of the k antigen in its heterozygous form is a minimum requirement. the presence of c w , lu a , wr a , and kp a is not mandatory on commercially available screening cells (cbo, 2011 we gathered routinely stored data on red cell transfusion dates, dates and results of antibody screens (including antibody specificity), patients' date of birth, sex and leucocyte counts from the hospitals' electronic laboratory information systems. in addition, we examined the medical charts of all cases and controls for the presence of various potential clinical risk variables during the alloimmunisation risk period, including dates of infection, the causative microorganisms, dates of fever (temperature ≥38á5°c), leucocyte counts, and c-reactive protein (crp) values. bacterial infections comprised tissue-invasive infections (i.e. involving an anatomic site location) and bacteraemia (i.e. involving positive blood cultures). tissue-invasive bacterial infections were considered present when confirmed by either a positive blood or tissue culture, or when a suspected clinical infectious phenotype was supported by an overtly disease-specific radiographic anomaly e.g. a clear lobar consolidation on a chest x-ray in a patient with fever and cough. we categorized these infections into 'mild' or 'severe' according to their expected degree of systemic inflammation. mild tissue-invasive bacterial infections included: routine (tip) cultures from central catheters, catheter-induced phlebitis, lower urinary tract infections, bacterial enteritis, skin and superficial wound infections, and upper respiratory tract infections. 'severe' tissue-invasive bacterial infections included: abscesses, intra-abdominal infections including spontaneously or secondarily infected abdominal fluid collections, arthritis, bursitis, myositis, fasciitis, infected haematoma, bacterial meningitis, deep wound or skin infections, endocarditis, mediastinitis, pericarditis, infected foreign material, lower respiratory tract infections, osteomyelitis, spondylodiscitis and upper urinary tract infections. bacteraemia were categorized according to their grampositive or gram-negative causative microorganism. for the qualification of a viral infection, a positive polymerase chain reaction (pcr) test demonstrating the replication of viral rna or dna was needed or, in case a pcr test was not performed, the clinical condition needed to be clearly virally induced, e.g. herpes labialis. viraemia and disseminated viral zoster infections were defined as 'disseminated viral infections', in contrast to 'local viral infections', which were restricted to one anatomic site location. the associations of various infections with the development of red cell alloimmunisation were evaluated using logistic regression analyses. for crude relative risk (rr) calculations, we conditioned on the matched variables, i.e. hospital and cumulative number of red cell units received. for multivariate analyses, we also conditioned on measured confounders, taking into account that a confounder meets the prerequisites of being associated with the exposure (i.e. infections) in the source population, is (a marker for) a causal risk factor of the outcome (i.e. alloimmunisation) and is not in the causal pathway between the exposure and the outcome (hernan et al, 2002; middelburg et al, 2014) . consequently, we used the following strategy. first, we identified a subset of covariates to be confounders of a given determinant based on their observed association with the determinant within the source population (i.e. the nonalloimmunised controls). such an association was defined as the implicated transfusion and alloimmunisation risk period. the last antigen-mismatched transfusion preceding the first serological detection of an antibody was defined as the 'implicated (or nth) transfusion' since this transfusion was the most likely to influenc alloimmunisation. to exclude possible boosting events, this implicated transfusion was required to precede the first positive screen by at least 7 days (i.e. lag period). an alloimmunisation risk period was then constructed starting 30 days before and finishing 7 days after this implicated transfusion. controls received at least the same number of red cell units as their matched case. a similar alloimmunisation risk period around the nth matched transfusion was constructed. a ≥3% difference in covariate presence between controls exposed and controls not exposed to the determinant. covariates in the causal pathway between the determinant and the outcome were not considered as confounders (hernan et al, 2002) . second, to be able to accurately control for confounders with low prevalences, we estimated a probability score for each determinant using logistic regression with the potential confounders as predictors (brookhart et al, 2013) . third, to minimize bias due to missing data on the confounders, we used multiple imputation. details on the used imputation model can be found in the data s1 and table si . finally, we evaluated the association between various types of infections and red cell alloimmunisation by subsequently entering the corresponding probability scores into the logistic regression model with alloimmunisation as the outcome and conditioning on the matched variables. we next assessed the association of level of crp values and leucocytosis as possible markers of inflammation with red cell alloimmunisation. leucocytosis was categorized as maximum measured leucocyte counts of 10-15, 15-20, 20-30 and >30 9 10 9 /l, and referenced to normal counts (4-10 9 10 9 /l). maximum measured crp values were categorized as 30-100, 100-200, 200-300 and >300 mg/l, and referenced to values ≤30 mg/l. missing crp and leucocyte values were multiply imputed using the same strategy as described above. while the likelihood that an increased inflammatory parameter has been recorded at least once increases with the number of measurements and thus with the duration of hospitalization, we repeated these analyses limited to parameters measured within the week following the implicated transfusion. as elevated crp levels and leucocytosis reflect various clinical conditions preventing causal inferences, they are presented here only as unadjusted rrs. as anti-e, anti-c w , anti-le a , anti-le b , anti-lu a , and anti-m can also form 'naturally' (e.g. directly in response to microbial epitope exposure (reid et al, 2004) , we evaluated a possible association between the presence of these antibodies and various types of infections using pearson's chi-square test. pvalues <0á05 were considered to be statistically significant. as we used an incidence-density sampling procedure to select controls (rothman, 2007) , we interpreted and present all odds ratios as rrs with 95% confidence intervals (95% ci). for some patients, the presence or absence of a certain type of infection could not be determined. these patients were left out of the corresponding analysis. regarding severe bacterial infections, we performed a sensitivity analysis in which these patients were alternately assigned to exposure and nonexposure of this infection. for patients with a suspected lower respiratory infection without conclusive or available cultures, we considered this infection to be due to a bacterial microorganism. although viral or (rarely) fungal pathogens may cause pneumonia, bacterial microorganisms are the most common cause in dutch hospitalised patients, with streptococcus pneumoniae and haemophilus influenzae alone representing 30-75% of causative pathogens (wiersinga et al, 2012) . finally, as contaminated blood cultures positive for coagulase-negative staphylococci (cns) might dilute an existing effect of gram-positive bacteraemia, we compared rrs for all gram-positive bacteraemia with those for non-cns gram-positive bacteraemia. among 54 347 newly-transfused patients, 24 063 were considered eligible (fig s1) of which 505 patients (2á1%) formed red-cell alloantibodies. thirty-seven of these alloimmunised patients (7á3%) only received units of which the cognate antigen was unknown. for these, we assumed the last nontested unit preceding the first positive screen to have elicited alloimmunisation. general and clinical characteristics of the 505 cases and their 1010 matched controls during the alloimmunisation risk period are presented in table i . among all cases and controls, 473 patients were diagnosed with at least one infection during the alloimmunisation risk period. of these, 417 suffered from bacterial infections, 53 from viral infections and 56 from fungal infections (table ii) . for 222 of 269 patients (82á5%) diagnosed with a severe tissue-invasive bacterial infection, the causal microorganism was identified by culture. for three of 53 virally-infected patients, no pcr test was performed during the alloimmunisation risk period. these patients were nevertheless included based on their clinical condition: one patient receiving an allogeneic stem cell transplantation with an outbreak of varicella zoster, one patient receiving chemotherapy for burkitt lymphoma with herpes labialis, and one patient with liver cirrhosis due to a chronic hepatitis c infection. identified confounders per alloimmunisation determinant are presented in tables sii and siii. as illustrated, control subjects with viral infections were younger, had received more red cell units, and were more often leucopenic as compared to those without viral infections. these differences were probably due to a higher frequency of haematological malignancies and associated treatment modalities. missing data for any identified confounder per determinant was at a maximum of 3á1%. crp values were not measured in 343 patients (22á6%) during the risk period (table si) . the number of cases and controls diagnosed per type of infection are presented in table iii . for some patients, the presence or absence of a certain type of infection could not be determined. the majority of these cases were due to an unestablished origin of the inflammatory condition (i.e. an infection or other inflammatory causes). in order to avoid misclassification, we omitted these patients from the corresponding analysis. mild bacterial infections were not associated with alloimmunisation. patients with a severe tissue-invasive bacterial infection tended towards increased alloimmunisation risks [adjusted rr 1á34 (95% ci 0á97-1á85); table iii ]. relative risks increased to significance when these infections were accompanied with long-lasting fever [adjusted rr 3á06 (95% ci 1á57-5á96) with fever present for at least 7 days, table iv ]. the timing of fever i.e. occurring close to the implicated transfusion or at any time point during the risk period did not influence rrs (table siv) . a sensitivity analysis on patients originally omitted from the analysis on severe bacterial infection (n = 47) did not change rrs (table sv) . given that alloantibodies against e, c w , le a , le b , lu a , and m can also form 'naturally', [e.g. in response to microbial epitope exposure rather than to transfusion-related red cell exposure (reid et al, 2004) ] we evaluated a possible association between the induction of these antibodies and various infections using pearson's chi-square test. the distribution of alloantibodies known to also occur 'naturally' did not differ between patients with and without severe bacterial infections (table v) . interestingly, patients with a gram-negative bacteraemia tended to demonstrate reduced alloimmunisation rates [adjusted rr 0á58 (95% ci 0á13-1á14)], while gram-positive bacteraemia was not associated with red cell alloimmunisation (table iii) . to exclude a potential dilution of an existing effect by contaminated blood cultures positive for cns, we also evaluated the association of non-cns gram-positive bacteraemia with alloimmunisation. rrs from this analysis were identical to the originally calculated rrs. any viral disease tended to be associated with increased red cell alloimmunisation incidences. the adjusted rr associated with disseminated viral infections was 2á41 (95% ci 0á89-6á53). the presence of fever did not influence rrs of viral infections (table iv) . †additionally adjusted for identified potential confounders (for details, see table siii ). only numbers of patients for whom the presence or absence of a given infection could be determined are presented. rr, relative risk; 95% ci, 95% confidence interval; nc, not computable. *adjusted for: number of transfused red cell units and hospital. †additionally adjusted for identified potential confounders (for details, see table siii ). fungal infections, as well as candidaemia and invasive aspergillus infections separately, were associated with heterogeneous rrs not reaching significance (table iii) . neither leucocytosis nor crp level was associated with red cell alloimmunisation. a sensitivity analysis on parameters determined within the week following the implicated transfusion did not change these results (table svi) . this study, the first of its kind, in transfused patients suggests a possible association between infectious conditions and red cell alloimmunisation. specifically, our observations suggest alloimmunisation to be influenced by the type and intensity of, and the patient's inflammatory response to infections. in summary, severe (tissue-invasive) bacterial and viral infections were associated with increased incidences of alloimmunisation [rrs 1á34 (95% ci 0á97-1á85) and 2á41 (95% ci 0á89-6á53)]. in contrast, gram-negative bacteraemia coincided with a twofold reduction of alloimmunisation risk [rr 0á58 (95% ci 0á13-1á14)]. our findings certainly require additional confirmatory research. however, they seem to be biologically plausible and are in line with prior animal experiment observations (hendrickson et al, 2006 (hendrickson et al, , 2007 (hendrickson et al, , 2008 smith et al, 2012) . first, long-lasting fever with severe bacterial infections was associated with a substantially increased risk [rr 3á06 (95% ci 1á57-5á96)]. here, persistence of fever could have reflected the most severe bacterial infections inducing a profound inflammatory response. alternatively, or additionally, fever might have been due to other concomitant inflammatory conditions. yet, both explanations are consistent with the 'danger model' (matzinger, 1994) which postulates that an immune response is facilitated by pathogen-associated molecular patterns or structures released from cells undergoing stress (matzinger, 1994; gallucci & matzinger, 2001; kawai & akira, 2010) . second, although the 95% ci encompassing 1 (i.e. a null effect) warrants firm conclusions, we observed substantially increased alloimmunisation rates in patients with systemic viral infections. murine experiments showed similar effects for poly(i:c) (hendrickson et al, 2006 (hendrickson et al, , 2007 (hendrickson et al, , 2008 smith et al, 2012) , a synthetic viral rna analogue that agonizes toll-like receptor (tlr) 3 (alexopoulou et al, 2001) . these poly(i:c) effects were attributed to an increased dendritic cell consumption of transfused cells with upregulation of costimulatory molecules, and activation and proliferation of naive cd4 + antigen-specific t-cells (hendrickson et al, 2007 (hendrickson et al, , 2008 ). an existing molecular mimicry between certain viral peptides and cd4 + t-cell red cell antigen epitopes was also suggested, although observed effects in polyomavirus infected mice did not reach statistical significance (hudson et al, 2010) . although we did not analyse the association between latent viral infections and red cell alloimmunisation, these might also be relevant. in addition, the assessment of possible different effects of rna and dna viruses was prevented by low event numbers. third, we observed a twofold alloimmunisation incidence reduction during gram-negative bacteraemia. analogous to viral infections, these findings require confirmatory research. yet, they again corroborate animal experiments showing significantly attenuated alloimmunisation responses upon lipopolysaccharide (lps) pretreatment in mice (hendrickson et al, 2008) . lps, an endotoxin in the outer cell membrane of gram-negative bacteria, strongly stimulates innate immunity by agonizing tlr4 on macrophages and dendritic cells. conversely, lps is also implicated in a transient, possibly self-protective immune paralysis, known as lps tolerance (lauw et al, 2000; weijer et al, 2002; gould et al, 2004) . restimulation with lps in this respect initiates blockage of cd4 + t cell functioning via impaired release of tumour necrosis factor a, interleukin (il)12 and il18 from monocytes and dendritic cells, together with a diminished upregulation of major histocompatibility complex class-ii and costimulatory molecules (mattern et al, 1998; gould et al, 2004) . while regulatory t cells selectively express tlrs (including tlr4), their lps-induced proliferation might also contribute to the observed effects in both mice and humans (caramalho et al, 2003) . finally, we cannot exclude an indirect role for gram-negative bacteraemia on red cell alloimmunisation due to their common association with other modulators. indeed, suppressed mitogenic b and t lymphocyte responses were observed following administration of antibiotics, including cephalosporins, an antibiotic class frequently used in the treatment of gram-negative bacterial infections (borowski et al, 1985; pomorska-mol et al, 2015) . in an intriguing contrast to the effects observed for gramnegative bacteraemia, we did not observe any association between gram-positive bacteraemia and red cell alloimmunisation. a common lower degree of acute inflammation evoked by gram-positive as compared to gram-negative bloodstream infections due to differing virulence mechanisms forms one hypothetical explanation (wang et al, 2003; gould et al, 2004; abe et al, 2010) . despite the rrs for fungal infections not differing significantly from those for gram-negative bacteraemia, the heterogeneous rrs for individual fungal microorganisms and the lack of other supportive evidence prevent tentative inferences. indeed, in contrast to our estimated rr, one report suggested neonatal alloimmunisation to be related to a disseminated histoplasmosis infection (hata et al, 2013) . the ultimate goal of our study would be to establish an accurate alloimmunisation prediction model, serving as a practical tool for risk-based extended matching. such a model would be most feasible when based on routinely measured patient parameters. in this perspective, we did not observe any association of the level of leucocytosis and crp with alloimmunisation, possibly due to the multifactorial nature of these parameters. other biomarkers, e.g. cytokine levels and immune cell subsets, might be better discriminative; however, they could not be evaluated in the current study. our study design, results and interpretations require additional remarks: first, our incidence-density sampling strategy guarantees that selected controls were similarly exposed as their matched cases (rothman, 2007) . hereby, our rrs are not influenced by transfusion burden, being a main determinant of red cell alloimmunisation (zalpuri et al, 2012b; evers et al, 2016) . second, by identifying the implicated transfusion, we could study conditions present at that given time. as the duration of alloimmunisation modulation is currently unknown and will also probably differ per risk factor, we chose a seemingly large risk period to precede the implicated transfusion. although one could argue that this strategy could possibly dilute some effects, on the other hand, it assures inclusion of most factors of influence at the time of exposure. for example, repeated lps exposure might induce a state of tolerance persisting for up to 30 days (cross, 2002) . in addition, a recent study showed that poly(i:c) facilitates red cell alloimmunisation for at least 14 days with its maximum effect reached 7 days after administration (elayeb et al, 2016) . as a validation of our chosen risk period length, a sensitivity analysis on infections diagnosed during the week preceding or following the implicated transfusion did not change our conclusions (data not shown). similarly, only the duration of fever accompanying severe bacterial infections, rather than its timing in the risk period, affected alloimmunisation. as we aimed to target the most likely first initiation of an alloimmune response, we limited the risk period to the first 7 days following the implicated transfusion. third, actual lag periods per antigen-specific antibody are currently unknown. as such, our chosen lag period of 7 days might not completely have prevented the exclusion of patients demonstrating recall responses, including women immunised due to prior pregnancies. direct antiglobulin tests were not performed on a routine basis shortly following transfusion and as such were of no help in identifying these patients. however, as non-rhd alloantibodies form in only 0á33% of first trimester pregnancies (koelewijn et al, 2008) , we believe that a substantial influence of previous pregnancies is unlikely. moreover, erroneously considering a substantial amount of boosting reactions as primary alloimmunisation events would have biased our rrs towards the null-effect. indeed, a sensitivity analysis in which we excluded the 53 patients in whom alloantibodies were discovered during the second week following their first antigenincompatible transfusion did not substantially change rrs (data not shown). in conclusion, we believe the eventual bias due to our choice of the lag period to be small. fourth, to avoid invalid inferences due to misclassification, we did not define patients with a non-established aetiology of their inflammatory phenotype as exposed patients. for example, for a vascular compromised patient diagnosed with osteomyelitis, wound cultures positive for staphylococcus aureus might have represented normal skin flora colonization of a primary ischaemic wound. consequently, the analysis on severe bacterial infections did not include this patient. a sensitivity analysis confirmed our results not to be affected by this possible misclassification bias. in conclusion, our data suggest a potential risk modifying influence of infection-associated inflammation on red cell alloimmunisation in transfused patients. alloimmunisation seems to be induced by severe bacterial or viral infections, 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pseudorabies the blood group antigen factsbook modern epidemiology. lippincott williams & wilkins incidence of alloantibody formation after abo-d or extended matched red blood cell transfusions: a randomized trial (match study) shot (2014) shot annual report and summary transfusion in the absence of inflammation induces antigenspecific tolerance to murine rbcs united kingdom blood services (2013) handbook of transfusion medicine prospective rbc phenotype matching in a stroke-prevention trial in sickle cell anemia: a multicenter transfusion trial cutting edge: bacterial lipoprotein induces endotoxin-independent tolerance to septic shock diminished interferon-gamma production and responsiveness after endotoxin administration to healthy humans anatomic therapeutic chemical index swab/ nvalt (dutch working party on antibiotic policy and dutch association of chest physicians) guidelines on the management of community-acquired pneumonia in adults does a febrile reaction to platelets predispose recipients to red blood cell alloimmunization? transfusion risk factors for alloimmunisation after red blood cell transfusions (r-fact): a case cohort study red-blood-cell-alloimmunization and number of red-blood-cell transfusions the authors would like to thank james zimring (bloodworks northwest, seattle, usa) for critically reviewing our manuscript. furthermore, we thank bert mesman and herman geerligs the authors declare that they have no conflict of interest relevant to the work presented in this manuscript. additional supporting information may be found in the online version of this article:data s1. study period per participating hospital and the used multiple imputation model. jjz and jgb designed the study. de, jt, and sz collected the data. de, jjz, ram, mh and jgb analysed and interpreted the data. de, jjz, mh, and jgb wrote the manuscript. all other authors revised and approved the final manuscript. key: cord-300230-a3jk6w90 authors: ding, ji-guang; sun, qing-feng; li, ke-cheng; zheng, ming-hua; miao, xiao-hui; ni, wu; hong, liang; yang, jin-xian; ruan, zhan-wei; zhou, rui-wei; zhou, hai-jiao; he, wen-fei title: retrospective analysis of nosocomial infections in the intensive care unit of a tertiary hospital in china during 2003 and 2007 date: 2009-07-25 journal: bmc infect dis doi: 10.1186/1471-2334-9-115 sha: doc_id: 300230 cord_uid: a3jk6w90 background: nosocomial infections are a major threat to patients in the intensive care unit (icu). limited data exist on the epidemiology of icu-acquired infections in china. this retrospective study was carried out to determine the current status of nosocomial infection in china. methods: a retrospective review of nococomial infections in the icu of a tertiary hospital in east china between 2003 and 2007 was performed. nosocomial infections were defined according to the definitions of centers for disease control and prevention. the overall patient nosocomial infection rate, the incidence density rate of nosocomial infections, the excess length of stay, and distribution of nosocomial infection sites were determined. then, pathogen and antimicrobial susceptibility profiles were further investigated. results: among 1980 patients admitted over the period of time, the overall patient nosocomial infection rate was 26.8% or 51.0 per 1000 patient days., lower respiratory tract infections (lrti) accounted for most of the infections (68.4%), followed by urinary tract infections (uti, 15.9%), bloodstream (bsi, 5.9%), and gastrointestinal tract (gi, 2.5%) infections. there was no significant change in lrti, uti and bsi infection rates during the 5 years. however, gi rate was significantly decreased from 5.5% in 2003 to 0.4% in 2007. in addition, a. baumannii, c. albicans and s. epidermidis were the most frequent pathogens isolated in patients with lrtis, utis and bsis, respectively. the rates of isolates resistant to commonly used antibiotics ranged from 24.0% to 93.1%. conclusion: there was a high and relatively stable rate of nosocomial infections in the icu of a tertiary hospital in china through year 2003–2007, with some differences in the distribution of the infection sites, and pathogen and antibiotic susceptibility profiles from those reported from the western countries. guidelines for surveillance and prevention of nosocomial infections must be implemented in order to reduce the rate. nosocomial infections, also called healthcare acquired infections or health care-associated infections, is defined by the cdc as a localized or systemic condition resulting from an adverse reaction to the presence of an infectious agent(s) or its toxin(s), without any evidence that the infection was present or incubating at the time of admission to the acute care setting [1] . nosocomial infections have become an important public health issue worldwide. nosocomial infections may result in an excess length of stay in hospital for up to 10 days and an increase in the costs of hospitalization. [2, 3] nosocomial infections pose a critical threat to patients, especially in the high-risk departments, such as the intensive care unit (icu). [4, 5] in industrialized countries, nosocomial infections occurs in 2-12% of hospitalized patients, with the rates being up to 21% in icu, while the rates are 3-18% in hospitalized patients with the rates being up to 54% in icu. [5] [6] [7] [8] [9] [10] [11] in china, there are more than 10,000 hospitals with significant differences among each other in the size, facilities, administration, teaching, research and academic levels. a few studies reported nosocomial infections in china, but these studies were limited in small sample sizes and short period of time, publication in chinese journals, with or without english abstracts. [11] [12] [13] recently, we carried out a retrospective study to determine the current status of nosocomial infections in a tertiary hospital in east china. all data on nosocomial infections between year 2003 and 2007 were retrieved and reviewed. the overall patient nosocomial infection rate, the incidence density rate of nosocomial infections, the excess length of stay, and distribution of nosocomial infection sites were determined. then, pathogen and antimicrobial susceptibility profiles were further investigated. the study was performed in the affiliated hospital of wenzhou medical university, which situates in zhejiang province, east china, with over 1000 beds and one mixed icu of 13 beds. since 1996, the hospital started an infection control program, including collection of data on infections acquired in the hospital. in the present study, data from january 2003 to december 2007 for patients in the icu were retrieved by the infection control team since completed raw data of the patients in icu were available only after 2003. this retrospective study was approved by the medical ethics committee of the third affiliated hospital, wenzhou medical college. all patients admitted to the icu for more than 48 hours were monitored for nosocomial infections, which were defined according to the american cdc. [1] infections developed within 48 hours of discharge from the icu were also considered to be icu-acquired unless there was an identified cause after discharge. the major nosocomial infections, including lower respiratory tract infections (lrtis), urine tract infections (utis), bloodstream infections (bsis) and gastrointestinal tract infections (gis) were defined as followings. lrtis refer to lower respiratory tract infection, other than pneumonia, i.e. bronchitis, tracheobronchitis, bronchiolitis, tracheitis, without evidence of pneumonia. bsis refers to laboratoryconfirmed bloodstream infections, utis refers to symptomatic urinary tract infections and gis refers to gastrointestinal tract (esophagus, stomach, small and large bowel, and rectum) infections excluding gastroenteritis and appendicitis. the detailed criteria to diagnose these nosocomial infections were described in the cdc documents [1] . data on the date and site of infection, patient demographic information and device use were collected for each infection. moreover, data on the isolated pathogens and their susceptibility testing to antimicrobial agents, if available, were also collected. the overall patient nosocomial infection rate was calculated by dividing the total number of patients with nosocomial infections by the total number of patients in the icu (×100) during the defined period of time (i.e. each year). the incidence density rate of nosocomial infections was calculated by dividing the total number of nosocomial infections by the total patient days (×1000) during the defined period of time. the total patients days were calculated by summating the days of each patient in the icu. in the meantime, the length of stay, which was defined as the overall days of a patient spent in the hospital including the icu and another department to which the patient was transferred from icu after stabilization of the conditions. the excess length of stay was then calculated by subtracting the average length of stay for patients without nosocomial infection from that of patients with nosocomial infections. statistical analyses were performed using spss software version 13.2 (spss inc., chicago, ill., usa). chi-square test and spearman's rank-correlation coefficients were applied where appropriate. for all analyses, a p value of less than 0.05 was considered statistically significant. from 2003 to december 2007, medical data of 1980 patients discharged from the hospital icu were colleted. the average length of stay was 9.95 days, giving 19700 patient-days. among these patients, 531 patients acquired a total of 1005 nosocomial infections, including 125 patients with two infections, 33 patients with three infections, 12 patients with four infections and one patient with five infections ( table 1) . the overall patient nosocomial infection rate was 26.8%, ranging from 22.6% to 33.2% among the 5 years. there was a significant difference in the infection rates among the 5 years (χ 2 = 10.395, p = 0.035). the incidence density rate of nosocomial infections was 51.0 per 1000 patient days, ranging from 38.5 to 59.2 among the 5 years. the excess length of stay was 9.4 days, ranging from 3. lower respiratory tract infections (lrtis) including bronchitis, tracheobronchitis and pneumonia, were the most common infections, occurring in 34.7% of the 1980 patients, followed by urine tract infections (utis) (8.1%), and bloodstream infections (bsis) (3.0%). among the 1005 nosocomial infections, lrtis accounted for 68.4%, followed by utis (15.9%), bsis (5.9%) and gastrointestinal tract infections (2.5%). most (76.0%) patients with nosocomial lrtis had received mechanical ventilation or tracheotomy before the infections, whereas 50.0% of nosocomial utis and 54.2% of nosocomial bsi were catheter associated (table 1) . there is no significant change in lrti, uti and bsi rates during the 5 years. the gi infection rate was significantly decreased from 5.5% in 2003 to 0.4% in 2007 (χ 2 = 12.603, p = 0.012), whereas nosocomial infections in other sites was increased significantly (χ 2 = 12.858, p = 0.012). the nosocomial infection rates at the surgical sites and skin and soft tissues remained under 2% (table 1) . pathogens were isolated and identified from 530 (52.7%) of 1005 nosocomial infections, or, in 338 (63.7%) of the 531 patients. the isolated pathogens responsible for nosocomial infections differed among the infection sites ( table 2 ). in patients with lrtis, acinetobacter baumannii and klebsiella pneumoniae were the most frequently isolated pathogens, followed by pseudomonas aeruginosa and staphylococcus aureus, accounting for more than half of the lrti related pathogen population. in patients with utis, the fungi, especially candida albicans, were the most com-mon pathogens, followed by escherichia coli. staphylococcus epidermidis, e. coli, and s. aureus were the first three most common pathogens for bsis. in addition, a. baumannii was commonly isolated in utis and bsis (table 2) . data on susceptibility testing were available for 3328 isolates, including 195 isolates of e. coli, 359 isolates of s. aureus, and 549 isolates of p. aeruginosa. overall, 79.3%, 80.0%, 82.3% and 77.8% of e. coli isolates were resistant to trimethoprim/sulfamethoxazole (tmp/smx) and ciprofloxacin, cefotaxime, and amoxicillin/clavulanic acid, respectively. all s. aureus isolates were sensitive to vancomycin, but 24.0%, 37.9%, 68.1% and 89.6% of isolates were resistant to nitrofurantoin, tmp/smx, rifampin and ciprofloxacin, respectively. in addition, 93.1%, 41.3% and 66.9% of p. aeruginosa were resistant to tmp/smx, ciprofloxacin and levofloxacin, respectively [see additional file 2]. all patients with nosocomial infections were treated with empirical antimicrobial therapies or according to the antimicrobial susceptibility test results, when available. the in the present study, the overall patient nosocomial infection rate in the icu was 26.8% during 2003 and 2007, which was higher than in the icus in many industrialized countries where the rates ranging from 7.7% to 16.5%, [14] [15] [16] and even higher than the rate (14.7%) observed in 55 icus of developing countries. [17] however, the rate is comparable with those reported in some latin american countries such as argentina and brazil [10] , and slightly lower than that reported in india. [18] over the 5 years, the lowest rate was reported in 2003 (22.6%), and the highest was reported in 2004 (33.2%). one plausible explanation is that in the early of 2003 the country was suffering from the outbreak of highly infectious pneumonia, namely severe acute respiratory syndrome (sars). due to the massive campaign to prevent the spread of sars, nosocomial infections were indirectly reduced. being fatigued from the campaign over the previous year, disinfection and sterilization procedures might be loosen in 2004, explaining the moderate rebound in 2004. however, the incidence density rate of nosocomial infections was the lowest in 2004, due to considerably longer stay of some patients in the icu ( table 1 ). the average length of stay in the hospital varied from 3.3 to 11.5 between 2003 and 2007, with the overall average being 9.4 days, which is generally in agreement with those (4.3-15.6 days) reported in european and the united states [19] [20] [21] , but much less than that reported in taiwan [22] . however, the it must be also mentioned that sars outbreak had some impact on the overall length of stay. although the average stay in the icu was only 6.6 days, the shortest among the 5 years, the average stay in the hospital was the longest for both patients with and those without nosocomial infections due to the isolation policy imposed in the special period of time. the distribution of nosocomial infections in the present study differed from that reported in the united states. we found that the lrtis were the most common infections in the icu, accounting for 68.4% of overall infections, whereas utis was the most frequently reported infections in the icus in the united states, with the rate of 31%, followed by pneumonia of 27%. [14] the proportions of utis and bsis in the present study were relatively lower than the data reported in the united states (15.9% vs. 31% and 5.9% vs. 19%, respectively). [14] although data from europe revealed same three most common infection sites as the present study did, the absolute proportion of lrtis was 47%, [6] which was lower than the rate in the present study. the common reasons proposed by studies in many western countries have suggested that nosocomial lrtis are mainly due to mechanical ventilation. [5, 14] in china, air pollution, high density of population, and improper health habits such as smoking may also account for the high rate of lrtis. the rate of lrtis slightly, but insignificantly, decreased from 2003 to 2007. there was no change in utis and bsis rates during the 5 years. notably, the gi rate significantly and stably decreased every year, suggesting an improvement in the environment and food sanitary in the region. since the lower respiratory tract and the urinary tract were the first two sites that nosocomial infections frequently occurred in the icu, constituting more than 80% of all nosocomial infections in 2003 and 2004, more efforts were later made to control these two kinds of infections, leading to decreased rates of lower respiratory tract and the urinary tract infections, and correspondingly increased rates of nosocomial infections at other sites. the present study showed three quarters of lrti patients received mechanical ventilation or tracheotomy, more than half of nosocomial uti and bsi cases were catheter associated. these findings are consistent with previous studies, [4, 9, 10, 14, 23] and indicate that the nosocomial infections are often associated with the use of invasive device. therefore, to effectively reduce nosocomial infections, the use of invasive device should be minimized and specific disinfection precautions taken during the device application. in the present study, pathogens were isolated from 52.7% of overall nosocomial infections or 63.7% of all patients with nosocomial infections. similar to the us report, gram-negative bacteria accounted for 53.2% of the lrtis in the present study, but the most frequently pathogens were a. baumannii and k. pneurnoniae in the present study whereas p. aeruginosa and s. aureus were the most common pathogens in the us report. [14] consistent with the us report, fungi was the most frequently pathogen for nosocomial utis; candida accounted for 54.7% of utis in the present study, suggesting a relatively narrow profile of pathogens in nosocomial utis. s. epidermidis was the most common pathogen for bsis in both the present study and the us report; however, e. coli, instead of enterococci, was the second common pathogen in the present study. [14] e. coli was the most common bacterial cause of nosocomial utis, and also frequently found in bsis and lrtis. it has been shown that the activity of beta-lactam antibiotics against e. coli is greatly reduced as a result of beta-lactamase production, but is restored by the addition of clavulanic acid. [24] in the present study, only 22.2% of e. coli isolates were sensitive to the formula amoxicillin combined with clavulanic acid, while 78.8% of e. coli isolates exhibited susceptibility to the combination in an uk study. [25] a high rate of resistance to tmp/smx (79.3%) was observed in these e. coli isolates, in contrast to the rate of 40% reported in uk [26] . in addition, there was a high proportion (80.0%) of e. coli isolates resistant to ciprofloxacin, whereas the rate was less than 10% in uk and the united states. [26, 27] these findings indicate that treatment with these antimicrobial agents for nosocomial infections caused by e. coli in china is likely to result in clinical failure in a substantial proportion of patients. we also found that a considerable number of p. aeruginosa isolates were resistant to fluoroquinolones, from 41.3% to ciprofloxacin to 66.9% to levofloxacin, which is comparable with the fluoroquinolone-resistant rates (49% to 64%) reported for patients in 55 icus of eight developing countries, [9] but much higher than that (30%) reported in the united states. [28] all s. aureus isolates in the present study were sensitive to vancomycin, which was similar to the observation by tsuji et al in japan. [29] in addition, we observed relatively low resistant rates to nitrofurantoin (24.0%) and tmp/smx (37.9%), which renders these antimicrobial agents suitable for empirical treatment for s. aureus infections. in china, the guidelines for surveillance and prevention of nosocomial infections was established in 1994, and modified in 2000. [30, 31] however, surveillance systems and control measures for nosocomial infections described in the guidelines were not completely implemented and executed in all hospitals, due to the imbalanced development and health care resources within the countries, and less attention to nosocomial infections in some hospitals. therefore, it is believed that the nosocomial infection rates must be higher in some rural hospitals or even nontertiary hospitals. in addition, due to empirical use or abuse of antibiotics, the proportion of antibiotic resistant pathogens for nosocomial infections in many lower level hospitals would also be higher than that reported in the present study. it is noticed that although the mortality rates in patients nosocomial infections were between 15%-20%, there was no mortality directly caused by nosocomial infections. the major reasons for this observation would be the fact that refractory nosocomial infections are relatively less encountered based on our susceptibility testing, which showed that most pathogens were sensitive to many most commonly used antibiotics, indicating that they can be effectively controlled. moreover, zhejiang is one of richest provinces in china where medical and healthcare systems are relatively well established and antiinfectious therapies are not a big problems. finally, it should be emphasized that our hospital is an tertiary infectious hospital with experience, methodologies and facilities to combat against various infections including nosocomial infections. the present study has some limitations, due to the retrospective nature. first, data on risk factors, except for the use of the medical device, that are potentially associated with nosocomial infections were not available. these factors may include the primary diseases for admission to the icu, patient resting posture (e.g. semirecumbent or supine body position), continuous prophylactic use of anti-peptic ulcer drugs, utilization of the alcohol-based handrubs and oral care, which need to be taken into consideration in the prospective studies. second, the data on the identification and isolation of the pathogens and their susceptibility were available only for half of the nosocomial infections. it would produce more accurate data if these numbers were increased. finally, the data on the clinical consequences were not available for most cases, making it impossible to compare the clinical outcomes between patients with and those without nosocomial infections. however, the present study was able to show that the length of stay in the hospital was significantly increased in patients with nosocomial infections, compared with those without the infections. in conclusion, there was a high and relatively stable rate of nosocomial infections in the icu of a tertiary hospital in china through year 2003-2007, with some differences in the distribution of the infection sites, and pathogen and antibiotic susceptibility profiles from those reported in the western countries. the guidelines for surveillance and prevention of nosocomial infections must be implemented national wide in order to reduce the rate. cdc definitions for nosocomial infections effect of intensive care unit nosocomial pneumonia on duration of stay and mortality prolongation of hospital stay and extra costs due to ventilator-associated pneumonia in an intensive care unit nosocomial infection rates in adult and pediatric intensive care units in the united states prevalence and risk factors for nosocomial lower respiratory tract infections in german hospitals selected aspects of the socioeconomic impact of nosocomial infections: morbidity, mortality, cost, and prevention the prevalence of nosocomial infection in intensive care units in europe. results of the european prevalence of infection in intensive care (epic) study. epic international advisory committee mortality attributable to nosocomial infections in the icu national nosocomial infections surveillance (nnis) system report, data summary from nosocomial infections in medical-surgical intensive care units in argentina: attributable mortality and length of stay prevalence rate of nosocomial infection in a general hospital surveillance of risk factors for nosocomial infection in patients in intensive care units study on the changing trends in national nosocomial infection transaction investigation results nosocomial infections in medical intensive care units in the united states nosocomial infections: prospective survey of incidence in five french intensive care units nosocomial infections in a neurosurgery intensive care unit device-associated nosocomial infections in 55 intensive care units of 8 developing countries epidemiology, risk factors and outcome of nosocomial infections in a respiratory intensive care unit in north india the estimation of the cost of nosocomial infection in an intensive care unit the impact of adverse patient occurrences on hospital costs in the pediatric intensive care unit excess length of stay, charges, and mortality attributable to medical injuries during hospitalization impact of nosocomial infection on cost of illness and length of stay in intensive care units national healthcare safety network (nhsn) report, data summary for amoxycillin/clavulanic acid: a review of its antibacterial activity, pharmacokinetics and therapeutic use a uk multicentre study of the antimicrobial susceptibility of bacterial pathogens causing urinary tract infection antimicrobial resistance in community and nosocomial escherichia coli urinary tract isolates identification and pretherapy susceptibility of pathogens in patients with complicated urinary tract infection or acute pyelonephritis enrolled in a clinical study in the united states from november gram-negative antibiotic resistance: there is a price to pay communityand health care-associated methicillin-resistant staphylococcus aureus: a comparison of molecular epidemiology and antimicrobial activities of various agents anonymous: guidelines for nosocomial infections control and prevention (draft) anonymous: guidelines for nosocomial infections control and prevention (draft) the authors acknowledge that the manuscript was edited by a professional company, medjaden biomedical services. the authors declare that they have no competing interests. j-gd, q-fs and k-cl were the principal investigators who designed and conducted the study, analyzed the data, performed literature research and prepared the manuscript. m-hz, x-hm and wn participated in the design of the study, contributed to the data analysis and made constructive comments on the manuscript. lh, j-xy, z-wr, r-wz, h-jz and w-fh participated in the design of the study, collected the all required original data, and generated results and tables that were the basis of the manuscript. all authors have read and approved the final manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/115/pre pub key: cord-310042-9z8rkzq8 authors: aysha, al‐ani; rentsch, clarissa; prentice, ralley; johnson, doug; bryant, robert v.; ward, mark g.; costello, samuel p.; lewindon, peter; ghaly, simon; connor, susan j.; begun, jakob; christensen, britt title: practical management of inflammatory bowel disease patients during the covid‐19 pandemic: expert commentary from the gastroenterological society of australia inflammatory bowel disease faculty date: 2020-07-12 journal: intern med j doi: 10.1111/imj.14889 sha: doc_id: 310042 cord_uid: 9z8rkzq8 the covid‐19 pandemic, caused by the novel coronavirus sars‐cov‐2, has emerged as a public health emergency and challenged healthcare systems globally. in a minority of patients, sars‐cov‐2 manifests with a severe acute respiratory illness and currently there are insufficient data regarding the virulence of covid‐19 in inflammatory bowel disease patients taking immunosuppressive therapy. this review aims to summarise the current literature and provide guidance on the management of inflammatory bowel disease (ibd) patients in the context of the covid‐19 pandemic in the australasian setting. the covid-19 pandemic, caused by the novel coronavirus sars-cov-2, has emerged as a public health emergency and challenged healthcare systems globally. it has rapidly spread across the world without regard for borders, manifesting as an acute respiratory illness that ranges broadly in severity from asymptomatic carriage to mild, non-specific symptoms to severe pneumonia, sepsis and death. 1 mortality is estimated to be between 1% and 2% with disease severity associated with advanced age, chronic respiratory illness, hypertension, diabetes and other comorbidities. 1 there is limited information on the impact of covid-19 on immunosuppressed patients, in particular, those with inflammatory bowel disease (ibd). ibd is a relapsing and remitting inflammatory condition of the bowel. a significant proportion of ibd patients are treated with long-term immunomodulator/immunosuppressive therapy which potentially places them at increased risk of infections and associated complications. practitioners and patients alike are therefore concerned about the risk and implications of covid-19 infection in the ibd patient, despite a paucity of evidence supporting an altered predisposition to disease or more severe disease course. as higher quality evidence gradually accumulates, this article aims to provide an interim practical guide for ibd management during this uncertain time. wuhan, china, in december 2019 and is transmitted via direct contact and exhaled droplets from an infected individual. 2 human-to-human transmission is enabled by the interaction of the sars-cov-2 spike (s)-protein with human angiotensin-converting enzyme 2 (ace2) receptor. 3 ace2 is expressed on multiple cell types throughout the body including alveolar type 2 (at2) cells in the lungs and enterocytes of the small intestine and colon. once the virus is attached to ace2 it uses the host serine protease tmprss2 for s priming allowing fusion of viral and cellular membranes and viral entry into the cell. 3 the median incubation period of covid-19 is 4-5 days, with the majority of patients developing symptoms within 2 weeks. 2 the most commonly reported symptoms include fever, dry cough and shortness of breath. 1 gastrointestinal symptoms include diarrhoea in 2-49.5% of patients and vomiting in 3.6-15.9% of patients. 4 gastrointestinal symptoms in covid-19 are important to note, as there is a subgroup of patients with mild disease who initially present with diarrhoea rather than respiratory symptoms, and this can lead to a delay in diagnosis. 5 the pathophysiology of diarrhoea in covid-19 has not been elucidated; however, virus rna has been detected in up to 50% of stool specimens and stool can remain persistently positive after clearance of respiratory tract samples in approximately 20% of patients. 6 in fact, the australian government is currently looking at methods of testing sewerage for sars-cov-2 rna as part of the australian wide monitoring programme to predict future spread and act as an early warning signal for imminent covid-19 outbreaks. 7 therefore, it is possible that enteric symptoms are caused by invasion of sars-cov-2 into ace2 expressing enterocytes of the gastrointestinal tract. the implications of gastrointestinal shedding are unknown, as a polymerase chain reaction (pcr) positive stool sample does not equate to viable virus, and whether the disease is transmissible via the faecal-oral route remains unclear. furthermore, whether gastrointestinal symptoms are more prevalent in patients with ibd is ill-defined, but if an ibd patient presents with worsening diarrhoea, especially in the context of respiratory symptoms and/or fevers, excluding sars-cov-2 infection is prudent. in suspected cases, diagnosis of covid-19 is via nucleic acid amplification testing (naat) of nasopharyngeal and oropharyngeal swabs. 2 serology testing and stool testing for sars-cov-2 are not currently widely available in australia. a suspected case of covid-19 can only be cleared following two consecutive negative covid-19 pcr swabs due to the potential of false-negatives. despite concerns regarding immunosuppression and consequent predisposition to infection, there is no evidence to suggest increased infection rates of covid-19 in ibd patients to date. reports from china and italy suggest very low infection rates in ibd patients and, at the time of writing, an international covid-ibd registry reported only 798 (466 crohn disease (cd); 329 ulcerative colitis (uc)/unspecified) cases worldwide, despite almost 3 million confirmed covid-19 cases. 8, 9 hence, expert consensus currently is that patients with ibd do not appear to be at increased risk of sars-cov-2 infection compared with the general population. 10 importantly, this should not negate attempts to minimise infection in ibd patients, particularly those with comorbidities including cardiovascular disease, hypertension, chronic pulmonary disease, diabetes and cancer which place them at increased risk of significant morbidity and mortality with covid-19 infection. 1 more specific risk factors for severe infection in immunocompromised patients include age >50 years, receipt of corticosteroids, lymphopenia and neutropenia. 2 mechanisms to protect these particularly vulnerable patients are crucial and include: • proactively reducing transmissionpatients should practice good hand hygiene by washing hands with soap and water for at least 20 s or use an alcohol based hand sanitiser, apply social distancing including working from and staying at home where possible and standing at least 1.5 m apart from people, as well as avoiding nonessential travel. • managing hospital and healthcare facility exposureevidence from china and italy suggest attendance at hospitals and healthcare facilities for non-covid-19 reasons may increase the risk of covid-19 exposure. 11 to reduce this risk, outpatient appointments should be moved to telehealth where possible and non-urgent pathology requests limited. elective surgery and endoscopy should be postponed, but when an urgent endoscopic procedure needs to occur, pre-screening for symptoms and exposure to covid-19 prior to endoscopy should occur. endoscopy should still be undertaken for acute severe ulcerative colitis, confirmation of a new diagnosis of ibd, cholangitis in primary sclerosing cholangitis and in an unresolved partial bowel obstruction. to limit pharmacy visits, prescriptions can be delivered via post; in australia there is currently a free delivery service available for vulnerable members of the community. • infusion accesspatients should continue to have access to infusions. to reduce the risk of transmission within infusion centres, patients should be screened for symptoms or risks of covid-19 prior to presenting and on attendance to the centre, with a temperature check on arrival. where possible the infusion centre should be moved to an 'off-site location' or 'clean' covid-19 free area of the hospital with a separate entrance. in addition, there should be 2 m between patient chairs and if feasible a single nurse per patient arranged to prevent infection spread. finally, infliximab infusions should be converted to a 30-60 min protocol where safe to do so to limit the duration patients are in the infusion centre. • optimising nutritionmalnutrition significantly increases the risk of infection in patients with ibd. 12 therefore nutritional status should be optimised and preferential use of exclusive enteral nutrition (een) for treatment of cd flares where appropriate and acceptable to patients should be considered. low vitamin d levels may increase susceptibility to covid-19 hence supplementation if levels are low is reasonable. 2 • reducing disease activitythere is evidence that moderate to severe disease activity increases the risk of infection in ibd patients. 12 active disease may also lead to corticosteroid use which can increase susceptibility and severity of covid-19 and/or hospitalisation which may inadvertently lead to covid-19 exposure. therefore, optimisation of medical therapy to maintain tight disease control is optimal. • smoking cessationsmoking may increase the chance of developing severe covid-19 symptoms, therefore smoking cessation encouragement and support should be a priority. 8 • vaccinationto reduce co-infection with influenza and other respiratory infections, influenza (quadrivalent inactivated vaccine) and pneumococcus (pcv13 and ppsv 23) vaccination should be provided and maintained in accordance with schedule recommendations. recommendations for the management of ibd medications during the pandemic are summarised in table 1 and a more detailed analysis can be found in a recently published review on the prevention, diagnosis and management of covid-19 in the ibd patient. 2 overall, medications should not be ceased without careful consideration of risk of disease flare, and corticosteroids should be avoided or exposure minimised. • there are no reports of increased risk of infection including serious or opportunistic infections with 5-asa medications. 13 • considered safe to start and continue. 2 corticosteroids • prednisolone and other systemic corticosteroids are associated with substantial increased risk of respiratory tract infections and other infections in ibd patients. 14 • they have also been associated with worse outcomes when used to treat middle eastern respiratory syndrome (mers), severe acute respiratory syndrome (sars) and influenza. 15 • avoid commencing prednisolone where possible. if induction agent required, alternatives include budesonide or een for cd and budesonide multimatrix (mmx) system for uc as well as topical steroids for distal disease. • if systemic steroids are required, we recommend rapid tapering where possible but balance this against risk of flare and advise against sudden cessation. • council patients to avoid self-commencing corticosteroids to control ibd symptoms, alternatively supporting ready to access medical advice. 3 immunomodulatorsthiopurines and methotrexate • thiopurines (azathioprine and mercaptopurine) are associated with an increased risk of serious and opportunistic infections and reduce immune response to viruses. 2 however, there is limited evidence to suggest an increased risk of either upper respiratory tract infections or pulmonary infections, and mercaptopurine has actually been shown to inhibit one of the proteases essential to viral maturation of mers-cov in vitro. 16 although no further animal based models exist and the studies have not been replicated for covid-19, it does raise the possibility that thiopurine use may not necessarily pose an increased risk from covid-19. • of note, thiopurines can cause lymphopenia. patients with lymphopenia caused by sars-cov-2 have a worse prognosis and have an increased risk of death associated with the virus. 1 hence, blood counts should be carefully monitored and thiopurine doses altered accordingly, where necessary. • a recent systematic review found that in the nonrheumatoid arthritis inflammatory disease population, methotrexate does not increase the risk of infection including respiratory infections. 17 • overall there is limited evidence of increased risk of infection with the use of immunomodulators. they appear to be relatively safe at the doses typically used for immune-mediated disease, and most patients will not require dose modification. 4 anti-tumour necrosis factor (tnf) therapies (infliximab, adalimumab, golimumab) • anti-tnf have been shown in multiple studies to increase the risk of upper and lower respiratory tract infections, as well as serious and opportunistic pulmonary infections. 2,18 • serious infection risk is most evident in patients using combination therapy with an immunomodulator and steroids. 18 • overall, anti-tnf are considered safe to continue during the pandemic. • in older patients in deep remission with good biologic levels, consider drug-holiday from immunomodulator if on combination therapy. • when initiating therapy, consider monotherapy with therapeutic drug monitoring and utilising subcutaneous formulations where possible to reduce hospital exposure. • it is not recommended to switch from intravenous to subcutaneous formulations due to risk of loss of response and consequent flare. 2 5 anti-interleukin-23 therapies (ustekinumab) • the risk of severe respiratory tract infections, severe infections or opportunistic infections does not appear to be increased in long term follow-up studies of ustekinumab in both ibd and psoriasis. 2 • ustekinumab is considered safe to start and continue during the pandemic. 6 anti-integrin (vedolizumab) • vedolizumab is a monoclonal antibody to the α 4 β 7 integrin that modulates gut lymphocyte trafficking. hence, there is a theoretical risk of increased susceptibility to gastrointestinal infections. however, respiratory tract infections, severe infections or opportunistic infections do not appear to be increased in long term follow-up studies of vedolizumab in ibd. 19 • vedolizumab is considered safe to start and continue during the pandemic. 7 jak-kinase inhibitor (tofacitinib) • tofacitinib impairs immunity to viral infections in long-term extension trials. • doses of 10 mg twice daily are associated with increased serious infection risk when compared to 5 mg twice daily. 20 • continue tofacitinib but ideally at the lower dose of 5 mg twice daily. • avoid commencing tofacitinib unless no other alternatives are available, to avoid potential side effects and frequent pathology monitoring. patient management if they are exposed to or develop covid-19 there are currently no evidence-based guidelines on medical management of ibd in a patient who becomes covid-19 positive. table 2 summarises our recommendations. based on the lack of data, in most patients we recommend temporary withholding of immunesuppressing therapies until the resolution of active infection as would be typical in the setting of any serious infection. in those exposed but with no symptoms, this should be weighed up against the risk of ibd flare. it is also important to note that many ibd medications may take months to be eliminated from the body so the utility of cessation in the short-term is likely to be limited. (table 2 ). there is currently no evidence to guide the recommencement of medication following exposure to or infection with sars-cov-2. most patients will develop symptoms within 14 days of exposure or testing positive for disease. of note, prodromal asymptomatic infection is increasingly being recognised in outbreak settings with a recent study of nursing home patients demonstrating that 24 of 27 (89%) asymptomatic patients who tested positive for sars-cov-2 developed symptoms within the subsequent 7 days. 21 therefore we recommend that ibd medications can be restarted after 14 days in exposed or asymptomatic infected patients provided they have not developed symptomatic illness. in those who develop covid-19, testing for clearance of virus before recommencing medications or accessing infusion centres may have limited utility as some patients shed virus for extended periods of time despite not being actively infected or infectious. relying on testing to clear these patients may result in unnecessary delays to ibd treatment. 6 in australia, covid-19 patients are cleared from isolation and considered no longer infectious 10 days from symptoms onset if they have clinical improvement and no fever for at least 3 days. european guidelines, however, suggest waiting 14 days in those who have been on immunosuppressive therapies due to the potential for prolonged shedding of virus. 22 therefore, in patients with mild to moderate covid-19, recommencing therapy after at least 14 days in those who are currently asymptomatic or have clinical improvement with no fever for at least 3 days is likely safe. in more severe cases, clinical judgement should be used, and a greater window between covid-19 improvement and medication recommencement may be prudent. serology testing is currently not widely available to help guide these decisions and the impact of immunosuppressive medications on seroconversion is as yet unknown. however, these tests may be utilised in the future. supporting the nutritional requirements of covid-19 affected patients is critical. this is of increased importance in the ibd patient secondary to the increased probability of premorbid malnutrition. early dietician review is therefore warranted. nutritional interventions are particularly important in those unable to maintain adequate oral nutritional intake due to a need for prolonged intubation or non-invasive respiratory support, and in those with increased gastrointestinal losses. the latter may occur in the setting of active ibd, or as a consequence of the virus itself. 4 enteral feeding via nasogastric tube (ngt) remains the preferred first line option, and should be considering within 24 h of admission for those requiring intensive care support. 23 delayed gastric emptying with elevated residual gastric volumes can occur in critically unwell covid-19 patients, increasing the risk of aspiration and therefore continuous rather than bolus feeding is preferable. 23, 24 in addition, prone positioning is often required for respiratory care and treatment in severe covid-19 and may hinder ngt feeding. 25 where necessary, nasojejunal tubes and parenteral nutrition are valid routes for nutritional support, although the former carries the risks of endoscopic placement and the latter requires intensive dietician input with risk of hyperglycaemia, refeeding syndrome and central line infections. 23, 24 of note, ngt insertion is considered an aerosol generation procedure 26 and thus appropriate personal protective equipment with full airborne precautions is recommended. 23 maintaining quality of care during the covid-19 pandemic the unintended immediate and longer term consequences of the covid-19 pandemic may be a loss of ibd control and an increased rate of flare. inappropriate cessation of medications poses risks to patients with ibd, further compounded by a potential lack of access to healthcare. maintaining quality ibd care is imperative. engagement with ibd services can be facilitated through ibd helplines, telemedicine clinics, as well as dissemination of accurate information via a regular ibd newsletter. while access to endoscopy for disease activity assessment is limited, objective monitoring may be undertaken using non-invasive tools such as faecal calprotectin and gastrointestinal ultrasound. utilisation of ibd-specific smart-phone applications as well as decision-aid tools may also be helpful where available and the covidsafe app, accessible in australia, may be useful to notify ibd patients of close contact with a covid-19 case. many patients with ibd are treated with long-term immunomodulating therapy and there is concern amongst patients and clinicians alike that this may predispose to an increased risk of covid-19. however, available data are reassuring and despite understandable anxiety, patients with ibd do not appear to be at increased risk of covid-19. in order to prevent covid-19 infection and its complications, optimisation of disease activity, nutrition, co-morbidities, smoking and vaccination status is important and where possible, corticosteroids should be avoided. if a patient with ibd does develop covid-19, ibd medications can be temporarily withheld and recommenced with timing of recommencement dependent on severity of covid-19 and baseline ibd disease. the mental health of medical workers in wuhan, china dealing with the 2019 novel coronavirus review article: prevention, diagnosis and managment of covid-19 in the ibd patient sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor review article: gastrointestinal features in covid-19 and the possibility of faecal transmission clinical characteristics of covid-19 patients with digestive symptoms in hubei, china: a descriptive, cross-sectional, multicenter study evidence for gastrointestinal infection of sars-cov-2 first confirmed detection of sars-cov-2 in untreated wastewater in australia: a ibd 798-804 © 2020 royal australasian college of physicians proof of concept for the wastewater surveillance of covid-19 in the community secure-ibd database public data update protection of 318 inflammatory bowel disease patients from the outbreak and rapid spread of covid-19 infection in wuhan international organization for the study of inflammatory bowel disease. management of patients with crohn's disease and ulcerative colitis during the coronavirus disease-2019 pandemic: results of an international meeting british society of gastroenterology guidance for management of inflammatory bowel disease during the covid-19 pandemic risk factors for opportunistic infections in patients with inflammatory bowel disease sulphasalazine and mesalazine: serious adverse reactions re-evaluated on the basis of suspected adverse reaction reports to the committee on safety of medicines the effect of corticosteroids on mortality of patients with influenza pneumonia: a systematic review and meta-analysis clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury thiopurine analogs and mycophenolic acid synergistically inhibit the papainlike protease of middle east respiratory syndrome coronavirus risk of infection with methotrexate therapy in inflammatory diseases: a systematic review and metaanalysis comparative risk of serious infections with biologic and/or immunosuppressive therapy in patients with inflammatory bowel diseases: a systematic review and metaanalysis the safety of vedolizumab for ulcerative colitis and crohn's disease real-world experience with tofacitinib in ibd at a tertiary center presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility european centre for disease prevention and control. technical report: guidance for discharge and ending isolation in the context of widespread community transmission of covid-19 -first update nutrition management for critically and acutely unwell hospitalised patients with covid-19 in australia and new zealand pdf 24 bapen's nasogastric tube safety special interest group. bapen statement on covid-19 and enteral tube feeding safety characteristics and outcomes of 21 critically ill patients with covid-19 in washington state nasogastric (ngt)/nasojejunal tube (njt) placement and aerosol generation (agp) key: cord-302056-wvf6cpib authors: benatia, d.; godefroy, r.; lewis, j. title: estimating covid-19 prevalence in the united states: a sample selection model approach date: 2020-04-30 journal: nan doi: 10.1101/2020.04.20.20072942 sha: doc_id: 302056 cord_uid: wvf6cpib background: public health efforts to determine population infection rates from coronavirus disease 2019 (covid-19) have been hampered by limitations in testing capabilities and the large shares of mild and asymptomatic cases. we developed a methodology that corrects observed positive test rates for non-random sampling to estimate population infection rates across u.s. states from march 31 to april 7. methods we adapted a sample selection model that corrects for non-random testing to estimate population infection rates. the methodology compares how the observed positive case rate vary with changes in the size of the tested population, and applies this gradient to infer total population infection rates. model identification requires that variation in testing rates be uncorrelated with changes in underlying disease prevalence. to this end, we relied on data on day-to-day changes in completed tests across u.s. states for the period march 31 to april 7, which were primarily influenced by immediate supply-side constraints. we used this methodology to construct predicted infection rates across each state over the sample period. we also assessed the sensitivity of the results to controls for state-specific daily trends in infection rates. results the median population infection rate over the period march 31 to april 7 was 0.9% (iqr 0.64 1.77). the three states with the highest prevalence over the sample period were new york (8.5%), new jersey (7.6%), and louisiana (6.7%). estimates from models that control for state-specific daily trends in infection rates were virtually identical to the baseline findings. the estimates imply a nationwide average of 12 population infections per diagnosed case. we found a negative bivariate relationship (corr. = -0.51) between total per capita state testing and the ratio of population infections per diagnosed case. interpretation the effectiveness of the public health response to the coronavirus pandemic will depend on timely information on infection rates across different regions. with increasingly available high frequency data on covid-19 testing, our methodology could be used to estimate population infection rates for a range of countries and subnational districts. in the united states, we found widespread undiagnosed covid-19 infection. expansion of rapid diagnostic and serological testing will be critical in preventing recurrent unobserved community transmission and identifying the large numbers individuals who may have some level of viral immunity. public health efforts to determine population infection rates from coronavirus disease 2019 (covid -19) have been hampered by limitations in testing capabilities and the large shares of mild and asymptomatic cases. we developed a methodology that corrects observed positive test rates for non-random sampling to estimate population infection rates across u.s. states from march 31 to april 7. we adapted a sample selection model that corrects for non-random testing to estimate population infection rates. the methodology compares how the observed positive case rate vary with changes in the size of the tested population, and applies this gradient to infer total population infection rates. model identification requires that variation in testing rates be uncorrelated with changes in underlying disease prevalence. to this end, we relied on data on day-to-day changes in completed tests across u.s. states for the period march 31 to april 7, which were primarily influenced by immediate supply-side constraints. we used this methodology to construct predicted infection rates across each state over the sample period. we also assessed the sensitivity of the results to controls for state-specific daily trends in infection rates. the median population infection rate over the period march 31 to april 7 was 0.9% (iqr 0.64 1.77). the three states with the highest prevalence over the sample period were new york (8.5%), new jersey (7.6%), and louisiana (6.7%). estimates from mod-1 els that control for state-specific daily trends in infection rates were virtually identical to the baseline findings. the estimates imply a nationwide average of 12 population infections per diagnosed case. we found a negative bivariate relationship (corr. = -0.51) between total per capita state testing and the ratio of population infections per diagnosed case. the effectiveness of the public health response to the coronavirus pandemic will depend on timely information on infection rates across different regions. with increasingly available high frequency data on covid-19 testing, our methodology could be used to estimate population infection rates for a range of countries and subnational districts. in the united states, we found widespread undiagnosed covid-19 infection. expansion of rapid diagnostic and serological testing will be critical in preventing recurrent unobserved community transmission and identifying the large numbers individuals who may have some level of viral immunity. social sciences and humanities research council. in december 2019, several clusters of pneumonia cases were reported in the chinese city of wuhan. by early january, chinese scientists had isolated a novel coronavirus (sars-cov-2), later named coronavirus disease 2019 (covid19) , for which a laboratory test was quickly developed. despite efforts at containment through travel 2 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 30, 2020. . https://doi.org/10.1101/2020.04. 20.20072942 doi: medrxiv preprint restrictions, the virus spread rapidly beyond mainland china. by april 7, more than 1.4 million cases had been reported in 182 countries and regions. our understanding of the progression and severity of the outbreak has been limited by constraints on testing capabilities. in most countries, testing has been limited to a small fraction of the population. as a result, the number of confirmed positive cases may grossly understate the population infection rate, given the large numbers of mild and asymptomatic cases that may go untested [1] [2] [3] [4] [5] . moreover, testing has often been targeted to specific subgroups, such as individuals who were symptomatic or who were previously exposed to the virus, whose infection probability differs from that in the overall population [6, 7] . 1 given this sample selection bias, it is impossible to infer overall disease prevalence from the share of positive cases among the tested individuals. a further challenge to our understanding of the spread of outbreak has been the wide variation in per capita testing across jurisdictions due to different protocols and testing capabilities. for example, as of april 7, south korea had conducted three times more tests than the united states on a per capita basis [8, 9] . large differences in testing rates also exist at the subnational level. for example, per capita testing in the state of new york was nearly two times higher than in neighboring new jersey [8] . because the severity of sample selection bias depends on the extent of testing, these disparities create large uncertainty regarding the relative disease prevalence across jurisdictions, and may contribute to the wide differences in estimated case fatality rates [10, 11] . in this study, we implemented a procedure that corrects observed infection rates among tested individuals for non-random sampling to calculate population disease prevalence. a large body of empirical work in economics has been devoted to the problem of sample selection and researchers have developed estimation procedures to 1 notable exceptions include the universal testing of passengers on the diamond princess cruise ship, and an ongoing population-based test project in iceland. correct for non-random sampling [12] [13] [14] [15] [16] [17] . our methodology builds on these insights to correct observed infection rates for non-random selection into covid-19 testing. our procedure compares how the observed infection rate varied as a larger share of the population was tested, and uses this gradient to infer disease prevalence in the overall population. because investments in testing capacity may respond endogenously to local disease conditions, however, model identification requires that we find a source of variation in testing rates covid-19 that is unrelated to the underlying population prevalence. to this end, we relied on high frequency day-to-day changes in completed tests across u.s. states, which were primarily driven by immediate supply-side limitations rather than the more gradual evolution of local disease prevalence. we used this procedure to correct for selection bias in observed infection rates to calculate population disease prevalence across u.s. states from march 31 to april 7. to evaluate population disease prevalence, we developed a simple selection model for covid-19 testing and used the framework to link observed rates of positive tests to population disease prevalence. we considered a stable population, denoting a and b as the numbers of sick and healthy individuals, respectively. let p n denote the probability that a sick person is tested and q n the probability that a healthy person is tested, given a total number of tests, n. thus, we have: cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 30, 2020. . https://doi.org/10.1101/2020.04. 20.20072942 doi: medrxiv preprint and the number of positive tests is: this simple framework highlights how non-random testing will bias estimates of the population disease prevalence. using bayes' rule, we can write the relative probability of testing as the following: q n p n = p r(sick|n)/p r(healthy|n) p r(sick|tested, n)/p r(healthy|tested, n) , which is equal to one if tests are randomly allocated, p r(sick|tested, n) = p r(sick|n). when testing is targeted to individuals who are more likely to be sick, we have p r(sick|tested, n) > p r(sick|n) and p r(healthy|tested, n) < p r(healthy|n), so the ratio will fall between zero and one. in this scenario, the ratio of sick to healthy people in the sample, p n a q n b, will exceed the ratio in the overall population, a b. we specified the following functional form for the relative probability of testing: which is in [0,1] for −a − bn ≤ 0. the term e −a−bn > 0 reflects the fact that testing has been targeted towards higher risk populations, with the intercept, −a, capturing the severity of selection bias when testing is limited. meanwhile, the coefficient b > 0 identifies how selection bias decreases with n as the ratio q n /p n approaches one. intuitively, as testing expands, the sample will become more representative of the overall population, and the selection bias will diminish. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 30, 2020. . https://doi.org/10.1101/2020.04. 20.20072942 doi: medrxiv preprint combining both equations, we have: we used the fact that the ratio of negative to positive tests is much larger than one to make the following approximation: 2 given a change in the number of tests conducted in a particular population, n 1 to n 2 , equation (2) implies the following change in the share of positive tests: our empirical model was derived from equation (3). we used information on testing across states i on day t to estimate the following equation: where n i,t is the number of tests on day t, s i,t is the share of positive tests, and pop i is the state population. the term u i,t is an error which we assumed to follow a gaussian distribution with mean zero and unknown variance. we restricted the model to a cubic approximation of the function in equation (4), since higher order terms were found to be statistically insignificant. this approximation is supported by graphical evidence depicted below. we estimated equation (4) by maximum likelihood. for model identification, we required that day-to-day changes in the number of tests be uncorrelated with the error term, u i,t . in practice, this assumption implies that daily changes in underlying population disease prevalence cannot be systematically related to day-to-day changes in testing. our identification assumption is supported by at least three pieces of evidence. first, severe constraints on state testing capacity have caused a significant backlog in cases, so that changes in the number of daily tests primarily reflects changes in local capacity rather than changes in demand for testing. second, because our analysis focuses on high frequency day-to-day changes in outcomes, there is limited scope for large evolution in underlying disease prevalence. finally, in robustness exercises, we augmented the basic model to include state fixed effects, thereby allowing for state-specific exponential growth in underlying disease prevalence from one day to the next. these additional controls did not alter the main empirical findings. to recover estimates of population infection rates,p i,t , in state i at date t, we combined the estimates from equation (4) and set n = pop i according to the following equation:p we then used the delta-method to estimate the confidence interval forp i,t . 7 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the analysis was based on daily information on total tests results (positive plus negative) and total positive test results across u.s. states for the period march 31 to april 7. these data were obtained from the covid tracking project, a site that was launched by journalists from the atlantic to publish high-quality data on the outbreak in the united stated [8] . the data were originally compiled primarily from state public health authorities, occasionally supplemented by information from news reporting, official press conferences, or message from officials released on facebook or twitter. we focused on the recent period to limit errors associated with previous changes in state reporting practices. we supplemented this information with data on total state population from the census [18] . (4), estimated across states for the period march 31 to april 7. becauseβ is negative, the upward sloping pattern implies a negative relationship between daily changes in testing and the share of positive tests. a symptom of selection bias is that variables that have no structural relationship with the dependent variable may appear to be significant [13] . thus, these patterns strongly suggest non-random testing, since daily changes in testing should be 8 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 30, 2020. . https://doi.org/10.1101/2020.04.20.20072942 doi: medrxiv preprint unrelated to population disease prevalence except through a selection channel. 3 table 2 reports the results that adjust observed covid-19 case rates for nonrandom testing based on the procedure described in section 2. for reference, column (1) reports the observed positive test rate on april 7, 2020. columns (2) and (3) the average estimates are similar to the april 7 estimates, albeit generally smaller in magnitude, suggesting continued spread of the disease in many states. in table 3 , we examined the robustness of the main estimates. to begin, we estimated modified versions of equation (4) that include state fixed effects. these models allow for an exponential trend in infection rates, thereby addressing concerns that underlying disease prevalence may evolve from one day to the next. we allowed each state to have its own specific intercept to capture the fact that the trends may differ depending on the local conditions. the results (reported in cols. 2 and 7) are virtually identical to the baseline estimates. moreover, the augmented model tends to produce more precise confidence intervals. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 30, 2020. we explored the sensitivity of the results to excluding days in which a large fraction of tests were positive. this specification addresses concerns that the functional form of the estimating equation may differ in settings in which the share of positive was large, due to the approximation in equation (2). we restricted the sample to observations in which fewer than 50% of tests were positive, and re-estimated equation (4) . table 3 , cols. 5,6,9 report the results. although the sample size is reduced, the predicted infection rates are similar in magnitude to the baseline estimates and have similar confidence intervals. in table 4 , we explored the relationship between the number of diagnosed cases and total population covid-19 infections implied by our estimation procedure. we compared the average population infection rates from march 31 to april 7 to the total number of diagnosed cases by april 12. because many individuals may not seek testing until the onset of symptoms, the latter date was chosen to capture the virus's typical five day incubation period [19, 20] . column (1) reports the total diagnosed cases by april 12; column (2) reports the total number of covid-19 cases implied by the estimates reported in table 2 (col. 4); and column (3) presents the ratio of total cases to diagnosed cases. the results reveal widespread undetected population infection. nationwide, we found that for every identified case there were 12 total infections in the population. there were significant cross-state differences in these ratios. in new york, where more than two percent of the population had been tested, the ratio of total cases to positive diagnoses was 8.7, the lowest in the nation. meanwhile, oklahoma had the highest ratio in the country (19.4) , and tested less than 0.6 percent of its population. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 30, 2020. . https://doi.org/10.1101/2020.04.20.20072942 doi: medrxiv preprint response to geographic differences in pandemic severity. instead, the patterns suggest that states that expanded testing capacity more broadly were better able to track population prevalence. and population prevalence. the similarity between these two series is notable, given that our estimates were derived from an entirely different source of variation from the cumulative case counts. nevertheless, observed case counts do not perfectly predict overall population prevalence. for example, despite similar rates of reported positive tests, michigan had roughly twice as many per capita infections as rhode island. these differences can partly be explained by the fact that nearly two percent of the population in rhode island had been tested by april 12, whereas fewer than one percent had been tested in michigan. together, these findings suggest that differences in state-level policies towards covid-19 testing may mask important differences in underlying disease prevalence. the high proportion of asymptomatic and mild cases coupled with limitations in laboratory testing capacity has created large uncertainty regarding the extent of the covid-19 outbreak among the general population. as a result, key elements of virus' clinical and epidemiological characteristics remain poorly understood. this uncertainty has also created significant challenges to policymakers who must trade off the potential benefits from non-pharmaceutical interventions aimed at curbing local transmission against their substantial economic and social costs. a number of recent studies have sought to estimate covid-19 disease prevalence and mortality in the united states and internationally [21] [22] [23] [24] [25] [26] . one approach has 11 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 30, 2020. other research has relied on bayesian modelling to infer past disease prevalence from observed covid-19 deaths, and apply sir models to forecast current infection rates. this approach requires fewer assumptions regarding the underlying parameter values. nevertheless, because these models 'scale up' observed deaths to estimate population infections, small differences in the assumed case fatality will have substantial effects on the results. this poses a challenge for estimation, given that there is considerable uncertainty regarding the case fatality rate, which may vary widely across regions due to local demographics and environmental conditions [27] [28] [29] [30] [31] . moreover, to the extent that there is significant undercounting in the number of covid-19 related deaths [32, 33] , these estimates may fail to capture the full extent of population infection. in this paper, we developed a new methodology to estimate population disease prevalence when testing is non-random. our approach builds on a standard econometric technique that have been used to address sample selection bias in a variety of different settings. our estimation strategy offers several advantages over existing methods. first, the analysis has minimal data requirements. the three variables used for estimation -daily infections, daily number of tests, and total population -are widely reported across a large number of countries and subnational districts. second, the model identification is transparent and depends only on a simple exclusion restriction assumption that daily changes in the number of conducted tests must be uncorrelated with underlying changes in population disease prevalence. this assumption is likely to hold in many jurisdictions where constraints on capacity are a primary determinant of 12 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 30, 2020. . we used this framework to estimate disease prevalence across u.s. states. we our findings are comparable to previous studies on u.s. population prevalence that find ratios of population infection to positive tests ranging from 5 to 10 by mid-march [22, 25] . despite a dramatic expansions in testing capacity in the intervening weeks, the vast majority of covid-19 cases remain undetected. our results are comparable to recent estimates of population prevalence in a number of european countries [21] . we found a nationwide 1.9 percent infection rate in early april, which is similar to the estimated prevalence in austria (1.1%), denmark (1.1%), and the united kingdom (2.7%) as of march 28. meanwhile, germany's 0.7% infection rate would rank in the lowest tercile of prevalence among u.s. states. the highest rates of infection in new york (8.5%), new jersey (7.6%), and louisiana (6.7%) are still lower than the estimated rates in italy (9.8%) and spain (15%). given the rapidly expanding availability of high frequency testing data at both the national and subnational level, in future research we plan to apply this methodology to compare infection rates across a broader spectrum of countries. there are several limitations to our study, which should be taken into account when interpreting the main findings. first, the estimation results depend on several functional form assumptions including a constant exponential growth rate in new infections and the specific functions governing how the number of available tests affect individual testing probability. as more data on testing become available, the increased sample sizes will allow future studies to impose weaker functional form assumptions through either semi-or non-parametric approaches. second, our analysis required an assump-13 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 30, 2020. . https://doi.org/10.1101/2020.04. 20.20072942 doi: medrxiv preprint tion that the underlying sample selection process was similar across observations. to the extent that decisions regarding who to test, conditional on the number of available tests, diverged across states or changed within states over the sample period, our model may be misspecified. finally, our analysis depends on the quality of diagnostic testing, and systematic false negative test results may affect the population disease prevalence estimates [34] [35] [36] . 4 as countries continue to struggle against the ongoing coronavirus pandemic, informed policymaking will depend crucially on timely information on infection rates across different regions. randomized population-based testing can provide this information, however, given the constraints on supplies, this approach has largely been eschewed in favor of targeted testing towards high risk groups. in this paper, we developed a new approach to estimate population disease prevalence when testing is non-random. the estimation procedure is straightforward, has few data requirements, and can be used to estimate disease prevalence at various jurisdictional levels. contributions db, rg, and jl conceptualized the study, analyzed the data, and drafted and finalized the manuscript. all authors approved of the final version of the manuscript. we declare no competing interests. this study was supported by funding from the social sciences and humanities research council (grant: sshrc 430-2017-00307). 4 provided that the rates of misdiagnosis were unrelated to the number of tests, these errors will not bias the coefficient estimates, but may reduce precision through classical measurement error [37] . 14 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 30, 2020. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 30, 2020. . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 30, 2020. . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 30, 2020. notes: columns (1) to (6) report the estimates and heteroskedasticity robust 95% confidence intervals for population prevalence of covid-19 on april 7 based on the methodology described in section 2. columns (7) to (9) report the the average estimates for population prevalence of covid-19 from march 31 to april 7. columns (3), (4) and (8) report results based on models that include state fixed effects. columns (5), (6) , and (9) report results based on models that restrict the sample to observations for which the share of positive cases was less than 0.5. in cases of incomplete testing data on april 7, population prevalence is reported for the closest day: * indicates prevalence on april 6, ** indicates prevalence on april 5, and *** indicates prevalence on march 31. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 30, 2020. . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 30, 2020. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 30, 2020. . https://doi.org/10.1101/2020.04.20.20072942 doi: medrxiv preprint epidemiological characteristics of 2143 pediatric patients with 2019 coronavirus disease in china sars-cov-2 infection in children evidence of sars-cov-2 infection in returning travelers from wuhan, china asymptomatic cases in a family cluster with sars-cov-2 infection. the lancet infectious disease presumed asymptomatic carrier transmission of covid-19 therapeutic and triage strategies for 2019 novel coronavirus disease in fever clinics centers for disease control and prevention: coronavirus (covid-19 the covid tracking project the many estimates of the covid-19 case fatality rate. the lancet infectious disease coronavirus covid-19 global cases the common structure of statistical models of truncation, sample selection and limited dependent variables and a simple estimator for such models the economics 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viral shedding of 2019-ncov infections. medrxiv working paper econometric analysis of cross section and panel data key: cord-289650-q2io8vgi authors: hammond, ffion r.; lewis, amy; elks, philip m. title: if it’s not one thing, hif’s another: immunoregulation by hypoxia inducible factors in disease date: 2020-07-06 journal: febs j doi: 10.1111/febs.15476 sha: doc_id: 289650 cord_uid: q2io8vgi hypoxia inducible factors (hifs) have emerged in recent years as critical regulators of immunity. localised, low oxygen tension is a hallmark of inflamed and infected tissues. subsequent myeloid cell hif stabilisation plays key roles in the innate immune response, alongside emerging oxygen‐independent roles. manipulation of regulatory proteins of the hif transcription factor family can profoundly influence inflammatory profiles, innate immune cell function and pathogen clearance and, as such, has been proposed as a therapeutic strategy against inflammatory diseases. the direction and mode of hif manipulation as a therapy is dictated by the inflammatory properties of the disease in question, with innate immune cell hif reduction being, in general, advantageous during chronic inflammatory conditions, while upregulation of hif is beneficial during infections. the therapeutic potential of targeting myeloid hifs, both genetically and pharmacologically, has been recently illuminated in vitro and in vivo, with an emerging range of inhibitory and activating strategies becoming available. this review focuses on cutting edge findings that uncover the roles of myeloid cell hif signalling on immunoregulation in the contexts of inflammation and infection, and explores future directions of potential therapeutic strategies. hypoxia inducible factors (hifs) are master transcriptional regulators of the cellular response to hypoxia, that have influential roles in innate immune cell behaviour during inflammation and infections [1] . hif activity is tightly regulated, both at the transcriptional and post-translational levels [2, 3] . the major point of control is regulation of protein degradation of the hif-α subunits. in normal oxygen conditions (normoxia) the alpha subunits are hydroxylated by prolyl hydroxylase domain (phd) enzymes leading to subsequent degradation by the proteasome (figure 1 ). phd enzymes require oxygen for their enzymatic activity, and so when oxygen levels drop, phds become inactive and hif-α is stabilised, translocating to the nucleus to form its transcription factor complex that activates downstream genes. multiple isoforms of the alpha subunit (hif-1α, hif-2α and hif-3α) contribute to an extra level of regulatory complexity [4, 5] , and while hif-1α is the most widely characterised, important roles of hif-2α in immunity are emerging [6] . the hif-3α gene is the least understood of the isoforms, with alternative transcriptional start and splice sites contributing to different hif-3α variants that can have opposing functions [7] . partially due to this complexity, the roles of hif-3α in immunity remain elusive. this article is protected by copyright. all rights reserved hif researchers were awarded the nobel prize in physiology or medicine in 2019 for their discoveries on the regulation of the cellular hypoxia response, with their medical research predominantly focusing on hifs' potential role as a therapeutic target to combat anaemia, due to hif's activating effect on red blood cell production [8] . other blood cells, especially those of the myeloid lineage of innate immunity, are exquisitely sensitive to hif modulation, due to their adaption to functioning in locations of low oxygen tension (for example wounds or infected tissues) [9] . the effects of hif modulation on immune cells are wide-ranging and context dependent. over the past decade hifs have emerged as attractive targets for immunoregulation and immunotherapy due to their ability to profoundly influence immune cell behaviour and function, combined with roles in regulating inflammatory phenotypes in other cell types of the diseased tissue milieu. here, we explore recent developments that illuminate hifs' roles in innate immunity and highlight hif signalling components as therapeutic targets in multiple disease settings. hifs are key regulators of myeloid innate immune cell function, influencing survival, migration and polarisation [9] . during infection and inflammation, hif-α is stabilised in immune cell populations, partially driven by the hypoxic tissue context of disease, alongside oxygen independent activation [10]. neutrophils are the first innate immune cell responders to wounds or infections in many disease contexts and are exquisitely sensitive to low oxygen levels. hif-1α stabilisation increases the lifespan of neutrophils and their bactericidal capabilities in multiple experimental models [11, 12] . neutrophil degranulation is an important process in pathogen control, but can cause extensive tissue damage when uncontrolled in chronic inflammatory diseases where tissue hypoxia is often a hallmark [13] . human neutrophils in hypoxia have augmented degranulation, while neutrophils with stabilised hif-α, after treatment with the pan hydroxylase inhibitor, dimethyloxalylglycine (dmog), interestingly do not have the same increased degranulation [14] . a dichotomy between physiological hypoxia and hif stabilisation is also observed in neutrophil extracellular trap (net) formation, a mechanism by which toxic components of the neutrophil (including histones, neutrophil elastase and granules) are ejected into the tissue microenvironment [15] . this article is protected by copyright. all rights reserved including chronic obstructive pulmonary disease (copd), asthma and post-covid-19 lung inflammation [16] [17] [18] . neutrophils produce fewer nets in hypoxic conditions, due to net formation requiring the oxygen-dependent respiratory burst [15] . hypoxia is even able to ablate net production when human blood-derived neutrophils are exposed to the most potent net inducing chemical, phorbol myristate acetate (pma) [15] . however, hif stabilisation in normoxia has been shown to increase neutrophil net formation through [23], although in other disease situations il-6 is associated with pro-inflammatory profiles [28] . macrophage behaviours during wound repair can be improved using hif directed therapies. in a mouse model of chronic kidney disease the phd inhibitor mk-8617, increased the infiltration of macrophages into damaged muscle, improving muscle repair and reducing muscle atrophy [29] . additionally, treatment of hif-α inhibitor yc-1, in a mouse injury model decreased numbers of pro-inflammatory macrophages in scar tissue, and reduced levels of proinflammatory cytokines in vivo [30] . these studies indicate that this article is protected by copyright. all rights reserved macrophage hif targeted therapies could allow a return to tissue homeostasis after injury/inflammation. chronic inflammation underpins diseases that are characterised by a hyperinflammatory profile with extensive immune cell-induced tissue damage. high levels of hypoxia and hif-1α are associated with many of these conditions, including asthma, copd, rheumatoid arthritis (ra), colitis and atherosclerosis [31] [32] [33] [34] . furthermore, hif signalling interplays with key inflammatory signalling pathways, including glucocorticoids, mtor and arachidonic acid [35] [36] [37] . targeting excessive hif can be beneficial in the outcome of chronic inflammatory diseases. in a mouse model of asthma, suppression of hif-1α with yc-1 reduced expression of il-5, il-13, myeloperoxidase (mpo) and inducible nitric oxide synthase (inos), which alleviated asthma symptoms [31] . in the highly-inflammatory, granulomatous condition of sarcoidosis, down-regulation of hif-1α through genetic or pharmacological means, reduced pro-inflammatory il-1β, il-17 and il-6 and improved disease outcomes [38] . however, downregulation of hif-1α can also be detrimental in some inflammation-related conditions. hif-1α deficiency in b cells exacerbates collageninduced arthritis via impairment of il-10 and b cell expansion, highlighting a need to target specific immune cell subsets [39] . stabilising hif-α subunits using hydroxylase inhibitors has been shown to be beneficial in some inflammatory conditions. dmog suppresses the progression of periapical bone loss and attenuated inflammatory cell infiltration in apical periodontitis [40] . dmog was also beneficial in a rat ischemia model, reducing infarction size and increasing il-4 and il-10 induced protection [41] . in a mouse model of multiple sclerosis, the dual peroxisome proliferator-activated receptor gamma (ppar) and cb 2 agonist, vce-004.8, was able to alleviate neuroinflammation and demyelination through inhibition of pro-inflammatory cytokines, via activation of hif [42] . these anti-inflammatory effects of hif are emerging, and are likely to be a diseasedtissue specific effect. dmog treatment of macrophages directly increases expression of pro-inflammatory factors such as inos and decreases anti-inflammatory factors such as arginase [43] . this can be highly detrimental in inflammatory conditions. for example in an ra mouse model, hif-1α stabilisation with cobalt chloride (cocl 2 ) in combination with il-17, is associated with increased disease severity [44] . stabilising hif-α in inflammatory diseases is therefore likely to be detrimental due to proinflammatory effects, although tissue and disease specific benefits may also occur. together these recent findings demonstrate the potential of hif regulation to treat chronic inflammatory conditions through modulation of the myeloid component. however, treatments that target hif can have pleiotropic effects, that differ in multiple cell types that are present in disease tissue contexts, and may require specific targeting. in tb host defence [46] . furthermore, hif-1α ko bmdms have compromised lipid droplet formation, sites of eicosanoid synthesis that are required for host tb defence [47] . during early infection, hif-1α stabilisation is beneficial for innate immune control of tb. in a zebrafish tb model, using the fish-adapted pathogen and close genetic relative of mtb, mycobacterium marinum (mm), hif-1α stabilisation increased myeloid production of il-1, vital for decreasing bacterial burden via neutrophil nitric oxide (no) production [48, 49] , alongside increasing proinflammatory macrophage tnfa production [50] . the hif-induced, host no response, has also shown to be important in a mouse model of tb infection, with hif-1α and inos in a positive feedback loop, balancing the inflammatory phenotype [51] . however, too much hif-1α during later stage tb infection can be detrimental, as hif-1α regulators prevent excess inflammation and host damage during this article is protected by copyright. all rights reserved active disease. il-17 has been shown to limit hif-1α expression in a murine model, with anti-il-17 leading to an increased number of hif-1α positive macrophages and larger tb lesions [45] . inhibition of hif-1α in these anti-il-17 mice improved infection outcome, reducing granuloma size and reversing excess inflammation [45] . together these recent data show critical roles of hif-1α in both innate immune cell tb control and in later stage granuloma pathogenesis. recent studies have added to the large bulk of evidence showing that hif stabilisation is critical for the control of multiple bacterial infections [52, 53] . helicobacter pylori infection of human gastric tissue stabilised hif-1α but not hif-2α, with hif-2α expression decreasing as disease severity increased [54] . hif-1α was shown to be crucial for bacterial killing of helicobacter pylori as hif-1α ko neutrophils possessed 66% more surviving bacteria and ko macrophages had 5 times more surviving bacteria compared to wildtype [54] . hif-1α ko mice are also more susceptible to listeria monocytogenes infection, possessing higher bacterial burdens and more severe pathological inflammation of the liver [55] . in a mouse model of uropathogenic escherichia coli (upec), stabilising hif-1α using the hydroxylase inhibitor akb-4924, reduced bacterial burden by ~40% and attenuated the virulence of a hyper-infective upec strain [56] . interestingly, activation of hif-1α in this way reduced the inflammatory profile in the murine bladder context, another example of hif-α stabilisation leading to reduced inflammation. the bactericidal properties of hif-1α stabilisation have now been moved into clinical settings, with promising results from cocl 2 containing bandages, that not only prevented bacterial survival and growth, but also promoted wound healing both in vitro and in vivo in a mouse infection model [57] . these recent studies highlight how host hif-1α could be stabilised to improve bacterial infection outcomes, particularly relevant due to rising occurrence of multi-drug resistant bacterial strains. fungal infections are an emerging global concern and recent studies have demonstrated important roles for hif in their pathogenesis. myeloid hif-1α ko mice were more susceptible to candida albicans infection and have reduced no and glycolytic activity [55] . promoting hif-1α stabilisation with the hydroxylase inhibitor cocl 2 promoted fungal death in vitro (human macrophages) and in vivo (mouse) indicating a therapeutic potential for hif-1α manipulation in candida infection [55] . activation of hif-1α has even this article is protected by copyright. all rights reserved emerged as being a natural mechanism by which commensal bacteria inhibit candida albicans colonisation in the gut [58] . infection of myeloid specific hif-1α ko mice with histoplasma capsulatum had an increased anti-inflammatory macrophage signature, increased fungal burden and decreased mouse survival, indicating important roles of hif in infection control [59] . however, alveolar macrophage (am) specific hif-1α ko mice have comparable survival compared to wt, suggesting a contribution from other cells of the myeloid lineage [59] . these studies indicate an opportunity to stabilise hif-1α in hard-to-treat fungal infections. however, as some fungi are capable of producing and releasing their own proline hydroxylases, the effectiveness of phd inhibitors as a therapy remains unclear [60] . some parasitic infections are able to circumvent host immunity by either failing to raise a proinflammatory response or, by mechanisms that actively reduce the host proinflammatory response. trypanosoma brucei suppresses host hif-1α through production of indolepyruvate, a ketoacid reported to be a cofactor of phds, promoting hif-1α degradation [61] . therefore, in trypanosome infections it may be beneficial to stabilise hif-1α therapeutically. conversely, leishmania donovani infection increases levels of hif-1α, and silencing hif-1α in murine peritoneal macrophages reduced both parasitic load and infectivity at 24 hours post infection [62] . bmdms from hif-1α deficient mice were more resistant to l. donovani infection, expressed more ros, and were parasitised less compared to wildtype [63] . however, a recent study has shown that l. donovani infected hif-1α deficient mice developed hypertriglyceridemia and lipid accumulation in splenic and hepatic myeloid cells, which impaired host defence [64] . together these studies suggest that parasites are able to manipulate hif-1α levels in a variety of ways as part of their circumvention of the host immune response, creating an opportunity to target hif-1α to improve infection outcome that has yet to be fully explored. due to the complexity of innate immune contributions to inflammatory and infectious diseases, targeting the correct hif proteins, in the correct cells, at the correct time may be important considerations for any therapeutic strategy. this article is protected by copyright. all rights reserved recent in vitro and in vivo model studies described here have largely focused on hydroxylase inhibition as the potential hif modulation therapy. the most successful hydroxylase inhibitor in human trials is roxadustat, (otherwise known as fg-4592). roxadustat has undergone phase 3 clinical trials for chronic kidney disease induced anaemia, and was able to successfully increase patient haemoglobin levels over a prolonged 8-18 week period [65] . other hydroxylase inhibitors used in clinical trials of various stages in recent years include, molidustat [66] , vadadustat (or akb-6548) [67] , and daprodustat (gsk1278863) [68] . recently, a study has shown that hif-α stabilisation can also be achieved by intrabody targeting of phd2 [69] . hif modulation via hydroxylase inhibition stabilises all hif-α isoforms, but there are emerging methods to modulate hif-α more directly. pt2385 is a hif-2α specific inhibitor that has been investigated in the first in-human safety study and was shown to be well tolerated with a favourable safety profile for patients with clear cell renal cell carcinoma (ccrcc) [70] , and may open the way for specific hif-α isoform stabilisation in future therapies against inflammatory diseases. it is interesting to note that current clinical trials for anaemia and ccrcc have used oral doses of hif targeting drugs, without delivery to specific cell types [65] . while there are no immediate indications that wholebody hif-drug delivery is detrimental in the short term, this review highlights that in complex inflammatory conditions targeting the wrong hif-α isoform in the wrong cell type could be detrimental to disease outcome. for example, it was recently shown in a model of chronic inflammation of the cornea (herpes stromal keratitis), that hif-1α was high in the myeloid lineage, whereas hif-2α was higher in epithelial cells, highlighting how different cell types in close proximity regulate hif signalling differently and may need to be targeted separately in disease [71] . the potential of hif-1α stabilisation to contribute to inflammatory and infectious comorbidities has recently been explored in zebrafish models, where it was shown that although hif-1α stabilisation delays neutrophil inflammation resolution, a host-protective effect against mycobacterial infection remains [72] . a potentially important step in immunoregulatory therapies would be cell specific targeting of hif drugs in a timely way. drug delivery methods, such as ph sensitive liposomes and liquid emulsion systems, have been used for timely delivery of hif targeting drugs to specific tissues in animal models of disease [73, 74] . emerging technologies, such as synthetic polymersomes that are preferentially accepted article taken up by innate immune cells, could be employed to deliver hif targeted drugs to myeloid cells during infection and inflammation [75] . gene therapy methods have also been explored, with nanoparticle delivery of sirna for hif-1α used to genetically manipulate hif in a murine model of hypoxic tumour growth [76] . in conclusion, hypoxia and hif stabilisation are hallmark characteristics of progressed tissue inflammation and infection. the effects of hif modulation on the innate immune response are profound, with recent findings illustrating that hif's effects can be complex and disease specific. studies in in vitro and in vivo models indicate that hif modulation during inflammation and infections may be promising therapeutic strategies against these hard-to-treat diseases. subtle fine-tuning of the innate immune system via hif manipulation to achieve a balance between protection against pathogens and hyperinflammation will be an important consideration to develop future therapies that target host immune cells during diseases of inflammation and infection. this article is protected by copyright. all rights reserved outcomes. generally, hif-1α stabilisation is beneficial in the context of bacterial infections, for example mycobacterium tuberculosis/marinum (blue column), however the level of hif-1α must be carefully balanced, with too little or too much resulting in excess inflammation and detrimental infection outcome. hif-1α knockouts have decreases in ifn [77] , il-1 [51] , and il-6 [51] leading to excess tissue damage and lower survival [46] . early hif-1α stabilisation increases inflammatory factors leading to myeloid control of infection [48] [49] [50] , while excessive hif-1α can lead to prolonged inflammation and larger tb lesions in later stage disease [51] . in the context of the fungal infection candida albicans (yellow column), hif-1α knockout decreases proinflammatory factors while increasing anti-inflammatory il-10 leading to increased fungal survival [55] . hif-1α stabilisation improves infection outcome, re-arming the host inflammatory response leading to increased fungal death [55, 58] . in the parasitic infection leishmania donovani (purple column), reducing hif-1α improves infection outcome, as the parasite itself upregulates host hif-1α, and stabilised hif-1α prevents cd8+ t cell expansion, exacerbating the infection further [62] [63] [64] 78] . each infection context is distinct, requiring a tailored and controlled hif-α response that is not uniform across infections. biophysica acta the pro-and anti-in fl ammatory properties of the cytokine interleukin-6 hypoxia-inducible factor-prolyl hydroxylase inhibitor ameliorates myopathy in a mouse model of chronic kidney disease pharmacological hif-inhibition attenuates postoperative adhesion formation hypoxia-inducible factor-1 α inhibition modulates airway hyperresponsiveness and nitric oxide levels in a balb / c mouse model of asthma myeloid cell hypoxia-inducible factors promote resolution of inflammation in experimental colitis correlation of serum levels of hif-1 α and il-19 with the disease progression of copd : a retrospective study mechanical activation of hypoxia-inducible factor 1 α drives endothelial dysfunction at atheroprone sites. arter bidirectional crosstalk between hypoxia-inducible factor and glucocorticoid signalling in zebrafish larvae glucocorticoids promote von hippel lindau degradation and hif-1 α stabilization accepted article this article is protected by copyright. all rights reserved hypoxia ameliorates intestinal inflammation through nlrp3/mtor downregulation and autophagy activation hif-1 a regulates il-1 b and il-17 in sarcoidosis hypoxia-inducible factor-1α is a critical transcription factor for il-10-producing b cells in autoimmune disease activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss hypoxia inducible factor 1 a plays a key role in remote ischemic preconditioning against stroke by modulating in fl ammatory hypoxia mimetic activity of vce-004 . 8 , a cannabidiol quinone derivative : implications for multiple sclerosis therapy phd2 is a regulator for glycolytic reprogramming in macrophages interleukin 17 under hypoxia mimetic condition augments osteoclast mediated bone erosion and expression of hif-1 α and mmp-9 interleukin-17 limits hypoxia-inducible factor 1 α and development of hypoxic granulomas during tuberculosis myeloid hif-1 a regulates pulmonary inflammation during experimental mycobacterium tuberculosis infection lipid droplet formation in mycobacterium tuberculosis infected macrophages requires ifn-γ / hif-1 α signaling and supports host defense plos pathogens: hypoxia inducible factor signaling modulates susceptibility to mycobacterial infection via a nitric oxide dependent mechanism hif-1 α − induced expression of il-1 β protects against mycobacterial infection in zebrafish hypoxia induces macrophage tnfa expression via cyclooxygenase and prostaglandin e2 in vivo nitric oxide modulates macrophage responses to mycobacterium tuberculosis infection through activation of hif-1 α and repression of nf-κ b the impact of hypoxia on the host-pathogen interaction between neutrophils and staphylococcus aureus interdependence of hypoxic and innate immune responses myeloid hif-1 is protective in helicobacter pylori − mediated gastritis hif1 α -dependent glycolysis promotes macrophage functional activities in protecting against bacterial and fungal infection role of hypoxia inducible factor-1 α ( hif-1 α ) in innate defense against uropathogenic escherichia coli infection cobalt-mediated multi-functional dressings promote bacteria-infected wound healing activation of hif-1α and ll-37 by commensal bacteria inhibits candida albicans colonization inverse correlation between il-10 and hif-1 α in macrophages infected with histoplasma capsulatum biochemical and genetic characterization of fungal proline hydroxylase in echinocandin biosynthesis trypanosoma brucei metabolite indolepyruvate decreases hif-1 α and glycolysis in macrophages as a mechanism of innate immune evasion leishmania donovani activates hypoxia inducible factor-1 α and mir-210 for survival in macrophages by downregulation of nf-κ b mediated proinflammatory immune response hif-1αlpha hampers dendritic cell function and th1 generation during chronic visceral leishmaniasis the absence of hif-1 a increases susceptibility to leishmania donovani infection via activation of bnip3 / mtor / srebp-1c axis roxadustat for anemia in patients with kidney disease not receiving dialysis. n. accepted article this article is protected by copyright. all rights reserved engl first-in-man -proof of concept study with molidustat : a novel selective oral hif-prolyl hydroxylase inhibitor for the treatment of renal anaemia clinical trial of vadadustat in patients with anemia secondary to stage 3 or 4 chronic kidney disease original investigation a novel hypoxia-inducible factor 2 prolyl hydroxylase inhibitor (gsk1278863) for anemia in ckd: a 28-day, phase 2a randomized trial intrabody against prolyl hydroxylase 2 promotes angiogenesis by stabilizing hypoxiainducible factor-1 α phase i dose-escalation trial of pt2385 , a first-in-class hypoxia-inducible factor-2 a antagonist in patients with previously treated advanced clear cell renal cell carcinoma development of inflammatory hypoxia and prevalence of glycolytic metabolism in progressing herpes stromal keratitis lesions hif-1alpha stabilisation is protective against infection in zebrafish comorbid models programmed delivery of cyclopeptide ra-v and antisense oligonucleotides for combination therapy on hypoxic tumors and for therapeutic targeted delivery of the hydroxylase inhibitor dmog provides enhanced ef fi cacy with reduced systemic exposure in a murine model of colitis therapeutic delivery of sirna silencing hif -1 alpha with micellar nanoparticles inhibits hypoxic tumor growth hif-1α is an essential mediator of ifn-γ-dependent immunity to mycobacterium tuberculosis key: cord-342464-6vk2oxo5 authors: edwards, michael r.; bartlett, nathan w.; hussell, tracy; openshaw, peter; johnston, sebastian l. title: the microbiology of asthma date: 2012-06-06 journal: nat rev microbiol doi: 10.1038/nrmicro2801 sha: doc_id: 342464 cord_uid: 6vk2oxo5 asthma remains an important human disease that is responsible for substantial worldwide morbidity and mortality. the causes of asthma are multifactorial and include a complex mix of environmental, immunological and host genetic factors. in addition, epidemiological studies show strong associations between asthma and infection with respiratory pathogens, including common respiratory viruses such as rhinoviruses, human respiratory syncytial virus, adenoviruses, coronaviruses and influenza viruses, as well as bacteria (including atypical bacteria) and fungi. in this review, we describe the many roles of microorganisms in the risk of developing asthma and in the pathogenesis of and protection against the disease, and we discuss the mechanisms by which infections affect the severity and prevalence of asthma. asthma is a chronic airway disease for which the symptoms are wheezing, breathlessness, chest tightness and coughing, with variable and often reversible airway obstruction accompanied by airway hyperreactivity. these symptoms result from a range of processes, including contraction of airway smooth muscle (asm), airway inflammation, airway infections, immunoglobulin e-mediated allergic responses, exposure to pollutants, exercise, stress and the stimulation of cholinergic and sensory nerves 1 . a key feature of asthma is decreased lung function, measured as a reduction in either the peak expiratory flow (pef) or the forced expiratory volume in 1 second (fev 1 ). some of the immediate symptoms of asthma are reversed by a short-acting inhaled β 2 agonist, but longer-term control requires inhaled anti-inflammatory glucocorticoids. worldwide, asthma affects ~300 million people, including both adults and children. it is associated with onset early in life and is increasing in incidence in the developed world. an interesting aspect of asthma is its heterogeneous, complex nature and the fact that it can present as a chronic, stable disease and as asthma exacerbations. asthma can be severe or mild, and can vary spontaneously in activity and greatly in terms of the time of onset or the response to therapy. indeed, whether asthma is one disease or a spectrum of related but subtly different conditions is often debated 2 , and asthma has recently been divided into separate pheno types, which are in turn subdivided into several endotypes 3 (table 1) . asthma exacerbations are often triggered by multiple factors that may work in an additive or synergistic manner. this complex pathogenesis has made it challenging to understand the cellular mechanisms that are responsible for asthma, to decipher candidate genetic predispositions and to identify causative agents. the causes of asthma are multifactorial, and epidemiological studies spanning three decades across five continents have proved that there is a strong association between respiratory infections and the risk and pathogenesis of asthma. in this review, we summarize the association of microorganisms with asthma, discuss the importance of such organisms for the disease and consider the idea that part of the cause and at least some of the patho genesis of asthma -in particular, of asthma exacerbations -result from microorganisms and their components. we also discuss the potential therapeutic value of micro organisms and their products for treating asthma and argue that future treatment and management of the disease must carefully consider the role of microorganisms. the hygiene hypothesis. the hygiene hypothesis posits that repeated exposure to diverse common infections (in particular, with bacteria, food-borne and oro faecal parasites 4 , and hookworms 5 ) and exposure to environmental microbiota during childhood 6, 7 are strongly associated with a healthy maturation of the immune system and with protection from the development of asthma and allergies later in life 8, 9 . our understanding of the importance of microorganisms in protection from allergies and asthma has been recently enhanced by a molecular analysis of the microbiome. populations that live in environments with diverse microbiological fluid or mucus in the airways, which gives rise to a typical 'wheezy' sound when breathing. flora are associated with a low incidence of asthma, whereas populations that live in environments with low micro biological diversity are associated with a higher incidence of asthma 7 . the incidence of bacteria and their components in different environments has been studied using culture-dependent and molecular methods [10] [11] [12] , and both qualitative and quantitative differences are found between countries with a high incidence of atopy and those with a low incidence of atopy 10 . evidence for the hygiene hypothesis is also supported by experiments in mice, whereby intranasal exposure to escherichia coli 13 or acinetobacter lwoffii str. f78 protects against allergic inflammation of the airways 14 . in addition, a link has been established between the fermentation of dietary fibre by the mouse intestinal microbiota and protection against inflammatory diseases, including asthma 15 . although these studies provide experimental support for the hygiene hypothesis, our understanding of the mechanistic basis of this phenomenon is still in its infancy. these studies have also promoted the use of bacterial components such as toll-like receptor (tlr) ligands and other pathogenassociated molecular patterns as potential therapies for asthma (table 2) . much evidence points to a relationship between severe lower respiratory infections with human respiratory syncytial virus (human rsv; referred to hereafter as rsv) or human rhinoviruses (rvs) early in life and the development of recurrent wheeze followed by asthma diagnosis in later childhood. rsv is a negative-sense single-stranded rna (ssrna) virus of the paramyxoviridae family. it is an important pathogen of young children (<2 years of age) and accounts for ~70% of severe infantile viral bronchiolitis cases 16 . serological monitoring shows that rsv infects nearly all children in their first 2 years of life; however, only ~40% of children exhibit clinical signs of infection in the lower respiratory tract. for unknown reasons, some children with bronchiolitis develop recurrent respiratory symptoms. rvs are members of the family picornaviridae, are composed of a positive-sense ssrna genome of approximately 7.1-7.5 kb and can be grouped into the major and minor groups on the basis of their host receptors: major group viruses bind intercellular adhesion molecule 1 (icam1), whereas minor group viruses bind low-density-lipoprotein receptor (ldlr). rvs can also be classified into three groups on the basis of nucleotide sequence identity (rv-a, rv-b and rv-c). the recently proposed rv-c group viruses 17, 18 have unique sequences at the icam1-and ldlr-binding sites, suggesting that they use a different (but currently unknown) receptor 19 . it is estimated that there are more than 100 serotypes of rvs, meaning that multiple serotypes may infect an individual throughout the year 20 . recent cohort studies suggest that lower respiratory infections by rvs are as important as infections by rsv in terms of the risk of later asthma development 21-23 . whether these viruses are true causative agents of asthma or (owing to shared risk factors) simply act as markers for an increased risk of asthma development, or even whether both possibilities are true, is unclear and much debated. rsv-mediated bronchiolitis is clearly associated with later asthma development 24,25 , perhaps owing to an inappropriate or overexuberant immune response to the infection 26 . although associations between rsv, wheeze and asthma development are evident, birth cohort studies have shown that 90% of nonatopic wheezers lose this phenotype at school age and have normal lung function, indicating that asthma and virus-induced wheeze may be distinct diseases 27 . the association of rsv with asthma is most clearly demonstrated in a long-term (18 years) study of a cohort of swedish children. the most recent update from this study 28 reports that, compared with controls, children who have rsv-mediated bronchiolitis in infancy are more likely to be asthmatic at 18 years old (39% versus 9%), show increased sensitization to perennial allergens (41% versus 14%) and have striking persistent and recurrent wheeze (30% versus 1%). children with a history of bronchiolitis have an enhanced interleukin-4 (il-4) (that is, t helper 2 (t h 2) type) response to rsv and cat allergens, whereas controls have an equally strong interferon-γ (ifnγ) (that is, t h 1 type) response to rsv antigens 29 . however, it is not yet clear whether the inflammation caused by viruses causes long-term respiratory disease or is associated with wheeze and asthma diagnosis because of shared risk factors. animal studies have further contributed to our understanding of this phenomenon 30 , and although many clinical studies are compatible with a direct effect of rsv on the later development of recurrent childhood wheeze, it has been hard to prove this direct causal relationship. the strongest evidence that human rsvmediated bronchiolitis has long-term effects comes from studies of palivizumab (synagis; medimmune), a monoclonal antibody that prevents infection by rsv in infancy. these studies suggest a causal link between infantile rsv-mediated disease and wheeze followed by asthma diagnosis in later childhood; in a study of a measure of the ratio of the probability that an event will occur in one group versus another. 191 palivizumab-treated and 230 control (untreated) premature babies who were followed for 24 months, the rates of recurrent wheeze were about 50% lower in those who had prophylactic treatment with palivizumab 31 . more recently, palivizumab prophylaxis was shown to be associated with a similar substantial protection against recurrent wheeze in children aged 2-5 years 32 . much debate has focused on which of these viruses are more important to asthma development, rsv or the rvs. the answer is likely to be complicated and may involve host genetics and susceptibility factors as well as the nature of the viruses themselves. it is possible that rsv and rvs contribute to asthma development in different ways. a recent study has shown that allergic sensitization precedes or is a risk factor for symptomatic infection with rvs, and both sensitization and rv infection are strongly correlated with the occurrence of asthma at 6 years of age 33 . by contrast, the role of rsv infection in this study seemed to be different, as sensitization did not increase the risk of infection in this cohort. in the absence of independently conducted, placebocontrolled prospective studies of specific prophylaxis measures, doubt will remain about the nature of the association between infections with these viruses in infancy and the later development of childhood asthma. however, the evidence obtained so far raises hope that preventing such infections will have long-term beneficial effects on asthma development. specific asthma phenotypes or endotypes stable asthma. microorganisms are now also thought to be important in the development and pathogenesis of stable asthma. 16s ribosomal rna gene sequencing was carried out on bronchial brushings or bronchoalveolar lavage (bal) from patients with stable asthma, patients with chronic obstructive pulmonary disease (copd) and healthy controls 34 , and revealed 5,054 bacterial sequences that were unique to the 43 subjects with asthma or copd, with each bronchus containing a mean of 2,000 bacterial genomes per cm 2 surface sampled. pathogenic proteobacteria, particularly haemophilus spp., were more frequent in the bronchi of adults with asthma or copd than in the bronchi of controls. similar increases in proteobacteria were found in children with asthma. in addition, there was a lack of members of the phylum bacteroidetes in individuals with asthma when compared with controls, suggesting a potential role for commensals as well as pathogens in influencing asthma 34 . it is thought that such commensals may stimulate the immune system such that pathogenic species are eliminated if and when infection occurs (fig. 1) . changes in the abundance and diversity of commensals can also affect subsequent infection by influenza viruses in mice 35 . the lower airway thus contains a characteristic microbiota that is quantitatively and qualitatively different in patients with stable asthma or copd than in control subjects 34 . this study challenges the previously held belief that in healthy individuals the lower airway is a sterile environment that does not support a complex microbial ecology. the diversity of bacteria that has now been revealed suggests that clinically important microorganisms which are unidentifiable by culture have roles in airway diseases such as asthma. invasive pneumococcal infection (ipi), which is caused by the common pathogen streptococcus pneumoniae, has also been linked to asthma. adults with stable asthma have been shown to have a greater incidence of s. pneumoniae carriage 36 , and asthma has been shown to be a risk factor for ipi in both adults 37,38 and children 39 . one study had a particularly important finding 37 : for low-risk asthma (as defined by the need for prescription medication for asthma) the odds ratio for ipi (versus no ipi) was 2.8-fold, whereas for high-risk asthma (as defined by hospitalization for asthma in the past 12 months) it was over 12-fold. together, these studies show the importance of asthma in the risk of invasive s. pneumoniae infections, and combined with the other studies they strongly advocate the need for antipneumococcal vaccination for individuals with asthma. atopic asthma is the most common form of asthma and is often characterized by eosinophils in bal and sputum. filamentous fungal species of the aspergillus, alternaria, cladosporium, penicillium and didymella genera (in the phylum ascomycota) produce spores that may act as allergens and initiate bronchial asthma in atopic individuals 40 . comparative genomics studies have identified proteins that act as allergens in 22 fungal species 41 , many of which are ubiquitous in the environment and commonly found on grains and cereals. the release and abundance of fungal spores is affected by environmental factors, including season, humidity, rainfall and temperature 42 . many fungal aeroallergens increase in abundance in the summer along with pollen levels and have been shown to be further increased by summer storms that include lightning strikes. indeed, fungal aeroallergens have been associated with asthma epidemics that occur during summer figure 1 | the microbiome is influenced by and may determine factors affecting asthma. the microbiome is regulated by diet, environmental factors (such as smoking and pollution), treatment, host genetics, and previous infections and host immunity. the diversity of the microbiome may in turn affect asthma development, as suggested by the hygiene hypothesis, and may influence the pathogenic species that contribute to stable and severe asthma and asthma exacerbations. an abnormal increase in the number of neutrophils in a body fluid such as bronchoalveolar lavage. a chronic, irreversible inflammatory condition of the airways, involving tissue destruction and neutrophilic inflammation. bronchiectasis has a wide range of causes and is associated with bacterial and fungal infections. it can occur in parallel with asthma. lightning strikes 43 , possibly owing to spore aerosolization and fragmentation to produce finer particles that easily find their way to the lower respiratory tract. neutrophilic asthma is a less common form of the disease that is characterized by neutrophils, with or without eosinophils, being the most abundant granulocyte in the airways of the patient 44 . respiratory viral infections have been associated with airway neutrophilia: asthmatic adults experiencing asthma exacerbations show increased airway neutrophilia 45 , including neutrophil elastase in their sputum 46 . respiratory viruses can induce neutrophil chemokine synthesis and release 45,47 , providing a mechanistic link between neutrophilic asthma and virus infections. bacterial pathogens such as haemophilus influenzae, staphylococcus aureus, moraxella catarrhalis and pseudomonas aeruginosa have also been associated with neutrophilic asthma, with one study showing an association between airway neutrophils, increased bacterial load and the neutrophil chemokine il-8 (also known as cxcl8) 48 . recently, a molecular approach was used to investigate the microbiota in poorly controlled asthma 49 , and the results suggest an association between the families comamonadaceae, sphingomonadaceae and oxalobacteraceae and airway hyper-reactivity. furthermore, this study shows that the bacteria which are found in the lung differ in health and disease and that the abundance of particular phylotypes, including members of the families comamonadaceae, sphingomonadaceae, oxalobacteraceae and others, is highly correlated with the degree of bronchial hyper-responsiveness (disease severity). fungi have also been associated with severe asthma 50,51 . in fact, severe asthma with fungal sensitization (safs) has been considered as an independent subgroup of severe asthma, and patients with safs may benefit from antifungal therapy. in a recent clinical trial of antifungal therapy in patients with safs, improvements were seen in antifungal-treated patients (versus placebo-treated patients) in terms of asthma-related quality of life, rhinitis score, lung function and total serum ige 52 . allergic bronchopulmonary aspergillosis. allergic bronchopulmonary aspergillosis (abpa) is a unique form of asthma that is caused by colonization of the lower respiratory tract with aspergillus spp. chronic infection with aspergillus fumigatus can lead to bronchiectasis 53 , a disease that can occur in parallel with asthma and is characterized by neutrophilic inflammation, epithelial destruction and degradation of the connective tissue matrix. in this disease, the fungus acts as both a source of allergen and a pathogen, and symptoms of abpa can be attributed to both functions. aspergillus spp. are widespread fungi and potent sources of allergens 54 , as shown by the reactivity of human ige to at least 12 allergens derived from these species 55 . recently, the genome of a. fumigatus was sequenced 56 , and nine new potential allergens were identified on the basis of sequence identity with other known allergens. the predicted and known allergens of a. fumigatus are diverse in nature and include secreted proteases, glucanases and cellulases. recent work suggests that some a. fumigatus allergens are 'hidden' from the immune system; hydrophobin (roda) is an immunologically inert surface protein on dormant conidia, and removal of roda through chemical or genetic means produces conidia that are able to activate the immune system, potentially explaining why fungal species often do not evoke immune responses in healthy individuals 57 . colonization by a. fumigatus results in sensitization to fungal allergens and, subsequently, leads to allergic and/or asthma symptoms, eosinophilic and neutrophilic pneumonia 53 , and lung damage caused by the extra cellular hyphae and lung inflammation. an infection model in mice using a bioluminescent a. fumigatus strain 58 revealed that infection results in extensive haemorrhagic bronchopneumonia, characterized by inflammatory lesions on the bronchi and bronchioles, underscoring the potentially aggressive nature of this pathogen in affected individuals. furthermore, a. fumigatus infection has been linked to ige sensitization, airway neutrophilia and decreased lung function in abpa, and antifungal therapy has also recently been shown to be effective in patients with abpa 59 . microorganisms in asthma exacerbations. respiratory viruses are the major viruses associated with asthma exacerbations, accounting for 50-60% of exacer bations in all age groups 60,61 (fig. 2) . rv-c may represent a group that causes more severe exacerbations, although how this occurs is unknown 18 . epidemiological studies have shown that, in the northern hemisphere, infections by rvs result in a marked increase in emergency room admissions owing to asthma exacerbations. in fact, this 'asthma epidemic' has been shown to coincide with labour day in canada 60 , which is in the third week of september, ~2 weeks after children return to school, highlighting the important role of school-age children as vectors of virus-induced asthma exacerbations. mouse models of infection with major and minor group rvs 62,63 and of rv-mediated exacerbations of airway allergen challenge 62,64 have recently been developed and used to demonstrate the ability of rv infection to augment airway inflammation caused by allergen sensitization and challenge. despite common bacterial infections first being suggested to have a role in asthma exacerbations as early as the 1940s 65 , it was not thought that such infections were important in the pathogenesis of asthma exacerbations 66 until recently. however, we now realize that patients with asthma have an increased susceptibility to respiratory bacterial infections 37-39 and increased carriage of pathogenic respiratory bacteria (identified by both culture methods 36 the atypical bacteria mycoplasma pneumoniae and chlamydophila pneumoniae are common respiratory pathogens and have also been associated with asthma, wheeze and asthma exacerbations in both adults and children 69-71 . detection rates for these bacteria can be as high as 80% in patients with these conditions 69 , and serological positivity rates for these atypical bacteria are often 40-60% 69,71 , indicating that viral and atypical bacterial infections probably interact to increase the risk of asthma exacerbations. the macrolide antibiotic clarithromycin has previously shown efficacy in patients with stable asthma who are known to be carrying either m. pneumoniae or c. pneumoniae; in such patients, the drug improves lung function and reduces tumour necrosis factor (tnf) production 72 . more recently, the ketomacrolide telithromycin has shown efficacy in patients with asthma exacerbations, resulting in substantial reductions in the asthma symptom score and improvements in lung function 71 . however, the effects of telithro mycin were found to be not necessarily related to c. pneumoniae or m. pneumoniae exposure, suggesting that telithromycin has additional protective properties that are independent of its antibacterial properties. the identification of key microorganisms that are linked with asthma development or asthma pathogenesis raises the important question of which properties of these microorganisms promote asthma. the microorganisms that commonly infect different areas of the lower airway are shown in fig. 3 . some of these microorganisms may be found systemically in more severe disease, as has been shown for rvs 73 . a key feature of a microorganism in promoting asthma is the ability to induce lung inflammation, injury, or repair and remodelling. many of the innate receptors that recognize respiratory fungi, viruses or bacteria, including tlrs, rig-i-like helicases and nod-like receptors, activate the nuclear factor-κb (nf-κb) family of transcription factors, which induce more than 100 pro-inflammatory and host response genes 74 . rsv and rvs induce a range of pro-inflammatory cytokines, chemokines, growth factors, adhesion molecules and mucins. mucins cause mucous plugging of the airway and may provide a substrate for bacterial colonization, whereas cytokines facilitate cellular chemotaxis, and activation and proliferation of immune cells in the infected airway 45,75 . microorganisms may damage and compromise the integrity of the airway by infecting airway epithelial cells, causing cell death 76 and shedding 77 . they can also affect epithelial permeability, leading to increased airway inflammation and creating opportunities for increased infection 78 , allergen uptake 79 or exposure to environmental pollutants, all of which are important contributing factors in asthma (fig. 4) . respiratory pathogens can also induce mediators that are required for airway repair, resulting in airway scarring or remodelling. these processes include angiogenesis, increased asm proliferation and hypertrophy, and thicker basement membranes owing to collagen and fibronectin deposition, as well as the generation of new lymphatic vessels that ultimately result in thicker airway walls and reduced lung function. whether or not viral or bacterial infections directly initiate this process is unclear; however, what is clear is that respiratory infections, including by mycoplasma pulmonis, rvs, rsv and a. fumigatus spores, can induce some of these pro-angiogenic mediators, including fibroblast growth factors and vascular endothelial growth factors [80] [81] [82] , and the structural proteins perlecan (also known as hspg) and collagen 83 , the building blocks of airway remodelling. asthma treatment often requires daily treatment with a range of immunomodulatory or immunosuppressive agents, such as macrolide antibiotics or glucocorticoids, suggesting another way that microorganisms interact with asthma. surprisingly, scant attention has been paid to the possible effects of such treatments on an individual's microbiota and their antimicrobial responses. however, some studies provide indirect evidence for an immunosuppressive effect. for example, increases in bacterial pneumonia have been noted in patients with copd who are treated with glucocorticoid therapy 84 . furthermore, an increased incidence of upper respiratory tract infections following treatment with the leuko triene receptor antagonist montelukast has been reported in young children with asthma 85 . it is also interesting to speculate whether the fact that asthma is a risk factor for ipi, as previously discussed, is directly attributable to asthma therapies such as glucocorticoids. conversely, there is some experimental evidence suggesting that the action of glucocorticoids confers benefits a substance that interferes with the association of two liquids or a solid and a liquid. detergents are surfactants that function by lowering the surface tension of liquids. substances that enhance adaptive immune responses (both b cell and t cell responses) to immunogens. adjuvants are not recognized by t cells or b cells themselves but may function by activating specific cells or acting as a long-term depot for slow and effective immunogen release. to antimicrobial immunity. in vitro, glucocorticoids have been shown to induce a number of antimicrobial peptides, pentraxins, collectins and complement proteins 86 . furthermore, clinical studies support the benefits of glucocorticoid use: in preschool children, the use of glucocorticoids for 2 years reduces asthma exacerbations (as measured by exacerbations that require systemic gluco corticoid administration and hospital admissions) 87 . although several studies support the use of macrolide antibiotics for the treatment of atypical pathogens in patients with asthma exacerbations 88 , it is important to consider how the microbiome is affected by macrolides or other antibiotics and how this might affect asthma and the host responses to infection. treatment of mice with the antibiotics vancomycin, neomycin, metro nidazole and ampicillin greatly changes their airway and gut microbiome and impairs their antiviral responses to influenza viruses 35 . in human studies, it is currently unclear whether the genera that seem to colonize the airways of patients with asthma or copd and of individuals without asthma or copd differ because of treatment-specific effects 34 . more research is needed to understand whether the microbiome of patients with asthma is actually a consequence of active therapy. further large clinical trials with such treatments are required to understand the relationships between microorganisms and asthma treatments. infections or environmental stresses may induce alterations in epithelial cells and underlying stromal cells, and these alterations may modify the response of these cells to further stimulation or may influence barrier function. for example, enhanced permeability caused by virusinduced cytopathology may lead to inhaled allergens, pollutants and other irritants having greater access to basal cells and the underlying airway tissue, which could be detrimental because submucosal antigen-presenting cells (apcs) are not restrained like their airway lumen counterparts. epithelial cells orchestrate innate immune regulation in the airway lumen, preventing an inflammatory response to non-injurious stimuli by controlling tlr signalling and phagocytosis through the expression of surfactant proteins 89 . epithelial cells also express mucin 1 (muc1), which suppresses alveolar macrophage responses by binding to tlrs 90 . il-10 and transforming growth factor-β (tgfβ) from airway epithelial cells can influence the immune milieu by raising the threshold of activation of airway macrophages 91 and driving the expression of the inhibitory receptor cd200r on alveolar macrophages. the ligand for cd200r, cd200 (also known as ox2), is expressed on the luminal aspect of the airway epithelium, and the interaction of these two proteins prevents the activation of alveolar macrophages in the presence or absence of inflammatory stimuli 92 . thus, macrophages in the airspaces are less proficient at antigen presentation than submucosal macrophages, are poorly phagocytic and secrete prostaglandins and tgfβ to reduce t cell responses, leading to reversible t cell inactivation 93 . alveolar macrophages also actively inhibit interdigitating dendritic cells (dcs) in the airways 94 , which in turn secrete il-10. apcs in the submucosal tissues are less restrained by these factors simply because the cells are not exposed to these epithelium-derived innate suppressors. should any of these pathways be altered through infection, the less restrained apcs may recognize innocuous antigens or allergens, ultimately contributing to inflammation and disease. recognition of respiratory microorganisms by pattern recognition receptors can inadvertently initiate or exacerbate an unrelated inflammatory condition. for instance, tlr agonists may act as microbial adjuvants that enhance the recognition of concurrent allergens 95 . the major cat allergen fel d 1 is often contaminated with tlr ligands derived from microorganisms found in the feline oral cavity, and the house dust mite often contains the commensal fungus aspergillus penicillioides, which activates tlr2 and tlr4. these allergen preparations can contain dna from gram-negative microorganisms 96, 97 . the relationship between tlrs and allergens has recently become more complex, with evidence showing that allergens themselves may be direct tlr agonists 98, 99 . molecular mimicry between allergens and microbial components 100 raises the idea that tlrmediated recognition of allergens may be an important part of sensitization to these molecules. infection also causes the recruitment of apcs in high numbers and for long periods following pathogen lymphoid tissue that develops only after stimulation with antigen, such as in the process of inflammation. the lymphocytes of tertiary lymphoid tissue are typically derived from primary or secondary lymphoid tissue. the body or bulk of a tissue, such as the lungs. the lung parenchyma typically encompasses the alveoli and cells within. clearance. for example, cells expressing cd11c (also known as integrin αx) display enhanced antigen presentation long after the resolution of an acute infection by influenza virus or rsv 101, 102 . such cells are long lasting 103 , suggesting that an enhanced mature apc compartment assists immunological responses on subsequent exposure to environmental allergens. this would also be facilitated by an increase in tertiary lymphoid tissue in the lung parenchyma, which is a common feature following respiratory tract infection 104 , in human asthma 105 and in experimental mouse models of asthma 106 . successive infections may also alter the lung environment and affect the delicate balance between host, pathogen and chronic airway disease. bacterial infections are known to follow influenza virus infections; indeed, many of the individuals who died in the influenza pandemics exhibited coexisting bacterial pneumonia 107 . this has implications for asthma: what is assumed to be a virusinduced asthma exacerbation may in fact include a secondary bacterial infection. data suggesting that this does occur have been reported for c. pneumoniae 69 , but further studies are required to determine whether secondary bacterial infections commonly follow viral infections in asthma. thus, by causing barrier disruption, viruses may initiate a cascade that, as described above, allows environmental bacteria better access to the underlying tissue (fig. 4) . microorganism and host factor synergy t helper 2 type immunity. t h 2 cells are cd4 + t cells that produce a defined array of cytokines -classically, il-4, il-5, il-9 and il-13 -and home to sites of inflammation. t h 2 cells are involved in defence against gastrointestinal nematodes and are also strongly implicated in the pathogenesis of asthma and asthma exacerbations. case control studies show a clear link between respiratory virus infection together with allergen exposure in sensitized children 108 and adults 109 in increasing the risk of hospital admissions due to asthma exacerbations. furthermore, experimental rv infection models have shown that patients with asthma have more severe lower respiratory tract symptoms and greater reductions in lung function than control patients, and have increases in bronchial hyper-reactivity 75 . exacerbation severity is strongly correlated with both t h 2 type cytokine production from bal cd4 + t cells and viral load. rv infection in mouse asthma models supports these observations, as rvs enhance t h 2 type cytokine production by sensitized mice that are simultaneously challenged with aeroallergen 62 . how viral infection can synergize with or act additively to t h 2 type immunity is shown in fig. 5. excessive t h 2 type responses are implicated in the pathogenesis of rsv-mediated bronchiolitis 110 , and increased production of il-5 by t cells at birth is associated with a greater risk of severe respiratory infection 111 . genetic associations also link polymorphisms in the t h 2 gene locus to severe rsv-mediated disease 112, 113 . increased t h 2 type responses are reported in peripheral blood mononuclear cells and sputum preceding acute asthma exacerbations in vivo and also in mixed t h 1-t h 2 cell populations during acute exacerbations in vivo 114 . , cxcl5 (also known as ena78) and cxcl8 (also known as il-8)), and t helper 1 (t h 1) type chemokines (cc-chemokine ligand 5 (ccl5; also known as rantes), cxcl10 (also known as ip10) and cxcl11 (also known as itac)), and increases epithelial permeability. neutrophils secrete several mediators that can contribute to inflammation and the integrity of the airway, including matrix metalloproteinases (mmps), elastase and reactive oxygen species (ros). epithelial cells normally express luminal cd200, which, when bound to its receptor, cd200r, on lumen macrophages, prevents the macrophage response to inflammatory stimuli. lumen macrophages are normally inhibitory, as they produce transforming growth factor-β (tgfβ), which inhibits inflammatory airway dendritic cells (dcs). a higher epithelial permeability or epithelial damage as a result of infection allows the unrestrained submucosal macrophages access to the allergen and to other stimuli, and this can promote overzealous inflammation. submucosal macrophages and epithelial cells are triggered by exposure to bacteria or viruses (and their products) to produce interleukin-1β (il-1β), il-6 and tumour necrosis factor (tnf), which promote inflammation. asm, airway smooth muscle; gm-csf, granulocytemacrophage colony-stimulating factor; ifnγ, interferon-γ; mhc, major histocompatibility complex; t h 1, t helper 1. also known as tarc), a chemokine that binds with high affinity to cc-chemokine receptor 4 (ccr4), which is abundantly expressed on t h 2 cells. in mice, rsv was found to induce ccl17 production, and this finding was reinforced in a t h 2-biasing model in which mice were first vaccinated with recombinant vaccinia virus expressing rsv major surface glycoprotein g and then infected with rsv 115 . in vitro, production of ccl17 by human becs infected with human rsv requires the presence of t h 2 type cytokines and is associated with activation of both nf-κb and stat6 (signal transducer and activator of transcription 6) 115 . recently, the signalling molecule endoplasmic reticulum ifn stimulator (eris; also known as tmem173) has been shown to induce stat6 activation in viral infections 116 , providing a potential mechanism for how viruses induce t h 2 type cytokines or chemokines. thymic stromal lymphopoietin (tslp) is a t h 2 cell-promoting cytokine that is produced by becs and can be induced by both bacterial (tlr2 and tlr9 activating) and viral (tlr3 and tlr8 activating) ligands 117 . this cytokine matures the dc network and, in humans, enhances the cytolytic activity of cd8 + t cells and the production of t h 2 type cytokines, leading to enhanced airway hyper-reactivity. furthermore, mast cells in the bronchial mucosa of patients with asthma express the tslp receptor. together with il-1, supernatants from tlr2-activated airway epithelial cells induce mast cell production of il-5 and il-13, which have pivotal roles in the development and maintenance of asthma 117 . both rv-and rsv-infected becs produce tslp. human rsv-induced tslp production stimulates dc expression of the t h 2-associated molecules ccl17 and ox40 ligand (ox40l; also known as tnfsf4) 118 . rv-induced tslp production is synergistically enhanced by il-4 (ref. 119 ). becs from patients with asthma also produce higher levels of tslp than becs from healthy individuals when polyinosinicpolycytidylic acid stimulation is used as a surrogate for viral infection 120 . thus, by producing tslp in response to infection, the airway epithelium may orchestrate the pathophysiology of asthma. il-25 is structurally related to members of the il-17 cytokine family but, like tslp, is another t h 2 cellamplifying cytokine that is expressed by a number of different cells, including becs. a recent study using a mouse rsv infection model demonstrated the importance of il-25 in regulating airway disease following viral infection. natural killer cell-depleted mice exhibit enhanced t h 2 type airway inflammation following rsv infection. using an il-25-neutralizing antibody, this rsv-specific t h 2 cell-skewed response was shown to be dependent on il-25 production by airway epithelial cells 121 . allergen-specific ige is a key mediator in allergic reactions and has a central role in the pathogenesis of asthma. il-4 and il-13 stimulate the production of ige, which binds allergen, allowing crosslinking of ige receptors on mast cells and basophils; this causes the release of mediators that have profound effects on airway inflammation and hyper-reactivity. analysis of nasal secretions from rsv-infected infants revealed the presence of rsv-specific ige, and ige levels were found to positively correlate with wheeze and higher levels of histamine 122 , although these studies have been hard to replicate. in mice, primary rsv infection is usually dominated by t h 1 type effects but may induce il-4 and il-13 expression in the lungs as well as the production of rsv-specific ige in serum under some conditions. transfer of rsv-specific ige to naive mice increases airway hyper-reactivity following rsv infection, an effect that is dependent on expression of high-affinity ige receptor 123 . rsv infection in neonatal mice also induces t h 2 type cytokine-dependent rsv-specific ige. re-infection with rsv 5 weeks later induces a t h 2 cell-skewed immune response that is reduced in magnitude by ige-specific antibody therapy 124 . a mechanism has been proposed to explain how ige against a related paramyxovirus, sendai virus (sev), enhances t h 2 cell-associated airway disease in mice. this suggested mechanism involves infection-induced upregulation of high-affinity ige receptor on conventional lung dcs. the authors speculate that the later appearance of sev-specific ige, after viral clearance, crosslinks ige receptors (with viral antigen) on conventional dcs, stimulating the production of ccl28, which in turn recruits activated il-13-producing t h 2 cells to the lung 125 . although there is still much to learn, it has become clear that understanding how respiratory infections interact with t h 2 type immunity, thereby increasing the severity of allergic airway inflammation, may provide opportunities for the development of novel strategies to treat asthma. therapies targeting t h 2 type immunity and ige (table 2) are already yielding encouraging results. the idea that patients with asthma might be more susceptible to viral infections than healthy individuals first surfaced through the study of co-habiting spouse pairs with and without asthma 126 . although individuals with and without asthma had the same incidence of rv infections, individuals with asthma had lower respiratory tract symptoms for longer than individuals without asthma, and had an increased severity of symptoms. cultured primary becs from adults with allergic asthma exhibit lower levels of rv-induced ifnβ (a type i ifn), lower levels of virus-induced apoptosis and higher rv loads than cells from control adults 127 . a deficiency in the antiviral type iii ifns, ifnλ1 (also known as il-29), ifnλ2 (also known as il-28a) and ifnλ3 (also known as il-28b), has also been described 128 . ifns may therefore be protective against rv-induced asthma exacerbations. however, two similar studies failed to see any difference between individuals with and without asthma regarding rv-induced ifn expression in similar model systems 129, 130 , suggesting that impaired ifn expression is a feature of a subset of patients with asthma. these studies raise the possibility of using ifnβ and ifnλs as therapeutics against asthma exacerbations. although epidemiological data clearly show that asthma is a risk factor for ipi, there are few studies that have considered a mechanistic rationale for this. genome-wide association studies in individuals with asthma or atopy do, however, highlight polymorphisms in innate immune genes such as those encoding tlrs and transcription factors, and such polymorphisms potentially explain the increased susceptibility of these individuals to viruses, bacteria and fungi [131] [132] [133] . deficiencies in the t h 1 type cytokines ifnγ and il-12 in patients with asthma 75 also suggest that impaired host defence responses to bacteria are probably implicated. an understanding of how these genetic and cytokine differences affect host susceptibility to infection may help in establishing new therapies for asthma. advances in molecular microbiology offer new avenues by which to discover the roles of microorganisms in asthma pathogenesis and exacerbation. there is now overwhelming evidence that asthma can be viewed as a chronic inflammatory condition which is initiated, triggered and maintained by the respiratory microbiota. microorganisms have roles in the onset and development of asthma but can also be protective (the hygiene hypothesis). it is not yet clear whether some organisms (especially rsv) actually cause atopic disease or are instead associated with atopy because individuals who are prone to atopy suffer more from infantile bronchiolitis. viruses undoubtedly cause most asthma exacerbations, but some non-viral pathogens may also play a part. certain fungi and bacteria have diverse roles in driving the pathogenesis and increasing the severity of asthma, and various microorganisms play different parts in different asthma endophenotypes. more research is needed, but our ultimate goal is to identify microbial targets that are amenable to manipulation with vaccines or anti microbial drugs and thus to reduce the burden of asthma on society. global strategy for asthma management and prevention: gina executive summary asthma: defining of the persistent adult phenotypes salmeterol: an inhaled β2-agonist with prolonged duration of action exposure to foodborne and orofecal microbes versus airborne viruses in relation to atopy and allergic asthma: epidemiological study asthma and current intestinal parasite infection: systematic review and meta-analysis the 'hygiene hypothesis' for autoimmune and allergic diseases: an update this study shows that microbial diversity is inversely related to the risk of developing asthma family size, infection and atopy: the first decade of the "hygiene hypothesis the coming-of-age of the hygiene hypothesis predominance of gram-positive bacteria in house dust in the low-allergy risk russian karelia diversity and seasonal dynamics of bacterial community in indoor environment culture-independent analysis of bacterial diversity in a child-care facility bacterial-induced protection against allergic inflammation through a multicomponent immunoregulatory mechanism maternal tlr signaling is required for prenatal asthma protection by the nonpathogenic microbe acinetobacter lwoffii f78 regulation of inflammatory responses by gut microbiota and chemoattractant receptor gpr43 the burden of respiratory syncytial virus infection in young children sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution this study shows the protective effects of the ketomacrolide antibiotic telithromycin in asthma exacerbations, suggesting that targeting atypical bacteria with anti-infectives protects against asthma exacerbations mycoplasma pneumoniae and chlamydia pneumoniae in asthma: effect of clarithromycin rhinovirus viremia in children with respiratory infections the family of five: tirdomain-containing adaptors in toll-like receptor signalling rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th1/2 cytokine and il-10 production rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells shedding of infected ciliated epithelial cells in rhinovirus colds dispersal of staphylococcus aureus into the air associated with a rhinovirus infection allergic airway inflammation is exacerbated during acute influenza infection and correlates with increased allergen presentation and recruitment of allergenspecific t-helper type 2 cells synergistic effects of fluticasone propionate and salmeterol on inhibiting rhinovirusinduced epithelial production of remodellingassociated growth factors respiratory syncytial virus infection provokes airway remodelling in allergen-exposed mice in absence of prior allergen sensitization pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation rhinovirus infection induces extracellular matrix protein deposition by primary bronchial epithelial cells and lung fibroblasts salmeterol and fluticasone propionate and survival in chronic obstructive pulmonary disease safety and tolerability of montelukast in placebo-controlled pediatric studies and their open-label extensions glucocorticoids enhance or spare innate immunity: effects in airway epithelium are mediated by ccaat/enhancer binding proteins long-term inhaled corticosteroids in preschool children at high risk for asthma the role of antibiotics in asthma pulmonary surfactant protein a regulates tlr expression and activity in human macrophages muc1 mucin is a negative regulator of toll-like receptor signaling inhibition of il-10 receptor function in alveolar macrophages by toll-like receptor agonists a critical function for cd200 in lung immune homeostasis and the severity of influenza infection differential role of cd80 and cd86 on alveolar macrophages in the presentation of allergen to t lymphocytes in asthma downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages lipopolysaccharide-enhanced, toll-like receptor 4-dependent t helper cell type 2 responses to inhaled antigen bacterial 16s ribosomal dna in house dust mite cultures the contribution of toll-like receptors to the pathogenesis of asthma short ragweed pollen triggers allergic inflammation through toll-like receptor 4-dependent thymic stromal lymphopoietin/ox40 ligand/ox40 signaling pathways house dust mite allergen induces asthma via toll-like receptor 4 triggering of airway structural cells allergenicity resulting from functional mimicry of a toll-like receptor complex protein sustained increases in numbers of pulmonary dendritic cells after respiratory syncytial virus infection pulmonary dendritic cells and alveolar macrophages are regulated by γδ t cells during the resolution of s. pneumoniae-induced inflammation essential contribution of monocyte chemoattractant protein-1/c-c chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia role of inducible bronchus associated lymphoid tissue (ibalt) in respiratory immunity aggregations of lymphoid cells in the airways of nonsmokers, smokers, and subjects with asthma interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma the 1918 influenza pandemic: insights for the 21st century study of modifiable risk factors for asthma exacerbations: virus infection and allergen exposure increase the risk of asthma hospital admissions in children synergism between allergens and viruses and risk of hospital admission with asthma: case-control study type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis interleukin-10/interleukin-5 responses at birth predict risk for respiratory infections in children with atopic family history peripheral blood cytokine responses and disease severity in respiratory syncytial virus bronchiolitis genetic association study for rsv bronchiolitis in infancy at the 5q31 cytokine cluster t-cell activation during exacerbations: a longitudinal study in refractory asthma respiratory syncytial virus synergizes with th2 cytokines to induce optimal levels of tarc/ccl17 activation of stat6 by sting is critical for antiviral innate immunity thymic stromal lymphopoietin is released by human epithelial cells in response to microbes, trauma, or inflammation and potently activates mast cells tslp from rsv-stimulated rat airway epithelial cells activates myeloid dendritic cells tlr3-and th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells double-stranded rna induces disproportionate expression of thymic stromal lymphopoietin versus interferon-β in bronchial epithelial cells from donors with asthma this study demonstrates the important role of natural killer cell-derived ifnγ in controlling il-25 production during human rsv infection the development of respiratory syncytial virus-specific ige and the release of histamine in nasopharyngeal secretions after infection the role of virus-specific immunoglobulin e in airway hyperresponsiveness virus-specific ige enhances airway responsiveness on reinfection with respiratory syncytial virus in newborn mice induction of high-affinity ige receptor on lung dendritic cells during viral infection leads to mucous cell metaplasia frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus this investigation shows the important protective role of ifnλ proteins in asthma exacerbations and that patients with asthma have impaired ifnλ expression and protein production rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma in vitro susceptibility to rhinovirus infection is greater for bronchial than for nasal airway epithelial cells in human subjects toll-like receptor heterodimer variants protect from childhood asthma irf-1 gene variations influence ige regulation and atopy polymorphisms in toll-like receptor genes and susceptibility to pulmonary aspergillosis viruses and bacteria in acute asthma exacerbations -a ga 2 len-dare systematic review t helper type 2-driven inflammation defines major sub-phenotypes of asthma lebrikizumab treatment in adults with asthma interleukin-4 receptor in moderate atopic asthma. a phase i/ii randomized, placebocontrolled trial effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyperresponsiveness, and the late asthmatic response randomized trial of omalizumab (anti-ige) for asthma in inner-city children the role of a soluble tnfα receptor fusion protein (etanercept) in corticosteroid refractory asthma: a double blind, randomised, placebo controlled trial serum vitamin d levels and severe asthma exacerbations in the childhood asthma management program study randomized trial of vitamin d supplementation to prevent seasonal influenza a in schoolchildren reversing the defective induction of il-10-secreting regulatory t cells in glucocorticoidresistant asthma patients probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial randomized placebo-controlled trial of lactobacillus on asthmatic children with allergic rhinitis microbial manipulation of immune function for asthma prevention: inferences from clinical trials treatment with the tlr7 agonist r848 induces regulatory t-cell-mediated suppression of established asthma symptoms toll-like receptor 7-triggered immune response in the lung mediates acute and long-lasting suppression of experimental asthma clinical and immunological effects of low-dose ifn-α treatment in patients with corticosteroid-resistant asthma il-28a (ifn-λ2) modulates lung dc function to promote th1 immune skewing and suppress allergic airway disease the authors declare no competing financial interests. key: cord-321835-qn33sx8x authors: bailey, emily s.; fieldhouse, jane k.; choi, jessica y.; gray, gregory c. title: a mini review of the zoonotic threat potential of influenza viruses, coronaviruses, adenoviruses, and enteroviruses date: 2018-04-09 journal: front public health doi: 10.3389/fpubh.2018.00104 sha: doc_id: 321835 cord_uid: qn33sx8x during the last two decades, scientists have grown increasingly aware that viruses are emerging from the human–animal interface. in particular, respiratory infections are problematic; in early 2003, world health organization issued a worldwide alert for a previously unrecognized illness that was subsequently found to be caused by a novel coronavirus [severe acute respiratory syndrome (sars) virus]. in addition to sars, other respiratory pathogens have also emerged recently, contributing to the high burden of respiratory tract infection-related morbidity and mortality. among the recently emerged respiratory pathogens are influenza viruses, coronaviruses, enteroviruses, and adenoviruses. as the genesis of these emerging viruses is not well understood and their detection normally occurs after they have crossed over and adapted to man, ideally, strategies for such novel virus detection should include intensive surveillance at the human–animal interface, particularly if one believes the paradigm that many novel emerging zoonotic viruses first circulate in animal populations and occasionally infect man before they fully adapt to man; early detection at the human–animal interface will provide earlier warning. here, we review recent emerging virus treats for these four groups of viruses. | the geographical location of first detections (with known reservoirs) for recently emerged adenoviruses (ads), enteroviruses (evs), coronaviruses, and influenza viruses. zoonotic (coronaviruses and influenza viruses) and non-zoonotic viruses (ads and evs) are shown. for zoonotic viruses, the hosts range from cattle, bats, chickens, camels, wild birds, cats, ferrets, goats, and humans (from left to right). the different sizes of the circles represent the number of human cases during the first outbreaks of the emerging respiratory viruses. human cases of adenoviral infections are shown in blue; human cases of enteroviral infections are shown in yellow; human cases of coronaviral infections are shown in green; and human cases of influenza viral infections are shown in red. zoonotic influenza introduction and epidemiology influenza viruses are rna viruses that are members of the orthomyxovirus family and classified into four types: a, b, c, and d (2) . as shown in table 1 , these four types of viruses are characterized by their immunologically distinct nucleoprotein and matrix protein antigens. influenza a and b viruses consist of hemagglutinin (ha), which binds a sialic acid receptor, allowing the virus to enter the host cell, and neuraminidase (na), which cleaves the sialic acid to release the virus. similarly, influenza c and d viruses contain ha-esterase fusion glycoproteins that also allow for the attachment of viral and cellular membranes. antigenic shifts (influenza a only) in ha, na, and the ha-esterase proteins contribute to the generation of novel viral strains. the host range of influenza viruses includes humans, birds, pigs, bats, and other livestock animals such as cattle and goats. the network of the influenza viral transmission is complex with both inter-and intraspecies transmission. as the viruses continue to change in their genetic sequences, ongoing research is imperative in investigating the ecology of these viruses at the human-animal interface to control further spread of infections and prevent the risk of future pandemics. influenza a virus h3n2 subtypes are frequently reported in swine, avian, and canine hosts that are responsible for highly infectious respiratory diseases in pigs and have been examined as a potential cause of influenza in humans. one study, examining the role of iav in pigs at usa agricultural fairs, reported an average influenza a prevalence of 77.5% among 161 swine across all seven fairs (3) . the genomic sequences of the viruses isolated from the swine were ≥99.89% similar to the h3n2 viruses isolated in humans. at these fairs, iavs were detected at least 1 day before symptoms of the virus were observed in humans, indicating that h3n2 was transmitted from pigs to humans in this case. since 2009, h1n1 virus has posed a significant threat to livestock workers and the greater community and has now become a seasonal influenza virus which circulates in humans. to explore the role of swine production facilities in the development of new swine-like influenza viruses, the spatiotemporal association between weekly influenza-like illnesses (ilis) in humans and the location of pig farms was investigated in north carolina over four influenza seasons (4) . analyses showed that the years of h1n1 pandemic, 2009-2010 and 2010-2011, were closely related with earlier peaking of ili cases. these findings suggest that increased exposure to pigs was associated with earlier observations of the greatest number of human h1n1 cases. in china, the transmission of influenza a between humans and pigs in six farms is being examined using a one health approach, taking into consideration the interconnectedness of humans, animals, and the environment (5) . findings suggest that both a(h1n1)pdm09-like and swine-lineage h1n1 and swine-lineage h3n2 viruses are circulating in swine workers and that these viruses likely reassort and cross species within the pig farms; as such, additional research is needed to understand the relationship between cross species transmission of viruses in humans and pigs. avian influenza viruses are the largest group of influenza a viruses reservoired in aquatic birds or poultry. although infrequently transmitted to humans, many cases have now been reported. human infection with avian influenza can lead to serious health conditions, including death. the first outbreak of an iav strain, h5n1, in humans occurred in hong kong sar, china, in 1997, infecting 18 humans (6). the first identified cases of human infection with h7n2, another avian influenza, occurred in north america with two human cases reported in 2002 (7) . another variation of the virus, h7n7, was the first avian influenza strain reported in europe; it infected 89 humans in 2003. in 2004, the first human cases of h10n7 infections were observed in africa (8) . it is important to note that h5n1 virus outbreak occurred again in 2004, 7 years after its first outbreak in humans, infecting more than 650 humans and causing more than 386 deaths worldwide (9) . the avian influenza viruses have continuously evolved, causing serious infections among humans across the world. h7n9 virus, a sporadic subtype of an avian influenza a virus, was first reported in humans in china in 2013. since the first outbreak, china has been experiencing epidemics annually, with a cumulative number of 1,562 reported cases, 40% of which have led to deaths as of september 2017 (10) . the incidence of the h7n9 infections has been increasing in both humans and poultry and in 2017 alone 764 infections have been reported (11). although h7n9 was first recognized as a low pathogenic avian influenza, two divergent lineages were detected in 2016-including a highly pathogenic avian influenza variant (12). according to the center for disease control and prevention (cdc), h7n9 is now recognized as the virus with the greatest potential to cause a pandemic due to its rapid genetic changes over the last 5 years. this further supports the need to improve disease control strategies and increase efforts to develop an effective vaccination strategy in the future as the spread of the h7n9 infection poses a threat to the poultry business. influenza d virus (idv) is a novel influenza virus that is structurally different from the other influenza viruses. idv was first isolated in 2001 from pigs in usa and since the first report, viral infection has been reported in various locations in usa, europe, and asia. in a serological study, cattle workers and non cattleexposed adults in florida were screened for idv antibodies (13) . of the cattle workers, 97% of idv seroprevalence was observed, while less than 20% was observed in non-cattle-exposed adults, suggesting a greater risk of idv infection for cattle workers. during a swine respiratory disease outbreak in northern italy in 2015, the idv genome was detected and isolated in both pigs and cattle herds (14) . the viral genome isolated from the pigs was closely related to the viral genome isolated in usa in 2011. additionally, the archived serum samples from 2009 had lower idv antibody titers compared to the serum samples collected in 2015. these findings suggest that the incidence of idv infections in pigs may have increased over time, and therefore, idv may pose a public health threat to the community. (17, 18) . recently, it has also been suggested that bats may play a role in the direct human transmission as bat sars-like coronaviruses have been identified in some species (19) . in the past decade, teams from the sabin vaccine institute and baylor college of medicine have been working toward the development of a vaccine for sars-cov. although initial reports indicated that a vaccine may be ready for human clinical trials in 2017, progress has been slow and few human sars vaccine trials have been conducted to date. middle east respiratory syndrome was first recognized in saudi arabia in 2012. many cases were linked to travel to or residence in countries in and near the arabian peninsula. symptoms include severe acute respiratory illness with fever, cough, and shortness of breath. there is limited human-to-human transmission of mers-cov, but exposure to camels is a risk factor for infection, with seroprevalences 15-23 times higher in camel exposed individuals (20) . despite this, major health care-associated transmission of mers-cov was reported in the middle east and korea, with outbreaks characterized by interhospital spread related to overcrowding and a lack of personal protective equipment (21) . the total number of worldwide cases reported to the who as of january 9, 2017 was 2,067 mers-cov cases (22). the cocirculation of covs in its animal reservoirs (camels and bats) raises important questions about the evolution of mers-cov. in a study conducted between 2014 and 2015 in saudi arabia, researchers found that dromedary camels share three cov species with humans, including betacoronavirus 1, mers-cov, and a cov 229e-related virus (23) . with the aim of reducing mers-cov transmission to humans, haagmans et al. developed a vaccine for camels using a poxvirus vehicle (24) . this vaccine has significantly reduced virus excretion among camels and conferred cross-immunity to camelpox infections (25) . enteroviruses are small, positive-sense, single-stranded rna viruses in the picornaviridae family. there are 12 species of evs found globally, including ev a-j (ev-a, b, c, d, e, f, g, h, and j) and rhinovirus a-c (rv-a, b, and c). with low replication fidelity and frequent recombination, evs have viral genetic diversity and a potential for cross-species infection. in february 2013, the international committee on taxonomy of viruses (ictv) approved changes to ev and rhinovirus species names after many of the human ev species were identified and isolated in nonhuman hosts. based on an analysis of picornaviridae hosts listed in the ictv database and subsequent studies of ev infection in non-human primates, there is growing evidence to indicate a potential for future zoonotic transmission between animals and humans (26, 27) . among the most important emerging respiratory viruses are ev68, ev71, coxsackieviruses, echoviruses, rhinoviruses, and polioviruses. enterovirus transmission occurs year-round, with seasonal peaks occurring in the summer and fall (june-october). infants less than 1 year of age are most susceptible to infection, and males are at an increased risk for infection until the age of 20 years (28) . the predominant mode of transmission is through a direct or indirect fecal-oral route; however, certain serotypes are transmitted via the respiratory route, in tears, and via fomites (29) . immunity to evs is serotype specific with most causing mild respiratory infections. rhinoviruses are small, single-stranded rna viruses in the picornavirus family that are responsible for more than half of all upper respiratory tract infections. in addition to exacerbating asthma and chronic obstructive pulmonary disease, rhinoviruses have also been associated with acute respiratory hospitalizations among children (30) . in a large prospective study of us pneumonias, rhinoviruses have been identified as the second most prevalent etiology of pneumonia in children after respiratory syncytial virus and the first most common etiology among adults (31) . there are more than 150 unique types of rhinoviruses. among the three genotypes (a, b, and c) types a and c are most often associated with increased morbidity and bacterial secondary infection. in animals, rhinovirus type c has been associated with morbidity in chimpanzees (32) . with an array of unique serotypes no vaccines or approved antiviral therapies have been commercially produced; however, experiments have suggested that vaccines and antiviral therapy may be possible (33, 34) . enterovirus d68 has caused sporadic respiratory disease outbreaks across asia, europe, and usa since 1960s; however, in 2014, a nationwide outbreak of d68 was associated with severe respiratory illness in usa, resulting in 14 deaths out of a known 1,150 cases (35) . the cdc found 36% of all evs tested during this outbreak were d68 and that patients with a history of asthma were found to be at a disproportionately increased risk of infection (36). one study of the 2014 outbreak found 59% of patients seen with ev-d68 in hospitals across missouri, illinois, and colorado were admitted to intensive care units and 28% received ventilator support (35) . in a study evaluating evs in non-human primates, ev-d68 was detected as a recombinant zoonotic strain (37) . while there are several strains of coxsackievirus and evs that can cause hand-foot-and-mouth disease (hfmd), ev71 is most commonly associated with severe disease outcomes. hfmd predominantly affects young children and is found worldwide but especially in the asia-pacific region. although ev71 is not typically detected in animals, recent research has indicated that it infects non-human primates (38) . various antiviral therapies are currently under study, including small molecules, monoclonals, and antivirals. vaccine candidates are also in development, with two vaccines currently available in china, which involve recombinant proteins, attenuated strains, inactivated whole-virus and virus-like particles, and dna vaccines (39) . first discovered in 1953 by rowe et al., ads are non-enveloped, double-stranded dna viruses with 57 unique serotypes, some of which are specific for attacking the respiratory track, conjunctiva, or gastrointestinal track (40) . key features of ad infections include various symptoms of disease, including rhinorrhea, nasal congestion, cough, sneezing, pharyngitis, keratoconjunctivitis, pneumonia, meningitis, gastroenteritis, cystitis, and encephalitis. illnesses may be asymptomatic, mild, or severe; however, immunocompromised patients and infants are at increased risk of severe morbidity and death. outbreaks of respiratory ad infection are common in both military recruits and other large training groups, such as police trainees. large persistent epidemics of ad type 4-associated respiratory disease have been documented in various military trainees (41) (42) (43) . in response to the increased disease burden from ad4 and ad7 in military recruits, teva has made a vaccine available to military recruits in usa (42) . despite a 12-year hiatus from use, in late 2011 oral ad4 and ad7 vaccines were reintroduced as an infection control measure for military recruits (42) . after reintroduction, military recruits experienced a 100-fold decline in ad disease burden, which accounted for the prevention of approximately 1 death, 1,100-2,700 hospitalizations, and 13,000 febrile ad cases per year among trainees (44) . outbreaks of ad in the general population have been characterized by infection due to novel viruses such as ad7h, ad7d2, ad14a, and ad3 variants. these novel viruses are sometimes associated with high attack rates and a high prevalence of pneumonia. severe mortality is also prevalent among patients with chronic disease and in the elderly. one of the most important novel serotypes, ad14, previously rarely reported, is now considered as an emerging ad type causing severe and sometimes fatal respiratory illness in patients of all ages (45) . beginning in 2005, ad14 cases were suddenly identified in four locations across usa (46) ; the strain associated with this outbreak was different than the original ad14 strain isolated in 1950s. the novel strain, ad14a, has now spread to numerous us states and is associated with a higher rate of severe illness when compared to other ad strains. novel ad species have also been recently detected in crossspecies infections from non-human primates to man in usa and between psittacine birds and man in china (47) . these cross-species infections indicate that ads should be monitored for their potential to cause cross-species outbreaks. in a recent review of the risks of potential outbreaks associated with zoonotic ad (48) , it was noted that intense human-animal interaction is likely to increase the probability of emergent cross-species ad infection. additionally, the recombination of advs with latent "host-specific" advs is the most likely scenario for adaptation to a new host, either human or animal. currently, there are no fda approved antivirals for ad infection; however, the best antiviral success has been seen with ribavirin, cidofovir, and most recently brincidofovir an analog of cidofovir (49) . as it is clear that many emerging respiratory viruses have zoonotic reservoirs, the design and implementation of effective control strategies are increasingly important. it has been suggested that avoiding direct contact with animals known to be zoonotic reservoirs for these viruses is one potential strategy (50); however, in populations where contact at the human-animal interface is common this may not be an acceptable solution. complex disease problems cannot be solved by one institution or one discipline; as such, this presents opportunities to incorporate the one health approach of working across disciplines to incorporate human, animal, and environmental health to solve complex problems. although some of the respiratory viruses described here are found almost exclusively in humans (ad strains), many of the most important emerging respiratory viruses are found at the human/animal interface. this suggests that strategies for novel virus detection should incorporate global surveillance at the human-animal interface to detect potentially emerging zoonotic viruses. this surveillance will require collaboration and cooperation among many stakeholders in order to address emerging and novel viral diseases. eb, jf, and jc conducted the literature review and wrote the manuscript; gg conceived the idea of the review and helped revise the manuscript to add important scientific content and refine the interpretation of the results. all the authors reviewed the final version of the manuscript and agreed to its submission. this work was supported in part by nih/niaid grant r01ai108993-01a1 (gregory gray pi). current infectious disease challenges centers for disease control and prevention. types of influenza viruses influenza a(h3n2) virus in swine at agricultural fairs and transmission to humans are people living near modern swine production facilities at increased risk of influenza virus infection? evidence for cross-species influenza a virus transmission within swine farms, china centers for disease control and prevention. h7n2 questions & answers avian influenza virus a (h10n7) circulating among humans in egypt global and local persistence of influenza a(h5n1) virus food and agriculture organization of the united nations serologic evidence of exposure to influenza d virus among persons with occupational contact with cattle influenza d in italy: towards a better understanding of an emerging viral infection in swine update: outbreak of severe acute respiratory syndrome -worldwide factors associated with nosocomial sars-cov transmission among healthcare workers in hanoi isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe 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illness from avian influenza h7n3, british columbia genome analysis of south american adenovirus strains of serotype 7 collected over a 7-year period outbreak of adenovirus genome type 7d2 infection in a pediatric chronic-care facility and tertiary-care hospital the 1998 enterovirus 71 outbreak in taiwan: pathogenesis and management human infection with influenza virus a(h10n8) from live poultry markets, china avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome in vitro susceptibility of adenovirus to antiviral drugs is speciesdependent key: cord-314500-89ovdnxl authors: dunachie, susanna; chamnan, parinya title: the double burden of diabetes and global infection in low and middle-income countries date: 2018-12-04 journal: trans r soc trop med hyg doi: 10.1093/trstmh/try124 sha: doc_id: 314500 cord_uid: 89ovdnxl four out of five people in the world with diabetes now live in lowand middle-income countries (lmic), and the incidence of diabetes is accelerating in poorer communities. diabetes increases susceptibility to infection and worsens outcomes for some of the world’s major infectious diseases such as tuberculosis, melioidosis and dengue, but the relationship between diabetes and many neglected tropical diseases is yet to be accurately characterised. there is some evidence that chronic viral infections such as hepatitis b and hiv may predispose to the development of type 2 diabetes by chronic inflammatory and immunometabolic mechanisms. helminth infections such as schistosomiasis may be protective against the development of diabetes, and this finding opens up new territory for discovery of novel therapeutics for the prevention and treatment of diabetes. a greater understanding of the impact of diabetes on risks and outcomes for infections causing significant diseases in lmic is essential in order to develop vaccines and therapies for the growing number of people with diabetes at risk of infection, and to prioritise research agendas, public health interventions and policy. this review seeks to give an overview of the current international diabetes burden, the evidence for interactions between diabetes and infection, immune mechanisms for the interaction, and potential interventions to tackle the dual burden of diabetes and infection. there are now 336 million people with diabetes living in low-and middle-income countries (lmic). 1, 2 diabetes increases susceptibility to infection and worsens outcomes for diseases such as tuberculosis (tb), 3 and the under-recognised tropical disease melioidosis. 4, 5 current international treatment guidelines for diabetes are based on research conducted in high-income countries focussed on preventing adverse cardiovascular outcomes and early death. there is a lack of evidence upon which to base guidelines for people living in lmic, where there is an increased burden of infectious diseases compared with high income countries. diabetes has traditionally been viewed as a 'disease of the wealthy', mostly found among elderly people in developed countries. now, however, diabetes affects every strata of society, and has become a fast-growing problem in poorer communities. the global burden of disease (gbd) study estimated there were 1.4 million deaths worldwide from diabetes in 2016, 6 representing a 31% increase from 2006. of the estimated 425 million people with diabetes worldwide, four-fifths currently live in lmic and increasing numbers of children and young adults have been diagnosed with the disease. 1 this number is projected to increase to 629 million by 2045, and most of the rising burden will occur in lmic ( figure 1 ). in addition, diabetes is more likely to be undiagnosed or poorly treated in lmic. 1 data from the ncd risk factor collaboration 2 show that agestandardised diabetes prevalence in adults has increased or remained unchanged since 1980 in every country. 2 importantly, the burden of diabetes has increased faster in lmic than in high-income countries. 2 this rising prevalence of diabetes in lmic is believed to be associated with many factors, including ageing populations, urbanisation, cultural and social changes, dietary changes, physical inactivity, changes in diagnostic criteria and screening practices, better treatment and survival, and increasing trends in overweight and obesity. classification of diabetes table 1 shows the current classification of diabetes by who and the american diabetes association (ada), 7, 8 which includes four clinical and aetiological classes: type 1 diabetes (t1dm), type 2 diabetes (t2dm), gestational diabetes mellitus, and other specific types of diabetes due to other causes. t1dm is caused by an autoimmune reaction which destroys the insulin-producing beta cells in the islets of the pancreas, leading to no or low production of insulin. t2dm is the most common type of diabetes, accounting for approximately 90% of all cases of diabetes worldwide. 1 it is characterised by an inadequate production of insulin and an inability of the body to respond fully to insulin, defined as insulin resistance. it is important to note that assigning a type of diabetes to an individual is often reliant on the circumstances present and additional testing at the time of diagnosis, and that many patients with diabetes do not easily fit into a single class. in lmic, it is often unknown which type of diabetes a person has, and therefore this review will use the general term 'diabetes', but >90% of patients with diabetes in lmic are believed to have t2dm. 1 table 2 summarises the modifiable and non-modifiable risk factors for t2dm, which is a disease caused by a complex interplay between genetic and environmental factors. 9 the rapid increase in the prevalence of diabetes over recent decades suggests that environmental and lifestyle factors might play an increasingly important role in the development of the disease. it is generally recognised that people with diabetes are at increased risk of infection and worse outcomes, 10, 11 including diabetic foot infection, urinary tract infections (especially from tuberculosis (tb) is a leading cause of disease and death worldwide, with an estimated 9 million cases of tb and 1.2 million deaths in 2017. 6 diabetes is associated with a threefold increased risk of developing tb, 3 and increased risk of death or treatment failure in tb. 12 the gbd group reported diabetes accounting for 10.6% of the tb mortality in hiv-negative people. 13 in 2017, 790 000 cases of tb were attributable to diabetes, 14 and the absolute numbers of people with tb-diabetes co-morbidity is now similar to people with tb-hiv co-infection. more than half of the world's tb cases occur in five countries, 15 which have significant prevalence rates and total numbers of diabetes cases in adults aged 20-79 years as follows: china (10.9%, 114 million), india (8.8%, 73 million), indonesia (6.2%, 10 million), philippines (6.2%, 3.7 million) and pakistan (6.9%, 7.5 million). 1 there is evidence that the presence of clinical tb disease drives stress hyperglycaemia, impacting on clinical outcomes and response to treatment. 16 as rates of diabetes continue to rise, and the tb epidemic continues, there is a pressing need for bidirectional screening in countries facing the double burden of tb and diabetes. work in india has shown success at screening newly diagnosed tb cases for diabetes, 17 due to the availability of a simple screening blood test (hba1c) and the use of existing systems established to screen tb cases for hiv. screening people with diabetes for tb is more difficult due to the reliance on symptom questionnaires followed by chest xray. a blood test to diagnose tb in this setting is highly desirable, but current interferon gamma release assays (igras) do not have sufficient sensitivity and specificity for this purpose. diabetes is also associated with higher rates of mycobacterium leprae. 18, 19 the greatest increased risk for infection in people with diabetes is seen for the grossly under-recognised tropical disease melioidosis, which is caused by the gram-negative bacterium burkholderia pseudomallei. people with diabetes have a twelvefold increased risk of melioidosis, and over half of all cases of melioidosis have diabetes. 20 b. pseudomallei is an environmental saprophyte with a predilection for rice paddy fields, and melioidosis is typically seen in adults in middle age and above, often in rice-farming communities. a broad range of clinical presentations are seen, including pneumonia, acute sepsis with bacteraemia, abscess formation in any organ site, chronic subacute disease and latency. 21, 22 transmission of the bacterium to humans occurs via three routes: inhalational, cutaneous via skin abrasions and ingestion of contaminated drinking water. 23 the disease is commonly diagnosed in southeast asia and northern australia, but is now known to be present in 45 countries across tropical regions, with an estimated annual 165 000 cases and 89 000 deaths. 24 if rates of diabetes continue to rise as predicted, coupled with an increased reliance on older people for rice farming due to exodus of the younger generation to urban areas, then the burden of melioidosis will also rise. studies in high-income countries have shown that people with diabetes have high rates of infection from many common bacteria, 25 but some bacterial species are more frequently reported in association with diabetes. diabetes is an established risk factor for invasive infection with staphylococcus aureus. 26 s. aureus is the commonest cause of tropical pyomyositis, an infection of skeletal muscle featuring intramuscular abscesses that is commonly seen in tropical regions and can account for 1-4% of acute admissions. 27 pyomyositis occurs less frequently in temperate zones, where diabetes is a known risk factor. 28, 29 while diabetes has been reported in some case reports of tropical pyomyositis, 30 further research is needed to establish the box 1. relationship between diabetes and susceptibility/increased disease severity for significant pathogens in low-and middle-income countries. see text for discussion of evidence. 31 diabetes was associated with around a threefold increased risk of infection with s. enteritidis following exposure in a us hospital outbreak. 32 people with diabetes have an increased risk of klebsiella infections, 33 especially klebsiella liver abscess in asia. 34, 35 scrub typhus is a febrile illness caused by the rickettsial group intracellular pathogen orientia tsutsugamushi. around a million cases a year occur in asia, 36 and it is believed to exist in other tropical regions outside asia. 37 diabetes was an independent risk factor for more severe disease in a prospective study of eschar-positive scrub typhus. 38 it remains to be established why diabetes confers much greater susceptibility to, and worse outcomes for some bacteria than to others. several of the bacteria most closely associated with diabetes, such as m. tuberculosis and b. pseudomallei, are predominantly intracellular bacteria. impairments in phagocyte function and adaptive t cell immunity in diabetes may contribute to increased susceptibility to intracellular pathogens. diabetes is associated with antimicrobial resistance (amr). diabetes status is associated with increased rates of drug resistance in tb, including multidrug-resistant tb. 39, 40 besides tb, people with diabetes are over-represented in cohorts with multi-drugresistant infections, 41 but there is a lack of evidence at present of higher rates of amr in bacterial isolates from people with diabetes compared with isolates from non-diabetics. what is clear is that increasing rates of amr will have a larger impact on people with diabetes, due to their higher risk of infection and increased need for healthcare exposure and interventions. dengue causes an estimated 101 million clinical cases per year and 37 800 deaths. 6 an association between diabetes and severe presentations of dengue is now broadly accepted. 42 however, studies are typically retrospective, often small in nature, and use varying definitions of severe dengue. in a metaanalysis of five case-control studies of acute dengue, diabetes was associated with an increased risk of a severe clinical presentation of dengue compared with either asymptomatic infection or non-severe acute dengue, 43 although given the limited data, the authors emphasised this was only suggestive of a link. this finding has been supported by further studies in malaysia 44 and china. 45 a systematic review 46 to evaluate the contribution of non-communicable diseases (ncds) to the development of severe dengue identified 16 relevant publications and gives a clear overview of this literature, but given the heterogeneity of the studies the authors concluded that the existing literature was inadequate for meaningful estimation of the impact of diabetes and other ncds on dengue severity. the following year a canadian group 47 reported a nearly three-fold higher prevalence of diabetes in 3236 patients in 22 studies with severe dengue than the prevalence of diabetes in 9067 subjects in 13 studies with non-severe dengue. the same analysis found hypertension, heart disease and obesity (conditions interrelated with diabetes) to be significantly more prevalent in severe dengue, and also reported a four-fold increased prevalence of diabetes in severe west nile fever cases by the same methodology, supporting an earlier study 48 . the authors acknowledge the multiple limitations of this approach, including the heterogeneity of the studies and publication bias for studies included. there is clearly a need for further high-quality epidemiological studies to further define the association. there is some evidence that diabetes is associated with more severe disease in chikungunya disease, 49 but there is a current lack of data for analysing the relationship with zika virus. other viral infections may be associated with diabetes. people with diabetes are an at-risk group for clinical illness, disease severity and death from influenza, 50 with a meta-analysis of 234 articles 51 showing diabetes to be a risk factor for death from pandemic influenza (predominantly h1n1), although there was a lack of high-quality cohort studies to demonstrate this in seasonal influenza. it is estimated that 260 million people are chronic carriers of the hepatitis b virus (hbv), 52 and hbv has recently been described as a neglected tropical disease. 53 a number of studies have shown a higher prevalence of hbv in people with diabetes, [54] [55] [56] and people with chronic hbv are reported to have an increased risk of developing diabetes, 57 although this has not been demonstrated to date in sub-saharan africa. 58, 59 in addition, diabetes is associated with disease progression of hbv, 60-62 which has also been reported for hepatitis c. 63 varicella zoster virus (vzv) causes chickenpox as a primary infection which can reactivate as herpes-zoster (shingles), especially in older people. diabetes is an established risk factor for shingles. 64, 65 diabetes is considered a risk factor for middle east respiratory syndrome (mers). a recent systematic review 66 identified 58 published works for analysis, with several reporting diabetes as a risk factor for infection and death, but meta-analysis to quantitate the increased risk of diabetes across the studies was not possible due to the small number of studies addressing this risk factor. people living with hiv (plhiv) who receive combined antiretroviral therapy (art) now have excellent long-term survival, but increased rates of metabolic disorders such as impaired glucose tolerance, hyperlipidaemia and body morphological changes (lipodystrophy syndrome) have been reported. 67 increased rates of insulin resistance in plhiv could occur due to the proinflammatory effects of chronic viral infection, direct effects of art, and also indirect effects of art such as dyslipidaemia and body fat distribution changes. although studies in high-income western countries have shown inconsistent results as to whether hiv infection increases the risk of t2dm or merely represents earlier diagnosis in a closely monitored population, 68 there is evidence that hiv increases the risk of diabetes in asian and african populations. a study in taiwan suggested plhiv to be as much as sixfold more likely to develop diabetes than the rest of the population, 69 and a recent thai study of plhiv showed diabetes developed at a younger age compared with the general population. 70 increased rates of diabetes have also been reported in plhiv in south africa, 71 tanzania 72 and ethiopia. 71 older art drugs such as stavudine and zidovudine are known to increase the risk of metabolic syndrome and diabetes, and are used less now, so future studies may not confirm this relationship. parasitic malaria remains one of the world's largest causes of mortality from infectious diseases, with an estimated 213 million cases and 720 000 deaths per annum. 6 in 2010, researchers reported a 46% increased risk of malaria in people with diabetes 73 in a case-control study in an urban setting in ghana. the malaria was predominantly p. falciparum, asymptomatic and diagnosed by pcr. the comparison of probability of malaria infection was made between patients with diabetes (n=675) and a control group (n=791) comprising patients attending hypertension clinics, patients attending other hospital clinics, and hospital staff. the groups were not matched in that the diabetes group were older by a mean of 7.6 years and had a socio-economic profile associated with greater poverty, and the adjusted odds ratio for diabetes as a risk factor for p. falciparum infection was just outside significance in a multivariate analysis adjusting for these parameters (adjusted or 1.36, 95% ci 0. 98-1.90 ). this report is very interesting, but there have been no further published clinical studies addressing the relationship between diabetes and malaria. if diabetes increases the risk of malaria this would be of huge significance on a global scale. as india is a country with a high burden of both diabetes and p. vivax malaria, 74 one might expect evidence of a relationship between diabetes and vivax malaria to emerge from research in this region. further prospective studies with well-matched cohorts, and research into the effect on outcomes for people with malaria is needed. diabetes has been linked to increased risk of cutaneous 75,76 and visceral leishmaniasis, 77 and diabetes and hyperglycaemia were more frequently reported in cardiomyopathy caused by trypanosoma cruzi (chagas disease) than in controls. 78 there are a lack of data on the relationship between diabetes and most neglected tropical diseases (reviewed 79 ) and prospective studies of people with diabetes in tropical regions are needed to evaluate this. some interesting findings have been reported for a lower risk of diabetes following helminth infections including schistosomiasis, strongyloides and filariasis, as reviewed by berbudi and colleagues. 80 the mechanism for such a protective effect could be by helminth infection inducing a shift towards type 2 (anti-inflammatory) immune responses, and reducing chronic low-grade inflammation. a mouse model 81 demonstrated greater insulin sensitivity following s. mansoni infection in mice fed a high-fat diet, with an increase in type 2 cytokines and the ratio of m2 to m1 macrophages in white adipose tissue. further research is evaluating whether helminth-derived molecules could be developed as novel therapeutic approaches to diabetes and metabolic syndrome. 82 in addition, a prospective randomised-control trial of helminth eradiation by albendazole therapy in indonesia 83 is underway evaluating the impact of helminth infections on insulin resistance. the mechanisms by which diabetes confers altered susceptibility to infections are likely to be via multiple effects on the human immune system. people with diabetes have altered skin flora, including increased colonisation with s. aureus. 84 breaches in the skin's integrity as a physical barrier to infection occur more commonly in diabetes due to the impact of chronic hyperglycaemia on peripheral nerves and vascular supply. diabetes and its treatment has an impact on the composition of the gut microbiome, 85 and the pivotal role of the gut microbiome in modulating human innate and adaptive responses via pathogen recognition receptor pathways and secretion of immunomodulatory molecules by gut bacteria is now emerging. 86 in vitro, mild hyperglycaemia may favour pathogen growth, and while the contribution of this to human susceptibility to infection is unknown, glycosuria favours urinary tract infections. in addition, hyperglycaemia has a number of immunosuppressive effects, including impairment of neutrophil degranulation, complement activation and phagocytosis. 50 hyperglycaemia does not, however, appear to be a key mechanism for the increased susceptibility to tb. 87 diabetes is associated with endothelial dysfunction, oxidative stress and chronic inflammation. 88 in infections like dengue, this may support a shift towards an excessive pro-inflammatory response, leading to the cytokine storm, shock, vasculopathy and coagulopathy that feature in severe dengue. 47 finally, people with diabetes have higher rates of hospital admissions, 1 which exposes them to hospital-acquired infections and the risk of amr. there is overlap between the spectrum of pathogens that people with diabetes have, notably increased susceptibility to such as s. aureus and invasive fungi, and the infections seen in chronic granulomatous disease (cgd), 89 which is congenital deficiency in phagocytic function. this suggests impairment of neutrophil and macrophage function in people with diabetes as a key mechanism of susceptibility. this is supported by studies showing impaired neutrophil migration, phagocytosis and intracellular killing in the host response to b. pseudomallei in people with diabetes. 90 however, people with cgd are not known to have increased susceptibility to viral infections, and therefore this impairment of phagocyte function is unlikely to be the only cause of increased susceptibility to infection in diabetes. the literature on humeral responses to infection in people with diabetes does not suggest that poor antibody response is a dominant mechanism for the increased susceptibility to infection seen in diabetes. most studies support adequate induction of antibodies in response to licenced antibody-inducing vaccines in people with diabetes [91] [92] [93] . higher antibody responses to influenza vaccine were seen in a us study of elderly people with diabetes compared with elderly non-diabetic people, 94 and antibodies induced by natural exposure to melioidosis were higher in those with diabetes. 95 such higher responses in diabetes could be due to chronic hyperactivation of the innate immune response in t2dm resulting in polyclonal b-cell stimulation and enhanced antibody production to stimuli. 96 beyond neutrophil changes, people with diabetes have alterations in the function of several cell types including macrophages, s. dunachie and p. chamnan natural killer cells, cd4 t cells and cd8 t cells function. 97 t cell responses are known to be important in host defence against intracellular pathogens, with intracellular antigens presented to cd8 t cells via the mhc class 1 antigen presentation pathway, and digested antigens from both extracellular and intracellular sources presented to cd4 t cells via the mhc class 2 system. hiv results in reduction of cd4 t cells, and the huge increased risk of tb seen in advanced hiv demonstrates the importance of cd4 cells in defence against tb. there is evidence of impaired antigenspecific t cell responses in people with diabetes in response to early tb 97 , b. pseudomallei 20 and vzv 98 , although in established tb the cellular response appears to be higher in diabetes. 97 in addition, differences in the regulation and orchestration of the immune response are seen in dengue. 99 for further detailed discussion of immune mechanisms, the reader is referred to a number of excellent reviews of immune impairment in diabetes. 97, 100 overall, diabetes can impair the immune system at a systemic, cellular and molecular level, but it is the alteration of t cell function that is most amenable to boosting with targeted vaccination strategies. acute infection is known to lead to hyperglycaemia as a consequence of the stress-response activation of the hypothalamicpituitary-adrenal axis to increase secretion of cortisol and other hormones which promote peripheral insulin resistance, alongside alteration of insulin-receptor signalling by pro-inflammatory cytokines (reviewed 101 ). sepsis-related hyperglycaemia may be a risk factor for future development of t2dm, 101 and stress hyperglycaemia induced by chronic infections such as tb may contribute to the global burden of diabetes. 16 concerted international efforts to stem the tide of advancing diabetes in lmic are urgently required. implementation of evidencebased approaches that are effective at both prevention and early intervention for diabetes are needed. such approaches require behavioural changes in diet and physical inactivity, raising public awareness of diabetes, and convincing policy-makers and the public alike of the benefits of early diagnosis for disease reversal and management. one of the targets for the un's sustainable development goal 3 (good health and well-being) is to reduce by one-third premature mortality from ncds through prevention and treatment by 2030, with action on diabetes essential. actions include implementation of national diabetes programmes and extension of health promotion activities. 1 tackling the interaction between diabetes and infection requires a greater understanding of the immune mechanisms underlying the altered susceptibility to infection seen in diabetes, and knowledge of how therapeutic management of diabetes impacts on risk of infection. it is likely that tight control of hyperglycaemia lowers the risk of infection, although this has yet to be comprehensively demonstrated in prospective studies. current international treatment guidelines for t2dm are based on research conducted in high-income countries focussed on preventing adverse cardiovascular outcomes and early death. there is a lack of evidence on which to base guidelines for people living in lmic, and choice of glucose-lowering therapy may impact on infection risk and outcomes. for example, there is emerging evidence for beneficial infection outcomes in people with diabetes taking metformin compared with other therapies, [102] [103] [104] and glyburide/glibenclamide has been associated with anti-inflammatory properties and lower mortality in melioidosis. 105 vaccination remains the cornerstone in controlling infectious diseases, and people with diabetes are prioritised as a high-risk group for vaccination against a range of pathogens including pneumococcus, influenza and vzv. development of highly efficacious vaccines against intracellular infections such as tb, melioidosis and leishmaniasis, to which people with diabetes are at greater risk, is an important priority, and progress requires consideration of how to overcome the specific immune impairments seen in diabetes. characterising the impact of diabetes on protective immunity is particularly important for melioidosis, where more than half of all cases occur in people with diabetes, and cost-effective implementation of a successful vaccine is likely to involve targeting this group. 106 b. pseudomallei therefore represents an exemplar pathogen for defining the immune deficits in diabetes, and how to overcome them. the collision of diabetes and the world's global infections is a highly neglected area. more research to clearly define the epidemiology and illuminate successful interventions needs prioritising. the majority of the evidence for associations between diabetes and specific infections relies on the use of relatively small retrospective case-control studies, and meta-analysis approaches are limited by the heterogeneity of patient-level factors across studies. diabetes is interrelated with obesity, hypertension and cardiovascular diseases that share similar risk factors and are each linked to adverse disease outcomes, rendering elucidation of the precise contribution of diabetes to excess morbidity and mortality difficult. the causal interaction between diabetes and infection rates and outcomes is also challenging. for some chronic viruses such as hbv and hiv, the association with diabetes may represent increased risk of diabetes by pathogenesis mechanisms resulting in insulin resistance related to chronic inflammation, rather than the other way around. high-quality, large prospective epidemiology studies are needed to quantitate the problem, alongside randomised controlled trials of interventions to define optimal treatment strategies in diabetes. raising awareness of the interaction of diabetes and infection with policy-makers, and ensuring the engagement of social scientists, health economists and the pharmaceutical industry in developing new strategies to fight this double burden in lmic is vital. authors' contributions: sd and pc wrote and reviewed the manuscript together. international diabetes federation worldwide trends in diabetes since 1980: a pooled analysis of 751 population-based studies with 4.4 million participants diabetes mellitus increases the risk of active tuberculosis: a systematic review of 13 observational studies the epidemiology of melioidosis in ubon ratchatani, northeast thailand melioidosis epidemiology and risk factors from a prospective whole-population study in northern australia gbd causes of death collaborators. global, regional, and national age-sex specific mortality for 264 causes of death, 1980-2016: a systematic analysis for the global burden of disease study world health organization. definition and diagnosis of diabetes mellitus and intermediate hyperglycaemia. geneva: world health organization american diabetes a. 2. classification and diagnosis of diabetes international diabetes federation: a consensus on type 2 diabetes prevention influence of diabetes and hyperglycaemia on infectious disease hospitalisation and outcome increased risk of common infections in patients with type 1 and type 2 diabetes mellitus impact of diabetes mellitus on treatment outcomes of patients with active tuberculosis the global burden of tuberculosis: results from the global burden of disease study geneva: world health organization stress hyperglycemia in patients with tuberculosis disease: epidemiology and clinical implications 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type 2 diabetes mellitus and increased risk for malaria infection epidemiology of plasmodium vivax malaria in india risk factors for anthroponotic cutaneous leishmaniasis in unresponsive and responsive patients in a major focus, southeast of iran reinfestation in cutaneous leishmaniasis: a new look at predisposing conditions visceral leishmaniasis: a threat to immunocompromised patients in non-endemic areas? freqüência de diabetes mellitus e hiperglicemia em mulheres chagásicas e não-chagásicas the global diabetes epidemic: what does it mean for infectious diseases in tropical countries? parasitic helminths and their beneficial impact on type 1 and type 2 diabetes chronic helminth infection and helminth-derived egg antigens promote adipose tissue transactions of the royal society of tropical medicine and hygiene m2 macrophages and improve insulin sensitivity in obese mice schistosoma infection and schistosoma-derived products modulate the immune responses associated with protection against type 2 diabetes helminth infections and type 2 diabetes: a cluster-randomized placebo controlled sugarspin trial in nangapanda nasal colonization with staphylococcus aureus in patients with diabetes mellitus disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota interactions between the microbiota and the immune system the effect of hyperglycaemia on in vitro cytokine production and macrophage infection with mycobacterium tuberculosis cellular and molecular mechanisms of endothelial dysfunction in diabetes chronic granulomatous disease human polymorphonuclear neutrophil responses to burkholderia pseudomallei in healthy and diabetic subjects effects of risk factors on anti-hbs development in hepatitis b vaccinated and nonvaccinated populations prospective clinical trial of hepatitis b vaccination in adults with and without type-2 diabetes mellitus safety, humoral and cellmediated immune responses to herpes zoster vaccine in subjects with diabetes mellitus young and elderly patients with type 2 diabetes have optimal b cell responses to the seasonal influenza vaccine antibodies in melioidosis: the role of the indirect hemagglutination assay in evaluating patients and exposed populations elevated b cell activation is associated with type 2 diabetes development in obese subjects immunological mechanisms contributing to the double burden of diabetes and intracellular bacterial infections comparison of varicella-zoster virus-specific immunity of patients with diabetes mellitus and healthy individuals increased production of interleukin-4, interleukin-10, and granulocyte-macrophage colony-stimulating factor by type 2 diabetes' mononuclear cells infected with dengue virus, but not increased intracellular viral multiplication the impact of diabetes on the pathogenesis of sepsis hyperglycemia in sepsis is a risk factor for development of type ii diabetes metformin and other glucoselowering drug initiation and rates of community-based antibiotic use and hospital-treated infections in patients with type 2 diabetes: a danish nationwide population-based cohort study metformin as adjunct antituberculosis therapy metformin use and severe dengue in diabetic adults glyburide is antiinflammatory and associated with reduced mortality in melioidosis melioidosis vaccines: a systematic review and appraisal of the potential to exploit biodefense vaccines for public health purposes acknowledgements: none. ethical approval: not required. key: cord-323551-22v2hn3v authors: galanti, m.; birger, r.; ud-dean, m.; filip, i.; morita, h.; comito, d.; anthony, s.; freyer, g. a.; ibrahim, s.; lane, b.; matienzo, n.; ligon, c.; rabadan, r.; shittu, a.; tagne, e.; shaman, j. title: rates of asymptomatic respiratory virus infection across age groups date: 2019-04-15 journal: epidemiol infect doi: 10.1017/s0950268819000505 sha: doc_id: 323551 cord_uid: 22v2hn3v respiratory viral infections are a leading cause of disease worldwide. a variety of respiratory viruses produce infections in humans with effects ranging from asymptomatic to life-treathening. standard surveillance systems typically only target severe infections (ed outpatients, hospitalisations, deaths) and fail to track asymptomatic or mild infections. here we performed a large-scale community study across multiple age groups to assess the pathogenicity of 18 respiratory viruses. we enrolled 214 individuals at multiple new york city locations and tested weekly for respiratory viral pathogens, irrespective of symptom status, from fall 2016 to spring 2018. we combined these test results with participant-provided daily records of cold and flu symptoms and used this information to characterise symptom severity by virus and age category. asymptomatic infection rates exceeded 70% for most viruses, excepting influenza and human metapneumovirus, which produced significantly more severe outcomes. symptoms were negatively associated with infection frequency, with children displaying the lowest score among age groups. upper respiratory manifestations were most common for all viruses, whereas systemic effects were less typical. these findings indicate a high burden of asymptomatic respiratory virus infection exists in the general population. respiratory viral infections are a leading cause of disease worldwide, affecting all age groups and representing a serious threat to human health, particularly for infants, older adults and the immunocompromised. the effects of infection on individuals can vary considerably and include completely asymptomatic manifestations, mild upper respiratory effects and severe symptoms requiring hospitalisation. the epidemiology of respiratory viral infections is generally analysed through passive symptom-based surveillance. when studying the burden of infection, many observational studies focus on severe outcomes, such as cardiac and pulmonary complications in hospitalised patients [1] [2] [3] [4] , or the role of respiratory viruses in the exacerbation of pre-existing respiratory conditions [5] [6] [7] . additionally, community-based longitudinal studies have generally been restricted to young children or households and involve the sampling of specimens to identify viruses after an index symptomatic episode occurs [8] [9] [10] . investigation of the prevalence and effects of respiratory viral infections in the broader population, not just among individuals seeking medical attention, is needed to more fully understand the burden of these infections within the community and to develop adequate preventive measures against these pathogens. in particular, the proportion of the population that is infected and yet does not develop symptoms must be determined to better quantify transmission risk, forecast future disease incidence and pathogen spread, and support public health response efforts. here, we document rates of asymptomatic respiratory virus infection through a large-scale community study across multiple age groups. we use data from a cohort of individuals who were tested weekly for respiratory viruses irrespective of symptom status. we combine these test results with participant-provided daily records of cold and flu symptoms and use this information to characterise symptom outcomes by virus and age category. we enrolled 214 individuals from multiple locations in manhattan borough, new york city. we refer to the participants as healthy as they were enrolled from the general population, as opposed to individuals seeking clinical care. the cohort included children attending two daycares, along with their siblings and their parents, teenagers and teachers from a high school, adults working at two emergency departments (a paediatric er and an adult er), and adults working at a university medical centre. the study period spanned 2 years from october 2016 to april 2018 with some individuals enrolled for a single cold and flu season (october-april) and others for the entire period. all individuals from the selected facilities who were willing to participate were enrolled in the study. enrolled individuals were asked to complete a baseline survey and provide two nasopharyngeal swab samples (one from each nostril). following this preliminary step, two nasopharyngeal samples were again collected weekly from each participant irrespective of symptoms. the baseline questionnaire completed at the time of enrolment included information on ethnicity, general health, daily habits, travel history and household structure. for the entire duration of the study, participants provided a daily report rating nine respiratory illness-related symptoms (fever, chills, muscle pain, watery eyes, runny nose, sneezing, sore throat, cough, chest pain), which were recorded on a likert scale (0 = none, 1 = mild, 2 = moderate, 3 = severe). participants were also asked to note if they had sought medical attention, stayed home or taken influenza-like illness (ili)-related medications as a consequence of their listed symptoms. parents provided consent, baseline questionnaire answers and the daily survey information for their enrolled children. a total daily score was generated for each participant by summing the scores of the individual symptoms (total daily score ranges from 0 to 27). details on the participants are summarised in table 1 . two nasopharyngeal samples per participant were collected on a weekly basis using minitip flocked swabs. both samples were stored jointly in 2 ml dna/rna shield (zymo research, irvine, ca, usa) at 4-25°c for up to 30 days and then stored at −80°c in two aliquots. nucleic acids were extracted from 200 µl of sample and 10 µl of internal control using the easymag nuclisens system (biomerieux, durham, nc, usa). samples were then screened for viruses using the esensor xt-8 respiratory viral panel (rvp; genmark dx, carlsbad, ca, usa) [4] , a multiplex pcr assay. the rvp system separately detects influenza a (any subtype, a/h1n1, a/h3n2, a/h1n1pdm2009) and b; rsv a and b; parainfluenza (piv) 1, 2, 3 and 4; human metapneumovirus (hmpv); human rhinovirus (hrv); adenovirus b/e and c; and coronaviruses (cov) 229e, nl63, oc43 and hku1. samples positive for a particular virus were identified by an electrical signal intensity of ⩾2 na/mm 2 (with the exception of cov oc43 for which positive results were identified by an intensity of ⩾25 na/mm 2 , per manufacturer specifications). we classified all specimens, irrespective of result, as symptomatic or asymptomatic according to the individual symptom score in the days surrounding the date of swab collection. we used multiple definitions as a standard for symptomatic infection does not exist. definitions are summarised in table 2 : definitions 1-4 consider a time window of 7 days around the day of the collection, whereas definitions 5-8 use a window of 11 days. while some definitions use raw metrics, definitions 4 and 8 normalise scores by the average symptom score for each participant (average weekly total symptom scores for each participant ranged from 0 to 39). we refer to infections that do not satisfy one or more specified definitions as asymptomatic infections. the association between reporting respiratory symptoms and testing positive was calculated with the χ 2 test. a 'symptomatic week' was defined as a calendar week where the total symptom score was ⩾10. analyses were conducted using the total number of positive samples, as well as the number of illness-events. we defined an illness-event as a group of consecutive weekly swab specimens for a given individual that were positive for the same virus (allowing for a 1-week gap to account for false negatives and temporary low shedding). the effects of different viruses and the severity of symptoms among different age groups were assessed using analysis of variance (anova). the χ 2 statistic was also used to assess pairwise differences. participants were divided into four groups: children (under 10 years of age), teenagers (10-17 years of age), adults with daily contact with children (parents and pediatric er doctors) and adults without daily contact with children. to assign adult participants to the correct category, we used information on household composition derived from the initial questionnaire. in the analysis of symptoms by virus, specimens positive for more than one virus were excluded. to analyse the specific effects of different viruses, we grouped symptoms into upper respiratory symptoms (runny nose, sneezing, sore throat, watery eye), lower respiratory symptoms (cough, chest pain) and systemic symptoms (fever, chills, muscle pain). of the 4215 nasopharyngeal samples collected, 737 (17.5%) were positive for one or multiple respiratory viruses. among the positive results, between 69% and 74% of the samples were classified as asymptomatic depending on the chosen definition (table 2) . overall, 55% of positive specimens were asymptomatic by all definitions. testing positive for one or more respiratory viruses was associated with an increased likelihood of being symptomatic (p < 0.0001); however, for the majority of symptomatic weeks (67%), rvp did not identify the presence of any respiratory virus. there was a weak association between pre-existing respiratory conditions (asthma, allergies) and the likelihood of experiencing symptomatic infections. however, the association was significant only for some definitions, and the effect on symptom score was marginally significant (p = 0.08 anova). coinfections accounted for 10% of positive samples and were found most frequently among children; they occurred throughout the year and were predominantly a combination of hrv and adenovirus. pairwise comparisons between single infections and coinfections across all eight definitions showed that testing positive for multiple viruses was not associated with more severe symptoms. among the viruses considered, influenza and hmpv were associated with significantly higher symptom scores than rsv, hrv, cov, adenovirus and piv. as shown in figure 1 , more than 50% of influenza and hmpv infections were classified as symptomatic by a majority of definitions, whereas the other viruses were mostly asymptomatic according to all definitions (p < 0.01 and p < 0.001 when comparing, respectively, the ratio of symptomatic influenza and hmpv infections to the other viral infections). the analysis of specific symptoms confirmed the higher severity of influenza and hmpv for lower, upper and systemic 2 m. galanti et al. symptoms (fig. 2a) . upper respiratory symptoms were most commonly associated with positive results for all viruses and for all age groups, followed by lower respiratory symptoms and then systemic symptoms ( fig. 2a and b) . infected children showed a similar percentage of upper and lower respiratory symptoms, and teenagers showed a similar percentage of lower respiratory and systemic symptoms. the majority of hmpv infections presented with both upper and lower respiratory symptoms. higher severity of symptoms was not associated with higher frequency of infection. figure 3 shows that while children were most frequently infected with a respiratory virus (they presented with the highest number of viral shedding events per season), they recorded (as reported by their parents) the lowest symptom scores on average. adults without daily contact with children reported the highest symptom score (the difference was significant only between children and adults without contacts with children, p = 0.003). this finding holds when controlling for length of infection, as longer-lasting infections were more frequent in children. host response to respiratory viruses is heterogeneous: some presumed infections, measured by detectable shedding of virus, exhibit no symptoms or signs of disease, whereas others result in more serious symptoms. when assessing the burden of an [11] [12] [13] . in [11] we showed that most individuals, particularly children and their close contacts, contract multiple respiratory infections per year. here, we analysed the symptoms reported by the same infected individuals and characterised them by virus and by age group. the presence of respiratory symptoms was associated with testing positive for one or more respiratory viruses. however, the majority of symptomatic manifestations were not paired with a positive rvp result. the origin of these symptomatic, negative rvp results could be due to allergies, bacterial infections or potential viral infections with pathogens that have not yet been identified or for which the viral panel does not test. one of the main goals of this analysis was to estimate the asymptomatic ratio, that is, the fraction of infections occurring without symptoms. the asymptomatic ratio is an important indicator for constraining both true respiratory virus prevalence within a community and the potential for further disease transmission [14] . asymptomatic carriers can, in fact, contribute to disease spread by generating ( possibly symptomatic) secondary infections. estimates of the asymptomatic ratio vary widely not only across diseases (from 95% of polio virus infections to <10% of measles), but also within the same disease due to different diagnostic testing procedures (pcr vs. serological tests [15] ) and sampling approaches (household vs. longitudinal studies). among respiratory viruses, the role of asymptomatic infection is poorly understood. for influenza alone, the prevalence of asymptomatic infection has been estimated to be as low as 9.4% and as high as 90% depending on the virus type, study, season and definition of asymptomatic infection [15, 16] . there is also some evidence that viral shedding correlates with symptom severity and that the contagiousness of asymptomatic individuals is less than for symptomatic persons [17, 18] . the rationale is that respiratory symptoms (coughing, sneezing, runny nose) help spread pathogens through droplet transmission, either inhaled or settled [19] . however, the absence of symptoms might bring asymptomatic infected individuals into greater contact with susceptible persons outside the home. this is particularly true for infants and toddlers whose behaviour, hygiene habits and close physical contact typically favour the spread of germs, especially in childcare settings. whether greater contact occurs and makes asymptomatic individuals effectively as contagious as symptomatic persons is not known. an additional difficulty is that a standard, accepted definition of symptomatic infection does not exist. further, perception of symptoms is highly subjective, and it may be difficult to assess whether a symptom is caused by the pathogen. for example, chronic symptoms can occurrunny nose is a common sign in children that does not necessary imply viral infectionand allergies can cause sneezing. fever, muscle pain and chills may also be caused by infections other than respiratory viruses. for these reasons, we adopted multiple definitions of 'symptomatic episodes', including personalised metrics. more than half of the rsv, adenovirus, piv, cov and hrv infections documented here were asymptomatic according to all definitions. influenza and hmpv infections caused significantly more symptoms than these other viruses; however, given its high prevalence, hrv was responsible for more than half of symptomatic episodes. in general, all viral infections presented with similar symptoms, with upper respiratory manifestations being more frequent, whereas systemic symptoms such as fever, muscle pain and chills were less typical. for surveillance in which ili (defined as fever plus cough and/or sore throat) is used to record respiratory infections among people seeking care, this constraint may mean that many viral infections go undetected. consistent with previous findings [20, 21] we did not find an association between viral co-infection and the likelihood of being symptomatic or presenting with more severe symptoms. regardless of the definition, our findings underscore the extremely high proportion of respiratory viral infections that are asymptomatic. further analysis is required to capture the role played by asymptomatic individuals in outbreak transmission dynamics, specifically the relative infectiousness of asymptomatic vs. symptomatic infections. there was considerable heterogeneity in individual immune response: some individuals seemed to be consistently less predisposed to developing respiratory symptoms upon viral infection, whereas others were always symptomatic when testing positive. infections in children were less symptomatic than in adults, even though children proved to be more frequently infected with respiratory viruses [11] . frequent infections may enhance the ability of the immune system to identify and respond to pathogens; hence, the group subjected to the fewest respiratory viral infections (adults without children) reported the highest average symptom scores. however, the apparent lower pathogenicity among children may simply be an artefact introduced due to parents reporting symptoms for their children. some symptoms, such as sore throat, muscle pain and chest pain, are difficult to identify in young children, and in fact they were less reported in this age category than among adults and teenagers. this possible bias in the reporting of symptoms is one limitation of our study. the daycare and workplace connections among some individuals in our cohort are another possible bias: some infections were likely caused by the same strain; thus the symptom profiles could be due to specific features of the pathogen rather than individual immune response. further, all the children in our cohort were attending daycare or were siblings of children attending daycare. interactions within the daycare setting could be a confounder in this analysis. future studies should investigate the genetic basis of heterogeneity in host response to respiratory virus infection in order to identify the regulatory pathways controlling reactions to these infections. moreover, longitudinal studies of this type, involving large networks of connected individuals, are needed to assess the role of asymptomatic infections in the transmission of respiratory viruses. the burden of respiratory syncytial virus infection in young children estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the global burden of disease study mortality associated with influenza and respiratory syncytial virus in the united states comparison of the biofire filmarray rp, genmark esensor rvp, luminex xtag rvpv1, and luminex xtag rvp fast multiplex assays for detection of respiratory viruses role of viral respiratory infections in asthma and asthma exacerbations respiratory viruses and exacerbations of asthma in adults role of viruses in exacerbations of chronic obstructive pulmonary disease molecular epidemiology of respiratory syncytial virus transmission in childcare viral shedding and clinical illness in naturally acquired influenza virus infections epidemiology of viral respiratory tract infections in a prospective cohort of infants and toddlers attending daycare longitudinal active sampling for respiratory viral infections across age groups asymptomatic summertime shedding of respiratory viruses asymptomatic shedding of respiratory virus among an ambulatory population across seasons estimating pathogen-specific asymptomatic ratios the fraction of influenza virus infections that are asymptomatic: a systematic review and meta-analysis household transmission of the 2009 pandemic a/h1n1 influenza virus: elevated laboratory-confirmed secondary attack rates and evidence of asymptomatic infections does influenza transmission occur from asymptomatic infection or prior to symptom onset? world health organization writing group (2006) nonpharmaceutical interventions for pandemic influenza, international measures dynamics of infectious disease transmission by inhalable respiratory droplets epidemiology of multiple respiratory viruses in childcare attendees infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis author orcids.m. galanti, 0000-0002-9060-1250.financial support. this work was supported by the defense advanced research projects agency contract w911nf-16-2-0035. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.conflict of interest. js and columbia university disclose partial ownership of sk analytics. all other authors declare no competing interests. key: cord-332737-iclruwmx authors: webley, wilmore c.; hahn, david l. title: infection-mediated asthma: etiology, mechanisms and treatment options, with focus on chlamydia pneumoniae and macrolides date: 2017-05-19 journal: respir res doi: 10.1186/s12931-017-0584-z sha: doc_id: 332737 cord_uid: iclruwmx asthma is a chronic respiratory disease characterized by reversible airway obstruction and airway hyperresponsiveness to non-specific bronchoconstriction agonists as the primary underlying pathophysiology. the worldwide incidence of asthma has increased dramatically in the last 40 years. according to world health organization (who) estimates, over 300 million children and adults worldwide currently suffer from this incurable disease and 255,000 die from the disease each year. it is now well accepted that asthma is a heterogeneous syndrome and many clinical subtypes have been described. viral infections such as respiratory syncytial virus (rsv) and human rhinovirus (hrv) have been implicated in asthma exacerbation in children because of their ability to cause severe airway inflammation and wheezing. infections with atypical bacteria also appear to play a role in the induction and exacerbation of asthma in both children and adults. recent studies confirm the existence of an infectious asthma etiology mediated by chlamydia pneumoniae (cp) and possibly by other viral, bacterial and fungal microbes. it is also likely that early-life infections with microbes such as cp could lead to alterations in the lung microbiome that significantly affect asthma risk and treatment outcomes. these infectious microbes may exacerbate the symptoms of established chronic asthma and may even contribute to the initial development of the clinical onset of the disease. it is now becoming more widely accepted that patterns of airway inflammation differ based on the trigger responsible for asthma initiation and exacerbation. therefore, a better understanding of asthma subtypes is now being explored more aggressively, not only to decipher pathophysiologic mechanisms but also to select treatment and guide prognoses. this review will explore infection-mediated asthma with special emphasis on the protean manifestations of cp lung infection, clinical characteristics of infection-mediated asthma, mechanisms involved and antibiotic treatment outcomes. incidence and etiology of childhood and adult onset asthma asthma incidence is highest in childhood and thereafter decreases and remains stable at~1-3 new cases per 1000 per year throughout late adolescence and adulthood [1] . in adult populations, the prevalence of active cases of childhood-onset asthma (coa) and adult-onset asthma (aoa) are approximately equal, or favor aoa [2] . reasons for this counterintuitive prevalence ratio include (1) the propensity for coa to remit more frequently than aoa and (2) the greater number of years of adulthood in which to accrue new cases [2] . of relevance to clinical management and population disease burden is the wide range of asthma severities, from mild intermittent to severe persistent; the most severe 20% of cases account for 80% of health care utilization and morbidity [3] . robust population-based data indicate that around half of adults with asthma remain suboptimally controlled, even when treated with currently available anti-inflammatory medications, and~15% of adults with active asthma are severely uncontrolled [4] [5] [6] . these data indicate the need for novel therapies that are effective in the most severe and treatment-resistant cases of asthma that account for the majority of morbidity, mortality and health care utilization. the emerging evidence that a wide variety of microbes are present in the lower airway and may play a role in asthma pathogenesis suggests that manipulating the airway microbiome may be a novel approach towards this goal. studies confirm the existence of an infectious etiology mediated by chlamydia pneumoniae (cp) [7] and possibly other viral [8] , bacterial [9] and fungal [10] microbes. among the various infections associated with asthma, the obligate intracellular respiratory pathogen cp is of particular interest, as it is associated with both asthma severity and treatment resistance [11] [12] [13] . although this review focuses on cp we will discuss mycoplasma pneumoniae (mp) briefly under treatment (section v). it is possible that microbes such as cp and mp that have been implicated in recurrent wheeze and asthma etiology may serve as cofactors for viral infections, but certainly appear to act independently in asthmatic disease. the etiology of asthma remains unknown and is almost certainly multifactorial. many "triggers" for asthma attacks are well known (e.g., allergens, viral respiratory infections, fumes, cold air, exercise) but underlying mechanisms for why some exposed individuals develop asthma while most do not remain elusive [14] . genetic studies have failed to locate a unique "asthma gene" and instead point towards complex multifactorial genetic and environmental factors [15] . a currently popular paradigm, the "hygiene hypothesis," posits that the increased incidence of allergies (hayfever and eczema) and asthma noted in recent decades, is associated with less exposure to childhood infections and bacterial products (e.g., endotoxin). emerging evidence supports the hygiene hypothesis for hayfever and eczema but not for asthma which appears instead to be related to infections throughout the life cycle [16] [17] [18] . the host lung and gut microbiome as they relate to asthma are active areas of research [19] . yet it must be pointed out that studies of bacterial rrna may fail to detect cp due to low copy numbers or sampling problems due to deep tissue intracellular locations for this species [20, 21] . an increasing number of studies have now confirmed that the host microbiome has a significant impact on the risk of asthma development. a study published in 2010 by hilty and colleagues using 16s rna clone-library sequencing showed that when compared with healthy controls, patients with asthma had significantly more pathogenic proteobacteria and fewer bacteroidetes [22] . careful assessment of both healthy controls and asthmatic patients has confirmed the presence of bacterial communities. however, the bacterial burden was significantly greater in patients with asthma than in the healthy controls [23] . the microbial burden was even greater in asthmatics with greater bronchial reactivity upon methacholine challenge. these patients showed marked improvement in bronchial reactivity to methacholine after 6 weeks on clarithromycin. importantly, greater bronchial reactivity also correlated with greater relative abundance of members of certain bacterial communities known to exhibit characteristics that contribute to asthma pathophysiology, including species capable of inducing nitric oxide reductase, produce sphingolipids or have the ability to metabolize steroid compounds [24, 25] . a recent study showed that 1-month old infants who had positive oropharynx cultures of streptococcus pneumoniae, moraxella catarrhalis, or haemophilus influenzae showed increased susceptibility for development of childhood asthma [19, 26] . another recent study concluded that the nasopharyngeal microbiome within the first year of life was a determinant for infection spread to the lower airways and predicted the severity of accompanying inflammatory symptoms, as well as risk for future asthma development. the authors showed that early asymptomatic colonization of the nasopharynx with streptococcus was a strong asthma predictor [27] . these authors also demonstrated that antibiotic usage disrupted this asymptomatic colonization and prevented asthmatic onset [27] . these findings support the hypothesis that colonization of the developing airway by certain microbes (both viral and bacterial) can significantly alter the airway architecture and overall immune function, influencing how the airway responds to a variety of insults [28] . these findings also suggest that antimicrobial agents may represent an effective therapeutic tool with the potential to curtail both the duration and severity of asthma exacerbations initiated by a variety of microbes and exposes the limitation of the hygiene hypothesis in this regard [26] . the microbiome studies cited here have not specifically targeted cp and mp in upper airways. studies that have specifically tested for these atypical organisms have reported positive detection [29, 30] . intracellular detection of cp in adenoid tissue of symptomatic children was extremely common [30] and raises questions regarding a potential for cp-microbiome interactions. infections in early life can act either as inducers of wheezing or as protectors against the development of allergic disease and asthma. many young children have wheezing episodes associated with early-life respiratory infections. the infections most likely to be associated with these wheezing episodes include respiratory syncytial virus (rsv), human rhinovirus (hrv), human metapneumovirus, parainfluenza viruses and coronavirus [31] . the hygiene hypothesis has proposed that, for some infants, frequent early life infections may protect against asthma [17] and this certainly appears to be the case for most infants, as wheezing episodes with respiratory infections diminish as the child ages. however, for others, early-life wheezing episodes may mark the beginning of asthma. regarding established asthma, many types of viral respiratory infections have been shown to have a significant influence. in fact, viral respiratory infections are diagnosed in 80% of episodes of asthma in both children and adults [32, 33] . the question then remains; what factors determine if a viral respiratory infection provokes the onset of chronic asthma? factors appear to include the type of virus and the viral infectious dose as well as host susceptibility factors leading to inflammation, airway cellular infiltration with neutrophils and eosinophils or the presence of allergens in the airway and their interactions with the host immune system. if this combination of host and pathogen factors results in airway inflammation and hyperresponsiveness, the outcome could be asthma. could cp play a key role in this complex scenario? a clue to the answer to this question was found in a secondary analysis [34] of data from a community-based pediatric viral respiratory infection study that identified viral infections in 80-85% of exacerbations [33] . one hundred and eight children with asthma symptoms completed a 13-month longitudinal study in which exacerbations were recorded, and cp pcr and cp-specific secretory iga (cp-siga) antibodies were measured both during exacerbations and during asymptomatic periods. cp pcr detections were similar between the symptomatic and asymptomatic episodes (23% v 28%, respectively). children reporting multiple exacerbations remained cp pcr positive (p < 0.02) suggesting chronic infection. cp-siga antibodies were more than seven times greater in subjects reporting four or more exacerbations compared to those who reported just one (p < 0.02). the authors suggested that immune responses to chronic cp infection may interact with allergic inflammation to increase asthma symptoms [34] . notably, mp was not found to be important in this study. emerging evidence links cp infection with both de novo asthma (asthma onset during/after an acute lower respiratory tract infection in a previously non-asthmatic individualalso referred to as the "infectious asthma" syndrome) and with asthma severity [11, 12, 35, 36] . this section will review what is known about cp in asthma initiation and severity, and the multiple experimentally established mechanisms that might mediate these associations. therapeutic implications are reviewed in section v. de novo wheezing during an acute lower respiratory tract infection is remarkably common [37] . most of these wheezing episodes appear to resolve without chronic sequelae but sometimes chronic asthma develops. surprisingly, clinical studies report that asthma onset after an acute respiratory illness is exceedingly common (up to 45% of adult-onset asthma cases [38] . this strong temporal association of respiratory infections and asthma onset has been confirmed in a population-based study [39] . the most reliable way to establish whether a specific respiratory pathogen can initiate asthma would be to perform large, long-term prospective microbiological and clinical cohort studies of the general (non-asthmatic) population. such a study would be very expensive and has not yet been undertaken. a second approach would be to perform prospective studies in selected non-asthmatic patients exhibiting "risk factors" for asthma in clinical settings [40] . if the selected "risk factors" do indeed identify people at higher likelihood of developing the "infectious asthma" syndrome, this type of study might be feasible. characteristics associated with cp/mp biomarkerpositive "infectious asthma" include patients with severe, treatment-resistant asthma, exhibiting a neutrophilic airway inflammation or test pcr positive for cp or mp. it should however, be noted that there is currently no test or set of tests that will definitively diagnose who will benefit maximally from azithromycin treatment. factors that predict risk in non-asthmatics for developing the "infectious asthma" syndrome include a previous history of self-limited lower respiratory tract illnesses such as acute bronchitis (often with wheezing) and/or pneumonia [35, 38, 39] . other risk factors may be operative but are poorly understood at this time. over a 10-year time period, hahn et al. [35] collected prospective cp microbiologic testing and clinical data on 10 patients with de novo wheezing. nine of these subjects exhibited an acute bronchitic illness and one had community-acquired pneumonia. all 10 met serological criteria for an acute primary (n = 8) or secondary (n = 2) cp infection. of the nine patients with acute bronchitis and wheezing, four improved without treatment and five progressed to chronic asthma. the patient with pneumonia was treated with a traditional short course of a macrolide with resolution of pneumonic infiltrate, yet developed chronic bronchitis and cp was isolated by culture from his sputum 6 months later. this type of study has not been replicated but raises several questions. cp is well known to cause protean manifestations of acute respiratory illness; these observations suggest that cp may also be capable of causing protean manifestations of chronic respiratory conditions (e.g., asthma, chronic bronchitis and copd, reviewed in [41] ). whereas some of the cp infected patients with de novo wheezing resolved their acute illness without treatment, others developed chronic sequelae; identification of underlying protective and promoting factors might help address the current asthma pandemic. once established, cp-associated asthma has been linked with increased severity in several studies. cook et al. [42] first identified cp biomarkers in what they referred to as "brittle asthma" (asthma that was hard to control and more severe than average). an accumulating body of evidence supports the association of cp infection with asthma severity [11, 12, 43] and with steroid resistant asthma [44] . multiple mechanisms support the biologic plausibility of these associations (reviewed in [45] ). exposure to cigarette smoke is an established factor tied to steroid resistance in asthma [46] . similar to cigarette smoke, cp induces pulmonary bronchial epithelial ciliostasis [47] . additionally cp infects alveolar macrophages and lung monocytes leading to enhanced production of tnf-α, il-1β, il-6 and il-8; infects human bronchial smooth muscle cells to produce il-6 and basic fibroblast growth factor (with potential effects on bronchial hyper reactivity and lung remodeling that have yet to be thoroughly investigated); and chronic infection exposes tissues to chlamydial heat shock protein 60 (chsp60) and bacterial lipopolysaccharide (lps) that have been associated with increased inflammation and asthma (reviewed in [48] ). lastly, cp-specific ige has been demonstrated to be strongly associated with severe persistent asthma (80% of cases) [43] and other chronic respiratory illnesses in children severe enough to justify undergoing bronchoscopy [49] . whereas exposure to recognized allergens can be mitigated, exposure to unrecognized bacterial "allergens" may result in chronic unrelenting exposures that could contribute to severity [43, 50] . it may prove difficult or even impossible to unravel exactly which mechanism(s) contribute to producing an "infectious asthma" phenotype. in regard to the involvement of cp in asthma pathogenesis, the controversy of whether the association is causal or coincidental can be settled in two ways: (1) patients diagnosed with asthma can be treated with the aim of evaluating the effects of antibiotics in ameliorating asthma symptoms compared to untreated of placebo controls and (2) animal models can be performed to evaluate the role of cp in asthma initiation and/or exacerbation. experimental animal inoculation studies may help to elucidate mechanisms underlying cp asthma pathogenesis. over the past three decades, animal models of asthma have been extensively utilized to elucidate mechanisms of the disease, determine the activities of genes of interest, investigate cellular pathways and predict the safety and efficacy of various drugs being considered for asthma treatment. initial murine models of chlamydial lung infections were carried out in adult mice and seemed to closely represent acute human asthma. these studies utilized the mouse pneumonitis biovar of c. trachomatis (mopn) since it is well known as a natural mouse pathogen [51] and would therefore represent the best choice for investigating host-pathogen interactions in this context. these early studies recorded extensive lung consolidation after 7 days of airway infection and found significant airway inflammation characterized by neutrophil infiltration in airway exudates [52] . these early studies also confirmed that multiple reinfections were required to induce symptoms of chronic asthma and that a th1 immune response contributing ifn-γ and subsequently activated macrophages was necessary to clear the infection [53, 54] . more recently, many studies have utilized neonatal mouse models for infectious asthma since early studies demonstrate that neonatal t cell immune responses in both mice and human are skewed toward a th2 cellular phenotype as a result of placental immune pressure. these th2 cells are much less effective in the immune response compared to their adult counterparts [55, 56] . horvat, et al. later demonstrated that neonatal chlamydial lung infection induced mixed t-cell responses that drive allergic airway disease (aad) using a balb/c mouse model with ovalbumin to induce aad [57] . further work from this group confirmed that chlamydial infection in neonatal and infant, but not adult mice, exacerbated the development of hallmark features of asthma in ovalbumin-induced allergic airways disease models. some of these notable features include increased mucus-secreting cell numbers, il-13 expression, and airway hyperresponsiveness [58] . studies from our own lab confirm that early-life chlamydial airway infection induces a th2 immune response, both airway eosinophilia and neutrophilia, and permanent alteration of lung structure and function with concomitant enhancement of the severity of allergic airways disease in later life [59] . we confirmed that neonatally infected mice never cleared the infection, showed dissemination to the liver and spleen through the peripheral circulation, and the development of chlamydia-specific ige antibodies in the infected neonates but not adult controls [59] . recently, hansbro et al. completed work using a bone marrow chimera reconstitution that clearly demonstrated that infant lung infection results in lasting alterations in hematopoietic cells, leading to increased severity of aad later in adult life [60] . a significant study by kaiko et al. [61] , demonstrated that infection of bone marrow-derived dentritic cells (bmdc) promoted th2 immunity and airways hyperreactivity in a mouse model. intratracheal passive transfer of infected bmdc but not uninfected control bmdc into naïve balb/c mice resulted in increased il-10 and il-13 in the bal fluid [61] . these animals also showed significant increases in airways resistance and a reduction in airways compliance compared to their uninfected counterparts. these are hallmarks of asthma and further confirm the role of chlamydial infection in asthma initiation and pathology, at least in mice. a further set of experiments by schröder et al. [62] demonstrated that adoptive transfer of lung dendritic cells from cp infected mice, but not from uninfected mice, produced eosinophilic airway inflammation after challenge with an exogenous allergen (human serum albumin) that was dose-, timing-, and myd88-dependent. taken together, these findings suggest it is plausible that cp infection solely of lung dendritic cells may be sufficient to induce an asthma "phenotype" that may demonstrate characteristics that are both "infectious" and "allergic". these animal model studies have added significantly to our understanding of the mechanisms involved in the inflammatory process of chlamydial infection leading to asthma initiation and exacerbation. it also appears that the damage caused by chlamydial airway infections over time leads to an exaggerated airway repair or airway wall remodeling. the major features of this type of response include epithelial cell shedding, goblet cell hyperplasia, hypertrophy and hyperplasia of the airway smooth muscle bundles, basement membrane thickening and increased vascular density through angiogenesis [63] . the functional and mechanical consequences of this type of aberrant repair leads to bronchial wall thickening which can uncouple the bronchial wall from the surrounding parenchyma, significantly enhancing airway narrowing and severe obstruction [63] . this type of airway damage might prove irreversible even with long-term inhaled steroid treatment. moreover, it is well documented that corticosteroid use drives cp out of a persistent state into active replication, since corticosteroids negatively impact several aspects of cell-mediated immunity while favoring the shift from a th1 towards a th2 immune response [64] . this shift in response significantly impedes the ability of the host to eradicate intracellular pathogens like cp and may lead to the release of chsp60 which exacerbates the inflammatory process [11] . there is also evidence that cp infection may promote airway remodeling by decreasing the ratio of mmp9 to timp1 secreted by inflammatory cells, and by altering cellular responsiveness to corticosteroids [65] . see fig. 1 the concept that asthma is a syndrome with different underlying etiologies is well accepted. the use of the word "phenotype" to describe asthma subtypes based primarily on the inflammatory composition of respiratory secretions and/or peripheral blood is more problematic. the original definition of "phenotype" referred to relatively stable somatic manifestations of underlying genetics (such as eye color) whereas current asthma inflammatory "phenotyping" is based on cross sectional sampling of a dynamic physiologic process (host inflammatory response) and does not account for the fact that inflammatory composition is not necessary a fixed characteristic [66] . in the context of a review that focuses on chlamydial infection we are reluctant to place too much emphasis on asthma phenotypes based on inflammatory cell compositions because well described host responses to acute, sub-acute and chronic chlamydial infections involve a wide array of inflammatory cells (including eosinophils, neutrophils and monocytes) the composition of which varies significantly at different temporal stages of the infection [67] . we have commented on some fairly well defined asthma categories but even these can change over time (e.g., mild asthma can become severe, stable asthma can become uncontrolled). the dynamic and often unpredictable nature of asthma symptomatology is one of the factors that make asthma research so challenging. historically asthma was categorized as either allergic or non-allergic but this distinction was put into question as early as the 1980s [68] . an early report of the association between cp and asthma did find independent associations of cp biomarkers, clinical allergy and asthma [40] yet in the clinical setting there is overlap between atopy and cp infection [69] . the animal models described earlier indicate that cp can promote both asthma and atopy, thus an absolute distinction between these two categories as indicators of differing underlying etiologies may not be warranted. macrolide treatment trials that examine subgroup responses are one approach to examining the predictive value of this and other subgroups. asthma has also been characterized as either "eosinophilic" or "neutrophilic" based on the cellular composition of respiratory secretions or bronchoalveolar lavage fluid (balf) [70] . simpson et al. [71] performed an rct of a macrolide (clarithromycin) in severe refractory asthma in adults and reported no overall benefit in the group as a whole. however, there was a positive effect in the pre-specified subgroup of patients with "neutrophilic" asthma as defined by sputum il-8 and neutrophil numbers. the predictive power of these findings is limited since it is unclear whether sputum composition is stable over time in severe refractory asthma (or any asthma, for that matter). the majority of people with asthma can be well controlled with conventional guideline-based anti-inflammatory treatments (mainly inhaled steroids, sometimes in combination with an inhaled long-acting bronchodilator) [72] . nevertheless, a significant minority of people with asthma is not well controlled by guideline treatments [73, 74] . the proportion of all people with "refractory" asthma (asthma that is not responsive to guideline therapies) has been estimated at between 5 and 15% but the contribution of refractory asthma to asthma morbidity and mortality is considerably greater, as the most severe 20% of asthma cases account for 80% of asthma morbidity and health care costs [3] . if patients with the "overlap syndrome" (asthma and copd) are included, the numbers of people with refractory disease increases significantly [75] . of the various novel therapies under consideration for refractory asthma [76] , macrolides appear to be one of the most promising. a 2013 metaanalysis of 12 randomized, controlled trials (rcts) of macrolides for the long term management of asthma in both adults and children found positive effects on peak expiratory flow rate (pefra measure of pulmonary function), asthma symptoms, asthma quality of life (aql), and airway hyper responsiveness (ahr), but not on forced expiratory flow rate in 1 s (fev1) [77] . the updated 2015 cochrane review of 18 rcts [78] reported positive benefits on asthma symptoms and fev1 but not on aql (ahr and pefr were not analyzed). a joint european respiratory society/american thoracic society (ers/ats) guideline on severe asthma recommends against the use of macrolides ("conditional recommendation, very low quality evidence") [79] . the ers/ats guideline states "this recommendation places a relatively higher value on prevention of development of resistance to macrolide antibiotics, and relatively lower value on uncertain clinical benefits." the inconsistent findings of the meta-analyses, along with the uncertainties surrounding the clinical benefits of macrolides, underscore the need for higher quality evidence. this section adds some evidence not included in the meta-analyses, reviews what is known about macrolide side effects (including the clinical consequences of resistance) and suggests research approaches to obtain better evidence. we conclude with some provisional recommendations for clinicians who may be approached by patients with new-onset, uncontrolled and/or refractory asthma who are asking for macrolide treatment. current evidence for all asthma treatments is limited due to selection bias initiated by researchers, clinicians, and even asthma patients themselves. researcher bias. the academic literature is replete with asthma efficacy studies lacking in generalizability [80] . the efficacy trials on which current asthma treatment guidelines are based systematically exclude >90% of people with asthma encountered in the general clinical population [81, 82] . only pragmatic effectiveness trials, with minimal exclusions, are able to provide evidence applicable to the general population [44] . clinician bias. a recent trial of azithromycin for acute exacerbations of asthma (aza-lea) is notable because over 95% of patients with an exacerbation were not eligible for enrollment primarily because they had received an antibiotic from a treating clinician [83] . an accompanying editorial speculated that one possible reason for the negative results of azalea was that clinicians were somehow able to identify and treat likely candidates, making them ineligible for the research [84] . be that as it may, azalea is an example of asthma research made less informative due to nonresearcher clinician behavior. patient bias. hahn et al. [44] performed a pragmatic trial of azithromycin for asthma (azmatics) in which the likely candidates excluded themselves from randomization. this unanticipated event occurred because azmatics was an internet-based trial; people with severe, refractory asthma identified themselves as likely candidates and contacted the pi for enrollment; but upon learning that they had a 50% chance of receiving placebo, they opted out of randomization in favor of receiving a comparable azithromycin prescription from their personal clinician [85] . rather than lose data on this "open-label" (ol) group, the study protocol was altered to include a third (ol) arm. randomized results were similar to azalea (negativesee fig. 2 ); however, ol subjects exhibited large and unprecedented improvements in symptoms and quality-of-life (qol) that persisted long after treatment was completed (figs. 2 & 3) . because the ol group was not randomized, these results do not appear in any meta-analysis of rcts; nevertheless they strongly suggest that future macrolide rcts should focus on the severe end of the asthma spectrum, as also recommended by others [42, 71, 86] , and preferably engage patient populations that are unlikely to want to opt out of randomization. macrolide mechanisms of action in asthma are thought to be directly anti-inflammatory, indirectly anti-inflammatory (i.e., antimicrobial), or both. it is difficult to invoke direct anti-inflammatory macrolide effects as responsible for large clinical benefits persisting to 9 months after treatment completion. antimicrobial effects, against specific respiratory pathogens or against the general lung microbiome, remain likely possibilities. circumstantial evidence suggests that macrolide treatment effects may, at least in part, be attributable to antimicrobial actions against chronic atypical infections [9, 87] . this issue is by no means settled and requires further research that may be challenging given the selection biases noted above coupled with likely low sensitivity of lung sampling leading to false negative diagnosis of, for example, chronic cp lung infection [20] . fig. 2 azithromycin improves asthma symptoms and patient quality of life. subjects with severe refractory asthma treated with azithromycin (open label) had fewer persisting asthma symptoms a and greater asthma quality of life b than groups with lesser asthma severity randomized to azithromycin or to placebo [44] azithromycin is generally well tolerated and is widely used for a variety of acute respiratory illnesses. concerns about adverse effects of azithromycin include development of antibiotic resistance, sudden cardiac death, hearing loss and effects on the host mcrobiome. development of resistance is a possibility whenever antibiotics are used; azithromycin is no exception. however, there are no reports of patient harm from resistant organisms in any cardiorespiratory trial performed to date [89] . rather, the only detectable clinical effects of azithromycin in these trials were decreased incidences of sinusitis, acute bronchitis and pneumonia, and less use of other antibiotics [88, 89] . sudden cardiac death attributable to azithromycin (1 in 4000 prescriptions in high cardiac risk patients) was plausibly documented in an epidemiologic study of a medicaid population in tennessee [91] . the same risk was also present for a quinolone (levofloxacin). subsequent population-based studies in average risk populations showed no increased risk of sudden death [92, 93] . mild hearing loss was reported in an excess of <1% of heart disease subjects randomized to 600 mg azithromycin once weekly for 12 months [88, 90] . hearing test changes leading to discontinuation of azithromycin occurred in 2.8% of 1142 severe copd subjects randomized to 250 mg azithromycin daily for 1 year [90] . the clinical significance of these hearing test changes is unclear. notably, it is likely that daily azithromycin dosing is unnecessary [94] and may lead to increased adverse events [91] . the prolonged half-life of azithromycin within cells, including within immune system cells, allows weekly dosing and may be preferable to daily dosing when targeting either immune cells or intracellular pathogens such as cp. although largely speculative at this time, it appears that macrolide effects against the lung microbiome may be potentially harmful or helpful in asthma. segal et. al. reported that an 8 week treatment with azithromycin did not alter bacterial burden but reduced α-diversity [95] . they also observed significant reduction in certain pro-inflammatory cytokines, which might explain the non-specific anti-inflammatory effects proven beneficial in copd and asthma [95] . published findings from slater et al. that specifically evaluated azithromycin effects on the lung microbiome revealed a significant reduction in bacterial richness in the airway microbiota [96] . importantly, reductions were most significant in three pathogenic genera: pseudomonas, haemophilus and staphylococcus [96] . overall, available data suggest that azithromycin treatment of severe asthma, while controversial, may benefit those with confirmed atypical bacterial infection [97] . resistance, adverse events including sudden death, hearing loss and changes in host microbiome should be monitored in future pragmatic trials. protean manifestations of chronic cp infection, that may include asthma, chronic bronchitis, copd, and the "overlap syndrome" (asthma and copd) argue in favor of pragmatic trials with broad inclusion criteria that include patients with lung multi-morbidity. at least nine domains distinguish pragmatic (or effectiveness) trials from explanatory (efficacy) trials (table 1 ) [98] . in the context of future rcts of macrolides for asthma, we fig. 3 asthma quality of life (aql) improvement scores at 12 months (9 months after completing azithromycin). the minimum clinically important score is ≥0.5; a score of 1.5 is considered a large important change [44] propose that the most important pragmatic domains are (1) broad eligibility to account for the protean clinical manifestations of both chronic reactive/obstructive lung disease and cp infections as discussed previously and (2) a comprehensive patient-centered primary outcome. asthma exacerbations are a current popular choice as a primary outcome because they are clinically relevant [99] . however, exacerbations are only one of many outcomes that are important to asthma patients [100] . compared to exacerbations, asthma quality-of-life (qol) more comprehensively measures patient-important outcomes. qol includes, but is not limited to, the adverse effects of exacerbations on patient well-being [100] and qol has proven robust in the sole pragmatic macrolide-asthma trial performed to date [44] (figs. 2 and 3 ). many patients in this pragmatic trial [44] had significantly decreased asthma qol at study entry and large important improvements in qol after azithromycin, but did not experience exacerbations. this significant subgroup would have been either possibly ineligible for inclusion or not counted as successes in a trial using exacerbations as the primary outcome. pragmatic trials primarily ask does this treatment work? explanatory trials primarily ask what is the mechanism? addressing target groups/mechanisms in pragmatic trials of macrolides is desirable and possible as secondary aims by specifying a priori hypotheses coupled with subgroup analyses. we recommend studying a wide array of biomarkers using this approach. it is notable that rcts of macrolides have been performed and/or macrolides are being recommended in the treatment of many chronic lung conditions (diffuse pan-bronchiolitis, cystic fibrosis, bronchiectasis, copd, post-transplant bronchiolitis obliterans) [101, 102] . a planned trial will test the effectiveness of azithromycin in patients with the "overlap syndrome" (asthma-copd) [103] . it is time to add asthma to the growing list of chronic respiratory conditions that are being evaluated by robust macrolide rcts that are pragmatic in nature. in the meantime, patients with severely uncontrolled and/or refractory asthma, or new-onset asthma are increasingly searching the internet for new information and are sometimes better informed than their doctor about current evidence regarding macrolides for asthma (hahn: personal observations). pending more robust data from asthma rcts that have yet to be performed, how should practicing clinicians respond when such patients request macrolide treatment? as stated above, the ers/ats guidelines on severe asthma recommend against the use of macrolides, albeit with caveats that the evidence for this recommendation is weak and provisional [79] . informal guidelines from a pulmonology research group state that they recommend macrolide treatment only for confirmed diagnoses of atypical lung infection [104] . from a practical standpoint, their recommendation limits treatment only to those who have undergone bronchoscopy; even then the diagnostic sensitivity is likely to be less than perfect due to sampling issues discussed earlier. both these recommendations have met resistance from patients who have read and understood the evidence (hahn: personal communication). we offer a third alternative recommendation, repeated word for word from the conclusion of the sole practice-based pragmatic trial of azithromycin for asthma conducted to date [44] : "pending further randomized trials, given the relative safety of azithromycin and the significant disease table 1 proposed design for a randomized trial of azithromycin for the long-term management of asthma. seven of nine precis-2 [98] domains are recommended as pragmatic and two as explanatory domain pragmatic or explanatory? a comments eligibility. who is selected to participate in the trial? pragmatic exclusions only for safety; comorbidities included. recruitment. how are participants recruited into the trial? pragmatic recruited from practice sites (emergency rooms, clinics). setting. where is the trial being done? pragmatic performed at the practice site. organization. what expertise and resources are needed to deliver the intervention? no extraordinary expertize required. flexibility: delivery. how should the intervention be delivered? total weekly oral dose can be administered on any schedule desired. flexibility: adherence. what measures are in place to make sure participants adhere to the intervention? adherence encouraged by frequent contacts by the research team and monitored by patient report and pill count. follow-up. how closely are the participants followed-up? explanatory 3-monthly study visits to collect non-routine information (e.g., spirometry, biomarkers, qol) primary outcome. how relevant is it to participants? pragmatic outcome is patient-centered (see text for discussion). to what extent are all data included? pragmatic intention-to-treat. a precis-2 grades on a scale from 1 (extremely explanatory) to 5 (extremely pragmatic). column 2 presents recommendations for which end of the spectrum is emphasized burden from severe refractory asthma, prescribing prolonged azithromycin therapy to patients with uncontrolled asthma may be considered by managing clinicians, particularly for patients who have failed to respond to conventional treatment and as an alternative to instituting immunomodulatory agents". interested clinicians and others wishing more information on patient experiences, scientific evidence and treatment alternatives are referred to a book on the subject [69] . evidence supports a complex interaction between host genetics/immune response and environmental factors (e.g., viral infections, microbiome) in the development, exacerbation and severity of asthma. emerging evidence from animal models and human studies points to chlamydia pneumoniae (cp) as a key player in this complex scenario. future research is required to unravel the quantitative 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services, national institutes of health, national heart, lung, and blood institute characterization of the severe asthma phenotype by the national heart, lung, and blood institute's severe asthma research program adherent uncontrolled adult persistent asthma: characteristics and asthma outcomes characteristics and outcomes of hedis-defined asthma patients with copd diagnostic coding unconventional treatment options in severe asthma: an overivew macrolides for the long-term management of asthma -a meta-analysis of randomized clinical trials macrolides for chronic asthma international ers/ats guidelines on definition, evaluation and treatment of severe asthma importance of evidence grading for guideline implementation: the example of asthma external validity of randomised controlled trials in asthma: to whom do the results of the trials apply? how representative are clinical study patients with asthma or copd for a larger "real life" population of patients with obstructive lung disease azithromycin for acute exacerbations of asthma: the azalea randomized clinical trial azalea trial highlights antibiotic overuse in acute asthma attacks an unanticipated effect of clinical trial registration chlamydia pneumoniae infection is not associated with unselected cases of acute asthma, but may be associated with chronic type 1 "brittle" asthma secondary outcomes of a pilot randomized trial of azithromycin treatment for asthma azithromycin for the secondary prevention of coronary events azithomycin for the secondary prevention of coronary heart disease events. the wizard study: a randomized controlled trial azithromycin for prevention of exacerbations of copd azithromycin and the risk of cardiovascular death use of azithromycin and death from cardiovascular causes lack of association between azithromycin and death from cardiovascular causes antibiotic prevention of acute exacerbations of copd randomised, double-blind, placebo-controlled trial with azithromycin selects for anti-inflammatory microbial metabolites in the emphysematous lung the impact of azithromycin therapy on the airway microbiota in asthma chronic infection and severe asthma the precis-2 tool: designing trials that are fit for purpose the role of macrolides in asthma: current evidence and future directions measuring quality of life in asthma role of long term antibiotics in chronic respiratory diseases long-term macrolide treatment for chronic respiratory disease rofumilast or azithromycin to prevent copd exacerbations (reliance) the role of atypical infections and macrolide therapy in patients with asthma not applicable. not applicable. data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. authors' contributions ww initiated the writing of the manuscript and contributed to the sections highlighting basic research and animal models. dh contributed to sections dealing mainly with clinical analyses, treatment and clinical trials. however, both authors contributed to its multiple revisions and formatting. both authors read and approved the final manuscript. ww is an associate professor of microbiology at the university of massachusetts amherst and director of the premed/prehealth advising center. he was the first to show a direct link between cultivable c. pneumoniae in the lower airway and pediatric asthma severity. ww has carried out animal model studies confirming a link between early life airway infection with c. pneumoniae and reactive airway disease. the authors declare that they have no competing interests. figures 2 and 3 were reproduced by permission of the american board of family medicine. ethics approval and consent to participate not applicable.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-290783-ipoelk4h authors: crouch, c. f. title: vaccination against enteric rota and coronaviruses in cattle and pigs: enhancement of lactogenic immunity date: 1985-09-30 journal: vaccine doi: 10.1016/s0264-410x(85)90056-8 sha: doc_id: 290783 cord_uid: ipoelk4h passive immunity against enteric viral infections is dependent upon the continual presence in the gut lumen of a protective level of specific antibodies. this article examines methods currently used to enhance the titre and duration of specific antibody in the mammary secretions of cows and pigs with particular reference to rotavirus and coronavirus infections. in addition, some of the potential problems to be found in attempting to produce vaccines against these viral infections are outlined neonatal diarrhoea is a complex disease associated with a number of infectious agents occurring either singly or in combination ~-3. in domestic animals economic losses are suffered, as a result of mortality (ranging between 0 and 80%), and also veterinary costs and decreased productivity of the survivors. the viral agents most commonly associated with this syndrome are rotavirus and coronavirus, both of which have been found to be primary pathogens in calves 4,~ and piglets 6-8. these viruses are most frequently isolated during the period from birth to weaning, and animals of this age have been the most intensively studied because of the frequency and severity of these infections. animals of all ages are, however, susceptible, with subclinical infections apparently common in both adult cows and pigs 9,'°. in neonatal calves the incidence of rotavirus and coronavirus associated diarrhoea is similar varying between 15 and 76% 3'~'-~3. the situation in neonatal piglets is less clear, rotavirus infections are apparently common 6.t4-tt, w.hilst transmissible gastroenteritis virus (tgev), the prototype enteric coronavirus in swine, is an example of a seasonal cold-weather disease, probably related to both the thermal sensitivity of the virus ~ and the effect of cold-stress on converting subclinical to clinical infections ~8. the pathogenesis of enteric rotavirus and coronavirus diseases of swine and cattle are similar. in contrast to tgev, however, rotaviruses appear to be confined to the alimentary tract, predominantly the small intestine, although there is some evidence in both lambs and piglets for infection of the large intestinal 9,2°. the infections are characterized by diarrhoea and dehydration caused by the functional and anatomical loss of the absorbtive cells of the intestine. the principal site of virus replication has been shown to be the intestinal villus epithelium. the infected cells are lost from the tips of the villi and are replaced with immature crypt cells. generally, there is a dimunition in the number and size of the villi and a progressive replacement of the epithelium with squamous and cuboidal cells which lack a brush bordep -6.~.2°-26. such immature cells have been shown to possess reduced levels of disaccharidases =~.28. the loss of the absorptive cells of the intestine is assumed to result in the observed malabsorption syndrome. this is further exacerbated by the decreased ability to utilize dietary lactose, resulting in its accumulation in the large intestine, thereby preventing further absorption of water by exerting an osmotic effect. as a result of the severity of these enteric viral infections during the first few weeks of life, passively acquired antibody is the major source of protection. in calves and pigs there is no selective transfer of immunogiobulins from the maternal to the foetal circulation during the last third of the pregnancy. instead, during the period immediately following birth, maternal immunoglobulin is acquired from the colostrum ofthe dam z9'3°. absorption of colostral immunoglobulins by the intestinal epithelial cells is a non-selective process 3t-34 lasting 24-48 h 33,3s. factors present in colostrum may influence the absorption of immunoglobulins 36,37 or help prevent their proteolytic degredation 38. in addition to immunogiobulins, colostrum and milk have recently been shown to contain functional immunocompetent cells including macrophages and t and b lymphocytes 39'~°. in contrast to colostral absorption, highly specific mechanisms operate in the colostrum-forming m a m m a r y glands of cattle and pigs causing large amounts of igg (relative to iga and igm) to concentrate in the coiostrum ' ' q ' . lgg passively acquired by the neonate from colostrum persists in the serum for several weeks protecting against systemic infection. in tgev infection of pigs '5 and rotavirus infection of calves 'g circulating antibody has been found to be of little value. resistance to these infections appears to be mediated instead by local immunity at the epithelial surface of the intestine. in cattle the selective transfer of igg~ from serum to milk continues throughout lactation, although at a reduced level when compared with colostrum formation. the concentration of all three classes ofimmunoglobulin is significantly reduced (30 to 60-fold) in milk and in consequence igg~ remains the primary immunoglobulin in bovine milk4 ~. in contrasl in pigs the concentration of lgg~ decreases about 30-fold during the first week of lactation, whilst that of secretory lga declines only about three-fold, leaving it to become the predominant class of immunoglobulin in swine milld t,'8. most adult cattle are seropositive for both rotavirus 49,s° and coronavirus s~ antibodies. there is a dramatic decline in these colostral antibody titres during the transition to milld 6'49"s~-55, reflecting this reduction in concentration of immunoglobulins. this partially explains the high incidence of rotavirus and coronavirus infections in calves older than five days, as the titres of passively derived protective antibody decline. despite the presence of one or more common antigens it has been demonstrated that rotaviruses isolated from different species can differ antigenically from each other s6-59. more recently it has been shown that different serotypes exist within isolates obtained from single species. the existence of at least two different serotypes of porcine rotavirus 6° and at least three distinct bovine serotypes 6~ have been described. bridger et al have suggested the occurrence ofintermediate bovine rotavirus types 62, although more work is essential to clarify this situation. some recent isolates possessing the distinctive morphology of rotaviruses have been found to lack the common group antigen. to date, these atypical rotaviruses have been isolated from humans, birds, calves, lambs and pigs 63~8. in pigs, preliminary results using two previously characterized atypical isolates 69,7° have indicated that these are distinct and do not share a common group antigen g2,~'. these observations have been extended by snodgrass et al ga who suggest the occurrence of at least four distinct groups ofrotaviruses based on their group antigen. the significance of the serotypic differences observed between rotaviruses in vitro still needs to be fully assessed in vivo. orbiviruses (also members of the reoviridae) possess many serotypes and require the use of multivalent vaccines 72. many of the cross-protection studies carried out using different rotavirus serotypes are contradictory and the data inconclusive. for example, in utero vaccination of calves with a bovine rotavirus was found to protect against diarrhoea caused by challenge with human rotavirus serotype 2, although challenge virus was still shed v3. in contrast, one out of three calves was protected against a bovine rotavirus challenge after vaccination with a human serotype 2 or an equine rotavirus tm and this animal shed no detectable virus. furthermore, piglets vaccinated with human rotavirus and challenged with porcine rotavirus were protected against the clinical disease but enhancement of lactogenic immunity:. c f. crouch shed virus ~s. using a more defined challenge system. evidence has been obtained indicating that rotavirus isolates from different animal species and of different serotypes show poor cross-protective properties in vivo tm. this observation has been confirmed and extended by studies in gnotobiotic calves and piglets showing that cross-protection only occurred between rotaviruses of the same serotype, and that even a minor serotype difference could be sufficient to affect cross-protection 6°,g~. further evidence for a lack of cross-protection between rotavirus serotypes can be obtained from studies of sequential infections, where subsequent rotavirus infections were found to be associated with different serotypes ~7. the situation with coronaviruses is simpler. to date, the coronaviruses isolated from mammals and birds have been grouped into four antigenic classes, where little or no cross-reactivity can be demonstrated between classes tm. tgev is antigenically distinct from bovine enteric coronavirus tm as well as from another as yet unclassified coronavirus causing diarrhoea in pigs (cv777) 8°. two approaches have been used in an attempt to provide calves with protection against rotavirus and coronavirus infections. the first approach involves oral vaccination with live attenuated virus in order to stimulate active immunity in the calf(scourvax ii, norden laboratories). the incidence of diarrhoea in neonatal calves orally vaccinated with attenuated rotavirus was found to be reduced 8~-83, but the vaccine was not proven to be effective in blind field trials s4-86. there are a number of limitations associated with this approach. these include the potential of the vaccine to regain virulence: a high incidence of seropositive adult animals, leading to the possibility of interference with vaccine virus replication by maternally derived (milk) antibodies: and the relative immaturity of the neonate's immune system, the second approach utilizes passive protection produced through lactogenic immunity, stimulated by maternal vaccination. attempts to vaccinate dams using an attenuated live vaccine (calf guard, norden laboratories) have failed to significantly enhance milk antibody titres ",8~ (table 1) , whilst inactivated, adjuvanted rotavirus preparations have been found to enhance levels of specific antibody in coiostrum and milk (tables 1 and 2) . a number of parameters need to be considered in attempting to optimize the enhancement of antibody production in mammary secretions. dose and form of vaccine. in considering inactivated vaccines, it ~s to be expected that relatively large amounts are necessary to achieve a satisfactory response. further. the process of inactivation may decrease the immunogenicity of some viral polypeptides. table 2 shows that no significant differences in milk antibody titres were obtained following vaccination of cows with rotavirus preparations containing either 200 or 800 elisa units (after inactivation) emulsified in freund's incomplete adjuvant. in contrast, if the same preparations were used. but adjuvanted with aluminium phosphate, the higher dose resulted in a greater antibody response. a similar result using an oil adjuvanted rotavirus vaccine has been previously reported ss. formaldehyde inactivated rotavirus vaccines have been used to successfully enhance milk antibody titres as compared with controls s8"-9°. other workers have reported increased antibody responses using/~-propriolactone as the inactivating agentol although saif et al found that antibody titres in mammary secretions were at least tenfold greater from cows vaccinated with binary ethylenimine inactivated rotavirus compared with those vaccinated with ,8-propriolactone inactivated rotavirus 53. adjuvant snodgrass et a188 found that oil-based adjuvants were more effective than alhydrogel for the enhancement of rotavirus antibody titres in mammary secretions. this concurs with the data presented in table1. most workers have demonstrated a satisfactory immune response following vaccination using oil-based adjuvants. generally freunds incomplete adjuvant (table2). route and timing of vaccination. to some extent the route and timing of vaccination are dependent upon the type ofcattle being farmed. thus the intramammary route used successfully by saifetal 53, whilst applicable to dairy cattle, may not be practical in beef cows. similarly. from an administrative viewpoint a single vaccination would be preferable to a regime utilizing several doses. the majority of studies have reported a significant increase in rotavirus antibody titres in mammary secretions using either subcutaneous or intramuscular injection of oiladjuvanted vaccines. all such vaccines have also proved to be effective when administered as either single or double doses injected prior to or at parturition 53.88-92 ( table 1) . the efficacy of immune milk as a mechanism for providing passive immunity against rotavirus challenge has been examined by a number of workers(table3). the alactogenic antibody originated from either vaccinated (vacc), control (cont) or normal cows (normal). bcalves were either suckled naturally (suckled) or fed a supplemented diet containing antibody (supp). ccalves were either challenged experimentally (exp) or naturally exposed under field conditions (field). d days after challenge edays after birth, r days after start of experiment. ~cows vaccinated with commercial vaccine nr, not reported results, however, are difficult to compare, due to variations in the feeding regime used for the immune milk and also the challenge systems used. the amount and the timing of the feeding oflactogenic antibody and the dose, virulence and serotype of the virus challenge strain used will all affect the apparent susceptibility of the calf to infection. further, in situations where a field challenge has been used. failure of protection may be due to infection by rotavirus serotypes other than those used in the vaccine, or possibly by other agents capable of causing diarrhoea. generally, these investigators reported either a reduced incidence of rotavirus shedding or diarrhoea or both. in only one study 92 did the feeding oflactogenic antibody fail to significantly affect the incidence or onset of diarrhoea. the majority of animals receiving passive immunity appear to be capable of developing active immunity during this period 93"95, consequently vaccination should lead to elimination of clinical disease rather than a delay in its onset investigation of the immunoglobulin isotypes associated with this protective antibody induced by vaccination in bovine milk and colostrum suggests that igg, plays the major role 95,96. these observations are in agreement with those discussed earlier concerning passive immunity in the bovine. in contrast to the bovine system, evidence suggests that milk or colostral immunoglobulin of the iga isotype is more effective than those of the igg isotypes at protecting piglets against infection by tgev 97-~°°. high persisting levels of lgg may, however, provide some degree of vaccine, vol. 3, s e p t e m b e r 1 9 8 5 2 8 7 enhancement of lactogenic immunity:. c e crouch protection against virus challeng~ ~. as a result of these observations` most studies have examined methods for optimising the stimulation of secretory iga antibodies in milk. the origin of tgev-specific iga found in mammary secretions remains somewhat obscure, although there is a good correlation with the presence of an infection in the intestinal tract 9~,99,1°°,'01. secretory iga in porcine milk is almost certainly locally produced in the mammary glan& °=-~°4. in order to explain this phenomenon, it has been suggested that specificallysensitized iga-secreting lymphocytes may migrate to the mammary gland following initial sensitization in the intestine s~-~°°. such an inter-relationship between the intestinal and the mammary immune systems has also been proposed in rabbits =°5 and humans ~°6. direct evidence for such migration, under the influence of pregnancy-associated hormones, has been obtained in micd °7. a summary of various investigations into the antibody response and efficacy of lactogenic immunity following different vaccination protocols is given in table 4 . reduced immunogenicity in pigs of cell culture attenuated tgev has been described ~°8. oral vaccination with a live, attenuated tge vaccine, whilst producing neutralizing antibody, did not stimulate good lactogenic immunity in suckling pigs ~°°,~°9.~'°. intramuscular vaccination of sows with live, attenuated tgev leads to the enhancement of specific igg levels in colostrum and milld~,"l higher titres oftgev-specific igg have been achieved using intramammary injection, with an associated increase in the protection provided to suckling pigs 9~. these results are supported by the observations of other workers "=-"5. feline infectious peritonitis virus (fipv) is a member of the same antigenic class as tgev and consequently the two viruses are serologically related. good levels of cross-protection, associated with high titres of tgev-specific neutralizing antibody have been reported in sows vaccinated orally with fipw ~. in contrast, the results of a more recent study have shown that whilst tgev neutralizing antibodies of the lgg subclass are stimulated in milk and colostrum, the survival rate for suckling pigs was low i". it may be possible to boost the level oflga in mammary secretions. preliminary investigations have revealed that specific secretory lga levels in milk can be enhanced by the parenteral injection, at parturition, of tgev or rotavirus into naturally infected (orally primed) animals "s."9. a similar approach also combining oral with parenteral antigen administration has been proposed as a means of providing lactogenic immunity against colibacillosis in pigs t=°. lgg can be induced readily in the mammary secretions of cattle, by intramuscular or subcutaneous injection of adjuvanted immunogen. in pigs however, whilst live, virulent virus is capable of inducing high levels in iga iri milk. it is apparent that the ideal candidate vaccine virus must be sufficiently attenuated to produce only mild or no disease in neonatal pigs, whilst retaining sufficient virulence to infect the intestinal tract of adult swine. more work is essential in the possible use of inactivated vaccines for the boosting of existing iga levels in mammary secretions. these may require prior natural infection of the sow, the incidence of which will vary between herds, with an associated affect upon the efficacy of such a vaccine. further investigation into the variety of strains and serotypes of rotaviruses is of obvious importance, as is the response to vaccination of cattle and swine by rotaviruses or coronaviruses. current data suggests that crossprotection between rotavirus serotypes is limited, although there is little information concerning the specificities of the antibodies induced by vaccination of previously infected animals. such animals naturally exposed to a variety of serotypes may produce a heterogeneous antibody response, capable of reacting with a broad spectrum of rotavirus serotypes. it is apparent that the enhancement of lactogenic immunity through the vaccination of the dam provides a suitable mechanism by which neonatal pigs and calves can be protected against rotavirus and coronavirus infections. the production of truly effective vaccines, however, awaits further work in some of the areas outlined above. acute undifferentiated neonatal diarrhoea in beef calves 1. occurrence and distribution of infectious agents a-l pathogenic relationships of rotavirus, escherichia colz and other agents in mixed infections in calves pathological and microbiological observations made on spontaneous cases of acute neonatal calf diarrhoea pathology of neonatal calf diarrhoea induced by a reo-like virus pathology of neonatal calf diarrhoea induced by a corona-like agent vet_ path the isolation of 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and lacteal secretions vaccination of cows with a combined rotavirus/ enterotoxigenic escherichia coil k99 vaccine to protect newborn calves against diarrhoea immune responses of pregnant cows to bovine rotavirus =mmunization passive immunity in calf rotavirus infections. maternal vaccination increases and prolongs immunoglobulin g 1 antibody secretion in milk comparison of different antigen preparat=ons as substrates for use in passive haemagglutin~tion and enzyme-linked immunosorbent assays for the detection of antibody against bovine enteric coronavirus` j. c/in. microbiol relation between viruses from acute gastroenteritis of children and newborn calves antigenic relat=onship between human and simian rotaviruses serological relationships between rotaviruses from different species as studied by complement fixation and neutralization morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice, and foals ljo 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gastroenteritis transfer of immunoglobulins igg, iga and igm to lacteal secretions in the parturient sow and their absorption by the neonatal piglet secretory iga and antibodies to escherichia coli in porcine colostrum and milk and their significance in the alimentary tract of the young pig the transfer of immunoglobulins igg, iga and igm from serum to colostrum and milk in the sow the induction and characteristics of secretory iga antibodies lidinjanson, (3. and sohi-akerlund, a. antibodyforming cells in human colostrum after oral immunization e hormonal induction of the secretory immune system in the mammary gland immunogenicity and distribution of transmissible gastroenteritis in pigs transmissible gastroenteritis in: diseases of swine immunity in tge of swine after infection and vaccination passive immunity in transmissible gastroenteritis of swine: intramuscular injection of pregnant swine with a modified live-virus vaccine experimental immun,zation of sows against transmissible gastroenteritis experimental immunization of sows with cell-cultured tge virus experimental immumzation of sows with inactivated transmissible gastro'enteritis (tge) virus. car~ j present status of products for use against transmissible gastroenteritis cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses efficacy of vaccination of sows with serologically related coronaviruses for control of transmissible gastroenteritis in nursing pigs passive immunity against enteric viral infections passiveimmunityagainstentericviral infections of piglets intestinal defence of the neonatal pig:-inter-relationships of gut and mammary function providing surface immunity against colibacillosis i would like to thank dr s.d. acres for his permission to include some ofthe data obtained during my employment at vido. key: cord-299918-0ahvoak4 authors: aykac, kubra; karadag‐oncel, eda; tanır basaranoglu, sevgen; alp, alpaslan; cengiz, ali bulent; ceyhan, mehmet; kara, ates title: respiratory viral infections in infants with possible sepsis date: 2018-09-24 journal: j med virol doi: 10.1002/jmv.25309 sha: doc_id: 299918 cord_uid: 0ahvoak4 background: knowledge of infections leading to sepsis is needed to develop comprehensive infection prevention and sepsis, as well as early recognition and treatment strategies.the aim of this study was to investigate the etiology of sepsis and evaluate the proportion of respiratory viral pathogens in infants under two years of age with possible sepsis. methods: the prospective study was performed in two years. multiplex reverse transcriptase polymerase chain reaction (rt‐pcr) was performed to detect viral pathogens. all patients who were included in this study had sepsis symptoms as defined by the surviving sepsis campaign. results: we compared 90 patients with sepsis into three groups as patients (n = 33) who had only viral positivity in nasopharyngeal swab, patients (17) had proven bacterial infection with or without viral infection, and patients (40) without the pathogen detection. human rhinovirus (16.7%) and influenza (7.8%) were the most commonly seen viruses. a cough was more common in the viral infection group than other groups ( p = 0.02) and median thrombocyte count was lower in the bacterial infection group than the others ( p = 0.01). patients having bacterial sepsis had the longest duration of hospitalization than the other groups ( p = 0.04). during winter and spring seaons, patients with sepsis had more viral infection; however, in summer and autumn period, patients were mostly in a state that we could not prove infection agents ( p = 0.02). conclusions: our results suggest that respiratory tract viruses may play an important role in patients with sepsis and they should be kept in mind, especially during winter and spring seasons. in overall infection, viral respiratory viruses as a single pathogen with a detection rate of 36.6% in sepsis etiology. sepsis results from a wide spectrum of infectious agents and is a very common cause of mortality and morbidity worldwide. it leads to 7.5 million annual deaths in children under 5 years of age. 1, 2 previously, in 2012, sepsis was defined as systemic inflammatory response syndrome (sirs) in the presence of or as a result of suspected or proven infection by the surviving sepsis campaign. infection could be bacterial, viral, or fungal. culture and nonculture-based diagnostic methods might be helpful for identification of these pathogens. 3 various viruses could cause sepsis, which are influenced by age and underlying immune status. [4] [5] [6] [7] respiratory viral infections (rvis) among infants can cause morbidity and mortality. 8 limited data exist on their proportion in sepsis. numerous cases of clinical deterioration sepsis remain without bacterial evidence. thus, viruses may also be considered as causative pathogens. [4] [5] [6] respiratory viruses are ubiquitous in the surroundings of the children and are the most common source of respiratory infection among healthy and immunocompromised individuals. several respiratory viruses may also cause severe respiratory disease mainly in infants but also in children. these viruses are well known as influenza viruses, respiratory syncytial virus (rsv), coronavirus (cov), human metapneumovirus (hmpv), parainfluenza viruses type 1 to 3 (piv 1-3), adenovirus (av), enteroviruses (ev), human rhinovirus (hrv), and human bocavirus (hbov). 9, 10 despite their clinical expansiveness, rvis are usually not considered to be of clinical significance among septic patients. influenza is one of the most common causes of viral sepsis in children with the highest rate of hospitalizations and the number of death. 11 the management of pediatric sepsis should be arranged according to the age, the immune capacity of child and severity, site, and the source of the infection. 1 currently, there are few studies about the etiology of nosocomial or community-acquired sepsis in children. 12, 13 the aim of the current study was to evaluate the proportion of respiratory viral pathogens in infants under two years of age with possible sepsis. statistical analyses were performed using ibm spss for windows version 20.0 (chicago, illinois). descriptive statistics were used to summarize the participants' baseline characteristics, including medians, minimum-maximum for continuous variables and numbers, and total percentages for categorical variables. the kruskal-wallis test was used to compare the baseline characteristics of categorical variables (age, duration of hospital day, and laboratory findings in tables). comparisons between groups for categorical variables (season, gender, and symptoms and clinical findings in tables) were made using the chi-square (χ 2 ) test. statistical significance was defined as p values less than 0.05. since we could not predict the patient count for the study at the beginning, we did a power analysis at the end of the study. when assessed for the presence of factors among seasons, the power was 84.5%. total of 90 infants with sepsis were enrolled in this study during the 24-months study period. nasopharyngeal swabs were collected from all the patients. we compared patients among three groups as patients (n = 33) who had only viral positivity in nasopharyngeal swab, patients (n = 17) who had proven bacterial infection (bacteremia and meningitis) with or without viral infection, and patients (n = 40) without pathogen detection (viral or bacterial). no patient had a urinary tract infection. the median age of the groups were 5 months (minimum-maximum; 1-23), 6 months (minimum-maximum; 1-20), and 3.5 months (minimum-maximum; 1-21), respectively. most patients were male in all three groups. no statistically significant differences were found between the groups in terms of gender (p = 0.19) and age (p = 0.36) ( table 1) . in viral infection group, 30.3% (n = 10) of patients had no underlying disease, the most frequent underlying disease was prematurity (24%, n = 8). in the bacterial infection group, gastrointestinal and immunodeficiency diseases were commonly seen at the same rate (23.5%, n = 4) and only %11.8 of patients had no underlying disease. in no proven infection agent group, neurological (25%, n = 10) and immunodeficiency (20%, n = 8) disease were most common underlying disease and only 12.5% of patients had no underlying disease. according to seasonal distribution, in winter and spring, patients with sepsis mostly had viral infection; however, in summer and autumn, patients were mostly in which we could not prove infection agents. the seasonal distribution was significantly different between the three groups (p = 0.02). empirical therapy was started to all patients and switched according to the test results and clinical status of patients. patients with bacterial sepsis had the longest duration of hospitalization (median; 28 days), although pediatric intensive care unit (picu) stay rates and infection-related mortality rates were not different between groups. overall mortality rate was 18.8% (n = 17) and the infection-related mortality rate was 12.2% (n = 11). four patients with rvis, five patients with no confirmed infection, and two patients with both bacteremia and rvi died. however, there was a statistically significant difference between groups in terms of duration of hospitalization (p = 0.04; table 1 ). symptoms and clinical findings of patients are presented in table 2 . a cough was the only symptom that had statistical significance between groups (p = 0.02), with the highest rate in the viral infection group (36.4%, p = 12). in laboratory findings of patients, median thrombocyte count was lower in the bacterial infection group than the others (p = 0.01). there was no difference in other parameters (white blood cell count, active prothrombin time, international normalized ratio, d-dimer, and c-reactive protein; table 3 in addition, we evaluated patients in two groups as has (40%, n = 36) and cas (60%, n = 54). no difference was found between two group in terms of age, sex distribution, season, duration of hospitalization, picu stay, infection-related mortality rate, and viral infection rate. however, 31.5% (n = 17) of patients in the cas group had no underlying disease, and in the has group all the patients had several underlying diseases such as prematurity (27.8%) and neurological diseases (19.4%). there was the statistically significant difference between groups with regard to bacterial bloodstream infection rate (p = 0.001). in the has group 33.3% (n = 12) patients had bacteremia, whereas in the cas group only 5.6% (n = 3) patients had bacterial infection. viral infection rates were equal in groups (table 4 ). twenty-seven (30%) patients received antibiotics before the sepsis diagnosis. we prospectively evaluated a group of infants for rvis from one month to two years with possible sepsis in our tertiary hospital. virus detection in pediatric departments is an important issue, which has been considered to contribute to successful antibiotic infections, mostly in winter, could be explained due to respiratory virus epidemics, which are often seen in winter-like influenza and photoperiodicity influence on leukocyte function and relative d vitamin deficiency in wintertime which has an important role in the modulation of both innate and cellular immune responses. [23] [24] [25] we found that rv infections were seen throughout the year, especially in autumn and spring when we evaluated only patients with rv infection. studies from our country indicated that rv infections were seen during all year. gülen et al found that rv infections were detected especially winter and spring and in another study rv infections seen more frequently in early spring and winter. 26, 27 the causes of sepsis are so important for the duration of hospitalization and the severity of the disease. in our study, patients had bacterial sepsis who had the longest duration of hospitalization (median; 28 days), although pediatric intensive care unit (picu) stay rates and infection-related mortality rates were not different between groups. there was the statistically significant difference between groups in terms of duration of hospitalization (p = 0.04). this could be related that patients with bacteremia had longer antibiotic treatment duration. in addition to this, patients with rvis had same infectious mortality rate and picu stay rate showed us that we should consider patients with sepsis in terms rvis due to the same rate of mortality and picu stay. miggins et al investigated critically ill patients in the usa and detected that viral infections may play a significant yet unrecognized role in overall outcomes of icu patients. 28 in our study, hrv and influenza were the most commonly seen viruses with the rate of 16.7% and 7.8%, respectively ( figure 1) . it was reported that influenza causes an increased risk of sepsis in the literature. 28 also, we know that hrv are associated with severe respiratory infections in patients with immunocompromised or chronic lung disease. 29 twenty-seven (30%) patients received antibiotics before the sepsis diagnosis. this could interfere with blood culture tests for bacteria and for this reason we had found, cdc recommends to obtain cultures before starting antimicrobial therapy. our study has also some limitations including the small sample size and one-center results because viral infection rates appear to vary substantially between different centers based upon limited prior reports in infants. another limitation is that detection of nucleic acid from the respiratory tracts of infants could represent false-positive results, prolonged shedding from a precede infection, or even colonization rather than acute infection. another limitation is that we did not obtain respiratory samples specifically at the beginning of clinical findings of sepsis in our outpatient population, we were able to collect the specimens upon admission to the emergency department. despite many of these limitations, this is the first infant population study that has documented rvis among patients with sepsis from our country. identification of the causative pathogen provides an opportunity to select the ideal treatment that improves survival rates, overall patient outcomes, reduces hospitalization duration, and overall hospital costs. 33 as well as further research should focus place of rvis in sepsis, whether viruses are a cause of sepsis or are they colonization in the nasopharynx. this could solve with a prospective study with serial quantitative pcr analyses in asymptomatic, septic, and ill but nonseptic children. we believe that our conclusion, which is having significant viral etiology in patients with sepsis is underdiagnosed by clinicians, is warranted. diagnosing rvis early may be of help for the clinical decision and treatment strategies. the authors have stated explicitly that there are no conflicts of interest in connection with this study. kubra aykac http://orcid.org/0000-0002-0974-4765 pediatric sepsis: important considerations for diagnosing and managing severe infections in infants, children, and adolescents pediatric sepsis in the developing world: challenges in defining sepsis and issues in post-discharge mortality surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock detection of respiratory viral infections in neonates treated for suspicion of nosocomial bacterial sepsis: a feasibility study clinical characteristics of children and adults hospitalized for influenza virus infection rhinovirus infection associated with serious illness among pediatric patients do viral infections mimic bacterial sepsis? 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seasonal variation in host susceptibility and cycles of certain infectious diseases time course and role of the pineal gland in photoperiod control of innate immune cell functions in male siberian hamsters seasonality of viral infections: mechanisms and unknowns prevalence and seasonal distribution of respiratory viruses during the 2014-2015 season in istanbul ten year retrospective evaluation of the seasonal distribution of agent viruses in childhood respiratory tract infections the potential influence of common viral infections diagnosed during hospitalization among critically ill patients in the united states human rhinoviruses and severe respiratory infections: is it possible to identify at-risk patients early? serious respiratory illness associated with rhinovirus infection in a pediatric population nosocomial viral respiratory infections pathogenesis of rhinovirus infection integrating rapid pathogen identification and antimicrobial stewardship significantly decreases hospital costs how to cite this article respiratory viral infections in infants with possible sepsis key: cord-312197-d5d8amk7 authors: edmond, karen; zaidi, anita title: new approaches to preventing, diagnosing, and treating neonatal sepsis date: 2010-03-09 journal: plos med doi: 10.1371/journal.pmed.1000213 sha: doc_id: 312197 cord_uid: d5d8amk7 karen edmond and anita zaidi highlight new approaches that could reduce the burden of neonatal sepsis worldwide. neonatal sepsis or septicaemia is a clinical syndrome characterized by systemic signs of circulatory compromise (e.g., poor peripheral perfusion, pallor, hypotonia, poor responsiveness) caused by invasion of the bloodstream by bacteria in the first month of life. in the pre-antibiotic era neonatal sepsis was usually fatal. case fatality rates in antibiotic treated infants now range between 5% and 60% with the highest rates reported from the lowestincome countries [1] . the world health organization (who) estimates that 1 million deaths per year (10% of all under-five mortality) are due to neonatal sepsis and that 42% of these deaths occur in the first week of life [2] . there are wide disparities in neonatal care between highand low-income countries. in high-income countries the major concern is the increasing numbers of extremely premature infants with high nosocomial infection rates due to multiresistant organisms in intensive care units. health facility infections are also a major problem in lowincome countries, but the more pressing issues are the high proportion of home deliveries in unclean environments predisposing to sepsis and ensuring that all neonates have access to effective interventions from health care providers in the first days of life 2 . indeed, new strategies that can prevent, diagnose, and treat neonates with sepsis are needed in both low-and high-income settings. distal risk factors for neonatal sepsis include poverty and poor environmental conditions. proximate factors include prolonged rupture of membranes, preterm labour, maternal pyrexia, unhygienic intrapartum and postnatal care, low birth weight, and prelacteal feeding of contaminated foods and fluids [3] [4] [5] . the bacteria that cause neonatal sepsis are acquired shortly before, during, and after delivery (figure 1 ). they can be obtained directly from mother's blood, skin, or vaginal tract before or during delivery or from the environment during and after delivery. streptococcus agalactiae (group b streptococcus, gbs) is the most common cause of neonatal sepsis in many countries, though low rates are reported from many low-income countries, especially those in south asia. [6] [7] [8] ; gramnegative bacilli (escherichia coli, klebsiella spp., pseudomonas spp., acinetobacter spp.) and gram-positive cocci (such as staphylococcus aureus and staphylococcus epidermidis) are other important causes [6] [7] [8] . however, there are many difficulties in interpreting aetiological neonatal sepsis data, because many studies report selected populations of high-risk infants. specimens from infants in the first 24 hours of life are also seriously under-represented, especially those from low birth-weight babies and babies born outside health facilities [6, [9] [10] [11] . intrapartum antibiotic prophylaxis against s. agalactiae has also led to a substantial change in the bacteria responsible for early onset neonatal sepsis; gramnegative bacilli and staphylococcus spp. predominate in countries implementing these programs [12] . there are also many other important neonatal infectious disease pathogens that are not associated with the sepsis syndrome including: treponema pallidum, rubella virus, herpes simplex virus, cytomegalovirus, toxoplasmosis, clostridium tetani, hiv, hepatitis b virus, and bordetella pertussis ( figure 1 ) [1, 7, 13] . these infectious pathogens cause serious morbidities in young infants and multifaceted disease syndromes including congenital anomalies, developmental disabilities, chronic liver disease, neonatal tetanus, and apnoea. they are also important causes of morbidity and mortality in older age groups. however, only pathogens that cause neonatal sepsis are discussed in this paper. neonates have a functionally immature immune system. they have extremely low immunoglobulin (ig) levels except for igg to specific maternal antigens transferred passively across the placenta during the last trimester of pregnancy [14, 15] . t cell function is relatively unimpaired but complement activity is half that of healthy adults. neonates have a low neutrophil storage pool, and their existing neutrophils have impaired capacity to migrate from the blood to sites of infection [16] . the basal expression of toll-like receptors (tlrs, receptors that detect the presence of microbes) is similar in the neonate and adult [17] . however, innate immune responses of neonatal mononuclear cells are characterised by markedly reduced release of the proinflammatory th1-polarizing cytokines tumour necrosis factor-alpha (tnf-a) and interferon-gamma (ifn-c) with relative preservation of anti-inflammatory th2-polarizing cytokines such as interleukin 6 (il6) [18] . these findings may reflect in utero requirements, including the avoidance of harmful inflammatory immune reactions [19] . these immunological problems are reflected in the clinical presentation of neonatal sepsis. neonates have a rapid and fulminant progression of septicaemic disease, nonspecific clinical signs of infection, and difficult-to-interpret laboratory results including haematological and immunological biomarkers of infection and inflammation. low birth-weight (preterm and small for gestational age) infants have even poorer functional immunity, and are especially at risk of sepsis [19] . however, neonates do have well-functioning cationic membrane-active antimicrobial proteins and peptides (apps) which have microbicidal properties [15, 19] . these apps can be found in the vernix caseosa covering the skin at birth, and in the neonatal gastrointestinal and respiratory tracts. many older studies have demonstrated that improving maternal health and nutri-tion before delivery is directly associated with improved neonatal health outcomes [3] . randomised controlled trials (rcts) of maternal protein-calorie and multiple micronutrient and supplementation have demonstrated significant improvements in rates of prematurity and birth weight and variable impact on mortality; but no studies have examined their impact on rates of neonatal sepsis [20, 21] . maternal immunisation is an important method of providing neonates with appropriate antibodies as soon as they are born [22] . this approach is less sensitive to obstacles in accessing the health care system than are other approaches, and examples of successful interventions include maternal tetanus toxoid and influenza immunisations [23, 24] . studies of maternal immunisation with s. agalactiae type iii conjugate vaccine have demonstrated excellent placental transfer and persistence of protective levels in 2month-old infants [22] . phase i and ii trials of other serotypes in nonpregnant women have also demonstrated safety and immunogenicity. a recent modelling study estimated that vaccination with s. agalactiae vaccine would prevent 4% of us preterm births and 60%-70% of neonatal s. agalactiae infections [25] . encouraging results are also emerging from studies of maternal immunisation with pneumococcal polysaccharide and conjugate vaccines [22, 26] . the vaccines all have excellent safety profiles. however, barriers to maternal immunisation include: liability issues for vaccine manufacturers in developed countries; education of the public and health care providers regarding the benefits of maternal immunisation; and poor ascertainment of data from lowincome countries [22] . there is strong evidence that clean delivery practices and handwashing during delivery reduces rates of neonatal sepsis in both home and health facility settings [27] [28] [29] . interventions to improve handwashing rates have been remarkably successful in research settings [30, 31] . the reasons for lack of successful scaleup of handwashing interventions into policy, programs, and behaviour change are less clear [32] . new studies from malawi and nepal indicate that maternal antisepsis interventions such as vaginal chlorhexidine during labour may have a significant impact on rates of neonatal mortality and sepsis in developing countries [33] . however, other studies from high-income countries have demonstrated little effect on rates of hiv or neonatal infections [34] . intrapartum antibiotic prophylaxis has been highly effective in reducing both early-onset neonatal bacterial and maternal sepsis in developed countries [35] . chemoprophylaxis in the us has halved the incidence of early-onset neonatal bacterial sepsis caused by s. agalactiae from 1.7 per 1,000 live births in 1993 to 0.6 per 1,000 in 1998 [36] . clear protocols are in place in high-income countries for the management of women with risk factors for neonatal sepsis [37] . risk factors for early-onset neonatal bacterial sepsis in low-income settings are probably similar to resource-rich settings, but have not been evaluated in the context of high rates of maternal undernutrition, anaemia, hiv, and malaria. there is also strong evidence that handwashing by health care providers after delivery can reduce neonatal sepsis and infection rates, especially in hospitals [27, 28] . there is less evidence for the importance of rigorous handwashing and use of antiseptics in mothers of their own infants. five key papers on preventing, diagnosing, and treating neonatal sepsis in high-income settings, studies have not shown an advantage of antibiotics or antiseptics over simply keeping the umbilical cord clean [2] . however, umbilical stump chlorhexidine cleansing has recently been shown to substantially reduce neonatal deaths in nepal [38] . other studies investigating the effects of chlorhexidine on prevention of omphalitis are currently underway in several countries [39] . there is emerging evidence that neonatal skin antisepsis preparations such as sunflower seed oil provides cheap, safe, and effective protection against nosocomial infections in hospitalized preterm neonates and infants in studies in south asia. application of chlorhexidine to neonatal skin has also been shown to be effective in reducing neonatal sepsis in studies from south asia [39, 40] . neonatal immunisation has long been considered an important method of reducing neonatal infections. however, response varies according to the antigen [15] . bcg, polio, and hepatitis b vaccines are highly immunogenic when given at birth [41] . however, maternal antibodies interfere with a neonate's response to measles vaccine when administered under six months. protein antigen vaccines (e.g., pertussis and tetanus toxoid) given at birth have been shown to produce poor responses compared to the same antigen given at two months of age and are associated with later tolerance [41] . studies also indicate that s. agalactiae and streptococcus pneumoniae vaccines are both likely to be ineffective when given in the neonatal period [15] . breastmilk contains secretory iga, lysozymes, white blood cells, and lactoferrin and has been shown to encourage the growth of healthy lactobacilli and reduce the growth of e. coli and other gramnegative pathogenic bacteria [15] . rcts that focused on increasing early initiation and exclusive breastfeeding rates demonstrated significant reductions in diarrhoea and acute respiratory infections in neonates and older infants in india [42] . other observational studies have demonstrated impact on infection specific mortality rates and all-cause mortality during the neonatal period [43] [44] [45] . neonatal micronutrient supplementation trials have focused on vitamin a supplementation. older studies have shown significant reductions in respiratory disease in low birth-weight infants after the administration of parenteral vitamin a [46] . more recently, trials of newborn vitamin a supplementation have shown encouraging reductions in neonatal mortality, and more trials are underway [47] . in high-income countries, clinical trials of immune stimulants such as granulocyte/ monocyte colony stimulating factor (gm-csf) to enhance the quantity and quality of neonatal neutrophils and monocytes appear promising but have not yet shown a significant clinical benefit [15] . the evaluation of recombinant apps as adjunctive therapy for neonatal infection are still under evaluation. the impact of tlr agonists to improve defences against microorganisms are also being evaluated [15] . neonatal clinical sepsis syndrome identification is difficult as the clinical signs of neonatal septicaemia can be very similar to those of other life-threatening diseases such as necrotising enterocolitis, hyaline membrane disease, and perinatal asphyxia [48, 49] . however, recent studies in middle-and low-income countries have provided seven danger signs which can be used to identify infants with very severe disease including neonatal sepsis (table 1 ) [49] . these signs provide high sensitivity and moderate specificity for detecting serious illness in newborns in low-resource settings and have now been incorporated into the new neonatal who integrated management of childhood illness (n-imci) guidelines. identification of neonatal sepsis before delivery also remains challenging. a combination of maternal risk factors and clinical signs and symptoms is currently used [50] . however, peripartum proteomic analysis of the amniotic fluid is now offering the opportunity for early and accurate diagnosis of early-onset neonatal sepsis in the select population of women undergoing amniocentesis in high-risk pregnancies [51, 52] . confirmation of pathogenic organisms allows targeted antibiotic therapy. however, identification of pathogenic organisms in neonates with sepsis syndrome is fraught with difficulties. bacterial load may be low due to mothers receiving antepartum or intrapartum antibiotics and because only small amounts of blood can often be taken from newborns [53] . contamination rates may also be very high due to the technical difficulties of sterile venipuncture in small babies. there may also be misinterpretation of the role of coagulase-negative staphylococci (e.g., s. epidermidis), as these organisms are both normal skin flora and pathogenic organisms in preterms and infants with indwelling blood vessel catheters [54] . automated blood culture systems have long been considered the gold standard for microbiological diagnosis. however, despite improvements in growth media and instrumentation, results of blood culture can be delayed by up to 48 hours [53, 55] . the condition of a neonate with true sepsis can deteriorate quickly, thus the most common approach is to initiate empiric broad-spectrum antibiotic therapy in all young infants with suspected bacterial infection [49] . a negative blood culture after 48 hours may allow cessation of antibiotic therapy in a well infant. while appropriately cautious, this practice leads to antibiotic exposure in a large number of newborns for whom antibiotic treatment may be unnecessary since blood cultures are positive in only 5%-10% of suspected sepsis cases, even at highly resourced facilities [56] . antigen detection techniques allow rapid detection and identification of microorganisms without culturing. the most commonly used commercially available test is the latex agglutination assay, which is based on specific agglutination by bacterial cell wall antigens of antibodycoated latex particles. however, these tests can only detect specific organisms such as s. agalactiae and are associated with high false positive and negative rates [57] . new urinary antigen tests for pneumococcus are more encouraging but are also associated with false positives from pneumococcal carriage [58] . the polymerase chain reaction (pcr) has been widely used in biomedical research laboratories for pathogen identification in neonatal sepsis and in some clinical hospital laboratories. the high sensitivity of pcr allows detection of bacterial dna even when concentrations are low [57] . conventional assays are being replaced by a newer ''real-time'' system, which is faster and associated with lower contamination rates because amplification and detection occur simultaneously in a closed system [59] . the real-time pcr is based on the measurement of a fluorescent signal generated during each amplification cycle. it produces quantitative results within 30 minutes and calculates bacterial load. broad-range real-time pcr uses a single primer to detect the universal bacterial genome (16s rna or 23s rna) which is a conserved ribosomal genome sequence across all bacterial genera [60] . broadrange real-time pcr can be used to distinguish bacterial septicaemic disease from other causes of neonatal illness such as asphyxia or complications of prematurity. however, it has been used with varying success in the analysis of whole blood for neonatal sepsis; specificity is generally high but sensitivity can be as low as 40% [60, 61] . in contrast, multiplex pcr involves the parallel amplification of different targets but is focused only on specific pathogens, and false negatives can occur if the aetiologic agent of interest is not included in the database [62] . real-time pcr is now often used to screen for microbial load, followed by sequence-based targeting and identification of pcr amplicons (pyrosequencing) [62] . this process can detect very small copy numbers of specific nucleic acid sequences. there is also a new commercially available multiplex pyrosequencing pcr assay which can identify up to 40 different bacterial and fungal pathogens directly from whole blood [63] . realtime pcr and pyrosequencing of the universal 23s rrna gene has also recently been used successfully in neonatal blood culture samples [64] . further tests on neonatal whole blood have been planned by a number of different research groups. the biggest problem with real time pcr testing is that the specimen must be collected with a sterile venipuncture, which may be difficult in young neonates. neonatal capillary heel prick specimens are easier to collect but highly contaminated by skin flora. there is also high potential for contamination of enrichment media, reagents, or the sample during collection and processing [61] other problems include low sensitivity due to competition from human dna in whole blood, especially if white cell counts are high. also, bacterial organisms require lysis before their dna can be available for analysis, and gram-positive organisms are difficult to lyse because of their resilient cell wall [61] . real-time pcr technologies are also expensive and currently can be used only by highly trained staff. important haematological tests include microscopic examination of the blood for white cells (total leucocyte count, differential, neutrophil count, and immature neutrophil to total neutrophil ratio). advantages are that these specimens do not require sterility and a heel prick specimen can be used. however many of these indices are falsely low in a septic neonate. biological biomarkers are human blood components that increase in response to infection. the most commonly used acute phase reactant is the c-reactive protein (crp). however, the crp takes 12-24 hours to increase to measurable levels; its half life is very long and it takes 5-7 days to normalize after eradication of the infectious agent. cytokines such as il6, il8, tnf-a, and procalcitonin have also been extensively studied [65, 66] . cytokines rise quickly after infection even in neonates, and are more sensitive to low concentrations of pathogens than crp [66] . however, cord and postnatal blood cytokine concentrations can be depressed in the presence of pregnancy-induced hypertension and can rise after induced vaginal or urgent cesarean delivery, delivery room intubation, muscular damage, and inflammation from other causes [57] . simultaneous measurement of multiple biomarkers may improve both sensitivity and specificity [66, 67] . however, biomarker assays are likely to be less acceptable to physicians who often place higher value on tests that confirm biological agents and allow targeting of antibiotic therapy [57] . microtechnologies, especially microfluidics, have provided the greatest recent contribution to the diagnosis of neonatal sepsis. microfluidics is the study of the behaviour, precise control, and manipulation of fluids geometrically constrained to submillimetre (nanolitre or picolitre) channels [68] . microfluidic technology uses the unique proprieties of continuous flow micro-volume channels: viscosity, surface tension, energy dissipation, and fluidic resistance, and also includes micro pneumatic pump and valve systems. one specific application of microfluidics is bacterial dna protein microarray hybridization [69] . in this test, dna probes specific to selected targets are spotted on a glass or silicon slide in a known order. target dna fragments are labelled with a reporter molecule, combined into a single hybrid, and measured using fluorescent signals [62, 68] . this technique has been used in the identification of the specific sepsis pathogen in bacterial meningitis, acute viral respiratory tract infections, and neonatal sepsis, and also in the detection of their antimicrobial resistance and virulence genes in research settings [63] . microfluidic technology has also allowed sample preparation and a number of different assays to be combined in small, disposable, single-use diagnostic cartridges or cards that have been called a ''lab on-achip'' or loc (figure 2 ) [68] . some locs have combined sample preparation, biomarkers, real-time pcr, and dna microarrays to provide information about indices of inflammation, pathogen identification, and antimicrobial susceptibility patterns at the point of care [68, 70] . locs have been reported to perform assays at sensitivity, specificity, and reproducibility levels similar to those of central laboratory analysers, but yet require little user input other than the insertion of the sample. single drops of blood, faeces, and saliva have all been tested with encouraging results. locs are currently being evaluated for use in sepsis, endocarditis, hiv, tuberculosis, severe acute respiratory syndrome (sars), and pneumonia [68] . however, they are not yet in clinical use nor licensed by regulatory authorities. as neonatal sepsis can be rapidly fatal if left untreated, highly effective antibiotic therapy must be used and delays in the provision of care must be minimised. treatment must be effective against the causative pathogen, safe for the newborn, and feasible to deliver reliably in the hospital or community setting. parenteral (intravenous or intramuscular) regimens for neonatal sepsis currently recommended by national paediatric associations are a combination of penicillin/ ampicillin and gentamicin, or third-generation cephalosporins (e.g., ceftriaxone or cefotaxime) for 10-14 days. these antibiotics are safe and retain efficacy when administered at extended intervals (e.g., twice daily or daily dosing) [56] . these regimens are very effective against streptococcus spp., but staphylococcus spp. can be highly resistant [71] . gram-negative antimicrobial susceptibility to ampicillin and gentamicin can also be poor, especially for klebsiella spp. [8, 71] . emerging e. coli resistance to ampicillin, gentamicin, and third-generation cephalosporins in hospital nurseries in both developed and developing countries is also causing increasing concern [8] . the potential for significant life-threatening toxicity among neonates associated with chloramphenicol makes it the least preferred empiric parenteral therapy [56] . oral antibiotic therapy must be considered in settings where referral is not possible and there are no health care providers trained to give parenteral antibiotics [72] . the incremental benefit of injectable over oral antibiotics is not known, and oral antibiotic therapy is better than no antibiotic therapy at all. a series of trials are currently evaluating the impact of home and clinic-based short course (7 days) intramuscular and oral antibiotic therapy for neonatal sepsis in low-income countries [72] . most data are available on the effect of oral cotrimoxazole in community-based treatment of serious neonatal bacterial infections from nepal and india. however, there are concerns about high resistance rates, and side effects such as neonatal jaundice have been reported [71] . oral amoxicillin is highly efficacious against streptococcus spp. and some gram-negative bacilli and has an excellent safety record. however, it has no anti-staphylococcus coverage and resistance is emerging in gram-negative bacilli such as e. coli. new, better-absorbed oral antibiotics are also being considered. the new second-generation cephalosporins (e.g., cefadroxil and cefuroxime) have an excellent safety profile, a spectrum of activity similar to cotrimoxazole, and may be more effective given the high resistance of neonatal pathogens to cotrimoxazole. ciprofloxacin also is increasingly accepted as safe in neonates and warrants further investigation for treatment of infections in newborns. however, the current cost of these agents and potential for exacerbating antimicrobial resistance may limit widespread use in developing countries [72] . poor maternal-neonatal health systems, low levels of care-seeking, and lack of access to sick newborns during the first day of life, when mortality risks are highest, are also important concerns [73] . recent studies have shown that community health workers can deliver antibiotic treatment to neonates with very severe infections at home safely and acceptably when hospitalization is not feasible [74] . trials are currently evaluating the effectiveness, quality of care, and coverage of these community health worker programmes in asia and africa [73] . barriers to large-scale implementation include high cost, poor staff training and retention, and difficulties with referral (e.g., lack of ambulances and poor institutional links). newborn sepsis is a major cause of child mortality across the world. industrialized countries have made remarkable progress in reducing newborn sepsis and sepsisrelated mortality by providing access to hygienic skilled delivery for all women, risk-based intrapartum antibiotic prophylaxis, and high-quality intensive care for newborns that need it. although resource constraints preclude whole-scale adoption of these strategies in developing countries, there are a number of low-cost proven interventions and promising approaches that have the potential to significantly reduce the burden of neonatal sepsis worldwide (table 2) . however, practicability of implementing these new advances must be considered. skilled attendance at delivery is increasing in low-and middle-income countries. thus, intrapartum approaches such as risk-based antibiotic prophylaxis and improved hand washing during delivery are likely to be both cost-effective and feasible in these countries. more challenges face the implementation of diagnostic technologies. it may take many years for technologies such as the ''lab-on-a-chip'' to be sufficiently robust and affordable for scale-up to low-income countries. homebased antibiotic treatment of neonatal sepsis also faces major obstacles to largescale implementation. concerns such as ''one law for the rich and another for the poor'' have already been raised. a careful assessment of the risks and benefits of new technologies and interventions is clearly needed. in low-income settings there are also difficulties with care-seeking for neonatal illnesses, and home visiting programs are needed to identify sick newborns early in life. neonatal sepsis is also one of the most rapidly fulminating clinical diseases, and many practitioners, including experi-enced neonatologists, administer parenteral antibiotics rather than wait for the results of any diagnostic tests. these practitioners rightly consider that the individual patient's health is more important than the potential risks of emerging antibiotic resistance. thus, front-line health workers and families must be partners in all research and evaluation planning. detailed assessment of end-user attitudes and preferences using formative and 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markers of bacterial infections predictive value of soluble immunological mediators in neonatal infection microfluidic diagnostic technologies for global public health microfluidic dna amplification-a review enabling a microfluidic immunoassay for the developing world by integration of on-card dry reagent storage antimicrobial resistance among neonatal pathogens in developing countries oral antibiotics in the management of serious neonatal bacterial infections in developing country communities management of newborn infections in primary care settings: a review of the evidence and implications for policy? effect of home-based neonatal care and management of sepsis on neonatal mortality: field trial in rural india key: cord-317548-ft7lkpzq authors: proud, david title: upper airway viral infections date: 2007-07-05 journal: pulm pharmacol ther doi: 10.1016/j.pupt.2007.06.004 sha: doc_id: 317548 cord_uid: ft7lkpzq upper airway viral infections (uri) are a major cause of absence from school and work. although morbidity is low in most of the subjects, the complications of uri, including otitis media, sinusitis and exacerbations of asthma and chronic obstructive pulmonary disease (copd) have an enormous health impact. despite the major health care consequences associated with these complications, our understanding of how uri trigger upper airway symptoms and cause exacerbations of lower airway diseases remains limited. this article reviews our current understanding of the pathogenesis of uri, and of viral exacerbations of asthma and copd, and considers host defense parameters that may regulate susceptibility to disease exacerbations. we will also consider current and potential therapeutic approaches for the treatment of uri and their lower airway complications. their complications uri, manifesting as the clinical syndrome we refer to as the common cold, is the most frequent acute respiratory illness experienced by humans. adults will experience 2 to 4 colds each year, while children experience 6 to 10. as a result of this, according to the centers for disease control and prevention, 22 million school days are lost annually in the united states due to colds. although common colds can be caused by a variety of different virus types, including coronaviruses, parainfluenza virus and respiratory syncytial virus (rsv), the predominant viral pathogens, particularly in the autumn season, are human rhinoviruses (hrv) [1] [2] [3] . although simple colds in healthy individuals are associated with little morbidity, it has long been known that rhinovirus infections can precipitate or exacerbate other diseases, including otitis media [4] , and sinusitis [5, 6] . more recently, growing evidence also has implicated uri as the predominant risk factor associated with exacerbations of both asthma and chronic obstructive pulmonary disease (copd). in the case of asthma, there is a clear temporal relationship between increase in hospitalizations for asthma exacerbations and outbreaks of uri [7, 8] , with a major spike in early september, which is the peak time for hrv infections. moreover, prospective studies using rt-pcr to assist in viral detection have demonstrated that common respiratory viruses are associated with up to 60% of asthma exacerbations in adults and over 80% of exacerbations in children [9, 10] . although several viral types were found during these exacerbations, the dominant pathogen detected was hrv. hrv also was the most common viral pathogen associated with asthma attacks in young children over 2 years of age presenting in the emergency room [11, 12] . there also has been a growing appreciation regarding the important role of uri in triggering exacerbations of copd [13] . recent studies indicate that about half of all copd exacerbations are associated with viral infections, and that hrv is, again, the dominant viral pathogen [14, 15] . interestingly, respiratory viral infections are associated with copd exacerbations that are more frequent, severe and have longer recovery times [14] . the ability of uri to serve as precipitants for exacerbations of asthma and copd has enormous consequences in terms of both health care costs and patient's quality of life. the total health care costs for asthma in the united states for the year 2000 was estimated at $12.1 billion [16] , and acute exacerbations account for half of the total health care costs for the disease [17, 18] . similarly, acute exacerbations of copd are a major cause of hospitalizations and death, and account for 70% of health care costs for the disease [19] . moreover, exacerbation frequency is a major determinant of health status and quality of life for copd patients [20] . despite the high incidence and serious complications of uri, the mechanisms by which viruses induce upper airway symptoms, or cause exacerbations of lower airway diseases, remain poorly understood. although it is possible that different viral types may induce these outcomes via variable mechanisms, it seems more likely that common aspects of viral pathogenesis dominate. given that hrv is the major viral pathogen associated with colds and exacerbations of asthma and copd, we will focus on the current status of our knowledge of the response to hrv infection as representative of viral pathogenesis, indicating differences with other viral types when appropriate. it is clear that uri are associated with increased airway inflammation. in particular, hrv infections lead to increased numbers of neutrophils and lymphocytes in the upper airways [21] [22] [23] . hrv infections also induce neutrophilic recruitment to the lower airways in subjects with asthma [24, 25] . consistent with this virally induced pattern, many acute asthma exacerbations seen in the clinical setting are characterized by increased levels of neutrophils and lymphocytes in the airways [26] [27] [28] . asthmatics who display this neutrophilic profile show a poor response to inhaled corticosteroids [29] . similarly, while stable copd is associated with a characteristic infiltration of the bronchial mucosa with cd8 + t lymphocytes and macrophages, severe exacerbations of copd are associated with increased neutrophilic and lymphocytic influx [30, 31] . it seems reasonable, therefore, to infer that viruses may trigger exacerbations of asthma and copd by enhancing already existing inflammation in the lower airways. the mechanisms by which viral infections are able to enhance upper, and lower, airway inflammation are not fully defined, but growing evidence supports the concept that viral modulation of epithelial function may initiate the inflammatory response. the airway epithelial cell is the primary target for inhaled pathogens and expresses receptors for several viral types. indeed, the epithelial cell is the only cell type in which hrv has been detected, thus far, by in situ hybridization [32, 33] , during in vivo infections. moreover, there is now strong evidence that, upon experimental nasal inoculation with hrv, virus spreads to infect epithelial cells in the lower airways [34, 35] , suggesting that epithelial infection may also directly initiate lower airway inflammatory responses. in contrast to viruses such as influenza and rsv, hrv infections do not cause overt epithelial toxicity [36, 37] . thus, while the cytotoxic effects of influenza and rsv may contribute to the severity of symptoms, it seems reasonable to assume that alterations of epithelial biology represent a common pathway of symptom development by multiple virus types. in support of this concept, infection of epithelial cells by hrv has been shown to generate a wide variety of proinflammatory chemokines and cytokines, including il-8 (cxcl8), ena-78 (cxcl5), ip-10 (cxcl10), rantes (ccl5), il-1, il-6 and il-11 [23, [37] [38] [39] [40] [41] [42] . given that several of these products also are detected in airway secretions during hrv infections in vivo [23, 38, [42] [43] [44] , it is likely that they contribute to recruitment and activation of inflammatory cells during infections. the ability to induce proinflammatory cytokine production from epithelial cells is also shared by other viruses. for example, influenza infection induces epithelial production of il-6, il-8 and rantes [45] , while rsv infections induce expression of a wide range of chemokine genes [46] . although there is a clear potential for these chemokines and cytokines to induce inflammation, the profile of products described clearly has the capacity to recruit a plethora of inflammatory cell types to the airways. despite this, a relatively selective cellular profile is seen during infections in vivo. it is unclear what other parameters regulate this limited pattern of inflammatory cell recruitment. it also must be acknowledged that our understanding of the mechanisms by which virally induced chemokine production occurs remains limited. in the case of hrv infections, some chemokines are induced quickly after viral exposure and do not seem to require viral replication [41, 47] , while other genes are not induced until several hours post-infection and are absolutely dependent upon replicating virus [23, 39] . although both article in press phosphatidylinositol 3-kinase and mitogen activated protein kinase pathways have been implicated in viral induction of chemokines [47] [48] [49] , the early signaling events induced by virus remain poorly elucidated. similarly, while the viral replication intermediate, double-stranded rna, and the rhinovirus 3c protease both have been implicated as mediators of late, replication dependent events [23, 50, 51] , the pathways by which such products induce responses are not well defined. moreover, while it is clear that viral induction of some cytokines and chemokines occurs via transcriptional pathways involving nf-kb and/ or interferon regulatory factor [23, 52] , our understanding of the control of transcriptional and post-transcriptional regulation of epithelial cytokine and chemokine production in response to viral infection also is limited and requires further study. delineating those aspects of signaling that may be unique for viral induction of chemokines may provide a rational basis for targeted interventions, while studies with selective chemokine or chemokine receptor antagonists will be required to provide a definitive answer on which are the key epithelial mediators involved in disease pathogenesis. once viral infection of the epithelium initiates a proinflammatory process, subsequent production of other mediators that are not of epithelial origin may further contribute to the inflammatory status of the airways. these mediators may be derived from plasma or from other cell types within the airway mucosa. of the mediators assessed thus far, some are relatively virus-specific, while others are observed with colds induced by multiple viral types. for example, while rsv infections have been reported to be associated with the generation of virus-specific ige and increased release of histamine into nasal secretions [53] , levels of histamine are not increased in airway secretions during hrv infections [22] . moreover, while older antihistamines that display anticholinergic and sedative properties do reduce rhinorrhea during colds, second generation antihistamines lacking these side effects are ineffective [54] . by contrast, other mediators, such as kinins and leukotrienes have been associated with infections due to more than one type of common respiratory virus [22, [55] [56] [57] . defining the role of each of these mediators in disease pathogenesis will, again, require studies with effective and selective antagonists. several factors are likely to play a role in determining the severity of the clinical outcome to upper airway viral responses, including the susceptibility of patients with asthma or copd to experience lower airway exacerbations. such factors include pre-existing immunity to a particular viral strain and, in the case of lower airways, the degree of disease control at the time of infection. another important factor is likely to be the variability of the individual host immune and antiviral response to infection. although both innate and specific immunity contribute to the host response to infection, it appears as though the innate response is dominant early after infection and is more likely to help regulate the symptomatic response. for example, while hrv infections elicit antigen-specific humoral and cellular immune responses, these are usually not detectable until after disease symptoms have resolved [58] . as may be expected based on its central role in viral infection, the epithelial cell is a significant contributor to the innate response to infection. as noted above, infected cells release several cytokines and chemokines that can recruit, and activate, cells of the immune system to the airways. in addition, epithelial production of nitric oxide (no) appears to play an important role in host antiviral responses [59] . infection of cultured epithelial cells with any of several common respiratory viruses leads to marked upregulation of inducible nitric oxide synthase (inos) and increased generation of no. a similar response occurs during experimental hrv infections in vivo, in that levels of epithelial inos induction correlate with levels of exhaled no. interestingly, in this study, subjects with the highest levels of exhaled no cleared virus more rapidly and had lower symptoms [60] . a rationale for this is provided by several studies showing that no exerts direct antiviral activity against several common respiratory viruses, in part by nitroslyating key thiol residues in viral proteases. moreover, it has been shown that no also inhibits virally induced generation of several cytokines and chemokines from epithelial cells [59] . it also should be noted that infection of epithelial cells with hrv induces the production of human b-defensin-2 (hbd-2) and hbd-3 [61] . these peptides are chemotactic for immature dendritic cells expressing ccr6, as well as other cell types contributing to the immune response, and likely play an important role in linking innate and specific immunity to hrv [62] . hbd-3 also can reduce the extent of influenza infections by blocking the fusion of the virus with cell membranes [63] . as viral replication progresses, and intact virus is released into airway secretions, it can interact with other cell types that may further contribute to the immune response. presumably, for example, dendritic cells initiate antigen presentation to t cells in the airways or lymph nodes to initiate the specific immune response. in addition, monocytes and macrophages may release additional cytokines, including interferons that can stimulate a variety of interferon (ifn)-stimulated genes (isgs) that collectively limit virus replication and spread. traditional approaches to mitigate the effects of uri have focused on symptomatic relief, although such approaches have generally had modest success. the topical anticholinergic, ipratropium bromide reduces rhinorrhea, an effect mimicked to some degree, as noted above, by older ''first generation'' antihistamines that are known to also have anticholinergic properties. similarly oral adrenergic drugs have modest benefit in terms of reducing nasal blockage, while topical agents have a greater efficacy but suffer from issues of rebound [54] . a somewhat greater reduction in total symptoms was observed in experimental hrv infections when a combination of ifn-a2b, together with the first generation antihistamine, chlorpheniramine, and ibuprofen was administered beginning 24 h after viral challenge [64] . although this represents an important proof of concept regarding the effectiveness of combining an antiviral compound with conventional compounds for symptomatic relief, the practical utility of this combination is limited by cost factors. there is also a growing literature on the use of ''natural' remedies for the treatment of colds. although the rationale for the use of zinc in the treatment of colds is not well established, multiple studies have evaluated the effectiveness of various zinc salts in this regard. overall, the results of these studies have been inconclusive. a recent, wellcontrolled trial, however, found that zinc salts had an extremely modest effect in reducing symptoms of experimental hrv infections and was ineffective in natural colds [65] . similarly, echinacea and ginseng have been widely reported as herbal remedies for colds. most of the trials to evaluate such agents have been small and have generated conflicting data. recent randomized trials with relatively large number of subjects reported modest efficacy of a ginseng extract in reducing the frequency of natural colds [66] , but found no significant effect of an extract of echinacea in experimental hrv infections [67] . a major issue in regard to trials of herbal remedies, of course, is that there is no standardization of extracts used across studies. indeed, given that the identity of any proposed ''active'' ingredient is unknown, it is impossible to know what to standardize. all of the above trials have been limited to analyzing effects on nasal symptoms, and have never evaluated effects on viral exacerbations of asthma or copd. given the data reported, however, it seems unlikely that they will provide any major benefit. indeed, an interesting question is whether drugs that are currently used for the treatment of asthma and copd have any beneficial effects during viral exacerbations. there is no doubt that the use of corticosteroids, long acting b-agonists, or leukotriene receptor antagonists, alone or in combination, to maintain optimal asthma control has proven efficacious in reducing number of asthma exacerbations, and the use of oral corticosteroids early in exacerbations helps prevent relapses. effects on basal inflammation, however, may not necessarily translate to effects on viral-specific inflammation, and there have been no defined studies of the effects of these medications during asthma or copd exacerbations of known viral origin. corticosteroids have little efficacy in hrv-induced colds [68] , and it is of interest that asthmatics who display prominent sputum neutrophilia, perhaps indicative of viral etiology, are poorly responsive to inhaled corticosteroids [29] . because of these limitations, alternative therapeutic approaches to virally induced airway disease continue to be sought. an obvious strategy is to use antiviral approaches. influenza vaccine is clearly effective in reducing upper airway symptoms, and in preventing lower disease exacerbations, induced by this virus during the winter months. unfortunately, vaccination approaches are not feasible for hrv, given the large number of viral serotypes, and have, thus far, proven unsuccessful in the case of rsv infections. neutralizing antibody prophylaxis has proven effective in preventing rsv-induced bronchiolitis but cost factors limit a broader utility and, again, major feasibility issues would arise with using this approach for hrv. selective antiviral approaches have shown more promise. in the case of influenza, neuraminidase inhibitors have been available for several years and have proven clinical efficacy in reducing development and severity of symptoms. at least two approaches also have been used to develop potential antiviral agents against hrv. the novel capsid-binding inhibitor, pleconaril, prevents viral uncoating. in natural colds, oral pleconaril (400 mg) administered three times daily led to a significant, but modest reduction in symptoms and also shortened the reported duration of colds [69] . concerns regarding effects on cytochrome p450 3a4, however, precluded further development as an oral treatment. the alternative antiviral approach used for hrv infections has targeted inhibition of the viral 3c protease, which is necessary for cleavage of the viral polyprotein and, thus, replication. topical administration of one 3c inhibitor, ruprintrivir, significantly inhibited symptoms in experimental hrv infections even when administered beginning 24 h after infection, although multiple daily dosing was required [70] . neither of these drugs has been evaluated for their ability to limit hrvinduced exacerbations of lower airway diseases. upper respiratory tract viral infections and their complications lead to a significant burden on health care systems throughout the world. current treatments are less than ideal, and a greater insight into the molecular basis by which common viruses induce both upper and lower airway symptoms is needed if alternative therapeutic approaches are to be developed rationally. the delineation of which specific cytokines and chemokines are key contributors to disease pathogenesis, and elucidation of signaling pathways selective for viral modulation of epithelial cell function may 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experimental rhinovirus infection efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers key: cord-297790-tpjxt0w5 authors: mandl, judith n.; schneider, caitlin; schneider, david s.; baker, michelle l. title: going to bat(s) for studies of disease tolerance date: 2018-09-20 journal: front immunol doi: 10.3389/fimmu.2018.02112 sha: doc_id: 297790 cord_uid: tpjxt0w5 a majority of viruses that have caused recent epidemics with high lethality rates in people, are zoonoses originating from wildlife. among them are filoviruses (e.g., marburg, ebola), coronaviruses (e.g., sars, mers), henipaviruses (e.g., hendra, nipah) which share the common features that they are all rna viruses, and that a dysregulated immune response is an important contributor to the tissue damage and hence pathogenicity that results from infection in humans. intriguingly, these viruses also all originate from bat reservoirs. bats have been shown to have a greater mean viral richness than predicted by their phylogenetic distance from humans, their geographic range, or their presence in urban areas, suggesting other traits must explain why bats harbor a greater number of zoonotic viruses than other mammals. bats are highly unusual among mammals in other ways as well. not only are they the only mammals capable of powered flight, they have extraordinarily long life spans, with little detectable increases in mortality or senescence until high ages. their physiology likely impacted their history of pathogen exposure and necessitated adaptations that may have also affected immune signaling pathways. do our life history traits make us susceptible to generating damaging immune responses to rna viruses or does the physiology of bats make them particularly tolerant or resistant? understanding what immune mechanisms enable bats to coexist with rna viruses may provide critical fundamental insights into how to achieve greater resilience in humans. an estimated ∼60% of emerging infectious diseases are caused by pathogens which originate from a non-human animal source, referred to as zoonoses (1) (2) (3) . moreover, the frequency of outbreaks caused by zoonotic pathogens has been increasing over time in the human population, with viruses being the most successful at crossing the species barrier (2) (3) (4) . given the impact of viral zoonoses on global public health, considerable resources have been invested into better understanding patterns in their emergence to improve predictions of where they might arise. one key variable in such predictions is to determine the animal reservoir populations within which these novel viruses can be maintained indefinitely (with or without disease) and which therefore act as sources for transmission to humans (5) . in some instances, epidemiological associations may provide clues to identifying a reservoir host species, and the detection of natural infection through seroconversion or the virus itself provides further evidence. recently, phylogenetic analyses have also been used to investigate viral origins-with a presence of greater diversity and of strains ancestral to those in humans being indicative of a virus circulating within a particular natural host population (6) . once identified, viral reservoirs have historically been critical levers through which to reduce human cases (5) . however, reservoir hosts may also provide us with fundamental insights into host-pathogen interactions and are a rich opportunity to examine the immunological processes that contribute to patterns governing which pathogens cross into humans, cause disease and why (7, 8) . this can be particularly informative as in many instances, the zoonotic viruses that are so pathogenic in humans do not cause disease in the reservoirs with which they coexist. bats have been confirmed as reservoir hosts for many viruses, several of which are associated with fatality rates as high as 90% among diagnosed human cases. it has long been appreciated that rabies and other lyssaviruses causing lethal encephalitis can be transmitted from numerous bat species (9, 10) . live marburg virus (marv) has been isolated from rousettus aegyptiacus fruit bats which, jointly with epidemiologic evidence and detection of viral rna, strongly suggests that r. aegyptiacus is a reservoir host of this filovirus (11) . the related ebolavirus (ebov) likely also circulates in african fruit bats, with a few species having been implicated so far-the mobility of which accounts for the sudden appearance of ebola in west africa during the 2014 outbreak, a region where ebolavirus had not previously been detected (12, 13) . the highly pathogenic henipaviruses, of which hendra virus emerged in australia and nipah virus in south-east asia via horse and pig intermediate hosts respectively, have been shown to be transmitted from pteropus bats (14, 15) . in china, horseshoe rhinolophus bats have been identified as the reservoirs for sars coronavirus via palm civet intermediate hosts, the cause of a large outbreak of atypical pneumonia across several countries that began in 2002 in china (16) (17) (18) . more recently, mers coronavirus that has caused lethal respiratory infections mostly in saudi arabia, likely transmitted via dromedary camels, was shown to be closely related to several bat coronaviruses, including those sequenced from neoromicia capensis, pipistrellus abramus, and vespertilio superans bats (19, 20) . moreover, additional viruses may continue to emerge from bats, as in the single case of sosuga virus infection in a wildlife biologist collecting bats in south sudan (21) . in addition to these emerging zoonotic viruses, bats may be the source of a number of viruses with which humans have older evolutionary associations. for instance, bats harbor viruses closely related to both mumps (rubula virus) and measles (morbilli virus) and have likely been donors of these viruses to other mammalian groups, possibly including humans (6, 22) . furthermore, both old and new world bats carry diverse hepadnaviruses, some of which are related to hepatitis b virus and can infect human hepatocytes (23) . hepaciviruses that are related to hepatitis c virus and pegiviruses that are related to human gb viruses were detected in the sera of many different bat species, and given the basal position of these bat viruses in phylogenetic trees, may also represent strains ancestral to those found in humans (24, 25) . the preponderance of links between bat and human pathogens has led to a debate about whether bats disproportionately contribute to emerging viral infections crossing the species barrier into humans (26) (27) (28) (29) (30) . given the diversity of the chiroptera order (figure 1) , we may simply see more bat viruses because there are so many (>1,300) species of bats (31) . however, even when accounting for the fact that they make up ∼20% of extant terrestrial mammals, bats are overrepresented as reservoir hosts of pathogens with a high potential for spilling into human populations (32, 33) . in fact, no known predictors that have been described to impact the likelihood of crossing the species barrier, including reservoir host ecology, phylogenetic relatedness to humans or frequency of reservoir-human contact, explain this pattern (32) . thus, why bats are such a frequent source of pathogenic human viruses remains a tantalizing mystery. among viruses, those that have genomes encoded by rna generally jump across species boundaries more frequently, presumably due to their inherently greater mutation rates that facilitate the rapid adaptation to replicating within new hosts (34) . interestingly, all pathogenic viruses that have made the jump to humans for which bat species may be reservoirs share the common feature that they have single-stranded rna genomes (with the exception of hepadnaviruses which have a dna genome but replicate via an rna intermediate). so far, available evidence suggests that bats remain disease-free when infected with the rna viruses they carry-even those highly pathogenic to humans-and are able to coexist with them without detectable fitness costs using measures such as changes in temperature, loss of body weight, or overt signs of inflammation (35) . indeed, so far only one rna virus studied which circulates in a bat population has been shown to consistently cause significant morbidity and mortality: tacaribe virus in the jamaican fruit bat (artibeus jamaicensis), which recent evidence suggests is not a reservoir host for this virus (36) . data from experimental rabies and lyssavirus infections suggests that rhabdoviruses may also cause disease in bats, although experimental infection outcome is very dependent on the infection route. intracerebral infection with different strains and in different bat species invariably led to death (37, 38) . in contrast, intramuscular infection led to muscle weakness, paralysis and visible histological cns lesions in 30% of experimentally infected flying foxes (pteropus poliocephalus) (39) . similarly, a subset of vampire bats (desmodus rotundus) experimentally infected intramuscularly with a high dose of rabies virus remained healthy despite viral shedding in the saliva and survived (40) . naturally infected bats are thought to either die or remain healthy and seroconvert, but transmission in freeranging populations remains incompletely understood (41) . while bats seem to be frequent hosts for rna viruses, current available data indicates that primates and humans disproportionately harbor dna viruses such as herpesviruses (32) . interestingly, it is these dna viruses that can persist in an individual which can also be found in isolated, small indigenous groups-perhaps suggestive of humans having a more ancient relationship with such dna viruses (42) . it may even be the case that persistent dna viruses in humans impact immune responses specifically to rna viruses, but this has not yet been examined. it is likely that differences in evolutionary history of pathogen exposure between bats and humans have led to distinct adaptations in anti-viral immune responses and the ability to tolerate certain infections without disease while being susceptible to others. importantly, bats differ in many aspects of their physiology and behavior from humans that may have direct or indirect effects on immune function. bats are a monophyletic mammalian group traditionally divided by morphological data into two suborders, the megabats and microbats, which more recent molecular data has revised into the yinpterochiroptera and yangochiroptera suborders (figure 1 ). bats possess a suite of traits that make them distinct from other mammals in a number of ways. these unique life history traits may play a role in understanding which pathogens bats have evolved to coexist with and why. in particular, such traits may explain the ability of bat populations to maintain particular viral pathogens indefinitely, and may have effects on immune function through specific energetic or evolutionary trade-offs we have yet to better define. despite the diversity of viruses carried by bats, they are not typically known to cause mass bat die-offs or reduce bats' remarkable longevity. in this respect, bats represent a potential opportunity for long-term persistence of viruses within a population and across generations. bats live significantly longer than similarly-sized terrestrial mammals and, despite their small size, are characterized as "slow" mammals in the slow-fast continuum (43, 44) . although their weights range from 2 grams to 2 kilograms, with respect to longevity bats group with large mammals such as humans and non-human primates (45) . aerial living has an obvious advantage in avoiding predation, but bats outlive even birds. for example, the brandt's bat (myotis brandtii) lives up to 41 years, compared to selasphorus platycercus, a bird species of similar size that lives for ∼14 years (45, 46) . thus, flight can only partially account for their extraordinarily long lives. initially, the longevity of some bats was attributed to seasonal hibernation, as temperate-zone species enter continuous torpor of up to 75 days, with a dramatic drop in metabolic rate such that small fat reserves can sustain them throughout the entire hibernating season (43) . however, even non-hibernating bat species live three times longer, on average, than predicted by their size, and heterothermy is not an accurate predictor of lifespan in other mammalian orders, suggesting that the driving force behind their surprising longevity is intrinsic to bats as a group (47) (48) (49) . like other "slow" mammals, bat females typically only have one offspring per year, perhaps because the volant lifestyles of bats make it difficult to rear more than one offspring, as pregnant females and those with recent births must navigate and forage with added weight; on average, neonatal bat pups are ¼ of their mother's weight (50) . the physical and energetic constraints of rearing multiple offspring may necessitate small litters, which would in turn require prolonged reproductive capability and enhanced longevity to ensure maintenance of the population over generations. thus, in bats, the dependence of colony survival as a whole may depend upon enhanced individual survival and delayed senescence (51) . genetic analyses of several bat species have shown differences in the growth hormone (gh)/insulin-like growth factor 1 (igf1) axis which in humans is associated with aging, resistance to diabetes and cancer (52) . the determinants of adult survival in bats have been historically difficult to identify, as this requires tracking individuals over many years, and until recently longitudinal studies of bat mortality were conducted using tagged bats, of which only a fraction were recovered (53) . recently, a 19year study of a colony of bechstein's bats demonstrated that unlike terrestrial mammals, survival could not be predicted by common indicators such as season, age, and body size. instead, the only accurate predictor of mortality was a single cataclysmic weather event that affected multiple countries in north-central europe. additionally, even the oldest female bats were reproductively capable, indicating that bat survival is primarily affected by catastrophic natural events rather than factors that normally dictate an individual's fitness (45) . molecular phylogenetic studies of bats suggest that there are massive gaps in bat fossil records. as bats are the second most diverse order of mammals, outnumbered only by rodents, the number of species unrepresented in the fossil records is staggering. over half of microbat and nearly all of megabat fossil histories are missing (31, 54) . the enormous incompleteness of the fossil records has made it difficult to identify when specific morphological traits of bats arose. as molecular phylogeny groups two echolocation-reliant microbat species with megabats (also called old world bats or pteropodids), which do not rely on echolocation, there is some debate as to whether echolocation first arose in the common ancestor of bats and was subsequently lost in megabats, or whether it arose twice, independently (31) . pteropodids have adaptations that enhance visual acuity at night (55), and they do not require echolocation for foraging (56) . there are multiple types of echolocation that can be partially delineated by species, but are more clearly categorized by the type of environment. divergent species that inhabit the same type of environment, such as those that hunt in large, open spaces, often use the same form of echolocation, suggesting that habitat has a greater influence on echolocation than phylogeny (31) . importantly, echolocation can result in the production of droplets or small-particle aerosols of oropharyngeal fluids, mucus, or saliva, thus facilitating transmission of viruses between individuals in close proximity (57, 58) . the unique navigation tactic of many bat species may inadvertently facilitate virus transmission among bats in the same habitat. bats are the only mammal capable of powered flight, which likely evolved ∼65 million years ago alongside birds following radical ecological changes that resulted in the extinction of the dinosaurs (54, 59) . during flight, bats consume approximately four times as much oxygen, and they have a markedly higher concentration of red blood cells compared to small terrestrial mammals (60). bat flight is markedly different from that of birds and insects, whose wing surfaces are typically composed of inflexible material, such as feathers or chitin. bat wings are constructed from live skin stretched across elongated arm and finger bones, making them extraordinarily malleable and sensitive to environmental cues (59) . the plasticity of bats' wings allows them to navigate and inhabit diverse ecospheres, contributing to their extensive speciation. moreover, the capability of powered flight can allow the efficient spread of viruses and thus the introduction of pathogens to which colonies may otherwise have remained naïve. as flight is extremely metabolically demanding, in addition to evolving the physical mechanisms required for flight, bats have also evolved necessary underlying molecular mechanisms. the mitochondrial respiratory chain accounts for nearly all atp required for mobility in eukaryotes, and genetic analysis of both micro-and megabat species revealed an enrichment of genes specific to the oxidative phosphorylation (oxphos) pathway. specifically, 4.9% of nuclear-encoded and 23% of mitochondrial oxphos genes have evidence of positive selection in bats, which is markedly higher than the expected 2% of orthologous genes in previous genome-wide studies that show evidence of positive selection (61) . genomic analysis of pteropus alecto and m. davidii suggests positive selection for the dna damage checkpoint pathway and changes in overlapping aspects of this pathway with the innate immune system, indicating that evolutionary adaptations important for flight may have secondarily affected bat immunity (62). as a group, bats exhibit the greatest diversity of social systems in mammals. tropical species are primarily responsible for this diversity, as temperate species are more restricted in their social behavior. generally, however, bats are extremely social creatures that tend to form dense roosting colonies (63) , and almost all temperate-zone species live in closed societies with very little infiltration of foreign bats into established roosts (63, 64) . in particular, female bats form maternity colonies in which males do not take part. as bats are capable of longdistance flight, dispersal barriers cannot explain the philopatry of females. instead, benefits such as knowledge of foraging areas and social thermoregulation likely selected for these colony types. additionally, there is evidence that forming closed societies limits the potential invasion of new pathogens, thereby protecting colony members that would otherwise be vulnerable to infection. for example, pseudogymnaoscus destructans has decimated north american bat populations that do not live in the type of closed societies observed elsewhere (64) . dna analysis of a closed society of bechstein's bats revealed extraordinarily high conservation of mitochondrial dna and relatively low conservation of nuclear dna, suggesting stable maternal populations within colonies and gene flow between colonies via promiscuous mating with males. it is possible that the mating patterns of temperate-zone species may allow transmission of pathogens between colonies via traveling males while the frontiers in immunology | www.frontiersin.org more insular females may allow viruses to persist throughout generations within a colony. an important commonality among pathogenic rna viruses in humans presenting with disease is that the host response is an important contributor to the disease process, with dysregulated and excessive innate immune responses being particularly important drivers of tissue damage during infection (8) . given the general absence of clinical signs of disease in bats infected with the same viruses that are so lethal in humans or other non-natural hosts infected experimentally, a critical question has been to understand whether bats might establish effective disease tolerance, thus maintaining fitness despite pathogen replication, or whether bats are more resistant to infection through more successful control of pathogen replication and what the contribution of the immune response is (65, 66) . the lack of many fundamental immunological tools enabling the probing of bat immune responses has meant that truly mechanistic studies of bat immunity have been very limited, although recently there has been some progress in establishing approaches such as flow cytometry to identify distinct bat immune cell populations (67, 68) . so far, studies of bat immunity have primarily taken one of three approaches, whereby each comes with important strengths and weaknesses that have to be kept in mind: (i) comparative genome studies, (ii) in vitro cell culture assays, and (iii) experimental infections. comparative genome studies have confirmed that the critical components of the innate and adaptive immune system are conserved in bats at the gene level and that bats have the machinery for innate responses to pathogen-associated molecular patterns (pamps), the production of anti-viral effector molecules such as type i interferons (ifn), t cell responses (variable t cell receptors, mhci and mhcii), and b cell responses [reviewed in (35) ]. interestingly, based on the 10 bat genomes sequenced so far, the only family of genes lost entirely in all of them are pyhin genes (69) . members of the pyhin family are dna sensors capable of recognizing foreign dna, including dna viruses and damaged self dna which can be generated by rna viral infection. recognition of dna results in production of ifn through interaction with stimulator of interferon genes (sting). the pyhin family also encode the only identified class of dna sensors capable of activating the inflammasome. it has been hypothesized that the absence of the pyhin family may allow bats to limit activation of the innate immune response to damaged self-dna generated by rna viral infection, thus avoiding excessive inflammation (69, 70) . genome comparisons highlighting contractions or expansions of specific gene families, specific genes under positive selection, or nonconserved sequence differences in critical protein domains can thus provide the basis for hypotheses worth testing further. however, it is important to note that much can be missed in absence of data on gene regulation, especially during infection when gene expression kinetics can make a critical difference to the infection outcome. moreover, the absence of a gene or gene family does not rule out that other proteins have evolved to compensate for their loss of function. thus, while whole genome analyses can provide a context for specific questions or be hypothesis-generating, on their own they cannot distinguish tolerance from resistance mechanisms. the repeated identification of signatures of positive selection in innate immune genes in particular, does however lend credence to the idea that bats have specific adaptations as a result of a long co-evolutionary history with viruses. cell culture assays with bat cell lines, or, in some instances, primary bat cells, have been used to assess whether bats are permissive for viral replication and to determine whether particular immune receptor signaling pathways are intact. as discussed below, such studies have probed the type i ifn pathway in particular, revealing some possible species-specific differences among bats (71) (72) (73) (74) (75) (76) (77) (78) (79) (80) (81) (82) (83) . however, it is important to note that in some instances immortalized cells can behave differently from primary cells and that such cultures may miss additional differences imposed by changes in cell localization, cell recruitment or cell-cell interactions in a whole animal. careful experiments measuring the quality, magnitude, and kinetics of immune responses in bats during infection and upon administration with defined stimuli for which we have comparative information from humans remain to be done to provide additional evidence that specific innate immune pathways are wired differently. experimental infections come with the enormous challenge of having to house and/or breed colonies of bats and to have biosafety-level 4 facilities in place to perform infections with viruses lethal to humans. moreover, some trial and error is involved in determining which route and dose leads to viral replication, establishing a source of the virus (humanadapted strains tend to replicate less well in bats than strains obtained from naturally infected bats), and amplifying this viral stock without extensive tissue culture passaging. studies to date have examined the kinetics of viral replication by quantifying the extent of viremia and dissemination to other tissues, and assessing changes in white blood cell counts, body mass, and temperature. given the generally low levels of viral shedding and short infectious periods observed so far it remains poorly understood how transmission occurs in the wild to sufficient levels that cross-species jumps occur. some infection experiments have also provided evidence that a particular bat species is unlikely to be a reservoir despite epidemiological evidence, for example for r. aegyptiacus and ebolavirus. certainly, once good experimental infection models are established, such studies have the potential to be hugely informative with regard to anti-viral immune responses elicited using, for instance, comparative transcriptome analyses. one drawback may be that experimental infections do not mimic the impact of chronic stress arising from the disruption of wildlife populations, which bats are particularly sensitive to jones et al. (84) . comparison of either cave-roosting or foliage-roosting species in areas of malaysian borneo designated as actively logged forest, recovering forest, or fragmented forest revealed varying impacts of habitat disturbance on stress and circulating white blood cells (85) . overall, the limited studies of bat immunity that have been done have focused largely on 2 species: p. alecto and r. aegyptiacus. we summarize this work below, but comparisons of observations made across species suggest that although a number of species appear to be capable of avoiding the pathological effects of rna virus infection, each bat species may have achieved this through distinct pathways, possibly involving changes to both increase pathogen replication control and to mitigate any immunopathology through decreased inflammatory responses and hence increased disease tolerance. the most well studied bat species with regard to antiviral immune responses is the australian black flying fox (p. alecto). this interest has stemmed from the fact that pteropid bats have been identified as the natural reservoirs for the deadly hendra and nipah viruses (86) , which continue to cause outbreaks [such as most recently in india in may 2018 (87)]. to date, several studies have examined the kinetics of viral infection in pteropus bats and the nature of transmission and replication in other susceptible species (88) (89) (90) (91) . in australia, all four species of pteropid bats (p. alecto, p. poliocephalus, p. scapulatus, and p. conspicillatus) have antibodies to hendra virus but only p. alecto and p. conspicillatus are considered to be the primary reservoir hosts (14, 92, 93) . in south east asia, both pteropus spp. occurring in malaysia have been found to be seropositive for nipah virus neutralizing antibodies, and the virus has been isolated from p. hypomelanus and p. vampyrus (15, 94) . experimental infections of pteroid bats with hendra or nipah virus result in sub-clinical infection with short periods of virus replication and shedding, and low antibody titres (88) (89) (90) (91) . upon subcutaneous infection of p. poliocephalus with hendra virus, viral antigen was detected by immunohistochemistry at 10 dpi in blood vessels of spleen, kidney and placenta (89) . similarly, oronasal hendra virus infection of p. alecto led to the presence of viral genome in lung, spleen, liver and kidney 3 weeks later, but virus isolation was unsuccessful at this timepoint (89, 91) . the malaysian flying fox, p. vampyrus and the australian species, p. poliocephalus demonstrate similarly short periods of viremia upon infection with nipah virus. in subcutaneously infected p. poliocephalus, virus was isolated from the kidney and uterus of bats euthanized at 7dpi, but no virus was isolated at any of the other timepoints examined (3, 5, 10, 12, or 14 dpi) and there was no evidence of antigen in any tissue by immunohistochemistry, including tissues collected at 7 dpi. in this study, low neutralizing antibodies were detected in all bats with the exception of one individual that developed a significant neutralizing antibody titre -possibly reflecting the fact that p. poliocephalus is not the natural host for nipah virus (90) . in p. vampyrus challenged by oronasal nipah inoculation, viral genome was detected in a throat swab at 4 dpi and a rectal swab of the same individual at 8 dpi but virus was undetectable in tissues collected at postmortem from all individuals (49, 50, or 51dpi), consistent with a short period of viremia. similar to previous studies, antibody titres were low in all p. vampyrus bats (91) . overall, these results are consistent with bats controlling replication rapidly, at least following experimental infections which involve higher doses of virus compared to what bats would likely be naturally exposed to in the wild. the absence of a robust antibody response also appears to be typical of all experimental hendra and nipah virus infections performed to date. since antibody responses are the only immune parameter that has been measured during experimental infections of bats so far, it is difficult to speculate on the mechanisms responsible for control of viral infections in vivo. pteropus alecto was among the first bat species to have its genome described in detail. genomic studies provided initial clues for possible differences in the innate immune system of bats, with evidence for selection of key innate immune genes and the expansion or contraction of specific immune gene families (62, 68, 95) . the mhci region is contracted (96) , as is the type i ifn locus, which in p. alecto contains fewer ifn genes than any other mammalian species sequenced, with only three functional ifn-α loci (68) . in contrast, pteropid bats have the largest and most diverse family of apobec (apolipoprotein b mrna editing enzyme, catalytic polypeptide-like) proteins identified in any mammal (95) . apobecs interfere with the replication of retroviruses by deaminating cytosine residues in nascent retroviral dna. this is notable, as bats are an important source of mammalian retroviruses, many of which have been transmitted to other mammals (97, 98) . apobec diversification may therefore have occurred to counteract the effect of retroviruses and possibly other viruses, as apobecs have been shown to restrict the replication of other virus families including hepadnaviruses, and parvoviruses (99, 100) . members of the apobeca3 protein family exhibit direct antiviral activity through dna cytosine deamination which results in hypermutation of the nascent retroviral dna which is then degraded or rendered non-functional (101) . the mechanism of antiviral activity against non-retroviruses remains largely unknown. for parvovirus adeno-associated virus, apobec meditated inhibition has been speculated to involve direct interaction with the viral dna or the replication machinery (102) . whether the expanded family of abobecs in bats have evolved other mechanisms to control dna and rna viruses remains to be determined. as apobecs can be induced by even low levels of type i ifn (103) , one hypothesis to be tested is that bats, through their multiple apobecs, are able to restrict viral replication without causing inflammation. pteropus alecto is the only bat species to date in which apobec genes have been mapped, and whether the expansion of this gene family extends to other bat species remains to be determined. in addition to the identification of putative immune pathways distinct in p. alecto through genome studies, differences have been identified in the activation of innate immune effectors in p. alecto from studies performed in vitro, primarily using cell lines derived from tissues including the kidney and lung. ifns are the first line of defense following viral infection and unsurprisingly, because of this, they have been the most extensively studied group of genes in bats. both type i (ifna and ifnb) and iii (ifnl) ifns are detectable in bat cells. curiously, a unique characteristic of pteropid bats is the constitutive expression of mrna for ifna and the signaling molecule, ifn regulatory factor 7 (irf7) in unstimulated tissues and cells [75, 68a] . constitutively expressed ifna and irf7 may allow bats to respond more rapidly to infection, thus avoiding the lag time between pathogen detection and response. furthermore, viral infection or stimulation with synthetic ligands result in little ifna induction in pteropid bat cells (68) . the constitutive expression of ifna has been described in two species of pteropid bats (p. alecto and cynopterus brachyotis) and is a first for any species. ifnb and ifnl are activated following stimulation of cells from p. alecto and p. vampyrus with synthetic ligands such as polyic (71) (72) (73) (74) . moreover, bat ifns demonstrate antiviral activity (68, (71) (72) (73) (74) 104) . however, viral infection of p. alecto splenocytes results in induction of ifnl but not ifnb, hinting at differences in the function of type i and iii ifns (74) . in humans and mice, ifnl has recently been demonstrated to have a role not only in controlling virus replication, but also in dampening damage-inducing neutrophil functions and in modulating tissue-damaging, transcriptionindependent responses such as production of ros (77, 80) . a hypothesis yet to be tested is whether upregulation of ifnl rather than ifnb has a similar function in bats. the endoplasmic reticulum (er) membrane protein, sting, is involved in induction of type i ifn by cytosolic dna (105) . stimulation of bat splenocytes with gmp-amp, which is produced following sensing of cytosolic dna by cgas, results in little induction of ifn compared to responses observed in mouse splenocytes (83) . bat sting contains an amino acid substitution of the highly conserved and functionally important serine residue s358 which may be responsible for dampening sting-dependent ifn activation in bat cells in response to dna. however, comparable levels of ifn induction in mouse and bat cells in response to the rna viral mimic polyic indicate that sting-associated inhibition of the ifn response does not extend to rna viruses (83) , thus the relevance to rna viruses in bats remains unknown. downstream of the induction of ifns, novel subsets of ifn stimulated genes (isgs) have been detected in unstimulated and stimulated pteropid bat cells indicative of a response that is less damaging to the host. furthermore, the isg response is elevated for a shorter period of time in p. alecto compared to human cell lines which again may be a strategy to avoid tissue damage (78, 81) . the less inflammatory profile of isgs may be the key to the ability of bats to tolerate higher ifn expression without adverse consequences. the balance between resistance and tolerance may therefore be achieved through careful selection of the pathways that are activated and shorter periods of activation or limited activation to prevent inflammation. in this regard, studies of the regulation of ifn signaling in bats is likely to provide important additional insights. a second bat species whose host responses to viral infections has been studied more recently is the egyptian fruit bat (r. aegyptiacus). marburg virus (marv) has been repeatedly isolated from this species with demonstrated seasonal pulses of active marv replication in juvenile bats living in caves in uganda (11, 106) . moreover, r. aegyptiacus were a suspected reservoir for ebolavirus (ebov) based on epidemiological evidence and detected seroreactivity to ebov, but no infectious virus has been isolated thus far from wild rousettus bats (107) . indeed, while cell lines from r. aegyptiacus are equally susceptible to marv and ebov (79, 108) , experimental infections of r. aegyptiacus seem to confirm that it is a reservoir for marv, but is unlikely to be the source of ebov spillover to humans. subcutaneous ebov infection results in very low viral replication, no viremia, little dissemination to other tissues, and no viral shedding, although some animals seroconvert, suggesting that r. aegyptiacus are unlikely to perpetuate ebov in the wild (109, 110) . in contrast, experimental marv infection of r. aegyptiacus resulted in acute viremia that peaked on days 5-6 post-infection (although generally at lower levels than in humans), oral shedding that peaked on days 7-8 postinfection, and dissemination to other tissues including spleen, liver, kidney and salivary glands (109, (111) (112) (113) . interestingly, viral replication was not associated with increases in white blood cell counts, any clinical signs of infection such as changes in body temperature or body weight, and infected tissues showed little evidence of inflammatory infiltrates (109) . in all experiments, viremia was cleared by day 13 and oral shedding ceased by day 19. intriguingly, a cohousing experiment resulted in marv transmissions to uninfected bats 4-7 months after experimental infection, raising the question of whether persistent infection with intermittent shedding is possible or whether very long latent periods without detectable viral replication could follow exposure (114) . upon secondary challenge of previously marv-infected bats, none showed any detectable viral replication or shedding, providing evidence that protective immunity is established (115) . unlike for pteropus bats, no constitutive expression of type i ifns has been detected in r. aegyptiacus (79) , but type i ifns are induced in r. aegyptiacus cell lines upon stimulation with sendai virus as seen in other mammals (82) . furthermore, in r. aegyptiacus the type i ifn genes are expanded, again in contrast to p. alecto (82), but like for p. alecto a number of genes in the type i ifn pathway or involved in innate immune recognition of pamps show signs of having been under positive selection (82) . whether positive selection of genes in either bat species is associated with tolerance remains to be determined, especially given that innate immune genes in humans have also been under positive selection (116) . a transcriptome study which generated 20 rna sequencing libraries from 11 tissues taken from 1 female and 1 male r. aegyptiacus found a reduced coverage of nk cell related genes compared to other mammals, but confirmed that in these bats the predominant t cells had an αβ t cell receptor, and showed that ige, igg, igm, and iga, as well as a number of pro-and anti-inflammatory cytokines, were all detectable (117) . the recently sequenced r. aegyptiacus genome revealed substantial differences in the repertoire of nk cell receptors, with this bat species entirely lacking functional killer cell immunoglobulin receptors (kirs) and with all killer lectinlike receptors (klrs) encoding either activating and inhibitory interaction motifs, or inhibitory interaction motifs only (82) . nk cells are important immune cell players in an antiviral response but without assessment of the consequences of these genomic differences it is difficult to draw any specific conclusions with regard to viral control or the magnitude of inflammation elicited upon infection with viruses like marv. nonetheless, these genomic data provide some interesting hypotheses to be tested in the future. some additional studies probing the induction of cytokines upon stimulation of bat cells with defined innate immune stimuli provides some evidence that innate immune recognition of viruses may be altered, leading to a reduction in proinflammatory responses. stimulation of kidney and myeloid cells from the big brown bat (eptesicus fuscus) with polyinosinicpolycytidylic acid (polyi:c) resulted in only limited activation of the inflammatory cytokine, tumor necrosis factor alpha (tnfα) compared to human cells which display a robust tnfα response. induction of tnfα is controlled by transcription factors, including the nf-kappa b (nf-κb) family which consists of five members, [rela (p65), relb, c-rel, nfκb-1 (p50), and nfκb-2 (p52)] which form homo-or hetero-dimers that are bound by molecules of the inhibitor of nfκb (iκb) family and retained in the cytoplasm of the cell in an inactivated state (118) . in e. fuscus, a potential repressor (c-rel) binding motif was identified in the tnfα promoter region which may explain the difference in induction of tnfα in e. fuscus cells. consistent with this hypothesis, partial knockdown of c-rel transcripts significantly increased basal levels of tnfα transcripts in e. fuscus cells (104) . the transcription factor, c-rel has also undergone positive selection in the bat ancestor which may indicate that this mechanism is common to other species of bats (62) . of note, low levels of tnfα induction have also been associated with tolerance in european bank voles which are a natural reservoir for puumala hantavirus (puuv) (119) . stimulation of macrophages from the greater mouse eared bat (myotis myotis) suggested that this species may have also evolved mechanisms to avoid excessive inflammation caused by cytokines. while high levels of tnfα, il1β, and ifnβ were produced in response to in vitro challenge with lipopolysaccharides (lps) and polyi:c, there was also a sustained, high-level transcription of the anti-inflammatory cytokine il-10, which was not observed in mouse macrophages (120) . furthermore, unlike in the mouse, m. myotis macrophages did not produce the proinflammatory and cytotoxic mediator, nitric oxide, in response to lps. the same study also showed evidence of bat specific adaptations in genes involved in antiviral and proinflammatory signaling pathways through comparison with other mammalian taxa, including rig-i, il1b, il-18, nlrp3, sting, and casp1, further supporting the evolution of adaptations associated with reducing inflammatory responses in bats (120). even less is known about immune responses of bats to nonviral pathogens than to viral pathogens, but it is clear that while anti-inflammatory responses may be characteristic of antiviral responses in bats, they are susceptible to disease upon infection with particular pathogens-in some instances due to dysregulated and damaging immune responses. one particular example of this is the emerging infectious disease, white nose syndrome (wns), that has decimated north american bat populations beginning in 2006, in what will likely rank as one of the most devastating wildlife diseases in history (121) (122) (123) . for reasons that remain poorly understood, the psychrophilic fungus pseudogymnoascus destructans (formerly geomyces destructans) causes no mass mortality in european bats despite being abundantly detected (124, 125) . indeed, evidence suggests that a single p. destructans genotype was introduced to north american bat species from europe (125) . in north america, p. destructans infection is not specific to a particular bat genus, replicating in many different bat species during hibernation and targeting the furless skin of the wings, ears, and muzzle (126) . distinct hypotheses have been proposed for why p. destructans is so deadly in north american bats, ascribing the impaired tolerance to infection compared to european bat counterparts to either physiological or immunological factors. on the one hand, more frequent arousal, electrolyte depletion, and dehydration are thought to contribute to mortality following infection (127, 128) . the destruction of wing tissue in wns results in a marked electrolyte imbalance, as the wings play a critical role in maintaining water levels, especially during hibernation, during which bats are particularly vulnerable to dehydration (129, 130) . dehydration catalyzes arousal in hibernating bats, which is extraordinarily metabolically costly and rapidly depletes the fat reserves necessary to survive until spring (127) . an alternative hypothesis posits that the restoration of the immune system following emergence from hibernation induces the fatal pathology of wns. during hibernation, destruction of cutaneous tissue is limited and infiltrating immune cells are entirely absent, yet in the weeks following arousal, infected bats exhibit overt wing damage and corresponding neutrophilic and lymphocytic infiltration (131) . hibernation does not preclude a localized immune response to p. destructans at the site of infection and transcriptomic analysis of infected tissue showed upregulation of some acute inflammatory genes in infected tissue (132, 133) . however, the observed immune responses likely occur during arousal periods, which are more common in infected bats. ultimately, immunosuppression during torpor allows p. destructans to colonize infected bats relatively unchecked (124) , and upon emergence from hibernation, the exuberant immune response may result in deadly immunopathology during wns (131) . in addition to general studies of immune cell recruitment and transcriptional responses during wns, body mass and white blood cell counts were examined following lps administration in four bat species (134) (135) (136) (137) . subcutaneous lps challenge in of pallas's mastiff bats (molossus molossus) led to a loss of body mass of ∼7% within the first day, but did not result in changes in circulating white blood cell counts or body temperature (135) . seba's short-tailed fruit bat (carollia perspicillata) also showed a decrease in body mass following lps challenge, but this was associated with increases in white blood cell counts as well as increases in derivatives of reactive oxidative metabolites (drom) (134) . subdermal lps challenge of fish-eating myotis (myotis vivesi) led to body mass decreases, increased resting metabolic rate and skin temperature (136) , while intraperitoneal lps challenge of wrinkle-lipped bats (chaerephon plicatus) caused an increase in circulating leukocytes, but did not result in a reduction in body mass compared to controls (137) . the differential responses to lps challenge suggest that the immune response to bacterial infection varies across species. of note, postmortem examinations of ∼500 dead bats comprising 19 species from germany revealed inflammatory lesions, many of which had evidence of underlying bacterial or parasitic infections, particularly in the lung (138) . bats have an array of unique life history characteristics that not only allow them to be particularly good reservoirs for viruses that are highly pathogenic in other species, but also appear to have shaped their immune systems. although research on bat antiviral immunity has focused on only a few species to date, at the genomic level, selection on genes is concentrated on the innate immune system across both suborders of bats. however, while these studies have provided a rich source of hypotheses, the majority remain to be tested at the functional level and many questions remain that cannot be answered from comparative genome studies. experimental studies to date have demonstrated some functional differences between bat species, with the common emerging theme that the overall antiviral response appears to converge on a lower inflammatory profile, with tight regulation of the cytokine and inflammatory response key to clearing viral infection without the pathological outcomes typically associated with infection. however, whether this is due to specific tolerance mechanisms that are at play or increased resistance to rna virus replication still remains unclear. fewer studies have examined the adaptive immune system than those probing innate immune pathways, but experimental infections with bat borne viruses have demonstrated that bats generate low or absent antibody responses which often wane rapidly. this is reminiscent of the response of another reservoir host, the sooty mangabey which is the natural reservoir for simian immunodeficiency virus (siv) and for yellow fever virus. sooty mangabeys given an attenuated yellow fever virus vaccine strain generate much lower, transient antibody responses as compared to humans or rhesus macaques. changes to innate immune responses are also evident in sooty mangabeys (139) . thus, intriguingly, different reservoir hosts may have arrived at similar solutions to avoid the pathological consequences that follow viral infection in non-natural hosts. despite the ability of bats to avoid disease associated with viral infection, this trait does not extend to all pathogens, as evidenced by the severe consequences associated with infection of north american bats with the fungus that causes wns. thus, the pathways associated with the control of other pathogens have not been under the same selection pressures as those responsible for controlling infections with rna viruses-or there are immunological trade offs involved which lead to greater susceptibilities to some pathogens than others. overall, it is clear that studying host-pathogen interactions in reservoir hosts has considerable potential to provide novel insights into host 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pseudogymnoascus destructans infection tolerated in europe and palearctic asia but not in white-nose syndrome initiates a casacde of physiological disturbances in the hibernating bat host frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome electrolyte depletion in white-nose syndrome bats wing pathology of whitenose syndrome in bats suggests life-threatening disruption of physiology evaporative water loss is a plausible explanation for mortality of bats from white-nose syndrome pathology in euthermic bats with white nose syndrome suggests a natural manifestation of immune reconstitution inflammatory syndrome the white-nose syndrome transcriptome: activation of anti-fungal host responses in wing tissue of hibernating little brown myotis immune responses in hibernating little brown myotis (myotis lucifugus) with white-nose syndrome inflammatory challenge increases measures of oxidative stress in a free-ranging, long-lived mammal no fever and leucocytosis in response to a lipopolysaccharide challenge in an insectivorous bat metabolic cost of the activation of immune response in the fish-eating myotis (myotis vivesi): the effects of inflammation and the acute phase response simulated bacterial infection disrupts the circadian fluctuation of immune cells in wrinkle-lipped bats (chaerephon plicatus) diseases in free-ranging bats from germany distinctive tlr7 signaling, type i ifn production, and attenuated innate and adaptive immune responses to yellow fever virus in a primate reservoir host all authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the handling editor declared a shared affiliation, though no other collaboration, with the authors jm and cs.copyright © 2018 mandl, schneider, schneider and baker. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-336456-wg8vfh6w authors: webb, glynn w.; kelly, sophie; dalton, harry r. title: hepatitis a and hepatitis e: clinical and epidemiological features, diagnosis, treatment, and prevention date: 2020-11-01 journal: clin microbiol newsl doi: 10.1016/j.clinmicnews.2020.10.001 sha: doc_id: 336456 cord_uid: wg8vfh6w hepatitis a and e are both ancient diseases but have only been properly recognized as being caused by distinct pathogens in modern times. despite significantly different genomic structures, both viruses employ remarkably similar strategies to avoid host detection and increase environmental transmission. there are millions of cases of acute viral hepatitis due to hepatitis a virus (hav) and hepatitis e virus (hev) each year, resulting in tens of thousands of deaths. the presentations can be clinically indistinguishable, but each virus also has a range of less common but more specific phenotypes. the epidemiology of hav is complex, and is shifting in countries that are making improvements to public health and sanitation. hev presents a significant public health challenge in resource-limited settings but has historically been incorrectly regarded as having little clinical relevance in industrialized countries. descriptions of a clinical syndrome recognizable as hepatitis can be found in sumerian medical texts from the third millennium before the common era. approximately 2,500 years later, hippocrates recorded the features of "epidemic jaundice," including clinical descriptions suggestive of fulminant hepatitis. by the middle ages, the idea that jaundice might be transmissible had emerged; in the 8th century, pope zacharias had men with jaundice quarantined to control the spread of the disease, although he had no understanding of the exact etiology of the condition. large epidemics of jaundice, variously called "catarrhal jaundice," "infectious hepatitis," "epidemic hepatitis," or similar, were mainly associated with military campaigns and were a significant cause of morbidity and mortality among troops in the napoleonic wars, the american civil war, and both world wars. it was during the second world war that the first evidence of the viral etiology of epidemic jaundice, and the existence of distinct forms of the condition, emerged. a series of experiments in germany, the united kingdom, and the united states throughout the 1940s used filtered materials from infected individuals to infect volunteers, or in some cases prisoners [1] . by the end of the decade, these studies, along with epidemiological studies, had elucidated two subtypes of viral hepatitis, distinguished by the primary route of transmission and period of incubation: orally transmitted "infectious hepatitis," with a short incubation period, and parenterally transmitted "serum jaundice," with a prolonged incubation period. these were later termed hepatitis a and hepatitis b, respectively. it was the former that would be blamed for the large waterborne outbreaks of hepatitis that had plagued humankind since antiquity. vol. 42, no. 21 november 1, 2020 www.cmnewsletter.com introduction by the end of the 1970s, however, evidence of another pathogen causing epidemics of hepatitis started to emerge. work done during the preceding decade at the u.s. national institutes of health had already demonstrated that most cases of transfusion-associated hepatitis were due to neither hepatitis a virus (hav) nor hepatitis b virus (hbv). the cause of these cases of non-a, non-b hepatitis would eventually be identified as hepatitis c virus (hcv). an outbreak of hepatitis in kashmir, india, in 1978 provided the first evidence of another hepatitis virus. the epidemic was waterborne, and large, with around 52,000 cases and 1,700 deaths [2] . the mode of transmission and clinical presentation were generally in keeping with hepatitis a; one key distinguishing feature was the excess morbidity and mortality seen among pregnant women. around the same time, another group was examining serum samples from three previous indian hepatitis outbreaks, including a large epidemic in delhi in 1955 [3] . in all cases, serological testing of infected individuals found no evidence of infection with hav. further outbreaks of non-transfusion-associated non-a, non-b hepatitis were identified in other parts of asia, north africa, and the middle east [3] . however, as no causative agent had been identified, it was impossible to determine if the same pathogen was involved in each outbreak. still, it was becoming increasingly clear that clinically apparent hepatitis a was actually very rare in developing countries [3] . a few years later, a russian virologist named mikhail balayan was investigating an outbreak of non-a, non-b hepatitis among soviet troops stationed in tashkent, the capital of what is now uzbekistan. balayan wanted to take clinical samples back to moscow for further study, but that would have meant refrigerating the samples. as the infrastructure to do this was lacking, balayan instead ingested a pooled filtrate of samples from his patients [3] . upon returning to moscow, he developed a case of acute hepatitis. electron microscopy of his stool identified viral particles that caused a hepatitis-like illness when experimentally inoculated into cynomolgus monkeys. crucially, these novel viral particles did not react with anti-hav igm. by the start of the 1990s, the genome of the novel non-transfusion-associated non-a, non-b hepatitis virus had been sequenced, and it had been named hepatitis e virus (hev) [3] . hav and hev can cause diseases that are clinically indistinguishable from each other, and despite being only distantly related, share some remarkable similarities in terms of the pathogenic strategies they employ. however, they are quite distinct in many other respects. this article reviews their similarities and differences, comparing the epidemiologies, clinical manifestations, diagnosis, treatment, and prevention of the two viruses. hav is a member of the family picornaviridae and was initially placed in the genus enterovirus. however, further study demonstrated that the virus was sufficiently different from other picornaviruses to be classified within its own genus, hepatovirus. hev was initially considered to be a member of the family calciviridae but was later reclassified as a member of the genus orthohepevirus, in the family hepeviridae. hav is one of nine species of hepatovirus and the only one known to infect humans. there are six genotypes of hav, three that infect humans and three affecting simians, but only one serotype [4] . the remaining members of the genus infect a range of species, including bats, hedgehogs, shrews, and rodents. phylogenetic analysis suggests that the genus originated in small mammals and that human hav has a rodent origin [4] , although a zoonotic reservoir no longer exists. the genus orthohepevirus contains four species (a to d). human disease is caused by orthohepevirus a, which has eight genotypes. the remaining three species are found in birds (hev-b), rodents and ferrets (hev-c), and bats (hev-d). two genotypes of orthohepevirus a are obligate human pathogens (hev1 and hev2), and two are endemic in a wide range of species and cause zoonotic infections in humans (hev3 and hev4). the remaining genotypes are primarily restricted to wild boar (hev5 and hev6) and camels (hev7 and hev8). however, human hev7 infection has been reported [5] , and hev5 is capable of infecting primates [6] . both hav and hev are non-enveloped icosahedral viruses. the lack of a lipid envelope offers both viruses a significant advantage in terms of their ability to spread in the environment, as demonstrated by the foodborne and waterborne outbreaks, which are synonymous with both hepatitis a and e [3, 7] . this is because a stable, naked protein capsid offers the fragile rna genome significant protection against harsh environmental conditions. in comparison, the transmission of enveloped viruses tends to require at least close contact between individuals, if not the exchange of bodily fluids. this is because the viral envelope contains virus-encoded glycoproteins called peplomers, which mediate interactions with cell surface receptors. without these peplomers, the virion is unable to gain access to a host cell, so it is vulnerable to anything that would disrupt the lipid bilayer, such as drying, solvents, or detergents. within a host, however, enveloped viruses have several advantages over naked virions. the envelope facilitates crossing the plasma membrane, allowing new virions to leave the cell without the need for cell lysis, and also protects the virus from the immune response by hiding antigens from neutralizing antibodies [8] . there is a significant body of evidence that suggests that although both hav and hev are non-enveloped viruses, they can also enjoy at least some of the benefits of enveloped viruses. hav and hev virions, which are shed in the stool, are naked protein capsids, ideally suited to their role of reaching new hosts across both time and distance in a potentially hostile environment [8] . however, hav and hev, which are isolated from the serum of individuals suffering an acute infection, are wrapped in a hijacked layer of host cell membrane, similar to those found on classical enveloped viruses but distinguished by the lack of any virusencoded proteins at the surface [8] . this allows circulating virions to avoid the immune response, as antigenic proteins are protected from neutralizing antibodies [8] . however, the lack of peplomers raises questions as to how these quasi-enveloped virions achieve entrance into host cells [8] . the world health organization estimates that there are 1.4 million cases of hepatitis a globally each year, resulting in approximately 7,000 deaths [9] . in comparison, there are an estimated 20 million hev infections each year, leading to 3.3 million symptomatic cases and around 44,000 deaths [10] . these figures are concerned only with parts of the world where hev is endemic and, as such, are likely to represent a gross underestimate of the actual global disease burden [11] . the primary route of transmission for hav is fecal-oral, primarily through direct person-to-person contact, but also via contaminated food or water. men who have sex with men are at increased risk of infection, as are any persons engaging in oral-anal sexual contact regardless of gender or sexual orientation [1] . parenteral transmission via contaminated blood products has been described [1] , and injecting drug users are at high risk [1] , with increased prevalence positively correlated with low incomes [1] . infected individuals shed virus in their stool for around 2 weeks before becoming symptomatic and typically for a few days after but may continue to do so for several weeks. even with good standards of hygiene and sanitation facilities, the rate of infection in close contacts of cases is high, suggesting very efficient interpersonal transmission. the rate and pattern of hav transmission vary widely between different parts of the world, primarily determined by socioeconomic factors. in regions with smaller family sizes, better sanitation facilities, and greater access to clean drinking water, rates of infection are lower. counterintuitively, lower rates of transmission do not equate to less disease. in resource-poor areas of high endemicity, such as africa, parts of asia, and south america, infection with hav in early childhood is widespread. in most cases, young children are asymptomatic or experience a very mild illness, and hav infection typically confers lifelong immunity. conversely, in high-income regions of low endemicity, like north america, western europe, japan, and australia, exposure in childhood is rarer. as a result, a much smaller proportion of the adult population has anti-hav antibodies. if hav is introduced, significant outbreaks can result, particularly in high-risk groups, such as men who have sex with men [12] , homeless people, and recreational drug users [13] . these outbreaks primarily affect adolescents and adults, who are more susceptible to becoming seriously ill. over the past few decades, improvements in hygiene and sanitation in some lowand middle-income countries have reduced hav transmission and increased the average age at infection [14] . this "epidemiological transition" shifts the epidemiological pattern closer to that seen in industrialized nations, producing a paradoxical increase in both morbidity and mortality associated with hepatitis a [15] . the epidemiology of hav depends upon the genotype involved and, by extension, the geographical region under examination. as mentioned above, human hepatitis e is predominantly caused by four of the eight genotypes of orthohepevirus a. hev1 and hev2 are similar to hav in that they are endemic in lower-income countries [16] , infect only humans, and are spread via the fecaloral route. however, unlike hav, infection with hev does not confer lifelong immunity. this results in both sporadic cases and periodic outbreaks, which occur periodically when anti-hev igg seroprevalence in the population drops below a critical threshold for herd immunity [17] . hev3 and hev4, in contrast, are zoonoses that infect a wide range of mammalian species; however, pigs constitute the primary viral reservoir [16] . hev3 causes locally acquired infections in europe, north america, australasia, and japan [18] . hev4 has historically been restricted to china and japan [11] . transmission between pigs occurs via the fecal-oral route, and infection is typically apathogenic in the animals. consumption of infected meat is the primary vector for human infection, and hev has been identified in retail pork products at the point of sale [16] . a range of other foods have also been implicated, with viruses isolated from shellfish, fruits, and vegetables [18] ; this is likely due to pig slurry contaminating watercourses or being used as fertilizer. more than two-thirds of zoonotic hev infections are asymptomatic [19] . of the remaining cases, only a small fraction are confirmed by serological or molecular testing; historically, most are either misdiagnosed or go unrecognized. in recent years, however, there has been a surge in reported cases. the number of laboratory-confirmed cases in europe increased by a factor of 10 between 2005 and 2015 [20] . in part, this likely reflects increased awareness of hev among clinicians, but in at least some countries, there has been an actual increase in incidence [21] . reports from several different countries have described parenteral hev transmission via infected blood products [11] . studies have also demonstrated viremia at the time of donation among healthy blood donors, with a wide variation in the rates of viremia in different countries, from 1:27 in india [22] to 1:74,131 in australia [23] . the extent of the contribution that transfusion-associated hev infection makes to the global burden of disease is unclear, but it certainly presents less risk than the primary modes of transmission. however, patients who are at risk of chronic infection or more severe hepatitis are over-represented in the cohort of patients who are most likely to receive blood products. this includes transplant recipients, immunocompromised patients, and pregnant women. in response to this, a growing number of european countries now routinely screen blood donations for hev [11] . in the last 2 years, evidence has emerged of a new zoonotic source of hev infection. as mentioned above, hev-c affects rodents. genotype 1 of hev-c (hev-c1) circulates in rats in europe, asia, and north america. a recent large prospective study in hong kong identified both acute and chronic hev-c1 infection in both immunocompetent and immunocompromised patients [24] . extrahepatic manifestations were also described; one patient developed meningoencephalitis and died, with hev-c1 rna found in their cerebrospinal fluid (csf) [24] . the presence of anti-hev-a antibodies does not appear to offer any protection, and molecular testing for hev-a does not detect hev-c1 due to sequence differences between the species. these findings suggest that hev-c1 could be prevalent around the world and yet be routinely missed, which would have important clinical implications and would pose a threat to the safety of blood products. clinical symptoms of hepatitis a typically occur following an incubation period of 14 to 28 days but can present as much as 50 days after exposure (table 1 ) [1] . the clinical presentation ranges from asymptomatic to fulminant hepatitis [25] . the disease course is typically more severe with increasing age; very young children often have no symptoms at all. the typical presentation involves two phases: a prodromal phase that lasts for 3 to 10 days and is characterized by malaise and myalgia, followed by an icteric phase [25] . the icteric phase involves a mixed hepatic and cholestatic jaundice associated with anorexia, nausea, and fatigue. this stage lasts between 1 and 3 weeks. acute liver failure occurs in approximately 0.3% of cases, although it is highly age dependent; in children and adults less than 40 years of age, the case fatality rate is between 0.1% and 0.3%, while in adults over 49 years of age, it is 1.8% [26] . co-existing chronic infection with hbv or hcv increases the chance of acute liver failure due to infection with hav [26] . a subset of patients with hepatitis a may present atypically [27] . up to 5% can develop cholestatic hepatitis, which includes a prolonged period of jaundice lasting 12 weeks or more [28] . the typical clinical course in these patients involves significant jaundice, pruritis, fever, weight loss, diarrhea, and malaise [27, 28] . liver function tests (lfts) show a cholestatic picture, with marked elevation of bilirubin and alkaline phosphatase and a mild to moderate rise in alanine aminotransferase (alt). generally, patients with cholestatic hepatitis require only supportive treatment and go on to make a full recovery. a further 10% of patients experience a relapse of hepatitis a in the 6 months following the primary infection [27, 29] . following an apparent initial recovery, there is a biochemical relapse that may or may not be accompanied by clinical deterioration. alt levels can exceed 1,000 iu/dl, and patients generally remain anti-hav igm seropositive through the course of the illness [29] . there are detectable levels of virus in the stool during relapses, so affected patients should be considered infectious. when symptoms recur, they are typically milder than the initial illness and of short duration, usually less than 3 weeks [29] ; however, it can take as long as a year for full biochemical resolution. it is not known what causes some patients to relapse, and no risk factors have been identified [29] . multiple relapses can occur, but most patients go on to make a full recovery. most patients who develop clinically apparent hepatitis e experience an acute, self-limiting illness lasting 4 to 6 weeks [11] . after an incubation period of between 2 and 10 weeks, patients develop features typical of hepatitis-jaundice, fatigue, fever, abdominal pain, nausea, and vomiting. in developing countries where hev1 and hev2 are predominant, young adults are the most commonly affected group. males are more likely to present clinically than women. overall mortality is between 0.2% and 4% [16] but is significantly higher in vulnerable groups, including children under 3 years of age [30] , individuals with pre-existing liver disease [31] , and pregnant women (see below). in higher-resource settings, locally acquired cases are caused by hev3 and hev4. there is significant heterogeneity in the clinical picture of acute infection in these areas; only a small minority of patients present with typical viral hepatitis as described above. despite this, hev is the most common cause of viral hepatitis in several european countries, including france, germany, and the united kingdom [32] . as with hev1 and hev2, genotypes 3 and 4 preferentially affect men, with a male-to-female ratio of around 3:1, although they tend to be older, with a median age of [33] . it this thought that this is a result of host factors rather than due to variations in exposure. pre-existing subclinical liver disease has been proposed as a potential risk factor, as both alcohol excess and diabetes, which are associated with hepatic steatosis and fibrosis, are over-represented among patients with acute hepatitis e [34] . progression to liver failure is generally uncommon; however, a small number of cases have been reported, and a german study identified hev as the most likely precipitant of acute liver failure in 10% of a cohort of 80 patients. individuals with pre-existing liver disease are at risk of decompensation or acute-on-chronic liver failure. however, hepatitis e is not as common a cause of decompensation in industrialized countries [35, 36] . this likely represents differences in the pathogenicity of the genotypes, which are found in different regions. one of the most important public health challenges related to acute hepatitis e infection, which most commonly occurs in developing countries, is the excess morbidity and mortality seen among pregnant women (table 1 ). in these parts of the world, where hev1 is the predominant genotype, around a quarter of pregnant women with acute hepatitis e die [37] . this highest-risk period is the third trimester, with 33% of women infected during that stage of pregnancy developing fulminant hepatic failure [38] . other than liver failure, the major causes of hev-related maternal death are obstetric complications, such as eclampsia or hemorrhage [38] . there is also a significant risk to both the fetus and neonates. vertical transmission is common [39] , and there is a substantial increase in the risk of intrauterine death, stillbirth, pre-term birth, and low birth weight [40] . fetal mortality is approximately 33%, including those who die as a result of maternal death, and neonatal mortality is around 8% [37] . the mechanisms that underlie the increased disease severity in pregnant women are not well understood. pathogenic differences between hev genotypes may play a part, as the rates high of mortality and morbidity are seen with hev1 and have not been described in the context of hev3 infection. both hev1 and hev3 are capable of infecting the decidua basalis (i.e., the uterine endometrium at the site of embryo implantation) and the placenta. however, hev1 replicates with greater efficiency in vitro in tissue explants and stromal cells from both tissues and is associated with increased tissue damage [41] . hev1 also affects the secretion profile of the maternal-fetal interface, increasing the release of pro-inflammatory factors [41] . both the viral load and serum inflammatory cytokine levels (tumor necrosis factor alpha, transforming growth factor î²1, interleukin 6 and interferon gamma [ifn-î³]) are higher in pregnant women than in non-pregnant women infected with hev1, and levels of the cytokines correlate positively with adverse pregnancy outcomes [41] . other studies have shown that high levels of hev rna are also associated with worse outcomes in pregnancy [41] . while hav does not cause chronic infection, persistent infection with hev has been documented. the majority of the literature on the subject describes solid organ transplant recipients, but chronic infection has been reported in other immunocompromised cohorts, as well, including in hiv-positive patients with cd4 + counts of <200/mm 3 , in individuals with hematological malignancies receiving chemotherapy and stem cell transplants, and in rheumatology patients treated with immunosuppressive drugs [11] . in almost all cases, chronic infection involves hev3 or hev4, but there has been a single report of chronic hev7 infection in a patient who had consumed camel meat and milk [5] . chronic infection is defined as hev replication persisting for 3 months or more [11] . around two-thirds of solid organ transplant recipients who are exposed to hev develop a chronic infection [11] . the risk is increased for those with immunosuppression and for those who are treated with tacrolimus. most chronic infections are asymptomatic, with only a mild or moderate rise in alt. some patients have normal lfts, and some remain seronegative for anti-hev antibodies despite active viral replication [11] . without treatment, the development of fibrosis can occur, with a risk of rapid progression to cirrhosis, decompensation, and death [11] . extrahepatic manifestations of hav infection are most commonly reported in patients who experience cholestatic hepatitis or relapsing hepatitis a. between 10 and 15% of patients develop a rash and/or arthralgia, but a range of rarer complications have also been described. they include vasculitis, cryoglobulinemia, and thrombocytopenia [42, 43] . a broad range of extrahepatic manifestations (table 2 ) have been reported in the context of both acute and chronic hev infection, the most important of which are neurological and renal complications. the mechanisms that underlie these manifestations are not currently understood, but both immune-mediated processes and direct viral tropism have been suggested. neurological injury is the most frequently reported extrahepatic complication of hev infection, with around 150 cases involving hev1 or hev3 currently described [44] . in all cases, it is the neurological signs and symptoms that dominate the clinical picture. patients are typically anicteric and have either normal liver enzymes or at most mild to moderately deranged lfts. a wide variety of neurological illnesses have been reported in association with hev (table 2 ), but the mostly strongly associated conditions are neuralgic amyotrophy (na), guillain-barrã© syndrome (gbs), and encephalitis/myelitis. na is an acute monophasic injury to the brachial plexus that results in pain, weakness, and sensory disturbance in the upper limbs. in a study of dutch and english patients with na, 10.6% had evidence of acute hev infection at the onset of their neurological illness [45] . hev-associated na has a characteristic clinical phenotype. the symptoms tend to be more severe, with more extensive bilateral damage to the brachial plexus than is seen in non-hev-associated cases [46] . an international multi-center study in europe that prospectively tested 464 patients with acute, non-traumatic neurological injury found evidence of hev infection in 2.4% of the patients. there were three cases of na; all three had evidence of infection, and all three displayed the characteristic clinical phenotype of hev-associated na [47] . gbs is an immune-mediated polyradiculopathy involving rapidly progressive muscle weakness, which can lead to respiratory failure. it is considered a post-infective condition, and campylobacter jejuni is the most common precipitant. the causative organism was not identified in over 50% of the cases [48] . the earliest reports of hev-associated neurological illness involved gbs, following the observation that around a third of dutch gbs patients had lft derangement without an apparent cause [44] . three case-control studies from the netherlands, bangladesh, and japan found evidence of recent hev infection at significantly higher rates among gbs patients than in control subjects [44] . twelve cases of hev-associated encephalitis/myelitis have been reported in europe, asia, and the u.s. [44] . five of them were chronically infected solid organ transplant recipients. five patients developed ataxia, which appears to be associated with worse outcomes; two of the ataxic patients died, and the remainder had a more significant long-term neurological deficit than those who did not have ataxia. six patients had hev rna in both blood and csf. in one case, the virus isolated from csf showed evidence of quasispecies compartmentalization, which may suggest the emergence of directly neurotropic strains [49] . renal injury has been described in the context of both acute and chronic hev infection. renal biopsies of patients with hevassociated renal impairment show evidence of membranoproliferative glomerulonephritis (mpgn), cryoglobulinemia, and membranous glomerulonephritis [41] . hev rna has also been isolated from the cryoprecipitate of an immunocompetent patient who presented with acute hev infection and mpgn [41] . once viral clearance is achieved, renal function and proteinuria improve in most patients [41] . the differential diagnosis of acute hepatitis is extensive. in addition to the five hepatitis viruses, there are several other viral causes, such as cytomegalovirus and epstein-barr virus. non-viral infectious causes include bacterial infections, such leptospirosis, rocky mountain spotted fever, and typhoid, or parasitic infection with liver flukes or roundworms. non-infectious causes include autoimmune hepatitis, systemic lupus erythematosus, drug-induced liver injury-iatrogenic or secondary to deliberate self-harmand toxins, most commonly alcohol. for several reasons, hev infection may not be considered a potential diagnosis. as it is still often incorrectly regarded as an "emerging" disease, many clinicians have limited knowledge of the disease. the heterogeneous presentation of hev infection, especially in developed countries, further compounds the problem. even when hev infection is considered, there are issues surrounding the availability and reliability of testing. in the u.s., there is no u.s. food, and drug administration-approved assay on the market. where they are available, there is significant variation in the sensitivity and specificity of tests [50] , and the previous "gold standard" test has been shown to substantially underestimate seroprevalence in comparison to later-generation assays [51] . in chronic hev infection, as previously described, patients are often seronegative for anti-hev antibodies, so molecular analysis must be used to detect hev rna directly (fig. 1a) . this type of analysis can be less useful in acute infection, as the period of viremia can be very narrow. however, virions are shed in the stool for a longer period. in contrast, highly sensitive and specific serological assays for anti-hav igm have been available for more than 40 years (fig. 1b) [1] . specific igm remains positive for a variable period of time following infection, disappearing from the sera of most patients within 7 months but remaining for up to a year in some individuals [1] . anti-hav igg is seropositive in both current and past infections, as it typically remains present in the serum for life. for both hepatitis a and acute hepatitis e, the majority of cases require no specific treatment. the minority of patients who develop fulminant hepatic failure should receive aggressive supportive therapy and be considered for liver transplantation. a small number of patients with severe, acute hepatitis e have been treated with ribavirin, producing a rapid resolution of liver enzyme derangement and viral clearance [11] . it is, however, difficult to draw conclusions from these reports due to the lack of controls and range of different treatment protocols employed. in chronic infections in immunosuppressed transplant recipients, the first-line treatment is the reduction of immunosuppressive drugs, particularly those that target t cells. around 33% of patients clear the virus without any other intervention [11] . for the remaining patients, ribavirin may be useful. the optimal duration of treatment is still to be fully determined, but the european association for the study of the liver guidelines suggest an initial 3-month course followed by a further 6-month course for those who fail to clear the virus after the first course [11] . a large retrospective study suggested that around 81% of patients achieve a sustained virologic response following one course of treatment, rising to nearly 90% after a second [52] . pegylated ifn-î± can be considered for liver transplant recipients who cannot tolerate ribavirin or fail to respond but is generally contraindicated in patients who have received other organs as the risk of rejection is increased [11] . in other immunosuppressed cohorts, successful treatment with ribavirin, ifn-î±, or a combination of the two has been described [11] . the main methods by which the spread of hav can be prevented are good hygiene practices, proper sanitation, case investigation and contact tracing during outbreaks, and active and passive immunoprophylaxis. thorough hand washing and careful food handling practices are of great importance. postexposure prophylaxis can be considered for close contacts of infected individuals, subject to local guidelines. this can consist of either vaccination or intramuscular immune globulin. the u.s. centers for disease control and prevention recommends vaccination for healthy people aged 12 months to 40 years; immune globulin for those aged over 40, although vaccination is acceptable; and immune globulin for children under 12 months, immunocompromised individuals, patients with chronic liver disease, and anyone with contraindications for vaccination [53] . screening for hav should be offered to at-risk individuals in sexual-health settings, mainly targeting men who have sex with men, intravenous drug users, and patients with hiv, hbv, or hcv disease [54] . patients who test negative should be offered vaccination [54] . in regions where hev1 and hev2 are endemic, the key prevention strategies surround improving sanitation facilities and access to clean, safe drinking water. in resource-rich settings, where zoonotic transmission is the major route of infection, proper preparation of food products is the primary preventive measure. meat products, in particular pork and game, should be thoroughly cooked [11] . anyone with risk factors for developing a more severe illness, such as those with pre-existing liver disease or immunosuppressed individuals, should take particular care to avoid uncooked meat. people whose employment brings them into contact with pigs, boar, or deer and their products should take care to minimize direct contact and use appropriate protective equipment. an effective vaccine against hev, designated hev-239, has been on the market in china for several years [11] . the vaccine has been designed to offer long-term protection against all genotypes of hev but has yet to be licensed outside china. hav and hev share many similarities but are distinguishable from each other in many ways. they occupy similar ecological niches and have developed similar characteristics and strategies despite not being closely related. their status as quasi-enveloped viruses has not been seen in any other virus and likely represents an adaptation to take advantage of the secretory pathways that are accessible from hepatocytes. both viruses present public health challenges to both high-and low-income countries. the changing epidemiological patterns that hav displays in response to socioeconomic progress in developing countries require careful attention. interventions, such as universal childhood vaccination programs, must be properly implemented and timed correctly. if a vaccination program introduced to a low-endemicity region achieved inadequate coverage, it would likely exacerbate the epidemiological transition it was intended to ameliorate. our understanding of hev, particularly the zoonotic genotypes, remains inadequate, and the covid-19 pandemic has brought the risks presented by emerging zoonotic infections into sharp focus. there is mounting evidence that hev is significantly underdiagnosed, particularly in the context of its many extrahepatic manifestations. further attention is required to elucidate the actual burden of disease presented by hev and to better understand the risks presented by potential novel zoonoses, such and hev-c1. hepatitis a: old and new discovery of hepatitis e: the epidemic non-a, non-b hepatitis 30 years down the memory lane hepatitis e: an emerging awareness of an old disease evolutionary origins of hepatitis a virus in small mammals chronic infection with camelid hepatitis e virus in a liver transplant recipient who regularly consumes camel meat and milk genotype 5 hepatitis e virus produced by a reverse genetics system has the potential for zoonotic infection new developments in an old disease naked viruses that aren't always naked: quasi-enveloped agents of acute hepatitis european association for the study of the liver. electronic address: easloffice@easloffice.eu; european association for the study of the liver. easl clinical practice guidelines on hepatitis e virus infection hepatitis a outbreak disproportionately affecting men who have sex with men (msm) in the european union and european economic area hepatitis a virus outbreaks associated with drug use and homelessness hepatitis a virus seroprevalence by age and world region increasing incidence of hepatitis a in korean adults transmission of hepatitis e virus in developing countries hepatitis e: an underestimated emerging threat high proportion of asymptomatic infections in an outbreak of hepatitis e associated with a spit-roasted piglet european centre for disease prevention and control. hepatitis e in the eu/eea hepatitis e virus (hev) in scotland: evidence of recent increase in viral circulation in humans hepatitis e virus infection may be transmitted through blood transfusions in an endemic area hepatitis e virus rna in australian blood donors: prevalence and risk assessment transmission of rat hepatitis e virus infection to humans in hong kong: a clinical and epidemiological analysis hepatitis a: clinical manifestations and management clinical course and management of acute hepatitis a infection in adults atypical clinical manifestations of hepatitis a prolonged intrahepatic cholestasis secondary to acute hepatitis a relapsing hepatitis a. review of 14 cases and literature survey hepatitis e epidemic hepatitis e virus (hev) infection in patients with cirrhosis is associated with rapid decompensation and death hepatitis e virus: assessment of the epidemiological situation in humans in europe, 2014/15 autochthonous hepatitis e in southwest england: natural history, complications and seasonal variation, and hepatitis e virus igg seroprevalence in blood donors, the elderly and patients with chronic liver disease host risk factors and autochthonous hepatitis e infection hepatitis e virus in patients with decompensated chronic liver disease: a prospective uk/french study hepatitis e infection in patients with severe acute alcoholic hepatitis hepatitis e during pregnancy: maternal and foetal case-fatality rates and adverse outcomes-a systematic review hepatitis e in pregnancy clinical course and duration of viremia in vertically transmitted hepatitis e virus (hev) infection in babies born to hev-infected mothers burden of hepatitis e virus infection in pregnancy and maternofoetal outcomes: a systematic review and meta-analysis clinical manifestations, pathogenesis and treatment of hepatitis e virus infections arthritis, vasculitis, and cryoglobulinemia associated with relapsing hepatitis a virus infection thrombocytopenia in hepatitis a-an atypical presentation hepatitis e virus and neurological injury neuralgic amyotrophy and hepatitis e virus infection clinical phenotype and outcome of hepatitis e virus-associated neuralgic amyotrophy hepatitis e virus infection and acute non-traumatic neurological injury: a prospective multicentre study the spectrum of antecedent infections in guillain-barrã© syndrome: a case-control study hepatitis e virus-induced neurological symptoms in a kidney-transplant patient with chronic hepatitis hepatitis e seroprevalence in europe: a meta-analysis two generations of "gold standards": the impact of a decade in hepatitis e virus testing innovation on population seroprevalence ribavirin for hepatitis e virus infection after organ transplantation: a large european retrospective multicenter study prevention of hepatitis a virus infection in the united states: recommendations of the advisory committee on immunization practices british association for sexual health and hiv. 2017 interim update of the 2015 bashh national guidelines for the management of the viral hepatitides key: cord-323463-osf6t7cw authors: cercenado, emilia; garau, javier; almirante, benito; ramón azanza, josé; cantón, rafael; cisterna, ramón; maría eiros, josé; fariñas, carmen; fortún, jesús; gudiol, francisco; mensa, josé; pachón, jerónimo; pascual, álvaro; luis pérez, josé; rodríguez, alejandro; sánchez, miguel; vila, jordi title: update on bacterial pathogens: virulence and resistance date: 2008-04-30 journal: enfermedades infecciosas y microbiología clínica doi: 10.1016/s0213-005x(08)76378-x sha: doc_id: 323463 cord_uid: osf6t7cw the present article is an update of the literature on bacterial pathogens. recognizing the interest and scientific and public health importance of infections produced by bacterial pathogens with new virulence mechanisms and/or new mechanisms of resistance to antimicrobial agents, a multidisciplinary group of spanish physicians and microbiologists organized a joint session and revised the most important papers produced in the field during 2006. each article was analyzed and discussed by one of the members of the panel. this paper focus on a variety of diseases that pose major clinical and public health challenges today; and include infections produced by community-acquired methicillin-resistant staphylococcus aureus and s. aureus small colony variants, infections produced by multiply resistant coagulase-negative staphylococci, pneumococcal infections, human listeriosis, meningococcal disease, haemophilus influenzae, pertussis, escherichia coli, esbl-producing organisms, and infections due to non-fermenters. after a review of the state of the art, papers selected in this field are discussed. although predictions during the 20th century indicated that the incidence of infectious diseases would diminish as a result of improvements in sanitation and by the introduction of many vaccines and antibiotics, at the beginning of the 21st century the rates of infections produced by new pathogens or by reemerging microorganisms possessing new virulence or resistance phenotypes is increasing, threatening the overall human health [1] [2] [3] [4] [5] . over the last 100 years we have moved from the pre-antibiotic era through the antibiotic era into the era of emerging infectious diseases. the discovery of new infectious diseases and the steady increase in the number of microorganisms that are resistant to multiple antimicrobial agents have altered the practice of medicine within the hospital, and are affecting the management of infections in the ambulatory care setting 6, 7 . it is in this scenario where community-acquired methicillin-resistant staphylococcus aureus (ca-mrsa) has emerged as the most common pathogen isolated from patients with skin and soft-tissue infections attending to the emergency departments in many united states and australian cities 8, 9 , and at present, its incidence is increasing in other parts of the world 10, 11 . despite the growing prevalence of mrsa in hospitals, these strains have been uncommon in the community. in many circumstances mrsa escape into the community when patients still harbouring the organisms are discharged or when hospital personnel go from work to home. these strains can be associated with infections that begin in the community, but the isolates are hospital-associated. however, there is now an increasing number of reports of mrsa infections in the community that have not escaped from the hospitals, and no longer can mrsa be considered as an exclusively nosocomial pathogen. there is molecular evidence that strains of mrsa have also evolved in the community, are well adapted to survive there, and are causing an epidemic outside the hospitals 12, 13 . ca-mrsa infections refer to mrsa infections in patients lacking established mrsa risk factors and without a previous history of mrsa infection or colonization: they do not have a medical history in the past year of hospitalization, healthcare-related admission (to a nursing home, skilled nursing facility or hospice), dialysis, surgery, or implantation of a permanent indwelling catheter or other medical devices 8, 14 . ca-mrsa tends to cause infections that occur in clusters or small outbreaks that affect otherwise healthy and unique populations such as children and young adults, australian aborigines, native americans, alaskan natives, prisoners, military recruits, men who have sex with men, college athletes and players competing in contact sports. recent antimicrobial exposure is less likely to be reported as a risk for mrsa infections in the community that in health care settings. most infections are mild and limited to skin and soft tissues, though rapidly fatal invasive infections such as necrotizing fasciitis and overwhelming pneumonia and sepsis accompanied by the waterhouse-friderichsen syndrome do occur and may serve as "sentinels" that first bring a more widespread community problem to the attention of public health personnel [15] [16] [17] [18] [19] [20] . because skin infections caused by these organisms often have necrotic centers, the disease can be misdiagnosed as a "spider bite". similar to the epidemiology of methicillinsusceptible s. aureus, ca-mrsa infections occur in children in different regions of the united states and throughout the world 11, [21] [22] [23] [24] . although minor skin and soft-tissue infections predominate, life-threatening invasive disease and death can result. in children, ca-mrsa can cause septic thrombophlebitis of the extremities and a "pelvic syndrome" consisting of septic arthritis of the hips, osteomyelitis of the pelvic bones, pelvic abscesses, and septic thrombophlebitis 6 . unlike hospital strains, which typically are resistant to multiple antimicrobial agents and share a common genotype with other hospital isolates, community acquired strains tend to be susceptible to non-betalactams and have genotypes distinct from hospital isolates in the same community 12 . two main clones (usa 400 and usa 300, belonging to st1 and st8 by mlst, respectively) have been described as responsible for the majority of infections caused by ca-mrsa throughout the united states 12, 25 . in other continents, the most frequent sts of ca-mrsa are the st30 for the southwest pacific clone, and the st80 for the european clone, although in europe the st8 and st30 clones have also been described 13 . the meca gene, the genetic determinant necessary for the expression of oxacillin resistance, resides in these strains on genetic elements (usually sccmec types iv and v) that are different from those encoding methicillin resistance in hospital-acquired mrsa, although in recent years sc-cmec type iv has been frequently found in nosocomial mrsa isolates mainly in europe 9, 26, 27 . another key difference is that almost all ca-mrsa isolates contain genes encoding the panton-valentine leukocidin, which is a cytotoxin that causes leukocyte destruction and tissue necrosis. although the exact role of panton-valentine leukocidin in the serious infections caused by these organisms is unclear, it is in general a marker for ca-mrsa 13, 28, 29 . a hypothesis in order to explain the origins of ca-mrsa is that the meca gene or the sccmec have been transferred horizontally to one or more previously oxacillin-susceptible s. aureus strains that occupy traditional community niches. this possibility does account for the distinct phenotypic and genotypic characteristics of ca-mrsa. the relatively long time lag from the appearance of mrsa in hospitals to its emergence in the community may in part be due to the low frequency of horizontal chromosomal gene transfer 26 . at present, the knowledge of ca-mrsa epidemiology is incomplete which adds to the challenge of controlling the infection. in addition, nasal colonization may not precede ca-mrsa infections and since nasal carriage is not the prime predictor of subsequent infection it is more difficult to identify and control populations that are at risk. colonization of the gastrointestinal tract and of household pets may act as additional reservoirs for ca-mrsa 30, 31 . moreover, ca-mrsa are now being introduced into hospitals, thus blurring the borders between community-acquired and hospital-acquired strains 25 . therapy for infections due to ca-mrsa includes appropriate drainage of skin and soft-tissue lesions, since milder infections often respond to local incision and drainage alone 15 . the clinician should know when to use drugs with activity against ca-mrsa (clindamycin, minocycline, doxycycline, trimethoprim-sulfamethoxazole or vancomycin), although optimal antimicrobial agent therapy is unknown. for severe and invasive infections therapy with vancomycin or linezolid should immediately start since antimicrobial agents may be ineffective when the patient receives treatment too late 6, 15 . the recognition that partial vancomycin resistance may result in some vancomycin treatment failures for serious mrsa infections, portends the near-term loss of this firstline drug as the treatment of choice. vancomycin-intermediate s. aureus (visa) and heterogenous visa (hvisa) have become a significant problem in many parts of the world [32] [33] [34] . visa/hvisa isolates can arise from fully vancomycin-susceptible s. aureus during persistent infection that fails to respond to glycopeptide therapy and are associated with significant phenotypic changes 35 . resistance may develop by different pathways, but these strains have a thickened cell wall with reduced peptidoglycan crosslinking leading to cell wall "clogging" with vancomycin. moreover, a marked reduction in autolytic activity and reduced cell wall turnover have been found in visa /hvisa strains 36 . several studies have demonstrated a number of metabolic pathways and regulatory genes that may contribute to resistance 37 . in particular, the agr twocomponent regulatory system has been linked to low-level vancomycin resistance in s. aureus, with reports suggesting that agr type ii strains and loss of agr function are associated with visa 38 . it has also been noted that many reported visa infections have involved biomedical devices, and biofilm formation on these devices could be an important initial step in the pathway to vancomycin resistance 39 . an "emerging" pathogen can be seen as a well-known pathogen for which newly discovered subpopulations of the parent strain are able to produce disease. staphylococcus aureus small-colony variants (scvs) fall into this category. this variant subpopulation is defective in electron transport, grow slowly, and produce colonies < 10% the size of the parent strain grown on the same medium for the same amount of time. most of these clinical isolates cannot synthesize menadione or hemin, and these results in a block of electron transport at the level of menaquinone or the cytochromes 40 . scvs might seem to be less virulent because of their slow growth and decreased production of coagulase and alpha-toxin. however, the intracellular location of scvs shields them from host defenses, their slow growth reduces the efficacy of cell wall-active antibiotics, and a decreased membrane potential protects them from positively charged antimicrobials 41 . scvs represent a subpopulation of s. aureus that can cause persistent and recurrent infections. while their overall prevalence has not been firmly established, problems in discovering and identifying these organisms may cause an underestimation or their prevalence 42 . coagulase-negative staphylococci can also be seen as an "emerging" pathogen since it has acquired new virulence factors and resistance to new antimicrobials 43 . these microorganisms can colonize indwelling catheters (including central venous catheters) and form biofilms, that can result in bloodstream infections. the organisms embedded in a matrix of extracellular polymeric substances that they have produced exhibit an altered phenotype with respect to growth rate and gene transcription. treatment of these infections with conventional antimicrobial agents alone is frequently unsuccessful due to the high tolerance of these agents in microorganisms comprising the biofilm 44 . several preventative and treatment approaches have been investigated for catheter-related infections including a recent renewed interest in the use of bacteriophages for mitigating biofilm formation on indwelling catheters 45 . recently, another cause of concern among coagulase-negative staphylococci is their resistance to new antimicrobial agents such as linezolid. although resistance to linezolid is very infrequent among staphylococci, these organisms are able to acquire de novo resistance among patients who have a long duration of hospitalization and that have received high amounts of linezolid. moreover, linezolid-resistant strains can be transmitted from patientto-patient with their establishment as part of the skin flora and may be a precursor of linezolid-resistant mrsa 46 . streptococcus pneumoniae is a cause of serious infections that are a major source of morbidity and mortality among all age groups in both the developed and the developing worlds. in addition, the emergence of strains with high-level resistance to penicillin and to other antimicrobial agents has raised concerns about the current and future effectiveness of antibiotic regimens. nevetheless, there is now evidence of decreasing resistance to some antimicrobials in some regions of the world. reduction in antimicrobial use in some community settings has been associated with a decline in resistance of pneumococci to some classes of antimicrobials, mainly beta-lactams. however, we are still witnessing a relentless increase in resistance to the macrolide class of antimicrobials 47 . fortunately, control of pneumococcal disease through vaccination with the 23-valent polysaccharide vaccine in adults has demonstrated a reduction in risk of bacteremic pneumococcal disease after vaccination, as well as decreased risk of death and complications of hospitalization, which reinforces the need to improve compliance with existing pneumococcal vaccination recommendations for adults. moreover, the introduction of the 7-valent pneumococcal conjugated vaccine not only prevents invasive disease in children but also appears to have resulted in a decline in the prevalence of resistant serotypes included in the vaccine, resulting in an overall decrease in the prevalence of pneumococcal resistance 48 . however, penicillin-nonsusceptible pneumococcal clones of nonvaccine serotypes have been reported as a cause of bacteremia and other infections. these clones may have been derived from capsular transformation of vaccine-related serotypes 49 . in recent years an upsurge in the incidence of human listeriosis seems to have occurred in some geographical areas. although most cases are foodborne, the epidemiology is complex. prevention of the disease must include dietary advice on avoiding high-risk foods routinely to pregnant women, to the elderly and to immunocompromised patients. listeriosis manifests primarily as abortion, septicemia, or central nervous system infections with a high case-fatality rate in all patient groups. listeria monocytogenes is the third most common cause of bacterial meningitis that occurs among immunocompromised patients and elderly individuals 50 . symptoms and signs of patients presenting with l. monocytogenes meningitis are not different from those found in the general population of patients with community-acquired bacterial meningitis, albeit with a longer prodromal phase; however typical cerebrospinal fluid findings predictive for bacterial meningitis might be absent, and gram stain has a low yield. in patients aged > 50 years old or with risk factors for l. monocytogenes meningitis, an amoxicillin-based empirical antimicrobial regimen is mandatory, since this bacterium is resistant to cephalosporins. despite the availability, for decades, of meningococcal vaccines, neisseria meningitidis remains a leading cause of meningitis, sepsis, and other serious infections in both industrialized nations and the developing world. group c meningococcal conjugate-vaccine effectiveness in the united kingdom declines from ~ 90% in the first year to 0% between 1 and 4 years after immunization in infants immunized at 2, 3, and 4 months of age and to 61% in toddlers given a single dose. a recent study 51 did not find evidence of lower immunity in children immunized as infants than as toddlers. on the basis of serum bactericidal activity and/or passive protection, 40-50% of both age groups are protected at 2-3 years after immunization, which is significantly greater than in unimmunized historical controls (< 5%). the study of snape et al 52 on antibody responses to either a reduced dose of meningococcal c polysaccharide vaccine, which is meant to simulate exposure to n. meningitidis, or meningococcal c conjugate vaccine in healthy 13-15-year-olds in the united kingdom who had been primed with a serogroup c conjugate vaccine 3-4 years previously, adds new data on the number of days required for antibody levels to increase after boosting with either of the 2 vaccines. in addition to susceptibility associated with flawed complement pathway function, several other pathways have also been convincingly linked to altered host susceptibility to meningococcal disease. the article by jack et al 53 breaks new ground in increasing the understanding of the human genetic basis for some host defence failures in meningococcal disease. surfactant protein (sp)-a and sp-d are pattern-recognition molecules of the respiratory tract that activate inflammatory and phagocytic defences after binding to microbial sugars. variation in the genes of the surfactant proteins affects the expression and function of these molecules. routine use of haemophilus influenzae type b (hib) conjugate vaccines has dramatically decreased the incidence of invasive hib disease in the western world. despite the effectiveness of the vaccine, an increase in the incidence of hib disease has recently been observed in some european countries. careful analysis of circulating hib strains is essential for prompt detection of any change in the properties of bacteria, enabling particular clones to overcome the host's immune response. in a recent italian study 54 , contrary to previous reports, neither increased genetic diversity of hib strains isolated from children nor the disappearance of individual clones was observed after the rountine immunization of infants against hib was established; however, an upward temporal trend in proportion of strains possessing multiple copies of the capsulation b locus was detected. the results of this study suggest that vaccine pressure may be positively selecting for strains that harbour amplified cap b sequences. interactions of nontypeable haemophilus influenzae (nthi) with human alveolar macrophages are implicated in the persistence of nthi in chronic obstructive pulmonary disease (copd). a recent study by sethi's group 55 confirmed the hypothesis that immunologic responses of alveolar macrophages to nthi are impaired in copd. the same group has described a phenotypic variant of nthi, haemophilus haemolyticus that frequently colonizes the airway of copd patients and is not found in normally sterile sites. these findings substantially strengthen the association of true h. influenzae with clinical infection and exacerbation of chronic bronchitis 56 . the control of pertussis infection is not optimal, because neither vaccination nor natural infection induces long-lived immunity. increasing proportions of reports of pertussis cases involve adolescents and adults. the aim of a recent us study was to define, among unimmunized control subjects, a yearly infection rate and to characterize the proportion of those infections that include prolonged coughs 57 . a similar analysis was attempted for acellular pertussis (ap)-vaccinated subjects. pertussis infections in older persons was found to be largely asymptomatic; ap boosters confer protection for adolescents and adults against symptomatic pertussis.the diagnosis of pertussis in older individuals is problematic because of the lack of specific clinical criteria, insensitivity of culture and pcr, and the limited availability of standardized serologic tests and criteria for diagnosis. a spanish study suggests that real time-pcr is the most sensitive and specific test available for the diagnosis of pertussis and that the direct fluorescent assay should be abandoned 58 . three recent studies in escherichia coli infections deserve comment. the first two deal with fluoroquinolone resistance in this species. the first shows the increasing prevalence of strains with reduced sensitivity to the quinolones in the gut of hospitalized patients 59 , and the second analyses the differences and similarities of fluoroquinolone-resistant e. coli from humans and from chicken. the similarities among the strains studied strengthen the hypothesis of an animal origin of the human strains of fluoroquinolone-resistant e. coli 60 . a third study addressed the progressive importance of infections caused by esbl-producing e. coli 61 . bacteremia caused by these organisms is increasing and given the difficulties encountered in its treatment,-frequent multiresistance to other traditionally used agents,-a reappraisal of current empirical regimens is in order. a very good example of the difficulties found treating esbl-producing organisms, is the case reported from israel, a patient with an esbl-producing klebsiella pneumoniae endocarditis that developed resistance to ciprofloxacin and piperacillin/tazobactam while on therapy 62 . the presence of esbl-producing enterobacteriaceae in the stools of humans, sewage and animals has been found to be high in areas where these studies have been done. from 20 to 100% of the farm animals examined in a recent spanish study harbour esbl-producing enteric bacteria 63 . the potential for dissemination of multi-resistance isolates is illustrated by two recent reports: the emergence of proteus mirabilis carrying the bla metallo-beta-lactamase gene, the first instance in this species 64 , and bla vim-2 , and bla vim-7 carbapenemase-producing pseudomonas aeruginosa 65 . among the latter, the emergence of resistance to carbapenems can be very rapid; when associated with resistance to polimixins the problem at clinical level can become insoluble 66 . acinetobacter baumannii has recently emerged as a major cause of hospital-acquired infection, because of its propensity to accumulate mechanisms of antimicrobial resistance that lead to pan-drug resistance and cause large nosocomial outbreaks that often involve multiple facilities. the problem is particularly serious in intensive care settings. an ex-cellent review has been published recently on the epidemiology and control of a. baumannii infections in health care facilities 67 . finally, although rare, communityacquired pneumonia due to a. baumannii is known to occur. a retrospective case-control study (cases: community-acquired pneumonia; controls: hospital acquired pneumonia) from hong-kong has been published 68 . below, a group of spanish physicians and microbiologists with an interest in the field of bacterial pathogens expand on the most remarkable papers published in this area during 2006. the following are the publications selected for discussion. these studies are an example of the ca-mrsa epidemic in the united states at present time. a population study conducted in three united states communities during 1997-1999 established the annual incidence of ca-mrsa to be 18-26 per 100.000, and most isolates were associated with clinically relevant infections. the current relevance of mrsa as a cause of skin and soft-tissue infections has been demonstrated in a prospective prevalence study involving patients presenting to emergency departments in 11 u.s. cities (moran gj et 12) . according to these observations, the distinction between healthcare and community-associated mrsa is rapidly blurring. these studies demonstrate: 1) an increase in the incidence of skin and soft-tissue infections as well as in the incidence of invasive infections due to ca-mrsa. 2) the current predominance of the genotype usa 300. 3) the migration of ca-mrsa strains into the healthcare settings. stapylococcus aureus is an infrequent cause of community-acquired pneumonia (cap), but it is a recognized cause of influenza-associated cap. mrsa commonly causes nosocomial pneumonia, but relatively few cases of mrsa cap have been reported. during the 2003-2004 influenza season, 17 cases of cap due to s. aureus were reported to the cdc from 9 states; 15 (88%) were caused by mrsa, and all isolates available for microbiological study presented genes for the panton-valentine leukocidine. thirteen were admitted to the icu, and death occurred in 5 patients. authors suggest that empiric therapy of severe cap during periods of high influenza activity should include coverage for mrsa. in a country with high incidence of ca-mrsa, as it is in the united states, empiric therapy of severe cap during periods of high influenza activity should include coverage for mrsa, even in patients without recognized risk factors for mrsa since they can be infected by a ca-mrsa strain. pyomiositis is an acute bacterial infection of skeletal muscle with localized abscess formation caused in 75-90% of cases by s. aureus. acute bacterial myositis is a less common muscle infection, but inflammation extends to more than one muscle group without distinct abscesses. although commonly caused by streptococcus pyogenes, myositis caused by s. aureus have been described. cases of pyomiositis and myositis have increased in pediatric patients since 2000 at texas children´s hospital, and this increase appears to correlate with the emergence of ca-mrsa. this study reviews the medical records of all patients admitted in this hospital from 2000 to 2005 with infective pyomyositis and myositis. forty-five cases were analyzed. forty percent had negative culture but 57.8% were caused by s. aureus. the number of cases increased from 2000 to 2005 as a result of an increase in the prevalence of ca-mrsa. the thigh and pelvis were the most commonly affected sites. fifteen out of 24 available s. aureus isolates were ca-mrsa and 9 were community-acquired methicillin-susceptible s. aureus (ca-mssa). by pfge, 16 isolates were found to be usa300, and 17 carried the panton-valentine leukocidin (pvl) genes. patients with ca-mrsa, usa300 and/or pvl-positive strains required more drainage procedures that did those with ca-mssa, non-usa300 and/or pvl-negative strains (81 vs. 40%, 82 vs. 29%, and 81 vs. 38%, respectively). the authors strongly consider coverage for mrsa in the empirical treatment regimen (vancomycin or clindamycin) of these infections in children. ca-mrsa is an increasing cause of pyomyositis and myositis in children. although in this study the infections caused by ca-mrsa, usa300, and pvl-positive isolates were more severe than those caused by ca-mssa, non-usa300, and pvl-negative strains, additional studies are needed to find out the virulence factors associated with muscle infection caused by ca-mrsa. the epidemiology of ca-mrsa among healthy children has been recently described. however, little is known about ca-mrsa in children with underlying medical conditions. the authors compare in this study the clinical and molecular epidemiology of ca-mrsa in children with and without risk factors for health care-associated infections (rf-hai). a 3-year retrospective cohort study of children with ca-mrsa infection was conducted. rf-hai, including hospitalization within the past year, indwelling medical devices or chronic medical condition, were identified by chart review. the authors identified 446 episodes of community-acquired s. aureus infections, of which 134 (30%) were caused by mrsa. during the 3-year study period, the proportion of s. aureus infections caused by mrsa rose from 15% (12 of 80) to 40% (93 of 235) (p < 0.001) with the increase noted predominately in children with skin and soft tissue infections. rf-hai were identified in 56 (42%) patients with ca-mrsa. among subjects with ca-mrsa, children with rf-hai were more likely to have had an invasive infection than healthy children (32 versus 5%; p < 0.001). ca-mrsa isolates from children with rf-hai were similar to those without rf-hai; all laboratory-retained ca-mrsa isolates harbored the sccmec type iv cassette, and almost all isolates were susceptible to cotrimoxazole and clindamycin. pfge revealed greater molecular diversity among ca-mrsa isolates recovered from children with rf-hai compared with those from otherwise healthy children (p = 0.001). additionally, ca-mrsa isolates from children with rf-hai were less likely to be pvl-positive (p < 0.001) and more likely to be resistant to 3 or more classes of antibiotics (p = 0.033). this study demonstrates that the borders between community-acquired and hospital-acquired mrsa strains are blurring, and suggests that ca-mrsa strains might have become endemic not only in adult health care facilities but also within paediatric facilities. ca-mrsa was first reported in western australia (wa) in the early 1990s from indigenous people living in remote areas. a statewide policy of screening all hospital patients and staff who have lived outside the state for mrsa has prevented the establishment of multidrug-resistant epidemic mrsa (memrsa), however, this study demonstrates that this policy has not prevented sccmec type iv and type v mrsa clones (community clones) from becoming established in wa. all mrsa isolated in wa from july 2003 to december 2004 were included in this study. isolates were recovered from clinical and infection control screenings. of the 4,099 mrsa isolates analysed (those sent to a reference center), 77.5% were ca-mrsa. using different molecular methods, a total of 22 different ca-mrsa clones were characterized. of these isolates, 55.5% were resistant to ≥ 1 non-beta-lactam antimicrobial drug. five pvl-positive ca-mrsa clones were identified. the australian policy that prevented the establishment of memrsa, has not prevented sccmec type iv and v mrsa clones, including non-memrsa and ca-mrsa, from becoming established in wa. the emergence of multidrug-resistant ca-mrsa clones and the detection of pvl toxin genes in clones previously reported as pvl-negative is a major public health concern. molecular typing is very important in tracing the origin of isolates and in designing antimicrobial drug prescribing policies for their control, particularly in the community. some studies have indicated that ca-mrsa strains are generally more virulent than hospital-acquired mrsa, a finding consisting with the ability of ca-mrsa to cause disease in individuals without predisposing risk factors. although the molecular basis for the enhanced virulence is not known, there is strong association between ca-mrsa infections and the presence of pvl. however, the role that pvl plays in the pathogenesis of ca-mrsa has not been tested directly. in this study the authors evaluated, in a mouse infection model, the role of pvl in the virulence of ca-mrsa as well as the lytic activity and the intracellular survival in human polymorphonuclear leukocytes (pmns) of isogenic strains of ca-mrsa, expressing or not pvl. they compared the virulence of pvl-positive with that of pvl-negative ca-mrsa representing the leading disease-causing strains. unexpectedly, strains lacking pvl were as virulent in mouse sepsis and abscess models as those containing the leukotoxin. isogenic pvlnegative (luks/f-pv knockout) strains of usa300 and usa400 were as lethal as wild-type strains in a sepsis model, and they caused comparable skin disease. moreover, lysis of pmns and pathogen survival after phagocytosis were similar between wild-type and mutant strains. although the toxin may be a highly linked epidemiological marker for ca-mrsa strains, the authors conclude that pvl is not the major virulence determinant of ca-mrsa. it may be that pvl contributes to specific pathologic conditions such as necrotizing pneumonia, a disease not tested in this study. probably future studies using other different animal models will help to understand the pathogenesis of ca-mrsa or to identify other factors responsible for the type and severity of disease caused by these emerging pathogens. the main limitation of this study is its retrospective nature, which cannot exclude bias. unrecognized common factors leading to fluoroquinolone therapy and mrsa acquisition cannot be ruled out. in addition, adverse events associated with increased use of those antimicrobials substituting the fluoroquinolones need to be considered. the authors investigate the association between the use of fluoroquinolones and the recovery of mrsa in hospitalized patients. four hospitals located in the same region of france with a similar pre-study incidence of mrsa are selected. the study manoeuvre consists of discontinuing the use of fluoroquinolones in one hospital for the study period, while the three others serve as controls without changing their antibiotic policies. as a consequence, the study hospital reduces the use of fluoroquinolones from 54 to 5 ddd per 1000 bed-days and increases the consumption of amoxicillin-clavulanate by 28%, ceftriaxone by 42%, gentamicin by 35%, amikacin by 25%, erythromycin by 65% and cotrimoxazole by 46%. during the intervention period the incidence of mrsa tended to be lower in the study hospital (odds ratio 0.82; 95% ci 0.68-0.99). no reduction in the rate of fluoroquinolone resistance occurred in gram-negative bacilli and the study centre suffered a nosocomial esbl-producing klebsiella pneumoniae outbreak during the intervention period. the observed reduction in mrsa rate is significant, although small, in this interestingly designed study. unfortunately, a spontaneous decrease of the incidence of mrsa, rather than due to the absence of the use of fluoroquinolones, may be responsible for the observed 18% reduction. in addition, a potential causal relation between the changes in antibiotic policy and the occurrence of an esblproducing k. pneumoniae outbreak is a matter of concern. howden bp, johnson pd, ward pb, stinear tp, davies jk. isolates with low-level vancomycin resistance associated with persistent methicillin-resistant staphylococcus aureus bacteremia. antimicrob agents chemother. 2006;50:3039-47. low-level vancomycin-resistant staphylococcus aureus (vancomycin-intermediate s. aureus -visa-and heterogenous visa -hvisa-) leads to glycopeptide treatment failure. the genetic changes leading to hvisa and visa have not been clearly determined. this study characterizes five clinical pairs of fully vancomycin-susceptible s. aureus (vssa) and hvisa/visa (vancomycin mics, 2 to 4 mg/l) isolates obtained before and after failed vancomycin therapy, in patients with bacteremia due to mrsa, in order to better understand the changes associated with this type of resistance. the hvisa/visa phenotype was associated with increased cell wall thickness, re-duced autolytic activity in four of five hvisa/visa strains, and a striking reduction in biofilm formation compared to the parent strains in all pairs. all five pairs were isogenic, and genomic dna microarray comparison suggested that major genetic changes are not required for the development of the resistant phenotype in these strains. a marked reduction in rnaiii expression of the agr gene was found in four pairs. this study performs a detailed analysis of the different mechanisms that could explain the conversion of a vssa into a visa strain. many of the phenotypic changes are consistent with previous reports; however, reduced autolytic activity is not essential for the expression of low-level vancomycin resistance in s. aureus. the use of pairs of clinically derived, isogenic strains of vssa and hvisa/visa will be a valuable resource to elucidate the genetic mechanism of low level glycopeptide resistance in the latter. staphylococcus aureus small-colony variants (scvs) are able to persist inside of host cells and to resist antibiotics. this characteristic is especially important when involved in implant-associated infections. the authors analyze 5 cases of hip-prosthesis-associated infections due to scvs, including their course prior to identification of the pathogen. the patients' mean age was 62.2 years. all patients experienced treatment failures prior to isolation of scvs, despite as many as 3 surgical revisions and up to 22 months of antibiotics. transmission electron microscopy performed on biopsy specimens from periprosthetic tissue revealed intracellular cocci in fibroblasts. all prostheses were removed without implanting a spacer, antimicrobial agents were administered for 5.5-7 weeks, and reimplantation of the prostheses was performed for 4 patients. this 2-stage exchange was associated with successful outcome, with a mean follow-up of 24 months. slow growth, atypical colony morphology, and unusual biochemical profile make clinical laboratory personnel likely to miss or misidentify scvs. in the case of a poor response to adequate antimicrobial and surgical treatment in implant-associated staphylococcal infections, the clinician and the clinical laboratory should consider special efforts to search for scvs. this study investigates an unusually high incidence of 4% linezolid resistance in coagulase-negative staphylococci (lr-cns) at their centre in the usa. they perform an analysis of linezolid use and a retrospective case-control study to identify the risk factors and epidemiological profile of patients in whom lr-cns had been identified. each of the 25 case patients were first matched with 4 randomly selected concomitant patients at the same hospital ward. in a second analysis, each case patient was matched with 2 patients with linezolid-susceptible cns isolated from clinical samples. the authors report that the use of linezolid had increased from 0.61 ddd per 100 bed-days in 2001 to 13.6 in 2005. in 19 of 25 (76%) patients with lr-cns they detected prior use of linezolid. lr-cns was identified in blood cultures of 5 patients, 14 times it was considered to be a contaminant and in 3 patients it was identified in 1 of 2 blood samples. all except one isolate (staphylococcus lugdunensis) were identified as staphylococcus epidermidis. mics were higher than 256 mg/l and genotyping revealed relatedness of strains from 21 (84%) patients, 15 of whom had been admitted to the same icu. compared to random controls, prior treatment with linezolid was significantly associated with acquisition of lr-cns (odds ratio 20.6, 5.8-76), as was admission to ward "c" (odds ratio 12.4, 3.4-45.5). patients with linezolid-susceptible cns had not been given linezolid (odds ratio 0.09, 0.01-0.57). this paper describes a clonal outbreak due to lr-cns associated with extensive use of linezolid. although the information provided is of interest, it is based on experience from only one centre, and it is a short series of 25 strains and only 5 clinical infections. this in vitro study investigates the effect of a bacteriophage on biofilm formation by staphylococcus epidermidis on hydrogel-coated foley catheters. they demonstrate that pre-treatment of catheters with the lytic s. epidermidis bacteriophage 456 prevents formation of biofilm. while new technologies, such us pre-treatment of catheter surfaces with antiseptic or antibiotic agents show promise as prophylaxis/treatment of catheter-related infections, the results of this study highlight the potential of phages for the reduction of biofilm development on biomedical implant surfaces and suggest future developments in catheter-associated infection prevention. in spite of the antibiotic treatment and intensive care, the mortality of community-acquired pneumonia (cap) remains high. on the other hand, pneumococcal vaccination reduces the incidence of pneumococcal bacteremia, which itself has a high fatality rate. the objective of this study was to evaluate the impact of prior 23-valent pneumococcal vaccination (pv23) on the early mortality and complications in hospitalized adults with cap. the study was carried out in 109 us teaching and community hospitals that share a common database. a total of 62,918 individuals with cap, identified by the incd 9th rev. codes 480.0-487.0 entered the study during a five-year period (1999) (2000) (2001) (2002) (2003) . data were obtained systematically using standardized definitions by trained nurses, concurrently with patient care, and validated monthly. variables included those necessary for calculation of the fine score, vaccination status (yes, 12%; no, 23%; unknown, 65%) and other comorbidities. no data on microbial etiology were recorded. statistical analysis was done with multivariable logistic regression models. vaccine recipients were less likely to die during hospitalization that were unvaccinated patients (adjusted or 0.50; 95% ci 0.43-0.59). this trend remained under varying assumptions about missing vaccination data. vaccination also had a positive effect on the risk of respiratory failure (or 0.67; 95% ci 0.59-0.76). length of stay was also reduced in vaccinated patients (p < 0.001). in conclusion, prior vaccination with the pv23 is associated with a 50% mortality reduction and a lower risk of complications, then reinforcing the efforts towards improving vaccination coverage in adults. the absence of microbiological data, and the high proportion of individuals with unknown vaccination status were the main weaknesses of this observational study. this is a case-control study on the effectiveness of the 7-valent pneumococcal conjugate vaccine (pcv7) to prevent invasive pneumococcal disease in children 3-59 months old, including those vaccinated with incomplete schedules. the study was carried out in several urban areas and two entire states of the usa. cases were identified by an active surveillance program operated by the cdc during 2001-2004. a total of 782 cases were included, and 3 controls per case, matched by age and zip code, were selected. a total of 485 cases were excluded, mostly those who had no isolate available for serotyping, or those whose parents refused to participate. serotyping (danish scheme), and susceptibility tests were done at cdc or at some state laboratories. matched odds ratio (or) was calculated by using conditional regression models controlled by underlying disorders, race, sex, and access to scheduled vaccinations, among other variables. effectiveness was defined as one minus the adjusted matched orx100%. a total of 45% of cases were caused by serotypes included in the pcv7 (vt), mostly 14 and 19f, of which 26% were in children who had received al least one dose of the vaccine, and 8% were in fully vaccinated. effectiveness in healthy children was 96% for vt strains, and 44% for vaccine-related serotypes [same serogroup but different type (vrt)]. lower effectiveness was observed in children with underlying diseases (81% and 35% for vt and vrt, respectively. the level of protection was also lower for strains with decrease susceptibility to penicillin. the pcv7 did not prevent serotype 19a infection. several schedules were more protective than no vaccination, being 3 doses plus a booster more protective than 3 doses alone. in summary, the pcv7 prevents invasive disease in both healthy and chronically ill children, even with various no standard schedules. this study opens new doors to prospective studies for investigating simpler vaccination schedules. the vaccine fails with some serotypes and is less efficient with penicillin non-susceptible strains. in this observational study from the children's hospital of philadelphia, the authors aimed to evaluate the effect of the pcv7 vaccine on the pneumococcal bacteremia (pnb) caused by vrt or by pneumococci non susceptible to penicillin (pnsp) in the post-licensure period of this vaccine. from january 1999 to may 2005, all episodes of pnb attended at the pediatric emergence unit were revised retrospectively, as well as bacteremias caused by other respiratory bacterial pathogens (orbp; haemophilus influenzae, neisseria meningitidis, and moraxella catarrhalis), that were used as a non-equivalent dependent variable with the purpose of statistical analysis. blood cultures were done according with the physician indication. serotyping was performed with the staten seruminstitut antisera collection, and mics to penicillin by using e-test strips (ab biodisk). pcv7 vaccination status was no recorded among variables entering the study. an overall 57% decrease per year in the incidence of pnb was observed, mainly due to the reduction of cases caused by vaccine type strains, although a shift in this trend was noted in 2004-05. the incidence of pnb caused by non-vaccine types and orbp remains stable, but that of vrt increased by 6% along the study period, being serotype 19a responsible for most cases. the percentage of pnsp strains also increased from 25 to 39% (p < 0.05). this study calls attention on the possibility of serotype replacement after vaccination with the pcv7, and on the emergence of pnb caused by pnsp strains, possibly related with selection of some serotypes (19a) for which the pcv7 has poor immugenicity. in addition to its retrospective nature, the study has other limitations. the absence of records on the previous pcv7 vaccination in the study population, the use of a non-equivalent dependent variable for control of biases, and changing criteria in performing blood cultures along the study period (confound-ing by indication) were the main weaknesses of the study, although the authors believe that some of these variables would be controlled in a certain extent. therefore, prospective multicenter studies on this subject would be welcome. after large-scale introduction of the pcv7 vaccine, two effects have become apparent. first, increasing pharyngeal colonization by serotypes not covered by the pcv7 (serotype replacement), and second, acquisition of a new nonvaccine capsule through recombination in naturally transformable clones (serotype swiching). authors from taiwan investigated the in vitro capacity of clinical strains of s. pneumoniae isolated in an area with low pcv7 coverage to become transformed (competence) in vitro. a total of 118 strains belonging to 7 serotypes were prospectively collected from taiwanese hospitals from 2003 to 2005. with the exception of serotype 3, all strains were included in the pcv7. competence studies were done with the use of two variants of the competence-stimulating peptide (csp 1 and 2) and the pdl278 plasmid as a vector. genetic diversity were also studied by pulse field gel electrophoresis (pfge). serotype 6b had the greatest competence, followed by types 14, 19f, 9v, 23f, 3, and 18c. serotype 6b also showed the highest genetic diversity and higher proportion of penicillin nonsusceptibility rates. conversely, strains belonging to types 3 and 18c showed the highest clustering, were mostly incompetent for transformation, and were 100% susceptible to penicillin. in spite of this qualitative association between serotype, competence and resistance, no correlation was observed between the level of competence and the degree of resistance to penicillin (or 0.9; 95% ci 0.58-1.25). in this way, serotype 23f was relatively incompetent, but had a clear trend to higher proportion of resistant strains, and with higher penicillin mics. this study shows how certain serotypes with lower capacity to be transformed are genetically more stable, and why resistance to penicillin is rare in strains belonging to incompetent serotypes all over the world. competence would be a necessary condition, but no sufficient, for explaining the genetic diversity and, secondarily the acquisition of penicillin resistance determinants in s. pneumoniae. streptococcus pneumoniae is the leading cause of cap in adults. bacteremic pneumococcal pneumonia (bpp) is among the most serious forms of pneumococcal disease. this population-based case-control study identifies clini-cal and demographic factors associated with macrolideresistant bpp in adults from 43 acute-care hospitals at the pennsylvania region. from december 1, 2000, to april 17, 2004 patients included in the study were individuals who 1) were 18 years or more; 2) had at least one blood culture that grew s pneumoniae drawn within 48 hours of hospital admission; 3) resided in one of the five counties surrounding pennsylvania, and 4) had a bacterial isolate confirmed in the laboratory as s pneumoniae. seventy-six patients had erythromycin-resistant infections and were selected as the case-patients for this study. the authors found that exposure to macrolides in the six months preceding infection, a history of influenza vaccination in the previous year, and hispanic ethnicity were all independently associated with an increased probability of erythromycin -resistant s. pneumoniae infection. among patients who reported taking antimicrobial agents in the 6 months preceding infection, failure to complete the course of prescribed drugs was associated with an increased probability of macrolide resistance ([or = 3.4]; 95% ci 1.2-9.9). however, most patients with macrolide-resistant infections did not report any prior antimicrobial drug exposures. the authors assume in the discussion potential biases that may have affected the assessment of different risk factors. future studies correlating duration of therapy with risk for colonization with macrolide-resistant pneumococci would be useful to further explore this phenomenon. the incidence of invasive pneumococcal disease (ipd) in children and adults increases in winter, however possible underlying causes have not been carefully studied. this ecological study correlated population-based data on ipd and respiratory virus activity in the year 2000 in metropolitan new south wales, australia, with climatic parameters. seven hospitals participated in the study from may to october. during a year with good separation of respiratory syncytial virus (rsv) and influenza virus activity, it was demonstrated in children a significant correlation with rsv activity and ipd activity, but no significant correlation between influenza virus activity and ipd. in adults the epidemic curves of rsv and influenza virus activity corresponded with peaks of ipd, but it was only possible to demonstrate a statistically significant correlation when the activity of both viruses was combined. of the climatic parameters there was a clear inverse relationship between weekly mean minimum and maximum temperatures and ipd activity in adults and children. data from other temperature geographic areas and data obtained after the introduction of vaccines are required to confirm these findings. probably a case-control study comparing the isolation of respiratory viruses in patients with ipd matched with age, sex, location and time with control subjects without ipd would be required to specifically examine the role that respiratory viruses play in predisposing to ipd. the effect of falling temperatures on the pathogenicity of s penumoniae has not been extensively studied and warrants further investigation. human metapneumovirus (hmpv) has been found to be associated with lower respiratory tract infections although the pathogenesis remains to be elucidated. there are few reports of respiratory tract infections due to bacterial coinfection with hmpv. this hypothesis-generating study, performed from march 1998 to october 2002, involved a cohort of 39,836 children in south africa randomized to receive the 9-valent pneumococcal polysaccharide-protein conjugate vaccine (pcv9) or placebo. these that were hospitalised (3.069) for lower respiratory tract infection (lrti) were tested for hmpv infection. by use of a nested reverse-transcription pcr assay targeted at amplifying a fragment of the hmpv fusion protein gene, 202 such infections were identified among 2715 episodes of lrti in children. in fully vaccinated children, the incidence of hospitalization for a least one episode of hmpvassociated lrti was reduced by 46% (p = 0.0002) overall, by 45% (p = 0.002) in human immunodeficiency virus (hiv)-uninfected children, and by 53% (p = 0.035) in hiv-infected children. there was a significant reduction in the incidence of clinical pneumonia among vaccine recipients overall (58%; p = 0.0001), in hiv-uninfected children (55%; p = 0.003), and in hiv-infected children (65%; p = 0.02). there was no significant reduction in the incidence of hospitalization for hmpv-associated bronchiolitis among vaccine recipients in the entire study population. the absence of sensitive tools to diagnose bacterial pneumonia has been a major obstacle in order to define the role of bacterial-virus coinfection in humans. it has been documented that approximately one-third of children with respiratory syncytial virus associated pneumonia may have pneumococcal coinfections. the results of the present study suggest that bacterial coinfections, particularly pneumococcal infections, are an essential part of the pathogenesis of most severe hmpv infections progressing to pneumonia. an implication of this observation is that children hospitalized with the diagnosis of hmpv-associated pneumonia should be treated with antibiotics, and a significant proportion of these hospitalisations may be prevented by vaccination with pneumococcal vaccine. nevertheless more information is needed in order to clarify the pathogenic mechanisms of the mixed infections. in this study, the authors describe the clinical features, complications, treatment and outcome of 30 episodes of community-acquired listeria monocytogenes meningitis in adults. these cases were included in a prospective nationwide observational cohort study of bacterial meningitis with a positive lcr culture, performed in the netherlands between october 1998 and april 2002. over the period of study, 696 cases of bacterial meningitis were diagnosed and these caused by l. monocytogenes represented 4%. the annual incidence l. monocytogenes meningitis was 0.07 cases per 100,000 adults. all patients were immunocompromised (67%) or > 50 years old. in 19 (63%) of 30 patients, symptoms were present for > 24 h, and the clinical presentation was subacute in 8 patients (27% had symptoms for ≥ 4 days). the classical triad of stiff neck, fever and alterations in mental status was present in 43% of the cases. gram staining of cerebrospinal fluid (csf) samples revealed the causative organism in 7 (28%) of 25 cases and a biochemical csf indicator of bacterial meningitis was present in 23 (77%). the initial antimicrobial therapy was amoxicillin based for 21 (70%) of 30 patients. the coverage of initial antimicrobial therapy was microbiologically inadequate for 9 (30%) of the patients. the mortality rate was 17% (5 patients), and 8 (27%) experienced an unfavourable outcome. inadequate initial antimicrobial therapy was not related to outcome. the study have limitations due to the selection of patients with a positive l. monocytogenes csf culture. the most severe patients (in which antimicrobial treatment is started before the csf is obtained or these with important neurological alterations) might have been excluded. nevertheless, this prospective study confirms that typical csf findings predictive for bacterial meningitis might be absent (biochemistry and gram stain), but in contrast with previous reports, patients with meningitis due to l. monocytogenes do not present with atypical clinical features. it is interesting to note the high rate of patients (83%) with hyponatremia and the development of a subarachnoidal hemorrhage in one patient, complications more frequently related with tuberculous meningitis. the authors review the microbiologic and epidemiologic data of 1,933 cases of human listeriosis reported in england and wales from 1990 to 2004. ten common-source outbreaks affecting 60 patients were excluded from the study. a significant increase was observed during the period 2001-2004 compared to 1990-2000. the majority of the cases were sporadic (1,873) and not pregnancy-related (1,155) . deaths were more frequent in the group of nonpregnancy-related cases (44 vs. 10%). the majority of the sporadic cases diagnosed between 2001 and 2004 occurred in patients > 60 years of age with bacteriemia without cns affectation and without relation to sex, race, season of the year, socioeconomic differences or underlying conditions. serotypes 4b and 1/2a were the most frequent throughout the entire study. the increase of cases of listeriosis shown in the second period analysed in this study could be due to an increased interest in reporting this illness. nonetheless, a change in the pathogenicity of l. monocytogenes could also explain the results. the advice to avoid high-risk foods for people of advanced age and for those that are immunocompromised, and not only for pregnant women, is a plausible message of this study. the immunized toddlers have a higher meningococcal antibody in serum samples than immunized infants. the authors determined meningococcal antibodies in serum samples obtained from children who were immunized with group c meningococcal vaccines 2-3 year earlier as infants or toddlers.the geometric mean serum antibody concentrations were higher in immunized infants or immunized toddlers than in un-immunized historic control (0.82 and 0.56 mcg/ml vs. 0.08 mcg/ml, respectively; p < .0001). the proportion of immunized infants who had bactericidal titers ≥ 1:4 (considered to be protective when measured with human complement was higher than of immunized toddlers (61 vs 24%; p < .01). even if threshold for protection is considered to be a serum bactericidal titer ≥ 1:8 then the respective percentages of serum samples with protective titers is 50% for immunized infants, compared with 16% for the immunized toddlers (p = .0006). when passive protective was tested, 50% of serum samples from immunized infants and 41% of serum samples from immunized toddlers conferred protection against group c bacteremia, compared with 3% of serum samples from unimmunized historic controls (p < .0001). there was no evidence of lower immunity in children immunized as infants than as toddlers. on the basis of serum bactericidal activity and/or passive protection, 40-50% of both age groups are protected at 2-3 years after immunization, which is significantly greater than in unimmunised historical controls (< 5%). the aims of this study were to determine hsba (human serum bactericidal assays) gmts (geometric mean titers) and the percentage of participants with an hsba titer ≥ 8 at 2-7 days after vaccination with menps. secondary endpoits were to determine the hsba gmt and geometric mean antibody concentration measured by elisa for the following populations: 1) all vaccine recipients (at day 0); 2) all recipients of menps and mencv (analyzed separately for values at both day 0 and day 28 after vaccinations), and groups 1-6 (analyzed separately for all days on which blood samples were obtained) this was a phase iv, open-label randomized comparative trail. healthy 13-15 year olds who were vaccinated with a single dose of menjugate (chiron vaccines), a meningococcal serogroup c-crm 197 glycoconjugate vaccine (mencv) in the 1999-2000. previous immunization status was determined by reference to the centralized immunization records of the relevant child health computer departments. subject numbers were prospectively randomized in blocks of 6 according to computer-generated blocked randomization scheme that allocated equal numbers of subjects to the 6 groups. participants randomized to groups 1, 2, 3, or 4 received the plain polysaccharide serogroup a and c meningococcal vaccine (menps), where persons randomized to groups 5 or 6 received mencv. a one-fifth dose of menps was used. blood samples of up to 10 ml were obtained prior vaccination, twice more in the week after vaccination and at day 26-28 after vaccination. serum samples were analyzed for menc sba titer using hsba. an hsba titer ≥ 8 was used to indicate a very conservative measure of protection. a total of 274 participants were randomized to 1 of 6 groups. one hundred seventy-one of these had been randomized to group 1-4 (received menps) and 89 randomized to groups 5 ot 6 (received mencv). an hsba titer ≥ 8 was measured in the serum samples of 74.6% of participants. no increase in hsba gmt was detected until 5 days after administration of menps or mencv. all participants demonstrated an hsba titer ≥ 8 at day 28 after vaccination. the hsba gmts observed 28 days following vaccination with mencv (4979.4) were higher than those observed following vaccination with menps (2370.9). elisa gmcs were similarly higher 28 days after vaccination with men c or menps (35.7 vs. 19.5, respectively) . this study shows persistence of sustained levels of bactericidal antibodies for at least 3 years after vaccination of adolescent with mencv. after challenge of immunized adolescents with menps, there was no increase in sba observed until days 5 after vaccination, indicating that immunological memory may be too slow to generate protection against this potentially rapidly invasive organism. the distribution of polymorphisms in three genes (sp-a1; sp-a2 and sp-d) in a cohort of patients with microbiologically proven meningococcal disease was studied. this study was realized in a cohort of patients with proven meningococcal disease. from july 1998 through november 1999, all whole-blood samples obtained from patients were stored. the serogroup of the infecting organism, the disease outcome and the age of patients were recorded. the control group comprised healthy volunteers. samples were chosen at random from the cohort for sp-a/sp-d genotyping. in total, 302 patients were genotyped for polymorphisms at all loci in sp-a2, and 303 for sp-a1. data for sp-d polymorphisms were available for 294 patients. in the control group, 218 and 222 individuals were typed successfully for sp-a2 and sp-a1 respectively, and 227 were typed for sp-d. two alleles of sp-a2 had a significant association with susceptibility to meningococcal disease. homozygosity for 1a was associated with an increased risk of meningococcal disease (or 7.4; 95% ci 1.3-42.4), with a 6.3% of the patient population homocygous for 1a compared with only 0.9% of the control population. homozygosity for the rare snp (sp-a2 at codon 223) was associated with an increased risk of meningococcal disease (or 6.7; 95% ci 1.4-31.5) and increased risk of death (or corrected for age 2.9; 95% ci 1.1-7.7). haplotype 6a/1a was higher in patients (4.1%) than in control subjects (1.5%; or 2.35 95% ci 1.28-4.31). gene polymorphism of the binding domain of surfactant protein a-2, sp-a2 at codon 223, increases susceptibility to meningococcal disease, as well as the risk of death. in the era of universal vaccination of children with the h. influenzae conjugate vaccine, it is critical to characterize the currently circulating strains for detection of any genetic change that could render the current vaccine no longer protective. in italy, this vaccine is included in the vaccine calendar since 1999, ant the uptake of the population exposed has been > 50% since 2000. the aims of this study were to evaluate the genetic and capsular structures of the available strains from patients with invasive disease from 1997 to 2003, to assess the influence of vaccination in the changes observed in these structures.. the genetic diversity of the 95 strains was analysed by pfge and capsular changes by southern blot. the 60 strains isolated pre licensure (1997-1998) was compared with the 35 strains isolated post licensure (1999) (2000) (2001) (2002) (2003) . the 95 strains had by pfge a total of 28 restriction patterns, with a clear predominante of 5 of them. it should be stressed that almost half of the strains had a restriction pattern indistinguishable from 40f, the dominant and endemic clone in italy since 1994. four strains were fromchildren that had received at least one dose of the vaccine. a high genetic homology was detected in 90.5% of the isolates. the amplification of the locus of capsulation b showed that there is a significant difference between the two analysed periods. thus, the strains from the vaccine period, including those from children previously vaccinated, had a number of copies higher than 2 in 54,3% of the cases, compared to 33,3% in the prevaccine period (p = 0,046). the results of this study indicate the importance of monitoring invasive strains. the use of molecular genetics applied to this end, has shown that vaccination has not conditioned the occurrence of genetic changes nor the disappearance of dominant clones; it has been followed, however, by relevant changes in the expression of the capsulation genes, that could explain, at least in part, the lack of immune response in some vaccinated children. non-typable haemophilus influenzae (nthi) is a frequent pathogen in patients with copd. when acute exacerbations are due to bacterial infection, nthi are the most frequent isolated organisms. in this report, 490 nthi isolates from a variety of respiratory sources, were studied. also, the microbiological results of 118 copd patients of monthly sputa collected prospectively or in case of exacerbation, collected during a period of 9 years, were evaluated. identification and typing were done by standard methods. phenotypic variants were identified as h. haemolyticus with 4 independent methods: análisis of rdna sequence, mlst, hybridization dna-dna y sequencing the gene encoding protein p6. of a total of 490 strains studied, 39.5% of sputum isolates and 27.3 of nasopharingeal isolates had genotypic characteristics of h. haemolyticus. none of the invasive isolates studied had the genotypic characteristics of h. haemolyticus. molecular typing of sputum isolates made possible to demonstrate a statistically significant difference in the acquisition of a new strain of h. influenzae as a causal agent of an exacerbation in patients with copd (44.5 vs 16.5%; p < 0.0001; rr 4.09 ic 95% 2.89-5.80). the differences in the case of h. haemolyticus were not significant (20.7 vs. 17.4%; p = 0.41; rr 1.24 ic 95% 0.74-2.07). from the results of this study, it can be inferred that h. haemolyticus is a frequent inhabitant of the respiratory tract of children and adults with copd, as a commensal, without pathogenic capacity. unfortunately, with the standard methodology used for identification of h. influenzae it is not possible the identification of the phenotypic variant h. haemolyticus, a fact that might have important clinical implications in the use of antibiotic treatment in many patients. the persistance of non-typable haemophilus influenzae (nthi) in patients with copd is thought to be related with interactions between this bacterium and human alveolar macrophages. the immune mechanisms that mediate this macrophage response are poorly known. the aims of this study were to demonstrate the importance of the alteration of the immune response of alveolar macrophages in the persistence of nthi in the lower airways in patients with copd. three groups of patients were included: esmokers with copd (n = 14), esmokers without copd (n = 15) and non-smokers (n = 9). respiratory and blood samples were used for separation of purified macrophages. phagocytosis of adherent cells and survival of intracellular nthi, three different strains of nthi from patients with copd were used. alveolar macrophages of copd patients had diminished phagocytosis as compared with with macrophages from the other two groups in front of the three strains. however, phagocytosis of nthi by blood macrophages was similar in all three groups studied. finally, intracellular survival of nthi was not altered in the alveolar macrophages of the patients with copd. the results of the study demonstrate the existence of an alteration in the immune response of alveolar macrophages of patients with copd vs. nthi, as sown by a diminution of phagocytosis, that explains the persistence of this organism in the airways. the absence of alterations in blood macrophages indicates the existence of a compartmentalised immune response in this population. however, intracellular lysis of nthi is not altered in alveolar macrophages of copd patients the control of pertussis infection is not optimal, because neither vaccination nor natural infection induces longlived immunity. the estimated annual incidence of clinical pertussis infection in usa in adolescents and adults is about 370-500 cases per 100,000 person-years. the lack of specific clinical criteria and the limited availability of standardized serologic criteria make difficult the diagnosis of pertussis infection. a study to determine the impact of vaccination in adolescents and adults, as a part of a national institutes of health-sponsored multicenter, prospective, randomized acellular pertussis vaccine (ap) efficacy trial in the us, was recently reported. usa. the study was performed in 2781 adults (15-65 yearsold) in which one-half of the subjects received ap vaccine and one-half received hepatitis a vaccine (control subjects). all patients were observed for 2 years for clinical illness (telephone calls every 14 days) and serological studies obtained at baseline, +1 month and +12 months after vaccine application. for serological evaluation iga and igg antibodies to pertusis toxin (pt), filamentous hemagglutinin (fha), pertactin (prn) (antigens all included in vaccine), and fimbriae 2/3 (fim) (not included in vaccine), were quantitated by elisa. seroconversion rates for each antigen (pt was the most specific) ranged from 0.4% to 2.7% in unvaccinated pa-tients. authors estimate that the infection rate among unvaccinated adults is close to 1% over an 11-month period. in vaccinated patients the infection rate and the efficacy of vaccine are difficult to obtain. only 0.47% of vaccinated patients had titers at 1 year that were equal to or higher than the titres 1 month after immunization and only 0.23% of patients a 3-fold cut-off. fim antibodies (antigen not included in vaccine) were significantly less common among vaccinated subjects than among controls (p < 0.046) suggesting that infection was less frequent (although marginally significant) in vaccinated patients. in vaccinated and unvaccinated patients symptoms probably related to pertussis infection were more common in patients who had serologic evidence of infection. the incidence of b. pertussis infection in adults is about 1% per year and is usually asymptomatic. b. pertussis boosters may confer protection for adolescents and adults against symptomatic pertussis infection and may reduce transmission to others. the diagnosis of pertussis infection is problematic because of the lack of specific clinical criteria, insensitivity of culture and pcr and the limited availability of standardized serology tests. the aim of this study was to determine the usefulness of several procedures, including real-time pcr, for the laboratory diagnosis of pertussis, and to investigate clonal relationships among clinical isolates of bordetella pertussis. the study was performed in one tertiary hospital in madrid, spain. during 15 months (august 2002 to october 2003) nasopharyngeal swabs were collected from paediatric and adult patients with symptoms of pertussis, and contact cases. the samples were processed by culture (regan-lowe medium), direct fluorescence assay (with polyclonal antibodies against wall antigens), and real-time pcr (light-cycler, primers bp1, region is481). most of the isolates were further characterized by pulsed-field gel electrophoresis. among 121 clinical samples corresponding to 117 patients, b. pertussis was detected in 17 samples by culture (14.1%), 30 samples (24.8%) by dfa and 41 samples (33.9%) by real-time pcr. real-time pcr diagnosed 26 and 24 more cases than culture and dfa, respectively. in relation to culture, sensitivity and specificity of pcr were 88.2% and 75%, respectively. authors consider that a probable partial immunity in patients may justify better results for pcr in relation to culture. seventeen clinical isolates were available for pfge analysis and 5 different genotypes were identified. fourteen isolates were included in two genotypes (genotype c and genotype e). real-time pcr applied to the diagnosis of pertussis provides more positive results than dfa and culture. on the basis of these results, dfa should no longer be used for the diagnosis of b. pertussis infection. a 2 to 5 years cycle is observed in relation to b. pertussis infection, independently of vaccine programmes. some clones are dominant and one of them has circulated at least since 1997. the aims of this study were to determine the prevalence of clinical isolates of e. coli resistant to the fluoroquinolones in hospitalizad patients, as well as the underlying mechanisms of resistance (mutations in genes gyra and parc), and tolerance to organic solvents as markers of of overexpression of the active efflux system acrab. e. coli with a levofloxacin mic > 0,125 mg/l were studied. of the 789 fecal samples analysed, 149 (18.9%) e. coli with diminished fluoroquinolone susceptibility were isolated (18.9%). of these 149 isolates, 102 had a levofloxacin mic > 8 mg/l, these strains had a mean of 3 mutations in genes gyra and parc and presented tolerance to organic solvents more frequently than the isolates with a levofloxacin mic < 8 mg/l that exhibited a mean of one mutation in gyra. the prevalence of isolates with tolerance to organic solvents varied during the period of study. no clonal dissemination was detected. the main conclusions of this study are that colonization of the gut by escherichia coli with reduced susceptibility to quinolones in hospitalizad patients is common, and that the resistant determinants to the quinolones vary over time. resistance to nalidixic acid can be used in the identification of strains of e. coli with mutations in gyra. comparative study of the molecular epidemiology of escherichia coli of humans (35 blood isolates and 33 faecal isolates) and chicken (49 faecal isolates) that were susceptible (n = 57) o resistant (n = 60) to ciprofloxacin, where an analysis of the filogenetic group, virulence genotype and the epidemiologic relationship with rapd and pfge were carried out. among human isolates, those resistant to ciprofloxacin were different of the ciprofloxacinresistant strains, while the sensitive and resistant isolates of chicken were indistinguishable. susceptible human isolates had more genes associated with virulence factors than resistant human isolates or susceptible or resistant chicken isolates. some resistant human isolates were very similar to some chicken isolates by rapd and pfge. this is another study that increases the degree of evidence to the hypothesis that ciprofloxacin resistant isolates can appear de novo (after selection pressure by enrofloxacin and other quinolones used in animal husbandry), in susceptible progenitors in the animal's gut, then be transmitted to humans via the food chain and, finally cause infections in humans with ciprofloxacin-resistant strains. this is a retrospective study of the predisposing factors, clinical presentation and evolution of 43 episodes of esbl-producing escherichia coli bacteraemia seen from january 2001 to march 2005 in a single institution; it represents a 8,8% of the total cases of e. coli bacteraemia seen during the same period. 70% of esbl-harbouring e. coli produced ctx-m types and the great majority was not clonally related; 49% were of nosocomial origin, 32% were from geriatric centers, and 19% from the community. the number of cases increased from 6 in 2001 to 16 in 2004. the mean age of the infected patients was 71, 70% were male, 33% had a foley catheter, 40% had urinary tract obstruction and 31 patients (72%) had received antimicrobials in the recent past (aminopenicillins, third generation cephalosporins, and fluoroquinolones).crude mortality was 21%. patients treated with a combination of β-lactam/β-lactamase inhibitor or a carbapenem had a mortality of 9% as compared with a mortality rate of 35% in those treated with cephalosporins or fluoroquinolones (p = 0.05). the failure rate of those treated with a cephalosporin was independent of the mic values; however the number of patients studied was small and this finding has o be interpreted with caution. in areas where the prevalence of esbl-producing e. coli is increasingly represented, therapeutic guidelines should be revised. given the characteristics of the population described, this suggerence is particularly important when sepsis is diagnosed in old males, carriers of a urinary catheter and those that had been treated with antimicrobials in the recent past. the auhors decribe the case of a 45 years old woman with a prosthetic mitral valve that had a klebsiella pneumoniae bacteraemia secondary to an infected intravenous catheter. the initial isolate produced an esbl, susceptible to ciprofloxacin (mic, 0.38 mg/l) and piperacillin/tazobactam (pt). patient was with a combination of ciprofloxacin and amikacin, and had recurrent bacteraemia 15 days later. this time the blood isolate was resistant to ciprofloxacin (mic, 6 mg/l) and an echocardiogram showed vegetations in the prosthetic valve. ciprofloxacin was replaced by piperacillin/tazobactam. however, after two weeks treatment bacteraemia persisted and repeat blood cultures grew pt resistant e. coli. finally, the patient underwent prosthetic valve replacement and was treated with meropenem. the three isolates were genotypically identical. the resistance to ciprofloxacin was associated to a mutation in gyra. the activity of pt against inolcula of 10 5 and 10 7 ufc/ml showed that with low inocula, the first two isolates were sensitive to pt, while the third isolate had an mic > 256 mg/l. using high inocula, all three isolates had an mic > 256 mg/l. no changes in activity were seen with meropenem in relation to the bacterial inoculum used. the three isolates were susceptible to cefoxitin and amikacin (mic, 8 mg/l). this case exemplifies two important points: first, the possibility of therapeutic failure with pt in case of infection by esbl-producing k pneumoniae and, second, the combination with amikacin does not avoid the emergence of resistance to the companion antibiotic, in this instance, ciprofloxacin. the mechanism of resistance to pt was not characterized; no mutations were identified in the gen bla that could explain the resistance to tazobactam, on the other hand, the susceptibility to cefoxitin ruled out the possibility of resistance due to a loss of a porin. this study analysed the presence of esbl-producing enterobacteriaceae in clinical samples, in faecal samples of the population seen in the emergancy department, in sewage waters, in faeces of farm animals, in faeces of patients with gastroenteritis, and in samples from food. faecal samples were cultured in a 2 mg cefotaxime containing medium. the study was carried out in 2003 and during the same period the estimated comsumption of antimicrobials in ambulatory care was measured. the prevalence of esbl-producing enterobacteriaceae was 1.9% in clinical isolates, 6.6% in faecal samples, and 31% in samples obtained in patients with gastroenteritis. their prevalence in samples from farm animals ranged from 20 and 100%, and in food was 0.4%. of note, 79% of food samples analysed had been previously cooked. the prevalence of faecal carriers was significantly higher in july to november as compared to the period of february to may. the lowest prevalence rates corresponded to the periods with highest antibiotic consumption. given the fact that amoxicilin-clavulanate was the most frequently used antimicrobial, the authors speculate on the possibility that its use could be a reason for a lower detection of esbl-producing strains. however, the impact of other variables such as the difference in alimentary habits (increased comsumption of of uncooked foods during the summer) should be considered. the prevalence of esbl-producing enterobacteriaceae in this area of spain is considerable in all different environments studied. these findings suggest that the wide dissemination of these strains in the community could be transmitted to humans via the food chain. metalo-beta-lactamase (mlb) producing enterobacteriaceae are increasingly recognized in some european countries, particularly vim derivatives. in greece, they have been identified in escherichia coli, enterobacter cloacae and in klebsiella pneumoniae. the last species is considered endemic in this country. vourli et al detected (between june 2004 and march 2005) in a single institution in greece seven proteus mirabilis isolates resistant to cefotaxime, ceftazidime and imipenem, but susceptible to aztreonam. the use of the double disk diffusion assay with edta and imipenem was useful to ascertain the production of a mlb, unlike the e-test. pulsed-field electrophoresis analysis revealed four related clones representing all these isolates indicating persistence of this strain in the hospital setting. in all cases, vim-1 metallo-beta-lactamase was identified. the corresponding bla vim-1 gene was detected in a chromosomally encoded integron platform widely disperse in greece and commonly associated with a plasmid. all p. mirabilis isolates were recovered form patients hospitalized during a prolonged period of time in three different wards, which indicate persistence over time and dispersion through the single institution. this is the first description of a p. mirabilis producing vim-1 that also illustrates the potential for dissemination of multi-resistance isolates. this article should also be considered as an alert of potential failure of the etest strips in the detection of mlb in certain enterobacteriaceae. metalo-beta-lactamases (mbls) in pseudomonas aeruginosa have been mainly described in asia and europe and remain scarce in the usa. aboufaycal et al. detected three multi-resistant p. aeruginosa isolates with a positive metallo-beta-lactamase test during a continuous surveillance study (the mystic study) performed in a single institution in usa (anderson cancer center) over a 7 year period (1999 to 2006). isolates were recovered from unrelated patients and displayed different pulsed-field electrophoresis patterns as well as different ribotypes suggesting an independent emergence, possibly under carbapenem usage. two of these isolates produced the vim-7 enzyme, in one of them simultaneously with oxa-45, whereas vim-2 enzyme was identified in the remaining one. in both vim-7 producing isolates, bla vim-7 gene was associated with an identical integron platform, which may suggest horizontal gene transfer processes. both patients were treated with carbapenems. interestingly, the vim-2 producing isolate was recovered form a patient previously treated with carbapenems in jordan. this study shows a well documented example of selection and spread of multi-resistant isolates from distant geographic areas, in this case, a vim-2 producing pseudomonas aeruginosa. an interesting point addressed by the authors was the phenotypic detection of mlbs in these p. aeruginosa isolates. in one them the double disc synergy approximation test with edta was negative with imipenem and in another one with meropenem. in addition, in two isolates this double disk diffusion assay was negative with ceftazidime. the use of 2-mercaptopropionic acid did not enhanced detection of mlb producing p. aeruginosa isolates. these results also illustrate the laboratory difficulties to detect the presence of mlbs in p. aeruginosa. pseudomonas aeruginosa is a nosocomial pathogen with resistance to antimicrobials, both intrinsic and acquired. the rise of resistance has evolved to the appearance of epidemic caused by strains only susceptible to polymyxins, but the risk factors associated to this fact are not well known. the present study was designed to know the risk factors associated to the development of infections caused by strains of p. aeruginosa only susceptible to polymyxins. a retrospective case-control study was carried-out in a tertiary hospital in athens, greece, including all the patients with a episode of p. aeruginosa bacteraemia (n = 70) between january 2002 and august 2005. cases were those with bacteraemia caused by strains only susceptible to polymyxins (n = 16) and controls those with bacteraemia caused by strains susceptible at least to polymyxins and carbapenems (n = 40). those patients with bacteraemia caused by p. aeruginosa strains susceptible to polymyxins and other antimicrobials, but resistant to carbapenems, were excluded (n = 14). the most common sources of bacteraemia were pneumonia, unknown, urinary tract infections, and intra-abdominal infections. different factors were evaluated, including the use of antimicrobials, considering only those that the patients had received during the hospitalization and at least for three days. mortality rates were 62.5% and 37.5% in cases and controls, respectively. a multivariable logistic regression model revealed that the only factor associated to the development of infec-tions caused by strains of p. aeruginosa only susceptible to polymyxins was the previous use of carbapenems (or 9; p = 0.001). this study proved the rapid emergence of resistance to carbapenems among patients infected by p. aeruginosa receiving this antimicrobial class. the exclusion of patients infected by strains susceptible to polymyxins but resistant to carbapenems is a major limitation of the study because the over-estimation of the use of carbapenems as a risk factor, prompted by the criteria to select the control group and to exclude patients of the study. also, the authors do not comment if the occurrence of infections by strains only susceptible to polymyxins was related to outbreaks nor they made molecular analysis of these strains, to be sure that there was not horizontal transmission of them. the objective of this manuscript was to review the non resolved aspects of the infections caused by acinetobacter baumannii. the genus acinetobacter consists of strictly aerobic, gram-negative cocobacillary rods. different studies have resulted in the description of 25 genomic species, 17 of the have been validated. in relation with the habitat, acinetobacter colonize skin of human beings (< 1% a. baumannii); recent studies have shown a. baumannii in unsuspected reservoirs as foods (fruits and vegetables) and arthropods (body lice); a. baumannii has been the cause of infections in traumatic injuries in iraq, kuwait, afghanistan, and vietnam, suggesting environmental contamination of wounds, although the source remains unknown; also, a. baumannii shows tolerance to the desiccation. a. baumannii develops rapidly antimicrobial resistance, related to the use of antimicrobials in hospitals, through multiple mechanisms: plasmids, integrons, transposons, and natural transformation; it results in the frequent appearance of strains resistant to almost all available antimicrobials. the most frequent infections caused by a. baumannii affect respiratory tract, surgical wounds, urinary tract, and bacteraemia. the crude mortality is high in some infections, as bacteraemia and ventilator-associated pneumonia, although they seem have non-attributable mortality in case-control studies. the frequency of infections and colonisations by a. baumannii differs among hospitals, and they are mostly nosocomial; the incidence in paediatric wards is low. the outbreaks of infections by a. baumannii are facilitated by its resistance to the desiccation and antimicrobial resistance; they occurred in all hospital areas, being more frequent in intensive care units; multiple risks factors have been identified, including wards with high density of colonized/infected patients, environmental contamination, and carriage by hands of staff members; the adherence to strict infection-control measures is useful. the complexity of the epidemiology of a. baumannii infections is highlighted by the existence of centres with endemic/epidemic situations in which multiple clones are responsible of colonisations/infections; also, there is coexistence of outbreaks with sporadic cases of infection. there has been transmission of strains among different sanitary centres in several countries. respect to the treatment, different clinical studies have shown the efficacy of sulbactam and colistin in the treatment of cases of infections caused by multi-drug resistant strains, although there have been contradictory results in experimental models in the case of colistin. in depth review of some selected aspects of acinetobacter baumannii infections. the authors identify several fields in which we need more knowledge: virulence of a. baumannii, identification of reservoirs, strategies for the control of multi-drug resistant strains, and the treatment of infections caused by these strains. acinetobacter baumannii is a nosocomial pathogen causing, among other infections, hospital-acquired pneumonia (hap). previously, there were some reports of communityacquired pneumonia (cap) caused by a. baumannii. the objective of this manuscript was to analyze the clinical and prognostic characteristics of cap by a. baumannii and to compare them with hap by this bacterium. the method was a retrospective case-control study (cases: cap; controls: hap) carried-out in a regional hospital in hong kong between july 2000 and december 2003. pneumonia was defined by clinical and radiographic criteria from the centers for disease control and prevention. a. baumannii was considered "definite pathogen" if isolated from blood or pleural fluid, and "probable pathogen" if isolated from sputum or tracheal aspirate cultures. nineteen cases (cap) and 74 controls (hap) were analyzed. compared with the hap, cap had more smokers (84 vs. 55%; p = 0.03) and more patients with chronic obstructive pulmonary disease (63 vs. 29%; p = 0.01). also, from a clinical point of view, cap had bacteraemia more frequently (31 vs. 0%; p < 0.001), as were the presence of acute respiratory distress syndrome (84 vs. 17%; p < 0.001), and disseminated intravascular coagulation (57 vs. 8%; p = 0.001). finally, the mortality rate at 30 days, was higher in cap than in hap (57 vs. 35%; p < 0.001). factors associated to mortality in cap were: bacteraemia, low platelet count, acidosis, and disseminated intravascular coagulation. these data are consistent with that from previous studies. one limitation of the results is that the number of polymicrobial pneumonias was high: 21% and 62% in cap and hap, respectively, resting specificity to the aetiologies of the cases and controls included in the study. also, it would be useful to know the percentage that cap by a. baumannii represents in the total of cases of cap attended in the hospital in the period of the study. finally, one hypothesis from the study is the possibility that only the severe cases of cap by a. baumannii were admitted to the hospital. the prospects of treatment failure in the chemotherapy of infectious diseases in the 1990s multiple mechanisms of antimicrobial resistance in pseudomonas aeruginosa: our worst nightmare? considerations in control and treatment of nosocomial infections due to multidrug-resistant acinetobacter baumannii community-acquired methicillinresistant 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between humans and a dog centers for disease control and prevention. reduced susceptibility of staphylococcus aureus to vancomycin-japan staphylococcus aureus with reduced susceptibility to vancomycin-united states vancomycin-resistant staphylococci and enterococci: epidemiology and control vancomycin treatment failure associated with heterogeneous vancomycin-intermediate staphylococcus aureus in a patient with endocarditis and in the rabbit model of endocarditis alterations of cell wall structure and metabolism accompany reduced susceptibility to vancomycin in an isogenic seris of clinical isolates of staphylococcus aureus resistance to autolysis in vancomycin-selected staphylococcus aureus isolates precedes vancomycin-intermediate resistance reduced susceptibility of staphylococcus aureus to vancomycin and platelet microbicidal protein correlates with defective autolysis and loss of accessory gene regulator (agr) function vancomycin in surgical infections due to methicillin-resistant staphylococcus aureus with heterogeneous resistance to vancomycin electron transport-deficient staphylococcus aureus small-colony variants as emerging pathogens variant subpopulations of staphylococcus aureus as cause of persistent and recurrent infections small colony variants in staphylococcal infections: diagnostic and therapeutic implications staphylococcus epidermidis: why is it so successful? biofilms: survival mechanisms of clinically relevant microorganisms susceptibility of staphylococcus epidermidis biofilm in csf shunts to bacteriophage attack key: cord-283138-18q23z8l authors: balasubramanian, s.; rao, neha mohan; goenka, anu; roderick, marion; ramanan, athimalaipet v title: coronavirus disease 2019 (covid-19) in children what we know so far and what we do not date: 2020-04-09 journal: indian pediatr doi: 10.1007/s13312-020-1819-5 sha: doc_id: 283138 cord_uid: 18q23z8l pediatric coronavirus disease-19 (covid-19) infection is relatively mild when compared to adults, and children are reported to have a better prognosis. mortality in children appears rare. clinical features of covid-19 in children include fever and cough, but a large proportion of infected children appears to be asymptomatic and may contribute to transmission. it remains unclear why children and young adults are less severely affected than older individuals, but this might involve differences in immune system function in the elderly and/or differences in the expression/function of the cellular receptor for severe acute respiratory syndrome coronavirus 2 (sars-cov-2)angiotensin converting enzyme 2 (ace2). laboratory findings and chest imaging may not be specific in children with covid-19. diagnosis is by reverse transcriptase-polymerase chain reaction (rt-pcr) testing of upper or lower respiratory tract secretions. this review additionally considers covid-19 in immunosuppressed children, and also suggests a management algorithm for the few children who appear to present with life threatening infection, including the potential use of antiviral and immunomodulatory treatment. the most significant threat to global child health from sars-cov-2 is unlikely to be related to covid 19 in children, but rather the socio-economic consequences of a prolonged pandemic. c oronavirus disease-2019 (covid-19) is a global health crisis. the clinical characteristics, disease progression and outcome in children and young adults appear significantly milder compared to older individuals. since first being reported in wuhan, china in december 2019, covid-19 has rapidly spread to affecting over 200 countries worldwide. children account for 1-5% of diagnosed covid-19 cases [1] ; although, many infected children may be asymptomatic and therefore not diagnosed without population screening. at the time of writing, in india, the number of virologically confirmed covid-19 positive cases is 5273 (149 deaths) as per the ministry of health and family welfare (mohfw) [2] . coronaviruses are a family of enveloped, single stranded, zoonotic rna viruses which can rapidly mutate and recombine, leading to novel viruses that can spread from animals to humans [3] . the precise events that led to the emergence of severe acute respiratory syndrome coronavirus-2 (sars-cov-2) causing covid-19 remain unknown. sars-cov-2 is transmitted through inhalation of respiratory droplets of an infected person and touching surfaces contaminated with the virus. in previous coronavirus epidemics, children globally accounted for 6.9% of sars 2002-3 infections and 2% of middle east respiratory syndrome (mers) infections. it appears that sars-cov-2 has a higher transmission capability compared with the closely related viruses causing sars 2002-3 and mers [4] . the true case fatality rate (cfr) of covid-19 infection is currently unknown due to lack of population-scale longitudinal data. thus, estimates of cfr currently vary between 0.5-5% [1, 5] . the sars-cov-2 virus utilizes angiotensin converting enzyme 2 (ace2) receptors as its cell surface receptor, similar to the sars 2002-3 virus. ace2 is expressed in highly byciliated epithelial cells in the human lungs and this receptor allows the virus to attach to the cell [6] . the ace2 receptor is also expressed in the intestines, potentially accounting for the gastrointestinal symptoms that commonly occur in the early stage of the illness. volume 57 __ may 15, 2020 balasubramanian, et al. severe covid-19 disease is characterized by three phases: the first being the viral phase; the second being the cytokine storm; and the third encompassing acute respiratory distress syndrome (ards), impaired cardiac function and death [7] . the cytokine storm appears to be driven by a dysregulated host immune response [8] and might contribute to mortality [9] . the profile of the cytokine storm associated with severe covid-19 disease is similar to that of secondary hemophagocytic lymphohistiocytosis (hlh), which is a rare complication of other viral infections (3.7-4.3%) [8] . secondary hlh is characterized by fulminant and fatal hypercytokinemia with multiorgan failure. in severe infection, lower peripheral lymphocyte counts (cd4 and cd8 t cells), higher interleukin (il) levels (il-6 and il-10), decreased interferon-gamma expression in cd4+ t cells and higher d-dimer and fibrin degradation products (fdp) levels, leading to increased thrombosis and multiorgan injury has been described. moreover, patients with severe infection may also have abnormal coagulation parameters, perhaps related to high expression of ace2 receptors in vascular endothelial cells. most infected children are likely to be secondary cases and acquire the infection after exposure to a covid-19 positive adult, although there are no longitudinal data to confirm this yet. intra-family transmission may be important [10] . an as yet unquantified proportion of children with covid-19 is asymptomatic and may contribute to transmission. it is unknown whether covid-19 is acquired by contact with infected feces [10, 11] . in a report of 10 children admitted for covid-19 with positive nasopharyngeal swabs, 8 of 10 children demonstrated persistently positive real time reverse transcriptase-polymerase chain reaction (rt-pcr) of rectal swabs after their nasopharyngeal testing had become negative [12] . it remains unclear whether the detection of virus by rt-pcr in fecal matter represents active viral replication or residual viral genomic material; however, it appears that viral shedding from the digestive tract might be greater and last longer than that from the respiratory tract [12] . multiple reports have demonstrated that children and young adults have a milder form of the disease compared to adults [13] . asymptomatic, mild and moderate infections comprise over 90% of all children who have tested positive for covid-19 with fewer severe and critical cases (5.9%) compared to adults (18.5%) [13] . the possible reasons for lower number and milder infections in children and young adults include lower exposure to virions, being isolated at home and minimal exposure to pollution and cigarette smoke contributing to healthier respiratory tracts. the elderly may be susceptible to severe covid-19 disease by their qualitatively different immune response, encompassed by the terms 'immunosenescence' and 'inflammaging' [14] . viral co-infection may be important in potentially leading to limited replication of the sars-cov-2 by direct virus-to-virus interaction and competition [15] . additionally, the distribution, maturation and functioning of viral receptors such as ace2 may be important in age-dependent susceptibility to severe covid-19 [13, 16] . due to smaller number of reported cases in children, it is at present challenging to delineate the clinical characteristics of children with severe covid-19 infection, combined with the lack of a clear biomarker to indicate severity of infection [17] . dong, et al. [13] , in the largest pediatric review of 2143 children, described that 13% of virologically confirmed children were asymptomatic. this makes epidemiological inference problematic since asymptomatic children are less likely to be tested and may still contribute to transmission. in addition, a significant proportion of children can also have coinfections with other viruses, and the detection of sars-cov-2 may therefore be clinically insignificant [11] . it has been proposed that the outcome for some children may be worse due to exposure to antenatal smoking and obesity [17] . another theory that has been postulated is the protective role of bacillus calmette-guérin (bcg) vaccine in covid-19. bcg vaccination has been associated with heterologous immunity to other pathogens, potentially by a phenomenon called 'trained immunity' involving innate cells such as macrophages, monocytes and epithelia [18] . trials are underway to understand if bcg vaccination may offer protection against covid-19. children of all ages can be infected with covid-19, with more cases reported in younger children and infants [13] . acknowledging the possible reporting biases discussed above, there is no age or sex preponderance [13] and the median age of infection is 6.7 years (rangenewborn to 15 years) [19] . the incubation period of covid-19 in children has been reported as 2 days (range-2 to 10 days) [1] . at the time of diagnosis, 13-15% of virologically positive children may be asymptomatic [13, 19] . the most common symptoms described at onset in children are fever (50%) and mild cough (38%) [10] . fever is present in about 40% of volume 57 __ may 15, 2020 balasubramanian, et al. covid-19 in children children [19] . other clinical features include sore throat, rhinorrhea, sneezing, myalgia, fatigue, diarrhea and vomiting. children may have more upper respiratory symptoms than lower respiratory symptoms [13] , and appear to recover in 1-2 weeks [20] . in the largest pediatric cohort to date, dong, et al. [13] describe suspected and confirmed cases based on symptoms, laboratory abnormalities, chest imaging, and rt-pcr/genomic analysis. the severity of covid-19 was divided into asymptomatic, mild, moderate, severe and critical. severe covid-19 accounted for 18 (2.5%) of virologically confirmed cases, and furthermore the definition of severe included children with only mild hypoxia. critical covid-19 was observed in 3 (0.4%) of virologically confirmed cases, defined by the presence of ards or organ failure. though data on chronology of complications and predictors of mortality is available in adults, there is insufficient data on predictors of mortality in children. the ministry of health and family welfare (mohfw) [2] in their updated guidelines (as of 7 april, 2020) has categorized patients into three groups -those with mild, moderate and severe illness, and have designated covid dedicated facilities for their treatment. rt-pcr testing of nose and throat swab for detection of sars-cov-2 nucleic acid has been recommended as the confirmatory test for covid-19 [21] . other alternative samples for rt-pcr include bronchoalveolar lavage or endotracheal aspirate. the government of india has now advised the use of antibody tests in patients with symptomatic influenza-like illness (ili) in 25 districts across the country, or 'covid hotspots' [22] . based on the results of the antibody test, confirmatory rt-pcr and clinical assessment, hospital treatment or home isolation measures are instituted, with contact tracing measures as per protocol. the limited data in children describes relatively lower rates of lymphopenia and elevated inflammatory markers compared to adults [1] . henry, et al. [23] summarized the findings from 12 studies on 66 children and reported normal leucocyte counts (69.2%), neutropenia (6.0%), neutrophilia (4.6%) and lymphopenia (3.0%). c-reactive protein (crp) and procalcitonin were high only in 13.6% and 10.6% of cases, respectively. slight elevation of liver transaminases is common [23] . it is recommended to monitor the lymphocyte count and crp as signs for severe infection, while using procalcitonin levels to detect potential bacterial co-infection [23] . chest x-ray findings in children appear to be nonspecific. children with mild disease should not routinely need computed tomography (ct) chest imaging in view of the high radiation exposure [24] . when ct is performed, ground glass opacities is seen in one third of patients [19] . peripheral distribution of lung lesions has been noted, with multilobar involvement [25] . consolidation with surrounding halo sign is considered typical of pediatric patients [26] . however, chest ct alone cannot accurately diagnose covid-19 due to similar radiological presentations with other infections. patients admitted with severe infection are known to have elevated plasma levels of il-2, il-7, il-10, granulocyte colony stimulating factor (gcsf), interferon-gamma-inducible protein 10 (ip10), monocyte chemoattractant protein 1(mcp1), macrophage inflam-matory protein 1-alpha (mip1a) and tumor necrosis factor (tnf) alpha [9] . in a study comprising of 150 confirmed covid-19 cases in wuhan, china, elevated ferritin (mean 1298 ng/ml vs 614 ng/ml; p<0.001) and il-6 levels (p<0.0001) were found in survivors compared to non-survivors [7] . these cytokines are produced by inflammatory macrophages which have been implicated in the cytokine storm. this is similar to previous outbreaks of mers and sars 2002-3 in terms of having high proinflammatory cytokines in patients with severe disease [27] . upon suspicion of covid-19 infection, immediate infection prevention control (ipc) measures must be instituted. standard precautions such as hand hygiene, use of personal protective equipment (ppe), safe waste management and cleaning and disinfection of equipment must be followed as per the guidelines issued by the mohfw [2] . for the few children who will require admission to a healthcare facility, the cornerstone of management is supportive therapy including adequate nutrition and calorie intake, fluid and electrolyte management and oxygen supplementation. communication with parents and alleviating anxiety is an important part of management. in adults with severe covid-19, early intubation and mechanical ventilation with lung protective strategies and prone positioning has been recommended [20] . antibiotics may be indicated if bacterial super-infection is suspected. there are no randomized clinical trial data to guide treatment of the very few children that present with lifethreatening covid-19 including severe pneumonia, volume 57 __ may in the absence of data from these trials, clinicians may be left in the difficult scenario of deciding whether to pursue treatment with antiviral drugs and immunomodulatory therapies for children with severe covid-19. a relatively new antiviral drug being tested in adults with covid-19 is remdesivir, which in combination with chloroquine has been found to inhibit sars-cov-2 growth in vitro [28] . interferon alpha-2b and oral lopinavir/ritonavir together with corticosteroids for complications and intravenous immunoglobulin for severe cases has been recommended in one report in china [29] . a hiv test should be performed before commencing antiviral treatment, in particular lopinavir/ritonavir. the mohfw has allowed off label use of hydoxychloroquine in combination with azithromycin in adults with severe disease and requiring intensive care [2] . however, these treatments are not currently recommended in children below the age of 12 years. corticosteroids are not routinely recommended and might exacerbate covid-19 associated lung injury [30] . ivermectin, the broad spectrum anti-parasitic agent, has in vitro antiviral action against sars-cov-2 [31] . owing to the cytokine storm syndrome in covid-19, there may potentially be a role of immunomodulators in treating patients with severe infections to ameliorate pulmonary inflammation and hopefully improve mortality. there is an established role of anakinra (il-1 blockade) in survival benefit of patients with hyperinflammation, without increased adverse events [8] . a multicenter randomized control trial (rct) of the il-6 receptor blocker, tocilizumab is in progress in china for adults with covid-19 pneumonia and raised il-6 levels (chictr2000029765) [32] . there may also potentially be a role of janus kinase inhibitors (jki), since these drugs block downstream inflammatory pathways and may alter cellular viral entry [33] . a suggested management algorithm based on the limited observational data from adults is depicted in figs. 1 and 2 . the common drugs used in covid-19 are detailed in table i covid-19 in children children with covid-19 are likely to need any specific therapy other than supportive treatment, and the decision to start antiviral or immunomodulatory treatment should therefore be made carefully in consultation with experts in pediatric infectious disease and immunology. given that severe covid-19 appears very rare in children, an important part of this assessment is ascertaining whether a positive rt-pcr for sars-cov-2 is a clinically important factor in explaining the child's condition, or whether more occult pathology may be responsible. for neonatal management of covid-19 infected mothers, it is recommended to have a separate room adjacent to the delivery room for neonatal resuscitation or for resuscitation staff to maintain atleast a 2 meter gap between the infected mother and newborn [34] . only essential personnel should attend the delivery with full ppe, with the mother following meticulous hand hygiene and wearing a mask. standard neonatal resuscitation measures are to be followed and positive pressure ventilation if needed should be provided by a selfinflating bag and mask rather than a t-piece resuscitator. if the baby requires intensive care, a single patient room is ideal preferably with negative pressure. the baby should be tested at 24 hours of life and repeat testing should be performed at 48 hours. antivirals/hydroxychloroquine/steroids or intravenous immunoglobulin (ivig)should not be administered to the newborn. the baby should then be tested every 48-72 hours until two consecutive negative tests. it is critical that breastfeeding shouldbe encouraged with the mother wearing a mask. the baby should be vaccinated prior to discharge from the hospital. data on children with immunocompromised conditions and covid-19 are scarce, but severe disease may be more common in adults with cancer [35] . despite concerns that immunocompromised children may have severe infection analogous to infection with adenovirus, rhinovirus, influenza, respiratory syncytial virus, and experience from previous pandemics (such as influenza h1n1),antiga,et al. [36] described that children who were immunocompromised were not at greater risk of severe covid-19, probably owing to the fact that a functional host innate immune response is the main driver for lung damage. in bergamo, among 200 transplant recipients including 10 inpatients, 100 with autoimmune liver disease and three undergoing chemotherapy for hepatoblastoma (inpatients), none had clinical pulmonary disease, despite the fact that 3 patients tested positive for sars-cov-2, suggesting that the immunocompromised may be protected by their weaker immune response. no data is available on severity of covid-19 infection in children with malnutrition, rheumatic heart disease or human immunodeficiency virus (hiv) positive children. several vaccines against sars-cov-2 are in development; however, it remains unclear when a successful vaccine might be rolled out. studies on factors responsible for immune dysregulation may provide insights into developing vaccines capable of inducing durable protective immunity and avoiding vaccinerelated adverse events. this unprecedented pandemic should prompt improved global surveillance of infectious diseases, as well as cooperation and communication so that the global society remains interconnected and limits the spread of this outbreak. lastly, we fear the greatest impact on children from covid-19 is likely to be delayed presentation of other childhood illnesses due to fear and ignorance amongst parents/families. this coupled with the impact of economic uncertainty on those in the low socioeconomic strata, is likely to have a greater adverse impact on child health in india in these uncertain times. contributors: sb, avr-initiated the preparation of the manuscript;nmr: substantial contribution to the conception and design of the work, and prepared and finalized the draft; sb, avr, ag, mr-substantial contributions to the acquisition, analysis, and interpretation of data for the work, sb, avr, ag, mr-revising it critically for important intellectual content;sb, nmr, ag, mr, avr: final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. funding: none; competing interests: none stated. systematic review of covid-19 in children show milder cases and a better prognosis than adults ministry of health and family welfare coronavirus infections in children including covid-19. pidj sars-cov-2/ : a storm is raging. jci estimating case fatality rates of covid-19 covid-19: knowns, unknowns, and questions. msphere why is covid-19 so mild in children? acta paediatr covid-19: consider cytokine storm syndromes and immunosuppression clinical features of patients infected with 2019 novel coronavirus in wuhan a case series of children with 2019 novel coronavirus infection: clinical and epidemiological features. cid covid-19 in children: initial characterization of the pediatric disease characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding epidemiological characteristics of 2143 pediatric patients with 2019 coronavirus disease in china the impact of immunosenescence on pulmonary disease virus-virus interactions impact the population dynamics of influenza and the common cold are children less susceptible to covid-19? jmii covid-19 infection in children bcg-induced cross-protection and development of trained immunity: implication for vaccine design.front immunol sars-cov-2 infection in children. nejm sars-cov-2 infection in children: transmission dynamics and clinical charateristics revised guidelines on clinical management of covid -19 advisory to start rapid antibody based blood test for covid-19 laboratory abnormalities in children with novel coronavirus disease 2019. cclm covid-19 in children: the link in the transmission chain radiographic and clinical features of children with 2019 novel coronavirus (covid-19) pneumonia. indian pediatr clinical and ct features in pediatric patients with covid-19 infection: different points from adults mers-cov infection in humans is associated with a pro-inflammatory th1 and th17 cytokine profile remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro clinical and epidemiological features of 36 children with coronavirus disease 2019 (covid-19) in zhejiang, china: an observational cohort study clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury the fda-approved drug ivermectin inhibits the replication of sars-cov-2 invitro chinese clinical trial registry. a multicenter, randomized control trial for the efficacy and safety of tocilizumab in the treatment of new coronavirus pneumonia (covid-19) baricitinib as potential treatment for 2019-ncov acute respiratory disease perinatal-neonatal management of covid-19 infection -guidelines of the federation of obstetric and gynecological societies of india (fogsi), national neonatology forum of india (nnf), and indian academy of pediatrics (iap) cancer patients in sars-cov-2 infection: a nationwide analysis in china coronaviruses and immunosuppressed patients bpaiig position statement: sars-cov-2 treatment guidance version 1.2 key: cord-303517-8971aq02 authors: cajamarca-baron, jairo; guavita-navarro, diana; buitrago-bohorquez, jhon; gallego-cardona, laura; navas, angela; cubides, hector; arredondo, ana maría; escobar, alejandro; rojas-villarraga, adriana title: sars-cov-2 (covid-19) in patients with some degree of immunosuppression date: 2020-10-16 journal: nan doi: 10.1016/j.reumae.2020.08.001 sha: doc_id: 303517 cord_uid: 8971aq02 background it is not clear whether patients with some degree of immunosuppression have worse outcomes in sars-cov-2 infection, compared to healthy people. objective to carry out a narrative review of the information available on infection by sars-cov-2 in immunosuppressed patients, especially patients with cancer, transplanted, neurological diseases, primary and secondary immunodeficiencies. results patients with cancer and recent cancer treatment (chemotherapy or surgery) and sars-cov-2 infection have a higher risk of worse outcomes. in transplant patients (renal, cardiac and hepatic), with neurological pathologies (multiple sclerosis (ms), neuromyelitis optica (nmods), myasthenia gravis (mg)), primary immunodeficiencies and infection with human immunodeficiency virus (hiv) in association with immunosuppressants, studies have shown no tendency for worse outcomes. conclusion given the little evidence we have so far, the behaviour of sars-cov-2 infection in immunosuppressed patients is unclear, but current studies have not shown worse outcomes, except for patients with cancer. in december 2019, a group of five patients with severe pneumonia of unknown origin were reported to have had contact with a seafood market in the city of wuhan, hubei province, china, as an epidemiological link. the chinese centre for disease control and prevention (china cdc), deployed a rapid response for the epidemiological and aetiological investigation of cases and identified a new coronavirus with the ability to cause severe lung disease that can rapidly progress to death in affected patients. 1, 2 given its rapid progression and the poor knowledge of the infection, how it behaves in patients with multiple comorbidities is not clear, especially patients with some degree of immunosuppression, due either to their underlying disease or to the use of immunosuppressants to manage it. in this review, we will focus on describing the literature on sars-cov-2 infection in patients with some degree of immunosuppression, other than rheumatological diseases, among these, cancer patients, transplant recipients, primary immunodeficiency, and hiv patients. initially the virus was termed the new coronavirus (2019-ncov) and variations of same. sars-cov-2 is the name currently used for the virus, which shares genetic similarities with the sars-cov virus. covid-19 (coronavirus disease 2019) is the name of the disease generated by sars-cov-2 infection. 3 j o u r n a l p r e -p r o o f epidemiology from december 18th to 29th, samples of bronchoalveolar lavage fluid were collected from patients hospitalized for severe pneumonia in the city of wuhan, the epicentre of the pandemic, and the new coronavirus was isolated. results for viruses such as severe acute respiratory syndrome (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), influenza, avian influenza, and other common respiratory pathogens were negative. 1, 3 on january 24, a case series of 41 confirmed sars-cov-2 patients treated at a wuhan hospital was released. most were male (73%), with a median age of 49 years, and less than half had comorbidities (32%) such as diabetes mellitus (20%), hypertension (15%), and cardiovascular disease (15%). of these patients, 66% had a history of exposure to the seafood market. 4 in another case series of 138 hospitalized patients in wuhan, china, 41% of the infected patients were presumed to have been infected by nosocomial transmission, 26% of the patients required icu hospitalization, and mortality was about 4.3%. 5 the infection rapidly escalated and was declared a public health emergency by the world health organization (who) on january 30, 2020. as of today, june 27, 214 affected countries have been reported, and the number of confirmed cases worldwide is close to ten million (9, 770, 954) , with a total of 493,898 deaths and 5,391,416 cases that have recovered. the country with the most confirmed cases is currently the united states, followed by brazil and russia. china, the first country affected, has a declining case curve and is now in twentieth place worldwide. 6 in the americas, there are 4,933,972 confirmed cases with 241,931 deaths; the united states and brazil together account for 75% of all cases and 75% of all deaths currently reported in the region. 7 infection in children is less frequent and most reported cases are among family members and, to a lesser extent, from close contact with infected patients. 8 j o u r n a l p r e -p r o o f the coronaviruses (cov) are a group of viruses discovered in 1960 that have a single strand of rna (~26-32 kb in length) that codes for structural, envelope, membrane and nucleotide proteins, as well as for non-structural proteins. 9 they belong to the coronaviridae family which in turn is part of a larger family, the nidovirals. the coronaviridae family is divided into two subfamilies: orthocoronavirinae and torovirinae. the former is classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. they are zoonotic viruses, bats have been acknowledged as natural hosts, but six types have been recognized as having the ability to infect humans: two alpha-coronaviruses (229e and nl63) and four betacoronaviruses (oc43, hku1, sars-cov and mers-cov). 1, 9, 10 in 2003, there was an outbreak of sars-cov that caused 794 deaths worldwide. in 2012, mers-cov was discovered in middle eastern countries with a fatality rate of 35.5%. 1 sars-cov-2 is a betacoronavirus, subgenus sarbecovirus and from the subfamily orthocoronavirinae with an envelope composed of a lipid bilayer derived from the host membrane. the genome encodes for spike glycoprotein (s), small envelope protein (e), membrane protein (m), and nucleocapsid protein (n). it also encodes accessory proteins that interfere with the host's immune response. 11 its name is due to its similarity to a crown, given the spherical morphology of the virus and the projections on its surface that correspond to the s protein, which is glycosylated and mediates the viral entry into the host cells. the m protein gives the shape to the viral particle and together with the e protein directs the assembly of the virus and its maturation. the n protein participates in the packaging of the viral rna during assembly. haemagglutinin is one of the accessory proteins, which binds to sialic acid in host glycoproteins, improving entry into the cell. 10 like sars-cov, sars-cov-2 uses the receptor for the angiotensin 2 converting enzyme (ace2) as a means of entry into the cell where it binds by means of the s protein, however, unlike the other viruses, sars-cov-2 binding is much stronger since this protein undergoes a residue substitution in its c-terminal domain that increases affinity for the receptor. 11, 12 the s protein has two subunits, s1 which determines the cell tropism and s2 which mediates the fusion of the virion to the membrane so that it can enter the cell where it rapidly translates two polyproteins that form the replication/transcription complex into a double-membrane vesicle; the virion contained in these vesicles fuses with the plasma membrane to be released later. 11 the viral genome found in cytoplasm acts as pathogen-associated molecular patterns (pamps) and are recognized by the molecular pattern recognition receptors (prrs) that are toll-like receptors (tlr 3, tlr7, tlr8 and tlr9). the rig-i receptor (retinoic-acid inducible gene-i), the cytosolic receptor mda-5 (melanoma differentiation-associated gene 5) and cgas (nucleotidyltransferase cyclic gmp-amp synthase) recognize viral rna and recruit adaptive molecules that trigger a response cascade leading to the activation of the nuclear transcription factor- and interferon regulatory factor 3 (irf3), producing interferon  and  and pro-inflammatory cytokines. 11 different elevated cytokines have been found in patients with covid-19: il-1, il2, il-4, il-7, il-10, il-12, il-13, il-17, macrophage colony-stimulating factor (mcsf), mcp-1, hepatocyte growth factor (hgf), ifn- and tnf-. this supports the fact that lung damage is secondary to a cytokine storm induced by the inflammatory response, resulting in the person entering a critical condition. 11, 13 the transmission dynamics are not yet fully known. the intermediate host between the natural reservoir and humans is unknown. however, it has been possible to confirm person-to-person transmission, which contributes to the rapid spread of the disease, and this is confirmed by the data found in the case series, which show that as of january 1, 55% of cases were linked to the seafood market in wuhan (china); however, of the cases reported after this date, only 8.6% had this link. 14 to date, person-to-person transmission has been considered to occur via respiratory droplets produced by coughing or sneezing. however, the presence of the virus has been detected in other fluids such as blood, faeces, and saliva. 15, 16 initially, it was believed that spread was by people with clinical manifestations. however, it has been shown that asymptomatic carriers also transmit, and some people have even been recognized as "super spreaders", infecting many people, including health workers. 3 vertical transmission of the virus in pregnancy has not been proven, however it is not known whether there is a risk during delivery through the vaginal canal. 17 clinical manifestations can range from being asymptomatic to acute respiratory distress syndrome and multiorgan dysfunction. the incubation period is estimated at 5.2 days; however, this can vary. 18 in the first case series of zhu et al. and ren et al. of patients treated at a hospital in hubei, the predominant symptoms were dry cough, dyspnoea, and fever. lung opacities consistent with a pneumonic process, worsening rapidly (two to four days) and requiring invasive mechanical ventilation, are recorded. 1, 2 in general, the most common clinical manifestations are fever (although not present in all cases), cough, odynophagia, fatigue, and myalgia. other less frequent symptoms are sputum production, headache, haemoptysis, and diarrhoea. cases of keratoconjunctivitis and fulminant myocarditis have also been described. 4, 5, 14 in early stages of the disease, chest x-ray can be normal, but as the disease progresses, bilateral ground glass opacity or consolidation can be found in more than 89% of patients. chest ct is much more sensitive than radiography. these findings can be found in asymptomatic patients. 13, 19 it has been reported that up to 17% of patients will have no radiological changes. 20 as for laboratory findings, lymphopenia was common, found in more than 80% of patients, with less frequent evidence of thrombocytopenia and leukopenia. a large proportion presented with elevated c-reactive protein, and in fewer cases, elevated alanine aminotransferase, aspartate aminotransferase, creatine kinase, and d-dimer were found. disturbances are pronounced in patients with severe disease. 20 in the different case series and epidemiological reports published since the appearance of sars-cov-2, comorbidities have been highlighted as risk factors associated with severity. it has also been established that patients admitted to the intensive care unit have more comorbidities than those in general hospitalization. the most prevalent underlying diseases are high blood pressure, diabetes mellitus, cardiovascular and cerebrovascular disease. 21, 22 the results of nine metaanalyses in patients with covid-19 published to date are summarised in tables 1 and 2. table 1 presents the prevalence of comorbidities in patients with covid-19, most of them hospitalized, and analysed through seven meta-analyses. in turn, table 2 shows the risk associated with severity, death, or fatality due to the presence of comorbidities, through seven meta-analyses. the meta-analysis by wang et al. is noteworthy, which identifies arterial hypertension, diabetes mellitus, chronic obstructive pulmonary disease (copd), cardiovascular and cerebrovascular disease as factors that negatively impact mortality. specifically, copd increases by 5.9 times 23 the risk of progression and deterioration of patients with covid-19. these data are supported by the results of another meta-analysis by zhao et al. where the severity of covid-19 is four times higher in copd patients; they also assessed the impact of active smoking, which increases the risk of severe covid-19 two-fold. 24 it has been widely demonstrated that patients suffering from diabetes mellitus are admitted more frequently to the intensive care unit and have a higher mortality, as shown in table 2 . the results of a primary study by roncon et al. are highlighted (or 3.21, 95% ci 1.82-5.64; p < .0001, i 2 = 16%). 25 these patients tend to present a more severe pneumonic process, with greater inflammatory response and tissue damage, which makes them more prone to cytokine storm leading to rapid deterioration, which is why patients with this history should be strictly monitored. 26 cardiovascular disease is a risk factor per se increased by covid-19 infection that generates or aggravates myocardial damage, but when associated with myocardial injury the results are usually fatal for patients. 27, 28 among other comorbidities, chronic kidney disease is associated with in-hospital mortality, as are cancer and cerebrovascular disease, demonstrated through two meta-analyses that included over fifteen thousand patients ( table 2) ; studies suggest that superficial fungal infections and psoriasis confer vulnerability to covid-19; a body mass index (bmi) > 40 kg/m2 is an independent risk factor for complications from the infection; and there are discouraging results regarding underlying neurological disease and sars-cov-2. [29] [30] [31] [32] in general, the presence of comorbidities should imply strict follow-up of patients to detect early complications; however, more attention should be paid to certain comorbidities where strong associations have been found with covid-19 infection and its severe outcomes. overall mortality is .5% to 4%, but it changes to 5% to 15% among patients requiring hospitalization and increases to 62% in critically ill patients. studies have shown two peaks, at 14 and 22 days. among the causes of death, respiratory failure prevails, followed by shock due to myocardial dysfunction and finally, the combination of the two. 33 it is important to maintain a high degree of diagnostic suspicion in patients with fever or respiratory symptoms who have travelled to affected areas or have had close contact with j o u r n a l p r e -p r o o f suspected or confirmed cases 14 days before the onset of symptoms. in this scenario, confirmation by molecular testing is required. real-time reverse transcription polymerase chain reaction is performed on specimens collected from the lower or upper respiratory tract if the former cannot be obtained. 34 there are several assays that are performed on serum or plasma for the detection of both viral proteins and antibodies. the most widely used are to detect immunoglobulin g (igg) and immunoglobulin m (igm) antibodies, which are produced in the second week of infection. 35 there are several therapeutic goals and they are directed at different levels: inhibition of virus entry into the cell, inhibition of fusion of the viral envelope to the membrane, transcription inhibition, inhibition of viral proteins and blockade of il-6 signalling to prevent the cytokine storm. 35 at first, chloroquine (clc) and hydroxychloroquine (hcq) were shown to block sars-cov-2 in vitro, with better results for the latter and therefore its use was indicated for the management of covid-19 infection. 36 promising results have been shown in case series; however, the studies have limitations such as the sample size used. however, preliminary results were recently published from the recovery study, 37 a randomized clinical trial to evaluate potential drugs for management of the infection in the united kingdom, in which they concluded no beneficial effect of the use of antimalarials in hospitalized patients, and therefore they stopped including patients for this treatment arm and the recommendation that it should not be used has been extended worldwide. additional results have recently been published in several articles on the efficacy and safety of the antimalarials chloroquine and hydroxychloroquine for the treatment of different phases of sars-cov-2 infection. however, the data are controversial, some not demonstrating efficacy or reporting a high number of adverse events, primarily associated with cardiac arrhythmias. 38 it is important to note that a number of criticisms and concerns have been raised regarding the accuracy of the data from these studies and they have therefore been withdrawn. 39 to date there are more than 200 clinical trials underway with hcq, and 80 with clq, 35 several of which are in prophylactic use in healthcare workers and others post-exposure. 36, 40 lopinavir/ritonavir has studies in sars and mers, the data published for covid-19 are reports and retrospective studies with which the effect cannot be established with certainty. there is a clinical j o u r n a l p r e -p r o o f trial of 199 patients with covid-19 with no difference in mortality, hospital discharge or recovery. however, despite this, in some centres it is still being used at doses of 400 mg/100 mg twice a day for 14 days. 35 ribavirin, like lopinavir/ritonavir, has activity against other coronaviruses and was considered to be a possible treatment for sars-cov-2; however, studies carried out for sars show limited, highdose activity leading to a high rate of haematological and hepatic adverse events, therefore its use is now limited. 35, 41 other antivirals such as oseltamivir were used in the first cases in hubei, china, because it was suspected to be seasonal influenza; they are now not indicated for use in sars-cov-2. 35 remdesivir is a nucleoside analogue that showed in vitro activity against sars-cov-2, used later in a 53-patient cohort in canada, the united states, europe, and japan, achieving a satisfactory response in 36 patients. 42 based on preliminary studies, some drug regulatory agencies (united states and japan), 43 conducted emergency approvals for use in hospitalized patients. results from ongoing studies are expected to evaluate efficacy and safety. currently umifenovir or arbidol is under study, an antiviral that aims to inhibit the interaction between protein s and ace2. 35 the use of corticosteroids is limited in sars-cov-2 infection to scenarios of chronic obstructive pulmonary disease exacerbation and refractory shock, taking into account previous studies in influenza pneumonia where they were associated with increased mortality. 35 recently, preliminary results from the recovery study 44 showed that the use of dexamethasone reduced mortality in one-third of critically ill patients who were on mechanical ventilation, while reduction was onefifth in those receiving non-invasive oxygen. definitive results are expected from this and the more than 10 clinical trials currently underway to define the particular subgroups that would benefit from this treatment. monoclonal antibodies against il-6 are another therapy studied, in phases of adult respiratory distress syndrome (ards), with promising results in small case series. 35 convalescent plasma is another therapy used as salvage in sars and mers. at the beginning of the pandemic, a case series of five critical patients from china, who were given convalescent plasma, showed improvement in their clinical status. more recently, several case series and preliminary trial results have demonstrated clinical benefits and decreased mortality with its use, 45 particularly in hospitalized patients with moderate to severe involvement; however, results from more than j o u r n a l p r e -p r o o f 200 clinical trials 46 are awaited to clarify the characteristics of the plasma, the donors, and the specific individuals who could benefit. 47 despite the abovementioned therapies, there is still no specific treatment and therefore the recommendations are symptomatic management in mild cases, supportive therapy in cases of critical illness and management of ventilation in cases of ards. 35 immunosuppression and sars-cov-2 given the suddenness of the pandemic, and its rapid spread, little is known about sars-cov-2 infection and certain types of condition or disease, this is the case for people with some type of immunosuppression (either primary, associated to underlying or pharmacological diseases), which given the physiopathogenesis of sars-cov-2 infection known so far, would raise two hypotheses: it could be a possible benefit, since this state of immunosuppression could avoid that uncontrolled immune response or "cytokine storm", but on the other hand, it is equally clear from previous studies that immunosuppressant use or status is associated with increased risk of infection. in epidemics such as the abovementioned sars-cov, immunosuppressed patients, especially transplant recipients, did not have worse outcomes than the general population. 48, 49 similar findings were presented in the mers epidemic, being male, advanced age and comorbidities such as diabetes mellitus, obesity, pulmonary pathology and renal disease being found as risk factors, and immunosuppression status not being associated as a factor of poor prognosis. 50 to date, the centres for disease control and prevention (cdc) and other international agencies 51 have included as poor prognostic factors patients with some degree of immunosuppression, including people with a history of cancer treatment, smokers, transplant recipients, people with immunodeficiencies, poorly controlled hiv or aids and people with prolonged use of steroids or immunosuppressive drugs, all based on previous studies that associate such diseases with respiratory infections, especially of viral aetiology. 52 current evidence of conditions associated with immunosuppression and sars-cov-2 infection j o u r n a l p r e -p r o o f c a n c e r on march 21, liang et al., 53 published a study collecting data from 575 hospitals in china, up until january 31, 2020, on patients with sars-cov-2, comparing those with a history of cancer and those without. they collected 1,519 patients, 18 (1%) with a history of cancer. the most frequent neoplasm was lung (five cases [28%]), and of the total cancer patients, four (25%) had undergone chemotherapy or surgery within the previous month, and the rest were cancer survivors, with strict follow-up. in terms of sociodemographic characteristics, the cancer patients were older, had a greater history of exposure to cigarettes, presented more polypnoea and had more severe pulmonary tomographic manifestations. in the analysis of outcomes, they showed that patients with a history of cancer and sar s-cov-2 infection were at greater risk of serious events (defined as the percentage of patients admitted to the intensive care unit requiring invasive ventilation or death) compared to patients without cancer (seven (39%) of 18 patients vs. 124 (8%) of 1,572 patients; p = .0003). in addition, patients who underwent chemotherapy or surgery in the last month had a higher risk (three (75%) out of four patients) of clinically severe events than those who had not undergone chemotherapy or surgery (six (43%) out of 14 patients). these data were confirmed by logistic regression (odds ratio (or) 5.34; 95% ci 1.80-16.18; p = .0026) after adjusting for other risk factors such as age and smoking history. in addition, the patients with cancer deteriorated more rapidly than those without cancer (median time to severe events 13 days (ci 6-15 vs. 43 days, 20-unreached; p <.0001). furthermore, desai et al., 54 recently published a meta-analysis, in which they included 11 studies, finding a prevalence of cancer in patients with covid-19 of 2% (ci 2.0%-3.0%; i2 = 83.2%). we should clarify that some authors consider that the current evidence is insufficient in this field, however, the number of research results has been increasing, showing similar results. 55, 56 taking into account the results mentioned, it can be stated that cancer and its recent treatment are bad prognostic factors for sars-cov-2 infection. therefore, special recommendations should be considered for these patients, such as postponing adjuvant chemotherapy or elective surgery in people with "stable" cancer, especially in endemic areas, adopting stricter personal protection measures for cancer patients or cancer survivors, and considering stricter surveillance or treatment when cancer patients are infected with sars-cov-2. 57 in general, decisions should be made on a "patient-to-patient" basis. 55, 58 t r a n s p l an t we highlight the study of a case series, 59 two heart and kidney transplants and one liver (paediatric population). the heart transplant patients were confirmed by pcr to be infected, one of them was 51 years old, came with immunosuppression with tacrolimus 2 mg per day and mycophenolate 1 g per day, and attended consultation for fever, fatigue and liquid stools, with characteristic findings of sars-cov-2 infection on chest tomography. he presented criteria of severe pneumonia, immunosuppression was discontinued, and he was managed with immunoglobulin (ivig) 10g/day and methylprednisolone 80 mg/day and made adequate medical progress. the second patient, 43 years old, in immunosuppression with tacrolimus 3.5 mg a day and mycophenolate 1 g a day, attended with fever and fatigue, had lymphopenia, did not require hospitalization, nor discontinuation of immunosuppression, and was managed with ceftriaxone and ganciclovir, with an adequate outcome. their adequate clinical course suggests that in patients with this type of transplant the disease has a similar presentation to non-transplanted patients. 59 it should be noted that in a series of seven cases 60 (two liver, three kidney, one lung and one heart) an initial attenuated inflammatory response was evident, suggesting that although patients with transplant immunosuppression may have higher susceptibility to sars-cov-2 infection, their clinical course could be similar to that of immunocompetent patients. with regard to the renal transplantation patients, until the time of the report, a 50-year-old patient remained in icu managed with lopinavir/ritonavir, requiring suspension of immunosuppression who came under management with tracrolimus, everolimus and prednisolone at intermediate doses. he was admitted for fever and vomiting that progressed to respiratory symptoms, and had thrombocytopenia, lymphopenia, and elevated d-dimer as factors of poor prognosis. the second, a 52-year-old, under immunosuppression with tacrolimus, mycophenolate, and prednisolone, consulted for fatigue, abdominal pain, dyspnoea, fever, and dry cough, presented lymphopenia and imaging findings typical of sars-cov-2 infection. he was managed with ivig (5 g, then 10 g/day x 11 days), methylprednisolone 40 mg/day and interferon α (5 million/u day), in addition to suspension of immunosuppression, and responded adequately to treatment. with regard to this type of transplant, the atypical presentation of the first case is noteworthy, and the adequate response of the second that could be associated with the use of multiple therapies, without it being possible conclude whether renal transplantation is associated or not with a worse prognosis. 61 gandolfini et al., 62 publish two cases of renal transplant and covid-19, a 75-year-old male and a 52-year-old female patient under management with tacrolimus, corticoids and mycophenolate, who developed severe pneumonia; in addition to suspending immunosuppressants, management with hydroxychloroquine, lopinavir/ritonavir and colchicine was started, due to the unavailability of tocilizumab. the administration of colchicine achieved an impact in decreasing il-6 serum levels, thanks to its interfering with inflammasome assembly which leads to the production of il-1b and other interleukins such as il-6. in italy, the paediatric liver transplant group of hospital papa giovanni xxiii bergamo followed up 700 liver transplants (two in the last three months), associated with autoimmune liver diseases (100 patients), three additionally in chemotherapy (for hepatoblastoma). of the total number of transplant recipients, three were confirmed to be infected with sars-cov2, and all remained asymptomatic without requiring hospitalization or suspension of immunosuppression. additionally, qin et al. 63 , report the case of a patient with hepatocellular carcinoma who underwent liver transplantation and suffered an undetected sars-cov-2 infection in the perioperative period; immunosuppression was initiated with tacrolimus and glucocorticoids; however, persistence of fever led to confirmation of sars-cov-2 infection; management with oseltamivir and immunoglobulin was initiated, and despite a prolonged convalescence, they did not present multiorgan failure, thus immunosuppression was maintained. the importance of sars cov-2 detection is highlighted for organ receptors and donors to reduce the transmission and risk of severe infection or rejection due to adjustments in immunosuppression. given the above, it is not clear whether transplantation and use of immunosuppressants in this context is a risk or severity factor for sars-cov-2 infection. likewise, in the event of sars-cov-2 infection, the adjustment or suspension of immunosuppressors should be assessed, and we should always seek to protect graft function with the administration of glucocorticoid doses and support measures, among others. 64 neurology is a continuously growing specialty. many diseases have a component that compromises autoimmune aggression to a greater or lesser extent and therefore go on to require immunosuppressant or immunomodulatory management. within the multiple entities, two diseases have become relevant in recent times, multiple sclerosis and optical neuromyelitis, due on the one hand to their physiopathological mechanism that involves neurodegeneration and inflammation by excessive activity of the immune system derived from antigenic epitopes and proinflammatory molecules, and on the other hand the use of therapies that trigger regulation of immune cells, affecting in some cases innate and adaptive immunity in most cases. if we consider that the response mechanisms to viral infections are based on inhibition of the infection by type i interferons and the death of the infected cells by nk lymphocytes (innate immunity), the generation of antibodies that block the union and entry of the virus into the cells, and the elimination of cells infected by cytotoxic t cells (adaptive immunity), the different drugs currently used could to a greater or lesser extent alter the immune response to sars-cov-2 infection, and this is why there are now different considerations when initiating or continuing therapies. 65 people with ms are at higher risk of admission to the intensive care unit due to infections, and higher mortality at one year after admission than the general population. the use of diseasemodifying therapies implies a higher risk of infections, however, to date there is no data to indicate that patients with ms are at higher risk of sars-cov-2 infection, or more severe infection. it is even possible that such disease-modifying therapies and their immunosuppressive effect may play a protective role during 19-covid infection by preventing or dampening hyperimmune activity that, in some cases, could lead to clinical deterioration; there is even a report of a patient with primary progressive multiple sclerosis receiving treatment with ocrelizumab and becoming infected with sars-cov-2, in the context of lymphopenia and hypogammaglobulinema expected for this type of treatment, without generating major clinical complications, this hypothesis is obviously limited for now only to academic deductions and limited information. 66, 67 in recent results of the multicentre registry covisep, 68 which includes information from 347 patients with ms and covid-19, it was demonstrated that age, obesity and highest score in the expanded disability status scale, were independent risk factors for severity of covid-19. it is suggested that people with ms and related disorders receiving immunotherapy continue to receive the therapy during mild viral infections. in those with documented mild sars-cov-2 infection, it may be reasonable to continue treatment. neurologists should have a lower threshold for suspending treatment in people taking therapies with greater immunosuppressant effects. 69 consideration should be given to suspending treatment in those who are hospitalized with severe or complicated sars-cov-2 infection. treatment may be restarted after four weeks or when symptoms have completely resolved, considering the risk of rebound of ms activity with s1p modulators and natalizumab. neurologists should alert intensive care physicians to the importance of fever management in people with ms. 70 in people with ms and disease-modifying treatment, the decision to start, continue, temporarily suspend, or defer doses should be individualized, 71 taking into account factors such as disease activity and the possibility of disease progression, as well as considerations of the mechanism of action of the drugs and their ability to deplete lymphocytes. recommendations from experts suggest not suspending first-line drugs (interferons, glatiramer acetate, teriflunomide, or dimethyl fumarate) and considering deferring therapies such as cladribidine and alemtuzumab based on their ability to deplete lymphocyte counts rapidly and aggressively. 72, 73 a survey of patients with nmosd or ms from china, from 10 centres, did not find an increased risk of infection by covid-19, suggesting as a possibility the role of self-care and protective measures taken by patients and their healthcare team, regardless of their condition and immunosuppression drug. 74 relapses in patients with nmosd can be devastating and patients should be encouraged to continue therapies for the prevention of attacks, including corticosteroids, azathioprine, mycophenolate mofetil, rituximab, tocilizumab and eculizumab. if there is a clinical need to discontinue or delay treatment in patients with nmosd, corticosteroids may be used in moderate doses to prevent relapses in the short-term, it is important to consider individualized therapy and comorbidities when deciding on management of this condition during the covid-19 pandemic. 69 my a s t h e n i a g r a v i s / l a mb e r t -e a to n my a s t h e n i c s y n d r o me ( mg / l e ms ) because most patients with myasthenia gravis (mg) are on immunosuppressant or immunomodulatory therapies and may also have muscle weakness and ventilatory failure, there is a theoretical concern that they may be at increased risk for infection or experience severe manifestations of sars-cov-2 infection. in a series of five cases 75 with mg hospitalized for covid-19 infection, a variable clinical course was demonstrated, with three requiring mechanical ventilation and one presenting mg crises, and although it is difficult to assess the latter due to intubation and sedation in two of the cases, none had a fatal outcome. two additional cases have been reported, one developing crises due to myasthenia 76 and the other with chronic refractory mg, 77 with good outcome, without complications or worsening of their baseline condition. there are numerous recommendations circulating that attempt to provide clarity and guidance. however, the differences between the recommendations have created confusion, because decision-making varies in different countries, and due to the lack of databases with an adequate number of patients. patients with mg/lems should continue their treatment and are advised not to discontinue any existing medications; there is no scientific evidence to suggest that symptomatic therapies such as pyridostigmine increase the risk of infection and they should not be discontinued unless there are other clinical reasons to do so, given the risk of increased disease activity and/or mg exacerbation or crisis. 1 with regard to certain therapies (immunoglobulins, plasmapheresis) there is no information pointing to increased risk of infection, however, the use of immunoglobulin should be based on the individual need of the patient and indiscriminate use should be avoided. in general, these therapies should be reserved for patients with acute exacerbation and if required as maintenance therapy on an exceptional basis, additional precautions should be taken. in patients with severe sars-cov-2 infection, temporary suspension of immunosuppression may need to be considered. it is important to note that decisions to intensify or change treatment should be individualized based on the relative severity of the sars-cov-2 infection. 78 there is very little data regarding the impact of sars-cov-2 infection on primary immunodeficiencies (pi), which is why several international organizations such as the european society for immunodeficiency (esid), 79 the reference centre for hereditary immunodeficiencies (le centre de référence déficits immunitaires héréditaires, ceredih) 80 organization for primary immunodeficiencies (ipop) 81 are collecting data through a survey of physicians in order to gather information and provide them better care. both the idf (immune deficiency foundation) 82 and the ascia (australasian society of clinical immunology and allergy) 83 and other agencies 84 have considered their patients' increased risk of severe respiratory infections or of experiencing a more severe disease course, however, they recognize that it cannot be said whether people with primary immunodeficiencies are at higher or lower risk of severe sars-cov-2 infection. ascia and ipopi promote measures to prevent the spread of the virus, social isolation, and call for early consultation with medical services when infection is suspected, and recommend maintaining continuity of medication, especially in those receiving immunoglobulin. 81, 83 virtual resources with patient and medical community education are available in the idf. these experts on the subject have theorized the possible effects of sars-cov-2 in different populations, 82 for example, in the group of t lymphocyte (tl) immunodeficiency (combined immunodeficiency, digeorge syndrome, among others) measures of isolation and protection must be maximised, since the action of these defence cells is necessary for the control of the virus; in b-lymphocyte deficiency (agammaglobulinaemia, common variable immunodeficiency) the risk of infection is not thought to be higher than in the community, except in patients with structural involvement at the lung level, and in the phagocytosis deficiency immunodeficiency group (neutropenia and chronic granulomatous disease) although neutrophils are not as important in controlling the virus, the possibility of co-infection needs to be considered, while the chronic granulomatous disease group is not thought to be at increased risk of infection or severe manifestation. 85 other recommendations according to the ipopi joint statement on the current coronavirus pandemic, are the use of pcr tests for diagnosis, since for some forms of pi there is no production of antibodies, and therefore tests based on immunoglobulins are not effective. in this same publication, as of april 5, 2020, 15 sars-cov-2 cases in different types of pi, exhibiting typical symptoms (fever, cough, and upper respiratory symptoms), 13 of which were under 45 years of age; seven required hospitalization (two developed adult respiratory distress syndrome) and all were under 45 years of age. in the most recent update, based on collected (unpublished) data, there does not appear to be an increased risk of sars-cov-2 infection, especially in its severe form, however, given the still limited information and risk for these patients, isolation and infection prevention measures should be maintained as much as possible. 81 another important aspect for these patients is the impact during the sars-cov-2 pandemic on their health-related quality of life (hrqol), requiring strict isolation and a remote care programme. in an italian cohort of 158 patients with pi due to a bl defect, two scales were evaluated, one specific to health-related quality of life, cvidqol (common variable immune deficiency quality of life), and another generic scale to assess anxiety and depression, ghq-12 (12-item general health questionnaire); finding that the remote care programme does not affect hrqol, however, in the group of patients at risk of anxiety/depression there is impaired quality of life, emphasizing the importance of individualizing each patient and psychosocial support. 86 hu during the stay at this institution infection was documented by nasopharyngeal sample associated with amplification of the betacoronavirus e gene and the specific sars-cov-2 rdrp gene by pcr, comorbidities such as hypothyroidism and asthma were also identified in these patients. all came under antiretroviral treatment, with cd4 cell count (> 400 cell/mm3 in four of the patients). prior classification according to the patient's clinical status as mild, moderate or severe, under the precept that protease inhibitors could have activity against coronavirus protease, cobicistat + darunavir was considered appropriate in two of them, starting ritonavir + lopinavir in the rest combined with hydroxychloroquine, azithromycin, corticosteroids, interferon β-1b and even tocilizumab, according to duration and progression. the survival of all is documented up to the time of publication and the conclusion is that patients in advanced stages of the disease should be guaranteed differential diagnosis by opportunistic pulmonary agents, inferring that they may have a poorer outcome, as well as an ominous prognosis. 89, 90 in a recent study of incidence and severity 91 of covid-19 involving 60 hiv clinics in spain (77,590 patients), 236 patients were diagnosed with covid-19, 151 were hospitalized, 15 required intensive therapy and 20 died. it was found that patients receiving tenofovir disoproxil fumarate (tdf)-emtricitabine (ftc) reverse transcriptase inhibitors had a lower risk of developing covid-19 and of hospitalization compared to groups receiving other treatments. there are isolated cases 92 in which patients coinfected with hiv and covid-19 in management with lopinavir and ritonavir had a favourable clinical response, considering that this reaction can have two effects: inhibition of sars-cov-2 replication, as well as inhibition of hiv replication, allowing a slight activation of the immune system capable of responding to sars-cov-2 without the progression of the patient to hyperinflammatory status, even highlighting that lymphopenia would not be considered a marker of poor prognosis but a protective immune effect, being considered an disturbance of the overactive response of the immune system, avoiding serious clinical manifestations. 92 other hypotheses raised as a favourable clinical response dependent on the patient's immune status, coinfection or history of opportunistic infections, mainly at the pulmonary level, the risk or benefit in relation to the use of glucocorticoids and even the benefit of introducing tocilizumab early were not omitted. 93 in patients with hiv and sars-cov-2 infection it can be concluded that the immune response, prognosis and outcome are highly variable and / or subjective according to the antiretroviral treatment in place, duration in relation to diagnosis and viral suppression, because hiv patients without treatment, newly diagnosed or with no viral suppression may have a compromised immune system (mediated by a low cd4 count), even being vulnerable not only to the worst outcomes due to sarv-cov-2, but alleged coinfection by other agents in pulmonary opportunists. patients with adherence to antiretroviral treatment, who have achieved viral suppression and do not have low cd4 count, will be affected by sars-cov-2 with the same chances as immunocompetent patients who develop mild manifestations, regardless of change in antiretroviral treatment or adjustment with ritonavir and lopinavir, however, it is important to emphasize the divergences in approach, management and choice versus antiretroviral treatment documented to date. for this type of patients, as well as those mentioned previously, recommendations have already been published on their management with covid-19. 94 the degree of pharmacological immunosuppression is assessed according to the patient's immunological risk, the type of protocol used, the type of target at which the drug or group of drugs is directed, and the type of disease for which it is indicated (e.g., neoplasms, transplants, immunological, etc.). although in all cases it is not easy to directly assess the degree of immunosuppression, some biomarkers have been developed that reflect the individual's response to immunosuppressants, which can range from general tests such as liver function enzymes, to the use of specific genotypes, including cell counts of specific lymphocyte populations, cytokines, leukocyte markers and target enzymes, among others. 95 in other cases, the degree is assessed according to the number of immunosuppressant drugs, dose and time employed, being low when a drug is used in low or moderate doses for a short time, and increasing to a higher degree when two or more immunosuppressants are used in combination, regardless of the dose. 96 there are many drugs used in different medical specialties that are associated with a certain degree of immunosuppression and that render patients vulnerable to one or another infectious process. however, in the field of sars-cov-2 infection, the data are scarce and unknown. as mentioned throughout the review, comorbidities are the most important risk factors compared to drugs used. the literature search predominantly reveals information about possible drug interactions that can occur with the treatment of covid-19, which can clearly also generate damage and worsen the clinical picture. to date there are no data on specific drugs that are associated to a greater or lesser degree with infection by the new coronavirus. 97 there is a warning about possible drug interactions between immunosuppressive drugs and those under investigation for the treatment of covid-19, generating alerts and guidelines for the development of this complex task by clinicians. remdesivir, an antiviral with promising results in the treatment of covid-19 as mentioned, has no data so far on possible drug interactions with immunosuppressive drugs, unlike chloroquine/hydroxychloroquine and lopinavir/ritonavir, which although their use is declining, were initially an active part of treatment, suggesting major interactions with calcineurin inhibitors, mtor (mammalian target of rapamycin inhibitors) and corticosteroids, especially lopinavir/ritonavir. 97 there are recommendations about discontinuing mycophenolate in critically ill transplant recipients, 98 which would also apply to covid-19, bearing in mind that during the h1n1 pandemic it was documented that this drug decreased the serological response in transplant recipients. 97 however, given the scarcity of data, this decision must be tailored to each patient in this special subgroup. in reference to calcineurin inhibitors (tacrolimus, cyclosporine), a dose minimization scheme is proposed, with the possibility of increasing the interval for its administration, suggesting safety in these regimens, particularly in individuals with kidney transplantation and covid-19. 99 multiple recommendations and guidelines have been generated around the use of immunosuppressors or cytostatics in the oncology field, 100 such as cyclophosphamide, doxorubicin, cytarabine, vinblastine, as well as immunotherapy and the use of biological drugs in the context of cancer, according to the type of neoplasm in the context of risk or presence of covid-19 infection, suggesting in general a decrease in dose, but always balancing individual cases according to the type of neoplasm, stage and immunosuppressive scheme proposed. 58 the recommendation is to avoid high doses of corticosteroids since they could, as observed in patients with mers-cov, prolong viral replication in patients with covid-19. 97 as mentioned above, their use would be reserved for specific subgroups of critically ill patients other drugs, not immunosuppressants, but associated with the physiopathogenesis of the disease, such as angiotensin-converting enzyme inhibitors or renin-angiotensin-aldosterone system j o u r n a l p r e -p r o o f inhibitors, have not been shown to increase the risk of sars-cov-2 infection, and conversely, their withdrawal could be harmful. 101 considering the evidence available to date on sars-cov-2 infection outcomes in patients with immunosuppression (either due to their disease or the use of immunosuppressants) its behaviour is not clear in this type of individuals. we can highlight that patients with cancer and recent treatment of cancer (chemotherapy or surgery) have a higher risk of worse outcomes, with faster deterioration than those without cancer, an increased risk of severity and mortality having been shown through two meta-analyses. with regard to transplant patients (kidney, heart and liver), patients with neurological disease associated with the use of immunosuppression (ms, nmods, mg), primary immunodeficiencies and hiv, studies have not shown a tendency to poorer outcomes than patients without these diseases or drugs and have sars-cov-2 infection, similar to that found in rheumatological diseases. 102 this could perhaps be explained in that the severity of sars-cov-2 infection has been associated with an aberrant inflammatory response (cytokine storm). for the time being and as more information is obtained, and based on the aforementioned literature and recommendations of societies, it is suggested that immunosuppressant therapy be continued, starting new therapies should be avoided as much as is possible (especially in endemic areas) and in case of infection, depending on its severity, the risk/benefit should be evaluated of suspending it during the period of infection. it should be noted that the presence of comorbidities, such as high blood pressure, diabetes mellitus and copd, increases the risk of severity, intensive care 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treated with immunosuppressive targeted therapies key: cord-331500-l3hkn2li authors: luyt, charles-edouard; bouadma, lila; morris, andrew conway; dhanani, jayesh a.; kollef, marin; lipman, jeffrey; martin-loeches, ignacio; nseir, saad; ranzani, otavio t.; roquilly, antoine; schmidt, matthieu; torres, antoni; timsit, jean-françois title: pulmonary infections complicating ards date: 2020-11-11 journal: intensive care med doi: 10.1007/s00134-020-06292-z sha: doc_id: 331500 cord_uid: l3hkn2li pulmonary infection is one of the main complications occurring in patients suffering from acute respiratory distress syndrome (ards). besides traditional risk factors, dysregulation of lung immune defenses and microbiota may play an important role in ards patients. prone positioning does not seem to be associated with a higher risk of pulmonary infection. although bacteria associated with ventilator-associated pneumonia (vap) in ards patients are similar to those in patients without ards, atypical pathogens (aspergillus, herpes simplex virus and cytomegalovirus) may also be responsible for infection in ards patients. diagnosing pulmonary infection in ards patients is challenging, and requires a combination of clinical, biological and microbiological criteria. the role of modern tools (e.g., molecular methods, metagenomic sequencing, etc.) remains to be evaluated in this setting. one of the challenges of antimicrobial treatment is antibiotics diffusion into the lungs. although targeted delivery of antibiotics using nebulization may be interesting, their place in ards patients remains to be explored. the use of extracorporeal membrane oxygenation in the most severe patients is associated with a high rate of infection and raises several challenges, diagnostic issues and pharmacokinetics/pharmacodynamics changes being at the top. prevention of pulmonary infection is a key issue in ards patients, but there is no specific measure for these high-risk patients. reinforcing preventive measures using bundles seems to be the best option. acute respiratory distress syndrome (ards) regroups a wide range of diseases whose consequence is lung inflammation, alveolar damage and pulmonary edema [1] . whatever the initial lung injury, patients with ards are prone to develop secondary pulmonary infection, namely ventilator-associated pneumonia (vap). recent data from the center for disease control and prevention suggest that vap rates are not dropping in the usa despite patients with ards exemplify the apparently paradoxical immune state of critically ill patients, whereby activated immune cells mediate organ damage while manifesting impaired antimicrobial defenses [6] . impaired cellular functions have been identified across both the innate and adaptive arms of the immune system [7, 8] , and appear to be stereotyped rather than specific to any precipitating cause of ards [9] . this apparently paradoxical state is due to the ability of pro-inflammatory and tissue damage molecules to drive immune dysfunction [9, 10] . dysfunctional immune cells are found in the lung as well as peripheral blood [9] . interestingly, lung mucosal immune defects are protracted after the cure from primary inflammation, thus increasing the susceptibility to hospital-acquired pneumonia and ards for weeks after systemic inflammation [11] . following experimental pneumonia, pulmonary macrophages and dendritic cells demonstrated prolonged suppression of immune functions which increased the susceptibility to secondary infection [12] . expansion of immuno-modulatory regulatory t cells (t reg ) is also seen and may mediate impaired innate as well as adaptive immune function [13] . patients with suspected vap, including those with ards, demonstrated impaired phagocytic function of alveolar neutrophils, which interestingly appeared to be mediated by different mediators than those driving dysfunction in the peripheral blood [9] . while we have a growing understanding of the mediators driving dysfunction, and the intracellular mechanisms which drive them [14] , we do not as yet have proven therapies although there are multiple potential agents [7] . when aiming at modulating immunity during inflammation, it is important to differentiate innate and adaptive immune cells responses. while exhaustion and apoptosis seem to be central to lymphocyte defects observed in critically ill patients [15] , some innate immune cells undergo reprogramming involving epigenetic reprogramming and increased cellular metabolism, a phenomenon so-called trained immunity, resulting in high production of inflammatory cytokines such as il-6 and tnfα during secondary immune challenge [16] . while glucocorticoids are classically considered as immunosuppressive drugs, it has been shown that they can prevent the immune reprogramming observed after inflammatory response [16] , thus limiting the susceptibility of patients admitted to the intensive care unit (icu) to respiratory complications such as pneumonia or ards and improving outcomes of patients with ards [17] . part of the complexity of pulmonary super-infections arises from the interaction between the injured host with their pulmonary microbiome. although considerably less abundant and diverse than the better studied gastrointestinal microbiome [18] , the pulmonary microbiome is increasingly well defined and undergoes significant changes during critical illness and ards [19] . the major role of respiratory microbiota on mucosal immunity and respiratory functions in health suggests that its alterations could be involved in the respiratory complications observed in critically ill patients [20] . indeed, mechanically ventilated patients experience a reduction in diversity of pulmonary microbes and an increase in enteric-type organisms, even in the absence of overt infection [21] . early alterations of the lung microbiome, notably increased bacterial burden and biofilm formation, enrichment with gut-associated bacteria and loss of diversity, are associated with the risk of ards and the duration of mv support in critically ill patients [22] . pre-existing dysbiosis, such as that induced by tobacco smoke, may also influence the development of ards following major trauma [23] . alongside changes in bacterial species, it is common to find reactivation of latent herpesviridae such as herpes simplex virus (hsv) and cytomegalovirus (cmv) [24] . the drivers of these changes are incompletely understood but are multi-factorial, with possible mechanisms illustrated in fig. 1 [13, 22, 25] . adding further complexity is the potential for microbes themselves to drive further immune dysfunction [26] . vap should therefore be conceptualized as less a de novo infection by an exogenous pathogen, but rather a dysbiotic response to critical illness with overgrowth of specific genera of bacteria [27] . appropriate antibiotic therapy targeting the dominant species, those frequently detected by culture, is key in certain patients but risks exacerbating dysbiosis and further harm to the patient [28] . what remains to be proven is whether interventions to restore symbiosis, i.e., to increase bacterial diversity rather than only eliminating dominant species, can improve outcomes [27] . although the experience of fecal transplantation in clostridium difficile associated diarrhea suggests that microbial transplantation may be an effective form of therapy [29] , negative experience of probiotics in pancreatitis and recent examples of 'probiotic' bacteria causing infections sound a note of caution [30, 31] . developing effective therapies for respiratory dysbiosis will require tools to profile the host peripheral pulmonary superinfections in ards patients considerably impact patients' prognosis which is favored by altered local and systemic immune defenses. the poor outcome of ards with pulmonary superinfections is probably related to the lack of early accurate diagnostic methods and difficulties in optimizing therapy. and pulmonary immune cell function and the pulmonary microbiome [8] . hyperoxia is common in patients receiving mv for ards. a secondary analysis of the lung safe trial [32] reported that 30% of the 2005 analyzed patients had hyperoxia on day 1, and 12% had sustained hyperoxia. while two randomized controlled trials found beneficial effect of avoiding hyperoxia [33, 34] , a recent large international multicenter trial demonstrated no effect of conservative oxygen therapy in a cohort of critically ill patients [35] . however, a subsequent sub-study raised the possibility of clinically important harm with conservative oxygen therapy in patients with sepsis [36] . oxygen toxicity is mainly related to the formation of reactive oxygen species (ros), especially during hypoxia/ re-oxygenation and long exposure to oxygen. high level of inspired oxygen is responsible for denitrogenation phenomena and inhibition of surfactant production promoting expiratory collapse and atelectasis [37] . absorption atelectasis occurs within few minutes after pure o 2 breathing. in mechanically ventilated patients, atelectasis seriously impairs cough reflex and mucus clearance resulting in abundant secretions in the lower airways and higher risk for vap. prolonged hyperoxia also impairs the efficacy of alveolar macrophages to migrate, phagocyte and kill bacteria, resulting in decreased bacterial clearance [38] . hyperoxemia markedly increased the lethality of pseudomonas aeruginosa in a mouse model of pneumonia [39] . additionally, o 2 can cause pulmonaryspecific toxic effect called hyperemic acute lung injury (hali) (fig. 2) . although earlier studies reported a link between high fio 2 and atelectasis, further studies are required to evaluate links between hyperoxia and mortality or vap. in a single center cohort study of 503 patients, among whom 128 (28%) had vap, multivariate analysis identified number of days spent with hyperoxemia [or = 1.1, 95% ci: (1.04-1.2) per day, p = 0.004], as an independent risk factor for vap. however, the study was retrospective, performed in a single center, and the definition used for hyperoxia (at least one pao 2 value > 120 mmhg per day) could be debated [40] . in the recent hypers2s randomized controlled trial [34] , the percentage of patients with atelectasis doubled in patients with hyperoxia compared with those with normoxia (12% vs. 6%, p = 0.04). however, no significant difference was found in vap rate between hyperoxia and control group (15% vs. 14%, p = 0.78). however, vap was not the primary outcome of this trial, and there is no clear definition of icu-acquired pneumonia. further well-designed studies are required to determine the relationship between hyperoxia and vap. prone position is recommended in patients with severe ards and is commonly used in this population. there is a rationale supporting a beneficial effect of prone position on the incidence of vap, as it facilitates secretion drainage and allows atelectasis resolution. previous human and animal studies have clearly showed a link between atelectasis and vap, and reported that efficient secretion drainage might result in lower incidence of vap [37] . on the other hand, prone position might facilitate microorganisms' dissemination and increase microaspiration of contaminated secretions. the results of studies on the relationship between prone position and vap should be interpreted with caution, because of some limitations such as observational design, small number of included patients and confounding factors. five recent studies were performed in patients with protective lung mv, including four randomized controlled studies and one large observational cohort. mounier et al. [41] reported no significant reduction of vap incidence in a large cohort (n = 2409) of hypoxemic patients positioned in the prone position, as compared to those who did not receive this intervention [hr 1.64 (95% ci 0.7-3.8)]. one randomized controlled trial reported reduced risk for vap in multiple trauma patients who were subjected to intermittent prone position, as compared to those who did not (p = 0.048) [42] . however, the incidence of vap was very high in the control group (89%), and the number of included patients was small (n = 40). three other randomized controlled trials reported no significant relationship between prone position and vap [4, 43, 44] . however, these studies lack information on efficient preventive measures of vap, such as the use of subglottic secretion drainage or continuous control of tracheal cuff pressure, and vap was not their primary outcome. in summary, available data do not support a significant relationship between prone position and vap, although it has demonstrated beneficial effects on mortality in severe ards. the diagnosis of lung infections in patients with ards is challenging [45] . the diagnosis of pneumonia, the dominant respiratory infection of concern in ards, is ultimately a histopathological diagnosis which requires the presence of airspace inflammation and an infecting organism. however, obtaining lung tissue for diagnosis is seldom practical or desirable in ventilated patients [5] . the clinical features of systemic inflammation and localizing chest signs such as crepitations and bronchial breathing are non-specific and insensitive. while radiological evidence of airspace infiltration is useful, the gold standard of computed tomography is not practical for most patients, leading practitioners to rely on plain radiographs and ultrasound, and even computed tomography cannot always reliably distinguish between infective and non-infective causes of airspace infiltration [5, 45] . use of clinical and radiographic criteria alone are likely to significantly overestimate the rate of pneumonia and lead to excessive, potentially harmful, use of antibiotics [28] . it is also important to recall that pneumonia itself is the commonest precipitant of ards, which, together with the bilateral radiographic alterations in ards patients, creates an additional challenge for the ascertainment of a "new or worsening pulmonary infiltrate", a condition required for clinical diagnosis of vap [5] . another challenge is the distinction between ventilator-associated tracheobronchitis (vat) and vap. vat is defined as a lower respiratory tract infection without involvement of the lung parenchyma (and therefore without new/progressive chest x-ray infiltrate). the distinction between vat and vap in ards patients remained challenging given the poor accuracy of chest radiograph to detect new infiltrates. obtaining samples from the lungs for microbiological culture is crucial to the establishment of infection. however, there is considerable variability in the timing and type of specimen obtained in practice [46] . the identification of infection can be complicated by colonization of the proximal airways, which happens rapidly after intubation and is frequent in ards patients [5] . it is important to differentiate between colonization (presence of bacteria, even at a high burden, in the respiratory tract without lung infection), a harmless phenomenon, and infection. although protected deep lung sampling by broncho-alveolar lavage or protected specimen brush reduces the risk of false positives relative to endotracheal aspirate, this has not been convincingly demonstrated to alter outcomes although observational data suggest they can safely reduce antibiotic use [47] . although falsepositive results from proximal colonization are a significant problem, intercurrent use of antibiotics is common in ards patients and increases the risk of false-negative culture. this is, increasingly, being addressed by the use of culture-independent molecular technique; however, the utility of the tools available is limited by their restricted range of organisms covered and the risk of over-sensitive detection of irrelevant organisms driving inappropriate use of antimicrobials [48] [49] [50] . physicians should be aware of this particular point and therefore interpret with caution the results of these tests. there are very few prospective studies demonstrating the impact of molecular diagnostics on patient management and the results of forthcoming trials are awaited. antigen detection in the lower respiratory tract can also aid diagnosis, especially with organisms such as aspergillus where culture and pcr are imperfect [51] . the value of aspergillus sp. and aspergillus fumigatus pcr is promising, but remain to be evaluated in ards patients. in patients with ards and bilateral radiographic infiltrates, there remains a question of which region to sample invasively. while trials have not been undertaken to answer this question definitively, observational data suggest that in the presence of bilateral infiltrates, unilobe sampling is sufficient and minimizes risk of lavage volume and duration of bronchoscopy [52] . the host response makes up the crucial second component of any infection syndrome, and therefore host biomarkers can be of use in diagnosing infection in ards. laboratory hematological features of inflammation, including leucocytosis, neutrophilia and elevated c-reactive protein, are not specific to infection and can occur in sterile precipitants of ards [53] . the inflammatory response in pneumonia is highly compartmentalized and alveolar cytokines and other alveolar markers are the most discriminant for pneumonia (table 1 ) [54] . notably, although alveolar cytokines demonstrated excellent assay performance, measurement of pulmonary cytokines did not alter antimicrobial prescribing in a recent randomized trial [55] . this illustrates that the challenges in diagnosis lie not only with the technology, but also the behavioral response to results. peripheral blood markers have the advantage of avoiding the need for bronchoscopic sampling and are therefore easier to obtain; however, they are generally less able to discriminate pneumonia from other infections table 1 summary of host-based biomarkers for diagnosis of pneumonia in ards ards acute respiratory distress syndrome, rct randomized controlled trial, strem soluble triggering receptor expressed on myeloid cells, vap ventilator-associated pneumonia, hla human leukocyte antigen interleukin-1/interleukin-8 validated in multi-center cohort [54] but did not influence practice in an rct [55] strem-1 initial report, but not validated in follow-up study [113, 114] exhaled breath markers experimental with technical variation currently limiting implementation [115] pentraxin-3 meta-analysis suggested alveolar levels superior to plasma levels with moderate diagnostic performance, no rct testing influence on practice [116] and many lack sensitivity and or specificity for infection (table 1 ). in summary, the diagnosis of pulmonary infection in ards is challenging, and existing techniques are imperfect and risk both inadequate and overtreatment. a combination of clinical, biological and radiological assessment, combined with microbiological sampling from the lungs, remains the current gold standard (fig. 3) . the development of molecular diagnostics focusing on both host and pathogen offers great promise, but their impact on patient management and outcomes remains to be convincingly demonstrated. the most common bacterial causes of vap include enterobacterales, pseudomonas aeruginosa, staphylococcus aureus, and acinetobacter among the general population of mechanically ventilated patients [56] . the pathogens associated with vap in ards are similar to those seen among non-ards patients who develop vap (fig. 4) [4, 52, 57] . moreover, patients with ards undergoing extracorporeal membrane oxygenation (ecmo) demonstrate the same breakdown of pathogens with pseudomonas aeruginosa and staphylococcus aureus predominating [58] . one important element, regardless of the specific causative bacteria seen in vap, is that antibiotic resistance is increasing in vap as well as in other nosocomial infections. in 2017, the tigecycline evaluation and surveillance trial described important european changes in antimicrobial susceptibility between 2004 and 2014, with increases in the rates of esbl-positive escherichia coli (from 8.9 to 16.9%), mdr acinetobacter baumannii complex (from 15.4 to 48.5%), esbl-positive klebsiella pneumoniae (from 17.2 to 23.7%), and methicillin-resistant staphylococcus aureus (mrsa) (from 27.5 to 28.9%) [59] . similar worrisome trends for bacterial susceptibility to available antimicrobials have been reported by other investigators as well [60, 61] . most worrisome is the increasingly recognized presence of resistance to new antibiotics specifically developed to treat vap [62] . prior antibiotic exposure and subsequent changes in the host's airway microbiome due to dysbiosis seem to drive the prevalence of antibiotic-resistant bacterial causes of vap (fig. 5) [22, 63] . the presence of invasive devices such as endotracheal tubes and antibiotic administration promote pathogenic bacterial colonization due to the overwhelming of local defenses, resulting in the development of an intermediate respiratory infection termed vat [64] . vat represents a compartmentalized host response associated with a better overall prognosis compared to vap, but vat can prolong the duration of mv and icu length of stay [65] . if the aforementioned response is not compartmentalized, progression to vap is likely and potentially other organ failure including ards may occur [66] . one of the major fears concerning nosocomial pulmonary infections in ards at the present and into the future is the increasing presence of novel pathogens and infections with microorganisms for which limited treatment options exist. as we increasingly treat older and more immunocompromised hosts with ards, the likelihood for emergence of novel pathogens and infection with pan-resistant microorganisms will increase. early identification of such emerging pathogens in ards is critical. the importance of early identification of novel pathogens is necessary to facilitate epidemiologic surveillance, curtailing pathogen spread, and providing early treatment as illustrated by recent nosocomial outbreaks of middle eastern respiratory syndrome coronavirus, sars-cov-2 and pan-resistant escherichia coli [67] [68] [69] [70] . in the future, metagenomic next-generation sequencing should allow earlier and more targeted treatments for novel pathogens causing ards or complicating the course of patients with ards. such technology will allow earlier pathogen identification and accelerate the workup and treatment for both infectious and noninfectious causes of diseases complicating ards [71] . although the majority of respiratory infections in ards patients are caused by bacteria, icu-induced immunoparalysis may induce infection with unusual pathogens. although invasive pulmonary aspergillosis (ipa) has been reported mainly in immunocompromised patients, lower respiratory tract colonization with aspergillus has been more frequently associated with ards than in other patients invasively ventilated in icu [72] . the mechanism of damage involves the combination of alveolar damage (induced by ards) and a dysregulation of the local immune response, together with sepsis-induced immunosuppression, innate immunity and antigen presentation impairment, accounting for the development of ipa in previously colonized patients [15, 73] . co-infection with influenza has been reported as a risk factor for ipa [74] . contou et al. reported isolation of aspergillus in the lower respiratory tract in almost 10% of patients with [4] . bar graphs depicting the percentages of the most frequently isolated microorganisms in icu-acquired pneumonia episodes for 2016 (red bars) and for patients with acute respiratory distress syndrome (ards) (blue bars). total number of isolates 16, 869 and 112, respectively ards (50% had putative or proven ipa) [75] . an important finding from this study was that the median time between initiation of mv and first sample positive for aspergillus spp. was only 3 days. moreover, a post-mortem study in ards patients found that 10% of deceased patients had ipa manifestations [76] . if aspergillus is identified as a pathogen in an immunocompetent patient, it is recommended to screen for any kind of immunosuppression (humoral, cellular or combined, complement, etc.). viruses may also be responsible for infection in ards patients. because of immunoparalysis following the initial pro-inflammatory response to aggression, latent viruses such as herpesviridae may reactivate in icu patients [7] . hsv and cmv are frequently recovered in lung or blood of icu patients (up to 50%, depending on the case mix), their reactivation being associated with morbidity and mortality [24, 77, 78] . however, the exact significance of these reactivations is debated: these viruses may have a true pathogenicity and cause lung involvement [24, 79] , thereby having a direct role in morbidity/mortality observed with their reactivation; or they may be bystanders, their reactivation being only secondary to disease severity or prolonged icu stay. to date, the answer is not known, data regarding a potential benefit of antiviral treatment being controversial. for hsv, the most recent randomized control trial found no increase in ventilator-free days in patients having received acyclovir, but a trend toward lower 60-day mortality rate (hazard ratio for death within 60 days post-randomization for the acyclovir group vs control was 0.61 (95% ci 0.37-0.99, p = 0.047) [80] . for cmv, two recent randomized clinical trials (rcts) were performed: the first one showed that valganciclovir prophylaxis in cmv-seropositive patients was associated with lower rate of cmv reactivation as respiratory microbiome dysbiosis is also demonstrated as a prerequisite for most cases of vap and vt compared to placebo, but not with better outcome [81] ; and the second one showed that, as compared to placebo, ganciclovir prophylaxis did not lead to lower il-6 blood level at day 14, but patients having received ganciclovir had trend toward lower duration of mv [82] . besides latent viruses, respiratory viruses (rhinovirus, influenza, adenovirus…) have been recently found to be responsible for nosocomial infection in ventilated or non-ventilated patients [83] . however, like herpesviridae, their true impact on morbidity/mortality is not known. in summary, hsv and cmv may cause viral disease in ards patients, and respiratory viruses may be responsible for hospital-acquired pneumonia; however, the true impact of these viral infections on outcomes remains to be determined. veno-venous extracorporeal membrane oxygenation (vv-ecmo) is now part of the management of refractory ards [84, 85] . these very sick patients are at high risk for developing typical icu-related nosocomial infections (e.g., vap or bloodstream infections), in addition to ecmo-specific infections, including localized infections at peripheral cannulation insertion sites. bizzarro et al. reported a high prevalence rate of nosocomial infection of 21% in a large international registry of ecmo patients [86] , pulmonary infection being the most frequently reported. this high prevalence may be explained by underlying comorbidities, concomitant critical illness, prolonged mechanical support, mv and icu stay as well as impairment of the immune system by the extracorporeal circuitry through endothelial dysfunction, coagulation cascade, and pro-inflammatory mediators release [87] . while the rate of pulmonary infection on ecmo has not been thoroughly compared with a population with the same critical illness but in the absence of ecmo, vap was reported in 32 out of 92 patients receiving ecmo (87% vv-ecmo) by grasseli et al. [88] . among 220 patients who underwent va-ecmo for > 48 h and for a total of 2942 ecmo days, 142 (64%) developed 222 nosocomial infections, corresponding to a rate of 75.5 infectious episodes per 1000 ecmo days. vap was the main site of infection with 163 episodes occurring in 120 patients after a median ± standard deviation of 7 ± 12 days [89] . vap and resistant organisms are therefore common in that population [88] [89] [90] . the duration of ecmo has been frequently associated with a higher incidence of vap [89, 91] , even if a causal relationship is impossible to establish. indeed, longer ecmo runs could be a direct consequence of infectious complications rather than a risk factor. however, it seems clear that ecmo patients who acquired vap had longer durations of mv and ecmo support and a higher overall icu mortality [88, 89, 91] . similarly, immunocompromised patients and older age were consistently found as risk factors associated with infections on ecmo [89, 92] . the clinical diagnosis of pulmonary infection in ecmo patients is challenging, since they may have signs of systemic inflammatory response, possibly triggered by the ecmo itself, whereas fever could be absent if the temperature is controlled by the heat exchanger on the membrane. in addition, the common application of an ultraprotective ventilation aiming to rest the lung on vv-ecmo and frequent pulmonary edema on va-ecmo make the interpretation of new infiltrates on chest-x ray, which are commonly used to suspect a vap, difficult. beyond the diagnosis challenge of pulmonary infection on ecmo, the changes of pharmacokinetics/pharmacodynamics (pk/pd) of antimicrobial agents could also contribute to delaying appropriate antimicrobial treatment and consequently increase the burden of infections. an increase in the volume of distribution by ecmo as well as the severity of the underlying illness and drug clearance impairment through renal or liver dysfunctions complicates the management of antibiotics and antifungal therapies [93] . while waiting for large in vivo studies aiming to report the respective pk/pd of antimicrobial agents on ecmo, avoiding lipophilic agents (i.e., more likely sequestrated on the ecmo membrane) [93] and therapeutic drug monitoring are warranted. apart from bacteremias/fungemias, most infections are in interstitial or tissue spaces and hence the efficacy of a drug should be related to drug concentrations and actions in those tissues [94] . drugs will cross the body membranes (move from intravenous compartment into tissue compartments) if there is an intrinsic "carrier mechanism", or if the compound is either a small molecule or is lipophilic [95] . hydrophilic antimicrobials are found in extravascular lung water, but for relevant lung tissue penetration the lipophilic drugs are most important [94] [95] [96] [97] . large molecules such as vancomycin, teicoplanin, aminoglycosides and colistin will have poor lung tissue concentrations when given intravenously (elf/plasma concentration ratio << 1) [95, 96] . betalactams penetrate into lung parenchyma better than other hydrophobic antibiotics [96] . elf/plasma concentration ratio for glycylcyclines (e.g., tigecycline) is around 1. lipophilic compounds such as macrolides, ketolides, quinolones, oxazolidinones, antifungals and antivirals will have good lung tissue concentrations (elf/plasma concentration ratio > 1) after intravenous administration [97] . oxazolidinones (linezolid), glycylcyclines (tigecycline) and sulfonamides (cotrimoxazole) may be effective in the treatment of mdr pathogens; however, there is no ards-specific lung pk (elf/plasma concentration) data for these drugs. although newer antimicrobials (ceftolazone-tazobactam, meropenem-vaborbactam, plazomicin) have activity against drug-resistant gram-negative pathogens, there are limited alternatives against drug-resistant acinetobacter baumaniii such as cefiderocol which is undergoing phase 3 clinical trials. the advent of newer generation of delivery devices and mdr organisms has led to a renewed interest in the field of nebulized antimicrobials [98] , although recent trials in pneumonia have failed to demonstrate clinical benefits [99, 100] . ards is often associated with multiple organ dysfunction syndrome. hence, the possibility of achieving high intrapulmonary concentrations with limited systemic side effects is appealing. although recent wellconducted rcts argued against systematic use of nebulized antimicrobials in nosocomial pneumonia [99, 100] it may still have a place in the treatment of severe lung infections due to mdr bacteria. in this view, selecting the correct antimicrobial formulation and dosing (table 2) is an essential first step, as well as the best device, namely vibrating mesh nebulizer [101] . clinical pk data available for some nebulized antibacterial, antiviral and antifungals confirm high pulmonary and low systemic exposure [102] . sputum pk studies report high variability and are difficult to interpret [102] . however, lung deposition of nebulized antimicrobials is influenced by many factors, including specific ventilator settings. ventilator settings and procedures usually recommended for improving aerosol delivery (high tidal volume, low respiratory rate and low inspiratory flow, systematic changes of expiratory fil-ters…) are difficult to implement in patients with ards, at least those with the most severe forms. ards is a heterogeneous lung condition causing inhomogeneous ventilation distribution potentially affecting drug delivery at the affected site. increased lung inflammation can also increase systemic concentrations by increased diffusion across the alveolo-capillary barrier, thus influencing the nebulized drug dosing [103] . further pk studies investigating nebulized antimicrobial in ards are required for recommending dosing regimens in this condition. areas of investigation such as pulmonary nanomedicine and targeted delivery using intracorporeal nebulization catheter, while still investigational, have the potential to overcome many of these barriers and enhance lung tissue antimicrobial concentrations [104] . nosocomial infections may contribute to the mortality related to ards given that such infections are responsible for worsening hypoxemia and causing sepsis. as such, the prevention of these infections must be reinforced to avoid straining the prognosis of patients suffering from ards. however, interpreting the vap prevention literature in this context is challenging because (1) no studies have been conducted expressly in ards patients; (2) several preventive measures have been shown to reduce the rate of pulmonary infection, but many less have demonstrated an impact on patient prognosis [105] . that being said, the general strategy for preventing pulmonary infection applies also in ards patients. however, some preventive measures deserve a special focus in the context of ards patients (fig. 6) : (1) oral care with chlorhexidine is suspected to worsen respiratory failure; (2) selective digestive decontamination (sdd) deserves to be discussed in such high-risk patients, as it has been proven to be effective in reducing mortality in icu patients and likely lowers vap rates. there is no single preventive measure that will completely avert pulmonary infection in patients suffering from ards and patients must be approached with a package or bundle of preventive measure [106] provided that an early weaning strategy is part of the bundle [107] . other preventive measures and notably some expensive medical devices such as automated endotracheal tube cuff pressure monitoring or endotracheal tube allowing subglottic secretion drainage have not been proven effective on patient's outcomes (mortality, duration of mv, antibiotic use), but could be dedicated to these high-risk patients. however, translating research into an efficient bundle of care to prevent pulmonary infection remains a challenge and behavioral approaches to implement the measures are as important as the measures themselves [108] . chlorhexidine-gluconate (chg) use for oral care in icu patients may be harmful despite previous consistent data showing its beneficial effect in preventing vap [109] . oral mucosa adverse events with 2% (w/v) chg mouthwash in icu are frequent, but often transient. adverse events described were erosive lesions, ulcerations, plaque formation (which are easily removed), and bleeding mucosa in 29 of 295 patients (9.8%) who received 2% (w/v) chg [110] . a systematic review and meta-analysis by labeau et al. in 2011 evaluated the effect of oral decontamination with chx [109] . twelve studies were included (n = 2341). overall, chx use resulted in a significant risk reduction of vap (rr = 0.67, 95% ci 0.55-0.94, p = 0.02). favorable effects were more pronounced in subgroup analyses for 2% chx (rr = 0.53, 95% ci 0.31-0.91) and for cardiosurgical patients (rr = 0.41, 95% ci 0.17-0.98). however, a recent metaanalysis suggested that oral chg paradoxically increased the risk of death, which may have resulted from toxicity of aspirated chg in the lower respiratory tract [111] . consequently, it remains unclear whether using chg for oral care affects outcomes in critically ill patients. selective digestive decontamination (sdd) remains definitely a matter of controversy [112] . on one hand, it reduces the mortality in mechanically ventilated patients, while on the other hand its use is limited by the potential fig. 6 prevention of pulmonary infections in ards patients: from highly recommended preventive measures to a cautious or even a not recommended use of inducing more bacterial resistance. however, in ards patients at high risk of mortality with high level of bacterial resistance, sdd deserves to be evaluated. the better understanding of ards phenotype may offer an opportunity to develop more selective preventive measures in the future. pulmonary superinfections of ards patients considerably impact patients' prognosis. it is favored by altered local and systemic immune defenses. the poor outcome of ards with pulmonary superinfections is probably related to the lack of early accurate diagnostic methods and difficulties in optimizing therapy. this article reviewed the available knowledge and revealed areas for future investigations in pathophysiology, diagnosis, treatment and prevention. potentials for improvements are numerous in all the fields: to improve knowledge about the host factors (both systemic and local) favoring superinfections. to identify early the disequilibrium between the host and the microbiota that may promote pneumonia in ards patients. to identify early criteria for suspicion of vap and vat. to determine the appropriate time to perform bacteriological samples, and in particular develop a morphological way to unmask areas of pneumonia at the bedside. to identify new diagnostic tests providing accurate and early diagnosis of pneumonia. to develop accurate early methods of pathogen identification and to distinguish patients infected and simply colonized (especially for viruses and fungi). to evaluate the impact of new molecular methods in diagnosing pneumonia in ards patients and improve prognosis. to evaluate the impact of tdm monitoring of antimicrobials on the prognosis of ards patients with pneumonia. to develop non-antibiotic therapies in the future, including vaccines, monoclonal antibodies and phage therapy. evaluate the benefit on antimicrobial consumption and prognosis of the use of sdd in ards patients in icus with a high level of bacterial resistance. acute respiratory distress syndrome changes in prevalence of health care-associated infections in us hospitals ventilator-associated pneumonia and icu mortality in severe ards patients ventilated according to a lung-protective strategy ventilator-associated pneumonia in ards patients: the impact of prone positioning. a secondary 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pneumonia-a systematic review diagnostic value of pentraxin 3 in respiratory tract infections: a meta-analysis. medicine (baltimore) 99:e19532 a biomarker panel (bioscore) incorporating monocytic surface and soluble trem-1 has high discriminative value for ventilator-associated pneumonia: a prospective observational study the current status of biomarkers for the diagnosis of nosocomial pneumonias maintenance treatment with inhaled ampicillin in patients with cystic fibrosis and lung infection due to methicillin-sensitive staphylococcus aureus use of inhaled ampicillin-sulbactam against multiresistant acinetobacter baumannii in bronchial secretions of intensive care unit patients aerosolized ceftazidime prophylaxis against ventilator-associated pneumonia in high-risk trauma patients: results of a double-blind randomized study aerosolized ceftazidime for prevention of ventilator-associated pneumonia and drug effects on the proinflammatory response in critically ill trauma patients nebulized ceftazidime and amikacin in ventilator-associated pneumonia caused by pseudomonas aeruginosa nebulized imipenem to control nosocomial pneumonia caused by pseudomonas aeruginosa levofloxacin inhalation solution (mp-376) in patients with cystic fibrosis with pseudomonas aeruginosa reduction of bacterial resistance with inhaled antibiotics in the intensive care unit aerosolized antibiotics and ventilator-associated tracheobronchitis in the intensive care unit aerosolized tobramycin in the treatment of ventilator-associated pneumonia: a pilot study for nebulized antibiotics in ventilator-associated pneumonia (2020) ventilator-associated pneumonia caused by multidrug-resistant gram-negative bacteria: understanding nebulization of aminoglycosides and colistin inhaled aztreonam lysine for chronic airway pseudomonas aeruginosa in cystic fibrosis key: cord-345222-otfnrarh authors: ciccarelli, simona; stolfi, ilaria; caramia, giuseppe title: management strategies in the treatment of neonatal and pediatric gastroenteritis date: 2013-10-29 journal: infect drug resist doi: 10.2147/idr.s12718 sha: doc_id: 345222 cord_uid: otfnrarh acute gastroenteritis, characterized by the onset of diarrhea with or without vomiting, continues to be a major cause of morbidity and mortality in children in mostly resource-constrained nations. although generally a mild and self-limiting disease, gastroenteritis is one of the most common causes of hospitalization and is associated with a substantial disease burden. worldwide, up to 40% of children aged less than 5 years with diarrhea are hospitalized with rotavirus. also, some microorganisms have been found predominantly in resource-constrained nations, including shigella spp, vibrio cholerae, and the protozoan infections. prevention remains essential, and the rotavirus vaccines have demonstrated good safety and efficacy profiles in large clinical trials. because dehydration is the major complication associated with gastroenteritis, appropriate fluid management (oral or intravenous) is an effective and safe strategy for rehydration. continuation of breastfeeding is strongly recommended. new treatments such as antiemetics (ondansetron), some antidiarrheal agents (racecadotril), and chemotherapeutic agents are often proposed, but not yet universally recommended. probiotics, also known as “food supplement,” seem to improve intestinal microbial balance, reducing the duration and the severity of acute infectious diarrhea. the european society for paediatric gastroenterology, hepatology and nutrition and the european society of paediatric infectious diseases guidelines make a stronger recommendation for the use of probiotics for the management of acute gastroenteritis, particularly those with documented efficacy such as lactobacillus rhamnosus gg, lactobacillus reuteri, and saccharomyces boulardii. to date, the management of acute gastroenteritis has been based on the option of “doing the least”: oral rehydration-solution administration, early refeeding, no testing, no unnecessary drugs. acute gastroenteritis (age), characterized by the onset of diarrhea with or without vomiting, continues to be a major cause of morbidity and mortality in children mostly in resource-constrained nations. although generally it is a mild and self-limiting disease, gastroenteritis is one of the most common causes of hospitalization and is associated with a substantial disease burden. 1, 2 according to the world health organization (who), diarrhea is defined as the passage of three or more loose or liquid stools per day, or more frequently than is normal for the individual. 3 when young children suddenly experience an episode of acute diarrhea, with or without vomiting, infectious gastroenteritis is by far the most common explanation. 4 viewed from a global perspective, gastroenteritis in children is of enormous public health importance. worldwide, about 10.6 million children still die every year before reaching their fifth birthday. gastroenteritis alone is responsible for almost 20% of the deaths. 4 in spite of the intense promotion of oral rehydration solution (ors) at the community level and the training of health care workers, diarrhea mortality remains unacceptably high: more than 2 million children aged less than 5 years die each year from gastroenteritis, almost all living in resource-constrained nations, where acute diarrhea represents a leading cause of child mortality, second only to pneumonia. 2 age causes 1.5 million visits to primary care providers each year and 220,000 hospital admissions for children under the age of 5 years; that is 10% of all the hospital admissions of children in the us. 5 in general, resource-constrained nations have a higher rate of hospital admissions compared to rich nations. 6 in the us, the admission rate is nine per 1000, per year, for children younger than 5 years old. 5 in england each year, 9.4 million cases of gastroenteritis occur in the community and 1.5 million present to their primary care doctor. 4 in europe, rotavirus infection accounts for more than 50% of hospitalizations for gastroenteritis and about one-third of emergency department visits. 2, 6, 7 not surprisingly, the economic burden of acute diarrhea is substantial, not only in management costs but also in indirect costs, such as absence from work by parents or caregivers of sick children. 2 the severity of acute diarrhea is related to etiology, with rotavirus infection disproportionately implicated in severe cases that frequently require hospitalization. 2, 8 worldwide, up to 40% of children aged less than 5 years with diarrhea are hospitalized with rotavirus: while most of the episodes are mild, about 10% of cases lead to dehydration requiring a doctor visit, and in resource-constrained nations, one in 250 children will die from this dehydration. [7] [8] [9] [10] [11] in europe, rotavirus infection accounts for more than 50% of hospitalizations for gastroenteritis and about one-third of emergency department visits. 2, 7 otherwise, some agents have been found predominantly in resource-constrained nations, including shigella spp, vibrio cholerae, and the protozoan infections. mode of transmission is mainly horizontal, through physical contact with an infected person or with their excretions. the pathogens, most frequently transmitted during the passage through the birth canal, are enteropathogenic escherichia coli, salmonella, and enterovirus. although rare, the passage of the germ can also occur transplacentally during bacteremia. several maternal infections are asymptomatic. horizontal transmission can occur through direct contact with siblings, parents, or health care workers. some cases of transmission through the ingestion of contaminated water or infant formula are also reported. some viral infectious agents, such as adenovirus and rotavirus, can be transmitted by air. 12 the relatively low incidence of the disease in newborns is the result of several factors: breastfeeding and the universal practice of giving birth at home in rural villages, and improvements in social and educational standards and medical care in advanced countries. 13, 14 otherwise, newborns are particularly susceptible to enteric infections in early life, due to reduced local and systemic immune response, absence of an adequate intestinal flora, and reduced gastric acidity. in the newborn, the protective role of gastric motility and of the intestinal mucus is still uncertain. other external factors contribute to the balance of the intestinal ecosystem: nutrition, type of delivery, hygiene habits, use of antibiotics in the mother and infant and the supplementation with probiotics and/or prebiotic oligosaccharides in the newborn. [15] [16] [17] the mortality risk for very low-birth-weight infants (less than 1500 g) due to acute diarrhea is 100 times higher than for infants of low or appropriate birth weight (more than 1500 g). acute diarrhea has several risks and complications; it may lead to lifethreatening dehydration and electrolyte disturbances. when diarrhea is not halted, there is a risk of disturbed digestion and absorption of nutrients with nutritional deterioration. 13 prevention is essential, and all health professionals should ensure caregiver education in the following main principles of prevention: 13 • full and exclusive breastfeeding that protects against intestinal infections and prevents exposure to environmental contamination. 18, 19 thriving breastfed babies under 6 months of age do not require water supplements, even in hot weather. 19 ,20 • provision of safe water for drinking and food preparation. • proper hand-washing hygiene after toilet use and before food preparation and feeding. • safe disposal of human and other waste. table 1a-c shows the main characteristics of the principal bacterial, viral, fungal, and parasitic enteropathogens, respectively: typical age of presentation, type of diarrhea, duration of symptoms, clinical features, transmission, and seasonality. not always well defined, because some agents use both these pathogenetic strategies to induce disease. in europe, the most common bacterial agent is either salmonella or campylobacter, depending on country. 6 aeromonas hydrophila and plesiomonas shigelloides (previously also known as aeromonas shigelloides) belonging to the family of vibronacee, can cause watery diarrhea. the pathogenic role of a. hydrophila as an enteric pathogen causing gastroenteritis is difficult to confirm, because of the frequency of other pathogens isolated with a. hydrophila in symptomatic and asymptomatic subjects. but a. hydrophila is recognized increasingly as a clinically significant enteric pathogen associated with diarrhea also in children younger than 2 years of age living in a rural community, and is linked to local drinking water sources. 21 campylobacter is the most common enteropathogen after 5 years of age, particularly in northern european countries. 6 c. jejuni and c. coli infections are endemic worldwide and hyperendemic in resource-constrained nations. infants and young adults are most often infected. 22 c. jejuni, followed by c. coli and c. lari, are the most common bacterial causes of acute diarrheal illnesses in rich nations. 22 c. jejuni has invasive properties, leading to epithelial ulceration and inflammatory infiltrates in the lamina propria, mainly in the colon, ileum, and jejunum. some c. jejuni isolates elaborate very low levels of cytotoxins, similar to shiga toxin. some isolates have been reported to elaborate an enterotoxin similar to cholera toxin. enterotoxin production has been more frequently observed in isolates from resource-constrained nations, where infection by c. jejuni has been associated with watery diarrhea. however, the clinical significance of the toxigenicity of these organisms is still unclear. 23 symptoms and signs of c. enteritis are not distinctive enough to differentiate it from illness caused by many other enteric pathogens. diarrhea is often associated with blood, but it can be difficult to distinguish from other invasive forms. a cholera-like illness with massive watery diarrhea may also occur. bacteremia is uncommon (less than 1%) in immunocompetent patients with c. jejuni infection. 22 newborn infection by campylobacter spp is rare; most cases were born to mothers with campylobacter diarrhea at the time of delivery. the transplacental passage of campylobacter fetus is responsible for abortion, premature birth, bacteremia, and meningitis. c. jejuni/coli infections can cause a series of complications as reactive arthritis, irritable bowel syndrome, and guillain-barré syndrome (gbs), an acute neurologic disease driven by autoimmunity and molecular mimicry in which the body stages a cell-mediated and humoral immunological response against peripheral nerve myelin. a recent systematic review of gbs estimated that 40%-70% of all cases are preceded by an acute infectious illness, of which 22%-53% are upper respiratory infections and 6%-26% are gastrointestinal infections, one of the most common being enteritis due to campylobacter. 24, 25 several studies have shown that patients with gbs (most of cases associated with the variant acute motor axonal neuropathy) have a recent history of infection due to c. jejuni. 26 clostridium difficile is a major nosocomial pathogen that causes a spectrum of intestinal disease from uncomplicated antibiotic-associated diarrhea to severe, possibly fatal, antibiotic-associated colitis. 27 in the last 5-7 years, a change in the epidemiologic pattern of c. difficile infection characterized by an increasing incidence and severity of infection has been observed. a few epidemiological studies recently conducted in the pediatric population demonstrated a twofold increase in the incidence of c. difficile infection in the last 5 years, but with no increase in the incidence of severe complications, such as the need for colectomy or mortality. 28, 29 the clinical presentation of c. difficile-associated disease can range from asymptomatic carriage in the gastrointestinal tract and mild diarrhea to potentially fatal pseudomembranous colitis. 30 diarrhea is watery and usually nonbloody, but approximately 5%-10% of patients have bloody diarrhea. fecal material typically contains excess mucus, and pus or blood may also be noted. 31 the disease may progress to a pseudomembranous colitis, possibly including intestinal perforation and toxic megacolon. neonatal infections by c. difficile can be asymptomatic, but usually display fever, diarrhea, and irritability within 48 hours after production of the toxins. 32 escherichia coli are the predominant nonpathogenic facultative anaerobe of human colonic flora and usually remain harmlessly confined to the intestinal lumen. some e. coli have evolved the ability to cause a broad spectrum of human diseases, and different types associated with enteric infections are classified into five groups according to their virulence properties and are briefly described here. enteroaggregative e. coli (eaec) serotypes exhibit a characteristic aggregative pattern of adherence and produce persistent gastroenteritis and diarrhea in infants and children in resource-constrained nations. 22 enteroinvasive e. coli serotypes have properties similar to invasive salmonella, but the presence of blood in the stool is less frequent. these can also produce an enterotoxin that cause watery diarrhea, resembling the effects of shigella in children and adults. enteropathogenic e. coli (epec) serotypes in the past were 33 serotypes number about 50, and in rich countries are primarily responsible for gastroenteritis with bloody diarrhea, severe abdominal pain, and cramps that resolve in a few days with an adequate oral rehydration. since 1982, gastroenteritis from shiga toxin-producing e. coli (stec), an e. coli strain with the capacity to produce a cytotoxin similar to that produced by shigella spp, has been identified as a significant health problem in the developed world. 34, 35 infections with stec, of which ehec o157 is the most well-known serotype, have been recorded in many regions, including north america, western europe, central and south america, the middle and far east, africa and australia; also, eaec serotype o104:h4 can produce shiga toxins (stec). [36] [37] [38] infections by stec are characterized by abdominal cramps and acute bloody diarrhea; 39 however, more serious sequelae may also result, including hemolytic uremic syndrome (hus), thrombocytopenia, and associated complications, which can lead to kidney failure and death in some individuals. 36, 40 most outbreaks and sporadic cases of bloody diarrhea and hus have been attributed to strains of stec serotype o157:h7. however, in europe and recently in the us, the role of non-o157 stec strains (eg, o26:h11/h-, o91:h21/h-, o103:h2, o104:h4, o111:h-, o111:nm, o113:h21, o121:h19, o128:h2/h-, and o145:h28/h-) as causes of hus, bloody diarrhea, and other gastrointestinal illnesses is being increasingly recognized. 33, 41 in many studies, a significant association between illness and the consumption of pink or undercooked hamburgers, pinkish ground beef, undercooked meat, or barbecued food has been demonstrated. 38, 41, 42 the natural reservoirs of stec are ruminant animals, especially cattle, and transmission to humans usually occurs via contaminated food or water. helicobacter pylori (previously named campylobacter pyloridis)-infected children may have no symptoms or a wide variety of symptoms, and rarely potentially life-threatening complications, such as gastroduodenal ulcers and bleeding. symptoms of ulcers may include pain or discomfort (usually in the upper abdomen), bloating, an early sense of fullness with eating, lack of appetite, nausea, vomiting, blood in the stools, and diarrhea. in the last 10 years, various studies have been performed investigating its role to modify the susceptibility to gastroenteritis in children. 43 a study reported an increased risk of chronic diarrhea, compared with healthy control subjects, among infected age-matched gambian children with malnutrition. 44 in a nested case-control design, the authors found an increased risk of severe cholera among h. pylori-infected subjects without vibriocidal antibodies. 45 similarly, in an urban slum, h. pylori infection was twice as common among subjects with typhoid fever (salmonella typhi) 46 than in neighborhood control subjects. another study reported increased diarrhea episodes among peruvian infants with recent h. pylori seroconversion. 47 conversely, in a thai orphanage, no association was found between seroconversion and diarrheal disease. 48 some investigators have even speculated that local inflammatory factors induced by infection may be protective. 49 in a cross-sectional study of elementary school-age children in germany, the infection seemed associated with a reduced frequency of diarrheal illnesses. 50 these epidemiological discrepancies could be explained, because h. pylori could be argued to increase or decrease susceptibility to enteric pathogens. depending on the age at acquisition and the anatomical site of colonization, for example, h. pylori decreases gastric acid secretion in some people, thereby potentially reducing the effectiveness of the gastric acid barrier to intestinal pathogens, but increases gastric acid secretion in others. 51 in this prospective study, h. pylori did not seem to increase the risk of gastroenteritis in people more than 2 years old. 43 these data have recently been confirmed. 52 klebsiella, enterobacter, citrobacter, and streptococcus group d have been isolated from feces of sick newborns and associated with intestinal disease, but there is insufficient evidence to define the pathogenic role of these agents. proteus and providencia, although rarely, may be responsible for intestinal infections in newborns. providencia species occur in normal feces and have been isolated from epidemic and sporadic causes of diarrhea, though their importance in the causation of diarrheal disease is not easy to assess. 53 pseudomonas aeruginosa can colonize (0%-10%) the intestine of the newborn during the first days of life. 54 clinically, the infection may be asymptomatic at first, later developing into grayish-blue stool color or watery diarrhea, profuse vomiting, and systemic symptoms. salmonella pass through the intestinal mucosa and multiply within the lamina propria, may invade mesenteric lymphonodes, and systemic spread of the organisms can occur, giving rise to enteric fever. 55 in fact, 3%-5% of infants may have extraintestinal symptoms. the colonization and initial invasion probably occur at the distal ileum, while the mucosal edema and cryptic abscesses are frequently in the colon. the diarrhea is due to secretion of fluid and electrolytes by the small and large intestines. the incubation period is usually 12-48 hours, rarely for a few days. 22 salmonella spp is normally acquired through the birth canal, and the mother can be an asymptomatic carrier. 55 shigella spp are the leading bacterial causes of diarrhea worldwide and are relatively common in children. they cause invasive gastroenteritis, and symptoms can take as long as a week to show up, but most often begin 2-4 days after ingestion. mild symptoms are self-limiting, but s. dysenteriae serotype 1 resistance to multiple antibiotics has the ability to elaborate the potent shiga toxin, which may lead to extraintestinal complications, including hus and death. 56 cases of neonatal infections from staphylococcus aureus are reported in the literature. the pathogen produces two enterotoxins, g and i, that cause atrophy of intestinal villi, diarrhea, and poor growth during the first weeks of life. vibrio cholerae causes watery diarrhea through the production of cholera toxin without invading the intestinal mucosa. yersinia enterocolitica is a common enteropathogen usually causing relatively mild disease. y. enterocolitica crosses the intestinal mucosa, replicates in peyer's patches, and children infected present acute diarrhea associated with fever and pharyngitis, chronic or recurrent diarrhea, or abdominal pain associated with mesenteric adenitis. this infection looks like salmonella infections, with feces containing mucus with or without blood. the pathogen, within phagocytes, can reach other sites through the bloodstream. some of the y. enterocolitica pathogenic biotypes express the yst gene encoding for an heat-stable enterotoxin that may contribute to the pathogenesis of diarrhea. 57, 58 viral enteritis (table 1b) viruses are responsible for approximately 70% of the episodes of acute gastroenteritis in children. viral gastroenteritis is of shorter duration than bacterial gastroenteritis and associated with an increased risk of vomiting and dehydration compared with those without viral infection. the severity of dehydration is significantly higher in children infected with either astrovirus or rotavirus group a. prolonged hospitalization is also more likely to occur with rotavirus infection. 4, 22, 55, 59, 60 enteric adenoviruses are a common cause of viral gastroenteritis in infants and young children. although there are many serotypes of adenovirus that can be found in the stool especially during and after typical infections of the upper respiratory tract, only serotypes 40 and 41 cause gastroenteritis and are very difficult to grow in tissue culture. 61 adenovirus directly infects intestinal enterocytes, causing villous hypoplasia and crypt hypertrophy. the virus causes a massive infiltration of the lamina propria of the villi by mononuclear cells. enteric adenovirus is associated with longer lasting diarrhoea, compared to other viral gents. 4, 22, 55 maternal antibodies are certainly protective; however, among premature and/or low-birth-weight infants the infection spreads rapidly and can be associated with a poor prognosis and high morbidity. 5 cytomegalovirus and herpes virus (cmv or hh5 and hhv6-7; family herpesviridae, subfamily betaherpesviridae) can cause gastrointestinal symptoms such as diarrhea or colitis with profuse hematochezia and bowel perforation. 62 most people who are infected with a non-polio enterovirus (ev; family picornaviridae, genus enterovirus) have no disease, but all ev may cause diarrhea. 63 human ev 68 (ev-d68) is a historically rarely reported virus linked with respiratory disease. in the last 3 years, a large increase in respiratory disease associated with ev-d68 has been reported, with documented outbreaks in north america, europe, and asia. 64 ev71 infections can be asymptomatic or can cause diarrhea, rashes, and hand, foot, and mouth disease. however, ev71 may be responsible for severe complications, including meningitis, encephalitis, cardiovascular and respiratory problems as pulmonary edema, or heart failure. cases of fatal ev71 encephalitis have occurred during outbreaks. most ev71 infections occur under 3 years of age. coxsackie (family picornaviridae, genus enterovirus)-a16 and ev71 are two of the major pathogens responsible for hand, foot, and mouth disease, but the most severe cases are associated with ev71. 65, 66 pleconaril, a viral agent active against enteroviruses, has demonstrated efficacy against neonatal infection with systemic symptoms. however, further confirmatory studies are needed. 67 results of phase i clinical trials suggest an ev71 vaccine has a clinically acceptable safety profile and immunogenicity. outbreaks of ev71 are a serious socioeconomic burden not only in the western pacific region. for this reason, an ev71 vaccine is now being tested and seems to submit your manuscript | www.dovepress.com have an acceptable safety profile and clinically acceptable immunogenicity. 68 human astrovirus (family astroviridae, genus mamastrovirus) may also be responsible for sporadic infections or epidemics, occasionally in newborns and children. 61, 63 human bocavirus (family parvoviridae, genus bocavirus), recently discovered, has been suggested to be involved in a large spectrum of clinical manifestations, including gastroenteritis. 63, 69 human coronaviruses (hcovs; family coronaviridae, genus coronavirus) are common causes of upper respiratory tract infections. a new coronavirus was found to be a causative agent of severe acute respiratory syndrome (sars). sars-hcov caused a serious lower respiratory tract infection with high mortality. diarrhea is common in this condition, and in one study was registered in 38.4% of patients. in the same study, sars-hcov was also isolated from intestinal tissue, and viral rna was detected in stool samples. moreover, non-sars hcovs can be found in stool samples of children with age. however, most of the hcov findings were coinfections with well-known enteric pathogens -norovirus and rotavirus. it is also difficult to determine whether hcovs in the respiratory tract in cases of age were primarily causing the respiratory or gastrointestinal symptoms. hcovs may also be found in occasional stool samples of children without gastroenteritis. these findings suggest that known hcovs may at most have a minor etiologic role in age of children. 70 human rotavirus (family reoviridae, genus rotavirus) in the past was considered to be responsible for the most severe episodes of diarrhea in children. 71, 72 there have been reports of epidemics in neonatal intensive care units caused by rotavirus or enterovirus that can determine cases of necrotizing enterocolitis or necrotizing enterocolitis-like symptoms: abdominal distention, bloody diarrhea, and septicemia secondary to enteric bacteria. improved diagnostic tools for norovirus (family caliciviridae, genus norovirus): have shown that it has a major role in both epidemic and sporadic cases of gastroenteritis. 59, 73 sapoviruses (family caliciviridae, genus sapovirus): mainly infect children younger than 5 years of age. 59 the illness is milder than that caused by noroviruses. 73 antibody prevalence studies show that virtually all children are infected with sapoviruses by the time they are 5 years of age, indicating that sapovirus infection is widespread, although the illness most likely is sporadic with a high rate of asymptomatic infection. 59, 74, 75 torque teno midi virus/small anellovirus (ttmdv/sav) is a member of the family anelloviridae. although human ttv infection is ubiquitous and several infecting genogroups of the virus have been identified, to date there is no consistent evidence of a link between ttv infection of humans and specific disease. [76] [77] [78] [79] in a recent hungarian study, viral shedding, molecular epidemiology, and genetic diversity of ttmdv/ sav were studied in human body fluids (nasopharyngeal aspirates of children with acute respiratory diseases and serum, stool and urine samples collected from eight healthy children with previous ttmdv/sav infection). in this study, shedding of ttmdv/sav and related viruses was detected in two other human body fluids, feces and urine, suggesting the existence of fecal-oral/urinary-oral transmission routes beyond the originally presumed blood-borne and later-suggested respiratory route. this finding extends the number of possible successful transmission routes. fungal enteritis (table 1c) the pathogenic role of candida in neonatal diarrhea is still difficult to prove. symptoms ascribed to candida-associated diarrhea in the literature include prolonged secretory diarrhea with abdominal pain and cramping but without blood, mucus, fever, nausea, or vomiting. 80 disseminated candida infection can cause intestinal symptoms similar to necrotizing enterocolitis, especially in premature infants and in infants treated with antibiotics (especially third-generation cephalosporins) and with central venous catheters or in surgical patients. 81, 82 candida infections frequently develop into systemic forms, and are a major cause of morbidity and mortality in neonatal intensive care units. 83 the incidence of candidemia in the neonatal intensive care unit is steadily increasing, with an estimated incidence of 1%-2% in very low-birth-weight infants and of 2%-23% in extremely low-birth-weight infants. 84 infection-associated mortality following candida bloodstream infections is as high as 40% (very low birth weight 2%-30%, extremely low birth weight 12%-50%), and neurodevelopmental impairment is common among survivors (extremely low birth weight 57%). [84] [85] [86] [87] [88] because invasive fungal infections are common and extremely difficult to diagnose, prevention (decrease of risk factors that contribute to increased colonization and concentration of fungal organisms like maternal vertical transmission or nosocomial acquisition) and antifungal prophylaxis should be considered. 89 parasitic enteritis -protozoan (table 1c) some waterborne protozoan parasites induce enteritis through their membrane-associated functional structures and virulence factors that alter host cellular molecules and submit your manuscript | www.dovepress.com dovepress dovepress signaling pathways, leading to structural and functional lesions in the intestinal barrier. 90 cryptosporidium parvum has a high infectivity with significant enteric disease: rarely is asymptomatic. there have been reported cases of infection in the first month of life; the passage of the maternal antibodies and breastfeeding are a protective factor against infection. 91 giardiasis is one of the intestinal protozoa that cause public health problems in most resource-constrained nations, as well as some resource-rich countries. many infected persons can be asymptomatic, leading to difficulties in the eradication and control of this parasite due to the number of potential carriers, such as school children. giardia lamblia is observed almost three times more in asymptomatic children than in symptomatic children. the first signs of acute giardiasis include nausea, loss of appetite and an upper gastro-intestinal uneasiness, followed or accompanied by a sudden onset of explosive, watery, foul-smelling diarrhea. stools associated with giardia infection are generally described as loose, bulky, frothy and/or greasy with the absence of blood or mucus, which may help distinguish giardiasis from other acute diarrheas. other gastro-intestinal disturbances may include: flatulence, bloating, anorexia, cramps. the acute stage usually resolves spontaneously in a few days. occasionally an acute infection will persist and lead to malabsorption, steatorrhea, loss of strength and weight loss. it has been estimated that about 200 million people are infected each year in africa, asia and latin america. in the resource-rich countries the prevalence rate of giardiasis is 2-5%. however, in resorce-constrained countries, giardia lamblia infects children early in life thus a prevalence rate of 15-20% in children younger than 10 years is common. children who are malnourished are more frequently infected. 92 cryptosporidium and giardia most often cause diarrhea in immunocompromised children or in children from resource-constrained nations, and diarrhea tends to be chronic in both settings. 60, 92, 93 although e histolytica generally causes bloody diarrhea, some studies have demonstrated that e histolytica could also be responsible for watery diarrhea, particularly in infants. that nondysenteric diarrhea is a common presentation of amebiasis in children less than 2 years of age but it was also reported among children aged 2-12 years of age. 94, 95 isospora belli is an opportunistic protozoan more frequent in developing countries of tropical and subtropical regions and should be monitored in both immunocompromised and immunocompetent patients with gastrointestinal complaints such as abdominal pain, nausea, and diarrhea. several helminths can also cause diarrhea, and their importance depends on geographic location, climatic conditions, poor sanitation, unsafe drinking water, and the immune status of the child. 6, 60 strongyloidiasis is an infection caused by the intestinal nematode strongyloides stercoralis. infected healthy individuals are usually asymptomatic; however, it can cause watery or chronic diarrhea, abdominal cramping, failure to thrive, and cachexia. it is potentially fatal in immunocompromised hosts, due to its capacity to cause an overwhelming hyperinfection. a screening assay for strongyloides infection in suspected patients is needed for early detection and successful cure. 96 trichuris trichiura infections are widespread globally, with prevalence and intensity-of-infection peaks in school age. nevertheless, as soon as infants start to explore their environment, thus coming into contact with contaminated soil, they are at risk of infection according to the levels of transmission in the area. the pathogen can cause inflammatory damage to mucosa, bloody diarrhea, iron deficiency, and anemia. most children with gastroenteritis do not require any laboratory investigations. many infants and children experience brief episodes of diarrhea, and are managed by their parents without seeking professional advice. even if advice is sought, health care professionals often consider that a clinical assessment is all that is required, and laboratory investigations are not undertaken. 4 however, there may be particular circumstances when investigations may be helpful in diagnosis. frequently the signs and symptoms are not sufficient to make an etiological diagnosis as they often are nonspecific. the localization of the pathogen in the small intestine or in the colon, the characteristics of the feces (table 2) , the clinical history of the disease, and environmental risk factors can support the diagnostic evaluation. it would be also important to be aware of any history of recent contact with someone with acute diarrhoea and/or vomiting and exposure to a known source of enteric infection (possibly contaminated water or food) or a recent travel abroad. 4 severe watery diarrhea in the absence of mucus, pus, or blood suggests secretory diarrhea or malabsorption (such as by noninvasive vibrio cholera, etec, or by rotavirus, adenovirus, or astrovirus), while the presence of blood and mucus are more indicative of an invasive germ, such as salmonella, shigella, campylobacter jejuni, or yersinia enterocolitica. 60 the presence of vomiting and fever with diarrhea is nonspecific and cannot help in the diagnosis. 4 it would be also important to be aware of any history of recent contact with submit your manuscript | www.dovepress.com 1 year and a few percent of adults. although these data support the potential for endogenous sources of human infection, there was early cir cumstantial evidence to suggest that this pathogen could be transmissible and acquired from external sources. 97 therefore the possibility of other disorders would require careful consideration in such cases of diarrhea and/or vomiting as shown in table 3 . regarding collection and transport of stool specimens, for stool culture for bacterial pathogens: one stool specimen is sufficient in most cases: • c. difficile toxin testing: one stool specimen is sufficient in most cases. • ova and parasite testing: specimen must be submitted in an appropriate preservative (sodium acetate-acetic acidformalin fixative). • viral pathogens: stool for viral pathogens are not routinely tested; for a suspected outbreak of viral gastroenteritis, one stool specimen submitted in a sterile container is sufficient in most cases. for the investigation of bacterial pathogens, stool specimens should be delivered to the laboratory as soon as possible, as a delay may compromise bacterial pathogen recovery. a single stool specimen, properly collected and promptly submitted, will identify most patients with a bacterial pathogen. additional stool specimens need to be submitted if the culture results are negative, symptoms persist, and other causes cannot be found. if there are concerns regarding timing or transport of the specimen, consult the laboratory. submit your manuscript | www.dovepress.com for physical and chemical study of feces, fecal ph less than or equal to 5.5 or the presence of reducing substances is a sign of intolerance to carbohydrates, mostly secondary to viral infection. for microscopic examination of stool, the presence of leukocytes is suggestive of an enteroinvasive infection, although the absence of leukocytes cannot exclude it. however, infections mediated by enterotoxins (etec, vibrio cholerae, and viruses) have no white blood cells in stool. for fecal culture, various culture media are used to isolate the bacteria. the clinical history, physicochemical characteristics of the stool, and laboratory tests thus allow the choice of an appropriate culture medium. in the presence of clinical signs and leukocytes in the stool, it is always necessary to perform culture for salmonella, shigella, and campylobacter. if stool cultures are not performed within 2 hours of sample collection, it is necessary to keep the stool at 4°c. blood cultures are examined for evidence of bacteremia. research of bacterial toxins is conducted through enzyme-linked immunosorbent assay (elisa). 42 the fecal rotavirus antigen is examined through elisa and latex agglutination test; search of adenovirus fecal antigen is performed using elisa. 98 polymerase chain reaction (pcr) or other molecular investigations are performed for viral research, especially on stools for norovirus, adenovirus, sapovirus, and human bocavirus, and pcr of stool and blood is used for the detection of enteroviruses. 63 a providencia genus-specific pcr method has been developed, and its specificity and sensitivity was evaluated to be 100% with various bacterial strains. 99 recently, pcr methods have been applied to investigate the prevalence of the virulence genes specific for five major pathogroups of diarrheagenic escherichia coli in primary cultures from feces of animals slaughtered for human consumption in burkina faso that revealed the common occurrence of the diarrheal virulence genes in feces of food animals. 100 another study investigated using pcr for the incidence, antimicrobial resistance, and genetic relationships of epec in children with diarrhea. 101 regarding parasitic infection and study of trophozoites or oocysts, antigen detection of stool through elisa is used; the serological study can be helpful in rare systemic infections. 60, 82, 91 for intestinal infection by enteroinvasive agents, it is possible to find low blood levels of albumin and high levels of alpha-l-antitrypsin in the stool, and an index of extended intestinal inflammation with dispersion of proteins. 22 parents, caregivers, and children should be informed that it is possible to prevent the spread of gastroenteritis using some simple rules: • wash hands with soap and water, especially after using toilet or changing diapers and before preparing, serving, or eating food • do not share towels used by infected children • children should not attend any school or other child-care facility while they have gastroenteritis; they can go back to school from at least 48 hours after the last episode of diarrhea or vomiting • children should not use swimming pools for 2 weeks after the last episode of diarrhea 102,103 • implementing rotavirus vaccination: the new rotavirus vaccines are safe and reduce the severity of infection and prevent deaths, but they do not prevent all cases of rotavirus diarrhea. 104 two live, oral, attenuated rotavirus vaccines were licensed in 2006: a pentavalent bovine-human recombinant vaccine and a monovalent human rotavirus vaccine. both vaccines have demonstrated good safety and efficacy profiles in large clinical trials in resource-rich countries and in latin america. 105, 106 immunization against rotavirus is recommended in europe and the us. 107, 108 the protective effects of breastfeeding against gastroenteritis infections have been demonstrated in several studies. 109, 110 antibacterial substances, such as lactoferrin, lysozyme, phagocytes, and specific secretory immunoglobulins plays a protective role. 111 the ligand-specific action of κ-casein inhibits helicobacter pylori adherence to the gastric mucosa. human milk also has antiviral action through the lactoferrin and products of digestion of lactoferrin and milk fatty acids. 112 all of these elements suggest that exclusive breastfeeding contributes to protection against common infections during infancy and lessens the frequency and severity of infectious episodes. 113 breastfeeding promotes the colonization of the intestinal ecosystem with a predominance of bifidobacteria and lactobacilli (probiotics) rather than coliforms, enterococci, and bacteroides that characterize the intestinal microflora of infants fed with formula. 114 some authors have demonstrated that the use of formulas supplemented with probiotics (bifidobacterium lactis and lactobacillus gg) have decreased the incidence (up to 57%) and severity of diarrhea. the probiotics, also submit your manuscript | www.dovepress.com dovepress dovepress defined as food supplements, improve intestinal balance, have beneficial effects on health, and are able to balance the intestinal ecosystem and reduce the duration and severity of diarrheal infections, especially in the course of rotavirus infections. the probiotics in the intestine determine resistance to colonization by other potentially pathogenic microbes through mechanisms of competition or inhibition, and the effects are expressed both on nonspecific innate and acquired immunity. 115 lactobacillus rhamnosus gg (lgg) is considered particularly effective in the management of age; this is confirmed by a recent cochrane review documenting that lgg reduced the duration of diarrhea, mean stool frequency on day 2, and the risk of diarrhea lasting $4 days. 1, 116 according to a recent cochrane review, saccharomyces boulardii reduces the risk of diarrhea lasting $4 days, and a more recent review confirmed that s. boulardii significantly reduced the duration of diarrhea and hospitalization. a recent randomized controlled trial evaluated the efficacy of treatment with lactobacillus reuteri dsm 17938 compared with placebo: the administration of l. reuteri reduced the duration of watery diarrhea, the risk of diarrhea on days 2 and 3, and the relapse rate of diarrhea. 1 it has been suggested that probiotics may decrease infant mortality and nosocomial infections because of their ability to suppress colonization and translocation of bacterial pathogens in the gastrointestinal tract. several meta-analyses evaluating probiotics in preterm infants suggest a beneficial effect for the prevention of necrotizing enterocolitis and death, but less for nosocomial infection. l. reuteri may reduce these outcomes because of its immunomodulation and bactericidal properties. a large, double-blinded, randomized controlled trial (rct) using l. reuteri was performed to test this hypothesis in preterm infants. this study suggested that although l. reuteri did not appear to decrease the rate of death or nosocomial infection, the trends suggest a protective role consistent with what has been observed in the literature: a protective role for mortality, nosocomial infection, and necrotizing enterocolitis. feeding intolerance and duration of hospitalization were significantly decreased in premature infants less than 1500 g. 117 the use of formulas supplemented with probiotics (particularly bifidobacterium lactis and lactobacillus gg) seems to decrease the incidence (up to 57%) and severity of infectious acute diarrhea. symbiotics, a combination of prebiotics and probiotics that beneficially affect the host by improving survival and implantation of live microbial dietary supplements in the gastrointestinal tract, has recently been evaluated by two european rcts for the management of age. these studies are promising, but presently it would not be appropriate to recommend the use of symbiotics until confirmatory data are available. 1 dehydration is probably the main complication of gastroenteritis in childhood. who classification of patients' hydration status is based on the presence of symptoms and signs. the presence of one of these signs or symptoms immediately classifies the patient as a more severe case. table 4 summarizes the who management of rehydration. 118, 119 all moderate and severe patients require close monitoring, but patients at the extreme ages of life, especially children under 18 months, require meticulous observation and immediate measures if their condition worsens. according to current who recommendations, oral rehydration therapy (ort) is considered the treatment of choice to replace fluid and electrolyte losses caused by diarrhea in children with mild to moderate dehydration. intravenous rehydration is the treatment of choice in cases of failure of ort, and it has to be reserved for patients with severe dehydration or who eliminate more than 10-20 ml/ kg/hour. in the 1960s, efforts by young scientists and researchers led to the development of ort for the treatment of dehydration that often accompanies acute attacks of diarrhea. many members of the research team responsible for this discovery were associated with noteworthy universities and medical research centers in the us. the goal of their research was to devise effective therapies for cholera-induced diarrhea; as a result of their hard work, they developed a new framework for treatment that would soon be adopted throughout the developing world as a key element in the overall strategy to combat acute diarrhea and the potentially fatal dehydration that accompanies the disease. in an article published in 1968 in the lancet, it was confirmed that cholera patients could be rehydrated orally with a simple solution of water, salt, and sugar, and equally importantly that field staff could easily be trained to administer the therapy. as stated in the article, ort also offers a practical treatment for large numbers of patients in the developing world who do not have access to traditional intravenous drip therapy. at first, studies on the efficacy of ort, when compared with intravenous therapy (ivt), were conducted only in patients with cholera. [120] [121] [122] following that, other studies established the effectiveness of ort in children with acute diarrhea from other causes. 123 some trials also compared the effectiveness of oral rehydration submit your manuscript | www.dovepress.com with ivt in children with different degrees and types of dehydration (mild, moderate, severe, and hypernatremic dehydration), and they concluded that the use of ors to rehydrate children is safe and that there are no significant differences in incidences of hyponatremia, hypernatremia, mean duration of diarrhea, weight gain, or total fluid intake if compared with ivt. in terms of outcomes, ort was associated with a higher risk of rehydration failure, while babies treated with ivt had a significantly longer stay in hospital and a higher risk of phlebitis, but no statistically significant differences were seen. 124 the main reason for this failure is that ors neither reduces the frequency of bowel movements and fluid loss nor shortens the duration of illness; moreover, the unpalatability of regular ors (strong salty taste) also decreases this acceptance. however, the most important aspect of treatment of gastroenteritis is the water and electrolyte balance. it must be adjusted according to serum electrolytes, body water content (greater the younger the child), and the water demand must be calculated on the weight of the newborn infant (table 5a and b). different ors are now commercially available (table 6 ). 125 recently, some companies have added probiotics to the saline solution to obtain a quick balance of intestinal bacterial flora. the infant should be monitored closely to check the status of nutrition and hydration. infants with moderate dehydration, suspected infected by ehec, with bloody diarrhea, or systemic symptoms should be hospitalized. these newborns are at high risk of secondary complications. during ort, milk-feeding is often temporarily suspended. among all the oral solutions, the ideal one has a low osmolarity (210-250 mosm/l) and a sodium content of 50-60 mmol/l to avoid high levels of serum sodium. the solution should be administered frequently and in small amounts to prevent vomiting. initial oral rehydration is 50-100 ml/kg in the first 4 hours. 126 also consider giving the ors via a nasogastric tube if patients are unable to drink it or if they vomit persistently and to monitor them by regular clinical assessment. in cases of failure of oral rehydration, it is necessary to establish an appropriate parenteral rehydration fluid and electrolyte solutions (table 7) . 127 pediatric presentations of racecadotril were first authorized in france in 1999, and today it is approved and widely used in seven european countries (france, spain, italy, portugal, greece, bulgaria, and romania) and outside europe. 128 this antisecretory drug is a peripherally acting enkephalinase inhibitor that reduces intestinal water and electrolyte hypersecretion acting on the enkephalins (neurotransmitters of the gastrointestinal tract) through the selective stimulation of delta receptors inhibit adenylate cyclase activity by reducing the intracellular concentration of camp, thus reducing the secretion of water and electrolyte in the intestinal lumen. the result is a reduction of water and electrolyte secretion without changes in intestinal motility. moreover, the action of racecadotril takes place only when there is a hypersecretion and has no effect on the activity secretory baseline. new data have reconfirmed that racecadotril is an effective adjunctive therapy to oral rehydration in watery diarrhea. 1 a recent individual patient data meta-analysis 129 assessed the efficacy of the use of racecadotril as an adjunct to ors compared with ors alone or with placebo. raw data from nine rcts involving 1348 children aged 1 month to 15 years with age were available for the analysis. two trials compared the effect of racecadotril with placebo 130, 131 with no treatment (two rcts), or with kaolin-pectin (two rcts). compared with placebo, racecadotril significantly reduced the duration of diarrhea after inclusion. almost two times more patients recovered at any time in the racecadotril group vs the placebo group (p , 0.001). there were no interactions between treatment and dehydration, rotavirus infection, type of study (outpatient/inpatient), or country. in the studies evaluating inpatients, the ratio of mean stool output racecadotril/placebo was reduced (p , 0.001). in outpatient studies, the number of diarrheal stools was lower in the racecadotril group (p , 0.001). in the responder analysis (defined as a duration of diarrhea of less than 2 days), the proportion of responders was significantly higher in the racecadotril group compared with the placebo group. by adjusting for dehydration and rotavirus, the absolute risk difference was 24.7% (95% confidence interval 19.8-29.7), and the associated number needed to treat was four. the secondary need for care in outpatients was significantly in favor of racecadotril in two studies. also, the need for ivt was lower in the racecadotril group compared with the placebo group. there was no difference in the incidence of adverse events between the groups. the results of this recent meta-analysis support the use of racecadotril as an adjunct to ors for the management of age in children. in addition, the safety of racecadotril in children has been demonstrated in clinical studies, including a large pre-and postaccess study showing that racecadotril has a favorable adverse-event profile in children. [128] [129] [130] despite racecadotril's proven safety and efficacy in treating acute watery diarrhea, its cost-effectiveness for infants and children has not yet been determined in europe. 128 the uk model highlights the potential savings arising from reduction in diarrhea duration and avoidance of reconsultation and referral rates in children with diarrhea. 128 children presenting with age often have high levels of vomiting that can interfere with the oral rehydration process, which could limit the success of the oral therapy. ondansetron is widely used in the pediatric emergency department for vomiting and age; it can help with the successful delivery of ort, thereby reducing the need to treat with ivt. a recent study evaluated the spectrum of diagnoses for which ondansetron is used in the pediatric emergency room. medical records of patients 3 months to 18 years of age given ondansetron for 2 years were retrospectively reviewed. patients without a primary discharge diagnosis of vomiting or gastroenteritis were defined as non-gastroenteritis, and they were compared to the gastroenteritis group. the non-gastroenteritis group includes 38% of the subjects, and they were older (8.3 vs 4.3 years) than the gastroenteritis patients. the most common primary diagnoses for non-gastroenteritis discharged patients were fever (15%), abdominal pain/ tenderness (13%), head injury/concussion (7%), pharyngitis (6%), viral infection (6%), migraine variants (5%), and otitis media (5%). although ondansetron is a widely accepted treatment for gastroenteritis submit your manuscript | www.dovepress.com in children -62% of total use -this study identifies a broader spectrum of primary diagnoses for which ondansetron is being used. 132 another study used rcts comparing antiemetics with placebo or no treatment in children and adolescents under the age of 18 years, for vomiting due to gastroenteritis. the proportion of patients with cessation of vomiting in 24 hours was 58% with intravenous ondansetron, 17% placebo, and 33% in the metoclopramide group (p = 0.039). in this case, the authors' conclusions were that oral ondansetron increased the proportion of patients who had ceased vomiting and reduced the number needing intravenous rehydration and so immediate hospital admissions. 1, 133 today, it is still unclear if in spite of an improvement in the vomiting, ondansetron worsens diarrhea. some trials report a statistically significant increase in its frequency as an adverse event. in cochrane reviews, diarrhea was reported as a side effect in four of the five ondansetron studies. [133] [134] [135] according to who, of the antiemetics available, those with the greatest evidence of efficacy in the prevention of nausea and vomiting (particularly in the treatment of postsurgery nausea and vomiting) were ondansetron and dexamethasone, ondansetron as first-line treatment with the addition of dexamethasone as required. 136 zinc is an important trace element, as over 300 enzymes require zinc for their activation and nearly 2000 transcription factors require zinc for gene expression. zinc is essential for epithelial barrier integrity, tissue repair, cell-mediated immunity, and immune function. zinc as an antioxidant and antiinflammatory agent is effective in gastrointestinal structure and function. 137 diarrhea is associated with significant zinc loss, and the use of zinc supplements can reduce the duration and severity of diarrhea in children. in areas where the prevalence of zinc deficiency or the prevalence of moderate malnutrition is high, zinc may be of benefit in children aged 6 months or more. the current evidence does not support the use of zinc supplementation in children below 6 months of age. 138 the who has recommended zinc supplementation in children with gastroenteritis. supplements should be started at the beginning of the symptoms. recommended doses and duration: • for children less than 6 months of age, 10 mg daily for 10 days • for children from 6 months to 5 years of age, 20 mg daily for 10 days. this therapy decreases the severity and reduces the number of episodes of diarrhea occurring within 2-3 months following the intake of zinc. 139 the physiological composition of intestinal microflora is essential to maintain an appropriate balance of microbiota and the intestinal barrier. probiotics, also defined as food supplements, improve the intestinal microbial balance of the host, have beneficial effects on health, prevent outbreaks of community-acquired diarrhea, reduce colonization of infants with pathogenic microorganisms, and reduce the duration and severity of diarrheal infections, balancing the intestinal ecosystem. in large clinical trials, lactobacillus reuteri, lgg, and saccharomyces boulardii have shown the best therapeutic effects (reducing mean duration and frequency of watery diarrhea and number of watery stools per day, and improving stool's consistency). [140] [141] [142] particularly, a recent randomized double blind study carried out in three italian pediatric centers showed that l. reuteri dsm 17938, taken together with a standard ors, significantly reduced the duration of watery diarrhea compared with placebo (2.1 ± 1.7 days vs 3.3 ± 2.1 days, p , 0.03). on days two and three of treatment, watery diarrhea persisted in 82% and 74% of the placebo and 55% and 45% of the l. reuteri recipients, respectively (p , 0.01, p , 0.03). moreover, children receiving l. reuteri dsm 17938 had a significantly lower relapse rate of diarrhea (15% vs 42%, p , 0.03). 143 the european society of gastroenterology, hepatology, and nutrition and the national institute for health and clinical excellence have suggested the use of probiotic strains with proven efficacy and in appropriate doses for the management of children with acute gastroenteritis as an adjunct to rehydration therapy. 144, 145 probiotics and symbiotics are of interest as they elicit healthpromoting properties to the host, release various soluble low-molecular-weight molecules of different nature (surface and exogenous proteins, peptides, amines, lectins, sirtuines, nucleases, other enzymes, bacteriocines, fatty and amino acids, lactones, nitric oxide, etc), are able to interact with corresponding cell receptors, to reply quickly by induction of special sets of genes, to support stability of host genome and microbiome, to modulate epigenomic regulation of gene phenotypic expression, and to ensure information exchange in numerous bacterial and bacteria-host systems. all this plays an important role in the control for many physiological, biochemical, and genetic functions in supporting host health. recently, probiotic l. reuteri strain atcc pta 6475 demonstrated the ability to potently suppress human tumor necrosis-factor production by lipopolysaccharide-activated monocytes and primary monocyte-derived macrophages from children with crohn's disease: the primary mechanism of probiotic-mediated immunomodulation is transcriptional regulation. 146 other researchers have confirmed these results, and it has been shown that l. reuteri produce biologically active small compounds, previously unknown, that can modulate host mucosal immunity. the identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immunomediated diseases. 147 antibiotic therapy bacteria most cases of age in children are viral, self-limited, and need only supportive treatment. antibacterial therapy serves as an adjunct, to shorten the clinical course, eradicate causative organisms, reduce transmission, and prevent invasive comsubmit your manuscript | www.dovepress.com dovepress dovepress plications. selection of antibacterials to use in acute bacterial gastroenteritis is based on clinical diagnosis of the likely pathogen prior to definitive laboratory results. antibacterial therapy should be restricted to specific bacterial pathogens and disease presentations. in general, infections with shigella spp and vibrio cholera should usually be treated with antibacterials, while antibacterials are only used in severe unresponsive infections with salmonella, yersinia, aeromonas, campylobacter, plesiomonas spp, and clostridium difficile. antibacterials should be avoided in ehec infection. 148 there is no evidence of benefit for antibiotics in nontyphoidal salmonella diarrhea in otherwise healthy people. the effects in very young people, very old people, and in people with severe and extraintestinal disease are not always so clear, and a slightly higher number of adverse events are noted in people who receive antibiotic treatment for nontyphoidal salmonella diarrhea. 149 however, empiric therapy may be appropriate in the presence of a severe illness with bloody diarrhea and stool leucocytes, and particularly in patients with risk factors (ill-fed or debilitated patients), the use of systemic antibiotics has been recommended (table 8) . 148, 150 the benefits and risks of adverse drug reactions should be weighed before prescribing antibacterials. moreover, a major concern is the emergence of antibacterial-resistant strains due to the widespread use of antibacterial agents. aeromonas spp produce a β-lactamase that induces resistance to penicillin and first-generation cephalosporins. in fact, several studies have demonstrated a relatively high resistance rates for cephalothin and for trimethoprimsulfamethoxazole; low rates of resistance has been found to third generation cephalosporin (cefotaxime), to ciprofloxacin and to chloramphenicol. with high levels of resistance to many antibiotics, resistance of a. hydrophila to ciprofloxacin is still very low, which may suggest that ciprofloxacin and other quinolone class antimicrobials may be considered as potential drugs for the treatment of bacterial diarrhea caused by a. hydrophila. 21, 151, 152 for campylobacter jejuni, antibiotics are initiated in cases of febrile diarrheas, especially those believed to have moderate to severe disease. considering the increased incidence of c. jejuni and the resistance of the great majority of isolated strains to quinolones, the administration of azithromycin empirically for acute diarrhea, when indicated, could be appropriate. 153, 154 moreover, erythromycin treatment of acute c. jejuni diarrhea demonstrated antibacterial efficacy by reducing the mean number of days until first negative stool culture. 4 according to recent studies, the management of clostridium difficile infections involves three basic principles: supportive care, discontinuing the precipitating antibiotic(s), and the initiation of effective anti-c. difficile therapy. discontinuation of the offending antibiotic may be sufficient for the resolution of mild symptoms and facilitates the reconstitution of the normal enteric flora. 154 for mild-moderate c. difficile infection in children, metronidazole is the drug of choice, with efficacy similar to vancomycin. for severe infection, oral vancomycin with intravenous metronidazole is recommended. linezolid also has a potential impact, and in adults, recurrence is less frequent with fidaxomicin than with vancomycin. [155] [156] [157] [158] a vaccine against c. difficile is desirable and being developed for prevention. 159 regarding enterobacteriaceae, an antibiotic-susceptibility profile indicated that enteropathogens are generally susceptible to meropenem and ceftriaxone, followed by amikacin and ciprofloxacin. almost all enteropathogens were resistant to ampicillin and amoxicillin. 160 epec infection is primarily a disease of infants younger than 2 years of age, often contracted during travel in hot countries. in moderate-severe forms of gastroenteritis caused by epec in nurseries, the use of antibiotics such as imipenem, amikacin, gentamicin, and fluoroquinolone seem to be useful in reducing morbidity, mortality, and time of excretion, but few studies have evaluated in a systematic manner the value of antimicrobials for the management of epec infection in children. 161, 162 etec has been reported to be the most important pathogen responsible in traveler's diarrhea. eaec also plays an important role in traveler's diarrhea. pathogen-and geographic-based approaches to traveler's diarrhea treatment should be encouraged. fluoroquinolones and nonabsorbable rifaximin are the drugs of choice for travelers to high-risk areas in which e. coli is the predominant etiologic agent (latin america, the caribbean [haiti and the dominican republic], and africa), leaving azithromycin for travelers to south and southeast asia as well as patients with febrile dysenteric illnesses acquired in any region. 163 antibiotic therapy for stec is complicated. the growth of o157:h7 in the intestinal tract leads to diarrhea, and patients would presumably benefit if antibiotic treatment eliminated the bacteria. however, systemic dissemination of shiga toxin type 2 produced by the bacteria in the intestinal tract can lead to life-threatening complications, including neurological damage and hus. antibiotic treatments that induce the phage lytic cycle, resulting in increased shiga toxin production, could lead to more serious disease. recent studies suggest that normal flora can have a profound impact on shiga toxin production. subinhibitory levels of antibiotics that target dna synthesis, including ciprofloxacin and sulfamethoxazole-trimethoprim, increased shiga toxin production, while antibiotics that target the cell wall, transcription, or translation did not. so ciprofloxacin and sulfamethoxazole-trimethoprim are not appropriate for treatment of o157:h7. in contrast, azithromycin significantly reduced shiga toxin levels, even when relatively high levels of o157:h7 were recovered. 164 moreover, the eradication rate reported in stec o104:h4 infections is 86%. azithromycin might be considered a potentially effective and safe antibiotic, and may be used safely for decolonization of stec o104:h4 long-term carriage. 165 numerous outbreaks, as well as sporadic cases of stec infections and hus, have been documented worldwide. there are numerous reports on stec o157:h7 as the most common serotype associated with hus, especially in children. several reports on non-o157 stec underline their potential to cause sporadic disease as well as epidemics. during 2011, there was a large outbreak in northern germany, with a satellite outbreak in western france caused by an eaec of serotype o104:h4 expressing a phage-encoded shiga toxin 2: clinicians were confronted with a large number of mainly adult patients with hus associated with severe hemolysis and neurological complications. 166 medical centers used varying therapeutic regimens, including plasmapheresis, glucocorticoids, and the submit your manuscript | www.dovepress.com dovepress dovepress anti-c5 monoclonal antibody eculizumab, but currently there is no effective prophylaxis or treatment available for stec infections and hus. 166 the probiotic escherichia coli strain nissle 1917 (ecn) seemed to have very efficient antagonistic activity on the ehec strains of serotype o104:h4 and o157:h7, with reduced growth of pathogens. 167 the 2011 outbreak strains perfectly showed the genome plasticity and evolution of e. coli as a result of horizontal gene transfer. these strains combine the virulence mechanisms of two pathotypes (eaec and ehec), leading to an improved ability to adhere to and infect host cells. furthermore, the acquisition of mechanisms mediating increased antibiotic resistance hampered patient treatment and recovery. these strains have conserved most of the virulence factors of an eaec strain, but several mobile genetic elements were responsible for the acquisition of new functions involved in high-frequency recombination, mobilization, and transfer of genes. despite the alternative mechanisms that have evolved to colonize and adapt to new niches, e. coli strains have maintained a core genome sequence, and therefore share several components that could be useful targets for a universal vaccine against e. coli. considering the increasing antibiotic resistance present among e. coli strains, which is derived from an uncontrolled use of antibiotics, vaccination is the most promising approach to control disease. 168 helicobacter pylori is a leading cause of chronic gastritis, peptic ulcers, nonulcer dyspepsia, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. eradication of the pathogen has a failure rate of more than 30% in pediatric patients, particularly because of poor compliance, antibiotic resistance, and occurrence of side effects. 169 treatment regimens generally include a proton-pump inhibitor (lansoprazole, omeprazole, pantoprazole, rabeprazole, dexlansoprazole, or esomeprazole), which allows the tissues damaged by the infection to heal, and two antibiotics to reduce the risk of treatment failure and antibiotic resistance. treatment requires several medications for 7-14 days. in pediatric patients, gastric mucosal lesion-caused h. pylori infection is a reversible process, and the eradication of this infection not only stops the activity of the inflammatory process but also restores the mucous membranes, reduces the incidence of recurrence of gastritis and peptic ulcer disease, and can lead to prevention of malignant disease in 70%-80% of cases. 170 adjuvant therapy with probiotics has been studied in recent years. 169 in a recent randomized, placebo-controlled, double-blind study, children on h. pylori eradication therapy receiving seven strains of probiotic in addition to the standard triple regimen (omeprazole + amoxicillin + claritromicin or omeprazole + amoxycillin + furazolidon) were compared with patients on the same triple regimen receiving placebo. the findings reported a significant reduction in treatment complications and an improved therapeutic outcome: the rate of eradication was significantly higher, and children had a lower rate of nausea/vomiting and diarrhea during treatment. 168 the long-term success of h. pylori eradication interventions for preventing gastric cancer, depends on the recurrence determinants (nonadherence and demographics) that are as important as a specific antibiotic regimen. 171 despite plesiomonas shigelloides seeming to be a minor cause of bacterial enteritis, the pathogen has been implicated in gastroenteritis outbreaks in tropical regions and in cases of traveler's diarrhea. antibacterials are used only in cases of severe and unresponsive infections: the most effective are ciprofloxacin and azythromycin, and partially trimethoprimsulfamethoxazole. with salmonella spp, antibiotics are initiated in cases of febrile diarrhea, especially in case of moderate to severe disease. the administration of azithromycin empirically for acute diarrhea, when indicated, could be appropriate. 4, 153 consistent evidence from several clinical trials suggests that antibiotic treatment (ampicillin, amoxicillin, cefixime, azithromycin, cotrimoxazole) did not shorten the duration of diarrhea or lead to an earlier resolution of clinical symptoms. intramuscular ampicillin protects children against relapse and reduces the carriage of salmonella infection significantly better than placebo, oral ampicillin, or amoxicillin. 4 antibiotics are usually avoided in mild shigella illness, because mild forms of shigella dysentery are said to be self limiting, some shigella strains are resistant to antibiotics, and their use may lead to increased resistance. if necessary, in severe cases, the choice of antibiotic to use as first line against shigella dysentery should be governed by periodically updated local antibiotic sensitivity patterns of shigella isolates. other supportive and preventive measures recommended by the who should also be instituted along with antibiotics (eg, health education and hand-washing). ciprofloxacin has been recommended by the who as the drug of choice for all patients with bloody diarrhea, irrespective of their ages. 172 according to a recent cochrane review, the authors did not find robust evidence to suggest that antibiotics of a particular class are better than those belonging to a different class. trials report that at various periods of time, different antibiotics have been effective against isolates of shigella dysentery in different parts of the world. these are ampicillin, cotrimoxazole, nalidixic acid, fluoroquinolones submit your manuscript | www.dovepress.com dovepress dovepress like ciprofloxacin, pivmecillinam, ceftriaxone, and azithromycin. however, limited data from a subgroup of studies suggest that a fluoroquinolone (ciprofloxacin) would be more effective than a beta-lactam (ampicillin) in reducing diarrhea among adults, and that beta-lactams would be more effective than fluoroquinolones in reducing diarrhea among children with proven shigella dysentery. emerging drug resistance to ciprofloxacin and second-line drugs such as pivmecillinam, ceftriaxone, and azithromycin is increasingly being reported in many parts of the world, as is multiple-drug resistance. 173, 174 in india, for example, several shigella strains (s. flexneri, s. dysenteriae, s. boydii, and s. sonnei) isolated from children # 15 years of age are resistant to ampicillin, cotrimoxazole, ciprofloxacin, and nalidixic acid. alternatives include ceftriaxone and azithromycin, but ceftriaxone is an injectable drug and azithromycin has limited therapeutic benefit, as organisms easily develop resistance to it. 175 it has been noted that all shigella spp utilize a type iii secretion system to translocate bacterial proteins -invasins ipaa-d and ipgd -into host eukaryotic cells to initiate infection. because they are common to all virulent shigella spp, they seem to be ideal candidate antigens for a subunit-based broad-spectrum protective vaccine for prevention of shigellosis. 176 vibrio cholerae strains from endemic outbreaks within the last decade revealed patterns of antibiotic resistance to ampicillin, tetracycline, and trimethoprim correlated with widespread therapeutic and prophylactic administration of antibiotics. 177 treating severe cases of cholera with antibiotics is important, but the continuing spread of resistance to the most important therapeutic agents is a matter of concern, as some strains have either intermediate resistance or are resistant to ceftriaxone, ciprofloxacin, and tetracycline. 178 the most common clinical manifestation of a yersinia enterocolitica infection is a self-limited gastroenteritis that resolved spontaneously within 2 weeks. 179 y. enterocolitica usually shows in vitro susceptibility to aminoglycosides, chloramphenicol, doxycycline, cotrimoxazole, third-generation cephalosporins, carabapenems, and fluoroquinolones. 180 recently, in a case-control study conducted among children aged less than 12 years, it was found that y. enterocolitica is generally susceptible to meropenem (100%), ceftriaxone (94%), and ciprofloxacin (94%), followed by ceftazidime (88%) and amikacin (81%). almost all y. enterocolitica was resistant to ampicillin. 57 fungal fungal infections of the gastrointestinal tract are not common in children, especially in immunocompetent ones. in the neonatal period, candida infections frequently develop into systemic forms. 82 fluconazole prophylaxis in infants , 1000 g (3 mg/kg twice a week), while intravenous access is required, appears to be safe and effective in preventing invasive candida infections with or without diarrhea, while attenuating the emergence of fungal resistance. 88, 181 new echinocandines -anidulafungin, caspofungin, and micafungin, recently introduced -seem to have some advantages over fluconazole and amphotericin b, as they better meet the needs of pediatric patients, neonates, and in particular preterm infants with invasive candidiasis and/or diarrhea from candida spp infection. micafungin, a dose-dependent candidacidal agent with excellent in vitro efficacy against most candida spp, including species resistant to amphotericin b, is approved for the treatment of invasive candidiasis in children, including preterm infants aged less than 3 months. efficacy and safety were demonstrated in comparison with liposomal amphotericin b and fluconazole. the most appropriate dose in children weighing less than 40 kg is 2 mg/kg/day in the treatment of invasive candidiasis and or gastroenteritis and 1 mg/kg/day as prophylaxis. in premature infants, the most appropriate doses to achieve appropriate levels in the brain parenchyma are 7 mg/kg/day in infants weighing more than 1,000 g and 10 mg/kg/day in those weighing less than 1,000 g, respectively. micafungin has few drug-drug interactions and an acceptable safety profile thus providing a promising drug in the prophylactic and therapeutic management of invasive candidiasis. micafungin has few drug-drug interactions, and an acceptable safety profile. [182] [183] [184] data from randomized trials conducted in pediatric and adult patients showed through a subgroup analysis that both caspofungin and micafungin are effective and well tolerated also in neonates. 185, 186 parasitic -protozoan the major causes of diarrhea worldwide in children are cryptosporidium parvum, giardia lamblia, and entamoeba histolytica: "the neglected parasitic disease." cryptosporidium is a diarrheagenic protozoan pathogen for children, and immunosuppressed individuals are disproportionately affected. until a few years ago, the most commonly used treatments, only partially effective, were paromomycin and azithromycin. recent investigations have focused on nitazoxanide, as it significantly shortens the duration of diarrhea and decreases mortality in malnourished children. nitazoxanide is not effective without an appropriate immune response, as in aids patients. 187, 188 giardia lamblia is a diarrheagenic protozoan pathogen most commonly treated with metronidazole (mtz) or tinidazole submit your manuscript | www.dovepress.com dovepress dovepress but failures occur in 10%-20% of cases. albendazole may be of similar effectiveness to metronidazole, may have fewer side effects, and has the advantage of a simplified regimen. nitazoxanide is a viable therapeutic option as an effective alternative to mtz in reducing the duration of diarrhea. 61, 188, 189 auranofin, a gold complex classified by the who as an antirheumatic agent, has been revealed to be active against multiple mtz-resistant strains blocking a critical enzyme involved in maintaining normal protein function. these results indicate that auranofin could be developed as an antigiardial drug, particularly against mtz-resistant strains. 190 entamoeba histolytica amebiasis is the fourth-leading cause of death and the third-leading cause of morbidity due to protozoan infections worldwide. in children, effective drugs for diarrhea are mtz and other nitroimidazoles. however, eradication of e. histolytica infection after completion of mtz requires additional therapy with luminal amebicides, such as paramomycin. nitazoxanide is a recent therapeutic advance, due to its action against luminal and invasive parasite forms. 191 nitazoxanide-treated patients had statistically shorter durations of diarrheal illness. auranofin could represent a promising therapy for amebiasis. 187 parasitic -helminths nematodes strongyloides stercoralis and trichuris trichiura are causes of diarrhea. strongyloides stercoralis can penetrate host skin and parasitize human intestines, leading to burning pain, tissue damage, ulcers, edema and obstruction of the intestinal tract, and diarrhea, as well as loss of peristaltic contractions. ivermectin is the first-choice therapy because of its higher tolerance, and albendazole is the second-choice therapy. 61, 192 trichuris trichiura is chiefly a tropical infection, and children are especially vulnerable to infection due to their high exposure risk. light infestations (, 100 worms) are frequently asymptomatic, but heavy infestations may cause mechanical or inflammatory damage to the mucosa, abdominal pain, profuse or chronic diarrhea, and bloody diarrhea. mebendazole seems to be the first-choice therapy and albendazole the second-choice therapy, while nitazoxanide shows no effect. an albendazole and nitazoxanide-albendazole combination showed only a minimal effect. there is a need to develop new anthelmintics against trichuriasis. 193 age remains a major problem in children and still represents one of the leading causes of illness costs and of deaths, as an estimated 2.5 million gastroenteritis deaths occur each year in children less than 5 years of age throughout the world, especially in resource-constrained countries. in rich countries, transmission occurs much more frequently from contaminated food compared to direct person-to-person contact, except for enteric viruses, which can also be transmitted by aerosol formation after vomiting. most cases of age in children are viral, self-limited, and need only supportive treatment. rehydration (oral or intravenous) with an appropriate fluid-and-electrolyte balance, with close attention to nutrition, remains central to therapy: this may turn into an additional benefit in limiting hospitalizations. intestinal infections often require drugs such as antiemetics, antidiarrheal agents, and probiotics that may deeply change the impact, severity, and duration of acute diarrhea. in cases of severe infectious diarrhea with a prolonged course, signs of inflammation, bloody stool, immunosuppression, and comorbidity, and in suspected outbreaks, fecal microbial analysis, should always be performed, and a specific therapy should be considered if indicated to shorten the clinical course, eradicate causative organisms, reduce transmission, and prevent invasive complications. selection of antibacterials to use in acute bacterial gastroenteritis is based on clinical diagnosis of the likely pathogen and on definitive laboratory results. based on epidemiological data and after collecting organic materials for etiological diagnosis (often a single fecal sample studied for etiologic agents is the customary way to make an etiologic diagnosis), an initial empiric therapy may be appropriate in case of a severe illness, particularly in infancy and the immunocompromised. in case of suspected ehec serotype o157 and eaec serotype o104:h4 (but also of shigella dysenteriae serotype 1), as it is estimated that 5%-8% of infected individuals will develop hus following stec infection with e. coli o157:h7 the most commonly involved serotype, antibiotics should be prescribed according to more recent guidelines. moreover, a major concern is the emergence of antibacterialresistant strains due to the widespread use of antibacterial agents: a continuous monitoring of antibiotic resistance in diarrhea-related bacterial pathogens is recommended. the benefits and risks of 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research agenda for helminth diseases of humans: intervention for control and elimination efficacy and safety of nitazoxanide, albendazole, and nitazoxanide-albendazole against trichuris trichiura infection: a randomized controlled trial submit your manuscript here: http://www.dovepress.com/infection-and-drug-resistance-journal infection and drug resistance is an international, peer-reviewed openaccess journal that focuses on the optimal treatment of infection (bacterial, fungal and viral) and the development and institution of preventive strategies to minimize the development and spread of resistance. the journal is specifically concerned with the epidemiology of antibiotic resistance and the mechanisms of resistance development and diffusion in both hospitals and the community. the manuscript management system is completely online and includes a very quick and fair peerreview system, which is all easy to use. visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors. key: cord-289697-g24xib4l authors: macdowell, ana l.; bacharier, leonard b. title: infectious triggers of asthma date: 2005-03-01 journal: immunol allergy clin north am doi: 10.1016/j.iac.2004.09.011 sha: doc_id: 289697 cord_uid: g24xib4l there is abundant evidence that asthma is frequently exacerbated by infectious agents. several viruses have been implicated in the inception and exacerbation of asthma. recent attention has been directed at the role of infections with the atypical bacteria mycoplasma pneumoniae and chlamydia pneumoniae as agents capable of triggering asthma exacerbations and potentially as inciting agents for asthma. this article examines the evidence for interaction between specific infectious agents and exacerbations of asthma, including the immunopathology of infection-triggered asthma, and the current therapeutic options for management. the rise in the incidence of atopic disease, including asthma, over the past several decades has not been limited to a particular geographic area and has occurred in developed and developing countries. several factors influence the development and severity of asthma, including atopy, environmental exposures, genetic predisposition, gene-environment interactions, stress, obesity, diet, socioeconomic status, and infection. the ''hygiene hypothesis'' [1, 2] has focused attention on the role of infection in the development of allergic disease. this hypothesis suggests that infections in early life can have a protective effect on the development of asthma and atopy. other researchers have suggested, however, that infection may be a cause for the onset and persistence of asthma. in this ''hit and run hypothesis,'' a pathogen promotes dysregulation of the immune system, leading to prolonged inflammatory responses even after the pathogen has been cleared [3] . thus, the role of infectious agents in the development of asthma is complex: evidence implicates infections as causal and protective with respect to asthma development. in addition to the potential role of infection in the inception of asthma, infection has been implicated as the most common precipitant of asthma exacerbations. several clinical observations have indicated that most asthma episodes are precipitated by factors other than allergen exposure. many asthma episodes are preceded by upper respiratory tract symptoms and may last several days to weeks, in contrast with allergen-induced asthma exacerbations, where exposure often leads to a rapid onset of symptoms with a recovery time of approximately 24 hours [4, 5] . infections have been linked to asthma exacerbations since the 1950s, and over the past several decades there has been extensive investigation 0889 of infectious agents as they relate to asthma development and exacerbations. in this article, we examine infections as triggers of asthma, with a focus on asthma exacerbations. respiratory tract infections (rtis) are the most common cause of acute illness in adults and children, with upper respiratory infections (uris) constituting the majority of such illnesses [6] . adults typically experience two to four uris per year, and children may have up to 12 uris per year [7] . rtis are the major cause of visits to primary care physicians [8] and are associated with significant work and school absenteeism, with an estimated 150 million lost workdays annually. consequently, rtis have great economic impact, with an estimated cost of $40 billion annually in the united states [9] . numerous viruses produce uris (table 1) , and because the symptom patterns are common between many viruses, it is difficult to determine clinically the specific viral etiology of an acute illness ( table 2) . viral infections commonly trigger asthma exacerbations, having been noted in nearly half of asthma exacerbations in adults [10] and in an even greater percentage of exacerbations in children. this was demonstrated in a 13-month study investigating the role of viral infections in asthma exacerbations in 114 children 9 to 11 years of age with asthma [11] . peak expiratory flow (pef) rate was performed twice daily, and upper and lower respiratory tract symptoms were recorded daily. virologic samples were obtained within 48 hours of an increase in upper or lower respiratory symptoms, a fall in pef by more than 50 l/min from the child's baseline, or if the parent subjectively felt that the child was developing a cold. evidence of a viral infection was detected in 80% to 85% of episodes with respiratory tract symptoms, fall in pef, or both. the highest detection rate occurred during reported episodes of wheeze, cough, and upper respiratory tract symptoms, together with a decline in pef. in addition, the severity of a respiratory illness may influence the outcome of a uri because more severe viral infections seem more likely than mild infections to lead to exacerbations of asthma [5] . viral infection has been noted more often during severe exacerbations of asthma than during milder exacerbations [12] . the advent of more sensitive diagnostic tools to detect specific infectious pathogens, such as detection of microbial dna or rna using the polymerase chain reaction (pcr), has strengthened the evidence for viruses as a primary triggering factor in asthma exacerbations [13] . a recent study confirmed a significant increase in the weighted average viral identification in patients of all ages with asthma exacerbation in studies that used pcr when compared with the pre-pcr studies [14] . the same study suggests that viral recovery occurs more often in asthmatic patients who are having an acute exacerbation than in asymptomatic asthmatics or nonasthmatic individuals. almost 100% of children are infected with rsv by 2 y of age. it is the most common cause of bronchiolitis and pneumonia in infants. varied -includes cough, coryza, fever, irritability, anorexia, wheezing, pharyngitis, vomiting, or diarrhea unknown by 5 y of age nearly 100% individuals have been infected most commonly causes respiratory illnesses such as acute bronchitis, including pharyngitis, and occasionally otitis media, which may be bullous. ten percent of infected individuals develop pneumonia within a few days that may last for 3-4 wk. causes disease only in humans; it is highly transmissible by droplets. epidemics occur every 4-7 y because immunity is not long lasting. the long incubation period (ranging from 1-4 wk) along with long asymptomatic carriage (for weeks to months) facilitates familial spread, which may continue for months. responsible for a variety of respiratory diseases including pneumonia, acute bronchitis, and, less commonly, pharyngitis, laryngitis, otitis media, and sinusitis. many infected patients are asymptomatic or mild to moderately ill. a prolonged illness may be present with cough persisting for 2-6 wk, sometimes with a biphasic course. assumed transmission is person-toperson, via infected respiratory secretions. recurrent infection is common, especially in adults. in tropical, less-developed areas, infection seems to occur earlier in life. in the united states, 50% of adults have antibodies by 20 y of age, with initial infection peaking between 5 and 15 y of age. another important observation that links viral infection with asthma exacerbation is the seasonal pattern of distribution of viral infections and asthma exacerbations, especially severe cases requiring hospitalization. in a 2-year study comparing asthma exacerbations due to seasonal allergens, other environmental triggers, and viral infections, a strong relationship was found between the seasonal incidence of asthma and viral infection, although there was no correlation with pollen and spore counts [15] . similarly, viral infections were the major identifiable risk factor for autumnal asthma exacerbations [16] . in addition to viral infections, rtis with atypical organisms, such as mycoplasma pneumoniae and chlamydia pneumoniae, precipitate a significant proportion of acute episodes of wheezing, contribute to the severity and persistence of asthma, and may serve as the initial insult that leads to development of asthma [17] [18] [19] . human rhinovirus (rv) causes nearly half of all upper respiratory illnesses. although rv infection was initially believed to be limited to the upper airways [20] , lower airway epithelial rv infection has been demonstrated [21] . although infection of the lower respiratory tract may occur, the mechanisms through which viral infections, including rv, provoke asthma are unclear but may include direct extension of upper rtis to the lower respiratory tract. the mechanism may be indirect and involve effects on airway responsiveness independent of the direct epithelial damage and inflammation associated with lower rtis (lrtis). rv infection can enhance the immediate and late-phase responses to allergen [22] , potentially augmenting the allergic inflammation within the airway and precipitating asthma exacerbations. rv infection can lead to profound exacerbation of asthma and is responsible for the majority of hospitalizations for childhood asthma, although less so in adults [20] . rv infections are associated with declines in lung function in asthmatics compared with normal subjects within 2 days after development of a rv infection [23] . rv infection augments airways hyper-responsiveness 4 days after experimental rv infection, an effect that was more pronounced in those with a severe cold [24] . the rise in airways hyper-responsiveness was accompanied by an increase in nasal interleukin (il)-8 in the rv-infected group at days 2 and 9; the increase in nasal il-8 at day 2 correlated significantly with the change in airway responsiveness at day 4. coronavirus is the second most common virus associated with asthma episodes in children and adults. infections due to coronavirus may be associated with less severe lower respiratory tract symptoms than infections with other viruses. this is suggested by the finding that coronavirus-associated asthma episodes in asthmatic school-age children were associated with smaller median declines in pef (56 l/min) compared with episodes triggered by other viruses (85.5 l/min) [11] . in a study of elderly adults, coronavirus was associated with lower respiratory illness in more than 40% of patients, and one quarter of patients consulted a medical practitioner and received antibiotics. more impressive was the observation that coronavirus infection produced a greater disease burden value than influenza or respiratory syncytial virus [25] . influenza virus triggers asthma exacerbations in all age groups [11, 26] . in addition, asthmatic individuals seem to be more susceptible to death associated with influenza infections, as observed in the asian pandemic in 1957 [27] . the time course of influenza-induced asthma exacerbations was examined retrospectively in 20 asthmatic children 8 to 12 years of age with acute respiratory symptoms [28] . fifteen of 20 patients had decreases in fev 1 n20% from baseline during the acute stage, beginning from onset of symptoms in all but one subject, whose fev 1 decreased during the incubation period. fev 1 decreased maximally on the second day of illness by an average of 30%. improvement began on the third day, and fev 1 returned to within 10% of normal between the seventh and tenth day. the rate of adenoviral infection declines with age until 9 years and then increases. the exception to this pattern is infection with serotype 7, whose infection rate increases with age [29] . infection is frequently associated with wheezing, as demonstrated in a retrospective chart review study [30] where wheezing was noted in 58.3% of nonasthmatic children under 2 years of age admitted to an intensive care unit with adenoviral acute lrti. in this study, the mortality rate was 16.7%, generally in the setting of infection with adenoviral serotype 7. adenoviral infection has been demonstrated during acute asthma episodes, but the frequency of adenoviral infection is substantially lower that the frequency for rhinovirus and coronavirus [31] . latent adenoviral infection may have a role in the genesis of asthma. furthermore, adenoviral shedding may be prolonged, lasting up to 906 days. when nasopharyngeal swabs from 50 asymptomatic asthmatic children and 20 healthy control subjects were examined by pcr, adenovirus dna was found in 78.4% of asthmatic children, compared with only 5% of healthy control subjects [32] . adenovirus has been recovered from bronchoalveolar lavage (bal) in children with asthma 12 months or more after acute infection [33] . in this study, bal was performed in 34 children (mean age of 5 years) with unfavorable responses to standard corticosteroid and bronchodilator therapy. adenoviral infectious triggers of asthma antigens were detected in bal fluid (balf) from 94% of subjects. repeat studies within 1 year showed that six of eight subjects were positive for adenovirus on two occasions and that three were positive when sampled three times. cultures of the balf were positive for adenovirus in all cultures performed, indicating that the virus was capable of replication. similar studies performed in control patients without persistent asthma failed to detect evidence of adenovirus. respiratory syncytial virus (rsv) infects almost 100% of children by 2 years of age and is the most common cause of bronchiolitis and pneumonia in infants [34] . in addition to causing acute lrti, rsv serves as a trigger for exacerbations of asthma and other chronic lung diseases. infants who experience severe rsv bronchiolitis seem to have increased frequencies of wheeze and asthma later in life. a comparison of several retrospective studies of children admitted for bronchiolitis found that the postbronchiolitis group had a significantly higher frequency of bronchial obstructive symptoms 2 to 10 years later and, when pulmonary function studies were performed, diminished fev 1 or increased bronchial reactivity compared with healthy control subjects [35] . these findings were confirmed in a prospective study when children hospitalized with confirmed rsv bronchiolitis were evaluated at 7.5 years of age and compared with age-and gender-matched control subjects [36] . by 7.5 years of age, the cumulative prevalence of asthma was 30% in the rsv group versus 3% in the control group, and current asthma was present in 23% of the rsv group versus 2% of the control group. however, the duration of the effect of rsv infection on asthma-related symptoms appears to be limited. in a prospective study of 1246 children enrolled at birth, 207 developed an rsv ltri not requiring hospitalization during the first 3 years of life [37] . when compared with a control group of children with no lrti documented during the first 3 years of life, the group with mild rsv lrti had a substantially increased risk of frequent wheeze at 6 years of age (odds ratio [or] 4.3), and the risk for frequent wheeze remained significantly increased at 11 years of age (or 2.4), at which time prebronchodilator fev 1 , but not postbronchodilator fev 1 , was significantly lower in the rsv group. by age 13 years, there were no significant between-group differences in terms of increased risk for frequent or infrequent wheezing. these studies demonstrate that rsv bronchiolitis is a significant independent risk factor for subsequent frequent wheezing, although this effect seems to decrease with age and may be dependent upon the severity of the rsv infection. similar to adenoviral infection, the persistence of rsv may underlie in part the sequelae of severe rsv disease. infection may lead to alteration in the patterns of local interferon, chemokine, and cytokine production [38] , potentially leading to chronic inflammation [39] . furthermore, the age at first viral infection may direct the pattern of disease later in life by generating a th2-biased memory response to rsv, which may direct responses to other antigens in the lung toward an allergic phenotype. this is suggested by a study in which mice infected with rsv at different ages (1, 7, 28, or 56 days) demonstrated stronger th2 responses in the group primed at the youngest age when reinfected with rsv at 12 weeks of age [40] . the parainfluenza viruses (piv) cause a spectrum of respiratory illness similar to that caused by rsv but result in fewer hospitalizations [41, 42] . most illnesses are limited to the upper respiratory tract [41] , although approximately 15% involve the lower respiratory tract, and 2.8 of every 1000 children with such infections required hospitalization [42] . although less common than rv or coronavirus infection, piv was detected in 14% of episodes of increased symptoms or decreased pef in school-aged children [11] . more frequent and severe wheezing has been correlated with elevated levels of ige antibody to rsv and piv in nasal secretions of children with bronchiolitis due to rsv and piv [43] . human metapneumovirus (hmpv) was identified in 2001 in respiratory samples from children with respiratory disease in the netherlands [44] . the clinical symptoms experienced by infected individuals are diverse and may consist of upper or lower respiratory tract symptoms ranging from otitis media to bronchiolitis, croup, pneumonia, and possibly exacerbations of asthma [45] . hmpv is responsible worldwide for community-acquired acute rtis affecting children and other age groups, with a mean age of illness of 11.6 months and a male predominance (male/female ratio 1.8:1). the broad epidemic seasonality and the evidence of genetic variability suggest that there may be more than one serotype of hmpv [44] . wheezing is part of the clinical symptomatology associated with hmpv infection. more than half of otherwise healthy children presenting with acute respiratory illness and evidence of hmpv infection experienced wheezing in one study [45] . in series of 19 children with evidence of hmpv infection, bronchiolitis was the most common diagnosis, and 50% of patients had wheezing [46] . both of these studies evaluated specimens collected from previously healthy children during an acute respiratory illness during which no other pathogen was identified and detected evidence of hmpv in 6.4% [46] and 20% [45] of the previously negative samples. although hmpv infection is often accompanied by wheezing, there have been conflicting reports linking hmpv infections and asthma exacerbations [47, 48] . nevertheless, bronchiolitis is a common cause for hospitalization, and given the increasing hospitalization rates over the past two decades [49] , it is possible that hmpv may be responsible for a portion of hospitalizations in children with infectious triggers of asthma 53 bronchiolitis and wheezing unrelated to rsv infection [46] . furthermore, coinfection with rsv and hmpv may augment the severity of bronchiolitis [47] . m pneumoniae and c pneumoniae initial evidence suggested that infection with m pneumoniae and c pneumoniae was associated with asthma chronicity. several case reports suggest associations between infections with atypical organisms with decreased expiratory flow rates and increased airway hyper-responsiveness in nonasthmatic individuals [50] and the onset of asthma symptoms in previously healthy nonasthmatic adults [51, 52] . most of these individuals present with complaints of malaise, shortness of breath of gradual onset, and wheezing, which typically resolve after treatment with macrolide antibiotics or oral corticosteroids [51] . symptoms may progress and persist, as illustrated by an adult male with fever, severe cough, shortness of breath, consolidation on chest radiograph, and evidence of m pneumoniae infection based on a rise in serum antibody titers who subsequently developed wheezing episodes with reversible airway obstruction and airway reactivity to methacholine [52] . infections with these organisms can persist for months, and animal studies show that m pneumoniae can be detected by pcr for up to 200 days after infection, even though the animals become antibody and culture negative by 70 days [53] . these reports suggest that m pneumoniae may serve as a cause of acute wheezing and a triggering factor for the onset of asthma. the most comprehensive evaluation of the role of m pneumoniae and c pneumoniae infections in patients with chronic asthma evaluated 55 adult patients with chronic asthma and 11 control subjects by using pcr, culture, and serology to detect m pneumoniae species, c pneumoniae species, and viruses from the nasopharynx, lung, and blood [54] . fifty-six percent of the asthmatic patients had positive pcr studies for m pneumoniae (n = 25) or c pneumoniae (n = 7), which were mainly found in balf or biopsy samples. only 1 of 11 control subjects had a positive pcr finding for m pneumoniae. cultures for these organisms were negative in all patients. a distinguishing feature between pcr-positive and pcr-negative patients was a significantly greater number of tissue mast cells in the group of patients who were pcr positive. of additional significance is the link of atypical infectious organisms with asthma exacerbations. in a serologically based prospective study, 100 adult patients hospitalized with exacerbations of asthma were compared with hospitalized surgical patients with no history of lung disease at any time or uri in the month before admission [55] . in this series, m pneumoniae was identified more often than any other pathogen in the asthmatic group (18 m pneumoniae, eight c pneumoniae, 11 influenza a, five influenza b, three piv-1, two piv-2, one piv-3, six adenovirus, two rsv, three s. pneumoniae, and five legionella spp.) and in the control group (three m pneumoniae). however, only 8 of the 18 patients had m pneumoniae identified as the sole infectious agent, making it difficult to ascertain the culpability of m pneumoniae as the cause of hospitalization. a study of 71 children with acute wheezing and 80 age-matched healthy children detected m pneumoniae in 22.5% and c pneumoniae in 15.5% of children with wheezing compared with 7.5% and 2.5%, respectively, in healthy control subjects [56] . when the children who were infected with either organism were treated with clarithromycin, improvement in the course of the disease was observed, further supporting the role of these atypical organisms in the exacerbation of asthma. these findings were recently confirmed in a french series, where m pneumoniae infection was found in 20% and c pneumoniae infection was found in 3.4% of children during an acute asthma exacerbation [19] . acute m pneumoniae infection was confirmed in 50% and c pneumoniae in 8.3% of patients experiencing their first wheezing episode. further studies are needed to confirm the association between infection and asthma exacerbation, to determine the prevalence of such infections in patients with acute exacerbations of asthma, and to examine if infection with these organisms modifies the severity of the exacerbation or the response to therapy. viral-induced wheeze (viw) is characterized by brief episodes of lower respiratory symptoms and decreased pulmonary function in the setting of an acute viral uri, interspersed with longer asymptomatic periods with normal pulmonary function [11, 57] . this differs from classic childhood asthma, which is characterized by chronic symptoms, with atopy being a major risk factor [58] . classic asthma and viw were considered two different entities until 1969, when a report suggested that the two groups have similar characteristics [59] and benefited similarly from the same prophylactic treatment [60] . in the 1990s, there was a division of the wheezing phenotypes, especially in children [58] . patients with viw alone seem to outgrow the symptoms by age 6; however, in some patients, the pattern of viw may continue into adulthood with less severe symptoms, negative methacholine challenges, and pulmonary functions that remain normal [61] . the inability to reliably differentiate between viw and asthma, especially in young children, complicates the evaluation of the influence of viral infections on exacerbations of wheezing. furthermore, this heterogeneity in wheezing phenotypes has implications in terms of the efficacy of therapies used to treat such episodes. viruses typically enter the body through contact with mucosal surfaces. the cell-specific distribution of viral receptors determines the viral tropism. once the viral particles are internalized, nucleic acids are released, and transcription and production of viral proteins starts. the viral genome is replicated, and virions are one of the earliest responses to viral infection is the production of ifns by different cell types; ifn-a is produced by leukocytes, ifn-b is produced by fibroblasts, and inf-g is produced by th1 cells and natural killer (nk) cells. ifns induce transcription of many genes, including two with direct antiviral activity, and lead to increased expression of mhc class i and ii genes. interferons are potent activators of antiviral effector cells such as nk cells, cd8 t lymphocytes, and macrophages. although the inflammatory process generated by virus infection is generally viewed as a th1 pattern with a predominance of interferons, especially inf-g, in atopy there is a predominance of the th2 cytokine profile. however, viral infections promote increased cytokine-mediated inflammation through direct induction of specific cytokines produced by different viral agents [62] . the ability of certain pathogens to stimulate the production of th2 cytokines [63] may explain why certain pathogens are more strongly associated with asthma exacerbation than others. viruses have been implicated in the inception of asthma because viral infections with a propensity for lower airway involvement during infancy have been associated with chronic lower respiratory tract symptoms and asthma [64] . this seems to be particularly relevant to rsv bronchiolitis, which has been demonstrated to be a significant independent risk factor for subsequent frequent wheezing [37] . the sequelae of severe rsv disease could be explained in part by viral persistence [39] . this has been supported by a recent study demonstrating the persistence of viral genomic and messenger rna in lung homogenates of balb/c mice up to 100 days post rsv infection, whereas virus could no longer be detected in balf after day 14 post-infection [65] . another possible mechanism by which a virus could promote asthma is by generating changes in patterns of pro-inflammatory cytokine production, which could facilitate virus persistence, as demonstrated with rsv [38] . viral infection may exert direct effects on airway cells. an increase in the production of il-10 by nonspecifically stimulated peripheral blood mononuclear cells during acute and convalescent phases of rsv infection requiring hospitalization has been demonstrated [66] . in animal studies, it was suggested that il-10 may have a direct effect in airway smooth muscle and in the regulation of airway tone [67] . although there is evidence supporting the role of viral infections in the development of asthma, further investigation is necessary to confirm this hypothesis because the mechanisms that could allow persistency or latency of viral infection are poorly understood. it has been hypothesized that asthmatic individuals have increased susceptibility to viral infections. some researchers have found an increased incidence of viral infections in asthmatic children when compared with nonasthmatics [14, 26] , a pattern that could be explained by the increased expression of icam-1, the receptor for rv, in asthmatics subjects [68] . however, this finding was not confirmed in a study that followed cohabitating couples consisting of an atopic asthmatic and a healthy nonatopic, nonasthmatic individual [23] . in this study, subjects completed daily diary cards of upper and lower respiratory tract symptoms and measured pef twice daily. nasal aspirates were taken and examined for rhinovirus every 2 weeks. rhinovirus was detected in 10.1% of samples from the asthmatics and 8.5% of samples from the nonasthmatic participants. after adjustment for confounding factors, asthma did not significantly increase the risk of infection with rhinovirus in asthmatic individuals (or 1.15). the effect of atopic status on the rate of viral infection is unclear; evidence exists suggesting no difference between the rate of viral infection between atopic and nonatopic individuals [69] or an even lower rate of viral infections among atopic individuals [15, 70] , although these studies did not have adequate statistical power to confirm this trend. there is an increased risk of acute wheezing when atopy is combined with viral infection when compared with atopy or virus infection alone [70] , and infants with a family history of atopy seem more likely to develop bronchiolitis with a higher rate of hospitalization [71] . even if asthmatics do not experience more frequent infections than nonasthmatics, it is possible that asthmatics have a higher incidence of symptoms when experiencing viral infections. during rhinoviral infection, there is a greater incidence of symptoms in asthmatics compared with nonasthmatics [72] . this is further suggested by a report that asthmatics experienced seroconversion to influenza a virus at the time of asthma exacerbation even in the absence of signs of respiratory infection [5] . although there is evidence supporting the role of infection in the genesis of asthma and allergy, a protective effect of infections in the development of atopy has also been postulated. an inverse relationship between infection and allergy was first noted when a study comparing white families with native americans reported that ige levels and the prevalences of asthma and eczema were higher in the white population, whereas helminthic, viral, and bacterial infections were more prevalent in the native americans [1] . it was observed that increased family size, often associated with more frequent infections in early childhood, had an inverse relationship with the prevalence of allergic rhinitis [2] and asthma [73] . this was further supported by studies reporting an inverse relationship between the age of day care entry and the diagnosis of asthma [74, 75] . one potential explanation for this pattern is that at birth there is a predominant th2 response, and, as exposure to infections occurs, there is a gradual shift toward a th1dominant response. however, if the skewing of the immune response to th1, which regulates response to viral infection, is impaired, a th2 response would infectious triggers of asthma predominate, favoring the development of allergy. ex vivo studies have shown that asthmatics exposed to viral infections lack the capacity to mount a strong th1 response [76, 77] . there is no clinically effective treatment for the common cold. as the mechanisms of viral-induced wheezing and asthma are elucidated, new forms of treatment may emerge. the involvement of many inflammatory pathways suggests that antiviral and anti-inflammatory therapies have potential roles for intervention after onset of symptoms; however, a combination of both therapeutic approaches may have the greatest impact. prophylaxis for the acquisition of viral infections, in the form of vaccination or pharmacologic therapy, offers the best hope of disease control. the major obstacle for treatment is the wide variety of organisms associated with uris, including viral and bacterial agents ( table 2 ). in addition, accurate and timely diagnosis is essential for the appropriate targeting of specific antiinfective therapies. the rapid rate of mutation of viruses leads to the emergence of resistant strains. in addition, there are difficulties with the delivery, expense, and efficacy of drugs [78] . treatment for viral rtis remains symptomatic, although future approaches will likely be directed toward reducing the inflammatory response elicited by the virus. vaccination remains the mainstay of prophylaxis against infections. however, with the exception of influenza, vaccine development for respiratory viruses has been slow and disappointing. influenza vaccine contains three strains (two a and one b) of inactivated virus, one or two of which are modified yearly based upon predictions of the upcoming viral strains. they are produced in embryonated hen eggs and are highly immunogenic, conferring protection in 70% to 80% of the vaccine recipients with minimal adverse effects. whole-cell influenza vaccine is no longer available, and the current vaccines consist of subvirion (prepared by disrupting the lipid membrane) or purified surface antigen. recently, a liveattenuated, cold-adapted, trivalent, intranasal influenza vaccine (flumist) has been introduced, but it is contraindicated in asthmatics [79] . a long-standing concern that influenza vaccination may trigger exacerbations of asthma was addressed in a multicenter, randomized, double-blind, placebocontrolled, crossover trial in 2032 patients with asthma (age range 3-64 years). this study confirmed the safety of the influenza vaccine in asthmatics by demonstrating that the frequency of exacerbations of asthma was similar in the 2 weeks after vaccination with the active influenza vaccine or placebo (28.8% and 27.7%, respectively) [80] . although yearly influenza vaccination is recommended as a routine element of asthma management [81] , a recent study generated concern about the usefulness of influenza vaccine in preventing influenza-related asthma exacerbations. this randomized, double-blind, placebo-controlled trial showed that the number, se-verity, and duration of influenza-related asthma exacerbation was similar between the group receiving influenza vaccination and the group receiving placebo over the course of one influenza virus season [82] . vaccinated children tended to have shorter exacerbations (by approximately 3 days) than nonvaccinated children. antiviral therapy targets the source of infection directly, decreasing the number of infectious agents and therefore reducing inflammatory process. the only licensed antiviral therapies are directed against influenza a (amantadine and rimantadine), influenza a and b (zanamivir and oseltamivir), and rsv (ribavirin). the neuraminidase inhibitors, zanamivir and oseltamivir, have an advantage over adamantanes, amantadine, and rimantadine because they have a broader spectrum and are effective against the a and b strains of influenza virus. the inhibition of neuraminidase, whose active site consists of 11 amino acids conserved in all naturally occurring influenza virus [83] , prevent cleavage of sialic acid from newly acquired membrane, leaving emerging virus inactive and thereby decreasing infectivity [84] . both neuraminidase inhibitors improve respiratory outcomes in patients with asthma and acute influenza infections [78] and have the added benefit of being effective in the prophylaxis against influenza infections [85] . although it is generally well tolerated, there are case reports of bronchospasm after treatment with inhaled zanamivir [86] ; however, it is difficult to separate these symptoms from the effects of the influenza infection. the disadvantage of current antiviral therapy is the specificity for influenza and the need for initiation of treatment within 48 hours of onset of infection. the toxicity profile of ribavirin, approved for use in severe rsv infections, limits its clinical use except in settings of severe illness in immunocompromised hosts. antibiotic use is appropriate if there is evidence of bacterial infection contributing to asthma exacerbations, although pyogenic lung infections rarely exacerbate asthma and are rarely associated with wheezing. although some macrolide antibiotics have been reported to have antiviral effects in vitro against rhinoviruses [87] , these effects have not been confirmed in vivo, and a recent cochrane review does not support the use of antibiotics for the treatment of the common cold [88] . the anti-inflammatory effects of macrolide antibiotics are not limited to their ability to interfere with corticosteroid metabolism [89] , as evidenced by inhibition of the neutrophil oxidative burst [90] , reduction of cytokine formation [91] , and reduction of icam-1 production [92] . asthmatic patients infected with m pneumoniae or c pneumoniae may benefit from prolonged treatment with clarithromycin, as evidenced by significant improvement in fev 1 [18, 93] . furthermore, in a double-blind, randomized, crossover study, 17 patients with stable mild or moderate asthma not evaluated for m pneumoniae or c pneumoniae received 200 mg of clarithromycin or placebo twice daily for 8 weeks. methacholine responsiveness improved in all the patients after 8 weeks of clarithromycin treatment [94] . improvement in airway hyperresponsiveness after 8 weeks of clarithromycin treatment was confirmed in a group of patients with asthma receiving concomitant therapy with inhaled corticosteroids who were not selected on the basis of infection with m pneumoniae or c pneumoniae [95] . it remains unclear as to the mechanism by which macro-infectious triggers of asthma lide antibiotics improve airway hyper-responsiveness in patients with asthma, but possibilities may include treatment of occult or chronic infection, interference with steroid metabolism, or the anti-inflammatory properties of this class of antimicrobials. although there are international guidelines for the management of asthma [81, 96] , there is a relative paucity of evidence regarding therapeutic strategies specifically for viw in asthmatics or healthy subjects. because most acute exacerbations of asthma are induced by viral infections and because many forms of asthma therapy, especially inhaled corticosteroids, reduce the frequency and severity of exacerbations, one would presume that the current treatment for chronic asthma would be efficacious in preventing viw. however, the varying phenotypes of wheezing, especially in childhood, seem to respond differently to such management approaches. this is particularly true for rsv-associated wheezing, which does not consistently respond to medications often used to treat asthma exacerbations, including bronchodilators and corticosteroids [97] . thus, despite the efficacy of inhaled corticosteroids in the control of asthma and reduction of exacerbations, patients continue to experience exacerbations, particularly in the setting of viral rtis. several treatment approaches have been investigated in an attempt to reduce the morbidity associated with wheezing associated with rtis. brunette et al [98] examined the effect of a short course of oral corticosteroid administered in an unblinded manner at onset of uri symptoms in a group of children with histories of recurrent wheezing in the setting of viral infections. over a 1-year period, the group receiving oral corticosteroids at the early signs of rtis experienced reductions in the frequencies of wheezing, emergency room visits, and hospitalizations. however, a recent double-blind, placebo-controlled trial evaluating the use of parent-initiated oral corticosteroids at the early signs of an episode of presumed viral-induced wheezing did not detect a difference between oral corticosteroid therapy and placebo in terms of symptom scores and rate of hospitalization [99] . thus, the role for the use of oral corticosteroids at the early signs of illness in children with recurrent viral wheezing is unclear, and additional investigation is required to determine the efficacy of this approach in the management and attenuation of wheezing episodes. the repeated use of systemic corticosteroids for such episodes remains a clinical concern. given the efficacy of inhaled corticosteroids (ics) in the daily management of asthma and their favorable safety profile when compared with systemic corticosteroids, the use of ics in the management of viw has been explored. although ics are effective in the management of persistent asthma, current evidence suggests a lack of efficacy in the regular use of ics in patients with mild viw [100, 101] . a recent meta-analysis concluded that the use of ics episodically for viral-triggered wheezing in children not using them as maintenance may decrease the rate of oral corticosteroid requirement [101] . in patients receiving daily ics therapy, the common clinical practice of doubling the dose of ics at the onset of an asthma exacerbation has been shown to be ineffective in preventing symptom progression [102] . however, a recent study in adults demonstrated the valuable effects of quadrupling the ics dose with acute asthma exacerbations [103] . these data suggest that corticosteroids, taken orally or inhaled, may be used as treatment and preventive therapy for asthma exacerbations in the setting of rtis. the cysteinyl leukotrienes (cyslts) have been identified as important mediators in the complex pathophysiology of asthma. cyslts are detectable in the blood, urine, nasal secretions, sputum, and balf of patients with chronic asthma. elevated cyslts have been detected in respiratory secretion of children with viral induced wheezing [104] . similar to elevated levels in asthmatics, 20 infants with prolonged or persistent wheeze (mean 14.9 months) and a history of viral illness at wheeze onset had significant elevations of leukotrienes in bal despite the fact that 12 of 20 infants were receiving daily ics therapy ( 450 mg/d) [105] . these findings suggest that, similar to asthma pathophysiology, cyslts play a role in the pathophysiology of viral-induced wheeze. additionally, based on the above study, the cyslts are not fully suppressed by the preferred standard antiinflammatory therapy, inhaled corticosteroids. thus, antagonism of the effects of cyslt using the leukotriene receptor antagonists may provide clinical benefit to patients with viw. the relative efficacies of these intervention strategies aimed at reduction of wheezing and asthma in the setting of rtis depend upon the wheezing phenotype and probably the timing of the initiation of therapy. investigation of other therapeutic approaches to viw is ongoing and may provide insight as to the optimal treatment approach for this challenging condition. infections have been implicated in asthma exacerbations and in the inception of asthma. several studies support the concept that viruses and atypical infectious agents may induce asthma exacerbations and contribute to the chronicity of asthma. the further elucidation of the mechanisms that underlie the interaction between infectious agents and asthma will lead to improvements in treatment and prevention of such exacerbations. studies 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efficacy of a short course of parent-initiated oral prednisolone for viral wheeze in children aged 1-5 years: randomised controlled trial effect of continuous treatment with topical corticosteroid on episodic viral wheeze in preschool children inhaled steroid for episodic viral wheeze of childhood treatment of acute asthmatic exacerbations with an increased dose of inhaled steroid low-dose budesonide with the addition of an increased dose during exacerbations is effective in long-term asthma control increased production of ifn-gamma and cysteinyl leukotrienes in virus-induced wheezing persistent wheezing in very young children is associated with lower respiratory inflammation key: cord-279483-gwikyux2 authors: wong, joshua guoxian; aung, aung-hein; lian, weixiang; lye, david chien; ooi, chee-kheong; chow, angela title: risk prediction models to guide antibiotic prescribing: a study on adult patients with uncomplicated upper respiratory tract infections in an emergency department date: 2020-11-02 journal: antimicrob resist infect control doi: 10.1186/s13756-020-00825-3 sha: doc_id: 279483 cord_uid: gwikyux2 background: appropriate antibiotic prescribing is key to combating antimicrobial resistance. upper respiratory tract infections (urtis) are common reasons for emergency department (ed) visits and antibiotic use. differentiating between bacterial and viral infections is not straightforward. we aim to provide an evidence-based clinical decision support tool for antibiotic prescribing using prediction models developed from local data. methods: seven hundred-fifteen patients with uncomplicated urti were recruited and analysed from singapore’s busiest ed, tan tock seng hospital, from june 2016 to november 2018. confirmatory tests were performed using the multiplex polymerase chain reaction (pcr) test for respiratory viruses and point-of-care test for c-reactive protein. demographic, clinical and laboratory data were extracted from the hospital electronic medical records. seventy percent of the data was used for training and the remaining 30% was used for validation. decision trees, lasso and logistic regression models were built to predict when antibiotics were not needed. results: the median age of the cohort was 36 years old, with 61.2% being male. temperature and pulse rate were significant factors in all 3 models. the area under the receiver operating curve (auc) on the validation set for the models were similar. (lasso: 0.70 [95% ci: 0.62–0.77], logistic regression: 0.72 [95% ci: 0.65–0.79], decision tree: 0.67 [95% ci: 0.59–0.74]). combining the results from all models, 58.3% of study participants would not need antibiotics. conclusion: the models can be easily deployed as a decision support tool to guide antibiotic prescribing in busy eds. supplementary information: the online version contains supplementary material available at 10.1186/s13756-020-00825-3. upper respiratory tract infection (urti) is one of the most cited reasons for use of antibiotics [1] . in the majority of urtis, the routine use of antibiotics is not recommended [1] [2] [3] [4] [5] . in the united states (u.s.), it was estimated that antibiotics have been prescribed for over 60% of uncomplicated urtis in adults and increasingly so for broad-spectrum antibiotics [6] [7] [8] [9] . between 2001 and 2010, 126 million (12.2%) emergency department (ed) visits in the u.s. were for acute respiratory tract infections, with almost half (47.9%) of patients with infections being administered antibiotics inappropriately [10] . from 2009 to 2010, adults had the highest rate of inappropriate antibiotic use for acute respiratory tract infections (urtis, influenza, and viral pneumonia), with 500 antibiotic prescriptions per 1000 ed visits for adults aged 20-64 years and 666 per 1000 visits for those aged > = 65 years [10] . in singapore, while primary care clinics are highly accessible in the community, there are individuals who preferred to seek care at the ed for urti, accounting for a substantial proportion of ed attendances [11] . urti accounted for 6-10% of ed visits by non-frequent attenders (1-4 ed visits in one year) and up to 25% of ed visits by frequent attenders (≥5 ed visits in one year) [12] . a previous study at an adult general hospital has reported that 24% of adult patients attending at ed for urti were inappropriately prescribed antibiotics, with the penicillin class of antibiotics being the most commonly prescribed [13] . studies have shown a strong link between antibiotic prescribing and antimicrobial resistance [6, 14, 15] . in addition, a population-wide study on us pharmacy records showed that antibiotic use and resistance appears to be closely linked to broadly distributed low-intensity prescribing [16] . as a consequence, antimicrobial resistance has risen to dangerously high levels globally. a global study estimated that escherichia.coli and klebsiella pneumoniae resistant to third-generation cephalosporin caused 6.4 million bloodstream infections and 50.1 million serious infections in 2014. carbapenem-resistant strains were estimated to cause 0.5 million bloodstream infections and 3.1 million serious infections [17] . antimicrobial resistance is associated with higher medical cost, prolonged hospital stays, increased mortality and economic burden [18, 19] . hence, there is an urgent need to ensure the prudent use of antibiotics for common illnesses predominantly of viral etiology such as urtis. in singapore, considerable efforts have been made to address antibiotic resistance [20] . although computerized decision support systems have been developed to guide antibiotic prescribing, they are largely based on guidelines drawn by expert consensus and not on actual data derived from local patients [21] . furthermore, most studies on antibiotic prescribing focus on understanding behaviors and perceptions or finding associative factors for antibiotic prescribing decisions [22] [23] [24] [25] . to date, prediction models to guide antibiotic prescribing has been confined largely to pediatric populations [26] [27] [28] . differentiating bacterial and viral infections is not straightforward in adult urtis. in uncertainty avoidance, physicians tend to over-prescribe antibiotics. in this study, we aim to develop prediction models based on local clinical and laboratory data to guide antibiotic prescribing for adult patients with uncomplicated urti with the ultimate goal of deploying them as an evidencebased clinical decision support tool for routine practice. seven hundred-fifteen patients were recruited from the ed at tan tock seng hospital (ttsh), the second largest adult hospital in singapore between june 2016 and november 2018. eligible patients were 21 years and above attending at ttsh ed for the first time with a primary diagnosis of uncomplicated urti (icd10-am j00-j06) within 30 days who provided informed consent. ttsh ed is the busiest ed in the country, attending to an average of 450 patients daily. at discharge from the emergency department, the patients were invited to participate in the study and consent was obtained. patients who were subsequently admitted were excluded from the study. a nasopharyngeal swab was taken to determine the presence of respiratory viruses using multiplex pcr (seeplex® rv15 ace detection). the panel detects 15 major respiratory viruses including adenovirus, bocavirus 1/2/3/4, coronavirus 229e/nl63 and oc43, enterovirus, influenza a and b, metapneumovirus, parainfluenza 1, 2, 3 and 4, respiratory syncytial virus a and b, and rhinovirus. we chose not to include the bacterial respiratory pcr panel in the study, as commensal bacteria are common in the upper respiratory tract and detection on pcr does not necessarily indicate a bacterial infection. a lower respiratory tract sample (such as sputum) was also not practicable for every participant. instead, we performed a point-of-care c-reactive protein (crp) test on a drop of capillary blood obtained from a finger prick (quikread go® crp). crp is widely used in clinical settings as a supportive test to diagnose bacterial infection [29] . our main outcome of interest was to identify patients for whom antibiotics were clearly not recommended (1 = nabx) from those for whom the physician should review the need for antibiotics (0 = rabx). we defined nabx as patients with a respiratory virus detected via pcr and crp < 20 mg/l or patients who did not have a respiratory virus detected via pcr and crp ≤ 5 mg/l [30] . patients who did not fall into these 2 categories were assigned to the rabx group. demographic, clinical and laboratory data documented as part of the patients' routine care were extracted from the hospital electronic medical records. these include age, gender, ethnicity, visit date, pre-existing comorbidities, respiratory symptoms, full blood count, kidney/liver panels, and biochemistry tests. according to comorbid status of the participants, charlson's comorbidity index was calculated [31] . additionally, epidemiologic data on smoking, influenza vaccination, travel history, and prior medical consultation and antibiotic consumption were obtained from an interviewer-administered questionnaire. descriptive statistics were performed and differences between the nabx and rabx groups compared using mann-whitney u-test for continuous variables and chisquared test for categorical variables. where appropriate, fisher's exact tests were used to account for small cell sizes. variables with more than 10% of data missing were excluded from the analysis. categorical variables with data missing were recoded as 0 under the assumption that presence of any clinical covariates would have been recorded. continuous variables were imputed according to their group medians. with ease of use in mind, we decided to perform predictive modeling using 3 methods that could subsequently be easily deployed for implementation: logistic regression, lasso regression and classification and regression trees (cart). the models were derived using 70% of the participants as training set. the optimal cutoffs for each model were decided by taking the predicted probability that achieved the highest sensitivity with specificity of at least 0.4. the final model performance was validated by calculating the area under the receiver operating characteristic curve (auc), sensitivity, specificity, positive predictive value and negative predicative values on the remaining 30% data. univariate analysis was performed on all 50 candidate variables. demographic factors, clinically relevant variables and significant variables from univariate models were fitted into the final multivariable model via stepwise elimination using a cutoff of p < 0.1. in stepwise regression, it is often difficult to tell the effect after removal of each variable. model selection may also be difficult in datasets with a huge number of variables. lasso regression addresses this by shrinking the coefficients of features that are less relevant or exhibit collinearity to zero. this reduces the problem of overfitting of prediction model and the variance without substantial increase in bias. we performed this by selecting a minimum optimal shrinking parameter of λ = 0.03971531 through a 10-fold cross validation of the training dataset, giving a set of coefficients governed by eq. 1. cart is a popular tool in supervised learning for classification as they are distribution-free and robust to outliers. unlike generalized linear models, classification trees make an excellent tool for overcoming problems due to multicollinearity and skewed covariates. it uses the gini index to iteratively split branches based on purity. this feature is an added benefit as important interactions can be easily detected. it also has the ability to identify patient subgroups that are more predictive than others. in our analysis, we created a maximum tree depth of 5 and a minimum of 10 subjects in a node before a split is attempted to prevent overfitting. the choice of the final tree size was decided by finding the number of splits that produce the smallest crossvalidation error. analyses were performed using r4.0.2 and stata 13.0 at a 5% significance level. lasso and cart models were developed using the glmnet, rpart and rattle packages in r [32] [33] [34] . the study participants were young, with a median age of 36 years (iqr: 28-51 years) and a slight preponderance of males (61.3%). (table 1 ) two-thirds (66.4%) had no pre-existing comorbidities and one-third (36.8%) had received influenza vaccination in the prior 12 months. almost two-thirds (60%) of the patients presented with fever. while 50.3% of the patients had nasal problems like running and blocked nose, 45.6% of them had a sore throat. almost half (47.8%) of the patients had a respiratory virus detected. influenza (20.6%) and rhinovirus (14.4%) were common respiratory viruses detected. influenza circulated year-round, with bimodal peaks observed in november and may-june, with rhinovirus dominating in the inter-influenza periods. (data not shown) the median crp level was 6 mg/l (iqr 4-19 mg/l) and its (fig. 1) . in total, 461 (64.5%) patients were classified as nabx (fig. 1) . baseline covariates were largely similar between patients in the rabx and nabx groups. patients were less likely to have prior consultation 14 days before the ed visit in the nabx group compared with the rabx group (49.5% vs 57.5%, p = 0.04). influenza vaccination uptake rates were similar in both groups (37.0% vs 36.7%, p = 0.675). there was no evidence of comorbidity being associated with antibiotic need, except those with steroid use and cancer. median time from earliest symptom onset to ed visit was similar between both groups at 4 days (iqr: 3-7 days). rabx patients were more likely to present with symptoms of fever, body ache, sore throat and vomiting. nabx patients were likely to display symptoms of shortness of breath and giddiness. patients in the rabx group had a higher median maximum body temperature and lower median systolic and diastolic blood pressures than those in the nabx group (table 1) . highest temperature and highest pulse rate were commonly identified to be important predictors in all logistic, lasso and cart models ( table 2 ; fig. 2 ). in addition, age, the presenting symptoms of fever, giddiness and shortness of breath were identified to be significant predictors in the final logistic regression model. similarly, indian ethnicity, fever, giddiness and cancer status were included in the lasso model. (table 2 (table 3 ). in addition, we looked at the corresponding metrics at a probability cut-off of 0.5. the models have marked improvement in sensitivity, but specificity fell below 0.5 (logistic: sen = 0.88, spe = 0.34; lasso: sen = 0.94, spe = 0.27; cart: sen = 0.95, spe = 0.29). detailed documentation on different probability cutoffs can be found in additional file 1. a qualitative study previously conducted in our hospital revealed that ed physicians were confident with their clinical decisions. however, doctors had a lower threshold for prescribing antibiotics for older patients who were immunocompromised and suffering from chronic conditions. junior physicians were observed to be uncomfortable not prescribing antibiotics for urti patients [22] . patients with bacterial and viral infections present with similar symptoms and differentiation of patients requiring antibiotics from those who do not is problematic. our algorithms developed using three rigorous statistical methods together with laboratorybased confirmatory tests served as a good guide for physicians in their decisions on antibiotic prescribing for urti patients. a recent cochrane systematic review provided evidence that patient satisfaction and clinical outcomes were similar between those for whom antibiotic prescribing was delayed and those not prescribed antibiotics at all. delayed prescribing of antibiotics has been found to be associated with marked reduction in antibiotic use [35] . our results showed that the performance of all 3 prediction models were similarly modest. while we tried to be pragmatic with our algorithms, we also carried out similar analysis on more complex classification trees and random forests, both of which showed minimal or no improvement in performance (auc0 .7). a recent systematic review showed that there was minimal improvement using machine learning techniques over traditional regression models [36] . relevant literature on prediction models for antibiotic prescribing in adults are limited and tended to focus on life-threatening infections. several clinical prediction models were built for pneumonia and serious bacterial infections in children mostly using either logistic regression or decision trees [27, 37, 38] . the findings from this study add to the limited knowledge on clinical decision support tools for antibiotic prescribing in an adult ed setting. we found that fever and pulse rate were significant factors in all 3 models. most studies on viral respiratory infections have focused on influenza with a high temperature identified as a significant risk factor in both younger and older adults [39] [40] [41] [42] . heart rate was found to be significant in a group of patients presenting with influenza-like illness at a hospital emergency department [43] . a significant proportion of patients with influenza infection present with tachycardia. this could be due to the physiologic response to fever although cardiac manifestations are not uncommon with complications of influenza [44] . shortness of breath and giddiness were also found to be significant predictors in two of our models. while these symptoms could be non-specific, we believe that it would have to be significant enough for adult patients to volunteer these symptoms to their physicians when they had them. as physicians often have to make antibiotic prescribing decisions based on subjective symptoms reported by their patients, we believe that our clinical decision support tool, developed from three different models, will provide physicians with a reliable tool when making antibiotic prescribing decisions for patients with urti at the point-of-care. there are a few limitations in our study. firstly, the ability to predict well is dependent on the richness of the data. our study is limited to the information obtained at the time that the patient medically attended at ed. knowledge on baseline vital signs and trajectories prior to ed visit may be important information that could improve our models. a 2017 study by stanford university on wearable devices detected that anomalies in skin temperature and heart rate corresponded to periods of high crp levels [45] . secondly, we did not consider laboratory parameters like full blood count, renal and liver function panels in our model as 48% of patients did not receive a full blood count, and even fewer had our data reflected this as 50% of patients had prior consultation although the time between the earliest symptom onset to ed visit was only 4 days on average. the local literature on vaccination uptake in the community is limited. to our knowledge, there is only one population health survey on influenza vaccination uptake in older adults done in 2013 [46] . the authors found that the influenza vaccination uptake in this population was only 15.2%. our patient cohort had a higher vaccination rate (37%) than in the community. however, this does not invalidate our findings and we believe that the impact on the generalizability of our models is minimal. nonetheless, our study had its strengths. we were able to take seasonality into account as the study spanned two years covering two influenza seasons each of northern and southern hemispheres. the use of pcr together with appropriate crp cutoffs were based on findings from several international studies and selected to be the most conservative estimates. the cutoff point for crp was set lower to increase sensitivity of the rabx group [30, 47, 48] . we also note that the proportion of positive viral pcr among the crp < 5 and crp > 20 groups were quite similar (fig. 1) . patients with high crp and positive viral pcr represent patients with secondary bacterial infection. in a sub-group analysis of 229 patients with complete blood count performed, those with high crp levels of > 20 and positive viral pcr were almost twice as likely to have leukocyte counts of > 9.6 × 10 9 /l as those with crp < 20 and positive viral pcr (39.3% vs. 20.2%, p = 0.003). this supports our exclusion of patients with high crp and positive viral pcr from the nabx group. comprehensive assessment of medical records was performed by two clinically trained individuals with standardization in data extraction methods and definitions to ensure data accuracy and consistency. analysing the data with 3 different methods not only allowed us to compare models but also allowed us to triangulate the findings from all our models. notably, maximum pulse rate and highest temperature were considered as important variables in all 3 models. finally, our models were either coefficient or rule based. they can easily be entered into an excel sheet or the hospital electronic system without the need to integrate complicated programming codes. combining the results from the three models, 58.3% of study participants would not need antibiotics. moving forward, physicians could use this tool as a useful complement to their clinical judgement in their practice to guide their decisions on antibiotic prescribing. antibiotics should be prescribed with caution even during low influenza periods as there are still other viruses circulating throughout the year. at the time of writing, we have developed a mobile application (app) named the "abx stew-ards" to provide clinical decision support for busy physicians practicing in the ed on antibiotic prescribing for urti (fig. 4) . ed physicians are required to fill in 9 parameters all on one screen. all fields are mandatory, and the app will provide a recommendation either to review the need for antibiotics or that antibiotics was not needed, based on the predicted outcomes of all 3 validated models. a validation study is underway. it is hoped that evidence-based clinical decision support tools accessible at the point-of-care can lead to better antibiotic prescribing decisions and the reduction of antibiotic resistance. antibiotics for the common cold and acute purulent rhinitis. cochrane database of systematic reviews excessive antibiotic use for acute respiratory infections in the united states principles of appropriate antibiotic use for treatment of acute respiratory tract infections in adults: background, specific aims, and methods antibiotics for acute bronchitis. cochrane database syst rev biomarkers as point-of-care tests to guide prescription of antibiotics in patients with acute respiratory infections in primary care. cochrane database of syst rev over-prescribing of antibiotics and imaging in the management of uncomplicated uris in emergency departments trends in emergency department antibiotic prescribing for acute respiratory tract infections ambulatory antibiotic prescribing for acute bronchitis and cough and hospital admissions for respiratory infections: time trends analysis antibiotic use for viral acute respiratory tract infections remains common antibiotic utilization for acute respiratory tract infections in u.s. emergency departments predictors of frequent attenders of emergency department at an acute general hospital in singapore frequent attenders at the emergency department: an analysis of characteristics and utilisation trends. proceed singapore healthcare antibiotic prescribing for patients with upper respiratory tract infections by emergency physicians in a singapore tertiary hospital effect of antibiotic prescribing in primary care on antimicrobial resistance in individual patients: systematic review and meta-analysis a systematic review and meta-analysis of the effects of antibiotic consumption on antibiotic resistance the distribution of antibiotic use and its association with antibiotic resistance estimating the number of infections caused by antibiotic-resistant escherichia coli and klebsiella pneumoniae in 2014: a modelling study attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the eu and the european economic area in 2015: a population-level modelling analysis clinical and economic impact of antibiotic resistance in developing countries: a systematic review and metaanalysis guidelines for antimicrobial stewardship training and practice implementation hurdles of an interactive, integrated, point-of-care computerised decision support system for hospital antibiotic prescription determinants of antibiotic prescribing for upper respiratory tract infections in an emergency department with good primary care access: a qualitative analysis multisite exploration of clinical decision-making for antibiotic use by emergency medicine providers using quantitative and qualitative methods medical and psychosocial factors associated with antibiotic prescribing in primary care: survey questionnaire and factor analysis factors predicting antibiotic prescription and referral to hospital for children with respiratory symptoms: secondary analysis of a randomised controlled study at out-of-hours services in primary care using machine learning to guide targeted and locally-tailored empiric antibiotic prescribing in a children's hospital in cambodia can clinical prediction models assess antibiotic need in childhood pneumonia? a validation study in paediatric emergency care predicting risk of serious bacterial infections in febrile children in the emergency department performance of a bedside c-reactive protein test in the diagnosis of community-acquired pneumonia in adults with acute cough contributions of symptoms, signs, erythrocyte sedimentation rate, and creactive protein to a diagnosis of pneumonia in acute lower respiratory tract infection a new method of classifying prognostic comorbidity in longitudinal studies: development and validation foundation m. an introduction to recursive partitioning using the rpart routines rattle: a data mining an introduction to glmnet delayed antibiotic prescriptions for respiratory infections. cochrane database syst rev a systematic review shows no performance benefit of machine learning over logistic regression for clinical prediction models clinical prediction model to aid emergency doctors managing febrile children at risk of serious bacterial infections: diagnostic study signs and symptoms for diagnosis of serious infections in children: a prospective study in primary carecommentary clinical signs and symptoms predicting influenza infection clinical differences between respiratory viral and bacterial mono-and dual pathogen detected among singapore military servicemen with febrile respiratory illness. influenza other respir viruses predictors of influenza among older adults in the emergency department clinical prediction rules combining signs, symptoms and epidemiological context to distinguish influenza from influenza-like illnesses in primary care: a cross sectional study c-reactive protein as predictor of bacterial infection among patients with an influenza-like illness the cardiovascular manifestations of influenza: a systematic review digital health: tracking physiomes and activity using wearable biosensors reveals useful health-related information factors associated with influenza vaccine uptake in older adults living in the community in singapore the course of c-reactive protein response in untreated upper respiratory tract infection diagnostic value of pct and crp for detecting serious bacterial infections in patients with fever of unknown origin: a systematic review and meta-analysis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ms. ong lay see and grace tin for collecting the data, staff of ttsh ed who helped with study recruitment, and patients who participated in the study.authors' contributions ac, dcl, and cko conceived the study and designed the study. ac obtained research funding. ac supervised the conduct of the trial and data collection. cko implemented the study including providing oversight of patient recruitment. jgw provided statistical oversight to the study. jgw, aah, and wxl analysed the data. jgw, aah, wxl, and ac drafted the manuscript, and all authors contributed substantially to its revision. ac takes responsibility for the paper as a whole. all author(s) read and approved the final manuscript. the online version contains supplementary material available at https://doi. org/10.1186/s13756-020-00825-3.additional file 1: appendix 1a. training diagnostic performance at different probability cutoffs. appendix 1b. 2 x 2 tables for the actual vs predicted values on the training and validation set using the best probability cutoff all authors have no conflict of interest related to the submitted work. key: cord-343690-rafvxgx1 authors: hartmann, katrin title: clinical aspects of feline retroviruses: a review date: 2012-10-31 journal: viruses doi: 10.3390/v4112684 sha: doc_id: 343690 cord_uid: rafvxgx1 feline leukemia virus (felv) and feline immunodeficiency virus (fiv) are retroviruses with global impact on the health of domestic cats. the two viruses differ in their potential to cause disease. felv is more pathogenic, and was long considered to be responsible for more clinical syndromes than any other agent in cats. felv can cause tumors (mainly lymphoma), bone marrow suppression syndromes (mainly anemia), and lead to secondary infectious diseases caused by suppressive effects of the virus on bone marrow and the immune system. today, felv is less commonly diagnosed than in the previous 20 years; prevalence has been decreasing in most countries. however, felv importance may be underestimated as it has been shown that regressively infected cats (that are negative in routinely used felv tests) also can develop clinical signs. fiv can cause an acquired immunodeficiency syndrome that increases the risk of opportunistic infections, neurological diseases, and tumors. in most naturally infected cats, however, fiv itself does not cause severe clinical signs, and fiv-infected cats may live many years without any health problems. this article provides a review of clinical syndromes in progressively and regressively felv-infected cats as well as in fiv-infected cats. feline leukemia virus (felv) and feline immunodeficiency virus (fiv) belong to the most common infectious diseases in cats. both are retroviruses, but felv is a γ-retrovirus, while fiv is classified as open access a lentivirus. although felv and fiv are closely related, they differ in their potential to cause disease. in the united states, prevalence of both infections is about 2% in healthy cats and up to about 30% in high-risk or sick cats [1, 2] . risk factors for infection include male gender, adulthood, and outdoor access [3, 4] . retroviral tests can diagnose only infection, not clinical disease. felv is more pathogenic than fiv. for a long time, felv was considered to account for most disease-related deaths, and to be responsible for more clinical syndromes than any other single agent in cats. it was proposed that approximately one third of all tumor-related deaths in cats were caused by felv, and an even greater number of cats died of felv-related anemia and secondary infections caused by suppressive effects of the virus on bone marrow and the immune system. today, these statements have to be revised, as in recent years the prevalence and consequently the importance of felv as a pathogen in cats have been decreasing. still, if present in closed households with other viruses, such as feline coronavirus (fcov), or fiv, felv infection has the greatest impact on survival [5] . the death rate of progressively felv-infected cats in multi-cat households has been estimated at approximately 50% in two years and 80% in three years [6, 7] , but is much lower today, at least for cats that are well taken care of and that are kept strictly indoors in single-cat households. a survey in the united states compared the survival of more than 1000 felv-infected cats to more than 8000 age-and sex-matched uninfected control cats and found that in felv-infected cats median survival was 2.4 years compared to 6.0 years for control cats [2] . despite the fact that progressive felv infection is associated with a decrease in life expectancy, many owners elect to provide treatment for their cats, and with proper care, felv-infected cats might live for many years with good quality of life. although fiv can cause an acquired immunodeficiency syndrome in cats ("feline aids") comparable to human immunodeficiency virus (hiv) infection in humans, with increased risk for opportunistic infections, neurologic diseases, and tumors, in most naturally infected cats, fiv does not cause a severe clinical syndrome. with proper care, fiv-infected cats can live many years and, in fact, can die at older age from causes unrelated to their fiv infection. in a follow-up study in naturally fivinfected cats, the rate of progression was variable, with death occurring in about 18% of infected cats within the first two years of observation (about five years after the estimated time of infection). an additional 18% developed increasingly severe disease, but more than 50% remained clinically asymptomatic during the two years [8] . fiv infection has little impact on a cat population and does not reduce the number of cats in a household [2] . thus, overall survival time is not shorter than in uninfected cats, and quality of life is usually fairly high over an extended period of time. both infections are chronic in nature and develop through different disease stages. characteristically for both infections, there is a long asymptomatic phase, in which cats do not show clinical signs. felv infection has different stages. recently, novel diagnostic tools, including very sensitive pcr methods providing new data on the course of felv infection, have questioned the traditional understanding of felv pathogenesis. cats believed to be immune to felv after infection were found to remain provirus-positive. antigen-negative, provirus-positive cats are frequently detected and their clinical relevance and role in felv epidemiology is still not fully understood. antigen-negative, provirus-positive cats are considered felv carriers. following reactivation, they can act as an infection source. as felv provirus is integrated into the cat's genome, it is unlikely to be fully cleared over time. antigen-negative, provirus-positive cats do not shed the virus, but reactivation with reoccurring virus shedding is possible [9, 10] . based on this information, a new classification has been proposed, in which the stages of felv infection are defined as abortive infection (comparable to the former "regressor cats"), regressive infection (comparable to the former "transient viremia" followed by "latent infection"), progressive infection (comparable to the former "persistent viremia"), and focal or atypical infection (table 1) [11] [12] [13] [14] . abortive infection. after infection, the virus starts initially to replicate in the local lymphoid tissue in the oropharyngeal area. in some immunocompetent cats (formerly called "regressor cats)", viral replication may be terminated by an effective humoral and cell-mediated immune response; these cats never become viremic. they have high levels of neutralizing antibodies. neither felv antigen nor viral rna or proviral dna can be detected in the blood at any time. abortive infection is likely caused when a cat is exposed to low doses of felv [15] . it is still unknown, how often this situation really occurs in nature, because studies using very sensitive pcr methods have found that in many of the formerly considered "regressor cats", the virus actually can still be found later in these cats when investigating tissue samples. thus, it appears likely that none or only very few cats can completely clear felv infection from all cells. regressive infection develops following an effective immune response. in regressive infection, virus replication and viremia are contained prior to or shortly after bone marrow infection. after initial infection, replicating felv spreads systemically through infected mononuclear cells (lymphocytes and monocytes). during this stage, cats have positive results on tests that detect free antigen in plasma (e.g., elisa). they shed virus, mainly with saliva. in cats with regressive infection, this viremia, however, is terminated within weeks or months (therefore formerly called "transient viremia"). in some cats, viremia may persist longer than three weeks. after about three weeks of viremia, bone marrow cells become infected, and infected hematopoietic precursor cells develop into infected granulocytes and platelets that circulate in the body. even if bone marrow cells become infected, a certain percentage of cats is able to clear viremia. however, they cannot completely eliminate the virus from the body, even if they terminate viremia because the information for virus replication (proviral dna) is present in bone marrow stem cells. this condition has been called "latent infection" (and is now a part of the regressive infection). the molecular basis of latency is the integration of a copy of the viral genome (provirus) into cellular chromosomal dna. although proviral dna remains present within the cellular genome, no virus is actively produced. thus, cats with regressive infection have negative results in all tests that detect felv antigen. during cell division, proviral dna is replicated and the information given to the daughter cells. thus, complete cell lineages may contain felv proviral dna. however, proviral dna is not translated into proteins, and no infectious virus particles are produced. therefore, regressively infected cats do not shed felv and are not infectious to others. sensitive pcr methods can detect provirus in the blood of cats with regressive infection that are antigen-negative. in a study in switzerland it was shown that in addition to the antigen-positive, provirus-positive cats, about 10% of the cat population that were negative for antigen were positive for proviral dna in the blood [16] . regressive infection can be reactivated because the information for producing complete viral particles is present and can potentially be reused when antibody production decreases (e.g., after immunosuppression). in cats with progressive infection, felv infection is not contained early in the infection. thus, extensive virus replication occurs, first in the lymphoid tissues, followed by the bone marrow and mucosal and glandular epithelial tissues. progressively infected cats remain persistently viremic. they are infectious to other cats for the remainder of their life. this condition has been called "persistent viremia" and is now classified as progressive infection. cats with progressive infection develop felv-associated diseases, and most of them will die within a few years. regressive and progressive infections can be distinguished by repeated testing for viral antigen in peripheral blood; regressively infected cats will turn negative at latest 16 weeks after infection, while progressively infected cats will remain positive. initially both, regressive and progressive infections are accompanied by persistence of felv proviral dna in the blood detected by pcr but later are associated with different felv loads when measured by quantitative pcr; regressive infection is associated with low, progressive infection with high virus load [11, 17] . focal infections or atypical infections have been reported in up to 10% of experimentally infected cats. focal infections or atypical infections may also be observed in natural infections, but are probably rare in the field. focal infections are characterized by a persistent atypical local viral replication (e.g., in mammary glands, bladder, eyes). this replication can lead to intermittent or low-grade production of antigen, and therefore, these cats can have weakly positive or discordant results in antigen tests, or positive and negative results may alternate [14] . experimental fiv infection also progresses through several stages, similar to hiv infection in people, including an acute phase, a clinically asymptomatic phase of variable duration, and a terminal phase sometimes called "feline acquired immunodeficiency syndrome" ("aids") [18, 19] . however, there is no clear distinction between these stages in naturally fiv-infected cats, and not all stages are apparent; therefore, the usefulness of this staging in natural fiv infection has been questioned. moreover, even cats in moribund condition with severe immunosuppression and secondary infections may fully recover with appropriate care and return to an asymptomatic stage. thus, different from hiv-infected people, cats classified as being in the "aids phase" (high virus load, severe clinical signs due to secondary infection) can recover and be asymptomatic again, and their virus loads can even decrease dramatically. clinical signs in both retrovirus infections are variable. after a long asymptomatic phase, cats can develop tumors, hematopoietic disorders, neurologic disorders, immunodeficiency, immune-mediated diseases, and stomatitis. the pathomechanism of these disorders is different in both retrovirus infections (table 2) . although felv was named after a tumor that first garnered its attention, most infected cats are presented to the veterinarian not for tumors but for anemia or immunosuppression. clinical signs associated with felv infection can be classified as tumors, immunosuppression, hematologic disorders, immune-mediated diseases, and other syndromes (including neuropathy, reproductive disorders, fading kitten syndrome). of 8642 felv-infected cats presented to north american veterinary teaching hospitals, various co-infections (including fiv infection, feline infectious peritonitis (fip), upper respiratory infection, hemotropic mycoplasmosis, and stomatitis) were the most frequent findings (15%), followed by anemia (11%), lymphoma (6%), leukopenia or thrombocytopenia (5%), and leukemia or myeloproliferative diseases (4%) [20] . the outcome of felv infection and the clinical course are determined by a combination of viral and host factors. some of the differences in outcome can be traced to properties of the virus itself, such as the subgroup that determines differences in the clinical picture (e.g., felv-b is primarily associated with tumors, felv-c is primarily associated with non-regenerative anemia). a study aiming to define dominant host immune effects or mechanisms responsible for the outcome of infection by using longitudinal changes in felv-specific cytotoxic t-lymphocytes (ctl) found that high levels of circulating felv-specific effector ctls appear before virus-neutralizing antibodies in cats that have recovered from exposure to felv. in contrast, progressive infection with persistent viremia has been associated with a silencing of virusspecific humoral and cell-mediated immunity host effector mechanisms [21] . probably the most important host factor that determines the clinical outcome of cats infected with felv is the age of the cat at the time of infection [22] . neonatal kittens develop marked thymic atrophy after infection ("fading kitten syndrome"), resulting in severe immunosuppression, wasting, and early death. as cats mature, they acquire a progressive resistance. when older cats become infected, they tend to have abortive or regressive infections or, if developing progressive infection, have at least milder signs and a more protracted period of apparent good health [7] . clinical signs in naturally fiv-infected cats usually reflect secondary diseases, such as infections and neoplasia, to which fiv-infected cats are considered more susceptible. fiv itself may cause some clinical features (e.g., neurologic signs) resulting from abnormal function or inflammation of affected organs. in experimental infection, an initial stage is sometimes noticed usually with transient and mild clinical signs, including fever, lethargy, signs of enteritis, stomatitis, dermatitis, conjunctivitis, respiratory tract disease, and generalized lymph node enlargement [23] . the acute phase may last several days to a few weeks, after which cats will enter a period in which they appear clinically healthy. this phase is usually not noticed by the owners in naturally infected cats. the duration of the following asymptomatic phase varies, but usually lasts many years. factors that influence the duration of the asymptomatic phase include the pathogenicity of the infecting isolate (also depending on the fiv subtype), exposure to secondary pathogens, and the age of the cat at the time of infection [24, 25] . in the last, symptomatic phase ("aids phase") of infection, the clinical signs are a reflection of opportunistic infections, neoplasia, myelosuppression, and neurologic disease. while felv-infected cats are 62-times more likely to develop lymphoma or leukemia than noninfected cats and felv plays a direct role in tumorgenesis, fiv-infected cats have about a five-fold increased risk of tumor development, and the role of fiv is usually indirect. lymphomas are the most common tumors in felv-and fiv-infected cats. while felv-infected cats have most commonly t-cell lymphomas, lymphomas in fiv are mostly of b-cell origin [26, 27] . felv is a major oncogene that causes different tumors in cats, most commonly lymphoma and leukemia, less often other hematopoietic tumors and rarely other malignancies (including neuroblastoma, osteochondroma, and others). the association between felv and lymphomas has been clearly established in several ways. first, these malignancies can be induced in kittens by experimental felv infection [28] [29] [30] . second, cats naturally infected with felv have a higher risk of developing lymphoma than uninfected cats [29, 31] . third, most cats with lymphoma were-at least in earlier times when prevalence of felv was still higher-felv-positive in tests that detected infectious virus or felv antigens. previously, up to 80% of feline lymphomas and leukemias were reported to be felv-related [32] [33] [34] [35] [36] [37] [38] . however, since the 1980s a reduction in the prevalence of viremia has been noted in cats with lymphoma [39] [40] [41] . the decrease in prevalence of felv infection in cats with lymphoma or leukemia also indicates a shift in tumor causation in recent years. whereas 59% of all cats with lymphoma or leukemia were felv antigen-positive in one german study from 1980 to 1995, only 20% of the cats were felv antigen-positive in the years 1996 to 1999 in the same university teaching hospital [41] . in a recent study in the netherlands, only four of 71 cats with lymphoma were felv-positive, although 22 of these cats had mediastinal lymphoma, which previously was strongly associated with felv infection [42] . a greater prevalence of lymphoma in older-age cats in now observed. the major reason for the decreasing association of felv with lymphoma is the decreasing prevalence of felv infection in the overall cat population as a result of felv vaccination as well as testing and elimination programs. however, prevalence of lymphomas caused by felv may be higher than indicated by conventional antigen testing of blood [43] . cats from felv cluster households had a 40-fold higher rate of development of felv-negative lymphoma than did those from the general population. felv-negative lymphomas have also occurred in laboratory cats known to have been infected previously with felv [44] . felv proviral dna was detected in lymphomas of cats that tested negative for felv antigen [43] , also suggesting that the virus may be associated with a larger proportion of lymphomas than previously thought. felv has been shown to incorporate cellular genes; several such transducted genes also present in regressively infected cells have been implicated in viral oncogenesis [44] [45] [46] . it is still unclear, how common regressive felv infection is responsible for felv-associated tumors in the field as study results have been controversial. proviral dna was detected in formalin-fixed, paraffin-embedded tumor tissue in 7/11 felv-negative cats with lymphoma [43] . however, other groups found evidence of provirus in only 1/22 [45] and in 0/50 felv antigen-negative lymphomas [47] . the most important mechanism by which felv causes malignancy is by insertion of the felv genome into the cellular genome near a cellular oncogene (most commonly myc), resulting in activation and over-expression of that gene. these effects lead to uncontrolled proliferation of these cells (clone). a malignancy results in the absence of an appropriate immune response. felv may also incorporate the oncogene to form a recombinant virus (e.g., felv-b, fesv) containing cellular oncogene sequences that are then rearranged and activated. when they enter a new cell, these recombinant viruses are oncogenic. in a study of 119 cats with lymphomas, transduction or insertion of the myc locus had occurred in 38 cats (32%) [48] . thus, felv-induced neoplasms are caused, at least in part, by somatically acquired insertional mutagenesis in which the integrated provirus may activate a proto-oncogene or disrupt a tumor suppressor gene. a recent study suggested that the u3-ltr region of felv transactivates cancer-related signaling pathways through production of a non-coding 104 base rna transcript that activates nf kappab [49] . twelve common integration sites for felv associated with lymphoma development have been identified in six loci: c-myc, flvi-1, flvi-2 (contains bmi-1), fit-1, pim-1, and flit-1. oncogenic association of the loci is based on the fact that c-myc is known as a proto-oncogene, bmi-1 and pim-1 have been recognized as myc-collaborators, fit-1 appears to be closely linked to myb, and flit-1 insertion was shown to be associated with over-expression of cellular genes, e.g., activin-a receptor type ii-like 1 (acvrl1). [50] . flit-1 seems to have an important role in the development of lymphomas and appears to represent a common novel felv proviral integration domain that may influence lymphomagenesis by insertional mutagenesis. among 35 felv-related tumors, 5/25 thymic lymphomas demonstrated proviral insertion within flit-1 locus, whereas 0/4 alimentary lymphomas, 5/5 multicentric lymphomas, and 1/1 t-lymphoid leukemia examined had rearrangements in this region. expression of acvrl1 mrna was detected in the two thymic lymphomas with flit-1 rearrangement, whereas normal thymuses and seven lymphoid tumors without flit-1 rearrangement had no detectable acvrl1 mrna expression [51] . fibrosarcomas that are associated with felv are caused by fesv, a recombinant virus that develops de novo in felv-a-infected cats by recombination of the felv-a genome with cellular oncogenes. through a process of genetic recombination, fesv acquires one of several oncogenes, such as fes, fms, or fgr. as a result, fesv is an acutely transforming (tumor-causing) virus, leading to a polyclonal malignancy with multifocal tumors arising simultaneously after a short incubation period. with the decrease in felv prevalence, fesv also has become less common. fesv-induced fibrosarcomas are multicentric and usually occur in young cats. strains of fesv identified from naturally occurring tumors are defective and unable to replicate without the presence of felv-a as a helper virus that supplies proteins (such as those coded by the env gene) to fesv. fibrosarcomas caused by fesv tend to grow rapidly, often with multiple cutaneous or subcutaneous nodules that are locally invasive and metastasize to the lung and other sites. solitary fibrosarcomas in older cats are not caused by fesv. these tumors are slower growing, locally invasive, slower metastasizing, and only occasionally curable by excision combined with radiation and/or gene therapy. they usually are classified as feline injection site sarcomas (fiss) caused by the granulomatous inflammatory reaction at the injection site, commonly occurring after inoculation of adjuvant-containing vaccines. it has been demonstrated that neither fesv nor felv play any role in the development of fiss [52] . a few other tumors have been found in felv-infected cats; some of them might have an association with felv, others likely have just been observed by chance simultaneously in an infected cat. iris melanomas, for example, are not associated with felv infections, although in one study three of 18 eyes tested positive for felv/fesv proviral dna [53] . in a more recent study, however, immunohistochemical staining and pcr did not find felv or fesv in the ocular tissues of any cat with this disorder [54] . multiple osteochondromas (cartilaginous exostoses on flat bones of unknown pathogenesis) have been described in felv-infected cats. although histologically benign, they may cause significant morbidity if they occur in an area such as a vertebra and put pressure on the spinal cord or nerve roots [55, 56] . in spontaneous feline olfactory neuroblastomas (aggressive, histologically inhomogenous tumors of the tasting and smelling epithelium of nose and pharynx with high metastasis rates), budding felv particles were found in the tumors and lymph node metastases, and felv dna was detected in tumor tissue [57] . the exact role of felv in the genesis of these tumors is uncertain. cutaneous horns are a benign hyperplasia of keratinocytes that have been described in felv-infected cats [58] , but the role of felv is also unclear. fiv-infected cats are about five times more likely to develop lymphoma or leukemia than noninfected cats [26, 27] . lymphomas (mostly b-cell lymphomas) [26, 27, 59, 60] , leukemias, but also several other tumors have been described in association with fiv infection [26, [61] [62] [63] [64] [65] [66] , including squamous cell carcinoma, fibrosarcoma, and mast cell tumor. fiv provirus, however, is only occasionally detected in tumor cells [67] [68] [69] [70] , suggesting a more indirect role in lymphoma formation, such as decreased cell-mediated immune surveillance or chronic b-cell hyperplasia [68, 71] . however, clonally integrated fiv dna was found in lymphoma cells from one cat that had been experimentally infected six years earlier, indicating the possibility of an occasional direct oncogenic role of fiv [67, 70, 72] . the prevalence of fiv infection in one cohort of cats with lymphoma was 50% [60] , much higher than the fiv prevalence in the population of cats without lymphomas, which is also supportive of a cause and effect relationship. fiv could alternatively increase tumor incidence by decreasing tumor immunosurveillance mechanisms. it also could promote tumor development through the immunostimulatory effects of replicating in lymphocytes. myelosupression and other hematopoietic disorders can occur in both, felv and fiv infection. it is, however, much more common and more severe in felv-infected cats. hematologic changes described in association with felv include anemia (non-regenerative or regenerative), persistent, transient, or cyclic neutropenia, platelet abnormalities (thrombocytopenia and platelet function abnormalities), aplastic anemia (pancytopenia), and panleukopenia-like syndrome. for the majority of pathogenic mechanisms in which felv causes bone marrow suppression, active virus replication is required. however, it has been demonstrated that in some felv antigen-negative cats, regressive felv infection without viremia may be responsible for bone marrow suppression. in a recent study including 37 cats with myelosuppression that tested felv antigen-negative in peripheral blood, 2/37 cats (5%) were found regressively infected with felv by bone marrow pcr (both had non-regenerative anemia) [73] . in these regressively infected cats, felv provirus may interrupt or inactivate cellular genes in the infected cells, or regulatory features of viral dna may alter expression of neighboring genes. additionally, cell function of provirus-containing myelomonocytic progenitor and stromal fibroblasts that provide bone marrow microenvironment may be altered. alternatively, felv provirus may cause bone marrow disorders by inducing the expression of antigens on the cell surface, resulting in an immune-mediated destruction of the cell. anemia is a major nonneoplastic complication that occurs in a majority of felv-infected cats [4] . anemia in felv-infected cats may have various causes. approximately 10% of felv-associated anemias are regenerative [74] , most felv-associated anemias, however, are non-regenerative and are caused by the bone marrow suppressive effect of the virus resulting from primary infection of hematopoietic stem cells and infection of stroma cells that constitute the supporting environment for hematopoietic cells. in vitro exposure of normal feline bone marrow to some strains of felv caused suppression of erythrogenesis [6] . in addition to the direct effect of the virus on erythropoiesis, other factors can cause nonregenerative anemia in felv-infected cats (e.g., anemia of chronic inflammation promoted by high concentration of cytokines). felv infection can cause decreased platelet counts. it also can be responsible for platelet function deficits, and the lifespan of platelets is shortened in some felvinfected cats. thrombocytopenia (resulting in bleeding disorders) can occur secondary to decreased platelet production from felv-induced bone marrow suppression or leukemic infiltration. platelets harbor felv, and megakaryocytes are frequent targets of progressive felv infection. immunemediated thrombocytopenia, which rarely occurs as a single disease entity in cats, often accompanies immune-mediated hemolytic anemia (imha) in cats with underlying felv infection. felv infection also can cause decreased neutrophil or lymphocyte counts. neutropenia is common in felv-infected cats [75] and generally occurs alone or in conjunction with other cytopenias. in some cases, myeloid hypoplasia of all granulocytic stages is observed, suggesting infection on neutrophil precursors. in some neutropenic felv-infected cats, an arrest in bone marrow maturation can occur at the myelocyte and metamyelocyte stages. it has been hypothesized that an immune-mediated mechanism is responsible in cases in which neutrophil counts recover with glucocorticoid treatment ("glucocorticoidresponsive neutropenia"). hematopoietic neoplasia ("myeloprolifertaive disorders"), including leukemia, can also cause bone marrow suppression syndromes by crowding out. myelodysplastic syndrome (mds), characterized by peripheral blood cytopenias and dysplastic changes in the bone marrow, is a pre-stage of acute myeloic leukemia. it was found that changes of the ltr region of the felv genome (presence of three tandem direct 47-bp repeats in the upstream region of the enhancer (ure)) are strongly associated with the induction of mds [76] . myelofibrosis, another cause of bone marrow suppression, is a condition characterized by abnormal proliferation of fibroblasts resulting from chronic stimulation of the bone marrow, such as chronic bone marrow activity from hyperplastic or neoplastic regeneration caused by felv. in severe cases, the entire endosteum within the medullary cavity can be obliterated. feline panleukopenia-like syndrome (fpls), also known as felv-associated enteritis (fae) or myeloblastopenia, consists of severe leukopenia (< 3000 cells/μl) with enteritis and destruction of intestinal crypt epithelium that mimics feline panleukopenia caused by feline panleukopenia virus (fpv) infection. however, fpv antigen has been demonstrated by ifa in intestinal sections of cats that died from this syndrome after being experimentally infected with felv [77] . fpv was also demonstrated by electron microscopy despite negative fpv antigen tests. it appears that this syndrome might actually not be caused by felv itself, as previously thought, but by co-infection with fpv. the syndrome also has been referred to as fae in cats with progressive felv infection because the clinical signs observed are usually gastrointestinal, including hemorrhagic diarrhea, vomiting, oral ulceration or gingivitis, anorexia, and weight loss [78, 79] . it is still unclear whether all theses syndromes have the same origin and are simply caused by co-infection with fpv (and even modified life fpv vaccines have been discussed) or if they are caused by felv itself [77] . although cytopenias caused by bone marrow suppression are a common finding in felv infection, these are rather uncommon in fiv-infected cats. during the acute phase of infection, fiv-infected cats can exhibit mild neutropenia, which resolves as the cat progresses to the asymptomatic phase of infection. clinically ill fiv-infected cats in a later phase of infection may have a variety of cytopenias, with lymphopenia being most common. lymphopenia is caused by direct replication of the virus in cd4 + lymphocytes. anemia and neutropenia (usually mild) may also be seen [4, 51] , although these abnormalities may be as much a reflection of concurrent disease as direct effects of fiv itself. a recent study in a high number (3784) of client-owned field cats compared hematologic parameters in fivinfected, felv-infected and uninfected cats [4] . anemia and thrombocytopenia were not significantly more common in fiv-infected versus uninfected cats. only neutropenia was significantly more often present, in about 25% of fiv-infected cats. soluble factors have been shown to inhibit bone marrow function in fiv-infected cats, and bone marrow infection has been associated with decreased ability to support hematopoietic potential in vitro or has been proposed as a mechanism underlying the development of cytopenias [51] . neurologic dysfunction may be present in felv-and in fiv-infected cats and is one of the few syndromes directly caused by the retrovirus. however, mechanisms of neurologic dysfunction are different with both viruses. in felv-infected cats, most neurologic signs are caused by lymphoma and lymphocytic infiltrations in brain or spinal cord leading to compression, but in some cases, no tumor is detectable with diagnostic imaging methods or at necropsy. in these cats, felv-induced neurotoxicity is suspected. anisocoria, mydriasis, central blindness, or horner's syndrome have been described in felv-infected cats without morphologic changes. in some regions (such as the southeastern united states), urinary incontinence caused by neuropathies in felv-infected cats has been described [80] . direct neurotoxic effects of felv have been discussed as pathogenetic mechanisms. felv envelope glycoproteins may be able to produce increased free intracellular calcium leading to neuronal death (this has also been described in hiv-infected humans). a polypeptide of the felv envelope was found to cause dosedependent neurotoxicity associated with alterations in intracellular calcium ion concentration, neuronal survival, and neurite outgrowth. the polypeptide from a felv-c strain was significantly more neurotoxic than the same peptide derived from a felv-a strain [81, 82] . neurologic signs in 16 cats with progressive felv infection consisted of abnormal vocalization, hyperesthesia, and paresis progressing to paralysis. some cats developed anisocoria or urinary incontinence during the course of their illness. others had concurrent felv-related problems such as myelodysplastic disease. the clinical course of affected cats involved gradually progressive neurologic dysfunction. microscopically, white-matter degeneration with dilation of myelin sheaths and swollen axons was identified in the spinal cord and brain stem of affected animals [80] . immunohistochemical staining of affected tissues revealed consistent expression of felv p27 antigens in neurons, endothelial cells, and glial cells, and proviral dna was amplified from multiple sections of the spinal cord [80] . these findings suggest that in some felv-infected cats, the virus may directly affect cns cells cytopathically. neurologic signs also have been described in both natural and experimental fiv infections [83] [84] [85] [86] [87] [88] . about 5% of symptomatic fiv-infected cats have a neurological disease as a predominant clinical feature. neurologic disorders in fiv infection seem to be strain-dependent [89] . both central and peripheral neurologic manifestations have been described, comparable to the changes in hiv-infected human beings. dementia in human patients with aids is often characterized by a slight decline in cognitive ability or behavior, changes that may be too subtle to be recognized in cats. neurological abnormalities seen in naturally infected cats tend to be more behavioral than motor. psychotic behavior, twitching movements of the face and tongue, compulsive roaming, dementia, loss of bladder and rectal control, and disturbed sleep patterns have been observed. other signs described include nystagmus, ataxia, seizures, and intention tremors [90] [91] [92] . abnormal forebrain electrical activity and abnormal visual and auditory-evoked potentials have also been documented in cats that appeared otherwise normal [24, 66, 93, 94] . although the majority of fiv-infected cats do not show clinically overt neurologic signs, a much higher proportion of infected cats have microscopic cns lesions. brain lesions may occur in the absence of massive infection, and abnormal neurologic function has been documented in fiv-infected cats with only mild to moderate histologic evidence of inflammation [8] . pathologic findings include the presence of perivascular infiltrates of mononuclear cells, diffuse gliosis, glial nodules, and white matter pallor. these lesions are usually located in the caudate nucleus, midbrain, and rostral brain stem [8] . mostly, abnormal neurologic function is the result of a direct effect of the virus on cns cells. neurologic signs upon fiv infection are highly strain-dependent. the virus infects the brain early, with virus-induced cns lesions sometimes developing within two months of experimental infection [8] . microglia and astrocytes are infected by fiv, but the virus does not infect neurons. however, neuronal death has been associated with fiv infection; in particular, forebrain signs are often a result of direct neuronal injury from the virus. the exact mechanism of neuronal damage by fiv is unclear but may include neuronal apoptosis, effects on the neuron supportive functions of astrocytes, toxic products released from infected microglia, or cytokines produced in response to viral infection. in vitro studies support the hypothesis that fiv infection may impair normal metabolism in cns cells, particularly astrocytes [8] . documented abnormalities of astrocyte function include altered intercellular communication, abnormal glutathione reductase activity that could render cells more susceptible to oxidative injury, and alterations in mitochondrial membrane potential that disrupt the energy-producing capacities of the cell [95] . astrocytes are by far the most common cell type of the brain and are important in maintaining cns neuronal vascular microenvironment. one of the most important functions of astrocytes is to regulate the level of extracellular glutamate, a major excitatory neurotransmitter that accumulates as a consequence of neuronal activity. excessive extracellular glutamate often results in neuronal toxicity and death. fiv infection of feline astrocytes can significantly inhibit their glutamate-scavenging ability, potentially resulting in neuronal damage [95, 96] . sometimes, neurologic signs may also be caused by opportunistic infections such as toxoplasmosis, cryptococcosis, or fip. the most clinically important consequence of both retrovirus infections is immunosuppression. immunosuppression can lead to secondary infectious diseases accounting for most clinical signs, but also can lead to decreased tumor surveillance mechanisms causing an increased risk of tumor development. it is important to realize that many of these secondary diseases in felv-and fivinfected cats are treatable. the mechanisms that cause the immunosuppression are different for the two infections. many felv-infected cats have concurrent bacterial, viral, protozoal, and fungal infections, but few controlled studies exist proving that these cats have a higher rate of infection than felv-negative cats. thus, although felv certainly can suppress immune function, it should not be assumed that all concurrent infections are a direct consequence of felv infection. progressively felv-infected cats develop immunosuppression similar to that in hiv-infected people. the exact mechanisms of how the virus destroys the immune system are poorly understood, as is why different animals have such varying degrees of immunosuppression. immunosuppression has been associated with non-integrated viral dna from replication-defective viral variants [97] . these pathogenic immunosuppressive variants, such as felv-t, require a membrane-spanning receptor molecule (pit1) and a second co-receptor protein (felix) to infect t lymphocytes [98] . the latter protein is an endogenously expressed protein encoded by an endogenous provirus arising from felv-a, which is similar to the felv receptor-binding protein of felv-b [99] . felv-infected cats may develop thymic atrophy and depletion of lymph node paracortical zones following infection. lymphopenia and neutropenia are common. in addition, neutrophils of viremic cats have decreased chemotactic and phagocytic function compared with those of normal cats. in some cats, lymphopenia may be characterized by preferential loss of cd4 + helper t cells, resulting in an inverted cd4/cd8 ratio (as typically seen in fiv infection) [100, 101] , but more commonly, substantial losses of helper cells and cytotoxic suppressor cells (cd8 + cells) occur [101] . many immune function tests of naturally felv-infected cats are abnormal, including decreased response to t-cell mitogens, prolonged allograft reaction, reduced immunoglobulin production, depressed neutrophil function, and complement depletion. il-2 and il-4 are decreased in some cats [7, 102] , but felv does not appear to suppress il-1 production from infected macrophages. ifn-γ may be deficient or increased. increased tnf-α has been observed in serum of infected cats and in infected cells in culture. each cytokine plays a vital role in the generation of a normal immune response, and the excess production of certain cytokines, such as tnf-α, can also cause illness. t-cells of felvinfected produce significantly lower levels of b-cell stimulatory factors than do those of normal cats (this defect becomes progressively more severe over time) [72] , but when b-cells of felv-infected cats are stimulated in vitro by uninfected t-cells, their function remains normal. primary and secondary humoral antibody responses to specific antigens are decreased and may occur delayed in felv-infected cats. in vaccination studies, felv-infected cats were not able to mount an adequate immune response to vaccines, such as rabies. therefore, protection in a felv-infected cat after vaccination is not complete and not comparable to that in a healthy cat;thus, more frequent vaccinations (e.g., every six months) have to be considered. in fiv-infected cats, immunosuppression usually occurs in later stages of the infection, and leads to predisposition for secondary infections. in a survey study of 826 naturally fiv-infected cats examined at north american veterinary teaching hospitals, the most common disease syndromes were stomatitis, neoplasia (especially lymphoma and cutaneous squamous cell carcinoma), ocular disease (uveitis and chorioretinitis), anemia and leukopenia, opportunistic infections, renal insufficiency, lower urinary tract disease, and endocrinopathies, such as hyperthyroidism and diabetes mellitus [78] . some of these problems, however, are most likely associated rather with the older age at which these cats presented (e.g., endocrinopathies, renal insufficiency) than with their fiv infection. infections with many different "opportunistic" pathogens of viral, bacterial, protozoal, and fungal origin have been reported in fiv-infected cats. few studies, however, have compared the prevalence of most of these infections in fiv-infected and non-infected cats, and thus, their relevancy as true secondary invaders is unclear. the most important immunologic abnormality shown in experimental [104] [105] [106] as well as in natural [107, 108] infection is a decrease in the number and relative proportion of cd4 + cells in the peripheral blood as well as in most primary lymphoid tissues [109] . loss of cd4 + cells leads to inversion of the cd4/cd8 ratio. in addition, an increase in the proportion of cd8 + cells also contributes to the inversion [104, 108, 110] , in particular a population referred to as "cd8 + alpha-hi, beta-low cells" [111] [112] [113] , a subset of cd8 + cells that may contribute to suppression of viremia in fivinfected cats. causes of cd4 + cell loss include decreased production secondary to bone marrow or thymic infection, lysis of infected cells induced by fiv itself (cytopathic effects), destruction of virusinfected cells by the immune system, or death by apoptosis (cell death that follows receipt of a membrane signal initiating a series of programmed intracellular events) [114] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] . the degree of apoptosis correlates inversely with the cd4 + numbers and the cd4/cd8 ratio [127] . fiv env proteins are capable of inducing apoptosis in mononuclear cells by a mechanism that requires cxcr4 binding [128] . ultimately, loss of cd4+ cells impairs immune responses, because cd4 + cells have critical roles in promoting and maintaining both humoral and cell-mediated immunity. a certain subset of cd4 + cells, the "treg" (for t-regulatory cells), also seems to play an important role, and treg cells with suppressive activity have been documented during early [129] and chronic fiv infection [130] . in fiv-infected cats, increased activity of treg cells could thus play a role in suppressing immune responses to foreign antigens or pathogens. in addition, treg cells are themselves targets for fiv infection [129, 131] , and may serve as a fiv reservoir during the latent stage of infection and be capable of stimulating virus production [132] . in addition, other immunologic abnormalities can be found. lymphocytes may lose the ability to proliferate in response to stimulation with mitogens or antigens, and priming of lymphocytes by immunogens may be impaired [105, [133] [134] [135] [136] [137] [138] [139] . lymphocyte function may be reduced by altered expression of cell surface molecules, such as cd4, major histocompatibility complex ii antigens, or cytokines and cytokine receptors [140] [141] [142] [143] [144] , or through over-expression of abnormal molecules, such as receptors [145] , leading to disrupted production of cytokines or receptor function. impaired neutrophil adhesion and emigration in response to bacterial products have been described in fiv-infected cats [146] [147] [148] . natural killer cell activity may be diminished [149] or increased [150] , in acutely or asymptomatically infected cats, respectively. changes in cytokine pattern include increased production of ifn-γ, tnf-α, il-4, il-6, il-10, and il-12 [151] [152] [153] [154] , but also differences in cytokine ratios (e.g., il-10/il-12 ratio) [155, 156] . in addition to a dysregulation of the immune system leading to immunosuppression, retrovirusinfected cats can also develop immune-mediated diseases caused by an overactive immune response. the most commonly seen immune-mediated response is a hypergammaglobulinemia which is caused by an excessive antibody response against the chronic persistent infection. the produced antibodies are not neutralizing and thus, may lead to antigen antibody complex formation. these immune complexes can deposit, usually in narrow capillary beds, leading to glomerulonephritis, polyarthritis, uveitis, and vasculitis. secondary immune-mediated diseases are more commonly seen in fiv-than in felv-infected cats. when comparing plasma electrophoretograms, felv-infected cats do not show hypergammaglobulinemia and hyperproteinemia significantly more often than non-infected cats, whereas in fiv-infection, hypergammaglobulinemia and hyperproteinemia occur significantly more commonly [4, 157] . nevertheless, immune-mediated diseases have been described in felv-infected cats as well. while humoral immunity to specific stimulation decreases during the course of felv infection, nonspecific increases of igg and igm have been noted. the loss of t-cell activity in combination with the formation of antigen antibody complexes promotes immune dysregulation [158] . immunemediated diseases described in felv-infected cats include imha [159] , glomerulonephritis [160] , uveitis with immune complex deposition in iris and ciliary body [161] , as well as polyarthritis [58] . chronic progressive polyarthritis can be triggered by felv; in about 20% of cats with polyarthritis, felv seems to be an associated agent [58] . measurement of felv antigen has shown that cats with glomerulonephritis have more circulating viral proteins than do other felv-infected cats. antigens that can lead to antigen antibody complex formation include not only whole virus particles, but also free gp70, p27, or p15e proteins [162, 163] . immune-mediated diseases observed in fiv-infected cats are caused by an excessive immune response leading to hypergammaglobulinemia [4, 104, 164] . hypergammaglobulinemia reflects polyclonal b-cell stimulation and is a direct consequence of fiv infection, because experimentally fiv-infected specific pathogen-free (spf) healthy cats also develop hypergammaglobulinemia [164] . increased igg as well as circulating immune complexes have been detected in fiv-infected cats [165] . chronic ulcero-proliferative gingivostomatitis is very common in retrovirus-infected cats, especially in those with fiv infection. in cats naturally infected with fiv, it is the most common syndrome (affecting up to 50%). it characteristically originates in the fauces and spreads rostrally, especially along the maxillary teeth. histologically, the mucosa is invaded by plasma cells and lymphocytes, accompanied by variable degrees of neutrophilic and eosinophilic inflammation. lesions are often painful, and tooth loss is common. severe stomatitis can lead to anorexia and emaciation. the cause of this syndrome is unclear, but the histologic findings suggest an immune response to chronic antigenic stimulation or immune dysregulation. circulating lymphocytes of cats with stomatitis have increased expression of inflammatory cytokines [103] , further implicating immune activation in the pathogenesis of this condition. this type of stomatitis is not always correlated with felv or fiv infection [166] , and is usually not seen in spf cats experimentally infected with felv or fiv, suggesting that exposure to other infectious agents also plays a role [167] . concurrent feline calicivirus (fcv) infection is often identified in the oral cavity of these cats, and experimental and naturally occurring co-infection of fiv and fcv infection results in more severe disease [168, 169] . felv can cause severe clinical syndromes, and progressive felv infection is associated with a decrease in life expectancy. still, many owners still elect to provide therapy for their felv-infected cats, and with proper treatment, felv-infected cats, especially in indoor-only households, may live for many years with good quality of life. diseases secondary to immunosuppression account for a large portion of the syndromes seen in felv-infected cats, and it is important to realize that many of these secondary diseases are treatable. in most naturally infected cats, fiv does not cause a severe clinical syndrome. most clinical signs in fiv-infected cats reflect secondary diseases, such as infections and neoplasia, to which fiv-infected cats are more susceptible. with proper care, fiv-infected cats can live many years and, in fact, commonly die at an old age from causes unrelated to their fiv infection. while long-term studies describing clinical outcomes of naturally occurring felv and fiv infection are lacking, modalities for treatment of secondary infections or other co-incident diseases are available, and by treating these symptomatically, the life expectancy and quality of life of felv-and fivinfected animals can be significantly improved. rare, direct influence of the virus (specific fiv strains), impairment of astrocyte function immunodeficiency common, several mechanisms, e.g., replication of virus in all bone marrow cells (including neutrophils), changes in cytokine pattern common, several mechanisms, e.g., decrease in cd4 + cells, changes in cytokine pattern immune-mediated diseases rare, e.g., immune-mediated hemolytic anemia sometimes, hyperglobulinemia common with immune complex deposition leading to e.g., glomerulonephritis and uveitis stomatitis common, multi-factorial disease very common, multi-factorial disease report of the national felv/fiv awareness project seroprevalence of feline leukemia virus and feline immunodeficiency virus infection among cats in north america and risk factors for seropositivity feline leukemia-virus infection and diseases hematology and serum biochemistry of feline immunodeficiency virusinfected and feline leukemia virus-infected cats long-term impact on a closed household of pet cats of natural infection with feline coronavirus, feline leukaemia virus and feline immunodeficiency virus feline viral neoplasia felv and non-neoplastic 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leukemia virus-associated enteritis and other relevant forms of feline enteritis feline leukemia virus-associated myelopathy in cats an oligopeptide of the feline leukemia virus envelope glycoprotein is associated with morphological changes and calcium dysregulation in neuronal growth cones felv envelope protein (gp70) variable region 5 causes alterations in calcium homeostasis and toxicity of neurons feline immunodeficiency virus: a neurotropic lentivirus feline immunodeficiency virus neurotropism: evidence that astrocytes and microglia are the primary target cells aids-associated encephalopathy with experimental feline immunodeficiency virus infection development of clinical disease in cats experimentally infected with feline immunodeficiency virus neurological abnormalities associated with feline immunodeficiency virus infection regional distribution of lesions in the central nervous system of cats infected with feline immunodeficiency virus neurovirulence in feline immunodeficiency 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feline retrovirus lymphocyte subset alterations and viral determinants of immunodeficiency disease induction by the feline leukemia virus felv-faids comparison of t-cell subpopulations in cats naturally infected with feline leukaemia virus or feline immunodeficiency virus the effects of feline retroviruses on cytokine expression cvt update: feline immunodeficiency virus. in kirk's current veterinary therapy xiii small animal practice, bonagura immunologic abnormalities in pathogen-free cats experimentally infected with feline immunodeficiency virus acquired immune dysfunction in cats with experimentally induced feline immunodeficiency virus infection: comparison of short-term and long-term infections early events in the immunopathogenesis of feline retrovirus infections lymphocyte population changes in cats naturally infected with feline immunodeficiency virus decline in cd4+ cell numbers in cats with naturally acquired feline immunodeficiency virus infection evaluation of t lymphocytes in captive african lions (panthera leo) infected with feline immunodeficiency virus infection with feline immunodeficiency virus is followed by the rapid expansion of a cd8+ lymphocyte subset expansion of cd8alpha+beta-cells in cats infected with feline immunodeficiency virus phenotypic changes in cd8+ peripheral blood lymphocytes in cats infected with feline immunodeficiency virus pathogenesis of a texas feline immunodeficiency virus isolate: an emerging subtype of clade b programmed cell death (apoptosis) as a mechanism of cell death in peripheral blood mononuclear cells from cats infected with feline immunodeficiency virus (fiv) apoptosis induced by tumor necrosis factor in cells chronically infected with feline immunodeficiency virus induction of apoptosis in a t lymphoblastoid cell line infected with feline immunodeficiency virus apoptosis and cd4+ lymphocyte depletion following feline immunodeficiency virus infection of a t-lymphocyte cell line spontaneous programmed 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with the virus spontaneous programmed cell death (pcd) process of lymphocytes of fiv-infected cats: cellular targets and modulation the role of in vitro-induced lymphocyte apoptosis in feline immunodeficiency virus infection: correlation with different markers of disease progression feline immunodeficiency virus envelope glycoprotein mediates apoptosis in activated pbmc by a mechanism dependent on gp41 function cd4+cd25+ regulatory t cells are infected and activated during acute fiv infection transforming growth factor-beta/transforming growth factor-betarii signaling may regulate cd4+cd25+ t-regulatory cell homeostasis and suppressor function in feline aids lentivirus infection different thresholds of t cell activation regulate fiv infection of cd4+cd25+ and cd4+cd25-cells preferential replication of fiv in activated cd4(+)cd25(+)t cells independent of cellular proliferation decrease in mitogen-induced lymphocyte proliferative responses in cats infected with feline immunodeficiency 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il-2-dependent andindependent feline lymphocyte lines: host cells range distinctions and specific cytokine upregulation t cells overexpressing interferon-gamma and interleukin-10 are found in both the thymus and secondary lymphoid tissues of feline immunodeficiency virus-infected cats cd8+ thymic lymphocytes express reduced levels of cd8beta and increased interferon gamma in cats perinatally infected with the jsy3 molecular clone of feline immunodeficiency virus constitutive expression of types 1 and 2 cytokines by alveolar macrophages from feline immunodeficiency virus-infected cats effect of feline immunodeficiency virus on cytokine response to listeria monocytogenes in vivo altered bone marrow dendritic cell cytokine production to toll-like receptor and cd40 ligation during chronic feline immunodeficiency virus infection plasma electrophoretogram in feline immunodeficiency virus (fiv) and/or feline leukaemia virus (felv) infections feline leukemia virus infection primary immune-mediated hemolytic anemia in 19 cats: diagnosis, therapy, and outcome membranous glomerulonephritis associated with leukaemia in cats ocular disease in felv-positive cats: 11 cases (1981-1986) circulating immune complexes associated with naturally occurring lymphosarcoma in pet cats detection of circulating immune complexes by a clq/protein a-elisa during the preneoplastic stages of feline leukemia virus infection polyclonal b-cell activation in cats infected with feline immunodeficiency virus serum concentration of circulating immune complexes in cats infected with feline immunodeficiency virus detected by immune adherence hemagglutination method evaluation of the association of bartonella species, feline herpesvirus 1, feline calicivirus, feline leukemia virus and feline immunodeficiency virus with chronic feline gingivostomatitis feline immunodeficiencyvirus chronic oral infections of cats and their relationship to persistent oral carriage of feline calici-, immunodeficiency, or leukemia viruses effect of chronic feline immunodeficiency virus infection on experimental feline calicivirus-induced disease many doctoral students have contributed to various parts of the author's publications cited in this paper and are acknowledged for all their hard work. the authors declare no conflict of interest. key: cord-318172-bdotp9ko authors: blanco, jorge c. g.; core, susan; pletneva, lioubov m.; march, thomas h.; boukhvalova, marina s.; kajon, adriana e. title: prophylactic antibody treatment and intramuscular immunization reduce infectious human rhinovirus 16 load in the lower respiratory tract of challenged cotton rats date: 2014-01-01 journal: trials in vaccinology doi: 10.1016/j.trivac.2014.02.003 sha: doc_id: 318172 cord_uid: bdotp9ko human rhinoviruses (hrv) represent the single most important etiological agents of the common cold and are the most frequent cause of acute respiratory infections in humans. currently the performance of available animal models for immunization studies using hrv challenge is very limited. the cotton rat (sigmodon hispidus) is a well-recognized model for the study of human respiratory viral infections. in this work we show that, without requiring any genetic modification of either the host or the virus, intranasal infection of cotton rats with hrv16 resulted in measurable lower respiratory tract pathology, mucus production, and expression of interferon-activated genes. intramuscular immunization with live hrv16 generated robust protective immunity that correlated with high serum levels of neutralizing antibodies. in addition, cotton rats treated prophylactically with hyperimmune anti-hrv16 serum were protected against hrv16 intranasal challenge. finally, protection by immunization was efficiently transferred from mothers to newborn animals resulting in a substantial reduction of infectious virus loads in the lung following intranasal challenge. overall, our results demonstrate that the cotton rat provides valuable additional model development options for testing vaccines and prophylactic therapies against rhinovirus infection. human rhinoviruses (hrv) represent the most important etiological agents of common colds and the most frequent cause of acute respiratory infections in humans [1] [2] [3] . hrv are single-stranded positive-sense rna viruses, members of the picornaviridae family, genus enterovirus which includes the three so far recognized species rhinovirus a, b and c and also species enterovirus a-j [4, 5] . the recent sequencing and analysis of all known hrv serotypes classified within species a and b and of genomic rna corresponding to species rhinovirus c allowed a better understanding of evolutionary relations among viruses classified within the 3 species and also of some of the structural implications of genetic variability in genes encoding capsid proteins [6, 7] . hrv is currently a frequently detected virus in association with hospitalizations for acute respiratory illness in young children and the elderly [8, 9] and also a frequent opportunistic pathogen of transplant recipients [10] . in addition, hrv infections have been linked to exacerbation episodes in asthmatic [11] , and chronic obstructive pulmonary disease (copd) patients [12] . due to the occurrence of more than 100 hrv serotypes with extensive sequence variability in the antigenic sites and the lack of animal models to test the efficacy of approaches to prevent or treat infection in vivo, no rhinovirus vaccines are available for use in humans. the lack of animal models has also limited the ability to conduct preclinical studies of antiviral compounds. limitations on animal modeling options for human rhinovirus infections are -at least in part -consequences of the high host species specificity of these viruses. chimpanzees have been successfully infected with hrv14 and hrv43, and gibbons with hrv1a, hrv2, and hrv14, but no overt illness was observed in the infected animals [13, 14] . intracranial injections of virus into monkeys, hamsters, or baby mice did not result in either infection http or disease [15, 16] . various balb/c and c57/bl mouse models have been recently developed and utilized for the study of aspects of minor-and major-group hrv-induced disease [17] [18] [19] [20] [21] . in both mouse models hrv infection was tested showing limited replication of the virus [17] . the [22] [23] [24] showing strong serological data suggesting cross protection. however their published data did not include proof of vaccine efficacy. thus, no successful immunization/challenge/protection studies in any animal model have been reported. the availability of new experimental small animal model options is therefore still a major pressing need in the vaccinology field. over the years, the cotton rat (sigmodon hispidus) has been shown to support replication of a broad spectrum of human viruses including respiratory syncytial virus (rsv) [25] , nonadapted strains of human influenza [26, 27] , and measles [28, 29] , among others [30] , providing modeling capabilities for the corresponding infections. in the present study, we evaluated the cotton rat as a model of human rhinovirus infection that could potentially facilitate the development and testing of vaccines and prophylactic therapies aimed against these important human viruses. stocks of hrv1b (atcc cat# vr-1645), and hrv16 (strain 11757, atcc cat# vr-283), were produced in hela ohio cells, a generous gift of dr. dean erdman (cdc, atlanta, ga, usa). cells were grown in minimal essential medium containing earle salts (e-mem), 10% fetal bovine serum (fbs); 1.5 g/ml na 2 co 3 , l-glutamine and penicillin/streptomycin and maintained in the same medium with 2% fbs. replication-deficient virus was generated by exposing the stock to ultraviolet light for 15 min on ice at 100 mj/cm 2 . infectious virus titers were determined by plaque assay under agarose overlay in monolayers of the same cell line as described below to control for ablation of infectivity. four to eight weeks old cotton rats of both genders were obtained from the inbred colony maintained at sigmovir biosystems, inc. (sbi). sera from sentinel cotton rats in the colony during the time period of all experiments tested seronegative for rhinovirus by neutralization assay, and seronegative by elisa to adventitious respiratory viruses (e.g. pneumonia virus of mice, rat parvovirus, rat coronavirus, sendai virus), and mycoplasma sp. oropharyngeal and fecal cultures tested negative for salmonella sp., klebsiella sp., pseudomonas sp., and citrobacter sp.). pups were obtained from mothers in the same colony. animals were housed in large polycarbonate cages, and fed a diet of standard rodent chow and water ad libitum. all experiments were performed following federal guidelines and protocols approved by sbi's institutional animal care and use committee (iacuc). cotton rats were infected intranasally (i.n.) or immunized intramuscularly (i.m.) with hrv16 under isoflurane anesthesia by application of 100 ll of solution (10 6 -10 7 pfus) per rat. i.m. immunization with 10 7 pfus of live hrv1b, uv-inactivated hrv16 (10 7 pfu), or lpol ò polio vaccine was carried out under the same conditions. serum was obtained by retro-orbital blood collection under isoflurane anesthesia. pups (3-5 days old) were infected i.n. with 20 ll of virus ($5 â 10 6 pfus). adult animals were euthanized by carbon dioxide asphyxiation. pups were euthanized by decapitation. tissue samples (left lung lobe, entire nose, and trachea) from adult animals were homogenized in 3 ml of infection medium (e-mem, 2% fetal calf serum, 1.5 g/ml sodium bicarbonate, 25 mm hepes, penicillin and streptomycin), whereas left lung lobes from pups were homogenized in 1 ml. infectious virus titers were determined by standard plaque assay and expressed as plaque forming units (pfu)/g of tissue. briefly 100 ll of pure or tenfold serial dilutions of tissue homogenate supernatant or virus stocks were plated in triplicate onto confluent monolayers of hela oh cells in 6-well plates. after 1 h adsorption with rocking every 10 min, monolayers were overlayed with 2 ml of 0.7% low melt agarose in e-mem containing 2% fbs. following incubation for 3 days at 33°c, monolayers were fixed with buffered formaldehyde and stained with crystal violet. neutralizing antibody titers were determined in a plaquereduction assay. serial fourfold dilutions of serum were incubated for 1 h at 37°c with $50 pfu of hrv16. virus incubated with pbs was used as control. neutralization mixes were then plated in quadruplicate onto confluent hela cell monolayers in 24-well plates, incubated at 33°c for 1 h and overlayed with 0.7% low melt agarose in mem. cells were incubated at 33°c and 5% co 2 for 72 h and then fixed with 1% buffered formaldehyde and stained with crystal violet for reading. rna was isolated from the lung lingular lobe using the rneasy kit (qiagen sciences). bronchoalveolar lavage (bal) was collected for total rna isolation from a dedicated group of infected animals by lavaging 3 times with a total 2 ml of pbs each. for mrna assays, cdna was prepared by using quantitect reverse transcription kit (qiagen sciences). each cdna reaction was diluted to match 1 lg of the initial rna per 100 ll of the final cdna volume. three microliters of diluted cdna were used for each qpcr reaction. a hrv16specific qpcr protocol was developed using primers that target the 5 0 utr of hrv-16 (genbank accession# l24917). for a quantitative assessment of viral replication, cdna for the negative stranded viral rna (as an indicator of active rna transcription), was synthesized by priming with 5 0 -ccctttcccaaatgtaacttagaagc-3 0 . realtime pcr quantification of both strands was performed using the forward primer 5 0 -tcaagcacttctgtttccccggt-3 0 and the reverse primer 5 0 -tcccatcccgcaattgctcattac-3 0 , generating a fragment of 370 bp. a plasmid containing the 5 0 utr of hrv16 was diluted to generate a copy number standard curve that allowed for the quantification of copies/g tissue of the negative replication intermediates. the assessment of cotton rat mx-1 and mx-2 mrna expression was carried out as previously described [30, 31] . the right lobes of the lung were dissected with the caudal 1/3 of the trachea attached. lung lobes were inflated to their normal volume with 10% neutral buffered formalin, immersed in the same fixative, and then processed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (h&e). additional sections were stained with alcian blue-periodic acid-schiff (ab-pas) for visualization of mucus and mucus-bearing cells. lung sections were evaluated for several indices of inflammation and cell changes in the conducting airway and parenchymal (alveolar) compartments (described more fully in the results). changes were assessed subjectively and scored blindly on a 0 to 4 scale for severity (absent, minimal, mild, moderate, marked), with validation of scoring done by two pathologists experienced in respiratory viral pathogenesis. graded findings included peribronchiolar inflammatory cell infiltrates, bronchiolitis, bronchiolar epithelial degeneration, mucous cell hyperplasia /hypertrophy, airway luminal mucous /other exudate, perivascular inflammatory cell infiltrates, alveolar septal infiltrates, and alveolar luminal inflammatory cell exudates. tracheas and nasal turbinates from selected, infected animals were also evaluated. viral titers, expression of mx genes, and production of the hrv16 replicative intermediate were calculated as geometric means ± standard error for all animals in a group at a given time post infection. student t-test was applied to determine statistically significant differences between two groups, using an unpaired, two-tailed test set at p < 0.05. pulmonary pathology scores were expressed as the arithmetic mean ± standard error for all animals in a group. significance of score differences between uninfected and infected groups at different days post-infection for each histological parameter was analyzed by one way kruskal-wallis anova and set at a value of p < 0.05. adult cotton rats were infected i.n. with 10 7 pfu of hrv16. groups of 5 animals were euthanized at 30 min, 2, 4, 6, 8, and 10 h, and at day 1, 2, and 4 post-infection (p.i.) (fig. 1a) . non-infected animals or animals inoculated with uv-inactivated hrv16 were used as controls. infectious hrv16 was recovered from the nose and trachea until day 1 p.i., and from the lung until day 2 p.i. no virus was detected in any of the tissues from animals euthanized on day 4 p.i. or in any of the tissues from uninfected controls (not shown). the highest virus titers were detected in the lungs (>10 7 pfu/g of tissue), followed by the nose and the trachea ($10 6 pfu/g of tissue). a brief plateau of viral loads was detectable in the nose and trachea between 6-10 h p.i. followed by a decrease and clearance of virus. infection with hrv16 at a lower dose (10 6 pfu/animal) resulted in lung viral titers of 5.9 â 10 5 ± -8.6 â 10 4 pfu/g at 8 h p.i. (n = 16), with no detectable infectious virus at 48 h p.i. (data not shown). we compared the viral load in the lung, between rats infected with the major group hrv16 and rats infected with the minor group hrv1b and found that the course of infection with the 2 viruses was very different (fig. 1b) . higher infectious virus titers were recovered from the lungs of animals infected with hrv16 than from animals infected with a similar dose of hrv1b, suggesting that the cotton rat is significantly more permissive to infection by hrv16. based on these results we only used hrv16 in all the subsequent experiments. the lung load of virus was also examined by determining production of the replication intermediate, negative strand viral rna [(à)vrna] by pcr (fig. 1c) . (à)vrna strand was detected in lungs of infected animals up to 48 h p.i. (fig. 1c , see also inset). animals inoculated with uv-inactivated hrv16 (10 7 pfu) showed undetectable negative strand hrv-16 rna. the apparent rapid kinetics of viral replication observed in vivo were consistent with data from one-step growth curves carried out in hela ohio cells showing that a complete replication cycle of hrv16 occurs in 6-10 h (fig. 1d) . we measured the expression of cotton rat mx1 and mx-2 genes in the lungs in response to hrv16 infection as evidence of presence of type i ifns. mx1 and mx2 are two ifn-inducible genes that mediate antiviral activity [31] [32] [33] . the activation of expression of mx-1 and mx-2 was detected in bal cells of hrv16-infected cotton rats at 6 h p.i. (fig. 1e) but not in either of the two subsequent time points (12 h and 24 h -data not shown), indicating that the induction of ifn was transient. analysis of the pathology associated with hrv16 infection was performed in the nose, trachea, and lung. no significant lesions were observed in the nasal turbinate sections. epithelial degeneration was present in the trachea and large pulmonary airways of hrv16-infected rats. infection was associated with direct and progressive damage of the ciliated columnar epithelium of the trachea that peaked on day 4 p.i. and often exposed the basal membrane ( fig. 2a) . lung pathology demonstrated mild but significant alveolitis (neutrophilic and histiocytic), and peribronchiolar infiltrates of neutrophils, macrophages, and lymphocytes (fig. 2b) . peak damage of the lung parenchyma (perivasculitis, alveolar septal infiltrates, and alveolitis) was recorded on day 1-2 p.i., whereas airway damage was predominantly seen on day 3 p.i. mucous cell hypertrophy/hyperplasia was evident in h&e-and ab-pas-stained lung sections as early as 1 day p.i. but continue elevated by day 4 p.i. (fig. 2c) . thus, hrv16 infection in the cotton rat reproduces aspects of human disease in the urt with detectable inflammation in the lower airways and lung parenchyma. in contrast, infection with hrv1b did not result in significant pathology. intramuscular immunization of adult rats with live hrv16 at a dose of 10 6 pfus in a priming (day 0) and boosting (day 21) schedule resulted in high serum levels of neutralizing antibodies at 42 days after the first immunization. surprisingly, that was not the case when the same amount of virus was instilled i.n. following an identical schedule. as shown in table 1 , all animals immunized intramuscularly showed neutralizing antibody titers >1280, whereas animals that underwent i.n. infection or re-infection with hrv16 showed low neutralizing antibody titers (<16). furthermore, when animals were immunized i.m. once with 10 7 pfus and challenged i.n. 21 days later with hrv16 (10 7 pfus), infectious virus was not detectable in the nasal turbinates or in the trachea, and a reduction (>3 log 10 ) in infectious virus titers was detected in the lung (fig. 3a) . as expected, intramuscular immunization with live hrv1b, or uv-inactivated hrv16 (10 7 pfu), or with a current polio vaccine (ipol) failed to confer measurable protection upon i.n. hrv16 challenge (fig. 3b) . the possibility that the observed reduction in viral titers in the lungs of animals vaccinated i.m. was caused by neutralization in vitro was evaluated by mixing an equal amount of hrv16-infected lung homogenate from a naïve animal with lung homogenates (h1, h2, h3) of individual vaccinated animals, all euthanized at 10 h p.i. as shown in fig. 3d , homogenate mixes yielded the amount of virus in the lung suspension predicted from dilution in the indicated proportions (1:2 and 1:5 respectively) without evidence of in vitro neutralization. this result indicated that the protection observed was due to bona fide immunity and not to neutralization of the virus ex vivo after homogenization of the tissue. we tested the efficacy of the prophylactic administration of immune (neutralizing antibody titer of 1280 against hrv16) or normal cotton rat serum to protect against hrv16 challenge. the neutralizing antibody titers detected in treated animals prior to challenge are shown in table 2 . all animals that received 500 ll of undiluted sera (neat) intraperitonealy or serum diluted 1:4, 24 h prior to challenge showed a reduction of lung viral titers of 2 and 1 log 10 , respectively, whereas animals that received more diluted immune serum (1:8), naïve/control cotton rat serum, or pbs, remained unprotected (fig. 4) . these data indicate that passive transfer of antibodies can be an effective prophylactic therapy against hrv infection. young female cotton rats that were immunized twice i.m. with live hrv16 (10 6 pfus) were bred to unimmunized males. newborn pups delivered from immunized or non-immunized females (the gestation time for s. hispidus is 4 weeks) were challenged at 3-5 days of age with hrv16 i.n. and euthanized at 10, 24, and 48 h p.i. pups from non-immunized mothers showed lung hrv16 titers consistent with those of naïve adult cotton rats challenged with hrv16 (fig. 5a) . pups from immunized mothers showed a statistically significant reduction in infectious virus titers (>3 log 10 ) and also a significant reduction in the viral load measured as copies of hrv16 (à)vrna (fig. 5b) . neutralizing antibody titers in the pups correlated with the level of lung protection. fully protected pups (all animals euthanized at 10 h, fig. 5 ) showed neutralization titers >8192, whereas pups euthanized at 24 or 48 h post infection harboring low but detectable virus titers showed neutralizing antibody titers of $256, suggesting a correlation of protection and serum neutralizing antibody titers as seen in the adults. these data indicate that maternal immunity conferred by immunization is transferable to newborn pups and sufficient for protection of offspring from viral infection and possible replication in the lung. the morbidity and mortality attributable to rhinovirus infection are considerable and result in billions of dollars of health care costs every year. despite the significance of the problem, no effective prevention of hrv infection or treatment of hrv-associated disease is currently available due in part to the lack of a reliable animal model suitable for protection studies. hrv16 is one of the 91 serotypes classified within the major group that use the well-characterized icam-1/cd56 receptor for attachment and entry [34] , and has been a model virus for studying transmission of rhinoviruses [35] , pathogenesis of the common cold [35, 36] , virus-induced asthma and copd [37] [38] [39] , and for the evaluation of anti-rhinovirus drugs in human volunteers because it reproducibly induces severe symptoms in human subjects. hrv16 has been recently used for in vitro studies aiming to define the molecular mechanisms of rhinovirus-induced inflammatory responses in airway epithelial cells [40, 41] . transgenic balb/c mice expressing a human/mouse icam-1 chimeric receptor show human disease-relevant outcomes including pulmonary inflammation and mucin production when infected i.n. with hrv16, despite the low levels of viral replication detectable by rt-pcr [17] . our data show that in contrast to the mouse, which requires genetic manipulation, the cotton rat appears to host infection and possibly replication of hrv16 in the urt and lrt. although the data from examination of viral load in the respiratory tract over time is certainly compatible with a non-permissive model of infection and clearance of the inoculum over time, the differences observed between the course of infection by hrv16 and hrv1b together with the detection of (à)vrna in infected tissues suggest that hrv16 may be actually replicating in the cotton rat. the refinement of our real time qrt-pcr protocols to increase specificity for the detection of the (à) strand viral rna will provide the ability to address this important issue and better describe the model. in contrast to what has been described for other respiratory viral infections, such as influenza [26, 27] , hrv infection of cotton rats resulted only in mild epithelial damage (vacuolar degeneration, apical blebbing in the bronchioles, sloughing in the trachea), consistent with previous observations in humans [42, 43] . unlike the laboratory mouse, which either lacks or has defective mx genes, the cotton rat has a set of fully functional mx genes. as described for the human mx counterparts [31, 32, 44, 45] , cotton rat mx genes are expressed in response to infection with influenza and rsv. as shown by the present study, transient mx gene expression was detected in infected coton rat bal cells 6 h post hrv16 infection. this result parallels the detection of mx1 (mxa) and mx2 expression in nasal epithelial scrapings obtained 8 h after experimental hrv16 infection of humans [46] . the results of the described experimental work show that hrv16 infection in the cotton rat reproduces aspects of hrvassociated human disease in the respiratory tract, causing detectable inflammation in the lower airways and lung parenchyma and mucus production, and inducing a transient expression of interferon-stimulated genes that merits further investigation. in our model of i.n. infection, the pulmonary infectious viral load is measurable within the 48 h following instillation and thus provides a powerful readout for assessment of protection by immunization and passive transfer of neutralizing antibodies. we found that i.m. inoculation of live virus but not uv-inactivated resulted in a significant decrease of viral loads in nasal turbinates, trachea, and lung of challenged animals suggesting that replication at the site of injection played a role in the resulting. a measurable protection against challenge was achieved with only one immunization (fig. 3) . the immunogenicity of the inoculum was likely dependent upon viral replication in the area of immunization since uv-inactivation of the virus completely abolished protection. the protection achieved with live virus was specific for hrv16 since no protection was conferred by immunization with live hrv1b. these results could be due to the apparent lack of replication of hrv1b in the cotton rat respiratory tract (data not shown), but are most likely the result of the lack of shared neutralizing epitopes between hrv16 and hrv1b. the spectrum of cross-reactivities of cotton rat anti-hrv16 neutralizing antibodies should be further investigated in view of the recent reports regarding the induction of in vitro cross-neutralizing antibodies after immunization with recombinant hrv14 and hrv89 vp1 [22] , and the weak induction of hrv1b binding antibodies but not neutralizing antibodies in the mouse by immunization with hrv16 vp0 [23] . previous studies conducted in cotton rats have demonstrated the passive transfer of maternal immunity against rsv to offspring [47] . in this study, we showed that the anti-hrv16 immunity elicited in mothers through i.m. immunization also conferred complete protection of their 3-5 days old pups against i.n. challenge with hrv. importantly, data from this experiment strongly suggest that newborn cotton rats also support viral replication in the lrt, setting the stage for future testing of potential antibody-based and other therapies for infants. the cotton rat is well-recognized for its pivotal role in the development of the immunoglobulin prophylactic treatments for rsv-associated disease, respigam ò and synagis ò [48] [49] [50] [51] . data from the present study demonstrate that passive transfer of cotton rat hyper-immune serum is also effective and that a neutralizing antibody titer of 320 was sufficient for a 2 log 10 reduction of viral load in the lung. without a doubt, the cotton rat model will be useful for determining the value of different arrays of anti-rhinovirus antibodies in an in vivo challenge experimental design. although the high diversity of serotypes that characterize this group of viruses remains a major challenge for the development of pan-crossreactive antibody-based intervention, the work conducted using our cotton rat model of hrv infection generated strong evidence that the parenteral route of immunization can be effective and is therefore disserving of additional investigation given the important questions that have been raised in the vaccinology field regarding the design strategies that will be required for effective multivalent hrv vaccines or therapeutic antibodies. importantly, our data highlight the large potential of the cotton rat to provide an experimental platform complementary to that offered by the available mouse models to further evaluate the immunogenicity of individual hrv capsid proteins [22, 23, 47] , some of the already identified anti-hrv antibodies with documented neutralizing activities in vitro [52] , antibodies targeting select domains of the virus receptor [53] , as well as other alternative antibody-based strategies under consideration [54] to support new developments in this area. in this work we show that the cotton rat hosts infection with hrv16 with infectious virus (and negative stranded vrna) loads detectable in the lung of infected animals within 48 h following intranasal infection. infection results in measurable pathology, mucus production, and expression of inflammatory mediators. while intranasal infection with hrv16 resulted in the production of low levels of neutralizing antibodies that conferred reduced protection after re-challenge, intramuscular immunization with live hrv16 resulted in strong type-specific protection that correlated with high levels of systemic neutralizing antibodies. in addition we demonstrated that passive transfer of antibodies generated in vaccinated cotton rats can protect naïve animals from infectious virus titers in the lung were determined by plaque assay at the indicated times p.i. (b) hrv16 (à) viral rna detection in lungs of newborn cotton rats. n = 6-8 animals/group where each group consisted of pups from 2 different mothers. ⁄ p < 0.05 in student t-test comparison between group of pups whose mothers were immunized vs groups of pups of the same age from unimmunized, naïve mothers. pulmonary infection. thus, these results demonstrate that the cotton rat is a suitable animal model for challenge studies to explore the development of anti-rhinovirus vaccines and anti-viral therapies. frequency and natural history of rhinovirus infections in adults during autumn fields virology human rhinoviruses virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses proposals for the classification of human rhinovirus species a, b and c into genotypically assigned types sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution analysis of the complete genome sequences of 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response mechanisms of immunity to respiratory syncytial virus in cotton rats quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats a direct comparison of the activities of two humanized respiratory syncytial virus monoclonal antibodies: medi-493 and rshzl9 reduction of respiratory syncytial virus hospitalization among premature infants and infants with bronchopulmonary dysplasia using respiratory syncytial virus immune globulin prophylaxis the impact-rsv study and group, palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants antibodies to the buried n terminus of rhinovirus vp4 exhibit cross-serotypic neutralization an anti-human icam-1 antibody inhibits rhinovirus-induced exacerbations of lung inflammation the application of prophylactic antibodies for rhinovirus infections we thank raymonde oue, jamall mckay, and susana canas for their technical support and charles smith, marta malache, and ana and freddy rivera for cotton rat husbandry. the present study has been supported national institutes of health, united states, grant ai101480 to j.c.g.b. key: cord-337105-jlmh79qv authors: jacob, fadi; pather, sarshan r.; huang, wei-kai; zhang, feng; hao wong, samuel zheng; zhou, haowen; cubitt, beatrice; fan, wenqiang; chen, catherine z.; xu, miao; pradhan, manisha; zhang, daniel y.; zheng, wei; bang, anne g.; song, hongjun; carlos de la torre, juan; ming, guo-li title: human pluripotent stem cell-derived neural cells and brain organoids reveal sars-cov-2 neurotropism predominates in choroid plexus epithelium date: 2020-09-21 journal: cell stem cell doi: 10.1016/j.stem.2020.09.016 sha: doc_id: 337105 cord_uid: jlmh79qv neurological complications are common in patients with covid-19. while sars-cov-2, the causal pathogen of covid-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood. here we investigated the susceptibility of human induced pluripotent stem cell (hipsc)-derived monolayer brain cells and region-specific brain organoids to sars-cov-2 infection. we found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. we optimized a protocol to generate choroid plexus organoids from hipscs and showed that productive sars-cov-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. together, our findings provide evidence for selective sars-cov-2 neurotropism and support the use of hipsc-derived brain organoids as a platform to investigate sars-cov-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the pathogen responsible for the covid-19 pandemic, has infected over 29 million people and has contributed to over 936,000 deaths world-wide this year so far (who, 2020) . while the disease primarily affects the respiratory system, damages and dysfunction have also been found in other organs, including the kidney, heart, liver, and brain (yang et al., 2020b) . neurological complications, such as cerebrovascular injury, altered mental status, encephalitis, encephalopathy, dizziness, headache, hypogeusia, and hyposmia, as well as neuropsychiatric ailments, including new onset psychosis, neurocognitive syndrome, and affective disorders, have been reported in a significant number of patients (mao et al., 2020; varatharaj et al., 2020) . viral rna has been detected in the brain and cerebrospinal fluid (csf) of some patients with covid-19 and concomitant neurological symptoms (helms et al., 2020; moriguchi et al., 2020; puelles et al., 2020; solomon et al., 2020) . despite numerous reports of neurological findings in patients with covid-19, it remains unclear whether these symptoms are a consequence of direct neural infection, para-infectious or post-infectious immune-mediated disease, or sequalae of systemic disease (ellul et al., 2020; iadecola et al., 2020) . variability in patient presentation and variable timing of testing for viral rna in the csf or brain complicate the interpretation of results in human patients. limited availability of autopsy brain tissue from patients with covid-19 and neurological symptoms and the inability to study ongoing disease pathogenesis in the human brain further underscore the need for accessible and tractable experimental models to investigate sars-cov-2 neurotropism, its functional impact, and to test therapeutics. classic animal models, such as rodents, are limited in their ability to recapitulate human covid-19 symptoms and usually require viral or transgenic mediated overexpression of human sars-cov-2 receptor angiotensin-converting enzyme 2 (ace2) to exhibit symptoms sun et al., 2020) . although cell lines, including many human cancer cell lines, have been used to study sars-cov-2 infection and test drug efficacy (hoffmann et al., 2020b; ou et al., 2020; shang et al., 2020; wang et al., 2020) , they do not accurately recapitulate normal human cell j o u r n a l p r e -p r o o f 4 behavior and often harbor tumor-associated mutations, such as tp53, which may affect sars-cov-2 replication or the cellular response to sars-cov-2 infection (ma-lauer et al., 2016) . furthermore, human tissue and organ systems contain multiple cell types with variable levels of ace2 expression and viral susceptibility, which are not adequately represented in these human cell lines. these limitations support the development of human cellular models for sars-cov-2 infection that better recapitulates the cellular heterogeneity and function of individual tissues. human pluripotent stem cell (hipsc)-based models provide an opportunity to investigate the susceptibility of various brain cell types to viral infection and their consequences. hipscs have been used to generate a variety of monolayer and three dimensional (3d) organoid cultures to study human diseases and potential treatments. for example, hipsc-derived neural progenitors in monolayer and brain organoids were instrumental in studying the impact of zika virus infection on human brain development and solidifying the link between zika virus infection of neural progenitor cells and microcephaly seen in newborns (ming et al., 2016; qian et al., 2016; tang et al., 2016) . additionally, these cultures were useful in screening for drugs to treat zikv infection (xu et al., 2016) . recently, hipsc-derived organoids have been used to model sars-cov-2 infection in many organs, including the intestine, lung, kidney, liver, pancreas, vasculature and brain (lamers et al., 2020; monteil et al., 2020; ramani et al., 2020; yang et al., 2020a; zhou et al., 2020) . these studies have shown that sars-cov-2 can infect and replicate within cells of multiple organs, leading to transcriptional changes indicative of inflammatory responses and altered cellular functions. here, we used hipsc-derived neurons, astrocytes, and microglia in monolayer cultures and region-specific brain organoids of the cerebral cortex, hippocampus, hypothalamus, and midbrain to investigate the susceptibility of brain cells to sars-cov-2 infection. we observed sparse infection of neurons and astrocytes, with the exception of regions of organoids with choroid plexus epithelial cells that exhibited high levels of infectivity. using an optimized protocol to generate choroid plexus organoids (cpos) from hipscs, we showed evidence of productive infection by sars-cov-2 and functional consequences at cellular and molecular levels. to investigate the susceptibility of human brain cells to sars-cov-2 infection, we tested various hipsc-derived neural cells in monolayer cultures and region-specific brain organoids generated using several established (qian et al., 2018; qian et al., 2016) and modified protocols (sakaguchi et al., 2015) ( figure 1a ). monolayer hipsc-derived cortical neurons, astrocytes, and microglia were exposed to either sars-cov-2 virus isolate or vehicle control for 12 hours and analyzed at 24, 48, and 120 hours post-infection (hpi) for immunolabelling using convalescent serum from a patient with covid-19 or sars-cov-2 nucleoprotein antibodies (figures s1a-c). we confirmed the identity of neurons, microglia, and astrocytes by immunostaining for markers map2, pu.1, and gfap, respectively (figures s1a-c). hipsc-derived cortical neurons were co-cultured on hipscderived astrocytes to improve survival, and analysis at 120 hpi showed rare infection of map2 + cells ( figure s1a ). at 48 and 120 hpi, hipsc-derived microglia showed no infection of pu.1 + cells ( figure s1b ), whereas hipsc-derived astrocytes showed sparse infection of gfap + cells ( figure s1c ). as a validation, we tested infection of primary human astrocytes and observed sparse infection at 24, 48, and 120 hpi ( figure s1d ). quantification of hipsc-derived and primary human astrocytes showed infection of 0.02% and 0.18% of all cells at 120 hpi, respectively ( figure s1e ). these results revealed the ability of sars-cov-2 to infect monolayer human cortical neurons and astrocytes, but not microglia, although infection of neurons and astrocytes was rare. to examine the neurotropism of sars-cov-2 in a model system that more closely resembles the human brain, we exposed cortical, hippocampal, hypothalamic, and midbrain organoids to sars-cov-2 or vehicle control for 8 hours and analyzed samples at 24 and 72 hpi. we confirmed the regional identity of cortical, hippocampal, hypothalamic, and midbrain organoids by immunostaining with the markers ctip2, prox1, otp, and th, respectively ( figure s1f ). sars-cov-2 nucleoprotein was detected sparsely in these organoids derived from two different hipsc lines in a range that averaged between 0.6% and 1.2% of all cells at 24 and 72 hpi (figures j o u r n a l p r e -p r o o f 6 1b, 1c, and s1g). co-immunolabeling with dcx and sars-cov-2 nucleoprotein identified most of infected cells as neurons in all organoids ( figure 1d ) and infection of some gfap + astrocytes was found in hypothalamic organoids ( figure s1h ). because these brain organoids contained mostly neurons, the relative susceptibility of neurons compared to astrocytes could not be assessed with certainty. the number of infected cells did not significantly increase from 24 to 72 hpi ( figures 1c), indicating that infection may not spread among neurons in brain organoids within this time window. during development, the choroid plexus develops adjacent to the hippocampus (lun et al., 2015) and some of our hippocampal organoids contained regions with choroid plexus epithelial cells, which were identified by transthyretin (ttr) expression ( figure 1e ). notably, we observed a greater density of infected cells in these regions ( figure 1e ). additionally, we observed colocalization of patient convalescent serum and double-stranded rna (dsrna) immunolabeling in regions with choroid plexus cells, further supporting sars-cov-2 infection ( figure 1f ). together, these results demonstrate that sars-cov-2 exhibits limited tropism for neurons and astrocytes of multiple brain regions, but higher infectivity of choroid plexus epithelial cells. to validate the higher susceptibility and further investigate consequences of sars-cov-2 infection of choroid plexus cells, we sought to generate more pure choroid plexus organoids from hipscs. the most dorsal structures of the telencephalon, including the choroid plexus, are patterned by high wnt and bmp signaling from the roof plate (lun et al., 2015) . an early in vitro study demonstrated the sufficiency of bmp4 exposure to induce choroid plexus fate from neuroepithelial cells (watanabe et al., 2012) . furthermore, exposure of human embryonic stem cell-derived embryoid bodies to the gsk3 antagonist chir-99021 and bmp4 was shown to generate 3d choroid plexus tissue (sakaguchi et al., 2015) . building upon these previous studies, we optimized a simple protocol to generate choroid plexus organoids (cpos) from hipscs ( figure 2a ). undifferentiated hipscs grown in a feeder-free condition were aggregated into embryoid bodies consisting of j o u r n a l p r e -p r o o f 7 approximately 5,000 cells each using an aggrewell plate ( figure 2a ). embryoid bodies were patterned to the anterior neuroectodermal fate using dual-smad inhibition combined with wnt inhibition ( to further characterize these cpos, we performed transcriptome analysis of cpos at 50 div by bulk rna sequencing (rna-seq). gene expression analysis confirmed the expression of choroid plexus markers, including ttr, aqp1, chloride intracellular channel 6 (clic6), keratin 18 (krt18), msx1, and lmx1a, in cpos at high levels comparable to published transcriptomes of adult human choroid plexus tissue (rodriguez-lorenzo et al., 2020) and at much higher levels than those in hippocampal organoids at 45 div ( figure 2d ). in addition, cpos expressed genes related to adherens junction, signaling, ion channels and solute transporters at high levels comparable to those in adult human choroid plexus tissue ( figure 2d ), indicating choroid plexus identity and function. at the whole transcriptome level, cpos, but not hippocampal organoids, showed high correlation to adult human choroid plexus tissue ( figure 2e ). we validated high levels of expression of a group of choroid plexus signature genes in cpos at 50 div and 100 div in comparison to hippocampal organoids at 20 div by qpcr ( figure s2d and table s1 ). given our initial finding of a high rate of infection of choroid plexus cells by sars-cov-2, we examined the expression of known sars-cov-2 receptors. rna-seq analysis showed expression j o u r n a l p r e -p r o o f 8 of ace2 and tmprss2 in cpos at levels similar to the adult human choroid plexus tissue, which were much higher than in hippocampal organoids ( figure 2f ). expression levels of nrp1, a newly identified receptor for sars-cov-2 (cantuti-castelvetri et al., 2020) , were similar in all three samples. immunohistology confirmed expression of ace2 at a much higher level in cpos than in hippocampal organoids ( figure 2g ). qpcr analysis also showed higher levels of ace2 and tmprss2 expression in cpos at 50 div and 100 div than in hippocampal organoids ( figure s2d ) together, these results show that our cpos exhibit a similar transcriptome as adult human choroid plexus tissue and express markers for choroid plexus epithelial cells and sars-cov-2 receptors, representing a suitable experimental model to study sars-cov-2 infection. next, we exposed cpos at 47 div and 67 div to either sars-cov-2 or vehicle control for 8 hours and analyzed samples at 24 and 72 hpi. upon sars-cov-2, but not vehicle treatment, many ttr + choroid plexus cells showed infection as identified by co-localization of patient convalescent serum and sars-cov-2 nucleoprotein immunolabeling in cpos derived from two independent hipsc lines ( figures 3a and s3a ). additionally, we observed co-localization of patient convalescent serum and abundant dsrna immunolabeling in ttr + choroid plexus cells ( figure s3b ). infected cells identified by sars-cov-2 nucleoprotein immunostaining also showed high levels of ace2 expression, consistent with ace2 being a critical cell entry receptor for sars-cov-2 ( figure s3c ). quantification revealed an average of 9.0% and 12.6 % of ttr + cells infected at 24 hpi and 11.9% and 18.6% of ttr + cells infected at 72 hpi for cpos infected at 47 div and 67 div, respectively, across two hipsc lines ( figures 3b and s3d ). an increase in the number of infected cells from 24 to 72 hpi suggested that infection could be productive and spread among cells ( figures 3b and s3d ). to confirm sars-cov-2 productive infection, we examined viral titers using culture supernatants and cpo lysates at 0, 24, and 72 hpi. there was a significant increase in titers of j o u r n a l p r e -p r o o f 9 infectious virus over time after the initial infection ( figure 3c ). the increase of infectious virus present in cpo lysates superseded the increase in culture supernatants, suggesting a slow release of infectious viruses into the culture medium. the increase in viral titers appeared to be bigger than the increase in the number of infected cells, suggesting that the relationship is not linear. consistent with this notion, exposure of cpos at 67 div to different amounts of sars-cov-2 showed a nonlinear increase in infection with higher viral titers ( figures s3e and 3f ). importantly, we observed infection using 10 3 ffu (focus forming units) with an estimated moi (multiplicity of infection) of 0.001, indicating very high susceptibility of cpos to sars-cov-2 infection ( figures s3e and 3f ). next, we examined the cellular consequence of sars-cov-2 infection of cpos. we observed the presence of syncytia in sars-cov-2 infected cells, which significantly increased from an average of 4.6% at 24 hpi to 10.5% by 72 hpi (figures 3d and 3e ). development of syncytia by cell-cell fusion mediated by interaction between sars-cov-2 spike protein and ace2 expressed on adjacent cells has been reported with sars-cov-2 infection of several cell types and is a major mechanism for viral spread (hoffmann et al., 2020a; matsuyama et al., 2020; ou et al., 2020) . in particular, the sars-cov-2 spike protein appears to facilitate membrane fusion at a much higher rate than sars-cov-1 (xia et al., 2020) . by examining individual confocal z-planes we could identify as many as 12 nuclei within a single infected cell at 72 hpi ( figure 3d ). interestingly, in addition to syncytia, we observed a significant increase in cell death in both infected and uninfected ttr + choroid plexus cells in cpos exposed to sars-cov-2 as measured by tunel immunolabeling ( figures 3f, 3g and s3g). cell death increased from an average of 1.4% to 5.9% in infected and from 1.9% to 5.4% in uninfected ttr + cells from 24 to 72 hpi in cpos infected at 67 div across two hipsc lines ( figure 3g ). tunel + uninfected cells tended to appear adjacent to infected cells, suggesting that infected cells may induce adjacent cells to die through an extrinsic mechanism ( figure 3f ). similar results were found for cpos infected at 47 div ( figure s3g ). to confirm the susceptibility of choroid plexus epithelial cells to sars-cov-2 infection, we examined primary human choroid plexus epithelial cells (phcpecs). phcpecs exposed to sars-j o u r n a l p r e -p r o o f cov-2, but not vehicle control, for 12 hours showed many infected cells by immunolabeling with sars-cov-2 nucleoprotein at 72 and 120 hpi ( figure s3h ). the number of infected cells increased with higher moi, with an average of 1.7% and 0.9% of cells infected at 72 and 120 hpi, respectively, using a moi of 5 ( figure s3i ). together, using cpos as a 3d human cellular model, our findings reveal sars-cov-2 tropism for choroid plexus epithelial cells that results in productive infection, increased cell syncytia that may promote viral spread through cell-cell fusion, and increased cell death among both infected and uninfected adjacent cells. to gain additional insight into functional consequences of sars-cov-2 infection in cpos at the molecular level, we performed rna-seq of sars-cov-2 and vehicle-treated 47 div cpos at 24 and 72 hpi. principal component analysis showed clustering of biological replicates within different treatment groups ( figure s4a ). after aligning reads to the sars-cov-2 genome, we detected high numbers of viral transcripts at 24 and 72 hpi, confirming that the virus was replicating within cpos ( figure 4a ). we also confirmed the expression of sars-cov-2 receptors ace2, tmprss2, and nrp1 in cpos at all time points ( figure 4b ). comparison of sars-cov-2 and vehicle-treated cpos revealed 1,721 upregulated genes and 1,487 downregulated genes at 72 hpi, indicating that sars-cov-2 infection leads to large-scale transcriptional dysregulation ( figure s4b and table s2 ). more detailed gene ontology (go) analysis of upregulated genes at 72 hpi showed enrichment for genes related to viral responses, rna processing, response to cytokine, cytoskeletal rearrangement, and cell death ( figure 4c and table s3 ). closer examination of upregulated genes revealed an increase in inflammatory cytokines ccl7, il32, ccl2 (mcp1), il18, and il8 ( figure 4d ). il32 plays important roles in both innate and adaptive immune responses by inducing tnf-alpha, which activates the signaling pathway of nf-kappa-b and p38 mapk. this signaling pathway has been shown to be activated following sars-cov-2 infection in j o u r n a l p r e -p r o o f 11 other cells (bouhaddou et al., 2020) . we validated the upregulation of p38 signaling by immunostaining of the phosphorylated form of p38 in infected cells ( figures s4c and s4d ). interestingly, quantification showed elevated levels of phospho-p38 in nearby uninfected cells, although to a lesser degree than in infected cells, suggesting a substantial non-cell autonomous effects ( figure s4d ). upregulation of many vascular remodeling genes ( figure 4d ), such as nppb and vcan, suggests that infected choroid plexus cells could signal the vasculature to promote leukocyte invasion (shechter et al., 2013) . go analysis of downregulated genes at 72 hpi, on the other hand, showed enrichment for genes related to ion transport, transmembrane transport, cilium, and cell junction ( figure 4c and table s3 ). closer examination of downregulated genes revealed a decrease in the expression of many transporters and ion channels, such as aqp1, aqp4 and slc22a8, which are important for normal csf secretory function ( figure 4d ) (brown et al., 2004; hladky and barrand, 2016) , suggesting functional deficits of choroid plexus cells. downregulation of many cell junction genes suggests a remodeling or break-down of normal csf-blood barrier function, which also occurs during brain inflammation (engelhardt and tietz, 2015) . additionally, the dramatic decrease in ttr production may result in decreased thyroxine transport to the csf, which has been shown to contribute to neuropsychiatric symptoms and "brain fog" or difficulty in concentrating in patients (samuels, 2014) . we validated downregulation of ttr levels in infected cells by immunostaining ( figures s4c and s4d ). we also observed downregulation of ttr levels in uninfected cells at 72 hpi, although to a lesser degree than in infected cells, again suggesting a non-cell autonomous effect ( figure s4d ). transcriptional dysregulation induced by sars-cov-2 in cpos at 24 hpi showed similar gene expression changes as at 72 hpi, although less severe in most cases ( figures 4d and s4e ). comparison of the list of dysregulated genes at 24 and 72 hpi revealed a substantial overlap of 42% and 32% of upregulated and downregulated genes, respectively ( figure s4f ). we validated the dysregulation of a subset of these genes by qpcr ( figure s4g ). j o u r n a l p r e -p r o o f 12 to investigate whether organoids from different tissues exhibit similar transcriptional responses upon sars-cov-2 infection, we compared sars-cov-2-induced transcriptional changes among cpos and published datasets from hepatocyte organoids (yang et al., 2020a) and intestinal organoids . surprisingly, we found little overlap among upregulated or downregulated genes in the three different organoid types, suggesting a predominantly cell typespecific response to sars-cov-2 infection ( figure 4e ). this finding supports the use of a diverse array of human model systems to investigate effects of sars-cov-2 infection. additionally, we found many more dysregulated genes in cpos compared to the other two organoid types, suggesting that sars-cov-2 may exhibit a greater impact on choroid plexus cells ( figure 4e ). together, these transcriptome analyses suggest that sars-cov-2 infection of choroid plexus cells leads to viral rna production and inflammatory cellular responses with a concomitant compromise of normal barrier and secretory functions as well as increased cell death. furthermore, responses to sars-cov-2 infection are largely tissue organoid specific, at least at the transcriptional level. using a platform of hipsc-derived monolayer neurons, microglia, astrocytes, and region-specific brain organoids, we showed limited tropism of sars-cov-2 for neurons and astrocytes with a particularly high rate of infection of choroid plexus epithelial cells. using an optimized protocol for generating cpos from hipscs that resemble the morphology, marker expression, and transcriptome of adult human choroid plexus, we revealed productive infection of sars-cov-2 in choroid plexus epithelial cells, which we confirmed using primary human choroid plexus epithelial cells. analysis of the consequence of sars-cov-2 infection of cpos showed an increase in both cell autonomous and non-cell autonomous cell death and transcriptional dysregulation related to increased inflammation and altered barrier and secretory function. our study provides an organoid platform to investigate the cellular susceptibility, pathogenesis, and treatment strategies of sars-j o u r n a l p r e -p r o o f 13 cov-2 infection of brain cells and further implicates the choroid plexus as a potentially important site for pathophysiology. notably, the choroid plexus is known to be a site of infection for several viruses and agents that affect the brain (schwerk et al., 2015) . although covid-19 primarily manifests as respiratory distress resulting from inflammatory damage to the lung epithelium, there is mounting clinical evidence implicating other organs, such as the kidney, intestine, and brain, in disease pathogenesis (renu et al., 2020) . the currently widely used cellular model systems, such as vero e6 cells or human cancer cell lines, do not adequately recapitulate the cellular diversity and breadth of the disease. although mice engineered to express human ace2 have provided an in vivo model to study sars-cov-2, the system relies on human ace2 overexpression and does not fully recapitulate symptoms of covid-19 in humans. recently, organoids have emerged as a model to study human cells and organogenesis in vitro (clevers, 2016) . organoids for various tissues have been used to better understand sars-cov-2 viral tropism and pathology monteil et al., 2020; ramani et al., 2020; yang et al., 2020a; zhou et al., 2020) . these studies support cell-type specific susceptibility to sars-cov-2 infection. our finding that dysregulated gene expression varies widely among hepatocyte, intestinal, and choroid plexus organoids infected with sars-cov-2 suggests unique responses in different cell types and highlights the need for diverse human cellular model systems when studying the disease. brain organoid models for sars-cov-2 infection are particularly useful since it is not feasible to take brain biopsies from patients with covid-19. the load of sars-cov-2 that may enter the brain is likely to be variable throughout the course of illness and may be difficult to accurately assess in brain samples. for example, viral particles or rna has been detected in the csf or brain of some but not the majority of patients with neurological symptoms (helms et al., 2020; moriguchi et al., 2020; puelles et al., 2020; solomon et al., 2020; virani et al., 2020) , although the amount of viral particles in the csf may be insufficient for detection . j o u r n a l p r e -p r o o f 14 brain organoids allow analyses of the acute and long-term impact of sars-cov-2 infection in real time, with the ability to introduce perturbations, such as therapeutic agents, to assess cellular responses. use of region-specific brain organoids, including cpos, expands the organoid toolset for investigating sars-cov-2 infection, and provides a platform for testing new therapeutics. building upon previous findings (sakaguchi et al., 2015; watanabe et al., 2012) , we optimized a cpo model that is simple, robust and reproducible with the initial goal to study sars-cov-2 infection. these cpos express high levels of sars-cov-2 receptors and exhibit productive infection of sars-cov-2 and pathogenesis at cellular and molecular levels. cpos may provide a better model system than primary human choroid plexus epithelial cells, which are not widely available and often lose some characteristics upon multiple rounds of expansion in culture and showed a lower infection rate than our cpos. unlike a very recently developed protocol that utilizes cultures with matrigel extracellular matrix (pellegrini et al., 2020) , our cpos do not obviously polarize or develop fluid-filled, cystic structures, but do offer the benefit of not relying on matrigel, which has batch-to-batch variation and can lead to increased inter-organoid variability. our protocol uses feeder-free hipscs to further improve organoid reproducibility, and cpos are cultured on an orbital shaker to enhance oxygen and nutrient diffusion throughout the organoids. with these improvements, cpos reproducibly generate projections of cuboidal cells and exhibit uniform expression of choroid plexus markers otx2, aqp1, and ttr in most of the cells within the organoids. they also exhibit a transcriptome similar to adult human choroid plexus tissue. importantly, these cpos express many transporters that are essential for csf secretion. these cpos provide a platform for future investigations of cell type-specific pathogenesis, mechanisms and treatment of sars-cov-2 infection. our cpo platform can also be used to model human choroid plexus development and associated brain disorders in the future (lun et al., 2015) . spread in the cns j o u r n a l p r e -p r o o f 15 ace2 has been identified as a key cell entry receptor for sars-cov-2 (hoffmann et al., 2020b). although moderate ace2 expression has been detected in various brain regions, particularly high levels have been reported in the choroid plexus in humans and mice . the high levels of ace2 expression in cpos may explain their higher susceptibility to sars-cov-2 infection compared to other brain organoids. moreover, the productive infection and high incidence of syncytia in infected choroid plexus cells may contribute to the viral spread. on the other hand, despite relatively low ace2 expression, we did detect sparse infection of neurons by sars-cov-2. recent studies point to the potential for other proteins that are expressed more highly in neural cells, such as nrp1, to serve as receptors for sars-cov-2. we did not observe an obvious increase in the number of infected neurons over time, suggesting that the virus may not spread efficiently from neuron-to-neuron. central nervous system disorders, including seizures and encephalopathy, have been reported with sars-cov-1 with detection of infectious virus and rna in the csf (hung et al., 2003; lau et al., 2004; xu et al., 2005) . a substantial portion of patients with covid-19 exhibit various neurological symptoms, which are highly variable, probably due to many factors (helms et al., 2020; moriguchi et al., 2020; puelles et al., 2020; solomon et al., 2020; virani et al., 2020) . how sars-cov-2 may enter the brain is currently unknown. the main hypotheses are either neuron-toneuron spread via bipolar cells located in the olfactory epithelium that extend axons and dendrites to the olfactory bulb, or a hematogenous route across the blood-csf-barrier (desforges et al., 2014) . neuron-to-neuron propagation has been described for other coronaviruses (dube et al., 2018; netland et al., 2008) , but it was not obvious in our study. our finding that the choroid plexus is particularly susceptible to productive infection with sars-cov-2, raises the possibility that choroid plexus epithelial cells could be infected from virus circulating in closely associated capillaries. sars-cov-2 may replicate within choroid plexus cells and shed viral copies into the csf. indeed, we detected increased viral load in culture supernatants overtime. alternatively, virus may enter the csf via the cribiform plate in nasal passages and then replicate in choroid plexus j o u r n a l p r e -p r o o f 16 cells, increasing viral availability in the central nervous system. viral propagation through the csf may also explain cases of spinal cord pathology in some patients with covid-19 (giorgianni et al., 2020; valiuddin et al., 2020) . more detailed analyses of autopsy brain samples from patients with covid-19 and better animal models of sars-cov-2 infection are necessary to understand potential routes of viral entry into the brain. the transcriptional dysregulation in cpos after sars-cov-2 infection indicates an inflammatory response and perturbed choroid plexus cellular function. our study further suggested both cell autonomous and non-cell autonomous impact of sars-cov-2 infection. many pro-inflammatory cytokines were upregulated, including ccl7, il32, ccl2, il18, and il8, which are important for recruiting immune cells to sites of infection. future studies are needed to validate the release of these cytokines by elisa analyses. notably, one case report examining inflammatory cytokines present in the serum and csf of a patient with covid-19 presenting with severe seizures identified ccl2 as the only cytokine enriched in the patient's csf compared to serum (farhadian et al., 2020) . ccl2 was one of the most highly upregulated genes after sars-cov-2 infection, suggesting that the choroid plexus may be the source of this cytokine in the csf. upregulation of vascular and extracellular matrix remodeling genes also suggests that during infection, choroid plexus cells can signal the adjacent vasculature to recruit inflammatory cells. downregulation of many genes important for csf secretion, including aqp1, and many ion transporters, such as atp1a2, may lead to altered csf production and composition. mutations in many of these dysregulated transporter genes have been implicated in neurological complications in humans, such as migraine and encephalopathy (murphy et al., 2018) . increased expression of cell death genes supports our finding of increased numbers of tunel + cells after sars-cov-2 infection. downregulation of many genes related to the cilium suggests an impaired cytoskeleton and dysfunctional sensing of the extracellular environment. there was also a significant downregulation of ttr, which is necessary for carrying thyroid hormone from the blood into the csf, and lowered ttr production by the choroid plexus has been associated with neuropsychiatric disorders, such as schizophrenia and depression (huang et al., 2006; sullivan et al., 1999) . furthermore, low csf thyroid hormone has been associated with many neurological symptoms, including slowing of thought and speech, decreased attentiveness, and reduced cognition, which have all been reported by patients with covid-19 (ellul et al., 2020) . our study implicates the choroid plexus epithelium as a potential target of sars-cov-2 infection and a contributor to covid-19 disease pathogenesis that warrants further investigation. our study demonstrates clear susceptibility of hipsc-derived neurons, astrocytes, and choroid plexus cells, as well as primary astrocytes and choroid plexus epithelial cells, to sars-cov-2 infection in vitro, however, a thorough analysis of brain samples from patients with covid-19 is necessary to confirm neural susceptibility in vivo. our methodology has a number of constraints that limit result interpretation. first, since brain organoids more closely resemble the developing fetal brain than the mature adult brain, there may exist important differences in sars-cov-2 susceptibility between immature and mature cells. there has been recent evidence of vertical transmission of sars-cov-2 from mother to fetus (dong et al., 2020; vivanti et al., 2020; zeng et al., 2020) , but the impact on the fetal brain remains unclear. brain organoids may serve as a useful model system to explore the potential effects of fetal sars-cov-2 exposure on brain development. second, direct exposure of brain organoid cultures to sars-cov-2 in the culture medium may not accurately recapitulate the physiological amount and duration of viral exposure in humans. different viral titers may reveal different viral tropism in vitro. to avoid the use of high titers that may cause j o u r n a l p r e -p r o o f 18 non-physiological viral infection, we exposed different brain region-specific organoids of the same estimated moi of 0.1 and found a differential infection rate. importantly, sars-cov-2 at an estimated moi as low as 0.001 can lead to detectable infection of cpos, indicating very high susceptibility of choroid plexus cells, at least in vitro. third, brain organoid models lack an intact blood-brain-barrier or blood-csf-barrier which may modulate sars-cov-2 access to the brain. our brain organoids also lack additional cell types, such as oligodendrocytes, microglia, stromal cells, immune cells, and endothelial cells, which may modulate susceptibility to infection and contribute to disease pathogenesis in vivo. immune cells, such as t cells and monocytes, have been shown to mediate host responses to sars-cov-2 infection, including tissue-specific inflammation (tay et al., 2020) . endothelial cells are major targets of sars-cov-2 and may be the primary cause of sars-cov-2-related effects in the brain with neurological symptoms being secondary to vascular changes and hypoxia (ellul et al., 2020) . future studies of sars-cov-2 susceptibility can extend to a more diverse selection of brain cells, as well as peripheral neurons. also see figure s2 and table s1. table s2 for the complete list of differentially expressed genes and table s3 for the complete list of go terms for differentially expressed genes. tmem.: transmembrane. (e) venn diagrams comparing the overlap of upregulated and downregulated genes following s-cov-2 infection in cpos, human hepatocyte organoids (yang et al., 2020a) , and human intestinal organoids . differentially expressed genes at both 24 and 72 hpi were combined for cpos and differentially expressed genes for both expansion and differentiation intestinal organoid types were combined for intestinal organoids. also see figure s4 and tables s1, s2 and s3. further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, dr. guo-li ming (gming@pennmedicine.upenn.edu) . this study did not generate new unique reagents. the access number for the rna-seq data reported in this study is deposited in geo (gse157852). human ipsc lines used in the current study were derived from healthy donors and were either obtained from commercial sources or previously generated and fully characterized (chiang et al., 2011; wen et al., 2014; yoon et al., 2014) . c1-2 hipscs were generated from fibroblasts obtained from atcc (crl-2097). c3-1 hipscs were generated from fibroblasts obtained from an individual in a previously characterized american family (sachs et al., 2005) . wtc11 hipsc were obtained from coriell (gm25256). ht268a hipscs were generated from dermal fibroblasts obtained from coriell (gm05659) (vu et al., 2018) , and pcs201012 hipscs were generated from dermal fibroblasts obtained from atcc (pcs-201-012) (beers et al., 2015) . generation of hipsc lines (bardy et al., 2015) . primary human astrocytes (sciencell research laboratories) were cultured and expanded for two passages, according to the supplier's instructions. after the second expansion, astrocytes were cryopreserved at 250,000 cells per vial. banked astrocytes were thawed directly onto 96 well imaging plates (greiner bio-one) coated with poly-l-ornithine (sigma-aldrich) at 2 âµg/cm 2 and laminin from engelbreth-holm-swarm murine sarcoma basement membrane (sigma-aldrich) at 5 âµg/ml at a density of 16,000 cells per well for shorter infection times (48 hours or less) and 8000 cell per well for 120 hour infections. primary human choroid plexus epithelial cells (sciencell research laboratories) were cultured according to the supplier's instructions. cells were either passaged once or plated directly out of thaw onto 96-well imaging plates coated with poly-l-ornithine at 2 âµg/cm 2 at a density of 4000 cells per well. all studies involving hipscs were performed under approved protocols of the university of pennsylvania and nih. refer to the key resources table for the source of each hipscs. all hipsc lines were confirmed to have a normal karyotype and were confirmed free of mycoplasma, acholeplasma, and spiroplasma with a detection limit of 10 cfu/ml by targeted pcr (biological industries). for cortical, hippocampal, hypothalamic, and midbrain organoid generation, hipscs were cultured on mouse embryonic fibroblast feeder (mef) cells in stem cell medium consisting of dmem:f12 supplemented with 20% knockout serum replacement, 1x mem-neaas, 1x glutamax, 1x penicillin-streptomycin, 1x 2-mercaptoethanol, and 10 ng/ml bfgf in a 5% co 2 , 37â°c, 90% relative humidity incubator as previously described (qian et al., 2018) . culture medium was replaced every day. hipscs were passaged every week onto a new tissue-treated culture plate coated with 0.1% gelatin for 2 hours and pre-seeded with î³-irradiated cf1 mef cells one day in advance. colonies were detached by washing with dpbs and treating with 1 mg/ml collagenase type iv for 30-60 minutes. detached colonies were washed 3 times with 5 ml dmem:f12 and dissociated into small clusters by trituration with a p1000 pipette. for cpo generation, hipscs j o u r n a l p r e -p r o o f 33 were maintained in feeder-free conditions on plates pre-coated with hesc-qualified matrigel (corning) using mtesr plus medium (stemcell technologies). culture medium was replaced every other day and hipscs were passaged every week by washing with dpbs and incubating with relesr reagent (stemcell technologies) for 5 minutes. detached hipscs were broken into smaller clusters by gentle trituration using a 5 ml pipette and seeded onto fresh coated plates. generation of cortical organoids from c3-1 hipscs was performed as previously described (qian et al., 2018; qian et al., 2016) . briefly, hipsc colonies were detached with collagenase type iv, for generation of hypothalamic organoids from c1-2 and c3-1 hipsc lines, hipsc colonies were processed similarly to those for cortical organoids followed by exposure to shh signaling and wntinhibition in induction medium with 50 ng/ml recombinant sonic hedgehog, 1 âµm purmorphamine, 10 âµm iwr1-endo and 1 âµm sag, and then transferred to differentiation medium for further culture. organoids were maintained with media changes every other day. at 70% confluence, undifferentiated hipscs (c1-2 and wtc11 lines) grown in feeder-free conditions were detached using relesr reagent and gently triturated into small clusters using a 5 ml pipette and pipette aid. hipsc clusters were centrifuged at 200 g for 3 minutes and the supernatant was aspirated and replaced with mtesr plus medium supplemented with 10 âµm y-27632. approximately 5,000 hipscs were aggregated into each eb by following instructions using the aggrewell 800 plate and cultured overnight in mtesr plus medium supplemented with 10 âµm y-27632. the next day (day 1), embryoid bodies were gently removed from the aggrewell plate using a p1000 pipettor with a wide-bore tip, washed three times with dmem:f12, and transferred to neural induction medium containing dmem:f12 supplemented with 20% knockout serum on day 30, medium was replaced with differentiation medium containing a 1:1 mixture of dmem/f12 and neurobasal supplemented with 1x n2 supplement, 1x b27 supplement w/o vitamin a, 1x penicillin/streptomycin, 1x non-essential amino acids, 1x glutamax, 10 ng/ml bdnf, and 10 ng/ml gdnf. differentiation medium was replaced every three days and organoids could be maintained for over 100 days. sars-cov-2 usa-wa1/2020 (gen bank: mn985325.1) (harcourt et al., 2020) viral isolate was obtained from bei resources. sars-cov-2 virus was expanded and titered using vero e6 cells. culture cells were infected in a 96-well plate with 100 âµl of medium per well using multiplicity (moi) of 0.2, 1, or 5, and vehicle-treated cells as controls. after virus exposure for 12 hours, cells were washed 3 times with fresh medium and returned to culture. brain organoids were infected in a 24well ultra-low attachment plate (corning) with 0.5 ml of medium per well containing vehicle, 10 3 , 10 4 , or 10 5 focus forming units (ffu) of sars-cov-2 for 8 hours on an orbital shaker rotating at approximately 100 rpm. after virus exposure, organoids were washed 3 times with fresh medium and returned to culture. for infection of organoids, we controlled the virus titer rather than the moi since it is not feasible to obtain an accurate cell count on each batch of organoids before infection. according to our estimate, each organoid contains approximately 250-500,000 cells, corresponding j o u r n a l p r e -p r o o f 36 to an estimated moi of 0.1-0.05 when using 10 5 ffu sars-cov-2 virus and infecting 4 organoids together. at experimental endpoints, brain organoids were placed directly in 4% methanol-free formaldehyde (polysciences) diluted in dpbs (thermo fisher scientific) overnight at 4 o c on a rocking shaker. after fixation, brain organoids were washed in dpbs and cryoprotected by overnight incubation in 30% sucrose (sigma-aldrich) in dpbs at 4â°c. brain organoids were placed in a plastic cryomold (electron microscopy sciences) and snap frozen in tissue freezing medium (general data) on dry ice. frozen tissue was stored at â��80â°c until processing. monolayer cells grown on glass coverslips were fixed in 4% methanol-free formaldehyde (polysciences), diluted in dpbs (thermofisher scientific) for 5 hours at room temperature, washed with dpbs, and stored at 4â°c until ready for immunohistology. serial tissue sections (25 âµm for brain organoids) were sliced using a cryostat (leica, cm3050s), and melted onto charged slides (thermo fisher scientific). slides were dried at room temperature and stored at â��20â°c until ready for immunohistology. for immunofluorescence staining, the tissue sections were outlined with a hydrophobic pen (vector laboratories) and washed with tbs containing 0.1% . tissue sections were permeabilized and nonspecific binding was blocked using a solution containing 10% donkey serum (v/v), 0.5% triton x-100 (v/v), 1% bsa (w/v), 0.1% gelatin (w/v), and 22.52 mg/ml glycine in tbst for 1 hour at room temperature. the tissue sections were incubated with primary antibodies (see the key resources table) diluted in tbst with 5% donkey serum (v/v) and 0.1% triton x-100 (v/v) overnight at 4â°c. after washing in tbst, tissue sections were incubated with secondary antibodies (see the key resources table) diluted in tbst with 5% donkey serum (v/v) and 0.1% triton x-100 (v/v) for 1.5 hours at room temperature. after washing with tbst, sections were washed with tbs to remove detergent and incubated with trueblack reagent (biotium) diluted 1:20 in 70% ethanol for 1 minute j o u r n a l p r e -p r o o f 37 to block autofluorescence. after washing with tbs, slides were mounted in mounting solution (vector laboratories), cover-slipped, and sealed with nail polish. monolayer cultures were imaged with a perkin-elmer opera phenix high-content automatic imaging system with a 20x air objective in confocal mode. at least 9 fields were acquired per well. images were analyzed using columbus image data storage and analysis system. brain organoid sections were imaged as z stacks using a zeiss lsm 810 confocal microscope or a zeiss lsm 710 confocal microscope (zeiss) using a 10x, 20x, 40x, or 63x objective with zen 2 software (zeiss). images were analyzed using either imaris 7.6 or imagej software. images were cropped and edited using adobe photoshop (adobe) and adobe illustrator (adobe). tissue culture supernatants and lysates from sars-cov-2 treated cpos were collected at 0, 24, and 72 hpi for 2 biological replicates per timepoint containing 4 organoids each. tissue culture supernatants were stored at -80â°c until titration. for lysates, organoids were washed with cold pbs, mechanically dispersed and disrupted by freeze-thaw cycles, and the clarified homogenate was stored at -80â°c until titration. viral titers were determined in vero e6 cells using an immune focus forming unit assay. briefly, vero e6 cells (3 x 10 4 cells/96-well) were inoculated with 100 âµl of serially diluted organoid supernatant or lysate in a 96-well plate at 37â°c and 5% co 2 for 20 hours. cells were fixed with 4% methanol-free paraformaldehyde overnight at 4â°c. infected foci were labeled using immunofluorescence staining for sars-cov-2 nucleoprotein. sars-cov-2 nucleoprotein positive cells were counted in each well. readouts were performed on wells with the highest dilution displaying observable infection. j o u r n a l p r e -p r o o f 38 to minimize variability due to sampling and processing, each biological replicate consisted of 3 organoids and replicates for all experimental conditions were processed in parallel for rnaextraction, library preparation and sequencing. at the desired experimental endpoints, organoids were homogenized in trizol (thermo fisher scientific) using a disposable pestle and handheld mortar and stored at -80 c until processing. rna clean-up was performed using the rna clean & concentrator kit (zymo research) after trizol phase separation according to the manufacturer's protocol. rna concentration and quality were assessed using a nanodrop 2000 (thermo fisher scientific). library preparation was performed as previously described with some minor modifications (weng et al., 2017) . about 300 ng of rna in 3.2 âµl was combined with 0.25 âµl rnase inhibitor (neb) and 1 âµl cds primer (5'-aagcagtggtatcaacgcagagtact30vn-3') in an 8-well pcr tube strip, heated to 70 âºc for 2 minutes, and immediately placed on ice. 5.55 âµl rt mix, containing 2 âµl of 5x smartscribe rt buffer (takara), 0.5 âµl of 100 mm dtt (millipore sigma), 0.3 âµl of 200 mm mgcl2 (thermo fisher scientific), 1 âµl of 10 mm dntps (takara), 1 âµl of 10 âµm tso primer (5'-aagcagtggtatcaacgcagagtacatrgrgrg-3'), 0.25 âµl of rnase inhibitor (neb), and 0.5 âµl smartscribe reverse transcriptase (takara) was added to the reaction. rt was performed under the following conditions: 42 âºc for 90 minutes, 10 cycles of 50 âºc for 2 minutes and 42 âºc for 2 minutes, 70 âºc for 15 minutes, and 4 âºc indefinitely. for cdna amplification, 2 âµl of the rt reaction was combined with 2.5 âµl of 10x advantage 2 buffer (takara), 2.5 âµl of 2.5 mm dntps (takara), 0.25 âµl of 10 âµm is pcr primer (5'-aagcagtggtatcaacgcagagt-3'), 17.25 âµl nuclease free water (thermofisher), and 0.5 âµl advantage 2 dna polymerase (takara). thermocycling conditions were as follows: 94 âºc for 3 minutes, 8 cycles of 94 âºc for 15 s, 65 âºc for 30 s, and 68 âºc for 6 minutes, 72 âºc for 10 minutes, and 4 âºc indefinitely. amplified cdna was purified using 0.8x ampure xp beads (beckman coulter), eluted in 15 âµl nuclease-free water, and quantified using qubit dsdna hs assay kit (thermo fisher scientific). cdna was fragmented by combining 100 pg cdna in 1 âµl nuclease free water, 2x td buffer (20 mm tris, ph 8.0; thermo j o u r n a l p r e -p r o o f 39 fisher scientific), 10 mm mgcl 2 , and 16% peg 8000 (milliporesigma), and 0.5 âµl tn5 (lucigen). the mixture was heated to 55 âºc for 12 minutes, and the reaction was terminated upon the addition of 1.25 âµl of 0.2% sds (fisher) and incubated at room temperature for 10 minutes. fragments were amplified by adding 16.75 âµl nuclease free water (thermo fisher scientific), 1 âµl of 10 mm nextera i7 primer, 1 âµl of 10 mm nextera i5 primer, and 25 âµl kapa hifi hotstart readymix (emsco/fisher). thermocycling conditions were as follows: 72 âºc for 5 minutes, 95 âºc for 1 minute, 14 cycles of 95 âºc for 30 s, 55 âºc for 30 s, and 72 âºc for 30 s, 72 âºc for 1 minute, and 4 âºc indefinitely. dna was purified twice with 0.8x ampure xp beads (beckman coulter) and eluted in 10 âµl of 10 mm tris, ph 8 (thermo fisher scientific). samples were quantified by qpcr (kapa) and pooled at equal molar amounts. final sequencing library fragment sizes were quantified by bioanalyzer (agilent) with an average size of ~420 bp, and concentrations were determined by qpcr (kapa). samples were loaded at concentrations of 2.7 pm and sequenced on a nextseq 550 (illumina) using 1x72 bp reads to an average depth of 40 million reads per sample. choroid plexus organoid raw sequencing data were demultiplexed with bcl2fastq2 v2.17.1.14 (illumina) with adapter trimming using trimmomatic v0.32 software (bolger et al., 2014) . alignment was performed using star v2.5.2a (dobin et al., 2013) . a combined reference genome consisting of gencode human genome release 28 (grch38.p12) and ensembl sars-cov-2 wuhan-hu-1 isolate genome (genome assembly: asm985889v3, gca_009858895.3; sequence: mn908947.3) was used for alignment. multimapping and chimeric alignments were discarded, and uniquely mapped reads were quantified at the exon level and summarized to gene counts using star -quantmode genecounts. all further analyses were performed in r v3.6.0. transcript lengths were retrieved from gtf annotation files using genomicfeatures v1.36.4 (lawrence et al., 2013) and raw counts were converted to units of transcripts per million (tpm) using the calculatetpm function in scater v1.12.2 (mccarthy et al., 2017) . a combined tpm normalization using both human and primer sequences are listed in figure s1 . about 0.5-1.5 âµg rna was used to generate the cdna for qpcr with superscriptâ�¢ iii first-strand synthesis system (thermo fisher scientific) according to manufacturer's protocol. for qpcr reaction, 2 âµl of cdna was mixed with 6 âµl of nuclease free water, 1 âµl of 10 mm forward primer, 1 âµl of 10 mm reverse primer and 10 âµl of sybr green qpcr master mix (thermo fisher scientific), and the qpcr reactions were performed on the steponeplus real-time pcr system (applied biosystems). thermocycling conditions were as follows: 95 âºc for 20 s, 44 cycles of 95 âºc for 3 s and 60 âºc for 30 s. the difference between the ct values (â��ct) of the genes of interest and actin gene was calculated for each experimental sample, and 2^(-â��â��ct) was used to calculate the fold-change in expression of the genes of interest between samples using 20 div hos as the reference for figure s2d and 72 hpi vehicle-treated cpos as the reference for figure s4g . shown as mean â± sem or mean + sd as stated in the figure legends . in all cases, the p values are represented as follows: * * * p < 0.001, * * p < 0.01, * p < 0.05, and ns, not statistically significant when p > 0.05. in all cases, the stated ''n'' value is either separate wells of monolayer cultures or individual organoids with multiple independent images used to obtain data points for each. no statistical methods were used to pre-determine sample sizes. for all quantifications of immunohistology, the samples being compared were processed in parallel and imaged using the same settings and laser power for confocal microscopy. for monolayer cultures, cells were segmented by first identifying nuclei, and then cytoplasmic area. mean cytoplasmic intensities for sars-cov-2 nucleoprotein immunostaining were then measured in the segmented cytoplasmic regions, and a mean cytoplasmic intensity threshold was determined to identify sars-cov-2 infected cells. cell numbers were then automatically counted for sars-cov-2 infected dapi + cells, and total dapi + cells. for brain organoids, immuno-positive cells were quantified manually using the cell counter function in imagej (nih) or imaris (bitplane). ming and colleagues tested sars-cov-2 neurotropism using monolayer neural cells and brain organoids generated from human pluripotent stem cells and show minimal neuron and astrocyte infection, but efficient choroid plexus infection, leading to cell death and functional deficits. ccl7 il32 ccl2 il18 il8 casp8 igfbp3 anxa3 anxa2 pawr plau tnc vcan thbs1 nppb ctgf tagln actg2 cdcp1 acta2 cd274 tnfaip6 itgb6 ywhae dynlt1 aqp1 aqp4 atp2b3 slc7a8 slc39a12 rgs9bp arr3 cdhr1 ush2a cngb1 slc22a8 slc5a5 atp1a2 atp8a1 atp2b2 anxa9 ptprc kcna1 slc17a8 camk4 apoe hpgd ttr gene ontology: tool for the unification of biology. the gene ontology consortium the pathogenicity of sars-cov-2 in hace2 transgenic mice neuronal medium that supports basic synaptic functions and activity of human neurons in vitro a cost-effective and efficient reprogramming platform for large-scale production of integration-free human induced pluripotent stem cells 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coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune crossreactivity with sars-cov human cns barrier-forming organoids with cerebrospinal fluid production multiorgan and renal tropism of sars-cov-2 generation of human brain region-specific organoids using a miniaturized spinning bioreactor brain-region-specific organoids using mini-bioreactors for modeling zikv exposure sars-cov-2 targets neurons of 3d human brain organoids coronaviruses pathogenesis, comorbidities and multi-organ damage -a review altered secretory and neuroprotective function of the choroid plexus in progressive multiple sclerosis a frameshift mutation in disrupted in schizophrenia 1 in an american family with schizophrenia and schizoaffective disorder generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue psychiatric and cognitive manifestations of hypothyroidism. current opinion in endocrinology, diabetes, and obesity 21 the choroid plexus-a multi-role player during infectious diseases of the cns cell entry mechanisms of sars-cov-2 orchestrated leukocyte recruitment to immuneprivileged sites: absolute barriers versus educational gates neuropathological features of covid-19 low levels of transthyretin in the csf of depressed patients a mouse model of sars-cov-2 infection and pathogenesis zika virus infects human cortical neural progenitors and attenuates their growth the trinity of covid-19: immunity, inflammation and intervention the gene ontology resource: 20 years and still going strong acute transverse myelitis associated with sars-cov-2: a case-report neurological and neuropsychiatric complications of covid-19 in 153 patients: a uk-wide surveillance study syndrome associated with sars-cov-2 infection. idcases transplacental transmission of sars-cov-2 infection neural stem cells for disease modeling and evaluation of therapeutics for tay-sachs disease remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro bmp4 sufficiency to induce choroid plexus epithelial fate from embryonic stem cell-derived neuroepithelial progenitors synaptic dysregulation in a human ips cell model of mental disorders an intrinsic epigenetic barrier for functional axon regeneration coronavirus disease (covid-19) situation dashboard inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen a human pluripotent stem cell-based platform to study sars-cov-2 tropism and model virus infection in human cells and organoids clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study encephalitis as a clinical manifestation of covid-19 modeling a genetic risk for schizophrenia in ipscs and mice reveals neural stem cell deficits associated with adherens junctions and polarity antibodies in infants born to mothers with covid-19 pneumonia infection of bat and human intestinal organoids by sars-cov-2 2020) was downloaded from the ncbi gene expression omnibus (geo: accession number gse137619). the data was aligned to grch38.p12 and raw counts tpm-normalized to parallel cpo and ho datasets. tpm expression values were log 10 (tpm + 1) transformed for plotting individual gene expression values 2014) and correlation analysis was performed by calculating spearman correlation coefficients in the space of shared genes across the whole transcriptome of all datasets differential gene expression analysis for cpos between 72 hpi vehicle, 24 hpi sars-cov-2 infection, and 72 hpi sars-cov-2 infection was performed using deseq2 sars-cov-2 viral transcripts were removed for differential gene expression analysis upregulated and downregulated genes for 24 hpi sars-cov-2 compared to 72 hpi vehicle, and 72 hpi sars-cov-2 compared to 72 hpi vehicle were filtered by adjusted p-value < 0.05. volcano plots for 24 hpi sars-cov-2 and 72 hpi sars-cov-2 were plotted using enhancedvolcano v1.7.8. shared and unique signatures for upregulated and downregulated genes between 24 hpi sars-cov-2 and 72 hpi sars-cov-2 were visualized using venndiagram v1 tpm + 1) normalized and converted to row z-scores per gene for visualization. for sars-cov-2 cross-organoid comparison analysis, a list of upregulated and downregulated differentially expressed genes (degs) after sars-cov-2 infection were downloaded for adult hepatocyte organoids at 24 hpi (yang et al., 2020a), intestinal organoids at 60 hpi in expansion (exp) medium (lamers et al., 2020), and intestinal organoids at 60 hpi in differentiation (dif) medium we thank members of ming f.j. contributed to histological analysis of various brain organoids and development of the cpo model. s.r.p. and f.z. contributed to rna-seq data analysis. f.z. contributed to qpcr validation. the authors declare no competing interests.j o u r n a l p r e -p r o o f key: cord-301276-eer1l8vg authors: sehrawat, sharvan; rouse, barry t. title: opinion: does the hygiene hypothesis apply to covid-19 susceptibility? date: 2020-07-09 journal: microbes infect doi: 10.1016/j.micinf.2020.07.002 sha: doc_id: 301276 cord_uid: eer1l8vg in this commentary we argue that the hygiene hypothesis may apply to covid-19 susceptibility and also that residence in low hygienic conditions acts to train innate immune defenses to minimize the severity of infection. we advocate that approaches, which elevate innate immune functions, should be used to minimize the consequences of covid-19 infection at least until effective vaccines and antiviral therapies are developed. in this commentary we argue that the hygiene hypothesis may apply to susceptibility and also that residence in low hygienic conditions acts to train innate immune defenses to minimize the severity of infection. we advocate that approaches, which elevate innate immune functions, should be used to minimize the consequences of covid-19 infection at least until effective vaccines and antiviral therapies are developed. currently, the world is experiencing a new coronavirus pandemic with no solution or end in sight. in some societies > 10% of the population has been exposed to severe acute respiratory syndrome-coronavirus 2 (referred to as covid19) , but epidemiologists inform us that 60-70% need to experience the infection if protective herd immunity is to be established (1) . we could minimize the need for natural herd immunity by using prophylactic vaccines, but the prospects of developing vaccines that confer long term effective immunity against covid-19 is uncertain. this new coronavirus is highly infectious with the outcome of infection extremely variable ranging from asymptomatic to fatal. however, the spectrum of disease in different societies varies markedly for reasons as yet not fully understood. for instance, in some developed countries, the reported fatality rate is far higher than in others. also evidence shows that patients infected in low socioeconomic conditions suffer low rates of severe and lethal infection compared to those raised in more hygienic circumstances (2) . all infections have variable consequences and others and we have discussed the many factors that influence the outcome of infection with a virus (3) (4) (5) . these variables include age when infected, dose and nature of infection, presence of co-morbidities when infected such as immune suppression, metabolic disease and cancer, genetic and epigenetic variables, composition of the microbiome in different locations as well as past infection and environmental experiences. it is relevant to identify how these various factors come into play during covid-19 infection since some are subject to manipulation by therapies that could lower the prospect of severe disease. one susceptibility issue that has received minimal consideration is the immune status established as a consequence of past environmental and microbial experiences particularly early in life. in the early 90s, the so-called hygiene hypothesis was formulated to explain the rising incidence of diseases such as allergy and autoimmune diseases occurring in the developed world (6, 7) . the hypothesis advocated that children exposed to certain environments such as farms, domestic pets and exposure to enteric parasites were less likely to develop allergies and some autoimmune diseases than those who experienced a more hygienic upbringing. indeed, at least in some western societies well before covid-19, the extensive use of hand sanitizers and frequent hand washings were encouraged in growing children. added to this heightened concern for intense hygienic practices has been the frequent exposure of children to antibiotics to ablate potential problems before they develop into clinical problems that most often amount to temporary inconveniences. the unintended consequence of these hyper-hygienic regimens was to change the basic status of non-specific immunity, perhaps in part a result of a change in the balance of commensal microorganisms in the gastrointestinal tract, skin and other surface locations (8) . the hygiene hypothesis has satisfactorily explained the increased frequency of some disease syndromes, but could it also explain why some individuals are more susceptible to the severe consequences of covid-19 infection than are others? we suspect that the hygiene hypothesis is a viable concept that applies to covid-19 susceptibility, but it could be a long time, even in this era of accelerated information gathering before epidemiologists could assemble evidence that early lifestyle can be related to later covid-19 susceptibility. however, it does seem likely that extensive exposure to multiple microbes in the environment, food and water, as commonly occurs among those that reside in depressed socioeconomic circumstances, may render them resistant to the more severe consequences of covid-19 infection. similarly, the numbers of recorded cases and deaths in other societies with low socioeconomic status have experienced far fewer cases than many developed countries (11). although the efficiency of recording all cases of covid-19 infection and its consequence varies markedly between different countries, gathering evidence supports the idea that the outcome of infection is more likely to be asymptomatic or mild in developing countries and rarely has lethal consequences. thus, the who statistics show lethality rates that vary from 0.3% to 10% with the latter occurring in some developed countries (2,11). severe disease necessitating hospitalization and oxygen supplementation is also more common in developed as compared to most developing countries. if as increasing evidence is showing covid-19 infection is of less severity in developing countries, how can this be explained? even more important, can the information be applied to reduce the consequences of infection in the developed world? the most likely explanation for the different manifestations is that repeated exposure to unhygienic conditions exposes persons to microbes that express multiple so-called pathogen associated molecular patterns that activate one or more aspects of innate immunity. this changed activation state, which may persist for months but will not remain indefinitely (12) , is often referred to as trained immunity and unlike adaptive immunity is not highly antigen-specific, is less robust and has minimal or no long-term memory (12, 13) . however, trained immunity's advantage is that the response generated against one set of microbes can have bystander immune protection against other infections. this phenomenon was fist advocated by the mackaness and nathan groups in the early 60s with the bystander immune effect being attributed to so-called activated or angry macrophages, terms seldom used these days (14, 15) . we now realize that bystander immunity can be attributed to many cell types such as natural killer cells, dendritic cells, innate lymphocytes, gamma delta t cells in addition to macrophages (12) . the fact that trained immunity can be induced raises the prospect of its exploitation to raise the threshold of resistance to covid-19 infection, a valuable maneuver during the time when we have yet to develop effective treatments or vaccines. the questions will include what approaches could be used to achieve trained immunity and how long they will be useful. some studies have already shown that communities that still use bacillus calmette-guérin (bcg) as a vaccine against tuberculosis may have lesser instances of severe covid-19 infection than those countries that do not use bcg (16) . this notion has been complemented by advocating the use of bcg to vaccinate persons in an attempt to protect them against . a recent letter by hiv pioneer bob gallo advocated using oral polio vaccine for a similar effect (17) . we consider that administration of galectin molecules, some of which can activate the innate immune system might also represent an approach to reduce the consequences of covid-19 infection (18) . it is also worth noting that galectin therapy also has the potential to reduce the severity of covid-19 once infected. thus, galectins such as galectin-9 can reduce inflammatory cytokine production and change the nature of the inflammatory response caused by some viral lesions from a tissue damaging reaction dominated by pro-inflammatory t cells to one where counter-inflammatory regulatory t cells predominate (19) . for reasons still unknown, clinical disease following covid-19 infection in children is uncommon in all communities. this might be linked to their having received multiple vaccines and their tendency to develop several minor infections which may have served to activate their innate immune systems. there also are recent claims that vaccination with mmr vaccines might confer protection against covid-19 as it is claimed to do for rubella because of cross reactivity (20) . other explanations for the apparent resistance of children to clinically evident covid-19 infection could include lower levels of the viral entry receptor and the tendency of children to generate less tissue damaging so-called type i inflammatory reactions to antigens. in conclusion, we surmise that frequent exposure of individuals to natural environmental microbes, live attenuated vaccines against other viruses or bacteria or certain natural ligands such as different types of lectins and saponins that can stimulate innate immune receptors all help to reduce the clinical consequences of covid-19 infection. moreover, we await with interest observations comparing the outcome of covid-19 infection in adults raised as infants in environments that that support the hygiene hypothesis compared to those raised in more hygienic circumstances. it seems relevant to us to employ innate immune activators in the face of infection at least until such time when effective vaccines are developed. could it also be that constant hand washing, taking antibiotics whenever we endure minor infections and over-sanitizing our environs is not necessarily such a good idea? herd immunity: understanding covid-19 coronavirus disease (covid-19) situation report immunity and immunopathology to viruses; what decides the outcome? genetic susceptibility to infectious diseases: big is beautiful, but will bigger be even better? human genetic determinants of viral diseases hay fever, hygiene, and household size the 'hygiene hypothesis' for autoimmune and allergic diseases: an update targeting the human microbiome with antibiotics, probiotics, and prebiotics: gastroenterology enters the metagenomics era situation report covid-19. 27 th trained immunity: a program of innate immune memory in health and disease bcg-induced trained immunity: can it offer protection against covid-19? cellular resistance to infection alterations of macrophage functions by mediators from lymphocytes bcg vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained immunity the case for a stopgap vaccine promotion of tissue inflammation by the immune receptor tim-3 expressed on innate immune cells role of tim-3/galectin-9 inhibitory interaction in viral-induced immunopathology: shifting the balance toward regulators homologous protein domains in sars-cov-2 and measles, mumps and rubella viruses: preliminary evidence that mmr vaccine might provide protection against covid-19 btr's viral immunology research is supported by nih grants ey05093 and ai142862 the authors declare no conflict of financial interest. key: cord-301904-mjfbvl5n authors: schultz-cherry, s. title: astroviruses date: 2014-11-28 journal: reference module in biomedical sciences doi: 10.1016/b978-0-12-801238-3.02539-3 sha: doc_id: 301904 cord_uid: mjfbvl5n astroviruses are positive-sense, single-stranded rna viruses. their genomes contain three open reading frames, but the exact number of encoded proteins remains unknown. astroviruses were originally identified in association with childhood diarrhea; subsequently, they have been identified as a common enteric virus infecting children under the age of 2. infection is not restricted to humans, however, and astroviruses have been found in widespread mammalian and avian species. generally, infection causes a mild, self-limiting gastroenteritis, although infection can result in nephritis, hepatitis, and encephalitis in certain host species. astrovirus pathogenicity and immune response is only poorly characterized and may differ between mammalian and avian species. in this article, the current knowledge of astroviruses is reviewed, including their molecular virology, viral evolution, pathogenesis, and immune response. astroviruses are enteric viruses first identified in the feces of children with diarrhea that predominantly impact the pediatric population. detection was originally based on a five-to six-pointed star morphology of virions by electron microscopy (em). however, only about 10% of viral particles display these structures; the remaining 90% of particles have a smooth surface and a size similar to other small, round-structured viruses like picornaviruses and caliciviruses. thus, accurate diagnostics were difficult to obtain and determining the true prevalence of astrovirus within a population was challenging. development of much more sensitive detection techniques like real time reverse transcription-polymerase chain reaction (rt-pcr), cell culture rt-pcr, and astrovirus-specific enzyme-linked immunosorbent assays (elisas) have made detection more accurate and specific, even allowing diagnosis of specific serotypes. utilizing these techniques, classic human astroviruses are known to be distributed worldwide and are associated with 2-9% of cases of acute, non-bacterial diarrhea in children although incidences as high as 61% have been reported. astroviruses can also be isolated in a subset of asymptomatic individuals, suggesting that a proportion of infected individuals shed the virus asymptomatically or for some time after the resolution of other symptoms of infection. asymptomatic carriers may be a major reservoir for astroviruses in the environment and could contribute to dissemination of the virus. the release of astroviruses into the environment is a concern due to the extreme stability of the virus. astroviruses are resistant to inactivation by alcohols (propanol, butane, and ethanol), bleach, a variety of detergents, heat treatment including 50 c for an hour or 60 c for 5 min, and uv treatment up to 100 mj cm à2 . human astroviruses are known to survive up to 90 days in both marine and tap water, with survival potential increasing in colder temperatures. studies have described the isolation of infectious virus from water treatment facilities. furthermore, astroviruses can be concentrated by filter-feeding shellfish like oysters and mussels in marine environments. astroviruses are transmitted fecal-orally, and contaminated food and water have been linked to astrovirus outbreaks. similarities, astroviruses were originally thought to belong to either the families picornaviridae or caliciviridae. however, the lack of a helicase and use of a frameshifting event during replication (discussed below) distinguish astroviruses so completely that, in 1995, the international committee on taxonomy of viruses (ictv) classified astroviruses as a unique family, astroviridae, which is now divided into two genera, mamastrovirus and avastrovirus. since 2008, the number of animal hosts that have been shown to be infected with astroviruses has significantly increased comprising at least 30 mammalian and 14 new avian species increasing the complexity of classification. originally, classification within each genus was based on the species of the host of origin. at present genera are divided into viral species or genotypes based on either the host range or genetic differences within the complete capsid sequence. on average the mean amino acid distance (p-dist) between both genera is 0.83 and within genera distances are 0.72 and 0.64 respectively. currently, ictv recognizes 19 species within the mamastrovirus genus (mastv) and 3 within the avastrovirus (aastv) genus but following the standardized criteria for classification, the two genera could be further divided into 33 and 11 genotype species respectively ( figure 2 ). this complexity is likely to increase in the future given the continual isolation of new astrovirus genotypes, identification of recombinant viruses, and due to the fact that it is common to find that a single host species may be susceptible to infection with divergent astrovirus lineages. there is also increasing evidence of cross-species transmission highlighting astroviruses zoonotic potential. astroviruses have been detected throughout the world. while the exact incidences of infection vary from study to study, community-acquired astroviruses are found in 2-9% of children with infectious gastroenteritis. in some developing countries, infection rates as high as 61% have been observed. in many cases, astroviruses are one of the most commonly detected viral pathogen in young children after rotavirus and norovirus. astrovirus infections are identified in up to 2% of asymptomatic figure 2 phylogenetic relationships between representative species and genotypes of the family astroviridae. the predicted nt sequences of the entire genomes of representative viruses were aligned using clustal w. the phylogenetic tree was generated using the neighbor joining algorithm as implemented by the mega 6 program. individuals. these data may underrepresent actual astrovirus infections, as studies generally survey individuals visiting medical care centers. because astrovirus disease is generally mild in humans (see the section discussing pathogenesis), hospital cases may represent only a slight proportion of actual infections in the community. in support of this, serological studies have demonstrated that up to 90% of children have been exposed to at least one strain of astrovirus by age 9. viral infection occurs with equal frequency in boys and girls and predominantly in children under the age of 2. infection is not restricted to young children, however, and has been noted in individuals of all ages, including immunocompetent adults and the elderly. immunodeficient individuals, particularly those that are hiv-positive, appear to be at an increased risk of astrovirus infection. astrovirus infection occurs year-round, but with the highest frequency during the autumn and early winter months. in tropical climates, infection correlates with the rainy season. these seasonal correlations likely reflect the indoor confinement of the population as well as the increased stability of astroviruses in cold, damp conditions. astrovirus outbreaks have also been associated with high-density environments, including childcare centers, primary and junior high schools, military recruiting centers, elderly care centers, and swimming pools. astrovirus as a cause of hospital-acquired viral diarrhea in young children is second only to rotavirus and norovirus, occurring at rates of 4.5-6%, and, in some studies, surpasses rotavirus in rates of nosocomial infections. four phylogenetic clades of human astroviruses (hastv) have been identified to date including the 8 serotypes of classic hastv (hastv 1-8 in mastv 1) and the novel hastv-mlb (mastv 6), hastv-va2/4 and hmo-a (mastv 8), and hastv-va1/3, hmo-b/c, and ps (mastv 9). the classic hastv 1-8 strains are widely recognized as a common cause of diarrhea in children, with all eight circulating globally to various levels. hastv-1 is by far the most prevalent serotype, comprising 25-100% of astroviruses in a region, and the most prevalent reactivity of antibodies detected, although serological surveys of all serotypes have not been undertaken. hastv-6, -7, and -8 are the least frequently detected, although three to four serotypes of hastv are often detected in a region at any given time. the differing prevalence of serotypes could be a reflection of severity; perhaps hastv-1 infection results in a higher frequency of hospital visits than other serotypes and is therefore overrepresented in hospital-based epidemiological studies. alternatively, serotypes may be restricted by region. for example, one mexican study identified hastv-1 as the predominant serotype throughout the country, but hastv-3 and -8 were prominent in select regions. intriguingly, there appears to be a decrease in classic hastv incidence in the last few decades. this could be due to differences in detection methodologies or it's possible that this could be due to displacement of classic hastv infections by those of the novel hastv strains. infections with the non-classic hastvs (hastv-mlb and hastv-va/hmo), which are genetically more closely related to animal viruses, were first detected in the stool of children with gastroenteritis. however, a case control study on hastv-mlb1 and classic hastvs in india showed that while classic hastvs were significantly associated with diarrhea, hastv-mlb1 was not and mlb1 titers in stool did not differ between symptomatic and asymptomatic individuals. hastv-mlb2 viruses have also been detected in the plasma of a child with upper respiratory infection suggesting that hastv-mlb pathogenicity may affect extra-enteric tissues and tropism may not be restricted to the gastrointestinal tract. similarly, the hastv-va/hmo viruses have been detected in pediatric gastroenteritis samples from several parts of the world and serological studies suggest that hastv-va is a highly prevalent human infectious agent. recent studies reported that an hastv-va1-like strain was detected in a patient with new-onset celiac disease and associated with extra-intestinal dissemination (including neural tissue) in immunocompromised children. together, these findings highlight the public health need to better understand the impact of different human astrovirus genotypes in human health requires. importantly, new diagnostic tests are desperately needed that detect all of the hastv genotypes. our current assays are primarily limited to detecting the classical hastv 1-8 strains. interestingly, astrovirus infection occurs quite frequently (up to 50%) as a co-infection with other enteric pathogens. the most frequent co-pathogens are noroviruses and rotaviruses, but infections with adenoviruses, parasites, and enteric bacteria are often detected as well. the importance of this in humans is not entirely clear. in a study specifically examining co-infections, astrovirus co-infection with rotavirus increased the duration of diarrhea and vomiting over either virus alone, although whether this difference was statistically significant is unknown. further, no studies to date have correlated disease severity with the duration of viral load or specific human astrovirus genotype. most animals are not routinely screened for astrovirus infection, so our knowledge of the prevalence of infection is limited to surveillance studies. through these studies we now know that there is remarkable genetic diversity amongst the mastvs and aastvs. astroviruses have been found in association with most animals examined, although the effect of infection varies with species (see below). while astroviruses were originally identified in humans, they have since been identified in both marine and land-based, companion and a variety of mammals including mink, bat, rabbits, mice, calves, sheep, piglets, dogs, red-tailed deer, kittens, and a variety of domestic and wild avian species. like hastvs, there appears to be large genetic diversity amongst the other mastv including ovine, bovine, and bat astroviruses. the best epidemiologically characterized animal astroviruses are the turkey astroviruses. surveillance of turkey flocks in the 1980s isolated astrovirus from 78% of diseased flocks, but only 29% of normal flocks. astroviruses were the first pathogen detected in many flocks and were most commonly detected in birds less than 4 weeks of age. similar to human infections, turkey astrovirus was frequently isolated with other pathogens, most commonly rotavirus-like viruses. the early age of infection and the prevalence of co-infections led one group to postulate that astrovirus infection may predispose birds to infection by other viruses. attempts at in vitro propagation of astroviruses have been met with varying degrees of success. the most successful techniques utilize cultured cells from the host species and provide exogenous trypsin in the culture. successful propagation of the classical hastv 1-8 strains was originally achieved by repeated passage through primary human embryonic kidney cells; it was later discovered that direct passage through the human intestinal cell line caco-2 would also yield infectious virus. propagation of porcine, bovine, and chicken astroviruses has been successful in their respective host cells in vitro. however, many astroviruses still have not been adapted to propagation in vitro for unknown reasons, while others lose infectivity with subsequent passages and therefore cannot be maintained continuously. this problem has been circumvented in some systems by passing the virus through an animal system, as is the case for the turkey astrovirus, in which highly concentrated virus can be obtained from infected turkey embryos in ovo. astroviruses contain one copy of positive-sense, single-stranded rna. the genome is approximately 6.8 (6.2-7.8) kb excluding the 3' polyadenylated tail and contains three open reading frames (orfs), orf1a, -1b, and -2, as well as 5 0 and 3 0 untranslated regions (utrs) (figure 3) . a vpg protein is covalently linked to the 5' end of the genome and the 5 0 and 3 0 utrs are highly conserved and are believed to contain signals important for genome replication. the classic hastvs, hastv-va/hmo, cat, ovine, porcine and avian astvs also contain a stem-loop ii secondary structure motif at the 3' end of their genomes of unknown function that is also found at the 3'end of the genomes of some members of coronaviruses, noroviruses, and rhinoviruses. the length of the orfs varies amongst the astrovirus strain with variations due largely to insertions and deletions present at the 3' end of the orf1a. a new orf, termed orfx, overlapping the 5' end of orf2 in the +1 reading frame has been described in the classic hastvs and some mammalian astroviruses. astroviruses initiate infection by binding to an unknown receptor. given the susceptibility of different cell lines for hastv infection (depending on the serotype and genotype) it is possible that astroviruses use a variety of attachment proteins or receptors including carbohydrate moieties. studies of hastv infection in the highly permissive caco-2 cells suggest that hastv enters the cell via clathrin-mediated endocytosis. rna uncoating likely occurs upon acidification and maturation of the endosome leading to release of the viral rna into the cytoplasm where orf1a and -1b are immediately translated by the host machinery. it has been estimated that the initial binding and virus uncoating steps takes $130 min. orf1a is 2.8 kb and encodes a polypeptide of approximately 110 kda. this polypeptide contains a variety of conserved motifs, including several putative transmembrane domains, a bipartite nuclear localization sequence (nls), and a serine protease motif. the translated polypeptide is cleaved by both cellular protease(s) and the viral protease into at least five peptides. the actual function of each protein remains largely unknown. the transmembrane domains may localize to the endoplasmic reticulum (er) membrane to facilitate replication, as all plus-strand rna viruses have been shown to replicate in association with a membrane. one peptide, nsp1a/4, colocalizes with the viral rna at the er membrane; mutations in nsp1a correlate with increased viral titers in vitro and in vivo, suggesting a role for this protein in viral replication. the role for the nls remains unclear; some reports suggest viral antigen is observed in the nucleus, while others find that it is excluded. recent studies demonstrated that some of the non-structural proteins may undergo posttranslational modifications including phosphorylation that could modulate viral protein-protein interactions. the second reading frame, orf1b, overlaps orf1a by 70 nucleotides and has no detectable start codon. intensive research has determined that orf1b is translated by a frameshift into the à1 frame. this frameshifting event is unique among plus-strand animal rna viruses and requires a highly conserved shifty heptameric sequence (a 6 c) as well as a downstream hairpin structure. this event, which occurs with frequencies up to 25% in cells, results in an orf1a/1b fusion peptide. cleavage near the 1a/1b border releases the orf1b gene product: the viral rna-dependent rna polymerase (rdrp). astrovirus polymerase is a super group 1 rdrp, a group which generally utilizes a vpg to initiate transcription. expression of the rdrp results in production of a minus-strand viral template. this generates multiple copies of the plus-strand genome as well as a polyadenylated subgenomic rna (sgrna) containing short 5 0 and 3 0 utrs and orf2. orf2 is in the 0 frame and overlaps orf1b slightly (four nucleotides) in human astroviruses. production of the capsid protein from a sgrna not only temporally restricts capsid production to later in the viral replication cycle, but also allows for massive capsid protein expression; it is estimated that sgrna is produced in tenfold excess of the viral genome by 12 h post infection (hpi). the sgrna is about 2.4 kb and encodes the single structural protein of approximately 87 kda. this peptide is cleaved by an intracellular protease to approximately 79 kda; mutational analyses suggest that this 8 kda stretch is required for efficient expression of the capsid protein. individual capsid proteins multimerize spontaneously to form icosahedral structures of about 32 nm (figure 1(b) ). positive-sense genomes are packaged into these viral-like particles (vlps), possibly through interactions with the first 70 amino acids of the capsid protein. the virions are released by an unknown mechanism, which may involve cellular caspases, after which the capsid undergoes an extracellular trypsin-mediated maturational cleavage. this increases infectivity up to 10 5 fold, condenses the virion to approximately 28 nm, and transforms the 79 kda capsid protein into at least three smaller peptides of approximately 34, 29, and 26 kda. computational predictions suggest that vp34 may comprise the core of the virion while vp29 and vp26 form spike-like projections that may be important for viral tropism and receptor binding. this is corroborated by studies suggesting that vp26 is only loosely associated with the virion. these spikes are also thought to be responsible for the star morphology visible by em (figure 1(a) ). finally, a subset of mammalian astroviruses contain an additional orf, orfx, that overlaps the 5' end of the orf2 in the +1 reading frame that could be translated through leaking scanning. the translation product of orfx has not been confirmed. little is known about the role of host cell signaling pathways in astrovirus replication. interaction of caco-2 cells with hastv results in the activation of the extracellular signal-regulated kinase (erk1/2) and the phosphoinositide-3 kinase (pi3k) pathways both of which are required for effective entry and productive replication. activation of erk1/2 is independent of productive viral replication; binding alone is sufficient. although clearly required for productive replication, the exact mechanism(s) by which these host kinases regulate the astrovirus life cycle remains unknown. examination of nucleotide changes and nonsynonymous amino acid changes from the whole genome and across species suggests that an ancient divergence between avian and mammalian astroviruses occurred approximately 310 million years ago and was followed by divergence within the mammalian viruses due to several cross-species transmissions. the phylogenetic tree topology suggests that inter-species transmissions may occur and that astroviruses have zoonotic potential. several of the mammalian and avian astrovirus species may infect more than one host indicating that cross-species transmission is a frequent event, especially in birds. further, it is clear that a variety of hastv genotypes can infect humans. as rna viruses, nucleotide mutations and recombination events are important in evolution. hastv genome mutation rates are similar to other rapidly evolving ss(+) rna viruses for example picornaviruses and estimated to undergo approximately 3.7 x 10 à3 nucleotide substitutions per site per year; although higher genetic variability occurs in the orf2 as compared to orfs 1a and 1b due to selective pressures or distinct evolutionary constraints. amongst the host species, enhanced accumulation of synonymous substitutions, particularly in orf2, have been observed in porcine, ovine, mink, and turkey astroviruses compared to those infecting humans. finally, recombination events are being increasingly identified amongst the astroviruses. natural recombinants have been identified between strains belonging to the same genotype or different serotypes of classic hastv. whether recombination occurs amongst strains belonging to distinct viral genotypes is unknown although there have been suggested events between classic hastv and california sea lion astrovirus and classic hastv and porcine astroviru. most recombination points have been described as upstream of the orf1b/orf2 junction region, but can be found at areas within each of the orfs. further work is needed to understand the impact of recombination on astrovirus evolution. astrovirus infection in mammals presents clinically as gastroenteritis. disease has been most closely studied in humans and, in volunteer studies, astrovirus-infected individuals develop diarrhea, the most prominent symptom, as well as vomiting, nausea, anxiety, headache, malaise, abdominal discomfort, and fever. hastv diarrhea is typically milder than those caused by rotaviruses or noroviruses. onset of symptoms at 2-3 days post infection (dpi) correlates with shedding of the virus in feces, although shedding can continue after resolution of other symptoms. astrovirus infection has also been associated with intussusception, although a causative role has not been established, and with necrotizing enterocolitis in premature infants. more recently, classic hastv infection were shown to spread systemically and cause severe disseminated lethal infections in highly immunocompromised children. as described above, the novel hastv-mlb and hastv-va/hmo have also been identified in extra-enteric tissues including the upper respiratory tract and brain as well as sera. further studies are needed to understand the tropism of astroviruses and the role of these viruses in pediatric infections. additionally, mink and bovine astrovirus infections have been associated with neurological complications including encephalitis. the earliest studies of astrovirus pathogenesis utilized gnotobiotic sheep and calves as models. in calves, astrovirus infection was localized to the dome epithelial cells overlying peyer's patches. these cells appeared flat or rounded and released cells were identified in the intestinal lumen. astrovirus infection in calves was shown to be specifically targeted to m cells and led to the sloughing of necrotic m cells into the intestinal lumen. enterocytes were never observed to be infected. specific tropism of the virus for immune cells suggests that astrovirus may have an immunomodulatory role in calves. while the virus replicated in these animals and could be detected in their feces, the calves displayed no clinical signs. in most bovine studies, viral infection is asymptomatic, although changes in the feces from solid and brown to soft and yellow were noted in one study. mild villus atrophy and slight changes in villus-to-crypt ratios have been noted but no changes in xylose absorption were observed. despite the lack of symptoms, viral shedding continued until the termination of the experiment. studies in sheep have shed more light on histological changes associated with infection. astrovirus-infected sheep developed a transient diarrhea as early as 2 dpi, but virus was detected at early as 14 hpi and initially confined to the lumenal tips of the intestinal villi. by 23 hpi, virus was observed coating the microvilli and infection had spread to the apical two-thirds of the villi. this correlated with sloughing of degenerate cells from the apical portion of the villi, which continued through 38 hpi. at this time, villus blunting was apparent in the ileum and midgut. furthermore, normal epithelial cells lining the villi were replaced with immature, cuboidal cells reminiscent of crypt cells. neither these immature cells nor crypt cells were ever observed to be infected, suggesting that only mature enterocytes are susceptible to infection. by 5 dpi, viral infection had cleared and intestinal histology had returned to normal. volunteer studies in humans have not explored the underlying causes of astrovirus pathogenesis; our knowledge is therefore limited to intestinal biopsies taken for other reasons, but generally support the observations described above. in a biopsy from a child shedding large quantities of astrovirus, slight histological changes, including mild villous blunting and irregular epithelial cells, were observed. infection increased distally through the small intestine. similarly to animal models, astrovirus infection was restricted to the apical two-thirds of intestinal villi and could be identified in infected cells. the recent characterization of murine astroviruses may afford a mouse model to study many aspects of astrovirus replication, tropism and immune response, although they did not suffer diarrhea and may not be an ideal model to understand disease pathogenesis. in vitro studies using differentiated caco-2 cells demonstrated that hastv infection leads to reorganization of the cytoskeleton and disruption of the epithelial tight junctions resulting in increased epithelial barrier permeability. notably, the increased barrier permeability was independent of viral replication; purified recombinant capsid protein alone was sufficient. similar results were obtained in turkey poults administered purified recombinant turkey astrovirus type-2 capsid protein. inoculated poults exhibited disruption of cellular tight junctions and epithelial barrier permeability and acute diarrhea suggesting that the astrovirus capsid protein may act as a novel viral enterotoxin. although this appears to be independent of increased cell death, particular strains of hastv have been shown to induce apoptosis late during the course of infection. further studies are required to understand the mechanism(s) for the increased barrier permeability and the role in disease pathogenesis. in avian species, astrovirus infection has a much broader range of disease than in mammals. while astrovirus does cause gastroenteritis in turkeys and chickens, it can also cause nephritis in chickens and a severe, often fatal, hepatitis in young ducklings. turkey astrovirus was the first discovered avian astrovirus and remains the best characterized in terms of pathogenesis, due in part to the development of the turkey as a small animal model. in these animals, virus could be detected from 1 to 12 dpi in the intestines. viral replication was limited to the enterocytes on the apical portion of the villi, but the virus could be detected throughout the body, including the blood. the development of viremia is rare among enteric viruses and its function remains unclear. infected turkeys developed a yellow, frothy, gas-filled diarrhea from 1 to 12 dpi. diarrhea occasionally contained undigested food, but never blood. the intestines of infected birds became thin walled, flaccid, and distended. despite these changes, histological examination suggested that only mild changes occur during infection. a mild crypt hyperplasia and shortening of the villi were noted from 4 or 5 to 9 dpi, and single degrading enterocytes could be identified. however, tunel staining suggested that the amount of cell death in infected intestines is similar to control birds. d-xylose absorption, a measure of intestinal absorption, was significantly decreased from 2 to 5 dpi in one study and up to 13 dpi in another. this effect was exacerbated in the presence of another enteric pathogen, turkey coronavirus. astrovirus infection also caused a significant growth depression in turkey poults by 5 dpi; infected birds never recovered from this, leading to flock unevenness. infected birds also demonstrated a transient (3-9 dpi) reduction of the thymus, which returned to normal by 12 dpi. avian infection by astroviruses can present with nonenteric symptoms as well. infection of ducklings with duck astrovirus causes a severe hepatitis. infected birds develop liver hemorrhage, swollen kidneys, and hepatocyte necrosis. on farms, infection leads to mortality rates of 10-25% in adult (4-6-week-old) ducks, but can reach 50% in ducklings under 14 days of age. in chickens, infection with the astrovirus avian nephritis virus (anv) results in discoloration of the kidney, development of renal lesions, and interstitial nephritis by 3 dpi. pathogenesis is age dependent, with 1-day-old chicks the most susceptible and adult birds the least. anv infection can result in mortality rates of up to 33%, although rates appear to be strain specific. the immunological response to astrovirus infection is poorly defined; however, observations in humans and animal models suggest that both the adaptive and innate responses play important roles in controlling and eliminating the virus. the humoral immune response likely plays a major role in astrovirus immunity. the biphasic infection pattern of young children and the elderly suggests that antibodies are protective during the middle of life. indeed, serological studies have indicated that approximately 50% of neonates have maternally acquired antibody to hastv, which wane by 4-6 months of age. children then acquire anti-hastv antibodies rapidly due to astrovirus exposure. by the age of 9, up to 90% of the population has been exposed to hastv-1. furthermore, volunteer experiments demonstrate that astrovirus exposure generally leads to an increase in anti-astrovirus antibody titer. while astrovirus antibodies protected individuals from symptoms associated with infection, virus was identified in the feces, suggesting that such antibodies do not necessarily prevent viral replication. additionally, immunoglobulin treatment has been attempted as a treatment for severe or chronic astrovirus infection. the results have been mixed and difficult to interpret, as the presence of astrovirus-specific antibodies in the immunoglobulin treatment was not always confirmed. seroprevalence studies also showed that the vast majority of healthy young adults have antibodies against the novel hastvs. cellular immunity may also play a role in controlling and/or preventing astrovirus infection. studies have demonstrated that most individuals possess hla-restricted, astrovirus-specific t cells. when stimulated with astrovirus in vitro, these cells produce tumor necrosis factor, interferon gamma, and occasionally interleukin (il)-5 but not il-2 or il-4. these cytokines are typical of the t-helper-type response thought to be important in controlling viral infections. individuals deficient in t and b-cell functions are unable to control infection, shedding virus to very high titers (! 10 14 particles ml à1 ) and for extended periods of time (up to 18 months) further supporting the importance of cellular immunity. finally, adaptive immunity has been shown to restrict astroviral replication during primary infections in the murine model. rag1 à/à mice, which are deficient in b and t cells, show significantly higher levels of viral shedding in the feces and higher genomic copies in intestinal and extra-intestinal tissue as compared to wild-type mice suggesting that viral dissemination is restricted by the murine adaptive immune response. viral replication is also higher in stat1 à/à highlighting a role for interferon in limiting astrovirus replication. experiments in a turkey model demonstrate that the adaptive response is not the only important immunological response. in this model, no increase in t cells (cd4 + or cd8 + ) could be demonstrated after tastv-2 infection. moreover, while infected turkeys produced a slight increase in antibody production, these antibodies were not neutralizing and did not prevent against future infection. however, it was noted that macrophages from tastv-2 infected turkeys produced significantly higher levels of nitric oxide (no) both in vivo and upon stimulation ex vivo. inhibition of no in vivo led to a significant increase in viral production, while addition of exogenous no decreased viral production to below the detection limit, suggesting that no is an important factor in controlling astrovirus infection. the importance of macrophages and their role in astrovirus infection has been corroborated by observations in astrovirus-infected lambs, where em showed virions within macrophages. furthermore, it is possible that astroviruses have a mechanism to combat this response, as macrophages in astrovirus-infected turkeys demonstrate a reduced ability to phagocytose. finally, the human astrovirus capsid protein inhibits the complement system, which is an important innate immune response against infection. purified recombinant capsid protein directly interacts with and inhibits the complement regulatory proteins c1 and mbl suggesting an ability to block both the classical and lectin complement pathways. intriguingly, a 15-amino acid sequence (residues 79-108) at the conserved n-terminal end of the capsid, which is highly conserved amongst a variety of mammalian astroviruses, is the minimal region required to inhibit complement. more work is needed to understand the role of complement inhibition in astrovirus pathogenesis. because astrovirus infection is generally mild and self-limiting in humans, treatment is generally restricted to fluid rehydration therapy. this can often be accomplished at home; thus, hospital admittance is rare. no vaccine is yet available for humans, and as noted above, immunoglobulin treatment for immunocompromised individuals has been met with varying degrees of success. more recently, authors have speculated that the use of probiotics may interfere with the biological cycle of enteric viruses and may be useful in treatment; however, more studies are needed to confirm these findings. additionally, no treatment for astrovirusinfected animals exists. the best solution, therefore, is prevention of transmission, which is best done in humans by conscientious hand and food washing. the stability of astroviruses and their resistance to inactivation make them difficult to eliminate after introduction. this is a significant problem in hospitals, where individuals are generally immunocompromised and therefore more susceptible to infection. one outbreak in a bone marrow transplant ward prompted the hospital to scrub the entire ward with warm, soapy water. however, surveillance of the subsequent inhabitants demonstrated fecal shedding of astroviruses, underscoring the difficulty in removing the virus. this is also a significant problem in commercial farming, where astrovirus infection of animals significantly decreases productivity. its introduction and maintenance in this environment can mean drastic financial losses. in each of these environments, early detection and thorough disinfection are keys to limiting transmission and controlling infection. human astroviruses astrovirus infections in humans and animals -molecular biology, genetic diversity, and interspecies transmissions astrovirus research: essential ideas, everyday impacts, future directions key: cord-343728-udjjijyu authors: muggia, victoria a.; puius, yoram a. title: nocardia ignorata infection in heart transplant patient date: 2020-11-17 journal: emerg infect dis doi: 10.3201/eid2611.202756 sha: doc_id: 343728 cord_uid: udjjijyu nan to the editor: we read with interest the recent description of pulmonary nocardia ignorata infection (1) . we report a similar infection in an orthotopic heart transplant recipient, which likely began as a pulmonary infection with dissemination to soft tissue, without known exposure. risk factors included tacrolimus, steroids, older age, and posttransplant intensive care unit admission (2) . the patient was a 66-year-old african american man with a history of ischemic cardiomyopathy. after implantation of a left ventricular assist device, infectious complications included enterococcus faecalis device infection and extended spectrum β-lactamase-producing (esbl) klebsiella urosepsis. the course after left ventricular assist device explantation and orthotopic heart transplant was complicated by tamponade requiring a pericardial window and an esbl klebsiella urinary tract infection treated with meropenem. because of leukopenia, pneumocystis prophylaxis was changed from trimethoprim/ sulfamethoxazole to atovaquone 2 weeks posttransplant. esbl klebsiella bacteremia recurred 6 weeks later, again treated with meropenem. the patient returned 6 months posttransplant with 10 days of cough and dyspnea. chest computed tomography demonstrated bilateral nodules with cavitation, bronchiectasis, and spiculation. we initially treated the patient with meropenem and doxycycline. results from severe acute respiratory syndrome coronavirus 2 swab test, respiratory pathogen panel, fungal studies, and sputum culture were nondiagnostic. we obtained no additional pulmonary samples. due to severe left calf pain, venous duplex was performed, revealing a nonvascular mass. the patient reported no trauma, soil contact, or recent travel. the abscess was aspirated, demonstrating branching gram-positive beaded rods. the isolate was identified by a reference laboratory (mycobacteria and nocardia laboratory, university of texas health center at tyler, tyler, tx, usa) by partial 16s rrna sequencing as a 99.51% match with nocardia ignorata, with susceptibilities identical to the isolate in rahdar et al. (1) . brain magnetic resonance imaging results were unremarkable. the patient's respiratory status and leg pain quickly her current research investigates the impact of social determinants of health needs and interventions on severe maternal morbidity and maternal mortality in an attempt to improve parity in maternal/neonatal health outcomes. isidore etienne nocard (1850-1903), a french veterinarian and microbiologist who discovered the bacteria in 1888 from a bovine farcy case. he named this filamentous, branching bacteria streptothrix farcinica (greek streptós-"twisted" and thrix "hair"). farcy (old french farcin), is a form of cutaneous glanders, characterized by superficial lymph node swelling and ulcerating nodule formation under the skin (late latin farcīminum "glanders," from latin farcīmen "a sausage," from farcīre "to stuff"). one year later, trevisan characterized and termed the bacteria nocardia farcinica, creating the genus nocardia. in 1890, eppinger isolated a similar organism from a brain abscess and called it cladothrix asteroides (greek kládos-"branch" and -thrix "hair") because of its star-shaped colonies (greek asteroeidēs "starlike"). blanchard renamed the organism nocardia asteriodes in 1896. additional taxonomic work in 1962 resulted in nocardia asteroides replacing nocardia farcinica as the type species for the genus nocardia. twisted hair bacteria (nocardia spp.) described by edmond nocard, from a bronchial alveolar lavage sample. nocardiosis is an opportunistic infection, commonly associated with pulmonary disease. nocardia are partially acid-fast, filamentous, branching bacilli (modified kinyoun acid-fast stain using weak acid [0.5% sulfuric acid] for decolorization and methylene blue counterstain, original magnification x1,000.) photograph courtesy of the author. improved and he was discharged on long-term trimethoprim/sulfamethoxazole and doxycycline. because of renal insufficiency, trimethoprim/sulfamethoxazole was switched to moxifloxacin after 2 weeks. chest radiograph results were improving 3 months later. associations between built environment, neighborhood socioeconomic status, and sars-cov-2 infection among pregnant women in new york city disparities in incidence of covid-19 among underrepresented racial/ethnic groups in counties identified as hotspots during georgia department of public health. covid-19 daily status report what obstetrician-gynecologists should know about population health understanding and enhancing the u.s. department of housing and urban development's zip code crosswalk files characteristics of women of reproductive age with laboratory-confirmed sars-cov-2 infection by pregnancy status-united states plant pests excluding bacteria the type species of the genus nocardia note on the disease of oxen from guadeloupe known as farcin nocardiosis: review of clinical and laboratory experience milan: zanaboni and gabuzzi; 1889. references 1 pulmonary nocardia ignorata infection in gardener, iran european study group for nocardia in solid organ transplantation. nocardia infection in solid organ transplant recipients: a multicenter european case-control study key: cord-296567-six7u615 authors: hussain, akhtar; cristina do vale moreira, nayla title: clinical considerations for patients with diabetes in times of covid-19 epidemic date: 2020-04-10 journal: diabetes metab syndr doi: 10.1016/j.dsx.2020.04.002 sha: doc_id: 296567 cord_uid: six7u615 nan dear sir. as of today's reports, the global number of confirmed cases of covid-19 has surpassed 150,000. the number of known cases is increasing by several thousand every day. on march 11, 2020, who publicly characterized covid-19 as a pandemic. the issue is of serious concern and deserves momentous attention. coronaviruses are a family of viruses that cause respiratory illnesses. most of them cause illness in animals, but seven known types of coronaviruses cause illness in humans. the coronavirus sars-cov-2 (severe acute respiratory syndrome-coronavirus-2) is one of those viruses -it causes the illness currently known as coronavirus disease-2019 (covid-19) . though we are still learning what exactly puts someone at greater risk of developing a severe illness with covid-19, early information indicates older patients and those with chronic medical conditions such as hypertension, diabetes and cardio-cerebrovascular diseases may be at higher risk (1) (2) (3) . infection caused by covid-19 is likely to disturb metabolic regulation. diabetic patients with covid-19 may face an altered immune response on the background of an already compromised health status owing to the diabetes-related complications and/or aging. the most important findings in patients with hyperglycemia and a viral infection were significant worsening of symptoms, which implies greater morbidity in these patients when compared to those without diabetes (3) . however, the pathophysiology of this association remains uncertain. it is not known whether hyperglycemia changes the virulence of the infection, or if the virus modifies the glycemic metabolism. what we know is that diabetic patients are more susceptible to infection, and this can impact on glucose metabolism (4). dm is not just a disorder of glucose metabolism, but a chronic inflammatory condition characterized by multiple changes in lipid, carbohydrate and protein profiles (5) . such inflammatory processes are due to hyperglycemia which leads to increased synthesis of glycosylation end products (ages), activates macrophages and other cells of the immune system, increase oxidative stress and promote the synthesis of pro-inflammatory cytokines, besides stimulating the synthesis of adhesion molecules that facilitate inflammation in the tissues (5) . the inflammatory process and its complications might provide a higher propensity to infections or a greater severity of these conditions. another important issue is how this inflammatory and immune response occur in diabetic patients who acquire a viral infection, as well as whether the virus itself interferes with insulin secretion or the glycemic control. at this stage, the biological mechanism of the relationship between covid-19 and diabetes is not known, but the association for the severity of cases and death is pronounced. we need to develop a hypothesis to explain the causal path underlying the more severe clinical presentation of covid-19 infection and subsequent death in diabetic patients. biochemical tests are also essential to clarify the molecular pathophysiology involved. the association of covid-19 and dm is of substantial public health importance and deserves proper attention, since a large and diverse population is being affected globally. nowadays this comorbidity poses a relevant threat to human health, and prospective well-designed studies to elucidate the biological mechanism and the best clinical management of this association are urgently needed. it was very useful to see the review published in the journal on diabetes and covid 19 infection [1] . in it you highlight that people with diabetes have a death rate perhaps around four times that of the background population, and i note that it has been suggested over 20-40 % of deaths in china/wuhan were in people with diabetes [2, 3] , clearly it is important to understand why. there will be some confounders here in the form of associations with other risk factors for dying with a covid-19 infection, notably age and cardiovascular disease, but these cannot explain all the excess. we need to understand this better to mitigate the risk. it is reported that most of the deaths are occurring in the context of pneumonitis [2, 3] , and in particularly onset of adult respiratory distress syndrome (ards), a condition in which excess fluid in the alveoli blocks gas exchange between air and blood. people with diabetes, and many with cardiovascular disease (cvd) without diabetes, have a very permeable vasculature, identified since the 1980s as albumin lead through the kidneys (micro-and macro-albuminuria), but even prior to that as a late blush over the retina with intravenous fluorescence marker injection [4] . this leaky vasculature is associated with vascular inflammation, metabolic syndrome, and steatohepatitis, as well as cvd per se. it would seem not too far a stretch, in the absence of further research as yet, to assume that prior enhancement of vascular permeability could account for the increased rate of ards and death in people with diabetes, particularly type 2 diabetes, and also some with cvd. aside from further research i would suggest that those already with albuminuria (a routine yearly test for people with diabetes), those with higher liver enzyme markers (alt), and perhaps those with diabetes associated dyslipidaemia (low hdl cholesterol) should take particular steps to self-isolate. it is unclear to me how fast vascular inflammation can be ameliorated by improved blood glucose control (nearly all glucose-lowering medications reduce microalbuminuria in time), but tight glucose control in infected persons with insulin would meanwhile seem sensible. very poor glucose control is further known to interfere with leukocyte/lymphocyte function [5] . dear editor, we recently published an article highlighting the special concerns while managing patients with diabetes in the times of covid-19 pandemic i . there have been some concerns about the use of angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers (arbs), which were not clarified in our publication ii . we are summarising the current evidence in this regard and will try to arrive at a reasonable conclusion. in the absence of a vaccine and an antiviral drug for the covid-19 infection, several therapeutic approaches are being studied. one such approach is the use of inhibitors of the renin angiotensin system, namely ace inhibitors and arbs. on the other hand, some concern has been raised about the fact that patients on these agents might be at an increased risk of infection by severe acute respiratory syndrome coronavirus-2 (sars cov-2). angiotensin converting enzyme-2 (ace-2) is the receptor for sars cov-2 as well as other coronaviruses and is expressed in type 2 alveolar epithelial cells and endothelium. the sglycoprotein on the surface of coronavirus binds to ace2. this leads to a conformational change in the s-glycoprotein and allows proteolytic digestion by host cell proteases (tmprss2) ultimately leading to internalization of the virion iii . viral s-glycoprotein, tmprss2 and ace-2 inhibition are potential targets of therapy and possibly vaccine development. as ace-2 is essential to coronavirus infection, its blockade is thought to be beneficial in preventing/treating this infection. a retrospective analysis found reduced rates of death and endotracheal intubation in patients with viral pneumonia who were continued on ace inhibitors iv . mice with coronavirus induced lung injury showed improvement when treated with losartan v . as far as cvid-19 infection is concerned, the data on ras activation or the effect of its blockade is limited at present. hypokalaemia could be a marker of ras activation and high incidence of hypokalaemia has been reported in patients with covid-19 infection vi . despite these small studies suggesting the benefit of drugs acting on ras, there is some data, albeit scarce, from animal models and human studies that treatment with ace inhibitors and arb could cause up regulation of ace2 vii . ibuprofen and thiazolidinediones have also been shown to do the same viii,ix . increased expression of ace2 could theoretically increase the risk of infection with sars cov-2. this could be a concern in people with diabetes who are at already elevated risk of infections because of many other factors. however, currently, there is no evidence to support this hypothesis. in view of lack of robust evidence for either benefit or harm, it is reasonable for patients to continue using ace inhibitors and arb, as recommended by european society of cardiology council on hypertension and european society of hypertension. x,xi . fortis cdoc hospital, chirag enclave, risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease prevalence and impact of cardiovascular metabolic diseases on covid-19 in china clinical considerations for patients with diabetes in times of covid-19 epidemic diabetes and infection: is there a link?--a mini-review subclinical inflammation in children and adolescents with type 1 diabetes ) 3. the novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) -china 2020 vascular complications of diabetes: mechanisms of injury and protective factors infections, immunity and diabetes clinical considerations for patients with diabetes in times of covid-19 epidemic are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? the novel coronavirus 2019 (2019-ncov) uses the sars-coronavirus receptor ace2 and the cellular protease tmprss2 for e ntry into target cells impact of angiotensin-converting enzyme inhibitors and statins on viral pneumonia angiotensin-converting enzyme 2 (ace2) mediates influenza h7n9 virusinduced acute lung injury hypokalemia and clinical implications in patients with coronavirus disease 2019 (covid-19) the vasoprotective axes of the renin-angiotensin system: physiological relevance and therapeutic implications in cardiovascular, hypertensive and kidney diseases ibuprofen attenuates cardiac fibrosis in streptozotocin-induced diabetic rats pioglitazone upregulates angiotensin converting enzyme 2 expression in insulin-sensitive tissues in rats with high-fat diet-induced nonalcoholic steatohepatitis x position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers xi esh statement on covid-19 key: cord-288505-v4dbswyk authors: roberts, m.t.m.; lever, a.m.l. title: an analysis of imported infections over a 5-year period at a teaching hospital in the united kingdom date: 2003-11-30 journal: travel medicine and infectious disease doi: 10.1016/j.tmaid.2003.10.002 sha: doc_id: 288505 cord_uid: v4dbswyk abstract background. imported infections are an important cause of morbidity and mortality in the united kingdom. methods. a 5-year analysis of cases seen in a large teaching and district general hospital in the eastern region of the uk was performed using ward records correlated with hospital coding data and hospital episode statistics from the department of health. results. a surprising number (301) and diversity of imported infections was diagnosed. prophylactic measures were, where assessable, generally inadequate. conclusions. these data warrant renewed efforts to educate travellers of the risks of infection acquired abroad. the continued rise in global travel along with emergence of new infectious diseases emphasises further the need for expanded infectious diseases services incorporating accessible travel advice services in the uk which are currently underprovided. the number of people travelling overseas from the united kingdom (uk) is estimated to be increasing at a rate of around 8% a year. in 2002 there were over 59 million visits abroad. 1 this fact coupled with increasing immigration, has led to a steady rise in the incidence of imported infections that includes diseases rarely seen in the uk, some of which are life-threatening. furthermore, these infections may have public health implications for the uk due to the risk of ongoing transmission to other people. the recent severe acute respiratory syndrome (sars) epidemic has highlighted the need for close monitoring of emerging infections and vigilance over all imported infections. 2, 3 moreover, the speed of international travel enhances the threat of diseases such as sars in the uk. the most frequently encountered, potentially fatal imported infection into the uk is malaria, with 1081 cases notified to public health in 2001. 4 due to under reporting this is believed to be an underestimate with the actual number of cases more likely closer to 2000 annually. most predictions suggest that this statistic is also set to rise further. like many other imported infections it can be a diagnostic challenge. cases of malaria often present as a 'fever in a returning traveller', which is a heterogeneous group of diseases. clinical assessment of imported infections has been recently reviewed by spira. 5 hospitals close to ports and major airports are traditionally accepted as seeing a disproportionate caseload of infections in returning travellers. we were interested in the caseload seen in a typical large uk hospital away from a major entry route since our impression was that the number of cases of imported infection seen in cambridge is substantial. a five-year 'look-back' was therefore carried out. the analysis includes all imported infections seen on the infectious diseases ward as recorded in the ward data book, at addenbrooke's hospital in cambridge, uk over a 5-year period 1998 -2002. the records were checked against hospital case coding and the hospital episode statistics data validation information from the department of health. all three gave a very similar data set. cambridge is a university town in the eastern region with a population of around 120,000. the population catchment area of addenbrooke's hospital is approximately 500,000, shared partly by two smaller district hospitals. there is a nearby international airport but with no transatlantic or longhaul flights. the ward is a 12-bedded dedicated infectious diseases unit in a large (1000 þ bed) teaching hospital, which acts as a local district general hospital but is also the regional infectious diseases referral centre. only cases directly seen, investigated and treated by the infectious diseases team were included. outpatient cases seen in clinics only were excluded. those infections which were self-limiting presenting to other physicians were not covered. also excluded was an analysis of sexually transmitted infections acquired abroad seen only by the sexually transmitted disease service. all cases of malaria were diagnosed initially by blood films, though non-falciparum cases additionally had an hrp-2 antigen test to further rule out falciparum infection in the advent of a dual infection being overlooked. a summary of imported infections seen between 1998 and 2002 on the infectious diseases ward at addenbrooke's hospital is shown (table 1) . cases are shown both as specific diagnoses, when a causative organism is determined, and grouped as symptom complexes (gastroenteritis, fever, rash etc.) or by haematological indices such as eosinophilia, where no specific pathogen was identified but the clinical picture was of an imported infectious disease. the data indicate clearly that imported infections are an important cause of disease seen at this teaching hospital in the uk despite being relatively distant from large international airports or coastal ports. given the relatively small population catchment it seems likely the diversity and number of diseases seen may have been bolstered by the large academic contingent associated with the large university population amongst whom are many postgraduate students from abroad. the number of imported infections necessitating hospital admission is surprisingly high, yet it is likely that this is 'the tip of an iceberg' with many cases treated by general practitioners or non-specialist hospital physicians in this and other local hospitals and some not seeking medical attention. in addition cases of sexually transmitted infection acquired abroad including some early presentations of hiv infection such as seroconversion illness are missing from this survey. the cases documented here represent a diverse group of diseases, a reflection in part of the wide geographical distribution over which people now travel. there is an increasing trend in the number of cases seen per annum as borne out by national statistics. malaria remains the most frequent imported infection throughout the study with 85 cases over 5 years, 28% of the total. when investigated as cases of fever in the returning traveller, this percentage rises further (fig. 1) . the strong concordance between local and reference laboratories supports well the current diagnostic approach. overall the increasing numbers of cases accorded a diagnosis, is an impressive reflection of the diagnostic services. all admissions to the infectious diseases ward are placed in negative pressure ventilated rooms that are regularly assessed to ensure that a secure flow is maintained. this is especially important for the rising numbers of suspected cases of tuberculosis often associated with the human immunodeficiency virus (hiv) infection. two cases of multi-drug resistant tuberculosis (mdrtb) were admitted over this period, one an imported infection in a refugee. these infections pose a particular threat to public health and require considerable resources to manage. the newer definition of suspected mdrtb for all cases of tuberculosis acquired outside the uk, means numbers and need for isolation beds is certain to rise. the introduction of molecular techniques for the early detection of rifampicin resistance is a useful and cost effective advance. 6 amongst the group of hiv diagnoses: one presented with a rash, fever and lymphadenopathy and was diagnosed with an hiv seroconversion illness and there were five children who acquired hiv through vertical transmission. gastroenteritis as a category is certainly underrecorded since many cases are managed by general physicians in separate rooms in the general medical wards of the hospital. ideally all of these infectious diarrhoea cases should receive the same infection control measures as on the specialist unit. the two cases of typhoid both had full screening of family members as directed by the public health physicians with no evidence of carriage. a detailed geographical history as part of the clinical assessment is vital since 90% of all cases of falciparum malaria were in travellers to africa and all seven cases of dengue fever originated in asia reflecting the higher endemicity of these infections in these tropical regions. the two cases of lyme disease were acquired in north america and western europe, respectively, with the former presenting as a carditis and the latter as localised cutaneous involvement of erythema chronica migrans and arthralgia, reflecting the differing pathogenesis of borrelia burgdorferi sensu stricto causing carditis, compared to strains more frequently found in europe, b. garinii and b. afzelii, which cause late neurological sequelae. a small group presented as asymptomatic cases of schistosomiasis, part of a nationwide rise mainly from lake malawi, as reported elsewhere, 7 and unusually a patient-led epidemic. in most of these figure 1 distribution by diagnosis of fever in the returning traveller. cases the diagnosis was made on antibody-based tests. three of the cases of malaria were second presentations of the same disease at a later admission possibly due to repeat exposure, poor compliance with medication or drug resistance and in two cases dual infection occurred. prophylaxis is recommended for travellers to malarious regions as set out by the hospital for tropical diseases guidelines. 8 it is noteworthy that only 22% of cases admitted received a full course of prophylaxis whilst 41% did not receive any prophylaxis (fig. 2) . similar data are reported from communicable disease control in the usa on a study of 4685 cases of imported malaria in which 56% took no chemoprophylaxis. 9 in our series there was one death in a 95-year old who had visited kenya in the month preceding admission without taking prophylaxis then contracted plasmodium falciparum malaria complicated by the development of pneumonia. in conclusion, this local analysis illustrates the surprising number and diversity of imported infections seen at a hospital away from major ports of entry despite which it largely mirrors the national situation. diagnosis and management of these conditions are optimised by having a specialist unit with experience. individual physicians in nonspecialist units are unlikely to see these conditions frequently to become familiar with their management. prevention is a key component of future efforts to contain these infections along with better diagnostics and enhanced surveillance. hence, there is a requirement for expertise and the need for all large centres to have clinical infectious diseases staff. the uk is underprovided compared with most countries especially the usa but also closer comparators like australia. the lack of prophylactic measures against malaria is very worrying. this suggests inadequate availability of advice or lack of knowledge by general practitioners and physicians or a lack of advice seeking by the general public. over the past decade there have been 100 deaths from malaria in the uk, hence this is the most important area to target preventative efforts. 4 this merits a department of health driven education effort. the first steps have been taken with the formation of the national travel health network and centre, which is a government, funded initiative launched in 2003 in response to this need. 10 london: hmso a major outbreak of severe acute respiratory syndrome in hong kong transmission dynamics and control of severe acute respiratory syndrome public health laboratory service assessment of travellers who return home ill a clinical, microbiological and economic analysis of a national service for the rapid molecular diagnosis of tuberculosis and rifampicin resistance in mycobacterium tuberculosis schistosomiasis in travellers returning from sub-saharan africa hospital for tropical diseases the national travel health network and centre (nathnac) the invaluable help of the microbiology and haematology laboratories at addenbrooke's hospital, cambridge along with support from the hospital for tropical diseases, london is acknowledged without whom diagnosis would not have been possible. key: cord-303299-p15irs4e authors: dzien, alexander; dzien-bischinger, christine; lechleitner, monika; winner, hannes; weiss, günter title: will the covid-19 pandemic slow down in the northern hemisphere by the onset of summer? an epidemiological hypothesis date: 2020-06-23 journal: infection doi: 10.1007/s15010-020-01460-1 sha: doc_id: 303299 cord_uid: p15irs4e the covid-19 pandemic has affected most countries of the world. as corona viruses are highly prevalent in the cold season, the question remains whether or not the pandemic will improve with increasing temperatures in the northern hemisphere. we use data from a primary care registry of almost 15,000 patients over 20 years to retrieve information on viral respiratory infection outbreaks. our analysis suggests that the severity of the pandemic will be softened by the seasonal change to summer. the infection caused by the human corona virus covid-19 (sars-cov2) resulted in a worldwide pandemic affecting several million people and causing severe disease and fatality mostly based on virus mediated lung failure [1, 2] . thus far, no effective therapy is available and, therefore, contact precautions, hygiene measures, contact tracing, social distancing and quarantine are the methods of choice to control the spread of this infection [3, 4] . however, epidemics with respiratory virus such as not only influenza but also human corona viruses are prevalent in the northern hemisphere over several months during the cold season and then disappear whereas influenza remains prevalent in tropical regions throughout the whole year [5] [6] [7] . the reasons for this decline are still incompletely understood. we thus questioned whether or not the covid-19 pandemic will be in part slowed down by the change from the cold to the warm seasons in the northern hemisphere. to assess local epidemics from respiratory infections over the course of a season, we retrospectively analyzed the records of 15,336 patients (8907 women and 6429 men) who visited a primary care internal medicine practice in innsbruck, austria, between july 1, 1999, and march 15, 2020. overall, our dataset included 145,740 diagnoses, of which 16,317 are assigned to acute respiratory diseases according to the international classification of diseases (icd-10; www.icd.who.int/brows e10/2019/en). in particular, we included viral and bacterial infections classified into diagnostic groups a and b as well as infectious diseases of the respiratory tract of groups j (pulmonology), h (ear, nose and throat) and r (general symptoms consistent with infections such as dyspnea or fatigue). based on this data, we first counted the number of patients who sought medical help because of serious respiratory infections. second, we related these counts to the total number of diagnoses within each month to obtain the share of viral infections. in the denominator of this ratio, we excluded malignancies (group c), neoplasia (d), diabetes mellitus 2 (e-11.9), arterial hypertension (i-10) and ischemic heart diseases (i-20). as the mentioned practice was sometimes closed due to holiday breaks, the share of viral infections might provide a more suitable figure on seasonal patterns than the simple count measure. figure 1 plots the counts of respiratory diseases over the course of the years. it shows a strong seasonal pattern with peaks around the winter months december to march. the strongest upward deviations are observed in the years between 2008 and 2010 and for 2015. figure 2 depicts the share of respiratory diseases and pools the information from fig. 1 into one graph. the smoothed lines represent a nonlinear time trend based on year-wise regressions with the relative contributions of respiratory diseases per month as the dependent variable. in particular, we included a linear and a quadratic time trend, which allows to calculate predicted values and, in turn, the hump-shaped graphs in fig. 2 . we also used more flexible functional forms, e.g., higher order polynomials, but it turned out that the quadratic specifications performed well in terms of overall fit; a pooled regression over all seasons led to an r 2 of about 60%. all the calculations are carried out with stata (version 16). the colored lines plot the seasons in which previous pandemic respiratory infections have been recorded (02/03 sars-cov, purple line; 04/05 h5n1, green line; 09/10 pandemic flu, blue line) [8] but not showing the absolute contributions of these pandemic infections to the total number of respiratory infections. the grey lines indicate seasons where no specific pandemic respiratory infections are recorded. in line with fig. 1 , we can see that the peak of respiratory diseases (including putatively covid-19) lies between february and april 2020 followed by a prominent and sustained reduction towards summer. if covid-19 would behave similar to other respiratory viruses causing respiratory infections including human corona viruses which peak during winter time and early spring, there is hope that the covid-19 pandemic can be slowed down by this seasonal trend [7, 9] . in those seasonal infections, no herd immunity is achieved during a specific season [7] . this would go in a line with the duration of the sars-cov-1 epidemic in 2002/2003, which also started in china, peaked in february to april and was terminated in summer 2003 although strict contact precaution measures were likewise the main secret of success [9] . however, pandemics with new viruses such as the influenza h1n1v can circulate independent of typical respiratory viral seasons throughout the whole year [10] . in line with this, the pandemic "spanish flu" presented even with a summer peak in scandinavia in the year 1918 preceding the major outbreak in the cold season of 1918/1919 in this area [11] . this might be the only one published exception of an influenza summer epidemic. the next few months will provide a definitive answer to which scenario will hold true for the control of covid-19 infections and how changes of temperature and social behavior will impact on the control of this pandemic. conflict of interest none of the authors has any conflict of interest in association with this manuscript. ethical statement an approval by an ethical committee was not applicable to this study. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. strategic who, technical advisory group for infectious h. covid-19: towards controlling of a pandemic covid-19, sars and mers: are they closely related? novel coronavirus: where we are and what we know successful containment of covid-19: the who-report on the covid-19 outbreak in china spatiotemporal circulation of influenza viruses in 5 african countries during 2008-2009: a collaborative study of the institut pasteur international network consecutive infections with influenza a and b virus in children during the 2014-2015 seasonal influenza epidemic seasonality of respiratory viral infections pandemic influenza control in europe and the constraints resulting from incoherent public health laws sars and other coronaviruses as causes of pneumonia epidemiologic characterization of the 1918 influenza pandemic summer wave in copenhagen: implications for pandemic control strategies acknowledgements open access funding provided by university of innsbruck and medical university of innsbruck. key: cord-326887-lyewg2c9 authors: bloomfield, sally f.; aiello, allison e.; cookson, barry; o'boyle, carol; larson, elaine l. title: the effectiveness of hand hygiene procedures in reducing the risks of infections in home and community settings including handwashing and alcohol-based hand sanitizers date: 2007-12-10 journal: am j infect control doi: 10.1016/j.ajic.2007.07.001 sha: doc_id: 326887 cord_uid: lyewg2c9 infectious diseases (id) circulating in the home and community remain a significant concern. several demographic, environmental, and health care trends, as reviewed in this report, are combining to make it likely that the threat of id will increase in coming years. two factors are largely responsible for this trend: first, the constantly changing nature and range of pathogens to which we are exposed and, secondly, the demographic changes occurring in the community, which affect our resistance to infection. this report reviews the evidence base related to the impact of hand hygiene in reducing transmission of id in the home and community. the report focuses on developed countries, most particularly north america and europe. it also evaluates the use of alcohol-based hygiene procedures as an alternative to, or in conjunction with, handwashing. the report compiles data from intervention studies and considers it alongside risk modeling approaches (both qualitative and quantitative) based on microbiologic data. the main conclusions are as follows: (1) hand hygiene is a key component of good hygiene practice in the home and community and can produce significant benefits in terms of reducing the incidence of infection, most particularly gastrointestinal infections but also respiratory tract and skin infections. (2) decontamination of hands can be carried out either by handwashing with soap or by use of waterless hand sanitizers, which reduce contamination on hands by removal or by killing the organisms in situ. the health impact of hand hygiene within a given community can be increased by using products and procedures, either alone or in sequence, that maximize the log reduction of both bacteria and viruses on hands. (3) the impact of hand hygiene in reducing id risks could be increased by convincing people to apply hand hygiene procedures correctly (eg, wash their hands correctly) and at the correct time. (4) to optimize health benefits, promotion of hand hygiene should be accompanied by hygiene education and should also involve promotion of other aspects of hygiene. the effectiveness of hand hygiene procedures in reducing the risks of infections in home and community settings including handwashing and alcohol-based hand sanitizers there can be no doubt that advances in hygiene during the 19th and 20th centuries, along with other aspects of modern medicine, have combined to improve both the length and quality of our lives. however, since the middle of the 20th century, following the development of vaccines and antimicrobial therapy, and with serious epidemics of the ''old'' infectious enemies such as diphtheria, tuberculosis, and others apparently under control, hygiene has tended to lose its prominent position, and the focus of concern has shifted to degenerative and other chronic diseases. nowhere has the decline in concern about hygiene been more evident than in the home and community. however, whereas advances in medicine and public health seemed, at one time, to offer the possibility that infectious diseases (id) might soon be a thing of the past, it is now clear that this is not the case. in the past 20 years, concern about id and the need for prevention through home and community hygiene has moved steadily back up the health agenda. between 1980 and 1992, deaths attributable to id increased by 22% in the united states alone, representing the third leading cause of death among us residents. 1 two factors are largely responsible for this trend: first, the constantly changing nature and range of pathogens to which we are exposed and, secondly, the changes occurring in the community, which affect our resistance to infection. to what extent our more relaxed attitudes to hygiene practice have contributed to these trends is not known, but poor hygiene is a significant factor for a large proportion of the gastrointestinal (gi), skin, and respiratory tract (rt) infections, which make up the greatest part of the id burden. prior to approximately 1980, common pathogens such as rotavirus, campylobacter, legionella, escherichia coli (e coli) o157, and norovirus were largely unheard of. whereas methicillin-resistant staphylococcus aureus (mrsa) and clostridium difficile (c difficile) were once considered largely hospital-related problems, this is no longer the case. now, community-associated mrsa (ca-mrsa) strains are a major public health concern in north america and, increasingly, in europe. most recently, the severe acute respiratory syndrome (sars) outbreak and concerns about avian flu have raised awareness of the potential for transmission of respiratory viruses via hands and surfaces. demographic trends mean that the proportion of the population in the community who are more vulnerable to infection is increasing, whereas trends toward shorter hospital stays and care in the community also demand increased emphasis on care of ''at-risk'' groups in the home who require protection from infection. in assessing the potential for reducing id transmission through hygiene practice, it is recognized that contaminated hands and failure to practice hand hygiene are primary contributors. in this report, we review the evidence base related to the impact of hand hygiene in reducing transmission of id in the home and community. this report focuses on developed countries, most particularly north america and europe, within the context of renewed public health concerns about ids and their impact on health and well-being. the review also evaluates the use of alcohol-based hand hygiene procedures as an alternative to, or in conjunction with, handwashing. these products are defined by a number of different terms in europe and north america (hand sanitizers, handrubs, and others). for the purposes of this report, we will refer to them as alcohol-based hand sanitizers (abhs). although this report focuses primarily on the home, it is recognized that the home forms a continuum with public settings such as schools, offices, and public transport and cannot be considered totally in isolation. nevertheless, the hand hygiene practice framework proposed in this review is largely also applicable to ''out of home'' settings. this report compiles data from intervention studies and considers it alongside risk modeling approaches based on microbiologic data. currently, there is a tendency to demand that, in formulating evidence-based policies and guidelines, data from intervention studies should take precedence over data from other approaches. although there are those who still adhere to this, it is accepted increasingly that, as far as hygiene is concerned, because transmission of pathogens is highly complex and involves many different pathogens each with multiple routes of spread, decisions regarding infection control must be based on the totality of evidence including microbiologic and other data. this document is intended for infection control and public health professionals who are involved in developing hygiene policies and promoting hygiene practice for home and community settings, including those involved with food and water hygiene, care of domestic animals, pediatric care, care of elderly adults, and care of those in the home who may be at increased risk for acquiring or transmitting infection. the purpose of the review is to provide support for those who work at the interface between theory and practice, particularly those involved in developing policies for the home and community, by providing a practical framework for hand hygiene practice together with a comprehensive review of the evidence base. in recent years, a significant amount of research has been done to identify strategies for changing hygiene behavior. whereas those who manage hygiene improvements often choose to promote hygiene by educating people on the links between hygiene and health, one of the lessons that has been learned is that traditional (cognitive) approaches can raise awareness but do not necessarily achieve the desired effects. if practices such as handwashing are to become a universal norm, a multidimensional promotion that engages the public is needed to persuade people to change their behavior. although we recognize that this aspect is fundamental, it is outside the scope of this report and is reviewed elsewhere. [2] [3] [4] [5] [6] whereas, in the past, research and surveillance largely focused on health care-associated and foodborne illnesses, increasing resource is now being allocated to generating data that give a better view of the extent to which infections are circulating in the community; how they are being transmitted; and how this varies from one region, country, or community to another. although the data in the following section represent a useful overview, we note that the data collection methods differed significantly from one study to another, which means that comparisons from different geographic locations must be interpreted with care. current trends in communicable ids in europe are described in more detail in the recent (2007) european communicable disease epidemiological report from the european centre for disease prevention and control (ecdc). 7 infectious gi disease and hygiene foodborne disease. rates of foodborne illness remain at unacceptably high levels, despite the efforts of food producers to ensure the safety of the food supply. raw meat and poultry and fruits and vegetables bought at retail premises may be contaminated with pathogens. good hygiene practices during food preparation in the home are therefore essential in preventing cross contamination of prepared foods from raw foods and preventing contamination of food by infected household members or domestic animals. the european food standards agency (efsa) 2005 report 8 and the 2007 ecdc report 7 cite campylobacteriosis as the most reported animal infection transmitted to humans. in 2005, reported campylobacter infections increased by 7.8% compared with the previous year, rising to an incidence rate of 51.6 cases per 100,000 people. the efsa states that the source of most human campylobacter infections is related to fresh poultry meat. on the other hand, salmonella infections fell by 9.5% in 2005 to an incidence of 38.2 cases per 100,000 (176, 395 reported cases). the 2003 world health organization (who) report 9 concluded that approximately 40% of reported foodborne outbreaks in the who european region over the past decade were caused by food consumed in private homes. the report cites several factors as ''critical for a large proportion of foodborne diseases'' including use of contaminated raw food ingredients, contact between raw and cooked foods, and poor personal hygiene by food handlers. united kingdom data show that food poisoning notifications reached a peak in 1997-1998 and has since declined but remains in excess of 70,000 per year. 10 in reality, the burden of food poisoning is much higher because most cases go unreported; according to the uk food standards agency, 11 the true number of cases is approximately 4.7 million per year. in 1999, mead et al 12 reported on food-related illness in the united states, using data from a range of sources including national surveillance and community-based studies. they estimated that foodborne illness in the united states causes 76 million illnesses, 500,000 hospital admissions, and 9000 deaths each year. most frequently recorded pathogens were campylobacter, salmonella, and norovirus, which accounted for 14.2%, 9.7%, and 66.5%, respectively, of estimated foodborne illnesses. data suggest that the total number of reported outbreaks has not declined substantially in recent years, ranging from 980 to 1400 outbreaks and between 20,000 and 80,000 cases per year for the years 2000 to 2005. 13 other infectious gi disease. from recent investigations, it is now recognized that a substantial proportion of the total infectious gi disease burden in the community is because of person-to-person spread within households, particularly for viral infections, where it is most often the cause. person-to-person transmission in the home can occur by direct hand-to-mouth transfer, via food prepared in the home by an infected person, or by transmission because of aerosolized particles resulting from vomiting or fluid diarrhea. apart from transmission by inhalation of airborne particles, these infections are preventable by good hygiene practice. the 2003 who report 9 stated that, of the total gi infection outbreaks (including foodborne disease) reported in europe during 1999 and 2000, 60% and 69%, respectively, were due to person-to-person transmission. in the united kingdom, it is estimated that up to 50% of gi infection results from person-to-person tranmsission. 11 a study of united kingdom outbreaks 14 suggested that 19% of salmonella outbreaks and more than half of e coli o157 outbreaks are transmitted by nonfoodborne routes. national surveillance systems vary in their methods of data collection but mostly focus on foodborne disease. inevitably, this means that data on gi illnesses relate mainly to large foodborne outbreaks in restaurants, hospitals, and others, whereas sporadic nonfoodborne cases in the general community go largely unreported. in the united kingdom, even when ''household'' outbreaks are reported, they mostly involve home catering for parties and other functions and are therefore mainly foodborne outbreaks. 15 because milder cases of gi illness often go unreported, this means that the overall gi infection burden, particularly that which is not foodborne, is unknown; the most informative data on the overall burden of infectious gi illness (both foodborne and nonfoodborne) in the community come from various community-based studies, which have been carried out in europe and the united states and are reviewed below. two large community studies have been carried out in europe: one in the united kingdom and the other in the netherlands. the uk study, carried out from 1993 to 1996 involving 460,000 participants in the community presenting to general practice, estimated that only 1 of 136 cases of gi illness is detected by surveillance. the study indicated that as many as 1 in 5 people in the general uk population develop gi illness each year, with an estimated 9.4 million cases occurring annually of which about 50% are nonfoodborne. 11, 16 it was estimated that, for every 1 reported case of campylobacter, salmonella, rotavirus, and norovirus, another 7.6, 3.2, 35, and 1562 cases, respectively, occur in the community; based on the number of laboratory reports, it is possible to estimate the true number of infections occurring in the community (table 1) . from the community study carried out in the netherlands between 1996 and 1999, 17 it was estimated that approximately 1 in 3.5 people experience a bout of infectious gi disease each year. campylobacter was detected most frequently (10% of cases), followed by ghiardia lamblia (5%), rotavirus (5%), norovirus (5%), and salmonella (4%). relative to the population of the netherlands (16 million), 650,000 norovirus gastroenteritis cases occur annually. 18 the us study of mead et al, 12 which also included data from community-based studies, indicated that the total number of cases of infectious gi illness annually is approximately 210 million (of which approximately 64% are nonfoodborne). they estimated that the number of episodes of acute gastroenteritis per person per year is approximately 0.79. from the available data, the authors were also able to estimate the proportion of total episodes that were nonfoodborne. as shown in table 2 , by far the most frequently reported causes of gi illness were norovirus, rotavirus, and campylobacter. for campylobacter, e coli, and norovirus, a significant proportion of cases was estimated as nonfoodborne, whereas, for hepatitis a (hav), shigella, and rotavirus, almost all cases were estimated as nonfoodborne. for salmonella on the other hand, only 5% of cases were considered as nonfoodborne. davis et al reviewed outbreaks of e coli o157 related to family visits to animal exhibits. 19 indications are that norovirus is now the most significant cause of infectious gi illness in the developed world, both outbreak related and endemic. 20, 21 currently, we are seeing increased outbreaks of norovirus, a major concern in japan 22 and also in europe. 23 expert opinion is that norovirus strains now circulating are more ''virulent'' and more easily spread from person to person via hands and surfaces or during food handling. 20 infection with hav is common worldwide, 24 and adenovirus is also a frequent cause of gastroenteritis. c difficile-associated disease now occurs with increasing frequency in the community, in which it usually affects persons receiving antibiotic therapy but also healthy individuals. 25 recently, a new strain (027) of c difficile has emerged in north america, causing infections in the community among individuals with no predisposing factors. 25 a recent study 26 indicated that exposure to a family member with helicobacter pylori gastroenteritis was associated with a 4.8-fold increased risk of infection in another family member and that infection most usually involved person-to-person transmission, associated with conditions of crowding and poor hygiene. using data from the 2006 e coli o157:h7 outbreak in 2006 in the united states associated with contaminated spinach, seto et al developed a model that showed that secondary person-to-person transmission was similar to that in previous e coli outbreaks (12%). the model suggests that even a modestly effective hygiene promotion strategy to interrupt secondary transmission (prevention of only 2%-3% of secondary illnesses) could result in a reduction of 5% to 11% of symptomatic cases. 27 respiratory tract infections are largely caused by viruses. in the united states, viruses account for up to 69% of respiratory infections. 28 the common cold is reported to be the most frequent, acute infectious illness to humans. 29 data from the united states suggest that the mean number of respiratory illnesses experienced per year in adults is approximately 1.5 to 3.0, and, in children under 5 years of age, it is approximately 3.5 to 5.5. 28 approximately 80% of upper rt infections are caused by rhinoviruses. other species causing acute rhinitis are coronaviruses, parainfleunza viruses (piv), respiratory syncytial viruses (rsv), and adenoviruses. 30 although colds are generally mild and self-limiting, they represent a significant economic burden because of loss in productivity and medical costs. furthermore, secondary infections produce complications, such as otitis media, sinusitis, or lower respiratory infections including pneumonia, with its risk of mortality, particularly in elderly adults. several studies have demonstrated that colds are also a trigger for asthma. 31 rsv is the major cause of viral rt infection in young children worldwide. child day care attendance in north america caries with it a very high risk of rsv infection within the first 2 years of life and accounts for 0.5% to 1.0% of hospitalized infants in the united states. 32 influenza is a more serious rt illness, which can cause complications that lead to increased physician visits, hospitalization, and death, the risks being highest among persons aged .65 years, children aged ,2 years, and persons who have medical conditions (eg, diabetes, chronic lung disease). [33] [34] [35] influenza must also be considered in terms of days absent from work and school and pressure on health care services. 35 an important aspect of influenza is the threat associated with the emergence of novel subtypes capable of causing an influenza pandemic. 36 according to bridges et al, 37 influenza epidemics in the united states result in an annual average of 36,000 deaths and 114,000 hospitalizations; among those with influenza who belong to an ''at-risk'' group, a significant proportion develop pneumonia, and up to 1 in 10 can die of related complications. in europe, the 2004-2005 influenza season annual report 38 showed that, of 25 countries, 15 recorded what is regarded as high activity (150 up to 3000 influenza-like or acute respiratory illnesses per 100,000 population). although data indicating the role of hands and other surfaces in the transmission of colds have been available for some time, it is only in the last few years that there has been any real awareness that hands and surfaces may also be a transmission route for flu viruses. 32 evidence that measures such as hand hygiene can reduce spread of rt infections comes from the sars outbreaks in hong kong, which coincided with the latter part of influenza season, when it was observed that, as extensive personal and community public health measures took place, influenza case numbers fell significantly, more so than usual for the time of year. 39 skin and wound infections are common in the home and community, but most are self-limited. because these infections, apart from s aureus infections go unreported, little or no data are available on the burden of skin and wound infections in the community. s aureus is the most common cause of infections of skin and soft tissue, which, in a small proportion of cases, lead to the development of bacteremia or pneumonia. 40 serious infections usually occur in health care facilitiesin patients who are immunocompromised-in which s aureus is mostly usually associated with wounds and intravenous devices and in which the antibioticresistant strain, mrsa, is a major concern. infected patients discharged from hospitals and health care workers (hcws) caring for mrsa-infected patients can bring mrsa into the home and pass it on to healthy family members, who become colonized, thereby spreading the organism into the community and facilitating the circulation of these strains. [41] [42] [43] mrsa colonization in an individual can persist for up to 40 months. 44, 45 in recent years, mrsa has been increasingly found to cause infections in healthy members of the community without apparent risk factors. 25 these ca-mrsa strains are different from health care-associated (hca) mrsa strains and are a concern because they equally infect children and young adults. these strains primarily cause skin and soft tissue infections but can also cause invasive infections such as sepsis, pneumonia, and osteomyelitis, which is some cases can be fatal. 25 some ca strains are known to produce panton-valentine leukocidin (pvl), which has been implicated as a virulence factor, 46 although opinion is, however, divided as to whether this is the case; whereas some studies support this notion, 47 others do not. 48 in the united states, ca-mrsa is now a significant concern. ca-mrsa strains have also now been detected in france, switzerland, germany, greece, the nordic countries, australasia, the netherlands, and latvia. 25 in the united kingdom, cases of ca-mrsa and pvlproducing strains have been reported, but the number of reported cases is still small. 25, 49 within the global population that affect our resistance to infection.''at-risk'' groups cared for at home include not only newborn infants whose immune system is not fully developed but also the rapidly increasing elderly population whose immune system is declining. ''atrisk'' groups include patients discharged recently from hospital, immunocompromised family members, and family members with invasive devices such as catheters. it also includes people whose immunocompetence is impaired as a result of chronic and degenerative illness or because they are undertaking certain drug therapies. all of these groups, together with those who carry hiv/aids, are increasingly cared for at home by a caregiver, who may be a household member. a survey of the united states and 3 european countries-germany, the netherlands, and the united kingdom-suggests that up to 1 in 5 of the population belongs to an ''at-risk'' group (table 3) . the data suggest that between 12% and 18% of the population of these countries are .65 years of age. in an intervention study of 148 patients with aids, it was found that patients assigned to the intensive handwashing intervention group developed fewer episodes of diarrheal illness (1.24 6 0.9 vs. 2.92 6 0.6 new episodes of diarrhea, respectively, during a 1-year observation period. 50 gi pathogens are now implicated as causative or contributory factors in the development of cancers and other chronic conditions; examples include hepatitis b virus (hepatocelluar carcinoma), 51 h pylori (peptic ulcer disease), 52 and campylobacter jejuni (guillain barrã© syndrome). 53 foodborne illness has been estimated to result in chronic sequelae in 2% to 3% of cases 54 ; a european commission report 55 cites evidence of chronic disease, such as reactive arthritis, following 5% of salmonella cases, with 5% of e coli o157 cases progressing to serious and even fatal complications. even mild viral infections can be predisposing factors to more severe and possibly fatal secondary bacterial infections. 56 in devising a strategy for home hygiene and producing hygiene practice advice, the international scientific forum on home hygiene (ifh) has developed an approach based on risk management that involves identifying the ''critical control points'' for preventing the spread of id in the home. risk management (also known as hazard analysis critical control points [haccp] ) is now the standard approach for controlling microbial risks in food and other manufacturing environments and is becoming accepted as the optimum means to prevent such risks in home and hospital settings. 57 the key feature of the ifh approach is that it recognizes the need to look at hygiene from the point of view of the family and the total range of problems it faces to reduce id risks, including food hygiene, personal hygiene (particularly hands) and hygiene related to the general environment (toilets, baths, hand basins, surfaces, and others), domestic animals, and family members at increased risk. adopting a holistic approach makes sense because all these issues are interdependent and based on the same underlying microbiologic principles. haccp also forms the basis for developing an approach to home hygiene that can be adapted to meet differing needs. indeed, it is only by adopting such a holistic approach that the causal link between hands and infection transmission in the home can be addressed properly because hand hygiene is a central component of all these issues. the ifh risk management approach to hygiene starts from the principle that pathogens are introduced continually into the home by people (who may have infection or may be asymptomatic), food, and domestic animals and also sometimes via the water or the air. additionally, sites at which stagnant water accumulates, such as sinks, toilets, waste pipes, or items, such as cleaning or face cloths, readily support microbial growth and can become a primary reservoir of infection; although microbial species are mostly those that represent a risk to vulnerable groups, primary pathogens can also be present. 58 so long as there are people, pets, and food in the home, there will always be the risk of pathogenic microbes. in many homes, there will also be at least one family member who is more susceptible to infection for one reason or another. within the home, there is a chain of events, as described in fig 1, that results in transmission of infection from its original source to a new recipient. to an extent, we can limit the exit and entry of pathogens from and into the body, but the link that we have most control over is that related to the ''spread of pathogens.'' the spread of infection can be interrupted by good hygiene practice, which includes adherence to hand hygiene recommendations and cleaning and disinfecting contaminated environmental surfaces. the risk-based approach to home hygiene is described in more detail by bloomfield and scott 59 and bloomfield. 60 they suggest that sites and surfaces in the home should be categorized into 4 main groups: reservoir sites, reservoir/disseminators, hands and hand and food contact surfaces, and other surfaces. risk assessment is then based on the frequency of occurrence of pathogenic contamination at that site, together with the probability of transfer from that site such that family members may be exposed. this means that, even if a particular environmental site is highly contaminated, unless there is a high probability of transfer from that site, the risk of infection transmission is low. from this, it is possible to determine the ''critical control points'' for preventing spread of infection. the data suggest the following: d for reservoir sites such as the sink waste pipes or toilets, although the probability of contamination (potentially pathogenic bacteria or viruses) is high, the risk of transfer is limited unless there is a particular risk situation (eg, a family member with enteric infection and fluid diarrhea, when toilet flushing can produce splashing or aerosol formation that can settle on contact surfaces around the toilet). 58,61 d by contrast, for reservoir sites such as wet cleaning cloths, not only is there high probability of significant contamination, but, by the very nature of their usage, they carry a high risk of disseminating contamination to other surfaces and to the hands. d for hands and hand contact and food preparation surfaces, although the probability of contamination is, in relative terms, lower, it is still significant, for example, particularly following contact with contaminated food; people; pets; or other contaminated surfaces such as door, faucet, and toilet-flush handles. because there is a constant risk of spread from these surfaces, hygiene measures are important for these surfaces. d for other surfaces (floors, walls, furniture, and others), risks are mainly due to pathogens such as s aureus and c difficile, which survive under dry conditions. because the risks of transfer and exposure are relatively low, these surfaces are considered low risk, but where there is known contamination, for example, soiling of floors by pets, crawling infants may be at risk. cleaning can also recirculate dust-borne pathogens onto hand and food contact surfaces. overall, this approach allows us to rank these various sites and surfaces (fig 2) according to the level of transmission risk; this suggests that the ''critical control points'' for breaking the chain of infection are the hands, together with hand and food contact surfaces, cleaning cloths, and other cleaning utensils. however, although this is a useful ''rule of thumb'' ranking, it is not a constant. for example, although risks from toilets, sinks, floors, and others relate mainly to the relatively lower risk of transfer from these sites to hands, hand and food contact surfaces, and cloths, this risk can increase substantially during occasions when an infected family member has fluid diarrhea or when a floor surface is contaminated with vomitus, urine, or feces. in the following section, we evaluate data indicating the extent to which the hands, both alone and in combination with other surfaces, are responsible for the spread of infection. the criteria for assessing causal inference of a link between hygiene practice and id risk reduction have been reviewed by aiello and larson. 62 establishing the potential health impact of a hygiene intervention such as hand hygiene requires examination of the evidence related to a range of criteria that should include the strength, consistency, and temporality (cause and effect) of the association, together with data on plausibility (biologic or behavioral rationale) and biologic gradient. aiello and larson recognize that, although a single factor such as the hands may be a ''sufficient cause'' of infection transmission, spread of infection frequently involves a number of ''component causes,'' which, together or independently, work to determine the overall risk. the risk assessment approach, as outlined above, indicates that the ''critical control points'' or ''component causes'' of infection transmission in the home are the hands, together with hand and food contact surfaces and cleaning cloths. based on plausibility, the role of the hands relative to other surfaces can be understood by mapping the potential routes of spread of gi, rt, and skin infections in the home as shown in fig 3. this suggests that, for all 3 groups of infections, the hands are probably the single most important transmission route because in all cases they come into direct contact with the known portal of entry for pathogens (the mouth, nose and, conjunctiva of the eyes) and are thus the key last line of defense. figure 3 shows, however, that, although in some cases the hands alone may be ''sufficient cause'' for transmission of an infection (eg, from an mrsa carrier, to hands, to the wound of a recipient), in other cases transmission involves a number of component causes (eg, from contaminated food, to a food contact surface, to hands, to the mouth of a recipient). what this means is that the transmission risk via the hands also depends on the extent to which surfaces become contaminated with pathogens during normal daily activities, ie, the risk of hand-to-mouth transfer will be increased if extensive transfer from raw food to food preparation surfaces also occurs. defining the importance of hand hygiene relative to other hygiene practices, such as surface and cloth hygiene, is difficult because of the close interdependence of these factors. although the focus of this review is the prevention of infection through hygiene practice, fig 3 shows that in some cases airborne transmission can operate independently, without involving the hands, whereas, for gi infection, transmission can operate independently via food. although handwashing intervention studies provide data supporting the causal link between hand contamination and id transmission, defining the importance of hand hygiene relative to other hygiene practices, such as surface and cleaning cloth hygiene, or the risks associated with airborne transmission is difficult because of the close interdependence of these factors. currently, such assessments can only be made on a qualitative basis, using microbiologic data (as in the following section) together with some limited epidemiologic data. in this section, we present epidemiologic and microbiologic data to support the causal relationship between hygiene and id risk. because the risks of hand transfer increase as the risks of contamination of other surfaces increases, data related to relevant surfaces are also included. in recent years, a range of studies has been published, many related specifically to the home, which indicate the extent to which id agents occur and are spread in home and community settings during normal daily activities and their potential to cause infection. these studies include assessments of frequency occurrence of sources of pathogens in the home, their rate of ''shed'' from an infected source into the environment, their rate of die away on hands and other surfaces, their rate of transfer via the hands to the mouth, nose, conjunctiva, and others and/or to ready-to-eat foods, and their the infectious dose. the infectious dose (ie, the number of particles to which the recipient is exposed), their immune status, and the route by which they are infected are key factors that determine the infection risk. the ''infectious dose'' varies for different pathogens and is usually lower for people who are ''at-risk'' than for healthy household members. transmission of infectious gi disease. risks from exposure to gi pathogens via the hands. as shown in fig 3 , exposure to gi pathogens can occur by direct hand-to-mouth contact or indirectly via contaminated food. in the home, food can be contaminated either directly by an infected food handler or indirectly by cross contamination via hands and surfaces from another source, which may be contaminated food, another infected household member (or carrier), or a household pet or farm animal. hand-to-mouth contact is a frequent occurrence, particularly among children; a study of mouthing behavior in 72 young children showed that children ,24 months of age exhibit the highest frequency, with 81 events/hour; for children .24 months of age, this was reduced but was still of the order of 42 events/hour. 63 the potential for transmission of pathogens from hands to ready-to-eat foods is supported by a number of studies: d in a model domestic kitchen, 29% of food preparation sessions using campylobacter-contaminated chicken resulted in positive campylobacter isolations from prepared salads, cleaning materials, and food contact surfaces. 64 d bidawid et al 65, 66 showed that touching lettuce with finger pads contaminated with hav and feline calicivirus (fcv), used as a surrogate for norovirus, for 10 seconds resulted in transfer of 9.2% and 18%, respectively, of the virus. based on the load for hav in feces (10 6 to 10 9 viral particles/g), an estimated 1300 particles were transferred to the lettuce. d rusin et al showed that, when volunteers' fingertips were inoculated with a pooled suspension of micrococcus luteus (m luteus), serratia rubidea (s rubidea), and bacteriophage prd-1 and held to the lip area, transfer rates were 40.99%, 33.97%, and 33.90%, respectively. 67 as stated above, the infection risk from oral consumption depends on the number of bacterial cells or viral particles that are consumed. table 4 shows that, for many of the commonly occurring gi pathogens, the infectious dose is relatively small. sources and spread of gi pathogens to the hands. figure 3 illustrates that the risk of exposure to gi pathogens via the hands depends on the extent to which these pathogens are brought into the home (either by infected people or pets or via contaminated food) and the extent to which they are spread via hands and other surfaces and by airborne transmission. relevant data from various sources, as summarized below, suggest that exposure to gi pathogens via the hands is a frequent occurrence during normal daily activities and that the numbers of organisms transferred by handto-mouth contact can be well within the numbers required to cause infection. household members who are infected, or who are carriers, are a primary source of infection in the home. pathogens that can be carried persistently by otherwise healthy people include salmonella species and c difficile. approximately 3% of adults (mainly those .65 years of age), and up to two thirds of babies, are known to carry c difficile in their gut, although it is not known what proportion are toxin producing. 25 people or animals that carry gi pathogens shed large numbers of organisms in their feces or when they vomit. a single vomiting incident following norovirus infection may produce 30 million viral particles, 72 and, at the peak of a rotavirus infection, .10 11 virions may be excreted per gram feces. 74 surfaces in the home may become contaminated by enteric organisms that are aerosolized during vomiting or by transfer of vomitus and fecal matter via hands. viruses aerosolized from flushing the toilet can remain airborne long enough to contaminate surfaces throughout the bathroom. 75 infectious agents introduced into the home via food include salmonella, campylobacter, listeria, and e coli o157. a variety of foods can act as a source of these organisms, including meat, fish and poultry products, dairy products, fruits, and vegetables. organisms in particles, and moisture or juices, from food will contaminate any surface they come into contact with. an salmonella species up to 10 6 but could be as low as 10-100 cells. 68 contamination may be amplified by transfer to foods, which are then stored incorrectly. campylobacter species 500 organisms can result in human illness. 69 oral dose for e coli 0157 may be as little as 10 cells. 70 in one outbreak, a median dose of ,100 organisms per hamburger was reported. 71 norovirus 10-100 units or even less. 72 may be as few as 10 particles. 73 ward et al showed that 13 of 14 adults became infected after consuming rotavirus (10 3 particles) picked up from a contaminated surface via the hands. 73 efsa survey 76 of salmonella in chicken indicates significant differences among eu member states, with isolation rates between 0% and 68.2%; the level reported for the united kingdom was 7.1% to 9.4%. the efsa also reported that up to 66% of samples from fresh poultry were positive for campylobacter. 77 in the united states, more than half of raw chicken is estimated to be contaminated with camplylobacter. 76 chapman et al 78 showed that 0.4% to 0.8% of meat products purchased from uk butchers were positive for e coli o157. in a recent study in canada, c difficile was isolated from 20% of 60 samples of retail ground meat purchased over a 10-month period, and 11 isolates were toxigenic. 79 the home is frequently a shelter to a range of different pets; more than 50% of homes in the englishspeaking world have cats and dogs, with 60 million cats and dogs in the united states. in the united states, up to 39% of dogs may carry campylobacter, and 10% to 27% may carry salmonella 80 ; cats are also carriers of these organisms. carriage of c difficile in household pets is quite common; up to 23% of pets are affected, although these mostly involve noncytotoxigenic strains. 25 kramer et al, 81 sattar et al, 82 and rzezutka and cook 83 reviewed data showing that gi pathogens can survive on surfaces for several hours and, in some cases, days, particularly on moist surfaces, although infectivity depends on the numbers that survive (table 5) . studies to quantify transfer between hands, foods, and kitchen surfaces 67, 84 showed that transfer rates were highly variable, ranging from as high as 100% to as low as 1%. transfer to hands was highest from nonporous surfaces but lower from surfaces such as carrots, sponges, and dishcloths (,1%). rusin et al 67 sampled volunteers hands after touching surfaces contaminated with m luteus, s rubidea, and phage prd-1. activities included wringing out a dishcloth/sponge, turning off a faucet, cutting up a carrot, making hamburger patties, holding a phone receiver, and removing laundry from the washing machine. transfer efficiencies for the phone receiver and faucet were 38% to 65% and 27% to 40%, respectively. paulson 85 showed that, when gloved hands were contacted for 5 to 10 seconds with surfaces such as cutting boards and doorknobs contaminated with fcv (log 5.9 particles), the log number of particles recovered from hands was 4.7 to 5.4. these laboratory studies are supported by a range of field studies showing spread via the hands and other surfaces during normal daily activities: d following preparation of salmonella and campylobacter-contaminated chickens in domestic kitchens, these species were isolated from 17.3% of hands and hand and food contact surfaces. isolation rates were highest for hands, chopping boards, and cleaning cloths (25%, 35%, and 60%, respectively, of surfaces sampled). 86 d in homes containing an infant recently vaccinated for polio (during which time shedding occurs in feces), virus was isolated from 13% of bathroom, living room, and kitchen surfaces. 87 most frequently contaminated were hand contact sites such as bathroom taps, door handles, toilet flushes, soap dispensers, nappy changing equipment, and potties. d following handshaking with a volunteer whose hands were contaminated from touching a viruscontaminated door handle, successive transmission from one person to another could be followed up to the sixth person. 88 d where fingers were contacted with noroviruscontaminated fecal material, the virus was consistently transferred via the fingers to melamine surfaces and from there to hand contact surfaces, such as taps, door handles, and telephone receivers. contaminated fingers sequentially transferred the virus to up to 7 clean surfaces. 89 d a study 90 with fcv showed survival for up to 3 days on telephone buttons and receivers, for 1 or 2 days on computer mouse, and for 8 to 12 hours on keyboard keys and brass disks representing faucets and door handles. the time for 90% virus reduction was ,4 hours on computer keys, mouse, and brass disks; 4 to 8 hours on telephone receivers; and 12 to 24 hours on telephone buttons. d in homes of infants with recurrent c difficile infection, 12% of environmental surfaces were positive for c difficile, and 1 of 4 other household members carried c difficile in stool. in a control home with no household carriers, none of 84 environment samples were positive for c difficile. 91 d in 4 out of 6 homes in which there was a salmonella case, the causative species was isolated from fecal soiling under the flushing rim and scale material in the toilet bowl for up to 3 weeks after notification of infection. 58 flushing toilets seeded with salmonella enteritidis resulted in contamination of hand contact surfaces such as toilet seats and toilet seat lids. these represent recent examples of studies that have been reported. these and other studies are also reviewed elsewhere. 82, [92] [93] [94] in developing hygiene policies for preventing gi infections, one of the difficulties is assessing risks associated with hand transmission relative to other risks such as inadequate cooking or storage of food or inhalation of infected vomit particles. gillespie et al 15 reported an evaluation of reported outbreaks linked to uk households for 1992 to 1999 that suggested, of the 85% of outbreaks designated as foodborne, cross contamination was implicated in 20% of outbreaks compared with 30% and 31% of outbreaks for which inadequate storage and cooking, respectively, were thought to be the cause. there were no data to suggest what percentage of cross contamination events involved the hands, and gillespie et al 15 expressed concern that most of the reported outbreaks were linked to home catering, thus not necessarily representative of normal daily routine. aerosol transmission can result from settling on hand and food contact surfaces, but, for norovirus, infection can sometimes result from direct inhalation of infected particles of vomit by people immediately adjacent to the person who vomits. the potential for airborne transmission of norovirus was demonstrated in studies in a restaurant and a primary school, in which close proximity to infected persons in the immediate aftermath of a vomiting attack was identified as a risk factor. 95, 96 transmission of rt infections. the last 2 years have seen an unprecedented global focus on developing strategies for preventing transmission of influenza. the who 97 is taking a lead on pharmaceutical interventions such as vaccines and antivirals but has also made recommendations for other interventions, 98 which include highlighting the importance of hygiene, and in particular hand hygiene, in minimizing spread in the home and community. risks from exposure to respiratory pathogens via the hands. as shown in fig 3, exposure to rt viruses can occur either by inhalation of infected mucous or inoculation of the nasal mucosa or eyes with viruscontaminated hands, which then cause infection via the mucous membranes and upper rt. rhinovirus and rsv are deposited into the front of the nose or into the eye (where they pass down the lacrymal duct), either on the end of the finger or possibly sometimes in aerosolized droplets. 99 rubbing the eyes and nose with the fingertips is a common occurrence; hendley et al found that 1 in 2.7 attendees of hospital rounds rubbed their eyes, and 33% picked their nose, within a 1-hour observation period. 100 a review of the data 94 (table 6 ) suggests that the infectious dose for respiratory viruses is relatively small. alford et al suggest that aerosolized doses of as little as 1 tcid 50 (tissue culture infective dose) of influenza virus could infect volunteers. 101 evidence for transmission of rhinovirus and rsv infections via contaminated hands comes from a number of studies: d a number of studies have demonstrated that selfinoculation by rubbing the nasal mucosa or conjunctivae with rhinovirus-contaminated fingers can lead to infection. 100, 102 over a period of 10 years, gwaltny and hayden performed intranasal challenges on 343 healthy young adults who had no antibody to the challenge, and infected 321 (95%). 103 after handling contaminated coffee cups and other objects, more than 50% of subjects developed infection. 104 hall et al showed that volunteers touching contaminated objects and/or the fingers of symptomatic individuals had a higher attack rate of colds if they touched their eyes or nose. 105 d in a 4-year family trial, hendley and gwaltney 104 found that prophylactic treatment of mothers' fingers with iodine reduced the incidence of rt infections. when illness occurred in the family, mothers were instructed to dip their fingers in iodine upon awakening in the morning, then every 3 or 4 hours or after activities that washed the iodine from the skin. the secondary attack rate in mothers was 7% in the iodine group and 20% in placebo families. no infections occurred in mothers after 11 exposures to an infected index case in the iodine group, compared with 5 infections after 16 exposures in the placebo group. d hall et al showed that infected infants excrete prodigious amounts of rsv in their nasal secretions for several days 105 and that rsv could be recovered from hands that had touched surfaces contaminated with secretions from infected infants. 99 hall and douglas found that close contact with symptomatic infants who were producing abundant secretions, or their immediate environment, was necessary for infection. 106 sources and spread of rt pathogens to the hands. figure 3 illustrates that the risk of exposure to rt pathogens via hands depends on the extent to which these pathogens are spread from an infected person during normal daily activities. relevant data come from various sources and are summarized below. taken together, the data suggest that, when a household member is infected, exposure of other household members via hands is likely to occur during normal daily activities and that the numbers of organisms involved are within those required to initiate infection if transferred to the eyes or nose. people infected with cold viruses shed large quantities of virus-laden mucus. droplets of nasal secretions generated by coughing, sneezing and talking can travel over a distance .3 m to contaminate surrounding surfaces. 37, 98, [107] [108] [109] up to 10 7 infectious influenza particles per milliliter has been detected in nasal secretions. 110 the mean duration of a cold is 7.5 days. viral shedding may occur 24 to 48 hours before illness onset but generally at lower titers than during the symptomatic period. titers generally peak during the first 24 to 72 hours of illness and decline within several days, with titers low or undetectable by day 5. children can shed virus for up to 3 weeks, whereas immunocompromised people may continue to shed virus for weeks to months. 98 infectious material can also be deposited directly on hands and tissues during sneezing and blowing the nose. contamination of hands can occur by handshaking or touching contaminated surfaces. pathogens shed into the environment from these sources can survive for significant periods and are readily spread around the home to and from the hands and via handkerchiefs and tissues, tap and door handles, telephones, or other hand contact surfaces: d gwaltney and hendley demonstrated that most subjects with experimental colds had rhinovirus on their hands and that virus could be recovered from 43% of plastic tiles they touched. 104 for people with rhinovirus colds, virus was found on 39% of hands and 6% of objects in their immediate environment. 100 opinion as to the importance of the hands relative to the airborne route for transmission of rhinovirus colds is divided. some investigators 30, 99, 103, 104 maintain that contamination of the hands followed by inoculation of the eyes or nose is of paramount importance; in fact, gwaltney et al found that it was exceedingly difficult to transmit virus orally or by kissing and found little evidence of droplet or droplet nuclei transmission. 100, 113 others maintain that the evidence favors droplet and droplet nuclei transmission as the most important mode of spread. 118 for rsv, there is general agreement that the hands are the primary route for the spread of infection. 32, 105, 106 for influenza, although more data are needed, it is increasingly accepted that not only airborne (both true airborne transmission involving droplet nuclei [,5 mm in diameter] and ''droplet transmission'' involving droplets .10 mm that deposit onto surfaces quite rapidly) but also surface (including hand) transmission come into play. 98, 119, 120 the relative contribution of each mode of transmission is unknown but appears to vary depending on the circumstances, symptoms, respiratory tract loads, and the viral strain. 121 data from animal studies and influenza outbreaks suggest that droplets generated when infected persons cough or sneeze are the predominant mechanism of airborne transmission, 37 although data supporting droplet nuclei spread are also available. 101, [122] [123] [124] it is possible, however, that influenza is less transmissible via hands and surfaces compared with rhinovirus and others because of its lower ability to survive outside a human or animal host. data suggest that, to some extent, airborne droplets and droplet nuclei cause infection as a result of settling on hand contact surfaces. the frequent occurrence of diarrhea and the detection of viral rna in fecal samples tested suggest that the h5n1 influenza virus may replicate in the human gut and could be a source of transmission via hands and surfaces. 125 at present, however, it is thought that this is unlikely. the growing evidence base related to the survival, transmission, and human exposure to rt viruses via hands and other surfaces is also reviewed elsewhere. 32, 35, 37, 82, 94, 99 transmission of skin and wound infections. risks from exposure to skin and wound pathogens via the hands. as shown in fig 3, exposure to skin pathogens such as s aureus can occur via the hands. exposure can produce colonization and/or infection that usually occurs in areas in which there are cuts, abrasions, and others that damage the integrity of the skin. where there are predisposing factors, the numbers of organisms required to produce infection may be relatively small. marples 126 showed that up to 10 6 cells may be required to produce pus in healthy skin, but as little as 10 2 may be sufficient in areas in which the skin is occluded or traumatized. risks associated with exposure to hca-mrsa and ca-mrsa are different. hca-mrsa usually affects elderly adults and those who are immunocompromised, particularly those with surgical or other wounds or who have indwelling catheters. for ca-mrsa, those at particular risk appear to be younger, generally healthy people who practice contact sports or other activities that put them at higher risk of acquiring skin cuts and abrasions. 127 us experience suggests that ca-mrsa may be more virulent than other strains and is easily transmissible within households and community settings (eg, schools, day care centers, sport teams) in which skin-to-skin contact or sharing of contaminated items (eg, towels, sheets and sport equipment) are vehicles for person-to-person transmission. 128 a case-control study 129 involving 55 cases of mrsa in a us prison showed that inmates who washed their hands #6 times per day had an increased risk for mrsa infection compared with inmates who washed their hands .12 times per day. sources and spread of skin and wound pathogens to the hands. figure 3 illustrates that the risk of exposure to skin pathogens via the hands depends on the extent to which people or animals colonized or infected with pathogenic strains are present in the home and the extent to which these pathogens are spread during normal daily activities. transfer of skin pathogens to the hands can occur either by direct contact with an infected source or indirectly via hand contact surfaces or the surfaces of clothing or household linens. relevant data, as outlined below, suggest that, when there is a person in the home who is infected or colonized with s aureus, exposure of other household members as a result of transfer via hands, surfaces, clothing, and others is likely to occur during normal daily activities and that the numbers of organisms involved are within the numbers of particles that could initiate an infection in a susceptible recipient. a study by kluytmans et al suggests that s. aureus is carried as part of the normal body flora in up to 60% of the general population, 130 although a 2006 us study 131 suggests that the carriage rate is much less (31.6%). in the united kingdom, indications are that the proportion of the general population carrying antibiotic-resistant strains of s aureus (either hca-or ca-mrsa) is somewhere between 0.5% and 1.5%, the majority being carriers of hca-mrsa who are .65 years of age and/or have had recent association with a health care setting. 132 although cases of ca-mrsa and pvlproducing mrsa have been reported, indications are that the prevalence of mrsa and pvl-producing strains circulating in the community is currently very small. 25 in the united states, although it is concluded that colonization rates for mrsa in the community are still low, it is nonetheless thought to be increasing. 127, 133 graham et al 131 report on an analysis of 2001-2002 data from the national health and nutrition examination survey (nhanes) to determine colonization with s aureus in a noninstitutionalized us population. from a total of 9622 participants, it was found that 31.6% were colonized with s aureus, of which 2.5% were colonized with mrsa. of persons with mrsa, half were identified as strains containing the sccmec type iv gene (most usually associated with ca-mrsa), whereas the other half were identified as strains containing the sccmec type ii gene (most usually associated with hca-mrsa). several other investigators have examined the epidemiology of mrsa in the us community; differences in the data suggest a sporadic distribution of ca-mrsa, with carriage rates ranging from 8% to 20% in baltimore, atlanta, and minnesota 127 up to 28% to 35% for an apparently healthy population in new york. 134 domestic pets can also be a source of s aureus, including mrsa and pvl-producing strains. [135] [136] [137] [138] [139] manian 136 described 2 dog owners suffering from persistent mrsa infection, who suffered relapses whenever they returned home from the hospital; further investigation revealed that the dog was carrying the same strain of mrsa. people who carry s aureus can shed the organism in large numbers most usually associated with skin scales. 140 kramer et al 81 review data showing that s aureus (including mrsa) can survive on dry surfaces for periods from 7 days up to 7 months. scott and bloomfield showed that, during a 4-hour drying period, up to 50% of s aureus inoculated onto laminate could be transferred to fingertips by contact. transfer to fingertips also occurred when a cloth contaminated with s aureus was used to wipe a clean surface. 141 studies in health care settings, as reviewed by bloomfield et al, 25 147 found that transmission of the mrsa strain from an index case to 2 siblings and the mother occurred at least 3 times, and one family member was colonized for up to 7 months or more. these represent recent examples of studies that show survival and transfer of mrsa around the home. these and other studies are also reviewed by bloomfield et al. 25 intervention studies to establish the causal link between hand hygiene and infectious disease in the home and community both observational and interventional study designs have been used to assess the relationship between hand hygiene and id transmission. by definition, observational studies are not randomized and must utilize careful methods to preserve internal validity. control of confounding and the potential for selection, recall, and other biases are also a concern, for example, individuals who wash their hands less frequently are also less likely to report symptoms. intervention studies on the other hand compare infection rates in groups in which handwashing is, or is not, promoted. intervention studies employing randomization of treatment groups have been considered the ''gold-standard'' in terms of reducing selection biases. these studies have the ability to ensure that randomized groups are similar, apart from treatment allocation and differences that occur by chance. for these reasons, we limit discussion to intervention studies, focusing on gi and rt illnesses, because these are the most common infectious illness symptoms in home and community settings. a range of intervention studies have been carried out to evaluate the causal link between handwashing and id transmission and have been reviewed in a series of papers to assess the consistency and strength of the link. [151] [152] [153] overall, these studies indicate a strong and consistent link between handwashing and gi disease and a significant link between handwashing and rt illnesses. for the most part, these studies have been carried out in child day care centers, schools, and military and other public settings in which the outcome is often measured against a high baseline level of infection. relatively few studies have been carried out in household settings in the united states and europe. difficulties associated with studying households in developed areas include fewer children under the age of 5 years, higher level of hygiene infrastructure, and difficulties in collecting data. given that there are likely fewer susceptible individuals clustered within household settings, the prevalence of gi and rt illnesses is relatively much lower, making it more difficult to detect a significant influence of hand hygiene on the occurrence of illness. whereas some intervention studies are not relevant to this review and have been omitted, others give useful insight into the potential impact of handwashing in the home and in the general community. studies that are included have been selected on the basis of whether transmission routes are likely to reflect those in the home, most particularly whether the relative rates of transmission via these routes (as shown in fig 2) are likely to be similar. for this reason, studies on gi infection in developing countries have been excluded; in these settings, limited access to sanitation means that rates of direct hand-to-mouth transmission from feces is high relative to other routes of transmission (eg, person-to-person transmission via hands, or inadequate food hygiene), compared to settings with adequate water and sanitation in which transmission is more likely to involve person-to-person transmission and transmission via food, rather than direct feces-to-hand-to-mouth. for gi illnesses, we have, therefore, focused on studies carried out in developed country communities, although, even for studies such as those in child daycare centers, in which food preparation is not undertaken by study participants, the data probably reflect mainly the impact on person-to-person transmission. for rt infections, studies conducted in both developed and developing countries are included on the basis that relative rates of airborne transmission versus transmission via hands are likely to be similar regardless of setting. in a recent review, aiello et al assessed the relationship between handwashing and gi outcomes 154 focusing on studies conducted in north america and europe. table 7 summarizes studies providing an effect estimate (risk ratio, rate ratio, and others) as well as 95% confidence intervals (95% ci). in all of the studies, handwashing with soap was the factor studied, although in some, this was combined with hygiene education measures. all studies assessing handwashing and hand hygiene education were conducted in school or day care settings. among the studies in table 7 , the reduction in gi illness associated with handwashing ranged from 210% to 157%. however, 3 of the 5 studies were not statistically significant, including the study that identified a value of 210%. the studies that gave statistically significant results all describe reductions close to 50%. overall, these reviews suggest a consistent causal relationship between handwashing and reduction in gi illness, although the findings are less consistent and of a lesser magnitude than in lesser developed settings in which studies considered statistically significant suggested reductions from 26% to 79%. 154 in assessing rt infections, the reviews of aiello and larson 151 and aiello et al, 154 mentioned above, 161 they reported that hand hygiene (handwashing, education, and waterless hand sanitizers) can reduce the risk of respiratory infection by 16% (95% ci: 11%-21%). these investigators have now updated their estimate with 2 further, more recent, studies that, when all studies are taken together, give a pooled impact on respiratory infection of 23%. 162 based on these studies, table 8 summarizes the results of community-based interventions (excluding health care-related and military settings) on rt illnesses. most studies were conducted in economically developed countries (83%, 5/6). the range of reduction in illness was 5% to 53%, but only 33% (2/6) of the studies were statistically significant. the results suggest that hand hygiene education and promotion of handwashing can reduce rates of rt illnesses, but the impact is less than for gi infections, although it must be borne in mind that the available data are more limited. 154 there are also several studies of handwashing that do not distinguish between gi and rt outcomes. 157, 167, 168 these studies measure outcomes such as illness-related absenteeism, making it difficult to assess the impact on specific disease etiologies. of these studies, only one reported a significant reduction (25%). 157 two were conducted in day care centers, and 1 was conducted in an elementary school. all 3 studies were conducted in economically developed areas (united states, sweden, and israel). 157, 167, 168 several methodologic issues must be considered for these studies. studies that use randomization are more likely to produce study groups with similar baseline characteristics. surprisingly, 40% of the 11 studies in tables 7 and 8 did not randomize. in some studies, randomization may not be an option (eg, in community settings) because the intervention is too complicated to randomize to multiple groups rather than assigning it to a single geographic area. controlling for potential confounding variables is also an important issue, for example, if a study did not control for age and included adults as well as children, the effect of a hygiene intervention may be diluted because adults are at lower risk for diarrheal disease compared with children. in randomized studies, adjustment for confounding in the statistical analysis may not be required if potential confounders associated with intervention and control groups appear balanced, for example, randomization of households in the same geographic area may produce intervention and control arms with the same age distributions, hygiene habits, and health profiles. as summarized in table 9 , of the 11 studies, only 50% (9/18) reported controlling for at least 1 potential confounding factor. although masking (also known as blinding) can be difficult to implement in hygiene studies because subjects, observers, and interviewers are usually aware of the intervention status, a few studies (2/18) were able to employ masking to reduce knowledge of the intervention. masking can reduce biases associated with knowledge of intervention, including changes in behaviors, practices, and data collection methods. for intervention studies, disregarding clustered sample design may cause bias. for example, a handwashing program in a day care center may affect a child's risk of disease through its individual-level effect (the effect of handwashing of a child on his or her own risk of disease) and through its group-level effect (the effect of centerwide handwashing on risk of disease, even if the child is not following the handwashing program). clustered interventions must take into account the grouped data structure in subsequent analyses or must analyze data at the in the section above, which describes the development of a risk-based approach to home hygiene, we evaluated how pathogens are introduced into the home and the chain of events that can lead to healthy household members becoming infected. an assessment of the microbiologic data related to each stage of the infection transmission cycle suggests that the critical control points for preventing the spread of infection in the home are the hands, hand contact surfaces, food contact surfaces, and cleaning cloths and utensils. intervention at the appropriate time (eg, during raw food handling, rather than as part of daily routine cleaning) is an equally fundamental part of a riskbased approach to hygiene. in practice, pathogens may be transmitted by more than one route, and it is impossible to achieve 100% hand hygiene compliance. therefore, interventions to reduce id transmission in the home must be multifaceted. key to preventing infection transmission via the hands (and other surfaces) is the application of effective hygiene procedures. because the evidence reviewed in the earlier sections shows that the ''infectious dose'' for many common pathogens such as campylobacter, norovirus, and rhinovirus can be very small (1-500 particles or cells), intuitively one must argue that, in situations in which there is significant risk, the aim should be to get rid of as many organisms as possible from critical surfaces. organisms can be removed from hands and other surfaces by the following: d physical removal using soap or detergent-based cleaning; or d microbes can be killed in situ by applying a disinfectant or sanitizer. in principle, handwashing using soap or detergent and water mechanically dislodge organisms, but, to be effective, it must be applied in conjunction with a rubbing process that maximizes release of microbes from the skin and a rinsing process that washes the organisms off the hands. although elimination of transient contamination from the hands by the application of a hygiene procedure is plausible, the evidence considered below suggests that, in practice, procedures vary considerably in the extent to which they achieve this. in this section, data on the efficacy of hand hygiene procedures are summarized. a range of test methods has been used to measure the efficacy of hand hygiene products and procedures. although these methodologies yield valuable data, the results can vary considerably depending on the method used. in 2004, sickbert-bennet et al 169 produced a study, based on published literature and their own data, which indicated that factors that affect efficacy measurements are as follows: use of experimental contamination versus normal flora, application method of test organism, type of hand hygiene agent, concentration of active ingredient, volume, duration of contact and application method of the agent, and study method (in vivo panel test vs in vitro suspension test). interpretation of data is made difficult by failure to compare multiple agents in the same study; because of these limitations, comparisons of results from different studies must be interpreted with care. in vivo ''panel test'' studies of the effectiveness of handwashing. in europe, the efficacy of handwashing is established by panel tests that determine the reduction in the number of organisms released from artificially contaminated hands. the test applicable to handwash products is the committee european normalisation hygienic handwash test en1499. 170 in this test, e coli is inoculated onto the hands and dried. the handwash product is applied to the hands with a rubbing action for either 30 seconds or 1 minute. the residual number of bacteria present on the hands is assessed pre-and postwash by a rinse sampling process and the log reduction determined. to make a claim that a product is a hygienic handwash, it must produce a log reduction in release of e coli from the hands at least equivalent to that produced by a reference soft soap product (mean, 2.76 log in 1 minute; range, 2.02-4.27). in the united states, handwashing is evaluated by a similar panel test using serratia marcescens as 171 the product, when evaluated by this method, must produce a 2-log reduction after 5 minutes. a range of studies, based on these methodologies, has been carried out to determine the efficacy of handwashing, and are reviewed by boyce and pittet, 172 kampf and kramer, 173 and sickbert-bennet et al. 169 from their assessment, kampf and kramer estimated that handwashing produced a mean reduction of up to 2.4 log within 1 minute. data from individual studies are summarized in table 10 and suggest that, for e coli, the greatest reduction is achieved within the first 30 seconds, ranging from 0.6 to 1.1 log after 15 seconds to 1.8 to 2.8 log after 30 seconds. extending the washing time to 1 minute produces a reduction of 2.6 to 3.23 log, but increasing the process for more than 1 minute does not appear to gain any additional reduction. relatively few data are available on the effectiveness of handwashing in removal of viruses, but the available data (table 10) suggest that handwashing may be less effective for viruses compared with bacteria. although panel test data suggest that handwashing efficacy is similar across a range of bacterial species, some field-based studies suggest that efficacy can vary quite significantly. in some cases, organisms can be attached to the hands too firmly and may not be removed by handwashing. a study of the spread of salmonella and campylobacter from contaminated chickens via hands during handling and preparation in a kitchen 176 showed that, although campylobacter were efficiently released from the hands by a 30second rub and rinse process, a 2-minute process was necessary to eliminate salmonella. the hand rinsing process is also important; cogan et al 86 showed that, following preparation of salmonella and campylobacter-contaminated chickens in domestic kitchens, 15.3% of hands and hand and food contact surfaces still showed evidence of contamination even after participants had carried out a washing-up routine with detergent and hot water and then used a cloth to wipe surfaces. sites contaminated most frequently were hands (20%); dishcloths, utensils, and tap handles (25%); and sink surrounds (30%). these results were confirmed in further studies 176, 177 in which, after cleaning up with a typical routine involving a bowl of hot soapy water and a cloth, although isolation rates from hands of participants were 5% (1/20) for campylobacter, 45% (9/20) of participants still had salmonella on their hands, and, on 3 occasions, counts recovered were .10 3 colony-forming units. in a further study in which participants cleaned up in the same way but then rinsed their hands under running water for 10 seconds, no samples were positive for campylobacter. however, 15% (3/20) still had low numbers of salmonella isolated from their hands. larson et al showed that the quantity of soap (1 ml and 3 ml) used can also have an impact on the microbial reduction achieved by handwashing. 178 bidawid et al 65, 66 studied the impact of handwashing in preventing transfer of hav and fcv from artificially contaminated finger pads to pieces of lettuce (table 11 ). touching the lettuce for 10 seconds resulted in transfer of 9.2% and 18%, respectively, of the virus. when finger pads were washed before the lettuce was touched, the amount of virus transferred was reduced to 0.39% and 0.4%, respectively. amounts of hav and fcv remaining on treated finger pads were 6.5% and 7%, respectively. surprisingly, virus transfer to lettuce when the finger pads were rinsed with water alone was between 0% and 0.3%, depending on the volume of water used for rinsing. barker et al showed that a thorough 1-minute handwash with soap was sufficient to eliminate norovirus from fecally contaminated hands to levels that gave negative reverse-transcription polymerase chain reaction assays. 89 however, schurmann and eggers 175 concluded that enteric viruses, particularly poliovirus, may be more strongly bound to the skin and that the inclusion of an abrasive substance in handwash preparations is needed to achieve effective removal. handwashing was also found to be ineffective in eliminating adenovirus from hands of a physician and patients. 179 for handwashing, a hand-rubbing time of 15 seconds with soap is generally recommended, although the data in table 10 indicate that 30 seconds to 1 minute is needed to achieve the optimum of 2-to 3-log reduction. in practice, it is doubtful whether people comply with even a 15-second handwash, although there are few data to confirm this. a study of 224 healthy another study with 52 office workers and students showed a mean log prewash count of 4.81 compared with 5.07 postwash. 181 kampf and kramer 173 also reviewed studies from health care settings in which increased bacterial counts were found on the hands after handwashing, and handwashing failed to prevent transfer of bacteria from hands to surfaces. although there are no data available to confirm this, increases in contamination may result from sweating induced by hot water, which flushes resident bacteria from the sweat glands onto the hand surface or aids detachment of bacteria attached to skin scales. it is important to bear in mind that, although soap and water removes contamination from the hands, soap itself has a limited antimicrobial effect, which means that contamination will be transferred to the sink. hospital studies show that pseudomonas aeruginosa and burkholderia cepacia 182 can form reservoirs of contamination in sink waste pipes and can be a source of infection at times when splashes of contaminated water come in contact with hands. mermel et al reported that hands of hcws became recontaminated from faucet handles during a shigella outbreak. 183 soap bars also have the potential to spread contamination from person to person via the hands. 88 efficacy of abhs abhs are formulations that contain either ethanol 1propanol or 2-propanol or a combination of these products. their antimicrobial activity is attributed to their ability to denature proteins. although products containing 60% to 95% alcohol are most effective, higher concentrations are less effective because proteins are not easily denatured in the absence of water. a range of in vivo and in vitro studies have been carried out to determine the effectiveness of abhs and are reviewed by boyce and pittet, 172 kampf and kramer, 173 and sickbert-bennet et al. 169 in vivo panel testing of abhs. in europe, the efficacy of abhs is established by panel tests that determine the reduction in the number of organisms released from artificially contaminated hands. the test applicable to abhs is the committee european normalisation hygienic handrub test en1500. 184 in this test, e coli is inoculated onto the hands and dried. the sanitizer is applied to the hands with a rubbing action for a specified period. the residual number of bacteria present on hands is assessed pre-and posttreatment by a rinse-sampling process and the log reduction determined. to claim that a product is a hygienic handrub, it must produce a log reduction at least equivalent to that produced by a reference product containing 60% vol/vol 2-propanol (mean, 4.24 log in 1 minute; range, 3.17-6.46). in vivo panel testing against bacterial strains. data from in vivo panel tests, summarized in table 12 , indicate that abhs show good and rapid activity against bacterial stains such as e coli and s aureus. efficacy is at least as good, if not better, than that achieved by handwashing with soap (table 10) ; log reductions obtained after a 30-second contact period were of the order of 3.4 to 3.7 or more compared with 1.8 to 2.8 for a 30-second handwashing process. boyce and pittet 172 conclude that, typically, log reductions of the release of test bacteria from artificially contaminated hands average 3.5 log after 30 seconds and 4.0 to 5.0 log after 1 minute. paulson et al 185 compared the efficacy of abhs containing 62% ethanol (contact time 5 minutes) with handwashing against s marcescens, which showed that handwashing (20 seconds rubbing followed by 30 seconds rinsing) produced a log reduction of 2.29 compared with 3.83 for the abhs. hammond et al 186 recorded a 2.84-log reduction for 62% ethanol against s marcescens in 10 seconds using the astm method. sickbert-bennet et al, 174 however, showed that exposure of s marcescens to 60% to 62% ethanol for 10 seconds produced only a 1.15-to 1.55-log reduction compared with 1.87-log reduction for handwashing for 10 seconds, when tested by the astm 1147 method. leischner et al 187 carried out in vivo tests that showed that alcohol gels were significantly less effective against c difficile spores (1.68-to 1.94-log reduction) compared with handwashing with chlorhexidine soap (2.46-log reduction). residual spores were readily transferred by handshaking following abhs use. 187 the reduction in spore counts is higher than expected in view of their known resistance to alcohol and may result from the friction associated with application of the gel rather than a bactericidal action; kampf and kramer 173 state that water alone can produce a reduction of 0.5 to 2.8 log within 1 minute for e coli. using the standard astm 1174 method, sickbert-bennet et al evaluated the effect of exposure time and volume of product used on the efficacy of 62% ethanol. 169 they showed that the use of 7 g of the abhs produced a higher log reduction compared with 3 g (2.7-to 3.8-log reduction compared with 1.0-to 1.8log reduction). rubbing the hands until dry (3-12 minutes) was more effective compared with a 10-second application (1.0-to 1.6-log reduction compared with 0.6-to 1.1-log reduction). two recent field studies indicate that an abhs is equally or slightly more effective than handwashing in reducing bacterial contamination on hands. davis et al 19 compared the reduction of bacterial counts on hands using soap and water or a 62% ethanol-based hand sanitizer (contact time 30 seconds) after animal handling at a us livestock event. there was no significant difference in the distribution of log reductions obtained using abhs compared with handwashing; log reductions in total count ranged from 21.4 to 1.4 and 23.0 to 3.5 for total coliforms. traub-dargtz et al carried out a study at 2 clinics in canada to evaluate the efficacy of handwashing compared with use of abhs (62% ethanol, contact time 30-60 seconds) on veterinary staff performing routine equine physical examinations. 188 mean bacterial load on hands increased by 0.36 and 0.91 log (for the 2 clinics, respectively) as a result of handling the animals, whereas the mean log reduction produced by handwashing with soap was less than 0.6, compared with 1.29 and 1.44 log (for the 2 clinics, respectively) produced by abhs. in vivo panel testing against viral strains. a number of in vivo studies have been carried out to determine the efficacy of abhs in reducing the release of viruses from hands. test methods were variants of the method of ansari et al 189 or the astm e1174 method, 171 in which the virus is applied to the fingertips and the efficacy of the product in reducing the numbers of viral particles recoverable from the hands determined. the residual number of viral particles present on the hands is assessed pre-and posttreatment and the log reduction determined. data collated by boyce and pittet 172 (table 13) indicate that ethanol at 60% to 80% produces a 0.8-to .3-log reduction against a range of viruses, the extent of the reduction depending on the viral strain, the nature and concentration of the alcohol, and contact time. data indicate that activity of abhs against viral strains is less than against bacterial strains and that ethanol has greater activity against viruses than 2-propanol. however, all of the strains referred to in table 13 are nonenveloped viruses, which are known to be more resistant to disinfectants than enveloped viruses. as far as hand hygiene in the home and community is concerned, however, this is key because many of the viral strains responsible for hygiene-related id commonly occurring in community settings (rotavirus, norovirus, rhinovirus, and adenovirus) are nonenveloped. that having been said, the data suggest that, although nonenveloped viruses such as hav and enteroviruses (eg, poliovirus) require 70% to 80% alcohol to be reliably inactivated, studies by sattar et al 194 in a number of these studies, handwashing with soap was also investigated. these studies 175, 189, 196 showed that the action of abhs against hav, polio, and rotavirus was significantly better than that achieved by handwashing with soap. however, in the test model used by ansari et al 189 and mbithi et al, 196 inoculated fingertips are exposed to soap solution or abhs by inverting them over a vial containing the product. in practice, handwashing involving rubbing and rinsing is likely to remove larger numbers of organisms from hands. in a further experiment, ansari et al 115 also demonstrated that 2-propanol (70%) was more effective (98.9% reduction after 10 seconds) than liquid soap (77% reduction) against rotavirus. mbithi et al showed that the log reduction of polio and hav virus (0.89-1.34) by application of 70% ethanol was sufficient to prevent transfer to another surface via the fingertips. 196 using similar methodology, bidawid et al 65, 66 studied the impact of ethanol hand sanitizers in preventing transfer of hav and fcv from artificially contaminated finger pads to pieces of lettuce. results (table 11) show that touching the lettuce for 10 seconds resulted in transfer of 9.2% and 18%, respectively, of the virus. when finger pads were treated with 62% ethanol or 75% ethanol (contact time 20 seconds) before the lettuce was touched, the amount of virus transferred was reduced to 0.64% and 0.46%, respectively, for hav and 2.1% and 1.2%, respectively, for fcv. although both 62% and 75% alcohol produced significant reductions in virus transfer, significant amounts of virus were found to remain on treated finger pads. in all cases, treatment with ethanol was less effective than handwashing. kampf and kramer 172 and boyce and pittet 173 suggest that, to achieve satisfactory activity against nonenveloped viruses, higher alcohol concentrations and extended contact times are needed. absolute ethanol reduced viral release from hands by 3.2 log, 80% ethanol by 2.2 log, and absolute 1-propanol by 2.4 log 197 but with a contact time of 10 minutes. schurmann and eggers 175 concluded that high alcohol-concentration products are effective against enteroviruses only under favorable conditions (large disinfectant/ virus volume ratio, low protein load). other studies also demonstrate superior activity of high ethanol concentrations against nonenveloped viruses such as polio, hav, and adenovirus. 198, 199 in vitro testing against bacteria, viruses, and fungi. whereas in vivo tests can be used to indicate the efficacy of products under use conditions, in vitro suspension tests are used to establish whether efficacy extends to a broad range of organisms. in vitro testing bacterial and fungal strains. alcohols have excellent and rapid activity against gram-positive and gram-negative vegetative bacteria and fungi when tested in vitro. 172, 173 a study by fendler et al 200 (table 14) shows the efficacy of an abhs containing 62% ethyl alcohol against a range of bacterial and fungal species, giving 4-to 6-log reduction in 15 to 30 seconds. in vitro testing against viral strains. data, as reviewed by boyce and pittet, 172 confirm that enveloped viruses such as herpes, influenza, piv, and rsv are very susceptible to alcohols. data from individual studies (table 15) suggest that activity against enveloped viruses is equivalent to that against bacterial strains. however, in agreement with in vivo data, alcohols tend to be less effective against nonenveloped viruses, although this is not the case for all strains. fendler et al 200 confirmed good activity for ethanol (62%) against piv and herpes viruses (.4-log reduction in 30 seconds) and some, but relatively less, activity against the nonenveloped rhinovirus, cocksackie virus, adenovirus, and hav (1-to 3-log reduction in 30 seconds). hammond et al 186 showed .5-log reduction against herpes and influenza virus but also .4.25-log reduction against rhinovirus type 16. there are no data on efficacy against rotavirus in vitro. in vitro tests suggest that alcohols are relatively effective against fcv, although gehrke et al 190 (table 15) found that 1-propanol was more effective than 2-propanol and ethanol. it was also found that these alcohols were less effective against fcv at 80% than at 50% and 70%. at this concentration (80%), 1-propanol, 2-propanol, and ethanol produced log reductions of only 1.9, 1.35, and 2.16, respectively. by contrast, duizer et al 201 showed that 70% ethanol produced less than a 2-log reduction for fcv after 8 minutes and a 3-log reduction after 30 minutes. these data are confirmed by a further study (mcneil-ppc unpublished) using in vitro suspension test methods as used to generate data in table 15 . the data (table 16) show that 62% ethanol gave a 3to 6-log reduction in 30 seconds against a range of nonenveloped viruses including not only rsv, piv, and influenza a and b but also against some strains of rhinovirus and echovirus. efficacy of abhs under conditions of soiling. alcohols are considered inappropriate when hands are visibly dirty or soiled because they fail to remove soiling. however, in a number of in vitro studies, in which the efficacy of abhs was determined in the presence and absence of soil (10% fetal calf serum or 0.3% bovine serum albumin), soil produced little or no loss of efficacy. 198, 202 larson and bobo showed that, in the presence of small amounts of protein material (eg, blood), ethanol and 2-propanol were more effective than soap in reducing bacterial counts on hands. 203 using the astm 1174 method, sickbert-bennet et al 169 showed that applying protein to hands did not produce any significant reduction in efficacy of abhs or handwashing but produced a modest but significant increase; log reductions for handwashing were 1.18 to 1.39 and 1.56 to 1.87 in the absence and presence of protein, respectively. log reductions for abhs were 0.18 to 1.07 and 1.35 to 1.55 in the absence and presence of protein, respectively. one of the problems in developing hygiene promotion policies is the lack of quantitative data on the relative health impact of different hygiene interventions. although intervention studies yield quantitative data on health impact, as discussed in section 4.2, the reliability of these estimates is difficult to confirm. by contrast, in vivo and in vitro tests are more economic to perform and can be used to determine relative efficacy of different procedures but give no assessment of how the contamination reduction on hands correlates with health impact. in an attempt to overcome these problems, haas et al have applied the technique of quantitative microbial risk assessment (qmra) to estimate the relative health benefits resulting from use of different hygiene procedures. 204 this approach involves using microbiologic data from the published literature related to each stage of the infection transmission cycle to calculate infection risk. in a recent study, 205 these investigators developed a model for studying the effect of hand contact with ground beef during food preparation, which was used to study the impact of handwashing and use of abhs in preventing subsequent transference from the hands to the mouth compared with no handwashing. pathogenic e coli and e coli o157:h7 were selected for this study because it is known from other investigations 206 that handling ground beef during home food preparation poses a risk of infection with e coli. to perform the risk assessment, data on the density of pathogens in ground beef, transference from beef to hands, removal by handwashing or abhs, rate of transfer from hand to mouth, and infectivity of ingested pathogens were obtained from the literature and, after screening for data quality, were used to develop probability distributions. for assessing log reductions produced by hand hygiene procedures, only in vivo panel testing data were considered. the median log reduction used in these calculations was 0.3 (range, 0.2-3.0) for handwashing and 4.3 (range, 2.6-5.8) for abhs. table 17 shows the estimates of the infection risk from handling raw beef, as obtained from the analysis. the authors note that these risks are conditional in the sense that they quantify the risk to an individual who has handled ground beef and who engages in hand-to-mouth activity. the probability that an individual will engage in such behavior is not known, and, therefore, a direct comparison with actual disease rates cannot be made. however, with some plausible assumptions, it was assessed that, assuming that there are 100 million individuals in the united states, each of whom handles ground beef once per month, this results in 1.2 3 10 9 contacts per year. assuming that 10% of these individuals contact hand to mouth after handling ground beef, this amounts to 1.2 3 10 8 incidents per year. for e coli o157:h7, using the median risk, this would result in an estimate ranging from 0.014 infections per year if all individuals washed their hands with soap following contact with ground beef to 0.7 infections per year if no handwashing is done. this would equate to a 98% median risk reduction for handwashing compared with no handwashing. if an abhs was used, this would result in an estimate of 0.00005 infections per year if all individuals used abhs following contact with ground beef. this would equate to a 99.9996% median risk reduction for use of abhs compared with handwashing. this study follows an earlier study by haas et al 207 to calculate risks associated with hand-to-mouth transfer after diaper changing of a baby infected with shigella. based on this model, it was calculated that the probability of acquiring infection was between 24 of 100 and 91 of 100 for those who used handwashing with soap after changing diapers. this was based on panel test data 205 conclude that quantitative microbial and id risk models offer a useful tool to assess the relative extent to which different hygiene procedures can impact on id risks. they concede, however, that, although risk modeling represents a promising approach, there are limitations to most models because of the multifactorial nature of infection transmission, the dynamic environment in which transmission takes place, and the paucity of data to specify model parameters. in an earlier section, we evaluated intervention study data to assess the strength of the causal link between hand hygiene and id transmission. in this section, we use these data to evaluate the effectiveness of handwashing as a hygiene measure and in relation to the effectiveness of using abhs as an adjunct or an alternative to handwashing. despite the methodologic limitations, the collective weight of evidence from intervention and microbiologic studies described earlier suggests that handwashing with soap can have a significant impact in reducing the incidence of gi and rt infection. the data, however, show that the health impact from handwashing promotion varies significantly according to the setting and outcome. statistically significant reductions ranged from 48% to 57% for gi illness and 20% to 51% for rt illness. although all studies were carried out in settings such as day care centers and schools, we believe that the modes of transmission in these settings and the relative rates of transmission of rt and gi infections are likely to reflect those occurring in the home. in 2004, meadows and le saux published a review of the effect of rinse-free hand sanitizers in elementary schools over a 20-year period. 208 they concluded, however, that the data were of poor quality and that more rigorous intervention trials are needed. in a more recent study, aiello et al examined the epidemiologic evidence for a relationship between waterless hand sanitizers and infections in the community setting over several decades. 154 in table 18 , we present the studies that specifically examined abhs. only studies with an effect estimate and 95% ci are presented. the type and content of the abhs varied across studies, for example, one study reported the use of a 60% isopropyl alcohol rinse and another study utilized an alcohol-based foam sanitizer. however, most studies used alcohol-based gels or other alcohol-based emollients. the alcohol content included ethanol and 2-propanol at concentrations ranging from 60% to 90%. of the 8 intervention studies, 7 were conducted in the united states (88%, 7/8) and 1 in finland (13%). most were conducted in child day care centers, elementary schools, or universities (88%, 7/8), and one was conducted in the household (13%, 1/8). outcomes included gi-related illnesses/symptoms and/or upper rt-related illnesses/symptoms examined as separate outcomes or in combination with other infectionrelated symptoms as part of a school absence-related definition of ''infectious-illness.'' of the 8 studies, 88% (7/8) reported significant results for at least one age group or outcome. the effect was stronger in younger compared with older age groups for studies providing age stratified data. the reduction in gi illness ranged from 0% to 59% for the 4 intervention studies that examined gi illnesses as separate outcomes. [209] [210] [211] 215 of these, all but one showed statistically significant reductions. 210 the study by uhari et al showed a significant reduction in gi illness only among children #3 years of age. 211 all but one of these studies was conducted in child care settings. 215 when evaluated separately, reductions in gi illness appeared more robust compared with the findings for upper rt illness. for upper rt illness, the reduction in infectious illness/symptoms ranged from 26% to 26%. only 2 of the 5 studies (44%) examining upper rt illness as a main outcome reported a statistically significant reduction. uhari et al reported a 13% reduction in rt illnesses among children #3years of age, but no significant effect in older children. 211 white et al reported a 20% reduction in upper rt illness among students using abhs in residence halls. 213 however, this study suffered from several methodologic shortcomings, including lack of control for clustered units, no randomization, no masking, and no monitoring of product use. all but one of the intervention studies included a hygiene education component, but, in 7 of these studies, this was only provided in the intervention arm. the level of education varied widely, ranging from basic information on when to use the abhs (ie, after sneezing and coughing, after use in the restroom, before lunch) to in-depth education programs 214 and biweekly instructional material designed to educate families on hand hygiene and infection transmission. 215 in all studies, abhs was promoted as a supplement to handwashing, or as an alternative to handwashing when soap was unavailable, and it is likely that the hygiene education would have had the effect of encouraging more frequent handwashing as well as use of abhs. although almost all studies indicated that hygiene education combined with promotion of abhs can reduce the risks of gi or rt illness, only 2 studies allowed any assessment of the independent effect of the abhs. of these 2 studies, both studies showed a significant reduction in illness-related absences (20% and 44%, respectively), but it is not clear whether the illnesses were predominantly gi or rt because these studies used a loose definition of absence-related illnesses. sandora et al 215 was the only study carried out in the household setting and, of all the studies, reported the highest reduction for gi illness. the trial involved 292 families with children enrolled in 26 child care centers. intervention families received a supply of abhs and biweekly hand hygiene educational materials for 5 months; control families received only materials promoting good nutrition. a total of 252 gi illnesses occurred during the study; 11% were secondary illnesses. the secondary gi illness rate was significantly lower (59%) in intervention families compared with control families (see table 18 ). a total of 1802 rt illnesses occurred during the study; 25% were secondary illnesses. although rt illness rates were not significantly different between groups, families with higher abhs usage had marginally lower rt illness rate than those with less usage (19% reduction). sandora et al 215 suggested that the difference may be due to heightened diligence associated with using abhs after a gi-related incident compared with an rt incident, such as sneezing. overall, based on relatively limited data available, the results in table 18 suggest that the impact of abhs promotion, as part of a hygiene education and promotion program in reducing the incidence of gi infections in young children, is similar to that observed for promotion of handwashing with soap. promotion of abhs in this manner also produced some reduction in the incidence of rt infections, which was less than that associated with promotion of handwashing alone. assessing whether there might be an added health benefit of using abhs above and beyond the effect of hygiene education is hampered by the fact that most studies used hygiene education and abhs in the intervention arm but did not provide an educational component to the control arm. several important methodologic issues were evident, although more recent studies have improved designs and conduct. in most studies, either parents or school personnel provided information on id among children in the study populations. in all but one study, 34 the parents, participants, or personnel monitoring and reporting infections were not masked as to their own or their child's intervention status. although masking of participants and interviewers to the intervention status is important, because it might influence reporting, it is often difficult to conduct masked hygiene interventions and may not be ethical. sandora et al determined that it was neither feasible nor ethical to mask subjects or interviewers because it is difficult to devise a formulation that could act as a ''placebo'' for abhs, and using a placebo abhs product might endanger the control group via inadequate hand hygiene. 215 in many of the abhs studies, especially the recent ones, efforts were made to control for potential confounding factors. however, many of the studies did not collect information on baseline hand hygiene practices (nor methods and frequency of cleaning/disinfecting soiled/contaminated environmental surfaces in homes) as well as abhs use. the studies also excluded participants who reported current abhs use in the home. furthermore, participants were asked to refrain from using abhs in settings outside the home. these are all important design strategies minimizing bias associated with noncompliance or differential usage. two of the 8 intervention studies failed to use systematic monitoring for hygiene practices, such as frequency of hand-sanitizing episodes, frequency of handwashing, or duration of handwashing. 212, 213 this is especially concerning because the study by sandora et al suggests that the quantity of abhs influences the risk of infection in a dose-response manner. 215 moreover, if frequency of handwashing and abhs use is not recorded, it is impossible to isolate the independent effects of abhs from that of handwashing on infection rates. in these studies of abhs use, surveillance measures included calculating use from monthly demand, total amount supplied, observation by research assistants, participant report, and reported use by primary caregivers in households. 186, 209, 212, 214, 215 overall, the microbiologic data, together with the intervention study data (both those involving abhs as well as those involving handwashing) as presented in this review, provide consistent evidence of a strong causal link between hygiene and the spread of infection in the home and community, and suggest that probably the single most important route for the spread of infection is the hands. if the data from intervention studies (summarized in table 19 ) are an accurate reflection of the true picture, it is suggested that, for up to 60% of gi illnesses, the hands are the ''sufficient,'' or a ''component'' (see earlier for definitions), cause of spread of infection. this correlates with microbiologic and other data reviewed in this report, which suggest that, although there is a tendency to assume that gi infections are mostly foodborne and result from inadequate cooking and inadequate storage of food, in reality, most gi infections in the home result from person-to-person spread or contamination of ready-to-eat foods within the home, much of which involves the hands as the sufficient or a component cause. for rt illnesses, the intervention study data (summarized in table 19 ) suggest that transmission via hands could be a sufficient or component cause of up to 50% of illnesses; whereas there has been a tendency to assume that the lower impact of hand hygiene on rt compared with gi infections is due to the fact that spread of rt pathogens is mainly airborne, the microbiologic and other data in this review correlate with the intervention data in suggesting that, for rt infections commonly circulating in the community, such as rhinovirus and rsv, the hands are the major route of spread. although up to 50% to 60% reduction in id risk was observed in some intervention studies, in other studies the reduction was much less. this variability could well be due to methodologic issues but could also be due to other factors within and between study communities. one possibility is that it relates to differences in the range of pathogens with differing modes of spread prevalent in different study groups, which means that hand hygiene has greater impact in some intervention groups than others. alternatively, the differences may reflect differing levels of hand hygiene compliance in different intervention groups. in some studies, the quality of the hygiene education, the manner in which the hygiene promotion was conducted, and the enthusiasm with which it was received may have given the intervention group a better understanding of what was required, with the result that they used better hygiene technique and were more likely to apply it at critical times. although there are no intervention study data to confirm this, the microbiologic data together with the qmra assessments suggest that even a relatively modest increase in log reduction on hands within a population could produce a significant increase in the health impact of a hand hygiene promotion campaign, which could, in turn, be achieved by addressing the issues in the next 2 sections. although panel tests carried out under controlled conditions showed that handwashing can reduce the numbers of bacteria and some viruses on the hands by up to 2-to 3-log within 30 seconds to 1 minute, in practice it is doubtful whether people wash their hands properly, even for the prescribed period of 15 seconds, to achieve this. at present, there is a paucity of data on the efficacy of handwashing in relation to how people actually wash their hands on a day-to-day basis, both in the duration of handwashing and handwashing technique; in most of the intervention studies described earlier no attempt was made to time handwashes or to determine residual levels of contamination on the hands after handwashing. microbiologic data suggest that, for some pathogens (eg, salmonella), mechanical removal by handwashing alone is inefficient. these data, together with the results of in vivo panel testing of the effectiveness of handwashing and of abhs, as described above, question the efficacy of handwashing in community-based groups and suggest that more work is needed to determine the efficacy of hand hygiene procedures under conditions normally encountered in the home and how hand hygiene procedures could be improved. for abhs, in vivo and in vitro testing suggest that these formulations are highly effective against bacterial pathogens and can produce a 3.5-log reduction on hands within 30 seconds and 4.0-to 5.0-log reduction after a 1-minute application against a wide range of species including salmonella. it is possible, however, that the potential for increased benefits against bacterial infections compared with handwashing may be offset by reduced efficacy against important nonenveloped viruses such as rotavirus, some strains of rhinovirus, and possibly also norovirus. it has been argued that the higher impact of abhs interventions against gi compared with rt infections is due to the fact that rt infections are predominantly viral. however, because the intervention data indicate that handwashing also supports a stronger reduction in gi diseases compared with rt diseases, this seems unlikely. some studies suggest that, to achieve satisfactory activity that includes all types of viruses, higher concentrations up to 80% ethanol should be advised. other studies suggest that the efficacy of abhs may be increased by increasing the volume of agent applied to the hands. 215 in formulating policies for hand hygiene, it would be convenient to be able to define what represents a ''safe'' residual level of contamination on the hands after hand hygiene, ie, sufficient to prevent infection transmission, but, because the infectious dose varies from one species to another and is dependant on the immune status of the recipient, this approach is untenable. the qmra approach, as outlined earlier in this review, however, demonstrates that strategies that produce an increase in the log reduction on hands from 2 to 3 to 4 are accompanied by a significant incremental reduction in the risk of infection in a given population and could thus be worthwhile. this suggests that the health impact from promoting hand hygiene could be increased by developing and promoting procedures for use in the home and community that increase the log reduction of contamination on the hands. this involves identifying products and procedures (both soap-based and waterless products or wipes) that achieve high levels of removal and/or ''kill'' (alone or in combination) of the full range of gram-negative and gram-positive bacteria and enveloped and nonenveloped viruses and that deliver hand hygiene under conditions that people are prepared to employ in their busy and mobile daily lives. it also suggests that, particularly for ''high-risk'' situations as outlined below, there is advantage to be gained by promoting handwashing followed by use of abhs to increase the log reduction. applying hand hygiene at the correct time: the need for hygiene education the data presented in this review suggest that the favorable health impact from promoting hand hygiene could be further increased by getting people to practice hand hygiene not just more frequently but also at the right time. a number of studies show the relatively poor understanding of the principles of hygiene that is present in the community. this may be one of the factors responsible for the higher risk reductions observed in intervention studies of gi compared with rt infections. for example, knowledge regarding the need for handwashing after coughing or sneezing may not be as pervasive as knowledge about handwashing after defecation, and it may be that people understand better when to wash their hands during food-associated activities but not, for example, while handling contaminated tissues. although some intervention studies described in this review involved a component of education, it was not possible to determine the extent to which hygiene education that enhanced people's understanding of infection transmission also enhanced health outcome. because visible soiling is an unreliable indicator of the presence of pathogens on the hands, people are unlikely to wash their hands at the correct time unless they have been taught to do so or have some awareness of the chain of infection transmission in the home, ie, they are aware of when their hands may be contaminated. whereas risks associated with food handling are largely confined to defined periods of time, for rt and skin infections (and person-to-person transmission of gi infection), the risk is ongoing and involves a large proportion of our ongoing daily activities. thus, whereas it is possible for hand hygiene advice associated with food hygiene to be rule based, this is not the case for other types of infections. in the event of a flu pandemic, the advice issued by the uk health protection agency 216 to ''wash hands frequently'' is unlikely to be effective unless people have some idea of the times when their hands are likely to be contaminated with flu virus. although current thinking about hygiene promotion tends toward a view that the most effective way to change behavior is by mass social marketing of single rule-based hygiene messages, 4 the data presented in this review suggest that the complexity and shifting nature of the id threat is such that a rule-based approach to hygiene is inadequate to meet current public health needs. the need is for an approach founded on awareness of the chain of infection transmission and how it differs for different groups of infections. hygiene education needs to be consistently incorporated as part of hand hygiene promotion programs if people are to properly understand the risks and adapt their behavior accordingly. based on the data presented in this review, we propose that, in promoting hand hygiene, significant improvements in health impact could be achieved by giving better guidance to people, first, on how to choose the best methods for hand hygiene (handwashing and/or use of abhs) based on the situation and showing them how to apply it properly and why this is important. secondly, it means stressing when it is important to apply hand hygiene, ie, what are the risk situations or critical control points at which hand hygiene needs to be applied. although the level of risk varies according to the occupants of the home (eg, presence of children, pets, ill people) and their immune status, based on the risk assessment approach as outlined earlier in this review, the critical control points or situations in which hand hygiene is indicated are as follows: d after using the toilet (or disposing of human or animal feces); d after changing a baby's diaper and disposing of the feces; d immediately after handling raw food (eg, chicken, raw meat); d before preparing and handling cooked/ready-to-eat food; d before eating food or feeding children; but also d after contact with contaminated surfaces (eg, rubbish bins, cleaning cloths, food contaminated surfaces); d after handling pets and domestic animals; d after wiping or blowing the nose or sneezing into the hands; d after handling soiled tissues (self or others', eg, children); d after contact with blood or body fluids (eg, vomit and others); d before and after dressing wounds; d before giving care to an ''at-risk'' person; and d after giving care to an infected person. in choosing the appropriate option for hand hygiene, there are 3 possibilities: either handwashing with soap, use of abhs (or other effective waterlessbased sanitizers), or handwashing followed by use of abhs. a possible framework for informing appropriate choice according to the particular situation is outlined in fig 4. this suggests that, in situations in the home and community that are ''standard risk'' (perhaps better described as situations not specifically regarded as ''high risk''), either handwashing or use of abhs may be chosen. within this, however, there are factors that advise, or in some cases dictate, choice, for example, handwashing is only an option when there is access to soap and water, whereas use of abhs is not an option when hands are heavily soiled (although people are likely to choose handwashing in this situation, prompted by the need to ''clean'' their hands). as discussed previously, there will always be situations in the home in which there is increased risk, either because there is a known source of infection or someone who is at increased risk of becoming infected. these situations are summarized in table 20 . these situations may relate to activities that are carried out routinely in the home, such as handling of raw meat and poultry, or involve household members such as pregnant women or young babies who are otherwise healthy but at increased risk of (or from) infection. they also relate to ''nonroutine'' situations such as a person in the home who is infected with a cold, or norovirus or other gi infections, or to situations in which there is someone who is at increased risk of infection as a result of underlying illness, immunosuppressive drug treatment, or needing catheter or wound care. although much of the ''health care'' carried out at home is done by trained caregivers, increasingly, there are situations in the home in which simple but risky actions are carried out by household members. in all of these ''increased risk'' situations, as outlined in table 20 , it is suggested that handwashing followed by use of an abhs should be encouraged. in persuading people to change behavior, one of the key factors is ''removing barriers to action.'' 217 lack of convenient access to a sink is a significant barrier to compliance, and time pressure is a barrier to getting people to wash their hands thoroughly. a key benefit of abhs is that they offer the means to apply hand hygiene in situations in which there is limited or no access to a soap and water. in home care situations, abhs offer an alternative to handwashing in situations in which other pressures mitigate against finding the time to visit the bathroom for handwashing, for example, when caring for a baby in the nursery or a sick person. they also offer a substitute for handwashing in ''out of home'' settings such as offices and public places, such as public transport or animal exhibits, at which access to soap and water is a particular problem and all of which offer frequent opportunities for hand transmission of infection. promoting use of abhs has the potential to get people to undertake hand hygiene more frequently and at critical times. in response to concerns about the possibility of a flu pandemic, the centers for disease control and prevention recommend the use of abhs for use as an alternative to handwashing. 218 in the event of a flu pandemic, it seems particularly important to encourage people to adopt good hand hygiene in public places. in health care settings, links between use of abhs and increased hand hygiene compliance and reduced infection rates has been observed. 219 in applying the framework outlined in fig 4, our intention is that this should not be regarded as an ''either handwashing or abhs'' situation; the fundamental aim should be to encourage more people to undertake hand hygiene procedures wherever possible at critical times. in view of the fact that hands are part of a complex system of infection transmission pathways, it must also be considered whether hand hygiene can, or should, be promoted in isolation. because people are reluctant to comply with handwashing, together with the microbiologic data showing the potential for transfer via hand and food contact surfaces and cloths to hands, which increase as the frequency occurrence of contamination of these surfaces increases, it would seem that, to maximize the health impact from hand hygiene promotion, it should be s56 vol. 35 no. 10 supplement 1 combined with promotion of hygiene in general, including hygienic cleaning of critical surfaces. if nothing else, this could raise awareness that hand contamination can arise from touching apparently clean surfaces. we are concerned that emphasis on handwashing alone without putting it within the context of other aspects of hygiene is encouraging the perception that handwashing is all that is required, ie, ''if you wash your hands you won't get sick.'' the aim of this report has been to review the evidence base for hand hygiene and develop a practical framework from it for promoting an effective approach to hand hygiene in home and community settings. provision of detailed guidelines for hand hygiene is outside the scope of this review. for such guidelines the reader is referred to the ifh guidelines and training resource on home hygiene. as part of its work in promoting home hygiene, the ifh has produced ''guidelines for prevention of infection and cross infection in the domestic environment'' and ''recommendations for selection of suitable hygiene procedures for use in the domestic environment.'' 220, 221 these documents are based on the concept of a risk-based approach and give detailed guidance on hand hygiene in the context of all aspects of home hygiene including food hygiene, general hygiene, personal hygiene, care of pets, and others. most recently, the ifh has also produced a teaching/self-learning resource on home hygiene. 222 this is based on the ifh guidelines and recommendations but is designed to present home hygiene theory and practice in simple practical language that can be understood by those with relative little infection control training or background. infectious diseases circulating in the community remain a significant concern, both in developed and developing countries. the global burden of id accounts for over 13 million deaths annually but, whereas the majority of deaths occurs in the developing world, infection also causes approximately 4% of deaths in developed countries. 223 although mortality from id has declined in the developed world, trends in morbidity suggest a change in the pattern of id rather than declining rates. several demographic, environmental, and health care trends, as reviewed in this report, are combining to make it likely that the threat of id will increase in coming years, rather than decline. one such factor is the rising proportion of the population in the community who are more vulnerable to infection. an important part of current european and us health policy is commitment to shorter hospital stays. a key requirement is to ensure that the increased health provision at home is not accompanied by an increase in id risks; otherwise, the cost savings gained by care in the community are likely to be overridden by costs of rehospitalization. even for the ''healthy community,'' id represents a significant economic burden because of absence from work and school and added health care costs. secondary infections can produce complications, and some infections may be associated with the development of diseases such as cancer or other chronic conditions, which can manifest at a later date. those responsible for ensuring that the public are protected from infection in health care facilities are now realizing that their ability to manage the problem is hampered by spread of pathogens such as mrsa, c difficile, and norovirus in the community and the home, and the number of infected people or carriers who come into their facilities, and are looking for ways to address this by engaging the public to adopt more rigorous standards of hygiene. one of the things that is apparent from newly emerging data, and that is reflected in this review, is the extent to which common infections circulating in the community are hygiene related. this suggests, in turn, that hygiene promotion could have a significant benefit in terms of improved public health and well-being; in particular, the data highlight the extent to which viruses (norovirus, rotavirus, rhinovirus, influenza, and other viruses) are responsible for hygiene-related diseases now circulating in the community. the main conclusions from this review are as follows: d id circulating in the home and community is a serious public health problem in the developed as well as the developing world. d good hygiene practice is key to reducing the burden of id in the home and community. d hand hygiene is a key component of good hygiene practice in the home and community and can produce significant benefits in terms of reducing the incidence of infection, most particularly for gastrointestinal infections but also for respiratory tract and skin infections. d decontamination of hands can be carried out either by handwashing with soap or by the use of waterless hand sanitizers, which achieve a log reduction in bacterial and viral contamination on hands by the removal of contamination or by killing the organisms in situ. the health impact of hand hygiene within a given community can be increased by using products and procedures, either alone or in sequence, that maximize the log reduction of both bacteria and viruses on hands. d the impact of hand hygiene in reducing id risks could be increased by convincing people to apply hand hygiene procedures correctly (eg, wash their hands correctly) and at the correct time. d to optimize health benefits, promotion of hand hygiene must be accompanied by hygiene education and should also involve promotion of other aspects of hygiene, for example, surface and cloth hygiene. this report highlights a number of areas in which additional data are needed: d further studies are needed to characterize the frequency of, and factors associated with, id transmission in noninstitutional settings such as the home. d further studies are needed to assess the relative efficacy of hand hygiene procedures in reducing hand contamination (handwashing with soap and use of abhs, involving different ''contact/application/rinsing'' times, and others). this includes the following: (1) in vivo panel tests to determine the reduction in bacteria and viruses on hands under controlled conditions. committee european normalisation or astm tests now provide standard test models for comparing the efficacy of handwashing with the use of waterless hand sanitizer products, under defined conditions. they provide an economic approach (relative to intervention studies) that can be used, alone or in combination with qmra, to inform hygiene policy and/or the design of intervention studies. (2) field studies to determine log reduction in counts on hands in relation to how people actually wash their hands or apply abhs in their homes. d additional data are needed to understand how, when, and why people practice hand hygiene at home and how this relates to their understanding of id transmission and risks. d intervention studies are needed to determine the health impact of hand hygiene promotion with hygiene education, compared with hygiene promotion without education. this should also include understanding how hand hygiene combines with surface hygiene to influence health outcome. d intervention studies are needed to determine the potential for an increase in health impact from promoting use of abhs in conjunction with handwashing (ie, handwashing followed by use of abhs) or as a supplement to handwashing (in situations in which access to water is limited), compared with the promotion of handwashing alone. trends in infectious disease mortality in the united states 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mothers-in-law can promote behaviour change a multifaceted approach to changing handwashing behaviour guidelines for prevention of infection and cross infection the domestic environment recommendations for selection of suitable hygiene procedures for use in the domestic environment home hygiene-prevention of infection at home: a training resource for carers and their trainers memorandum on the threat posed by infectious diseases. need for reassessment and a new prevention strategy in germany. rudolf schulke foundation. wiesbaden: mph-verlag gmbh the authors thank dr. michele pearson, centers for disease control and prevention, atlanta, ga, for her very valuable and extensive contributions to the preparation of this review. key: cord-274763-i6e3g3te authors: liu, wen-kuan; liu, qian; chen, de-hui; tan, wei-ping; cai, yong; qiu, shu-yan; xu, duo; li, chi; li, xiao; lin, zheng-shi; zhou, rong title: epidemiology of hbov1 infection and relationship with meteorological conditions in hospitalized pediatric patients with acute respiratory illness: a 7-year study in a subtropical region date: 2018-07-16 journal: bmc infect dis doi: 10.1186/s12879-018-3225-3 sha: doc_id: 274763 cord_uid: i6e3g3te background: human bocavirus 1 (hbov1) is an important cause of acute respiratory illness (ari), yet the epidemiology and effect of meteorological conditions on infection is not fully understood. to investigate the distribution of hbov1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of china. methods: samples from 11,399 hospitalized pediatric patients (≤14 years old), with ari were tested for hbov1 and other common respiratory pathogens using real-time pcr, between july 2009 and june 2016. in addition, local meteorological data were collected. results: of the 11,399 patients tested, 5606 (49.2%) were positive for at least one respiratory pathogen. two hundred forty-eight of 11,399 (2.2%) were positive for hbov1 infection. co-infection was common in hbov1-positive patients (45.2%, 112/248). a significant difference in the prevalence of hbov1 was found in patients in different age groups (p < 0.001), and the peak prevalence was found in patients aged 7–12 months (4.7%, 56/1203). two hbov1 prevalence peaks were found in summer (between june and september) and winter (between november and december). the prevalence of hbov1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. conclusions: this study provides a better understanding of the characteristics of hbov1 infection in children in subtropical regions. data from this study provide useful information for the future control and prevention of hbov1 infections. human bocavirus 1 (hbov1), which belongs to family parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 [1, 2] . hbov1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. the prevalence of hbov1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness (ari), according to different studies [3] [4] [5] [6] [7] . serological and nucleic acid test results are generally consistent [8] [9] [10] [11] , showing hbov1 infection is very common. hbov1 can cause both upper respiratory illness (uri) and lower respiratory illness (lri) [12] [13] [14] [15] [16] [17] [18] . infection with hbov1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms [15, 19] . in some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms [18, 20] . clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications [18] [19] [20] [21] [22] . in some cases, patients develop severe respiratory injury symptoms, which can be fatal [21, 23] . hbov1 can be detected in fecal samples [24] , blood samples [25, 26] , urine [27, 28] , cerebrospinal fluid [29] [30] [31] , river water [32] and sewage [33, 34] , indicating that hbov1 may be associate with a variety of diseases. current in vitro studies modeling tissue-like airway epithelial cells cultures show hbov1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy [35] [36] [37] , similar to lung injury tissue changes in vivo. there is currently no vaccine or specific treatment for this virus; prevention and treatment of hbov1-related diseases still require further research. the prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses [38] . studying the epidemiology of hbov1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. in this study, we investigated the epidemiology of hbov1 infection in children (≤14 years old) hospitalized with ari in a subtropical region in china over a 7-year period. in addition, we collected climate data to determine if there was a relationship between hbov1 prevalence and meteorological conditions. this study will add to existing epidemiological data on hbov1 and its relationship with climate conditions in subtropical regions and will play a positive role in hbov1 control and prevention. the study sites were three tertiary hospitals in guangzhou, southern china (longitude: e112°57′ to e114 03′; latitude n22°26′ to n23°56′). inclusion criteria were pediatric patients (≤14 years old) who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. chest radiography was conducted according to the clinical situation of the patient. throat swab samples were collected from the enrolled patients between july 2009 and june 2016 for routine screening for respiratory viruses, mycoplasma pneumoniae (mp), and chlamydophila pneumoniae (cp). the samples were refrigerated at 2-8°c in viral transport medium, transported on ice and analyzed immediately or stored at − 80°c before analysis, as described previously [15, 39] . meteorological data for guangzhou, were collected from july 2009 to june 2016, from the china meteorological administration, including the monthly mean temperature (°c), mean relative humidity (%), rainfall (mm), mean wind speed (m/s), mean air pressure (hpa), mean vapor pressure (hpa), sunshine duration (h). real-time pcr for hbov1 and common respiratory pathogen detection dna and rna were extracted from the respiratory samples using the qiaamp dna mini kit and qiaamp viral rna mini kit (qiagen, shanghai, china), respectively, in accordance with the manufacturer's protocols. taqman real-time pcr for hbov1 was designed based on the conserved region of the np1 gene, as described previously [15] . common respiratory pathogens, including respiratory syncytial virus (rsv), influenza a virus (infa), influenza b virus (infb), four types of parainfluenza (piv1-4), adenovirus (adv), enterovirus (ev), human metapneumovirus (hmpv), four strains of human coronavirus (hcov-229e, oc43, nl63 and hku1), human rhinovirus (hrv), mp and cp were detected simultaneously as previously reported [40] . data were analyzed using chi-squared test and fisher's exact test in spss 19.0 (spss inc., chicago, il, usa). correlation with climate data was analyzed using multiple linear regression analysis. all tests were two-tailed and a p value < 0.05 was considered as statistically significant. eleven thousand three hundred ninety-nine pediatric patients (≤14 years old) hospitalized with ari were enrolled in the study between july 2009 and june 2016. the male-to-female ratio was 1.82:1 (7361:4038) and the median age was 1.75 years (interquartile range 0.75-3.83). overall, 86.5% (9857/11399) of patients were under the age of 5 years. all the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 (49.2%) were positive for one or more of those pathogens (table 1) , and had a median age of 1.50 years (interquartile range 0.67-3.00). the male-to-female ratioes were 1.94: 1 (3698:1908) in pathogen-positive patients and 1.72: 1 (3663:2130) in pathogen-negative patients (p = 0.002). two hundred forty-eight of 11,399 patients (2.2%) tested positive for hbov1 infection. of the hbov1-positive patients, 112 (45.2%) were co-infected with other pathogens, most frequently with rsv (11.7%, 29/248) ( table 1 ). the median age was 1 year (interquartile range 0.75-1.83). the male-to-female ratio was 2.54:1 (178:70) in hbov1-positive patients and 1.81:1 (7183:3968) in hbov1-negative patients (p = 0.019). to clarify the age distribution of hbov1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. there was a significant difference in the prevalence of hbov1 in patients in different age groups (p < 0.001) and the peak prevalence was found in patients aged 7-12 months (4.7%, 56/1203) (fig. 1) . in this study, we monitored the prevalence of hbov1 in patients (≤14 years old) hospitalized with ari from july we collected meteorological data for guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of hbov1. guangzhou, which is located in southern china (longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′), has a maritime subtropical monsoon climate. between july 2009 and june 2016, the mean temperature was 21.8 ± 5.8°c (mean ± standard deviation), humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hpa and vapor pressure was 21.3 h ± 7.4 hpa. between 2009 and 2016, the mean temperature from may to september was greater than 25°c (fig. 3) . for multiple linear regression analysis of hbov1 prevalence and meteorological conditions correlation, independent variables of mean air pressure (adjusted r 2 = 0.793, p < 0.001) and mean vapor pressure (adjusted r 2 = 0.929, p < 0.001), which linearly associated with mean temperature, and rainfall (adjusted r 2 = 0.278, p < 0.001), which strongly correlated with mean relative humidity, were excluded. the independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. the effect of temperature had a delay therefore mean temperature in the preceding month (mean temperature 1 month before) was also included as an independent variable in the analysis ( table 2) . both regression models were established (p < 0.001) and the adjusted r 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. hbov1 prevalence was positively correlated with temperature (coefficient = 0.259 in the current temperature model (p = 0.002), coefficient = 0.328 in mean temperature in the preceding month model (p < 0.001)). conversely, hbov1 prevalence was negatively correlated with relative humidity (coefficient = − 0.126 in the current temperature model (p = 0.024), coefficient = − 0.083 in the temperature delay model (p = 0.039)) ( table 2 ). ari is one of the most common human diseases, predominantly caused by different respiratory viruses [41, 42] . one of these viruses, hbov1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas [3] [4] [5] [6] [7] 43] . in general, the prevalence of viruses varies because of factors such as multiple linear regression analysis was performed using hbov1 monthly prevalence as the dependent variable, monthly mean temperature (or mean temperature in the preceding month), mean relative humidity, mean wind speed and sunshine duration as the independent variables data captured in bold are highly significant geographical location, climatic conditions, population and social activity [38] . epidemiology of hbov1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring [15, 16, 39, 44] . to describe the epidemiology of hbov1 in guangzhou, we collected throat swabs from 11,399 children (≤14 years old), hospitalized with ari and monitored hbov1 and other common respiratory pathogens over a 7-year period (table 1 ). in the current study, 86.5% (9857/11399) of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ari, consistent with previous reports [45, 46] . overall, 49.2% (5606/11399) of patients tested positive for one or more respiratory pathogens, 2.2% (248/11399) of patients were tested with hbov1 infection (table 1) . a higher prevalence of hbov1 was detected in male patients compared with female patients (p = 0.019), consistent with previous reports [15, 16, 39, 44] . co-infection with hbov1 and other pathogens is common [14, 15] . in our study, 45.2% (112/248) of hbov1-positive patients also tested positive for other pathogens (table 1 ). this may be partly caused by coinciding epidemics of hbov1 and other pathogens. in our study, the hbov1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference (fig. 2) . current research shows that hbov1 infection can lead to the collapse of the first line of defense of airway epithelium [35] [36] [37] , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. the characteristics of the hbov1 infection are likely to be a good model for studying the effects of co-infections. in this study, there was a significant difference in prevalence of hbov1 in patients of different ages (p < 0.001). the majority of hbov1 infections occurred in patients under 2 years old and the peak frequency of hbov1 infection occurred in patients aged 7-12 months (fig. 1) , consistent with previous serological and epidemiological reports on the virus [8-11, 15, 16, 39, 44] . this might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. the distribution of hbov1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. many factors affect the epidemiology of pathogens, such as geographical location and local climate. guangzhou, a central city and main transport hub in southern china, is located in a subtropical region. guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period (fig. 3) . in this study, two hbov1 peaks were observed (fig. 2) . the large prevalence peaks of hbov1 infection occurred between june and september of each year, which are the summer months in guangzhou, with mean temperatures of higher than 25°c (fig. 3) . small peaks of hbov1 infection occurred in winter, between november and december of each year. this seasonal distribution is similar to the prevalence in subtropical regions reported previously [47] , but different from the hbov1 epidemics in temperate regions, which mostly occur in winter and spring [15, 16, 39, 44] , as well as from tropical regions, such as india, where no obvious epidemic season has been found [48] . to analyze the correlation between hbov1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with hbov1 monthly prevalence as the dependent variable and mean temperature (or mean temperature in the preceding month), mean relative humidity, mean wind speed and sunshine duration as the independent variables (table 2) . both regression models were established (p < 0.001) and the adjusted r 2 value (0.373) of the temperature dorp 1 month model was greater than the adjusted r 2 value (0.231) of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. both of the models showed that the prevalence of hbov1 was significantly correlated with temperature and relative humidity ( table 2 ). in detail, hbov1 prevalence was positively correlated with temperature, that is consistent with previous reports [47, 49] . conversely, hbov1 prevalence was negatively correlated with relative humidity, this was different from a previous report in suzhou [47] , which may be related to guangzhou high humidity (mean monthly relative humidity was 77.2 ± 7.3%) (fig. 3) . it is common for pathogen prevalence to fluctuate over time because of a variety factors. in this study, hbov1 prevalence was relatively low in 2013 to 2014. it might be partly related to the relatively higher mean relative humidity during this period (fig. 3) . climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between hbov1 infection and wind speed or sunshine duration were found in this study (p > 0.05) ( table 2) . some limitations of this study should be noted. first, because our study mainly focused on hbov1 circulation in hospitalized patients with ari, hbov1 in outpatients and the asymptomatic population were not included. second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. third, the study was only conducted in three hospitals and may not be representative of the overall population. our study has provided a better understanding of the epidemiology of hbov1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of hbov1 infections. cloning of a human parvovirus by molecular screening of respiratory tract samples human parvoviruses virological and clinical characterizations of respiratory infections in hospitalized children detection of human bocavirus type 1 infection in panamanian children with respiratory illness detection of human bocavirus in nasopharyngeal aspirates versus in broncho-alveolar lavage fluids in children with lower respiratory tract infections human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection detection of bocavirus in saliva of children with and without respiratory illness b-cell responses to human bocaviruses 1-4: new insights from a childhood follow-up study seroepidemiology of human bocavirus defined using recombinant viruslike particles seroepidemiology of human bocaviruses 1-4 the genomic and seroprevalence of human bocavirus in healthy chinese plasma donors and plasma derivatives human bocavirus: current knowledge and future challenges spontaneous pneumomediastinum as a complication in human bocavirus infection human bocavirus-the first 5 years detection of human bocavirus from children and adults with acute respiratory tract illness in guangzhou, southern china comorbidity and high viral load linked to clinical presentation of respiratory human bocavirus infection epidemic and molecular evolution of human bocavirus in hospitalized children 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response, and co-infection with other respiratory viruses in children with acute respiratory infection human bocavirus in patients with encephalitis identification of human bocaviruses in the cerebrospinal fluid of children hospitalized with encephalitis in china detection of human bocavirus in the cerebrospinal fluid of children with encephalitis detection and quantification of human bocavirus in river water mixed viral infections causing acute gastroenteritis in children in a waterborne outbreak frequent detection of highly diverse variants of cardiovirus, cosavirus, bocavirus, and circovirus in sewage samples collected in the united states in vitro modeling of human bocavirus 1 infection of polarized primary human airway epithelia establishment of a reverse genetics system for studying human bocavirus in human airway epithelia human bocavirus 1 infects commercially available primary human airway epithelium cultures productively are meteorological parameters associated with acute respiratory tract infections? epidemiology of acute respiratory infections in children in guangzhou: a three-year study epidemiology and clinical presentation of the four human parainfluenza virus types global burden of childhood pneumonia and diarrhoea viral etiology of hospitalized acute lower respiratory infections in children under 5 years of age -a systematic review and meta-analysis human bocavirus capsid messenger rna detection in children with pneumonia human bocavirus-1 primary infection and shedding in infants respiratory virus surveillance and outbreak investigation respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology clinical and epidemiological profiles of lower respiratory tract infection in hospitalized children due to human bocavirus in a subtropical area of china human bocavirus infection in children with acute respiratory tract infection in india clinical and epidemiologic profile of lower respiratory tract infections associated with human bocavirus we thank the study volunteers for their generous participation. we thank yinghua zhou, haiping huang, jing zhang and jing ma for their technical assistance. . the sponsors of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. key: cord-324398-68je1l3o authors: kashiwazaki, hiromi; nomura, risa; matsuyama, shutoku; taguchi, fumihiro; watanabe, rihito title: spongiform degeneration induced by neuropathogenic murine coronavirus infection date: 2011-01-23 journal: pathol int doi: 10.1111/j.1440-1827.2010.02639.x sha: doc_id: 324398 cord_uid: 68je1l3o soluble receptor‐resistant mutant 7 (ssr7) is isolated from a highly neurovirulent mouse hepatitis virus (mhv) jhmv cl‐2 strain (cl‐2). srr7 exhibits lower virulence than its maternal strain in infected mice, which is typically manifested in a longer lifespan. in this study, during the course of infection with srr7, small spongiotic lesions became apparent at 2 days post‐inoculation (pi), they spread out to form spongiform encephalopathy by 8 to 10 days pi. we recently reported that the initial expressions of viral antigens in the brain are detected in the infiltrating monocyte lineage and in ependymal cells. here, we demonstrate that the next viral spread was observed in glial fibrillary acidic protein‐positive cells or nestin‐positive progenitor cells which take up positions in the subventricular zone (svz). from this restricted site of infection in the svz, a large area of gliosis extended deep into the brain parenchyma where no viral antigens were detected but vacuolar degeneration started at 48 h pi of the virus. the extremely short incubation period compared with other experimental models of infectious spongiform degeneration in the brain would provide a superior experimental model to investigate the mechanism of spongiotic lesions formation. transmissible spongiform encephalopathies (tse) are characterized by neuronal loss, vacuolation, and, in most cases, accumulation in the central nervous system of causative agents such as abnormal prion protein (prp sc ). 1 however, in sheep scrapie, there is no significant neuronal loss and relationships between different magnitudes, topographical and cytological forms of prp sc accumulation, and clinical signs are not evident. 2 furthermore, it has long been recognized that vacuolation is not always a prominent feature of the pathology of scrapie-affected sheep and mice with neurological disturbances. 3 therefore, questions still remain to be answered, especially regarding the mechanisms and roles of spongiform degeneration in tse, which show variable neurological and neuropathological features depending upon infectious agents and infected species. 4 the common neuropathology observed in and around the spongiotic area is astroglial and microglial cell activation after infection with prp sc , 1, 5 or with other infectious agents such as neuropathogenic murine retroviruses. 6, 7 the scrapie-infected cell homogenate triggers the recruitment of microglial cells by interacting with both neurons and astrocytes through upregulation of the expression levels of a broad spectrum of neuronal and glial chemokines very soon after the infection, before amyloid aggregates of the pathological prion protein become apparent. 5 a8 virus, a neuropathogenic murine retrovirus strain isolated from friend leukemia virus, 8, 9 causes spongiosis accompanied by microgliosis in the rat brain after infection at birth. 7 the viral antigens are mainly detected in the blood vessel walls of infected brains, 10, 11 or in the endothelial cells of brain tissue culture. 12 the viral antigens on blood vessel walls are distributed throughout the whole brain and spinal cord of infected rats and are not correlated with the distribution of spongiotic areas. a closer association of the lesions with viral antigen expression has been observed in the infected and activated microglia. 6 in this report, we describe a neuropathogenic strain of mouse hepatitis virus (mhv)-induced spongiform encephalopathy after the infection of mice. we recloned the srr7 (h2) virus (see materials and methods) from viral stock prepared as a soluble receptor-resistant (srr) mutant, clone srr7, 13 isolated from a highly neuropathogenic mhv jhm strain, cl-2. 14,15 srr7 (h2) induced spongiosis within a short incubation period, at 48 h post-inoculation (pi). furthermore, the viral antigens, which are detectable during the early phase of infection (between 12 and 24 h pi) in the inflammatory cells, appeared in meninges and ependymal cells without spreading into the brain parenchyma including the virus injection sites. 16 they were detected in the nestin-positive immature cells beneath the ependymal cells at 48 h pi when spongiosis became apparent, in spongiotic lesions no viral antigens were detectable. in addition, in the subventricular zone (svz) beneath the ependymal cells, glial fibrillary acidic protein (gfap)-positive astrocytes projecting cell processes to the ependymal cell layer were found to be infected. in the developing mouse brain, the migrating neuroblasts (type a cells) advance as chains through tubes defined by the processes of slowly proliferating svz astrocytes, 17 commonly referred to as type b cells, which develop as the primary neuronal precursors in the svz of adult mice, 18 besides contributing to the niche environment together with macrophages and endothelial cells. 19 the neuronal stem progenitor cells in developing brains are targets of infection for viruses such as cytomegalovirus (cmv), 20 coxsackievirus, 21 and simian immunodeficiency virus. 22 in adult brains, neural precursor cells in the ependymal wall are susceptible to murine cmv infection. 20 however, none of the viruses induce spongiotic lesions. possible mechanisms of spongiogenesis after infection with srr7 to neural precursor cells in the ependymal wall are discussed. specific pathogen-free inbred balb/c mice purchased from charles river (tokyo, japan) were maintained according to the guidelines set by the ethics committee our university. at 5 weeks old, each mouse was injected with 1x10 2 of the srr7 virus into the right frontal lobe under deep anesthesia. since srr7 is isolated from the cl-2 virus, 13 the virus has long been maintained by several passages on dbt cells. 23 in order to eradicate the possible contamination of a mutant virus emerging during long and repeated passages, the virus stock was recloned by limiting dilution. the virus was used to infect dbt cells in the wells of 96-well tissue culture plates at a concentration of 0.1-0.01 plaque-forming units (pfu) per well. at 24 h after infection, the culture supernatant in wells in which a single and distinct plaque was observed was transferred to naïve dbt cell culture medium in 24-well plates. after three passages to obtain an adequate amount of the cloned viral stock for further experiments, the s proteincoding region of each clone was sequenced after amplification of the s gene employing a reverse transcription polymerase-chain reaction (rt-pcr), as described previously. 24 a clone, designated as srr7 (h2), was selected for further experiments because it gave rise to a high titer viral production in dbt cells without showing changes in the amino acid sequence in the region of the s protein, although point mutations of the coding region for the s protein were detected at positions 528 (a to c) and 1578 (t to c) as silent mutations, without resulting amino acid substitutions. the stability of the recloned virus regarding virulence and pathogenicity was tested and compared with those obtained from 15 and 21 mice infected in 2007 and 2009, respectively. dbt cells were grown in dulbecco's modified minimal essential medium (dmem; nissui, tokyo, japan) supplemented with 5% fetal bovine serum (fbs; japan bioserum, hiroshima, japan) and cultured at 37°c with humidity in 5% co2, as described previously. 25 after exsanguination of the infected animals under deep anesthesia, removed parts of the brain were frozen for viral titration and the remaining portions were fixed in 4% paraformaldehyde buffered with 0.12 m phosphate (pfa) to obtain paraffin-embedded sections for histological staining with he or he and luxol fast blue, and for enzyme immunohistochemistry. 7 viral antigens were visualized using the rabbit polyclonal antibody, sp-1, or mouse monoclonal antibody (mab). 25, 26 rat antimouse f4/80 either unlabeled or biotin-conjugated (serotec, oxford, uk and ebioscience, san diego, ca, usa, respectively), rat antimouse cd11b biotin-conjugated (bd pharmingen, san diego, ca, usa), rabbit or mouse anti-gfap (santa cruz biotechnology inc., santa cruz, ca, usa and sigma, tokyo, japan, respectively) were used for cell identification. as a second or third application, biotinylated donkey antirabbit igg (amersham, tokyo, japan), goat antimouse igg (cappel, solon, oh, usa), biotin-conjugated donkey antimouse igg (rockland, gilbertsville, pa, usa), rabbit peroxidase antiperoxidase complexes (cappel,), or avidin-peroxidase conjugate (molecular probe, eugene, or, usa) was used, as described previously. 16 after deparaffinization, sections were incubated with 50% normal horse or 50% normal mixed serum (fetal calf, calf, pig, and horse) diluted in phosphate buffered saline (pbs) prior to the first antibody application to block non-specific antibody binding. the non-specific activity of endogenous peroxidase was blocked after primary antibody incubation by incubating sections with 0.3% h2o2 in methanol. washes in pbs were carried out between each step. for the peroxidase reaction, 0.2 mg/ml 3,3′-diaminobenzidine tetrahydrochloride (dab) (wako, osaka, japan) in 0.1 m tris buffer (ph 7.6) was used to obtain brown staining. for double staining, horseradish peroxidase localization was revealed using 4-chloro-1naphthol (wako) substrate, resulting in purple staining. the degree of spongiform neurodegeneration was scored as follows: 6 0, no lesions; 1, less than 20 vacuoles in the total area; 2, 20 to 100 vacuoles counted in the light microscopic field at 10 ¥ magnification (field (x10)); 3, clusters consisting of over 100 vacuoles spread within one field (x10); 4, more than two clusters consisting of over 100 vacuoles in the area, or clusters of vacuoles occupying over 30% of the total area. intermediate scoring between each of the five established scores was permitted by adding a value of 0.5 to the lower score. to score the cns pathology, five areas were selected: the frontal hemisphere, thalamus, cerebellum, pons, and spinal cord. at desired time points after infection, mice were deeply anesthetized, followed by a two-stage perfusion. mice were first perfused with 1 ml of pbs followed by 10 ml of paraformaldehyde (4% paraformaldehyde in phosphate buffer (ph 7.2)) from the aorta via the left ventricle. brains were fixed in 4% pfa overnight, followed by immersion in pbs for 1 h on a rotator (iuchi seieido inc., osaka, japan). the tissues were then rinsed stepwisely in pbs or sterilized water supplemented with increasing concentrations of sucrose (wako), followed by a final rinse with 25% sucrose. all steps were performed at 4°c in a rotator (taab laboratories, reading, uk). tissues were isolated and embedded in oct compound (sakura, tokyo, japan). tissue blocks were frozen in dry ice. sections of 10 mm were cut using a cryostat (sakura), air dried, and stored at -80°c until stained. fluorescence double immunostaining was performed using combinations of rabbit anti-nestin antibody (santa cruz biotechnology, inc.) with mouse monoclonal antibody h-4. cryosections were treated with pbs containing 0.05% tween 20 (sigma), 1% bovine serum albumin (bsa) (sigma), 0.1% sodium azide, goat antimouse igg (cappel), and 5% horse serum for blocking. after incubation in a combination of primary antibodies for 1 h at room temperature, the sections were thoroughly washed with pbs supplemented with 0.1% bsa and 0.05% tween 20. they were then incubated with fitc-antirabbit igg antibody (abcam, tokyo, japan) or biotin-antimouse igg antibody (rockland) and avidin-alexa fluor 568 (molecular probe) for 30 min at room temperature. stained sections were mounted with gold antifade reagent (invitrogen, carlsbad, ca, usa) and examined using a fluorescent microscope (keyence, osaka, japan). when we published our first report on the neuropathology induced by cl-2 and srr7, 25 we were already aware that spongiotic lesions were induced by srr7 infection in mice. however, we did not describe this neuropathology because the occurrence of the change was not constant (data not shown) when we used viruses derived from the original stock 13 and maintained in our laboratory through passages in dbt cells. the highly mutative nature of mhv 27 causes a tendency in mutant viruses to revert to the original neurovirulent phenotype. 28 srr7, a mutant isolate from cl-2 as a ssr strain, has, to some extent, been showing slightly higher neurovirulence, like its maternal strain (data not shown), whereas averaged neurovirulences and neuropathological characteristics have shown no significant changes. therefore, we started recloning the virus strain and obtained srr7 (h2), which had the same amino acid sequence as the original isolate 13 in the region of the viral surface protein (s protein; see materials and methods). although there could be mutations in the other coding or non-coding regions, which might influence the neurovirulence or host reactions, [29] [30] [31] after examination of the viral genome sequence coding s protein, we decided to use srr7 (h2) to study spongiosis induced by viral infection because srr7 differs from cl-2 only in one amino acid sequence in the s protein-coding region, 23 and, more importantly, the virus exhibited the same induction rate of spongiosis with a similar intensity between the experiments performed in 2007 and 2009 (fig. 1) . after the recloning of srr7 in 2007, we performed our experiments using viruses in five passages after the initial cloning. incidences of spongiotic degeneration and virulence in mice infected with srr7 were compared from the experiments carried out in 2007 and 2009 (fig. 1) , because we considered the virus to be highly mutative (see discussion). all of the 15 and 21 mice infected in 2007 and 2009, respectively, survived for the initial 3 days and died within 12 days pi (data not shown). furthermore, in all of the neuropathologically examined animals, large spongiotic lesions composed of various sizes of vacuole became apparent 4-5 days after viral inoculation mainly in the pons, cerebellum, and thalamus (figs 1,2) , the lesions were less extensive in the frontal area (fig. 1 ) among the animals examined and compared in 2007 and 2009. when spongiosis occupied an extended area (fig. 2h ) scored as grade 4 (see materials and methods), we designated the spongiotic lesion as extensive spongiosis (exspongi) to distinguish this from less extensive spongiotic lesions scored grade 1 or 2, and named vacuolar lesions (vaclesions). in vaclesions each vacuole looked smaller and some vacuoles showed back-to-back attachment forming bunches (fig. 2b,j) , each of which consisted of less than 100 vacuoles. the exspongi exhibited several neuropathological characteristics. first, the lesion was fairly well-demarcated (fig. 2h) . second, the lesions were accompanied by few or no inflammatory reactions. this phenomenon occurs rather rarely in viral infections with apparent destructive lesions. at a lower magnification of exspongi, many small nuclei were observed (fig. 2h) , many of which were pyknotic and devoid of cytoplasm at a higher magnification (fig. 2i) . we were unable to characterize the remainder, except for a few macrophages present in the lesions (black arrows in fig. 2i ). around the blood vessels in and around the exspongi, almost no inflammatory cell exudation was observed either at the capillary level or at a larger size (thin white arrows in fig. 2h-j) , including in the area with myelinated fibers (fig. 2j) . the third neuropathological characteristic of exspongi was that neurons looking viable were found in the lesion (white arrowheads in fig. 2h,i) , where vacuoles attached to the neurons directly. the neurons were also well-preserved in vaclesions around the exspongi (fig. 2h,j) . the close association of small vacuoles with thick eosinophilic walls and the neurons (white arrowheads in fig. 2i ) resembles the vacuoles formed in murine retroviral infection, 9 in which the vacuoles are shown as enlarged presynaptic bulbs. 8 the viral antigens were more strongly expressed around the exspongi than inside (fig. 2 m,n) . although the initial viral antigen expression was detected in the inflammatory cells in the meninges as early as 12 h pi, followed by infection to the choroid plexus and ependymal cells, as reported previously 16 and shown in fig. 2a ,c, they were eliminated from ependymal cells and the svz in the latter phase of infection (figs 2 m,3 ). in addition, facing the ventricles a repaired line of ependymal cells with flattened cytoplasm and few cilia was observed (data not shown). another trace of initial events could be detected as the pronounced activation of gfap-positive glial cells in and around the svz. the gliosis was found to extend deep into the brain parenchyma and again in the middle of the exspongi (black arrowheads in fig. 2k ) gfap-antigen expression was less extensive than that seen in the surrounding vaclesions. even in this area with mild gliosis, astroglial foot processes around the blood vessels were an outstanding feature (fig. 2l) , indicating that a key component of the blood brain barrier had been well-preserved in the exspongi during the course of the disease, which conspicuously contrasts with pathologies involving an inflammatory perivascular cuff induced by infection with other strains of jhm virus, where there is a disappearance of astrocytes around blood vessels with inflammation followed by degeneration of the astrocytes with swollen cytoplasm around the blood vessels in the initial phase of cell infiltration. 32 to study vacuolar formation during the early phase of infection, we investigated infected mice at 48 h pi, because the viral antigens are distributed only in the restricted area 16 at this time while vaclesions become apparent (fig. 2b,g) . vacuolar degeneration was already detectable before 48 h pi, but the number of vacuoles and magnitude were so low that it was sometimes difficult to distinguish from an artifact produced during the preparation of sections (data not shown). in fig. 2a (the dotted area with the letter b and arrow) a distinct vaclesion developed (figs 2b,3) , but viral antigens were not detectable in this area or in the area between the boxed area and ependymal cells which expressed the viral antigens. occasionally viral antigenbearing cells in the svz were observed extending their cytoplasm to the ventricle (black arrow in fig. 2c) . we considered these to be b cells, gfap-positive precursor cells resident in the svz, so we carried out double staining of serial sections next to the stained section (fig. 2c) . as shown in fig. 2e , a gfap-positive cell projecting its cytoplasm to the ventricular surface was infected. nestin-positive precursor cells in the svz were also infected (fig. 2o) . astroglial activations were most prominent in the area near the very restricted site of infection around the ventricle, already during this early stage of infection gliosis was found extending deep into the brain parenchyma, like in the later stage but in a less extensive manner (fig. 2d) . interestingly, in the cerebellum, the area of gliosis abruptly ended at the border of the granular layer (indicated by black arrowheads in figures 2d and f) . the vaclesion also finished there. at a lower magnification, the activated astrocytes looked scattered and unrelated to the vacuoles observed in the region of gliosis (fig. 2f) , but a higher magnification revealed fine fibrous or dot-like structures exhibiting gfap-antigens facing the vacuolar walls (fig. 2g) . the distribution of spongiotic lesions showed a predilection for the brainstem and cerebellum (figs 1,2 ) without forming spongiosis in the svz (figs 2,3) , although our previous report demonstrated that the viral antigens in the brain appear in the choroid plexus and svz, including ependymal cells, during the early phase of infection after the initial emergence in infiltrating cells of meninges at 12 h pi, without spreading into the brain parenchyma including the site of injection. 16 in addition, the frontal area where the viruses were inoculated contained less extensive lesions compared to the brainstem (fig. 1 ) and the brain cortex facing the subarachnoid space where infected inflammatory cells are abundant is not the initial site of invasion of srr7 into the brain parenchyma. 16 furthermore, the brain cortex including the frontal area was not the main area of lesions induced by infection of srr7 during the later phase. 16, 25 it is indicated that the viral spread into the brain starts from the junctional area between the arachnoid and choroid plexus where viral antigens are detected during the initial phase of infection. 16 therefore, our studies on the relationship between the viral antigen distribution and formation of vacuolar degeneration have mainly focused on the periventricular and surrounding areas in the brainstem. surprisingly, at 48 h pi, we found large vaclesions where viral antigens were not detected ( fig. 2b-g) . on the same section as the vaclesions were detected and on successive serial sections, viral antigens were found only in restricted areas, as in the svz, where gfap-positive cells with a peculiar shape, projecting narrow cytoplasm to the ventricular surface and leaving their perikaryon beneath the surface-lining ependymal cells, were found to have been infected (fig. 2c,e) . the shape and localization of these astrocytes corresponded to b 18 or b1 19 cells, which serve as both stem and niche cells in the svz and play critical roles in adult neurogenesis. b cells bear gfap and have a long basal process that terminates on blood vessels and an apical ending at the ventricle surface, with long cilia projecting into the ventricle, 18 which can be a target of infection via infected monocytes that have infiltrated the ventricle during the initial phase of infection. 16 it is not plausible for the infected gfap-positive cells in the svz to have a direct effect on spongiogenesis, because the vaclesion is located a long way from the svz. instead, these cells might have triggered a chain reaction of astrogliosis which spread from the svz reaching the deep white matter of the cerebellum (fig. 2d,f) through the pathway of neurogenesis or the blood supply, given this area of gliosis ended abruptly at the edge of the granular layer (fig. 2d,f) . it is presumed that b cell-originating migratory cells ascend in close contact with the basal lamina along with blood vessels in adulthood. 19 the cerebellar cortex receives its blood supply from the meninges that cover the cerebellum, in a different way from the cerebellar white matter. in addition, the granular layer is formed from granular neurons that have migrated from the external granular layer during embryonic and postnatal stages in mice. 33 those astrocytes activated in a large area after being triggered by infection of the svz must have communicated with each other, leading to vacuole formation away from the infection sites. this cell-to-cell communication can be facilitated by direct contact between cells through an elongated cytoplasmic projection, as reported for the immunological synapse between leukocytes, especially between dendritic cells and lymphocytes. 34 fine projections of astrocytes are detected in vaclesions, where, at a higher magnification, gfap-positive foot processes were shown to be closely attached to the vacuolar walls (fig. 2g) . alternatively, signals can be spread by means of cytokine production, which is observed in all kinds of cell constituting the cns (central nervous system), including astrocytes and neurons. 35 the contribution of microglia 36, 37 in this context should not be ignored, but the activation of microglia monitored by f4/80 or cd11b expression was not prominent at 48 h pi. 16 furthermore, it has been reported that neurodegenerative progression can be protected by microglia and monocyte lineages 38 which produce neurotrophins. 39, 40 the finding that srr7 infection did not induce the prominent activation of microglia during the early phase of infection is markedly different from the spongiosis induced by infection with the neuropathogenic murine retroviral strain a8-v 8 . the a8-v-induced vacuoles showed close contact with either infected or non-infected microglia, with many activated microglia around the lesions. 6, 7 however, it takes a prolonged incubation period, 4-6 weeks after infection with a8-v, although a much shorter time compared with prion diseases, to observe neuropathologically apparent vaclesions. after such a long incubation period, there are several stages of neuropathological lesions and infectious agents either related or unrelated to the induction of spongiosis observed in the section can be scattered anywhere in the infected brain, leading to controversial findings concerning the correlation between distributions of infectious agents and lesions, typically reported in several prion diseases. 1 in srr7 infection, already at 72 h pi, the widespread distribution of viral antigens is observed, 25 which may disguise lesions produced by the indirect effects of infected cells. with the characteristic neuropathology of spongiosis where inflammatory cell infiltration was suppressed to the minimum level both in the initial phase ( fig. 2a-g) and during the latter phase (fig. 2h-n) like in other spongiotic lesions induced by different kinds of infectious agents, 1,7 srr7infection of mice would provide a superior experimental model to investigate the mechanism of how spongiotic lesions are formed, which remains unclear in spite of the long history of scientific research since infectious spongiotic encephalopathy was first reported 41 because of its extremely short incubation period compared with other experimental models of infectious spongiform degeneration in brains, minimizing many events inevitably induced after infection. cellular pathogenesis in prion diseases classical sheep transmissible spongiform 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protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants characterization of a variant virus selected in rat brains after infection by coronavirus mouse hepatitis virus jhm neurovirulence of six different murine coronavirus jhmv variants for rats cytopathy of an infiltrating monocyte lineage during the early phase of infection with murinecoronavirus in the brain subventricular zone astrocytes are neural stem cells in the adult mammalian brain for the long run: maintaining germinal niches in the adult brain neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain roles of neural stem progenitor cells in cytomegalovirus infection of the brain in mouse models coxsackievirus targets proliferating neuronal progenitor cells in the neonatal cns simian fetal brain progenitor cells for studying viral neuropathogenesis soluble receptor potentiates 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residues in the putative viral cyclic phosphodiesterase ns2 comparative analysis of coronavirus jhm-induced demyelinating encephalomyelitis in lewis and brown norway rats the cells and molecules that make a cerebellum correlation of a dynamic model for immunological synapse formation with effector functions: two pathways to synapse formation distribution and phenotype of dendritic cells and resident tissue macrophages in the dura mater, leptomeninges, and choroid plexus of the rat brain as demonstrated in wholemount preparations microglial activation in chronic neurodegenerative diseases: roles of apoptotic neurons and chronic stimulation expression of proinflammatory cytokines and its relationship with virus infection in the brain of macaques inoculated with macrophage-tropic simian immunodeficiency virus microglia activated with the toll-like receptor 9 ligand cpg attenuate oligomeric amyloid beta neurotoxicity in in vitro and in vivo models of alzheimer's disease neurotrophin secretion from cultured microglia autocrine activation of cultured macrophages by brainderived neurotrophic factor degenerative disease of the central nervous system in new guinea; the endemic occurrence of kuru in the native population this work was financially supported in part by grants from the ministry of education, culture, sports, science and technology. key: cord-304720-0lgup7yj authors: robbins, r.c.; almond, g.; byers, e. title: swine diseases and disorders date: 2014-08-21 journal: encyclopedia of agriculture and food systems doi: 10.1016/b978-0-444-52512-3.00134-0 sha: doc_id: 304720 cord_uid: 0lgup7yj swine diseases and disorders that are significant in modern, commercial swine production systems are organized by body system; the reader will need to know basic anatomy and physiology. the industry significance, etiology, epidemiology, pathogenesis, clinical signs, postmortem and histpathologic lesions, diagnostic testing, and generic treatment, control, and prevention are described. diseases of a particular system are summarized in a differential diagnosis table. r 2014 elsevier inc. all rights reserved. autogenous vaccines vaccine made from microorganisms isolated from the animal it will be used on; in the united states these are killed and, by permit, are extended for use in a farm, production system, or region. farrow process of parturition; location where a pig is born and stays till weaning, usually 3-4 weeks of age. grow-finish phase of production that follows the nursery period where pigs reach slaughter weight; used to describe pigs from 10 to 30 weeks of age. histopathology; -ic the science and microscopic examination of formalin-fixed and paraffin-embedded sections of diseased tissues. lesion visible (microscopic or macroscopic) deviation from normal. national animal health monitoring service a program administered by united states department of agriculture-animal and plant health inspection service. nursery phase of production that begins after weaning, used to transition pigs from the farrowing house to finishing; used to describe pigs from 3 to 10 weeks of age. oie world organization for animal health created on 25 january 1924. pathognomonic characteristic of a specific disease or disorder that, when present, is sufficient to make a diagnosis. veterinary diagnostic laboratory location where samples are submitted and tests are run to determine the cause of disease. in an approach to investigating any suspected disease or disorder in swine production, a history should be gathered first. important history to understand from caretakers includes: age of pigs affected, duration of clinical signs, morbidity rate, mortality rate, treatments administered, response to treatments, and any other important information regarding previous diagnoses or disease in the affected group of animals. this is also the time to examine any production records that have been kept on the affected group of swine as well as previous groups for comparison. records include but are not limited to: where the animals originated from; number in the herd; age; daily mortality; number treated; name of treatment, route of delivery and dose; feed and water usage; high-low temperatures; and vaccinations received or administered. after examining the production records and obtaining a history, proceed with a visual examination of the herd. typically, it is a biosecurity custom to observe youngest groups first; however, in cases of suspected infectious diseases, it may be best to begin with the healthiest group advancing in order of increasing severity or prevalence. often, a definitive diagnosis is not achieved without an extensive clinical and pathological investigation. a postmortem examination, or necropsy, of affected pigs should occur last. any pigs recently deceased of natural causes should be examined to establish trends, with the understanding that submission of tissues from these animals may not yield valuable diagnostic results. tissues for diagnostic evaluation should be collected from clinically affected pigs that are euthanized immediately before necropsy. sampling of five or more pigs may be required to obtain a valuable diagnosis. when investigating signs referable to the central nervous system (cns), it is important to preserve brain and spinal cord tissue for microscopic evaluation in cases of neurological disease; therefore, blunt force trauma and brain penetration by captive bolt are not preferred methods of euthanasia. at minimum, fresh and formalin-fixed tissue samples should include: brain, tonsil, heart, lung, lymph nodes, spleen, kidney, liver, and intestine. additional samples that may be beneficial for diagnosis include: premortem whole blood and ethylenediaminetetraacetic acid-chelated blood (for serum chemistry and complete blood count), spinal cord, intact stifle and hock joints (remove the leg at the hip), intact eyeball with optic nerve attachment, urine, feed, and water. consult a diagnostic lab regarding any additional samples that may be required in determining an etiologic diagnosis. the etiologic diagnosis should be based on consistent history, signs, and pathology derived from a list of differential diagnoses that are most common or most likely to occur in that herd or production system. a treatment, control, or prevention program should be formulated simultaneously. before using any chemical, pharmaceutical, or biologic in swine intended for food, know the domestic use guidelines, importer requirements or producer-packer agreements regarding withdrawal times, residue and tolerance limits, prescribing guidelines, and prohibited substances. this section will focus on a practical approach to investigating signs of neurological disease in swine summarized in table 1 . it is important to determine if clinical signs are consistent with cns or peripheral nervous system lesions (pns). common cns signs in pigs include behavioral abnormalities (most commonly stupor), ataxia, loss of righting, seizures or seizure-like activity (paddling), nystagmus, and blindness. musculoskeletal disorders may clinically confuse or complicate perceived pns signs and must be differentiated from each other. streptococcus suis is a gram-positive cocci with 35 reported serotypes. observational studies implicate sows as carriers and piglets are colonized as they pass through the birth canal (amass et al., 1996) . disease occurs most frequently during the suckling and postweaning period. commingling pigs from different herds, concurrent infection with porcine reproductive and respiratory syndrome (prrs), and other stress factors may increase the risk of developing s. suis meningitis (villani, 2002; thanawongnuwech et al., 2000) . variable morbidity and mortality: mortality depends on early recognition and treatment. clinical signs of s. suis meningitis include paddling, recumbency, nystagmus, and seizure. isolation of s. suis from the lung, nasal secretions, or tonsil from normal pigs is clinically insignificant. in contrast, s. suis isolation from cerebrospinal fluid (csf), meninges, joints, endocardium, or serosal surfaces with or without lesions is relevant (pijoan, 1994) . few to no gross lesions may be observed during necropsy. early recognition of clinical signs followed by injection with an antimicrobial that s. suis is susceptible is the most effective means of treatment. administering an antimicrobial that s. suis is susceptible to in the drinking water has been proposed to control morbidity (villani, 2002) . antimicrobial susceptibility patterns for s. suis isolates from regional diagnostic laboratories can be used to assist in selection of an appropriate antimicrobial while diagnostic tests are pending; ceftiofur is effective . commercial and autogenous vaccines are available but due to s. suis serologic diversity may not be effective . haemophilus parasuis (hps), also called glässer's disease, causes bacterial meningitis, arthritis, and polyserositis similar to s. suis. infections are not clinically or grossly distinguishable from s. suis. definitive diagnosis is by bacterial isolation. however, hps is a fastidious gram-negative rod and culture media must be supplemented with v factor for successful isolation. owing to the difficulty in isolating hps, polymerase chain reaction (pcr) tests are a suitable alternative (oliveira et al., 2001) . like s. suis, isolation from the airways has little significance unless lesions are present (hoefling, 1994) . antimicrobial susceptibility testing identifies ceftiofur or florfenicol that are typically effective first choice therapeutics (oliveira, 2007b) . prevention may be achieved with medicated early weaning. edema disease results when a fimbrial (f18 or f4) and shiga-like toxin (stx-2e) positive strain of escherichia coli successfully attaches to brush border receptors releasing toxin that damages blood vessels including those of the blood-brain barrier causing edema and encephalomalacia. edema disease most commonly affects rapidly growing pigs, 2 weeks postweaning. morbidity is moderate to high and mortality is high. acute death of robust pigs, ataxia, eyelid swelling, and diarrhea are typical clinical signs (rademacher, 2001) . at necropsy, edema may be observed in the mesentery between the loops of the spiral colon and in the cardiac region of the gastric mucosa. stomachs are usually full of feed. bacteriologic isolation of a β-hemolytic strain of e. coli from affected pigs with meningoencephalitis is not sufficient for a diagnosis. genotyping is necessary to confirm that the e. coli isolated was f18 or f4 and stx-2e positive and thus capable to induce such lesions. there is no effective treatment. vaccination using an avirulent live culture of e. coli postweaning, thorough cleaning and disinfection between groups, and use of genetically resistance breeds that lack the fimbrial receptor are preventatives (fairbrother and gyles, 2006) . pseudorabies (prv), also known as aujezsky's disease, is caused by a herpesvirus. prv was eradicated from the us commercial swine herd in 2004 (usda aphis, 2008) . feral swine are potential reservoirs. cattle, sheep, dogs, and cats can also be infected with prv. high morbidity is due to large quantities of virus shed in saliva and nasal secretions for several weeks following infection. mortality is inversely related to age approaching 100% in neonates. clinical signs are also age dependent. neonates may die without signs. suckling and recently weaned pigs are those that commonly exhibit ataxia, tremors, excess salivation, and seizures. at necropsy, the brain appears congested and hemorrhagic. necrotic foci occur in the spleen, liver, lung, lymph node, and specifically tonsils. histopathologic lesions are characterized by nonsuppurative meningitis and intranuclear inclusion bodies. pcr, virus isolation (vi), immunohistochemistry (ihc), or fluorescent antibody can be used to confirm the diagnosis. no specific treatment is available. vaccination and eradication are effective for control (usda aphis, 2008) . in areas free of prv, suspicion of the disease should be reported to state and federal agencies as required. congenital tremors result when hypomyelination or demyelination of the brain and spinal cord. clinical signs are clonic muscle contractions that cause a general tremor of the entire body. pigs are affected at birth but severity subsides with note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). age (dewey, 2006) . mortality is variable and is the sequela of malnutrition because piglets are unable to nurse. there is no known treatment or prevention. hypoglycemia occurs when piglets fail to nurse the sow. this condition is observed within 24 h afterbirth. there is a low morbidity but high mortality. pigs may appear disoriented, ataxic, recumbent, or dead. on necropsy, affected piglets will have empty stomachs. assigning an employee to attend farrowing to ensure piglets nurse will reduce incidence. water deprivation, also referred to as salt poisoning, is an idiopathic disease resulting from a period of inadequate water intake (carson, 2006) . high morbidity with variable mortality occurs. the disease is suspected when there is a history of power outage or poor management (thacker, 2000) . fighting over water access is the first clinical sign and occurs within hours. dog-sitting, opisthotonous, convulsions, and fighting over water follow and develop after 24 h without water. removal of the brain from an affected animal reveals edema and eosinophilic meningoencephalitis with perivascular cuffing and this is pathognomonic (gudmundson and meagher, 1961) . serum or csf with a sodium level above160 meq l à1 may also be used for supporting evidence (osweiler and hurd, 1974) . treatment of swine showing signs with an anti-inflammatory is variably effective. when water is restored, limit intake to short, 10-15 min intervals until all animals have had a chance to drink and fighting has ceased after which water can be provided ad libitum. prevention is daily observation to ensure each animal can access water, adequate water delivery system, and equipping the facility with a generator or alternative method to deliver water during power outages. gastrointestinal diseases and disorders can occur in all ages of swine as summarized in table 2 . most digestive diseases are referable to the gastrointestinal tract and result in diarrhea and occasional vomiting. diarrhea is the result of an intestinal dysfunction caused by malabsorption, excessive secretion, or effusion. unfortunately, this is not an exclusive characterization of diarrhea and overlap occurs (moeser and blikslager, 2007) . rather, differentials for diarrhea should be referable to age at onset and site of infection. gastric ulcers are noninfectious and result when glandular mucosa specifically the pars esophagea is traumatized by gastric acid. gastric ulcers have a wide variety of causes but are most commonly associated with small feed particle size (ayles et al., 1996) and interruption of feed intake whether caused by disease or poor management. it is common to see signs consistent with gastric ulceration increase following an acute prrs or influenza outbreak. morbidity and mortality vary with the scope of the underlying cause. clinical signs include regurgitation, vomiting, pallor or jaundice, and acute death. an acutely dead pig with blood in its stomach is indicative of an active ulcer and is sufficient evidence for a diagnosis. in chronic cases, ulceration causes hyperplasia resulting in stricture of the pars esophagea and regurgitation. feeding a coarse ground diet for 3 weeks significantly decreases severity (ayles et al., 1996) but is impractical in modern production facilities. rotavirus is a nonenveloped rna virus with a doublelayered capsid allowing it to remain stable and infective in the environment for months and intrinsically resistant to some disinfectants. four serogroups infect swine: a, b, c, and e (the latter only reported from the united kingdom). in addition, infections with particular serogroups vary by age: type c mostly in suckling piglets and type a predominately in nursery pigs (stephenson et al., 2013) . type a is the most prevalent serogroup. severity of disease decreases with age and is self-limiting. the virus infects and destroys villous enterocytes resulting in villous atrophy. in response, crypt cells fill in the gaps but, because they are incapable of absorption, suckling piglets quickly loose body condition and have a gaunt or wasted appearance. neither clinical signs nor gross lesions are pathognomonic although loops of small intestine appear thinwalled with moderate to large amounts of watery contents. a histopathologic report of blunted villi and crypt hyperplasia is suggestive of rotavirus infection. infection with rotavirus can be confirmed by pcr or electron microscopy (em). enzymelinked immunosorbent assay (elisa) is also available but limited to detection of serogroup a. ihc and em detect rotavirus and confirm its role in pathology, but both tests lack table 2 common gastrointestinal diseases and disorders of pigs note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). sensitivity. treatment is supportive by administration of oral rehydration solutions. acidifiers and antibiotics are sometimes administered to control secondary bacterial infections. treatment success is variable and depends on the degree of malnourishment. prevention among neonatal piglets is through ingestion of lactogenic virus neutralizing antibody from the sow, which is stimulated by administering feedback of rotavirus positive piglet feces or intestines (arruda et al., 2011) or a modified-live commercial type a vaccine no less than 3 weeks before farrowing. a modified-live commercial type a vaccine is also available for pigs. it does not induce cross-protection for other serogroups and may be cost-prohibitive. tge is caused by tgev, a coronavirus that is heat labile at temperatures above 21 1c, prone to dessication and photosensitization (bay et al., 1952) . the epidemic form causes acute disease in all age groups within as little as 18 h of infection. morbidity and mortality is high, approaching 100%, in an epizootic outbreak. the severity of disease is age dependent but all ages will develop diarrhea (moeser and blikslager, 2007) . postweaning infections result in high morbidity but low mortality; the most significant economic losses at this time are caused by reduced average daily gain, market weights, and overall system efficiency. necropsy reveals that the small intestine and colon are fluid filled, the small intestinal wall is thin almost translucent, and lacteals are empty. necrosis and atrophy are observed throughout the length of the villus. the colon and cecum are spared. the endemic form occurs when susceptible animals are introduced to the herd or after maternal antibody wanes. prior exposure to porcine respiratory coronavirus (prcv) may cause false-positive antibody test results. a tgev/prcv differential elisa is available. in an outbreak, sows are fed tissue of diarrheic pigs to stimulate herd immunity and new introductions of animals are stopped. after the exposure and a subsequent cool-down period of 4-6 months or after clinical signs cease, sentinels can be introduced and monitored for seroconversion (saif and sestak, 2006) . absence of seroconversion indicates successful elimination of tgev. commercial vaccines are available but should be used with caution and only when elimination is not an option. ped is caused by pedv, a coronavirus that causes signs and histolopathologic lesions indistinguishable from tgev. unlike tgev, pedv is more environmentally resistant making elimination more difficult. the disease has been described in europe, asia, and, as of 2013, the united states. prevalence of the enzootic form is approximately 50% (chae et al., 2000) . morbidity approaches 100% and mortality is 80% or more in a naïve sow herd resulting in 3-5 weeks of production losses. clinical signs appear within 12 h; piglets develop a watery, fetid diarrhea leading to dehydration, metabolic acidosis, and death before caretakers are able to humanely euthanize them. vomiting also occurs. the severity of disease is age dependent but all ages will develop diarrhea. postweaning infections result in a high morbidity but low mortality; most significant economic losses at this time are caused by reduced average daily gain, market weights, and overall system efficiency. viral shedding occurs up to 10 days postinfection. reproductive failure and inefficiency is a sequela of an outbreak (olanratmanee et al., 2010) . tgev/pedv differential pcr is available to confirm a presumptive diagnosis of ped. serum can be submitted for elisa or immunofluorescent antibody but collected no sooner than 3 weeks after diarrhea was o bserved. immunoprophylaxis using egg antibody or hyperimmune serum and supportive care including electrolyte administration have been used for treatment. in an outbreak, sows are fed tissue and feces from diarrheic pigs to stimulate herd immunity (olanratmanee et al., 2010) . hygiene is the key to reducing environmental contamination. preventing introduction of virus into a herd with biosecurity alone may not be sufficient because the virus has been found in aerosol up to 10 miles from a positive farm (goede et al., 2013) . porcine coccidiosis is most often caused by isospora suis. farm hygiene, specifically farrowing rooms, and sow infestation influence the persistence of disease; however, age at infection rather than infectious dose has the greatest impact on severity (worliczek et al., 2009) . the prepatent period is approximately 5 days. morbidity is variable and mortality is low. pasty diarrhea, unthrifty to potbellied appearance of 7-21 day old pigs, and below average wean weight is suspicious for coccidiosis. on necropsy, the small intestine often is thickened and the mucosal surface is necrotic and has an adherent pseudomembrane. histopathologic examination of the affected portion of the intestine reveals larvae in the lamina propria. sensitivity of fecal flotation is moderate. there is no effective treatment. prevention is by oral administration of an anticoccidial (maes et al., 2007) . heat treatment (flaming) of flooring may reduce environmental contamination. concrete, rubber coated and plastic flooring in the farrowing crates are difficult to clean and disinfect so removal may be the only option. swine dysentery (sd) is a spirochete of the genus brachyspira that is an oxygen-tolerant anaerobe giving it the ability to survive for long periods of time in manure, pits, and lagoons (schwartz et al., 2012) . rodents, particularly mice, are known vectors and can serve as reservoirs. brachyspira hyodysenteriae is the species known for causing sd. other species of brachyspira have been recently described in dysentery-like disease (burrough, 2012) . incubation is 10-14 days but disease occurs in a 3-4 week cycle. administration of tiamulin or lincomycin in the feed or water may alter the time to onset of signs after exposure. morbidity is high and mortality is low to moderate characteristically causing disease in only the finisher and mature groups. economic significance is mostly lost performance due to reduced daily gain and feed conversion. the specific mechanism of pathogenesis is not well understood but the spirochete does not invade the lamina propria. clinical signs are the presence of mucohemorrhagic diarrhea containing flakes of frank blood or appearing as a generalized brick red to rust color. lesions are mostly observed in the spiral colon where epithelial sloughing and mucosal invasion cause necrosis resulting in the formation of a pseudomembrane. the colonic walls may be thickened due to vascular congestion and mucosal hyperplasia. bacterial culture produces strong β-hemolysis. pcr test for confirmation and speciation is recommended for any isolate with characteristic growth. introduction of infected pigs and contaminated equipment or facilities are the source of infection. pleuromutilins, like tiamulin, and the lincosamide, lincomycin, are effective for treatment. however, if the environment remains contaminated, clinical signs will recur. depopulation has resulted in successful eradication (harms, 2011) . medicated elimination that combines thorough pulse medication with tiamulin, cleaning and disinfection, and employment of an aggressive rodent control program is also effective (burrough and sexton, 2013) . escherichia coli is a gram-negative rod that infects all ages of swine but must express virulence factors to cause diarrhea. escherichia coli colonize the small intestine by fimbria that binds receptors on the villous surface of enterocytes. enterotoxigenic e. coli (etec) then produce toxin(s) that increase osmolality leading to diarrhea (moeser and blikslager, 2007) . etec is subdivided by fimbria, toxin, and age of pig affected. neonatal diarrhea (nd) is most common in pigs 0-7 days of age. the onset of postweaning diarrhea (pwd) caused by f18 is delayed, occurring 5-14 days postweaning, compared to that caused by f4 and its severity is indirectly related to wean age. clinical signs are profuse diarrhea, rapid dehydration leading to emaciation, or death due to metabolic acidosis. fluid filled and hyperemic sections of jejunum and ileum may be present at necropsy but few consistent gross lesions occur. intestinal contents have a distinctly alkaline ph. isolation of large numbers of e. coli and with dense layers of rod-shaped bacteria covering villi seen on histopathology in samples from pigs with diarrhea is sufficient for diagnosis of e. coli but not etec. genotyping is necessary to determine fimbria and toxin types, which are essential to confirm diagnosis of etec. treatment of affected pigs/litters/groups includes administration of antibiotics and oral rehydration solution or electrolytes to correct hyperkalemia (kiers et al., 2006) . control and prevention of nd is by passively derived lactogenic immunoglobins from vaccinated females (kohler, 1974) . prevention of pwd include selection of genetically resistant breeds lacking k88 and f18 receptors, administration of an oral avirulent live culture to stimulate active immunity or competitively exclude field strains (genovese et al., 2000) , feeding 2500 ppm zinc oxide postweaning and probiotics. immunity and exclusion is unique to each fimbria; vaccines should include the prevalent genotype(s) causing the diarrhea. clostridium perfringens type a (cpa) is a gram-positive bacillus and inefficient sporulator. sows are regarded as the source of neonatal infection. frequency of cpa diarrhea is on the rise in the usa. in uncomplicated cases, mortality is low whereas morbidity is high and below average weaning weights result. cases of cpa diarrhea are associated with the expression of α and β 2 toxin. cpa is cultured from the stomach and upper third of small intestine but does not bind intestinal epithelium causing few to no histologic lesions. because of its ubiquitous nature and prevalence among health pigs, cpa may be an opportunist and its role as a primary cause of neonatal enteritis is not definitive. large numbers (3 þ or 4 þ ) of gram-positive bacilli cultured from feces or intestinal contents of diarrheic pigs is suggestive of cpa. genotyping by pcr to confirm presence of cpb2 gene in cpa isolates and rule out of other causes of nd are supportive to the diagnosis (bueschel et al., 2003 ). treatment has variable success rates and is limited to administration of empirically selected antibiotics and oral rehydration solutions to affected piglets. control of cpa enteritis is best accomplished by preventing other causes of nd. following a thorough cleaning, sporicidal disinfectant should be applied to farrowing crates and equipment between litters and be allowed to dry before reloading. feeding of bacitracin to sows has resulted in significant increases in weaning weights (schultz, 2007) . a commercial cpa toxoid vaccine is available (hammer et al., 2008) . autogenous whole cell vaccines are also in use. if vaccine is unavailable, feedback might be considered but should be pursued with caution (robbins and byers, 2013a) . cpc is a gram-positive bacillus and inefficient sporulator. sows are regarded as the source of infection. pathogenesis of type c is due to expression of β toxin leading to necrosis of intestinal epithelium resulting in hemorrhagic diarrhea or acute death of piglets less than 3 days of age. gross necropsy reveals hemorrhagic and blood-filled loops of small intestine. a pseudomembrane may form on the luminal surface, and intestinal mucosa is edematous. gross and histopathologic lesions in the presence of large numbers (3 þ or 4 þ ) of grampositive bacilli cultured from feces or intestinal contents warrant a presumptive diagnosis. genotyping by pcr to confirm presence of the cpb gene is confirmatory (songer and uzal, 2005) . treatment of affected piglets is unrewarding due to the rapid and debilitating course of this disease. prevention is accomplished by vaccination of gestating females with a commercial toxoid and ensuring piglets consume sufficient colostral antibodies to result in protection. clostridium difficile is a gram-positive bacillus that easily sporulates making it environmentally resistant to many disinfectants. clostridium difficile associated diarrhea leads to a 10-15% reduction in wean weights (songer and uzal, 2005) . although more than a third of piglet diarrhea involves c. difficile, it is the better known to cause healthcare-associated infections among humans. the pathogenesis of c. difficile infections is in response to the expression of toxins a and b. a watery diarrhea occurs in 1-7 day old piglets. mesocolonic edema may be observed at necropsy. clostridium difficile is difficult to culture and can be isolated from healthy piglets. therefore, volcano lesions on histologic exam and confirmation of toxins in fecal contents by antigen elisa are diagnostic. treatment is ill-defined but is likely similar to that for cpa enteritis, because it is likely to be initiated based on clinical signs, which are similar. autogenous vaccines are used to aid in prevention but efficacy is unclear. ppe, commonly referred to as ileitis, is the general categorization of infections caused by lawsonia intracellularis, an obligate intracellular bacterium. because the bacteria cause lesions in the ileum, ppe is also referred to as ileitis. seroprevalence in grow-finish herds can reach 100%. ppe can further be divided into four clinical forms (kroll et al., 2005) . porcine intestinal adenomatosis (pia) is most common in 6-20 week pigs and causes little mortality. porcine hemorrhagic enteritis (phe) affecting pigs 28 weeks of age and older including breeding swine and can be associated with increased mortality and dark, bloody stools. necrotic enteritis (ne) and subclinical ileitis, the most common form, occur among postweaning pigs. in all forms, transmission is by the fecaloral route. crypt enterocytes infected with l. intracellularis become hyperplastic. the altered ratio of villous and crypt enterocytes leads to malabsorption and subsequent increases in feed conversion and time to reach market weights. pia results in variable degrees of thickened ileum that can be found at necropsy. the ileal lumen may contain a blood clot in phe or pseudomembrane in ne. when diarrhea ranging in color from normal (pia, ne, and subclinical) to dark-red or black (phe) is observed, ppe should be considered as a possible cause. subclinical ileitis usually causes no clinical signs (gebhart, 2007) . histopathologic lesions containing intracellular s-shaped organisms are suggestive of lawsonia infection but ihc should be used to confirm diagnosis. pcr is helpful to detect infection and is highly specific but moderately sensitive. cross-sectional or longitudinal serologic profiling using a widely available elisa is the best tool for determining timing of exposure. treatment is with effective antibiotics, such as tylosin, administered by injection or in the feed or water. control is by administration of a commercially available modified-live oral vaccine before infection or feeding antibiotics when infection is known to occur. vaccination should take place at least 8 weeks before seroconversion (walter et al., 2004) . salmonellosis causing gastrointestinal disease in swine is most commonly associated with the species typhimurium. salmonella typhimurium is commonly isolated from swine. isolation of multidrug resistance strains of s. typhimurium from swine at slaughter have garnered attention from public health and food safety professionals and it is this that make this infection significant to the pork industry (foley et al., 2008) . some european union member states have implemented meat-juice serologic monitoring at slaughter to assess on-farm salmonella control programs. pathogenesis of s. typhimurium is similar to salmonella cholerasuis by invading enterocytes and subsequently macrophages leading to an infectious carrier state. initial infection causes inflammation and cytokine release that result in watery, yellow diarrhea containing feed particles. button ulcers may be visible on the mucosal surfaces of the colon and cecum on gross necropsy examination and, on histopathology, can be found to extend into the lamina propria. bacterial isolation without using enrichment media and the presence of histopathologic lesions is consistent with a diagnosis of salmonella enteritis. treatment is with antibiotics administered symptomatically to diarrheic pigs. antibiotic susceptibility of the isolate should be considered before initiating treatment. rearing pigs on slatted floors, decreasing stocking density, and acidification of digesta are effective in reducing the prevalence of salmonella infections in swine (funk and gebreyes, 2004; boyen et al., 2008) . cross-protection with s. cholerasuis vaccine has been reported and reduces carcass colonization (husa et al., 2009) . whipworm infestations of swine are the result of trichuris suis infection. pigs kept on pasture, in outdoor lots, or facilities with a history of t. suis diagnosis are at greatest risk for disease (pittman et al., 2010a) . the prepatent period is 6-7 weeks. the egg is not immediately infective, which requires 3-4 weeks in the environment. the infectious larva hatches from the egg and invades enterocytes in the small intestine and cecum. the entire life cycle of t. suis is completed in the intestine. ulcerations in the mucosa and damage to capillary blood supply of intestinal epithelium lead to hemorrhage, anemia, and hypoalbuminemia. clinical signs are depressed weight gain, increased feed conversion, bloody diarrhea, ill thrift, and death. adult worms imbedded in the ileum, cecum, or proximal colon are sufficient for diagnosis of whipworms. eggs are intermittently shed and thus not a reliable method of diagnosis (pittman et al., 2010a) . treatment and control are synonymous and require administration of an effective anthelmintic like fenbendazole. prevention is by steam sanitation and drying; however, eggs are resistant to common disinfectants and remain infective for years. the porcine integument or skin, like that of other domestic species, serves as a protective barrier between fragile internal tissues and harsh external hazards. skin is comprised of layers (from external to internal): epidermis, dermis (superficial and deep), and subcutis. blood vessels, hair follicles, sebaceous glands, and muscles are found in the dermis. notably, the pig's skin does not contain sweat glands; therefore, modern swine facilities are outfitted with evaporative cooling systems for thermal regulation in hot climates. skin diseases and disorders can be the result of viral or bacterial infections, parasite infestations, immunologic reactions, and idiopathic or iatrogenic causes that are summarized in table 3 by their various macroscopic and microscopic lesions. greasy pig is a skin disease of swine caused by a toxin produced by staphylococcus hyicus. a break in the skin is the typical sequela. gilt litters reportedly have a higher incidence of this disease, presumably due to deficient maternal immunity. all ages of pigs may be affected but suckling and nursery pigs are most likely to develop disease. affected pigs develop focal crusts on the face, neck, and axillary region, and the crusts may coalesce as the disease progresses. affected areas are greasy to touch and may appear black due to dirt adhering to it. if pigs are untreated or fail to respond to treatment, the trunk and extremities may become involved. pyrexia and lethargy can be observed in severe cases and are followed by growth reduction. gross appearance of affected skin is rarely confused with other skin conditions of swine. submission of formalin-fixed skin sections that include the junction of affected and unaffected layers of epidermis and dermis for histopathologic examination is needed for a diagnosis. the pathognomonic histologic lesion is exudative epidermitis. table 3 common integument diseases and disorders of pigs greasy pig x x x erysipelas x x porcine dermatopathy and nephropathy syndrome (pdns) x x x sarcoptic mange x x the first column provides the diseases. the remaining columns represent the type of lesion that occurs. the s. hyicus can be cultured from the surface of clinically normal skin sections. treatment includes topical application of antimicrobials or disinfectants. unaffected pigs with direct contact with affected pigs should also be treated to control spread. in cases where pigs exhibit systemic signs, administration of an injectable antimicrobial and anti-inflammatory is warranted. in the united states, no antimicrobials are labeled for the treatment, control or prevention of s. hyicus so all antimicrobial therapy is extra-label. prevention should focus on facility hygiene and include a soap degreaser and disinfectant regimen to reduce contamination. in addition, scarification of the skin of breeding age females with the farmspecific s. hyicus strain can reduce disease incidence in suckling pigs (murray and rademacher, 2008) . erysipelas or diamond skin disease is caused by a soilborne gram-positive bacterium, erysipelothrix rhusiopathiae. this zoonotic pathogen is transmitted by migratory fowl, turkeys, and pigs. humans may become sickened when direct contact with blood from affected animals contaminates an open wound (brooke and riley, 1999) . the finding of lesions at slaughter results in partial or complete carcass condemnation (bender et al., 2011) . the disease is most common in growing, finishing, and breeding age swine. bacterial emboli lodge in blood vessels causing vasculitis, thrombosis, and ischemia leading to lameness, abortions in gestating females, and raised, red to purple rhomboid skin lesions for which erysipelas is best known. skin biopsies from the affected area should include epidermis and dermis, but histologic lesions are only supportive. bacteriologic isolation or pcr identifying e. rhusiopathiae confirms the diagnosis. treatment with β-lactam antibiotics including penicillin is effective. commercial bacterins and avirulent live cultures are available for prevention (wood, 1984) or in the face of outbreaks to prevent the chronic form. pdns has been associated with porcine circovirus type 2 (pcv2) infection, but any disease process resulting in ischemia could cause result in pdns. the condition is characterized by red to purple discoloration of skin that begins on the caudal surface of the hind limbs and the ventral surface of the abdomen resulting from ischemia. on necropsy, gross examination of the kidney cortex may be speckled with pinpoint, white foci caused by infarcted blood vessels. pig of any age can be affected with pdns, but it is more commonly observed during growing and finishing stages. submission of fresh and formalin-fixed skin sections that include the junction of affected and unaffected layers of epidermis and dermis is required. there is no specific treatment or prevention; rather, diagnose the underlying cause to determine appropriate therapy ( figure 1) . sarcoptic mange is the result of an allergic reaction to the saliva of ectoparasites, sarcoptes scabiei. mange may also be caused by demodex phylloides. mortality is low and morbidity is moderate. economic losses are the result of reproductive inefficiency, growth reduction, and carcass condemnation. infestation and subsequent clinical signs in the breeding herd, most notably an incessant scratching, develop following the purchase of infested genetic replacements. in addition, growing pigs placed in facilities that previously housed infected swine or facilities that reuse straw bedding or have solid wood partitions may also become infested. the mite is rare in modern, high-health swine operations. the burrowing mite causes red pustules and flaking skin. individual pigs may develop signs in as few as 3 weeks but a herd may not show signs for several months. in the chronic stage, thick crusts develop at the corners of and inside the ears. examination of a scraping from the crusts will reveal the mite (averbeck and stromberg, 1993 ). an elisa test is used to determine prior exposure and determine success of eradication programs. treatment can be applied topically using an antiparasitic, such as amitraz, to temporarily alleviate clinical signs. control and eradication programs utilize feeding or injection of ivermectins (mohr, 2001) . the musculoskeletal system is comprised of tendons, ligaments, muscles, and bones. disorders and disease of this system are typically characterized by lameness. lameness is any deviation in normal locomotion including favoring a limb or failure to bear weight on the limb. neurologic conditions, which also cause changes in locomotion, may be ruled out by postmortem examination of articular surfaces and diagnostic testing. investigation of musculoskeletal diseases and disorders should always start with the claws that are easily traumatized causing pain resulting in lameness. flooring and genetics also influence the incidence of lameness. common musculoskeletal diseases and disorders of swine can be divided into osteopathies and myopathies and summarized in table 4 . mycoplasma hyosynoviae colonizes upper airways and tonsils resulting in a carrier state. transmission is vertical from sow to pigs and lateral between pigs (ross and spear, 1973) . m. hyosynoviae is most often diagnosed during the grow-finish phase. morbidity is variable but mortality is low. clinical signs are a stiff gait and difficulty in standing, most often the stifle or elbow and less frequently the hock, hip, and shoulder. signs often occur 2-3 weeks after a stressful event; lesions begin to resolve 7 weeks postinfection. the affected joint contains yellow or blood-tinged effusion with moderate villous proliferation but is not always observed despite lameness and does not necessarily correlate with presence of histopathologic lesions. aseptic collection of synovial fluid by needle aspiration or sterile swab or submission of the affected joint intact is recommended for diagnosis. pcr is the most sensitive test; culture requires special media and lacks sensitivity (gomes neto et al., 2012) . histopathologic examination of formalin-fixed synovium reveals nonsuppurative fibrinous polyarthritis and lymphoplasmacytic perivascular synovitis. elisa is also available. lincomycin has historically been an effective therapeutic choice (burch and godwin, 1984) . treatment should be initiated when lameness is first observed; however, spontaneous resolution is common. no commercial vaccines are available. mycoplasma hyorhinis is a ubiquitous bacterium that is an early colonizer of upper airways. transmission is vertical from sow to pigs and then between pigs postweaning (rovira, 2009) . infection can progress to polyarthritis, polyserositis, and otitis in the pre-or postweaning phases; arthritis develops postweaning. clinical signs include lameness, arthritis, and fever that develop 3-10 days after septicemia occurs and persists for 10-14 days (gomes neto et al., 2012) . disease may become chronic resulting in ill thrift, reduced growth, and death. articular surfaces may be eroded. in cases of lameness, synovial fluid and formalin-fixed synovium can be submitted. alternatively, the entire affected leg can be submitted; disarticulate above the infected joint keeping the affected joint intact. submission of fibrin or fibrin covered tissue(s) should be included for pcr testing to differentiate m. hyorhinis from other bacteria that form fibrin on serosal surfaces like hps and s. suis (rovira, 2009 ). histopathology reveals fibrinosuppurative inflammation in affected tissues. treatment is empirical. erysipelas is the result of a chronic e. rhusiopathiae infection causing arthritis and endocarditis that follows the initial septicemia. lameness and joint swelling is mostly noticeable in hock and carpal joints. lameness may also occur in stifle and elbow but swelling cannot be appreciated. synovial fluid appears serosanguinous and can be submitted for testing by bacterial culture or pcr. alternatively, the entire affected leg can be removed to prevent contamination; disarticulate the leg above the infected joint. histopathologic examination of formalin-fixed synovium reveals a proliferative synovitis. other lesions that occur are nonsuppurative fibrinous polyarthritis and erosion of cartilage that can progress to pannus and ankylosis. treatment with β-lactamase antibiotics including penicillin is effective. an anti-inflammatory is added to a treatment program for pain management. commercial bacterins or avirulent live cultures are available for control and prevention. oc is the result of a delay in ossification of articular cartilage, and represents the most common lesion among culled sows. morbidity is most often reported in adult and breeding age pigs (dewey et al., 1993) . mortality is variable and is the result of humane euthanasia because the animal becomes nonambulatory. oc causes lameness, pain, and joint swelling. a noninfectious lameness most often affects the distal part of the humerus or femur. lesions are typically bilateral and symmetrical. diagnosis is made by ruling out other causes of lameness. ricketts occurs as a result of phosphorus deficiency, vitamin d deficiency, or secondary to iron toxicity but is not caused by dietary calcium deficiency. the condition should be suspected when there is an increase in nonambulatory pigs and broken bones during the finishing stage particularly at and immediately before marketing. occasionally, joint swelling in the nursery stage is observed. rachitic rosary (enlargement of costochondral junctions) and soft bones are observed on necropsy. if rickets are present, a bone ash analysis of the second rib will be below normal. feed analysis can identify low levels of vitamin d or phosphorus. low levels of vitamin d or phosphorous serum chemistry also will occur (madson et al., 2012) . supplementation of vitamin d is the only reported treatment and response that may be considered diagnostic. prevention includes proper diet formulation for the stage of production. mhd is a noninfectious disease of muscle caused by deficiency of vitamin e or selenium. it can occur if pigs are fed grain grown in selenium deficient soils (dewey, 2006) . clinical signs are limited to acute death of large, robust pigs. on necropsy, the heart muscle has a mottled appearance. feed analysis, response to vitamin e supplementation, and ruling out other causes support diagnosis of vitamin e/selenium deficiencies like mhd (hooser, 1996) . supplementation with selenium is impractical in the united states because of environmental regulations, and overzealous supplementation may cause toxicosis. splayleg is a noninfectious, congenital condition resulting from delayed myofibril development with no known cause. splayleg has low morbidity and mortality as long as it is identified and corrected before it leads to starvation or being crushed by the sow. treatment includes the use of nonslip note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). flooring in farrowing crates and application of harness or tape that holds the rear legs under the pig until it is strong enough to walk on its own. reproductive failure occurs when insemination fails to result in pregnancy or pregnancy fails to produce viable pigs due to infectious and noninfectious causes summarized in table 5 . reproductive failure should be considered when a low conception or farrowing rate, irregular returns to estrus, abortions, stillbirths, or mummies persist at an abnormal rate. infertility occurs when fewer than four embryos are present at the time of maternal recognition of pregnancy resulting in a regular return to estrus and reduced conception rate for that breeding group. irregular returns to estrus result from embryonic death or early term abortion after implantation but before calcification of the fetuses. embryonic death of some or all of the embryos will result in low total born or irregular return to estrus, respectively. early term abortion also will reduce farrowing rates. mummies and stillborns can occur any time after calcification of the fetuses. the normal rates for mummies and stillborns are o0.5 and o1 pig per litter, respectively. late-term abortions are classified as those occurring after 70 days of gestation. total abortion rate should remain o2% of a breeding group. these are general guidelines; thus, familiarity with the herd's normal reproductive performance is the most sensitive means to identify a reproductive problem. prrs is, at this time, known only to occur among swine. the estimated cost of prrs to the us pork industry is us$664 million annually (holtkamp et al., 2013) . prrs usually results when susceptible swine are infected with either the leylystad or north american strains of prrs virus (prrsv), a member of the arteriviridae family. viremia lasts up to 42 days, but shedding of infectious virus can last much longer (murtaugh and genzow, 2011) . prrsv is most commonly transmitted by introduction of infected swine or contaminated fomites, use of contaminated semen, and aerosol. the pathogenesis of the reproductive form is believed to be arteritis of fetal umbilical cords during gestation (lager and halbur, 1996) . swine may show no signs when reinfected with a homologous strain. conversely, infection with a heterologous strain will reproduce lesions and disease but is usually less severe than that of naïve swine (murtaugh and genzow, 2011) . clinical signs of prrs in a breeding herd start with an epidemic of abortions followed by an increase in low viable piglets, stillbirths, and mummies. abortions result due to fetal death or pyrexia of the gestating female. sows and gilts may be anorexic, pyrexic, or lethargic. periparturient females may become agalactic. in severe outbreaks of prrs, sow mortality also increases. in utero infection of feti can result in persistently infected piglets (rossow, 1998) . prewean mortality commonly increases and may remain above the herd average for weeks. diagnosis can be made by submitting lung, spleen, and lymph node from fetuses or low viable piglets. whole fetuses can also be submitted but should be refrigerated to prevent autolysis. lesions are not pathognomonic so confirmatory testing such as pcr, ihc, or vi should be conducted. tissues and thoracic fluid from stillbirths, aborted, or mummified feti can be submitted but may result in false negatives. serum collected from aborted sows or low viable piglets and tested for prrsv by pcr is another option for diagnosis. prrsv elisa indicates previous exposure but is not useful in a previously exposed herd. treatment of prrs is supportive. anti-inflammatories to reduce fever and antibiotics for control and treatment of secondary bacterial pneumonia may be necessary. the most common methods for control include depopulationrepopulation and herd closure and rollover, also called loadclose-homogenize, using commercial vaccine or herd-specific live virus exposure (corzo et al., 2010) . periods of closure vary based on facility capacity but a minimum of 180 days is recommended. commercial modified-live and killed vaccines are available but do not prevent infection and should be used in accordance with label and domestic guidelines. ppv is sometimes described by the acronym smedi (stillborns, mummies, embryonic death, and infertility). ppv is an enzootic infection of swine breeding herds in the united states. the virus is ubiquitous and is transmitted through ingestion of infected feces, afterbirth, or fetal tissue. the disease most commonly affects gilts and younger parity sows (christianson, 1992) . the pathogenesis is through damage to table 5 common reproductive diseases and disorders of pigs note: the first column provides the diseases. the remaining columns represent the clinical signs. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). the placental epithelium resulting in fetal death. clinical signs of ppv range from low total born, mummies of various sizes, irregular returns to estrus, and females diagnosed pregnant but fail to farrow. diagnosis is based on vaccination history, clinical signs, and pcr testing of mummified fetuses. ppv elisa may provide diagnostic value if acute and convalescent serum samples are used. there is no effective treatment for ppv; however, commercial killed vaccines are available and very effective. exposure of unbred females to tissue or cull sows from a seropositive herd has been used for immunization when vaccine is unavailable. leptospirosis is caused by infection by spirochete bacteria. leptospira species may be zoonotic (leptospira canicola, l. icterohemorrhagiae), swine-adapted (l. pomona and l. bratislava), or incidentally infect swine (l. grippotyphosa and l. hardjo). infection has been associated with exposure of swine to contaminated soil or untreated surface water, and exposure to urine from infected vectors, such as rodents. infected swine can become carriers resulting in chronic disease. the pathogenesis is due to bacteremia resulting in transplacental infection followed by fetal death. clinical signs include pyrexia, low conception rate, abortion, stillbirths, and low viability pigs resulting in increased prewean mortality. diagnosis is made using dark field microscopy or ihc performed on tissues, particularly kidney, of aborted feti or stillbirths. paired or matched serology for hemagglutination inhibition (hi) testing may be useful if suspected. treatment with antibiotics, such as chlortetracycline, may be pursued (henry et al., 1993) . commercial killed bacterins are available to aid in prevention and should be given at least semiannually to breeding stock (christianson, 1992) but may not be available in all countries. for example, federal regulations prohibit the use of these bacterins in france and the netherlands (figure 2) . pcv2 is a ubiquitous virus in swine facilities. pigs become infected with pcv2 through ingestion (oral nasal contact). in addition, breeding females can become infected via insemination with contaminated semen (madson et al., 2009a) . gilts and low parity sows are affected most often, whereas boars show no clinical signs. pcv2-associated reproductive failure may occur in conjunction with ppv. infection results in variable lengths of viremia. pcv2-reproductive failure is due to transplacental infection of fetuses. clinical signs depend on the stage of gestation when the infection occurs. embryonic death, early term abortions, stillbirths, mummies, low total born, or low viable pigs can result from infection. mummies may vary in size, like ppv, and measuring crown to rump length is useful to determine the time when that fetus was infected. pcv2-reproductive failure is diagnosed by the presence of viral antigen confirmed by ihc or deoxyribonucleic acid confirmed by pcr along with the presence of lesions in fetal tissue notably myocardial mineralization. pcr testing of fetal thoracic fluid is sufficient to diagnose in utero infection of piglets (madson and opriessnig, 2011) . commerical killed baculovirus vectored vaccines are available and effective for prevention of disease but not infection or viremia (madson et al., 2009b) . prv or aujezsky's disease virus was eradicated from the us commercial swine herd in 2004; a comprehensive review is available (usda aphis, 2008). prv is a member of the herpesviridae family and, like other herpesviruses, infection can result in a carrier state or latency within nervous tissue with the potential for recrudescence. the pathogenesis of prv results from viremia, and then replication and necrosis of epithelial tissue including the placenta (christianson, 1992) . the period of viremia gives prv time to cross the placenta and cause fetal death. clinical signs following acute infection include embryonic death, abortion, mummies, and stillborns. necrotic foci can be found in fetal spleen, liver, lung, and lymph node. histopathology is not definitive; ihc is required to confirm presence of antigen. diagnosis may also be made through serology; a commercial elisa test is available and can differentiate between exposure to the gene-deleted vaccine and wild-type virus used extensively in the us eradication. commercial prv vaccines are available but only should be used in accordance with federal guidelines. brucellosis is a zoonotic infection caused by the bacteria, brucella suis biovars 1 and 3. brucella suis is transmitted through direct contact with susceptible swine, ingestion of infected tissue, or fluids including milk and contaminated semen. pathogenesis of b. suis is initiated when the mucosal epithelium is penetrated, thereby resulting in bacteremia that commonly persists for 5 weeks and results in placentitis among other lesions. clinical signs of infection in gilts and sows include abortion with or without vaginal discharge, whereas boars have reduced libido and fertility. bacteriologic isolation of b. suis from vaginal discharge or tissue confirms diagnosis. serology reflects prior exposure (or vaccination) to b. suis but not for diagnosis of acute disease. the us commercial swine herd is brucellosis free. swine erysipelas (se) is a zoonotic, gram-positive bacterium, which is ubiquitous among swine. erysipelothrix rhusiopathiae is the sole causative species. carrier swine shed the bacteria in saliva, nasal discharge, and feces. infection may result from direct contact with carriers, exposure to infected facilities or soil (wood, 1984) . a bacteremia lasting several days precedes lesions. reproductive failure is most often due to abortion but infertility and low total born following high fevers or endometritis at the time of breeding is also possible. clinical signs including rhomboid skin lesions, high fevers, lethargy, inappetence, withdrawal, and response to treatment with penicillin of affected sows and gilts are suggestive of acute and subacute se. serology is available; availability is by veterinary diagnostic laboratory (vdl) and value is limited when vaccine is in use. bacterial culture and histopathologic examination of fetal tissue is unrewarding for diagnosis of se but is helpful to rule out other causes of abortion. in chronic se, culture of e. rhusiopathiae from vulvar discharges was successful (gertenbach and bilkei, 2002) . treatment involves injections of antibiotics and anti-inflammatories. commercial vaccines are available and effective. co poisoning induces hypoxia resulting in an increased number of stillborns (hooser, 1996) . concentrations of 4250 ppm are toxic. malfunctioning heating units or poorly ventilated farrowing rooms are the cause. diagnosis is done by ruling out infectious causes of stillbirths. fetal blood or thoracic fluid can also be measured for co concentrations. zearalenone is a luteotropic mycotoxin produced by fusarium rosea. it binds estrogen receptors resulting in irregular returns to estrus, signs of estrus in prepubertal gilts, and reduced litter size (hooser, 1996) . diagnosis is by detection of elevated levels in feed samples. however, definitive diagnosis is rarely possible because the contaminated feed has long been consumed by the time reproductive failure occurs. aas and seasonal infertility is a noninfectious cause of reproductive failure. the declining photoperiod and temperature fluctuations during the fall months result in declining progesterone levels. high-ambient temperature experienced during lactation and the postweaning period are suspected but not confirmed as a cause. diagnosis is done by ruling out infectious causes and careful assessment of management, facilities, and reproduction records (rueff, 2000) . modern facilities that utilize gestation crates and evaporative cooling systems may improve but not prevent infertility during the fall months (leman, 1992) . the respiratory system can be simply divided into upper and lower portions. the upper portion includes the nasal cavity and sinuses, throat, trachea, and bronchi for air conduction. the lower portion is the lung comprised of bronchioles and alveoli responsible for air exchange. the respiratory system is commonly involved in numerous infectious diseases of swine summarized in table 6 . the most notable infectious agents are the viral pathogens, prrs and pcv2, which cause primary pathologic lesions to both the respiratory and the immune system. this damage to the immune system often leads to respiratory or systemic disease incited by secondary infectious agents. such mixed respiratory infections can occur at any age, and, when they occur in growing and finishing pigs, are termed porcine respiratory disease complex (prdc). multifactorial respiratory disease can obscure histopathologic lesions complicating the diagnostic process. app is a host-adapted, fastidious, and gram-negative encapsulated rod that is transmitted vertically from sow to piglet. morbidity and mortality are strain-specific; virulence varies with expression of apxi and apxii toxins. inhalation of strains of app expressing apx toxins results in lung lesions within 24-36 h. the disease is economically significant because mortality occurs during the later part of the finishing phase, usually just before slaughter. clinical signs of fever, lethargy, dyspnea, and acute death are common. pigs found dead may have a frothy, blood-tinged discharge from the nose and mouth. focal hemorrhage occurs in the diaphragmatic lung lobe, which is firm, and its appearance is likened to that of a bull's eye. fibrinous, necrotizing bronchopneumonia containing streaming leukocytes is a key histopathologic feature. bacterial culture is difficult and requires nicotinamide adenine dinucleotide (nad)-supplemented media so diagnosis is traditionally made on finding characteristic postmortem and histopathologic lesions. a pcr test is also available. in an outbreak, the entire population should receive antimicrobial therapy parentally. unlike many other gram-negative bacteria, app is sensitive to a variety of antimicrobials; at iowa state university vdl 490% of isolates were sensitive to ceftiofur, enrofloxacin, florfenicol, tiamulin, tilmicosin, and tulathromycin. prevention is aimed at eliminating carrier swine through depopulation or by pulse medication (marsteller and fenwick, 1999) . actinobacillus suis causes a hemorrhagic, necrotizing pneumonia during nursery and grow-finish phases. actinobacillus suis infection has similar clinical signs and pathologic appearance to app. affected pigs are frequently observed in a dog-sitting position with elbows abducted. unlike app, lung lesions are random in their distribution and petechial table 6 common respiratory diseases and disorders of pigs postweaning nursery postweaning grow-finish adult note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). hemorrhages may be seen in other organs due to the septicemia that follows a. suis infection (yaeger, 1995) . a pcr test is available to help differentiate disease from app and hps (oliveira, 2007a) . like app, outbreaks should be treated by parental delivery of an antimicrobial; however, treatment of only those individual pigs with clinical signs is usually sufficient. autogenous vaccines can be used for control but response is variable because most pigs are already seropositive at the time of vaccination. ascaris suum, the swine roundworm, is the most common parasitic infection of swine. the reduced growth performance and liver condemnations are responsible for economic losses (stewart and hoyt, 2006) . the prepatent period is 40-53 days. adult roundworms are present in the manure but it is the migration of larvae through the lungs, occurring 8-10 days after ingestion of an infective egg that causes respiratory signs. a persistent cough and dyspnea result due to verminous pneumonia. the liver develops whitish spots, called 'milk spots' that are the cause of condemnations but resolve within 25 days (stewart and hoyt, 2006) . the presence of eosinophils is suggestive of a parasite infestation. treatment and control is accomplished using anthelmintics: dichlorvos, fenbendazole, levamisole eliminate adults and larvae; piperazine kills only adults. proper cleaning and disinfection particularly removing fecal material between groups reduces potential for exposure but it is virtually impossible to get rid of a. suum once a premise is infested (pittman et al., 2010b) . it is necessary to prevent access to contaminated soil. ar is described in two forms: progressive (par) and nonprogressive (npar). par is caused by toxigenic strains of pasteurella multocida, whereas npar is the result of toxigenic strains of bordetella bronchiseptica. in both forms, the bacteria attach to cilia in the nasal passages and the cytotoxin production causes hypoplasia of nasal turbinates. clinical signs include sneezing, deviated snouts, and, in cases of par, bloody nasal discharge occurring in a large number of grow-finish pigs. mortality is low but the reduced growth that results due to ar makes it economically important. beacause the cytotoxins are responsible for ar, isolation of either bacterium from nasal passages is not sufficient for diagnosis. in addition, b. bronchiseptica and p. multocida colonize the lung leading to bronchopneumonia causing cough and dyspnea in pigs postweaning, often part of prdc (hansen et al., 2010) . therefore, examination of nasal turbinates at slaughter is the recommended method for diagnosis of ar (gatlin et al., 1996) . transmission is vertical; therefore, prefarrow vaccination of sows can protect piglets up to 16 weeks of age. if vaccination does not prevent ar, depopulation of the herd may be necessary. hps is also called glässer's disease. there are 15 serovars identified and prefer to colonize the nose (macinnes et al., 2007) . hps may not actually result in pneumonia but does cause signs of respiratory disease including nasal discharge and dyspnea. in addition, fever, lethargy, and acute death are observed. on necropsy, one or all of the pleural, pericardial, epicardial, and peritoneal serosal surfaces become covered in fibrin. effusion commonly occurs. histopathologic lesions are described as fibrinopurulent. definitive diagnosis is by bacterial isolation on culture media supplemented with v factor. owing to the difficulty in isolating hps, pcr testing is now available (oliveira et al., 2001) . isolation from the airways in the absence of lesions has little significance (hoefling, 1994) . ceftiofur, enrofloxacin, or tulathromycin delivered parenterally to affected animals are effective therapeutic drugs. use of water-soluble antimicrobials is for control. maternal immunity, medicated early weaning, and controlling infections with prrs, pcv2, and influenza postpone or prevent disease onset (rapp-gabrielson et al., 2006) . commercial and autogenous vaccines are available but may experience limited efficacy due to serologic diversity; controlled exposure to low dose, live virulent culture is another option (oliveira et al., 2004) (figure 3) . mh is known to infect pigs in production systems worldwide causing reduced growth performance and mortality. the disease is classified as enzootic pneumonia or a component of prdc. both manifestations of mh cause paralysis of the mucociliary escalator resulting in a severe cough and dyspnea known as thumping. vertical and lateral transmission can occur; but, owing to its slow rate of transmission between pigs, the disease primarily occurs in grow-finish pigs (meyns et al., 2004) . in addition, time of colonization with mh and disease severity are directly related (fano et al., 2007) . on necropsy, well-demarcated (red to purple lobular consolidation occurs in the apical) diaphragmatic, and accessory lung lobes is visible. histopathologic lesions characteristic of mh is bronchopneumonia with lymphocytic perivascular, peribronchial, and peribronchiolar cuffing. because mh is difficult to isolate, pcr is the most sensitive method of detection. elisa is available and is helpful in establishing herd status but must be interpreted in the context of vaccination as tests do not distinguish between antibodies produced subsequent to vaccination or field infection. treatment of affected pigs with parental antimicrobials like enrofloxacin, tulathromycin, or lincomycin or administration of water-soluble lincomycin, tiamulin, or tetracyclines to affected groups is effective in outbreaks. control can be achieved through pulse-medication in feed of chlortetracycline (thacker et al., 2006) beginning figure 3 epicarditis, heart, nursery pig. fibrin gives surface a granular appearance, caused by hps infection. note the enlarged (draining) mediastinal lymph nodes located cranial to the base of the heart and the excess thoracic fluid (reddish-brown) indicative of septicemia. courtesy dr. glen almond. 1 week before the historical onset of disease . commercial vaccines are whole cell bacterins marketed to reduce lesions but do not prevent disease or slow transmission rate. simultaneous infection with prrsv reduces efficacy of mh vaccination thacker, 2000) . eradication from the herd is preventative but practically difficult to accomplish. pcvad is any disease process where pcv2 infection results in lesions and includes pmws (ellis et al., 1998) and pdns. infection with pcv2 is widespread. morbidity and mortality is variable, often dependent on the occurrence of secondary infections and their virulence. survivors of pcvad remain stunted, owing to the economic significance of this collection of diseases. clinical signs include wasting, dyspnea, depression, ill thrift, and diarrhea. lungs are wet, heavy, and fail to collapse; pulmonary edema and lymphadenopathy also can be found at necropsy. histopathology results include presence of interstitial pneumonia, lymphoid depletion, enteritis, nephritis, and dermatitis. for a diagnosis of pcvad the following must occur: pcv2 antigen within characteristic lesions and lymph nodes are depleted (sorden, 2008) . ihc is used to confirm presence of pcv2 antigen within the histopathologic lesion. pcr has little value in diagnosing pcvad unless the herd is considered free. commercial vaccines are very effective and available with flex labels for administration to sows and pigs and as 1 or 2 doses (chae, 2012) . nonvaccinated, subclinically infected pigs have poorer weight gain compared to their vaccinated counterparts (kristensen et al., 2011) ; therefore, it is part of most vaccination protocols by us pork producers ( figure 4) . prrs is the result of infection with the leylystad or north american strain of prrsv. the estimated cost of prrs to the us pork industry is us$664 million annually (holtkamp et al., 2013) . prrsv is the most commonly diagnosed viral respiratory pathogen at vdls (gauger, 2009) . infection is observed to increase susceptibility to other infections, particularly opportunistic bacteria. this apparent increased susceptibility to secondary and opportunistic infections is the result of the pathologic process in which prrsv recruits and replicates in pulmonary alveolar macrophages, and then disseminates systemically (rossow, 1998) . clinical signs are nonspecific including fever, lethargy, and dypsnea but not cough. signs also depend on the type of secondary infection(s) present. lungs fail to collapse and appear heavy, wet, and gray on postmortem examinations. lymphadenopathy is caused by hyperplasia of germinal centers. interstitial pneumonia, alveoli are lined with hyperplastic type ii pneumocytes and contain necrotic debris, whereas the lining of bronchi and bronchioles is normal (rossow, 1998) . vasculitis also occurs. pcr is the most sensitive method for confirming infection. owing to the genetic diversity of prrsv, sequencing of the orf5 region is a common adjunct to pcr testing. sequences are then used to create dendrograms for use by production systems pursuing prrs control and epidemiologic investigations (murtaugh, 2012) . prrsv elisa is helpful for establishing herd status; national animal health monitoring service reports that a large percentage of us herds are seropositive. treatment is limited to maintaining pig comfort, minimizing stress, and controlling secondary infections. commercial modified-live vaccines (mlv) are available and administration during the nursery phase significantly reduces mortality and improves growth performance during the grow-finish phase of production (robbins et al., 2013b) . mlv vaccines do replicate and should not be used in negative populations. salmonella cholerasuis is the swine-adapted salmonella from the c1 serogroup and, unlike s. typhimurium, is not a foodborne pathogen. ingestion or inhalation of the bacteria causes a septicemia resulting in low to moderate morbidity with high mortality within 1-2 days of infection that occurs postweaning, predominately during the grow-finish phase (baskerville and dow, 1973) . signs include high fevers (440 1c), lethargy, dyspnea, acute death, and cyanotic extremities and abdomen. the latter makes it impossible to differentiate clinically from classical swine fever virus (csfv). pleuropneumonia, interlobular edema, mediastinal, and tracheobronchial lymphoadenopathy, and occasionally white foci in the liver are apparent postmortem (turk et al., 1993) . acute histopathologic lesions that form in the lung are purulent bronchitis, lobular necrosis, and abscessation, whereas paratyphoid nodules are observed in the liver. isolation is best achieved from the draining lymph nodes, lung, or liver using selective culture media. serogrouping and typing is necessary for speciation and diagnostic confirmation. owing to the rapid onset of disease, parental treatment is recommended. salmonella cholerasuis isolates are commonly susceptible to ceftiofur. increased hygiene particularly eliminating access to waste and vaccination is preventive (husa et al., 2009) . siv is more accurately described as influenza, to encompass the infections occurring in swine, avian, and human species. influenza virus is classified by its hemagglutinin and neuraminidase proteins; the three predominant strains in pigs are h1n1, h1n2, and h3n2. rapid transmission and onset are characteristic; in the experimental inoculation of one nonvaccinated nursery age pig resulted in 10.66 more becoming infected (romagosa et al., 2011) . virus is shed for 3-5 days and uncomplicated lesions resolve 28 days postinfection (gramer, 2007) . nasal discharge, fever, and lethargy occur but resolve quickly. cough and dyspnea can last up to 2 figure 4 pulmonary edema, lung, grow-finish pig. interlobular edema associated with pcvad, ventral portion of apical and diaphragmatic lung lobes is consolidated (purple) due to secondary bacterial infection. courtesy dr. glen almond. weeks postinfection. pcr and vi detect virus for diagnosis of clinical cases. elisa and hi detect antibodies; elisa is helpful in establishing herd status, whereas hi is best for vaccination timing and measuring postvaccination titers (allerson et al., 2008) . necrotizing bronchitis, bronchiolitis, and alveolitis as lesion resolves affected areas appear vacuolated. pigs recover quickly so treatment should focus on maintaining pig comfort, minimizing stress, and controlling secondary infections. all licensed vaccines are killed; commercial and autogenous products are in use in the united states. vaccination reduces lung lesions and rate of transmission, but does not prevent infection and is complicated by antigenic shift and drift. in the united states, it is typical to vaccinate the sows rather than pigs to control disease and infection (allerson et al., 2013) . trade diseases are those listed by the oie. when one of these diseases is suspected or confirmed, it results in closure of international market access, which would be economically devastating to import-export businesses. the primary method for managing diseases that affect trade is to prevent their introduction. foot-and-mouth disease (fmd) is caused by a picornavirus, fmdv, which causes mucosal lesions exclusively in cloven hoofed species. clinical signs are excessive salivation, anorexia, and lameness causing high morbidity but low mortality. gross lesions are vesicles at cutaneous junctions, on the snout, or in the oral cavity. similar lesions can be caused by seneca valley virus, vesicular stomatitis, swine vesicular disease, and vesicular exanthema of swine; therefore, any blister in swine warrants diagnostic investigation. fmdv is highly transmissible within and between species. african swine fever (asf) is caused by asfv, currently classified as an iridovirus. soft ticks can act as reservoirs or vectors. current outbreaks are reported throughout eastern europe and russia that have been associated with improper garbage feeding. the virus damages blood vessels resulting in clinical signs and gross lesions consistent with septicemia; including red to purple skin discoloration and enlarged spleen, liver, and lymph nodes. excess blood and fluid in body cavities may occur. classical swine fever, historically referred to as hog cholera, is caused by csfv, a pestivirus, eradicated from the united states in 1972. transmission is associated with infected feeding, uncooked or undercooked garbage containing pork or pork by-products to swine. the virus remains infectious for months when refrigerated and years when frozen. clinical signs are nonspecific and are easily confused with s. cholerasuis. the virus replicates rapidly in tonsils, which makes it the ideal tissue to collect for diagnosis of csfv. see also: animal health: ectoparasites. animal health: foot-and-mouth disease. animal health: global antibiotic issues. slum livestock agriculture. vaccines and vaccination practices: key to sustainable animal production. zoonotic helminths of livestock the impact of maternally derived immunity on influenza a virus transmission in neonatal pig populations maternally derived antibody transfer to piglets following siv vaccination streptococcus suis colonization of piglets during parturition model for characterizing oral controlled exposure in a field setting sarcoptic mange in swine effect of dietary particle size on gastric ulcers, assessed by endoscopic examination, and relationship between ulcer severity and growth performance of individually fed pigs pathology of experimental pneumonia in pigs produced by salmonella cholera-suis some properties of the causative agent of transmissible gastroenteritis in swine erysipelothrix spp. genotypes, serotypes, and surface protective antigen types associated with abattoir condemnations non-typhoidal salmonella infections in pigs: a closer look at epidemiology, pathogenesis and control erysipelothrix rhusiopathiae: bacteriology, epidemiology and clinical manifestations of an occupational pathogen prevalence of cpb2, encoding beta2 toxin, in clostridium perfringens field isolates: correlation of genotype with phenotype use of tiamulin in a herd of pigs seriously affected with mycoplasma hyosynoviae arthritis brachyspira-associated colitis − update and review swine dysentery: diagnostic criteria and elimination strategies toxic minerals, chemicals, plants, and gases commercial porcine circovirus type 2 vaccines: efficacy and clinical application prevalence of porcine epidemic diarrhea virus and transmissible gastroenteritis virus infection in korean pigs stillbirths, mummies, abortions, and early embryonic death control and elimination of porocine reproductive and respiratory syndrome virus diseases of the nervous and locomotor systems clinical and postmortem examination of sows culled for lameness isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome postweaning escherichia coli diarrhea and edema disease effect of mycoplasma hyopneumoniae colonization at weaning of disease severity in growing pigs salmonella challenges: prevalence in swine and poultry and potential pathogenicity of such isolates risk factors associated with salmonella prevalence on swine farms the quantitation of turbinate atrophy in pigs to measure the severity of induced atrophic rhinitis porcine respiratory disease complex trends and diagnostics for practitioners tests to diagnose subclinical ileitis competitive exclusion treatment reduces the mortality and fecal shedding associated with enterotoxigenic escherichia coli infection in nursery-raised neonatal pigs erysipelas: potential involvement in urogenital disease of the sow detection of porcine epidemic diarrhea virus in air samples at varying distances to epidemic farms in oklahoma mycoplasma-associated arthritis: critical points for diagnosis siv: an update on circulating strains, advances in diagnostic tests and interpretation of test results sodium salt poisoning of swine efficacy of antimicrobial treatments and vaccination regimens for control of porcine reproductive and respiratory syndrome virus and streptococcus suis coinfection of nursery pigs serological evaluation of a clostridium perfringens type a toxoid in a commercial swine herd an investigation of the pathology and pathogens associated with porcine respiratory disease complex in denmark practitioner experiences with swine dystentery leptospira pomona: a case report in growing swine and breeding stock the various forms of haemophilus parasuis assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers swine toxicosis. swine health production a comparison of the safety, cross-protection, and serologic response associated with two commercial oral salmonella vaccines in swine effect of osmolality on net fluid absorption in non-infected and etec-infected piglet small intestinal segments protection of pigs against neonatal enteric colibacillosis with colostrums and milk from orally vaccinated sows a meta-analysis comparing the effect of pcv2 vaccines on average daily weight gain and mortality rate in pigs from weaning to slaughter proliferative enteropathy: a global enteric disease of pigs caused by lawsonia intracellularis gross and microscopic lesions in porcine fetuses infected with porcine reproductive and respiratory syndrome virus optimizing farrowing rate and litter size and minimizing nonproductive sow days prevalence of actinobacillus pleuropneumoniae, actinobacillus suis, haemophilus parasuis, pasteurella multocida, and streptococcus suis in representative ontario swine herds effect of porcine circovirus type 2 (pcv2) on reproduction: disease, vertical transmission, diagnostics and vaccination rickets: case series and diagnostic review of hypovitaminosis d in swine reproductive failure experimentally induced in sows via artificial insemination with semen spiked with porcine circovirus type 2 effect of porcine circovirus type 2 (pcv2) vaccination of the dam on pcv2 replication in utero control of mycoplasma hyopneumoniae infections in pigs effects of toltrazuril on the growth of piglets in herds without clinical isosporosis actinobacillus pleuropneumoniae disease and serology quantification of the spread of mycoplasma pneumoniae in nursery pigs using transmission experiments mechanisms of porcine diarrheal disease sarcoptic mange control/eradication and their impact on pig performance: a literature review and comparison of different protocols and monitoring programs successfully implemented worldwide with ivomec s products for swine system wide sick pig management immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) use and interpretation of sequencing in prrsv control programs impact of porcine epidemic diarrhea virus infection at different periods of pregnancy on subsequent reproductive performance in gilts and sows update on actinobacillus suis diagnosis, epidemiology, and control: on the path from good to great development of a pcr test to diagnose haemophilus parasuis infections evaluation of haemophilus parasuis control in the nursery using vaccination and controlled exposure determination of sodium content in serum and cerebrospinal fluid as an adjunct to diagnosis of water deprivation in swine diagnosis of streptococcus suis infections trichuris suis in finishing pigs: case report and review prevalence of internal parasites in a production system: part ii-finishing pigs diagnostic approaches to swine central nervous system disorders haemophilus parasuis what do we really know about feedback to gestating dams? prrsv control in finisher pigs, a large scale barn study in a high dense area in usa vaccination of influenza a virus decreases transmission rate in pigs role of the sow as a reservoir of infection for mycoplasma hyosynoviae porcine reproductive and respiratory syndrome review of mycoplasma hyorhinis diagnostic approaches to reproductive failure in pigs. swine health production transmissable gastroenteritis and porcine respiratory coronavirus effect of bmd s in sow gestation/lactation diets on clostridial infection, piglet pre-weaning performance, and sow body condition effect of waste environment on survival of brachyspira hyodysenteriae clostridial enteric infections in pigs update on porcine circovirus and postweaning multisystemic wasting syndrome (pmws). swine health production rotavirus and undifferentiated diarrhea in suckling piglets: what's new and diagnostics criteria internal parasites efficacy of a chlortetracycline feed additive in reducing pneumonia and clinical signs induced by experimental mycoplasma hyopneumoniae challenge effect of vaccination on the potentiation of porcine reproductive and respiratory syndrome virus (prrsv)-induced pneumonia by mycoplasma hyopneumoniae water in swine nutrition pathogenesis of porcine reproductive and respiratory syndrome virus-induced increase in susceptibility to streptococcus suis infection pleuropneumonia in missouri swine pseudorabies (aujeszky's disease) and its eradication: a review of the u.s. experience a retrospective evaluation of actions taken to control streptococcus suis infection serologic profiling and vaccination timing for lawsonia intracellularis swine erysipelas − a review of prevalence and research age, not infection dose, determines the outcome of isospora suis infections in suckling pigs actinobacillus suis septicemia: an emerging disease in highhealth herds key: cord-286683-mettlmhz authors: ortiz-prado, esteban; simbaña-rivera, katherine; gómez-barreno, lenin; rubio-neira, mario; guaman, linda p.; kyriakidis, nikolaos c; muslin, claire; jaramillo, ana maría gómez; barba-ostria, carlos; cevallos-robalino, doménica; sanches-sanmiguel, hugo; unigarro, luis; zalakeviciute, rasa; gadian, naomi; lópez-cortés, andrés title: clinical, molecular and epidemiological characterization of the sars-cov2 virus and the coronavirus disease 2019 (covid-19), a comprehensive literature review date: 2020-05-30 journal: diagn microbiol infect dis doi: 10.1016/j.diagmicrobio.2020.115094 sha: doc_id: 286683 cord_uid: mettlmhz abstract coronaviruses are an extensive family of viruses that can cause disease in both animals and humans. the current classification of coronaviruses recognizes 39 species in 27 subgenera that belong to the family coronaviridae. from those, at least seven coronaviruses are known to cause respiratory infections in humans. four of these viruses can cause common cold-like symptoms. those that infect animals can evolve and become infectious to humans. three recent examples of these viral jumps include sars cov, mers-cov and sars cov-2 virus. they are responsible for causing severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers) and the most recently discovered coronavirus disease during 2019 (covid-19). covid-19, a respiratory disease caused by the sars-cov-2 virus, was declared a pandemic by the world health organization (who) on 11 march 2020. the rapid spread of the disease has taken the scientific and medical community by surprise. latest figures from 20th may 2020 show more than 5 million people had been infected with the virus, causing more than 330,000 deaths in over 210 countries worldwide. the large amount of information received daily relating to covid-19 is so abundant and dynamic that medical staff, health authorities, academics and the media are not able to keep up with this new pandemic. in order to offer a clear insight of the extensive literature available, we have conducted a comprehensive literature review of the sars cov-2 virus and the coronavirus diseases 2019 (covid-19). the viral membrane contains the spike (s) glycoprotein that forms the peplomers on the virion surface, giving the virus its 'corona'or crown-like morphology in the electron microscope. the membrane (m) glycoprotein and the envelope (e) protein provide the ring structure. within the virion interior lies a helical nucleocapsid comprised of the nucleocapsid (n) protein complexed with a single positive-strand rna genome of about 30 kb in length [16] . the first genome of sars-cov-2 named wuhan-hu-1 (ncbi reference sequence nc_045512) was isolated and sequenced in china in january 2020 [12, 16] . the sars-cov-2 genome has similarities to other viruses: approximately 96% similarity to the bat coronavirus batcov rath13; an estimated 80% similarity with sars-cov [16] , and an estimated 50% identity with mers-cov [17, 18] . sars-cov-2 has a positive-sense single-stranded rna genome. it is approximately 30,000 bases in length and comprises of a 5′ terminal cap structure and a 3′ poly a tail. according to wu et al. [19] , this novel coronavirus (ivdc-hb-01/2019 strain) has 14 open reading frames (orfs) encoding 29 proteins. the 5' terminus of the genome contains the orf1ab and orf1a genes. orf1ab is the largest gene and encodes the pp1ab protein that contains 15 non-structural proteins named nsps (nsp1-nsp10 and nsp12-nsp16). orf1a encodes the pp1a protein and also has 10 nsps (nsp1-nsp10) [19] . the 3' terminus of the genome contains four structural proteins: spike (s) glycoprotein; envelope (e) protein; membrane (m) glycoprotein and nucleocapsid (n) phosphoprotein. it also contains 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14) [20] (figure 3b ). the global scientific community from 58 countries have united to study this novel coronavirus by sequencing and submitting 12,059 sars-cov-2 genomes to the global initiative on sharing all influenza data (gisaid) (https://www.gisaid.org/) between december 2019 and april 2020 [21, 22] . sars-cov-2 has accumulated mutations in its rna genome as the outbreak progresses. as an intracellular obligate microorganism, the coronavirus exploits the host cell machinery for its own replication and spread. since virus-host interactions form the basis of diseases, knowledge about their interplay is of great importance, particularly when identifying key targets for antivirals. j o u r n a l p r e -p r o o f ace2 is a type i membrane protein that participates in the maturation of angiotensin, a peptide hormone that controls vasoconstriction and blood pressure [30] . in the respiratory tract, ace2 is widely expressed on the epithelial cells of alveoli, trachea, bronchi, bronchial serous glands [31] , and alveolar monocytes and macrophages [32] . xu et al. reported the [33] rna-seq profiling data of 13 organs with para-carcinoma normal tissues from the cancer genome atlas (tcga; https://www.cancer.gov/tcga) and 14 organs with normal tissue from fantom5 cage (https://fantom.gsc.riken.jp/). these were used to validate the expression of the human cell receptor ace2 in the virus and may indicate the potential infection routes of sars-cov-2 [34] . interestingly, the ace2 receptor is expressed more in oral cavity than lung. this potentially could indicate that susceptibility and infectivity of sars-cov-2 is greater from oral mucosa surfaces [33] . following the binding of the rbd in the s1 subunit to the receptor ace2, sars-cov-2 s protein is cleaved by the cell surface-associated transmembrane protease serine 2 tmprss2, which activates s2 domain for membrane fusion between the viral and cell membrane [35] . a functional polybasic (furin) cleavage site was found at the s1-s2 boundary through the insertion of 12 nucleotides [13, 29, 36] . the s673, t678 and s686 residues of o-linked glycans flank the cleavage site and are unique in sars-cov-2 [29] . in addition to the s glycoprotein -ace2 receptor complex, wang following the release and uncoating of viral rna to the cytoplasm, coronavirus replication starts with the translation of orf1a and orf1b into polyproteins pp1a and pp1ab via a frameshifting mechanism (figure 4 ) [38] . subsequently, polyproteins pp1a and pp1ab are processed by internal viral proteases, including the main protease m pro , a potential drug target whose crystal structure was recently determined for sars-cov-2 [15] . polyprotein cleavage yields 15 mature replicase proteins, which assemble into a replicationtranscription complex that engages in negative-strand rna synthesis. both full-length and multiple subgenomic negative-strand rnas are produced. the former serves as template for new full-length genomic rnas and the latter template the synthesis of the subgenomic mrnas required to express the structural and accessory protein genes residing in the 3′proximal quarter of the genome [37] . coronavirus rna replication occurs on a virusinduced reticulovesicular network of modified endoplasmic reticulum (er) membranes [39] . the assembly of virions is quickly ensued with the accumulation of new genomic rna and structural components. the n protein complexes with genome rna, forming helical structures. then, the transmembrane m protein, localized to the intracellular membranes of the er -golgi intermediate compartment (ergic) , interacts with the other viral structural proteins (s, e and n proteins) to allow the budding of virions [40, 41] . following assembly and budding, virions are transported in vesicles and eventually released by exocytosis. normal immune responses against the majority of viruses involve a rapid containment phase mediated by innate immunity components and -if necessary-a delayed, yet more sophisticated adaptive immunity phase that should be able to eradicate the pathogen andhopefully-generate long-lasting immunological memory. the former includes antiviral type i interferons (ifns), macrophage and neutrophil activation that leads to proinflammatory cytokine production and nk cells on the other hand, anti-viral adaptive immune responses involve a virus-tailored coordinated attack by antigen specific cd8+ cytotoxic t cells (ctls), the th1 subset of cd4+ t helper cells that orchestrates the j o u r n a l p r e -p r o o f immune response against viruses and other intracellular pathogens, specific antibody producing plasma cells, and finally the production of memory t and b cell subsets. immune responses following sars-cov-2 infection can be a double-edged sword. a rapid and robust type i ifn orchestrated response can lead to virus clearance and -given that antiviral lymphocytes are activated and expanded-immune memory. conversely, a late activation of innate immunity, possibly owing to is usually associated with severe pathology that can lead to pneumonia, ards, septic shock, multi-organ failure and, eventually, death. in this line, a delayed type i ifn response and inefficient sars-cov-2 clearance by alveolar macrophages can promote excessive viral replication that can lead to severe pathology accompanied by increased viral shedding and, thus, viral transmissibility. accordingly, in patients whose immune system is weakened or otherwise dysregulated, such as older men with comorbidities, severe covid-19 is clearly more likely to occur [42] [43] [44] . a recent study has demonstrated that the average duration of sars-cov-2 viral shedding was 20 days after covid-19 onset, raising a debate as to the optimal time of patient isolation [45] . however, in terms of transmission viral shedding seems to be more relevant in the early phases of the infection as it can precede covid-19 symptoms by 2-3 days whilst up to 50% of infections are associated with viral shedding by asymptomatic cases [46] [47] [48] therefore, individuals that mount efficient containment-phase immune responses accompanied by decreased inflammatory responses will not experience infection-or immune response-mediated overt manifestations, but may be important silent spreaders of sars-cov-2. type i ifns are mainly produced by plasmacytoid dendritic cells (pdcs) and have a plethora of antiviral effects such as blocking cell entry and trafficking of viral particles, inducing rnase and dnase expression to degrade virus genetic material, enhancing presentation of viral antigens by mhc-i, inhibiting protein synthesis, inducing apoptosis of j o u r n a l p r e -p r o o f infected cells and activating anti-viral subsets such as macrophages and cytotoxic nk cells and t lymphocytes [49] . pathogen recognition receptors like cytosolic rig-i and mda-5 [50, 51] or endosomal toll like receptors (tlrs) 7 and 8 that recognize viral rna [52] are responsible for the activation of signaling cascades that activate the transcription factors nf-kb, interferon regulatory factor (irf) 3 and irf7 that translocate to the nucleus and induce proinflammatory cytokines and type i interferon (ifn) production. in turn, type i ifns activate the downstream jak-stat signal pathway resulting in expression of ifnstimulated genes (isgs) [53, 54] . our experience from sars-cov and mers-cov infection has shown that delayed type i ifn production and excessive recruitment and activation of infiltrating proinflammatory cells (neutrophils and monocytes-macrophages) are possible mediators of lung dysfunction and bad prognostic factors for the outcome of the infection. delayed type i ifn production allows for highly efficient viral replication that, in turn, results in recruitment of hyperinflammatory neutrophils and monocytes. therefore, the pathogen recognition receptors (prrs) of these proinflammatory cells recognize high numbers of their ligands and subsequently secrete excessive amounts of proinflammatory cytokines that lead to septic shock, lung pathology, pneumonia or acute respiratory distress syndrome. it has been shown that in severe cases both sars-cov and mers-cov fruitfully employ an immune evasion mechanism whereby early type i ifn responses to viral infection are dampened [54] . this can be achieved by blocking signaling both upstream, as well as downstream of type i ifn expression. sars-cov can inhibit irf3 nuclear translocation, whereas mers-cov can impede histone modification [56] . additionally, both viruses can inhibit ifn signaling by decreasing stat1 phosphorylation [57] . due to the many sequence similarities of sars-cov-2 with sars-cov and mers-cov it would be enticing to speculate that similar mechanisms are also present, however further studies are needed to shed light to this hypothesis. hyperactivated neutrophils and monocytes-macrophages are the usual source of the cytokine storm. in this aspect, absolute neutrophil counts and neutrophil to lymphocyte j o u r n a l p r e -p r o o f ratio (nlr) were strongly associated with disease severity in a large cohort of covid-19 patients and were proposed as markers of adverse disease prognosis [58] . interestingly, the increased amounts of proinflammatory cytokines in serum associated with pulmonary inflammation and extensive lung damage described both in sars [59] and mers diseases [60] were also reported in the early study of 41 patients with covid-19 in wuhan [41] . evidence shows that the leading cause of covid-19 mortality is respiratory failure caused by acute respiratory distress syndrome (ards). there is an association with a cytokine storm mediated by high-levels of proinflammatory cytokines including il-2, il-7, il-10, g-csf, ip-10, mcp-1, mip-1a and tnf-α. ards was associated with increased fatality and subsequent studies confirmed il-6 and c-reactive protein are significantly upregulated in patients that died compared to convalescent patients [56] moreover, a recent study of 452 patients in wuhan identified that severe cases showed significantly higher cytokines and chemokines such as tumor necrosis factor-α (tnf-α), il-2, il-6, il-8 and il-10 expressed [58] . in accordance with these findings, therapeutic strategies are being tested. a phase 3 randomized controlled trial of il-1 blockade (anakinra) in sepsis has shown significant survival benefit in patients with hyperinflammation, without apparent increased adverse events [61] . currently, a multicenter, randomized controlled trial of tocilizumab (il-6 receptor blockade, licensed for cytokine release syndrome), is being trialed in patients with covid-19 pneumonia presenting with high levels of il-6 in china (chictr2000029765) [62] . moreover, several clinical trials are exploring if the well-established antiviral [63] and anti-inflammatory effects of hydroxychloroquine will be effective in treating patients with covid-19 as has previously been suggested for sars-cov infection [64] . this has also been demonstrated in vitro for sars-cov-2 [65] . in contrast, janus kinase (jak) inhibition has been proposed as a potential treatment in order to reduce both inflammation and cellular viral entry in covid-19 [66] . thus, it comes as no surprise that in a recent correspondence, lancet authors have identified the following potential therapeutic options for cytokine storm syndrome including ards the use of corticosteroids, selective cytokine blockade (eg, anakinra or tocilizumab) and jak inhibition [67] . j o u r n a l p r e -p r o o f virus presentation to the different t cell subsets stands on the crossroads between innate and adaptive immune responses. studies on sars-cov and mers-cov [72] presentation have identified several susceptibility and protection conferring hla alleles. the dearth of similar data regarding sars-cov-2 antigen presentation to t cells and possible virus evasion mechanisms of this process suggests it is a virgin investigation field to be explored. apart from the sustained inflammation and cytokine storm, lymphopenia has been implicated as a major risk factor for ards and mortality in the context of covid-19 [73] . similar findings were described for sars-cov infected patients who had considerable decreases of cd4+ t and cd8+ t cells [69] . however, in convalescent patients specific tcell memory responses to sars-cov were still found six years post infection [74] . showed no reactivity with viral antigens. however, the small number of sera used (n=4) implies that further investigation is needed to corroborate these results [78] . nonetheless, since we are currently in early stages of sars-cov-2 pandemic more studies need to be carried out to shed light on antibody persistence (both igm and igg) and protective effects. recently, macaques re-challenged with sars-cov-2 after a primary infection did not show signs of re-infection, suggesting that protective immunity and memory responses were fruitfully mounted. this finding can also impact vaccine production strategies [79] . importantly, covid-19 convalescent sera was shown to hold promise as a passive immune therapy alternative to facilitate disease containment [80] . to the best of our knowledge, at j o u r n a l p r e -p r o o f least one pharmaceutical company, takeda, is preparing to purify antibody preparations from covid-19 convalescent sera against sars-cov-2 [81] . a recently published case report of a patient with mild-to-moderate covid-19 revealed the presence of an increased activated cd4+ t cells and cd8+ t cells, antibody-secreting cells (ascs), follicular helper t cells (tfh cells), and anti-sars-cov-2 igm and igg antibodies, suggesting that both cellular and humoral responses are important in containing the virus and inhibiting severe pathology [82] . antibody dependent enhancement (ade) is a mechanism whereby non-protective antibodies produced during an infection with an agent either mediate increased uptake of this agent into target cells or cross-recognize a different pathogen and facilitate its entrance to target cells [83] . evidence emerging over the past two decades suggests that antibodies against different coronavirus can cross-react to some extent and mediate ade [84] . ade in the context of sars-cov was thought to be mediated by antibodies produced against 229e-cov [85] and was contemplated as contributing to high mortality rates in china [86] . the described mechanism suggests that low affinity or low title anti-spike protein antibodies rather than neutralizing the virus result in fc receptor mediated infection of immune cells, further aggravating the dysregulation of anti-sars-cov immune responses [87] . indeed, in vitro as well as in vivo experimental models have shown that ade hinders the ability to manage inflammation in the lung and elsewhere. this may lead to ards and other hyperinflammation-induced clinical manifestations also observed in several of the documented cases of severe covid-19 [88, 89] . while the molecular and immunological host response to sars-cov-2 infection has not yet been fully elucidated to confirm ade is occurring, anti-sars-cov-2 antibodies have been shown to partially cross-react with sars-cov, suggesting enhancement is a possibility. with this in mind, ade in populations previously exposed to other coronavirus can partially explain the geographic discrepancies observed in covid-19 pathogenesis and severity. finally, ade can have several implications in vaccine development as low-affinity or low-titer antibody producing vaccines can increment susceptibility rather than confer protection against the pathogen as has previously been described for a dengue vaccine [90] [91] [92] . j o u r n a l p r e -p r o o f detection methods based on nucleic amplification are often used in the case of sars-cov, mers-cov and other viruses, because have high sensitivity and specificity, particularly in the acute phase of infection [93] . case identification and surveillance of covid-19 spread although rt-qpcr assay is considered the gold-standard method to detect viruses such as sars-cov and mers-cov [94, 95] , currently available rt-qpcr assays targeting sars-cov-2 have important considerations. firstly, due to the genome similarity of j o u r n a l p r e -p r o o f sars-cov-2 to sars-cov (82% of nucleotide identity [96] ), some of the primer-probe sets described by different groups and listed in the who coronavirus disease (covid19) technical guidance [97] , have cross-reaction with sars-cov and other bat-associated sars-related viruses, therefore, it is important to run confirmatory tests. loop-mediated isothermal amplification (lamp) is a one-step isothermal amplification reaction that couples amplification of a target sequence with four to six primers, to ensure high sensitivity and specificity, under isothermal conditions (63-65°c), using a polymerase with high strand displacement activity [129] . in the case of an rna sample, lamp, is preceded by the reverse transcription of the sample rna. rt-lamp has been used before for the detection of various pathogens [130] . including sars-cov-2 and other respiratory viruses [131, 132] . recently, it received emergency use authorization (eua) from the u.s. j o u r n a l p r e -p r o o f serological tests also, called immunoassays, are rapid and simple alternatives for screening of individuals that have been exposed to sars-cov-2 based on the qualitative or quantitative detection of sars-cov-2 antigens and/or anti-sars-cov-2 antibodies. there are several types of serological tests available, including elisa (enzyme-linked immunosorbent assay), iift (indirect immunofluorescence test), lateral flow immunoassays and neutralization tests. immunoassays assays are very useful because they allow to study the immune response(s) to sars-cov-2 in a qualitative and quantitative manner. in addition, help to determine the precise rate of the infection [78, 133] , and to determine more precisely the fatality rate of the infection [78] . several sars-cov-2 targeted serological tests are commercially available or in development [134] . a recently developed kit, reported a sensitivity of 88.66% and specificity of 90.63% [135] using sars-cov-2 igg-igm combined antibody rapid (within 15 minutes) test [135] . despite their simple and fast readout and their potential for being used outside laboratory environments (bedside, small clinics, airports, train stations, etc.), serological tests have a critical disadvantage; given the fact that antibodies specifically targeting the virus would normally appear after 6 days or longer [136] after the illness onset [137] , tests based on this principle have a lag period of approximately 4 to 7 days post-infection. during this lag period, infected and non-infected individuals will both result in a negative output. in addition, it is important to highlight that because serological tests depend on the ability to produce antibodies, intrinsic immunological differences and/or responses between individuals, can significantly affect the outcome of these tests. recently, some commercially available immunoassays received ce mark for professional use [138, 139] , and therefore are registered as in vitro diagnostic devices. currently, there are a plethora of antibody tests for covid-19 with variable performance (sensitivity varying from 45 to 100%, specificity from 96 to 100%, reviewed in (foundation for innovative new diagnostics) [134] . different manufacturers of serological assays declare that their assays have no cross reactivity to other human coronaviruses and other respiratory viruses. however, despite the data provided by manufacturers, recent studies highlighted that several of the commercially available tests have sensitivity and/or specificity issues that should be considered for using and analyzing results of many of these tests [140] [141] [142] [143] [144] . j o u r n a l p r e -p r o o f as mentioned before, immunoassays -particularly tests detecting anti-sars-cov-2 igm and/or igg-indicate that the person has been exposed to the virus. in the case of other viral infections, having antibodies targeting a pathogen has often been considered an indication of having immunity against that pathogen [145] . based on this idea, some governments have suggested using serological tests, to determine who has developed immunity against sars-cov-2 and provide positive individuals a "risk-free certificate" or "immunity passports", which would enable them to travel or to return to work, assuming that they are protected against re-infection [146]. however, based on the limited knowledge of how immunity to this virus works [147] , there is not enough evidence to declare a person immune, or to confirm that people who have anti-sars-cov-2 antibodies are protected from a re-infection. even though covid-19 can be diagnosed using qpcr as the gold standard, inadequate access to reagents and equipment has slowed disease detection even in developed countries such as the us. several low cost and rapid tests using different approaches have been described. unlocking) technique for the detection of covid-19 and the detectr (developed by mammoth biosciences) prototype rapid detection diagnosis kit using crispr to detect the sars-cov-2 in human samples have been described [148] . the use of rna aptamers, have recently emerged as a powerful background-free technology for live-cell rna imaging due to their fluorogenic properties upon ligand binding, a technology that could be of use to diagnose sars-cov-2 infection [149] . finally the use of next generation sequence (explify®) might be used to detect and identify bacterial, viral, fungal, and parasitic pathogens by their unique genome sequences [107] . in covid-19 symptomatic infection, the clinical presentation can range from mild to ventilation assistance [151] . the spectrum of symptoms of covid19 infection are characteristic of a mild disease in most of the cases, however, it is important to point that the progression could lead to a severe respiratory distress. asymptomatic infection (while incubation occurs) was described both in the first cases in wuhan and in other cohorts. a group of isolated patients were screened for sars-cov-2, where 17% (629 cases) were positive for the test, and half of these cases had no symptoms. on the other hand, there are reports of cases without overt symptoms in which there were ground glass images in the chest tomography in up to 50% of patients [152] . of the asymptomatic cases studied in wuhan city, the 2.5% of people exposed developed specific symptoms in 2.2 days, and the remaining 97.5% were symptomatic in the following 11.5 days (ci, 8.2 to 15.6 days). the median estimated incubation period was 5.1 days (95% ci, 4.5 to 5.8 days) [153] . some patients with initially mild symptoms had symptom progression over the course of one week [154] . the descriptive studies available so far have concluded that the majority of cases are mild infections (more than 80% of cases); with up to 15% of patients being sever j o u r n a l p r e -p r o o f in most cohorts, and less than 5% have been considered as critical cases with high vital risk [155] . in a study describing 138 patients with covid-19 pneumonia in wuhan, the most common clinical characteristics at the onset of the disease were described. this is consistent with other international cohorts (table 3 ) [150] . it is important to note that fever is not always present and up to 20% of patients could had a low grade temperature between 37.5 to 38 degrees celsius or normal temperature [156] . if these patients required hospitalization, 89% developed a fever during the course of the illness. rarer accompanying symptoms included headache without warning signs, odynophagia and rhinorrhea. gastrointestinal symptoms such as nausea and watery diarrhea were relatively rare [151] . dyspnea develops after a median of 5 to 8 days from the onset of symptoms. it is important to notice that, if dyspnea is an important clinical finding, not all the patients with this j o u r n a l p r e -p r o o f symptom will develop severe respiratory distress or require oxygen supplementation [150] . according to world health organization (who) guidelines, covid-19 infection can present as pneumonia without signs of severity, and could be managed in the outpatient setting. this is applicable to those patients who do not need supplemental oxygen [157] . as previously mentioned, the most serious manifestation of covid 19 infection is pneumonia, characterized by cough, dyspnea, and infiltrates on chest images; the latter is indistinguishable from other viral lung infections. acute respiratory distress syndrome (ards) is a major complication of covid pneumonia in patients with severe disease. this develops in 20% after a median of eight days. mechanical ventilation is implemented in 12.3% of cases [158] . in different case reports, the need for supplemental oxygen via the nasal cannula was required in approximately 50% of hospitalized patients. 30% required non-invasive mechanical ventilation, and less than 3% required invasive mechanical ventilation with or without extracorporeal membrane oxygenation (ecmo) [159] . it is important to mention that the proportion of severe cases is highly dependent on the study population and may be related to the epidemiological behavior of the infection in each country. additionally, the number of people tested will influence the denominator. in italy, the average age of people infected with covid-19 is between 60 and 65 years, and 16% of those hospitalized require admission to the intensive care unit (icu) [160]. the who recommendations had established that severe covid-19 disease could be defined by the following parameters in table 4 [157] . [162] . among the established risk factors for the development of ards is age greater than 65 years, diabetes mellitus and hypertension, in at least 40% of patients [151] . it should be clarified that, although advanced age is identified as a risk factor for a severe infection, those of any age may suffer from severe illness from covid-19. the descriptions made so far of the patients from china have determined that almost 90% of the patients were between the ages of 30 and 79 years (cohort of 44,500 cases) [155] . in other population settings, such as in the united states, more than 60% of confirmed the clinical characteristics of symptomatic cases and their severity has been described. in addition to the symptoms reported by the patients, the findings on physical examination may be absent during mild coivd-19 infection. those with moderate to severe covid-19 infection have various signs during pulmonary auscultation, however the most common findings include: wet rales; global decrease in respiratory sounds and increased thrill [164] . early recognition is essential to classify cases as potential cases and initiate one of the most important measures to contain the pandemic, isolation. 2. anyone who has resided or been traveling in areas where widespread community transmission has been reported. 3. any patient who has had potential exposure through attending events or has spent time in specific settings where cases of covid-19 have been reported. the scenarios described respond to the context of a high suspicion of covid-19 infection. the world health authorities (cdc, who) continually update these contexts. there have been multiple case definitions and clarifications regarding when to perform a covid-19 test: • they have pointed out the importance of fever, cough and dyspnea as sentinel symptoms, since these should form part of the clinical judgment that guides doctors. this allows to expand the group of suspicious patients. • in cases of severe respiratory distress of undetermined etiology and that do not meet the previously indicated criteria, a screening for covid-19 would be indicated. • in areas of limited resources, the suggestion is to prioritize cases that require hospital care, and in this way guide the epidemiological fence to order isolation and protect the most vulnerable people (chronically ill and over 65 years of age), as well as test those with the greatest possibility of exposure (travelers and health personnel). currently, there is no laboratory data profile that is framed in covid 19 infection. from a cohort of 43 patients confirmed with covid 19, these findings were classified as mild, moderate and severe disease [165] . high levels of d-dimer and more severe lymphopenia have been associated with mortality due to a prothrombotic state that determines multi-organ failure. in general, leukopenia and / or leukocytosis can be found in the interpretation of blood biometry, however, the most widely described finding is lymphopenia [166] . it should be considered that in the context of viral pneumonia biomarkers such as procalcitonin and pcr are not useful, as often these biomarkers are in the normal range for patients with covid-19. among other findings, descriptive studies have reported considerable elevations of lactate dehydrogenase and ferritin as well as alteration in aminotransferases; although elevation ranges for these parameters have not been established [167] . about the imaging findings, covid 19 viral pneumonia has similar features on imaging to other viral infections. although computed tomography (ct) is the test of choice, it is not useful for a definitive diagnosis due to the wide variety of images that can be found in patients with covid 19 infection. this statement is derived from a large cohort of more than 1000 wuhan patients, where rt-pcr confirmation of covid 19 and chest ct images of these patients were correspondingly analyzed. ct images were determined to have a sensitivity of 98%; however, the specificity was only 25% [168] . in general, the majority of descriptive studies concur that the finding of ground glass opacifications is most common. it is typically basal and bilateral, and rarely associated with underlying consolidation. a multicenter chinese study that retrospectively reviewed the ct scans of 101 patients found that 87% had typical ground-glass images and up to 53% had this finding along with consolidations. these findings were more frequent in the most j o u r n a l p r e -p r o o f severe and older age groups of patients [169] . pleural effusion (4%), and lymphadenopathy (2.7%) [170] . the emergence and outbreak of sars-cov-2, the causative agent of covid-19, has rapidly become a global concern that highlights the need for fast, sensitive, and specific tools to monitor the spread of this infectious agent. diagnostic protocols to detect sars-cov-2 using real-time quantitative polymerase chain reaction (rt-qpcr) were listed on the world health organization (who) website as guidance, however, various institutions and governments have chosen to establish their own protocols that might not be publicly available or listed by who. there are important challenges associated with close surveillance of the current sars-cov-2 outbreak. firstly, the rapid increase of cases has overwhelmed diagnostic testing capacity in many countries, highlighting the need for a high-throughput, scalable pipelines for sample processing [171, 172] . secondly, given that sars-cov-2 is closely related to other coronaviruses [96] , some of the currently available nucleic acid detection assays can result in false positives [173] . thirdly, critical concern for molecular detection is the low sensitivity reported for rt-qpcr assays [168] and serological tests [135] , particularly in the early stages of infection. additionally, most of the available rt-qpcr assays require sample processing and equipment only available in diagnostic and/or research laboratories. it is important to mention that coinfection is a possibility, as some reports from italy and in spain, less than 1% of cases in a cohort correspond to plwh, which have had a satisfactory evolution and less than half required an intensive care unit [177] . in the us, of the 5,700 hospitalized patients in the new york area, only 47 patients had hiv, while in san francisco, data was published on 1,233 people who had diagnosed with sars-cov-2 infection, of which less than 3% had hiv and none of them developed severe covid-19 [178] . despite the existence of in vitro studies on the efficacy of the use of lopinavir / ritonavir, it is currently known that its effect in cases of moderate and severe covid-19 is null, and therefore at the moment no recommendation can be given nor how treatment, much less as prophylaxis [179] . this clarification is important given that there is a belief that plwhs could be protected if they take antiretroviral therapy. therefore, current recommendations for plwhs are to maintain antiretroviral therapy with the goal of controlling hiv as well as following the same standards of care as the general population to avoid acquiring a sars-cov-2 infection [180] . regarding sars-ncov infection in pregnant women, there is currently limited evidence about the effect of the virus on the mother or fetus. however, due to the physiological changes typical of pregnancy, especially on the immune system (immunosuppression) and the cardiopulmonary system, pregnant women are thought to be more susceptible to developing severe symptoms when they acquire the viral respiratory disease. in 2009, when influenza a h1n1 infection occurred, only 1% of the infected population were pregnant, yet they accounted for 5% of infection-related deaths [181] . pregnancy (25%) [182] . in another study of 11 pregnant patients infected with mers-cov, 9 presented adverse results (91%), 6 neonates were admitted to the neonatal intensive care unit (55%) and three of them died (27%) [183] . however, it is important to note the small sample size which could increase the risk of bias and low power of the study. with information obtained so far from the wuhan sars-cov-2 outbreak, the infection appears to be less severe for pregnant women, compared to previous sars-cov and mers-cov outbreaks [181] . however, it is important to take into account that the data from covid-19 infection should be monitored with a doppler ultrasound every two weeks, due to the risk of developing intrauterine growth restriction [187, 188] . the time of termination of the pregnancy, as well as the method, also depend on several factors, including gestational age, maternal condition in relation to sars-cov-2 infection, presence of maternal comorbidities, and fetal condition. decisions must be made collaboratively during multidisciplinary team discussions, with individualized management plans established for each patient [189] . a diagnosis of covid-19 alone is not an indication for the termination of pregnancy, rather it should be made in combination with consideration of morbidity and mortality of both the fetus and mother. after delivery, the use of corticosteroids is recommended for antenatal fetal lung maturation, with betamethasone or dexamethasone [190] ; taking special care in critically nursing patients, as this may worsen their condition, and may delay delivery, which is necessary for the management of these patients [187, 191] . the symptoms children present with are similar to adults, as is the incubation period ranging from 1 to 14 days (mean of 5.2). a cough is the most frequent presenting symptom (65%) followed by fever (60%). there is a higher occurrence of gastrointestinal symptoms including diarrhea (15%), nausea, vomiting (10 %) and abdominal pain. these gastrointestinal symptoms are usually more variable in children than adults and are sometimes the only clinical manifestation in associations with fevers. [192, 193] . the clinical progression and disease severity in pediatric patients is markedly different from that of adults. over 90% of affected children are asymptomatic or have mild to moderate disease [192] . the majority of serious cases in children are related to those with significant comorbidities such as heart disease, immunosuppression, etc. to date of this review, only a few cases of children without underlying comorbidities have died as a result of covid 19 have been reported. this difference of severity of illness between adults and j o u r n a l p r e -p r o o f children has not been clarified, however, several theories have been postulated. these include that children express more ace2 receptors in their lungs which confer some protection to severe injuries such as those caused by rsv and which would decrease dramatically with age [194, 195] . immunological factors may also influence outcomes, as in childhood we are most exposed to frequent challenges including recent seasonal viruses such as rsv in the winter months. most likely, it is multifactorial and depends on factors from both the host and the virus itself [195] . abnormal radiological (ct) findings are found in asymptomatic children and consist of bilateral lung lesions (50%). elevated crp (creactive protein), procalcitonin pct (80%), and liver enzymes are present in most affected children, unlike adults in whom pct is not a reliable marker. virus elimination via the stool even after the negativity in the nasopharyngeal mucosa and the disappearance of symptoms makes them a potential source of contagion through the fecal-oral route [196] . patients with cancer are generally more susceptible to infections than healthy people, because they have a state of systemic immunosuppression that is exacerbated during chemotherapy or radiotherapy [197] . in china, according to national surveillance data, coronavirus infection occurs in 1.3% of patients with malignant tumors. this is a much higher proportion than the general incidence of 0.3% [198] . p<0.0001) even after adjusting for age [197] . further research, completed in a tertiary hospital in wuhuan -china similarly found that 25% of patients with cancer and sars-cov-2 infection died, most of them over 60 years of age [199] . due to these findings, it has been proposed by many international entities that during the pandemic, for prevention, an individualized plan based on the patient's specific conditions is required, with the aim to minimize the number of visits to health institutions.  for early-stage patients with need of post-surgical adjuvant chemotherapy, especially those whose clinical, pathologic, and molecular biologic staging suggest a better prognosis, the start time of adjuvant chemotherapy may be delayed up to 90 days after surgery without affecting the overall effect of treatment [200] .  for patients with advanced cancer, the main approach should be to minimize hospitalization in covid-19 positive installations. replacing the existing intravenous treatment regimen with oral chemotherapy during this special period may be considered, to ensure that treatment is not interrupted for a long time during the pandemic [201] . however, if there is a suspicion of covid-19 infection in this population group, the same updated diagnostic guidelines and the corresponding management should be followed depending on their severity of illness. moreover, an individualized follow-up plan should be outlined due to higher likelihood of complications in this group of population [202] . it should be noted that patients attending out-patient appointments for cancer have higher levels of anxiety, depression and other mental health problems than the general population. studies have shown that approximately 50% of malignant tumor survivors have a moderate to severe fear of tumor recurrence [203] . for this reason, psychologist surveillance of outpatients in quarantine or during hospitalization should be considered. reported complications derived from covid-19 describe a severe disease that requires management in an intensive care unit (icu) in approximately 5% of proven infections. j o u r n a l p r e -p r o o f appear to be most susceptible to the life-threatening complications. the risk of patient-topatient transmission in the icu is currently unknown, therefore adherence to infection control precautions is paramount [204, 205] . progressive deterioration of respiratory function is undoubtedly the most common and lifethreatening complication of the infection. the prevalence of hypoxic respiratory failure in covid-19 patients is 19%, and it can progress to acute respiratory distress syndrome (ards), with the need of mechanical ventilation support at 10.5 days on average. one study found that between 10 and 32% of hospitalized patients require admission to the icu due to respiratory deterioration [205] . as respiratory complications are the most common cause of severe deterioration, early identification of them will undoubtedly help in timely support. support provided should be adapted to take into account risk factors such as advanced age, neutrophilia and organic dysfunction for the development of ards. the diagnostic support of pulmonary tomography is undoubtedly a valid tool; images in patients with different clinical types of covid-19 have characteristic manifestations, but it can become an operational problem due to the difficulty in performing imaging on critically ill patients. on the contrary, lung ultrasound at the bed-side could provide an alternative to radiographs and tomography during the diagnosis of covid-19 [206, 207] . since more than 70% of hospitalized patients will require supplemental oxygen, it is recommended that oxygen should be started when pulse oximetry values fall below 90%. an upper-limit of 96% saturation should be established, since higher values have been shown to be harmful [208, 209] . hemodynamic deterioration has a variability of presentation, this depends on the study population and the definition [223] , the presence of shock in the intensive care unit may be present between 25 to 35% [204, 224] . cardiomyopathy related to viral infection is one of j o u r n a l p r e -p r o o f the main causes of hemodynamic detriment, occurring in up to 23% of patients with covid-19 [43] . hemodynamic failure is one of the main causes of death in these patients, with percentages of up to 40%, inconclusive risk factors are associated to date such as diabetes, hypertension, lymphopenia, and elevation of d-dimer [225] . acute kidney injury (aki) is present in up to 12% of critically ill patients, podocytes and proximal tubule cells are potential host cells for sars-cov-2, caused by the virus induced cytopathic effect. the diagnosis is based on markers of early kidney injury and urinary output [210] . initial management of shock is based on fluid resuscitation, based on the application of dynamic parameters to predict response to fluids, such as variation in stroke volume (svv), variation in pulse pressure volume (ppv) and change in stroke volume with passive leg elevation or fluid challenge above static parameters [225] . variables such as skin temperature, capillary refill time and/or serum lactate measurement are currently valid tools to assess shock. the volume of liquids used in resuscitation should be restricted and administered in relation to dynamic assessment. a liberal water resuscitation strategy is not recommended, rather a balance of crystalloids over colloids as resuscitation liquids should be encouraged and avoiding the use of hydroxyethyl starches, albumin, dextrans or gelatins [226, 227] . indirect evidence suggests that the target mean arterial pressure (tam) for patients with septic shock is 65 mmhg using vasoactive support [228] . the recommendation of norepinephrine use as the first agent is maintained. if norepinephrine is unavailable, vasopressin or epinephrine could be used, avoiding the use of dopamine as the initial vasopressor due to the potential development of arrhythmias [229, 230] . in patients with covid-19 and shock with evidence of cardiac dysfunction and persistent hypoperfusion despite fluid resuscitation and norepinephrine use, dobutamine as inotropic is recommended. given the development of refractory septic shock, the suggestion of the use of hydrocortisone in continuous infusion is maintained, as indirect evidence, this in favor of reducing the length of stay in the icu and the resolution time of the shock [229] . according to the investigative mission of the who in china, the case-fatality rate ranged other reports from china have coincided with this clinical risk profile, for example, a study that included 41 confirmed cases, 12 patients who had ards had as main underlying diseases: diabetes and high blood pressure. of these cases, 6 patients died [156] . according to who, the recovery time is estimated to be two weeks for patients with mild infections and three to six weeks for those with serious illnesses. on the other hand, cdc established that people who had symptoms in the mild to moderate spectrum and maintained home isolation have a resolution of 3 days after the fever decrease, and there was a substantial improvement in respiratory symptoms, even without use of medications. isolation may be limited to 7 days from resolution of symptoms, however, it must be adapted to the population circumstances of the epidemic [158] . the evolution of the epidemiological curve in covid-19 outbreak makes consider containment strategies in china primarily, and other countries based on nonpharmaceutical interventions (npis). according to the who, the most effective measure is hands washing [231] . combination as public health measures reduced contact rates in the population and therefore reduce virus transmission (table 6 ) [232] . table 6 non -pharmacological measures. increasing the level of hand cleanliness to 60% in places with a high concentration of people, like all airports in the world would have a reduction of 69% in the impact of a potential disease spreading [233] . the epidemiological evolution of the covid-19 pandemic through phases has required the application of specific measures according to the time or phase in which the virus is found in each country. the evolution in the non-pharmacological measures has been as variable as the pharmacological ones, in such a way, since january to march, it was ensured that the use of face masks was limited only to people who had contact with epidemiological foci, not to healthy people [234] . this concept was also reinforced by cdc, in order to optimize the use of masks for health workforce. definitely the course of the pandemic was changing rapidly, which also demanded the change from containment measures to mitigation. the recommendations in the current context remain regarding the use of a facial mask in the community, but its optimization is important for health professionals. the use of the mask is not a substitute for handwashing and social distancing measures, as these ones together allow avoiding viral particles in aerosols or drops, as a low cost and accessible measure for general population [235, 236] . there is still non-specific information for the recommendation of masks, in general, having in several studies claims that surgical or cotton cloth masks do not prevent the spread of the sars-cov-2 virus [237] . the evidence about the transmission of the virus in the asymptomatic period also changed the containment measures, suggesting the community use of masks. it is from this that the recommendations for the rational use of masks arise since in some j o u r n a l p r e -p r o o f countries the massive use of n95 masks was reported, masks indicated for the use of medical personnel [238] . regarding to this non pharmaceutical recommendations, the studies suggest to priories the resources on vulnerable population, in endemic areas, older people, adult with comorbidities and health workforce. studies are still needed on the duration of the protective effect of the masks and above all the possibility of their reuse for resource optimization. meanwhile the most important recommendation continues to be its use in addition to hand hygiene and social distancing [238] . therapeutic j o u r n a l p r e -p r o o f this effect is reinforced by azithromycin. there were the best results in terms of viral load reduction, even though is mentioned some limitations in the study like small sample size, a short long-term outcome follow-up, and dropout of six patients from the study [246] . concerning to mortality rates, a study was conducted in new york with 1376 patients (0.63-1.85). thus, it concludes that the use of hcq is not associated with either a decreased or increased clinical impairment, intubation or death [247] results reinforced by other study in 1438 hospitalized patients with covid-19 diagnosis in new york city, whom received treatment with hydroxychloroquine, azithromycin, or both drugs was not associated with significantly reduction in mortality [248] . relating to safety of this drug, in a study carried out in 200 patients in which the theoretical complications of the use of hcq and its combination with macrolides (azithromycin) were assessed by serial electrocardiograms, the following results were obtained. in 5% of patients (10) received chloroquine, 95% (191) received hcq, and 59% (119) [252] . supportive therapies in immune regulation, together with the use of antivirals, are important to take into account, especially in those patients in a serious and critical state, in which they could improve the clinical response and perhaps avoid residual lung injuries. the convalescent plasma is extracted from recovered individuals from an infection, being an antibody transfer medium to provide passive immunity (neutralizing antibodies and globulin). the goal is to provide a rapid immune response until the patient can develop their own active immune response in the hope that there will be clinical improvement [253] . improvement in most individuals, as well as viral suppression 7 days after treatment [255] . in the same country, at the shenzhen hospital, 5 cases of patients with severe covid-19 were reported who met criteria for acute respiratory distress syndrome (ards), who were administered convalescent plasma (titration greater than 1: 1000 and neutralizing antibodies greater than 40). it was found that clinical recovery occurred approximately 12 days after the transfusion (4 patients) and 3 of the 5 patients were discharged 55 days after admission. it is important to mention that this group of patients also received antivirals, methylprednisolone and all the necessary support measures in intensive care [256] . other drugs like ivermectin, nitazoxanide, and others have been studied in the context of covid-19 treatment, but the results are inconsistent. all of the clinical trials evidence, supporting or against the use of mentioned drugs are detailed in (supplementary table 2 ). this review summarized some drug repurposing agents previously known to has efficacy against other virus like sars-cov, mers-cov, influenza. actually, exist some new drugs with high potential action on targets for covid-19 therapeutics. it is important to notice that there is no specific treatment for the coronavirus approach. in context of the scientific evidence exposed and the particular clinical features of each patient, the reader will be able to make the best clinical and therapeutic decisions. when it comes to vaccine design and manufacturing, the main objectives are to ensure its safety, its efficacy in activating specific adaptive immune responses and the production ofideally-long term memory. thus, eliciting protective immune responses including neutralization antibodies and/or ctl generation is of paramount importance. huge challenges need to be tackled in order to minimize the long and cumbersome process of vaccine generation. among them, candidate antigen targets need to be identified, immunization routes and delivery systems investigated, animal models set, adjuvants optimized, scalability and production facility considered, target population selected, and vaccine safety and long-term efficiency evaluated. currently there are no approved vaccines against any human coronavirus, suggesting that their generation is quite novel. several candidate vaccines against sars-cov had shown promise reaching phase i or phase ii clinical trials [258, 259] , but the rapid containment of sars-cov expansion rendered them redundant, did not allow for a test population for phase iii trials and, therefore, put their further assessment to a halt. ctl memory could last up to 11 years after infection [260] . these data suggest that vaccine strategies employing viral structural proteins that can elicit effective, long-term memory t cell responses could yield fruitful results. on the other hand, the s1 spike protein region containing the ace receptor binding domain (rdb) is the obvious option when neutralizing antibody responses are considered [261] [262] [263] . indeed, a candidate sars vaccine antigen consisting of the rbd of sars-cov spike protein was created and found it could elicit robust neutralizing antibody responses and long-term protection in vaccinated animals [264] . the fact that covid-19 convalescent sera shows potential as a therapeutic approach [80] aligns with the theory that efficient b cell responses are mounted and lead to production of protective antibodies. two different groups, using an immunoinformatic approach mapped several ctl and b cell epitopes on different proteins of the virus [265, 266] . moreover, various ctl epitopes were found to be binding mhc class i peptide-binding grooves via multiple contacts, illustrating their probable capacity to elicit immune responses [265, 266] . consequently, these identified b and t cell epitopes could be potential targets for therapeutic vaccines. however, important safety considerations should be taken into account before releasing a new vaccine in the market. previous studies on macaque models have shown that a vaccineinduced anti-spike protein antibody at the acute stage of sars-cov infection can provoke severe acute lung injury [267] . similar observations of sars-cov vaccine-induced pulmonary injury have also been described in multiple several murine and monkey animal models [268] . an additional factor that needs to be checked in phase ii and iii trials is that the vaccine does not cause ade of the pathogen, as has previously been described. such concerns have risen in the context of a dengue vaccine [269] . the pharmaceutical companies that are currently on a race to produce a vaccine for covid-19 along with the vaccine developing strategies they are using are summarized in table 8 and figure 7 . as can be easily deduced from table 8 hospitalization and admission to already heavily charged icus due to these pathologies that could prove critical for weaker health systems that would struggle to carry the burden of combined outbreaks. moreover, vaccinating health care workers is crucial for reducing the risk of absence due to disease, thereby strengthening the healthcare workforce and minimizing the risk to infect covid-19 hospitalized patients with additional pneumoniacausing pathogens. lastly, covid-19 patients vaccinated for influenza and streptococcus pneumoniae allow their immune system to focus on one pathogen and, therefore, give it a better fighting chance against sars-cov-2 infection [276] . high risk groups prioritized for vaccination for these two pathogens include pregnant women, persons with immunocompromised immune systems (either due to congenital or acquired immunodeficiencies), children, adults ≥ 65 years and health care professionals. j o u r n a l p r e -p r o o f heat or chemical treatment inactivation. f) attenuated live pathogen vaccine strategies consist in administering a live pathogen that due to cell culture passaging has lost its virulence. they usually elicit robust and long-term memory immune responses without the need to administer an adjuvant. g) in dna vaccines the dna codifying a highly immunogenic antigen is administered and captured by professional antigen presenting cells (apcs) leading to antigen production and presentation by these cells. h) moderna's vaccine candidate already in phase i clinical trials uses an mrna vaccine approach whereby the genetic information codifying for the s protein of sars-cov-2 is delivered in lnps to enhance absorption by apcs. once uptaken by apcs the mrna induces the expression of s antigen that is subsequently mounted on and presented by mhc molecules to elicit adaptive immune response. numerous studies confirm that climate has an impact on virus (i.e., influenza, coronavirus, etc.) spread through manipulating the 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zinc for the prevention and treatment of sars-cov-2 and other acute viral respiratory infections date: 2020-08-01 journal: adv integr med doi: 10.1016/j.aimed.2020.07.009 sha: doc_id: 310239 cord_uid: mmvuij3k nan zinc may potentially reduce the risk of sars-cov-2 infections and shorten the duration and severity of illness, including recovery from stroke, through several mechanisms. indirect evidence from systematic reviews have found zinc supplementation is effective for the prevention of acute respiratory infections in young children and zinc lozenges may reduce the duration of the common cold in adults. safety concerns associated with high doses or prolonged intake of zinc include anosmia (loss of smell) and copper deficiency. as of the 9 june 2020, the preliminary findings of a rapid review of zinc for the prevention or treatment pending any definitive evidence, clinicians might consider assessing the zinc status of people with chronic disease co-morbidities and older adults as part of a sars-cov-2 clinical work-up, as both groups have a higher risk of zinc deficiency/insufficiency and poorer outcomes from sars-cov-2. supplementation might be indicated for those with low or borderline low results, low dietary intake and/or increased needs. the global covid-19 pandemic has prompted an urgent search for pharmaceutical and traditional, complementary and integrative medicine (tcim) interventions. data from all countries indicate that the case fatality and morbidity rates from sars-cov-2 increases with age and for those with noncommunicable chronic disease co-morbidities. [1] [2] [3] [4] notably, zinc deficiency/insufficiency is prevalent in populations aged over 71 years, [5] [6] [7] [8] [9] , in people with chronic diseases [10] [11] [12] including diabetes, [10, 12, 13] and cardiovascular diseases [10, 12] and hospitalised patients following stroke [14] -see box 1. [ insert box 1] j o u r n a l p r e -p r o o f south-east asia, sub-saharan and central and south american regions, however, marginal deficiencies are also prevalent in developed regions. [33, 34] assessment of zinc status is notoriously difficult due to absence of sensitive and precise biochemical indicators. the most reliable methods involve combining a clinical assessment with laboratory tests assessing tissue concentrations of zinc in plasma or hair. [35] clinical manifestations of mildmoderate zinc deficiency include recurrent infections, slow tissue repair, rough skin, mental lethargy, irritability, headaches and reduced lean body mass. [36] assessment of dietary zinc with validated food frequency instruments may help identify dietary insufficiency [37] however zinc status is still likely to be underestimated due to individual physiological characteristics. [31] for instance, whilst zinc insufficiency/deficiency is known to diminish antibody and cell-mediated immunity in humans that in turn increases the risk of infections, this may only become apparent upon immune system provocation. [38, 39] through several mechanisms, zinc has the potential to reduce the risk of viral respiratory tract infections, including sars-cov-2, and shorten the duration and severity of illness. the authors of a recent non-systematic narrative review of the underlying mechanisms postulate that along with its direct antiviral properties, zinc has the potential to reduce inflammation, improve mucocillary clearance, prevent of ventilator-induced lung injury, and modulate antiviral immunity. in vitro studies have demonstrated that zinc can inhibit the enzymatic activity and replication of sars-cov rna polymerase and may inhibit angiotensin-converting enzyme 2 (ace2) activity. [40, 42, 43] the antiviral effects of zinc are also hypothesised to potentiate the therapeutic effects of chloroquine, [44] , as chloroquine acts as a zinc ionophore increasing zn 2+ influx into the cell. [40] zinc may also modify the host's response to an infection as it is an essential co-factor element with a broad range of functions in the body. zinc has an essential role in immune and airways function, wound healing and tissue repair that in turn, may delay or prevent recovery from viral respiratory illnesses. [45] [46] [47] [48] [49] [50] [51] other consequences of zinc deficiency include an increased risk of vitamin a deficiency that is also critical for immune function, due to carrier proteins and activation enzymes being dependant on sufficient zinc status. [52] the potential role of zinc as an adjuvant therapy for sars-cov-2 may be broader than just antiviral and/or immunological support. zinc also plays a complex role in haemostatic modulation acting as j o u r n a l p r e -p r o o f an effector of coagulation, anticoagulation and fibrinolysis . [53, 54] zinc is also essential for neurological function and normalisation of zinc intake has been shown to improve neurological recovery following stroke. [14] the effectiveness of zinc in preventing or treating sars-cov-2 infections is yet to be systematically evaluated and, along with other nutritional supplements, was not mentioned in a recent narrative review of tcim for the treatment of coronavirus disease 2019 . [55] the findings of systematic reviews of related populations are promising; however, the reviews are limited by population, intervention, or are out of date. [56] [57] [58] a 2016 cochrane review of six rcts concluded zinc supplementation was effective for the prevention of pneumonia in children aged two to 59 months. [57] unlike an earlier review in 2000 of seven rcts with adult participants and one rct with children, [59] an updated 2011 systematic review of 13 rcts found a dose-dependent effect of zinc lozenges compared to placebo controls for reduced duration of common colds in adults. [60] daily dosages less than 75mg of zinc had no significant effect on duration of colds, however, daily dosage over 75mg reduced the duration of colds by 42% (95% ci: 35% to 48%). in a subsequent 2017 systematic review of seven rcts of zinc lozenges with a daily dose >75mg, a smaller reduction of 33% (95% ci 21% to 45%) in the duration of common colds was found. [61] no differences in duration were found for daily doses of 192-207mg compared to doses of 80-92mg. other formats of zinc for preventing or treating upper respiratory infections were examined in three cochrane systematic reviews, however, all were withdrawn. [56, 62, 63] a protocol for the systematic review of zinc for prevention and treatment of common colds was withdrawn in 2019 due to noncompletion within the editorial time-frame. [64] search strategy: the primary objective of this rapid review was to assess the effects of zinc on the incidence, duration and severity of acute upper or lower respiratory tract infections caused by sars-cov-2 infection in people of any age and of any zinc status when used as a preventive supplement or as a therapy. the secondary objectives are to assess the effects of zinc on the incidence, duration and severity of assessed rob and extracted data for each study. primary studies included were randomized controlled trials (rcts) and quasi-randomised controlled trials. there were no date nor language restrictions, however, studies published in languages other than english or chinese are yet to be translated. included were any zinc conjugates, such as salts or amino-chelates as a single ingredient, in any form (e.g. tablet, syrup, lozenge, gel, spray, liquid), dose and duration, administered via oral, intranasal, sublingual, transdermal, intramuscular or intravenous routes. excluded excluded were systematic reviews, non-randomised studies of interventions and studies without a concurrent control, such as case series and case reports. excluded were people with respiratory tract infections or other upper/lower respiratory illnesses when the cause was confirmed not to be a viral infection, or a non-viral cause is common. excluded were co-interventions and zinc administered alongside other nutraceuticals, herbs or pharmaceuticals unless both the intervention and control groups received the co-intervention. the exception were co-ingredients with the primary purpose to facilitate absorption (e.g. vitamin b12) or cellular retention (e.g. vitamin b6 or magnesium) of zinc. the a total of 1,625 records were retrieved from the database searches, of which 1,182 records remained after duplicates were removed. a further 981 records were excluded at title and abstract screening, and 80 following full-text screening (due to ineligible study design n=29, population n=11 or intervention n=32; full-text not available n=7; or awaiting translation n=1), leaving 121 records reporting 122 primary studies (86 published in english and 35 in chinese). one study published in spanish is pending translation. four trials specific to sars-cov-2 were included, all of which are currently ongoing, and the investigators have been contacted are yet to report their results. a further 15 ongoing trials were excluded as the interventions used zinc in combination with other nutraceuticals (most commonly vitamin c and d) and/or as an agonist (additive) to hydroxychloroquine. as such, the independent effects of zinc cannot be determined. of the remaining 118 published studies, none investigated zinc for prevention or treatment of acute respiratory infections caused only by a coronavirus infection. most of the studies (79%) evaluated zinc for treating or preventing upper and/or lower acute respiratory infections in children. (table 1 ). all the studies of adult participants were for acute upper respiratory infections i.e. the common cold (table 1) , of which 21 were naturally occurring infections and six inoculated the participants with human rhinovirus species. the prevention effect of zinc was assessed in a variety of ways, mostly as the incidence or recurrence of respiratory infections as reported by study clinicians, the participants' physician or other healthcare workers, parents or self-reports, hospitalisation and/or laboratory tests. treatment effects for severity and duration included time to symptom resolution, fever or respiratory distress, time in hospital, viral shedding, and self or clinician reported clinical severity. a wide range of zinc formulations and dosages were used, including lozenges, nasal gels and sprays, and oral zinc delivered in syrup, tablet or capsule formats. only one study evaluated intravenous zinc. is a four-arm pragmatic rct comparing zinc gluconate only, zinc gluconate and vitamin c, vitamin c only, and usual care (standard prescribed medication/supplements). the dose of zinc gluconate is 50mg daily, taken at bedtime. the primary outcome is the number of days required to reach a 50% reduction in symptom severity score (derived from a composite self-rating score of fever, cough, shortness of breath and fatigue rated on a 0-3 scale). secondary outcomes are time to symptom resolution for each symptom, total symptom composite score at day 5, proportion requiring hospitalisation, use of prescribed adjunctive medicines, and adverse events. methodological limitations include subjective primary outcome measures from unblinded participants, potential uncertainty around the quality and quantity of the ingredients in the supplements, [68] a potentially insufficient dose of elemental zinc and that the usual care group may use any combination of readily available prescribed medications / supplements, including zinc or vitamin c. strengths of the pragmatic design include a capacity to inform 'real-world' decisions about any benefits and risks of additional zinc supplementation using products that are readily available compared to usual care alone. the second study, "high-dose intravenous zinc (hdivzn) as adjunctive therapy in covid-19 positive critically ill patients: a pilot randomized controlled trial" (actrn12620000454976), is being conducted in a hospital setting in australia. hdivzn is a two-arm, double-blind rct comparing intravenous zinc chloride (0.5mg/kg/d) or placebo in 250ml saline bags infused daily over 3-6 hours for seven days. hdivzn aims to recruit 160 patients who are hospitalised with sars-cov-2 infection. the primary outcome is oxygenation. secondary outcomes are concerned with feasibility, including adequacy of blinding, availability/delivery/storage of the zinc infusions and per-patient costs. methodological strengths include blinding and the use of an objective primary outcome measure. limitations include not assessing any other clinical outcomes listed in the core outcome set (cos) for clinical trials on covid-19. [4] the dose of zinc, approximately 50% more than the minimum daily requirement and without an intracellular transporter co-factor, may be insufficient to effect change of the outcome measurements. [69] given this is a single-centre trial located in australia with a low incidence of sars-cov-2, as of the 14 th june 2020, no eligible participants had been recruited to the study; and, according to the investigator a/professor ischia, "due to the low numbers of covid-19 infections, the trial is unlikely to reach full recruitment to achieve its desired statistical power." [70] prevention of sars-cov-2 is being evaluated in a multicentre trial of 660 military health professionals exposed to sars-cov-2 and located in tunisia (nct04377646: a study of hydroxychloroquine and zinc in the prevention of covid-19 infection in military healthcare workers (covid-milit)). participants will be randomized to one of three study arms; either hydroxychloroquine and zinc, hydroxychloroquine and placebo, or two placebo controls. in covid-milit, hydroxychloroquine 400 mg will be administered at day 1 and day 2, then as a weekly dose for up to 2 months. zinc will consist of 15mg per day for up to two months. the primary outcome is the frequency of infection at two months, secondary outcomes are frequency of ten symptoms and adverse events. the low dose of zinc will provide minimum intake required for health. the treatment of sars-cov-2 with either hydroxychloroquine plus zinc compared to hydroxychloroquine alone will be evaluated in 80 hospitalised adults with confirmed sars-cov-2. this study, registered on the iranian clinical registry (irct20180425039414n2; the effect of zinc on the treatment and clinical course of patients with sars-cov2 (covid-19)), is being conducted at the amin hospital in isfahan. participants will be randomised to either hydroxychloroquine 200 mg every 12 hours plus zinc 220mg twice daily, or to hydroxychloroquine alone during their hospital stay. outcomes include mortality rates, length of hospital stay and the clinical course of sars-cov-2 (fever, shortness of breath, cough, blood oxygenation (sao2) and hemodynamic parameters). the treatment j o u r n a l p r e -p r o o f study was designed to ensure that all study participants diagnosed with sars-cov-2 received treatment. preliminary findings of this rapid systematic review found limited direct evidence evaluating zinc for the prevention or treatment of sars-cov-2, as the results of the four registered rcts that were identified are pending. once available, the findings from the covidatoz trial that is evaluating the comparative effectiveness of zinc supplements against vitamin c and usual care for treatment of mild to moderate symptoms of community-based sars-cov2-19 infections, will be relevant to the general population who can self-prescribe, along with a wide range of health practitioners who provide tcim advice. the findings from the hdivzn trial that is evaluating the efficacy and safety of intravenous zinc infusions for hospitalised patients may provide safer and less expensive therapeutic options compared to pharmaceuticals currently being evaluated. delivery of the intervention, however, requires medical oversight that will restrict its application to hospital settings and perhaps a few primary care settings. the two comparative effectiveness studies will not explain the preventative or treatment effects of zinc as a stand-alone therapy, however they will explain the potential benefits of zinc adjunct to hydroxychloroquine in populations at high risk of zinc deficiency, [34] for the prevention of sars-cov-range of functions in the body that modulate immunity, respiratory tract inflammation, coagulation and neurological function to name a few. [14, [38] [39] [40] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] pending any definitive evidence, it might be reasonable for clinicians to consider assessing the zinc status of people with chronic disease co-morbidities and older adults as part of a sars-cov-2 clinical work-up, as both groups have a higher risk of zinc deficiency/insufficiency and poorer outcomes from sars-cov-2. zinc status can be assessed by taking a diet and clinical history (see box 1), clinical examination and laboratory tests. plasma zinc may be more reliable than serum zinc and whilst hair mineral analysis is another option a timely result may not be available. [35] for prevention of sars-cov-2 and most importantly for general health, given that zinc supplements are readily available, they may be indicated for people with low or borderline low results, low dietary intake and/or increased needs. to optimise safety, a daily dose lower than the tolerable upper limits (<7mg for children aged 1-3 years up to 22mg for those aged 15-17 years) should be used along with dietary modifications whenever possible. in adults, doses up to the no observed adverse effect level (noael) of 50 mg/day should be considered. [28] at this stage, it is unclear if there is any additional benefit from supplementing zinc for the prevention of sars-cov-2 or other viral respiratory infections in low risk populations nor for people with normal zinc status. it is also unclear if there are any benefits from supplementing with zinc for the treatment of sars-cov-2. there is limited indirect evidence from viral upper respiratory infections that zinc lozenges with a daily dose of >75mg of zinc may shorten the duration of the common cold. however, there are risks with higher doses above the noael including permanent loss of smell. [28] therefore, a daily dose higher than 100mg of elemental zinc in a lozenge is probably not advisable, as it is questionable whether there are any additional therapeutic effects. [61] disclaimer: this article has not been peer-reviewed; it should not replace individual clinical judgement. the views expressed in this rapid review are the views of the authors and not necessarily from the host institutions. the views are not a substitute for professional medical advice. publications [41] j o u r n a l p r e -p r o o f di napoli r: features, evaluation and treatment coronavirus (covid-19). in: statpearls the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak diabetes is a risk factor for the progression and prognosis of covid-19 a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) low zinc status: a new risk factor for pneumonia in the elderly? mineral intakes of elderly adult supplement and nonsupplement users in the third national health and nutrition examination survey rink l: correlation between zinc status and immune function in the elderly prevalence of zinc deficiency and its clinical relevance among hospitalised elderly. archives of gerontology and geriatrics zinc intake of the us population: findings from the third national health and nutrition examination survey discovery of human zinc deficiency: its impact on human health and disease zinc fluxes and zinc transporter genes in chronic diseases. mutation research/fundamental and molecular mechanisms of mutagenesis serum zinc level and coronary heart disease events in patients with type 2 diabetes copper, chromium, manganese, iron, nickel, and zinc levels in biological samples of diabetes mellitus patients normalization of zinc intake enhances neurological retrieval of patients suffering from ischemic strokes the nutrition crsp: what is marginal malnutrition, and does it affect human function? plant breeding: a long-term strategy for the control of zinc deficiency in vulnerable populations. the american journal of clinical nutrition dietary calcium, phytate, and zinc intakes and the calcium, phytate, and zinc molar ratios of the diets of a selected group of east african children. the american journal of clinical nutrition effect of vegetarian diets on zinc status: a systematic review and meta-analysis of studies in humans effect of gestational zinc deficiency on pregnancy outcomes: summary of observation studies and zinc supplementation trials effects of maternal zinc supplementation on pregnancy and lactation outcomes. food and nutrition bulletin zinc deficiency in pregnancy and fetal-neonatal outcomes and impact of the supplements on pregnancy outcomes the dynamic link between the integrity of the immune system and zinc status zinc in health and chronic disease. the journal of nutrition, health & aging alcoholism causes alveolar macrophage zinc deficiency and immune dysfunction. american journal of respiratory and critical care medicine practice patterns of naturopathic physicians: results from a random survey of licensed practitioners in two us states recent aspects of the effects of zinc on human health ineffectiveness of zinc gluconate nasal spray and zinc orotate lozenges in common-cold treatment: a double-blind, placebo-controlled clinical trial. alternative therapies in health and medicine european commission health & consumer protection directorate-general: scientific committee on food estimating the global prevalence of zinc deficiency: results based on zinc availability in national food supplies and the prevalence of stunting national risk of zinc deficiency as estimated by national surveys use of national food balance data to estimate the adequacy of zinc in national food supplies: methodology and regional estimates indicators of zinc status at the population level: a review of the evidence zinc: the missing link in combating micronutrient malnutrition in developing countries assessing zinc in humans. current opinion in clinical nutrition and metabolic care as: discovery of zinc for human health and biomarkers of zinc deficiency. in: molecular, genetic, and nutritional aspects of major and trace minerals a short 18 items food frequency questionnaire biochemically validated to estimate zinc status in humans reprogramming of the immune system during zinc deficiency zinc supplementation of young men alters metallothionein, zinc transporter, and cytokine gene expression in leukocyte populations zinc and respiratory tract infections: perspectives for covid-19 (review) zinc and respiratory tract infections: perspectives for covid-19. permission to reprint figure 1 papain-like protease 2 (plp2) from severe acute respiratory syndrome coronavirus (sars-cov): expression, purification, characterization, and inhibition zn(2+) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture does zinc supplementation enhance the clinical efficacy of chloroquine/hydroxychloroquine to win todays battle against covid-19? zinc modulates cytokine-induced lung epithelial cell barrier permeability zinc modulates airway epithelium susceptibility to death receptormediated apoptosis apoptosis in the normal and inflamed airway epithelium: role of zinc in epithelial protection and procaspase-3 regulation new insights into the role of zinc in the respiratory epithelium zinc metabolism in airway epithelium and airway inflammation: basic mechanisms and clinical targets. a review impact of zinc metabolism on innate immune function in the setting of sepsis. international journal for vitamin and nutrition research zinc: mechanisms of host defense. the journal of nutrition effect of zinc deficiency on hepatic enzymes regulating vitamin a status zinc homeostasis in platelet-related diseases zinc: an important cofactor in haemostasis and thrombosis complementary, alternative and integrative medicine (cam) for the treatment of coronavirus disease 2019 (covid-19): an overview: licensed under a creative commons attribution 4.0 international license zinc for the common cold zinc supplementation for the prevention of pneumonia in children aged 2 months to 59 months efficacy of zinc against common cold viruses: an overview zinc and the common cold: a meta-analysis revisited zinc lozenges may shorten the duration of colds: a systematic review. the open respiratory medicine journal zinc lozenges and the common cold: a meta-analysis comparing zinc acetate and zinc gluconate, and the role of zinc dosage zinc for the common cold withdrawn: zinc for the common cold zinc for preventing and treating the common cold protocol for a rapid review of zinc for the prevention or treatment of covid-19 and other coronavirusrelated respiratory tract infections in humans rob 2: a revised tool for assessing risk of bias in randomised trials a revised tool for assessing risk of bias in randomized trials the contents of herbal and dietary supplements implicated in liver injury in the united states are frequently mislabeled hdivzn trial progress update on human rhinovirus and coronavirus infections key: cord-309642-wwaa6ls0 authors: potgieter, leon n.d. title: pathogenesis of viral infections date: 1986-11-30 journal: veterinary clinics of north america: small animal practice doi: 10.1016/s0195-5616(86)50129-7 sha: doc_id: 309642 cord_uid: wwaa6ls0 the article considers factors that influence pathogenesis, initiation of infection, dissemination of virus within a host, lytic viral infections, viral immunosuppression, viral immunopathology, and viral oncogenesis. genetic characteristics of the host1 9 · 97 · 133 or environmental influences. genetic host factors include animal species, breed, and organ or tissue susceptibility. 7 · 18 · 84 · 133 such restrictions function at the cellular level either as the presence or absence of appropriate cell surface receptors (in some instances, they have been shown to be inherited as dominant alleles in a mendelian manner) 9 · 18 · 26 · 46 · 68 · 97 ·u 9 · 120 or the intracellular hospitality of the cell (several genetic host restrictions on virus replication have been identified).18·32·59·80·82·108·109·120·126 restricted growth of several dna viruses in some cells results in transformation without production of progeny viruses. 34 to a degree, the animal's ability to respond immunologically or by interferon production is an innate property of the host. 33,36,67.79,91,97,120,121.126 nongenetic factors include passive immunity, acquired immunity, age, stress, trauma, hormonal levels, nutritional status, environmental temperature, pregnancy, and concurrent infections (which may either enhance or interfere with a virus infection). 13,18,20,33,4o,47,67,79,121,139 virus factors certain virus properties materially affect the manner in which disease is elicited. cytocidal viruses such as alphaherpesviruses effectively inhibit cellular metabolism, 3 · 4 · 21 · 61 · 105 ·ii 4 · 122 · 137 · 138 and others result in pathophysiologic permeability of cell membranes. 88 certain viruses contain cytotoxins, which may be structural proteins (such as the penton adenovirus protein) or induce production of cytotoxic substances. 3,4,u,us,121,122,125,137 noncytocidal viruses may result in steady-state infections, 2 · 35 · 67 · 81 · 85 • 90 but these in turn can result in perturbations of the immune response. 2, 5, 15, 35, 43, 4 s-so,oo, 79 · 81 · 87 · 9 5, 98 ·m· 136 cell-mediated destruction of tissues, immune complex disease, autoimmunity, and disseminated intravascular coagulation are possible sequelae. the presence of the enzyme reverse transcriptase in virions of members of the family retroviridae imparts two important characteristics to these viruses-the capacity for latency as "proviruses" and for transformation of the cell. 90 • 135 · 140 · 141 strain variation in virulence and in tissue tropism is well recognized. 33 · 84 · 102 ·uo.u 9 · 134 these phenomena are undoubtedly related to variations in the nucleotide sequences of the viruses. 33 ·u 0 · 134 some viruses, as a result of their genomic structure or their particular replication strategy, induce interferon production in the host more efficiently, which in turn may result in greater resistance to the virus. 79 · 121 several instances have been documented in which viruses modify cell function without detectable cell injury. 84 · 85 examples include disease as a result of virus modulation of immunocompetent cells and cells with endocrine functions. 16 · 62 · 8 4-8b,lll of particular concern in viral pathogenesis is the tissue tropism of the virus, which is determined by complex interactions of virus structure, cell receptors, cell metabolism, intracellular hospitality, and virus replication strategy. 18,26,27,32,59,68,73,oo,82,109,12o,121,134 the host is more vulnerable if cell destruction occurs in vital tissues or organs such as the heart, central nervous system, immunocompetent cells, liver, endothelium, and certain endocrine tissues. 33, 37, 54, 62, 10, 79, 83, 85, 134, 139 whether a virus initiates a successful infection in a particular host is often dependent on the amount of inoculum that the host is exposed to and the route by which virus gains entrance into the body. 33 · 47 · 79 · 128 for such viruses, a certain threshold amount of virus is required for successful infection, and, in some instances, the severity of the ensuing disease may be determined by dose of the virus. as alluded to earlier, the first barriers that viruses must penetrate are various epithelia of the skin and its mucosal extensions, alimentary canal, respiratory tract, and the urogenital tract. 33 · 47 · 79 · 128 these epithelia may have various products that aid in resistance to virus, such as film, mucus, keratin (skin), acidity (stomach), and so forth. these barriers must be overcome by viruses to initiate infections. some viruses (orthomyxoviruses, for example) penetrate mucus by enzymatic action (neuraminidase); others are more phresistant and may more readily infect the intestinal tract, and some viruses initiate infection after mechanical injury to epithelia. 18 · 33 · 79 ·u 5 · 121 cell surface receptors characteristic for a particular species and for a particular tissue may determine species (or breed) and organ susceptibility.18·46·68·97·u5·120 these receptors are genetically specified. in most instances, the chemistry of cell receptors is not well characterized. it is best known for the ortho-and paramyxoviruses for which it is a neuraminic acid-containing mucoprotein. 18 ·u 5 others include lipoglycoproteins (picornaviruses), histocompatibility complex antigens (some alphaviruses), and fe and complement receptors on macrophages. 18 · 27 · 39 a 2 · 93 · 94 · 97 the latter receptors on some flavi-, alpha-, bunya-, rhabdo-, and reoviruses result in enhanced infections in the presence of subneutralizing concentrations of antibody or heterologous strain antibody that promotes attachment to fe receptors. receptors that are ubiquitous on the surface of cells of various species (such as neuraminic acid-containing glycoproteins) are not important determinants of host range or tissue tropism. 26 · 121 receptors for picornaviruses often do determine these phenomena. 18 · 120 a receptor map has been made for four groups of nonenveloped viruses, and it is clear that even unrelated viruses may share the same receptor. 68 viruses that share the same receptor can interfere with one another since they compete for the receptor (homologous interference). 33 · 120 host range and receptor specificity are particularly important in the retroviridae family. 33 · 120 · 121 for instance, the susceptibility of certain genetic strains of chickens to various oncovirinae depends on the nature of cell surface receptors for the largest viral envelope glycoprotein. these receptors are specified genetically and are inherited as dominant alleles in a mendelian manner. the viral protein and cell receptor have clinical significance because occupation of a receptor by an avirulent oncovirus will block infection with a highly oncogenic virus. another factor in the initiation of infection should be considered. attachment of ortho-and paramyxoviruses to receptors is not important in determining host range and tissue tropism, but it appears that the step of penetration does determine these phenomena. 18 · 58 · 59 · 108 · 121 certain peplomeres of paramyxoviruses have a fusion (f) protein that, after attachment, results in the fusion of the viral envelope with the plasma membrane, al-lowing the viral rna to enter the cell to initiate virus replication. the fusion protein exists as a precursor and is not activated until cleaved by specific cellular proteases probably on the surface of the plasma membrane. cell susceptibility is determined by the presence of the appropriate proteases. a similar event occurs with the orthomyxoviruses, but in this virus group, cleavage of the hemagglutinin facilitates penetration (but does not affect attachment to cells). 58 • 59 • 108 · 121 however, the cleavage of the hemagglutinin occurs during assembly of the virions late in the replication cycle. thus, virion infectivity is dependent on the virus undergoing a full replication cycle in susceptible cells that contain the appropriate protease. a novel concept with regard to receptors on cell surfaces is that cells infected with viruses may permit adherence of certain pathogenic bacteria, leading to bacterial colonization. 112 the phenomenon appears to be mediated by virus-induced receptors on the surface membrane of cells and may be one mechanism of the often-encountered secondary bacterial infections associated with viral diseases. some viruses initiate infections as a result of mechanical injuries of epithelial barriers. 8 • 33 • 79 this can occur as a result of insect bites, which is particularly important for the arthropod-borne viruses (many of the togaviridae, reoviridae, bunyaviridae, and so on). iatrogenic introduction of viruses with needles and so forth is not uncommon. rabies virus constitutes a classical example of a disease initiated by a wound and influenced by dose of virus. 28 • 44 · 128 the latter virus is deposited in a wound usually as a result of a bite from an animal with salivary secretion of virus. if sufficient virus is present, nerve endings are immediately penetrated. however, often the virus first replicates in local muscle cells, which allows for amplification of the infectious dose and penetration of the nerve endings followed by centripetal spread along the axoplasm of nerves to the central nervous system. variations in pathogenesis occur when this general progression of virus is delayed or stopped at some point. replication of virus in muscle cells at the inoculation site may be responsible for variations in incubation period. survival may occur when infection is localized for some reason at the inoculation site or peripheral nervous system. for a variable period of time (before the virus enters the nerves), it is susceptible to antibody. the respiratory tract is a very common site at which virus infections are initiated, usually as the result of airborne infections. 8 • 33 • 51 • 52 • 79 droplets often originate from the respiratory tract and mouth of infected animals, but aerosols of urine, fecal material, and so on can infect the respiratory tract. small droplets of less than 100 j.lm in diameter dry to droplet nuclei of i to 3 j.lm in diameter that remain suspended for long periods and easily gain entrance to bronchioli and alveoli. viruses that resist desiccation remain viable in droplet nuclei for extended periods of time. film and mucus afford some protection, but myxoviruses that are entrapped by receptor-like mucoproteins in mucus may be released by the peplomere enzyme neuraminidase, thereby allowing the virus to move on and finally become attached to a cell surface receptor. 96,113,ns,121,132 a number of viruses remain localized in the respiratory tract during the course of infection-some in the upper respiratory tract (rhinoviruses, some herpesviruses) and others in the lower respiratory tract (parainfluenza virus). 8 · 33 · 79 although restricted to the respiratory tract, some viruses (influenze, for example) still cause generalized clinical signs such as fever, malaise, and muscle pains as a result of the products and inflammation associated with cell injury and destruction. 33 · 79 · 125 many viruses become rapidly disseminated throughout the body after the primary infection of the respiratory tract (canine distemper virus is a good example). 8 • 33 · 62 · 79 usually there is no evidence of respiratory tract infection in the initial phase of these diseases and the animal usually is not infectious during this phase. the virus spreads via macrophages or lymphatics to regional lymph nodes before spreading further. 33 generally, viruses initiate infection in epithelial cells of the respiratory tract, but some viruses achieve this by successfully infecting alveolar macrophages. 29 · 51 · 52 infection of alveolar macrophages has important sequelae in the pathogenesis of viral respiratory tract disease. 51 · 52 · 104 viral respiratory tract disease is a consequence of mechanical and biochemical injury to epithelial cells and alveolar macrophages, which can, in the most severe instances, result in secondary bacterial infection, pneumonia, and death. 17 · 29 · 40 · 51 · 52 · 104 · 117 denuded respiratory tract, impaired mucociliary escalator, and the growth medium provided by exudate have been thought to enhance bacterial growth and colonization in the respiratory tract. however, these mechanisms may not be as important as originally thought. injury to various biocidal mechanisms may be of greater consequence. 17 · 40 · 52 · 53 the alveolar macrophage, which is of critical importance in pulmonary resistance to bacterial colonization, may be affected in several ways either as direct consequence of virus replication in this cell or as a result of immunemediated cytotoxicity directed at the virus-infected macrophage. the latter is of particular consequence. it occurs late in the disease process when the immune response is initiated and when macrophages are ingesting virusladen cell debris. not only is the number of alveolar macrophages reduced by viral infection, the function of the remaining cells may be impaired.17·40·51-53·124·126·130·131 evidence suggests that such macrophages have suppressed immunologic (fe) and nonimmunologic membrane receptor binding activity, fe and nonspecific receptor-mediated phagocytic ingestion, phagosome-lysosome fusion, intracellular killing, and bacterial degradation. some of these observations are the result of low levels of lysosomal enzymes and impaired biochemically mediated killing mechanisms. the alveolar macrophage may be impaired also because of hypoxia (it is an aerobic cell) and reduced surfactant levels. 40 · 124 the latter facilitates phagocytosis and is produced by type-ii alveolar pneumocytes. viral injury to the latter causes reduced production of surfactant in the lung. as alluded to earlier, certain viruses (such as influenza virus) promote bacterial colonization by altering the plasma membrane of infected cells, which facilitates bacterial adherence to the surface of such cells. 112 the phenomenon appears to be the result of virus-induced receptors on the cell surface. some viruses appear to produce disease primarily by suppressing immunity, which is relatively sequestered from the systemic immune func-tions. 44 • 51 • 52 • 56 • 62 • 129 apart from the pulmonary macrophages, pulmonary cellmediated immunity is derived from bronchus-associated lymphoid tissue and augmented by the influx of blood monocytes and neutrophils. locally synthesized lgg, primarily synthesized in the lung, or transudated serum immunoglobulins function as opsonins, whereas secretory iga (primarily from the upper respiratory tract) prevents bacterial adherence and colonization and probably aggregates bacterial particles to facilitate mucociliary clearance. various viruses attach and replicate in epithelia of the mouth, pharynx, tonsils, and (or) the gastrointestinal tract. 33 · 79 • 121 however, the stomach, abomasum, and intestine are not very hospitable to viruses because of an unfavorable ph and the presence of bile. enteric viruses usually are tolerant of a low ph. certain viruses (rotaviruses, for example) remain restricted to the gastrointestinal tract. 8 · 33 • 74 • 76 secretory immunity (and not humoral immunity) is protective against such viruses. 20 • 116 • 121 many other viruses that invade intestinal epithelia subsequently become disseminated with varying frequency (enteroviruses). 8 • 133 • 142 certain parvoviruses (canine parvovirus ii and feline panleukopenia virus) initiate infection in the pharynx-tonsil area, spread by the blood stream to various organs, and finally infect and destroy the crypt cells of the intestinal tract. 99 the intestines thus become infected by a very circuitous route. villous atrophy results because of impaired replacement of enterocytes, which are derived from the crypt cells. central nervous system infection by the coronavirus, hemagglutinating encephalomyelitis virus of swine, occurs as a result of nerve tract migration from the gastrointestinal tract to peripheral ganglia. infection of neurons that regulate peristaltic functions of the intestinal tract results in gastrointestinal disease and subsequent starvation. 116 certain viruses such as rinderpest and african swine fever initiate submucosal infection without infecting epithelial cells and appear to have the capacity to pass through the epithelial barrier. 12 ·m some enteric viruses destroy the absorbtive columnar enterocytes on the tips of intestinal villi. 66 • 74 • 76 the upper portion of the intestine is first affected, but infection often spreads throughout the entire length of the intestine. the affected cells are desquamated and are replaced by cuboidal or even squamous cells, resulting in a dramatic atrophy of the villi. these target cells are important for the digestion of disaccharides and the absorption of macrosaccharides, and contribute to osmoregulation. replacement of these cells occurs by m'igration of undifferentiated cells from the crypts, which are resistant to infection. however, the new villous tip cells are immature (contain thymidine kinase instead of sucrase, which is an enzyme profile similar to crypt cells) and result in a disturbed sodium transport system with a net extracellular fluid-to-lumen flux of sodium ions. pathophysiologic changes include loss of water, sodium, chloride, bicarbonate, and potassium. metabolism of glucose and lactate becomes severely disturbed, and the hypoglycemia, lactic acidosis, and an elevated efflux of potassium to hypovolumic plasma may lead to acute shock, heart failure, and death. the disease is more severe in neonates because of their milk diet and their dependence on readily available nutrients, and because replacement of sloughed epithelial cells is slower. the inert superficial protective layers of the intact skin usually are impervious to virus infection. 8 • 33 • 79 • 128 injury to the integument can allow several viruses to penetrate into susceptible tissues. trauma, insect bites, needles, and so on may be responsible for the introduction of virus (see earlier discussion). viruses that commonly initiate infection in this manner include most of the arboviruses, rabies, papular stomatitis, and some herpesviruses. some viruses penetrate directly through mucosal epithelia. 12 • 121 infection initiated in the urogenital tract few viral venereal diseases, affecting primarily the genital tissues, have been described. 33 • 79 the principal diseases in this category include those caused by herpesviruses and perhaps genital papillomaviruses of various species. 33 • 79 • 141 however, the potential of infection occurring in the genital tract is high because many viruses may be present in semen. several viruses are capable of establishing infection in the placenta of pregnant animals and subsequently result in congenital infections. vertical infection occurs when a virus is transmitted to progeny with the germ cell. the latter occurs primarily with retroviruses in which viral genetic material becomes incorporated in host dna. 141 it is difficult to consider the establishment of viral disease in a fetus without considering the infection of the dam, because the latter usually is infected for a variable time before the fetus. transplacental infection is perhaps the principal route of fetal infection by a virus. 78 • 79 it may occur by a variety of mechanisms depending on the nature of placentation of the dam and the nature of the virus. 33 • 79 in some instances, the virus appears to have selective pathogenicity for the fetus, because disease may not occur in the dam (for example, some strains of bluetongue virus). 54 • 55 • 79 perhaps a primary infection of the fetus is one that is transmitted with the germ cells. this apparently occurs only with retroviruses, in which viral genetic material (provirus) is passed along as a cellular gene insert. 11 8. 1 35,14 1 fetal infection is a prominent feature of several viruses. the outcome of the disease in the fetus depends on both virus and host factors. some cytolytic viruses (for example, some herpesviruses and parvoviruses) uniformly result in fetal death, whereas other viruses (bluetongue virus, bovine virae diarrhea virus) may not. 33 • 75 • 78 • 79 the nature of the disease in the fetus with some of the latter viruses may depend on the age of the fetus. 54 • 75 this has been quite well documented with porcine parvovirus, bluetongue virus, and bovine viral diarrhea virus. the fetus is very vulnerable in the first third of pregnancy, and infection at this gestational age often results in death, but the fetus becomes progressively resistant to the effect of virus infection. infection in the last third of pregnancy may not have serious consequences. however, frequently the fetus is partially resistant only during the middle third of pregnancy, and infection during this stage may result in various lesions. the latter may include malformations of the central nervous system such as cerebellar hypoplasia (feline panleukopenia, bovine viral diarrhea virus), hypomyelinization (bovine viral diarrhea virus, border disease), hydranencephaly (bluetongue virus, bovine viral diarrhea virus), porencephaly (bluetongue virus, bovine viral diarrhea virus), and hydrocephalus (parainfluenza virus, japanese encephalitis virus). porencephaly is a later manifestation than hydranencephaly in lamb fetuses infected with bluetongue virus. stillbirths, weak neonates, and skeletal malformations may also occur. the progressive development of resistance in a fetus is correlated with the ontogeny of the immune response. some viruses (such as rubella virus) reduce the mitotic rate of infected fetal tissues and thereby affect organ development. 14 • 69 • 78 offspring may be stillborn or runted. infection of the fetus at certain stages of gestation by a number of viruses (pestiviruses, arenaviruses) may result in "immunotolerant" offspring that are persistently viremic and without a detectable immune response. 33 • 38 • 67 • 71 in some instances, superinfection of such animals with the same virus or certain strains of the same virus results in serious, often fatal, systemic disease that may be immune-mediated. fetal disease can occur as a result of viral infections of the dam, even if the fetus itself is not infected. 19 • 31 • 78 • 79 changes in the placental circulation (vasoconstriction, congestion, and hemorrhage) as a result of viral placentitis can rapidly and severely affect the fetus. coxsackie b3 virus-induced pancreatic acinar atrophy in pregnant mice results in malnutrition owing to the mices' inability to digest and metabolize protein. fetal wastage and growth retardation are two sequelae of this condition. the hyperthermia associated with some viral infections in pregnant animals (for example, influenza) can cause abortions, stillbirths, and malformations (anencephaly, microencephaly, hydroencephaly) of fetuses. local extension of viral lesions occurs in susceptible hosts. 33 · 79 • 109 • 121 it may occur by virus release and dispersal from infected cells to neighboring susceptible cells. in some instances, lysis of infected cells is necessary before virus is liberated. herpesviruses, paramyxoviruses, and others may spread from cell to cell by a fus·ion mechanism. virus-induced cell proliferation, as occurs with poxviruses, papovaviruses, and retroviruses, is another mechanism of lesion extension. dissemination of the virus from the initial focus of infection occurs by several mechanisms. 12 · 33 • 77 • 79 • 80 • 121 the first step in generalized dissemination of a virus is the spread from the local lesion to the regional lymph node, either free in lymph or, more frequently, within carrier cells such as lymphocytes or phagocytic cells. virus concentration may be amplified by replication in the regional lymph node; secondary spread of the virus in blood vessels, either free or, more likely, within carrier cells, to other tissues and organs in the body may occur. this secondary spread of the virus may be detected clinically as the second part of a biphasic fever. in many instances, the critical event in viral dissemination is the successful infection of phagocytes. 77 • 80 the macrophage provides an important resistance mechanism of viral infections, and virulent viruses overcome their antiviral action. impaired macrophage function markedly enhances the susceptibility of animals to viruses. neonatal and corticosteroid-treated animals may be more susceptible for this reason. it is likely that several mechanisms of antiviral action exist in macrophages. 80 • 119 often virus penetration occurs in both susceptible and resistant macrophages, but uncoating of the virus is blocked in resistant macrophages, thereby terminating virus replication. various relatively sequestered organs may become infected with certain viruses. the central nervous system (cns) has nonfenestrated vasculature, tightly packed cellular components, and lacks a conventional lymphatic system. 54 • 55 the endothelial cells are joined by tight junctions and are surrounded by dense basement membranes, which in turn are tightly packed against astrocytic footplates. these structural barriers impede virus invasion of the cns from the bloodstream, but transendothelial migration of an infected lymphocyte and endothelial injury (either as a result of the virus infection or some other cause) may result in cns infection. 33 • 54 • 55 the traffic of leukocytes into the cns is very limited under normal circumstances. some viruses are transported across the cells in pinocytotic vesicles and are deposited in the cytoplasm of the adjacent astrocytes. the cns may become infected by virus migration along nerve trunks (herpesviruses, rabies, hemagglutinating encephalomyelitis virus). 28 • 33 • 45 • 128 several mechanisms have been described 33 • 128 : movement within the central axoplasm, along endoneural spaces, and cell-to-cell infection of schwann cells. both centripetal and centrifugal spread along nerve trunks to and from the cns are possible. rabies virus infects salivary glands by the latter route. viral disease of the cns requires that the virus either has the capacity to invade these tissues or that its entry is facilitated by some unrelated event. 54 • 55 virus infection that leads to injury by direct or indirect mechanisms of oligodendrocytes results in demyelinization. 54 • 55 • 62 • 134 neurologic dysfunction may develop in the absence of obvious cell injury. persistent canine distemper virus infections of rat glioma cells cause a reduction of betaadrenergic receptors. some viruses specifically infect neurons of the cns. virus specificity may be so restricted that only certain subpopulations of neurons become infected. 54 in humans, poliomyelitis viral infection is restricted mainly to motor neurons. rabies virus, during the early stages of infection, is confined primarily to neurons of the limbic system. this facilitates transmission by biting because the cortical-neurons are not involved early, and instead of seizures and motor deficits, alertness and aberrant behavior are the predominant clinical signs. virus selectivity determines also the development of hydranencephaly and porencephaly in newborn lambs infected in utero with bluetongue virus. the virus selectively destroys the germinal cells of the subventricular zone, the precursors of the neurons and glial cells of the forebrain. before the development of the cortical mantle in early gestation, necrosis and hydranencephaly occur owing to the destruction of these cells. after the formation of the cortical mantle before midgestation, porencephaly develops because the glial cell precursors are destroyed, resulting in focal white matter necrosis. several parvoviruses, which replicate only in cells in s-phase mitosis, selectively destroy the germinal cells of the cerebellum, resulting in hypoplasia of the cerebellum with abnormal foliation, depleted granular cells, and aberrant synaptic organization. 54 this condition, which occurs in cats after infection as neonates or in late gestation, is known as spontaneous ataxia of kittens and is the most common neurologic disease in cats. for many years, it was thought to be an inherited condition. it has become evident that viruses can injure endocrine tissues specifically with the concomitant deficit in endocrine secretion. 7 • 35 · 84 • 85 • 133 • 142 in some instances, functional impairment develops without obvious cell injury. certain picornaviruses cause selective destruction of beta-pancreatic cells, resulting in diabetes. 133 • 142 evidence suggests that this may occur in humans also. 142 impaired growth hormone production, growth rate, and glucose metabolism result from selective noncytopathic infection of anterior pituitary gland cells with lymphocytic choriomeningitis virus in mice. 84 • 85 immunosuppression vastly increases the potential of a virus to advance from a localized to a generalized infection. 62 • 10° complex interactions occur between various microorganisms in natural infections. 33 • 63 • 64 • 100 • 101 a virus that induces immunosuppression in an animal can significantly enhance the severity and change the nature of a concurrent viral disease. one report suggested that canine parvovirus, which is lymphocytolytic, may enhance the neuropathogenicity of modified live canine distemper vaccine virus. 63 bovine viral diarrhea virus, which also affects lymphocyte function, greatly enhances the dissemination of a herpesvirus, infectious bovine rhinotracheitis virus. 100 various host-and virus-related restrictions determine tissue or organ invasion by a virus. cell surface receptors and the virus penetration step have already been discussed. 18 • 46 • 68 • 100 • 101 intracellular hospitality for the particular virus may determine whether virus replication (either partial or complete-with progeny) occurs in a particular cell. 18 • 80 • 109 · 121 defective interfering particles are viruses of subgenomic size that contain most or all of the normal viral proteins. 24 • 33 they lack complete genomes and are unable to replicate in the absence of the parental virus. paradoxically, they interfere with the replication of the parental virus, apparently because the defective particles with their smaller genomes have a replication advantage and are able to sequester the replicase systems. defective interfering particles have been demonstrated in most groups of viruses. they may mediate the cessation of a viral disease or may convert an acute, lytic disease to a persistent or chronic infection. evidence indicates that the generation of defective interfering particles is controlled by host cells. blockage of host dna synthesis blocks generation of these particles when vesicular stomatitis virus free of defective interfering particles is used as the infecting virus. defective interfering particles are produced in such cells if they are introduced with the parenteral virus. this evidence, together with evidence that the defective particles can alter the pathogenesis of viral infections, suggest that cells may have evolved mechanisms to protect themselves against viruses that successfully enter and penetrate. the most rapidly produced defense against viruses is a family of proteins secreted by tissue cells in response to various stimuli such as viruses, bacteria, foreign cells, foreign macromolecules, and several other compounds. 6 • 36 • 121 interferons act indirectly by stimulating surrounding cells to produce protein(s) that, in turn, may regulate virus replication, the immune response, cell growth, and other functions. interferons are unexpressed genetic functions of mature cells and may have a normal role in cell regulation. certain viruses are much more efficient in eliciting interferon production than others, which, of course, affects the course of the virus infection. 33 • 121 generally, the most potent interferon stimulators are also the most susceptible to interferon. 33 · 121 there are many examples of exquisitely sensitive genetically controlled expression of viral disease functional within the host cell. an example of \iral genetic control of pathogenesis is that of reoviruses in newborn mice. 134 reovirus type 1 produces nonfatal infection of ependymal cells after intracerebral inoculation, whereas type 3 results in fatal encephalitis with neuronal destruction. the tropism for ependymal cells or neurons seems to be regulated by a single gene (the s genome segment) that codes for the sigma-1 capsid protein. another genome segment (m2) determines the ability of these viruses to initiate local or systemic infection in newborn mice after oral inoculation. an example of host genetic control of pathogenesis at the intracellular level is the fv-1 system in mice, which determines susceptibility to murine leukemia viruses. 33 this mechanism operates after penetration but before integration of viral genetic material in host dna. the cellular gene, known as fv-1, is present on chromosome 4, and the virus determinant with which it interacts to determine this tropism is the p30 viral protein. this mechanism is illustrated by murine leukemia viruses that preferentially infect cells from ~ih swiss mice (n-tropic) or balb/c mice (b-tropic). cells may be destroyed when they become infected with certain viruses, particularly dna viruses. several mechanisms are responsible for this effect on cells. 3, 4, 11, 20, 25, 33, 41, 61, 88, 95, 105, 114, 121, 122, 125, 138 in some instances, cell metabolism is drastically altered. macromolecular synthesis may be inhibited by a variety of mechanisms and may occur at the level of replication, transcription, or translation. certain viruses 'interfere with host dna replication. vaccinia virus mrna competes with cellular mrna. this virus may also result in disruption of polysomes and impaired rna processing. poliovirus inhibits the initiation factor in mrna translation. increased permeability of virusinfected cells causes increased intracellular sodium ions, which favor viral mrna translation. the rate of formation of viral products may be greater than their release, which has an adverse effect on cellular metabolism. the virus may deplete substrates essential for vital cellular functions, and the physical presence of viral products in a cell has an adverse effect on cell metabolism. complete or partial replication of a virus is necessary for cell injury in many instances, but virus replication does not necessarily result in cell injury, particularly with many rna viruses. several viruses produce a toxin-like substance that is either a direct effect of a virus-induced product or a secondary effect caused by the activation of lysosomal enzymes by a virus. viral cytotoxins have been described for poxviruses and adenoviruses (the penton capsid protein which reversibly modifies cell membranes from the outside). these cytotoxins may be either structural virion components (preformed) or induced during virus replication. vaccinia cytotoxin has been identified as a surface tubular virion protein, whereas adenovirus cytotoxin is contained in the penton capsomere (the hexon capsomeres and fiber antigen inhibit macromolecular synthesis). the cytotoxic effect is markedly amplified in certain virus infections. t lymphocytes infected with dengue virus produce a cytotoxin that induces macrophages to produce a cytotoxic factor. 41 the exact mechanism by which various viruses affect cell metabolism and produce cytopathogenesis is not well known. however, it is known that poxviruses rapidly cut off host macromolecular synthesis, disaggregate host polysomes, interfere with processing of rna, and redistribute lysosomal enzymes. one hypothesis is that the cytopathic and pathophysiologic changes may be due to a common cause-an alteration of the permeability of cell membranes. leakiness of cell membranes occurs during infection with viruses from several virus families and usually precedes cytopathogenesis. certain physiologic and clinical manifestations of virus infections can be attributed to membrane leakiness. these include excess respiratory tract mucus production with rhinovirus infections, loss of vision in corneal keratitis caused by herpesvirus infections, and excessive loss of water and electrolytes from the gastrointestinal tract during rotavirus infections. one of the most important aspects of infectious diseases that is receiving increasing recognition is the interaction and synergism of pathogenic microorganisms in the manifestation of the pathologic state. in many instances of enhanced disease due to microorganism interaction, it is the result of virus-induced immunosuppression. 52 • 53 • 62 -65 • 99 -101 a virus-causing immunosuppression can enhance dramatically diseases caused by viral, bacterial, or protozoan infections that normally are relatively innocuous. viral immunosuppression may facilitate dissemination of microorganisms in a host and promote persistent or chronic infections. the cellular basis and consequences of viral-induced immunodeficiency are discussed in detail elsewhere in this issue. a brief summary will be presented here. the most readily recognized types of viral immunosuppression are those infections that result in destruction of immunocompetent cells such as macrophages, neutrophils, and lymphocytes. 17 however, in the majority of instances of viral immunosuppression, cytocidal infection of immunocompetent cells is not recognized, and the phenomenon appears to be the result of impaired function.1·10·16·17·51-53·57·62·65·106·107·m virus-induced immune modulation has been recognized in macrophages, neutrophils, various subsets oft lymphocytes, and b lymphocytes. many of the immune responses inhibited by viruses may be attributed to alteration of macrophage function (see the discussion on the antibacterial activity of alveolar macrophages). influenza virus, lymphocytic choriomeningitis virus, bovine viral diarrhea virus, and several other viruses are capable of affecting phagocytic cells. several studies have indicated suppression of phagocytic, chemiluminescence, and chemotactic responses of macrophages and neutrophils. delayed wound healing in mice has also been attributed to viral alteration of macrophage function. paradoxically, impaired function of some macrophages (such as the alveolar macrophage) may be due to immune-mediated injury as a result of virus-specific cytotoxicity directed against the virus-laden macrophage. virus-induced modulation of macrophages conceivably could alter the outcome of numerous cellular interactions, either due to impaired direct participation in lymphocyte functions or because of interference of secretion of regulatory factors. functional impairment of lymphocytes by viruses may be the result of changes in cell surface receptors, competition between virus and immunogen for protein or dna synthetic machinery, generation of suppressor interferon, interruption of the cellular communication network in the immune response, and stimulation of suppressor t cells. 10 • 16 · 62 · 65 · 103 ·m as has been observed for macrophages, viral infection in lymphoid cells may result in the production of a lymphocytotoxic immune response, which may lead to the premature senescence of t cells. another mechanism that may occur in some viral infections is virus-induced alterations in normal lymphocyte traffic and recirculating patterns in the host. this results in redirecting immunocompetent cells away from lymph nodes and spleen and decreasing the immunologic reserve of lymphatic tissues. an active area of investigation is immunosuppression by retroviruses, which seems to be mediated by the pl5e envelope protein of some of these viruses (particularly feline leukemia virus). 22 · 62 · 65 · 86 · 123 · 127 impaired immunologic functions associated with exposure to pl5e include monocyte chemotaxis, lymphocyte blastogenesis, erythroid colony formation, macrophage accumulation, and tumor immunity. it has been suggested that pl5e causes immunosuppression by blocking the production of interleukin 2 by lymphocytes. virus-induced immunosuppression is often accompanied by multiple deficits of the immune response. 62 during certain viral infections, there is both an activation and increase of suppressor t cells, which, in turn, suppress autologous t-cell proliferation to antigens as well as b-cell antibody production. 62 · 65 · 103 ·m certain paramyxoviruses cause silent infections of lymphocytes, which fail to generate natural killer (nk) cell activity (an important antiviral mechanism) or produce antibodies. 16 · 65 several viruses have been identified that inhibit interferon production in a host, resulting in increased susceptibility to other viruses. 62 • 121 host-damaging immune responses in viral infections have been reviewed adequately in the literature. 2 • 95 • 111 the mechanisms and consequences of viral immunopathology will be summarized in this section. generally, host immune responses are responsible for recovery from virus infections but, occasionally, they can initiate or enhance cell injury. certain viruses, such as herpesviruses and picornaviruses, are cytolytic, and disease results from direct destruction of tissue cells. in such instances of viral infection, the immune response can limit infection by destroying infected cells before progeny virus is assembled and released. immune-mediated cytolysis can be deleterious to the host if it occurs late in the replication cycle of the virus, because then it serves only to release infectious virus. noncytolytic viruses often do not cause direct cell injury, but immune responses may injure cells persistently infected with such viruses. a chronic inflammatory response may occur when such cells are not rapidly destroyed by immune processes. such responses frequently are harmful to the host, especially if they interfere with the function of certain tissues and organs. immunopathologic chronic inflammation usually involves antigen-sensitized t lymphocytes and/or antibody immune complexes that also activate complement with inflammatory consequences. viral immunopathology may be divided into two categories: lesions due to antibodies, either directly or with other nonspecific effector mechanisms (for example, antibody-dependent cellular cytotoxity-adcc) or lesions due to specific cellular immune responses. the classical example of virus antibody immunopathology is immune complex disease. 2 • 1 ,· 25 • 49 • 50 · 87 • 95 • 136 • 137 the development of immune complex disease is associated with circulating antibody-antigen complexes in serum, deposition of the smaller complexes (in instances of antigen excess) in tissues with limiting basement membranes (glomeruli, arterioles, choroid plexus, joints, and so on) to produce injury, and the presence of antigen, antibody, and complement components at the site of injury. the initial step in immune complex disease is the release of vasoactive substances, such as histamine and serotonin, and the subsequent increase in vascular permeability. serotonin is released from platelets when they react with immune complexes in the presence of complement. other mechanisms of immune complex-mediated vascular permeability, such as kinins generated from activated hageman factor, may also occur. such platelets may contribute also to immune complex lesions by causing capillary thrombosis. increased vascular permeability facilitates trapping of immune complexes, usually those of small size that develop in moderate antigen excess and in the walls of arterioles and capillaries. the trapped complexes activate complement locally and chemotactically attract polymorphonuclear cells. the circulating and fixed macrophages can degrade immune complexes, but the phlogogenic effect with anaphylotoxins (and the consequent release of vasoactive amines), neutrophil release of cathepsins and proteolytic basic proteins, and subsequent activation of hageman factor (which leads to kinin and plasmin production) cause direct vessel wall injury. arteritis, glomerulonephritis, arthritis, and choriomeningitis are common sequelae of immune complex disease. immune complex disease is particularly prevalent in infections with noncytopathic viruses such as those that cause equine infectious anemia, aleutian disease of mink, and feline infectious peritonitis (fip), but it may also cause some of the lesions of cytocidal viruses (for example, canine distemper virus). evidence indicates that fip is an immune complex disease. 49 • 50 • 136 • 137 humoral immunity is not protective in this disease, but a functional cell-mediated immunity appears to prevent lesions caused by fip virus. impaired cellular immunity is associated with manifestation of the disease (the effusive form with greatly deficient cell-mediated immunity, and the noneffusive form with a partially impaired cellular immunity. 91 • 92 thus, in this disease, we have paradoxical mechanisms of immunopathogenesis. one facet (the humoral response) is enhanced, whereas another (cell-mediated) immunity, may be impaired. this may be the reason fip is associated frequently with feline leukemia virus, a powerful suppressor of lymphocyte function. virus-infected cells may be destroyed by antibody-dependent cellular cytotoxicity (adcc). 95 in this synergistic action of specific antibody and effector cells, the immunologic specificity is provided by the antibody molecules (only minute quantities are required), whereas the effector cells act nonspecifically. the latter must bear fe receptors and include t cells, b cells, killer cells, macrophages, and neutrophils. complement-assisted cytotoxicity occurs when complement enhances adcc. interferon also has been reported to enhance adcc. 95 complement-mediated cytotoxicity involves virus-infected cell destruction by complement following activation by the classical and alternative pathways with specific immunoglobulins. 95 activation of complement by the alternative pathway by virus-infected cells, in the absence of antibody, may also occur. a special case of antibody-induced host injury is the enhancement, by specific antibody, or infectivity of certain viruses of host cells. this has been described for flaviviruses (for example, dengue virus) and for fip virus, for which target cells are macrophages. 49 although mononuclear cells appear to be the only cells that support replication of dengue virus, the latter is not internalized efficiently in the host cell unless complexed with non-neutralizing antibody, which attaches to fe receptors on the cells. some investigators have speculated on the possible allergic immunopathologic responses to viruses. 87 • 89 • 95 the potential for lge-mediated type i hypersensitivity in virus infections exists, but little evidence has been presented to support this hypothesis. an interesting hypothesis has been proposed to explain certain antibodymediated hypersensitivities in virus infections. 87 • 89 it envisages a viral infection (such as dengue virus) in a host with pre-existing parasitic infection leading to depletion of suppressor t lymphocytes. the resultant augmented production of igg and ige could then result in type iii and type i hypersensitivities, respectively. t-lymphocyte cytotoxicity is an immunologically specific and highly effective lysis of cells with viral antigen expression on the plasma membrane. 2 • 23 • 95 ·m in some species (most notably mice), genetic restriction oftcell cytotoxicity occurs in that there is a requirement, not only for specific antigen binding, but also for recognition of certain antigens of the major histocompatibility region. 2 the cytolytic event requires contact between the effector and the target cell. the former can lyse a number of target cells in sequence, but the number of immune t cells at a particular site may not be high; for this reason, indirect t-cell events such as macrophage recruitment may be more important in immunity and immunopathology. cytotoxic lymphokines (lymphotoxins) are soluble products oft cells following reaction with viral antigen. lymphotoxins cause cytolysis by affecting the permeability of the plasma membrane of cells. natural or normal killer (nk) cells are lymphocytes with cell surface fe receptors that nonspecifically destroy certain virus-infected and neoplastically transformed cells. interferon and complement can facilitate cytolysis by t lymphocytes and thus enhance viral immunopathology. macrophages can be directly cytotoxic, but this appears to be a nonspecific event. they may be localized at sites of specific antigen by t cellmediated and phlogogenic recruitment. evidence is accumulating that certain viruses may elicit tissue-reaction antibodies and t lymphocytes, which results in tissue and organ injury. 5 • 25 • 35 • 43 • 48 • 62 in some autoimmune diseases, the organ and tissue specificity is quite broad, whereas in other instances, it is very restricted to specific cell types. a polyendocrine disease affecting several hormones as a result of virus-induced autoimmunity has been described. 35 • 43 several mechanisms of the pathogenesis of viral pathogenesis have been described. one possibility is the virus-induced access of the immune system to sequestered antigens, such as the cns (protected by the blood-brain barrier), which are normally "immunologically privileged" sites and are not normally monitored by recirculating lymphocytes. this may occur during the acute phase of the disease. subacute or chronic demyelinating encephalomyelitis that occurs with some strains of canine distemper may be the result of such a mechanism. 62 another mechanism that is plausible is virus-induced changes in host cell membrane. the expression of new host antigens such as embryonic antigens or alloantigens is induced. a related mechanism may be the result of virus antigens expressed at the cell surface that share antigens with the host cell. the immune response to either the new or cross-reactive antigens is capable of reacting with normal tissues. the final mechanism is one that has been alluded to already-that of loss of immune regulation, such as impaired suppressor cell function. tolerance to self-antigens may be maintained by suppressor cell activity, at least in part. diminished activity of these cells then results in increased responsiveness to foreign antigens and an immune response to self-antigens. a special case of autoimmune disease may occur in mucosal disease and bluetongue virus-associated hemorrhagic disease in cattle that have "tolerant" infections as a result of in utero infections. 67 superinfection with a heterologous strain or an overwhelming dose of a homologous strain may induce an immune response to the "tolerant" viral antigens that are present in most tissue cells and thereby induce cell injury. much of the discussion of viral pathogenesis has focused on structural and functional injury of cells. another virus-induced response of cells is that of immortalization due to oncogenic transformation. the mechanism by which various viruses transform cells depends on the nature of the virus. oncogenic dna viruses belong to several virus families, but oncogenic rna viruses are members of the retroviridae. viral oncogenesis is a complex phenomenon and is an area receiving considerable attention by the scientific community. transformed cells have altered morphology, changed behavior, and altered biochemistry. often new membrane proteins and glycoproteins appear (for example, tumor-and/or virus-specific surface antigens and fetal antigens). co-carcinogens have been identified that appear to promote transformation by certain viruses. immunosuppression, a prominent feature of infection by several viruses, may be an indirect oncogenic mechanism of these viruses. it is still not clear which of the complex of changes induced in cells by viruses cause transformation and which are secondary events. at best, this review can only summarize the information on viral oncogenesis, and in order to achieve this, the subject material is greatly oversimplified. for a more detailed description of viral oncogenesis, please consult the reviews by dues berg, 3° fenoglio and lefkowitch, 34 weiss, 135 generally, dna viruses replicate in cells and destroy the cell after progeny is produced. cells that support virus replication in this manner are known as permissive cells. however, in some cells (nonpermissive cells), virus replication is not completed and progeny virions are not produced. on rare occasions, these nonpermissive cells become transformed. defective virus may also, on rare occasions, transform both permissive and nonpermissive cells. an essential event for dna virus-induced transformation appears to be integration of viral dna into host dna. poxviruses seem to be the exception. indeed, poxvirus-induced tumors consist of hyperplastic rather than trans-formed cells. neither the cellular site of integration nor the location on virus dna is unique or even specific, and integration seems to be the result of random "illegitimate" recombination between nonhomologous regions of the virus and the host. there is insufficient evidence that oncogenes are involved in dna virus transformation. although the entire viral dna genome may be integrated in transformed cells, all transformed clones contain at least part of the early region of the viral genome. various early functions seem to initiate and/or maintain the transformed phenotype, whereas late viral functions do not have a role in transformation. thus, many dna viruses mediate transformation through the action of some viral gene products, which generally are required for the virus to complete its normal cytolytic cycle. it is not known exactly how the transformation genes originate, but, unlike the retroviruses, there is little evidence that they are derived from normal host genes. however, there is some evidence for limited transcription from virus dna sequences into flanking host sequences. in addition, recent transfection experiments with burkitt's lymphoma cells resulted in the discovery of an oncogene of cellular origin. this oncogene is activated in burkitt's lymphoma. furthermore, one locus in the human genome has some homology with the gene-enhancer sequences of a human papovavirus, indicating that some of the oncogenic enhancer dna sequences of dna viruses can be evolutionarily related to host cell sequences. it appears also that dna virus oncogenes can interact with retroviral (or cellular) oncogenes. a synergistic interaction of the "ras" oncogene and an adenoviral oncogene has been reported. viral dna can mediate transformation through several possible mechanisms. viral dna integrated at certain sites in host dna could act as a mutagen, thereby destroying the control of cellular genes. perhaps integrated viral genetic material may contain promoters of viral gene expression and also coincidentally affect host gene expression. finally, viral dna may specify protein(s), which when synthesized, may directly cause transformation of the cell. as mentioned earlier, several viral early proteins have been identified that initiate and/or maintain transformation. the large t and small t antigens of sv40 virus are proteins that function in this manner. co-carcinogens promote oncogenesis by several dna viruses. the flavinoid (quercetin) from bracken fern is associated with progression of upper alimentary tract papillomas to carcinomas in cattle. burkitt's lymphoma, a malignent b-cell lymphoma of humans, is associated with infection by the herpesvirus epstein-barr virus. the most likely co-factor is holoendemic malaria. aflatoxin appears to be a co-factor in primary liver carcinoma associated with hepatitis b virus. the oncovirinae subfamily of the retroviridae, due to its unique intracellular biology, has an exquisitely well-developed mechanism for cell transformation. 30 • 34 • 116 • 118 • 141 the enzyme, reverse transcriptase, constitutes the fundamental basis of the molecular biology of these viruses. the diploid rna genome usually contains four major genes in a specific sequence. the gag gene, which is closest to the 5' end, codes for a poly protein, a precursor of four structural proteins of the nucleoid. the next gene, pol, codes for the reverse transcriptase, whereas the env gene codes for the two glycoproteins on the surface of the envelope. the final major gene codes for a phosphoprotein with protein kinase activity and is responsible for neoplastic transformation. the latter gene, known as the virus oncogene, is inconsistently present on oncoviruses. virus replication is initiated when virus binds to specific receptors and penetrates into a cell. in permissive cells, virus progeny is produced and the host cell may or may not become neoplastic. nonpermissive cells may be transformed occasionally, but viral replication is never completed. defective viruses may efficiently transform both permissive and nonpermissive cells. within a few hours, virion reverse transcriptase synthesizes linear double-stranded dna copies of the haploid genome. linear dna molecules migrate to the nucleus and become circularized. the latter becomes integrated into cellular dna, a step essential for retrovirus replication and gene expression. integrated virus dna is known as the provirus, and the number of copies in the haploid host genome varies from 1 to 100. the integrated provirus retains the topography of the viral genome and is subjected to expression and control by host mechanisms. proviruses can be transmitted vertically and thus inherited in germ cells as genes by mendelian genetics. viral gene expression relies on the hospitality of the host cell. transcription of proviral dna is catalyzed by cellular rna polymerase ii. expression may be governed by genetic determinants of the cell that are closely linked but separable from the provirus. two general types of oncoviruses exist. the sarcoma-type viruses transform fibroblasts in vitro, have an oncogene, and usually are highly oncogenic. often they fail to produce progeny virus because of a deletion of the env function and are known as replication defective (rd). coinfection with a related virus containing intact env activity may provide the deficient envelope glycoproteins to allow full maturation and progeny sarcoma virus but with surface antigen specificity of the helper (or associated) virus. prior infection or occupation of cell surface receptors by an oncovirus with identical envelope characteristics will result in interference and prevent superinfection by the second virus. the initial virus is known as the resistance-inducing factor. leukemia viruses do not seem to have an oncogene and cannot transform fibroblasts in vitro. such viruses are transformation defective (td). thus, the transforming segment, the oncogene, is not essential for virus replication and is not of intrinsic importance to the virus. however, many of these viruses are leukemogenic in animals after a prolonged incubation period and seem to be responsible for many of the leukemias and lymphomas of various animals. the mechanism of leukemogenesis is not known, but it is likely that an indirect mechanism occurs with these viruses. perhaps these viruses recombine with other viral genomes, but more likely, because of their insertion in the vicinity of cellular oncogenes, activate the latter. cellular oncogenes are very similar to those of sarcoma viruses, and it seems plausible that the latter may have acquired oncogenes by transduction during past interactions with their host cells. various hypotheses on oncovirus oncogenesis have been proposed as the information on the biology of these viruses became available. the virogene-oncogene theory was proposed in 1969 by heubner. temin introduced in 1972 the protovirus concept for viral oncogenesis and the existence of endogenous oncoviruses. however, considerable evidence now exists to support the cellular oncogene theory, which contains some of the concepts of the two hypotheses oftemin and heubner. an oversimplified explanation of this hypothesis is that most cells contain the so-called oncogenes, a highly conserved host gene with a useful function. this gene product seems to be a protein kinase catalyzing the phosphorylation of certain proteins and has been implicated in regulating growth of normal cells by activity on cell surfaces. this unique protein kinase differs from the normal cell kinases in that it phosphorylates tyrosine instead of serine or threonine. a large number of viral oncogenes and cellular equivalents have now been identified. they are distinguished by the prefix "c" for cell oncogenes and "v" for viral oncogenes. viral oncogenes are probably of cellular origin and may be merely a passenger acquired by sarcoma viruses from the host dna. viruses with oncogenes are directly oncogenic, whereas oncoviruses without oncogenes are indirectly tumorogenic because they interfere with a cellular oncogene at or near the site of provirus integration. two hypotheses have been proposed to explain the mechanism of oncogene transformation; the mutational hypothesis and the dosage hypothesis. the former requires that the viral oncogene differs, as a result of mutation, from the parent cellular oncogene and, upon expression of the gene, neoplastic transformation occurs instead of its normal regulatory function. furthermore, in the instance of nononcogene-bearing leukemia viruses, a similar mutation occurs in cellular oncogenes during integration. proto-oncogene (inactive) activation by one-point mutation has been best documented in the family of "ras" oncogenes. the dosage (or amplification) hypothesis requires that excessive production of the gene product occurs after infection with a sarcoma virus and formation of one or multiple copies of the provirus in the cell. nononcogene-bearing leukemia viruses probably stimulate cellular oncogene activity because some are inserted as proviruses in the immediate vicinity of this gene. recent evidence suggests that the latter theory may be accurate. normal cellular control prevents the oncogene's transforming capacity. however, when an oncovirus includes an oncogene in its genome, the oncogene is removed from its controlled environment, resulting in increased production during viral replication. enhanced activity of the oncogene probably is due to the viral promoter. an unexpressed oncogene (proto-oncogene), when introduced with its normal promoter into host cells, does not cause transformation; however, transformation occurs when the viral promoter accompanies the cellular oncogene in transfection experiments. the activation of normal cell oncogenes by leukemia viruses is probably due to the viral promoter inserted in the vicinity of these genes. the delayed tumor induction of leukemia viruses may be due to random integration of the viral genome. thus, the location of the viral promoter at the appropriate site for affecting the cellular oncogene is an inefficient event. the appropriate point of integration of the promoter into the cellular dna varies, and evidence indicates that the site can be some distance from the target oncogene. another mechanism of retroviral neoplasia is related to immunosuppression induced by some of these viruses. immune surveillance may be severely 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10.1016/s0020-7519(99)00076-4 sha: doc_id: 308816 cord_uid: nux087gc cryptosporidium species are coccidian parasites with a large capacity to reproduce and to disseminate. several species are known to infect farm animals, although the economic importance of cryptosporidiosis is highly host species dependent. this paper reviews the impact of cryptosporidial infections in livestock and poultry. for different farm animals, the cryptosporidium spp. that occur, as well as their clinical and pathological features, and their interactions with other pathogens, are described. in addition, data concerning the prevalence, the transmission and the epidemiology of the disease are mentioned and a description of the economic losses associated with cryptosporidiosis in each of the hosts is given. cryptosporidiosis seems to be mainly a problem in neonatal ruminants. cryptosporidium parvum is considered to be an important agent in the aetiology of the neonatal diarrhoea syndrome of calves, lambs and goat kids, causing considerable direct and indirect economic losses. avian cryptosporidiosis is an emerging health problem in poultry, associated with respiratory disease in chickens and other galliformes, and with intestinal disease in turkeys and quails. because of limited availability of effective drugs, the control of cryptosporidiosis relies mainly on hygienic measures and good management. the genus cryptosporidium was named at the beginning of this century, but was only recognised as a potential cause of disease in 1955, when it was found to be associated with diarrhoeic turkeys [1] . although cryptosporidium was subsequently found in a broad range of farm animals, its impact was neglected until the early 1980s when it was found to be a common, serious primary cause of outbreaks of diarrhoea in certain farm mammals [2, 3] . the fact that cryptosporidium was found to infect humans [4] , and could cause a life-threatening disease in immunode®cient people, especially aids patients [5] , as well as the association of cryptosporidium with waterborne-related human outbreaks of diarrhoea [6] , has certainly given the parasite a more widespread recognition. it has encouraged the scienti®c work on cryptosporidium in many domains, such as in the veterinary ®eld, to some extent because animal husbandry is seen as a threatening source of infection for humans by the release of tremendous numbers of resistant oocysts in surface waters. recent studies have revealed the heterogeneity of the cryptosporidium sp. infecting both humans and animals, making the causal connection between animal and human cryptosporidiosis far more complex [7] . nevertheless, the interest for cryptosporidiosis in the veterinary ®eld arises even more from the fact that it concerns a harmful, dicult to control disease of many farm animals, that results in signi®cant economic losses. this point will be addressed in this review. the life cycle of cryptosporidium is direct and monoxenous and it follows the patterns described for other enteric coccidia, which include a merogonic cycle with two generations of meronts, a gametogonic cycle with macrogametes, microgametes and zygotes and a sporogony (outlined in [8] ). compared with eimeria spp., the life cycle of cryptosporidium is characterised by a number of peculiarities, some of them of major importance for the establishment and spread of the infection and for the treatment of the disease: (i) exposure of cryptosporidium oocysts to reducing conditions, pancreatic enzymes and bile salts, results in a high percentage of excystation. however, in contrast to most other coccidia, cryptosporidium oocysts can liberate their sporozoites in warm aqueous solutions without any of the aforementioned special stimuli [9] . this spontaneous excystation explains, in part, the ability of cryptosporidium to infect tissues other than the intestine, such as the conjunctiva of the eye [10±13] and the respiratory tract [10, 14±19]; (ii) the parasite develops inside the epithelial cell of the digestive or respiratory tract, although on the edge of the host cell cytoplasm and separated from it by a feeder organelle membrane. this intracellular extracytoplasmatic location is unique for the coccidia and might play a major role in the failure of many antimicrobial agents to inhibit the growth of cryptosporidium [20] ; (iii) two stages can cause auto-infection: the recycling type i meronts and the thin-walled oocysts. consequently, in the absence of a protective immune response cryptosporidium may persist inside a single host, even without further exposure to exogenous oocysts; (iv) the thick-walled oocysts are already fully sporulated when they leave the body with the faeces and are therefore immediately infectious. thus, cryptosporidium seems to have an extraordinary reproductive ability. in addition, the oocysts can travel a considerable distance following runo [21] , can survive for a relatively long time in an aqueous environment [22, 23] , and are infectious to a wide range of animals, thus having many potential excretors [20] . as a result, this parasite undoubtedly has an exceptional capacity to disseminate. infection by cryptosporidium spp. in cattle were ®rst reported in the early 1970s [24±26]. however, because of the association with other viral or bacterial enteropathogens, the role of cryptosporidium spp. as primary enteropathogens was uncertain until 1980, when tzipori et al. [2] attributed an outbreak of neonatal diarrhoea to cryptosporidial infection alone. in the following years methods to free the infective oocysts from other contaminating pathogens became available, which permitted the experimental demonstration that cryptosporidium was capable of causing clinical diarrhoea in calves [27, 28] . in cattle two species of the genus cryptosporidium can be distinguished: cryptosporidium parvum infecting the distal small intestine, and cryptosporidium muris infecting the abomasum. substantial dierences in the size and shape of c. parvum (5.0 mmâ 4.5 mm and sperical [29, 30] ) and c. muris (7.4 mmâ5.6 mm and ovoid [30] ) oocysts enables the two species to be distinguished readily on microscopical examination. only c. parvum has been associated with neonatal diarrhoea. cryptosporidium muris is much less prevalent and was only found in weaned calves or adult cattle [31±33] and c. muris infection is considered to be clinically mild, aecting weight gain [34] and milk production [35] . the kinetics of oocyst shedding of experimentally c. parvum infected neonatal calves, revealed a prepatent and a patent period ranging from 3± 6 and 4±13 days, respectively [36] . in practice, oocyst excretion has been described as early as 3 days of age, which means that calves are already susceptible for infection during or shortly after birth [37±39] . calves raised in isolation from cryptosporidium remain susceptible to infection at older age, but the clinical signs become less severe [40] . in neonates, a great variability was observed in the severity and duration of diarrhoea due to cryptosporidiosis, even when the animals were exposed to similar conditions. in most calves diarrhoea has already began 3±5 days p.i., and lasted from 4 to 17 days [36] . in fattening units, where the prevalence of c. parvum is known to be high (93%), up to 38% of the calves developed a liquid diarrhoea, that could be attributed to cryptosporidiosis [41] . cryptosporidial diarrhoea is associated with the excretion of tremendous numbers of oocysts [42] . high mortality due to cryptosporidiosis has been reported, even in the absence of other enteropathogens [43] , and this would occur more often in the belgian blue±white, and the french limousin and charolais meat breeds [44] . cryptosporidium infections are mainly concentrated in the distal small intestine but lesions were also found in the caecum and colon [43, 45] , and occasionally in the duodenum [43] . the pathological ®ndings associated with cryptosporidium are a mild to moderate villous atrophy, villous fusion, and changes in the surface epithelium (reviewed in [46] ). further, in®ltration of mononuclear cells and neutrophils was seen in the lamina propria [43] . after primary exposure, a/b t-cells, both cd4 + and cd8 + , and g/d t-cells accumulated in the intestinal villi, while in challenged immune animals only an increase in the number of cd8 + t-cells was found [47] . respiratory cryptosporidiosis has been reported [19] , but can be considered to be of less economic importance than the enteritic form. the neonatal diarrhoea syndrome, both in large and small (see section 4.3) ruminants, is a clear example of a multifactorial disease governed by a wide range of factors related to the animal, conditions of the environment and husbandry, and a variety of viruses, bacteria and protozoan parasites. in calves, enterotoxigenic escherichia coli (etec), with thermolabile and/or thermostable enterotoxins and with colonisation factors [48] , are recognised as a common cause of diarrhoea in calves under 3 days old. this age-dependence is caused by a diminished adherence of etec to enterocytes after the ®rst few days of life [49] . between 4 days and 6 weeks of age digestive problems can mostly be attributed to c. parvum and/or a variety of viruses, with rotavirus, coronavirus and bovine viral diarrhoea (bvd) virus being the most important. data from the veterinary and agrochemical research centre in brussels, on the proportional occurence of viral enteropathogens and c. parvum in samples from diarrhoeic calves (fig. 1) , highlighted the increasing importance of cryptosporidium. during the period july 1982±june 1983 cryptosporidium infections made up only 6.7% (3.9% as a single pathogen, 2.8% in association) of the digestive problems, whereas the impact of cryptosporidiosis was considerably greater in 1992±1993. indeed, almost 40% of the diarrhoeic calves shed c. parvum; in 30.7% of the cases cryptosporidium was the only pathogen present, and in 8.9% it was found in association with a viral infection. for comparison: rotavirus, coronavirus and bvd-virus were found as a single pathogen in, respectively, 11.2, 23.7 and 8.9% of the diagnosed samples. the decreased number of cases of rotavirus, shown in fig. 1 , is probably due to the use of commercialised rotavirus vaccines that became available halfway during the studied period (vanopdenbosch, personal communication). the proportional occurrence of these pathogens may dier according to geographic parameters and the studied period, but in the last decade several studies described c. parvum as the most commonly detected enteropathogenic agent in calves [50±52]. attaching and eacing e. coli have been implicated in diarrhoea and dysentery in 2-to 8-weekold calves [53, 54] , with a calculated mean age of 5 weeks [55] . verotoxigenic e. coli, a group of bacteria that produces potent cytotoxins known as verocytotoxins, were found to be occasionally associated with calf diarrhoea [56] . they were detected in 6.0 and 9% of diarrhoeic calves in germany [57] and spain [58] , respectively. there are reports of viruses other than the aforemen-tioned ones, populating the intestine of young calves (picobirnavirus [59] , calici-and astrovirus [60] , adenovirus [61] , enterovirus [62] and parvovirus [63] ), but their pathogenicity is still uncertain. giardia infections have also been found to be exceptionally frequent in suckling and weanling calves, although the role of this protozoan parasite in the aetiology of diarrhoea in calves remains unclear [39] . finally, the problems related to clinical salmonellosis are only minor in dairy, beef [64] and veal units [65] . cryptosporidium parvum oocysts were detected in bovines ranging from 3 days old to adults, although the prevalence was signi®cantly higher in suckling calves [39, 66±68] . in a spanish epidemiological study on the prevalence and age distribution of c. parvum infections, as many as 44.4% of calves aged 3±4 days were infected, but infection rates peaked (76.7%) at 6±15 days of age. prevalence was also high in weanling calves aged 1.5±4 months (14%), fattening calves and heifers 4±24 months old (7.7%) and adults (17.8%). however, cryptosporidium infection was only statistically associated with diarrhoea in suckling calves [39] . the transmission occurs by the faecal-oral route. the majority of adult cattle can be described as excretors of c. parvum oocysts when sensitive detection methods are used. nevertheless, their importance in the transmission of the disease remains questionable, since oocyst excretion by adult cattle was similar in herds with serious problems of cryptosporidial neonatal diarrhoea and in those without [68] . so far, in cattle, no increased output of c. parvum oocysts around parturition has been observed [67±69]. however, these ®ndings should be considered with great caution, as it was shown that more frequent samplings and more sensitive detection methods in sheep revealed a periparturient rise in oocyst score (requejo-fernandez ja, pereira-bueno j, pilar-izquierdo m, rojo-vazquez fa, ortego-mora lm. periparturient rise in ovine cryptosporidiosis. in: proc cost 820 wg3 meeting. brussels, 1997, p. 5). nevertheless, infected newborn calves excrete oocyst numbers of the order of 10 6 to 10 7 g à 1 of faeces [42] and were considered to be a more dangerous source of infection. infection can rapidly spread from calf to calf when animals are communally housed and overcrowded, or from cow to calf via the udders when they are contaminated with infected calf faeces in the lying area of the dams [46] . this might explain the association between cryptosporidial diarrhoea and the farm type and/or its speci®c hygienic conditions. cryptosporidium infections were more common in single or multiple suckler beef herds [70] and dairy farms with multiple-cow maternity facilities [71] . in the so-called fattening units, where calves are purchased through markets, almost 100% of the animals become infected during transit from the market to the rearing unit or soon after their arrival [41] . other potential risk factors are herd size [71] and season. in a canadian study of beef calves, higher prevalence was found in winter and spring, the period related to calving season and consequently the period with the greatest number of calves in the high risk group (1-to 3-weeksold) [72] . however, in american dairy farms, where calvings tend to be year-round, and environmental contamination level is less subjected to¯uctuations, cryptosporidiosis was more prevalent in the summer [71] . little information is available on dierences in infectivity, excretion patterns, virulence or immunogenicity among dierent isolates of c. parvum from cattle or other sources [36] . this is at least partially due to the lack of single oocyst cloned isolates since all reported isolates are heterogenic mixtures. the economic losses due to cryptosporidial infections of neonatal calves are related to diarrhoea: dehydratation, growth retardation and to a lesser extent mortality [43] . diarrhoeic problems of calves demand special care: feeding of electrolyte solutions, i.v.¯uid therapy, drug administration, hygienic measures, etc. which are costly as well as labour and time-consuming. in belgium, mortality due to the neonatal diarrhoea syndrome is estimated between 5±10% (vanopdenbosch, personal communication). cryptosporidium parvum is considered as the most commonly found enteropathogen in calves during their ®rst weeks of life [50±52]. the parasite frequently acts alone, but the losses are more severe when concurrent infections occur (vanopdenbosch, personal communication) , as has been demonstrated for viral and bacterial enteropathogens [73] . in sheep, infection by cryptosporidium was ®rst described in australia in 1-to 3-week-old lambs with diarrhoea [25] . its role as a primary aetiological agent was con®rmed in the early 1980s in studies on experimental infections in the absence of other enteropathogenic agents [3, 74] . since then, cryptosporidium has been attributed an increasingly important role in neonatal diarrhoea syndrome in this domestic species and is currently associated with high morbidity rates and, depending on environmental conditions and the presence of other intestinal pathogens, mortality [46, 75, 76] . in goats, infection by this agent was also ®rst described in australia, in a 2-week-old kid with diarrhoea [77] . since then, the infection has been diagnosed in outbreaks of diarrhoea in goat kids in several european countries [78±80] and is now considered to be one of the principal enteropathogens in these animals [76] . only one of the two species of the genus cryptosporidium described in domestic ruminants, c. parvum, has been associated with diarrhoea in small ruminants. in a recent work, a bovine isolate of c. muris did not produce infection in goats [81] . in lambs, the prepatent period oscillated between 2 and 7 days [82] and around 4 days in goat kids [83] . this period increases with a reduction in the dose of the infectious agent [84] or with an increase in the age of the animal [85] . the main clinical manifestations of cryptosporidiosis in neonate small ruminants are: apathy and depression, anorexia, abdominal pain, and mainly diarrhoea accompanied by the shedding of a large number of oocysts [74, 83, 85±88] . the faeces are usually yellow, have a soft or liquid consistency and give o a strong unpleasant odour. in experimental infections in lambs [85] the dry weight of the faeces can drop from 24 to 10%. in milder cases of the disease the animals have diarrhoea for 3 to 5 days and in more severe cases for 1 to 2 weeks. the diarrhoea usually coincides with the period of oocyst shedding. the duration of the oocyst shedding depends on factors such as the age or immune status of the animals. the kinetics of the shedding is usually similar in experimentally and naturally infected animals. after the ®rst 2 or 3 days of the patent period there is a progressive increase in the number of oocysts shed which reaches a maximum 5± 6 days p.i. and drops sharply between days 10 and 15 p.i. [83, 85, 89] . in addition to the diarrhoea and oocyst shedding the animals character-istically manifest anorexia [74, 83, 85] which results in weight loss and retarded growth during the ®rst few weeks of life [90] . studies on the development of the lesions over the incubation period and the clinical course of the disease [74, 83] have shown that, over this period, the parasite mainly proliferates in the jejunum and the ileum. after the start of oocyst shedding (3 days after the start of infection) the lesions can spread to other parts of the small and large intestine. a recent study showed that the most frequent aetiologic agent involved in outbreaks of diarrhoea in lambs was c. parvum (65% of the outbreaks and 45% of the individuals) followed by e. coli potentially pathogenic (61% of the outbreaks and 30% of the individuals). other agents such as rotavirus were less important (7% of the outbreaks and 2% of the individuals). in goat kids, c. parvum was present in 40% of the outbreaks and 42% of the individuals, followed by e. coli (36 and 22%), rotavirus (14 and 22%), clostridium perfringens (20 and 11%) and salmonella spp. (7 and 3%) [76] . in a parallel study, it has been shown that e. coli strains isolated from diarrhoeic lambs and kids older than 4 days (mainly between 7 and 15 days old) are not generally toxigenic and belong to a large number of serogroups [91, 92] . in small ruminants older than 4 weeks, infections by eimeria spp. are increasingly important accompanied by dietary changes and situations of stress [93] . the role of other intestinal protozoa such as giardia sp. is controversial and although infection by this parasite is often diagnosed during the ®rst month of life [94] it is not clearly associated with the production of diarrhoea [39, 95] . the prevalence of infection by c. parvum in lambs and goat kids has been studied in both outbreaks of diarrhoea and in randomly selected farms (table 1) , although more research has been done on cattle. in outbreaks of diarrhoea, morbidity can be very high in lambs [98, 100] and goat kids [86, 102±104] . mortality increases when the disease is associated with concurrent infections or de®ciencies in nutrition or husbandry [82, 87, 100] . in goat herds in france and hungary, c. parvum was considered to be the predominant aetiological agent in neonate goat kids with diarrhoea [79, 102] . in spain, in a study on 97 sheep farms and 31 goat farms, all randomly selected, corresponding to a total of 2204 lambs and 367 goat kids under 5 weeks old, ock prevalence was 47 and 36% and individual prevalence was 15 and 11% for lambs and goat kids, respectively [80] . infection occurs mainly by the ingestion of oocysts previously eliminated with the faeces of infected neonates or asymptomatic adult carriers [100] . daily excretion of oocysts by infected lambs can exceed 2â10 9 and more than 10 10 oocysts are shed during the patent period [85, 89] . moreover, adult sheep can act as asymptomatic carriers shedding small numbers of oocysts to the environment which was shown to increase in number in the perinatal period and contribute to maintaining the infection between lambing periods. between 75 and 100% of lambs born in this environment become infected in the ®rst few weeks of life [105] . in a recent study, the viability of oocysts shed was shown to be greater between days 5 and 11 p.i. than between days 11 and 15 p.i. [106] . other possible sources of infection, such as farm rodents, should also be considered [107] . the in¯uence of the dose of the infectious agent on the course of the disease has been studied by several authors. there do not seem to be any dierences between natural or experimental infections with respect to clinical symptoms, oocyst shedding or serum antibody responses [108] . however, the prepatent period is a few days longer in experimental infections with low infectious doses [84] . in gnotobiotic lambs the minimum infectious dose can even correspond to a single oocyst and the average infectious dose is around ®ve oocysts [84] . at present, no intraspeci®c dierences in pathogenicity have been found between isolates obtained from domestic ruminants. however, in the laboratory of one of the authors (ortega-mora, personal communication), two dierent isolates obtained from diarrhoeic ruminants showed signi®cant dierences in the number of oocysts shed and the severity of the diarrhoea after experimental infection in lambs. however, further research is necessary to con®rm this ®nding. in both natural and experimental infections the clinical process that accompanies infection has been reported to be more common in lambs and goat kids under 30 days old [76, 82, 85] . extension of the prepatent period and reduction of oocyst shedding occur as the age of the lambs at infection increases. in a recent study, diarrhoea was only present in lambs infected at 6 days old and in a small percentage of lambs infected at 28 days old, but not in lambs infected at 56 days old. the size of the speci®c serum (igg and iga) and faecal (iga) humoral response was similar and apparently independent of the age, suggesting the participation of mechanisms other than the humoral response in the development of protection [85] . however, a greater intestinal mucus production and increased glucoprotein concentration in the mucus was observed in animals infected at 28 or 56 days old. the kinetics of the iga in the intestinal mucus response varied with the age of the animal at infection, the most rapid responses and highest titres occurred in animals which were older when infected (quintanilla-gozalo a, wright s, pereira-bueno j, rojo-vazquez fa, ortega-mora lm. changes in the intestinal mucus during infection by cryptosporidium parvum in lambs. in: proc 1995 cost 820 annual workshop. prague, 1995, p. 70). in natural infections, the majority of the infected lambs and kids were also less than 2 weeks old and the proportion of infected animals underwent a pronounced decrease in animals more than 3 weeks old [76, 80, 109] . the economic losses associated with this disease are not only due to the resulting mortality, but also to the retarded growth of the animals, the cost of drugs, veterinary assistance and the increased labour involved. in the absence of other enteropathogens, mortality is higher in lambs than in calves [88] and morbidity can reach 100% [46, 75] . the anorexia is very pronounced at the start of the process in both lambs [74, 82, 85] and goat kids [83, 90] . in lambs naturally infected with c. parvum, there was a mean weight dierence of 2 kg at 4 weeks old compared with non-infected control lambs of this age (ortega-mora, personal communication). first reports of a cryptosporidium sp. in avian species were from tyzzer [110] . he found a parasite in the caecal epithelium of chickens with structural similarities to c. parvum, found in mice. at present, only two cryptosporidium spp. are recognised as valid in avian hosts: (i) cryptosporidium meleagridis, ®rst reported in turkeys [1] ; and (ii) cryptosporidium baileyi, isolated from broiler chickens [111] . cryptosporidium meleagridis and c. baileyi can be distinguished by morphological dierences between their oocysts (length and width) [112] . there is possibly a third species from bobwhite quail [113, 114] and fourth species from ostrich [115] . infection by cryptosporidium spp. has been detected in over 30 species of birds including domesticated chickens, turkeys, ducks, geese, quails, pheasants, peacocks, and a wide variety of wild and captive birds [116, 117] . cryptosporidium meleagridis may infect the intestinal tract, bursa of fabricius (bf) and cloaca of turkeys [118] and chickens [112] , but infection was only associated with illness, including diarrhoea and moderate mortality in turkeys [119] . cryptosporidium baileyi may infect the respiratory tract (larynx, trachea, primary and secondary bronchi, air sacs), bf and cloaca of chickens [120, 121] , turkeys [122] and ducks [123] . it is the most prevalent cryptosporidium sp. in poultry and the most commonly associated with disease in chickens. following experimental in-oculation, c. baileyi can develop infections in many anatomic sites of its avian hosts, and it seems that the route of oocyst administration strongly determines the site where the infection ®nally establishes (reviewed in [124] ). a cryptosporidium sp. was responsible for intestinal disease, including diarrhoea, in quails [113, 114] . sometimes, the disease can be severe and mortality can reach 90% in young quails [125] . naturally occurring cryptosporidiosis in chickens usually manifests as respiratory disease, and only occasionally as intestinal or renal disease [124] . symptoms (depression, anorexia, emaciation, coughing, sneezing, dyspnoea), pathological consequences (excessive mucoid exudate, local in¯ammation, airsacculitis, deciliation, epithelial hypertrophy and hyperplasia), as well as increased mortality are most often associated with respiratory cryptosporidiosis [15, 126, 127] . the primary respiratory disease potential of c. baileyi was proven in the absence of other detected pathogens, and the negative eect of cryptosporidiosis on growth performance and carcass pigment was clearly shown [121] . severe air sac disease [15, 121] which resulted in heavy carcass condemnation at processing, places c. baileyi among the agents to be considered in the respiratory disease complex [121] . the infection of immunocompetent birds is self-limiting. two-week-old broiler chicks inoculated orally with c. baileyi oocysts and capable of clearing the endogenous stages from the cloaca and/or bf, were resistant to subsequent oral challenge [128] . furthermore, there is an age-related resistance to clinical disease. older chickens are less susceptible to c. baileyi infection, exhibiting a longer prepatent and a shorter patent period [129±131]. unlike younger (7-day-old) chicks [121] , food conversion and body weight gain were not in¯uenced by c. baileyi infection in 26-day-old broilers [132] . in practice, infections with cryptosporidium are known to occur only in chickens less than 11 weeks of age [133] , and not in adult birds [127, 134] . when appropriate diagnostic tools are used, cryptosporidium spp. appear to be present wherever avian hosts are raised commercially [135] . cryptosporidium baileyi has been reported in domestic chickens from asia, australia, europe, north america [136] and north africa [137] . the prevalence of cryptosporidium spp. in chickens and other galliformes has been studied both in outbreaks of respiratory and/or intestinal disease and in randomly selected¯ocks ( table 2) . histological observations demonstrated a similar incidence of cryptosporidium infection in broilers and commercial egg-layer-type pullets [134] . however, limited epizootiological data suggest that the infection in the latter [126] is underreported compared with that in broilers [136] . goodwin et al. [148] found that 41% (23/56) of the northern georgia broiler¯ocks had c. baileyi tracheitis. parasitism rates among c. baileyi-infected¯ocks ranged from a low of 10% to a high of 60%. cryptosporidium baileyi tracheitis was very highly correlated to severity of tracheitis, negatively correlated with average body weight, and correlated with airsacculitis and condemnations. this study indicated that c. baileyi infection rates in georgia were much higher than previously suspected. avian cryptosporidiosis has been reported in concurrence with immunosupressive viruses like reovirus [113] , marek's disease virus (mdv) [149± 151], infectious bursal disease virus [139, 150] , and chicken anemia virus [152] . in chickens, the synergistic eect of c. baileyi and these viruses was experimentally con®rmed (reovirus [153] [154] , chicken anaemia virus [155] ). marek's disease virus may induce the establishment of the parasite in inhabitual sites ( [156] , abbassi h, coudert f, cherel y, brugere-picoux j, naciri m. experimental reproduction of renal cryptosporidiosis (c. baileyi) in spf chickens after oral inoculation of parasite. in: aaap meeting. baltimore, 1998, pp. 210±211). the severity of respiratory disease induced by c. baileyi can be enhanced by other respiratory pathogens, such as infectious bronchitis virus and e. coli (157, hoerr fj, blagburn bl, lindsay ds, giambrone jj. interactions of cryptosporidium with other infectious pathogens in chickens. in: proc of the 124th annual meeting of the am vet med assoc, chicago, usa, 1987, p. 134). moreover, c. baileyi infection aects the development of humoral immunity to heterologeous antigens [121, 158, 159] , and can diminish cellmediated immunity, as shown by decreased delayed hypersensitivity indices of infected chickens [121] . chickens can become infected by ingestion or inhalation and/or aspiration of oocysts present in the environment (litter, faeces, water, breeding materials, dust, etc.). as few as 100 oocysts can result in intestinal or respiratory infections [135] . since c. baileyi can infect a variety of wild birds they must be considered as possible sources of infection. although c. baileyi does not infect rodents (mice and rats) or insects, they may serve as mechanical carriers of oocysts [135] . hygienic conditions and management strongly in¯uences the incidence and persistance of avian cryptosporidiosis. the disease was shown to be more prevalent in¯ocks with defective hygiene [137] . goodwin et al. [145] , suggested that the increase of intestinal and respiratory cryptosporidiosis infection from 1% (63/6050) during 1974±1984 to 6% (157/2622) during 1984±1988, could be explained in part by the relatively widespread practice of cleaning out poultry houses less frequently. the increased use of`built-up litters' could increase exposure to and infection of chicks by cryptosporidium [145] . in quails, rigorous clean up and disinfection with hypochlorite acid was eective in preventing continued infection, morbidity and mortality attributed to cryptosporidium sp. [125] . furthermore, outbreaks of cryptosporidiosis seem to be related to climatologic parameters. it was found that the percentage of cryptosporidiosis cases in winter was signi®cantly lower than in other seasons [134] . dierences in the incidence between geographic locations were related to the number of days of frost [147] . for a long time, avian cryptosporidiosis was considered to be an opportunistic disease, but it is now recognised as a ®rst order disease [120, 121, 127] . the economic losses associated with this disease are due to poor¯ock performance (growth retardation and increased consumption index) and/or mortality [121, 140, 141, 148] . in addition, cost of therapy, although most drugs are inecient [122, 125, 142, 160] , and carcass condemnation at processing due to air sacculitis [15, 121] , should also be taken into account. when c. baileyi occurs concurrently with immunosuppressive or other respiratory pathogenic agents, the consequences are more serious, and the losses more important [15, 127, 157] . vaccination with the attenuated serotype 1 mdv can enhance the severity of subsequent cryptosporidial respiratory disease, resulting in unexpected losses (abbassi h, couder f, cherel y, brugere-picoux j, naciri m. eect of cryptosporidium baileyi on the development of vaccinal immunity to marek's disease in spf chickens. in: 5th avian immunology research group meeting. turku, 1998, p. 7.4). thus, the role this parasite plays in the pathogenesis of respiratory disease and the related production losses could be unexpect-edly large. therefore, avian cryptosporidiosis should not be neglected or overlooked during diagnosis. cryptosporidial infection was ®rst reported in pigs by bergeland [161] and kennedy et al. [162] . thereafter, naturally occurring cryptosporidiosis in pigs has been described worldwide [163] . experimental infections of piglets resulted only in diarrhoeic problems when the animals were inoculated before 2 weeks of age [164±166]. in older piglets, experimental cryptosporidium infection did not cause clinical manifestations [ 167± 169] . the prevalence of cryptosporidiosis was reported to be very low in nursing piglets [168, 170, 171] . several studies demonstrated that naturally occurring cryptosporidiosis is delayed until after weaning [146, 169, 171±173] . the infections are mostly asymptomatic and usually of low intensity, and no statistical association between infection and clinical symptoms has been found [171, 174] . previous studies have con®rmed that diarrhoea in suckling and weaned piglets is usually a multifactorial problem, where mixed infections between c. parvum and e. coli or rotavirus are frequent. it has been suggested that cryptosporidium is not an important primary agent of diarrhoea in piglets but it may be a copathogen in the multifactorial aetiology [163] . recently, genetic analysis of the cryptosporidium isolates from pig herds revealed that the animals harboured two distinct genotypes: a porcine genotype and a bovine genotype with distinct virulence in nude mice [175] . the zoonotic potential of the porcine genotype is still uncertain and requires further study. in commercial rabbits, digestive disorders are the predominant cause of mortality. mainly weaned rabbits of 4 to 8 weeks of age are aected, although another peak may occur in suckling rabbits of 8 to 12 days old [176] . experimental cryptosporidium infection of rabbits resulted in high mortality and liquid diarrhoea in suckling 3-day-old rabbits, but no mortality and very discrete diarrhoea in weaned animals [177] . the parasite was found in 2±11% of weaned diarrhoeic belgian rabbits [176] . in general, ®eld outbreaks of cryptosporidiosis in suckling rabbits are rarely detected and in weaned rabbits the parasite causes only subclinical enteritis. when concurrent infections with immunosuppressive agents or respiratory pathogens occur, the impact of cryptosporidium infection can be more signi®cant (peeters, personal communication). aetiological treatment of cryptosporidiosis has only recently been made possible. drugs demonstrated to be partially eective in the treatment and prophylaxis of cryptosporidiosis in ruminants include halofuginone lactate, paromomycin and decoquinate (table 3) . however, the commercial availability of some of these products is still a problem in many countries. in poultry, so far none of the tested drugs showed a satisfactory anti-cryptosporidial activity, unless at toxic dosage [122, 125, 142, 160] . recently, it was shown that the oral infection of hens with c. baileyi at the beginning of their laying period, resulted in partial protection of their progeny [185] . the importance of colostrum in protecting neonatal ruminants against infection by c. par-vum is a very valuable point to address. in ®eld conditions, passively acquired antibodies did not protect calves [44, 186] and lambs against naturally acquired infection [108] . however, both calves [187] and lambs [188] fed with colostrum from immunised mothers with high titres of speci®c antibodies were partially protected against infection. immunoprophylaxis of cryptosporidiosis is more thoroughly discussed in another contribution of this special issue of the international journal for parasitology [189] . from a perspective of disease control, preventive hygiene measures are the most important tools in the struggle against cryptosporidiosis in farm animals, the objective being to destroy external forms of the parasite and to prevent their transmission among animals and from the environment to the host [46] . in ruminant husbandry, the destruction of oocysts in the pens and buildings used for parturition by applying moist heat and/or chemical disinfectants, the use of abundant clean straw beds, avoidance of high stocking rates in the parturition area and the separation of healthy and ill animals during outbreaks of diarrhoea, in addition to the administration of appropriated supplies of colostrum to neonates, all help to prevent outbreaks of cryptosporidiosis and to minimise mortality and morbidity in infected herds. [184] * per kg body weight. cryptosporidiosis is mainly a problem in neonatal ruminants. cryptosporidium parvum is the most commonly found enteropathogen during the ®rst weeks of the life of calves, lambs and goat kids and is considered to be an important agent in the aetiology of the neonatal diarrhoea syndrome. the parasite frequently acts alone, but the losses are more pronounced when concurrent enteropathogens are present. economic losses associated with cryptosporidiosis are retarded growth and mortality, and a number of hard to estimate costs resulting from interventions necessitated by diarrhoeic problems. especially in small ruminants, the direct losses due to mortality caused by cryptosporidiosis alone was reported to be high. because of the limited availability of eective drugs, hygienic measures and good management are the most valid weapons in the struggle against this disease. avian cryptosporidiosis is an emerging health problem. in chickens and other galliformes, cryptosporidiosis is mostly manifest as respiratory disease, although in turkeys and quails enteric forms of the disease were also reported to be responsible for morbidity and mortality. it was alarming to ascertain the unexpectedly high prevalence of c. baileyi in chicken¯ocks and the enhanced pathogenicity when immunosuppresive or other respiratory pathogens are present, or when birds are subjected to vaccination against other pathogens. in pigs and rabbits, cryptosporidium spp. were reported to cause only clinical disease when administered experimentally to nursing neonates, but in these farm animals naturally occurring cryptosporidiosis is mostly asymptomatic. cryptosporidium meleagridis (sp.nov.) an outbreak of calf diarrhoea attributed to cryptosporidial infection intestinal lesions in spf lambs associated with cryptosporidium from calves with diarrhoea acute enterocolitis in a human being infected with the protozoan cryptosporidium human cryptosporidiosis in immunocompetent and 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cryptosporidium sp. chez les lapereaux avant et apres sevrage. in: 4eâ mes journeâ es de la recherche cunicole en france ecaciteâ du lactate d'halofuginone dans le traitement de la cryptosporidiose chez l'agneau the eect of halofuginone lactate on experimental cryptosporidium parvum infections in calves speci®c serum and local antibody responses against cryptosporidium parvum during medication of calves with halofuginone lactate paromomycin is eective as prophylaxis for cryptosporidiosis in dairy calves chemoprophylaxis of cryptosporidium parvum infection with paromomycin in kids and immunological study the eect of varying levels d. eccox 1 on experimental cryptosporidia infections in holstein bull calves evaluation of decoquinate to treat experimental cryptosporidiosis in kids assessment of maternal immunity to cryptosporidium baileyi in chickens eect of colostral antibody on susceptibility of calves to cryptosporidium parvum infection ecacy of hyperimmune bovine colostrum for prophylaxis of cryptosporidiosis in neonatal calves treatment of experimental ovine crytosporidiosis with ovine or bovine hyperimmune colostrum speculation on whether a vaccine against cryptosporidiosis is a reality or fantasy key: cord-299786-wuve0tjz authors: anderson, robert title: manipulation of cell surface macromolecules by flaviviruses date: 2004-02-27 journal: adv virus res doi: 10.1016/s0065-3527(03)59007-8 sha: doc_id: 299786 cord_uid: wuve0tjz cell surface macromolecules play a crucial role in the biology and pathobiology of flaviviruses, both as receptors for virus entry and as signaling molecules for cell–cell interactions in the processes of vascular permeability and inflammation. this review examines the cell tropism and pathogenesis of flaviviruses from the standpoint of cell surface molecules, which have been implicated as receptors in both virus–cell as well as cell–cell interactions. the emerging picture is one that encompasses extensive regulation and interplay among the invading virus, viral immune complexes, fc receptors, major histocompatibility complex antigens, and adhesion molecules. flaviviruses comprise a rich and diverse family of agents that infect a variety of hosts and cause a wide spectrum of disease. three disease types are recognized for flaviviruses, namely encephalitis, hemorrhagic fever, and fever-arthralgia-rash. disease distinctions are not absolute, and overlapping pathologies among various flavivirus members are often observed. the ability of flaviviruses to cause such divergent clinical syndromes, associated with virus replication in a number of different organs, has profound implications for the types of cell surface molecules the virus recognizes as receptors. mutational analyses of the flaviviral e protein have demonstrated a striking ability of flaviviruses to adapt to different cells and receptors. given the considerable homologies among them, flaviviruses show a remarkable capacity to cause vastly different diseases with a minimum of alterations in the e protein. the cell surface molecules, which act as receptors for flaviviruses, are only starting to be identified. in addition to providing the molecules involved in virus attachment and penetration, the host cell erects a battery of surface structures that mediate communication with other cells and trigger host defense and pathological processes. many of these are modulated by flavivirus infection and contribute to the overall picture of pathogenesis. the flavivirus e protein is a multifunctional protein involved in cell receptor binding (anderson et al., 1992; chen et al., 1996; he et al., 1995) and virus entry via fusion with a host cell membrane (rice, 1996) . some of the functional activities of the e protein, notably membrane fusion, are regulated by interaction with a second viral protein, prm. it is believed that the association of prm with e stabilizes certain ph-sensitive epitopes on the e protein, thereby preventing the conformational changes that normally occur at acidic ph and activate the fusogenic activity of the e protein guirakhoo et al., 1992; heinz et al., 1994) . in addition to its normal role in flavivirus assembly, the prm protein has also been included in novel recombinant formulations in which it is generally coexpressed with the e protein; the resultant e/prm complexes have been shown to be immunogenic and protective as vaccines against challenge with several flaviviruses, including japanese encephalitis virus (mason et al., 1991) , yellow fever virus (pincus et al., 1992) , dengue virus (fonseca et al., 1994) , and tick-borne encephalitis (tbe) virus . in tbe virus, the majority of extracellular virus is largely free of prm protein due to a late intracellular processing event that generates a carboxy-terminal fragment designated m and which together with the e and c proteins are believed to constitute the protein components of the mature virus particle (heinz et al., 1994) . cleavage of prm to m enhances low ph-dependent virus-cell fusion (guirakhoo et al., 1991) and infectivity (guirakhoo et al., 1992; heinz et al., 1994; randolph et al., 1990; shapiro et al., 1972; wengler, 1989) . dengue virions containing prm are still infectious (randolph et al., 1990) and bind to permissive cells in a manner that can be blocked using e-specific antibodies wang et al., 1999) . virus particles containing mainly e and prm also show antibody-enhanced binding to fc receptor-bearing k562 cells as well as to platelets . thus, in addition to being requisite precursors to mature virus particles, virus particles containing prm possess many properties associated with mature virus particles. flaviviruses appear to gain entry to the cell by the endocytic pathway (rice, 1996) . at low ph, the e protein undergoes a conformational change involving dissociation of the e dimer (stiasny et al., 1996) , thereby exposing a hidden fusion peptide, followed by reorganization of e into a trimer , in which the fusion peptide is brought close to the membrane-anchoring carboxy terminus (ferlenghi et al., 2001) . remarkably similar structural features and conformational rearrangements have been noted between the flavivirus e protein and the alphavirus e1 lescar et al., 2001; pletnev et al., 2001; strauss and strauss, 2001) , suggesting a common evolutionary origin for these two virion surface proteins. considerable homology exists among flaviviral e proteins, raising the possibility that different flaviviruses may have similar receptorbinding motifs. for example, many mosquito-borne flaviviruses contain an rgd sequence (e.g., residues 388-390 of the murray valley encephalitis virus e protein), which has been implicated in virulence (lobigs et al., 1990) and receptor binding by analogy with integrin-binding motifs (rey et al., 1995) . mutagenesis studies of the yellow fever virus (van der most et al., 1999) and murray valley encephalitis virus (hurrelbrink and mcminn, 2001 ) rgd motifs, however, have cast doubt on the role of integrins in flavivirus attachment or entry. studies with tbe virus have identified important determinants for pathogenicity within the suspected receptor-binding site on the upperlateral surface of domain iii (mandl et al., 2000) . acquisition of heparan sulfate-binding mutations by passaging tbe in cell culture has also implicated amino acids in this region in receptor binding (mandl et al., 2001) . the selection of virus mutants on the basis of weak binding to brain membranes has been used with several neurotropic flaviviruses (holbrook et al., 2001; ni and barrett, 1998; ni et al., 2000) and has identified a variety of mutations within domain iii as well as other regions of e. for dengue virus, blocking of virus cell binding correlates more closely to virus neutralization for mab 3h5 than for mab 1b7 . this may suggest that mab 3h5 neutralizes dengue virus predominantly by blocking virus-cell attachment, whereas mab 1b7 neutralizes dengue virus largely by a postattachment mechanism. the mab 3h5-binding site on the dengue viral e protein has been partly characterized (hiramatsu et al., 1996; megret et al., 1992; trirawatanapong et al., 1992) and probably encompasses, at a minimum, residues 383-385 (hiramatsu et al., 1996) within domain iii. more recent data involving a larger number of monoclonal antibodies indicate that mabs that interact with domain iii are in fact the most effective blockers of virus-cell attachment (crill and roehrig, 2001) . a putative heparan sulfate-binding site on the dengue-2 e protein is also located within this region , and comparative sequencing of dengue type 2 genomes has implicated amino acid 390 of the e protein as a major determinant of pathogenicity (leitmeyer et al., 1999) . the ph-dependent conformational ''hinge'' region (between domains i and ii) of the e protein has also been implicated in virulence, receptor interaction, and/or membrane fusion (hurrelbrink and mcminn, 2001; lee et al., 1997; monath et al., 2002) . further mutagenesis studies will undoubtedly help define the sites of the e protein involved in flavivirus-cell macromolecule recognition. transmission of flaviviruses to humans generally occurs via the bite of an infected mosquito or tick. in the case of dengue, inoculated virus is thought to first replicate in skin langerhans (dendritic) cells (palucka, 2000; taweechaisupapong et al., 1996a taweechaisupapong et al., , 1996b wu et al., 2000) . dendritic cells have also been shown to be involved in the transport of intradermally inoculated west nile virus to local draining lymph nodes, with a subsequent accumulation of leukocytes . it is likely that dendritic cells will prove to be efficient carriers of a wide number of flaviviruses from their cutaneous site of infection to lymphoid and possibly other tissues. given the importance of dendritic cells in initiating immune responses (banchereau et al., 2000) , they probably play a pivotal role in stimulating host defense against invading flaviviruses. dengue virus infection of immature myeloid dendritic cells has been shown to induce their maturation accompanied by the expression of major histocompatibility complex (mhc) class i and ii antigens; the costimulatory molecules cd40, cd80, and cd86; and the dendritic cell marker cd83 (libraty et al., 2001) . such changes were seen in both dengue-infected and bystander cells, indicating that upregulation of cell surface molecules could be a consequence of virus infection as well as virusinduced cytokine expression. similarly, langerhans cells infected with west nile virus, as well as an alphavirus, semliki forest virus, express increased cell surface mhc class ii and appear to undergo maturation to a cell type similar to lymphoid dendritic cells (johnston et al., 1996) . the efficient presentation of both mhc class i-and ii-associated viral peptides on the surface of dendritic cells permits the generation of potent cytotoxic and helper t cell responses (see also section v,a). monocytes and macrophages have long been recognized as major targets of flavivirus replication in the human host (halstead, 1989; scott et al., 1980) . they are also important host cells for the antibody-enhanced replication of certain flaviviruses (see section iv,c). because of their presence in the circulation, blood monocytes may be particularly important to the pathogenesis of hemorrhagic viruses, such as dengue. because most of the pathological changes associated with dengue virus are hemostatic in nature, it is suspected that blood cells, particularly virus-infected blood monocytes, orchestrate many of these effects. dengue virus-infected human monocytes have been shown to be potent sources of vasoactive cytokines such as tumor necrosis factor (tnf)and interleukin (il)-1 (chang and shaio, 1994) . monocytes are also known producers of several other vasoactive mediators, including il-6, platelet-activating factor (paf), prostaglandins, thromboxanes, leukotrienes, and nitric oxide (bulger and maier, 2000; funk, 2001; lefer, 1989; maruo et al., 1992; montrucchio et al., 2000; szabo and billiar, 1999) , any of which could have powerful effects on endothelial cell physiology. a crucial aspect in understanding dengue pathogenesis will be the identification of additional vasoactive mediators, which trigger the key dysfunctional events in vascular integrity. various tissue macrophages are undoubtedly important in the pathogenesis of flaviviral diseases but have, to date, not received much attention. skin mononuclear cells, pulmonary, splenic, and thymic macrophages and liver kupffer cells have been recognized carriers of viral antigen (halstead, 1989) . in the liver, virus or viral antigen has been found in kupffer cells and hepatocytes in infections with yellow fever (monath et al., 1989) and dengue (bhamarapravati et al., 1967; hall et al., 1991; halstead, 1989; rosen and khin, 1989) . destruction of kupffer cells, possibly by apoptosis, has been reported in the liver of some patients with fatal dengue (huerre et al., 2001) . primary cultures of kupffer cells apparently undergo an abortive infection with dengue virus in which viral antigen but no progeny virus is produced (marianneau et al., 1999) . many flaviviruses invade either visceral or central nervous system tissues following initial replication in dendritic cells, monocytes, or macrophages. often this necessitates a transfer of virus across blood vessel endothelial layers. for neurotropic flaviviruses, endothelial cells of the cerebral microvasculature constitute a barrier that must be overcome in order to gain access to the central nervous system. how this occurs remains uncertain. transendothelial passage of virus may direct infection of cerebral microvascular endothelial cells, may transport across the endothelial layer, or both (dropulic and masters, 1990) . japanese encephalitis virus has been observed electron microscopically to traverse mouse cerebral endothelial cells by transcytosis (liou and hsu, 1998) . alternatively, virus may spread from blood vessels to the olfactory neuroepithelium and from there to olfactory neurons (mcminn et al., 1996; monath et al., 1983) . even normally nonneurotropic flaviviruses may occasionally invade the central nervous system under certain conditions. modulation of the blood-brain barrier by anesthetics (ben-nathan et al., 2000) or lipopolysaccharide (lustig et al., 1992) has been reported to facilitate neuroinvasion by a normally noninvasive strain of west nile virus. flaviviruses may also trigger the production of soluble factors that perturb the integrity of the blood-brain barrier, leading to increased leakage of proteins and cells into the central nervous system (chaturvedi et al., 1991) . these studies indicate that even nonneurotropic flaviviruses may infect tissues of the central nervous system or otherwise affect the integrity of the blood-brain barrier under special circumstances. transendothelial migration of individual leukocytes (e.g., lymphocytes, monocytes, neutrophils, eosinophils) is regulated in a highly specific manner by the differential expression of selected adhesion molecules on endothelial cells (reviewed in crockett, 1998; lowell and berton, 1999) . flaviviruses, including dengue and west nile (shen et al., 1997) viruses, activate endothelial cell adhesion molecule expression by either direct (virus-mediated) or indirect (cytokine-mediated) mechanisms (see section v,c). in the presence of leukocyte-attracting chemokines, such virus-triggered activation of the vascular endothelium may contribute toward the migration of leukocytes into extravascular tissues. in addition to being a mechanism for virus dissemination, this process may also be a factor in phenomena such as leukopenia and particularly neutropenia (loss of circulating leukocytes, neutrophils) often observed in flavivirus, particularly dengue, infection (reviewed in halstead, 1989) . due to the lack of suitable animal models for severe dengue disease, i.e., dengue hemorrhagic fever (dhf) or dengue shock syndrome (dss), there are difficulties in assessing the roles of such events, particularly the identification of adhesion molecules mediating the transendothelial migration of neutrophils using blocking antibodies against specific integrins, as has been performed for other disease states (doerschuk et al., 1990; gao et al., 1994; issekutz and issekutz, 1993; laberge et al., 1995; springer, 1995) . the hallmark feature of increased vascular permeability in hemorrhagic flavivirus (e.g., dengue) infection suggests that vascular endothelial cells may mediate the fluid leakage and hemorrhaging that occur in dhf/dss. endothelial cells line the inner surface of blood vessels and play essential roles in maintaining an antithrombogenic surface and regulating vascular permeability. increased vascular permeability can arise from a variety of mediators associated with acute inflammation and shock (bulger and maier, 2000; funk, 2001; lefer, 1989; michel, 1988; montrucchio et al., 2000; schnittler et al., 1990) . it is thought that vascular permeability is largely controlled by changes in endothelial cell-cell contact, which result in gap formation, thus allowing for fluid exchange between blood and interstitial tissue fluid (michel, 1988 ). an electron microscopic study of endothelium from dhf biopsy samples revealed the occasional presence of gaps (sahaphong et al., 1980) , thus providing evidence that endothelial cell features may indeed be perturbed during dhf/dss. although dengue virus infects endothelial cells in vitro (andrews et al., 1978; avirutnan et al., 1998; killen and o'sullivan, 1993) , there is no evidence that endothelial cell infection occurs clinically, as neither virus particles nor viral antigen has been detected in the endothelium of tissue specimens (halstead, 1988 (halstead, , 1989 sahaphong et al., 1980) , in contrast to that seen in cases of ebola (zaki et al., 1999) or hantaan hemorrhagic fever (gavrilovskaya et al., 1999; wang et al., 1997) . it is likely that dengue virus mediates endothelial cell activation via an indirect route, involving blood monocytes, which are a major cell target for dengue virus infection (halstead et al., 1977b; scott et al., 1980) . a major candidate event in such a route is the activation of endothelial cell adhesion molecules by a factor(s) (particularly tnf-) produced by dengue virus-infected blood monocytes . tnf is a key cytokine in a variety of normal and pathological immune responses, including immunoregulation, regulation of cell proliferation, cytotoxicity, and in the mediation of endotoxic shock (fiers, 1991; tartaglia and goeddel, 1992; tracey and cerami, 1993; vassalli, 1992) . monocyte-derived tnf-appears to play a pivotal role in dengue-associated endothelial cell activation and may be an important effector in the manifestation of dhf/dss. support for the clinical significance of this observation comes from observations of elevated tnf levels in the sera of patients with severe dengue disease (green et al., 1999b; hober et al., 1993; vitarana et al., 1991; yadav et al., 1991) . taken together, current evidence indicates that dengue virus represents a rather unique group of viruses that target monocytes, thereby triggering the production of factors such as tnf-, which in turn affect other cell targets, including endothelial cells. while the overall picture of endothelial cell dysfunction in dhf/dss is obviously more complex than can be explained by any single factor, the role of tnf in dengue pathogenesis would seem to merit particular attention. current knowledge of endothelial cell responses observed in endotoxic shock may be instructive for the understanding of vascular leakage in dhf/dss. plasma leakage induced by endotoxin (lipopolysaccharide, lps) from gram-negative bacteria encompasses a complex cascade of processes, including activation and functional alteration of endothelial cells. major mediators of endothelial cell perturbation in endotoxic shock are lps itself, as well as cytokines such as tnf-and il-1 (bevilacqua, 1993) . these factors can modulate endothelial cell function to varying degrees by activating cytokine and vasoactive factor release (rink and kirchner, 1996; shanley et al., 1995) , upregulating adhesion molecule expression (bevilacqua, 1993; luscinskas et al., 1991; moser et al., 1989; smith et al., 1989) , and mediating transendothelial migration of specific leukocytes luscinskas et al., 1991; morzycki et al., 1990; moser et al., 1989; smith et al., 1989) . additional factors, particularly lipid mediators such as paf, leukotrienes, thromboxanes, and prostaglandins, may contribute to further endothelial cell dysfunction, including vascular leakage (bulger and maier, 2000; funk, 2001; lefer, 1989; montrucchio et al., 2000) . while the involvement of these vasoactive mediators is recognized in endotoxic shock, more needs to be learned of their role in the vascular dysfunction that occurs in severe dengue disease. although lymphocytes are potently involved in the host response and immunopathology of flavivirus (especially dengue) diseases, their role as virus-permissive host cells is unclear. dengue virus has been identified in circulating b cells from acutely ill dengue patients by immunocytochemistry and by recovery of infectious virus after passage in mosquitoes . in vitro studies showed that cells and cultured cell lines of both b and t cell derivation could be infected with dengue virus (bielefeldt-ohmann et al., 2001; kurane et al., 1990; marchette and halstead, 1978; mentor and kurane, 1997; sung et al., 1975; takasaki et al., 2001; theofilopoulos et al., 1976) . continued passage of dengue virus in lymphoblastoid (raji) cells can give rise to dengue virus variants capable of replication in human lymphocytes (brandt et al., 1979) . interestingly, lymphocytes do not appear to undergo antibody-enhanced dengue virus infection (brandt et al., 1979; kurane et al., 1990) , even though b cells do have fc receptors (dijstelbloem et al., 2001 ; see section iv,c). the initial stages of pathogenesis for neurotropic flaviviruses appear to be common for flaviviruses in general in that the virus progresses from the subcutaneous site of inoculation to lymph nodes, followed by viremia and replication in extraneural tissues. invasion into the central nervous system is marked by high virus titers in the brain and detectable virus or viral antigen in neurons (albrecht, 1968) . cell destruction in tick-borne encephalitis may be less extensive than that seen in herpes simplex type 1 encephalitis (studahl et al., 2000) , although this is variable and may involve considerable inflammation (chu et al., 1999; matthews et al., 2000; suzuki et al., 2000) . susceptible cell types include both neurons and glial cells (chu et al., 1999; ramos et al., 1998; steele et al., 2000) . as notorious producers of vasoactive mediators, mast cells have been a source of controversial speculation for years in dengue pathogenesis. cells resembling degranulated mast cells have been reported in skin perivascular infiltrates from dhf/dss cases (bhamarapravati et al., 1967) . dengue patients showed elevated levels of urinary histamine (a major granule product of mast cells), which correlated with disease severity (tuchinda et al., 1977) , suggesting that mast cells may have a contributory role in the pathogenesis of dengue. although antihistamine treatment does not resolve shock in severely dengue-diseased patients (halstead, 1989) , histamine is only one of several potent vasoactive factors produced by mast cells (benyon et al., 1991; bradding et al., 1993; galli et al., 1984; grabbe et al., 1994; marshall and bienenstock, 1994; moller et al., 1991 moller et al., , 1993 moller et al., , 1998 nilsson et al., 1995; schwartz and austen, 1984) , some of which could cause vascular dysfunction in dengue infection. dhf/dss patients have been reported to have elevated serum levels of ige (pavri et al., 1979) , which has been speculated to relate to ige-triggered histamine release in the manifestation of shock (pavri and prasad, 1980) . mast cells reside mainly in the tissues, often closely associated with blood vessels (alving, 1991; anton et al., 1998; pesci et al., 1996; pulimood et al., 1998; selye, 1966; selye et al., 1968) . they are present in large numbers in the skin (marshall et al., 1987) , where transmission of insect-borne flaviviruses occurs. basophils, however, comprise about 1% of total circulating cells and would be accessible to virus in the blood. dengue virus infects basophil/mast cell-like ku812 cells in an antibody-enhanced manner, coupled with the release of vasoactive cytokines, il-1 and il-6 (king et al., , 2002 . this cell line, which can be differentiated easily toward either a basophil or mast cell phenotype (saito et al., 1995) , may provide further insights into potential roles for basophils and mast cells in dengue disease. dengue patients show increased serum levels of anaphylatoxins c3a and c5a (malasit, 1987) , which can attract (nilsson et al., 1996) and activate (kownatzki, 1982) mast cells. among the expected mast cell secretion products would be vasoactive factors, including histamine, which has been detected in elevated amounts in the urine of dengue patients (tuchinda et al., 1977) . evidence for platelet involvement in dengue pathogenesis comes from at least two (probably related) sources. first, thrombocytopenia (loss of circulating platelets) is one of the most consistent clinical features of severe dengue infection (halstead, 1989) . second, viral immune complexes have been detected on platelets from dengue patients phanichyakarn et al., 1977a) . functional studies on platelets in dengue-diseased individuals have been sparse, but include a markedly reduced half-life (mitrakul et al., 1977) , deficient adp release (mitrakul et al., 1977) , increased adhesiveness (doury et al., 1976) , increased tagging by complement fragments (malasit, 1987) , and increased release of -thromboglobulin and platelet factor 4 (srichaikul et al., 1989) . there is also evidence for platelet activation in dengue patients (doury et al., 1976; krishnamurti et al., 2001; srichaikul et al., 1989) . although these results relate to a variety of platelet functions, they do indicate a general alteration in platelet physiology, which is consistent with platelet involvement and triggering of thrombocytopenia in dengue disease. dengue virus has been recovered from washed patient platelets (scott et al., 1978) , and virus has been reported to bind to platelets in the absence of antibody as assayed using immunofluorescence and immunoperoxidase techniques (funahara et al., 1987) . however, the levels of antibody-independent bound virus are very low compared to the levels of virus bound in the presence of dengue-specific antibodies . as noted earlier, dengue immune complexes have been demonstrated on platelets from dengue patients phanichyakarn et al., 1977a) . weiss and halstead (1965) originally proposed the possibility that dengue virus interactions with platelets might be involved in the thrombocytopenia observed in severe dengue disease. the finding that dengue virus binding to platelets is dependent on a virus-specific antibody is consistent with epidemiological and experimental data linking preexisting host antibodies to an increased risk of dhf/dss (reviewed in halstead, 1990) . several other viruses have been shown to bind directly to platelets (bik et al., 1982; danon et al., 1959; forghani and schmidt, 1983; larke and wheelock, 1970; lee et al., 1993; zucker-franklin et al., 1990) . platelet association may stabilize or protect blood-borne viruses (larke and wheelock, 1970) and may function as a mechanism of hematogenous dissemination (forghani and schmidt, 1983) . virus binding to platelets has been suggested to be a contributing mechanism to thromobocytopenia arising from infections with vaccinia (bik et al., 1982) , chikungunya (larke and wheelock, 1970) , and rubella (bayer et al., 1965) . thrombocytopenia in these virus infections is generally much milder than that observed in severe dengue disease. levels of dengue virus in the blood can exceed 10 7 infectious units/ml (gubler, 1988; monath, 1994) . such high viremic titers are likely necessary to ensure infection and transmission of the obligate mosquito intermediary host (monath, 1994) . assuming a reasonable particle:infectivity ratio of 100:1, virus particle titers in blood may rival normal platelet counts (3 â 10 8 /ml). such parity between numbers of virus particles and platelets suggests that antibodyenhanced binding of virus to platelets may have a profound effect on platelets. circulating virus-immune complexes are detected in dhf/ dss, and levels of immune complexes have been correlated with severity of disease (ruangjirachuporn et al., 1979) and some of these are platelet associated phanichyakarn et al., 1977a) . these observations suggest that sufficient binding of virus immune complexes to platelets may occur to tag the majority of circulating platelets. such an event could lead to immune clearance by the reticuloendothelial system, thereby precipitating the thrombocytopenia frequently associated with severe dengue disease. it is likely that molecules other than fc receptors on the platelet surface may mediate antibody-enhanced binding of dengue virus . drug-induced thrombocytopenias provide interesting examples in this regard. it is known that given the appropriate accessory ligand (i.e., drug), igg can bind to platelets through either the fc receptor or other surface proteins. a variety of clinical thrombocytopenias are known that involve an immune component in pathogenesis. many of these reflect activities of host antibodies, which react with proteins on the surface of platelets. these antibodies may be autoimmune in nature (i.e., antibodies that bind to platelet surface molecules) or dependent on a third party ligand (drug or protein), which then induces binding of the antibody-ligand complex to either the platelet fc receptor or to another surface protein. for example, a number of individuals are susceptible to drug-dependent thrombocytopenia when administered drugs such as heparin or quinine/quinidine (aster, 1989; hackett et al., 1982) . while heparindependent antibodies bind to the platelet fc receptor (adelman et al., 1989; chong et al., 1989a chong et al., , 1989b kelton et al., 1988) , quinine/quinidine-dependent antibodies bind to platelet protein heterodimers gpiib/iiia and gpia/ix (berndt et al., 1985; chong et al., 1983; christie et al., 1987; devine and rosse, 1995) . this latter category of immune-mediated thrombocytopenia may be relevant to the understanding of dengue-associated thrombocytopenia, as patient antibodies mediate dengue virus binding to platelets via a platelet surface protein other than the fc receptor . communication between platelets and endothelial cells is a frequent intermediate step in certain events such as platelet adhesion, aggregation, and regulation of vascular permeability. how this occurs in dengue infection and what the effects are on endothelial cell function are unknown. binding of viruses to platelets can have potentially profound immunological effects [e.g., the stimulation of tgf-release by platelets bound by epstein-barr virus (ahmad and menezes, 1997) ]. in light of reports of altered platelet function in dengue patients, discussed earlier, there is a tantalizing need to determine the immunological consequences of antibody-enhanced dengue virus binding to platelets in terms of platelet as well as endothelial cell physiological responses. many products of complement activation can also be deposited on platelets (devine, 1992) . in view of evidence for complement activation in severe dengue disease (halstead, 1989; malasit, 1987) , binding of complement products might play a role in the immune destruction of platelets leading to thrombocytopenia. platelets display surface receptors, e.g., c1q receptor ghebrehiwet, 1987, 1998) , membrane cofactor protein (seya et al., 1986) , and decay-accelerating factor (devine et al., 1987) , for specific components of complement activation. in addition, the platelet surface can act as a substrate for the deposition of c3dg and c5b-9 (devine, 1992) . fragments of c3 have been detected on the platelets of dhf/dss patients (malasit, 1987) . in addition to immune complex deposition on platelets, thrombocytopenia associated with dhf/dss might also arise by the immune destruction of platelets through antiplatelet autoantibodies. antiplatelet autoantibodies have been reported in the sera of dengue patients (lin et al., 2001) , although they have also been detected in patients recovering from a variety of viral infections (imbach, 1994) . antiplatelet antibodies are strongly linked to the pathogenesis of immunemediated thrombocytopenias, such as idiopathic thrombocytopenic purpura (winkelstein and kiss, 1997) . while this brief discussion of cell targets for flaviviruses is by no means complete, it highlights some of the major interactions as they relate to pathogenesis. because pathogenesis is probably best understood for dengue, fig. 1 illustrates the interactions of hemorrhagic flavivirus (e.g., dengue) with cell targets both within and outside the vascular system. glycosaminoglycans and proteoglycans (i.e., proteins bearing glycosaminoglycans) are important cell surface molecules involved in a variety of ligand recognition and cell signaling processes (gallo, 2000) . because glycosaminoglycans are widely distributed on cells, they are attractive candidates as virus receptors. some degree of specificity (i.e., virus tropism) may arise from the compositional heterogeneity of glycosaminoglycans, as well as quantitative differences in the degree of expression on various cell types. flaviviruses seem to share, with a large number of virus families, the ability to bind glycosaminoglycans (birkmann et al., 2001; dechecchi et al., 2000 dechecchi et al., , 2001 duisit et al., 1999; feldman et al., 1999 feldman et al., , 2000 giroglou et al., 2001; goodfellow et al., 2001; heil et al., 2001; hsiao et al., 1999; hulst et al., 2000 hulst et al., , 2001 lin et al., 2000; liu and thorp, 2002; patel et al., 1993; rue and ryan, 2002; shukla et al., 1999; shukla and spear, 2001) . glycosaminoglycans such as heparin and its structural analogues have been investigated for their ability to bind dengue virus and thereby to gain insights as to the structural requirements for dengue receptors. potential glycosaminoglycan-binding motifs have been identified on the dengue viral e protein at two sites, the best characterized of which appears to be composed of amino acids 188, 284-295, and 305-310 and which may also play a role in virus-cell attachment . heparin (minimum of 10 carbohydrates) and an uncharacterized highly sulfated heparin sulfate isolated from bovine liver were found to show the best binding to dengue e protein . attachment of dengue virus to human hepatoma cells has also been reported to be inhibited by heparin (hilgard and stockert, 2000) . a further study involving a panel of natural and synthetic polyanionic, sulfated compounds suggested that binding of the dengue e protein required a highly sulfated (and highly charged) oligosaccharide with a minimum size of 39å and a high degree of structural flexibility (marks et al., 2001) . the role of glycosaminoglycans in natural (i.e., nontissue cultureadapted) strains of flaviviruses needs to be studied further. it has long been recognized that dengue virus passaged in various host cell types can give rise to virus variants with altered cell specificity (brandt et al., 1979; halstead et al., 1984a halstead et al., , 1984b halstead et al., , 1984c . passage-dependent mutations of the dengue virus e protein at a number of different amino acid residues have been documented (lee et al., 1997) . following passage of tbe virus in cultured bhk-21 cells, virus mutants were selected that contained more positively charged amino acids in the putative receptor-binding region of the e protein, resulting in dependence on cell surface heparan sulfate (mandl et al., 2001) . such mutants were diminished in their neurovirulence in mice as well as in their replication in primary chicken cells and plaque formation in porcine kidney cells (mandl et al., 2001) . a large number of other viruses have also been shown to undergo loss of virulence upon adaptation to cell culture associated with heparan sulfate utilization (bernard et al., 2000; byrnes and griffin, 2000; klimstra et al., 1998 klimstra et al., , 1999 lee and lobigs, 2000; neff et al., 1998; sa-carvalho et al., 1997) . cd14 and the toll-like receptor (tlr) pattern recognition receptors are involved in the innate response to lipopolysaccharide and other microbial products (diamond et al., 2000; imler and hoffmann, 2000) . a role for cd14 and tlr4 has been found for respiratory syncytial virus (rsv) (kurt-jones et al., 2000) , suggesting that these receptors may have a broader involvement in host response than previously thought. a possible role for cd14 in dengue infection has been postulated on the basis of inhibition of dengue virus infection of human monocytes with bacterial lipopolysaccharide (chen et al., 1999) . however, this has been disputed (bielefeldt-ohmann et al., 2001) and requires further investigation. as indicated earlier, flaviviruses are capable of initiating infection of appropriate host cells through as yet largely unidentified primary receptors. in addition, a number of flaviviruses are capable of using subneutralizing levels of virus-specific antibodies to attach to and gain entry to cells bearing fc and/or complement receptors (cardosa et al., 1983; halstead, 1982; halstead and o'rourke, 1977a; schlesinger and brandriss, 1981a ) by a process known as antibody-dependent enhancement (ade) of infection (table i) . ade has been documented for dengue , west nile (peiris and porterfield, 1979) , yellow fever (schlesinger and brandriss, 1981b) , tick-borne encephalitis (phillpotts et al., 1985) and japanese encephalitis (cecilia and ghosh, 1988) viruses. early work with dengue virus and monocytes differentiated between trypsin-sensitive and trypsinresistant cell surface molecules as the putative receptors for antibody-independent and antibody-dependent infection, respectively (daughaday et al., 1981) . to date, dengue virus appears to be the only flavivirus in which strong evidence exists for antibody-dependent enhancement as a major contributing factor to severe disease (halstead, 1980; thein et al., 1997) . severe dengue disease, encompasing conditions known as dengue hemorrhagic fever/dengue shock syndrome, involves several well-defined hemostatic abnormalities, including the leakage of plasma into interstitial spaces, as well as thrombocytopenia and bleeding (halstead, 1990; kurane et al., 1994) . the potential to cause severe hemorrhagic disease is a general property of dengue viruses and is not limited to any one viral serotype (gubler, 1998; rigau-perez et al., 1998) . although different strains of dengue may influence the severity of hemorrhagic symptoms (leitmeyer et al., 1999; rico-hesse et al., 1997) , it is also generally accepted that pathogenesis depends on immunopathological processes (rothman and ennis, 1999) . thus the roles of prior immunity, antibody-enhanced virus infection, and immune-mediated pathologic effects on the vascular system are key points in understanding the pathogenesis of dengue hemorrhagic disease. while the pathogenesis of severe dengue disease is not completely understood, it is clear from laboratory and epidemiological studies that a considerable risk factor is prior immunity. severe dengue disease, dhf/dss, rarely occurs in seronegative individuals suffering their first dengue infection, but instead occurs in individuals who have preexisting dengue viral antibodies, either from a previous infection or from passive antibody transfer, e.g., following maternal transmission of antibodies to the fetus (kliks et al., 1988 (kliks et al., , 1989 . estimates suggest that 99% of children suffering from dhf/dss have preexisting immunity from a prior dengue virus infection (halstead, 1988) . consequently, from this and other studies, it has been calculated that prior exposure to dengue increases the risk for hemorrhagic disease in a okayama et al. (2000) , anselmino et al. (1989) , and tuijnman et al. (1993) . b from . c from wu et al. (2000) and libraty et al. (2001) . d from king et al. (2000) . e abortive infection, but expressing viral antigen (marianneau et al., 1999) . f from littaua et al. (1990) and kontny et al. (1988). second dengue infection by at least 15-fold (halstead, 1980; thein et al., 1997) . preexisting serum antibodies can potentiate virus infection by the mechanism of antibody-dependent enhancement, giving rise to amplified virus replication and to an increased potential for the development of hemorrhagic symptoms (halstead, 1989) . viremic titers are higher in secondary dengue infections in both humans (gubler et al., 1979) and experimental monkeys (halstead et al., 1973) . antibody-enhanced dengue virus infection of human blood monocytes is necessary for the production of endothelial cell activators , thereby providing a link between antibody-dependent enhancement and alteration of endothelial cell properties, which might contribute to vascular permeability in dengue infection. for certain other viruses, e.g., influenza (tamura et al., 1993) and hiv (takeda et al., 1990 (takeda et al., , 1992 , distinct ''neutralizing'' and ''antibodyenhancing'' epitopes have been identified on the respective viral attachment proteins. surprisingly, no systematic approach has yet been undertaken to identify regions on the e protein that are essential for ade, even though this issue was raised as a challenge to research on dengue many years ago (halstead, 1988) . human fc receptors are currently categorized into three classes: fcri (cd64), fcrii (cd32), and fcriii (cd16). while fcri shows high affinity for monomeric igg, fcrii and fcriii bind monomeric igg poorly and are more likely involved in binding immune complexes (dijstelbloem et al., 2001) . fcrii is the most widely distributed, being expressed on most circulating leukocytes (van de winkel and anderson, 1991) . monocytes express all three fcrs to varying degrees (van de winkel and anderson, 1991) , although fcri and fcrii predominate, whereas fcriii appears to be limited to a subpopulation ($10%) of monocytes (anderson et al., 1990; passlick et al., 1989) . fcriii constitutes the major fcr on macrophages (fanger et al., 1989) , although fcri and fcrii are also present (tuijnman et al., 1993; van de winkel and anderson, 1991) . it is also important to recognize that fcr expression on cells, including macrophages, can vary depending on the microenvironment (tomita et al., 1994) . although strong evidence exists for fcr involvement in ade of dengue virus, the participating fcrs in vivo have not yet been identified rigorously. in cultured cell lines (monocytic u937 or erythroleukemic k562 cells), fcri (kontny et al., 1988) and fcrii (littaua et al., 1990) have been shown to mediate ade of dengue virus infection. that fcri has the ability to mediate ade of dengue has been demonstrated using cos cells transfected with fcri (schlesinger and chapman, 1999) . dengue and dhf patients show elevated serum levels of interferon (ifn)(kurane et al., 1991) . because ifn-can upregulate both mhc class i and ii molecules as well as fc r (particularly fc ri) expression in monocytes (erbe et al., 1990; perussia et al., 1983) , the chances for ade may be increased, thereby creating a vicious cycle involving positive cytokine feedback and virus amplification (kurane and ennis, 1992) . ifn-has been shown to enhance ade of dengue virus infection of human monocytic u937 cells (kontny et al., 1988) , although any enhancing effect on dengue infection of peripheral blood monocytes may be negated by the antiviral properties of ifn(sittisombut et al., 1995) . mast cells and basophils express mainly fc rii (anselmino et al., 1989; okayama et al., 2001a; wedi et al., 1996) and some (ifn--inducible) fc ri (okayama et al., 2000 (okayama et al., , 2001b as well as the highaffinity fc eri for ige (guo et al., 1992; sperr et al., 1994) . as noted previously, the basophil/mast cell ku812 cell line exhibits antibodyenhanced dengue virus infection and produces vasoactive cytokines . although fc r-mediated ade of flaviviruses has been examined extensively as a mechanism for virus amplification, the biological consequences for the participating host cell are not well understood. because fc r-mediated cell signaling is complex, the functional effects of virus-antibody interactions with cell surface fc rs need to be investigated. monocytes infected with dengue virus in the presence of antibody release cytokines such as tnf. induction of tnf-requires infectious virus , suggesting that virus replication (or perhaps expression of one or more crucial viral genes) is responsible for the stimulation of tnf-release. therefore, in this case, the fc r is likely facilitating antibody-enhanced virus replication rather than providing a signal triggered by virus binding to the fc r. similarly, antibody-enhanced dengue virus infection of ku812 basophil/mast cells produces il-1, il-6 (king et al., , 2002 , and selected chemokines (king et al., 2002) . suppressive effects of antibody-enhanced flavivirus or alphavirus infection on monocyte cytokine secretion have also been reported (lidbury and mahalingam, 2000; yang et al., 2001) . both activating (fcri, fcriia, and fcriiia) and inhibitory (fcriib) forms of fcrs exist, which mediate signal transduction via a cytoplasmic immunoreceptor tyrosine-based activation motif (itam) or inhibitory (itim) motif, respectively (dijstelbloem et al., 2001) . the itam and associated molecules are necessary for the endocytosis of fcr-bound immune complexes (amigorena and bonnerot, 1999 ) and therefore play a likely role in the initiating events of antibody-enhanced flavivirus infection. although not necessary for fcrii, an accessory subunit (homo-or heterodimeric or chains) is required for signaling through fcri and fcriiia (ravetch, 1994) . a further fcr (fcriiib) lacks transmembrane and cytoplasmic domains and is instead anchored to the cell surface membrane via a glycosylphosphatidylinositol (gpi) linkage (selvaraj et al., 1988; simmons and seed, 1988) . it apparently does not participate in signal transduction and has been speculated to sequester and accumulate immune complexes at specific sites on the cell surface (huizinga et al., 1988; selvaraj et al., 1988) . the roles of activating and inhibitory fcrs in viral ade have not yet been ascertained. activating fcrs are expressed on monocytes, macrophages, granulocytes, natural killer (nk) cells, and platelets but not on most lymphocytes (dijstelbloem et al., 2001) . inhibitory fcrs, however, are found on b cells, dendritic cells, and macrophages (dijstelbloem et al., 2001) . interestingly, ade of dengue virus is best documented for monocytes/macrophages and related cell lines (halstead, 1989) . in contrast, lymphocytic cells (brandt et al., 1979; kurane et al., 1990) and dendritic cells (wu et al., 2000) do not appear to support antibody-enhanced dengue virus infection. whether this is due to differential expression of activating versus inhibitory fcrs remains to be investigated. fcrs for ige (primarily the high-affinity fceri) are expressed on cells such as monocytes, macrophages, mast cells, basophils, and dendritic cells and are structurally related to fcrs (ravetch, 1994) . their role in binding ige and/or immune-complexed flaviviruses, such as dengue, remains unexplored. similarly unexplored is the potential role of the neonatal fc igg receptor (fcrn), structurally related to mhc class i and involved in igg transport across cells (ghetie and ward, 2000) . in addition to being expressed on certain epithelial and endothelial cells, fcrn is also expressed functionally on monocytes, macrophages, and dendritic cells (zhu et al., 2001) . in addition to the fcr, the antibody-complexed flavivirus has been shown to be taken up by a macrophage cell line using the complement receptor-3 (cardosa et al., 1983) . in the case studied-west nile virus infection of mouse p388d1 macrophages-ade was mediated by the presence of antiviral igm and was inhibited with a cr3-blocking antibody. this mode of ade was, however, found to be quantitatively less productive than the more commonly studied route of ade, i.e., involving fcr-mediated uptake route of igg-virus complexes (cardosa et al., 1983) . the recent demonstration of dc-sign as a functional dengue virus receptor on human dendritic cells represents an important advance in the definitive identification of flavivirus receptors (navarro-sanchez et al., 2003; tassaneetrithep et al., 2003) . several studies have identified cell surface proteins that bind flaviviruses, generally assayed by virus overlay blots of sds-page-resolved cell proteins (table ii) . further work is required to confirm the involvement of these and other proteins as receptors in flavivirus infection. a number of flaviviruses are able to stimulate the expression of cell surface molecules. notable among these are adhesion molecules and major histocompatibility antigens. multiple mechanisms appear to be involved, including virus-and cytokine-dependent pathways. flavivirus infection of a number of cell types causes an increase in cell surface mhc class i expression (king and kesson, 1988; king et al., 1989; libraty et al., 2001; liu et al., 1989; lobigs et al., 1996; shen et al., 1995a shen et al., , 1997 . evidence for both virus-dependent (lobigs et al., 1996) and cytokine-dependent (libraty et al., 2001; shen et al., 1997) mechanisms has been reported. one process appears to be driven by the amount of flaviviral peptides generated by proteolysis and imported into the transporter associated with antigen processing (tap), which results in increased cell surface expression of peptideloaded mhc class i (momburg et al., 2001) . the upregulation of mhc class i molecules by flaviviruses is perhaps reminiscent of that observed in infections by coronaviruses (suzumura et al., 1986) but stands in contrast to the virus-manipulated downregulation of mhc class i by viruses such as herpesviruses (jennings et al., 1985; ploegh, 1998) , adenoviruses (sparer and gooding, 1998) , poxviruses (boshkov et al., 1992) , and hiv (scheppler et al., 1989) . although enhanced chu and ng (2003) mhc class i expression would be expected to lead to greater cytotoxic t (tc) cell-mediated cytolysis, it would render cells less susceptible to recognition by nk cells. evidence has been presented that flavivirusinfected cells in fact show reduced susceptibility to nk cells at the cost of enhanced tc cell-mediated lysis (lobigs et al., 1996) . it has been suggested that such a response may permit flaviviruses to evade an early nk cell response and thereby allow for substantial amplification of virus during the viremic phase of infection (momburg et al., 2001 ). nevertheless, evidence shows that nk cells are activated during dengue infection (green et al., 1999a) , and nk cell-mediated cytotoxicity has been reported to correlate with the severity of disease (homchampa et al., 1988) . dendritic cells also undergo upregulation of mhc class i molecules following infection with dengue virus (libraty et al., 2001) . compared to other antigen-presenting cells, dendritic cells have superior t cellstimulating activities (mckinney and streilein, 1989; timares et al., 1998) . because antigen presentation via dendritic cell mhc class i can provoke exceptionally strong proliferation in cd8-bearing t cells (bhardwaj et al., 1994; elbe et al., 1994; mckinney and streilein, 1989) , much of the overall cytotoxic t cell response arising in flavivirus infection may be dictated at the level of the dendritic cell. west nile virus infection induces mhc class ii expression in mouse macrophages (shen et al., 1995a) , mouse astrocytes (liu et al., 1989) , rat schwann cells (argall et al., 1991) , and human myoblasts (bao et al., 1992) . upregulation of dendritic cell mhc class ii occurs in response to dengue (libraty et al., 2001) and west nile (johnston et al., 1996) virus infection. given the potent ability of dendritic cells to activate t cells (banchereau et al., 2000) , the communication between dendritic cell mhc class ii-peptide complexes and recognition molecules on cd4-expressing t cells should provide insights into some of the molecular processes underlying t cell activation. adhesion molecules are expressed on a variety of cells and mediate a spectrum of processes (ley, 2001; roebuck and finnegan, 1999; springer, 1995) . from the standpoint of flaviviruses, the most significant processes likely concern adhesion molecules on vascular endothelial cells, as these cells regulate permeability as well as transendothelial migration of leukocytes (springer, 1995) . of particular importance are intercellular adhesion molecule 1 (icam-1; cd54), vascular cell adhesion molecule-1 (vcam-1; cd106), and e-selectin (cd 62e), which are upregulated on the surface of the endothelium by inflammatory cytokines, cellular stress, and virus infection (roebuck and finnegan, 1999) . in the case of dengue, activation of endothelial cells occurs in vitro via tnf-released from antibody-enhanced dengue virus infection of monocytes . such activation involves upregulation of adhesion molecules e-selectin, icam-1, and vcam-1. evidence that similar activation processes occur in vivo comes from clinical studies showing elevated serum levels of tnf(green et al., 1999b; hober et al., 1993; vitarana et al., 1991; yadav et al., 1991) and soluble vcam-1 (murgue et al., 2001) in dengue-and dhf/dss-infected patients. surprisingly, serum levels of soluble icam-1 were actually found to be lower than those of control subjects, although this may reflect plasma protein loss through leakage (bethell et al., 1998) . moreover, the function of soluble forms of icam-1 remains unclear, and their expression appeazrs to be regulated differently from that of membranebound icam-1 (komatsu et al., 1997; van den engel et al., 2000) . two phases of icam-1 upregulation have been noted in west nile and kunjin virus infection of human embryonic fibroblasts, namely an early ($2 h postinfection) virus-dependent process and a later ($24 h postinfection) event that is mediated by type 1 interferon (shen et al., 1995b) . for neurotropic flaviviruses, such as west nile virus in the mouse, the development of encephalitis has been correlated with viremia (weiner et al., 1970) , suggesting virus penetration of the blood-brain barrier. the endothelium of the brain microvasculature normally represents a block between circulating virus and the central nervous system. expression of endothelial cell adhesion molecules, thereby facilitating leukocyte adherence and diapedesis through the endothelium, may be an important mode of dissemination of virus-infected monocytes or other leukocytes into the brain. west nile virus infection of human endothelial cells causes the upregulation of e-selectin, icam-1, and vcam-1 (shen et al., 1997) , which could mediate the transendothelial migration of leukocytes. upregulation of these adhesion molecules was observed to occur early (2-4 h) in infection and appeared to be triggered by the virus rather than by cytokines (shen et al., 1997) . further studies are required to clarify the role of endothelial cell adhesion molecule expression in the neuroinvasion of certain flaviviruses. assuming such a role is confirmed, it will be incumbent to identify the mechanisms by which either free or cell-borne flaviviruses are stimulated to cross the vascular endothelial layer. for virusinfected leukocytes, such stimulation likely arises, at least in part, from chemokines produced by cells of the central nervous system. astrocytes infected with je virus have been reported to release chemokines (rantes and mcp-1), which may play a role in the transendothelial migration of leukocytes (including those possibly carrying virus) across the blood-brain barrier (chen et al., 2000) . thus, once neural infection is initiated, the process could be amplified by the production of leukocyte-attracting chemokines at the site of infection. complement activation is well documented in dengue disease (nishioka, 1974; phanichyakarn et al., 1977b; russell et al., 1969) , with peak activation and the production of c3a and c5a occurring at the time of vascular leakage and/or shock (malasit, 1987) . complement activation is likely to be largely mediated by immune complexes consisting of igg and virus (bokisch et al., 1973a (bokisch et al., , 1973b shaio et al., 1992; sobel et al., 1975) , although the low levels of circulating immune complexes detected in patients have stimulated thought as to other possible mechanisms (malasit, 1987) . receptors for c3a and c5a are found on a wide variety of cells, including many human peripheral blood leukocytes (chenoweth and hugli, 1978; fureder et al., 1995; kretzschmar et al., 1993; nilsson et al., 1996; van epps and chenoweth, 1984) . c5a receptors have been reported on endothelial cells, although at lower levels than myeloid cells (zwirner et al., 1999) . although endothelial cells do not appear to be major targets for dengue virus in vivo (halstead, 1988 (halstead, , 1989 sahaphong et al., 1980) , endothelial cells infected with dengue virus in vitro can become a substrate for deposition of c3dg and c5b-9, provided the dengue antibody is present (avirutnan et al., 1998) . the presence of complement activation products on the endothelial cell surface could be a contributing factor to vascular permeability . furthermore, anaphylotoxins and/or deposition of sublytic c5b-9 on the endothelial cell surface has the potential to activate the expression of adhesion molecules (foreman et al., 1994) , cytokines (saadi et al., 2000) , chemokines (selvan et al., 1998) , cyclooxygenase-2 (bustos et al., 1997) , tissue factor , heparan sulfate proteoglycan proteinases (ihrcke and platt, 1996) , and even functional or morphological changes such as permeability loss and gap formation . thus, in addition to being activated by leukocyte-derived cytokines , endothelial cells may also be coaxed toward a more permeability-enhancing state by virus infection and virusmediated complement deposition. at present, the lack of evidence for in vivo infection of endothelial cells by dengue virus would suggest that the cytokine-mediated pathway is dominant. figure 2 shows a model illustrating the potential role of endothelial cell perturbation by monocyte-derived cytokines and complement activation products in initiating vascular permeability and leukocyte extravasation in severe hemorrhagic flavivirus disease. or by deposition of c5b-9 and other products of complement activation (avirutnan et al., 1998) . c5b-9 is represented as a membrane attack complex pore structure, although the deposition of c5b-9 on dengueinfected cells appears associated with sublytic, rather than lytic, responses (avirutnan et al., 1998) . increased adhesion molecule expression, along with uncharacterized vasoactive factors, can lead to endothelial leakage and can mediate rolling, adhesion, and transendothelial migration of leukocytes into extravascular tissues. similar processes may also contribute to the invasion of cell-borne neurotropic flaviviruses through the endothelial blood-brain barrier. much remains to be learned about the primary receptors for flaviviruses, though much knowledge has been gained about the initial interactions of flaviviruses with cell surface structures. the ability of flaviviruses to affect cell entry through heparan sulfate-type proteoglycans, as well as their dexterity to adjust mutationally to different receptors, depending on host cell type, illustrates the plasticity of the viral e protein to adapt to changing conditions and to ensure successful virus replication. beyond this, certain flaviviruses, notably dengue virus, are masters at exploiting host antibody and fc receptor-bearing cells to dramatically amplify viral replication. flavivirus replication is coupled to altered cellular expression of cytokines, chemokines, and cell surface molecules, which shape the host response and immunopathogenesis 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tumor necrosis factors elevated tumour necrosis factor in dengue fever and dengue haemorrhagic fever prm-and cell-binding domains of the dengue virus e protein antibodyenhanced binding of dengue-2 virus to human platelets transmission electron microscopic study of the hemorrhagic spots in patients with epidemic hemorrhagic fever in the early stage human hmc-1 mast cells exclusively express the fc gamma rii subtype of igg receptor experimental encephalitis following peripheral inoculation of west nile virus in mice of different ages studies of hemostasis in thai hemorrhagic fever cell-associated west nile flavivirus is covered with e+pre-m protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release immunohematologic disorders human skin langerhans cells are targets of dengue virus infection dengue haemorrhagic fever and dengue shock syndrome: are they tumour necrosis factor-mediated disorders? antibodydependent enhancement of heterotypic dengue infections involved in suppression of ifn-gamma production a novel immunohistochemical assay for the detection of ebola virus in skin: implications for diagnosis, spread, and surveillance of ebola hemorrhagic fever mhc class i-related neonatal fc receptor for igg is functionally expressed in monocytes, intestinal macrophages, and dendritic cells internalization of human immunodeficiency virus type 1 and other retroviruses by megakaryocytes and platelets expression of the anaphylatoxin c5a receptor in non-myeloid cells key: cord-285467-uxfk6k3c authors: ragni, enrico; mangiavini, laura; viganò, marco; brini, anna teresa; peretti, giuseppe michele; banfi, giuseppe; de girolamo, laura title: management of osteoarthritis during covid‐19 pandemic date: 2020-05-21 journal: clin pharmacol ther doi: 10.1002/cpt.1910 sha: doc_id: 285467 cord_uid: uxfk6k3c the pandemic spread of the new covid‐19 coronavirus infection in china first, and all over the world at present, has become a global health emergency due to the rapidly increasing number of affected patients. currently, a clear relationship between covid‐19 infection incidence and/or complications due to chronic or occasional treatments for other pathologies is still not clear, albeit covid‐19 pandemic may condition the treatment strategy of complex disorders, as osteoarthritis (oa). importantly, oa is the most common age‐related joint disease affecting more than 80% of people older than the age of 55, an age burden also shared with the highest severity in covid‐19 patients. oa patients often show a large array of concomitant pathologies such as diabetes, inflammation and cardiovascular diseases that are again shared with covid‐19 patients and may therefore increase complications. moreover, different oa treatments such as nsaids, paracetamol, corticosteroids, opioids or other molecules have a wide array of iatrogenic effects, potentially increasing covid‐19 secondary infection incidence or complications. in this review we critically analyse the evidences on either negative or positive effect of drugs commonly used to manage oa in this particular scenario. this would provide orthopaedic surgeons at first, and physicians, pharmacologists and clinicians at general, a comprehensive description about the safety of the current pharmacological approaches and a decision making tool to treat their oa patients as the coronavirus pandemic continues. due to the expected residency of the covid-19 pandemic in the next months/years, the conservative therapeutic approach for the treatment of patients affected by osteoarthritis (oa) would need an adjustment not to expose patients to additional risks. the purpose of this review is, beyond presenting an overview of the most prescribed molecules in everyday practice and those envisioned as future therapeutic options, to provide orthopaedics with some guidelines on the management of osteoarthritic patients during this covid-19 era. in particular, the susceptibility to covid-19 life threatening complications and the potential increment of the sars-cov-2 morbidity/mortality incidence will be discussed to provide a roadmap to orthopaedic surgeons about the safety of treatments as well as the possible need for their discontinuation. this is this article is protected by copyright. all rights reserved especially important since many patients will need to receive multiple drugs over the course of their disease and more in general as the coronavirus pandemic continues. also, since the discussed oa comorbidities and therapeutic options are also faced by the general population with oaunrelated inflammatory and/or age-related pathologies, the proposed indications will be a useful outline for general practitioners and specialized clinicians of other branches of medicine in the era of covid-19 pandemic. osteoarthritis is the most common degenerative disease of the joint that impairs quality of life and leads to important disability (1) . although the disease pathophysiology is still poorly understood and under investigation, it is accepted that the origin of oa is multifactorial. inflammation, biomechanical alterations and the immune response play an important role (2) . indeed, risk factors are sex, obesity, genetic factors and mechanical factors (3) . in the development of oa, the whole joint undergoes a complex remodeling, which in turn ends in degeneration. common histopathological findings in oa are articular cartilage damage, subchondral bone sclerosis and osteophyte formation, joint capsule hypertrophy and periarticular muscle dysfunction (4), as well as inflammation of the synovium. synovitis is in fact a hallmark of oa, characterized by increased vascularization, infiltration of macrophages and lymphocytes and villous hyperplasia (4) . the inflamed synovium secretes several cytokines and chemokines, which sustain inflammation and contribute to cartilage degeneration and subchondral bone changes. among cytokines, the most studied are interleukin 1 β (il-1β) and tumor necrosis factor α (tnfα), which can activate cartilage matrix degeneration by activation of toll-like receptors (tlrs) (5) . moreover, il-15 and il-17 are also secreted by the synovium lymphocytes and are associated with oa progression by inducing chemokine production by synovium fibroblast and chondrocytes (5) . in articular cartilage, the degenerative process is initiated by biomechanical stress and inflammation (6) . both these stimuli activate the canonical nf-ĸb, stress-induced and mapk pathways, which trigger the inflammatory cascade and matrix degradation via mmps (especially mmp-13), nos-2, cox-2, hif-2α, adamts-4,5 (4) . in addition, chondrocytes undergo hypertrophy through the activation of the canonical wnt signaling pathway and the consequent upregulation of β-catenin (7) . lastly, oa is also associated to an increased chondrocyte apoptosis: cell death may be caused by a hmgb-1-mediated mitochondrial dysfunction, which leads to secretion of ros, prostaglandins and nitric oxide and, ultimately, to oxidative stress. the products this article is protected by copyright. all rights reserved of this catabolic process (i.e. small pieces of collagen, fibronectin and proteoglycan) amplify the inflammatory response in cartilage and synovium by inducing innate immune responses through the complement pathway modulation (8) . eventually, subchondral bone is also subjected to profound changes in oa that essentially lead to sclerosis, microfractures and osteophyte formation. the excessive and repetitive mechanical stress causes some initial microfractures, which trigger bone remodeling through the opg/rank/rankl triad, mmps, il-6 and il-8 (4). at the same time, vascular endothelial growth factor (vegf) expressed by hypertrophic chondrocytes maintains bone remodeling by recapitulating endochondral bone formation. moreover, recent data show that sclerostin, a wnt pathway inhibitor, is downregulated in some subchondral areas, causing local bone sclerosis (9) . finally, osteophyte formation represents an attempt to restore the normal mechanical loading through endochondral bone formation and it is triggered by transforming growth factor β (tgfβ) and bone morphogenetic protein 2 (bmp-2), which are released by synoviocytes and chondrocytes (10). hence, in the osteoarthritic joint the cross talk between these three main tissues causes a vicious cycle that progressively sustains and amplifies the inflammatory and degenerative processes. the covid-19 pandemic represents an unprecedented and largely unanticipated challenge for healthcare systems and professionals worldwide. it is caused by sars-cov-2 infection, a novel coronavirus of zoonotic origin, and symptoms include fever, dyspnoea, fatigue, dry cough, olfactory and gustatory dysfunction, lymphopenia and, in the most severe cases, interstitial pneumonia with alveolar damage (11) . according to john hopkins coronavirus research center, 2.3 million people has been infected since the beginning of sars-cov-2 outbreak, with more than 155,000 confirmed deaths at april 20 th , 2020 (12) . nevertheless, some estimations outnumber the confirmed cases by orders of magnitude, with the possible prevalence of infection up to the 10% of the population in some countries (13) , and it is highly likely that a wide portion of individuals suffering from other common conditions, such as osteoarthritis, may have being infected by sars-cov-2. in particular, oa is more frequent in the elder population, a category that is at high risk of infection, given the more frequent need for health care and hospitalization, as well as more subjected to severe or fatal outcomes following sars-cov-2 infection (14). this article is protected by copyright. all rights reserved the molecular mechanisms of sars-cov-2 infection has been partially elucidated by recent reports that identified the angiotensin-converting enzyme 2 (ace2) as the host cell surface receptor allowing for the viral infection (15) . this protein is expressed in a number of tissues including alveolar epithelial cells, vascular endothelium, oral mucosa and it is responsible for the cleavage of angiotensin i and angiotensin ii (16) , playing a regulatory function in the heart (17) and possibly a protective role in lung diseases (18) . covid-19, similarly to other viral infections such as sars-cov in general and h5n1, causes a decrease of ace2 expression, partially explaining the severity of the lung damage in the pathology (19, 20) . then, the treatment of pathologies such as hypertension, requiring ace-inhibitors administration, may accelerate the progression of the pathology, even if the evidences are still insufficient to balance the cost/benefit equilibrium towards the suspension of these therapies in all patients undergoing this treatment (21) . in this frame of cost/benefit balance, ace-inhibitors also lead to upregulation of ace2 expression (22), eventually dealing with a double-edge sword by increasing protection for lung tissue function at the cost of potentially increased infectivity. beside the initial phase of sars-cov-2 infection, covid-19 pathology may exhibit three grades of increasing severity, from early infection to pulmonary involvement and eventual systemic hyperinflammation (23) . usually the grades are associated with the upregulation of pro-inflammatory cytokines, such as il-1β and il-6, il-2, il-8 and tnfα, or chemokines, that are significantly elevated in those patients with a more severe disease (23) . the high levels of these cytokines have been also reported to be inversely related to the absolute lymphocytes count (24) . since an effective immune response against viral infections depends on cytotoxic t cells activation (25) , experimental evidence supports the observation that overexpression of inflammatory cytokines like il-6 during the viral immune response might be associated with a decreased viral clearance by impairing the polarization and functionality of th1 and cd8 cells (26), contributing to the worsening of the covid-19 symptoms, and their management may appear an intriguing therapeutical approach. overall, the administration of drugs for the control of inflammation, inhibiting the response of the immune system, may be detrimental in the initial phases of the viral infection, reducing the ability of the body to react to the presence of sars-cov-2, as observed in patients chronically treated for rheumatoid arthritis (27) . on the other hand, this action may result beneficial in the reduction of the cytokines and chemokines excess, responsible for the worsening of the clinical picture. indeed, drugs managing cytokines which are known to increase during the covid-19 infection, as il-6 and tnfα, were postulated as possible effective treatments to counteract the this article is protected by copyright. all rights reserved immunopathological manifestations of the covid-19 infection based either on in vitro and in vivo data, as metronidazole (reducing several inflammatory cytokines like il-6 and tnfα) (28), or on preliminary good response, to be evaluated with caution and confirmed, in the treatment of a small cohort of covid-19 patients, as tocilizumab (a monoclonal antibody against il-6) that was used in combination with methylprednisolone (29) . therefore, in the context of covid-19 patients, the governance of the cytokine crossroad and inflammation is one of the major unmet needs, together with the adjunctive chronic or acute co-morbidities and the effects of drugs administrated for their management. predisposing comorbidities in oa patients at the moment, no studies have investigated a potential relationship between respiratory viral infections and the development of oa, as described for parainfluenza and coronavirus and the incidence of rheumatoid arthritis (30) . similarly, looking the other way round, there is no documented increased risk of respiratory infections for oa patients compared with the general population. comorbidities are another factor tipping the balance towards an increase of morbidity and mortality during infections. in oa patients, several concomitant disorders such as obesity, low muscle mass, hyperuricemia in women, diabetes, hypertension and cardiovascular diseases (cvd) are present with greater ratio than in the general population (31) . a recent meta-analysis showed that the most prevalent covid-19 comorbidities were hypertension, cardiovascular diseases and diabetes mellitus (21, 32) , and their presence increased life threatening complications. in this frame, it was recently reported that obesity may be a trigger to covid-19 morbidity and mortality (33) . similar outcomes are expected also for diabetes patients (34) , following what was stated for the two earlier cov infections, severe acute respiratory syndrome (sars) in 2002 (35) and the middle east respiratory syndrome (mers) in 2012 (36) . consistently, a recent report indicated diabetes as a risk factor significantly associated with covid-19 unfavourable clinical outcomes (37) . also, arterial hypertension may be associated with increased risk of mortality in hospitalized covid-19 infected subjects (38) . regarding cvd, pre-existing cardiovascular pathologies increase the morbidity and mortality of covid-19, and covid-19 itself causes serious cardiac sequelae (39) . as a consequence, although a direct relationship between covid-19 mortality and morbidity in oa patients has not been reported yet, the presence of oa-related concomitant disorders might trigger the life-threatening risks for oa patients in case of sars-cov-2 infection. this article is protected by copyright. all rights reserved this shall prompt orthopaedics and clinicians in general to evaluate with extreme care the clinical conditions of oa patients not only under the perspective of oa symptoms management but also for undercurrent comorbidities, naturally occurring or oa-treatment-related, that, in the era of covid-19 pandemic, may strongly affect patients outcomes more than the net combination of sars-cov-2 infection and oa. this paradigm is valid also for other pathologies characterized by comorbidities similar to those herein discussed or other conditions reported to affect covid-19 trajectory. international and national guidelines recommend nsaids for the treatment of severe pain and musculoskeletal pain in oa patients (40) . nsaids are the most commonly prescribed drugs, used by 60% of oa patientstaking medication in europe (41) and more than 50% across us (42) . nsaids may be divided into non-selective (nsnsaids), targeting both cox-1 and cox-2, and cox-2 selective (snsaids). cox-pathway inhibition leads to decreased production of prostanoids and decreased recruitment of polymorphonuclear neutrophils to the inflammatory site (43) . in general, nsaids have been associated with higher frequencies of gastrointestinal, renal and cvd negative outcomes, with the degree of cox-1 and cox-2 inhibition, and not cox-2 selectivity, being responsible for the increased risk (44) . as previously mentioned, cvd and covid-19 are directly linked, and the development of kidney failure during hospitalization in patients with covid-19 is frequent and associated with mortality (45). regarding a major covid-19 outcome like respiratory tract infections, including complicated pneumonia, pleural effusions, and peritonsillar abscess, nsaids use mainly resulted in an increase of complications. a recent review associated pre-hospital nsaids exposure with higher risks of a protracted and complicated course of pneumonia, including those in intensive care units (46) . another population-based study in northern denmark evaluated nsaids use as a prognostic factor for clinical outcomes in hospitalized patients with pneumonia (47) . all current users, including long-term users, showed an increase in the adjusted rate ratios (arrs) of at last, regarding the role of nsaids in viral infections, there is not a clear indication due to lack of clinical evidences. in rats, ibuprofen induced the overexpression of ace2 (50) , and this effect might theoretically worsen the covid-19 infection (21) . nevertheless, to date, no conclusive evidence in favour or against the use of nsaids during the treatment of covid-19 patients is available (51, 52) . therefore, a pragmatic and cautionary approach would suggest the clinicians to carefully consider nsaids use as the first line option for managing symptoms, if not absolutely necessary, due to both respiratory and cardiovascular complications in several settings. regarding pain patients, as oa patients, that are not sars-cov-2 infected, they may be reassured by their physicians on the safety of nsaids continuation, because there is nothing conclusive to show the potential for an increased incidence of viral infection, and especially of covid-19 (53). conversely, chronic pre-hospital nsaids exposure might increase complications like in all other patients, and oa patients with sars-cov-2 infection under nsaids treatment should be monitored with additional care and nsaids use considered only when strictly necessary. (table i and figure 1 ) the 2011 national health and wellness survey (nhws) showed that on 3,750 patients from five eu countries with self-reported peripheral joint oa, 47% of patients reported prescription medication, with paracetamol ranging from 0% in germany up to 6% in spain (54) . similarly, in the usa, paracetamol was taken by approximately 10% of patients participating to the osteoarthritis initiative (55). the relevance of paracetamol is its use for the longest duration (mean 84 months) and usually for more than 20 days per month (54), due to its safety at correct dose. its exact mechanism of action remains to be determined, although its effect on the prostaglandin production also at the level of central nervous system has been often hypothesized. paracetamol has similar effects to those of the selective cox-2 inhibitors, but without any antiinflammatory capacity (56) . it is generally considered to be safer than nsaids, albeit recently increased risk of adverse outcomes with frequent paracetamol dosing was published, including mortality, cvd, and renal adverse events (57) . moreover, acute liver injury (ali) resulting in relevant liver function abnormalities (bilirubin ≥ 3 mg/dl, alanine aminostransferase (alt) > 5xuln, alkaline phosphatase > 2xuln) is not uncommon with therapeutic doses of paracetamol this article is protected by copyright. all rights reserved in patients without other possible causes of liver injury (58) , as well as a general alteration of liver functionality (alt > 3xuln) even in healthy subjects without ali symptoms or laboratory evidence of hepatic failure (59) . also, the development of liver diseases during hospitalization in patients with covid-19 is high and associated with mortality (60) . therefore, how underlying liver conditions may influence the onset of hepatic complications in patients with covid-19 and their association with the use of drugs needs to be meticulously evaluated. regarding respiratory tract infections, a paucity of data is reported and is related to paracetamol effect on disease complications rather than incidence. in the previously mentioned trial for ibuprofen (61) , paracetamol showed a better performance with only 12% of patients this article is protected by copyright. all rights reserved 3) corticosteroids (cs) cs are potent multitargeting anti-inflammatory drugs (65) . in oa patients, they are administered both systemically and, more often, intra-articularly (ia). among others, prednisone is the most prescribed systemic steroid (66) and other few molecules have fda (methylprednisolone, triamcinolone, betamethasone and dexamethasone) or ema (methylprednisolone, triamcinolone) labels for intra-articular injections (67) . cs have both antiinflammatory and immunosuppressive effects, and their mechanism of action is complex. it includes inhibition of accumulation of inflammatory cells, metalloproteases and metalloprotease activators, and synthesis and secretion of pro-inflammatory factors (68) . long-term systemic (oral or parenteral) use of these agents is associated with adverse events like, among others, diabetes and hyperglycaemia, osteoporosis, superinfection, cvd and immunosuppression (69) . for intraarticular administration, adverse events are less likely, probably due to serum cortisol levels decreasing within hours and recovery to baseline in 1-4 weeks. nevertheless, ia cs resulted in reduction of inflammatory markers like c-reactive protein and erythrocyte sedimentation rate that can last for months, and in a transient increase in blood glucose levels in diabetic oa patients (70) , despite this treatment often shows short-term benefits. to avoid this pitfall, triamcinolone acetonide extended release, produced using microsphere technology, was recently approved by fda given the significant improvement over placebo and even reduced systemic exposure compared with immediate-release triamcinolone (71) . in the context of pneumonia, a recent cochrane review analysed 28 studies evaluating systemic cs therapy, given as adjunct to antibiotic treatment, versus placebo or no corticosteroids for adults and children with community acquired pneumonia (72) . the combined therapy after infection reduced mortality and morbidity in adults with severe pneumonia, and morbidity, but not mortality, for adults and children with non-severe pneumonia. hyperglycaemia was indicated as main adverse event, as also emerged in another review covering 4 clinical trials for pneumonia patients (73). additionally, in 50 patients with respiratory syncytial virus infection, those who received steroids had an impaired antibody response, although no significant differences in viral load peak were reported (78) . conversely, other studies supported the use of corticosteroids at low-to-moderate dose in patients with sars infection. in a restrospective study on 401 patients, cs were shown contributing to lower overall mortality, instant mortality, and shorter hospitalization stay (p < 0.05) (79) . also, in a prospective cohort study enrolling 2,141 patients with influenza a (h1n1)pdm09 viral pneumonia, low-to-moderate-dose (25-150 mg/day) cs were related to this article is protected by copyright. all rights reserved injections that are usually administered on a cadence basis of few months, semesters or yearly, the presumable interruption in the hospitals and clinics of non-life-saving treatments during the pandemic should avoid even the smallest and still unreported risk. again, systemic use, new or continuing pre-infection therapy, during covid-19 infection should be carefully monitored for potential complications and, if possible, reduced at minimum. (table i and figure 1) opioids may be a valuable treatment option for severe oa pain when other analgesics are contraindicated (e.g. allergic patients or gi problems) or insufficient to control pain. opioids may be divided in weak, such as codeine and tramadol, or strong, among which morphine, fentanyl and oxycodone and their analogous molecules are the most common (85) . in general, opioids should be administered with care since they may interfere with the innate and acquired immune response (86) , are associated with respiratory depression (87) , and increase the incidence and severity of infections of the airway's tracts, including pneumonia (88) . moreover, opioid systems impair and modulate immune responses induced by the influenza virus that, on one hand, might be beneficial for controlling viral immunopathogenesis, but, on the other hand, may lead to delayed viral clearance (89) . aware of these premises, individual molecules differ in their effect (90) . a group of them (i.e. morphine, fentanyl and remifentanil) was described as immunosuppressive (90) and their use associated to increased pneumonia incidence compared to molecules with no immunosuppressive activity (88, 91) . also, weak opioids were associated with a reduced risk of notably, considering the different administration routes, no differences in the overall adverse event profile emerged between transdermal opiates and oral treatments (94) . nevertheless, the risk for adverse outcomes due to opioid abuse remains, since more than 20% of oa patients receiving prescriptions has a risk factor for misuse (95) , and associated adverse events (constipation, this article is protected by copyright. all rights reserved nausea/vomiting) (96). being aware of the several molecules used in oa management, we will below report available information about two widely prescribed weak opioids, with and without immunosuppressive activity, due to their possible reduced interaction with respiratory tract infections and their preferential use in place of paracetamol or nsaids. tramadol is a weak analgesic opioid, without immunosuppressive activity (90) , that is recommended to manage pain in oa patients by both the american academy of orthopaedic surgeons (97) and american college of rheumatology guidelines (98) because of the relatively small number of deaths from each specific cause, often between 1% and 0.1% per cohort, most associations were not statistically significant. codeine is a weak analgesic opioid with immunosuppressive activity. in us, codeine was among the five most prescribed opioid to manage oa pain in the 2003-2008 period (102) . in a double-blind randomized placebo control trial of controlled codeine release for oa treatment in 103 patients, constipation, somnolence and dizziness were the most significant side effects (103) . although being a weak opioid, but consistently with its immunosuppressive activity, chronic codeine use was associated with higher pneumonia incidence compared to non-use (or of 1.93 [1.22-3 .06]) (104) . again, pneumonia risk was closer to null for use begun more than 90 days prior to index date (1.27 [0.91-1.77]). eventually, in chronic consumers, no pattern was seen for pneumonia risk in relation to estimated daily dose (104) , although the risk of adverse events due to abuse or misuse, like dependence and /or constipation, remains. this article is protected by copyright. all rights reserved regarding covid-19, it is appropriate to postulate that chronic pain patients, as oa patients, on strong (and/or immunosuppressive) opioids could potentially be more susceptible to sars-cov-2 infection complication like pneumonia, whereas weak opioids might have reduced side effects and infections susceptibility and be therefore preferable. nevertheless, at present, there is not a clear indication for or against opioids discontinuation in relation to increased covid-19 infection incidence, but surveillance in case of strong drugs should be conducted. (table i and disease-modifying osteoarthritis drugs (dmoads) are molecules targeting key tissues in the oa pathophysiology process and aiming to prevent structural progression, control inflammation, and relieve pain (105) . currently, no dmoads have been licensed for use in the treatment of oa but several putative dmoads are in phase ii development. in particular, mabs and inhibitors directed against oa-related cytokines such as tumor necrosis factor α (tnfα) (106) (107) (108) (109) , nerve growth factor (ngf) (110) (111) (112) or interleukin molecules like il-1α/β (113) (114) (115) , are under investigation, due to their regular use as biological disease-modifying anti-rheumatic drugs (bdmards) for the management of rheumatoid arthritis inflammation (116) . in a report comparing safety outcomes of bdmards in rheumatoid arthritis in 42 observational studies, general safety profile of bdmards emerged with very sporadic cases of cardiovascular and infection incidence (117). moreover, in a study aimed at evaluating the incidence of influenza-like illness (ili) in a group of patients suffering from chronic inflammatory rheumatism and treated with bdmards, ili resulted higher than the value reported in the general population, although no important complications or hospitalizations have been reported (118) . similarly, very low rate or absence of adverse events was observed for tested dmoads, like tnfα (119-121), ngf (111, 112) and il-1α/β (114, 115, 122) inhibitors, suggesting an overall safety profile. their use is therefore envisioned as a cutting-edge approach with lower risks, readily available as soon as efficacy data will be available. further, and increasing the interest in the field, recent studies indicated a possible link between these treatments and the positive management of covid-19 infection. for tnfα inhibitors, tnfα production has been associated with tnfαconverting enzyme (tace)-dependent shedding of the ace2 ectodomain, crucial for the penetration of the virus into the cell (123) . as a consequence, tnfα inhibitors might interfere with sars-cov infection incidence and the consequent organ damage (124) , via tnfα inhibition and this article is protected by copyright. all rights reserved down-regulation of ace2 expression and shedding, as recently showed in the gut (125) . for these reasons, a clinical study for the efficacy and safety of adalimumab injection in the treatment of patients with severe novel covid-19 pneumonia is ongoing in china (chictr2000030089). moreover, a potential role for il-1β inhibitors or blockers could be envisioned from data showing an activation of the nlrp3 inflammasome by sars-cov (126) , with sars-cov infected patients having elevated serum levels of il-1β (23) . similarly to other sars-cov, also covid-19 triggers inflammasome activation, especially within lymphoid cells, and patients have increased serum il-1β (127) . consistently, a chinese study demonstrated that inhibition of proinflammatory cytokines such as il-1β might be a beneficial strategy for the treatment of sars infections (128) . further, a phase iii rct of il-1β blockade in sepsis showed significant survival benefit in patients with hyperinflammation (129) . nevertheless, at present, there is no evidence for il-1β blockers in the treatment of covid-19 patients. the literature, however, did suggest a potential role for the reduction of pro-inflammatory markers, such as il-1β, which are elevated as part of the immune response and may have a role in the severe lung damage associated with human coronaviruses. under the same paradigm, il-6 proinflammatory cytokine is under investigation as target for covid-19 therapy, particularly in patients developing ards with severe hyperinflammatory response characterized by high increases of plasma il-6 and crp levels. in very preliminary results of a recent report, il-6 blocker tocilizumab appeared to be an effective treatment option in covid-19 patients, although used in combination with glucocorticoids (29) . in conclusion, in the covid-19 patients with concomitant oa, for which traditional treatments still have a wider and documented consistency and safety, more data about mabs efficacy/safety and the completion of dmoads phase ii studies will lay the foundation for the use of these cutting edge and possibly safer therapeutics as first line option in the near future, when the pandemic is expected to remain a threat. (table i and figure 1 ) the data reported in this review partially identify the effects of commonly used drugs on both infection incidence of covid-19 related pathologies and disease complications (table i and this article is protected by copyright. all rights reserved side effects and more specificity to reduce pro-inflammatory markers, thus preventing or reducing the most severe outcomes of disease. the introduction of some mabs in compassionate use for covid-19 patients showed encouraging results, which can indicate the real consistence with this previously mentioned hypothesis, but, again, this still needs to be studied and confirmed by large epidemiological studies. in this view, we underline the crucial role of the orthopaedic registries as an effective collection of evidences in this prominent field. to face the pandemic at present times, on a daily basis clinicians decide the first-line pharmacological therapy, whether or not discontinue an existing therapy, and the efficacious alternative when the previous approach fails. both patients' characteristics (comorbidities) and previous treatments' iatrogenic effects are essential to drive these decisions and reflect perceived differences in safety across drugs for incidence and complications. the currently available data indicate that oa therapies are safe and there is not any clear indication to avoid prescription or suggest discontinuation of existing pharmacological therapies due to covid-19 infection incidence or complications. nevertheless, we are convinced that particular attention should be used for oa patients, especially if hospitalized. in our opinion, specialists and clinicians should carefully consider each single patient profile and balance the cost/benefit ratio of current or new therapies (table i and figure 1 ). often, anti-inflammatory and anti-pain drugs are prescribed for weak to moderate symptoms caused by everyday life movements that are largely reduced, if not absent, during home isolation or hospitalization. therefore, drugs reported to have the strongest supposed influence on secondary infections or complications should be prescribed only when benefits overcome the potential harm. in this frame, another crucial and still largely underestimate factor for the choice of the most appropriate therapy and its continuation/discontinuation is the stage of the covid-19 infection (figure 1 ). in absence of symptoms or in the early/middle stage, especially in younger patients with absence of relevant comorbidities, continuation of oa therapies is strongly recommended since approximately 80% of affected individuals will end in this stage without complications leading to hospitalization/intensive care. in the mild/late phase of infection, characterized by a state of high levels of inflammation leading to further clinical deterioration and potential involvement of extrapulmonary sites, the cost/benefit ratio of oa therapies has to be evaluated with care, especially for cs or strong/immunosuppressive opioids. in general, with a few exceptions that deserve cost/benefit considerations, oa patients should be reassured to continue their treatment even during covid-19 outbreak. this would prevent disease flares that can contribute to increase patient burden, disability, poor quality of life, and healthcare this article is protected by copyright. all rights reserved use. at the same time, all the physicians are encouraged to keep up to date on new evidences that will emerge from the future epidemiological studies and that may modify the existing knowledge. this article is protected by copyright. all rights reserved prevalence of doctor-diagnosed arthritis and arthritis-attributable activity limitation ---united states role of inflammation and the immune system in the progression of osteoarthritis osteoarthritis: a disease of the joint as an organ the role of inflammation in the pathogenesis of osteoarthritis a framework for the in vivo pathomechanics of 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therapeutic target? sars-coronavirus open reading frame-8b triggers intracellular stress pathways and activates nlrp3 inflammasomes the deadly coronaviruses: the 2003 sars pandemic and the 2020 novel coronavirus epidemic in china interleukin-1 receptor blockade is associated with reduced mortality in sepsis patients with features of macrophage activation syndrome: reanalysis of a prior phase iii trial* authors want to thank all health-care workers of irccs istituto ortopedico galeazzi for their exceptional work during this unexpected world challenge. key: cord-288945-c9ow1q5c authors: spengler, ulrich title: liver disease associated with non-hepatitis viruses date: 2019-11-01 journal: encyclopedia of gastroenterology doi: 10.1016/b978-0-12-801238-3.65782-3 sha: doc_id: 288945 cord_uid: c9ow1q5c hepatitis is commonly associated with certain viruses labeled as “hepatitis” viruses. however, many other viral infections can also affect the liver ranging from mild asymptomatic elevations of aminotransferases to fulminant hepatic failure. this article will provide a brief overview on a variety of different viral infections that may be associated with significant liver pathology at least under certain conditions, for example, immunosuppression. this overview discusses key virological features, clinical presentation of associated liver disease and provides some information on diagnosis and an outline of treatment options. thus, the overview can provide first orientation when infectious hepatitis is encountered in a patient that cannot be explained by the usual hepatitis viruses. the liver is a highly perfused organ and the first filter for blood coming from the intestines. thus, the liver is particular prone to become involved in blood-borne infections. apart from the so-called hepatitis viruses, many other viruses, primarily targeting other extrahepatic tissues, also lead to liver damage and hepatitis. damage can range from asymptomatic elevations of aminotransferases to fulminant hepatic failure depending on the virus and the host's immune response. when the immune system controls infection poorly, direct infection of hepatocytes and liver necrosis may occur. this situation applies to patients under severe immunosuppression or infections with particularly virulent agents such as the viruses that cause hemorrhagic fevers. alternatively, liver cells may become victims of collateral damage without direct infection when cytolytic cd8 þ t effector lymphocytes are expanded outside the liver and then recruited via liver-resident macrophages such as kupffer cells presenting viral antigens (polakos et al., 2006; schumann et al., 2000) . this type of liver damage is particularly associated with respiratory viruses such as influenza virus. however, since many viral infections expand cd8þ t lymphocytes, this by-stander mechanism may affect the liver more often (murali-krishna et al., 1998) . here, we provide a brief overview over viral infections primarily not designated as hepatitis viruses which may lead to liver disease and hepatic complications. this overview will largely focus on the hepatic aspects of viral infections. thus, readers interested in more detailed information should consult a comprehensive textbook in microbiology or clinical infectious diseases. approximately 8% of travelers to the developing world require medical care during or after travel, and fever is the underlying problem in 28% of them (wilson et al., 2007) . physicians evaluating returned travelers frequently suspect rare or exotic diagnoses. although exotic diseases, in particular viral hemorrhagic fevers are a severe threat in certain regions of the world, liver disease due to exotic infections such as ebola virus, rift valley fever or lassa fever have been reported only sporadically but do not represent a frequent health problem in returning travelers. viral hemorrhagic fevers share some epidemiological and clinical features and cause rather similar liver pathology. most viruses are transmitted via arthropod vectors. the viruses cause small vessel damage in multiple organs resulting in overt hemorrhage. the spectrum of diseases and their geographical distribution are listed in table 1 . much attention has been paid to abnormal liver function and altered hepatic pathology. nevertheless, clinically significant liver disease or death from liver failure are rare complications except in yellow fever. dengue fever is among the top three etiological agents, accounting for approximately 6% of febrile illnesses in the traveler (wilson et al., 2007) . of note, although malaria is the leading cause of systemic febrile illness worldwide, apart from sub-saharan africa and central america travelers returning from tropical or sub-tropical regions had dengue fever more frequently than malaria. chikungunya fever is an emerging novel virus infection recently expanding in asia and africa, which also causes fever, myalgia, arthralgia and skin rash in increasing numbers of patients. table 1 viral hemorrhagic fevers affecting the liver the dengue virus complex comprises four antigenically related but distinct flaviviruses termed dengue virus serotypes 1 through 4 (den-1 to . dengue viruses are transmitted by aedes aegypti mosquitos in epidemic and endemic outbreaks and cause acute infections. three to six days after a mosquito bite the virus spreads via the blood-stream, and among the various organs it can be isolated frequently from liver samples (rosen and khin, 1989) . dengue virus infection usually causes a flu-like illness with a rash-dengue fever (fig. 1 ). hepatomegaly and elevated serum aminotransferases, which are usually mild, are common in dengue virus infections (wahid et al., 2000) . clinically more severe diseases, for example, dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) can follow from secondary infection with dengue virus of different serotype. in 2009, the world health organization issued new guidelines and introduced a revised classification scheme consisting of the following categories: dengue without warning signs, dengue with warning signs, and severe dengue (world health organization, 2009 ). however, there are no reliable warning signs for severe dengue. in dhf there are widespread petechial hemorrhages together with multiple organ damage; in dss, which mostly affects children below the age of 15, there is extensive capillary leakage and severe fluid depletion leading to hypovolemic shock. if untreated, mortality approaches 50%. in fatal cases of dhf the liver is enlarged, pale from steatosis and shows multifocal hemorrhages. laboratory diagnosis of dengue virus infection is done by detection of viral components, for example, pcr in serum or indirectly by serology. the sensitivity of each approach depends on the duration and course of the patient's illness when the patient presents for evaluation. currently vaccines against dengue virus are being developed but have not become licensed, yet. there is no direct antiviral therapy available against the dengue viruses. thus, treatment is supportive, requiring meticulous fluid management and intensive care in dss. details for treatment are summarized in recent who guidelines (world health organization, 2012). chikungunya is an arthropod-borne toga virus initially endemic to west africa, which next spread to the indian ocean islands and southeast asia (charrel et al., 2007) . chikungunya fever was originally considered a disease of tropical and subtropical regions, until an outbreak in northern italy was recorded in 2007 (rezza et al., 2007) . since then chikungunya infections have become exported to other western countries, the americas and caribbean region via international travel. of note, dengue and zika viruses are transmitted by the same mosquito vectors as chikungunya. thus, the viruses can co-circulate in the same geographic region, and coinfections have been documented. transmission of chikungunya via blood products has been reported in france, where a nurse was infected by exposure to blood from an infected patient. chikungunya, might also be transmitted inadvertently by organ transplantation since viremia can exceed high levels prior to onset of symptoms. the infection begins abruptly with high fever, symmetric polyarthralgia and macular or maculopapular skin rash (taubitz et al., 2007) . pruritus and bullous skin lesions have also been described. previously chikungunya fever has been considered a self-limited disease. however, severe complications also comprising acute viral hepatitis and deaths have been reported in the recent outbreaks; particularly in elderly patients (> 65 years) and people with pre-existing chronic medical problems. some patients have persisting symptoms for a variable length of time after the acute illness. manifestations include arthritis/arthralgia, edematous polyarthritis of fingers and toes, morning pain and stiffness, and severe tendosynovitis. clinically chikungunya fever must be differentiated from dengue fever, which shares many symptoms and features. the diagnosis of chikungunya virus infection should be suspected in a patient with acute onset of fever and polyarthralgia who had possible exposure by travel to or residency in an epidemiological risk area. the diagnosis of chikungunya is established by detection of chikungunya viral rna via real-time reverse-transcription polymerase chain reaction (rt-pcr) or chikungunya virus serology (pan-american health organisation, 2017). chikungunya igm antibodies become detectable by elisa 5 days after the onset of symptoms. for certain regions simultaneous testing for dengue and zika virus infection is recommended. there exists no specific antiviral therapy for acute chikungunya virus infection. treatment consists of supportive care and includes rest, fluid replacement, and eventually the use of nonsteroidal antiinflammatory drugs (nsaids) or acetaminophen to relieve pain and fever. in patients suspected to have dengue virus coinfection nsaids should be avoided before the patient becomes afebrile for >2 days, in order to avoid bleeding complications associated with severe dengue virus infection. chikungunya infection cannot be prevented by vaccination. hanta viruses are examples of emerging viruses, which belong to the genus hantavirus within the bunyaviridae family. they are negative-sense single-stranded rna viruses, primarily harbored in rodents and shed in rodent urine, saliva and feces. the virus is inhaled by man as aerosols from dried rodent excreta, or in unusual circumstances, transmitted by bites (nichol et al., 2000) . hantaviruses exist in multiple serotypes worldwide, which differ in their virulence. some are considered apathogenic, while certain isolates can produce two distinct severe syndromes in humans: the hanta virus cardiopulmonary syndrome, mostly due to isolates in the americas; and the hemorrhagic fever with renal syndrome caused by isolates (seoul virus, dobrava virus, puumala virus, hantaan virus) in europe and asia (plyusnin et al., 2001) . in some instances, patients with hanta virus hemorrhagic fever suffered from severe acute hepatitis, whereas renal damage was rather mild (wong et al., 1987; chan et al., 1987; lledó et al., 2003) . hanta virus has also been incriminated as a potential cause of cryptogenic hepatitis in southwestern china. however, this hypothesis has not been confirmed by antibody studies in japan. hanta viruses, however, may still play some role, because hanta virus infection has been observed to trigger autoimmune liver disease (yotsuyanagi et al., 1998) . thus, this mechanism may contribute to community-acquired hepatitis (martin et al., 2008) . nevertheless, the precise role of hanta virus infections for human liver disease still awaits clarification. by the time symptoms are evident, patients uniformly have antiviral igm antibodies and most have igg antibodies. diagnostic assays to detect hanta virus antibodies include enzyme-linked immunosorbent assay (elisa), immunoblot test (sia), western blot, indirect immunofluorescence (ifa), complement fixation, and hemagglutinin inhibition as well as neutralization assays. hanta virus strains associated with hemorrhagic fever and hepatorenal syndrome are sensitive to ribavirin in vitro. a prospective, randomized, double-blind, placebo-controlled trial in the people's republic of china reported a sevenfold decrease in mortality among ribavirin-treated patients with serologically confirmed hanta virus disease (huggins et al., 1991) . however, ribavirin appeared to be less effective in hanta virus cardiopulmonary syndrome. thus, it has not yet been approved, but nevertheless may be tried as rescue treatment in emergency situations (sidwell and smee, 2003) . yellow fever is a member of the flaviviridae family and constitutes a single-stranded plus strand rna virus. it comprises a single conserved serotype and seven major genotypes reflecting distinct regions in west africa, central-east africa and south america (vasconcelos et al., 2004; barnett, 2007) . yellow fever virus is transmitted by a variety of different aedes vectors and causes endemic and epidemic outbreaks in africa and south america. yellow fever can be prevented by vaccination, and thus, has become rare in travelers. the spectrum of yellow fever virus infection ranges from subclinical infection to a life-threatening disease with fever, jaundice, renal failure and hemorrhage. usually yellow fever initially presents as an acute, flu-like illness of sudden onset with fever, myalgia and headache, which cannot be distinguished easily from other acute infections such as severe malaria, leptospirosis, fulminant viral hepatitis or dengue hemorrhagic fever. between 48 and 72 h after onset, aminotransferases start to rise in blood heralding the development of jaundice. the degree of liver abnormalities at this stage predicts the severity of liver disease during the course of the illness later on. next, a period of apparent remission lasting up to 48 h may follow the initial infection. patients with abortive infection recover at this stage. about 15% of patients will enter the third stage of intoxication characterized by the return of fever, prostration and organ dysfunction. patients suffer from nausea, vomiting, or epigastric pain, and develop jaundice and oliguria. bleeding can occur from the mouth, nose, eyes or stomach. serum aspartate transferase (ast) levels usually exceed those of alanine transferase (alt). the outcome of yellow fever infection is determined during the second week after onset, when many patients either rapidly recover, while between 20% and 50% of the patients, who have progressed to the stage of intoxication, will ultimately die from circulatory shock. convalescence may be associated with fatigue over several weeks, and occasionally jaundice and elevated aminotransferases may persist for months. the diagnosis of yellow fever is confirmed by demonstration of specific igm by elisa, by pcr or by isolating the virus from the blood. polymerase chain reaction (pcr) testing in blood and urine can detect the virus in early stages of the disease. in later stages, testing to identify antibodies is recommended. liver biopsies should be avoided due to a high risk of bleeding complications. there is currently no specific anti-viral drug to treat yellow fever but specific care to manage dehydration, liver and kidney failure as well as fever improves outcomes. ribavirin inhibits yellow fever virus in vitro. however, the extremely high concentrations of the drug needed cannot be achieved in vivo. recently there have been attempts to treat liver failure resulting from yellow fever infection by high urgency liver transplantation (song et al., 2019) . however, outcomes after liver transplantation were mixed and the few survivors had frequent postoperative bacterial and cytomegalovirus infections. a highly active attenuated live-vaccine is available, which induces seroconversion rates >95% and provides a high level of protection. due to potential risks associated with a live virus vaccine children below the age of 9 months, pregnant women and immunosuppressed individuals should not receive the vaccine, nor should subjects be vaccinated who are allergic to egg proteins. infrequently two serious vaccine-related complications may occur: a form of encephalitis termed yellow fever-associated neurotropic disease and a syndrome resembling natural infection designated as yellow fever vaccine-associated viscerotropic disease. adenoviruses have a worldwide distribution and cause febrile diseases. over 50 serotypes can be distinguished which are further subdivided into the six subgoups a through f. typical syndromes comprise conjunctivitis, upper respiratory tract infections such as pharyngitis and coryza, pneumonia and otitis media. in young children an acute diarrheal illness is caused by subgroup f type 40 and 41 adenoviruses. adenoviruses can persist in human tissue over prolonged periods (garnett et al., 2009) , and can cause a variety of clinical syndromes in immunocompromised individuals including severe hepatitis (kojaoghlanian et al., 2003) . in particular, transmission of latent adenovirus with the donated organ is a risk factor for adenoviral hepatitis in pediatric liver transplantation (michaels et al., 1992) . adenoviral hepatitis occurs in congenital and acquired immunodeficiency syndromes and resembles severe necrotizing hepatitis associated with herpes simplex virus infection. patients develop extensive areas of liver cell necrosis (fig. 2) , massively elevated aminotransferases, and a severe coagulopathy (south et al., 1982; janner et al., 1990; krilov et al., 1990) . ultimately, outcomes may be fatal. liver biopsy specimens may reveal typical intranuclear inclusion bodies in necrotic areas of the liver, but biopsies carry an extremely high bleeding risk. viral isolation and pcr techniques help to identify the causative viral strain. to date a proven therapy or vaccine to prevent adenoviral hepatitis does not exist, but ribavirin may be helpful in selected cases (wulffraat et al., 1995) . liver damage in fatal influenza has been considered immune-mediated, because high cytokine levels were detected (murali-krishna et al., 1998; peiris et al., 2004) . moreover, volunteers infected experimentally with intranasal influenza a/kawasaki/86 (h1n1) transiently developed elevated aminotransferases (polakos et al., 2006) . since the rise in liver enzymes occurred after pyrexia had settled, it was concluded that the host's immune responses rather than viral infection caused damage to the liver. immune mediated liver damage may also be the cause for elevated aminotransferases in other viral respiratory infections such as respiratory syncytial virus (peiris et al., 2004) . however, cardiovascular failure and hepatic ischemia must be considered in as alternative factors in patients with severe respiratory infections ( fig. 3) (eisenhut et al., 2004) . influenza viruses represent three genera in the orthomyxoviridae family. generally influenza a viruses is associated with more severe disease in humans than influenza viruses b and c. influenza a is further subdivided with respect to genetic variation in its hemagglutinin (h) and neuraminidase (n) genes. influenza viruses commonly cause a self-limited acute respiratory infection with fever, rhinorrhea, sore throat and occasionally gastrointestinal symptoms. therefore, aminotransferases are not monitored routinely. in the 2004 h1n5 influenza outbreak, however, about 60% of patients with pneumonia had deranged liver function tests with gastrointestinal symptoms such as vomiting, abdominal pain and diarrhea on initial presentation (yuen and wong, 2005) . although molecular evidence for viral liver disease was not found, autopsy revealed hepatic centro-lobular necrosis in some cases . in influenza virus infection the clinical presentation is dominated by fever and respiratory symptoms. the diagnosis be established by detecting viral antigen or antibodies, but nowadays the gold standard has become detection of viral rna in throat washings by pcr. influenza virus infection can be treated and prevented by neuraminidase inhibitors such as oseltamivir and zanamivir, which have been recommended especially for treatment in influenza h1n5 infection by the who in 2010 (schünemann et al., 2007) . however, their benefit is limited due to the appearance of resistant isolates in recent outbreaks. a preventive vaccine is adapted annually to the circulating strains. severe acute respiratory syndrome (sars) is caused by a novel coronavirus (sars-coronavirus, sars-cov), which caused outbreaks of severe infections of the lung and gastrointestinal tract in the far east and canada (ksiazek et al., 2003; drosten et al., 2003; lee et al., 2003; poutanen et al., 2003) . there was also other organ involvement. middle east respiratory syndrome coronavirus (mers-cov) first appeared in the arabian peninsula and meanwhile has occasionally been observed also in few travelers returning from risk areas. apart from viral pneumonia other internal organs may become affected including hepatitis (alsaad et al., 2018) , particularly when mers-cov hits patients with concomitant diseases, for example, diabetes mellitus. sars-cov and mers-cov have been detected in masked palm civets, dogs and cats as well as camel and thus represent zoonotic infections in man. approximately 25% of patients with sars had elevated liver enzymes at the onset of infection, and further 45% of patients with normal liver enzymes at initial presentation developed elevated aminotransferases later on, so that overall up to 70% of patients showed elevated liver enzymes during their illness (booth et al., 2003; choi et al., 2003; wong et al., 2003; chan et al., 2005) . jaundice was observed in <10% of cases. in most patients aminotransferases started to rise toward the end of the first week and peaked at the end of the second week. with resolution of sars aminotransferases normalized spontaneously in the majority of patients. severe liver damage (alt >5 times the upper limit of normal) was observed more frequently in male patients, and those with significant other comorbidities or elevated serum creatinine levels (chan et al., 2005) . diagnosis of sars-cov and mers-cov infection is suspected in persons with typical symptoms who had contact to risk areas. the diagnosis is confirmed in certified specialized microbiology labs by pcr from respiratory fluids. hygienic prevention measures have to be respected when handling samples and caring for patients with suspected coronavirus pneumonia. during sars outbreaks both ribavirin and kaletra (baby dose ritonavir/lopinavir) were tested as experimental therapy but showed limited success. herpesviruses form a large family of dna viruses, which comprises eight members that can cause disease in man (table 2 ). herpes simplex virus (hsv), varizella zoster virus (vzv), epstein-barr virus (ebv), cytomegalovirus (cmv) and human herpesvirus type 6 or 7 (hhv6, hhv7) can directly affect the liver and are infections in the human population usually acquired during childhood or adolescence. hhv8 can be transmitted sexually and presumeably also vertically from mother to child but has a more limited prevalence. in severely immunosuppressed patients hhv8 can cause kaposi sarcoma and body cavity lymphoma. herpesviruses persist life-long and can reactivate liver disease in immunosuppressed patients later on. primary herpes simplex infection produces characteristic oral (hsv-1) or genital (hsv-2) vesicular lesions. symptoms can be severe with fever and malaise but primary infections are frequently asymptomatic. fulminant hepatitis is a complication both of hsv-1 and hsv-2 infection (pinna et al., 2002) . organ transplantation and treatment for hematological malignancies are the most frequent underlying predispositions (johnson et al., 1992) . further individuals at risk include neonates, patients on steroids, hivinfected patients, and patients with cancer or myelodysplastic syndromes (pinna et al., 2002; johnson et al., 1992; kusne et al., 1991; zimmerli et al., 1988; frederick et al., 2002) . rarely fatal hsv-hepatitis has also been reported in immunocompetent adults (goodman et al., 1986) . varicella zoster virus (herpesvirus type 3) causes chicken pox; and shingles when latent infection is reactivated. after primary infection there is replication of varicella zoster virus in the epithelia of gut, respiratory tract, liver and endocrine glands. secondary viraemia then leads to infection of the skin and causes the typical rash. liver disease is rare and limited to patients with severe immunodeficiency. hsv-related hepatitis has a high (>80%) mortality and resembles septic endotoxic shock; jaundice is not always present (kusne et al., 1991) . patients suffer from fever, anorexia, nausea, vomiting, abdominal pain, leucopenia, and coagulopathy. typical oral or genital vesicular lesions may be present in only about 30% of patients (pinna et al., 2002) . some patients have disseminated further extrahepatic involvement, for example, lung, lymphnodes, spleen, and adrenal glands. in severe varicella zoster virus infection hepatic lesions are similar to herpes simplex hepatitis. varicella zoster virus has also been reported to trigger severe autoimmune type hepatitis (al-hoamoudi, 2009). the diagnosis of hsv-related hepatitis must be rapidly established. serologic assays are of little use. herpes simplex and varicella zoster viruses are detected preferentially by the polymerase chain reaction (finström et al., 2009) , or occasionally by viral isolation or immunofluorescence staining. prompt systemic treatment with acyclovir reduces hsv-associated morbidity and serious complications in hiv-infected patients. antiviral acyclovir prophylaxis has markedly reduced hsv re-activation after organ transplantation (seale et al., 1985; pettersson et al., 1985) . acyclovir resistance occurs in about 5% of immunocompromised patients and is negligible (<0.5%) in immunocompetent subjects (tyring et al., 2002) . valacyclovir is a prodrug of acyclovir, and famciclovir a prodrug of penciclovir, which have similar antiviral mechanisms as acyclovir. thus hsv isolates resistant to acyclovir are also resistant to these drugs (levin et al., 2004) . cidofovir and foscarnet are alternative choices to treat acyclovir-resistant hsv but are less well tolerated (safrin et al., 1991) . treatment and prophylaxis of varicella zoster hepatitis is similar to herpes simplex viruses because acyclovir also inhibits replication of varicella zoster virus. in immunocompetent hosts cytomegalovirus (cmv) infection may be rather asymptomatic but occasionally causes transient minimally symptomatic acute disease. congenital cytomegalovirus infection occurs in <2% of newborns and is encountered in (kylat et al., 2006) . when newborns or immunocompromised patients, for example, hiv infection, cancer, solid organ or bone marrow transplantation become cmv-infected, they may develop serious disease. cmv-related liver disease represents the commonest cause of viral hepatitis after organ transplantation. infection may result from re-activation of endogenous virus under immunosuppression, infection from the transplanted organ or blood transfusion of a cmv-positive donor. in liver transplantation, most cmv disease occurs at 1-4 months after transplantation. cmv infection is also a potential factor triggering acute and chronic rejection. vice versa, rejection therapy with corticosteroid boluses may induce endogenous cmv reactivation. although cmv re-activation is a frequent complication also in hiv-positive patients with advanced immunodeficiency (cd4 counts <200/ml), involvement of the liver seems to be rather rare (palmer et al., 1987) , but cmv occasionally causes bile-duct necrosis and a so-called hiv-cholangiopathy, a sclerosing cholangitis encountered in patients with terminal hiv-immunodeficiency (bonacini, 1992) . in about 10% of immunocompetent subjects primary cytomegalovirus infection produces an infectious mononucleosis-like syndrome, which is associated with elevated aminotransferases and a mild hepatitis (fig. 4) . liver histology may show focal hepatocyte and bile duct damage with lymphocytic infiltration into the sinusoids and occasionally epithelioid granulomas without necrosis, while cmv inclusion bodies or cmv immunostaining are only rarely seen (snover and horwitz, 1984) . fetal cmv infection has also been associated with obstructive biliary disease and neonatal hepatitis with giant cell transformation, cholestasis and viral inclusion bodies (finegold and carpenter, 1982) . in liver biopsies hepatocytes are swollen and may contain basophilic granules in the cytoplasm. a typical intranuclear amphophilic inclusion body can be present, resembling an "owl's eye" (fig. 3c ). both nuclear and cytoplasmic inclusions are full of virions (desmet, 1983) . however, in posttransplantation cmv hepatitis cytomegalovirus inclusion bodies are scanty. instead small foci of necrosis and inflammation (microabscesses) may be present (fig. 3b) . de novo appearance of cmv igm antibodies or a fourfold rise in igg antibodies herald cmv infection in immunocompetent individuals. however, serology is unreliable in immunocompromised patients and is replaced by quantitative molecular dna amplification assays (humar et al., 1999; caliendo et al., 2003) . meanwhile most transplant centers perform cmv surveillance by weekly quantitative determination of cmv dna. in most transplant units organ recipients at high risk for acquiring cmv disease receive immune prophylaxis with cmvhyperimmune antibodies and antiviral drugs (paya, 2001) . however, cmv infection and disease may still develop. cmv hepatitis should be treated promptly in patients with immunodeficiency. intravenous ganciclovir or oral valganciclovir over 3 weeks is the treatment of choice. drugs must be continued in reduced doses as chemoprophylaxis, if prolonged immunosuppression is anticipated (crumpacker, 1996) . fortunately, ganciclovir resistance is still rare (martin et al., 2008) , so that the more toxic alternatives cidofovir and foscarnet are rarely needed. epstein-barr virus (ebv) is shedded in oral secretions, and most primary ebv infections occur in adolescents. ebv accounts for 90% acute infectious mononucleosis syndromes. it persists life-long in a latent state, which results from a dynamic interplay between viral evasion strategies and the host's immune responses. while-unlike other herpesviruses-ebv reactivation-associated liver disease is not a prominent feature of persistent ebv infection, this herpesvirus is a potent cause for various malignancies such b-and t cell lymphomas, hodgkin lymphoma, and nasopharyngeal carcinoma. ebv has also been associated with an aggressive lymphoproliferative disease after liver transplantation. ebv intrauterine infection may lead to diverse congenital anomalies also comprising biliary atresia (goldberg et al., 1981) . however, only few pregnant women are susceptible, thus intrauterine ebv infection is rare. in infants and young children primary infection is frequently asymptomatic, while in adults it results in the infectious mononucleosis syndrome. patients develop malaise, headache, low-grade fever, before the more specific symptoms such as pharyngitis/ tonsillitis, swelling of cervical lymphnodes and moderate to high-grade fever occur. nausea, vomiting, and anorexia are frequent findings. a mild clinical hepatitis accompanies infectious mononucleosis in approximately 90% of patients (fig. 5) . splenomegaly is found in about half of patients, but hepatomegaly and jaundice are infrequent findings. patients show peripheral blood lymphocytosis with characteristic large abnormal lymphocytes in their blood smears. the vast majority of patients recover over 2-4 weeks, but fatigue may persist over several months after infection. ebv does not infect hepatocytes but lymphoid tissue. thus, liver damage is due to immune-mediated pathology and-when exceptionally done-biopsy specimens show diffuse lymphocytic infiltrates in the sinusoids but only occasionally focal apoptotic hepatocytes (fig. 3a ) (purtilo and sakamoto, 1981) . moreover, ebv-related immune activation can lead to several complications: patients with x-linked lymphoproliferative disease (xlp) caused by a mutation in the sh2d1a gene on the x chromosome are particularly vulnerable to the epstein-barr virus and may suffer fatal infections with extensive liver necrosis (seemayer et al., 1995) . in patients with severe immunodeficiency lymphomatoid granulomatosis is a further unusual complication of epstein-barr virus infection leading to granuloma formation in multiple organs including the liver, which may require interferon-alpha antiviral therapy (wilson et al., 1996) . in patients with hiv infection an ebv-associated lymphoproliferative disorder with hepatic infiltration of immunoblasts (beissner et al., 1987) , and a hemophagocytic syndrome have been reported (albrecht et al., 1997) . epstein-barr virus is also the major causative agent for the so-called posttransplant lymphoproliferative disease (ptld), which after organ transplantation may result in lymphocytic infiltration of the liver and other organs ranging from benign polyclonal b cell proliferation to malignant b cell lymphoma (hanto, 1995) . ptld occurs more commonly in children than in adults, depending on the degree of immunosuppression. it is primarily a complication in ebv-negative organ recipients, who develop primary ebv infection under immunosuppression owing to a graft from an ebv-positive donor. finally, ebv is a potent risk factor to develop lymphoma in later life even in patients without overt immunosuppression (fig. 6) . the clinical suspicion of epstein-barr virus infection is confirmed by detection of heterophilic or ebv-specific antibodies in infectious mononucleosis and quantitative polymerase chain reaction assays in patients with lymphoproliferative disorders (bruu et al., 2000; weinberger et al., 2004) . liver biopsy is not recommended for routine diagnostics. treatment of epstein-barr virus infection is primarily supportive. corticosteroid therapy can ameliorate symptoms. however, this option should be only considered in individuals with immune-mediated life-threatening complications, for example, imminent liver failure. because of theoretical concerns to suppress the immune system in an infection with a potentially oncogenic virus, corticosteroids are not recommended in general. acyclovir inhibits the ebv dna polymerase, and antiviral therapy with this drug has shortened virus shedding but failed to demonstrate a convincing clinical benefit even in severe acute epstein-barr virus infection (torre and tambini, 1999) . acyclovir antiviral therapy is not effective against latent ebv-infection. thus, reduction in immunosuppression, anticancer chemotherapy, and b-cell depleting antibodies are needed to treat ebv-related lymphoproliferative disorders. human herpesviruses types 6 (hhv6) and 7 (hhv7) hhv-6 exists in two variants, hhv6-a and hhv-6b, which infect t cells and various other cells types expressing the cd46 receptor (santoro et al., 1999) . although genetically clearly distinct from hhv-6, hhv-7 is another b-herpesvirus that shares many features with hhv-6. primary infection with either virus commonly occurs at young age and can lead to a febrile illness known as exanthema subitum or roseola infantum (leach, 2000) . pityriasis rosea reflects primary infection with hhv-7. hvv-6 also integrates into the host's genome and is transmitted via the germline. hhv6 and hhv-7 can reactivate each other (tanaka-taya et al., 2000) as well as cytomegalovirus leading to symptomatic cmv-disease in liver transplantation (humar et al., 2000) . the full spectrum of diseases caused by chronic hhv-6 and -7 infection is still unclear, but these viruses are putatively involved in a variety of different syndromes such as encephalitis, multiple sclerosis, pneumonitis, an infectious mononucleosis-like syndrome, postinfectious drug hypersensitivity as well as lymphoproliferative disorders and systemic disease in immunocompromised patients (stoeckle, 2000) . hhv-6 can cause severe and fatal hepatitis in neonates, children and adults (mendel et al., 1995; chevret et al., 2008; härmä et al., 2003) . hepatitis due to infection and reactivation of hhv-6 and hhv-7 can also complicate organ transplantation (dockrell and paya, 2001; härmä et al., 2006; ohashi et al., 2008) . in addition, hhv-6 has been associated with autoimmunity and giant-cell hepatitis and giant-cell transformation of bile duct cells (potenza et al., 2008) . hhv6-igm antibodies develop within a week after infection but are an unreliable marker, because false-positive test results in about 5% of healthy controls. beyond that serology does not distinguish between hhv-6a and hhv-6b variants and may cross-react with hhv7. the preferred method to diagnose hhv-6 and -7 infection is by quantitative dna amplification assays (deback et al., 2008) . detecting high viral loads in liver specimens or hhv-6 viremia is associated with approximately twofold increased mortality after liver transplantation (pischke et al., 2012) . in immunocompetent patients hhv-6 and -7 cause a benign self-limited infection, which does not require specific antiviral treatment. unlike hhv-6b both hhv-6a and hhv-7 are relatively resistant against ganciclovir, while foscarnet acts against all three viruses (yoshida et al., 1998; de clercq et al., 2001) . cidofovir may be a therapeutic alternative, but some resistant hhv-6 isolates have been identified (bonnafous et al., 2008) . human herpesvirus 8 is a g-herpesvirus, which has potential for malignant transformation. although primary hhv-8 infection can cause rash and fever in children and immunocompromised individuals, the onset of hhv-8-related diseases usually occurs several years after hhv-8 acquisition: kaposi sarcoma, body cavity lymphoma, and multicentric castleman's disease are the typical presentations of hhv-8 infection but bone marrow aplasia and multiple myeloma has also been described in association with hhv-8 infection (lee and henderson, 2001) . in autopsy studies kaposi sarcoma involved the liver in approximately 20% of patients with aids and was usually part of a widespread cutaneous and visceral disease. due to highly active antiretroviral combination therapy, kaposi sarcoma has become a rare complication of hiv infection. however, fulminant hepatic kaposi sarcoma may occur after organ transplantation (cahoon et al., 2018, fig. 7) . macroscopically there are dark-red tumors on the skin, the liver capsule and the parenchyma. under the microscope the typical lesion is a mesh of spindle-cell-like tumor cells and dilated thin-walled vessels (glasgow et al., 1985) . hepatosplenomegaly, fever, and weight loss are typical features of multicentric castleman's disease, a pre-malignant proliferation of b-lymphocytes (fig. 8a ). lymphocytes in multicentric castleman's disease and kaposi sarcoma seem to cooperate with each other, and thus the two hhv-8-related lesions occasionally occur within the same lymphnode (naresh et al., 2008) . ascites and pleural effusions are the hallmark of hhv-8-related body cavity lymphoma possibly giving rise to a false initial diagnosis of decompensated liver cirrhosis. however, abundant lymphoma cells in the aspirated fluids provide a pivotal diagnostic hint (fig. 8b ). hhv-8 can also cause solid organ lymphoma involving the liver (cesarman and knowles, 1999) . the diagnosis of hhv-8 associated malignancies is established from biopsies via their characteristic histopathological features. the virus itself is detected by various antibody assays or polymerase chain reaction (pcr) assays (chiereghin et al., 2017) . reconstitution of immune function is the primary goal for treatment of hhv-8 associated diseases. this can be achieved by highly active antiretroviral therapy in hiv infection and alternatively by antiproliferate m-tor (mammalian target of rapamycin) inhibitors for immune suppression in transplantation (barozzi et al., 2009) . immune stimulation with imiquimod and interferon-alpha (babel et al., 2008; van der ende et al., 2007) has been attempted. oncological therapy with liposomal anthracyclines or paclitaxel in kaposi sarcoma (di trolio et al., 2006; stebbing et al., 2003) , or rituximab in the case of castleman's disease and lymphoma are further potent treatment options (bower et al., 2007) . ganciclovir, cidofovir, foscarnet, adefovir and lobucavir but not acyclovir can block hhv-8 replication in vitro, and in a controlled crossover trial valganciclovir reduced oropharyngeal shedding of hhv-8 by 80% (casper et al., 2008) . this overview addresses the currently most relevant viral infections involving the liver. however, it provides only an outline and is in far not exhaustive. in rare instances, hepatitis occurred in the context of infections with enteroviruses (sun and smith, 1966) , measles (khatib et al., 1993) and rubella viruses (mclellan and gleiner, 1982) as well as parvovirus b19 (yoto et al., 1996; hayakawa et al., 2007) . the reader will find details of these rare infections in microbiology textbooks. beyond that, international travel and global warming are likely to introduce new exotic infections, which must be considered in the differential diagnosis of severe hepatitis. this problem is illustrated by the recent autochthonous crimean-congo hemorrhagic fever (cchf) virus infections in spain (negredo et al., 2017) , which did not occur in this country before. thus, hepatologists must be constantly prepared to face new challenges. epstein-barr-virus-associated hemophagocytic syndrome. a cause of fever of unknown origin in hiv infection severe autoimmune hepatitis triggered by varicella zoster infection histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection-clinicopathological and ultrastructural study development of kaposi's sarcoma under sirolimus-based immunosuppression and successful treatment with imiquimod yellow fever: epidemiology and prevention indirect antitumor effects of mammalian target of rapamycin inhibitors against kaposi sarcoma in transplant patients fatal case of epstein-barr virus-associated lymphoproliferative disorder associated with a human immunodeficiency virus infection hepatobiliary complications in patients with human immunodeficiency virus infection clinical features and short term outcome of 144 patients with sars in the greater toronto area rare tumor entities associated with persistent hhv8 infection. (a) castleman's disease in a lymphnode of hiv-positive patient who developed malaise, fever and cutaneous kaposi sarcoma. castleman's disease is a hhv8-associated pre-malignant lymphoproliferative disorder, which can progress to b-cell lymphoma. (b) tumor cells of hhv8-associated body cavity lymphoma brief communication: rituximab in hiv-associated multicentric castleman disease evaluation of 12 commercial tests for detection of epstein-barr virus-specific and heterophile antibodies risk of kaposi sarcoma after solid organ transplantation in the united states comparison of molecular tests for detection and quantification of cell-associated cytomegalovirus dnd valganciclovir for suppression of human herpesvirus-8 replication: a randomized, double-blind, placebo-controlled, crossover trial the role of kaposi's sarcoma-associated herpesvirus (hshv/hhv8) in lymphoproliferative diseases haemorrhagic fever with renal syndrome involving the liver clinical significance of hepatic derangement in severe acute respiratory syndrome chikungunya outbreaks-the globalization of vectorborne diseases human herpesvirus-6 infection: a prospective study evaluating hhv-6 dna levels in liver from children with acute liver failure multicenter 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virus and herpes simplex virus in various clinical samples by the use of different pcr assays fatal disseminated herpes simplex virus infection in a previously healthy pregnant woman latent species c adenoviruses in human tonsil tissues clinical and pathologic findings of the liver in acquired immune deficiency syndrome (aids) utero epstein-barrvirus (infectious mononucleosis) infection herpes simplex hepatitis in apparently immunocompetent adults classification of epstein-barr virus associated posttransplant lymphoproliferative diseases: implications for understanding their pathogenesis and developing rational treatment strategies human herpesvirus-6 and acute liver failure pre-transplant human herpesvirus 6 infection of patients with acute liver failure is a risk factor for post-transplant human herpesvirus 6 infection of the liver a clinical study of adult human parvovirus b 19 infection prospective, double-blind, concurrent, placebo-controlled clinical trial of intravenous ribavirin therapy of hemorrhagic fever with renal syndrome clinical utility of quantitative cytomegalovirus viral load determination for predicting cytomegalovirus disease in liver transplant recipients human herpesvirus-6 is associated with cytomegalovirus reactivation in liver transplant recipients fatal adenovirus infection in a child with acquired immunodeficiency syndrome hepatitis due to herpes simplex virus in marrow-transplant recipients measles associated hepatobiliary disease: an overview the impact of adenovirus infection on the immunocompromised host disseminated adenovirus infection with hepatic necrosis in patients with human immunodeficiency virus infection and other immunodeficiency states a novel coronavirus in patients with severe acute respiratory syndrome herpes simplex virus hepatitis after solid organ transplantation clinical findings and adverse outcome in neonates with symptomatic congenital cytomegalovirus (sccmv) infection human herpesvirus-6 and -7 infections in children: agents of roseola and other syndromes emerging viral infections a major outbreak of severe acute respiratory syndrome in hong kong resistance of herpes simplex virus infections to nucleoside analogues in hiv-infected patients hantavirus infections in spain: analysis of sera from the general population and from patients with pneumonia, renal disease and hepatitis change over time in incidence of ganciclovir resistance in patients with cytomegalovirus retinitis acute hepatitis in an adult with rubeola fulminant hepatitis in neonates with human herpesvirus 6 infection adenovirus infection in pediatric liver transplant recipients counting antigen-specific cd8 t cells: a reevaluation of bystander activation during viral infection lymphnodes involved by multicentric castleman disease among hiv-positive individuals are often involved by kaposi sarcoma autochthonous crimean-congo hemorrhagic fever in spain emerging viral diseases human herpesvirus 6 infection in adult living related liver transplant recipients the liver in aids disease tool for the diagnosis and care of patients with suspected arboviral diseases. washington dc: pan-american health organisation prevention of cytomegalovirus disease in recipients of solid-organ transplants re-emergence of fatal human influenza a subtype h5n1 disease prophylactic oral acyclovir after renal transplantation five cases of fulminant hepatitis due to herpes simplex virus in adults high intrahepatic hhv-6 virus loads but neither cmv nor ebv are associated with decreased graft survival after diagnosis of graft hepatitis hantavirus infections in europe kupffer cell-dependent hepatitis occurs during influenza infection hhv-6a in syncytial giant-cell hepatitis identification of severe acute respiratory syndrome in canada epstein-barr virus and human disease: immune response determines the clinical and pathological expression infection with chikungunya virus in italy: an outbreak in a temperate region ut. recovery of virus from the liver of children with fatal dengue: reflections on the pathogenesis of the disease and its possible analogy with that of yellow fever a controlled trial comparing foscarnet with vidarabine for acyclovir-resistant mucocutaneous herpes simplex infection in the aids patient cd46 is a cellular receptor for human herpesvirus 6 importance of kupffer cells for t-cell-dependent liver injury in mice who rapid advice guidelines for pharmacological management of sporadic human infection with avian influenza a (h5n1) virus prevention of herpesvirus infections in renal allograft recipient x-linked lymphoproliferative disease: twenty-five years after discovery viruses of the bunya-and togaviridae families: potential as bioterrorism agents and means of control liver disease in cytomegalovirus mononucleosis: a light microscopical and immunoperoxidase study of six cases liver transplantation for fulminant hepatitis due to yellow fever fatal adenovirus hepatic necrosis in severe combined immune deficiency paclitaxel for anthracycline-resistant aids-related kaposi's sarcoma: clinical and angiogenic correlations the spectrum of human herpesvirus 6 infection: from roseola infantum to adult disease hepatitis associated with myocarditis. unusual manifestation of infection with coxsackie virus group b, type 3 reactivation of human herpesvirus 6 by infection of human herpesvirus 7 chikungunya fever in travelers: clinical presentation and course pathology of fatal human infection associated with avian influenza a h5n1 virus acyclovir for treatment of infectious mononucleosis: a metaanalysis valacyclovir for herpes simples virus infection: long-term safety and sustained efficacy after 20 years' experience with acyclovir complete clinical and virological remission of refractory hiv-related kaposi's sarcoma with pegylated interferon alpha genetic divergence and dispersal of yellow fever virus a comparison of the pattern of liver involvement in dengue hemorrhagic fever with classic dengue fever quantitation of epstein-barr virus mrna using reverse transcription and real-time pcr association of lymphomatoid granulomatosis with epstein-barr viral infection of b lymphocytes and response to interferon-alpha 2b fever in returned travelers: results from the geosentinel surveillance network temporal pattern of hepatic dysfunction and disease severity in patients with sars dengue: guidelines for diagnosis, treatment, prevention and control world health organization (2012) handbook for clinical management of dengue recovery from adenovirus pneumonia in a severe combined immunodeficiency patient with intravenous ribavirin comparison of antiviral compounds against human herpesvirus 6 and 7 human parvovirus b19 infection associated with acute hepatitis acute exacerbation of autoimmune liver disease associated with hantaviral infection human infection by avian influenza a h5n1 disseminated herpes simplex type 2 and systemic candida infection in a patient with previous asymptomatic hiv infection histology slides were kindly provided by prof. dr. hans-peter fischer, department of pathology, rheinische friedrich-wilhelms university of bonn, germany. key: cord-267816-84z9fp2u authors: magdi, mohamed; rahil, ali title: severe immune thrombocytopenia complicated by intracerebral haemorrhage associated with coronavirus infection: a case report and literature review date: 2019-07-12 journal: eur j case rep intern med doi: 10.12890/2019_001155 sha: doc_id: 267816 cord_uid: 84z9fp2u immune thrombocytopenic purpura (itp) is an autoimmune disorder that causes isolated thrombocytopenia. many viruses have been identified as triggering the autoimmune process, including hiv, mcv, ebv, parvovirus, rubella and measles. however, itp in association with coronavirus infection has not previously been reported. we describe the case of a healthy man who presented with severe itp complicated by intracranial haemorrhage following upper respiratory tract infection. an infection screen revealed coronavirus infection. learning points: coronavirus can cause severe immune thrombocytopenic purpura (itp). intracerebral haemorrhage is an uncommon presentation of itp. intravenous immunoglobulin and steroids are very effective treatments for severe itp. immune thrombocytopenic purpura (itp) is a rare haematological disorder formerly known as idiopathic thrombocytopenic purpura before the autoimmune mechanism was identified and found to be triggered by many, mostly viral, infectious agents. most cases of itp are benign with only minor mucosal bleeding as the main presentation. we present a case of severe thrombocytopenia complicated by intracranial haemorrhage following infection with a coronavirus, which has not previously been reported. a 24-year-old healthy man presented to the emergency department with prolonged gum bleeding and a 2-day history of a diffuse skin rash on his extremities and abdomen. he was in his usual state of health until 1 week before presentation when he developed fever and a runny nose. he reported no joint pain, oral ulcers or raynaud's phenomenon. he denied intake of any medications. his family history was not contributory. upon arrival at the emergency department, the patient was awake and oriented, with blood pressure 127/72 mmhg, heart rate 90 bpm, temperature 37.3°c and respiratory rate 18 breaths/min. physical examination revealed signs of gum bleeding. examination of the skin was remarkable for petechiae over the chest and limbs. the remainder of the systemic examination was unremarkable. laboratory findings were as follows: haemoglobin: 13 g/dl; wbc: 6.8×103/mm 3 ; platelets: 1,000/µl; ptt: 28.2 sec; pt: 11.8 sec; inr: 1.02. a peripheral blood smear was normal. respiratory viral panel pcr was positive for coronavirus hku1. autoimmune work-up including ena and anca serology was negative. frequent platelet transfusions, intravenous immunoglobulin 1 g/kg for 3 days and intravenous dexamethasone 40 mg for 4 days were initiated. the next day, the patient developed a headache and vomiting, and his level of consciousness deteriorated to a glasgow coma scale score of 10/15. urgent ct of the head showed a large left frontal intracerebral haemorrhage extending into the ventricles with a midline shift of 12 mm. after 2 days, the platelet count rose to 100,000/µl and then 400,000/µl. the patient`s level of conscious and muscle power gradually recovered. after 1 month of rehabilitation, he was discharged in a good state of health. itp is characterized by isolated low levels of circulating platelets (plt <100,000/µl) secondary to autoimmune destruction of platelets or inhibition of synthesis. it varies from mild to severe disease with lethal sequelae. the severe form includes bleeding requiring treatment which usually occurs with a platelet count <20,000/µl. itp may present acutely or chronically; the chronic form is defined as thrombocytopenia of more than 6 months' duration since initial clinical presentation. acute itp is common in children (<10 years) in contrast to the chronic form which is more common in adults. the exact mechanism of itp is poorly understood, with many hypotheses claiming that viral infection triggers the disease after which preformed antibodies cross-react with platelet antigens [1, 2] . clinical presentation varies from the more common petechiae, purpura and mucous membrane bleeding (epistaxis or gum bleeding) to the rare severe gastrointestinal or intracranial bleeding, which has been reported in 1.4% of patients [3] . viruses thought to cause itp include hiv, hcv, cmv, ebv, herpes viruses and vzv [4] [5] [6] . in 1992, wright [7] reported a case of severe thrombocytopenia secondary to asymptomatic cmv infection. in 2004, hamada et al. [8] described a patient with severe thrombocytopenia associated with varicella zoster infection whose platelet count returned to normal after antiviral treatment. interestingly, zea-vera and parra [9] reported a case of itp exacerbation that was secondary to zika virus infection. an association between itp and some bacterial infections such as tuberculosis and helicobacter pylori has been documented [10] . however, a connection between itp and infection with coronavirus, even though the virus is common, has not previously been reported in the literature. infection with coronavirus (cov) has been associated with severe acute respiratory syndrome (sars). haematological changes in patients with sars are common and notably include lymphopenia and thrombocytopenia. the development of thrombocytopenia may involve a number of mechanisms. although the development of autoimmune antibodies or immune complexes triggered by viral infection may play a significant role in inducing thrombocytopenia, sars-cov may also directly infect haematopoietic stem/progenitor cells, megakaryocytes and platelets, inducing their growth inhibition and apoptosis [11] . in contrast, we report severe thrombocytopenia following mild coronavirus upper respiratory tract infection. coronavirus infections were considered benign until the sars outbreak of 2003 when the their virulence attracted increased attention and new group members like cov.hku1 were identified. cov.hku1 was discovered in 2005 in hong kong in an adult with chronic pulmonary disease [12] and is now considered to be associated with acute respiratory infections. coronavirus is not a well-known cause of immune thrombocytopenia even though it may possibly cause severe itp. therefore, a respiratory viral panel test should be used in the initial assessment of a patient with immune thrombocytopenia. characterization of autoantibodies against the platelet glycoprotein antigens iib/iiia in childhood idiopathic thrombocytopenia purpura varicella-associated thrombocytopenia: autoantibodies against platelet surface glycoprotein v severe bleeding events in adults and children with primary immune thrombocytopenia: a systematic review cytomegalovirus can make immune thrombocytopenic purpura refractory viral infection of megakaryocytes in varicella with purpura idiopathic thrombocytopenic purpura after human herpesvirus 6 infection severe thrombocytopenia secondary to asymptomatic cytomegalovirus infection in an immunocompetent host a case of varicella-associated idiopathic thrombocytopenic purpura in adulthood zika virus (zikv) infection related with immune thrombocytopenic purpura (itp) exacerbation and antinuclear antibody positivity effects of eradication of helicobacter pylori infection in patients with immune thrombocytopenic purpura: a systematic review thrombocytopenia in patients with severe acute respiratory syndrome characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia key: cord-353190-7qcoxl81 authors: nicklas, werner; bleich, andré; mähler, michael title: viral infections of laboratory mice date: 2012-05-17 journal: the laboratory mouse doi: 10.1016/b978-0-12-382008-2.00019-2 sha: doc_id: 353190 cord_uid: 7qcoxl81 viral infections of laboratory mice have considerable impact on research results, and prevention of such infections is therefore of crucial importance. this chapter covers infections of mice with the following viruses: herpesviruses, mousepox virus, murine adenoviruses, polyomaviruses, parvoviruses, lactate dehydrogenase-elevating virus, lymphocytic choriomeningitis virus, mammalian orthoreovirus serotype 3, murine hepatitis virus, murine norovirus, murine pneumonia virus, murine rotavirus, sendai virus, and theiler’s murine encephalomyelitis virus. for each virus, there is a description of the agent, epizootiology, clinical symptoms, pathology, methods of diagnosis and control, and its impact on research. in interpreting the microbiological status of laboratory animals, it must be understood that infection and disease are not synonymous. infection refers to the invasion and multiplication of microorganisms in body tissues and may occur with or without apparent disease. disease refers to interruption or deviation from normal structure and function of any tissue, organ or system. many of the infections with which we are concerned may not cause discernable disease in many strains of mice. however, they may cause inapparent or subclinical changes that can interfere with research. such interference often remains undetected, and therefore modified results may be obtained and published. the types of interference of an agent with experimental results may be diverse. there is no doubt that research complications due to overt infectious disease are significant and that animals with clinical signs of disease should not be used for scientific experiments. but clinically inapparent infections may also have severe effects on animal experiments. there are numerous examples of influences of microorganisms on host physiology and hence of the interference of inapparent infections with the results of animal experiments. many microorganisms have the potential to induce activation or suppression of the immune system, or both at the same time but on different parts of the the laboratory mouse ó 2012 elsevier ltd. all rights reserved. isbn 978-0-12-382008-2 doi: 10.1016/b978-0-12-382008-2.00019-2 immune system, regardless of the level of pathogenicity. all infections, apparent or inapparent, are likely to increase interindividual variability and hence result in increased numbers of animals necessary to obtain reliable results. microorganisms, in particular viruses, present in an animal may contaminate biological materials such as sera, cells or tumours [1, 2] . this may interfere with in vitro experiments conducted with such materials and may also lead to contamination of animals [3] . mouse antibody production (map) testing or polymerase chain reaction (pcr) testing of biologics to be inoculated into mice is an important component of a disease prevention programme. finally, latent infections may be activated by environmental factors, by experimental procedures, or by the combination and interaction between various microorganisms. for all these reasons, prevention of infection, not merely prevention of clinical disease, is essential. unfortunately, research complications due to infectious agents are usually considered artefacts and published only exceptionally. information on influences of microorganisms on experiments is scattered in diverse scientific journals, and many articles are difficult to find. to address this problem, several meetings have been held on viral complications on research. the knowledge available is summarized in conference proceedings [4, 5] and has later repeatedly been reviewed [6] [7] [8] . viral infections of mice have been studied in detail, and comprehensive information on their pathogenic potential, their impact on research, and the influence of host factors such as age, genotype, and immune status on the response to infection is available. the nomenclature and taxonomy of viruses is described based on recent nomenclature rules by the international union of microbiological societies [9] and the universal virus database of the international committee on the taxonomy of viruses (http://www. ictvdb.org). retroviruses are not covered in this chapter because they are not included in routine health surveillance programmes and cannot be eradicated with the methods presently available. this is because most of them are incorporated in the mouse genome as proviruses and thus are transmitted via the germline. the ability to accurately determine whether or not laboratory animals or animal populations have been infected with a virus depends on the specificity and sensitivity of the detection methods used. most viral infections in immunocompetent mice are acute or short term, and lesions are often subtle or subclinical. the absence of clinical disease and pathological changes has therefore only limited diagnostic value. however, clinical signs, altered behaviour or lesions may be the first indicator of an infection and often provide clues for further investigations. serology is the primary means of testing mouse colonies for exposure to viruses, largely because serological tests are sensitive and specific, are relatively inexpensive and allow screening for a multitude of agents with one serum sample. they are also employed to monitor biological materials for viral contamination using the map test. serological tests detect specific antibodies, usually immunoglobulin g (igg), produced by the host against the virus and do not actually test for the presence of the virus. an animal may have been infected, mounted an effective antibody response and cleared the virus, but remains seropositive for weeks or months or for ever, even though it is no longer infected or shedding the agent. active infection can only be detected by using direct detection methods such as virus isolation, electron microscopy or pcr. meanwhile, pcr assays have been established for the detection of almost every agent of interest. they are highly sensitive and, depending on the demands, they can be designed to broadly detect all members of a genus or only one species. however, good timing and selection of the appropriate specimen is critical for establishing the diagnosis. in practice, combinations of diagnostic tests are often necessary, including the use of sentinel animals or immunosuppression to get clear aetiological results or to avoid consequences from false-positive results. reports on the prevalence of viral infections in laboratory mice throughout the world have been published frequently. in general, the microbiological quality of laboratory mice has constantly improved during the last decades, and several agents (e.g. herpesviruses and polyomaviruses) have been essentially eliminated from contemporary colonies due to advances in diagnostic methodologies and modern husbandry and rederivation practices [10] [11] [12] [13] [14] [15] . they may, however, reappear, since most have been retained or are still being used experimentally. furthermore, the general trend towards better microbiological quality is challenged by the increasing reliance of biomedical research on genetically modified and immunodeficient mice, whose responses to infection and disease can be unpredictable. increasing numbers of scientists are creating genetically modified mice, with minimal or no awareness of infectious disease issues. as a consequence, these animals are more frequently infected than 'standard' strains of mice coming from commercial breeders, and available information on their health status is often insufficient. frequently they are exchanged between laboratories, which amplifies the risk of introducing infections from a range of animal facilities. breeding cessation strategies that have been reported to eliminate viruses from immunocompetent mouse colonies may prove to be costly and ineffective in genetically modified colonies of uncertain or incompetent immune status. it must also be expected that new agents will be detected, although only occasionally. infections therefore remain a threat to biomedical research, and users of laboratory mice must be cognizant of infectious agents and the complications they can cause. two members of the family herpesviridae can cause natural infections in mice (mus musculus). mouse cytomegalovirus 1 (mcmv-1) or murid herpesvirus 1 (muhv-1) belongs to the subfamily betaherpesvirinae, genus muromegalovirus. murid herpesvirus 3 (muhv-3) or mouse thymic virus (mtv) has not yet been assigned to a genus within the family herpesviridae. both are enveloped, double-stranded dna viruses that are highly host-specific and relatively unstable to environmental conditions such as heat and acidic ph. both agents are antigenically distinct and do not cross-react in serological tests, but their epidemiology is similar [16] . mcmv-1 is very uncommon in european and american colonies of laboratory mice and is found at a very low rate [11] or reported as not found [14, 15] . seropositivity has, however, been reported from asian countries [17, 18] . testing for mtv is not frequently reported, and no sample tested positive in recent studies [11] . the data available suggest that the prevalence of both viruses in contemporary colonies and thus their importance for laboratory mice is negligible. however, both mcmv-1 and mtv are frequently found in wild mice, which may be coinfected with both viruses [8, [19] [20] [21] . mouse cytomegalovirus 1 (mcmv-1) or murid herpesvirus 1 (muhv-1) natural infection with mcmv-1 causes subclinical salivary gland infection in mice. like other cytomegaloviruses, mcmv-1 is strictly hostspecific. it persists in the salivary glands (particularly in the submaxillary glands) and also in other organs [22] [23] [24] . the virus can be cultured in mouse fibroblast lines like 3t3 cells, but primary mouse embryo fibroblasts are more sensitive to infection and produce higher virus titres. however, passage in cell culture results in its attenuation. to maintain virulence, the virus is best propagated by salivary gland passages of sublethal virus doses in weanling mice of a susceptible strain (e.g. balb/c) [25] . most information concerning the pathogenesis of mcmv-1 infection is based on experimental infection studies. these results are very difficult to summarize because the outcome of experimental infection in laboratory mice depends on various factors such as mouse strain and age, virus strain and passage history [26] , virus dose and route of inoculation [24] . in general, newborn mice are most susceptible to clinical disease and to lethal infection and develop higher levels of resistance with increasing age. infection of neonates leads to abnormal brain development [27, 28] . virus replication is observed in newborn mice in many tissues and appears in the salivary glands towards the end of the first week of infection when virus concentrations in liver and spleen have already declined. resistance develops rapidly after weaning between days 21 and 28 of age. experimental infection of adult mice results in mortality only in susceptible strains and only if high doses are administered. not even intravenous or intraperitoneal injections of adult mice usually produce signs of illness in resistant strains [29] . mice of the h-2 b (e.g. c57bl/6) and h-2 d (e.g. balb/c) haplotype are more sensitive to experimental infection than are mice of the h-2 k haplotype (e.g. c3h), which are approximately 10-fold more resistant to mortality than those of the b or d haplotype [24] . subclinical or latent infections can be activated by immunosuppression (e.g. with cyclophosphamide or cortisone) or critical illness such as sepsis [30] . reactivation of mcmv-1 also occurs after implantation of latently infected salivary glands into prkdc scid mice [31] . immunodeficient mice lacking functional t cells or natural killer (nk) cells, such as foxn1 nu and lyst bg mice are more susceptible than are immunocompetent animals. experimental infection in prkdc scid mice causes severe disease or is lethal, with necrosis in spleen, liver and other organs, and multinucleate syncythia with inclusion bodies in the liver [32] . similarly to aids patients infected with human cytomegalovirus, athymic foxn1 nu mice experimentally infected with mcmv-1 also develop adrenal necrosis [33] . the virus also replicates in the lungs, leading to pneumonitis, whereas replication and disease are not seen in heterozygous (foxn1 nu/þ ) littermates [34] . the pathogenesis of mcmv-1 infection in immunocompetent and in immunocompromised mice, as well as the role of the immune system, have been reviewed by krmpotic et al. [35] . the most prominent histological finding of cytomegaloviruses is enlarged cells (cytomegaly) of salivary gland epithelium with eosinophilic nuclear and cytoplasmic inclusion bodies. the inclusion bodies contain viral material and are found also in other organs such as liver, spleen, ovary and pancreas [24] . depending on inoculation route, dose, strain, and age of mice, experimental infections may result in inflammation or cytomegaly with inclusion bodies in a variety of tissues, pneumonitis, myocarditis, meningoencephalitis or splenic necrosis in susceptible strains [8, 24, 36] . virus is transmitted by the oronasal route, by direct contact and is excreted in saliva, tears and urine for several months. the virus is ubiquitous in wild house mice worldwide. they serve as a natural reservoir for infection and can even be infected with different virus strains [37] . the virus is most frequently transmitted horizontally through mouse-to-mouse contact but does not easily spread between cages. sexual transmission and transmission with tissues or organs is also possible. the virus does not cross the placenta in immunocompetent mice, although infection of pregnant females results in fetal death or resorption and wasting of borne pups. however, fetal infection is possible by direct injection of mcmv-1 into the placenta [38] and also occurs by transplacental transmission in mice with severe immunodeficiency [39] . vertical transmission is also possible by milk during lactation [40] . it is generally assumed that mcmv-1 has a very low prevalence in contemporary colonies of laboratory mice. the risk of introduction into facilities housing laboratory mice is very low if wild mice are strictly excluded. monitoring is necessary if populations of laboratory mice may have been contaminated by contact with wild mice. as for other viruses, different serological tests, including multiplex fluorescent immunoassay (mfia) [41] , are used for health surveillance of rodent colonies. as the virus persists, direct demonstration of mcmv-1 in infected mice is possible by pcr [42] [43] [44] or by virus isolation using mouse embryo fibroblasts (3t3 cells). although mcmv-1 does not play a significant role as a natural pathogen of laboratory mice, it is frequently used as a model for human cytomegalovirus infection [45] . these aspects have been discussed in detail by shellam et al. [24] . mcmv-1 has also been used as a vaccine vector aiming at a disseminating mouse control agent by inducing immunocontraception in mice [46] . the virus is known to influence immune reactions in infected mice [47, 48] and may therefore have an impact on immunological research [6, 8] . mtv was detected during studies in which samples from mice were passaged in newborn mice. unlike other herpesviruses, mtv is difficult to culture in vitro and is usually propagated by intraperitoneal infection of newborn mice. the thymus is removed 7-10 days later, and thymus suspensions serve as virus material for further studies. the prevalence of mtv is believed to be low in laboratory mice, and for this reason, and also due to the difficulties in virus production for serological assays, it is not included in many standard diagnostic or surveillance testing protocols. limited data are available indicating that it is common in wild mice [8, 49] . further, mtv obviously represents a significant source of contamination of mcmv-1 (and vice versa) if virus is prepared from salivary glands, since both viruses cause chronic or persistent salivary gland infections and can coinfect the same host. all mouse strains are susceptible to infection, but natural or experimental infection of adult mice is subclinical. gross lesions appear only in the thymus and only if experimental infection occurs at an age of less than about 5 days. infection results in nuclear inclusions in thymocytes and their almost complete destruction within 2 weeks. virus is present in the thymus but may also be found in the blood and in salivary glands of surviving animals. salivary glands are the only site yielding positive virus isolations if animals are infected as adults. the virus persists here and is shed via saliva for months. mtv also establishes a persistent infection in athymic foxn1 nu mice, but virus shedding is reduced compared to euthymic mice, with virus recovery possible only in a lower percentage of mice [50] . pathological changes caused by mtv occur in the thymus, and reduced thymus mass due to necrosis in suckling mice is the most characteristic gross lesion [36] . lymphoid necrosis may also occur in lymph nodes and spleen [51] , with necrosis and recovery similar to that in the thymus. in mice infected during the first 3 days after birth, necrosis of thymus becomes evident within 3-5 days, and the animals' size and weight are markedly reduced at day 12-14. intranuclear inclusions may be present in thymocytes between days 10-14 after infection. the thymus and the affected peripheral tissues regenerate within 8 weeks after infection. regardless of the age of mice at infection, a persistent infection is established in the salivary glands, and infected animals shed virus for life. several alterations of immune responses are associated with neonatal mtv infection. there is transient immunosuppression, attributable to lytic infection of t lymphocytes, but activity (e.g. response of spleen cells to t-cell mitogens) returns to normal as the histological repair progresses [51] . selective depletion of cd4þ t cells by mtv results in autoimmune disease [52, 53] . information about additional influences on the immune system is given in textbooks [6, 8] . in experimentally infected newborn mice, oral and intraperitoneal infections similarly result in thymus necrosis, seroconversion and virus shedding, suggesting that the oral-nasal route is likely to be involved in natural transmission [54] . the virus spreads to cagemates after long periods of contact. it is transmitted between mice kept in close contact, and transmissibility from cage to cage seems to be low. mtv is not transmitted to fetuses by the transplacental route, and intravenous infection of pregnant mice does not lead to congenital damage, impairment in size or development, or abortion [55] . mtv and mcmv-1 do not cross-react serologically [16] . serological monitoring of mouse populations for antibodies to mtv is possible by indirect immunofluorescent assay (ifa) testing, which is commercially available; enzyme-linked immunosorbent assays (elisa) tests have also been established [41, 56] . elisa and complement fixation yield similar results [57] . serological tests based on recombinant proteins and direct detection of virus by pcr are currently not possible because the genome of the virus has not yet been sequenced. it must be noted that the immune response depends on the age at infection. antibody responses are not detectable in mice infected as newborns, whereas adult mice develop high titres that are detectable by serological testing. if neonatal infection is suspected, homogenates of salivary glands or other materials can be inoculated into pathogen-free newborn mice followed by gross and histological examination of thymus, lymph nodes and spleens for lymphoid necrosis [49] . alternatives to the in vivo infectivity assay for detecting mtv in infected tissues include a competition elisa [58] and map testing, although this is slightly less sensitive than infectivity assays [59] . there is very little experience of eradication methods for mtv because of its low prevalence in contemporary mouse colonies. methods that eliminate other herpesviruses will likely eliminate mtv. procurement of animals of known negative mtv status is an appropriate strategy to prevent infection. strict separation of laboratory mice from wild rodents is essential to avoid introduction of the virus into laboratory animal facilities. murid herpesvirus 2 (muhv-2) or rat cytomegalovirus infects rats and is also a member of the genus muromegalovirus. murid herpesvirus 4 (muhv-4) is a member of the genus rhadinovirus in the subfamily gammaherpesvirinae and is also known as mouse herpesvirus strain 68 (mhv-68). other murid herpesviruses are not yet assigned to a subfamily within the family herpesviridae. among these is murid herpesvirus 3 (mouse thymic herpesvirus), but also murid herpesvirus 5 (field mouse herpesvirus) which infects voles (microtus pennsylvanicus), murid herpesvirus 6 (sand rat nuclear inclusion agent), and murid herpesvirus 7 [60] . furthermore, a gammaherpesvirus of house mice (mus musculus) has been described recently which is clearly distinct from mhv-68 [61] . experimental infection of laboratory mice with mhv-68 is a frequently used model system for the study of human gammaherpesvirus pathogenesis, e.g. of kaposi's sarcoma-associated herpesvirus or epstein-barr virus (ebv) [62, 63] which are members of the same subfamily. they are also important models to study viral latency and immune mechanisms controlling latency [64] [65] [66] . mus musculus is not the natural host for this virus; it was first isolated in slovakia from bank voles (myodes glareolus). additional closely related strains (mhv-60, mhv-72) exist from the same host species, and similar strains (mhv-76, mhv-78) were isolated from wood mice (apodemus flavicollis and apodemus sylvaticus). apodemus sp. seem to be the major hosts for mhv-68 in great britain [67] . different virus strains exhibit different genetic and biological properties and also differ in their pathogenicity, e.g. for prkdc scid mice [68] . infections in laboratory mice take the same course as in their natural hosts [69] . there are, however, some differences as, e.g. higher virus levels are reached in the lungs of balb/c mice, and wood mice develop higher titres of neutralizing antibodies [70] . house mice develop an acute infection in the lungs after intranasal infection. a latent infection develops within 2 weeks and the virus persists lifelong in epithelial cells in the lungs and also spreads to the spleen and other organs (e.g. bone marrow, peritoneal cells) where it persists in different cells of the immune system. it behaves like a natural pathogen in inbred strains of mice and persists without causing disease. the mousepox (ectromelia) virus (ectv) is a member of the genus orthopoxvirus belonging to the family poxviridae. it is antigenically and morphologically very similar to vaccinia virus and other orthopoxviruses. poxviruses are the largest and most complex of all viruses, with a diameter of 200 nm and a length of 250-300 nm. mousepox (ectromelia) virus contains one molecule of double-stranded dna with a total genome length of nearly 210 000 nucleotides [71] . it is the causative agent of mousepox, a generalized disease in mice. experimental transmission to young rats (up to 30 days of age) is possible [72] . unlike various other orthopoxviruses, ectromelia virus does not infect humans [73] . the virus is resistant to desiccation, dry heat and many disinfectants. it is not consistently inactivated in serum heated for 30 min at 56 c [3, 74] and remains active for months when maintained at 4 c in fetal bovine serum [75] . effective disinfectants include vapour-phase formaldehyde, sodium hypochlorite and iodophores [8, 76] . historically, ectv has been an extremely important natural pathogen of laboratory mice. the virus was widespread in mouse colonies worldwide and can still be found in several countries. between 1950 and 1980 almost 40 individual ectromelia outbreaks were reported in the usa. the last major epizootic in the usa occurred in 1979-80 and has been described in great detail [77] . severe outbreaks were also described in various european countries [78] [79] [80] . a more recent outbreak in the usa, which resulted in the eradication of almost 5000 mice in one institution, was described by dick et al. [81] . another recent and well-documented case of mousepox was published by lipman et al. [3] . few additional but unpublished cases of ectromelia have been observed since then; the latest report of an outbreak was published in 2009 [74] . in general, positive serological reactions are occasionally reported from routine health surveillance studies [17] but the virus is extremely rare in european and american colonies of laboratory mice [13] [14] [15] . natural infections manifest differently, depending on many factors. mousepox may occur as a rapidly spreading outbreak with acute disease and deaths, or may be inconspicuous with slow spreading and mild clinical signs and may therefore be very difficult to diagnose [81] . the mortality rate can be very low in populations in which the virus has been present for long periods. the infection usually takes one of three clinical courses: acute asymptomatic infection, acute lethal infection (systemic form) or subacute to chronic infection (cutaneous form) [8, [81] [82] [83] . the systemic or visceral form is characterized clinically by facial oedema, conjunctivitis, multisystemic necrosis and usually high mortality. this form is less contagious than the cutaneous form because the animals die before there is virus shedding. the cutaneous form is characterized by typical dermal lesions and variable mortality. the outcome of infection depends on many factors including strain and dose of virus; route of viral entry; strain, age, and sex of mouse; husbandry methods and duration of infection in the colony. while all mouse strains seem to be susceptible to infection with ectv, clinical signs and mortality are strain-dependent [84] [85] [86] . acute lethal (systemic) infection occurs in highly susceptible inbred strains such as dba/1, dba/2, balb/c, a and c3h/hej. immunodeficient mice may also be very susceptible [87] . outbreaks among susceptible mice can be explosive, with variable morbidity and high mortality (>80%). clinical disease may not be evident in resistant strains such as c57bl/6 and akr, and the virus can be endemic in a population for long periods before being recognized. furthermore, females seem to be more resistant to disease than males, at least in certain strains of mice [84, 85] . killer cells are necessary to control mousepox infections [88] . mice that are resistant to mousepox may lose their resistance with increasing age, most likely due to the decreased number and activity of nk cells [89] . the mechanisms determining resistance versus susceptibility are not fully understood but appear to reflect the action of multiple genes. the genetic loci considered to be important include h2d b (termed rmp3, resistance to mousepox), on chromosome 17 [90] ; the c5 genes (rmp2, on chromosome 2); rmp1, localized to a region on chromosome 6 encoding the nk cell receptor nkr-p1 alloantigens [91] ; the nitric oxide synthase 2 locus on chromosome 11 [92] ; and the signal transducer and activator of transcription 6 locus on chromosome 10 [93] . mousepox infections are controlled for several days during the initial course of infection by the complement system until the adaptive immune system can react. loss of the complement system results in lethal infection [94] . clearance of the virus by the immune system is dependent upon the effector functions of cd8þ t cells while nk cells, cd4þ t cells and macrophages are necessary for the generation of an optimal response [95, 96] . t-and b-cell interactions and antibodies play a central role during recovery from a secondary infection [97] . mousepox (ectromelia) virus usually enters the host through the skin with local replication and extension to regional lymph nodes [8, 82, 86] . it escapes into the blood (primary viraemia) and infects splenic and hepatic macrophages, resulting in necrosis of these organs and a massive secondary viraemia. this sequence takes approximately 1 week. many animals die at the end of this stage without premonitory signs of illness; others develop varying clinical signs including ruffled fur, hunched posture, swelling of the face or extremities, conjunctivitis and skin lesions (papules, erosions or encrustations mainly on ears, feet and tail; figure 3 .2.1). necrotic amputation of limbs and tails can sometimes be seen in mice that survive the acute phase, hence the common gross lesions of acute mousepox include enlarged lymph nodes, peyer's patches, spleen and liver; multifocal to semiconfluent white foci of necrosis in the spleen and liver; and haemorrhage into the small intestinal lumen [36, 81, 86, 87] . in animals that survive, necrosis and scarring of the spleen can produce a mosaic pattern of white and red-brown areas that is a striking gross finding. the most consistent histological lesions of acute mousepox are necroses of the spleen (figure 3.2. 3), lymph nodes, peyer's patches, thymus and liver [3, 36, 81, 86, 87] . occasionally, necrosis may also be observed in other organs such as ovaries, uterus, vagina, intestine and lungs. the primary skin lesion, which occurs about a week after exposure at the site of inoculation (frequently on the head), is a localized swelling that enlarges from inflammatory oedema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop 2-3 days later as the result of viraemia ( natural transmission of ectv mainly occurs by direct contact and fomites [8, 82, 98, 99] . the primary route of infection is through skin abrasions. faecal-oral and aerosol routes may also be involved [98] . in addition, the common practice of cannibalism by mice may contribute to the oral route of infection [99] . intrauterine transmission is possible at least under experimental conditions [100] . virus particles are shed from infected mice (mainly via scabs and/or faeces) for about 3-4 weeks, even though the virus can persist for months in the spleen of an occasional mouse [8, 99] . cage-to-cage transmission of ectv and transmission between rooms or units is usually low and largely depends on husbandry practices (e.g. mixing mice from different cages). importantly, the virus may not be transmitted effectively to sentinel mice exposed to dirty bedding [3] . various tests have been applied for the diagnosis of ectromelia. previous epidemics were difficult to deal with because of limited published data and information on the biology of the virus and the lack of specific and sensitive assays [101] . in the 1950s, diagnosis relied on clinical signs, histopathology and animal passages of tissues from moribund and dead animals. culture of the virus on the chorioallantoic membrane of embryonated eggs was also used. serology is currently the primary means of routine health surveillance for testing mouse colonies for exposure to ectv. the methods of choice are mfia, elisa and ifa; they are more sensitive and specific than the previously used haemagglutination inhibition (hi) assay [41, 102, 103] . serological tests based on virus particles detect antibodies to orthopoxviruses and do not distinguish between ectv and vaccinia virus or other orthopoxviruses, respectively. vaccinia virus is commonly used as an antigen for serological testing to avoid the risk of infection for mice. thus, false-positive serological reactions may be found after experimental administration of replication-competent vaccinia virus. it has been shown that even cage contact sentinels may develop antibodies, and vaccinia virus leading to seroconversion may even be transmitted by dirty bedding [104] . confirmation of positive serological results is important before action is taken because vaccinia virus is increasingly prevalent in animal facilities as a research tool (e.g. for vaccination or gene therapy). as observed in different outbreaks, serological testing is of little value in the initial stages of the disease. for example, in the outbreak described by dick et al. [81] depopulation was nearly completed before serological confirmation was possible. for this reason, negative serological results should be confirmed by direct detection methods (pcr, immunohistochemistry, virus isolation) or by histopathology, especially when clinical signs suggestive of mousepox are observed. pcr assays to detect different genes of poxviruses in infected tissues have been used [3, 81, 105] . other pcr tests which were developed to detect smallpox virus have also been shown to detect ectromelia virus and can be used as well [106, 107] . the key to prevention and control of mousepox is early detection of infected mice and contaminated biological materials. all institutions that must introduce mice from other than commercial barrier facilities should have a health surveillance programme and test incoming mice. perhaps even more important than living animals are samples from mice (tumours, sera, tissues). the virus replicates in lymphoma and hybridoma cell lines [108] , and such cells or material derived from them may therefore be a vehicle for inadvertent transfer between laboratories. the last three published outbreaks of ectromelia were introduced into the facilities by mouse serum [3, 74, 81] . lipman et al. [3] found that the contaminated serum originated from a pooled lot of 43 l that had been imported from china, but in both other cases, serum was obtained from animals in the usa. because mouse serum is commonly sold to the end user in small aliquots (a few millilitres), it has to be expected that aliquots of the contaminated lot may still be stored in freezers. these published cases of ectromelia outbreaks provide excellent examples of why testing should be performed on all biological materials to be inoculated into mice. in the case of ectromelia virus it was shown that pcr is more sensitive, and map testing failed to detect contamination [74] . eradication of mousepox has usually been accomplished by elimination of the affected colonies, disinfection of rooms and equipment, and disposal of all infected tissues and sera. while culling of entire mouse colonies is the safest method for eradication of mousepox, it is not a satisfactory method because of the uniqueness of numerous lines of genetically modified animals housed in many facilities. several studies indicate that mousepox is not highly contagious [75, 84, 99] and that it may be self-limiting when adequate husbandry methods are applied. therefore, strict quarantine procedures along with cessation of breeding (to permit resolution of infection) and frequent monitoring, with removal of clinically sick and seropositive animals, are a potential alternative. the period from the last births until the first matings after cessation of breeding should be at least 6 weeks [99] . sequential testing of immunocompetent contact sentinels for seroconversion should be employed with this option. in the past, immunization with live vaccinia virus was used to suppress clinical expression of mousepox. vaccination may substantially reduce the mortality rate, but it does not prevent virus transmission or eradicate the agent from a population [109, 110] . after vaccination, typical pocks develop at the vaccination site, and infectious vaccinia virus is detectable in spleen, liver, lungs and thymus [111] . vaccination also causes seroconversion so that serological tests are not applicable for health surveillance in vaccinated populations. it is therefore more prudent to control mousepox by quarantine and serological surveillance than by relying on vaccination. mortality and clinical disease are the major factors by which ectv interferes with research. severe disruption of research can also occur when drastic measures are taken to control the infection. the loss of time, animals and financial resources can be substantial. experimental mousepox infections are frequently used as a model to study various aspects of smallpox infections of humans [112] [113] [114] . mousepox shares many aspects of virus biology and pathology, and models the course of human smallpox. experimental mousepox infections are used to study vaccination procedures [115, 116] or anti-poxvirus therapies [117] . murine adenoviruses (madv) are non-enveloped, double-stranded dna viruses of the family adenoviridae. two distinct strains have been isolated from mice. the fl strain (madv-1) was first isolated in the usa as a contaminant of a friend leukaemia [118] and has been classified as a member of the genus mastadenovirus. the k87 strain (madv-2) was isolated in japan from the faeces of a healthy mouse [119] and has not yet been assigned to a genus. both strains are considered to represent different species [120] [121] [122] . they are host species specific and are not infectious for infant rats [123] . madv-1 can be cultured in vitro in mouse fibroblasts (e.g. 3t6 or l929 cells), madv-2 is usually cultured in vitro in a mouse rectum carcinoma cell line (cmt-93). in laboratory mice, seropositivity to adenoviruses was reported to be very low [11, 14, 15, 17, 18, 124] or negative [12, 13] . antibodies to madv are also found in wild mice [21, 125] and in rats [21, 126] . neither virus is known to cause clinical disease in naturally infected, immunocompetent mice. however, madv-1 can cause a fatal systemic disease in suckling mice after experimental inoculation [118, 127, 128] . disease is characterized by scruffiness, lethargy, stunted growth and often death within 10 days. experimental infection of adult mice with madv-1 is most often subclinical and persistent but can cause fatal haemorrhagic encephalomyelitis with neurological symptoms, including tremors, seizures, ataxia and paralysis in susceptible c57bl/6 and dba/2j mice [129] . balb/c mice are relatively resistant to this condition. athymic foxn1 nu mice experimentally infected with madv-1 develop a lethal wasting disease [130] . similarly, prkdc scid mice succumb to experimental infection with madv-1 [131] . gross lesions in response to natural madv infections are not detectable. occasional lesions observed after experimental infection with madv-1 include small surface haemorrhages in the brain and spinal cord of c57bl/6 and dba/2j mice [129] , duodenal haemorrhage in foxn1 nu mice [130] and pale yellow livers in prkdc scid mice [131] . histologically, experimental madv-1 infection of suckling mice is characterized by multifocal necrosis and large basophilic intranuclear inclusion bodies in liver, adrenal gland, heart, kidney, salivary glands, spleen, brain, pancreas and brown fat [8, 36, 127, 132] . in experimentally induced haemorrhagic encephalomyelitis, multifocal petechial haemorrhages occur throughout the brain and spinal cord, predominantly in the white matter, and are attributed to infection and damage to the vascular epithelium of the central nervous system (cns) [129] . histopathological manifestations in madv-1-infected prkdc scid mice are marked by microvesicular fatty degeneration of hepatocytes [131] . in contrast to madv-1, the tissue tropism of madv-2 is limited to the intestinal epithelium. naturally or experimentally infected mice develop intranuclear inclusions in enterocytes, especially in the ileum and caecum [8, 36, 133] . transmission of madv primarily occurs by ingestion. madv-1 is excreted in the urine and may be shed for up to 2 years [134] . madv-2 infects the intestinal tract and is shed in faeces for only a few weeks in immunocompetent mice [135] ; immunodeficient mice may shed the virus for longer periods [136] . murine adenovirus infections are routinely diagnosed by serological tests. however, there is a one-sided cross-reactivity of madv-1 with madv-2 [137] . serum from mice experimentally infected with madv-1 yielded positive reactions in serological tests with both viruses, while serum from mice infected with madv-2 reacted only with the homologous antigen [138] . smith et al. [126] reported that sera might react with madv-1 or madv-2 or both antigens. occasional reports of mice with lesions suggestive of adenovirus infections and negative serology (with madv-1) indicate that the infection may not be detected if only one virus is used as an antigen [139] . it is therefore usual to test sera for antibodies to both madv-1 and madv-2. the commonly used methods are ifa, elisa and mfia. the low prevalence in colonies of laboratory mice indicates that madv can easily be eliminated (e.g. by hysterectomy derivation or embryo transfer) and that barrier maintenance has been very effective in preventing infection. the low pathogenicity and the low prevalence in contemporary mouse populations are the main reasons why adenoviruses are considered to be of little importance, which is also indicated by the fact that recent publications about murine adenoviruses are very rare. however, the viruses might easily be spread by the exchange of genetically modified mice and therefore re-emerge. only a few influences on research attributable to madv have been published. for example, it has been shown that madv-1 significantly aggravates the clinical course of scrapie disease in mice [140] . natural infections with madv could also interfere with studies using adenovirus as a gene vector. a novel murine adenovirus classified as a mastadenovirus has recently been isolated from a striped field mouse (apodemus agrarius) [141] . it was cultured in vero e6 cells and named madv type 3 (madv-3). it revealed the highest similarity to madv-1 but it represents a separate serotype. however, there is some cross-reactivity between madv-3 and both other mouse viruses [142] . in addition to serological and antigenic differences it also shows a unique organotropism and infects predominantly the heart tissue of c57bl/6n mice after experimental infection. experimentally infected mice show no clinical signs. the virus is not easily transmitted from experimentally infected mice to contact sentinels [142] . polyomaviridae are enveloped, double-stranded dna viruses. two different agents of this family exclusively infect mice (mus musculus), and both belong to the genus polyomavirus. murine pneumotropic virus (mptv) was formerly known as 'newborn mouse pneumonitis virus' or 'k virus' (named after l. kilham who first described the virus). the second is murine polyomavirus (mpyv). both are related, but antigenically distinct, from each other [143] , and also viruslike particles from the major capsid protein (vp1) do not cross-react [144] . they are enzootic in many populations of wild mice but are very uncommon in laboratory mice. even older reports indicate that both have been eradicated from the vast majority of contemporary mouse colonies, and their importance is negligible [8] . seropositivity to these viruses was not reported in a recent survey conducted in the usa [13] , and other publications also indicate that these viruses do not presently play a significant role in laboratory mice [11, 14, 15] . because of their low prevalence, neither virus is included in the list of agents for which testing is recommended on a regular basis by felasa [145] . although polyomavirus genes, especially those of sv40, are widely used in gene constructs for insertional mutagenesis, very few reports have been published on spontaneous or experimental disease due to mpyv or mptv in the last 10-15 years. the reader is therefore referred to previous review articles for details [8, 146] . natural infections with mptv are subclinical. the prevalence of infection is usually low in an infected population. the virus may persist in infected animals for months and perhaps for life depending on the age at infection and is reactivated under conditions of immunosuppression. virus replicates primarily in endothelial cells, but renal tubular epithelial cells are the major site of viral persistence [147, 148] . clinical signs are observed only after infection of infant mice less than 6-8 days of age. infected pups suddenly develop respiratory symptoms after an incubation period of approximately 1 week, and many die within a few hours of onset of symptoms with an interstitial pneumonia caused by productive infection of and damage to pulmonary endothelium (figure 3.2.5). endothelial cells in other organs are also involved in virus replication [148, 149] (figure 3 .2.6). in older suckling mice, mptv produces a more protracted infection, and the virus or viral antigen can be detected for as long as 4 months. in adult animals, the virus produces a transient asymptomatic infection. even in immunodeficient foxn1 nu mice, experimental infection of adults is clinically asymptomatic, although virus is detectable for a period of several months [150] . in vitro cultivation of mptv is difficult. no susceptible permanent cell line is known to support growth. it can be cultured in primary mouse embryonic cells, but viral titres are not sufficient for use in serological assays [151] . for this reason, the hi test using homogenates of livers and lungs of infected newborn mice is still frequently used, but ifa and elisa tests are also available [152] . furthermore, a pcr test for demonstration of mptv in biological samples has also been published [153] . mpyv was first detected as a contaminant of murine leukaemia virus (mulv) when sarcomas developed in mice after experimental inoculation of contaminated samples. it has later been shown to be a frequent contaminant of transplantable tumours [1] . natural infection of mice is subclinical, and gross lesions including tumours are usually not found. tumour formation occurs when mice are experimentally infected at a young age or when inoculated with high virus doses. development of tumours may be preceded by multifocal necrosis and mortality during the viraemic stage [36] . parotid, salivary gland and mammary tumours are common, and sarcomas or carcinomas of kidney, subcutis, adrenal glands, bone, cartilage, teeth, blood vessels and thyroid also occur. virus strains vary with regard to the tumour types or lesions that they induce, and mouse strains vary in their susceptibility to different tumour types. those of c57bl and c57br/cd lineage are considered to be the most resistant strains; athymic foxn1 nu mice are considered to be most susceptible; c3h mice are particularly susceptible to adrenal tumours and a mice tend to develop bone tumours. immunosuppression or inoculation into immunodeficient strains (e.g. foxn1 nu ) also supports the growth of tumours. on the other hand, experimental infection of adult immunocompetent mice does not result in tumour formation because the immune response suppresses tumour growth, and newborn immunocompetent mice develop runting only if inoculated with high virus doses [154] . after experimental intranasal infection, mpyv initially infects the respiratory tract followed by a systemic phase in which liver, spleen, kidney and colon become infected [155] . the virus is shed in faeces and in all body fluids, and transmission occurs rapidly by direct contact between animals, but also between cages in a room. further, intrauterine transmission has been documented after experimental infection [156] . mpyv persists in all organs in prkdc scid mice while viral dna is detectable in immunocompetent mice after experimental infection for only a limited period of about 4 weeks [157] . however, virus may persist and can be reactivated by prolonged immunosuppression [158] or during pregnancy, at least in young mice [159] . it has been shown that interferon-gamma is an important factor of the host defence against tumour formation and mpyv infection [160] . biological materials of mouse origin are likely to be the most common source of contamination of laboratory mice, emphasizing the importance of map or pcr screening of biological materials to be inoculated into mice. the most frequently used tests for health surveillance of mouse colonies are elisa, mfia and ifa; in addition, the hi test is still used. latent infections can be detected by intracerebral inoculation of neonate mice or by map testing, but direct demonstration of virus in biological samples is also possible by pcr testing [153] . while mpyv infections are of low importance for laboratory animal medicine, the virus is used in models of persistent virus infection [161, 162] . virus-like particles from both murine polyomaviruses have been used as a vector for gene therapy or vaccines [163, 164] . parvoviruses are non-enveloped small viruses (approximately 20 nm in diameter) with a singlestranded dna genome of approximately 5000 nucleotides. murine parvoviruses are members of the family parvoviridae, genus parvovirus. they are remarkably resistant to environmental conditions like heat, desiccation, acidic and basic ph-values. up to date, two distinct species that infect laboratory mice are officially listed: the minute virus of mice (mvm), previously named mice minute virus (mmv), and the mouse parvovirus (mpv). non-structural proteins (ns-1 and ns-2) are highly conserved among both viruses whereas the capsid proteins (vp-1, vp-2, vp-3) are more divergent and determine the serogroup [165] . both viruses require mitotically active cells for replication. severe clinical signs are therefore not found in mature animals because of the lack of a sufficient number of susceptible cells in tissues. general aspects of rodent parvovirus infections and their potential effects on research results have been reviewed [6, 8, [166] [167] [168] [169] [170] . already in the mid-1980s mouse colonies were identified that gave positive reactions for mvm by ifa but not by hi tests. it was subsequently shown that these colonies were infected with a novel parvovirus, initially referred to as 'mouse orphan parvovirus'. the first isolate of mpv was detected as a contaminant of cultivated t-cell clones interfering with in vitro immune responses [171] and was named 'mouse parvovirus'. it does not replicate well in currently available cell cultures, and sufficient quantities of virus for serological tests are difficult to generate. hitherto, only very few isolates of mpv have been cultured and subsequently characterized on a molecular basis [165, 172] . on the basis of epidemiological analyses, further parvoviruses were recently identified in mice, sequenced, and tentatively named serially mpv-2 and mpv-3 [173] , mpv-4 (genbank fj440683) and mpv-5 (genbank fj441297). in addition, several variants are published for mpv-1 [172, 174, 175] . at present, mpv is among the most common viruses found in colonies of laboratory mice. the prevalence of sera positive for parvoviruses ranged from 1% to nearly 10% in western europe and north america, with the majority of sera being positive for mpv in studies differentiating between the two parvovirus species [12, 14, 15, 176] . these prevalence data are based on testing at commercial laboratories and do not reflect that, despite highly specific and sensitive test methods, enzootic parvovirus infections are difficult to detect due to virus-associated characteristics [169, 170] . a recent survey conducted in the usa showed that during a 24-36 month period mouse parvoviruses were detected at almost all facilities that responded to a questionnaire, with mpv being more often diagnosed than mvm [13] . clinical disease and gross or histological lesions have not been reported for mice naturally or experimentally infected with mpv. infections are subclinical even in newborn and immunocompromised animals [177, 178] . in contrast to many other viruses infecting mice, viral replication and excretion is not terminated by the onset of host immunity. tissue necrosis has not been observed at any stage of infection in infected infant or adult mice [177, 178] . humoral immunity to mpv does not protect against mvm infections, and vice versa [179] . serological surveys have indicated that mpv naturally infects only mice, with the exception that mpv-3 shows genetic similarity to hamster parvovirus, suggesting that a cross-species transmission has occurred, where the mouse probably served as the natural host [173, 180] . differences in mouse strain susceptibility to clinical mpv infection do not exist. however, seroconversion seems to be strain-dependent. after experimental infection with mpv-1b, seroconversion occurred in all c3h/hen mice, fewer balb/c, dba/2 and icr mice, and seroconversion could not be detected in c57bl/6 mice [181] . upon mpv-1f inoculation, antibody response was absent in balb/carc mice [182] . diagnosis of mpv infection by pcr testing of small intestine and mesenteric lymph nodes also depended on the mouse strain. mpv dna was detected in all mouse strains evaluated except dba/2 even though seroconversion was detected in these mice. after oral infection, the intestine is the primary site of viral entry and replication. the virus spreads to the mesenteric lymph nodes and other lymphoid tissues, where it persists for more than 2 months [178] , and seems to be excreted via the intestinal and the urinary tract. after experimental inoculation of weanling mice, mpv is transmitted to cagemates by direct contact for 2-6 weeks [177] , and transmission by dirty bedding is also possible. these results implicate a role for urinary, faecal, and perhaps respiratory excretion of virus. another study showed that naturally infected mice might not transmit the virus under similar experimental conditions [183] . serology is a useful tool to identify mpv infections in immunocompetent hosts, but reaching a diagnosis based on serological assays may be difficult and requires a good knowledge of the available techniques. neither the virion elisa nor hi is a practical screening test for mpv because they require large quantities of purified mpv, which is difficult to obtain. diagnosis of mpv infections has long been made on the basis of an mvm hi-negative result coupled with an mvm ifa-positive result. ifa provides the opportunity to detect both serogroup-specific vp proteins as well as ns proteins that are conserved among mouse parvoviruses. a generic rodent parvovirus elisa using a recombinant ns-1 protein as antigen has been developed [184] , but mpv ifa and mpv hi assays are more sensitive techniques than the ns-1 elisa and the mvm ifa [181] . in contrast, elisa tests that use recombinant vp-2 provide sensitive and serogroup-specific assays for the diagnosis of mpv infections in mice [176, 185] , although considerable cross-reactivity with heterologous capsid antigens exists [173] . nevertheless, when using the elisa technique, one needs to consider that mpv-2 may not consistently be detected by mpv-1 vp-2 elisa [168, 173] , especially when antibody titres are low (own observations). therefore, elisas using mpv-2 vp-2 and mpv-3 vp-2 antigens are also used for diagnostics. as parvovirus diagnostics using recombinant assays should be based on a combination of antigens, bead-based mulitplex assays are a convenient extension of traditional elisa, allowing the use of multiple antigens simultaneously. in immunodeficient mice that do not generate a humoral immune response, pcr assays can be used to detect mpv [186, 187] and other parvoviruses. mpv has been shown to persist for at least 9 weeks in the mesenteric lymph nodes [178] . this tissue is considered the best suited for pcr analysis, but spleen and small intestine can also be used with good success [181] . for antemortem detection, shedding of parvoviruses can also be detected by pcr of faecal samples [188] . the virus persists sufficiently long in mesenteric lymph nodes so that pcr assays may also be used as a primary screening tool for laboratories that do not have access to specific mpv antigen-based serological assays. the pcr is further a good confirmatory method for serological assays and has also been described for the detection of parvoviruses in cell lines and tumours [189] . in addition, the map test has been reported as a sensitive tool to detect mpv [183] . given the high environmental stability of the virus and the potential fomite transmission, together with the long virus persistence in infected animals, spontaneous disappearance from a mouse population (e.g. by cessation of breeding) is unlikely. eradication of infection is possible by elimination of infected animals and subsequent replacement with uninfected mice, and the agent can be eliminated from breeding populations by embryo transfer or by hysterectomy. it should be noted that recent studies suggest a risk of virus transmission by embryo transfer, though successful sanitation of immunodeficient mice was achieved despite antibody response in recipients and progeny after embryo transfer [190, 191] . although there are few published reports of confounding effects of mpv on research, it is lymphocytotropic and may perturb immune responses in vitro and in vivo. infections with mpv have been shown to influence rejection of skin and tumour grafts [192] . mvm is the type species of the genus parvovirus. the virus was intermediately named mice minute virus (mmv). it was originally isolated by crawford [193] from a stock of mouse adenovirus, and this prototype isolate was later designated mvmp. its allotropic variant was detected as a contaminant of a transplantable mouse lymphoma [194] and designated mvmi because it exhibits immunosuppressive properties in vitro. both variants have distinct cell tropisms in vivo and in vitro. mvmp infects fibroblast cell lines and does not cause clinical disease [195, 196] . both strains are apathogenic for adult mice, but the immunosuppressive variant is more pathogenic for neonatal mice than is mvmp. a third strain, the cutter strain mvmc, was isolated from bhk-21 cells [172] . in contrast to these three strains detected as cell culture contaminants, an isolate was obtained from naturally infected mice with a b-cell maturational defect maintained at the university of missouri and therefore denominated mvmm [173] . serological surveys show that the mouse is the primary natural host [19, 125, 197] , but the virus is also infective for rats, hamsters [168, 198] , and mastomys [199] during fetal development or after parenteral inoculation. natural infections are usually asymptomatic in adults and infants, and the most common sign of infection is seroconversion. kilham and margolis [200] observed mild growth retardation a few days after experimental infection of neonatal mice with mvmp. studies of transplacental infection yielded no pathological findings in mice [201] . the immunosuppressive variant, but not the prototype strain, is able to produce a runting syndrome after experimental infection of newborn mice [195] . depending on the host genotype, experimental infections of fetal and neonatal mice with mvmi produce various clinical presentations and lesions. infection in c57bl/6 mice is asymptomatic, but the virus causes lethal infections with intestinal haemorrhage in dba/2 mice. infection of strains such as balb/c, cba, c3h/he and sjl is also lethal and mice have renal papillary haemorrhage [196] . the mvmi also infects haematopoietic stem cells and mediates an acute myelosuppression [202, 203] . because of its dependence on mitotically active tissues, the fetus is at particular risk for damage by parvoviruses. mvm and other parvoviruses may have severe teratogenic effects and cause fetal and neonatal abnormalities by destroying rapidly dividing cell populations, often resulting in fetal death. adult prkdc scid mice develop an acute leukopenia 1 month after experimental infection with mvmi and die within 3 months. the virus persists lifelong in the bone marrow of these mice [204] . during a natural concurrent outbreak of mvmm and mpv, a runting syndrome with lymphohistiocytic renal inflammation and inclusion bodies in cells resembling splenic haematopoietic progenitor cells was reported in b-cell (ighm)deficient mice [205] . mmv is shed in faeces and urine. in faecal samples, mvm was detected for up to 4-6 weeks by pcr [206, 207] , although shorter periods (9-12 days) have been observed [208] . notably, shedding re-occurred after immunosuppression by irradiation [207] . contaminated food and bedding are important factors in viral transmission because the virus is very resistant to environmental conditions. direct contact is also important and the virus does not easily spread between cages. routine health surveillance is usually conducted by serological methods. unlike mpv, mvm can easily be cultured in cell lines so that antigen production for hi and elisa (using whole purified virions) is easy. hi is a highly specific diagnostic test whereas ifa always exhibits some degree of cross-reactivity with mpv and other closely related parvoviruses. elisa is probably the most frequently used test, but depending on the purity of the antigen preparation, cross-reactions with mpv may occur due to contamination with non-structural proteins that are common to both viruses. this problem can be avoided by the use of recombinant vp-2 antigen [176] . by using serological methods, one needs to consider that the mouse strain has a considerable effect on seroconversion so that an antibody response might not be detectable despite infection; while c57bl/6j mice showed good antibody response, seroconversion was observed only in some balb/c, akr/n, dba/2j, fvb/n and c3h/hen, but not in nmri and icr mice upon contact exposure to mvmi-inoculated mice [206] . viral detection is also possible by pcr in biological materials, organs (intestine, mesenteric lymph node, kidney, spleen) and faeces from infected animals [187, 189, 206, 207, 209] . although mvm was not thought to cause persistent infection in immunocompetent mice, recent data show that it can be detected in spleens for up to 16 weeks after exposure in some mouse strains [207] . therefore, pcr may be considered as a confirmatory method for serology. the virus can be eliminated from infected breeding populations by caesarean derivation or by embryo transfer. however, certain precautions such as careful washing and accompanying testing need to be minded, as mvm has been detected in reproductive organs and gametes and this virus firmly attaches to the zona pellucida or might even cross it [210, 211] . in experimental colonies, elimination of infected animals and subsequent replacement with uninfected mice is practical if careful environmental sanitation is conducted by appropriate disinfection procedures. it is important that reintroduction is avoided by exclusion of wild mice and by strict separation from other infected populations and potentially contaminated materials in the same facility. admission of biological materials must be restricted to samples that have been tested and found to be free from viral contamination. both allotropic variants of mvm have been used as models for molecular virology, and their small size and simple structure have facilitated examination of their molecular biology and expedited understanding of cell tropism, viral genetics and structure. the significance for laboratory mouse populations was considered low or uncertain because natural infections are inapparent. however, various effects on mouse-based research have been published [6, 7, 166, 167, 170] . because of their predilection for replicating in mitotically active cells, they are frequently associated with tumour cells and have a marked oncosuppressive effect [212] . special attention is also necessary for immunological research and other studies involving rapidly dividing cells (embryology, teratology). in addition, mvm is a common contaminant of transplantable tumours, murine leukaemias and other cell lines [1, 2, 213] . lactate dehydrogenase-elevating virus (ldv) is a single-stranded rna virus of the genus arterivirus belonging to the family arteriviridae. the genome organization and replication of ldv and other arteriviruses, their cell biology and other molecular aspects have been reviewed by snijder and meulenberg [214] . ldv has repeatedly been detected in wild mice (mus musculus), which are considered to be a virus reservoir [215, 216] . after infection of mice, virus titres of 10 10 -10 11 particles per ml serum are found within 12-14 h after infection. the virus titre drops to 10 5 particles per ml within 2-3 weeks and remains constant at this level for life. it persists in infected mice for the whole lifetime although it stimulates various immune mechanisms [216] [217] [218] [219] . the virus can be stored in undiluted mouse plasma at à70 c without loss of infectivity, but it is not stable at room temperature and is very sensitive to environmental conditions. only mice and primary mouse cells are susceptible to infection with ldv. it replicates in a subpopulation of macrophages in almost all tissues and persists in lymph nodes, spleen, liver, and testes tissues [220] . as suitable cell systems have not been available for virus production, routine serology has not been easily possible so that testing for ldv was not included in serological health monitoring programs. the prevalence of ldv in contemporary colonies of laboratory rodents is likely to be very low but detailed information about its prevalence comparable to most other agents is not available. ldv was first detected during a study of methods that could be used in the early diagnosis of tumours [221] . it produces a persistent infection with continuous virus production and a lifelong viraemia despite ldv-specific immune reactions of the host [217]. ldv has been found in numerous biological materials that are serially passaged in mice such as transplantable tumours including human tumours or matrigel prepared from such materials [1, 2, 222, 223] , monoclonal antibodies or ascitic fluids [224] , or infectious agents (e.g. haemoprotozoans, k virus, clostridium piliforme). these materials are contaminated after serial passage in an infected and viraemic mouse. contamination with ldv leads to the infection of each sequential host and to transmission of the virus by the next passage and remains associated with the specimen. it is therefore the most frequently detected contaminant in biological materials [1, 2] . infection with ldv is usually asymptomatic, and there are no gross lesions in immunocompetent as well as in immunodeficient mice. the only exception is poliomyelitis with flaccid paralysis of hindlimbs developing in c58 and akr mice when they are immunosuppressed either naturally with ageing or experimentally. it has been shown that only mice harbouring cells in the cns that express a specific endogenous mulv are susceptible to poliomyelitis [225] . the characteristic feature of ldv infection is the increased activity of lactate dehydrogenase (ldh) and other plasma enzymes [8, 226] , which is due to the continuous destruction of permissive macrophages that are responsible for the clearance of ldh from the circulation. as a consequence, the activity of plasma ldh begins to rise by only 24 h after infection and peaks 3-4 days after infection at 5-10-fold normal levels, or can even be up to 20-fold in sjl/j mice. the enzyme activity declines during the next 2 weeks but remains elevated throughout life. antigen-antibody complexes produced during infection circulate in the blood and are deposited in the glomeruli [226] . in contrast to other persistent virus infections (e.g. lymphocytic choriomeningitis virus), these complexes do not lead to immune complex disease and produce only a very mild glomerulopathy. the only gross finding associated with ldv infection is mild splenomegaly. microscopically, necrosis of lymphoid tissues is visible during the first days of infection. in mouse strains that are susceptible to poliomyelitis, ldv induces lesions in the grey matter of the spinal cord and the brainstem. ldv is not easily transmitted between mice, even in animals housed in the same cage. fighting and cannibalism increase transmission between cagemates, most likely via blood and saliva. infected females transmit the virus to their fetuses if they have been infected few days prior to birth and before igg anti-ldv antibodies are produced, but developmental and immunological factors (e.g. gestational age, timing of maternal infection with ldv, placental barrier) are important in the regulation of transplacental ldv infection [227, 228] . maternal immunity protects fetuses from intrauterine infection. immunodeficient prkdc scid mice also transmit virus to their offspring during chronic infection [229] . an important means of transmission is provided by experimental procedures such as mouse-to-mouse passage of contaminated biological materials or the use of the same needle for sequential inoculation of multiple mice. in principle, serological methods such as ifa may be used for detecting ldv infection [230] but they are not of practical importance. circulating virus-antibody complexes interfere with serological tests, and sufficient quantities of virus for serological tests are difficult to generate because ldv replicates only in specific subpopulations of primary cultures of murine macrophages and monocytes for one cell cycle [226] . however, it is meanwhile possible to use recombinant viral proteins of ldv as antigens [231] in elisa and mfia tests so that routine testing by serology is possible. in the past, diagnosis of ldv infection has primarily been based on increased ldh activity in serum or plasma of mice. ldv activity in serum or plasma can be measured directly, or samples (e.g. plasma, cell or organ homogenates) are inoculated into pathogen-free mice and the increase in ldh activity within 3-4 days is measured. an 8-10-fold increase is indicative of ldv infection. detection of infectivity of a plasma sample by the induction of increased ldh activity in the recipient animal is the most reliable means of identifying an infected animal. however, it is important to use clear non-haemolysed samples because haemolysis will (falsely) elevate activities of multiple serum or plasma enzymes, including ldh. this assay was usually included in a 'map test', but antibody detection similar to other viruses was not involved for reasons mentioned earlier. persistent infection makes ldv an ideal candidate for pcr detection in plasma or in organ homogenates [232, 233] . however, reports exist that pcr may produce false-negative results and should be used cautiously [234] . just as important as detecting ldv in animals is its detection in biological materials. this may be done by assay for increased ldh activity after inoculation of suspect material into pathogenfree mice [1, 2] or by pcr [232, 233, [235] [236] [237] . ldv spreads slowly in a population because direct contact is necessary. therefore, ldv-negative breeding populations can easily be established by selecting animals with normal plasma ldh activity. embryo transfer and hysterectomy derivation are also efficient. the presence of ldv in experimental populations may be indicative of contaminated biological materials. in such cases, it is essential that the virus is also eliminated from these samples. this is easily achieved by maintenance of cells by in vitro culture instead of by animal-to-animal passages [238] . due to the extreme host specificity of the virus, contaminated tumour samples can also be sanitized by passages in nude rats [223] or other animal species. another method to remove ldv from contaminated cells, which is based on cell sorting, has recently been described [239] . ldv is a potential confounder of any research using biological materials that are passaged in mice. once present in an animal, the virus persists lifelong. the most obvious signs are increased levels of plasma ldh and several other enzymes. ldv may also exhibit numerous effects on the immune system (thymus involution, depression of cellular immunity, enhanced or diminished humoral responses, nk cell activation, development of autoimmunity, and suppression of development of diabetes in nod mice); [218, 219, 224, [240] [241] [242] [243] [244] and enhance or suppress tumour growth [6, 7, 226] . interaction with other viruses has also been described [245] . lymphocytic choriomeningitis virus (lcmv) is an enveloped, segmented single-stranded rna virus of the genus arenavirus family, arenaviridae. it can easily be propagated in several commonly used cell lines like bhk-21 cells. however, cells are not lysed and a cytopathic effect (cpe) is not visible. the virus name refers to the condition that results from experimental intracerebral inoculation of the virus into adult mice and is not considered to be a feature of natural infections. mice (mus musculus) serve as the natural virus reservoir [246] , but syrian hamsters are also important hosts [247] . additional species such as rabbits, guinea-pigs, squirrels, monkeys and humans are susceptible to natural or experimental infection [248] . natural infection of callitrichid primates (marmosets and tamarins) leads to a progressive hepatic disease that is known as 'callitrichid hepatitis' [249, 250] . antibodies to lcmv have been found in wild mice in europe [251, 252] , africa [253] , asia [254] , australia [125] and america [255] . thus, it is the only arenavirus with worldwide distribution. infection with lcmv is rarely found in laboratory mice [248] . seropositivity to lcmv in laboratory mice was reported to be low during the last decade [11, 15, 17, 124] or negative [12] [13] [14] . in addition to laboratory mice and other vertebrate hosts, the virus has frequently been found in transplantable tumours and tissue culture cell lines from mice and hamsters [2, 256] . despite the low prevalence in laboratory mice, seropositivity to this zoonotic agent should raise serious concern for human health. lcmv is frequently transmitted to humans from wild mice and is also endemic to a varying degree in the human population [257] [258] [259] [260] [261] due to contact with wild mice. it has also been transmitted to humans by infected laboratory mice [262] and by pet and laboratory syrian hamsters [263] [264] [265] [266] . in addition, contaminated biological materials are important sources of infections for humans, and several outbreaks of lcm among laboratory personnel have been traced to transplantable tumours [267, 268] . transmission of lcmv to humans also occurred repeatedly by organ transplantation and was most likely transmitted to organ donors by close contact with infected pets [266, 269] . lcmv can cause mild-to-serious or fatal disease in humans [262, 270, 271] . congenital infection in humans may result in hydrocephalus, or fetal or neonatal death [272] . in mice, clinical signs of lcmv infection vary with strain and age of mouse, strain and dose of virus, and route of inoculation [8, 248, 251] . two forms of natural lcmv infection are generally recognized: a persistent tolerant and an (acute) non-tolerant form. the persistent form results from infection of mice that are immunotolerant. this is the case if mice are infected in utero or during the first days after birth. this form is characterized by lifelong viraemia and viral shedding. mice may show growth retardation, especially during the first 3-4 weeks, but they appear otherwise normal. infectious virus is bound to specific antibodies and complement, and these complexes accumulate in the renal glomeruli, the choroid plexus, and sometimes also in synovial membranes and blood vessel walls. at 7-10 months of age, immune complex nephritis develops with ruffled fur, hunched posture, ascites and occasional deaths. this immunopathologic phenomenon is called 'late onset disease' or 'chronic immune complex disease'. the incidence of this type of disease varies between mouse strains. gross lesions include enlarged spleen and lymph nodes due to lymphoid hyperplasia. kidneys affected with glomerulonephritis may be enlarged with a granular surface texture or may be shrunken in later stages of the disease process. microscopically, there is generalized lymphoid hyperplasia and immune complex deposition in glomeruli and vessel walls, resulting in glomerulonephritis and plasmacytic, lymphocytic perivascular cuffs in all visceral organs [36] . the non-tolerant acute form occurs when infection is acquired after the development of immunocompetence (in mice older than 1 week). these animals become viraemic but do not shed virus and may die within a few days or weeks. natural infections of adults are usually asymptomatic. surviving mice are seropositive and in most cases clear the virus to below detection levels of conventional methods. however, virus may persist at low levels in tissues (particularly spleen, lung and kidney) of mice for at least 12 weeks after infection as determined by sensitive assays such as nested reverse transcriptase (rt)-pcr or immunohistochemistry [273] . such non-lethal infection leads to protection against otherwise lethal intracerebral challenge. protection from lethal challenge is also achieved by maternally derived anti-lcmv antibodies through nursing or by the administration of anti-ldv monoclonal igg2a antibodies [274] . in experimentally infected mice, the route of inoculation (subcutaneous, intraperitoneal, intravenous, intracerebral) also influences the type and degree of disease [248] . intracerebral inoculation of adult immunocompetent mice typically results in tremors, convulsions and death due to meningoencephalitis and hepatitis. neurological signs usually appear on day 6 after inoculation, and animals die within 1-3 days after the onset of symptoms, or recover within several days. the classic histological picture is of dense perivascular accumulations of lymphocytes and plasma cells in meninges and choroid plexus. while infection following subcutaneous inoculation usually remains inapparent, reaction of mice to intraperitoneal or intravenous inoculation depends on the virus strain and on the mouse strain. infection by these routes primarily causes multifocal hepatic necrosis and necrosis of lymphoid cells. athymic foxn1 nu mice and other immunodeficient mice do not develop disease but become persistently viraemic and shed virus. as a general rule, all pathological alterations following lcmv infection are immune-mediated; and mice can be protected from lcmvinduced disease by immunosuppression [275] . lcmv disease is a prototype for virus-induced t-lymphocyte-mediated immune injury and for immune complex disease. for detailed information on the pathogenesis, clinical and pathological features of lcmv infection, the reader is referred to review articles [248, 276, 277] . in nature, carrier mice with persistent infection serve as the principal source of virus. intrauterine transmission is very efficient, and with few exceptions all pups born from carrier mice are infected. furthermore, persistently infected mice and hamsters can shed large numbers of infectious virions primarily in urine, but also in saliva and milk. the virus can replicate in the gastric mucosa after intragastric infection [278, 279] . gastric inoculation elicits antibody responses of comparable magnitudes as intravenous inoculation and leads to active infection with lcmv, indicating that oral infection is possible, e.g. by ingestion of contaminated food or by cannibalism. a self-limiting infection frequently results from infection of adult mice. the virus does not spread rapidly after introduction in populations of adult mice, and the infectious chain usually ends. however, if the virus infects a pregnant dam or a newborn mouse, a lifelong infection results, and soon a whole breeding colony of mice may become infected if the mice live in close proximity (which is the case under laboratory conditions). the virus is not easily transmitted to dirty-bedding sentinels, and it is important that colony animals or animals having had direct contact with a population are tested to exclude lcmv infection [280] . lcmv is most commonly diagnosed by serological methods such as mfia, ifa and elisa [281] . all strains show a broad cross-reactivity and are serologically uniform. however, subclinical persistent infections may be difficult to detect because they may be associated with minimal or undetectable levels of circulating antibody. it is important that bleeding of mice is done carefully because of a potential risk due to viraemic animals. historically, direct viral detection was performed by inoculating body fluids or tissue homogenates into the brain of lcmv-free mice or by subcutaneous injection into mice and subsequent serological testing (map test). more recently, pcr assays have been developed for the direct detection of viral rna in clinical samples or animals [282] [283] [284] . both map test and pcr can also be used to detect contamination of biological materials [235, 237] . specifically for exclusion of contamination by lcmv, it was requested by different authorities that virus is inoculated intracerebrally at a lethal dose 3-4 weeks after administration of the material to be tested. in case of contamination by lcmv and subsequent seroconversion, animals survive the challenge infection. vertical transmission of lcmv by transuterine infection is efficient so that this virus cannot reliably be eliminated by caesarean rederivation [280] . caesarean derivation may be effective if dams acquired infection after the development of immunocompetence (non-tolerant acute infection) and subsequently eliminated the virus, but such a strategy is difficult to justify in light of lcmv's zoonotic potential. in breeding colonies of great value, virus elimination might be possible soon after introduction into the colony by selecting non-viraemic breeders. this procedure is expensive and time consuming and requires special safety precautions. fortunately, infections of laboratory mice with lcmv are very uncommon. however, once lcmv has been detected in animals, or in biological materials, immediate destruction of all contaminated animals and materials is advisable to avoid risk of human infection. foxn1 nu and prkdc scid mice may pose a special risk because infections are silent and chronic [268] . cages and equipment should be autoclaved, and animal rooms should be fumigated with disinfectants such as formaldehyde, vaporized paraformaldehyde, hydrogen peroxide or other effective disinfectants. prevention of introduction into an animal facility requires that wild mice cannot get access to the facility. similarly important is screening of biological materials originating from mice and hamsters because these can be contaminated by lcmv. finally, it has been shown that the virus can also be introduced into a population by mice with an undetected infection [280] . appropriate precautions are necessary for experiments involving lcmv, or lcmv-infected animals or materials. biological safety level (bsl) 2 will be considered to be sufficient in most cases. bsl 3 practices may be considered when working with infected animals owing to the increased risk of virus transmission by bite wounds, scratching or aerosol formation from the bedding. animal biosafety level (absl) 3 practices and facilities are generally recommended for work with infected hamsters. appropriate precautions have been defined for different bsls or absls by cdc [285] . lcmv is frequently utilized as a model organism to study virus-host interactions, immunological tolerance, virus-induced immune complex disease, and a number of immunological mechanisms in vivo and in vitro [286] [287] [288] . accidental transmission may have a severe impact on various kinds of experiments [6, 7, 248, 251] and also affect infection with other agents [289] . mammalian orthoreoviruses (mrv) are nonenveloped, segmented double-stranded rna viruses of the family reoviridae, genus orthoreovirus. they have a wide host range and are ubiquitous throughout the world. the designation reo stands for respiratory enteric orphan and reflects the original isolation of these viruses from human respiratory and intestinal tract without apparent disease. the term 'orphan' virus refers to a virus in search of a disease. mammalian orthoreovirus can be grouped into three serotypes, numbered 1-3. mammalian orthoreovirus-3 (synonyms: hepatoencephalomyelitis virus; echo 10 virus) infection remains prevalent in contemporary mouse colonies and has been reported in wild mice [20, 125, 290] . a study in france reported antibodies to mrv-3 in 9% of mouse colonies examined [10] . in more recent studies in north america and western europe, such antibodies were detected in 0.01-0.2% of mice monitored [11, 14, 15] . schoondermark-van de ven et al. [12] found antibodies to mrv-3 in 0.6% of mouse samplings from western european institutions; and in a survey conducted by carty [13] , about 6% of responding institutions in the usa reported mrv-3 infection in their mouse colonies. in addition, contamination of mouse origin tumours and cell lines by mrv-3 has been reported many times [2, 8, 290] . experimentally, mrv-3 infection of infant mice has been used to model human hepatobiliary disease, pancreatitis, diabetes mellitus and lymphoma [8, 291] . the literature on mrv-3 infections in mice is dominated by studies on experimentally infected animals. the virus can cause severe pantropic infection in infant mice [290] [291] [292] . after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes and blood vessels. following oral inoculation, reoviruses gain entry by infecting specialized epithelial cells (m cells) that overlie peyer's patches. the virus then becomes accessible to leukocytes and spreads to other organs by way of the lymphatic system and the bloodstream. neural spread to the cns has also been well documented [293, 294] . the mechanisms of viral pathogenesis and their interactions with the host cell as well as the host's immune response are reviewed in detail by tyler et al. [295] , schiff et al. [296] and ward et al. [291] . natural infection by mrv-3 in a mouse colony is usually subclinical, although diarrhoea or steatorrhoea and oily hair effect in suckling mice may be noted [8, 36, [290] [291] [292] . the latter term has been used to describe the matted, unkempt appearance of the hair coat that results from steatorrhoea due to pancreatitis, maldigestion and biliary atresia. in addition, runting (attributed to immune-mediated destruction of cells in the pituitary gland that produce growth hormone), transient alopecia, jaundice (due to excessive bilirubin in the blood, which is attributed to the liver pathology, especially biliary atresia) and neurological signs such as incoordination, tremors or paralysis may develop. when present in natural infections, clinical signs and lesions are similar to but milder than in experimental neonatal infections. early descriptions of naturally occurring disease may have been complicated by concurrent infections such as mhv (murine hepatitis virus) or murine rotavirus a (murv-a)/epizootic diarrhoea of infant mice (edim) virus that contributed to the severity of the lesions especially in liver, pancreas, cns and intestine. the outcome of mrv-3 infection depends on age and immunological status of mouse, dose of virus and route of inoculation. adult immunocompetent mice typically show no clinical signs and have no discernible lesions even in experimental infections. mucosal and maternally conferred immunity are considered to be important in protection from or resolution of disease [297, 298] . experimental infection of adult prkdc scid mice is lethal [299] . depending on the route of inoculation, experimental infection of adult foxn1 nu mice is subclinical or results in liver disease [299, 300] . histological findings reported to occur after experimental mrv-3 infection of neonatal mice include inflammation and necrosis in liver, pancreas, heart, adrenal, brain, and spinal cord; lymphoid depletion in thymus, spleen, and lymph nodes; and hepatic fibrosis with biliary atresia [36, [290] [291] [292] 298] . transmission of reoviruses probably involves the aerosol as well as the faecal-oral route [8, 291] . fomites may play an important role as passive vectors because reoviruses resist environmental conditions moderately well. serological screening with mfia, elisa or ifa is in widespread use for detection of antibodies to mrv-3 in diagnostic and health surveillance programmes. both elisa and ifa detect cross-reacting antibodies to heterologous mrv serotypes that can infect mice [301] , although a recent report indicates that some ifa-positive mrv infections in mice may not be detected by commonly used elisas [302] . the hi test does not detect such cross-reacting antibodies but is prone to give false-positive results due to nonspecific inhibitors of haemagglutination [301, 303] . rt-pcr methods for the detection of mrv-3 rna [304, 305] or mrv rna [302, 306] are also available. reports on contamination of mouse origin tumours and cell lines by mrv-3 and its interference with transplantable tumour studies [307, 308] emphasize the importance of screening of biological materials to be inoculated into mice by map test or pcr. natural seroconversion to mrv-3 without clinical disease is also observed in laboratory rats, hamsters and guinea-pigs [8, 290] . caesarean derivation and barrier maintenance have proven effective in the control and prevention of mrv-3 infection [8, 291] . the virus may interfere with research involving transplantable tumours and cell lines of mouse origin. it has the potential to alter intestinal studies and multiple immune response functions in mice. in enzootically infected colonies, protection of neonates by maternal antibody could complicate or prevent experimental infections with reoviruses. it could further complicate experiments that require evaluation of liver, pancreas, cns, heart, lymphoid organs and other tissues affected by the virus. the term murine hepatitis virus (mhv; commonly referred to as 'mouse hepatitis virus') designates a large group of antigenically and genetically related, single-stranded rna viruses belonging to the family coronaviridae, genus coronavirus. they are surrounded by an envelope with a corona of surface projections (spikes). mhv is antigenically related to rat coronaviruses and other coronaviruses of pigs, cattle and humans. numerous different strains or isolates of mhv have been described. they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens, by use of monoclonal antibodies, or by sequencing [309] . the beststudied strains are the prototype strains mhv-1, mhv-2, mhv-3, jhm (mhv-4), a59, and s, of which mhv-3 is regarded as the most virulent. like other coronaviruses mhv mutates rapidly, and strains readily form recombinants, so that new (sub)strains are constantly evolving. strains vary in their virulence, organotropism and cell tropism [310] . based on their primary organotropism, mhv strains can be grouped into two biotypes: respiratory (or polytropic) and enterotropic. however, intermediate forms (enterotropic strains with tropism to other organs) also exist. murine hepatitis virus is relatively resistant to repeated freezing and thawing, heating (56 c for 30 min) and acid ph but is sensitive to drying and disinfectants, especially those with detergent activity [8] . given the environmental conditions present in mouse rooms, mhv might remain infective for several days, at low humidity (20% relative humidity) or low temperatures (4 c) even for weeks on surfaces [311] . mus musculus is the natural host of mhv. it can be found in wild and laboratory mice throughout the world and is one of the most common viral pathogens in contemporary mouse colonies. while polytropic strains have historically been considered more common, this situation is thought to have reversed. monitoring results for research institutions across north america and europe indicate that the prevalence of mhv has decreased in the past, though it seems to have remained quite stable since the 1990s [11, 12] . recently 1.57% of north american laboratory mouse serum samples tested positive [15] . in europe, prevalence rates ranged from 3.25% to 12% [12, 14, 15] . a retrospective study in france covering the period from 1988 to 1997 reported antibodies to mhv in 67% of mouse colonies examined [10] , and a survey performed in 2006 revealed that almost half of north american research institutions detected mhv in their mouse populations [13] . suckling rats inoculated experimentally with mhv had transient virus replication in the nasal mucosa and seroconversion but no clinical disease [312] . similarly, deer mice seroconverted but showed no clinical disease after experimental infection [313] . mhv is also a common contaminant of transplantable tumours [1, 2] and cell lines [314, 315] . the pathogenesis and outcome of mhv infections depend on interactions between numerous factors related to the virus (e.g. virulence and organotropism) and the host (e.g. age, genotype, immune status, and microbiological status) [8, 36, 309, 310, 316, 317] . mhv strains appear to possess a primary tropism for the upper respiratory or enteric mucosa. those strains with respiratory tropism initiate infection in the nasal mucosa and then may disseminate via blood and lymphatics to a variety of other organs because of their polytropic nature. respiratory (polytropic) strains include mhv-1, mhv-2, mhv-3, a59, s and jhm. infection of mice with virulent polytropic mhv strains, infection of mice less than 2 weeks of age, infection of genetically susceptible strains of mice or infection of immunocompromised mice favour virus dissemination. virus then secondarily replicates in vascular endothelium and parenchymal tissues, causing disease of the brain, liver, lymphoid organs, bone marrow and other sites. infection of the brain by viraemic dissemination occurs primarily in immunocompromised or neonatal mice. additionally, infection of adult mouse brain can occur by extension of virus along olfactory neural pathways, even in the absence of dissemination to other organs. in contrast, enterotropic mhv strains (e.g. livim, mhv-d, mhv-y) tend to selectively infect intestinal mucosal epithelium, with no or minimal dissemination to other organs such as mesenteric lymph nodes or liver. all ages and strains are susceptible to active infection, but disease is largely age related. infection of neonatal mice results in severe necrotizing enterocolitis with high mortality within 48 h. mortality and lesion severity diminish rapidly with advancing age at infection. adult mice develop minimal lesions although replication of equal or higher titres of virus occurs compared with neonates. the age-dependent decrease in severity of enterotropic mhv disease is probably related to the higher mucosal epithelium turnover in older mice, allowing more rapid replacement of damaged mucosa. another factor that is of considerable importance to the outcome of mhv infections is host genotype. for example, balb/c mice are highly susceptible to enterotropic mhv disease while sjl mice, at the other end of the spectrum, are highly resistant [318] . unlike in polytropic mhv infection where resistance is correlated with reduced virus replication in target cells [319] , enterotropic mhv grows to comparable titres in sjl and balb/c mice at all ages [318] . therefore, the resistance of the sjl mouse to disease caused by enterotropic mhv seems to be mediated through an entirely different mechanism than resistance to polytropic mhv. furthermore, mouse genotypes that are susceptible to disease caused by one mhv strain may be resistant to disease caused by another strain [316] . it is therefore not possible to strictly categorize mouse strains as susceptible or resistant. the genetic factors determining susceptibility versus resistance in mhv infections are as yet poorly understood. both polytropic and enterotropic mhv infections are self-limiting in immunocompetent mice. immune-mediated clearance of virus usually begins about a week after infection, and most mice eliminate the virus within 3-4 weeks [316, 318, 320] . humoral and cellular immunity appear to participate in host defences to infection, and functional t cells are an absolute requirement [321] [322] [323] [324] . therefore, immunodeficient mice such as foxn1 nu and prkdc scid mice cannot clear the virus [317, 325] . similarly, some genetically modified strains of mice may have deficits in antiviral responses or other alterations that allow the development of persistent mhv infection [326] . recovered immune mice are resistant to reinfection with the same mhv strain but remain susceptible to repeated infections with different strains of mhv [327] [328] [329] . similarly, maternal immunity protects suckling mice against homologous mhv strains but not necessarily against other strains [329, 330] . however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine [330] . therefore, the susceptibility of young mice to infection significantly increases at weaning. most mhv infections are subclinical and follow one of two epidemiological patterns in immunocompetent mice [8, 310] . enzootic (subclinical) infection, commonly seen in breeding colonies, occurs when a population has been in contact with the virus for a longer period (e.g. several weeks). adults are immune (due to prior infection), sucklings are passively protected, and infection is perpetuated in weanlings. epizootic (clinical) infection occurs when the virus is introduced into a naive population (housed in open cages). the infection rapidly spreads through the entire colony. clinical signs depend upon the virus and mouse strains and are most evident in infant mice. typically, they include diarrhoea, poor growth, lassitude, and death. in infections due to virulent enterotropic strains, mortality can reach 100% in infant mice. some strains may also cause neurological signs such as flaccid paralysis of hindlimbs, convulsions and circling. adult infections are again usually asymptomatic. as the infection becomes established in the colony, the epizootic pattern is replaced by the enzootic pattern. in immunodeficient (e.g. foxn1 nu and prkdc scid ) mice, infection with virulent polytropic mhv strains is often rapidly fatal while less virulent strains cause chronic wasting disease [317] . in contrast, adult immunodeficient mice can tolerate chronic infection by enterotropic mhv, with slow emaciation and diarrhoea, or minimal clinical disease [316, 325] . subclinical mhv infections can be activated by a variety of experimental procedures (e.g. thymectomy, whole body irradiation, treatment with chemotherapeutic agents, halothane anaesthesia) or by coinfections with other pathogens (e.g. eperythrozoon coccoides, k virus; reviewed in [8, 309] ). in most natural infections, gross lesions are not present or are transient and not observed. gross findings in neonates with clinical signs include dehydration, emaciation, and in contrast to edim, an empty stomach [309, 331, 332] . the intestine is distended and filled with watery to mucoid yellowish, sometimes gaseous contents. haemorrhage or rupture of the intestine can occur. depending on the virus strain, necrotic foci on the liver [36, 309, 332] and thymus involution [331, 333] may also be seen in susceptible mice. liver involvement may be accompanied by jaundice and haemorrhagic peritoneal exudate. splenomegaly may occur as a result of compensatory haematopoiesis [334] . histopathological changes in susceptible mice infected with polytropic mhv strains include acute necrosis with syncytia in liver, spleen, lymph nodes, gut-associated lymphoid tissue, and bone marrow [8, 36, 309, 316] (figure 3.2.7) . recently, pulmonary inflammation has been observed in susceptible mouse strains (c3h/hej and a/j) after intranasal inoculation with polytropic mhv-1 [335, 336] . neonatally infected mice can have vascular-oriented necrotizing (meningo)encephalitis with demyelination in the brainstem and periependymal areas. lesions in peritoneum, bone marrow, thymus and other tissues can be variably present. mice can develop nasoencephalitis due to extension of infection from the nasal mucosa along olfactory pathways to the brain, with meningoencephalitis and demyelination, the latter of which is thought to be largely t-cell mediated [324] . this pattern of infection regularly occurs after intranasal inoculation of many mhv strains but is a relatively rare event after natural exposure. syncytium arising from endothelium, parenchyma or leukocytes is a hallmark of infection in many tissues including intestine, lung, liver, lymph nodes, spleen, thymus, brain and bone marrow. lesions are transient and seldom fully developed in adult immunocompetent mice, but they are manifest in immunocompromised mice. highly unusual presentations can occur in mice with specific gene defects. for example, granulomatous peritonitis and pleuritis were found in interferongamma-deficient mice infected with mhv [337] . histopathological changes caused by enterotropic strains of mhv are mainly confined to the intestinal tract and associated lymphoid tissues [8, 36, 309, 316] . the most common sites are terminal ileum, caecum and proximal colon. the severity of disease is primarily age-dependent, with neonatal mice being most severely affected. these mice show segmentally distributed areas of villus attenuation, enterocytic syncytia (balloon cells) and mucosal necrosis accompanied by leukocytic infiltration. intracytoplasmic inclusions are present in enterocytes. erosions, ulceration, and haemorrhage may be seen in more severe cases. lesions can be fully developed within 24-48 h, but are usually more severe at 3-5 days after infection. surviving mice may develop compensatory mucosal hyperplasia. mesenteric lymph nodes usually contain lymphocytic syncytia, and mesenteric vessels may contain endothelial syncytia. pathological changes in older mice are generally much more subtle and may only consist of transient syncytia. an occasional exception seems to occur in immunodeficient animals such as foxn1 nu mice, which can develop chronic hyperplastic typhlocolitis of varying severity [325] , but other agents such as helicobacter spp. may have been involved. in general, enterotropic mhv strains do not disseminate, but hepatitis and encephalitis can occur with some virus strains in certain mouse genotypes. in t-cell deficient mice, multisystemic lethal infection was observed after experimental infection with the enterotropic strain mhv-y [338] . mhv is highly contagious. it is shed in faeces and nasopharyngeal secretions and appears to be transmitted via direct contact, aerosol and fomites [8, 309] . vertical (in utero) transmission has been demonstrated in experimental infections [339] but does not seem to be of practical importance under natural conditions. mhv was transmitted by ovarian transplantation after reproductive organs became infected [340] . however, risk of mhv transmission by sperm or oocytes (ivf) or by embryo transfer seems to be low, though thorough washing of gametes and embryos is required [211, [340] [341] [342] . diagnosis during the acute stage of infection can be made by histological demonstration of characteristic lesions with syncytia in target tissues, but clinical signs and lesions can be highly variable and may not be prominent. suckling, genetically susceptible or immunocompromised mice are the best candidates for evaluation. active infection can be confirmed by immunohistochemistry [343] or by virus isolation. virus recovery from infected tissues is difficult but can be accomplished using primary macrophage cultures or a number of established cell lines such as nctc 1469 or dbt [301] . these cells, however, may not be successful substrates for some enterotropic mhv strains. virus in suspect tissue can also be confirmed by bioassays such as map testing or infant or foxn1 nu mouse inoculation [301, 344] . amplification by passage in these mice increases the likelihood of detection of lesions and antigen, or virus recovery. other direct diagnostic methods that have been successfully utilized to detect mhv in faeces or tissue of infected mice include monoclonal antibody solution hybridization assay [345] and a number of rt-pcr assays [346] [347] [348] [349] . because of the transient nature of mhv infection in immunocompetent mice, serology is the most appropriate diagnostic tool for routine monitoring. multiplex fluorescent immunoassay, elisa and ifa are well established and sensitive, and all known mhv strains cross-react in these tests [301, 350, 351] . the magnitude of antibody response depends on mhv strain and mouse genotype [319, 352] . dba/2 mice are poor antibody responders whereas c57bl/6 mice produce a high antibody titre and are therefore good sentinels. antibody titres remain high over a period of at least 6 months [327, 329] . infected mice may not develop detectable antibodies for up to 14 days after initial exposure [350] . in such cases, a direct diagnostic method, as discussed above, may be useful. another drawback of serology is that mice weaned from immune dams can have maternal antibodies until they are 10 weeks of age [353] . this may impact serological monitoring because the possibility must be considered that low positive results are due to maternally derived passive immunity. because the virus can be transmitted by transplantable tumours and other biological materials from mice, including hybridomas [354] and embryonic stem cells [355, 356] these materials should also be routinely screened for mhv contamination. mouse inoculation bioassay, map test and rt-pcr can be used for this purpose. therefore, surveillance programmes should combine careful evaluation of clinically ill animals, testing of biological materials and routine health monitoring. soiled-bedding sentinel mice, which are frequently used for routine monitoring, are likely well suited for detecting enterotropic strains of mhv, but might not indicate the presence of less contagious respiratory strains of mhv [309] equally well. the mouse strain used as sentinel should be considered as a critical factor. furthermore, duration of mhv shedding and stability of the virus, which seems to be lower in static microisolator cages than in ivc cages, might interfere with detection. the amount of bedding transferred seems not to be as critical as for, e.g. parvoviruses, at least for enterotropic strains [357] . use of contact and exhaust air sentinels and testing of exhaust filters by pcr was also shown to be effective at detecting mhv [358] . the best means of mhv control is to prevent its entry into a facility. this can be accomplished by purchasing mice from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance programme. control of wild mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbour virus are also important measures to prevent infection. if infection occurs, the most effective elimination strategy is to cull the affected colony and obtain clean replacement stock. however, this is not always a feasible option when working with valuable mice (e.g. genetically modified lines, breeding stocks). caesarean derivation or embryo transfer can be used to produce virus-free offspring, and foster-nursing has also been reported to be effective [359] . quarantine of an affected colony with no breeding and no introduction of new animals for approximately 2 months has been effective in immunocompetent mice [360] . the infection is likely to be terminated because mhv requires a constant supply of susceptible animals. this method works best when working with small numbers of mice. large populations favour the development of new mhv strains that may result in repeated infections with slightly different strains [361] . it may be practical to select a few future breeders from the infected population and quarantine them for approximately 3 weeks [317] . this can be achieved in isolators, or in individually ventilated cages if proper handling is guaranteed. after this interval, breeding can resume. the 3-week interval should permit recovery from active infection, and the additional 3-week gestation period effectively extends the total quarantine to 6 weeks. it is advisable to select seropositive breeders because the possibility of active infection is lower in such animals. the breeding cessation strategy may not be successful if immunodeficient mice are used because they are susceptible to chronic infection and viral excretion [325] . genetically engineered mice of unclear, unknown or deficient immune status pose a special challenge because they may develop unusual manifestations of infection or may be unable to clear virus. rederivation is likely to be the most cost-effective strategy in such situations. along with the measures described, proper sanitation and disinfection of caging and animal quarters, as well as stringent personal sanitation, are essential to eliminate infection. careful testing with sentinel mice should be applied to evaluate the effectiveness of rederivation. if transplantable tumours are contaminated with mhv, virus elimination can be achieved by passage of tumours in athymic foxn1 rnu rats [362] . mhv is one of the most important viral pathogens of laboratory mice and has been intensively studied from a number of research perspectives (e.g. as a model organism for studying coronavirus molecular biology or the pathogenesis of viral-induced demyelinating disease). numerous reports document the effects of natural and experimental infections with mhv on host physiology and research, especially in the fields of immunology and tumour biology (reviewed in [6-8, 310, 316, 317] ). noroviruses are non-enveloped, single-stranded rna viruses with high environmental resistance and belong to the family caliciviridae, genus norovirus. they were first identified after an outbreak of acute gastroenteritis at a school in norwalk (ohio, usa) in 1968 and cause about 90% of non-bacterial epidemic gastroenteritis in humans. noroviruses found in animals include bovine, porcine and murine noroviruses. noroviruses are not known to cross species. murine norovirus (mnv) is endemic in many research mouse colonies and currently the most commonly detected viral agent in laboratory mice [14, 15, 363] . in the hitherto largest survey [15] , about 32% of mouse serum samples examined had antibodies against mnv. the first norovirus to infect mice was described in 2003 [364] . experimental inoculation studies with this murine norovirus (mnv-1) show that duration of infection and disease manifestation vary depending on the mouse strain [363] [364] [365] . in immunocompetent strains, mnv infection is variable in length (e.g. !7-14 days in 129s6 mice, !5 weeks in hsd:icr mice) and does not induce clinical signs. infection is associated with mild histopathological alterations in the small intestine (increase in inflammatory cells) and spleen (red pulp hypertrophy and white pulp activation) of 129s6 mice. in certain immunodeficient strains, however, infection can cause lethal systemic disease (encephalitis, vasculitis, meningitis, hepatitis and pneumonia in interferon-alpha-beta-gamma-receptor-deficient and stat1 tm1 mice) or persist without symptoms (!90 days in rag1 à/à and rag2 à/à mice). these findings indicate that components of the innate immune system are critical for resistance to mnv-1 induced disease. consistent with this hypothesis, it was demonstrated that mnv-1 replicates in macrophages and dendritic cells [366] . meanwhile, many additional strains of mnv with diverse biological properties were isolated [367, 368] . an analysis of 26 mnv isolates revealed 15 distinct mnv strains that comprise a single genogroup and serotype [368] . experimental inoculation studies show that several mnv strains are able to persist in various tissues (small intestine, caecum, mesenteric lymph node, spleen) of immunocompetent (c57bl/6j, hsd:icr, jcl:icr) and immunodeficient (cb17-prkdc scid ) mice with viral shedding in faeces for the duration of at least 35-60 days [367] [368] [369] . murine norovirus is transmitted via the faecaloral route and is efficiently transferred to sentinel mice by soiled bedding [370, 371] . mnv infection can be detected directly by rt-pcr on faecal pellets or tissue specimens (see above) and indirectly by serology (mfia, elisa, ifa) [363, 367, 369] . detection is facilitated by high stability of mnv rna in faeces (at least 2 weeks at room temperature) [371] and by broad serological cross-reactivity among different strains of mnv [367, 368] . embryo transfer [370] and hysterectomy [369] are most likely effective means of eliminating mnv from mouse colonies. since 1-to 3-day-old pups are resistant to infection, elimination of mnv may also be achieved by transferring neonates from infected dams to uninfected foster dams ('cross-fostering') [372] . this transfer should ideally be performed within 24 h after birth. mnv is used as a surrogate to evaluate resistance of human noroviruses to disinfectants. the impact of mnv on animal experiments remains to be evaluated. recent studies show that mnv is immunmodulatory and may alter disease phenotypes in mouse models of inflammatory bowel disease [373] [374] [375] and other experimental mouse models [376, 377] . murine pneumonia virus, commonly referred to as 'pneumonia virus of mice' (pvm), is an enveloped, single-stranded rna virus of the family paramyxoviridae, genus pneumovirus. it is closely related to human respiratory syncytial virus (hrsv). the virus name is officially abbreviated as 'mpv' according to the international union of microbiological societies [9] ; however, the former designation 'pvm' will be used in this chapter to avoid confusion with the official abbreviation of mouse parvovirus (mpv). pvm infection remains prevalent in contemporary colonies of mice and rats throughout the world. a serological survey in france demonstrated antibodies to pvm in 16% of mouse colonies examined [10] . in more recent studies in north america and western europe, the prevalence of pvm-specific antibodies in mice ranged between 0% and 0.1% [11, 14, 15] . schoondermark-van de ven et al. [12] found antibodies to pvm in 0.2% of mouse samplings from western european institutions. antibodies to pvm have also been detected in hamsters, gerbils, cotton rats, guinea-pigs and rabbits [8, 378, 379] . experimentally, pvm infection of mice is used as a model for hrsv infection and has therefore been extensively studied (reviewed by rosenberg and domachowske [380] ). in immunocompetent mice, natural infection with pvm is transient and usually not associated with clinical disease or pathological findings [8, 379, 381] . however, natural disease and persistent infection may occur in immunodeficient mice [382] [383] [384] . in particular, athymic foxn1 nu mice seem to be susceptible to pvm infection, which can result in dyspnoea, cyanosis, emaciation and death due to pneumonia [383, 384] . similar clinical signs have been reported for experimentally infected immunocompetent mice [385] . necropsy findings in naturally infected foxn1 nu mice include cachexia and diffuse pulmonary oedema or lobar consolidation [384] . pulmonary consolidation (dark red or grey in color) has also been found after experimental infection of immunocompetent mice [381] . histologically, natural infection of foxn1 nu mice with pvm presents as interstitial pneumonia [383, 384] . experimental intranasal inoculation of immunocompetent mice can result in rhinitis, erosive bronchiolitis and interstitial pneumonia with prominent early pulmonary eosinophilia and neutrophilia [381, 386] . hydrocephalus may result from intracerebral inoculation of neonatal mice [387] . susceptibility to infection is influenced by age and strain of mouse, dose of virus, and a variety of local and systemic stressors [8, 379, 386] . in terms of the extent of the alveolar inflammatory response, 129/sv and dba/2 mice are susceptible to pvm infection, while balb/c and c57bl/6 mice are relatively resistant [386] . in terms of the control of viral replication, mice of strains 129/sv, dba/2, balb/c and c57bl/6 are susceptible to pvm infection, while sjl mice are relatively resistant. pvm is labile in the environment and rapidly inactivated at room temperature [8, 379] . the virus is tropic for the respiratory epithelium [382, 385] , and transmission is exclusively horizontal via the respiratory tract, mainly by direct contact and aerosol [8, 379] . therefore, transmissibility in mouse colonies is low, and infections tend to be focal enzootics. serology (mfia, elisa, ifa or hi) is the primary means of testing mouse colonies for exposure to pvm. immunohistochemistry has been applied to detect viral antigen in lung sections [382, 384] ; however, proper sampling (see chapter 4.4, 'health management and monitoring') is critical for establishing the diagnosis due to the focal nature of the infection. an rt-pcr assay to detect viral rna in respiratory tract tissues has also been reported [388] . however, the use of direct methods requires good timing because the virus is present for only up to about 10 days in immunocompetent mice [381] . embryo transfer or caesarean derivation followed by barrier maintenance can be used to rear mice that are free of pvm. because active infection is present in the individual immunocompetent mouse for only a short period, strict isolation of a few (preferably seropositive) mice with the temporary cessation of breeding might also be successful in eliminating the virus [8, 378] . pvm could interfere with studies involving the respiratory tract or immunological measurements in mice. in addition, pvm can have devastating effects on research using immunodeficient mice because they are particularly prone to develop fatal disease [383, 384] or become more susceptible to the deleterious effects of other agents such as p. murina [389] . murv-a/edim (commonly referred to as 'mouse rotavirus' or 'epizootic diarrhoea of infant mice virus') is a non-enveloped, segmented double-stranded rna virus of the family reoviridae, genus rotavirus. it is antigenically classified as a group a rotavirus, similar to rotaviruses of many other species that cause neonatal and infantile gastroenteritis [291] . murv-a/edim infection remains prevalent in contemporary mouse colonies and appears to occur worldwide. large commercial laboratories found 0.6% to 9% of mouse sera from north american and european facilities to be positive for antibodies against murv-a/edim [11, 12, 14, 15] , and up to 30% of mouse colonies in the usa were identified as affected in a survey performed in 2006 [13] . experimentally, murv-a/edim infection in mice is used as a model for human rotavirus infection, especially in investigations on the mechanisms of rotavirus immunity and in the development of vaccination strategies [390] . clinical symptoms following murv-a/edim infection range from inapparent or mild to severe, sometimes fatal, diarrhoea. 'epizootic diarrhoea of infant mice' describes the clinical syndrome associated with natural or experimental infection by murv-a/edim during the first 2 weeks of life [8, 36, 291, 391, 392] . diarrhoea usually begins around 48 h after infection and persists for about 1 week. affected suckling mice have soft, yellow faeces that wet and stain the perianal region (figure 3.2.8) . in severe instances, the mice may be stunted, have dry scaly skin, or are virtually covered with faecal material. morbidity is very high but mortality is usually low. gross lesions in affected mice are confined to the intestinal tract. the caecum and colon may be distended with gas and watery to paste-like contents that are frequently bright yellow. the stomach of diarrhoeic mice is almost always filled with milk, and this feature has been reported to be a reliable means to differentiate diarrhoea caused by rotavirus from the diarrhoea caused by mhv infection. histopathological changes may be subtle even in animals with significant diarrhoea (figure 3.2.8 ). they are most prominent at the apices of villi, where rotaviruses infect and replicate within epithelial cells; the large intestinal surface mucosa may also be affected. though inflammation is minimal, the lamina propria may be oedematous, lymphatics may be dilated and mild leukocytic infiltration in the large intestinal mucosa and submucosa has been observed in a recent outbreak of disease [36, 393] . hydropic change of villous epithelial cells is the hallmark finding of acute disease. the villi become shortened, and the cells that initially replace the damaged cells are less differentiated, typically cuboidal instead of columnar, and lack a full complement of enzymes for digestion and absorption, resulting in diarrhoea due to maldigestion and malabsorption. undigested milk in the small intestine promotes bacterial growth and exerts an osmotic effect, exacerbating damage to the villi. intestinal fluid and electrolyte secretion is further enhanced by activation of the enteric nervous system [394] and through the effects of a viral enterotoxin called nsp4 (for non-structural protein 4) [395] . it is hypothesized that nsp4 is released from virus-infected cells and then triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. susceptibility to edim depends on the age of the host and peaks between 4 and 14 days of age [8, 36, 291, 391, 392] . mice older than about 2 weeks can still be infected with murv-a/edim, but small numbers of enterocytes become infected, there is little replication of virus and diarrhoea does not occur. the exact reason for this agerelated resistance to disease is unknown. pups suckling from immune dams are protected against edim during their period of disease susceptibility [396] . in general, the infection is self-limiting and resolves within days. successful viral control and clearance is promoted by an intact immune response [396] [397] [398] [399] , and some immunodeficient mice (e.g. prkdc scid and rag2 tm1fwa mice) may shed virus for extended periods or become persistently infected [400, 401] . protection against murv-a/edim reinfection is primarily mediated by antibodies [396, 397] . murine rotavirus-a/edim is highly contagious and transmitted by the faecal-oral route [8, 291, 391] . dissemination of the virus occurs through direct contact or contaminated fomites and aerosols and is facilitated by the general property of rotaviruses that they remain infectious outside the body, show resistance to inactivation (e.g. low ph, non-ionic detergents, hydrophobic organic liquids, proteolytic enzymes), and are shed in high quantities (>10 11 particles/g faeces) [291] . murv-a/edim is stable at à70 c but otherwise tends to be susceptible to extreme environmental conditions, detergents and disinfectants containing phenols, chlorine or ethanol [291] . mfia, elisa and ifa are in widespread use for detection of serum antibodies to murv-a/ edim in diagnostic and health surveillance programmes; other assay systems such as those using latex agglutination are also used [402] . as murv-a/edim shares the vp6 protein determined group a antigen, for example, with human, simian or bovine rotavirus strains, commercially available elisa assays utilizing polyclonal or monoclonal antibodies have been used to detect rotavirus antigen in mice; however, great care must be taken in interpreting the results because some feeds have been reported to cause false-positive reactions with certain elisa kits [403] . electron microscopy of faeces of diarrhoeic pups should reveal typical wheel-shaped rotavirus particles, 60-80 nm in diameter. rt-pcr also can be used to detect rotavirus rna in faecal samples [404] . good timing is critical for establishing the diagnosis from faeces because virus is shed for only a few days in immunocompetent mice. embryo transfer or caesarean derivation followed by barrier maintenance is recommended for rederivation of breeding stocks [8] . in immunocompetent mice in which infection is effectively cleared, a breeding suspension strategy for 8-10 weeks combined with excellent sanitation, filter tops and conscientious serological testing of offspring and sentinel mice has also been reported to be effective, and prolongation of breeding cessation up to 12 weeks resolved infection even in immunocompromised mice [393] . murv-a/edim has the potential to interfere with any research using suckling mice. it may have a significant impact on studies where the intestinal tract of neonatal or infant mice is the target organ. the infection also poses a problem for infectious disease and immune response studies, particularly those involving enteropathogens in infant mice [405] . a disease-induced stress-related thymic necrosis may occur and alter immunology experiments [36] . in addition, runting could be interpreted erroneously as the effect of genetic manipulation or other experimental manipulation. sendai virus (sev) is an enveloped, singlestranded rna virus of the family paramyxoviridae, genus respirovirus. it is antigenically related to human parainfluenza virus 1. the virus was named after sendai, japan, where it was first isolated from mice. historically, infections were relatively common in mouse and rat colonies worldwide. in addition, there is evidence that hamsters, guinea-pigs and rabbits are susceptible to infection with sev [8, 301, 406, 407] ; however, some apparently seropositive guinea-pigs may in fact be seropositive to other parainfluenza viruses instead of sev. a study in france reported antibodies to sev in 17% of mouse colonies examined [10] . a low rate of seropositive mice (0.2%) was found in a survey in north america [11] . schoondermark-van de ven et al. [12] also found antibodies to sev in 0.2% of mouse samplings from western european institutions. in more recent surveys in north america and western europe, sev infection was not detected [13] [14] [15] , indicating that sev, like most viruses, has meanwhile been eliminated from the majority of mouse colonies. sev can contaminate biological materials [1] . sev is pneumotropic and can cause significant respiratory disease in mice. the pneumotropism is partially a consequence of the action of respiratory serine proteases such as tryptase clara, which activate viral infectivity by specific cleavage of the viral fusion glycoprotein [408] . in addition, the apical budding behaviour of sev may hinder the spread of virus into subepithelial tissues and subsequently to distant organs via the blood. two epidemiologic patterns of sev infection have been recognized, an enzootic (subclinical) and epizootic (clinically apparent) type [8, 379, 409] . enzootic infections commonly occur in breeding or open colonies, where the constant supply of susceptible animals perpetuates the infection. in breeding colonies, mice are infected shortly after weaning as maternal antibody levels wane. normally, the infection is subclinical, with virus persisting for approximately 2 weeks, accompanied by seroconversion that persists for a year or longer. epizootic infections occur upon first introduction of the virus to a colony and either die out (self-cure) after 2-7 months or become enzootic depending on colony conditions. the epizootic form is generally acute, and morbidity is very high, resulting in nearly all susceptible animals becoming infected within a short time. clinical signs vary and include rough hair coat, hunched posture, chattering, respiratory distress, prolonged gestation, death of neonates and sucklings and runting in young mice. breeding colonies may return to normal productivity within 2 months and thereafter maintain the enzootic pattern of infection. factors such as strain susceptibility, age, husbandry, transport and copathogens are important in precipitating overt disease. dba and 129 strains of mice are very susceptible to sev pneumonia, whereas sjl/j and c57bl/6/j and several outbred stocks are relatively resistant. resistance to sev infection is under multigenic control with epistatic involvement [410] . there is no evidence for persistent infection in immunocompetent mice, but persistent or prolonged infection may occur in immunodeficient mice and can result in wasting and death due to progressive pneumonia [411, 412] . clearance of a primary sev infection is mediated by cd8þ and cd4þ t-cell mechanisms [413, 414] . heavier than normal, consolidated, plumcolored or grey lungs are a characteristic gross finding in severe sev pneumonia [8, 36, 379, 409] . lymphadenopathy and splenomegaly reflect the vigorous immune response to infection. histologically, three phases of disease can be recognized in susceptible immunocompetent mice: acute, reparative and resolution phases [36, 409] . lesions of the acute phase, which lasts 8-12 days, are primarily attributed to the cellmediated immune response that destroys infected respiratory epithelial cells and include necrotizing rhinitis, tracheitis, bronch(iol)itis and alveolitis. epithelial syncytiae and cytoplasmic inclusion bodies in infected cells may be seen early in this phase. alveoli contain sloughed necrotic epithelium, fibrin, neutrophils and mononuclear cells. atelectasis, bronchiectasis and emphysema may occur as a result of damage and obstruction of airways. the reparative phase, which may overlap the acute phase but continues through about the third week after infection, is indicated by regeneration of airway lining epithelium. adenomatous hyperplasia and squamous metaplasia (with multilayered flat epithelial cells instead of normal columnar cells) in the terminal bronchioles and alveoli are considered to be a hallmark of sev pneumonia. mixed inflammatory cell infiltrates in this phase tend to be primarily interstitial, rather than alveolar, as they are in the acute phase. the resolution phase may be complete by the fourth week after infection and lesions may be difficult to subsequently identify. residual, persistent lesions that may occur include organizing alveolitis and bronchiolitis fibrosa obliterans. alveoli and bronchioles are replaced by collagen and fibroblasts, foamy macrophages and lymphoid infiltrates, often with foci of emphysema, cholesterol crystals and other debris, which represent attempts to organize and wall off residual necrotic debris and fibrin. lesions are more severe and variable when additional pathogens such as mycoplasma pulmonis are present [8] . otitis media has also been reported in natural infections with sev although some of these studies have been complicated by the presence of other pathogens [415] . sev has been detected in the inner ear after experimental intracerebral inoculation of neonatal mice [416] . sev is extremely contagious. infectious virus is shed during the first 2 weeks of infection and appears to be transmitted by direct contact, contaminated fomites and respiratory aerosol [8, 379] . serology (mfia, elisa, ifa, or hi) is the approach of choice for routine monitoring because serum antibodies to sev are detectable soon after infection and persist at high levels for many months, although active infection lasts only 1-2 weeks in immunocompetent mice. the short period of active infection limits the utility of direct methods such as immunohistochemistry [382] and rt-pcr [388, 417] . although sev is considered to be highly contagious, studies have shown that dirty bedding sentinel systems do not reliably detect the infection and that outbred stocks may not seroconvert consistently [418, 419] . map testing and rt-pcr can be used to detect sev in contaminated biological materials. sev infection in mouse colonies has proved to be one of the most difficult virus infections to control because the virus is highly infectious and easily disseminated. depopulation of infected colonies is probably the most appropriate means of eliminating the virus in most situations. embryo transfer, or caesarean derivation, followed by barrier maintenance, can also be used to eliminate the virus [8, 379] . a less effective alternative is to place the infected animals under strict quarantine, remove all young and pregnant mice, suspend all breeding and prevent addition of other susceptible animals for approximately 2 months until the infection is extinguished, and then breeding and other normal activities are resumed. vaccines against the virus have been developed [8, 379, 409] , but these probably do not represent a practical means to achieve or maintain the seronegative status of colonies that is in demand today. sev has the potential to interfere with a wide variety of research involving mice. reported effects include interference with early embryonic development and fetal growth; alterations of macrophage, nk-cell, and t-and b-cell function; altered responses to transplantable tumours and respiratory carcinogens; altered isograft rejection; and delayed wound healing (reviewed in [6] [7] [8] ). pulmonary changes during sev infection can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other agents. they could also affect the response to anaesthetics. in addition, natural sev infection would interfere with studies using sev as a gene vector. theiler's murine encephalomyelitis virus (tmev), or murine poliovirus, is a member of the genus cardiovirus in the family picornaviridae. members of this genus are non-enveloped viruses with single-stranded rna. the virus is rapidly destroyed at temperatures above 50 c. it is considered to be a primary pathogen of the cns of mice and can cause clinical disease resembling that due to poliomyelitis virus infections in humans. antibodies to tmev have been identified in mouse colonies and feral populations worldwide, and mus musculus is considered to be the natural host of tmev [420] . the best-known and most frequently mentioned tmev strain is gdvii, which is virulent for mice. infant or young hamsters and laboratory rats are also susceptible to intracerebral infection. the original isolate is designated to (theiler's original) and represents a group of tmev strains with low virulence for mice. many additional virus strains have been isolated and studied, and they all fall in the broad grouping of to and gdvii. a similar virus strain has also been isolated from rats, but in contrast to mouse isolates, this virus is not pathogenic for rats and mice after intracerebral inoculation [421] . recently, another rat isolate has been characterized and shown to be most closely related to, but quite distinct from, other tmev viruses [422] . antibodies to tmev (strain gdvii) have been detected in guinea-pigs and are considered to indicate infection with another closely related cardiovirus [423] . seropositivity to tmev was reported in approximately 48% of french mouse colonies in a retrospective study [10] . in more recent studies, the prevalence of tmev infections was found to be lower. schoondermark-van de ven et al. [12] detected antibodies to tmev in 2.2% of mouse samplings from western european institutions. in a survey conducted by carty [13] , about 9% of responding institutions in the usa reported tmev infection in their mouse colonies. further surveys in north america and western europe revealed antibodies in 0.09-0.26% of mice monitored [11, 14, 15] . tmev is primarily an enteric pathogen, and virus strains are enterotropic. in natural infections, virus can be detected in intestinal mucosa and faecal matter, and in some cases it is also found in the mesenteric lymph nodes. however, histological lesions in the intestine are not discerned. virus may be shed via intestinal contents for up to 22 weeks, sometimes intermittently [424] , and transmission under natural conditions is via the faecal-oral route, by direct contact between mice, as well as by indirect contact (e.g. dirty bedding). the host immune response limits virus spread, but it does not immediately terminate virus replication in the intestines. virus is cleared from extraneural tissues, but persists in the cns for at least a year. clinical disease due to natural tmev infection is rare, with a rate of only 1 in 1000-10 000 infected immunocompetent animals [36] . in immunodeficient mice, especially in weanlings, clinical signs may be more common and mortality may be higher [425] . this group of viruses usually causes asymptomatic infections of the intestinal tract. they may spread to the cns as a rare event where they cause different neurological disease manifestations. the most typical clinical sign of tmev infection is flaccid paralysis of hindlimbs. the animals appear otherwise healthy, and there is no mortality. experimental infection in mice provides models of poliomyelitis-like infection and virusinduced demyelinating disease including multiple sclerosis [426] . after experimental infection, tmev causes a biphasic disease in susceptible strains of mice. the acute phase is characterized by early infection of neurons in the grey matter. encephalomyelitis may develop during this phase and may be fatal, but most animals survive and enter the second phase of the disease at 1-3 months after the acute phase. this phase is characterized by viral persistence in the spinal cord white matter, mainly in macrophages, and leads to white matter demyelination. persistence and demyelination occur only in genetically susceptible mouse strains, while resistant strains clear the infection after early grey matter encephalomyelitis through a cytotoxic t lymphocyte response. the severity and nature of disease depend on virus strain, route of inoculation, host genotype and age [8, 36, 427] . in general, virus isolates with low virulence produce persistent cns infection in mice whereas virulent strains are unable to cause persistent infection. intracerebral inoculation results in the most severe infections, but the intranasal route is also effective. experimental intracerebral infections with virulent fa and gdvii strains of tmev are more likely to cause acute encephalomyelitis and death in weanling mice 4-5 days after inoculation ('early disease'). death may be preceded by neurological manifestations of encephalitis such as hyperexcitability, convulsions, tremors, circling, rolling and weakness. animals may develop typical flaccid paralysis of hindlimbs, and locomotion is possible only by use of the forelimbs. interestingly, the tail is not paralyzed. experimental infections with low-virulence virus strains (e.g. to, da, ww) are more likely to cause persistent infection with development of mild encephalomyelitis followed by a chronic demyelinating disease after a few months ('late disease'). these virus strains infect neurons in the grey matter of the brain and spinal cord during the acute phase of viral growth, followed by virus persistence in macrophages and glial cells in the spinal cord white matter. sjl, swr and dba/2 strains are most susceptible to this chronic demyelinating disease. cba and c3h/he are less susceptible strains, and strains a, c57bl/6, c57bl/10 and dba/1 are relatively resistant [428] . differences in humoral immune responses play a role in resistance to tmev infection [429] , but genetic factors are also important. several genetic loci implicated in susceptibility to virus persistence, demyelination, or clinical disease have been identified, including the h-2d region of the major histocompatibility complex [430] . furthermore, the age at infection influences the severity of clinical disease. in infant mice, intracerebral infection with low-virulence virus strains (e.g. to) is often lethal. young mice develop paralysis after an incubation period of 1-4 weeks while adult mice often show no clinical signs of infection. the only gross lesions are secondary to the posterior paralysis and may include urine scald or dermatitis due to incontinence of urine and trauma to paralyzed limbs, or wasting or atrophy of the hindlimbs in long-term survivors. tmev infects neurons and glial cells, and histological changes in the cns include nonsuppurative meningitis, perivasculitis and poliomyelitis with neuronolysis, neuronophagia and microgliosis in the brainstem and ventral horns of the spinal cord [36] . demyelination in immunocompetent mice is considered to be immunemediated. susceptible strains develop a specific delayed-type hypersensitivity response which is the basis for inflammation and demyelination. this reaction is mediated by t cells that release cytokines leading to recruitment of monocytes and macrophages as a consequence of infection of macrophages and other cns-resident cells [431] [432] [433] . protection from chronic demyelinating disease is possible by vaccination with live virus given previously by subcutaneous or intraperitoneal inoculation [434, 435] . early immunosuppression at the time of infection, e.g. by treatment with cyclophosphamide or antithymocyte serum, inhibits or diminishes demyelination. immunosuppression in mice chronically infected with tmev leads to remyelination of oligodendrocytes [436] . further details related to the pathogenesis of tmev infections and the role of immune mechanisms have been reviewed by yamada et al. [437] , kim et al. [432] and lipton et al. [433] . experimental infection of foxn1 nu mice results in acute encephalitis and demyelination. demyelination associated with minimal inflammation and neurological signs, including the typical hindlimb paresis, develop 2 weeks after inoculation, and most animals die within 4 weeks. in foxn1 nu mice, demyelination is caused by a direct lytic effect of the virus on oligodendrocytes [438] . demyelination and lethality are reduced after administration of neutralizing antibodies [439] . histopathological changes in prkdc scid mice are very similar to those in foxn1 nu mice [440] . young mice born in infected populations usually acquire infection shortly after weaning and are almost all infected by 30 days of age. intrauterine transmission to fetuses is possible during the early gestation period, but a placental barrier develops during gestation and later prevents intrauterine infection [441] . all tmev isolates are closely related antigenically and form a single serogroup, as determined by complement fixation and hi [427] . hemelt et al. 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phenotype different roles for cd4þ and cd8þ t lymphocytes and macrophage subsets in the control of a generalized virus infection correlates of protective immunity in poxvirus infection: where does antibody stand? transmission of mouse-pox in colonies of mice mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. iii. experimental transmission of infection and derivation of virus-free progeny from previously infected dams intrauterine infection of mice with ectromelia virus mouse pox threat serological detection of ectromelia virus antibody evaluation of an enzyme-linked immunosorbent assay for the detection of ectromelia (mousepox) antibody administration of vaccinia virus to mice may cause contact or bedding sentinel mice to test positive for orthopoxvirus antibodies: case report and follow-up investigation specific detection of mousepox virus by polymerase chain reaction real-time pcr system for detection of orthopoxviruses and 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experimentally exposed in vivo-derived embryos antineoplastic activity of parvoviruses experience with viral contamination in cell culture the molecular biology of arteriviruses lactic dehydrogenase virus isolation of lactate dehydrogenase-elevating viruses from wild house mice and their biological and molecular characterization lactate dehydrogenaseelevating virus induces systemic lymphocyte activation via tlr7-dependent ifnalpha responses by plasmacytoid dendritic cells distinct gamma interferon-production pathways in mice infected with lactate dehydrogenase-elevating virus lactate dehydrogenase-elevating virus replication persists in liver, spleen, lymph node, and testis tissues and results in accumulation of viral rna in germinal centers, concomitant with polyclonal activation of b cells transmissible agent associated with 26 types of experimental mouse neoplasms from bench to cageside: risk assessment for rodent pathogen contamination of cells and biologics lactic dehydrogenase virus (ldhv) contamination in human tumor xenografts and its elimination contamination of a monoclonal antibody with ldh-virus causes interferon induction infection of central nervous system cells by ecotropic murine leukemia virus in c58 and akr mice and in in utero-infected ce/j mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus lactate dehydrogenase-elevating virus regulation of transplacental virus infection by developmental and immunological factors: studies with lactate dehydrogenaseelevating virus transplacental lactate dehydrogenase-elevating virus (ldv) transmission: immune inhibition of umbilical cord infection, and correlation of fetal virus susceptibility with development of f4/80 antigen expression regulation of maternal-fetal virus transmission in immunologically reconstituted scid mice infected with lactate dehydrogenase-elevating virus immunofluorescent antibody response to lactic dehydrogenase virus in different strains of mice characterization of lactate dehydrogenase-elevating virus orf6 protein expressed by recombinant baculoviruses enzymatic amplification of lactate dehydrogenase-elevating virus detection of lactate dehydrogenase-elevating virus in transplantable mouse tumors by biological assay and rt-pcr assays and its removal from the tumor cell false negative results using rt-pcr for detection of lactate dehydrogenase-elevating virus in a tumor cell line comparison of the sensitivity of in vivo antibody production tests with in vitro pcr-based methods to detect infectious contamination of biological materials detection and typing of lactate dehydrogenase-elevating virus rna from transplantable tumors, mouse liver tissues, and cell lines, using polymerase chain reaction comparison of the mouse antibody production (map) assay and polymerase chain reaction (pcr) assays for the detection of viral contaminants relationship between the lactic dehydrogenase-elevating virus and transplantable murine tumors removal of lactate dehydrogenase-elevating virus from human-in-mouse breast tumor xenografts by cell-sorting infection of mice with lactate dehydrogenase-elevating virus leads to stimulation of autoantibodies suppression of development of diabetes in nod mice by lactate dehydrogenase virus infection natural killer cell activation after infection with lactate dehydrogenase-elevating virus effects of various adjuvants and a viral infection on the antibody specificity toward native or cryptic epitopes of a protein antigen lactate dehydrogenase-elevating virus infection at the sensitization and challenge phases reduces the development of delayed eosinophilic allergic rhinitis in balb/c mice suppression of acute anti-friend virus cd8þ t-cell responses by coinfection with lactate dehydrogenase-elevating virus mammalian reservoirs of arenaviruses risk to humans through contact with golden hamsters carrying lymphocytic choriomeningitis virus (author's transl) lymphocytic choriomeningitis virus first outbreak of callitrichid hepatitis in germany: genetic characterization of the causative lymphocytic choriomeningitis virus strains arenavirus-mediated liver pathology: acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin-6 expression and hepatocyte proliferation lymphocytic choriomeningitis virus spatial and temporal dynamics of lymphocytic choriomeningitis virus in wild rodents, northern italy antibodies to lymphocytic choriomeningitis virus in wild rodent sera in egypt seroepidemiological survey of lymphocytic choriomeningitis virus in wild house mice in china with particular reference to their subspecies lymphocytic choriomeningitis virus infection and house mouse (mus musculus) distribution in urban baltimore contamination of transplantable murine tumors with lymphocytic choriomeningitis virus human-rodent contact and infection with lymphocytic choriomeningitis and seoul viruses in an inner-city population seroprevalence of lymphocytic choriomeningitis virus in nova scotia lymphocytic choriomeningitis virus infection in a province of spain: analysis of sera from the general population and wild rodents mouse-tohuman transmission of variant lymphocytic choriomeningitis virus exposure to lymphocytic choriomeningitis virus lymphocytic choriomeningitis outbreak associated with nude mice in a research institute laboratory studies of a lymphocytic choriomeningitis virus outbreak in man and laboratory animals lymphocytic choriomeningitis virus in southern france: four case reports and a review of the literature lymphocytic choriomeningitis in laboratory personnel exposed to hamsters inadvertently infected with lcm virus pet rodents and fatal lymphocytic choriomeningitis in transplant patients outbreak of lymphocytic choriomeningitis virus infections in medical center personnel virus zoonoses and their potential for contamination of cell cultures transmission of lymphocytic choriomeningitis virus by organ transplantation lymphocytic choriomeningitis virus: an unrecognized teratogenic pathogen lymphocytic choriomeningitis virus: reemerging central nervous system pathogen lymphocytic choriomeningitis virus: emerging fetal teratogen persistence of lymphocytic choriomeningitis virus at very low levels in immune mice mechanisms of antibody-mediated protection against lymphocytic choriomeningitis virus infection: mother-to-baby transfer of humoral protection murine hepatitis caused by lymphocytic choriomeningitis virus. ii. cells involved in pathogenesis biology and pathogenesis of lymphocytic choriomeningitis virus infection lymphocytic choriomeningitis infection of the central nervous system murine infection with lymphocytic choriomeningitis virus following gastric inoculation timed appearance of lymphocytic choriomeningitis virus after gastric inoculation of mice lymphocytic choriomeningitis infection undetected by dirty-bedding sentinel monitoring and revealed after embryo transfer of an inbred strain derived from wild mice detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by elisa using purified recombinant nucleoprotein development of a reverse transcription-polymerase chain reaction assay for diagnosis of lymphocytic choriomeningitis virus infection and its use in a prospective surveillance study detection of lymphocytic choriomeningitis virus by use of fluorogenic nuclease reverse transcriptase-polymerase chain reaction analysis quantitative pcr technique for detecting lymphocytic choriomeningitis virus in vivo centers for disease control and prevention and national institutes of health mechanisms of humoral immunity explored through studies of lcmv infection lymphocytic choriomeningitis virus and immunology viral persistence: parameters, mechanisms and future predictions stage of primary infection with lymphocytic choriomeningitis virus determines predisposition or resistance of mice to secondary bacterial infections reovirus type 3 infection, liver, mouse the mouse in biomedical research. diseases reovirus infection in laboratory rodents direct spread of reovirus from the intestinal lumen to the central nervous system through vagal autonomic nerve fibers type 3 reovirus neuroinvasion after intramuscular inoculation: direct invasion of nerve terminals and age-dependent pathogenesis reoviruses and the host cell orthoreoviruses and their replication passive immunity to fatal reovirus serotype 3-induced meningoencephalitis mediated by both secretory and transplacental factors in neonatal mice infectivity, disease patterns, and serologic profiles of reovirus serotypes 1, 2, and 3 in infant and weanling mice reovirus-induced liver disease in severe combined immunodeficient (scid) mice. a model for the study of viral infection, pathogenesis, and clearance histopathological characterization of the naturally occurring hepatotropic virus infections of nude mice detection methods for the identification of rodent viral and mycoplasmal infections reverse transcriptionpolymerase chain reaction detection and nucleic acid sequence confirmation of reovirus infection in laboratory mice with discordant serologic indirect immunofluorescence assay and enzyme-linked immunosorbent assay results diagnosis of murine infections in relation to test methods employed reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease detection of reovirus type 3 by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction detection of reovirus by reverse transcription-polymerase chain reaction using primers corresponding to conserved regions of the viral l1 genome segment isolation of a non-pathogenic tumour-destroying virus from mouse ascites an oncolytic virus recovered from swiss mice during passage of an ascites tumour mouse hepatitis virus enterotropic mouse hepatitis virus effects of air temperature and relative humidity on coronavirus survival on surfaces asymptomatic infection of mouse hepatitis virus in the rat effects of experimental infection of the deer mouse (peromyscus maniculatus) with mouse hepatitis virus isolation of a latent murine hepatitis virus from cultured mouse liver cells induction of lytic plaques by murine leukemia virus in murine sarcoma virus-transformed nonproducer mouse cells persistently infected with mouse hepatitis virus mhv-s mouse hepatitis virus biology and epizootiology the cellular and molecular pathogenesis of coronaviruses enterotropic coronavirus (mouse hepatitis virus) in mice: influence of host age and strain on infection and disease response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus jhm duration of mouse hepatitis virus infection: studies in immunocompetent and chemically immunosuppressed mice effective clearance of mouse hepatitis virus from the central nervous system requires both cd4þ and cd8þ t cells role of cd4þ and cd8þ t cells in mouse hepatitis virus infection in mice antibody prevents virus reactivation within the central nervous system mouse hepatitis virus enterotropic mouse hepatitis virus infection in nude mice persistent transmission of mouse hepatitis virus by transgenic mice duration of challenge immunity to coronavirus jhm in mice virus strain specificity of challenge immunity to coronavirus duration and strain-specificity of immunity to enterotropic mouse hepatitis virus passively acquired challenge immunity to enterotropic coronavirus in mice epizootic coronaviral typhlocolitis in suckling mice isolation of mouse hepatitis virus from infant mice with fatal diarrhea thymus involution induced by mouse hepatitis virus a59 in balb/c mice adverse effects of mouse hepatitis virus on ascites myeloma passage in the balb/ej mouse murine hepatitis virus strain 1 produces a clinically relevant model of severe acute respiratory syndrome in a/j mice tolllike receptor 4 deficiency increases disease and mortality after mouse hepatitis virus type 1 infection of susceptible c3h mice granulomatous peritonitis and pleuritis in interferon-gamma gene knockout mice naturally infected with mouse hepatitis virus pathogenesis of enterotropic mouse hepatitis virus in immunocompetent and immunodeficient mice vertical transmission of mouse hepatitis virus infection in mice tissue distribution and duration of mouse hepatitis virus in naturally infected immunocompetent icr (cd-1) and immunodeficient athymic nudenu mouse strains used for ovarian transplantation and in vitro fertilization rederivation of inbred strains of mice by means of embryo transfer risk assessment of mouse hepatitis virus infection via in vitro fertilization and embryo transfer by the use of zona-intact and laser-microdissected oocytes mouse hepatitis virus immunofluorescence in formalin-or bouin's-fixed tissues using trypsin digestion comparison of isolation in cell culture with conventional and modified mouse antibody production tests for detection of murine viruses monoclonal antibody solution hybridization assay for detection of mouse hepatitis virus infection detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction detection of mouse hepatitis virus by the polymerase chain reaction and its application to the rapid diagnosis of infection detection of rodent coronaviruses by use of fluorogenic reverse transcriptase-polymerase chain reaction analysis an immunofluorescence test for detection of serum antibody to rodent coronaviruses simultaneous detection of antibodies to mouse hepatitis virus recombinant structural proteins by a microsphere-based multiplex fluorescence immunoassay differences in antibody production against mouse hepatitis virus (mhv) among mouse strains maternally-derived passive immunity to enterotropic mouse hepatitis virus mouse hepatitis virus: molecular biology and implications for pathogenesis maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus replication of murine coronaviruses in mouse embryonic stem cell lines. in vitro reliability of soiled bedding transfer for detection of mouse parvovirus and mouse hepatitis virus efficacy of three microbiological monitoring methods in a ventilated cage rack rederivation of mhv and mev antibody positive mice by cross-fostering and use of the microisolator caging system elimination of mouse hepatitis virus from a breeding colony by temporary cessation of breeding evolution of mouse hepatitis virus (mhv) during chronic infection: quasispecies nature of the persisting mhv rna the elimination of mouse hepatitis virus by temporary transplantation of human tumors from infected athymic nude mice into athymic nude rats (rnun/rnun) development of a microsphere-based serologic multiplexed fluorescent immunoassay and a reverse transcriptase pcr assay to detect murine norovirus 1 infection in mice stat1-dependent innate immunity to a norwalk-like virus murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by stat1-dependent interferon responses replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages persistent infection with and serologic cross-reactivity of three novel murine noroviruses murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence molecular detection of murine norovirus from experimentally and spontaneously infected mice naturally occurring murine norovirus infection in a large research institution soiled-bedding sentinel detection of murine norovirus 4 the use of cross-foster rederivation to eliminate murine norovirus, helicobacter spp., and murine hepatitis virus from a mouse colony impact of murine norovirus on a mouse model of ibd virus-plus-susceptibility gene interaction determines crohn's disease gene atg16l1 phenotypes in intestine murine norovirus: an intercurrent variable in a mouse model of bacteria-induced inflammatory bowel disease investigation of the impact of the common animal facility contaminant murine norovirus on experimental murine cytomegalovirus infection effects of murine norovirus infection on a mouse model of diet-induced obesity and insulin resistance mouse adenovirus, k virus, and pneumonia virus of mice sendai virus and pneumonia virus of mice (pvm) pneumonia virus of mice: severe respiratory infection in a natural host pneumonia virus of mice infection, lung, mouse, and rat persistence of pneumonia virus of mice and sendai virus in germ-free (nu/nu) mice fatal pneumonia with terminal emaciation in nude mice caused by pneumonia virus of mice respiratory disease and wasting in athymic mice infected with pneumonia virus of mice pathogenesis of pneumovirus infections in mice: detection of pneumonia virus of mice and human respiratory syncytial virus mrna in lungs of infected mice by in situ hybridization differential resistance/susceptibility patterns to pneumovirus infection among inbred mouse strains experimental pneumovirus infections: 1. hydrocephalus of mice due to infection with pneumonia virus of mice (pvm) detection of sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis lethal exacerbation of pneumocystis murina. pneumonia in severe combined immunodeficiency mice after infection by pneumonia virus of mice handbook of animal models of infection mouse rotavirus murine rotavirus infection, mouse successful sanitation of an edim-infected mouse colony by breeding cessation role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhoea age-dependent diarrhoea induced by a rotaviral nonstructural glycoprotein the immunology of rotavirus infection in the mouse murine model of rotavirus infection evidence that resolution of rotavirus infection in mice is due to both cd4 and cd8 celldependent activities ifn-l determines the intestinal epithelial antiviral host defense persistent rotavirus infection in mice with severe combined immunodeficiency role of b cells and cytotoxic t lymphocytes in clearance of and immunity to rotavirus infection in mice comparison of methods for detection of serum antibody to murine rotavirus identification of nonspecific reactions in laboratory rodent specimens tested by rotazyme rotavirus elisa removal of inhibitory substances from human fecal specimens for detection of group a rotaviruses by reverse transcriptase and polymerase chain reactions synergistic rotavirus and escherichia coli diarrhoeal infection of mice infection of rabbits with sendai virus pathogenesis of sendai virus infection in the syrian hamster determinants of organ tropism of sendai virus sendai virus infection, lung, mouse, and rat multigenic control of resistance to sendai virus infection in mice naturally-occurring sendai virus infection of athymic nude mice signs and lesions of experimental sendai virus infection in two genetically distinct strains of scid/beige mice cooperation between cytotoxic and helper t lymphocytes in protection against lethal sendai virus infection. protection by t cells is mhc-restricted and mhc-regulated; a model for mhc-disease associations delayed clearance of sendai virus in mice lacking class i mhc-restricted cd8þ t cells naturally occurring sendai virus disease of mice affinity of sendai virus for the inner ear of mice detection of nucleoprotein gene of sendai virus in the lungs of rats by touchdown nested reverse transcription polymerase chain reaction the effectiveness of a microisolator cage system and sentinel mice for controlling and detecting mhv and sendai virus infections the efficacy of a dirty bedding sentinel system for detecting sendai virus infection in mice: a comparison of clinical signs and seroconversion serological evidence that mus musculus is the natural host of theiler's murine encephalomyelitis virus comparison of mhg virus with mouse encephalomyelitis viruses genetic analysis of a theiler-like virus isolated from rats a serological indication of the existence of a guineapig poliovirus duration and patterns of transmission of theiler's mouse encephalomyelitis virus infection a spontaneous outbreak of theiler's encephalomyelitis in a colony of severe combined immunodeficient mice in the uk axonal loss results in spinal cord atrophy, electrophysiological abnormalities and neurological deficits following demyelination in a chronic inflammatory model of multiple sclerosis the theiler's murine encephalomyelitis viruses susceptibility of inbred mice to chronic central nervous system infection by theiler's murine encephalomyelitis virus role of the humoral immune response in resistance to theiler's virus infection the genetics of the persistent infection and demyelinating disease caused by theiler's virus infection with theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via nf-kappab activation: potential role for the initiation of demyelinating disease innate immune response induced by theiler's murine encephalomyelitis virus infection cardioviruses: encephalomyocarditis virus and theiler's murine encephalomyelitis virus effect of immunization with theiler's virus on the course of demyelinating disease protection of sjl/j mice from demyelinating disease mediated by theiler's murine encephalomyelitis virus immunosuppression promotes cns remyelination in chronic virus-induced demyelinating disease pathogenesis of theiler's murine encephalomyelitis virus mechanism of theiler's virus-induced demyelination in nude mice survival of athymic (nu/nu) mice after theiler's murine encephalomyelitis virus infection by passive administration of neutralizing monoclonal antibody vacuolar neuronal degeneration in the ventral horns of scid mice in naturally occurring theiler's encephalomyelitis evolution of the placental barrier to fetal infection by murine enteroviruses enhanced detection of theiler's virus rna copy equivalents in the mouse central nervous system by real-time rt-pcr spontaneous demyelinating myelopathy in aging laboratory mice key: cord-310942-191m0e65 authors: boga, jose antonio; coto‐montes, ana; rosales‐corral, sergio a.; tan, dun‐xian; reiter, russel j. title: beneficial actions of melatonin in the management of viral infections: a new use for this “molecular handyman”? date: 2012-04-18 journal: rev med virol doi: 10.1002/rmv.1714 sha: doc_id: 310942 cord_uid: 191m0e65 melatonin (n‐acetyl‐5‐methoxytryptamine) is a multifunctional signaling molecule that has a variety of important functions. numerous clinical trials have examined the therapeutic usefulness of melatonin in different fields of medicine. clinical trials have shown that melatonin is efficient in preventing cell damage under acute (sepsis, asphyxia in newborns) and chronic states (metabolic and neurodegenerative diseases, cancer, inflammation, aging). the beneficial effects of melatonin can be explained by its properties as a potent antioxidant and antioxidant enzyme inducer, a regulator of apoptosis and a stimulator of immune functions. these effects support the use of melatonin in viral infections, which are often associated with inflammatory injury and increases in oxidative stress. in fact, melatonin has been used recently to treat several viral infections, which are summarized in this review. the role of melatonin in infections is also discussed herein. copyright © 2012 john wiley & sons, ltd. the methoxyindole melatonin (n-acetyl-5-methoxytryptamine) is a secretory product of the pineal gland. it was first reported as a skin lightening agent in amphibians [1, 2] . further investigations showed that another function, supported by its direct effects in regions containing high densities of melatonin receptors, such as the circadian pacemaker (the suprachiasmatic nucleus) and the pars tuberalis, is to regulate and reset circadian rhythms as well as to be involved in the measurement of day length, an environmental variable used for seasonal timing of reproduction, metabolism and behavior in species responding to photoperiodic changes [3] [4] [5] [6] [7] . in recent decades, melatonin has been reported to possess numerous additional functions and act in neural and non-neural tissues or cells that express melatonin receptors that are at lower densities than in the suprachiasmatic nucleus. thus, melatonin is involved in sleep initiation, vasomotor control, anti-excitatory actions, immunomodulation including possessing anti-inflammatory properties, antioxidant actions, and actions on energy metabolism, influences on mitochondrial electron flux, regulation of the mitochondrial permeability transition pore (mtptp), and mitochondrial protection against free radicals [8] [9] [10] [11] [12] [13] . deficiencies in melatonin production or melatonin receptor expression and decreases in melatonin levels (such as those that occur during aging) are likely to contribute to numerous dysfunctions [14] [15] [16] . in fact, several clinical trials have shown that melatonin is efficient in preventing cell damage under acute (sepsis, asphyxia in newborns) and chronic states (metabolic and neurodegenerative diseases, cancer, inflammation, aging) [17] [18] [19] [20] [21] [22] . in humans, the efficacy of melatonin as a treatment of ocular diseases, cardiovascular diseases, sleep disturbances and several other pathologies, as well as a complementary treatment in anesthesia, haemodialysis, in vitro fertilization and neonatal care, has been assessed and reported to be beneficial [23] . likewise, melatonin reduces the toxicity and increases the efficacy of a large number of drugs whose side effects are well documented [24] . the beneficial effects of melatonin are explained by its properties as a potent antioxidant, a modulator of apoptosis and a positive regulator of immune functions [25] [26] [27] [28] [29] . these actions suggest the potential to treat viral infections, which usually cause inflammatory injury and elevated oxidative stress [30, 31] . a number of reports examining the ability of melatonin to protect against viral infections have been published, as summarized in the following section. encephalomyocarditis virus (emcv) is a highly pathogenic and aggressive virus that causes encephalitis and myocarditis in rodents. administration of melatonin prevented paralysis and death of mice infected with sublethal doses of emcv [32] . melatonin also has a protective effect in mice infected with semliki forest virus (sfv), a classic encephalitis arbovirus, that invades the cns and whose replication in the mouse brain eventually leads to death. melatonin administration not only reduced the death rate but also significantly postponed the onset of the disease. furthermore, the level of virus in the blood in melatonin-treated mice was lower than in non-treated mice [33] . although attenuated west nile virus (wnv) strain wn-25 is an encephalitis virus that does not invade the brain and does not normally cause encephalitis, exposure of mice to various stressful stimuli induces wn-25 encephalitis. melatonin counteracts the immunodepressive effect of stress exposure and prevents the stress-related encephalitis and death of wn-25 infected mice [33] . venezuelan equine encephalomyelitis (vee) is an important human and equine disease caused by vee virus (veev), a mosquito-borne organism. outbreaks have occurred in northern south america from the 1920s to the 1970s with thousands of people and horses, donkeys and related species being infected. mice have been used as an animal model for this condition, because veev-infected mice show excitation and hypermotility followed by hypomotility, paralysis, coma and death. melatonin administration protects mice infected with veev by decreasing the virus load in brain and serum, reducing mortality rates, delaying the onset of the disease and deferring the time to death. furthermore, in surviving mice treated with melatonin, the veev-mediated igm antibody titres are highly elevated [34] . aleutian mink disease is a natural condition caused by persistent infection with the aleutian mink disease virus (amdv). animals in the progressive state of the disease show a marked hypergammaglobulinemia, because of high titers of non-neutralizing admv antibodies. this is thought to cause lesions in the kidney, liver, lungs and arteries. melatonin implants reduced mortality in admv-infected mink [35] . the findings in these reports document the ability of the melatonin to protect against viral infections [ table 1 ]. the potential protective mechanisms include melatonin acting as a free radical scavenger, an antioxidant enzyme inducer, a positive regulator of immune functions and an inhibitor of inflammation, as well as a regulator of programmed cell death (pcd) [ table 2 ]. free radicals are molecules formed naturally during many metabolic processes. they contain an unpaired electron in their valence orbital that makes them unstable and reactive. these reactive agents damage essential molecules in cells including lipids, proteins and dna [36, 37] . among these reactants, the superoxide anion radical (o 2 • à ), nitric oxide (no•) and especially their derivatives, the hydroxyl radical (•oh) and peroxynitrite (onoo à ), are highly biologically damaging elements produced in the host during microbial infections [38] [39] [40] [41] . phagocytes, such as neutrophils and macrophages are assumed to be the major generators of free radicals. elevated levels of o 2 • à are although ifn-g is the major cytokine inducing inos and no• overproduction in the pathogenesis of these viral infections, inos expression is downregulated by il-4, il-10 and transforming growth factor-b (tgf-b) [60] [61] [62] . ifn-g is known to be associated with type 1 helper t cell (th1) responses, and il-4 and il-10 are induced by type 2 helper t cell (th2) responses; no• biosynthesis catalyzed by inos is precisely regulated by a polarized th1-th2 balance. in other viral diseases, viral replication or viral components directly induce inos without mediation by pro-inflammatory cytokines. thus, the hiv envelope glycoprotein gp41 triggers inos expression in human astrocytes and murine cortical brain cells in culture [63, 64] . rsv directly upregulates inos in human type 2 alveolar epithelial cells (a549 cells) [65] . free radicals are produced to eliminate the pathogenic agent or to kill the virus-infected cells by a non-specific response. thus, antiviral effects of no• have been described for some dna viruses such as murine poxvirus (ectromelia virus) and herpes viruses including hsv, ebv and some rna viruses such as coxsackie virus [66] [67] [68] [69] [70] [71] . the toxic oxygen and nitrogen-based reactants, unfortunately, cannot discriminate between exogenous invading pathogens and the host cells themselves, and therefore, they also damage the host. to minimize such self-damage during the elimination of pathogens, the host employs several primitive tactics; it uses recruited phagocytes for the physical containment of pathogens in infectious foci. most bacteria, for example, can be phagocytosed and confined to septic foci, which are typically abscesses or granulomas. under these conditions, free radicals can affect bacteria rather selectively with the surrounding normal tissue remaining mostly intact. in viral infections, in contrast, free radical mediators cause non-specific oxidative/nitrosative damage in virus-infected tissue and produce oxidative stress; this occurs when the virus cannot be confined to limited areas by the non-specific host defense [56, 58, 72] . thus, no• has appreciable antiviral actions on several types of viruses including ortho-and paramyxovirus, murine vaccinia virus, coronavirus (mouse hepatitis virus), lymphocytic choriomeningitis virus, murine emcv, tickborn encephalitis virus (tbe-v) [73] [74] [75] [76] [77] [78] ; also, no• and its derivatives, especially onoo -, can be considered pathogenic in some viral infections. indeed, no• inhibition or lack of no• generation reduces the pathological consequences of viral pneumonia in mice caused by influenza virus, sev and hsv-1, hsv-1-induced encephalitis in rats, emcv-induced carditis and diabetes, and murine encephalitis induced by flavivirus (murray valley encephalitis virus, tbe-v) [55, 57, 74, [78] [79] [80] [81] [82] . a similar pathogenicity with a lack of antiviral effects has been observed for o 2 •in several experimental models of virus-induced pneumonia including those caused by influenza virus and cmv [43] [44] [45] 56, 72, 83, 84] . hcv-induced oxidative stress is emerging as a key step and a major initiator in the development and the progression of liver damage [85] . ns3, one of the non-structural proteins of hcv, was reported to induce reactive oxygen species by nadph oxidase in neutrophils [86] . high-risk human papilloma virus (hpv), which causes cervical cancer, promotes inos-dependent dna damage, leading to dysplastic changes and carcinogenesis [87] . epstein-barr virus is a herpes virus that infects the majority of the world population, generally during childhood; it has been linked to the genesis of a number of lymphoproliferative diseases and neoplasia such as the african burkitt lymphoma, nasopharyngeal carcinoma or gastric carcinoma. early stages of ebv infection generate oxidative stress either in b lymphocytes or in epithelial cells, so contributing to pathology [88] . influenza a virus causes a respiratory disease, which ranges from mild upper respiratory tract illness with or without fever to severe complications such as pneumonia. the latter disease results in respiratory failure, acute respiratory distress syndrome, multi-organ failure and even death. an abrupt increase in o 2 • à production occurs during phagocytosis, which induces injury in non-infected cells. these o 2 • à -mediated pathways contribute to a portion of the extensive tissue injury observed during severe influenza-associated complications [56] . to protect themselves against free radicalmediated damage, cells have developed an antioxidant defense that includes enzymatic and non-enzymatic mechanisms. free radical generation and a functionally efficient antioxidant defense system must be in equilibrium to avoid cellular damage caused by radicals and their derivatives. enzymes involved in the elimination of free radicals include the superoxide dismutases (sod), catalase (cat) and glutathione peroxidase (gpx). in addition to the enzymatic antioxidant system, organisms possess non-enzymatic free radical scavengers, which directly remove toxic reactants because of their electron donating ability. the best known nonenzymatic antioxidants are vitamin e (a-tocopherol), vitamin c (ascorbate), glutathione (gsh), b-carotene and, as recently described, melatonin [25] . several radical scavengers have been efficacious in ameliorating the severity of viral diseases. n-acetylcysteine, a gsh precursor, inhibits hiv in vitro [89] as did the natural thiol antioxidant, alpha-lipoic acid [90] . glutathione administration to hiv seropositive individuals by aerosol treatment can correct the glutathione deficiency [91] . the combination of several antioxidants with antiviral drugs synergistically reduces the lethal effects of influenza virus infections [92] . thus, any agent that functions as a direct radical scavenger and also stimulates antioxidative enzymes could have utility in the treatment of patients with severe complications of viral infections. melatonin is a powerful and effective •oh scavenger, which provides protection against oxidative damage of cell components. it also scavenges the peroxyl radical to a lesser degree generated during lipid peroxidation with an activity that, in some situations, is reportedly greater than that of vitamin e [22, [93] [94] [95] [96] . also, melatonin directly detoxifies the onoo à and possibly peroxynitrous acid (onooh) [97] . in vivo, melatonin stimulates several antioxidative enzymes including gpx, cat and sod, thereby potentiating its antioxidant properties [98] [99] [100] [101] . melatonin can cross anatomical barriers, including the placenta and the blood-brain barrier [102, 103] , and easily enter cells [104] . splenocytes infected with veev generated less of no•, when treated with melatonin; this finding suggests that the indoleamine protected mice infected with the veev by a mechanism involving a reduction in no• concentrations in tissue [105] . elevated production of no• and lipid peroxidation products were also found in supernatants and cellular elements of veev-infected neuroblastoma cell cultures. both no• and lipid peroxidation were decreased by melatonin treatment in a timedependent manner with an associated reduction in inos expression [106] . production of brain and serum nitrite, as well as neural lipid peroxidation products, was increased in veev-infected mice. melatonin treatment curtailed nitrite concentrations in the brain and serum of infected mice and lowered lipid peroxidation products [107] . respiratory syncytial virus is a common cause of bronchiolitis, a severe lower respiratory tract affliction that infects nearly all infants by age three worldwide. mice inoculated intranasal with rsv showed elevated oxidative stress due to rises in no• and •oh. also elevated malondialdehyde (mda) and decreases in gsh and sod activities were observed. pre-administration of melatonin in vivo resulted in marked reduction of acute lung oxidative injury induced by rsv, suppressed mda, no• and •oh generation, and restored gsh and sod levels in the lungs of rsv-infected mice [31] . rabbit hemorrhagic disease virus (rhdv) causes bleeding in the respiratory system, liver, spleen, cardiac muscle,and occasionally in the kidneys of infected rabbits with mortality over 90% in adults [108] . the activity and mrna expression of the antioxidants enzymes gpx, glutathione-s-transferase (gst) and mn-sod were significantly reduced in the liver of rhdvinfected rabbits used as a model of fulminant hepatic failure; these changes were reduced by melatonin administration in a concentrationdependent manner. melatonin treatment also caused a rise in protein expression of the nuclear factor erythroid 2 (nrf2), a transcription factor that plays a critical role by binding to the antioxidant response element in the promoter region of a number of genes encoding for antioxidant and detoxifying enzymes in several types of cells and tissues [109] . the activation of nrf2 during prevention of oxidative liver injury by melatonin in rats treated with dimethylnitrosamine has been reported [110] . during the early phase of infection and depending on the nature of the infected cells and the infecting virus, early innate defense mechanisms may be triggered to limit the extent of viral spread. the first mechanism to limit the extent of viral spread is the recognition of pathogenassociated molecular patterns (pamps), which are mostly viral nucleic acids, or their synthetic analogs produced during the viral infection, by a large repertoire of pattern recognition receptors (prrs), including toll-like receptors (tlrs), nodlike receptors (nlrs), rig-i-like receptors (rlrs) and aim2-like receptors (alrs) [111] [112] [113] [114] . such recognition initiates signaling cascades that culminate in the activation of transcription factors including nuclear factor kappa b (nf-kb), activating transcription factor 2 (atf-2), activating protein-1 (ap-1) and interferon regulatory factors 3 (irf3) and 7 (irf7). these stimulate the expression of type i ifn genes that are synthesized in most cell types and especially in plasmacytoid dendritic cells (pdc) [115] . all ifns bind to specific ubiquitously expressed cell surface receptors and induce a large number of interferon-stimulated genes (isg), whose encoded proteins mediate the antiviral effects of interferons. among these isgs, dsrna-activated protein kinase (pkr) primarily inhibits replication of rna viruses such as vesicular stomatitis virus (vsv), emcv, wnv, hcv and dna viruses including hsv-1 [116] . another group of isgs is the 20-50-oligoadenylate synthetases (oas) that requires dsrna for its activation and is a major antiviral effector against picornaviruses (e.g. emcv) and influenza a virus, as well as other rna viruses [117] . non-specific ssrna cleavage also occurs after induction of isg20, a 30-exoribonuclease, which contributes to inhibition of rna viruses such as vsv [118] . an additional, non-enzymatic mechanism of translation inhibition is pursued by the isg56/ifit family proteins, which act against hcv [119] [120] [121] . another ifn-induced protein is the human mxa, which is a key component in innate defense against orthomyxoviruses such as influenza virus as well as measles virus, vsv, hanta virus and sfv [116, 122] , the viperin (cig5), which might interfere with viral budding of enveloped viruses, such as cmv, hcv, and influenza virus [123] , and the nucleic acid-editing enzymes apobec3g and à3 f, which inhibit retroviruses [124] . a second mechanism is the triggering of effector functions of cellular components of the innate immune system, such as granulocytes, natural killer cells (nk) and natural killer t cells (nkt cells), macrophages, and dendritic cells, which are normally rapidly recruited and/or activated at the site of virus infection, causing a local inflammation [125] . during this early phase, activated nk cells release ifn-g, which is not stimulated by viral pamps but by il-12 and il-18 released by activated macrophages [126] . all of the cellular components of the innate immune system can participate in the antiviral response by killing infected cells, by producing chemokines (including eotaxin, rantes, mcp-1, il-8) that recruit inflammatory cells into the infected tissue and by producing antiviral and immunoregulatory cytokines (including tnf-a, il-1, il-3, il-4, il-5, il-6, il-12, il-18, gm-csf) that enable the adaptive immune response to recognize infected cells and perform antiviral effector functions [127] [128] [129] [130] . lymphocytes are cells of this adaptive immune system. among them, two subsets of cd4 + t cells, th1 and th2, play a key role in antiviral immunity. after being stimulated by antigen presenting cells, th1 cells produce il-2, tnf-a and ifn-g, which possess antiviral activities and regulate activation of cd8 + cytotoxic t cells, whereas th2 cells produce il-4, il-5, il-10 and il-13, which stimulate b cells to produce antibodies [131] . despite the fact that virus-specific th2 cells can be detected following primary infection by any virus, virus-specific th1 cells are usually much more abundant and reach very high numbers at the peak of the acute infection [132] . moreover, their frequencies remain elevated following resolution of the infection. melatonin is synthesized in lymphoid organs, such as the bone marrow, thymus and lymphocytes [133] [134] [135] , and there are high affinity membrane melatonin receptors as well as nuclear binding sites in circulating lymphocytes, spleen cells and thymocytes [136] [137] [138] . melatonin is known to activate both innate and adaptive immune responses leading to an increase in immune responsiveness and regulation of several immune functions [27, 28, [139] [140] [141] [142] [143] . melatonin has properties as an inflammatory regulator, because it differentially modulates pro-inflammatory enzymes, and controls the production of inflammatory mediators such as cytokines and leukotrienes. the timing of its pro-inflammatory and anti-inflammatory effects suggests that melatonin might promote early phases of inflammation, on the one hand, and contribute to its attenuation on the other hand, to avoid complications of chronic inflammation [144] . melatonin enhances the production of il-1, il-6, tnf-a and il-12 from the monocytes [145] and of il-2, ifn-g and il-6 from cultured human peripheral blood mononuclear cells [137] . it has been suggested that melatonin and ifn-g create an immunoregulatory circuit responsible for the antiviral, antiproliferative and immunomodulatory actions of . this cytokine increases serotonin and melatonin levels in lymphocytes and macrophages. the early stimulation in the production of ifn-g by melatonin suggests that earlier treatment with this indoleamine could increase the antiviral activity of ifn-g [147] . in addition to stimulating the production of several cytokines that regulate immune function, melatonin enhances immune function by directly stimulating polymorphonuclear cells, macrophages, nk cells and lymphocytes [148] . recently, considerable attention has been focused on the fact that melatonin treatment has been found to augment cd4+ t cells in lymph nodes of rats [149] . consequently, melatonin is considered an immunoenhancing agent [141, 150] . in retrovirus-infected people and mice, whereas th1 cytokine (il-2 and ifn-g) production declines, th2 cytokine (il-4, il-5, il-6, and il-10) production increases [151] [152] [153] . the excessive th2 cytokines suppress th1 cells, causing anergy of cell-mediated immunity, thus allowing the retrovirus as well as normal flora to reproduce and promote free radical generation by macrophages [154] . female c57bl/6 mice infected with the lp-bm5 mlv develop murine aids. treatment with melatonin, alone or with dehydroepiandrosterone (dhea), prevented retrovirus-induced reduction in b-cell and t-cell proliferation and in th1 cytokine secretion, as well as overproduction of th2 cytokines and tnf-a [155] . in fact, melatonin alters the balance of th1 and th2 cells mainly towards th1 responses increasing the production of th1 cytokines [156] . a link between melatonin and the immune system has been also reported in patients infected with hiv-1. although mean serum il-12 levels in hiv-1-affected individuals did not significantly differ from healthy controls, the il-12 levels of hiv-1 patients with advanced disease (cdc stage c) were significantly lower than those of patients in less advanced cdc stages b and a. taking into account that serum il-12 levels run parallel with serum melatonin concentrations as the disease advances, a relationship between immune function and melatonin has been suggested; a reduction in serum melatonin could possibly affect il-12 production thereby contributing to the progress of hiv-1 infection [157] . the protective effect of melatonin against veev by regulation of the immune system has been described by bonilla et al. [158] . the endogenous production of ifn-g, il-1b and tnf-a, but not of il-2 and il-4, is stimulated in veev-infected mice treated with melatonin [159] . nevertheless, the average mortality obtained during neutralization experiments with the corresponding anticytokine antibody suggests that although neither tnf-a nor ifn-g is essential for the protective effect of melatonin observed in murine veev infection, il-1b induced by melatonin treatment is a target cytokine to promote the immune enhanced state. this in turn causes the viral clearance or helps generate an earlier immune response against the veev infection [160] . in contrast, in the brain of veev-infected mice, melatonin stimulates the endogenous production of il-1b but reduces the concentration of tnf-a [158] . il-1b is considered one of the earliest host mediators during infectious diseases of the cns and its role in infectious processes of the brain parallels its role in the peripheral immune system [160] . although il-1b deficiency is protective against fatal sindbis virus infection [161] , mice deficient in il-1b have increased susceptibility to influenza virus [162] . in poxvirus animal models, the viral induction of this cytokine is also beneficial for the host [163] . the increase in il-1b levels detected in blood and in brain of veev-infected mice after melatonin treatment also plays a protective role, possibly by neuronal support and protection by inducing nerve growth factor secretion by astrocytes [164] . this supplies a trophic factor for many neuronal cell types in times of stress such as that produced by veev infection. the significant reduction in the concentration of brain tnf-a induced by melatonin in veevinfected mice likely diminishes the inflammatory response caused by the migration of granulocytes and macrophages to inflammatory sites within the cns [165] . these cells are recruited by colonystimulating factors produced by astrocytes stimulated by tnf-a and as a consequence of alterations in blood-brain barrier (bbb) permeability caused by the adhesive properties of astrocytes stimulated by tnf-a. tnf-a is known to induce intercellular adhesion molecules on neighboring endothelial cells [166] , alter bbb permeability and promote inflammatory cell infiltration into the cns. by reducing adhesion molecule production, which melatonin is known to do [167] , the indole would protect the brain infected with veev. respiratory syncytial virus bronchiolitis in infants is characterized by a massive infiltration of inflammatory cells into the airways. of the diverse intracellular signaling pathways, rsv is recognized by tlr3, which initiates a signaling cascade that culminates in the activation of the transcription factor nf-kb; nf-kb is a central mediator of rsv-induced airway inflammation in vivo [145, 168, 169] . rsv infection of raw264.7 macrophages time-dependently stimulates the rapid activation of tlr3 and nf-kb, as well as subsequent nf-kb dependent genes, many of which encode for pro-inflammatory cytokines and chemokines including tnf-a and il-1b. melatonin decreases tlr3-mediated downstream gene expression in rsv-infected macrophages in a dose-dependent and time-dependent manner. such inhibition of nf-kb activity, as well as of tnf-a in serum, seems to be the key event required to explain the reduction in inflammatory gene expression caused by melatonin [31, 170] . as obligate intracellular parasites, viruses are dependent on the host for each stage of replication and, therefore, constantly interface with multiple components of the host cell machinery, including cellular receptors and uptake pathways, gene expression mechanisms and the cell division apparatus. viral utilization of these systems likely causes cell stress and activates death-signaling pathways or alters expression of genes that control cell survival, evoking pcd [171, 172] . apoptosis is one type of pcd, which is dependent on cleavage of important cellular factors by effector caspases such as caspase-3 and caspase-7. two major pathways govern the activation of such effector caspases. in the intrinsic pathway, intracellular stresses sensed by the bh3-only members of the bcl-2 family promote the formation of the apoptosome by activation of caspase-9 through release of proapoptotic molecules such as cytochrome c and smac/diablo from the mitochondria. the apoptosome directly activates effector caspases. in the extrinsic pathway, occupation of death receptors such as fas and tumor necrosis factor receptor (tnf-r) by death ligands including fasl and tnfa forms a death-inducing signaling complex (disc). this results in the activation of the initiator caspase, caspase-8, which directly mediates effector caspase activation and causes cell death. the ability of melatonin to modulate apoptosis and to differentially regulate the expression of pro-apoptotic and anti-apoptotic mediators has been reported in many studies [29, [173] [174] [175] [176] [177] . rhdv infection induces liver apoptosis with increased caspase-3 expression and activity [178, 179] . these effects are attenuated by melatonin in a concentration-dependent manner. anti-apoptotic actions of melatonin on the intrinsic pathway were related to a reduced expression of bax and cytosolic cytochrome c release, increased expression of bcl-2 and bcl-xl, and inhibition of caspase-9 activity. melatonin treatment also has effects on extrinsic pathway resulting in a reduction in caspase-8 activity, tnf-r1 expression and phosphorylated janus kinase (jnk) expression, and increased expression of cellular fliceinhibitory protein (c-flip), an inhibitor of caspase-8 [179] . these findings show that inhibition of apoptotic mechanisms contributes to the beneficial effects of melatonin in rabbits with experimental infection by rhdv and supports a potential hepatoprotective role of melatonin in fulminated hepatic failure. autophagy is a type of pcd characterized by the formation of autophagosomes to remove excessive proteins and thereby maintains homeostasis within the cell. autophagy is now recognized as a component of both innate and adaptive immune responses to bacterial and viral pathogens [180] . varicella zoster virus infection provides an excellent example of autophagy in humans, because abundant autophagosomes are easily detected in the skin vesicles of both varicella and zoster [181] . autophagy is also found during viral replication of hcv [182] , rabbit calicivirus [183] and poliovirus [184] . given that melatonin modulates autophagy through redoxsensitive transcription factors [185] , the role of melatonin in such viral infections involving autophagy should be examined. beneficial effects of melatonin when combined with several drugs, such as doxorubicin, cisplatin, epirubicin, cytarabine, bleomycin, gentamicin, cyclosporin, indometacin, acetylsalicylic acid, ranitidine, omeprazole, isoniazid, iron and erythropoietin, phenobarbital, carbamazepine, haloperidol, caposide-50, morphine, cyclophosphamide and l-cysteine have been reported [24] . recently, a single blind randomized study showed a higher percent of a complete regression of symptoms of hsv-1 infection after a treatment with melatonin plus sb-73 (an extract of aspergillus sp. with antiherpetic properties) compared with the treatment with acyclovir alone [186] . effects of melatonin to increase the efficacy of other antivirals should be studied. melatonin is an endogenously produced and ubiquitously acting molecule [187] [188] [189] . because of its highly diverse actions, this indoleamine has potential to combat a wide variety of pathophysiological conditions [190] [191] [192] [193] [194] ; it has been tested in numerous clinical trials [23] with the outcomes of the treatments always being beneficial. because of its essential and basic actions on cell physiology, melatonin qualifies for the moniker "molecular handyman," as indicated in the title of this review. in relation to viral infections, 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antioxidant actions melatonin promotes puromycin-induced apoptosis with activation of caspase-3 and 5 1 -adenosine monophosphate-activated kinase-alpha in human leukemia hl-60 cells programmed cell death in the pathogenesis of rabbit hemorrhagic disease melatonin attenuates apoptotic liver damage in fulminant hepatic failure induced by the rabbit hemorrhagic disease virus autophagy during common bacterial and viral infections of children varicella-zoster virus infection induces autophagy in both cultured cells and human skin vesicles autophagy proteins promote hepatitis c virus replication structural and functional analysis of virus factories purified from rabbit vesivirus-infected vero cells remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles melatonin modulates autophagy through a redox-mediated action in female syrian hamster harderian gland controlling cell types and gland activity regression of herpes viral infection symptoms using melatonin and sb-73: comparison with pineal melatonin: cell biology of its synthesis and of its physiological interactions melatonin: a multitasking molecule a survey of molecular details in the human pineal gland in the light of phylogeny, structure, function and chronobiological diseases sirtuins, melatonin and circadian rhythms: building a bridge between aging and cancer melatonin, cardiolipin and mitochondrial bioenergetics in health and disease melatonin as a therapeutic tool in ophthalmology: implications for glaucoma and uveitis drug-mediated ototoxicity and tinnitus: alleviation with melatonin matrix metalloproteinases in health and disease: regulation by melatonin the authors have no competing interest. jab is a researcher of isciii/ficyt. his stay at uthscsa has been subsidized by isciii (ba 11/ 00084). key: cord-332344-upsn0zb4 authors: jeswin, joseph; xie, xiao-lu; ji, qiao-lin; wang, ke-jian; liu, hai-peng title: proteomic analysis by itraq in red claw crayfish, cherax quadricarinatus, hematopoietic tissue cells post white spot syndrome virus infection date: 2016-02-01 journal: fish shellfish immunol doi: 10.1016/j.fsi.2016.01.035 sha: doc_id: 332344 cord_uid: upsn0zb4 to elucidate proteomic changes of hpt cells from red claw crayfish, cherax quadricarinatus, we have carried out isobaric tags for relative and absolute quantitation (itraq) of cellular proteins at both early (1 hpi) and late stage (12 hpi) post white spot syndrome virus (wssv) infection. protein database search revealed 594 protein hits by mascot, in which 17 and 30 proteins were present as differentially expressed proteins at early and late viral infection, respectively. generally, these differentially expressed proteins include: 1) the metabolic process related proteins in glycolysis and glucogenesis, dna replication, nucleotide/amino acid/fatty acid metabolism and protein biosynthesis; 2) the signal transduction related proteins like small gtpases, g-protein-alpha stimulatory subunit, proteins bearing pdzor 14-3-3-domains that help holding together and organize signaling complexes, casein kinase i and proteins of the map-kinase signal transduction pathway; 3) the immune defense related proteins such as α-2 macroglobulin, transglutaminase and trans-activation response rna-binding protein 1. taken together, these protein information shed new light on the host cellular response against wssv infection in a crustacean cell culture. white spot syndrome virus (wssv) is an enveloped dna virus with a wide host range in crustaceans such as shrimp, crab and crayfish [1, 2] . in shrimp aquaculture wssv infection will result in collapse of the whole shrimp stock within 3e10 days [3] . after the emergence of the wssv from 1990s, researchers have been focusing on the virus and the immune response of the host. for example, quantitative determination of the mrna pool in infected cells or animals versa controls has been widely used [4, 5] . with the virus characterized and some knowledge regarding the immune response of the host accumulated [6] , a comprehensive approach to investigate how the host physiological system responds to the viral invasion is the important next step to develop efficient control of the infection. proteomics is a means of comprehensive interpretation used to describe more direct molecular responses than conventional studies assessing the messenger rna [7] . hence, researchers employed proteomic approaches such as twodimensional electrophoresis for studying host protein changes during wssv proliferation [8, 9] . recently, itraq combined with liquid chromatography-tandem mass spectrometry is used to directly quantify and compare the protein expression levels of samples with great efficiency and accuracy [10] . such a global approach helps to correlate different host proteins participating in biological processes, cellular components and molecular function in response to a pathogenic infection. as one of the principle targets by wssv, crayfish hpt cells have been set up as a good model for investigation of wssv infection [11] . intriguingly, the crayfish hpt cells were lately proved by wu et al. [12] to be a better cell model for wssv replication than hemocytes which could not afford the virus for proper replication and viral assemble. previously, we have investigated the transcript alteration of hpt cells from red claw crayfish, cherax quadricarinatus, towards wssv challenge using subtractive library screening. we noticed that numerous genes, with differential expression between wssv infection and mocked infection, were present with annotated functions in immune defense, cytoskeletal organization, signal transduction, stress, metabolism and homeostasis [13] . to further identify proteins or pathways altered during viral infection, here we report proteomic responses of crayfish hpt cells by itraq at both early (1 hpi) and late (12 hpi) stages post wssv infection accordingly. these differentially expressed proteins provide us with better understanding of the host's coordinated effort to defend the wssv infection. adult healthy red claw crayfish were collected from crayfish farms in zhangpu, fujian, china. the crayfish were acclimatized for one week at least in well-aerated tanks and only intermolting animals were sacrificed for hpt cells preparation as described by s€ oderh€ all et al. [14] . the isolated hpt cells were cultured in six well plates with 85e90% confluence using l-15 medium containing 5 mm 2-mercaptoethanol, 1 mm phenylthiourea, 2 mm l-glutamine and a crude astakine preparation [15] . the cells were then inoculated, 1 hpi as early infection stage and 12 hpi as late infection stage accordingly, with 10 5 copies of wssv (chinese isolate, wssv-cnaf332093) kindly provided by prof. xun xu from the third institute of oceanography, state oceanic administration, china. uv-inactivated wssv was used as mocked infection. the cells in duplicates above were harvested by scraping in culture medium with a rubber policeman and collected by centrifugation at 4200 rpm for 5 min at 4 c. the cell pellets were then lysed with ripa lysis buffer (thermo scientific) and the supernatant was collected after centrifugation followed by protein purification using a 2-d clean-up kit (bio-rad), and protein concentration was determined by lowry method using the rc dc protein assay kit (bio-rad). for itraq assays, two independent samples were pooled and each pool was divided into duplicates. total protein (100 mg) of each sample solution as prepared above was digested with trypsin gold (promega) at a ratio of protein:trypsin ¼ 30:1 at 37 c for 16 h. thereafter, peptides were dried by vacuum centrifugation and reconstituted in 0.5 m triethyl ammonium bicarbonate and processed according to the manufacturer's protocol for 8-plex itraq reagent (applied biosystems). two technical replicates were prepared for itraq labeling as suggested by the manufactory instructions. the proteins from the infected (1 hpi labeled with 114/ 118; 12 hpi labeled with 116/121) and mock-infected samples (1 hpi labeled with 113/117; 12 hpi labeled with 115/119) were labeled with itraq tags (fig. s1 ). the tags employed n-hydroxysuccinimide chemistry which consists of three functional groups: an amine-reactive group and an isotopic reporter group (n-methylpiperazine) linked by an isotopic balancer group (carbonyl) for the normalization of the total mass of the tags. the labeled peptide mixtures were pooled and dried by vacuum centrifugation and then reconstituted in buffer a (25 mm nah 2 po 4 in 25% acetonitrile, ph 2.7) followed by separation on a strong cation exchange chromatography column (4.6 â 250 mm, 5 mm, ultremex scx, phenomenex, usa). the peptides were next treated with an isocratic elution at a flow rate of 1 ml/min with buffer a for 10 min followed by a gradient from 5 to 60% of buffer b (25 mm nah 2 po 4 , 1 m kcl in 25% acetonitrile, ph 2.7) for 27 min, and then a gradient elution from 60 to 100% of buffer b for 1 min. the system was then maintained at 100% buffer b for 1 min before equilibrating with buffer a for 10 min prior to the next injection. fractions were collected every 1 min and the eluted peptides were pooled into 20 fractions followed by desalting on a strata x c18 column (phenomenex) and vacuum-dried. each fraction was resuspended in buffer a (5% acetonitrile, 0.1% formic acid) and centrifuged at 20,000 g for 10 min, and the final concentration of peptide was about 0.5 g/l on average. ten microlitre of supernatant was loaded on a lc-20ad nanohplc (shimadzu, japan) by the autosampler onto a 2 cm c18 trap column. then, the peptides were eluted onto a 10 cm analytical c18 column packed in-house. the samples were loaded at 8 ml/min for 4 min, then the 35 min gradient was run at 300 nl/min starting from 2 to 35% buffer d (95% acetonitrile, 0.1% formic acid), followed by 5 min linear gradient to 60% and by 2 min linear gradient to 80%. after that, the samples were maintained at 80% buffer d for 4 min, and finally return to 5% in 1 min. data acquisition was performed with a triple tof 5600 system (ab sciex) fitted with a nanospray iii source and a pulled quartz tip as the emitter (new objectives, ma). data was acquired using an ion spray voltage of 2.5 kv, curtain gas of 30 psi, nebulizer gas of 15 psi, and an interface heater temperature of 150 c. the ms was operated with a rp of greater than or equal to 30,000 fwhm for tof ms scan. for ida, survey scans were acquired in 250 ms and as many as 30 product ion scans were collected if they exceeded a threshold of 120 counts per second. total cycle time was fixed at 3.3 s. the q2 transmission window was 100 da for 100%. four time bins were summed for each scan at a pulser frequency value of 11 khz through monitoring of the 40 ghz multichannel tdc detector with four anode channel detect ion. a sweeping collision energy setting of 35 ± 5 ev, coupled with itraq adjust rolling collision energy, was applied to all precursor ions for collision induced dissociation. dynamic exclusion was set for half of peak width (15 s) and then the precursor was refreshed off the exclusion list. peptide sequence data were processed using mascot software version 2.3.02 to obtain the information of associated proteins. a false discovery rate of less than 1% was used as cut off. the proteins were further classified using gene ontology (go) and pathway enrichment analysis (http://www.geneontology.org). to determine the alteration of protein expression induced by wssv infection, protein expression fold changes were calculated relatively in wssv infected hpt cells compared to those of mock-infected cells. student's t-test was used to identify statistically significant changes in protein expression (p < 0.05). the quantitative proteins ratios of !1.3 were considered as up-regulation and proteins with a label ratio of 0.77 [16, 17] were considered as down-regulated proteins in wssv infected hpt cells versus mock-infected cells. rna isolation was performed by genelute mammalian total rna isolation kit (sigma-aldrich) according to manufacturers protocol. briefly, reverse transcription was done with 1 mg of total rna using superscript ii reverse transcriptase (invitrogen) according to instructions of manufacturer. real-time pcr was performed on abi 7500 using faststart universal sybr master (roche) and the primers used were included in supplementary information (tabel s2). the expression level of mrna was normalized by 16s rrna. the data was presented as relative expression levels (means ± sd, n ¼ 3), and subjected to one-way analysis of variance (anova) followed by fisher's least significant difference test. the significant difference between wssv treated and control (uv treated wssv) samples was represented by one asterisk for p < 0.05. to get insight into the proteins differentially expressed at both early stage (1 hpi) and late stage (12 hpi) of wssv infection as recent publications reported that these two time points have been clearly defined accordingly of wssv replication in crayfish hpt cells [12, 13] , itraq analysis was employed for quantification followed by protein profiling, which resulted in 594 protein hits in mascot. the functional enrichment analysis of go annotations for biological processes showed that the identified proteins were classified into 23 categories (fig. 1) , in which proteins related to cellular process (17.14%) and metabolic process (15.41%) were the most abundant molecules followed by cellular component organization biogenesis (6.43%), biological regulation (6.37%), regulation of biological process (5.85%), developmental process (5.44%), response to stimulus (4.90%), signaling transduction (2.95%), negative regulation of biological process (2.23%) and others. apparently, the protein expression in hpt cells showed variation at both early and late stages of infection if compared to those of mock-infected cells (tables 1e3). among these proteins, alpha aminoadipic semialdehyde synthase, ribonucleoside-diphosphate reductase and thymidylate synthase in metabolism; casein kinase i isoform alpha, camp-dependent protein kinase, pdz domain containing protein gipc1 and rab-1 involved in signal transduction were found for the first time to be involved in the host response to wssv infection. furthermore, the cytoskeletal associated proteins like calponin-3 and beta chain spectrin were also found to be significantly expressed towards wssv infection. here we focus on the brief interpretation of diverse functional classes related to metabolic processes, signal transduction proteins and immune related proteins, which are putatively key molecules involved in metabolism and host defense against cellular stress caused by wssv infection. in addition, numerous proteins were expressed in wssv infected cells but without significant difference in up-or down-regulation when compared to a mock infection (table s1 ). viruses require host cellular metabolism for their energy needs and successful replication. wssv infection perturbs the expression of at least 14 enzymes action in glycolysis, glucogenesis, the pentose phosphate pathway, metabolism of nucleotide, amino acid and fatty acid, dna replication and protein biosynthesis. an increase in activity of metabolic pathways associated with glycolysis, the pentose phosphate pathway, nucleotide biosynthesis, glutaminolysis and amino acid biosynthesis were observed in shrimp hemocytes post wssv challenge [18] . in our present study, phosphoglycerate mutase 2 with a role in glycolysis was downregulated at both 1 hpi and 12 hpi post infection. further studies with glucose starved media should be done to ascertain its role in wssv replication and possible therapeutic applications. the pentose phosphate pathway fulfills two essential functions, the formation of pentose phosphate for synthesis of nucleotides, rna, dna and the generation of nadph for biosynthetic reactions to maintain glutathione in the reduced state and protecting the cell from damaging reactive oxygen intermediates. glucose-6phosphate dehydrogenase, the key enzyme of pentose phosphate pathway showed increased activity in wssv infected litopenaeus vannamei [19] . interestingly, in our study the key enzyme of the oxidative pentose phosphate pathway, 6-phosphogluconate dehydrogenase, was up-regulated at 12 hpi whereas in penaeus monodon it was down-regulated during yhv infection [20] . this difference may reflect alternative energy utilization of these two viruses. viruses use several strategies to ensure adequate supply of nucleotide by expressing virus encoded enzymes that boost nucleotide synthesis and increasing the flux of pentose phosphate pathway [21à23]. wssv-infection perturbs the nucleotide biosynthesis, particularly on nucleoside and nucleotide metabolic enzymes involved in producing monomer intermediates for dna synthesis such as ribonucleoside-diphosphate reductase, thymidylate synthase and gmp synthase. the first two enzymes were down-regulated at 1 hpi whereas gmp synthase, an atp-dependent enzyme involved in purine nucleotide synthesis, and thymidylate synthase was over-expressed at 12 hpi(in comparison to its expression at 1 hpi with live virus infection, table 3 ). a similar increase in nucleotide biosynthesis was observed during wssv infection in l. vannamei [18] . the relative increase of nucleosides at 12 hpi when compared to 1 hpi implies that the dna replication machinery might be sequestered to produce viral dna. recently pharmaceutical compounds are proved to inhibit rna, dna and retroviruses by targeting pyrimidine biosynthesis [24] . broadspectrum antivirals targeting nucleotide biosynthesis could be further tested in vitro such as in the hpt cell cultures. antiviral drugs targeting nucleotide biosynthesis is commonly used against human viruses and this might be extended to wssv. differential expression of enzymes associated with amino acid metabolism was observed in our present study. alpha aminoadipic semialdehyde synthase, involved in the lysine degradation pathway was downregulated at 1 hpi while increased to control levels at 12 hpi. adenosylhomocysteinase, a key enzyme in the regulation of the intracellular concentration of s-adenosylhomocysteine (sah) was up-regulated at 12 hpi (table 3) . sah is an intermediate in the synthesis of cysteine and adenosine. an increased intracellular concentration of sah inhibited vaccinia virus replication in murine cells [25] . aminoacyl-trna synthetases (arss) are highly conserved for efficient and precise translation of genetic codes. evidence is accumulating that several arss form a macromolecular protein complex that would work as a molecular hub linked to the multiple signaling pathways [26] . the up-regulation of bifunctional aminoacyl-trna synthetase at 12 hpi (table 3) implies that protein synthesis might be elevated during later stages of the wssv infection. recent studies in crayfish found that there was a decrease in long chain fatty acids during wssv infection at 12 hpi and later it increased at 24 hpi [27] . the knowledge of virus induced changes in cell metabolome may lead to potential use of inhibitors of metabolic enzymes to treat wssv infection. in total eleven proteins related to signal transduction were differentially expressed in hpt cells upon wssv-infection. three of those are gtp-binding proteins (rab-1, ras opposite, gtp-alpha g s ). gtp binding proteins regulate a wide variety of cell functions acting as biological timers, for instance, that initiate and terminate specific cell functions. in addition they play key roles in determining spatial locations of specific cell functions. rab-1 regulates protein transport from endoplasmic reticulum to golgi apparatus. rab gtpase is essential for the control of intracellular membrane trafficking in all eukaryotic cells and further affects the ability of phagocytic cells to scavenge pathogen. this is in synchrony with the observed upregulation of rab-1 protein during 1 hpi of the current study. in penaeus monodon, rab 7 was found to bind to rvp28 of wssv. the in vivo neutralization assay of wssv infected shrimp in the presence of anti-rab7 antibody revealed less mortality compared to that of wssv alone [28] . depletion of rab-1 significantly decreased the replication of hepatitis c virus in a human hepatoma cell line [29] . the upregulation of rab-1 suggests its role in wssv pathogenesis. g-protein alpha stimulatory subunit (ga s ) is activated by g-protein coupled receptors located in the cell membrane. ga s stimulates adenylate cyclase to produce camp [30] a ubiquitous second messenger that enables cells to detect and respond to extracellular signals [31] . the intracellular camp-level affects the camp-dependent protein kinase (pka) and consequently many metabolic processes of the cell. in our present study wssvinfection caused over expression of ga s at 12 hpi. more in depth investigation is needed to understand its role during a late infection stage of wssv infection. adapter molecules or domains such as gipc1 and 14-3-3 are involved in protein-protein interaction. in the current study the strongest response was the expression of gipc1, a scaffolding protein that regulates cell surface receptor expression and trafficking. viruses usually use carboxyl-terminal pdz domain-binding motif (pbm) that mediates interactions with cellular pdz proteins. gipc1 contains such a pdz domain, a protein-protein interaction module typically found in cytoplasmic and membrane adapter proteins that are involved in a variety of cellular processes of significance to viral infection, for instance maintenance of cell-cell junctions, cellular polarity, and signal transduction pathways [32] . gipc1 was in control level at first hour after viral challenge and later upregulated at 12 hpi, implying that the gipc1 might have modulated the cellular process in favor of wssv replication and dissemination in the cells. to reveal the role of gipc1 during wssv infection, protein-protein interaction studies should be undertaken in the future. 14-3-3 epsilon protein is known for its ability to bind multiple cellular protein ligands. it interacts with a wide range of proteins for functions like cell cycle control, regulation of cell death and apoptosis etc. in addition 14-3-3 epsilon can interact with a diverse array of viral products, and thereby re-direct cellular processes [33à35]. for example, hepatitis c virus core protein can interact with 14-3-3 epsilon protein [33] . also severe acute respiratory syndrome corona virus nucleocapsid protein was phosphorylated and localized in the cytoplasm by 14-3-3-mediated translocation [36] . in our study 14-3-3 epsilon was at control levels at 1 hpi and up-regulated at 12 hpi and therefore indicative of an established viral infection. further studies are therefore needed to elucidate the interaction of 14-3-3 with viral proteins. there was a trend of down regulation observed in proteins that assemble the proteasome (proteasome subunit alpha type, 26s proteasome regulatory subunit rpn2). the main function of proteasome is to degrade unneeded or damaged proteins. there is a high stoichiometric imbalance of host target proteins over those encoded by the virus. to overcome this, many viruses have evolved mechanisms by which their cellular target proteins are directed to the 26s proteasome and subjected to proteolytic degradation [37] . recently a plethora of viral proteins have been shown to direct host-cell proteins for proteolytic degradation. these activities are required for various aspects of viral life cycle like viral entry [38] , replication [39] , enhanced survival [40, 41] , and to viral release [42] . similar to our study, infection of enterovirus 61 in rhabdomyosarcoma cells resulted in down-regulation of proteasome subunit alpha type 2 [43] . the cellular signal transduction is largely controlled by protein phosphorylation. several protein kinases were differentially expressed during wssv infection such as protein casein kinase i (ck1) isoform alpha, a serine/threonine kinase, which was upregulated at 12 hpi. it inhibits hyperphosphorylation of nonstructural protein 5a of hepatitis c virus. the nonstructural (ns) protein 5a is a phosphoprotein and experiments indicated that the phosphorylation state of ns5a is important for the outcome of viral rna replication [44] . similarly, in our study the up-regulation of ck1 might be indicative of its interaction with wssv proteins. wssv envelope proteins like vp28 and vp19 were previously reported to be threonine phosphorylated [45] . further investigations are needed to understand the role of casein kinase i isoform alpha. camp-dependent protein kinase (pka) is involved in the regulation of glycogen, sugars and lipid metabolism. its down-regulation at 12 hpi (table 3 ) might affect the production of essential metabolites during infection. activation of camp can block apoptosis induced by herpes simplex virus 1 associated genes [46] . as the wssv infection is nearly established at 12 hpi, wssv might suppress camp activation for promoting apoptosis in infected cells, spreading viral infection to nearby cells. the mitogen-activated protein kinase (mapk) cascade pathways have global role in the intracellular signaling network pathway. mitogen-activated protein kinase kinase (map2k/mek) phosphorylates mitogen-activated protein kinase (mapk/erk) are part of the protein kinase cascade starting with an extracellular stimulant ending with the expression of transcription factors in the nucleus. it is thought that a viral infection acts as an extracellular stimulant that activates this pathway. rna silencing of p38 mapk homologue ivp38 caused increase in wssv replication on l. vannamei [47] . in our study mapk was in control levels at 1 hpi but down-regulated at 12 hpi while map2k down-regulated at initial period and up-regulated at latter time point (table 3 ). the significant expression of mapk and map2k should be further investigated. among these proteins, nine immune related proteins were differentially expressed during this period of infection within 12 hpi. two of these proteins, a2-macroglobulin and transglutaminase, are well known to play a role in coagulation of hemolymph during pathogen infection. for instance, a2macroglobulin is considered as a broad range proteinase inhibitor involved in phagocytosis [48, 49] . silencing of a2-macroglobulin clearly reduced phagocytosis of chryseobacterium indologenes by hemocytes both in vitro and in vivo in hard tick ixodes ricinus [50] . according to the itraq quantification, no change in protein expression was found for a2-macroglobulin at 1 hpi but its expression was clearly up-regulated at 12 hpi, suggesting that a2macroglobulin might have a role during late infection hours of wssv in crayfish hpt cells. transglutaminase has a regulatory function in antimicrobial peptide production in kuruma shrimp. silencing of transglutaminase caused the down regulation of antimicrobial peptides such as crustin and lysozyme, thus, negatively affecting on the host's defense against pathogen infection [51] . additionally, it has been documented that in a freshwater crayfish transglutaminase prevents hematopoietic stem cells from migrating into the hemolymph [52] . as hemocytes are particularly important for innate immune defense in invertebrates, this prevention on release of matured hpt cells from the hematopietic tissue is likely to have an impact on host immunity. in our present study transglutaminase expression was down-regulated at 1 hpi if compared to those of mock-infection, implying that transglutaminase might act a critical role during virus infection. further functional studies on transglutaminase against wssv infection in red claw crayfish both in vitro and in vivo will benefit the elucidation of an important role of this enzyme. sp€ aetzle is the well-known component of toll signaling pathway, which plays a key role in regulating innate immune responses in invertebrates. gram positive bacteria, fungi and certain viruses can activate toll signaling pathway initiating an intracellular signaling cascade to promote the expression of immunerelated genes such as antimicrobial peptides through the activation of the nf-kb family-dorsal proteins [53] . it has been shown that key components of the toll pathway, like toll [54] and dorsal [55] , are activated during viral infection in shrimp. by recognition of high mannose residues expressed on the surface of viruses or virus surface glycoproteins, toll like receptors are able to mediate virus entry into host cells, in which binding of ligand sp€ aetzle to toll is necessary to initiate this pathway [56] . recently, transcriptomic changes of sp€ aetzle was also reported during wssv and vibrio infection in fenneropenaeus chinensis [57] . protein sp€ aetzle was upregulated in crayfish hpt cells at 12 h post wssv infection (table 3) . recent studies in crayfish hpt shows that by 12 hpi progeny wssv virions will be successfully generated [12] . the presence of progeny virions at this stage might be the reason for up regulation of protein sp€ aetzle. further functional studies are needed to understand the late expression of sp€ aetzle during wssv infection. antioxidant enzymes regulate the concentrations of radical oxygen species, for instance, glutathione peroxidase detoxifies lipids and hydrogen peroxidase while glutathione-s-transferase marks xenobiotics for cellular degradation. this antioxidant activity usually is increased upon pathogenic infection. we found that glutathione peroxidase and glutathione-s-transferase were obviously affected by wssv infection in crayfish hpt cells, in which the former expression was promoted at 1 hpi and the latter was enhanced at 12 hpi. an increased glutathione peroxidase activity at early hours and an up-regulated glutathione-s-transferase activity at late hours were also found in l. vannamei post wssv infection [58] . increased oxidative damage will occur in response to the challenge when rate of free radical formation exceeds its removal by antioxidant system [58] . faulty antioxidant defense causes the failure of sensitive cellular and subcellular components leading to cell death [59] . these findings clearly indicated that antioxidant response was modulated during wssv infection in crustaceans [58] . invertebrates and plants are capable of suppressing viral infection through rna silencing mediated by risc. as a key component of risc, trans-activation response rna-binding protein (trbp) was found to be associated with dicer and argonautes, both of which are involved in rna interference and microrna pathways [60] . combination of trbp with eukaryotic initiation factor 6 leads to an antiviral rna interference pathway in shrimp marsupenaeus japonicus [61] . knockdown of a shrimp tudor staphylococcal nuclease (pmtsn), one of the risc associated protein involved in post transcriptional regulation, resulted in reduced inhibition of yhv infection mediated by rnai of yhv in penaeus monodon [62] . in our studies we found that tar rna-binding protein isoform 1 and tsn, both involved in risc, were down-regulated at 12 hpi. this implied that wssv-infection might negatively affect the immune defense against this viral infection via rnai process, but how this effect is elicited and modulated during wssv replication needs further exploration. to validate the differentially expressed proteins identified by itraq, a randomly selected group of proteins transcriptional level was confirmed by quantitative real-time pcr. as shown in fig. 2 , the gene expression levels were in agreement to our itraq results. among these tested genes, tudor staphylococcal nuclease and thymidylate synthase involved in metabolic process group showed similar gene expression trend to our proteomics data. briefly, tudor staphylococcal nuclease showed a significant decrease at 12 hpi, while thymidylate synthase was significantly down-regulated at 1 hpi but up-regulated at 12 hpi. in regarding to the signal transduction group, the transcriptional level of mapkk, mapk-14, 14-3-3 epsilon and ras opposite were analyzed. mapkk was significantly decreased at 1 hpi but increased at late infection stage, and mapk-14 was clearly down-regulated at late hours of infection. ras opposite showed a decrease on initial hours while 14-3-3 epsilon was obviously enhanced at late hours of infection. in case of immune-related and cytoskeletal group, both transglutaminase and calponin-3 showed significant decline at initials hours post wssv infection, which were in well consistent with our itraq result. the wssv infection in the crayfish hpt cells obviously altered the host cellular expression of proteins related to metabolic processes, signal transduction, immunity and cytoskeletal organization. wssv utilized host metabolic system for its energy needs, replication and dissemination inside the host cells. the virus managed to alter important signal transduction proteins those function as gtp binding, adapter molecules, protein biosynthesis and protein phosphorylation. the protein expression in early hours exposes vulnerability of host defense mechanism to the viral infection. the role of many of the identified proteins in viral infection remains unknown, some of those are first observations and will require systematic studies to show the mechanism of action. by identifying a series of proteins that are affected by viral infection this study opens new opportunities in two directions: 1) to better understand host-virus interactions and 2) to select targets to reduce vulnerability of the host toward the challenge. the proteins identified and host physiological changes elucidated can be further utilized for developing putative drug targets for controlling this virus. protective immunity induced by oral immunization with a rotavirus dna vaccine encapsulated in microparticles white spot syndrome baculovirus (wsbv) detected in cultured and captured shrimp, crabs and other arthropods a handbook of shrimp pathology and diagnostic procedures for disease of cultured penaeid shrimp discovery of immune molecules and their crucial functions in shrimp immunity shrimp 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potential pathogen chryseobacterium indologenes transglutaminase regulates immune-related genes in shrimp s€ oderh€ all, i. s€ oderh€ all, transglutaminase activity in the hematopoietic tissue of a crustacean, pacifastacus leniusculus, importance in hemocyte homeostasis the host defense of drosophila melanogaster molecular cloning, characterization and expression analysis of two novel tolls (lvtoll2 and lvtoll3) and three putative spatzle-like toll ligands (lvspz1-3) from litopenaeus vannamei a dorsal homolog (fcdorsal) in the chinese shrimp fenneropenaeus chinensis is responsive to both bacteria and wssv challenge toll-like receptor 4-mediated activation of p38 mitogen-activated protein kinase is a determinant of respiratory virus entry and tropism identification and molecular characterization of a spaetzle-like protein from chinese shrimp (fenneropenaeus chinensis) magall on-barajas, antioxidant enzyme activity in pacific whiteleg shrimp (litopenaeus vannamei) in response to infection with white spot syndrome virus a selenium-dependent glutathione peroxidase (se-gpx) and two glutathione s-transferases (gsts) from chinese shrimp (fenneropenaeus chinensis) trbp, a regulator of cellular pkr and hiv-1 virus expression, interacts with dicer and functions in rna silencing trbp and eif6 homologue in marsupenaeus japonicus play crucial roles in antiviral response tudor staphylococcal nuclease from penaeus monodon: cdna cloning and its involvement in rna interference supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2016.01.035. key: cord-298227-av1ev8ta authors: kähler, christian j.; hain, rainer title: fundamental protective mechanisms of face masks against droplet infections date: 2020-06-28 journal: j aerosol sci doi: 10.1016/j.jaerosci.2020.105617 sha: doc_id: 298227 cord_uid: av1ev8ta many governments have instructed the population to wear simple mouth-and-nose covers or surgical face masks to protect themselves from droplet infection with the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in public. however, the basic protection mechanisms and benefits of these masks remain controversial. therefore, the aim of this work is to show from a fluid physics point of view under which circumstances these masks can protect against droplet infection. first of all, we show that the masks protect people in the surrounding area quite well, since the flow resistance of the face masks effectively prevents the spread of exhaled air, e.g. when breathing, speaking, singing, coughing and sneezing. secondly, we provide visual evidence that typical household materials used by the population to make masks do not provide highly efficient protection against respirable particles and droplets with a diameter of 0.3–2 μm as they pass through the materials largely unfiltered. according to our tests, only vacuum cleaner bags with fine dust filters show a comparable or even better filtering effect than commercial particle filtering ffp2/n95/kn95 half masks. thirdly, we show that even simple mouth-and-nose covers made of good filter material cannot reliably protect against droplet infection in contaminated ambient air, since most of the air flows through gaps at the edge of the masks. only a close-fitting, particle-filtering respirator without an outlet valve offers good self-protection and protection against droplet infection. nevertheless, wearing simple homemade or surgical face masks in public is highly recommended if no particle filtrating respiratory mask is available. firstly, because they protect against habitual contact of the face with the hands and thus serve as self-protection against contact infection. secondly, because the flow resistance of the masks ensures that the air stays close to the head when breathing, speaking, singing, coughing and sneezing, thus protecting other people if they have sufficient distance from each other. however, if the distance rules cannot be observed and the risk of inhalation-based infection becomes high because many people in the vicinity are infectious and the air exchange rate is small, improved filtration efficiency masks are needed, to take full advantage of the three fundamental protective mechanisms these masks provide. at present, humanity is threatened by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic. the risk of severe infection with the virus depends heavily on physical factors of the infected persons and the quality of the medical system. according to a recent study the estimated infection fatality ratio (ifr), averaged over all age-groups including those who don't have symptoms, is between 0.2% -1.6% with an average of 0.66% (verity et al., 2020) . these numbers look small, and the fatality risk may seem acceptable, and therefore the danger is often marginalized. this is surprising considering that the apollo crew, the space shuttle astronauts and the allied soldiers during the 2003 iraq war took a deadly risk of this magnitude. only very few people take such risks voluntarily and with full consciousness. for comparison, the lethal risk of a fatal accident with a commercial aircraft was 1:7700000 in 2008 and even such a small risk is not taken by some people. considering that the ifr of the seasonal flu is about 0.04 -0.1% (centers for disease, 2010) or even much lower (wong et al. 2013 ) the mortality rate of sars-cov-2 appears to be significantly higher than for influenza flu. although the numbers for sars-cov-2 are quite preliminary and the estimates may drop over time (verity et al., 2020 , faust, et al., 2020 it is quite clear that the strategy of herd immunization of the population is not an option, as the number of victims would be far too high. great hopes for coping with the pandemic currently rest on the development of a vaccine. unfortunately, it is completely uncertain when an effective and well-tolerated vaccine will be generally available to contain the pandemic. drugs such as chloroquine, remdesivir, lopinavir and ritonavir are also considered to be great sources of hope in the fight against the coronavirus disease 2019 (grein at al., 2020) . however, even if one of the drugs should prove to be effective, there is no guarantee that the drug can be made available to the world population in sufficient quantities. in addition, it is possible that, despite the use of drugs, going through a severe course of disease can lead to lifelong neuropsychiatric sequelae (troyer et al., 2020 and zandifar & badrfam, 2020) or cause other diseases (ackermann et al., 2020 and varga et al., 2020) . containing the pandemic is therefore the only viable way to quench the spread of the virus. but containing the pandemic is a difficult task as about 44% of sars-cov-2 infections are caused by people with a presymptomatic and asymptomatic course of infection (he et al., 2020) . therefore, due to the absence of symptoms, many people do not know that they are infected and are spreading the virus and these people make it very difficult to trace the transmission chains. furthermore, about 10% of infected people are responsible for 80% of infections (kupferschmidt, 2020 and lloyd-smith et al., 2005) . people who have many social contacts at work or in their private lives and who do not protect themselves and others sufficiently by observing the rules of distance and hygiene, or who consider the risk of the virus to be low, appear to be a serious problem in the actual pandemic. for these reasons, the government must act at various levels to avert great harm to the population. the effectiveness of the containment strategy depends on 1. how societies are able to protect themselves personally against infection through hygiene, social distance and technical aids such as protective masks, glasses, gloves, 2. how well the infrastructure is in place to identify the infection chains and effectively contain the spread, e.g. through mobile data collection, isolation or a lockdown, 3. how well the seriously ill can be treated in hospitals. in view of these prospects, it seems necessary for the time being to prevent the spread of the virus and to treat those infected as well as possible. in order to ensure the latter, the capacities of the health system must not be overloaded. but it is clear that this condition means that the pandemic will last for years without a vaccine. to not overload the medical system, governments are pursuing the concept of containment by means of a lockdown because it proved successful in st. louis during the spanish flu of 1918. this approach is quite effective when the population obeys the rules, but the impact on the state, economy and society is devastating when the lockdown lasts longer than a few weeks. therefore, this concept is not a viable way to contain the pandemic in the long term. consequently, it is necessary to fight the infection where it occurs. understanding the transmission pathways is the key to finding effective measures to block the infection and to reliably protect healthcare workers and the population. contact infection were initially assumed to be the main transmission route of sars-cov-2. today, hygiene measures and the avoidance of shaking hands effectively prevent this path of infection. droplet infection is currently assumed to be the main transmission route over short distances . since this path of infection is via the air, the rules of distance are effective (soper, 1919 and wells et al., 1936) . but it is also known that sars-cov-2 can remain infectious in aerosols for more than 3 hours, at least under laboratory conditions at high humidity (van doremalen et al., 2020 and pyankov et al., 2018) . it is therefore conceivable that infections can also occur under special conditions over long distances, provided that the local virus concentration reaches the minimum infection dose due to poor air exchange in rooms. a significant proportion of the aerosol exhaled by humans has a diameter of less than 10 μm (johnson et al., 2011) when breathing, speaking, singing and coughing. it is also known that the size and number of droplets increases with the volume of the voice (asadi et al. 2019 and loudon & roberts, 1968) and it is known that upper respiratory tract diseases increase the production of aerosol particles (lee et al., 2019) . water droplets of this size evaporate within a few seconds at normal humidity (liu et al. 2014 and rensink 2004) . droplets with a diameter of 10 μm for instance are evaporated after about 1 s at 50% relative humidity and larger droplets sink quickly to the ground and evaporate (marin et al., 2016 . if the viruses are released as "naked" viruses together with the salt after the droplets have evaporated, the spatial concentration decreases rapidly over time, as the viruses no longer move in a correlated manner but quickly separate due to the chaotic turbulent flow motion. the viral load thus decreases rapidly in time and space, making infections over long distances or long periods of time increasingly unlikely. for this reason it is most important to understand the transmission of the virus over short distances. hygiene regulations and social distancing are very effective in blocking short distance infections. during the lockdown, the distance rules can usually be adhered to, but what happens when the actual lockdown is over and the people meet again in a confined space? then additional effective and efficient protection is essential to stabilise infection rates. since the viruses are spread by contact and droplet infection, technical devices are required that effectively intervene in the chain of infection and effectively block infection. an effective protection is the respiratory mask as known since 100 years (soper, 1919) . the sars outbreak in hong kong suggested that the use of simple face masks may have contributed to an overall reduction in the incidence of viral respiratory infections lo et al., 2005) . another study has shown that even a simple surgical mask can effectively reduce sars infection (seto et al., 2003) . these results are supported by recent articles (leung et al., 2020b , howard et al., 2020 . it was surprizing that for months, who, the cdc and many public health professionals in europe advised against wearing face masks unless someone has covid-19 or cares for someone who has covid-19 (feng et al., 2020 and leung et al., 2020a) . this recommendation was based on three allegations. first, it was said that there is no scientific evidence that face masks can protect against droplet / aerosol infections. second, it was argued that the population will not be able to wear the masks properly. third, the statement that people will feel safe when wearing masks and then become careless and take risks was frequently made. at the same time, these experts have stressed that health professionals urgently need face masks to protect themselves effectively. this contradiction has created uncertainty among the population and called into question the credibility of the experts. it is a fact that particle filtration masks are recognized as legal occupational safety equipment and that the wearing of these masks in contaminated areas is required by labor law. there is therefore no doubt that these masks, when used correctly, provide effective protection within the specification range. the effectiveness of simple mouth-and-nose covers and surgical masks is less well accepted. the international council of nurses (icn) estimates that, on average, 7% of all confirmed cases of covid-19 are among healthcare workers (icn, 2020) . this illustrates that surgical masks may not provide the reliable protection against droplet infection, as anticipated. it is therefore very important to distinguish clearly between the different mask types when talking about their protective function. unfortunately, this was not done sufficiently by the virologists and politically responsible persons in the initial phase of the pandemic. also the second argument is questionable. why should the people of western societies not be able to protect themselves as many people in east asian countries have long been doing? many people in east asian countries have already recognized through numerous pandemics that proper masks work effectively. it does not seem right to regard the western population as unteachable or even incapable. the third argument is also false, because the opposite is true according to scientific studies (kimberly et al., 2020 , scott et al., 2007 and ruedl et al., 2012 . if people protect themselves personally, they have dealt with the danger and therefore they benefit from the protection of the safety device and from the less risky behaviour due to insight. the reason why these facts were not appreciated by the experts is due to the attempt to prevent competition for protective masks between medical personnel and the public. in the meantime, the general perception of the protective effect of face masks has become generally accepted. in the usa, the cdc has changed its guidelines and recommended that the public wear fabric face masks. in other countries, too, it is now recommended to protect themselves with suitable masks. however, it is recommended by governments and professionals to wear only simple mouse-and-nose covers that can be manufactured by the people themselves or surgical masks to avoid distribution battles with medical staff for certified and comfortable particle filtration masks. but the big question is, how effectively these homemade mouth-and-nose covers and surgical masks can protect against droplet infection. the answer is highly relevant to guide public behaviour (leung et al., 2020a) . one study suggests that a surgical face mask and masks made of dense cotton fabrics apparently cannot effectively prevent the spread of sars-cov-2 into the environment through the coughing of patients with covid-19 (bae et al., 2020) . another study suggests that any mask, no matter how efficiently it filters or how well it is sealed, has minimal effect unless used in conjunction with other preventive measures such as isolation of infected cases, immunisation, good respiratory etiquette and regular hand hygiene (kwok et al., 2015) . these findings contradict the results in , lo et al., 2005 and seto et al., 2003 . due to the contradiction, it is understandable that experts in the media have expressed the opinion that there is no scientific evidence for the effectiveness of masks and therefore the wearing of masks in public was not recommended for a long time. the fallacy of politicians and virologists, however, was to generalize the results obtained with simple mouth-and-nose covers to all masks without differentiation. in order to clarify whether or to what extent these masks offer effective protection against droplet infection and to understand why research results differ on this simple scientific question, we have carried out these tests. first, we analyse the flow blockage caused by surgical masks when coughing, as this is essential for the protection of others and because coughing is a typical symptom of covid-19. second, we qualify the effectiveness of different filter materials and masks to determine the protection ability against droplets. finally, we prove the effect of gap flows at the edge of surgical and particle filtrating respiratory masks. in contrast to the medical studies cited, we apply engineering research methods of fluid mechanics. the use of this research approach has several reasons: firstly, the detachment of droplets in the lungs and throat and their convective transport through the mouth into the atmosphere until inhalation as well as the deposit and evaporation of droplets is a purely fluid mechanical process. secondly, the effective blocking of the flow with suitable masks is a research subject of fluid mechanics. thirdly, the filtering of particles from an air stream with the aid of suitable materials is also a purely fluid mechanical problem as well as the gap flow. finally, this approach also has the advantage that the results are reproducible in a statistical sense, since the boundary conditions are well defined. we are not studying whether an infection really occurs in a special case, but whether an infection is physically possible in general. in the first sets of the experiments, outlined in section 3.1, the flow field generated by coughing without and with a surgical mask is examined as coughing sets the air strongly in motion and because coughing is a typical symptom of covid-19. to measure the flow field quantitatively in space and time we use particle image velocimetry (piv) (raffel et al., 2018) . for the measurements a 8 m long testing room with a cross section of 2 m × 2 m was seeded with dehs (di-ethyl-hexyl-sebacat) tracer particles with a mean diameter of 1 μm (kähler et al., 2003) . dehs was used as these droplets do not evaporate as quickly as water droplets. the tracer particles provided by a seeding generator (pivtec gmbh, germany) were illuminated in a light-sheet generated with a frequency doubled nd:yag laser (spitlight piv 1000-15, innolas laser gmbh, germany). the light-sheet was oriented normal to the mouth opening and parallel to the symmetry axis of the body and the longitudinal axis of the room. the light scattered by the tracer particles were recorded with back illuminated scientific cmos cameras (pco.edge 5.5, pco ag, germany) equipped with zeiss distagon t* lens with a focal length of 35 mm and 50 mm. the triggering of the system components was achieved with a programmable timing unit (lavision gmbh, germany). the recorded series of images were evaluated with a commercial computer program (davis, lavision gmbh, germany). these quantitative piv measurements allow to determine the area that can be contaminated due to the exhaled air, the velocity of the exhaled droplets and the turbulence properties of the flow. in the second set of experiments, discussed in section 3.2, common household materials currently used by the population and some medical staff to make simple masks at home were tested but also a surgical mask and a ffp3 mask to visualize their filtering properties. the tested materials are given in table 1 . for the investigation, a test set-up was installed which largely fulfilled the officially prescribed test conditions in europe (din en 149). the materials were installed one after the other in a fixed position in front of the inlet of a rectangular flow channel with a cross-section of 0.1 m × 0.1 m, as shown in fig. 1 . the material was held in place with a special clamping device that seals tightly to the duct to avoid leakage flows. to explore the filtering performance of the different materials the movement of small aerosol droplets passing through the media was observed visually in front of and behind the filter material with a digital camera. we only use droplets whose diameter is less than 2 μm, since the removal of the smallest droplets in an air stream is the greatest challenge in mask development. if these droplets can be effectively filtered out effectively, then all droplets larger than 2 μm can also be filtered. the droplets were generated from dehs with an aerosol generator (agf 2.0, palas gmbh). dehs was used again as these droplets are long lasting. consequently, bias errors due to evaporation effects can be neglected. a nd:yag double-pulse laser (evergreen 200, quantel, france) was used to illuminate the droplets. the output beam was fanned out with a few lenses to form a 1 mm thin light-sheet. the light-sheet was located in the middle of the flow channel parallel to the flow direction as indicated in fig. 1 . the scattered light emitted by the illuminated aerosol in the light-sheet plane was recorded with a highly sensitive pco edge 5.5 scmos camera equipped with a zeiss distagon t* lens with a focal length of 50 mm. the triggering of the system components and the data recording was realized again with the software davis from lavision. the flow velocity was driven by the pressure difference between the atmosphere and the flow box. the flow rate through the filter material was adjusted approximately according to the din en 149 test standard (90 liter/minute). the volume flow rate and the movement of the droplets through the filter material was measured optically with high spatial and temporal resolution using piv. to calculate the volume flow rate the average flow velocity within the light-sheet plane in the flow channel was measured and it was assumed that this velocity is homogeneous over the cross section of the channel. this assumption is justified as the filtering materials are homogeneous and the inflow condition is constant across the filtering material. with the know size of the cross section the volume flow rate can be calculated. the pressure drops provided in table 1 are calculated from the measured pressure drops and the volume flow rate. it is assumed that the pressure loss is proportional to the square of the volume flow rate. the pressure drop across the filter material was measured with a testo 480 (testo se & co. kg, germany) pressure transducer with an uncertainty of about 3 pa for the third set of experiments, analysed in section 3.3, simple flow visualizations using smoke were performed in order to demonstrate the effect of the gap around the mask edge. a person exhaled air seeded with tracer particles while wearing surgical and ffp2 masks. in the first series of experiments, one person performed a single severe cough while the piv system was measuring the flow field data. the video in the supplementary material shows the temporal evolution of the process. the results displayed in this subsection show instantaneous velocity fields of various independent timeresolved flow field measurements. color-coded is the magnitude of the local flow velocity and the vectors indicate the direction of the flow movement at a given time step. in areas where the flow movement remains close to zero over the whole recording time (blue colour), no droplets can penetrate as only the flow can move the particles to other areas. large droplets with a diameter of one millimetre or more, such as those produced when sneezing (lok, 2016) , can fly ballistic over long distances, and occasionally ballistic flying droplets are produced when certain sounds are spoken. but sneezing is not a typical covid-10 symptom so that this will not we considered here. the small droplets that are normally produced when breathing, speaking, singing and coughing are immediately slowed down and then move with the flow velocity of the ambient air. it is therefore important to study the air set in motion by exhalation. furthermore, the small droplets are particularly dangerous because they can be inhaled deep into the lungs. figure 2a shows that the spread of the exhaled air forms a cone like shape similar to a free turbulent jet (see video). the flow velocity is reaching values up to 1 m/s near the mouth, but due to the widening of the cone caused by the turbulent mixing and entrainment (reuther et al., 2020) the flow velocity decreases in streamwise direction. the widening of the area in motion reduces the viral load significantly with distance. a single strong cough sets the air in motion over a distance of less than 1.5 m in the experiments. distances of more than 1.5 m can be considered safe according to these results, since no droplets can reach such large distances when accelerated by a single cough. however, if the cough lasts longer, greater distances can be achieved, as shown in fig. 2b . for this reason, it is important to dynamically increase the distance to a person if the coughing stimulus is about to last longer. the results in fig. 2c illustrate how the spread of the airflow from the mouth during coughing is very effectively inhibited by a surgical mask. physically the mask ensures that the directional jet like air movement with high exit velocity from the mouth is converted into an undirected air movement with low velocity behind the mask. this is because the exhaled air increases the pressure inside the mask compared to the atmosphere outside, and the pressure difference creates a flow movement in all directions. this effect is of utmost importance for limiting the virus load in the environment. the results show that even a simple mouth-and-nose cover or a surgical mask can effectively protect other people in the vicinity because the mask prevents the droplets from spreading over a wide area. a simple mask with sufficient flow resistance therefore provides very effective protection for people 1 m 1 m 0.3 m 0.3 m in the surrounding area when infected and wearing the mask. wearing a mask is therefore absolutely useful to protect others according to our quantitative measurements. figure 2d shows the spread of exhaled air when speaking. it can be clearly seen that a greater spread of the exhaled air appears than when coughing with a mask. consequently, wearing a mask during normal face-to-face conversations and of course also when talking on the smartphone in a human environment is extremely useful to stop the transmission of the sars-cov-2 infection via droplets. it must also be taken into account that persons with a presymptomatic or asymptomatic course of infection will infect other persons most likely during face-toface conversations. a mask will therefore make an effective contribution to suppressing this significant path of infection. in this section we want to find out if the material of simple mouth-and-nose covers, surgical masks and ffp3 masks can protect the user from droplet infection, if the surrounding air is contaminated with sars-cov-2. in this case, the mask material must have good filtering properties to stop small droplets that typically occur when speaking, singing and coughing. since large droplets are easily filtered out by simple materials, we focus on small droplets in the range between 0.3 and 2 μm because they are produced in large fractions when speaking, singing and coughing and they can penetrate deep into the lungs. the droplets were distributed approx. 400 mm in front of the filter materials. in order to make the motion of the droplets and the filtering ability of the materials clearly visible an inhomogeneous droplet distribution was generated. the flow direction is from left to right and the flow state of the incoming air is laminar. if the intensity of the scattered light emanating from the droplets is large in front of the filter material (left image) and close to zero behind the filter material (right image), the droplets are almost completely filtered out through the material. if, on the other hand, no significant reduction in intensity can be detected behind the filter material, the filter effect is negligible. the area of the filter mount and the channel edges are not shown in the following images, since no relevant flow and droplet information is visible in these areas. the results presented are qualitative, but intended to be this way to provide readers with visual evidence of the particle penetration through different candidate filter media. a better impression of the filter efficiency is obtained by viewing the second video in the supplementary material. the comparison of the two pictures in fig. 3 (left) shows that almost all droplets pass the tested surgical face mask unhindered. consequently, this mask does not provide serious self protection against droplet infection. only a mixing of the droplet distribution takes place due to the porosity of the filter material. it is fatal that medical personnel are often so poorly protected by these masks. but it is also fatal for patients if clinical staff with a presymptomatic or asymptomatic course of infection uses these masks. even worse than the surgical face masks is the hygiene mask, see fig. 3 (right). this mask is designed for catching larger objects such as hair and spook, but tiny droplets, such as those produced when talking, singing and coughing, cannot be filtered out of the air stream by the hygiene mask. it should also be noted that the flow resistance of the hygiene mask is so low that even the protective mechanism described in subsection 3.1 does not function effectively. figure 4 reveals the effectiveness of particle filtering with toilet paper with 4 layers, paper towel, coffee filters, and microfibre cloth which also offer no serious protection against droplets in this size range. only very large droplets are retained by these materials and therefore these materials are suitable for their intended use, but not as filter material for small droplets. it is therefore strongly discouraged to make masks from these materials with the aim of protecting oneself from infection. furthermore, a very strong fleece was tested, which serves as a protective coating on ironing boards. the material is 4 mm thick, completely opaque and has a pressure drop of about 35 pa. however, a filter effect is not visible, as indicated in fig. 5 (left) . the droplet clouds flow almost unfiltered through the fleece. even several layers of a dense fabric do not have a proper filtering effect on the considered droplet sizes, which escape mainly when breathing, speaking, singing and coughing. good results could only be achieved with the material of a vacuum cleaner bag with fine dust filter properties, see fig. 5 (right) . despite the small droplets used in these tests, almost all droplets are reliably filtered out. consequently, also no larger droplets will be able to pass through the material. according to the manufacturer swirl, the material filters 99.9% of fine dust down to 0.3 μm diameter. this vacuum cleaner bag with fine dust filter therefore has better filtering properties than all tested materials and masks and even an ffp2 protective mask has poorer filtering properties, as it only has to filter out 94% of the fine dust down to 0.6 μm to meet the specifications (uvex, 2020). the material of vacuum cleaner bags with fine dust protection is therefore very well suited as a self-protecting mask if only the filter effect is considered. however, because vacuum cleaner bags are not certified clinical products, they may contain unhealthy ingredients that kill bacteria and harmful fibers that may leak from the bag material. it is therefore uncertain whether this material is suitable in practice as a material for a respirator mask. figure 6 (left) illustrates the filtering capabilities of an ffp3 mask under the test conditions. nearly all droplets are filtered out as expected. therefore, this mask type is very well suited to protect people from an infection by means of aerosols even when the environment is strongly contaminated with infectious droplets. recently, some hospitals in the usa make use of halyard h600 material to protect their employers from aerosol infection. the test result of the material is displayed in fig. 6 (right). it is clearly visible that the filtering capacity of the material is not sufficient to protect people from infection by aerosols if the environment is contaminated with the sars-cov-2. figure 7 shows with different resolution microscopic images of the halyard h600 material. it is composed to fibers but the density might not be sufficient to filter the particles used in our investigation. there are also tiny holes in the pockets visible, which could be the reason why the aerosol passes through the material, as the flow resistance at the holes is low compared to the other parts of the material. the flow tests clearly show that apart from the vacuum cleaner bag and the ffp3 mask, the filter effect of the tested materials is not sufficient to protect against droplet infection reliably if the environment is contaminated with sars-cov-2. even masks routinely used by medical staff in hospitals and doctor's offices have almost no significant filtering effect on the droplet sizes typically produced when breathing, speaking, singing and coughing. the results are therefore in good agreement with the results from (leung et al., 2020b and davies et al., 2013 and kwok et al., 2015 . but why has wearing these masks been shown to provide effective protection against infection with the virus in the sars epidemic, as shown in , lo et al., 2005 and seto et al., 2003 ? because a mask is important not only because of its filtering ability, but to limit the droplet propagation as discussed in section 3.1. so in combination with distances these mask can protect if only a few people are infected in the surrounding. the results in (leung et al., 2020b and bae et al., 2020) are correct, but they do not consider the full performance of masks, but only a partial aspect. therefore, the conclusions in the articles are not universal. the findings in and lo et al., 2005 and seto et al., 2003 are understandable when the full performance of masks in blocking infections is considered. unfortunately, wearing a simple mouth-and-nose cover may be less comfortable than wearing a particle filtering face mask. in effect, this can promote a smear infection. since all these transmissions of infection are possible in daily life, wearing a comfortable mask is essential to block human-to-human transmission by smear and droplet infection. to ensure the best possible protection, a particle filtering mask should be used if the number of infected persons in the environment and the viral load in the room is unknown. at present, social distancing practices and universal masking wearing seem to be the best methods of containing viral pandemic without stricter lockdown policies and without vaccines. some recent studies show that even the simple materials we have tested have some filtering ability (davies et al., 2013 , drewnick, 2020 , konda et al., 2020 and van der sande et al., 2008 , we do not question these results, although the pressure drops in one study is anomalously low (see supporting information in konda et al., 2020) , but we state explicitly that a material that does not have an adequate filtering ability equivalent to an ffp2/n95/kn95 mask cannot be recommended as a filter material for self-protection against droplet infection. statistically speaking, every loss of performance leads to an increase in the number of infected people and thus to an increase in the number of death. it is therefore very dangerous to recommend materials with some filtering properties as possible materials for self-protection masks. but there is another important aspect that will be discussed next. according to the previous section one might argue that a mouth-and-nose cover or surgical mask made of a good filter material would provide good protection against infection when infected people are in the vicinity or the room is contaminated with viruses. but that will not usually be the case. air takes the path of least resistance. as these masks do not seal tightly enough with the face, droplets can flow unhindered past the edge of the mask when inhaled and exhaled and reach the lungs or the environment. if the mask does not fit properly, this will even be the rule. this is illustrated in fig. 8 were a person is exhaling air during an easy exhalation without physical exertion (left), strong breathing during physical exertion (middle) and when coughing (right). the first video in the supplementary material shows the animated sequences. the analysis shows that it is very important do differentiate between mouth-and-nose cover, surgical mask and particle filtering respirator mask because they differ substantial in their fundamental protection properties. face masks can offer three fundamental different kind of protection: 1. they effectively prevents a smear infection, as the wearers of the masks no longer perform their habitual grip on the face and thus no longer bring the virus from the hand into the mouth or nose (howard et al., 2020) . 2. the flow resistance of the mask greatly limits the spread of viruses in the room. this significantly reduces the risk of infection in the vicinity of an infected person (protection of third parties). 3. the inhalation of droplets containing viruses can be prevented by using a tight-fitting mask with particle filtering properties (self-protection). the first fundamental protection mechanism can be reached by all face masks if they fit well and sit comfortably. if not, the user will touch the face even more than usual to correct the fit of the mask. as this can increase the risk of smear infection, a good fit of the mask is very important. the first and second fundamental protection mechanisms are fulfilled by all masks that have sufficient flow resistance. if the mask is worn and a candle can easily be blown out despite the mask, the mask does not fulfil this function and should not be used. all three fundamental protection mechanisms can be only achieved with ffp2/n95/kn95 or better particle filtering respirator mask. typical materials currently used by the public to build masks reduce the risk of smear infection and effectively prevent the widespread spread of viruses in the environment. therefore, the use of these mouse-and-nose covers and surgical masks are very important to prevent smear infection and droplet infection to others if the distance is not too close. as these masks do not have a significant particle-filtering protective effect against droplets that are typically produced when breathing, speaking, singing, coughing and sneezing they should not be used if the environment is contaminated, like in hospitals, even when the distance rules are followed. to achieve effective self-protection in a virus-contaminated environment, masks with particle filtering properties (ffp2/n95/kn95) are absolutely necessary from our point of view. if a large number of infected persons are present and distance rules cannot be achieved, a very good particle filtration mask (ffp3 or better) is strongly recommended. if these general rules are followed and all people use suitable particle-filtering respirators correctly, the transmission of viruses via droplets / aerosols can be effectively prevented. otherwise, these types of masks would never have received certification, nor would they be a core component of the personal protective equipment in hospitals and other environments. therefore, proper face masks can save lives while maintaining social life and securing the economy and the state. but universal masking alone is not enough for two reasons: first, many people are not very good at following rules consistently. therefore, it is advisable to observe the rules of hygiene and distance and to be careful even when wearing a mask. in the event of a car accident, the occupants are also protected by various devices (bumpers, crumple zone, safety belts, airbags, head and legroom, autonomous assistance systems, ...). second, some people are extremely bad at following rules, either because they do not want to or because they simply cannot. these people can become superspreaders. therefore, the early detection of sources of infection and their isolation remains important beside universal masking and the rules of hygiene and distance. pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 aerosol emission and superemission during human speech increase with voice loudness effectiveness of surgical and cotton masks in blocking sars-cov-2: a controlled comparison in 4 patients estimates of deaths associated with seasonal influenza ---united states physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis. the lancet testing the efficacy of homemade masks: would they protect in an influenza pandemic? aerosol and 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upper respiratory tract infection the impact of community psychological responses on outbreak control for severe acute respiratory syndrome in hong kong mass masking in the covid-19 epidemic: people need guidance. the lancet respiratory virus shedding in exhaled breath and efficacy of face masks evaporation and dispersion of exhaled droplets in stratified environment superspreading and the effect of individual variation on disease emergence respiratory infections during sars outbreak where sneezes go singing and the dissemination of tuberculosis solutal marangoni flow as the cause of ring stains from drying salty colloidal drops surfactant-driven flow transitions in evaporating droplets survival of aerosolized coronavirus in the ambient air reducing transmission of sars-cov-2. science, eabc6197 particle image velocimetry verdunstung akustisch levitierter schwingender tropfen aus homogenen und heterogenen medien effect of the intermittency dynamics on single and multipoint statistics of turbulent boundary layers interfacial flows in sessile evaporating droplets of mineral water does risk compensation undo the protection of ski helmet use? professional and home-made face masks reduce exposure to respiratory infections among the general population testing the risk compensation hypothesis for safety helmets in alpine skiing and snowboarding effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars). the lancet the lessons of the pandemic are we facing a crashing wave of neuropsychiatric sequelae of covid-19? neuropsychiatric symptoms and potential immunologic mechanisms. brain, behavior, and immunity endothelial cell infection and endotheliitis in covid-19 estimates of the severity of coronavirus disease 2019: a model-based analysis clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan air-bone infection case fatality risk of influenza a (h1n1pdm09): a systematic review covid-19: considering the prevalence of schizophrenia in the coming decades identifying airborne transmission as the dominant route for the spread of covid-19 the authors would like to thank stefan ostmann for conducting the mask experiments presented in section 3.3 and amirabas bakhtiari for taking the microscopic images in figure 7. (www.preprints christian j. kähler (prof. dr.) and rainer hain (dr.) • a simple mouth-and-nose cover or a surgical mask is able to effectively limit the spread of air and aerosol when breathing, speaking, singing, coughing and sneezing. • wearing a mask is therefore a very useful contribution to contain a pandemic by protecting people in the vicinity from droplet infection.• however, a mouth-and-nose cover or a surgical mask does not fit tightly enough on the face to protect against droplet infection.• only a particle-filtering half-mask that fits tightly offers protection against droplet infection.• common household materials, however, do not have a sufficient filter effect to protect against droplet infection. key: cord-273326-gmw8gl2r authors: saiz, juan-carlos; de oya, nereida jiménez; blázquez, ana-belén; escribano-romero, estela; martín-acebes, miguel a. title: host-directed antivirals: a realistic alternative to fight zika virus date: 2018-08-24 journal: viruses doi: 10.3390/v10090453 sha: doc_id: 273326 cord_uid: gmw8gl2r zika virus (zikv), a mosquito-borne flavivirus, was an almost neglected pathogen until its introduction in the americas in 2015, where it has been responsible for a threat to global health, causing a great social and sanitary alarm due to its increased virulence, rapid spread, and an association with severe neurological and ophthalmological complications. currently, no specific antiviral therapy against zikv is available, and treatments are palliative and mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration. nevertheless, lately, search for antivirals has been a major aim in zikv investigations. to do so, screening of libraries from different sources, testing of natural compounds, and repurposing of drugs with known antiviral activity have allowed the identification of several antiviral candidates directed to both viral (structural proteins and enzymes) and cellular elements. here, we present an updated review of current knowledge about anti-zikv strategies, focusing on host-directed antivirals as a realistic alternative to combat zikv infection. since the beginning of the 21st century, a number of infectious disease threats have emerged that demand a global response. among them, severe acute respiratory syndrome virus, avian influenza in humans, pandemic influenza a (h1n1), middle east respiratory syndrome coronavirus, chikungunya virus, and ebola virus have been the most threatening ones. nonetheless, the emergency of a vector-borne virus, zika virus (zikv), which is responsible for congenital malformations and other neurological and ophthalmological disorders, was hard to predict. zikv is a mosquito-borne virus belonging to the spondweni serocomplex in the genus flavivirus of the family flaviviridae [1] . the virus has been isolated from various mosquito species, although it seems that the natural transmission vectors are mosquitoes of the genus aedes [2, 3] . besides mosquito bites, viral direct human-to-human transmission can occur perinatally, sexually, and through breastfeeding and blood transfusion [4] . the zikv genome is a single-stranded rna molecule (≈10.7 kb) of positive polarity encoding a single open reading frame (orf) flanked by two untranslated regions at the 5 and 3 ends [5] . zikv was first isolated from the serum of a monkey in 1947, and one year later from aedes africanus mosquitoes caught in the same area, the zika forest [6] . until it was detected in asia in the 1980s, the virus had been confined to africa. later on, human outbreaks were reported in the pacific islands, micronesia in 2007 and, then, in french polynesia in 2013 [4] . the natural course of zikv infection was usually asymptomatic or produce a relatively mild illness and an uneventful recovery [7] , hence, the virus was considered an almost neglected pathogen until its recent introduction into the americas in 2015, when it became a threat to global health, showing increased virulence, rapid spread, since the recent outbreak in 2015 in the americas, a quite high number of possible antiviral candidates are being tested in vitro and in vivo. however, until now, no specific therapy has been approved against any flavivirus [17] , including zikv [18] , and, thus, current treatments are mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration [15] . nevertheless, it should be noted that some commonly used drugs, such as acetylsalicylic acid, are contraindicated in zikv-infected patients, since they increase the risk of internal bleeding, and other arboviruses (dengue or chikungunya viruses) that can co-infect the patients may produce hemorrhages [3] . due to the natural course of zikv infection, which is usually asymptomatic or produce a relatively mild illness and an uneventful recovery, when facing anti-zikv strategies, a very important point to take into account is the main target population that would benefit from it, namely immunocompromised patients and pregnant women and their fetuses [4] . in this sense, only for some of the tested drugs their safety profiles are known [19] . however, in cases of food and drug administration (fda) (https://www.drugs.com/) category b compounds (animal reproduction studies have failed to demonstrate a risk to the fetus and there are no adequate and well-controlled studies in pregnant women), or even in those of category c (animal reproduction studies have shown an adverse effect on the fetus and there are no adequate and well-controlled studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks), or d (there is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks), their use in pregnancy can be contemplated if the potential benefit outweighs the risks. even more, some of the assayed compounds cross the placenta and, thus, can also benefit the fetus. nonetheless, if used, this should be done in an individualized way, conditioning dosage and timings, and always under a clinician's control where the patient is informed of the pros and cons. current search for zikv antivirals is being conducted with different approaches: by screening of compounds libraries; by the repurposing of drugs of known active efficacy against other diseases now in use in clinical practice, many of which display broad-spectrum activity; and by testing natural products. two different strategies can be applied when pursuing for antivirals, those searching for compounds directed to viral targets (direct-acting antivirals) and those aimed to target cellular components needed for the viral life cycle (host-directed antivirals). among the virus-directed drugs tested [18, 20] are those acting against the viral rna-dependent rna polymerase (non-structural protein 5 (ns5)) catalytic domain, including nucleoside analogs and polymerase inhibitors; the methyltransferase catalytic domain of the ns5 responsible for transferring the mrna cap; the ns2b-ns3 trypsin-like serine protease needed for proper processing of the viral polyprotein; and the ns3 helicase. the crystal structures of all these proteins have already been resolved and will certainly help to find new antivirals [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] . in the same way, structures from other viral proteins are also available that could help to design zikv therapeutic alternatives, such as those of the capsid c protein [33], whose destabilization may impair zikv multiplication, the ns1 [34,35], an immuno-modulator, or the envelope glycoprotein [36-38], which mediates cell binding and endosomal fusion, constitutes a major target for neutralizing antibodies, and could be also the target for virucidal compounds [39] . on the other hand, it has also been reported that passive transfer of neutralizing antibodies to pregnant mice suppresses zikv multiplication, inhibits cell death, reduces the number of progenitor neuronal cells, and prevents microcephaly [40, 41] . likewise, administration of monoclonal antibodies (mabs) recognizing the domain iii of the zikv-e protein protect mice of lethal zikv challenge [42, 43] and other mabs are able to bind and neutralize zikv, including those directed against the e dimer epitope [44] . human polyclonal antibodies produced in transchromosomal bovines also protect mice from zikv lethal infection, eliminated zikv induced tissue damage in the brain and testes, and protected against testicular atrophy [45] . thus, administration of therapeutic antibodies seems to also be a potential strategy against zikv. nevertheless, it should be noted that, although still controvertial in the case of zikv infection [46] , the well-known antibody dependent enhancement effect (ade) [47] , of which dengue virus (denv) is the prototypic model, may potentiate the risk of disease exacerbation. flaviviruses have small rna genomes (around 10.7 kb in length) and thus require many host factors and co-option of cellular metabolic pathways to successfully infect host cells and propagate efficiently [48] . this offers an opportunity to search for host targets as therapeutic tools that, in many instances, as they are shared by different members of the flaviviridae family, can be envisaged as pan-flaviviral antivirals [48] [49] [50] . this strategy can be directed to host factors implicated in infection, pathogenesis, and in the immune response, as it has been shown for denv and the west nile virus (wnv) [51] . in addition, their effect would be less prone to the emergence of mutants that will escape their action, as often occurs with drugs targeting viral components. consequently, this kind of approach could ideally lead to the discovery of broad spectrum antivirals that could provide low cost but effective tools for the control of flaviviral threats. different approaches are being used to identify potential host factors as therapeutic targets against flaviviruses including the analyses of transcript levels (e.g., next generation rna sequencing) for altered expression patterns during infection, proteome changes, kinases activities variations, and protein-rna interactions (e.g., two-hybrid screenings and affinity chromatography). likewise, functional analysis can be applied by overexpressing cdnas or by rnai-mediated loss of function screens using dsrna, sirna, or shrna libraries, although it should be noted that in some cases downregulation is inefficient and some genes have redundant functions [51] . replicons may also be used to specifically assay replication activity [52, 53] . theoretically, host-acting antivirals can be directed to any molecule or pathway implicated in the different steps of the viral life cycle, from early events (binding, entry, and fusion), to the formation of the replication complex, and the viral maturation and egress. the first step of zikv infection is its binding to the cellular receptor ( figure 1 ). several molecules have been proposed as a zikv receptor (members of the tyro3/axl/mer (tam) family of receptor tyrosine kinases, t-cell immunoglobulin and mucin domain (tim) and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (dc-sign)) that are expressed in different neuronal and non-neuronal permissive cell types. these molecules are also receptors for other viruses, including flaviviruses such as denv and wnv, regulate several cellular activities (adhesion, migration, proliferation, and survival, release of inflammatory cytokines, antigen uptake, and signaling), and play important roles in the host's response to infection [54] . however, elimination of a known receptor does not necessarily result in complete protection from viral infection, since flaviviruses use different receptors and, thus, there is always redundancy and alternatives. for instances, inhibiting, downregulating, knocking-down, or ablating axl, although in some cases they reduce zikv infection, they do not completely abolish it, pointing to the use of different cell surface receptors on different cell types [55] [56] [57] [58] . different molecules have been shown to inhibit zikv infection at the entry step ( figure 1 ). r448 (an axl kinase inhibitor) and myd1 (an axl decoy receptor) compromises, but do not completely abolish, zikv infection of glial cells [57] . r448, as well as cabozantinib, an inhibitor of axl phosphorylation, that are currently in clinical trials for anticancer activities, significantly impairs zikv infection of human endothelial cells in a dose-dependent manner by affecting a post-binding step [59] . likewise, curcumin, a widely used food additive and herbal supplement, reduces zikv infection in cell culture inhibiting cell binding while maintaining viral rna integrity [60] , as does suramin, an anti-parasitic that interferes with attachment to host cells and with virion biogenesis by affecting glycosylation and maturation [61, 62] . once zikv binds to the cell receptor, like other flaviviruses, it is internalized through clathrin-mediated endocytosis and transported to the endosomes with the involvement of cellular actin and microtubules to establish a productive infection ( figure 1 ) [57] . after internalization, to start translation and replication, the viral genome is released inside the cytoplasm by fusing the viral envelope with the membranes of the cellular endosomes, a process triggered by acidic ph inside them [63, 64] . nanchangmycin, an insecticide and antibacterial polyether, inhibits zikv multiplication and, although the exact mechanism of action has not been completely elucidated, it probably targets axl and blocks clathrin-mediated endocytosis [65] . acid endosomal ph triggers rapid conformational changes on viral envelope protein that result in its fusion with endosomal membrane in a ph-dependent manner, thus allowing nucleocapsid release to the cytoplasm for genome uncoating ( figure 1 ). the optimal ph for conformational rearrangements and viral fusion is 6.3-6.4, and these processes are likely dependent on the presence of cholesterol and specific lipids in the target membrane [66] . these processes can be potentially druggable, and in fact, arbidol, a broad-spectrum antiviral and immunomodulatory use for human influenza a and b infections, inhibits zikv multiplication in cell culture probably because it intercalates into membrane lipids leading to the inhibition of membrane fusion between virus particles and plasma membranes, and between virus particles and the membranes of endosomes [67] . chlorpromazine, an antipsychotic drug that also inhibits clathrin-mediated endocytosis, reduced zikv infection, confirming the requirement for clathrin-mediated endocytosis of zikv [68] . in addition, 25-hydroxycholesterol (25hc) is increased in zikv-infected human embryonic cells and brain organoids, and reduces viremia and viral loads without affecting viral binding, but blocking internalization and suppressing viral and cell membranes fusion [69] . even more, 25hc reduces mortality and prevents microcephaly in zikv-infected mice, and also decreases viral loads in the urine and serum of treated non-human infected primates [69] . daptomycin, a lipopeptide antibiotic that inserts into cell membranes rich in phosphatidylglycerol, which suggests an effect on late endosomal membranes enriched in this lipid, has also been described as a zikv inhibitor [70] . the dependence on endosomal acidification for zikv infection also provides a host target suitable for antiviral intervention. for instance, obatoclax (or gx15-070), an anti-neoplastic and pro-apoptotic inhibitor of the bcl-2 that targets cellular mcl-1, impairs zikv endocytic uptake by reducing the ph of the endosomal vesicles in cell culture, and thereby most likely inhibits viral fusion [71, 72] . however, obatoclax, which presents a low solubility, has not produced satisfactory results in clinical trials for hematological and myeloid diseases. saliphenylhalamide (saliphe), which targets vacuolar adenosine triphosphatase enzyme (atpase) and blocks the acidification of endosomes, inhibits zikv multiplication in human retinal pigment epithelial cells [71] that are natural targets for zikv infection [12] . similar results were found by adcock et al. (2017) with saliphe using a different screening [73] ; however, they reported that, contrary to that described by others [65] , other compounds that interfere with the endocytic pathway, such as dynasore, that blocks clathrin-mediated endocytosis, or monensin, a cation transporter, were either toxic for the cells used or did not show any anti-zikv activity, as neither did chloroquine (cq). these contradictory results are probably explained by the different methodologies, cell types, and, to a lower extent, viral strains used to analyze the antiviral activities of the compounds and suggest that compounds showing different activities should be carefully evaluated before going further with investigations. in this line, and contrary to above mentioned report [73] , cq, an fda-approved anti-inflammatory 4-aminoquinoline and an autophagy inhibitor widely used as an anti-malaria drug that is administered to pregnant women at risk of exposure to plasmodium parasites, was shown to have anti-zikv activity in different cell types (vero cells, human brain microvascular endothelial cells (hbmecs), and human neural stem cells (nscs)), affecting early stages of the viral life cycle, possibly by raising the endosomal ph and inhibiting the fusion of the envelope protein to the endosomal membrane [74, 75] . cq has been shown to reduce placental and fetal zikv infection [76] , and also attenuate zikv-associated morbidity and mortality in mice and protect the fetus from microcephaly [77] . even more, cq attenuated vertical transmission in zikv-infected pregnant interferon signaling-competent swiss jim lambert (sjl) mice, significantly reducing fetal brain viral loads [78] . similarly, cq, and other lysosomotropic agents (ammonium chloride, bafilomycin a1, quinacrine, mefloquine, and n-tert-butyl isoquine (gsk369796)) that neutralize the acidic ph of endosomal compartments, block infection of a human fibroblast cell line and vero cells [68, 75] . additionally, by medicinal chemistry-driven approaches, a series of new 2,8-bis(trifluoromethyl)quinoline and n-(2-(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives have been proved to inhibit zikv replication in vitro with a higher potency than chloroquine or mefloquine [79, 80] . more recently, by screening fda-approved drugs using a cell-based assay, it has been shown that amodiaquine, another antimalarial drug, also has anti-zikv activity in cell culture by targeting early events of the viral replication cycle [81] . niclosamide, a category b antihelmintic drug approved by fda, was capable of inhibiting zikv infection, and although its antiflaviviral effect has been associated to its ability to neutralize endolysosomal ph and interfere with ph-dependent membrane fusion, in the case of zikv, it seems that it was affecting other post-entry steps [82] . in addition, recently, it has been reported that niclosamide decreases zikv production, partially restores differentiation, and prevents apoptosis in human induced nscs; even more, it can partially rescue zikv-induced microcephaly and attenuate infection in a developed humanized zikv-infected embryo model in vivo [83] . likewise, tenovin-1, which represses cell growth and induces apoptosis in cells expressing p53 by inhibiting the protein-deacetylating activities of sirt1 and sirt2 and, thus, affects endosome functions, potently inhibits zikv infection in primary placental fibroblast cells [65] . iron salt ferric ammonium citrate (fac) also inhibits zikv infection through inducing viral fusion and blocking endosomal viral release by promoting liposome aggregation and intracellular vesicle fusion [84] . overall, these studies evidence the potential of targeting viral entry to combat zikv. once zikv-rna is released from the endosomes in the cytoplasm, it acts as mrna to synthesize the negative-strand viral rna that directs positive-strand rna synthesis (figure 1 ) [4] . silvestrol, a natural compound isolated from the plant aglaia foveolata that it is known to inhibit the asp-glu-ala-asp (dead)-box rna helicase eukaryotic initiation factor-4a (eif4a) required to unwind structured 5 -untranslated regions and thus impairing rna translation, exerts a significant inhibition of zikv replication in a549 cells and primary human hepatocytes [85] . n-(4-hydroxyphenyl) retinamide (fenretinide or 4-hpr), an activator of retinoid receptors that inhibits the proliferation of cancer cells and can induce apoptosis, inhibits zikv in cell culture and significantly reduces both serum viremia and brain viral burden in mice by decreasing the rate of viral rna synthesis, though not via direct inhibition of the activity of the viral replicase [86] . zikv relies on polyamines for both translation and transcription [87] , so that, drugs targeting the polyamine biosynthetic pathway, such as difluoromethylornithine (dfmo or eflornithine), an fda-approved drug that is used to treat trypanosomiasis, hirsutism, and some cancers, as well as diethylnorspermine (denspm) limit viral replication in bhk-21 cells [88] . zikv replication and particle morphogenesis take place associated with a virus-induced organelle-like structure derived from the membrane of the endoplasmic reticulum (er) (figure 1 ) [4] . de novo synthesized positive strand-rna, once packaged, form enveloped immature virions in the er, enter the secretory pathway and, then, in the trans-golgi network, the prm is cleaved before the virus is released from the infected cell ( figure 1 ) [89, 90] . er-membrane multiprotein complexes, such as the oligosaccharyltransferase (ost) complex, have been reported to be critical host factors for flavivirus multiplication. in this regard, it has been shown that the n-linked glycosylation inhibitor-1 (ngi-1) chemical modulator of the ost complex blocks zikv-rna replication in different cell types [91] . similarly, the host er-associated signal peptidase (spase) is an essential, membrane-bound serine protease complex involved in cleavage of the signal peptides of newly synthesized secretory and membrane proteins at the er and also for processing of the flavivirus prm and e structural proteins [92] . it has also been reported that cavinafungin, an alaninal-containing lipopeptide of fungal origin, potently inhibits growth of zikv-infected cells [93] . nitazoxanide, a broad-spectrum antiviral agent approved by the fda as an antiprotozoan and with potential activity against several viruses in clinical trials (rotavirus and norovirus gastroenteritis, chronic hepatitis b, chronic hepatitis c, and influenza), also inhibits virus infection targeting a post-attachment step, most likely virus genome replication [94] . likewise, brefeldin a, a penicillium sp. product that inhibits protein transport from the er to the golgi apparatus, inhibits zikv multiplication [95] , as does emetine, an anti-protozoal agent that inhibits both zikv ns5 polymerase activity and disrupts lysosomal function [96] . zikv infection leads to cell-death by inducing host caspase-3 and neuronal apoptosis during its propagation [97] . thereby, bithionol, a caspase inhibitor, inhibits zikv strains of different geographical origin in vero cells and human astrocytes [98] . similarly, by using a drug repurposing screening of over 6000 molecules, it was found that emricasan, a pan-caspase inhibitor that restrains zikv-induced increases in caspase-3 activity and is currently in phase 2 clinical trials in chronic hepatitis c virus (hcv)-infected patients, protected human cortical neural progenitor cells (npc) in both monolayer and three-dimensional organoid cultures, showing neuroprotective activity without suppression of viral replication [82] . additionally, bortezomib, a dipeptide boronate proteasome inhibitor approved for treatment of multiple myeloma and mantle cell non-hodgkin's lymphoma that regulates the bcl-2 family of proteins, has also been described as a zikv inhibitor [70] . similarly, different cyclin-dependent kinase (cdk) inhibitors, such as (alphas)-4-(acetylamino)-alpha-methyl-n-(5-(1-methylethyl)-2-thiazolyl)benzeneacetamide (pha-690509), reduced zikv-infection and propagation [82] . however, cdk inhibitors should not be suitable for the treatment of pregnant women but could be useful for the treatment of other non-pregnant patients, preventing the complications associated with zikv infection. the need for specific host lipids for flavivirus replication and particle envelopment make lipid metabolism a potential target for an antiviral search [66, 99] , and, even though manipulating a major metabolic pathway such as lipid biosynthesis can be envisaged as a dangerous antiviral approach due to the undesirable effects that could be detrimental for the host, current use of drugs such as ibuprofen and aspirin (cyclooxygenase-2 (cox-2) inhibitors) or statins (3-hidroxi-3-metil-glutaril-coa (hmg-coa) reductase inhibitors) highlights the feasibility of lipid-based therapeutics [100, 101] . accordingly, inhibition of key enzymes involved in fatty acid synthesis, such as acetyl-coa carboxylase (acc) [102] , and fatty acid synthase (fasn) [103] [104] [105] , are potential targets for anti-zikv therapy. in this line, we have reported that nordihydroguaiaretic acid (ndga) and its derivative tetra-o-methyl nordihydroguaiaretic (m4n or terameprocol), two compounds that disturb the lipid metabolism probably by interfering with the sterol regulatory element-binding proteins (srebp) pathway, inhibit the infection of zikv and wnv, likely by impairing viral replication, as did other structurally unrelated inhibitors of the srebp pathway, such as 4-[(diethylamino)methyl]-n-[2-(2-methoxyphenyl)ethyl]-n-(3r)-3-pyrrolidinyl-benzamide dihydrochloride (pf-429242) and fatostatin [106] . in the same way, the dependence on cholesterol for different processes during flavivirus infection also provides a suitable target for antiviral strategies. as mentioned above, 25hc reduces viremia and viral loads in vitro, and also reduces mortality and prevent microcephaly in mice, and decreases viral loads in the urine and serum in non-human infected primates [69] . lovastatin and mevastatin are hypolipidemic agents (hmg-coa inhibitors) belonging to the family of statins that are widely used for lowering cholesterol in patients with hypercholesterolemia and have been previously shown to present antiviral activity against dengue and hepatitis c viruses. both agents have been proposed as therapeutic candidates against zikv [107] . in fact, lovastatin attenuates nervous injury in animal models of gbs [108] . likewise, imipramine, an fda-approved antidepressant, inhibits zikv-rna replication and virion production in human skin fibroblasts, probably by interfering with intracellular cholesterol transport [109] . regarding sphingolipid metabolism, which has been involved in flavivirus infection [66] , treatment with the neutral sphingomyelinase inhibitor gw4869 reduced zikv production by affecting viral morphogenesis [110] as described for other flaviviruses [111] . finally, since adenosine monophosphate-activated protein kinase (ampk) is a master regulator of lipid metabolism, its activation by pf-06409577 or metformin reduced zikv infection by impairing viral replication [112, 113] . thus, targeting lipid metabolism could provide therapeutic alternatives for the discovery of host-directed antivirals against zikv. the ns5 protein is the viral rna-dependent rna polymerase responsible for the rna synthesis that also inhibits interferon (ifn) signaling by acting over the signal transducer and activator of transcription 2 (stat2) protein [114] , being, thus, a major target for antiviral design. besides the proven antiviral activities of different nucleosides analogs and inhibitors of the zikv-ns5 [18] , several inhibitors of the biosynthesis of nucleosides (purines and pyrimidines) also impair zikv replication (figure 1 ). ribavirin is an inhibitor of the inosine monophosphate dehydrogenase (impdh) with antiviral activity to several rna viruses [115] , but its mechanism of action is not entirely clear. it may act as a guanosine synthesis inhibitor, a viral cap synthesis inhibitor, a viral rna mutagen, and as an inducer of lethal mutagenesis [116] [117] [118] . by using a cell-based assay, no antiviral activity of the drug was initially observed [73] but, later on, it was reported that although no activity against zikv was detected in vero cells, the drug did inhibit virus multiplication in human cell lines, including liver huh-7 and rhabdomyosarcoma (rd) cells [119] . further studies have confirmed an inhibitory activity of ribavirin against zikv strains of different geographical origin in various types of cells, such as human neural progenitor cells (hnpcs), human dermal fibroblasts (hdfs), human lung adenocarcinoma cells (a549), and even in vero cells [120] [121] [122] . still more, the drug was shown to abrogate viremia in zikv-infected stat-1-deficient mice [121] , which lack type i ifn signaling, are highly sensitive to zikv infection, and exhibit a lethal outcome. two other inhibitors of impdh, merimepodib (mmpd or vx-497) [123] and mycophenolic acid (mpa) [65, 70, 124] also inhibit zikv-rna replication in different cell types, including huh-7 cells, human cervical placental cells, and neural stem and primary amnion cells. however, other authors [73] have described that mpa have little effect on zikv replication and showed significant cell toxicity. likewise, azathioprine, another inhibitor of purine synthesis and immunosuppressant, impaired zikv replication in hela and jeg3 cells [70] ; nonetheless, its use in pregnant women is not recommended. the above described contradictory results stress again the differences that drug treatments may have as a consequence of the different viral strains, cell types, and methodologies used to assess them. as with the inhibitors of purine biosynthesis, compounds inhibiting the synthesis of pyrimidines have also effect on zikv replication (figure 1 ). so that, the virus was highly susceptible to brequinar and cid 91632869 treatments in cell culture [73] . however, it should be noted that it has been reported that brequinar, as well as dd264, antiviral activity may not be due to pyrimidine deprivation, but rather to the induction of the cellular immune response [125, 126] . similarly, other inhibitors of the pyrimidine synthesis, such as gemcitabine, an activator of cellular caspases [65, 71] , and, although with a lower efficiency probably due to its lower solubility, 6-azauridine and finasteride, a 4-azasteroid analog of testosterone that inhibit type ii and type iii 5α-reductase and is being tested for benign prostatic hyperplasia and male pattern baldness, reduce zikv replication [73, 107] . several other compounds have been shown to have anti-zikv activity by inhibiting viral entry and/or rna synthesis, although their mechanisms of action have not yet been fully elucidated. among them are antiparasitics such as ivermectin (used mainly against worms infections) and pyrimethamine (a folic acid antagonist that inhibits the dihydrofolate reductase and, thus, dna and rna synthesis, is classified as a pregnancy category c, and was initially used to treat malaria and now toxoplasmosis and cystoisosporiasis) [70] ; antibiotics such as azithromycin that prevents infection, replication, and virus-mediated cell dead [55] , and kitasamycin (a natural product from streptomyces narbonensis that inhibits protein biosynthesis) [107] ; drugs used to prevent chemotherapy-induced nausea and vomiting as palonosetron (a fda-approved 5-ht3 antagonist) [107] ; antidepressants like sertraline (a selective serotonin reuptake inhibitor) [107] and cyclosporine (that is also use for rheumatoid arthritis, psoriasis, crohn's disease, nephrotic syndrome, and in organ transplants, is believed to lower the activity of t-cells, and is currently in clinical trials for tis possible use in ameliorate neuronal cellular damage) [70] . similarly, after chemical screening, it was found that hippeastrine hydrobromide (hh), an active component of traditional chinese medicine, and amodiaquine dihydrochloride dihydrate (aq), an fda-approved drug for treatment of malaria, inhibit zikv infection of human pluripotent stem cell-derived cortical npcs and in adult mouse brain in vivo even when the infection was already ongoing but, again, their mechanisms of action are not known [127] . besides drugs that act against host targets directly implicated in the viral cycle, there are compounds that can prevent undesirable effects of zikv infection. in this regard, zikv infection leads to massive neuronal damage, especially of neural progenitor cells, and neurodegeneration [128] [129] [130] , via both direct replication in neuronal cells and possibly through increased excitotoxicity via over activation of n-methyl-d-aspartate receptor (nmdar)-dependent neuronal excitotoxicity in nearby cells. memantine, a pregnancy category b fda-approved drug widely used to treat patients with alzheimer's disease, as well as other nmdar blockers (dizocilpine, agmatine sulfate, or ifenprodil), prevents neuronal damage and death and intraocular pressure increase induced by zikv infection in infected mice, but it does not affect virus replication, pointing to its possible use to prevent or minimize zikv-related microcephaly during pregnancy [131] . ebselen (ebs), an antioxidant that reduces oxidative stress and improves histopathological features in a testicular injury study model and is currently in clinical trials for various diseases, showed minor effects in reducing zikv progeny production and viral e protein expression and on overall survival and viremia level of challenged ag129 mice; however, it should be noted that ebs reduced some zikv-induced effects, such as testicular oxidative stress, leucocyte infiltration, and production of pro-inflammatory response, whereas, in a model of male-to-female mouse sperm transfer, the drug improved testicular pathology and prevented the sexual transmission of zikv [132] . ifns play a key role in the elimination of pathogens and they are release upon the activation of the innate immune response by infecting viruses. in this way, zikv infection induces ifn signaling pathways and further activates cytoplasmic retinoic acid inducible gene 1 protein (rig1)-like receptors (rlrs) and several type i and iii ifn-stimulated genes, driving to the subsequent activation of the janus kinase (jak)/stat innate immune pathway that confer resistance to zikv infection [54] . different studies showed that ifn-α, ifn-β, and ifn-γ inhibit zikv replication in cell culture [124, 133, 134] , and that treatment of pregnant mice with ifn-λ reduced zikv infection [135] . in addition, ifitm1 and ifitm3, which are interferon-induced transmembrane proteins, impair early stages of zikv infection. even more, ifitm3 prevents zikv-induced cell death [136] . likewise, it has been reported that an interferon-activating small molecule (1-(2-fluorophenyl)-2-(5-isopropyl-1,3,4-thiadiazol-2-yl)-1,2-ihydrochromeno [2,3-c] pyrrole-3,9-dione (avc) strongly inhibits replication of zikv in cell culture [137] . however, it is also known that the virus is capable of evading type i ifn responses by acting over the jak/stat signaling pathway [114, [138] [139] [140] , and that type i ifns might be mediators of pregnancy complications, including spontaneous abortions and growth restriction [141] . in any case, use of ifn against zikv, alone or in combination with other antivirals, deserve further studies. by screening a library of known human micrornas (mirnas), small, noncoding rnas (sncrnas) that modulate gene expression post-transcriptionally and regulate a broad range of cellular processes, several mirnas were found to inhibit zikv by increasing the capability of infected cells to respond to infection through the interferon-based innate immune pathway [142] . another alternative is intervening over epigenetic regulation by using epigenetics modulators. for instance, histone h3k27 methyltransferases (ezh1 and ezh2) suppress gene transcription and it has been shown that inhibitors such as 1-[(2s)-butan-2-yl]-n-[(4,6-dimethyl-2-oxo-1h-pyridin-3-yl)methyl]-3-methyl-6-(6-piperazin-1-ylpyridin-3-yl)indole-4-carboxamide (gsk-126) reduce zikv multiplication in cell culture through the activation of cellular antiviral and immune responses [143] . in any case, further studies are needed to evaluate the potential therapeutic capability of these immunomodulators against zikv infection. a great effort is being lately made to find compounds to fight zikv infection by applying different approaches, from repurposing of drugs with known antiviral activity to the screening of bioactive molecules from different libraries, as well as natural products. however, most of the already tested drugs have been found to inhibit viral replication in vitro, and only a few have been tested in vivo. hence, since, in many instances, the results will be difficult to extrapolate to humans, it would be hard for most of the tested antivirals to complete the entire drug development pipeline. in addition, it should be remarked that many drugs could have untoward effects and, thus, careful evaluation should be conducted before using them in clinical practice, as the main target populations for anti-zikv therapy will be pregnant women and patients with other medical complications. many of the already tested drugs are directed against viral structural and enzymatic proteins, including, for instance, anticancer and anti-inflammatory molecules, antibiotics, and antiparasitics; however, it is well known that this approach can easily lead to the appearance of resistance. since flaviviruses require many host factors and co-option of cellular metabolic pathways to successfully infect host cells and propagate efficiently, this offers an opportunity to search for host targets as therapeutic tools that, in many instances, can be broad spectrum agents, and which effect would be less prone to the emergence of mutants that will escape their action. because of that, and even though manipulating host metabolic pathways can be seen as dangerous due to the undesirable effects that could be detrimental for the host, its success for other diseases make of them a realistic option for the treatment of zikv infection. phylogeny of the genus flavivirus potential of selected senegalese aedes spp. mosquitoes (diptera: culicidae) to transmit zika virus zika virus zika virus: the latest newcomer full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses zika virus. i. isolations and serological specificity zika virus outbreak on yap island, federated states of micronesia neurological manifestations of zika virus infection the history of zika virus detection of zika virus in urine zika virus 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zika virus infection in mice axl-mediated productive infection of human endothelial cells by zika virus curcumin inhibits zika and chikungunya virus infection by inhibiting cell binding polysulfonate suramin inhibits zika virus infection suramin inhibits zika virus replication by interfering with virus attachment and release of infectious particles molecular mechanisms of flavivirus membrane fusion acid-dependent viral entry screening bioactives reveals nanchangmycin as a broad spectrum antiviral active against zika virus lipids and flaviviruses, present and future perspectives for the control of dengue, zika, and west nile viruses arbidol (umifenovir): a broad-spectrum antiviral drug that inhibits medically important arthropod-borne flaviviruses infection by zika viruses requires the transmembrane protein axl, endocytosis and low ph 25-hydroxycholesterol protects host against zika virus infection and its associated microcephaly in a mouse model a screen of fda-approved drugs for inhibitors of zika virus infection obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism obatoclax inhibits alphavirus membrane fusion by neutralizing the acidic environment of endocytic compartments evaluation of anti-zika virus activities of broad-spectrum antivirals and nih clinical collection compounds using a cell-based, high-throughput screen assay chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models antiviral activities of selected antimalarials against dengue virus type 2 and zika virus inhibition of autophagy limits vertical transmission of zika virus in pregnant mice fda-approved drug, prevents zika virus infection and its associated congenital microcephaly in mice repurposing of the anti-malaria drug chloroquine for zika virus treatment and prophylaxis -(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives, synthesized by thermal and ultrasonic means, are endowed with anti-zika virus activity (trifluoromethyl)quinoline analogs show improved anti-zika virus activity, compared to mefloquine the antimalarial drug amodiaquine possesses anti-zika virus activities identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen niclosamide rescues microcephaly in a humanized in vivo model of zika infection using human induced neural stem cells antiviral effects of ferric ammonium citrate inhibition of zika virus replication by silvestrol antiviral activity of n-(4-hydroxyphenyl) retinamide (4-hpr) against zika virus interferon-induced spermidine-spermine acetyltransferase and polyamine depletion restrict zika and chikungunya viruses inhibition of polyamine biosynthesis is a broad-spectrum strategy against rna viruses a structural perspective of the flavivirus life cycle post-translational regulation and modifications of flavivirus structural proteins a small-molecule oligosaccharyltransferase inhibitor with pan-flaviviral activity a crispr screen defines a signal peptide processing pathway required by flaviviruses the natural product cavinafungin selectively interferes with zika and dengue virus replication by inhibition of the host signal peptidase pediatric drug nitazoxanide: a potential choice for control of zika identification of novel antiviral of fungus-derived brefeldin a against dengue viruses emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry zika virus infects human cortical neural progenitors and attenuates their growth bithionol blocks pathogenicity of bacterial toxins, ricin, and zika virus targeting host lipid synthesis and metabolism to inhibit dengue and hepatitis c viruses the structure of mammalian cyclooxygenases present status of statin therapy modification of the host cell lipid metabolism induced by hypolipidemic drugs targeting the acetyl coenzyme a carboxylase impairs west nile virus replication dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis west nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4-phosphate lipids dengue virus infection perturbs lipid homeostasis in infected mosquito cells antiviral activity of nordihydroguaiaretic acid and its derivative tetra-o-methyl nordihydroguaiaretic acid against west nile virus and zika virus zika antiviral chemotherapy: identification of drugs and promising starting points for drug discovery from an fda-approved library lovastatin attenuates nerve injury in an animal model of guillain-barre syndrome imipramine inhibits chikungunya virus replication in human skin fibroblasts through interference with intracellular cholesterol trafficking zika virus propagation and release in human fetal astrocytes can be suppressed by neutral sphingomyelinase-2 inhibitor gw4869 the composition of west nile virus lipid envelope unveils a role of sphingolipid metabolism in flavivirus biogenesis direct activation of adenosine monophosphate-activated protein kinase (ampk) by pf-06409577 inhibits flavivirus infection through modification of host cell lipid metabolism suppression of zika virus infection and replication in endothelial cells and astrocytes by pka inhibitor pki 14-22 zika virus targets human stat2 to inhibit type i interferon signaling ribavirin-current status of a broad spectrum antiviral agent broad-spectrum antiviral activity of the imp dehydrogenase inhibitor vx-497: a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon rna virus error catastrophe: direct molecular test by using ribavirin extinction of hepatitis c virus by ribavirin in hepatoma cells involves lethal mutagenesis efficacy of the broad-spectrum antiviral compound bcx4430 against zika virus in cell culture and in a mouse model in vitro susceptibility of geographically and temporally distinct zika viruses to favipiravir and ribavirin ribavirin inhibits zika virus (zikv) replication in vitro and suppresses viremia in zikv-infected stat1-deficient mice favipiravir and ribavirin inhibit replication of asian and african strains of zika virus in different cell models an impdh inhibitor, suppresses replication of zika virus and other emerging viral pathogens a sensitive virus yield assay for evaluation of antivirals against zika virus inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity discovery of a broad-spectrum antiviral compound that inhibits pyrimidine biosynthesis and establishes a type 1 interferon-independent antiviral state high-content screening in hpsc-neural progenitors identifies drug candidates that inhibit zika virus infection in fetal-like organoids and adult brain the brazilian zika virus strain causes birth defects in experimental models zika virus impairs growth in human neurospheres and brain organoids zika virus disrupts neural progenitor development and leads to microcephaly in mice n-methyl-d-aspartate (nmda) receptor blockade prevents neuronal death induced by zika virus infection ebselen alleviates testicular pathology in mice with zika virus infection and prevents its sexual transmission zika virus infectious cell culture system and the in vitro prophylactic effect of interferons type iii interferons produced by human placental trophoblasts confer protection against zika virus infection gestational stage and ifn-lambda signaling regulate zikv infection in utero the ifitms inhibit zika virus replication a novel agonist of the trif pathway induces a cellular state refractory to replication of zika, chikungunya, and dengue viruses zika virus inhibits type-i interferon production and downstream signaling zika virus antagonizes type i interferon responses during infection of human dendritic cells axl promotes zika virus infection in astrocytes by antagonizing type i interferon signalling type i interferons instigate fetal demise after zika virus infection a microrna screen identifies the wnt signaling pathway as a regulator of the interferon response during flavivirus infection inhibitors of the histone methyltransferases ezh2/1 induce a potent antiviral state and suppress infection by diverse viral pathogens key: cord-271752-h05sten7 authors: pérez-arellano, josé luis; górgolas-hernández-mora, miguel; salvador, fernando; carranza-rodríguez, cristina; ramírez-olivencia, germán; martín-echeverría, esteban; rodríguez-guardado, azucena; norman, francesca; velasco-tirado, virginia; zubero-sulibarría, zuriñe; rojo-marcos, gerardo; muñoz-gutierrez, josé; ramos-rincón, josé manuel; sánchez-seco-fariñas, m. paz; velasco-arribas, maría; belhassen-garcía, moncef; lago-nuñez, mar; cañas garcía-otero, elías; lópez-vélez, rogelio title: executive summary of imported infectious diseases after returning from foreign travel: consensus document of the spanish society for infectious diseases and clinical microbiology (seimc) date: 2018-03-31 journal: enfermedades infecciosas y microbiología clínica doi: 10.1016/j.eimc.2017.02.009 sha: doc_id: 271752 cord_uid: h05sten7 abstract in a global world, knowledge of imported infectious diseases is essential in daily practice, both for the microbiologist–parasitologist and the clinician who diagnoses and treats infectious diseases in returned travelers. tropical and subtropical countries where there is a greater risk of contracting an infectious disease are among the most frequently visited tourist destinations. the seimc considers it appropriate to produce a consensus document that will be useful to primary care physicians as well as specialists in internal medicine, infectious diseases and tropical medicine who help treat travelers returning from tropical and sub-tropical areas with infections. preventive aspects of infectious diseases and infections imported by immigrants are explicitly excluded here, since they have been dealt with in other seimc documents. various types of professionals (clinicians, microbiologists, and parasitologists) have helped produce this consensus document by evaluating the available evidence-based data in order to propose a series of key facts about individual aspects of the topic. the first section of the document is a summary of some of the general aspects concerning the general assessment of travelers who return home with potential infections. the main second section contains the key facts (causative agents, diagnostic procedures and therapeutic measures) associated with the major infectious syndromes affecting returned travelers [gastrointestinal syndrome (acute or persistent diarrhea); febrile syndrome with no obvious source of infection; localized cutaneous lesions; and respiratory infections]. finally, the characteristics of special traveler subtypes, such as pregnant women and immunocompromised travelers, are described. in a global world, knowledge of imported infectious diseases is essential in daily practice, both for the microbiologist-parasitologist and the clinician who diagnoses and treats infectious diseases in returned travelers. tropical and subtropical countries where there is a greater risk of contracting an infectious disease are among the most frequently visited tourist destinations. the seimc considers it appropriate to produce a consensus document that will be useful to primary care physicians as well as specialists in internal medicine, infectious diseases and tropical medicine who help treat travelers returning from tropical and sub-tropical areas with infections. preventive aspects of infectious diseases and infections imported by immigrants are explicitly excluded here, since they have been dealt with in other seimc documents. various types of professionals (clinicians, microbiologists, and parasitologists) have helped produce this consensus document by evaluating the available evidence-based data in order to propose a series of key facts about individual aspects of the topic. the first section of the document is a summary of some of the general aspects concerning the general assessment of travelers who return home with potential infections. the main second section contains the key facts (causative agents, diagnostic procedures and therapeutic measures) associated with the major infectious syndromes affecting returned travelers [gastrointestinal syndrome (acute or persistent diarrhea); febrile syndrome with no obvious source of according to the world tourism organization, there were around 1,184 million international tourist arrivals in 2015, some 50 million more than in 2014 (an increase of 4.4%). international tourist arrivals for the year 2016 were forecast to increase by 4%, both worldwide and regionally. regional growth was expected to be highest in asia and the pacific (between 4% and 5%) and the americas (between 4% and 5%), followed by europe (between 3.5% and 4.5%), and africa and the middle east (between 2% and 5%). 1 familitur data showed that international tourist movements made by spaniards were 11.8 million in 2014, 1,140,000 of which were to the african continent, 764,000 to america (central caribbean and south) and 520,000 to asia. 2 the most frequently visited tourist destinations include tropical and sub-tropical countries where there is a higher risk of contracting an infectious disease. 3 travel to economically less developed countries frequently involves exposure to biological agents that cause infections or to infectious diseases transmitted by different routes (digestive, respiratory, mucocutaneous, vector, and so on). one point that should be highlighted from the outset is the possibility that an infection in the international traveler could also be caused by cosmopolitan agents found within our own country, an example of which would be sexually transmitted diseases (stds), a set of infectious conditions that has been insufficiently studied among travelers. the differential diagnosis therefore should always include diseases that have a restricted geographic distribution as well as those with a global presence. furthermore, the severity of the clinical pictures presented here varies a good deal, so that a special section has been included on managing the seriously ill patient. finally, there are certain situations that are physiological (such as pregnancy) or pathological in nature (for example, the immunocompromised patient, whether or not associated with hiv infection) that have special characteristics that warrant further discussion. from a medical point of view, these guidelines will be useful to primary care physicians as well as specialists in internal medicine, infectious diseases and tropical medicine who treat travelers returning from tropical and sub-tropical areas with infections. the target population in this document is adults with infections imported after returning from international travel. the prevention of imported diseases and infections imported by immigrants are explicitly excluded here, since these have been considered in recent eimc reviews. also left out here in a general sense are other noninfectious illnesses among travelers, although certain aspects will be mentioned in particular sections. various types of professionals (clinicians, microbiologists and parasitologists) have helped produce this consensus document by evaluating the evidence-based information available and making recommendations about the following aspects: • general evaluation of the returned traveler with a potential infection -the need to evaluate the asymptomatic traveler -the main syndromes associated with imported infectious diseases -evaluation of the traveler with severe infectious disease -evaluation of the traveler with potentially transmissible diseases and isolation precautions • main infectious syndromes in the returned traveler -acute or persistent diarrhea -fever of unknown origin -localized cutaneous lesions -respiratory infections -eosinophilia -neurological infections -urinary tract infections • special characteristics of the pregnant traveler • special characteristics of the immunocompromised traveler general methodology of the document a systematic review of the bibliography was performed to evaluate all data concerning the causes, diagnostic methods and therapeutic options for infections imported by travelers. a search of the pubmed database was performed using the following selection criteria: articles published between 1968 and march 2016, in english or spanish, and limited to humans only. the search terms used were "travel*" associated with each of the items explored (e.g. "fever", "diarrhea", and so on). this search was complemented with a review of the cochrane database of systematic reviews, using "travel*" as the key term, and also of the international guidelines dealing with each of the separate aspects evaluated. the search was conducted using prisma reporting criteria, 4 and was reviewed by the contributors in the first instance, then by those coordinating the text version. a total of 436 publications were selected, eliminating those that were duplicated or not relevant. the specific set of references selected for each section may be requested from the contributors. the recommendations were based on the international standards used in the consensus guidelines of the infectious disease society of america (idsa) and the appraisal of guidelines research & evaluation (agree) instrument. 5 the coordinators and authors of the document issued a consensus version, which was published on the seimc web site between 13 november 2016 and 14 december 2016 for external review. the final submitted article was returned with approval for publication. the management board of the seimc will designate coordinators to review this document in the next 5 years. this section indicates the main internationally accepted definitions used by the world tourism organization. with respect to duration, three types may be distinguished: short-term (<3 weeks), medium-term (3 weeks to 3 months), and long-term (>3 months). depending on the purpose of the trip, there are two main groups: trips for personal reasons and those undertaken for professional reasons. trips for personal reasons can be further sub-divided into: (i) leisure, recreation and holidays; (ii) visiting friends and relatives (vfr); (iii) shopping trips; (iv) trips for educational/training purposes; (v) health tourism; (vi) for religious reasons and pilgrimages, and (vii) travel for cooperation and humanitarian aid. travel for professional reasons includes travel for business/conferences and military purposes. the selected key facts (kf) are indicated in the following sections. the need to evaluate the asymptomatic traveler kf1. systematic evaluation is not indicated for all international travelers in the absence of clinical signs and symptoms (a-ii). kf2. immigrant travelers visiting friends and relatives may benefit from evaluation, even if they are asymptomatic (c-ii). kf3. long-term travelers (>3 months), those to high-risk areas and/or including high-risk activities may benefit from a directed evaluation (b-ii). kf4. travelers who have been in contact with freshwater sources in endemic areas, or who have walked barefoot on contaminated soil may benefit from screening for schistosomiasis and strongyloidiasis respectively (a-ii). kf5. any traveler who has engaged in risky sexual practice without protection may benefit from serological testing for the detection of stis, hiv, hepatitis b and c and syphilis (a-ii). kf6. health aid workers exposed to patients with active tuberculosis may benefit from the tuberculin skin test or interferongamma release assays (igras) (b-ii). principal syndromes associated with imported diseases by travelers kf7. in overall terms, the most common syndromes affecting travelers who return home feeling ill are gastrointestinal (acute or persistent diarrhea), fever of unknown origin, localized skin lesions and respiratory infections (a-ii). kf8. the relative frequency of these syndromes varies depending on the geographic area or region visited (b-ii). kf9. the severity of these syndromes is variable. fever of unknown origin or associated with other symptoms (such as diarrhea or respiratory problems) accounts for the majority of hospital admissions (b-ii). evaluation of the traveler with serious infectious disease kf10. the initial evaluation of the international traveler with severity criteria should be carried out at three levels: assessment of vital functions, syndromic evaluation and diagnostic strategy (b-iii). kf11. the immediate evaluation of hemodynamic stability should necessarily include blood pressure, respiratory rate, oxygen saturation, diuresis, heart rate and level of consciousness. other variables to be taken into account are body temperature, the presence of edema, capillary refill and the presence of ileus (a-iii). kf12. the syndromic picture should be clearly established, since this will make it possible to select the most appropriate diagnostic tests and prognostic scales (b-iii). kf13. the analytical determinations that should be ordered for the seriously ill patient include blood count, biochemical tests including serum transaminase, bilirubin and blood coagulation, renal function, glycemia, arterial blood gas, and an analysis of urine. when possible, c-reactive protein and procalcitonin levels should also be requested (a-iii). kf14. the diagnostic strategy requires tests that are able to rule out malaria and dengue fever, and at least two sets of blood cultures, plus serological tests to detect rickettsial diseases, q fever, hiv, hbv or hcv infection (a-iii). kf15. it is recommended that a specialist in tropical medicine or infectious diseases assess the patient as quickly as possible (c-iii). kf16. in the returned traveler, the clinician should initially evaluate not only the individual disease, but also the possibility that it may involve a current public health alert (a-iii). kf17. the recommended methods (in spain) are by phoning the departamento de alertas de salud publica (department of public health alerts) of the appropriate autonomous community, or by consulting the web page of the ministerio de sanidad, servicios sociales e igualdad (ministry of health) (a-iii). kf18. different isolation precautions will be applied depending on the clinical syndrome and the traveler's travel itinerary (b-iii). kf19. high-level isolation units (hliu) for patient management are indicated for confirmed and suspected cases of specific viral hemorrhagic fevers, highly pathogenic emerging respiratory diseases, multidrug-resistant tuberculosis (mdr-tb) and outbreaks of potentially serious transmissible diseases (pstd) caused by unknown agents (a-iii). kf20. a basic pillar of control of pstds involves the selection, education and training of staff. this should be regarded as one more isolation precaution (b-iii). kf21. restricting the use of invasive tests is also an isolation precaution. the selection of tests and the staff involved should be agreed by protocols adapted to the center where the patient is being treated (b-iii). kf22. all travelers transferred from foreign hospitals should be regarded as potential carriers of multidrug-resistant organisms and should be proactively screened (by rectal smear) (a-iii). diarrhea (acute or persistent) kf23. most cases of acute traveler's diarrhea are caused by bacterial pathogens. enterotoxigenic escherichia coli (etec) is the most frequently identified causative agent worldwide (a-iii). kf24. in a significant percentage (30-50%) of cases of acute traveler's diarrhea, an etiological diagnosis cannot be made (a-iii). kf25. there are notable geographical variations with respect to the etiology of acute traveler's diarrhea, independent of the length of the trip (a-iii). kf26. for acute traveler's diarrhea, microbiological studies should be restricted to patients who present fever, dysentery, choleriform diarrhea, or who are dehydrated, immunosuppressed or have significant comorbidities (a-iii). kf27. the diagnostic method of choice for acute traveler's diarrhea is the conventional stool culture or cultures on selective media (depending on clinical suspicion) together with serial blood cultures if there is fever, although the diagnostic yield is low (a-iii). kf28. for any traveler with fever and acute diarrhea arriving from an endemic area, malaria should be ruled out with the appropriate methods (b-iii). kf29. before traveling, the patient should be given information about the main self-treatment measures to be taken in case of diarrhea, and told to seek medical care in the presence of high fever, severe abdominal pain, bloody diarrhea, uncontrolled vomiting, or if self-treatment is ineffective (a-iii). kf30. for previously healthy adults, rehydration with conventional liquids, especially associated with loperamide, should be enough in cases of mild diarrhea (a-i). kf31. rehydration and restoration of electrolyte balance with antidiarrheal drugs and non-absorbable antibiotics (rifaximin) is indicated for moderate diarrhea, and for the old or immunocompromised with no previous history of invasive disease (a-iii). kf32. for severe diarrhea with obvious signs of dehydration, intravenous rehydration is recommended to restore the fluid and electrolyte balance (a-iii). kf33. the use of antidiarrheal agents is contraindicated in the presence of invasive disease (a-iii). kf34. the most useful drugs for the treatment of invasive diarrhea are fluoroquinolones or azithromycin, chiefly in single doses (a-i). kf35. the pathogenesis of persistent traveler's diarrhea may fall into one of three major groups: persistent infection or coinfection; post-infectious syndromes (transient lactose intolerance, post-infectious irritable bowel syndrome, small intestinal bacterial overgrowth (sibo) and tropical sprue); or an underlying gastrointestinal disease unmasked during or after the trip (a-iii). kf36. the most common infections in persistent traveler's diarrhea are due to protozoan pathogens, for which the diagnostic method of choice is the standard comprehensive parasitology profile, using specific stains based on clinical suspicion, and antigen detection methods and pcr, as available, for increased sensitivity (a-iii). kf37. diagnosis of post-infectious irritable bowel syndrome is exclusively clinical (rome criteria iii-iv) and it is especially important to determine the state of digestive health before travel and to consider at an early stage whether there are clinical and analytical signs of alarm/organicity (a-iii). kf38. the incidence of tropical sprue may be underestimated. its main differential diagnosis is with celiac disease. upper endoscopy (egd) to examine the jejunum and biopsy are frequently required to differentiate them (a-iii). kf39. some authors recommend empirical therapy with nitroimidazoles if giardia intestinalis is highly suspected, even if specific studies are negative (c-iii). causative agents kf40. the most common causes of fever of unknown origin in the returned traveler are, in order of frequency: malaria, arbovirus (e.g. dengue, chikungunya, zika) and bacterial infections (typhoid fever, rickettsial infections, q fever and leptospirosis) (a-ii). kf41. even though they are rare, serious diseases that are highly contagious, such as viral hemorrhagic fevers (e.g. ebola, lassa, marburg, rift valley fever, crimean-congo hemorrhagic fever) should always be considered (c-iii). kf42. the traveler's provenance, period of incubation and specific risk exposures should provide guidance as to the etiology of the febrile process (a-ii). kf43. travelers who present with fever of unknown origin after visiting a tropical or sub-tropical area should seek immediate medical attention (a-ii). kf44. if there is any possibility of viral hemorrhagic fever (vhf), this should be investigated using appropriate biosafety measures and techniques that require the least handling possible (rapid tests or pcr) (a-ii). a significant proportion of fever episodes post-travel either do not lead to a specific diagnosis (a-ii) or are due to cosmopolitan infections (a-ii). kf46. if there is no risk of vhf, malaria should be ruled out in the first instance using microscopy techniques and rapid diagnostic tests (a-ii). kf47. if the acute phase of an arbovirus is suspected (<10-15 days of clinical evolution), the patient should be tested for the presence of (ns1) antigens or viral rna in blood and urine (a-ii). kf48. if arbovirus in later stages is suspected (>15 days of clinical evolution), the serologic response (principally igm) should be studied and cases confirmed by neutralization tests (a-ii) . kf49. the diagnosis of bacterial infection responsible for fever of unknown origin is based, in the acute phase, on isolating the bacterial organism or using molecular biology techniques, and in later phases, on serologic studies of paired serum samples (a-ii). kf50. treatment (etiological or symptomatic) should be based on identifying the causative agent (a-ii). kf51. if there is a high probability of malaria and a diagnosis cannot be made, or will be delayed for more than 3 h with no alternative diagnosis, administration of empirical antimalarial therapy is recommended (a-ii). kf52. patients with a likely diagnosis of severe acute schistosomiasis or neuroschistosomiasis are treated with corticosteroids in combination with praziquantel (b-ii). kf53. in complicated or severe cases of malaria, use of ceftriaxone plus doxycycline is recommended while waiting for confirmation of diagnosis (c-iii). causative agents kf54. various cosmopolitan infections, such as superficial mycoses (e.g. pityriasis versicolor, cutaneous candidiasis or dermatophytosis) and some ectoparasitic infections (such as scabies) are more frequent in travelers (a-ii). kf55. classic bacterial infections constitute the leading cause of consultation for skin lesions and specifically, for those due to certain strains of methicillin-resistant s. aureus (mrsa) that produce panton-valentine leukocidin (pvl) (a-ii). kf56. the primary morphology of the lesion (e.g. papules, pustules, nodules, ulcers, blisters), configuration (e.g. linear) and distribution (exposed versus covered areas, specific parts of the body) are helpful for diagnosis (a-ii). kf57. in most cases, diagnosis is clinical, and dermoscopy is useful for some entities (scabies, cutaneous larvae migrans, furuncular myiasis and tungiasis) (b-iii). kf59. in an imported dermatosis that develops slowly, cutaneous leishmaniasis, mycobacterial infection or a subcutaneous mycosis should be suspected. histological examination and identification of the causative agent with molecular biology techniques or mass spectrometry is essential (a-ii). kf60. for many imported dermatoses, treatment is symptomatic with oral antihistamines (diphenhydramine, hydroxyzine, or loratadine), topical antipruritic therapy (calamine lotion) and topical corticosteroids (low potency for the genitals, medium potency for the trunk or extremities) (a-ii). kf61. the treatment of choice for the main imported dermatoses that are linear in pattern (cutaneous larva migrans (ctm), cutaneous larva currens and gnathostomiasis) is ivermectin, with albendazole an alternative option (a-i). kf62. surgical removal (associated with antibacterial tetanus chemoprophylaxis where appropriate) is the treatment of choice for furuncular myiasis, tungiasis, and d. repens infections (a-ii). causative agents kf63. the most common causes of respiratory infection in the local environment are also the most common among travelers, with exotic imported infections being much less frequent (a-ii). kf64. in general, viral and bacterial infections are more frequent than those caused by fungi and parasites (b-ii). kf65. most respiratory infections in travelers are mild, affect the upper respiratory tract and are caused by viruses (a-ii). kf66. the most serious respiratory infection in travelers is pneumonia, and the most common causes of it in the traveler are similar to those in the autochthonous population, although with a greater incidence of infections due to s. aureus and legionella spp. (a-ii). kf67. during short trips, the risk of tuberculosis is low and depends on the incidence of the disease in the countries visited (b-iii). kf68. the main causes of respiratory conditions associated with eosinophilia are acute schistosomiasis, löffler's syndrome and paragonimiasis (b-iii). kf69. travelers may also have non-infectious problems that have respiratory manifestations such as pulmonary embolism and mefloquine toxicity (b-iii). kf70. most respiratory infections during and after travel do not require diagnostic confirmation because they are mild, self-limited upper respiratory tract infections (a-ii). kf71. the diagnostic methods for pneumonia are no different from those used on autochthonous patients (b-ii). kf72. in patients with pneumonia, the decision as to whether a patient with pneumonia should be admitted to hospital or the icu should be based on severity scales used in the autochthonous population (a-ii). kf73. procalcitonin levels can guide the clinical judgment of the attending physician in prescribing antibiotics for respiratory infections (b-ii). kf74. if tuberculosis is suspected, or any other infection transmitted via respiratory secretions (such as the mers-cov coronavirus), respiratory isolation and droplet precautions are recommended (a-i). kf75. most respiratory infections during and post-travel do not require specific treatment because they are mild and self-limiting. treatment may be given for the symptoms (b-iii). kf76. for pneumonia in travelers, antibiotic therapy is initially empiric and should include drugs effective against the most common community-acquired pathogens, including legionella spp. (b-ii). kf77. in travelers with influenza and severity criteria or risk factors, treatment with oseltamivir or zanamivir is recommended (b-ii). kf78. if the traveler with tuberculosis has come from a country with a high incidence of antimicrobial resistance, it would be reasonable to evaluate treatment with at least 4 drugs and rapid dna-based tests to detect mutations associated with resistance (b-iii). kd79. the causes of imported eosinophilia vary a good deal and depend on the characteristics of the patient, particularly whether the person is a traveler or an immigrant, and on the geographical destination, itinerary and length of exposure (b-ii). kd80. when a patient presents with imported eosinophilia of non-filarial etiology, it is important to rule out infection due to parasites, principally helminths (a-ii). kd81. in patients with imported eosinophilia, the cause is identified as a parasite in 60-75% of cases (b-ii). kd82. the three most frequently found parasitic infections are strongyloidiasis, schistosomiasis and soil-transmitted helminth infections, as well as filariasis in the case of equatorial guinea (b-ii). kd83. all returning travelers with eosinophilia should request a stool examination to search for parasites using a concentration method and strongyloides stercoralis serology (b-ii). kd84. travelers returning from africa should request tests for diagnosing schistosomiasis (examination of the urine sediment and feces), and schistosoma spp. serology (b-ii). kd85. in cases where a diagnosis cannot be made with direct techniques, serological methods are a useful diagnostic tool, although their possible drawbacks should not be forgotten (b-iii). kd86. when the cause cannot be identified, the empiric treatment for imported eosinophilia should be a combination of oral ivermectin plus albendazole. praziquantel should be added for cases with possible epidemiological exposure to schistosomiasis (b-iii). kd87. before starting empiric ivermectin therapy, the possibility of loa loa infection should first be ruled out (a-ii). kd88. when empiric anthelmintic treatment is administered, the patient should receive clinical follow-up with monitoring of peripheral blood eosinophil counts, since some parasites, like trichuris spp. and schistosoma spp., have developed resistance leading to anthelmintic treatment failure (a-ii). causative agents kf89. the most frequent causes of neurological disease in travelers are malaria, viral infections and bacterial meningitis (a-ii). kf90. generally speaking, the main clinical manifestations affect the brain and/or meninges, and more rarely, the spinal cord and peripheral nervous system (b-iii). kf91. the main manifestation in the peripheral nervous system is guillain-barré syndrome, which is associated with infection due to campylobacter jejuni, dengue and, more recently, the zika virus (b-ii). kf92. any international traveler who returns with fever, severe headaches, sensitivity to light and/or a stiff neck should be evaluated urgently to rule out the presence of meningitis or encephalitis (a-iii). kf93. the cerebrospinal fluid (csf) analysis can adopt four different patterns: normal, raised neutrophils, raised lymphocytes, raised eosinophils, which is useful for differential diagnosis (a-ii). kf94. direct ophthalmoscopy is recommended for all patients with a diagnosis or suspicion of cerebral malaria, given its high prognostic and diagnostic value for malarial retinopathy (a-ii). kf95. the possibility of infection with extended-spectrum betalactamase (esbl)-producing enterobacteriaceae should always be considered in travelers with urinary tract infections, principally those acquired in south and southeast asia (a-iii). kf96. in the presence of non-specific complaints and/or hematuria, urinary schistosomiasis (s. haematobium) should be considered in travelers returning from traditionally endemic areas (and other more recently reported ones, such as corsica) (a-ii). the pregnant traveler kf97. pregnant women are much more susceptible to certain infectious diseases, such as traveler's diarrhea, listeriosis, typhoid fever and malaria (a-ii). kf98. various diseases that are transmitted by eating or drinking contaminated food or water (traveler's diarrhea, hepatitis e, listeriosis and typhoid fever) are more severe in pregnant women (a-ii). kf99. mother-to-child transmission of the dengue and chikungunya viruses can occur if the mother has fever in the days close to and during birth, while the main complications of zika appear in the first trimester of the pregnancy (a-ii). kf100. azithromycin is the antibiotic of choice for traveler's diarrhea and typhoid fever in pregnancy (b-ii). the immunocompromised traveler kf101. the course of malaria in immunocompromised patients (especially those infected with hiv and with low cd4+ lymphocyte counts) tends to involve more severity criteria than in non-immunocompromised individuals. early diagnosis and treatment is essential, therefore, if there is clinical suspicion (a-iii). kf102. immunocompromised individuals who present with traveler's diarrhea should be given the parasitology stool test, including the modified kinyoun stain for detecting coccidian species, such as cryptosporidium spp, cystoisospora belli and cyclospora cayetanensis (b-iii). kf103. s. stercoralis hyperinfection syndrome and disseminated strongyloidiasis are more frequently found in immunosuppressed patients (corticotherapy, transplants, htlv-1 coinfection) and have very high mortality rates. early diagnosis and treatment with ivermectin at a dose of 200 g/kg/per day for at least 7 days is required (a-iii). world tourism organization (unwto) familitur características demográficas, quimio-profilaxis antimalárica e inmunoprofilaxis en 6.783 viajeros internacionales atendidos en una unidad monográfica declaración prisma: una propuesta para mejorar la publicación de revisiones sistemáticas y meta-análisis the agree collaboration. appraisal of guidelines for research & evaluation (agree) instrument the authors declare no conflict of interest. supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.eimc.2017.02.009. key: cord-289139-5ljqnc39 authors: mengelle, c.; mansuy, j.m.; pierre, a.; claudet, i.; grouteau, e.; micheau, p.; sauné, k.; izopet, j. title: the use of a multiplex real-time pcr assay for diagnosing acute respiratory viral infections in children attending an emergency unit date: 2014-09-03 journal: j clin virol doi: 10.1016/j.jcv.2014.08.023 sha: doc_id: 289139 cord_uid: 5ljqnc39 background: the use of a multiplex molecular technique to identify the etiological pathogen of respiratory viral infections might be a support as clinical signs are not characteristic. objectives: the aim of the study was to evaluate a multiplex molecular real-time assay for the routine diagnosis of respiratory viruses, to analyze the symptoms associated with the pathogens detected and to determine the spread of virus during the period. study design: respiratory samples were collected from children presenting with respiratory symptoms and attending the emergency unit during the 2010–2011 winter seasons. samples were tested with the multiplex respifinder(®) 15 assay (pathofinder™) which potentially detects 15 viruses. results: 857 (88.7%) of the 966 samples collected from 914 children were positive for one (683 samples) or multiple viruses (174 samples). the most prevalent were the respiratory syncytial virus (39.5%) and the rhinovirus (24.4%). influenza viruses were detected in 139 (14.4%) samples. adenovirus was detected in 93 (9.6%) samples, coronaviruses in 88 (9.1%), metapneumovirus in 51 (5.3%) and parainfluenzae in 47 (4.9%). rhinovirus (40%) was the most prevalent pathogen in upper respiratory tract infections while respiratory syncytial virus (49.9%) was the most prevalent in lower respiratory tract infections. co-infections were associated with severe respiratory symptoms. conclusion: the multiplex assay detected clinically important viruses in a single genomic test and thus will be useful for detecting several viruses causing respiratory tract disorders. acute respiratory infections (aris) are more prevalent than any other form of infectious disease in children. they range from mild upper respiratory tract problems to serious lower respiratory infections such as bronchiolitis and pneumonia. viruses are the main pathogens and they account for many emergency hospital admissions [1, 2] . clinical signs and symptoms overlap between different viruses, but also between viruses and bacteria, making etiological e-mail address: mengelle.c@chu-toulouse.fr (c. mengelle). 1 both first authors equally contributed to this work. diagnosis based on clinical presentation alone difficult and sometimes leading to overuse of antibiotics. techniques involving culture, fluorescent detection of antigens or immunochromatography have been replaced by nucleic acid tests (nats). due to numerous viruses that might be involved, many monoplex nucleic acid tests are necessary to identify the pathogen(s) responsible for a respiratory disorder. this strategy is thus expensive and time consuming. the use of multiplex assays should significantly reduce hands-on time and cost, and rapidly provide reliable results. the multiplex ligation-dependent probe amplification technology (mpla)-respifinder ® respiratory assay [3] recently became commercially available. this assay is approved for in vitro diagnosis in europe and canada and can detect up to 15 respiratory viruses. this prospective study was done to evaluate this multiplex technique for use in clinical diagnosis. all the samples taken from http://dx.doi.org/10.1016/j.jcv.2014.08.023 1386-6532/© 2014 elsevier b.v. all rights reserved. children attending the emergency unit of the toulouse university hospital suffering from aris were collected prospectively and analyzed. clinical data related to the viruses detected were also analyzed, as was the spread of seasonal respiratory viruses for the winter following the influenza a h1n1pdm09 epidemic (october 2010 to march 2011). nasopharyngeal swabs (virocult ® kitnia, labarthe inard, france), aspirates or nasal washes were prospectively collected from children under 15 with symptoms of aris (see below) who attended the emergency unit of the toulouse university hospital between october 1, 2010 and march 31, 2011 and sent to the virology department for analysis. paediatricians completed a specific questionnaire related to the symptoms, including fever and upper respiratory manifestations (rhinitis, pharyngitis, otitis, sore throat, cough) and presence of symptoms of lower respiratory infections (bronchiolitis, pneumonia, acute flu and flu syndrome). whether or not a child had been vaccinated against influenza was also recorded. the collected samples were diluted in 1 ml minimum essential medium (gibco ® -life technologies, rockville, md, usa) and nucleic acids were extracted with the magna pure 96 tm instrument using the magna pure 96 dna and viral na small volume kit ® (roche diagnostics, meylan, france) according to the manufacturer's instructions (extracted volume: 200 l, elution volume: 100 l). extracts were analyzed using the respifinder ® 15 assay (pathofinder tm , maastricht, netherlands), a multiplex ligationdependent probe amplification (mlpa) technology [3] . this assay can detect 15 viruses: influenza viruses (iv) types a and b, parainfluenza viruses 1 to 4 (piv), respiratory syncytial viruses a and b (rsv), rhinovirus (rv), coronaviruses 229e, oc43 and nl63 (cov), human metapneumovirus (mpv) and adenovirus (adv). the test also includes a probe for detecting the avian influenza virus a h5n1. an internal control for pcr inhibitors detection was included in each test. a positive flu a sample and a negative sample were used as controls. samples that were positive for influenza a were subtyped with the realtime ready inf a/h1n1 detection set (roche diagnostics, meylan, france) on the light cycler 480 tm system (roche diagnostics, meylan, france). data were analyzed using stata tm v9.0 software (statacorp, texas, usa). qualitative variables were analyzed with the chisquared test. p values of less than 0.05 were considered significant. logistic regression was used to determine the odds ratios (or) of age and co-infections linked to severe respiratory symptoms. a total of 914 children, of whom 509/914 (55.7%) were male were enrolled in the study between october 1, 2010 and march 31, 2011 and provided 966 samples. the mean age was 1.6 ± 2.6 years and the median age was 7.3 months [range: 0.2-186], but 572/914 (62.6%) children were under 1 year old. the 914 children in the study group included 232/914 (25.4%) with upper respiratory tract infections (243 samples) defined as rhino-pharyngitis or sore throat, with or without otitis media. 682 children (723 samples) suffered from lower respiratory infections defined as bronchiolitis (338/914; 37%), pneumonia or bronchopneumonia (109/914; 11.9%), acute asthma (78/914; 8.5%) and flu or flu-like syndrome (158/914; 17.3%). 76 (8.3%) children were suffering from a chronic (n = 10) or a congenital disorder (n = 66): respiratory, haematological, neurological, cardiac disorders. 25/914 (2.7%) children had been vaccinated in the fall against flu, and 16/25 (64%) of them were suffering from a chronic or congenital disorder. we found co-infections in 24 (9.96%) samples from children with upper respiratory tract symptoms and in 144 (19.9%) samples from children with lower respiratory infections (p < 0.01). the most frequent co-infections in cases of severe respiratory symptoms involved rsv (associated with rv, adv, or cov) and rv associated with adv. 25% of the cases of bronchiolitis involving rsv were co-infections. rsv was more prevalent in children under 1 year old (48.2% vs 24.8%; p < 0.0001), while rv was not (26.4% vs 21.1%; ns). adv (14.8% vs 3.4%) and iv (24.4% vs 9.2%; p < 0.0001) were more frequent in children older than 6 months. co-infections were more frequent in younger children (1.3 years vs 1.7 years; p < 0.05). four parameters were included in the multivariate analysis: vrs infection, presence of co-infection, rv infection and age under 1 year. independent factors associated with severe respiratory symptoms were vrs infection (adjusted or, 3.8; 95% ci, 2.64-5.68), co-infections (adjusted or, 2.5; 95% ci, 1.55-3.94) and rv infection (adjusted or, 1.2; 95% ci, 0.85-1.83). age under 1 year was not associated with such symptoms. the frequency of positive samples varied from 71.4% (5/7 positive samples; week 40, 2010) to 98.4% (60/61 positive samples; weeks 1, 2011; p < 0.05) (fig. 3a) . all the viruses in the panel were detected in our patients, but their distributions varied (fig. 3b) . rv was the first of the three main viruses to appear and was detected throughout the study. rsv appeared in october and reached epidemic peak in week 51 of 2010; iv did not appear until december and reached a plateau between weeks 1 and 7 of 2011. iva was predominant until week 7 of 2011, while ivb was the most prevalent thereafter, decreasing slowly until the end of march. adv, mpv and cov were most frequently detected during the winter months, although piv was detected throughout the study. the availability of molecular assays has made laboratory diagnosis more efficient and has led to the improved detection of a broad spectrum of respiratory viruses [4, 5] . this study evaluates the use of a new multiplex molecular technique for detecting up to 15 respiratory viruses in routine practice. we collected respiratory samples from a group of children suffering from acute respiratory infections and the results obtained were analyzed in comparison to the clinical manifestations. the spread of the viruses was also analyzed during this winter period. a very high percentage of the samples were positive giving a virus signature in nearly 90% of cases, and all the viruses in the test panel were detected. these results are similar to those for young children and infants obtained by others (88-92%) [6] [7] [8] , whereas the rate of detection in adults was lower (43%) [9] . rv, rsv, and iv were the three most prominent pathogens detected, while adv, cov, mpv and piv were less frequent (<10% of cases). as expected, rv was the most frequently pathogen detected in upper respiratory infections [10] , but it was also found in lower respiratory infections, as was described recently by o'callaghan-gordo [11] . rsv remained however the most prominent agent in lower respiratory infections as published previously in bronchiolitis [12] and pneumonia [13] . adv, cov, mpv and piv also accounted for lower respiratory tract aris as shown previously: adv infection was shown to lead to severe chronic disease and to the increase in the mortality rate in children [14] , whereas cov [15] , mpv [16] and piv [17] can be responsible for severe lower respiratory tract infections requiring hospitalization, especially among the youngest. flu and flu syndromes were preferentially linked to influenza viruses and older children seemed to be more susceptible to these viruses. we found a relationship between an influenza virus infection and severe infection due to hyperthermia, but not with more severe respiratory symptoms, in contrast to the situation in adults [18] . the use of multiplex assays has demonstrated that infections with multiple viruses are common [4] ; the frequency of such infections can be as high as 27%, 30% or 46% [5, 19, 20] . this was exactly the case for our children: all the viruses in the panel were detected in co-infections, not only rsv, but also adv and covs. martin et al. [21] described similar results. they detected adv in association with other viruses in 52% of samples and cov in 50%. they also showed that multiple virus infections were correlated with less severe disease. the relationship between co-infections and illness severity remains uncertain and may depend upon the virus detected. dual infections involving rsv and rv [22] or rsv and mpv can lead to severe bronchiolitis [23] whereas co-infections without rsv do not [24] . we also analyzed the way viral infections spread during the winter. rsv and iv were epidemic while the frequency of the other viruses changed little. our study carried out in the winter following the influenza ah1n1pdm09 pandemia shows that iv and rv infections had returned to their pattern. this has also been described in germany by gröndahl et al. [25] who showed that the ah1n1pdm09 outbreak had a great impact on the spread of other respiratory viruses, but that the virus infections had return to their usual epidemiologic characteristics the year after. this new multiplex technique dramatically shortens hands-on time. the results for 15 viral pathogens can be obtained in about one day. it is also much simpler than conventional routine techniques, which need many and various steps and a wide range of technical competence (culture, pcr, immunofluorescence). while multiplex assays are more expensive than monoplex pcrs, laboratories can still choose to perform single pcrs in a strategic stepwise fashion. this strategy may appear to be less expensive but it needs regular revision to take into account the spread of virus(es). moreover, if several monoplex pcrs are needed, this then can become more expensive than multiplex pcrs. it must also be remembered that diagnosis of co-infections is more uncertain due to a smaller number of viruses tested. in conclusion, our results indicate that multiplex pcr techniques can be used in the routine etiological diagnosis of respiratory infections. the mlpa-respifinder ® 15 assay revealed that a high percentage of samples from children attending the emergency unit with aris during the autumn and winter of 2010/2011contained at least one virus, and that co-infections were very common. rsv and rv were the most prevalent pathogens, particularly in the youngest children, and co-infections were associated with more severe respiratory symptoms. the epidemiology of viruses responsible for aris had returned to its usual characteristics one year after the ah1n1pdm09 pandemic. none. none of the authors of this manuscript has any commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy). not required. rates of hospitalisation for influenza, respiratory syncytial virus and human metapneumovirus among infants and young children spectrum and frequency of illness presenting to a pediatric emergency department relative quantification of 40 nucleic acid sequences by multiplex ligationdependent probe amplification performance of a rapid molecular multiplex assay for the detection of influenza and picornaviruses pathogen chip for respiratory tract infections a prospective study of agents associated with acute respiratory infection among young american indian children comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness viral etiology of common cold in children prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate do rhinoviruses cause pneumonia in children? lower respiratory tract infections associated with rhinovirus during infancy and increased risk of wheezing during childhood. a cohort study multiple viral respiratory pathogens in children with bronchiolitis viral pneumonia lower respiratory infections by adenovirus in children. clinical features and risk factors for bronchiolitis obliterans and mortality severity and outcome associated with human coronavirus oc43 infections among children burden of human metapneumovirus infection in young children viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection adult hospitalizations for laboratory-positive influenza during the comparison of the luminex xtag respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections epidemiology of viral respiratory tract infections in a prospective cohort of infants and toddlers attending daycare multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children severe lower respiratory tract infection in infants and toddlers from a nonaffluent population: viral etiology and co-detection as risk factors dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response the 2009 pandemic influenza a(h1n1) coincides with changes in the epidemiology of other viral pathogens causing acute respiratory tract infections in children the english text was checked by dr owen parkes. key: cord-280060-gzby85u9 authors: rello, jordi; manuel, oriol; eggimann, philippe; richards, guy; wejse, christian; petersen, jorgen eskild; zacharowski, kai; leblebicioglu, hakan title: management of infections in critically ill returning travellers in the intensive care unit—ii: clinical syndromes and special considerations in immunocompromised patients() date: 2016-04-28 journal: int j infect dis doi: 10.1016/j.ijid.2016.04.020 sha: doc_id: 280060 cord_uid: gzby85u9 this position paper is the second escmid consensus document on this subject and aims to provide intensivists, infectious disease specialists, and emergency physicians with a standardized approach to the management of serious travel-related infections in the intensive care unit (icu) or the emergency department. this document is a cooperative effort between members of two european society of clinical microbiology and infectious diseases (escmid) study groups and was coordinated by hakan leblebicioglu and jordi rello for esgitm (escmid study group for infections in travellers and migrants) and esgcip (escmid study group for infections in critically ill patients), respectively. a relevant expert on the subject of each section prepared the first draft which was then edited and approved by additional members from both escmid study groups. this article summarizes considerations regarding clinical syndromes requiring icu admission in travellers, covering immunocompromised patients. over the last 20 years, the increase in international travel, which has been intensified by the availability of low-cost flights, has facilitated the movement of an increased number of patients from areas with endemic diseases to distant regions. as a consequence, cities around flight hubs have been and are exposed to the rapid dissemination of imported infections, as was reported in the initial dissemination of hiv infection in north america, and more recently in the 2009 influenza pandemic. similarly, outbreaks of cholera have been reported in travellers after long distance flights, and tourism has also been associated with the dissemination of infections such as measles, rubella, diphtheria, typhoid fever, and chicken pox, in addition to malaria and haemorrhagic fevers. poor health conditions and crowding are associated with tuberculosis (tb), diarrhoea, tetanus, and other infectious events, which may be imported by migrants from areas devastated by war. immunocompromised patients encompass a growing population with increased susceptibility to infectious complications. because they live longer and have a better quality of life than ever before, they may have more opportunity to travel and potentially encounter travel-associated infections. it has been estimated that up to one third of solid-organ transplant (sot) recipients may travel to resource-limited countries within the first year post-transplant. 1 in a survey in north american transplant centres, up to 44% of haematopoietic stem cell transplant (hsct) recipients reported travel outside the usa and canada after transplantation. 2 a international journal of infectious diseases 48 (2016) 104-112 significant number of immunocompromised patients may also be migrants who may return to their countries of origin to visit friends and relatives, and may acquire travel-associated infections. the increased use of monoclonal antibodies for therapy in immunological and oncological diseases has created another at-risk population, although the actual risk of travel-associated infection in these patients is not well established. 3 data on the real risk of infection in immunocompromised travellers relative to the general travel population are scarce, 4 and particularly the risk of developing an illness severe enough to warrant admission to an intensive care unit (icu). the repatriation of immunocompromised patients from hospitals in destination countries also carries the risk of contamination of the receiving hospital with multidrug-resistant (mdr) microorganisms, which requires specific infection control measures. 5 this article also addresses certain specific syndromes, such as pneumonia and acute respiratory distress syndrome (ards) occurring after travel. this position paper is the second escmid consensus document on this subject and aims to provide intensivists, infectious disease specialists, and emergency physicians with a standardized approach to the management of serious travel-related infections in the icu or emergency department. this document is a cooperative effort between members of two european society of clinical microbiology and infectious diseases (escmid) study groups and was coordinated by hakan leblebicioglu and jordi rello for esgitm (escmid study group for infections in travellers and migrants) and esgcip (escmid study group for infections in critically ill patients), respectively. a relevant expert on the subject of each section prepared the first draft, which was then edited and approved by additional members from both escmid study groups. this article summarizes considerations regarding clinical syndromes requiring icu admission in travellers, covering immunocompromised patients. (table 1) the risk of infection in sot recipients varies according to multiple factors, namely the type of organ transplanted, the time from transplantation, and the type and dose of immunosuppressive drugs received. 6 during the first month post-transplant, infectious complications are mainly healthcare-associated. the most profound immunosuppression occurs between months 2 to 6; historically, this is the period in which most opportunistic infections were diagnosed, including herpesvirus infections (cytomegalovirus), pneumocystis jirovecii pneumonia, and invasive fungal infections. 7 however, with the use of universal antiviral preventive strategies and long-term co-trimoxazole prophylaxis, opportunistic infections are currently rarely seen. after 6-12 months, the risk of infection decreases significantly and infections over this period are usually community-acquired, except in the case of increased immunosuppression (due to allograft rejection or dysfunction) or in the case of chronic surgical complications. because the incidence of infection is higher early after transplantation, it is recommended to avoid travel during the first year. 8 hsct recipients are at increased risk for bacterial and fungal infections during the engraftment period in the first month posttransplant. in the case of graft-versus-host disease, cellular immunosuppression is the mechanism responsible for the development of viral infections (particularly cytomegalovirus, adenovirus, and bk virus) and invasive fungal infections. 9 after the second year post-transplant it is considered that the degree of immunosuppression is non-significant if the patient has not developed chronic complications. while the use of biological agents for the therapy of rheumatological and autoimmune diseases has increased considerably over recent years, data on the risk of infection are mainly limited to the use of anti-tumour necrosis factor (tnf) agents. several large cohort studies found patients receiving anti-tnf therapy to be at greatest risk of developing skin infections, although the overall risk of severe infections was similar to that of patients receiving other non-biological therapies. 10 a study from the netherlands assessed the risk of infection in 75 travellers receiving biological agents relative to their travelling companions. 4 immunocompromised patients were at significantly higher risk of developing skin infections, fatigue, and abdominal pain, but not fever, diarrhoea, or respiratory infections. of note, no serious infection developed during or after the trip in these patients. patients on anti-tnf therapy have an increased risk of developing mycobacterial infections, with several cases of disseminated tb with a fatal outcome reported in the literature. 11 cutaneous leishmaniasis has also been reported in patients on anti-tnf treatment. 12 the risk of bacterial and fungal infections in patients with an oncological condition is increased during the administration of chemotherapy and/or radiotherapy and/or immunotherapy, particularly during the period of neutropenia. 13 in contrast, the risk of infection is generally considered not to be increased some months after the conclusion of chemotherapy and in patients receiving hormone therapy. patients with haematological conditions, such as lymphoma or hodgkin disease, may, however, have some degree of cellular immunosuppression even months after the remission of the disease. asplenic patients are at significantly higher risk of infection with encapsulated bacteria, namely streptococcus pneumoniae, neisseria meningitidis, haemophilus influenzae, and capnocytophaga canimorsus. 14 thus, appropriate vaccination with conjugated vaccines is an essential preventive strategy in these patients. other potentially life-threatening infections that are more common in asplenic patients include salmonellosis, babesiosis, and malaria. while the risk of infection is higher during the first month following splenectomy, the increased risk persists for years. the risk of infection in asplenic patients depends on the underlying condition, being higher in patients with haematological diseases and in those in whom immunization may not be fully successful or is associated with suboptimal protection over long periods of time. such patients may be instructed to start empirical antibiotics targeted at encapsulated bacteria immediately if any clinical signs or symptoms of infection ensue. 15 there are few studies that have addressed the epidemiology and clinical manifestations of malaria in immunocompromised patients. the incidence of malaria was reported to be less than 1% in hsct recipients in an endemic country (pakistan); however, data on other immunosuppressive conditions and in travellers are missing. 15 despite the lack of prospective studies, it appears that malaria is associated with more severe outcomes in immunocompromised patients than in the general population. a recent systematic review found that up to 45% of published cases of malaria in sot recipients had at least one criterion for severe malaria (o. manuel, personal communication). importantly, malaria may develop through transmission from the organ donor, and as such there may not be a travel history. 16 a case of cerebral malaria with >50% parasitemia has been reported in a patient receiving infliximab; 17 however such severe disease can occur in patients not on biologicals as well. malaria can also be more severe in splenectomized patients due to the lack of clearance of intraerythrocytic parasites. the successful treatment of severe malaria in immunocompromised patients has been reported with the use of erythropheresis and artesunate. 18 the choice of the preventive strategy for malaria in immunocompromised travellers should be individualized, favouring antimalarial prophylaxis in patients travelling to intermediate-risk and high-risk regions. several cases of severe dengue in immunocompromised patients have been reported, mostly in patients in endemic countries. in a series of kidney transplant recipients in india, up to 37% of patients diagnosed with dengue had a severe course and died. 19 all presented with fever, thrombocytopenia, myalgia, and retro-ocular pain. in contrast, in a series of eight patients receiving biologicals who were diagnosed with dengue, none developed severe infection. 20 dengue fever was reported to be a frequent cause of febrile neutropenia in haematological patients in india, but this was not associated with worse outcomes. 21 early diagnosis is essential in immunocompromised travellers with clinical manifestations suggestive of dengue in order to initiate early appropriate supportive therapy. aggressive volume replacement within the first 24 h of icu admission is important to limit the development of multiple organ dysfunction syndrome and increase the probability of survival. travel-related fungal infections in immunocompromised patients are uncommon, but potentially associated with a severe course and increased mortality. 22 invasive travel-related fungal infections that have manifested with a severe course in sot recipients and hiv-infected individuals include disseminated penicillium marneffei infection, aspergillosis, histoplasmosis, and coccidioidomycosis. 23 in patients receiving monoclonal antibodies, severe travel-associated histoplasmosis has been associated with a 50% mortality rate, and in another report, malignancy was a risk factor for acquiring a cryptococcus gattii infection. 24 importantly some of these infections may have a long incubation period so the travel history may be underreported. as such, a detailed travel history should be sought in immunocompromised travellers who develop fever associated with pulmonary lesions and/or localized cutaneous or subcutaneous disease, and these patients should be investigated promptly and aggressively for the diagnosis of invasive fungal infections. 22 the risk of tb is increased in transplant patients, hiv-infected individuals, and in patients receiving biologicals, but the risk of travel-acquired tb in immunocompromised patients is not well established. screening for latent tb infection after travel to endemic regions in these patients might, however, identify patients at risk of developing active tb. leptospirosis is a common cause of fever in returning travellers, and can be associated with severe complications. in a series of nine hiv-infected patients with leptospirosis, 67% presented with severe sepsis and the mortality was 22%. 25 data on the severity of leptospirosis in other immunocompromised populations are lacking. nocardiosis in sot recipients is associated with a high incidence of disseminated disease, particularly with central nervous system involvement. strongyloidiasis in immunocompromised patients is a rare but potentially life-threatening condition. donor-derived or travelacquired infestation with strongyloides stercoralis is associated with a high mortality. 26 cases of chagas disease (trypanosoma cruzi) either as a consequence of reactivation of a latent infection not identified at the time of transplant (because an unrecorded travel history or stay in an endemic area) or by transmission through the organ donor, can also be associated with a high mortality. 27 furthermore, immunocompromised patients may be particularly susceptible to severe forms of west nile virus infection and tick-borne encephalitis, all of which should be actively sought in the workup of patients with central nervous system symptoms after returning from endemic areas. there have also been case reports of severe disease from other travel-associated infections, such as salmonellosis, vibrio parahaemolyticus, and visceral leishmaniasis in immunocompromised patients. there are many causes of respiratory failure and of ards. those that are specific to certain geographic regions and that may appear unexpectedly in travellers are less common, but are nevertheless extremely important because appropriate therapy requires a correct diagnosis, and some infections may have epidemic potential. the infectious causes in particular may not be recognized immediately because they may be out of their usual geographical context. those that can cause ards will be discussed briefly below. table 2 summarizes the main recommended antimicrobial regimens for specific organisms involved in ards in returning travellers. cap is the most likely cause of acute respiratory failure in returning travellers. the usual pathogens, such as s. pneumoniae, h. influenzae, mycoplasma pneumoniae, chlamydophila pneumoniae, legionella pneumophila, and viruses such as influenza and respiratory syncytial virus, are the most common culprits. however aspiration must be considered in the elderly and in those who have become inebriated whilst on holiday. less common pathogens such as staphylococcus aureus, avian influenza viruses such as h7n9 and h5n1, the middle east respiratory syndrome coronavirus (mers-cov), and gram-negative rods such as burkholderia pseudomallei must also be considered, as well as a few other pathogens that do not usually cause pneumonia, such as malaria. influenza viruses such as h1n1 and h3n2, which are currently circulating, are perhaps the most common travel-related infections, particularly in the unvaccinated, those travelling across hemispheres, and where the available vaccine does not cover a particular strain effectively. influenza is an acute illness manifested by pyrexia, cough, chills, myalgia, and fatigue. there can, however, be more severe complications, specifically pneumonia, especially in pandemic years. in the 2009 influenza a(h1n1)pdm09 pandemic, more than 18 500 deaths were reported, with global estimates 15 times higher. the primary risk factors were age (young to middle age; >60% were aged <65 years), morbid obesity, pregnancy, and an immunocompromised status. influenza also increases the risk of bacterial pneumonia, particularly that caused by s. pneumoniae and s. aureus. those with severe disease deteriorate acutely after 4-5 days, with profound hypoxemia, shock, and often multiple organ dysfunction syndrome. the pathological findings are of an intense inflammatory/ haemorrhagic pneumonia, the severity of which seems to be influenced by the presence or absence of associated bacterial cap. 28 any patient with the above features, particularly if unvaccinated or having travelled to another hemisphere during the winter season, should be investigated for influenza, with diagnosis based on throat swab or nasal wash and a commercial kit based on antigen or rt-pcr. 29 unfortunately there is very low uptake of influenza vaccine even amongst healthcare workers, and as such there remains a large pool of susceptible individuals. although not yet reported to have been transmitted from humans, avian influenza h5n1 and h7n9 remain a potential threat, particularly in southeast asia. travellers who have had contact with birds in the affected areas and who present with otherwise unexplained ards should be screened. h5n1 has been reported from 17 countries and is currently most prevalent in egypt. 30 staphylococcus aureus pneumonia is usually a fulminant disease associated with rapid onset respiratory failure, frequently progressing to multiple organ dysfunction, shock, and death. complications are frequent and include pulmonary necrosis and abscess and empyema formation, particularly if the strain is a producer of panton-valentine leukocidin (pvl) toxin, a cytotoxin responsible for leukocyte destruction and tissue necrosis. risk factors are colonization or infection with s. aureus and a preceding influenza-like illness (ili). leucopenia (2.5 â 10 9 /l) is characteristic and may be an inverse biomarker of pvl burden. 31 both methicillin-sensitive s. aureus (mssa) and methicillinresistant s. aureus (mrsa) can cause cap. the latter is primarily a problem of recognition, as the organism is not prevalent in all countries and standard guideline-based therapies for pneumonia do not cover mrsa. the sensitivity profile of community-acquired mrsa differs from that of hospital-acquired mrsa in that it may be susceptible to macrolides, quinolones, clindamycin, and trimethoprim-sulfamethoxazole. therapy consists of appropriate antimicrobial therapy such as linezolid (possibly in preference to vancomycin, particularly if the strain is a pvl-producer), vancomycin, or ceftaroline. 32 pneumonia is the most common presentation of legionnaire's disease or legionellosis, and it may be severe, leading to multiorgan failure and death. characteristic clinical findings are relative bradycardia, hyponatremia, elevation in serum creatinine kinase, diarrhoea, confusion, and impaired liver and kidney function. 33 the recommended treatment regimen is macrolides or fluoroquinolones. 34 ten years after the severe acute respiratory syndrome (sars) epidemic that affected almost 8000 people and caused 775 deaths, mers-cov, a new coronavirus of the same family, appeared in saudi arabia and subsequently spread to nine countries in or near the arabian peninsula and 14 countries elsewhere. 35 on march 10, 2015, the world health organization global case count was 1060 laboratory-confirmed cases with 394 deaths (37%). all cases were resident in or had travelled to the middle east, most to saudi arabia, or had been in contact with travellers returning from these areas. mers-cov differs from sars-cov in that it binds to different receptors, and camels are thought to be the primary reservoir host, although the means of transmission from these animals is poorly understood. whereas transmission can occur between humans, the epidemic potential appears to be less. the disease is not always severe and symptoms range from an ili to severe pneumonia requiring mechanical ventilation. the most severely affected patients have mostly had comorbidities such as diabetes, renal failure, and chronic lung disease, or have been immunocompromised. 36 there is no specific antiviral treatment available for mers-cov infection. management is primarily supportive, directed towards the prevention of respiratory complications and infection control. corticosteroids are not currently recommended in this setting. it is still advised that those travelling to the middle east and who are at increased risk of severe disease should avoid contact with camels and their secretions, and avoid drinking raw camel milk (which will also prevent infection with brucella). all travellers should practice good hand and food hygiene, particularly where camels are present. a number of gram-negative pathogens may cause pneumonia and ards, in particular in relation to aspiration, or in association with ventilation where pathogens such as pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, escherichia coli, and other enterobacteriaceae are of concern. 37 the latter include the extended-spectrum beta-lactamase (esbl)-and carbapenemaseproducers (such as those producing new delhi metallo-blactamase 1 (ndm-1)), which may be acquired during 'medical tourism'. 38 diagnosis requires a high index of suspicion and testing for the specific genes responsible for enzyme production. treatment remains a challenge due to deficiencies in the antibiotic pipeline. burkholderia pseudomallei is also a gram-negative bacillus endemic in southeast asia, northern australia, and possibly the indian subcontinent, southern china, hong kong, and taiwan. infection results from inoculation of contaminated soil and surface water through skin abrasions, with subsequent haematogenous spread. horizontal transmission also occurs, as well as transmission through the inhalation of polluted water. it is the most common cause of fatal community-acquired bacteraemia and pneumonia in certain areas of north-eastern thailand, as well as in darwin, australia. 39 travellers from endemic areas, especially in the wet season and particularly if there are comorbidities, are at risk. variable disease severity and the range of presentations (pneumonia, abscesses, osteomyelitis, and arthritis) make diagnosis a challenge. about 50% of patients present with pneumonia (which may appear as nodular infiltrates or air space consolidation), often with septic shock. the diagnosis is made when b. pseudomallei is cultured, but specific media are required. ceftazidime or meropenem with or without high-dose co-trimoxazole are the drugs of choice. although tb may occur in any patient, it seldom causes respiratory failure over a short period of time. yet, a recent prospective study from south africa reported that 1.5% of adults with active tb may require mechanical ventilation because of refractory hypoxemia, which in high tb prevalence countries translates into a significant burden of disease. 40 in this setting, 15% of tb suspects had confirmed tb, and it should be considered if the travel history involves relevant exposure. standard smear microscopy or culture are used for diagnosis, or rapid pcr if available (genexpert mtb/rif), which has been shown to have increased sensitivity and shorten the time to treatment. 41 where there is a high clinical suspicion of tb, empiric therapy should be initiated after adequate sampling has been obtained, particularly in the case of life-threatening or disseminated infection. initial therapy with four drugs (isoniazid, rifampicin, pyrazinamide, and ethambutol) is generally recommended where there is a low prevalence of resistance, and depending on the patient's origin and the results of the rapid detection of rpob gene mutations. patients with mdr-or xdr-tb need to be treated with second-line agents including aminoglycosides, quinolones, para-aminosalicylic acid, cycloserine, and clofazimine, and new drugs such as bedaquiline, linezolid, and delamanid. malaria, which is a frequent travel-related disease, may also lead to ards in severely affected patients. 41 increased alveolar capillary permeability may result in pulmonary oedema and respiratory failure either at presentation or after treatment. pregnant women are particularly at risk. slide microscopy and rapid antigen tests are the standard diagnostic tools, and the treatments of choice are the parenteral artemisinins, although resistance is emerging. non-infectious causes of bilateral pulmonary infiltrates with respiratory failure must be differentiated from infectious causes. these include cardiogenic pulmonary oedema, inflammatory pulmonary diseases such as cryptogenic organizing pneumonia (cop) and fibrosis, alveolar haemorrhage (including idiopathic granulomatous polyangiitis, lupus, and vasculitis), and ards from conditions such as eosinophilic pneumonia, pancreatitis, inhalational injury, and trauma. to identify these, clinical expertise is critical, along with the use of biomarkers (such a c-reactive protein, procalcitonin, and pro-b-type natriuretic peptide), serology to exclude autoimmune diseases, and imaging including echocardiography. if mechanical ventilation alone is inadequate, the use of neuromuscular blockade, recruitment techniques including prone ventilation, and veno-venous extracorporeal membrane oxygenation (ecmo) may improve oxygenation and the outcome. the berlin classification of ards severity is universally accepted and should be utilized to determine the site of therapy. 42 the acute physiology and chronic health evaluation (apache) score provides additional information regarding icu and hospital outcomes. 43 for pneumonia, the most frequently used scores are the pneumonia severity index (psi) and curb-65. to evaluate mortality risk in tb patients, the tbscore is useful and has been shown to predict the outcome. 44 haemorrhagic symptoms and fever can be caused by many infections due to bacteria, viruses, and parasites. disseminated intravascular coagulation (dic) may be a manifestation of severe septicaemia and can be caused by almost all gram-positive and gram-negative bacteria. dic is particularly present in septicaemia with n. meningitidis (figure 1 ), but is also seen in patients with s. aureus and s. pneumoniae bloodstream infections (figures 2 and 3 ). numerous viruses may also cause haemorrhagic symptoms, and these include dengue virus, crimean-congo haemorrhagic fever virus, ebola virus, yellow fever virus, hanta virus, and others (table 3) . it is most important to establish whether the patient has a history of travel within the past 4 weeks to areas where viral haemorrhagic fevers are endemic immediately at admission (table 3 ). if the history and the clinical features are suggestive, further details should be obtained, as shown in table 4 , and the patient should be evaluated as to whether isolation is necessary. in most countries where haemorrhagic fever viruses occur, malaria is also endemic, and a malaria test (rapid diagnostic test or microscopy) should be performed immediately and at the same time as blood cultures for bacterial infections are obtained. once malaria has been excluded, treatment should be started to cover a broad range of bacterial infections until such time as the diagnosis is confirmed. an example of gangrene related to severe staphylococcal septicaemia is shown in figure 2 . rapid assessment of the patient and isolation are key to limiting healthcare-associated transmission. the mers-cov outbreak in south korea illustrates how rapidly infections can spread in overcrowded hospitals. 45 most mers-cov cases in saudi arabia have also been linked to transmission in hospitals, 46 as was the case with sars-cov 47 and with crimean-congo haemorrhagic fever. 48 training of paramedical staff, nurses, and physicians, as well as guidelines for the recognition and rapid assessment of febrile patients at the initial point of contact, are essential, as is an isolation area for febrile patients with a relevant travel history. dic is an acquired condition of the vascular system leading to an uncontrolled systemic activation of the coagulation pathway. the generation of thrombin and fibrin may cause thrombotic occlusions of blood vessels, and hence organ injury and failure. 49 this is accompanied by an inflammatory reaction, further augmenting the coagulation process. dic frequently accompanies systemic inflammatory response syndrome (sirs), severe sepsis, trauma, and other conditions as diverse as anaphylaxis and heat stroke. [49] [50] [51] [52] [53] [54] the systemic activation of the clotting system is associated with the consumption of both coagulation factors and platelets, and as such, various combinations of platelet count, prothrombin time, activated partial thromboplastin time (aptt), a decrease in anti-thrombin (at) and protein c, as well plasma levels of fibrin and d-dimers have been used for the diagnosis. 55 a more standardized approach can be achieved by using the scoring system of the international society of thrombosis and haemostasis. 56 the successful therapy of dic is only possible when the underlying cause is identified and treated. the substitution of coagulation factors is currently unclear due to the lack of appropriate randomized placebo-controlled trials. the use of antifibrinolytics during dic should be avoided, as this drug class may lead to the deposition of fibrin in the vascular walls. 57 overall, the prevalence of dic during viral haemorrhagic fever is high and contributes to morbidity and mortality. early and effective treatment against the viral infection, if available, reduces the detrimental complications of dic. parameters for assessing dic and haemolysis are provided in table 5 . questions to be asked if the patient has travelled in an area where haemorrhagic fever occurs does the patient have a fever (>38 8c) or history of fever in the previous 24 hours? and has the patient cared for/come into contact with body fluids of/handled clinical specimens (blood, urine, faeces, tissues, laboratory cultures) from a live or dead individual or animal known or strongly suspected to have vhf? has the patient received a tick bite and/or crushed a tick with their bare hands and/or travelled to a rural environment where contact with livestock or ticks is possible in a cchf endemic area? has the patient lived or worked in basic rural conditions where lassa, ebola, or marburg fever is endemic, i.e., west/central africa or south america? has the patient travelled to any local area where a vhf outbreak has occurred? cchf, crimean-congo haemorrhagic fever; vhf, viral haemorrhagic fever. patients with suspected severe sepsis should be managed according to standard guidelines. 58 these include broad-spectrum antibiotics, aggressive initial fluid replacement, blood pressure support if needed, the correction of acidosis, and oxygenation by intubation and mechanical ventilation as needed. echocardiography is essential to evaluate cardiac function and any vegetations on the cardiac valves. in the initial stages it is difficult to differentiate between a viral haemorrhagic fever, severe bacterial sepsis, and severe malaria. travel patterns and risk behavior in solid organ transplant recipients international travel patterns and travel risks for stem cell transplant recipients international travel in the immunocompromised patient: a cross-sectional survey of travel advice in 254 consecutive patients symptoms of infectious diseases in immunocompromised travelers: a prospective study with matched controls multidrug-resistant bacteria without borders: role of international trips in the spread of multidrug-resistant bacteria infection in solid-organ transplant recipients impact of antiviral preventive strategies on the incidence and outcomes of cytomegalovirus disease in solid organ transplant recipients travel medicine and transplant tourism in solid organ transplantation hematopoietic stem cell transplantation: an overview of infection risks and epidemiology rates of serious infection, including site-specific and bacterial intracellular infection, in rheumatoid arthritis patients receiving anti-tumor necrosis factor therapy: results from the british society for rheumatology biologics register tuberculosis associated with infliximab, a tumor necrosis factor alphaneutralizing agent anti-tumour necrosis factor-induced visceral and cutaneous leishmaniasis: case report and review of the literature bacterial infections in low-risk, febrile neutropenic patients post-splenectomy and hyposplenic states the stem cell transplant program in pakistan-the first decade posttransplant malaria: first case of transmission of plasmodium falciparum from a white multiorgan donor to four recipients overwhelming parasitemia with plasmodium falciparum infection in a patient receiving infliximab therapy for rheumatoid arthritis donor-transmitted malaria after heart transplant managed successfully with artesunate dengue virus infection in renal allograft recipients: a case series during 2010 outbreak dengue fever in patients under biologics dengue fever as a cause of febrile neutropenia in adult acute lymphoblastic leukemia: a single center experience fungal infections in immunocompromised travelers donorderived fungal infections in organ transplant recipients: guidelines of the american society of transplantation, infectious diseases community of practice risk factors for cryptococcus gattii infection leptospirosis and human immunodeficiency virus co-infection among febrile inpatients in northern tanzania donor-derived strongyloides stercoralis infection in solid organ transplant recipients in the united states trypanosoma cruzi fatal reactivation in a heart transplant recipient in switzerland critically ill patients with influenza a(h1n1)pdm09 virus infection in 2014 rapid diagnostic testing for influenza: information for health care professionals avian influenza a(h7n9) virus. geneva: who severe community-onset pneumonia in healthy adults caused by mrsa carrying the panton-valentine leukocidin genes ceftaroline fosamil for the treatment of staphylococcus aureus bacteremia secondary to acute bacterial skin and skin structure infections or community-acquired bacterial pneumonia clinical features and predictors of mortality in admitted patients with community-and hospitalacquired legionellosis: a danish historical cohort study legionnaires disease and the updated idsa guidelines for community-acquired pneumonia middle east respiratory syndrome (mers) interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk aetiological agents of ventilator-associated pneumonia and its resistance pattern-a threat for treatment risk factors for infections with extended-spectrum beta-lactamase-producing escherichia coli and klebsiella pneumoniae at a tertiary care university hospital in switzerland melioidosis: a review a randomised controlled trial of the impact of xpert-mtb/rif on tracheal aspirates nested within a burden of disease study in south african intensive care units managing malaria in the intensive care unit acute respiratory distress syndrome: the berlin definition. ards definition task force severity scoring in the critically ill: part 1-interpretation and accuracy of outcome prediction scoring systems tbscoreii: refining and validating a simple clinical score for treatment-monitoring patients with pulmonary tuberculosis middle east respiratory syndromeadvancing the public health and research agenda on mers-lessons from the south korea outbreak mers-cov outbreak in jeddah-a link to health care facilities cluster of severe acute respiratory syndrome cases among protected health-care workers-toronto, canada probable crimean-congo hemorrhagic fever virus transmission occurred after aerosol-generating medical procedures in russia: nosocomial cluster disseminated intravascular coagulation prospective validation of the international society of thrombosis and haemostasis scoring system for disseminated intravascular coagulation disseminated intravascular coagulation in trauma patients disseminated intravascular coagulation (dic) in cancer the obstetric patient and disseminated intravascular coagulation disseminated intravascular coagulation guidelines for the diagnosis and management of disseminated intravascular coagulation. british committee for standards in haematology towards definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation prevention and treatment of major blood loss surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012 the authors declare no conflicts of interest. jr and hl designed the manuscript: om and pe wrote the immunocompromised host section, gr and cw wrote the ards section, and cw, ep and kz wrote the haemorrhagic fever section. om and jr assembled the final version. all authors read and approved the last version of the manuscript. key: cord-327493-v2iatbol authors: kwon, hyo jin; rhie, young jun; seo, won hee; jang, gi‐young; choi, byung min; lee, jung hwa; lee, chang‐kyu; kim, yun kyung title: clinical manifestations of respiratory adenoviral infection among hospitalized children in korea date: 2013-08-05 journal: pediatr int doi: 10.1111/ped.12108 sha: doc_id: 327493 cord_uid: v2iatbol background: the objective of our study was to understand the epidemiological and clinical features of respiratory adenoviral infections among children at a single institution over the course of several years. methods: from january 2005 to april 2009, 1836 children (≤15 years old) who had been admitted to korea university ansan hospital were tested for acute respiratory infection. the patients who were positive for an adenovirus infection were enrolled in this study, and their medical records were retrospectively reviewed. results: adenoviruses were isolated from 310 patients. the male to female ratio was 1.6:1 and mean age was 32 ± 24 months. children under 5 years of age had the highest prevalence. in 2007, adenovirus infections occurred endemically throughout the year. the clinical diagnoses were primarily upper respiratory tract infections (45.4%), lower respiratory tract infections (48.1%), and neurologic disease (5.2%). associated symptoms, signs and laboratory findings included fever (91.9%), cough (83.9%), pharyngeal injection (62.3%), rale (32.6%) and elevated c‐reactive protein (93.9%). the most common radiologic findings were perihilar and peribronchial infiltrates (42.6%). co‐infections were observed in 29 cases. the mean durations of hospitalization and fever were 6.2 ± 6.5 and 4.8 ± 3.1 days, respectively. the lengths of hospitalization were similar for patients admitted for upper respiratory tract infections with severe morbidity and those admitted for lower respiratory tract infections. no children in the study died. conclusion: our study demonstrates that respiratory adenovirus infections are an important cause of hospitalization in young children, and contribute to a significant morbidity. adenovirus is a major cause of acute respiratory illness in children worldwide. 1, 2 adenovirus-related deaths in children are rare, but adenovirus-attributable hospitalizations in young children and high-risk populations are quite high. 3, 4 a previous study found a high burden of hospital admission among children with adenoviral infection. 5 the spectrum of adenoviral infection in children ranges from subclinical illness to complicated disease involving multiple organs, with pneumonia being one of the most common presentations. although most infections are selflimited, adenovirus can be associated with severe conditions in both immunocompromised and immunocompetent individuals. 6, 7 sporadic serotype analysis and some case reports have been performed in korea since 1995. 8, 9 however, little information on the epidemiology and clinical characteristics of respiratory adenoviral infection, especially in hospitalized children, has been published. the study was performed to more fully characterize the epidemiological pattern, clinical features and complications associated with hospitalization for adenoviral infection in korean children. nasal aspirate specimens were collected from patients presenting with acute respiratory symptoms at ansan hospital, which is affiliated with korea university and serves the communities of ansan city, a city neighboring seoul. nasal aspirates were routinely tested to identify adenovirus, parainfluenza, respiratory syncytial virus (rsv) and influenza a/b by viral culture using three standard cell lines (hep-2, mdck and llc-mk2). we retrospectively analyzed data from hospitalized children 15 years of age or younger who had a laboratory-confirmed adenovirus infection between january 2005 and april 2009. study approvals were obtained from the institutional review boards of korea university ansan hospital. clinical, laboratory and radiological data were extracted from electronic medical records. clinical information regarding diagnosis, management during hospitalization, intensive care unit (icu) stay, the need for mechanical ventilator assistance and any underlying conditions were obtained from the hospital records. the statistical analysis was performed using spss version 12.0 (spss, chicago, il, usa). data were presented as numbers (percentage), mean ϯ sd or median (range) as appropriate. comparisons between groups were carried out using the mann-whitney u-test. a p-value of < 0.05 was considered to be significant in the analysis. from january 2005 to april 2009, a total of 1836 specimens were collected from patients admitted with respiratory viral infection. of these samples, 310 episodes were confirmed as adenoviral infections, an overall proportion of 16.9% (310/1836). ages ranged from 1 month to 11.3 years, and the mean age was 32 ϯ 24 months. the patients in this study were grouped by age as 0-<1, 1-<2, 2-<3, 3-<4, 4-<5, and ն5 years. about 90% of all the adenovirus-related cases occurred primarily in children aged less than 5 years, and the peak incidence was in the 1-<2year group (23.5%) (fig. 1 ). when the <1-year-old group was divided into two subgroups, the subgroup younger than 6 months represented only 8.4% of the total cases, while the 6-<12 month subgroup was 14.2%. the ratio of boys to girls was 1.6:1. twenty-six patients (8.4%) had one or more underlying conditions, including asthma, chronic neurologic disease and/or prematurity. the annual detection (detection rate, numbers of positive/ numbers of specimen) of adenovirus was as follows: 68 cases diagnoses at admission were based on clinical, laboratory, and radiographic information. adenovirus was associated with a wide variety of diagnoses, ranging from upper respiratory tract infections (urti) to severe pneumonia and encephalitis ( table 1 ). the most frequent presentation was lower respiratory tract infections (lrti) (149/310, 48.1%), followed by urti (141/310, 45.4%). among the lrti, the admitting diagnosis was pneumonia for 107 patients (107/310, 34.5%), acute bronchiolitis in 20 (20/310, 6.5%), and acute bronchitis in 22 (22/310, 6.1%). urti included pharyngitis (92/310, 29.6%), otitis media (26/310, 8.4%), sinusitis (13/310, 4.2%) and croup (10/310, 3.2%). twenty patients (6.5%) presented with other diagnoses, including febrile convulsions, neonatal sepsis, gastroenteritis and encephalitis. over 90% of adenoviral infections were accompanied by fever, which was the most frequent clinical finding. the mean duration of fever was 4.8 ϯ 3.1 days (range 0-15 days). a high fever (ն39°c) was described in one-third of all patients, and prolonged fever (ն10 days) was reported in 6.8% of cases. other common features were cough (83.9%), rhinorrhea (63.2%) and sputum (61.3%). respiratory distress was seen in 19 cases (6.1%). gastrointestinal symptoms were also commonly found. sixty-five (21%) patients presented with diarrhea, anorexia was seen in 63 (20.3%), and vomiting was found in 10 cases (3.2%). other clinical symptoms included seizure (8.4%), headache (4.5%) and skin rash (2%). crackles on auscultation were heard in 32.6% (101/310) of patients. wheezing was noted in 37 (12%) patients, and retraction of the chest wall was seen in 14 (4.5%). other abnormal findings on physical examination were pharyngeal injection (62.3%), redness of the tympanic membrane (12.6%) and conjunctival injection (8.7%). in addition, cervical lymphadenopathy was detected in eight (2.6%) patients, and hepatosplenomegaly was found in two (0.6%). leukocytosis was detected in 69 (22.3%) patients, and leukopenia was seen in four (1.3%) ( table 2) . 10 elevated c-reactive protein (crp) and erythrocyte sedimentation rate (esr) were the most common laboratory findings (93.9%). liver enzyme, aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels were elevated in about 10% of the patients. co-infections with other pathogens were identified in 29 cases. the most common cause was mycoplasma pneumoniae (m. pneumoniae) (12/29). influenza a (2/29) and b (4/29), parainfluenza virus (2/29), rsv (2/29) and rotavirus (7/29) were also concomitantly identified with adenovirus infection. five cases of rotavirus infection were identified as hospital-acquired infection, while other bacterial isolates were not detected at the time of admission. chest radiography was performed in all patients at admission, and abnormal findings were found in 42.6% (132/310); 104 (35.5%) had perihilar or peribronchial infiltrates, 23 (7.4%) had lobar infiltration or consolidation, three (1%) had hyperinflation and two (0.6%) had pleural effusion. the mean duration of hospital stay was 6.2 ϯ 6.5 days (range 3-98 days). the majority of patients (93.9%, 291/310) received antibiotics. two or more antimicrobial agents were used in 34.2% of patients. the most commonly used agents were ampicillin/ sulbactam (80.8%), third-generation cephalosporin (24.7%), aminoglycoside (16.2%) and macrolide (8.6%). oxygen supplementation was provided to 11 patients (3.6%). five patients (1.6%) were admitted or transferred to the icu, and three of them received mechanical ventilation. the characteristics of icu patients are detailed in table 3 . two of the icu patients were admitted in 2005, while the remaining three patients were admitted in 2007. all were male, and had no underlying conditions, except one who was born prematurely. the first case was diagnosed with severe pneumonia, which progressed to ischemic-hypoxic encephalopathy. the second case was pneumonia complicated with bronchiolitis obliterans. the third case was a 23-month-old premature infant with bronchopulmonary dysplasia who required ventilator care for 24 days. the fourth case was pneumonia with a long-lasting fever (15 days) that was improved without complication. the fifth case was admitted for fever and seizure, and adenovirus from csf was identified. no deaths were reported. duration of hospital stay and fever were used as parameters for determining disease severity. duration of hospital stay was significantly longer in children with lrti (p < 0.001) and co-infection (p = 0.001). however, the length of hospitalization was not related to age, sex or pre-existing condition. on the other hand, prolonged fever was associated with underlying disease (p = 0.026) and asthma (p = 0.016). age, sex, diagnosis and co-infection, including m. pneumoniae, were not related to the duration of fever. this study revealed that adenoviral infection accounted for a relatively high proportion (16.9%) of all respiratory virus infections in hospitalized children compared to the results of previous reports. 11, 12 other pathogens included rsv (48%), influenza (19%), and parainfluenza (16.1%). in our study, 81.3% of the adenoviral infections occurred in young children between 6 months and 5 years of age. similar demographic features have been reported in other studies. 13, 14 according to pereira's report, children aged 5 years had higher antibody positivity against adenovirus, and most infections happened in children younger than 5 years. 15 infants less than 6 months of age are known to have a neutralizing antibody by maternal transmission, which appears to be protective during the first 6 months of life. the frequency of infection was low in infants under 6 months of age; however, a severe case, such as encephalitis, as reported in this study, may occur. the male to female ratio was 1.6:1. the tendency for male predominance has been described in the literature concerning adenovirus infection, as well as in studies of other respiratory viruses. [16] [17] [18] we observed that more cases of adenoviral infectious disease occurred in 2007. adenovirus was the most common in the spring and summer of 2007, but occurred throughout the year in other years examined in this study. it seems that adenovirus shows a seasonal variation, with sporadic epidemics. in korea, small outbreaks of adenovirus infection were reported in the summer of 1995 and in the spring of 1996. 19 in a temperate climate, such as korea, adenovirus infections are known to occur in the spring, early summer and winter. 20 our data showed similar findings, illustrating a slight tendency toward variations in the seasons in which adenovirus was detected more frequently. in one study, 21 adenoviral diseases in children were characteristically accompanied by a high and persistent (mean 5.4 days) fever. over one-third of the children in our study showed a high fever despite the supportive treatment, and a prolonged fever (ն10 days) was found in 6.8% of cases. larrañaga et al. 22 reported that 70% of hospitalized children with adenoviral infection had pneumonia, while our results revealed a significant proportion of patients (45.4%) with urti. the prolonged fever associated with adenovirus infection highlights the primary cause for hospital treatment in patients with urti. also, many patients appeared to have gastrointestinal symptoms with hepatic involvement, as determined by a mild to high elevation of liver enzymes. adenovirus was detected in the csf of one infant who was diagnosed with encephalitis. according to our study, the clinical manifestation of adenovirus infection varied, with multiple organ involvement. several studies have demonstrated that adenovirus is unique among the common respiratory viruses in that it can involve other organs, resulting in conjunctivitis, gastroenteritis, acute hemorrhagic cystitis and meningoencephalitis. 23, 24 although the white blood cell count was within the normal range in most patients (76.4%), elevated crp and esr levels were shown in 93.9% of all the patients. elevated crp and esr levels are also generally thought to be related to bacterial infection, as they are inflammatory markers. others have reported that adenoviral infection typically results in elevated esr and crp levels, unlike what is seen in other viral diseases. 25, 26 therefore, it is not surprising that many patients were initially treated with antibiotics. the clinical use of antigen detection tests is expected to make earlier diagnosis of adenoviral infection, and to reduce unnecessary treatment of antibiotics. the relation to co-infections has been reported by some authors. korppi et al. reported that mixed infection was common (55%) in children with adenovirus infection, and bacterial co-infection was demonstrated in 45% of patients. 27 another study noted that co-infection with measles was a risk factor for mortality in the acute stage of adenovirus respiratory infections. 28 in our study, the most common cause of co-infection was m. pneumoniae. we found no significant difference in disease severity between adenovirus-infected patients with co-infection and those without. when more than one pathogen is identified with adenovirus, it is difficult to determine whether it is true co-infection because adenovirus can persist in the respiratory or gastrointestinal tract after active infection. further study will be necessary to determine whether co-infection can affect the disease course and prognosis. there were several limitations to our study. first, the serotypes of adenovirus were not determined. therefore, we were unable to make more refined observations regarding differences in age distributions, clinical characteristics and determinants of severity based on the serotypes. second, nearly all the children received antibiotic treatment, and the judicious use of antibiotics and resistant development is becoming increasingly important in korea. finally, we did not test for human bocavirus, coronavirus, metapneumovirus, or aerobic bacteria; therefore, some co-infections may have been overlooked. in conclusion, our study contributes to critical epidemiological baseline on respiratory adenoviral infection in korean children, and highlights the importance of adenovirus as a major cause of hospitalization. adenovirus was more frequently detected in young children and was associated with significant morbidity. prolonged fever was associated with urti and necessity of hospitalization, as well as lrti. large-scale investigation through a multicenter approach is required to determine the optimal monitoring and treatment strategies and to achieve a better understanding of the clinical course of adenoviral infection in children. multicentered study of viral acute lower respiratory infections in children from four cities of argentina, 1993-1994 genetic heterogeneity of the hexon gene of adenovirus type 3 over a 9-year period in korea adenovirus bronchiolitis in manitoba: epidemiologic, clinical, and radiologic features severe diffuse adenovirus 7a pneumonia in a child with combined immunodeficiency: possible therapeutic effect of human immune serum globulin containing specific neutralizing antibody burden of viral respiratory disease hospitalizations among children in a community of seoul severe adenovirus bronchiolitis in children adenovirus: an increasingly important pathogen in paediatric bone marrow transplant patients ten cases of severe adenoviral pneumonia in the spring 1995 comprehensive serotyping and epidemiology of human adenovirus isolated from the respiratory tract of korean children over 17 consecutive years (1991-2007) nathan and oski's hematology of infancy and childhood, 7th edn the association of newly identified respiratory viruses with lower respiratory tract infections in korean children clinical picture and epidemiology of adenovirus infections (a review) adenovirus infection in hospitalized immunocompetent children adenovirus infections in hospitalized patients in israel: epidemiology and molecular characterization adenovirus infections genotype prevalence and risk factors for severe clinical adenovirus infection, united states risk factors associated with severe influenza infections in childhood: implication for vaccine strategy interleukin-9 polymorphism in infants with respiratory syncytial virus infection: an opposite effect in boys and girls clinical characteristics of acute viral lower respiratory tract infections in hospitalized children in seoul, 1996-1998 adenoviral diseases in children: a study of 105 hospital cases adenovirus surveillance on children hospitalized for acute lower respiratory infections in chile (1988-1996) lower respiratory tract infections due to adenovirus in hospitalized korean children: epidemiology, clinical features, and prognosis disseminated adenovirus disease in immunocompromised and immunocompetent children the differentiation of classic kawasaki disease, atypical kawasaki disease, and acute adenoviral infection: use of clinical features and a rapid direct fluorescent antigen test serum c-reactive protein in children with adenovirus infection mixed infection is common in children with respiratory adenovirus infection lower respiratory infections by adenovirus in children. clinical features and risk factors for bronchiolitis obliterans and mortality we thank the doctors and laboratory staff of korea university ansan hospital for their participation in the retrospective adenoviral infection study. we also thank our collaborators at myung moon pediatrics for their support. key: cord-292521-tpb12dkq authors: howard, john; thompson, thomas z.; macarthur, rodger d.; rojiani, amyn m.; white, joseph title: widely disseminated cryptococcosis manifesting in a previously undiagnosed human immunodeficiency virus (hiv)-positive 18-year-old date: 2020-10-12 journal: am j case rep doi: 10.12659/ajcr.924410 sha: doc_id: 292521 cord_uid: tpb12dkq patient: male, 18-year-old final diagnosis: disseminated cryptococcosis in previously undiagnosed hiv symptoms: diarhea • lymphadenopathy medication: — clinical procedure: — specialty: general and internal medicine • pathology objective: unusual clinical course background: after initial infection with hiv, loss of cd4+ t cells progresses along a predictable timeline. the clinical latency stage lasts an average of 10 years, until the cd4+ t cell count falls below 200 cells/ul or the patient develops an aids-defining opportunistic infection/cancer. this report describes an unusual opportunistic infection in a young patient with no prior clinical evidence of hiv infection. case report: an 18-year-old man presented with fever, abdominal pain, and dyspnea for the previous 2 weeks and was symptomatically treated for gastroenteritis. he presented 2 weeks later with extreme fatigue, and a ct scan revealed diffuse lymphadenopathy. he was transferred to a regional hospital, but upon arrival and prior to detailed investigative work-up, he developed cardiac arrest. despite maximal resuscitative efforts, he died approximately 8 h after admission. at autopsy, diffuse lymphadenopathy, splenomegaly, and pulmonary congestion were noted. disseminated cryptococcal infection involving almost every organ system was identified at autopsy. a postmortem hiv-1 antibody test was positive. the cause of death was severe immunodeficiency as a result of advanced hiv infection resulting in disseminated cryptococcal infection, with cerebral edema, herniation, and respiratory failure. conclusions: this patient’s non-specific symptoms in conjunction with his rapid decline made arriving at a correct diagnosis challenging. only during autopsy was the disseminated fungal infection discovered, leading to suspicion of hiv infection. hiv autopsies are not uncommon, but the clinical history is usually known beforehand. this case report highlights the importance of considering hiv-related conditions in patients presenting with this array of symptoms, as well as to alert healthcare providers and staff to the need for increased biosafety precautions. in most cases, following primary infection with hiv, the loss of cd4+ t cells continues over a fairly predictable, extended period of time. the period during which the body's defenses attempt to bring the virus under control, at least to a more stable set point, is defined as the clinical latency stage, which lasts an average of 10 years, until the cd4+ t cell count falls below 200 cells/µl or the patient develop an aids-defining opportunistic infection/cancer. the patient described here had no clinical evidence of hiv infection or other manifestation of the disease. because he remained asymptomatic before presentation, there was no indication of a need for hiv testing. his clinical presentation is highly unusual in that his initial non-specific gastrointestinal symptoms and fatigue did not in any way raise suspicion of hiv infection, and his subsequent diffuse lymphadenopathy was suspected to be lymphoma. the identification of disseminated cryptococcosis at autopsy in the absence of prior clinical evidence of hiv infection led to a postmortem diagnosis of aids. this case may have been acute retroviral syndrome; however, since the patient had no prior testing or evidence of hiv infection, it is impossible to confirm that diagnosis [1] . this case report presents important information by defining the very extensive dissemination of cryptococcosis and discussing the implications for differential diagnosis. additionally, patient care and exposure at autopsy are important considerations in the absence of a prior hiv diagnosis. an 18-year-old african american man presented to the hospital with complaints of fever, nausea, dyspnea, and abdominal pain over the previous 2 weeks. he was subsequently symptomatically treated for gastroenteritis without additional workup, and blood culture, stool analysis, and colonoscopy were not performed. this case occurred before the present covid-19 pandemic. this was his first clinical episode and he otherwise appeared to be healthy, with no evidence of cachexia. he had no history of recent travel, prior surgery, or blood transfusion. there was no social history of sexual activity or preference. he returned 2 weeks later with extreme fatigue to the point of being unable to walk, at which point a ct of his chest, abdomen, and pelvis was ordered, which revealed diffuse lymphadenopathy. a lymphoma was suspected, and the patient was admitted for work-up (day 1). a complete blood count (cbc) was also ordered, which demonstrated a leukocytosis (15 200 cells/µl) with an absolute and relative neutrophilia (90%). soon after admission, before the patient could undergo additional investigative studies including lymph node biopsy or complete pulmonary work-up, he rapidly declined and developed respiratory distress requiring intubation. he was transferred to a regional medical center for a heightened level of care. upon evening arrival, he exhibited altered mental status, prompting a chest x-ray that revealed bilateral hilar adenopathy and right lower-lobe predominant consolidations. subsequent x-rays over the next several hours showed increasing levels of pulmonary edema. another cbc was ordered, which showed a further increase in wbc (16 900 cells/µl), with 93% neutrophils (15 700 cells/µl) and 5% lymphocytes (850 cells/µl), as well as hemoglobin and hematocrit values of 11.5 qm/dl and 36.2%, respectively. a complete metabolic panel revealed elevated ast (147 iu/l) and alt (62 iu/l), as well as extremely high crp (6.40 mg/dl) and procalcitonin (5.33 ng/ml) levels. the following morning (day 2), he had a sharp decline in hemodynamic status and had a cardiac arrest secondary to ventricular tachycardia. he received multiple rounds of advanced cardiac life support over the next 4 h, but died despite maximal resuscitation efforts. an unrestricted autopsy was performed on this generally normal-appearing man, with no evidence of cachexia, no external wounds, rash, icterus, or other obvious pathology. autopsy findings demonstrated diffuse lymphadenopathy observed in the mediastinal, retroperitoneal, mesenteric, and inguinal groups, splenomegaly, and pulmonary congestion. histologic examination of submitted tissues revealed disseminated cryptococcal infection involving the spleen ( figure 1a hiv is spread through contact with certain human body fluids, after which the virus invades and attacks the host's immune system. hiv infects host cd4 + t cells via interaction between an hiv envelope-bound protein called env, and host cell cd4, ccr5, and cxcr4 receptors [2] . the hiv protein env extends from the virion and consists of 2 glycoproteins, gp120 and gp41, which make up the cap and stem, respectively, of env [2] . gp120 is required for binding to host cd4 receptors, while gp41 plays a role in promoting fusion of the viral envelope and host cell plasma membrane after receptor binding [2] . the hiv virus then uses host cell machinery to reproduce, destroying these cells in the process. cd4 + t cells are a vital part of the human immune system; therefore, their destruction by the hiv virus weakens the body's overall immune capabilities. after initial seroconversion due to infection with hiv, progressive loss of cd4+ t cells follows a fairly predictable timeline. there is typically a clinical latency stage, followed by clinical evidence of aids. the clinical latency stage develops as the body's immune response is able to substantially, but not completely, lower the viral load. the clinical latency stage for patients who are not receiving treatment lasts an average of 10 years, until the cd4+ t cell count falls below 100-200 cells/µl. infected individuals not receiving treatment eventually progress to the third stage of hiv infection, known as aids, when their cd4 + t counts drop below 200 cells/µl or they develop an aids-defining opportunistic infection/cancer. the acute hiv infection stage, also known as acute retroviral syndrome (ars), typically presents (in 50% or more of patients) with symptoms 2-4 weeks after seroconversion [1] . symptoms during this stage can last from a few days to several weeks, and fall under 4 broad categories as defined by braun et al. [3] : neurological, constitutional, gastrointestinal, and skin/mucosal (table 1) . typical ars is defined as the presence of a fever plus at least 1 of the 17 most common symptoms associated with ars, or, in the absence of fever, at least 2 of the 17 symptoms listed in table 1 [3] . during the typical presentation of ars, the cd4 + t cell count usually falls rapidly as the virus replicates and may be followed by acute infections or other manifestations. in our patient, both of these possibilities remain viable options. however, because the patient presented to us at the autopsy stage, we are unable to confirm the precise pathogenesis. he was not previously known to be hiv-infected, and was unable to give any specific history to the health care providers. it is possible that the patient had latent hiv infection for many years prior, but was asymptomatic and not cachexic, and developed disseminated cryptococcosis when his immunocompromised status crossed a threshold level with a cd4 + t cell count of less than 100-200 per µl. on average, progression from initial presentation to a cd4 + t cell in this range takes about 10 years. however, higher hiv rna levels are associated with faster decline in cd4 + t cell counts, such that our patient's decline could have occurred in less than 5 years. many individuals in the usa become infected with hiv as early as age 13 years. another possibility is that our patient could have been congenitally infected. while less likely, the possibility also remains that this represents the acute retroviral syndrome presenting 6 weeks or so after initial exposure to hiv. ec state that treatment should proceed in 3 stages: induction, consolidation, and maintenance. induction begins with interval treatments of iv amphotericin b in conjunction with oral flucytosine for a minimum of 2 weeks [7] . after moderate clinical improvement, treatment can move to the consolidation stage, in which fluconazole (400-800 mg daily) is indicated for at least 8 weeks. finally, the dosage of fluconazole can be decreased to a maintenance dose (200 mg/day), which should be taken for at least 1 year [5] . despite advances in both hiv and cryptococcosis treatment, ec remains a major cause of mortality in hiv-positive individuals, with mortality rates for untreated individuals ranging from 22% to 96% at 10-12 weeks, and 100% for untreated cryptococcal meningoencephalitis [8, 9] . cryptococcal meningoencephalitis can result in death by causing cerebellar tonsillar herniation leading to cardiopulmonary collapse, as was seen in our patient. cryptococcal infection is most commonly due to cryptococcus neoformans and cryptococcus gattii. the most common site of infection is the lungs, with macroscopic, tan-to-white nodules that can demonstrate central necrosis. when dissemination occurs, the brain and skin are the most common secondary infection sites. skin lesions can appear as molluscum contagiosum-like raised, umbilicated papules or weeping, ulcerated lesions. brain lesions are often cystic and periventricular. microscopically, immunocompetent patients demonstrate granulomas with multinucleated giant cells containing the fungal organisms. these granulomas can become necrotizing, with surrounding histiocytes. in immunocompromised patients, inflammation is minimal, with expansion within alveolar spaces or production of cryptococcomas in other organs. the fungal organisms are often variable in size, with a thick, refractile-appearing capsule. we present the case of an 18-year-old man with no evident prior clinical disease presenting with symptoms of fever, nausea, dyspnea, and abdominal pain, which were followed 2 weeks later by diffuse lymphadenopathy. he died shortly thereafter and at autopsy showed disseminated cryptococcal infection and hiv positivity. the time from initial presentation to death was only 16 days. the patient's non-specific symptoms in conjunction with his rapid decline made arriving at a correct diagnosis challenging. only during autopsy was the disseminated fungal infection discovered, leading to suspicion of hiv infection. hiv autopsies are not uncommon, but the clinical history is often known beforehand. this case report highlights the importance of considering hiv-related conditions in patients presenting with the discussed array of symptoms. early diagnosis can greatly improve patient outcomes and alert healthcare providers and staff to the need for increased biosafety precautions. clinical suspicion of hiv infection should be high for individuals in at-risk groups, as the presentation can be highly variable or atypical. lack of understanding of acute hiv infection among newly-infected persons-implications for prevention and public health: the nimh multisite acute hiv infection study: ii hiv: cell binding and entry. cold spring harb perspect med frequency and spectrum of unexpected clinical manifestations of primary hiv-1 infection centers for disease control and prevention: aids and opportunistic infections cryptococcosis -guidelines for the prevention and treatment of opportunistic infections in adults and adolescents with report of 12 cases and review of the literature treatment of cryptococcal meningitis in peruvian aids patients using amphotericin b and fluconazole clinical presentation, natural history, and cumulative death rates of 230 adults with primary cryptococcal meningitis in zambian aids patients treated under local conditions long-term mortality and disability in cryptococcal meningitis: a systematic literature review according to the cdc, the most prevalent opportunistic infections for hiv-positive individuals living in the usa are candidiasis of the bronchi, trachea, esophagus, or lungs, coccidioidomycosis, extrapulmonary cryptococcosis, and cytomegalovirus [4] . extrapulmonary cryptococcosis (ec) in immunocompromised hosts can affect any organ in the body, but it most commonly manifests as cryptococcal meningoencephalitis, with associated symptoms including fatigue, headache, altered mentation, stiff neck, and fever [5] . other associated symptoms include skin rash, dyspnea, and cough, which are indicative of disseminated cryptococcal infection [6] . our patient did not have any skin manifestations. the diffuse lymphadenopathy was not investigated because of the unstable condition of the patient and his subsequent cardiac arrest. however, at autopsy, the nodes were seen to be severely involved with cryptococcosis, with no evidence of lymphoma. the guidelines for treatment of none. key: cord-267947-dnv2xl0h authors: gornet, jean-marc; linh tran minh, my; leleu, florian; hassid, deborah title: what do surgeons need to know about the digestive disorders and paraclinical abnormalities induced by covid-19? date: 2020-04-24 journal: j visc surg doi: 10.1016/j.jviscsurg.2020.04.017 sha: doc_id: 267947 cord_uid: dnv2xl0h abstract the symptoms associated with covid-19 are mainly characterized by a triad composed of fever, dry cough and dyspnea. however, digestive symptoms have also been reported; at first considered as infrequent, they in fact seem to affect (to some extent) more than half of patients. the symptoms are mainly manifested by anorexia, diarrhea, nausea and/or vomiting and abdominal pain. even though prognosis is associated with lung injury, digestive symptoms seem significantly more frequent in patients presenting with severe covid-19 infection. digestive forms, which may be isolated or which can precede pulmonary symptoms, have indeed been reported, with diarrhea as a leading clinical sign. the main biological abnormalities that can suggest covid-19 infection at an early stage are lymphopenia, elevated crp and heightened asat transaminases. thoraco-abdominal scan seems useful as a means of on the one hand ruling out digestive pathology not connected with coronavirus and on the other hand searching for pulmonary images compatible with covid-19 infection. no data exist on the interest of digestive endoscopy in cases of persistent digestive symptoms. moreover, the endoscopic surgeons may themselves be at significant risk of contamination. fecal-oral transmission of the infection is possible, especially insofar as viral shedding in stools seems frequent and of longer duration than at the ent level, including in patients with negative throat swab and without digestive symptoms. in some doubtful cases, virologic assessment of stool samples can yield definitive diagnosis. in the event of prolonged viral shedding in stools, a patient’s persistent contagiousness is conceivable but not conclusively established. upcoming serology should enable identification of the patients having been infected by the covid-19 epidemic, particularly among previously undetected pauci-symptomatic members of a health care staff. resumption of medico-surgical activity should be the object of a dedicated strategy preceding deconfinement. coronaviruses are rna-enveloped viruses transmitted from one human being to another. among them, three distinct types of coronavirus are responsible for severe lung diseases. sars-cov was responsible for an epidemic occurring in asia from 2002 to 2004 (sars-cov-1). mers-cov provoked an epidemic in the middle east in 2012. sars-cov-2, which appeared in china and is responsible for the current pandemic, is primarily the virus responsible for the 2019 coronavirus disease, which is why it is known as covid-19. over only a few weeks, the medical community has found itself compelled to learn about the semiology and the short-term natural history of the covid-19 infection; this has been done essentially on the basis of the literature from china, initial epicenter of the epidemic. the mortality rate of the infection has been evaluated at 3.6% in china and at 1.5% of the population contaminated outside of china. according to the projections of the world health organization, the estimated mortality rate throughout the world will be 5.7% [1] . up until now, no curative treatment has been validated. three patient profiles have appeared: pauci-symptomatic infection with initially high viral load; secondary respiratory aggravation on the 10 th day notwithstanding reduced viral load, symptomology suggesting ill-adapted host immune response; and rapidly progressive infection with multi-system organ failure and persistence of high viral load [2] . while the general and respiratory signs are known by one and all, the same cannot be said for digestive organ injury. the objective of this paper is to describe covid-19-related digestive disorders, thereby informing physicians liable to enter into contact with patients presenting these types of symptoms. the coronavirus spike protein is capable of binding to the receptor of the angiotensin-converting enzyme 2 (ace2), entering into the infected cell, and interacting with the serine protease tmprss2, thereby provoking viral replication in the contaminated tissue. ace2 plays a role in regulation of inflammatory response and is strongly expressed in the epithelial cells of the enterocytes of the proximal and distal small intestine. this helps to explain the highly elevated incidence of digestive disorders, particularly diarrhea, associated with the covid-19 infection [3] . in the one reported case of endoscopic biopsy for diarrhea ascribed to covid-19, note was taken of lymphoplasmocytary infiltration and an edema of the chorion as well as ace2 and a cytoplasmic viral capsid protein in the stomach, the duodenum and the rectum [4] . as a result, one may hypothesize the ingestion of contaminated food as a possible avenue of contamination. in the original series by huang et al. reporting a series of 41 patients hospitalized in a hospital center in wuhan, the incidence of digestive disorders seemed anecdotal, limited to diarrhea occurrence in 3% of the patients [5] . another, large-scale chinese study (1099 patients) seemed to confirm these findings, reporting incidence of nausea and/or vomiting (5%) and diarrhea (3.8%) [6] . the study also provided interesting epidemiological data including median age (47 years), comorbidities (23.7%), median incubation time (4 days), medical staff affected (3.5% of the cohort), normal pulmonary imagery in non-severe patient (17.9%), utilization of oxygen therapy (41.3%), need for mechanical ventilation (6.1%) and risk factors for severe forms (age and comorbidities). while possible, a purely digestive form was initially considered as rare. at the outset of the epidemic, there was a reported case of a 22-year-old female patient presenting with isolated febrile diarrhea along with normal blood test and negative fecal culture; on the other hand, chest scan revealed bilateral pneumopathy suggesting covid-19 [7] . throat swab confirmed the diagnosis, and during an 18-day hospitalization the diarrhea progressively improved, with no development of extra-digestive signs. however, recent and consistent data suggest that in point of fact, digestive disorders are more frequent. in a recent retrospective study involving 1141 patients presenting with documented infection, the frequency of initially isolated digestive disorders came to 16%, whereas in 96% of cases, injured lungs appeared on ct-scan [8] . in these patients, the symptoms observed were the following: anorexia (98%), nausea (73%), diarrhea (37%), diffuse abdominal pain (25%), nausea and vomiting (20%), abdominal pain and diarrhea (9%), all of these symptoms (7%). mortality (3.8%) was exclusively associated with acute respiratory insufficiency. it bears mentioning that the diarrhea reported in the different studies was invariably fluid, that the severe hydroelectrolytic disorders secondary to the diarrhea were not clearly described, and that no case of mucohemorrhagic diarrhea secondary to viral infection has been reported. moreover, up until now abdominal pains have not been clearly characterized in the literature, and do not appear specific. the most recent chinese epidemiological data report that the incidence of digestive disorders during infection progression is 79%, including (in descending order): anorexia, diarrhea, nausea/vomiting, abdominal pain and bleeding in the digestive tract [9] . in a cohort study of 305 patients from wuhan, diarrhea with median duration of 4.1 days +/-2.5 days (1 to 14 days) was observed in half of the patients while they were hospitalized. vomiting seemed more frequent in children than in adults. the study by jin et al compared the evolution of a group of 577 patients without digestive symptoms with that of a group of 74 patients having presented with at least one of the three following digestive symptoms: diarrhea (defined as the passage of more than 3 stools a day) with negative fecal culture, nausea and vomiting [10] . familial contamination (31% vs 20%) and a more severe form of the disease (23% vs 8%) entailing a need for in-hospital resuscitation (6.8% vs 2.1%) were significantly more frequent in the "digestive symptoms" group. from a clinical standpoint, fever ≥ 38.5, asthenia, dyspnea and headaches were significantly more frequent in the "digestive symptoms" group. from a biological standpoint, lymphopenia and elevated asat and crp were likewise significantly more frequent in the "digestive symptoms" group. productive cough and elevated ldh were the two main predictive factors for evolution toward a severe form of covid-19 in patients presenting with digestive symptoms. schematically speaking, one may distinguish two clinical presentations in an endemic zone. some patients without any known infection have aspecific, isolated, acute inaugural digestive symptoms suggestive of covid-19 infection. other patients have had covid-19 infection revealed and documented by respiratory signs, and present secondarily with digestive symptoms. in these cases, it is important not to be misled by digestive illness unrelated to covid-19, especially in the event of persistent fever. particular attention should be paid to diarrhea so as to avoid overlooking another possible cause (medication, clostridium difficile-associated disease….). it also bears mentioning that infected patients tend to have a large number of digestive hemorrhages with usual causes; this may stem from a frequently observed need for effective anticoagulation, which is related to a heightened and documented risk of thromboembolic complications associated with covid-19. the main data in the literature are summarized in table 1 . covid-19 viremia seems anecdotal. for example, a study published in nature reports no case of viremia in patients having undergone iterative testing [11] ; these findings are in agreement with previously observed data on middle east respiratory syndrome coronavirus (mers-cov) and severe acute syndrome-related coronavirus sars-cov, which were responsible for other recent epidemics. as concerns the safety of blood donations in patients having been affected by the disease, these data are reassuring. serology seems to be a reliable means of detecting infected patients. a recent study showed that 100% of the infected patients tested presented with positive elisa ig g [12] . even though no validated kit presently exists, this method is clearly likely to emerge in routine practice, particularly as a means of detection in caregivers who may have unknowingly contracted the disease. fecal-oral transmission of the corona virus has been a known fact for a number of years. in a study published in 2003, 100% of patients presented with viral excretion in stool specimens and 67% still had a detectable virus 3 weeks after the onset of clinical symptoms [13] . this has also been reported in covid-19, with a well-documented case of positive rt-pcr results in stools (during 7 days after hospital admission there were also 4 other negative rt-pcr test results, 2 on throat swabs, and the other 2 on sputum) in a patient presenting with non-severe bilateral pneumopathy [14] . moreover, it appears that viral excretion in stool specimens from coronavirus patients is a frequent occurrence. in a study involving 73 patients, rna virus was found in the stools of 53.4% of the population, with viral clearance lasting from 1 to 12 days [4] . that much said, only 43.5% of the patients with positive results in stools had diarrhea. it bears mentioning that while stool analysis showed persistent viral rna, in one quarter of cases the initially positive throat swab became negative. that is one reason why the criterion for healing currently utilized in china (negativization of 2 rt-pcr at an interval of least 24 hours on a throat swab) may soon be reevaluated [15] . on this subject, we do not presently know whether the quantity of residual virus in a patient's stools is associated with persistent contagiousness. for the protection of family members and others residing in the same home, it is nonetheless recommended to proceed to daily cleaning and disinfection (applying ready-to-use concentrated household bleach tablets or an equivalent household disinfectant) of the toilets used by infected patients for as many as fourteen days after disappearance of respiratory symptoms. a number of biological abnormalities have been consistently reported in several studies and need to be better known insofar as they may draw attention to infection in a patient with scant symptomatology. in the series by guan et al [6] , the frequency of the main biological abnormalities was: lymphopenia < 1500/mm 3 (median value 1000/mm3): 83.2%, thrombopenia < 150 000/mm 3 : 36.2%, leucopenia < 4000/mm 3 in the series by luo et al. [8] involving 183 patients with initially isolated digestive disorders, the authors observed leucopenia (mean value 2700/mm 3 ), lymphopenia (mean value 530/mm 3 ), elevated crp (mean value 18.7 +/-6.8 mg/l), minimal hepatic cytolysis (asat 65.8 +/-12.7 and alat 66.4 +/-13.2) and normal renal function. abnormal liver function tests have been reported in most of the studies. in the series by shi et al, it was suggested that patients presenting with symptoms for less than a week showed less elevated transaminase levels than those with symptoms from 1 to 3 weeks [16] . moreover, the abnormalities observed in hepatic tests were more pronounced in patients presenting with a severe infection, particularly those hospitalized in intensive care [17] . in the absence of available histological data, the presence of viral rna in the liver has yet to be demonstrated. one may suppose that liver dysfunction is multifactorial (drug toxicity, inflammatory cascade, hypoxemia). it should nonetheless be noted that notwithstanding the presence of the ace2 receptor in the cholangiocytes, the patients did not present with intrahepatic cholestasis, which is associated with the virus (absence of elevated gamma gt and pal in study patients without preexisting liver disease). as regards intensive care patients, up until now acute liver failure has not been reported as a complicating factor in severe covid-19 infections. that much said, and even though as of now it is not documented in the literature, cirrhotic patients may be particularly at risk of severe covid-19 infection. the afef (société française d'hépatologie) recommends sick leave for cirrhotic patients unable to telework and systematic thoracic ct-scan in the event of any decompensated cirrhosis, the objective being to avoid overlooking possible covid-19 infection. up until now, no relevant data has been reported in the literature. even though frequency of occurrence remains unknown, a clinical picture of banal gastroenteritis can contain scanographic abnormalities such as thickening and/or parietal contrast. in one series, it was estimated that in a systematic analysis of 446 pathology scanners, thickness of the jejunum and/or the ileum was probably infectious in 25% of cases [18] . a recent retrospective analysis of abdominal imagery from patients admitted to hôpital saint-louis in paris with documented covid-19 infection did not seem to show parietal abnormalities (personally obtained data). on the other hand, in some patients presenting with digestive symptoms we observed unusual small-intestinal and/or colic fluidic stasis (figures 1 and 2) . it also bears mentioning that a clinical presentation highlighting digestive disorder may in some cases preclude recognition of a progressive or persistent viral infection. for example, a 67-year-old woman monitored in our unit for metastatic colon cancer who had been undergoing chemotherapy was hospitalized for fever and abdominal pain related to streptococcal septicemia that was complicating the evolution of sigmoid fistula with probable loco-regional recurrence. systematic thoracic slices revealed an aspect typical of covid-19 infection (figure 3 ). thoracic ct scan fostered suspicion of the infection, which was confirmed by throat swab. during questioning, the patient reported a dry cough (2 days) and ansomia with ageusia (4 days). notwithstanding rapid apyrexia (48 hours) under antibiotic treatment, her respiratory state quickly deteriorated, leading finally to her death on d9 of hospitalization. this case illustrates the potential interest of systematic thoracic ct scan in a patient presenting with digestive disorders accompanied by fever. up until now, no data has been reported in the literature. the extreme contagiousness induced by the aerosolization of saliva droplets or coughing during upper gastrointestinal (ugi) endoscopy and the potential contagiousness of stools in lower gastrointestinal (lgi) endoscopy are such that these procedures should be limited to emergency endoscopy [19] . in clinical practice, the indications are as follows: digestive hemorrhage with deglobulization, impaction of foreign bodies, symptomatic digestive stenosis and biliary obstruction, necrosectomy or drainage of pancreatic fluid collection, sigmoid volvulus. even though the société française d'endoscopie digestive recommends continuation (to the greatest possible extent) of colonoscopy in the event of positive fecal blood test and as a means of exploring iron deficiency anemia, it seems illusory to hope to apply these recommendations in highly endemic areas. in ile de france, for example, virtually all public and private structures limit implementation of these procedures to dire, life-threatening emergencies. with our current level of knowledge, cibd patients are not more at risk of this infection than the general population [20] . up until now, no case of severe covid-19-related infection has been reported in cibd patients exposed to either immunosuppressive therapy (azathioprine, methotrexate) or a monoclonal antibody (anti-tnf, ustekinumab, vedolizumab). it is consequently recommended not to suspend preventive immunomodulatory treatments; discontinuation would place the patients at risk of progressive disease relapse with an additional loss of opportunity due to the difficulties of obtaining emergency treatment during a sanitary crisis. a worldwide register of cibd patients with covid-19 infection has been set up by the ioibd (international organization for the study of inflammatory bowel diseases), which should be providing supplementary details over the months to come. two studies seem to that show cancer patients at an advanced age when diagnosed have a particularly high risk of covid-19 and that their prognosis may be less favorable than for non-cancer patients [21, 22] . that much said, among the 3114 patients analyzed in the two studies, only 30 presented with cancer, including 11 undergoing treatment (chemotherapy +/-immunotherapy n=5, chemotherapy and/or surgery during the previous month n = 4, radiotherapy n = 2) and not a single reported case of digestive cancer. even though these data remain highly fragmentary, digestive cancer patients are probably significantly at risk of covid-19 infection, particularly those currently undergoing intravenous chemotherapy, who risk contamination during their care pathway (repeated hospitalizations, imaging examinations and blood tests in medicalized structures…). moreover, given that the mean age of cancer diagnosis in the most frequent tumor locations approximates 70 years, whether undergoing treatment or being monitored this population is clearly particularly at risk of developing a severe infection. for example, among the 6 infected patients in our cohort (mean age 62.6 years, ongoing chemotherapy n = 5, palliative home treatment n = 1, colorectal cancer n = 4, pancreatic cancer n =1, esophageal cancer n =1), 3 died of respiratory failure and 2 required oxygen therapy. it is mentioned in the french national thesaurus of digestive cancerology that existing chemotherapy modalities ought to be revised, taking into closer account the risk/benefit ratio (expert agreement). this is singularly relevant, especially insofar as intensive care admission criteria for patients presenting with severe covid-19 infection are exceedingly strict. that is one reason why patients presenting with incurable metastatic cancer have limited access to mechanical ventilation. it is also necessary to customize treatments according to the level of contamination in the health care structure accommodating the patient. as regards these two types of "at risk" population, in the event of covid-19 infection the getaid (groupe d'etude thérapeutique des affections inflammatoires du tube digestif) and the tncd (thésaurus national de cancérologie digestive) recommend suspension of all treatment, until full resolution of symptoms. even though this approach has yet to be validated, it would seem judicious immediately prior to resumption of treatment to ascertain negativation of at least a throat swab either at the conclusion of quarantine (ambulatory patient) or following resolution of symptoms (hospitalized patient). in the overwhelming majority of cases involving any type of digestive pathology, teleconsultation is preeminent. even though there have been no dedicated studies on the subject, its acceptability by actively monitored patients seems satisfactory, and use of the telephone appears to suffice (personally obtained data). among patients necessitating hospitalization, in some centers systematic search for covid-19 infection is carried out, even in the absence of symptoms, to avoid potential risk of dissemination in a hospital structure. the covid-19 epidemic necessitates continual updating of our knowledge pertaining to the infection. a multiplicity of publications and their free availability online illustrates the intense activity of the medical community on this subject. even though they are not clinically highlighted, digestive disorders are in all likelihood more frequent than initially reported. they can precede other symptoms or be present in isolation. biological abnormalities, particularly lymphopenia, moderately elevated crp and transaminases, can suggest clinical diagnosis. digestive imagery is only marginally contributory. that much said, pulmonary injury can be fortuitously discovered on low thoracic slices in abdominal ctscan. that is one reason why some authors have discussed the possible interest of systematic thoracic ct-scan when abdominal injury is indicated, especially in the event of fever. resumption of medicalsurgical activity as the epidemic recedes will be difficult, given (a) the multiplicity of endoscopic procedures and surgical interventions requiring rescheduling and (b) the numerous delays in treatment of non-covid patients who, as they are confined, have not been consulting during the height of the epidemic. following resolution of the first epidemic wave, a screening strategy aimed at limiting the risk of new contaminations will call for discussion on patients suffering from digestive pathology necessitating hospital treatment. real estimates of mortality following covid-19 infection clinical and virological data of the first cases of covid-19 in europe: a case series diarrhoea may be underestimated: a missing link in 2019 novel coronavirus evidence for gastrointestinal infection of sars-cov-2 clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of coronavirus disease 2019 in china sars-cov-2 induced diarrhoea as onset symptom in patient with covid-19 don't overlook digestive symptoms in patients with 2019 novel coronavirus disease (covid-19) review article: gastrointestinal features in covid-19 and the possibility of faecal transmission epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (covid-19) with gastrointestinal symptoms virological assessment of hospitalized patients with covid-2019 molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study covid-19 disease with positive fecal and negative pharyngeal and sputum viral tests responding to covid-19: perspectives from the chinese society of gastroenterology radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study liver injury in covid-19: management and challenges etiology of small bowel thickening on computed tomography prevention of nosocomial sars-cov-2 transmission in endoscopy: international recommendations and the need for a gold standard what should gastroenterologists and patients know about covid-19? sars-cov-2 transmission in patients with cancer at a tertiary care hospital in wuhan, china cancer patients in sars-cov-2 infection: a nationwide analysis in china key: cord-294468-0v4grqa7 authors: kasilingam, dharun; prabhakaran, s.p sathiya; dinesh kumar, r; rajagopal, varthini; santhosh kumar, t; soundararaj, ajitha title: exploring the growth of covid‐19 cases using exponential modelling across 42 countries and predicting signs of early containment using machine learning date: 2020-08-04 journal: transbound emerg dis doi: 10.1111/tbed.13764 sha: doc_id: 294468 cord_uid: 0v4grqa7 covid‐19 pandemic disease spread by the sars‐cov‐2 single‐strand structure rna virus, belongs to the 7(th) generation of the coronavirus family. following an unusual replication mechanism, it’s extreme ease of transmissivity has put many counties under lockdown. with uncertainty of developing a cure/vaccine for the infection in the near future, the onus currently lies on healthcare infrastructure, policies, government activities, and behaviour of the people to contain the virus. this research uses exponential growth modelling studies to understand the spreading patterns of the covid‐19 virus and identifies countries that have shown early signs of containment until 26(th) march 2020. predictive supervised machine learning models are built using infrastructure, environment, policies, and infection‐related independent variables to predict early containment. covid‐19 infection data across 42 countries are used. logistic regression results show a positive significant relationship between healthcare infrastructure and lockdown policies, and signs of early containment. machine learning models based on logistic regression, decision tree, random forest, and support vector machines are developed and show accuracies between 76.2% to 92.9% to predict early signs of infection containment. other policies and the decisions taken by countries to contain the infection are also discussed. coronaviruses, though uncommon, are serious pathogens responsible for infections that posit flu-like symptoms in infected individuals. these symptoms sometimes resemble the cold and cough symptoms caused by the rhinovirus. recently, the family has added its seventh generation coronavirus -sars-cov-2 (chengxin et al., 2020) . the virus shares 79% identity to severe acute respiratory syndrome (sars) and 50% identity to middle east respiratory syndrome (mers) epidemic outbreak in 2003 and 2012 (salute, 2020) . sarc-cov-2 that causes covid-19 mutated to transmit from animal to human. this virus is believed to have transferred to humans through bats from a meat market in wuhan, china (rajendran et al., 2020; shereen et al., 2020) . in march 2020, who declared the covid-19 to be a pandemic; a pandemic being described as an infection that has spread across countries and international borders rather than within a local region or neighbouring countries. the sarc-cov-2 is a deadly corona virus that is transmitted readily between humans and already infected more than 530,000 people all over the world in 198 countries as on 26 th march 2020 which led global shutdowns (who, 2020) . the fatality rate has varied among countries and age groups. until june 2020, the fatality rate averaged 5.5% with italy recording the highest of 14.49%. the fatality rate of us, germany, and india were 5.56%, 4.72%, and 2.86% respectively until june 2020 (our world in data, 2020) . of the total deaths, less than 5% belonged to the age group of less than 45 years thereby indicating that the younger population is much more resilient to the covid-19 (worldmeters, 2020) . while these fatality rates are significantly less than those of mers (34.4%) and sars (9.5%) (petrosillo et al., 2020) , covid-19 has severe transmissivity because of the possibility of asymptomatic people being carriers and spreaders of the virus (daw et al., 2020) . the reproduction number r 0 for sarc-cov-2 has been found to be between 2.06 to 2.52 (sheng et al., 2020) . a value of r 0 greater than 1 indicates that the disease can invade the human population and higher the value, the easier is it's spread. sarc-cov-2 is the largest single-strand rna virus known to the humankind; while other viruses have a single protein spike for attachment to the human cell, this coronavirus family has 10 to 12 spike proteins, which makes it easier for the virus to attach itself to the ace-2 protein in humans (paraskevis et al., 2020) . the virus follows an unusual double step replication mechanism, which leads to high rates of proliferation (luan et al., 2020) . the this article is protected by copyright. all rights reserved incubation period is typically 2 to 14 days, and the infected person often does not have serious symptoms, rather showing common symptoms associated with flu and pneumonia (rodeny, 2020) . general symptoms of pneumonia include fever, cough, chest pain, shortness of pain, fatigue, headache, myalgia, and arthralgia (sattar sba, 2020) . in addition to symptoms of pneumonia, covid-19 infected individuals may experience a loss of taste or smell, nausea, congestion, and diarrhoea (cdc, 2020) . there are a few drugs that are being recommended and used to manage the symptoms of covid-19, but there has, as yet, been no vaccines that are proven to be effective against the coronavirus family, including covid-19 (sexton et al., 2016; gautret et al., 2020) . in the absence of vaccines, it is imperative to check transmission of the virus by alternative ways (dey et al., 2020) . policy changes in pandemic and epidemic situations involve social distancing, lockdowns, travel restrictions, awareness campaigns etc. it has been speculated in past research that environmental conditions of countries like temperature and humidity also sometimes play a significant role in controlling pandemics (lin et al., 2020) . quantitative covid-19 impact analyses are scarce in literature, given the recency of the pandemic and more studies in this area are necessary, given the seriousness of the infection. epidemics are assumed to have an exponential growth at an early stage and the number of infections reduces over time, due to interventions like lockdowns, travel restrictions, awareness programs, etc. mathematical modelling studies using exponential growth analysis coupled with machine learning could provide a better prediction model for covid-19 transmission (keeling and danon, 2009; siettos and russo, 2013; victor, 2020) . such models must incorporate the various precautionary measures taken during the viral outbreak. the objective of the research is to develop a mathematical model using exponential growth analysis coupled with machine learning, to predict worldwide covid-19 early containment signs. models have been developed based on data collected from 42 countries. the objectives of this work are twofold. first, it seeks to identify countries that were successful in early containment of the covid-19 virus. secondly, the research aims at building supervised machine learning models with high accuracies for predicting signs of early containment with infrastructure availability, environmental factors, infection severity factors, and government policies of countries as independent variables. in the process of modelling, the significance of the above variables in containing the infection at early stages is this article is protected by copyright. all rights reserved also studied. this report also includes a discussion on other activities undertaken by the governments of various nations to flatten the infections curve and their corresponding effectiveness. covid-19 is believed to have originated in an animal meat market in wuhan, china and it is thought to have been transmitted from bats (shereen et al., 2020) . within few months, the virus has rapidly spread across the world, through transmissions of fluids and aerosol particles between humans. initially, all diagnosed cases outside china had a travel history to the wuhan market. soon, community transfer caused exponential increases in infections in countries like italy, us, uk, korea, japan, etc. the ability of the sars-cov-2 virus to double replicate with the spike protein, has posed significant challenges to the development of vaccines (shereen et al., 2020) . while hydroxychloroquine and azithromycin have been recommended by some researchers, to treat covid-19-infected people, there haven't been too many clinical trials to validate the claim (gautret et al., 2020) . thus, until a scientifically validate cure or vaccine is developed, countries have to rely on preventive measures to contain the spread. this, in turn, depends on epidemiological studies that can predict spreading patterns so that policymakers can take appropriate protective measures. several viruses including sars have been reported to be vulnerable to hot temperatures, which results in differences in spreading patterns across geographic locations (zhang et al., 2020) . however, such geographic variations have not yet been analysed for covid-19. other factors like government policies and interventions, infrastructure availability, and the severity of the infection itself can affect the ability of a country to contain epidemics and pandemics. this research seeks to explore all the above factors. the climatic conditions such as temperature and humidity play an important role in both airborne and aerosol virus transmissions. the 30-year human relationship with the influenza virus has proven that the mortality rate is directly related to temperature and humidity (lowen and steel, 2014) . hence, in order to minimize transmission of diseases, isolation wards in hospitals generally tend to have optimized pressure, temperature, and humidity (who, 2014) . research on the virus in the diamond cruise ship off the coast of japan showed that a one-degree rise in temperature and a one percent increase in pressure this article is protected by copyright. all rights reserved could reduce the reproduction number r 0 down by 0.0224 -0.0383. it must be mentioned that the generalizability of the study is questionable because the ship was a contained environment and the results may not apply to the real world (sheng et al., 2020) . certain studies in china and indonesia have investigated the relationship between the temperature and the spread of infection and resultant deaths and have reported a low to medium level of correlation (tosepu et al., 2020; yueling ma et al., 2020) . relative humidity was found to have low to no correlation with infection spread or deaths. global warming has also been a reason for recent temperature increases and certain studies indicate that this can reduce flu based viral infections (national research council, 2001; actuaries, 2010; dincer et al., 2010) . however, these statements need to be further validated. while the spread of virus may be affected by climatic conditions, once the virus enters the human body, it is independent of the outside environment. however, since the virus lives outside the human body for a period of at least 12 hours under normal conditions (richard, 2020) , it is necessary to study the effects of the environmental on the spreading patterns itself. social distancing, although a new terminology for the 21 st century, is not a new approach to epidemic control. it was used by the united kingdom in 1918 to control the influenza virus outbreak that caused about 100 million deaths. social distancing involves the avoidance of mass gatherings and distancing of at least six feet between people. such measures are combined with enhanced personal hygiene through regular hand wash, and wearing a protective mask for flu-like outbreaks (yu et al., 2017; leung et al., 2018) . this is done primarily because flu causing viruses are spread through aerosols generated from saliva and nasal fluid, which can be transmitted across distances as much as three feet. the average lifetime of covid-19 viruses in the outer environment is believed to be about 12 hours, which increases transmissivity because aerosols from infected people can settle on doorknobs, lifts, transports, hotels, malls etc. and stay active for a long time, thus increasing the window of transmission. direct physical contacts, such as hand-shaking, are also avenues of transmission of the virus. the reduction of social contact has been proven to significantly reduce flu-like diseases (maharaj and kleczkowski, 2012) . the closure of schools and malls flattened the this article is protected by copyright. all rights reserved spread curve during the influenza pandemic in 2009 (rashid et al., n.d.; moh, 2014) . thus, governments worldwide have stressed on social distancing and quarantining measures for at least 14 daysthe typical incubation period of covid-19 virus -to contain its spread (prem et al., 2020) . lockdown is a preventive strategy taken by local, central or global administration during the spread of epidemic or pandemic diseases and involves stopping transportation between cities, provinces or counties. the world has so far seen four major pandemics, viz., plague in the 14 th century, influenza in 1918, sars in 2009, and the current covid-19 in 2019 as reported by who (porta, 2008; east et al., 2020; pi, 2020) . in all these cases, lockdowns were implemented by various countries to control the outbreaks. china announced lockdown as early as january 2020, to flatten the curve of the covid-19 infections over time. in march, most countries around the globe announced lockdowns of local transport, office, industries, city and national borders to contain the virus (callaway et al., 2020) . although quarantine centres for the infected are available in hospitals, large-scale infections necessitate self-quarantines and lockdown measures, in addition to the hospital-based quarantines (wuhan, 2020) . during epidemic and pandemic viral outbreaks, the availability of and access to health care infrastructure such as hospitals, beds, healthcare workers, clinical equipment, first aid kits, ventilators, and protective equipment are vital to pandemic management (bambas and drayton, 2000; persoff et al., 2018) . during the massive influenza outbreak of 1918, even developed countries had inadequate health care infrastructure, which further expanded the outbreak (george, 2008). the ebola outbreak in west africa became uncontrollable due to lack of infrastructure facilities (paweska et al., 2017) . after the outbreak, who in south africa had asked the hospitals to report their available facilities to plan for future infections optimally (murrin, 2018) . innovative measures have been recommended, to create necessary healthcare infrastructure during pandemic and epidemic situations by converting schools, colleges, theatres, and stadiums into hospitals and quarantine centres (wimberly, 2018; nuzzo et al., 2019) . health care workers supported by ngos, youth, and volunteers also play a significant role in containing outbreaks (itzwerth, 2013) . hence studying health care this article is protected by copyright. all rights reserved infrastructure availability across countries can predict covid-19 containment at an early stage. predictive modelling using machine learning and growth models can provide actionable insights to policy makers and governments to contain epidemic and pandemic infections (thompson et al., 2019) . during the onset of an epidemic, it is crucial to use exponential growth models to understand the infection rates and with proper policy implementations and behavioural changes among the susceptible group of the population, the slope reduces and the curve flattens over time (keeling and danon, 2009 ). for other outbreaks like smallpox, ebola, sars, and influenza, various studies have used mathematical and statistical modelling to understand the growth of infections (dietz, 2002; nishiura, 2011; kerkhove and ferguson, 2020) . in fact, the centres for disease control and prevention has an exclusive book with established procedures for analysing disease outbreaks, stressing on the importance of the such modelling studies. (dicker, 2006) in outbreaks, epidemiologists generally use the exponential growth model at the onset of an outbreak and proceed with prediction and classification techniques like regression, decision trees, neural networks deep learning, etc. to forecast outbreaks. (sameni, 2020; victor, 2020) . there are few studies on modelling and predicting containment of covid-19 so far (lin et al., 2020; prem et al., 2020) . the research work reported in this paper, sought to integrate crucial variables concerning infrastructure, environment, policies, and severity of the disease to predict initial signs of containment. the study used a machine learning and exponential growth model. the variables used as part of the predictive mode were, doctors per 1000 population, beds per 1000 population, average temperature, average humidity, days since official lockdown, percentage of lockdown days, total cases per million population, deaths per million population, days since the first contact, and percentage of serious cases of infected. data associated with the variables were collected from different official sources for a total of 42 counties with respect to covid-19 infections as on 26 th march 2020. this accounts for 448,989 covid-19 cases comprising of 84.78% of the total infections this article is protected by copyright. all rights reserved worldwide. the daily number of infections, recovery, and deaths were collected from the website of the who. the data for infrastructure-centred variables like the number of hospitals and the number of doctors were taken form (world bank, 2020) . environmentbased variables like average temperature and humidity since the onset of covid-19 was taken from (weather underground, 2020). day-wise covid-19 case distributions extracted from who were used to identify countries that showed sign of containment of the virus based on a novel exponential growth modelling approach. raw data from the sources were also consolidated and the variables physicians per thousand individuals, hospitals per thousand individuals, percentage of lockdown days since the first contact, cases per million population, deaths per million population, days since the first case, serious cases per thousand infections, average temperature since the first infection, and average humidity since the first infection were calculated to train the machine learning models. most epidemic and pandemic diseases grow exponentially in the initial stages of the outset in a country (ma, 2020) . a popular modelling technique that demonstrates this is the susceptible-infectious-recovered (sir) model (kermack et al., 1927) . if s denotes the fraction of susceptible individuals to a pandemic, i indicates the fraction of infectious people, r is the fraction of recovered patients, β indicates the transmission rate per infectious individual, and the recovery rate is denoted by γ, the infectious period is exponentially distributed with a mean of 1/ γ as shown below. linearizing this about the disease-free equilibrium, we get the following. hence from the above expression, if − > 0, then the infection function i(t) grows exponentially about the disease-free equilibrium point. in addition to this, at the onset of the infection, ≈ 1 and hence the incidence rate = also grows exponentially. hence, modelling the initial stages on a pandemic like covid-19 is both relevant and crucial in understanding the growth of the infection. although sub-exponential and polynomial modelling have worked in cases of outbreaks like ebola, hiv, and foot and mouth diseases (chowell et al., 2016) , they generally work well with proceeding generations. for pandemics like covid-19, the exponential growth model is relevant and the use of least-squares at the initial stages can afford precise insights. figure 1 shows the analysis plan to achieve the objectives of the research. this article is protected by copyright. all rights reserved the exponential growth model assumes that the onset of any outbreak follows an exponential distribution. however, due to government interventions, medical innovations, behavioural changes etc, at a later stage, the growth curve flattens and rate of infections gradually reduces (kermack et al., 1927) . to identify such signs, we looked at the infections in the last seven-day period and the deviation of the data points from the modelled exponential curve was captured using the mean absolute percentage error metric. based on the errors and the direction in which the actual data points were to the modelled growth curve, the countries were classified according to whether they showed initial sign of containment or not. in line with the objectives of the study, classifiers were built based on a set of independent variables to predict if a country that has covid-19 infections showed early signs of infection containment as a reflection of policy implementations and behaviour changes. logistic regression was used to understand the list of independent variables significantly affecting the infection containment and their corresponding importance in the model. then, to predict signs of early containment, machine learning algorithms like logistic regression, decision trees, random forest and support vector machines were used and their corresponding accuracies are compared. for all models, cross-validation was done in 5 folds to address overfitting. logistic regression by le cessie and van houwelingen, (1992) is a generalized linear model (glm) and is one of the most widely used classifiers. according to (kondofersky and theis, 2018) , when there is binary response, as in this research, by using logistic regression one typically aims at estimating the conditional probability . as with simple linear regression, bearing equation = + , estimating the dependent variable y directly, the logistic regression estimates p( = 1) using the following equation. this article is protected by copyright. all rights reserved as with multiple linear regression, logistic regression can also handle multiple independent variables and its probability estimate can be represented as follows. = 1 1 + −( 1 1 + 2 2 + 3 3 ……+ the conditional probability ( = 1| = ) can be calculated using the odds ratio /(1 − ). the significance of the beta coefficient values ( 1 , 2 , 3 , … , ) in the above equation can be tested to see if their corresponding independent variables ( 1 , 2 , 3 , … , ) are influencers of the dependent variable. a wald test is generally conducted to evaluate the statistical significance of the coefficients in the model. since logistic regression falls under the category of glm, the significance of each independent variable in predicting the outcome of the dependent variable, sign of early containment, can be studied. a decision tree is a decision support model that illustrates the consequences, chance, and event outcomes of certain decisions. decision trees are used as a predictive model to make statistical conclusions about an item's target value, based on observations. in this tree structure, leaves represent class labels and branches represent conjunctions of features that lead to those class labels. there are both classification trees where the response variable takes on a set of categorical values and regression trees where the response variable takes on a set of continuous values. the collective name for such trees is classification and regression trees (cart), first introduced and developed by (breiman et al., 1984) in classification and regression trees. decision trees use two metrics namely entropy and information gain to arrive at the final tree. entropy is the measure of the total amount of uncertainity in the dataset and is given as follows: s -the data set for which entropy is to be calculated cset of classes in the data set s p(c)ratio of number of elements in class c to the number of elements in set s this article is protected by copyright. all rights reserved when the entropy value is equal to zero, the dataset s is perfectly classified. the information gain metric is defined as the measure of the difference in the entropy from before to after the dataset s is split based on an atribute a and is given as follows. step 1: compute entropy for the dataset step 2: for every feature in the dataset, compute the following i. calculate the entropy for all the categorical values ii. find the average information entropy for current attributes iii. calculate the gain for curret attributes step 3: select the attribute with the highest gain step 4: repeat from step 1 till the desired tree is achieved introduced by (breiman, 2001) , random forest is a statistical supervised machine learning technique that we used for both regression and classification. this is an ensemble learning technique that uses an averaged combination of many decision trees for the final prediction. the technique of averaging a statistical machine learning model is called bagging and it improves stability and avoids overfitting (hastie et al., 2009) . normally, decision trees are not competitive to the best-supervised learning approaches in terms of prediction accuracy since they tend to have high variance and low bias. this is because building two different decision trees can yield in two different trees. bagging is therefore well suited for this article is protected by copyright. all rights reserved decision tress since it reduces the variance. the idea behind random forests is to draw bootstrap samples from the training data set and then build several different decision trees on the different training samples. this method is called random forest because it chooses random input variables before every split when building each tree. by doing this, each tree would have reduced covariance, which, in turn, would lower the overall variance even further (hastie et al., 2009) . the random forest algorithm has two stagesrandom forest creation followed by random forest prediction. the steps involved in the stages are as follows. step 1: randomly select 'k' features from the total 'm' features available in the dataset where k << m step 2: using the best split point, calculate the node 'd', among the selected 'k' step 3: split the node into daughter nodes using the best split step 4: repeat steps 1 to 3 until 'l' nodes are reached step 5: repeat steps 1 to 4 for 'n' number times to create a forest of 'n' number of trees stage ii: random forest prediction step 1: using the features and applying the rules of randomly selected decision tree, predict the outcome and store it as a predicted target step 2: calculate the votes for each predicted target step 3: the highest voted predicted target will be the prediction of the random forest algorithm the objective of support vector machine (svm) is to find a line that best separates the data into multiple groups. this is achieved by an optimization process supported by the data in the training set. these instances are called support vectors and they form a crucial role in the classification process (flake and lawrence, 2002) . finally, few datasets can be separated with just a straight line. sometimes a line with curves or even polygonal regions must be marked. this is achieved with svm by projecting the data into a higher-dimensional space to draw the lines and make predictions. svms calculate a maximum margin around the boundary that ultimately results in a homogenous partition. the ultimate goal is to establish a this article is protected by copyright. all rights reserved margin as wide as possible. in order to so, a lagrange multiplier has to be constructed as follows and maximized. ( , ) = + table 3 shows the result for logistic regression with early containment as the dependent variable. of all the independent variables, the availability of beds in hospitals and the percentage of lockdown days significantly and positively affected the signs of early this article is protected by copyright. all rights reserved containment. other variables did not significantly influence the dependent variables. the model had an accuracy of 78.57% in the classification. the true positive and false negative rates were found to be 78.6% and 21.6% respectively. precision and recall values were 0.788 and 0.786. the f1 score and roc values were found to be 0.786 and 0.755 respectively. a j48 decision tree was constructed for predicting early infection containment with the independent variables listed in figure 1 . the batch size was set to 10 and a confidence factor was selected as 0.25. the minimum number of objects on the tree was set as 2. the accuracy of the tree was found to be 80.95%. the variables in the decision tree were percentage lockdown days, days since official lockdown, and death rate per million population. the decision tree is shown in figure 4 . the true positive and false negative rates were found to be 81% and 25.4% respectively. precision and recall values were 0.857 and 0.81. the f1 score and roc values were found to be 0.796 and 0.852 respectively. a random forest ensemble algorithm was created with 100 combined trees. the batch size was selected as 10 and the depth of the trees was set to unlimited. other metrics for the random forest algorithm are given in table 4 . this model reported a high accuracy figure of 92.9% in correctly classifying countries that showed signs of early containment. the true positive and false negative rates were found to be 92.9% and 8.1% respectively. precision and recall values were 0.929 and 0.929. the f1 score and roc values were found to be 0.928 and 0.993 respectively. in order to make predictions for signs of early containment, an svm was modelled this article is protected by copyright. all rights reserved on 5-fold cross-validation with the data for all the algorithms and models, it can be inferred that the random forest design produces the minimum error and maximum accuracy as reported in table 4 . it outshines all the other machine learning algorithms constructed in the study. j48 decision tree, logistic regression and svm produce almost similar levels of accuracy in predicting the sign of containment of covid-19. this research is one of the first of its kind to integrate exponential growth modelling with machine learning techniques for predicting the spread of covid-19. the research presents machine learning models based on variables such as infrastructure, environment, policies, and the infection itself, to predict early signs of containment in the country. for the purpose, disease data from 42 leading countries in covid-19 infections were taken and exponential growth modelling was used to see if the countries showed signs of containment. then with the sign of the early containment of the infection as a dependent variable, supervised machine learning predictive models including logistic regression, decision tree, random forest, and support vector machine were developed. this research can directly be of use to countries and policymakers to understand if their proposed interventions are effective in containing infections even during early stages. (tosepu et al., 2020; yueling ma et al., 2020) . however, the long-term effect of environmental factors on the infection rates may prove to be significant. decision tree analysis also shows that early signs of containment are possible if the number of lockdown days is at least 33.7% of the days since the first contact to contain the infection. if that is not the case, countries show recovery signs if the lockdown is at least 10 days or more. for countries with a lockdown period less than 10 days, variable depicting the number of deaths per million population plays a significant role in containing the infection. this variable is indirectly related to the health care infrastructure of countries like beds, physician, ventilators, icus etc. hence in any pandemic situation, governments must be proactive and frame policies even at the onset, thereby reducing the risk of spread, which would ultimately lead to early containment. this also emphasises on the need for resilient health care infrastructure to contain infections at an early stage. the machine learning models random forest and support vector machines were able to classify the countries with respect to their signs of early containment with an accuracy of 92.9 and 76.2 percentages, respectively, proving random forest to be the best machine learning algorithm for the problem studied. although this research applies data from only 42 countries, the proposed models with their corresponding hyper parameters can be extended to predict early containment for the other countries as well. similarly, although these models were built only for the covid-19 pandemic, they can serve as a base for other future pandemics that have similar characteristics and reproduction numbers thereby giving governments the necessary information to take timely actions to protect both people and the economy. this article is protected by copyright. all rights reserved act called covid-19 act which has proven to be effective to contain the infection (library of congress, 2020). the number of hospital beds per 1000 population of austria was also high, which facilitated early recovery. chile has implemented sanitary barriers and intense screening mechanisms to track and quarantine the infected (us embassy, 2020). in addition to tough quarantine measures, denmark closed down schools and also announced lockdown in march. employers were also instructed to not cut salaries of the employees on quarantine thereby encouraging social distancing and hence containing the infection (carstensen, 2020a) . japan, south korea, and singapore did not announce any lockdowns. south korea used processes that led to early detection of the covid-19 and quarantining the infected, thus stopping spread. they also predicted the movement of viruses and tactical interventions were taken to minimize spread (npr, 2020). singapore had a ready infrastructure with isolation wards in place during the sars outbreak and was readily equipped, which led to early containment of covid-19. strong community engagement messages and communications from the government also led to better pandemic management in singapore (fisher, 2020a) . most other countries that showed early signs of recovery rigorously followed lockdowns, social distancing, travel restrictions, and testing to contain infections. another reason for the countries like japan, korea and austria to contain the infection was the presence of availability of strong health care infrastructures in these countries to address the infections. the various actions taken by the government to control the transmission of covid-19 are shown in table 5 . countries like italy, brazil, india, malaysia, pakistan, united kingdom etc. do not have the necessary health care infrastructure to support mass admission of covid-19 patients and hence need to rely on intense lockdowns to contain the infections. the increase in the number of covid-19 cases in the us and the inability to contain it is also due to late lockdown decision of the government post-outbreak. the percentage of lockdown days since the first infection continues to be low for these countries to be on a recovery path against the infection. with time, there is a high probability that the infection will be contained. however, in the long run, these countries must invest in improving health care facilities to reduce causalities during pandemics. countries must be prepared for epidemics and pandemics and proactive policies and infrastructure as in the case of singapore can save more lives than reactive measures. it is evident that covid-19, unlike sars, will not be controlled by environmental factors and any future outbreaks will still rely on healthcare infrastructures, timely lockdowns, and social distancing for containment. this article is protected by copyright. all rights reserved there is no conflict of interest with the authors. no funding received. the data is openly available in world health organisation report. the research confined to the highest level of ethics. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved gesley, 2020) brazil employees at the airport were asked to wear a mask. borders were closed for flights from affected countries (cdcp, 2020) canada all travellers were forced to self-isolate for 14 days upon entry to control the outbreak (gc, 2020) chile screening in the airport was enhanced and people with symptoms were iran followed strict social distancing and lockdown (duddu, 2020) ireland invested in massive testing facilities. treated all patients equally irrespective of their income strata. all hospitals operated on a not for profit basis (bbc, this article is protected by copyright. all rights reserved 2020) used technology to track the movement of infected individuals with their mobiles and quarantined the people who came in contact with the individual (lomas, 2020) italy though italy closed borders during the onset, lack of proper testing facilities caused a massive outbreak. this was followed by a strict lockdown (gary, 2020) japan managed the outbreak with rules and excellent medical infrastructure. (japan, 2020) luxembourg quarantined people over 60 years to reduce casualties (piscitelli, 2020) malaysia the banned entry of people from infected countries followed with additional screening measures in the airport. promoted personal hygiene and eventually ended with a lockdown. (world, 2020a) netherlands travellers returning from affected countries were advised to visit doctors and medical facilities if symptoms were felt. post outbreak, the country went under lockdown. (world, 2020b) norway travel bans and closure of schools, public services like gums, malls, theatres etc. (norway panorama, 2020) formed a team to monitor situations and take necessary actions on a daily basis. (pakistan, 2020) portugal employed strict lockdown (ivo oliveira, 2020) qatar proper tracking, and strict screening and testing of travellers (health, 2020) republic of korea proactively built a centralized testing and quarantine facility before an outbreak in the country. china's reports triggered this action (beaubien, 2020) romania lockdown and border closing (gherasim, 2020) singapore with previous experience from sars pandemic, the country had a proper infrastructure facility with negative pressure room for pandemic control. the testing was done rigorously and the infected were not let back into society. migrants from other countries were not allowed to work until the pandemic is controlled. (fisher, 2020b) slovenia used innovative ways to spread covid-19 control messages before going into lockdown. (slovenija, 2020) south africa immediately implemented entry and exit to affected countries. declared as a this article is protected by copyright. all rights reserved national disaster and went for the lockdown to prevent a major outbreak (fihlani, 2020) spain local movement controlled by social distancing. travel to an affected country completely banned. enhanced medical attention at arrival to control the spread. (kate mayberry, 2020) after closing school, colleges and non-essential business, the country used their military and civilian support to enhance infrastructure and healthcare needs to contain the infection (keystone, 2020) the united kingdom people with symptoms were asked to self-quarantine. cancelled overseas travels and only tested people who were admitted. followed social distancing, lookdown, isolation and house quarantined. the country did not force people for testing. (yong, 2020) united states of america enforce travel restriction and implemented mandatory quarantine in new york. a level of screening and lockdown was implemented. 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bacterial pneumonia homology-based identification of a mutation in the coronavirus rna-dependent rna polymerase that confers resistance to multiple mutagens 2020: estimation of the reproductive number of novel coronavirus ( covid-19 ) and the probable outbreak size on the diamond princess cruise ship : a data-driven analysis covid-19 infection: origin, transmission, and characteristics of human coronaviruses mathematical modeling of infectious disease dynamics 2020: statnews, ecuador becomes the latest country to eye compulsory licensing for covid-19 products gov of slovenija accepted article this article is protected by copyright. all rights reserved 2019: detection , forecasting and control of infectious disease epidemics : modelling outbreaks in humans , animals and plants 2020: correlation between weather and covid-19 pandemic in jakarta 2020: u.s embassy in chile, global level 4 health advisory 2020: mathematical predictions for covid-19 as a global pandemic 2020: wunderground infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care covid-19) outbreak situation report of the who-china joint mission on coronavirus disease 2019 (covid-19). world health organization who, 2020c: report of the who-china joint mission on coronavirus disease 2018: digitalcommons @ unmc pandemic planning : estimating disease burden of pandemic influenza to guide preparedness planning decisions for nebraska medicine physicians (per 1,000 people) [online] available at accepted article this article is protected by copyright 2020a: garda world, malaysia: new travel restrictions introduced february 6 /update 2020b: garda world, netherlands: government confirms first case of covid-19 nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study 2020: locked down wuhan and why we always overplay the threat of the new kenan malik china ' s reaction to the coronavirus outbreak may have the opposite effect to what ' s needed 2020: tthe atlantic, the u.k.'s coronavirus 'herd immunity' debacle effects of reactive social distancing on the 1918 influenza pandemic effects of temperature variation and humidity on the death of covid-19 in sars-cov-2 turned positive in a discharged patient with covid-19 arouses concern regarding the present standard for discharge 2020: estimating the effective reproduction number of the 2019-ncov in china this article is protected by copyright. all rights reserved key: cord-312803-fxuynxjd authors: gómez-ríos, manuel ángel; casans-francés, rubén; abad-gurumeta, alfredo; taboada-lópez, elena title: preventing infection of patients and healthcare workers should be the new normal in the era of novel coronavirus epidemics: comment date: 2020-06-16 journal: anesthesiology doi: 10.1097/aln.0000000000003373 sha: doc_id: 312803 cord_uid: fxuynxjd nan w e read with great interest the editorial by bowdle et al. 1 we wish to describe what spanish anesthesiologists and healthcare professionals are experiencing with the first pandemic of the twenty-first century, caused by a new coronavirus. the disease is highly contagious and has therefore spread faster than previous coronavirus infections. the disease has surpassed the capacity of even the most solvent healthcare systems. the natural tendency is to collapse, making it inevitable to ration health resources. the situation in our country, spain, which currently presents the steepest infection curve, is particularly striking. all spanish governments to date have boasted about the excellence of the national health service, considering it the "jewel in the crown." and rightly so, given the high standard of clinical results and quality of care, even in times of budget constraints. this has largely been achieved at the cost of substandard working conditions (understaffing, extended shifts, and poor pay) and cutbacks on resources to protect staff from occupational risks. unfortunately, it has taken a coronavirus to reveal the extent of these shortcomings, and it comes as no surprise that 12,300 spanish health professionals have so far been infected, with 2,000 infections registered today. this represents 15% of total infections, a far higher percentage than countries such as italy (8.67%), china (4.12%), or the united states (1.42%). our patients have been protected-a source of pride for all-but our healthcare professionals, the foundation of our system, have been sorely neglected. this has an enormous impact. our colleagues are "falling like flies," reducing the number of healthcare workers on duty and our capacity to treat our patients, and producing further infections in patients and colleagues. staff numbers are severely depleted, and we are now reduced to recalling retired doctors and recruiting trainees and even medical students. there are two reasons for this: (1) personal protective equipment, which was scarce even at the start of the outbreak, is now entirely lacking, and (2) symptomatic healthcare workers cannot be polymerase chain reaction-tested, so the authorities have to allow them to continue working. at the start of the outbreak, hospital departments went to great lengths to draw up local protocols to ensure the highest quality of care for patients with coronavirus disease 2019 (covid-19) . however, many of these protocols are infeasible due to lack of material resources. anesthesiologists perform high-risk procedures such as endotracheal intubation, with the consequent risk of contamination from secretions, blood, droplets, and aerosols. 2,3 these procedures warrant special measures and should be performed using appropriate personal protective equipment for airborne precautions. 1,2 however, we have no appropriate masks, hazmat suits, goggles, or face shields. the safety of healthcare workers and the enforcement of stringent precautions to control infection should be our highest priority. but our day-today reality is far removed from these laudable principles. preventing infection of epidemics expert recommendations for tracheal intubation in critically ill patients with novel coronavirus disease 2019 chinese association of anesthesiologists: perioperative management of patients infected with the novel coronavirus: recommendation from the joint task force of the chinese society of anesthesiology and the chinese association of anesthesiologists unauthorized reproduction of this article is prohibited. <zdoi the authors declare no competing interests. key: cord-295873-kykyubdq authors: morikawa, saeko; kohdera, urara; hosaka, taisuke; ishii, kousuke; akagawa, shohei; hiroi, satoshi; kase, tetsuo title: seasonal variations of respiratory viruses and etiology of human rhinovirus infection in children date: 2015-10-22 journal: j clin virol doi: 10.1016/j.jcv.2015.10.001 sha: doc_id: 295873 cord_uid: kykyubdq background: using the polymerase chain reaction (pcr) method it is possible to detect uncultivable viruses and discover multiple viral infections. however, the clinical importance of these findings in relation to symptoms is not known. objectives: the seasonal fluctuations of respiratory viruses and the clinical outcomes of single infections and dual infections were investigated. study design: nasal aspirate samples were obtained from outpatients and inpatients of a children’s hospital and these samples were subjected to real-time pcr to detect 16 respiratory viruses. seasonal variations of the 16 viruses and the clinical outcomes such as wheezing, the need for oxygenation and prolonged hospitalization of patients with single viral infections and multiple infections were determined for the 5 most often detected viruses. results: among 512 specimens analyzed, one or more viruses were detected in 424 (83%) specimens. two or more viruses were detected in 160 samples (31% of all samples). the epidemic peaks of the viruses did not coincide with each other. rhinoviruses were the most frequently detected viruses and their coinfection rates were also higher. however, the disease severity in the lower respiratory tract did not differ in most respiratory viral infections regardless of whether there was single infection or dual infection with a rhinovirus and other respiratory virus. conclusions: seasonal distribution was seen for each virus. there were no significant differences in clinical symptoms in the children studied. because the infection of rhinoviruses is the common occurrence in children, it is hypothesized that the factors related to disease severity are mainly the underlying conditions of the children. respiratory tract infections are frequently seen in children and a significant number of these infections are caused by viral pathogens [1, 2] . especially for infants, viral respiratory infections carry a high risk for severe symptoms resulting in hospitalization. there is a abbreviations: pcr, polymerase chain reaction; rs virus, respiratory syncytial virus. * corresponding author. e-mail addresses: morikawa@iph.pref.osaka.jp (s. morikawa), kohdera@nakano-kodomo.or.jp (u. kohdera), thosaka@pd6.so-net.ne.jp (t. hosaka), kousuke. 22 .ishii@gmail.com (k. ishii), shohei@mbk.nifty.com (s. akagawa), hiroi@iph.pref.osaka.jp (s. hiroi), kasetetsuo@iph.pref.osaka.jp (t. kase). strong correlation between viral bronchiolitis in infants and wheezing later in childhood [3] . however, most children show mild symptoms during viral respiratory infections involving only the nose and upper respiratory passages. moreover, clinically useful antivirals do not exist for most such viruses and it is thought that for viral respiratory infections it is not necessary to examine the pathogen. recently, nucleic acid amplification tests such as pcr are increasingly being used to diagnose viral respiratory tract infections. several studies have shown that most common respiratory viruses have epidemic seasons in many areas [4, 7] . pcr makes it possible to detect uncultivable viruses such as human bocavirus and rhinovirus c and discover concurrent viral infections. however, the clinical importance of these findings with regard to symptoms is not known. some reports indicate that human "classical" subtypes http://dx.doi.org/10.1016/j.jcv.2015. 10 .001 1386-6532/© 2015 elsevier b.v. all rights reserved. table 1 the monthly variation of viruses detected in nasal aspirates during the study period. 0 0 4 4 2 1 3 0 1 0 0 0 1 16 (3.1) parainfluenzavirus 2 0 0 0 1 0 0 0 0 0 0 0 0 0 1 (0.2) parainfluenzavirus 3 5 17 9 6 2 0 0 0 0 0 0 0 2 41 (8.0) parainfluenzavirus 4 0 0 1 3 8 2 0 0 0 0 0 0 0 14 (2.7) rsvirus 2 3 3 3 3 6 8 7 5 0 2 8 2 of coronavirus, oc43, nl63, 229e and hku-1, have low impacts on respiratory health [8, 9] . in this study, separate real-time pcr assays were used to detect 16 respiratory viruses in nasal aspirates taken from pediatric patients and we investigated the seasonal fluctuations of the respiratory viruses. we also compared the clinical outcomes such as wheezing, the need for oxygenation and prolonged hospitalization, for patients with single and multiple viral infections. from week seventeen 2013 to week sixteen 2014, nasal aspirate samples were obtained from outpatients and inpatients of a children's hospital. their symptoms were systematically recorded by the attending physicians. written informed consent was obtained from the parents. of the 513 samples obtained, 1 specimen was excluded because of withdrawal of approval. the median age of the patients was 1y (range 0-14 years). age groups were: 0 year 35.9% (n = 184), 1 year 32.4% (n = 166), 2 years 11.9% (n = 61), 3years 7.2% (n = 37), 4 years 5.6% (n = 30), and ≥5 years 6.8% (n = 35). the proportion of females was 41.4%. each sample was amplified using primers and probes specific for each of the targets as previously described [10] . briefly, nucleic acids were extracted from 200 l specimens using the magtration system with a magdea viral dna/rna 200 kit (precision system science co., ltd., chiba, japan) with a 50 l elution volume. rt reactions were performed using a revertra ace qpcr rt kit (toyobo co., ltd., osaka, japan) following the manufacturer's instructions. the cdna was then amplified using realtime pcr master mix (toyobo) with a total volume of 25 l. the sensitivity of each of the realtime pcr methods was reported previously [10] . enteroviruses and rhinoviruses were genotyped by direct sequencing. amplification of the vp4/vp2 region of the enterovirus or rhinovirus for typing was performed with semi-nested rt-pcr as previously described [11] . the purified pcr products were subjected to direct sequencing with a bigdye terminator v1.1 kit as per the manufacturer's instructions (applied biosystems, ca, usa). sequence analysis was performed using the dnadynamo program (blue tractor software, uk). using mega5.2 (tamura et al., 2011, ver5.2.2), we employed the neighbor-joining method [12] to construct phylogenetic trees from the vp4/vp2 region (420 nt) sequences of prototype isolates of each rhinovirus type commonly used in epidemiologic studies of human rhinoviruses retrieved from genbank [13] [14] [15] and new types proposed previously [13, 16, 17] . genotypes were assigned on the basis of their clustering with known prototype reference strains. the kruskal-wallis test, mann-whitney u-test and fisher's exact test were used for comparisons. for all analyses, a p-value of less than 0.05 was considered significant. statistical analysis was performed using spss v16.0 (spss inc., tokyo, japan). among the 512 specimens analyzed, one or more viruses were detected in 424 (83%) specimens (table 1) . two or more viruses were detected in 160 samples (31% of all samples). only one specimen included 5 distinct viruses (human metapneumovirus, rhinoviruses were found most often (n = 192, 37.5% of all samples and 45.3% of positive samples) followed by adenoviruses (n = 86, 16.8% of all samples) and human metapneumovirus (n = 68, 13.3%). influenza virus types a and b were detected in the winter and human metapneumovirus was detected during the spring months. human bocavirus and parainfluenza virus type 3 were found during the spring and early summer. parainfluenza virus type 1 and parechovirus were detected mainly in the summer. the detection of rs virus increased in the autumn. genetically conserved regions of both enteroviruses and rhinoviruses were detected by real-time pcr all year round with a high proportion of positive samples. genotyping revealed the presence of enteroviruses in the summer and a decrease in rhinovirus a in the winter. on the other hand, rhinovirus c was detected in the winter months. adenoviruses were detected mainly in the summer and winter (fig. 1 ). next, we evaluated the prevalence of multiple infections by the viruses. for the human bocavirus, parechovirus and rhinoviruses a and c, the rates of coinfection were high compared with other respiratory viruses. rhinoviruses were the most frequently detected viruses and their coinfection rates were also higher than those of the other viruses. therefore, we compared the clinical symptoms caused by five types of viruses, adenoviruses, human bocavirus, rs virus, parainfluenza virus type 3, and human metapneumovirus, which were detected most often after rhinoviruses, and rhinovirus single infections and symptoms in cases with dual infections including rhinoviruses. there was no significant difference between the number of days of hospitalization caused by rhinoviruses and the other five viruses. the number of days in the hospital of patients in whom rs virus was detected was longer than that of patients infected with human metapneumovirus (table 2) . for the five viruses discussed above, we compared the number of hospitalization days of the cases with single infections by the each 5 viruses with those having dual infections with a rhinovirus and those with dual infection with a virus other than a rhinovirus. the number of days of hospitalization of the children with parainfluenza virus type 3 infection alone was shorter than for children with paranfluenza virus and rhinovirus dual infection. on the other hand, children with infection by human metapneumovirus alone spent fewer days in the hospital than those with dual infections by human metapneumovirus and a respiratory virus other than a rhinovirus (table 3) . we next compared the requirement for oxygenation and the presence of wheezing of the children with single infections and dual infections with a rhinovirus or other respiratory virus. the patients with dual infections with an adenovirus and rhinovirus needed significantly more oxygenation than those with an adenovirus infection or dual infection with an adenovirus and other respiratory virus. however, the severity of the lower respiratory tract disease for which the requirement of oxygenation was assumed and the presence of wheezing as an index did not differ among most respiratory viral infections, regardless of whether they were single infections or dual ones with a rhinovirus and other respiratory virus (table 4 ). in this study, separate real-time pcr assays were used to detect 12 rna viruses and two dna viruses, and real-time reverse transcription (rt) pcr was used to detect influenza viruses a and b. of the 512 samples analyzed, 424 were positive 1 virus or more. the overall viral detection rate was 83%, which was much higher than in similar past reports [5] [6] [7] 18] . the reason may be that our method had many detection targets. furthermore, the higher detection rates among young children likely correspond to the higher incidence of viral respiratory tract infections in children, although other factors such as pre-existing immunity might also have played a role [4] . since the specimens from children aged 1 year or younger accounted for 68% of those studied, it is considered that the rate of viral detection and the rate of concurrent infections became higher than in past reference data. the largest number of viruses detected in one sample was 5 in the nasal aspirate from a 9-month-old girl. when comparing the number of viruses detected per specimen, it was found that the specimens from younger patients tended to include more than one virus (data not shown). seasonal distribution was seen for each virus. the epidemic peak of each virus was about the same as in another report from japan [19] . seasonal influenza virus type a migrates globally between epidemics and is reintroduced every winter season in temperate climates [20] , although the underlying cause of the seasonality of the other respiratory viruses remains unknown. it has been suggested that rhinovirus infections could reduce subsequent rs virus and influenza virus type a infections by inducing an interferon response, thereby creating an undesirable environment for these viruses [21, 22] . in this study, the peaks for the various viral epidemics did not coincide. thus, it is thought that some kind of interference by viruses may influence epidemics of respiratory viruses. in this study, human rhinoviruses were the most common viruses. rhinoviruses are thought to be mainly associated with the common cold, causing mild respiratory symptoms [23] . these viruses are classified into three species and divided into more than 160 serotypes or genotypes. thus they are among the mostly commonly detected viruses in respiratory specimens of children [10] . however, recent reports suggest that rhinovirus infections may induce and/or exacerbate asthma and be responsible for lower respiratory tract infections with severe symptoms [24, 25] . based on the sequence data, rhinovirus c was detected mainly in the winter, whereas rhinovirus a was detected all year round, with a high proportion of positive samples in june (44% of the samples). although rhinovirus b was detected, its seasonality was not clear. however, it became clear that there was a difference in the epidemic seasons of rhinoviruses a and c. furthermore, the detected rhinoviruses consisted of 32 genotypes of group a, 5 genotypes of group b and 21 genotypes of group c, suggesting that multiple genotypes were brought into the area and that epidemics of some of them might occur at the same time (supplementary table 1 ). rhinovirus a consists of 80 serotypes and b consists of 32 types, including genotypes, and there are now 55 rhinovirus c genotypes proposed [17] . it is not clear whether the genotypes of the rhinoviruses detected in this study cause severe illness. we also compared the clinical symptoms of single infections and dual infections by rhinoviruses and other respiratory viruses of the children infected by one of the five most commonly detected respiratory viruses. the results revealed that there were no significant differences in the number of days of hospitalization, the necessity for oxygen inhalation or the existence of wheezing between the children with single infections and those with dual infections. in former reports that evaluated the impacts of rhinoviruses on lower respiratory infections, there were only marginal differences between the different rhinovirus groups and between single rhinovirus infection and rhinovirus coinfection [26, 27] . though there was no significant difference in the number of hospitalization days of patients with single infections by rhinoviruses or other respiratory viruses, our data suggested the importance of rhinoviruses as a potential cause of pediatric pneumonia. recently, our group evaluated the prevalence of rhinovirus infections among asymptomatic children [10] . rhinoviruses were often detected in their throats at a time without any symptoms. since rhinoviruses do not exist in the upper respiratory tract for a long time even if a child does not show symptoms, these were "active" asymptomatic infections rather than persistent infections. in conclusion, rhinoviruses are causative agents of various conditions ranging from asymptomatic infection to lower respiratory tract infection and pneumonia. rhinovirus coinfection with other respiratory viruses is not responsible for more severe symptoms, so it is hypothesized that the factors related to disease severity are mainly the underlying conditions of the children. respiratory viral infection in infants: causes, clinical symptoms, virology, and immunology frequency of viruses associated with acute respiratory infections in children younger than five years of age at a locality of mexico city wheezy babies-wheezy adults? review on long-term outcome until adulthood after early childhood wheezing seasonal variations of 15 respiratory agents illustrated by the application of a multiplex polymerase chain reaction assay, scand clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial viral etiology of respiratory infections in children in southwestern saudi arabia using multiplex reverse-transcriptase polymerase chain reaction ten years' experience with year-round active surveillance of up to 19 respiratory pathogens in children high incidence but low burden of coronaviruses and preferential associations between respiratory viruses human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls detection of respiratory viruses in gargle specimens of healthy children molecular diagnosis of human enteroviruses by phylogeny-based classification by use of the vp4 sequence the neighbor-joining method: a new method for reconstructing phylogenetic trees proposals for the classification of human rhinovirus species a, b and c into genotypically assigned types prospective genotyping of human rhinoviruses in children and adults during the winter of molecular epidemiology of human rhinovirus c in patients with acute respiratory tract infections in osaka city proposals for the classification of human rhinovirus species c into genotypically assigned types picornaviridae study group comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children epidemiological study of respiratory viruses detected in patients under two years old who required admission because of lower respiratory disease phylogenetic analysis reveals the global migration of seasonal influenza a viruses do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? does viral interference affect spread of influenza? viral etiology of common cold in children, finland emerg wheezing rhinovirus illness in early life predict asthma development in high-risk children the abcs of rhinoviruses, wheezing and asthma impact of rhinoviruses on pediatric community-acquired pneumonia human rhinovirus in the lower respiratory tract infections of young children and the possible involvement of a secondary respiratory viral agent the authors would like to thank maki otsuka for helpful technical assistance in amplification and kim barrymore for editing the manuscript. this work was supported by grant-in-aid for scientific research (c) grant number from the japan society for the promotion of science. none declared. this study was approved by the osaka prefectural institute of public health ethical committee (no. 1302-05-01) . supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2015.10.001. key: cord-285785-29ohzeug authors: chen, xiaolan; ali abdalla, bahareldin; li, zhenhui; nie, qinghua title: epigenetic regulation by non-coding rnas in the avian immune system date: 2020-08-12 journal: life (basel) doi: 10.3390/life10080148 sha: doc_id: 285785 cord_uid: 29ohzeug the identified non-coding rnas (ncrnas) include circular rnas, long non-coding rnas, micrornas, ribosomal rnas, small interfering rnas, small nuclear rnas, piwi-interacting rnas, and transfer rnas, etc. among them, long non-coding rnas, circular rnas, and micrornas are regulatory rnas that have different functional mechanisms and were extensively participated in various biological processes. numerous research studies have found that circular rnas, long non-coding rnas, and micrornas played their important roles in avian immune system during the infection of parasites, virus, or bacterium. here, we specifically review and expand this knowledge with current advances of circular rnas, long non-coding rnas, and micrornas in the regulation of different avian diseases and discuss their functional mechanisms in response to avian diseases. about 1% of the mammalian genome has the potential to encode proteins, which means a huge amount of non-coding rnas (ncrnas) exist in mammalian transcripts [1] . the huge amount of ncrnas indicated that they are very important biological molecules in epigenetic regulation. although the current studies on ncrnas are not elaborate, its important functions for various biological processes have been discovered, especially its extensive roles in different kind of diseases. as a pillar of the economy and people's livelihood, poultry industry becomes important for our life, the meat and egg of chicken is directly associated with the human health. but the threat of disease infection to poultry farming has never been absent, which means it requested from us to develop the poultry host immunity to prevent the infection of avian diseases. many factors have an impact on the chicken innate and acquired immunity, such as environment, nutrition, and genetics. as the most complicated factor, genetics has its special power to improve the host defense. genetic improvement is the most effective strategy to deal with disease invasion, and is a complex biological process of multilevel regulation, in which ncrnas played their unique role. numerous studies have been conducted on ncrnas analysis in avian diseases; therefore, summarizing the roles of ncrnas in the regulation of avian immunity is helpful for us to investigate the new potential function of ncrnas in avian immunity. the classical recognition of ncrnas is that they are the products of genome dna transcription but they lack the ability to encode a functional protein [2] . however, with the continuous development of the technology of high-throughput sequencing, the implementation of ribosome sequencing (ribo-seq) has given the evidence that most of the ncrnas can be bound to ribosome, especially the large circular rnas are special endogenous ncrna molecules that contain a closed loop structure. previously, we knew that the majority of identified circrnas were from only exonic region or only intronic region or both exonic and intronic region, and relatively fewer circrnas produced by intergenic regions, antisense transcripts of known transcripts, untranslated regions (utrs), and other regions in eukaryotes [25] . so circrnas are mainly classified in exonic circular rna (ecirna), intronic circular rna (cirna), and exon-intron circular rna (eicirna). but recently, circrnas from mitochondria dna were newly investigated, this kind of circrna was called meccirnas [26] . because circrnas have no free 5 cap and 3 poly a tail, it makes them not easily degraded by rnase. sometimes, they are more stable than other rnas and even expressed more abundantly than parental gene in eukaryotes [27] . as a kind of stably existing rna, several circrna functional mechanisms have been investigated, such as acting as mirna sponges [28] (figure 1g ), interacting with rna binding protein (rbp) [29, 30] (figure 1h ) and translating to a functional peptide (figure 1i ) [31, 32] . among them, circrna as mirna sponge is the most well studied. it is presented that circrnas have a certain amount of mirna biding cites and could arrest mirna and then release mirna targets [33] . the interaction with rbp makes circrnas possess various regulatory functions. for instance, circrna interacts with small nuclear ribonucleoprotein u1 subunit (u1 snrnp) in the nucleus to regulate the transcript of rna [34] ; circrna binds to eukaryotic initiation factor 4e (eif4e) and eukaryotic initiation factor 4g (eif4g) to regulate the translation of mrna [29] ; quaking (qki) protein interacts with the pre-mrna of gene and promotes the biogenesis of circrna [35] ; methylation-related protein-yth n6-methyladenosine rna binding protein 2 (ythdf2) interacts with circrna and activates the m(6)a-induced degradation of circrna [30] . as the most novel functional mechanism, circrna translation was mainly discovered in circrna which has internal ribosome entry site (ires) or m6a modification [36] . ires allows direct recruitment of initiation factors and ribosomes on the rna and initiates the circrna translation through cap-independent manner [37, 38] . it is discovered that some endogenous circrnas that have m (6) a motifs have the ability to translate and the abundance of m (6) a motifs could affect the translation efficiency of circrna [36] . however, it requires the involvement of eukaryotic translation initiation factor 4 gamma 2 (eif4g2) and m (6) a reader yth n6-methyladenosine rna binding protein 3 (ythdf3) and will be affected by other m (6) a modification-related proteins. it can be enhanced by methyltransferase like 3/14 (mettl3/14) and suppressed by demethylase fto [36] . mirnas are a class of small ncrnas that were~22 nucleotides in length. the classical role of mirna is binding to the 3 utr (figure 1j ) of the mrnas and then induce the degradation of mrna or inhibit the translation of mrna [39] . notably, at the rna world, mirna plays a key role in the whole ncrna regulatory network. it becomes the central of the cerna network and could bind lncrnas/circrnas with mrnas as both circrnas and lncrnas have the mirna response element (mre site) and could act as a mirna sponge [28] . lncrna/circrna-mirna-mrna axis is a powerful regulatory mechanism and was extensively investigated in various biological process of different species. with the deep study of molecular biology, mirna was given several new functions. first, it could regulate the expression of circrna by recruiting argonaute (ago) protein to interact with circrna ( figure 1k ). for instance, mir-671 regulates the production of a circcdr1 via cleaving the cdr1 antisense transcript in an ago2-mediated manner [40] . mirna-1224 negatively regulates circrna-filip1l expression through binding and splicing pre-circrna-filip1l, which also requires the ago2 protein [41] . additionally, the m7g methylation of mirna showed another functional mechanism of mirna. it is evidenced that the m7g methylation of let-7 mirna induced by methyltransferase like 1 (mettl1) could regulate the process of lung cancer [42] . moreover, it was investigated that some pri-mirnas in plants have the ability to encode a peptide (figure 1l ) that is termed as mirna-encoded peptide (mipep). the translation of the pri-mirnas leads to the accumulation of their corresponding mirnas, then causing the downregulation of mirna targets. for instance, pri-mir-171b in medicago truncatula (m. truncatula) contains two orfs, orf1 and orf2; orf1 could encode functional peptide, termed as mipep171b. the overexpression of mipep171b or the application of a synthetic mipep171b could increase the abundance of mir-171b [43] . similar functional mechanism was found in other pri-mirnas, such as mir-160b, mir-164a, mir-169d, mir-171e, mir-319a, and mir-165a in m.truncatula or arabidopsis thaliana (a. thaliana) [43, 44] . lastly, mirna-derived sequences that are identical with pre-mirnas could regulate lncrna splicing through overlapping the pre-transcript of lncrna ( figure 1m ). specifically, hsa-mir-99b regulates the splicing of the primary transcript of linc01129 through complementary combination with its exon-intron junction [45] . in spite of the underlying mechanisms of those newly identified mirna functions warrant further analysis, these novel functions provided us the new area to study the potential role of mirnas. the important roles of lncrnas, circrnas, and mirnas in regulating the pathogenicity of many diseases have been mainly discovered in humans. the critical roles of ncrnas in avian inflammation and autoimmune regulation were also investigated. this review sheds light on the regulatory mechanism of lncrnas, circrnas, and mirnas on chicken immunity and be helpful for identifying potential ncrna biomarkers for avian diseases avian invasion diseases are mainly caused by parasites, viruses, or bacterial infections. they infect chickens by specific manners and cause serious damage to chicken organs, thus influencing livability and body health of chickens. the direct consequence of this damage is the decrease of egg and meat production, which can bring huge financial losses to the poultry industry. according to the research progress on ncrnas, we selected the related diseases researches in avian to summarize here. avian leukosis virus (alv) is a retrovirus belonging to a retroviridae family. alv includes seven subgroups of a, b, c, d, e, j, and k, among which, alv-j is the most pathogenic subgroup with high transmission and pathogenicity [46, 47] . the clinical manifestation of alv-j-infected chicken including pathogenic tumors, immune tolerance, delayed growth and also higher susceptibility to a secondary infection [46, 48] . several studies have been focused on the expression profile and the molecular regulatory mechanisms of circrnas during the infection of alv. thousands of circrnas were identified in various types of alv-infected organs, and the number of differentially expressed circrnas (decircrnas) are calculated, ranges from dozens to thousands [49] [50] [51] [52] . gene ontology (go) term and kyoto encyclopedia of genes and genomes (kegg) pathway analysis showed that the decircrnas were involved in immune-related go items and kegg pathways. main go terms include regulation of b-cell activation [49] , b-cell differentiation [49, 50] , alpha-beta t-cell activation [49] , and main kegg pathways have signaling pathways of mtor [49, 50] , tgf-beta [50] , toll-like receptor [50] , rig-i-like receptor [50] , jak-stat [50] , insulin [50] and erbb [50] , mapk [52] , wnt [52] , and b-cell receptor [52] . many circrna-mirna-mrna cerna networks were characterized [49] [50] [51] [52] . even through so many circrnas were identified, only few circrnas were validated for their localization or functional mechanism. one exon-intron decircrna, circhrh4 localizes in cytoplasm and was abundantly expressed in different chicken tissues and cells [50] . circ-vav3 can interact with mir-375 and upregulate the expression of yes-associated protein 1 (yap1), a target gene of mir-375. circ-vav3/mir-375/yap1 axis participated in tumorigenesis through activating epithelial-mesenchymal transition (emt) by altering the expression of emt markers, including vimentin, zinc finger e-box binding homeobox 1 (zeb1), matrix metallopeptidase 2 (mmp2), n-cadherin, fibronectin, and e-cadherin [51] . as we know from the current research process, the role of circrnas related to the infection of alv largely remain on the early stage of the characterization, expression profile, and associated pathways. only a few researches have explored the single circrna molecular function in response to the alv-infection, which inspired us to study the specific functional mechanism of single circrna in regulating alv-infection. similar to circrna, a few researches have been focused on the lncrna expression during alv infections. rna-seq had been performed on both chicken tissues and cells to screen the alv-associated lncrnas. for instance, differentially expressed lncrnas (delncrnas), differentially expressed mirnas (demirnas), and differentially expressed mrnas (demrnas) between infected and non-infected tissues [53, 54] , chick embryo fibroblasts cells (cef cells) [55] or chicken primary monocyte-derived macrophages (mdms) [56] , were identified. some important immune-related pathways were enriched, including toll-like receptor, nodlike receptor, rig-i receptor, and jak-stat signaling pathways [55] . the co-expression network analysis among those demrnas, demirnas, and delncrnas revealed that the delncrnas could interact with immune-related mirnas and genes to exhibit their role in diseases and cancers process [53] [54] [55] [56] . specially, some lncrna-mirna-mrna interaction networks participated in the regulation of cyclin d3 (ccnd3) and suppressor of cytokine signaling 5 (socs5) through jak -stat signaling pathway [56] . in addition, several lncrnas (xloc_672329, aldbgalg0000001429, xloc_016500, and aldbgalg0000000253) ( table 1) were predicated to cis-regulate cholesterol 25-hydroxylase [ch25h)/cytokine inducible sh2 containing protein (cish)/interleukin 1 beta (il-1β)/cd80 molecule (cd80) to participate in host antiviral responses [56] . cd80 participate in host antiviral responses [56] the current findings of lncrna involvement in the infection of alv are just simply focused on the lncrna characterization, expression profile, and associated pathways, however, still more proper investigations become necessary. unlike circrnas and lncrnas, many studies have been conducted to explore not only the expression profile but also the underlying functional mechanism of mirnas involved in alv infection. mirna microarray or small rna sequencing revealed many mirnas were aberrantly expressed after alv infection [57] [58] [59] [60] . among the demirnas, several of them were verified by qrt-pcr analysis in one or more kinds of alv-infected materials, including mir-221 [57] [58] [59] [60] , mir-222 [57, 59, 60] , mir-1456 [57] , mir-1704 [57] , mir-1777 [57] , mir-1790 [57] , mir-2127 [57] , let-7b [57, 60, 61] , let-7i [57, 60, 61] , mir-125b [57] [58] [59] [60] , mir-375 [57, 60] , mir-458 [57] , mir-193a [58] , mir-193b [58, 59] , mir-148a [59] , mir-27b [59] , mir-34a [59] , mir-130a [59] , mir-23b [62] and mir-34b-5p [63] . obviously, the expression of mir-221/mir-222, let-7b/i, mir-375, mir-125b, and mir-193b were changed in not only one type of materials after alv-infection, indicating their important roles in response to the alv. the qrt-pcr-verified demirnas were involved in immune-related pathways by analyzing the target genes of the demirnas. of which, mir-221/222 may have effect on oncogenicity [57] and exist in several common pathways (mapk [58] , oocyte meiosis [58] , wnt [58] , and the chronic myeloid leukemia pathway [61] ). in addition, mir-221 was also involved in antigen presentation and apoptosis pathways [59] . mir-1456, mir-1704, mir-1777, mir-1790, and mir-2127 may have effect on oncogenicity [57] . mir-125b was related to the oocyte meiosis and mapk signaling [58] , and may have effect on tumor suppression [57] . mir-193a was involved in wnt signaling and oocyte meiosis pathway, while mir-193b was included in wnt signaling and oocyte meiosis pathway [58] . let-7b/i was related to the chronic myeloid leukemia pathway [61] . let-7b/i, mir-375, and mir-458 may be involved in tumor suppression [57] . the underlying mechanisms of some demirnas involved in alv infection were revealed ( table 2 ). for mir-221 and mir-222, they have multiple roles in the regulation of alv infection. on one hand, mir-221/mir-222 could facilitate df1 (chicken fibroblast cell line) cell proliferation and migration, and could inhibit the expression of apoptosis-related gene, bcl-2 modifying factor (bmf), leading df1 cells not easy to undergo apoptosis [64] . on the other hand, mir-221/222 could promote cell proliferation to help alv replication in df1 cells. because the depression of g1/s switch and over proliferation induced by alv-j for df1 cells required high expression of mir-221 and mir-221, mir-221 and mir-221 can downregulate the expression of cdkn1b (cyclin dependent kinase inhibitor 1b)], which have the ability to arrest the cell cycle and inhibit the cell proliferation process via the cdkn1b-cdk2 (cyclin dependent kinase 2) /cdk6 (cyclin dependent kinase 6) pathway [65] . to sum up, mir-221 and mir-222 could promote the alv progression in df1 cells through inhibiting apoptosis and facilitating cell proliferation, migration, and growth. in addition, the expression of mir-23b is up-regulated in spleen after alv-j infection. moreover, mir-23b could enhance alv-j replication by regulating interferon regulatory factor 1 (irf1) [62] . meanwhile, mir-34b-5p is another validated demirna in spleen after alv-infection and it could target melanoma differentiation-associated gene 5 (mda5) to promote alv-j replication by facilitating the cell migration and proliferation [63] . marek's disease virus (mdv) has three serotypes: serotype 1 (mdv-1), serotype 2 (mdv-2), and turkey herpesvirus (hvt) [66] . only mdv-1 has an oncogenic /pathogenic/tumorigenic effect [48] . marek's disease (md) is caused by mdv. the chickens infected by mdv will cause immunologic suppression, t-cell lymphoma, and neurologic diseases, resulting in tissue or cell damage in chickens. if the mdv susceptible chickens are infected with mdv, the mortality can reach up to 100%, which brings massive loss to the poultry industry [67] . to our knowledge, one circrna sequencing research has been performed with three types of spleens: first, spleens from mdv infection-induced tumors; second, spleens from the survivors (without any lesion) after mdv infection; third, spleens from non-infected chickens. about 2169 circrnas were detected in this research, of which, 113 circrnas were differentially expressed. circrna-mirna-mrna networks showed circzmym3 could not only interact with 7 mirnas but also target immune-related genes, such as swap70 and ccl4, because swap70 and ccl4 shared the same target site with mir-214 and circzmym3. mir-155 was predicted to interact with circgtdc1, circmyo1b, gata binding protein 4 (gata4), peripherin 2 (prph2), eomesodermin (eomes), rho related btb domain containing 1 (rhobtb1), and serine palmitoyltransferase, small subunit b (sptssb) [68] . chicken md resistant line 6 3 and md susceptible line 7 2 are model animals for the investigation of responsible biomarkers or clinical diagnosis during the infection of mdv [69] . expression analysis and bioinformatical functional annotation through rna sequence by using line 6 3 and line 7 2 showed that lncrnas were aberrantly expressed and were involved in immune-related pathways that indicate that lncrnas participate in regulating mdv infection [70] . comparing the expression of the rnas between infected and noninfected chicken bursa, 425 delincrnas and 387 de genes were found in line 6 3 , while 636 delincrnas and 2383 de genes were identified in line 7 2 . co-location analysis for the 30 delincrnas associated with immune response found that most of the neighboring genes of these lincrnas were also associated with immune response. of them, one candidate lincrna, termed linc-satb1, was positively related to inflammatory/defense/external stimulus response and lymphocyte activation. in addition, the expression of linc-satb1 was correlated with special at-rich binding protein-1 (satb1), which is known to have the ability to regulate chromatin structure and t-cell development/activation [71] . it implies that lnc-satb1 may participate in immune response to md by regulating satb1 [70] . lncrna could participate in mdv through interacting with both host mdv susceptible/resistant gene. for example, linc-galmd1 was a delincrna of mdv non-infected and infected chickens [72] . igll1 (immunoglobulin lambda-like polypeptide 1) has distinct expression between line 6 3 and line 7 2 . it has lower expression in line 6 3 chickens but higher expression in line 7 2 chickens after infection of mdv, which implies that igll1 could be a line-specific or susceptible gene in response to md [72] . meanwhile, linc-galmd1 expression level had a positive correlation with igll1 which indicated that linc-galmd1 could potentially regulate md by interacting with igll1 [72] . some lncrnas were predicted to interact with md-resistant candidate genes [73] . mstrg.360.1 was correlated with c-x-c motif chemokine ligand 12 (cxcl12), tnf receptor superfamily member 6b (tnfrsf6b), swap70, cytotoxic t-lymphocyte associated protein 4 (ctla4), and histone deacetylase 9 (hdac9) [73] . mstrg.6725.1 may interact with ctla4 and joining chain of multimeric iga and igm (jchain). mstrg.6754.1 was associated with jchain and ctla4 [73] . mstrg.15539.1 was associated with swap70, hdac9, cd72 molecule (cd72), and jchain [73] . mstrg.7747.5 were strongly correlated with cd8b molecule (cd8b), hdac9, cd72, and insulin-like growth factor level (igf-i) [73] . lncrna directly regulates mdv gene expression is another approach for chicken host to response to md. it was evidenced by the upregulation of viral gene-meq (an essential gene for mdv progression), induced by downregulation of linc-galmd1 [72] . lncrnas act as an upstream regulator to modulate mirna expression, thus exhibiting specific function during md viral infection was also an approach for lncrna to regulate mdv infection. linc-galmd3, which has a higher expression level in cd4 + t cells after mdv infection, was found to be located on the upstream of the gene transcribes mir-223 [74] . the loss of function of linc-galmd3 will suppress mir-223 expression and mdv replication. additionally, deg analysis of rna-seq between the linc-galmd3 knockdown and control msb1 (mdcc-msb1) cells showed that about 27 degs were also mir-223 target genes, which implied the cis regulatory mechanism of linc-galmd3 on mir-223 [74] . the linc-galmd3-mir-223-target degs interaction network could be used for further investigation during mdv infection. the selected lncrnas, their targets, and their functions during the mdv infections were shown in table 3 . numerous researches have been focused on the exploration of mirna expression profile and their potential role during the infection of mdv. hundreds of mirnas were significantly differentially expressed between mdv-infected and noninfected groups [75] [76] [77] [78] [79] [80] . let7 [75] , mir-199a-1 [75, 77] , mir-26a [75, 77] , mir-181a [75, 77] mir-16 [75] mir-223 [76] , mir-150 [76] , mir-155 [76, 79] , mir-221 [77] , mir-199 [77] , mir-15b [78] , mir-456 [78] , let7i [78] , mir-762 [79] , mir-29b [79] , mir-140-3p [81] , mir-199-3p [81] , and mir-221-5p [81] , were verified by qrt-pcr/northern blot analysis. the classical functional mechanism of mirnas in regulating mdv infection is targeting the 3 utr of their target genes and inhibiting the transcription of the mrna or interfering their translation [82] . mir-221/mir-222 were significantly upregulated after mdv infection. the cyclin-dependent kinase (cdk) inhibitors 27 kip1 has direct effect on cell cycle and cell proliferation [82] . conserved binding site between mir-221/mir-222 and 27 kip1 was verified in several species and their target relationship has been confirmed in several cancer cells [82] . in avian, mir-221/mir-222 would also target 27 kip1 to regulate msb1 cell proliferation [83] . retinoid acid receptor-related orphan receptor alpha (rora) is a suppressor for tumor and could be involved in immunity, inflammation metabolism [84] , and tumor initiation [85] . in mbs1 cells, mir-155 could increase the proliferation, invasiveness, and reduce apoptosis by targeting rora [86] . target interactions of mir-181a with myb proto-oncogene like 1 (mybl1)/ insulin like growth factor 2 mrna binding protein 3 (igf2bp3), and mir-26a with eukaryotic translation initiation factor 3 subunit a (eif3a), were confirmed by luciferase reporter assays [77] . furthermore, mir-181a was verified to inhibit proliferation of msb1 by targeting mybl1 [87] . never in mitosis gene a (nima)-related kinase 6 (nek6) is another verified target gene of mir-26a and showed opposite expression pattern with mir-26a between mdv-infected spleens and noninfected controls. mir-26a would exhibit suppression to msb1 cell proliferation through inhibiting nek6 [88] . mir-103-3p decreases cell migration by targeting transcription factor dp-2 (e2f dimerization partner 2) (tfdp2) and cyclin e1 (ccne1) [89] . for mir-130a, it could inhibit msb1 cell proliferation and migration by targeting homeobox a3 (hoxa3) and myod family inhibitor domain containing (mdfic) [90] . mir-219b could target b-cell chronic lymphocytic /lymphoma 11b (bcl11b) and suppresses the proliferation, migration, and invasion of msb1 cells [91] . as a homologous to mdv-encoded mirna to participate in regulate the process of mdv infection is another mirna regulatory mechanism in avian. this was discovered for mir-155, mir-29b, and mir-221. mir-155 shared seed sequence with the same target genes with mdv-induced mirna, mdv-1-mir-m4 [92] . the shared target genes including pu.1, hivep2 (hivep zinc finger 2), cebpβ (ccaat enhancer binding protein beta), pdcd6 (programmed cell death 6), bcl2l13 (bcl2 like 13), rreb1 (ras responsive element binding protein 1), gpm6b (glycoprotein m6b), map3k7ip2 (tgf-beta activated kinase 1/map3k7 binding protein 2), and c-myb (myb proto-oncogene, transcription factor) [93, 94] . many of these genes are known to have a specific role in regulating tumor cell proliferation/apoptosis and tumor formation. additionally, mdv-1-mir-m4 is essential for mdv infection due to the deletion of mdv-1-mir-m4 or a 2 nucleotides mutation on its seed region will lead to an inhibition for the induction of lymphomas. this inhibition could be rescued by gga-mir-155, thus in turn implied mir-155 is a crucial regulator for lymphomas. toll-like receptor 3 (tlr3) is crucial for innate and adaptive immunity. it was abundantly expressed in mdv-infected cef cells, resulting in replication inhibition of the rb1b strain of mdv. however, such inhibition would alter both mdv1-mir-m4-5p and mir-155 [95] . mir-29b shared a seed sequence with mdv2-mir-m21, and expression of mdv2-mir-m21 may ensure viral proliferation in host cells. mir-221 shared a seed sequence with both hvt-mir-h14-3p and mdv1-mir-m32 [92, 96, 97] . as the homologous mirnas of virus-encoded mirnas, some host mirnas share the same biding site and even same target genes with virus-encoded mirnas to influence the biological function of virus-encoded mirnas may be a new strategy to resist virus invasion. the interaction of host mirnas with virus-encoded gene is also a regulatory mechanism for mirnas during the virus infection. for instance, the viral oncoprotein meq could regulate the expression of mir-21 by binding to the promoter region of mir-21. meanwhile, mir-21 could target chicken programmed death cell 4 (pdcd4) to suppress growth and apoptosis escape of tumor cells [98] . it provided a viral gene-host mirna-host gene regulation mechanism involved in the mdv infection. meanwhile, mir-155 was found to target viral env transcripts and could significantly lower the env transcripts abundance in msb1 and cef cells [99] . the selected-verified mirnas and their target genes and their functions during the mdv infections are presented in table 4 . infectious bursal disease (ibd) is a virus disease of young chickens which is caused by avian infectious bursal disease virus (ibdv). ibdv has negative effective on t cells proliferation, causing immunologic suppression in chicken [100, 101] . moreover, ibd has caused death and vaccination failure to other disease in chicks [101] . dendritic cells (dcs) have special role in both innate and acquired immune during virus infection [102] . a microarray study on ibdv-stimulated dcs and non-stimulated dcs revealed 114 lncrnas, 18 mirnas, and 965 mrnas were differentially expressed after stimulated with ibdv [103] . functional annotation of the delncrnas, demirnas, and de genes showed that there was an association with cellular response to starvation, protein localization, the rna biosynthetic process, etc. these were involved in jak-stat/mapk/mtor/neurotrophin/ccr5/interleukin-17 (il-17) signaling pathways, as predicted by a pathway analysis [103] . mirnas could regulate viral infection through regulating the ibdv genome sequence. ibdv as a double stranded virus has two segments (segment a and b) and encodes five viral proteins, vp1-vp5, which play different roles in the infection process of ibdv to the host. of them, vp1 is required for both ibdv replication and virulence [104] . as the target of tumor suppressors in human [105, 106] , mir-21 could suppress ibdv replication via inhibiting the expression of vp1 in avian [107] . similarly, mir-454 targeting ibdv genomic segment b to inhibit ibdv replication [108] . mir-130b could inhibit ibdv replication through targeting ibdv segment a [109] . apart from targeting the viral genome sequence, mirna could also regulate the ibdv infection through targeting regulators of type i interferon (ifn), as ifn is a crucial cellular molecule to combat viral infection and interferon regulatory factor-dependent pathways play important roles during the infection of various disease. socss (suppressors of cytokine signaling) gene family was known as one kind of negative regulators of ifn. specially, the inhibition of socs6 can increase the expression of ifn-β. mir-454 could suppress the ibdv replication through suppression of socs6 [108] . similarly, mir-130b could facilitate ifn-β expression through binding to the host socs5 to inhibit ibdv replication [109] . for mir-155, it was found to suppress ibdv replication through targeting not only socs1 but also another type i ifn negative regulator, tank (tnf receptor-associated factor family member-associated nf-κb activator) [110] . on the contrary, mir-9 could inhibit ifn expression by inhibiting irf2 and then promoting the replication of ibdv [111] . mir-142-5p was founded to decrease the activity of the ifn-β by directly by targeting the mda5 and then promoting ibdv replication through irf7 (interferon regulatory factor 7) signaling pathway [112] . additionally, mir-2127 has been found to promote ibdv replication by suppressing p53 translation and attenuating p53-mediated innate immune response against ibdv infection [113] . more novelty, ibdv alters the expression of responsible mirnas through inducing the demethylation of the mirna promoter is a new strategy for ibdv to protect themselves for survival from the immune responses induced by related-mirnas. for instance, ibdv infection could induce the promoters of pre-mir-27 and pre-mir-16-2 demethylation, thus upregulating mir-27b-3p and mir-16-5p expression [114, 115] . furthermore, mir-27b-3p could increase the expression of chicken ifn-β, nf-κb, and irf3 (interferon regulatory factor 3) by targeting socs3 and socs6, thus inhibiting ibdv replication [114] . mir-16-5p increased the caspase-9/3 activity through targeting bcl2, thus promoting the ibdv-induced apoptosis [115] . avian infectious bronchitis virus (ibv) belongs to coronavirus. it mainly replicates in chicken epithelial cells and could infect both meat type and commercial type of small or old chickens [116, 117] . the investigation of the host responsible mrnas and lncrnas in ibv-infected dcs revealed thousands of mrnas were differentiated expressed in ibv-infected avian dcs and noninfected cells [118] . with 1093 up-regulated and 845 down-regulated. mirnas and lncrnas interaction provided further information for the candidate therapeutic biomarkers in response to ibv infection [118] . mirna transcriptome analysis in chicken kidneys showed 58 de mirnas were found and were shown to be mostly associated with immune response, catalytic activities, metabolic processes, and gene expression [119] . among them, mir-1723, mir-7b, mir-222b-3p, mir-1782, mir-6516-3p, mir-202-5p, mir-1559-3p, mir-449a, mir-1454, and mir-1563 were verified by qrt-pcr [119] . as a predicted demirna by sequence analysis, mir-146a-5p was also found to promote ibv replication of by targeting tumor necrosis factor receptor superfamily member 18 (tnfrsf18) and il-1 receptor associated kinase-2 (irak2) [120] . as another demirna, mir-30d was showed to inhibit ibv replication through suppressing ubiquitin-specific protease 47 (usp47) in hd11 (avian macrophage-like cells) cells [121] . newcastle disease is caused by a single-stranded rna virus, newcastle disease virus (ndv), resulting in a high mortality rate in infected chicken worldwide [122] . only few studies have been focused on ncrna investigation associated with nd. one study performed mrna-seq and small rna-seq to identify the interaction pairs of mrna-mirna involved in nd. 1069 mirna-mrna pairs were found. among them, the interaction of mir-203a and transglutaminase 2 (tgm2) were confirmed by both qrt-pcr and dual-luciferase reporter assay [123] . additionally, mir-19b-3p could promote the expression of inflammatory cytokines and suppress ndv replication by targeting ring finger protein 11 (rnf11) and zinc-finger protein, mynd-type containing 11 (zmynd11), which are two negative moderators of nf-κb signaling [124] . mir-455-5p could suppress ndv replication by directly targeting socs3 [125] . mir-375 inhibited ndv growth through targeting the m gene of ndv and host drosophila-like rna binding protein 4 (elavl4) [126] . orthomyxoviridae family [127] . other viruses in this family include influenza b and influenza c virus, but only influenza a virus has been found in birds [127, 128] . although avian influenza causes only mild illness, it still can be very dangerous because of its high transmission among birds, and even among human beings [129, 130] . several rna-seq researches had revealed the expression profile and associated pathways in response to aiv infection in different tissues and cells in avian [131] [132] [133] [134] . according to the bioinformatics analysis of the expression and the associated pathways of the demirnas in those researches, mir-34a, mir-122-2, mir-122-1, mir-206, mir-146a, mir-155, mir-1719, mir-1599, mir-1594, mir-451, mir-146, mir-1576, mir-1636, mir-206, mir-142-5p, mir-17-5p, mir-19b mir-133c, mir-1710, mir-181a/b, mir-30b/c/e, and mir-455 were considered to be strong candidate mirnas related to the immune response of aiv infection in chickens [131] [132] [133] [134] . the genome of type a influenza includes eight segments and each segment-encoded protein is crucial for viral replication and pathogenesis [133] . mirnas interact with virus genes is also one approach for mirna to control or resist aiv. many chicken host mirnas have been analyzed to have target relationship with avian influenza [135] . specially, the non-structural protein 1 (ns1) is one protein of segment 8 of h5n1 genome that is directly associated with the pathogenicity of the influenza strain. mir-1658 could regulate the aiv infection by targeting the ns1 gene of h5n1 genome [135] . moreover, pb1 (polymerase pb1), pb1-f2 (pb1-f2 protein), and n40 were three proteins encoded by segment 2 of h5n1 virus. mir-1710, mir-133c, and mir-146c were predicted to target pb1-f2, pb1, and n40 proteins, implying that they potentially regulate h5n1 infecting by interacting with virus genes [133] . reticuloendotheliosis, a reticuloendotheliosis virus (rev)-induced disease, is highly prevalent in avian and human [136] . it can cause chronic b cell and t-cell lymphomas [137] , delayed growth, immunodepression, and tumor in birds [138] . to the best of our knowledge, only few studies related to ncrnas have been investigated during re infections. high-throughput sequencing has been conducted in rev-infected and non-infected tissues or cells and the results identified many candidate demirnas involved in rev infection. these demirnas could target immune-related genes and participate in immune-related pathways [138] [139] [140] . among them, mir-2945, mir-106-3p/5p, mir-29b/3p/, mir-7b, mir-16c-5p/, mir-122-5p, mir-155, mir-18a-5p, mir-147, mir-184-3p, mir-222b-5p/3p, mir-145b-5p, mir-20b-5p, and mir-1b-3p expressions were verified by qrt-pcr. go term analysis showed that these mirnas are related to apoptotic process, intracellular signal transduction, cell death regulation. also, many important kegg pathways were enriched, such as mapk signaling, endocytosis, focal adhesion, apoptosis, cell cycle pathways, mtor signaling, cell growth and death, and immune system. specially, of so many demirnas, the underlying regulatory mechanisms of mir-155 and novel-72 were verified and indicated that they play distinct role in response rev infection. for mir-155, it showed a high expression level during rev infection in cef cells. it has been demonstrated that mir-155 could inhibit apoptosis by targeting caspase-6 and accelerate cell cycle through inhibiting foxo3a and jarid2 (jumonji, at rich interactive domain 2) expression during the rev infection [141, 142] . for novel-72, it showed a significantly lower expression in rev-infected cells. four predicated target genes of novel-72, pdpk1 (phosphoinositide dependent protein kinase-1), mtor (mammalian target of rapamycin), s6k1 (s6 kinase 1), and eif4e (eukaryotic initiation factor 4e) of mtor signaling pathway were related to cell proliferation and survival, indicating that novel-72 could be involved in mtor signaling pathway to mediate the cell fate during rev infection [139] . the selected-verified mirnas and their target genes and their functions during the ibdv, ibd, ndv, aiv, and rev infections were listed below (table 5 ). foxo3a, jarid2 inhibit apoptosis and accelerate cell cycle during the rev infection [141, 142] as avian virus disease has been well studied, the mechanisms that mirnas participate in avian virus diseases are clear now. there are four functional mechanisms have been reported: (1) direct interaction with the host gene ( figure 2a ) [82] ; (2) acts as homologous to virus-encoded mirnas and shares the same target gene with virus-encoded mirnas (figure 2b ) [92] [93] [94] [95] [96] [97] 143] ; (3) interacts with virus genomic gene (figure 2c) [98, 99, [107] [108] [109] ; (4) alters the expression of responsible mirnas through inducing the demethylation of the mirna promoter to protect virus themselves to survival from the immune responses induced by related-mirnas (figure 2d ) [114, 115] . chicken coccidiosis is an epidemic parasitic disease can be caused by nine eimeria spices, including eimeria tenella, necatrix, maxima, acervuline, brunetti, mivati, hagani, praecox, and mitis. they chicken coccidiosis is an epidemic parasitic disease can be caused by nine eimeria spices, including eimeria tenella, necatrix, maxima, acervuline, brunetti, mivati, hagani, praecox, and mitis. they are infecting chicken intestinal epithelial cells. among them, eimeria tenella, eimeria maxima, and eimeria necatrix have the higher pathogenicity. one rna sequence analysis of differentially expressed lncrnas, circrnas, and mrnas has been performed in chicken small intestines during eimeria necatrix infection. this rna sequence analysis obtained 1543 demrnas, 95 delncrnas, 13 decircrnas [144] . functional annotation for lncrnas, mirnas, and circrnas were correlated with chicken host immune defense and pathogenesis during e. necatrix infection [144] . in addition, mirna expression was compared between naturally or eimeria maxima and eimeria acervulina -infected ross 308 broilers using rna sequencing. among demirnas, mir-122-5p, mir-144-3p, and mir-205b were verified by qrt-pcr [145] , however, their potential roles during avian coccidiosis need further investigations. the regulatory mechanisms of ncrnas in response to coccidiosis remain largely unknow. cryptosporidium baileyi (c. baileyi) belongs to cryptosporidium species in birds [146] . it can infect in the epithelial cells of bursa or respiratory tract, causing respiratory diseases [146, 147] . in broiler chickens, it can cause high mortality and morbidity [148] . one research focused on the identification of lncrnas, circrnas, and mrnas involved in c. baileyi infection in chickens' tracheal tissues. it found 124 lncrnas, 104 circrnas, and 1317 mrnas were differentially expressed. go analysis found that the targets and source genes of mrnas and lncrnas are all involved in immune response and immune system process, while kegg analysis showed that they are related to intestinal immune network for iga production, cell adhesion molecules (cams), cell cycle, and the cytokine-cytokine receptor interaction. the circrnas were significantly enriched in pathways associated with tight junction and glycerolipid metabolism, nucleotide sugar metabolism, and amino sugar [149] . however, the underlying molecular mechanism of ncrnas in response to c. baileyi infection needs further investigation. campylobacter jejuni (c. jejuni) is a foodborne pathogen that causes human diarrhea on consuming chicken products which are contaminated by c. jejuni [150] . two studies on ncrnas associated with c. jejuni have been reported [138, 151, 152] . solexa sequencing for cecal tissue from c. jejuni inoculated and non-inoculated spf chicken showed 4 mirnas, mir-155, mir-1416-5p, mir-19b-3p, and mir-19a-3p were significantly differentially expressed [151] . in addition, mir-30b, mir-30c, mir-148a, and mir-1416-5p were able to interact with the socs3 in response to c. jejuni inoculation which indicated they may mediate c. jejuni infection through regulating socs3 [152] . necrotic enteritis is usually caused by clostridium perfringens with symptoms of weight depression, appetite loss, and even cause death to chickens [153] . small rna-seq showed many mirnas were differently expressed after challenged by ne [154] [155] [156] . mir-215, mir-194, mir-217, mir-200a, mir-216a, mir-200b, mir-216b, mir-34b, mir-429, mir-1684, mir-9-5p, mir-196-5p, mir-20b-5p and let-7d were confirmed by qrt-pcr [154, 156] . some mirnas were correlated with immune-related mrna expression levels, such as mir-216 and tgfβr2 (transforming growth factor beta receptor 2), mir-30b/mir-30c/mir-455-5p and socs3, mir-181a/b and cxcl14 (c-x-c motif chemokine ligand 14) , mir-429 and tnfsf11β (tnf superfamily member 11 beta), mir-223 and hsp90β1 (heat shock protein 90 beta family member 1), mir-1329 and nfkbiz (nfkb inhibitor zeta), mir-1674 and arhgef (ferm, arh/rhogef and pleckstrin domain protein 1), mir-30e/ mir-32 and serpinf1 (serpin family f member 1) [155] . many immune-related pathways were identified for demirna targets, such as mapk, erbb, notch, tgf-β, jak-stat, cytosolic etc. specially, jak-stat as the key pathway, 20 genes of this pathway showed marked differential expression, strongly suggesting the crucial role of jak-stat pathway in regulating ne [157] . the mir-200a-3p could regulate inflammatory factor and mapk signaling pathway-related gene in response to ne. for instance, it regulated the expression of il-1β, ifn-γ, il-12p40 (interleukin 12b), il-17a (interleukin 17a), litaf (lipopolysaccharide induced tnf factor), and zak (also known as map3k20-mitogen-activated protein kinase kinase kinase 20), map2k4 (mitogen-activated protein kinase kinase 4), and tgfβ2 (transforming growth factor beta 2) of mapk signaling pathway [158] . the mir-10a could inhibit the expression of myd88-dependent pathway-related genes, including traf6 (tnf receptor associated factor 6), tak1 (tgf-beta activated kinase 1 (map3k7) binding protein 1), nf-κb1 (nuclear factor kappa b subunit 1), and downstream genes of the myd88-dependent pathway related genes, including il-1β, ifn-γ, il-12p40, tnfsf15 (tnf superfamily member 15), and litaf. it strongly indicated that mir-10a regulates the ne through toll-like receptor pathway [159] . salmonella enterica serovar enteritidis (se) is the most common serotype of the salmonella bacteria. chicken se infection is mainly caused by ingesting water or consuming food contaminated with salmonella bacteria. circrnas have been identified through next-generation sequencing during se infection [160] . total of 62 circrnas were significantly differentially expressed (30 upregulated and 32 downregulated) between the control and treated groups. the immune-related pathway associated with the de circrnas including, adrenergic signaling in cardiomyocytes, herpes simplex infection, and signaling pathway of mapk/ p53/vegf/notch. three immune-related genes, txndc9 (thioredoxin domain containing 9), jag2 (jagged canonical notch ligand 2), and nfatc2 (nuclear factor of activated t cells 2), generated 11 de circrnas involved in the b cell receptor signaling pathway, b cell proliferation, drug biological process, and cytokine production [160] . in addition, mir-1306-5p regulated the immune response to se by inhibiting the toll-interacting protein (tollip), which is an up-regulator for inflammatory cytokines-nf-κb, il-6 (interleukin 6), tnf-α, and il-1β (interleukin 1-beta) [161] . mir-101-3p and mir-155 could alter the expression of their target genes, lrrc59 (leucine rich repeat containing 59) and irf4 (interferon regulatory factor 4), and decrease the expression of pro-inflammatory cytokines during se infection [162] . in addition, mir-1662, mir-1416-5p, and mir-34a-5p showed opposite expression pattern with the immune-related target genes of bcl10 (b-cell cll/lymphoma 10), tlr21 (toll-like receptor 21), notch2 (notch 2), tlr1la (toll-like receptor 1 family member a), and thbs1 (thrombospondin 1), indicating their important roles in se infection [163] . salmonella typhimurium (s. typhimurium) commonly occurred in avian and humans [164] . it is a major food-borne pathogen and has an impact on the microbiological safety of eggs which is also a risk factor to humans [165] . a previous study revealed 14 mirnas that were significantly altered by the infection of s. typhimurium [166] . among them, mir-3525, mir-215-5p, mir-193a-5p, mir-122-5p, and mir-375 were verified by qrt-pcr. the predicted demirnas target genes were enriched in immune system development, stress-activated mapk cascade, the regulation of camp-dependent protein kinase activity, and the regulation of immune system process (such as, mapk and wnt signaling pathways) [166] . the selected-verified mirnas and their target genes and their functions during the bacterial infections were listed below (table 6 ). ncrnas are recognized as powerful regulatory molecules and have been largely investigated in various disease process of different species, including avians. the unique functional mechanisms of mirna, lncrna, and circrna were revealed. although the underlying functional mechanism of ncrnas has been revealed in many species, it is still beginning to emerge in response to avian disease, except for mirnas. researches on circrnas and lncrnas are basically performed on the analysis of their expression profile and associated pathways. ncrnas have been found to play roles during the infection of various avian diseases through different approaches. for lncrnas and circrnas, they mainly exhibit their function through lncrna/circrna-mirna-mrna axis, however, in some cases they could also regulate the immune process by directly interacting with rna binding proteins or virus gene sequence. mirnas can regulate various aspects of immune response process. in avian, some specific mirnas were identified to be differentially expressed in many different types of chicken diseases from virus, parasite, and bacteria, such as mir-155 [68, 110, 141, 142] , mir-221/222 [60, 83, 119, 138] , mir-130 [59, 90, 109] , mir-30 [121, 152, 155] , mir-34 [59, 63, 132, 154, 163] , let-7 [57, 81, 155] , mir-181 [77, 134, 155] , mir-16 [80, 115] , mir-455 [125, 134, 155] , etc. it indicated their extensive roles in regulating avian immunity. especially, mir-155 has been reported to play important roles in response to different kinds of avian diseases, such as alv [68] , md [76] , ibd [110] , avian influenza [132] , reticuloendotheliosis [141, 142] , campylobacter jejuni [151] , and also salmonella enterica serovar enteritidis [162] . the functional mechanisms of ncrnas in regulating avian immune system can be categorized into three mechanisms: first, ncrnas interact with pathogenic gene of virus/parasite/bacterium; second, ncrnas act as a homologous to the product of pathogen; third, ncrnas directly interact with host resistant and susceptible genes. epigenetic regulation by long noncoding rnas ribosome profiling provides evidence that large noncoding rnas do not encode proteins classification and function of small open reading frames the translation of non-canonical open reading frames controls mucosal immunity peptides/proteins encoded by non-coding rna: a novel resource bank for drug targets and biomarkers origins and mechanisms of mirnas and sirnas small rnas as guardians of the genome subtractive hybridization identifies novel differentially expressed ncrna species in ebv-infected human b cells non-coding rnas in development and disease: background, mechanisms, and therapeutic approaches long noncoding intronic rnas are differentially expressed in primary and metastatic pancreatic cancer molecular mechanisms of long noncoding rnas a novel role for xist rna in the formation of a repressive nuclear compartment into which genes are recruited when silenced h19 imprinting control region methylation requires an imprinted environment only in the male germ line the human xist gene: analysis of a 17 kb inactive x-specific rna that contains conserved repeats and is highly localized within the nucleus transcription of two long noncoding rnas mediates mating-type control of gametogenesis in budding yeast the non-coding rna terra is a natural ligand and direct inhibitor of human telomerase intergenic transcription is required to repress the saccharomyces cerevisiae ser3 gene methylation of adenine residues in dna of eukaryotes the p53-induced lincrna-p21 derails somatic cell reprogramming by sustaining h3k9me3 and cpg methylation at pluripotency gene promoters particle, a triplex-forming long ncrna, regulates locus-specific methylation in response to low-dose irradiation long noncoding rnas: functional surprises from the rna world control of myogenesis by rodent sine-containing lncrnas a natural antisense transcript regulates zeb2/sip1 gene expression during snail1-induced epithelial-mesenchymal transition but not its lncrna products, induces imprinted igf2r silencing circular rnas are a large class of animal rnas with regulatory potency identification of meccirnas and their roles in the mitochondrial entry of proteins circular rnas in the mammalian brain are highly abundant, conserved, and dynamically expressed kinetic expression analysis of the cluster mdv1-mir-m9-m4, genes meq and vil-8 differs between the lytic and latent phases of marek's disease virus infection translation of yes-associated protein (yap) was antagonized by its circular rna via suppressing the assembly of the translation initiation machinery endoribonucleolytic cleavage of m6a-containing rnas by rnase p/mrp complex circ-znf609 is a circular rna that can be translated and functions in myogenesis novel role of fbxw7 circular rna in repressing glioma tumorigenesis natural rna circles function as efficient microrna sponges exon-intron circular rnas regulate transcription in the nucleus the rna binding protein quaking regulates formation of circrnas extensive translation of circular rnas driven by n6-methyladenosine ires-mediated cap-independent translation, a path leading to hidden proteome how are circrnas translated by non-canonical initiation mechanisms? conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microrna targets mirna-dependent gene silencing involving ago2-mediated cleavage of a circular antisense rna microrna-1224 splicing circularrna-filip1l in an ago2-dependent manner regulates chronic inflammatory pain via targeting ubr5 mettl1 promotes let-7 microrna processing via m7g methylation primary transcripts of micrornas encode regulatory peptides translational dynamics revealed by genome-wide profiling of ribosome footprints in arabidopsis small rna sequences derived from pre-micrornas in the supraspliceosome myotropic avian leukosis virus subgroup j infection in a chicken a novel subgroup of exogenous avian leukosis virus in chickens the long view: 40 years of marek's disease research andavian pathology circular rna alterations are involved in resistance to avian leukosis virus subgroup-j-induced tumor formation in chickens circular rna and mrna profiling reveal competing endogenous rna networks during avian leukosis virus, subgroup j-induced tumorigenesis in chickens circular rna vav3 sponges gga-mir-375 to promote epithelial-mesenchymal transition expression patterns of novel circular rnas in chicken cells after avian leukosis virus subgroup j infection integrated host and viral transcriptome analyses reveal pathology and inflammatory response mechanisms to alv-j injection in spf chickens comprehensive transcriptome analysis reveals competing endogenous rna networks during avian leukosis virus, subgroup j-induced tumorigenesis in chickens gene expression profile and long non-coding rna analysis, using rna-seq, in chicken embryonic fibroblast cells infected by avian leukosis virus long non-coding rna and microrna profiling provides comprehensive insight into non-coding rna involved host immune responses in alv-j-infected chicken primary macrophage aberrant expression of liver microrna in chickens infected with subgroup j avian leukosis virus differential expression of micrornas in avian leukosis virus subgroup j-induced tumors subgroup j avian leukosis virus infection of chicken dendritic cells induces apoptosis via the aberrant expression of micrornas expression of dysregulated mirna in vivo in df-1 cells during the course of subgroup j avian leukosis virus infection temporal changes of microrna gga-let-7b and gga-let-7i expression in chickens challenged with subgroup j avian leukosis virus microrna-23b promotes avian leukosis virus subgroup j (alv-j) replication by targeting irf1 mir-34b-5p suppresses melanoma differentiation-associated gene 5 (mda5) signaling pathway to promote avian leukosis virus subgroup j (alv-j)-infected cells proliferaction and alv-j replication role of gga-mir-221 and gga-mir-222 during tumour formation in chickens infected by subgroup j avian leukosis virus avian leukosis virus subgroup j promotes cell proliferation and cell cycle progression through mir-221 by targeting cdkn1b immunological tolerance in chickens hatching from eggs injected with cell-associated herpesvirus of turkey (hvt) marek's disease virus: lytic replication, oncogenesis and control genome-wide analysis of circular rnas involved in marek's disease tumourigenesis in chickens the national registry of genetically unique animal populations: usda-adol chicken genetic lines the conservation and signatures of lincrnas in marek's disease of chicken linc-galmd1 regulates viral gene expression in the chicken integrated analysis of lncrna and mrna repertoires in marek's disease infected spleens identifies genes relevant to resistance long intergenic non-coding rna galmd3 in chicken marek's disease differential expression of micrornas in marek's disease virus-transformed t-lymphoma cell lines a systematic analysis of mirna transcriptome in marek's disease virus-induced lymphoma reveals novel and differentially expressed mirnas mirna expression signatures induced by marek's disease virus infection in chickens distinct expression pattern of mirnas in marek's disease virus infected-chicken splenic tumors and non-tumorous spleen tissues microrna profile of marek's disease virus-transformed t-cell line msb-1: predominance of virus-encoded micrornas the inhibitory effects of gga-mir-199-3p, gga-mir-140-3p, and gga-mir-221-5p in marek's disease tumorigenesis the cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy micrornas 221 and 222 target p27kip1 in marek's disease virus-transformed tumour cell line msb-1 retinoic acid-related orphan receptors (rors): regulatory functions in immunity, development, circadian rhythm, and metabolism rorα, a potential tumor suppressor and therapeutic target of breast cancer effect of gga-mir-155 on cell proliferation, apoptosis and invasion of marek's disease virus (mdv) transformed cell line msb1 by targeting rora chicken gga-mir-181a targets mybl1 and shows an inhibitory effect on proliferation of marek's disease virus-transformed lymphoid cell line gga-mir-26a targets nek6 and suppresses marek's disease lymphoma cell proliferation chicken gga-mir-103-3p targets ccne1 and tfdp2 and inhibits mdcc-msb1 cell migration chicken gga-mir-130a targets hoxa3 and mdfic and inhibits marek's disease lymphoma cell proliferation and migration gga-mir-219b targeting bcl11b suppresses proliferation, migration and invasion of marek's disease tumor cell msb1 sequence conservation and differential expression of marek's disease virus micrornas ortholog encoded by the oncogenic marek's disease virus marek's disease virus microrna designated mdv1-pre-mir-m4 targets both cellular and viral genes activation of toll-like receptor 3 inhibits marek's disease virus infection in chicken embryo fibroblast cells roles of avian herpesvirus micrornas in infection, latency, and oncogenesis micrornas of gallid and meleagrid herpesviruses show generally conserved genomic locations and are virus-specific the oncogenic microrna oncomir-21 overexpressed during marek's disease lymphomagenesis is transactivated by the viral oncoprotein meq expression of the env gene from the avian endogenous retrovirus alve and regulation by mir-155 infectious bursal disease: a complex host-pathogen interaction infectious bursal disease virus of chickens: pathogenesis and immunosuppression dendritic cells and the control of immunity genome-wide profiling of chicken dendritic cell response to infectious bursal disease the enhanced virulence of very virulent infectious bursal disease virus is partly determined by its b-segment microrna-21 targets the tumor suppressor gene tropomyosin 1 (tpm1) microrna-21 targets tumor suppressor genes in invasion and metastasis overexpression of microrna gga-mir-21 in chicken fibroblasts suppresses replication of infectious bursal disease virus through inhibiting vp1 translation gga-mir-454 suppresses infectious bursal disease virus (ibdv) replication via directly targeting ibdv genomic segment b and cellular suppressors of cytokine signaling 6 (socs6) microrna gga-mir-130b suppresses infectious bursal disease virus replication via targeting of the viral genome and cellular suppressors of cytokine signaling 5 gga-mir-155 enhances type i interferon expression and suppresses infectious burse disease virus replication via targeting socs1 and tank gga-mir-9* inhibits ifn production in antiviral innate immunity by targeting interferon regulatory factor 2 to promote ibdv replication gga-mir-142-5p attenuates irf7 signaling and promotes replication of ibdv by directly targeting the chmda5 s 3 untranslated region gga-mir-2127 downregulates the translation of chicken p53 and attenuates chp53-mediated innate immune response against ibdv infection gga-mir-27b-3p enhances type i interferon expression and suppresses infectious bursal disease virus replication via targeting cellular suppressors of cytokine signaling 3 and 6 (socs3 and 6) epigenetic upregulation of chicken microrna-16-5p expression in df-1 cells following infection with infectious bursal disease virus (ibdv) enhances ibdv-induced apoptosis and viral replication global distributions and strain diversity of avian infectious bronchitis virus: a review whole-genome characterization of uruguayan strains of avian infectious bronchitis virus reveals extensive recombination between the two major south american lineages microarray analysis of infectious bronchitis virus infection of chicken primary dendritic cells microrna transcriptome analysis in chicken kidneys in response to differing virulent infectious bronchitis virus infections mir-146a-5p promotes replication of infectious bronchitis virus by targeting irak2 and tnfrsf18 gga-mir-30d regulates infectious bronchitis virus infection by targeting usp47 in hd11 cells delayed newcastle disease virus replication using rna interference to target the nucleoprotein common microrna-mrna interactions in different newcastle disease virus-infected chicken embryonic visceral tissues gga-mir-19b-3p inhibits newcastle disease virus replication by suppressing inflammatory response via targeting rnf11 and zmynd11 microrna gga-mir-455-5p suppresses newcastle disease virus replication via targeting cellular suppressors of cytokine signaling 3 mir-375 has contrasting effects on newcastle disease virus growth depending on the target gene evolution and ecology of influenza a viruses overview of influenza viruses a review of avian influenza in different bird species the translation into spanish of the oie manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees): problems, solutions and conclusions identification of differentially expressed mirnas in chicken lung and trachea with avian influenza virus infection by a deep sequencing approach integrated analysis of microrna expression and mrna transcriptome in lungs of avian influenza virus infected broilers identification of chicken pulmonary mirnas targeting pb1, pb1-f2, and n40 genes of highly pathogenic avian influenza virus h5n1 in silico xiang, m. micrornas in avian influenza virus h9n2-infected and non-infected chicken embryo fibroblasts in-silico search of virus-specific host micrornas regulating avian influenza virus ns1 expression poultry oncogenic retroviruses and humans retrovirus-induced disease in poultry analysis of microrna expression profile in specific pathogen-free chickens in response to reticuloendotheliosis virus infection integrative analyses of transcriptome sequencing identify functional mirnas in the chicken embryo fibroblasts cells infected with reticuloendotheliosis virus integrated analysis of mirna and mrna expression profiles in spleen of specific pathogen-free chicken infected with avian reticuloendotheliosis virus strain snv reticuloendotheliosis virus strain t induces mir-155, which targets jarid2 and promotes cell survival regulatory mechanism of microrna-155 in chicken embryo fibroblasts in response to reticuloendotheliosis virus infection microrna 29b functions in acute myeloid leukemia genome-wide analysis of differentially expressed profiles of mrnas, lncrnas and circrnas in chickens during eimeria necatrix infection. parasites vectors diagnosis of sub-clinical coccidiosis in fast growing broiler chickens by microrna profiling the life cycle of cryptosporidium baileyin. sp. (apicomplexa, cryptosporidiidae) infecting chickens cryptosporidium baileyiinfection associated with an outbreak of ocular and respiratory disease in otus owls (otus scops) in a rehabilitation centre pathobiology of cryptosporidiosis (c. baileyi) in broiler chickens genome-wide analysis of differentially expressed profiles of mrnas, lncrnas and circrnas during cryptosporidium baileyi infection campylobacter jejuni-an emerging foodborne pathogen chicken cecal micrornas in the response to campylobacter jejuni inoculation by solexa sequencing correlation between mirnas and target genes in response to campylobacter jejuni inoculation in chicken identification and cloning of two immunogenic clostridium perfringens proteins, elongation factor tu (ef-tu) and pyruvate:ferredoxin oxidoreductase (pfo) of c. perfringens modulation of micrornas in two genetically disparate chicken lines showing different necrotic enteritis disease susceptibility differential regulation of microrna transcriptome in chicken lines resistant and susceptible to necrotic enteritis disease distribution and differential expression of micrornas in the intestinal mucosal layer of necrotic enteritis induced fayoumi chickens differentially expressed jak-stat signaling pathway genes and target micrornas in the spleen of necrotic enteritis-afflicted chicken lines microrna gga-mir-200a-3p modulates immune response via mapk signaling pathway in chicken afflicted with necrotic enteritis microrna gga-mir-10a-mediated transcriptional regulation of the immune genes in necrotic enteritis afflicted chickens cecal circrnas are associated with the response to salmonella enterica serovar enteritidis inoculation in the chicken chicken gga-mir-1306-5p targets tollip and plays an important role in host response against salmonella enteritidis infection splenic microrna expression profiles and integration analyses involved in host responses to salmonella enteritidis infection in chickens cecal micrornaome response to salmonella enterica serovar enteritidis infection in white leghorn layer salmonella typhimurium dt104: a virulent and drug-resistant pathogen a critical review ofsalmonellatyphimurium infection in laying hens involvement of micrornas in probiotics-induced reduction of the cecal inflammation by salmonella typhimurium key: cord-307899-427a7i3h authors: bittle, james l.; muir, susie title: vaccines produced by conventional means to control major infectious diseases of man and animals date: 1989-12-31 journal: advances in veterinary science and comparative medicine doi: 10.1016/b978-0-12-039233-9.50005-6 sha: doc_id: 307899 cord_uid: 427a7i3h publisher summary this chapter reviews the development of some of vaccines and their use in controlling such major diseases as diphtheria, rinderpest, newcastle disease, smallpox, pertussis, yellow fever, rabies, etc. park–williams number 8 (pw8) strain is used to make diphtherial toxoid for vaccines. as a source of toxin, it is rendered nontoxic by incubation with formalin under alkaline conditions. the product's retention of antigenicity, enabling it to induce antitoxin antibodies, makes it an excellent pediatric vaccine. vaccine against rinderpest virus was developed by koch in 1897 by administering bile from infected cattle. animals that survived were permanently immune. formalinand chloroform-inactivated vaccines were developed using tissues from the infected animals. for the control of newcastle disease, a number of attenuated live-virus vaccines have been developed which are widely used to control the disease. the bl strain, the lasota strain, and the f strain are used to immunize birds of all ages by different routes, including by addition to drinking water and by spraying. protection against rabies correlates with sn antibody, which can be assessed by a number of tests. pasteur's classical vaccine, developed from infected spinal cord tissue dried at room temperature for 3–14 days, was given in a series of 21–28 inoculations beginning with material dried the longest and progressing through material dried for only 3 days. the first parvovirus shown to be a filterable agent was feline panleukopenia virus (verge and christoforoni, 1928) , which was not characterized until much later (johnson, 1967a,b; johnson et al., 1967) . the virus affects most members of the family felidae, causing enteritis and bone marrow hypoplasia that results in severe leukopenia. the virus infects kittens in utero and postnatally causes cerebellar hypoplasia and ataxia (kilham and margolis, 1966) . immunization. the first panleukopenia vaccines were produced by infecting susceptible cats and harvesting their tissues. filtrates from these tissue suspensions were treated with chemical inactivants such as formalin. two inoculations of this type of vaccine produced longlasting protection (leasure et al., 1934; enders and hammond, 1940) . however, subsequent vaccines, produced in primary feline kidney cell culture and inactivated with formalin, proved safer and more effective (bittle et al., 1970; davis et al., 1970 ). an attenuated live-virus vaccine was also very effective in inducing immunity and protection (slater and kucera, 1966) . concern for the wide-scale use of an attenuated live-virus vaccine centers on the premise that shedding may spread the virus to other species, possibly allowing the emergence of pathogenic variants in these species. attempts to immunize cats orally with attenuated live-virus vaccines have not been successful, but intranasal aerosol application has been effective (scott and glauberg, 1975; schultz et al., 1973) . three members of the parvovirus family are known to infect dogs: minute virus of canines, a defective canine adeno-associated virus, and the pathogenic canine parvovirus. canine parvovirus is related to feline panleukopenia virus and may have originated from one of the mammalian parvoviruses. in young dogs, the virus causes leukopenia and severe intestinal disease with necrosis of crypt epithelium in the small intestine. myocarditis occasionally develops, causing sudden heart failure in young dogs. in 1978 an epizootic of this disease occurred in the united states, causing high mortality (eugester, 1978) . immunization. because of the close relationship between feline panleukopenia virus and canine parvovirus, inactivated feline panleukopenia vaccine initially was used to protect susceptible dog populations (appel et al., 1979a) . later, inactivated and attenuated vaccines produced with the canine virus proved to be much more effective (pollack and carmichael, 1983; appel et al., 1979b) . these parvovirus vaccines have been formulated with other vaccines, including canine distemper and canine adenovirus vaccines, and are administered routinely to young dogs. porcine parvovirus has a distant serologic relationship to other parvoviruses, but it causes disease only in swine. the virus, which is widespread, causes abortions, fetal death, and infertility in sows infected early in gestation. immune sows reinfected during gestation give birth to normal piglets. immunization. numerous reports have shown the effectiveness of inactivated porcine parvovirus vaccines in swine (suzuki and fujsaki, 1976; mengeling et al., 1979; wratthal et al, 1984; fujisaki et al., 1978; joo and johnson, 1977) . for example, mengeling et al. (1979) demonstrated the value of a vaccine in which the virus was inactivated with acetylethyleneimine. an attenuated live-virus vaccine has been described (paul and mengeling, 1980) in which a porcine isolate was attenuated by 120-165 passages in a swine testicular cell line. this vaccine effectively induced antibodies and protected challenged animals. the vaccine virus did not cross the placenta; however, it did kill fetuses when inoculated in utero. the virus was shed in feces, and so could be transmitted to unvaccinated animals. the inactivated vaccines now used in united states have been effective in controlling porcine parvovirus infection. vaccination is recommended for gilts and sows before breeding. adenoviruses cause significant disease in dogs, foxes, and man, but have also been isolated from cattle, swine, goats, sheep, horses, turkeys, and chickens, where they produce mild infections, mainly associated with the respiratory and intestinal tracts. there are at least 80 different adenoviruses, 43 of which occur in man. each adenovirus has a narrow host range. there are two canine adenoviruses. canine adenoviruses 1 (cav-1) causes infectious canine hepatitis, which at one time was widespread, but now has been controlled by vaccination. infected dogs also develop corneal opacities following this infection, as a result of the formation of immune complexes and uveitis within the anterior chamber of the eye (carmichael et al., 1975) . in foxes, cav-1 produces a rapidly fatal encephalitis. canine adenovirus 2 (cav-2) causes respiratory disease in dogs, but neither hepatitis nor encephalitis in dogs or foxes. the respiratory disease varies depending on the strain of virus and bacterial superinfection. cav-2 may be transmitted by aerosol, whereas cav-1 is spread by other direct means such as contact with urine or saliva from infected animals. cav-1 and cav-2 are oncogenic in experimentally infected hamsters (sarma et al., 1967; dulcac et al., 1970) . immunization. dogs that recover from natural cav-1 infection are immune for a long period. the first cav-1 vaccines were produced by formalin inactivation of tissue homogenates from infected dogs. cav-1 was first adapted to tissue culture by cabasso et al. (1954) and by fieldsteel and emery (1954) . the latter modified the virus by serial passage in porcine and canine tissue cultures; the resulting vaccine immunized dogs and did not produce clinical signs of infection except for occasional corneal opacity similar to that caused by natural infection. dogs immunized with cav-1 vaccines are also protected against cav-2 (appel et al., 1973) . the immunity produced by the attenuated live-virus cav-1 vaccines is long lasting and has drastically reduced the incidence of the canine disease. although cav-2 is closely related to cav-1, when it is inoculated parenterally into dogs, it does not cause disease, although the virus is shed from the respiratory tract (appel et al., 1973) . such dogs become immune to both cav-1 and cav-2. an attenuated live-virus vaccine containing cav-2 is now being widely used in place of older cav-1 vaccines (bass et al., 1980) ; this has resulted in a much lower incidence of corneal opacity in recipients. however, because of the oncogenicity of adenoviruses in other hosts, the respiratory shedding of cav-2 virus should be a concern. in man, adenoviruses mainly produce disease of the respiratory tract which varies in severity depending on the virus and the age of infected individuals. the viruses cause acute pharyngitis in infants and children, pharyngitis and conjunctivitis in children, and acute respiratory disease (ard) in military recruits and institutionalized young adults. pneumonia may also occur, expecially following ard. a vaccine consisting of adenovirus types 3, 4, and 7, grown in monkey kidney cell culture and inactivated with formalin, was introduced in 1958 for use in u.s. military recruits. the vaccine was effective in reducing ard in this population (sherwood et al., 1961) . however, the vaccine was withdrawn from use in 1963 because of concern for possible oncogenicity of the adenoviruses, and of the sv 40 virus present in the monkey kidney cell culture. a subsequent adenovirus, type 4, was passed in human tissue and was, therefore, devoid of sv 40 genomic material. this virus, encapsulated in enteric-coated capsules, proved to be a safe and effective vaccine edmonson et al., 1966 ). an adenovirus 7 vaccine, prepared in the same way and given simultaneously with adenovirus 4 vaccine, was equally effective (top et al., 1971) . the administration of a vaccine with only one of these viruses was not effective in controlling ard caused by any of several different adenoviruses, but formulation of a vaccine with both viruses proved to be broadly cross-protective and have had a major influence in controlling ard in u.s. military recruits. over 70 herpesviruses produce disease in man and animals. these viruses have an affinity for epithelial tissues and nervous system tissues. these tropisms lead to specific disease syndromes involving the respiratory and urogenital tracts, and the central and peripheral nervous systems. the viruses often cause persistent infections, which can be latent and can be reactivated. herpesvirus disease are generally much more severe in young humans and animals. some herpes-viruses, including epstein-barr virus (ebv) in humans and marek's disease virus in fowl, are associated with malignancies. the presence of specific antibodies may prevent or modify the clinical disease but does not prevent infection. vaccines have been developed for those herpesviruses causing major diseases in animals; however, despite the seriousness of human herpesvirus diseases, including those caused by herpes simplex virus, ebv virus, cytomegalovirus, and varicella virus, progress has been slow in developing vaccines for humans. this stems from concern over possible persistence and oncogenicity of vaccine viruses. in the past few years, several attenuated live-virus varicella vaccines have been tested and found safe and efficacious (takahashi et al., 1974; asano et al., 1977; arbeter et al, 1983) . since the initial recognition of infectious bovine rhinotracheitis (ibr) in the early 1950s and the later recognition of another manifestation of the disease, infectious pustular vulvovaginitis, this bovine herpesvirus has been acknowledged as a major problem in livestock. the respiratory disease varies from mild to severe, and herd mortality can be as high as 10% in an acute outbreak. the virus may cause abortions in pregnant cattle, and the genital disease results in a chronic vulvovaginitis but not abortions, apparently due to a lack of viremia. immunization. attenuated live-virus vaccines were developed by serial passage of field isolates in bovine kidney cell cultures. such vaccine viruses have reduced virulence when administered intranasally and do not produce disease when administered parenterally york et al., 1957) . vaccine virus does not spread from vaccinated to unvaccinated contact animals. the widescale use of such vaccines has controlled this disease effectively. when administered by the intranasal route (todd et al., 1971) , these vaccines had the advantage of producing more rapid protection, but long-term protection was no greater than with perenterally administered vaccines (mckercher and crenshaw, 1971) . because of the possibility that the attenuated live-virus vaccines given parenterally might cause abortion, their use has been restricted to nonpregnant animals. a formalin-inactivated vaccine has also been developed, but requires multiple inoculations, and the serum neutralizing (sn) antibody response is low (matsuaba et al., 1972) . however, inactivated vaccines are used especially in dairy cattle because of concern that the attenuated live-virus vaccines may cause abortion. four herpesviruses affect horses: equine rhinopneumonitis virus (ehv-1) causes abortion which may be epizootic, and also, occasionally, rhinopneumonitis; ehv-2 is cytomegalovirus found in buffy coat cells of most horses, but its role in causing disease is unknown; ehv-3 causes equine coital exanthema, a urogenital tract disease; and ehv-4 is the main cause of equine rhinopneumonitis. ehv-1 and -4 are related antigenically, whereas types 2 and 3 are distinct (sabine et al., 1981; studdert and blackney, 1979) . immunization. the first vaccine for equine rhinopneumonitis was developed by doll and bryans (1963) , who adapted an ehv-1 isolate to suckling hamsters. this vaccine produced a mild disease, but induced protective immunity against the more serious respiratory disease and abortion that occurs in older animals. however, this vaccine sometimes caused abortions, and the virus was known to spread from vaccinated to unvaccinated horses. a more attenuated strain of ehv-1 has been used widely and is considered to be reasonably effective (mayr, 1970) . this strain was attenuated by passage in hamsters and in pig kidney cell culture before adaptation to an equine cell line (gerber et al., 1977) . this vaccine induces low levels of sn antibodies, and protects against respiratory disease but not against abortion. a formalin-inactivated vaccine, emulsified in an oil adjuvant, has also been found safe and effective in preventing respiratory disease. this vaccine, in controlled field trials, lowered the abortion rate significantly (bryans, 1980; bryans and allen, 1981) . the attenuated live-virus and the inactivated vaccines contain only ehv-1, but apparently there is enough cross-protection induced by repeated vaccinations to protect against the ehv-4 respiratory disease. this herpesvirus is unusual in that it occurs naturally in many species-cattle, sheep, goats, swine, dogs, cats, rats, and mice. it produces a fatal disease in all of these species except adult swine, in which the disease is mild. in each species except swine the primary sign is intense puritis resulting in the animal biting the affected area. infection rapidly spreads to the central nervous system, leading to paralysis and death. in adult swine, the signs of infection are mild, usually of a respiratory nature, but abortions follow in approximately 50% of pregnant sows. in young pigs, especially neonates, the infection may be fatal. since the disease is prevalent only in swine, this is the only animal for which a vaccine has been developed. in examining a virulent strain of pseudorabies virus, bartha and kojnok (1963) found two plaque sizes. the small plaque size variant, called k, occurred naturally and had reduced virulence for rabbits. further studies with this strain passaged in chick embryo or calf testicle cell culture produced a safe vaccine for swine. one dose induced partial immunity and a second dose yielded good immune responses in all recipients. mcferran and dow (1975) adapted the κ strain to vero cells and showed that one dose of a vaccine prepared from this passage material was protective. pigs vaccinated with this vaccine did not shed the virus. although the vaccine did not prevent infection, it prevented clinical disease. this attenuated live-virus vaccine is used widely. feline rhinotracheititis virus, which was first isolated in 1957, produces a widespread respiratory disease in cats (crandell and maurer, 1958) . the virus may also cause fetal death. immunization. an attenuated live-virus vaccine was developed by passage of a field isolate in feline kidney cells (bittle and rubic, 1975) . the vaccine is given parenterally and is safe and effective (scott, 1977) . low levels of sn antibodies persist for at least 6 months. vaccinated cats are resistant to intranasal instillation of virulent virus; they may be reinfected, but do not develop clinical disease (bittle and rubic, 1976; kahn et al., 1975) . the vaccine has controlled this important respiratory pathogen and, when combined with a feline calicivirus vaccine, has drastically reduced the incidence of respiratory disease in this species. this herpesvirus is highly contagious and causes lesions in the larynx, trachea, and bronchi of infected fowl. the infection causes the formation of an exudate that produces the characteristic respiratory distress and rattling in severely affected birds. birds that recover from this disease are immune for a long period, but may remain as carriers and a source of virus for reinfection of flocks. immunization. the earliest method of immunization was developed by beaudette and hudson (1933) , who applied virulent virus from tracheal exudate to the mucosa of the bursa of fabricius and the cloaca with a stiff brush. this produced a local infection and a solid systemic immunity. the use of fully virulent virus caused occasional outbreaks of disease, particularly when the scarification was not properly done or insufficient virus was present, and birds did not become immune. the virus was propagated on the chorioallantoic membrane of embryonated eggs by burnet (1934) , and this became a source of vaccine material. other methods of vaccination involved intranasal instillation (benton et al., 1958) and feeding in drinking water (zamberg et al., 1971) . attenuated strains of ltv have been developed by serial passage in cell culture (gelenczei and marty, 1964) and by feather follicle passage (molgard and cavett, 1947) . attenuated strains isolated from outbreaks or selected from passage are now used in preference to virulent virus. marek's disease virus causes lymphoproliferative disease in chickens, occurring in three forms: neural, ocular, and visceral (the latter mainly in young birds) (sevoian and chamberlain, 1962; biggs and payne, 1963) . sevoian was the first to provide evidence that marek's tumors were caused by a virus and were transmissible. the virus has been established as a gamma herpesvirus (churchill and biggs, 1967; soloman et. al., 1968) . immunization. fatalities from marek's disease caused major economic losses in the poultry industry until a vaccine was developed for its control. this was accomplished by churchill et al. (1969) , who attenuated by serial passage a virus isolated from chickens that is parenterally administered at 1 day of age. thereafter, okazaki et al. (1970) selected a herpesvirus from turkeys (hvt) that was relatively avirulent in chickens, and zander et al. (1972) and schat and calnek (1978) , selected a natural avirulent strain from chickens. these three vaccines are effective, but the hvt strain has been more widely used because it can be obtained from infected cells and can be lyophilized (calnek et al., 1970) . the vaccines are given parenterally to chicks at hatching and produce good protection (80-100%) against virulent virus challenge (purchase et al., 1972) . viruses of the family poxviridae infect most domestic animals and man. from the standpoint of immunoprophylaxis, the most important poxviruses are: orthopoxvirus, smallpox (variola), mousepox (ectromelia); avipoxvirus, fowlpox, pigeonpox; capripoxvirus, sheeppox, goatpox; leporipoxvirus, myxomatosis virus; parapoxvirus, contagious pustular dermatitis virus. all these poxviruses cause serious disease in their primary host species and some may infect other species. each of the poxviruses causes characteristic vesicular lesions on the skin and mucous membranes, with the exception of myxomatosis virus which produces hyperplastic lesions in the form of myxomas and fibromas. ectromelia (mousepox) is caused by a virus closely related to vaccinia virus and produces a serious disease of laboratory mice. vaccination with vaccinia apparently will reduce the morbidity and mortality of mousepox in a colony, but it will not prevent infection and may act to maintain a silent reservoir of virus (buller and wallace, 1985) . sheeppox is one of the most serious pox diseases, occurring in europe, the middle east, and africa, but it is controlled by vaccination (aygun, 1955; sabban, 1955) . goatpox occurs mainly in the middle east and africa; a goatpox vaccine has been reported to also immunize against sheeppox (rafyi and ramyar, 1959) . contageous pustular dermatitis virus is unrelated to sheeppox, but causes a pox-like disease in young lambs in which vesicles form around the skin of lips, nostrils, and eyes. boughton and hardy (1935) showed that animals could be protected by scarification with dried contageous pustullar dermatitis virus material similar to that use with vaccinia. myxomatosis virus causes a fatal disease of domestic rabbits and may be spread by direct contact or by blood meals of insects such as mosquitos and fleas (myers et. al., 1954) . mckercher and saito (1964) developed a safe and efficacious attenuated live-virus vaccine by passage of the virus in rabbit kidney cell culture. this virus, once the cause of epidemics that decimated entire cities, has now been eradicated. the control was brought about by worldwide vaccination and isolation of infected persons. another factor in the control of smallpox was that variola virus had no other host except man. immunization. material from lesions of smallpox-infected individuals had been used for centuries to infect susceptible persons so they would develop a mild form of the disease and become resistant. this variolation was dangerous but much safer than natural exposure to the smallpox virus. jenner (1798) practiced an improved form of this method by using cowpox virus (vaccinia) to inoculate susceptible persons, as described in chapter 6. most vaccinia vaccines were produced by scarifying the skin of a calf with infected material and harvesting the lymph from the crusted lesions as aseptically as possible. this was stored in 40% glycerol to stabilize the virus and preservatives were added to destroy bacteria. sheep and rabbits were also used similarly for vaccine production. vaccinia virus was also adapted to grow in embryonated eggs (goodpasture et al., 1935) . vaccinia virus is very stable, can be produced at a low cost, and is simple to administer. these factors played a major role in allowing the wide-scale use of vaccinia for the eradication of smallpox. fowlpox virus occurs mainly in chickens and produces pox lesions on the wattles, comb, mouth, nostrils, and eyes. the disease is spread mainly by direct contact with infected birds and blood-sucking parasites such as mosquitoes. although it is a fairly resistant virus, it is not otherwise very transmissible. immunization. fowlpox vaccine was originally made by scarifying cockrel combs with virulent virus and harvesting the exudate. johnson (1929) demonstrated that dried exudate would produce immunity when scarified in the wing web or applied to the thigh skin free of feathers. fowlpox virus was later cultivated on the chorio allontoic membrane by goodpasture et al. (1931) and used as a source of vaccine material. later the virus was adapted to tissue culture. an attenuated live-virus fowlpox vaccine produced in cell culture may be used in 1-day-old chicks (siccardi, 1975) . another vaccine, an attenuated strain (hp-1) developed by mayr et al. (1976) , is given orally to 5-day-old chicks, then repeated after 3 to 4 months. hepadnaviridae is a new-found family of viruses containing hepatitis β virus of man as well as three similar but distinct viruses that infect woodchucks, beechey ground squirrels, and pekin ducks. these viruses have many of the same ultrastructural, molecular, and biological features and their surface antigens cross-react to a small, variable degree. the host range appears to be specific for each virus. hepatitis β infects man and certain higher primates, including the chimpanzee and gibbon. infection with these hepadnaviruses results in subacute hepatitis, which often becomes progressive and chronic. the most important of these viruses is hepatitis β virus, which produces a chronic disease in man (blumberg et al., 1967; prince, 1968) . hepatitis β virus is transmitted by blood, saliva, and semen, but also from mother to offspring, the latter route accounting for as much as one-third of persistent infections. the disease is usually selflimiting, but in 5-10% of patients the infection becomes chronic, with the virus persisting in a carrier state. there are over 200 million chronic carriers of this virus worldwide. a late sequela in chronic carriers is hepatocellular carcinoma. it is estimated that 40-60% of malignancies in africa are the result of hepatitis b-induced oncogenesis. immunization. the development of a vaccine for hepatitis β was hampered by the difficulty of growing the virus in cell culture. krugman et al. (1970) was the first to show that hepatitis β virusinfected serum could be heat-inactivated and retain its antigenicity. they also showed that this inactivated serum given parenterally could protect subjects exposed to virulent hepatitis β virus. this led to the use of plasma from infected but healthy virus carriers as the source of antigen. carriers produce large quantities of the hepatitis β virus, along with its outer coat protein. by purifying and inactivating the coat protein, a safe and effective vaccine was developed (hilleman et al., 1978) . the coat protein, naturally formed into 22-nm particles, was purified by ammonium sulfate concentration, isopycnic banding, rate zonal separation and enzymatic digestion. the purified protein particles were then inactivated with 1:4000 formalin. the particles induce good levels of protective antibody when given in a series of three injections (symuness et al., 1980) . however, the high cost of this vaccine has limited its use. newer vaccines produced by recombinant dna methods are now being used, as described in other chapters. the four genera in the family picornaviridae are: aphthovirus, rhinovirus, enterovirus, and cardiovirus. viruses in the first three genera cause important diseases in domestic animals and man, whereas viruses in the fourth infect rodents. the picornaviruses in general have a primary affinity for superficial tissues especially of the digestive and respiratory tracts. viruses in the first three genera also have an ability to mutate, thus yielding many serotypes. rhinoviruses cause clinical disease in man, horses, and cattle. no vaccines have been developed for the infections of humans because of the multiplicity of viral serotypes. the number of serotypes in horses (three) and cattle (two) is fewer; nevertheless, no vaccines are available. over 100 enteroviruses exist; of these, vaccines have been developed only for poliomyelitis viruses, avian encephalomyelitis, and duck hepatitis viruses. other viruses in this group either are of low pathogenicity or the number of serotypes is so large as to preclude the development of vaccines. the exception is human hepatitis a virus, which causes a serious disease and has one serotype; the development of both inactivated virus and attenuated live-virus vaccines is in progress (hilleman et al., 1982; provost et al., 1983) . fmd was the first animal disease shown to be caused by a virus (loeffler and frosch, 1898) . fmd viruses cause one of the most economically important diseases of animals and its control is critical to the world's supply of animal protein. the viruses are widespread and occur in many cattle producing regions of the world. the viruses also affect other cloven-footed animals including sheep, swine, and goats. fmd virus infection produces vesicles on oral mucous membranes, including the tongue, gums, and dental pads, but also on the skin including the interdigital spaces and teats. these vesicles on the mucous membranes coalesce and erupt, leaving large denuded areas. the mucous membrane and skin lesions can incapacitate an animal for weeks, thus severely disrupting its productivity. the viruses are highly contagious and persist for long periods in infected animals. animals that recover from natural infection are immune for approximately one year. vallee and carre (1922) showed that there was more than one fmd virus; seven serotypes with over 60 subtypes have now been identified, making the development of effective vaccines difficult. immunization. the first fmd vaccine for cattle was reported in 1925 and consisted of a formalinized emulsion of vesicular epithelium (vallee et al., 1925) . a similar but improved version called the schmidt-waldmann vaccine followed and contained vesicle material from the tongue epithelium of infected cattle. this material was treated with formalin and used with aluminum hydroxide (schmidt, 1936; waldmann and klobe, 1938) . another advance was described by frenkel (1947) , who infected superficial layers of bovine tongue epithelium in culture and inactivated the newly replicated virus with formalin to produce a more uniform product. although this method is used today in some areas of the world, most fmd vaccines are now produced by growing the virus in baby hampster kidney cells (mac-pherson and stoker, 1952; mowat and chapman, 1962; capstick et al., 1962) . the imines replaced formalin as an inactivant in most fmd vaccines after brown et al. (1963) showed that viral inactivation was more complete, and safer vaccines could be produced by this process. all inactivated fmd vaccines contain more than one serotype, including the serotypes most common in the area in which the vaccines are to be used. although inactivated vaccines that are produced and used properly can effectively lead to the control fmd, their stability could be improved, thereby lowering their cost. this is discussed further in the chapter by brown. attenuated live-virus fmd vaccines have been developed (henderson, 1978) but are not used because of the fear that the virus might persist in animals and in meat and milk products from animals (hyslop, 1966 (hyslop, -1967 . there are three polio viruses and minor variants of each. the viruses infect man by entry into the upper alimentary tract, infecting cells, and spreading via the blood to the central nervous system, producing neuronal destruction in the medulla and spinal cord. the degree of paralysis that follows infection depends on such factors as virus strain and virus tropism. the vast majority of persons infected with wild polio viruses show no apparent clinical disease. paralysis occurs only in an estimated 1% of infected individuals; polio 1 virus is responsible for at least 85% of cases. immunization. early attempts to develop inactivated poliovirus vaccines were hampered by not knowing that there are three distinct viruses. the differentiation of the three viruses by bodian (1949) and kessel and pait (1949) was a major step toward controlling the disease. enders et al. (1949) found that poliovirus would grow in extraneural tissues of human origin and thus laid the groundwork for the development of poliovirus vaccines. the first vaccine (salk) contained all three polio viruses grown in monkey kidney cell culture and inactivated with formalin (salk et al., 1954) . this vaccine, introduced in 1953, reduced the incidence of paralytic poliomyelitis 80-90% where it was used; however, multiple doses were required and intestinal tract infection was not prevented, thus allowing the virus to continue to spread. there was an intense effort in the 1950s to develop an attenuated live-virus vaccine that could be administered orally, and could protect the intestinal tract, thus breaking the chain of transmission. koprowski, sabin, and cox each developed vaccine strains of reduced neurovirulence that underwent extensive laboratory and field studies (koprowski et al., 1956; sabin, 1955; cox et al., 1959) . the strains developed by sabin were finally licensed in the united states; they produced rapid immunity as well as protection of the intestinal tract while preventing spread to unvaccinated, susceptible persons in contact with vaccinées. this improved the overall level of immunity in communities. with the widescale use of oral poliomyelitis vaccine, the incidence of paralytic disease in the united states has dropped to less than 10 cases per year. the occasional reaction to the attenuated vaccine is discussed in the chapter by hogle and filman. avian encephalomyelitis was first discribed and shown to have a viral etiology by jones (1932 jones ( , 1934 . the virus is widespread and affects young chickens (1-3 weeks old). characteristic clinical signs are ataxia and tremors of the head and neck. extensive neuronal degeneration occurs in the anterior horn of the cord and in the medulla and pons. the virus may affect older laying birds, causing a drop in egg production. flocks that have survived an outbreak or subclinical infection during the growing period are resistant to further infection (schaaf and lamoreux, 1955) . moreover, infected chickens 16-20 weeks of age undergo only mild disease, providing an opportunity for vaccination (schaff, 1958) . calnek and taylor (1960) successfully immunized immature birds with an attentuated live-virus vaccine delivered in drinking water. a number of vaccines have been developed including the 1143 strain (calnek, 1961) , the nsw-1 strain (westbury and senkovic, 1978) , the philips duphar strain (folkers et al., 1976) , and a strain grown in chicken pancreas cell culture (miyamae, 1978) . inactivated ae virus vaccines have been developed for use in susceptible breeding flocks that are in production (schaaf, 1959; calnek and taylor, 1960; butterfield et al., 1961; macleod, 1965) . two viruses in the family caliciviridae cause significant disease in animals, vesicular exanthema virus in swine and sea lions and feline calicivirus. caliciviruses have also been isolated from humans, calves, reptiles, nonhuman primates, birds, dogs, and fish, but do not produce significant disease in these animals. vesicular exanthema virus caused a disease in swine closely resembling fmd (traum, 1933) . eradication of this disease followed the discovery that the main source of contagion was uncooked infected meat in garbage fed to swine, and the consequent enforcement of garbage cooking laws. fastier (1957) first isolated a calicivirus from a domestic cat and showed that it produced an upper respiratory tract infection. a large number of viral isolates, with different neutralization patterns, were made from clinically ill cats (crandell et al., 1960; bittie et al., 1960) . these serotypes were later shown to have a common antigen and are now considered a single serotype (povey and ingersoll, 1975) . this virus is widespread, having been isolated from cats in many countries. the virus produces a disease that is generally mild, but, if allowed to progress, the lesions may extend from the upper respiratory tract into the lung causing pneumonia and death (kahn and gillespie, 1971) . immunization. cats that recover from natural infection and have neutralizing antibodies can be reinfected, but they do not have recurrent clinical disease. an attenuated live-virus calicivirus vaccine has been prepared by serial passage at low temperature; this vaccine virus produces only mild clinical signs in recipients (bittle and rubic, 1976) . this attenuated live-virus vaccine is administered parenterally; it induces high levels of neutralizing antibody and protects vaccinated cats challenged intranasally with both homologous and heterologous strains (kahn et al., 1975; scott, 1977) . immunity from vaccination persists for at least 6 months and probably longer. the calicivirus is combined with feline rhinotracheitis and feline panleukopenia to make a multivalent vaccine that is routinely used in domestic cats (bittle and rubic, 1975) . inactivated vaccines have also been licensed and are used in multivalent vaccines. the family reoviridae is divided into three genera: reovirus, rotavirus, and orbivirus. infections cause by member viruses are common in mammals and birds. reoviruses are commonly isolated from dogs, cats, sheep, cattle, horses, mice, rats, rabbits, birds, and man. only in birds is the disease serious enough to warrant control with vaccines. the reoviruses are commonly found in sewage, and the mode of transmission is thought to be the fecal-oral route. avian reovirus. in chickens and turkeys, reoviruses produce a widespread disease called viral arthritis (tenosynovitis). this disease of the synovial membranes, tendon sheaths, and myocardium was first recognized by dalton (1967) and by olsen and solomon (1968) . viral arthritis occurs primarily in meat-producing birds, and in acutely affected flocks there is a high rate of condemnation. there are at least five avian reovirus serotypes, but they are antigenically unrelated to the mammalian reoviruses. immunization. vaccination of breeding stock is an effective way to control viral arthritis. van der heide et al. (1976) used an attenuated live-virus vaccine and cessi and lombardini (1975) used an inactivated vaccine in laying hens to protect chicks with maternal antibody. this eliminated transmission and protected susceptible day-old chicks. rotaviruses produce acute gastroenteritis in many species, especially in newborns, including newborn calves, foals, lambs, piglets, puppies, monkeys, and humans. the clinical signs are similar in all species; in each there is acute diarrhea followed by dehydration and rapid loss of weight. secondary bacterial infection may exaggerate the symptoms and also cause pneumonia. the viruses infect epithelial cells of villi, causing desquamation and loss of absorptive function, resulting in diarrhea, rapid dehydration, and emaciation. secretory antibody is very important in protecting the epithelial surface of the small intestine (snodgrass and wells, 1976) ; antibody contained in colostrum is protective when in high titer. a. bovine rotavirus. bovine rotovirus causes a rapidly spreading disease in neonatal calves (mebus, 1969) . the antigenic relationship of the bovine rotavirus and other rotaviruses isolated from children, calves, piglets, mice, and foals is very close (woode et al., 1976) . immunization. an attenuated live-virus vaccine developed by mebus et al. (1976) is administered in two doses to cows prior to calving. this is meant to stimulate colostral antibody, which is passed on to the nursing calves. this vaccine has also been combined with an attenuated live-virus coronavirus vaccine and entero-toxigenic e. coli vaccine to prevent calf scours. b. porcine rotavirus. leece et al. (1976) isolated a rotavirus from piglets with fatal diarrhea. additional reports of this disease showed that it was widespread and warranted the development of a vaccine for its control. immunization. early attempts to immunize pigs by oral administration of a bovine attenuated live-virus rotavirus vaccine were unsuccessful (leece and king, 1979) . presently an attenuated porcine rotavirus vaccine containing two serotypes, a 1 and a 2 , is licensed in the united states and is being used in combination with a transmissible gastroenteritis (tge) vaccine. the vaccines are administered to pregnant sows by both parenteral and oral routes. at least two doses of vaccine are given orally, 5 and 3 weeks before farrowing, and one dose is given parenterally 1 week before farrowing. this induces antiviral colostral antibody for the protection of suckling piglets. the member viruses of this genus replicate in arthropods as well as in vertebrates. the most important viruses are the bluetongue viruses, and african horse sickness viruses. colorado tick fever virus, the only virus in this genus that infects man, is transmitted by the bite of infected ticks. the disease is usually benign, and infection produces long-lasting immunity. a. bluetongue virus. bluetongue viruses infect ruminants and are transmitted by culicoides gnats. the most serious disease is in sheep, which develop fever, depression, oral lesions, pneumonia, and lame-ness. mortality can be high, especially in lambs. ewes infected early in gestation may produce lambs with hydrocephalus and other congenital deformities. although cattle rarely have clinical bluetongue disease, in utero transmission can occur, resulting in congenital deformities. of the 24 distinct bluetongue viruses, 5 occur in the united states. infection with one bluetongue virus confers resistance to reinfection with that same virus for several months, but cross-protection against infection with other viruses is minimal. a multivalent attenuated live-virus vaccine developed in south africa by serial passage of several different viruses in sheep proved difficult to standardize. a more uniform vaccine was later developed by alexander et al. (1947) ; it contained strains of at least five viruses, attenuated by passage in chick embryos, and gave broad protection against the multiple viruses seen in the field. a similar vaccine was developed by mckercher et al. (1953) , who isolated bluetongue virus in the united states and also attenuated a strain of serotype 10 by serial passage in chick embryos (mckercher et al., 1957) . recently mcconnell and livingston (1982) have been attempting to incorporate more bluetongue virus strains into multivalent attenuated live-virus vaccines. inactivated vaccines for bluetongue would have the advantage of greater safety than attenuated live-virus vaccines. they would eliminate the possible chance of reversion to virulence, and the chance of vaccine-associated abortion and birth defects. such vaccines are under development (stott et al., 1979) . 6. african horse sickness virus. african horse sickness virus causes an acute disease in equine animals in africa, the middle east, and asia. it was shown to be caused by a virus (mcfadyean, 1900) and has been more thoroughly characterized by breeze et al. (1969) . the viruses are transmitted to horses by culicoides species and affect principally the vasculature of the respiratory tract causing edema of the lungs, head, and neck. the viruses also cause cardiac lesions. immunization. some animals that recover from natural infection may be reinfected, so immunity is not permanent. a spleen pulp vaccine inactivated with formalin was made by dutoit et al. (1933) and administered in multiple doses. later, an attenuated live-virus vaccine was developed by serial intracerebral passage of a field isolate in mice (alexander and dutoit, 1934) . however, because there are nine african horse sickness viruses, it has been necessary to adapt each to mice and to combine them in a polyvalent vaccine. such vaccine has been effective in protecting horses. the virus that causes ibd was first isolated by winterfield and hitchner (1962) using embroyonated eggs. it causes a disease of chickens in commercial poultry-producing areas. the virus affects mainly young birds 3-6 weeks of age, with clinical signs of diarrhea and dehydration. the lesions arise in lymphoid tissues such as the bursa of fabricius, thymus, and spleen, producing immunosupression with the associated opportunistic infections. immunization. both attenuated live-virus and inactivated vaccines have been developed to control ibd. the vaccines are used mainly for immunizing breeder flocks to confer passive immunity through the yolk sac of the egg. maternal antibody protects chicks for 1-3 weeks. if breeder flocks are boosted with oil-adjuvant-inactivated vaccines, maternal antibody may last longer. there are several types of attenuated live-virus vaccines with varying degrees of virulence. these vaccines are administered in water, etc., to chicks from 1 day to 2-3 weeks of age in broiler-breeder flocks, followed by vaccination with an inactivated product at approximately 16-18 weeks of age (lukert and hitchner, 1984 ). the four genera in the family togaviraidae are: alphavirus, pestivirus, rubivirus, and arterivirus. each of these genera contain important pathogens. the alphavirus genera include: all cause encephalitis in horses and humans. in horses, the mortality rate of eee in over 80%, and that from wee is about 20-30%. the main mode of transmission is by culicine mosquitoes; however, vee has been transmitted from horse to horse by contact with body fluids. immunization. infections with togaviruses produce viremia, longterm humoral antibody responses, and immunity. early vaccines were made from formalin-inactivated infected animal brain tissue. the cultivation of both wee and eee in the chick embryo by higbee and howitt (1935) made possible the development of successful inactivated vaccines (beard et al., 1938) . more recent vaccines for wee and eee are produced in tissue culture. an attenuated live-virus vee vaccine, first developed for humans, is also used for horses in endemic areas (berge et al., 1961; mckinney et al., 1963) . the vaccine virus is grown in primary chick embryo cell cultures; it induces long-lasting immunity. an inactivated vee vaccine has also been developed and is combined with wee and eee vaccines in a trivalent formulation. a. hog cholera (hc) virus. hog cholera virus and bovine virus diarrhea (bvd) virus antigenically are closely related pestiviruses, but are specific in the diseases they cause in swine and cattle, respectively. hc virus produces an acute febrile disease marked by multiple hemorrhages, necrosis, and infarcts in internal organs. lethargy, vomiting, and encephalomyelitis are seen in a high percentage of infected animals during an outbreak and mortality is high. immunization. passive protection with convalescent swine serum from swine has been used effectively for short-term control of outbreaks for many years (dorset et al., 1908) . antiserum and either virulent or partially attenuated virus strains were also used to establish active immunity. although there is only one antigenic type of hc virus, variant biotypes have arisen that are more difficult to protect against with standard vaccines. the presence of neutralizing antibody correlated well with protection. an inactivated virus vaccine prepared from defibrinated swine blood taken during the acute phase of the disease and treated with crystal violet or phenol was safe, but required multiple doses (mcbryde and cole, 1936) . attenuation of hc virus was first accomplished by passage in rabbits (baker, 1946; koprowski et al., 1946) . tissue culture attenuated live-virus vaccines eventually replaced the rabbit vaccine; the latter produced a rapid and long-lasting immunity. a large number of different attenuated live-virus hc vaccines with different characteristics have been used over the years, but residual pathogenicity, shedding, and spread of vaccine viruses have remained problems. a vaccine containing bvd virus was tested in swine, based on evidence that this virus could block replication of hc virus (sheffy et al., 1961) . however, the bvd vaccine did not protect against all strains of hc virus (tamoglia et al., 1966) . formerly, control of hc was difficult because hc virus persists in infected meat scraps fed to swine in uncooked garbage. however, since 1969 no vaccines have been used in the united states. by controlling the transport of swine and cooking all garbage used as feed, the disease has been eradicated from the united states, and several european countries. b. bovine virus diarrhea (bvd) virus. bvd virus causes a widespread disease of cattle, especially in young stock. clinical signs, which vary in severity, include scouring, ulcerations of the oral cavity, and abortion. young animals that recover often remain stunted and unproductive for long periods. bvd viral strains differ in their cytopathic effects in tissue culture; cattle infected with noncytopathogenic strains during the first 3 months of gestation can transmit the virus to the fetus, which may be born viremic and immunologically tolerant. later exposure to cytopathogenic strains, naturally or by vaccination, can cause offspring to develop the more severe form of the disease (bolin et al., 1985) . a cytopathogenic strain of virus isolated from a calf and designated oregon (c24v) strain (gillespie et al., 1960) became less pathogenic for calves after 32 passages in bovine kidney tissue culture . this has been the standard vaccine strain and has been used widely for many years. for cattle never exposed to bvd antigen, this vaccine strain is safe and effective; however, persistently infected cattle may react strongly to vaccination with the cytopathic strain of bvd virus, causing a mucosal disease syndrome. it is important to identify and eliminate persistently infected cattle from herds. rubella virus. in man, rubella virus causes a generally mild exanthematous disease, with malaise and respiratory symptoms. complications include arthritis, thrombocytopenia purpura, and encephalitis. gregg's observation (1941) that rubella virus produces fetal abnormalities if infection occurs early in pregnancy emphasized the destructive effects of this virus and the need to develop a means to protect against infection. immunization. natural exposure to rubella virus evokes nasopharyngeal antibody, which is important in preventing reinfection. antibody, especially igg antibody in mother's plasma, is important in preventing fetal infection. two groups isolated rubella virus in tissue culture (parkman et al., 1962; weller and nova, 1962) , allowing the first attempts to develop vaccines. both inactivated and attenuated live-virus vaccines were tried before the latter evolved as superior products. the attenuated live-virus vaccines were developed using different cell culture systems, including monkey kidney (parkman et al., 1966) , duck embryo (buynak, 1968), rabbit kidney (peetermans and huygelen, 1967) , canine kidney (musser and hilsabeck, 1969) , and human diploid cells (plotkin, 1967) . the vaccines now being used are more than 95% effective in inducing protective levels of antibody that persist for at least 9 years. the annual incidence of rubella in the united states has dropped from 56,000 reported cases in 1970 to less than 700 in 1985. equine arteritis virus. equine arteritis virus was first isolated by doll et al. (1957) . the disease caused by this virus is characterized by edema of limbs, stiffness, and swelling in the tissues surrounding the eye, and abortion. immunization. horses that recover from infection develop longlasting immunity. an effective attenuated live-virus vaccine was developed by serial passage of bucyrus field strain virus in primary equine and rabbit kidney cell culture and an equine dermal cell line (doll et al., 1968; wilson et al., 1962; mccollum, 1986 ). the vaccine has been shown in challenge trials to protect recipients for as long as 24 months (mccollum, 1986) ; it does not cause any clinical manifestations and it is not spread to susceptible horses in contact with vaccinated recipients. yellow fever virus causes acute hepatitis and hemorrhagic fever in man, characterized by jaundice, shock, and renal damage. transmission is by mosquitoes belonging to the aedes genus throughout tropical areas of south america and africa. the virus is maintained in a transmission cycle between mosquitoes and monkeys, with man being infected when he enters a territory in which the monkey-mosquito cycle exists. immunization. an attenuated live-virus yellow fever vaccine was developed by passage of the virulent asibi strain in mouse brain and cell culture until it had lost its pathogenicity for monkeys and man (theiler, 1951) . the vaccine virus, termed 17d, is propagated in embryonated eggs. the vaccine, given as a single dose, is extremely safe and efficacious, providing immunity for at least 10 years. the family orthomyxovirus comprises the influenza viruses, the cause of acute, highly contagious respiratory disease in man, horses, swine, and birds. two structural viral proteins (nf and m) divide this family into three distinct genera: a, b, and c. the viruses, especially influenza a virus, undergo genetic réassortaient, which allows variant viruses to emerge. two viral proteins, the hemagglutinin and neuraminidase, both located on the surface of virions, are important in inducing immunity. vaccines have been widely used for controlling influenza with reasonably good success. since immunity is more closely related to local secretory iga antibody than to serum antibody, it is difficult to stimulate and maintain protection with the presently used inactivated vaccines. with human infections, the type a viruses undergo occasional antigenic shifts and drifts, after which the antigens in the vaccine may not be representative of those viruses found in the field. this potential antigenic variability, as well as animal reservoirs for this virus, are responsible for the pandemics associated with this virus. as an example, the acute respiratory disease of swine caused by a type a strain of virus was first recognized in 1918 during the human influenza epidemic and is believed to have been transmitted from swine from humans. both swine and humans are susceptible to the swine virus. however, the disease in swine occurs sporadically and has not been enough of a problem to warrant the use of vaccines in that species. influenza is a respiratory infection with systemic manifestations that include fever, chills, muscular aches, etc. the severity of the disease depends on the virus strain and the susceptibility of the population. persons that have recovered from an influenza infection are usually immune to rechallenge with the homologous virus. however, the change of a few amino acids in hemagglutinin may give rise to antigenic drift and reinfection of populations. immunization. because of the epidemic threat of influenza viruses, careful surveillance for new strains is carried out in many parts of the world. the strains that public health officials predict will be the cause of the next winter's epidemics are then scheduled for vaccine production. for vaccine production, virus is grown in the allantoic cavity of embryonated eggs and is purified and concentrated by zonal centri-fugation. the virus is inactivated with formalin, ß-propiolactone, or irradiation. the quantity of viral antigen per dose is standardized before use. the vaccine usually contains several type a viruses and a type β virus. these inactivated whole virus vaccines produce protective levels of antibody in approximately 85% of primed recipients and in 60% of the unprimed recipients. antibody levels are maintained for approximately 1 year in primed individuals. attenuated live-virus influenza vaccines have been used extensively in the ussr with varying results. problems of adverse reactions, inconsistent potency, and questionable appropriateness of strains make it difficult to evaluate the effectiveness of these vaccines. more recent attempts to develop attenuated live-virus vaccines involve genetic reassortment, a method that offers considerable promise (reviewed by wright and karzon, 1987) . influenza in horses resembles the disease in man and swine. the two type a influenza viruses of importance in the horse are: a/equi l/prague/56 type 1 and a/equi 2/miami/63 type 2. the disease spreads rapidly through susceptible horses, and those that recover are protected for only a short time. recovery from infection with one virus type does not provide immunity against the other. immunization. vaccines for equine influenza are produced in essentially the same manner as human influenza vaccines. formalinactivated vaccines contain both equine type 1 and 2 viruses and one of several adjuvants, as described by bryans et al. (1973) . vaccines of this type are widely used and effectively control equine influenza. the family paramyxoviridae contains several viruses that cause significant disease in animals. the family is composed of three genera that include the following viruses for which vaccines have been developed. these viruses are transmitted by the respiratory route and are antigenically rather stable. parainfluenza viruses infect humans, rodents, swine, dogs, and cattle. these viruses, by themselves, cause mild upper respiratory tract disease, but when combined with other viral and bacterial pathogens may cause a more severe syndrome. parainfluenza types 1, 2, 3, and 4a/4b infect humans, especially young children. type 3 is considered the most pathogenic, causing a bronchitis and pneumonitis. vaccines for parainfluenza of rodents (sendai virus infection), dogs (canine parainfluenza), and cattle (bovine parainfluenza) have been developed. there is no licensed parainfluenza vaccine for man. a. sendai virus. sendai virus, first isolated during attempts to recover human respiratory viruses in mice (kuroya et al., 1953) , is a parainfluenza type 1 virus that causes respiratory disease in mice, rats, hamsters, and swine. the disease occurs either in an acute-short duration form or a chronic-persistent, clinically inapparent form. spread is by either direct contact or by aerosol. in mouse colonies, this disease is difficult to control because the virus is so highly infective. immunization. formalin-inactivated vaccines have been effective in controlling the disease in mice and rats (fukumi and takeuchi, 1975; eaton et al, 1982; tsukui et al, 1982) . additionally, a temperature-sensitive mutant strain of sendai virus has been used as an aerosol-delivered vaccine in mice. it suppresses virus replication, but the vaccine virus spreads throughout the colony and makes it difficult to monitor for wild virus strains (kimura et al, 1979) . b. canine parainfluenza virus. outbreaks of mild respiratory disease in laboratory dogs have been attributed to parainfluenza type 2 virus (binn et al, 1968; crandell et al, 1968) . when other respiratory agents such as mycoplasma and bordetella bronchiseptica were given intranasally after exposure to this parainfluenza virus, more severe respiratory signs occurred (appel and percy, 1970) . this encouraged efforts to develop a vaccine for canine parainfluenza virus. immunization. an attenuated live-virus parainfluenza vaccine has been shown to protect dogs against aerosol challenge with virulent virus (emery et al, 1976) . the vaccine produces no untoward effects and has been combined with canine distemper and canine adenovirus vaccines in multivalent formulations. c. bovine parainfluenza virus. a parainfluenza type 3 virus isolated from cattle can also cause mild respiratory disease (reisinger et al, 1959) . the virus, when combined with other respiratory pathogens including pasteurella and infectious bovine rhinotracheitis virus, causes the severe pneumonia syndrome, called "shipping fever." immunization. an attenuated live-virus parainfluenza type 3 vaccine administered parenterally induces good levels of antibodies and affords protection against experimental challenge (mohanty and lillie, 1964; thorsen et al., 1969) . this vaccine has been combined with infectious bovine rhinotracheitis virus and bovine virus diarrhea vaccines in multivalent formulations. an inactivated vaccine, requiring two inoculations, induces high hemagglutination-inhibition titers and lessens the severity of the disease in cattle challenged with the same virus (gale et al., 1963) . mumps virus causes an acute infection in man with parotitis as the main clinical manifestation, although the central nervous system and other organs including the testes and ovaries can be affected. mumps virus has a limited host range; in addition to man, only certain monkey species and laboratory rodents can be infected. immunization. recovery from natural infection with mumps virus confers long-term immunity. early experiments with formalininactivated virus derived from infected parotid glands of monkeys showed that monkeys and humans could be immunized (enders et al., 1945; stokes et al., 1946) . habel (1946) found that chick embryo grown virus could be inactivated with ultraviolet light or formalin and would induce protection in monkeys. a similar vaccine was later shown to induce protection in man (habel, 1951; henle et al., 1951) . poor antibody responses to multiple inoculations of this type of vaccine encouraged the search for a more effective vaccine. a mumps virus strain ( jeryl lynn) has been attenuated by passage in chick embryos (weibel et al., 1980) . the vaccine is immunogenic in 93-98% of subjects, and neutralizing antibodies persist for at least 10 years. the annual incidence of mumps in the united states has been reduced from 160,000 cases in 1968 to less then 3,000 in 1985 by application of this vaccine. as presently used, mumps vaccine is combined with measles and rubella vaccines in a pediatric formulation called mmr. newcastle dibcctse is one of the most serious widespread diseases affecting poultry. the disease was first described by kranevelt (1926) and shortly thereafter by doyle (1927) , who named it after the area in england where an outbreak occurred and showed that its cause was a filterable virus. the disease has several forms causing mainly respiratory, enteric, and central nervous system manifestations. the morbid-ity and mortality vary depending on the virus strain. burnet (1942) described the hemagglutinating property of the virus, which has been very helpful in its quantitation and immunodiagnosis. immunization. a number of attenuated live-virus vaccines have been developed which are widely used to control the disease. the bl strain (hitchner and johnson, 1948) , the lasota strain (winterfield et al., 1957) , and the f strain (asplin, 1952) are used to immunize birds of all ages by different routes, including by addition to drinking water and by spraying. vaccines containing inactivated virus do not produce long-lasting immunity but may be used in certain situations when only short-term immunity is needed, such as when boosting immunity is needed in laying flocks. increasing the antigen content and using oil-emulsion adjuvants improves the quality of these inactivated vaccines (stone et al, 1980; zanella and marchi, 1982) . measles is a highly contagious disease of humans, occurring mostly in children, causing exanthemata and sometimes more serious manifestations including encephalomyelitis. the virus has two principal immunogens, the hemagglutinin and the fusion protein (norrby et al., 1975) . the immunity produced by natural infection is long lasting. immunization. the growth of measles virus in chick embryo fibroblasts by enders and peebles (1954) paved the way for the development of vaccines. a formalin-inactivated measles virus vaccine was shown to induce partial immunity. however, some vaccinated children later exposed to measles virus, either naturally or as attenuated live-virus measles vaccine, developed atypical measles (atypical rashes, edema of hands and feet, and respiratory disease). it was later found that formalin-inactivated vaccine failed to stimulate antibody to the fusion protein: consequently the virus could spread from cell to cell, causing the atypical manifestations of disease (norrby et al., 1975 ). an attenuated live-virus vaccine was developed from the edmonston strain of measles virus by passage first in human cell culture, then in the amnionic sac of embryonated hen's eggs, and finally in chick embryo cells (milovanovic et al., 1957) . this vaccine (enders passage level b) was effective in inducing immunity but produced some adverse effects (katz and enders, 1959; stokes et al., 1962) . further attenuation by growth at lower temperature yielded an equally effective vaccine that produced fewer side effects (schwarz, 1964; hilleman et al., 1968) . attenuated live-virus measles vaccine has been combined with mumps and rubella vaccines. measles vaccine usage has reduced the incidence of measles in the united states from 440,000 cases in 1960 to 3,000 in 1985. canine distemper virus affects most carnivores, causing respiratory, gastrointestinal, and central nervous system disease. the mortality rate in dogs is about 20%. dunkin and laidlaw (1926) first described the disease in detail and confirmed the viral etiology proposed earlier by carre (1905) . immunization. dunkin (1926, 1928a,b) prepared a vaccine by treating virus derived from spleens of infected dogs with formalin. initial administration of this vaccine, followed 2 weeks later with a small dose of virulent virus, usually produced only a mild disease with solid immunity. this approach was replaced with inactivated virus vaccines, given in multiple doses, which served as the main means of controlling the disease from 1930 to 1950. green (1945) serially passaged canine distemper virus in ferrets and produced the first attenuated live-virus vaccine; however, this vaccine caused disease in some dogs. the adaptation of canine distemper virus to the chorioallantoic membrane of embryonated eggs by haig (1948) was a major step in developing an attenuated live-virus vaccine. cabasso and cox (1949) applied this method and after 28 passages showed that the virus lost virulence for ferrets but retained its immunizing property for dogs. rockborn (1958) adapted a strain of canine distemper virus to cell culture, and this method is now widely used to produce attenuated live-virus vaccines. rinderpest is an acute highly contagious disease of ruminants characterized by erosions and necrosis of the intestinal mucosa. the disease is epizootic in parts of africa and asia, causing great losses of cattle and buffalo. immunization. koch in 1897 developed one of the first means of immunizing cattle against rinderpest by administering bile from infected cattle. animals that survived were permanently immune. formalin-and chloroform-inactivated vaccines were developed using tissues from infected animals. these vaccines were safe, but required two or three doses and protection lasted less than a year (walker et al., 1946) . rinderpest virus has been adapted to several foreign hosts, including goats (daubney, 1949) and rabbits (nakamura et al., 1938) and has been attenuated by passage in these animals. tissues of these animals have been used to produce vaccines in many countries. however, on continued passage, the seed strains tend to lose their immunogenicity, and vaccines become contaminated with adventitious agents from the foreign host. the kabete strain of rinderpest virus was adapted to grow on the chorioallantoic membrane of embryonated eggs, becoming attenuated for cattle after 19 passages . this vaccine and another containing a lapinized strain of virus adapted to embryonated eggs have been used widely in africa (nakamura and miyamoto, 1953) . more recently, the kabete strain was adapted to bovine kidney cell culture and after 70 passages became avirulent for cattle. this vaccine strain is safe and efficacious in most cattle breeds (plowright and ferris, 1962) . protection persists for at least 4 years. over 150 million doses of this vaccine have been used in africa, with good success (maurer, 1980) . bovine rsv produces a rhinitis and catarrhal bronchiolitis in cattle (mohanty et al., 1975; jacobs and edington, 1975; paccaud and jacquier, 1970) . the virus appears to be widespread having been isolated in europe, the united states, and asia. human and bovine virus are related antigenically (paccaud and jacquier, 1970) ; however, the cattle virus is not known to infect man. immunization. nasal secretory antibody is protective but the disease may occur in the presence of serum antibody. an inactivated bovine rsv vaccine is combined with vaccines for infectious bovine rhinotracheitis, bovine virus diarrhea, and bovine parainfluenza components in a multivalent formulation. the efficacy of the rsv component in this formulation is unclear. of the viruses in the family rhabdoviridae that cause disease in man and domestic animals, the most important is rabies virus. others include vesicular stomatitis viruses (vsv) and bovine emphemeral fever virus (befv). vsv occurs sporadically and epizootically, affecting horses, cattle and swine in the united states. there is a formalininactivated vaccine (gearhart et al., 1987) , but it is used rarely. befv is an arthropod-transmitted disease of cattle occurring mainly in africa, but also in asia and australia; it is controlled by immunization with attenuated live-virus vaccines (van der westhuizen, 1967; inaba et al, 1973; spradbrow, 1977; theodoridis et al, 1973) . rabies is an infection of the central nervous system; the disease can occur in most mammals and is usually fatal. there are only a few documented cases of human survivors. after isolating the virus, pasteur (1881) developed a vaccine for its control. historically rabies virus has been considered as a single serotype. but now shared antigens have been found in other viruses in africa of which two, mokola and duvenhage, may be associated with human disease (shope et al. y 1970) . mokola virus causes a rabies-like disease in dogs and cats in zimbabwe (foggin, 1983) . immunization. protection against rabies correlates with sn antibody, which can be assessed by a number of tests. pasteur's classical vaccine, developed from infected spinal cord tissue dried at room temprerature for 3-14 days, was given in a series of 21-28 inoculations beginning with material dried the longest and progressing through material dried for only 3 days (pasteur 1881) . even though the last inoculum was virulent enough to cause rabies, the earlier inoculations conferred sufficient immunity to protect the recipients. this method of producing a vaccine was successful in most instances but caused the disease occasionally and was eventually replaced by chemically inactivated vaccines prepared from infected brain tissue. although effective, these vaccines gave rise to undesirable side-effects because they contained a myelin-related encephalitogen present in brains from mature animals. substitution of brain tissue from immature animals such as suckling mice, rats, and rabbits with their lesser myelin antigenic content greatly reduced these post-vaccination reactions. the adaptation of the flury strain of rabies virus to growth in chick embryos led to the development of attenuated live-virus vaccines produced in this tissue (leach and johnson, 1940; koprowski and cox, 1948; black, 1950, 1954) . the growth of rabies virus in tissue culture has further improved rabies vaccines (kissling, 1958; cabasso et al., 1965; emery et al., 1968; brown et al., 1967; fenje, 1960; abelseth, 1964 ). yet, despite their benefits, the attenuated rabies vaccines occasionally caused rabies, particularly in cats. therefore, suckling mouse brain and tissue culture again became the substrates of choice to produce inactivated rabies virus vaccines for animals. in humans the requirement for a safe substrate is more exacting than in animals. for this reason, duck embryos proved better than brain tissue to produce rabies virus (peck et al., 1956) . after inactivation with ß-propriolactone (bpl), this virus was an improved product for humans, although the allergenic effects from duck embryo tissue still present a problem. therefore the adaptation of the pm rabies virus strain to human diploid cells and inactivation with bpl (wiktor et al., 1964) was a further improvement. this vaccine is less reactive and more effective for pre-and post-exposure use in humans than any other yet made (bahmanyar et al., 1976) . a similar vaccine produced in bhk cells is also beneficial in animals. retroviruses have an rna genome, a portion of which encodes the unique enzyme reverse transcriptase. this enzyme imparts to retroviruses the ability to make rna-directed dna copies of their genome, which can then act as a transposable element and can be integrated into the host cell dna. thus, once a cell is infected, it may escape immune surveillance and destruction and the host animal may be infected for life. the retroviruses thus constitute a considerable challenge to traditional vaccine approaches, as discussed further in the chapters by arlinghaus and by nathanson and gonzalez-scarano. many retroviruses infect mammalian species, from mouse to man. most notable are the c-type retroviruses, including the primate, murine, and feline leukemia viruses, as well as human t-cell leukemia viruses types i and ii; the b-type retroviruses, particularly mouse mammary tumor virus; and the lentiviruses, including caprine infectious anemia virus, visna virus, equine infectious anemia virus, feline immunodeficiency virus, bovine immunodeficiency virus, and human immunodeficiency viruses (hiv-1 and hiv-2). in recent years, as hiv has become a major threat, massive efforts have been directed to developing an efficacious vaccine. so far, all attempts have met with failure. in fact, there are only two retrovirus vaccines that have been proven effective: a formalin-inactivated whole virus preparation of the primate saids type d retrovirus, which is capable of protecting monkeys from a lethal challenge (marx et al., 1986) , and the commercially available vaccine for feline leukemia. felv commonly infects cats in urban areas, usually by the oralnasal route. kittens under 6 months of age are particularly susceptible. about 1% of infected cats develop persistent anemia from which myeloproliferative disease and hypoplastic anemia may follow. the immunosuppression caused by felv infection may predispose to severe chronic opportunistic infections. because cats that develop neutralizing antibody are usually immune to infection, vaccines have been developed and tested with that goal in mind. immunization. the problem in developing a vaccine for feline leukemia was to find immunogens that could be used without exposing animals to oncogenic materials. early studies with inactivated whole virus were unsuccessful (yohn et al., 1976) . although attenuated live-virus vaccines induce sufficiently high levels of neutralizing antibodies to be protective ( jarrett et al., 1974; pedersen et al., 1979) , their oncogenic potential makes them unacceptable. efforts to develop vaccines containing only viral proteins, such as envelope protein, have had variable results. however, cultivation of felv in fl 74transformed cells, followed by treatment to release viral and cell proteins, yields a vaccine that stimulates antibodies to both viral and cell membrane components. a commercial vaccine using this method of antigen production has been approved for use in the united states; it is based on studies done by olsen and lewis (1981) . subsequently, the efficacy of this vaccine has been disputed (pederson and ott, 1985) . viruses in the family coronaviridae cause important diseases including avian infectious bronchitis, transmissible gastroenteritis of swine (tge), feline infectious peritonitis (fip), and human coronavirus infections. other coronaviruses may cause disease in calves, dogs, mice, rats, turkeys, horses, and parrots, but the diseases are of less importance. coronavirus diseases usually follow a similar pattern, except for fip. fip is a chronic debilitating disease manifested as fibrinous peritonitis and pleuritis. the infection may be inapparent, but is fatal in a small proportion of infected cats. the immune response to fip virus seems to mediate the disease; the immune response is not protective and antibody levels are higher in diseased animals. immune complexes have also been demonstrated in renal glomeruli of cats with fip. bovine coronavirus causes acute diarrheal disease in neonatal calves (mebus et al., 1973 ). an attenuated live-virus vaccine is being used in combination with an attenuated live-virus rotavirus vaccine to control calf diarrhea. the vaccine is administered to pregnant cows near the end of gestation and stimulates colostral antibodies that offer protection to nursing calves. with the exception of avian infectious bronchitis, most coronavirus infections have been difficult to control with vaccines. perhaps this is because primary lesions are in mucous membranes of the respiratory and gastrointestinal tracts, sequestered from immune reactivity. coronaviruses produce about 15% of common colds in man, second only to rhino viruses. there are two groups of human coronaviruses that are antigenically distinct. ibv is a highly contagious respiratory infection of young chickens. the virus may also infect older birds, causing a decrease in egg production. the disease was first shown to be caused by a virus by bushnell and brandley (1933) . beaudette and hudson (1937) propagated the virus in chick embryos, making possible the quantification of the virus and the means for attenuation. there are a number of serotypes of ibv, making the development of an effective vaccine difficult. immunization. immunity following natural infection may last up to 1 year, depending on the serotype and the severity of challenge. van roekel et al. (1950) first developed an immunization procedure; he used a field strain of virus to infect 7-to 15-week-old birds before they start to lay, inoculating a few of the birds and allowing infection to spread naturally through the flock. today, there are a number of attenuated live-virus vaccines licensed in the united states. there is good protection (90-100%) against homologous virus strains and about 40% against heterologous strains (hofstad, 1981) . reduced pathogenicity may be associated with reduced immunogenicity, so a balance must be maintained. the attenuated live-virus vaccines are administered by the usual labor-saving devices of spraying, dusting, or placing the vaccine in drinking water. the wide-scale use of ibv vaccines has significantly reduced the economic loss caused by this disease. tge is an often fatal disease of pigs under 2 weeks of age. the main lesion is enteritis, resulting in malabsorption, diarrhea, and dehydration. tge virus is serologically related to fip virus, but the diseases have entirely different characteristics. there is one serotype of tge virus and one serotype of fip virus. high levels of maternal tge antibody in sows' colostrum protect piglets if fed continuously. immunity of this type has been produced by feeding sows tissues containing virulent tge virus several weeks prior to gestation. the effects of this virus are relatively mild in older animals. attenuated live-virus vaccines administered parenterally to pregnant swine in the latter part of the gestation period produce colostral antibodies. apparently, in sows previously exposed to tge, this vaccine produces sufficient immune responsiveness to be of value. the vaccine is also used orally in pigs 1-3 days old to induce local immunity. the effectiveness of this use of the vaccine has not been thoroughly demonstrated. the genus corynebacterium is a heterogeneous grouping with its species placed together largely on the basis of similar cell wall components (goodfellow and minnikin, 1981) . these species share a basic cell wall chemistry (barksdale, 1981) of which the mycolic acids (silva and ioneda, 1977) , especially trehalose dimycolate, are frequently used as potent adjuvants in immunization protocols. two corynebacteria-c. pyogenes and c. pseudotuberculosis-are important in veterinary medicine. the former is frequently associated with ruminate suppurative conditions and abscesses, but it rarely affects man. infections with this organism are sporadic, because it is an opportunist that gains entry through wounds and abrasions. it may also be seen as a secondary invader in devitalized tissues; e.g., vaccination site abscesses. the efficacy of vaccines, toxoids, and antisera against c. pyogenes is equivocal; little is known about immunity to the bacterium. corynebacterium pseudotuberculosis causes caseous lymphadenitis of goats and sheep. it is recognized as a worldwide problem and a serious cause of economic loss to the goat industry (burrell, 1981; ashfaq and campbell, 1980) . as with c. pyogenes, the responsible bacterium, c. pseudotuberculosis, is primarily an opportunist entering wounds or abrasions, where it causes local inflammation before settling in the regional lymph nodes. immunization. cell-mediated immunity is necessary for acquired resistance and protection against c. pseudotuberculosis (jolly, 1965; tashjian and campbell, 1983; irwin and knight, 1975) . killed and autogenous vaccines and a toxoid vaccine have been used in attempts to immunize against the bacterium (cameron, 1972; brogden et al., 1984; nairn et al., 1977; burrell, 1978; anderson and nairn, 1984; brown et al., 1986) ; however no one vaccine has proven highly efficacious. diphtheria, characterized by the formation of a tightly adherent pseudomembrane on the pharyngeal mucous membranes of the throat and trachea, is a highly contagious disease of man caused by the bacterium c. diphtheriae. the bacterium can also be isolated from the pharyngeal mucosa of normal individuals. the organism produces a lethal protein exotoxin (gill and pappenheimer, 1971; collier and kandel, 1971) . immunization. successful immunization against c. diphtheriae actually protects against the diphtherial exotoxin. because diphtheria toxin is produced in high yield by the park-williams number 8 strain (pw8), pw8 is used to make diphtherial toxoid for vaccines. as a source of toxin it is rendered nontoxic by incubation with formalin under alkaline conditions. the product's retention of antigenicity, enabling it to induce antitoxin antibodies, makes it an excellent pediatric vaccine. it is commonly utilized in combination with antigens from c. tetani and b. pertussis. the most important species of the bacillus genus, b. anthracis, is the organism responsible for the disease anthrax in both man and animals. anthrax was the first bacterial disease ever to be reported, being described by davaine in 1863. koch in 1876 reproduced the disease via animal inoculation and in 1881 pasteur successfully vaccinated against anthrax. in animals, natural infection usually occurs by ingestion of spores that germinate in the mucosa of the esophagus or the intestinal tract. herbivorous animals, especially cattle, horses, sheep, and goats, are highly susceptible to the disease, usually the result of grazing in infected pastures or consuming infected foods. in man, anthrax is manifested in three forms: cutaneous (malignant carbuncle), pulmonary (woolsorter's disease), and gastrointestinal with cutaneous being the most common. death results from the combined effects of an extracellular toxic protein complex (vodkin and leppla, 1983) comprised of three components: edema factor, protective antigen, and lethal factor (leppla, 1982; stephen, 1981) . effective vaccines require all three components. immunization. the attenuated pasteur vaccine has been supplanted in veterinary medicine by stable spore vaccines, carbo-zoo vaccine, or stern vaccine (jackson et al., 1957) prepared from avirulent, nonencapsulated variants of b. anthracis. the viable bacterial spores are suspended in 10% saponin. immunity is attributed to the development of antibodies to the toxins released from growing bacteria. vaccines of killed bacteria provide little immunity, since no bacterial toxins are produced; hence, no antitoxin antibodies are generated. purified protective antigen (complex toxin) is both antigenic and immunogenic and has been used as a vaccine for humans. it is prepared by aluminum potassium sulfate precipitation of sterile b. anthracis culture filtrates and has proven highly efficacious. erysipelothrix is found in soil, water, and decaying vegetative material and carcasses. the major species of interest is e. rhusiopathiae, which has 20 serotypes (norrung, 1979) . the bacterium, most notable for causing swine erysipelas, is capable of invading the tissues of both man and animal. fatally affected animals develop welt-like, discolored cutaneous lesions, and numerous hemorrhagic lesions in thoracic and abominai viscera; chronic debilitating arthritis predominates in surviving animals. immunization. the principal vaccines used to control erysipelas are formalin-killed, alum-adsorbed, whole-cell culture vaccines. these combinations of soluble bacterial glycoprotein and whole killed bacterial cells are usually produced from strains of serotype 2, which possess highly antigenic soluble cell wall glycoproteins. animals immunized with cell-free culture fluids develop agglutinins to the whole bacteria (white and verway, 1970) . such vaccines are highly effective in controlling swine erysipelas. the pathogenic clostridia invade both man and many animal species of veterinary interest, in which they cause such diseases as tetanus (c. tetani), gas gangrene (c. perfringens, c. septicum, c. oedematous), botulism (c. botulinum), enterotoxemia, and dysentery (c. perfringens). the clostridia are widely distributed in soil and water and are common inhabitants of the intestinal tracts of animals and humans. additionally, the bacteria can often be isolated from infected wounds. vaccination is not routinely practiced against all clostridial organisms, notably c. botulinum. the toxins of c. botulinum, which exert their effects upon the nervous system (schantz and sugiyama, 1974) , are as potent as those of c. tetani. the lethal dose of the toxin, however, is less than that required to induce an antibody response. clostridium perfringens has five serotypes, a-e, classified according to the production of lethal exotoxins. types a and c are pathogenic for man, whereas all five serotypes can affect animals (see table i ). immunization. the exotoxins of c. perfringens are antigenic proteins that can be detoxified for use in vaccines. the existence of common capsular antigens, which elicit cross-reactions between the serotypes, demonstrates the considerable heterogeneity of this group. ewes and lambs are frequently vaccinated against c. perfringens enterotoxemias. effective vaccines employ type-specific alumprecipitation or formalinized toxoids (smith and matsuoka, 1959; kennedy et al, 1977) . clostridium tetani elaborates potent neurotropic exotoxins (tetanospasmin and tetanolysin) that may be lethal for susceptible species such as man, horses, mules, swine, cattle, and sheep. birds are not naturally susceptible to the bacterium. tetanus toxin is one of the most poisonous toxins known. it acts only on the nervous system and its effect characteristically causes spastic paralysis and generalized convulsions. immunization. protective antitoxin blood levels are obtained by immunizing both humans and susceptible animals with alumprecipitated or absorbed tetanus toxoid (chodnik et al., 1959) . ramon and lemetayer (1931) first introduced the concept of active immunization against tetanus when they used formalinized tetanus toxoid precipitated with alum to vaccinate horses. clostridium tetani vaccines are very effective at inducing long-lasting immunity in both man and domestic animal species. a serious, often fatal disease has been successfully controlled with these vaccines. clostridium novyi possesses four antigenic types, a, b, c, and d; type a is the most common clinical pathogen. types a and β are responsible for gas gangrene both in man and animals (elder and miles, 1957) . in areas where sheep simultaneously carry a heavy liver-fluke infestation, exposure to c. novyi is often associated wtih hepatic necrosis and subcutaneous edema. migrating flukes produce foci of hepatic necrosis suitable for the germination of spores and the subsequent elaboration of lethal toxins (williams, 1962) . immunization. effective vaccinations for c. novyi in animals employ chemically inactivated, detoxified, and adjuvanted suspensions of alum-precipitated formalinized whole broth cultures. clostridium chauvoei, which is the etiologic agent for the disease "blackleg" and is pathogenic for animals only, occurs primarily in ruminant species. protective antigens and toxins with hemolytic and necrotizing activity are formed in susceptible animals ( jayaraman et al., 1962) . the necrotizing toxin may effect fatal toxemia with degenerative foci of myonecrosis. immunization. immunity to c. chauvoei can be produced via vaccination with its alum-precipitated formalinized cultures (chandler and gulasekhuram, 1970) . clostridium septicum, in contrast to c. chauvoei, is pathogenic for both man and animal. in man, it is associated with gas gangrene and in affected animals, primarily ruminants, it is the agent most closely identified with the diseases malignant edema and braxy. the organism produces four lethal necrotizing, hemolyzing toxins that cause an increase in capillary permeability and myonecrosis. immunization. immunity to c. septicum is induced with injection of formalinized bacterial cultures. the antitoxin provides homologous protection and additionally protects against c. chauvoei. animals are often vaccinated with mixtures of clostridial species; i.e., novyi, chauvoei, septicum, perfringens, and sordelli in one combination vaccine. these are highly efficacious vaccines and are routinely used in veterinary medicine. although infections with mycobacterium tuberculosis primarily occur through inhalation of the tubercle bacillus, ingestion of large numbers of the bacilli in contaminated milk or infectious sputum can readily produce disease in susceptible species. the bacterium is pathogenic in man, but can also cause disease in monkeys, pigs, and occasionally in cattle, dogs, and parrots. the disease may be asymptomatic or produce severe, debilitating pulmonary lesions. if infection is not restricted by the immune system, the disease may be fatal (comstock, 1982; bloom and godal, 1983) . since bacteriocidal mechanisms of the normal macrophage prevent m. tuberculosis from multiplying intercellularly (goren, 1977; goren et al., 1976) , protective immunity depends on cell-mediated immunity (lagrange, 1984) . mycobacterium bovis, closely related to m. tuberculosis, and m. avium causes disease primarily in cattle and birds. they can, however, be contagious to man, sheep, and pigs. immunization. immunoprophylaxis for tuberculosis is based on vaccination with an attenuated, relatively avirulent strain of m. bovis that does not produce lesions. the strain is known as bcg or the bacillus of guérin (1924, 1926) . worldwide, this is one of the most widely used human vaccines, as it has proven efficacious in controlling a severe disease. additionally, bcg has been used for nonspecific enhancement of resistance against tumors and other infec-tions. the cell wall of m. tuberculosis is a potent immunostimulant when used in freund's adjuvant. although streptococci may be normal inhabitants of the gastrointestinal tract, they may also be pathogenic for both man and animals. on the basis of characteristic cell wall components, the streptococci are traditionally divided into lancefield groupings (lancefield, 1934) . streptococcus agalactiae, a streptococcus group β organism, causes severe mastitis in the bovine species and has been identified as a major cause of serious neonatal infections in man (eickoff et al., 1964) . capsular antigens form the four major type-specific antigens (la, lb, ii, and iii), with type iii organisms being most commonly associated with neonatal meningitis. in infants, early-onset disease occurs within the first days of life and is characterized by sepsis and pneumonia. the mortality rate is high. late-onset disease occurs around 1 month of age and is characterized by meningitis (einstein et al., 1982) . immunization. there is a direct correlation between the absence of maternal igg antibody to type iii antigen and the incidence of neonatal infection. thus, susceptibility to the bacterium is related to the absence of significant levels of maternal serum antibody being transferred transplacental^ to the fetus. current vaccine developments are directed toward maternal immunization with type iii antigen (einstein et al., 1982) . streptococcus pneumoniae, the etiologic agent of pneumococcal pneumonia in human infants and adults, may cause septicemia, meningitis, and inner ear infections. aerosol transmission of the bacterium, often in association with viral upper respiratory infections, is the major mode of transmitting s. pneumoniae infections. streptococcus pneumoniae possesses a capsular polysaccharide capable of deterring phagocytosis, thus enhancing the virulence of the bacterium. of over 80 types of the bacterium identified, 10 serotypes are most frequently associated with the disease. a polyvalent pneumococcal vaccine prepared from soluble purified capsular polysaccharides of the 23 most predominant s. pneumoniae serotypes has proven effective in adults. the capsular polysaccharides are well-tolerated and highly immunogenic; signifi-cant rises in protective serum antibody titers are achieved following vaccination (kasper et al., 1982) . however, vaccination of infants has not proven beneficial, because they develop no higher antibody titers to the bacteria than do unvaccinated infants (ginsburg, 1986) . enterotoxigenic pathogenic strains of escherichia coli may cause severe, potentially fatal, diarrheal disease in both man and domestic species, particularly neonatal cattle and swine. the capsular (k) antigens are cell-surface proteins and/or polysaccharides associated with virulence. the k88 antigen mediates adhesion to the microvillus of intestinal epithelial cells; production and release of enterotoxin follow (bywater, 1970; lonroth et al., 1979) . escherichia coli neonatal enteritis of newborn calves is also a serotype-specific disease (myers and guinée, 1976) . ail important colostral antibodies in both swine and cattle are anti-k antibodies (usually k99) (myers and guinée, 1976; moon et ah, 1978) . immunization. vaccination of gilts, sows, heifers, and cows with vaccines prepared from the k88 or other pilus-associated antigens has reduced morbidity and mortality from e. coli nec natal enteritis of newborn piglets and calves (nagy et ah, 1978; rutter, 1975; kohler et ah, 1975; childrow and porter, 1979; myers and guinée, 1976) . to prepare the porcine vaccines, bacterial strains specific for the herds to be vaccinated are used to immunize animals 3 weeks prior to parturition, thereby generating specific, protective colostral antibodies. recombinant dna technology, discussed in the chapter by collett has introduced the potential to construct e. coli vaccine strains that would afford considerably better protection than those currently available. salmonella species are a major cause of invasive enteric infections in humans and domestic animals, with domestic poultry constituting the largest reservoir of salmonella organisms in nature. normally, infection occurs through the oral route. salmonella is a facultative intracellular pathogen; therefore, cell-mediated immunity is more important than humoral immunity in resistance to the disease, salmonellosis (fields et ah, 1986a,b; dougan et ah, 1987; woolcock, 1973) . salmonella typhi, the only salmonella species that has a capsular polysaccharide (vi antigen), is the etiologic agent of typhoid fever, a serious and common disease in underdeveloped areas (edelman and levine, 1986) . this pathogen infects humans only; there is no suitable animal model for typhoid fever. immunization. few vaccines have been developed for salmonella, and most are of low efficacy with undesirable side-effects. live vaccines are more effective than killed ones in promoting better immunity (levine et ah, 1983; dougan et ah, 1987; roantree, 1967) . with respect to s. typhi, vaccines containing the inactivated bacteria offer only limited and transient protection with undesirable side-effects (levine, et ah, 1983) . the attenuated strain of s. typhi, ty-21a, requires multiple doses to achieve 60-70% protection (hirschel et ah, 1985) . consequently, typhoid fever has not been controlled by immunization, although the vi antigen has recently been hailed as the agent of a preventative vaccine (robbins and robbins, 1984) without adverse side-effects (acharya et ah, 1987) . yersinia pestis is the etiologic agent of plague or "black death" in man, a highly fatal disease with fever and purulent lymphadenopathy. although not a disease of domestic animals, rats, ground squirrels, and other rodents may be affected. the bacterium is spread by the rat flea, xenopsylla cheopis, from rat to rat and from rat to man. immunization. the most widely used vaccine for the prevention of y. pestis infection is haffkine's vaccine, first developed in 1897. this vaccine is prepared from heat and phenol-killed virulent cultures. formalin-killed virulent bacteria are also successful, as are living avirulent strains (grasset, 1946) . the is no evidence that any vaccine protects against pneumonic plague, the most contagious and fatal form of the disease. furthermore, vaccine protection is only recommended for plague research workers. disease control is primarily dependent upon eradication of rodent carriers of y. pestis. pasteurella multocida and p. haemolytica are common commensals of the mucous membranes of the respiratory tract and oropharynx of healthy cattle, sheep, swine, dogs, and cats. when the bacteria multiply unchecked, they can penetrate the oral and/or respiratory mucosa, where they quickly grow and overpower the host's defense systems. pasteur first described this bacterium as the etiologic agent of fowl cholera; it is also associated with bovine pneumonia, swine plague, and an epizootic hemorrhagic septicemia in ungulates. the bacterium's heat-stable antigens have been used as serologic indicators in the gel diffusion precipitin test to define its 16 serotypes (brogden et al., 1978) . immunization. pasteur's successful bacterial vaccine to fowl cholera, and the first vaccine ever used, consisted of avirulent cultures of the p. multocida attenuated by prolonged growth on artificial medium. killed p. multocida vaccines are prepared from virulent immunogenic strains of the bacterium. the organisms are suspended in formalinized saline, incorporated into an adjuvant, and injected subcutaneously (heddleston et al., 1974) . these vaccines induce substantial immunity to fowl cholera. additionally, live vaccines for oral administration have been developed for use in the poultry industry (dougherty et al., 1955; heddleston et al., 1974; oison, 1977; bierer and derieux, 1972) . pasteurella multocida is usually mixed with modified live or killed bovine rhinotracheitis virus, parainfluenza 3 virus, bovine viral diarrhea virus, and p. hemolytica bacteria in combination vaccines to protect against pasteurella pneumonia in cattle. bovine pneumonic pasteurellosis (shipping fever) is a severe fibrinous pneumonia of feedlot cattle usually associated with biotype a, serotype 1 infections with this organism. immunization. administration of either killed or live vaccines has been of limited efficacy in controlling shipping fever. partial protection from experimental disease follows immunization of cattle with either live p. haemolytica by aerosol or parenteral routes confer et al., 1984) or lyophilized p. haemolytica vaccines consisting of chemically altered, streptomycin-dependent, or modified live organisms given intramuscularly or intradermally (confer et al., 1986) . humoral antibody responses appear to correlate strongly with protection against experimental disease (confer etat., 1985; mckinney et al., 1985) . for example, purified p. haemolytica lipopolysaccharide stimulates specific antibody formation and has protected calves challenged with the bacterium from developing the disease (hilwig et al., 1985) . these obligate parasites, restricted to respiratory and pharyngeal mucous membranes, cause important diseases in porcine (h. pleuropneumonia, h. suis, andi/. parasuis), equine (h. equigenitalis), bovine (h. somuns), and avian (h. gallinarum, h. paragallinarum) hosts. most hemophilus species require two factors, hemin (x) and nicotinamide adenine dinucleotide (v), for growth. antigens associated with protection and virulence have been described fori/, paragallinarum (yamamoto, 1984) , the etiologic agent of infectious coryza in chickens. birds that have recovered from natural infection possess varying degrees of immunity to re-exposure (page et al., 1963) ; immunity is serotype-specific. adjuvanted vaccines containing multiple bacterial serotypes are prepared from chicken embryos or formalinized bacterial broth cultures and are effective vaccines in preventing infectious coryza in chickens. this bacterium is the etiologic agent of the porcine disease, contagious pleuropneumonia, which is characterized by severe multifocal, necrotizing pneumonia with venous thrombosis and associated serofibrinous pleuritis (didier et al., 1984) . the disease is of considerable economic importance to the swine industry, being most prevalent in situations where swine are raised under intensive management conditions. hemophilus pleuropneumoniae possesses major and minor antigens that are both common and serotype specific (gunnarson et al., 1978; gunnarson, 1979; mittal et al., 1982) . since high antibody titer apparently provides little protection from the disease, cell-mediated immunity may be important in protection from infection (rapp and ross, 1984; rosendal et al., 1981) . immunization. no adequate immunoprophylaxis against contagious pleuropneumonia is currently available, although many vaccines have been tried (nielsen, 1976; henry and marstellar, 1982; christensen, 1982; masson et al., 1982) . prior infection with one serotype provides protection from heterologous serotypes (nielsen, 1976) . the bacterial strains used in vaccines are serotype specific and, while not preventing the disease, can reduce its severity (christensen, 1982) . hemophilus somnus is the cause of infectious meningoencephalitis, a disease with low morbidity but high mortality in cattle. whole or sonicated bacterial cells and bacterial protein are immunogenic (noyer et al., 1976) and efficacious bacterins foster protective immunity in calves (williams et al., 1978) . the bacterins of h. somnus, adjuvanted with aluminum hydroxide, are prepared from highly immunogenic strains of the bacterium and grown in serum-free media for use as vaccines. the species in this genera, b. pertussis, b. bronchiseptica, and b. parapertussis, can be either parasites or, as in swine and dogs, common inhabitants of the upper respiratory tract. these small, serologically related bacilli produce a dermonecrotic toxin. infection is by aerosol transmission with bacteria adherent to tracheal cilia (bemis et al., 1977) . local, not serum, antibody concentration is important in clearance of the infection. the etiologic agent of whooping cough, β. pertussis, produces two distinct hemagglutinins, leukocytosis-promoting factor-hemagglutinin (lpf-ha) and filamentous hemagglutinin (fha), and various toxins (pertussis toxin [pt] and dermonecrotic toxin). fha is involved in bacterial adherence to the respiratory mucosa, whereas pt is believed to be the major protective (sato and sato, 1984) and pathogenic antigen (steinman et al., 1985) . immunization. although efficacious, the safety of the human vaccines currently in use, suspensions of killed whole cells containing protective antigens, is open to question (robinson et al., 1985) . undesirable side-effects such as screaming, collapse, encephalopathy, and other serious neurological complications have been reported in association with b. pertussis vaccinations (dick, 1978) . the potencies of whole cell vaccines correlate with the antigenic content of pt (reiser and germanier, 1986) . bordetella bronchiseptica is an obligate parasite of the upper respiratory tract of both dogs and pigs. in dogs the bacterium frequently invades the lungs as a sequela to canine distemper (caused by a morbillivirus), causing an often fatal bronchopneumonia. the bacterium is also associated with mild to severe tracheobronchitis, "kennel cough," in dogs (bemis et al., 1977) . in pigs, a deformation of the bony structures of the nasal area (atrophic rhinitis) and reduction of the total volume of nasal turbinates commonly follow the bacterial infection (ross et al., 1963) . degenerative changes in the osteoblasts and osteocytes may be caused by elaboration of a dermonecrotic toxin (dnt), which is released from b. bronchiseptica after colonization or multiplication of the organisms on the nasal mucosa (nakai et al., 1985) . the release of dnt from p. multocida type d is thought to exacerbate the disease. immunization. vaccines to control canine b. bronchiseptica infections are commonly incorporated into combination packages containing attenuated live-virus canine distemper, and canine adeno and parainfluenza viruses. to control atrophic rhinitis, avirulent live, or inactivated organisms alone or in combination with p. multocida, erysipelothrix rhusiopathiae and!?, coli have been utilized in vaccines. the organisms in this group, b. abortus, b. suis, β. melitensis, β. canis, and b. ovis, cause the disease brucellosis in domestic animals and man. the bacterium may localize in the reproductive tract which, in the female, can lead to fetal death with subsequent abortion. brucellosis, due to b. abortus or b. melitensis, is a zoonotic disease, readily transmitted from animal to man. the potentially severe consequences of brucellosis, fetal death and abortion, in the pregnant cow and epididymitis and sterility in the bull result in significant economic loss to the cattle industry. the primary source of infection is infected animals, whose mammary and/or genital secretions may contain the bacterium. calves can become infected in utero; however, the main portals of infection are oral mucosa, nasopharynx, and conjuctiva of exposed animals. immunity to b. abortus is dependent upon cell-mediated immunity, as the presence of serum antibodies, although a significant indicator of infection, does not correlate with the immune status of the host (fitzgeorge et al., 1967; kaneene, et al., 1978; swiderska et al., 1971; montaraz and winter, 1986) . most humans who contract brucellosis have been exposed either to b. melitensis, the etiologic agent of malta or mediterranean fever, or b. abortus. brucella melitensis, found in the milk of infected sheep and goats, may produce fatal disease when ingested by humans. brucellosis of sheep and goats mimics the disease as it is seen in cattle, with fetal death and abortion occurring in ewes and does and epididymitis in rams and billies. brucella ovis infects sheep, causing late fetal death and abortion in pregnant ewes and epididymitis in rams, such as b. abortus does in cattle. immunization. brucella abortus (strain 19) is currently used as the vaccine of choice for control of brucellosis of cattle in the united states. this is a viable, smooth strain that, while posing virtually no threat for cattle, may cause disease in man. the major objections to the vaccine are this pathogenicity for humans and the difficulty of differentiating vaccinated from naturally infected animals since persistent serum antibodies are induced by the vaccine. killed b. melitensis in adjuvant or live avirulent strains have been used for vaccines to induce a high degree of immunity in sheep and goats. brucella ovis bacterins in adjuvant, as well as b. melitensis vaccines, have been used to protect animals from the disease b. ovis causes, since the antigens of these two pathogens are cross-protective (diaz et al., 1967) . pathogenic members of this genus are associated with venereal disease, fetal death, and abortion in cattle. this bacterium is transmitted to uninfected cattle by coitus or artificial insemination and is an obligate parasite of the genital tract. immunization. stimulation of opsonizing antibodies of the igg class by systemic vaccination with adjuvanted vaccines is effective in preventing natural infection in bulls (bouters et al., 1973) and infertility in cows (corbeil et al., 1974; hoerlin and kramer, 1964) , and prevents the carrier state (wilkie and winter, 1971 ). unlike c. fetus venerealis, this bacterium is contracted by ingestion and is not transmitted venereally. both sheep and cattle can be infected; however, the disease is most severe in pregnant ewes, which undergo a high percentage of abortions or premature births within a flock. immunization. in sheep, vaccination with polyvalent adjuvanted vaccines is efficacious in preventing disease (thompson and gilmour, 1978) . the most important organism of this genus, v. cholera, causes severe, acute enteritis in humans and nonhuman primates. the etiologic agent of cholera in humans, v. cholera, causes a potentially fatal diarrheal disease. infection results in the production of a powerful enterotoxin in the small intestine, which stimulates an increase in cyclic amp in intestinal epithelial cells and causes a profuse outpouring of isotonic fluids. vibrio possesses immunogenic heat-labile flagellar (h) protein and heat-stable (o) lipopolysaccharide somatic antigens. the cholera toxin is immunologically and functionally similar to the heat-labile enterotoxin of e. coli (yamamoto et al., 1984) . it consists of six light subunits (l) that assist in toxin adherence to intestinal cell receptors and one heavy subunit (h), which is the toxic entity. immunization. present cholera vaccines are administered by parenteral or oral routes. parenteral vaccines consist of formalin/phenolinactivated bacteria, whereas oral vaccines employ killed or live bacteria. vaccines given by either route provide protection for approximately 3-9 months. the predominant immune mechanism is antibacterial rather than antitoxic (levine et al., 1979) ; antibacterial antibodies prevent attachment of the bacterium, whereas antitoxic antibodies inhibit toxin adherence to cell receptors. because current vaccines often produce adverse side-effects (feeley, 1970) , synthetic and semi-synthetic vaccines are currently under investigation. for the latter, a nonpyrogenic, bivalent cell-surface protein-polysaccharide conjugate is being investigated (kabir, 1986) . h. leptospira spp. the pathogenic genera of this family can penetrate the gastrointestinal mucosa and abraded epidermis. leptospires are transmitted through contact with the urine of animal carriers, either directly or in contaminated water or soil. rodents are the primary reservoir of the bacteria which, due to its ability to synthesize urease, colonizes the renal nephron and subsequently is shed into the urine. leptospirosis causes economically serious disease in cattle and swine by causing fetal death, abortion, and infertility. recovery from infection with one serotype lends immunity only to that serotype. this immunity is predominantly humoral, since agglutinins (igm) are responsible for the initial clearance of the bacteria; neutralizing antibodies (igg) are also protective (hanson, 1974; negi et al., 1971) . canine leptospirosis infections may be severe, occurring more commonly in male dogs than in females. man is a dead-end host for leptospires; infection in man is accidental and usually related to occupational exposure. immunization. killed, multivalent, leprospira vaccines protect against clinical disease in cattle and swine; however, in pigs, immunity does not protect against renal colonization (stalheim, 1968) . dogs can be vaccinated successfully with formalin or phenol-killed vaccines that contain antigens from the two most common infecting serotypes, l. canicola and l. icterohemorrhagica (kerr and marshall, 1974) . vaccines for humans, prepared from chemically inactivated cells of leptospires, have been used extensively in certain areas of the world. neisseria meningitidis neisseria meningitidis is a frequent cause of endemic purulent meningitis in human infants and adults, although the incidence of the disease is substantially higher in young infants (hoffman, 1986) . bacterial invasion of the meninges is usually hematogenous from the upper respiratory tract and is a life-threatening affliction. neisseria meningitidis has been classified into at least nine groups (a, b, c, w-135, x, y, z, l, 29-e) on the basis of its capsular polysaccharides (morse, 1986) . immunization. protection against meningococcus meningitis results primarily from the presence of antibodies against the capsular polysaccharide of n. meningitidis (frasch et al., 1982) . group a and c polysaccharide vaccines are especially effective against disease in children over two years of age and in adults. epidemic typhus fever has afflicted mankind since ancient times. it is an acute highly infectious disease with the potential for explosive epidemics in man. significant outbreaks of the disease have been intimately associated with war and famine. the disease is characterized by sustained high fevers, headache, panencephalitis, a diffuse maculopapular skin rash, and toxic vascular damage. the fatality rate may be high. the etiologic agent, r. prowazeki, is transmitted from person to person by the human body louse pediculus humanus corporis. infection is established by inoculating infected louse feces into the skin by scratching. immunization. although no etiologic relationship has been demonstrated between r. prowazeki and the bacterial strain proteus 0x19, these two species share a common polysaccharide antigen (castaneda, 1934) . the sera of infected typhus patients agglutinates proteus 0x19 and this test is now standard (weil-felix reaction) for diagnosis of the acute disease. additionally, r. prowazeki has two major antigenic components-one heat labile and the other heat stable (craigie et al., 1946; topping et al., 1945) . typhus fever vaccine contains killed organisms propagated in the yolk-sac membranes of developing chick embryos (cox, 1948) . this vaccine not only diminishes the symptoms of typhus in immunized persons, but it also greatly reduces the mortality rate (gilliam, 1946) . the usefulness of the attenuated (madrid ε strain) vaccine is hampered because, under appropriate conditions, the strain may revert to virulence (brezina, 1982) . the prokaryotes proc. world vet. congress, 17th proc. annu. conf. aust. vet. assoc tuberculosis in bacterial infections in humans: epidemiology and control symposium on rickettsial diseases vm/sac new trends and developments in vaccines vaccines '87: modern approaches to new vaccines principles and practice of cholera control vaccines '86 proc. natl. acad the prokaryotes proc. natl public health rep viral hepatitis viral hepatitis proc. west. states food anim. conf vaccination against foot and mouth disease an inquiry into the cause and effects of the vanidae vaccinae vaccines '86 a system of bacteriology the mycobacteria: a source book proc. natl. acad. sei. u.s.a. 79 diseases of poultry bovine medicine and surgery proc. int. conf. equine infect. dis proc. annu. conf. aust. vet. assoc proc. natl. acad. sei. u.sa. 60 proc. conf. res. work. anim. dis vaccines '86 proc. 65th annu. meet. u.s. livestock sanit. assoc proc. natl. acad proc. 69th annu. meet. u.s. livestock sanit. assoc the virus in yellow fever proc. int. vet. congr vaccines '85 diseases of poultry were useful references in writing this chapter. key: cord-276348-vr5fit8r authors: ogra, pearay l. title: respiratory syncytial virus: the virus, the disease and the immune response date: 2004-01-31 journal: paediatric respiratory reviews doi: 10.1016/s1526-0542(04)90023-1 sha: doc_id: 276348 cord_uid: vr5fit8r abstract rsv is the primary cause of hospitalisation in the first year of life for children in most parts of the world, and nearly 100% of children in the usa are infected with the virus by 2 to 3 years of age. the agent is an enveloped rna virus with a non-segmented single-stranded negative-sense genome. the viral genome encodes 8 structural and 2 non-structural proteins. important structural proteins include the fusion (f) protein and the attachment (g) protein which are essential for viral penetration and attachment to the host cells. both proteins are important in development of immune responses. the virus is estimated to cause 3000 to 4000 deaths annually. primary infections are as a rule symptomatic. the spectrum of clinical manifestations ranges from mild upper tract illness, infection in middle ear which progresses to acute otitis media, croup, to apnoea in premature infants, pneumonia and bronchiolitis. premature babies born at 30–35 weeks of gestation, infants with cyanotic congenital heart disease, hiv-infected subjects, and patients on intensive immunosuppressive therapy especially after bone marrow transplant are considered to be at risk for increased mortality and morbidity during rsv infection. the virus does not normally replicate outside of the bronchopulmonary tree and the infection is exquisitely restricted to the respiratory mucosa. however, development of extrapulmonary disease has been observed in certain t and b cell immunodeficiency states. the association of rsv with asthma and reversible reactive airway disease in early childhood has attracted significant attention. recurrent wheezing for up to 5 to 7 years of age and established airway disease has been observed in a significant number of children with a strong family history of allergy, after primary infection or reinfection with rsv. immune response to primary infection is relatively small but on reinfection, a significant booster effect with sustained immunologic reactivity is observed in serum and respiratory mucosa. both cd4and cd8-specific as well as th1and th2-cell specific immune responses have been observed during human infection. in addition, proinflammatory as well as immunoregulatory cytokines and chemokines are induced in the respiratory tract after natural and induced (in vitro) infection. significant progress has been made in understanding the role of th1 vs. th2, ige, viral induced cytokines and chemokines in the mechanisms of pathogenesis of the disease, development of wheezing and in the prevention and treatment of the infection and its sequelae. respiratory syncytial virus (rsv) is one of the commonest human viral infections, and virtually every child is infected by the third birthday. because of its restricted mucosal immunopathology, and frequent association with bronchial hyperreactivity and development of wheezing, rsv has served as an important model to investigate mechanisms of mucosal immune responses and development of mucosal disease following infection. the importance of rsv in bronchopulmonary disease and development of bronchial hyperreactivity has been the focus of several recent symposia [kimpen jl, simoes eaf. am j respir crit care med 2001; 163:s1–s6]. this brief report will only summarise, based on selected references, the historical landmarks of its discovery and current understanding of the mechanisms of immunity, and their possible role in the pathogenesis of bronchopulmonary disease. s120 p.l. ogra rsv was discovered in 1956, when a group of chimpanzees in a colony outside of washington, dc (usa) were noted to have developed cold-like illness. morris and colleagues recovered a cytopathic agent from one of these chimpanzees, who had an upper respiratory tract illness with coryza, runny nose, and malaise. they named this agent "chimpanzee coryza agent"(cca). 2 these investigators examined the entire colony, and nearly 100% of the chimpanzees were found to be infected. an interesting observation was that the human contacts working with the chimpanzees were also infected and exhibited mild upper respiratory tract illness and coryza, somewhat less severe than observed in the chimpanzees. subsequent studies identified two major isolates of the virus recovered from other patients with upper respiratory tract illnesses. the long strain, commonly used in laboratory studies, was recovered from the bronchopulmonary washings of a child with bronchopneumonia, and the schneider strain was recovered from a patient with croup. 3, 4 on the basis of the cytopathology of this agent in tissue culture with formation of syncytia, and the similarities between the isolates recovered from the monkeys and the long and the schneider strains recovered in humans, chanock and colleagues coined the term "respiratory syncytial virus" to incorporate all available isolates, and provided a classic description of the disease in children. 3, 4 shortly thereafter, beem and colleagues described in detail the epidemiology of rsv infection during community outbreaks. 5 the agent is a pleomorphic, enveloped, cytoplasmic virus containing single-stranded, negativesense rna. the rna is associated with viral proteins, consisting of a nucleocapsid core that is packaged within a lipid envelope. rsv is classified in the genus pneumovirus, which belongs to the family paramyxoviridae. the paramyxoviridae family also includes two other genera, paramyxovirus (containing, e.g., parainfluenza virus types 1, 2 and 3 and mumps virus) and morbillivirus. the genera are differentiated by the diameter of the helix, the number of genes, and the nature of their surface glycoproteins. the surface g glycoprotein of the virus lacks neuraminidase and hemagglutinin. complementary dna (cdna) cloning has identified ten different viral genes, each coding for a single protein. the sequences of each gene have been described. the characteristics of these genes differentiate rsv from the other members of paramyxoviridae as reviewed previously. 6 eight of the ten rsv proteins are present in infected cells and in the virions, and therefore are structural proteins. the disulfide-bonded glycoprotein (f, fusion protein) and the large glycoprotein (g, attachment protein) are surface proteins and are the major antigenic determinants of the virus. these proteins induce protective antibodies. the g protein mediates viral attachment. the f protein mediates viral penetration and syncytium formation. the small hydrophobic protein (sh), the matrix protein (m), and the m2 protein are envelope-associated proteins. the nucleoprotein (n), the phosphoprotein (p), and the large nucleoprotein (l) are present in the rsv nucleocapsid. ns1 and ns2 are non-structural proteins; they are found only in infected cells but not in virions. 6 rsv displays minimal antigenic heterogeneity. however, two major groups a and b, with antigenic differences on the g, f, n, and p proteins, have now been identified. the g protein is the most variable protein, with only 53% homology in the amino-acid sequences between the proteins of the a and b groups. in contrast, the f and n proteins have a high degree of genetic and antigenic homology between the two groups. both f and g proteins have several distinct antigenic sites. 6 recent data have shown considerable genetic diversity among groups a and b. the g-protein sequences may differ 20% in different group-a lineages and 9% in different group-b lineages. the replication of the virus includes the following specific events. the virus attaches the cell through g protein. the viral envelope fuses with the plasma membrane of the host cell through the f protein. after penetration, the nucleocapsid of the virus is released into the cellular cytoplasm, where the replication takes place. the viral rna serves as a template for messenger rna. the messenger rna serves as a template for translation of viral proteins, and complementary rna serves as a template for transcription of virion rna. the viral antigens can be detected in 9 hours in cell culture, and the infectious virus in 11 to 13 hours. human rsv replicates in several animal species, including mice, cotton rats and chimpanzees, and to a smaller extent in guinea pigs and ferrets. 6 the clinical picture of rsv infection varies according to age. the primary infection at 6 weeks to 2 years of age is usually symptomatic and involves the lower respiratory tract. asymptomatic primary rsv infection in children is rare. repeated infections in older children are usually less severe. respiratory tract infections are frequently associated with expiratory wheezing (variously referred to as bronchiolitis or wheezy bronchitis, asthma), pneumonia and acute otitis media. rsv infections in neonates differ from those in older children. such neonates do not often exhibit wheezing, and apnoea may be the only symptom of infection. the mortality in healthy children is extremely low, but life-threatening infections are common in immunocompromised patients and in patients with cardiac abnormalities. pneumonia is the most common manifestation in elderly subjects. isolated upper respiratory tract infections associated with rsv have been noted, especially in older children and adults during re-exposure. the common symptoms are rhinorrhea, nasal congestion, pharyngitis, and cough. some studies suggest that common colds induced by rsv may be more prolonged and severe than those induced by other viruses. 6, 7 the term bronchiolitis has been used as a diagnosis of a specific clinical symptom complex since 1940. the diagnostic criteria vary between different centers in the usa and in other countries. in general, bronchiolitis is a clinical presentation in infants less than 12 months old in whom a first attack of an acute illness, after a brief prodrome of upper respiratory symptoms, is characterised by wheezing, dyspnea, respiratory distress, poor feeding, tachypnea (>50/min), and radiologic evidence of hyperaeration of the lung. fine crepitation can usually be heard by auscultation of the chest. 6, 7 in the majority of patients, the symptoms and signs resolve within a few days to a week after the onset of illness. in sicker infants, the duration of hospitalisation may last up to 7 days. in the past infants under 6 weeks old and those with underlying illnesses often needed longer hospitalisation. although rsv is the most common etiologic agent of bronchiolitis and virtually the only agent that induces epidemics, other respiratory viruses can also induce bronchiolitis. these include parainfluenza type 1 and 3 virus, rhinoviruses, adenoviruses, coronaviruses, and influenza a virus. adenoviruses can induce very severe bronchiolitis and with high mortality. numerous follow-up studies have shown that up to 75% of the patients with rsv bronchiolitis exhibit recurrent wheezing or pulmonary function abnormalities years later. the clinical symptoms gradually decrease and disappear usually during the following 10 years. in some of these patients, the symptoms continue and they are classified as having asthma. a diagnosis of asthma has been established in up to 92% of patients followed prospectively for 5−10 years after bronchiolitis. even after milder lower respiratory tract rsv disease, increased morbidity was documented through the third and fourth year of life. however, normal pulmonary function was found between the ages of 8 and 12 years. 6, 7 it is not clear whether rsv can induce long-lasting or permanent damage to the small airways and in the growing lung. it is possible that development of bronchiolitis during primary infection may be restricted to subjects already genetically and anatomically at risk for pulmonary hyperreactivity. this possibility is strongly supported by the findings that pre-existing diminished lung function measured very early in life (before any respiratory illness) was found to be a risk factor for recurrent wheezing. 7, 8 although rsv infection is rare in the first 4 weeks of life, epidemics in neonates have been described. rsv infection has been observed in 20−30% of babies studied in a neonatal unit and in 35% of those hospitalised for 6 days or longer. 9 in this study, 61% of the babies had respiratory illness, and of these, approximately half had upper respiratory tract infection, the other half had pneumonia. when pneumonia during the first month of life was studied, rsv was found to make up 55% of all isolates. 9 it is now well-documented that rsv infection occurs commonly in adults as well as children. in a family study, 17% of the adults living with infected children also became infected. 8 in adults, rsv infection can be asymptomatic or can induce mild to moderate upper respiratory tract symptoms. in healthy adults, the infection is rarely severe or fatal. the symptoms include fever for 1 to 4 days, nasal congestion, rhinorrhea, sore throat, ear pain, and cough lasting 10 days or longer. the average duration of virus shedding is 5 days. based on clinical features, rsv infection cannot be differentiated from other agents associated with cold-like symptoms. adults who are immunocompromised, institutionalised, or aged, or who have some underlying illness (especially chronic pulmonary disease) are at risk of severe rsv pneumonia. the occurrence of pneumonia in long-term care facilities varies from 5% to 67%, with mortality rates ranging from 0 to 53%. the chest radiograph usually reveals patchy changes, diffuse consolidation or interstitial infiltrates. during outbreaks, rsv must be included in the differential diagnosis of fever with evidence of pulmonary infiltrates in immunocompromised adults. children at increased risk from rsv infection include young infants with prematurity, bronchopulmonary dysplasia, congenital heart disease, congenital or acquired immunodeficiency, subjects with hematologic malignancies, patients with bone-marrow or organ transplants, and cystic fibrosis. as mentioned earlier, premature infants are more likely to have apnoeic spells, atelectasis/infiltrates, and hyperinflation as seen on the chest radiograph, and may require oxygen therapy and mechanical ventilation. consequently, these patients need longer hospitalisation. 10 some studies suggest that intubation increases the risk for fatal illness. rsv infection is a major reason for re-hospitalisation of children with bronchopulmonary dysplasia. in these patients, a large number of siblings, parental smoking and recent need for home oxygen therapy are major risk factors. 6 clinicians have long been aware that rsv infection may be particularly severe, long lasting, and sometimes fatal in children with congenital immunodeficiency diseases, especially those with both t-and b-cell defects. animal studies have suggested that t-cell mediated cellular immunity is responsible for terminating rsv infection. no reports are available of possible increased severity of rsv disease in children with isolated hypogammaglobulinemia. increased morbidity and mortality have, however, been documented in children undergoing chemotherapy. severe rsv infection has been reported in children who underwent liver transplantation. the risk factors appeared to be acquisition of infection soon after transplantation and pre-existing lung disease. 10 recently, rsv infection has been studied in human immunodeficiency virus (hiv)-infected children who experienced pneumonia and prolonged viral carriage. up to 25% of such children may develop fatal illness. 10, 11 congenital heart disease is another well-established risk factor for severe rsv infection. cardiac function is not depressed in patients with normal hearts who have rsv infection. infants with heart disease and rsv infection often need more treatment in the intensive care unit and more ventilator therapy than those without congenital heart disease. the mortality in infants with heart disease has been estimated to be about 37%. even higher mortality (73%) has been reported in patients with pulmonary hypertension. 6,10,11 the mortality associated with primary rsv infection in otherwise healthy children is estimated to be 0.005% to 0.020%. 6 in hospitalised children, mortality rates are estimated to range from 1% to 3%. 3 however, considerably higher mortality rates have been observed in children with cardiopulmonary abnormalities and in immunosuppressed subjects. due to the ubiquitous nature of rsv infection, even a low mortality rate may have marked impact on the total mortality of young children. the temporal patterns of respiratory viral isolations from ten laboratories in the usa with that of deaths of children has suggested that rsv isolations were clearly associated with the respiratory deaths of children 1 to 11 months old and with influenza in children 24 to 59 months of age. a significant correlation has been shown to exist between the occurrence of sudden infants death syndrome (sids) and rsv infections. 12, 13 rsv has been demonstrated in the lungs of up to 25% of infants who died from sids. 13 prolonged apnoea, which is a major sign of newborn rsv infection, may explain some of these deaths. at present, however, the role of rsv in sids in not fully understood. community-acquired respiratory viruses are important causes of potentially serious acute respiratory illnesses in hospitalised bone-marrow transplant (bmt) recipients. approximately one-third of such patients are infected with one of these viruses, although multiple viruses have been isolated continuously during these winter study periods. 10 the morbidity and mortality associated with these infections were substantial. more than half (58%) of these infections may be complicated by pneumonia, with an associated mortality of over 50%. in immunocompromised patients with rsv infection, the pneumonias are almost exclusively viral in origin and may be associated with a mortality of 100% if not treated promptly. many of the pneumonias in patients infected with other viruses, such as influenza virus, appeared to be either self-limited or appeared to have a substantial bacterial component, as judged by their favourable response to antibacterial therapy. however, fatal viral pneumonias occur as well. it is noteworthy that only a few infections due to parainfluenza virus and adenovirus are observed during these winter seasons and that their potential to cause fatal viral pneumonia has not been appreciated. 10, 11 in bmt recipients with an acute respiratory illness, community-acquired respiratory viruses must be considered seriously. other recent studies have also suggested extremely high morbidity and mortality with rsv infection in patients with leukemia and hematologic malignancies. 10 rsv infection is followed by the development of both serum and mucosal igm, iga and igg antibodies however in a few studies, igm antibodies against rsv were found to remain detectable for at least 1 year. 6 rsv-specific igg antibody response can be detected in most patients; it reaches maximum values in 20 to 30 days after the onset of symptoms. again, lower responses in young infants have been reported. igg responses occur mainly in igg1 and igg3 subclasses, indicating the antigenic nature of the protein moieties of the f and g proteins of rsv. one year after the primary infection occurs, rsv-specific igg levels appear to decline to low levels. after re-infection, a booster effect is noted, with high titers of igg detectable within 5 to 7 days. 14 the serum iga response occurs several days later than igm and igg responses. 15 interestingly, iga can be found free and cell-bound in nasopharyngeal secretions of patients with rsv infections. free anti-rsv iga appears within 2 to 5 days after infection, and peak titers are obtained between 8 and 13 days. the nasopharyngeal iga response is greater in children older than 6 months. a mucosal immune response to rsv has also been demonstrated by rsv-induced antibody response in vitro in tonsillar lymphocytes. 16 furthermore, nasal secretions contain free rsv-specific ige and cell-bound ige during rsv infection. 14 several studies have demonstrated specific antibody responses to major rsv structural proteins. antibody responses to the f protein of rsv are often cross-reactive with both rsv strains tested, whereas antibody responses to the g protein are subgroup specific. similar findings were reported for group-specific antibody responses to primary and secondary rsv infections. these observations suggest that primary and secondary infection with group-a viruses can induce cross-reactive neutralising antibody responses to group-b viruses. rsv infection induces specific cell-mediated immune responses, including lymphocyte transformation, cytotoxic t-cell responses, and antibodydependent cellular cytotoxicity responses. 1 a number of mechanisms have been proposed to explain the association of rsv infection with increased bronchial reactivity and wheezing (table 1) . these include anatomic restrictions of the neonatal bronchial tree, and tissue damage produced by the infection itself as a result of direct cytopathology of the mucosa. during the course of acute infection, there is sloughing of the respiratory epithelium with exposure and activation of irritant receptors, which induce neurogenic stimulation of bronchial smooth muscle and development of bronchial spasm. there is loss of inhibitory mediators and cholinergic neural activity as observed during parainfluenza and influenza virus infections. recent in vitro studies have demonstrated that rsv significantly augments the proinflammatory effects of substance p by upregulating the expression and density of its specific receptor nk1 on target cells. substance p is a neuropeptide which has been shown to exhibit significant bronchoconstrictor effects in experimental animal studies. 17 because of the anatomy of airway in the neonate, these neurogenic factors may be more important in the induction of bronchial hyperreactivity in early infancy and childhood than later in life. 17 there is good evidence from both clinical studies and experimental models with rsv, that early respiratory infections may contribute to early systemic sensitisation to other antigens or allergens in a genetically prone or atopic child (table 2) . such children may start building up homocytotropic immune responses to environmental allergens very early on in life. 18, 19 antibody-mediated immune responses are to a large extent protective. igg, iga and igm do not seem to contribute to the development of disease. in fact, they seem to be important in protection. high levels of maternal antibody are indeed protective against disease. however, the magnitude of such immune response in the early years is low. it has been observed that the primary immune responses against rsv are relatively ineffective, s124 p.l. ogra table 2 characteristics of cellular immune reactivity in children and adults relative to atopic susceptibility susceptibility response atopic th 2 response to common inhalant allergens non-atopic low levels of th 1 response cord blood low levels of th 1 response to food and inhalant allergens (evidence of in utero sensitization) atopic early childhood boosted th2 non-atopic early childhood deviated to th 1 but when such children get reinfected, they develop significant boosting, particularly for the igg and iga responses. 6 it is important to recognise that virtually all children who get infected with rsv develop virus-specific ige homocytotropic antibody in the respiratory tract. such ige activity is predominantly cell-bound to the mucosal epithelial of the respiratory tract. 20 in general, there is not much free ige detectable in respiratory excretions, unless children are wheezing or they have bronchiolitis or pneumonia. it seems that most children produce ige to rsv early in life, but it is the amount, the persistence and the duration of this response which is critical in determining which patients are going to develop bronchiolitis and wheezing. it has been observed that persistent virus-specific ige response in respiratory mucosa is an important element in the development of immunopathology for both rsv and parainfluenza viruses. 20 in the last decade there has been considerable interest in examining the role of cell-mediated immune responses in the mechanism of immunopathology in the respiratory tract. both cd4+ and cd8+ cells and th 1 and th 2 types of cd4+ cells have been implicated in the development of disease especially during rsv, rhinovirus and influenza virus infections. 1, 18, 19 in addition to ige antibodies and their interaction with mast cells and the subsequent release of inflammatory mediators, the interaction between rsv and the respiratory epithelium also results in the release of several other mediators. these proteins are very important in mobilising other cells to the site of disease. studies conducted by our group during the past decade have demonstrated the release of leukotrienes, eosinophil degranulation byproducts and epithelial cell-derived cytokines and chemokines during the course of rsv infection in in vitro and/or in vivo settings. 20−23 there is a wide spectrum of cytokines, chemokines and arachidonic acid metabolites, which are generated during the course of rsv infection. many of these products are critical in recruiting cells to the site of disease. furthermore, during the course of infection, there is induction of several cell-adhesion molecules and homing ligands (cd11b, icam-1, e-selectin) necessary for inflammatory and immune cells to be mobilised to the site of disease, to rollover, bind, and stick to the virus-infected tissues. they also induce expression of antigen-presenting molecules like hla class i and ii. thus, there is a complex array of events set in motion by rsv infection of respiratory epithelium, which mobilise inflammatory and possibly immunoregulatory cells to the site of disease in the mucosal epithelium. 24 considering the large number of mechanisms proposed as explanations for the disease caused by the virus it is important to attempt to separate the primary cause and the subsequent effects of specific cell-virus interactions in mucosal tissues. is rsv disease a true consequence of all these different pathogenic mechanisms proposed, or are they set in motion as a result of the activation of different immunoregulatory pathways during the replication of the virus in the mucosa? studies to date have identified a number of other viral-or host-induced events during rsv-associated clinical disease. clearly, all these events cannot be the cause of the disease seen after infection with rsv. in order to provide a unifying base to the multiplicity of phenomena observed, it has been proposed that rsv infection may trigger the activation of a "master switch" of genetic control, regulating the expression of one or more cellular functions identified above. previous studies in molecular biology have described in detail the role of transcription factors in activating certain genes or groups of genes. there are a number of nuclear factors, which mobilise gene activation processes and activate many cellular functions. rsv has been shown to induce activation of several such transcription factors. recently garafalo et al. and others have shown that rsv activates the gene promoters for nf-il-6 and nf-úb. such transcription factors regulate the synthesis of a variety of cytokines and chemokines and other important immunomodulating proteins. these include tnf-a, il-1b, il-2, il-6, gm-csf, and g-csf; adhesion molecules icam-1, vicam-1 and e-selectin; and chemokines, il-8, mip-1a, mcp-1, and eotaxin. these molecules are important in initiating the inflammatory cascade and in mobilising cells to the site of disease. 24, 25 it appears that rsv, during the course of replication at the level of the cell, initiates activation of certain transcription processes which may have a profound effect on expression of many mediators of immunoregulation and inflammation. other viruses such as influenza, rhinoviruses and adenoviruses have been shown to induce a similar activity. recent studies suggest that the ultimate expression of allergy involves a multiplicity of genetic and environmental factors. failure to switch off expected th2 phenotype may be a key factor in eventual development of allergic sensitisation. a number of other environmental factors in early infancy and childhood may also determine the outcome of allergic sensitisation by shifting cellular immune response towards immunologic hyperactivity (table 3). it is thus proposed that a balance between the expression of different pro-inflammatory cytokines and chemokines, and the development of th 1 vs th 2 or cd 4 vs cd 8 cellular response following rsv infection may ultimately determine the degree of pathology or the level of protection against bronchopulmonary disease. respiratory syncytial virus and reactive airway disease recovery of cytopathogenic agent from chimpanzees with coryza recovery from infants with respiratory illness of virus related to chimpanzee coryza agent (cca): i. isolation properties and characterization recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (cca): ii. epidemiologic aspects of infection in infants and young children respiratory syncytial virus neutralizing antibodies in persons residing in chicago respiratory syncytial virus respiratory syncytial virus and parainfluenza virus respiratory syncytial virus infections within families neonatal respiratory syncytial virus infection respiratory syncytial virus infections in immunocompromised adults respiratory syncytial virus pneumonia in hospitalized adult patients with leukemia respiratory viruses and sudden infant death secretory component and sudden-infant death syndrome the antibody response to primary and secondary infection with respiratory syncytial virus: kinetics of classspecific responses immunoglobulin class-specific antibody response in respiratory syncytial virus infection measured by enzyme immunoassay characteristics of in vitro production of mucosal antibody to respiratory syncytial virus in tonsillar tissue lymphocytes neural mechanisms of respiratory syncytial virus-induced inflammation and prevention of respiratory syncytial virus sequelae th1 and th2 cd4+ cells in the pathogenesis of allergic diseases the development of respiratory syncytial virusspecific ige and the release of histamine in nasopharyngeal secretions after infection interleukin-1a mediates the enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus inducible translational regulation of the nf-il6 transcription factor by respiratory syncytial virus infection in pulmonary epithelial cells characteristics of il-6 and tnf-a production by respiratory syncytial virus-infected macrophages in the neonate clinical aspects of bronchial reactivity and cell-virus interaction transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the rela transcription factor as a mechanism producing airway mucosal inflamation key: cord-290674-1kdc6xk8 authors: hershenson, marc b. title: rhinovirus-induced exacerbations of asthma and copd date: 2013-02-21 journal: scientifica (cairo) doi: 10.1155/2013/405876 sha: doc_id: 290674 cord_uid: 1kdc6xk8 over the past two decades, increasing evidence has shown that, in patients with chronic airways disease, viral infection is the most common cause of exacerbation. this review will examine the evidence for viral-induced exacerbations of asthma and chronic obstructive lung disease and the potential mechanisms by which viruses cause exacerbations. attention will be focused on rhinovirus, the most common cause of respiratory exacerbations. exacerbations due to rhinovirus, which infects relatively few cells in the airway and does not cause the cytotoxicity of other viruses such as influenza or respiratory syncytial virus, are particularly poorly understood. while the innate immune response likely plays a role in rhinovirus-induced exacerbations, its precise role, either adaptive or maladaptive, is debated. because current treatment strategies are only partially effective, further research examining the cellular and molecular mechanisms underlying viral-induced exacerbations of chronic airways diseases is warranted. readers of this paper who care for patients with asthma will be familiar with the following scenario. a patient with recurrent cough and wheeze presents to the clinic for evaluation. the patient states that the only time she has respiratory symptoms is fall, winter, and spring following "colds. " she has no symptoms in the summer, or between colds. she denies she has asthma and does not feel the need to take inhaled steroids even though she has been prescribed them in the past. my response to this patient, and the premise of this review, is that many patients have asthma which is only triggered by viral upper respiratory tract infections. nevertheless, the role of viral infections in the exacerbation of asthma, chronic obstructive disease (copd), and other chronic airways diseases was almost completely ignored until the late 1990s, when more sensitive methods of viral detection were developed. while the emphasis of allergic sensitization in the pathogenesis in asthma is strongly justified, serious exacerbations of this disease are more likely to relate to viral infection rather than allergen exposure (table 1) . exacerbations constitute the major cause of morbidity and mortality in asthma and copd, and therefore vigorous attention towards the problem of exacerbations is warranted. what is the evidence for viral-induced exacerbations of asthma and copd, and why was the role of viral infection ignored for so long? it was once thought that rhinovirus and other cold viruses did not infect the lower airway. before the advent of polymerase chain reaction (pcr), viruses were rarely cultured from the bronchoalveolar secretions of immunocompetent individuals, even after experimental infection [1] . early studies showed that replication of human rhinovirus (rv), the most prevalent respiratory virus, was temperature restricted, with optimal growth below body temperature, at 33 ∘ -35 ∘ c [2] [3] [4] . exacerbations of chronic airways disease following upper respiratory tract infections were explained by theories linking nasal (upper respiratory tract) and bronchial (lower respiratory tract) disease. the most common explanation was that nasal blockage and mouth breathing lead to inspiration of cold, dry, and unfiltered air, triggering an asthma attack. however, other workers in the field blamed a nasal-bronchial reflex mechanism involving trigeminal and vagal nerves (first proposed in 1919). [5] [6] [7] [8] . (this theory was also used to explain how nasal allergies promote asthma.) more recently, release of proinflammatory mediators from the nose and bone marrow into the circulation has been proposed [9] . (1) viral infection (most commonly rhinovirus) (2) allergen exposure (tree, grass, and weed pollens; mold; animal dander; dust mites; and cockroach particles) (3) exercise (4) irritants including environmental tobacco smoke exposure, smoke from wood-burning appliances or fireplaces, strong odors from perfumes, cleaning agents, air pollution, and occupational dust or vapors (5) medications (aspirin and other anti-inflammatory drugs, and beta blockers) (6) changes in weather (cold air, changes in temperature, and humidity) (7) gastroesophageal reflux (8) sinusitis in contrast to the study showing temperature restriction, subsequent studies showed that many rv strains replicate at body temperature. [4, 10] . for that matter, the temperature of the lower airways may decrease to 33-35 ∘ c during periods of increased minute ventilation (i.e., exercise) or cold temperature [11] . however, it was the advent of pcr for the detection of respiratory viruses which really changed the understanding of exacerbations. pcr-based studies examining the prevalence of virus identification among various cohorts of patients with chronic airways disease consistently show a higher prevalence of viral infection during exacerbations. outpatient children who are sick with asthma exacerbations show anywhere from 62-81% positivity for viral infection versus only 12-41% of children who are well [12, 13] . picornaviruses (primarily rv) were detected in 65% of cases, coronaviruses in 17%, influenza and parainfluenza viruses in 9%, and rsv in 5% [12] . similar studies have been performed in hospitalized children, adult outpatients, and hospitalized adults [14] [15] [16] [17] [18] [19] [20] [21] . adults seem to show a slightly lower prevalence of viral infection during exacerbations. finally, 22 to 64% of patients with copd exacerbations have virus identified by pcr versus 12 to 19% of nonexacerbating subjects [22] [23] [24] [25] [26] . across all of these studies, rv makes up approximately 50% of the viruses isolated ( table 2 ). the prevalence of rhinovirus may be even higher depending on the time of year. a recent study detected on rv in 82% of all children admitted to an emergency room for acute asthma between january and july [27] . together, these studies suggest that viral infections cause exacerbation of asthma and copd. additional information that viruses indeed cause attacks of chronic airways disease comes from an analysis of emergency department presentations for asthma and copd over the course of a calendar year. exacerbations of asthma in children peak after school return from summer vacation (in north america, the first week of september), consistent with an infectious cause [28] . this "epidemic" of asthma exacerbations in children is primarily associated with fall rhinovirus it is now clear that rhinovirus can indeed infect the lower airways. following experimental infection, rv has been detected in the lower airways by immunostaining, pcr, and in situ hybridization for positive-strand viral rna [29] [30] [31] [32] . a study from the university of wisconsin [30] was highly instructive. these investigators infected adult control and asthmatic subjects with rv16 and then stained biopsy tissue for rv16 capsid protein by immunohistochemistry. rv staining was clearly seen in the cytoplasm of cells in the epithelium ( figure 1 ). however, staining was patchy and in some samples only 1 or 2 cells were positive. other investigators have similarly noted that rv infects relatively few cells in the airway [33, 34] . researchers from imperial college, london [31] performed in situ hybridization of biopsies from experimentally infected patients and found positive-strand viral rna in the epithelium. interestingly, there was also sparse staining of cells in the subepithelial layer (there was insufficient detail to determine cell type). until recently there was little evidence that rhinovirus replication occurred in the lower airways. however, the wisconsin group [35] , examining sputum of experimentally infected controls and adults, showed persistence of viral rna up to 14 days after infection; this duration of infection could only occur with viral replication. (nevertheless, the amount of viral replication in the airways remains uncertain.) also, patients with asthma who had an exacerbation following experimental infection had higher levels of viral rna in their sputum compared to asthmatics that did not experience an exacerbation, further evidence that viruses do indeed cause exacerbations. if indeed viral infections do cause exacerbations of asthma and copd (and the preponderance of evidence suggests that this is so), what is the proposed mechanism by which viruses cause exacerbations? rhinovirus, unlike influenza scientifica 3 and other viruses, causes minimal cytotoxicity [36, 37] . also, the amount of epithelial damage does not correlate with the severity of the symptoms, suggesting that symptoms are produced by processes independent of the severity of direct virus-induced damage to the epithelium. while selected studies have demonstrated cytotoxicity in various cell culture models [38] , epithelial cell death is considered unlikely to contribute in vivo to rv-induced exacerbations of chronic airways disease. the current explanation is that rhinovirus infection induces the release of chemokines from airway epithelial cells, thereby attracting inflammatory cells to the airways. in patients with preexisting airway inflammation, the influx of additional inflammatory cells caused by rv infection would lead to additive or synergistic effects and an exacerbation of airways disease. there is ample evidence that rhinovirus infection induces airway epithelial cells to express proinflammatory chemokines. many studies of cultured airway epithelial cells show that rv increases expression of neutrophil-, eosinophil-, and t-cell-attracting chemokines, including cxcl8, ccl5, ccl11, and cxcl10 [10, [39] [40] [41] [42] [43] [44] [45] [46] [47] . a recent study by proud and colleagues [48] examined the effect of experimental rhinovirus infection on nasal epithelial cell gene expression in situ. the most highly upregulated genes were those encoding chemokines (ccl2, ccl8, cxcl11, cxcl10, cxcl13, cxcl9, and ccl20). we have obtained similar results in the lungs of rv-infected mice (table 3) . consistent with these results, experimental infections of asthmatic subjects have consistently shown increased number of airway neutrophils, lymphocytes and eosinophils [37, [49] [50] [51] . a number of clinical studies suggest that rv potentiates preexisting allergic inflammation [16, 37, [52] [53] [54] [55] . however, the notion that viral infection simply augments asthmatic airway inflammation may not explain why asthmatics suffer manifestations of lower airways disease after colds while normal individuals do not. could asthmatics tissue was stained for rv16 capsid protein (red) and dapi, a nuclear stain. note that some cells are stained with rv and others are not. (c) tissue was stained with mouse igg, a negative control, and dapi. note the absence of red staining. antibody against rv16 and tissue blocks were generously provided by wai-ming lee, nizar jarjour, and jim gern (university of wisconsin figure 2 : a mechanism to explain defective innate immunity in asthma. the differentiation of th1 and th2 t-helper cell lineages is mutually antagonistic. th2 cells produce il-4 which blocks th1 differentiation and th1 cells produce ifn-which blocks th2 differentiation. thus, individuals with an immune system skewed towards th2 tend not to produce eosinophils and ige-producing b cells, but not th1 cells. the relative lack of ifn-limits the antiviral response. airway epithelial damage [56, 57] and mucus metaplasia, both of which occur in asthma, may provide rhinovirus with increased access to more readily infected basal cells [58] and goblet cells [59] , thereby increasing viral replication. interferon responses, which have been shown to affect the outcome respiratory viral infections in animal models, may also be deficient in patients with asthma. how might this be the case? it is well known that many allergic asthmatics show a skewing of their t cell differentiation towards the th2 phenotype. since the differentiation of th1 and th2 t-helper cell lineages is mutually antagonistic (e.g., il-4 blocks th1 differentiation) [60] , allergic asthmatics with th2 polarization may have a deficiency in their interferon response ( figure 2 ). during induced colds, asthmatic subjects with strong peripheral blood monocytes and sputum cell interferon gamma responses showed milder cold symptoms and more rapid viral clearance [61, 62] . also following experimental rhinovirus infection, viral clearance and airway function correlate with blood and bronchoalveolar (bal) cd4 cell interferon gamma production [63] . epithelial and bal cells from asthmatic subjects show reduced type i and type iii interferon responses to rhinovirus infection ex vivo [64, 65] . together, these data suggest that asthmatics may be more susceptible for rv infection. on the other hand, other investigators have not found differences in interferon expression or viral clearance between controls and asthmatics. two groups have failed to show reduced interferon responses in cultured airway epithelial cells from subjects with asthma [66, 67] . perhaps most importantly, a difference in viral copy number or titer between controls and asthmatics has not been demonstrated following experimental rv infection [35] . to address the question of whether preexisting allergic airways disease alters the response to viral infection, and whether the interferon response to rhinovirus is required for viral clearance (or part of a maladaptive response to a relatively innocuous infection), we developed a mouse model of human rv infection [68] [69] [70] [71] [72] [73] . before describing this model, however, i like to say a little bit about the basic biology of rhinovirus. rv is an rna virus from the picornaviridae family. rhinovirus is composed of a single strand of positivesense rna enclosed in a small icosahedral capsid. there are greater than 100 known serotypes of rhinovirus. the major subgroup (88 serotypes) utilizes icam-1 as a receptor [74] ; the minor group (11 serotypes) uses the family of low density lipoprotein receptors (ldl-r, vldl-r, and ldl-r related protein) [75] . other surface molecules, including tlr2, may serve as a coreceptor and also be required for viral responses [76] . rhinoviruses may also be classified not only on the basis of their host receptor but according to their resistance to antiviral drugs. according to this classification, there are 74 type a viruses and 25 type b viruses [77] . a third group of rhinoviruses, type c, has recently been discovered [78] [79] [80] . rhinoviruses have also been classified according to their genome sequences [81] . interestingly, rv16, a major group virus commonly used for experimental human infection, and rv1, which has been used in animal models of rhinovirus infection (see below), are closely related. at this time, the only cell type conclusively shown to be infected by rv in humans is the airway epithelial cell. typically, rv infects small clusters of cells in the epithelial layer [30] . rhinovirus is internalized by clathrin-mediated endocytosis [82] [83] [84] [85] . the endosome acidic ph triggers viral uncoating and rna insertion. rv replication occurs entirely in the cytoplasm. during viral replication, negative sense rna and doublestranded rna intermediates are formed. dsrna produced during viral infection represents an important stimulus for the host innate immune response. dsrna is recognized and engaged by three pattern recognition receptors, toll-like receptor (tlr)-3, localized to the endosomal and plasma membranes, and cytoplasmic proteins rig-i, and mda-5 which are intracellular receptors for viral dsrna [86, 87] . returning to the problem of animal models, major group viruses do not bind to murine cells, owing to a lack of homology between the human and mouse icam-1. however, it was subsequently shown that minor group viruses infect la-4 mouse tracheal epithelial cells [88] . we [68] [69] [70] [71] [72] 89] and others [90] therefore infected mice with a minor group virus, rv1b, by the intranasal route. because of as-yet undefined factors which limit viral replication in the mouse, infection is followed by a steady reduction in viral copy number and titer. however, careful studies of viral copy number show a spike in positive-strand vrna approximately 24 hours after infection and a small amount of negative-strand vrna indicating viral replication. we also found a robust interferon response to infection, implying viral replication. immunofluorescence showed infection of airway epithelial cells and, interestingly, occasional subepithelial cells resembling monocytes. airways of infected mice showed neutrophilic inflammation which decreased with time, as well as subsequent lymphocytic infiltration, a pattern classically associated with viral infection. when we examined respiratory system resistance changes in response to methacholine administration, we found a small but significant increase in airways responsiveness in rhinovirus-infected mice which persisted at least four days after infection. interestingly, mice infected with uvirradiated replication-deficient virus showed modest airway inflammation and responsiveness one, but not four, days after infection. these data suggest that rhinovirus could induce a state of airways hyperresponsiveness without viral replication in the lungs, at least under some circumstances. consistent with this, uv irradiation inhibits but does not abolish major group rv-induced il-8 release in cultured airway epithelial cells [43, 91, 92] . we next set out to develop a model of viral-induced asthma exacerbation [72] . we employed a commonly used model of allergic airways disease. mice were treated with intraperitoneal and intranasal ovalbumin and then infected with rhinovirus. mice treated with ovalbumin alone demonstrated a significant increase in airways responsiveness, with respiratory system resistances higher than those obtained after rhinovirus infection. when we combined ovalbumin and rhinovirus treatments, airways hyperresponsiveness increased significantly over that generated by either ovalbumin or rhinovirus alone (figure 3 ). this state of airways hyperresponsiveness persisted at least 4 days after infection. when we examined airway inflammation, we found additive or synergistic increases in airway neutrophils, eosinophils, macrophages, lymphocytes, and chemokines such as ccl11 and ccl2. we also found that the combination of ovalbumin and rhinovirus caused additive increases in the th2 cytokines il-4 and il-13. when we performed immunohistochemistry to look for the source of ccl11 production, we expected to find ccl11 expression in rhinovirus-infected airway epithelial cells. however, to our surprise, we found that the main source of ccl11 was airway mononuclear cells. when we performed fluorescence microscopy using labeled antibodies against rhinovirus, ccl11 and cd68, a macrophage surface marker, we confirmed ample colocalization of rv1b, ccl11, and cd68, indicating ccl11 expression by cd68-positive macrophages. while most cells were in the subepithelium, we also found cd68-positive macrophages in the epithelial layer and the airway lumen. in follow-up studies, we also found colocalization of cd68, rv1b, and il-4 in subepithelial cells. several studies have examined the infection of monocytic cells by rhinovirus in vitro [93] [94] [95] [96] [97] [98] . however, the former data represent the first demonstration of rhinovirus infection of monocytes in vivo. at this point, we do not know whether colocalization represents replicative infection of airway macrophages, or simple phagocytosis of the virus. a small amount of viral replication has been noted in rvinfected peripheral blood monocyte-derived macrophages, but not in bal-derived macrophages [93, 94] . to address this further, we isolated bronchoalveolar lavage macrophages from control and ovalbumin-sensitized and -challenged mice and infected them with rhinovirus ex vivo. macrophages from control mice showed ample th1 cytokine (tnf-, il-12p70) expression in response to rv infection, but little or no th2 cytokine (il-4, il-13, ccl11) production. in contrast, bal cells from ovalbumin-sensitized and -challenged mice showed brisk th2 responses to ex vivo infection, but decreased th1 responses. this pattern of cytokine expression was consistent with a change in phenotype from m1 to alternatively activated m2 macrophages [99] . we confirmed this by measurement of m2 markers such as arginase-1, ym-1, and mgl-2. furthermore, flow cytometry showed high levels of cd11b expression, indicative of exudative macrophages found after lung infection or injury. to determine the requirement of these macrophages for the observed airways hyperresponsiveness of rhinovirusinfected and ovalbumin-sensitized and -challenged mice, we depleted the macrophages using clodronate liposomes. administration of clodronate decreased the number of airway macrophages, but not neutrophils or lymphocytes. however the lungs of mice receiving clodronate showed reduced levels of ccl11, eosinophils, il-13, and airways responsiveness, demonstrating that macrophages are responsible for rv-induced airway responses in mice with preexisting allergic airways disease. the production of th2 cytokines by lung macrophages is consistent with recent work showing that, in addition to th2 helper t cells, innate immune cells may contribute to the production of th2 cytokines in asthma [100] [101] [102] [103] [104] [105] [106] [107] . finally, our unpublished experiments indicate that, under certain circumstances, m1-polarized macrophages may also be targeted by rv infection. taken together, our data suggest that, rather than causing epithelial infection or damage, rv elicits asthma exacerbations by infecting inflammatory cells, in particular cells of the monocyte/macrophage lineage. but is there any evidence that rhinovirus infects inflammatory cells in humans? with the help of jim gern, nizar jarjour, and others from the university of wisconsin, we have begun to address this question. we obtained biopsy specimens from control and asthmatic subjects experimentally infected with rhinovirus. early unpublished results indicate colocalization of rv and cd68 in epithelial and subepithelial cells. returning to the question of antiviral innate immunity in asthma, we also employed our animal model to address this issue. does the inflammatory response to rhinovirus promote viral clearance and prevent more severe viral-induced disease? or is the inflammatory response to rhinovirus a maladaptive response to an innocuous stimulus, leading to exacerbations of lower airways disease? these questions have important ramifications for treatment. one often hears the statement that individuals "need to boost their immune response" to fight off colds or cold-induced asthma attacks. the imperial college group has championed the idea that the interferon response is deficient in asthmatics [64, 65] , thereby explaining viral-induced exacerbations. in this case, boosting of the immune response with interferons might be helpful. to test the hypothesis that the immune response to rhinovirus may be counterproductive, we infected mice with defects in tlr and mda-5 [89] , molecules which recognize double-stranded rna on the cell surface or in the cell cytoplasm, respectively. as noted above, these signaling pathways are responsible for double-stranded rnastimulated interferon production. our previous work showed that mda-5, an rna helicase, is required for rhinovirusinduced interferon production in cultured airway epithelial cells [73] . tlr3 knockout mice had normal interferon responses to rhinovirus infection. however, mda5 knockout mice showed a delay in type i interferon responses and a significant and persistent reduction in type iii interferon lambda production. however, despite these deficiencies, mda5 knockout mice showed only small increases in rv vrna and titer. further, rv-infected knockout mice showed less, rather than more, airway inflammation. similar results were found in rhinovirus-infected ovalbumin-sensitized and -challenged knockout mice. these data call into question the interferon requirement for rv clearance and suggest that the proinflammatory response to rhinovirus infection is indeed maladaptive. indeed, a recent study found that interferonlambda concentrations were higher in rhinovirus-infected infants with wheezing compared to those without wheezing [108] . what about viral-induced copd exacerbations? since copd differs from asthma in several key respects, the pathogenesis of virus induced exacerbations may be different in this disease. copd differs from asthma in at least the following ways. the key trigger for copd is cigarette smoke rather than aeroallergens, air pollutants, or infection. copd encompasses two distinct processes, chronic bronchitis, and emphysema, which result in structural changes that limit airflow. the copd airway epithelium undergoes squamous metaplasia. in copd, macrophages and cd8-positive cell play key roles rather than th2 cells, eosinophils, or mast cells. copd pathogenesis appears to be dominated by tgf-, tnf-, il-1 , and il-6. the lower airways of 25 to 50% of patients with copd are colonized by bacteria, particularly haemophilus influenzae, streptococcus pneumonia,and moraxella catarrhalis. [109, 110] . finally, oxidative stress resulting either from inhaled oxidants or inflammatory cells plays a significant role. as mentioned above, about one half of copd exacerbations are associated with viral infections, the majority of which are due to rv [22] [23] [24] [25] [26] . copd exacerbations may also be associated with the isolation of the new bacterial strain [110] . thus, interactions between virus, bacteria, and host may play a role in the pathogenesis of copd exacerbations. recent studies by the imperial college london, in which patients with copd were experimentally infected with rhinovirus [111] , have provided new information about rvinduced exacerbations in these patients. compared to controls, copd patients show increased sputum neutrophils in response to experimental rv infection. other investigators have shown increased neutrophils, eosinophils, macrophages, and lymphocytes in the airways of copd patients experiencing exacerbations [112] [113] [114] [115] [116] [117] [118] . unlike asthma patients, copd patients experimentally infected with rv show increased sputum viral copy number versus controls, and viral load correlates with sputum neutrophils and interleukin-8 levels [111] . bal cells from patients with copd show reduced interferon-alpha and -beta responses to rhinovirus infection ex vivo. together, these results provide ample evidence of altered innate immunity against viruses. these data reinforce in vitro data showing that exposure of lung cells to cigarette smoke extract reduces cxcl10, interferon-beta, and rig-i expression in response to influenza virus infection [119] . viral infection may also attenuate the antibacterial innate immune response in copd. for example, rhinovirus exposure impairs immune responses to bacterial products in human alveolar macrophages. we have shown that rv infection of mucociliary-differentiated airway epithelial cells cultured at air-liquid interface causes barrier dysfunction which allows translocation of haemophilus influenzae across the epithelium [69] . we have also shown that elastase-and lps-treated mice with a lung phenotype similar to copd show delayed clearance of rhinovirus in vivo, similar to humans with copd [71] . cystic fibrosis is a chronic airways disease with similarities to asthma and copd. cf may also be exacerbated by viral infection. for example, viruses were detected in 46% of patients with exacerbations of cystic fibrosis, compared to only 18% of patients in stable condition [120] . rv has been identified in 13 to 58% of cystic fibrosis patients with acute respiratory illness and was associated with increased respiratory symptoms, worse airway function and secondary bacterial infection, compared to uninfected patients [120] [121] [122] [123] . infancy: exacerbations of preexisting airways disease or a factor in asthma development? over the past decade, data from a birth cohort of highrisk infants with a positive family history of asthma (nicknamed the childhood origins of asthma, or coast) have shown that wheezing-associated illness with rv is the most important risk factor for asthma development [124, 125] . the relative risk of asthma development in infants with wheezing associated with rhinovirus infection was far higher than that of infants with allergen sensitization or rsv infection alone. also, in infants hospitalized for respiratory infectionassociated wheezing, infection with rv was associated with asthma development [126] in contrast to respiratory syncytial virus (rsv), which was negatively associated [127] . together, these data suggest two possibilities, either that rv infection causes asthma, or that rv infections may simply reveal a preexisting tendency for asthma. more recent data suggest that the latter is likely; prospective characterization of the coast birth cohort demonstrated that allergic sensitization precedes hrv wheezing and that the converse is not true [128] . also, it was recently shown that children developing asthma by age seven had a lung function deficit and increased bronchial responsiveness as neonates [129] , suggesting that asthma precedes rv infection. if this is the case, then wheezingassociated illnesses due to rhinovirus are essentially viralinduced asthma exacerbations. is it possible that, under the right circumstances, for example, the appropriate genetic background and allergen exposure, rhinovirus infection in early life modulates the immune response, increasing the likelihood of asthma development? a positive family is a known risk factor for asthma development, and it has recently been found that infants of mothers with asthma are more likely to have severe respiratory tract infections with rv [130] . to address this possibility, we infected baby mice with rhinovirus at 2 to 3 days of life. unlike mature mice, rhinovirus-infected neonatal mice showed mucus metaplasia and airways hyperresponsiveness which lasted for one month after infection [131] . this is reminiscent of data from washington university st. louis in mature mice following infection with sendai virus [132] . however, the finding of a developmental difference in response to rhinovirus, an infection that does not cause cytotoxic effects, warrants further investigation. studies using neutralizing antibody and an il-4 receptor knockout mouse showed that the effect of rv was dependent on il-13. we also found increased production of il-13 by invariant natural killer t (inkt) cells. the notion that early rhinovirus infection could modulate future immune responses, leading to allergic airway inflammation, has been bolstered by the discovery of novel epithelial-derived cytokines which promote th2 differentiation. these cytokines, thymic stromal lymphopoietin (tslp), il-25, and il-33, play a key role in the maturation of th2 cells via dendritic cell (dc) activation [133] [134] [135] [136] [137] [138] , as well as the activation of mast cells and inkt cells [139] . moreover, tslp, il-25, and il-33 induce il-13 production by two novel innate effector leukocytes that mediate th2 immunity-type 2 innate lymphoid cells (also called natural helper cells or nuocytes) [100] [101] [102] [103] [104] [105] [106] and type 2 myeloid cells [107] . respiratory viral infections cause exacerbations of asthma and copd. this is evidenced by the increased identification of viruses in the respiratory tract during exacerbations compared to stable periods. also, epidemic cycles of asthma and copd exacerbations occur after school return and around the christmas holiday, consistent with an infectious cause. it has also been well demonstrated that viruses infect and replicate in the lower airways. finally, the severity of viralinduced exacerbations correlates with viral load. viral infection of the epithelium increases expression of chemokines, leading to influx of inflammatory cells and increased airway inflammation. innate immunity to viral infection may be decreased in asthma, owing 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modulation of gene expression in bronchial epithelial cells from subjects with asthma human rhinovirus 1b exposure induces phosphatidylinositol 3-kinasedependent airway inflammation in mice rhinovirus disrupts the barrier function of polarized airway epithelial cells cxcr2 is required for neutrophilic airway inflammation and hyperresponsiveness in a mouse model of human rhinovirus infection elastase-and lpsexposed mice display altered responses to rhinovirus infection rhinovirus infection of allergen-sensitized and -challenged mice induces eotaxin release from functionally polarized macrophages role of doublestranded rna pattern recognition receptors in rhinovirusinduced airway epithelial cell responses the major human rhinovirus receptor is icam-1 members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus rhinovirus attenuates nontypeable hemophilus influenzae-stimulated il-8 responses via tlr2-dependent degradation of irak-1 two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children multiplex masstag-pcr for respiratory pathogens in pediatric nasopharyngeal washes negative by conventional diagnostic testing shows a high prevalence of viruses belonging to a newly recognized rhinovirus clade distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution internalization of human rhinovirus 14 into hela and icam-1-transfected bhk cells the clathrin endocytic pathway in viral infection elevated endosomal ph in hela cells overexpressing mutant dynamin can affect infection by ph-sensitive viruses human rhinovirus type 2 is internalized by clathrin-mediated endocytosis the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda-5, and inhibit its activation of the ifn-promoter mouse respiratory epithelial cells support efficient replication of human rhinovirus mda5 and tlr3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation bafilomycin a 1 inhibits rhinovirus infection in human airway epithelium: effects on endosome and icam-1 phosphatidylinositol 3-kinase is required for rhinovirus-induced airway epithelial cell interleukin-8 expression rhinovirus enters but does not replicate inside monocytes and airway macrophages rhinovirus replication in human macrophages induces nf-b-dependent tumor necrosis factor alpha production the role of p38 mapk in rhinovirus-induced monocyte chemoattractant protein-1 production by monocyticlineage cells human rhinovirus induces robust ip-10 release by monocytic cells, which is independent of viral replication but linked to type i interferon receptor ligation and stat1 activation rhinoviruses induce interleukin-8 mrna and protein production in human monocytes respiratory virus induction of alpha-, beta-and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells alternative activation of macrophages: an immunologic functional perspective innate production of t 2 cytokines by adipose tissue-associated c-kit + sca-1 + lymphoid cells nuocytes represent a new innate effector leukocyte that mediates type-2 immunity innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity human il-25-and il-33-responsive type 2 innate lymphoid cells are defined by expression of crth2 and cd161 innate lymphoid cells responding to il-33 mediate airway hyperreactivity independently of adaptive immunity innate il-13-producing nuocytes arise during allergic lung inflammation and contribute to airways hyperreactivity lung natural helper cells are a critical source of th2 cell-type cytokines in protease allergen-induced airway inflammation interleukin-25 induces type 2 cytokine production in a steroid-resistant interleukin-17rb + myeloid population that exacerbates asthmatic pathology a mechanistic role for type iii ifn-1 in asthma exacerbations mediated by human rhinoviruses nontypeable haemophilus influenzae in the lower respiratory tract of patients with chronic bronchitis new strains of bacteria and exacerbations of chronic obstructive pulmonary disease experimental rhinovirus infection as a human model of chronic obstructive pulmonary disease exacerbation relation of sputum inflammatory markers to symptoms and lung function changes in copd exacerbations biopsy neutrophilia, neutrophil chemokine and receptor gene expression in severe exacerbations of chronic obstructive pulmonary disease changes in bronchial inflammation during acute exacerbations of chronic bronchitis bronchial inflammation in acute bacterial exacerbations of chronic bronchitis: the role of leukotriene b4 granulocyte inflammatory markers and airway infection during acute exacerbation of chronic obstructive pulmonary disease cellular and structural bases of chronic obstructive pulmonary disease mmp-9, timp-1 and inflammatory cells in sputum from copd patients during exacerbation cigarette smoke extract suppresses the rig-iinitiated innate immune response to influenza virus in the human lung the role of respiratory viruses in cystic fibrosis effect of respiratory virus infections including rhinovirus on clinical status in cystic fibrosis effects of upper respiratory tract infections in patients with cystic fibrosis prevalence and impact of respiratory viral infections in young children with cystic fibrosis: prospective cohort study rhinovirus illnesses during infancy predict subsequent childhood wheezing wheezing rhinovirus illnesses in early life predict asthma development in high-risk children rhinovirus-induced wheezing in infancy-the first sign of childhood asthma? predictors of asthma three years after hospital admission for wheezing in infancy evidence for a causal relationship between allergic sensitization and rhinovirus wheezing in early life influence of maternal asthma on the cause and severity of infant acute respiratory tract infections interaction between asthma and lung function growth in early life neonatal rhinovirus infection induces persistent mucous metaplasia and airways hyperresponsiveness persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease nk cell deficiency predisposes to viral-induced th2-type allergic inflammation via epithelial-derived il-25 overexpression of smad2 drives house dust mite-mediated airway remodeling and airway hyperresponsiveness via activin and il-25 beyond inflammation: airway epithelial cells are at the interface of innate and adaptive immunity il-33-activated dendritic cells induce an atypical t 2-type response a role for tslp in the development of inflammation in an asthma model tlr3-and th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells differential role of thymic stromal lymphopoietin in the induction of airway hyperreactivity and th2 immune response in antigen-induced asthma with respect to natural killer t cell function this work is supported by nih grant hl081420. key: cord-277818-8w15dz20 authors: jaichenco, andre l.; lima, luciana cavalcanti title: infectious disease considerations for the operating room date: 2018-02-09 journal: a practice of anesthesia for infants and children doi: 10.1016/b978-0-323-42974-0.00050-1 sha: doc_id: 277818 cord_uid: 8w15dz20 the risk of infection transmission by anesthesia providers in their work area environment is reviewed. the dynamics of transmission and the strategies for preventing infection transmission in health care institutions are discussed. anesthesiologists have long been patient safety advocates and have taken on increasing responsibility for preventing health care–associated infections. anesthesia providers practice in a nonsterile environment within the operating room and have an impact on bacterial transmission and infection rates. understanding the characteristics of transmission elements provides the practicing anesthesiologist with methods to protect susceptible patients and themselves to avoid spreading infection. it is vital to have in place proper systems to remove contaminated air to minimize the risk of airborne pathogens being transmitted by children. preoperative patient skin and other bacterial reservoir decontamination and hand hygiene by anesthesia providers reduces contamination of the work area and iv access ports. hand hygiene is a well-known and effective solution to the problem of bacterial transmission within and across patients and is considered the most important and cost-effective individual intervention in the prevention of health care–associated infections in children and health care providers compliance with the current “5 moments” world health organization guidelines could make a major inroad into reducing provider hand and workspace contamination. surgical antimicrobial prophylaxis is an essential tool to reduce the risk of postoperative infections, and the anesthesia team plays a central role in ensuring the proper timing of drug administration. protocols, although effective, require continuous feedback and revision. the presence of a susceptible host is an important element in the chain of infection that paradoxically results from advances in current medical therapies and technology (e.g., children undergoing organ transplantation or chemotherapy, or extremely premature neonates) and the presence of children with diseases that compromise their immune systems (e.g., aids, tuberculosis, malnutrition, or burns). the organism may enter the host through the skin, mucous membranes, lungs, gastrointestinal tract, genitourinary tract, or the bloodstream via iv solutions, after laryngoscopy, or from surgical wounds. organisms may also infect the individual because of work accidents with cutting or piercing devices. the development of the rabbit hole that is the perioperative environment is not well understood by the majority of our general pediatric colleagues. similarly, the rabbit hole of the primary care clinic or the pediatric inpatient ward is not well understood by the majority of our anesthesiology colleagues. a pediatric patient may repeatedly enter the rabbit hole over the course of a hospital admission, a journey fraught with dangers of airway mishaps, respiratory and/or cardiac arrests, hemorrhage, profound anxiety and stress experienced by the young patient and his or her family, as well as infection risks. 1 anesthesiologists have long been patient safety advocates. it is not surprising that anesthesia providers in the 21st century have taken on increasing responsibility for preventing health careassociated infections (hais), including surgical site infections (ssis). anesthesia providers practice in a nonsterile environment within the operating room (or) and frequently contact areas of the patient known to have a high rate of contamination such as the axilla, nares, and pharynx. there are two recognized but poorly implemented interventions: preoperative patient skin and other bacterial reservoir decontamination and hand hygiene by anesthesia providers. 2 anesthesia providers have an impact on bacterial transmission and infection rates. specifically, anesthesiologists are known to contaminate their work environment within the or. contamination of the work environment includes contamination of intravenous (iv) access ports. without encouragement, anesthesiologists perform hand hygiene less frequently than once per hour during a case, but with reminders, the rate of hand hygiene is more frequent. improved hand hygiene reduces contamination of the work area and iv access ports from 32% to 8%, which in turn significantly reduces hais. 3, 4 the transmission of infection depends on the presence of three interconnected elements: a causative agent, a source, and a mode of transmission ( fig. 50.1 ). understanding the characteristics of each element provides the practicing anesthesiologist with methods to protect susceptible patients and themselves to avoid spreading infection. there has always been concern about the transmission of infectious agents to the patient from the anesthesiologist and vice versa. 5 in addition, there are many sites within the hospital environment where moist or desiccated organic material with the membranes. droplets remain suspended for only a short duration and distance from the source, but this may be affected by temperature, humidity, force of expulsion, and air currents. larger particle sizes contact the mucosa of the upper airway, whereas aerosols are capable of penetrating into the lower respiratory tract. infectious agents vary in their affinity for receptors in different regions of the respiratory tract. 9,10 when a person coughs, the exhaled air may reach a speed of up to 965 km/hour (600 mph). 11 however, because the droplets are relatively large, they tend to descend quickly and remain suspended in the air for a very brief period, thus obviating the need for special handling procedures for the or air. examples of droplet-borne diseases include influenza, respiratory syncytial virus (rsv), severe acute respiratory syndrome (sars), diphtheria, haemophilus influenzae, neisseria meningitidis, mumps, pertussis, rhinovirus, rubella, and ebola. droplet precautions include communication of infectious risk infection is influenced by the host defense mechanisms that may be classified as either nonspecific or specific: ■ nonspecific defense mechanisms include the skin, mucous membranes, secretions, excretions, enzymes, inflammatory responses, genetic factors, hormonal responses, nutritional status, behavior patterns, and the presence of other diseases. ■ specific defense mechanisms or immunity may occur because of exposure to an infectious agent (antibody formation) or through placental transfer of antibodies; artificial defenses may be acquired through vaccines, toxoids, or exogenously administered immunoglobulins. microorganisms are transmitted in the hospital environment through a number of different routes; the same microorganism may also be transmitted via more than one route. in the or, the three main routes of transmission are through the air and by direct and indirect contact. airborne infections that may infect susceptible hosts are transmitted via two mechanisms: droplets and droplet nuclei. droplet contamination is considered a direct transmission of organisms because there is a direct transfer of microorganisms from the colonized or infected person to the host. this generally occurs with particles whose diameters are greater than 5 µm that are expelled from an individual's mouth or nose, mainly during sneezing, coughing, talking, or during procedures such as suction, laryngoscopy, and bronchoscopy ( fig. 50.2 ). transmission occurs when the microorganism-containing droplets, expelled or shed by the infected person (source), are propelled a short distance (usually not exceeding 60 cm or about 2 feet through the air) and deposited on the host's conjunctivae or oral or nasal mucous droplet nuclei result from the evaporation of droplets while suspended in the air. unlike droplets, the nuclei have an outer layer of desiccated organic material and a very small diameter (1-5 µm) and remain suspended in air indefinitely. the microorganisms contained within these nuclei may be spread by air drafts over great distances, depending on the environmental conditions (dry and cold atmosphere, with limited or no exposure to sunlight favoring the spread). 12 in contrast to droplets, which are deposited on mucous membranes, droplet nuclei may enter the susceptible host by inhalation; examples of droplet nuclei-borne diseases include tuberculosis, varicella, and measles, zoster, smallpox, sars, and middle eastern respiratory syndrome. 10 direct and indirect contacts are the most significant and frequent methods of hospital infection transmission. this type of disease transmission involves direct physical contact between two individuals. the physical transfer of microorganisms from an infected or colonized person to a susceptible host may occur from child to health care provider or from health care provider to child during professional practice (e.g., venous cannulation, laryngoscopy, burn care, or suction of secretions). health care providers working in the or may be exposed to skin contamination by body fluids. this is an issue of grave concern because of the potential exposure of health care providers to patients with unrecognized infections, especially hepatitis b virus (hbv), hepatitis c virus (hcv), and human immunodeficiency virus (hiv). hepatitis b is a highly infectious virus that requires a small amount of blood (10 −7 -10 −9 ml) to transmit the disease. the incidence of skin contamination of anesthesiologists and related personnel by blood and saliva is substantial. one study examined 270 anesthetic procedures during 7 consecutive days. the blood of 35 patients (14%) contaminated the skin of 65 anesthesiologists in 46 incidents. of these contamination events, 28 (61%) occurred during venous cannulation. of anesthesiologists who had been contaminated by blood, 5 of 65 (8%) had cuts in the skin of their hands. 13 the importance of this observation is that seroconversion of health care providers has been reported after skin contamination by infected blood from hiv carriers 14 and hbv infection after blood splashing into health care workers' (hcws') eyes. 15 scabies, pediculosis, and herpes simplex are among the diseases most frequently transmitted by direct contact. [16] [17] [18] [19] [20] [21] [22] [23] meticulous hand washing before and after every patient contact and routine use of barriers such as gloves and eye protection are essential basic methods for protecting ourselves even during routine procedures such as starting an iv line or performing laryngoscopy. 4 indirect contact involves the transmission of microorganisms from a source (animate or inanimate) to a susceptible host by means of a vehicle (e.g., an intermediary object) contaminated by body fluids. tables 50.2 and 50. 3 provide examples of diseases associated with bodily fluids to which hcws may be exposed. the vehicle for transmission may be the hands of a health care provider who is not wearing gloves or a provider who fails to wash his or her hands after providing care to a child. 3, [24] [25] [26] this type of contact can also come from health care providers who touch (with or without gloves) contaminated monitoring or other patient care devices (e.g., blood pressure cuffs, stethoscopes, electrocardiographic cables, or ventilation systems [respirators, corrugated tubes, y-pieces, valves]) that are used without proper cleaning or disinfection between each use. [27] [28] [29] knowledge about the transmission of the spread of bacteria from patients to hcws' hands and to the hospital environment ( fig. 50 .3) has driven many interventions that have reduced patient risks for developing hais. 30 disease transmitted blood hbv, hiv, hcv, cmv, ebv, nanbh seminal fluid hiv, hbv, cmv vaginal discharge hiv, hbv, cmv saliva and sputum hsv, tb, cmv, respiratory diseases cerebrospinal fluid encephalopathic organisms (see table 50 30 characterization of the transmission dynamics of frequently encountered gram-negative bacteria in the anesthesia work area environment 32 demonstrates that the spread follows an epidemiologic pattern similar to that seen in icus and inpatient wards: from patient, to environment and hcws' hands, and to other patients ( fig. 50.4) . in this report, provider hands were less likely to serve as a transmitter of infection than contaminated environmental or patient skin surfaces. these findings have clinical implications for the risk of colonization and subsequent hcis-for example, ssis. this calls attention to the need to develop and enforce strict hand hygiene guidelines for personnel who are providing anesthesia care, but more importantly the need to increase compliance with environmental disinfection of the or (between cases and terminal cleaning), and to study further the directions of the spread of pathogens in the or and anesthesia work areas. this study unequivocally underscores our need to improve cleaning procedures in the or and equipment surfaces to reduce infection risk. there are also reports of equipment, fomites, and drugs (mainly propofol) that have resulted in hospital-acquired infections. 20, [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] propofol is widely used for both inpatient and outpatient anesthesia. this hypnotic agent is a nutrient-rich drug. it is hypothesized that propofol increases bacterial contamination of iv stopcocks and may compromise safety of iv tubing sets when continued to be used after propofol anesthesia. there is a covert incidence of iv stopcock bacterial contamination during anesthesia that is aggravated by the prior presence of propofol. propofol may increase the risk for postoperative infection because of bacterial growth in iv stopcock dead spaces. 52 other facets that may also contribute to infection include the following: ■ up to 40% of anesthetic equipment in direct or indirect contact with a child (blood pressure cuffs, cables, oximeters, laryngoscopes, monitors, respirator settings, and horizontal and vertical surfaces) may be contaminated with blood because of inadequate cleansing procedures between uses. 6,7,27,53,54 ■ in some institutions, up to 8% of the bain circuits that were reused without previous sterilization were contaminated. 55 ■ contamination of syringe contents has occurred with glass particles during ampule opening, which in turn may compromise the sterility of the contents, presumably because of the passage of bacteria contained on glass particles into the solution. 56-58 ■ iv tubing has both blood contamination as well as contamination by blood from syringes used to inject medications. this can occur with the absence of visible blood reflux in the tubing or syringe. simply replacing the needle on a syringe that will be reused is ineffective in preventing cross-infection; it is essential to not use the same syringe in multiple patients. 59 ■ refilling both glass and plastic syringes several times has also been shown to result in contamination of the contents; single use is therefore recommended. 59 studies on vancomycin-resistant enterococci established the importance of a domino effect of contamination in intensive care units (icus) and inpatient wards: spread of vancomycin-resistant enterococci that colonize patients' gastrointestinal tracts ("rectal carriage"), to patients' skin, to the hospital environment, to hands of hcws, and then to other patients. the skin contamination of patients with enteric organisms inspired the rather graphic description, the patient's "fecal patina." 31 also referred to as a "stool veneer," this coating with enteric organisms is limited not only to patients' skin but also extends to surfaces in the surrounding environment that are touched, and thereby contaminated, by patients and by hcws. the environmental contamination spreads out from the patient in a target-like concentric pattern, with the densest contamination closest to the rectum of patients who have rectal carriage of the problem bacteria. this interplay among the blood of a patient in an advanced disease stage or with a higher hiv viral load; a deep percutaneous injury; a procedure wherein the sharp was in the vein or artery of an infected source patient; an injury with a hollow-bore, blood-filled needle; and limited or delayed access to postexposure prophylaxis. after exposure, the risk of infection varies for specific bloodborne pathogens. for hbv, if the source patient has active hbv and the hcp do not already have immunity, the risk for infection after percutaneous injury is between 1% and 30%. if the source patient has active hcv, the risk of hepatitis c transmission is approximately 1.8% (range 0%-7%) after a percutaneous injury. if the source patient has hiv infection, the risk of hiv transmission is approximately 0.3% after a percutaneous exposure and 0.09% after a mucous membrane exposure. the risk of hiv transmission for an exposure with nonintact skin has not been determined and is estimated to be less than the risk after a mucous membrane exposure. 78 anesthesia staff lacking hbv protective antibodies are at great risk for acquiring the disease. 79, 80 these infection rates underscore the need for the use of "safe" needles and the need to advocate the use of "needleless" systems even though they are significantly more expensive. 81, 82 this also emphasizes the need for meticulous handling and disposal of needles and other sharp instruments, as well as the use of special "sharps boxes" designed to minimize accidental needlesticks (e.g., "mailbox"-type boxes that do not allow the hand to enter the disposal area). [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] the u.s. centers for disease control and prevention (cdc) has estimated that in the united states there are approximately 385,000 cutting and piercing accidents annually among hcp in hospitals; 25% of these occur in the or. 77 however, the actual prevalence is thought to be much greater, because many of these events are unreported. the distribution of these accidents among anesthesiologists is shown in fig. 50 .5a; the distribution of the items most frequently associated with cutting and piercing injuries in health care providers is shown in fig. 50 .5b. should such an accident occur (e.g., needle puncture, exposure to nonintact skin, or mucous membrane ■ needles that have been used for spinal or epidural anesthesia were contaminated with coagulase-negative staphylococci (15.7%), yeasts (1.5%), enterococci (0.8%), pneumococci (0.8%), and micrococci (0.8%), suggesting that there may be needle contamination despite standard skin preparation and cleansing. 68 it is unclear whether these skin organisms can be transmitted and cause an infection during administration of a neuraxial block. ■ blood and saliva frequently contaminate the skin of anesthetic personnel during routine anesthetic practice. 13 ■ violations of contemporary guidelines for preventing infections (e.g., hand washing, wearing gloves, surgical masks, ocular protection, scrubs, or syringe reuse) by anesthesiologists are frequent. 4 anesthesia staff are aware that they work in a potentially infectious environment, but they commonly do not adopt appropriate protective measures to reduce infections in both themselves and their patients (11%-99%). 27,69-71 percutaneous contamination from a cutting or piercing accident is the most effective means to transmit bloodborne pathogens. evidence suggests that this is the main route of hiv, hbv, and hcv infection, [72] [73] [74] especially if the injury is caused by hollow-bore needles that were used to draw blood or establish iv access. 75, 76 over 20 other bloodborne pathogens have been transmitted by this means, including those causing herpes, malaria, and tuberculosis. 77 the risk of exposure to blood and bloodborne pathogens is greater for health care personnel (hcp) than for people who do not work around blood. an exposure to infected blood, tissue, or other potentially infectious body fluids can occur by percutaneous injury or contact with mucous membrane or nonintact skin. the risk of infection after an exposure depends on a number of variables and appears to be greater with exposure to a larger quantity of blood or other infectious fluid; prolonged or extensive exposure of nonintact skin or mucous membrane to blood or other infectious fluid or concentrated virus in a laboratory setting; exposure to institutional administrative measures aimed at developing, implementing, and monitoring specifically designed accident prevention policies and procedures are important for reducing and preventing transmission of infectious agents in health care centers. to this end, centers should consider the following: 77,99,100 ■ include infection control as a major goal in the organizational mission statement and implement safety programs, both for patients and hcws. ■ provide sufficient administrative and financial support to carry out this mission. ■ provide sufficient administrative and financial support for the microbiology laboratory and implement an infection surveillance plan, especially for postsurgical infections. ■ establish a multidiscipline cross-functional team (e.g., a team manager, an epidemiologist, a representative from industrial health, and a person trained in quality control) to identify health and safety issues within the institution, analyze trends, assess outcomes, implement interventions, and make recommendations to other members of the organization. ■ provide sufficient administrative and financial support to develop and implement education programs for health care providers, patients, and their families. one positive example of such education is that anesthesiologists who have read the cdc's universal precaution guidelines for the prevention of occupational transmission of hiv and hbv have developed better hygienic practices. 69 ■ provide hcws with hepatitis a and b vaccine and document that an appropriate immunologic response was achieved. provide hepatitis a and b immune globulins (haig, hbig) for those exposed who do not have established immunity. 8 ■ provide a health care service for employees for counseling and postexposure prophylaxis should an exposure to hiv occur. 101 , there are now specific recommendations regarding immediate assessment of risk, assessment of the exposure source (chart review, inform the patient that an accident has occurred and ask permission to determine hbv, hcv, and hiv serologic status), and rapid initiation of appropriate antiviral treatment of the hcw. 82 it is advised to obtain as much information regarding the patient as possible-if the patient is known-to (1) obtain a sample of blood from the patient for determination of potential carrier state (table 50 .4) and (2) report to the health service for immediate institution of prophylaxis and follow-up ( air is delivered to each or from the ceiling, with downward movement toward several exhaust or return ducts near the floor. this design helps provide steady movement of clean air through the breathing and working zones. the aia has specific guidelines for the location of outside fresh air inlets to minimize contamination from exhaust systems and noxious fumes. a greater air inflow rate and a larger air-inlet area are desirable for contaminant control, but these approaches are detrimental to the thermal comfort of the staff and patient. 108 the aia recommends an air-change rate in an or of 20 to 25 air changes per hour (ach) for ceiling heights between 9 feet wound contamination in the or is the result of the patient's skin flora and bacteria shed on airborne particles from the or personnel. room ventilation affects the distribution of these airborne particles in four ways: total ventilation (dilution), air distribution (directional airflow), room pressurization (filtration barrier), and filtration (contaminant removal). as the air flows of the room increase, the greater the dilutional effect on airborne particles. balancing this phenomenon is important because while increased flow increases the effectiveness of air exchange, the resultant turbulent flow increases microbial distribution throughout the room. low-velocity unidirectional flow minimizes the spread of microbes in the room. directional flow can be inward, from the outside into the or (negative pressure), or outward, from the or to the outside (positive pressure). negative-pressure ventilation is used for highly infective rooms in the hospital (e.g., isolation rooms for tuberculosis patients), and positive-pressure ventilation is used for protective environments (e.g., ors and pep step 1: treat exposure site • use soap and water to wash areas exposed to potentially infectious fluids as soon as possible after exposure. • flush exposed mucous membranes with water. • flush exposed eyes with water or saline solution. • do not apply caustic agents, or inject antiseptics or disinfectants into the wound. step 2: report and document standard precautions 117 assume that any person or patient is potentially infected or colonized by microorganisms that could be transmitted and cause an infectious process. standard precautions must be implemented with all patients and include the following: ■ universal precautions-blood and body fluid precautions, developed to reduce bloodborne pathogen transmission ■ body substance isolation, designed to reduce the risk of pathogen transmission by moist body substances standard precautions are used to reduce the transmission of all infectious agents from one person to another, thus protecting health care providers and children against exposure to the most common microorganisms. standard precautions are implemented for any contact with blood and body fluids, secretions, and excretions (except sweat), whether or not they contain visible blood, as well as for any contact with nonintact skin, mucous membranes, and intact skin that is visibly soiled with blood and/ or body fluids. prevention is primary. all hcps should be familiar with standard precautions: wash hands frequently and thoroughly before and after patient care; use personal protective equipment: gloves, gowns, boots, shoe covers, eyewear, masks, and shields, as appropriate for the patient care situation; gloves must be worn when any kind of venous or arterial access is being performed; use sharps with caution: plan ahead (use sharps in a safe environment with a sharps container nearby), dispose of used sharps in puncture-proof receptacles immediately after use, do not recap needles, and use safety devices if available. all hcps should be vaccinated with the hepatitis b vaccine series and should undergo testing for hbsab response after completion of the series to document adequate protection. employees who have not gone through the vaccination series previously should be offered the hepatitis b series through their employer at no cost. 118 summaries of standard precautions, droplet precautions, airborne precautions, and contact precautions are available on line. 100, [119] [120] [121] hand washing overall hand hygiene compliance across health care providers remains less than 50%, with anesthesia providers identified as a particularly noncompliant group (one study found a compliance rate of only 23%). 122 bacterial contamination of anesthesia providers has been directly linked to high-risk bacterial transmission events to iv stopcocks and 30-day postoperative infections. 24 the vast majority of ssis are caused by staphylococcus aureus. transmission of specific staphylococcal phenotypes within and between patients is a major contributor to ssis and hais. 3, 4, 123 the role of anesthesia-provider hand contamination in transmission of enterococcus to the workstation and patient biome is concerning, even though it was not associated with actual infection, because of rising rates of antibiotic-resistant organisms and the observation that enterococcus is becoming a more prevalent pathogen. 3, 124 two approaches are indicated: improved methods of patient reservoir decontamination and more effective and frequent decontamination of provider hands. hand hygiene is a well-known and effective solution to the problem of bacterial transmission within and across patients. compliance with the current "5 moments" world health organization guidelines could make a major inroad into reducing provider hand and workspace contamination. one study found that only 20% of anesthesia providers demonstrated complete knowledge regarding who hand hygiene guidelines. 125 failure of providers to recognize prior contact with the environment and prior contact with the patient as hand hygiene opportunities contributed to this low percentage. several cognitive factors were associated (2.74 meters) and 12 feet (3.66 meters). some controversy exists between engineers and clinicians over the need for laminar airflow ventilation in the or to further minimize airborne infection. careful mathematical analyses of airflow suggest that laminar airflow is not necessary. clinical studies are confirmatory. similarly, the use of ultraviolet light to cleanse the room air is no longer recommended. 109 table 50 .8 shows the 2003 healthcare infection control practices advisory committee and cdc general recommendations for ventilation system specifications for the or. 12 children with tuberculosis require special consideration because of the high risk of occupational transmission of mycobacterium tuberculosis, 110, 111 especially after the emergence of multidrug-resistant strains (table 50.9 ). an easy preventive measure is to screen all children before coming to the or to determine recent exposure to infectious disease such as measles, mumps, rubella, and chickenpox because these infections can pose a significant risk to hcws and patients, especially those who are immunocompromised. 73, 106 another potential source for airborne spread of pathogens is through the anesthesia circuit; this may be reduced by the use of circuit filters. however, at present there are no regulatory requirements to use such devices, and performance characteristics vary widely. 28 • pep should be initiated within 2 hours of the exposure. • the eficacy of pep initiation is thought to diminish after 24 to 36 hours following an exposure. • if the fourth-generation combination hiv ag/ab assay is used to test the source patient, hiv follow-up testing can be completed 4 months after exposure. hand washing is considered the most important and costeffective individual intervention in the prevention of hais in children and health care providers. 126 its importance in medical practice had not been universally accepted, despite the pioneering work by oliver wendell holmes 127 (1843) and ignaz semmelweis 128 with a reduced risk of incomplete knowledge, including providers responding positively to washing their hands after contact with the environment, disinfecting their environment during patient care, believing that they can influence their colleagues, and intending to adhere to guidelines. these results suggest that anesthesia providers have knowledge deficits pertaining to opportunity-based hand hygiene in the intraoperative arena 125 hiv-positive class 1: asymptomatic hiv infection or known low viral load (e.g., <1500 ribonucleic acid copies/ml). hiv-positive class 2: symptomatic hiv infection, acquired immunodeficiency syndrome, acute seroconversion, or known high viral load. if drug resistance is a concern, obtain expert consultation. initiation of pep should be delayed pending expert consultation, and because expert consultation alone cannot substitute for face-to-face counseling, resources should be available to provide immediate evaluation and follow-up care for all exposures. the recommendation "consider pep" indicates that pep is optional; a decision to initiate pep should be based on a discussion between the exposed person and the treating clinician regarding the risks versus benefits of pep. g if pep is offered and administered and the source is later determined to be hiv-negative, pep should be discontinued. recommendations for surgical hand preparation are as follows: remove rings, wristwatch, and bracelets before beginning surgical hand preparation (ii); artificial nails are prohibited (ib); sinks should be designed to reduce the risk of splashes (ii); if hands are visibly soiled, wash hands with plain soap before surgical hand preparation (ii); remove debris from underneath fingernails using a nail cleaner, preferably under running water (ii); brushes are not recommended for surgical hand preparation (ib); surgical hand antisepsis should be performed using either a suitable antimicrobial soap or suitable alcohol-based handrub, preferably with a product ensuring sustained activity, before donning sterile gloves (ib); if the quality of water is not assured in the operating theatre, surgical hand antisepsis using an alcohol-based handrub is recommended before donning sterile gloves when performing surgical procedures (ii); when performing surgical hand antisepsis using an antimicrobial soap, scrub hands and forearms for the length of time recommended by the manufacturer, typically 2-5 minutes. long scrub times (e.g., 10 minutes) are not necessary (ib); when using an alcohol-based surgical handrub product with sustained activity, follow the manufacturer's instructions for application times. apply the product to dry hands only (ib); do not combine surgical hand scrub and surgical handrub with alcohol-based products sequentially (ii); when using an alcoholbased handrub, use sufficient product to keep hands and forearms wet with the handrub throughout the surgical hand preparation procedure (ib); after application of the alcohol-based handrub as recommended, allow hands and forearms to dry thoroughly before donning sterile gloves (ib). at present, alcohol-based handrubs are the only known means for rapidly and effectively inactivating a wide array of potentially harmful microorganisms on hands. the who recommends alcohol-based handrubs based on the following factors: evidencebased, intrinsic advantages of fast-acting and broad-spectrum microbicidal activity with a minimal risk of generating resistance to antimicrobial agents; suitability for use in resource-limited or remote areas with lack of accessibility to sinks or other facilities for hand hygiene (including clean water, towels, and so on); capacity to promote improved compliance with hand hygiene by making the process faster and more convenient; economic benefit by reducing annual costs for hand hygiene, representing approximately 1% of extra costs generated by an hci; minimization of risks from adverse events because of increased safety associated with better acceptability and tolerance than other products. 118 after hand washing, it is very important to dry the hands properly with appropriate paper towels, hot air flow, or both, because the level of pathogen transmission from a hcw's hands to a patient is greatly increased if the hands are wet. 141 sterile cloth towels are most frequently used in ors to dry wet hands after surgical hand antisepsis. several methods of drying have been tested without significant differences between techniques. 118 transmission may also occur from patients' wet sites, such as groins or armpits, or when a hcw gets his or her hands wet when opening parenteral solutions. it is critical for health institutions to establish written procedures and protocols to support adherence to the recommended hand hygiene practices. wearing clean or sterile gloves while caring for children is an effective means of reducing hais. gloves remain a supplementary barrier to infection that should not replace proper hand hygiene. more frequent (on average, 22 opportunities per patient-hour). the greatest adherence rate (59%) was observed in pediatrics, where the average intensity of patient care was smaller than elsewhere (on average, 8 opportunities per patient-hour). the results suggest that full adherence to guidelines is unrealistic and that easy access to hand hygiene at the point of patient care, (i.e., in particular, alcohol-based handrubbing) could help improve adherence to hand hygiene. perceived barriers to adherence with hand hygiene practice recommendations include skin irritation caused by hand hygiene agents, inaccessible hand hygiene supplies, interference with hcw-patient relationships, patient needs perceived as a priority over hand hygiene, wearing of gloves, forgetfulness, lack of knowledge of guidelines, insufficient time for hand hygiene, high workload and understaffing, and the lack of scientific information showing a definitive impact of improved hand hygiene on hai rates. lack of knowledge of guidelines for hand hygiene, lack of recognition of hand hygiene opportunities during patient care, and lack of awareness of the risk of cross-transmission of pathogens are barriers to good hand hygiene practices. furthermore, some hcws believed that they washed their hands when necessary even when observations indicated that they did not. the risk of pathogen transmission via the hands is proportional to the power of the number of times a child is touched. 140 table 50 .10 presents 1. wash hands with soap and water when visibly dirty or visibly soiled with blood or other body fluids (ib) or after using the toilet (ii). 2. if exposure to potencial spore-forming pathogens is strongly suspected or proven, including outbreaks of clostridium difficile, hand washing with soap and water is the preferred means (ib). 3. use an alcohol-based handrub as the preferred means for routine hand antisepsis in all other clinical situations described in terms 4(a) to 4(f) listed below, if hands are not visibly soiled (ia). if alcohol-based handrub is not obtainable, wash hands with soap and water (ib). 4. perform hand hygiene: a. before and after touching the patient (ib); b. before handling an invasive device for patient care regardless of whether or not gloves are used (ib); c. after contact with body fluids or excretions, mucous membranes, non-intact skin, or wound dressings (ia); d. if moving from a contaminated body site to another body site during care of the same patient (ib); e. after contact with inanimate surfaces and objects (including medical equipment) in the immediate vicinity of the patient (ib); f. after removing sterile (ii) or nonsterile gloves (ib). 5. before handling medication or preparing food, perform hand hygiene using an alcohol-based handrub or wash hands with either plain or antimicrobial soap and water (ib). 6. soap and alcohol-based handrub should not be used concomitantly (ii). gloves protect patients by reducing health care provider hand contamination and the subsequent transmission of pathogens to other children, provided the gloves are changed after providing care to each child. additionally, when the use of gloves is combined with cdc standard precautions, they protect the health care provider against exposure to bloodborne infections or infections transmitted by any other body fluids, such as excretions, secretions (except sweat), mucous membranes, and nonintact skin. examination gloves are single-use and usually nonsterile. sterile surgical gloves are required for surgical interventions. some nonsurgical care procedures, such as central vascular catheter insertion, also require surgical glove use. in addition to their sterile properties, these gloves have characteristics of thickness, elasticity, and strength that differ from other medical gloves. the use of gloves in situations when their use is not indicated represents a waste of resources without necessarily reducing crosstransmission. the wide-ranging recommendations for glove use have led to very frequent and inappropriate use. indications for gloving and glove removal are shown in table 50 .11. situations that require and that do not require glove use are presented in fig. 50 .6. ranked consensus recommendations for the use of gloves, categorized according to the cdc/hicpac system, include the following 118, 142, 143 : ■ wear gloves in case of contact with blood or any other potentially infecting body fluid, such as excretions, secretions (except sweat), mucous membranes, and nonintact skin (ic). ■ remove the gloves immediately after providing care to a child. staff should not wear the same pair of gloves to take care of more than one child, nor should they touch the surfaces of any equipment, monitoring devices, or even light switches. ■ alcohol-based handrub dispensers and clean glove boxes (at least two sizes) should be in place near every patient care site (e.g., on top of every anesthesia cart, medication cart, or in the nursing station). ■ disposable gloves should not be washed, resterilized, or disinfected (ib). if gloves are reused, appropriate reprocessing methods should be in place to ensure the physical integrity of the gloves and their full decontamination (ii). ■ sterile gloves are much more expensive than clean, disposable gloves and should be used only for certain procedures, such as when hands are in contact with normally sterile body areas or when inserting intravascular or urinary catheters. clean gloves should be used during any other procedure, including wound dressing. ■ latex-free gloves should be worn when caring for children at risk for latex allergy. surgical antimicrobial prophylaxis is an essential tool to reduce the risk of postoperative infections, and the anesthesia team plays a central role in ensuring the proper timing of drug administration. 147, 148 the aim of the perioperative administration of antibiotics is to obtain plasma and tissue drug concentrations exceeding the minimal inhibitory concentration of those organisms most likely to cause an infection. this will reduce the microbial load of the intraoperative contamination; it is not the intent to cover all possible pathogens, because this can lead to the selection of drug-resistant bacteria. there have been few studies regarding the effectiveness of prophylactic guidelines for prevention of ssis in children. currently, prophylactic antibiotic guidelines exist for certain subsets of the pediatric surgical population, but there are no global recommendations, and the guidelines that exist are mostly based on studies from adults or from expert opinion. a retrospective study suggested that the appropriate use of antibiotic prophylaxis was a vital modifiable risk factor and may be the easiest factor to influence. primary failure to administer the correct dose of antibiotics at the appropriate time resulted in an almost 2-fold increase in the risk of developing an ssi. the importance of correct antibiotic usage and dosing plays a major role in decreasing risk of ssis in children. recommendations are provided for adult (age ≥19 years) and pediatric (age 1-18 years) patients. the guidelines do not specifically address newborn (premature and full-term) infants (table 50. although pediatric-specific prophylaxis data are sparse, available data have been evaluated for specific procedures. selection of antimicrobial prophylactic agents mirrors that in adult guidelines, with the agents of choice being first-and second-generation cephalosporins, reserving the use of vancomycin for patients with documented β-lactam allergies. while the use of a penicillin with a β-lactamase inhibitor in combination with cefazolin or vancomycin and gentamicin has also been studied in pediatric patients, the number of patients included in these evaluations remains small. as with adults, there is little evidence supporting the use of vancomycin, alone or in combination with other antimicrobials, for routine perioperative antimicrobial prophylaxis in institutions that have a high prevalence of methicillin-resistant s. aureus (mrsa). vancomycin may be considered in children known to be colonized with mrsa and decreases mrsa infections. 150 mupirocin is effective in children colonized with mrsa, but choice, alternative antibiotics should be administered to those children at risk of anaphylaxis to β-lactams, based on their history or diagnostic tests (e.g., skin testing). however, the incidence of severe allergic reactions to first-generation cephalosporins in children with reported allergy to penicillin is rare (but not zero) 158, 159 ; furthermore, skin testing does not reliably predict the likelihood of adverse reactions to cephalosporins in those with reported allergy to penicillin. [160] [161] [162] there is no evidence of any risk of cross-reactivity between penicillin and second-and thirdgeneration cephalosporins. for the most part, "allergies" to oral antibiotics that appear on children's charts (rash, vomiting, gastrointestinal disturbances) are reactions to the additives in the antibiotic formulation, including food dyes, fillers, and other compounds, or a manifestation of the underlying infection. iv administration of small test doses of the pure antibiotic in a fully monitored (and anesthetized) child will determine whether the child is at risk for an allergic reaction to the antibiotic. in the case of surgical procedures where antibiotic prophylaxis is mainly directed at gram-positive cocci, children who are truly allergic to β-lactams (cephalosporins) should receive either vancomycin or clindamycin. 163 however, in those children where the history is consistent with either an ige-mediated penicillin allergy (urticaria, angioedema, anaphylaxis, bronchospasm) or a severe non-igemediated reaction (interstitial nephritis, toxic epidermal necrolysis, hemolytic anemia, or stevens-johnson syndrome) it is advisable to switch out the cefazolin. cross-sensitivity occurs when the r1 side chains of the penicillins and cephalosporins are similar, which perhaps surprisingly is not the case with cefazolin. cephalosporins with r1 side chains similar to penicillins include cephalexin, cefaclor, and cefadroxil. the risk associated with use of first-or second-generation cephalosporins with dissimilar side chains, or third-or fourth-generation cephalosporins, "appears to be very low in patients with mild-to-moderate reactions to penicillin g, ampicillin, or amoxicillin. dismissing cefazolin use when there is a vague history of any penicillin allergy should be reconsidered." 155 indications for prophylactic antibiotics surgical wounds are classified into four categories (table 50 .13). the use of antibiotic prophylaxis for postoperative infections is well established for clean-contaminated procedures. within the clean category, prophylaxis has been traditionally reserved for surgical procedures involving a foreign body implantation or for any surgical procedure where an ssi would be catastrophic (e.g., cardiac surgery or neurosurgical procedures). however, there is evidence that postoperative infections resulting from procedures not involving prosthetic elements are underreported; estimates show that more than 50% of all complications occur after the patient is discharged and are thus unrecognized by the surgical team. therefore antibiotic prophylaxis is also recommended for certain procedures, such as herniorrhaphy. 164, 165 the direct and indirect costs of these complications may not affect the hospital budget; however, they represent a substantial cost for the community at large. in the case of contaminated or dirty procedures, bacterial contamination or infection is established before the procedure begins. accordingly, the perioperative administration of antibiotics is a therapeutic, not a prophylactic, measure. the use of antibiotics in children has implications not only for the response to the current treatment but also to future treatments. thus all medical professionals are jointly responsible for the rational use of antibiotics. protocols, although effective, require continuous feedback on their acceptance and ssi results. 166 no surgical protocol can replace there are limited data supporting its use perioperatively. 151, 152 most recommendations for adults are the same for pediatric patients. dosing recommendations in pediatric patients are limited and have been extrapolated from adult data; therefore nearly all pediatric recommendations are based on expert opinion. pediatric efficacy data are few. fluoroquinolones should not be routinely used for surgical prophylaxis in pediatric patients because of the potential for toxicity in this population. the same principle of preoperative dosing within 60 minutes before incision has been applied to pediatric patients. additional intraoperative dosing may be needed if the duration of the procedure exceeds two half-lives of the antimicrobial agent or there is excessive blood loss during the procedure. as with adult patients, single-dose prophylaxis is usually sufficient. if antimicrobial prophylaxis is continued postoperatively, the duration should be less than 24 hours, regardless of the presence of intravascular catheters or indwelling drains. there are sufficient pharmacokinetic studies of most agents to recommend pediatric dosages that provide adequate systemic exposure and, presumably, efficacy comparable to that demonstrated in adults. therefore the pediatric doses recommended in guidelines are based largely on pharmacokinetic data and the extrapolation of adult efficacy data to pediatric patients. because few clinical trials have been conducted in pediatric surgical patients, strength of evidence criteria have not been applied to these recommendations. with few exceptions (e.g., aminoglycoside dosages), pediatric doses should not exceed the maximum adult recommended dosages. generally, if a dose is calculated on a milligram-per-kilogram basis for children weighing more than 40 kg, the calculated dosage will likely exceed the maximum recommended dose for adults; adult dosages should therefore be used for larger children. 153 the timing of antibiotic prophylaxis the 2013 revised policy paper on prophylactic antibiotics developed jointly by the american society of health-system pharmacists (ashp), the infectious disease society of america, the surgical infection society, and the society for healthcare epidemiology of america states: successful prophylaxis requires the delivery of the antimicrobial to the operative site before contamination occurs. thus, the antimicrobial agent should be administered at such a time to provide serum and tissue concentrations exceeding the minimum inhibitory concentration (mic) for the probable organisms associated with the procedure, at the time of incision, and for the duration of the procedure. 154 current evidence suggests that for most β-lactams, a bolus dose at 15 to 45 minutes before incision is ideal and provides maximum interstitial fluid concentrations at the time of initial bacterial seeding (see table 50 .12). because diffusion distances from capillary to pathogen are greater in obese patients, for this patient subset initiating antibiotic infusion 30 minutes or longer before incision is warranted on theoretical grounds. 155 the initial β-lactam bolus dose should be followed by additional doses at every 1 to 2 half-lives per the ashp guidelines. 154 the use of a ssi prevention bundle in pediatric patients improves compliance with preincision antibiotic administration and decreases the ssi infection rate. 156 allergy to β-lactams several studies have shown that the true incidence of allergy to antibiotics is less than that reflected in medical charts. 157 for surgical procedures where cephalosporins are the prophylaxis of the judgment of the medical professional; clinical reasoning must be tailored to the individual circumstances. finally, children with congenital heart disease and a subgroup of those with repaired congenital heart disease may require bacterial endocarditis prophylaxis (see also tables 16.2 and 16. 3). 167 preventing the transmission of pathogenic microbes during anesthesia infection control and anesthesia: lessons learned from the toronto sars outbreak fecal patina in the anesthesia work area intensive care unit environments and the fecal patina: a simple problem? transmission dynamics of gram-negative bacterial pathogens in the anesthesia work area serratia marcescens bacteremia traced to an infused narcotic postoperative infections traced to contamination of an intravenous anesthetic, propofol staphylococcus aureus bloodstream infections among patients undergoing electro-convulsive 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suzanne; small, j. david; geratz, joachim dieter; alexander, lorraine k.; baric, ralph s. title: an experimental model for myocarditis and congestive heart failure after rabbit coronavirus infection date: 1992-01-17 journal: j infect dis doi: 10.1093/infdis/165.1.134 sha: doc_id: 289406 cord_uid: 54vyzxjf in a model for virus-induced myocarditis and congestive heart failure, rabbit coronavirus infection was divided into acute (days 2–5) and subacute (days 6–12) phases on the basis of day of death and pathologic findings. during the acute phase, the principal histologic lesions were degeneration and necrosis of myocytes, myocytolysis, interstitial edema, and hemorrhage. the severity of these changes increased in the subacute phase. pleural effusion and congestion of the lungs and liver were also present at this time. myocarditis was detected by day 9 and peaked by day 12. heart weights and heart weight-to-body weight ratios were increased, and dilation of the right ventricular cavity became prominent early in infection and persisted. in contrast, dilation of the left ventricle occurred late in the subacute stage. virus was isolated from infected hearts between days 2 and 12. these data suggest that rabbit coronavirus infection progresses to myocarditis and congestive heart failure. viruses have long been recognized as important etiologic agents of heart disease in humans and experimental animals [1-3). epidemiologic evidence suggests that after viral infection, 2%-5% of a human population experience some degree of cardiac involvement [2, 3) . in humans and experimental animals, viruses commonly linked to heart disease include the picornaviruses, paramyxoviruses, myxoviruses, alphaviruses, and coronaviruses [2] [3] [4] [5] . viral infection of the heart may result in degeneration and necrosis of myocytes and lead to inflammation of the heart muscle. myocarditis may result in arrhythmias, conduction disturbances, circulatory collapse, and acute congestive heart failure [3] [4] [5] [6] [7] . acute viral infection of the heart muscle may also be an important factor in the development ofidiopathic dilated cardiomyopathy [8] [9] [10] [11] . coxsackie b virus and encephalomyocarditis virus (both enteroviruses) infections in mice are the best-characterized model systems for virus-induced heart disease [i, 5) . the exact mechanism for their pathogenesis is still controversial; however, considerable evidence suggests that the disease is primarily immune-mediated rather than the result of direct viral cytotoxicity to myocytes [12, 13] . both coxsackie band encephalomyocarditis virus infections in mice may progress to myocarditis and congestive heart failure, and some survi-vors may progress to a dilated cardiomyopathy later in life [5, [14] [15] [16] . the mechanisms by which viruses outside the enterovirus family cause heart disease are unclear. a model for virus-induced cardiomyopathy has also been described in rabbits [17] . rabbit cardiomyopathy is characterized by pulmonary edema, degeneration and necrosis of myocytes, and right ventricular dilation. similar findings have also been reported in rabbits infected with pleural effusion disease virus [18, 19) . the etiologic agent for rabbit cardiomyopathy is probably a rabbit coronavirus (rbcv) antigenically related to the human coronavirus strain 229e [ i 7] . we determined whether infection with rbcv would result in myocarditis and the development of congestive heart failure. animals and virus. rabbit coronavirus (rbcv) was originally obtained from a stock maintained by one of the authors (1.d.s.). viral stocks were diluted to 10 3_104 ridso/ml and stored at -140°c. male new zealand white rabbits (franklin rabbitry, wake forest, nc), weighing 2.5-3.0 kg, were housed at room temperature (21-24°c) and given water and rabbit diet (agway; grandville milling, creedmoor, nc) ad libitum. the animals were inoculated intravenously via the marginal ear vein with 0.2 ml of the 10 3-104 rid so viral stock. body weight and rectal temperature were recorded daily. animals were observed for signs of infection: dullness of the sclerae, severe congestion of the conjunctivae and irides, rectal temperatures >39°c, and weight loss. to assay viral titers in the heart muscle, moribund animals were intravenously injected with 50 mg/kg sodium pentobarbitol, and the hearts were perfused and washed extensively with pbs. two hundred micrograms of left ventricle were ground in 0.8 ml of pbs and centrifuged at 12,000 g for 10 min in an eppendorf centrifuge (fisher scientific, norcross, ga). the suexhibited moist rales and wheezing during the subacute stage. rabbits that died on days 10-12 had pleural effusion, pulmonary edema, ascites, enlarged hearts, dilated right and left ventricular cavities, and congestion in the lungs and liver. from these observations, we divided rbcv infection into an acute phase that was characterized by dilation of the right ventricle and pulmonary edema (days 2-5) and a subacute phase that was characterized by dilation of both ventricles and heart failure (days 6-12). heart weight and heart weight-to-body weight ratios during rbcv infection. body weight, heart weight, and heart weight-to-body weight ratios were measured on days 2-5, when myocardial degeneration and necrosis and pulmonary edema became apparent, and days 6-12, when heart failure was evident. body weights slowly decreased during the course of infection and were notably decreased (......,0.4 kg) during the subacute phase of the disease (data not shown). control rabbits sacrificed on days 2-5 (acute, n = 10) or days 6-12 (subacute, n = 10) had heart weights of 6.1 ± 0.3 g and 6.1 ± 0.5 g, respectively. after rbcv infection, heart weights were significantly increased, to 8.4 ± 1.4 g during the acute stage (n = 12) (p < .00 i) and 8.7 ± 1.6 g during the subacute phase of infection (n = 14) (p < .001). heart weight-to-body weight ratios were 0.0022 ± 0.0002 in the uninfected controls and were increased significantly, to 0.0031 ± 0.0003 (p < .00 i) and 0.0035 ± 0.0006 (p < .00 i) during the acute and subacute phases of infection, respectively. dimensions of the cardiac walls during rbcv infection. changes in the size of the heart and, in particular, dimensions of the ventricles were evident after rbcv infection (figure 2). to conclusively document the anatomic changes in the heart during infection, the thickness of the ventricular wall was measured through the coronal axis at the midpoint of the ventricles. the thickness of the right wall in uninfected controls was pernatant was serially diluted and inoculated into the marginal ear vein of rabbits. histologic studies. animals dying from rbcv infection were necropsied within 12 h of death. alternatively, moribund animals were sacrificed as described above. body weights were obtained to the nearest o. 10 kg. the heart was separated from the pericardial sac, trimmed of fat and extraneous tissue, and flushed with pbs. the heart was weighed to the nearest 0.1 g; the chambers were filled with 10% phosphate-buffered formalin (bf) and immersed in 10% bf for 24-48 h. the heart was removed and sectioned transversely at the widest dimensions of the ventricles. after additional fixation in bf, four paraffin-embedded 6-~m sections were cut at 150-~m intervals and stained with hematoxylin-eosin (h&e) stain. selected sections were also stained by masson's trichrome and the von kossa stains. sections ofthe lung, liver, thymus, kidney, and spleen were also stained with h&e. morphometric studies. a computerized zeiss videoplan-l digital morphometric system (carl zeiss, thornwood, ny) was used to measure the dimensions of the cardiac walls and cavities and to examine the area within each ventricular section. to measure the thickness of the right and left ventricular walls and interventricular septum, 15-20 measurements were taken at regular intervals across the ventricles. two or three different cardiac sections were measured from each animal, and the standard deviations were calculated and recorded. the dimensions of the ventricular cavities were measured at 15-20 points across the midpoint in each ventricle and then averaged between three or four consecutive cross-sections in the same animal. large differences between consecutive cross-sections were not detected. papillary muscle projections complicated measurements of the left ventricular cavity and wall. consequently, the area in each left and right ventricle cross-section was measured two or three times and averaged between different cross-sections in the same animal. animals dying during the acute or subacute phase ofinfection were grouped accordingly, and mean values were averaged and reported as mean ± so. student's t test for unpaired observations was used to evaluate the statistical significance of differences in cardiac dimensions. zealand white rabbits were inoculated with 0.2 ml of a 10 3 -10 4 rioso/ml stock and examined daily for clinical signs of infection. consistent with earlier studies [ i 7], mortality rates peaked at 4 days after infection, decreased, and then increased again between day 6 and 8. no animals died after 12 days after infection, and the overall mortality rate approached 64% (27% acute; 37% subacute) (figure i). animals dying early from infection had enlarged hearts characterized by striking dilation of the right ventricular cavity and accompanied by pulmonary edema. pleural effusion and congestion of the lungs and liver were occasionally present during the acute phase but were more commonly observed between days 6-9 after infection. clinically, rabbits between the uninfected controls and animals dying during the acute phase of the disease (p = .445). however, the thickness of the left ventricular wall was significantly reduced, by --15%, during the subacute phase (p < .05) (figure 3b) . dimensions of the ventricular cavities during rbcv infection. in uninfected controls, the width of the right ventricular cavity was --3100 jlm ( figure 4a ). in infected animals, the width of the right ventricular cavity was significantly increased, by --105% during the acute stage of to obtain an additional estimate of the dilation of the right and left ventricular cavities during infection, we averaged the area in several consecutive right and left ventricular cavity sections from animals dying during the acute or subacute phase. areas within the right and left ventricular cavity were similar between control animals sacrificed on days 3-5 or 6-12 ( figure 5a, b) . after rbcv infection, the area in the right ventricular cavity increased by --150% during the acute stage (p < .001) and 254% during the subacute phase (p < .001) ( figure 5a ). areas within the left ventricular cavity were not altered significantly during the early stages of infection (p = .278), but significant increases, of --30%, were noted during the subacute phase (p = .025) (figure 5b). pathologic findings during rbcv infection. rabbits were divided into two groups according to the day of death and pathologic findings in the host. with minor variations, lesions were similar to those previously reported [17] . the heart was the principal target organ, often with red streaks present on the epicardial and endocardial surfaces. pulmonary edema was always present, and accumulations of 10often present in the thorax. the frequency and volume of pleural effusion increased during the subacute period. late in the subacute period, a small amount of ascites fluid was seen in some animals. accentuation of the hepatic lobules was present in some rabbits dying during the subacute period; however, the liver margins remained sharp. microscopically, myocardial lesions in rabbits dying during the acute phase consisted of widening of intercellular spaces, scattered infiltrates of small numbers of heterophiles (rabbit neutrophils), increased granularity of myocyte cytoplasm, hemorrhage, and occasional degeneration and necrosis of myocytes ( figure 6a) . only rarely were foci of frank hemorrhage and calcification seen during this period. lesions were seen equally in the right and left ventricles and in the interventricular septum. as rabbits survived beyond 5 days, lesions progressed in size, number, and maturity. in several rabbits, necrotic foci had varying degrees of calcification ( figure 6b ). lesions were equally present in the left and right ventricles, interventricular septum, and papillary muscle. no lesions were seen in the heart valves or the vessels. interstitial edema increased. in addition to increased numbers of heterophils, macrophages and lymphocytes were seen. myocarditis was usually diffuse to focal and peaked by day 10-12 ( figure 6c ). alveolar epithelial cells were swollen. in a few rabbits of the subacute group, much of the intraalveolar fluid had been readsorbed, leaving fibrinous strands, macrophages, and heterophils. interalveolar blood vessels were distended ( figure 6d ). lymphoid tissue adjacent to bronchi was within normal limits, and bronchial epithelium was unremarkable. liver lesions were subtle during the acute phase. sinusoids were slightly distended, especially in areas surrounding central veins. in several rabbits the first row or two of hepatocytes surrounding some of the central veins were rounded up, hyalinized with indistinct nuclei, and deeply eosinophilic. with increased time to death in the subacute stage, b sinusoids became more distended, individual hepatocytes were compressed, the number of hepatocytes undergoing coagulative necrosis around the central veins increased, and the necrotic areas became increasingly hypercellular. portal triads were normal ( figure 6e) . isolation of infectious virus. infectious virus was isolated from the hearts of animals dying between days 2 and 12. virus was first detected in the heart muscle by day 2 (10 2-103 rid 50/g). peak' titers in the heart muscle occurred on days 3-5 (10 5-106 rid 50/g), and significant titers were detected viral infection of the heart muscle may result in degeneration and necrosis of myocytes, myocarditis, and congestive heart failure [15, 7] . viral infection may also be an important factor in the development ofidiopathic dilated cardiomyopathy [5, [20] [21] [22] . the incidence and mechanisms by which viruses cause heart disease in humans and animals are unclear, because these agents are rarely recovered from the patients and heart tissue at biopsy. since many different viral infections may result in cardiac damage, model systems are needed to examine the basis for virus-induced heart disease. rbcv infection results in degeneration and necrosis of myocytes, myocarditis, interstitial edema, hemorrhage, increased heart weight and heart weight-to-body weight ratios, and dilated ventricles. although dry weights of the hearts were not determined, pathologic findings suggest that the increase in heart weight is probably caused by interstitial edema. animals dying in the subacute stage of the disease develop congestion in the lungs and liver, suggesting that a significant percentage of these animals probably die from heart failure. manifestations of both left-and right-sided heart failure are clearly evident in the subacute phase of infection [4, 6, 7, 21] . previous studies in our laboratory clearly demonstrated the presence ofviral antigen in regions ofmyocardial degeneration and infectious virus in the hearts of infected animals, supporting the idea that changes in the myocardium are most likely caused by viral replication in the heart muscle [17] . the pathogen for this disease is morphologically and antigenically related to the group i human and porcine coronaviruses [17] . rbcv is also related to the etiologic agent responsible for pleural effusion disease in rabbits [17] [18] [19] 23] . both viruses have similar isolation histories and antigenic properties and produce similar diseases in vivo. here we demonstrated that infection with rbcv results in myocarditis. rabbits normally require 8-10 days to develop a strong lymphocytic infiltration after infection [24] . by the dallas classification system [25] , focal to diffuse myocarditis with fibrosis is clearly present by day 9 and reaches peak levels on days 10-12. in previous studies, little lymphocytic infiltration was noted because most of the animals dying from rbcv or pleural effusion disease virus infection were examined before day 9 [17, 18, 26] . it seems likely that pleural effusion disease virus infection also results in a significant percentage of animals dying from heart failure, since degeneration and necrosis of myocytes, pulmonary edema, pleural effusion, dilated ventricles, and congestion of the lungs, liver, and spleen are common [18, 26] . in humans, virus-induced myocarditis and congestive heart failure have been reported after infection with herpesvirus, enterovirus, paramyxovirus, myxovirus, alphavirus, flavivirus, and others [1-3, 27, 28]. complications associated with coronavirus infections in humans include myocarditis and perimyocarditis [4] . during the subacute phase of infection, myocardial lesions observed in rabbits closely resemble those of acute myocarditis in humans. lesions are distributed throughout the ventricles and consist of necrotic fibers surrounded by macrophages and lymphocytes. adjacent areas of the myocardium appear normal. the endocardium and pericardium are spared. later, necrotic fibers are replaced with connective tissue or calcification, and the number of macrophages and lymphocytes are reduced [1, 25] . the most extensively studied models for virus-induced myocarditis and congestive heart failure are in mice inoculated with enteroviruses (encephalomyocarditis virus or coxsackie b virus) [1, 5, 21] . there are also model systems for influenza virus-induced metabolic alterations in the heart [29] , reovirus-induced myocarditis in mice [3o], and parvovirus-induced myocarditis in dogs [31] . in rabbits, poxvirus infections may also result in degeneration and necrosis of myocytes and myocarditis [32] . rabbit coronavirus infection is similar to the encephalomyocarditis virus model. early in that infection, animals are probably dying from acute heart failure caused by a complete atrioventricular block [15, 33] . in the subacute stage of infection, animals die from myocarditis-induced congestive heart failure [13, 15] . the most notable difference between the two models is that myocarditis, myocyte necrosis, and calcification appear to be much more extensive after encephalomyocarditis virus infection [14, 15] . in the case of encephalomyocarditis and coxsackie b virus infections, maximum inflammation and cardiac necrosis occur after the clearance of virus from the heart, suggesting that direct viral cytotoxicity to myocytes is oflimited importance [i] . rather, the preponderance of data suggest that cardiac damage is immune-mediated [12, 13, [34] [35] [36] [37] [38] [39] . the pathogenic mechanisms for myocyte injury after rbcv infection are unclear. early in infection, significant myocyte damage correlates with high viral titers in the heart and occurs before the presence of significant inflammatory infiltrates. heterophils and macrophages that are occasionally present early in infection probably represent a nonspecific response to necrotic cell injury. these data suggest that myocardial injury may initially result from direct viral infection, similar to findings reported early in canine parvovirus infection [31] . we have described a model system for virus-induced myocarditis and congestive heart failure in rabbits. these data provide the underlying foundation for future studies examining the mechanism of rbcv-induced heart disease in rabbits. viral myocarditis abelmann who viral myocarditis and its sequelae abelmann who virus and the heart coronavirus infections ofman associated with diseases other than the common cold viruses and the heart: viral myocarditis and cardiomyopathy viral heart disease manual of acute cardiac disorders heart disease: a textbook ofcardiovascular medicine pathologic anatomy of the cardiomyopathies diagnosis and natural history of congested (dilated) cardiomyopathies a. incidence of congestive cardiomyopathy involvement of t lymphocytes in the pathogenesis of coxsackie virus b3 heart disease immunologic behavior of lymphocytes in experimental viral myocarditis: significance of t lymphocytes in the severity of myocarditis and silent myocarditis in balb/c-nu/nu mice an animal model ofcongestive (dilated) cardiomyopathy: dilation and hypertrophy of the heart in the chronic stage in dba/2 mice with myocarditis caused by encephalomyocarditis virus an experimental model for congestive heart failure after encephalomyocarditis virus myocarditis in mice a mouse model of dilatedtype cardiomyopathy due to coxsackievirus b3 rabbit cardiomyopathy associated with a virus antigenically related to human coronavirus strain 229e pathogenicity and persistence of pleural effusion disease virus isolates in rabbits pleural effusion disease in rabbits. properties of the aetiological agent dilated (congestive) cardiomyopathy: a syndrome of severe cardiac dysfunction with remarkably few morphological features of myocardial damage animal models of congestive heart failure and congestive (dilated) cardiomyopathy due to viral myocarditis in mice antibodies to coxsackie b virus in congestive cardiomyopathy pathogenetic observations on pleural effusion disease in rabbits effect of cortisone administration on host-parasite relationships in early experimental syphilis myocarditis, a histopathologic definition and classification pleural effusion disease in rabbits. clinical and post mortem observations myocarditis and cardiomyopathy after arbovirus infections (dengue and chikungunya fever) detection of enterovirus rna in myocardial biopsies from patients with myocarditis and cardiomyopathy using gene amplification by polymerase chain reaction sequential metabolic alterations in the myocardium during influenza and tularemia in mice the reovirus m i gene, encoding a viral core protein, is associated with the myocardic phenotype of a reovirus variant experimental viral myocarditis: parvoviral infection of neonatal pups susceptibility of the heart of the rabbit to specific infection in viral diseases electrocardiographic findings in experimental myocarditis in dba/2 mice: complete atrioventricular block in the acute stage, low voltage of the qrs complex in the subacute stage and arrhythmias in the chronic stage coxsackievirus b3 myocarditis. identification of different pathogenic mechanisms in dba/2 and balb/c mice coxsackievirus b-3 myocarditis in balb/c mice. evidence for autoimmunity to myocyte antigens differences in cytolytic t cell response ofbalb/c mice infected with myocarditic and nonmyocarditic strains of coxsackievirus group b, type 3 autoantibodies specific for the cardiac myosin isoform are found in mice susceptible to coxsackievirus b3-induced myocarditis coxsackievirus-b'i-induced myocarditis: virus receptor antibodies modulate myocarditis monoclonal antibody to coxsackievirus b4 reacts with myocardium we thank gillian harris and edna lennon for excellent secretarial assistance and james e. hall, sheila peel, mary c. schaad, and robert e. johnston for editorial comments. key: cord-260700-u12aa739 authors: kainulainen, leena; vuorinen, tytti; rantakokko-jalava, kaisu; österback, riikka; ruuskanen, olli title: recurrent and persistent respiratory tract viral infections in patients with primary hypogammaglobulinemia date: 2010-06-10 journal: j allergy clin immunol doi: 10.1016/j.jaci.2010.04.016 sha: doc_id: 260700 cord_uid: u12aa739 background: the occurrence of respiratory tract viral infections in patients with primary hypogammaglobulinemia has not been studied. objective: we conducted a prospective 12-month follow-up study of respiratory tract infections in 12 adult patients with primary hypogammaglobulinemia. methods: nasal swab samples and induced sputum samples were taken at the onset of acute respiratory tract infection and every 3 months thereafter. samples were tested for bacteria and viruses. pcr tests were performed for 15 respiratory tract viruses. in case the results for rhinovirus were positive, follow-up nasal swab samples were taken every 2 weeks until rhinoviral pcr results became negative. patients completed symptom diaries, which were collected every month. the spouses of the patients served as healthy control subjects. results: during the 12-month period, the 12 patients had 65 episodes of acute respiratory tract infections, and the 11 spouses had 12 acute episodes (p < .001). respiratory tract viruses were found in sputum in 54% of the infections. rhinovirus was the most common virus. in more than half of our patients, rhinoviral pcr results stayed positive for more than 2 months. the most long-acting persistence with the same rhinovirus was 4 months. conclusions: despite adequate immunoglobulin replacement therapy, patients with primary hypogammaglobulinemia have increased susceptibility to respiratory tract viral infections. rhinoviral infections are frequent and prolonged. patients with primary hypogammaglobulinemia typically experience recurrent bacterial infections. 1 it is generally considered that patients with hypogammaglobulinemia are not more prone to viral infections than immunocompetent subjects. enteroviruses, however, are an exception. systemic enterovirus infections (echoviruses, coxsackieviruses, and vaccine-related polioviruses) have caused severe morbidity and high mortality rates in patients with hypogammaglobulinemia. 2, 3 fatal enterovirus-induced meningoenchephalitis has been described in case reports in patients with primary x-linked agammaglobulinemia (xla). however, chronic enterovirus-induced meningitis has become less common in recent years since the use of higher immunoglobulin doses. 4 the occurrence of other picornaviruses, such as rhinoviral infections in patients with primary hypogammaglobulinemia, is not known. thus far, respiratory tract viral infections have not been studied in patients with primary hypogammaglobulinemia. using modern diagnostic techniques, we wanted to study the occurrence of respiratory tract infections, especially viral infections, in patients with primary hypogammaglobulinemia who were receiving regular immunoglobulin replacement therapy. twelve adult patients with primary hypogammaglobulinemia participated in the study (table i) . two patients had xla, and 10 had common variable immunodeficiency (cvid). their ages ranged from 32 to 79 years (median, 47.5 years). cvid was characterized by decreased serum immunoglobulin levels (>2 sds less than the age-adjusted mean), defective in vitro antibody formation, and exclusion of other known causes of humoral immune defects. the diagnosis of xlawas based on early-onset, very low serum immunoglobulin concentrations; male sex; and a lack of circulating mature b lymphocytes in the peripheral blood and mutation analysis of the bruton tyrosine kinase (btk) gene. 5 all patients were receiving regular immunoglobulin replacement therapy (dose, 400-600 mg/kg/mo). the trough serum igg concentration was greater than 5 g/l in all patients; 9 patients had serum igg levels of 6.0 g/l or greater. no patients were receiving prophylactic antibiotic treatment at the beginning of the study. all patients were nonsmokers. eleven spouses of the 12 patients served as healthy control subjects. in 3 families there were school-aged children. one of the patients was a widow and lived alone. respiratory tract infections. the study was carried out at turku university hospital, finland. informed consent was obtained from the patients and healthy control subjects, and the study was approved by the joint commission on ethics of the turku university hospital and the university of turku. the primary outcome variable was an acute episode of respiratory tract infection (the common cold) defined on the basis of new symptoms (ie, excessive sneezing, sore throat, nasal discharge, nasal obstruction, and cough). the secondary outcome variable was viral cause of this acute respiratory episode. we searched for 15 viruses: adenovirus; coronaviruses oc43, 229e, hku1, and nl63; enteroviruses; human bocavirus; human metapneumovirus; influenza a and b viruses, parainfluenza type 1, 2, and 3 viruses; rhinovirus and respiratory syncytial virus (rsv). at the first visit and follow-up visits every 3 months, patients were examined (l.k.). four nasal swabs were taken, and the patients provided induced sputum samples. at the onset of respiratory tract infection, the patients were asked to contact the investigator (l.k.) if they presented with new symptoms of respiratory tract infection. the patient was examined (l.k.) within 2 days. three nasal swabs were taken, and the patients provided an induced sputum sample. enteroviral and rhinoviral pcr, as well as viral and bacterial cultivation, were performed from the swab and sputum specimens. if rhinoviral pcr results were positive in the nasal swab samples, sputum samples, or both, new nasal swabs were taken every 2 weeks until rhinoviral rna results became negative. the research nurse taught the patient to take nasal swabs at home, and swabs were sent in vials by post to the laboratory. 6 if the bacterial culture of the sputum sample was positive, the patient received antibiotic treatment. a new induced sputum sample was taken after the antibiotic treatment. antibiotic treatment was also given in some patients with bacterial culture-negative acute infections. samples from healthy control subjects. if the spouse of the patient had acute symptoms of respiratory tract infection, she or he took nasal swabs at home according to the instructions of the research nurse and sent the vials by post. in cases in which the rhinoviral rna results were positive, new nasal swabs were taken every 2 weeks until they became negative. symptom diary. patients and spouses completed a common symptom diary, recording fever, nasal discharge, cough, sputum amount and color, absenteeism from work, and antibiotic treatments. the diaries were returned monthly and checked by the research nurse. if questions arose, the research nurse called the patient or the spouse. nasal swab. nasal swabs were obtained from the nostrils at a depth of 2 to 3 cm by using a sterile cotton swab. 6 the nasal swab for respiratory tract viral pcr was inserted into a dry vial. the nasal swab for viral culture was inserted into a vial containing 2.5 ml of viral transport medium (5% tryptose phosphate broth, 0.5% bsa, and antibiotics in pbs). the nasal swab for the bacterial culture was inserted into a culture medium vial. induced sputum sample. sputum production was induced by means of inhalation of 5% hypertonic saline solution according to the method of zar et al. 7 the quality of the sputum sample was assessed by means of light microscopy. the samples were classified in 6 categories, of which classes 4 to 6 (ie, those with >25 leukocytes and <10 squamous cells per field) were further processed. the samples were cultured on standard media, and potential respiratory tract pathogens were identified and tested for antimicrobial susceptibility. viral analysis. nasal swab samples were analyzed immediately for viral cultivation and pcr analysis for enteroviruses and rhinovirus. 6 sputum samples for viral cultivation were stored at 2708c until analysis. viral cultivation from nasal swabs and induced sputum specimens was performed according to standard roller tube methods in llc-mk2, hela ohio, and a549 cells and in human foreskin fibroblasts. nasal swabs were eluted by vortexing with 1 ml of pbs. nucleic acids were extracted from 220-ml aliquots of the specimens with the qiaamp minelute virus spin kit (qiagen, hilden, germany), according to the manufacturer's instructions, and from 550-ml induced sputum specimens with the nuclisens easymag automated extractor (biomerieux, boxtel, the netherlands) with an elution volume of up to 55 ml. extracts from nasal swabs and sputum specimens were analyzed for enteroviruses, rhinovirus, and rsv according to standard pcr protocols used in the virus diagnostic laboratory of the department of virology, turku university. extracts from the sputum specimens were also analyzed for adenovirus and human bocavirus by using pcr assays in accordance with standard protocols of the department of virology, turku university, and for human metapneumovirus by using the amplitect quantification kit for metapneumovirus genomes (ame biosciences, toroed, norway). a multiplex pcr assay (the seeplex respiratory virus detection assay; seegene, inc, seoul, korea) was used for detection of 13 respiratory tract viruses (adenovirus, influenza a and b viruses, coronaviruses oc43/hku1 and 229e/nl63, rhinovirus, human metapneumovirus, rsv a and b, and parainfluenza type 1, 2, and 3 viruses). rhinovirus-positive cdnas were amplified for sequence analysis, as described earlier. 6 the primers from the 59 noncoding region generate 397-bp-long amplicons, which were sequenced in the dna sequence service laboratory of the turku center for biotechnology. the partial sequences were aligned with clustalw software. viruses with greater than 98% sequence homology were considered the same type. 6 the phylogenetic tree was computed by using the jukes-cantor algorithm and the neighbor-joining method. phylogenetic analyses were conducted by using the mega4 program and the bootstrap consensus tree inferred from 1000 replicates. statistical analysis were performed with spss for windows software (version 16.0; spss, inc, chicago, ill). a paired-samples t test was conducted to compare respiratory tract infection episodes in patients and spouses and to compare respiratory tract viral infections and rhinoviral infections, respectively. (table ii ). in 7 patients the rhinoviral pcr results were positive for 8 or more weeks in nasal swabs taken every 2 weeks. in a patient with xla rhinovirus, pcr results stayed positive for 5 months. the same rhinoviral type, with more than 98% partial 59 nucleotide sequence homology, was found for 4 months (fig 2) . in a patient with cvid, the same rhinoviral type persisted for 6 weeks (score of 98% similarity). the majority of rhinoviral infections occurred during the fall months (fig 1) . adenovirus was found in the sputum samples of 5 patients 9 times. in 1 patient adenovirus was found 3 times during 8 months; (table ii) . the mean number of respiratory tract infections during the 12-month period per patient was 5 (range, [1] [2] [3] [4] [5] [6] [7] [8] , and the mean number of received antibiotic treatments was 4 (range, 1-8). the most common clinical signs and symptoms of acute respiratory tract infections were cough and rhinitis and increased sputum volume in patients with chronic cough. fever (>37.08c) was present in 14 of 65 episodes. there were no differences in the symptoms in patients with viral infection or viral-bacterial coinfection (data not shown). the mean duration of symptoms was 11 days. two patients required hospitalization because of pneumonia. results for rhinovirus and adenovirus were positive in sputum before pneumonia in 1 patient. bacteria were not found in the blood or sputum of the patients with pneumonia. six of the 11 spouses had 12 acute respiratory tract infections during the 12-month period. five had no infections. rhinovirus was found in only 1 (8%) of 12 episodes, and results were negative in a control sample 2 weeks later. parainfluenza viruses were detected in 3 episodes. in 1 family both the patient and the spouse had parainfluenza type 1 viral infections at the same time. there was a significant difference in the number of respiratory tract infection episodes in patients and spouses: patients had 4.4 episodes more than the spouses (95% ci, 2.6-6.1; p < .001). patients had also significantly more viral infections than the spouses (mean difference, 2.7; 95% ci, 1.3-4.2; p 5 .002) and more rhinoviral infections (mean difference, 1.8; 95% ci, 0.9-2.8; p 5 .002), respectively. our prospective 12-month study has 2 new observations. first, despite adequate immunoglobulin replacement therapy, most patients with primary hypogammaglobulinemia had increased susceptibility to respiratory tract viral infections. second, many patients specifically had recurrent and persistent rhinoviral infections. in this study patients with hypogammaglobulinemia had a median of 5 episodes of respiratory tract infection in a year, which was significantly more than seen in their spouses. it is well established that otherwise healthy adults have annually 1 to 2 episodes of respiratory tract infection. 8 half of the 65 respiratory episodes in our patients were associated with respiratory tract viruses, showing the increased susceptibility to respiratory tract viral infections. there are no previous studies focused on respiratory tract viruses in acute or chronic respiratory tract infections in patients with hypogammaglobulinemia. we have earlier searched for respiratory tract viruses in bronchoalveolar lavage fluid and maxillary sinus lavage samples at the time when patients with hypogammaglobulinemia were free from acute infection. 9, 10 an interesting observation in this study was that patients with primary hypogammaglobulinemia had recurrent and persistent rhinoviral infections. rhinovirus was the most common pathogen in respiratory tract infections, and results for it were positive in one third of the infections. rhinovirus was found both as a sole pathogen and also together with bacteria. rhinoviruses have been shown to induce proinflammatory response in the lower airways. [11] [12] [13] chronic inflammation might predispose patients to a progression of pulmonary changes, even if the immunoglobulin replacement therapy is adequate. 14 previously, chronic rhinoviral infection with the same virus in the lower respiratory tract has been described in immunocompromised lung transplant recipients. 15 in immunocompetent adults rhinoviral clearance is usually rapid, on average 1 to 2 weeks. 7, 16 in immunocompetent children rhinoviral shedding has been shown to persist for up to 3 weeks, but prolonged shedding for months has not been documented in immunocompetent subjects. 17 consequently, continuous viral rna detection for months suggests active replication and persistent infection. in more than half of our patients, rhinoviral pcr results were positive for more than 2 months. frequent rhinoviral infections raise the question of the possible role of the absence of secretory iga in the defense against rhinoviruses. our patients also had adenoviral, metapneumoviral, and coronaviral infections. it was of interest that 5 patients had a positive adenoviral pcr result in sputum samples, and in 3 patients it was found more than once. four of the patients with recurrent adenovirus had prominent bronchiectasis. there are several studies showing that persistent or latent adenovirus might contribute to chronic respiratory diseases. 18, 19 furthermore, in 4 episodes adenovirus was found concomitantly with other viruses or bacteria. in the present study enteroviruses were found on 6 occasions in the respiratory tract, although the initial presumption was that the patients would have recurrent enteroviral infections because it is known that patients with primary immunodeficiency are unusually susceptible to enteroviral infections. chronic enteroviral encephalitis is frequently a fatal complication in patients with agammaglobulinemia. 20, 21 in a review of 90 cases of chronic enteroviral infections in patients with primary immunodeficiency, enteroviruses were usually isolated from the cerebrospinal fluid, and the major signs and symptoms were neurological. echoviruses were the most common enteroviruses. in only 5 patients were enteroviruses documented in the respiratory tract. 3 a more recent report of 201 patients with xla also showed that enteroviruses were the most common causes in central nervous system infections. 22 several vaccine-and non-vaccine-associated poliomyelitis case reports have been published, and patients with hypogammaglobulinemia have been reported to chronically excrete poliovirus. [23] [24] [25] patients with primary hypogammaglobulinemia appear to have a more severe clinical course of certain viral infections, such as hepatitis c virus, herpes simplex virus, and varicella zoster virus, than immunocompetent subjects. [26] [27] [28] wheat et al 29 studied the prevalence of human herpesvirus 8 infection in patients with cvid and found that patients with granulomatous or lymphocytic interstitial lung disease had a markedly higher prevalence of human herpesvirus 8 than patients without granulomatous/lymphocytic interstitial lung disease. these data further suggest that antiviral competence is not intact in a group of patients with primary hypogammaglobulinemia. acute respiratory tract infections were common even if patients had a trough serum igg concentration of more than 6.5 g/l. repeated antibiotic treatments were considered necessary in many patients. this is in agreement with the observations of a recent follow-up study in italy that showed that patients with primary hypogammaglobulinemia tend to have recurrent bacterial bronchitis despite replacement therapy. 30 mixed viral-bacterial coinfection was detected in one fourth of the patients at the onset of acute infection. viral-bacterial infection might induce more severe inflammation in the lower respiratory tract than sole viral or bacterial infection. 31 it has recently been shown that rhinovirus induces impairment of the antibacterial host defense and thereby predisposes the patient to coinfections with bacteria and probably with other viruses and furthermore to development of pulmonary changes. 32 the mechanisms of increased susceptibility to respiratory tract viral infections in hypogammaglobulinemic patients are unclear. in patients with xla, btk is defective. btk is a cytoplasmic nonreceptor tyrosine kinase and a member of the tec family. btk is important in toll-like receptor (tlr) 8 and tlr9 signalling. both tlr8 and tlr9 are important in the activation of host defense against viral and bacterial infections. defective triggering of tlrs leads to impaired production of proinflammatory cytokines, such as tnf-a and il-6. tlr8 recognizes single-stranded rna. tlr9 recognizes unmethylated cpg motifs that exist in both viral and bacterial dna. patients with xla have impaired il-6, which might explain their susceptibility to viral infections. 33 patients with cvid also presented with prolonged persistence of rhinovirus. impaired il-6 and il-10 production, as well as tlr9 activation defects, have been demonstrated in patients with cvid. 34 this might partly explain the persistence of rhinovirus. our study has some limitations that should be taken into account in interpreting the results. the number of patients was small. the spouses did not visit the study clinic. nasal swabs were done based on self-assessment, and their quality might not be comparable with those taken by the research nurse. in addition, we used cotton swabs, and flocked swabs might have provided a better yield. rhinoviral shedding in healthy spouses could not be properly studied because only 1 spouse had rhinoviral infection. furthermore, association of rhinoviral pcr positivity with clinical illness is not yet fully understood. in conclusion, our study shows that in addition to bacterial lower respiratory tract infections, patients with primary hypogammaglobulinemia are prone to recurrent respiratory tract viral infections. especially rhinoviral infections were frequent and prolonged. recurrent and persistent respiratory tract viral infections, often in association with bacteria, most probably predispose patients to chronic pulmonary changes. adequate immunoglobulin replacement therapy is not effective in preventing respiratory tract viral infection, and other kinds of treatments are needed. unfortunately, the availability of antivirals is still very limited. however, our observations suggest that the possibility of respiratory viruses should be investigated by means of pcr more often in patients with hypogammaglobulinemia because viral infection often explains the respiratory signs and symptoms of the patient. infections in 252 patients with common variable immunodeficiency failure to clear persistent vaccine-derived neurovirulent poliovirus infection in an immunodeficient man encephalomyelitis in primary hypogammaglobulinemia enteroviral infections in primary immunodeficiency (pid): a survey of morbidity and mortality international union of immunological societies. primary immunodeficiency diseases rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infection sputum induction as a diagnostic tool for community-acquired pneumonia in infants and young children from a high hiv prevalence area epidemiology of viral respiratory infections bacteria and viruses in maxillary sinuses of patients with primary hypogammaglobulinemia viruses and bacteria in bronchial samples from patients with primary hypogammaglobulinemia rhinovirus infects the lower airways relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection the presence of rhinovirus in lower airways of patients with bronchial asthma pulmonary abnormalities in patients with primary hypogammaglobulinemia chronic rhinoviral infection in lung transplant recipients enhanced severity of virus associated lower respiratory tract disease in asthma patients may not be associated with delayed viral clearance and increase viral load in the respiratory tract picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season latent adenoviral infection in the pathogenesis of chronic airway obstruction amplification of inflammation in emphysema and its association with latent adenoviral infection persistent and fatal central-nervous-system echo virus infections in patients with agammaglobulinemia chronic enteroviral meningoenchephalitis in agammaglobulinemic patients x-linked agammaglobulinemia: report on a united states registry of 201 patients antigenic analysis of polioviruses isolated from a child with agammaglobulinemia and paralytic poliomyelitis after sabin vaccine administration paralytic poliomyelitis in a child with agammaglobulinemia failure to clear persistent vaccine-derived neurovirulent poliovirus infection in an immunodeficient man x-linked agammaglobulinemia: an analysis of 96 patients long-term outcome of chronic hepatitis c virus infection in primary hypogammaglobulinaemia common variable immunodeficiency: clinical and immunological features of 248 patients possible role of human herpes virus 8 in the lymphoproliferative disorders in common variable immunodeficiency longterm follow-up and outcome of a large cohort of patients with common variable immunodeficiency incidence and characteristics of viral community-acquired pneumonia in adults rhinovirus exposure impairs immune responses to bacterial products in alveolar macrophages signalling by toll-like receptors 8 and 9 requires bruton's tyrosine kinase tlr9 activation is defective in common variable immunodeficiency clinical implications: respiratory tract viruses should be investigated by means of pcr more often in patients with primary hypogammaglobulinemia. bacterial and viral coinfections might predispose patients to chronic pulmonary changes. speirs ultimately gave up on championing the theoretical aspects of his interpretation, which had become contested and controversial. nonetheless, the unique images he had recorded through radioisotope labeling and electron and inventive phase microscopy remain a noteworthy contribution to the archival collections of functional cytology and histopathology. 1. speirs rs. cells involved in hypersensitivity and immunity. blood 1963;22:363-7.electron micrograph of eosinophil in close contact with vacuolated mononuclear cell. the membranes appear to be broken in places, with cytoplasm flowing from one cell into the other. a nearby eosinophil granule is undergoing change s (provided by dr r. s. speirs).electron micrograph of rosette of eosinophils attached to macrophage (provided by dr r. s. speirs). key: cord-278873-x6i5tiju authors: reddy, vidhatha; kollhoff, alexander l; murase, jenny e; martires, kathryn title: management guidelines for pregnant healthcare workers exposed to infectious dermatoses date: 2020-04-18 journal: int j womens dermatol doi: 10.1016/j.ijwd.2020.04.004 sha: doc_id: 278873 cord_uid: x6i5tiju exanthematous diseases are frequently of infectious origin, posing risks, especially for pregnant healthcare workers (hcws) who treat them. the shift from cell-mediated (th1 cytokine profile) to humoral (th2 cytokine profile) immunity during pregnancy can influence the mother’s susceptibility to infection and lead to complications for both mother and fetus. the potential for vertical transmission must be considered when evaluating the risks for pregnant hcws treating infected patients, as fetal infection can often have devastating consequences. given the high proportion of women of childbearing age among hcws, the pregnancy-related risks of infectious exposure are an important topic in both patient care and occupational health. contagious patients with cutaneous manifestations often present to dermatology or pediatric clinics, where female providers are particularly prevalent, as a growing number of these physicians are female. unfortunately, the risks of infection for pregnant hcws are not well defined. to our knowledge, there is limited guidance on safe practices for pregnant hcws who encounter infectious dermatologic diseases. in this article, we review several infectious exanthems, their transmissibility to pregnant women, the likelihood of vertical transmission, and the potential consequences of infection for the mother and the fetus. additionally, we discuss recommendations with respect to avoidance, contact and respiratory precautions, and the need for treatment following exposure. hcws. this review identifies various infectious exanthems that pregnant hcws may be exposed 30 to and summarizes current available evidence regarding risk of transmission. specifically, we 31 discuss parvovirus b19, hand, foot and mouth disease, mycoplasma-induced rash and mucositis, 32 measles, herpes simplex virus, varicella-zoster virus, and pityriasis rosea. we also provide 33 guidelines for each disease in order for pregnant hcws and hcws of reproductive potential to 34 appropriately minimize risk and pursue work-up and treatment, if necessary, following exposure. 35 finally, given the ongoing global coronavirus disease 2019 (covid-19) pandemic caused by 36 sars-cov-2 (severe acute respiratory syndrome coronavirus 2), we also summarize available 37 safety guidelines for pregnant hcws who are working during this time. 59 data on the risk of transmission to hcws, however, is conflicting (adler et al., 1993) . following exposure to infected patients. one single-center study at children's hospital of 63 philadelphia found elevated risk of infection between exposed and unexposed staff (bell et al., 64 1998 ). however, a cohort study of 87 hcws exposed to two patients with parvovirus b19-65 induced aplastic crisis found no significant increase in parvovirus b19-specific immunoglobulin 66 m (igm) and immunoglobulin g (igg) antibodies when compared with unexposed health care 67 workers in the same facility (ray et al., 1997) . while the risk of transmission to hcws has not been definitively identified, preventing 69 the transmission of pvb19 infection is important as it can lead to adverse pregnancy outcomes. pvb19 infection carries a 9% excess risk of miscarriage within the first 20 weeks of gestation, 71 and 2.9% risk of fetal hydrops between weeks 9 and 20 (miller et al., 1998 vesicular rash affecting the hands, feet, and oral mucosa. as one of the most common pediatric 91 exanthems, hfmd is routinely seen by pediatric and dermatology hcws, presenting a potential 92 concern for occupational exposure. all hcws who are exposed to vzv should be monitored daily during days 8-21 after the etiology of pr is unclear. various clinical and epidemiological features of pr 318 support a viral origin, including its self-limiting course, low recurrence rate, occasional 319 household clustering, possible seasonal variation, prodromal symptoms, response to acyclovir, 320 and higher prevalence during states of impaired immunity (e.g., pregnancy). there is a well-321 established association between pr and human herpesviruses 6 and 7 (hhv-6/7) (drago et al., since pr is self-limiting, management is generally limited to reassurance and the 360 treatment of symptoms with emollients, antihistamines, and occasionally topical steroids. when caring for patients with pr. although pr is not thought to be contagious, and women of 374 childbearing age have likely already been exposed to hhv-6/7, this recommendation is based 375 upon the uncertainty surrounding its etiology and the potential danger of infection to the fetus. both pregnant hcws and patients have expressed concern regarding potential complications 387 from covid-19, although the disease appears to disproportionately affect men compared to the cdc's recommendations for the vaccination of hcws are summarized in table 3 . parvovirus b19 infections among school and hospital employees during endemic periods pityriasis rosea-like eruption associated with ondansetron use in 453 pregnancy measles in pregnancy: maternal morbidity and perinatal outcome immunization in special clinical circumstances red 460 book: 2012 report of the committee on infectious diseases varicella-zoster infections red book: 2012 report of the committee on infectious diseases notes from the field: outbreak of hand, foot, and mouth disease caused by 468 coxsackievirus a6 among basic military trainees -texas pregnancy outcome following rubella 471 vaccination: a prospective controlled study human parvo virus b19 474 infection among hospital staff members after contact with infected patients guideline for 479 infection control in healthcare personnel additional evidence that pityriasis rosea is associated with 482 reactivation of human herpesvirus-6 and -7 the acquisition of herpes simplex virus during pregnancy mouth disease caused by coxsackievirus a6--minnesota mycoplasma pneumoniae-induced rash and 489 mucositis as a syndrome distinct from stevens-johnson syndrome and erythema multiforme: a systematic 490 review early developmental outcomes of children with congenital 492 hhv-6 infection infections in pregnant women. the journal of infectious diseases interim infection prevention and control recommendations 496 for measles in healthcare settings transmission and clinical features of enterovirus 71 infections in 500 household contacts in taiwan human parvovirus b19 infection in healthcare workers placental massive perivillous fibrinoid deposition associated with 584 report of a case, and review of the literature atypical hand, foot, and mouth disease caused by 587 parvovirus b19: a review immunization of health-care personnel: recommendations of the advisory committee on immunization 591 practices (acip) association of covid-19 infection with pregnancy outcomes in 594 healthcare workers and general women. clinical microbiology and infection management of an obstetric health care provider with acute 597 parvovirus b19 infection implementation of hospital policy for healthcare workers and 600 patients exposed to varicella-zoster virus hand-foot-and-mouth disease caused by coxsackievirus a6 on the 603 rise a case-controlled study comparing clinical course and outcomes of 606 pregnant and non-pregnant women with severe acute respiratory syndrome. bjog: an international 607 parvovirus b19 infection in human pregnancy outbreak of parvovirus b19 infection among anesthesiology and surgical fellows association of state laws healthcare workers' influenza vaccination rates clinical manifestations and outcome of sars-cov-2 infection during 618 pregnancy pityriasis rosea: an update on etiopathogenesis and 620 management of difficult aspects updated recommendations for use of varizig -united states a report of three cases and review of intrauterine herpes 625 simplex virus infection a case of macrolide-refractory 627 mycoplasma pneumoniae pneumonia in pregnancy treated with garenoxacin centers for disease control and prevention. 630 prevention of measles, rubella, congenital rubella syndrome, and mumps immediate and long term outcome of human 634 parvovirus b19 infection in pregnancy epidemiology, outcome and control of varicella-zoster infection gestational pityriasis rosea: suggestions for 639 approaching affected pregnant women 208-guidelines for the management of herpes simplex virus in pregnancy. 641 journal of obstetrics and gynaecology canada hand, foot, and mouth disease in an adult torch infections notes from the field: severe hand, foot, and mouth disease associated with coxsackievirus maternal, fetal, and neonatal outcomes associated with measles during 654 pregnancy: namibia spontaneous abortion after hand-foot-and-mouth disease caused by 657 coxsackie virus a16 pityriasis rosea in pregnancy: a case report outcome after maternal varicella infection in the first 20 weeks 661 of pregnancy virus infections in neonates exposed to the virus at the time of vaginal delivery to mothers with 664 recurrent genital herpes simplex virus infections coxsackievirus a6 associated hand, foot and mouth disease in adults: clinical presentation and review of 668 the literature coronavirus disease 2019 (covid-19) 670 and pregnancy: what obstetricians need to know nosocomial 673 exposure to parvovirus b19: low risk of transmission to healthcare workers increased risk of 677 human parvovirus b19 infection in day-care employees: a cohort study among pregnant workers during an 678 epidemic in finland covid-19: doctors in final trimester of pregnancy should avoid direct patient contact pregnancy and susceptibility to infectious diseases. infectious 682 diseases in the congenital varicella syndrome an analysis of 38 pregnant women with covid-19, their newborn infants, and maternal-685 fetal transmission of sars-cov-2: maternal coronavirus infections and pregnancy outcomes clinicopathologic analysis of 688 atypical hand, foot, and mouth disease in adult patients clinical presentations of parvovirus b19 infection intrauterine viral infections 3 immunization of health-care personnel: recommendations of the 695 advisory committee on immunization practices (acip) guideline for isolation precautions: preventing 697 transmission of infectious agents in health care settings low birth weight and maternal virus diseases: a prospective study of rubella, 700 measles, mumps, chickenpox, and hepatitis congenital malformations following chickenpox, measles, mumps, and hepatitis. results of a 703 cohort study varicella in the fetus and newborn. seminars in fetal and neonatal medicine hand-foot-mouth disease type-specific antibodies to herpes simplex virus type 2 710 (hsv-2) glycoprotein g in pregnant women, infants exposed to maternal hsv-2 infection at delivery, and 711 infants with neonatal herpes human parvovirus b19 nosocomial outbreak in healthcare 715 personnel in a paediatric ward at a national tertiary referral centre in thailand risk factors for parvovirus b19 infection in pregnancy adverse pregnancy and neonatal outcomes associated with 722 ureaplasma urealyticum and u. parvum: a 723 systematic review and meta-analysis protocol diagnosis and management of pityriasis rosea update on hand-foot-and-mouth disease mandatory influenza vaccination for all healthcare personnel: a review on 731 justification, implementation and effectiveness. current opinion in pediatrics pityriasis rosea is associated with systemic active infection with 734 both human herpesvirus-7 and human herpesvirus-6 varicella vaccine exposure during pregnancy: data from 10 years of 736 the pregnancy registry pregnancy and perinatal outcomes of women with severe acute 738 respiratory syndrome world health organization. varicella and herpes zoster vaccines: who position paper disseminated herpesvirus infection during pregnancy maternal outcomes in pregnancies affected by varicella zoster key: cord-309478-yhmgopmr authors: jin, ying-hui; huang, qiao; wang, yun-yun; zeng, xian-tao; luo, li-sha; pan, zhen-yu; yuan, yu-feng; chen, zhi-min; cheng, zhen-shun; huang, xing; wang, na; li, bing-hui; zi, hao; zhao, ming-juan; ma, lin-lu; deng, tong; wang, ying; wang, xing-huan title: perceived infection transmission routes, infection control practices, psychosocial changes, and management of covid-19 infected healthcare workers in a tertiary acute care hospital in wuhan: a cross-sectional survey date: 2020-05-11 journal: mil med res doi: 10.1186/s40779-020-00254-8 sha: doc_id: 309478 cord_uid: yhmgopmr background: many healthcare workers were infected by coronavirus disease 2019 (covid-19) early in the epidemic posing a big challenge for epidemic control. hence, this study aims to explore perceived infection routes, influencing factors, psychosocial changes, and management procedures for covid-19 infected healthcare workers. methods: this is a cross-sectional, single hospital-based study. we recruited all 105 confirmed covid-19 healthcare workers in the zhongnan hospital of wuhan university from february 15 to 29, 2020. all participants completed a validated questionnaire. electronic consent was obtained from all participants. perceived causes of infection, infection prevention, control knowledge and behaviour, psychological changes, symptoms and treatment were measured. results: finally, 103 professional staff with covid-19 finished the questionnaire and was included (response rate: 98.1%). of them, 87 cases (84.5%) thought they were infected in working environment in hospital, one (1.0%) thought their infection was due to the laboratory environment, and 5 (4.9%) thought they were infected in daily life or community environment. swab of throat collection and physical examination were the procedures perceived as most likely causing their infection by nurses and doctors respectively. forty-three (41.8%) thought their infection was related to protective equipment, utilization of common equipment (masks and gloves). the top three first symptoms displayed before diagnosis were fever (41.8%), lethargy (33.0%) and muscle aches (30.1%). after diagnosis, 88.3% staff experienced psychological stress or emotional changes during their isolation period, only 11.7% had almost no emotional changes. arbidol (umifenovir; an anti-influza drug; 69.2%) was the drug most commonly used to target infection in mild and moderate symptoms. conclusion: the main perceived mode of transmission was not maintaining protection when working at a close distance and having intimate contact with infected cases. positive psychological intervention is necessary. infection in medical institutions happens easily when a new epidemic occurs. according to who daily situation report, after the coronavirus disease 2019 (covid19) outbreak, 22,073 covid-19 cases among healthcare workers have been reported to the who as of wednesday, 8 april 2020 [1] . the number of healthcare workers infected by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) reached 1716 china wide on february 11, 2020; among them, 1502 cases were in hubei province; with 1102 in wuhan [2] . as of early march, the infected number increased to 3300 and at least 22 died in china, and there were over 2600 infected with 13 died in italy as of 20 march, 2020 [3] [4] [5] . reports from medical staff describe physical and mental exhaustion, the torment of difficult triage decisions, and the pain of losing their patients and colleagues, all in addition to the infectious risk. healthcare workers are the pillar for fighting against this pandemic; hence, preventing them to be infected is a big challenge for maintaining a strong fighting force, high morale, and energy for fighting. now to protect healthcare workers away from infection has aroused great concern, many experts published opinions [3, 4, [6] [7] [8] [9] [10] to appeal to worldwide for paying attention to protect health care workers from woefully unprepared status. on 2 march 2020, lai et al. [11] reported mental health of 1257 healthcare workers treating patients exposed to covid-19 in 34 hospitals in china. results showed that they experienced psychological burden, especially nurses, women, those in wuhan, and frontline healthcare workers directly engaged in the diagnosis, treatment, and care for patients with covid-19. another qualitative study released 18 february 2020 suggested that maintains healthcare workers' mental health is essential to better control infectious diseases [10] . obviously, there is no study concerned about the infected healthcare workers with covid-19 and their mental health. therefore, we conducted this epidemiological investigation and aimed to explore the route of sars-cov-2 infection, influence, and management procedures of healthcare workers. we also believe our work will provide factual evidence and lessons learned can be used to prevent such infections in the future. this is a cross-sectional and single center study, using data of all 105 infectious healthcare staff from zhongnan hospital of wuhan university. 105 healthcare staff became infected before january 30, 2020, our study period was from february 15 to 29, 2020; and no new staff became infected until now. this study was reviewed and approved by the committee for ethical affairs of this hospital. all 105 healthcare professionals who were recorded in the division of medical affairs of confirmed covid-19 diagnosis were invited to finish the questionnaire. confirmation diagnosis was according to the diagnostic criteria of the national health committee of the people's republic of china (cnhc) [12] . we divided the departments represented in this study into two groups: high risk of nosocomial infection departments (hrds) and low risk of nosocomial infection departments (lrds) according to general principles for the management of nosocomial infection control of cnhc [13] and infection control expert opinion from this project group. hrds included departments of respiratory medicine, infectious diseases, emergency, clinical laboratory, anesthesia surgery, operating room, and intensive care unit; all other departments were categorized as lrds (fig. 1a) . all staffs were working in their normal departments and no one had been redeployed to other areas at the time of the survey. we employed a self-administered electronic tool to collect data using our validated questionnaire which included informed consent (see additional file 1). after readability and content validity test, the testretest reliability coefficient of our electronically administered questionnaire was 0.82 (1 = perfect repeatability) [14] . we also cross-checked information was from other sources. epidemiologic data were confirmed through phone calls to all participants, and we checked electronic patient records for collecting treatment information. additionally, our investigators also held tele-interviews with the department directors of infected ones where necessary, to determine exposure status of all staff in the week before sars-cov-2 infection. we conceptualized a research framework initially based on the following: 1) online interview or a free response set of questions to explore infection prevention and control experiences of covid-19 among frontline clinicians from the office of nosocomial infection control. 2) policy documents, guidelines or consensus for this epidemic or general prevention of infectious disease, such as notice on the issuance of a program for prevention and control of sars-cov-2 infection from the cnhc. samples presented were all from zhongnan hospital of wuhan university. zhongnan hospital of wuhan university is a 3500 bedded tertiary acute teaching hospital which has more than 800 doctors and 1800 nurses. this hospital is equipped with 46 clinical and medical technology departments, 17 teaching and research departments and 1 laboratory. zhongnan hospital of wuhan university is one of the key hospitals at the epicentre of this outbreak, and had treated 952 hospitalized patients with covid-19 between january 1 and march 12. samples selection in this study was not limited by departments of professional staff. we contacted all infected professional staff according to the hospital's records (n = 105). electronic questionnaires were sent to all infected staff member and send a message by mobile phone to reminder, and they were free to choose whether take part in or not; if unwilling, the can click "do not agree to participate in this research" bottom (see additional file 1). categorical variables were described as frequencies and percentages, continuous variables were showed as mean ± standard deviation (sd) or median (25-75% percentile) as appropriate. to assess the changes before and after covid-19 outbreak, mcnemar tests were performed for the change of training level, wilcoxon signed rank sum tests were conducted for the mastery level for knowledge of protective procedures which were measured using a likert scale. all analyses were carried out using the sas software (version 9.4 ts1m6, sas institute inc., cary, nc, us). twosided p values less than 0.05 were considered statistically significant. the data were visualized by microsoft 2019. all 105 infected medical staff with covid-19 in our hospital was invited, finally 103 cases agreed with the consent and finished our questionnaire was included for analysis (response rate: 98.1%), table 1 presented their basic characteristics. of them, 71 worked in lrds and 32 in hrds (fig. 1a) , the median age was 35.0 years, 41 were doctors (39.8%), 55 were nurses (53.4%), and 7 were medical technicians (6.8%). 39 (37.9%) were males and 64 (62.1%) were females. figure 1b presented the perceived routes of infection. among the 103 responders, 87 (84.5%) cases thought they were infected by the working environment in hospital, one (1.0%) thought infection was due to the laboratory environment with biological specimens of suspected or confirmed patients, and 5 (4.9%) thought they were infected in daily life or community environment. of the 87 staff who thought they were infected by the working environment in hospital found, 64 (73.6%) had close contact with confirmed patients, 15 (17.2%) had close contact with suspected patients, and 36 (41.4%) were exposed to their confirmed colleagues (fig. 1c) . among 103 infected staff, 46 (44.7%) had worked more than seven hours a day in an environment that posed a risk of them becoming infected (fig. 1d ). the top three perceived infection routes were: droplet transmission, contact transmission, and aerosol transmission (fig. 1e ). for nurses, the top three perceived infection causing procedures were sputum suction care, basic nursing, and pharyngeal swab collection (swab of throat) (fig. 1f ). for doctors, the top three procedures were physical examination, tracheal intubation, and manual ventilation before intubation (fig. 1g ). forty-three staff perceived protective equipment problems as the cause of their infection. of them, 44.2% believed infection was caused by inadequate provision of protective equipment, and another 32.6% considered it was due to insufficient protection provided by the personal protective equipment (ppe) they had (wearing only surgical mask to contact confirmed cases). use of protective equipment before covid-19 outbreak table 2 shows healthcare staff's retrospective account of the level of use protective equipment in their routine work before this outbreak. 77.7% staffs always strictly followed hand hygiene, and 53.4% always strictly followed the procedure for wearing and removing of protective equipment. among 103 staff, 66.0% always wore masks and 51.5% wore gloves in their routine work. the utilization of more sophisticated protective equipment in routine work was no higher than that of common protective equipment. we found there were table 2 also demonstrated the use of protective equipment in lrds and hrds. in routine work, utilization of masks (96.9% vs. 52.1%) and gloves (78.1% vs. 39.4%), strict hand hygiene procedure (81.3% vs. 76.1%), and wearing and removing protective equipment (84.4% vs. 39.4%) in hrds were much higher than those in lrds. for more sophisticated protective equipment, the utilization in routine work in hrds was also much higher than those in lrds. figure 2a and b presented types, layers, and length of masks and gloves usage by all participants (frequency), lrds and hrds (percentage). in their routine work, medical surgical masks were more often used than other types, and staffs in lrds were more likely to use medical and disposable surgical masks. however, those in hrds tended to use kn95 and n95 respirators. medical rubber examination gloves were used frequently. single layer and one-time use of both mask and glove were the main choices. of the 103 responders, symptoms were fever (48.5%), lethargy (42.7%), muscle aches (35.9%), and dry cough (34.0%) with a few of them having gastrointestinal symptoms (such as diarrhea, nausea and vomiting). in addition, the top three initial symptoms displayed before diagnosis were fever (41.8%), lethargy (33.0%), and muscle aches (30.1%), see fig. 3a . as shown in fig. 3b , among the infected cases who knew their results, 39.5% had decreased lymphocytes and 24.1% reported decreased white blood cell, but 31.8% reported increased c-reactive protein. platelet, alanine aminotransferase, aspartate aminotransferase, creatine kinase, and d-dimer remained almost normal. a total of 102 (99.1%) staff had ct chest examination, of which 82 were abnormal. all the 103 (100%) infected ones had nucleic acid test and 82.0% had at least one positive result or at least one suspected positive result; however, 18.0% of them had negative results. when confirmed as covid-19, all the 103 cases were divided into 2 classes, of which 98 cases presented with mild/moderate clinical stages and 5 were severe clinical stages, we had on one presenting as a critical stage. in mild/moderate cases, 12 of them received oxygen therapy, 86 received antiviral therapy, 23 received traditional chinese medicine (tcm) treatment, three developed into severe or critical stages after admission, but none were transferred to icu before the study closed. among the 86 staff receiving antiviral treatment, the most common antiviral drug was oseltamivir; some also received interferon, lopinavir / ritonavir and ribavirin, but none received peramivir. the majority of them received arbidol (umifenovir; an anti-influza drug used in china; 69.2%), followed by immunoglobulin (19.2%) and vitamin c (16.4%). all eight severe/critical stage cases received hormone and antibiotic treatment, three received interferon, three received antiviral therapy, one received noninvasive mechanical ventilation, and none of them received tcm treatment, extracorporeal membrane oxygenation (ecmo), or endotracheal intubation. all of 103 cases were isolated immediately after diagnosis: 47 were in the designated hospital, 38 at home, six at centralized isolation points, and 12 in other places (hotel, infection department in hospital and a mixture). of the 38 who isolated at home, 76.3% wore a mask at home, 89.5% did not go out during the isolation period, and 71.1% changed their masks in less than 8 h, 68.4% of them lived alone during home isolation, most took their temperature regularly at home, washed and disinfected their hands frequently, and had independent eating utensils. of the 65 who were not isolated at home, 95.4% received medical treatment immediately after diagnosis, and 98.5% were not accompanied by their family members during hospital isolation; 96.9% of them thought their quarantine location was disinfected daily, and 98.5% were satisfied with the isolation environment. prevention and control knowledge training before and after the outbreak before the outbreak of the 103 responders, 78 knew the covid-19 transmission route; and knew most of prevention and control information of covid-19 (with a correct rate > 90%), whereas the accuracy rate for "the window should be closed tightly in the general ward" (usually more than one patients in one sickroom in china) was only 57.3% (table 3) . compared with before outbreak, there was a focus on "wearing protective clothing", "wearing goggles or face shield", "isolation of suspected infectious patients", and "wearing isolation clothes". the training in these areas was significantly intensified (p < 0.05). conversely the training intensity of "hand hygiene" (98.1% before vs. 92.2% after) and "wearing gloves" (96.1% before vs. 90.3% after) was significantly decreased somewhat after outbreak. (table 4) . compared with before outbreak, the level of awareness of information related to "isolation of suspected infectious patients", "environmental cleaning and disinfection", "wearing goggles or face shield", "wearing protective clothing", and "wearing isolation clothes" has been significantly increased since the outbreak (p < 0.001), while the high level of awareness of "hand hygiene" and "wearing gloves" were only slightly enhanced (table 5) . results of these staffs' opinions regarding improvements needed for fighting nosocomial infection showed that: 84.5% chose "medical staff protection", followed by "emergency plan and work flow" (68.0%) and "pay attention to the health of medical staff" (66.0%); more than half believed further attention was needed in "infection outbreak management", figure 4 presents their psychological status. before they were diagnosed with infection, 49.5% said that they were fully aware of the seriousness of the situation, 64.1% remained neutral, 31.1% were anxious, 20.4% maintained an optimistic attitude, and only a few were fearful or pessimistic. after diagnosis, 88.3% staff experienced psychological stress or emotional changes during their isolation period, only 11.7% had almost no emotional changes. of the 88.3% ones, their psychological stress or emotional fortunately, they all took effective measures to control their emotions or stress, 75.8% staffs had actively expressed their psychological stress. the ways they used include self-regulation, communicating with others on wechat, and video call with family or colleagues. protecting medical professionals from infection is crucial to success in fighting covid-19. however, cluster outbreaks occurred in many departments in this study, for 9 departments had more than five cases. more than half of the infected staff had close contact with confirmed or suspected patients in their department or work environment before they knew the true status of sars-cov-2. during early january 2020, patients having fever and coughs were scatted throughout many hospitals across wuhan, just when influenza and common pneumonia rates are high and they were all treated in the same way. at that time most medical staffs usually used ordinary medical masks without any other medical equipment during these patients' diagnosis and treatment [15] . reports showed human-to-human transmission of sars-cov-2 was confirmed by the infection of 15 healthcare professionals after close contact with infected patients in a wuhan hospital [16] . for nearly a month, neither the public nor healthcare professionals were clear about all the characteristics of covid-19. during that time many medical staffs were not taking enhanced personal protective measures. our results were confirmed repeatedly by cross-checking information from the departments' directors of infected hospital workers and participants. lack of effective protective measures during the early stage is an important risk factor, and the main reason for transmission was failure to protect people working at a close distance and having intimate contact with infected patients. the prevention and control plan before diagnosis can help to explain this. only a fifth of infected staffs were routinely equipped with protective face shields, clothing or shoe covers. most of them just wore the usual protective equipment. in addition, improper use of ppe (e.g., repeated use of masks) is also a factor involved in increasing the infection risk [4] . with the continuous occurrence of confirmed infections during january 2020, the hospitals and government of wuhan introduced strategies and policies to improve the management of patients and protection of medical staff. the personal protection awareness of medical staff increased rapidly and the number of confirmed cases decreased rapidly during february 2020. we conducted an in-depth telephone interviews for cases confirmed in february 2020. for infected healthcare professionals beyond the concentrated outbreak period in january 2020, we cannot rule out that they may have experienced a longer incubation period before they were diagnosed as covid-19. in addition, some confirmed cases had worked in the frontline for a long time maybe leading to a lowered resistance to infection. the interview information showed that the last case we included is a frontline nurse who had been engaged in combating covid-19; she claimed that she was fully equipped by uniform standards at work, and she had frequently conducted cpr for confirmed patients before her own infection was confirmed. she believed that she was infected by confirmed patients, but was not sure about exactly when or which patient was involved. moreover, the first group of infected medical staff also forms part of the chain of transmission of nosocomial infections. 39 cases in our study suggested that their route of infection may be "close contact with colleagues who were subsequently", but they were infected during the early stage of epidemic. generally, we believe that physical examination poses a relatively lower infection risk than tracheal intubation or tracheotomy, but physical examination was selected by the highest proportion of infected doctors. in our study, nine infected doctors in the hepatopancreatobiliary surgery department, which is a lrd for nosocomial infection, reported that physical examination is the most likely cause of their infection. from tracking their questionnaire data we found that inadequate protective equipment and insufficient protection of protective equipment were frequently selected. the percentage of staff selecting "personal protective equipment used more often" was higher in hrds than lrds in routine work. this is maybe the reason why more infected cases appeared in lrds. however, we can only hypothesize why this occurred, because normally people don't use sophisticated infection control procedures routinely in lrds and to do so would be poor use of resources and also frightening for the public. as the above emphasizes, the main mode of transmission was failure to maintain protection when working at a close distance and having intimate contact with infected cases. for epidemics, like covid-19, protection for low exposure risk procedures should be upgraded, and medical protective masks, protective face shield/ screens and protective clothing are also needed especially in the emergency or infectious diseases department or when caring for high risk patients [12] . training in knowledge and skills for prevention and control covid-19 for healthcare providers is important [10] , especially as professor chen wang believes that sars-cov-2 may turn into a chronic disease and coexist with humans like the flu [17] . therefore, in order to cope with the long-term existence of sars-cov-2 and possible public health emergencies in future, health care workers should receive annual training on the use of personal ppe and additional education during surge events [4] . the learning and dissemination of epidemic prevention knowledge and skills should be increased not only for medical areas but for all the public. the areas of personal protection, emergency planning and work flow should be improved. shortages of ppe that intensify fears of coronavirus exposure at work contribute to the psychological distress or other illness. stable and adequate ppe supply is highly recommended even the time without pandemic. administrators in hospital mustensure that their staff feel safe, respected, prepared, and supported [4] . in addition, basic rules should be stressed, such as emphasizing hand hygiene, installing barriers to limit contact with patients at triage, prioritizing respirators for aerosol generating procedures and self-monitoring for signs of illness and self-isolating and reporting illness to managers, if it occurs [18, 19] . to sustain and restore frontline health care workers, health care organizations need to monitor the mental health outcomes of healthcare personals over time and prioritize the mental and physical health needs and recovery of first-line healthcare professionals [20] . understanding how workers are exposed to the virus will be essential to protect frontline healthcare staff. but, little knowledge is known about the presence of the virus contamination on surfaces in hospitals and concentration of virus in the air. this lack of evidence means we are using a precautionary approach which often results in our applying all available controls all the time. it also means that resources are used unnecessarily, possibly leading to shortages for workers in particular e. willingness to express psychological stress or emotional changes; f. methods to regulate their stress or mood changes; a-b, psychological status before diagnosis; c-f, psychological status after diagnosis environments where they could provide protection [21] . research about better ways of protecting workers from the virus is still needed. the major limitation of this study is estimating the exact reason of infection for some cases is not an easy thing, and some memory biases maybe exist among participants. participants usually pondered over the causes of infection. they could overstate the protection measures they were using before their diagnosis because they are reluctant or unwilling to believe that this is how they got infected. effective protective measures were generally lacking in hospitals in the early stage of covid-19. the main factor affecting transmission was not using protecting equipment when working at a close distance and having intimate contact with infected persons. most staff experienced psychological stress or emotional changes during their isolation period a after diagnosed. protective equipment should be upgraded in hospital at the onset of a new disease especially for staff conducting procedures involving close contact and caring for high risk patients. also, learning from these lessons is expected to help the chinese government and other parts of the world to better respond to future unexpected infectious disease outbreaks. supplementary information accompanies this paper at https://doi.org/10. 1186/s40779-020-00254-8. over 22,000 healthcare workers infected by covid-19: who xi: protection, care for health workers a priority covid-19: protecting health-care workers protecting health care workers against covid-19-and being prepared for future pandemics li wenliang, a face to the frontline healthcare worker. the first doctor to notify the emergence of the sars-cov-2, (covid-19), outbreak protecting health care workers during the covid-19 coronavirus outbreak -lessons from taiwan's sars response how to train the health personnel for protecting themselves from novel coronavirus (covid-19) infection during their patient or suspected case care managing mental health challenges faced by healthcare workers during covid-19 pandemic covid-19 and the risk to health care workers: a case report mental health care for medical staff in china during the covid-19 outbreak factors associated with mental health outcomes among health care workers exposed to coronavirus disease 2019 national health commission of the people's republic of china. accreditation regulation of control and prevention of healthcare associated infection in hospital development and preliminary validation of questionnaire for infection process and prevention of 2019 novel coronavirus infection in medical staffs reflections on the present response to the pneumonia associated with a novel coronavirus (2019-ncov) emergence of sars-like coronavirus poses new challenge in china covid-19) outbreak: rights,roles and responsibilities of health workers, including key considerations for occupational safety and health interim infection prevention and control recommendations for patients with suspected or confirmed coronavirus disease 2019 (covid-19) in healthcare settings mental health needs of health care workers providing frontline covid-19 care protecting workers from covid-19 we extend special thanks for the contribution of consultant experts and healthcare practitioners from all hospitals for their participation.authors' contributions xhw, xtz had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis; yhj, xtz designed this study; yhj, qh, lsl, bhl hz, td and yw collected all questionnaire data and conducted qualitative research; qh, zyp, yfy, zmc, xh and zsc analysed and interpreted data of this study; yhj, qh, lsl and yyw drafted the manuscript; xhw, yfy, nw, mjz and llm made critical revision of the article for important intellectual content; xhw got the funding for this study; xhw, xtz and yw gave administrative, technical or logistic support for conducting this study; all authors agreed with the results and conclusions of this article. all authors read and approved the final manuscript. this work was supported by the emergency science and technology project in hubei province (2020fca008). the authors had complete control over the design, conduct, and analysis of the study and the decision to submit the manuscript for publication. the datasets used and/or analyzed during the current study are available from the corresponding author on request.ethics approval and consent to participate written informed consent was obtained from each participant (see additional file 1). this study was reviewed and approved by the committee for ethical affairs of zhongnan hospital of wuhan university. not applicable. the authors declare that they have no competing interests. key: cord-269095-lwank6hk authors: jirru, ermias; lee, stefi; harris, rebecca; yang, jianjun; cho, soo jung; stout-delgado, heather title: impact of influenza on pneumococcal vaccine effectiveness during streptococcus pneumoniae infection in aged murine lung date: 2020-06-11 journal: vaccines (basel) doi: 10.3390/vaccines8020298 sha: doc_id: 269095 cord_uid: lwank6hk changes in innate and adaptive immune responses caused by viral imprinting can have a significant direct or indirect influence on secondary infections and vaccine responses. the purpose of our current study was to investigate the role of immune imprinting by influenza on pneumococcal vaccine effectiveness during streptococcus pneumoniae infection in the aged murine lung. aged adult (18 months) mice were vaccinated with the pneumococcal polyvalent vaccine pneumovax (5 mg/mouse). fourteen days post vaccination, mice were instilled with pbs or influenza a/pr8/34 virus (3.5 × 10(2) pfu). control and influenza-infected mice were instilled with pbs or s. pneumoniae (1 × 10(3) cfu, atcc 6303) on day 7 of infection and antibacterial immune responses were assessed in the lung. our results illustrate that, in response to a primary influenza infection, there was diminished bacterial clearance and heightened production of pro-inflammatory cytokines, such as il6 and il1β. vaccination with pneumovax decreased pro-inflammatory cytokine production by modulating nfҡb expression; however, these responses were significantly diminished after influenza infection. taken together, the data in our current study illustrate that immune imprinting by influenza diminishes pneumococcal vaccine efficacy and, thereby, may contribute to increased susceptibility of older persons to a secondary infection with s. pneumoniae. people worldwide are living longer, and it is estimated that, by 2050, the proportion of the world's population over 60 years of age will nearly double. natural lung aging is associated with molecular and physiological changes that cause alterations in lung function, diminished pulmonary remodeling and regenerative capacity, and increased susceptibility to acute and chronic lung diseases. in addition, diminished immune function and age-associated changes in primary vaccine responses contribute to weakened long-lasting antibody responses to pathogenic stimuli. as the aging population rapidly grows, it is essential to examine how alterations in cellular function and cell-to-cell interactions of pulmonary resident cells and systemic immune cells contribute to a higher risk of increased susceptibility to infection. influenza epidemics still remain a leading cause of morbidity and mortality worldwide, with the highest incidence of hospitalization and death occurring in persons >65 years of age. age-associated alterations in immune surveillance-specifically, decreased innate and adaptive immune responses and dysfunctional immune regulation-can be responsible for increased lung pathology to infectious stimuli [1] [2] [3] [4] [5] [6] . a primary influenza infection, through virally imprinting on immune responsiveness, can have a significant direct or indirect influence on secondary infections and vaccine efficacy [7, 8] . bacteria, especially streptococcus pneumoniae, are the most common pathogens that commonly cause pneumonia in the elderly [2] . upregulation of epithelial cell surface receptors in response to chronic inflammation can increase bacterial adhesion and accumulation in the aged lung and correlate with an age-associated increased in host susceptibility to pneumonia [2, 9] . depressed clearance mechanisms, such as cough, oral and mucociliary clearance, and swallowing disorders, can also increase susceptibility [10] [11] [12] [13] . immune imprinting by a viral infection has the potential to influence host responsiveness to subsequent infections. many studies have illustrated the role of a primary influenza infection in modulating host responses to a subsequent strain of influenza [7, 8, [14] [15] [16] [17] [18] . it has been suggested that negative antigenic interaction, or pre-immunity from a person's first influenza strain, due to changes in the memory immune responses, may have deleterious effects on clinical outcomes during a secondary infection or host vaccine memory responsiveness [19] . the timing between influenza infection and post-viral pneumococcal infection as well as the dose of bacteria administered can greatly impact the host outcomes of a secondary infection [17, [20] [21] [22] [23] . specifically, the initiation of influenza-virus-mediated lung inflammation has been shown to directly correlate with bacterial growth, and high dosages of bacteria have been associated with increased mortality during secondary infection [17, [20] [21] [22] [23] [24] . previous work has illustrated that, as early as day 3 post influenza, bacterial clearance from the lung was significantly altered [25] . progression of influenza and viral-mediated changes in the immune response contribute to increased virus-mediated damage to the lung and resulted in greater susceptibility to a secondary pneumococcal infection [18, 24, [26] [27] [28] [29] . further, recent work has demonstrated that, in the absence of influenza, young adult mice immunized with pneumococcal-specific vaccines are highly efficient in pulmonary clearance of the bacteria [30, 31] . in the presence of influenza, vaccine protection was dramatically reduced [17, 30, 31] . vaccine variations, such as immunization with pneumococcal surface protein a (pspa), engineered live-attenuated vaccines, and dual vaccine/cytokine administration, have been shown to fully protect influenza-infected mice against a physiologically relevant dose of s. pneumoniae [31] [32] [33] . taken together, the cascade of innate and adaptive immune responses to an immune imprinting event can greatly impact host susceptibility to secondary infection [7] . for adults >65 years of age, the 23-valent pneumococcal polysaccharide vaccine (ppv23) pneumovax and the 13-valent pneumococcal conjugate vaccine (pcv13) prevnar are two vaccines available for protection against pneumococcal infections. while there have been conflicts in pneumococcal vaccine effectiveness, a recent meta-analysis illustrated that pneumovax exhibited a weak protective effect on all-cause pneumonia among immunocompetent adults and persons over 65 years of age as well as high-risk persons (19-64 years of age) [34] . while multiple studies have illustrated that vaccination in persons >60 years with prevnar can result in improved immunogenicity against multiple s. pneumoniae serotypes, these antibody titers were found to decline after a year and were similar to titers observed post pneumovax vaccination [35] [36] [37] [38] . in addition, combined administration of prevnar prior to pneumovax can elicit a greater immune response than multiple dosages of prevnar, which only demonstrated a modest increase [37, 39] . as recent work has illustrated differential efficacy of prevnar vaccination in modulating the immune responses of adult mice to post-influenza infection with a serotype 3 strain of streptococcus pneumoniae, we chose to examine the impact of influenza infection on pneumovax responses during a secondary bacterial infection [30, 40] . specifically, the purpose of our current study was to investigate the role of immune imprinting by influenza on pneumococcal vaccine effectiveness during s. pneumoniae infection in the aged murine lung. aged adult (18 months) mice were vaccinated with the pneumococcal polyvalent vaccine pneumovax (5 mg/mouse). mice were instilled with pbs or influenza a/pr8/34 virus (3.5 × 10 2 pfu) 14 days post vaccination. on day 7, control and influenza-infected mice were instilled with pbs or s. pneumoniae (1 × 10 2 cfu, atcc 6303) and antibacterial immune responses were assessed in the lung. our results illustrate that, in response to a primary influenza infection, there was diminished bacterial clearance and heightened production of pro-inflammatory cytokines, such as il6 and il1β. vaccination with pneumovax decreased pro-inflammatory cytokine production by modulating nf-ҡb expression; however, these responses were significantly diminished after influenza infection. taken together, the data in our current study illustrate that immune imprinting by influenza diminishes pneumococcal vaccine efficacy and, thereby, may contribute to the increased susceptibility of older persons to a secondary infection with s. pneumoniae. young adult (3-4 months) and aged adult (18-20 months of age) male and female balb/c mice were purchased from the nia rodent facility (charles river laboratories). upon receipt, mice were handled under identical husbandry conditions and fed certified commercial feed. body weights were measured daily, and mice were humanely euthanized if they lost more than 15% of their starting body weight. the iacuc at weill cornell medicine approved the use of animals in this study, and methods were carried out in accordance with the relevant guidelines and regulations. no animals were used in the study if there was evidence of skin lesions, weight loss, or lymphadenopathy. influenza viral stock (material #: 10100374, batch #: 4xp170531, eid 50 per ml: 10 10.3 ) was purchased from charles river laboratories (norwich, ct, usa). all mice were anesthetized with isoflurane (5% for induction and 2% for maintenance) prior to intranasal instillation with 3.5 × 10 2 pfu of influenza (50-µl volume in pbs). tcid 50 was calculated using the viral toxglo assay (promega, madison, wi, usa). briefly, bal was diluted in 3.16-fold serial dilutions and plated for 24-48 h on >80% confluent mdck cells. upon visualization of cytopathic effect, atp detection reagent was added and luminescence was measured. values were calculated by plotting net relative luminescence unit (rlu) values after subtracting average blank wells against viral dilution. the tcid50 value is the reciprocal of the dilution that produced a 50% decline in atp levels compared to untreated controls. validated regression analysis was performed using graphpad prism (graphpad software, san diego, ca, usa). s. pneumoniae (6303, atcc manassas, va, usa) was grown on 10% sheep blood agar plates (bd biosciences, san jose, ca, usa) overnight at 37 • c, 5% co 2 . colonies were collected on an inoculating loop and added to 10 ml of thy (todd hewitt broth + 5% yeast extract) in a 125-ml polystyrene flask. flasks were incubated at 37 • c, 5% co2 and 200 rpm for 3-4 h. colony-forming units were quantified by dilution of samples in pbs, and titers were determined by colony counts × dilution. all mice were intranasally instilled with 1 × 10 3 colony-forming (cfu) units of s. pneumoniae (50-µl vol in pbs) after anesthetization with isoflurane (5% for induction and 2% for maintenance). pneumovax vaccination: pneumovax (ppv-23) vaccine was purchased from henry schein medical (newburgh, ny, usa). mice were vaccinated with 100 ml of vaccine (5 mg) via subcutaneous injection on day 0. bronchoalveolar lavage (bal): bal was collected using previously published methods [41] . briefly, 0.8 ml of pbs was slowly injected and aspirated 4 times prior to saving the recovered lavage fluid on ice. lavage was clarified at 1500 rpm for 10 min at 4 • c. lung tissue collection: at selected time points of infection, lung tissue was collected from control and influenza-infected young and aged adult mice. tissue was snap frozen or placed into allprotect (qiagen, germantown, md, usa) for future analysis. histology: mice were euthanized and right lung tissue was collected for downstream analysis. to maintain architecture, the left lung was distended with 1% low-melting agarose and placed into cold formalin [42] . tissue samples were processed and h&e stained by the translational research program at wcm pathology and laboratory of medicine. images were scanned using the evos fl auto imaging system (thermofisher scientific, fair lawn, nj, usa). for all animal experiments, we used 5-10 mice per group, and experiments were repeated at least three times. culture supernatants, lung homogenates, and bal were analyzed for il1β and il6 production using elisa kits purchased from thermofisher scientific. protein levels in bal were calculated using the biorad protein assay (biorad, hercules, ca, usa) per manufacturer's instructions. igm and igg antibody elisas were performed similarly to previously described methods [33] . briefly, serum from pbs and pneumovax-vaccinated mice was serially diluted in pbs + 1% bsa and incubated on elisa plates precoated with 50-ml of 15 mg/ml solution of unconjugated pps3 (atcc) in 0.05 m carbonate-bicarbonate buffer, ph 9.6 (sigma aldrich, st. louis, mo, usa). after overnight incubation, the plates were washed, and specific antibody titers were detected with anti-mouse igm or igg secondary antibodies (ready-set go, mouse igm and igg kits, thermofisher scientific). rna samples were extracted using the automated maxwell rna extraction protocol (madison, wi). samples were quantified and a 260/280 ratios were recorded. samples were reverse-transcribed using the first stand synthesis kit and quantified using quantitect primer assays, and rt 2 profiler tm assays (mouse antibacterial response pamm-148z) were used to assess gene expression (qiagen). all reactions were performed in triplicate. relative levels of messenger rna (mrna) were calculated by the comparative cycle threshold method, and either β-actin or β2 m mrna levels were used as the invariant control for each sample. fold change expression values were assessed by ingenuity pathway analysis (ipa) (qiagen). survival analysis between groups was calculated using the mantel-cox test. comparison of groups was performed using a two-tailed t-test and comparisons between groups were verified by one-way anova. all samples were independent and contained the same sample size for analysis. all data were analyzed using graphpad prism. statistical significance was considered as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. most data generated during this study are included in this published article. the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. as viral imprinting has the potential to influence susceptibility to secondary infections, the purpose of our current study was to determine if host immune responses initiated during influenza can impact pneumococcal vaccine efficacy against a secondary bacterial infection. to understand how a primary infection with influenza might modulate host susceptibility to s. pneumoniae, we infected young (3 months) and aged adult (18-20 months) mice with influenza (a/puerto rico/8/1934, h1n1) prior to secondary instillation with s. pneumoniae on day 3, 5, or 7 of influenza infection ( figure 1a) . to examine the impact of viral titer on host susceptibility to secondary s. pneumoniae, we examined viremia in bal samples collected from young and aged adult mice at select time points during influenza. when compared to young, there were significantly increased viral titers detected in aged adult lung at all time points of infection ( figure 1b) . in contrast to young adult mice, there was a significant increase in lethality in aged mice, which corresponded with the duration of influenza infection ( figure 1c,d) . specifically, there was increased lethality in aged mice in response to a secondary s. pneumoniae infection on day 3 of influenza ( figure 1d ). lethality continued to increase, with aged adult mice becoming highly susceptible to secondary s. pneumoniae infections at day 7 post influenza ( figure 1d ). examination of bacterial titers illustrated that there were significantly higher levels of s. pneumoniae present in bal isolated from uninfected and influenza-infected (day 3 post infection) aged mice ( figure 1e ). interestingly, when compared to aged adults, at day 5 post influenza there was a significant increase in bacterial titers in young adult lung ( figure 1e ). however, by day 7 of influenza infection, there was an abundance of s. pneumoniae present in aged adult lung, with levels significantly higher than in young ( figure 1e ). taken together, these results illustrate that a primary infection with influenza can directly impact host susceptibility to s. pneumoniae, with increased bacterial titers and lethality in aged adult mice. to expand our initial findings, we next examined the histopathological changes that occurred in young and aged adult lung in response to primary influenza and the impact of these alterations on host responses to secondary s. pneumoniae. in response to influenza, there was cellular infiltration in both young adult and aged adult murine lung, with marked levels of immune cells recruited to the aged lung ( figure 2a) . as the course of influenza infection progressed, there was a continued increase in inflammation and cellular recruitment in the aged lung (figure 2a) . in response to primary and secondary s. pneumoniae, there was also a marked increase in inflammatory damage to the alveolar capillary barrier, resulting in increased permeability and intra-alveolar edema in the aged lung ( figure 2b ). in sum, in response to influenza, there are increased changes in histopathology in aged adult murine lung, with increased inflammation, cellular infiltration, and inflammatory damage occurring post viral s. pneumoniae. based on these findings, we next investigated the impact of influenza on pneumococcal vaccine efficacy during s. pneumoniae infection in aged lung. specifically, aged adult mice were vaccinated with pneumovax (5 mg/mouse) on day 0 ( figure 3a ). mice were subsequently instilled with pbs or influenza on day 14 post vaccination ( figure 3a ). after seven days of influenza infection, aged mice were intranasally instilled with s. pneumoniae, and lung tissue was collected at 24 h post-secondary infection ( figure 3a ). to examine if pneumovax treatment altered cellular recruitment or viremia, total cell numbers and viral titers in bal isolated from pbs-and pneumovax treated-mice were quantified ( figure 3b,c) . when compared to pbs controls, there was no significant increase in cellular recruitment or viral titers in aged adult mice vaccinated with pneumovax on day 7 post influenza ( figure 3b,c) . to determine if vaccination with pneumovax resulted in igm and igg antibody titers, we collected serum from pbs-and pneumovax-treated mice at select time points post immunization and quantified igm and igg titers against pneumococcal polysaccharide serotype 3 (pps3) [33] . there was a detectable increase in a-pps3 igm and igg antibodies detected in aged adult serum post vaccination (figure 3 d,e) . based on these findings, we next examined if treatment with pneumovax altered cellular recruitment during primary and secondary s. pneumoniae infection. as shown in figure 3f , when compared to primary infection, there was a significant increase in total cell numbers during a secondary, post-viral s. pneumoniae infection. despite this increase, there was no significant difference in cells present in bal collected from pbs-and pneumovax-treated aged adult mice ( figure 3f ). in response to influenza infection, there was a significant increase in bacterial titers present in aged lung ( figure 3g ). secondary s. pneumoniae titers in aged mice vaccinated with pneumovax were significantly lower than the titers observed in unvaccinated mice ( figure 3g ). when compared to aged mice receiving only a primary infection with s. pneumoniae, the presence of influenza significantly decreased vaccine efficacy and resulted in augmented s. pneumoniae titers in aged lung ( figure 3g ). as protein-rich edema can interact with alveolar surfactants and result in decreased pulmonary compliance, we next examined if there were changes in protein levels present in bal collected from control and infected aged mice. despite vaccination, there was increased bal protein in aged lung in response to influenza, which was significantly heightened in response to secondary s. pneumoniae ( figure 3h ). taken together, our results illustrate that influenza contributes to decreased bacterial clearance and increased lung injury in both untreated and pneumovax-vaccinated mice. we next examined if a primary infection with influenza altered pro-inflammatory cytokine production in aged lung in response to s. pneumoniae. there was a significant increase in il-1β and il-6 mrna ( figure 4a ,b) as well as il-1β and il-6 cytokine production ( figure 4c ,d) in aged lung in response to secondary post-influenza infection with s. pneumoniae. to investigate the impact of influenza on pneumococcal vaccine efficacy, we evaluated inflammatory cytokine production in response to primary and secondary post-influenza s. pneumoniae infection. in response to pneumovax, there was a significant decrease in il-1β and il-6 mrna ( figure 4a ,b) as well as il-1β and il-6 cytokine production ( figure 4c ,d) in aged lung during primary and secondary s. pneumoniae infection when compared to unvaccinated controls. despite improvement in inflammatory cytokine production by pneumovax, these outcomes were directly impacted by influenza, with significantly higher levels of il-6 mrna and cytokine production being detected in aged lung during secondary s. pneumoniae infection ( figure 4b,d) . in sum, our results demonstrate that there is augmented il-1β and il-6 production in aged lung in response to a post-influenza infection with s. pneumoniae. while these levels are decreased in response to pneumovax vaccination, they remained significantly elevated in post-influenza s. pneumoniae-infected mice. as il-6 production is controlled by changes in nfҡb signaling, we next investigated the impact of influenza on the expression of several components of the nfҡb pathway. we first examined the impact of primary influenza infection on changes in the expression pattern of the nfҡb inhibitor ikkβ. there was a significant decrease in ikkβ mrna expression in aged lung during a secondary post-influenza infection with s. pneumoniae ( figure 5a ). vaccination with pneumovax increased ikkβ mrna expression during primary and secondary s. pneumoniae infection ( figure 5a ). despite an improvement with pneumovax vaccination, when compared to primary s. pneumoniae, ikkβ mrna expression in aged lung remained significantly lower during a secondary post-influenza infection ( figure 5a ). we next evaluated the impact of a primary infection with influenza on the expression of nfҡb inhibitor iҡbα. there was a significant decrease in iҡbα mrna expression in aged lung during a secondary post-influenza infection with s. pneumoniae ( figure 5b ). vaccination with pneumovax increased iҡbα mrna expression during both primary and secondary s. pneumoniae infections ( figure 5b ). despite an improvement with pneumovax vaccination, when compared to primary s. pneumoniae, iҡbα mrna expression in aged lung remained significantly lower during a secondary post-influenza infection ( figure 5b ). based on these findings, we next examined the impact of influenza on nfҡb mrna expression. there was a significant increase in nfҡb mrna expression in aged lung during a secondary post-influenza infection with s. pneumoniae ( figure 5c ). vaccination with pneumovax decreased nfҡb mrna expression during both primary and secondary s. pneumoniae infections ( figure 5c ). despite an improvement with pneumovax vaccination, when compared to primary s. pneumoniae, nfҡb mrna expression in aged lung remained significantly elevated during a secondary post-influenza infection ( figure 5c ). taken together, our results illustrate that, in response to influenza, there was an increase in nfҡb gene expression detected in aged lung during secondary s. pneumoniae infection. vaccination with pneumovax improved the expression of nfҡb inhibitors, ikkβ and iҡbα; however, these levels were decreased when compared to expression levels in the aged lung during primary s. pneumoniae infection. the purpose of our current study was to investigate the role of immune imprinting by influenza on pneumococcal vaccine effectiveness during streptococcus pneumoniae infection in the aged murine lung. our results illustrate that, in response to a primary influenza infection, there was diminished bacterial clearance and heightened production of pro-inflammatory cytokines, such as il-6 and il-1β. vaccination with pneumovax decreased pro-inflammatory cytokine production by modulating nf-ҡb expression; however, these responses were significantly diminished after influenza infection. taken together, the data in our current study illustrate that immune imprinting by influenza diminishes pneumococcal vaccine efficacy and, thereby, may contribute to increased susceptibility of older persons to a secondary infection with s. pneumoniae. for our studies, we chose to use the fda-cleared vaccine pneumovax to examine the impact of influenza on vaccine efficacy to secondary s. pneumoniae infection. for adults >65 years of age, the 23-valent pneumococcal polysaccharide vaccine (ppv23) pneumovax and the 13-valent pneumococcal conjugate vaccine (pcv13) prevnar are two vaccines available for protection against pneumococcal infections. while there have been conflicts in pneumococcal vaccine effectiveness, a recent meta-analysis illustrated that pneumovax exhibited a weak protective effect on all-cause pneumonia among immunocompetent adults and persons over 65 years of age as well as high-risk persons (19-64 years of age) [34] . while multiple studies have illustrated that vaccination in persons >60 years with prevnar can result in improved immunogenicity against multiple s. pneumoniae serotypes, these antibody titers were found to decline after a year and were similar to titers observed post pneumovax vaccination [35] [36] [37] [38] . in addition, combined administration of prevnar prior to pneumovax can elicit a greater immune response than multiple dosages of prevnar, which only demonstrated a modest increase [37, 39] . as recent work has illustrated differential efficacy of prevnar vaccination in modulating the immune responses of adult mice to a post-influenza infection with a serotype 3 strain of streptococcus pneumoniae, we chose to examine the impact of pneumovax on these responses [30, 40] . given these findings, it would be plausible that other vaccines, designed with specific bacterial components, such as pneumococcal surface protein a (pspa) and pneumolysin (ply) or liposomal encapsulation of polysaccharides (leps), may improve efficacy to post-influenza s. pneumoniae infection [31, [43] [44] [45] . future work will need to evaluate if additional vaccine types can prove efficacious and further reduce inflammatory cytokine production while improving bacterial clearance post-influenza infection. in response to pneumovax, in agreement with previous studies, we detected pps3-specific igm and igg in serum collected from aged adult mice by day 14 post vaccination [33, 46] . based on these findings, we chose to examine the impact of influenza on modulating vaccine efficacy against a secondary pneumococcal infection. it is important to note that, at day 14 post vaccination, the effector phase of the adaptive immune response was not completely cleared after vaccine administration. while we observed efficacy of pneumovax vaccination in decreasing the production of proinflammatory cytokines, such as il-6, and decreased bacterial titers during secondary s. pneumoniae infection, an increased duration of time after vaccination, when all effector immune cells have rested into a memory phenotype, may further improve these responses. neuraminidases (na) are glycoside hydrolase enzymes that, through the cleavage of glycosidic linkages, can facilitate the mobility of virus particles through the respiratory tract mucus as well as aid in viral elution from infected cells. influenza viral na has been previously shown to be an important factor in viral-bacterial synergism [24, 29] . increased sialic acid cleavage by viral na can promote greater pneumococcal adherence and bacterial invasion [47, 48] . this synergistic relationship can aid in successful invasion of the lower respiratory tract by s. pneumoniae during influenza infection [48] . of note, s. pneumoniae na, which plays an important role in biofilm formation, can also help facilitate pneumococcal pathogenesis and respiratory tract colonization [49] . given the properties of viral and bacterial neuraminidases, it is possible that the increased bacterial burden detected in pneumovax-treated lung after secondary s. pneumoniae infection was due to a greater ability of the bacteria to bind to the respiratory epithelium. despite bacterial suppression from the antibody response, increased sialic acid cleavage by influenza na during the primary infection can contribute to augmented bacterial titers during a secondary infection with s. pneumoniae when compared to pneumovax-treated lung during a primary s. pneumoniae infection alone. influenza virus, respiratory syncytial virus, parainfluenza virus, adenovirus, and coronavirus are commonly detected in patients with community-acquired pneumonia. unfortunately, at present, it is not fully clear to what extent these pathogens contribute to disease development or predispose a patient to secondary infections [50, 51] . while our work has focused on influenza, additional viral pathogens have the propensity to alter vaccine efficacy. it is therefore plausible that, while our results illustrate a specific mechanism by which inflammatory cytokine production is altered in response to influenza, similar inhibitory mechanisms might underlie decreased vaccine efficacy against secondary pneumococcal infections. future studies will need to be performed to examine if altered nfҡb signaling is a common mechanism that underlies increased inflammation during secondary post-viral pneumococcal infections. it is important to note that the timing between influenza infection and post-viral pneumococcal infection as well as the dose of bacteria administered can greatly impact the host outcomes to a secondary infection [17, [20] [21] [22] [23] . initiation of influenza-virus-mediated lung inflammation has been shown to directly correlate with bacterial growth, and high dosages of bacteria were associated with increased mortality during secondary infection [17, [20] [21] [22] [23] . in agreement with previous studies, results from our current work further illustrated that, as early as day 3 post influenza, bacterial clearance from lung was significantly altered in young adult lung, with significantly increased titers present in aged adult lung [25] . influenza-mediated changes in the immune response contributed to increased virus-mediated damage to the lung and heightened susceptibility to secondary pneumococcal infection [18, [26] [27] [28] . in our model of influenza and secondary s. pneumoniae infection, we detected a similar phenotype that occurred by day 7 of influenza, with more extensive damage observed in the aged adult lung. these results extend the findings from our previous work, which illustrated that an age-associated decrease in nlrp3 inflammasome activation contributed to increased morbidity and mortality of aged adult mice to secondary s. pneumoniae at day 14 post influenza [52] . similar to previously published studies in young adult mice, we also observed a dramatic reduction in vaccine efficacy against s. pneumoniae in the presence of influenza [17, 30, 31] . based on these results, it is possible that the immune priming response that occurs during influenza can have a direct negative impact on adaptive vaccine responses in aged adult lung. future work will need to be performed to evaluate if antibody titers can directly modulate host-pathogen interactions. previous work has illustrated that there is a relationship between the lung permeability index (lpi) and total protein measured in the bronchoalveolar lavage, with increased protein leak into the lungs being indicative of a continuing event or injury [53] . results of our current study illustrate that, despite vaccination, there was increased protein-rich edema present in the aged lung in response to influenza that was significantly heightened in response to secondary s. pneumoniae. it is possible that, despite pneumococcal vaccination, extensive viral-mediated damage to the epithelium by influenza can augment protein leakage into the lungs. it is important to note that, while we examined protein levels in bal fluid collected from pbs-and pneumovax-treated mice during primary and secondary s. pneumoniae infection, there are multiple types of protein and lipids present within each bal sample. pulmonary surfactant is a complex mixture of lipids and proteins that consists of lipids (90%) and proteins (10%) [54] . both hydrophilic surfactant protein (sp), sp-a and sp-d, and hydrophobic proteins, sp-b and sp-c, play important roles in modulating innate immune responses at the alveolar barrier and maintaining the biophysical activity of pulmonary surfactant [54, 55] . in the current study, we focus on a potential mechanistic pathway that may underlie increased susceptibility of aged lung to secondary s. pneumoniae infection. as sp-b has been previously shown to play an important role in preventing respiratory failure during acute lung injury, future work will need to be performed to examine the exact surfactant protein composition of bal collected during primary and secondary s. pneumoniae and the impact of pneumococcal vaccination on these protein concentrations [56, 57] . in summary, the results of our current study illustrate that immune imprinting by influenza diminishes pneumococcal vaccine efficacy and, thereby, may contribute to increased susceptibility of older persons to a secondary infection with s. pneumoniae. • primary influenza infection increases susceptibility of aged adult mice to s. pneumoniae. cellular senescence in aging and age-related 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increased susceptibility to secondary streptococcus pneumoniae infection the lung permeability index: a feasible measurement of pulmonary capillary permeability the role of lipids in pulmonary surfactant pulmonary surfactant: functions and molecular composition sp-b deficiency causes respiratory failure in adult mice structure and properties of surfactant protein b this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. key: cord-300747-fnli688g authors: calvo, cristina; garcía‐garcía, maría luz; pozo, francisco; carballo, daniel; martínez‐monteserín, eduardo; casas, inmaculada title: infections and coinfections by respiratory human bocavirus during eight seasons in hospitalized children date: 2016-05-06 journal: j med virol doi: 10.1002/jmv.24562 sha: doc_id: 300747 cord_uid: fnli688g the human bocavirus (hbov) has been identified in respiratory infections in children in a large number of studies. despite this, the pathogenic role of the hbov is under discussion. the main objectives of the study were: to determine the incidence of hbov in hospitalized children; to describe the main clinical features of the positive children; and to compare the data with those from other viral infections in the same population. a prospective study was performed between 2005 and 2013 including children up to 14‐year old with respiratory infection admitted to the severo ochoa hospital (spain). nasopharyngeal aspirates were taken from 3,275 patients and were tested for hbov and other 15 respiratory viruses by rt‐nested pcr. hbov was detected in 319 patients (9.9%); 80 cases as a single pathogen, and 239 cases (75%) as coinfections with other viruses. the hbov was the fourth most common virus detected, behind respiratory syncytial virus (39.8%), rhinovirus (30.6%), and adenovirus (15%). the most common clinical diagnosis, in cases that hbov was detected as a single pathogen was asthma exacerbation followed by pneumonia. a seasonal distribution was shown, with higher positivity rates in december and january. children affected by hbov were older than children infected by other viruses. differences in terms of clinical diagnosis were found, bronchiolitis diagnosis was lower compared with the other viruses, and hbov was associated with diagnosis of pneumonia, with increased use of antibiotics (41.8%), and radiographic infiltrates (47%). these findings could suggest a pathogenic role of hbov in respiratory infections in children under 14 years of age. j. med. virol. 88:2052–2058, 2016. published 2016. this article is a u.s. government work and is in the public domain in the usa. human bocavirus (hbov), a dna virus classified in the parvoviridae family, was first described in 2005 [allander et al., 2005] , and has been detected in up to 18% of hospitalized children with respiratory diseases [jartti et al., 2012] . however, it has also been found in children with mild infections [martin et al., 2015] and in asymptomatic ones [garc ıa-garc ıa et al., 2008] . up to 75% of the hbov infections are coinfections with one or more viruses [calvo et al., 2007] . a prolonged viral shedding has been described, about 75 days in outpatients [martin et al., 2010] , and up to 4.5 months in hospitalized children [blessing et al., 2009] , which probably explains hbov detection in asymptomatic cases [von linstow et al., 2008] . although the pathogenic role of hbov has been questioned, serologic diagnosis of hbov has recently confirmed significant increases in igg antibodies in children with pneumonia. these results support the idea that it is a true pathogen in respiratory tract infections in children . unfortunately, this technique is not yet available in many laboratories and requires an invasive procedure for diagnosis. in this context, we would like to contribute to the knowledge of hbov infections with a long-term prospective study. our aim was to determine the incidence of the hbov in hospitalized children, its epidemiological profile and to describe the main clinical features of the infected children. we also compared the clinical characteristics of single hbov infections with other prevalent single viral infections in the same population, in an attempt to differentiate the characteristics of each infection. the study population comprised all children between the first month of life and 14 years of age with a respiratory tract disease admitted to the secondary public hospital severo ochoa (legan es, madrid), between september 2005 and august 2013. the study was approved by the medical ethics committee. prior to sample collection, an informed consent was obtained from parents or legal guardians. all patients were evaluated by an attending physician. clinical characteristics of patients were analyzed. during the hospital stay, and as part of the study, a physician filled out a study-questionnaire with the clinical data. upper respiratory tract infection (urti) was diagnosed in patients with: rhinorrhea and/or cough and no signs of wheezing, dyspnea, crackles or bronchodilator use, with or without fever. asthma was diagnosed on the basis of the national asthma education and prevention program guidelines [naepp, 2002] . all other episodes of acute expiratory wheezing were considered to be recurrent wheezing. acute expiratory wheezing was considered to be bronchiolitis when it occurred for the first time in children aged less than 2 years. laryngotracheobronchitis was associated with inspiratory dyspnea and wheezing. laryngitis was associated with inspiratory dyspnea without wheezing. cases with both focal infiltrates and consolidation in chest x-rays were, in the absence of wheezing, classified as pneumonia. clinical specimens consisted of a nasopharyngeal aspirate (npa) taken from each patient at admission (monday-friday). all clinical specimens were sent to the respiratory virus and influenza unit at the national microbiology center (isciii, madrid, spain), for virological investigation. npas were processed within 24 hr after collection. upon receipt, three aliquots were prepared and stored at à70˚c. three rt-nested pcr assays were performed to detect a total of 16 respiratory viruses. in these assays, the reverse transcription (rt) and first amplification round were carried out in a single tube using the qiagen onestep rt-pcr kit (qiagen, hilden, germany). influenza a, b, and c viruses were detected by using previously described primer sets only to amplify influenza viruses in a multiplex pcr assay [coiras et al., 2003] . a second multiplex pcr was used to detect parainfluenza viruses 1-4, human coronaviruses 229e and oc43, enteroviruses and rhinoviruses (rv) [coiras et al., 2004] . presence of respiratory syncytial virus (rsv) a and b types, human metapneumovirus (hmpv), human bocavirus (hbov), and adenoviruses were investigated by a third multiplex rt-nested pcr-brq method [calvo et al., 2010] . values were expressed as percentages for discrete variables, or as mean and standard deviation for continuous variables. clinical characteristics of patients with single infections associated to hbov were compared with those associated with coinfections of hbov with other respiratory viruses. single hbov infections were also compared with single infections by rsv, rv, and hmpv. clinical characteristics and laboratory variables were compared using the student t-test, the mann-whitney u-test, the x 2 test, and fisher's exact test. a two-sided value of p < 0.05 was considered statistically significant. results were adjusted for age. all analyses were performed using the statistical package for the social sciences (spss), version 21.0. a total of 3,275 cases of respiratory diseases were analyzed during the study period. one or more viruses were detected in 76.5% of the samples. multiple infections were present in 29% (726) of the positive cases. the most frequent identified virus was rsv (39.8%), followed by rv (30.6%), and adenovirus (hadv); 15%. hbov was the fourth in frequency and it was detected in 319 cases (12.7% of the positives cases). figure 1 . out of 319 cases, 80 (25%) were single detections, and 239 (75%) were detected in coinfection with other viruses, mainly rsv, rv, and hadv ( fig. 1) . circulation of hbov was higher in autumn (60% of cases between november and december). clinical characteristics of patients with respiratory episodes associated to single hbov detections were analyzed (table i ).these childrenś group mean age was 21 months (standard deviation; 24 months). fever was present in 68.8% of cases, and hypoxia in 52% of them. recurrent wheezing or asthma was diagnosed in 58.8% of cases, followed by pneumonia (22.5%). x-ray infiltrates were present in 47% of the patients. clinical cases associated to single hbov detections were compared with the 239 cases of hbov coinfections (table i) . diagnosis was the only difference between groups. cases with single hbov detections were diagnosed as pneumonia more frequently (p < 0.0001) than coinfections. one patient with wheezing and with single hbov detection had a concomitant urinary tract infection and a bacteremia. eight patients had two different admissions during the study period associated to hbov (table ii) , with an interval between clinical episodes of 15 days to 10 months. they were asymptomatic between episodes and we did not perform any viral determination during this period. one of these patients had two different admissions more than 2 years apart. other single viral infections the clinical characteristics of the 80 patients with single hbov detection were compared with the single infections associated with rsv, rv, and hmpv in the same period. hbov versus rsv. rsv single infections were present in 665 children and it was the most frequently identified virus. it was observed that hbov affects more males (66% vs. 52%, p ¼ 0.02) at a later age (25 vs. 9 months, p < 0.0001), and was associated with distinct clinical features. episodes of wheezing or asthma and pneumonia were more frequent and bronchiolitis was less frequent and it was more characteristic in rsv infections (p ¼ 0.001). hypoxia (71% vs. 52%, p ¼ 0.001), duration of hypoxia and days of stay were significantly higher in rsv infections. leukocytes and c-rp were significantly higher in hbov infections and also the proportion of patients treated with antibiotics (41% vs. 17%, p < 0.001). hbov circulation (fig. 1 ) was mainly in november and december (>60% of cases), coinciding with rsv (>60% in these 2 months). however, rsv was also frequently identified in january with 22.7% of its cases (table iii) . hbov versus hmpv. hmpv single infections were present in 108 children during the study period. this group comprised children that were younger than hbov-positive children (14 vs. 25 months, p ¼ 0.002). diagnosis of pneumonia was more frequent in hbov children who also received more antibiotics (41% vs. 26%, p ¼ 0.026). leukocytes in blood and crp were also significantly higher in hbov group. nevertheless, hospitalization was longer in the hmpv group (p ¼ 0.06). hmpv was mainly detected in spring (march-may, fig. 1) (table iv) . hbov versus rv. rv single infections were present in 555 cases, and it was the second most prevalent virus. both groups were of a similar age. the proportion of children with fever was higher in hbov infections (68% vs. 41%, p < 0.0001). as with the other comparisons, diagnosis of pneumonia was more frequent in hbov group and these children also had fever more frequently and received more antibiotics (41% vs. 20%, p ¼ 0.0001). no other differences were found between them. rv circulated throughout the year, with a higher incidence in autumn ( fig. 1 ; table v) . hbov is, in our experience, the fourth virus identified in respiratory infections in hospitalized children. however, in only 25% of the cases it was detected as a single pathogen, with the remaining cases being characterized as coinfections. the single infections were characterized by the presence of high fever, wheezing, a significant percentage of pneumonia, hypoxia, moderate leukocytosis and elevated crp, all of which lead to frequent antibiotic treatment. children were on average 2-year old and infections occurred mainly in november and december. although hbov seasonally coincides with rsv, clinical characteristics of both viruses are quite different. rsv affects younger children, causing bronchiolitis and it is less frequently associated with radiographic infiltrates, it does not induce leukocytosis, and crp is normal. hmpv circulate in spring, and clinical features are similar to those of rsv and different to hbov infections. rhinovirus circulates throughout the year, children have a similar age to the hbov group, have also wheezing and infiltrates, but fever is less frequent. all these data suggest that hbov infections have their own characteristics and support the role of this virus as an actual pathogen. although some authors [martin et al., 2010] do not find differential clinical data in outpatients positive for hbov, our series describes a set of characteristic clinical data in hospitalized children, different from single infections caused by other viruses as rsv or hmpv. a potential explanation for the differential clinical characteristics found in our patients might lie in the greater severity of hospitalized patients versus ambulatory ones. furthermore, the number of patients analyzed by martin could make it difficult to find distinguishing characteristics. our series included a large number of patients and this allows us to draw conclusions on clinical data for a homogeneous population. although there are very few studies comparing the clinical characteristics of hbov infections with other viruses, some authors such as moriyama et al. [2010] also find, clear differences between hbov and rsv. younger age, bronchiolitis, and hypoxia were more frequent in rsv group as is the case in our series. we have not found specific comparisons between hbov and rv or hmpv, with the exception of previous work by our group [calvo et al., 2007] and the study of zhang in china with a small group of different viruses [zhang et al., 2013] . they could not find any characteristic data of hbov infection. many other studies have confirmed the relatively high frequency of hbov infections in hospitalized children and their association with wheezing episodes [do amaral de leon et al., 2013] and pneumonia [esposito et al., 2013; wang et al., 2015] . coinfections are as frequent as 75-80% also in previous studies [calvo et al., 2007; fry et al., 2007; martin et al., 2010; broccolo et al., 2015] , and this might suggest that the symptoms may be caused by other viruses. in addition, hbov is able to persist and therefore could be more often detected as an accompanying pathogen. several authors have demonstrated that hbov persist in different cells [l€ usebrink et al., 2011] , intestinal tissues [kapoor et al., 2011] , lung and tumors [schildgen et al., 2013] , and in tonsillar tissue in children [falcone et al., 2015] . moreover, byington et al. [2015] detected persistent hbov in children, with and without symptoms, lasting longer than with other viruses. therefore, the positivity of hbov does not imply causality [storch, 2015] . furthermore, it is worth mentioning that huang et al. [2012] and dijkman et al. [2009] have shown that hbov induces a cytopathic effect in human airway epithelial cell cultures, and it is very unlikely that a harmless bystander would be able to produce a cytopathic effect. prolonged hbov shedding, found by martin [2010] and others [blessing et al., 2009] , could also be the cause of detection in coinfection with other viruses, and their presence in asymptomatic children. more recently, in an outpatient population martin et al. [2015] , found that children infected with hbov were more likely to experience recurrent cough episodes and to visit a healthcare provider during the 14 days following the time of initial detection of hbov. sequences of the samples from 12 of the 48 children that were positive for hbov-1 were not 100% identical; and therefore, were considered to be hbov reinfections that contributed to long-term shedding. although hbov sequences were not determined in our patients, these two facts could help to explain recurrent infections that we found in a percentage of patients in our series, which had more than one admission, with hbov positive detection in a short period of time. although it might be expected that viral load could differ between acute infections or asymptomatic eliminations, quantification of viral load has been of no help to differentiate symptomatic from asymptomatic infections. in our opinion, this might happen because the technique for viral load measurement is not appropriate for respiratory secretions, where the standardization of the sample is difficult. this might also explain conflicting results in different studies [martin et al., 2010; ghietto et al., 2015; principi et al., 2015] . although not specifically included in this study, we ourselves have failed to find differences in viral load between symptomatic and asymptomatic patients. still, severity of infection might be correlated to viral load in some studies as [moesker et al, 2015] that reported a higher viral load in hbov-positive patients admitted to an intensive care unit. this study has some strength as its prospective nature, the long study period, the number of patients studied, both single and multiple detections, and the comparison with the most prevalent viruses. it also has some limitations, since we do not have a control group. however, we have confirmed in a previous work of our group, that symptomatic children in our setting have significantly higher hbov positivity than healthy children [garc ıa-garc ıa et al., 2008]. we do not have serial samples collected to assess the shedding of hbov. this prevents us from knowing if recurrent cases had a negative sample between episodes. we do not have serology and therefore, we cannot confirm that infections are acute and that hbov is the causal agent. serology has demonstrated that hbov has a pathogenic role in respiratory infections [kantola et al., 2008; s€ oderlund-venermo et al., 2009; don et al., 2010] , but it has only been available in the last few years and only in some laboratories. it is remarkable, that in the study by don et al. [2010] , serology confirms the pathogenic role of hbov in children with pneumonia, which is one of the most frequent diagnoses found in our series. other studies [ghietto et al., 2015] , match our series and find no clinical differences between single infections and coinfections with hbov, which may also suggest the role of this virus in multiple infections. in summary, we present a long prospective study of respiratory hbov infections, with a considerable number of patients and compared with other viral infections, in which clinical features specific to this virus are shown. in the future, prospective studies, probably including both serologic studies and shedding of the virus, may help to clarify the remaining doubts about hbov infections in children. cloning of a human parvovirus by molecular screening of respiratory tract samples prolonged detection of human bocavirus dna in nasopharyngeal aspirates of children with respiratory tract disease human bocaviruses possible etiologic role in respiratory infection community surveillance of respiratory viruses among families in the utah better identification of germs-longitudinal viral epidemiology (big-love) study clinical characteristics of human bocavirus infections compared with other respiratory viruses in spanish children detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays human bocavirus can be cultured in differentiated human airway epithelial cells clinical and epidemiologic profile of lower respiratory tract infections associated with human bocavirus serologically verified human bocavirus pneumonia in children impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses human bocavirus dna in paranasal sinus mucosa human bocavirus a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection comorbidity and high viral load linked to clinical presentation of respiratory human bocavirus infection establishment of a reverse genetics system for studying human bocavirus in human airway epithelia human bocavirus-the first 5 years serodiagnosis of human bocavirus infection bocavirus episome in infected human tissue contains non-identical termini serologic diagnosis of human bocavirus infection in children detection of head-to-tail dna sequences of human bocavirus in clinical samples frequent and prolonged shedding of bocavirus in young children attending daycare human bocavirus 1 primary infection and shedding in infants human bocavirus infection as a cause of severe acute respiratory tract infection in children distinctive clinical features of human bocavirus in children younger than 2 years expert panel report: guidelines for the diagnosis and management of asthma update on selected topics-2002 bocavirus infection in otherwise healthy children with respiratory disease the human bocavirus is associated with some lung and colorectal cancers and persists in solid tumors editorial commentary: plethora of respiratory viruses and respiratory virus data clinical and epidemiologic characteristics of human bocavirus in danish infants: results from a prospective birth cohort study incidence of viral infection detected by pcr and real-time pcr in childhood community-acquired pneumonia a meta-analysis viral etiology and clinical profiles of children with severe acute respiratory infections in china key: cord-285270-amh99u0j authors: husain, shahid; mooney, martha l.; danziger-isakov, lara; mattner, frauke; singh, nina; avery, robin; ison, michael; humar, atul; padera, robert f.; lawler, leo p.; fisher, andy; drew, richard j.; gould, kate f.; sole, amparo; studer, sean; munoz, patricia; singer, lianne g.; hannan, margaret title: a 2010 working formulation for the standardization of definitions of infections in cardiothoracic transplant recipients date: 2011-03-17 journal: j heart lung transplant doi: 10.1016/j.healun.2011.01.701 sha: doc_id: 285270 cord_uid: amh99u0j nan in the absence of standardized diagnosis and the presence of unique clinical syndromes, it is not surprising that considerable differences exist in the number of reported incidences of disease and the outcomes of various infections in cardiothoracic transplant (cttx) recipients. publications to date have employed variable and heterogeneous definitions of cttx-related infections, thereby limiting the comparison between the types and incidence of infections and the generalizability of these data across transplant centers. currently, there are no standard international definitions for infections uniquely related to cttx, with the exception of chagas disease and toxoplasmosis. 1 the purpose of the present working formulation is to provide consensus-derived expert opinion of definitions for infections in cttx for epidemiologic, research and registry data use. standard definitions of infections specifically related to cttx will allow for meaningful comparison of the type and incidence of these infections between different types of cttxs, different regimens of immunosuppression and between different transplant centers, thereby improving the reporting of infection-related morbidity and mortality after cardiothoracic transplantation. the definitions proposed herein are suitable for epidemiologic investigations and are intended to facilitate clinical decision-making. the definitions described in what follows have been reviewed and approved by a multidisciplinary working group of the international society for heart and lung transplantation (ishlt). these definitions were adapted from surveillance definitions of healthcare-associated sinusitis, tracheobronchitis and pneumonia, used in reporting to the national healthcare safety network (nhsn) and the centers for disease control and prevention's (cdc) surveillance system for patient and healthcare personnel safety. 2 definitions of invasive fungal infection (ifi) were based on those proposed by the european organization for research and treatment of cancer and the mycoses study group of the national institutes of health (eortc/msg), 3 whereas definitions from the american society of transplantation and other source documents represented the foundation for defining viral infections. 1, 4 bacterial infection overview bacterial infections are a major contributor of complications in the early post-transplant period in heart-and lung-transplanted patients. 5, 6 some bacterial infections (e.g., pre-transplant colonization or donor-derived infections) have unique issues and implications in cttx recipients 5, [7] [8] [9] [10] [11] [12] ; therefore, the definitions for these infections for epidemiologic, research or ishlt registry purposes are specifically addressed herein. many other bacterial infections (e.g., methicillin-resistant staphylococcus aureus or vancomysin-resistant enterococcus) are present similarly across hospitalized patients and solid-organ transplant (sot) recipients and are therefore not directly addressed in what follows. the existing literature in cttx has largely classified bacterial infections as "early" (e.g., post-operative) or "late" onset after transplantation, allowing transplant clinicians to determine the source of these infections and focus prevention strategy and early empirical antibiotic treatment regimens on the temporal onset of these infections. a further timeline is used to classify all infections diagnosed in the hospital setting as nosocomial, with onset 48 hours after the patient is admitted to the hospital, and communityacquired infection, with onset at the time of admission or within 48 hours of admission. the latter definitions of infection may be artificial in the setting of cttx as some infections, although related to healthcare and immunosuppression, may not occur within the established time-line of nosocomial infections. to fully appreciate the impact of the potential source of infection, we propose using the categories of nosocomial (after 48 hours of hospitalization) and community-acquired (prior to 48 hours of hospitalization) with the added category of community-acquired "transplant-related" infections. this category would include infections by pathogens acquired by the cttx patient prior to time of transplantation and that are clearly related to the immunocompromised state of the cttx patient after transplantation that may increase the risk for specific bacterial pathogens that are not common in the community. these pathogens may be related to the donor or the recipient via pre-transplant colonization of the respiratory or gastrointestinal (gi) tract and can be therefore regarded as "transplant-related" in this setting. 12, 13 it is also to be noted that community-acquired pneumonia may be transplant-related if caused by organisms that are typically associated with transplants (e.g., fungal, multidrug-resistant or atypical bacteria). respiratory bacterial infections occur frequently in lung transplant recipients. in one study, 72 episodes per 100 lung transplants per year were reported. 14 the definitions of bacterial pneumonia present significant challenges in cttx. frequent use of empirical anti-bacterial agents prior to specimen collection and the possibility of concurrent allograft rejection make the use of standard guidelines, as presented by the centers for disease control and prevention (cdc) for healthcareassociated infections (hcais), difficult to apply. 2 in addition, some microbiologic diagnostic procedures may not be routinely practiced at many transplant centers and this may limit the employment of diagnostic criteria for infections that require quantification of bacterial colonyforming units per milliliter in the bronchoalveolar lavage (bal) samples. this methodology has not been validated in the immunocompromised host and is not standardized across institutions. further, the thresholds proposed may underestimate the episodes of bacterial pneumonia in the cttx population, 20 where early empirical intervention with anti-microbial agents prior to obtaining the samples with suspected pneumonia is common practice. presence of endobronchial stents in lung transplant recipients further complicates the picture in defining various clinical syndromes. for these reasons, a specific classification of bacterial pneumoniae in cttx recipients is proposed based on radiographic findings, clinical symptoms, microbiology and histopathology (including consideration of acute rejection in lung transplant patients). in lung transplant recipients, the use of differential cytology in bal may be helpful. [21] [22] [23] the predominance of neutrophils with intracellular bacteria (hematoxylin-eosin and gram stain) is more suggestive of the presence of a bacterial pneumonia than a high proportion of lymphocytes or eosinophils in bal. on the other hand, a lymphocytic or eosinophilic bal could indicate an acute graft reaction, although cytomegalovirus (cmv), other viruses and atypical pathogens would need to be ruled out. acute rejection (ar) of the graft in lung transplant recipients presents a significant consideration in the diagnosis of all pneumonias, including those due to bacteria. there are frequent clinical scenarios where distinction between rejection and infection is critically dependent upon histopathologic findings. in many cases, evidence for infection and rejection coexist. therefore, in the setting of lung transplantation, the diagnosis of bacterial (or any) pneumonia is more precisely defined by the confirmation or exclusion of ar when microbiologic criteria are not met. 24 the determination of ar requires histologic examination. if an ar is documented and clinical and laboratory criteria for bacterial pneumonia are also fulfilled, the diagnosis of ar and concomitant pneumonia is possible. 24 accordingly, pneumonia should be indicated as pneumonia combined with an ar. • direct examination by light microscopy [gram stain, modified acid-fast bacilli (afb) stain for nocardia spp, afb stains for mycobacteria]. • culture (including rapid culture methods for legionella spp, mycobacteria spp and prolonged culture periods for detection of bacteria-causing infective endocarditis). • bal cell analysis: rule-out contamination with ͻ1% epithelial cells, 20 definitions of bacterial pneumonia and colonization in cttx are given in table 1a , whereas the definitions of tracheobronchitis are given in table 1b . 1. ventilator-associated pneumonia should be designated when reporting data. a distinction should be made between non-invasive and invasive ventilation. 2. aspiration pneumonia should be considered if the criteria are fulfilled for pneumonia (table 1a ). the cause of this type of pneumonia should be noted. 3. multiple or concurrent episodes of post-transplant pneumonia may occur. to determine a new episode in a single patient, resolution of the initial infection must be determined by clinical, laboratory or histologic evidence. the isolation of a new pathogen alone is not indicative of a new episode of pneumonia. in contrast, a second pneumonia may develop in a patient after single lung transplantation. here, the contralateral lung may develop an "independent" pneumonia by another organism. 4. sputum samples are frequently contaminated with airway colonizers (e.g., coagulase-negative staphylococcus and enterococcus spp), and therefore must be interpreted cautiously. 5. the interferon-gamma release assay (igra) serum test is not recommended for diagnosis of acute tracheobronchitis disease, although a positive result is an indication of latent disease or recent infection and a useful screening test if baseline igra testing is performed prior to transplantation. 25 6. episodes of airway colonization are not recorded as infections. 7. histologic representation of chronic graft rejection may not impact the diagnosis of bacterial pneumonia. therefore, it is not included as criteria for pneumonia definition. 26 8. the definition of "possible pneumonia" category allows recording of pneumonia after lung transplantation even if required diagnostic procedures were missed, which may occur with prior anti-microbial treatment or delay in diagnostic testing, etc. 9. in lung transplant recipients, it is desirable to always give additional information if evidence of acute graft rejection exists either by clinical or by histopathologic diagnosis. 10. it is possible to have concurrent infections-pneumonia with sinusitis or anastomotic tracheobronchitis. 11. quantification of organisms in bal is not considered essential for the diagnosis of ventilator-associated pneumonia (vap). 27 however, invasive diagnostics may help withdraw anti-bacterial therapy, which may prevent further emergence of multi-resistant organisms in future. 28,29 12. the category of bacterial tracheobronchitis is classified into probable and proven categories. they can only be diagnosed in the presence of bronchoscopic findings. we have refrained from using the term microbiogically negative tracheobrochitis as it requires more evidence. 13. endobronchial stent-associated tracheobronchitis or bronchial anastomotic infections, both fungal and bacterial, are categorized as probable (table 2 ). 14. no attempt is made to redefine atypical mycobacterial infections or pulmonary tracheobronchitis in lung transplant recipients and the use of existing definitions from european and north american societies are encouraged until further data emerge. 30 -33 15 . it is preferable to document the use of antibiotics in patients with pneumonia at the time of culture data collection. cardiothoracic transplant recipients are at an increased risk for viral infections with severe clinical sequelae. some viral infections have unique considerations and implications in cttx recipients. the definitions for these viral infections are specifically addressed herein and may be used for epidemiologic, research or registry purposes in cttx recipients. many other non-respiratory viral infections present similarly across sot recipients. diagnosis and management of these viral infections have been addressed adequately in other guidelines 1 and therefore will not be addressed herein. respiratory viral infections, including newly emerging viruses, occur frequently in lung transplant recipients. 34 some epidemiologic studies have suggested an association between respiratory viral infection and the development of bronchiolitis obliterans syndrome (bos). [35] [36] [37] [38] [39] [40] these studies yielded mixed results and the association between respiratory virus infection and bos remains unclear. 41, 42 the recent availability of molecular diagnostics, including pcr and multiplex gene techniques for the recovery of many viruses simultaneously from a single specimen, increased the recovery of pathogens in respiratory infections that previously were considered to be of undetermined etiology. 42 other viruses have been identified and are of uncer definitions for cmv infection and disease, especially for use in research, have been reported in the literature and used in other studies. 1, 4 the methodology for cmv recovery has shifted at many centers over the past decade from conventional viral culture methods and antigenemia toward quantitative molecular diagnostics, including pcr and hybrid capture assays. [45] [46] [47] however, the issues related to the recovery of cmv in bal fluid in the absence of histopathologic evidence of cmv remain unresolved, 48 and investigations are ongoing to resolve this issue. asymptomatic viral shedding in the upper oropharynx by cmv is distinguished from active cmv disease in these definitions and will assist in further assessing the role of cmv in cttx. in early studies, the recovery of cmv by viral culture in the absence of tissue diagnosis was considered diagnostic of cmv pneumonitis. 49, 50 however, further studies did not suggest that cmv recovery from bal was predictive of subsequent cmv pneumonitis. [51] [52] [53] [54] [55] with the advent of sophisticated molecular diagnostics, the recovery of cmv from bal became more specific and reproducible compared with conventional or shell-vial culture, 53 and additional studies suggested that cmv viral load in bal fluid may be correlated with invasive cmv pneumonia. 2, 56, 57 however, the utility of cmv viral load in bal in predicting cmv pneumonitis remains uncertain in studies to date. • molecular diagnostics (from whole blood, plasma, bal or tissue). -quantitative dna pcr or hybrid capture assays. -qualitative pcr. -genotypic resistance testing. • antigen pp65. • viral culture (conventional or shell-vial centrifugation). • in situ immunohistochemistry. • serology: not recommended for diagnosis. definitions for respiratory viral infections are given in table 3a and 3b. cttx recipients in general and lung transplant recipients in particular have the highest risk of mold infection. 58 recently published data suggest the cumulative incidence rate at 1 year to be 8%. 58, 59 among mold infections, the overwhelming majority of infections are due to aspergillus spp, followed by scedopsorium spp and zygomycetes. 60 despite the advancement in anti-fungal therapy, mortality remains at approximately 29% in aspergillus infections. 60 candida species was noted to be a major pathogen during the early cttx period, although it is rarely seen in lung transplant recipients in the current era. 61 although the incidence of invasive candidiasis has remained low in lung transplant recipients, this was the most common fungal infection noted in heart transplant recipients. 60 fungal infections in lung transplant recipients have certain characteristics that make them unique compared with other sot recipients as well as other immunocompromised hosts. this includes the presence of certain risk factors, such as airway ischemia and native lung or unique clinical syndromes, including tracheobronchitis, bronchial anastomotic infection and colonization-syndromes observed only in lung transplant recipients ( figure 2 ). 62 rejection syndromes in lung transplant recipients further complicate the clinical presentation. diagnosis of fungal infection based on histology alone may not be as accurate due to the concomitant presence of acute or chronic rejection in these individuals. 24 similarly, it is not only the unique clinical syndromes of fungal infection that set them apart from the other immunocompromised hosts, but the diagnostic utility of non-invasive testing also is different. serum galactomannan has markedly lower sensitivity (30%) in lung transplant recipients as compared with other immunocompromised hosts. 63, 64 similarly, the sensitivity of other serologic markers such as serum cryptococcal or coccidiodal antigen, histoplasma urine antigen may be variable. [65] [66] [67] [68] the use of bal for gm has resulted in sensitivities of ͼ66% when a 0.58 or 0.66 optical density (od) index was used as a cutoff. 69,70 a higher cutoff (1.5 od) yielded better results in one study. 71 we suggest the use of bal gm in the diagnosis of invasive aspergillosis (ia) in lung transplant recipi-ents. similarly, fungal pcrs, especially from bal specimens, are more likely to be less sensitive for the diagnosis of disease than bal specimens from other immunocompromised hosts owing to colonization of the airways. cell wall components of fungi have also been used in the diagnosis of fungal infections. currently available ␤-glucan is non-specific and is negative in cases of cryptococcosis and zygomycosis. 72 in a recent study of lung transplant recipients, serum ␤-d-glucan sensitivity was reported to be 93%, whereas specificity was merely 71%. 73 the mycoses study group (msg) and the european organization for research and treatment (eortc) recently updated the definitions of fungal infections in immunocompromised hosts. 3 these definitions represent an excellent attempt to standardize the reporting of fungal infections in studies. however, they fail to address the unique nature of clinical syndromes in lung transplant recipients, particularly colonization, tracheobronchitis and bronchial anastomotic infections. 8, 74, 75 also, the radiologic presentation of invasive mycoses in cardiothoracic organ transplant recipients may not conform to the classical "halo sign" presentation in neutropenic or stem a in the absence of biopsy categorize as probable: in the presence of histologic findings of both acute rejection and fungal invasion it should be classified as acute rejection with proven fungal infection. b the presence of mosaic appearance and ground-glass opacity may represent development of bronchiolitis obliterans syndrome or obliterative bronchiolitis. c isolation of non-pathogenic molds in culture (e.g., cladosporium spp, phialemonium, chaetomium, cunninghamella, syncephalastrum, curvularia, dactylaria, graphium or phialophora) or other non-pathogenic fungi [e.g., penicillium (non-marnefii), paecilomyces or basidiomyctes] do not qualify for the "probable" category. they should only be considered in the "proven" category. cell transplant recipients ( figure 2 ). 76 moreover, the definitions do not account for the differences in the sensitivity of serologic tests, particularly galactomannan in lung transplant recipients. 69, 70, 77, 78 in addition, the category of possible fungal infection might not be applicable in lung transplant recipients owing to a multitude of possible diagnoses in these patients. the american society of transplantation (ast) also put forward a set of definitions to be used in the study of these infections in sot recipients. 1 the ast definitions do take into account some unique clinical syndromes in lung transplant recipients but lack the detailed description of clinical syndromes. reported studies of fungal infections in lung transplant recipients used diverse definitions. 14,79 -83 the following sets of definitions are proposed to standardize the reporting of fungal infections, particularly mold and yeast (endemic mycoses, candida spp and cryptococcus spp) infections in cttx recipients, especially among general and lung transplant recipients. the isolation of non-pathogenic molds or other non-pathogenic fungi in bal or sputum is not believed to satisfy the microbiologic criteria for the diagnosis of probable invasive fungal infections in these patients without histologic confirma-tion (tables 4a and 4b) . however, these definitions of fungal infections do not address pneumocystis jiroveci infection, which has previously been adequately defined for use in cttx. 1 • direct examination by light microscopy (gram, giemsa and calcofluor stains). • culture. • histopathology: routine stains (hemotoxylin-eosin), special (gomori methenamine silver, mucicarmine, periodic acid-schiff), direct immunofluorescence and in situ hybridization). histopathologic diagnosis is useful in establishing the diagnosis of endemic fungi because of their distinctive morphology. 3 however, confusion may occur when attempting to differentiate the hyaline molds that commonly cause invasive disease. 84 fusarium spp and scedosporium spp cannot be distinguished from aspergillus spp in tissue sections and even the zygomycetes, which are morphologically quite distinct from fusarium spp, the presence of mosaic appearance and ground-glass opacity may represent development of bronchiolitis obliterans syndrome or obliterative bronchiolitis. a in the absence of biopsy categorized as probable: in the presence of histologic findings of both acute rejection and fungal invasion it should be classified as acute rejection with proven fungal infection. b isolation of non-pathogenic molds in culture (e.g., cladosporium spp, phialemonium, chaetomium, cunninghamella, syncephalastrum, curvularia, dactylaria, graphium or phialophora) or other non-pathogenic fungi [e.g., penicillium (non-marnefii), paecilomyces or basidiomyctes] do not qualify for the "probable" category. they should only be considered in the "proven" category. definitions of fungal pneumonia, tracheobronchitis, bronchial anastomotic infection and colonization in cttx are given in tables 4a, 4b, 4c , and 4d, respectively. non-cttx-specific infections, such as urinary tract infection (uti), surgical site infection (ssi), bloodstream infection (bsi), infective endocarditis (ie), clostridium difficile infection (cdi) and skin and soft tissue infections (sstis), are not included herein. 2,86 -94 the consensus opinion of the ishlt id council encourages the use of previously published international definitions for these infections, which have been well established outside of the cttx population. the use of these standard definitions will allow for intercenter comparisons of rates and types of infections 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surveillance of clostridium difficile-associated disease clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 update by the infectious diseases society of america diagnosis and management of complicated intra-abdominal infection in adults and children: guidelines by the surgical infection society and the infectious diseases society of america practice guidelines for the diagnosis and management of skin and soft-tissue infections key: cord-299261-ew99nraq authors: cipriano, l. e.; haddara, w. m. r.; zaric, g. s.; enns, e. a. title: impact of university re-opening on total community covid-19 burden date: 2020-09-18 journal: nan doi: 10.1101/2020.09.18.20197467 sha: doc_id: 299261 cord_uid: ew99nraq purpose: post-secondary students have higher than average contacts than the general population due to congregate living, use of public transit, high-density academic and social activities, and employment in the services sector. we evaluated the impact of a large student population returning to a mid-sized city currently experiencing a low rate of covid-19 on community health outcomes. we consider whether targeted routine or one-time screening in this population can mitigate community covid-19 impacts. methods: we developed a dynamic transmission model of covid-19 subdivided into three interacting populations: general population, university students, and long-term care residents. we parameterized the model using the medical literature and expert opinion. we calibrated the model to the observed outcomes in a mid-sized canadian city between march 1 and august 15, 2020 prior to the arrival of a relatively large post-secondary student population. we evaluated the impact of the student population (20,000 people arriving on september 1) on cumulative covid-19 infections over the fall semester, the timing of peak infections, the timing and peak level of critical care occupancy, and the timing of re-engaged social and economic restrictions. we consider multiple scenarios with different student and general population covid-19 prevention behaviours as well as different covid-19 screening strategies in students. results: in a city with low levels of covid-19 activity, the return of a relatively large student population substantially increases the total number of covid-19 infections in the community. in a scenario in which students immediately engage in a 24% contact reduction compared to pre-covid levels, the total number of infections in the community increases by 87% (from 3,900 without the students to 7,299 infections with the students), with 71% of the incremental infections occurring in the general population, causing social and economic restrictions to be re-engaged 3 weeks earlier and an incremental 17 covid-19 deaths. scenarios in which students have an initial, short-term increase in contacts with other students before engaging in contact reduction behaviours can increase infections in the community by 150% or more. in such scenarios, screening asymptomatic students every 5 days reduces the number of infections attributable to the introduction of the university student population by 42% and delays the re-engagement of social and economic restrictions by 1 week. compared to screening every 5 days, one-time mass screening of students prevents fewer infections, but is highly efficient in terms of infections prevented per screening test performed. discussion: university students are highly inter-connected with the city communities in which they live and go to school, and they have a higher number of contacts than the general population. high density living environments, enthusiasm for the new school year, and relatively high rates of asymptomatic presentation may decrease their self-protective behaviours and contribute to increased community transmission of covid-19 affecting at-risk members of the city community. screening targeted at this population provides significant public health benefits to the community through averted infections, critical care admissions, and covid-19 deaths. the covid-19 pandemic presents a substantial public health challenge for local, national, and international communities because the virus is highly transmissible, 1-3 including prior to symptom onset, 4, 5 and infections initially present with a wide range of non-specific and sometimes mild symptoms. 6, 7 the relatively high rate of hospitalization and need for critical care among severe cases can quickly overwhelm community health care resources and result in substantial mortality. 8, 9 many communities initially responded to covid-19 with school closures and stay-at-home orders, which included closure of university campuses and conversion of all in-person instruction to online formats. over the summer, universities began announcing plans for the fall term. some universities opted to operate fully online for the fall. 10 others announced plans to partially or fully re-open campus and welcome students back for in-person instruction with covid-19 mitigation strategies in place. these strategies included polices around mask wearing, limiting large gatherings, access to covid-19 testing, reduced dormitory occupancy, and accommodations for isolating and quarantining students. 11 while universities have the autonomy to make decisions about the level of on-campus activities offered to their students, their decisions have implications for the communities in which these campuses are located. university students live, work, and socialize both on and off campus, resulting in significant potential for on-campus covid-19 outbreaks to spill over into the community and vice versa. 12 infectious diseases can spread rapidly through a university campus, as evidenced by outbreaks of serogroup b meningococcal disease, mumps, 13 and the novel h1n1 influenza virus that emerged in 2009. 12, [14] [15] [16] surges of covid-19 cases have already been observed on the first campuses to re-open this fall, 17 prompting many universities to abruptly change their fall semester plans. 18 after 177 students tested positive for the novel coronavirus linked to at least four separate clusters, the university of north carolina abruptly moved all undergraduate classes online after only a week of in-person instruction. 19 high-density housing, including multiple roommates and shared facilities like bathrooms, as well as high levels of social activity puts the university population at particular risk for infectious disease transmission. 15 furthermore, past experience with the 2009 h1n1 pandemic indicates compliance with recommended public health precautions may be sub-optimal; despite public health guidance not to attend classes and other activities while ill during the h1n1 outbreak at a us university, half of students indicated interacting with a symptomatic individual in a classroom and nearly one-quarter indicated interacting with a symptomatic individual at a party or social activity. 15 given the unique features of university communities, several studies have modeled covid-19 transmission dynamics specifically on university campuses and evaluated potential mitigation strategies. 18, [20] [21] [22] [23] [24] these studies used mathematical models of viral transmission dynamics, tailored to reflect a university context, in order to evaluate testing and contact tracing strategies, largely focusing on the question of how frequently to test asymptomatic students. all analyses concluded that frequent testing (sometimes multiple times a week) would be needed to contain covid-19 outbreaks on campus. high frequency testing has been adopted by a number of public and private universities including, for example, the university of illinois (twice per week by saliva testing 25 ), colby college (twice per week by nasopharyngeal swab 26 ), cornell university (twice weekly for students and faculty with student contact by self-collected anterior nares sampling 27 ) , and harvard university (one to three days a week for students, staff, and faculty by saliva testing with frequency depending on types of on-campus activity 28 ). however, most universities do not have the resources or infrastructure to support high-frequency testing. 26 furthermore, while some of these studies did include infections among students arising from offcampus community contact, these studies did not consider the impact of university student infections and university administration prevention and management decisions on the broader community in which that campus is situated. university students themselves may be at lower risk of severe covid-19 disease due to age, but high infection rates on campus may spill over into the broader community, whose members are at higher risk for adverse covid-19 outcomes. it is therefore important to quantify the expected impact of the arrival of a relatively large number of university students on the broader community in terms of incremental infections, hospitalizations, and covid-19 mortality. through the many interactions between the student population and the city in which they reside, covid-19 mitigation policies targeted at university student communities and adopted by university leaders may have substantial public health implications for those in the surrounding community. we developed a dynamic transmission model of covid-19 to estimate the health impacts and health care resource demand in a covid-19 outbreak in a representative mid-sized city with a relatively large destination college campus. we assumed a city experiencing a low level of covid-19 activity going into the semester and explore how the on-campus arrival of the student population impacts covid-19 health outcomes in the community. we consider different assumptions about student behaviours related to physical distancing and mask wearing, as well as the mitigating effects of targeted routine and one-time covid-19 screening in the university population. we developed a dynamic compartmental model to simulate infection dynamics and health resource use of a representative mid-sized city with a population of 500,000 going into fall after experiencing low rates of covid-19 infections in the summer. we divided the population into three sub-populations: long-term care (ltc) residents, university students, and the general population (everyone other than ltc residents and university students). we evaluated covid-19 health outcomes in the city between august 15 and december 31 (4.5 months) with and without the introduction of 20,000 university students on september 1. we explored how the covd-19 risk and prevention behaviours of the general population and the student community affect the incremental covid-19 burden attributable to the arrival of the student population. under different scenarios of community physical distancing effort and routine testing in students, we calculated the number of infected individuals, peak hospital resource demand, and number of deaths over time. institutional ethics review was not required for this modeling study as human subjects were not involved. a schematic of the model is presented in figure 1 . in the model, susceptible individuals may become infected through interaction with infected individuals who may or may not be aware of their infection status. infection has a pre-symptomatic phase in which an infected individual can transmit the infection to others. 4, 5, 29 individuals may become aware of their infection status through symptom-based surveillance, contact tracing, or routine testing of asymptomatic and mildly symptomatic individuals. individuals aware of their infection status with mild or moderate symptoms isolate at home to reduce disease transmission. some patients develop severe symptoms requiring hospitalization or critical symptoms requiring mechanical ventilation (mv) in an intensive care unit or renal replacement therapy (rrt). patients receive medically indicated care, unless resource demand exceeds capacity. when hospital capacity for a medically indicated resource has been reached, patients receive the next-best available care. we estimated model parameters, including the duration of time spent in each health state, the infectiousness of covid-19, demand for hospital resources and disease mortality conditional on disease severity, and the effectiveness of covid-19 prevention strategies using the peer-reviewed literature, pre-published reports, and expert opinion ( table 1) . we calibrated uncertain model inputs to the observed hospitalization and mortality outcomes in london, ontario, canada, a mid-size city with a large university population, between march 1 to august 15. full details about the model structure and input parameter estimates are presented in the supplemental methods. we first establish the epidemic starting conditions in the city on august 15 before the potential arrival of students for fall semester. based on calibration to covid-19 outcomes in london, on, at the start of the simulation, 40 individuals in the general population had active covid-19 infections, 13.8 were exposed but not yet infectious, and 2,476 individuals had already recovered; thus, 493,937 individuals were susceptible at the beginning of the simulation. in sensitivity analysis, we vary the number of active covid-19 infections in the general population at the start of the simulation. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09. 18.20197467 doi: medrxiv preprint in our analysis, 0.7% (3,500 individuals) of the population live in long-term care (ltc). based on the model calibration we estimated that 75 ltc residents were recovered as of august 15. we assumed that there were no active infections in ltc residents on august 15. we assume that 20,000 university students arrive on september 1. in the base case, we conservatively assume that no students are infected with covid-19 upon arrival to campus. we vary this assumption in sensitivity analysis. a schematic of the health states and transition times for infected individuals is presented in appendix figure 1 . we assumed a mean incubation period, the time from exposure to symptom-onset, of 5.6 days (observed median of 5.1 days [95%ci 2.2, 11.5]). 30 in total, we estimated the average duration of infectiousness in asymptomatic and mild or moderate infections to be 10 days, including 2.5 days prior to symptom onset in individuals who do become symptomatic. 4, 31, 32 we assumed that patients with severe and critical symptoms remain infectious until recovered, but that patient isolation protocols prevent transmission for those admitted to hospital. we estimated hospital length of stay and mortality based on a report of over 20,000 hospitalized patients in the uk. 33 we estimated length of stay and mortality for patients receiving critical care using the uk intensive care national audit and research centre report describing the care and outcomes of 10,118 critical care covid-19 patients. 34 for ltc residents who are and are not hospitalized, we estimated the case fatality rate to be 47.4% and 25.5%, respectively, based on the observed outcomes in 680 canadian ltc covid-19 patients. 35 combining the assumptions about disease severity and severityspecific mortality rates, the overall infection fatality rate in our model was 25.1% for ltc residents, 1.3% for the general population, and 0.06% for university students. among hospitalized cases, the fatality rate was 24.2% for the general population, and 5.2% for university students. we did not consider mortality from causes other than covid-19 in the model. general population: based on an extrapolation of the 2008 polymod study in europe to reflect network structure of the canadian population, the average number of contacts per person in canada is 12.6 per day, of which 1.0 contacts is aged 20-24. 36 assuming that the university student population adds 20,000 individuals aged 20-24 to a community with an otherwise typical canadian age distribution, university students would comprise 38% of the population of people aged 20 to 24 in the community. as a result, we assume that a person in the 'general population' has contact with 0.38 university students per day. we calculated the average number of ltc contacts by calculating the number that would balance the staff and visitor contacts estimated for ltc residents, resulting in 0.1 ltc contacts per person in the general population. long-term care residents: we estimated that there are 19.9 resident-resident contacts per day (95% ci: 11.3 -28.5) and 13.7 resident-staff contacts per day (95%ci: 11.4, 15.9) using a canadian study of longterm care residents and staff (personal communication: s. moghadas). 37, 38 this study did not capture . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint resident-visitor contacts; we assumed 0.48 visitors per day based on the distribution of visit frequency in the 2012 ohio nursing home family satisfaction survey. 39 thus, in total, ltc residents have 14.2 contacts with the general population each day. we assumed no direct contact with university students. university students: in our base case analysis, we assume that students have 23.7 contacts per day, based on a study at the university of antwerp, 40 and that 60% of those contacts are with other university students based on the age distribution of student's reported contacts, 40, 41 with the remainder being with members of the 'general population' which includes staff and faculty of the university. because studies evaluating university student contact patterns often occur during late fall and winter months, the contact patterns identified may not be fully representative of student contact rates at other times of the year. in sensitivity analysis we explored higher rates of student-student contacts in the first few weeks of the semester. using exponential regression, we empirically estimated a basic reproduction number, r0, of 3.0 in the general population based on ontario's reported cases between march 7 to march 22. 42 using an average duration of infectiousness of 10 days and an average number of close contacts per person of 12.6, 36 we calculate the probability of transmission between a susceptible and an infected person in close contact, in the absence of any interventions, to be 0.024. interventions such as physical distancing, which reduces the average number of contacts between susceptible and infected people, and mask wearing, which reduces the probability of disease transmission between contacts, can reduce the expected number of infections. we assume the effectiveness of cloth masks in reducing disease transmission is 40% based on a german study evaluating the effectiveness of real-world mask use. 43 for people who are aware of their infection status and in home isolation, we assume a 90% reduction in contacts, which is at the high end of observed adherence to quarantine instructions in past epidemics. 44, 45 we subdivided the general population into two groups based on their intensity of covid-19 prevention behaviours. based on behaviours reported in an angus reid poll of canadians, taken in the first week of august, 46 we estimated that 'high-intensity physical distancers', representing 50% of the general population initially, reduce their average number of contacts by 75% (from 12.6 to 3.2 contacts per day) and that 86% of their remaining contacts are protected by a cloth mask. we assumed that the remainder of the population are not reducing their contacts, but are using a cloth mask to protect 38% of those contacts. 46 in the base case, we assumed that university students initially reduce their contacts by 24% (from 23.7 to 18.0 contacts), which approximates a 75% reduction in contacts for the 32% of 18-24 year-olds who reported substantial covid-19 prevention effort in the angus reid poll (32% x 75% = 24% reduction). in this same survey, 57% of 18-24 year-olds reported wearing a mask indoors with people outside their household and we use this as the base case level of mask wearing in the university student population. 46 responsive behaviour triggers: we assumed that the general population and university students respond to covid-19 outcomes in the community. in practice, this response may be voluntarily adopted due to public concern over reported increases in covid-19 cases, hospitalizations, and/or deaths or . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint imposed through policies that re-establish the social and economic restrictions utilized in the earlier phases of the pandemic. we included two triggers that would cause both the general population and university students to increase their adoption of protective behaviors: a high level of covid-19 patients in critical care and a high number of covid-19 deaths. the critical care trigger was set based on critical care capacity. the overall critical care capacity in ontario is 14.2 beds per 100,000 population. 47 thus, in a city of 500,000, normal critical care capacity would be about 70 beds. while additional capacity can be created by seconding resources and personnel from other hospital services, 48 based on expert opinion, substantial reductions in the provision of other types of health care (such as cancelling elective surgeries) would need to be undertaken if more than 30 critical care beds were used by covid-19 patients. therefore, we set one of the responsive behaviour triggers to activate when there are 15 covid-19 patients in critical care, representing 50% of the capacity available to covid-19 patients without modifying access to other health care services. in the base case, the proportion of the general population who are 'high intensity physical distancers' increases by 0.5% each day if the number of covid-19 patients in critical care exceeds 15 and by an additional 1.0% each day if the number of covid-19 deaths in the past 10 days exceeds 10, up to a maximum of 80% participation in high-intensity physical distancing. similarly, we assume students' reduction in contacts increases at the same rate in response to the same triggers, but up to a maximum of a 50% reduction in contacts (23.7 contacts to 11.9 contacts). for the general population and university students, we assumed the minimum time from symptom onset to diagnosis to be 2.1 days, which is consistent with the minimum time needed to self-assess, seek medical attention, and receive diagnostic results. 7 the observed median time to diagnosis through symptom-based surveillance alone was 4.6 days (95%ci: 4.2, 5.0) and symptom-based surveillance in combination with contact tracing efforts was 2.9 days (95%ci 2.4, 3.4) in shenzhen, china. 49 from this, we estimated that symptom-based surveillance and contact tracing results in a daily probability of diagnosis of 15.8% for symptomatic infections and a daily probability of detection (from contact tracing) of 4.1% for asymptomatic infections. this combination of assumptions resulted in approximately 22% of infected individuals being identified, consistent with the overall rates of diagnosis implied by preliminary serology data in ontario. 50 we considered policy alternatives of routine screening for covid-19 in university students at various screening frequencies, including every 14, 10, 7, 5, and 3 days. we also considered the value of a onetime universal screening three weeks after student arrival. we identified the date of the one-time testing by identifying the date that minimized the number of total infections over the semester. we assumed that testing will be performed with the standard covid-19 nasopharyngeal swab followed by pcr analysis with a test sensitivity of 72.1%. 51, 52 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint without the introduction of the student population, the base case assumptions for the general population and ltc residents leads to a total of 3,900 infections over 4.5 months (august 15 to december 31). in this scenario, infections, hospitalizations, and daily deaths do not peak until the new year (appendix table 1 ). demand for critical care (mechanical ventilation or renal replacement therapy) peaks at 27 beds early in the new year, and a total of 31 covid-19 deaths occur between august 15 and december 31 (60 deaths by january 31, 2021). the timing and magnitude of the city's covid-19 outbreak, excluding any impacts from students, is determined by the initial number of covid-19 infections in the community, the level of participation in physical distancing, the responsiveness of the community to increasing critical care cases and covid-19 deaths, and the proportion of contacts that are protected with mask wearing (appendix figures 5-7) . in the base case, we conservatively assumed that students would bring no undiagnosed infections of covid-19 to the community and would immediately engage in physical distancing efforts that resulted in a similar average contact reduction to the general population (reduction of 24%, from average of 23.7 contacts to 18.0 contacts). even so, university students continue to have a higher average number of contacts than members of the general population. as a result, in this base case scenario, the introduction of students to the community increases the total number of infections by 3,399 infections, representing an 87% increase (from 3,900 to 7,299) (figure 2) . only 28% (960) of these incremental infections occur in the student population (representing 4.8% of students becoming infected). of the remaining 72% of incremental infections, 2,428 occur in the general population (0.5% of the susceptible general population), and 11 occur in long-term care (0.3% of the susceptible ltc population). the larger absolute increase in infections in the general population occurs due to the connectivity of the university community with the general population and the relative size of the general population. the increase in infections among ltc residents, despite our assumption that there are no direct contacts between university students and ltc residents, occurs due to the increase in infections in the general population. the higher number of infections results in an increase in hospitalizations, demand for critical care, and covid-19 deaths. peak critical care occupancy increases by 48% (from 27 to 40 beds). these outcomes include the mitigation effects of the responsive behaviour of the community to seeing high levels of covid-19 hospitalizations. the introduction of students to the community also moves up the timing of responsive behaviours, with the threshold of 15 covid-19 patients in critical care being reached 3 weeks earlier (appendix table 2 ). if some students arrive exposed or asymptomatically infected, the total number of infections occurring over the course of the semester increases. for example, if 10 students arrive infected, the number of infections increases by 1,047 over the base case. the impact of students arriving already exposed or infected in the community is most substantial on the timing of peak infections, peak hospitalizations, peak critical care utilization, and the timing of responsive behaviour triggers. compared to the scenario without the introduction of the student population, responsive behaviours are triggered 6.5 weeks earlier if 10 students are infected when they arrive (appendix table 3 ). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint our estimates of average student contacts per day were based on surveys completed at times other than during the first few weeks of the school year, which normally involve a large number of social activities. even if muted, the first few weeks of the new academic year may still result in substantially more contacts than a typical pre-covid era day. therefore, we explored the consequences associated with students having an increased number of student contacts for one or two weeks upon arrival to the community. for example, if students simply delay implementing physical distancing for two weeks, the total number of infections attributable to the introduction of the students into the community increases by 4,713 infections (from 3,900 to 8,613 infections). in this scenario, the arrival of students would result in a 120% increase in the number of infections the community. as in the base case, the majority of these incremental infections occur in the general population (figure 2 ). if students have twice the pre-covid era number of contacts with other students for two weeks (37.9 contacts per day, 28.4 of whom are other students), the total number of infections in the community increases by 6,394 infections representing a 164% increase in the number of infections the community would expect without the students and leading to an additional 72 covid-19 deaths (appendix figure 8 , appendix table 4 ). delays in the implementation of contact reductions, or short-term increases in the number of student-student contacts increases demand for critical care resources and shortens the time until critical care beds dedicated to covid-19 patients exceeds 30 beds (figure 2b) . the impact on total infections is mitigated by the earlier activation of responsive behaviour triggers, which occurs 8.3 weeks after the arrival of the students and 8 weeks earlier than without the arrival of the student population. because young people have a high rate of asymptomatic and mild presentation, routine testing of students has been proposed for university campus settings. testing students every 28 days results in very little reduction in the number of infections but requires a large number of tests (714 students tested per day). testing every 14 days, as is recommended for staff at long-term care facilities, 53 reduces the number of infections in the student population from 960 to 735, a 23% reduction, and reduces the overall number of infections due to the introduction of the university student population by 22% (from 3,399 to 2,653) (figure 3) . more frequent testing reduces infections further. testing students every 5 days reduces the number of infections among the student population by 44% (from 960 to 541) and reduces the total number of infections due to the introduction of the university student population to the community by 41% (from 3,399 to 2,007). routine testing has greater impact in the scenarios when students engage in a shortterm increase in the number of contacts early in the term. in the scenario in which students double their student contacts for the first two weeks of term, testing every 5 days reduces the number of infections in the student population by 47% (from 1,990 to 1,047), reduces the total number of infections due to the introduction of the university student population by 42% (from 6,394 to 3,732), and delays the activation of responsive behaviour triggers by 1 week (appendix table 5 ). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint routine testing of students also averts critical care admissions and covid-19 mortality in the general population, because approximately two-thirds of averted infections are prevented in the general population ( table 2 ). in the base case analysis, in which students immediately reduce their contacts compared to a pre-covid-19, routine screening every 5 days averts 16.5 critical care admissions and 12.8 deaths. in scenarios in which students have short term increases in their contact behaviours or lower levels of contact reduction, routine testing every 5 days averts more than 29 critical care admissions and greater than 20 covid-19 deaths. sensitivity analysis revealed that routine testing of university students was more valuable when students have a higher rate of asymptomatic infections ( table 2 ) and in scenarios in which the differences in transmission risk between the university students and the general population were greater. for example, in a scenario in which the city had a high level of engagement in physical distancing routine screening of the student population can avert a large number of infections because in these scenarios the city expects very little covid-19 transmission without the introduction of the student population ( figure 3c) . conversely, in scenarios in which the city is engaged in a low level of physical distancing and so expects a large number of infections with or without the student population, the difference in risk profile between the city and the university populations decreases, as does the benefits of targeting prevention efforts at the university population. routine testing to identify and isolate asymptomatic infections for the purposes of reducing community transmission risk requires a large number of tests each day and may strain community testing resources. we also evaluated the benefits of a one-time universal screening event occurring three weeks after the students arrive. compared to routine testing every 5 days, which would require more than 400,000 tests to be performed over the semester, this strategy would only require 20,000 tests. through the isolation of identified cases, one-time testing is able to immediately decrease the daily number of new infections in the student population and, so, indirectly in the general population (appendix figure 9 ). in the case that students double their contacts with other students for a period of two-weeks, this strategy prevents 361 infections (122 infections in students, 238 infections in the general population, and 1 infection in ltc residents), 4.0 critical care admissions, and 3.1 covid-19 deaths ( table 2) . one-time screening does not significantly impact the timing of peak infections, resource utilization, or the time that responsive behaviour triggers are activated (appendix table 5 ). we performed extensive sensitivity analysis exploring the impact of general population and student population covid-19 prevention behaviours on the incremental impact of introducing students into the community. the negative impacts of introducing the student population can be partially mitigated through high uptake of covid-19 preventive behaviours in the student population including high rates of contact reduction or if the rate of mask wearing significantly exceeds the level reported by 18 to 24 year-olds (appendix figure 10 , appendix table 6 ). for example, if students immediately reduce their contacts by, . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint on average, 30% (from 23.7 to 16.6 contacts per day) and wear masks to protect 65% of their remaining contacts, the incremental number of infections attributable to the arrival of the student population can be reduced to 1,698 infections (from 3,900 to 5,598), representing only a 44% increase over the number of infections the community would expect without the students, and delays the activation of responsive behaviour triggers by 1 week. the magnitude of the impact of the introducing the student population is also determined by the covid-19 prevention behaviours of the general population. counter-intuitively, the relative impact of introducing the student population is greatest when the prevention efforts by the general population are high (appendix figure 11) . for example, when 60% of the general population are participating in high-intensity physical distancing, without students the number of new infections per day is nearly constant over time, resulting in a very low level of cumulative infections over the semester (total of 543 infections). introduction of the students results in 939 additional infections, more than doubling the total number of infections expected in the city without the addition of the student population (appendix table 2 ). in such a scenario, because the student population is an important determinant of city outcomes, the impact of routine covid-19 screening in the student population is greater ( figure 3c ). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint in this analysis, we consider the covid-19 impacts of the re-opening of a destination university in a midsized city with varying epidemiological contexts. without the return of university students, we devised several scenarios involving moderate to severe fall covid-19 waves based on difference levels of preventive behaviours in the general population. the return of a large number of students always worsens these waves, even under the conservative assumptions that arriving students do not introduce any new infections to a community and that they immediately adopt substantial covid-19 prevention behaviours. this is because university students have nearly double the number of contacts as the general population due to residing in shared or congregate living situations, working in the service sector, and higher levels of social activity. in the scenarios we considered, this increase in infections was substantial, potentially doubling of the total number of covid-19 infections in the city over the fall. notably, we found that more than two-thirds of the incremental infections attributable to the arrival of the student population occurred in the general population and, as a result, substantially impacted the city's health care resource needs, covid-19 mortality in the community, and accelerated the need for responsive behaviour which may be in the form of re-engaged social and economic restrictions. our base case finding that the return of university students would increase the number of infections by 87% is likely conservative. in this analysis, we assumed that no students arrive already infected, students do not engage in short term increases in contacts upon arrival, and that students respond to adverse community covid-19 outcomes by increasing the intensity of their prevention behaviours at the same rate as the general population. at the very least, it may take time for students to fully adopt protective measures; moving into dorms, orientation events, and start of semester social events (even if not officially sanctioned by the university) may result in higher-than-normal levels of contact for at least the first few weeks. in the analyses in which we consider short-term increases in the average number of student-student contacts, we show that a higher level of contact for just the first one or two weeks can dramatically increase the total number of infections experienced by the city over the semester. our analysis found that the majority of the increase in infections due to the arrival of students occurred in the general population, not in the student population itself. while university campuses may seem like isolated bubbles, the general community and university students are intertwined, as staff and faculty interact with students on campus and students interact with others off-campus in work, living, and social settings. previous studies modeling university populations did not account for infections in the broader off-campus community. [20] [21] [22] [23] [24] however, we have shown that including the general population when modeling covid-19 transmission on university campuses is critical, since this population bears the brunt of the incremental morbidity and mortality burden of covid-19. as a result, university policies that either discourage student return to the community, 54 such as offering coursework fully online, or mitigate covid-19 risks for students returning to campus, such as screening for covid-19 symptoms and routine covid-19 testing in asymptomatic individuals, can have substantial impacts on the city's covid-19 burden. imposing restrictions on students' off-campus social behavior may be practically difficult, necessitating modified behaviors in the general population in response to university reopening, such as additional reductions in social contacts to balance the increased risk of returning university students. for example, in the base case, the increase in infections due to student arrival could be mitigated if the proportion of the general population engaged in high-intensity physical distancing increased by 4.6% (from 50% to 54.6%). this illustrates the idea of "risk budgets", where increased risk . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09. 18.20197467 doi: medrxiv preprint in one domain of a community necessitates reducing risk in another to keep covid-19 impacts below desired thresholds. 55 our analysis indicates that routine testing of all students every 5 days can avert a substantial number of infections, critical care admissions, and covid-19 deaths. in the base case, we estimate that testing every 5 days prevents 16.5 critical care admissions and 12.8 deaths and, in the scenario in which students double their contacts with other students for two weeks, we estimate that testing every 5 days prevents 29.1 critical care admissions and 22.6 deaths. using the simplifying assumption that all deaths averted will be in 60-year-olds and a willingness to pay of $50,000 per life-year-gained, the economic value of these deaths averted is $13.4 million to $23.6 million, translating to a value of $33 to $59 per test. this calculation underestimates the benefits of testing by not accounting for savings due to averted critical care admissions and the community economic benefits of delaying social and economic restrictions; despite this, because we estimate the lab cost of nasopharyngeal testing for covid-19 at $80 per test at our center, high-frequency routine testing is likely only cost effective using batch testing strategies. alternatively, one-time universal testing of students after an initial burst of social activity among students may be more feasible operationally and economically. we estimated that this strategy can prevent 360 infections, 4.0 critical care admissions, and 3.1 covid-19 deaths corresponding to an approximate economic value of $3.3 million or $164 per test. this strategy is most effective at changing the trajectory of new infections if testing occurs after a short-term period of high social activity and is less effective if students have consistent but lower levels of contact reductions (e.g., immediately reducing their contacts, but by only 15% instead of the 24% in the base case). an important limitation of our analysis is that we assume students with a covid-19 diagnosis will be willing and able to self-isolate effectively. however, it may be challenging for students to isolate from roommates or refrain from using shared facilities, like bathrooms and kitchens, without dedicated university-organized isolation facilities. 56, 57 furthermore, adherence to isolation guidance may be low, especially if the majority of infections in university students are asymptomatic or mild. during the h1n1 pandemic, a survey of symptomatic university students found that only 41% of students followed recommendations to stay home until well. 16 in the base case, we also assume that students are equally responsive as the general population to covid-19 outcomes in the community reducing their contacts in response to high numbers of critical care hospitalizations and deaths. in reality, university students may be less aware of the impacts of covid-19 on hospital resources and less concerned about covid-19 generally given their lower risk of adverse outcomes. the extent and speed with university students respond to hospitalizations and deaths in the local community will impact the number of infections experienced by the community and the benefits of routine testing in the student population. compared to other modeling studies of covid-19 on university campuses, the total number of infections and the number of infections averted by testing we estimate over the semester are modest. this is because we assume that both university students and the general population will increase their self-protective behavior (physical distancing) in response to high numbers of covid-19 hospitalizations and deaths, either through individual decision-making or adaptive community policies. these adaptive behaviors are more realistic than assuming a population will maintain the same behavior no matter the severity of local covid-19 conditions. thus, in our analysis, testing is being layered onto a robust and reactive mitigation response. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint our model includes only three sub-populations and so does not include many other aspects of agestructured mixing or age-dependant health outcomes. the model does not estimate the impact of covid-19 patient utilization on the provision or effectiveness of other health care services and the model does not account for death from causes other than covid-19. 58 the model includes community transmission by stratified random mixing but does not include additional imported index cases from other cities, which may occur into the general population or the student population, nor does the model include the stochastic consequences of super-spreading events. especially early on in an epidemic or when cases have been brought to very low levels, dynamics are sensitive to random outcomes in the number of new infections resulting from each case (e.g., 'patient 31' in south korea 59 and 'patient one' in italy 60 ). we developed a model-based analysis to estimate the impact of a relatively large student population on the covid-19 outcomes of a mid-sized city with relatively few cases of covid-19 prior to the return of students. our analysis is relevant to a number of mid-sized cities in north america with relatively large university and college populations. because university students have substantially more contacts than the general population, due to congregate living environments, high-density social activities, and disproportionate employment in the service sector, the introduction of university students substantially increases the number of covid-19 infections and decreases the time until responsive behaviours are activated. substantial uncertainty exists in the level of contact reduction that students will choose, or is feasible given their living, transit, and work situations. public health interventions, such as routine testing, targeted at this population prevents infections in the entire population, improving community health related and unrelated to covid-19. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint figure 1. model schematics of (a) covid-19 health states and (b) close contact interactions between population subgroups. the number of contacts between groups indicated on the schematic represent the average number of contacts per day in a pre-covid-19 era. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. table 4 . end of the two-week burst in contacts. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . in panel (a), students have an average 24% reduction in contacts compared to normal student social interaction behaviour (average of 23.7 contacts reduced to 18.0 contacts) immediately upon arrival with no shortterm increase in contacts; in panel (b), students double their contacts with other students for the first two weeks and then implement a 24% reduction in their contacts; in panel (c), students double their contacts with other students for the first two weeks and then implement a 24% reduction in contacts and 60% of the general population is participating in high-intensity physical distancing (compared to 50% in the base case and other scenarios presented in this figure). other outcomes for these scenarios are reported in appendix table 5 . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint 49 from this, we estimated that symptom-based surveillance and contact tracing results in a daily probability of diagnosis of 15.8% and the daily probability of detection from contact tracing of 4.1% in asymptomatic infections. c. among critical care patients, we estimate the ratio of patients requiring renal replacement therapy (rrt) to mechanical ventilation (mv) based on the uk intensive care national audit and research centre (icnarc) report describing the care and outcomes of 10,118 critical care covid-19 patients in the uk. in this report, 7,277 patients required mv and 2,673 required rrt, resulting in a ratio of 0.37 rrt patients per mechanical ventilation patient. 34 d. in canada, based on 63,800 covid cases in people who were not residents of long-term care facilities reported between february 23 and june 21, 20.3% of hospitalized patients received critical care; 35 this is also consistent with rates of critical care observed in the uk (22% overall hospitalized patients go to icu). 33 therefore, we estimate the ratio of 3.92 hospitalized without critical care patients per critical care patient. e. initially estimated using the same process as is described in footnote d. adjusted in calibration process to better fit the observed data (see supplemental methods). f. median and iqr presented in the cited primary work were transformed to mean (95%ci range) assuming a gamma distribution. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint table 2. infections averted in the general population with 5-day testing and one-time testing of students compared to a policy of no routine asymptomatic testing (symptom-based surveillance and contact tracing only). scenarios vary the proportion of infections in the student population that are asymptomatic and timing and level of students contact reductions. we calculate the expected number of critical care admissions averted and covid-19 deaths averted to be 1.7% and 1.32% of general population infections averted which includes hospitalizations and deaths which may occur after december 31 to all individuals infected prior to december 31. one-time testing three weeks after student arrival compared to no routine testing . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20197467 doi: medrxiv preprint high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2 pattern of early human-to-human transmission of wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia temporal dynamics in viral shedding and transmissibility of covid transmission interval estimates suggest presymptomatic spread of on behalf of the oxford covid-19 evidence service team. in patients of covid-19, what are the symptoms and clinical features of mild and moderate cases? : centre for evidence-based medicine report 8: symptom progression of covid-19. imperial college london covid-19 response team baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region epidemiology, clinical course, and outcomes of critically ill adults with covid-19 in new york city: a prospective cohort study us centers for disease control and prevention. considerations for institutions of higher education extensive geographical mixing of 2009 human h1n1 influenza a virus in a single university community college campus outbreaks require timely public health response how the h1n1 influenza epidemic spread among university students in japan: experience from shinshu university transmission of 2009 pandemic influenza a (h1n1) at a public university--delaware non-pharmaceutical interventions during an outbreak of 2009 pandemic influenza a (h1n1) virus infection at a large public university schools briefing: university outbreaks and parental angst college professors made models showing how bad covid-19 would be on campus. some administrators ignored them outbreaks drive u.n.c. chapel hill online after a week of classes. the new york times assessment of sars-cov-2 screening strategies to permit the safe reopening of college campuses in the united states covid-19 mathematical modeling for cornell's fall semester high covid-19 transmission potential associated with re-opening universities can be mitigated with layered interventions entry screening and multi-layer mitigation of covid-19 cases for a safe university reopening colleges plan for coronavirus testing, but strategies vary. wcvb news covid-19 and reactivation planning: surveillance testing coronavirus testing & tracing: unobserved self-administered testing risk for transportation of 2019 novel coronavirus disease from wuhan to other cities in china the incubation period of coronavirus disease from publicly reported confirmed cases: estimation and application virological assessment of hospitalized patients with covid-2019 features of 20 133 uk patients in hospital with covid-19 using the isaric who clinical characterisation protocol: prospective observational cohort study statistics canada. detailed preliminary information on confirmed cases of covid-19 (revised), public health agency of canada projecting social contact matrices in 152 countries using contact surveys and demographic data the effect of individual movements and interventions on the spread of influenza in long-term care facilities individual movements and contact patterns in a canadian long-term care facility the predictors of and motivations for increased family involvement in nursing homes social mixing patterns for transmission models of close contact infections: exploring self-evaluation and diary-based data collection through a web-based interface who mixes with whom? a method to determine the contact patterns of adults that may lead to the spread of airborne infections ontario ministry of health and long-term care. covid-19 case data face masks considerably reduce covid-19 cases in germany changes in contact patterns shape the dynamics of the covid-19 outbreak in china how to improve adherence with quarantine: rapid review of the evidence covid-19 compliance: one-in-five canadians making little to no effort to stop coronavirus spread critical care readiness: expanding nursing staff to support covid-19 epidemiology and transmission of covid-19 in 391 cases and 1286 of their close contacts in shenzhen, china: a retrospective cohort study blood tests indicate one per cent of ontario's population had covid-19 variation in false-negative rate of reverse transcriptase polymerase chain reaction-based sars-cov-2 tests by time since exposure comparative accuracy of oropharyngeal and nasopharyngeal swabs for diagnosis of covid-19. centre for evidence-based medicine ontario ministry of health and long-term care. covid-19 testing for long-term care home staff winter term will be online: provost's letter close the bars. reopen the schools. vox college quarantine breakdowns leave some at risk. the new york times a university had a great coronavirus plan, but students partied on. the new york times delayed access or provision of care in italy resulting from fear of covid-19 the korean clusters. reuters graphics updated coronavirus: inquiry opens into hospitals at centre of italy outbreak the guardian outbreak investigation of covid-19 among residents and staff of an independent and assisted living community for older adults in infections in residents of a long-term care skilled nursing facility asymptomatic sars-cov-2 infections: a living systematic review and meta-analysis probability of symptoms and critical disease after sars-cov-2 infection effect of acute renal failure requiring renal replacement therapy on outcome in critically ill patients acute renal failure in intensive care units--causes, outcome, and prognostic factors of hospital mortality; a prospective, multicenter study. french study group on acute renal failure key: cord-288930-h13cxuh3 authors: lim, faye j; wake, zoe v; levy, avram; tempone, simone; moore, hannah c; richmond, peter c; de klerk, nicholas; conway, nicholas t; keil, anthony d; effler, paul v; smith, david w; blyth, christopher c title: viral etiology and the impact of codetection in young children presenting with influenza-like illness date: 2016-07-20 journal: j pediatric infect dis soc doi: 10.1093/jpids/piw042 sha: doc_id: 288930 cord_uid: h13cxuh3 background: children with acute respiratory tract infection (arti) frequently exhibit virus-virus codetection, yet the clinical significance of arti remains contentious. using data from a prospective cohort of children with influenza-like illness, we examined the virology of arti and determined the clinical impact of virus-virus codetection. methods: children aged 6 to 59 months who presented to a tertiary pediatric hospital between influenza seasons 2008 and 2012 with fever and acute respiratory symptoms were enrolled, and nasal samples were collected. respiratory viruses were identified by culture and polymerase chain reaction. we compared demographics, presenting symptoms, and clinical outcomes of children with a single-virus infection and those in whom 2 or more viruses were detected (virus-virus codetection). we used logistic regression models and estimated marginal means to calculate the adjusted odds ratios and probabilities of symptom presentation, prescription of antibiotics, and hospitalization. results: of 2356 children, a virus was detected in 1630 (69.2%) of them; rhinovirus (40.8%), influenza (29.5%), and respiratory syncytial virus (26.4%) were detected most commonly. two or more viruses were detected in 25% of these children. after we adjusted for demographic factors, children with virus-virus codetection had greater odds of presenting with cough (adjusted odds ratio [aor], 1.9; 95% confidence interval [ci], 1.2–3.1) and rhinorrhea (aor, 1.8; 95% ci, 1.1–2.9) than those with a single-virus infection, although both symptoms were common. children with influenza and respiratory syncytial virus combined had the highest probability of hospitalization (55%; 95% ci, 35%–73%), which was significantly greater than for those with influenza infection alone (22%; 95% ci, 16%–29%). conclusions: overall, virus-virus codetection has limited impact on clinical severity among children with influenza-like illness. however, infection with specific pathogen pairs might be associated with more severe outcomes. routine diagnostics to identify specific viruses should be restricted to common pathogens. acute respiratory tract infections (artis) in children place a significant burden on families and the community. commonly recognized viral pathogens that cause arti include influenza viruses, respiratory syncytial viruses (rsvs), parainfluenza viruses, human rhinoviruses, adenoviruses, and coronaviruses [1, 2] . advances in laboratory diagnostic techniques have resulted in the discovery of new viruses, including human metapneumovirus (hmpv) and polyomaviruses [3, 4] , yet a number of these pathogens have an uncertain pathogenicity [5, 6] . codetection can be defined as the detection of 2 or more pathogens in a single sample. with the improved sensitivity, availability, and affordability of modern diagnostics, virus-virus codetections are increasingly being found. the incidence of virus-virus codetection has been reported to be between 15% and 45%, depending on age, location, and testing methods [7] [8] [9] . the clinical significance of codetection in patients with arti remains contentious; the literature has described negligible to deleterious effects [9, 10] . with this study, we describe the virology of arti in children aged 6 months to 4 years who presented to a tertiary pediatric hospital in australia with influenza-like illness during influenza season. this study also enabled us to specifically examine the impact of virus-virus codetection on clinical symptoms and outcomes. western australia (wa) spans 2.5 million km 2 and has a population of approximately 2.5 million people, 7% of whom are younger than 5 years [11] . princess margaret hospital for children (pmh) is the only tertiary pediatric hospital in the state and is located in metropolitan perth, where approximately 80% of the population resides [12] . commencing in 2008, the western australia influenza vaccine effectiveness (waive) study was an observational cohort study established to determine the effectiveness of inactivated influenza vaccine. patient recruitment was conducted at pmh (and at selected general practices in metropolitan wa in 2008-2009). because of the small numbers recruited and differences in presentations, data from children who presented to the general practices were removed from our analyses. patient recruitment coincided with the annual influenza seasons. the start and end of the influenza seasons were defined by the infectious diseases surveillance unit at pathwest laboratory medicine wa by using a combination of indicators, including the weekly proportion of positive laboratory influenza test results. as a guide, 2 consecutive weeks with more than 10% positive influenza test results often coincides with the beginning of influenza season in wa. additional details on study design are described elsewhere [13] . all children 6 to 59 months of age who presented to pmh with a history of fever (according to parental report) or with a measured temperature of greater than 37.5°c at presentation and with at least 1 acute respiratory symptom within the previous 96 hours were eligible for enrollment. all children transited through the pmh emergency department. a proportion of these children were subsequently admitted to the hospital, and the remainder of them were discharged home from the emergency department. children with a known immunodeficiency disorder, with current or recent immunosuppressive treatment, or who received immunoglobulin in the previous 3 months were excluded from the study. patient demographics, medical history, and presenting symptoms were collected through a parental questionnaire. comorbidities recorded included prematurity, asthma, and chronic cardiac, neurological, and respiratory conditions. influenza vaccination status was determined by parental report and confirmed through the australian childhood immunisation register or by contacting immunization providers. vaccination status for other vaccines was not collected. a follow-up questionnaire regarding illness outcomes, including details of hospital admission(s), use of antibiotics, and time to recovery, was given to families to complete within 7 to 10 days after enrollment. a retrospective review of medical records was undertaken when hospitalization data were recorded incorrectly or missing. no follow-up was conducted for antibiotic use if data were missing. samples were collected from the children at enrollment with midturbinate nasal swabs (copan diagnostics, inc., murrieta, california). if a nasopharyngeal aspirate had already been collected by hospital staff as part of clinical care, that sample was used in lieu of a nasal swab. viral culture (madin-darby canine kidney cells, diploid lung fibroblasts) and multiplex tandem polymerase chain reaction (pcr) were used to detect all viruses except picornaviruses and hmpv [14, 15] . picornaviruses were detected by using nested pcr [16] targeting the 5′ untranslated region of the picornavirus genome, and sequencing was used to assist with the identification of rhinoviruses and enteroviruses. hmpv was tested by using an immunofluorescent assay (simulfluor hmpv immunofluorescent assay [millipore, temecula, california]) and pcr. all patients were subjected to the same panel of tests, and testing methods were consistent throughout the study period with the exception of testing for hmpv, which was based on clinical need. although both immunofluorescence and pcr assays were used throughout the study period, pcr testing was more common in later years. for all viruses (except hmpv), positive viral detection was defined as detection by viral culture and/or pcr. positive detection of hmpv was defined as detection by immunofluorescence and/or pcr. all influenza types/subtypes (i.e., influenza a/ h1n1, a/h3n2, and b) were grouped for analysis. similarly, subgroups of parainfluenza viruses (i.e., parainfluenza types 1-4) were grouped together for analysis. infection was defined as the detection of 1 or more viruses (i.e., rhinovirus, influenza, rsv, parainfluenza, adenovirus, coronavirus, and/or hmpv). codetection was defined as detection of 2 or more viruses in a single diagnostic sample. prematurity was defined as less than 37 weeks of gestation at birth. out-of-home care was defined as attendance at a playgroup, mothers' group, day care center, kindergarten, or preschool. hospital length of stay refers to the duration from admission to discharge. symptoms investigated included cough, rhinorrhea, wheeze, dyspnea, rash, diarrhea, and vomiting, and the outcomes investigated were antibiotic prescription and hospital admission. data cleaning and analyses were performed in microsoft excel, epibasic [17] , and spss version 23 (spss, inc., chicago, illinois). categorical variables were compared by using pearson's χ 2 tests. logistic regression models were used to calculate odds ratios (ors) with 95% confidence intervals (cis) to compare those with single infection to those with virus-virus codetection. dependent variables were symptoms (e.g., presence of cough or rhinorrhea) and outcome variables (e.g., hospitalization or use of antibiotics). we calculated adjusted ors (aors) by including the following covariates in the logistic regression models: age, sex, aboriginal status, prematurity, presence of comorbidities, outof-home care, and household smoking. age was included as a categorical variable in the models (6-11 months, 12-23 months, 2 years, 3 years, and 4 years [reference group]). covariates were selected on the basis of known epidemiological or clinical risk factors for codetection. data from all patients were included in the adjusted models unless they had data on 1 or more covariates missing. to investigate the impact of specific pathogen pairs, analyses were repeated for the most common pathogen pairs. estimated marginal means of logistic regression models were used to calculate probabilities with 95% cis for antibiotic prescribing and hospitalization for those infected with common pathogen pairs. this study was approved by the pmh human research ethics committee (approval 1673/ep), the western australian aboriginal health ethics committee (approval 212 06/08), and the university of western australia research ethics committee (approval ra/4/1/6456). of the 2356 patients enrolled, the majority of them (n = 1848 [78.4%]) were enrolled when they presented to the pmh emergency department. of these patients, 6.3% (n = 117) were subsequently admitted to the hospital. the median age was 22.0 months (interquartile range, 14.0-35.0), 54.9% were male, and 5.7% were of aboriginal or torres strait islander decent. children born preterm accounted for 13.5% (n = 319) of the patients. children with comorbidities accounted for 15.1% (n = 355) of this cohort. of those who had 1 or more comorbidity, asthma (n = 218 [61.4%]) and other chronic respiratory conditions (n = 54 [15. 2%]) were the most common. of 2356 patients, questions relating to outcomes (e.g., antibiotics use) were completed for 52.8% (n = 1244). although parents were requested to complete these questions 7 to 10 days after enrollment, the mean time to completion was 19.3 days (range, 0-149 days; median, 10 days). data on antibiotic prescription after enrollment were available for 51.0% (n = 1201) of the patients, 483 (40.2%) of whom were prescribed antibiotics. combining data from the questionnaires and a review of the hospital records resulted in near-complete data on hospitalization (99.4% [n = 2341]); 610 (26.1%) were hospitalized. of those who were admitted to the hospital, the median length of stay was 2 days (interquartile range, 1-3 days). overall a greater proportion of children with multiple viruses detected were younger than 2 years than those with a single-virus infection (65.4% vs 51.2%, respectively; p < .001; table 1 ). those with codetection also had greater odds of presenting with cough and rhinorrhea than those with a single-virus infection, although both symptoms were common in both groups ( table 2 ). this effect remained after adjusting for other covariates. it should be noted that, although less common, diarrhea was observed more frequently in children with viral codetection. there were no significant differences between patients with a single-virus infection and those with virus-virus codetection in the odds of being prescribed antibiotics (aor, 1.1; 95% ci, 0.8-1.5) or being hospitalized (aor, 1.1; 95% ci, 0.8-1.4) ( table 2) . we then selected the 3 most common pathogens (rhinovirus, influenza, and rsv) and investigated associations of infection with specific pathogen pairs with antibiotic prescriptions and hospitalization. after adjusting for other covariates, patients with both influenza and rsv detected had a 52% probability (95% ci, 28%-76%) of being prescribed antibiotics, and there was a trend toward more frequent prescription than those with influenza or rsv infection alone (figure 2) . similarly, the probability of being hospitalized was highest in those with influenza and rsv detected (probability, 55%; 95% ci, 35%-73%) and was significantly greater when compared with those with influenza infection alone (probability, 22%; 95% ci, 16%-29%) ( figure 3 ) and with a trend towards increased hospitalization observed compared with rsv infection alone (probability, 43%; 95% ci, 36%-51%). this is one of the largest single-site prospective studies of children up to 4 years of age that specifically investigated the incidence of and clinical outcomes associated with virus-virus codetection. our findings indicate that although differences in demographics, risk factors, and symptoms are identifiable, in general, virus-virus codetection is unlikely to be associated with more severe clinical illness among young children with influenza-like illness. infection with specific pathogen pairs might be associated with an increased probability of hospitalization, as was observed with influenza and rsv. this finding has implications in pediatric healthcare facilities, where the isolation of all children with acute respiratory viral infection is difficult during periods of peak respiratory virus activity and cohorting of children is frequently required before the availability of diagnostic test results. we detected small differences between the symptoms presented by patients with a single-virus infection and those presented by patients with virus-virus codetection. however, these symptoms were common and therefore likely to be of little clinical relevance. in contrast, the clinical outcomes chosen (i.e., antibiotic use and hospitalization) were more indicative of disease severity, but they are subject to clinical judgement and therefore can be less sensitive measures of disease severity. accordingly, we observed no significant differences in the these results are consistent with data from previous systematic reviews, which found negligible differences between outcomes in children and adults with virus-virus codetection compared to peers with single-virus infection [18, 19] . however, the results of additional analyses of pathogen pairs suggest that some combinations of specific viral pathogens, such as influenza and rsv, are potentially more significant than others. this result corroborates data from our recently completed systematic review in which we specifically investigated clinical outcomes in children with codetection; we found no differences overall, but the results suggest that some pathogen-specific effects might be present [20] . our data suggest that future research in this area should segregate analysis according to specific pathogen pairs when the numbers allow. we chose to exclude bocavirus and enterovirus detections from the analyses because their pathogenicity in arti is still not well established. bocavirus is often implicated in both symptomatic and asymptomatic codetection and is thought to have a prolonged period of shedding [6] ; both of these features might confound any associations between codetection and clinical severity. in contrast, the results of studies on the role of enteroviruses in arti are suggestive of pathogenicity [21] ; however, the numbers in those studies were small. for these reasons, detections of both viruses were excluded from the analyses presented here. repeat analyses including these viruses did not change the overall findings (supplementary tables 1 and 2 ). an important consideration when interpreting these findings is that active (and pathogenic) infection and viral shedding cannot be distinguished. prolonged viral shedding for some respiratory viruses, particularly rhinovirus, has been well documented [22, 23] . quantitative analysis might be of assistance in distinguishing these clinical states but has not yet become commonplace in the diagnostic laboratory for respiratory viruses. one limitation of our study is that only children who presented to 1 hospital with influenza-like illness and fever were eligible for enrollment. as a consequence, it is possible that these children were at the more severe end of the disease spectrum, which might have biased our results. during the course of this study, there was a shift from using an antigen-based assay to using pcr for detecting hmpv, although both methods were used throughout the study period. we elected to include detections from both methods but acknowledge that differences in the performance of these methods would mean that potential cases of hmpv might have been missed in earlier samples. these changes, and clinical discretion in testing for hmpv, may explain the proportion of hmpv detections in this cohort, which was lower than that in other studies [24, 25] . additional limitations of this study include missing outcomes data, particularly for antibiotic prescription. in addition, data on diagnosis at discharge were not collected, which might have helped to indicate the severity of symptoms. moreover, despite enrolling nearly 2500 children, the number of patients infected with specific pathogens and pathogen pairs was relatively small. future studies using routinely collected, linked administrative data might assist in addressing both issues. nonetheless, ours was one of the largest single-site studies to specifically investigate the effects of virus-virus codetection in young children by using a wide panel of tests for respiratory pathogens. our results are similar to those reported elsewhere, which adds to the validity of the findings [26] . we conclude that the impact of virus-virus codetection on disease severity in children who present with influenza-like illness is likely to be limited to those infected with specific pathogen pairs. therefore, routine screening for virus-virus codetection in this population should be restricted to those with common respiratory pathogens, and efforts to reduce cross infection should focus on these specific pathogens. detection of respiratory viruses by molecular methods epidemiology of viral respiratory infections the role of infections and coinfections with newly identified and emerging respiratory viruses in children presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection human rhinovirus c: age, season, and lower respiratory illness over the past 3 decades human bocavirus-the first 5 years single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-[gamma] response multipathogen infections in hospitalized children with acute respiratory infections evaluation of viral co-infections in hospitalized and non-hospitalized children with respiratory infections using microarrays multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children rates calculator, version 9.5.5. perth, western australia: health information centre, department of health article22012-13?opendocument&tabname=s ummary&prodno=3218.0&issue=2012-13&num=&view=. accessed november 13 effectiveness of trivalent flu vaccine in healthy young children duplex realtime reverse transcriptase pcr assays for rapid detection and identification of pandemic (h1n1) 2009 and seasonal influenza a/h1, a/h3, and b viruses an economical tandem multiplex real-time pcr technique for the detection of a comprehensive range of respiratory pathogens improved detection of rhinoviruses in nasal and throat swabs by seminested rt-pcr epibasic version 2.0 clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis single and multiple respiratory virus infections and severity of respiratory disease: a systematic review systematic review and meta-analysis of respiratory viral coinfections in children global reemergence of enterovirus d68 as an important pathogen for acute respiratory infections duration of rhinovirus shedding in the upper respiratory tract in the first year of life persistence of rhinovirus and enterovirus rna after acute respiratory illness in children use of an innovative webbased laboratory surveillance platform to analyze mixed infections between human metapneumovirus (hmpv) and other respiratory viruses circulating in alberta (ab) human metapneumovirus in hospitalized children in epidemiology of respiratory viral infections in children enrolled in a study of influenza vaccine effectiveness acknowledgments. we acknowledge peter jacoby for his assistance with the logistic regression analyses and gabriela willis for her assistance with cross-checking the data, and we thank members of the vaccine trials group, particularly christine robins, who managed the study. we also thank all the parents and children who participated in the waive study. potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. supplementary materials are available at journal of the pediatric infectious diseases society online. author contributions. c. c. b., p. c. r., p. v. e., and d. w. s. conducted the waive study; p. c. r., d. w. s., and c. c. b. conceptualized this study; a. l. and s. t. conducted the laboratory work; z. v. w. and n. t. c. conducted the preliminary data cleaning and analyses; f. j. l. conducted data cleaning and analyses with assistance from h. c. m., n. d. k., and c. c. b.; and f. j. l. and z. v. w. jointly wrote the first draft of the manuscript. all authors critically revised and approved of the final version of the manuscript. key: cord-301677-b6mnn27h authors: soleimanian, saeede; yaghobi, ramin title: harnessing memory nk cell to protect against covid-19 date: 2020-08-20 journal: front pharmacol doi: 10.3389/fphar.2020.01309 sha: doc_id: 301677 cord_uid: b6mnn27h the worldwide struggle against the coronavirus disease 2019 (covid-19) as a public health crisis continues to sweep across the globe. up to now, effective antiviral treatment against covid-19 is not available. therefore, throughout virus infections, a thorough clarification of the virus-host immune system interactions will be most probably helpful to encounter these challenges. emerging evidence suggests that just like sars and mers, covid-19 primarily suppresses the innate immune system, enabling its stable propagation during the early stage of infection. consequently, proinflammatory cytokines and chemokines have been increasing during infection progression associated with severe lung pathology. it is imperative to consider hyper inflammation in vaccine designing, as vaccine-induced immune responses must have a protective role against infection without leading to immunopathology. among the front-line responders to viral infections, natural killer (nk) cells have immense therapeutic potential, forming a bridge between innate and adaptive responses. a subset of nk cells exhibits putatively increased effector functions against viruses following pathogen-specific and immunization. memory nk cells have higher cytotoxicity and effector activity, compared with the conventional nk cells. as a pioneering strategy, prompt accumulation and long‐term maintenance of these memory nk cells could be an efficacious viral treatment. according to the high prevalence of human cytomegalovirus (hcmv) infection in the world, it remains to be determined whether hcmv adaptive nk cells could play a protective role against this new emerging virus. in addition, the new adaptive-like kir+nkg2c+ nk cell subset (the adaptive-like lung tissue residue [tr]nk cell) in the context of the respiratory infection at this site could specifically exhibit the expansion upon covid-19. another aspect of nk cells we should note, utilizing modified nk cells such as allogeneic off-the-shelf car-nk cells as a state-of-the-art strategy for the treatment of covid-19. in this line, we speculate introducing nkg2c into chimeric antigen receptors in nk cells might be a potential approach in future viral immunotherapy for emerging viruses. in this contribution, we will briefly discuss the current status and future perspective of nk cells, which provide to successfully exploit nk cell-mediated antiviral activity that may offer important new tools in covid-19 treatment. world health organization declared the outbreak of coronavirus disease 2019 (covid-19) a public health concern on 30 january 2020 (velavan and meyer, 2020) . this novel coronavirus disease pandemic caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) that has led to a global struggle to cope with this public health crisis (van bavel et al., 2020) . sars-cov-2 virus preferentially impacts the cells in the respiratory system, but the direct effects of its damage on other organs including heart, liver, brain, and kidneys have also been reported in patients with sars-cov-2 infection (prasanna and abilash, 2020 ). an unanswered question has been currently raised why sars-cov-2 can result in a wide variety of clinical manifestations ranging from: asymptomatic, mild, moderate, to severe states in covid-19 patients, a major challenge in the management of medical decisions and interventions (gandhi et al., 2020; meini et al., 2020; michelen et al., 2020) . the severe cases of covid-19, have accompanied pneumonia, which can progress to acute respiratory distress syndrome (ards), sepsis, septic shock, and multi-organ failure (particularly kidney, heart, and liver damage) (cao, 2020) . obviously, the pathogenesis of sars-cov-2 is largely related to its interplay with the host. indeed, the interaction between sars-cov-2 and host antiviral immunity, including innate and acquired immune response, should be investigated (ahmadpoor and rostaing, 2020; ranucci et al., 2020) . on the basis of current covid-19 data, cytokine storm and therefore the development of ards are due to the exaggerated immune responses leading to severe lung damage . up to now, there are no specific and effective covid-19 therapies. at this time, it is our opinion that immunotherapies based on immunomodulation and counterbalancing of inflammatory cytokine could reduce inflammation and inflammatory associated lung injury. in this regard, natural killer (nk) cells as essential front-line responders to many viral infections in humans have been proposed for a suitable therapeutic approach in severe covid-19 patients, and several clinical trials have begun (market et al., 2020) . in this study, we considered this new approach to enhance patient's survival using nk cells with memory features and immunomodulatory potential and discussed also the aspects of this proposed treatment. coronaviruses(covs) belong to the coronaviridae family and are characterized by a positive-sense single-strand ribonucleic acid (rna). the coronavirus genome is highly susceptible to mutations that result in genetic drift and evade immune recognition (kikkert, 2020) . consistent with this notion, sars-cov-2, as a new member of the genus beta coronaviruses, can escape from immune detection during the infection (channappanavar and perlman, 2017; prompetchara et al., 2020) and worsening disease outcome could be associated with the immune-escape mechanisms behind these chronic diseases. indeed, the immune homeostasis is disrupted by sars-cov-2 leading to declined responsiveness of host immune regulatory system, macrophage function, and alterations in lymphocyte subsets (merad and martin, 2020; wan et al., 2020) . accumulating evidence suggests that the stimulated immune response by sars-cov-2 infection results in two phases; first, immune defense-based protective phase at the early stage, and second, severe-stage inflammation-driven damaging phase (alberici et al., 2020) . once the sars-cov2 enters the body, the innate immune system performs as a first responder for the detection of viral infections. innate immune cells produce proinflammatory cytokines to inhibit viral replication and induce the adaptive immune response (koyama et al., 2008) . among the initial immune responses in the fight against respiratory rna viruses, such as coronavirus, detection of evolutionarily conserved microbial structures, known as pathogen-associated molecular patterns (pamps) are ensured through germline-encoded pattern recognition receptors (prrs) (bowie and unterholzner, 2008) . prrs including: tlr3, tlr7, tlr8, mda-5, and rigi receptors are produced by tissue resident macrophages and dendritic cells (dcs) (kumar and carmichael, 1998; kawai and akira, 2006) . this results in the activation of transcription factors involved in the production of type i interferons (ifns) (ifn-a/b). in this line, type i ifns have a critical role in concert with pattern prr signaling to prime innate and adaptive antiviral responses such as stimulating natural killer (nk) cells, macrophages, and production of proinflammatory cytokines (samuel, 2001; murira and lamarre, 2016) . the immunomodulatory strategies that are used by beta coronaviruses can lead to dysregulated ifn responses (blanco-melo et al., 2020) . similar to sars and mers, sars-cov-2 replicates silently in host cells with suppressed ifns response, leading to high viral loads (acharya et al., 2020; rokni et al., 2020) . by contrast, following limited production of ifns, the robust production of pro-inflammatory cytokines and chemokines may occur after an enhanced recruitment of neutrophils to the lungs (channappanavar et al., 2019) . the virus actively subverts ifns to manipulate the host cytokine environment for its benefit. during the early phase, a specific immune response is required to clear the virus and to impede disease progression to severe stages. when a protective immune response is impaired, the virus will propagate and cause massive damage to the organs expressing the surface receptors angiotensin-converting enzyme 2 (ace2). these are recognized by alveolar macrophages and epithelial cells, triggering the generation of pro-inflammatory cytokines and chemokines including: il-2, il-7, il-10; granulocyte colony-stimulating factor (g-csf), interferon gamma-induced protein 10 (ip-10), and monocyte chemoattractant protein-1 (mcp-1) in the damaged cells . moreover, such evidence has demonstrated elevated levels of macrophage inflammatory protein 1 alpha (mip-1a), tumor necrosis factor-alpha (tnf-a), as well as chemokines (c-c motif chemokine ligand 2: ccl2, ccl3, ccl5, c-x-c motif chemokine ligand 8: cxcl8, cxcl9, cxcl10) ( figure 1 ) in the severe stage of this viral disease (di lernia, 2020) . increasing concentration of these proteins results in the production of ifng by t cells and monocytes during virus infection (mardani et al., 2020; tay et al., 2020; yuan et al., 2020) . hence, excessive cytokine/chemokine release can cause hyperinflammation, which leads to severe complications of the disease including pneumonitis, acute lung injury (ali), ards, shock, vascular hyper permeability, organ failure, and death (henderson et al., 2020; mehta et al., 2020) . thus, it is essential to diagnose the infection during the first phase before the cytokine storm is initiated (nicholls et al., 2003; mehta et al., 2020) . in addition, the protective defense during the early stages of viral infections is of utmost importance to rein the spread of the viruses and it is imperative to boost immune responses (anti-sera or pegylated ifn-a) at this stage. in this regard, an experimental trial has established that recombinant human ifn-a (rhifn-a) nasal drops ( figure 1 ) in susceptible healthy people are able to potentiate protection against covid-19 pandemic (shen and yang, 2020) . thus, the timing of treatment administration in infection control is a key to yield protective responses (channappanavar and perlman, 2017; wu et al., 2020) . evaluation of diagnostic accuracy for covid-19 is still in progress, so an authentic interpretation of the findings in this area is essential in guiding patient care. indeed, clear pattern of inflammatory and immune biomarker abnormalities is needed to discriminate the stages of infection, which may potentially aid in addition to molecular detection of virus. the detection of both sars-cov-2 nucleic acid and specific antibodies to viral proteins have thus far become significant for primary diagnosis infection and immunity in covid-19 patients, respectively. two types of reliable diagnostic tests have been thus far commonly utilized including reverse transcriptase-polymerase chain reaction (rt-pcr) and immunoglobulin m (igm) and igg antibody testing (nalla et al., 2020; . the rt-pcr has been the key diagnostic test using upper respiratory tract specimens or nasopharyngeal swabs. at an early stage, before the launch of adaptive response, igm has been further detected in patient blood after 3-6 days and then igg responses that are important as a long-term immunity and immunological memory, reported after 8 days . notably, both asymptomatic and symptomatic patients have shown variations in the course of pcr positivity and seroconversion (sethuraman et al., 2020) . accordingly, identification of hematologic, biochemical, and especially immunologic biomarkers can be helpful in guiding patient care decisions. assessment of biomarkers including neutrophil and lymphocyte counts, neutrophil-to-lymphocyte ratio (nlr), crp, troponin t (tnt), d-dimer, il-6, il-2r,il-8,il-10 (henry et al., 2020) , ferritin, lactic acid dehydrogenase (ldh), and procalcitonin (pct) as a predictive of disease progression beside clinical manifestations and molecular diagnosis can be beneficial for making accurate decisions to fulfill antiviral and anti-inflammatory interventions (fang et al., 2020; henry et al., 2020) . figure 1 | schematic representation of the progression of covid-19 and potential interventions. sars-cov-2 enters cells through interactions with receptors that include angiotensin converting enzyme ii (ace2), a cell surface protein on the cells. the increased concentrations of the serum cytokines and chemokines is correlated with the disease severity and adverse clinical outcome. there are some proposed therapeutic options in the management of cytokine storm and pulmonary injury, including: use of cytokine activated mesenchymal stem cells, vitamin b3, and hyaluronidase. inhibition of tnf-a, il-1, or il-6 to manage covid-19-induced cytokine release syndrome (crs) is also recommended. a few weeks after covid-19 outbreak, several science groups started to design a vaccine for this novel disease. sars-cov and mers-cov immunization studies have also become a pattern for targeting antigen selection in designing vaccines (du et al., 2009; al-amri et al., 2017) . moving forward, knowledge of developing effective therapeutics for covid-19 is necessary to make a balance between inflammatory cytokine secretion and antiviral properties in immune cell therapy. therefore, controlling deleterious rather than protective role of lymphocyte responses should be taken into account. in this regard, recent evidence has suggested some effective strategies including blocking il-6 (tocilizumab) (tanaka et al., 2016; monteleone et al., 2020) , il-1 (anakinra), and tnfs to calm inflammatory storm (fu et al., 2020; shi et al., 2020) . selective cytokine blockade could be a potential treatment to reduce the mortality of severe covid-19 (shakoory et al., 2016; coomes and haghbayan, 2020; mehta et al., 2020) . moreover, another therapeutic option has been janus kinase (jak) inhibitor, tofacitinib, denoting that blocking jak/stat signaling can reduce cytokine storm as an anti-il-6 operator (henderson et al., 2020; liu et al., 2020) . it should be noted that despite the benefits of these anti-inflammatory agents, there are some disadvantages, such as tendency for general infections and the development of chronic inflammatory disorders, thus more experimental investigations should be done to clarify doubts (jones and ding, 2010; mcgonagle et al., 2020; soy et al., 2020) . furthermore, in response to inflammatory cytokines, lungs are filled with a clear liquid jelly. actually, the nature of the clear jelly is hyaluronan (ha). thus, inhibition of hyaluronan synthase (has) and elimination of this jelly by hyaluronidase has been recommended (modig and hällgren, 1989 ). in addition, numerous studies have suggested that vitamin b3 (i.e., niacin or nicotinamide) can be highly effective in preventing lung tissue injury in animal models with bleomycin-induced lung injury ( figure 1 ), which might be another approach to supply this food supplement to covid-19 patients (nagai et al., 1994; shi et al., 2020) . these treatment approaches play auxiliary roles in severe patients of covid-19. likewise, on the basis of new findings, the potential benefits or disadvantages of anti-cytokine therapy has implications for other therapeutic strategies such as cellular therapy in patients with severe inflammatory responses. cellular therapy as an effective treatment for severe covid-19 patients is a rapidly evolving field with novel constructs being developed. various clinical sites in china have revealed that mesenchymal stem cells (mscs) in severe covid-19 infection can be nominated as a helpful mode in the suppression of hyperactive immune response and promotion of tissue repair. as well, mscs with ifng as licensed-mscs can be more effective (wang et al., 2014; wang et al., 2018) . besides, current investigations have strongly indicated that t-cell response can be a crucial strategy for the control of sars-cov and mers-cov and probably proper for sars-cov-2; nevertheless, cytokine storm should be controlled in order not to cause lung pathology. indeed, one of the biggest obstacles in severe covid-19 cell therapy approach (such as chimeric antigen receptor: car t-cell) is the cytokine release syndrome (crs) affecting patients with severe conditions (maude et al., 2014) . as well, ifn-g production is indispensable for resistance against virus infection, but t-cells as a major source of ifn-g in the adaptive immune response take days to develop a prominent ifn-g response (chen g. et al., 2020) . remarkably, it is important to consider it in vaccine designing, as vaccine-induced immune responses need to play a protective role against infection without leading to immunopathology with timely management of treatments. it is noteworthy that the abolition of viruses requires the immune system to respond through a programmed regulatory system, subsequently to implement tolerance and to inhibit excess response. indeed, it must be balanced between sufficient inflammatory reactions against pathogen and overactive cytokine stimulation. among the immune cells, nk cells endowed with significant strategies for self-tolerance status while allowing efficacy against viral attacks. consistent with this context, education is one of the immunomodulatory process during nk cell development, operating not as an on-off switch but as a rheostat, tuned by a quantitative influence on individual nk cells (orr and lanier, 2010; vivier et al., 2011) . intriguingly, this pattern has implications for the use of nk cells in therapeutic settings and affects interpretations of how nk cells control virus infections and regulate autoimmunity. additionally, nk cells act as a rheostat by removing activated cd4+ and cd8+ t cells in viral infections such as hiv and hcv, thus preventing t cellmediated autoimmunity (lodoen and lanier, 2005; sun and lanier, 2008; frutoso and mortier, 2019) . based on this knowledge we will comment on immune modulating treatment options that are being a proper strategy for the current covid-19 crisis. nk cells are at the forefront of the antiviral response (tang et al., 2013) and they are known as a regulator of immunological procedures including viral defense and immunological homeostasis. actually, nk cells make use of various recognition modes in viral infections. the functional outcome of nk cells is determined by integrating both activating and inhibitory signals which regulate nk cell activity (lanier, 2005; duev-cohen et al., 2016) . virus-infected cells expressing defined ligands can be directly targeted through activating receptors such as nk group 2-member d (nkg2d), nk cell p46-related protein (nkp46), and nkp30 on nk cells. as well, release of cytokines such as infs, il-12, or il-18 by accessory cells can also alert bystander nk cells during viral infection (cerwenka and lanier, 2001; french and yokoyama, 2003; hammer et al., 2018b) and render nk cells to proliferate and produce cytokine such as ifn-g (zwirner and domaica, 2010) . another mode of recognition includes expressing fc receptors (i.e., cd16 or fcgriii), by nk cells identifying infected cell bound to antibodies in the surface of infected target cells, called antibody-dependent cell-mediated cytotoxicity (adcc), resulting in the release of cytotoxic factors such as perforin and proteases known as granzyme. therefore, nk cells can also kill virus-producing cells through this important property (vanderven et al., 2017) . virus-infected cells usually escape t-cell immune surveillance through down-regulating the expression of major histocompatibility complex (mhc) class i to compromise for antigen presentation pathway, making viruses difficult to be recognized by t-cells (pircher et al., 1990; mcmichael, 1998) . in addition, several lines of evidence have further supported the notion that nk cells play a crucial role in the first checkpoints of encountering antigens and trigger proinflammatory immune responses and they provide a critical response against infectious agents during the time that the acquired immune system is still being mustered (andoniou et al., 2006; hu et al., 2019) . these features suggest that nk cells can serve as the major antiviral effector cells wherein virusinfected cells should develop mechanisms of escaping t-cell surveillance (zander et al., 2019) . likewise, they are at the interface between innate and adaptive immunity, adjusting the activity of other immune cells through the release of cytokines including activating (e.g., ifn-g and tnf-a) as well as inhibitory cytokines (e.g., il-4 and il-10) for different purposes. for instance, nk cells promote the maturation of dendritic cell (dc) through the release of ifn-g and tnf-a (shariat et al., 2014; moravej et al., 2017) . additionally, nk cells are considered the important effectors in shaping the subsequent immune responses. they increase or decrease function of immune cells through ifng or il-10 secretion, respectively (vivier et al., 2011; della chiesa et al., 2019; monica et al., 2019) . the diversity of nk cell function and phenotype is accordingly an issue of growing attractiveness. with reference to cytolytic activity, cytokine secretion, and organ-specific localization, three main subsets of nk cells can be consequently identified. large amounts of cytokines such as ifng are thus produced by the cd56bright/cd16low subset in response to activation, whereas cd56low/cd16high cells are the major cytotoxic populations. cytotoxicity and cytokine secretion are further regulated by making a balance between inhibitors and activating receptors and the effector function of nk cells also relies on the incorporation of activating and inhibitory receptors (björkström et al., 2010) . a third nk cell subset is cd16+/cd56neg nk cells with different features because of less functionality and lower expression of natural cytotoxicity receptors (ncrs). evidence also suggests that cd56low nk cells originate from cd56high ones ( figure 2 ). the given subset is expanded in viral infection and then counterbalances the reduction in cd56dim/cd16pos cytolytic nk cell subset (mikulak et al., 2017) . in general, these various recognition modes trigger nk cells activation, and then their cytotoxicity function that results in the lysis of pathogen-infected target cells (hammer et al., 2018b) . although there are numerous ways to recognize viral antigens and peptides by nk cells, the implications of such cells should be considered in the control of viral infections. therapeutic strategies for covid-19 based on the present investigations of sars-cov and mers-cov infections may be helpful in offering potential treatment targets for fighting this pandemic. in this regard, the national research project for sars, beijing group, has reported that in the cohort study of 221 patients with sars who were admitted to hospitals, determining the total number of nk cells and cd158b+ (kir2dl3) nk cells in 72 cases of severe sars were significantly lower than those in mild cases (national research project for sars, beijing group, 2004) . more importantly, this study has shown that percentage of cd158b+ nk cells in patients with m pneumonia infection and sars patients (with positive clinical criteria and negative anti-sars coronavirus) wasn't significantly altered as compared to the percentages of nk cells in sars cases (with positive for both clinical criteria and anti-sars coronavirus), which indicates that the nk cells monitoring should be helpful in differentiating true sars from false sars and m pneumonia infection. due to the greatest similarity of covid-19 to sars-cov, nk cells monitoring can be investigated for this novel coronavirus (novel-cov) (bai et al., 2020; chen n. et al., 2020) . in addition, one of the covs in pigs, i.e., transmissible gastroenteritis virus (tgev) had been first described in 1946 in which increasing infection in young pigs could result from lack of nk cell activity against tgev-infected cells. studies have further suggested that ifn might be needed to activate nk cells in newborn pigs, contributing to resistance to challenges with tgev (cepica and derbyshire, 1984; lesnick and derbyshire, 1988; loewen and derbyshire, 1988; annamalai et al., 2015) , and they have further revealed that nk cells might play a significant role in fighting covs. other studies, consistent with this context, have shown severe cases of sars patients with a lower level of nk cells than those in milder cases (cui et al., 2003; peiris et al., 2003; wong et al., 2003) . likewise, in the study by j chen et al. in a pneumonia model of sars in mice, mimicking features of the human disease, illustrated that mice depleted of both cd4 and cd8t cells, had the ability to control sars-cov replication in the lungs, suggesting an immune mechanism independent of t cells, and a role for innate antiviral response and nk cells, in viral clearance. it should be noted that at the early phase of infection, nk cells, macrophages, and plasmacytoid dcs (pdcs) could migrate into the lungs. this can occur following the first wave of enhanced expression of cytokines including tnf-a, il-6, and cxcl10, ccl2, ccl3, and ccl5, as virus titers, produced by airway epithelial cells and alveolar macrophages (chen et al., 2010) . indeed, the activation of the innate immune system and nk cells has a significant role in the primary phase of infection in the control of sars-cov replication. this approves the hypothesis that in critically ill patients with sepsis, the activation of nk cells is an important element to combat infections. but, continuous activation of nk cells in sepsis causes exhaustion and hyporesponsiveness of nk cells which results in hyper inflammatory response due to reduced nk cell numbers (guo et al., 2018) . on the contrary, in the context of other respiratory viral infections like respiratory syncytial virus (rsv) (li et al., 2012) , influenza a virus (abdul-careem et al., 2012; zhou et al., 2013) , nk cells appear to cause enhanced inflammation and immunopathology during infections. furthermore, during influenza infection, stimulation, and migration of lymphocytes such as nk cells to the lungs, influence the ifn-g production and hyper inflammatory response. increased recruitment of hyper-responsive nk cells would thus far worsen the infection outcomes (vivier et al., 2011; scharenberg et al., 2019) . collectively, considering this duality of nk cell roles, we must be aware of the potential and challenges mainly the timing of cell therapy and the stage of infection to finely tune nk cell activity to facilitate viral clearance while impeding the harm of inflammatory responses. remarkably, the total number of nk cells, as well as b-cells and t-cells, can decrease significantly in patients with covid-19 particularly in severe cases, compared to non-severe ones (cao, 2020) . in this respect, several studies have identified reduced numbers of nk cells in the peripheral blood of covid-19 patients, which is related to the severity of the disease (masselli et al., 2020; vabret et al., 2020; wang et al., 2020b) . in the study by fan wang et al. on covid-19, the incidence rate of lymphopenia had been reported by 72% that was consistent with sars and mers. cd4+ t-cells, cd8+ t-cells, and b-cells had been also decreased in 29, 29, and 25% of patients, respectively. but, nk cells has been prominently reduced in 59% of the cases (higher incidence rate than others) (wang et al., 2020b) . accumulating evidence had supported the fact that nk cells had reduced in covid-19 patients as compared with noninfected cases (chen g. et al., 2020; wen et al., 2020; wilk et al., 2020) . of note, a latter study by feng wang et al. (wang et al., 2020a) has been reported that cd4+ t cells were increased in patients with covid-19, whereas the number of nk cells was decreased in the severe stage of infection. the important finding of this study is that the pathogenesis of severe sars-cov-2 infection is associated with the hyper function of cd4+ and cd8+ t cells but nk cell hyporesponsiveness. as well, new findings suggest that during covid-19 infection, production of igg-subclasses igg1 and igg3 may trigger nk-cell mediated adcc through the igg-fc-receptor (fcgr) iiia, and nk cells utilize the fc domain of bound igg to recognize extracellular virions or surface antigens expressed on infected cells. the lysis of infected cells and cytolytic functions by nk cells might be stimulated by this interaction (dokun et al., 2001; acharya et al., 2020; bonam et al., 2020) . additionally, a recent study found adcc mediated by nk cells, can contribute to viral control in covid-19 patients. they observed efficient sars-cov-2 s glycoprotein (s309-and s306), triggering adcc in sars-cov-2 s-glycoprotein-transfected cells (pinto et al., 2020) . interestingly, nk cells have a considerable role in inducing the lysis of virus-infected cells since the cytotoxicity of such cells is regulated by activating and inhibitory receptors. an increased nk group 2 member a (nkg2a) expression as a heterodimeric inhibitory receptor can also result in functional exhaustion of nk cells in patients with sars-cov-2 infection. indeed, the total number of nk cells declines during the covid-19 and the number of nk cells is simultaneously increased with the reduced expression of nkg2a after recovery (yaqinuddin and kashir, 2020; qin et al., 2020) , while this is associated with significantly decreased expression of the activation markers including cd107a, ifn-g, il-2, and tnf-a in t and nk cells of patients with severe covid-19 (antonioli et al., 2020; zheng et al., 2020) . in this respect, a study by vabret et al. revealed crosstalk between monocytes and nk cells that results in nk cell recruitment from the peripheral blood to the lungs and killing of sars-cov-2-infected cells. majority of human lung nk cells are non-resident and the expansion of cxcr3 ligands (cxcl9-11)-producing monocytes causes nk cell infiltration into the lung of covid-19 disordered patients (chu et al., 2020; vabret et al., 2020) . however, a question remains at this time how the sars-cov2 virus alters the number and function of nk cells. it is possible that nk cell redistribution in infected sites (nk cell infiltration into the lung) or apoptosis of these cells results in their reduction (mckechnie and blish, 2020) . moreover, elevated il-6 and il-10 levels in covid-19 cases can suppress nk cell activity. further, moderating the expression of the activating receptor nkg2d through il-6 production leads to impairment of nk cell activity. (osman et al., 2020) . in addition to elevated il-6 and il-10, the higher levels of tgf-b is responsible for suppressing nk cell antiviral activity through down-modulation of nkg2d (lee et al., 2004; huang et al., 2005) . thus, therapies targeting il-6, il-10, and tgf-b could be investigated as means of improving nk cell antiviral immunity in cases of covid-19. however, the utility of therapeutic tools to boost nk cell functions should, thus, be carefully considered in severely affected patients (rajaram et al., 2020) . over recent years, a novel concept has emerged indicating that the present paradigm that t-cells and b-cells are the only factors of adaptive immunity should be reconsidered. although, nk cells have been respected for their ability to mediate spontaneous cytotoxicity during innate immune responses in an antigen-independent manner, it has become recently clear that they display long-lived immunological memory responses (lee et al., 2015) . in mice, it has been initially described that cytomegalovirus infection drives adaptive features of nk cells with altered effector function, where nk cells bearing ly49h receptors expanded and provided stronger responses after a secondary encounter with the virus. intriguingly, human cytomegalovirus (hcmv) infection can also prompt emergence and expansion of adaptive or memory-like nk cells with more potent cytotoxicity in humans (lam and lanier, 2017) . moreover, adaptive nk cells are characterized by expression of the activating receptor nkg2c and the absence of the inhibitory receptor nkg2a. notably, non-classical mhc-i molecule, hla-e is a ligand for both but, nkg2a has the higher affinity for this ligand engagement in competition with nkg2c. thus, the inhibition of nkg2a expression is an essential element for expansion of nkg2c+nkcells with the more cytolytic activity (guo et al., 2018) . this distinct population of nk cells also provides stronger protective immunity against hcmv via adcc in the presence of anti-hcmv antibodies. moreover, adaptive nk cells are characterized by expression of cd57 (maturation marker), kirs, and the absence of the inhibitory receptor nkg2a and can further produce inflammatory cytokine and cytolysis in response to target cell recognition (lopez-verges et al., 2010; lopez-vergès et al., 2011) . intriguingly, adaptive nk cells additionally share notable similarities in epigenetic patterns with cd8+ effector t-cells and remarkable matches in adaptive cell differentiation as well (tesi et al., 2016) . recenly, it has been also indicated that uterine mucosa, liver, skin, adrenal gland, colorectal and visceral adipose tissues, as well as secondary lymphoid compartments (slcs), encompass cd56bright/cd16neg/dim nk cells, but cd56dim/cd16 bright cells settle in tissues such as lung and breast (carrega et al., 2014; melsen et al., 2016) . it should be noted that localization of nk cell subsets changes in various pathological conditions e.g. in the presence of tumors or virus-infected cells (nikzad et al., 2019) . even with the acquisition of new chemokine receptors by nk cells in a tumor or virus-infected cell microenvironments, nk cells may be changed functionally and phenotypically (das and khakoo, 2015; brillantes and beaulieu, 2020) . most importantly, nk cells differentiate into long-lived memory ones. for instance, in humans, infection with hcmv is associated with lasting expansions of different nk cell subsets expressing nkg2c display memory features that can result in robust recall responses (loṕez-botet et al., 2014) . in addition, in vaccination, expression of nkg2c might represent one potential early determinant to differentiate between responders and low responders. it can be also a driving force to promote efficacious adaptive responses at the postvaccination stage. indeed, vaccine responsiveness can be associated with changes in nk cell phenotype and functionality (riese et al., 2020) . in addition, nk cells present in the lung are often enriched in cd56dim/cd16bright cells but recently, during the hcmv infection, a novel adaptive killer-cell ig-like receptor (kir)+/nkg2c+/nk cell subset with a cd49a+/cd56bright/ cd16-tissue-resident (tr) nk cell phenotype (figure 2 ) has been identified in human lungs (cong and wei, 2019; dogra et al., 2020) . considering this immunological aspect, the development of trnk cells and the emergence of their adaptive feature have also demonstrated much broader diversity than before, and the generation of different types of long-lived adaptive nk cells has added further attractiveness and complexity to this field (freud et al., 2017; mazarzaei et al., 2019) . open questions about effective functions of memory nk cells against sars-cov-2 as mentioned above, another important consideration in expanding strategies to target nk cells is related to the findings that unique tr or tissue-specific (ts) nk cell populations can display adaptive features. recently, marquardt et al. have identified a new adaptive-like kir+/nkg2c+ nk cell subset with a cd49a+/cd56bright/cd16−trnk cell phenotype in human lung and in peripheral blood of hcmv-seropositive individuals, mentioned above (marquardt et al., 2019) . the adaptive-like lung trnk cells have been thus found to be hyper-responsive towards target cells (hervier et al., 2019) . this novel subset of human lung nk cells contributes to unique defense mechanisms in the context of the respiratory infection at this site (cong and wei, 2019) . with regard to this novel adaptive-like kir+/nkg2c+ nk cell subset, several interesting issues remain to be solved: i. can sars-cov-2 infection specifically drive the expansion of adaptive-like trnk cells? ii. is disease progression in hcmv-seropositive patients involved with sars-cov-2 infection compared with hcmv-seronegative ones can be different? consistent with this concept, based on clinical appearance and blood parameters during infection, we have observed that the severity of clinical manifestation in coinfection of covid-19 and hcmv in kidney transplant patients was less severe compared with renal transplant patients with only covid-19 (unpublished data). iii. is it possible to have a cross-reactivity of hcmv-specific nk cells ( figure 1 ) with a protective role against new viruses (such as sars-cov2), even unrelated ones? consistent with this hypothesis, previous studies have demonstrated that during acute influenza infection, adaptive-like nk cells could contribute to host immune response by promoting ifn-g production. these cells might be clinically effective in the defense of emerging respiratory viral infections (goodier et al., 2016; scharenberg et al., 2019) . thus, how to target these populations might be beneficial in generating protective immunity against pathogens that gain entry through or colonize lung tissues such as novel cov. a wave of investigations revealed that human nk cells presented adaptive immune responses upon immunization or infection. therefore, nk-mediated recall responses may further allow for development of immunization-based approaches in viral treatments (geary and sun, 2017; brillantes and beaulieu, 2020) . nkg2c+cd57+nkg2a-adaptive nk cells have shown effector activity in the settings of hantavirus pulmonary syndrome (hps), chikungunya virus (chikv), and type i human immunodeficiency virus (hiv-1). notably, the emergence and expansion of memory nk cells have been identified during hiv, hps, and chikv infections in hcmvseropositive patients. indeed, evidence suggests that hcmv is responsible for the expansion of nkg2c+nk cells upon additional viral infections (brunetta et al., 2010; petitdemange et al., 2011; beźiat et al., 2012) . in light of the clinical implications of hcmv-induced adaptive nk cell expansions, scholars have recently reported that the expansion of hcmv-adaptive nk cells in hematopoietic stem cell transplantation may be critical to decrease the risk of relapse and enhancing graft-versus-leukemia (gvl) reaction in patients (cichocki et al., 2014) . during this public health crisis, recent findings have correspondingly indicated a variation of covid-19 susceptibility in patients, highlighting an urgent need for comprehensive risk evaluation based on memory immune responses and crossreactivity patterns (hamiel et al., 2020) . this concept is consistent with a study that has proposed the hypothesis that the resultant immune response against prior influenza infection would, at least in part, foster immunity against sars-cov-2 due to this cross reactivity between flu and sars-cov-2, suggesting the fluinduced immune response could be useful in reducing the severity of covid-19 (salem and el-hennawy, 2020; salman and salem, 2020) . in this line, hcmv infection is a common global disease with higher prevalence rates in developing countries including iran (yaghobi et al., 2005; saadi et al., 2013; marin et al., 2016) . furthermore, hcmv seropositivity has been associated with higher frequencies of nkg2c+ nk cells in peripheral blood as compared with frequencies in hcmv seronegative individuals (davis et al., 2015) . indeed, nkg2c+ nk cells display improved adcc functionality and ifn-g production following exposure to antibody-coated hcmv infection cells (hwang et al., 2012) . significant factors can accordingly contribute to modifying viral disease outcomes. co-infection with another infectious agent including hcmv can be thus considered in terms of the influence of cross-reactivity in immune responses (wedemeyer et al., 2001; dapalma et al., 2010) . accordingly, what remains to be determined is whether hcmv adaptive nk cells can play a protective role against non-hcmv viral infections or not (mirahmadizadeh et al., 2019) like this newly emerging virus (moss, 2020) . comprehensive studies are necessary to test this hypothesis to sort out the relative influence that hcmv adaptive nk cells exert on sars-cov-2 pathogenicity in patients with detectable levels of sars-cov-2 genomic rna (figure 1) . a growing body of literature also suggests a footprint of adaptivelike nk cells in other infectious agents. moreover, memory or memory-like nk cells may expand in response to various viral and bacterial infections and some immunization strategies may arise in this way. of note, in some experimental models, memory nk responses are clearly pathogen-specific (brillantes and beaulieu, 2020) . for instance, nkg2c+ nk cells have expanded during the hcmv infection that result from the interaction between hcmv-derived peptide, ul40, in the context of hla-e, and the activating receptor cd94-nkg2c (hammer et al., 2018a) . in others, nk cells may need to be reprogrammed due to inflammatory signals during infection or immunization protocol. using hiv-envelope (env)-vaccinated humanized bone marrow-liver-thymus (blt) mice, nikzad et al. had found that human nk cells could mediate robust vaccination-dependent recall responses, indicating antigen specificity and longevity (nikzad et al., 2019) . isolating hiv-env-primed hepatic nk cells from blt mice had further indicated reduced expression of cd16, cd57, kir2dl1, and kir2dl5. it had also revealed an enhanced expression of adhesion molecules e.g. cd2, cd62l, nkg2a, and cxcr6, concluding that hepatic nk cells had been induced through dcbased hiv-env vaccination ( figure 2 ) and had similarly altered the expression levels of different surface markers of nk cells (paust et al., 2010; ackerman et al., 2012; jost and altfeld, 2013) . in addition, a study had shown that decades after initial varicellazoster virus (vzv) exposure to varicella-zoster virus (vzv) skin test antigen in individuals experiencing vzv, adaptive nk cells had been isolated from blister fluid associated with tissueresidency including cd56hi, cd16low, and more frequently expressed cxcr6, nkg2d, cd69, and cd62l. thus, human nk cells exhibit adaptive immune responses upon immunization or infection (nikzad et al., 2019; peppa, 2019) . to date, in cancer patients boosting in vivo nk cell-mediated antitumor activity by exposure to pro-inflammatory cytokines such as il2, il15, and il18 have been identified to imprint longlived memory-like features and to enhance nk cell survival and proliferation. in addition, il-12 as a new prospect improves cytokine production and cytotoxicity by nk cells (vacca et al., 2013; uppendahl et al., 2019) . use of adaptive nk cell capabilities in cellular immunotherapies particularly in viral infection also requires clinical efforts for a better understanding of their features. it will be interesting to see in future studies whether there is a memory nk response against sars-cov-2 antigen-vaccinated in lung tissue samples and peripheral blood or not. during covid-19 emergency, efforts to confront infections have been primarily focused on cellular therapy approaches through allogeneic nk cells. up to now, cellularity, as a clinical-stage cell therapeutics company (nj, usa; www. celularity.com) has produced an allogeneic and off-the-shelf cryopreserved nk cell, known as cynk-001, derived from placental stem cells for treating leukemia and multiple myeloma (ilic and liovic, 2020) . promising therapeutic strategies have been also developed for patients with moderateto-severe covid-19. indeed, inducing a robust immune response by allogeneic off-the-shelf nk cells may control the infection. notably, the biology of nk cells has indicated a possibility that immunotherapy can be used as an off-the-shelf treatment for future pandemic infections and re-emerging viruses (li h. et al., 2020) . researchers are also testing cynk-001 because nk cells that may be able to boost immunity in covid-19 patients as the first cell therapy are awaiting the food and drug administration (fda) approval for trials in this disease ( figure 2) . however, it is conceivable that destroying infected respiratory cells by nk cells may drive side effects such as hyper-inflammation, as the first cell therapy awaiting fda approval for testing cynk-001 in covid-19 patients. it is hoping that nk cell therapy might help patients suffering from this pandemic. despite the fact that hyper-inflammation has not been observed while treating cancer patients with cynk-001 (ilic and liovic, 2020) , the mechanisms to induce the virallydriven hyper-inflammation may be different. thus, it should be considered as a well-organized clinical trial to tune antiviral effects by escalating doses slowly while monitoring for toxicity evidence and side effects. a challenge in this regard is discovering whether covid-19 has the ability to evade nk cells or not. however, in the global battle against covid-19, every combat can be a valuable clue for the next step. even though nk cells may fail to fight against covid-19 efficiently, that lesson can be valuable to investigators. future work will need to be directed to understanding how longlived antigen-specific nk memory responses can be targeted towards improved human health via the development of novel clinical diagnostic approaches, therapeutic agents, or immunotherapies. cellular immunotherapy by cell engineering is also a relatively novel approach. a significant evolution of cell therapy can be thus represented by nk cells engineered with car-targeting antigens (car-nk) (souza-fonseca-guimaraes et al., 2019). car-nk cells may also characterize an off-the-shelf tool based on viral immunogenic and conserved domains and even applied singly or combined with other therapeutic approaches for viruses (mazarzaei et al., 2019) . novel strategies can be now used to manipulate nk cell function, targeting their major inhibitory checkpoints (bi and tian, 2019) such as kir (pende et al., 2009 ), nkg2a (andréet al., 2018 , and programmed cell death protein 1 (pd-1) (lipson et al., 2013) . currently, using monalizumab (namely, nkg2a monoclonal antibody), in rheumatoid arthritis (ra) and several neoplastic disorders might be an efficient target as a new anti-sars-cov-2 strategy that potentially reverses the inhibition of the innate immune system induced by the virus and leads to improved nk cells cytotoxicity at the early stage of the disease. in addition, cc motif chemokine receptor 7 (ccr7) acquisition by trogocytosis to optimize nk cell traffic to lymph nodes may improve the successful outcome of alloreactive nk cells (pesce et al., 2016) . recently, nkg2d-ace2 car-nk cell therapy is being recruited for patients infected with sars-cov-2 (nct04324996). it should be noted that nkg2d-ace2 car-nk cells produce il-15 to ensure nk cell long-term survival and release gm-csf-neutralizing scfv to suppress cytokine storm. these cells also express ace2 receptor to compete with sars-cov2 for binding sites on the susceptible cells, including alveolar epithelial cells (bonam et al., 2020) . in this line, introducing nkg2c-ace2 into chimeric antigen receptors in nk cells to enhance effector functionality might be a potential approach in future viral immunotherapy for emerging and re-emerging viruses. in addition, novel technologies such as crispr/cas9 now provide a new approach to gene editing (e.g. insertion of activating receptors or deletion of inhibitory checkpoints) . a recent report has further utilized induced pluripotent stem cell (ipsc) as an efficient off-theshelf source of expanding nk cells to optimize nk-car engineering . in conclusion, a promising approach to better management of respiratory diseases, as one of the leading causes of death worldwide, may be based on remodeling nk cells phenotypic and their functional features. it is hoped that the hypotheses addressed in this review will be employed as a stimulus for further research to help combat covid-19 as deadly contagious disease of increasing incidence around the world. the clarification of these ideas about utilizing and modifying nk cells will further allow future dissection of potential strategies 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with acute respiratory distress syndrome comparative analysis of a double primer pcr assay with plasma, leukocytes and antigenemia for diagnosis of active human cytomegalovirus infection in bone marrow transplant patients innate immunity in covid-19 patients mediated by nkg2a receptors, and potential treatment using monalizumab, cholroquine, and antiviral agents the correlation between viral clearance and biochemical outcomes of 94 covid-19 infected discharged patients + t cell help is required for the formation of a cytolytic cd8+ t cell subset that protects against chronic infection and cancer covid-19: melatonin as a potential adjuvant treatment functional exhaustion of antiviral lymphocytes in covid-19 patients nk cells exacerbate the pathology of influenza virus infection in mice cytokine regulation of natural killer cell effector functions the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 soleimanian and yaghobi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-350186-fogm1gkg authors: mammas, ioannis n.; greenough, anne; theodoridou, maria; kramvis, anna; christaki, iliana; koutsaftiki, chryssie; koutsaki, maria; portaliou, dimitra m.; kostagianni, georgia; panagopoulou, paraskevi; sourvinos, george; spandidos, demetrios a. title: current views and advances on paediatric virology: an update for paediatric trainees date: 2015-11-24 journal: exp ther med doi: 10.3892/etm.2015.2890 sha: doc_id: 350186 cord_uid: fogm1gkg paediatric virology is a bold new scientific field, which combines paediatrics with virology, epidemiology, molecular medicine, evidence-based medicine, clinical governance, quality improvement, pharmacology and immunology. the workshop on paediatric virology, which took place on saturday october 10, 2015 in athens, greece, provided an overview of recent views and advances on viral infections occurring in neonates and children. it was included in the official programme of the 20th world congress on advances in oncology and the 18th international symposium on molecular medicine, which attracted over 500 delegates from the five continents. during the workshop, the topics covered included the challenges of vaccine implementation against human papillomaviruses in countries under financial crisis, strategies for eradicating poliomyelitis and its 60th vaccine anniversary, as well as the debate on the association between autism and vaccination against measles, mumps and rubella. among the non-vaccine related topics, emphasis was given to viral infections in prematurely born infants and their long-term outcomes, new paediatric intensive care management options for bronchiolitis related to respiratory syncytial virus, the clinical implications of hepatitis b virus and cytomegalovirus genotyping, the ebola virus threat and preparedness in paediatric emergency departments, oral, oropharynx, laryngeal, nasal and ocular viral infections and merkel cell polyomavirus as a novel emerging virus of infancy and childhood. in this review, we provide selected presentations and reports discussed at the workshop. the history of paediatric infectious diseases closely parallels the history of paediatrics, as infections remain the major causes of childhood morbidity and mortality (1, 2) . viral paediatric infectious diseases are characterised by a great heterogeneity of clinical manifestations due to the unique characteristics of the neonatal period and childhood. almost 50 years ago, paediatric virology was not considered an isolated discipline and was included in the paediatric infectious diseases section of the scientific field of paediatrics (3) . however, during the past two decades, new advances in the field of clinical virology and molecular medicine have expanded the level of knowledge on the prevention, diagnosis and treatment of viral infections occurring in infancy and childhood. for this reason, although additional subspecialists in several fields may be diminishing (4) , paediatric virology as a bold new scientific field can combine paediatrics with virology, epidemiology, molecular medicine, evidence-based medicine, clinical governance, quality improvement, pharmacology and immunology (fig. 1) . to date, paediatric infectious diseases professionals have achieved a distinct role within the scientific field of paediatrics and their contribution to tertiary paediatric care is valued (2) . their current clinical and research aims are to resolve important public health issues in both developed and developing countries. furthermore, new emerging diseases, such as severe acute respiratory syndrome (sars), west nile virus (wnv) and ebola virus (ev) infections, require new prevention strategies and therapeutic protocols. in addition, paediatric infectious diseases professionals are involved in specialised paediatric care and in the follow-up of children with a history of viral infections, such as viral meningitis or acquired immunodeficiency syndrome (aids), who survive to adolescent years and beyond, requiring advanced medical care and technological services. moreover, newfound social issues have arisen, including the recent anti-vaccination wave, the financial crisis and the unprecedented immigration occurring globally and in mediterranean countries, in particular (5) . these developments and changes definitely highlight the demand for the continuous education of paediatric health professionals, informing them of the ongoing advances in paediatric care and management strategies involved in the discipline of paediatric virology (6,7). the 20th world congress on advances in oncology and the 18th international symposium on molecular medicine was held in athens (greece) and was attended by >500 participants from the five continents (8). this annual scientific meeting attracted its largest number of delegates to date and over a period of three days, up-to-date basic and clinical research findings on molecular medicine and oncology were presented and discussed. for the first time this year, during the third day of the meeting, participants had the chance to participate in the workshop on paediatric virology, with the theme 'paediatric virology: from the lab to clinical practice' (fig. 2 (10) and chaired by professor maria theodoridou, professor of paediatrics at the 'aghia sophia' children's hospital in athens (greece), this idea was re-enforced by a group of young paediatric trainees and junior researchers, leading to the creation of the paediatric virology study group (pvsg). the fundamental aim of the pvsg was to provide an array of scientific and educational activities, bringing together virology with paediatrics within a single entity. covering basic sciences and clinical medicine, the pvsg was open to scientists interested in the field of paediatric virology, either as basic scientists, researchers and virologists or as medical students, paediatric trainees, general paediatricians, paediatric infectious diseases physicians and allied health professionals. six years later, on october 2015, the pvsg had the great honour to coordinate the 'workshop on paediatric virology' as an official session of the 20th world congress on advances in oncology and the 18th international meeting in molecular medicine. this workshop was dedicated to promoting international scientific exchange and cooperation of both clinicians and researchers on paediatric virology and improving biomedical research and paediatric medical practice on viral infections occurring in childhood. in this review, we present an update on selected topics presented at the workshop. viral infections and long-term outcomes of prematurely born infants. respiratory syncytial virus (rsv) is a common respiratory pathogen in young children (11) . between 1 and 2% of infected children will require hospitalisation and up to 8% of those hospitalised will require mechanical ventilation. numerous studies (12) (13) (14) (15) (16) have demonstrated that infants born at term, who were previously healthy, subsequent to an rsv lower respiratory tract infection (lrti), develop chronic respiratory morbidity. there are many risk factors for severe acute rsv infection, which include being born prematurely and having developed bronchopulmonary dysplasia (bpd). greenough et al (12) have demonstrated that up to school age, those prematurely born children, who had developed bpd and an rsv lrti requiring hospitalisation in infancy, had increased healthcare utilisation and worse lung function, particularly affecting small airways, than children with bpd, who had not been hospitalised due to rsv. healthcare utilisation in the first two years after birth was also shown to be increased even in moderately prematurely born infants (32-35 weeks of gestation), who had were hospitalised due to rsv (13) . in two prospectively followed cohorts, one very prematurely born cohort (14) and the other, which included pre-term infants of a wide range of gestational ages (24-35 weeks), respiratory morbidity was greater during infancy in those who had had an rsv lrti (15, 16) . in the very prematurely born cohort, subsequent respiratory morbidity was increased after an rsv lrti regardless of whether hospitalisation was required or the infant had bpd (14) . the prematurely born infants, who required hospitalisation due to an rsv lrti, had poorer premorbid lung function (17) . it has also been identified that prematurely born infants may have a genetic predisposition to developing rsv lrtis. in addition, single nucleotide polymorphisms (snps) in several genes were found to be associated with chronic respiratory morbidity during infancy and reduced lung function at one year following an rsv lrti (18) . other respiratory viral infections also adversely influence respiratory outcomes during infancy in those born prematurely, including human metapneumovirus (hmpv) and human rhinovirus (hrv), particularly hrv-c (19) . poliomyelitis is an acute infectious disease affecting humans, occurring particularly in children, which is caused by small ribonucleic acid (rna) viruses of the enterovirus group of the family picornavidae (20) (21) (22) (23) (24) (25) (26) . three antigenically distinct strains (strains 1, 2 and 3) are known, with-type 1 accounting for 85% of cases. the clinical manifestations of the disease vary greatly. most cases are asymptomatic; paralytic illness is rare, affecting <1% of infected individuals. the year 2015 is the 60th anniversary since jonas salk launched the inactivated polio vaccine (ipv), enabling children to be protected against the crippling disease of poliomyelitis. with the development of the oral polio vaccine (opv) by albert sabin in 1961, the world was given the tools, with which to stop outbreaks, strengthen and build immunity, to ensure that children can grow up without the threat of polio. the combination of opv and ipv led to the eradication of polio in the americas, the western pacific and europe. today, 80% of the world population lives in polio-free regions. nevertheless, pakistan, afghanistan and nigeria are countries where polio is still categorised as an endemic viral infection. it should be noted that in 2013-2014 an upsurge of polio in areas, which were considered polio-free, occurred. the confirmed circulation of wild-type poliovirus (wpv) in israel and the outbreak of acute flaccid paralysis (afp) in syria mean that there is a high risk of the disease being reintroduced into europe. europe should implement a prevention policy, which is based on enhancing the vaccination of resident and refugee populations, strengthening surveillance and being prepared to rapidly respond to the identification of polio. in greece, there is a national action plan, which includes programmes to sustain high levels of polio immunisation coverage, afp surveillance and actions in the event of a suspected or confirmed poliomyelitis case. fighting polio with vaccination has been one of the most successful public health programmes in history, reducing the number of polio cases by 99%, making possible the expectation towards disease eradication. even though a successful vaccine against hbv has been implemented in 184 countries, the eradication of hbv is still not on the horizon (27) . according to the world health organization (who), there are 240 million chronic carriers of the virus, globally (28) . the risk of developing chronic hepatitis ranges from >90% in newborns of hepatitis b e antigen (hbeag)-positive mothers, 25-35% in children under the age of 5 and <5% in adults (29) . hbeag, a non-particulate viral protein, is conventionally considered a marker of hbv replication (30) . this is the only antigen of hbv, which can cross the placenta (31), leading to the specific unresponsiveness of helper t cells to the hepatitis b capsid protein (hbcag) and hbeag in newborns of hbeag-positive mothers (32) . hbeag is tolerated in utero and acts as a tolerogen after birth (33) . thus, perinatal transmission is frequent when mothers are hbeag-positive, whereas it occurs significantly less frequently when mothers are hbeag-negative (34) (35) (36) . sequence heterogeneity is a characteristic of hbv, the prototype member of the family hepadnaviridae (37) . based on an intergroup divergence of >7.5% across the complete genome, hbv has been classified phylogenetically into at least 9 genotypes. with between ~4 and 8% intergroup nucleotide divergence across the complete genome and good bootstrap support, genotypes a-d, f, h and i are further classified into subgenotypes. hbv genotypes and in some cases subgenotypes have a distinct geographical distribution both globally and locally (37) . the different genotypes/subgenotypes can develop different mutations in the regions of the hbv genome, which code for hbeag and the envelope proteins. these differences can be related to the role of the hbv genotypes, to their mode of transmission, to the clinical manifestation of the disease following hbv infection and to their response to antiviral therapy. thus, the genotypes/subgenotypes of hbv may be responsible for the different modes of transmission and natural history of infection in children, noted in different regions of the world, where distinct genotypes/subgenotypes prevail. despite the invention of the pap smear test by george n. papanicolaou almost 90 years ago, hpv is considered the most frequent carcinogen in humans, causing approximately 530,000 cases of cervical cancer per annum, with the majority of cases occurring in developing countries (38) (39) (40) . the link between this deoxyribonucleic acid (dna) virus and cervical cancer was firstly suspected in the early 1970s by professor harald zur hausen, who in 2008 received the nobel prize in physiology and medicine (41) . in the same year, two vaccines against hpv, the bivalent hpv 16/18 and the quadrivalent hpv 6/11/16/18, were implemented into clinical practice in several countries, worldwide (42) . however, despite the significant calculated expected health benefits following the implementation of hpv vaccination programmes, current trends indicate low hpv vaccination uptake among female adolescents in several countries in europe (43) (44) (45) (46) . these studies have also focused on potential socio-demographic factors, which influence parents' decisions to decline hpv vaccination to their daughters, including age, ethnicity, receipt of childhood vaccines, knowledge, attitudes, parental and community acceptability. of note, in greece, a country under financial crisis since 2010, in which several health quality indexes have already deteriorated (47) , financial reasons have also been elucidated behind non-vaccination against hpv (38) . since data on hpv vaccination among female adolescents in greece remain limited, the first pilot cross-sectional questionnaire-based study, known as the eleftheria study, was expected to assess hpv vaccination uptake among female adolescents in greece during the period between 2008-2014 and to investigate socio-demographic reasons for declining hpv vaccination (38) . it was designed by the first department of paediatrics at the university of athens and the department of clinical virology at the university of crete and its name was inspired by 'eleftheria', the name of the first adolescent, who was recruited in the study. the preliminary findings of this project stress the need to provide and maintain health insurance coverage to children in countries under financial crisis (38) . for this reason, urgent health policy responses to the recent financial collapse in greece are required as the full impact of austerity measures unfolds in the coming years. children and ebola infection. ev was first discovered in 1976 and has caused three outbreaks, two simultaneously in 1976 and one in 2014 (48) . the exact mode of transmission of ev remains unknown; however, it seems to be transmitted from the biological fluids of wild animals and subsquently through human-to-human contact (49) . according to the who, the 2014 west african outbreak claimed a case fatality, which ranged from 25 to 90% and a total number of roughly 10,000 fatalities; the number of children is unknown (49) . this fact makes it a potential lethal tool in bio-warfare. if used in residential areas or within the public transportation system, as a biological agent, ev can easily affect the welfare and life of children. emergency departments (eds) should be ready for such attacks, especially catering for the needs of paediatric victims (50, 51) . carley's consensus statement for formulating major incident plans can be quite helpful (52) . local tertiary paediatric hospitals or peadiatric units should play an active role by coordinating the care of children. planning for such events involving children should be carried out by experienced clinicians in paediatrics. approximately 10-15% of the equipment used in general hospitals should be suitable for peadiatric victims of all ages. staff training for major incidents should be regular and should include caring for children. while triaging, it is advisable to use modified adult scoring systems. in particular, when triaging a large number of children, the eichelberger modification to the triage revised trauma score should be used (48) . if possible, children's assessment and treatment, as well as transfers, when needed, should be provided by experienced paediatric teams. if allowed by the senior clinician, parents could possibly stay on site near their children. during hospitalisation and following discharge, adequate support should be provided to the families (52) . an ed should be ready to accommodate the needs of children following an ev attack by offering personal protective equipment (ppe) to staff, appropriate decontamination and isolation facilities and antidotes, if available, as well as general paediatric equipment. staff should always suspect bioterrorism if a few or several people present with unusual or unexpected symptoms or if their symptoms do not conform with a certain medical diagnosis. identify, isolate and inform is the current standard approach to ev suspicion (53) . hopefully, none of this will be actually needed in the near future. viral bronchiolitis, mainly caused by rsv infection, remains the leading cause of infant admission to the picu (54, 55) . recent laboratory tools have also confirmed many other viruses to account for bronchiolitis, either as co-infection with rsv or as the only aetiological pathogen. such viruses are hrv, hmpv, adenovirus, as well as influenza and parainfluenza viruses (56) . even following the anti-rsv post-prophylaxis era, rsv seems to be the most common cause of bronchiolitis in infancy and is responsible for severe clinical manifestations, longer hospitalisation and picu admissions (57) . severe bronchiolitis is indicated by persistently increased respiratory effort (tachypnoea, nasal flaring, intercostal, subcostal or suprasternal retractions, accessory muscle use and grunting) hypoxaemia, apnoea or acute respiratory failure. risk factors for severe disease and/or complications of bronchiolitis, even death, are considered to be prematurity (gestational age <37 weeks), age <12 weeks, chronic pulmonary disease, particularly bpd (also known as chronic lung disease), congenital and anatomic defects of the airways, congenital heart disease, immunodeficiency and neurologic disease (58) . though it is a common and old clinical entity, there is a wide variety in clinical practice and the need to clarify each of the therapeutic means has to be done with large, well-designed clinical studies. when it comes to the treatment of critically ill children, there is a paucity of evidence-based guidelines. fluid replacement, oxygen supplementation and close monitoring are the first steps in the management of the disease. the role of epinephrine and nebulised bronchodilators in combination with systemic corticosteroids is questioned, but can be given on an individualised basis (59) . nebulised hypertonic saline is a new therapeutic agent, which has not been investigated for possible use in the picu. although it is recommended, its use remains to be established. other therapies, such as heliox, surfactant and ribavirin, are also being examined, with controversial results thus far. as little seems to have been achieved when it comes to treatment, a lot of emphasis has been given to the prevention of the illness. hygiene measures and education of caregivers, hospital personnel, nurses and doctors on limiting transmission and elimination of the environmental risk factors are strongly recommended in published guidelines (59) . palivizumab prophylaxis is recommended for infants with risk factors, such as chronic illnesses, prematurity and immunodeficiency, as well as other conditions, which are being clarified at the revised published guidelines (59) . since the burden of rsv bronchiolitis is high, research must aim for the development of an anti-rsv vaccine, as well as new antiviral agents. autism is a severe neurodevelopmental disorder affecting the paediatric population (60) . autism spectrum disorders (asd) include disorders, such as psychomotor regression, language impairment and behavioural social withdrawal, placing patients with asd in permanent need for healthcare and social support (61) . earlier reports have associated vaccination against mmr with the occurrence of asd in children (62) , thus leading in particularly low vaccination coverage. as a result, outbreaks regarding the vaccine preventable strains have reappeared throughout europe (63-65), asia (66,67) and the united states (68, 69) . extensive research around the issue has emerged, soundly dissociating mmr vaccination from any asd occurrence, even in high-risk populations (70) (71) (72) . however, the loss of credibility of the mmr vaccine remains a concern. this can be partially explained by failure on behalf of the scientific community to effectively communicate: i) the limitations and bias of the original study of wakefield et al (62) in 1998, ii) the mounting evidence supporting the lack of a causal relationship between mmr vaccine receipt and autism onset, as proven by large epidemiological studies (70) (71) (72) and iii) adverse effects of vaccination in the general setting of coincidental, rather than causal associations. another contributing factor must be attributed to a powerful influence by the public media, such as television, newspapers and internet, regarding mmr vaccination, ultimately leading to a subsequent negative public health response. in the future, more effective communication strategies are required to reassure parents of vaccine safety and importance. the ocular manifestations of viral infections in neonates and children vary greatly and can range from innocuous to vision threatening (73) . the majority of viral conjunctivitis in children are caused by adenovirus, a dna virus, which can cause a range of human diseases, including upper respiratory tract infection. viral conjunctivitis is associated with epidemic keratoconjunctivitis, pharyngoconjunctival fever and acute haemorrhagic conjunctivitis (74) . signs include eyelid oedema and tender pre-auricular lymphadenopathy, prominent conjunctival hyperaemia, follicles and punctate epithelial keratitis. in viral infections in children, the involvement of the anterior segment is mild and self-limited; spontaneous resolution usually occurs within 2-3 weeks, except in cases of congenital infection, which are often associated with significant alterations in ocular structures. neonatal conjunctivitis (also known as ophthalmia neonatorum) is defined as conjunctival inflammation developing within the first month of life. it is the most common type of infection in neonates, occurring in up to 10% of neonates. it is often identified as a specific entity distinct from conjunctivitis in older infants as it is often the result of infection transmitted from the mother to the infant during delivery (74) . molluscum contagiosum ocular infection in children is caused by a specific double-stranded dna poxvirus, which typically affects otherwise healthy children with a peak incidence between the ages of two and four. transmission occurs through contact, with subsequent autoinoculation. presentation is with chronic unilateral ocular irritation and mild discharge, while lesions are usually self-limiting. primary infection with herpes simplex virus (hsv) is usually associated with eyelid and periorbital vesicles, papillary conjunctivitis, discharge and lid swelling. dendritic corneal ulcers can also be present, particularly in patients with atopic infection can lead to eczema herpeticum, which can be very severe (75) (76) (77) . varicella-zoster virus (vzv) is a serious, but rare, viral infection in children, in which prolonged inflammation may lead to corneal thinning or perforation, glaucoma and cataract formation (74) . involvement of the posterior structures mostly related to hsv and vzv is potentially sight-threatening. retinal or optic nerve involvement should be suspected in any child, who complains of an acute onset of blurred vision in the absence of anterior segment inflammation or opacities in the ocular media. optic neuropathy may occur as an isolated sign, although it is more often associated with a more generalised involvement of the central nervous system (77, 78) . while specific therapy is not always available, the early diagnosis of ocular viral disease in children should aid in the amelioration of acute symptoms and in the prevention of long-term complications. otorhinolaryngologists versus hpv-associated lesions. hpv is responsible for many benign lesions of the airway tract and the genital area in the adult population, as well as for cancer of the larynx and oral cavity, particularly subtypes 16 and 18 (79, 80) . a recent study (81) also revealed an increased frequency of hpv infections in neonates and children. otorhinolaryngologists are the specialists, who treat children with hpv-related lesions localised in the oral cavity, the oropharynx and larynx. these lesions are mostly benign. there are four types of hpv-related lesions concerning the upper airway track: i) squamous papilloma, ii) verruca vulgaris, iii) focal epithelial hyperplasia and iv) condyloma acuminatum. in children, the most common clinical expression of hpv is recurrent respiratory papillomatosis (rrp). it causes hoarseness of the voice and sometimes the lesions can cause obstruction of the upper airway track. in adults, the clinical behaviour differs, as the disease requires fewer surgical excisions than in children. in children, the lesions caused by hpv often need many surgical procedures before they become extinct and quiescent. inverted papilloma, which is a type of squamous papilloma, is strongly associated with hpv subtypes 6 and 1 and its incidence in children is twice as high compared to adults (82) . fortunately, the majority of these lesions can be surgically removed and provide the young patient with a good prognosis. microsurgery, transoral laser microsurgery (tlm) and endoscopic endonasal approach (eea) are some of the most frequent surgical methods of excision. a review of recent studies dealing with hpv lesions in children revealed only a few cases, which have been treated surgically. the most common approach of surgical treatment is using tlm. however, laser treatment can cause several complications, such as stenosis, burns of the airway tract and scars. an alternative to tlm are microdebriders (83) . microdebriders provide a more accurate excision, removing only the affected tissue and preserving the healthy tissue. the preservation of healthy epithelium is very useful, when repeated interventions are needed, particularly in children. when the lesion causes obstruction of the airway tract, a tracheotomy is necessary to keep the airway open. however, it should be avoided and performed only when it is an emergency because of the danger of spreading the disease to the respiratory tract (84) . in the future, the association between hpv-related lesions in children and cancerous lesions in adults needs to be considered carefully, revealing the importance of vaccination in children against hpv (85) . infection (86) . other agents, such as cmv, toxoplasma and adenovirus, produce a similar illness. the incubation period ranges from 33 to 49 days (87) . clinical presentation is usually prolonged (average 16 days) and ranges from a non-specific flu-like illness to the more distinctive triad 'fever, pharyngitis, lymphadenopathy-splenomegaly'. other clinical manifesta-tions include fatigue, hepatitis and eyelid oedema. possible complications are meningoencephalitis, haemolytic anaemia, thrombocytopenia, rash, conjunctivitis, haemophagocytic syndrome, myocarditis, neurologic diseases, pancreatitis, parotitis, pericarditis, pneumonitis, psychological disorders and splenic rupture. laboratory findings include the elevation of liver enzymes and lymphocytosis with a marked increase in the number of atypical lymphocytes in the peripheral blood (87) . additionally, immunophenotypic alterations of lymphocytes have been described in the various phases of ebv infection (88) . more specifically, a reduction of b-lymphocytes and an increase in the number of cd3 + cd8 + , t-lymphocytes, with a subsequent decrease in the cd3 + cd4 + /cd3 + cd8 + ratio is noted (89) . in the study by panagopoulou et al (86) , which was presented at the workshop, researchers aimed to examine whether there is an association between the immunophenotypic alterations and the variability of the clinical presentation of im. although several studies (89) (90) (91) have examined the immunophenotype of lymphocytes in ebv infection, very few (89) have correlated these with the clinical course. the presented study (86) showed that the immunophenotypic analysis of cytotoxic t cells provides important information on the physiology of the immune response to ebv infection. additionally, it may potentially play a predicting role, providing information on the expected clinical course, potential complications and the time to recovery from ebv infection. mcpyv is a newly-discovered small, human dna virus, which causes a widespread, previously unrecognised, human infection in adulthood and childhood (92, 93) . it is classified in the family polyomaviridae, a group of non-enveloped, double-stranded dna viruses with icosahedral symmetry, which can infect a variety of vertebrates, including humans and can cause malignant tumours upon their inoculation into heterologous hosts (94) . first described in january 2008, the prototype sequence of mcpyv has a 5,387 base pair genome and contains the early region, which encodes the large tumor (lt) antigen and the small tumour (st) antigen and the alternative tumor antigen open reading frame (alto), the late region, which encodes vp 1, vp 2 and vp 3 and a non-coding regulatory region. of note, the genome of mcpyv has been detected in approximately 80% of merkel cell carcinoma (mcc) cases (95) . although mcc is a relatively rare, highly aggressive, human skin cancer of neuroendocrine origin, its worldwide incidence has increased over the past twenty years from 500 to 1,500 cases per year. in the majority of mcc cases, mcpyv is integrated into the host genome in a monoclonal manner and the viral t antigen has truncating mutations, which render the t antigen unable to initiate the dna replication required to propagate the virus (94) . recent serological data using enzyme-linked immunosorbent assay (elisa) techniques have suggested that mcpyv infection is common in childhood and occurs during early childhood, after the disappearance of specific maternal antibodies against mcpyv (93) . mcpyv dna has been detected in nasopharyngeal aspirate samples collected from children, indicating the presence of mcpyv in the upper respiratory tract of children (96, 97) . mcpyv is acquired in childhood through close contact involving saliva and the skin (93) . as to date, the mode of mcpyv transmission has not been fully elucidated, the precise role of the respiratory secretions in mcpyv transmission in childhood requires further investigation. the respiratory tract system is involved as a 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prevention examining inequalities in the uptake of the school-based hpv vaccination programme in england: a retrospective cohort study have health trends worsened in greece as a result of the financial crisis? a quasi-experimental approach the challenge of modern biowarfare: ed preparedness for paediatric victims of the ebola virus ebola virus outbreak, updates on current therapeutic strategies american college of emergency physicians; emergency nurses association; society for academic emergency medicine: ethical issues in the response to ebola virus disease in united states emergency departments: a position paper of the american college of emergency physicians, the emergency nurses association, and the society for academic emergency medicine patients under investigation for ebola virus disease in the united states: hospital preparedness planning and alternate care facilities planning for major incidents involving children by implementing a delphi study ebola virus disease the clinical profile of infants with rsv bronchiolitis in picu management of infants with severe bronchiolitis who were admitted to two picus what is the clinical relevance of respiratory syncytial virus bronchiolitis?: findings from a multi-center, prospective study a comparison of characteristics and outcomes in severe human metapneumovirus and respiratory syncytial virus infections in children treated in an intensive care unit risk factors for requiring intensive care among children admitted to ward with bronchiolitis clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis mmr vaccination and autism: an update autism: definition, neurobiology, screening, diagnosis ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children spotlight on measles in italy: why outbreaks of a vaccine-preventable infection continue in the 21st century mumps outbreak and laboratory diagnosis measles outbreak in outbreaks of mumps: an observational study over two decades in a single hospital in korea nationwide rubella epidemic -japan an outbreak of measles in an undervaccinated community epidemiology and the economic assessment of a mumps outbreak in a highly vaccinated population autism occurrence by mmr vaccine status among us children with older siblings with and without autism early exposure to the combined measles-mumps-rubella vaccine and thimerosal-containing vaccines and risk of autism spectrum disorder vaccines are not associated with autism: an evidence-based meta-analysis of case-control and cohort studies ocular viral infections in neonates and children ocular viral infections maternal and neonatal herpes simplex virus infections pediatric herpes simplex virus infections: an evidence-based approach to treatment neonatal herpes simplex virus type-1 central nervous system disease with acute retinal necrosis chorioretinitis in infants hpv infections in children: current surgical treatment of hpv lesions found in the oral cavity, oropharynx, larynx and nose cavity hpv-associated head and neck cancer: a virus-related cancer epidemic the prevalence of human papilloma virus (hpv) infections in oral squamous cell carcinomas: a retrospective analysis of 88 patients and literature overview human papillomavirus in the oral cavities of children and adolescents. oral surg oral med oral pathol oral radiol endod juvenile recurrent respiratory papillomatosis: still a mystery disease with difficult management recurrent respiratory papillomatosis: current and future perspectives a: hpv vaccination in head and neck hpv-related pathologies correlation between the clinical course and the immunophenotypic profile of t-cytotoxic lymphocytes in children with infectious mononucleosis progress and problems in understanding and managing primary epstein-barr virus infections detailed analysis of epstein-barr virus-specific cd4+ and cd8+ t cell responses during infectious mononucleosis study of the immunophenotype of peripheral blood lymphocyte subsets in children with epstein-barr virus and cytomegalovirus infection: association with outcome phenotypic and functional separation of memory and effector human cd8+ t cells comparative immunophenotypic features of ebv-positive and ebv-negative atypical lymphocytosis merkel cell polyomavirus (mcpyv): a novel emerging virus of infancy and childhood merkel cell polyomavirus infection in childhood: current advances and perspectives novel human polyomaviruses -re-emergence of a well known virus family as possible human carcinogens the novel human polyomaviruses hpyv6, 7, 9 and beyond merkel cell polyomavirus in respiratory tract secretions merkel cell polyomavirus dna in immunocompetent and immunocompromised patients with respiratory disease the authors would like to thank all of the chair persons and the speakers of the workshop on paediatric virology for their continued support in providing the most up-to-date information to the symposium. the authors would also like to thank the participants, who attended the event and provided feedback so that further study on improving the program can be achieved for the future. last but not least, this symposium would not have been possible without the dedicated hard work and strong commitment from the congress secretary carol kalogridis, the members of the pvsg and the whole symposium organising team for developing the scientific program, organising, coordinating and guaranteeing the success of this scientific event. key: cord-302277-c66xm2n4 authors: bakaletz, lauren o. title: developing animal models for polymicrobial diseases date: 2004 journal: nat rev microbiol doi: 10.1038/nrmicro928 sha: doc_id: 302277 cord_uid: c66xm2n4 polymicrobial diseases involve two or more microorganisms that act synergistically, or in succession, to mediate complex disease processes. although polymicrobial diseases in animals and humans can be caused by similar organisms, these diseases are often also caused by organisms from different kingdoms, genera, species, strains, substrains and even by phenotypic variants of a single species. animal models are often required to understand the mechanisms of pathogenesis, and to develop therapies and prevention regimes. however, reproducing polymicrobial diseases of humans in animal hosts presents significant challenges. there is now compelling evidence that many infectious diseases of humans (fig. 1) and animals (table 1) are caused by more than one microorganism. the mixed microbial nature of these diseases has been recognized since the early 1920s but there has been renewed interest in this topic since the 1980s 1 , signalled by the publication of four important reviews from 1982 to the present date. polymicrobial diseases (see box 1 for nomenclature) can be caused by the synergistic or sequential action of infectious agents from either the same or different kingdoms, genera, species, strains or substrains, or by different phenotypic variants of a single species 6 . polymicrobial diseases share underlying mechanisms of pathogenesis, such as common predisposing factors (box 2) , but each disease has unique aspects. although the molecular mechanisms of some polymicrobial infections are known, other polymicrobial diseases are not well understood. owing to their complexity, the study of polymicrobial infections requires a multidisciplinary approach and specific in vitro methodologies and animal models. the development of assay systems and treatment and prevention regimes is needed. multiple diverse in vitro systems have been used to study polymicrobial diseases (box 3) . although in vitro methods are crucial for understanding polymicrobial diseases, rigorous, reproducible and relevant animal models of human diseases are essential for the prevention and treatment of these co-infections [7] [8] [9] [10] .all animal models of human diseases have inherent limitations but they also have important advantages over in vitro methods, including the presence of organized organ systems, an intact immune system and, in inbred mice, specific genetic backgrounds, and the availability of many reagents for characterizing the immune response to sequential or co-infecting microorganisms. the availability of mice with specific genetic backgrounds can have a pivotal role in understanding the mechanisms of pathogenesis of polymicrobial diseases, as exemplified by studies on septic peritonitis 11-17 , periodontal disease 18 and lyme arthritis 19, 20 . understanding the molecular mechanisms underlying polymicrobial diseases of veterinary importance has also been facilitated by the use of animal models. these veterinary systems are useful examples for those researchers attempting to develop animal models of complex human diseases. so far, most animal models for human polymicrobial diseases are rodents, usually mice, but also rats, gerbils, cotton rats and chinchillas. other animal models include non-human primates, which are useful for modelling diseases that are caused by microorganisms with a restricted host range. for most human viral co-infections of clinical importance, good animal models and culture systems are lacking and are urgently required. this review provides an overview of the pathogenesis of selected polymicrobial diseases, the molecular basis for some of these co-infections and describes animal models that have been developed to mimic these diseases. human co-infections with multiple hepatotropic viruses from the hepatitis virus group are well documented. co-infection with multiple hepatitis viruses is possible owing to their similar routes of transmission and ability to chronically infect the host. hepatitis a virus (hav) co-infection of individuals that are chronically infected with hepatitis b virus (hbv) and/or hepatitis c virus (hcv) results in a disease of increased severity and risk of death. moreover, hbv-hcv co-infection occurs in 10-15% of hbv patients, and hepatitis g virus (hgv)-hcv co-infection occurs in 10-20% of individuals with chronic hcv infection; however, hbv-hepatitis d virus (hdv) coinfection occurs only in the setting of co-infecting hbv. viral interference, in which replication of one virus is suppressed by another virus, is an intriguing aspect of triple hbv-hcv-hdv infection -hdv can suppress both hbv and hcv replication 29 . in a retrospective study of patients with hepatitis virus co-infections, hdv was dominant by rt-pcr detection of hdv rna in triple co-infections, but in dual co-infections there were alternating dominant roles for either hbv or hcv. multiple hepatotropic viral infections are associated with reduced hcv replication but increased pathology. patients with dual or triple co-infections have more severe liver disease pathologies than patients that are in cattle, infections with bovine viral diarrhoea virus (bvdv) can be clinically asymptomatic or can cause severe symptoms. the outcome depends on whether the primary infection occurred in utero or after birth 21 and whether the primary infection was with a cytopathic or non-cytopathic biotype of bvdv. milder, congenital persistent infection follows foetal infection with noncytopathic bvdv. death, or culling from the herd within 1 year of birth after failure to thrive, is common; however some persistently infected calves seem healthy at birth and survive for several years. mucosal disease follows congenital persistent infection and is due to coinfection with cytopathic and non-cytopathic bvdv in utero. conversely, acute bovine diarrhoeal disease is induced by primary post-natal infection with either of in some instances production of a virulence factor by one microorganism can increase the risk of infection or colonization by a second microorganism. this might include sharing virulence factors, such as adhesins; for example, h. influenzae shows enhanced adherence when pretreated with bordetella pertussis adhesins. infection with a microorganism that results in an impaired immune system predisposes the affected individual to infection with other microorganisms, or can allow infection of a niche that is usually protected in the body. [56] [57] [58] [59] [60] [61] . brdc pathology results from the effects of pathogen and host virulence factors. m. haemolytica produces multiple virulence factors, including a leukotoxin of the repeat in toxin (rtx) family that activates pmns, induces production of inflammatory cytokines, results in cytoskeletal changes and causes apoptosis. leukotoxin-activated pmns are crucial to pathogenesis and inflammatory mediators released by neutrophils are thought to be essential because inflammation and most of the pathology in brdc is absent human viruses, in contrast with rodent hosts, the use of greater primates for modelling human viral disease is limited by differences in the clinical presentation of disease -some diseases are asymptomatic in primates -and the expense of using primate models in research 40 . given the difficulties of modelling diseases caused by individual viruses, it is not surprising that models of virus co-infections, such as hiv and hcv, have not been established. a variety of small animal and lower-order nonhuman primate model systems have been developed to model human viral co-infections. mice and ferrets have been used to study interference between influenza a virus (iav) strains, as well as interference between cold-adapted influenza a and b vaccine reassortants and wild-type viruses [41] [42] [43] . murine hosts have been used to study how one retrovirus can block infection by a second retrovirus 44 , and to define the role of the tissue tropisms of helper viruses on the disease specificity of a co-infecting oncogene-containing retrovirus such as the type of tumour that is induced 45 . balb/c and nih swiss mice have been used as models to analyse a putative pathogenic interaction between a murine leukaemia virus and a polyomavirus 46 . rabbits have been used to produce models of mixed htlv-1 and hiv-1 co-infection 47 and co-infection with htlv types i and ii 48 . rhesus and pig-tailed macaque monkeys have been used to model co-infection with simian immuno deficiency virus (siv) and simian acquired immunodeficiency syndrome retrovirus type 1 (srv-1) 49 . more recently, macaques have been used to define the susceptibility to co-infection with two human hiv-2 isolates 50 . in this model, co-infections were established in macaques that were simultaneously exposed to both viruses, whereas in macaques that were sequentially challenged, co-infections were only observed if challenge with the second hiv-2 isolate occurred early after challenge with the first hiv-2 isolate and before full seroconversion. chimpanzees have also been used to study hiv-1 subtype b strain co-infections 51 there are several new in vitro methods, including: • genomic sequencing of individual microorganisms and mixed microbial ecosystems and the use of meta-genomics to study the genomes of uncultured microbial communities. • molecular phylogenetic studies, such as genotyping or 16s rrna analyses, to determine the genetic relatedness or diversity of microbial community members. • genome-wide transcription profiling using microarrays to assess the rates of transcription during polymicrobial infection. • fluorescence-based imaging and detection methods such as laser confocal microscopy using fluorescent probes, fluorescent in situ hybridization (fish) using species-specific 16s rrna-directed oligonucleotide probes and the use of transcription and translation reporter gene constructs. • analyses of inter-genera bacterial signalling such as quorum sensing. • use of biofilm chambers and continuous culture flow cell reactors to study polymicrobial diseases. • co-infections of cell lines, tissue and organ cultures and extracted teeth. • laser capture microdissection of colonized infected tissues. the mechanisms of synergy between pathogens in om have been analysed using in vitro methods and animal models (reviewed in ref. 7 ). briefly, viral infection compromises the protective functions of the eustachian tube, alters respiratory-tract secretions, damages the mucosal epithelial lining, interferes with antibiotic efficacy, modulates the immune response and enhances bacterial adherence 77 and colonization 78 to predispose the host to bacterial om. influenza and parainfluenza viruses have neuraminidases that remove sialic acids from host-cell glycoproteins, which results in the exposure of receptors for pneumococci. the activity of neuraminidases allows the adherence of and/or colonization by s. pneumoniae 79 , which is one of the primary aetiological agents of acute om. although all upper respiratory tract viruses can disrupt the host respiratory tract defences, each virus has a specific pathology. not surprisingly, there are specific partnerships between viruses and bacteria in om. in the chinchilla model (fig. 2) iav predisposes the host middle ear to s. pneumoniae-induced or pneumococcal om and adenovirus infection predisposes the host middle ear to nthi om. iav does not predispose the chinchilla host to nthi-induced om, nor does adenovirus predispose the host to either m. catarrhalis-induced om 80 or to pneumococcal om 78, 81 .virus and bacteria synergy seems to be maintained in adults and children. the oropharynges of 15% of adults with experimental iav infection were heavily colonized with s. pneumoniae six days after viral challenge 82 , whereas isolation rates for other middleear pathogens were unaffected. in children, s. pneumoniae is cultured more often from middle-ear fluids that contain iav than from those that are culture-positive for either rsv or parainfluenza virus 83 . cystic fibrosis polymicrobial diseases. upper respiratory viruses predispose the host to bacterial invasion of the lower respiratory tract and are often detected in patients with copd 84 or cystic fibrosis (cf). in addition to bacterial factors, host determinants also have a role in co-infections of the cf lung. cf patients do not have a higher incidence of viral disease compared with non-cf individuals, but viral disease produces more significant pathology. it has been proposed that cf patients have impaired innate immunity, which allows increased virus replication and upregulated cytokine production. in turn, this results in increased bacterial colonization of the lung. zheng and co-workers 85 showed that increased virus replication in cf patients is due to the absence of the antiviral nitric oxide synthesis pathway. this was attributed to impaired activation of signal transducer and activator of transcription (stat1), which is an important component of the antiviral defences of the host. compromising innate immunity provides a mechanism for the severity of viral disease in cf and the establishment of bacterial co-infections. expression of virulence determinants by pseudomonas aeruginosa, a pathogen of cf patients, can depend on signals produced by other bacteria. transcriptional profiling in vitro coupled with research in an animal model showed that adding exogenous signalling molecules, in neutrophil-depleted animals. in pigs, porcine respiratory disease complex (prdc) is a similar disease complex that is caused by co-infection with one of several porcine respiratory tract viruses and members of the pasteurellaceae family [62] [63] [64] [65] [66] . porcine reproductive and respiratory syndrome. prrs is caused by prrsv co-infection with multiple bacterial pathogens including streptococcus suis type ii 67 , bordetella bronchiseptica 68 , mycoplasma hyopneumoniae 69 and actinobacillus pleuropneumoniae 70 . in turkeys, pems is caused by turkey coronavirus, avian pneumovirus or newcastle disease virus co-infection with enteropathogenic escherichia coli 71, 72 . 1) . despite the diverse spectrum of diseases and anatomical niches, there are common underlying mechanisms involved in these co-infections. often, viral disruption of host defences has a role in the development of bacterial co-infections. in otitis media, which is a middle ear infection, a synergistic interaction that results in disease owing to co-infection with an upper respiratory tract virus and three bacterial species -streptococcus pneumoniae, nontypeable haemophilus influenzae (nthi) and moraxella catarrhalis -is well documented. however, certain viruses such as respiratory syncytial virus (rsv) and rhinovirus seem to predispose affected individuals more often to bacterial om. the saying that children ''get a cold and a week later develop om'' is substantiated by epidemiological data that indicate a seasonal influence on the coincidence of 'colds' and om, as well as evidence for a peak incidence of virus isolation that is coincident with, or immediately preceding, peak incidence of om (fig. 2) . in the recent finnish om cohort study and finnish om vaccine trial, the relationship between viruses and om was supported by data that showed the presence of a virus in either nasopharyngeal aspirates or middle-ear fluid specimens in 54% or 67% of om cases in these studies, respectively 76 . rhinovirus was the most commonly isolated virus, followed by enterovirus and rsv. a specific virus was detected in two-thirds of all cases of acute om in young children, but only those viruses that are tested for can be detected, so this figure is likely to underestimate the proportion of acute om events with viral co-infection. a sequential inoculation model has been developed in mice to probe the mechanisms of the interaction between s. pneumoniae and iav. mice infected simultaneously with s. pneumoniae and iav displayed gradual weight loss and increased mortality, commensurate with an additive effect. conversely, mice infected with s. pneumoniae seven days after iav infection uniformly died within 24 hours and had significant bacteraemia -lethality was due to overwhelming pneumococcal septicaemia 89 . this model is being used to define the molecular mechanisms of the lethal synergy of iav with s. pneumoniae 90 . the activity of viral neuraminidase was found to be crucial to this synergistic relationship 91 and, in common with om, has an important role in predisposing both the upper and lower respiratory tracts to invasion by s. pneumoniae. such as autoinducer-2, or the production of this molecule by the oropharyngeal bacterial flora upregulated the expression of genes that encode virulence factors 87 . modulation of gene expression by interspecies communication between normal flora and pathogenic bacteria could therefore have a role in polymicrobial diseases. periodontitis. some herpesviruses, including human cytomegalovirus (hcmv), epstein-barr virus type 1 (ebv-1) and hsv, have been implicated in the pathogenesis of a severe and highly aggressive form of periodontitis through co-infection with porphyromonas gingivalis 87, 88 . hcmv and hsv were detected at significant levels using pcr in periodontal disease and were shown to be good predictors of the presence of p. gingivalis. ebv-1 was not linked to isolation of p. gingivalis but was also predictive of active disease 87 . a common theme has emerged from these models that upper respiratory tract viruses of both animal and human hosts can predispose the respiratory tract to infection by pasteurellaceae in brdc and prdc in animals and periodontitis, sinusitis, copd and om in humans. a mouse model has been developed to evaluate the role of respiratory dendritic cells (rdcs) in viral-bacterial co-infections 105 . rdc migration from the lungs to the secondary lymph nodes after infection with pulmonary virus is monitored by the use of a fluorescent dye. after inoculation with influenza virus, the rate of rdc migration to the draining peribronchial lymph nodes increased, but this only occurred during the first 24 hours after virus infection. after 24 hours, rdcs did not migrate, despite virus replication and pulmonary inflammation. moreover, viral infection suppressed additional rdc migration in response to either a second pulmonary virus infection or administration of bacterial cpg dna. in addition to suppressed rdc migration, there was also suppression of an antiviral pulmonary cd8 + t-cell response. it seems likely that the transient suppression of rdc migration and the delayed development of an effective adaptive immune response to a second infection might be another mechanism by which influenza virus predisposes the host to bacterial co-infection. atrophic rhinitis. infection with more than one bacterial species is common in animals and man. in pigs, atrophic rhinitis (ar), which is characterized by severe atrophy of the nasal turbinates, is caused by co-infection with strains of b. bronchiseptica and heat-labile toxin-producing strains of p. multocida [106] [107] [108] . p. multocida can adhere to respiratory tissues, but co-infection with b. bronchiseptica allows more efficient colonization by p. multocida. p. multocida produces a dermonecrotic toxin called pmt (for p. multocida toxin), which interferes with normal bone modelling in both the nasal turbinates and long bones in swine, and is distinct from the b. bronchiseptica dermonecrotic toxin (dnt). in porcine models, pmt causes a more serious form of ar known as progressive ar, whereas b. bronchiseptica infection alone induces a milder, or non-progressive, form of the disease. bacterial co-infections of humans include orofacial infections 109 , adenotonsillitis 110 , persistent osteomyelitis 111 , peritonitis 112 , chronic sinusitis 113 , abscesses 114, 115 , necrotizing fasciitis and approximately one-third of urinary tract infections (utis) in the elderly 116 and in renal transplant patients 117 . two important bacterial co-infections are periodontitis and vaginosis. periodontitis. periodontal disease causes tooth loss and is associated with systemic vascular diseases such as atherosclerosis and carotid coronary stenotic artery disease 118 . periodontitis in an expectant mother can contribute to both low birth weight and pre-term labour 119 . periodontal disease is initiated by the formation of a a murine model has been developed to reproduce the pathogenesis of human meningococcaemia, which often results in serious symptoms or death 92 . in this model, adult balb/c mice are infected intranasally with a mouse-adapted iav, then seven or ten days later, are co-infected with neisseria meningitidis. fatal meningococcal pneumonia and bacteraemia occurred in mice challenged at seven, but not ten, days after iav infection. meningococcal pneumonia and bacteraemia did not develop in mice that were not co-infected with iav. susceptibility to lethal infections correlated with peak interferon-γ production in the lungs and decreased iav load and production of il-10, which indicates that transient iav-induced modulation of host immunity has a role in susceptibility to n. meningitidis co-infection. the only viral-bacterial co-infection model for om is the chinchilla. before 1980, most om studies in the chinchilla used inoculation of pathogens directly into the middle ear. although this induces disease in almost all of the animals inoculated and is therefore extremely useful for studies of therapeutics and surgical intervention strategies, it bypasses all of the early steps in the development of the pathogenesis of the disease, including colonization of the nasopharynx, ascension of the eustachian tube and initiation of infection in the middle ear. giebink and co-workers 93 , developed a clinically relevant model in which chinchillas were challenged intranasally with both s. pneumoniae and iav. this study showed that 4% of the chinchillas that were infected with iav alone, and 21% of those inoculated with s. pneumoniae alone, developed om, but of the animals that were coinfected with both microorganisms,~67% developed om (fig. 2) . this model has been useful in defining the molecular mechanisms of iav predisposition to pneumococcal om [94] [95] [96] and to investigate the role of pneumococcal virulence determinants in om 81 . to study the pathogenesis of om mediated by nthi, a chinchilla model that uses a co-challenge method was developed. in this model, adenovirus infection can predispose the chinchilla to nthi invasion of the middle ear 97 (fig. 2) ; however, iav infection has no effect. this adenovirus-nthi co-infection model has been used to study the mechanisms of adenovirus predisposition to nthi-induced om 98, 99 , to identify new nthi virulence determinants 100 and to assay the relative efficacies of different nthi-derived vaccine candidates for om [101] [102] [103] . a cotton rat model of rsv and nthi co-infection has also been developed to study co-infections of the respiratory tract 104 . in the cotton rat, colonization of the respiratory tract with nthi increased to a maximum level four days after infection with rsv and colonization was increased compared with rats that had not been infected with rsv. nthi colonization of the respiratory tract was increased by rsv co-infection and, although the mechanisms underlying this relationship are not understood, this model might be useful to determine the mechanisms of rsv predisposition to bacterial om. bacterial formed, in terms of microbial constituents, is the main predictor for periodontal disease. actinobacillus actinomycetemcomitans, p. gingivalis and bacteroides forsythus are the main periodontal pathogens. periodontal disease covers a range of clinical symptoms and there are multiple forms of periodontitis in children and adults. in individuals under 20 years of age, a. actinomycetemcomitans is the main bacterial pathogen, whereas in adults aged 35 years or older, periodontitis has been linked to p. gingivalis and b. forsythus. spirochaetes, especially treponema denticola, have been implicated in periodontitis despite the fact that most oral spirochaetes have not been successfully cultured. recent studies using molecular phylogenetic techniques have implicated new bacterial biofilm on the tooth surface followed by bacterial invasion of gingival tissues 119 . teeth have a non-shedding surface and are located in a warm, moist environment, so are a particularly suitable niche for biofilm formation by the oral microbial flora. in periodontitis, coaggregation -a process in which genetically distinct bacteria become interconnected by specific adhesins -is central to the formation of complex multispecies biofilms 120 (fig. 3) . in dental plaque, primary colonizers such as streptococcus gordonii, and other oral streptococci that express adhesins, provide a film on which other bacterial colonizers assemble the biofilm. it was thought that the abundance of plaque that formed was responsible for the induction of periodontitis but, at present, the favoured hypothesis is that the quality of the plaque periodontal disease. in most individuals, periodontal pathogens trigger an inflammatory response that effectively prevents microbial colonization and invasion of adjacent gingival tissues. however, individuals that have specific il-1 polymorphisms that result in increased levels of il-1 expression are predisposed to periodontal disease. using this criterion, a mouse model of polymicrobial-induced osteoclastogenesis, bacterial penetration, leukocyte recruitment and softtissue necrosis has been developed to clarify the role of cytokines in periodontal disease. in this model, the dental pulp of the first mandibular molars is exposed by surgically clipping the mesial cusps and then a mixture of putative oral pathogens is inoculated into the dental pulp (fig. 4a) . by monitoring the size of osseous lesions, tissue necrosis, osteoclastogenesis, osteoclastic activity, inflammatory cell recruitment and bacterial penetration into tissue periodontal disease, pathogenic mechanisms can be investigated 135 . il-1 or tnf receptor signalling does not seem to be required for bacteria-induced osteoclastogenesis and bone loss in this model, but does have a crucial role in protecting the host against anaerobic co-infections. a rat model of periodontitis was developed to test adherent (rough) and non-adherent (smooth) variants of a. actinomycetemcomitans for virulence, as well as to assess phenotypic reversion in vivo 136 . in this model, the normal flora of the oral cavity of sprague-dawley rats is reduced by antibiotic treatment, after which rats are inoculated with a. actinomycetemcomitans by either normal ingestion of food layered with bacterial cultures, oral swabbing or gastric lavage (fig. 4b) . when clinical isolates of a. actinomycetemcomitans were compared with laboratory-adapted variants, fine et al. 136 found that the clinical strains were more efficient at colonization and persisted longer in the rat oral cavity than laboratory strains. rough variants were more efficient colonizers of the rat oral cavity than smooth variants, regardless of the method of inoculation, although feeding was the preferred method owing to the similarity with human disease. importantly, rats that were orally infected with a. actinomycetemcomitans by feeding developed immunoglobulin g (igg) antibodies to the bacteria and had bone loss that was typical of periodontitis. this model has not been used to study the process of bacterial co-infection in periodontitis, but has been used to identify a gene locus that is important in virulence and which mediates tight adherence by a. actinomycetemcomitans 137 . a primate model (macaca fascicularis) of periodontal disease uses silk ligatures tied around the posterior teeth to induce plaque accumulation and the initiation of periodontitis 138 . so far, this model has only been used for single pathogen studies, but is considered to be a relevant animal model of periodontal disease owing to the similarity of clinical and histological features with those of periodontal disease of humans, and because, in this model, periodontal destruction is clearly triggered by bacterial infection 139 . species or phylotypes -including members of the uncultivated bacterial division tm7 -in periodontitis, dental caries and halitosis [121] [122] [123] [124] [125] [126] [127] . in many of these studies, not only were new species and phylotypes identified, but bacteria that are known to be oral pathogens were found to be numerically minor, which was expected because ~50% of oral flora have not been cultivated 123 . in localized juvenile periodontitis (ljp), the leukotoxin of a. actinomycetemcomitans, like that of m. haemolytica, is the best-studied virulence factor. this leukotoxin selectively kills pmns and macrophages in vitro, and pmns and macrophages are important components of the host defence in vivo. expression of this leukotoxin is variable among a. actinomycetemcomitans isolates and the leukotoxin-expression phenotype correlates with differences in the promoter region of the leukotoxin gene operon 128 . a subset of leukotoxin-overproducing strains are more virulent and are associated with ljp in humans. neutrophil abnormalities seem to be an important predisposing condition for periodontal disease. in addition, loss of tooth attachment and bone resorption, which are important events in periodontal disease, occur together with increased il-1 and tumour-necrosis factor (tnf) activities. the production of il-1 and tnf (both of which are pro-inflammatory cytokines) has been correlated with the spread of inflammatory cells to connective tissues, the loss of connective tissue attachment, osteoclast formation and the loss of alveolar bone. an overzealous host response to periodontal pathogens, resulting in excessive production of il-1 and tnf, is hypothesized to be responsible for much of the damage that occurs in periodontal disease 129 . ial co-infection. the mucosal environment of the vagina is influenced by developmental and hormonal changes 131 . the most common bacterial constituents of the vaginal microflora are lactobacilli, including lactobacillus crispatus and lactobacillus jensenii 131 . when these hydrogen peroxide (h 2 o 2 )-producing lactobacilli are outcompeted by anaerobic and facultatively anaerobic members of the vaginal flora, bv develops with a concomitant rise in vaginal ph, which further promotes the growth of the resident lactobacilli. bv is common, occurring in 5-51% of the global female population 130 and the role of lactobacilli in the maintenance of vaginal homeostasis has been well studied. women with stable bacterial colonization have a reduced risk of developing bv 132 . normal vaginal flora has a role in defence against the acquisition of other pathogenic microorganisms, including those that are responsible for sexually transmissible diseases (stds), and bv is a strong predictor of std acquisition 133 . compared with subjects with normal vaginal flora, subjects that have bv are more likely to test positive for neisseria gonorrhoea and chlamydia trachomatis. recently, bv has also been found to be associated with an increased risk of hsv-2 infection 134 . candida species. co-infection with multiple candida strains and substrains are also found. regardless of the co-pathogens, mycotic co-infections of the oral and vaginal cavities, on indwelling prosthetic devices, or systemic infection of the blood can present significant therapeutic challenges. difficulty in treating some of these infections is partly attributed to the formation of biofilms by candida spp. 152 biofilm formation on devices such as prosthetic heart valves and catheters has been studied in vitro 95 . when cultured on a variety of catheter materials, candida spp. form biofilms comprising a matrix of microcolonies of both the yeast and the filamentous hyphal forms. in studies of mixed microbial populations, candida spp. form biofilms with several bacterial species, including staphylococcus epidermidis and oral streptococcal species. the receptor for candida albicans co-aggregation with s. gordonii is a complex cell surface polysaccharide that is expressed on the surface of the bacterium. the interaction between yeast cells and oral streptococci or other bacteria has important implications for the mechanisms of yeast infections of the oral cavity, in addition to promoting biofilm formation on a variety of surfaces. in the oral cavity, candida-bacterial interactions are responsible for denture stomatitis, angular cheilitis and gingivitis, and also have a role in periodontitis 153 . although the role of pathogenic enterococci and their role in peritonitis is not understood, many putative virulence factors have been identified using animal models. available animal models include systemic infection in mice and compartmentalized infection in rats, and the bacterial virulence factors that have been identified using each model differ 140 . this indicates that both host and pathogen factors contribute to peritonitis and, perhaps, that the animal models are quite different. nevertheless, these models have identified a role for cytokines in septic shock, a protective role for il-10 against lethal shock 141 , a role for stat4 in the mortality seen in bacterial co-infection sepsis 142 and helped to define the role of the classical pathway of complement activation in defence against polymicrobial peritonitis 143 . animal models of bacterial co-infection peritonitis and/or sepsis can involve any of the following methods for induction of infection: peritoneal implantation of microbe-filled gelatin capsules 140, 141 ; intraperitoneal injection of faecal suspensions 17 or caecal ligation and puncture 112, [142] [143] [144] [145] [146] [147] [148] [149] [150] [151] . candida and mixed infections. as defined by soll and his colleagues 6 bacterial suspensions that are being tested for the ability to cause periodontal disease are injected into the dental pulp and the mouse is monitored for signs of periodontal disease by methods that include examination of osseous lesions, tissue necrosis, inflammatory cell recruitment, bacterial tissue penetration and osteoclastogenesis. b | in the rat model bacteria are grown using standard laboratory procedures and washed 3 times with phosphate-buffered saline (pbs) supplemented with 3% sucrose. the rats are pretreated with antibiotics and the mouth is swabbed with chlorhexidine to deplete the oral flora. the bacterial suspension is mixed into the rat's food so that this animal model replicates, as far as possible, the natural route of infection for periodontal disease. after daily inoculation the rats are assessed for bacterial colonization and bone loss. using this model, different strains of bacteria and the contribution of different virulence loci can be tested. cfu, colony-forming units. of these genetic manipulations 159 . a murine host has been used as a model of systemic candidiasis 160 and there is also a murine model for c. glabrata-induced vaginitis 161 . in the c. glabrata model, the increased susceptibility of non-obese diabetic mice to c. glabratainduced vaginitis compared with their non-diabetic counterparts indicates a link between susceptibility to diabetes and infection with c. glabrata. in addition to studies of candida genetics and pathogenicity 162 , this model is useful for the evaluation of the relative efficacy of antimycotic agents and probiotics for the prevention of vaginitis. an animal model of haematogenously disseminated candidiasis has recently been developed 163 that can investigate the role of phenotype switching in candidiasis. in this model (fig. 5) mice were injected with engineered c. albicans strains in which the transition between yeast and filamentous forms is under the control of a doxycycline-regulated promoter. mice that were infected with strains that switched to the filamentous form died, whereas those infected with strains that could not switch from the yeast to the filamentous form survived, despite the fact that the fungal burdens in both groups were nearly identical. these data indicate that the filamentous form is important for mortality but that the yeast form of c. albicans is important for dissemination to deeper tissues. parasite-parasite co-infections. several human diseases have mixed parasitic aetiologies, including co-infections with plasmodium spp. and nematodes. in some studies, co-infection with a helminth seemed to confer protection against severe complications of malaria 164 , but this is not always the case. when infected controls with a low helminth burden were compared with those with circulating helminth schizonts, co-infection with ascaris lumbricoides was found to be associated with protection from cerebral malaria. in addition, a later study showed a significant association between ascaris infection and the risk of co-infection with plasmodium falciparum and plasmodium vivax, indicating that pre-existing ascaris infection might increase host tolerance to coexisting plasmodium spp. 165 subsequently, helminthinfected patients were found to be more likely to develop falciparum malaria compared with those that were not co-infected 166 . moreover, the risk of developing falciparum malaria increased with the number of coinfecting helminth species. collectively, these findings indicate that a helminth-mediated helper t cell 2 (t h 2) shift (an immune response that is biased towards that which is characteristic of a t h 2-mediated response) might have a complex impact on malaria co-infection -decreasing antisporozoite immunity but inducing a protective outcome against severe complications of malaria. although the underlying mechanism is less clear, mwatha et al. 169 showed that exposure of schistosoma mansoni-infected children to p. falciparum had a significant influence on the severity of hepatosplenomegaly (enlargement of the liver and spleen) that was observed in co-infected children. a new category of polymicrobial diseases has been proposed for candida spp. in which the infection is due to phenotypic heterogeneity 154 . in addition to the hypha-bud transition, c. albicans has a reversible, high-frequency phenotype switch that can be identified by differences in colony morphology. c. albicans cells of two phenotypic phases have different virulence characteristics. the ability of this human pathogen to rapidly switch between phenotypes could be a higher-order pathogenic trait. support for this hypothesis comes from studies in which strains that cause deep tissue mycoses were shown to switch at higher frequencies than those that cause superficial infections. furthermore, pathogenic c. albicans strains that were isolated from the oral cavity switch at higher rates than commensal strains that were isolated from the same site. a clinically relevant example of the role of both phenotype and mating-type switching in disease was characterized by brockert et al. 155 , who investigated oral cavity and vaginal isolates of c. glabrata in three patients with vaginitis. the results of this study showed that switching occurs at sites of infection, that different switch phenotypes of the same strain can dominate in different anatomical locations in the same host and that mating-type switching occurs in vivo. these co-infections can cause disease in the lower respiratory tract. in cf patients, clinical specimens that also harbour c. albicans contain nine times the amount of p. aeruginosa compared with patients that do not harbour c. albicans 156 . moreover, sputum samples of 6-70% of cf patients contain c. albicans in addition to p. aeruginosa. hogan and kolter 157 showed that p. aeruginosa forms a dense biofilm on c. albicans filaments in vitro and, in doing so, kills the fungus. p. aeruginosa fails to bind to, or kill, the yeast form of c. albicans. it is unclear if a similar relationship between these two pathogens functions in vivo but, as several p. aeruginosa virulence factors that are important in human disease are also involved in killing the fungal filaments, this co-culture system could prove useful for the study of the pathogenesis of p. aeruginosa-induced disease. owing to the ability of candida spp. to switch between bud and hypha (or hypha-like) forms as well as to switch phenotype, all animal models of candida infection are likely to represent one or another of the multiple polymicrobial states that have been proposed for this microorganism. a rat model of oral colonization has been used to compare the relative pathogenicity of different candida strains as well as to determine the effect of chemotherapeutic immunosuppression on the ability of candida spp. to switch from a commensal to an invasive phenotype 158 . a rat model of oral candidiasis has also been developed and used to assay isogenic derivatives of a virulent c. albicans strain for the biological consequences co-infection with schistosoma species and plasmodium species has been modelled in field voles and mice since 1956 (ref. 172) , with conflicting observations concerning the ability of one parasite to suppress the capacity of the other to infect the host 173, 174 . results obtained seem to depend on the plasmodium species used as well as the immune status of the host. s. mansoni is, however, a potent inducer of a t h 2 dominant response, not only to itself but also to other bystander antigens that are present in a host, so it does have an influence on the clinical outcome in these co-infections 175, 176 . synergistic interactions between specific protozoans and helminths are often ascribed to the immunosuppression that is characteristic of protozoan infections 177 and which is observed in the mouse, which is the main model for these infections a mouse model has been used to model the arthritis and carditis that can occur in co-infections with b. microti and b. burgdorferi 19 . co-infection resulted in a significant increase in symptoms of arthritis. this increase was correlated with a reduction in concentrations of the cytokines il-10 and il-13. a mouse model for tick-borne lyme arthritis mediated by co-infection with b. burgdorferi and a causative agent of human granulocytic ehrlichiosis (hge) has been developed 178, 179 (fig. 6) . co-infection results in increased titres of both pathogens and more severe arthritis than does infection with b. burgdorferi alone. co-infection resulted in reduced concentrations of il-12, ifn-γ and tnf-α and increased concentrations of il-6. ifn-γ expression in macrophages was suppressed, which might indicate a reduction in phagocytic activity in co-infection. these models will allow us to define the modulation of host immune responsiveness that occurs in those individuals that are simultaneously or sequentially infected with multiple tick-borne pathogens 179 . co-infections can arise as a result of the virus-induced immunosuppression that is characteristic of a subset of human viral pathogens, the best characterized of which is hiv. schistosomiasis is a chronic helminth infection that is caused by s.mansoni. in hcv and s. mansoni co-infection, there is a higher incidence of viral persistence and accelerated damage to the liver than when the patient is infected with either infectious agent alone. in a recent study, stimulation of cd4 + t cells with hcv antigens produced a type 1 cytokine profile in patients infected with hcv, whereas in patients that were co-infected with hcv and s. mansoni, a type 2 cytokine predominance was evident despite the fact that t cells that were recovered from both patient populations responded in the same manner to stimulation with schistosomal antigens 168 . the helminth-induced inability to generate an hcv-specific cd4 + /t h 1 t-cell response has been shown to have a role in the persistence and severity of hcv infection, which indicates that the induction of a strong cellular immune response through new therapeutic approaches might limit subsequent liver damage in those individuals with chronic hcv infection 169 . parasite-bacteria co-infections. one example of coinfection with a parasite and bacterium in humans is that of borrelia burgdorferi (the causative agent of lyme disease) and the intra-erythrocytic parasite babesia microti. both of these pathogens are transmitted by the tick vector ixodes scapularis. co-infection can occur by a bite from a single tick carrying multiple pathogens, or from multiple tick bites. the first cases of lyme borreliosis and babesiosis co-infection were reported in the mid 1980s, with parasite-bacteria co-infection rates of up to 33% among those with confirmed tick-borne infection in certain populations. although ticks can also harbour the human pathogen anaplasma phagocytophilum, lyme borreliosis and babesiosis coinfection accounts for ~80% of polymicrobial disease in the eastern united states. consistent with the theme for other co-infections involving a parasite, patients that harbour both of these pathogens had more severe and longer-lasting symptoms than those with lyme borreliosis alone 170 (nug) and is characterized by a surface biofilm of mixed microbial flora overlying a subsurface flora comprising dense aggregates of spirochaetes. in contrast to nug, high levels of yeast and herpes-like viruses were observed using transmission electron microscopy examination of tissues recovered from the former patient group. herpes-like particles were observed in 56.5% of biopsies obtained from hiv-infected patients with nup. these findings correlate well with those of contreras and co-workers 183, 184 in which co-infection with herpesvirus was associated with high levels of periodontopathic bacteria. the role of viruses in the pathogenesis of nup or periodontitis is not known but, in addition to inducing immunosuppression, it has been suggested that viruses might promote the overgrowth of bacterial pathogens and/or induce the release of tissuedestroying cytokines by host cells 185 . 32, 180 . in addition to systemic diseases, localized infections with candida spp., such as thrush in the oral cavity, are common co-infections in hiv-infected individuals 181 . the commensal oral flora acquires an invasive phenotype in the hiv-infected host, and c. albicans is indicative of a defect in host t-cell immunity in hiv infection 182 . oropharyngeal candidiasis develops iñ 20-50% of hiv-infected patients and often precedes the development of a more invasive candida infection, oesophageal candidiasis. the progressive immunosuppression that is characteristic of hiv infection provides a mechanism for the development of oesophageal candidiasis, which is a reportable aidsdefining opportunistic illness. another disease of the oral cavity in hiv-seropositive patients is necrotizing ulcerative periodontitis (nup), which is a disease that is characterized by ulcerated gingival papillae 122 figure 6 | animal model for lyme disease and human granulocytic ehrlichiosis (hge) co-infection. these diseases share a tick vector, ixodes scapularis, and to analyse whether ehrlichia sp. and borrelia burgorferi (the causative agents of hge and lyme disease, respectively) co-infection leads to increased severity of spirochaete-induced lyme arthritis a mouse model has been developed. mice are infected intradermally with either spirochaetes (b. burgdorferi cultured in vitro) or hge (blood culture from a scid mouse, see inset panel) 178 . arthritis and presence of the two pathogens can then be determined through histopathology, pcr to detect bacterial dna and by assessing immune responses. ticks were allowed to feed on all groups of mice to assess transmission of the pathogens. after feeding, pcr (hge) and immunofluorescence (b. burgdorferi) were used for pathogen detection. and not due to either increased apoptosis or altered distribution of these cells between the spleen, blood or lymphatics. the ability of measles virus to suppress both innate and adaptive immune responses is thought to be responsible for the increased susceptibility to bacterial co-infection. the future of polymicrobial disease research molecular methods are now being used together with conventional culture techniques to determine the identity of the full complement of microorganisms that are involved in co-infections and to determine the interactions between these microorganisms. as a result, additional diseases of polymicrobial origin will be identified. this will necessitate the development of new animal models and new in vitro methods for the study of polymicrobial diseases. uncovering the molecular mechanisms that are involved in the pathogenesis of complex diseases might show that changes in lifestyle, such as smoking cessation or dietary changes, could prevent co-infections. developing methods to disrupt biofilms are one target for researchers. new antimicrobials and vaccine candidates for both the predisposing and the co-infecting microorganisms will be sought. therapeutic approaches for polymicrobial diseases might include the use of probiotics for the treatment or prevention of vaginal infections, gastroenteritis, inflammatory bowel disease, utis and periodontitis. moreover, advances in nanotechnology and biomedical engineering will allow the development of new ways to deliver these therapeutic or preventative agents in a disease-or site-specific manner such as the design and use of 'intelligent implants' 193 . these indwelling devices might be embedded with sensors to detect the biofilm-forming microorganisms and signal the release of antimicrobial agents stored in an internal reservoir. as the organizers of the first satellite conference on diseases of mixed microbial aetiology (see the online links box) stated -polymicrobial diseases are ''a concept whose time has come'' 1 . virus cannot present antigen to t cells and have a diminished capacity to secrete ig or proliferate. susceptibility to bacterial co-infection is likely owing to these underlying immune defects that result in the hallmark of measles virus infection -inhibition of the proliferation of cd4 + and cd8 + t cells [186] [187] [188] [189] . a primate model of hiv-induced immunosuppression that developes cutaneous leishmaniasis has been developed in rhesus macaques. in this model, macaques are chronically infected with siv, and then co-infected with leishmania major metacyclic promastigotes by intradermal injection. lesion size, parasite load and siv viraemia are measured weekly. this model has been used to assay both the synergistic relationship between these two pathogens and the responsiveness to, and relative protective efficacy of, cpg oligodeoxynucleotides delivered to co-and mono-infected macaques 190 . recently, a rhesus monkey model for siv predisposition to mycobacterium leprae co-infection has been developed, which showed that co-infection increases the susceptibility to leprosy regardless of the timing between the two infections 191 . as mentioned earlier, measles virus-induced immunosuppression often leads to bacterial co-infection. to understand the mechanisms of co-infection, a murine model of combined measles virus and listeria monocytogenes infection was developed 192 . in this model system, transgenic mice expressing the human measles virus receptor cd46 are co-infected with measles virus and l. monocytogenes, or are challenged with the bacterial pathogen alone. mice co-infected with measles virus were more susceptible to infection with l. monocytogenes and this susceptibility corresponded with a reduction in the macrophage and pmn populations in the spleen, as well as a reduction in ifn-γ production by cd4 + t cells. a reduction in cd11b + macrophages and ifn-γ producing t cells was found to be due to reduced proliferative expansion introduces and provides a concise overview of polymicrobial diseases the role of microbial interactions in infectious disease mechanisms of bacterial superinfections in viral pneumonias respiratory viral infection predisposing for bacterial disease: a concise review molecular pathogenesis of pneumococcal pneumonia polymicrobial diseases polymicrobial diseases regulation of gene expression by cell-to-cell communication: acylhomoserine lactone quorum sensing bacterial biofilms: an emerging link to disease pathogenesis endotoxemia and polymicrobial septic peritonitis resistance to acute septic peritonitis in poly(adp-ribose) polymerase-1-deficient mice interaction between the innate and adaptive immune systems is required to survive sepsis and control inflammation after injury cd40 contributes to lethality in acute sepsis: in vivo role for cd40 in innate immunity role of the classical pathway of complement activation in experimentally induced polymicrobial peritonitis mice lacking monocyte chemoattractant protein 1 have enhanced susceptibility to an interstitial polymicrobial infection due to impaired monocyte recruitment provided the background for the development of an animal model of parasitic co-infection, as well as demonstrating the potential importance of mouse strain -genetic background -in 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characteristics of women colonized by these species hydrogen peroxide-producing lactobacilli and acquisition of vaginal infections bacterial vaginosis is a strong predictor of neisseria gonorrhoeae and chlamydia trachomatis infection association between acquisition of herpes simplex virus type 2 in women and bacterial vaginosis interleukin-1 and tumor necrosis factor receptor signaling is not required for bacteria-induced osteoclastogenesis and bone loss but is essential for protecting the host from a mixed anaerobic infection provided the background for the development of an animal model of the relative bacterial colonization and persistence by single phenotypic variants of a bacterium tight-adherence genes of actinobacillus actinomycetemcomitans are required for virulence in a rat model inflammation and tissue loss caused by periodontal pathogens is reduced by interleukin-1 antagonists non-human primates used in studies of periodontal disease pathogenesis: a review of the literature 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pseudomonas-candida interactions: an ecological role for virulence factors the relative pathogenicity of candida krusei and c. albicans in the rat oral mucosa avirulence of candida albicans auxotrophic mutants in a rat model of oropharyngeal candidiasis in vivo pathogenicity of eight medically relevant candida species in an animal model a murine model of candida glabrata vaginitis the protective immune response against vaginal candidiasis: lessons learned from clinical studies and animal models engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of candida albicans during infection provided the background for the development of a murine model of fungal co-infection that allowed the demonstration of the importance of the ability of candida albicans to switch morphology from a yeast to a filamentous form in pathogenesis ascaris lumbricoides infection is associated with protection from cerebral malaria contemporaneous and successive mixed plasmodium falciparum and plasmodium vivax infections are associated with ascaris lumbricoides: an immunomodulating effect? intestinal helminth infections are associated with increased incidence of plasmodium falciparum malaria in thailand specific cellular immune response and cytokine patterns in patients coinfected with hepatitis c virus and schistosoma mansoni kinetics of intrahepatic hepatitis c virus (hcv)-specific cd4 + t cell responses in hcv and schistosoma mansoni coinfection: relation to progression of liver fibrosis associations between anti-schistosoma mansoni and anti-plasmodium falciparum antibody responses and hepatosplenomegaly, in kenyan schoolchildren epidemiology and impact of coinfections acquired from ixodes ticks. vector borne zoonotic dis coinfection in patients with lyme disease: how big a risk? some aspects of concomitant infections of plasmodia and schistosomes. i. the effect of schistosoma mansoni on the course of infection of plasmodium berghei in the field vole (microtus guentheri) infection of mice concurrently with schistosoma mansoni and rodent malarias: contrasting effects of patent s. mansoni infections on plasmodium chabaudi, p. yoelii and p. berghei suppression of schistosome granuloma formation by malaria in mice altered immune responses in mice with concomitant schistosoma mansoni and plasmodium chabaudi infections schistosoma mansoni infection cancels the susceptibility to plasmodium chabaudi through induction of type 1 immune responses in a/j mice heterologous synergistic interactions in concurrent experimental infection in the mouse with schistosoma mansoni, echinostoma revolutum, plasmodium yoelii, babesia microti, and trypanosoma brucei coinfection with borrelia burgdorferi and the agent of human granulocytic ehrlichiosis alters murine immune responses, pathogen burden, and severity of lyme arthritis coinfection with borrelia burgdorferi and the agent of human granulocytic ehrlichiosis suppresses il-2 and ifn-γ production and promotes an il-4 response in c3h/hej mice tuberculosis in hiv-infected patients: a comprehensive review a tem/sem study of the microbial plaque overlying the necrotic gingival papillae of hiv-seropositive, necrotizing ulcerative periodontitis molecular epidemiology of candida albicans strains isolated from the oropharynx of hiv-positive patients at successive clinic visits relationship between herpesviruses and adult periodontitis and periodontopathic bacteria herpesviruses in human periodontal disease herpesviruses: a unifying causative factor in periodontitis? modulation of immune system function by measles virus infection: role of soluble factor and direct infection modulation of immune system function by measles virus infection. ii. infection of b cells leads to the production of a soluble factor that arrests uninfected b cells in g 0 /g 1 manganese superoxide dismutase induction during measles virus infection suppression of antigen-specific t cell proliferation by measles virus infection: role of a soluble factor in suppression cpg oligodeoxynucleotides protect normal and siv-infected macaques from leishmania infection interactions between mycobacterium leprae and simian immunodeficiency virus (siv) in rhesus monkeys this paper reviews how virus-induced immunosuppression of both innate and adaptive immune responses can provide an underlying mechanism for bacterial co-infection this paper introduces the concept of one approach to treat or prevent polymicrobial diseases by using bioengineering and nanotechnology to specific anatomic sites and crucial time points in the disease course for intervention and/or prevention communication among oral bacteria. microbiol the author would like to thank j. neelans for help with manuscript preparation. discusses our increasing understanding of the link between biofilms and the pathogenesis of human disease, as well as identifying the need for relevant animal models with which to study these infectious states. 10. brogden key: cord-310840-h49dx92d authors: eslamy, hedieh k.; newman, beverley title: pneumonia in normal and immunocompromised children: an overview and update date: 2011-09-30 journal: radiologic clinics of north america doi: 10.1016/j.rcl.2011.06.007 sha: doc_id: 310840 cord_uid: h49dx92d pneumonia is an infection of the lung parenchyma caused by a wide variety of organisms in pediatric patients. the role of imaging is to detect the presence of pneumonia, and determine its location and extent, exclude other thoracic causes of respiratory symptoms, and show complications such as effusion/empyema and suppurative lung changes. the overarching goal of this article is to review cause, role of imaging, imaging techniques, and the spectrum of acute and chronic pneumonias in children. pneumonia in the neonate and immunocompromised host is also discussed. normal and immunocompromised children: an overview and update hedieh k. eslamy, md, beverley newman, md* pneumonia is an infection of the lower respiratory tract, involving the lung parenchyma. the world health organization estimates that there are 150.7 million cases of pulmonary infection each year in children younger than 5 years, with as many as 20 million cases severe enough to require hospital admission. 1 in north america and europe, the annual incidence of pneumonia in children younger than 5 years is estimated to be 34 to 40 cases per 1000, and decreases to 7 cases per 1000 in adolescents 12 to 15 years of age. 2, 3 the mortality in pediatric patients caused by pneumonia in developed countries is currently low (<1 per 1000 per year). 3 however, pneumonia is still the number one cause of childhood mortality in developing countries. 1, 4 the overarching goal of this article is to review cause, current role of imaging, imaging techniques, and the spectrum of acute and chronic pneumonias in children. pneumonia in the neonate and immunocompromised host is also discussed. infectious agents causing pneumonia in children include viruses, bacteria, mycobacteria, mycoplasmas, fungi, protozoa, and helminths. etiologic diagnoses of pneumonia are not so easy to determine or so accurate as is sometimes implied. in addition, proof of the cause of pneumonia is not obtained in most cases. there is a great deal of overlap in the radiographic appearance of pneumonias caused by different organisms. imaging is usually poor at predicting the broad category (eg, bacterial vs viral) of infectious agent, let alone the specific agent. preexisting lung disease may not only predispose to pulmonary infection but also modify the appearance of pulmonary consolidation. furthermore, because the lungs can respond to a diverse disease processes in only a limited number of ways, it is common for the radiographic features of both acute and chronic infectious pneumonia to overlap considerably with many noninfectious lung diseases. such noninfectious lung diseases are identified as pneumonia mimics in this article. viral pneumonia is rare in the neonatal period, because of conferred maternal antibody protection, whereas bacterial pneumonia is most frequently caused by pathogens acquired during labor and delivery, and is more prevalent in premature babies. with decreasing maternal antibody levels, viral pneumonia occurs at a peak between 2 months to 2 years of age. bacterial infections become relatively more common in older children from 2 years to 18 years of age. 5 the lung response to an infective antigen seems to be more agespecific than antigen-dependent (ie, bacteria vs viral). therefore, lobar and alveolar lung opacities are more common in older children and are more frequently caused by bacterial infections, whereas interstitial opacities are seen in all age groups, and are relatively nonspecific as to the type of causative organism. 6, 7 the role of imaging, including chest radiographs, ultrasound (us) and computed tomography (ct), is to detect the presence of pneumonia, determine its location and extent, exclude other thoracic causes of respiratory symptoms, and show complications such as parapneumonic effusion/ empyema and suppurative lung complications. 5 although magnetic resonance (mr) imaging is not routinely used for evaluating pneumonia in children, it is a promising imaging modality particularly for children with chronic lung conditions who require repeat imaging studies. frontal and lateral chest radiographs are the mainstay, and often the only, imaging needed in pediatric pulmonary infection. this imaging can be supplemented with other views such as lateral decubitus or other imaging modalities as the circumstances warrant. decubitus views are not useful when an entire hemithorax is opacified because layering fluid cannot be identified without any adjacent air. the main use of us is to identify, quantify, and characterize a parapneumonic effusion/empyema, as well as provide image guidance for drainage and identify residual collections after treatment. 8, 9 operator availability and expertise are important factors in making us a useful tool for evaluating pulmonary infection. although intrapulmonary fluid-filled cavities and even lung abscesses within consolidated lung can be identified on us, ct provides a more global view of the disease process. ct is often used to further evaluate: (1) suppurative lung complications and to differentiate these from parapneumonic effusion/empyema; (2) patients with recurrent or chronic pneumonia and concern for an underlying lesion; and (3) immunocompromised children with noncontributory or confusing chest radiographs and clinical findings that could be secondary to lung infection. 5 close attention to ct technique is crucial for imaging evaluation of pneumonia in pediatric patients. ct with low radiation dose technique should be carefully performed in all cases. eighty to 120 kvp with weight-based low milliampereseconds coupled with radiation dose modulation techniques is appropriate in most children for evaluation of pneumonia. multiple ct image acquisitions are usually not needed and the scan field of view should be tailored to the area of interest (especially if following a specific lesion serially over time) to further decrease the overall radiation dose. 10 occasionally, it may be useful to acquire additional expiratory scans to assess air trapping, which is an early imaging finding associated with small airway disease. in this situation, often at least 1 or both ct acquisitions can be obtained using a high-resolution ct (hrct) gap technique. to obtain optimal ct imaging at peak inspiration and close to expiratory residual volume, controlled ventilation (cvict) in infants and young children ( 5 years old) or spirometer-controlled ct in older children may be needed. 11 young children have little intrinsic tissue contrast. therefore, intravenous contrast is almost always needed for ct imaging of infection especially if mediastinal delineation is required. the exception is when hrct is used only for evaluating lung parenchymal and airway disease. breath-holding is usually desirable but can be adapted on a case-by-case basis depending on the needs of the study and the ability of the child to cooperate. however, for the study to be interpretable, gross patient motion should be absent. sedation or anesthesia may be required in infants, young children, or children with intellectual disability. delays between induction of anesthesia and scanning need to be minimized to prevent the potential for lung atelectasis with anesthesia. the anesthesiologist needs to pay close attention to techniques for preventing atelectasis or recruiting lung before the ct imaging. 12 peltola and colleagues 13 recently published their experience with mr imaging of lung infections in children using free-breathing t2-weighted, short tau inversion recovery, and t1-weighted with fat saturation precontrast and postcontrast sequences. their study showed that lung parenchymal, pleural, and lymph node inflammatory abnormalities can be characterized by mr imaging in children with lung infection. therefore, mr imaging might potentially be used to further evaluate suspected, acute complications of pneumonia. 13 children with chronic lung conditions and recurrent infection, such as cystic fibrosis, who are often subjected to substantial radiation exposure from repeated ct studies, would benefit the most from mr imaging evaluation of the lungs instead of ct. although mr imaging may not provide as much detail compared with ct especially with early, small or subtle changes ( fig. 1) , there are promising indications of a role for mr imaging in pulmonary infection. [13] [14] [15] there are several different descriptions of basic patterns of lung diseases on chest radiographs. in this review article, we adopt the one described by hansell and colleagues. 16 almost all of these are seen as part of the spectrum of infectious lung disease ( table 1) . pneumonia and bronchiolitis are both common in infants and have overlapping clinical and imaging features. many studies, particularly those in the developing world, use the term acute lower respiratory tract illness and make no attempt to differentiate pneumonia from bronchiolitis. 17 bronchiolitis occurs in children less than 2 years of age, who typically present with cough, coryza, and wheezing. bronchiolitis is a major cause of morbidity and mortality in infants. 18 respiratory syncytial virus (rsv) is the most common cause 2) . such imaging findings are related to diffuse airway inflammation and partial (air trapping) or complete (atelectasis) airway obstruction. 19 similar changes are seen in older children (>2 years of age) with bronchitis although the features of diffuse small airway obstruction are less common in these older children with larger airways. pneumonia can be divided into several syndromes based on clinical presentation, imaging appearance, underlying predisposition, and cause. pneumonia syndromes that are discussed in this article include acute focal pneumonia, atypical pneumonia, miliary or nodular pneumonia, progressive or fulminant pneumonia, aspiration pneumonias, pulmonary infiltrates with eosinophilia (pie), and chronic or recurrent pneumonia. 20 neonatal pneumonia is briefly highlighted separately. pneumonia in immunosuppressed individuals is included in the general discussion of pneumonia syndromes and then specifically reviewed with regard to the different infections associated with various types of immunodeficiency. acute and chronic complications of pneumonia are also reviewed. characteristics that are typical for acute focal pneumonia include fever more than 38.8 c (102 f), a toxic appearance, and a focal opacity on chest radiographs. pleuritic chest pain in lower-lobe pneumonia is sometimes referred to the abdomen and may be mistaken clinically for an acute abdominal condition. acute focal pneumonia is most often caused by bacterial infection with streptococcus pneumonia. other causes of acute focal pneumonia are summarized in box 1. the chest radiograph of acute focal pneumonia usually shows a dense, typically more peripheral airspace opacity, which may appear segmental, lobar, or spherical ( figs. 3 and 4) . [21] [22] [23] in a febrile child with a spherical density on a chest radiograph, the most likely diagnosis is a round pneumonia but the possibility of an underlying neoplasm may be considered. round pneumonias tend to be solitary, have well-defined borders, and are often located in the perihilar region or posteriorly in the lungs. the radiograph should be carefully scrutinized for features of consolidation such as air bronchograms as opposed to those of a mass such as vascular/airway displacement or bony erosion. a second view such as a lateral radiograph may be helpful because a round pneumonia is often less masslike in appearance on an orthogonal view. this is one of the few scenarios in which radiologic follow-up after about 2 weeks may be useful to document interval resolution of acute pneumonia. 22 acute respiratory distress may be secondary to an intrathoracic mass causing airway or lung compression, especially when there is complete opacification of a hemithorax on radiographs (fig. 5) . intrapulmonary masses including both benign and malignant entities may present clinically with acute superinfection. in addition, other conditions or anatomic variants may be mistaken for pneumonia when a chest radiograph is obtained in a child with a fever and respiratory symptoms. atypical features in pneumonia include prominent extrapulmonary features (eg, headache, sore throat, and pharyngeal exudates), minimal or disparate chest signs on physical examination, subacute onset, nonfocal lung opacity on chest radiographs, lack of clinical response to antibiotics, lack of substantial leukocytosis, and a slow disease course. common infectious causes of atypical pneumonia are summarized in box 2. on chest radiographs, the pulmonary opacity is seen as either airspace, reticular (linear), or bandlike opacities in a nonfocal, patchy, or mottled distribution, with various degrees of density, usually without a single dense area of consolidation (fig. 6 ). most patients with atypical pneumonia can be classified into one of the following subgroups or a combination of two of them based on findings on chest radiographs: 20 acute interstitial pneumonia chest radiographs show a patchy, nonfocal reticular pattern. causes of acute interstitial pneumonia include self-limited viral infections and other pathogens. subacute minimal patchy pneumonia chest radiographs show 1 or more patches of minimal foci of airspace opacity. the most common causes of subacute minimal patchy pneumonia are mycoplasma pneumoniae, chlamydia pneumoniae, and adenoviruses. chest radiographs show a dense focal airspace opacity that is segmental or subsegmental. most of the other features of acute focal pneumonia are absent. tuberculosis needs to be excluded in these patients. most children exposed to mycobacterium tuberculosis do not develop active disease but can have latent foci that may reactivate at a later date particularly if they become immunosuppressed or debilitated. primary infection of mycobacterium tuberculosis is more likely in infants with local spread from the initial parenchymal/lymph node complex to form larger single or multifocal parenchymal lesions, typically with prominent hilar and mediastinal lymph node involvement ( fig. 7) and occasional pleural or pericardial disease. the primary focus as well as involved nodes may cavitate with liquefaction of the caseous material and ultimately calcification (see fig. 7 ). enlarged lymph nodes may encroach on adjacent bronchi and cause bronchial narrowing with resultant air trapping or collapse in the distal lung (fig. 8) . distant spread to other organs may occur either via lymphatics or hematogenously (including military lung involvement). 24, 25 infections with more than 1 organism may cause the atypical pneumonia pattern, resulting in confusing persistence of the illness or prominent findings in another organ system. an example of this situation is influenza infection with superimposed typical or atypical pneumonia (see fig. 6b ). the more common mimics that simulate the appearance of atypical pneumonia syndromes are summarized in box 3. miliary or nodular pneumonia is characterized by chest radiographic findings of multiple miliary or larger nodular opacities. miliary pneumonia in pediatric patients is seen most commonly in tuberculous and fungal infections (fig. 9 ). nodular pneumonia (including reticular and reticulonodular patterns) in pediatric patients is seen in septic emboli, viral pneumonia, lymphocytic interstitial pneumonia associated with epstein-barr virus (ebv) infection with underlying human immunodeficiency virus (hiv) infection, and some fungal and bacterial infections (box 4; figs. 10 and 11). 25, 26 septic pulmonary emboli usually occur secondary to a focal staphylococcus aureus infection (eg, right-sided bacterial endocarditis, septic thrombophlebitis, osteomyelitis, soft tissue infection, or urinary tract infection). the pulmonary nodules in septic emboli may cavitate (see fig. 11 ). 27 mimics of the pattern of miliary or nodular pneumonia are summarized in box 5. pneumonia is deemed progressive when it becomes radiologically and clinically worse despite antibiotic therapy that should be effective against the presumed cause. in this situation, the cause is often nonbacterial pathogens and mimics should also be carefully considered. fulminant pneumonia is defined as a severe bilateral pneumonia with an unusually rapid progression clinically or radiologically, over 24 to 48 hours after initial presentation. a common cause of progressive or fulminant pneumonia is the influenza virus during an epidemic. uncommon infectious causes of this pattern and mimics are summarized in boxes 6 and 7, respectively ( fig. 12 ). 20 aspiration pneumonia refers to the pulmonary consequences of abnormal entry of fluid, particulate matter, or endogenous secretions into the lower airways. aspirated material can be relatively inert, toxic, or oropharyngeal secretions. the most commonly aspirated materials in children include oropharyngeal secretions, gastric contents, water, hydrocarbon, lipid, and foreign bodies. radiographic pulmonary opacities related to aspiration may have an upper rather than lower lobe distribution when the child aspirates in the supine position. bacterial aspiration pneumonia is an infectious process caused by the inhalation of oropharyngeal secretions that are colonized by pathogenic bacteria. the basic defect leading to bacterial aspiration pneumonia is failure of the normal oropharyngeal defense mechanisms. the patient typically has a depressed state of consciousness, abnormal swallowing, a neuromuscular defect that prevents adequate coughing, or an abnormal connection between the airway and esophagus (such as an h-type tracheoesophageal fistula). acute lung aspiration (mendelson syndrome) is an acute chemical injury caused by inhalation of gastric contents. in neurologically normal children, gastric aspiration usually occurs as a complication of anesthesia. the diagnosis of acute aspiration is mainly clinical and usually involves witnessed inhalation of vomitus or tracheal suctioning of gastric contents. 28, 29 chronic lung aspiration (cla) is repeated passage of food, gastric reflux, or saliva into the subglottic airways that causes chronic or recurrent respiratory symptoms. cla may present with chronic cough, wheeze, noisy breathing, choking during feeding, recurrent episodes of pneumonia or bronchitis, and failure to thrive. chronic aspiration often results in progressive lung disease, recurrent pneumonia, chronic airway inflammation, bronchiectasis, and respiratory failure. it is a major cause of death in children with severe neurologic disorders (fig. 13) . pulmonary aspiration may occur as a result of swallowing dysfunction, gastroesophageal reflux, and inability to adequately protect the airway from oral secretions or a combination of these. anatomic conditions that predispose to aspiration lung disease include esophageal stricture or obstruction (eg, vascular ring, foreign body, achalasia), cleft palate, tracheoesophageal fistula (fig. 14) , laryngeal cleft, and bronchobiliary fistula. 28, 29 aspiration related to near-drowning occurs when fluid enters the lungs without being prevented by laryngospasm. it typically manifests as pulmonary edema radiographically. 30 in a recent series of 83 children, secondary infections from aspiration related to near-drowning were rare. 31 hydrocarbon pneumonia is an acute, intense chemical pneumonitis after unintentional aspiration of volatile hydrocarbon compounds. most cases of hydrocarbon pneumonia occur in children. chest radiographs typically show bilateral, scattered pulmonary densities with middle and lower zone predominance. such densities may become confluent and progress to acute respiratory distress syndrome (ards) and respiratory failure. they typically worsen over the first 72 hours and then clear over the next few days. however, occasionally radiographic changes may take weeks to months to be cleared. obstructive emphysema, pneumatoceles, subsegmental, or segmental atelectasis may also be seen. 32 lipoid pneumonia is a rare form of pneumonia caused by inhalation or aspiration of a fatty substance. oral administration of various oils is a common cultural practice, including mineral oil, olive oil, shark liver oil, cod liver oil, coconut oil, and ghee. such oily materials can readily slide into the airway even in normal infants and young children without eliciting a cough reflex and are poorly removed by cilia. lipoid pneumonias are typified by mild, subacute, or chronic clinical findings with accompanying marked radiographic changes. chest radiographs of children with lipoid pneumonia typically show bilateral parahilar illdefined, airspace opacities. in a series of 7 pediatric patients, ct showed dense consolidation surrounded by ground-glass opacity with a geographic lobular distribution. 33 within the dense consolidations, areas with relatively low attenuation were identified in only 1 patient. therefore, low-density consolidation in the posterior lungs is an infrequent ct finding in the diagnosis of lipoid pneumonia in children (fig. 15 ). interlobular septal thickening in areas of ground-glass opacity (ie, crazy paving pattern) has also been described in children with lipoid pneumonia. 33 lipoid pneumonias may be complicated by superimposed infection especially with atypical mycobacteria. slow recovery usually takes place with cessation of the oil administration. there may be residual scarring/fibrosis especially with animal rather than vegetable oils. 34, 35 foreign body aspiration can also result in pneumonia. accidental aspiration of both organic and nonorganic foreign bodies is a cause of childhood morbidity and mortality, requiring prompt recognition and early treatment to minimize the potentially serious and sometimes fatal consequence. eating is the most common circumstance during which it occurs, with small food items being the most common foreign bodies aspirated. coughing, choking, acute dyspnea, and sudden onset of wheezing are the most common symptoms. clinical signs of foreign body aspiration have low positive predictive values. chest radiographs are the initial imaging modality for patients with clinically suspected tracheobronchial aspiration of a foreign body. chest radiographs may show air trapping, atelectasis, a radiopaque foreign body (rare), or be normal (fig. 16) . 36 when the routine inspiratory chest radiograph is unhelpful or confusing, inspiratory and expiratory radiographs (in a cooperative child) or bilateral decubitus views (in a younger child unable to follow breathing instruction) are useful in confirming focal or unilateral air trapping. in selected cases, ct (possibly integrated with virtual bronchoscopy) may be considered to exclude a foreign body. ct evaluation may avoid bronchoscopy or provide the exact location and postobstructive complications of the foreign body before bronchoscopy. 37 an underlying chronic unrecognized airway foreign body should be considered among other causes of recurrent or chronic pneumonia, particularly in the pediatric population (see fig. 16 ). pie syndrome comprises a group of heterogeneous disorders having the common findings of lung disease and eosinophilia in the peripheral blood, bronchoalveolar lavage fluid, or pulmonary interstitium. pie syndrome is rare in children. a subclassification for the pie syndromes in children is summarized in box 8. 38, 39 infectious causes of pie syndrome are uncommon and include chlamydia trachomatis (especially in infants less than 3 months of age), allergic bronchopulmonary aspergillosis (in asthmatics and cystic fibrosis), parasitic larvae in lungs (toxocara, ascaris, and others), and fungi (eg, cryptococcus, candida species). 20 the radiographic findings in pie syndromes tend to be nonspecific. chest radiographs may show interstitial, alveolar, or mixed (interstitial and alveolar) infiltrates, which tend to be bilateral and diffuse. certain pie syndromes may be associated with more specific findings. the classic radiographic appearance of chronic eosinophilic pneumonia is characterized by peripheral infiltrates with sparing of the central lung zones. this radiographic appearance has been described as the "photographic negative of pulmonary edema." 40 bronchiectasis with mucoid impaction is generally present on chest radiographs or ct in patients with allergic bronchopulmonary aspergillosis (fig. 17) . 41 acute eosinophilic pneumonia is frequently associated with small bilateral pleural effusions. imaging is often helpful in determining the extent of disease, localizing the potential sites for lung biopsy, and in assessing response to therapy once treatment has begun. 38 chronic pneumonia is defined as a pulmonary opacity that does not improve within 1 month. it is best classified from the anatomic pattern, as focal, interstitial, with hilar lymphadenopathy, or with cysts, cavities, or spherical masses. spherical masses, with or without cavitations, are often features of an infectious cause. infectious causes and mimics of this pattern are summarized in boxes 9 and 10 (see fig. 16 ; figs. [18] [19] [20] . 42 obstructive atelectasis may both mimic and predispose to chronic pneumonia. it may have many underlying causes, including foreign body, mucoid impaction, narrowed bronchus, and extrinsic bronchial compression by cardiovascular anomalies, lymphadenopathy, tumor, or postpneumonic inflammatory changes. anomalies of the lung, mediastinum, and diaphragm that may mimic an acute or chronic pneumonia pattern include atypical thymus, diaphragmatic eventration and hernia, tracheal bronchus, lung hypoplasia, and congenital bronchopulmonary malformations (bpms). 20 several of these lesions, such as the bpms, predispose to recurrent or chronic infection but differentiating an infected from uninfected lesion may be difficult or impossible on imaging. sometimes having previous imaging for comparison is helpful in terms of features such as the new presence of fluid in a previously air-filled cavity or perilesional consolidation. recurrent pneumonia is defined as more than 1 episode within a 1-year period or more than 3 episodes in a lifetime. many children with a chronic pulmonary lesion (especially a congenital anomaly) are believed to have recurrent pneumonias if chest radiographs are taken only during a febrile illness. recurrent pneumonias may be either focal or interstitial (linear). underlying abnormalities that may predispose to recurrent focal pneumonia include chronic aspiration (see section on aspiration pneumonia), congenital heart disease, bronchopulmonary foregut malformations (including bpms with enteric-respiratory tract fistula), airway abnormalities (foreign body, stenosis, bronchiectasis, cystic fibrosis, immotile cilia disease), paralysis or eventration of the diaphragm, and congenital, acquired, and iatrogenic immune deficiencies. 43, 44 recurrent interstitial pneumonia may be secondary to asthma, hypersensitivity pneumonitis, or pneumonias in children with aids (including pneumocystis jiroveci pneumonia, lymphoid interstitial pneumonitis, and recurrent streptococcus pneumoniae infection) (see fig. 10 ). 20 neonatal lung infections can be generally classified into 3 types depending on the initial source of neonatal infection: transplacental, perinatal, and postnatal (including nosocomial) infections. transplacental infections enter the fetus hematogenously via the umbilical cord. most infants affected with transplacental infections typically recurrent aspiration caused by tracheoesophageal fistula without esophageal atresia in a 6day-old girl who presented with recurrent episodes of apnea, cyanosis, and choking desaturations. barium esophagram shows an oblique connection (red arrow) coursing anterosuperiorly from the esophagus to the trachea at the level of the thoracic inlet. contrast has opacified the central tracheobronchial airways. inferior to the fistula, there is a focal mild narrowing of the esophagus (blue arrow), raising concern for a congenital esophageal stricture. manifest systemic and multiorgan disease rather than a primary lung infection. the most common transplacental infection is caused by cmv, which manifests as a diffuse reticulonodular pattern. 45 other less common, transplacentally acquired pneumonias include rubella, syphilis, listeria monocytogenes, and tuberculosis. perinatal infections can be acquired via ascending infection from the vaginal tract (most commonly group b streptococcus or escherichia coli), transvaginally during the birth process, or nosocomially in the neonatal period. 46 radiographic findings in neonatal pneumonia are nonspecific in differentiating between various etiologic pathogens, as well as differentiating pneumonia from other causes of respiratory distress (eg, transient tachypnea of the newborn, surfactant deficiency disease, and meconium aspiration). the most common radiographic manifestation of neonatal pneumonia is bilateral coarse perihilar reticular densities with possible scattered airspace opacities (fig. 21) . solitary lobar consolidations are uncommon. 47 there is an association between group b streptococcal pneumonia and an ipsilateral diaphragmatic hernia. 48 chest radiographs in group b streptococcal sepsis can mimic the diffuse ground-glass opacity seen in surfactant deficiency disease. however, the presence of this finding in a full-term infant or the presence of cardiomegaly or pleural effusions may help to differentiate group b streptococcal infection from surfactant deficiency disease. 49 pneumonia caused by chlamydia trachomatis occurs in about 10% of infants born to women who carry this organism in their genital tract and becomes symptomatic more than 2 to 3 weeks after birth. chlamydia pneumonia is characterized by hyperinflation and bilateral diffuse reticular perihilar densities that are disparate with relatively mild clinical symptoms. 50 concomitant conjunctivitis, which used to be a useful clue to the cause, is prevented by the routine instillation of antibacterial eye drops at birth. neonates with chlamydia pneumonia frequently have accompanying eosinophilia. bordetella pertussis has recently resurfaced to produce epidemics of infection, probably related to waning community immunity. the clinical presentation of pertussis in the newborn may lack some features (characteristic whooping cough and fever) typical of the disease in older children. the clinical presentation of the most severely affected newborns may be dominated by marked respiratory distress, cyanosis, and apnea. mortality caused by pertussis usually results from secondary pneumonia, encephalopathy, cardiac failure, or pulmonary hypertension. a suggested mechanism for pulmonary hypertension that may develop in newborns with bordetella pertussis infection is formation of leukocyte thrombi in pulmonary venules secondary to hyperleukocytosis. 51, 52 the classic radiographic appearance in pertussis is the shaggy heart with diffuse peribronchial cuffing related to airway inflammation. however, chest radiographic findings such as hyperaeration, atelectasis, segmental consolidation, and lymphadenopathy are usually nonspecific. recurrent pulmonary infection, with bacterial, viral, or occasionally fungal pathogens, are frequent problems in neonates undergoing prolonged hospitalization and complex treatments, especially in premature infants with chronic lung disease. radiographic alterations caused by infection may be subtle when superimposed on chronic lung changes. 47 pneumonia is a common disease in the immunocompromised host. immunocompromise may be congenital (congenital immunodeficiencies), acquired (hiv/aids, malnutrition) or iatrogenic (during chemotherapy for cancer or after tissue transplantation). immunodeficient states can result in: (1) humoral immunodeficiency (hypogammaglobulinemia, functional b-lymphocyte deficiency accompanying hiv infection); (2) cellular immunodeficiency (severe malnutrition, late stages of aids, some congenital immunodeficiencies such as digeorge syndrome); and (3) neutrophil dysfunction and neutropenia (chronic granulomatous disease, pure neutropenia). iatrogenic immunodeficiencies may be a combination of neutropenia or neutrophil dysfunction, innate or drug-induced defective lymphocyte function, and drug-induced breaks in the oral and intestinal mucosal barriers. 53 the causes of pneumonia in the immunocompromised host consist not only of the same agents that cause pneumonia in the normal host but also of several opportunistic agents depending on the type and severity of immunodeficiency as well as temporal pattern after chemotherapy or transplant. in an immunocompromised child with a noncontributory chest radiograph and clinical findings that could be attributed to a lung infection, chest ct is often required for evaluation of a possible lung infection. in this situation, there are 4 major advantages of chest ct over chest radiographs. first, the presence, pattern, and extent of the disease process are better visualized. second, more than 1 pattern of abnormality may be detected, suggesting dual pathologic entities. third, invasive diagnostic procedures (eg, bronchoscopy or needle aspiration) can be more precisely planned. fourth, ct also allows for increased sensitivity in assessment of the response to treatment. 54, 55 although the radiographic or ct appearance might not be specific for a pathogen, knowledge of the clinical setting in combination with the type and severity of immunodeficiency and imaging pattern may narrow the differential diagnosis. a commonly encountered clinical issue is the possibility of fungal infection in immunocompromised children. the hallmark ct finding of fungal infections is the presence of pulmonary nodules. such pulmonary nodules are often clustered peripherally and can show poorly defined margins, cavitation, or a surrounding halo of ground-glass opacity (ct halo sign) (fig. 22) . the ct halo sign is a nonspecific finding and represents either hemorrhage around a nodule or neoplastic or inflammatory infiltration of the lung parenchyma. in the immunocompromised host, the ct halo sign can be seen most commonly with fungal infections (eg, aspergillosis, mucormycosis, or candida) but also in viral infections (eg, cmv infection, herpes infection), organizing pneumonia, and pulmonary hemorrhage. 54, [56] [57] [58] [59] [60] [61] microorganisms associated with severe pneumonia in immunodeficiency states are summarized in box 11. lip is a form of subacute pneumonia seen in several states of immunologic dysregulation, particularly in children with hiv infection. lip is characterized by micronodules of proliferating lymphoid tissue associated with infection by ebv. chest radiographs in lip show a characteristic diffuse, bilateral nodular, or reticulonodular pattern 19 . wegener granulomatosis mimicking chronic cavitary pneumonia in a 15-year-old male who presented with hemoptysis and respiratory distress. initial chest radiograph showed bilateral confluent airspace opacities secondary to diffuse pulmonary hemorrhage (not shown). contrast-enhanced ct obtained 9 days after initial presentation shows multifocal consolidation with cavitation (arrow), ground-glass opacities, and a right pneumothorax. (see fig. 10 ). other recognized associated imaging findings of lip include consolidation, mediastinal adenopathy, and bronchiectasis. 26, 58, 62 radiologic features in common hiv-associated infections in pediatric patients are summarized in table 2 . in pediatric patients with iatrogenic deficiencies of the immune system, pneumonia is commonly caused by opportunistic bacteria and fungi, acquired nosocomially or from resident mucosal flora (fig. 23) . in addition, after solid-organ transplantation although total immunoglobulin levels are normal, children and adolescents may be susceptible to encapsulated bacteria (eg, streptococcus pneumoniae, haemophilus influenzae). viruses that commonly cause pneumonia in healthy hosts (eg, rsv, influenza, parainfluenza viruses, human axial contrast-enhanced ct shows multiple bilateral pulmonary nodules, some have rims of groundglass opacity (arrow), which is also known as the ct halo sign. microorganisms associated with severe pneumonia in immunodeficiency states in pediatric patients metapneumovirus, and adenovirus) display greater virulence in both children and adults after solid-organ or human stem cell transplantation, particularly when cellular immunity is profoundly suppressed. in the posttransplant setting, ebv can cause progressive pulmonary disease in the form of posttransplantation lymphoproliferative disease. 53 in 2 studies of hrct in bone marrow transplant recipients, the most useful distinguishing feature was the presence of large nodules and visualization of the halo sign, suggestive of fungal infection. 63, 64 after solid-organ transplantation, nosocomially acquired bacteria predominate as a cause of pneumonia in the first month. later, viruses, especially cmv and adenovirus, as well as listeria, nocardia, and aspergillus, may be the cause. after more than 6 months after solid-organ transplantation, community-associated bacterial pneumonia becomes more common. 53 a variety of noninfectious pulmonary processes can present with acute or subacute clinical findings mimicking pulmonary infection. 54 these findings include alveolar hemorrhage, pulmonary edema, drug reaction, idiopathic interstitial pneumonia, benign and malignant lymphoproliferative disorders, constrictive bronchiolitis, bronchiolitis obliterans with organizing pneumonia, and chronic graft-versus-host disease. the ct findings of many of these entities are nonspecific. 54, [57] [58] [59] [60] [61] abbreviations: alveolar, focal of diffuse alveolar pattern; interstitial, focal or diffuse interstitial pattern; ln, lymphadenopathy; 1 and 11, refer to relative frequency of radiographic finding with each organism/disease. acute complications of pneumonia can be categorized as suppurative lung parenchymal complications and pleural complications. suppurative lung parenchymal complications span a spectrum of abnormalities and include cavitary necrosis, lung abscess, pneumatocele, bronchopleural fistula (bpf), and pulmonary gangrene. the name given to the suppurative process depends on several factors including the severity and distribution of the process, condition of the adjacent lung parenchyma, and temporal relationship with disease resolution. 65, 66 cavitary necrosis cavitary necrosis represents a dominant area of lung necrosis associated with a variable number of thin-walled cysts. characteristic findings on ct for cavitary necrosis include loss of normal lung architecture, poor parenchymal enhancement, loss of the lung-pleural margin, and multiple thin-walled fluid-filled or air-filled cavities (fig. 24) . cavitary necrosis is seen earlier in chest us or ct compared with chest radiography because these cavities need to be filled with air to be visible on chest radiographs. such cavities filled with air are accomplished only after communication with bronchial airways. most pediatric patients can be managed successfully with conservative treatment. follow-up chest radiographs typically show complete or near-complete resolution of the cavitary necrosis (see fig. 24 ). 65, 66 lung abscess lung abscess is a severe complication of pneumonia in children, mostly occurring in the presence of predisposing factors, such as congenital or acquired lung abnormalities, or immunodeficiency. lung abscess represents a dominant focus of suppuration surrounded by a well-formed fibrous wall. the predominant pathogens isolated from primary lung abscesses in children include streptococcal species, staphylococcus aureus and klebsiella pneumoniae. children with a lung abscess have a significantly better prognosis than adults with the same condition. 67 lung abscesses are uncommon in immunocompetent children. on contrast-enhanced ct, a lung abscess appears as a fluid-filled or airfilled cavity with a thick definable, enhancing wall (fig. 25) . 65, 66, 68 although lung abscess in children has been managed successfully for many years with prolonged courses of intravenous antibiotics, the evolution of interventional radiology has seen the accelerated use of percutaneously placed pigtail drainage catheters using us and ct guidance. 67 pneumatocele pneumatocele is a term given to thin, smoothwalled air-filled cysts seen at imaging and may represent a later or less severe stage of resolving or healing lung necrosis (see fig. 24 ). pneumatoceles are most often caused by severe lung infection from staphylococcal pneumonia. however, they may be seen with other bacterial infections including streptococcus pneumonia and after hydrocarbon aspiration. on ct, thin-walled small or large cysts containing air with or without fluid are identified. the wall of a pneumatocele does not enhance. the surrounding lung may be opacified but does not typically show findings of lung necrosis. 65, 66 pneumatoceles usually resolve spontaneously over time although pneumatoceles may be atypically persistent in children with hyper-ige syndrome. large pneumatoceles containing fluid can be a source of ongoing infection and may occasionally require drainage. bronchopleural fistula bpf is defined as a communication between the lung parenchyma or airways and the pleural space. central bpfs (ie, main or lobar bronchi communicating with the pleural cavity) most often develop after traumatic injury to large airways or leak from the bronchial stump after pneumonectomy or lobectomy. the main causes for peripheral bpfs (ie, segmental or more distal airways or lung parenchyma communicating with the pleural cavity) are necrotizing pulmonary infection (ie, cavitary necrosis), trauma, lung surgery, and malignancy. 69, 70 presence of air in the pleural space before aspiration or drainage attempts is suggestive of either a peripheral bpf or infection with a gas-producing organism. multidetector ct with thin-section axial and multiplanar reformation images may show a fistulous tract between the pleural space and peripheral airway or lung parenchyma in peripherally located bpfs (fig. 26) . 70 pulmonary gangrene pulmonary gangrene is a rare complication of severe lung infection with devitalization of lung parenchyma and secondary infection. 71 the primary feature that distinguishes pulmonary gangrene from necrotizing pneumonia and lung abscess is the extent of necrosis and the fact that thrombosis of large vessels plays a prominent role in the pathogenesis. chest imaging shows lobar consolidation with bulging fissures and is followed by tissue breakdown to form many small cavities, which subsequently coalesce into a single large cavity occupying the entire lobe. such a large cavity is filled with fluid and irregular pieces of sloughed lung parenchyma. however, these findings are not invariably present. surgical resection of necrotic tissue is often necessary for proper management of children with pulmonary gangrene. [71] [72] [73] [74] the differential diagnosis of suppurative lung complications includes an underlying cystic axial contrast-enhanced t1-weighted mr image of the brain shows a rim-enhancing brain abscess in the right occipital lobe. the patient underwent right lower lobectomy and craniotomy for resection of these lesions. congenital bpm that has become secondarily infected. prior and follow-up imaging may aid in the distinction. the presence of large, welldefined cysts early in the course of the illness or a systemic arterial supply to the lung may be helpful in suggesting an underlying bpm, although a chronic inflammatory process in the lower lobe can acquire some systemic vascular supply from diaphragmatic vessels. pleural complications from acute pneumonia include parapneumonic effusion and empyema. parapneumonic effusion is defined as a pleural effusion in the setting of a known pneumonia. it may be simple or complicated based on the absence or presence of the infecting organism within the pleural space, respectively. 75 empyema is defined as thick purulent pleural effusion. it may be free-flowing or loculated. progression of a pleural effusion to empyema occurs through 3 stages: exudative, fibrinopurulent, and organization. parapneumonic effusions complicate pneumonia in 36% to 56% of cases in pediatric patients. 76 empyema complicates an estimated 0.6% of all childhood pneumonias. 77 chest radiographs can often detect a parapneumonic collection, although some fluid, especially in a subpulmonic location, may not be visible and is often seen better on a decubitus film. in cases with complete or almost complete opacification of a hemithorax with or without contralateral mediastinal shift, additional erect or decubitus views are unhelpful in defining the quantity or nature of the pleural fluid. us is most helpful in this situation because it can readily distinguish a parapneumonic collection from extensive consolidation or an underlying mass. the us determination of the echogenicity of the pleural collection (anechoic or echogenic) and showing fibrin strands, septations, loculations, or fibrinous pleural rind is helpful in determining appropriate therapy (see fig. 26 ). treatment options for parapneumonic effusions/ empyemas include antibiotics alone, simple tube drainage, chest drain insertion with fibrinolytics, or surgery (eg, video-assisted thoracoscopic surgery or open thoracotomy with decortication). although imaging techniques are used as a guideline, they do not always accurately stage empyema, predict outcome, or guide decisions regarding surgical versus medical management. 75 ct provides a more global overview of pleural and pulmonary abnormality from acute pneumonia, but is poor at differentiating parapneumonic effusion from empyema in pediatric patients. findings on ct, in patients with parapneumonic effusion/ empyema, include: (1) enhancement and thickening of visceral and parietal pleura; (2) thickening and increased density of extrapleural subcostal tissues; and (3) increased attenuation of extrapleural subcostal fat. 78 loculation can be inferred by the presence of a lenticular fluid collection or nondependent air. septations are usually not appreciated on ct (see fig. 26 ). pleuropulmonary infection may occasionally spread to involve the chest wall, including soft tissues and adjacent bones. mycobacterium tuberculosis, aspergillus, and actinomyces are the most common organisms in this scenario. chronic complications or consequences of pneumonia include parenchymal scarring, bronchial wall thickening, bronchiectasis, a predisposition to asthma, constrictive bronchiolitis, fibrothorax and a trapped lung, fibrosing mediastinitis, constrictive pericarditis, and pleural thickening. for practical purposes, bronchiectasis, constrictive bronchiolitis, fibrothorax and trapped lung, and fibrosing mediastinitis are discussed in the following sections. bronchiectasis is defined by the presence of permanent and abnormal dilation of the bronchi. this condition usually occurs in the context of chronic airway infection causing inflammation. bronchiectasis is nearly always diagnosed using hrct. the main diagnostic features of bronchiectasis on hrct are: (1) internal diameter of a bronchus that is wider than its adjacent pulmonary artery; (2) failure of the bronchus to taper peripherally; and (3) visualization of bronchi in the outer 1 to 2 cm of the lung zones (see fig. 17 ; figs. 27 and 28). a wide variety of factors predisposing to the development of bronchiectasis have been identified, including hereditary (cystic fibrosis, ciliary dyskinesia), infective, immunodeficiency (antibody deficiency), obstructive (intrabronchial foreign body), and systemic causes. causes most commonly associated with bronchiectasis are childhood infections, including pneumonia, pertussis, complicated measles, and tuberculosis (eg, mycobacterium tuberculosis and mycobacterium avium complex). [79] [80] [81] constrictive bronchiolitis (bronchiolitis obliterans) constrictive bronchiolitis (bronchiolitis obliterans) is characterized by the presence of concentric narrowing or obliteration of the bronchioles caused by submucosal and peribronchiolar fibrosis. a common cause of constrictive bronchiolitis is previous childhood infection, resulting in the so-called swyer-james syndrome, identifiable as asymmetric hyperlucent lung on chest radiographs. whereas the process may appear unilateral on chest radiographs, there is usually bilateral but asymmetric abnormality on ct (see fig. 28 ). central bronchiectasis and a characteristic mosaic appearance with patchy expiratory air trapping are seen on hrct. causes and associations of constrictive bronchiolitis include previous infections (viral including adenovirus, rsv, influenza, parainfluenza; mycoplasma and pertussis), collagen vascular diseases, previous transplant, toxic fume exposure, ingested toxins, drugs, and cryptogenic constrictive bronchiolitis. 82 pleural fibrosis can result from a variety of inflammatory processes (box 12). the development of pleural fibrosis follows severe pleural inflammation, which is usually associated with an exudative pleural effusion. fibrothorax and trapped lung are 2 uncommon consequences of pleural fibrosis (see fig. 27 ). 83 fibrothorax represents the most severe form of pleural fibrosis. with a fibrothorax, there is dense fibrosis of the visceral and parietal pleural surfaces, leading to fusion of these membranes, contracture of the involved hemithorax (and ipsilateral mediastinal shift), and reduced mobility of the lung and thoracic cage (see fig. 27 ). decortication is the only potentially effective treatment of fibrothorax in patients with severe respiratory compromise. 83, 84 a trapped lung is characterized by the inability of the lung to expand and fill the thoracic cavity because of a restrictive, fibrous, visceral pleural peel (see fig. 27 ). restriction of lung parenchymal expansion and subsequent negative pressure in the pleural space result in filling of the pleural space with pleural fluid (usually a transudate). the diagnosis of a trapped lung implies chronicity, stability over time, and a purely mechanical cause for the persistence of a fluid-filled pleural space. patients with a trapped lung usually do not experience improvement in dyspnea after thoracentesis. in symptomatic patients, decortication should be considered. the underlying lung parenchyma should be assessed before decortication. if the trapped lung is severely diseased and fibrotic, decortication is unlikely to result in lung reexpansion and the procedure does not provide symptomatic benefit. in contrast, lung entrapment is the result of an active inflammatory process or malignancy in the pleural space, leading to a restricted pleural space. pleural fluid from lung entrapment is an exudate, and symptoms in patients with lung entrapment typically improve after thoracentesis. 83, 85 fibrosing mediastinitis fibrosing mediastinitis is a rare condition characterized by proliferation of fibrous tissue within the mediastinum. symptoms are related to compression of the central airways, superior vena cava, pulmonary veins, pulmonary arteries, and esophagus. the most common cause of this disorder is fungal infection, especially histoplasma capsulatum in the united states. 86 pneumonia is an infection of the lung parenchyma caused by a wide variety of organisms in pediatric patients. imaging evaluation plays an important role in children with pneumonia by detecting the presence of pneumonia and determining its location and extent, excluding other thoracic causes of respiratory symptoms, and showing complications such as effusion/empyema and suppurative lung changes. clear understanding of the underlying potential cause, current role of imaging, proper imaging techniques, and characteristic imaging appearances of acute and chronic pneumonias can guide optimal management of pediatric patients with pneumonia. who child health epidemiology reference group. global estimate of the incidence of clinical pneumonia among children under five years of age pneumonia: an eleven-year study in a pediatric practice incidence of community-acquired pneumonia in the population of four municipalities in eastern finland pneumonia: the leading killer of children what imaging should we perform for the diagnosis and management of pulmonary infections? differentiation of bacterial and viral pneumonia in children aetiology of community-acquired pneumonia in children treated in hospital ultrasound of the chest in children (mediastinum excluded) us in the diagnosis of pediatric chest diseases multidetector ct in children: current concepts and dose reduction strategies computed tomography scanning techniques for the evaluation of cystic fibrosis lung disease increased inspiratory pressure for reduction of atelectasis in children anesthetized for ct scan magnetic resonance imaging of lung infections in children follow-up of acute pulmonary complications in cystic fibrosis by magnetic resonance imaging the role of advanced imaging techniques in cystic fibrosis follow-up: is there a place for mri? basic patterns in lung disease the epidemiology of acute respiratory tract infection in young children: comparison of findings from several developing countries susceptibility to bronchiolitis in infants the chest radiograph in acute bronchiolitis moffet's pediatric infectious diseases: a problem-oriented approach the many radiologic facies of pneumococcal pneumonia round pneumonia: imaging findings in a large series of children review of new and newly discovered respiratory tract viruses in children. pediatr emerg care tuberculosis in children: an update tuberculosis in children: an update chest radiographic features of lymphocytic interstitial pneumonitis in hiv-infected children clinical and radiographic spectrum of septic pulmonary embolism 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papillomatosis with pulmonary parenchymal spread. case report and review of the literature recurrent pneumonia in children: a case report and approach to diagnosis assessment of the child with recurrent chest infections radiological imaging of the neonatal chest intra-amniotic infection and premature rupture of the membranes imaging of medical disease of the newborn lung neonatal radiology. acquired diaphragmatic hernia with group b streptococcal pneumonia radiographic findings in early onset neonatal group b streptococcal septicemia chlamydia trachomatis in children pertussis with severe pulmonary hypertension in a newborn with good outcome-case report malignant pertussis in the pediatric intensive care unit principles and practice of pediatric infectious diseases revised reprint fungal pulmonary infections after bone marrow transplantation: evaluation with radiography and ct investigation and management of a child who is immunocompromised and neutropoenic with pulmonary infiltrates ct halo sign: the spectrum of pulmonary diseases thoracic mycoses from opportunistic fungi: radiologic-pathologic correlation thoracic disease in children with aids childhood leukemia: diagnostic accuracy of bedside chest radiography for severe pulmonary complications acute lung disease in the immunocompromised host: ct and pathologic examination findings cytomegalovirus pneumonia in transplant patients: ct findings pulmonary infections in hiv-positive children pulmonary infections after bone marrow transplantation: high-resolution ct findings in 111 patients pulmonary infections following bone marrow transplantation: high-resolution ct findings in 35 paediatric patients pneumonia in children: decreased parenchymal contrast enhancement-ct sign of intense illness and impending cavitary necrosis cavitary necrosis complicating pneumonia in children: sequential findings on chest radiography lung abscess in children lung abscess in a child with mycoplasma pneumoniae infection peripheral bronchopleural fistula: ct evaluation in 20 patients with pneumonia, empyema, or postoperative air leak multi-detector row computed tomographic evaluation of bronchopleural fistula: correlation with clinical, bronchoscopic, and surgical findings gangrene of the lung: treatment in two stages massive pulmonary gangrene pulmonary gangrene. a complication of bacterial pneumonia pulmonary gangrene complicating bacterial pneumonia imaging of parapneumonic pleural effusions and empyema in children therapy of parapneumonic effusions in children: videoassisted thoracoscopic surgery versus conventional thoracostomy drainage management of empyema in children ct appearance of parapneumonic effusions in children: findings are not specific for empyema an investigation into causative factors in patients with bronchiectasis the pathophysiology of bronchiectasis bronchiectasis: an update bronchiolar disorders: a clinical-radiological diagnostic algorithm pleural fibrosis physiological evaluation of results of pulmonary decortication trapped lung sclerosing mediastinitis mimicking anterior mediastinal tumor key: cord-275166-qduf08kp authors: assane, dieng; makhtar, camara; abdoulaye, diop; amary, fall; djibril, boiro; amadou, diop; niokhor, diouf jean baptiste; amadou, diop; cheikh, loucoubar; ndongo, dia; mbayame, niang; lamine, fall; bouh, boye cheikh saad title: viral and bacterial etiologies of acute respiratory infections among children under 5 years in senegal date: 2018-02-13 journal: microbiol insights doi: 10.1177/1178636118758651 sha: doc_id: 275166 cord_uid: qduf08kp acute respiratory infections (aris) are the leading cause of infectious disease–related morbidity, hospitalization, and morbidity among children worldwide. this study aimed to assess the viral and bacterial causes of ari morbidity and mortality in children under 5 years in senegal. nasopharyngeal samples were collected from children under 5 years who had ari. viruses and bacteria were identified using multiplex real-time reverse transcription-polymerase chain reaction and conventional biochemical techniques, respectively. adenovirus was the most prevalent virus (50%; n = 81), followed by influenza virus (45.68%, n = 74), rhinovirus (40.12%; n = 65), enterovirus (25.31%; n = 41), and respiratory syncytial virus (16.05%; n = 26), whereas streptococcus pneumoniae (17%; n = 29), moraxella catarrhalis (15.43%; n = 25), and haemophilus influenzae (8.02%; n = 13) were the most commonly isolated bacteria. virus pathogens seem more likely to be more prevalent in our settings and were often associated with bacteria and s. pneumoniae (6%; 16) coinfection. respiratory tract infections (rtis) such as acute otitis media, sinusitis, bronchitis, and community-acquired pneumonia are a leading cause of infectious disease-related morbidity, hospitalization, and mortality among children worldwide, particularly in low-income countries. 1 according to world health organization (who), the prevalence of hospitalized children under 5 years with acute respiratory infections (aris) is estimated to be 20% and 90% of those were due to pneumonia. 2 in addition, the number of childhood deaths annually related to aris is very important and is estimated between 1.9 and 2.2 million, and 70% of the deaths took place in africa and southeast asia which are the most. 1, 3 bacteria and viruses have been reported as the main causes of aris. in children under 5 years, aris are caused mainly due to viruses; respiratory syncytial viruses (rsvs), parainfluenza viruses, influenza virus a and b, and human metapneumovirus (hmpv) are the most common viruses isolated. 4, 5 however, primary infections with viral pathogens can predispose to secondary bacterial infections, and the most frequently isolated bacteria in aris include streptococcus pneumonia and haemophilus influenzae. 6 these bacteria were increasingly resistant to the most commonly used antibiotics for ari treatment, 7 leading to increase in mortality rates, hospital durations, and health care-associated costs. 8 in resource-limited countries, bacteria have been the main cause of aris. this could be explained by the inaccessibility of molecular diagnostic tools thus leading to inadequate antibiotics prescription and consequently contributed to a rapid increase in antimicrobial resistance among bacteria causing rtis. 9, 10 in senegal, few studies on viral and bacterial etiologies of rtis are available in pediatric settings. respiratory tract infections are mainly empirically treated with antibiotics on a simple suspicion of bacterial infection. indeed, this could be one of the major causes of high morbidity and mortality rates. the aim of this study was to investigate the viral and/or bacterial infections associated with aris in children under 5 years. this study has been approved by the ethics committee for research of the cheikh anta diop university of dakar. samples and information for questionnaires have been collected after patient's informed consent. bacteria were isolated from appropriate culture media and incubation conditions prior identification following microbiological standard procedures based on morphological, cultural, and biochemical or antigenic characters. strains were identified if the bacterial load was at least 10 5 cfus/ml. data were analyzed using r statistical software version 3.3.1. 11 over a period 1 year, 162 children with aris were included in this study including 97 (59.9%) men. overall, 30 children were under 6 months of age, 26 were 6 to 12 months old, 31 were 12 to 24 months old, 53 were 24 to 60 months old, and 23 were 60 to 112 months old. table 1 shows the distribution of major viruses isolated during this study period, the frequency of single or coinfection, and the number of infections stratified by age group. among selected virus species, adenovirus was the most prevalent (50%, n = 81), followed by influenza virus (45.68%, n = 74), rhinovirus (40.12%, n = 65), enterovirus (25.31%, n = 41), and rsv (16.05%, n = 26). single infection with adenovirus was very rare (3.7%, n = 6). however, adenovirus was associated with other viruses and bacteria in 25.31% (n = 41) and 4.94% (n = 8), respectively. in addition, adenovirus/other viruses/bacteria coinfections have been detected in 16.05% (n = 26) of children. influenza virus infections were associated with single-virus coinfections (33.3%, n = 54) or virus and bacteria coinfections (12.35%, n = 20). however, influenza virus single-infected children were not detected. low prevalence of both rhinovirus and enterovirus single infections was observed (1.85%, n = 3). these infections were most often associated with single virus or virus and bacteria coinfections. adenovirus, influenza virus, and enterovirus infections were more prevalent among children aged from 24 to 60 months. the prevalence of rsv infections associated with other viruses or other viruses and bacteria was 19.14% and 1.85%, respectively; however, no rsv single infection was detected. children aged from 0 to 6 months were more likely to be more affected with rhinovirus and rsv infections. the distribution of major bacterial clinical strains is shown in table 2 . among isolated bacterial species, s. pneumoniae was the most prevalent bacteria (17%) followed by m. catarrhalis (15.43%) and h. influenzae (8.02%). bacterial single infections were also very rare: s. pneumoniae (2%), m. catarrhalis (2%), and h. influenzae (1%). no bacteria/bacteria coinfections were detected. however, coinfections with bacteria and viruses had been observed in children having aris and coinfection with s. pneumoniae (16.66%) was most frequent followed by m. catarrhalis (14, 19%) and h. influenzae (4.32%). the contribution of viral and bacterial pathogens to the clinical syndrome is depicted in tables 3 and 4 , respectively. acute respiratory infections may be caused by viruses, bacteria, or both. thus, determination of the causative agent is crucial to limit the abuse of antibiotics. in our study conducted over a period of 1 year, we showed that viruses were the most common pathogens detected in aris. similar findings have also been reported in a study conducted in united states by obasi et al 12 in 2013. several studies conducted in other countries such as burkina faso, 10 zambia, 9 and niger 13 provided evidence of the role of viruses in upper and lower airway diseases. in senegal, adenovirus was the most common pathogen detected from aris in children. this study confirms previous results reported between 2013 and 2015 in senegal by niang et al. 14 the overall detection rate of adenovirus was 50% among the 162 selected children. this adenovirus infection prevalence seems very high compared with rates documented from other countries, namely, ghana, 15 burkina, 10 and zambia. 9 however, our results are in agreement with data reported in cameroon. overall, this confirms the overall high prevalence of adenovirus ari in africa. 16 in addition to adenovirus, influenza virus, rhinovirus, and enterovirus are the most common viruses detected, as reported in other studies. 10, 17 our study revealed viral codetection is frequent in aris. similar results were found in other countries. 9,10,15 viral codetection in clinical settings is becoming more common since the introduction of molecular-based multiplex tests. although the clinical significance of these findings remains unclear, this seems to have no impact in disease severity. 18 in this study, we also found that aris were associated with bacterial pathogens; among those, s. pneumoniae (17.9%), m. catarrhalis (15.43%), and h. influenzae (8%) were the most common. in contrast with our findings, high rate (56%) of s. pneumoniae detection was observed in niger among children having aris. 13 in our study, prevalence of mono-infections with s. pneumoniae (2%), m. catarrhalis (2%), or h. influenzae (1%) was very low. thus, this low prevalence of bacterial pathogens in aris proves that antibiotics should no longer be systematically used in the treatment of aris. however, among low-prevalence bacterial isolates, codetections with viruses were more frequent and s. pneumoniae (16, 6%) coinfection was the most frequent in our findings. similar results were reported in other studies. 19, 20 there results highlight the pivotal role of viruses in aris because primary infection with viral pathogens can predispose children to subsequent bacterial infections. 21 our study has limitations; among others, the sample size which does not really allow to determine the direct association between viral or bacterial infection and disease severity. moreover, a case-control study would be more suitable to determine the exact role of viruses in aris pathogenesis. in conclusion, this study reports the profile of viral and bacterial pathogens among children under 5 years hospitalized with aris in senegal. the high prevalence rates of viral infections and its clinical impact highlight the need to implement a systematic surveillance program for a better management of aris in children, particularly in resource-limited settings. estimates of wide distribution of child deaths from acute respiratory infections world health organization. acute respiratory infections in children rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human para influenza virus types 1, 2 and 3 by multiplex quantitative reverse transcription polymerase chain reaction-enzyme hybridization assay (hexaplex) development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses acute bacterial sinusitis complicating viral upper respiratory tract infection in young children antibiotic resistance of bacteria responsible of acute respiratory tract infections in children attributable hospital cost and length of stay associated with health care-associated infections caused by antibiotic-resistant gram-negative bacteria identification of viral and bacterial pathogens from hospitalized children with severe acute respiratory illness in lusaka, zambia viral aetiology of respiratory tract infections in children at the paediatric hospital in ouagadougou (burkina faso) r: a language and environment for statistical computing. r foundation for statistical computing detection of viral and bacterial pathogens in acute respiratory infections viral and bacterial aetiology of severe acute respiratory illness among children <5 years of age without influenza in niger respiratory viruses in patients with influenza like illness in senegal: focus on human respiratory adenoviruses respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana viral etiology of severe acute respiratory infections in hospitalized children in cameroon viral aetiology of respiratory infections in children in south-western saudi arabia using multiplex reverse transcriptase polymerase chain reaction clinical disease severity of respiratory viral co-infection versus single viral infection: systematic review and analysis viral and bacterial detection in acute respiratory infection in children under five years bacterial and viral etiology in hospitalized community acquired pneumonia with molecular methods and clinical evaluation high nasopharyngeal pneumococcal increased by viral co-infection, is associated with invasive pneumococcal pneumonia the authors are grateful to cea-samef (dakar, senegal), micro csb system (dakar, senegal), and institute pasteur dakar (dakar, senegal) who have contributed to the success of this study. key: cord-267402-kca05rvz authors: south, kieron; mcculloch, laura; mccoll, barry w; elkind, mitchell sv; allan, stuart m; smith, craig j title: preceding infection and risk of stroke: an old concept revived by the covid-19 pandemic date: 2020-07-24 journal: int j stroke doi: 10.1177/1747493020943815 sha: doc_id: 267402 cord_uid: kca05rvz anecdotal reports and clinical observations have recently emerged suggesting a relationship between covid-19 disease and stroke, highlighting the possibility that infected individuals may be more susceptible to cerebrovascular events. in this review we draw on emerging studies of the current pandemic and data from earlier, viral epidemics, to describe possible mechanisms by which sars-cov-2 may influence the prevalence of stroke, with a focus on the thromboinflammatory pathways, which may be perturbed. some of these potential mechanisms are not novel but are, in fact, long-standing hypotheses linking stroke with preceding infection that are yet to be confirmed. the current pandemic may present a renewed opportunity to better understand the relationship between infection and stroke and possible underlying mechanisms. the sars-cov-2 global pandemic at the time of writing, the global number of confirmed severe acute respiratory syndrome coronavirus 2 (sars-cov-2) cases is approaching 9 million, with over 470,000 reported fatalities. the current novel coronavirus outbreak began to receive worldwide media attention in early january 2020 with the earliest cluster of cases traced back to december 2019 in the city of wuhan in china. by 30 january the world health organization (who) declared the outbreak a ''public health emergency of international concern'' and, after cases were reported in 210 countries, the outbreak was recognized by who as a pandemic on 11 march 2020. sars-cov-2 is a member of the betacoronavirus genus of the coronaviridae family of enveloped, singlestranded rna viruses, several of which are known to cause mild respiratory disease in humans. it was named because of its similarity to sars-cov, the virus responsible for an epidemic in 2002-2003 that infected approximately 8000 people with almost 800 fatalities. both sars-cov and sars-cov-2 cause acute respiratory symptoms but due to enhanced rates of transmission derived from transmission from asymptomatic individuals 1 and a high level of early viral shedding in the upper respiratory tract, 2 this recent pandemic has attained a large global impact. angiotensin-converting enzyme 2 (ace2), the ''receptor'' for host cell entry of sars-cov-2, 3 is most prominently expressed on the surface of lung alveolar epithelial cells, venous and arterial endothelial cells, arterial smooth muscle cells and enterocytes of the small intestine. 4 notably, considering the possible the possible relationship between respiratory tract infection and the incidence of stroke, particularly ischemic stroke, is not a new concept. early case-control studies identified respiratory tract infections as a significant risk factor across all age groups despite adjusting for other known vascular risk factors. 14 a large caseseries analysis of uk medical records identified a significant risk of either first stroke or recurrent stroke associated with a diagnosis of acute respiratory tract infection. 15 this risk was highest in the first few days after infection, steadily declining thereafter but remaining elevated over baseline for some time. the incidence ratio of first stroke was found to be 3.19 (95% ci 2.81 to 3.62) within three days of infection and 2.09 (95% ci 1.89 to 2.32) within 14 days. a later retrospective case-crossover study of administrative data in the us, focusing on respiratory tract infections defined using centers for disease control and prevention criteria as ''influenza-like illness'', identified a similar risk of ischemic stroke within 15 days of infection (odds ratio 2.88, 95% ci 1.86 to 4.47). 16 large, systematically collated datasets are not yet available for the current sars-cov-2 pandemic and, as such reliable estimates of the associated risk of stroke have not yet been published. this is also true of the previous sars pandemic that only affected 8000 individuals. although, an approximate stroke incidence rate of 1 per 42 sars patients was determined from a small, retrospective single-center analysis. 17 for now, assumptions on the prevalence of stroke among covid-19 patients are based on small, single center observational studies, 18 which estimate an incidence rate of approximately 5% among the most severe cases. in a larger single center study of 3556 covid-19 patients the estimated stroke incidence rate was much lower at 0.9%. 19 it is likely that any estimation of stroke incidence will be confounded by under-reporting; both in severe infection with competing risk of mortality and milder infections (and strokes) not presenting to hospital or primary care. common features of covid-19 pathogenesis and early pathology of ischemic stroke as we begin to better understand covid-19 there are clearly aspects of its pathogenesis and disease course that are implicated in the initiation, or in the very early pathophysiology, of ischemic stroke. what follows herein is a detailed summary of the current literature surrounding covid-19, encompassing the immune and inflammatory responses to infection, thrombotic manifestations and vascular consequences of infection with a focus on possible mechanisms by which these elements may contribute to acute stroke events. activation of type i interferon (ifn), production of pro-inflammatory cytokines (interleukin-6 (il-6), tumor necrosis factor (tnf)) and the induction of ifn-stimulated genes 20 (figure 1(1) ). the immediate immunomodulatory impact of type 1 ifn (and the subsequent type 2 ifn (ifn-c) response) manifests as an accumulation of pro-inflammatory monocytes and macrophages in the alveoli. 21 recruited macrophages are themselves prominent sources of il-6, monocyte chemoattractant protein-1 (ccl2), il-8 and ifn, 22 thereby contributing to further influx of myeloid cells (figure 1 (1)), including neutrophils, which are an indispensable component of the inflammatory response to infection but also a major contributor to lung pathology. 23 extensive neutrophil extravasation into the alveolar space has been identified in post-mortem tissue of covid-19 patients 24 and neutrophil activation has long been associated with disease pathologies involving ards, and correlates with the extent of lung damage and with cytokine levels (tnfa, il-6 and il-8). 25 the release of pro-inflammatory cytokines (type i ifn and il-6) also aids the initiation of the adaptive immune response to viral infection, through the maturation of lung resident conventional or monocytederived dendritic cells (dcs). 26 these dcs migrate to draining lymph nodes and elicit a robust activation and proliferation of naı¨ve, antigen-specific cd8 þ (''cytotoxic'') t cells and naive cd4 þ (''helper'') t cells. 27 in a cohort of 128 sars patient samples, a high frequency of cd8 þ t cells, th1 and th2 cd4 þ t cells and polyfunctional cd4 þ t cells were identified in blood from patients with severe disease. 28 early indications from covid-19 patients suggest a similar clonal infection of the lower respiratory tract begins with the binding of sars-cov-2 to ace2 on the surface of type ii alveolar pneumocytes (1). the immediate type i interferon response recruits macrophages, monocytes, and neutrophils to the alveoli. propagation of the innate immune response is directed by th1 and th17 cd4 þ t cells (2) as neutrophils and pro-inflammatory monocytes are targeted to the site of infection (3) . endothelial activation, by either the inflammatory environment or by direct viral infection, upregulates key cell adhesion molecules allowing further infiltration of pro-inflammatory monocytes, cytotoxic t cells and activated neutrophils (4) . endothelial activation also elicits tissue factor release, endovascular recruitment of neutrophils releasing neutrophil extracellular traps (nets) (4) and von willebrand factor (vwf) exocytosis from weibel palade bodies (5) all of which contribute to the development of microvascular thrombosis. these local immune and pro-coagulant responses may result in systemic release of multiple cytokines and chemokines and ultra large vwf multimers, hyper-activation of circulating platelets and the embolization of vwf/platelet-rich thrombi (6) . increased pro-inflammatory and pro-coagulant factors in the plasma could be sufficient for in situ thrombus formation in the cerebral vasculature (7) and this may be exacerbated by infection and/or activation of the cerebral endothelium and local release of vwf and tissue factor (8) . endothelial activation would be expected to facilitate recruitment of neutrophils, monocytes and macrophages to the vessel lumen and induce a local inflammatory response in the surrounding brain parenchyma thereby polarizing microglia (9) . small vessel occlusion, by thromboemboli or by in situ thrombosis due to endothelial dysfunction, causes hypoperfusion of brain tissue (10) . ultimately, this combination of tissue hypoperfusion and the pro-inflammatory action of infiltrating and brain resident immune cells is the origin of stroke brain injury (11) . expansion of cd4 þ and cd8 þ t cells, 29 and differentiation of th1 30 and th17 cells. 31 a paradoxical, but common, characteristic of sars-cov-2 infection is the development of lymphopenia, observed also in the sars-cov, mers-cov and h 1 n 1 /h 7 n 9 influenza pandemics. 32 in covid-19 patients, a marked decrease in the number of peripheral cd4 þ and cd8 þ t cells, accompanied by hyperactivity of these populations, 31 has been suggested as an effective predictor of disease severity. 33 it has been postulated that this may be the result of virus-mediated dysfunction of lymphatic tissues, cytokine-mediated lymphocyte apoptosis or the direct killing of lymphocytes by viral infection. 6 it could also be, simply, the redistribution of lymphocytes to the infected tissue and/ or lymph nodes. the net result of lymphopenia, particularly in combination with enhanced granulocytosis, is an increase in the neutrophil to lymphocyte ratio (nlr). increased nlr is already evident in covid-19 cohorts and has been suggested as a potential prognostic marker of more severe illness and increased risk of mortality. 34 this is an important observation given that nlr also has prognostic value for determining stroke risk. 35 the exaggerated immune response (''the cytokine storm'') the cumulative consequence of leukocyte recruitment and activation is the accumulation of cytokines, both in the lung tissue and in the circulation. in severe covid-19 patients, this response is exaggerated, resulting in a ''cytokine storm'', in which aberrant cytokine expression and disproportionate inflammation results in persistent acute lung injury extending beyond the time of peak viral load. cell populations of particular relevance to the development of a cytokine storm are pro-inflammatory (cd14 þ , cd16 þ , il-6 hi ) monocytes and pathogenic (gm-csf þ , ifn-c þ ) th1 lymphocytes, which appear to predominate in the circulation of covid-19 patients in icu, 30 and th17 lymphocytes which are prevalent in influenza. 36 together these cells propagate a second wave of immune cell infiltration, 30 polarization of lung-resident macrophages to a proinflammatory phenotype 37 and cytokine production ( figure 1 (2) and (3)). macrophage polarization rapidly elevates the levels of circulating cytokines, most notably il-6 ( figure 1(6) ). in all of the covid-19 cohorts included in a recent meta-analysis, il-6 levels were significantly elevated (approximately 3 fold) in patients with complicated disease compared to those with non-complicated disease. 38 in the largest of the individual studies (n ¼ 452), the median plasma concentration of il-6 was 25.5 pg/ml, 39 in line with levels observed in severe sars-cov 40 and mers-cov infection. 41 the exaggeration of peripheral immune responses and ensuing inflammation is likely to be one of the key aspects of covid-19 pathogenesis that could result in cerebrovascular events. this is highlighted by the observation that il-6 is a predictor of stroke risk. 42 it is possible that hyperinflammation may contribute to the progression of two key stroke risk factors, atherosclerosis and atrial fibrillation (af). in the case of chronic atherosclerosis, viral infection is thought to drive the progression of atheromatous plaques through enhanced macrophage and t-cell responses 43 within the developing lesion, although this may not occur within a timeframe that is relevant to covid-19 related stroke. however, the release of ifn-c, tnf-a and other destabilizing factors can then expose the plaque's thrombogenic core 44 inducing plaque rupture which is more likely to be influenced by acute inflammatory conditions. there is also some evidence that plaque development and rupture is influenced directly by viral infection of vascular cells. 45 similarly, peripheral immune responses to viral infections are thought to contribute to the pathogenesis of af through the release of reactive oxygen species and myeloperoxidases from neutrophils and the local release of tnf-a and il-1b from macrophages. 46 viral infection may also be an important factor in the development of non-valvular af through upregulation of monocyte tlr2 and il-6 release. 47 covid-19 appears to share many aspects of its pathology with previous viral pandemics including the prevalence of thrombotic complications. during the 2009 h 1 n 1 influenza pandemic, the incidence of overt thrombotic manifestations (deep vein thrombosis (dvt) or pulmonary embolism (pe)) was approximately 6%. 48 during the 2002-2003 sars pandemic, the incidence of dvt was estimated to be as high as 20% with a further 11% of patients developing pe. 49 in a larger sars cohort, thrombotic abnormalities were identified in over 60% of patients. 50 studies of aberrant coagulation in covid-19 patients are often reporting on small cohorts and conclusions have been conflicting. in a cohort of 183 patients with severe covid-19, hemostatic abnormalities were associated with fatality, with 70% of non-survivors meeting the criteria for disseminated intravascular coagulation (dic), including a progressive increase in pt and d-dimer. 51 however, in a smaller cohort of icu patients a state of hypercoagulability, not consistent with overt dic (increased d-dimer without associated bleeding), was reported indicating extensive pulmonary vascular thrombosis. 52 this is further supported by a small autopsy series which identified thrombotic microangiopathy that was completely restricted to the lungs. 24 it is, therefore, still unclear if sars-cov-2 infection has a direct impact on hemostatic mechanisms or whether the thrombotic manifestations are purely the result of dic, secondary to systemic inflammation, or sepsis-induced coagulopathy. there is some emerging consensus regarding the rate of incidence of venous thrombosis (in the absence of overt dic) in covid-19 patients, and it is extraordinarily high. klok et al. recorded an incidence of total thrombotic complications of 31% (95%ci 20-41%, n ¼ 184), of which 81% were pe. 53 the incidence of unspecified venous thrombosis in two smaller studies was 25% (n ¼ 81) 54 and 69% (n ¼ 26). 55 in a larger cohort of patients (n ¼ 362), the incidence of thromboembolic events was 16.7% in icu patients and 6.4% across all covid-19 patients in general wards. 56 in all of these studies the incidence of vte is despite all patients having received, at least, a prophylactic dose of anticoagulants. there is clear evidence, from post-mortem lung pathology, of extensive thrombosis in the alveolar capillaries and small vessels in response to covid-19 infection. 24 the composition of these thrombi includes fibrin deposits, platelet aggregates, cd4 þ cell aggregates, and partially degenerated neutrophils (figure 1(4) ). fibrin deposition in response to inflammation is initially driven by elevated c-reactive protein (crp) and inflammatory cytokines. 57, 58 local tissue factor release, generating thrombin, coupled with elevated plasma concentrations of fibrinogen 59 results in deposition of fibrin which persists due to the concomitant suppression of fibrinolysis by crp-mediated release of plasminogen activator inhibitor-1 60 and thrombin activatable fibrinolysis inhibitor. 61 any other pro-coagulant and/or anti-coagulant factors that may be specifically influenced by sars-cov-2, thereby further contributing to the hypercoagulable state, are not yet known and, as most are synthesized in the liver, it may be unlikely that infection will alter their transcription. a comprehensive analysis of the plasma concentrations of coagulation factors in patients with covid-19, or indeed any viral infection, seems to be lacking at present from the literature. the presence of platelet-containing thrombi in the lungs of covid-19 patients indicates the involvement of other thromboinflammatory pathways that upregulate endothelial platelet recruitment and aggregation (figure 1(5) ). this is likely to be initiated by an increase in il-6, il-8, and tnf-a which stimulate the exocytosis of von willebrand factor (vwf) from weibel-palade bodies. 62 the vwf strings released unfold under rheological shear forces and capture platelets through the glycoprotein ib-ix-v complex, a process down-regulated by the protease adamts13. the synthesis of adamts13 is known to be inhibited by ifnc, il-4 and tnf-a and, through an as yet unknown mechanism, il-6 inhibits adamts13 activity at the endothelium. 62, 63 therefore, under inflammatory conditions, there is an amplification of vwf-mediated platelet capture. as an acute phase reactant, vwf (particularly ultra large vwf multimers) has long been associated with acute inflammation 64 and acute viral infection of the respiratory tract. 65, 66 it is also highly likely that vwf/adamts13 imbalance plays an important role in covid-induced thrombosis with some small studies already identifying vwf antigen and vwf activity in covid-19 patients as high as 600-800% of the normal range. 67, 68 notably, an imbalance in the vwf/adamts13 axis is an established risk factor for the incidence of ischemic stroke 69 and is already implicated in stroke complicating other viral infections. 70 the formation of platelet-rich thrombi could be further exacerbated by a local hyper-activation of platelets, the consumption of which would account for mild thrombocytopenia observed in many covid-19 patients and linked to higher rates of mortality. 71 platelet activation and aggregation is likely to be induced by the action of locally generated thrombin through par1/par4 signaling on platelets. the recruitment of platelets from the circulation may also be supplemented by local production of platelets by pulmonary megakaryocytes. these have been observed, in post-mortem lung pathology, actively producing platelets in the alveolar capillaries. 24 the significance of platelets in covid-19 pathology is highlighted by the possible therapeutic benefit of anti-platelet therapies. 72 importantly, platelet activation and subsequent degranulation may be an early contributor to the exacerbated immune/inflammatory response to sars-cov-2 infection through numerous mediators of leukocyte and endothelial function. 73 platelet recruitment of leukocytes to the developing thrombus would explain the notable presence of cd4 þ t cell aggregates 24 and neutrophil extracellular traps (nets), 74 both of which further contribute to both the proinflammatory and pro-coagulant environment. recently it has become apparent that stroke-causing thrombi are often rich in vwf, platelets, leukocytes and nets and that this composition is associated with tissue plasminogen activator resistance. 75 formation of thrombi with this composition requires the upregulation of multiple thromboinflammatory components, many of which (described above) are influenced by covid-19. this, together with the incontrovertible role of hypercoagulation in ischemic stroke pathology, 76 forms perhaps the strongest argument for a causative link between covid-19 and stroke. international journal of stroke, 15 (7) vasculitis and endothelial dysfunction emerging evidence suggests that covid-19 pathology may be considered, at least in part, a vascular disease and that the effects of sars-cov-2 on endothelial function may go beyond the release of tissue factor and vwf already described. 77 ace2 is expressed in venous and arterial endothelial cells, arterial smooth muscle cells, and pericytes. 4 a recent post-mortem case series has indicated that sars-cov-2 is capable of productively infecting and damaging endothelial cells across multiple tissue beds (figure 1(4) and (8)), although, the authors concede that these observations are not conclusive. 78 hyperactivation of the endothelium during viral infection is known to induce the loss of tight junctions, vessel permeability and, subsequently, pulmonary hemorrhage, and alveolar edema. 79 in the later stages of covid-19 progression, as the disease becomes more severe, it is possible that complement activation may also contribute to vasculitis. this mechanism has been observed previously in animal models of coronavirus infection 80 and pulmonary biopsies from a small number of covid-19 patients indicate the presence of complement activation. 81 in addition to inflammation of the vessel wall, complement may contribute to vascular dysfunction through the initiation of microvascular thrombosis. all of these processes could be expected to occur in the endothelium of multiple organs, including the brain, where alterations of the vascular environment are a key contributor to the development of ischemic brain injury. the possibility of sars-cov-2 neurotropism is intriguing. many other viruses, including coronaviruses, are capable of infecting cns related cell types including neurons, microglia, astrocytes, and oligodendrocytes. in covid-19 patients, cns infection may account for the high incidence of neurological manifestations (as high as 88% of severe cases 82 ) which include headaches, nausea, impaired consciousness, acute cerebrovascular disease, and seizures. 83 conversely, these manifestations may simply reflect the remote effects of systemic inflammation. a likely route of cns infiltration is through peripheral nerve terminals, particularly the olfactory bulb. in a humanized mouse model of sars-cov-1 infection, nasal inoculation was followed by dissemination of the virus from the olfactory epithelium to the axons of the olfactory bulb, through the pyriform cortex to the brain stem. 84 this may also explain the widespread incidence of anosmia occurring in covid-19 cohorts. there is a strong association between acute cns infection (e.g. meningitis) and the incidence of stroke 85 thought to result from vasculitis and a related hypercoagulant state. in the case of systemic thrombosis, an important distinction yet to be made is whether all thrombotic events are originating in a pro-coagulant micro-environment within the lungs or if there is a systemic procoagulant state facilitating in situ thrombus formation in the brain or embolism to the brain from elsewhere, such as the peripheral arterial or venous system (e.g. via patent foramen ovale). the latter is certainly possible through the presence of highly reactive ultra-large vwf multimers, hyper-activated platelets and upregulated cell adhesion molecules (through activation of the endothelium), all of which would be expected in the vasculature of the brain (figure 1(7) ). endothelial dysfunction, particularly vasoconstriction, may occur as a result of direct infection of either endothelial cells or smooth muscle cells (figure 1(8) ) and may enhance the shear-dependent formation of vwf-platelet aggregates in the smaller vessels (figure 1(10) ). neutrophils, circulating in high numbers and in an activated state, may contribute further to in situ thrombus formation in the brain through the formation of nets 86 (figure 1(7) ). the majority of strokes being reported in covid-19 patients are large vessel occlusions, 18 which is indicative of a thromboembolic source; however, in situ thrombosis cannot be ruled out. given the high incidence of myocardial injury (myocarditis, ischemia, pericarditis, etc.) associated with severe covid-19 disease/treatment, 87 secondary stroke in covid-19 patients may also have a cardioembolic source. is covid-19 directly contributing to stroke incidence? as approximately 65% of observed strokes in covid-19 patients are conventionally cryptogenic, 19 it may be tempting to prematurely assume a relationship; however, it is very important to emphasize that there is no direct evidence for a causal link between covid-19 and stroke. standardized case reporting and the application of bradford hill criteria will be essential in defining causality. 88 thrombotic and cerebrovascular complications are not uncommon in critically ill patients due to systemic inflammation, prolonged immobility, intermittent af, sedation, mechanical ventilation, and central catheter placement, but can be effectively prevented by international journal of stroke, 15 (7) prophylactic anti-coagulants. 89 this is not the case in covid-19 (and the previous sars outbreak) and a recent retrospective cohort study has suggested an incidence of stroke 7-8 times higher in patients hospitalized with covid-19 infection compared with those hospitalized by influenza, 90 supporting the possibility of a sars-cov-2-driven hyper-coagulant state. 17 platform trials of anticoagulant or immunomodulatory therapies in hospitalized patients with covid-19 present a unique opportunity for gaining insights into the causal role of inflammation and thrombosis in stroke risk. however, many of these trials focus on short-term outcomes and it is unclear whether there would be sufficient power to detect differences in stroke events, even if incident stroke was recorded as an a priori outcome measure. comorbidities that are common to both covid-19 and stroke (hypertension, diabetes, obesity, etc.) may explain, at least some, coincidence of the two pathologies. [91] [92] [93] obesity, in particular, is emerging as a prominent risk factor in the development of severe covid-19 disease and is generally associated with increased incidence and increased severity of respiratory viral infection. 94, 95 this is perhaps unsurprising given the existence of low grade inflammation in obese patients 96 which undoubtedly contributes to the initiation of ards. notably, the cytokine il-33 is persistently elevated in obese individuals and is capable of stimulating endothelial cells to release pro-coagulant tissue factor 97 which may expose them to more severe covid-19 disease and/or stroke. of course, not all covid-19 patients who go on to have strokes will have these comorbidities, and this is especially true of younger patients. 18 it is in these apparently healthy, young individuals that a causal link between covid-19 and cerebrovascular complications may be the most logical explanation, particularly in asymptomatic individuals with significant and undiagnosed inflammation. up until this point, we have discussed the incidence of stroke complicating covid-19 only in the context of the most severe and often critical cases as it does appear to be a delayed complication. it could, however, be argued that the strongest case for a causal link would be the incidence of stroke in individuals with sub-clinical sars-cov-2 infection, which has been identified in a number of cases. 18 the true extent of community infection is not known due to a failure, in most countries, to introduce widespread testing and contact tracing, although this is changing rapidly. it has been estimated that in china as many as 86% of cases were undocumented 1 and this is likely to be echoed in other epicenters of the outbreak and will become apparent when a reliable serological test becomes widely implemented. asymptomatic or mildly symptomatic individuals are also highly unlikely to undergo any kind of medical examination, especially in light of the imposed lockdown measures. therefore, we do not yet know the extent of systemic inflammation or the likelihood of aberrant coagulation in these mild or asymptomatic patients and whether or not they are at increased risk of stroke. an interesting study of infected individuals aboard the diamond princess cruise ship, one of the earliest clusters outside of china, has given some insight into the extent of asymptomatic infection within an isolated, and comprehensively tested population. 98 of the 454 confirmed infections on board, almost half were asymptomatic. follow-up chest imaging, performed on 76 of these asymptomatic individuals, revealed a 54% incidence of abnormal ct findings (mostly ground-glass opacities). this is not a trivial observation as such abnormalities are indicative of established infection and advanced inflammation. applied to the general population this could indicate that as many as 25% of all infected individuals may have undetected inflammation and may be at risk of thrombotic complications, including stroke. a proven, direct link between covid-19 and stroke may only arise when a vaccine is deployed which then results in a reduced stroke incidence in already at-risk groups, as is thought to be the case with the influenza vaccine. 99 recent meta-analyses have shown influenza vaccination is associated with a reduced risk of stroke; however, further prospective studies, particularly large, multicenter randomized controlled trials, are required to definitively show this link. until then, the mere possibility of a causal link will undoubtedly require some adaptation of current clinical practices to better manage their coincidence. this is likely to include changes to neurovascular imaging protocols, thrombolytic administration, and the application of mechanical thrombectomy. some of these proposed changes have been outlined in an international panel report published recently in ijs. 100 a better understanding of the mechanisms underlying a link between covid-19 and stroke will help to adapt these clinical practices further, particularly in regard to the use of thrombolytic, anticoagulant, and anti-platelet therapies. the sars-cov-2 pandemic is by no means the first viral infection to be linked to an increased incidence of stroke. research interest in this phenomenon has peaked upon the emergence of past epidemics, giving us some insight into possible mechanisms, but has dissipated as the threat is contained (as was the case with sars and mers). this represents a missed opportunity to gain valuable insight on the general link between infection and stroke. international journal of stroke, 15 (7) similarly, we may have missed the first opportunity to study the present pandemic as the number of new cases in the main epicenters of the outbreak begin to decline. studies of sars-cov-2 so far have been generated at a staggering rate, possibly at the expense of scientific rigor. many have been small, case series studies that could be viewed as less insightful than larger, collated clinical datasets. it is gradually becoming clear from the early epicenters of the outbreak (e.g., china, south korea) that sars-cov-2 has not yet been fully contained and, with any potential treatments or vaccine still months away, a second wave remains possible. in addition, the implications of any potential causal relationship between sars-cov-2 and stroke risk on the primary and secondary prevention of stroke is yet to be determined. finally, the impact of covid-19 combined with seasonal influenza on stroke risk remains uncertain. in the meantime, the research community should be preparing to employ large systematic clinical studies and establishing animal models of covid-19 to confirm the causative mechanisms by which stroke might occur. only then we will be prepared for the next viral threat and the cerebrovascular risk it may pose. searches were performed in pubmed using the search terms ''covid-19'', ''sars-cov-2'', ''covid-19, stroke'', ''sars-cov-2, stroke'', and ''viral infection, stroke''. searches were performed periodically from 13 april 2020 to 4 june 2020. search results were screened for relevance by multiple authors. expedited publications and those on pre-print servers were scrutinized by multiple authors for scientific rigor before inclusion. the author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: msv elkind will receive royalties from uptodate for a chapter related to covid-19 and stroke. the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was funded by medical research foundation fellowship (mrf-076-0004-rg-sout-c0756) awarded to k substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov-2) temporal dynamics in viral shedding and transmissibility of covid-19 sars-cov-2 cell entry 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doi: 10.1016/j.idc.2017.10.009 sha: doc_id: 267139 cord_uid: r8rg0iqq millions of children travel annually, whether they are refugees, international adoptees, visitors, or vacationers. although most young travelers do well, many develop a febrile illness during or shortly after their trips. approaching a fever in the returning traveler requires an appropriate index of suspicion to diagnose and treat in a timely manner. as many as 34% of patients with recent travel history are diagnosed with routine infections, but serious infections such as malaria, enteric fever, and dengue fever should be on the differential diagnosis due the high morbidity and mortality in children. millions of children travel annually, whether they are refugees, international adoptees, visitors, or vacationers. [1] [2] [3] [4] in 2015, the international tourism organization reported 1.2 billion overseas trips. 5, 6 although most young travelers do well, many develop febrile illnesses during or shortly after their journeys. 7 in a study of european children, 53% of all pediatric patients with travel-related infections were visiting friends and relatives (vfrs), 43.4% were tourists, and 2.4% were immigrants. 8 most illnesses are selflimited childhood infections that do not require subspecialist consultation. however, 28% of 24,920 ill american travelers sought care at travel clinics after returning home. 9 additionally, young children with fevers can present a diagnostic dilemma because they may not report symptoms and can be at risk for severe disease, such as malaria. as awareness of tropical illnesses rise in parents, such as the increase in multidrug-resistant bacteria worldwide or the emergence of epidemics with zika virus in south america, families may be more anxious about serious infections as an etiologic factor of fevers. approaching fevers in the returning traveler requires an appropriate index of suspicion to diagnose and treat the child in a timely manner. this article offers a framework on how to address these issues by discussing diseases based on geography, incubation period, and affected organ systems, as well as risk factors, diagnostic techniques, and resources. a thorough history is an important initial step when evaluating a pediatric traveler with a fever ( table 1) . discussing a detailed travel itinerary develops a timeline of exposures that can be unique to an urban or rural setting ( table 2) . many children receive vaccinations and/or antimicrobial prophylaxis, but reported adherence does not preclude an illness with a particular pathogen. up to 75% of travelers do not adhere to the recommended malaria prophylaxis. 10 many travel vaccines, including typhoid vaccine, provide only partial protection despite proper administration of these immunizations. 11 a medically complex individual may have sought care outside of the united states due to necessity or medical tourism, which can increase the risk of infection through body fluid exposures. multidrug-resistant pathogens can also be associated with health care exposure. up to half of hospitalized children in zimbabwe are colonized with extended spectrum beta lactamase producing enterobacteriaceae on admission to the hospital, 12 a problem that is increasingly seen worldwide. underlying medical conditions, such as asplenia or immunosuppression from chemotherapy, may predispose children to overwhelming infections and sepsis. refugee children from countries such as syria are susceptible to vaccine-preventable diseases such as polio due to infrastructure breakdown. 13 fever is a common and anxiety-provoking sign for parents that can be exacerbated by overseas travel. up to 34% of patients with recent travel history are diagnosed with routine infections. 3 of the 82,825 cases of infection in travelers from 1996 to 2011 reported to geosentinel, a worldwide data collection network on travel-related diseases, 4% of cases were considered to be life-threatening. 14 a study in swiss children showed that 0.45% of emergency room visits were due to travel-related morbidities with fever and gastrointestinal symptoms being the most common complaints in 63% and 50% of patients, respectively. 8 the temporality of travel to the onset of fever can offer important clues to the etiologic factors of fevers ( table 3) . because the causes and clinical outcomes associated with fevers in pediatric travelers vary from self-limited to deadly, a systems-based approach can lead to prompt diagnosis and treatment that evaluates for the most likely and serious diseases early in the illness course. according to geosentinel, 91% of patients with an acute, life-threatening illness will present with fever. 14 there are a broad range of potential tropical infections, including malaria, dengue fever, and enteric fever. the incidence of emerging infections such as zika virus and chikungunya are not yet known. in both adults and children, pneumonia, sepsis, meningococcemia, and urinary tract infections that were acquired at home or overseas should be on the differential diagnosis. the initial workup of a febrile child without a clear source will be based on the history, physical examination, and risk factors but commonly includes a complete blood count, liver function tests, creatinine, urinalysis, and blood cultures. 1, 3 malaria smears are also frequently helpful. other tests to consider include serologies for dengue fever scaggs huang & schlaudecker or other potential etiologic agents, polymerase chain reaction for zika virus or other pathogens, chest radiographs, and cultures of the urine and stool. patients with altered mental status may require head imaging and lumbar puncture. the most common and concerning causes of fever in a returning pediatric traveler are highlighted next. [50] [51] [52] fever in the returning traveler plasmodium falciparum malaria is one of the most common tropical infections. approximately 15% to 20% of all imported malaria cases are diagnosed in the pediatric population in industrialized countries each year. 3 malaria is transmitted via the nocturnal-feeding anopheles genus of mosquito. children who are vfrs are more likely to become infected with malaria than traditional tourists. 3 nonimmune children are also susceptible to severe malaria from other malaria strains such as plasmodium vivax 15 and many young patients can present with atypical symptoms such as abdominal pain and vomiting. 16 older children may present with paroxysmal fever, fatigue, myalgias, headache, abdominal pain, back pain, hepatosplenomegaly, and hemolytic anemia. additionally, severe malaria is more common in children after the first month of travel due to the incubation period of p falciparum (7-90 days), especially in those who visited sub-saharan africa. 17, 18 overall, sub-saharan africa is one of the most common geographic regions for acquisition, comprising 71.5% of cases according to a geosentinel study of travelers migrating or returning to canada from 2004 to 2014. 19 malaria should remain on the differential diagnosis for up to a year in an acutely ill, febrile child after travel to an endemic area where p vivax and p ovale strains are present. 17 interestingly, 20% of malaria cases can be acquired during trips as short as 2 weeks with less utilization of pretravel services being a contributing factor. 19 a minimum of 3 thick and thin blood smears must be performed before malaria can be excluded, preferably collected during febrile episodes. the specificity of blood smears is high but the sensitivity can be low depending on the experience of the individual interpreting the slides. 17 rapid diagnostic tests that detect specific proteins or lactate dehydrogenase are alternatives for diagnosis at medical centers with limited experience in microbiologic evaluation for malaria. 20 the result should be confirmed, however, through the state public health department. in general, a febrile child without a localizing source or splenomegaly, thrombocytopenia, or indirect hyperbilirubinemia, in addition to exposure to an endemic area, should be presumptively approached as having malaria until an alternative diagnosis can be made. 21 treatment of malaria is well-established by the centers for disease control and prevention (cdc) guidelines. children with acidosis, hypoglycemia, hyperparasitemia, end-organ dysfunction, and severe anemia meet the criteria for severe malaria and require prompt administration of parenteral medication. there is a growing body of evidence that artesunate may reduce mortality compared with quinidine and is becoming more common as first-line therapy in pediatric patients. 22, 23 artesunate must be obtained through the cdc malaria hotline (1-770-488-7788) because it is not routinely available in the united states. 24 quinidine may be initiated until the medication arrives. completion of therapy with an oral regimen for uncomplicated chloroquine-resistant p falciparum, such as atovaquone-proguanil, can be offered when the child is able to tolerate the medications and the parasite burden has decreased to less than 1%. severe disease is less common in p vivax and p ovale and infection can be treated with chloroquine or hydroxychloroquine in most areas outside of indonesia and papua new guinea. enteric fever accounts for 18% of the 3655 cases with life-threatening tropical diseases reported to geosentinel. most recorded cases were from the indian subcontinent and in vfrs. 1 infection with salmonella typhi and salmonella paratyphi are clinically indistinguishable with fever, abdominal pain, nausea, vomiting, myalgias, and arthralgias. diarrhea is greater than 2.5 times more common in infants than older children or adults, 25 although constipation can also be seen. patients can exhibit a fever in the returning traveler typhoid mask with dull features and confusion, as well as a stepladder fever progression with rising temperatures over time in untreated individuals. relative bradycardia and rose spots are also classic signs. 25 complications such as gastrointestinal bleeding are more common in young children who have been ill for 2 weeks or more. 1 transmission is fecal-oral, and humans, especially adults, may be chronic carriers. diagnosis of enteric fever is confirmed through cultures. the most sensitive sterile site is bone marrow (80%-95%). blood culture has the highest yield during the first week of illness (70%), and stool cultures are more sensitive as the duration of illness increases. 26 stool studies should be performed on all fellow travelers, and they must be monitored for signs of illness. other abnormal laboratory findings include transaminitis and a normal or decreased white blood cell count. the antimicrobial of choice for treatment varies based on the area in which the infection was acquired because multidrug resistance is increasing. empiric treatment with ceftriaxone or fluoroquinolones is typically recommended. strains in latin america and the caribbean can be susceptible to ampicillin and trimethoprimsulfamethoxazole. south and southeast asian serovars more frequently require azithromycin or cefixime. 27, 28 children with multidrug-resistant strains have more complications such as myocarditis and shock than children infected with susceptible strains but case fatality is similar (1.0% vs 1.3%, respectively). 29 relapse of infection can occur despite appropriate therapy, with the highest mortality in young children (6%). 29 dengue remains an important cause of fever in travelers returning from all tropical regions except africa. 30 the prevalence is rising, even in the united states, with 50 to 100 million global cases reported yearly and 22,000 deaths, primarily in children. 31 risk factors are dissimilar from those for malaria because transmission occurs in urban areas during the daytime due to the vector aedes aegypti, whereas malaria transmission is more common in rural areas from dusk to dawn with the anopheles species mosquito. 32 some patients may be asymptomatic, whereas others have hemorrhagic fever and shock. the illness presents as 3 distinct phases: (1) febrile phase over 3 to 7 days characterized by myalgias, headache, retroorbital pain, and rash; (2) critical phase of 24 to 48 days with plasma leakage; and (3) convalescent phase. 32 a rising hemoglobin and gallbladder wall thickening due to increased vascular permeability suggests the development of severe dengue in children. repeat infections with a different strain may lead to more severe disease. 31 serologies are most commonly used for diagnosis, although some rapid diagnostic tests are available. in cases in which infection is unclear, it may be helpful to repeat serologies 2 weeks after initial testing to monitor for an increase in titers. other common laboratory findings include leukopenia and thrombocytopenia. 33 treatment consists of hydration and avoidance of salicylate-containing products to decrease the risk for bleeding. 32 children who develop severe dengue with hemorrhage and shock may require blood products. no antivirals or vaccines are currently available. in recent years, arboviral illnesses transmitted via infected aedes aegypti mosquitos have caused epidemics of zika virus and chikungunya in south america. a european study of travelers returning from brazil in 2013 to 2016 reported that of the 29% of patients with travel-related complaints, 6% had dengue fever, 3% had chikungunya, and 3% had zika virus infection. 34 the prevalence of yellow fever, which is seen throughout low-resource settings and shares the same vector, has remained stable. 35 these infections are difficult to distinguish clinically with fever, retroorbital pain, conjunctivitis, and myalgias. knowledge on perinatal infection with zika and the neurodevelopmental sequelae of affected infants is rapidly evolving. 36 a canadian study found that 5% of travelers developed neurologic complications such as guillain-barre syndrome with zika, suggesting there is much to learn with this disease in nonperinatally acquired infections. 37 at this time, treatment is primarily supportive. additional tropical diseases associated with fevers are outlined in table 4 . vomiting and diarrhea are common complaints in returning travelers. up to 40% of children less than 2 years of age may develop diarrhea, with 15% requiring medical services. 38 fevers, nausea, and vomiting can be seen with norovirus that occurs worldwide and is frequently associated with contaminated food and water on cruise ships. 39 rotavirus, however, is one of the most frequent causes of diarrheal illnesses worldwide and is a common cause of infant mortality in low-resource settings. 5 the hepatitides present with a broad range of disease from mild abdominal pain and vomiting to fulminant liver failure, although serious complications are uncommon in pediatric travelers. 40 community-acquired clostridium difficile is uncommon in children but infection should be considered if the patient received recent antimicrobials. 41 geosentinel data reported that 2% of patients diagnosed with clostridium difficile after travel were 10 to 19 years of age. 42 there are many other causes of both febrile and nonfebrile gastrointestinal illness in children ( table 5) . in the pediatric population, common respiratory infections may be seen on return from international trips including pharyngitis, sinusitis, otitis, and pneumonia from pathogens commonly seen in the united states, such as streptococcus pneumoniae and rhinovirus. 4, 43 local epidemiology of infections can be helpful in diagnosis and management and is available through the cdc. in some tropical regions, influenza may occur throughout the year and should hence remain on the differential for patients who warrant treatment with oseltamivir. 44 mycobacterium tuberculosis is an important etiologic factor of lower respiratory tract disease worldwide and should be considered in children with risk factors or who do not recover with antimicrobials for bacterial pneumonia. 26 of note, children younger than 3 years of age are more likely to present with miliary tuberculosis or neurologic involvement than adult patients. there are also many other less common causes of febrile respiratory tract infections ( table 6) . children who present with dysuria, hematuria, and fevers may require urinalysis and culture to evaluate for urinary tract infection and/or pyelonephritis. gross hematuria with the passage of clots in an afebrile child with exposure to freshwater in africa, the middle east, china, and southeast asia should be tested for the helminth parasite from the genus schistosoma via serologies or microscopic identification of eggs in stool. 45 praziquantel is the treatment of choice and may improve anemia and nutrition in some children. 46 patients who may have early disease or a high parasite burden may require a repeat treatment. 45 children who are at risk for sexual abuse and adolescents should undergo testing for sexually transmitted infections such as chlamydia trachomatis and neisseria gonorrheae. rashes are a source of concern for parents without the context of travel and may be even more worrisome after going abroad. the differential diagnosis includes typical childhood illnesses, such as roseola or staphylococcal cellulitis, in addition to tropical infections. a study of canadian travelers from 2009 to 2012 found that cutaneous larva migrans (13%) and skin and soft tissue infections (12.2%) were some of the most common infectious dermatologic complaints among tourists. 47 in countries where vaccination rates are low, varicella zoster virus or rubella may cause disease, especially in young children who have not completed their immunization series. measles remains an important risk, with tourists comprising 44% of the 94 cases reported to geosentinel from 2000 to 2014, and 13% of patients being younger than 18 years of age, although this may represent underreporting due to the surveillance system's primarily adult focus. 48 petechiae on the extremities in an illappearing child may indicate a serious systemic process such as meningococcal or rickettsial infection. there are many other infections with primarily dermatologic manifestations that may not cause fevers ( table 7) . 49 as the numbers of children who travel abroad continues to increase, clinicians need to remain up-to-date on potential etiologic factors for febrile illnesses on families' return home. after ruling out life-threatening disorders that can be acquired locally or internationally, physicians are able to develop a focused diagnosis and management plan best suited to the patient's clinical picture. there is a growing body of resources to assist clinicians, such as the cdc (www.cdc.gov/travel/) and geosentinel (www.istm.org/geosentinel) for data on epidemiology, geography, and other risk factors. in the future, physicians will need to be prepared to deal with the global epidemic of antimicrobial drug resistance, evolving epidemics and pandemics caused by emerging pathogens, reemerging infections due to vaccine hesitancy or international conflicts, and medical tourism in both healthy and medically complex children. fever after international travel etiology and outcome of fever after a stay in the tropics imported malaria in children: a review of clinical studies evaluation of the sick child following travel to the tropics estimate of worldwide rotavirusassociated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis international tourist arrivals up 4% reach a record 1.2 billion in 2015 spectrum of disease and relation to place of exposure among 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malaria? south east asian quinine artesunate malaria trial (seaquamat) group. artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial artesunate versus quinine in the treatment of severe falciparum malaria in african children (aquamat): an open-label, randomised trial artesunate is available to treat severe malaria in the united states an appraisal of the clinical features of pediatric enteric fever: systematic review and meta-analysis of the age-stratified disease occurrence evaluation and management of illness in a child after international travel enteric (typhoid) fever in travelers azithromycin versus ceftriaxone for the treatment of uncomplicated typhoid fever in children systematic review of the global epidemiology, clinical and laboratory profile of enteric fever seasonality, annual trends, and characteristics of dengue among ill returned travelers dengue in travelers arboviral and other illnesses in travellers returning from brazil yellow fever vaccines and international travelers clinical impact of non-congenital zika virus infection in infants and children surveillance report of zika virus among canadian travellers returning from the americas incidence and clinical features of traveler's diarrhea in infants and children traveler's diarrhea: updates for pediatricians epidemiology of travel-associated and autochthonous hepatitis a in austrian children analysis of clostridium difficile associated diarrhea in pediatric patients with antibiotic-associated diarrhea clostridium difficile infection in returning travellers bordetella pertussis infections in travelers: data from the geosentinel global network update: influenza activity-united states and worldwide schistosomiasis in travellers and migrants single dose metrifonate or praziquantel treatment in kenyan children. ii. effects on growth in relation to schistosoma haematobium and hookworm egg counts dermatoses among returned canadian travellers and immigrants: surveillance report based on cantravnet data measles in the 21st century, a continuing preventable risk to travelers: data from the geosentinel global network dermatologic conditions of the ill returned traveler: an analysis from the geosentinel surveillance network b-virus and free-ranging macaques fever on return from abroad. in: acute medicine -a practical guide to the management of medical emergencies approach to fever in the returning traveler the yellow book: health information for international travel key: cord-288494-6qybdxc4 authors: liao, qiaohong; ip, dennis k m; tsang, tim k; cao, bin; jiang, hui; liu, fengfeng; zheng, jiandong; peng, zhibin; wu, peng; huai, yang; lau, eric h y; feng, luzhao; leung, gabriel m; yu, hongjie; cowling, benjamin j title: a clinical prediction rule for diagnosing human infections with avian influenza a(h7n9) in a hospital emergency department setting date: 2014-08-05 journal: bmc med doi: 10.1186/s12916-014-0127-0 sha: doc_id: 288494 cord_uid: 6qybdxc4 background: human infections with avian influenza a(h7n9) virus are associated with severe illness and high mortality. to better inform triage decisions of hospitalization and management, we developed a clinical prediction rule for diagnosing patients with a(h7n9) and determined its predictive performance. methods: clinical details on presentation of adult patients hospitalized with either a(h7n9)(n = 121) in china from march to may 2013 or other causes of acute respiratory infections (n = 2,603) in jingzhou city, china from january 2010 through september 2012 were analyzed. a clinical prediction rule was developed using a two-step coefficient-based multivariable logistic regression scoring method and evaluated with internal validation by bootstrapping. results: in step 1, predictors for a(h7n9) included male sex, poultry exposure history, and fever, haemoptysis, or shortness of breath on history and physical examination. in step 2, haziness or pneumonic consolidation on chest radiographs and leukopenia were also associated with a higher probability of a(h7n9). the observed risk of a(h7n9) was 0.3% for those assigned to the low-risk group and 2.5%, 4.3%, and 44.0% for tertiles 1 through 3, respectively, in the high-risk group. this prediction rule achieved good model performance, with an optimism-corrected sensitivity of 0.93, a specificity of 0.80, and an area under the receiver-operating characteristic curve of 0.96. conclusions: a simple decision rule based on data readily obtainable in the setting of patients’ first clinical presentations from the first wave of the a/h7n9 epidemic in china has been developed. this prediction rule has achieved good model performance in predicting their risk of a(h7n9) infection and should be useful in guiding important clinical and public health decisions in a timely and objective manner. data to be gathered with its use in the current evolving second wave of the a/h7n9 epidemic in china will help to inform its performance in the field and contribute to its further refinement. electronic supplementary material: the online version of this article (doi:10.1186/s12916-014-0127-0) contains supplementary material, which is available to authorized users. human infections with novel avian-origin influenza a (h7n9) virus were first identified in march 2013, [1, 2] mainly in the eastern provinces of china [3] . a total of 131 human infections, dominated by severe illness and mortality, were confirmed in the spring wave in mainland china from march through may 2013 [4, 5] . with the adoption of suitable public health measures including closure of live poultry markets, few cases were identified over the summer months [6] . however, h7n9 has resurged in this winter, 2013-2014 [7] . in the context of preparing and responding to further waves of this evolving epidemic, the importance of early detection, diagnosis, isolation and reporting of a(h7n9) infections has been repeatedly emphasized [8] . accurate and objective risk prediction can help physicians to guide clinical management and inform triage decisions * correspondence: yuhj@chinacdc.cn † equal contributors 1 for optimizing the utilization of valuable clinical and public health resources that may easily be overwhelmed during an epidemic. however, no simple and reliable decision tool has yet been available for predicting the risk of a (h7n9) influenza in an objective and timely manner. our objective in this study was to develop a clinical prediction rule that would accurately identify patients with a(h7n9) influenza on their first presentation to a hospital emergency setting and to evaluate the predictive performance of this rule. we analyzed two databases that contained clinical and basic laboratory data from two groups of patients presenting similarly with acute respiratory infections to hospitals in china, including 121 laboratory-confirmed a(h7n9) cases and 2,603 patients who suffered from acute respiratory infections other than a(h7n9) influenza. outpatient clinics or emergency departments in hospitals represent a typical first step for patients with acute respiratory infections in china to present to the healthcare system, owing to the coverage of national health insurance programs and the lack of standalone primary healthcare clinics in either the public or private sector as an alternative [9] . the a(h7n9) database consisted of patients who presented clinically with symptoms of acute respiratory infection to hospitals in different provinces of china and were subsequently confirmed to have a(h7n9) virus infection between march and may 2013. in china, all laboratory-confirmed infections with a(h7n9) viruses are reported to the chinese center for disease control and prevention (china cdc, beijing, china) through a national surveillance system [10] . diagnostic confirmation of a(h7n9)virus infection was done either by the isolation of a(h7n9) virus or a positive real-time reversetranscription polymerase chain reaction (rt-pcr) assay for a(h7n9) in a respiratory specimen [10] . case definitions, surveillance for identification of influenza a(h7n9) cases, and laboratory test assays are described in previous reports [10] . the second database consisted of patients who presented similarly with acute respiratory infections to hospitals in jingzhou city, hubei province of china from 4 january 2010 through 30 september 2012. their clinical details were captured by a surveillance program for severe acute respiratory infection (sari) conducted by china cdc in four surveillance hospitals in jingzhou during that period [11] . all patients admitted to these four surveillance hospitals were screened by either a nurse or physician. a patient was defined as having sari if they had on their clinical presentation an elevated temperature (rectal or axillary temperature ≥37.3°c) and at least one sign or symptom of acute respiratory illness, including cough, sore throat, tachypnea, difficulty breathing, abnormal breath sounds on auscultation, sputum production, hemoptysis, chest pain or chest radiograph consistent with pneumonia. nasopharyngeal swabs were collected from these patients and tested for seasonal influenza and avian influenza h5n1 viruses by rt-pcr. as these samples were all collected before the first h7n9 human case was identified, they were not tested for a(h7n9) virus. for patients included in either of these databases, clinical information on the medical history and physical examination findings on their initial clinical presentation was abstracted retrospectively from their original medical records using a standard data abstraction sheet by trained nursing and medical officers at each hospital. patients younger than 14 years of age from both databases were not included in this study, as there are few patients <14 years of age with confirmed a(h7n9) virus infection, and their presenting symptoms, the approach of symptom ascertainment, overall clinical course and disease severity, disease epidemiology, and even health care seeking pattern and pathway were generally very different from that in adults and elderly persons [3] [4] [5] . additional information collected included age, sex, history of any poultry exposure, including exposure to live poultry markets within two weeks of symptom onset, influenza vaccination history, smoking history, pregnancy, history of underlying medical conditions, height (m), weight (kg) and clinical symptoms. the results of investigations performed on presentation included chest radiography (signs of consolidation and pneumonia), simple hematologic blood analysis including hemoglobin level (hgb) and leukocyte count (wbc), and c-reactive protein (crp). because we did not have sufficiently complete data (>60%) on some laboratory determinations for the entire sample (including platelet count, other serum biochemistry tests and clotting profile), we excluded these determinations from the analysis for all patients. the collection of data from confirmed a(h7n9) cases was determined by the chinese national health and family planning commission as part of public health investigations of emerging influenza outbreaks and was exempted from institutional review board assessment. collection of data of sari cases was approved by the ethical review committee of the china cdc. therefore, informed consents from the cases were not required. a two-step regression model was used to develop the prediction rule, so as to simulate the decision making process in the setting of a clinic or hospital emergency room where a patient first presents [12, 13] . in step 1, basic variables obtainable from the medical history and physical examination were employed to identify a subgroup of patients who were more likely to have a(h7n9) and, therefore, needed additional investigation and workup. variables used in this stage included age group (<60 years, ≥60 years), sex, poultry exposure history, influenza vaccination history, smoking history, and history of underlying chronic diseases, pregnancy, major presenting symptoms and physical findings. in step 2, simple radiologic and laboratory variables were added to significant predictors from step 1 (those having a p-value less than 0.05) to further refine the identification of subgroups having higher risk for a(h7n9). we used a univariate logistic regression model to identify significant (p ≤ 0.05) predictors of a final diagnosis of a(h7n9) and then entered them into a multivariable logistic regression model with backward selection. we removed variables that had a p-value greater than 0.05. interaction terms were tested as candidate variables, but none of these terms entered the final models. we derived each prediction rule using all available information on patients. we used multiple imputations [14, 15] (20 imputations) in the derivation process to make the most of all available non-missing data while preserving the uncertainty from the missing data in the results [16, 17] [see additional file 1: appendix]. a score-based prediction rule for a final diagnosis of a (h7n9) was then developed for each step from the final logistic regression equations using a regression coefficientbased scoring method [13] . a simple integer-based point score was assigned for each predictor variable, which was calculated by dividing the corresponding β-coefficients by the absolute value of the smallest coefficient in the final model and rounding up to the nearest integer. the overall risk score for each patient was calculated by adding the scores for each component together [18] . aiming to be used as a screening tool to capture as many of the a/h7n9 cases as possible, a cutoff was specified with a priori sensitivity of 0.99 in step 1 and 0.95 for the overall model (steps 1 and 2). total risk scores above the cutoff threshold were then categorized in tertiles as a risk prediction rule for ease of clinical implementation. performance of the risk prediction rule in predicting a(h7n9) infection was examined by sensitivity, specificity, likelihood ratios for both positive and negative test results, and area under the receiveroperating characteristic (roc) curve [19] . calibration was evaluated by using the hosmer-lemeshow chi-square statistic (p > 0.05 for all models) [20] . regression models were tested for possible overfitting by using linear shrinkage estimators [13, 21] . the prediction rule was internally validated with samples of the same size resampled with replacement from the original derivation data set using the bootstrap method [22] . the model was refitted as the original model derivation process on these bootstrap samples for 1,000 iterations [13, 21] to determine the degree of performance deterioration to be expected when applied on an independent sample of patients [21] . we also estimated the optimism-corrected estimates to correct for the absolute magnitude of bias for each performance index [23] . we performed all analyses with r software, version 3.0.1. as of the end of may 2013, 131 confirmed a(h7n9) cases were officially reported in mainland china. among these patients, 121 patients of 14 years of age or older presenting with acute respiratory infection to an emergency department and requiring hospitalization for medical reasons were included in this study [5] . two cases younger than 14 and eight cases having mild disease and confirmed by routine testing through sentinel influenzalike illness surveillance were excluded from the present study [9] . during the surveillance period from 4 january 2010 through 30 september 2012, 90,890 patients had been hospitalized in the four surveillance hospitals in jingzhou city. among these, 25,406 (28%) patients met the sari case definition within 24 hours of hospital admission. ninety percent (22, 777) were <14 years of age and excluded from the present study. among the 2,603 included patients, 2,310 (89%) had a nasopharyngeal swab specimen collected, and 430 (19%) tested positive for influenza viruses by rrt-pcr, including 258 (60%) with influenza a(h3n2), 36 (8%) with a(h1n1) pdm09 and 136 (32%) with influenza b. table 1 shows the demographic and clinical characteristics of these two groups of patients on presentation to the emergency hospital. on univariate analysis, factors that were associated with an increased risk of a(h7n9) infection included older age (≥60 years), male sex, history of poultry exposure, smoking history, history of underlying medical conditions, fever, cough, haemoptysis, shortness of breath and diarrhea (table 1 ). in step 1 of the clinical prediction rule, male sex and history of poultry exposure were independently associated with a final diagnosis of a(h7n9) on multivariable analysis. the presence of three respiratory symptoms, namely fever, haemoptysis and shortness of breath, were also independently associated with a final diagnosis of a(h7n9) ( table 2 ). other factors became insignificant (age group, underlying medical illnesses, cough and diarrhea) and were not retained. fifty-eight percent of the cohort with a total score less than the threshold of 43 was assigned to the low-risk group and did not proceed to step 2 [see additional file 2: figure s1 ]. in step 2, two additional laboratory findings including pneumonia/consolidation on chest radiograph and leukopenia (wbc <4,000/μl) were each independently associated with an increased risk of a(h7n9). on the other hand, finding of leukocytosis (wbc >11,000/μl), or an abnormally low (hgb < 12 g/dl) or high hemoglobin level (hgb ≥16 g/dl) were each independently associated with a decreased risk of a(h7n9). all of the previous five factors entered in step 1 had remained statistically significant after adding these two factors. no statistical evidence of overfitting, as demonstrated by linear shrinkage estimation (shrinkage factor, 0.981 (95% confidence interval (ci), 0.976 to 0.984) for step 1 and 0.967 (95% ci, 0.960 to 0.972) for step 2), was seen in either multivariable regression model. for the ease of use in a setting of a clinical consultation, the magnitude of association of each of these factors with a(h7n9)virus infection was quantified by a point scoring system as shown in table 3 . a total score of 68 or greater would indicate the presence of a high risk for a(h7n9) infection, with a prespecified sensitivity of 95% overall. forty-five percent of patients considered in step 2 were further assigned to the low-risk category [see additional file 2: figure s1 ]. the magnitude of the scores had good diagnostic utility. when stratified by tertiles with the two cut-points of 70 and 90, a gradation with increasing level of risk for a (h7n9) infection was demonstrated. the corresponding risk of a(h7n9) infection was 0.3% (95% ci, 0.0% to 0.6%) for those assigned to the low-risk group (in steps 1 or 2), 2.5% (95% ci, 0.5% to 4.5%) for tertile 1 (risk score, 68 to 70), 4.3% (95% ci, 2.2% to 6.4%) for tertile 2 (risk score, 71 to 90) and 44.0% (95% ci, 37.4% to 50.8%) for tertile 3 (risk score >90) in the high-risk group. a similar gradation of risk was also observed in the validation analysis ( figure 1 ). this clinical prediction rule achieved good discriminative ability. it gave a sensitivity of 0.93 and a specificity of 0.80 (optimism-corrected estimates) in the derivation process, which were broadly maintained, respectively, at 0.96 and 0.75 in the bootstrap internal validation process (table 4 ). on the other hand, the optimism-corrected area under the roc curve was 0.96 from both the derivation and the internal validation processes (figure 2 ). likelihood ratios for a positive result with an assignment to the highrisk group after step 2 were moderately strong at 4.62 and 3.85 for the derivation and internal validation processes, respectively. likelihood ratios for a negative result with assignment to the low-risk group after step 1 or step 2 were 0.090 and 0.056 for the derivation and internal validation processes, respectively. our study presents a decision rule for objectively predicting a(h7n9) infection in adult patients presenting with severe respiratory illness. factors of particular importance in the prediction rule, including poultry exposure history, fever, shortness of breath and leukopenia, agreed generally with findings reported from previous epidemiological and clinical studies [4] . we had chosen the model with the best performance in terms of both the high sensitivity and area under the roc curve, which were also maintained in the validation samples, to identify patients having a high risk for the infection at their initial clinical presentation so as to optimize resources during an epidemic. as generally recognized by previous reports, most laboratory-confirmed cases of a(h7n9) have had a high risk of disease progression and fatality [3, 5] . early initiation of antiviral treatment and provision of a suitable level of intensive care have been identified as important factors in determining the final outcome of patients hospitalized with a(h7n9) virus infection [24] . our decision tool allows an initial risk assessment to be performed by frontline physicians in a setting where simple laboratory and radiographic examination may not be readily available, based only on simple information obtainable from the history and physical examination. in a setting with greater resources, the risk estimation of those deemed to have a non-trivial risk at step 1 could be further refined by the availability of simple radiographic and laboratory testing results. the scoring system also helps to categorize those being predicted as high-risk into different risk strata to facilitate further clinical decision-making (including the need for further work up, admission decision and ward allocation, initial treatment regimen, level of care and monitoring, and so on) before a definitive diagnosis based on rt-pcr can be available, often at a much later time. despite the current belief that a(h7n9) virus may not be readily transmitted from person to person, [3, 10] the existence of limited person-to-person transmission in a close contact setting [25] also carries an implication for this risk stratification tool to better inform isolation decision and practice. depending on resource availability and surge capacity, patients assigned to different risk groups may need to be handled differently. generally, however, patients assigned to the low-risk group in either steps 1 or 2 should have little risk implication to justify their admission at that particular juncture either for monitoring or hospitalized care unless having other indications for admission. this strategy can help to reduce the demands on inpatient beds and testing capacity. on the other hand, persons having a total risk score exceeding the threshold (≥68) in step 2 should be considered for admission for further assessment and possible initiation of treatment. the provision of different levels of monitoring and treatment to such patients could be guided by the magnitude of the total risk score, which is predictive of the eventual risk of confirmed a(h7n9) infection. allocation of isolation facilities including individual negative-pressure isolation rooms may also be informed by the individual risk score in case there is any enhancement in human-to-human transmissibility. our study included all cases of clinically confirmed a (h7n9) infection that presented with a picture of acute respiratory infection in a hospital setting in the first epidemic in china from march through may. our control consisted of a suitable sample of patients who clinically presented in a comparable setting within the same geographical area and were captured by a large-scale surveillance network, in a period immediately prior to the a (h7n9) epidemic. although controls had not been directly tested for a(h7n9) virus, their recruitment in a period during which human a(h7n9) infection was absent or at least very unlikely [26] should have helped to avoid their potential contamination by unascertained a(h7n9) cases. however, our study does suffer from a number of potential limitations. first, as the h7n9 positive cases and the control patients were not gathered from exactly the same setting, subtle biases may still be introduced by potential variability in the degree of exposure ascertainment or data documentation in the two different settings. although this problem should have been partly addressed by excluding any variables that were not measured or were generally missing from either group, some measured variables may have been more thoroughly ascertained for a(h7n9) cases, especially in an epidemic setting. second, a small proportion of all confirmed a(h7n9) cases were either asymptomatic or only mildly symptomatic and had not been hospitalized [9] . because of the very different clinical picture, our prediction rule may not be applicable for assessing the risk of a(h7n9) infection in this group of patients. third, as our study had already included almost all confirmed cases of a(h7n9) reported during the initial epidemic in china, the decision rule could only be internally validated by bootstrapping but lacked a suitable sample for external validation. as a result, the actual performance and utility of this prediction rule in future epidemics remains to be determined. ideally, our rule can be further validated in the current evolving second obtained from internal validation by using a bootstrap analysis in which the cohort was resampled 1,000 times with replacement.likelihood ratio for a positive test result refers to the likelihood of assignment to the high-risk group after step 2. likelihood ratio for a negative test result refers to the likelihood of assignment to the low-risk group after steps 1 or 2. ci, confidence interval; roc, receiver-operating characteristic. wave of epidemic in chinawithin a setting where all cases presenting with acute respiratory infection (ari) can be captured and ascertained with a definitive testing for a/h7n9 irrespective of their initial risk stratification status. this can inform the assessment of its performance in the field and contribute to its further refinement. finally, as the prediction rule was derived mainly based on data of the a/h7n9 virus with little or no humanto-human transmission potential, its performance may become very different should the virus acquire the ability to do so. other factors that may affect performance of the model in future waves of the evolving epidemic may include variation in disease severity, changing risk perception and health care seeking behavior. while the prediction of absolute risk for having a(h7n9) infection could be affected by variation in local factors, robustness of the model for separating higher from lower risk should probably be preserved. when being applied in the field, frontline physicians should remain alert concerning the potential limitations associated with practice guidelines and our decision rule should only supplement, but never supersede, the physician's judgment in equivocal or borderline cases. human infection with a novel avian-origin influenza a (h7n9) virus compiling of comprehensive data of human infections with novel influenza a (h7n9) virus comparative epidemiology of human infections with avian influenza a h7n9 and h5n1 viruses in china: a population-based study of laboratory-confirmed cases clinical findings in 111 cases of influenza a (h7n9) virus infection human infection with avian influenza a h7n9 virus: an assessment of clinical severity effect of closure of live poultry markets on poultry-to-person transmission of avian influenza a h7n9 virus: an ecological study human infection with avian influenza a(h7n9) virus re-emerges in china in winter 2013 novel h7n9 influenza virus shows low infectious dose, high growth and efficient contact transmission in the guinea pig model detection of mild to moderate influenza a/h7n9 infection by china's national sentinel surveillance system for influenza-like illness: case series preliminary report: epidemiology of the avian influenza a (h7n9) outbreak in china the substantial hospitalization burden of influenza in central china: surveillance for severe, acute respiratory infection, and influenza viruses a prediction rule to identify low-risk patients with community-acquired pneumonia hospital authority sars collaborative group: a clinical prediction rule for diagnosing severe acute respiratory syndrome in the emergency department multiple imputation: a primer regression modeling strategies: with applications to linear models, logistic regression, and survival analysis statistical analysis with missing data multiple imputation after 18+ years presentation of multivariate data for clinical use: the framingham study risk score functions the meaning and use of the area under a receiver operating characteristic (roc) curve applied logistic regression predicting mortality among patients hospitalized for heart failure: derivation and validation of a clinical model an introduction to the bootstrap. chapman & hall/crc pr multivariable prognostic models: issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors clinical, virological, and histopathological manifestations of fatal human infections by avian influenza a(h7n9) virus probable person to person transmission of novel avian influenza a (h7n9) virus in eastern china, 2013: epidemiological investigation serologic study for influenza a (h7n9) among high-risk groups in china submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank staff members of the bureau of disease control and prevention and health emergency response office of the national health and family planning commission and provincial and local departments of health for providing assistance with administration and data collection; staff members at county-, prefecture-, and provincial-level cdcs in the provinces where human a(h7n9) cases occurred for providing assistance with field investigation, administration and data collection. the views expressed are those of the authors and do not necessarily represent the policy of the china cdc. our prediction rule currently represents an important evidence-based decision tool for the triage of suspected cases of a(h7n9) infection when they first present clinically in an emergency department or primary care settings. this decision tool will be most useful in an evolving epidemic when the health system's surge capacity could be overwhelmed by the number of patients seeking care. with the current re-emergence of the a(h7n9) epidemic as the second wave in china, it would be a very timely and practical tool for helping both physicians working on the frontline to make important clinical decisions and public health professionals and health administrators to optimize the proper allocation of limited resources. additional file 1: appendix. details on the multiple imputation and validation processes involved in building the risk prediction model [14] [15] [16] [17] 23] .additional file 2: figure s1 . flowchart of influenza a(h7n9) infection stratified by risk categories. china cdc: chinese center for disease control and prevention; ci: confidence interval; crp: c-reactive protein; hgb: hemoglobin; roc: receiver-operating characteristic; rt-pcr: real-time reverse-transcription polymerase chain reaction; sari: severe acute respiratory infection; wbc: leukocyte count.competing interests dkmi reports receipt of research funding form hoffmann-la roche inc. bjc reports receipt of research funding from medimmune inc. and sanofi pasteur, and consults for crucell nv. gml has received speaker honoraria from hsbc and clsa. the authors report no other competing interests.authors' contributions hy and dkmi designed and supervised the study. ql, hj, fl, jz, zp, yh, lf, bc, zg and hy collected the data. ql, dkmi, tkt, hy, bjc, pw, ehyl and gml analyzed and interpreted the data. dkmi drafted the manuscript. all authors critically revised and approved the final manuscript. key: cord-291711-usvvad21 authors: radermecker, m. title: atopie et infections date: 2000-01-31 journal: revue française d'allergologie et d'immunologie clinique doi: 10.1016/s0335-7457(00)80034-9 sha: doc_id: 291711 cord_uid: usvvad21 summary there are obviously important and reciprocal relationships between atopy and infections. atopy predisposes to infections of the target organ, i.e. the site of the allergic reaction. as allergic inflammation prepares the terrain for these infections, it is particularly important to fight against allergic inflammation by eliminating allergens and irritants from the atopic subject's environment. in some cases, topical anti-inflammatory drugs, such as cromoglycate or corticosteroids spray or ointment, must be used. reciprocally, infections of the target organ accentuate atopic diseases by superimposing their specific inflammatory phenomena onto those of the allergic reaction, by amplifying this reaction and by creating hyperreactivity of the target organ. the possible role of chlamydia pneumoniae infections in the pathogenesis of certain severe forms of chronic asthma could attribute a place to macrolides in the treatment or even the prophylaxis of asthma. atopy predisposes to infections of the target organ, i.e. the site of the allergic reaction. as allergic inflammation prepares the terrain for these infectious, it is particularly important to fight against allergic inflammation by eliminating. allergens and irritants from the atopic subject's envaronment. in some cases, topical anti-inflammatory drugs, such as cromoglycate or corticosteroids spray or ointment, must be used. reciprocally, infections of the target organ accentuate atopic diseases by superimposing their specific inflammatory phenomena onto those of the allergic reaction, by amplifying this reaction and by creating hyperreactmty of the target organ. the possible role of chlamydia pneumomae infections in the pathogenesis of certain severe forms of chronic asthma could attribute a place to macrolides in the treatment or even the prophylaxis of asthma. l'atopie est la predisposition g6n~tique qu'ont certains individus (environ 30 % de la population) fi developper des ige globulines specifiques vis-fi-vis de substances normalement prdsentes dans l'atmosph&re ou l'alimentation (pollen, acariens, proteines animales ou v~g~tales, etc. [13] . elles ont toutes un 6quivalent clinique non allergique d'ttiologie mal connue mais avec une offgine infectieuse suspectte dans certains cas. contrairement aux sujets souffrant d'immunoddficience congdnitale ou acquise ou de mucoviscidose, lesquels sont pr~disposts aux infections pulmonaires graves et aux septictmies, l'enfant et l'adulte atopiques ne sont anormalement sensibles qu'aux infections de l'organe de choc c'est-~-dire sitge de la rtaction allergique. puisque les allergtnes sont surtout atriens, ce sont presque exclusivement les voies adriennes hautes, la conjonctive et la peau qui seront particulitrement touchtes. la simple observation clinique d~montre d~jfi la rtalit6 de cette association: la dermatite artpique prtdispose aux infections staphylococciques et virales de la peau et l'allergie respiratoire haute ou basse se complique souvent &exacerbations infectieuses surtout virales (rhino-pharyngite, sinusite ou bronchites). cette prdsomption clinique est parfaitement corroborte par des 6tudes fipiddmiologiques. les sujets allergiques souffrant de rhinite ou d'asthme font des infections respiratoires hautes plus frdquentes, plus durables et plus stv~res que les sujets non atopiques [10, 11] . quelles sont les raisons de cette susceptibilit~ anormale de l'organe de choc aux infections? l'absence de tout dtficit immunitaire important chez les atopiques et le fair que la propension aux infections ne porte que sur l'organe de choc (conjonctive, nez, bronche, peau) suggtrent dtj~ un trouble local des mtcanismes de d6fense qui pourrait 6tre secondaire fi l'inflammation allergique chronique. l'inflammation de la muqueuse respiratoire entraine une hyperstcrttion muqueuse, une r~duction de l'activitd ciliaire, une desquamation de la muqueuse pouvant expliquer des ulctrations, un mauvais drainage des sficrttions, une obstruction des voies a~riennes et/ou de l'ostium des cavit&s sinusales. par ailleurs, au cours de la r&action allergique, les cellules ~pith~liales et endoth~liales de la muqueuse respiratoire expriment la moldcule d'adhtsion icam-1 qui est le rtcepteur natutel pour 90 % des rhinovirus humains [5] . une hypothtse toute difftrente permettant d'expliquer la susceptibilit~ des atopiques aux infections de l'organe de choc serait l'existence d'une susceptibilit& anormale de leurs leucocytes /t toute une s~rie d'actions inhibitrices de l'histamine. outre son action pro-inflammatoire bien connue et lite ~ la stimulation des r~cepteurs h1 et h2 des muscles lisses, de l'endoth6lium capillaire, de l'tpithelium bronchique et de certaine glandes, l'histamine inhibe, fi des concentrations physiologiques, diverses fonctions des leucocytes humains. i1 est bien d~montr6 in vitro et in vivo que l'histamine peut par un effet h2 inhiber une strie de fonctions des cellules inflammatoires telles que cytotoxicitt, proliftration des lymphocytes, stcr&ion de tnf par les lymphocytes et les monocytes, le chimiotactisme des polynucl~aires et leur s~crttion d'enzymes lysosomiales [2] . on peut d~s lors penser que l'histamine, un des principaux m6diateurs libtras au siage des r6actions allergiques, exerce, par ses effets h2-anti-inflammatoires, une action inhibitrice sur les lymphocytes et les polynucl6aires diminuant ainsi les m~canismes locaux de dafense de l'organe de choc. chez l'asthmatique comme chez le bronchiteux chronique, ce serait donc les l~sions tissulaires secondaires ~t l'inflammation des bronches qui seraient le principal responsable de la propension de celles-ci aux infections. cette conclusion semble bien corrobor~e par les faits cliniques montrant que toute inflammation se complique souvent d'infection. ainsi, l'ec-z~ma de contact, les brfilures du second et troi-si~me degrd se compliquent souvent d'infections staphylococciques, la bronchite chronique fait le lit des infections locales par le trio infernal (pneumocoque, hemophilus influenzae, moraxella catharalis), l'ilfiite de crohn s'infecte souvent, l'ulc~re gastro-duodtnal est souvent associ6 & une infection de la muqueuse gastrique/t helicobacterpylori, les coronaropathies artfirioscl~reuses ~t chlamydia pneumoniae, etc. frick et al. [3] furent les premiers ~t sugg6rer en 1977 l'existence d'une association entre infection respiratoire virale et l'apparition d'une sensibilisation r6aginique chez l'enfant. plusieurs 6tudes r6alis6es chez l'animal et l'homme montrent qu'une infection exp6rimentale/t l'aide de diff6rents virus respiratoires est capable d'amplifier la r6ponse immunologique ~t ige et la r6action allergique cons6cutive ~t la r6administration d'un antigene [1, 4, 12] . chez l'enfant, les infections ~t virus syncytial respiratoire ou ~t parainfluenza entrainent la production d'ige sp6cifiques anti-virales et comportent un risque 6vident d'~volution ult6rieure vers l'asthme ou l'atopie [14, 15] . classiquement, les infections virales activent les lymphocytes t en privil6giant la production de cytokines th1 telles que l'interf6ron et l'interleukine 2 qui stimulent fortement l'activit6 des macrophages, des lympbocytes t cytolytiques et des lymphocytes nk en vue d'61iminer le virus de forganisme. certains virus pourraient favoriser la sensibilisation ige d6pendante en privil6giant la commutation des lymphocytes vers le type th2 et la production de cytokines proallergiques correspondantes (il4, il5). on peut 6galement concevoir que les 16sions 6pith61iales provoqu~es par certains virus respiratoires puissent favoriser la r6tendon et la p6n6tration des allergenes, la production d'ige globulines et finalement la r6action allergique. enfin, certains virus (cmv, ebv, hiv, etc.) peuvent stimuler la production isotypique d'ige. l'aggravation de la maladie atopique par une infection de l'organe de choc est particuli6rement bien document6e dans l'asthme. les infections bronchiques surtout virales sont une cause fr6quente d'exacerbations asthmatiques chez l'enfant et l'adulte. ellesjalonnent de fafon p6jorative le d6cours de la maladie et parfois en marquent le d6but. les rhinovirus sont de loin les principaux agents responsables des exacerbations asthmatiques infectieuses. d'autres virus respiratoires (coronavirus, virus syncytial, parainfluenza, influenza) et les chlamydia pneumoniae jouent un r61e moins important [6, 7j. comment expliquer ce r61e asthmog6ne des infections virales respiratoires ? on sait que les infections naturelles ou exp6rimentales par les virus parainfluenza ou influenza peuvent provoquer chez l'homme des sympt6mes respiratoires (toux, expectoration, bronchos-pasme), une augmentation des rdsistances des petites voies aeriennes et une hyperreactivit6 bronchique qui pent persister plusieurs semaines apres l'infection. les virus respiratoires et notamment les rhinovirus peuvent infecter les voies adriennes basses et y induire une inflammation comportant un effet cytopathique pour l'epithelium respiratoire et la lib&ration de cytokines et de mediateurs proinflammatoires. on pense que cette inflammation d'origine infectieuse en se superposant ~t l'inflammarion allergique preexistante peut aggraver les phenomenes d'obstruction bronchique dejfi presents. par ailleurs, l'inflammation liee/~ l'infection virale peut entra~ner une amplification de la reaction allergique et une hyperreactivit& bronchique durable [4] . les m~canismes responsables de cette hyperreactivitd sont mal connus et avancds principalement sur la base d'dtudes realisees in vitro on chez l'animal. les lesions 6pitheliales pourraient favoriser la pendtration des allergenes, reduire l'inactivation des tachykinines par les endopeptidases et, par le biais de la mise /t nu des fibres vagales afferentes, augmenter le tonus cholinergique des bronches et favoriser les rdflexes bronchoconstricteurs. i1 ne faut pas oublier que dans 20 fi 30 % des cas d'asthme de l'adulte ou de l'enfant, on ignore totalement la cause de l'inflammation bronchique qui est pourtant, comme dans l'asthme allergique, dependante des cellules t auxiliaires et qui comporte 6galement un recrutement et une activation des mastocytes/basophiles et des ~osinophiles. cette forme d'asthme pourrait avoir une origine infectieuse virale ou bactdrienne. certains travaux de la derniere decennie suggerent le r61e potentiel d'une infection chronique fi chlamydia pneumoniae dans la genese de certaines formes d'asthme non allergique saveres [8, 9] . en conclusion, en raison des relations reciproques 6troites liant atopie et infections, il importe d'essayer de prevenir ~t tout prix les infections de l'organe de choc. dans tous les cas, on recherchera et au besoin on corrigera toute anomalie locale favorisant les infections comme par exemple un foyer infectieux chronique orlou des polypes obstruant le drainage des fosses nasales ou des sinus. puisque c'est l'inflammation allergique m&me latente de l'organe de choc qui fait le lit de l'infection, on tentera de la diminuer par l'6viction des allergenes et des irritants, et surtout par l'utilisation precoce des madicaments anti-inflammatoires topiques tels que le cromoglycate ou les corticost6-roides en spray ou en pommade. en diminuant la r6action inflammatoire allergique au niveau de l'organe de choc, ces m~dicaments augmentent sa r&sistance aux infections. la rarefaction voire la disparition des exacerbations infectieuses darts les maladies atopiques est, comme la suppression de • m. radermecker / la dyspn6e nocturne dans l'asthme, un excellent crit6re de la bonne &volution de la maladie. le r61e possible des infections gt chlamydia pneumoniae dans la gen6se de certains asthmes non allergiques pourrait voir attribuer aux macrolides une place dans le traitement de ces affections. virus as precipitants of asthma symptoms : physiology and mechanisms clan exp allergy e -histamine-induced inhibition of neutrophfl chemotaxls and t-lymphocyte proliferation in man virus infection associated with onset of allergic sensitization in infants the effects of rhmovirus infecnons on allergic airways responses am the major human rhinovirus receptor is icam-1 the role of asthma and atopy in the susceptibility to respiratory viral lnfectmn in children commumty study of the role of viral infections in exacerbations of asthma in 9-11 year old children salkku e -chlamy&a pneumomae and chronic lung disease etal -greater frequencyofviral respiratory infections in asthmatic children as compared with their nonasthmatic siblings atopy and environmental factors in upper respiratory infections: an epidemlologlcal survey on 2304 school children, l'nt parainfluenza-3-virus infection enhances allergic sensitizanon in the guinea pig learned histamine release. scwnce asthma and immunoglobulin e anubodies after respiratory svncytial varus bronchiohtis : a prospective cohort study wlth matched controls predictive value of iespiratory syncltial vxrus-specafic ige response for recurrent wheezing following bronchiohus key: cord-278364-58d8kfdf authors: mohapatra, s. title: sterilization and disinfection date: 2017-03-31 journal: essentials of neuroanesthesia doi: 10.1016/b978-0-12-805299-0.00059-2 sha: doc_id: 278364 cord_uid: 58d8kfdf sterilization and disinfection are the basic components of hospital infection control activities. every day, a number of hospitals are performing various surgical procedures. even more number of invasive procedures are being performed in different health care facilities. the medical device or the surgical instrument that comes in contact with the sterile tissue or the mucus membrane of the patient during the various processes is associated with increased risk of introduction of pathogens into the patient's body. moreover, there is chance of transmission of infection from patient to patient; from patient or to health care personnel, and vice versa; or from the environment to the patient through the improper sterilized or disinfected devices. hence, medical personnel, laboratory people and the health care providers should have better knowledge regarding these techniques to prevent the spread of these pathogens. to define the desired level of antimicrobial killing for different devices. 12 he categorized the devices into three categories depending on the potential risk of transmission of infectious agents: critical, semicritical, and noncritical (table 59 .1). this simple classification of spaulding's needs to be revised, as it did not address the devices that come in contact with the mucous membrane (e.g., endoscope), biopsy forceps touching the breach sterile tissue, heat-sensitive items, and prions. the centers for disease control and prevention (cdc) in 1991 proposed an additional category to spaulding's classification as "environmental surfaces" to represent the surfaces that usually do not come in contact with patient. 13 environmental surfaces can be further subgrouped as clinical contact surfaces (medical equipment or high-touch surfaces) and housekeeping surfaces. cdc defines clinical contact surfaces as the areas that act like reservoirs of microorganisms, e.g., hands of health care workers. high-touch surfaces such as telephone, light switch board, bedrails, computer, door handle, and medical equipment like ventilator, x-ray machines, and hemodialysis machines are the contacting equipment that subsequently contact the patient. cdc had issued guidelines for hand washing and hospital environmental control. different llds and ilds that can be used to disinfect the clinical contact surfaces were approved by the environmental protection agency (epa). the housekeeping surfaces such as walls, floor, and sinks carry very low risk of transmission of infection. so, disinfection of such surfaces is less frequent in comparison to the previous one. the antimicrobial spectra of different methods are different from each other ( fig. 59.1) . hence, health care personnel should have adequate knowledge for the selection and recommendation of different sterilization and disinfection methods (tables 59.2 and 59.3) . a brief knowledge about the compatibility, toxicity, odor, and irritability due to various agents/methods is essential and useful for achieving adequate decontamination. the various chemicals used for the process of antisepsis or skin disinfection are chloroxylenols, anilides, hexachloraphene, polymeric biguanides, alexidine, diamidines, and triclosan. sterilization, disinfection, and cleaning in health care facilities include disinfection and cleaning of environmental surfaces with/without cleaning and reprocessing the medical equipment. the former includes mainly the noncritical items such as surfaces, floors, and high-contact surfaces (sinks, telephones, switches board, bed railings, trolleys etc.). it is observed that regular cleaning of all these housekeeping surfaces dramatically reduces the transmission of the infection. • the space for cleaning and other work should be clearly demarcated and separated by walls. • the hospital staffs should be properly trained regarding the cleaning and decontamination practices of hospital surfaces. • the staffs should wear personal prophylactic equipment (ppe), i.e., gowns, gloves, masks, and boots. there must be separate area for removing ppe. • cleaning and decontamination of the hospital surfaces having spilled blood is done as per the recommendation of occupational safety and health administration/world health organization/cdc. • ild or disinfectants with tuberculocidal activity should be used for blood spill in the hospital surfaces. • for decontamination of small amount of blood spills (<10 ml), sodium hypochlorite solution is used with a dilution of 1:100. for spill >10 ml, sodium hypochlorite with 1:10 dilution is used for the first application. the organic matter should be cleaned with absorbent material, and final disinfection may be done using sodium hypochlorite solution with 1:100 dilution. the cleaning and disinfection of medical equipment depends on their physical nature, character of the material it is made up of, lumen size, etc. thorough cleaning is preferred before the use of the disinfectants as cleaning effectively removes majority of the microbes from the equipment. • staffs should be properly educated and trained regarding the cleaning procedure, physical and chemical nature of the instruments, nature of disinfectants, etc. all the staffs during the process should use ppe. • dry organic materials are difficult to remove from the instrument. hence, drying should be avoided by immersing the equipment in the detergent or disinfectant solution prior to cleaning. the soaked matter can be cleaned by manual scrubbing and rubbing with brush or automated scrubber and thoroughly washed with water under pressure. avoid prolonged or overnight soaking of the devices. • the time of exposure, and concentration of the detergent or disinfectant, should be properly maintained as mentioned in the literature. too low concentration may not work effectively to remove the organic materials or microorganisms. • the ph of the disinfectant should be properly obtained as per the manufacturer's instruction. delicate articles should be processed in neutral ph. • enzymes like proteases may be added to the solution to fasten the cleaning action. enzymatic cleaners with neutral ph are preferred to avoid the damage of the articles. for example, in case of flexible endoscope, neutral ph detergent with enzymatic action is preferred. a new nonenzyme product [hydrogen peroxide based, us food and drug administration (fda) cleared] has been found to be very effective as cleaning agent. patients care equipment are divided into three categories (critical, semicritical, and noncritical) depending on the intended use and risk of transmission of infection. the cleaning and reprocessing protocol for each category are detailed in tables 59.4-59.6. devices that come in contact with the sterile parts of the body are included in critical items category. they carry the highest risk of transmission of infection. hence, sterilization is the method of choice for the reprocessing of these items (heat stable). the fda has approved ethylene oxide (eto), plasma sterilization, and liquid sterilization with glutaraldehyde or paa in heat-sensitive items. 22,23 all packed sterile items should be kept with proper precaution to avoid environmental contamination. items that come in contact with the mucous membrane of the skin are included in this category. these items should be processed by either heat sterilization or hld after cleaning (table 59.5) . all the semicritical items should be rinsed with sterile water or alcohol. forced air drying after the rinsing process drastically reduces the rate of contamination. it is found that cleaning also reduces the transmission of infection in human immunodeficiency virus (hiv)-contaminated instruments. items are found to be germ free when soaked in 2% glutaraldehyde for 20 min after the cleaning process. opa, glutaraldehyde, and automated process using paa are the three disinfectants commonly used for the reprocessing of endoscopes. items that come in contact with the intact skin are included within noncritical items. these include clothing, floors, high-touch surfaces, furniture, baths, bed pans, weighing scale, brushes, beddings, crockery, earphones, mobiles, and trolleys. the risk of transmission of infection with these items is observed to be the lowest. however, they contribute to the transmission of infection in indirect way. for example, methicillin-resistant staphylococcus aureus (mrsa), and vancomycin-resistant enterococci (vre) are commonly isolated with the patient's belonging and can be easily transmitted to other patient by health care worker's hand causing infection. these items do not need sterilization; however, they should be regularly cleaned and disinfected with lld to decrease the transmission of infective organisms (table 59 .6). respiratory apparatus such as ventilators, humidifiers, nebulizers, pulmonary screening devices, anesthetic equipment, laryngoscope and its blade, and suction equipment are most important in the icu setup because of its association with the risk of transmission of infection. proper cleaning and infection preventive measures should be followed while handling these instruments as they are highly associated with the transmission of infection from one patient to other. they come in contact with the mucous membrane of the body, are included in the semicritical item category, and are sterilized or disinfected with the hld. ventilators are important sources of hospital-acquired infection. this artificial airway is associated with increased chance of aspiration of the bacteria causing infection. mechanical ventilators are directly not associated with the infection, but their internal circuits (includes the filter, tubing, humidifier, etc.) and the fluids are the potential source of infection. as per the cdc guidelines, the permanent circuits should be replaced with sterile ones, when there is visible soiling or mechanical obstruction. it has been also seen that changing interval of tubing at 7, 14, and 30 days drastically reduces the transmission of infection. in case of detachable circuits, it should be dismantled, cleaned, and disinfected. • clean the equipment regularly and cover the ventilator, when it is not in use. • use sterile water to fill the humidifier as tap water causes introduction of microorganisms like burkholderia cepacia and legionella spp. • use ppe, and mask during handling these equipment. discard all disposals and perform hand hygiene after each handling. • tubings of the ventilator are infected with the secretion of the patient. the condensate from the inspiratory lines may spill to the tracheobronchial tree of the patient or into the nebulizer while handling, changing, or manipulating the ventilator circuit. • the effluent from the ventilator may contaminate the environment and can reenter through ventilator to the patient's airway increasing the chance of infection. • select hepa filters for both the inspiratory and expiratory limbs of ventilator circuits. • do not allow the condensate to drain back into the patient's airway or back to into humidifiers. • change the disposable parts of the ventilator after each use, and decontamination should be done after 48 h for the reusable items. • clean the visible soiling; sterilize the parts with autoclaving/low-temperature sterilization. • infant ventilators should be sterilized with eto. • toxic residues should be removed after each cycle of sterilization by flushing with air and oxygen. for proper humidification, use sterile water in place of tap water to fill the humidifiers. the fluid should be dispensed aseptically without entering or touching by hand. on the other hand, heat-moisture exchanger (hme) can be used. it absorbs the heat and humidity from the expired air of the patient and stores it. during the inhalation process, the cold dry gas entering to the ventilator absorbs this heat and moisture, thereby reducing the formation of condensate. hme should be exchanged in case of gross contamination, mechanical dysfunction, or in between patients. humidifiers can be cleaned using 70-90% alcohol. no antiseptic should be added to the water used for the humidifier. use sterile water for nebulization. the remaining fluid or medication should be handled aseptically. after every use, the nebulizer cap should be made dry or flushed with 70-90% alcohol before filling with water. the mouthpiece and mask should also be cleaned with warm water and dried before every use. anesthetic equipment such as face mask, ambu bag, tubings, and endotracheal tubes should be regularly cleaned. in patients suspected of tuberculosis, disposable face mask and tubings should be used. ambu bag should be kept covered to avoid exposure to dust particles. the bags should be changed in case of visible soiling or secretion. do not routinely sterilize or disinfect the internal machinery of pulmonary function test machines. they should be wiped and disinfected with hld in between patients. all the screening devices (inspiratory force manometer, tidal volume/vital capacity devices, and peak flow meters) should be discarded after single use. endoscopes are very useful tools for diagnostic as well as therapeutic processes. many outbreaks are reported with the contaminated endoscopes due to faulty reprocessing processes. reprocessing of endoscopes remains the most challenging task in the health care facilities. although the incidence of infection associated with the contaminated endoscope is quite low (∼1 in 1.8 million procedures), its frequency is found higher in comparison to other devices. the bioburden depends on the body cavity it is intended to visualize. for example, endoscopes used for gastrointestinal tract harbor 10 5 cfu-10 10 cfu/ml compared with 6.4 × 10 4 cfu/ml in bronchoscopes. it also depends on the material it is made up of. most of the flexible endoscopes (e.g., flexible bronchoscope, gastroscope, duodenoscope, sigmoidoscopes) are found to be heat sensitive and unable to withstand many chemicals. flexible endoscopes have multiple channels, small lumen, as well as honeycombed and blind ends, which are very difficult to clean. the endoscopes can acquire contamination from the patients, hospital environment, or water supply. it has been noticed that procedures such as endoscopic retrograde cholangiopancreatography (ercp) are associated with many iatrogenic infection. when the endoscopes touch the sterile tissue, they are classified as critical items and sterilization or hld is the ideal procedure for reprocessing. endoscopes coming in contact with the mucus membrane are classified as semicritical items, and hld should be used for the reprocessing. with proper cleaning, the level of bioburden seems to be decreased by 4-6 log 10 . many reports showed that with proper cleaning decontamination could be achieved from hiv also. few advice the application of 20% glutaraldehyde for 20 min after the cleaning step. the fda has approved a list of hlds/sterilants for reprocessing endoscopes. the following norms should be followed by the staffs to reduce the faults during reprocessing. • each person who reprocesses should be properly trained, use ppe (gowns, gloves, goggles, face mask, etc.) during the process • they should be properly educated about the chemical and biological hazards and comply with the manufacturer's instruction. • determine which process of sterilization is suitable for your endoscope. if it is heat stable, use autoclave/lowtemperature sterilization methods such as hydrogen peroxide gas plasma or paa, whereas liquid sterilants/ hlds can be used for heat-sensitive scopes. in general, reprocessing of the scopes includes five steps after a leak testing: cleaning, disinfection, rinsing, drying, and storing. cleaning can be done by completely immersing the scopes in the detergent/disinfectant solution. all the components should be dismantled before immersion. the internal and external surface of the scopes should be gently cleaned with the help of brush or soft cloth. brushes should be applied to the orifices and internal surfaces to remove the organic residues. all the items should be cleaned thoroughly, and the disinfectant/detergent should be discarded after cleaning. determine whether the scope can be suitable for automatic washer with sterilizers. these systems contain paa/hydrogen peroxide plasma-based (hpp), which are highly effective for killing vegetative and spore forms. hence, precleaning step is not required with these systems. first, the endoscope and its accessories should be completely immersed into the disinfectant/sterilant. all the channels should be profused with the disinfectant. the air pockets should be removed for adequate contact of the sterilant with surfaces. the time of exposure, concentration, and temperature should be maintained as per the instruction. the disinfectants that should not be used for endoscopes are chlorine compounds, iodoforms, quaternary ammonium compounds, phenols, and alcohols. opa for 12 min was found to be more advantageous than glutaraldehyde. low-temperature sterilization can be achieved with eto, but it is very lengthy, toxic, and expensive. after the treatment with the sterilant, the endoscopes should be thoroughly washed with sterile water/filtered water to remove all the residual chemicals. the scopes should be dried flushing 70-90% alcohol and forced air. drying should be done after disinfection and before storage to reduce the contamination. the disinfected endoscopes should be dried, capped, and kept vertically for the prevention of contamination. if automated washer is used, regular maintenance and disinfection of automated washer should be done. protocol should be developed to know whether the endoscopes are properly cleaned and disinfected or not. after the reprocessing, it can be used up to for 1/2 weeks with the maintenance of proper storage condition. there are many issues regarding nonendoscopic transmission of various infections. 36 there were many outbreaks due to the mishandling of various parts of endoscopes [e.g., intravenous (iv) tubings, needles, or syringes]. there is no consensus on the microbiological finding especially when the numbers of isolated bacteria are very small/isolation of environmental contaminants like coagulase-negative staphylococcus aureus or bacillus sp. alfa et al. have given <100 cfu/ml as the benchmark of a reprocessed endoscope channel. it has been also observed that pathogens like m. tuberculosis and p. aeruginosa are increasingly resistant to the commonly used hld, i.e., glutaraldehyde. now many rapid test methods such a strip detecting the residual protein, hemoglobin carbohydrate within the channels, and bioluminescence-based tests are available. along with the new technology of hlds such as improved hydrogen peroxide, improved automated endoscopic reprocessor are also introduced, e.g., evotech (fda cleared). the evotech machine reduces the manual cleaning process. all the scopes are put inside it after cleaning, and it checks all the leaks, disinfects, and promotes drying by alcohol flush. laparoscope is one of the popular equipment that enters into the sterile space. it is rigid, easy to clean, and disinfect. hence the risk of transmission of infection is also found less. it is disinfected with the hlds. the gastrointestinal endoscopes such as duodenoscopes are used in the ercp process. they are commonly found to be associated with introduction of iatrogenic infection. all the gastrointestinal endoscopes must be reprocessed after every patient because of high bioburden. since majority of them are heat sensitive, they should be sterilized with hpp or paa. similarly, bronchoscope should be reprocessed depending upon the material used. in general, the flexible bronchoscopes are sterilized with hpp or paa, and rigid bronchoscopes are sterilized with steam sterilization. all other flexible scopes such as the thoracoscope, laryngoscope, sinuscope, and esophagoscope are sterilized with hlds. in general, routine cleaning and disinfection of the hospital surface and floors should be done using epa-registered hospital detergent/disinfectant. these procedures are very important in places like the icu, ots, and emergency rooms, where there is likely chance of getting contamination with the blood/other body fluids. routine environmental disinfection remains crucial particularly in case of respiratory syncytial virus infection, severe acute respiratory syndrome complex, norovirus infection, clusters of clostridium difficile or infection with mrsa. disinfection should be done daily for all areas with patients in contact isolation (patients infected with mrsa). norovirus infection is highly contagious, hence items contaminated with norovirus infection vomit should be immediately disinfected with concentrated bleach or oxygen-related compound is needed. emerging pathogens and multidrug-resistant bacteria are one of the major concerns in medical facilities. emergence of antibiotic resistance in certain bacteria, e.g., multidrug-resistant m. tuberculosis, mrsa, vre, extended spectrum of β-lactamases, and metallo-β-lactamase producers are also important causes of various infection. except prions, most of the aforementioned pathogens are susceptible to different sterilization and disinfection procedures (see list of fda-approved chemicals in table 59 .7). hence, standard sterilization methods and epa-registered disinfectants should be used for all the equipment, devices, and surfaces irrespective of the patient status (known to be infected/not infected) to prevent the transmission of these infections. a sporicidal germicide such as glutaraldehyde/opa/paa/glutaraldehyde with phenol/h 2 o 2 is required for the proper elimination of c. difficile spores. all the equipment, devices, and surfaces should be treated in the same way as infected regardless of the whether the patient is known to be infected with hiv, hbv, hcv, or m. tuberculosis. these should be sterilized or disinfected with hlds approved by the epa or fda (see table 59 .7). among the llds, isopropyl alcohol is one of the common disinfectants used for small surface areas at a concentration of 60-95%. it has no action on spores, but it is active against bacteria, viruses, fungi, and m. tuberculosis. iodophore and phenolic compounds are ilds, which are used to disinfect various surgical and medical instruments. the exposure time is about 10 min. they were found to be inactive against bacterial spores. cjd has become a major crisis for the medical field because of lack of curative treatment. cjd is a highly infectious disease caused by the infection by prions (altered protein fragment). high-risk tissues include the neurological tissues such as the brain, spinal cord, and eye (retina, optic nerve). prions are resistant to the routine sterilization methods (includes heat sterilization and chemical sterilants). there are special guidelines for the sterilization and decontamination of the instruments exposed to patients with cjd. risk assessment of the patient is done with the following criteria. • known or suspected case of cjd or other transmissible spongiform encephalopathy • unusual progressive dementia with myoclonus and ataxia • family history of cjd/gerstmann-sträussler-scheinker disease/fatal familial insomnia, dura matter transplant • cadaver-derived pituitary hormone injection, etc. all the critical and semicritical devices exposed to the aforementioned surfaces are considered as infective and should be disinfected with proper care. sodium hydroxide (1n naoh, 121°c for 30 min) along with steam sterilization is used. instruments that do not tolerate this above method, must be cleaned twice, treated with various chemicals such as paa, iodophors, 3% sodium dodecyl sulfate, or 6m urea and autoclaved at 121°c for 30 min. the noncritical items are difficult to clean and should be discarded. hospital-acquired infection or nosocomial infection (ni) is defined as the localized or systemic infection acquired during the hospital care due to the adverse reactions to the presence of an infectious agent(s) or its toxin, which was neither present nor in incubating period during the time of admission. on the other hand, it can be defined as the infection that appeared after 48 h of admission to the hospital. over 1.4 million people acquire infection from the hospital worldwide. 43, 44 in developed countries, the incidence of ni varies from 5% to 25%. 45, 46 the incidence of ni in pediatric icu is estimated to be about 6.1-29.6%. 14 across the world, the incidence of ni (1%) was found lowest in the netherlands. 14 nowadays, health care-associated infection (hai) has become a major arm of ni, which indirectly reflects the colonized pathogens or exposure to variety of infectious agent through contaminated devices. the infection is usually mild, but sometimes it may become severe and life threatening. majority of infection occur in the icus, nurseries, and surgical units. additionally, with the increase in the length of stay, the treatment procedures become very expensive. in europe, hais contribute approximately 16 million extra stays in the hospital and 37,000 deaths per year. 47 the incidence of hai varies from 5.1% to 11.6% in developing countries in comparison to 7.1% in europe. 47 death rate varies from infection to infection. for example, hai due to blood stream infection and ventilator-associated pneumonia increase the death rate as well as the extra cost due to hospital stay. it was estimated that death rate due to hai is more than death rate due to aids, breast cancer, and road traffic accidents. hai is considered as the eighth most common cause of death in the united states. the mortality rate also varies depending on the patient population and health care setting. hais can be transmitted by the medical devices or the unclean hands of the health care workers. the infection caused by devices (such as catheter, ventilator) are catheter-related blood stream infection (cr-bsi), catheter-associated urinary tract infection (ca-uti), ventilator-associated pneumonia (vap), etc. infection due to c. difficile can be transmitted among the patients due to mal hand hygiene practice of the health care worker. the various bacterial pathogens causing hai are mrsa, vre, vancomycin-resistant staphylococcus aureus, m. tuberculosis, acinetobacter spp., p. aeruginosa, b. cepacia, c. difficile, clostridium sordellii, escherichia coli, and klebsiella spp. infections due to hepatitis virus, hiv, influenza virus, and norovirus are the common infections acquired in the hospital settings. the infections in operating rooms (ors) mainly appear as surgical site infections (ssis). the route of entry of the pathogens can be either air born or by contact of the contaminated instruments and hands of health care provider. 49, 50 the majority of ssis occur due to transfer of bacteria from air to the wound and less frequently with the contact. the bacteria from the patient get into the air and later settle down on the surface of the floor/instruments/health care provider's hand, etc. ventilation of the ots dramatically reduce the incidence of postoperative infections. it is also observed that the number of airborne infection increases with the number of members and movements inside the ot setup. the patients, in the icu, have severe illness, impaired defense mechanism, and on the invasive devices (e.g., ventilators, iv catheter, urinary catheter). 14 extreme of age, multiple trauma, burn, and abdominal surgery are the commonest risk factors for the patients to acquire ni during their stay. prolong hospitalization, immobilization, overcrowding, low patient-nurse ratio, and poor infection control practices enhance the infection rate in the icus. 14 common infections that occur in the icus are vap, cr-bsi, cr-uti, ssi, c. difficile-associated diarrhea, etc. devicerelated infections are most common among all. ssis are the commonest hai infections that occur in the ot due to faulty preventive protocols. these infections are difficult to treat and life threatening also. hence, recommendation of guidelines and application of the methods are required to prevent ssis in the ors. designing the ors is a complex task and should be done as per the recommendations. at least one anesthesiologist should be in the team while designing ors. the aim is to give maximum benefit to the patients arriving to the ors for various diagnostic, therapeutic, and palliative procedures. the ors should be separated from the main hospitals. the floors and walls should be smooth and covered with antistatic material, so that lesser amount of dust particles can be absorbed. broadly, the ors can be described in three sections. from outer to inner disposal zone, clean zone, and aseptic zone. the innermost protective zone should have maximum negative pressure and least bacteriological count in comparison to other zones. the inner negative pressure allows the free flow of germ-free air from outside to inside. outer/disposal zone: outer disposal area of the ors. 2. clean zone: a semirestricted area contains store room, anesthetic room, recovery room, scrub room, and rest room for the staffs. the exit of the clear zone should be through the outer zone. 3. aseptic zone: it should be restricted to the working team. it includes the ot and the area for storage of sterile equipment. in the ors, air flow system or ventilation removes the majority of airborne bacteria. ors should be designed properly for adequate ventilation. the air flow system is composed of ventilation, direction of air flow, maintenance of pressure, air filtration, etc. clean filtered air and adequate ventilation of indoor air are the two key factors in reduction of airborne infection. ventilation helps in maintenance of the temperature and humidity. the or's temperature should be kept 1°c lesser than the outside and maintained in between 18 and 24°c. about 40-60% of relative humidity should be maintained inside the ors for the comfort of the staff. ventilation causes exchange of air, and thereby removes all the bacteria released from the patient during the operative procedure and dilutes the anesthetic gases from the ors. the direction of air flow should remain from the ors to the main corridor. it has been observed that turbulent air flow exchanges the air more efficiently than low-velocity unidirectional flow. the anesthetic gases should be removed other than the main exhaust. filters with an efficiency of 20-40% are adequate at the entry side. hepa filters, which can remove particles of size >0.3 μm with efficiency of >99.97% are used as the second-line filters near the ors, icus, burn wards etc. air from the hepa filters flow unidirectionally (vertically or horizontally) causing few airborne bacteria at the operation site. exponential laminar air flow has been designed to overcome the limitation. the recommended air change rates (achs) in ors and recovery room are 20 ach/h and 12-15 ach/h, respectively. in ultraclean ventilated theaters, the minimum ach should be 37 ach/h. cleaning of the ot complex should be done either with vacuum or by wet mopping. try to keep the surfaces and floors dry, when the room is in use. the equipment inside the room should be covered during the cleaning process. all the inanimate objects (like tables, chairs, trolleys, sink door handler, etc.) should be cleaned with an epa-approved lld detergent. mopping should be regularly done at the beginning of each day before the ot process is going to start. the ot tables, trolley tops, lamps, etc., should be cleaned between use for each patient using lld. routine bacteriological surveillance of ors should be done for monitoring the presence of various airborne pathogens. the rate of microbial contamination in a well-designed, properly filtered, ventilated, and disinfected ot is much less. in conventionally ventilated theaters, microbiological sampling is done in the following way. close all the doors of the ors and keep empty for 1 h. using an air sampler, >250 l [recommended amount is 1 m 3 air (100 l)] air should be exposed to the no-selective culture medias. at least two samples should be taken per or. the colony count should not exceed 50-150 cfu/m 3 . as per the uk guidelines, with a 5-min exposure, the standard bacterial count should not exceed 35 bacterial and/or fungal particles per cubic metre of ventilating air. similarly, the geneva guidelines takes the standard of 25 cfu/m 3 for an empty or and 180 cfu/m 3 for 5-min exposure. the working condition of hepa filters should be checked first. in properly functioning hepa filters, the amount of airborne microbial contamination is very low. in unidirectional air flow zone, microbiological sampling is done from the four corners of the perimeter zone, one from the center and four from the inner corners of the inner zone. this zone should not contain more than 0.5 cfu/m 3 . nis or hais are a major cause of morbidity and mortality in the medical setup. the patients in the icus are observed to be more ill, receiving multiple high antibiotics, on multiple devices, and exposed to multiple antibioticresistant colonizers. the incidence of hais is found to be five to six times higher in the icus than in the general wards. the overall rate of icu infection varies from 5% to 35% of which approximately 25% are hospital-acquired infections. the most common infections observed in the icu setup are ventilator-associated infection (vap), cr-bsi, ca-uti, ssi, c. difficile-associated diarrheas, etc. among these infections device-related infection (vap, ca-bsi, ca-uti) constitute 80%. various risk factors for the development of infections are divided into host-related and hospital-related factors. the host-related factors include severity of diseases, extreme of ages, immunocompromised conditions (malignancy/transplant/organ failure/hiv infection), burn injury, trauma, and extensive surgeries. the factors related to the hospital or treatment are device related, treatment on immunosuppressive drugs, multiple blood transfusion, hemodialysis, parenteral nutrition, prolong immobilization, etc. since most of the infections are caused by the colonizers, the common pathogens isolated in these infections are mrsa, vre, esbl-producing gramnegative bacteria, stenotrophomonas maltophilia, fluconazole-resistant candida spp., etc. hais in the icus can be prevented by the application of recommended preventive measures. the hai rate has been reduced to one-third with proper applicability of infection control programs. one should have adequate knowledge about the risk factors, source of infection, type, and causation of infections to plan and establish the preventive strategies. bundle approach is an evidence-based group approach of preventive measures, which is found to be more effective when executed together. studies confirm that combined interventions result in better outcome than individual. these bundle approaches are small (three to five), straightforward practices performed in groups. these are mainly applied for the nis such as cr-bsi, vap, ca-uti, and ssi. however, the rate of reduction of these infections also depends on the baseline rate of infection of that health care center, staff adherence to the tactics, and the preventive measures chosen for the bundle. cr-bsi is an important cause of mortality and morbidity in patients admitted to the icus. the number increases with the increase in handling, duration of insertion, number of manipulation, number of lumens, etc. studies have proved that a large proportion of infections are preventable by applying the various control measures. all the elements of the bundle must be executed at the same point of time. these include hand washing, full-barrier precaution during the insertion of central line catheter, cleaning of the skin with chlorhexidine solution every time, frequent infusion of heparin or heparinlike substance through total parenteral nutrition to prevent the formation of fibrin, removal of unnecessary catheters, and avoidance femoral site catheterization. vap is another important cause of icu-acquired infection. it not only causes mortality but also is responsible for substantial cost of treatment. various recommendations for the prevention of vap include (1) appropriate cleaning, disinfection, and sterilization of ventilator equipment, (2) maintenance of ventilator circuits, and (3) routine care of patients requiring ventilation. 14 the cleaning, disinfection, and sterilization of the equipment have been discussed earlier in the chapter. the preventive measures taken for the patients to reduce the rate of vap are use of orotracheal intubation, noninvasive ventilation, minimization of the duration of ventilation. the patient should be maintained in semirecumbent position (30-45 degrees elevation of head of the bed). regular oral care should be performed. ca-uti is another common cause of infection in the icus 14 . staff must be trained enough to differentiate asymptomatic bacteriuria from infection due to catheterization. regular education regarding repeated hand washing before and after catheterization, maintenance of a closed system, maintaining free urine flow, and securing the catheter position correctly should be given to the health care workers for the prevention of these infections. ssi is a very common and life-threatening infection during icu stays. 14 there are various evidence-based guidelines recommended by the cdc. preoperative patient preparation, management of the colonizers, correct surgical technique, adequate antimicrobial prophylaxis, adequate sterilization and disinfection of the environmental surfaces, proper aseptic techniques, as well as washing surgical hands before and after the operative procedure are various preventive measures that can be taken to reduce the incidence of ssis. about one-third of the infections in the icus can be prevented by implementation of recommended preventive measures. apart from education of the health care personnel and standard precaution measures, other precautions like contact precaution, airborne precaution, and droplet precaution should be taken to prevent various infections. antibiotic-associated diarrhea due to c. difficile, and infection due to e. coli (o157:h7), shigella spp., hepatitis a, rotavirus, etc., can be transmitted by contact with infected patients or items. similarly infections transmitted by droplet such as tuberculosis, varicella, measles, viral hemorrhagic fever, and influenza can be prevented by taking adequate precaution. use of antibiotics should be restricted in the icu setup. minimum precautions like heating the water before use, regular cleaning, and maintenance of water tanks can be done to prevent water-borne infections. to conclude, 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challenge 2005-2006 'clean care is safer care nosocomial infections in adult intensive care units prevention of health care-associated-infections (hai) and antimicrobial resistance (amr) in europe the prevalence of nosocomial infection in intensive care units in europe. results of the european prevalence of infection in intensive care (epic) study. epic international nosocomial infection control committee the relative importance of routes and sources of wound contamination during general surgery. ii. nonairborne the relative importance of routes and sources of wound contamination during general surgery. ii. airborne key: cord-308184-w8ewm8ve authors: sarzi-puttini, piercarlo; marotto, daniela; antivalle, marco; salaffi, fausto; atzeni, fabiola; maconi, giovanni; monteleone, giovanni; rizzardini, giuliano; antinori, spinello; galli, massimo; ardizzone, sandro title: how to handle patients with autoimmune rheumatic and inflammatory bowel diseases in the covid-19 era: an expert opinion date: 2020-05-05 journal: autoimmun rev doi: 10.1016/j.autrev.2020.102574 sha: doc_id: 308184 cord_uid: w8ewm8ve • a correct patient risk stratification is of paramount importance for the proper management of economic and human resources. • it's fundamental to prioritize clinical trials evaluating dosing, prophylaxis, and treatment with immunosuppressant in covid-19 in order to avoid either an overuse and a treatment shortage. • future controlled studies may highlight in our patients a potential preventive role of immunosuppressant therapies in the development of severe forms of covid-19. • despite the overall risk of infection in rheumatic and gastroenterological diseases a conclusive association between these diseases and covid −19 remains questionable. since december 2019, the new coronavirus infections covid-19 has spread like a rising tide overwhelming the world. this unabated spread led the world health organisation (who) to classify it as a pandemic on march 11,2020 a global public health emergency that will also have serious social and economic repercussions [1] . the last time the who declared a pandemic was for h1n1 swine flu in 2009. at the time of writing, covid-19 has killed more than 30,100 people and, unfortunately, this number will continue to rise. while awaiting the development and distribution of a vaccine, scientists around the world are racing against time in the search for possible therapeutic strategies based on those used in previous health emergencies [2] . everyone, from governments to clinicians and entire populations, feels disoriented before this unknown enemy, and so scientific societies have been drawing up constantly updated guidelines in a bid to aid physicians caring for patients with autoimmune rheumatic and inflammatory bowel disease [3] [4] [5] [6] [7] [8] [9] . the highly contagious and phenotypically diverse sars-cov-2 virus has a direct impact on everybody's daily life, particularly in the case of already vulnerable patients [10] . preliminary recommendations regarding changes in the treatment of the patients with autoimmune diseases attending our rheumatology and gastroenterology units. patients with rheumatic and inflammatory bowel diseases (ibds) may be at higher risk of infection [6, 7, 11, 12] . disease activity, co-morbidities, immunosuppressive drugs including glucocorticoids (gcs), disease-modifying antirheumatic drugs (dmards), conventional synthetic (csdmards), biological (bdmards), targeted synthetic dmards (tsdmards), and the biological agents currently available for treating patients with ibd are all considered risk factors for infective complications. in addition, there are the risks inherent to the individual diseases and their treatments. the overall risk is increased if more than one of these drugs are taken, therefore preventing infections is a crucial part of the management of rheumatic and gastroenterological patients [13] [14] [15] [16] [17] . for example, before starting tumour necrosis factor (tnf)-α blocking agents or biological agents in general, quantiferon-tb is required for the diagnosis of latent or active tuberculosis, and it is rheumatological diseases and ibd are immune disorders that are associated with an increased risk of developing opportunistic and community-acquired infections such as respiratory virus infections. they are therefore also at risk of high co-morbidity and mortality rates, particularly those receiving immunosuppressive therapy [11, 12, 16] . this has raised concerns about the potential risk of covid-19 infection in ibd patients (particularly those who are taking immunosuppressants or biological drugs) because of the high morbidity and mortality rates observed in the old and frail with co-morbidities. however, to the best of our knowledge, the only data concerning the risk of sars-cov-2 infection in ibd patients come from just one study [19] , and there are no data concerning the risk in rheumatic patients. the observational study of ibd patients was carried out in wuhan, china, during the peak of the outbreak (between january 1,2020 and february 8,2020) involved 318 patients with a mean age of 39 years who answered a questionnaire covering concerns about their clinical condition and disease relapses. as a result of the early warning and strict preventive measures, none of the patients developed any significant clinical manifestation of covid-19 infection, not even those being treated with corticosteroids (12.6%), immunosuppressants (11%) and biological agents (6%), or those with co-morbidities. at the end of the study period, none of them reported any significant respiratory symptoms or other clinical manifestations attributable to viral infection, and none had a confirmed diagnosis of covid-19. an international pediatric and adult registry designed to monitor and report the outcomes of [ 24] . unfortunately, there are no data concerning the risk of relapse of ibd and related symptoms in patients with sars-cov-2 infection but, given that worsening symptoms or relapsing ibd are usually characterised by diarrhoea, nausea, vomiting and fever, these may mimic and hide a covid-19 manifestation. as this could be misinterpreted as a pure relapse, it would be appropriate to investigate other more specific symptoms of covid-19, such as headache, dyspnoea, or changes in smell and taste before planning an examination or treating such patients. at the time of writing, italy has the third highest number of covid-19 cases after usa and spain it is essential to promote the basic protective measures against covid-19 recommended by the who for everybody [25] :  stay at home and follow the directions of the local health authority;  maintain social distancing of at least two meters from anyone;  wash hands frequently and thoroughly with soap and water or clean them using an alcoholbased hand rub;  avoid touching the eyes, nose and mouth  practice respiratory hygiene by covering the mouth and nose with a bent elbow or tissue when coughing or sneezing (and immediately dispose of the used tissue);  seek medical care early if symptomatic. in addition, people should be educated to wear a mask in order to avoid infecting other people, avoid public toilets, and use gloves for shopping and all other outside activities. given the speed at which the pandemic is evolving, all of our patients have been asked to keep themselves informed and been told that our units would provide them with behavioural indications that will enable them to comply fully with all of the provisions issued by the italian government and regional institutions. all patients should be warned not to discontinue or reduce their treatment without consulting their rheumatologist unless they have specific symptoms. the first challenge for rheumatologists is to decide whether to interrupt or continue a treatment: although it is true that the treatment is designed to control disease activity, it is also undeniable that the same treatment may expose patients, such other infection, to an increased risk for covid-19. it is known that infections are a major concern in patients with overt inflammatory arthritis as they can contribute to disease flares [26] . one prospective cohort study of patients with inflammatory polyarthritis showed that the risk of hospitalisation because of infection is 2-4 times higher than in the healthy population [27] . similarly, an analysis by the us corrona registry of rheumatoid arthritis (ra) patients has shown that each 0.6 unit increase in the 28-joint disease activity score (das28) corresponds to a 25% increase in the rate of infections requiring hospitalisation and a 4% increase in the rate of outpatient infections [28]. j o u r n a l p r e -p r o o f although there are no published data supporting it, we prefer to discontinue the use of rituximab (rtx) infusions because of the related risk of infections. rtx is a monoclonal antibody that acts by binding the cd20+ surface antigen, thus leading to the depletion of cd20-positive b cells. it was originally approved for the treatment of a broad variety of hematological disease such as b-cell lymphoma, but it has been increasingly used in various autoimmune diseases in which b cells are thought to play a key role, and is now also approved for the treatment of severe active ra and granulomatosis with severe active polyangitis and microscopic polyangitis. in addition to infections. which remain a major concern for patients receiving rtx, another rare but potentially fatal complication associated with rtx infusion is cytokine release syndrome (crs), a life-threatening condition generated by uncontrolled immune activation that leads to multi-organ failure and eventually death [29] . covid-19 can induce such a "cytokine storm" [2, [30] [31] [32] of over-active effector t cells and the bulk production of pro-inflammatory cytokines, thus leading to acute lung injury (ali) and ards [33] . cyclophosphamide is a highly potent immunosuppressant that has demonstrated efficacy in autoimmune diseases. given its increased risk of opportunistic infections and potential haematological alterations such as leukopenia and thrombocytopenia we prefer to discontinue it although, at now, there aren't data supporting it [34, 35] . another important and much debated issue is how to deal with the use of corticosteroids, which do not seem to add any benefit in terms of the clinical outcome of covid-19, and may actually slow virus clearance. we therefore consider it appropriate to reduce their administration to the minimum effective dose, but not below 5-10 mg of prednisone per day. a great tumult arose about the use of nsaids after the french minister of health''s statement advising against the use of ibuprofen in patients with covid-19. the european medicines agency (ema) has reiterated that up to now there is no scientific evidence of a link between ibuprofen and the worsening of covid-19, although the situation will have to be monitored over time [36] . we believe that it is best to be prudent and that it is up to each individual rheumatologist to choose which nsaid or analgesic to use for each specific case. we therefore recommend their use on demand, albeit for as short a time as possible. a recent study found that chloroquine, a long used antimalarial drug, had a potent in vitro antiviral effect in a covid-19 assay by increasing the endosomal ph required for virus/cell fusion and j o u r n a l p r e -p r o o f interfering with the glycosylation of cell receptors, thus adding to its well-known immunemodulating action [37] . chloroquine is being considered for inclusion in the next versions of the guidelines for the prevention, diagnosis and treatment of covid. 19 [3] . although the available data do not suggest that there is a specific risk of sars-cov-2 infection and morbidity in ibd patients receiving immunosuppressive treatment, it is known that opportunistic infections have deleterious effects on such patients, which suggests that the risks and benefits of the treatment should be balanced before continuing its administration. there is evidence showing that ibd patients have impaired innate mucosal immunity, but they should not be considered as having altered immunocompetence per se. the current guidelines recommend screening patients who have to start immunosuppressive or biological therapy for some, but not all viruses: for example, screening for hbv, hcv and hiv is recommended, but screening for herpes simplex virus (hsv), patients should be vaccinated against vzv using inactivated vaccine before starting any immunomodulatory therapy because of the greater risk of developing severe viral infection [12] . given that there is no vaccine or specific treatment for covid-19, it seems that the only preventive measure that could be taken is to discontinued immunosuppressive treatment while bearing the associated risks in mind. there are no data that contraindicate the use of oral or rectal mesalazine, which can and should be continued if it is already being used. although the chinese experience suggests that the short-term use of low-dose steroids (≤0.5-1 mg/kg for seven days) may be beneficial in controlling overwhelming inflammation and cytokinerelated lung injury as much as possible, it is better to avoid systemic steroids, especially if they are any patient who develops symptoms of any infection such as fever, cough and/or shortness of breath should be tested (pharyngeal and/or nasal swab, sputum, bronchoalveolar lavage fluid) and given medical care. as sars-cov-2 preferentially proliferates in type ii alveolar cells, a higher positive rate of nucleic acids is found in the lower respiratory, and so a specimen taken from this area is to be preferred. however, an initial negative test should be repeated on subsequent days because peak viral shedding occurs 3-5 days after disease onset. in the case of a positive test, immunosuppressive treatment with traditional dmards other than chloroquine or hydroxychloroquine, biological dmards (bdmards), small molecules, and biological agents for ibd should be discontinued throughout the course of the infection. we believe that a temporary suspension of even one month does not worsen disease activity, whereas, at now, given the well-known infectious risk relating to these therapies, continuing immunosuppressive therapy would lead to the possibility of a rapid evolution of the infection. we resume biological agents after patients have undergone two negative tests and their full blood counts, creatinine, bilirubin, albumin, ldh, ast/alt, ck, crp, il-6, troponin t and ferritin levels, pro-thrombin (inr) and lipid profiles have normalized. our prudent attitude, guided by current scientific evidence on immunosuppressant infectious riskrelated, does not preclude the possibility that future data will instead highlight the protective role of these therapies in the development of severe forms of covid-19 [43] . in accordance with the provisions of the italian government and regional institutions, all deferred medical services were suspended except for those required for urgent clinical conditions (within three days) and those that could only be delayed for ten days. consequently, all of the scheduled clinical and instrumental evaluations of stable patients in regular follow-up were re-scheduled, although is still possible for patients to interact with their dedicated rheumatology and gastroenterology team via phone interviews and social networks, thus making it possible to monitor their clinical condition [44] . they can also send in the results of routine laboratory tests and report any problems with their clinical condition or current therapies. electronic intensive care unit (e-icu) monitoring programs, which allow nurses and physicians to monitor the status of sicker patients are ideal, but patients who report symptoms suggesting a recurrence are preferably assessed by means of a telephone interview in order to clarify the severity of the episode and make therapeutic decisions [45] . patients complaining symptoms suggesting a severe relapse are invited to visit the hospital (while strictly complying with all of the necessary procedures) with the aim of assessing the need for hospitalization, and the consequent diagnostic and therapeutic procedures. patients on the surgical list were contacted by our reference surgeon and re-scheduling was considered, while ensuring a non-covid path for urgent interventions. the following procedures were defined for patients enrolled in controlled clinical trials: -they can only be admitted to hospital if they are being treated with intravenously administered drugs; -if they are being treated with subcutaneous drugs and unpostponable visit is planned, the sponsor is asked to have the drug delivered to their home; -the possibility of carrying out laboratory tests at trusted laboratories close to each patient's home is verified. the de novo enrolment of patients with rheumatic disease and/or ibd is continued only if the study drug is the only therapeutic alternative. for the more than 1000 patients administered biological drugs at our rheumatology and gastroenterology units, the following general measures have been taken: -they are received by a nurse who provides them with an alcohol solution for hand cleansing, a mask and gloves; -they then undergo the planned outpatient examination by a doctor from the rheumatology or gastroenterology team; -infusions are administered in special rooms with armchairs separated by more than one meter (as mortality is higher among elderly patients [46] , they are allocated single rooms in order to avoid any other possible contact); -if necessary, the time interval between infusions is extended. in the case of subcutaneous biological therapies, lombardy's regional government has activated alternative means of delivering the medicines for home use in order to limit the need for patients lombardy but unable to attend a regional hospital for objective reasons, the ats schedules the collection of drugs from the hospital pharmacy and their delivery to each patient's home by a courier; in the case of patients resident, domiciled or simply present in the territory of the ats who are being treated at a hospital outside lombardy and for whom movement is not recommended, the ats coordinates with hospital facilities in lombardy in order to guarantee the continuation of the therapeutic program. some recent studies have reported that chloroquine has an antiviral effect on covid-19 assays, and chloroquine and its analogue hydroxychloroquine are well known to rheumatologists. the repositioning of old drugs to antiviral treatment is a remarkable strategy because the drugs have well-known safety profiles, side effects, schedules and drug interactions [37, 47] . recent findings have shown that hydroxychloroquine is three times more potent than chloroquine [48] .  despite the overall risk of infection in rheumatic and gastroenterological diseases a conclusive association between these diseases and covid -19 remains questionable. possible world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) covid-19, cytokines and immunosuppression: what can we learn from severe acute respiratory syndrome? simit vademecum available nhs clinical guide for the management of rheumatology patients during the coronavirus pandemic available from are my patients with rheumatic diseases at higher risk of covid-19? second european evidence-based consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease infection and malignancy in rheumatic diseases update of eular recommendations for vaccination in adult patients with autoimmune inflammatory rheumatics diseases eular recommendations for vaccination in adult patients with autoimmune inflammatory rheumatic diseases risk of serious and opportunistic infections associated with treatment of inflammatory bowel diseases protection of 318 inflammatory bowel disease patients from the outbreak and rapid spread of covid-19 infection in wuhan, china. the lancet mechanisms and management of chimeric antigen receptor t-cell therapy related toxicities epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (covid-19) with gastrointestinal symptoms manifestations of digestive system in hospitalized patients with novel coronavirus pneumonia in wuhan, china: a single-center, descriptive study review article: gastrointestinal features in covid-19 and the possibility of faecal transmission clinical characteristics of covid-19 patients with digestive symptoms in hubei, china: a descriptive, cross-sectional, multicenter study who -coronavirus disease (covid-19) advice for the public available from 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nephropathy: a pairwise and network meta-analysis int immunopharmacol infection risk in systemic lupus erythematosus patients: susceptibility factors and preventive strategies european drugs agency to review safety of ibuprofen remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro on the use of corticosteroids for 2019-ncov pneumonia potential benefits of precise corticosteroids therapy for severe 2019-ncov pneumonia clinical course of covid-19 in a series of patients with chronic arthritis treated with immunosuppressive targeted therapies ann rheum dis virtually perfect? telemedicine for covid-19 telehealth for global emergencies: implications for coronavirus disease 2019 (covid-19) case-fatality rate and characteristics of patients dying in relation to covid-19 in italy hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) hydroxychloroquine and azithromycin in patients with severe covid-19 infection natural small molecules as inhibitors of coronavirus lipid-dependent attachment to host cells: a possible strategy for reducing sars-cov-2 infectivity? infection and rheumatoid arthritis: faraway, so close! autoimmun rev scarpa r could sars-coronavirus-2 trigger autoimmune and/or autoinflammatory mechanisms in genetically predisposed subjects? autoimmun rev interferons at age 50: past, current and future impact on biomedicine what is the role of rheumatologists in the era of covid-19? key: cord-268729-n7slf5tx authors: wissinger, e l; saldana, j; didierlaurent, a; hussell, t title: manipulation of acute inflammatory lung disease date: 2008-05-07 journal: mucosal immunol doi: 10.1038/mi.2008.16 sha: doc_id: 268729 cord_uid: n7slf5tx inflammatory lung disease to innocuous antigens or infectious pathogens is a common occurrence and in some cases, life threatening. often, the inflammatory infiltrate that accompanies these events contributes to pathology by deleterious effects on otherwise healthy tissue and by compromising lung function by consolidating (blocking) the airspaces. a fine balance, therefore, exists between a lung immune response and immune-mediated damage, and in some the “threshold of ignorance” may be set too low. in most cases, the contributing, potentially offending, cell population or immune pathway is known, as are factors that regulate them. why then are targeted therapeutic strategies to manipulate them not more commonplace in clinical medicine? this review highlights immune homeostasis in the lung, how and why this is lost during acute lung infection, and strategies showing promise as future immune therapeutics. supplementary information: the online version of this article (doi:10.1038/mi.2008.16) contains supplementary material, which is available to authorized users. the mucosal immune system must maintain composure in the presence of an onslaught of antigenic and potentially pathogenic material. exposed to the outside world with, in most cases, only a single epithelial cell barrier protecting them, our mucosal surfaces have developed a sophisticated system of immune exclusion, ignorance and tolerance. the best characterized of these are described in the gastrointestinal tract. an understanding of immunity in the respiratory tract has lagged behind that of the gut, and although numerous key components have emerged, the sequence of events from initial inhalation to immune pathology in the lower respiratory tract is still unclear. despite best efforts to maintain immune homeostasis, respiratory inflammatory disease is common and significantly life threatening. this review will highlight mechanisms that maintain lung immune homeostasis and current therapeutic efforts to contain infection-induced exaggerated acute inflammation once it occurs. the respiratory tract includes the nasopharyngeal cavity, trachea and larynx, bronchi, bronchioles, and finally the alveoli. organized lymphoid tissue is embedded in some, but importantly not all, of these stages in the respiratory tree. similarly, draining lymph nodes are associated with only a few of these sites. the cellular composition, requirements for activation, and expansion dynamics of respiratory tract associated lymph nodes are virtually similar to any other lymph node and will therefore not be discussed in detail here. we will focus on the regulation (or de-regulation) of immune cells embedded in the respiratory tract itself. considering the total surface area of the respiratory tract constitutive, embedded organized lymphoid tissue is actually quite rare ( figure 1 ). organized structured lymphoid tissue exists in the nasal cavity of rodents (nasal associated lymphoid tissue, nalt) as paired lymphoid structures at the entrance to the pharyngeal duct, but identical structures in man remain elusive (for a review, see reference bienenstock and mcdermott 1 ). organized lymphoid follicles are observed in post-mortem specimens extracted from 150 children that contain occasional germinal centers, which are associated with lymphocytes in the overlying nasal epithelium and the presence of high endothelial venules. however, in adults such lymphoid tissue is disseminated across the whole nasal mucosa, 2 and is analogous to the less well-organized diffuse lymphoid tissue (termed d-nalt) lining the nasal passages of mice. 3 in man, diffuse nalt develops after birth, likely in response to antigen, and b-and t-cell responses parallel those that occur in lymph nodes. the waldeyer ' s ring comprising the nasopharyngeal (upper midline in nasopharynx, adenoids), paired tubal (around openings of auditory tube), paired palatine (either side of the oropharynx), and lingual (under the mucosa of the posterior third of the tongue) tonsil(s) are thought of as analogous structures to nalt, but are located outside of the respiratory tract and probably also contribute to gastrointestinal immunity. experiments with mice show that, unlike peripheral lymphoid organs, nalt develops independently of lymphotoxin-. however, its structure and function are perturbed in lymphotoxin--knockout mice, possibly due manipulation of acute inflammatory lung disease el wissinger 1 , j saldana 1 , a didierlaurent 1 , 2 and t hussell 1 inflammatory lung disease to innocuous antigens or infectious pathogens is a common occurrence and in some cases, life threatening. often, the inflammatory infiltrate that accompanies these events contributes to pathology by deleterious effects on otherwise healthy tissue and by compromising lung function by consolidating (blocking) the airspaces. a fine balance, therefore, exists between a lung immune response and immune-mediated damage, and in some the " threshold of ignorance " may be set too low. in most cases, the contributing, potentially offending, cell population or immune pathway is known, as are factors that regulate them. why then are targeted therapeutic strategies to manipulate them not more commonplace in clinical medicine? this review highlights immune homeostasis in the lung, how and why this is lost during acute lung infection, and strategies showing promise as future immune therapeutics. to impaired expression of cxcl13, c -c chemokine ligand19 (ccl19), and ccl21, which are crucial for the recruitment and placement of lymphocytes and dendritic cells (dcs). 4 the only other organized lymphoid structure described to date located within the respiratory tract is bronchus-associated lymphoid tissue (balt) (reviewed by bienenstock and mcdermott 1 ). whether it routinely contributes to primary immune responses or maintenance of t-and b-cell memory in the respiratory tract is not known. 5,6 however, a recent study in mice lacking peripheral lymphoid organs suggests that balt can initiate anti-influenza immunity and provide sufficient t cells to mediate protection against a second infection. 7 humoral immune responses elicited by balt are primarily mediated by immunoglobulin a (iga) and igg produced both locally and by balt-derived b cells that traffic to distant mucosal sites. 8, 9 similarly located t-cell responses have been noted. on the basis of these findings, balt can be thought of as functionally analogous to mucosal lymphoid aggregates in the intestine. present in up to 40 % of children and adolescents (to age 20), balt is rare in the lungs of healthy adults. 10,11 although originally described at the bifurcations of the bronchi, immediately beneath the epithelium, 12,13 in the absence of antigen balt is rare 14 and may be controlled or limited by regulatory t cells. 15 inflammation in the lung is associated with balt neogenesis and is described in a variety of pulmonary (moyron-quiroz et al 7 and references therein) and non-pulmonary 16 inflammatory conditions. homeostatic chemokines, including ccl19 and ccl21, in mice are required for development of such inducible balt (ibalt). 4 the extent of ibalt appears to depend on the level of inflammation in the local microenvironment, and suggests that it is developed when required. mice lacking oxidoreductases, that protect from oxidative stress, display heightened cellularity and inflammatory cytokines and ibalt is more prevalent. 17 whether it remains associated with larger airways and persists long after resolution of inflammation is still uncertain. intriguingly, mice lacking peripheral lymph nodes and spleen, but retaining ibalt, clear influenza infection (albeit slower) and survive higher doses of virus than do immune-competent mice. such lymphotoxin-knockout mice show slower generation of influenza-specific t cells that eventually reach wild-type levels, similar to antibody isotype switching to igg and t-cell cytokine production and effector function. this indicates that immune responses generated in ibalt although slower are protective and potentially less pathologenic. 7 this may represent a qualitative difference between local and peripherally derived immune cells or simply reflect the reduced magnitude of immunity when ibalt is the only inducing immune compartment. regardless, in the case of lung immunity, secondary lymphoid tissues are not essential for the maintenance of immunological memory, since a pulmonary infection with influenza virus is handled equally as efficiently in their absence. 18 to date no studies have shown that organized embedded lymphoid tissue such as ibalt and nalt contribute directly to inflammatory pathology in the lung. they may initiate production of immune t and b cells that then track to less organized lung immune compartments, 7,19 -22 but their neogenesis (ibalt) or continued presence (nalt), per se , is not associated with pathology. instead, pathological lung inflammation is attributed to those compartments that lack organized lymphoid structures, the airways and lung parenchyma. this could also be said of inflammation in the gut; peyer ' s patches and mesenteric lymph nodes may not be directly associated with pathology, whereas the lamina propria is. what the lamina propria and lung parenchyma have in common is a loose scattering of non-organized immune cells and a vast surface area of potentially non-professional antigen-presenting cells (apcs); the epithelium. the epithelium expresses constitutive mhc class i and, when inflamed, mhc class ii and b7 molecules. 23, 24 it can, therefore, process and present antigen and activate t cells, but can it turn them off? we believe this non-professionalism and lack of immune-cell organization leads to immune dysregulation. during inflammation, the mediastinal lymph nodes and ibalt expand in an organized and precisely compartmentalized manner. although low frequencies of antigen-specific cells can be observed in these sites, 25 the ratio of immune-cell subsets does not significantly change. contraction of lung-associated lymph nodes is also well controlled; again cell proportions are retained. in lung compartments devoid of organized lymphoid tissue however, immune cells are recruited in droves by a chemotactic gradient 26 -28 from infected epithelium and / or tissue resident and alveolar macrophages. once in the lung parenchyma or the airways, they do not form structures analogous to ibalt or lymph nodes, and it is here that pathological damage occurs, toll-like receptors ligands dominate, and inflammatory cytokines are produced by the infiltrate in abundance. unlike the lung-associated lymph nodes, the airways and lung parenchyma, therefore, experience dramatic shifts in their cellular composition. this is illustrated graphically in figure 2 . the approximate cellular composition in nalt, airways, and lung is shown in homeostasis and at the peak of respiratory infections by three very different pathogens, influenza virus, the bacterium streptococcus pneumoniae , and the fungus cryptococcus neoformans . obviously at other stages of the infection, slightly different cells will dominate, but only the peak of inflammation is presented for clarity. for example, natural killer cells dominate in the airways at days 3 -4 of a viral infection. 29 at its peak of activity, the influenzainfected airway and lung is dominated by cd8 + and cd4 + t cells, 25,30 -32 whereas during s. pneumoniae infection, macrophages, neutrophils, and t cells are more abundant. 33, 34 c. neoformans in c57bl / 6 mice induces an eosinophil-dominated response in the lung and airways. 35 -37 this infiltrate in the air spaces and lung for all pathogens is dramatically different to the same sites in homeostasis that contain few lymphoid cells but a prominent macrophage population. 38 -40 note that the nalt, despite being infected with all three pathogens, does not substantially alter the proportion of immune-cell subsets present, and the same is also true for the relatively non-infected lung draining lymph nodes. this lack of control and excessive response is only observed in a minority, but when it occurs it is life threatening. for most of us, respiratory pathogens are cleared by non-inflammatory means, including iga that does not fix complement well, but in dimeric form agglutinates and physically excludes antigen by a process known as immune exclusion. 41, 42 what, therefore, goes wrong in a minority? to address some of these questions, we need to understand how immune homeostasis is maintained in health in these non-organized lung compartments (reviewed by holt 43 ) and what pathways contribute to immune pathology. epithelial cells contribute a multitude of strategies to maintain lung immune homeostasis (for a review, see reference holt 43 ). in addition to barrier function, they secrete a variety of antimicrobial substances (surfactant protein c, mucins, and antimicrobial peptides), affect airway smooth-muscle, dc, and memory t-cell activation via nitric oxide production; 44, 45 assist in cell recruitment via production of cytokines and chemokines; 46, 47 and prolong cell survival by secreting stimulating factors such as granulocyte -macrophage colony-stimulating factor. 48, 49 raz and co-workers 50,51 highlight an interesting pathway critical for maintaining alveolar macrophage homeostasis, involving integrin v 6 that localizes these cells next to epithelial expressed transforming growth factor-(tgf-). this may explain why these cells are refractory to migration to the draining lymph nodes. 52 for inflammation to proceed, this inhibitory pathway must be overcome, which is mediated by a toll-like receptor-induced conformational change of macrophages, disruption of tgf-signaling, and reduced integrin expression. we often assume that innate immunity is inactive in the absence of antigen. however, the work of raz et al . clearly shows that active suppression is required for homeostasis. this is also observed in mice lacking components of nadph oxidase 53 that have heightened basal levels of airway macrophage activation due to loss of feedback inhibition. active suppressive mechanisms, therefore, set a " threshold of ignorance " . those that succumb continually to inflammatory lung disease may, therefore, have dysregulated homeostatic pathways or the threshold, which antigen must exceed to induce inflammation, set too low. in the cases of tgf--mediated suppression of alveolar macrophages, homeostasis is overcome by cleavage of the integrin tethering it to the respiratory epithelium. homeostasis is restored when macrophage-released matrix metalloproteinases transform latent tgf-into its active form. 51 it may, therefore, be possible to harness these pathways artificially to dampen inflammatory lung disease with the caveat that pathogen clearance may be affected by such a global antiinflammatory strategy. 54 macrophages, particularly in the airways, have long been known to have an immune-suppressive phenotype. renewal is achieved primarily via local cell proliferation; recruitment via ccl2:ccr2 does occur, although such cells may take days to mature into the classical immune-suppressive phenotype. 55 in addition to shielding the immune system from inhaled antigens, 56 they display poor phagocytic activity 57 and tend not to migrate well to draining lymph nodes. macrophages held in homeostasis also affect other cell types that may otherwise be proinflammatory within the respiratory tract (for a review, see reference holt 43, 58 ) . dc migration to the draining lymph nodes is enhanced upon macrophage depletion, 52 and t-cell-mediated inflammatory disease ensues to antigens that would otherwise have been ignored, 59 most likely due to the usual direct suppressive influence that alveolar macrophages have on dc function. 39 both myeloid and plasmacytoid dcs (pdcs) are present within the lung, both increase and are recruited rapidly during inflammation, and are attracted by chemokines and cytokines produced by epithelial cells and alveolar macrophages. 43,60 -62 myeloid dc responses are similar to counterparts found elsewhere in the body. pdcs, however, also appear to play a tolerogenic role in the respiratory tract. they have poor apc activity 63 (but once activated enhance cd8 + t-cell responses in vivo 64 and possibly cd4 + t cells at distant sites 65 ), promote inhalation tolerance, 66 and can protect from development of allergic airway disease (reviewed by de heer et al , 66 hammad and lambrecht, 67 and lambrecht 68 ). during acute respiratory viral infections, pdcs perform dual functions of promoting viral clearance by secretion of type i interferon (ifn), and limit inflammation by induction of interleukin-10 (il-10) (reviewed by grayson and holtzman 69 ). their role in limiting lung inflammation can be clearly seen in respiratory syncytial virus (rsv)infected mice where pdc depletion leads to increased viral replication and enhanced immunopathology in the lungs 70, 71 . dcs encounter antigen predominantly in the lung parenchyma, although microbial sampling, via dendrite projections through the epithelial cells into the airway lumen, may induce activation, maturation, and migration, arming them to support potent t-cell responses. 72 quite what returns these cells to homeostasis is unknown but may involve the level of toll-like receptor signals and / or the influence of surface expressed inhibitory receptors such as cd200r. 73 airway epithelial cells may also control dc activity via tgf-analogous to alveolar macrophages, 51 although this has not been proven. whether dcs actually transmigrate into the airspaces is still controversial. 74 -76 ccr2 assists their transit across the endothelium and ccr6 their subsequent migration into the airway. 74 the recently described cd103 + ( e 7), cd207 + , cd11b lo dcs express all of the requisite chemokine receptors to draw them into the airways, 77 but their presence in this compartment has not been proven. clearly, harnessing the homeostatic pathways described above may help to resolve ongoing inflammation. however, it is equally likely that immune cell types and pathways brought into the lung with inflammatory cells during inflammation also provide a therapeutic opportunity. it would not be possible to cover all of the pathways attempted to limit lung inflammation. we will, therefore, restrict our analysis to acute infectious events (i.e., not asthma) and to targeted therapeutic strategies (i.e., not global anti-inflammatories such as corticosteroids or experiments in gene-deleted animals). immune-mediated pathology can be manipulated at any stage of its generation. from a clinical perspective, however, after onset of identifiable symptoms would be the most beneficial. for this reason it is often resolution of inflammation that is targeted, which can include modulation of cell survival, successive waves of recruitment, and ongoing innate immunity. it is still unclear whether immune excess in the respiratory tract stems from over-exuberant recruitment, proliferation within the airspaces, and / or accumulation in the absence of clearance of innate and adaptive immune cells. evidence suggests that the airspaces may not support efficient t-cell proliferation 78 -80 despite memory t cells acquiring bromodeoxyuridine staining at the same rate as secondary lymphoid organs. 25, 80 it is likely that the inflammatory cytokine and chemokine cascade that ensues upon infection 81 -86 will prolong immunecell survival, but at the same time enhance their recruitment, either directly or by altering vascular or epithelial permeability. 87, 88 an inflammatory environment is also associated with the highest expression of " late co-stimulatory molecules " on t cells that prevent activation-induced cell death and rely on cognate ligands expressed on apcs for signalling. ox40 (cd134) is one such late co-stimulator that is restricted to recently activated t cells, whereas the ligand (ox40l) is expressed on a number of cell types, predominantly apcs. 89 stimulation through ox40 promotes cd4 and cd8 t-cell survival, clonal expansion, 90 inhibition of regulatory t cells, 91 and enhanced immunity to a variety of pathogens. 36,92 -94 however, during acute influenza virus infection of mice, transient blockade of ox40 is more beneficial; alleviating illness and pathology to influenza, without review compromising pathogen clearance or immunological memory. 95 a permanent absence of multiple late co-stimulators, however, can compromise immunological memory. 96 -98 manipulation of one late co-stimulatory pathway, therefore, leaves others intact to seed the memory t-cell pool. 97 this suggests that a full compliment of late co-stimulators may actually be prolonging t-cell survival and contributing to pathology. inflammatory cytokines, such as those abundantly expressed in the airways during infection, are known to increase ox40l on apcs. 99 due to lack of immune organization in the airways, late co-stimulatory molecules and their ligands may be downregulated too late to avoid bystander tissue damage. the observation of ox40l on inflamed endothelium highlights the difficulty in separating the effects of immune modulators on cell survival vs. cell recruitment, 100, 101 especially when ligation of ox40l on endothelial cells induces secretion of chemokines. 101,102 4-1bb, like ox40, is restricted to late-effector t cells and its absence or blockade impairs cd8 + t-cell responses to influenza, 103 although the effect on associated respiratory pathology is not yet known. care must be taken, however, since not all inducible co-stimulators result in a beneficial outcome when blocked. during lung influenza infection, icos blockade impairs respiratory t cells to such an extent that the virus escapes clearance. 32 this is similar to treatment of influenza-infected mice with a ctla4-ig fusion protein (that blocks cd28 binding to b7 molecules) 104 and cd40l-knockout mice infected with c. neoformans 105 (where macrophage antimicrobial strategies are impaired). it is likely that targeting cd27 may also produce untoward side effects, since it is expressed on resting na ï ve and memory t cells and is crucial for the formation cd8 + t-cell responses. 96, 106 perhaps the defining line for therapeutic potential should be placed between those late co-stimulators that absolutely depend on t-cell receptor and constitutive cd28 signaling for their expression and those that can appear in a bystander fashion in the presence of inflammatory cytokines. the likely effect of co-stimulatory blockade and the site where manipulation may have the most effect is shown in figure 3 . in addition to late co-stimulatory molecules, a number of other pathways affect the longevity of lung inflammation during acute infection. whether apoptosis is beneficial or harmful depends on the specific infection and the dominant cell type that mediates its clearance. 107 as a general rule, apoptosis favors the host in chronic and acute intracellular bacterial and viral infections (as the process clears the pathogen), but is detrimental for extracellular bacteria. 107 for example, during infection of rats with pneumocystis , alveolar macrophage apoptosis delays clearance of the organism that can be improved by administering caspase-9 inhibitors. 108 similarly, apoptosis of airway epithelial cells via fas / fas ligand is essential to prevent dissemination of pseudomonas aeruginosa . 109 however, during influenza infection it may be more advantageous to reduce cell survival, especially in the case of tnf producing cd8 + t cells. tnf receptor-ii and very late antigen--1 synergize to protect cd8 t cells in the influenza virus infected airways from apoptosis, 110 whereas engagement of qa-1b by cd94 / nkg2a transmits a negative signal that limits immune pathology. 111 it may, therefore, be possible to resolve t-cell inflammation before bystander tissue damage occurs by blocking or enhancing these surface receptors once the viral load has reduced. this would only be useful, however, if the strategy specifically targeted a defined cell population, since apoptosis of airway epithelial cells and leukocytes may be linked to the pathology observed in those infected with highly pathogenic avian influenza. 112, 113 furthermore, apoptosis leading to systemic lymphopenia, 114 observed during influenza infection, may assist virus propagation and survival. other respiratory pathogens, such as cytomegalovirus and parainfluenza virus, use antiapoptotic strategies to prolong survival of the cells they infect. 115 chlamydia blocks apoptosis by affecting the release of cytochrome c from mitochondria. 116 the extracellular pathogens pseudomonas cepacia figure 3 the location of the dominant site of action of co-stimulatory molecule blockade during acute lung infection. this model assumes that antigen specific t cells are primed in the lung-associated lymph nodes (e.g., mediastinal). as such blockade of cd28 on t cells using the b7 competitor ctla4:ig will affect t-cell priming in organized lymphoid tissue ( a ). the same is also likely to be true for icos that is induced rapidly after t-cell receptor and cd28 signalling. by contrast, ox40 and 4-1bb are expressed at very low levels in the lung-associated lymph nodes but upregulated in the lung parenchyma and airways, most likely due to re-recognition of antigen in situ and / or the inflammatory cytokine environment. blockade will, therefore, impact these sites rather than the lymph nodes ( b ). note that ox40 and 4-1bb blockade may also affect primed t-cell migration, as their respective ligands are present on inflamed endothelium ( c ). in the airways, the dominant effect will be to allow activation-induced cell death to progress; in the parenchyma, however, it may be a combination of activation-induced cell death ( d ) and migration. and s. pneumoniae cause apoptosis of neutrophils and airway epithelial cells ( table 1 ) , 117 respectively, to aid survival. modulation of apoptosis is, therefore, complicated; what may benefit the host for one infection would compromise it to another. the recruitment and trafficking of leukocytes in response to inflammation is a tightly regulated process that can tip the balance between protection from infection and immune mediated damage in the lung. in brief, it involves slow rolling (concomitant to the activation of leukocytes), an increase in integrin expression and avidity to regulate the rolling arrest, adhesion strengthening as well as spreading, and intravas-cular crawling culminating in paracellular or trans-cellular migration. 118, 119 since excessive cell recruitment is a feature of many acute lung infections, targeting specific molecules involved in any of the above-mentioned events may be beneficial. blocking monoclonal antibodies against the integrin very late antigen-4 (natalizumab) 120 and lymphocyte function-associated antigen-1 (efalizumab) 121 are currently used to treat inflammatory autoimmune disorders and crohn ' s disease, but are also immunosuppressive and have not been studied in the context of acute respiratory infection. 122 the potential benefit of blocking chemokines or their receptors using competitive blockers or antagonistic compounds is well described (reviewed by glass et al 123 ransohoff 124 ). secreted chemokines bind glucosaminoglycans on endothelial cells, forming chemoattractant gradients that direct cells to inflammatory sites, 125 and are classified into constitutive homeostatic and inflammatory (requiring a proinflammatory stimulus such as ifn-, tnf, or microbial products) chemokines. 126 chemokine receptors are transmembrane g-protein-coupled molecules that trigger a signal transduction event resulting in activation and firm adhesion of the migrating cell. 127 the interactions between chemokines and their receptors are functionally redundant; many chemokines bind the same receptor and one chemokine can bind several receptors. as such it can be difficult to design reliable therapeutics that disrupt the interaction of one particular chemokine with its receptor. 128 therapeutic administration of antibodies that block macrophage inflammatory protein-2 during influenza infection reduces neutrophil recruitment by 49 % and improves lung pathology without altering viral clearance. 129 however, this strategy requires testing in co-infection models, since neutrophils are critical for clearance of most respiratory bacteria that commonly cause secondary pneumonia in the presence of influenza virus. 130, 131 rantes (ccl5) is another potential target produced by respiratory epithelial cells during a variety of viral infections. rantes induces ccr1-, ccr3-, and ccr5-expressing t cell, eosinophil, and monocyte recruitment to the lung. 132 during rsv infection, ccl5 is expressed by infected epithelia and resident macrophages and secreted into the airway during the first 48 h of infection. production is then taken over by newly recruited t cells. blocking this chemokine with a competitive inhibitor (met-rantes) 133 during a primary rsv infection reduces lung immunopathology. however, heightened cell recruitment occurs during homologous rsv re-challenge, suggesting that manipulation during the first infection severely compromises immunological memory. 134 the same strategy has been tested in a mouse model of pneumonia virus infection. this highly lethal mouse pathogen induces a disease closely resembling severe human rsv infection in man, that is abrogated by co-administration of the antiviral agent rivabirin and met-rantes. 135 in contrast to inhibition of chemokines during virus-induced lung inflammation, some infections may require their administration. p. aeruginosa lung infection causes airway neutrophil infiltration that rapidly apoptose and become toxic. administration of recombinant monocyte chemoattractant protein-1 / ccl2 recruits and activates lung macrophages that clear apoptotic neutrophils before they cause pathology. 136 ifninducible protein 10 (cxcl10) is critical not only for clearance of klebsiella pneumoniae , 137 but is also produced during lung viral infection where neutralization may be beneficial. 138 many chemokine receptors are also increased during respiratory viral infection; cxcr3, for example, is upregulated during murine gammaherpes virus b8 infection, and its absence delays viral clearance. 139 antagonism of ccr1 reduces mortality of pneumovirus infection of mice, 140 and absence of ccr1 also prevents the rsv-induced exacerbation of asthma. 141 relatively little has been accomplished in this area, especially therapeutically, however, due to the paucity of reagents available for chemokine-receptor blockade. another method for altering lymphocyte migration is to manipulate sphingosine 1-phosphate receptors that are required for egress of lymphocytes from the thymus and peripheral lymph nodes, and impact on vascular permeability. fty720 is a novel synthetic immunosuppressive drug that inhibits lymphocyte emigration from lymphoid organs by binding and activating sphingosine 1-phosphate receptors. 142 sphingosine 1-phosphate is known to play a role in endotoxin-induced lung injury by affecting endothelial barrier function, 143 and is currently in trial for a variety of inflammatory disorders, including transplantation, 144 but is yet to be tested in acute lung infection. modulation of lung innate immunity represents another potential therapeutic target. however, once again, few studies have tested manipulating it after the onset of symptoms during acute respiratory infection; most examine the impact of an innate immunity-associated molecule using gene-depleted animals or prior to infection. manipulation of innate immunity may seem to be akin to " shutting the stable door after the horse has bolted, " since innate immunity is activated first and drives subsequent adaptive immune responses. however, each epithelial cell or macrophage infected sends off the next wave of an immune response. at the time of clinical presentation, therefore, both innate and adaptive immunity will be in full swing. manipulating innate immunity at this stage will assist the resolution process, but whether inhibition or activation is required will depend on the pathogen. one way to temper innate immunity is to mature the lung microenvironment or instill probiotic microbes that would compete with the survival of pathogenic microorganisms. oral administration of lactobacillus casei during lung s. pneumoniae infection is protective, resulting in more rapid clearance, a shorter period of septicemia, and decreased s. pneumoniae load in the lungs. this benefit is attributed to increased neutrophils, myeloperoxidase, and il-10 that limits lung tissue damage and is likely mediated by migration of mature apcs from the gut to the lung that are better equipped for bacterial clearance. 145 prior infection in the lung also has a beneficial effect on some subsequent acute respiratory infections through modification or maturation of the microenvironment. 146 -151 prior influenza infection, for example, reduces subsequent toll-like receptor responsiveness of alveolar macrophages for prolonged periods of time. 152 administration of microbial products, such as cpg dna or a modified bacterial labile toxin (ltk63), also protects against an array of subsequent respiratory pathogens, 153,154 as do chronic or acute infections in distant sites. 155 -157 the ability of pathogen-derived proteins to modify acute respiratory infections, however, is yet to be tested therapeutically. resveratrol, a polyphenolic compound found in red wine, inhibits nuclear factor-b activation, decreases mortality and pro-inflammatory cytokines (tnf, il-1 , and il-6) to serratia marcescens pneumonia in rats. though this strategy increases neutrophil numbers, they resolve more rapidly. 158 similarly, review an acidic polysaccharide compound from cordyceps militaris , an insect-borne fungus, has anti-viral properties in a murine influenza infection model. 159 intranasal administration of the fungal polysaccharide decreases mortality and influenza viral titers, while increasing lung pro-inflammatory cytokines. in vitro , influenza infected macrophage cell lines treated with resveratrol display enhanced inducible nitric oxide synthase and nitric oxide (no), suggesting that this compound may function through non-specific stimulation of alveolar macrophages in vivo . 159 enhanced innate and adaptive immunity and reduced microbial load is also observed upon therapeutic administration of retinoic acid in mice infected with mycobacterium tuberculosis . 160 the increased numbers of macrophages, natural killer and t cells, and increased expression of ifn-, tnf, inducible nitric oxide synthase, il-10, and cxcl10 may also benefit other acute respiratory bacteria or fungi, though during viral infection it would be predicted to be detrimental. acute respiratory infections are yet to be examined. a critical pathway for clearance of pathogens and infected lung epithelial cells is via no and reactive oxygen and nitrogen species. no contributes to host defence and mediates both proand anti-inflammatory effects (reviewed in 161, 162 ) . as with many of the therapeutic treatments discussed, the timing and extent of modulation of no and associated free radicals is critical. while there is no clear consensus in the literature, there are a few examples of successful intervention in this pathway. in rats with p. aeruginosa pneumonia, treatment with inhaled no post-infection improves bacterial clearance through direct bactericidal effects, increased recruitment of neutrophils to the airways 163 or enhanced endothelial permeability. 164 during k. pneumoniae infection of rats, inhaled no also suppresses bacterial replication and decreases lung intercellular adhesion molecule-1 expression, myeloperoxidase activity, tnf levels, and nuclear factor b activity. thus, no has many paracrine effects on inflammatory cells, but also promotes bacterial clearance. 165 during viral infection, the impact of no manipulation is less clear. inhibition of no during rsv infection reduces pulmonary inflammation and bystander tissue damage, but viral replication increases. 166 similar therapeutic strategies have been employed to reduce local concentration of reactive oxygen species and reactive nitrogen species, which are produced by infected lung epithelium and macrophages. during rsv infection of mice, administration of the antioxidant, butylated hydroxyanisole, decreases illness scores, weight loss, and lung neutrophil recruitment. again a multi-factorial attenuation of inflammation occurs. 167 the same is observed in influenza-infected mice treated with a free-radical scavenger, manganese superoxide dismutase, within 48 -96 h of infection. this enzyme has potent anti-inflammatory properties, resulting in less lung consolidation and improved arterial oxygen saturation, presumably as a result of decreased tissue damage. 168 reduced tissue damage also occurs in mice lacking superoxide dismutase or treated therapeutically with a manganic porphyrin that scavenges reactive oxygen species. 53 therefore, inhibition of reactive oxygen species / reactive nitrogen species and no is beneficial for acute respiratory viral infection, but likely to be detrimental for concurrent respiratory bacteria. modulation of immune-receptor signaling is in its infancy with regards to acute respiratory infection and may be limited by (a) the toxicity / safety profile of available drugs, (b) formulation challenges for in vivo delivery, (c) a lack of specificity due to shared receptor associations, and (d) problematic pharmacokinetics (sustained blockage will be detrimental for protection against infection and drugs may have to be delivered locally to prevent systemic effects). the only truly therapeutic manipulation, to date, is abrogation of airway fluid clearance during rsv infection, caused by interaction of uridine triphosphate with purinergic receptors, by post-infection administration of an active metabolite of leflunomide, a77-1726. leflunomide restores airway fluid clearance, providing symptomatic relief and reduced lung inflammation and hypoxemia, without impairing viral replication or clearance. 169 although signaling molecules associated with pattern recognition and cytokine receptors are well described, little is known about the complexity of innate pathways induced by a whole pathogen, especially in the lungs. 170 controlling the signaling pathways leading to exuberant inflammation is of major interest, but a balance needs to be struck to maintain host defence against infection. a feasible approach might be by treating patients at the peak of inflammation where immune mediators are in excess and using drugs with a short half-life. this is a promising strategy, but, to our knowledge, no drugs targeting signaling components are reported efficacious in human lung infection, although they are under development. 171 a comprehensive review of relevant inhibitors of inflammatory signaling pathways can be found elsewhere. 172 receptors recognizing pathogens such as toll-like receptors or nod-like receptors, but also tnf and il-1 , which are often involved in amplification of the inflammatory response, are potent activators of nf-b. 173 nf-b induction is a key factor triggering inflammation in the influenza-infected epithelium. 174 mice treated with a cell-permeable peptide that reduces nf-b levels, via an effect on i-kappa-b kinase beta, reduces pulmonary rsv-induced inflammation. 175 again, this strategy may negatively affect respiratory bacteria, since intra-tracheal adenovirus delivery of a dominant nf-b inhibitor impairs clearance of respiratory p. aeruginosa , 176 despite the reduced inflammation observed with purified bacterial products. 177, 178 peroxisome proliferator-activated receptor-is a nuclear receptor involved in the stress response during lung injury and attenuates inflammatory responses by inhibiting nf-b. 179, 180 many steroids target peroxisome proliferator-activated receptor-and show encouraging results in models of lung inflammation. 172 a series of natural and synthetic ligands for these receptors have been developed and rsv-specific responses in human lung epithelial cell lines are reduced by some of these agonists. 181 mitogen-activated protein kinase p38 and c-jun-n-terminal kinase pathways may also provide future suitable targets. activation of the p38 pathway is often associated with induction of nf-b (in toll-like receptor responses, for example) and is thought to maintain inflammatory responses by stabilizing cytokine mrna. 182 inhibiting the mitogen-activated protein kinase pathway may, therefore, favor termination of inflammation. inhibitors of p38 reduce the epithelial disruption caused by rsv and bordetella pertussis . 183, 184 again, inhibition of this pathway, to our knowledge, although tested in asthma, 185 has not been tested in lung infection models. another approach to reduce pathogen-induced immunopathology during acute infection is to target signaling molecules involved in cell migration. downstream of chemokine receptors is the phosphatidylinositol 3-kinase, which activates protein kinase c and rho gtpases. 186 phosphatidylinositol 3-kinase inhibitors reduce the recruitment of neutrophils and t cells in vivo . 187 -189 although this treatment is efficient in alleviating chronic inflammation such as asthma, 190 it is not validated for acute microbial infection. in addition, use of phosphatidylinositol 3-kinase inhibitors may interfere with development of the innate response to bacteria. 191 with the identification of each new cytokine, a series of papers describing their manipulation in models of acute infection has followed. all of them cannot be detailed here due to space constraint, but our discussion can be limited to those that have been tested therapeutically in acute lung infection models. the first, type-i ifn, plays such an important role in limiting viral replication that they have developed strategies to avoid it. during rna virus lung infection ifn-is produced predominantly by alveolar macrophages (or, to a lesser extent, pdcs) whose depletion impairs viral clearance. 192 dosing with ifn-and a double-stranded rna ifn-inducer, 4 h after sars coronaviruas infection, reduces lung viral titers. 193 similarly, rsv or human metapneumovirus-infected balb / c mice, treated intranasally with recombinant ifn-, have reduced lung viral titers and inflammatory disease as compared with untreated controls. 194 prophylactic treatment of sars coronavirus-infected macaques with pegylated ifn-significantly reduces viral load and pulmonary damage; post-exposure treatment is effective, although producing intermediate results. 195 recombinant ifn-, therefore, appears beneficial for reducing viral replication and associated pathology when administered early after infection. the influence of concomitant bacterial infection requires examination. other early innate cytokines important in respiratory viral and bacterial infections include il-1, il-12, and tnf. their blockade or promotion, however, is complicated by the fact that respiratory bacteria tend to require them for clearance. for example, rhinovirus induces il-1-receptor antagonist, il-1ra, from airway epithelial cells, which facilitates resolution of inflammation. 196 however, il-1ra enhances bacterial outgrowth in the lungs of mice with pneumococcal pneumonia, without the benefit of reducing the host response. 197 therefore, this pathway is predicted to be good for one lung infection but bad for another. blockade of il-12 is another example where neutralization benefits the severity of lung viral infection, 198, 199 but impairs clearance of lung histoplasma capsulatum 200 and legionella pneumophila 201 infections. the list continues with neutralization of tnf, which benefits immune pathology induced by influenza and rsv infection, 81, 202 but not respiratory bacteria or fungi, 203, 204 especially if treatment is prolonged (although this will depend on the precise strategy used 205 ). webster and coworkers reported recently that absence of tnf and il-6 is of no benefit during murine influenza h5n1 infection. 206 however, up to 50 % survival was observed in some experiments and the gene-depleted animals used may harbor other developmental abnormalities. viruses induce local production of ifn-by t and non-t cells in the respiratory tract, and its neutralization not only reduces local lung cellularity and systemic humoral responses to influenza virus infection in mice, 207 but may also delay viral clearance. 208 ifn-is also required for clearance of s. pneumoniae . 209 it would appear that the new cytokine on the block, il-17, may also present opposing effects in viral and bacterial lung infection. suitable reagents are yet to be developed for il-17 neutralization, but in vivo blockade of il-23p19 alone, or in combination with il-23 / il-12p40 (required for th17 development), significantly reduces mycoplasma pneumoniae -induced il-17 and subsequent bacterial clearance, possibly via reduced neutrophil activity. 210 k. pneumoniae clearance also depends on il-17, 211 but the influence of il-17 or il-23 neutralization on respiratory viral infection is unknown. administration of immune suppressive cytokines has been considered for infection induced lung inflammatory disease and encountered similar problems. il-10 neutralization increases survival of mice infected with k. pneumoniae . 212 in contrast, influenza induces indoleamine 2,3-dioxygenase and il-10 production, which may limit lung inflammation. however, treatment with an indoleamine 2,3-dioxygenase inhibitor (that would reduce il-10) induces a 20-fold reduction in lung s. pneumoniae load. 213 intranasal il-10 treatment of rsv-infected mice reduces lung nuclear factor b dna-binding activity, chemokine gene expression, and airway inflammation. 175 similarly, administration of tgf--encoding plasmid reduces inflammation to viral and fungal lung pathogens, but, without exception, prevents their clearance. 54 although a plethora of strategies have been used to modulate lung inflammation during acute infection, few are tested therapeutically after the onset of clinical symptoms, and even less are tested in models of common co-existing lung pathogens. hundreds of immune modulators are, therefore, beneficial during influenza infection, but what of the bacteria that sometimes accompany them? would immune therapeutics work best in combination with antibiotics? equally, selection of immune modulators requires precision in determining exactly what the patient is infected with. in the absence of this knowledge, we may apply a beneficial strategy to one supposed infection, but create an altogether different type of problem. several gaps remain in our knowledge of how immune homeostasis is maintained in the respiratory tract, inflammatory pathways that overcome review them, and the precise effector / memory phenotype of immune cells within the airways and lung parenchyma. controversy also still surrounds the potential of " acute " respiratory infections to persist, since detection of pathogen genome is common, but few studies have been able to demonstrate classical reactivation long after the primary infection. should persistence exist then, depending on the nature of the persisting organism, immune modulators may cause their reactivation. the development of sensitive tools for pathogen detection, and elucidation of specific gene expression patterns in patients with acute infection, 214 mean that future use of targeted immune modulators is not impossible as long as we are able to strike a balance between immune 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clearance but does not switch the response to a t helper cell 2 phenotype role of interferon-gamma in valpha14+ natural killer t cell-mediated host defense against streptococcus pneumoniae infection in murine lungs il-23-dependent il-17 production is essential in neutrophil recruitment and activity in mouse lung defense against respiratory mycoplasma pneumoniae infection cutting edge: roles of toll-like receptor 4 and il-23 in il-17 expression in response to klebsiella pneumoniae infection neutralization of il-10 increases survival in a murine model of klebsiella pneumonia infl uenza-induced expression of indoleamine 2,3-dioxygenase enhances interleukin-10 production and bacterial outgrowth during secondary pneumococcal pneumonia gene expression patterns in blood leukocytes discriminate patients with acute infections comparison of murine nasal-associated lymphoid tissue and peyer's patches isolation and characterization of mouse nasalassociated lymphoid tissue role of type 1 t helper cells in the resolution of acute streptococcus pneumoniae sinusitis: a mouse model key: cord-296635-8r3tm966 authors: breed, andrew c.; breed, martin f.; meers, joanne; field, hume e. title: evidence of endemic hendra virus infection in flying-foxes (pteropus conspicillatus)—implications for disease risk management date: 2011-12-14 journal: plos one doi: 10.1371/journal.pone.0028816 sha: doc_id: 296635 cord_uid: 8r3tm966 this study investigated the seroepidemiology of hendra virus in a spectacled flying-fox (pteropus conspicillatus) population in northern australia, near the location of an equine and associated human hendra virus infection in late 2004. the pattern of infection in the population was investigated using a serial cross-sectional serological study over a 25-month period, with blood sampled from 521 individuals over six sampling sessions. antibody titres to the virus were determined by virus neutralisation test. in contrast to the expected episodic infection pattern, we observed that seroprevalence gradually increased over the two years suggesting infection was endemic in the population over the study period. our results suggested age, pregnancy and lactation were significant risk factors for a detectable neutralizing antibody response. antibody titres were significantly higher in females than males, with the highest titres occurring in pregnant animals. temporal variation in antibody titres suggests that herd immunity to the virus may wax and wane on a seasonal basis. these findings support an endemic infection pattern of henipaviruses in bat populations suggesting their infection dynamics may differ significantly from the acute, self limiting episodic pattern observed with related viruses (e.g. measles virus, phocine distemper virus, rinderpest virus) hence requiring a much smaller critical host population size to sustain the virus. these findings help inform predictive modelling of henipavirus infection in bat populations, and indicate that the life cycle of the reservoir species should be taken into account when developing risk management strategies for henipaviruses. hendra virus (hev) and nipah virus (niv) are paramyxoviruses of the genus henipavirus with pteropid bats (i.e. flying-foxes; pteropus sp., family pteropodidae) being the primary wildlife reservoir [1] . evidence of henipavirus infection has been found across the range of pteropid bats from eastern australia, southeast asia, bangladesh, india and madagascar [2] . henipavirus infection has also been found to be present in eidolon helvum, a species of fruit bat (family pteropodidae) that occurs throughout sub-saharan africa [3, 4] . the potential to cross species boundaries from bats to domestic animals and humans causing fatal infection appears to be a consistent feature of henipaviruses wherever they have caused disease (australia and asia). given that over two billion people live in the area where pteropus or eidolon bats are present, even sporadic spillover to humans may result in a significant number of human infections. henipaviruses have the potential to infect a wide range of mammalian species, and hendra virus has spread from flying-foxes to horses in australia on at least 20 reported separate occasions (five involving horse-human transmission), most recently in 2011 [5, 6, 7] . seven humans have become infected with hev via contact with infected horses, resulting in four fatalities [5, 8, 9] . in peninsular malaysia and singapore during 1998 and 1999, nipah virus infected pigs and humans resulting in the death of over 100 humans and the culling of over one million pigs [10] . since that time, there have also been at least 10 outbreaks of niv disease in humans in bangladesh and india, with the resultant death of over 140 people. there is also clear evidence of humanto-human transmission of niv [11, 12] . in spite of the major health concerns, the knowledge of the epidemiology and ecology of these viruses is limited [1, 13, 14] . how the viruses are maintained in bat populations is not fully understood, nor is how the viruses avoid extinction as their host species become immune. in addition, whether these viruses are predominantly horizontally or vertically transmitted is also uncertain [12, 15, 16] , with the apparent viral latency and recrudescence in some human hev and niv infections suggesting that henipavirus infection dynamics may differ significantly from the closely related morbilliviruses [17] . a previous study by plowright et al. [14] on the infection dynamics of hev in the little red flying-fox, pteropus scapulatus, in the northern territory of australia suggested that viral transmission may be predominantly horizontal, with pregnancy and lactation suggested as risk factors for infection. however, plowright et al. [14] sampled multiple colonies over time, leaving the possibility that sampling was not confined to a single population. here, we focus on the transmission of hev in a single colony of flying-foxes over a 25-month period that was approximately 10 km from the location of a spillover of infection to a horse and human in october 2004 [5, 8] . we sampled the spectacled flying-fox, pteropus conspicillatus, a species which is restricted in distribution to the wet tropics bioregion of north queensland [18] . we present data on the infection dynamics of hev within this flying-fox colony over the 25-month period. we investigated the association of antibody response to the lifecycle stage of the host and the hypothesis that hev is maintained by episodic infection with periodic virus outbreaks taking place. a total of 521 pteropus conspicillatus were sampled over the six sampling sessions with an overall seroprevalence to hendra virus of 56% (95% c.i. 51-60). the logistic regression model that included age, sampling session, sex, reproductive status and weight best fitted variance in seroprevalence, thus these variables were analysed in more depth (daic c = 0.00; v i = 0.57; all other models daic c .2 and v i ,0.2; appendix 1 in supplementary information). models that included two-and/or three-way interactions had daicc.5, thus were not investigated further. weight and forearm length predictor variables were highly correlated (r 2 = 0.72), but the model that included forearm length and not weight had limited support (table s1) . temporal variation. seroprevalence steadily increased over the six sampling sessions from 44.7% in january 2005 to 69.4% in february 2007 ( figure 1 ; table s2 ). variation with sex and reproductive status. seroprevalence of female bats did not differ significantly from male bats (female: 58.7%; male: 53.7%; log binomial regression p = 0.25; table 1 ). however, pregnant females had a significantly higher seroprevalence than both males and all other female bats (pregnant females: 70.3%; all other bats: 54.6%), and were 1.3 times more likely to have antibodies to hev than the rest of the bats sampled (95% ci: 1.03-1.61; log binomial regression p,0.05). bats sampled in early lactation had a significantly higher seroprevalence than male and all other female bats (early lactation: 75.0%; all other bats: 54.9%). hence, those in early lactation were 1.4 times more likely to have hev antibodies than the others sampled (95% ci: 1.05-1.78; log binomial regression p,0.05). when seroprevalence was compared among the reproductive categories of adult females, pregnant bats and those in early lactation had a significantly higher seroprevalence than nonreproductive adult females (pregnant: 26 variation with age. seroprevalence was highest in bats of the adult category (60.3%), followed by juveniles (58.3%), while sub-adults had significantly lower seroprevalence (39.8%) than both adults (logistic regression p,0.001) and juveniles (logistic regression p,0.05; figure 2 ; table s3 ). variation with bodyweight and forearm length. seroprevalence did not show statistically significant variation with bodyweight and forearm length although a non-significant linear trend was observed between seroprevalence and bodyweight (table s4 ). hendra virus antibody titre levels were determined by serial dilution of the sera in the virus neutralisation test. significant differences in titre levels were found according to sampling session (kruskal-wallis test p,0.0001), bodyweight (kruskal-wallis test p,0.0001), pregnancy status (wilcoxon test p,0.001) and sex (wilcoxon test p,0.01; table s5 ). age, forearm length and lactation status were not significant risk factors for antibody titre level (kruskal-wallis and wilcoxon tests p.0.05). antibody levels varied greatly across the sampling sessions with animals sampled in january 2005 having a median titre of 15 (mean rank 25.7) followed by consistent increases to a peak median titre of 80 (mean rank 205.6) in september 2006, followed by a drop to 30 (mean rank 58.8) in february 2007, the final sampling session. individuals of greatest bodyweight (.850 g) had the lowest antibody titres with a median of 20 (mean rank of 113.3). females had significantly higher antibody titres than males (median titres 40 and 20; mean ranks 161.2 and 134.2, respectively) with the highest titres observed for pregnant females (median titre 80; mean rank 197.2). those in any stage of lactation did not show a significantly higher antibody titre than the rest of the bats sampled. previous studies have suggested that henipaviruses are maintained in flying-fox populations through episodic infection in a metapopulation structure, and do not persist endemically within a single population [14, 19] (see figure 1 , panel c). our findings do not support this hypothesis, but support an alternative pattern of endemic infection in the population. this endemic infection dynamic is consistent with a study on viral excretion of niv in pteropus lylei in thailand, where seasonal excretion of virus was observed to occur from the same small colonies each year [12] . our findings on hev antibody titre levels show a peak in september 2006 (median titre = 80; mean rank = 205.6) when all adult female bats sampled were at a late stage of pregnancy. this is plausibly consistent with a ''boosted'' immune response subsequent to the previous sampling session (march 2006: median titre = 40; mean rank = 73.96). the following sampling session showed a decrease in titre level (february 2007: median titre = 30; mean rank = 58.8), but an increase in seroprevalence from 62.1% in september 2006 to 69.4%. this finding is consistent with a period of increased viral transmission during late pregnancy that had resolved by the time the majority of females were lactating, as evidenced by the consequent increased seroprevalence. indeed pourrut et al., [20] have suggested that altered immune function in late pregnancy may cause a transient surge in viral replication of filoviruses in african fruit bats. our finding that late pregnancy and lactation were risk factors for hev seropositivity are concordant with results presented by plowright et al. [14] on p. scapulatus. furthermore, the reproductive cycle in other bat species has been linked to seropositivity and viral activity of filoviruses, coronaviruses, lyssaviruses and astroviruses [20, 21, 22] . our study identified the highest seroprevalence in the first few weeks of lactation, indicated by a seroprevalence of 75.0% in females carrying pups ( figure 2 ). our study also supports the conclusions of field (unpublished data) and plowright et al. [14] that maternal transfer of hev antibodies to juveniles likely occurs, evidenced by the higher seroprevalence of juveniles (58.3%) than sub-adults (39.9%), and a correspondingly higher seroprevalence of adults (60.3%). these findings are consistent with horizontal transmission of the virus, however the observed seroprevalence pattern does not preclude the occurrence of vertical transmission. vertical transmission may contribute to viral persistence in bat populations, and there is evidence that vertical transmission of hev occurs from experimental infection studies of flying-foxes and guinea pigs [15] and from natural infection in wild flying-foxes (field unpublished data) [16] . numerous viruses can be transmitted both horizontally and vertically (e.g. transplacentally), including human polyoma virus, bovine viral diarrhoea virus, feline leukaemia virus and parvoviruses (porcine, canine and feline) [17] . for females to be classified as adult in our study they must have shown signs of prior lactation (i.e. enlarged nipples; figure 2), and hence a previous pregnancy and lactation. our finding that hev seroprevalence in early lactation was significantly higher than adult females that were not pregnant or lactating (early lactation; 75.5%; not pregnant or lactating: 48.7%; p-value = 0.047) is evidence for a decline in hev seroprevalence in females in the life stage following lactation. such a decline suggests that detectable immunity to hev is not long lived in p. conspicillatus, and the pattern seen may reflect seasonal variation in response to repeated exposure. this variation is contrary to the assumption that hev induces long-lived detectable immunity in p. conspicillatus and p. poliocephalus (e.g. [14] ), and suggests that the transmission dynamics of henipaviruses may be different to those of the closely related morbilliviruses. indeed, the mechanism of survival of henipaviruses at the population level appears more likely to be one of endemic infection, perhaps similar to that found in bovine viral diarrhoea virus, classical swine fever or some herpes viruses utilising persistent infection, or vertical transmission, as found in arenaviruses or retroviruses [17] . these patterns of infection require much smaller critical host population sizes, in contrast to viruses that demonstrate an acute self-limiting episodic infection pattern determined by: a build-up of susceptibles, introduction of virus, and environmental conditions that promote spread (e.g. measles, newcastle disease virus or canine distemper virus; [17] ; see figure 1, panel b) . a previous serial cross-sectional study by plowright et al. [14] over a 16-month period on little red flying-foxes, p. scapulatus, sought to determine the factors that drive hev spillover. their study suggested that age, sex, body size, pregnancy, lactation, season and mating behaviour were possible risk factors for hev infection, and that horizontal transmission was the major mode of transmission between individuals. they also reported a rapid seroprevalence decline between two successive sampling sessions. however, given their sampling at multiple locations, the expansive geographic distribution and highly nomadic nature of p. scapulatus [23] , it is plausible that they were not sampling the same population over time. in contrast, the species in our study, p. conspicillatus, is not a nomadic species and has a restricted distribution to the wet tropics of northeast queensland [18] . furthermore, our study was conducted within 10 km of a location where an hev outbreak occurred in october 2004 [5] , and we collected samples from a single colony of p. conspicillatus on six separate occasions over a 25-month period. consequently, we are confident that we followed the infection dynamics of a single population of p. conspicillatus over the study period. nonetheless, interpretation of results from studies in wild animal populations should be made with care. capture of bats by mist-netting provides a statistically non-random sample of the population, and the practicalities of sampling from a roost site of many thousands of individuals also precludes following individuals over time. to counter these issues, we sought to investigate potential bias and confounding effects where possible. future studies on henipavirus infection dynamics in wild bats may benefit from: permanent marking of individuals to identify possible repeated capture and sampling of some animals; improved diagnostic capabilities to increase the probability of detection of viral shedding; and improved telemetry methods to enhance the understanding of movement of individuals between roost locations. our findings do not support the episodic infection hypothesis for hev persistence in our study population. rather we suggest that endemic infection of p. conspicillatus occurs, perhaps with periodic pulses of viral transmission associated with late pregnancy and early lactation. the consistent increase in seroprevalence over the duration of our study, together with increasing titres over the first five sessions followed by a drop in titre in the last session, also suggest the presence of inter-annual factors may be affecting viral transmission. an increase in viral transmission associated with pregnancy in flying-foxes is plausibly concordant with the temporal pattern of some hev incidents in horses in australia [5] , and of niv outbreaks in humans in bangladesh and india [12] . this pattern suggests that the risk of henipavirus transmission from flying-foxes to domestic animals and/or humans is higher during the gestation period of flying-foxes. thus, it is plausible that spillover risk may be uniformly spatially distributed wherever pregnant flying-foxes are present. the observed spatial clustering of henipavirus incidents may be confounded by: surveillance intensity (passive surveillance is the only method used, with heightened awareness of disease likely in areas where previous incidents have occurred); variation in flying-fox population density (there is evidence of increasing movement of flying-foxes in eastern australia into urban and rural areas [24] ); variation in horse density and husbandry practices; and as yet unidentified predisposing ecological or environmental factors (e.g. climate). a scenario of persistent henipavirus infection with viral latency and recrudescence in flying-foxes has been proposed by field (unpublished data) and sohayati et al., [25] . viral latency and recrudescence has also been shown to occur in a human hev case [26] , and at least 12 human niv cases [27] . this infection dynamic could lead to the endemic infection pattern seen in our study. however, it is plausible that different social structures of host populations (e.g. panmixia, metapopulation, seasonal aggregation) may favour different mechanisms of maintaining infection at the population level (e.g. predominantly horizontal transmission, predominantly vertical transmission, seasonal explosive outbreaks, repeated viral excretion by persistently infected individuals). consequently, population structures and mechanisms of maintenance of infection may reflect the biology of the host species and level of ecological disruption, rather than only the biology of the virus. as such, it is likely that different host populations may have varying levels of risk of infection spillover to domestic animals and/or humans. an improved understanding of the infection dynamics of henipaviruses in bat populations should facilitate the development of effective risk management strategies for disease spillover from bats to domestic animals and/or humans. here we show that hev infection in a population of pteropus conspicillatus is likely to be endemic rather than episodic, as previously proposed for hev in flying-foxes. we also present evidence for seasonal viral activity suggesting that immunity to the virus may wax and wane on a seasonal basis. these findings should inform disease risk management and approaches for modelling henipavirus infection in bat populations. if the ongoing threat that these viruses pose to public health is to be mitigated, further work is required to clarify the principal mode(s) of transmission of henipaviruses in bats, and to comprehensively determine how these viruses persist in their reservoir hosts. all study animals were captured and sampled at the gordonvale roost site (145u46.749e, 17u4.869s ). this site is in a 4.5 hectare mixed woodland and forest remnant, surrounded by sugarcane plantations and suburban housing. it has been permanently occupied by pteropus conspicillatus for at least ten years, usually containing 40,000 to 60,000 individuals, constituting approximately 20% of the australian population of this threatened species [18, 28] . this is the closest known flying-fox colony to the property where the hev spillover event occurred in october 2004 [5] . the australian population of p. conspicillatus is geographically isolated from other populations of this species in northern papua new guinea and the moluccan islands of indonesia [29] . flying-foxes were caught in mist nets, anaesthetised with isoflurane (isoflurane, laser animal health pty limited) and oxygen via an anaesthetic machine using the protocol of johnson et al. [30] for sampling. data collected were sex, bodyweight, forearm length, approximate age and reproductive status according to descriptions detailed in table s6 . animals were marked with coloured acrylic lacquer on their hind claws to prevent resampling within the same session, and were then released at the site of capture after recovery from anaesthesia. age classification of sampled bats was performed as described by hall and richards ([23] ; figure 3 ; table s6 ). bats being carried by their mother were classified as juvenile (estimated age 0 to 3 months old; figure 3 , a). free flying bats that lacked signs of sexual maturity (e.g. small or non-descended testes in males; lack of enlarged nipples in females) were classified as sub-adults (estimated age 3 months to 2 years; figure 3 , b, c). bats that showed signs of sexual maturity (e.g. large and descended testes in males; visibly enlarged nipples indicating a previous pregnancy and suckling of young in females) but did not show signs of severe wear on all molar teeth were classified as adults (estimated age 2 to 8 years; figure 3 , d, e, f). bats that showed signs of severe molar wear on all molar teeth, including at least two molars worn to the level of the gingiva, were classified as aged (estimated age 8 years and older; figure 3 , g). female bats were further classified according to their reproductive status (table s6 ). if a foetus could be palpated while anaesthetised bats were classified as pregnant (this is likely to represent females in the last trimester of pregnancy; [31] ). bats from which milk could be expressed from their teats and were captured carrying a young were classified as early lactating. bats from which milk could be expressed from their teats, but were not carrying young, were classified as late lactating (p. conspicillatus carry their young continuously for the first month after birth, after which time the young are left in a crèche while the females forage for the remainder of lactation [20] ). bats that were not classified into any of the previous three categories, which would include females in earlymid pregnancy, were classified as non-reproductive. blood samples were collected by venepuncture of the propatagial vein with a 23 or 25 gauge needle and 1 ml or 3 ml syringe depending on the animal size. blood was allowed to clot in 2 ml tubes for 24 hours before centrifugation and separation of serum and storage at 4uc. antibody titres to hendra virus were determined by virus neutralisation test (vnt) according to [32] at the australian animal health laboratory (geelong, victoria); the world organisation for animal health (oie) reference laboratory for hendra and nipah virus diseases. the tests were carried out under biosafety level 4 conditions as hendra virus is a dangerous human pathogen with a high case fatality rate and for which there is no vaccine or effective treatment [33] . a serum sample was considered positive if it neutralised 100 tcid 50 of hev at a dilution of 1:10 or greater in the vnt, since bat sera at lower dilutions have produced a high rate of toxic or non-specific reactions on neutralisation tests. to investigate the association of potential risk factors with serostatus, data were analysed by multivariate logistic regression. we used akaike's information criterion corrected for small sample sizes (aic c ) for model selection [34] . models were ranked according to the difference between the best-fitting model and the aicc value of model i (daicc). the strength of evidence for alternative models was measured by aicc weights (v i ). for the potential risk factors identified by multivariate logistic regression to be important in explaining variation in serostatus, we performed log binomial regression analyses to further analyse their associations with serostatus. the relative risk of being seropositive was determined for these predictor variables. due to the smaller sample sizes, fisher's exact test was used to investigate the hypothesised effect of reproductive status on serostatus in adult females, where serostatus of non-reproductive adult females was compared with those categorised as either pregnant, early lactation or late lactation. as titre levels were not assumed to conform to an a priori distribution, two measures appropriate for comparing titre data among groups where serial dilution of sera produce logarithmic dilutions were used. these measures were the median titres with an interquartile range, to indicate statistical dispersion, and the mean rank titres, which indicates the mean rank value for the titres of animals within a particular category when all animals are ranked according to titre level. subsequently, non-parametric models were fitted to the data. for risk factors with two levels, a simple wilcoxon test was performed. for risk factors with three or more levels, a kruskal-wallis test was performed. all animal work followed the guidelines of the american society of mammalogists guidelines [31] table s1 model selection results for the effects of risk factors and the seroprevalence. the model selection statistics are: number of parameters in model (k), akaike's information criterion corrected for small sample sizes (aic c ), difference between model i and the model with the smallest aic c (daic c ), aic c weights (v i ) and evidence ratios (v i /v j ). only models with daic c ,5 are shown. (doc) figure 3 . features for classification of age categories of pteropus conspicillatus used in this study. age classification features (highlighted by a red circle in b, c, d, e) for both sexes. key features include: juvenile bats (a) carried by their mother (estimated age 0 to 3 months old); sub-adult bats (b, c) were free flying that lacked signs of sexual maturity, including the lack of enlarged nipples for females (b) or small or non-descended testes for males (c; estimated age 3 months to 2 years); adults bats (d, e, f) showed signs of sexual maturity, including visibly enlarged nipples indicating a previous pregnancy and suckling of young in females (d) or large and descended testes in males (e), and but did not show signs of severe wear on all molar teeth (f; estimated age 2 to 8 years); aged bats (g) showed signs of severe molar wear on all molar teeth, including at least two molars worn to the level of the gingiva (estimated age 8 years and older). doi:10.1371/journal.pone.0028816.g003 the natural history of hendra and nipah viruses nipah virus: impact, origins, and causes of emergence evidence of henipavirus infection in west african fruit bats henipavirus rna in african bats epidemiological perspectives on hendra virus infection in horses and flying foxes hendra virus outbreak with novel clinical features queensland government, department of primary industries and fisheries hendra virus infection in a veterinarian human hendra virus encephalitis associated with equine outbreak nipah virus: a recently emergent deadly paramyxovirus person-to-person transmission of nipah virus in a bangladeshi community a longitudinal study of the prevalence of nipah virus in pteropus lylei bats in thailand: evidence for seasonal preference in disease transmission. vector borne zoonotic diseases the ecology of hendra virus and australian bat lyssavirus reproduction and nutritional stress are risk factors for hendra virus infection in little red flying foxes (pteropus scapulatus) experimental hendra virus infection in pregnant guinea-pigs and fruit bats (pteropus poliocephalus) isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus fenner's veterinary virology landscape-scale redistribution of a highly mobile threatened species, pteropus conspicillatus (chiroptera, pteropodidae), in response to tropical cyclone larry volant viruses: a concern to bats, humans and other animals spatial and temporal patterns of zaire ebolavirus antibody prevalence in the possible reservoir bat species amplification of emerging viruses in a bat colony ecology of rabies virus exposure in colonies of brazilian free-tailed bats (tadarida brasiliensis) at natural and man-made roosts in texas flying foxes: fruit and blossom bats of australia latitudinal range shifts in australian flying-foxes: a re-evaluation evidence for nipah virus recrudescence and serological patterns of captive pteropus vampyrus fatal encephalitis due to novel paramyxovirus transmitted from horses relapsed and late-onset nipah encephalitis dietary variation in spectacled flying foxes (pteropus conspicillatus) of the australian wet tropics field anaesthesia of three australian species of flying fox some aspects of female reproduction in the greyheaded flying-fox, pteropus poliocephalus (megachiroptera: pteropodidae) laboratory diagnosis of nipah and hendra virus infections hendra and nipah virus diseases. manual of diagnostic tests and vaccines for terrestrial animals model selection and multimodel inference for assistance with fieldwork and provision of photographs we thank les hall, carol de jong, tim kerlin, jonathan lee, lana little, jenny maclean, maria marklund, billie roberts, jack shield, craig smith, justin westrum and the australian quarantine and inspection service. for assistance with diagnostic test result interpretation we thank kim halpin and greer meehan. key: cord-279864-5ouuu49v authors: hou, jing; lv, dachao; sun, yuexia; wang, pan; zhang, qingnan; sundell, jan title: children’s respiratory infections in tianjin area, china: associations with home environments and lifestyles date: 2020-06-07 journal: int j environ res public health doi: 10.3390/ijerph17114069 sha: doc_id: 279864 cord_uid: 5ouuu49v children spend most of their indoors time at home, which may have substantial influence on their health. we conducted a cross-sectional study in the tianjin area, china to quantify the incidence of respiratory infections among children, and its association with home environments and lifestyles. the lifetime-ever incidences of croup, pneumonia and ear infection among children aged 0–8 in tianjin area was 9.2%, 28.7% and 11.6%, respectively. the incidence of common cold infections more than twice per year was 31.3%. home environments and lifestyles included strong risk factors for childhood respiratory infections. perceived dry air had the greatest association with childhood common colds (population attributable fraction (paf = 15.0%). modern floor covering had the greatest association with croup (paf = 14.7%) and ear infection (paf = 34.5%), while infrequent bedding sun-curing had the greatest association with pneumonia (paf = 18.7%). condensation (a proxy of poor ventilation) accounted for 12.2% of the incidence of croup (paf = 12.2%) and frequent common colds (paf = 8.4%). our findings indicate that factors related to “modern” home environments and lifestyles are risks for childhood respiratory infections. modifying such factors might reduce the incidence of respiratory infections among children. respiratory infection was the leading cause of global children mortality, accounting for 15-16% of under-five mortality, second only to prematurity [1] . in 2017 alone, an estimated 1.6 million children under age 5 died worldwide from respiratory infections, including 39,000 deaths in china [2] . there is no evidence for a robust relationship between respiratory infections and genetic or racial factors [3] . people spend most of their time indoors, especially children [4, 5] . infants and young children, compared to adults, are more vulnerable to environmental exposure because their immune systems are not fully developed [6] . household activities and environmental exposure at home are suspected risk factors for respiratory infections among children, especially in low income countries [7] . therefore, a global study on home environments and children's health was launched in eight countries/areas in 2000 [8, 9] , wherein children's health outcomes, home environments, lifestyles, socialeconomic status, and biological factors were investigated systematically [10] . home environmental exposures include chemical exposure (such as volatile organic compounds from building materials and decoration; no x from natural gas cooking), dampness and mold, and environmental tobacco smoke (ets) pre-and post-natally. many studies [11] [12] [13] [14] [15] have shown that dampness and mold in buildings are significantly associated with an increase in respiratory infections. people living in damp houses have been shown to be more likely to suffer from respiratory infections partly because of exposure to higher concentration of fungi and dust mites in damp environments [16, 17] . exposure to home indoor air pollutants such as ets [18] and chemical sources [19] have also been reported to be associated with respiratory infections among children. in addition to home environmental exposure, previous research has also shown that lifestyle factors and biological factors are important determinants of children's health [20] . sun-curing bedding and daily cleaning room are associated with fewer respiratory infections [21] . holberg et al. [22] and sun et al. [23] have demonstrated that daycare attendance was associated with more respiratory illness, asthma and allergy. occupancy level and type of daycare facility have been identified as important determinants of infection risk [24, 25] . the mode of infant delivery was found to affect early respiratory microbiota development, and may also play an important role in respiratory health later in life [26] . in addition, many studies in different countries have suggested that exclusive and prolonged breastfeeding was protective against respiratory infections [27] [28] [29] . with china's increasing wealth and urbanization in the past 40 years, chinese homes and lifestyles have changed dramatically. in urban areas, traditional chinese residential buildings, pingfang residences, have been replaced by high-rise apartments [10] . new furnishing materials have come into use [30] . homes have been tightened in order to save energy, resulting in poor ventilation [31] . along with this urbanization, a "modern" lifestyle has been adopted. it is less convenient for residents in urban areas to sun-cure bedding. fast food has become popular. childbirth tends to be cesarean delivery. breastfeeding time has been reduced, and daycare is being started earlier. in view of these changes in home environments and lifestyles in modern china, the main aim of this article is to study associations of respiratory infection outcomes in children with their home environments and lifestyles. this study is part of the china, children, homes and health (cchh) project [9, 32] . it was conducted from 2013 to 2015 in the tianjin metropolis, that is, the city of tianjin and its satellite city cangzhou. the tianjin metropolis is in northeast china, as shown in supplementary figure s1. metropolitan tianjin occupies an area of 11,920 km 2 and has a population of 15 million. cangzhou's area is 13,420 km 2 and its population is 7 million. in 2014, the gdps per capita for tianjin metropolis and cangzhou were usd 16,500 and usd 5800, respectively [33] . the average outdoor air temperature for spring, summer, autumn and winter are 13.4 • c, 25.7 • c, 13.6 • c and −1.7 • c, respectively, in tianjin and cangzhou [34] . cchh in tianjin metropolis consisted of two phases: phase i, a cross-sectional study and phase ii, a case-control study. in phase i, we analyzed data obtained from a questionnaire survey of children's health, demographic information, home environment and lifestyle. we selected daycares and primary schools from a list provided by the local municipal education commission, through a stratified random sampling method. finally, 24 institutes in urban, 6 in suburban and 9 in rural areas were involved in the survey. questionnaires were sent to daycare centers and primary schools, from where they were delivered to parents who responded to the questionnaires. the details of the questionnaire are provided in supplementary materials-questionnaire. the demographic information for investigated children included gender, age, family allergic history, home location and household income. questions related to home environment were about indoor dampness; building characteristics and indoor furnishings; exposure to environmental tobacco smoke; and pets at home. lifestyles referred to food habits; outdoor activity; home cleaning frequency; and children's daycare attendance. we also investigated biological factors for children such as their delivery mode, birth weight; and length of breastfeeding. questions on respiratory infections are as follows: • has your child ever had croup? (possible responses: yes; no) • has your child ever had doctor diagnosed pneumonia? (possible responses: yes; no) • has your child ever had ear infections? (possible responses: no; yes, 1-2 times; yes, 3-5 times; yes, >5 times) • in the last 12 months, how many times did your child have a common cold? (possible responses: none; 1-2 times; 3-5 times; 6-10 times; >10 times) all statistical analyses were performed with ibm spss statistics 22 (international business machines corporation (ibm), armonk, ny, usa). we accepted p-values < 0.05 as statistically significant. we first analyzed children's respiratory infections and their distribution by gender, age, family allergic history, home location and annual household income. the associations of childhood respiratory infections with home environments, lifestyles and biological factors were analyzed in univariate logistic regression models. odds ratios were calculated in logistic regression models with adjustment for gender, age, family allergic history, home location, household income and outdoor pollution (indicated by pm 10 concentrations, as shown in supplementary table s1 ). then, correlation coefficients among factors that reached significant levels (p < 0.05) were investigated by kendall correlation analyses. if the correlation coefficient was larger than 0.4, one factor in the pair was selected, and associations of the selected factors with respiratory infections were included in multivariate logistic regression models. finally, we put all the significant predictors in multivariate logistic regression models (using a forward conditional method) to identify the most important risk factors for respiratory infections among children. population attributable fraction (paf) is an estimate of the fraction of population with the health outcome that can be attributed to a particular risk factor or exposure. we calculated paf for a given exposure using the following formula [35] : where p 0 is the cumulative proportion of unexposed persons who develop the disease over the interval and p t is the cumulative proportion of the total population developing disease over the specified interval. the research office at tianjin university granted ethical approval for this study (no. 21207097). we received 7865 questionnaires, a response rate of 78%. ages were not reported for 204 children, while 295 children did not fit into the age range (0 to 8 years old). therefore, there are 7366 children in the final analysis. among these 7366 children, 52% were boys and 48% were girls. table 1 shows that children's respiratory infections had significant associations with family allergy history and home locations (p < 0.05). croup was reported more often for boys. more wealthy urban children had more infections compared to less wealthy rural children. dampness indicators and odors were defined as follows: (1) visible mold in child's room; (2) visible damp in child's room; (3) suspected moisture problem in child's home; (4) peeling or discolored floor covering in child's room; (5) flooding in child's room; (6) condensation on windowpane in winter in child's room; (7) perceived moldy odor; (8) perceived dry air. table 2 presents adjusted odd ratios (aor) of dampness problems for respiratory infections. we found that living in a damp room was associated with increased odds of respiratory infections. both pneumonia and the common cold were related to condensation on windowpanes. perceived dry air was a significant risk factor for all respiratory infections among children. the adjusted odds ratios of building characteristics for respiratory infections are presented in supplementary table s2 . infections (especially pneumonia) were associated with new apartments, modern floor covering, modern wall covering and use of air conditioners (ac). meanwhile, exposure to new furniture and redecoration in the child's early life due to home renovation was a risk factor for all respiratory infections. the adjusted odds ratio of environmental tobacco smoke (ets) and pet-keeping for respiratory infections are presented in supplementary table s3 . early life (i.e., during the first year of children's life or during pregnancy) exposure to ets, especially mother's smoking, increased the risks of children's respiratory infections. pet-keeping was a risk factor for infections. the adjusted odds ratios of daycare attendance for the studied respiratory infections among children are presented in supplementary table s4 . the type of childcare, starting age of daycare, exposure time and occupancy levels in daycare centers were analyzed. children in daycare centers were more susceptible to pneumonia and common colds than children who were mostly at home. children in daycare centers with more than 30 children have higher morbidity of pneumonia, ear infection and common colds. adjusted odds ratios for food habits, outdoor activity and cleaning habits for respiratory infections are presented in table 3 . spending ≥3 h per day watching tv was a strong risk for the common cold. children living in a frequently cleaned room with good ventilation and frequently sun-cured bedding had fewer respiratory infections. table 4 presents adjusted odds ratios of biological factors for respiratory infections among children. cesarean delivery is significantly associated with croup, pneumonia and ear infection. children who were not born on due week with less birth weight had a higher morbidity of pneumonia. factors that reached significant levels in univariate models were tested for correlation, as shown in supplementary table s5 . mold spots were correlated to damp spots. modern floor covering was correlated to building type. current pet keeping and environmental tobacco smoke were correlated to early life status. one factor in each of these paired variables was selected to be added to multivariate logistic regression models. finally, the variables mold spot, suspected moisture, floor moisture, condensation, moldy odor, perceived dry air, floor covering, cooling system, home renovation, early life smoking exposure, early life pets keeping, childcare type, size of daycare center, tv watching, room cleaning frequency, sun-curing bedsheets frequency, way of delivery, birth week and birth weight were used in the multivariate logistic regression model. multivariate analysis of associations of respiratory infections with these home environment, lifestyle and biological factors is shown in table 5 . dampness, condensation on windowpanes, moldy/perceived dry air, modern decoration materials and less frequency of sun-curing bed sheets were the greatest risk factors for croup, pneumonia, ear infections and common colds. in addition, attending day-care in a large class increased the risk of common colds and ear infections. the population attributable fractions (pafs) of home environment, lifestyle and biological factors for infections among children are shown in table 6 . the top contributor to childhood common colds is perceived dry air, while it is infrequent sun-curing bedding for pneumonia, and modern wall covering for croup and ear infection. condensation, a secondary attribution factor, contributes 12.2% and 9.2% to croup and frequent common colds, respectively. large class size is a risk factor for ear infection and common colds. in this study, a multivariate regression model was applied to identify the greatest home environmental, lifestyle and biological risks for children's infections. it was found that modern floor covering, perceived dry air (a proxy of indoor pollution), condensation on windowpanes (a proxy of poor ventilation), less sun-curing bedsheets and cesarean delivery are significantly associated with childhood infections. these factors might affect the development of the immune system and/or the transmission of infectious pathogens. with respect to biological factors, we found that cesarean delivery and not being born on the due day were significant risk factors with high population attributable fractions (paf) for childhood pneumonia. bosch et al. pointed out that cesarean delivery affects early respiratory microbiota development, thereby possibly increasing the frequency of respiratory infections later in life [26] . in our study, the cesarean delivery rate was 68% in tianjin metropolis, higher than the national level of 46% [36] and the recommended value by who of 15% [37] . our previous study showed that the tianjin modern urban area had a significantly higher cesarean delivery rate compared to the rural area close to tianjin [10] . in addition to cities having a higher cesarean delivery rate, city dwellings have increasingly modern materials being used for indoor furnishings and decoration [38] . china is currently the largest producer of wood-based panels, coating and furniture in the world. numerous new building furnishing materials have emerged in people's daily life. in the present study, we found that children who live in an urban apartment decorated with modern floor and wall coverings were more likely to have had respiratory infections. renovation during the first year of children's life was also a risk. considerable evidence has demonstrated that the chemical emissions of modern indoor materials (new wall coverings, new furniture, new synthetic carpets) are associated with increases in respiratory infections among children or infants [19] . home renovation and modern decoration materials are positively associated with high concentrations of formaldehyde, volatile organic compounds (vocs) and semi-vocs [39, 40] . these indoor pollutants may be associated with developmental delays in children, reduced activity of the immune system and direct toxicity [41, 42] . as a comparison, suburban or rural children who lived in pingfang dwellings with less "modern" redecoration and materials had fewer respiratory infections. it is interesting to find that perceived dry air had strong associations with pneumonia and common colds. the reported rate of "perceived dry air" is high in tianjin-53.1% in our study. "perceived dry air" has been shown to not necessarily be due to physically dry indoor air, but rather to polluted air [43, 44] . in spaces with poor ventilation, pollutants could not be efficiently removed [45] , which might irritate the respiratory tract and reflect in more complaints of dry air [44] . in this study, condensation on the child's room windowpanes in winter was a strong risk for respiratory infections among children. condensation on windowpanes in winter has been reported to be associated with insufficient ventilation in homes [31, 46] . chinese homes do not have a mechanical ventilation system. most of residential buildings in china still depend on the "natural ventilation" of open windows and infiltration to passively introduce outdoor air to indoors. in tianjin,~90% of homes closed the child's bedroom window at night in winter. thus, ventilation is mainly through infiltration, with a median value of 0.30 h -1 [31] . in such tight buildings, the poor ventilation rate could not efficiently dilute airborne-transmitted pathogens and subsequently results in their accumulation in our occupied spaces. a study conducted in army trainees found that rates of febrile acute respiratory diseases were significantly higher among trainees in modern (energy efficient design) barracks (an adjusted relative risk of 1.51) [47] . milton estimated that in offices with lower ventilation, relative risk for short-term sick leave was 1.53 (95% ci 1.22-1.92) [48] . a study conducted in chinese students' dormitories indicated a clear dose-response relationship between ventilation rate and common cold infections [49] . consistent with studies in america [50] and sweden [51] , our analysis demonstrated that daycare attendance was a significant risk factor for common colds among children in tianjin. daycare occupancy level and weekly exposure time are measures of contact with other children. we found that children spending more time in daycare or in higher occupancy daycare centers were susceptible to pneumonia, ear infections and common colds. attending daycares with higher occupancy level was a contributor for common cold infections ( table 6 , paf = 9.0%), next to poor ventilation in homes (indicated by condensation on windowpanes [52] ). more frequent contact with people may be a reasonable explanation for this observation. for respiratory infection viruses that are transmitted by small particle aerosols such as influenza, adenovirus, rhinovirus, and coronavirus, airborne transmission is considered the predominant transmission pathway [53, 54] ; however, contact (direct or indirect) can also spread these pathogens [55] . a study on the transmission of common colds in offices indicated that workers sharing offices have a significant risk of common colds compared to those working in single rooms (adjusted odds ratio-1.35) [56] . overcrowding in a large urban jail was reported to be a significant risk factor (p = 0.03) for an outbreak of pneumococcal disease among inmates [57] . currently in china, approximately 250 million families (accounting for 55% of the population in china) live in cities. rapid urbanization and modernization has led china to experience a dramatic change in both indoor environments and lifestyles in the past two decades [30] . modern high-rise apartments are constructed tightly and decorated with modern materials. the result is insufficient ventilation which causes increased concentrations of indoor-generated pollutants, including chemical compounds and airborne transmitted pathogens. it is almost impossible to sun-cure bedding frequently in modern urban buildings. it is common for children to attend daycare centers before three years old as both parents work in most cases. meanwhile, more health issues related to indoor environments are being observed. respiratory infections (as in this article), asthma and allergies [10] are more frequent in modern society, especially in homes with higher annual incomes (see table 1 ). in a previous study, we showed that indoor pollution increased significantly with increasing household income [40] . wealthier homes had more modern decoration materials (such as laminated wooden flooring) and household chemical products, all of which were associated with higher concentrations of modern chemical compounds (e.g., phthalates) [40] . as societies develop economically, ways to live a high-quality and healthy life need to be studied. parallel cchh studies performed in other cities have found similarly high rates of respiratory infections in children, especially in urban areas [21, 32] . the incidence of lifetime-ever pneumonia among children in modern cities (like tianjin-28.7%) are substantially higher than those in usa and european cities [58] . pneumonia is an infectious disease caused by bacteria or virus. the high infection rate of pneumonia in china may be partially due to insufficient vaccine coverage with the pneumococcal conjugate vaccine (pcv) and the hemophilus influenzae type b (hib) vaccine [58] . however, the incidence and severity or duration of pneumonia could be influenced by environmental factors [59] . our analysis in the present study is hierarchical so as to identify the important factors among a variety of possible risk factors. the most prominent risk factors from multivariate analysis were related to modern home environments and lifestyles. the higher rates of childhood respiratory infections are linked with a combination of biological, environmental and behavioral factors, which affect the development of immune system and the breeding and transmission of pathogens. in this study, surveys were performed using a stratified random sampling method. the response rate was 78%, which reduces the likelihood of selection bias. therefore, the strong association of infections with home environments and lifestyles is unlikely to be due to bias. modern floor covering, perceived dry air, condensation on the windowpanes in winter, and infrequent sun-curing of bedding are the strongest risks for childhood respiratory infections. "modern" home environments and lifestyles play an important role in the incidence of respiratory infections among children. increasing ventilation rate, frequent sun-curing of bedding and avoiding pollutants from furnishing and decoration materials might be effective measures to reduce the incidence of childhood respiratory infections. supplementary materials: the following are available online at http://www.mdpi.com/1660-4601/17/11/4069/s1, questionnaire: china-child-home-health, figure s1 : locations of tianjin metropolis and cangzhou city, china, table s1 : outdoor pm 10 concentrations in tianjin area (i.e., tianjin metropolis and cangzhou city) in 2013-2014, µg/m 3 table s2 : associations between building characteristics and respiratory infections among children, table s3 : associations between environmental tobacco smoke exposure, pet-keeping and respiratory infections among children, table s4 : associations between daycare and respiratory infections among children, monitoring 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diseases in army trainees risk of sick leave associated with outdoor air supply rate, humidification, and occupant complaints in china, students in crowded dormitories with a low ventilation rate have more common colds: evidence for airborne transmission childhood upper respiratory tract infections: to what degree is incidence affected by day-care attendance? respiratory illness in preschool children with different forms of day care sbs symptoms in relation to dampness and ventilation in inspected single-family houses in sweden viral pneumonia in children rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infections the common cold shared office space and the risk of the common cold an epidemic of pneumococcal disease in an overcrowded, inadequately ventilated jail high pneumonia lifetime-ever incidence in beijing children compared with locations in other countries, and implications for national pcv and hib vaccination lifetime-ever pneumonia among pre-school children across china-associations with pre-natal and post-natal early life environmental factors we would like to express our special appreciation to louise b. weschler, who polished our language and clarified our expressions. the authors declare no conflict of interest. key: cord-267531-tqqj4cy0 authors: he, ying; lin, guang-yu; wang, qiong; cai, xiao-ying; zhang, yin-hui; lin, chuang-xing; lu, chang-dong; lu, xue-dong title: a 3-year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in shenzhen, china date: 2014-05-14 journal: influenza other respir viruses doi: 10.1111/irv.12257 sha: doc_id: 267531 cord_uid: tqqj4cy0 background: the epidemiology of local viral etiologies is essential for the management of viral respiratory tract infections. limited data are available in china to describe the epidemiology of viral respiratory infections, especially in small–medium cities and rural areas. objectives: to determine the viral etiology and seasonality of acute respiratory infections in hospitalized children, a 3-year study was conducted in shenzhen, china. methods: nasopharyngeal aspirates from eligible children were collected. influenza and other respiratory viruses were tested by molecular assays simultaneously. data were analyzed to describe the frequency and seasonality. results: of the 2025 children enrolled in the study, 971 (48·0%) were positive for at least one viral pathogen, in which 890 (91·7%) were <4 years of age. the three most prevalent viruses were influenza a (iav; 35·8%), respiratory syncytial virus (rsv; 30·5%) and human rhinovirus (hrv; 21·5%). co-infections were found in 302 cases (31·1%), and dual viral infection was dominant. rsv, hrv and iav were the most frequent viral agents involved in co-infection. on the whole, the obvious seasonal peaks mainly from march to may were observed with peak strength varying from 1 year to another. conclusions: this study provides a basic profile of the epidemiology of acute respiratory viral infection in hospitalized children in shenzhen. the spectrum of viruses in the study site is similar to that in other places, but the seasonality is closely related to geographic position, different from that in big cities in northern china and neighboring hong kong. acute respiratory tract infections (artis) are a persistent and pervasive public health problem in both developed and developing countries. they cause a great burden of disease worldwide. especially in developing countries including china, artis, mainly pneumonia, are the leading cause of death among children under the age of 5 years. 1,2 a great variety of pathogens can cause artis, and viruses have been considered as the predominant pathogens in this children population. 3, 4 the most frequently reported viruses include respiratory syncytial virus (rsv), influenza viruses a and b (iav, ibv), parainfluenza viruses (pivs), human rhinovirus (hrv) and adenovirus (adv), which are responsible for most episodes of artis in children. 1 in the past decade, several new viruses associated with artis such as human metapneumovirus (hmpv), novel strains of coronaviruses (sars-cov, hcov-nl63 and hkui), human bocavirus (bov), wu polyomavirus (wupoyv) and ki polyomavirus (kipoyv) have been discovered in human respiratory tract specimens. among them, some have been identified to be causative pathogens of artis. 1, 4, 5 currently, there are no approved vaccines or medications available for most of the respiratory viruses. 1 a better understanding of the epidemiology of viral respiratory tract infections in children plays a key role for the prevention, control and treatment of artis. studies showed that many viral respiratory infections exhibited predictable seasonal variations. however, the epidemiological profiles of viral respiratory infections from different climate zones or different countries in the same climate zone may be varied. [6] [7] [8] [9] [10] [11] [12] china is a large country crossing three climate zones, and great differences in climate are found from region to region. a better understanding of the epidemiology of artis in different regions could be helpful to develop effective surveillance, prevention and treatment strategies. although some studies on the epidemiology of artis have recently been reported in big cities such as beijing, shanghai and hong kong, [13] [14] [15] [16] the epidemic characteristics of viruses in artis are still not well established all around china, especially in other cities and rural areas. shenzhen is the largest migratory city of china with high population density and population mobility. it is located in southern china at 22°27 0 -22°52 0 n and 113°46 0 -114°37 0 e, immediately north of hong kong, with a typical subtropical monsoon climate. the annual average temperature and relative humidity of shenzhen are about 23°c (12-33°c) and 77%, respectively. the purpose of this study is to investigate the prevalence, seasonality and clinical characteristics of acute viral respiratory infections in hospitalized children in shenzhen and to provide insights into etiologies of artis in local infants and children. a consecutive 3-year prospective study from july 2007 to june 2010 was conducted in shenzhen, a coastal city neighboring hong kong. four hospitals including a children's hospital were chosen for the study. selected patients with artis admitted to the pediatric wards were enrolled. the inclusion criteria were as follows: less than 14 years old, acute fever (t ≥ 38°c), with any one of respiratory symptoms (such as sore throat, cough, wheezing and dyspnoea/ tachypnoea), normal or low leukocyte count, the onset of illness within 3 days before hospitalization. the diagnosis of pneumonia was based on the guideline of the management of childhood community acquired pneumonia (cap) issued by the chinese medical association in 2006. 17 in the guideline, the clinical symptoms and signs for the diagnosis of childhood cap include fever, cough, tachypnoea (defined according to different age), difficulty breathing and/or lower chest wall indrawing. x-ray evaluation has been carried out when necessary. the study protocol was approved by the medical ethical committees of the hospitals. written informed consent was obtained from the parents or legal guardians of the children. nasopharyngeal aspirates (npa) were obtained by trained personnel following standard operating procedures within 24 hour after admission. the specimens were transported immediately to the laboratory by sterile viral transport media, then divided into aliquots and immediately frozen at à80°c until further processing. total viral nucleic acids (dna and rna) were extracted from 200 ll of npa specimen using the axyprep body fluid dna/rna miniprep kit (axygen, union city, ca, usa), according to the manufacturer's instructions. purified dna and rna were stored at à80°c in aliquots for further pcr analysis. for each specimen, assays for ten common and newly identified viruses were performed. briefly, wupoyv and bov were tested using monoplex pcrs described previously. 18, 19 other viruses were tested using the luminex platform and multiplex xtag tm respiratory viral panel assay (rvp assay) according to the manufacturer's instructions. 20 all multiple infection samples were retested. if there was discordance between two tests, the sample was confirmed by monoplex pcr. statistical package for the social sciences (spss) for windows version 11.0 (spss inc. chicago, il, usa) was used. for comparison of categorical data, chi-square or fisher's exact test was used. all tests were two-tailed, and a p value below 0á05 was considered statistically significant. a total of 2025 specimens were obtained from 2025 eligible patients ranging from 15 days to 14 years old with a median age of 12 months, in which 89á6% of patients were < 4 years old. there were 964 (47á6%) females and 1061 (52á4%) males included. of all hospitalized children enrolled in this study, 84á0% involved lower respiratory infection and 16á0% had upper respiratory tract infection (table 1) . among 971 positive cases, 572 (58á9%) were diagnosed as pneumonia. about 971 of the 2025 cases (48á0%) were positive for at least one viral pathogen. among them, iav, rsv, hrv and pivs were detected in 348 (35á8%), 296 (30á5%), 209 (21á5%) and 169 (17á4%) cases, respectively. single infection was observed in 669 (68á9%) cases, and multiple infection was found in 302 (31á1%). our results also showed that rsv, iav and hrv were the main pathogens in single viral infection cases ( table 1) . the monthly positive rates varied from 32á5% to 75á0% with a mean of 45á1% ( figure 1 ). in the year 2009, when influenza a (h1n1) was pandemic worldwide, the positive rate started to increase in march and the highest positive rate 75á0% was observed in may. among the 971 positive cases, a total of 1335 viral pathogens were detected. the most frequently detected pathogen was iav (26á1%, 348/1335), followed by rsv (22á2%, 296/1335), hrv (15á7%, 209/1335), piv1 and piv3 (12á7%, 169/1335) ( about 302 co-infection cases were identified, accounting for 14á9% of all 2025 hospitalized children. during the h1n1 outbreak from march to august 2009, co-infection cases and co-infection rate increased significantly ( figure 2 ). 143 of 302 (47á4%) co-infection cases were detected during that time. among them, 121 cases were involved in iav infection, including 90 dual infection cases. of all co-infection cases, 247 (81á8%), 49 (16á2%) and 5 (1á7%) were infected with two, three and four potential viral pathogens, respectively. one multiple infection with five viruses was detected in a rsv iav hrv bov piv3 adv hmpv wupoyv piv1 ibv total a total cases 163 172 85 55 51 49 37 26 24 7 669 bronchitis 12 19 7 7 5 2 2 3 5 0 62 bronchiolitis 45 19 20 7 13 9 8 2 5 0 128 pneumonia 93 97 55 28 24 28 25 18 14 5 387 urti 13 37 3 13 9 10 2 3 0 2 *case number and percentage in all enrolled children. **incidence rate in this age group significantly higher than the other age groups. ***no significant difference between these three age groups. †no significant differences between these two age groups, but significantly lower than the other age groups. 6-month-old infant. iav, rsv and hrv were the three most frequently found viruses in co-infection and detected in 176, 133 and 124 cases with co-infection rates of 50á6%, 44á9% and 59á3%, respectively ( table 2) . various multiple infection patterns were observed in the study. a total of 152 (50á3%) co-infection cases involved at least two viruses of rsv, hrv and iav. co-infection rate of each individual virus detected varied significantly. the lowest and highest co-infection rates were observed in wupoyv (33á3%) and ibv (66á7%), respectively. 91á4% (276/302) of co-infection cases were tested in the age group of 4 years old or younger ( table 2) , but among all age groups, no statistical difference in co-infection rate was found (v 2 = 1á83, p = 0á8721). gender-specific difference in co-infection rate was not observed (v 2 = 2á17, p = 0á1404). there was no significant difference in co-infection rates between picu and non-picu cases. similarly, no significant difference in clinical symptoms was observed between co-infection and single cases (data not shown). in general, respiratory viruses were detected more often in the period of march to may than in other months (55á4% and 40á6%, respectively, v 2 = 28á06, p = 0á0000), and obvious seasonal peaks were observed during those months with peak strength varying from 1 year to another. a weaker seasonal peak could also be distinguished in some winter months in different years ( figure 1) . the seasonality profile of each individual virus detected was diversified. a seasonal distribution of iav can be observed from late spring to summer (mainly march to may) and sometimes in fall (october, november or december). a wide seasonal peak of iav infection was detected from march to august 2009 ( figure 3a ). although rsv was tested almost a whole year, two yearly peaks were identified. one was found in november and/or december and the other stronger one was found in march to may of the year. the peak duration in 2009 was longer than those in other years. the seasonal trends of hrv and pivs were similar to that of rsv, but the peaks of these three viruses fluctuated and shifted mildly ( figure 3b ). although ibv and adv had a low detection rate in the study, similar seasonality was observed and their infection peaks were mainly in midwinter. peaks in spring and summer were also observed in some years ( figure 3c ). our investigation did not find regular seasonality in bov infections. a sudden increase in bov infection was recorded in april and may 2010. although the positive rate of hmpv infection was only 4á8%, regular seasonality was observed from march to may of each year. of 39 patients with wupoyv infection, 36 were detected after july 2008. our data implied that peak months of wupoyv infection were from march to may ( figure 3d ). the positive rates of viral infections in male and female were 52á5% and 47á5%, respectively. no significant gender difference was revealed (v 2 = 0á012, p = 0á9118). the distribution of viral agents and infection patterns in different age groups are shown in table 2 . of all 971 positive children, 890 (91á7%) were 4 years old or younger. the positive rate in this age group was significantly higher than that in children more than 4 years old (v 2 = 8á26, p = 0á0041). children under 6 months were the most susceptible to respiratory viral pathogens with a positive rate of 14á8% (table 2) . very few long-term prospective studies were performed for viral etiologies of artis among hospitalized children. in this present study, the infection frequency, seasonality, co-infection pattern and clinical features of viral respiratory infections were investigated based on prospective analysis of three consecutive year's data from hospitalized children with artis. our results provided a distinctive epidemiological profile of viral respiratory infections in hospitalized children with artis in the study areas, which was different from those in the big cities in northern china such as beijing and shanghai and also different from that in adjacent hong kong. overall, 48á0% of our cases were positive for respiratory virus infections, which resembled the latest study in the same city. 21 a similar incidence rate has been obtained in neighboring regions 13, 22 and other cities such as rome 23 and milan, 24 but it was different from other studies. [10] [11] [12] in china, the overall positive rate reported varied from 27á3 to 74á8% depending on different areas and detection methods. 15, 16, [25] [26] [27] [28] [29] [30] [31] the rate of respiratory viral infections varied worldwide, and many factors such as geographic distribu-tion, study design and detection protocols could lead to these variations. 1, 7, 8, 32 in our study, leukocyte count was used as an indicator of inclusion criteria and it probably affected the positive rate. viruses not considered in the study, for example coronaviruses, would underestimate the positive rate. most studies showed that rsv or hrv was the most prevalent viruses in children with viral respiratory tract infection. 1 in this study, iav was the most frequently detected respiratory virus, followed by rsv and hrv. iav (h1n1) outbreak in 2009 could explain this shift. data showed that about 60% of iav infections were detected during the outbreak period. studies showed that the h1n1 outbreak could change viral distribution patterns. 24, 29, 33 regardless of the iav (h1n1) outbreak, rsv and hrv were the two most common viral pathogens in artis, which was consistent with most previous studies. 1, 10, 15, 16, 22, [25] [26] [27] [28] [29] our study further confirmed the importance of rsv and hrv in children with artis, especially in children < 4 years of age. 10, 14, 23 our results also showed that 12á7% of viral pathogens detected were piv1 and piv3, which implied that pivs played an important role in children with artis. similar findings were obtained in the studies conducted in shanghai, 14, 34 changsha, 26 harbin, 30 hong kong 13 and rome. 23 the prevalence of piv3 was twofold higher than that of piv1, particularly in infants, which was similar with other reports, 25, 26, 30, 35 implying that infants could be more vulnerable to infection with piv3 than piv1. hmpv has been proven to be one of the main viral pathogens responsible for artis in children. 5 the positive rate found in the study was consistent with previously published results. 10, 36, 37 in china, the infection rate of hmpv varied from 3á2 to 10á6%. 22, 26, 28, 29, 31 the seasonality of hmpv in this study was mainly from march to may, similar to that in hong kong, 36 but different from other places. 5, 37 in our study, 4á9% of cases were positive for bov, which coincided with 5á0% in hong kong 38 and higher than guangzhou 39 and eastern guangdong. 22 our result suggested that bov might be present throughout the year with no seasonal distribution. however, seasonal distribution was noted from september to february in hong kong 38 and may and june in guangzhou. 39 the use of multiple pcr made it possible to simultaneously detect a broad spectrum of viruses with excellent sensitivity, at the same time, with increased viral detection rate and co-infection rate for artis. 12,40 among our positive cases, co-infection rate was 31á1%, which was similar to 27á9% reported by do et al. 10 co-infection rate reported elsewhere varied widely from 25á4 to 47á9%. 40 the relatively lower co-infection rates ranging from 0á24 to 26á9% were reported in the studies conducted in various cities of china. 22, [25] [26] [27] [28] [29] [30] [31] in most of these studies, immunofluorescence kits were used to test a lower number of respiratory viruses. it was worth to note that in the study by peng et al. 32 in wuhan, china, 69á5% of co-infection rate was reported with immunofluorescence kit. these variations might be attributed to geographic differences, diagnostic methods for viral agents and study design. 12, 32, 34, 41, 42 pathogens in those negative patients need to be further investigated as only ten common and newly identified viruses were included in our study, which might underestimate positive rate or coinfection rate. it was notable that the correlation between co-infection rate and positive rate was not observed. of multiple infections, dual infection was predominant in this study whether or not considering the iav (h1n1) outbreak in 2009, which was consistent with previous studies. 28, 32, 42, 43 similar with the studies conducted in the cities of guangzhou and wuhan, china, 28, 29 our study showed that iav, rsv and hrv were the main viruses involved in multiple infections. high co-infection rate between these three viruses could be explained from the overlap of their seasonal distributions. a variety of predominant multiple infection patterns between respiratory viruses were observed in different studies. 12, 32, 42, 43 for example, it was shown in martin et al.'s study 43 that adv and coronaviruses were the most common co-infection pattern. our study showed that rsv and hrv were the two most viruses involved in multiple infection, followed by iav and pivs, regardless of iav infection in the h1n1 outbreak period. it was difficult to explain the variations of coinfection patterns based only on seasonal distribution. a recent study suggested that co-infection patterns were not random and certain pathogens had higher frequency of coinfection. 41 as molecular assays only detect nucleic acid and positive result does not mean the presence of the pathogen, when studying co-infection patterns of respiratory viruses, the ability to differentiate the real causative pathogens needs to be solved first. viral load detection could provide some clues for solving this issue. 43, 44 although high co-infection rates have been reported in various studies, the associations among multiple infections, hospitalization rate and severity of artis were still not clear with inconsistent results in different studies. 42, 43, 45 our data suggested that multiple infection had less association with the severity of disease, consistent with peng et al.'s study. 32 the relationship between co-infection rates and age group was also investigated in our study, and little correlation was observed. several previous studies observed that co-infection rates were more frequent in a certain age group, but results were varied. 32, 43 in contrast to temperate region, where most viruses had winter-spring seasonality, the respiratory viral infections in tropical and subtropical regions appeared mainly to be spring-summer seasonality. 9 in this study, due to the high detection rate and similar seasonality of rsv, hrv, iav, piv and hmpv, an overall spring-summer seasonality of viral respiratory infections in children was concluded. studies conducted in hong kong showed that a clear seasonal peak was from april to september, 36, 46 with a longer duration than our study. the overall seasonality in this study was also different from the studies conducted in northern or central cities of china, in which the seasonality of most viruses presented in autumn-winter and/or winter-spring. 15, [25] [26] [27] 30 the winter-spring seasonality was also observed in guangzhou, a city about 150 kilometers north of shenzhen. 28 different seasonal onset and duration were observed in various studies conducted in (sub-) tropical regions. in these studies, ambient temperature, humidity and rainfall were widely used to explain these differences in seasonality, but inconsistent results were observed. 9, 46, 47 although most studies demonstrated that the seasonality of viral respiratory infections was correlated with increased rainfall, effects of climate factors such as humidity and temperature on the seasonality were complex and interactive. 9, 46, 48 the study areas have four indistinct seasons, and the coldest month usually emerges in january (average 12°c). during the period from march to may, the weather featured warm ambient temperature (average 18-25°c), high relative humidity (average 85%-90%) and increasing rainfall. these meteorological conditions were perhaps conducive to viral survival. 9, 48 in addition, intensive temperature fluctuations during seasonal alternation could increase the susceptibility to infections. 49 as reported in other studies in temperate, tropical and subtropical regions, viral infection rates in children population showed an inverse correlation with age, with younger individuals experiencing higher viral infection rates. 3, 4, 6, 9, 24 our results suggested that children younger than 4 years of age, particularly <6 months, were at higher risk of hospitalization for artis, compared with older children. this was particularly substantiated in rsv infection. our presumption was supported by other studies. 14,25-28 of course, this speculation needed to be validated by the population-based study. the findings reported elsewhere suggested that more males than females were affected by artis, which were not observed in our study. notably, our study occurred over a span of 3 years, which included the iav (h1n1) outbreak in 2009. the impact of the outbreak on the results should be considered. data showed that the detection rate of iav increased significantly and co-infection rate during outbreak months was much higher than average co-infection rate. unfortunately, we did not type these influenza strains based on the original study design. it was most likely that these strains contributed to the relatively high proportion of iav. relatively higher single and multiple infections of rsv, hrv and pivs were also observed during the outbreak of iav. increased susceptible population and awareness, intensive testing and altered patient and physician behavior could lead to these increases. these factors could partly explain the relatively high proportion of pneumonia cases in the study. furthermore, studies showed that the outbreak of iav (h1n1) could increase the risk of other viral infections such as rsv and hrv. 24, 33 other limitations also existed in this study. first, molecular methods allowed the detection of only viral nucleic acid even without virus replication, which complicates the interpretation of positive detection results. second, the subtype identification of some common respiratory viruses such as iav and hrv was not performed in our study, particularly during the iav (h1n1) outbreak in 2009. in summary, despite those aforementioned limitations, this three consecutive years' surveillance would provide a basic profile of the spectrum, seasonality, age and gender distribution, co-infection patterns as well as clinical association of viral respiratory infections in hospitalized children in the study sites. it could help the prediction, prevention and control of artis in children. respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology causes of deaths in children younger than 5 years in china in 2008 respiratory virus infection in infants and children emerging respiratory agents: new viruses for old diseases? ten years of human metapneumovirus research occurrence of respiratory virus: time, place and person epidemiology of respiratory syncytial virus infection in northern taiwan variation in timing of respiratory syncytial virus outbreaks: lessons 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in pediatric inpatients in southeast china pediatric hospitalization of acute respiratory tract infections with human bocavirus in hong kong detection of human bocavirus from children and adults with acute respiratory tract illness in guangzhou, southern china coinfections with influenza and other respiratory viruses evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections mixed respiratory virus infections multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection evaluation of viral co-infections in hospitalized and nonhospitalized children with respiratory infections using microarrays epidemiology of respiratory syncytial virus infection among paediatric patients in hong kong: seasonality and disease impact incidence of common respiratory viral infections related to climate factors in hospitalized children in hong kong the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus exposure to cold and respiratory tract infections we are most grateful to the clinicians and nurses for their assistance in sample collection. we also thank dr. yi-wei tang for reviewing this manuscript and dr. linda chui, dr leo su for their help in improving english in this paper. none of the authors has a conflict of interest. key: cord-283405-aozxvxxs authors: vermillion, meghan s.; klein, sabra l. title: pregnancy and infection: using disease pathogenesis to inform vaccine strategy date: 2018-02-01 journal: npj vaccines doi: 10.1038/s41541-017-0042-4 sha: doc_id: 283405 cord_uid: aozxvxxs vaccination is the mainstay of preventative medicine for many infectious diseases. pregnant women, unborn fetuses, and neonates represent three at-risk populations that can be simultaneously protected by strategic vaccination protocols. because the pathogenesis of different infectious microbes varies based on tissue tropism, timing of infection, and host susceptibility, the goals of immunization are not uniform across all vaccines. mechanistic understanding of infectious disease pathogenesis and immune responses is therefore essential to inform vaccine design and the implementation of appropriate immunization protocols that optimize protection of pregnant women, fetuses, and neonates. vaccination significantly reduces the health burden of many infectious disease, especially in high-risk populations. pregnant women, unborn fetuses, and neonates represent three populations of high-risk individuals that can all be simultaneously protected from vaccine-preventable infectious disease with strategic maternal immunization protocols. infectious microbes that pose significant health risks during pregnancy can be divided into three broad categories, based on the pathogenesis and disease outcome (fig. 1) , with some microbes falling within more than one category. first are maternal infections, which are defined by heightened disease severity in pregnant females, but with rare or inconsequential transmission and disease in the fetus. second are fetal or congenital infections, which are characterized by mild or no disease in pregnant females, but occasional vertical transmission and severe congenital disease in the fetus. third are neonatal and infant infections, which are not considered to pose significant risk to pregnant women or unborn fetuses, but can cause severe, and sometimes fatal disease in neonates and infants that lack protective maternal immunity following birth. the vaccination strategies employed differ for micobes within each of these categories and vary based on the at-risk individual (i.e., mother, fetus, and/or neonate/infant), the timing of the greatest risk of infection (i.e., early pregnancy, late pregnancy, or post-natal), and on the duration of protective immunity following vaccination. in this review, we discuss evidence to suggest that immunization strategies for pregnant women should be tailored to optimize protection for the mother, fetus, neonate, infant, or all individuals. we review vaccine-preventable infections during pregnancy and the current vaccination strategies employed to reduce the burden of infectious diseases, including influenza. further, we examine novel vaccine platforms and consider how their application may provide safe alternatives for enhancing protection of pregnant women. finally, we discuss vaccine development and prevention strategies for combatting emerging infectious diseases, including zika, that pose a threat to pregnant women and their fetuses. owing to physiologic and immunologic changes that support pregnancy and tolerance of a semi-allogenic fetus, 1 pregnant women demonstrate increased susceptibility to certain infectious agents including hepatitis e, varicella zoster, and influenza viruses. infection with these viruses during pregnancy results in severe maternal disease, increased maternal mortality and associated pregnancy complications, which are observed most frequently during the third trimester and peripartum period. for example, the case fatality rate among pregnant women infected with hepatitis e virus is estimated to be 5-25%, 2,3 compared with 1-3% in the general population. 4 approximately 28% of cases of varicella pneumonia in adults reported from 1965-1989 were from pregnant women 5 ; and pregnant women infected with the pandemic 2009 h1n1 influenza a virus (iav) were reportedly 4 times more likely to be hospitalized or die than the general population. 6 overall, vertical transmission of these viruses is relatively uncommon, but adverse pregnancy outcomes, including spontaneous abortion and pre-term birth can still occur as an indirect consequence of maternal inflammation. 7, 8 reports during the 2009 h1n1 pandemic in australia and new zealand indicated that among pregnant women who were hospitalized with suspected h1n1 iav infection, 50% of their infants required intensive care, and 10% were either stillborn or died shortly after birth; only 2 infants, however, had detectable 2009 h1n1 infection. 9 the primary goal of vaccination strategies for protecting against maternal infections is the generation of protective maternal immunity either prior to or during early pregnancy. optimally, vaccination should prevent or reduce disease by inducing sterilizing immunity (i.e., immunity that completely prevents infection). despite reported reductions in antiviral proteins during pregnancy, 10 studies comparing vaccine responses between pregnant and nonpregnant women find no difference in either the magnitude or duration of antibody responses against influenza a viruses. [11] [12] [13] in fact, surveillance data from taiwan reveal that influenza vaccination during pregnancy results in higher levels of seroprotection than does vaccination prior to conception, 14 with no effect of gestational age on vaccineinduced antibody responses. 15 as of 2004, the centers for disease control (cdc) advisory committee on immunization practices (acip) categorizes pregnant women as a target population for receiving the inactivated influenza vaccine and recommends that pregnant women be immunized during any trimester. 16 several studies evaluating adverse vaccine reactions in pregnant women have concluded that there is no link between pregnancy complications or adverse fetal outcomes among women who are vaccinated during pregnancy. [17] [18] [19] [20] although the live attenuated intranasal influenza vaccine is not recommended for pregnant women, accidental administration during pregnancy was not associated with an increased risk of adverse reactions. 17, 21 despite the plethora of data that support the benefits and safety of influenza vaccination during pregnancy, coverage remains low, with a less than 50% maternal vaccination rate during the 2010-2011 influenza season in the united states. 22 misconceptions about the safety and benefits of influenza vaccination represent the largest barriers to vaccine acceptance among pregnant women. 23 varicella zoster virus (vzv) is another vaccine-preventable infection associated with increased severity during pregnancy. 24 vzv is an alpha herpes virus and the causative agent of varicella or chickenpox. in temperate climates, seroprevalence among individuals over 30 years of age is estimated to be 95%, with almost 90% of infections occurring prior to 15 years of age. 25 the first modified-live vaccine against varicella zoster virus was licensed in the united states in 1995, and is now recommended for children over 12 months of age. 26 primary vzv infection during pregnancy is therefore uncommon, as most women of childbearing age have been either infected or immunized. in women who have not been previously exposed, however, primary vzv infection between weeks 8 through 26 of gestation is associated with a 2% risk of congenital transmission and disease in the offspring. 27 because all licensed vzv vaccines contain live-attenuated virus, their use during pregnancy is contraindicated. instead, the cdc recommends that nonpregnant women of childbearing age be vaccinated against vzv at least one month prior to conception. 26 as a herpes virus, infection with vzv is life-long, and reactivation occurs in approximately 10-30% of individuals, which results in a painful skin condition known as shingles or herpes zoster. 28 reactivated vzv, however, is not associated with increased disease severity or congenital infection during pregnancy. 24 acute viral hepatitis caused by hepatitis e virus (hev) is an emerging infectious disease that causes severe disease in pregnant women, with a fatality rate of up to 30% in endemic regions. 29 in addition to heightened maternal disease severity, hev infection during pregnancy is associated with increased rates of premature birth and prenatal mortality. 30 although vertical hev transmission rates are high, with estimates between 23-50%, 31 the relative contributions of fetal hev infection to adverse perinatal outcomes is unclear. 32 a recombinant hev subunit vaccine has been developed and proven safe and effective following completion of phase ii and iii clinical trials, 33 but commercial use is currently limited to china. furthermore, the vaccine is not approved for use in pregnant women despite being 100% efficacious in participants receiving all three doses. 34 additional hev vaccine candidates are being tested in preclinical pregnant animal models, and one recombinant hev vaccine has been shown to be safe and highly immunogenic in pregnant mice. 35 additional studies in a susceptible animal model are needed to confirm efficacy following virus challenge. developing fetuses are extremely vulnerable to both infectious and noninfectious insults. certain infectious agents that are often clinically silent in healthy adults can cause severe birth defects if fig. 1 infectious microbes that cause maternal, congenital, or postnatal complications. the infectious microbes are categorized according to the mechanism of transmission and disease, and the population at greatest risk for severe outcome during or after pregnancy. infection with some pathogens (e.g., sars coronavirus, hepatitis e virus, and ebola virus) during pregnancy cause severe disease in pregnant women, but are not transmitted to offspring. other infectious microbes (e.g., toxoplasma gondii, rubella virus, parvovirus b19, cytomegalovirus, and zika viruses) infect and cause mild or asymptomatic disease in pregnant females, but can be vertically transmitted to the fetus and congenital complications. another category of microbes (e.g., bordetella pertussis, clostridium tetani, and respiratory syncytial virus) pose the largest risk to neonates after birth. many infectious microbes (e.g., listeria monocytogenes, plasmodium spp., hiv, vzv, influenza viruses, chlamydia trachomatis, gbs, treponema pallidum, and herpes viruses) may cause overlapping syndromes depending on the timing of infection during pregnancy. understanding the pathogenesis of infectious diseases during pregnancy should inform vaccine design and the implementation of appropriate immunization protocols that optimize protection of pregnant women, fetuses and neonates they breach the placental barrier during critical developmental periods during pregnancy. an increasing number of pathogens are being recognized for causing congenital disease, and what was originally designated as the torch complex (toxoplasma gondii, "other," rubella virus, cytomegalovirus, and herpes simplex virus) is now expanded to include other infectious agents including zika virus. development of congenital disease can depend on the timing of infection during gestation, the infectious burden, and the pathogenesis in the fetus. the congenital syndrome for each pathogen is characterized by a variety of different developmental abnormalities, and commonly impact hearing, vision, and central nervous system function. 36 for many congenital infections, the timing of infection during gestation determines the relative risk to the fetus and dictates the spectrum of disease that results. for example, while infection with rubella virus during the first 9 weeks of gestation is associated with an 85% risk of congenital rubella syndrome (crs), this is reduced to a 52% risk between 9-12 weeks, and minimal risk for infections occurring after 16 weeks of gestation. 37 in contrast, the risk of congenital toxoplasmosis has been demonstrated to be highest during third trimester pregnancy, which is hypothesized to be due to differential expression of placental toll-like receptors, including tlr6, within first compared with third trimester trophoblast cells. 38 similarly, maternal infection with listeria monocytogenes is typically associated with adverse pregnancy outcomes during the third trimester, 39 though infection during the first trimester in nonhuman primates also leads to rapid fetal demise. 40 the primary goal of vaccination strategies for protecting against fetal infections is generation of protective maternal immunity prior to pregnancy. because congenital infections can occur in the absence of maternal symptoms, vaccines against congenital agents should ideally provide complete sterilizing immunity. rubella is included in a live-attenuated combination vaccine for measles, mumps, and rubella (mmr), which confers lifelong protective immunity. 41 because of the long duration of protective immunity following rubella vaccination, target populations include children and adolescent girls. however, incomplete vaccination coverage can lead to paradoxical increases in crs due to an increase in the average age of infection, 42 and so it is also recommended that unvaccinated women of childbearing age be counseled to receive the rubella vaccine at least one month prior to conception. 43 the implementation of large-scale rubella vaccination programs has resulted in sufficient population-level immunity, significant reductions in crs, 44 and elimination of rubella virus from several developed countries, including the united states. 45 following successful implementation of mmr vaccination programs, cytomegalovirus (cmv) has emerged as the most common congenital viral infection in the developed world. 46 the incidence of congenital cmv varies based on geographic region and socioeconomic status, but overall birth prevalence is estimated to be 0.64%, which is similar to the incidence of down syndrome and fetal alcohol syndrome. 47 in contrast to rubella virus, however, there is currently no licensed vaccine available for cmv, and with seroprevalence approaching 100% in some developing countries, 48 vaccine development has been identified as a priority public healthcare goal. 49, 50 while cmv infection of healthy adults is usually asymptomatic, adaptive immune responses are insufficient to clear the infection, which results in lifelong latent infection of myeloid precursor cells. 51 although latent or reactivated cmv is less likely to cause congenital infection than a primary cmv infection during pregnancy, 52,53 preconception immunity does not completely eliminate transplacental transmission and congenital disease. moreover, pregnant women with latent cmv infection are still susceptible to primary infection with different cmv strains, which have been shown to have distinct virulence patterns. 54, 55 overcoming the challenges associated with latent infections and strain variability are significant hurdles in the development of an effective cmv vaccine, and despite significant advances in our knowledge of cmv pathogenesis, the precise immune targets that constitute fetal protection remain unknown. of the several cmv vaccine candidates that have been tested, none have provided complete protection against infection, and all have failed to protect against reactivation of latent cmv. 56 more research on the pathogenesis cmv infection is needed to define immunological correlates of protection against cmv transmission during pregnancy to inform vaccine development. although not associated with congenital disease, hepatitis b virus (hbv) is another vaccine-preventable infection that can cross the placenta during pregnancy. mother-to-child-transmission remains the most common route of infection in endemic regions, 57 and women with active viral replication have up to a 90% chance of vertical transmission. 58 of those that are infected perinatally, up to 90% develop chronic hbv infection. 57 since the initial recommendation of routine hbv vaccination of children in 1991, the rate of new hbv infections has significantly declined in the united states, but chronic hbv remains prevalent in sub-saharan africa and east asia. although combined passive and active immunoprophylaxis of infants has significantly reduced perinatal hbv infection, perinatal transmission occurs in up to 20% of infected mothers. 59 to augment neonatal prophylactic strategies, the cdc acip recommends that pregnant women who are identified as being at risk for hbv infection be vaccinated with the recombinant hbv vaccine. 60 immunity following receipt of the hbv vaccine is long-lived, with anti-hbv antibodies persisting in most adults for at least 20 years. 61 because of the long-term protection conferred by the hbv vaccine, immunization is not necessary for pregnant women who have already been vaccinated and are at low risk of infection. 60 owing to the limited exposure to foreign antigen and blunted innate immune responses in utero, the neonatal immune system is immature at birth, making neonates (i.e., less than one month of age) particularly susceptible infections. 62 infectious diseases are responsible for over 60% of child mortality, and over 40% of these deaths occur within one month of age. 63 during the neonatal period of immune system maturation, protection against pathogens relies primarily on passive immunity from maternal-derived igg antibodies. in humans, most maternal antibodies are transferred into the fetal circulation through the placenta prior to birth, which contrasts with most veterinary species, in which maternal antibody is transferred via colostrum immediately following birth. regardless of species, vaccination during pregnancy increases circulating maternal antibodies and enhances transfer to the fetus/neonate. 14 the goal of vaccination strategies for protecting against neonatal infections is generation of robust maternal antibody responses during pregnancy to enhance placental transfer. further, because neonatal protection is exclusively conferred by maternal-derived antibody, vaccines aimed at protecting infants should prioritize induction of humoral over cellular immune responses, with the induction of igg1 being most important because this igg isotype is associated with the highest placental transport efficiency in females. 64 moreover, the kinetics of maternal vaccine-induced antibody response, the efficiency of placental antibody transfer, and the half-life of the antibody in the neonate should inform the optimal timing of vaccination during pregnancy. because the peak antibody response is typically observed 1-3 weeks following immunization, vaccination during pregnancy as opposed to before conception is likely to result in the greatest benefit to the neonate. further, the efficiency of placental antibody transfer in females increases throughout gestation, with less than 8% maternal igg transferred to the fetus in the first 16 weeks of gestation, 65 significantly more transferred during the second and third trimesters, and at delivery fetal igg often exceeds maternal levels. 66 vaccination of females during the second and third trimesters of pregnancy is most likely to generate the greatest level of protection in the neonate, but the precise timing for maximum protection is debated. controversy over the timing of pertussis vaccination in pregnancy has been reviewed elsewhere, 67 with some reports claiming peak cord blood antibody concentrations following vaccination in the second trimester, and others reporting peak antibody concentrations following vaccination in the third trimester. antibody avidity also influences the efficiency of placental transfer, with higher avidity antibodies crossing the placenta with greater efficiently than low avidity antibodies. 68 more consideration should be given to the development of high avidity antibodies in the timing of vaccination during pregnancy, as protective immunity in the infant depends on both the concentration and avidity of the maternal-derived antibody. the half-life of maternal antibodies in infants also must be considered in vaccine development and administration. maternalderived igg1 is reported to have a half-life of approximately 48 days in serum, 69 and depending on serum antibody titers present at birth, this translates into protective immunity for approximately the first 3-9 months of life for most infant pathogens. 70 the half-life of the antibody also dictates the vaccination schedules for infants, as the presence of maternalderived antibody interferes with vaccine efficacy, and it is not until maternal-derived antibody has waned below a certain threshold that an infant can mount its own active vaccine response. the goal of the infant vaccine series is to time vaccination to coincide with the time that maternal-derived antibody drops below the threshold at which it can neutralize the vaccine antigen. because the precise timing of these events is unpredictable, infant vaccination schedules are designed so that vaccines are administered in a series that spans the duration of this window, and minimize susceptibility to natural infection. in the united states, infant vaccines are recommended at 2, 4 and 6 months of age. 41 bordetella pertussis is a vaccine-preventable respiratory pathogen of significant public health importance, and it is a major cause of mortality in infants lacking protective maternal immunity. vaccination of women during pregnancy, however, significantly enhances the transfer of maternal antibody to the fetus, 71,72 and these newborns are 11 times more likely to have protective antibody titers at birth compared with those born from women who were not vaccinated during pregnancy. 71 inactivated pertussis antigen is combined with tetanus and diphtheria toxoids in a single vaccine (tdap), which the cdc acip recommends for all pregnant women, regardless of previous vaccine history. 73 in contrast to vaccine formulations that contain killed whole b. pertussis organisms, the tdap vaccine contains only select antigens and confers relatively weak and only transient protective immunity that declines after 1 year. 74 vaccination of women either prior to conception or during early pregnancy does not provide adequate neonatal protection against pertussis. 75 consequently, the cdc considers the third trimester to be the optimal time to administer the tdap vaccine to pregnant women. 73 adverse events reported following tdap vaccination are generally mild, and there are no reported risks of adverse pregnancy outcomes related to tdap vaccination during pregnancy. 76 despite consistent evidence that supports the benefit and safety of tdap vaccination during pregnancy, coverage remains low, with an estimated 42% of pregnant women receiving the tdap vaccine in the united states in 2013. 77 receipt of the tdap vaccine during pregnancy also confers protection against neonatal tetanus, which is associated with case fatality approaching 100% in the absence of medical care. 78 disease is caused by the toxin produced by clostridium tetani, and infection occurs most commonly due to contamination of the umbilical stump following delivery. consequently, the incidence of disease is much greater in developing countries, where maternal vaccination is scarce and perinatal hygiene practices are poor. 79 in 1989, the world health assembly called for the elimination of neonatal tetanus, which has inspired an initiative to improve vaccination coverage and birth hygiene in 59 countries with high disease prevalence. as part of this initiative, immunization standards have been expanded and recommend that pregnant women with unknown or inadequate vaccination history receive two doses of the toxoid-containing vaccine, administered one month apart. 80 maternal anti-tetanus antibodies are passively transferred to the fetus, and it is estimated that maternal immunization reduces neonatal tetanus mortality by 94%. 81 respiratory syncytial virus (rsv) is the most common respiratory viral pathogen of newborns and infants, and accounts for 50-90% of acute bronchiolitis and 5-20% of pneumonia cases in hospitalized children less than 2 years of age. 82 rsv is also reported to cause severe disease and hospitalization in pregnant women when infection occurs during the third trimester, 83, 84 and therefore dually qualifies as a maternal infection as well. a licensed vaccine against rsv is currently unavailable, but several vaccine candidates have shown promise in various animal models. [85] [86] [87] [88] given the importance of this pathogen during early life, vaccine development strategies have focused on maternal immunization, with three maternal vaccines currently in clinical trials. 89 maternal vaccination against rsv has direct and indirect benefits to the neonate; neonates are directly protected through passive transfer of maternal antibody through the placenta, and they are indirectly protected because a vaccinated mother is less likely to transmit the infection to her infant. 90 vaccination of pregnant women is controversial, and immunization with live (i.e., replication-competent) viral or bacterial vaccines is generally contraindicated due to the theoretical risk of congenital infection and teratogenic effects from the vaccine strains. however, in a report of over 2000 pregnant women who were unknowingly immunized with live attenuated rubella vaccine, there were no cases of vaccine-associated congenital rubella infection, 91 and live virus strains of influenza or yellow fever viruses administered to pregnant women also have no link with pregnancy complications. 21, 92 vaccination with inactivated vaccines such as influenza and tdap during pregnancy have low uptake, with concerns of safety among both patients and their healthcare providers being a primary barrier. the safety of vaccine adjuvants is debated, and although neither the tdap nor seasonal influenza vaccine recommended during pregnancy contain adjuvants, retrospective studies evaluating safety of the adjuvanted pandemic h1n1 influenza vaccine in pregnant women found no relationship with adverse pregnancy outcomes. 93 the conservative approach to vaccination protocols for pregnant women stems from the lack of controlled safety and efficacy studies for this population. for ethical reasons, pregnant women are exempted from almost all clinical and vaccine trials, and heath care providers are less likely to endorse prophylactic treatments for which safety and efficacy profiles have not been adequately characterized. whereas study in pregnant women is not possible, pre-clinical testing in animal models may provide a useful alternative, and vaccine preclinical trials in pregnant animal models may provide information to inform healthcare policies for pregnant women. although there are some differences in the length of gestation, placental structure, and fetal development between humans and animal models, many structural and functional parallels exist, [94] [95] [96] which serve as tractable platforms for evaluating the safety and efficacy of various therapies during pregnancy. similar to humans, pregnant mice, rats, and rabbits have a hemochorial placenta, and their relatively short gestation and large litters are advantageous for performing high throughput screening of candidate therapeutics for safety and efficacy. preclinical behavioral testing of rodent offspring has proven to be a promising avenue for identifying and predicting adverse effects associated with prenatal drug exposure in children. 97 both rodent and rabbit models have been instrumental in testing teratogenic effects of artemisinin-based combination therapies for treating malaria in pregnant women. these studies concluded that drug-related teratogenic effects are limited to the first trimester, which supports the world health organization (who) recommendation that artemisinin may be administered only during the second or third trimester in pregnant women. 98, 99 one limitation of mouse and rat models, however, is their inability to recapitulate certain elements of human congenital disease. for instance, because murine cmv is not transmitted vertically as it is in humans, other animal models, including guinea pigs and nonhuman primates, are required for studying this aspect of disease pathogenesis. studies in pregnant nonhuman primates have been instrumental for the identification of cd4 + t cell responses as critical for early control of cmv infection and transmission during pregnancy, 100 and studies in guinea pigs have demonstrated that a single-cycle infectious cmv vaccine induces immune responses similar to natural infection and protects against congenital infection. 101 guinea pigs are also a useful model of chlamydial genital infection in humans. experimental venereal infection with chlamydophila caviae mimics disease associated with c. trachomatis in humans, including both sexual and perinatal transmission. guinea pigs have therefore served as a useful model for testing candidate vaccines and treatments. 102 rabbits continue to serve as an important model of venereal infection with treponema pallidum, the causative agent of syphilis, which is associated with congenital disease in humans. while natural infection in rabbits is associated with the species-specific t. paraluiscanuculi, rabbits can be experimentally inoculated with human t. pallidum, and have been instrumental in testing the efficacy of candidate vaccines. 103 many mammalian species, including rodents, 104 ruminants, 105 and nonhuman primates, 40, 106 are susceptible to infection with listeria monocytogenes and demonstrate similar fetal complications when infection occurs during pregnancy. studies in various animal models have uniquely contributed to our understanding of placental listeriosis and serve as a platform for evaluating prevention strategies. 107, 108 finally, mice, 85 cotton rats, 86 guinea pigs, 88 and sheep 87 are all susceptible to infection with rsv, and vaccination of pregnant animals has facilitated the development and testing of maternal immunization strategies for protecting against neonatal rsv. based on preliminary studies in guinea pigs, 88 an experimental rsv recombinant f nanoparticle vaccine is now being evaluated in third-trimester pregnant women (clintrials.gov, nct02247726). beyond the direct modeling of human congenital infection in animals, information can also be gained from the study of related veterinary pathogens. for example, bovine viral diarrhea virus (bvdv) is an important reproductive pathogen that infects cattle worldwide, and infection during pregnancy causes congenital infection and disease. persistently infected animals serve as reservoirs within a herd and can have a huge agricultural financial impact. 109 as a result, significant resources have been dedicated to the development and optimization of bvdv vaccines and vaccine protocols, considering variables such as the type and timing of vaccination on immune response and protection against challenge. 110 the information gleaned from these studies may inform vaccine development and optimization protocols for related pathogens in pregnant women, for which similar studies cannot ethically be performed. zika virus (zikv) is a unique flavivirus that causes mild or subclinical disease in pregnant women, 111, 112 but can have devastating effects for the fetus and neonate. infection during pregnancy is linked with spontaneous abortion and a variety of birth defects, including microcephaly and impaired neurocognitive function. 113 since its initial discovery in african macaques in 1947, zikv has expanded its geographical range and evolved into separate virus lineages, with environmental pressures resulting in the emergence of virulent substrains, raising concerns about vaccine escape mutants once a vaccine is approved. 114 the combination of its unique pathogenesis, diverse modes of transmission, and rapid global spread has increased efforts toward development and licensing of a zikv vaccine. a basic understanding of zikv biology and pathogenesis is essential for development of an effective vaccine against zikv. although we can extrapolate some biological information from related flaviviruses, such as yellow fever, west nile, and dengue viruses, zikv has unique characteristics following in vivo infection, which pose significant challenges to vaccine design. unlike other flaviviruses, zikv has a tropism for reproductive tissues, including the testes, semen, and sperm in males 115, 116 and the placenta in pregnant females, [117] [118] [119] which is hypothesized to contribute to sexual and vertical modes of transmission, respectively. in addition to unique tissue tropisms, zikv persists in reproductive tissue following clearance of systemic viremia. in males, zikv rna can be detected for months following recovery from symptomatic infection, 120, 121 and virus persistence in the placenta of pregnant women is hypothesized to contribute to prolonged viremia in this population. 122 evidence of virus persistence suggests that zikv may have evolved mechanisms for evasion of host immune responses when infection occurs in certain immune-privileged tissues. scenarios involving persistent zikv infections should be considered in developing and testing candidate vaccines. considering the potential for viral persistence in the semen, vaccinating men may serve as an additional strategy to reduce transmission to the fetus, as pregnant women may be infected by their sex partners. characterization of the immune response to zikv infection is essential for determining correlates of protection for vaccine efficacy. following infection of both humans and nonhuman animals, zikv induces neutralizing antibodies against the zikv e protein, which prevent fetal infection and demise when administered to pregnant mice. 123 further, 26 mhc class i epitopes have been identified that conferred protection against zikv challenge in immunocompetent mice. 124 to date, 45 candidate zikv vaccines have been developed, and as of february 2017, nine have entered phase i clinical trials. 125 vaccine candidates have been developed using diverse platforms, including dna, mrna, and purified inactivated and live-attenuated virus, many of which have been tested in non-pregnant mouse and nonhuman primate models for their ability to generate immune responses that mimic responses to natural infection and protected against zikv challenge. a candidate dna plasmid vaccine induced robust cellular immunity and neutralizing antibody responses in both nonhuman primates and immunocompetent mice, and conferred complete protection against lethal zikv challenge in type i interferon receptor deficient (ifnar â��/â��) mice. 126 lnp-mrna vaccines induced similar protective immunity, which was characterized by high neutralizing antibody titers and sterilizing immunity against zikv challenge in non-pregnant mice and nonhuman primates. 127, 128 whether these candidate vaccines induce protective immunity in pregnant females that is sufficient to prevent fetal and neonatal infections 117 requires further evaluation. also, whether pre-existing immunity to other flaviviruses that co-circulate with zikv, including dengue virus and west nile virus, affects the efficacy of zikv vaccines in pregnant females should be considered in both preclinical animal models and human clinical trials. conventional vaccines are formulated from either live, attenuated pathogen strains or from inactivated pathogens, but there are notable disadvantages to each of these platforms. live, attenuated vaccines are replication-competent with the potential of becoming virulent and causing adverse effects in individuals with weakened immune systems. due to the unknown risk to the developing fetus, live virus vaccines are not recommended for use in pregnant women. inactivated vaccines, on the other hand, are not associated with a risk of reacquisition of virulence, but they tend to induce a weaker host immune response. 129 efforts to balance safety with immunogenicity have led to the development of several novel vaccine technologies, including replicationdeficient nanoparticle-based vaccines and self-assembling recombinant virus-like particles (vlps), replication-competent recombinant viral vectors, and single-cycle infectious viruses that can infect, but not replicate in host cells. nanoparticle delivery platforms, including liposomes and synthetic polymers, can be engineered to enhance selective tissue homing for high potency, targeted delivery of antigen in its native conformation. 130 recombinant vlps combine highly immunogenic surface viral proteins with encapsulated adjuvants that are devoid of infectious nucleic acid, but induce strong cellular and humoral immune responses. 131 both nanoparticle and vlp vaccines are devoid of genetic material and are therefore replication incompetent, enhancing safety in vulnerable individuals. although current production yields and costs associated with these technologies may prohibit large-scale use, these platforms may be well-suited for targeted use in high-risk populations, including pregnant women. since the first nanoparticle-based vaccine was licensed for hepatitis b virus in 1981, the technology has been applied to develop licensed vaccines against human papilloma virus, hepatitis e virus, and malaria. 131 demonstration of safety and efficacy during pregnancy has not yet been documented. recombinant viruses can be engineered to combine the antigenic genes of one virus with the structural genes of another. this targeted manipulation of the virus genome is used to remove virulence genes to enhance safety and alter envelope proteins to change cell tropism. recombinant viruses can be engineered to retain the ability to infect and replicate in the host, while preserving infectious potential and enhancing the generation of innate and adaptive immune responses that mimic natural infection. recombinant virus vaccines, however, warrant careful consideration of the safety of the vector itself, especially in pregnant women. once the safety and efficacy profiles of a viral vector platform have been established, engineering new antigenic targets into the viral genome are relatively simple, and do not require extensive re-validation, as safety and efficacy is most influenced by the vector virus. this can significantly reduce the time to develop and manufacture vaccines against new viral pathogens. 132 viruses from many different families can be used as vectors provided they can infect the host and elicit a productive immune response without causing disease. of note, poxviruses are practical vectors due to ease of growth and manipulation in vitro, wide host range, and robust induction of protective immune responses. although vaccinia virus vectors are contraindicated during pregnancy due to risk of disseminated disease, recent testing of a raccoonpoxvirus-vectored rabies vaccine in pregnant mice proved safe and effective. 133 other novel vaccine platforms are based on genetic engineering and creation of targeted loss-of-function mutations in the viral genome. reverse genetics technology has contributed the identification of the sequences within the viral genomes that are essential for infection and replication in vivo. it is now possible to engineer recombinant virus vaccines with targeted mutations in a cost-and time-efficient manner. in contrast to inactivated vaccines, reverse genetics allows for controlled manipulation of the viral genome that targets a specific process in the virus life cycle. this enables production of a vaccine strain with maximum efficacy and safety, and represents another promising alternative to conventional attenuated or killed virus vaccines. among the safest recombinant vaccine approaches are the single-cycle infectious (sci) viruses, which have been developed from influenza a virus (iav) backbones by deleting or truncating viral proteins necessary for completion of the virus life cycle within the host. 134 such genetic modifications render the virus replication-incompetent, but capable of infecting and inducing an immune response in the host. 135 the infectious capacity of sciiav vaccines results in strong cellular and humoral immune responses, without the risks and adverse effects associated with live-attenuated vaccine strains. studies in nonpregnant mice have demonstrated that a single dose of sciiav vaccine confers protection against heterosubtypic lethal challenge without any adverse effects. [136] [137] [138] [139] [140] [141] similar safety and efficacy profiles of sciiav have been replicated in ferret and pig models, 136, 142 but studies in pregnant animals have not been performed. compared with the risks associated with live virus vaccination and concerns of efficacy with inactivated vaccines, novel vaccine platforms, such as nanoparticle-based technologies, vlps and replication-deficient viruses have proven benefits. 130 additional safety and efficacy studies in pregnancy models are warranted to validate and expand the use of these vaccine platforms for pregnant women. (table 1) conclusions strategic immunization of women, either prior to or during pregnancy, can eliminate or substantially reduce the risk of maternal, fetal, and neonatal infection and disease. the effectiveness of an immunization protocol depends on both the efficacy of the vaccine in inducing protective immune responses and on the timing of vaccine delivery during pregnancy to synchronize the peak vaccine response with the period of greatest susceptibility in the host. optimization of vaccination protocols to achieve this goal requires an understanding of the mechanisms of infection and pathogenesis of disease during pregnancy. successful implementation of vaccine protocols for pregnant women requires consideration of additional challenges, such as the frequency of unplanned pregnancies and access to prenatal health care. human surveillance data provide correlative clues of the character of specific infections, but mechanistic understanding requires additional study in comparative 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and meta-analysis immunology of pediatric hiv infection parvovirus b19 infection in human pregnancy consequences of confirmed maternal rubella at successive stages of pregnancy severe acute respiratory syndrome (sars) in neonates and children sars during pregnancy, united states microbiology laboratory and the management of mother-child varicella-zoster virus infection initial description of the presumed congenital zika syndrome the authors thank the johns hopkins fisher center discovery program for funding. all authors (m.s.v. and s.l.k.) were involved in the conception and drafting of this review, and approved the manuscript before it was submitted by the corresponding author. competing interests: the authors declare no competing financial interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-302619-3hbbpmnt authors: strausbaugh, l. j. title: emerging health care-associated infections in the geriatric population. date: 2001 journal: emerg infect dis doi: nan sha: doc_id: 302619 cord_uid: 3hbbpmnt the increasing number of persons >65 years of age form a special population at risk for nosocomial and other health care-associated infections. the vulnerability of this age group is related to impaired host defenses such as diminished cell-mediated immunity. lifestyle considerations, e.g., travel and living arrangements, and residence in nursing homes, can further complicate the clinical picture. the magnitude and diversity of health care-associated infections in the aging population are generating new arenas for prevention and control efforts. the elderly have defective host defenses that compromise their ability to ward off infectious agents; factors influencing immunocompetence include immune senescence, changes in nonadaptive immunity, chronic diseases, medications, malnutrition, and functional impairments. t-lymphocyte production and proliferation decline with age, resulting in decreased cell-mediated immunity and decreased antibody production to new antigens (3) (4) (5) . thinning skin, enlarged prostate, diminished cough reflex, and other anatomic or physiologic accompaniments of aging are changes in nonadaptive immunity that render the elderly more vulnerable to infection. chronic diseases-cancer, atherosclerosis, diabetes mellitus, dementia-predispose to certain types of infection. medications such as sedatives, narcotics, anticholinergics, and gastric acid suppressants may further suppress innate defenses. malnutrition, which reduces cellmediated immunity, is common in nursing home residents (4) and may be more common in the geriatric community at large than is generally realized (6) . finally, functional impairments (e.g., immobility, incontinence, dysphagia) can complicate aging and enhance susceptibility to infection. these impairments may necessitate the use of urinary catheters, feeding tubes, and other invasive devices that magnify susceptibility. alone or in combination, these defects in host defense(s) place geriatric populations in the forefront of nosocomial infection statistics. data from the national nosocomial infections surveillance system for the period 1986-1990 indicated that persons 65 years of age accounted for 54% of all nosocomial infections (7) . similarly, gross and colleagues observed a decade-specific risk for nosocomial infection of 10 per 1,000 discharges from birth through the fifth decade. however, this risk steadily rose from the fifth decade onward, exceeding 100 infections per 1,000 discharges in patients >70 years of age (8) . finally, saviteer and coworkers, who reported a similar increase in nosocomial infections after the fifth decade (9), calculated daily nosocomial infection rates of 0.43% and 0.63% for persons aged <60 years and >60 years, respectively. the higher infection rates in the elderly were not attributable to increased lengths of stay. geriatric patients, like transplant recipients, may be compared to "sentinel chickens"-the first to be affected by new or emerging infections in hospitals and other health-care environments that care for adult patients. for example, the mean age of affected patients in a nosocomial outbreak of gastroenteritis caused by a small round-structured virus was 65 years (10) . the problem of tuberculosis (tb) deserves particular mention in the context of waning cell-mediated immunity. the elderly have not only this risk factor but also higher frequencies of latent infection, stemming from exposures during an era when tb was more prevalent. tb is the most the increasing number of persons >65 years of age form a special population at risk for nosocomial and other health care-associated infections. the vulnerability of this age group is related to impaired host defenses such as diminished cell-mediated immunity. lifestyle considerations, e.g., travel and living arrangements, and residence in nursing homes, can further complicate the clinical picture. the magnitude and diversity of health care-associated infections in the aging population are generating new arenas for prevention and control efforts. commonly reported notifiable disease in persons >65 years of age (3) . in 1995, 23% of reported cases in the united states occurred in this age group. elderly persons living in the community have twofold increased rates of active disease. as a health care-associated infection in this age group, tb comes to the fore in hospital and nursing home outbreaks (11) . elderly persons living in long-term care facilities have fourfold increased rates of active tb. the combination of decreased cell-mediated immunity and high prevalence of latent infection suggests that tb will continue to reemerge in geriatric populations. decreased cell-mediated immunity may also predispose geriatric patients to nosocomial cryptosporidiosis. a microbiologic review for a 325-bed hospital in rhode island identified 36 patients with cryptosporidiosis (12); 13 of these patients were in the 63-to 93-year age group (mean 77 years). in seven of these older patients, nosocomial acquisition was suspected. in addition, outbreaks of this disease have occurred in elderly nursing home residents (13) . thus, cryptosporidium may be an emerging health care-associated infection in the aged. the lifestyles of the elderly may entail additional risk factors for both acquiring and transmitting health careassociated infections. in western countries retired persons use their increased leisure time to travel, including domestic trips to visit family, cruises or tours to foreign countries, or volunteer work in developing countries, which put elderly travelers at risk for infections. in addition, recreational activities such as golfing, spelunking, hunting, and gardening may bring the elderly into contact with unusual pathogens. volunteer work, visiting ill friends in the hospital, and other patterns of socialization also expose the geriatric population to infections that may be transmitted or acquired in the health care setting. several factors specifically related to health care deserve attention in this regard. the first concerns outpatient visits. the elderly spend increased amounts of time visiting their physicians, potentially exposing themselves to various contagious diseases in the health-care environment. they also make frequent use of food services and providers of prepared foods, which carry some risk for transmitting foodborne diseases. these infections may then enter the health-care system and lead to secondary cases. adult daycare centers and home care services, which have proliferated under medical auspices in recent years, provide additional avenues for geriatric populations to acquire health careassociated infections. the impact of these lifestyle factors on nosocomial and other health care-associated infections has not been well documented. several observations provide examples of the potential influence of these factors. a recent report from taipei described a nosocomial outbreak of malaria resulting from contamination of a computed tomography injection device with blood from a returning traveler (14) . likewise, a 1998 outbreak of influenza in alaska and the yukon territories, where 60,000 to 70,000 tourists visit each summer, further delineated the potential role of travel (15) . prospective surveillance in 1998 identified 2,199 cases of acute respiratory illnesses in 12 hospitals and clinics in alaska and the yukon territory. among these illnesses, 35% of cases in tourists and tourism workers met criteria for influenzalike illness and 3.2% for pneumonia. median ages were 60 years for all persons with acute respiratory illnesses and 72 years for all persons with pneumonia. fifty of the persons with pneumonia required hospitalization. the role of lifestyle factors related to health care has received little attention, but one recent publication illustrates the potential problem. a 4-year study of acute respiratory illnesses in three senior day-care centers documented the annual occurrence of viral respiratory infections in 16 to 43 elderly participants and 6 to 23 staff (16) . identified pathogens included influenza a, influenza b, respiratory syncytial virus, coronavirus, parainfluenza virus, and rhinovirus. of special importance, an educational campaign stressing the importance of handwashing combined with use of a portable virucidal foam product cut the infection rate by 50% during the fourth year. this article describes a new setting for health care-associated infections and confirms that traditional approaches to prevention still apply. the spectrum of living arrangements for geriatric populations ranges from private residences in the community to skilled nursing homes. between these extremes are retirement homes, assisted living facilities, foster and group homes, chronic disease hospitals, and other arrangements that provide for the needs of persons with sustained self-care deficits (4) . little is known about the role that these arrangements play in the overall scope of health careassociated infections. however, during the last 15 years several studies have examined the problem of health careassociated infections in skilled-nursing homes (2, 4) . nursing homes are residential facilities for persons who require nursing care and related medical or psychosocial services (4) . approximately 90% of nursing home residents fall into the geriatric age range. as a group, nursing home residents exhibit virtually all the risk factors for infections associated with the geriatric population. as a consequence, infections occur commonly in this setting, and emerging health care-associated infections are no exception. three types of endemic infections occur regularly in all these facilities: urinary tract infections, lower respiratory tract infections-principally pneumonia, and various skin and soft tissue infections (4) ( table) . in the united states, the overall rates for nursing home-acquired infection are 3 to 7 infections per 1,000 resident day, or 1.6 to 3.8 million infections per year (4). occasionally, new etiologic agents crop up as causes of these endemic infections. for example, in a 2-year serologic study of selected pathogens causing respiratory tract infections and febrile episodes in two canadian long-term care facilities, orr and colleagues identified a positive serologic response to chlamydia pneumoniae in 9.4% of 224 febrile episodes (17) . these positive responses were associated with 12% of respiratory infections, including 5 of 30 pneumonias and 6.5% of infections of unknown origin. these data suggest that c. pneumoniae may be an emerging health care-associated infection in this setting. outbreaks also account for a proportion of the health care-associated infections observed in nursing homes (2, 4) . respiratory infections and gastroenteritis occur most frequently. although no national data on frequency of occurrence are available, published reports suggest that outbreaks are not uncommon. during 1970 to 1984, outbreak reports constituted approximately one-third of publications on infections in long-term care facilities (18) . from 1975 to 1987, the centers for disease control and prevention (cdc) received reports from 26 states about 115 foodborne outbreaks in nursing homes (19) . of the 106 outbreaks investigated by cdc's hospital infections program during the last decade, 6% occurred in long-term care facilities (20) . emerging pathogens account for some of the outbreaks in nursing homes. during the last decade, streptococcus pyogenes-the "flesh-eating" bacterium-was identified in nursing homes (21) . more recently, a foodborne outbreak of gastroenteritis caused by both salmonella heidelberg and campylobacter jejuni was reported (22) . loeb and colleagues recently described an outbreak of respiratory illness caused by l. sainthelensi in two canadian nursing homes (23) . these and other reports emphasize the vulnerability of frail, elderly residents who share common sources of air, food, water, and health care in nursing homes. health care-associated infections caused by antimicrobial drug-resistant bacteria have caused both endemic infections and outbreaks in nursing homes in the united states. the frequent movement of patients between hospitals and nursing homes undoubtedly facilitates the transfer of resistant microbes (24) . during the last 2 decades, gramnegative uropathogens with multidrug resistance and methicillin-resistant s. aureus have received the most attention (25) . gram-negative enteric bacilli have recently become resistant to fluoroquinolones and extended-spectrum cephalosporins (26) . in addition, vancomycin-resistant enterococci and penicillin-resistant pneumococci have been identified in long-term care facilities (27) (28) (29) . the appearance of the latter organism, which is seldom regarded as a nosocomial pathogen, again underscores the unique situation of this health-care setting. because of the frequent interchange of patients between hospitals and nursing homes, infections caused by antimicrobial drug-resistant bacteria will continue to emerge in geriatric populations. recognition of such threats has prompted new interest in the prevention and control of infections associated with longterm care facilities. recent guidelines have addressed requirements for infection control programs, as well as influenza, antimicrobial use, and antimicrobial resistant pathogens (25, (30) (31) (32) . although reports from the 1980s described numerous deficiencies in infection control practices in nursing homes, recent reports have been more encouraging (4, 33, 34) . a survey of 136 long-term care facilities in new england indicated that 98% had persons dedicated to infection control activities for a median of 8 hours per week (33) . nevertheless, protection of the vulnerable elderly residents in nursing homes merits additional attention, and changes in nursing home licensure and certification requirements may be needed at both state and national levels (35) . surveillance activity in less conventional care settings is a necessary first step in evaluating potential hazards. the vulnerable geriatric population plays a leading role in the scope of nosocomial and health care-associated infections. as the world's population ages, its role is likely to increase. as health care continues to move beyond hospital walls, the spectrum of health care-associated infections in the elderly will continue to expand, reflecting their multiple risk factors for infectious diseases. infection control practitioners and hospital epidemiologists are well advised to follow and study the aging population in the evolving health-care system. undoubtedly, they will find new opportunities to prevent health care-associated infections. in addition, they may be able to develop strategies to prevent the diverse contagions of the elderly from entering hospitals. dr. strausbaugh is hospital epidemiologist and staff physician, va medical center, portland, oregon; and professor of medicine, school of medicine, oregon health sciences university, portland, oregon. he is also the project director for the infectious diseases society of america emerging infections network, a cooperative agreement program sponsored by the centers for disease control and prevention. his research interests include surveillance for emerging infectious diseases and infection and antimicrobial resistance in long-term-care facilities. bacterial meningitis in the elderly prevention and control of nosocomial infections infections in the elderly epidemiology and prevention of infections in residents of long term care facilities clinical immunology of aging geriatric medicine and gerontology nosocomial infections in elderly patients in the united states, 1986-1990 nosocomial infections: decade-specific risk nosocomial infections in the elderly-increased risk per hospital day a viral gastroenteritis outbreak associated with person-to-person spread among hospital staff tuberculosis in elderly persons cryptosporidiosis: an unrecognized cause of diarrhea in elderly hospitalized patients diarrhea among residents of long-term care facilities a nosocomial outbreak of malaria associated with contaminated catheters and contrast medium of a computed tomographic scanner update: outbreak of influenza a infection-alaska and the yukon territory evaluation of a handwashing intervention to reduce respiratory illness rates in senior day-care centers serological study of responses to selected pathogens causing respiratory tract infection in the institutionalized elderly infections and infection risk in residents of long-term care facilities: a review of the literature, 1970-1984 foodborne disease outbreaks in nursing homes healthcare-associated outbreaks in the 90s: hospital infections program cdc group a streptococcal outbreaks in nursing homes a mixed foodborne outbreak with salmonella heidelberg and campylobacter jejuni in a nursing home two nursing home outbreaks of respiratory infection with legionella sainthelensi methicillin-resistant staphylococcus aureus in a nursing home and affiliated hospital: a four year perspective antimicrobial resistance in long-term-care facilities multiply antibiotic-resistant gram-negative bacilli in a long-termcare facility: a case-control study of patient risk factors and prior antibiotic use colonization with vancomycin-resistant enterococcus faecium: comparison of a long-term-care unit with an acute care hospital vancomycin-resistant eneterococcus faecium in a long-term care facility an outbreak of multidrug-resistant pneumococcal pneumonia and bacteremia among unvaccinated nursing home residents infection prevention and control in the longterm-care facility prevention of influenza in long-term-care facilities antimicrobial use in long-term-care facilities infection control programs in skilled nursing longterm care facilities a team approach to infection prevention and control in the nursing home setting infection control programs in long-term-care facilities: structure and process key: cord-272117-erzpz3c0 authors: downey, jeffrey; pernet, erwan; coulombe, françois; divangahi, maziar title: dissecting host cell death programs in the pathogenesis of influenza date: 2018-04-18 journal: microbes infect doi: 10.1016/j.micinf.2018.03.005 sha: doc_id: 272117 cord_uid: erzpz3c0 influenza a virus (iav) is a pulmonary pathogen, responsible for significant yearly morbidity and mortality. due to the absence of highly effective antiviral therapies and vaccine, as well as the constant threat of an emerging pandemic strain, there is considerable need to better understand the host–pathogen interactions and the factors that dictate a protective versus detrimental immune response to iav. even though evidence of iav-induced cell death in human pulmonary epithelial and immune cells has been observed for almost a century, very little is known about the consequences of cell death on viral pathogenesis. recent study indicates that both the type of cell death program and its kinetics have major implications on host defense and survival. in this review, we discuss advances in our understanding of cell death programs during influenza virus infection, in hopes of fostering new areas of investigation for targeted clinical intervention. influenza viruses cause the most recurring respiratory disease in humans, with outbreaks likely occurring since at least the middle ages [1] . present estimates indicate that each year, seasonal influenza affects 5e10% of the world's population, resulting in 3e5 million cases of severe illness and between 250 000 to 500 000 deaths [2] . in addition to seasonal outbreaks, influenza pandemics sporadically emerge, causing markedly higher mortality. in 1918, the worst pandemic in recorded history caused more than 50 million deaths worldwide [3] . since then, three other pandemics have occurred: first in 1957, then again in 1968 and finally in 2009 [4] . currently, there are major concerns surrounding the emergence of the new highly pathogenic avian influenza (hpai) viruses of h5n1, h7n9, h10n8, or the more recent h5n8dthe only hpai strain to cross from eurasia to america [5e9]. one of the key characteristics of highly infectious iav and hpai viruses is that they are poorly glycosylated and, thus, able to bypass the upper airway defense mechanisms to reach the lower respiratory tract, leading to pneumonia [10] . histopathologic studies of influenza autopsies from 1918 outline the key changes characteristic of severe iav viral pneumonia. these include "… varying degrees of acute intra-alveolar edema and/or hemorrhage, and diffuse alveolar damage in addition to necrotizing bronchitis and bronchiolitis" [11] . similarly, in the recent 2009 h1n1 outbreak, viral antigen could be found in the alveolar space, suggesting pandemic strains of iav have the capacity to infect distal areas of the lung and directly cause alveolar damage. these occurrences collectively predispose to a clinical condition termed acute respiratory distress syndrome (ards), which may occur as a complication of primary viral pneumonia [12] . ards is precipitated by cell death among the epithelialeendothelial barrier (eeb) of the distal lung, barrier rupture, fluid leakage into the alveolar lumen and respiratory insufficiency. hence, understanding and disrupting the strategies employed by the virus to damage the lower airways is critical in preserving pulmonary architecture. the most distal structure of the lung, the alveolus, is constantly patrolled by alveolar macrophages (am4) and is delimited by the eebda bi-cellular layer composed of type i and type ii alveolar epithelial cells (aec-i and aec-ii), facing the air space, and endothelial cells that form the pulmonary capillaries. the identification of the infective target cells of severe pulmonary viruses within the alveolus is critical in evaluating the potential effects of cell damage on lung function. for example, ex vivo infection of human lungs with middle east respiratory syndrome coronavirus (mers-cov)d a recent zoonotic virus with a fatality rate of 35e50% in humansdshowed that aec-i, aec-ii and endothelial cells can all be infected and killed [13e15] . in addition, while mers-cov productively replicates in human macrophages and t lymphocytes, it is also cytotoxic in these cells [16, 17] . interestingly, the tropism of the virus appears to have a significant impact on severity of disease. for instance, in comparison to mers-cov that infects both structural cells and leukocytes and causes high mortality, severe acute respiratory syndrome (sars)-cov only infects structural cells, causing less mortality [17] . in iav infection, a number of reports identify aec-ii as the primary replicative niche in the human lung for highly pathogenic strains, while low-pathogenicity strains fail to penetrate the lower airways [18e22] . hpai also infects human endothelial cells in vitro and some evidence suggests that infection of the endothelium may occur in vivo [23, 24] . in addition, iav antigens are found in human am4 and the virus can productively infect and kill human macrophages [18,19,10,23,21,25e27] . thus, similar to the highly pathogenic mers-cov, highly virulent iav shows tropism for both human alveolar structural cells and leukocytes, highlighting the severity of pandemic strains. am4 within the airways are the first leukocytes to encounter the majority of pulmonary pathogens and their initial response to infection is critical in "setting the tone" and orchestrating an effective immune response. studies examining how am4 are altered following pulmonary viral infection suggest that the loss of eeb integrity, reduction in regulatory mediators and activation of multiple pattern-recognition receptors (prr) lead to these cells becoming pro-inflammatory, rather than regulatory [28] . upon infection with iav, am4 initiate the immune response by producing chemokines and cytokines, including type i interferons (ifn-i; primarily composed of ifn-a/b), as well as instructing adaptive immune responses [25, 29] . if adequately regulated, these responses lead to effective immunity, clearance of the virus with minimal immunopathology and return to pulmonary homeostasis. if dysregulated, however, these responses can have dramatically deleterious effects, marked by impaired viral clearance, increased immunopathology and enhanced susceptibility to infection. thus, pulmonary immune responses must be tightly regulated to effectively eliminate viruses that reach the distal airways, while minimizing immunopathology in order to maintain optimal gas exchange [29] . the death of host cells as a result of infection has been recognized for over a century and it is now understood that pathogeninduced cell death may occur through a variety of complex mechanisms. initially, cell death was discussed in dichotomy as either: (1) apoptosis, considered an activedor programmeddprocess that preserves the plasma membrane and leads to the deletion of cells with little tissue disruption and no inflammation; or (2) necrosis, a passive form of cell death leading to complete disruption of the plasma membrane and spillage of cellular contents, causing inflammation and tissue damage [30] . however, this simplification of the cell death program has altered considerably over the last couple of decades. there are now several distinct forms of programmed cell death that have been described in addition to apoptosis, including pyroptosis, paraptosis and necroptosis [31] . interestingly, some of these cell death modalities have been observed in autopsies of iav-infected humans, particularly hpai cases [11, 32] ; yet, the consequences of these cell death programs on host immunity and the pathogenesis of iav are not fully understood. therefore, a more complete characterization of the forms of cell death occurring in iav infected lungs may define why certain cell types are more, or less, susceptible to iav-induced cell death and how this impacts viral propagation. furthermore, different forms of cell death may have distinct consequences on the virulence of iav strains by differentially modulating the immune response. this suggests that regulated modulation of cell death programs during iav infection may be an appealing target for anti-iav therapies. in this review, we focus on the current state of knowledge of cell death during influenza infection and explore research avenues that aim to dissect cell death programs, as well as their ensuing consequences in the pathogenesis of this threatening virus. considering the mechanisms of cell death have been extensively reviewed elsewhere [33, 34] , herein we focus on severe influenza infection and the immunological effects of influenza-induced cell death in the lung structural cells (e.g. epithelial cells) as well as immune cells (e.g. macrophages). finally, we suggest that manipulation of specific cell death programs at different stages of infection may offer avenues for novel therapy to iav by both sequestering viral replication and promoting host resistance mechanisms through immunomodulation. iav utilizes epithelial cells as its primary replicative site and all pulmonary epithelial cells are permissive to infection and subsequent replication. however, proximity of the replication to distal portions of the lung is strongly associated with an increase in virulence capacity of iav strains. for example, seasonal influenza strains, or those of low pathogenicity, replicate primarily in the upper airways and are rapidly killed in the pharynx and proximal lung by host resistance mechanisms, causing minor acute disease. on the contrary, hpai and highly pathogenic h1n1 strains bypass immune mechanisms of the upper airways to replicate in the distal portions of the lung and this distal tropism is highly correlated with primary viral pneumonia and mortality [11] . altered tropism of iav to the lower airways is thought to be mediated by two mechanisms. first, iav infects cells by its hemagglutinin (ha) binding to terminal sialic acids on the plasma membrane of cells, attached either via an a2,3 or a2,6 linkage. both a2,3 and a2,6 linkages are present throughout the human lung; however, preferential binding of a2,3 linked residues by iav hemagglutinin favors more distal and severe infections. second, glycosylation of iav hemagglutinin is inversely correlated with pathogenicity, as highly glycosylated hemagglutinins are more efficiently trapped and cleared from the upper airways by ciliary motion [35] , while poorly glycosylated iav bypass this host defense mechanism and access the lower airways [35] . for instance, the pandemic 1918 h1n1 strain exhibited only 1 glycosylation site on its hemagglutinin protein and hpai strains are known to preferably bind a2,3 residues, due to their enhanced expression on avian epithelial cells. in the lower airways, it was shown in a human lung explants model that approximately 90% of influenza positive cells are aec-ii cells, with a minor contribution of am4 and other cell types [21] . interestingly, iav replication is enhanced in aec-ii compared to bronchial epithelial cells [36] . therefore, highly pathogenic strains of iav have mechanisms to reach their preferred replicative niche in aec-ii in the distal lung, suggesting that maintenance of pulmonary epithelial cells is beneficial to the virus in promoting its replication. however, considering that iav is also lytic in epithelial cells of humans [37] and mice [38] hints that at certain time points after infection, cell death may be involved in preventing or facilitating viral propagation, escape and reinfection. therefore, the consequences of cell death in structural cells, and in particular aec-ii, on host immunity to iav must diverge depending on the kinetics of the infection. apoptosis comes from the ancient greek word meaning "falling off" and is the most characterized form of programmed cell death. proceeding in a non-inflammatory manner, apoptosis likely evolved to meet the growing demand to remove aging or damaged cells from the body as more complex and multicellular organisms appeared and, as a result, the apoptotic machinery remains evolutionarily conserved. indeed, at homeostasis in an adult human, it is estimated that between 100 and 200 billion cells are being cleared daily via apoptosis [39] . this number can then augment dramatically in developmental stages and aging. thus, the physiological demand for cellular turnover in a non-inflammatory manner is essential to maintain homeostasis. the signature of early apoptosis involves condensing of chromatin within the nucleus, compartmentalization of cellular organelles and the shrinking of the cell membrane. ultimately, these compartments bleb off in the form of apoptotic bodies, which are phagocytosed and degraded by macrophages and other phagocytic cells in a process known as efferocytosis [39] . importantly, the cell membrane of apoptotic cells remains intact throughout and the cell shrinks rather than ruptures, as appearing in necrotic forms of cell death. thus in apoptotic cells, there is no leakage of cytosolic contents to initiate an inflammatory response [33] . there are two major pathways that lead to apoptosis: intrinsic and extrinsic, both of which primarily converge on a family of proteins called caspases that cleave cellular substrates and lead to the morphological signature of apoptosis [40] . intrinsic apoptosis is initiated by the mitochondrion in response to internal cellular stress and involves activation of caspase 9 by cytochrome c. extrinsic apoptosis, on the other hand, is dependent upon death ligands of the tnf superfamily like trail or fasl binding their cognate cell surface receptors and the subsequent activation of caspase 8 [41, 42] . in addition to its physiological role, apoptosis is involved in promoting resistance or susceptibility to intracellular pathogens. for example, virulent mycobacterium tuberculosis (mtb) contains an anti-apoptotic gene (nuog) that diminishes both innate and adaptive immunity to promote the bacterial growth [43] . in contrast to mtb, apoptosis induced by listeria monocytogenes is an immune evasion strategy, allowing the bacteria to disseminate [44] . thus, it appears that apoptosis can be both protective and detrimental to the host depending on the pathogen. interestingly, both extrinsic and intrinsic pathways of apoptosis were shown to be activated in influenza-infected cells [45] . this observation is well established, being described in human autopsies for almost a century, beginning with the 1918 pandemic, where pronounced epithelial desquamation, hyalination and sloughing were noted [37] . experimentally, apoptosis of iav-infected epithelial cells was shown to be dependent upon viral replication, as an inactivated virus failed to induce apoptosis in mice [46] and human cells [47] . moreover, the magnitude of epithelial cell apoptosis was positively associated with iav strain pathogenicity in vivo [32, 23, 48] and in vitro [38, 49] in both mice and humans. however, whether apoptosis promotes host resistance or iav dissemination remains to be determined. logically, apoptosis of epithelial cells could represent a resistance strategy to iav, as it eliminates the intracellular niche required for viral replication. support for the protective capacity of apoptosis is that critical host antiviral factors actively induce epithelial cell apoptosis. upon viral infection, ifn-i (ifn-b) is rapidly produced, following recognition of the viral genome by pattern-recognition receptors (prr). in iav infection, rna sensing is predominantly mediated by the cytosolic retinoic acid-inducible gene (rig-i), which interacts with the mitochondrial antiviral signaling protein (mavs) at the mitochondrial outer-membrane to initiate ifn-i production mainly via the transcription factor interferon regulatory factor 3 (irf3) (reviewed in ref. [50] ). ifn-i then signals through a heterodimeric receptor complex of ifnar1 and 2, leading to sustained ifn-i (largely ifn-a) production and an upregulation of a large subset of genes, together termed interferon stimulated genes (isgs). isgs then serve to halt viral replication through both direct or indirect mechanisms. interestingly, both ifn-i and isgs have been shown to induce apoptosis. following iav infection, ifn-i signaling activates caspase-dependent apoptosis in aec-ii cells [51] , and the pharmacological inhibition of these caspases blocks apoptosis and enhances viral replication. additionally, several classical apoptosis proteins, including caspase 8 and trail, are isgs upregulated by ifn-i signaling. thus, iav sensing causes the release of ifn-i that induces epithelial cell apoptosis directly through caspase activation downstream of the ifnar1/2 complex, and indirectly by promoting the transcription of pro-apoptotic isgs. other isgs have been equally implicated in promoting apoptosis in iav-infected cells as an antiviral mechanism. for example, protein kinase r (pkr) is an isg known to inhibit iav replication through a variety of mechanisms. pkr can directly sense dsrna generated during viral replication to induce fas expression and fadd-dependent apoptosis [52] , as well as inhibit host and viral protein translation through the phosphorylation of eif2a [53] in iav-infected cells [54] . moreover, pkr has been shown to increase ifn-i production in fibroblasts directly in response to another rna virus: semliki forest virus [55] . thus, it appears pkr and other isgs contribute to antiviral immunity by promoting ifn-i responses, restricting viral protein translation and inducing apoptosis. in addition to being directly antiviral, apoptosis also protects against iav infection through the resolution of the immune response. in response to hpai, bronchiolar epithelial cell apoptosis limits excess pro-inflammatory cytokine (ie. tnf) release to dampen immunopathology and avoid the onset of ards [56] . furthermore, a recent study has implicated club cells, a subset of bronchiolar epithelial cells, in augmenting immunopathology to an h1n1 iav virus, by failing to undergo apoptosis following viral clearance [57] . therefore, the protective capacity of apoptosis in structural cells is both antiviral and immunomodulatory. given the importance of apoptosis in antiviral immunity, it is not surprising that iav has encoded factors to block its induction. the principal iav virulence factor, non-structural protein 1 (ns1), is rapidly transcribed upon infection and is the first protein expressed in infected cells. ns1 blocks early apoptosis in epithelial cells and fibroblasts by inhibiting ifn-i induction [58] and activating the host pro-survival pi3k/akt pathway to facilitate viral replication [59] . importantly, these functions are critical in viral fitness, as strains lacking a functional ns1 are severely impaired in their virulence. taken together, all the above studies highlight the importance of apoptosis of structural cells in promoting immunity to influenza, by limiting the intracellular niche necessary for viral replication. however, the notion that apoptosis solely represents a hostdriven antiviral strategy is confounded by the fact that iav encodes both anti-and pro-apoptotic factors. for example, h5n1 ns1 facilitates airway epithelial apoptosis [60, 49] , suggesting a strainor expression level-specific role for this protein in iav pathogenesis. furthermore, recent study has characterized h1n1 iav nucleoprotein (np) as a pro-apoptotic viral protein in human airway epithelial cells [61] that targets the host's anti-apoptotic protein ap15 [62] . in fact, host apoptotic machinery has been shown to be essential in promoting new virion production in epithelial cells in vitro [63e65], in a caspase-3[66] and -9dependent manner [67] , and in vivo by iav-manipulation of annexin-a1 [68] . these findings outline iav as an effective regulator of the host's apoptotic machinery in structural cells, capable of both inducing and blocking apoptosis to further its pathogenesis. the paradoxical role of apoptosis in immunity to iav, which appears to both prevent and permit viral dissemination, can perhaps be explained by the kinetics of the apoptotic response in epithelial cells (fig. 1) . immediately upon infection, it is beneficial for iav to block epithelial cell apoptosis to avoid destroying its replicative niche and this is primarily mediated by viral ns1. early blockage of apoptosis by iav is counteracted by host mechanisms, such as ifn-i signaling, to induce apoptosis and resist viral replication [69] . yet, following initial replication cycles, at later time points, iav must activate apoptotic pathways to generate new infectious virions, promote budding at the cell surface and facilitate subsequent rounds of infection in neighboring cells. thus, pharmacological inhibition of apoptosis in humans during the later stages of infection may offer appealing therapeutic avenues, either by blocking pro-apoptotic pathways [65] or enhancing antiapoptotic proteins [64] . interestingly, neutralization of proapoptotic trail or fas signaling post-iav infection in aec-ii cells decreased iav load [70] . similarly, mice treated with decoy fas in vivo to block fasl signaling were protected from lethal iav infection, when compared to untreated mice [71] . our understanding of the interplay between influenza, host apoptotic machinery and resistance mechanisms has increased exponentially recently. however, much of our knowledge still derives from study in vitro using human or mouse cells and, thus, the exact effects of these pathways on disease outcome remain to be determined. like apoptosis, the observation that iav causes necrosis in epithelial cells has long been established. yet, the impact of iavinduced epithelial cell necrosis on the host immune response, and the factorsdviral or host-deriveddinvolved in this process are just beginning to become known. canonically, necrosis was considered a passive form of cell death associated with increased injury or disease severity. however, it is now well established that many forms of necrosis are, in fact, programmed. necrotic cell death is delineated from apoptosis by a disruption of the cell membrane and spilling out of cytosolic contents that act as dangerassociated molecular patterns (damps). these damps are sensed by neighboring cells, resulting in a robust inflammatory response [72, 73] , thus, generating a potential positive feedback loop of tissue damage and immunopathology. the observation that hpai [11] and pandemic h1n1 often induce necrosis in epithelial cells, while seasonal h3n2 appears only to cause it in immune compromised hosts [48] , may at least partly explain the association between severity of disease and necrosis of the epithelium. moreover, other strains of highly pathogenic viruses such as dengue virus [74] or mers-cov [75] appear to induce considerable necrosis in epithelial cells to promote viral replication and propagation, while less virulent viruses like rhinovirus, or sars-cov induce limited or no necrosis [76, 75] . in vitro, h1n1 iav induces necrosis in human lung bronchiolar epithelial cells, coupled with massive release of proinflammatory cytokines and neutrophil chemoattractant cxcl8, which is highly suggestive of a link between necrosis of the epithelium and ards [77] . collectively, these observations promote the notion that epithelial cell necrosis is detrimental to the host and is consequential of severe infection. the most characterized form of programmed necrosis is ripk3dependent programmed necrosis (termed necroptosis). necroptosis is dependent upon the formation of the necrosome, which consists of ripk1 and ripk3, interacting through their rip homotypic interaction motifs (rhim). typically, ripk1 and ripk3 are cleaved by caspase-8, causing their inactivation and leading to extrinsic apoptosis. however, when caspase-8 activity is compromised, the necrosome is activated to induce necroptosis. known mediators downstream of the necrosome include mixed lineage kinase domain like pseudokinase (mlkl). mlkl is a substrate for ripk3 and induces necroptosis by a combination of the assembly of a pore-forming complex at the plasma membrane, or as a platform for ca 2þ -mediated necrosis [78] . although the exact executioner mechanisms of necroptosis remain unknown, recent study has implicated the plasma membrane repair machinery escrt iii as a potential mediator [79] . necroptosis regulates a variety of inflammatory processes, including ischemic heart disease, bacterial infection and embryogenesis [80] . interestingly, there is accumulating evidence for a role of necroptosis in promoting immunity to viral infection. seminal work showed necroptosis to be essential in antiviral defense, as ripk3 à/à mice succumbed to vaccinia virus infection [81] . moreover, herpes simplex viruses (hsv-1/2) and murine cytomegalovirus (mcmv) encode inhibitors of necroptosis, ul39 and m45 respectively, suggesting that viruses may block necroptosis as part of their pathogenesis [82, 83] . although no iav inhibitors of necroptosis have yet been identified; most recently, ripk3-dependent necroptosis of murine fibroblasts and alveolar epithelial cells was demonstrated to be critical in promoting iav clearance from the lungs and, as a result, ripk3-deficient mice were highly susceptible to iav infection coupled with enhanced viral loads [84] . thus, this study hints at a protective role for necroptosis in immunity to iav. however, human studies are required to investigate the potential clinical targeting of the necroptotic pathway in protection against iav infection. certainly, apoptosis and, more recently, necrosis have dominated study of iav-mediated cell death. however, the role of other forms of cell death in immunity to infectious diseases, such as iav, is beginning to be recognized. pyroptosis is a form of rapid inflammatory cell death with cell membrane rupture and dna damage, which is dependent upon caspase-1 and certain nod-like receptors. pyroptosis is coupled with proinflammatory il-1b and il-18 release from pyroptotic cells, which contributes in part to its inflammatory nature [85] . caspase-1 activation and subsequent cytokine release induced by iav in murine lung fibroblasts has been shown to inhibit viral pathogenesis [86, 87] . yet, whether caspase-1 activation in these models led to pyroptosis, or rather defined a pyroptosis-independent role for caspase-1 was not carefully elucidated. thus, more study of the role of caspase-1 activation and pyroptosis in immunity to iav is needed. although epithelial cells constitute the major replicative niche for iav, the lung is a complex organ and thus other structural cells can be infected with iav. pulmonary fibroblasts are the most substantial subset of structural cells in the lung interstitium, playing a significant role in the repair process to injurious exposure and pulmonary inflammatory disorders, such as asthma and copd [88] . fibroblasts are used extensively in iav studies as a model of lung structural cells. as in epithelial cells, fibroblasts are sensitive to both iav-induced apoptosis [58] and necroptosis [84] in vitro. however, the contribution of fibroblasts in immunity to iav infection is not well understood. whether fibroblasts are a target for iav infection in vivo and/or whether their response is analogous to, or completely independent of, epithelial cells, requires further investigation. understanding the role of fibroblasts in the pathogenesis of iav may provide additional targeted therapy against iav infection. similarly, endothelial cells represent a substantial compartment of the lung and in vitro infection of human endothelial cells led to productive iav replication and increased vascular leak, mediated by apoptosis [89] , particularly upon infection with hpai (e.g. h5n1) [90] . hpai has been shown to disseminate from the lung systemically upon infection in mice [91] . this has equally been observed in humans, where viral rna was found in multiple organs, including the brain and placenta, of fatal cases of h5n1 [92, 93] . the ability of hpai viruses to disseminate to other tissues is predominately mediated by changes to the cleavage sites of the viral ha protein, which is essential for viral entry into cells. in the case of low virulence seasonal strains of iav, ha cleavage occurs at a single basic arginine by trypsin-like proteases found exclusively in cells of the respiratory tree and gastrointestinal tract. on the other hand, hpai ha proteins contain polybasic cleavage sites that permit cleavage by furin proteases that are ubiquitously expressed. thus, this allows hpai strains to disseminate and cause systemic infection [94] . additionally, pandemic and highly virulent strains of the virus, including hpai and the 1918 h1n1 strain, are known to completely exhaust the replicative niche of epithelial cells of the lung over the course of infection as a by-product of overly exuberant replication and failure of immune response to control viral propagation. thus, it is appealing to associate the infection of endothelial cells by hpai viruses with subsequent systemic dissemination, made possible by the polybasic cleavage sites. however, this is complicated by the fact that no virus was found in endothelial cells of fatal cases of h5n1 infection [93] , yet virus was found in hyaline membranes and endothelial cells of fatal cases of the 2009 h1n1 pandemic, where dissemination to other organs was not noted [23] . as there is no known evolutionary advantage for iav to disseminate to other organs, a greater level of study is required to confirm what role infection of endothelial cells in humans plays and whether endothelial cell death is a pro-host or pro-pathogen strategy. during influenza virus infection, the pulmonary inflammatory response is characterized by early infiltration of leukocytes, including neutrophils and mononuclear cells and later by adaptive immune lymphocytes [95, 96] . autopsy reports of humans infected with hpai and pathogenic strains of h1n1 exhibited severe pulmonary leukopenia, which together constituted important clinical features of severe infection and fatal cases [97, 32] . importantly, this leukopenia was mainly contributed to iav-induced death of these cells at the site of infection, rather than failed recruitment or effector function in the lung [98e100]. interestingly, pulmonary iav infection has also been shown to have profound effects on bone marrow hematopoiesis in an ifn-i-dependent manner [101] . thus, the observed leukopenia could alternatively be explained by other mechanisms, such as exhaustion of the bone marrow hematopoietic stem cells due to severe infection. hematopoietic stem cell exhaustion is a phenomenon often observed during chronic bacterial or viral infections [102, 103] , but remains poorly characterized in iav infections. nevertheless, these findings illustrate the importance of preserved leukocyte viability and function in host defense to highly pathogenic strains of iav and activation of the cell death program in these cells may represent a strategy of immune evasion by iav. although iav has been shown to contribute to cell death in the majority of leukocytesdincluding neutrophils, dendritic cells and lymphocytesdthis review will focus on cell death in pulmonary monocyte/macrophages, given their critical role in the initial response to the infection. pulmonary macrophage populations change considerably during iav infection. at homeostasis, tissue resident am4 constitute the vast majority of cells in the airways and are the first leukocytes to encounter the virus, following replication in epithelial cells [29] . am4 can then be readily infected [104, 105] and activated to secrete a spectrum of cytokines and chemokines that orchestrate both innate and adaptive immune responses to iav and other pulmonary viral infections [106, 25, 107] . over the course of infection, am4 populations are diminished, while inflammatory monocytederived macrophages (md-m4, also called exudate macrophages) expand and then contract during the resolution phase [108] . both am4 and md-m4 have been linked to host protection and disease pathogenesis depending on the magnitude of activation of the immune response, which is directly associated with disease tolerance [108] . invariably, loss of am4 causes increased susceptibility to infection, due to reduced production of antiviral ifn-i (mainly ifn-b) leading to uncontrolled viral replication and increased immunopathology [106, 25, 107, 109] . thus, am4 represent a logical target for iav to induce cell death and, indeed, several studies have reported early apoptosis post-infection in am4 [110e112]. similarly, recruited m4 are highly permissive to iav infection and susceptible to iav-induced cell death [26, 113] . collectively, these findings suggest that in contrast to the biphasic role of epithelial cell apoptosis in preventing or promoting pathogenesis, highly virulent iav rapidly infects and induces early death in pulmonary m4 to suppress antiviral responses. apoptosis occurs in m4 during iav infection and can be either triggered intrinsically by viral proteins or activated extrinsically by host factors in the pulmonary microenvironment. while the induction of apoptosis by viral proteins has been studied extensively in structural cells, fewer studies have investigated it in m4 [114, 115, 112, 116, 117] . interestingly, influenza virus contains an essential gene, polymerase basic protein 1-frame 2 (pb1-f2, a product of an alternate reading frame of the pb1 gene), which increases apoptosis-mediated pathogenicity in iav [118] . functionally, pb1-f2 has been shown to be the main inducer of apoptosis in monocytes/m4, but not epithelial cells [116, 112] . the pro-apoptotic effect of pb1-f2 is elicited by its translocation to the mitochondrion [119] . in mitochondria, pb1-f2 interacts with components of the permeability transition pore complex (ptpc) ant3 and vdac1 [120] to disrupt the mitochondrial inner membrane potential, followed by cytochrome c release and the induction of apoptosis, which subdues the m4 antiviral capacity and promotes host susceptibility [116] . iav-induced apoptosis in m4 is not solely caused by viral protein expression and may result from the activation of the extrinsic pathway by host factors [110, 115, 121] . interestingly, iav-infected m4 are the main source of the key antiviral cytokine: ifn-i [25, 122, 116] and can induce m4 cell death via the upregulation of isgs. moreover, infection of human m4 with hpai strains induces trail release to trigger the apoptotic cascade [123, 27] . equally, upregulation of fas and secretion of fasl at later time points postinfection have been shown to induce apoptosis selectively in recruited m4 [115, 121] , a process which is thought to contribute to immune contraction and the return to homeostasis. of note, although tnf has been reported to induce apoptosis in iav-infected epithelial cells, it does not appear to mediate m4 apoptosis [45] . thus, during iav infection, activation or inhibition of apoptotic pathways are cell-type specific and can either promote or prevent iav pathogenesis. therefore, this complex network certainly needs to be interrogated more intensely. the outcome of iav-induced m4 apoptosis is still controversial, as it may favor both the host and the virus. nonetheless, much of the literature suggests a host-detrimental effect of m4 apoptosis. for instance, hpai and pandemic h1n1 influenza viruses hijack innate antiviral responses by inducing apoptosis in m4 to decrease m4-antiviral cytokines (primarily ifn-i) and promote viral replication [27, 123, 124] . considering residential am4 are the first immune cells encountered by the influenza virus and are critical in mounting an immune response, it is not surprising that iav efficiently infects and kills am4 to suppress their antiviral responses. thus, shortly after severe iav infection, the original pool of resident am4 decreases, resulting in enhanced viral replication [110, 105] . similarly, limiting am4 cell death through the exogenous administration or overexpression of gm-csfdthe critical growth and maintenance factor for am4dis protective, leading to lowered pulmonary viral load, decreased immunopathology and increased survival of mice after iav infection [125, 109] . in addition to inhibiting the early antiviral responses, iav-induced m4 apoptosis might skew the balance from a protective to pathological immune response. during iav infection, it has been shown that mice lacking type i ifn signaling die from an uncontrolled inflammatory response, as ifn-i signaling was essential to induce anti-inflammatory il-10 production by "regulatory" m4 [126] da subset of m4 known to be more susceptible to iav-induced apoptosis [113] . mechanistically, iav-induced m4 apoptosis is mainly mediated by the viral protein pb1-f2 and its expression has been linked to delayed viral clearance [120] . expression of the 1918 pb1-f2 protein in a mouse-adapted h1n1 strain resulted in early increased viral titers and death of infected m4 [127] . to prevent pb1-f2-induced early m4 apoptosis, we have recently shown that the host mitochondrial nlr-member nlrx1 directly interacts with pb1-f2 within mitochondria to disarm its apoptotic function. in iavinfected nlrx1 à/à mice, or m4 in vitro, higher levels of apoptosis were observed compared to wt m4. this was associated with reduced production of ifn-i and increased viral loads in vitro and in vivo [116] . thus, by inducing m4 cell death, iav hijacks the host's key antiviral responses, resulting in both enhanced iav replication/ dissemination and impairment of the anti-inflammatory response, which drives immunopathology and potentially ards (fig. 2) . however, recent findings indicate that in some cases, apoptosis of pulmonary m4 may be beneficial for the host. indeed, early apoptosis of hpai-infected porcine am4 correlated to decreased viral replication and magnitude of the inflammatory response [112] . aside from viral proteins, our laboratory recently demonstrated that iav induces production of the host eicosanoid prostaglandin e 2 (pge 2 ) to directly inhibit early production of ifn-i, which increased viral loads through reduced m4 apoptosis. thus, whether apoptosis is protective or pathological seems, again, to be connected to the kinetics of its induction. early post-infection, m4 viability is essential to promote antiviral immunity and this is countered by iav proteins such as pb1-f2. later, however, m4 apoptosis becomes protective, as the apoptotic vesicles stimulate adaptive immunity by promoting cross-presentation [25] . to conclude, apoptotic cell death of macrophages appears to be a double-edged sworddboth beneficial and detrimental to the host, depending on iav virulence factors and host mediators. further work is needed with closer inspection of the kinetics of expression of viral proteins (e.g. pb1-f2, ns1) as well as host factors (eg. nlrx1, ifn-i, eicosanoids) and their link with kinetics of m4 cell death (early versus late induction) to fully understand whether timing or, rather, specific pathways or types of apoptosis dictate this complex phenomenon. despite apoptosis being the main m4 death modality observed in iav infections, there is also evidence of necrosis [128, 32] and, specifically, of the more recently characterized ripk3-mediated necroptosis. however, iav-induced macrophage necrosis has been poorly studied in vivo and most of our knowledge is confined to in vitro observation. at low multiplicities of infection (moi), necrosis is not substantially induced in m4 [116, 26, 129] . in addition, we and others have recently shown that iav does not induce ripk3mediated necroptosis in m4, both in vivo and in vitro, but, rather, ripk3 plays a crucial role in the production of il-1b [129] and ifn-i [130] and, as a result, mice deficient in ripk3 are extremely susceptible to iav infection. however, following severe infection with a high viral titer, iav induces necroptosis through the ripk1eripk3ecaspase 8 complex in vitro [86] , suggesting a potential role for ripk3-mediated necroptosis in depletion of m4 during infection with highly virulent strains. synergy between these two observations may come from the pleiotropic function of ifn-i, which has been also shown to induce m4 necroptosis in vitro or in vivo during bacterial infections [131e133]. thus, it can be envisioned that dysregulated ripk3-mediated ifn-i production in severe infection may, in fact, become detrimental to the host by inducing necroptosis of pulmonary m4, suggesting a threshold for protective ifn-i production and deleterious immunopathology induced by m4 necroptosis. of note, pathways known to induce ifn-i in response to iav and other viruses, such as tlr3 [134] , pkr [132] and dna-dependent activator of ifn-regulatory factors (dai) [135] have also been shown to trigger necroptosis, advocating a potential link between type i ifn production and necroptosis induction. finally, iav-infected macrophages produce il-1b through inflammasome activation and caspase-1 cleavage, which are characteristics of pyroptotic cell death [136, 129] . however, there was no difference in cell death in iav-infected nlrp3 à/à (lacking the nlrp3 inflammasome) and wt m4, in vitro [86] , suggesting that pyroptosis does not occur in m4 during iav infection. certainly, further investigation is required to fully understand the precise mechanisms involved in this process. the lung represents a diverse microenvironment filled with numerous cellecell interactions with high turnover under homeostatic and pathological conditions. in particular, epithelial cells of the ebb and am4 exist in close proximity and are known to regulate each other's functions at homeostasis and upon infection. over the last ten years, we have gained appreciable knowledge of how pulmonary macrophages induce apoptosis in infected epithelial cells. in 2008, herold et al. showed for the first time that secretion of trail by m4 induced apoptosis of aec and contributed to lung injury during iav infection [137] . further investigation also revealed that secretion of ifn-i, and specifically ifn-b, by infected am4 was responsible for autocrine trail expression and epithelium injury [138] . most importantly, an upregulation of trail expression and secretion was observed in am4 from pandemic h1n1-infected individuals [138] . in addition to potentiating lung injury, this could potentially lead to viral dissemination by promoting efferocytosis. efferocytosis is the phagocytosis of apoptotic bodies released from dying cells by neighboring m4 [139] . recently, annexin a1, a key protein involved in efferocytosis [140, 141, 148] , was shown to facilitate iav replication and dissemination in vivo [68] . interestingly, tcherniuk et al. showed that newly formed viral particles contained annexin a1 on their membranes, increasing their infectivity and viral replication through enhanced uptake [142] . in addition, activation of formyl peptide receptor 2/lipoxin a4 receptor (fpr2/alx) via annexin a1, increased pulmonary viral load and susceptibility to infection [142] . hence, one can envision a model wherein the induction of epithelial cell apoptosis by am4derived ifn-i leads to the presence of annexin a1-expressing viral particles on the surface of infected cells. these new viral particles are, in turn, recognized and taken up by bystander m4, infecting them and preventing their antiviral responses. this leads to increased viral replication that subsequently enhances the magnitude of the immune responsedthus, continuing this vicious cycle of lung injury/reinfection. this may potentially explain how hpai and pandemic h1n1 viruses strip the pulmonary epithelium and leave the host leukopenic, as seen in fatal cases. therefore, discovery of factors that can mitigate this pathological cycle would be of clinical significance in combatting highly pathogenic strains of iav. despite influenza virus infections likely dating back to the middle ages and considerable burden on global health, there is no effective therapy against iav. to prevent infection, vaccination strategies are deployed worldwide. however, the protection conferred by vaccination is highly variable, due to the ability of the virus to shift antigens, leading to vaccine mismatch and a decrease in efficacy. antiviral therapies, like neuraminidase inhibitors (i.e. oseltamivir) that block viral egress to potentially ameliorate disease are currently in use. despite considerable efficacy in murine models in lowering viral titers and improving disease outcome [143, 144] , its protective effect in humans is controversial and limited benefit has been shown in the majority of patients [145, 146] . additionally, highly resistant strains of iav to oseltamivir have recently emerged that raises growing concern [147] . thus, these factors collectively suggest that the dogma of only targeting the virus and not the host's immune response in the generation of anti-iav drugs needs to be revisited. indeed, there are several "proof-of-concept" studies highlighting the promising therapeutic potential of targeting host cell death programs during iav-infection. first, anti-trail treatments selectively attenuated epithelial cells apoptosis, lung injury and increased survival of mice after iav infection. secondly, we recently demonstrated that targeted inhibition of mpges-1, the key enzyme in pge 2 production, selectively regulated pulmonary m4 apoptosis to boost antiviral and immunoregulatory properties. importantly, mpges-1 inhibition was effective even after three days of iavinfection, coinciding with the onset of symptoms in humans. in line with this, a recent study by hung et al. showed a reduction in viral titers, hospital stay and mortality when individuals infected with h3n2 iav were given a combinatory therapy of clarithromycin (antibiotic) enaproxen (cox inhibitor) e oseltamivir (neuraminidase inhibitor) [147] , compared to oseltamivir treatment alone, highlighting the importance of combining immunomodulatory therapies and those directly targeting the pathogen. additionally, we suggest ripk3 as an attractive target for immunomodulatory therapy. recently, the antiviral capabilities of ripk3 have been shown to be at least twofold, by both inducing epithelial cell necroptosis and promoting ifn-i responses from pulmonary m4 to potentiate survival to lethal iav infection. therefore, agonists of ripk3 may promote viral clearance via both epithelial cells and macrophages and ultimately confer survival to severe disease. this shows that combining treatments that differentially target structural cell and pulmonary m4 can significantly improve the outcome of influenza virus infection. the constant exposure of humans to potentially life-threatening respiratory pathogens presents ongoing challenges to clinicians. the knowledge gained from the studies of cell death programs in epithelial cells and macrophages during iav infection provide significantly better understanding of host mechanisms involved in protective or pathological immunity to iav infection. however, further investigation is still required to identify the kinetics of cell death during influenza infection, which appears to be critical in regulating antiviral and anti-inflammatory responses. in addition, most of our knowledge comes from in vitro experiments and thus a need for greater tools to investigate cell death in vivo and discovery of new cellular markers of the various cell death programs will be required for developing a novel therapy in influenza virus infection. . all authors declare that no 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hospitalized for influenza a (h3n2) infection: an open-label randomized, controlled, phase iib/iii trial annexin1 regulates dc efferocytosis and cross-presentation during mycobacterium tuberculosis infection key: cord-282142-76jr4p7n authors: wang, yun; wang, yiliang; han, xiaoxue; ye, jiazhuo; li, ruiman title: potential effect of covid-19 on maternal and infant outcome: lesson from sars date: 2020-08-07 journal: front pediatr doi: 10.3389/fped.2020.00511 sha: doc_id: 282142 cord_uid: 76jr4p7n the coronavirus disease 2019 (covid-19), caused by sars-cov-2, is highly infectious and its ongoing outbreak has been declared a global pandemic by the who. pregnant women are susceptible to respiratory pathogens and the development of severe pneumonia, suggesting the urgent need to assess the potential maternal and infant outcome of pregnancy with covid-19. the intrauterine vertical transmission potential of sars-cov-2 also remains controversial. herein, we discuss the potential effect of covid-19 on maternal and infant outcomes based on current studies, including those published in chinese, in a total of 80 mothers with covid-19 and 80 infants. we also comprehensively explored the mother-to-child transmission routes of sars-cov-2, in particular the route of intrauterine vertical transmission. given sars-cov-2 is a sister to sars-cov, of the sars-related coronavirus species, we made a comprehensive comparison between them to learn from experiences with sars. although there is no evidence supporting the intrauterine vertical transmission of sars-cov-2, our comprehensive analysis suggests that the adverse maternal and infant outcomes caused by covid-19 cannot be underestimated. further, we speculated that the inconsistency between nucleic acids and serological characteristics igm to sars-cov-2 of infants' specimens may be caused by the disruption of the amniotic barrier by the inflammatory factors induced by sars-cov-2 infection. our review is beneficial to understand the effect of sars-cov-2 on maternal and infant outcomes. at the beginning of december 2019, a cluster of pneumonia cases with unknown causes were reported (1, 2) . a subsequent high-throughput sequencing revealed that the pneumonia epidemic resulted from a novel beta coronavirus tentatively named "2019 novel coronavirus" (2019-ncov) that was subsequently termed "severe acute respiratory syndrome coronavirus 2" (sars-cov-2) (3) (4) (5) . sars-cov-2 is a sister to sars-cov, of the sars-related coronavirus species (4) (5) (6) (7) (8) . pneumonia caused by sars-cov-2 was correspondingly termed "coronavirus disease 2019" (covid-19) (4, 5) . the ongoing outbreak of covid-19 has posed a great threat to global human health, which has been declared by the who as a global public health emergency (1, 2) . as shown by the center for systems science and engineering at johns hopkins university (last updated on 07/07/2020), the global cumulative number of confirmed cases has reached 11,779,263, with 6,758,547 cures, and 540,948 deaths (9) . due to the high transmissibility of covid-19, the prevention, and control of covid-19 infection has become a major concern (4, 5) . pregnant women are more susceptible to respiratory pathogens and the development of severe pneumonia than the general population, especially so for those with chronic diseases or maternal complications (4) . the physiologic changes in pregnancy, including altered cell-mediated immunity, and alterations in pulmonary function, may confer the susceptibility and severity of pneumonia to pregnant women (6, 10, 11) . pneumonia arising from infectious etiology is the most common non-obstetric infectious condition that occurs in pregnant women (6, 12, 13) . in particular, universal sars-cov-2 screening for women admitted for delivery found that all women with positive test results were asymptomatic at the time of testing (14, 15) . therefore, the effect of sars-cov-2 infection on maternal and infant outcomes needs to be explored, especially the intrauterine vertical transmission potential of covid-19. moreover, in the use of a reverse transcriptase-polymerase chain reaction (rt-pcr) and the specific antibody to sars-cov-2 of neonate samples remains controversial (4, 5, (16) (17) (18) (19) (20) . given sars-cov-2 is a sister of sars-cov, it is important for us to learn from the experience of preventing and controlling sars-cov among pregnant people. in this review, we made a comprehensive comparison of sars-cov-2 and sars-cov in genetic, infection, transmission, and clinical characteristics. based on such a comparison, we summarized the potential maternal, and infant outcomes from pregnancy with covid-19 or sars. further, we discuss the potential of mother-tochild transmission of sars-cov-2, in particular the possibility of intrauterine vertical transmission. the guidelines for those women with sars-cov-2 infections during pregnancy and puerperium prepared by numerous experts were also briefly presented (21) . considering the ongoing global public health emergency, we believe that our review is important for understanding the mother-to-child transmission potential of sars-cov-2 and its implication for the safe management of covid-19 in pregnancy. the pathogen contributing to the covid-19 epidemic was tentatively named "2019-ncov" (3, 22) . based on phylogeny, taxonomy, and established practice, the coronavirus study group (csg) of the international committee on taxonomy of viruses formally recognizes this virus as a sister to sars-cov and designates it as sars-cov-2 (8) . a coronavirus is spherical, enveloped, and the largest of the positive-strand rna abbreviations: sars-cov-2, severe acute respiratory syndrome coronavirus 2; sars-cov, severe acute respiratory syndrome coronavirus 1; sars, severe acute respiratory syndrome; covid-19, 2019 novel coronavirus disease; ace2, angiotensin-converting enzyme 2; lga, large-for-gestational-age; sga, smallfor-gestational-age; ttn, transient tachypnea of the newborn; ncpap, nasal-continuous positive airway pressure. virus and sars-cov-2 is the seventh member of enveloped rna coronaviruses with the ability to infect humans (2, 6) . the coronaviruses currently known to infect humans include hcov-229e and hcov-nl63 (alphacoronavirus genus), hcov-oc43, hcov-hku1, mers-cov, sars-cov, and sars-cov-2 (betacoronavirus genus) (23) . sars-cov is the pathogen that caused the sars epidemic from 2002 to 2003 (24) . there were 8,422 cases and 916 deaths in 29 countries, with most of them having occurred in mainland china by 31 july 2003 (24) . given the great similarity between sars-cov and sars-cov-2, a comprehensive comparison between these two viruses can help us learn from the sars epidemic to control and prevent covid-19. their comparisons were mainly presented according to their clinical and viral characteristics ( table 1 ). in general, there was a 79.5% similarity in the whole genome between sars-cov and sars-cov-2, while only a 74.9% similarity in the gene coding spike glycoprotein (3, 22, 25) . both sars-cov-2 and sars-cov spread rapidly from humanto-human transmission (7, (28) (29) (30) (31) (32) . sars-cov can be spread via respiratory droplets, secretions, nosocomial contacts, and mechanical aerosols, such as the aerosols arising from the flushing of toilets (33) (34) (35) . sars-cov-2 seems to spread more easily among humans than sars-cov, which may result from the various modes of transmission and its high affinity with its receptor angiotensin-converting enzyme 2 (ace2) ( table 1) . the latest pilot experiment confirmed that 4 out of 62 stool specimens (6.5%) tested positive to sars-cov-2, and another 4 patients who tested positive toward sars-cov-2 in rectal swabs also had sars-cov-2 detected in their gastrointestinal tract, saliva, or urine (7) . the results suggest the possibility of transmission via aerosols arising from the flushing of toilets. in particular, sars-cov-2 can be detected in esophageal erosion and bleeding sites in cases with severe peptic ulcers after symptom onset (7) . although these results only suggest the existence of sars-cov-2 nucleotides fragments in these samples, sun et al. (36) reported that urine samples of covid-19 patients can isolate sars-cov-2 with the infectious ability, suggesting the existence of infectious viral particles in these samples. moreover, the gastrointestinal tract highly expressed ace2 as indicated by the human protein atlas, which may explain the existence of sars-cov-2 in urine and stool specimens (7) . indeed, the 20-30fold higher affinity of sars-cov-2 spike glycoproteins binding to ace2 than the sars-cov spike protein may also enable the rapid transmission of covid-19 (25) (26) (27) . collectively, the mounting routes of transmission and high affinity with ace2 might jointly contribute to the rapid spread of sars-cov-2. however, covid-19 exhibited a lower-case fatality rate than sars (7) . the median incubation period of sars-cov is also longer than sars-cov (7) . despite the high phylogenetic homogeneity between sars-cov-2 and sars-cov, there are still some clinical characteristics differentiating covid-19 from sars. the symptoms of those infected with sars-cov have been more common in respiratory out-patient clinics and wards (37, 38) . after analyzing the 1,099 covid-19 patients, guan et al. (7) found that the typical radiological finding on chest computed tomographies is groundglass opacity with a ratio of 50.00%. consistent with previous publications (1, 32, 39) , the most common clinical characteristics of covid-19 are fever (87.9%) and cough (67.7%), but not the gastrointestinal symptom that is more frequently observed in sars (7). the absence of fever in covid-19 seems to be more frequent than in sars-cov (1%), as fever occurred in only 43.8% of covid-19 patients on initial presentation (40) , implying the limitation of focusing on fever detection in defining surveillance cases (41) . although there were relatively few cases of patients infected with sars during pregnancy based on previous clinical studies and case reports, there were more than 100 cases of sars-cov infections that occurred in pregnant women as estimated by the who (24) . sars-cov infection during pregnancy was associated with a risk of adverse maternal and neonatal complications, including intrauterine growth restriction, preterm delivery, spontaneous miscarriage, severe maternal illnesses, such as, admission to the intensive care unit (icu), renal failure, and disseminated intravascular coagulopathy, and death (4, 6, 13, (42) (43) (44) (45) (46) . in detail, a case-control study found that the clinical characteristics of sars in pregnant women were similar to those reported for non-pregnant patients with sars (47) . however, all the pregnant women with sars required endotracheal intubation, and six were admitted to the icu, whereas the intubation rate and icu admission rate in the non-pregnant group was only 17.5 and 12.5%, respectively (47) . there were three deaths among pregnant women with sars, while no deaths obstetrical ultrasounds revealed a low-lying placenta (placenta previa), but the pregnancy was otherwise normal. the cesarean section was performed at 38 weeks gestation due to the placenta previa and a healthy baby girl was delivered (48, 49) . antibodies against sars-cov were tested positive from the maternal serum, umbilical cord blood, and breast milk. no viral rna was detected in specimens of maternal serum and whole blood, or in swabs from the maternal nasopharynx and rectum, post-delivery placenta, umbilical cord blood, amniotic fluid, and breast milk. however, no clinical specimens were available for testing from the infant in this study (48) . another 38-year-old woman was exposed to sars-cov in the same hotel as the aforementioned patients (50) . the serum samples taken on days 28 and 64 post-onset of illness tested positive for antibodies against sars-cov. her pregnancy continued and was unremarkable except for developing elevated glucose levels. due to the preterm rupture of membranes and fetal distress, this patient underwent a cesarean section at 36 weeks gestation and obtained a healthy baby boy. the mother's serum samples at the time of delivery were positive for antibodies against sars-cov, but both umbilical cord blood and placenta were negative. also, breast milk sampled 12 and 30 days after delivery were negative for sars-cov antibodies. the specimens, including maternal blood, stool, nasopharynx samples, and umbilical cord blood of the infant, were negative for sars-cov rna. consistently, the stool samples from the neonate obtained on days-of-life 12 and 30 were negative for sars-cov rna. yudin et al. (51) reported a 33-year-old pregnant woman who was admitted to the hospital at 31 weeks' gestation due to sars. following a 21-day stay in the hospital, the antibody against sars-cov tested positive, while she had a normal labor delivery. together, there were no cases of vertical transmission identified among the pregnant women with sars-cov infection (24, 43, 45, 52) . however, the effect of sars on maternal outcomes seems to be associated with the stage of pregnancy when the onset of sars-cov occurs (44, 53) . wong et al. (44) found that the sars-cov infections present during the first trimester of pregnancy was more likely to cause spontaneous miscarriages, while infections present after 24 weeks of pregnancy developed into delivered preterm. current research involving pregnancy with covid-19 were listed in table 2 . results seems to be inconsistent between antibody-based serological characteristics and rt-pcr-based virologic evidence of infants. specifically, a retrospective study published in the lancet from (5) reported that the clinical characteristics of sars-cov-2 infection in pregnancy were similar to those reported for non-pregnant adults with a sars-cov-2 infection. in brief, the typical symptoms, including fever (in seven of nine patients), cough (in four), myalgia (in three), malaise (in two), and sore throat (in two), were observed in these patients, while none of them developed severe covid-19 pneumonia or died. all patients underwent a cesarean section and their live births had a 1-min apgar score of 8-9 and a 5min apgar score of 9-10 (5). the samples of amniotic fluid, cord blood, neonatal throat swab, and breastmilk samples from six patients tested negative for sars-cov-2 (5), suggesting no intrauterine vertical transmission of sars-cov-2 in the nine pregnant covid-19 patients. however, this study enrolled only nine pregnant women with covid-19, and sample collection was successful in only six infants (5) . another study from chen et al. reported four pregnant women with covid-19 (16) . all mothers recovered from covid-19 and had no critical maternal illness, although one mother suffered severe dyspnea after delivery which required respiratory support, and one developed anemia and dyspnea after admission. of note, none of the three infants whose parents provided consent to be diagnosed tested positive for sars-cov-2 from throat swab samples or developed serious clinical symptoms such as fever, cough, or diarrhea. however, two newborns had a rash, which disappeared spontaneously without treatment; a newborn from the mother with placenta previa was considered to suffer from transient tachypnea of the newborn and was supported by non-invasive mechanical ventilation for 3 days. of note, a study published in jama pediatrics indicated three neonates born to a pregnant woman with covid-19 tested positive for sars-cov-2 by qrt-pcr (20) . however, as indicated by the medical record, the throat swab sample of the neonate was collected at more than 48 h after delivery. no direct testing of intrauterine tissue samples, such as amniotic fluid, cord blood, or placenta, was collected to detect sars-cov-2, which is critical for confirming that the sars-cov-2 infection in the neonate was due to intrauterine transmission (20) . therefore, intrauterine sars-cov-2 infection remains uncertain. recently, two studies published in jama from separate research teams in china reported that three neonates may have acquired sars-cov-2 in utero from mothers with covid-19 based on the elevated igm antibodies to sars-cov-2 in neonates (17, 19) . specifically, the study from zeng et al. made a retrospectively review for six pregnant women with covid-19 (19) . all these mothers had mild clinical manifestations and performed cesarean deliveries in their third trimester. of note, all six newborn babies had a normal 1-and 5-min apgar score and none of them presented any symptoms of covid-19. however, serological characteristic results indicated that two infants had sars-cov-2-specific igg and igm concentrations higher than the normal level (<10 au/ml). given that igm is not usually transferred from mother to fetus because of its larger macromolecular structure under normal conditions (57), the author speculated that the neonates may have been infected with sars-cov-2 in utero from mothers with covid-19. however, all neonatal throat swabs and blood samples had negative rt-pcr test results. moreover, this study is limited by the small sample size, lack of cord blood, placenta, amniotic fluid, mother's vaginal secretions, and breast milk and by incomplete information on the outcome of the infants (19) . similar to the case mentioned above, another study from lan et al. reported that an infant girl born to a mother with covid-19 (34 weeks, 2 days of gestation) may have acquired sars-cov-2 in utero due to the elevated igm antibodies to sars-cov-2 (17). however, both the infant's nasopharyngeal swabs and breast milk sampled 3 days after delivery had a negative rt-pcr test result of sars-cov-2. moreover, all neonates had a normal 1-and 5-min apgar score. the mother's vaginal secretions obtained at delivery also tested negative for sars-cov-2. however, this study is limited by the single case, and the lack of amniotic fluid and placenta. there was also no detailed information regarding the pregnancy stage of the onset of covid-19. in summary, there was no positive rt-pcr result in the neonate specimens obtained within 24 h post-birth (5, 14, 16, 17, 19) , implying no virologic evidence for congenital infection. however, the serological characteristics of infants reported three neonates with elevated igm antibodies to sars-cov-2 born to a mother with covid-19, suggesting a possible vertical transmission of sars-cov-2 from mother to newborn (17, 19) . indeed, the virologic evidence for supporting the utero transmission should be diagnosed based on rt-pcr test results of the samples from neonates but not igm detection with a high incidence of its false-positive and false-negative results (58, 59) . a reasonable explanation for such inconsistency may be the disruption of the placenta or amniotic barrier caused by the inflammatory mediators from mothers that, induced by sars-cov-2, facilitates the cross of igg and igm. in detail, lga, large-for-gestational-age; sga, small-for-gestational-age; ttn, transient tachypnea of the newborn; ncpap, nasal-continuous positive airway pressure. the placenta is a barrier to viral infection (60) . the damage of the placenta by sars-cov-2 may represent an important link in the vertical transmission according to the experience from sars-cov. the two placentas from women who were recovering from sars-cov infection in the third trimester of pregnancy had abnormal weights and pathologies (53) . by contrast, in the case of covid-19, whether the placentas from those pregnant while infected with covid-19 were abnormal or damaged in most of these studies are unknown (5, 16, 17, 19) . indeed, a study reported that there were various degrees of fibrin deposition inside and around the villi with local syncytial nodule increases in three placentas from those pregnant while infected with covid-19, especially a placenta with massive infarction (55) . however, these three placenta samples tested negative for the nucleic acid of sars-cov-2, suggesting no virologic evidence in the placenta (55) . however, another study revealed that sars-cov-2 invasion of the placenta in a woman with covid-19 in the second trimester through molecular and immunohistochemical assays and electron microscopy (61) . moreover, the public antibody-protein profiles resident in human protein atlas (hpa) revealed enrichment of the sars-cov-2 receptor ace2 in the placenta and ovary (62) . collectively, the possibility of sars-cov-2 infection acquired from the uterus cannot be excluded, highlighting the potential for severe morbidity among pregnant women with covid-19. an editorial published in jama holds that sars-cov-2 can theoretically be transmitted in the uterus, especially given that virus' nucleic acid has been detected in blood samples (59) . however, nucleic acids do not represent infectious particles. indeed, it had been revealed that inflammatory mediators, including il-6, il-1î², and tnf-î±, cause severe dysfunction of the amniotic barrier via decreasing the expression of tight junctions-associated factors claudin-3 and claudin-4 and inducing apoptosis of the amniotic epithelial cells (63) . of note, il-6 has prominent pro-inflammatory properties (64) . il-6 was significantly increased in all infants from mothers with covid-19 (19) and the clinical and immunological features suggested that both the concentration of il-6 and tnf-î± are higher in severe covid-19 patients than in moderate patients (65) . the elevated igm antibody to sars-cov-2 in the blood was not observed in all neonates, which may be associated with the different levels of inflammatory mediators among them. collectively, in addition to the possibility of false-positive and false-negative results of igm (59), disruption of the placenta barrier and amniotic barrier caused by inflammatory mediators causing the elevated igm concentration also needs to be further investigated. however, determination of the level of ordinary igg but not specific to sars-cov-2 in neonate blood would be a crucial indicator explaining the disruption of the placenta and amniotic barrier. in general, the routes of mother-to-child transmission of sars-cov-2 mainly include intrauterine vertical transmission, birth, or breastfeeding. there is currently no evidence supporting the intrauterine vertical transmission of both sars-cov and sars-cov-2 based on the discussion above (4-6, 42, 51) . however, all the pregnant women recruited in these studies were in their third trimester. of note, the effect of sars-cov-2 on the infant and maternal outcome may be closely associated with their pregnancy stage during the virus infection, which was observed in both sars-cov and rubella (44, 66) . therefore, the possibility of intrauterine transmission in pregnancy with sars-cov-2 infection in the first or second trimester of pregnancy cannot be overlooked. the potential damage caused by inflammatory factors (above) also needs to be assessed. for the transmission during birth, most of the people pregnant while infected with covid-19 discussed above underwent a cesarean section to deliver the live births in current studies, three neonatal from which exhibited early-onset infection with sars-cov-2 (20) . by contrast, there were ten patients with covid-19 who performed vaginal delivery, all infants from which tested negative for sars-cov-2 (16, 20, 54) . of note, such low transmitted cases were greatly based on the comprehensive protective methods. indeed, the samples of vaginal mucosa and shedding in birth canals are crucial samples indicating whether sars-cov-2 could be transmitted during vaginal delivery. there were few studies that collected vaginal secretion (1/80) or infant blood (12/80); all tested negative for sars-cov-2 (5). further, as revealed by hpa tissue atlas, vaginal secretion expresses virtually no ace2 (62) , implying that sars-cov-2 may not infect the tissue. together, the risk of sars-cov-2 transmission by vaginal delivery seems low, although more definitive evidence is required. finally, to determine the potential of sars-cov-2 transmission via breastfeeding, several studies collected and analyzed breast milk samples (7/63) from patients with covid-19 pneumonia after their first lactation (5, 17) . however, these samples tested negative for sars-cov-2, suggesting no evidence supporting the breastfeeding transmission of sars-cov-2 (5). of note, such results were similar to pregnancies with sars-cov infection. no viral rna was detected in the specimens of umbilical cord blood, amniotic fluid, and breast milk from those pregnant while infected with sars-cov (48) (49) (50) . indeed, the antibody against sars-cov can be tested from the umbilical cord and breast milk (48) (49) (50) . based on such experiences from sars-cov, the antibody against sars-cov-2 derived from pregnancy may penetrate the placental barrier to orchestrate antiviral defense in the fetus to combat sars-cov-2, which needs to be further determined. in summary, there was a low possibility for mother-to-child transmission of sars-cov-2 if adequate protective measures were taken. however, the most crucial point is the potential effect of covid-19 on maternal and fetal outcomes, rather than whether sars-cov-2 can be acquired from the uterus; however, the determination of mother-to-child transmission potential is also important. that said, the effect of covid-19 on maternal and fetal outcomes should be paid considerable attention. according to the experience from sars, although no mother-to-child transmission was observed in sars, sars-cov infection was associated with a high risk of severe maternal illness, maternal death, and spontaneous miscarriages (4, 6, 13, (42) (43) (44) (45) (46) . indeed, maternal pneumonia is closely associated with a high incidence of various adverse obstetrical outcomes, including the premature rupture of membranes, preterm labor, intrauterine fetal demise, intrauterine growth restriction, and neonatal death (67) (68) (69) . further, although observed in a few cases, covid-19 may be related to the adverse maternal and infant outcome, including premature births, fetal distress, abnormal fetal liver function, rapid heart rate, etc. ( table 2) . to address the safety issues for the obstetrical management and delivery of pregnant women with covid-19, the advice for those women with sars-cov-2 infections during pregnancy and puerperium was prepared by numerous experts from the fields of obstetrics and gynecology, pediatrics, infectious diseases, and critical care (21, (70) (71) (72) (73) (74) (75) . similar to the recommendations for the non-pregnant, early isolation, early diagnosis, and early management are still the core criteria of prevention and control transmission for pregnant women with suspected and probable sars-cov-2 infection. these recommendations mainly include: 1. at times of covid-19 outbreaks, all pregnant patients should be assessed for travel history or contact with people from the worst-hit areas of the epidemic within 2 weeks. the definition of a case with suspected covid-19 should be focused on the clinical symptoms of covid-19; 2. pregnant women with labor-confirmed sars-cov-2 infection should be treated centrally according to the designation by the department of medical administration. the corresponding risk of adverse pregnancy outcomes contributed by covid-19 should be informed to the patients; 3. a chest radiograph, especially the computed tomography, is crucial for evaluating the development of covid-19; 4. pregnant women with suspected or probable covid19 should be informed to the cdc and placed in an isolation room or a negative pressure room if it is available; 5. prenatal examination and delivery of pregnant women with a sars-cov-2 infection should be carried out in negative pressure isolation or on an isolation ward. the management medical staff should wear protective equipment; 6. the timing of childbirth should be based on the specific conditions of the mother and child, the gestational week, and the childbirth conditions. the delivery mode depends on obstetric indication; 7. the specific anesthesia method for sars-cov-2-infected pregnant women who require surgical delivery can be general anesthesia and regional anesthesia, which should be performed based on the professional anesthesiologist; 8. given that the possibility of the intrauterine vertical transmission of sars-cov-2 cannot be excluded, all newborns from pregnant patients with suspected or confirmed covid-19 should be isolated for at least 14 days and should not be breastfed during this period until a sars-cov-2 infection is ruled out or cured. the mothers should squeeze milk regularly to ensure lactation. an expert team consisting of obstetricians, nurses, pediatricians, infection control specialists, respiratory therapists, and anesthesiologists should jointly manage pregnant women with covid-19 and their newborn baby; 9. pregnant women with covid-19 should be managed by fixed staff, including obstetrics, neonatal, and other related professionals. the healthcare workers caring for pregnant covid-19 patients should not care for other patients. all healthcare workers should be daily monitored for fever and cough symptoms of covid-19. such individuals should be isolated if they were confirmed or suspected of covid-19; 10. all health care personnel, trainees, and support staff should be trained in infection control management and containment to prevent the spread of sars-cov-2. yuw, yiw, and jy: conception and design, collection and/or assembly of references, data analysis, interpretation, and manuscript writing. xh and rl: conception and design, manuscript writing, and final approval of manuscript. all authors read and approved the final manuscript. clinical features of patients infected with 2019 novel coronavirus in wuhan a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding what are the risks of covid-19 infection in pregnant women? clinical characteristics 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comprehensive investigation of the mrna and protein level of ace2, the putative receptor of sars-cov-2, in human tissues and blood cells inflammatory mediators weaken the amniotic membrane barrier through disruption of tight junctions cytokine release syndrome in severe covid-19 clinical and immunological features of severe and moderate coronavirus disease 2019 vauloup-fellous c. rubella and pregnancy: diagnosis, management and outcomes antepartum pneumonia in pregnancy risk factors associated with the increasing prevalence of pneumonia during pregnancy pneumonia during pregnancy: has modern technology improved maternal and fetal outcome? novel corona virus disease (covid-19) in pregnancy: what clinical recommendations to follow? safe delivery for covid-19 infected pregnancies management of newborns exposed to mothers with confirmed or suspected covid-19 isidog recommendations concerning covid-19 and pregnancy recommendations for the management of newborn with suspected or confirmed coronavirus disease-19 brief clinical recommendations: management tactics for pregnant women and women in labor and childbirth with suspected or confirmed covid-19 infection funding this work was supported by grants from the research and industrialization of medical devices for digital childbirth (no. 2015b020233010). key: cord-290385-0smnl70i authors: chan, jasper f.w.; choi, garnet k.y.; yip, cyril c.y.; cheng, vincent c.c.; yuen, kwok-yung title: zika fever and congenital zika syndrome: an unexpected emerging arboviral disease date: 2016-03-03 journal: j infect doi: 10.1016/j.jinf.2016.02.011 sha: doc_id: 290385 cord_uid: 0smnl70i unlike its mosquito-borne relatives, such as dengue, west nile, and japanese encephalitis viruses, which can cause severe human diseases, zika virus (zikv) has emerged from obscurity by its association with a suspected “congenital zika syndrome”, while causing asymptomatic or mild exanthematous febrile infections which are dengueor rubella-like in infected individuals. despite having been discovered in uganda for almost 60 years, <20 human cases were reported before 2007. the massive epidemics in the pacific islands associated with the zikv asian lineage in 2007 and 2013 were followed by explosive outbreaks in latin america in 2015. although increased mosquito breeding associated with the el niño effect superimposed on global warming is suspected, genetic changes in its rna virus genome may have led to better adaptation to mosquitoes, other animal reservoirs, and human. we reviewed the epidemiology, clinical manifestation, virology, pathogenesis, laboratory diagnosis, management, and prevention of this emerging infection. laboratory diagnosis can be confounded by cross-reactivity with other circulating flaviviruses. besides mosquito bite and transplacental transmission, the risk of other potential routes of transmission by transfusion, transplantation, sexual activity, breastfeeding, respiratory droplet, and animal bite is discussed. epidemic control requires adequate clearance of mosquito breeding grounds, personal protection against mosquito bite, and hopefully a safe and effective vaccine. globalisation and urbanisation with increasingly frequent and large-scale movements of humans, animals, and commodities by aviation and water transport has led to the spread of previously geographically-restricted microbes and vectors to distant and isolated places. 1 recent examples of emerging viruses that have spilled over to other continents from their original localities via exportation of travelrelated cases include coronaviruses (severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus), influenza viruses, and ebola virus. 2e11 moreover, global warming and climate changes have redefined the geographical distributions of important vectors of arthropod-borne viruses (arboviruses), such as the aedes mosquitoes, and facilitated the global spread of these viruses. dengue virus (denv), west nile virus (wnv), and chikungunya virus (chikv), have been introduced (wnv in 1999 and chikv in 2013) and/or spread rapidly in the western hemisphere in the past two decades. 12 zika virus (zikv) is an arbovirus that was little known before it caused a large outbreak on yap island of the federated states of micronesia in 2007. 13 even then, zikv was not considered as an important emerging pathogen because clinical disease was generally mild. the recent report of a possible association between zikv infection and an epidemic of microcephaly among neonates in brazil has attracted global attention. 14 the rapid spread of zikv beyond africa and asia to the americas and europe, and the potentially novel "congenital zika syndrome" outbreak have led the world health organisation (who) to declare the zikv epidemic as a global public health emergency on 1 february 2016. 15 it would therefore be important to review the current knowledge on the epidemiology, virology, clinical manifestations, and laboratory diagnosis of zikv infection, and most importantly, to formulate clinical management options with special reference to perinatal care and control measures based on comparisons made with other mosquito-borne arboviruses. important historical and epidemiological events zikv (strain mr 766) was first isolated from the blood of a febrile sentinel rhesus monkey (macaca mulatta), rhesus 766, during a study on yellow fever virus (yfv) in zika forest of uganda in april 1947 (table 1) . 16 in 1948, zikv was isolated from aedes africanus mosquitoes caught in zika forest, suggesting that the virus might be mosquito-borne. in 1954, zikv was isolated from the serum of a 10-year-old nigerian girl who had fever and headache, implying its role as a possible human pathogen. 17 further virological and/or serological evidence of human zikv infection was reported in african (uganda, tanzania, egypt, central african republic, sierra leone, and gabon) and asian (india, malaysia, the philippines, thailand, vietnam, and indonesia) countries. 18e26 zikv infection remained relatively restricted geographically with less than 20 sporadic cases reported in these areas in the first 60 years after its discovery. 13, 27 in 2007, zikv emerged outside africa and asia for the first time and caused a major outbreak on yap island of the federated state of micronesia. 13 over 70% of the yap residents who were !3 years were infected within 4 months. 13 the attack rate of zikv infection in this outbreak was 14.6 per 1000 residents (range, 3.5e21.5 per 1000 residents). subsequently, another major outbreak was reported in french polynesia in october 2013. an estimated 30,000 humans (>11% of the french polynesian population) were infected by zikv. zikv infection then spread from french polynesia to other pacific islands including new caledonia, cook islands, vanuatu, and solomon islands. 27e30 the first cases of human zikv infection in the western hemisphere occurred on easter island, chile, in february 2014, possibly originating from french polynesia during the annual tapati festival. 31 phylogenetic analysis revealed that the ns5 gene sequence of the chilean strains had !99.8% nucleotide and 100% amino acid identity to the french polynesian strains. 31 the epidemic continued to expand rapidly and autochthonous human cases were reported in many latin american countries in the ensuing 2 years. 32 brazil stands out as the hardest hit latin american country with an estimated 500,000e1,500,000 cases of zikv infection since march 2015. 33 based on the close phylogenetic relationship between the south american strains and asian and oceanic strains of zikv, the virus might have been introduced into brazil by asian travellers during the world cup or participants from the oceanic countries of the va'a world sprint championship canoe race in the summer of 2014. 30,34e36 the climate changes associated with el niño in north and eastern south america in 2015 on the background trend of global warming might have facilitated the rapid spread of aedes mosquitoes and zikv. 37 currently, >30 countries in africa, asia, south america, oceania, and micronesia have reported autochthonous cases of human zikv infection. 38 travel-related cases from endemic and epidemic regions were also reported in europe, north america, australia, and japan. 39e50 more worryingly, the brazil health ministry reported the detection of an unusual increase in the number of cases of neonates with microcephaly in northeastern brazil in october 2015, coinciding with the expanding zikv infection epidemic. 14 over 3000 suspected cases including some fatal cases were reported during the second half of 2015 alone. 14 this represented a >20-fold increase in the rate of microcephaly as compared to previous years. 32 on 24 november 2015, the french polynesia health authorities also reported an unusual increase in the number of foetal and neonatal central nervous system malformations in 2014 and 2015. 51 like other flaviviruses, zikv is mainly transmitted by mosquitoes. in addition to the sylvatic (enzootic) transmission cycle between the haematophagous mosquito vectors and susceptible primary vertebrate hosts, the recent large-scale epidemics suggest that zikv is also adapting to an urban transmission cycle. 52, 53 among the various mosquito species, aedes (stegomyia) mosquitoes appear to be the most important vector for zikv transmission, although some anopheles, culex, eretmapodites, and mansonia species have also been proposed as possible vectors (table 2) . 25,52,54e69 the animal reservoirs of zikv are unclear. non-human primates including m. mulatta, cercopithecus aethiops, c. ascanius schmidti, c. mona denti, c. albigena johnstoni, chlorocebus sabaeus, colobus abyssinicus, erythrocebus patas, and pongo pygmaeus, and other mammals including zebras, elephants, and rodents, have been suggested as possible vertebrate hosts of zikv in africa and asia, based on virological and/or serological evidence of infection. 16,54,65,68,70e72 ae. africanus is the first mosquito species from which zikv was isolated, and is likely an important vector in the sylvatic transmission cycle of zikv. 16, 54 inoculation of unfiltered supernatant of zikv-infected ae. africanus into mice and rhesus macaques led to clinical disease and/or neutralising antibody response. 16, 54 ae. hensilli is the most commonly found mosquito species on yap island, but no virus isolate was made from field-collected mosquitoes to ascertain its role as a vector for zikv transmission during the outbreak. 67 ae. aegypti and ae. albopictus, which have much wider geographical distributions than other aedes mosquitoes, are considered to be more important vectors in the urban transmission cycle of zikv. 73 these aedes mosquitoes are highly susceptible to zikv infection in vitro with potential for further transmission after an extrinsic incubation period of 5e10 days. 58, 63 they bite both indoors and outdoors, and mostly during daytime. 74 non-vector-borne transmission routes of zikv have been proposed (table 3) . like other arboviruses, blood transfusion-related transmission of zikv is possible, especially in endemic regions or where blood products obtained from infected travellers immediately returning from endemic regions are used. zikv rna was detected in the blood of 2.8% of the donors in french polynesia during the epidemic. 75 sexual transmission of zikv appears highly probable, especially in patients presenting with haematospermia with infectious viral particles and rna in semen. 76, 77 notably, no other arboviruses have been associated with haematospermia or isolated from human semen. 76 this might further complicate the control of the zikv epidemic, since most infected patients are asymptomatic. inadvertent sexual transmission of zikv to the female partner may then lead to virus transmission to the foetus, which may be potentially associated with severe congenital anomalies. besides transplacental transmission, perinatal transmission of zikv may also occur during delivery, via breastfeeding, and/or close contact after birth via exchange of saliva and other bodily fluids. zikv rna could be detected in breast milk and saliva of infected women, although replicative virus particles have not been demonstrated 78, 79 perinatal transmission of other arboviruses, including denv, chikv, wnv, and yfv, has also been reported. 80e90 other suspected routes of transmission of zivk infection are those reported for other flaviviruses. these include mucocutaneous exposure to the virus in infected blood or via monkey bite, haemodialysis, or organ transplantation. 91e96 particularly, as zikv may be shed in the urine of infected patients for more than 30 days, the risk to recipients of donated kidneys from donors at or returning from endemic areas has to be considered. 40, 41, 49, 50, 97 it is unknown whether zikv could be transmitted via respiratory droplets as viral rna could occasionally be detected in nasopharyngeal swab and saliva samples. 40, 78, 79, 96 virology and pathogenesis zikv is an enveloped, positive-sense, single-stranded rna virus belonging to the genus flavivirus in the family flaviviridae. it is closely related to spondweni virus and the 2 viruses represent the only members of their clade within the mosquito-borne cluster of flaviviruses ( fig. 1) . 98, 99 phylogenetic analysis suggests that zikv has likely emerged between 1892 and 1943 in uganda. 66 the two major lineages of zikv are the african (subdivided into west and east african) and asian lineages, which are responsible for causing the majority of infections in africa and asia (as well as the pacific and americas), respectively. 34, 69, 100 the single-stranded rna genome of zikv has a size of 10,794 nucleotides encoding 3419 amino acids, with 2 flanking untranslated regions (5 0 and 3 0 utrs) and a single long open reading frame encoding a polyprotein, which is cleaved into capsid (c), precursor of membrane (prm), envelope (e), and 7 non-structural (ns) proteins 66, 101 reverse transcription-polymerase chain reaction (rt-pcr) using primers targeting the e or ns5 gene is a key laboratory diagnostic tool for zikv infection in the recent outbreaks. 13, 102, 103 the e protein is a major virion surface protein that is involved in receptor binding and membrane fusion. the domain iii of e protein contains a panel of antigenic epitopes that are important targets of serological assays, neutralising antibodies, and vaccines. 104, 105 loss of the n154 glycosylation site in the e protein may be associated with adaptation to mosquito vectors and thus facilitate transmission. 66 a single amino acid mutation in the e protein (e1-a226v) of chikv has been reported to be associated with increased fitness of the virus in ae. albopictus and allows chikv to disseminate in regions lacking the typical ae. aegypti vector. 106 the recent spread of the asian lineage of zikv to oceania and the americas may be associated with significant ns1 codon usage adaptation to human housekeeping genes, which could facilitate viral replication and increase viral titres. 107 mutations in the e and ns1 genes should be detected in zikv strains causing the current epidemic. when an infected aedes mosquito bites an infected patient, it ingests a blood meal containing zikv. as in other flaviviruses, zikv likely replicates in the midgut epithelial cells and subsequently the salivary gland cells. after an extrinsic incubation period of 5e10 days, zikv can be found in the mosquito's saliva which can then infect human. 58, 63 moreover, the virus can likely be vertically transmitted transovarially as other flaviviruses. when the mosquito's saliva containing zikv is inoculated into human skin, the virus can infect epidermal keratinocytes, skin fibroblasts in the subcutaneous layer, and the langerhans cells. 108 the keratinocytes and fibroblasts contain axl, tyro3, and tim-1, which can serve as attachment factors or receptors for zikv. the langerhans cells contain dc-sign, which can also serve as a receptor for virus entry. 108 zikv infection of primary skin fibroblasts is associated with the upregulation with tlr3 mrna expression, and enhanced transcription of rig-i and mda5, which are known innate immune responses to rna virus infection. this is followed by enhanced expression of interferon-alpha and -beta, and their downstream pathways of immune activation. both types i and ii interferons can suppress the viral load of infected cells. moreover, zikv is capable of increasing its replication by the induction of autophagy in host cells. thus, autophagy inhibitors can decrease the viral load of infected cells. 109 infected cells of human skin explant exhibits cytoplasmic vacuolation, pyknotic nuclei, and oedema in the stratum granulosum. 108 after replication in endemic/epidemic areas: + universal nucleic acid testing of blood donors. + temporary discontinuation of blood donation (importation of blood products from blood blank centres in non-endemic regions). non-endemic/epidemic areas: + pre-donation questionnaire to identify donors with recent travel history to endemic/epidemic areas. + deferral of blood donors who have travelled to endemic areas within the preceding !14 days. + self-reporting of symptoms after blood donation ( these local tissue cells and the regional lymph nodes, zikv may then disseminate from the lymphatics and bloodstream to reach other organs/tissues, including the central nervous system, the skeletal muscles, myocardium, and perhaps transplacentally to the foetus. zikv was highly neurotropic in infected suckling mice. the brains of infected suckling mice show neuronal degeneration, cellular infiltration, and softening in the brain with virus replication in astroglial cells and neurons on histopathological examination. 16, 54, 110 moreover, evidence of inflammation in skeletal muscles and myocardium has also been demonstrated in infected suckling mice. 111 axl and tyro3 are members of the tam family of receptor tyrosine kinases (rtks). they are also present in neurons and under the influence of gonadotropin releasing hormone (grh), which in turn may affect neuronal survival and migration. furthermore, flaviviruses such as yfv may persist for up to 159 days after intracerebral inoculation in rhesus macaques. 112 the neurotropism and persistence of zikv may therefore partially explain microcephaly and predominantly neurological complications and foetal anomalies in this suspected entity of congenital zikv infection. most patients with zikv infection are asymptomatic. in the outbreak of zikv infection on yap island, only 18% of cases were estimated to be symptomatic. 13 the incubation period of zikv infection is unclear, but is estimated to be similar to other mosquito-borne flaviviruses (2e14 days). 76, 113 the clinical syndromes of symptomatic zikv infection can be broadly divided into zika fever and congenital infection ("congenital zika syndrome") ( table 4 ). zika fever is an acute "dengue fever-like" illness characterized by low-grade fever (37.8e38.5 c), rash, retroorbital headache, bilateral non-purulent conjunctivitis, myalgia, and arthritis/arthralgia with periarticular oedema of the small joints of hands and feet. 13 the rash in zika fever is typically described as a generalized, erythematous, maculopapular rash that spreads downward from the face to the limbs. 30 less commonly, some patients may have more prominent systemic symptoms including high-grade fever, chills, rigours, sore throat, hypotension, and cervical, submandibular, axillary, and/or inguinal lymphadenopathies. 74 digestive tract symptoms including nausea, vomiting, diarrhoea, constipation, abdominal pain, and aphthous ulcers may also be present. 13, 17, 19 patients with genitourinary symptoms including haematuria, dysuria, perineal pain, and haematospermia often have detectable viral rna or infectious virus particles in urine and/or semen. 76, 77 haematological and biochemical laboratory parameters are usually normal. however, some patients may have transient and mild leucopenia, neutropenia, lymphopenia or activated lymphocytes, monocytosis, thrombocytopaenia, and elevated serum levels of lactate dehydrogenase, aspartate aminotransferase, g-glutamyl transferase, fibrinogen, ferritin, c-reactive protein, and erythrocyte sedimentation rate during the viraemic phase. 29 associated with restoration of normal number of peripheral immune cells and normal function of antigen-presenting cells. 45 notably, the clinical manifestations of zika fever are non-specific and may mimic those seen in infectious diseases caused by other arthropod-borne pathogens, especially denv and chikv. some suggest that zika fever may be distinguished from dengue fever and chikungunya fever by more prominent oedema of the extremities, less severe headache and malaise, and milder degree of thrombocytopaenia seen in the former. 13, 115 moreover, haemorrhagic complications seen in dengue fever have not been reported in zika fever, and arthralgia in zika fever is less severe than that in chikungunya fever. 13 however, none of these features are pathognomonic and laboratory confirmation is required to exclude co-infections with these arboviruses and other causes of acute febrile illness in returned travellers from endemic regions, such as malaria. zika fever is usually self-limiting with most clinical manifestations resolving completely within 3e7 days. 13, 49, 116 no death, hospitalisation, or haemorrhagic complication was reported during the outbreak on yap island. 13 however, some patients may experience more protracted symptoms and other non-haemorrhagic complications. zika fever-related rash usually resolve within the first week, but may last for up to 14 days and may be pruritic. 13 other exanthematous diseases, such as denv, chikv, rubella virus, measles virus, parvovirus b19, adenovirus, enterovirus, and rickettsial infection, should be excluded. the median duration of arthralgia is 3.5 days, but some patients may develop persistent or recurrent arthralgia for more than a month after symptom onset, mimicking the post-infectious chronic arthritis seen in chikungunya fever and lyme disease. 13, 76 lymphadenopathies may be present for 2 weeks after symptom onset, and alternative diagnoses such as infectious mononucleosis-like syndrome, streptococcus pyogenes infection, and toxoplasmosis should be considered in refractory cases. 45 a post-infection asthenia appears to be frequent and further investigations may be necessary to determine possible association between zikv infection and chronic fatigue syndrome. 13, 117, 118 immune-thrombocytopenic purpura and cardiac complication have also been reported in a few cases. 114 jaundice was observed in patients with virological and/or serological evidence of zikv infection in eastern nigeria in the 1950s who had co-infections (malaria and microfilaraemia) and a patient with sickle cell anaemia. 17, 119 a possible association between zikv infection and severe neurological complications has been proposed during the recent epidemics in oceania and south america, during which the incidence of guillainebarré syndrome has increased by 8e20 times in french polynesia. 115,120 74/ 8750 (0.8%) patients with suspected zikv infection in the french polynesia outbreak developed neurological syndromes after presenting with a zika fever-like illness. 120 forty-two of these 74 (56.8%) patients were diagnosed with guillainebarré syndrome. 12, 51, 121 similarly, guil-lainebarré syndrome has been reported among patients with zika fever-like illness in south america. 51, 121 other neurological complications potentially linked to zikv infection include encephalitis, meningoencephalitis, myelitis, paraesthesia, vertigo, facial paralysis, and 48,76,114,115,120e122 suspected fatalities due to zikv-related guillainebarré syndrome have been reported. 123 while the neurotropism of zikv may partially explain these neurological manifestations, more details and serial studies on their cerebrospinal fluid and magnetic resonance images by case-control studies are required to ascertain their association. zika fever-related death appears to be extremely rare but a number of probable cases have been reported, especially among immunocompromised patients and neonates with suspected congenital zikv infection. 51,119,121 a small number of patients with coinfection with denv or hiv did not appear to have more severe disease. 29, 124 further studies should be conducted to identify patients who are at risk of severe disease or death. microcephaly (head circumference !2 standard deviations below the mean for sex and gestational age at birth) is the most prominent and commonly reported clinical feature of suspected congenital zika syndrome. 14, 125 besides microcephaly, neonates and foetuses with suspected congenital zikv infection also had other malformations (table 4 ). general features included low birth-weight, redundant scalp skin, anasarca, polyhydramnios, and arthrogryposis. neurological abnormalities included cerebral lesions, polymalformative syndromes, brainstem dysfunction, and absence of swallowing. 51 ophthalmological defects included cataract, asymmetrical eye sizes, intraocular calcifications, macular atrophy (well-defined macular neuroretinal atrophy and/or macular pigment mottling and foveal reflex loss), optic nerve hypoplasia, iris coloboma, and lens subluxation. 122, 126, 127 notably, other features characteristic of intrauterine infections, such as hepatosplenomegaly, rash, and chorioretinitis have not been reported. 14 ultrasonographic examination revealed cerebral atrophy, intracranial calcifications especially over the white matter of frontal lobes, caudate, lentostriatal vessels, cerebellum, or around the lateral and fourth ventricles, dysgenesis of corpus callosum, vermia, and thalami, enlarged cisterna magna, asymmetrical cerebral hemispheres, severe unilateral ventriculomegaly, displacement of the midline, and thinning of the parenchyma on the dilated side, pons and brainstem. 14,51,121,128 zikv particles and rna may be detected by electron microscopy and rt-pcr, respectively, in autopsied samples. 125 two important questions concerning congenital zikv infection remain unanswered. the first question is whether zikv is indeed the cause of microcephaly and other congenital anomalies in these patients. severe consequences have been reported for materno-foetal transmission of other arboviruses, such as dengue virus (preterm delivery, foetal death, low birth-weight, prematurity, acute foetal distress during labour), wnv (chorioretinitis and focal cerebral destruction), and chikv (encephalopathy and haemorrhagic fever). 80, 82, 86 preliminary analysis in the current epidemic of microcephaly has not yet completely excluded other infectious or environmental aetiologies. 51 moreover, there is some virological evidence to support the association between congenital zikv infection and these anomalies. zikv rna has been detected by rt-pcr in the amniotic fluid of 2 pregnant women whose foetuses had ultrasonographic evidence of microcephaly, in the blood and foetal tissues of a neonate with microcephaly and other congenital anomalies who died within the first 5 min of birth, and in the neonatal brain tissues of a few cases of full-term miscarriages and neonates with microcephaly. 14, 51, 121, 125 however, there is still no large-scale prospective cohort or caseecontrol study to demonstrate a causal link between the presence of zikv in the foetus and the congenital anomalies after exclusion of other infectious and toxic causes. some have suggested that the apparent microcephaly surge might be attributable to the intense search for cases due to the heightened awareness of a possible association with the zikv outbreak or the use of larvicide. 129 furthermore, detailed investigations for exclusion of other pathogens associated with congenital malformations have only been reported in a small number of cases. 14, 125 microcephaly is well reported in congenital cytomegalovirus, rubella virus, and varicella zoster virus infection. chorioretinitis and intracranial calcifications are common in congenital cytomegalovirus infection and toxoplasmosis, but the latter is more commonly associated with hydrocephalus. cataract and cardiac anomalies are characteristic of congenital rubella syndrome, although cataract can also be found in congenital herpes simplex virus infection. thus, the diagnosis of congenital zika syndrome would depend on the exclusion of these "torch" infections in future studies using clinical criteria, histopathological findings, and serological, molecular and conventional cell culture techniques. if zikv is eventually confirmed to be the cause of these congenital anomalies, the second key question would be whether congenital zika syndrome actually comprises a wider spectrum of varying clinical severities than that seen in the reported cases. as with other congenital infections, it is possible that the reported cases of microcephaly represent only the tip the iceberg, focussing on the more severely affected patients, and that the timing of infection is likely to be important in determining the severity and outcome of the affected foetus. early infection during the first or even second trimester may be associated with congenital anomalies or even intrauterine death. 14, 125, 130 indeed, preliminary data suggested that the greatest risk of microcephaly or congenital anomalies in the affected neonates appears to be associated with zikv infection in the first trimester of pregnancy. 51 of 35 mothers with infants born with microcephaly, 57% and 14% had a rash during the first and second trimester of pregnancy, respectively. 14 besides neurological defects, cardiac and muscular abnormalities should also be excluded, as suckling mice infected with zikv developed evidence of central nervous system infection, myositis and myocarditis. 111 some suspected cases of congenital zika syndrome developed severe arthrogryposis. 14, 51, 121, 128 it is possible that intrauterine zikv infections that occur at a later stage of the pregnancy may present differently, either with less severe manifestations, such as mental retardation, sensorineural deafness, and/or ophthalmological lesions, or as full-term miscarriages. 14 neonates with probable perinatal transmission of zikv infection appear to have mild disease and favourable outcome. 78 further investigations should be conducted to better define the spectrum of manifestations in different gestational stages of congenital zikv infection. definitive diagnosis of zikv infection requires laboratory confirmation as there are no pathognomonic clinical, biochemical, or radiological features that reliably distinguish zika fever from other arboviruses, and congenital zikv infection from other infective, toxic, or genetic causes of congenital anomalies. successful isolation of zikv in viral culture, the gold-standard of laboratory diagnosis of viral infections, mainly depends on the timing of specimen collection and viral loads in the specimens. zikv has been isolated in vero and vero e6 cells inoculated with infected patients' serum, urine, and/or semen samples (table 5) . 40, 75, 77 however, infectious virus particles were not recovered by culture in most specimens with low viral loads. a positive serum immunoglobulin (ig) m or 4-fold rise in the titre of neutralising antibodies in paired serum samples collected approximately 2 weeks apart also establishes the diagnosis of zikv infection. igm may be detected by enzyme-linked immunoassay on as early as day 3 of symptom onset and may last for over 2 months. 40, 49 igm antibodies to denv and wnv usually persist for 3 months and 5 months, respectively. 131e134 the major limitation of these serological tests is possible cross-reactivity with other flaviviruses. neutralising antibodies detected by plaque-reduction neutralisation test may be more specific than igm detection by elisa for primary zikv infection, but may also have indeterminate results for secondary infection, including patients with previous vaccination against or exposed to other flaviviruses. 13, 40, 102, 120 this is especially problematic in areas where there is cocirculation of multiple flaviviruses with the same aedes mosquito vectors. 13, 64, 102, 120 patients with primary zikv infection and past denv infection are more likely to have higher titre (usually !4-fold) of igm and/or neutralising antibodies against zikv than against denv or other flaviviruses. 13, 102 a positive serum denv ns1 antigen test without serial increase in igm or the combination of a positive igm response to denv and lack of an igg seroconversion in the convalescent-phase serum sample should prompt the clinician to investigate for another flavivirus such as zikv. moreover, co-infections with other mosquito-borne arboviruses, such as denv, chikv, wnv, and japanese encephalitis virus, are always possible and should be excluded by more extensive laboratory testing if clinically indicated. rapid and accurate diagnosis of zikv infection during the recent epidemics has mainly been achieved by the application of rt-pcr using primers that target the e or ns5 gene of zikv. 13, 102, 103, 135, 136 alternatively, rt-pcr sequencing using universal primers that target the conserved regions in the genomes, such as the ns5 gene, of multiple flaviviruses, may allow simultaneous detection of >50 different flaviviruses. 137 serum samples should be collected in the early phase of the disease, because viraemia is usually shortlived (usually 5 days, rarely up to 11 days) and may be low-level ( 10 2 copies/ml). 97, 102 alternatively, urine and semen samples may have higher viral rna loads table 5 advantages, limitations, and uses of different diagnostic tests and types of specimens for laboratory diagnosis of zikv infection. 14,40,41,49,50,75e79,96,97,102,121,125,127,137e139,153 may be useful to exclude concomitant infections in patients with persistent or atypical rash. may be useful to exclude concomitant infections in patients with persistent or atypical rash. may be useful to exclude concomitant infections in patients with persistent or recurrent arthritis. may be useful to exclude concomitant infections in patients with persistent or recurrent arthritis. may be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia. may be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia. other tissues brain, liver, spleen, and pooled visceral (kidney, lung, and heart) tissues were positive in a fatal case (an adult male with co-morbidities and immunosuppressive treatment). may be useful to exclude concomitant infections in patients with unusually severe or fatal infection. abbreviations: rt-pcr, reverse transcription-polymerase chain reaction; zikv, zika virus. (>10 6 copies/ml) than serum samples, and may be persistently positive for >30 days and !62 days after symptom onset, respectively. 97, 138 in a few cases, zikv rna has also been detected in saliva and nasopharyngeal swab samples of patients whose serum samples tested negative for zikv. these samples should therefore also be collected in suspected cases of zikv infection. 40, 78, 79, 96 collection of amniotic fluid should be considered in pregnant women with positive zikv test result or if the foetuses show ultrasonographic evidence suggestive of congenital zikv infection. 14, 102, 128 cerebrospinal fluid, placental, and/or umbilical cord tissues from neonates with suspected congenital zikv infection should be sent for virological and/or histopathological examinations to establish the diagnosis. 14,121,125 zikv rna may also be detected in organ tissues in the rare cases of suspected zikv-related deaths. 121 future studies should aim to better stratify the clinical use of these tests and to develop point-of-care tests (eg: antigen tests) that can be widely used in less developed regions without the facilities and expertise for molecular or serological tests. treatment is usually not required for patients with asymptomatic or uncomplicated zika fever. the mainstay of treatment is supportive as there are no specific anti-zikv antiviral agents. acetaminophen may be used to relieve fever and arthralgia. anti-histamines may help to control pruritus. adequate rehydration for fluid loss through sweating, vomiting, and insensible losses should be encouraged. aspirin should be avoided due to the risks of bleeding in those with thrombocytopaenia and developing reye's syndrome in children less than 12 years of age. nonsteroidal antiinflammatory drugs are also contraindicated in cases where denv and chikv infections cannot be confidently excluded in order to avoid haemorrhagic complications. potential neurological complications, especially guillainebarré syndrome, should be diagnosed promptly to allow early use of intravenous immunoglobulins and/or plasmapheresis. the risk of immune enhancement should also be considered if convalescent-phase plasma therapy with neutralising antibodies against zikv is used for treatment of severe cases. virological testing and foetal ultrasound to exclude zikv infection and foetal microcephaly or intracranial calcifications should be offered to pregnant women who develop zika fever-like symptoms during or within 2 weeks of travel to areas with zikv transmission. 139 besides collecting the appropriate specimens for virological tests, serial foetal ultrasound examinations should be performed every 3e4 weeks to monitor foetal anatomy and growth in suspected cases of congenital zikv infection. foetal ultrasound and/ or aminocentesis should also be offered to asymptomatic and seropositive pregnant women with history of travel to affected areas. after delivery, serum should be collected either from the umbilical cord or directly from the neonate within 2 days of birth for rt-pcr, igm and/ or neutralising antibodies against zikv. 140 comprehensive physical examination including measurement of the occipitofrontal circumference, length, and weight, evaluation for neurological abnormalities, dysmorphic features, hepatosplenomegaly, rash, ophthalmological lesions, and auditory defects, and laboratory testing for torch screening should be performed. 140 the affected child and the family should be managed and counselled by a multidisciplinary team consisting of paediatric neurologist, clinical geneticist or dysmorphologist, infectious disease specialist, medical social worker, and other relevant specialists. 140 long-term follow-up to monitor physical, intellectual, and functional progress of the child should be offered. 140 both vector control and personal preventive measures are important for interrupting the transmission of zikv. systematic mosquito surveillance and control programs should be established and coordinated by health authorities. 51 mass sanitation campaigns to eliminate mosquito breeding sites in household and high-risk areas such as garbage collection points, construction sites, illegal dumping grounds, and invalid car fields should be organised. mosquitoes should be removed with a radius of at least 400 m around areas with high population densities, such as schools, transport terminals, churches, and healthcare facilities. in areas where autochthonous or imported cases of zikv are detected, the use of adulticide through spraying to remove infected adult mosquitoes should be considered. residents in or travellers to affected areas should stay indoor with air conditioning, window and door screens if possible, wear long sleeves and pants, use permethrintreated clothing and gear, and use insect repellents when outdoor. 140 most environmental protection agency (epa)registered insect repellents, including n,n-diethyl-mtoluamide (deet), should be safe for pregnant and lactating women (20% deet), and children (10% deet) aged >2 months. 141 individuals returning from affected areas to non-affected regions should continue to use insect repellents for at least an additional 14 days to prevent local non-infected mosquitoes from the acquisition of virus from the asymptomatically infected returned travellers. this will serve to interrupt the mosquito-human-mosquito transmission chain. hospitalised laboratory-confirmed cases should be managed in designated wards to avoid mosquito bites. the effects of other novel mosquito-control measures, such as the wolbachia biological control approach, should be evaluated. 142 other animals such as rodents should also be investigated as potential animal reservoirs and controlled as findings indicate. non-vector-borne transmission of zikv may be prevented by specific measures (table 3) . concerning blood transfusion, universal nucleic acid testing of blood donors is recommended. the use of universal primers that can simultaneously detect multiple arboviruses such as denv and zikv should be considered. temporary discontinuation of blood donation should be considered during an outbreak situation. in non-endemic areas, pre-donation questionnaire to identify donors with recent travel history to regions with reported cases of zikv infection and deferral of blood donation from these donors until at least 14 days after returning from affected regions should be implemented. most transfusion-related transmissions of arboviruses are associated with asymptomatic infections, and symptomatic donors who were rt-pcr-positive for zikv usually developed symptoms between 3 and 10 days after blood donation. 143 newer pathogen reduction technologies for blood products should be considered. 143 similarly, donated organs, especially kidneys, from individuals with travel history to affected areas should be tested for zikv as the virus may persist in the genitourinary tract for an undetermined period. 40, 41, 49, 50, 97 barrier methods should be used to prevent sexual transmission through infected semen. male returned travellers should continue the use of condom with pregnant sex partner throughout the whole duration of pregnancy. future studies should evaluate the duration of virus shedding in semen and the infectiousness of rnapositive semen samples, in order to determine how long barrier methods should be used by men returning to nonendemic regions. some regional authorities have advised women to avoid pregnancy until the epidemic is over. 144 pregnant women or those planning for pregnancy should defer travelling to regions with reported cases of zikv infection. if such travel was unavoidable, they should strictly comply with personal protective measures to avoid mosquito bites. further studies are needed to determine the risk of zikv transmission by breast milk and saliva. other less common transmission routes, including mucocutaneous exposure to infected bodily fluid during laboratory and patient-care procedures, and bites by infected primates should be avoided with strict compliance to infection control measures. in the laboratory setting, zikv can be killed by potassium permanganate, ether, and heat (>60 c), but it is not effectively neutralised with low concentration (10%) of ethanol. 54 no zikv vaccine is available currently. because the moratorium for pregnancy may be impractical for some people, a safe and effective zikv vaccine is urgently needed. some realistic approaches include liveattenuated or killed vaccine from human cell lines (as in the case of yfv and japanese encephalitis vaccines), attenuated chimeric vaccine (denv vaccine using the yfv vaccine backbone, currently in phase iii clinical trial), dna and recombinant protein vaccine. 145 suitable animal models for evaluation of these potential vaccine candidates should be developed for zikv infection. the role of passive immunisation before and after exposure to zikv should also be assessed in future studies. the zikv 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and by the donations of hui hoy and chow sin lan charity fund limited and mr. larry chi-kin yung. the authors declare no conflict of interest. key: cord-272194-h7xnr389 authors: wiegers, hanke m. g.; van nijen, lisa; van woensel, job b. m.; bem, reinout a.; de jong, menno d.; calis, job c. j. title: bacterial co-infection of the respiratory tract in ventilated children with bronchiolitis; a retrospective cohort study date: 2019-11-06 journal: bmc infect dis doi: 10.1186/s12879-019-4468-3 sha: doc_id: 272194 cord_uid: h7xnr389 background: viral bronchiolitis is the most common cause of respiratory failure requiring invasive ventilation in young children. bacterial co-infections may complicate and prolong paediatric intensive care unit (picu) stay. data on prevalence, type of pathogens and its association with disease severity are limited though. these data are especially important as bacterial co-infections may be treated using antibiotics and could reduce disease severity and duration of picu stay. we investigated prevalence of bacterial co-infection and its association with disease severity and picu stay. methods: retrospective cohort study of the prevalence and type of bacterial co-infections in ventilated children performed in a 14-bed tertiary care picu in the netherlands. children less than 2 years of age admitted between december 2006 and november 2014 with a diagnosis of bronchiolitis and requiring invasive mechanical ventilation were included. tracheal aspirates (ta) and broncho-alveolar lavages (bal) were cultured and scored based on the quantity of bacteria colony forming units (cfu) as: co-infection (ta > 10(^5)/bal > 10(^4) cfu), low bacterial growth (ta < 10(^5)/bal < 10(^4) cfu), or negative (no growth). duration of mechanical ventilation and picu stay were collected using medical records and compared against the presence of co-infection using univariate and multivariate analysis. results: of 167 included children 63 (37.7%) had a bacterial co-infection and 67 (40.1%) low bacterial growth. co-infections occurred within 48 h from intubation in 52 out 63 (82.5%) co-infections. h.influenza (40.0%), s.pneumoniae (27.1%), m.catarrhalis (22.4%), and s.aureus (7.1%) were the most common pathogens. picu stay and mechanical ventilation lasted longer in children with co-infections than children with negative cultures (9.1 vs 7.7 days, p = 0.04 and 8.1vs 6.5 days, p = 0.02). conclusions: in this large study, bacterial co-infections occurred in more than a third of children requiring invasive ventilation for bronchiolitis and were associated with longer picu stay and mechanical ventilation. these findings support a clinical trial of antibiotics to test whether antibiotics can reduce duration of picu stay. bronchiolitis is a common respiratory condition in young children associated with a high morbidity. it is caused by viral infections, respiratory syncytial virus (rsv) being the commonest cause in hospitalized children [1] . the clinical picture of these infections ranges from mild symptoms to respiratory distress requiring hospitalization for supportive therapy [2] . about 10% of hospital admissions for rsv infection are so severe that invasive ventilation in a pediatric intensive care unit (picu) is needed [3] . although several risk factors for a more severe course of bronchiolitis have been identified, including cardiac disease, chronic lung disease, prematurity, this only partly explains why some children with bronchiolitis have severe respiratory failure and require mechanical ventilation [4] . previous studies suggested that bacterial co-infections may be associated with a more severe course, though definitive evidence is lacking [5] [6] [7] [8] . this is of particular interest as current treatment strategies for severe bronchiolitis are only supportive, whereas bacterial coinfections can be treated with antibiotics and thus could reduce morbidity. the aim of this study is to investigate the prevalence and types of bacterial co-infection of the respiratory tract in children with bronchiolitis requiring invasive ventilation. we further investigated whether bacterial co-infection was associated with a prolonged duration of mechanical ventilation and picu stay. finally, we analysed whether bacterial co-infection can be predicted using clinical or laboratory markers such as c-reactive protein (crp). this is a retrospective cohort study of children requiring invasive ventilation in the picu of the emma children's hospital/academic medical center amsterdam, between december 2006 and november 2014. the picu is a 14-bed, tertiary unit, serving the greater amsterdam area in the netherlands. patients less than 2 years of age who were clinically diagnosed with bronchiolitis and required invasive mechanical ventilation were included. in the netherlands patients are referred to the picu if they (are expected to) require (non) invasive ventilation. diagnoses were scored on discharge by the attending picu consultant and stored in an automated database containing all picu admissions. a standardised questionnaire was completed using data from the electronic patient chart that automatically recorded vital signs, ventilator settings; and collected medication given (metavision®). an electronic patient chart was used to gather demographic data, previous medical history, physical examination on admission, laboratory results, chest x-ray on admission, microbiological results, and outcome parameters. the mechanical ventilation duration was defined as the cumulative period of invasive ventilation and non-invasive ventilation. if extubation failed the cumulative periods of (non-)invasive ventilation was used. nasal specimens were collected and used for dna extraction and multiplex pcr as previously described [9] . the multiplex platform included influenza viruses (a and b), enterovirus, adenovirus, respiratory syncytial virus (a and b), rhinovirus (a-c), human metapneumovirus; parainfluenza viruses (1-4), parechovirus, human bocavirus and coronaviruses (hcov; hku1, nl63, 229e, and oc43). with every extraction and pcr, three controls were run. a minority of children had previously been tested in other hospitals for rsv using pcr or antigen tests. these data were used if multiplex results from our hospital were not available. viral testing was performed by the discretion of attending physicians. in some children no viral testing was performed at all. the cultures performed in this study were ordered by the attending physicians in case of clinical suspicion of a bacterial (super)infection. during the study period two sampling techniques were used: tracheal aspirates and mini-broncho-alveolar lavage [10] . sampling was performed the endotracheal tube without previous installation of normal saline (tracheal aspirate) or by mini broncho-alveolar lavage. tracheal aspirate was the primary sampling method. the mini-bal was performed as part of an observative study on cytokine levels in bronchiolitis. bacterial cultures were performed according to standard operating procedures at our laboratory. in brief, 10 μl of specimen were inoculated on standard media (cled, colombia, sheep blood and chocolate agars) and incubated for 1-2 days. bacterial colonies were counted and identified by vitek (biomerieux) or malditof (bruker). other diagnostics x-rays were scored and reported by attending radiologists. plasma levels of c-reactive protein, were analyzed on modular p800 and modular analytics e170 systems (roche). leukocytes were determined on an automated hematology cell counter. to distinguish between colonisation and infection we used quantitative culture results, represented by the number of cfu per ml. we distinguished 3 categories based on the american thoracic society definitions in adults and thorburn et al. [5, 11] . to distinguish between colonisation and infection we used the quantity of cfu/ml [5, 11] to distinguish 3 categories. no bacterial growth: no growth or growth of commensal bacteria only. low bacterial growth: presence of pathogens in the bal <10 4 cfu/ml or trachea aspirates <10 5 cfu/ml. bacterial co-infection: presence of pathogens in the bal ≥10 4 cfu/ml or trachea aspirates ≥10 5 cfu/ml. to distinguish between bacterial co-infections present on admission and those associated with ventilation we used the following definitions. early infections (non-ventilatorassociated) apply to cultures taken within 48 h of intubation, whilst late infections (ventilator -associated) refer to samples taken more than 48 h after intubation [11] . data was entered and analysed in an anonymised database spss 22.0 (spss inc., chicago, illinois, usa). for comparison of continuous variables we used independent ttests and for comparison of categorical variables chisquare test and fisher exact test were used. all pvalue reported are two-sided and values less than 0.05 was considered significant. potential predictors of duration of ventilation and picu stay were assessed using univariate and linear regression analysis. logistic regression was used to assess potential predictors of bacterial co-infection. variables were included in multivariate analyses if p-values of univariate associations were < 0.1. multivariate models included potential confounding factors (e.g. previous antibiotic use). the study adhered to the strobe guidelines. medical ethical approval was waived as it concerned retrospective analysis of anonymised patient data. of 251 picu admissions for bronchiolitis, 189 (75.3%) required invasive ventilation and were included in the study. the mean age was 2.9 months, 168 patients (88.9%) were less than 6 months of age and 108 (57.1%) were boys. twenty-five patients had an underlying medical condition (13.3%) and 53 (31.0%) were prematurely born (table 1) . data on antibiotic use prior to picu referral was available in 101 children, 51 of these received antibiotics (50.5%). one patient died due to pulmonary hypertension. rsv testing was performed in 162 (85.7%) patients and was detected in 126 (77.8% table 1 ). in 97 (51.3%) children a full multiplex platform was performed and 24 children (24.7%) had presence of two of more viral pathogens (table 1) . in 167 of 189 (88.4%) patients one or more cultures (bal or tracheal aspirates) were performed, whereas in 22 patients there was no culture performed (11.6%). demographic data or underlying conditions were not different in these groups (data not shown). among the 167 cultured children, the overall occurrence of bacterial co-infections was 63 (37.7% of children cultured, or 33.3% of all 189 children). low bacterial growth was found in 67 children (40.1%). in the 63 children with a bacterial co-infection 52 (82.5%) coinfections were detected within 48 h from intubation and were typed as early infection (table 2) . thirty-four co-infections were detected by tracheal aspirate (54.0%), and 29 (46.0%) by mini-bal. patients who received antibiotics prior to picu admission had more often negative cultures as compared to patients who did not receive antibiotics (43.2% versus 11.6%, p = 0.001). bacterial co-infections were found in 36.0% of 111 children with a proven rsv infection as compared to 21.9% in the rsv-negative group (7/32) and 66.7% in those who had no viral test performed (16/24; p = 0.002). in 63 children with a bacterial co-infection a total of 85 pathogens were isolated including h. influenza (40.0%), s. pneumoniae (27.1%), m. catarrhalis (22.4%), and s. aureus (7.1%) ( table 3) . enterobacteriaceae were only seen as late co-infection. in 67 patients with low bacterial growth the distribution of pathogens was comparable to the co-infection group (data not displayed). only s. aureus was more common in low bacterial growth as compared to the coinfection group (25.4% vs 7.1%, p = 0.02). s. pneumoniae was found in 13.5% of rsv-positive patients (15/111), 3.1% in rsv-negative patients (1/32) and in 29.3% of patients who had no viral test performed group (7/24, p = 0.02). children with a bacterial co-infection required a mean duration of ventilation of 8.1 days compared to 6.5 days in children with a negative culture result (p = 0.02; table 4 ). picu length of stay was 9.1 and 7.7 days in children with and without a co-infection respectively (p = 0.04). no difference was noted in duration of ventilation nor picu stay between the groups with coinfection and low bacterial growth. early co-infections were not associated with a longer duration of ventilation or picu stay as compared to children with negative cultures (7.5 vs 6.5, p = 0.12 and 8.5 vs 7.6, p = 0.21 respectively). late co-infections were associated with a longer duration of ventilation and picu stay (11.1 vs 6.5, p < 0.001 and 12.4 vs 7.6, p < 0.01). a high crp was associated with bacterial co-infection in a multivariate analysis including antibiotic use prior to picu admission (p = 0.001, table 5 ). a crp level of 40 mg/l or more had a sensitivity of 85.7% and a specificity of 56.8% to detect a bacterial co-infection. in this large and concise study assessing the prevalence and relevance bacterial co-infections of the respiratory tract in children with bronchiolitis requiring invasive ventilation, bacterial co-infections were identified in at least a third of patients. in our population bacterial coinfections were associated with a longer duration of ventilation and picu stay. crp was associated with bacterial co-infection and may be used to identify children at increased risk. in our study more than a third (37%) of the children with bronchiolitis requiring invasive ventilation had a bacterial co-infection. potentially this number may have been higher as we used: a) a very strict definition of bacterial coinfection, ignoring 40% of children with low bacterial growth; b) 50% of children received antibiotics prior to sampling, which may have decreased detection of bacterial co-infections. we may also have overestimated the prevalence of bacterial co-infections as we used a clinical diagnosis of bronchiolitis whilst some children may have had a primary bacterial infection. this hypothesis is less likely though as the prevalence of bacterial co-infections in the rsv-positive group was comparable to the overall prevalence (36 and 37% respectively). the prevalence of bacterial co-infection is in line with previous studies that reported bacterial co-infections occurred in 20-45% of children with bronchiolitis admitted to picu's [1, 2, [5] [6] [7] [8] [12] [13] [14] [15] . like our study, most data were retrospective and had similar limitations such as previous antibiotic use. a prospective study reported that 22% had a co-infection and 21% had low bacterial growth/possible co-infection [5] . also, in this study more than 50% of children had received antibiotics prior to airway sampling. most co-infections were detected within 48 h, a cut off that is used to distinguish pre-existing infections from ventilator acquired pneumonias (vap) [11] . although it is not possible to distinguish pre-existing co-infections from infections acquired by our invasive techniques the fact that most co-infections occurred within these 48 h suggest that these infections are not likely introduced by intubation or colonization of endotracheal tubes. in our setting most bacterial isolates were common airway pathogens such as h. influenza, s. pneumoniae, m. catarrhalis and to a lesser extent s. aureus. this corroborates with data from bronchiolitis studies in liverpool [5] and zurich [6] , however in our setting s. pneumoniae was more and s. aureus less common than in other studies. enterobacteriaceae were solely identified as late coinfections; which is in line with previous data on vap [16] . in our setting empirical treatment with amoxicillin (with or without clavulanic acid) would be appropriate for early infections, however in late co-infections an antibiotic with a broader coverage should be considered. in this study we identified an association between bacterial co-infection and duration of picu stay and mechanical ventilation. the difference was 2 days which can be considered clinically relevant. three other studies reported on duration of ventilation and the presence of bacterial co-infections in children with bronchiolitis requiring ventilation. kneyber et al. found a trend towards longer duration of mechanical ventilation in children with bacterial co-infections as compared to those without (14.3 vs. 10.6 days, p = 0.09) [7] ; thorburn et al. reported that bacterial infection contributed to longer mechanical support [5] and hennus et al. confirmed that mechanical ventilation and bacterial co-infection were positively correlated [15] . the association between bacterial growth and a prolonged duration of mechanical ventilation suggests that bacterial co-infection could be of clinical importance. alternatively, prolonged ventilation may predispose to increased prevalence of bacterial infections (ventilator associated pneumonia) [16] . in our study the difference in duration of ventilation was only significant in the late co-infections which corroborates with the latter theory. a similar trend was observed in the early co-infections which could suggest that both hypotheses are correct. the association between co-infections and length of ventilation in our and other studies justify a trial to assess the role of antibiotics in reducing duration of ventilation and picu stay in children with a bronchiolitis requiring invasive ventilation. ideally the design of such a study should include a curative and preventive arm to evaluate both the role of pre-existing and ventilator associated bacterial pneumonia. except for a study assessing the role of antibiotics to prevent picu admissions [10] , such a study has not been performed so far. the use of prophylactic antibiotics in ventilated patients has been studied in adults using selective digestive decontamination (sdd). d'amico et al. showed that sdd given to adults admitted to icu reduced vap and overall mortality [17] . the data on the overall effect of all ventilated children admitted to picu is conflicting however, but sdd appears to be effective in controlling respiratory infections [18] . no studies have been performed in the current subgroup of bronchiolitis patients admitted to picu. in this study crp was a useful predictor of bacterial coinfection, which corroborates with previous data [2] . that study used a much lower cut-off of 11 mg/l, which in practice does apply to most children admitted with a bronchiolitis and may not be very discriminative. other studies did not find an association between crp and bacterial co-infection in bronchiolitis patients [5, 10] . unlike these studies we used the maximum crp during admission, which may explain this discrepancy. crp is known to have a twofold increase every 6 h, and therefor crp dynamics could be used to guide antibiotic use in this population awaiting culture results. several limitations apply to our study, firstly we may have underestimated the prevalence of bacterial co-infections as we used a) a strict definition and b) the majority of the patients received antibiotics prior to sampling. these factors potentially may have contributed to an underestimation of the actual prevalence and underline that the number of bacterial co-infections in children with viral bronchiolitis in picu is high. alternatively we may have overestimated the role of bacterial co-infections in children with bronchiolitis as we recruited children based on the clinical discharge diagnosis of the attending picu consultant. although 4 of 5 children had a proven viral infection some children may have had a primary bacterial lower airway infection. this is less likely though as also in the group with proven rsv infection the co-infection rate was 36% as compared to 37% in the overall study. secondly, not every patient who was admitted to the picu had a culture and cultures were performed based on clinically suspicion. this may mean that selection bias applied. however, the culture rate was nearly 90% and even if all children that were not cultured would have negative cultures still a third would have had a co-infection. thirdly, this was a retrospective and observational study, therefore we can only detect associations and not prove causal relationships. however, it justifies a prospective randomized controlled trial which could test the role of (curative or preventive) antibiotics to reduce duration of ventilation. fourthly, we have used different sampling techniques for bacterial cultures. however, we have applied strict definitions and corrected for this using the definition from thorburn [5] et al. and international ats guidelines [11] . until prophylactic or curative use of antibiotics have been tested in a double blind, randomized controlled trial, the use of antibiotics may be restricted to children with a suspicion of bacterial co-infection. especially in young children and children with an increased crp (> 40 mg/l) cultures should be taken and antibiotics covering normal airway pathogens should be considered. in case late co-infection is suspected empiric antibiotics should cover gram-negative bacteria and s. aureus. future studies should prove if prophylactic or curative use of antibiotics could reduce duration of ventilation and picu stay in bronchiolitis patients. in this large and concise study on bacterial co-infections in children with bronchiolitis we have found that bacterial infections are common, associated with prolonged ventilation and a raised crp. bacterial co-infections of the respiratory tract occurred in at least 37.7% of children with a bronchiolitis requiring invasive ventilation. in most cases bacterial co-infection was present within 48 h after intubation and was caused by common airway pathogens: h. influenza, s. pneumoniae, m. catarrhalis. future studies should focus on the potential beneficial role of preventive or curative antibiotics to reduce duration of ventilation and picu stay. risk of bacterial infection in previously healthy respiratory syncytial virus-infected young children admitted to the intensive care unit the use of c-reactive protein in predicting bacterial co-infection in children with bronchiolitis bacteraemia and antibiotic use in respiratory syncytial virus infections pediatric investigators collaborative network on infections in canada (picnic) prospective study of risk factors and outcomes in patients hospitalized with respiratory syncytial viral lower respiratory tract infection high incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (rsv) bronchiolitis pulmonary and systemic bacteria co-infections in severe rsv bronchiolitis concurrent bacterial infection and prolonged mechanical ventilation in children with respiratory syncytial virus lower respiratory tract disease empiric antibiotics are justified for children with respiratory syncytial virus lower respiratory tract infection presenting with respiratory failure: a prospective study and evidence review development and evaluation of a four-tube real time multiplex pcr assay covering fourteen respiratory viruses, and comparison to its corresponding single target counterparts azithromycine does not improve disease course in hospitilized children with respiratory syncytial virus (rsv) lower respiratory tract disease: a randomized equivalence trial infectious diseases society of america. guidelines for the management of adults with hospital-acquired, ventilator associated, and healthcare-associated pneumonia risk of concurrent bacterial infection in preterm children hospitalized due to respiratory syncytial virus infection respiratory syncytial virus morbidity, premorbid factors, seasonality, and implications for profhylaxis survey of severe respiratory syncytial virus infection in kyoto prefecture from mechanical ventilation drives inflammation in severe viral bronchiolitis ventilator-associated pneumonia: diagnosis, treatment, and prevention antibiotic prophylaxis to reduce respiratory tract infections and mortality in adults receiving intensive care prevention of nosocomial infection in a pediatric intensive care unit (picu) through the use of selective digestive decontamination publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable.authors' contributions hw and ln performed data extraction. jc was an independent reviewer. jc, rb, and jw conceived the study. lab work and interpretation was supervised by mj. the article was written by all (hw, ln, jw, rb, mj and jc). all authors read and approved the final manuscript. not applicable. the anonymized datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate ethical approval was waived by the medical ethics review committee of the amc as it was retrospective analysis. not applicable. the authors declare that they have no competing interests. key: cord-271076-436nxsua authors: paul-pierre, pastoret title: emerging diseases, zoonoses and vaccines to control them date: 2009-10-30 journal: vaccine doi: 10.1016/j.vaccine.2009.06.021 sha: doc_id: 271076 cord_uid: 436nxsua abstract vaccination, when available, is undoubtedly the most cost-effective means of preventing and controlling, and even eradicating, infectious diseases. in recent years vaccination has also been used for other purposes in animal health, production and welfare, e.g. immunocastration. vaccination of animals serves many different purposes, such as controlling animal infections and infestations, thus improving animal health and welfare; controlling anthropozoonoses and food poisoning in humans, thereby protecting public health; solving problems associated with antibiotic and anthelmintic resistance; helping to leave food-producing animals free of chemical residues; protecting the environment and biodiversity and ensuring animal farming sustainability. the problem is nevertheless more complex when facing emerging or re-emerging infections particularly zoonotic ones. vaccination, when available, is undoubtedly the most costeffective means of preventing and controlling, and even eradicating, infectious diseases. unfortunately the problem is more complex when facing emerging or re-emerging infections particularly zoonotic ones. for instance canine parvovirosis was a real emergence in animal health [1, 2] ; the first step was to vaccinate dogs with a vaccine directed against feline panleukopaenia since the two causative viruses are antigenically nearly identical. this first step was rapidly followed by the development of vaccines, either inactivated or attenuated, specifically directed against canine parvovirosis. some situations are more problematic when facing the outbreaks of diseases caused by viruses showing broad antigenic diversity such as foot-and-mouth disease virus or bluetongue virus; in this latter case it is even more difficult due to the fact that the infection is transmitted by a culicoides vector from the family ceratopogonidae (biting midges). it took two years in northern europe before inactivated vaccines against serotype 8 of bluetongue virus were available [3] . in northern america, the spectacular spread of west nile virus infection, another vector transmitted disease, in humans and horses, was rapidly followed by the development of several vaccines, including a dna-based vaccine for horses. one solution to be ready to vaccinate in face of an outbreak of a re-emerging infection is to stockpile vaccines as exemplified by foot-and-mouth virus vaccines as concentrated antigens. stockpiling is also envisaged for the possible pandemic of avian influenza h5n1 in humans [4] (pandemic pre* tel.: +33 1 44151859; fax: +33 1 42670987. e-mail address: pp.pastoret@oie.int. paredness) or to mitigate the risk of bio-agro-terrorisms. for time being it seems more appropriate to combat the h5n1 influenza infection at the animal source to reduce human exposure. to prevent nipah virus (henipavirus) infection in pigs a vaccine has recently been developed but, unfortunately, in countries like bangladesh, humans are directly infected by the reservoir, a fruit bat species. animals may be vaccinated against certain infections not for their own sake, but to prevent human contamination. one of the best example being wildlife vaccination against terrestrial rabies by the oral route using baits [5] . animal vaccination may also be used to prevent food poisoning in humans; a vaccine against escherichia coli 0157:h7 has for example recently been conditionally approved for cattle in the united states. the changes following globalisation, climatic change [6, 7] , and the opening of previously closed ecosystems, have considerably modified the pattern of endemic (or enzootic) infections/diseases, and contributed to the emergence of new agents that are pathogenic for humans and domestic animals. emerging infections is a collective name for infections that have been identified and taxonomically classified recently. in humans, in the final quarter of the twentieth century, more than 30 such conditions were recognised [8] . zoonoses are defined as infectious diseases that can be transmitted naturally between humans and wild or domestic animals. these infections are particularly important in the context of emerging infectious diseases of humans as the majority of these are of zoonotic origin; a comprehensive review by cleaveland et al. [9] identified 1415 species of infectious organisms known to be pathogenic to humans, including 217 viruses and prions, 538 bacteria and rickettsia, 307 fungi, 66 protozoa and 287 helminths. out of these, 868 (61%) were classified as zoonotic and 175 pathogenic species were considered to be associated with emerging diseases. of 175 emerging pathogens of this group, 132 (75%) were zoonotic [40] , the vast majority of which coming from wildlife. wildlife obviously constitutes an important potential of new pathogenic agents for humans and domestic animals [10] . this paper will mainly focus on viruses, mammals and birds. nowadays, the total number of viruses identified reaches approximately 5000 species [11] , but the likely number could exceed 130,000 according to the first estimates. even this number is most probably an underestimate (e.g. a well-known mammal species like human beings harbours at least 8 different herpesviruses, and 5 different herpesviruses have been identified in cattle until now), but if one takes into account the estimated number of 130,000, only 4% of viruses have already been identified. moreover, it does not take into account their extreme variability, particularly rna viruses, leading to populations of quasi-species. if one considers that there are 5416 recognised mammal species and that, for instance, herpesviruses have been isolated from all classes of vertebrates and even from oysters, one must admit that the world of viruses is huge. the use of viral metagenomics will help to identify more viruses [12] . for mammalians, 5416 different species have already been recognised, whereas the expected number of species is estimated to be around 5500; for mammalian species we are, therefore, nearly at the end of the inventory, since 99% of the species are already known. the inventory of mammalian species was first established in 1982, when only 4170 species were recognised; the same inventory established in 1993 contained 4629 different species. in 2005, as already mentioned, the complete list of mammal species consisted of 5416 species [13] . this increase in number seems to be paradoxical and even contradictory if one takes into account the extinction of some species during the same period of time. this increase in number can be accounted for when one considers that each phenotype of newly discovered species is listed separately and, more importantly, that the advent of modern molecular technology allows for the discrimination of species according to their genotypes and increasingly detailed comparisons of species limits and evolutionary relationships (taxonomic revision). among mammals, there are 2277 species of rodents pertaining to 481 genera. since 1993, 128 new rodent species have been recognised. the rodents therefore compose 42% of recognised mammal species. this number is particularly important if one takes into account the fact that the order of rodents harbours, and is the reservoir of numerous zoonotic infections. among the most spectacular are hantaviroses [14] ; some african sciuridae species (funisciurus spp., heliosciurus spp.), are the reservoir of monkeypox [15] . recently, the introduction of one of these species into the united states nearly provoked an ecological disaster, due to the transmission of the virus to indigenous rodent species (prairie dogs) [16] . to date, the order of chiroptera contains 1116 species, pertaining to 202 genera; 49 new species have been identified since 1993. bats make up therefore 20.6% of the total number of mammal species. this is worrying, since bats have been the source of many emerging diseases, many of them being previously unknown. for instance, insectivorous and frugivorous bats are the reservoir of the archeolyssaviruses, from which all lyssavirus strains derive, even the strains responsible for terrestrial rabies. frugivorous bats are the reservoir of newly discovered viruses such as nipah and hendra (henipavirus), responsible for numerous human fatalities, of the coronavirus responsible for the epidemics of the severe acute res-piratory syndrome (sars) and, most probably, of filoviridae such as the virus responsible for ebola disease in africa. there are approximately 10,000 species of birds. in 1990, 9672 species were recognised, pertaining to 2058 genera [17] , 5712 of which are passerines (1162 genera) and 3960 are non-passerines (896 genera). birds, previously dinobirds, descend from dinosaurs and are therefore further removed from us than mammals but they are, nevertheless, reservoirs of zoonotic infections. the recent epidemics of west nile virus infection in the americas is a good example. the problem arising from highly pathogenic avian influenza, particularly its strain h5n1, is equally worrying, since avian infection, which is already zoonotic, may be responsible for a new human pandemic, similar to the one following the first world war. the virus responsible for this pandemic, which caused more human fatalities than the war itself, was recently reconstituted [18] [19] [20] and is highly virulent when inoculated to non-human primates. it is noteworthy that wildlife biodiversity hot-spots are mainly found in tropical and sub-tropical regions, such as sub-saharan africa, indonesia and south america. through selection, man has created a number of different breeds of domestic animals, e.g. there are approximately 700 recognised breeds of cattle worldwide [21] , but many of these are on the verge of extinction (less than 100 breeding cows). there is therefore currently a swift erosion of genetic variability in cattle that is really worrying. there are more than 300 recognised dog breeds, showing a remarkable phenotypic and genotypic variability. for instance a survey of the adaptive humoral response of different dog breeds following vaccination against rabies within the british pet scheme showed that there was a significant variation in response between breeds after vaccination [22] . there obviously exists a large variation of responses between breeds after vaccination which could be used for the selection, marker assisted or not, of good or bad responders to vaccination. poultry lines selected according to their humoral adaptive immune response have already been obtained [23] . as a matter of fact, the differences in the susceptibility of breeds to some infections or infestations has been well observed by breeders, for instance the "resistance" of the n'dama cattle breed to trypanomosis. the mechanisms allowing emergence or re-emergence of infections are numerous [24] . a key factor is the extreme variability of viruses (particularly rna viruses) leading to generation of populations of quasi-species, which allows them to easily cross the species barrier. viruses evolve far quicker than their hosts, by several mechanisms: point mutations, deletions, recombination, reassortment and acquisition of cellular genes. moreover, viruses have co-evolved with their natural hosts, often leading to unapparent infections. in animal health, the spectacular emergence of canine parvovirosis in 1978, which resulted in a disastrous epizooty within the dog population worldwide, was the result of a mutation of another parvovirus which is responsible for feline panleukopaenia [1, 25] , whereas dogs and cats were living peacefully before, without inter-specific transmission. another source of emergence is the opening of previously closed ecosystems, which leads to new contacts between unrelated species and shows that a species previously unknown to be susceptible to an infection (because of the lack of opportunity to be infected) is in fact fully susceptible. a recent illustration is given by the emergence of bluetongue serotype 8, in northern europe, transmitted by a culicoides vector culicoides dewulfi previously unknown to be susceptible to the infection [26] . invasive species or migratory species may also be responsible for emergence, as could be the deliberate or accidental release of foreign species in a new environment, as exemplified by the introduction of monkeypox virus in the united states. other possible sources of emergence include veterinary biologicals, climate change and globalisation with its 5 ts (trade, transport, travel, tourism and terrorism). in animal health, in face of an emergency, there still exist two possibilities, either mass slaughtering of animals or vaccination. unfortunately vaccines are not always available; for instance in face of an outbreak of african swine fever and in the absence of a vaccine, the only solution is to kill the infected animals as quickly as possible, and to destroy the carcasses, in order to avoid the transmission of infection to uninfected premises. anyway, the pigs will die from a disease which provokes nearly 100% mortality. it is even more true when facing a really emerging disease that moreover is zoonotic such as nipah virus infection [27] for which no vaccine was available yet, because the causative agent was previously unknown; the only solution is once again to kill and destroy the infected and in-contact animals. a vaccine has recently been developed to prevent nipah virus infection in pigs [28] ; unfortunately in countries like bangladesh, man is mainly at risk due to direct contact with the reservoir, a fruit bat, or contact with its secretions. in other cases, when re-emergence of a previously well-known infection and when a vaccine is already available one may have the choice; either slaughtering or vaccination [39] . foot-and-mouth virus infection gives an excellent example. footand-mouth virus is represented by seven serotypes, further divided into numerous sub-types. preventive vaccines are available as highly purified concentrated antigens stockpiled in liquid nitrogen [29] ; being highly purified, they allow the differentiation between vaccinated or infected animals (even if previously vaccinated) thanks to a companion diagnostic test based on the detection of antibodies directed against non-structural proteins. unfortunately this technology only allows certification of freedom at the herd level. in the two recent outbreaks in united kingdom, the choice was to slaughter the animals. following the dramatic outbreak of foot-and-mouth disease in united kingdom, and to a lesser extend in france and in the netherlands, the european union lightened its regulation and is nowadays more prone to consider emergency vaccination as an alternative to slaughtering. if preventive vaccines are used in an emergency situation they could still be improved by conferring an early onset of protection. whenever viruses are represented by several serotypes, an infection can re-emerge in a previously vaccinated population against another serotype than the wild circulating one. for instance, an influenza pandemic can emerge in a human population with a herd immunity against seasonal flu. in case of highly pathogenic avian influenza (particularly strain h5n1), one may choose to kill the birds or to vaccinate depending on the situation [30] . there are some instances where vaccination is the only reasonable option, particularly when facing arthropod-borne virus infections. among arthropod-borne infections, there are infections caused by viruses represented only by one serotype, such as west nile virus or rift valley fever virus and others caused by viruses presenting multiple serotypes such as bluetongue virus. the majority of animal infections involve only two partners: the pathogen and the host, which has at its disposal its genetic background (natural resistance to infection) [31] and its immune system. besides these infections or infestations, some vectorial infections are arthropod-borne and mainly transmitted by biting arthropod. these infectious systems are therefore more complex, because the vectors must be competent and able to multiply the pathogenic agent. in northern america there was the spectacular spread of west nile virus infection in humans and horses, rapidly followed by the development of several vaccines for horses, including a dna-based one. vaccination seems to be the only option since the reservoir is to find among birds and the infection is transmitted by mosquitoes. it is therefore nearly impossible to control the infection by other mean than vaccination without highly detrimental effect on the environment [32] . rift valley fever is expanding its range in africa [33] . until it was introduced into saudi arabia and yemen in 2000; rift valley fever tended to be confined to sub-saharan africa. the disease has recently occurred in madagascar. as of july 2008, at least 20 people reportedly died as a result of the infection, and the disease has claimed the lives of thousands of animals since the beginning of the year 2008. since the disease is transmitted by mosquitoes, extreme weather events might create the necessary conditions for rift valley fever to expand its geographical range northwards and cross the mediterranean and arabian seas, with an unexpected impact on the animal and human health of newly affected countries [34] . once again, vaccination is the best way to prevent the disease; an attenuated vaccine exists for sheep but is still abortigenic and should be improved. an arthropod-borne disease caused by a virus with multiple serotypes is typically bluetongue (24 serotypes) and perhaps a new one [35] . until 2006, bluetongue was only observed in the mediterranean regions of europe and only serotypes 1, 2, 4, 9, and 16 were involved. the disease appeared unexpectedly in northern europe in 2006 and serotype 8 was involved, which is typically a sub-saharan serotype. cattle and sheep herds were fully susceptible to the infection; moreover, the strain involved was particularly virulent in cattle. bluetongue is transmitted by a biting midge, a member of the family ceratopogonidae, the culicoides. in the mediterranean region, the main species involved in the transmission is culicoides imicola, which originated in africa and asia and extended its range towards the north of its previous distribution, probably due to climate change. however climatic change does not seem to be responsible for the extension of the infection in northern europe, since the main culicoides species involved is culicoides dewulfi, a typically nordic species, whose transmission competence was unknown until bluetongue appeared in northern regions; in fact, this potential vector competence had not previously had the opportunity to be expressed due to the lack of bluetongue virus. the biology of the larval stage of culicoides impedes the control of the vector without damaging the environment. the most sensible option is vaccination of domestic ruminants with an inactivated vaccine containing serotype 8. in developed countries, partly as a result of overproduction, public concern for food security has been replaced by a major concern about food safety [36] . this concern has increased following the bse (bovine spongiform encephalopathy) crisis. people are concerned about food-borne infections, the presence of drug residues following treatment of food-producing animals and the possible transfer of antibiotic resistance from bacteria causing disease in livestock to those which affect man [37] . veterinary vaccines may help to solve some of these problems. the best example of a veterinary vaccine used for public health purposes is the vaccination of wildlife against rabies; the primary goal was not to protect wildlife species from rabies but to prevent human exposure and the disease in human populations [5, 38] . being considered as products working by natural mechanisms, vaccines, except for some of their excipients, do not need to have an mrl (maximum residue limit) determination associated with a withdrawal period. in fact, since vaccine protection works after a lag period, the use of vaccines intrinsically contains a withdrawal period. veterinary vaccines can be used to prevent food poisoning as demonstrated by the "in ovo" vaccination of poultry against salmonellosis, in order to decrease carcass contamination. more recently a vaccine against escherichia coli 0157:h7 has been conditionally approved for cattle in the united states. a vaccine against sheep cysticercosis has been developed experimentally and may lead to the development of similar vaccines to control bovine cysticercosis and thus taenia saginata infestation in humans. bacterial resistance to antibiotics is an emerging problem for both the animal and public health sector. several antibacterial vaccines used in veterinary medicine disappeared after the second world war, and were replaced by the use of antibiotics. the resistance to antibiotics in the animal health sector with possible implications for human health, as well as the resistance of several parasites to anthelmintics may lead to the reappearance or the appearance of antibacterial and antiparasitic vaccines. even if other pathways such as the selection of food-producing animals for genetic resistance to diseases are followed, the story of marek's disease in chickens demonstrates that vaccines are often more economical to procure an animal's resistance to pathogens. canine haemorrhagic enteritis: detection of viral particles by electron microscopy frã©quence en belgique de l'infection ã  parvovirus chez le chien, avant et aprã¨s l'observation des premiers cas cliniques bluetongue in northern europe. world animal health publication stockpiling prepandemic influenza vaccines: a new cornerstone of pandemic preparedness plans largescale eradication of rabies using recombinant vaccinia-rabies vaccine climate change and biodiversity how the biodiversity sciences may aid biological tools and ecological engineering to assess the impact of climatic changes all creatures great and minute: a public policy primer for companion animal zoonoses diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergency infectious diseases: preparing for the future virus taxonomy; eight report of the international committee on taxonomy of viruses. virology division international union of microbiological societies viral metagenomics mammal species of the world-a taxonomic and geographic reference monkeypox outbreak traced to wisconsin pet dealer distribution and taxonomy of birds of the world characterization of the reconstructed 1918 spanish influenza pandemic virus the 1918 flu virus is resurrected aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus cattle breeds, an encyclopedia. doetinchem: misset uitgeverij factors influencing the antibody response of dogs vaccinated against rabies genetic and phenotypic correlation between antibody response to escherichia coli, infectious bursa disease (ibdv), and newcastle disease virus (ndv), in broiler lines selected on antibody response to escherichia coli la faune sauvage et les maladies ã©mergentes the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses regulatory issues surrounding the temporary authorisation of animal vaccination in emergency situations: the example of bluetongue in nipah virus infection of pigs in peninsular malaysia recombinant nipah virus vaccines protect pigs against challenge antigen and vaccine bank: technical requirements and the role of the european antigen bank in emergency foot and mouth disease vaccination control strategies for highly pathogenic avian influenza: a global perspective animal genomics for animal health west nile virus and north america: an unfolding story rift valley fever the impact of climate change on the epidemiology and control of rift valley fever genetic characterization of toggenburg orbivirus, a new bluetongue virus, from goats in switzerland. emerging infectious diseases, personal communication veterinary vaccines for animal and public health initiative aims to merge animal and human health science to benefit both the development and use of a vaccinia-rabies recombinant oral vaccine for the control of wildlife rabies: a link between jenner and pasteur risk factors for nipah virus encephalitis in bangladesh ecological sources of zoonotic diseases key: cord-312797-hohzjx74 authors: hamelin, marie-ève; abed, yacine; boivin, guy title: human metapneumovirus: a new player among respiratory viruses date: 2004-04-01 journal: clin infect dis doi: 10.1086/382536 sha: doc_id: 312797 cord_uid: hohzjx74 the human metapneumovirus (hmpv) is a newly described member of the paramyxoviridae family belonging to the metapneumovirus genus. since its initial description in 2001, hmpv has been reported in most parts of the world and isolated from the respiratory tract of subjects from all age groups. despite the fact that prospective and case-control studies have been limited, the epidemiology and clinical manifestations associated with hmpv have been found to be reminiscent of those of the human respiratory syncytial virus, with most severe respiratory tract infections occurring in infants, elderly subjects, and immunocompromised hosts. additional research is needed to define the pathogenesis of this viral infection and the host's specific immune response. part of the pneumovirinae subfamily within the paramyxoviridae family. initial electron microscopic examination of hmpv isolates revealed morphological characteristics consistent with paramyxoviruses. pleomorphic, spherical, and filamentous particles could be observed (figure 1) [6] . spherical enveloped particles vary in size, with a mean diameter of 209 nm. the nucleocapsid has a length varying from !200 to ∼1000 nm and a diameter of 17 nm. hmpv isolates were initially found to grow on tertiary monkey kidney and llc-mk2 (rhesus monkey kidney) cells, with poor or no viral growth on other cell lines, including madin darby canine kidney, vero (african green monkey kidney), human laryngeal carcinoma (hep-2), human foreskin fibroblast, human rhabdomyosarcoma, transformed human kidney (293), human lung adenocarcinoma (a-549), and human colon adenocarcinoma (ht-29) [4, 7] . in addition, hmpv isolates did not exhibit hemagglutinating activity when tested with human, turkey, chicken, or guinea pig rbcs, which is consistent with other members of the pneumovirinae subfamily [4, 7] . intranasal inoculation of ferrets and guinea pigs with hmpv isolates did not cause any clinical symptoms [4] . also, experimentally infected birds (juvenile turkeys and chickens) did not show clinical signs or virus replication over a 3-week period. on the other hand, there was evidence of efficient hmpv repfigure 1 . negative-stain electron micrographs of human metapneumovirus (hmpv). center image shows 5 pleomorphic hmpv particles; note the projections along the periphery of the viruses. upper right and lower left insets show the nucleocapsid and the filamentous rodlike particle, respectively. staining was done with 2% phosphotungstic acid. bar markers represent 100 nm. from [6] ; reproduced with permission from the university of chicago press. lication in the respiratory tract of experimentally infected monkeys (cynomolgus macaques) associated with mild upper respiratory tract signs [4] . thus, in contrast to apv, hmpv is a respiratory pathogen of primates. the hmpv genome consists of a single negative strand of rna of ∼13 kb containing genes coding presumably for a nucleoprotein (n), phosphoprotein (p), matrix (m) protein, fusion (f) protein, transcription elongation factor/rna synthesis regulatory factor (m2), small hydrophobic (sh) surface protein, major attachment (g) glycoprotein, and major polymerase (l) subunit, arranged in the order 3 -n-p-m-f-m2-sh-g-l-5 , similar to apv [8, 9] . two major differences exist between hmpv and hrsv genomes: the gene order is different, and hmpv does not contain nonstructural genes (figure 2) [10] . the possible relevance of the absence of these viral proteins, which have been associated with ifn antagonism by hrsv [11] , deserves additional study. for hrsv, the g and f surface proteins are known as the major protective antigens [11] , and this also needs to be verified for hmpv. phylogenetic analyses have demonstrated that, among members of the pneumovirinae subfamily, hmpv was most closely related to apv serotype c, the avian metapneumovirus that emerged in the united states in the late 1990s. for instance, the amino acid sequence identity between hmpv strains and apv-c and hrsv representatives varies from 66% to 89% and from 23% to 43%, respectively, when comparing the n, p, m, and f genes [4, 12] . the sh and g amino acid sequences of hmpv show important variability and share only 28% and 21% identity with their respective apv-c counterparts [8, 13, 14] . it is possible that this important divergence may account for the specific host range of these 2 related metapneumoviruses. genetic analysis on a large number of hmpv isolates has identified 2 major groups and 2 minor genetic clusters within each group [6, 7] . however, additional investigations are still required to determine whether these genotypes represent different antigenic groups. recently, the complete nucleotide sequences of hmpv isolates belonging to these 2 major groups were determined [9] . nucleotide and amino acid sequence identities between the 2 hmpv groups were found to be 80% and 90%, respectively, which is similar to what has been reported with hrsv a and b genotypes. since its initial report by the dutch researchers in 2001, hmpv has been found in most parts of the world, with reports from north america (united states and canada), europe (united kingdom, france, germany, italy, spain, and finland), asia (hong kong and japan), and australia (table 1). the virus has also been identified in hiv-infected and nonimmunocompromised children from south africa [15] . the few seroprevalence surveys from the netherlands [4] , japan [16] , and israel [17] have indicated that virtually all children are infected by 5-10 years of age. in addition, studies have shown that hmpv is not a new pathogen, with serological evidence of human infection dating from 1958 in the netherlands [4] and viral isolation for the past 10-20 years in europe and canada [4, 7] . cases of severe hmpv infection in adults [7] and of reinfection in immunocompromised subjects [18] suggest that, despite universal infection in childhood, new infections can occur throughout life due to incompletely protective immune responses and/or acquisition of new genotypes. surveys have indicated that hmpv has a seasonal distribution overlapping hrsv circulation, with most cases reported during the winter/ early spring months (table 1) . although most studies have limited their surveillance to the typical respiratory virus period, our group has shown that 87% of hmpv isolates recovered in cell culture in quebec, canada, were recovered during the period of december through may [7] . in addition, we found that outbreaks of hmpv infection tend to peak in early spring over a 4-6 week period, slightly later than outbreaks of hrsv infection, which also are more spread out in time [19] . however, additional studies over multiple years are needed to better define the seasonality of hmpv infection. the role of hmpv as the cause of arti has been evaluated in many studies, mostly using molecular detection methods (table 1) . young hospitalized children have been best studied, and hmpv has been generally found in 5%-10% of arti cases in that population. however, in one study from italy, the number of hmpv infections varied widely over a 3-year period and were associated with 7%-43% of hospitalizations for arti [20] . the relative role of hmpv in respiratory syndromes of adults has been less studied. in one study from rochester, new york, hmpv was detected by serological tests and/or rt-pcr in 4.5% of young and elderly adults with arti [21] . interestingly, evidence of hmpv infection was also found by serological testing in almost the same percentage (4.1%) in asymptomatic adults, raising the question of the causative role of hmpv in arti in adults. recent studies by our group indicate that hmpv is rarely identified by rt-pcr in npas obtained from asymptomatic young children, with a rate significantly lower than that of subjects with arti (!1% vs. 6%) [19, 22] . it is interesting to note that the rates of detection of hmpv have been generally higher in retrospective than prospective studies (table 1) , an observation consistent with some selection bias. thus, to better define the role of hmpv in various respiratory conditions, large prospective studies using appropriate controls need to be conducted. in addition, testing of all clinical samples (i.e., not only those samples that are found to be negative for other viruses) must be performed. nevertheless, most studies have so far indicated that hmpv is a frequent cause of severe arti, especially in young children. table 2 summarizes the clinical findings of the first 28 hmpvinfected canadian subjects retrospectively identified through our virology laboratory. symptoms of both upper and lower respiratory tract infections have been associated with hmpv in young children, although most reports are biased towards description of the most severe symptomatology in hospitalized subjects. in young hospitalized children, the clinical features associated with hmpv infections are very similar to those of hrsv [19, [23] [24] [25] . diagnoses of bronchiolitis, with or without pneumonitis, have been most commonly reported. a substantial proportion (up to 50%) of infected children also has concomitant otitis media [19] . acute wheezing and asthma exacerbation have been associated with hmpv in some [23, 26] but not all [27] studies. compared with hrsv infections, hmpv cases tend to occur at an older age [19, 26, 28] . the clinical outcome after hrsv infection has been more severe than that after hmpv infection when looking at the proportion of patients with hypoxemia, those with pneumonia, and those admitted to the intensive care unit in studies from canada [19] and europe [25, 28] . in contrast, researchers from hong kong found that hmpv infection was associated with a longer hospital stay and more cases of pneumonia than was hrsv infection [26] . the latter group reported an estimate of 442 hmpvassociated hospitalizations per 100,000 children aged р6 years. children with underlying medical conditions may have moresevere hmpv disease, leading to hospitalization. in studies from north america, 25%-33% of hmpv cases occurred in children with underlying conditions, such as prematurity, cardiopulmonary problems, and immunosuppression [7, 29] . hmpv has been associated with flulike illnesses and colds in healthy adults (table 2) [7] . stockton et al. [30] identified hmpv in 9 (2.2%) of 408 nasal and throat swabs obtained from subjects with flulike illnesses (i.e., patients who tested negative for influenza and hrsv rna) seen by general practitioners in england. eight of the 9 infected patients in that study were adults, and 6 of these patients also had clinical evidence of lower respiratory tract involvement. falsey et al. [21] found a higher rate of hmpv illness among young adults, although older adults experienced more dyspnea and wheezing than did younger adults, and those with cardiopulmonary conditions were ill for nearly twice as long as younger adults. among 10 retrospectively identified, infected patients aged 165 years who were hospitalized at our institution, 4 developed pneumonitis and 2 died (table 2) [7] . of note, all 10 patients had у1 underlying illness, such as leukemia/lymphoma and chronic cardiovascular, pulmonary, or neurologic diseases. collectively, available data in-dicate that the clinical presentation of hmpv is very similar to that of hrsv, with more severe outcomes in infants, elderly subjects, and persons with underlying diseases [31] . the detection of hmpv in special settings merits further discussion. preliminary data suggest that, similar to hrsv, hmpv infection may have a more fulminant course in severely immunocompromised individuals. our group reported the death of a 17-month-old girl with acute lymphoblastic leukemia due to severe pneumonitis and respiratory insufficiency [18] . although no autopsy was performed, an npa obtained before death revealed the presence of hmpv only. of interest, this child had presented with a first episode of bronchiolitis caused by hmpv 1 year earlier, and the 2 viral isolates were of different lineages (groups), highlighting the possibility of rapid reinfections in immunocompromised hosts. similarly, hmpv was the sole pathogen identified in the npa obtained from a hematopoietic stem cell transplant recipient who died of progressive respiratory failure after an upper respiratory prodrome [32] . the presence of a copathogen may also lead to more-severe hmpv infections. a report from england identified hmpv in 21 (70%) of 30 bronchoalveolar fluid specimens obtained from infants with severe hrsv bronchiolitis requiring ventilatory support, raising the possibility that hmpv might be a determinant of hrsv disease severity [33] . unfortunately, no data regarding dual infections in less severe hrsv disease were available for comparison. in contrast, the rate of bronchopneumonia was not altered by the presence of a second respiratory virus in italian infants with hmpv infection [20] . finally, the possible synergistic effect between hmpv and a new coronavirus has been recently postulated during outbreaks of severe acute respiratory syndrome (sars) in canada and hong kong [34, 35] . in a study of 48 patients with probable sars in hong kong, chan et al. [34] found evidence of hmpv infections by nested pcr in 25 cell cultures (52%) inoculated with npa samples. in fact, hmpv was more frequently isolated than the sars coronavirus in the latter study. this report is surprising, considering the results of experimental infections in macaques, in which severe multifocal pulmonary consoli-dation was induced by the sars coronavirus only, with no exacerbation after subsequent infection with hmpv [36] . hmpv growth in cell culture is fastidious; this may be one reason for its late identification. most studies have reported reliable cytopathic effects only in tertiary monkey kidney or llc-mk2 cells [4, 6, 7] . the cytopathic effect is variable, with some strains inducing hrsv-like syncytia formation and others inducing focal rounding and cell destruction ( figure 3) . typically, the cytopathic effect is displayed more than 10-14 days after inoculation (mean time was 17 days in our laboratory). confirmation of hmpv cytopathic effect is achieved by rt-pcr testing of infected supernatants in the absence of commercially available antibodies. a recent report by chan et al. [34] indicated that hmpv could replicate more efficiently in human laryngeal carcinoma (hep-2) cells, despite the absence of cytopathic effect. in that instance, presence of hmpv was confirmed by direct testing of hep-2 cell culture supernatants by rt-pcr or subsequent passage on llc-mk2 cells to observe cytopathic effect. because of the unavailability of rapid antigen detection tests and because of the slow and restrictive viral growth, rt-pcr has become the method of choice for the diagnosis of acute hmpv infection. most pcr protocols reported to date have relied on amplification of the l, n, or f gene (reviewed in [37] ), with primer sequences mainly derived from the prototype strain 001 from the netherlands (genbank accession number af371337). because of the existence of 2 hmpv lineages with significant genetic variability within each group, hmpv detection may be underestimated when inadequate primers are selected for pcr amplification. we have recently reported that rt-pcr assays targeting the n and l genes, which code for 2 internal viral proteins, are best suitable for hmpv diagnosis [37] . rapid and sensitive hmpv assays based on the real-time pcr methodology have recently been described, allowing amplification and detection of this pathogen in р2 h [37, 38] . serological testing only permits a retrospective diagnosis. because infection is almost universal in childhood, a seroconversion or a у4-fold increase in antibody titers must be demonstrated to confirm recent infection. the few serological surveys for hmpv were based on an indirect immunofluorescence assay using hmpv-infected cells [4, 16] . a home-brew elisa method has also been developed using cell lysates of hmpv [21] . clearly, simpler elisa tests using viral proteins possibly derived from the 2 main groups are needed to conduct large serological surveys in many parts of the world. no vaccines, chemotherapeutic agents, or antibody preparations are currently approved for the prevention or treatment of hmpv infection. recently, wyde et al. [39] showed that ribavirin and a polyclonal intravenous immunoglobulin (ivig) preparation had equivalent in vitro activity against hmpv and hrsv. in contrast, no activity against hmpv was conferred by palivizumab, a humanized monoclonal antibody directed against the f protein of hrsv. despite the unavailability of animal studies and the toxicity related to ribavirin administration, the combined use of ivig and ribavirin could be envisaged for treating severe hmpv infection in immunocompromised patients, as has been reported for hrsv infection [40, 41] . furthermore, until an effective vaccine is developed, another interesting strategy could consist in the development of hightitered ivig preparations with activity against hmpv. the recent identification of a presumably old viral pathogen is an exciting development in the field of respiratory viruses. available data indicate that hmpv appears to be a significant cause of both upper and lower respiratory tract infections in young children. hmpv reinfections seem to be frequent and could also lead to devastating complications in elderly subjects and immunocompromised hosts, although more studies with adequate control groups are needed to confirm this. overall, the epidemiological and clinical features of hmpv infection appear to be similar to those of hrsv, although differences have been noted. it is important to mention that many fun-damental questions related to viral pathogenesis and the host's specific immune response still remain to be answered. the tucson children's respiratory study ii: lower respiratory tract illness in the first year of life etiology of community-acquired pneumonia: impact of age, comorbidity, and severity prospective comparative study of viral, bacterial and atypical organisms identified in pneumonia and bronchiolitis in hospitalized canadian infants. pediatr a newly discovered human pneumovirus isolated from young children with respiratory tract disease metapneumoviruses in birds and 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infection in young children and genetic heterogeneity of the viral isolates children with respiratory disease associated with metapneumovirus in hong kong asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children human metapneumovirus as a cause of community-acquired respiratory illness human metapneumovirus infection in the canadian population human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease human metapneumovirus in severe respiratory syncytial virus bronchiolitis human metapneumovirus detection in patients with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada koch's postulates fulfilled for sars virus comparative evaluation of real-time pcr assays for detection of the human metapneumovirus molecular assays for detection of human metapneumovirus comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro prevention and treatment of respiratory syncytial virus and parainfluenza viruses in immunocompromised patients combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients evidence of human metapneumovirus in australian children human metapneumovirus and community-acquired respiratory illness in children human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children key: cord-286328-ap0wfjhq authors: lewis, toby c.; henderson, tiffany a.; carpenter, ashley r.; ramirez, ixsy a.; mchenry, christina l.; goldsmith, adam m.; ren, xiaodan; mentz, graciela b.; mukherjee, bhramar; robins, thomas g.; joiner, terence a.; mohammad, layla s.; nguyen, emily r.; burns, mark a.; burke, david t.; hershenson, marc b. title: nasal cytokine responses to natural colds in asthmatic children date: 2012-11-26 journal: clinical & experimental allergy doi: 10.1111/cea.12005 sha: doc_id: 286328 cord_uid: ap0wfjhq background: the mechanisms by which viruses induce asthma exacerbations are not well-understood. objective: we characterized fluctuations in nasal aspirate cytokines during naturally-occurring respiratory viral infections in children with asthma. methods: sixteen children underwent home collections of nasal aspirates when they were without cold symptoms and again during self-reported respiratory illnesses. the presence of viral infection was ascertained by multiplex pcr. cytokines were measured by multiplex immune assay. mrna expression for selected markers of viral infection was measured by rt-pcr. a cumulative respiratory symptom score was calculated for each day of measurement. generalized estimated equations were used to evaluate associations between viral infection and marker elevation, and between marker elevation and symptom score. results: the 16 patients completed a total of 37 weeks of assessment (15 “well” weeks; 22 self-assessed “sick” weeks). viral infections were detected in three of the “well” weeks and 17 of the “sick” weeks (10 rhinovirus, 3 coronavirus, 2 influenza a, 2 influenza b, 2 respiratory syncytial virus, 1 parainfluenza). compared to virus-negative well weeks, nasal aspirate ifn-γ, cxcl8/il-8, cxcl10/ip-10, ccl5/rantes, ccl11/eotaxin-1, ccl2/mcp-1, ccl4/mip-1β, ccl7/mcp-3 and ccl20/mip3α protein levels increased during virus-positive sick weeks. only a subset of cytokines (ifn-γ, cxcl8, ccl2, ccl4, ccl5 and ccl20) correlated with self-reported respiratory tract symptoms. while many aspirates were dilute and showed no mrna signal, viral infection significantly increased the number of samples that were positive for ifn-λ1, ifn-λ2/3, tlr3, rig-i and irf7 mrna. conclusions & clinical relevance: we conclude that, in children with asthma, naturally-occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, ifns and ifn-responsive genes. epidemiological studies have uncovered a strong association between viral infections, especially those caused by rhinovirus, and exacerbations of asthma attacks [1] [2] [3] . however, the mechanisms by which viruses induce exacerbations of chronic airways disease are not well understood. in response to viral infection, the airway epithelium, and to a lesser extent immune cells such as monocytes, macrophages, and dendritic cells [4] [5] [6] [7] , release pro-inflammatory cytokines, interferons (ifns), and antimicrobial substances, which in turn, promote clearance of microorganisms, and activation of the adaptive immune system. however, in patients with chronic respiratory illnesses, the innate immune response may also be responsible for disease exacerbation [8] . although the inflammatory response to experimental rhinovirus infection has been monitored [9] [10] [11] [12] [13] [14] , few studies have examined the innate immune response of patients with asthma to natural colds. increases in respiratory tract cxcl8/il-8, ccl5/rantes, ccl19/ mip-1a, il-10, ccl7/mcp-3, and ccl13/mcp-4 have been detected [15] [16] [17] [18] . to further examine the innate immune response to viral infection in children with asthma, we measured nasal aspirate cytokine levels in 16 asthmatic children before and after upper respiratory tract infections. in contrast to the previous studies, we examined at least three time points before and in association with symptomatic illness. we evaluated the associations between viral infection and cytokine expression, and between cytokine expression and symptom score. we also examined the effects of upper respiratory tract infection on mrna levels of selected markers of viral infection, including ifns. finally, we evaluated a new method of virus detection using a single polymerase chain reaction-ligation detection reaction (pcr-ldr) multiplex assay. we hypothesized that respiratory viral infection of children with asthma causes robust elaboration of pro-inflammatory cytokines and ifns, and that the level of pro-inflammatory cytokines correlates with symptom severity. we conducted an observational cohort study of 16 children with asthma. children were eligible for the study if they received care from a university of michigan physician for asthma, were aged 6-18 years, and lived within a 30 mile radius of ann arbor. outpatients were enrolled for 3-6 months, with the goal of assessing cytokines at baseline and in association with at least one viral-induced exacerbation during that period. initial consent and first evaluation was conducted at the university of michigan, but all subsequent evaluations were performed in the participant's home. this study was approved by the university of michigan institutional review board (approval number hum00018442). all clinical investigations were conducted according to principles expressed in the declaration of helsinki (http://www.wma.net). at the initial assessment, parents or guardians completed an extensive questionnaire, which included standardized questions about presence and frequency of asthma symptoms, an inventory of a child's asthma medications and current usage, and queries about environmental exposures, and child and family demographic information. baseline asthma severity was assessed using national asthma education and prevention program guidelines [19] incorporating an adjustment for asthma controller therapy use [20] . we performed home measurements of respiratory symptoms and collected nasal secretions (for detection of viral rna by pcr and host biomarkers by pcr and elisa) on 3 days during a week when children were healthy (not reporting upper respiratory tract infection or asthma symptoms), and again during a week when they experienced cold or flu-like symptoms. families were given a calendar and a simple respiratory symptom scale and asked to mark the level of their symptoms. we used a modified version of the respiratory symptom score developed by the child origins of asthma study [21] , which assessed fever, cough, nasal symptoms, wheezing, difficulty breathing, waking up at night with cough, and interference with usual activities. when the patient experienced a symptomatic respiratory illness, as defined by a symptom score of two or higher on a scale of 0-13, the family notified the study staff and scheduled a visit within 48 h of the beginning of symptoms (defined as day 1 of the illness). information regarding impact of respiratory symptoms on daily activities and recent use of asthma medicines was also collected. as noted above, study technicians visited the home every 2-3 days to retrieve additional nasal washing samples, for a total of three visits during the week following reporting of symptoms. the three specific days of the week selected for analysis were based on the convenience of the subjects and laboratory technician. although specimens were most often collected on days 1, 4, and 7 of a given week, specimen collection was sometimes compressed into a 5 or 6-day period. to collect a nasal lavage sample, we utilized a protocol developed by powell and shorr [22] and modified by james gern (university of wisconsin, personal communication). two squirts of isotonic 0.65% sodium chloride were instilled into the child's nostrils (estimated at < 1 ml per nostril) using a commercially available nasal saline spray (b.f. ascher, lenexa, ks, usa). the subject then blew their nose into a zippered plastic bag, and 3 ml of m4rt viral transport medium (remel, lenexa, ks, usa) was added. in general, samples were collected in the presence of study staff and were transported from homes to the laboratory within 3 h of collection in a cooler with ice packs. however, due to logistical issues scheduling home visits, families were also provided with kits for independent collection of nasal washings specimens at home. if a visit could not be scheduled within 48 h, families were instructed to collect the sample, double bag the sample, place in a tightly sealed collection box, and to keep it in the refrigerator until the staff could collect the sample. we required a 1 week interval between sample collections for sequential illnesses. nasal aspirates were homogenized using a handheld homogenizer to allow pipetting of viscous samples and frozen at à70°c to allow for batched analysis. viral rna and dna were extracted using a minelute kit (qiagen, valencia, ca, usa). samples were assessed for the presence of viral rna or dna via pcr using the seegene seeplex rv-12 detection kit (seegene, rockville, md, usa). this kit detects human parainfluenza viruses 1, 2, and 3, human metapneumovirus, human coronavirus 229e/nl63, and oc43, human adenovirus, influenza viruses a and b, human respiratory syncytial virus (rsv) a and b, and human rhinovirus a. we also analysed specimens for respiratory viruses using a novel polymerase chain pcr-ldr multiplex assay [23] . the original system, which was designed to detect influenza viruses, was expanded to include parainfluenza 1, 2, 3, 4a and 4b, coronaviruses 229e and oc43, influenza a and b, rhinoviruses a, b, and c, adenoviruses a-e, metapneumovirus, and rsv a and b. we have detected all the viruses included in this multiplex system in clinical samples (i.e. nasal aspirates), with the exception of the adenoviruses, for which we used laboratory positive controls. the viral sequences for the multiplex assay are listed in table 1 . protein levels of ifn-c, cxcl8/il-8, cxcl10/ip-10, ccl5/rantes, ccl11/eotaxin-1, ccl2/mcp-1, ccl4/ mip-1b, ccl7/mcp-3, ccl19/mip-3b, and ccl20/mip-3a were determined using a magnetic bead-based multiplex immune assay (bio-rad, hercules, ca, usa). our interest on the above cc chemokines was prompted by gene array results from rv1b-infected, ovalbumin-sensitized, and -challenged mice (data not shown). ifn-a and ifn-b levels were measured using standard elisa (r&d systems, minneapolis, mn, usa). aspirates were homogenized and mrna extracted as described above, using the rneasy or the rneasyplus kit (qiagen). mrna was analysed for cxcl8/il-8, cxcl10/ ip10, ccl5/rantes, ccl11/eotaxin, ifn-k1, ifn-k2/3, ifn-a, ifn-b, icam-1, tlr3, mda-5, rig-i, and irf7 using quantitative real time pcr using specific primers and probes. signals were normalized to gapdh. we assessed feno in exhaled breath on 3 days during a week when the child was well, and on at least 3 days during a viral infection. to measure feno, we employed the niox mino (aerocrine, new providence, nj, usa). because feno measurement can be influenced by diet and exercise, participants were asked to refrain from eating or drinking within 1 h of their exhaled breath assessment. triplicate samples were obtained to assess reliability. mean, standard deviation and interquartile range were used to describe measured values of nasal cytokine levels, nasal mrna, and symptom score before and during viral illnesses. box plots were used to represent the change in biomarkers at different points of the cold relative to the baseline. whiskers of the boxplots represent the minimum and maximum values. distributions of these continuous outcome variables were examined and appropriate transformations taken to achieve normality. the effects of viral illness, as defined by the seegene kit results, on biomarker protein levels, and of biomarker level on symptom score were determined using a generalized mixed models (proc mixed), with an auto-regressive correlation structure using sas statistical software (sas, cary, nc, usa). we evaluated and adjusted for relevant covariates such as age, gender, ethnicity/race, and nasal steroid use. other potential explanatory variables, such as family income, baseline asthma severity, tobacco smoke exposure, and oral antihistamine use were evaluated, but not included in final models as they were not significant predictors. for all mrna outcomes except ifn-b, levels were undetectable in a large number of samples. we therefore categorized our results into detectable/non-detectable levels, and analysed as a binary variable using the generalized estimating equation (gee) approach with the tobit link [24] . we analysed ifn-b mrna levels as a continuous variable using the gee approach with identity link. unadjusted models are shown. sixteen children with physician-diagnosed asthma were recruited. participants ranged in age from 6 to 16 years old, were 69% boys, 36% non-white, and reported symptoms or medication use consistent with persistent asthma ( table 2) . participants completed a total of 37 weeks of assessment ( table 3 ). the current report focuses on the 28 weeks where there was concordance between selfreported illness and viral detection using the seegene kit (16 virus-positive 'sick' weeks, and 12 virus-negative 'well' weeks). we re-tested eight positive and seven negative samples (by seeplex) using the pcr-ligation detection multiplex assay. twelve of 15 samples were concordant between the two assays. two samples were positive by seeplex and negative by pcr-ldr. one sample showed rsv by seeplex assay and coronavirus oc43 by pcr-ldr. symptom scores, which qualitatively evaluated the severity of illness, rose during the sick weeks (fig. 1a) . respiratory symptoms were highest at the first assessment during sick weeks, 1-3 days after initial report of illness. children with confirmed viral illnesses experienced significant increases in cough, wheeze, and chest tightness, indicative of asthma exacerbation (table 4) . selected nasal aspirate cytokines were measured using magnetic bead-based multiplex immune assay. there were significant increases in all cytokines examined during weeks of reported symptoms, with the single exception of ccl7 (table 5 , fig. 1b) . this pattern was more striking when samples with documented viral infections were compared to those confirmed to be virus-negative. unadjusted estimates of the association of confirmed viral infection and cytokine level were highly significant (not shown) and did not change appreciably when adjusted for age, gender, race, and nasal steroid use. changes in cytokines over the course of the illness are shown in fig. 2 . in most cases, cytokines increased during the first 2 days of infection and persisted throughout the week. we also examined the relationship between cytokines and symptom score. unadjusted estimates of the association of confirmed viral infection and cytokine level with symptom score were significant for ifn-c, cxcl8, ccl5, ccl20, ccl2, and ccl4 (table 6) . cxcl10, ccl11, ccl19, and ccl7 cytokine levels did not correlate with symptom score. we also attempted to measure ifn-a and ifn-b protein levels in the nasal aspirates by elisa. ifn-a levels over the lower detection limit (12.5 pg/ml) were found in only five of 116 specimens (all virus-positive weeks). for ifn-b, levels over the detection limit (25 pg/ml) were found in only 12 samples (five virus-negative, seven virus-positive). many aspirates were dilute and a number of specimens showed no mrna signal (table 7) . nevertheless, we detected transcripts for icam-1, cxcl-10, ifn-k1, cxcl-8, rig-i, mda-5, tlr3, ifnk2/3, irf7, ifn-a, and ifn-b from patients both before and during viral infections. we could not detect ccl5 and ccl11 mrna in any specimens. finally, infection significantly increased the number of samples that were positive for cxcl8, ifn-k1, ifn-k2/3, tlr3, rig-i, and irf7 mrna. we measured feno before and during virus-positive sick weeks (fig. 3) . there was no significant change in feno measurement with viral infection. viral infections are the most frequent cause of asthma attacks [1] [2] [3] . in theory, chemokine production by virus-infected epithelial cells induces the recruitment of inflammatory cells to the airways, which in turn elaborate cytokines and mediators capable of increasing airway responses. data suggest that immune cells may also be infected by respiratory viruses and produce chemokines [7, [25] [26] [27] [28] [29] [30] . in the present study, we found that, during natural colds, respiratory tract cytokine levels significantly increase in children with asthma. levels of a subset of cytokines correlated with the degree and time course of respiratory symptoms. the chemokines detected are responsible for recruitment of a broad array of inflammatory cells including neutrophils, monocytes, macrophages, and eosinophils. finally, we detected changes in nasal lavage specimens, which were collected at home on a repeated basis, both before and in association with infection. although we did not measure cytokine levels in normal subjects, these data show that children with asthma are apparently capable of a robust cytokine response to viral infection, even during mild exacerbations. in addition, home collection of nasal lavage specimens appears to be a practical tool for studying the natural history of viral infection in children. our results expand upon prior work characterizing cytokine responses detectable in respiratory secretions of asthmatics in association with viral illnesses. pizzichini et al. [15] found that, compared to 21 days after infection, sputum levels of cxcl8, eosinophil cationic protein and fibrinogen from six adult asthmatics were significantly elevated 4 days after the start of a coldinduced exacerbation. furthermore, cxcl8 levels correlated with the number of sputum neutrophils. teran et al. asthma [16] found that, compared to asymptomatic periods, levels of major basic protein, ccl5 and ccl19 were increased in the nasal aspirates of 26 children during acute viral-induced exacerbations. grissell et al. asthma [17] found that compared to a group of stable asthmatics, levels of ccl5 and il-10 were significantly elevated in the induced sputa of older children and adults suffering from acute viral-induced asthma exacerbations, along with neutrophils and eosinophils. cxcl8 and ccl19 were also increased, although not significantly. finally, santiago et al. [18] found that compared to control samples, ccl7 and ccl13 were increased in the nasal aspirates of asthmatic children 12 -24 h after upper respiratory tract infections. chemokine levels correlated with macrophages in the nasal aspirate and upper respiratory symptoms scores. in the present study, in which patients were used as their own controls, we confirmed the above changes in cxcl8, ccl5, ccl19, and ccl7, and provide new data showing significant increases in cxcl10, ccl11, ccl2, and ccl4. importantly, we found that increases in these chemokines were sustained throughout the week following viral infection. we also examined the relationship between cytokines and respiratory symptom score. interestingly, we found that some cytokines correlated with symptom score (ifn-c, cxcl8, ccl5 , ccl20, ccl2, and ccl4) whereas others did not (cxcl10, ccl11, ccl19 or ccl7). although we did not examine lower airway cytokine levels, it is tempting to speculate that the former set of cytokines is responsible for the acute asthmatic response to viral infection, whereas the latter set may modulate future immune responses. based on differences in ifn-b and ifn-k production between cells isolated from controls and asthmatic subjects [31] [32] [33] , it has been proposed that patients with asthma are prone to rhinovirus-induced exacerbations due to deficient ifn production on the other hand, other in vitro studies showed no differences in rhinovirus-induced gene expression in epithelial cells isolated from asthmatic and healthy subjects [34, 35] , and con-trol and asthmatic subjects experimentally infected with rhinovirus show no difference in respiratory tract viral titre or copy number [14] . we found that viral infection during natural colds significantly increased the levels of ifn-c protein and ifn-k mrna in the nasal aspirates. viral infection also significantly increased the expression level of rig-i and irf-7, each of which are inducible by type i ifns [36, 37] . together, these data suggest that asthmatic children are capable of a functional ifn response. on the other hand, we failed to detect ifn-a or ifn-b protein in nasal aspirates, and there was no significant change in mrna levels during natural colds. detection of type i ifns in respiratory secretions is made difficult by the low sensitivity of commercially available assays, physiologically low levels of these cytokines in biological fluids (particularly in fluids without sufficient numbers of plasmacytoid dendritic cells) and presence of natural inhibitors (e.g. soluble receptors). also, we could not compare the results from our experimental subjects to children without asthma. our finding that irf7 mrna is elevated after respiratory viral infection in children with asthma is validated by a recent study analysing patterns of gene expression in nasal lavage samples from children experiencing picornavirus-induced asthma exacerbations [38] . consistent with our previous work examining the requirement of irf7 for rhinovirus-induced gene expression in cultured airway epithelial cells [39] , coexpression analysis demonstrated irf7 to be a major hub connecting ifn-mediated responses in virus-induced asthma exacerbations. in this study, we piloted a new pcr-ldr-based method for respiratory virus detection. twelve of 15 samples were concordant between the two viral detection techniques; in two cases, samples were positive by seeplex and negative by pcr-ldr. the precise cause of the discrepancy between the two tests is not certain, but the seeplex test, which relies on visual detection of bands on agarose electrophoresis gels, may be liable to false positives. we also optimized our pcr-ldr test for maximum specificity, with the goal of avoiding false positives. this may have resulted in a reduction in sensitivity if copy number of viral nucleic acid was low. we measured the effect of natural colds on feno. previous studies have shown feno to be a reliable measure of eosinophilic airway inflammation, steroid responsiveness, and clinical control in children and adults with allergic asthma [40] [41] [42] [43] . feno levels have also been shown to increase during emergency steroid treatment of acute asthma exacerbations [44, 45] . in contrast, we found that despite increases in airway cytokines and respiratory symptoms, there was no increase in feno during natural colds in children relative to their baseline state. these data are consistent with the notion that airway inflammation in the context of viral-induced asthma exacerbations is different in character than that associated with chronic asthma. there are several limitations to our study. first, although nasal sampling has the advantage of increased safety and decreased participant burden, sputum speci-mens more closely reflect the response of the lower airways to viral infection. however, as noted above, levels of ifn-c, cxcl8, ccl5, ccl20, ccl2, and ccl4 significantly correlated with respiratory symptoms, including lower respiratory tract symptoms such as cough, wheeze, chest tightness, and shortness of breath, each of which are indicative of asthma exacerbation. in addition, previous studies have found similar changes in nasal aspirate and sputum cytokine concentrations following viral exacerbations of asthma [16, 17] . finally, observations demonstrate consistent effects of nasal and bronchial provocation [46] . second, we did not measure inflammatory cells in the nasal aspirates. we therefore could not assess the functional effects of chemokine release in our subjects. previous studies have shown recruitment of neutrophils, eosinophils, and macrophages to the respiratory tract following rhinoviral infection of asthmatic subjects [9, 15, 17, 18, [47] [48] [49] [50] . also, we cannot determine the cellular source of the mrnas measured in our study. pilot analysis showed primarily epithelial cells and neutrophils in the nasal aspirates, suggesting that epithelial cells were the primary source of mrna. third, as noted above, we did not compare responses of asthmatic subjects to controls. a recent study showed that asthmatics and non-atopic subjects experience similar colds, including chemokine responses, following experimental rhinovirus infection [17] . fourth, although we were able to detect significant differences in many cytokines following upper respiratory tract infection, a fraction of the samples were too dilute to detect mrna. (there may have been other reasons for the loss of rna, for example sample ribonucleases). dilution may also affect the concentration of measured biomarkers. we did not adjust measurements by total protein content, as vascular leakage due to inflammation may alter protein concentrations in children with upper respiratory infections [51] . in our experience, total protein is difficult to measure in many nasal (and tracheal) aspirate specimens, even when cytokines are readily detectable, and therefore normalization for total protein does not increase the reliability of the concentrations. finally, studying naturally occurring colds in children using home-based methods inherently involves some variability in sample timing and handling that is not present in the controlled environment of the laboratory. yet this approach also offers an opportunity to observe 'real world' scenarios. our ability to detect statistically significant changes in specific mrnas and proteins using the current study design suggests that this is a viable approach for use in future investigations, for example, studies examining whether an early adjustment of asthma medications could abort or temper the impact of respiratory viral infections. we conclude that in children with asthma, naturally occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, ifns, and ifn-responsive genes. cytokine responses are sustained the first week following viral infection. future analyses of controls and asthmatics which combine the determination of cytokine responses and asthma health outcomes will provide further insight into the cellular pathways responsible for virus-induced asthma exacerbations. respiratory viruses and exacerbations of asthma in adults community study of role of viral infections in exacerbations of asthma in 9-11 year old children persistence of rhinovirus rna after asthma exacerbation in children respiratory syncytial virus infection of human mononuclear leukocytes in vitro and in vivo effect of granulocyte/macrophage colony-stimulating factor on human monocytes infected with influenza a virus. enhancement of virus replication, cytokine release, and cytotoxicity characterization of human metapneumovirus infection of myeloid dendritic cells rhinovirus infection of allergen-sensitized and -challenged mice induces eotaxin release rom functionally polarized macrophages mda5 and tlr3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects interleukin-1beta and interleukin-1ra levels in nasal lavages during experimental rhinovirus infection in asthmatic and non-asthmatic subjects quantitative and qualitative analysis of rhinovirus infection in bronchial tissues rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th1/2 cytokine and il-10 production gene expression profiles during in vivo human rhinovirus infection: insights into the host response similar colds in subjects with allergic asthma and nonatopic subjects after inoculation with rhinovirus-16 inflammatory indices in induced sputum: a feasibility study rantes, macrophage inhibitory protein 1-a and the eosinophil product major basic protein are released into upper respiratory secretions during virus-induced asthma exacerbations in children il-10 gene expression in acute virus-induced asthma role of monocyte chemotactic protein-3 and -4 in children with virus exacerbation of asthma lung and blood institute. national asthma education and prevention program expert panel report 3: guidelines for the diagnosis and management of asthma. bethesda, md: national heart, lung and blood institute identification of gaps in the diagnosis and treatment of childhood asthma using a community-based participatory research approach rhinovirus illnesses during infancy predict subsequent childhood wheezing improved method for collection of nasal mucus an integrated microfluidic device for influenza and other genetic analyses linear mixed models for longitudinal data rhinovirus enters but does not replicate inside monocytes and airway macrophages rhinovirus replication in human macrophages induces nf-jb-dependent tumor necrosis factor alpha production the role of p38 mapk in rhinovirus-induced monocyte chemoattractant protein-1 production by monocytic-lineage cells human rhinovirus induces robust ip-10 release by monocytic cells, which is independent of viral replication but linked to type i interferon receptor ligation and stat1 activation rhinoviruses induce interleukin-8 mrna and protein production in human monocytes respiratory virus induction of alpha-, beta-and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus role of deficient type iii interferon-lambda production in asthma exacerbations relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection in vitro susceptibility to rhinovirus infection is greater for bronchial than for nasal airway epithelial cells in human subjects rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma genes transcriptionally modulated by interferon alpha2a correlate with the cytokine activity central role of interferon regulatory factor-1 (irf-1) in controlling retinoic acid inducible gene-i (rig-i) expression interferon regulatory factor 7 is a major hub connecting interferon-mediated responses in virusinduced asthma exacerbations in vivo role of double-stranded rna pattern recognition receptors in rhinovirus-induced airway epithelial cell responses changes in the dose of inhaled steroid affect exhaled nitric oxide levels in asthmatic patients correlation between exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness in patients with mild asthma exhaled nitric oxide correlates with airway hyperresponsiveness in steroid-naive patients with mild asthma noninvasive assessment of airway inflammation in children: induced sputum, exhaled nitric oxide, and breath condensate expired nitric oxide levels during treatment of acute asthma corticosteroids decrease exhaled nitric oxide in children with acute asthma inhalation of swine dust induces cytokine release in the upper and lower airways role of nasal interleukin-8 in neutrophil recruitment and activation in children with virusinduced asthma experimental rhinovirus 16 infection. effects on cell differentials and soluble markers in sputum in asthmatic subjects the effect of an experimental rhinovirus 16 infection on bronchial lavage neutrophils rhinovirus-induced airway inflammation in asthma. effect of treatment with inhaled corticosteroids before and during experimental infection upper airway sampling the authors declare no conflicts of interests. key: cord-300187-fr6tme32 authors: kearns, shawn title: infectious hepatopathies in dogs and cats date: 2009-11-26 journal: top companion anim med doi: 10.1053/j.tcam.2009.06.004 sha: doc_id: 300187 cord_uid: fr6tme32 this article serves to review the various infectious diseases that affect the liver primarily or as a part of systemic infection. although bacterial infections are probably the most common cause of infectious hepatitis, the clinician should be aware of other potential organisms and other commonly involved systems. therefore, this article includes a description of common bacterial, mycobacterial, viral, fungal, protozoal, parasitic, and rickettsial diseases in dogs and cats. alimentary flora circulates to the liver under various clinical conditions. these bacteria are extracted by kupffer cells, killed by neutrophils, or excreted in bile in healthy clinical states. a low-flow, low-pressure perfusion of hepatic sinusoids may allow superior removal of bacteria by phagocytes, and pressure differentials in the biliary system may limit retrograde access of enteric organisms. 2, 4, 5 changes in this sinusoidal flow may decrease the effectiveness of phagocytosis when portal flow is compromised. bowel disease, cholestasis, immunosuppression, and altered gut motility result in altered portal circulation, and the subsequently unchecked bacterial access to the liver may result in bacterial hepatitis or cholangiohepatitis. common isolates implicated in bacterial hepatitis and cholecystitis include escherichia coli, enterococcus spp, bacteroides spp, streptococcus spp, and clostridium spp. 6 cultures can be obtained by liver aspirate, liver biopsy, and cholecystocentesis. a combination of liver and gall bladder samples (fig 1) may increase the likelihood of identification of the offending organism(s). surgical or laparoscopic biopsies may be more rewarding for culture growth compared with aspirates. 6 in suspected cases, broad-spectrum antibiotics for common enteric isolates should be initiated pending specific culture results. focal micro-and macro-abscesses have also been documented in dogs and cats. 7, 8 predisposing causes include alterations in blood flow, trauma, ascending biliary infections, liver lobe torsions, 9 immunocompromised clinical states, 10 and neoplasia. 7, 11 microabscesses are often identified in association with extrahepatic infection and sepsis. 7, 12 ultrasound has greatly enhanced the early diagnosis of hepatic abscesses. 8 greater than 50% of solitary abscesses are polymicrobial. antimicrobial treatment should be directed at both anaerobes and aerobes regardless of whether anaerobic cultures are negative if a polymicrobial hepatic infection is documented. 2 bacterial isolates in hepatic abscesses are similar to those identified in diffuse bacterial hepatic disease. however, clinically rare isolates including klebsiella, listeria, salmonella, brucella, yersinia pseudotuberculosis, actinomyces, nocardia, and pasturella have also been documented. 13 focal abscesses may require surgical drainage and antibiotic therapy. treatment in all cases must be implemented for a minimum of 6 to 8 weeks. leptopirosis is an extremely common nonenteric bacterial infection in the canine liver. leptospires are thin, filamentous, spiral-shaped motile bacteria with a lipopolysaccharide outer envelope. direct transmission occurs via contact with infected urine, venereal and placental tissues, or fluids. indirect transmission can occur through contaminated water sources, soil, food, or bedding. the organism can stay stable for several months with the right environmental conditions. the organism initially penetrates the mucous membranes and rapidly multiplies after entry into the vascular space. dissemination and replication occur in many tissues, including the liver. however, the organism tends to persist in the kidney and can be shed for weeks to months after infection. certain serovars are more frequently associated with hepatic involvement and include leptospira icterohaemorrhagiae and l. pomona. young dogs (ͻ6 months of age) seem to develop signs of hepatic dysfunction more frequently in disease outbreaks. 14 profound hepatic dysfunction may occur without significant histologic changes because of subcellular damage produced by bacterial toxins. the endothelial damage, subsequent thrombosis, and possible disseminated intravascular coagulation seen in acute disease may contribute to hepatic damage. chronic hepatitis has been reported as a sequelae to leptospiral infection. 15, 16 diagnosis is usually made based on clinical signs and serologic titers. however, leptospirosis polymerase chain reaction (pcr) performed be-fore treatment may increase testing sensitivity given vaccinal interference and delayed seroconversion in the acute phase. 17 penicillins are the treatment of choice in the acute phase and must be followed by appropriate antibiotics to eliminate the carrier state. alternatively, doxycycline may be used for both the acute and carrier states. bartonella spp are gram-negative fastidious bacteria and are well adapted for the intracellular environment. a recent case report documented b. henselae and b. clarridgeiae dna in the liver of 2 dogs with granulomatous inflammation. both had a positive clinical response to azithromycin and demonstrated biochemical reduction in hepatocellular enzymes. 18 another dog with peliosis hepatitis (a rare vascular condition characterized by multiple, randomly distributed blood-filled cavities throughout the liver) had b. henselae dna amplified from multiple hepatic specimens by pcr. 19 helicobacter canis has been isolated from the liver of a single dog with hepatitis. 20 helicobacter spp have also been amplified from hepatic tissue in cats with cholangiohepatitis. further studies are required to determine whether these organisms are associated with inflammatory liver disease. these organisms are difficult to culture, and this failure may reflect the fastidious nature of these bacteria. pcr positivity may reflect the presence of intestinal helicobacter from the enterohepatic circulation or transient colonization rather than a true disease association. 21 francisella tularensis (tularemia) is a pleomorphic, gramnegative, nonspore-forming bacillus. this disease frequently occurs as a result of exposure to ticks or wildlife. macrophages are the primary host cells, and bacteremia with multiorgan involvement is common. lungs, spleen, liver, and skin are common sites for embolic spread, resulting in microabscesses and granulomatous disease. puppies and young cats appear more susceptible to infection, and dogs are generally more resistant to infection. clinical findings include depression, oral/lingual ulceration, regional or generalized lymphadenomegaly, hepatosplenomegaly, panleukopenia with severe toxic neutrophil changes, hyperbilirubinemia, and bilirubinuria. [22] [23] [24] examination for evidence of microscopic agglutinating antibody is most frequently used for diagnosis, although indirect fluorescent antibody testing may be useful as well. 24 aminoglycosides are the primary treatment in humans. however, tetracyclines (doxycycline), chloramphenicol, and quinolones are commonly used in dogs and cats. unfortunately, clinical relapse is common with these antibiotics. tyzzer's disease (clostridium piliforme) is caused by a flagellated, spore-forming, gram-negative intracellular parasite. although spores have been identified in rodent species, interspecies transmission via ingested feces has not been documented. however, spontaneous disease has been documented in dogs and cats. [25] [26] [27] [28] colonization of the liver results in multifocal, periportal hepatic necrosis and may result from a currently unidentified toxin. 29 minimal inflammation may be present despite extensive necrosis. 30 death usually occurs within 24 to 48 hours once the organism is in the liver. [31] [32] [33] [34] [35] [36] [37] rhodococcus is a soil-borne pleomorphic, gram-positive bacteria normally associated with suppurative infections in figure 1 . fine-needle aspirate and cytology from the gallbladder of a cat with cholangiohepatitis. the aspirate consists predominantly of bacteria of mixed type. the bacteria are frequently present in chains (black arrow). also, note dark brown-staining amorphous material (bile pigment: yellow arrow). the finding of bacteria in cytologic specimens of bile is considered abnormal. the following organisms were cultured from the bile: escherichia coli, streptococcus pneumoniae, an anaerobic bacterial rod, prevotella oralis, and a gram-positive rod that could not be classified. courtesy of the pathology department, angell animal medical center, boston, massachusetts. domestic livestock. inhalation from soil or wound inoculation are the suspected routes of transmission. disseminated infection and death have been reported in a single dog. 38 clinical reports are rare in cats. mycobacterium spp are aerobic, nonspore-forming, nonmotile bacteria with a wide host affinity and pathogenic potential. they are typically classified based on growth in culture and by the pathologic production of tubercles or granulomatous disease. mycobacterium tuberculosis and m. bovis are the most pathogenic, and humans are reservoirs for these species. aerosolized organisms in sputum are considered the primary mode of transmission. however, m. bovis can be acquired via uncooked meats and wildlife reservoirs. mycobacterial disease is often subclinical in dogs and cats, but signs may be associated with granuloma formation in various organs. 39, 40 nontuberculous mycobacterium, including those in the mycobacterium avium complex, are saprophytic opportunistic organisms primarily implicated in disseminated disease in cats [41] [42] [43] [44] [45] and occasionally in dogs. 46 -53 no clear associations have been identified with retroviral diseases. canine and feline breeds with potentially increased susceptibility include basset hounds, 51 miniature schnauzers, 53 siamese, 45 and abyssinians. 42 dogs with m. avium complex-induced disease will often demonstrate extensive granulomatous disease of the intestine, spleen, liver, and mesenteric lymph nodes. animals undergoing immunosuppressive drug therapy with inhibition of cell-mediated immunity may be at risk for disseminated disease, including renal transplant patients. 43 acidfast cytology can demonstrate bacilli, although false negatives can occur. negative bacterial images may be identified on routine stains (fig 2) . pcr may provide greater sensitivity and safety than culture. 52 combination therapies are often required, because organisms build resistance quickly, particularly with disseminated disease. although not a risk for immunocompetent individuals, dogs and cats infected with saprophytic mycobacterium pose a risk for immunodeficient people. mycobacterium lepraemurium was considered the main causative agent for feline leprosy until recently. however, m. visibilis has been associated with feline multisystemic granulomatous mycobacteriosis, resulting in diffuse cutaneous disease and widespread dissemination to multiple internal organs. 54 organisms responsible for disseminated fungal infections include histoplasma capsulatum, coccidioides immitis, blastomyces dermatitides, aspergillosis sp, cryptococcussp, and sporothrix schenckii. most are dimorphic, saprophytic, opportunistic fungi that exist in the mycelial stage in the environment. spores are produced in the mycelial stage and be-come pathogenic on inhalation, ingestion, or inoculation. dissemination occurs via the hemolymphatic system. specific environmental conditions are required for the individual organisms, and this dictates their geographic range. histoplasma capsulatum is located primarily in the temperate and subtropical areas of the world. organisms are phagocytized by mononuclear cells and replicate intracellularly once they are inhaled and converted to the yeast phase. the primary clinical signs in dogs are associated with the gastrointestinal system (diarrhea, tenesmus, mucous, fresh bloods in stools). clinical signs in cats are vague. dissemination to other visceral organs (including the liver) has been documented in both species. [55] [56] [57] clinically affected animals are usually young (1-4 years of age). diagnosis is usually achieved with fine-needle aspirate or exfoliative cytology of affected organs. aspergillosis is primarily associated with rhinitis. however, several reports have documented systemic infections in german shepherds and in non-shepherd breeds. aspergillus terreus 58 -62 and a. deflectus 63, 64 have been most frequently implicated in systemic infection. predisposing factors include optimal climatic conditions, access to a partial strain, or subtle defects in mucosal immunity. 65 disseminated aspergillosis has also been documented in cats. 66, 67 neurologic deficits, spinal column pain, urinary system disorders, and respiratory pathology are the primary presenting clinical signs. prototheca is a saprophytic, achlorophyllous alga found in the southeastern united states. three species of prototheca have been identified, but p. zopfii is the only one associated with disseminated disease. the organism is associated with sewage, slime flux of trees, and animal waste. transmission generally occurs through ingestion or penetration of injured skin or mucosa. disease can develop with diminished host resistance or concurrent diseases. 68 concomitant large intestinal diarrhea and ocular signs should prompt clinical consideration of prototheca infection. dissemination via blood or lymph to other organs including the liver is common. various stages of development of the organism may be identified on cytology or histopathology. urine culture and sediment are also useful in organism identification. 69 this disease is invariably fatal, although disease progression may be delayed with various antifungal and antibacterial agents. 70 -73 coccidioides immitus is a dimorphic fungus with preference for the alkaline sandy soil environment found in the lower sonoran life zone in the southwestern united states, western mexico, and central and south america. mycelia are produced during rainfall, but arthroconidia develop with soil drying and become airborne under dry and windy conditions. inhalation is the primary mode of infection in dogs and cats. the spherule (tissue parasitic form) undergoes division with eventual rupture. the severity and extent of clinical disease depend on immunocompetence and range from a mild, asymptomatic, pulmonic form to severe, life-threatening disseminated disease. dissemination most commonly involves the axial and appendicular skeleton and overlying skin. tissues from abdominal viscera, the central nervous system (cns), pericardium, myocardium, and prostate can also be involved. 74, 75 cytology or histology may reveal spherules, although diagnosis is often made based on history, clinical signs, and positive serology. antigens for sero-testing commonly use tube precipitin and complement fixation with agar gel immunodiffusion. sporothrix schenckii causes a chronic granulomatous disease of worldwide distribution. infection is usually the result of trauma and inoculation with infective conidiophores. the skin is the primary target organ. however, disseminated disease has been reported, particularly in the cat. no clear dissemination pattern has been identified because of low case numbers, but affected organs include the internal lymph nodes, liver, lungs, eyes, bone, muscles, and cns. 76, 77 diagnosis is frequently made by cytology. blastomyces dermatitidis is found primarily in mississippi, missouri, the ohio river valley, the mid-atlantic states, and some canadian provinces. growth of the organism requires sandy, acidic soil with some proximity to water. preferred sites for dissemination include the skin, eyes, bones, and lymph nodes, although dissemination to the liver has been reported. 78, 79 cryptococcus neoformans has a worldwide distribution. inhalation may be the primary mode of infection, and sites of infection tend to be areas of the body with cooler temperatures, including the respiratory passages and subcutaneous tissues. the fungus is occasionally disseminated to the kidneys and rarely to the liver. 80 treatment of most disseminated fungal infections involves the use of triazoles, including itraconazole and fluconazole, as well as amphoterocin b. 59,81-85 clinical signs may resolve in many cases, but relapses occur and patients with severe clinical illness generally have a poor prognosis. leishmania, transmitted by the sandfly (lutzomyia in the new world, phlebotymus in the old world), frequently causes cutaneous and visceral lesions in the dog. promastigotes transmitted by the female sandflies become amastigotes in the vertebrate and are phagocytized by mononuclear cells. the organism travels through hemolymph organs to remote dermal sites and other organs. clinical signs will not develop in all exposed animals, and the immune response at the time of infection appears important in determining development of disease. leishmania infection should be considered in dogs from endemic areas with marked hyperglobulinemia or in those with a travel history to endemic areas. mild increases in liver enzymes are often noted. however, unlike the kidneys, the liver is not a primary target organ. infection can be associated with chronic hepatitis. 86 definitive diagnosis is made by demonstration of organisms on cytology or histopathology, or by serology, culture, or pcr. amphotericin b in a soybean oil lipid emulsion has been intravenously administered for higher clinical cure success rates and greater numbers of negative posttreatment parasitologic tests compared with other treatments. other less successful treatment options include allopurinol and the pentavalent antimonials. 87 hepatozoon canis is a worldwide protozoal disease reported in domestic dogs and is most prevalent in subtropical and temperate climates. the primary vector is the rhipicephalus sanguineous tick, which is primarily located in warm and temperate regions. transmission occurs through ingestion of ticks containing mature protozoal oocyts. sporozoites are released in the intestine on tick ingestion and penetrate the gut wall, invade mononuclear cells, and disseminate. target organs include the bone marrow, spleen, and lymph nodes but can involve other internal organs such as the liver, kidney, and lungs. 88, 89 the most striking clinicopathologic abnormality is leukocytosis with evidence of parasitemia of the white cells on peripheral blood smears. clinical findings can range from incidental hematologic findings to severe life-threatening illness. hepatitis, glomerulonephritis, and pneumonitis have all resulted from h. canis infection. 90 coinfections with other protozoal diseases (toxoplasma, leishmania, and babesia spp) or other tick-borne diseases (ehrlichia spp) and immunosuppressive states can predispose animals to clinical illness. the hepatitis is associated with developing meronts within the liver and their associated neutrophilic and mononuclear inflammation. hepatozoon has also been documented in felines. [91] [92] [93] microscopic detection of gamonts in peripheral blood smears is the most frequently used diagnostic test. imidocarb is the treatment of choice in dogs. subcutaneous or intramuscular injections are administered every 14 days until gamonts are no longer visualized in the leukocytes. a new species, hepatozoon americanum, was identified in 1997, with the amblyomma maculatum tick as its definitive host. 94 this emerging disease has spread to the north and the east since its initial identification in the gulf coast region. clinical signs are often severe, even in the absence of other diseases or in the presence of immunosuppression. waxing and waning clinical signs are attributed to repeated cycles of asexual reproduction and pyogranulomatous inflammation. the primary site of infection for the merozoites is the cardiac and skeletal muscle. however, single zoites can enter circulation and reproduce asexually at distant locations. 95 diagnosis is most often made with muscle biopsy, although a recent study has identified promise in the use of pcr testing. 96 an enzyme-linked immunosorbent assay has been developed with sporozoites as the antigen. 97 no treatment effectively eliminates the tissue stages of h. americanum. however, treatment with trimethoprim-sulfadiazine, clindamycin, and pyrimethamine followed by long-term administration of decoquinate resulted in extended survival times and an excellent quality of life. 98 the microsporidial parasite encephalitozoon cuniculi is an obligate intracellular protozoan. infection likely occurs by inhalation or ingestion of spores from contaminated urine or feces shed by infected hosts. the organism undergoes asexual reproduction or binary fission after infecting host cells and ruptures, leading to infection of new cells or shedding of resistant spores into the environment. typical organs of localized infection include the kidney, liver, lungs, and brain with resultant granulomatous inflammation. 99, 100 cats and older dogs are not commonly affected, and renal disease predominates in young dogs. cytological examination of fluids (particularly urine) is important in making a diagnosis in animals with disseminated disease as other tests are commercially unavailable. cytauxzoon felis is a tick-borne protozoal disease of domestic and wild cats. the bobcat is the natural reservoir in north america and is usually asymptomatic despite persistent erythroparasitemia. the tissue phase of infection consists of the development of large schizonts in mononuclear phagocytes. the schizonts line the lumens of vessels in most organs, eventually leading to vessel occlusion. merozoites are released into blood or tissue fluid once the host cells rupture and infect red blood cells. late-stage parasitemia can often be detected on blood films at about 1 to 3 days before death. most clinical signs, including those associated with liver abnormalities, are due to schizont-associated mechanical obstruction. however, parasite by-products may also be toxic, pyogenic, and vasoactive. the anemia is regenerative but mild in comparison with clinical icterus. this may be useful in differentiating this infection from hemotropic mycoplasmas. demonstration of piroplasms in wright's-stained or giemsa-stained blood films most frequently provides a definitive diagnosis. histopathology reveals schizont-laden mononuclear phagocytes in the veins of the lungs, liver, and spleen. the prognosis is generally considered poor, but different geographic strains may have varying virulence. 101 treatment with diminazene or imidocarb has been somewhat successful. 102 toxoplasma gondii is an obligate intracellular coccidian parasite that infects almost all warm-blooded animals. do-mestic cats are the definitive hosts and excrete the infective oocyts. three stages of the life cycle are considered infectious, including oocyst sporozoites, tissue cyst tachyzoites, and tissue cyst bradyzoites. transmission can occur through ingestion of oocysts or tissue cysts and via congenital transmission. other reported modes include lactation, transfusions, and transplantation. 103 a higher frequency of disease is reported in dogs and cats fed raw meat or those in a rural/ feral environment. the extra-intestinal life cycle is the same in all hosts, and sporozoites encyst in the intestinal lumen, penetrate cells, and divide into tachyzoites. the tachyzoites can form cysts in the cns, muscle, and visceral organs, and may persist for the life of the host. clinical signs were diverse in 100 cats with histologically confirmed toxoplasmosis, and more than 90% had pulmonary, cns, and liver manifestations. 104, 105 in dogs, disseminated infection is most often associated with canine distemper, other infections including ehrlichiosis and immunosuppression, or vaccination with live attenuated vaccines. 106 clinical cases in cats have been seen with steroids, cyclosporine use, hemotropic mycoplasms, and viral disease. [107] [108] [109] liver and lung involvement is associated with quicker mortality than other organ involvement. tachyzoites may be detected on cytology of various organs and body fluids. however, diagnosis is most frequently based on clinical signs, serology (immunoglobulin g, immunoglobulin m), and response to treatment. clindamycin is the treatment of choice. neospora caninum is a protozoan similar to toxoplasma. dogs and coyotes are considered definitive hosts, and deer and cattle are intermediate hosts. the predominant mode of transmission is transplacental in the dog, and clinical signs are usually secondary to exacerbation of a congenital infection. acute phases of infection include widespread dissemination to many organs, including the liver, whereas chronically infected animals are restricted to muscular and neuronal sites. 103 serology and muscle biopsy often provide a diagnosis, although tachyzoites may be detected in other parasitized tissue or body fluid. 110 sarcocystis canis is an apicomplexan protozoan with no particular geographic distribution. infection results in disseminated disease, including protozoal hepatitis. 111, 112 many reports involve puppies, suggesting the presence of congenital infection. however, the life cycle is still unknown. sarcocystis canis is the only sarcocystis species known to form schizonts in canine tissue. infectious canine hepatitis (ich) is caused by adenovirus type 1. this is the only virus with primary tropism for the liver. 113 infection leads to severe hepatic necrosis and can also cause ocular and renal changes. the virus localizes in the tonsils after oronasal exposure, spreads to regional lymph nodes, and disseminates via the thoracic duct. hepatic parenchymal cells and vascular endothelial cells are the prime targets of viral localization, and injury leading to centrilobular to panlobular hepatic necrosis ranges from self-limiting to fatal. most affected dogs are less than 1 year of age and unvaccinated. severely affected dogs can become moribund and die within hours of disease onset and with few predictive clinical signs. if patients survive the acute phase, they may develop clinical signs including vomiting, diarrhea, and abdominal pain. 114, 115 those that survive may go on to develop chronic hepatitis and fibrosis, likely secondary to self-perpetuating liver inflammation rather than chronic infection. 116 diagnosis is frequently made based on clinical signs and serology, although the virus can be isolated in cell cultures. this disease is rarely encountered because of the high efficacy of vaccination. canine acidophil hepatitis is believed to be caused by a viral agent. however, the specific agent is not yet identified. disease has been reproduced via injections of sterile liver homongenates from spontaneously affected animals. acute infections can lead to acute to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. diagnosis is made on histology because acidophils are scattered throughout lesions. this disease has only been reported in great britain. 117, 118 canine herpesvirus causes tissue necrosis and localized mucosal or generalized systemic infections in young or immunocompromised animals. the virus only infects dogs because of specific cell-surface receptors. replication occurs via viral dna synthesis within the host nucleus. transmission occurs through direct contact with mucosal secretions from the respiratory or genital tract of animals. factors predisposing to infection in puppies include hypothermia and a poorly developed immune system. newborns can acquire disease in utero, during passage through the birth canal, during contact with infected littermates, from oronasal secretions of the dam, and from fomites. puppies less than 1 week of age are more susceptible to generalized fatal infections. dissemination leads to hemorrhagic necrosis in several organs including the adrenal glands, kidney, liver, lungs, and spleen. clinical signs include loss of interest in nursing, loss of body weight, soft yellow-green feces, abdominal discomfort, and dullness. a marked increase in alanine aminotransferase is often noted on biochemistry profile. definitive diagnosis is by viral isolation. 119 feline leukemia virus is a single-stranded retrovirus that replicates in many tissues. clinical illness is generally related to the hematopoietic system and the immune system. feline leukemia virus has also been associated with icterus and various inflammatory and degenerative liver diseases including focal liver necrosis. 120 feline infectious peritonitis (fip) is a feline coronavirus that has undergone frequent rna mutations, resulting in an ability to enter and replicate in macrophages. an immunemediated vasculitis occurs if the virus is not eliminated. affected cats develop signs related to target organ lesions (kidney, liver, cns, intestine) or due to fluid redistribution. abnormal liver enzymes can occur because of hepatitis, hepatic lipidosis, or prehepatic sequalae of vasculitis, erythrocyte destruction, and hypoxia. hyperbilirubinemia is common and usually secondary to vasculitis in the liver. 121 histopathology is required for definitive diagnosis but is sup-ported by history, physical examination, and laboratory findings. a new pcr test may also prove useful in the diagnosis of fip. 122 treatment is generally unrewarding. conflicting information exists on the usefulness of feline recombinant interferon, although it may be beneficial for a subpopulation of fip-infected cats. 123, 124 rickettsial diseases the most common agents encountered in dogs with clinical evidence of liver involvement include the ehrlichia sp, rickettsia rickettsii, and borrelia burgdorferi. these organisms can infect either hepatocytes or endothelial cells. hepatic involvement in erhlichia infections occurs in more than 80% of human patients, leading to mild transient increases in transaminases. 125 liver injury may be related to organism proliferation in hepatocytes and stimulation of immunologic and nonspecific inflammatory mechanisms. rocky mountain spotted fever is vasculotropic in nature and can cause moderate increases in transaminases. experimental evidence with borrelia suggests direct hepatic invasion by the spirochetes in conjunction with cellular and humoral immunologic mechanisms. 126 an association with borrelia was observed and confirmed with liver biopsy in 2 dogs. lesions were consistent with lobular dissecting hepatitis and mixed multifocal inflammation leading to focal pyogranulomas in the other. 2 chronic cholangitis associated with liver fluke infestation in endemic areas is primarily observed in cats and less frequently in dogs. most infections are due to opistorchus and metorchis, which require 2 intermediate hosts. the first hosts are water snails, and the second hosts include a wide variety of fish with encysted metacercariae. the final host acquires infection by ingestion of fish, and the young liver flukes migrate to the liver through the bile ducts. this results in bile duct thickening and dilation. rarely, cysts may be formed as well. 127 a slight to moderate inflammation may be seen both within the ducts and in the portal areas. although eosinophils may be present, they are usually limited in numbers. the number of liver flukes and eggs within the dilated bile ducts varies markedly, and often only limited evidence of liver flukes or eggs is identified. platynosomum concinnum is a trematode of the feline biliary system. terrestrial snails, lizards, toads, and terrestrial isopods act as intermediate hosts based on geographic location. disease is most prevalent in the tropical and subtropical climates. clinical cases involve adult indoor or indoor-outdoor cats. the severity of clinical signs is proportional to the number of adult flukes as well as to the duration of parasitemia. early diagnosis can be difficult. however, diagnosis is easier when eggs have been identified in the bile. 128 treatment of p. concinnum and liver fluke infections is best accomplished with praziquantel. there are many infectious diseases that ultimately affect the liver. few, however, have primary tropism for hepatic tissue. testing should be directed based on signalment, geographic locale, and primary presenting complaint. cytology and/or histopathology of the liver will most frequently provide a definitive diagnosis in clinical situations with liver involvement. the prognosis is guarded with many disseminated infections. toxic, metabolic, infectious, and neoplastic liver diseases infectious diseases of the dog and cat detection of portal and systemic bacteremia in dogs with severe induced hepatic disease and multiple portosystemic shunts diseases of the gallbladder and biliary tree cholangitis/cholangiohepatitis complex in the cat bacterial culture results from liver, gallbladder, or bile in 248 dogs and cats evaluated for hepatobiliary disease: 1998-2003 hepatic abscesses in cats: 14 cases hepatic abscesses in 13 dogs: a review of the ultrasonographic findings, clinical data and therapeutic options liver lobe torsion and liver abscess in a dog hepatic abscesses associated with diabetes mellitus in two dogs hepatocellular carcinoma with secondary abscessation in a cat hepatic abscesses in dogs hepatic abscesses in dogs: 14 cases (1982-1994) infectious diseases of the dog and cat chronic active hepatitis in dogs associated with leptospires chronic hepatitis associated with leptospiral infection in vaccinated beagles clinical application of a polymerase chain reaction assay for diagnosis of leptospirosis in dogs detection of bartonella henselae and bartonella clarridgeiae dna in hepatic specimens from two dogs with hepatic disease peliosis hepatis in a dog infected with bartonella henselae helicobacter canis isolated from a dog liver with multifocal necrotizing hepatitis association of helicobacter with cholangiohepatitis in cats feline tularemia on tularemia in a cat acute tularemia in three domestic cats bacillus piliformis infection in an adult dog tyzzer's disease in kittens with familial primary hyperlipoproteinaemia tyzzer's disease in a puppy tyzzer's disease in puppies infectious diseases of the dog and cat the liver in systemic disease. an innocent bystander naturally occurring tyzzer's disease as a complication of distemper and mycotic pneumonia in a dog tyzzer's disease in a dog tyzzer's disease in an adult cat naturally occurring tyzzer's disease in a cat naturally occurring tyzzer's disease in a puppy tyzzer's disease complicated with distemper in a puppy tyzzer's disease in puppies vapa-negative rhodococcus equi in a dog with necrotizing pyogranulomatous hepatitis, osteomyelitis, and myositis mycobacterium tuberculosis complex infection in a dog putative transmission of mycobacterium tuberculosis infection from a human to a dog disseminated mycobacterium avium infection in a cat disseminated mycobacterium avium infection in young cats: overrepresentation of abyssinian cats disseminated mycobacterium avium complex infection following renal transplantation in a cat tuberculosis in cats disseminated mycobacterium avium complex infection in three siamese cats fatal mycobacteriosis with hepatosplenomegaly in a young dog due to mycobacterium avium a case of disseminated tuberculosis in a dog caused by mycobacterium avium-intracellulare systemic mycobacterium smegmatis infection in a dog disseminated mycobacterium avium infection in a dog disseminated mycobacterium avium complex infection in a dog tuberculosis in five basset hounds systemic mycobacterium avium infection in a dog diagnosed by polymerase chain reaction analysis of buffy coat disseminated mycobacterium avium infection in three miniature schnauzer litter mates histologic and genotypic characterization of a novel mycobacterium species found in three cats atypical histoplasma capsulatum infection in a dog disseminated histoplasmosis in dogs: 12 cases (1981-1986) disseminated histoplasmosis in cats: 12 cases (1981-1986) disseminated aspergillosis in two dogs in israel long-term survival of four dogs with disseminated aspergillus terreus infection treated with itraconazole disseminated aspergillus terreus infection in a dog disseminated aspergillosis in a dog with diskospondylitis and neurologic deficits disseminated aspergillosis in a dog systemic mycosis due to aspergillus deflectus in a dog disseminated aspergillosis attributable to aspergillus deflectus in a springer spaniel canine disseminated aspergillosis systemic aspergillosis and mucormycosis in 23 cats feline disseminated aspergillosis altered immune function in a dog with disseminated protothecosis urinary tract manifestations of protothecosis in dogs more than meets the eye: subretinal aspirate from an acutely blind dog disseminated protothecosis causing acute blindness and deafness in a dog disseminated protothecosis in a dog infectious diseases of the dog and cat deep mycotic infections in cats disseminated coccidioidomycosis in a dog pathology of sporotrichosis in 10 cats in rio de janeiro disseminated sporotrichosis in a cat ocular changes in a cat with disseminated blastomycosis legendre am: blastomycosis, in greene ce fatal disseminated cryptococcosis and concurrent ehrlichiosis in a dog disseminated opportunistic fungal disease in dogs: 10 cases infectious diseases of the dog and cat cryptococcosis coccidioidomycosis and paracoccidioidomycosis infectious diseases of the dog and cat chronic hepatitis associated with canine leishmaniosis (leishmania infantum): a clinicopathological study of 26 cases initial and long term efficacy of a lipid emulsion of amphotericin b desoxycholate in the management of canine leishmaniasis hepatozoon canis infection in two dogs canine hepatozoonosis in oklahoma hepatozoon canis infection feline hepatozoonosis granulomatous cholangiohepatitis in a cat due to a protozoan parasite resembling hepatozoon canis hepatozoon species infection in domestic cats: a retrospective study a new hepatozoon species from dogs: description of the causative agent of canine hepatozoonosis in north america characterization of stages of hepatozoon americanum and of parasitized canine host cells diagnosis of canine hepatozoon spp. infection by quantitative pcr an indirect enzymelinked immunosorbent assay for diagnosis of american canine hepatozoonosis treatment of dogs infected with hepatozoon americanum: 53 cases (1989-1998) mammalian microsporidiosis experimental encephalitozoonosis in neonatal dogs cats surviving natural infection with cytauxzoon felis: 18 cases (1997-1998) administration of diminazene aceturate or imidocarb dipropionate for treatment of cytauxzoonosis in cats toxoplasmosis and neosporosis infectious diseases of the dog and cat acute primary toxoplasmic hepatitis in an adult cat shedding toxoplasma gondii oocysts fatal toxoplasmosis in five cats acute toxoplasmosis following renal transplantation in three cats and a dog feline immunodeficiency virus predisposes cats to acute generalized toxoplasmosis neonatal toxoplasmosis in littermate cats histologically confirmed clinical toxoplasmosis in cats: 100 cases (1952-1990) neospora caninum associated with septic peritonitis in an adult dog fatal cutaneous and visceral infection in a rottweiler dog associated with a sarcocystis-like protozoan fatal hepatic sarcocystosis in a puppy with eosinophilia and eosinophilic peritoneal effusion canine viral diseases viral hepatitis of dogs (rubarth's disease) infectious canine hepatitis (hepatitis contagiosa canis use of polymerase chain reaction and immunohistochemistry for detection of canine adenovirus type 1 in formalin-fixed, paraffin-embedded liver of dogs with chronic hepatitis or cirrhosis a new transmissible agent causing acute hepatitis, chronic hepatitis, and cirrhosis in dogs persistent hepatitis and chronic fibrosis induced by canine acidophil cell hepatitis virus canine herpesvirus frequency and significance of feline leukemia virus infection in necropsied cats feline infectious peritonitis and feline enteric coronavirus feline infectious peritonitis: typical findings and a new pcr test use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis effect of feline interferonomega on the survival time and quality of life of cats with feline infectious peritonitis ehrlichial diseases of humans: emerging tickborne infections host-pathogen interactions in the immunopathogenesis of lyme disease severe cholestatic liver disease secondary to liver fluke key: cord-263749-bbhh5xb1 authors: larenas-linnemann, désirée; rodríguez-pérez, noel; arias-cruz, alfredo; blandón-vijil, maría virginia; del-río-navarro, blanca e.; estrada-cardona, alan; gereda, josé e.; luna-pech, jorge a.; navarrete-rodríguez, elsy maureen; onuma-takane, ernesto; pozo-beltrán, césar fireth; rojo-gutiérrez, maría isabel title: enhancing innate immunity against virus in times of covid-19: trying to untangle facts from fictions date: 2020-10-09 journal: world allergy organ j doi: 10.1016/j.waojou.2020.100476 sha: doc_id: 263749 cord_uid: bbhh5xb1 introduction in the light of the current covid-19 pandemic, during which the world is confronted with a new, highly contagious virus that suppresses innate immunity as one of its initial virulence mechanisms, thus escaping from the first-line human defense mechanisms, enhancing innate immunity seems a good preventive strategy. methods without the intention to write an official systematic review, but more to give an overview of possible strategies, in this review article we discuss several interventions that might stimulate innate immunity and thus our defense against (viral) respiratory tract infections. some of these interventions can also stimulate the adaptive tand b-cell responses, but our main focus is on the innate part of immunity. we divide the reviewed interventions in: 1) lifestyle related (exercise, >7 hours sleep, forest walking, meditation/mindfulness, vitamin supplementation); 2) non-specific immune stimulants (letting fever advance, bacterial vaccines, probiotics, dialyzable leukocyte extract, pidotimod) and 3) specific vaccines with heterologous effect (bcg vaccine, mumps-measles-rubeola vaccine, i.e.). results for each of these interventions we briefly comment on their definition, possible mechanisms and evidence of clinical efficacy or lack of it, especially focusing on respiratory tract infections, viral infections and eventually a reduced mortality in severe respiratory infections in the intensive care unit. at the end a summary table demonstrates the best trials supporting (or not) clinical evidence. conclusion several interventions have some degree of evidence for enhancing the innate immune response and thus conveying possible benefit, but specific trials in covid-19 should be conducted to support solid recommendations. context of the covid-19 pandemic, extrapolated evidence from previous studies shows that regular, 128 moderate to vigorous exercise improves immune competency across the lifespan and may help to 129 reduce the frequency of upper-respiratory tract infections (urtis).(9, 10) importantly, this has been 130 observed also in older individuals (>65 year-olds) who regularly exercise, whose urtis incidence per 131 year has been inversely associated with the energy expenditure utilized during physical activity. conversely, some studies report that during prolonged periods of strenuous exercise (like marathons) 155 and after approximately 2 weeks following intense competitions, there is an immunodepression span 156 (the "open window" period), related to a higher rate of urti-like symptoms (mainly self-reported) vs. 157 those who undergo lower physical activity. (21, 22) this theory, attributed to the consequences of 158 psychological stress and excessive training,(23, 24) is however highly debated today in the light of a 159 better understanding of the variability in the intensity-related response to physical activity, which 160 depends largely on the functional capacity of the immune system, so up-to-date perceptions on this 161 topic tend to discard the concept of exercise-induced immunosuppression. in fact, it is widely accepted 162 that, when performed regularly, during and after exercise the immune system is in a heightened state of 163 immune surveillance and regulation.(25) 164 during the covid-19 quarantine it is important to stay active and to exercise regularly (gradually 165 increasing intensity), not only as a way to keep a healthy lifestyle, but also as an immunoprotective and 166 immunoregulatory activity. it is possible that the older adults (who are at higher risk of a severe sars-167 cov-2 infection) may obtain the greatest exercise-induced benefits to their immunological health. 168 169 2.2. forest bathing (shinrin yoku) 170 in a series of several, subsequent small studies, japanese investigators in cooperation with experts from 171 the stanford university have shown that walking in a forest seems to enhance nk cell activity, partly 172 stimulated by volatile substances produced by trees (particularly phytoncides: antimicrobial essential 173 oils from trees). the first experiment showed in vitro that phytoncides activate nk cell activity and 174 augment nk cell granules content.(26) then they confirmed this in peripheral blood lymphocytes of 175 subjects submitted to a prospective trial of a 3-day, 2-night trip to the forest including three 2-hour 176 walks.(27) in a third case-control trial they demonstrated that the effect was not related to the walking 177 j o u r n a l p r e -p r o o f 8 tour, as they compared the rise in nk-cell activity after a 3-day tour to a small city with that after a 3-day 178 tour to the forest. both tours included three 2-hour walks, but nk-cell activity was only enhanced after 179 the forest trip. moreover, nk-cell activity stayed elevated one week and still somewhat 30 days after the 180 tour. ( other studies have shown that sleep deprivation, both complete or only selective to the rapid eye 198 movement (rem) stage, modifies various components of the innate immune system, such as dendritic 199 cells (dcs) and the percentage of some subpopulations of t cells (cd4+, cd8+ and nk) and cytokine 200 levels (ifn-γ, tnf-α and il-1).(32-34) specifically, in a clinical study in women 77 hours of sleep 201 deprivation (total and rem phase) resulted in a proinflammatory state and affected the cellular immune 202 response, presenting changes in the production of ifns and in the phagocytic activity. sleep deprivation 203 also decreased lymphocytic blastogenesis, nk cell activity, and regulation of il-1 and il-2.(35) 204 j o u r n a l p r e -p r o o f 9 moreover, in the elderly, a sleep pattern with poor quality (self-reported) was related to increased levels 205 of il-6 and this association was not explained by other factors, such as depressive symptoms, stress or 206 loneliness, but exclusively by sleep disturbance as an independent factor.(36) 207 as for the clinical evidence, an adequate sleep pattern also appears to be beneficial in modulating the 208 adaptive immune response. spiegel the previously mentioned interventions are potential options that could improve the immune system 303 against sars-cov-2, but more evidence is required.(70) see table 2. 304 zinc is an essential trace element for the proper functioning of multiple biological processes within the 306 human body, and its contribution to immunity is not an exception.(72) 307 the complete mechanism by which zinc could decrease the number or severity of viral infectious 308 processes in general and of covid-19 in particular is not exactly understood yet; however, effects have 309 been observed on the binding of the viral agent to the mucosa and on its replication, as well as on the 310 regulation of the inflammatory process;(73) enhanced benefits have been hypothesized when co-311 administered with other medications such as (hydroxy)chloroquine that could function as a ionophore, 312 facilitating the entrance of zinc into the cells.(74) the human body's ability to store zinc is known to be 313 low; its deficiency compromises the immune system, as has been evidenced occasionally by thymic 314 atrophy, lymphopenia and altered lymphocyte responses. is not rock-solid yet, could be considered options that might help enhance the innate immune system 604 and thus worthwhile, especially, as most lack the risk for serious adverse events. together with the 605 techniques focusing on reduction of the viral load, such as social distancing, equipment for personal 606 protection and adequate hand hygiene, improving our first-line defense seems vital. finally, we add in 607 exercise regular exercise has a probable immunoprotective and immunoregulatory effect. older adults may obtain the greatest exercise-induced benefits for their immunological health. forest walks might be beneficial to boost innate immunity, particularly in times of a pandemic; evidence is limited, but there are no direct health-related safety issues. the potential benefit of adequate sleep patterns overweighs the low level of evidence available up to date mindfulness a constant program of a professionally guided meditation strategy could be beneficial to achieve an effective stress reduction and probably to reduce the burden of urtis, mainly in older adults the published study results so far have not been conclusive. subjects with vitamin deficiency would likely benefit most from supplementation. the intervention could be considered relatively safe a cochrane collaboration systematic review showed daily doses of 0.2mg reduced the duration of common cold. in ards patients very high iv doses might be beneficial. more data are needed. all 4 meta-analyzes have reported beneficial effects of zinc supplementation in children for the prevention of pneumonia. important to restore the redox balance. adverse effects are considered mild a modest efficacy of some probiotics (lactobacillus and bifidobacterium strains) was found in reducing the incidence and duration of viral respiratory infections though it might seem of benefit, evidence of the reduction of infections is very scarce and quality trials are lacking. with the use of pidotimod there is a lower recurrence of respiratory tract infections as compared to conventional treatment and less use of antibiotics we encourage to wait for the results of the ongoing rct before using bcg vaccination for the prevention of covid-19 heterothe immune stimulation produced by heterologous vaccines enhances the response to other pathogens, different from the one in the vaccine and was associated with reduced all-cause infant mortality (e.g. measles vaccine, bcg). influenza: the once and future pandemic estimation of the basic reproduction number, average incubation time, 615 asymptomatic infection rate, and case fatality rate for covid-19: meta-analysis and sensitivity analysis preliminary prediction of the basic 618 reproduction number of the wuhan novel coronavirus 2019-ncov sars coronavirus pathogenesis: host innate immune responses and viral 620 antagonism of interferon covid-19: consider 622 cytokine storm syndromes and immunosuppression is a "cytokine storm" relevant to covid-19? targets of t cell 626 responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals therapeutic potential of b-1a cells in covid-19 moderate to vigorous 630 physical activity and risk of upper-respiratory tract infection physical activity, 632 stress, and self-reported upper respiratory tract infection the symptomatology of upper respiratory tract 634 infections and exercise in elderly people exercise and the aging 636 immune system moderate exercise enhances the production of 638 interferon-gamma and interleukin-12 in peripheral blood mononuclear cells acute exercise 641 mobilises cd8+ t lymphocytes exhibiting an effector-memory phenotype exercise-induced redistribution of t 644 lymphocytes is regulated by adrenergic mechanisms t-regulatory cells exhibit 646 a biphasic response to prolonged endurance exercise in humans exercise and psychosocial factors 649 modulate immunity to influenza vaccine in elderly individuals signaling via trif contributes to a protective innate immune response to severe acute respiratory 653 syndrome coronavirus infection influence of 655 exercise training and age on cd14+ cell-surface expression of toll-like receptor 2 and 4 exercise amelioration of depression-like behavior in ovx mice 658 is associated with suppression of nlrp3 inflammasome activation in hippocampus does exercise increase the risk of upper respiratory 664 tract infections exercise, infection, and immunity exercise and airway injury in athletes debunking the myth of exercise-induced immune suppression: 669 redefining the impact of exercise on immunological health across the lifespan phytoncides (wood 672 essential oils) induce human natural killer cell activity forest bathing enhances 675 human natural killer activity and expression of anti-cancer proteins visiting a forest, but not 678 a city, increases human natural killer activity and expression of anti-cancer proteins effect of phytoncide 681 from trees on human natural killer cell function using psychoneuroimmunity against covid-19 galphas-684 coupled receptor signaling and sleep regulate integrin activation of human antigen-specific t cells number and function of circulating human antigen 687 presenting cells regulated by sleep rem sleep deprivation in rats results in 689 inflammation and interleukin-17 elevation effects of acute and chronic sleep loss on 691 immune modulation of rats the bidirectional relationship between sleep and immunity 694 against infections sleep disturbance and 696 older adults' inflammatory responses to acute stress effect of sleep deprivation on response to immunization. 698 sleep habits and susceptibility to the 700 common cold behaviorally assessed sleep and susceptibility 702 to the common cold psychological stress and susceptibility to the common cold relationships between mindfulness practice and levels of mindfulness, 706 medical and psychological symptoms and well-being in a mindfulness-based stress reduction program meditation or exercise for 709 preventing acute respiratory infection: a randomized controlled trial epidemic influenza and 711 vitamin d vitamin d and the intracrinology of innate immunity the interplay between vitamin d and viral 715 infections vitamin d in allergic disease: shedding light on a complex problem unexpected actions of vitamin d: new perspectives on the regulation of 719 innate and adaptive immunity vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths. 722 nutrients administration in ventilated intensive care unit patients: a pilot double blind randomized controlled 725 trial vitamin d and respiratory tract 727 infections: a systematic review and meta-analysis of randomized controlled trials vitamin d 730 supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of 731 individual participant data effect of vitamin d3 supplementation on 733 respiratory tract infections in healthy individuals: a systematic review and meta-analysis of 734 randomized controlled trials vitamin d supplementation for the prevention 736 of childhood acute respiratory infections: a systematic review of randomised controlled trials the 2011 report on 739 dietary reference intakes for calcium and vitamin d from the institute of medicine: what clinicians need 740 to know vitamin-d and covid-19: do deficient risk a poorer outcome? lancet diabetes 745 endocrinol vitamin d: deficiency, sufficiency and toxicity intravenous vitamin c for reduction of cytokines storm in acute respiratory 749 distress syndrome vitamin c is an essential factor on the anti-viral 751 immune responses through the production of interferon-alpha/beta at the initial stage of influenza a 752 virus (h3n2) infection vitamin c and common cold-induced asthma: a systematic review and statistical 754 analysis acid potentially regulates immune and inflammatory response associated 757 with coronavirus infections: a perspective from system biology analysis hypovitaminosis c and 759 vitamin c deficiency in critically ill patients despite recommended enteral and parenteral intakes. crit 760 care functional role of 763 dietary intervention to improve the outcome of covid-19: a hypothesis of work vascular endothelialitis, thrombosis, and angiogenesis in covid-19 modulation of host defence 768 against bacterial and viral infections by omega-3 polyunsaturated fatty acids flavonoids from houttuynia cordata attenuate 771 h1n1-induced acute lung injury in mice via inhibition of influenza virus and toll-like receptor signalling individual risk management 774 strategy and potential therapeutic options for the covid-19 pandemic the clinical benefits of chinese patent 776 medicines against covid-19 based on current evidence traditional chinese medicine in the treatment of 778 cov-2): a review and perspective pharmacological perspective: glycyrrhizin may be an efficacious therapeutic 781 agent for covid-19 the role of zinc in antiviral immunity zinc and 785 respiratory tract infections: perspectives for covid19 (review) improving the efficacy of chloroquine and hydroxychloroquine against 787 sars-cov-2 may require zinc additives -a better synergy for future covid-19 clinical trials zinc in human health: effect of zinc on immune cells role of zinc administration in prevention of childhood diarrhea 791 and respiratory illnesses: a meta-analysis low zinc status: a new risk factor for pneumonia in the 793 elderly? prevention of diarrhea and 795 pneumonia by zinc supplementation in children in developing countries: pooled analysis of randomized 796 controlled trials. zinc investigators' collaborative group zinc supplementation for the prevention of acute lower 798 respiratory infection in children in developing countries: meta-analysis and meta-regression of 799 randomized trials zinc lozenges may shorten the duration of colds: a systematic review fever and the thermal regulation of immunity: the immune 803 system feels the heat fever-like thermal conditions 805 regulate the activation of maturing dendritic cells conservation of 807 il-6 trans-signaling mechanisms controlling l-selectin adhesion by fever-range thermal stress from fever to immunity: a new role for igfbp-6? activation of the sympathetic nervous 814 system modulates neutrophil function the regulation of interleukin-6 implicates skeletal muscle as an integrative 816 stress sensor and endocrine organ human monocyte stimulation by 818 experimental whole body hyperthermia functional expression 820 of trpv channels in t cells and their implications in immune regulation the acute phase 822 protein haptoglobin regulates host immunity passive heat therapy protects 826 against endothelial cell hypoxia-reoxygenation via effects of elevations in temperature and circulating 827 factors community-acquired pneumonia in the elderly: association of 829 mortality with lack of fever and leukocytosis the effect of antipyretic 831 therapy upon outcomes in critically ill patients: a randomized, prospective study the value of sanitarium treatment in respiratory diseases washington, d.c.: review and 836 herald publishing assn antipyretic drugs in patients with fever and infection: literature review the effect of antipyretic 841 medications on mortality in critically ill patients with infection: a systematic review and meta-analysis population-level effects of suppressing fever therapeutic targeting of trained 846 immunity bcg-induced trained 848 immunity in nk cells: role for non-specific protection to infection trained immunity: a 852 program of innate immune memory in health and disease human dendritic cells activated with mv130 induce th1, th17 and il-10 responses via ripk2 and myd88 855 signalling pathways mv140, a 857 sublingual polyvalent bacterial preparation to treat recurrent urinary tract infections, licenses human 858 dendritic cells for generating th1, th17, and il-10 responses via syk and myd88 efficacy of whole-cell killed bacterial vaccines in preventing 864 pneumonia and death during the 1918 influenza pandemic sublingual therapeutic 866 immunization with a polyvalent bacterial preparation in patients with recurrent respiratory infections: 867 immunomodulatory effect on antigen-specific memory cd4+ t cells and impact on clinical outcome primary prevention of severe lower 870 respiratory illnesses in at-risk infants using the immunomodulator om-85 trained immunity in newborn 873 infants of hbv-infected mothers probiotics and the gut immune system: indirect regulation beneficial effects of 877 probiotic consumption on the immune system immunobiotic strains modulate toll-like receptor 3 agonist induced innate 879 antiviral immune response in human intestinal epithelial cells by modulating ifn regulatory factor 3 880 and nf-kappab signaling type i/ii 882 interferon in hiv-1-infected patients: expression in gut mucosa and in peripheral blood mononuclear 883 cells and its modification upon probiotic supplementation prospective study of probiotic 885 supplementation results in immune stimulation and improvement of upper respiratory infection rate probiotics and paraprobiotics in viral infection: 888 clinical application and effects on the innate and acquired immune systems probiotics and covid-19: one size does not fit all effectiveness of probiotics on the duration 893 of illness in healthy children and adults who develop common acute respiratory infectious conditions: a 894 systematic review and meta-analysis probiotics for preventing acute upper respiratory tract infections serum and 898 fecal profiles of aromatic microbial metabolites reflect gut microbiota disruption in critically ill patients: 899 a prospective observational pilot study is diet partly 901 responsible for differences in covid-19 death rates between and within countries? the cellular transfer of cutaneous hypersensitivity to tuberculin in man cd8+ t cells produce a 906 dialyzable antigen-specific activator of dendritic cells immune memory by dialyzable leukocyte extract from a cd8+ t cell line early differentiation of human cd11c(+)nk cells with gammadelta t cell activation properties 912 is promoted by dialyzable leukocyte extracts adjuvant treatment with a dialyzable leukocytes extract contributes to maintain 915 hpv-infected women free of low-grade cervical lesions dialyzable leukocyte extracts activate tlr-2 on monocytes the dialyzable leukocyte extract transferon(tm) inhibits tumor growth and brain 921 metastasis in a murine model of prostate cancer dialyzable leukocyte extract (transferon) administration in sepsis: 924 experience from a single referral pediatric intensive care unit innate immune system to improve survival traits in high risk pathogen scenarios adjuvant treatment using transfer factor 929 for bronchogenic carcinoma: long-term follow-up immunostimulants in respiratory diseases: focus on pidotimod effect of pidotimod on phagocytosis and 933 intracellular killing of staphylococcus aureus by human circulating polymorphonuclear neutrophils and 934 alveolar macrophages pidotimod stimulates natural killer cell 936 activity and inhibits thymocyte cell death pidotimod shows a chemokine-like activity through cxc chemokine receptor 3 (cxcr3) pidotimod promotes 941 functional maturation of dendritic cells and displays adjuvant properties at the nasal mucosa level immunomodulatory effects 944 of pidotimod in adults with community-acquired pneumonia undergoing standard antibiotic therapy pidotimod, an immunostimulant in pediatric recurrent respiratory 947 tract infections: a meta-analysis of randomized controlled trials safety and immunogenicity of early 949 bacillus calmette-guerin vaccination in infants who are preterm and/or have low birth weights: a 950 systematic review and meta-analysis treating bcg-induced disease in 952 children neonatal bcg vaccination against hospitalization due to respiratory infection and sepsis. clin infect 955 dis early bcg-denmark 957 and neonatal mortality among infants weighing <2500 g: a randomized controlled trial the efficacy of bacillus calmette-guerin 960 vaccinations for the prevention of acute upper respiratory tract infection in the elderly bcg 963 vaccination enhances the immunogenicity of subsequent influenza vaccination in healthy volunteers: a 964 randomized, placebo-controlled pilot study bcg vaccination protects 966 against experimental viral infection in humans through the induction of cytokines associated with 967 trained immunity bcg vaccine protection from severe coronavirus 969 disease 2019 (covid-19) sars-cov-2 rates in bcg-vaccinated and unvaccinated young 971 adults bcg-induced trained immunity: can it offer protection against covid-19? 973 heterologous immunity: role in natural and vaccine-induced resistance to 975 infections vaccination and heterologous immunity: 977 educating the immune system non-specific effect of vaccines: immediate 979 protection against respiratory syncytial virus infection by a live attenuated influenza vaccine. front 980 microbiol vitamin d supplementation and risk of respiratory tract infections: a meta-982 analysis of randomized controlled trials vitamin d supplementation for preventing infections 984 in children under five years of age effect of vitamin c 986 infusion on organ failure and biomarkers of inflammation and vascular injury in patients with sepsis 987 and severe acute respiratory failure: the citris-ali randomized clinical trial allergic rhinitis and its impact on asthma (aria) guidelines for allergic rhinitis based on grading of 991 recommendations assessment, development and evaluation (grade) and real-world evidence enteral omega-3 994 fatty acid, gamma-linolenic acid, and antioxidant supplementation in acute lung injury association of bcg, dtp, and measles containing vaccines with childhood mortality: systematic review. 998 days (enteral supplementation of n-3 fatty acids, γ-linolenic rice unlike the normal anti-viral response with increased ifn type i and iii and with it the activation of genes 1006 stimulated by ifn in adjacent cells and thereby increasing its anti-viral defense, the coronavirus has 1007 mechanisms that lower this anti-viral innate defense mechanism by interfering with ifn production and 1008 its effects. in addition, chemotactic molecules are released in a viral infection that attract macrophages 1009 (m∅), natural killer (nk) cells, and neutrophils. this reaction is not fully achieved during early infection 1010 with coronavirus, so the initial innate immune response appears incomplete and slow. after this first 1011 innate response, adaptive immunity is triggered via activation of dendritic cells (dc) that stimulate 1012 specific th1 lymphocytes, which in turn activate cytotoxic t cells (tccell) to eliminate infected cells, in 1013 the more advanced stages of the infection, along with plasma cell development and the production of 1014 antiviral igm and igg antibodies (not shown). however, in some patients with covid-19 at this stage a 1015 dysregulated activation of macrophages (i.e. by il6) is seen, causing the feared cytokine storm. 1016 the conversion of 25 ohd 2 to 1,25-(oh) 2 d 3 can be done directly in some cells of the immune system 1018 such as macrophages. the macrophage, like kidney cells, contains the enzyme 1-alpha hydroxylase that 1019 is capable of transforming vitamin d into its active form, calcitriol. 1020exposure of the macrophage to some pathogens induces the production of cp27b (step 1) (which allows 1021 25 ohd to enter the mitochondria for transformation into its active form 1,25-(oh) 2 d 3 ), (step 2) as well 1022 as vitamin d receptor (vdr) (step 3), which by binding to 1,25-(oh) 2 d3 increases the production of 1023 cathelicidin. (step 4) 1024the antimicrobial activity of vitamin d appears to be primarily dependent on the induction of 1025 cathelicidins, which perform numerous functions that enhance both innate and adaptive immunity; help 1026 improve the digestion process within the phagolysosome through a non-oxygen dependent mechanism 1027 key: cord-305085-bv7udg9k authors: lawrence, robert m. title: chapter 13 transmission of infectious diseases through breast milk and breastfeeding date: 2011-12-31 journal: breastfeeding doi: 10.1016/b978-1-4377-0788-5.10013-6 sha: doc_id: 305085 cord_uid: bv7udg9k nan a large body of evidence clearly demonstrates the protective effects of breastfeeding and documents the transmission of specific infections to infants through breast milk. the fear and anxiety that arise with the occurrence of any infectious disease are even greater in the situation of the breastfeeding mother-infant dyad. uncertainty and lack of knowledge often lead to proscribing against breastfeeding out of fear, which then deprives the infant of the potential protective, nutritional, and emotional benefits of breastfeeding exactly at the time when they are most needed (see the discussion of immunologic benefits of human milk in chapter 5). decisions concerning breastfeeding in a mother with an infectious illness should balance the potential benefits of breastfeeding versus the known or estimated risk for the infant acquiring a clinically significant infection via breastfeeding and the potential severity of the infection. documenting transmission of infection from mother to infant by breastfeeding requires not only the exclusion of other possible mechanisms of transmission but also the demonstration of the infectious agent in the breast milk and a subsequent clinically significant infection in an infant that was caused by a plausible infectious process. the first step is to establish the occurrence of a specific infection (clinically or immunologically evident) in a mother and demonstrate the persistence of the infectious agent such that it could be transmitted to the infant. isolation or identification of the infectious agent from the colostrum, breast milk, or an infectious lesion of the breast is important but not necessarily proof of transmission to an infant. epidemiologic evidence of transmission must be considered, including identifying characteristics of the organism that relate an isolate from an infant to the maternal isolate. infectious organisms can reach the breast milk either by secretion in the fluid or cellular components of breast milk or by contamination of the milk at the time of or after expression. a reasonable mechanism of infection via breast milk should be evident and proved through either animal or human studies. demonstration of a subclinical or clinically evident infection in an infant should follow these outlined steps. exclusion of other possible mechanisms of transmission (exposure to mother or other persons/ animals via airborne, droplet, arthropod, or vector modes of transmission or through direct contact with other infectious fluids) would complete the confirmation of transmission of infection via breastfeeding. it is essential to exclude prenatal or perinatal transmission of infection to a fetus/infant, but doing this can often be difficult. clinical case reports or studies confirming the isolation of an infectious agent from the milk are important. to determine a reasonable estimate of the risk for infection via breast milk, larger epidemiologic studies are needed that compare infection rates in breastfed infants versus formula-fed infants, robert m. lawrence addressing the issues just identified. timing of breastfeeding is important relative to the timing of maternal infection and to the presence of a pathogen in colostrum or breast milk. the duration of breastfeeding is another important variable to consider in the estimate of risk because shedding of a pathogen in breast milk may be intermittent. these considerations are only some of the variables to be taken into account, in general, to assess the risk for transmission of an infectious agent from mother to infant via breast milk or breastfeeding. efforts to prove transmission of infection in a particular maternal-infant dyad can be just as difficult and must consider many of the same factors. this chapter focuses on a discussion of specific, clinically relevant, infectious agents and diseases, with reasonable estimates of the risk for infection to infants from breastfeeding. the basic tenet concerning breastfeeding and infection is that breastfeeding is rarely contraindicated in maternal infection. 243 the few exceptions relate to specific infectious agents with strong evidence of transmission and to the association of an infant' s illness with significant morbidity and mortality. the risk or benefit of breastfeeding relative to immunization of a mother or infant is discussed for certain microorganisms. appendix d addresses drugs in breast milk and includes table d-1, on antiinfective agents, and chapter 5 reviews how breastfeeding may protect against infection. chapter 21 addresses specific concerns relating to banked breast milk and includes standards developed by the human milk banking association of north america to guide the appropriate handling of banked human milk relative to possible infectious agents. isolation precautions have undergone some revisions in terminology and conceptualization. 143 understanding that the transmission of microorganisms can occur with a known infection and with unrecognized sources of infection, recommendations have been made for standard precautions to be applied to all patients to protect health care workers from potentially infectious body fluids. additionally, precautions based on the predominant modes of transmission have been recommended to protect against infection through the airborne route, direct contact, or contact with droplets. although these precautions are intended to be used in clinical situations to protect health care workers, they may be applied in certain situations to the mother-infant dyad to prevent transmission of infectious agents from one to the other or to other hospitalized mothers and infants. these precautions are useful most often when a mother and infant are still hospitalized. the use of such precautions within the home is not meant to limit breastfeeding. they are intended to allow breastfeeding in the majority of cases and to facilitate the continuation of breastfeeding with some additional safeguards in certain situations, after short temporary periods of stopping breastfeeding, and when to safely use expressed breast milk (see appendix f). standard precautions include preventing contact with blood, all body fluids, secretions and excretions, nonintact skin, and mucous membranes by (1) careful handwashing before and after every patient contact; (2) use of gloves when touching body fluids, nonintact skin, or mucous membranes or any items contaminated with body fluids (linens, equipment, devices, etc.); (3) use of nonsterile gowns to prevent contact of clothing with body fluids; (4) use of masks, eye protection, or face shields when splashing with body fluids is possible; and (5) appropriate disposal of these materials. standard precautions should be applied to all patients regardless of actual or perceived risks. the centers for disease control and prevention (cdc) does not consider breast milk a body fluid with infectious risks and thus these policies do not apply to breast milk. (see section on misadministration of breast milk later in this chapter as a possible exception to this concept.) in considering breastfeeding infant-mother dyads and standard precautions, body fluids other than breast milk should be avoided, and only in specified situations should breast milk also be avoided. in general, clothing or a gown for the mother and bandages, if necessary, should prevent direct contact with nonintact skin or secretions. avoiding infant contact with maternal mucous membranes requires mothers to be aware of and understand the risks and to make a conscious effort to avoid this type of contact. the use of gloves, gowns, and masks on infants for protection is neither practical nor appropriate. the recommendations concerning the appropriateness of breastfeeding and breast milk are addressed for specific infectious agents throughout this chapter. human immunodeficiency virus (hiv) infection is an example of one infection that can be prevented by the use of standard precautions, including avoiding breast milk and breastfeeding. the recommendations concerning breastfeeding and hiv and the various variables and considerations involved are discussed later. airborne precautions are intended to prevent transmission via droplet nuclei (dried respiratory particles smaller than 5 mcm that contain microorganisms and can remain suspended in the air for long periods) or dust particles containing microorganisms. airborne precautions include the use of a private room with negative-air-pressure ventilation and masks at all times. in the case of pulmonary tuberculosis (tb), respiratory protective devices (requiring personal fitting and seal testing before use) should be worn. airborne precautions are recommended with measles, varicella or disseminated zoster, and tb. breastfeeding in the presence of these maternal infections is prohibited for the infectious period. this is to protect against airborne transmission of the infection from the mother and to allow the infant to be fed the mother' s expressed breast milk by another individual. the exception to allowing breast milk would be local involvement of the breast by varicella-zoster lesions or mycobacterium tuberculosis, such that the milk becomes contaminated by the infectious agent. transmission via droplets occurs when an individual produces droplets that travel only a short distance in the air and then contact a new host's eyes, nose, mouth, or skin. the common mechanisms for producing droplets include coughing, sneezing, talking (singing or yelling), suctioning, intubation, nasogastric tube placement, and bronchoscopy. in addition to standard precautions applied to all patients, droplet precautions include the use of a private room (preferred) and a mask if within 3 feet (0.9 m) of the patient. droplet precautions are recommended for adenovirus, diphtheria, respiratory infections, haemophilus influenzae, neisseria meningitidis or invasive infection, influenza, mumps, mycoplasma, parvovirus, pertussis, plague (pneumonic), rubella, and streptococcal pharyngitis, pneumonia, or scarlet fever. the institution of droplet precautions with a breastfeeding mother who has these infections should be specified for each particular infection. this may require some period of separation for the infant and mother (for duration of the illness, for short-term or complete treatment of the mother, for the infectious period) with use of expressed breast milk for nutrition in the interim. prophylactic treatment of the infant, maternal use of a mask during breastfeeding or close contact combined with meticulous handwashing, and the mother's avoidance of touching her mucous membranes may be adequate and reasonable for certain infections. contact precautions are meant to prevent transmission of infection via direct contact (contact between the body surfaces of one individual with another) and indirect contact (contact of a susceptible host with an object contaminated with microorganisms from another individual). contact precautions include cohorting or a private room, gloves and gowns at all times, and handwashing after removal of gown and gloves. contact precautions are recommended for a long list of infections, such as diarrhea in diapered or incontinent patients with clostridium difficile infection, escherichia coli o157:h7, shigella, rotavirus, hepatitis a, respiratory illness with parainfluenza virus or respiratory syncytial virus (rsv), multidrug-resistant (mdr) bacteria (e.g., enterococci, staphylococci, gramnegative organisms), enteroviral infections, cutaneous diphtheria, impetigo, herpes simplex virus (hsv) infection, herpes zoster (disseminated or in immunocompromised individuals), pediculosis, scabies, staphylococcus aureus skin infection, viral hemorrhagic fevers (e.g., ebola, lassa), conjunctivitis and abscesses, cellulitis, or decubitus that cannot be contained by dressings. 94 for a breastfeeding infant-mother dyad, implementation of precautions for each of these infections in a mother requires meticulous attention to gowning and handwashing by the mother and a specialized plan for each situation. each of these transmission-based precautions can be used together for organisms or illnesses that can be transmitted by more than one route. they should always be used in conjunction with standard precautions, which are recommended for all patients. the red book: report of the committee on infectious diseases by the american academy of pediatrics (aap) 96 remains an excellent resource for infection control guidelines and recommendations to prevent transmission in specific situations and infections. routine culturing of breast milk or culturing breast milk to screen for infectious agents is not recommended except when the milk is intended as donor milk to another mother' s child directly or through human milk banks. see chapter 21 for specific bacterial count standards for raw donor milk and for pasteurization of donor milk. breastfeeding and the expression of or pumping of breast milk (referred to as expressed breast milk) for later use are not sterile activities. in general expressed breast milk should not contain large numbers of microorganisms (less than 10 4 for raw milk and less than 10 6 for milk to be pasteurized), nor should it contain potential pathogens such as s. aureus, β-hemolytic streptococci, pseudomonas species, proteus species, or streptococcus faecalis or faecium. few studies have examined "routine" culturing of milk and the significance of specific bacterial colony counts relative to illness in infants. the studies have been primarily concerned with premature or low-birth-weight (lbw) infants who remain hospitalized and are commonly fed via enteral tubes. a study from canada tested 7610 samples of milk for use in 98 preterm infants. 242 the study did not identify any adverse events in the infants attributed to organisms growing in the milk samples, and routine bacteriological testing of expressed breast milk was not recommended. a study from chicago examined gram-negative bacilli in the milk used in premature infants. 48 samples were tested before feeding and from the nasogastric tubes during feeding. milk samples from before feeding were less likely to contain gram-negative bacilli (36%) than milk samples from the nasogastric tubing (60%). feeding intolerance was observed when there were more than 10 3 colony-forming units per milliliter (cfu/ml), and episodes of sepsis were identified when the bacterial counts in the milk were greater than or equal to 10 6 cfu/ml. this study recommended the routine bacteriologic testing of expressed breast milk. another study from arkansas focused on contamination of feeding tubes during administration of expressed breast milk or formula. 277 ten infants in the neonatal intensive care unit (nicu) were exposed to greater than 10 5 gram-negative bacteria in their feeding tubes. the three infants who were fed expressed breast milk with contamination at greater than 10 5 organisms remained well, but the seven formula-fed infants with high levels of bacterial contamination in the feeding tubes developed necrotizing enterocolitis. the gram-negative bacteria with high level contamination in the feeding tubes were either enterobacter or klebsiella in all cases. many nicus consider 10 5 to 10 6 cfu/ml as the significant bacterial count for gram-negative bacilli in breast milk that places premature and lbw infants at greater risk for infection. even less data are available concerning specific bacterial colony counts for gram-positive organisms and the risk to the infant. generally less than 10 3 gram-positive organisms per ml of milk is considered acceptable, with only case reports and no controlled trials to support this cutoff. when the presence of an infectious illness in an infant and/or the breastfeeding mother' s breast when breast milk is seriously considered as a possible mechanism of transmission to the infant, culturing breast milk to identify the organism may be warranted and useful. more important than hurrying to culture breast milk is the careful instruction of mothers on the proper technique for collecting expressed breast milk, storing it, and cleaning the collection unit. the reinforcement of proper technique from time to time, especially when a question of contamination arises, is equally important. many small reports comment on the contamination of breast milk with different collection methods. relative comparisons suggest decreasing contamination of expressed breast milk when collected by the following methods; drip milk, hand pumped milk, manual expression, modern electric pumped milk. one group from malaysia published results showing no difference in contamination between milk collected by electric pump versus manual expression when collected in the hospital. expressed breast milk collected at home by breast pump had higher rates of contamination with staphylococci and gram-negative bacteria. 46 discussion continues about the need to discard the first few milliliters of milk to lower bacteria numbers in expressed breast milk without any evidence to suggest if this is truly necessary. 62, 337 no evidence shows that cleansing the breast with anything other than tap water decreases the bacterial counts in cultured expressed breast milk. 414 if an infant is directly breastfeeding, collecting milk for culture by manual expression and trying to obtain a "midstream" sample (as is done with "midstream" urine collection for culture) is appropriate. if an infant is being fed expressed breast milk, collecting and culturing the milk at different points during collection (utilizing the same technique the mother uses [manual expression, hand pump, or electric pump]) and administration is appropriate. this might include a sample from immediately after collection, another of stored expressed breast milk, and a sample of milk from the most recent infant feeding at the time the decision to culture is made. please see box 13-1 for the basic steps in culturing expressed breast milk. interpretation of such culture results can be difficult and should involve a pediatric infectious disease expert, a microbiologist, and hospital epidemiologist. additional organism identification is often required, utilizing antibiogram patterns or molecular fingerprinting by various techniques to correlate a bacterial isolate from breast milk with an isolate causing disease in infant or mother. misadministration of breast milk, also known as misappropriation, breast milk exposure, and accidental ingestion of breast milk, and other terms, is a medical-legal issue when it occurs in a hospital. this scenario occurs when one infant receives breast milk from another mother by mistake. this occurrence can be very distressing to the families (recipient patient, recipient parent, and donor mother) and medical staff involved. the actual risk for transmission of an infectious agent to an infant via a single ingestion of expressed breast milk (the most common occurrence) from another mother is exceedingly low. in this scenario, the cdc recommends treating this as an accidental exposure to a body fluid, which could be infectious. 84 bacterial, fungal, or parasitic infection from the one exposure is highly unlikely. the concern is about viral pathogens, known to be blood-borne pathogens, which have been identified in breast milk and include but are not limited to hepatitis b virus (hbv), hepatitis c virus (hcv), cytomegalovirus (cmv), west nile virus, human t-cell lymphotropic virus (htlv), and hiv. most hospitals have protocols for managing the situation from both the infection control/prevention and the medical-legal perspectives. these protocols advise informing both families about what occurred, discussing the theoretical risks of harm from the exposure, and reviewing test results and/ or recommending testing to determine the infectious status of each mother relative to the above mentioned viruses. hcv is not a contraindication to breastfeeding and west nile virus infection in lactating women is rare. 74, 177 neither infection has a documented effective form of prevention or acute treatment. testing either mother (donor or of recipient infant) for these agents is not warranted. prenatal testing for hiv is more commonplace throughout the world. the incidence of hiv among women of childbearing age is low, although it varies significantly by geographic location, and the hospital or locale-specific incidence would be important to know to estimate risk. most women and medical staff are aware that hiv can be transmitted by breastfeeding; therefore breast milk from hiv-positive women is rarely if ever stored in hospitals. the risk for transmission of hiv via breastfeeding is due to the volume of feedings over months (estimated at 400 to 500 feedings in the first 2 months of life) compared with the small "dose of exposure" from one or two "accidental feedings." transmission of hiv from a single breast milk exposure has never been documented. immunologic components in breast milk, along with time and cold of storage, inactivate the hiv in expressed breast milk. for these reasons, the risk for transmission of hiv via expressed breast milk consumed by another child is thought to be extremely low. htlv-i/ii infection in childbearing women is uncommon except in certain geographic regions (japan, africa, the caribbean, and south america). transmission of htlv via breast milk does occur and, like hiv, appears to be related to the volume and duration of breastfeeding. limiting the duration of breastfeeding is effective in decreasing transmission. 407, 409, 446 freezing and thawing expressed breast milk decreases the infectivity of htlv-i. 11 in areas of low prevalence, a positive test in a mother should be suspected to be a false positive test, and retesting with both antibody and polymerase chain reaction (pcr) testing should be performed. for these reasons the transmission of htlv-i/ii via accidental expressed breast milk exposure is thought to be extremely low. although the majority of women are cmv positive by childbearing age and cmv transmission occurs via breastfeeding, the risk for cmv in a full-term infant is low. premature or lbw infants are at greater risk for developing disease with cmv infection. freezing expressed breast milk (at −20° c) for 3 to 5 days significantly decreases the infectivity of cmv. here again the risk for cmv transmission from a single accidental exposure to cmv-positive expressed breast milk is extremely low. with a discussion of theoretical risk should be a discussion of possible preventive interventions, such as vaccination or antimicrobial postexposure prophylaxis. if donor mothers are positive for hbv, it is appropriate to give recipient infants hepatitis b virus immunoglobulin (hbig) and hbv vaccines if they have not already received them. if a box 13-1. culturing breast milk 1. wash hands as per routine. 2. wash breast with warm tap water and a clean washcloth. 3. manually express breast milk ("midstream" collection is not required) or attach breast pump flange (previously cleaned as per routine) for collection and collect milk. 4. place a 3 to 5 ml sample of expressed breast milk in a sterile container with a nonleakable top. 5. deliver to the labatory in less than 1 hour or refrigerate at 4° c until delivery. before sending samples to the viral lab or for nucleic acid/ po lymerase chain reaction (pcr) testing, confirm that the laboratory will accept and process the sample as requested and that the appropriate collection container and prelaboratory management of the specimen are utilized. 6 324 it may also be appropriate to consult a pediatric infectious disease specialist. additional important components of the hospital-based protocols for managing accidental expressed breast milk exposure include ongoing psychosocial support for the families and staff, documentation of medical discussions with the families, investigative steps, consents and interventions, and the demonstration of ongoing infection control efforts to prevent additional events of misadministration of breast milk. microorganisms produce a whole spectrum of clinical illnesses affecting mothers and infants. many situations carry the risk for transmission of the involved organism from a mother to the infant, or vice versa; in general, however, infants are at greater risk because of such factors as inoculum size and immature immune response. as always, an infection must be accurately diagnosed in a timely manner. empiric therapy and initial infection control precautions should begin promptly based on the clinical symptoms and the most likely etiologic agents. when dealing with a maternal infection, clarifying the possible modes of transmission and estimating the relative risk for transmission to the infant are essential first steps to decision-making about isolating a mother from her infant and the appropriateness of continuing breastfeeding or providing expressed breast milk. breastfeeding infrequently is contraindicated in specific maternal infections. 243 often the question of isolation and interruption of breastfeeding arises when symptoms of fever, pain, inflammation, or other manifestations of illness first develop in a mother and the diagnosis is still in doubt. a clinical judgment must be made based on the site of infection, probable organisms involved, possible or actual mechanisms of transmission of these organisms to the infant, estimated virulence of the organism, and likely susceptibility of the infant. additionally, by the time the illness is clearly recognized or diagnosed in a mother, the infant has already been exposed. given the dynamic nature of the immunologic benefits of breast milk, continuation of breastfeeding at the time of diagnosis or illness in a mother can provide the infant protection rather than continued exposure in most illnesses. stopping breastfeeding is rarely necessary. many situations associated with maternal fever do not require separation of mother and infant, such as engorgement of the breasts, atelectasis, localized nonsuppurative phlebitis, or urinary tract infections. appendix f lists a number of clinical syndromes, conditions, and organisms that require infection control precautions in hospitals. this appendix also includes short lists of possible etiologic agents for these conditions and appropriate precautions and recommendations concerning breastfeeding for different scenarios or organisms. this chapter considers specific infectious agents that are common, clinically significant, or of particular interest. bacillus anthracis, a gram-positive, spore-forming rod, causes zoonotic disease worldwide. human infection typically occurs due to contact with animals or their products. three forms of human disease occur: cutaneous anthrax (the most common), inhalation anthrax, and gastrointestinal (gi) disease (rare). person-to-person transmission can occur as a result of discharge from cutaneous lesions, but no evidence of human-to-human transmission of inhalational anthrax is available. no evidence of transmission of anthrax via breast milk exists. standard contact isolation is appropriate for hospitalized patients or patients with draining skin lesions. the issue of anthrax as a biologic weapon has exaggerated its importance as a cause of human disease. the primary concerns regarding anthrax and breastfeeding are antimicrobial therapy or prophylaxis in breastfeeding mothers and the possibility that infant and mother were exposed by intentional aerosolization of anthrax spores. the cdc published recommendations for treatment and prophylaxis in infants, children, and breastfeeding mothers. 72 the recommendations include the use of ciprofloxacin, doxycycline, amoxicillin, and several other agents without discontinuing breastfeeding. little available is information on ciprofloxacin and doxycycline in breast milk for prolonged periods of therapy or prophylaxis (60 days) and possible effects on infants' teeth and bone/cartilage growth during that time period. depending on the clinical situation and sensitivity testing of the identified anthrax strain, other agents can be substituted to complete the 60-day course. the cdc has approved the use of ciprofloxacin and doxycycline for breastfeeding women for short courses of therapy (less than several weeks). simultaneous exposure of infant and mother could occur from primary aerosolization or from spores "contaminating" the local environment. in either case decontamination of the mother-infant dyad' s environment should be considered. breastfeeding can continue during a mother' s therapy for anthrax as long as she is physically well. open cutaneous lesions should be carefully covered and, depending on the situation, simultaneous prophylaxis for the infant may be appropriate. considerable justifiable concern has been expressed because of the reports of sudden infant death from botulism. infant botulism is distinguished from food-borne botulism from improperly preserved food containing the toxin and from wound botulism from spores entering the wound. infant botulism occurs when the spores of clostridium botulinum germinate and multiply in the gut and produce the botulinal toxin in the gi tract. 17 the toxin binds presynaptically at the neuromuscular junction, preventing acetylcholine release. the clinical picture is a descending, symmetric flaccid paralysis. not every individual who has c. botulinum identified in the stool experiences a clinical illness. the age of infants seems to relate to their susceptibility to illness. the illness is mainly in children younger than 12 months of age; the youngest patient described in the literature was 6 days old. 17 most children become ill between 6 weeks and 6 months of age. the onset of illness seems to occur earlier in formula-fed infants compared with breastfed infants. when a previously healthy infant younger than 6 months of age develops constipation, then weakness and difficulty sucking, swallowing, crying, or breathing, botulism is a likely diagnosis. the organisms should be looked for in the stools, and electromyography may or may not be helpful. in a group reviewed by arnon et al, 19 33 of 50 patients hospitalized in california were still being nursed at onset of the illness. a beneficial effect of human milk was observed in the difference in the mean age at onset, with breastfed infants being twice as old as formula-fed infants with the disease. the breastfed infants' symptoms were milder. breastfed infants receiving iron supplements developed the disease earlier than those who were breastfed but unsupplemented. of the cases of sudden infant death from botulism, no infants were breastfed within 10 weeks of death. all were receiving iron-fortified formulas. in most cases, no specific food source of c. botulinum can be identified, but honey is the food most often implicated, and corn syrup has been implicated in infants older than 2 months of age. honey may contain botulism spores, which can germinate in the infant gut. however, botulin toxin has not been identified in honey. it has been recommended that honey not be given to infants younger than 12 months of age. this includes putting honey on a mother' s nipples to initiate an infant' s interest in suckling. arnon 18 reviewed the first 10 years of infant botulism monitoring worldwide. the disease has been reported from 41 of the 50 states in the united states and from eight countries on four continents. the relationship to breastfeeding and human milk is unclear. in general the acid stools (ph 5.1 to 5.4) of human milk fed infants encourage bifidobacterium species. few facultative anaerobic bacteria, or clostridia, existing as spores, are present in breastfed infants. in contrast, formula-fed infants have stool phs ranging from 5.9 to 8.0, with few bifidobacteria, primarily gram-negative bacteria, especially coliforms and bacteroides species. c. botulinum growth and toxin production decrease with declining ph and usually stops below ph 4.6. breast milk also contains additional protective immunologic components, which purportedly have activity against botulinum toxin. 269 the relationship between the introduction of solid foods or weaning in both formula-fed and breastfed infants and the onset of botulism remains unclear. for a breastfed infant, the introduction of solid food may cause a major change in the gut with a rapid rise in the growth of enterobacteria and enterococci followed by progressive colonization by bacteroides species, clostridia, and anaerobic streptococci. feeding solids to formula-fed infants minimally changes the gut flora as these organisms already predominate. although more hospitalized infants have been breastfed, sudden-death victims are younger and have been formula fed, which supports the concept of immunologic protection in the gut of a breastfed infant. much work remains to understand this disease. clinically, constipation, weakness, and hypotonicity in a previously healthy child constitute botulism until ruled out, especially with recent dietary changes. at this time, no reason exists to suspect breastfeeding as a risk for infant botulism, and some evidence suggests a possible protective effect from breastfeeding. breastfeeding should continue if botulism is suspected in mother or infant. brucella melitensis has been isolated in the milk of animals. foods and animals represent the primary sources of infection in humans. brucellosis demonstrates a broad spectrum of illness in humans, from subclinical to subacute to chronic illness with nonspecific signs of weakness, fever, malaise, body aches, fatigue, sweats, arthralgia, and lymphadenitis. in areas where the disease is enzootic, childhood illness has been described more frequently. the clinical manifestations in children are similar to those in adults. 259 infection can occur during pregnancy, leading to abortion (infrequently), and can produce transplacental spread, causing neonatal infection (rarely). the transmission of b. melitensis through breast milk has been implicated in neonatal infection. 259, 260 there have been eight cases of brucellosis in infants that were possibly associated with breastfeeding, but brucella was not isolated from the breast milk in any of those cases.* one case of brucellosis in an infant caused by breast milk transmission, with b. melitensis isolated from the breast milk, before antibiotic treatment was given to the mother has been documented. 415 additionally, brucella melitensis has been cultured from women with breast lumps and abscesses. 295 only one of six women described in this report was lactating at the time of diagnosis, and no information about the infant was given. brucellosis mastitis or abscess should be considered in women presenting with appropriate symptoms and occupational exposure to animals, contact with domestic animals in their environment, or exposure to animal milk or milk products (especially unpasteurized products). the breast inflammation tends to be granulomatous in nature (without caseation) and is often associated with axillary adenopathy; occasionally systemic illness in the woman is evident. treatment of brucellosis mastitis or abscess should be treated with surgery or fine needle aspiration as indicated and 4 to 6 weeks of combination antibiotic therapy with two or three medications. temporary interruption of breastfeeding with breast pumping and discarding the milk to continue stimulation of milk production is appropriate. breastfeeding should then continue after an initial period of 48 to 96 hours of therapy in the mother. acceptable medications for treating the mother while continuing breastfeeding include gentamicin, streptomycin, tetracycline, doxycycline, trimethoprim-sulfamethoxazole, and rifampin (see appendix d). chlamydial infection is the most frequent sexually transmitted disease (std) in the united states and is a frequent cause of conjunctivitis and pneumonitis in an infant from perinatal infection. the major determinant of whether chlamydial infection occurs in a newborn is the prevalence rate of chlamydial infection of the cervix. 364 chlamydial immunoglobulin a (iga) has been found in colostrum and breast milk in a small number of postpartum women who were seropositive for chlamydia. no information is available on the role of milk antibodies in protection against infection in infants. 389 it is not believed that chlamydia is transmitted via breast milk. use of erythromycin or tetracycline to treat mothers and oral erythromycin and ophthalmic preparations of tetracyclines, erythromycin, or sulfonamides to treat suspected infection in infants are appropriate during continued breastfeeding. separating infants from mothers with chlamydial infections or stopping breastfeeding is not indicated. simultaneous treatment of mothers and infants may be appropriate in some situations. corynebacterium diphtheriae causes several forms of clinical disease, including membranous nasopharyngitis, obstructive laryngotracheitis, and cutaneous infection. complications can include airway obstruction from membrane formation and toxinmediated central nervous system (cns) disease or myocarditis. the overall incidence of diphtheria has declined even though immunization does not prevent infection but does prevent severe disease from toxin production. fewer than five cases are reported annually in the united states. transmission occurs via droplets or direct contact with contaminated secretions from the nose, throat, eye, or skin. infection occurs in individuals whether they have been immunized or not, but infection in those not immunized is more severe and prolonged. as long as the skin of the breast is not involved, no risk for transmission exists via breast milk. no toxin-mediated disease from toxin transmitted through breast milk has been reported in an infant. breastfeeding, along with chemoprophylaxis and immunization of affected infants, is appropriate in the absence of cutaneous breast involvement (see appendix f). maternal infection with neisseria gonorrhoeae can produce a large spectrum of illness ranging from uncomplicated vulvovaginitis, proctitis, pharyngitis, conjunctivitis, or more severe and invasive disease, including pelvic inflammatory disease, meningitis, endocarditis, or disseminated gonococcal infection. the risk for transmission from mother to infant occurs mainly during delivery in the passage through the infected birth canal and occasionally from postpartum contact with the mother (or her partner). risk for transmission from breast milk is negligible, and n. gonorrhoeae does not seem to cause local infection of the breasts. infection in neonates is most often ophthalmia neonatorum and less often a scalp abscess or disseminated infection. mothers with presumed or documented gonorrhea should be reevaluated for other stds, especially chlamydia trachomatis and syphilis, because some therapies for gonorrhea are not adequate for either of these infections. with the definitive identification of gonorrhea in a mother, empiric therapy should begin immediately, and the mother should be separated from the infant until completion of 24 hours of adequate therapy. treatment of the mother with ceftriaxone, cefixime, penicillin, or erythromycin is without significant risk to the infant. single-dose treatment with spectinomycin, ciprofloxacin, ofloxacin, or azithromycin has not been adequately studied but presumably would be safe for the infant given the 24-hour separation and a delay in breastfeeding without giving the infant the expressed breast milk (pump and discard). doxycycline use in a nursing mother is not routinely recommended. careful preventive therapy for ophthalmia neonatorum should be provided, and close observation of the infant should continue for 2 to 7 days, the usual incubation period. empiric or definitive therapy against n. gonorrhoeae may be necessary depending on an infant' s clinical status and should be chosen on the basis of the maternal isolate' s sensitivity pattern. the mother should not handle other infants until after 24 hours of adequate therapy, and the infant should be separated from the rest of the nursery population, with or without breastfeeding. haemophilus influenzae type b can cause severe invasive disease such as meningitis, sinusitis, pneumonia, epiglottitis, septic arthritis, pericarditis, and bacteremia. shock can also occur. because the increased utilization of the h. influenzae type b conjugate vaccines, invasive disease caused by haemophilus has decreased dramatically, more than 95%, in the united states. most invasive disease occurs in children 3 months to 3 years of age. older children and adults rarely experience severe disease but do serve as sources of infection for young children. children younger than 3 months of age seem to be protected because of passively acquired antibodies from the mothers, and some additional benefits may be received from breast milk. transmission occurs through contact with respiratory secretions, and droplet precautions are protective. no evidence suggests transmission through breast milk or breastfeeding. evidence supports that breast milk limits the colonization of h. influenzae in the throat. 185 in the rare case of maternal infection, an inadequately immunized infant in a household is an indication to provide rifampin prophylaxis and close observation for all household contacts, including the breastfeeding infant. expressed breast milk can be given to an infant during the 24-hour separation after the mother' s initiation of antimicrobial therapy, or if the mother' s illness prevents breastfeeding, it can be reinitiated when the mother is able (see appendix f). although uncommon in the united states, leprosy occurs throughout the world. this chronic disease presents with a spectrum of symptoms depending on the tissues involved (typically the skin, peripheral nerves, and mucous membranes of the upper respiratory tract) and the cellular immune response to the causative organism, mycobacterium leprae. transmission occurs through long-term contact with individuals with untreated or multibacillary (large numbers of organisms in the tissues) disease. leprosy is not a contraindication to breastfeeding, according to jeliffe and jeliffe. 202 the importance of breastfeeding and urgency of treatment are recognized by experts who treat infants and mothers early and simultaneously. no mother-infant contact is permitted except to breastfeed. dapsone, rifampin, and clofazimine are typically and safely used for infant and mother regardless of the method of feeding (see appendix d). listeriosis is a relatively uncommon infection that can have a broad range of manifestations. in immunocompetent individuals, including pregnant women, the infection can vary from being asymptomatic to presenting as an influenza-like illness, occasionally with gi symptoms or back pain. severe disease occurs more frequently in immunodeficient individuals or infants infected in the perinatal period (pneumonia, sepsis, meningitis, granulomatosis infantisepticum). although listeriosis during pregnancy may manifest as mild disease in a mother and is often difficult to recognize and diagnose, it is typically associated with stillbirth, abortion, and premature delivery. it is thought that transmission occurs through the transplacental hematogenous route, infecting the amniotic fluid, although ascending infection from the genital tract may occur. 122 early and effective treatment of a woman can prevent fetal infection and sequelae. 206, 257 neonatal infection occurs as either early-or late-onset infection from transplacental spread late in pregnancy, ascending infection during labor and delivery, infection during passage through the birth canal, or, rarely, during postnatal exposure. no evidence in the literature suggests that listeria is transmitted through breast milk. treatment of the mother with ampicillin, penicillin, or trimethoprim-sulfamethoxazole is not a contraindication to breastfeeding as long as the mother is well enough. expressed colostrum or breast milk also can be given if the infant is able to feed orally. the management of lactation and feeding in neonatal listeriosis is conducted supportively, as it is in any situation in which an infant is extremely ill, beginning feeding with expressed breast milk or directly breastfeeding as soon as reasonable. n. meningitidis most often causes severe invasive infections, including meningococcemia or meningitis often associated with fever and a rash and progressing to purpura, disseminated intravascular coagulation, shock, coma, and death. transmission occurs via respiratory droplets. spread can occur from an infected, ill individual or from an asymptomatic carrier. droplet precautions are recommended until 24 hours after initiation of effective therapy. despite the frequent occurrence of bacteremia, no evidence indicates breast involvement or transmission through breast milk. the risk for maternal infection to an infant after birth is from droplet exposure and exists whether the infant is breastfeeding or bottle feeding. in either case the exposed infant should receive chemoprophylaxis with rifampin, 10 mg/kg/dose every 12 hours for 2 days (5 mg/kg/dose for infants younger than 1 month of age), or ceftriaxone, 125 mg intramuscularly (im) once, for children younger than 15 years of age. close observation of the infant should continue for 7 days, and breastfeeding during and after prophylaxis is appropriate. the severity of maternal illness may prevent breastfeeding, but it can continue if the mother is able, after the mother and infant have been receiving antibiotics for 24 hours. a period of separation from the index case for the first 24 hours of effective therapy is recommended; expressed breast milk can be given during this period. respiratory illness caused by bordetella pertussis evolves in three stages: catarrhal (nasal discharge, congestion, increasing cough), paroxysmal (severe paroxysms of cough sometimes ending in an inspiratory whoop, i.e., whooping cough), and convalescent (gradual improvement in symptoms). transmission is via respiratory droplets. the greatest risk for transmission occurs in the catarrhal phase, often before the diagnosis of pertussis. the nasopharyngeal culture usually becomes negative after 5 days of antibiotic therapy. chemoprophylaxis for all household contacts is routinely recommended. no evidence indicates transmission through breast milk, with similar risk to breastfed and bottle-fed infants. in the case of maternal infection with pertussis, chemoprophylaxis for all household contacts, regardless of age or immunization status, is indicated. in addition to chemoprophylaxis of the infant, close observation and subsequent immunization (in infants older than 6 weeks of age) are appropriate. despite chemoprophylaxis, droplet precautions and separation of mother and infant during the first 5 days of effective maternal antibiotic therapy are recommended. expressed breast milk can be provided to the infant during this period. staphylococcal infection in neonates can be caused by either s. aureus or coagulase-negative staphylococci (most often s. epidermidis) and can manifest in a wide range of illnesses. localized infection can be impetigo, pustulosis in neonates, cellulitis, or wound infection, and invasive or suppurative disease includes sepsis, pneumonia, osteomyelitis, arthritis, and endocarditis. s. aureus requires only a small inoculum (10 to 250 organisms) to produce colonization in newborns, most often of the nasal mucosa and umbilicus. 193 by the fifth day of life, 40% to 90% of the infants in the nursery will be colonized with s. aureus. 126 the organism is easily transmitted to others from mother, infant, family, or health care personnel through direct contact. outbreaks in nurseries were common in the past. mothers, infants, health care workers, and even contaminated, unpasteurized, banked breast milk were sources of infection. 298, 326 careful use of antibiotics, changes in nursery layout and procedures, standard precautions, and cohorting as needed decreased the spread of s. aureus in nurseries. now the occurrence of methicillin-resistant s. aureus (mrsa) is again a common problem, requiring cohorting, occasionally epidemiologic investigation, and careful infection control intervention. there are numerous reports of mrsa outbreaks in nicus.* the significance of colonization with staphylococcus and the factors leading to development of disease in individual patients are not clear. the morbidity and mortality related to s. aureus infection in neonates is well described. 192, 195, 219 management of such outbreaks has been reviewed. 147, 250 little has been written about the role of breastfeeding in colonization with s. aureus in nicus, wellbaby nurseries, or at home. mrsa is an important pathogen worldwide. community-acquired mrsa is different from hospital-acquired mrsa. community-acquired mrsa is usually defined as occurring in an individual without the common predisposing variables associated with hospital-acquired mrsa, lacking a mdr phenotype (common with hospital-acquired mrsa), frequently carrying multiple exotoxin virulence factors (such as panton-valentine leukocidin toxin), as well as carrying the smaller type iv staphylococcal cassette cartridge for the meca gene on a chromosome (hospital-acquired mrsa carries types i-iii staphylococcal cassette cartridge) and as being molecularly distinct from the common nosocomial strains of hospital-acquired mrsa. community-acquired mrsa is most commonly associated with skin and soft tissue infections and necrotizing pneumonia and less frequently associated with endocarditis, bacteremia, necrotizing fasciitis, myositis, osteomyelitis, or parapneumonic effusions. community-acquired mrsa is so common, it is now being observed in hospital outbreaks. 24, 144, 164, 358 community-acquired mrsa transmission to infants via breast milk has been reported. 34, 144, 210, 253, 286 premature or small-forgestational-age infants are more susceptible to and at increased risk for significant morbidity and mortality due to mrsa due in part to prolonged hospitalization, multiple courses of antibiotics, invasive procedures, and intravenous (iv) lines, their relative immune deficiency due to prematurity and illness, and altered gi tract due to different flora and decreased gastric acidity. therefore colonization with mrsa may pose a greater risk to infants in nicus in the long run. full-term infants develop pustulosis, cellulitis, and soft tissue infections, but rarely has invasive disease been reported. 82, 132, 298 fortunov et al 132 from texas reported 126 infections in term or late-preterm previously well infants including 43 with pustulosis, 68 with celluliltis or abscesses, and 15 invasive infections. family history of soft tissue skin infections and male sex were the only variables associated with risk for infection; cesarean delivery, breastfeeding, and circumcision were not. 132 nguyen et al 298 reported mrsa infections in a well-infant nursery from california. the eleven cases were all in full-term boys with pustularvesicular lesions in the groin. the infections were associated with longer length of stay, lidocaine injection use in infants, maternal age older than 30 years, and circumcision. breastfeeding was not an associated risk factor for mrsa infection. 298 the question of the role of circumcision in mrsa outbreaks was addressed by van howe and robson. 426 they reported that circumcised boys are at greater risk for staphylococcal colonization and infection. 426 others report that s. aureus carriage in infants (and subsequent infection) is most likely affected by multiple variables including infant factors (antibiotics, surgical procedures [circumcision being the most common], duration of hospital stay as a newborn), maternal factors (previous colonization, previous antibiotic usage, mode of delivery, length of stay), and environmental factors (mrsa in the family or hospital, nursery stay versus rooming-in, hand hygiene).* gerber et al 147 from the chicago area published a consensus statement for the management of mrsa outbreaks in the nicu. the recommendations, which were strongly supported by experimental, clinical, and epidemiologic data, included using a waterless, alcohol-based hand hygiene product, monitoring and enforcing hand hygiene, placing mrsa-positive infants in contact precautions with cohorting if possible, using gloves and gowns for direct contact and masks for aerosolgenerating procedures, cohorting nurses for care of mrsa-positive infants when possible, periodic screening of infants for mrsa using nares or nasopharyngeal cultures, clarifying the mrsa status of infants being transferred into the nicu, limiting overcrowding, and maintaining ongoing instruction and monitoring of health care workers in their compliance with infection control and hand hygiene procedures. evaluation of the outbreak could include screening of health care workers and environmental surfaces to corroborate epidemiologic data and laboratory molecular analysis of the mrsa strains if indicated epidemiologically. the use of mupirocin or other decolonizing procedures should be determined on an individual basis for each nicu. s. aureus is the most common cause of mastitis in lactating women. 317, 394, 395, 436 recurrence or persistence of symptoms of mastitis is a well described occurrence and an important issue in the management of mastitis. communityacquired mrsa has been associated with mastitis as well. 342 pasteurization, s. aureus was not detected in any of the 6820 samples of expressed breast milk. colonization of one infant with mrsa was identified, but no mrsa infections were identified in any of the hospitalized infants in the nicu during the 18 months of the study. 26 novak et al 300 identified mrsa in 57 of 500 samples (11%) of expressed fresh-frozen milk from 500 different donors from five brazilian milk banks. only 3 of the 57 samples were positive with high-level bacterial counts of mrsa: greater than 10,000 cfu/ml. these were the only samples that would not have been acceptable by bacteriological criteria according to brazilian or american criteria for raw milk use. they did not investigate other epidemiologic data to identify possible variables associated with low or high level contamination of expressed breast milk with mrsa. 300 management of an infant and/or mother with mrsa infection relative to breastfeeding or use of breast milk should be based on the severity of disease and whether the infant is premature, lbw, very-lowbirth-weight (vlbw), previously ill, or full term. full-term infants who themselves or their mothers develop mild to moderate infections (impetigo, pustulosis, cellulitis/abscess, mastitis/breast abscess, or soft tissue infection) can continue breast feeding after a short period of interruption (24 to 48 hours). during this time, pumping to maintain the milk supply should be supported, an initial evaluation for other evidence of infection should be done in the maternalinfant dyad, the infected child and/or mother should be placed on "commonly" effective therapy for the mrsa infection, and ongoing observation for clinical disease should continue. the mother and infant can "room-in" together in the hospital, if necessary, with standard and contact precautions. culturing the breast milk is not necessary. empiric therapy for the infant may be chosen based on medical concerns for the infant and the known sensitivity testing of the mrsa isolate. appropriate antibiotic choices include short-term use of azithromycin (erythromycin use during infancy [less than 6 weeks of age], or breastfeeding associated with an increased risk for hypertrophic pyloric stenosis), sulfamethoxazoletrimethoprim (in the absence of g6pd deficiency and older than 30 days of age), clindamycin, and perhaps linezolid for mild to moderate infections. infants in nicus (premature, lbw, vlbw, and/ or previously ill), who themselves or their mothers have a mrsa infection, should have the breast milk cultured and suspend breastfeeding or receiving breast milk from their mother until the breast milk is shown to be culture negative for mrsa. the infant should be treated as indicated for their infection or empirically treated if symptomatic (with pending culture results) and closely observed for development of new signs or symptoms of infection. pumping to maintain the milk supply and the use of banked breast milk are appropriate. the infant should be placed on contact precautions, in addition to the routine standard precautions. the infant can be cohorted with other mrsa-positive infants with nursing care cohorted as well. for the mother with mrsa infection, she should be instructed concerning hand hygiene, the careful collection, handling, and storage of breast milk, contact precautions to be used with her infant, and the avoidance of contact with any other infants. the mother can receive several possible antibiotics for mrsa that are compatible with breastfeeding when used for a short period. if the mother remains clinically well, including without evidence of mastitis, but her breast milk is positive for mrsa greater than 10 4 cfu/ml, empiric therapy to diminish or eradicate colonization would be appropriate. various regimens have been proposed to "eradicate" mrsa colonization, but none have been proven to be highly efficacious. these regimens usually include systemic antibiotics with one or two medications (rifampin added as the second medication), nasal mupirocin to the nares twice daily for 1 to 2 weeks with routine hygiene, with or without the usage of hexachlorophene (or similar topical agent or cleanser) for bathing during the 1 to 2 week treatment period. there is no clear information concerning the efficacy of using similar colonization eradication regimens for other household members or pets in preventing recolonization of the mother or infant. before reintroducing the use of the mother' s breast milk to the infant at least two to three negative breast milk cultures should be obtained after completion of therapy. routine screening of breast milk provided by mothers for their infants in nicus for the presence of mrsa is not indicated in the absence of mrsa illness in the maternal-infant dyad, an mrsa outbreak in nicus, or a high frequency of mrsa infection in a specific nicu. one case of staphylococcal scalded skin syndrome was reported by katzman and wald 208 in an infant breastfed by a mother with a lesion on her areola that did not respond to ampicillin therapy for 14 days. subsequently the infant developed conjunctivitis with s. aureus, which produced an exfoliative toxin, and a confluent erythematous rash without mucous membrane involvement or nikolsky sign. no attempt to identify the exfoliative toxin in the breast milk was made, and the breast milk was not cultured for s. aureus. the child responded to iv therapy with nafcillin. this emphasizes the importance of evaluating mother and infant at the time of a suspected infection and the need for continued observation of the infant for evidence of a pyogenic infection or toxin-mediated disease, especially with maternal mastitis or breast lesions. this case also raises the issue of when and how infants and their mothers become colonized with s. aureus and what factors lead to infection and illness in each. the concern is that staphylococcus can be easily transmitted through skin to skin contact, colonization readily occurs, and potentially serious illness can occur later, long after colonization. in the case of staphylococcal scalded skin syndrome or toxic shock syndrome (tss), the primary site of infection can be insignificant (e.g., conjunctivitis, infection of a circumcision, or simple pustulosis), but a clinically significant amount of toxin can be produced and lead to serious disease. toxic shock syndrome can result from s. aureus or streptococcus pyogenes infection and probably from a variety of antigens produced by other organisms. tss-1 has been identified as a "superantigen" that affects the t lymphocytes and other components of the immune response, producing an unregulated and excessive immune response and resulting in an overwhelming systemic clinical response. tss has been reported in association with vaginal delivery, cesarean delivery, mastitis, and other local infections in mothers. mortality rate in the mother may be as high as 5%. the case definition of staphylococcal tss includes meeting all four major criteria: fever greater than 38.9° c, rash (diffuse macular erythroderma), hypotension, and desquamation (associated with subepidermal separation seen on skin biopsy). the definition also includes involvement of three or more organ systems (gi, muscular, mucous membrane, renal, hepatic, hematologic, or central nervous system); negative titers for rocky mountain spotted fever, leptospirosis, and rubeola; and lack of isolation of s. pyogenes from any source or s. aureus from the cerebrospinal fluid (csf). 368 a similar case definition has been proposed for streptococcal tss. 451 aggressive empiric antibiotic therapy against staphylococci and streptococci and careful supportive therapy are essential to decreasing illness and death. oxacillin, nafcillin, first-generation cephalosporins, clindamycin, erythromycin, and vancomycin are acceptable antibiotics, even for a breastfeeding mother. the severity of illness in the mother may preclude breastfeeding, but it can be reinitiated when the mother is improving and wants to restart. standard precautions, but allowing breastfeeding, are recommended. staphylococcal enterotoxin f has been identified in breast milk specimens collected on days 5, 8, and 11 from a mother who developed tss at 22 hours postpartum. 428 s. aureus that produced staphylococcal enterotoxin f was isolated from the mother' s vagina but not from breast milk. infant and mother lacked significant antibody against staphylococcal enterotoxin f in their sera. the infant remained healthy after 60 days of follow-up. staphylococcal enterotoxin f is pepsin inactivated at ph 4.5 and therefore is probably destroyed in the stomach environment, presenting little or no risk to the breastfeeding infant. 35 breastfeeding can continue if the mother is able. coagulase-negative staphylococcal infection (the predominant isolate is staphylococcus epidermidis) produces minimal disease in healthy, full-term infants but is a significant problem in hospitalized or premature infants. factors associated with increased risk for this infection include prematurity, high colonization rates in specific nurseries, invasive therapies (e.g., iv lines, chest tubes, intubation), and antibiotic use. illness produced by coagulasenegative staphylococci can be invasive and severe in high-risk neonates, but rarely in mothers. there are reports of necrotizing enterocolitis associated with coagulase-negative staphylococcus. at 2 weeks of age, for infants still in the nursery, s. epidermidis is a frequent colonizing organism at multiple sites, with colonization rates as high as 75% to 100%. serious infections with coagulase-negative staphylococci (e.g., abscesses, iv line infection, bacteremia/sepsis, endocarditis, osteomyelitis) require effective iv therapy. many strains are resistant to penicillin and the semisynthetic penicillins, so sensitivity testing is essential. empiric or definitive therapy may require treatment with vancomycin, gentamicin, rifampin, teicoplanin, linezolid, or combinations of these for synergistic activity. transmission of infection in association with breastfeeding appears to be no more common than with bottle feeding. as with s. aureus infection control includes contact and standard precautions. occasionally, during presumed outbreaks, careful epidemiologic surveillance may be required, including cohorting, limiting overcrowding and understaffing, surveillance cultures of infants and nursery personnel, reemphasis of meticulous infection control techniques for all individuals entering the nursery, and, rarely, removal of colonized personnel from direct infant contact. s. epidermidis has been identified as part of fecal microbiota of breastfed infants. 203 s. epidermidis has also been identified in the breast milk of women with clinical evidence of mastitis. 107 nevertheless, s. epidermidis is rarely associated with infection in full-term infants. conceivably breast milk for premature infants could be a source of s. epidermidis colonization in the nicus. the other factors associated with hospitalization in a nicu noted previously presumably play a significant role in both colonization and infection in premature infants. the benefits of early full human milk feeding potentially outweigh the risk for colonization with s. epidermidis via breast milk. 348 ongoing education and assistance should be provided to mothers about the careful collection, storage, and delivery of human breast milk for their premature infants. 353 s. pyogenes (β-hemolytic group a streptococcus [gas]) is a common cause of skin and throat infections in children, producing pharyngitis, cellulitis, and impetigo. illnesses produced by gas can be classified in three categories: (1) impetigo, cellulitis, or pharyngitis without invasion or complication; (2) severe invasive infection with bacteremia, necrotizing fasciitis, myositis, or systemic illness (e.g., streptococcal tss); and (3) autoimmune-mediated phenomena, including acute rheumatic fever and acute glomerulonephritis. gas can also cause puerperal sepsis, endometritis, and neonatal omphalitis. significant morbidity and mortality rates are associated with invasive gas infection; mortality rate is 20% to 50%, with almost half the survivors requiring extensive tissue débridement or amputation. 347 infants are not at risk for the autoimmune sequelae of gas (rheumatic fever or poststreptococcal glomerulonephritis). transmission is through direct contact (rarely indirect contact) and droplet spread. outbreaks of gas in the nursery are rare, unlike with staphylococcal infections. either mother or infant can be initially colonized with gas and transmit it to the other. in the situation of maternal illness (extensive cellulitis, necrotizing fasciitis, myositis, pneumonia, tss, mastitis), it is appropriate to separate mother and infant until effective therapy (penicillin, ampicillin, cephalosporins, erythromycin) has been given for at least 24 hours. breastfeeding should also be suspended and may resume after 24 hours of therapy for the mother. group b streptococcus (gbs, streptococcus agalactiae) is a significant cause of perinatal bacterial infection. in parturient women, infection can lead to asymptomatic bacteriuria, urinary tract infection (often associated with premature birth), endometritis, or amnionitis. in infants, infection usually occurs between birth and 3 months of age (1 to 4 cases per 1000 live births). it is routinely classified by the time of onset of illness in the infant: early onset (0 to 7 days, majority less than 24 hours) and late onset (7 to 90 days, generally less than 4 weeks). infants may develop sepsis, pneumonia, meningitis, osteomyelitis, arthritis, or cellulitis. early-onset gbs disease is often fulminant, presenting as sepsis or pneumonia with respiratory failure; three quarters of neonatal disease is early onset. type iii is the most common serotype causing disease. transmission is believed to occur in utero and during delivery. colonization rates of mothers and infants vary between 5% and 35%. postpartum transmission is thought to be uncommon, although it has been documented. risk factors for early-onset gbs disease include delivery before 37 weeks' gestation, rupture of membranes for longer than 18 hours before delivery, intrapartum fever, heavy maternal colonization with gbs, or low concentrations of anti-gbs capsular antibody in maternal sera. 95 the common occurrence of severe gbs disease before 24 hours of age in neonates has lead to prevention strategies. revised guidelines developed by the aap committees on infectious diseases and on the fetus and newborn 95 have tried to combine various variables for increased risk for gbs infection (prenatal colonization with gbs, obstetric and neonatal risk factors for early-onset disease) and provide intrapartum prophylaxis to those at high risk ( figure 13 -1) the utilization of these guidelines and intrapartum prophylaxis across the united states has decreased the incidence of early-onset disease by approximately 80%. in 2005, the incidence of early-onset disease was 0.35 cases per 1000 live births. 95 late-onset gbs disease is thought to be the result of transmission during delivery or in the postnatal period from maternal, hospital, or community sources. dillon et al 112 demonstrated that 10 of 21 infants with late-onset disease were colonized at birth, but the source of colonization was unidentified in the others. gardner et al 141 showed that only 4.3% of 46 children who were culture negative for gbs at discharge from the hospital had acquired gbs by 2 months of age. anthony et al 15 noted that many infants are colonized with gbs, but the actual attack rate for gbs disease is low and difficult to predict. acquisition of gbs through breast milk or breastfeeding is uncommon. cases of late-onset gbs disease associated with gbs in the maternal milk have been reported. 58, 214, 313, 366, 438 some of the mothers had bilateral mastitis, at least one had delayed evidence of unilateral mastitis, and the others were asymptomatic. it was not clear when colonization of the infants occurred or when infection or disease began in the infants. the authors discussed the possibility that the infants were originally colonized during delivery, subsequently colonized the mothers' breasts during breastfeeding, and then became reinfected at a later time. butter and demoor 56 showed that infants initially colonized on their heads at birth had gbs cultured from their throat, nose, or umbilicus 8 days later. whenever they cultured gbs from the nipples of mothers, the authors also found it in the nose or throat of the infants. byrne et al 58 presented a review of gbs disease associated with breastfeeding and made recommendations to decrease the risk for transmission of gbs to infants via breastfeeding or breast milk. some of their recommendations included confirming appropriate collection and processing procedures for gbs cultures 370 in medical facilities to decrease false-negative cultures, reviewing proper hygiene for pumping, collection, and storage of expressed breast milk with mothers, reviewing the signs and symptoms of mastitis with mothers, and utilizing banked human milk as needed instead of mother' s milk. when a breastfed infant develops late-onset gbs disease, it is appropriate to culture the milk. (see discussion of culturing breast milk earlier in this chapter.) consider treatment of the mother to prevent reinfection if the milk is culture positive for gbs (greater than 10 4 cfu/ml), with or without clinical evidence of mastitis in the mother. withholding the mother' s milk until it is confirmed to be culture negative for a pathogen is appropriate and should be accompanied by providing ongoing support and instruction to the mother concerning pumping and maintaining her milk supply. serial culturing of expressed breast milk after treatment of the mother for gbs disease or colonization would be appropriate to insure the ongoing absence of a pathogen in the expressed breast milk. there are reports of reinfection of the infant from breast milk. 23, 225 eradication of gbs mucosal colonization in the infant or the mother may be difficult. some authors have recommended using rifampin prophylactically in both the mother and infant at the end of treatment to eradicate mucosal colonization. 23 (see chapter 16 for management of mastitis in the mother.) a mother or infant colonized or infected with gbs should be managed with standard precautions 94 while in the hospital. ongoing close evaluation of the infant for infection or illness and empiric therapy for gbs in the infant are appropriate until the child has remained well and cultures are subsequently negative at 72 hours. occasionally, epidemiologic investigation in the hospital will utilize culturing medical staff and family members to detect a source of late-onset gbs disease in the nursery. this can be useful when more than one case of late-onset disease is detected with the same serotype. cohorting in such a situation may be appropriate. selective prophylactic therapy for colonized infants to eradicate colonization may be considered, but unlike gas or staphylococcus infection, gbs infection in nurseries has not been reported to cause outbreaks. no data support screening all breastfeeding mothers and their expressed breast 4 cbc including wbc count with differential and blood culture. 5 applies only to penicillin, ampicillin, or cefazolin and assumes recommended dosing regimens. 6 a healthy-appearing infant who was ≥ 38 weeks' gestation at delivery and whose mother received ≥ 4 hours of iap before delivery may be discharged home after 24 hours if other discharge criteria have been met and a person able to comply fully with instructions for home observation will be present. if any one of these conditions is not met, the infant should be observed in the hospital for at least 48 hours and until criteria for discharge are achieved. milk for gbs as a reasonable method for protecting against spread of gbs infection via expressed breast milk. selective culturing of expressed breast milk may be appropriate in certain situations. the face of tuberculosis (tb) is changing throughout the world. in the united states the incidence of tb rose during 1986 through 1993 and has been declining since then. 60 increased rates of tb were noted in adults between 25 and 45 years of age, and because these are the primary childbearing years, the risk for transmission to children increased. tb during pregnancy has always been a significant concern for patients and physicians alike. 340 it is now clear that the course and prognosis of tb in pregnancy are less affected by the pregnancy and more determined by the location and extent of disease, as defined primarily by chest radiograph, and by the susceptibility of the individual patient. untreated tb in pregnancy is associated with maternal and infant mortality rates of 30% to 40%. 365 effective therapy is crucial to the clinical outcome in both pregnant and nonpregnant women. tb during pregnancy rarely results in congenital tb. any individual in a high-risk group for tb should be screened with a tuberculin skin test (tst). no contraindication or altered responsiveness to the tst exists during pregnancy or breastfeeding. interpretation of the tst should follow the most recent guidelines, using different sizes of induration in different-risk populations as cutoffs for a positive test, as proposed by the cdc. 68 figure 13 -2 outlines the evaluation and treatment of a pregnant woman with a positive tst. 398 treatment of active tb should begin as soon as the diagnosis is made, regardless of the fetus' gestational age, because the risk for disease to mother and fetus clearly outweighs the risks of treatment. isoniazid, rifampin, and ethambutol have been used safely in all three trimesters. isoniazid and pyridoxine therapy during breastfeeding is safe, although the risk for hepatotoxicity in the mother may be a concern during the first 2 months postpartum. 391 congenital tb is extremely rare if one considers that 7 to 8 million cases of tb occur each year worldwide and that less than 300 cases of congenital tb have been reported in the literature. as with other infectious diseases presenting in the perinatal period, distinguishing congenital infection from perinatal or postnatal tb in infants can be difficult. postnatal tb infection in infancy typically presents with severe disease and extrapulmonary extension (meningitis, lymphadenopathy, and bone, liver, spleen involvement). airborne transmission of tb to infants is the major mode of postnatal infection because of close and prolonged exposure in enclosed spaces, especially in their own household, to any adult with infectious pulmonary tb. potential infectious sources could be the mother or any adult caregiver, such as babysitters, day care workers, relatives, friends, neighbors, and even health care workers. the suspicion of tb infection or disease in a household with possible exposure of an infant is a highly anxiety-provoking situation ( figure 13 -3). although protection of an infant from infection is foremost in everyone' s mind, separation of the infant from the mother should be avoided when reasonable. every situation is unique, and the best approach will vary according to the specifics of the case and accepted principles of tb management. the first step in caring for the potentially exposed infant is to determine accurately the true tb status of the suspected case (mother or household contact). this prompt evaluation should include a complete history (previous tb infection or disease, previous or ongoing tb treatment, tst status, symptoms suggestive of active tb, results of most recent chest radiograph, sputum smears, or cultures), physical examination, a tst if indicated, a new chest radiograph, and mycobacterial cultures and smears of any suspected sites of infection. all household contacts should be evaluated promptly, including history and tst with further evaluation as indicated. 68 continued risk to the infant can occur from infectious household contacts who have not been effectively evaluated and treated. an infant should be separated temporarily from the suspected source if symptoms suggest active disease or a recent tst documents conversion, and separation should continue until the results of the chest radiograph are seen. because of considerable variability in the course of illness and the concomitant infectious period, debate continues without adequate data about the appropriate period of separation. 278 this should be individualized given the specific situation. hiv testing and assessment of the risk for mdr tb should be done in every case of active tb. sensitivity testing should be done on every mycobacterium tuberculosis isolate. table 13 -1 summarizes the management of the newborn infant whose mother (or other household contact) has tb. initiation of prophylactic isoniazid therapy in the infant has been demonstrated to be effective in preventing tb infection and disease in the infant. therefore continued separation of infant and mother is unnecessary after therapy in both mother and child has begun. 114 the real risk to an infant requiring separation is from airborne transmission. separation of the infant from a mother with active pulmonary tb is appropriate, regardless of the method of feeding. however, in many parts of the world, after therapy in the mother and prophylaxis with isoniazid in the infant has begun, the infant and mother are not separated. with or without separation, the mother and infant should continue to be closely observed throughout the course of maternal therapy to ensure good compliance with medication by both mother and infant and to identify, early on, any symptoms in the infant suggestive of tb. tuberculous mastitis occurs rarely in the united states but does occur in other parts of the world* and can lead to infection in infants, frequently involving the tonsils. a mother usually has a single breast mass and associated axillary lymph node swelling and infrequently develops a draining sinus. tb of the breast can also present as a painless mass or edema. involvement of the breast can occur with or without evidence of disease at other sites. evaluation of extent of disease is appropriate, including lesion cultures by needle aspiration, biopsy, or wedge resection and milk cultures. therapy should be with multiple anti-tb medications, but surgery should supplement this, as needed, to remove extensive necrotic tissue or a persistently draining sinus. 16 neither breastfeeding nor breast milk feeding should be done until the lesion is healed, usually 2 weeks or more. continued anti-tb therapy for 6 months in the mother and isoniazid for the infant for 3 to 6 months is indicated. in the absence of tuberculous breast infection in the mother, transmission of tb through breast milk has not been documented. thus even though temporary separation of infant and mother may occur pending complete evaluation and initiation of adequate therapy in the mother and prophylactic isoniazid therapy (10 mg/kg/day as a single daily dose) in the infant, breast milk can be expressed and given to the infant during the short separation. breastfeeding can safely continue whether the mother, infant, or both are receiving anti-tb therapy. anti-tb medications (isoniazid, rifampin, pyrazinamide, aminoglycosides, ethambutol, ethionamide, p-aminosalicylic acid) have been safely used in infancy, and therefore the presence of these medications in smaller amounts in breast milk is not a contraindication to breastfeeding. although conflicting, reports indicate that breastfeeding by tst-positive mothers does influence infants' responses to bacille calmette-guérin notes: 1 further workup should always include evaluation of tb status of all other household (or close) contacts by tuberculin skin testing (tst), review of symptoms, physical examination, and chest x-ray (cxr). sputum smears and cultures should be done as indicated. 2 separation should occur until interpretation of cxr film confirms absence of active disease, or, with active disease, separation should continue until individual is no longer considered infectious: three negative consecutive sputum smears, adequate ongoing empiric therapy, and decreased fever, cough, and sputum production. separation means in a different house or location, not simply separate rooms in a household. duration of separation should be individualized for each case in consultation with tb specialist. 3 this assumes no evidence of breast involvement, suspected tb mastitis, or lesion (except in status 5, when breast involvement is considered). risk to infant is via aerosolized bacteria in sputum from the lung. expressed breast milk can be given even if separation of mother and infant is advised. 4 tst positive, no symptoms or physical findings suggestive of tb, negative cxr film. 5 prophylactic therapy: isoniazid 10 mg/kg/day, maximum 300 mg for 6 months; pyridoxine 25 to 50 mg/day for 6 months. empiric therapy: standard three-or four-drug regimens for 2 months, and treatment should continue for total of 6 months with isoniazid and rifampin when organism is shown to be sensitive. suspected multidrug-resistant (mdr) tb requires consultation with tb specialist to select optimum empiric regimen and for ongoing monitoring of therapy and clinical response. vaccine, the tst, and perhaps the m. tuberculosis bacillus. despite efforts to identify either a soluble substance or specific cell fractions (gamma/delta t cells) in colostrum and breast milk that affect infants' immune responsiveness, no unified theory explains the various reported changes and no evidence has identified a consistent, clinically significant effect. 39, 213, 319, 367 viral infections arboviruses arboviruses were originally a large collection of viruses grouped together because of the common mode of transmission through arthropods. they have now been reclassified into several different families: bunyaviridae, togaviridae, flaviviridae, reoviridae, and others. they include more than 30 human pathogens. these organisms primarily produce either cns infections (encephalitis, meningoencephalitis) or undifferentiated illnesses associated with fever and rash, severe hemorrhagic manifestations, and involvement of other organs (hepatitis, myalgia, polyarthritis). infection with this array of viruses may also be asymptomatic and subclinical, although how often this occurs is uncertain. some of the notable human pathogens include bunyaviridae (california serogroup viruses), hantavirus, hantaan virus, phlebovirus (rift valley fever), nairovirus (crimean-congo hemorrhagic fever), alphavirus (western, eastern, and venezuelan equine encephalomyelitis viruses, chikungunya virus), flavivirus (st. louis encephalitis virus, japanese encephalitis virus, dengue viruses, yellow fever virus, tick-borne encephalitis viruses), and orbivirus (colorado tick fever). other than for crimean-congo hemorrhagic fever and for reported cases of colorado tick fever associated with transfusion, direct person-to-person spread has rarely been described. recent outbreaks of chikungunya virus infection in reunion island and in india described infection in young infants probably secondary to vertical spread from mother to infant transplacentally. 146, 339, 422 a few cases of early fetal deaths were associated with infection in pregnant women. the cases of vertical transmission occurred with near-term infection in the mothers, and the infants developed illness within 3 to 7 days of delivery. 146, 339 no evidence for transmission via breast milk or breastfeeding is available. little evidence indicates that these organisms can be transmitted through breast milk. the exceptions to this include evidence of transmission of two flaviviruses via breast milk, west nile virus, and yellow fever vaccine virus. standard precautions are generally sufficient. with any of these infections in a breastfeeding mother, the severity of the illness may determine the mother' s ability to continue breastfeeding. providing the infant with expressed breast milk is acceptable. (see the discussion of west nile virus and yellow fever vaccine virus later in this chapter.) in general, treatment for these illnesses is supportive. however, ribavirin appears to decrease the severity of and mortality from hantavirus pulmonary syndrome, hemorrhagic fever with renal failure, and crimean-congo hemorrhagic fever. ribavirin has been described as teratogenic in various animal species and is contraindicated in pregnant women. no information is available concerning ribavirin in breast milk, with little information available on the use of iv or oral ribavirin in infants. arenaviruses are single-stranded ribonucleic acid (rna) viruses that infect rodents and are acquired by humans through the rodents. the six major human pathogens in this group are (1) lymphocytic 6 sensitivity testing should be done on any positive culture. 7 isoniazid 10 mg/kg/day for 3 to 9 months depending on mother's or contact's status; repeat tst at 3 months and obtain normal cxr in infant before stopping isoniazid. before beginning therapy, workup of infant for congenital or active tb may be appropriate. this workup should be determined by clinical status of infant and suspected potential risk, and may include tst after 4 weeks of age, with cxr, complete blood count, and erythrocyte sedimentation rate, liver function tests, cerebrospinal fluid analysis, gastric aspirates, sonography/computed tomography of liver/spleen, and chest if congenital tb is suspected. 8 breastfeeding is proscribed when separation of mother and infant is indicated because of risk for aerosolized transmission of bacteria. expressed breast milk given to infant via bottle is acceptable in absence of mastitis or breast lesions. 9 consult with tb specialist about mdr tb. empiric therapy will be chosen based on the most recent culture sensitivities of index patient or perhaps suspected source case, if known, as well as medication toxicities and other factors. 10 tb mastitis usually involves a single breast with associated axillary lymph node swelling and, infrequently, a draining sinus tract. it can also present as a painless mass or edema of breast. 11 with suspected mastitis or breast lesion caused by tb, even breast milk is contraindicated until lesion or mastitis heals, usually 2 weeks or more. 12 patient has a documented, recent tst conversion but has not been completely evaluated. evaluation should begin and cxr done and evaluated in less than 24 hours to minimize separation of this person from infant. further workup should proceed as indicated by symptoms, physical findings, and cxr results. choriomeningitis virus, (2) lassa fever virus, (3) junin virus (argentine hemorrhagic fever), (4) machupo virus (bolivian hemorrhagic fever), (5) guanarito virus (venezuelan hemorrhagic fever), and (6) sabia virus. the geographic distribution of these viruses and the illness they cause are determined by the living range of the host rodent (reservoir). the exact mechanism of transmission to humans is unknown and hotly debated. 25, 69, 131 direct contact and aerosolization of rodent excretions and secretions are probable mechanisms. lymphocytic choriomeningitis virus is well recognized in europe, the americas, and other areas. perinatal maternal infection can lead to severe disease in the newborn, but no evidence suggests transmission through breast milk. 28, 224 standard precautions with breastfeeding are appropriate. lassa fever (west africa) and argentine hemorrhagic fever (argentine pampas) are usually more severe illnesses with dramatic bleeding and involvement of other organs, including the brain. these fevers more frequently lead to shock and death than do the forms of hemorrhagic fever caused by the other viruses in this group. person-to-person spread of lassa fever is believed to be common, and transmission within households does occur. 212 this may relate to prolonged viremia and excretion of the virus in the urine of humans for up to 30 days. 330 the possibility of persistent virus in human urine, semen, and blood after infection exists for each of the arenaviruses. the possibility of airborne transmission is undecided. current recommendations by the cdc 69 are to use contact precautions for the duration of the illness in situations of suspected viral hemorrhagic fever. no substantial information describes the infectivity of various body fluids, including breast milk, for these different viral hemorrhagic fevers. considering the severity of the illness in mothers and the risk to the infants, it is reasonable to avoid breastfeeding in these situations if alternative forms of infant nutrition can be provided. as more information becomes available, reassessment of these recommendations is advisable. a vaccine is in clinical trials in endemic areas for junin virus and argentine hemorrhagic fever. preliminary studies suggest it is effective, but data are still being accumulated concerning the vaccine' s use in children and pregnant or breastfeeding women. cytomegalovirus (cmv) is one of the human herpesviruses. congenital infection of infants, postnatal infection of premature infants, and infection of immunodeficient individuals represent the most serious forms of this infection in children. the time at which the virus infects the fetus or infant and the presence or absence of antibodies against cmv from the mother are important determinants of the severity of infection and the likelihood of significant sequelae (congenital infection syndrome, deafness, chorioretinitis, abnormal neurodevelopment, learning disabilities). 234 about 1% of all infants are born excreting cmv at birth, and approximately 5% of these congenitally infected infants will demonstrate evidence of infection at birth (approximately five symptomatic cases per 10,000 live births). approximately 15% of infants born after primary infection in a pregnant woman will manifest at least one sequela of prenatal infection. 96 various studies have detected that 3% to 28% of pregnant women have cmv in cervical cultures and that 4% to 5% of pregnant women have cmv in their urine. 120, 172 perinatal infection certainly occurs through contact with virus in these fluids but usually is not associated with clinical illness in fullterm infants. the lack of illness is thought to result from transplacental passive transfer of protective antibodies from the mother. postnatal infection later in infancy occurs via breastfeeding or contact with infected fluids (e.g., saliva, urine) but, again, rarely causes clinical illness in full-term infants. seroepidemiologic studies have documented transmission of infection in infancy, with higher rates of transmission occurring in daycare centers, especially when the prevalence of cmv in the urine and saliva is high. cmv has been identified in the milk of cmv-seropositive women at varying rates (10% to 85%) using viral cultures or cmv deoxyribonucleic acid (dna) pcr. 172, 301, 397, 430 cmv is more often identified in the breast milk of seropositive mothers than in vaginal fluids, urine, and saliva. the cmv isolation rate from colostrum is lower than that from mature milk. 172, 396 the reason for the large degree of variability in identification of cmv in breast milk in these studies probably relates to the intermittent nature of reactivation and excretion of the virus in addition to the variability, frequency, and duration of sampling of breast milk in the different studies. some authors have hypothesized that the difference in isolation rates between breast milk and other fluids is caused by viral reactivation in cells (leukocytes or monocytes) in the breast leading to "selective" excretion in breast milk. 301 vochem et al 430 reported that the rate of virolactia was greatest at 3 to 4 weeks postpartum, and yeager et al 455 reported significant virolactia between 2 and 12 weeks postpartum. antibodies (e.g., secretory iga) to cmv are present in breast milk, along with various cytokines and other proteins (e.g., lactoferrin). these may influence virus binding to cells, but they do not prevent transmission of infection.* several studies have documented increased rates of postnatal cmv infection in breastfed infants (50% to 69%) compared with bottle-fed infants (12% to 27%) observed through the first year of life 120, 281, 397, 430 in these same studies, full-term infants who acquired cmv infection postnatally were only rarely mildly symptomatic at the time of seroconversion or documented viral excretion. also, no evidence of late sequelae from cmv was found in these infants. postnatal exposure of susceptible infants to cmv, including premature infants without passively acquired maternal antibodies against cmv, infants born to cmv-seronegative mothers, and immunodeficient infants, can cause significant clinical illness (pneumonitis, hepatitis, thrombocytopenia).* in one study of premature infants followed up to 12 months, vochem et al 430 found cmv transmission in 17 of 29 infants (59%) exposed to cmv virolactia and breastfed compared with no infants infected of 27 exposed to breast milk without cmv. no infant was given cmv-seropositive donor milk or blood. five of the 12 infants who developed cmv infection after 2 months of age had mild signs of illness, including transient neutropenia, and only one infant had a short increase in episodes of apnea and a period of thrombocytopenia. five other premature infants with cmv infection before 2 months of age had acute illness, including sepsis-like symptoms, apnea with bradycardia, hepatitis, leukopenia, and prolonged thrombocytopenia. 430 vollmer et al 431 followed premature infants with early postnatal cmv infection acquired through breast milk for 2 to 4.5 years to assess neurodevelopment and hearing function. none of the children had sensorineural hearing loss. there was no difference between the 22 cmv-infected children and 22 matched premature control cmv-negative infants in terms of neurologic, speech and language, or motor development. 431 neuberger et al 296 examined the symptoms and neonatal outcome of cmv infection transmitted via human milk in premature infants in a case-control fashion; 40 cmv-infected premature infants were compared with 40 cmv-negative matched premature infants. neutropenia, thrombocytopenia, and cholestasis were associated with cmv infection in these infants. no other serious effects or illnesses were found directly associated with the infection including intraventricular hemorrhage, periventricular leukomalacia, retinopathy of prematurity, necrotizing enterocolitis, bronchopulmonary dysplasia, duration of mechanical ventilation or oxygen therapy, duration of hospital stay or weight, gestational age, or head circumference at the time of discharge. exposure of cmv-seronegative or premature infants to cmv-positive milk (donor or natural mother' s) should be avoided. 379 various methods of inactivating cmv in breast milk have been reported, including holder pasteurization, freezing (−20° c for 3 days), and brief high temperature (72° c for 10 seconds). 120, 135, 155, 393, 455 one small, prospective study suggests that freezing breast milk at −20°c for 72 hours protects premature infants from cmv infection via breast milk. sharland et al 379 reported on 18 premature infants (less than 32 weeks) who were uninfected at birth and exposed to breast milk from their cmv seropositive mothers. only one of 18 (5%) infants became positive for cmv at 62 days of life, and this infant was clinically asymptomatic. this transmission rate is considerably lower than others reported in the literature. cmvseronegative and leukocyte-depleted blood products were used routinely. banked breast milk was pasteurized and stored at −20° c for various time periods and maternal expressed breast milk was frozen at −20° c before use whenever possible. the infants received breast milk for a median of 34 days (range 11 to 74 days) and they were observed for a median of 67 days (range 30 to 192 days). breast milk samples pre-or postfreezing were not analyzed by pcr or culture for the presence of cytomegalovirus. 379 buxmann et al 57 demonstrated no transmission of cmv in 23 premature infants receiving thawed frozen breast milk until 33 weeks (gestational age + postnatal age) (less than or equal to 31 weeks gestational age) born to 19 mothers who were cmv-igg negative. cmv infection was found in five premature infants of 35 infants born to 29 mothers who were cmv-igg positive and who provided breast milk for their infants. three of the five children remained asymptomatic. one child development a respirator-dependent pneumonia and the second developed an upper respiratory tract infection and thrombocytopenia in association with their cmv infections. 57 yasuda et al 454 reported on 43 preterm infants (median gestational age 31 weeks) demonstrating a peak in cmv dna copies, detected by a real-time pcr assay, in breast milk at 4 to 6 weeks postpartum. thirty of the 43 infants received cmv dna-positive breast milk. three of the 30 had cmv dna detected in their sera, but none of the three had symptoms suggestive of cmv infection. much of the breast milk had been stored at −20° c before feeding, which the authors propose is the probable reason for less transmission in this cohort. 454 lee et al 248 reported on the use of maternal milk frozen at −20°c for a minimum of 24 hours before feeding to premature infants in a nicu; 23 infants had cmv-seropositive mothers and 39 infants had cmv-seronegative mothers. two infants developed cmv infection, which was symptomatic. they were both fed frozen thawed milk from cmv-seropositive mothers. 248 others have reported individual cases of cmv infection in premature infants despite freezing and thawing breast milk. 268, 314 simple freezing and thawing of breast milk does not completely prevent transmission of cmv to premature infants. the efficacy of freezing and thawing breast milk for varying lengths of time to prevent cmv infection in premature infants has not been studied prospectively in a randomized controlled trial. eleven of 36 neonatal units in sweden (27 of which have their own milk banks) freeze maternal milk to reduce the risk for cmv transmission to premature infants. 314 a prominent group of neonatologists and pediatric infectious disease experts in california who recognize the significant benefits of providing human milk to premature and lbw infants recommend screening mothers of premature infants for cmv igg at delivery and, when an infant' s mother is cmv igg positive at delivery, using either pasteurized banked human milk or frozen then thawed maternal breast milk for premature infants until they reach the age of 32 weeks. 445 in consideration of the low rates of cmv virolactia in colostrum 169, 397 and the predominant occurrence of virolactia between 2 and 12 weeks (peak at 3 to 4 weeks) postpartum, 430,455 they reasonably propose beginning colostrum and breast milk feedings for all infants until the maternal cmv serologic screening is complete. they appropriately recommend close observation and follow-up of premature infants older than 3 weeks of age for signs, symptoms, and laboratory changes of cmv infection until discharge from the hospital. 445 cmv-seropositive mothers can safely breastfeed their full-term infants because, despite a higher rate of cmv infection than in formula-fed infants observed through the first year of life, infection in this situation is not associated with significant clinical illness or sequelae. dengue viruses (serotypes dengue 1 to 4) are flaviviruses associated primarily with febrile illnesses and rash; dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. the mosquito aedes aegypti is the main vector of transmission of dengue virus in countries lying between latitudes 35 degrees north and 35 degrees south. more than 2.5 billion people live in areas where transmission occurs; dengue virus infects over 100 million individuals a year and casuses approximately 24,000 deaths a year. 159, 163 although dengue hemorrhagic fever and dengue shock syndrome occur frequently in children younger than 1 year of age, they are infrequently described in infants younger than 3 months of age. 167 there are also differences in the clinical and laboratory findings of dengue virus infection in children compared with adults. 222 boussemart et al 49 reported on two cases of perinatal/prenatal transmission of dengue and discussed eight additional cases in neonates from the literature. prenatal or intrapartum transmission of the same type of dengue as the mother was confirmed by serology, culture, or pcr. phongsamart et al 333 described three additional cases of dengue virus infection late in pregnancy and apparent transmission to two of the three infants with passive acquisition of antibody in the third infant. sirinavin et al 386 reported on 17 cases in the literature of vertical dengue infection, all presenting at less than 2 weeks of age, but no observations or discussion of breast milk or breastfeeding as a potential source of infection were published. watanaveeradej et al 439 presented an additional three cases of dengue infection in infants documenting normal growth and development at follow-up at 12 months of age. it has been postulated that more severe disease associated with dengue disease occurs when an individual has specific igg against the same serotype as the infecting strain in a set concentration, leading to antibody-dependent enhancement of infection. the presence of preexisting dengue serotype specific igg in an infant implies either previous primary infection with the same serotype, passive acquisition of igg from the mother (who had a previous primary infection with the same serotype), or perhaps acquisition of specific igg from breast milk. watanaveeradej et al 439 documented transplacentally transferred antibodies against all four serotypes of dengue virus in 97% of 2000 cord sera at delivery. follow-up of 100 infants documented the loss of antibodies to dengue virus over time with losses of 3%, 19%, 72%, 99%, and 100% at 2, 4, 6, 9, and 12 months of age, respectively. no evidence is available in the literature about more severe disease in breastfed infants compared with formula-fed infants. no interhuman transmission of dengue virus in the absence of the mosquito vector and no evidence of transmission via breast milk are known. only one report of a factor in the lipid portion of breast milk, which inhibits the dengue virus, is available, and no evidence for antibody activity against dengue virus in human breast milk is known. 127 breastfeeding during maternal or infant dengue disease should continue as determined by the mother' s or infant' s severity of illness. epstein-barr virus (ebv) is a common infection in children, adolescents, and young adults. it is usually asymptomatic but most notably causes infectious mononucleosis and has been associated with chronic fatigue syndrome, burkitt lymphoma, and nasopharyngeal carcinoma. because ebv is one of the human herpesviruses, concern has been raised about lifelong latent infection and the potential risk for infection to a fetus and neonate from the mother. primary ebv infection during pregnancy is unusual because few pregnant women are susceptible. 149, 189 although abortion, premature birth, and congenital infection from ebv are suspected, no distinct group of anomalies is linked to ebv infection in fetus or neonate. also, no virologic evidence of ebv as the cause of abnormalities was found in association with suspected ebv infection. culturing of ebv from various fluids or sites is difficult. the virus is detected by its capacity to transform b lymphocytes into persistent lymphoblastoid cell lines. pcr and dna hybridization studies have detected ebv in the cervix and in breast milk. one study, which identified ebv dna in breast milk cells in more than 40% of women donating milk to a breast milk bank, demonstrated that only 17% had antibody to ebv (only igg, no igm). 204 another study examining serologic specimens from breastfed and bottle-fed infants showed similar seroprevalence of ebv at 12 to 23 months of age (36/66 [54.5%] and 24/43 [55.8%]) in the breastfed and bottle-fed children, respectively. 236 the question of the timing of ebv infection and the subsequent immune response and clinical disease produced requires continued study. differences exist among the clinical syndromes that manifest at different ages. infants and young children are asymptomatic, have illness not recognized as related to ebv, or have mild episodes of illness, including fever, lymphadenopathy, rhinitis and cough, hepatosplenomegaly, or rash. adolescents or young adults who experience primary ebv infection more often demonstrate infectious mononucleosis syndrome or are asymptomatic. chronic fatigue syndrome is more common in adolescents and young adults. burkitt lymphoma, observed primarily in africa, and nasopharyngeal carcinoma, seen in southeast asia, where primary ebv infection usually occurs in young children, are tumors associated with early ebv infection. 246 these tumors are related to "chronic" ebv infection and tend to occur in individuals with persistently high antibody titers to ebv viral capsid antigen and early antigen. the questions of why these tumors occur with much greater frequency in these geographic areas and what cofactors (including altered immune response to infection associated with coinfections, immune escape by ebv leading to malignancy, or increased resistance to apoptosis secondary to ebv gene mutations) may contribute to their development remain unanswered. 21, 289 it also remains unknown to what degree breast milk could be a source of early ebv infection compared with other sources of ebv infection in an infant' s environment. similar to the situation of postnatal transmission of cmv in immunocompetent infants, clinically significant illness rarely is associated with primary ebv infection in infants. more data concerning the pathogenesis of ebv-associated tumors should be obtained before proscribing against breastfeeding is warranted, especially in areas where these tumors are common but the protective benefits of breastfeeding are high. in areas where burkitt lymphoma and nasopharyngeal carcinoma are uncommon, ebv infection in mother or infant is certainly not a contraindication to breastfeeding. marburg and ebola viruses cause severe and highly fatal hemorrhagic fevers. the illness often presents with nonspecific symptoms (conjunctivitis, frontal headache, malaise, myalgia, bradycardia) and progresses with worsening hemorrhage to shock and subsequent death in 50% to 90% of patients. personto-person transmission through direct contact, droplet spread, or airborne spread is the common mode of transmission. however, the animal reservoir or source of these viruses in nature for human infection has not been identified. attack rates in families are 5% to 16%. 330 no postexposure interventions have proved useful in preventing spread, and no treatment other than supportive is currently available. a recent report documented the presence of ebola virus in numerous body fluids including in breast milk. one acute breast milk sample on day 7 after the onset of illness and a "convalescent" breast milk sample on day 15 from the same woman were positive for ebola virus by both culture and pcr testing. 30 in the same study, saliva remained virus positive for a mean of 16 days after disease onset, urine was positive for a mean of 28 days, and semen for a mean of 43 days after the onset of disease. no information is available concerning the risk for transmission of these viruses in breast milk or additional risks or benefits from breastfeeding. contact precautions are recommended for marburg virus infections and contact and airborne precautions for ebola virus infection. given the high attack and mortality rates, these precautions should be carefully instituted and breastfeeding not allowed. if any other suitable source of nutrition can be found for an infant, expressed breast milk should also be proscribed for the infant of a mother with either of these infections for at least 3 weeks postrecovery. the diagnosis of hepatitis in a pregnant woman or nursing mother causes significant anxiety. the first issue is determining the etiology of the hepatitis, which then allows for an informed discussion of risk to the fetus/infant. the differential diagnosis of acute hepatitis includes (1) common causes of hepatitis, such as hepatitis a, b, c, and d; (2) , igm anti-hbcag, anti-hcv) as the initial diagnostic tests. simultaneous consideration of other etiologies of acute liver dysfunction is appropriate depending on a patient' s history. if the initial diagnostic tests are all negative, subsequent additional testing for anti-hepatitis d virus (hdv), hcv rna, hepatis g virus (hgv) rna, anti-hepatis e virus (hev), or hev rna may be necessary. if initial testing reveals positive hbsag, testing for anti-hdv, hbeag, and hbv dna is appropriate. these additional tests are useful in defining the prognosis for a mother and the risk for infection to an infant. during the diagnostic evaluation, it is appropriate to discuss with the mother or parents the theoretic risk for transmitting infectious agents that cause hepatitis via breastfeeding. the discussion should include an evaluation of the positive and negative effects of suspending or continuing breastfeeding until the exact etiologic diagnosis is determined. the relative risk for transmission of infection to an infant can be estimated and specific preventive measures provided for the infant (table 13-2) . hepatitis a virus (hav) is usually an acute selflimited infection. the illness is typically mild, and generally subclinical in infants. occasionally, hav infection is prolonged or relapsing, extending 3 to 6 months, and rarely it is fulminant, but hav infection does not lead to chronic infection. the incidence of prematurity after maternal hav infection is increased, but no evidence to date indicates obvious birth defects or a congenital syndrome. 372, 464 hav infection in premature infants may lead to prolonged viral shedding. 349 transmission is most often person to person (fecal-oral), and transmission in food-borne or water-borne epidemics has been described. transmission via blood products and vertical transmission (mother to infant) are rare. 440 transmission in daycare settings has been clearly described. infection with hav in newborns is uncommon and does not seem to be a significant problem. the usual period of viral shedding and presumed contagiousness lasts 1 to 3 weeks. acute maternal hav infection in the last trimester or in the postpartum period could lead to infection in an infant. symptomatic infection can be prevented by immunoglobulin (ig) administration, and 80% to 90% of disease can be prevented by ig administration immune serum globulin within 2 weeks of exposure. hav vaccine can be administered simultaneously with ig without affecting the seroconversion rate to produce rapid and prolonged hav serum antibody levels. transmission of hav via breast milk has been implicated in one case report, but no data exist on the frequency of isolating hav from breast milk. 440 because hav infection in infancy is rare and usually subclinical without chronic disease and because exposure has already occurred by the time the etiologic diagnosis of hepatitis in a mother is made, no reason exists to interrupt breastfeeding with maternal hav infection. the infant should receive ig and hav vaccine, administered simultaneously. hepatitis b virus (hbv) infection leads to a broad spectrum of illness, including asymptomatic seroconversion, nonspecific symptoms (fever, malaise, fatigue), clinical hepatitis with or without jaundice, extrahepatic manifestations (arthritis, rash, renal involvement), fulminant hepatitis, and chronic hbv infection. chronic hbv infection occurs in up to 90% of infants infected via perinatal and vertical transmission and in 30% of children infected between 1 to 5 years of age. given the increased risk for significant sequelae from chronic infection (chronic active hepatitis, chronic persistent hepatitis, cirrhosis, primary hepatocellular carcinoma), prevention of hbv infection in infancy is crucial. transmission of hbv is usually through blood or body fluids (stool, semen, saliva, urine, cervical secretions). 94 vertical transmission either transplacentally or perinatally during delivery has been well described throughout the world. vertical transmission rates in areas where hbv is endemic (taiwan and japan) are high, whereas transmission to infants from hbv carrier mothers in other areas where hbv carrier rates are low is uncommon. 399 transmission of hbv to infants occurs in up to 50% of infants when the mothers are acutely infected immediately before, during, or soon after pregnancy. 462 hbsag is found in breast milk, but transmission by this route is not well documented. beasley 31 and beasley et al 32 demonstrated that although breast milk transmission is possible, seroconversion rates are no different between breastfed and nonbreastfed infants in a long-term follow-up study of 147 hbsag-positive mothers. hill et al 176 followed 101 breastfed infants and 268 formula-fed infants born to women who were chronically hbsag positive. all infants received hepatitis b immunoglobulin at birth and a full series of hepatitis b vaccine. none of the breastfed infants and nine of the formulafed infants were positive for hbsag after completion of the hbv vaccine series. breastfeeding had occurred for a mean of 4.9 months (range 2 weeks to 1 year). transmission, when it does happen, probably occurs during labor and delivery. another report from china followed 230 infants born to hbsag-positive women. the infants received appropriate dosing and timing of hbig and hbv vaccine. at 1 year of age, anti-hbs antibody was present in 90.9% of the breastfed infants and 90.3% of the bottle-fed infants. 437 risk factors associated with immunoprophylaxis failure against vertical transmission of hbv include hbeag-seropositive mothers and elevated hbv dna "viral loads" in the mothers. 392 in 2009 the aap committee on infectious diseases stated that "that breastfeeding of the infant by a hbsag-positive mother poses no additional risk for acquisition of hbv infection by the infant with appropriate administration of hepatitis b vaccine and hbig." 96 screening of all pregnant women for hbv infection is an essential first step to preventing vertical transmission. universal hbv vaccination at birth and during infancy, with administration of hepatitis b immunoglobulin (hbig) immediately after birth to infants of hbsag-positive mothers, prevents hbv transmission in more than 95% of cases. breastfeeding by hbsag-positive women is not contraindicated, but immediate administration of hbig and hbv vaccine should occur. two subsequent doses of vaccine should be given at appropriate intervals and dosages for the specific hbv vaccine product. this decreases the small theoretic risk for hbv transmission from breastfeeding to almost zero. when acute peripartum or postpartum hepatitis occurs in a mother and hbv infection is a possibility, with its associated increased risk for transmission to the infant, a discussion with the mother or parents should identify the potential risks and benefits of continuing breastfeeding until the etiology of the hepatitis can be determined. if an appropriate alternative source of nutrition is available for the infant, breast milk should be withheld until the etiology of the hepatitis is identified. hbig and hbv vaccine can be administered to the infant who has not already been immunized or has no documented immunity against hbv. 400 if acute hbv infection is documented in a mother, breastfeeding can continue after immunization has begun. acute infection with hcv can be indistinguishable from hepatitis a or b infection; however, it is typically asymptomatic or mild. hcv infection is the major cause of blood-borne non-a, non-b hepatitis (nanbh). chronic hcv infection is reported to occur 70% to 85% of the time regardless of age at time of infection. sequelae of chronic hcv infection are similar to those associated with chronic hbv infection. bortolotti et al 47 the two commonly identified mechanisms of transmission of hcv are transfusions of blood or blood products and iv drug use. however, other routes of transmission exist because hcv infection occurs even in the absence of obvious direct contact with significant amounts of blood. other body fluids contaminated with blood probably serve as sources of infection. transmission through sexual contact occurs infrequently and probably requires additional contributing factors, such as coinfection with other sexually transmitted agents or high viral loads in serum and other body fluids. studies of transmission in households without other risk factors have demonstrated either low rates of transmission or no transmission. the reported rates of vertical transmission vary widely. in mothers with unknown hiv status or known hiv infection, the rates of vertical transmission were 4% to 100%, whereas the rates varied between 0% and 42% in known hiv-negative mothers. 113 these same studies suggest that maternal coinfection with hiv, hcv genotype, active maternal liver disease, and the serum titer of maternal hcv rna may be associated with increased rates of vertical transmission. 263, 307, 461 the correlation between hcv viremia, the hcv viral load in a mother, and vertical transmission of hcv is well documented. 288, 355, 406, 456 the clinical significance and risk for liver disease after vertical transmission of hcv are still unknown. the timing of hcv infection in vertical transmission is also unknown. in utero transmission has been suggested by some studies, 125 whereas intrapartum or postpartum transmission was proposed by ohto et al 308 when they documented the absence of hcv rna in the cord blood of neonates who later became hcv rna positive at 1 to 2 months of age. more recently, gibb et al 150 reported two pieces of data supporting the likelihood of intrapartum transmission as the predominant time of vertical transmission: (a) low sensitivity of pcr for hcv rna testing in the first month of life with a marked increase in sensitivity after that for diagnosing hcv infection in infants and (b) a lower transmission risk for elective cesarean delivery (without prolonged rupture of membranes) compared with vaginal or emergency cesarean delivery. 150 another group, mcmenamin et al, 275 analyzed vertical transmission in 559 mother-infant pairs. the overall vertical transmission rate was 4.1% (18/441), with another 118 infants not tested or lost to follow-up. comparison of the vertical transmission rate was no different for vaginal delivery or emergency cesarean in labor versus planned cesarean (4.2% vs. 3.0%). this held true even when mothers had hepatitis c rna detected antenatally (7.2% vs. 5.3%). the authors did not support planned cesarean delivery to decrease vertical transmission of hepatitis c infection. no prospective, controlled trials of cesarean versus vaginal delivery and the occurrence of vertical hepatitis c transmission are available. the risk for hcv transmission via breast milk is uncertain. anti-hcv antibody and hcv rna has been demonstrated in colostrum and breast milk, although the levels of hcv rna in milk did not correlate with the titers of hcv rna in serum. 36, 162, 256, 355 nevertheless, transmission of hcv via breastfeeding (and not in utero, intrapartum, or from other postpartum sources) has not been proven in the small number infants studied. transmission rates in breastfed and nonbreastfed infants appear to be similar, but various important factors have not been controlled, such as hcv rna titers in mothers, examination of the milk for hcv rna, exclusive breastfeeding versus exclusive formula feeding versus partial breastfeeding, and duration of breastfeeding.* zanetti et al 461 including 23 infants whose mothers were seropositive for hcv rna. eight infants in that study were infected with hcv, their mothers had both hiv and hcv, and three of these eight infants were infected with both hiv and hcv. the hcv rna levels were significantly higher in the mothers coinfected with hiv compared with those mothers with hcv alone. overall, the risk for hcv infection via breastfeeding is low, the risk for hcv infection appears to be more frequent in association with hiv infection and higher levels of hcv rna in maternal serum, no effective preventive therapies (ig or vaccine) exist, and the risk for chronic hcv infection and subsequent sequelae with any infection is high. it is therefore appropriate to discuss the theoretic risk for breastfeeding in hcv-positive mothers with the mother or parents and to consider proscribing breast milk when appropriate alternative sources of nutrition are available for the infants. hiv infection is a separate contraindication to breastfeeding. additional study is necessary to determine the exact role of breastfeeding in the transmission of hcv, including the quantitative measurement of hcv rna in colostrum and breast milk, the relative risk for hcv transmission in exclusively or partially breastfed infants versus the risk in formulafed infants, and the effect of duration of breastfeeding on transmission. the current position of the cdc is that no data indicate that hcv virus is transmitted through breast milk. 83 therefore breastfeeding by a hcvpositive, hiv-negative mother is not contraindicated. infants born to hcv rna-positive mothers require follow-up through 18 to 24 months of age to determine infants' hcv status, regardless of the mode of infant feeding. infants should be tested for alanine aminotransferase and hcv rna at 3 months and 12 to 15 months of age. alanine aminotransferase and anti-hcv antibody should be tested at 18 to 24 months of age to confirm an infant' s status: uninfected, ongoing hepatitis c infection, or past hcv infection. hepatitis d virus (hdv) is a defective rna virus that causes hepatitis only in persons also infected with hbv. the infection occurs as either an acute coinfection of hbv and hdv or a superinfection of hbv carriers. this "double" infection results in more frequent fulminant hepatitis and chronic hepatitis, which can progress to cirrhosis. the virus uses its own hbv rna (circular, negative-strand rna) with an antigen, hdag, surrounded by the surface antigen of hbv, hbsag. hdv is transmitted in the same way as hbv, especially through the exchange of blood and body fluids. hdv infection is uncommon where the prevalence of hbv is low. in areas where hbv is endemic, the prevalence of hdv is highly variable. hdv is common in tropical africa and south america as well as in greece and italy but is uncommon in the far east and in alaskan inuit despite the endemic occurrence of hbv in these areas. 390 transmission of hdv has been reported to occur from household contacts and, rarely, through vertical transmission. no data are available on transmission of hdv by breastfeeding. hdv infection can be prevented by blocking infection with hbv; therefore hbig and hbv vaccine are the best protection. in addition to hbig and hbv vaccine administration to the infant of a mother infected with both hbv and hdv, discussion with the mother or parents should include the theoretic risk for hbv and hdv transmission through breastfeeding. as with hbv, once hbig and hbv vaccine have been given to the infant, the risk for hbv or hdv infection from breastfeeding is negligible. therefore breastfeeding after an informed discussion with the parents is acceptable. hepatitis e virus (hev) is a cause of sporadic and epidemic, enterically transmitted nanbh, which is typically self-limited and without chronic sequelae. hev is notable for causing high mortality rate in pregnant women. transmission is primarily via the fecal-oral route, commonly via contaminated water or food. high infection rates have been reported in adolescents and young adults (ages 15 to 40 years). tomar 416 reported that 70% of cases of hev infections in the pediatric population in india manifest as acute hepatitis. maternal-neonatal transmission was documented when the mother developed hepatitis e infection in the third trimester. although hev was demonstrated in breast milk, no transmission via breast milk was confirmed in the report. five cases of transfusion-associated hepatitis e were reported. 416 epidemics are usually related to contamination of water. person-to-person spread is minimal, even in households and day care settings. although ig may be protective, no controlled trials have been done. animal studies suggest that a recombinant subunit vaccine may be feasible. 344 hev infection in infancy is rare, and no data exist on transmission of hev by breastfeeding. no evidence of clinically significant postnatal hev infection in infants or of chronic sequelae in association with hev infection and no documented hev transmission through breast milk is available. currently no contraindication exists to breastfeeding with maternal hev infection. ig has not been shown to be effective in preventing infection, and no vaccine is available for hev. hepatitis g virus (hgv) has recently been confirmed as a cause of nanbh distinct from hepatitis viruses a through e. several closely related genomes of hgv, currently named gbv-a, -b, and -c, appear to be related to hcv, the pestiviruses, and the flaviviruses. epidemiologically, hgv is most often associated with transfusion of blood, although studies have identified nontransfusion-related cases. hgv genomic rna has been detected in some patients with acute and chronic hepatitis and a small number of patients with fulminant hepatitis. gbv-c/hgv has also been found in some patients with inflammatory bile duct lesions, but the pathogenicity of this virus is unconfirmed. hgv rna has been detected in 1% to 3% of healthy blood donors in the united states. 8 feucht et al 128 described maternal-to-infant transmission of hgv in three of nine children. two of the three mothers were coinfected with hiv and the third with hcv. none of these infants developed signs of liver disease. neither the timing nor the mode of transmission was clarified. lin et al 255 reported no hgv transmission in three mother-infant pairs after cesarean delivery and discussed transplacental spread via blood as the most likely mode of hgv infection in vertical transmission. wejstal et al 442 reported on perinatal transmission of hgv to 12 of 16 infants born to hgv viremic mothers, identified by pcr. hgv did not appear to cause hepatitis in the children. 442 fischler et al 130 followed eight children born to hgv-positive mothers and found only one to be infected with hgv. that child remained clinically well, while his twin, also born by cesarean delivery and breastfed, remained hgv negative for 3 years of observation. five of the other six children were breastfed for variable periods without evidence of hgv infection. ohto et al 309 examined hgv mother-to-infant transmission. of 2979 pregnant japanese women who were screened, 32 were identified as positive for gbv-c/hgv rna by pcr; 26 of 34 infants born to the 32 hgv positive women were shown to be hgv rna positive. reportedly, none of the infants demonstrated a clinical picture of hepatitis, although two infants had persistent mild elevations (less than two times normal) of alanine aminotransferase. the viral load in mothers, who transmitted hgv to their infants, was significantly higher than in nontransmitting mothers. infants born by elective cesarean delivery had a lower rate of infection (3 in 7) compared with infants born by emergency cesarean delivery (2 of 2) or born vaginally (21 of 25) . in this study, hgv infection in breastfed infants was four times more common than in formula-fed infants, but this difference was not statistically significant because only four infants were formula fed. the authors report no correlation between infection rate and duration of breastfeeding was seen. testing of the infants was not done frequently and early enough routinely through the first year of life to determine the timing of infection in these infants. 309 schröter et al 371 reported transmission of hgv to 3 of 15 infants born to hgv rna positive mothers at 1 week of age. none of 15 breast milk samples were positive for gbv-c/hgv rna, and all of the children who were initially negative for hgv rna in serum remained negative at follow-up between 1 to 28 months of age. 371 the foregoing data suggest that transmission is more likely to be vertical, before, or at delivery rather than via breastfeeding. the pathogenicity and the possibility of chronic disease due to hgv infection remain uncertain at this time. insufficient data are available to make a recommendation concerning breastfeeding by hgv-infected mothers. herpes simplex virus types 1 and 2 (hsv-1, hsv-2) can cause prenatal, perinatal, and postnatal infections in fetuses and infants. prenatal infection can lead to abortion, prematurity, or a recognized congenital syndrome. perinatal infection is the most common form of infection (1 in 2000 to 5000 live births, 700 to 1500 cases per year in the united states) and is often fatal or severely debilitating. the factors that facilitate intrapartum infection and predict the severity of disease have been extensively investigated. postnatal infection is uncommon but can occur from a variety of sources, including oral or genital lesions and secretions in mothers or fathers, hospital workers and home caregivers, and breast lesions in breastfeeding mothers. a number of case reports have documented severe hsv-1 or hsv-2 infections in infants associated with hsvpositive breast lesions in the mothers. 116, 161, 338, 403 cases of infants with hsv gingivostomatitis inoculating the mothers' breasts have also been reported. in the absence of breast lesions breastfeeding in hsv-seropositive or culture-positive women is reasonable when accompanied by careful handwashing, covering the lesions, and avoiding fondling or kissing with oral lesions until all lesions are crusted. breastfeeding during maternal therapy with oral or iv acyclovir can continue safely as well. inadequate information exists concerning valacyclovir, famciclovir, ganciclovir, and foscarnet in breast milk to make a recommendation at this time. breastfeeding by women with active herpetic lesions on their breasts should be proscribed until the lesions are dried. treatment of the mothers' breast lesions with topical, oral, and/or iv antiviral preparations may hasten recovery and decrease the length of viral shedding. human herpesvirus 6 (hhv-6) is a cause of exanthema subitum (roseola, roseola infantum) and is associated with febrile seizures. hhv-6 appears to be most similar to cmv based on genetic analysis. no obvious congenital syndrome of hhv-6 infection has been identified, although prenatal infection has been reported. 118 seroepidemiologic studies show that most adults have already been infected by hhv-6. therefore primary infection during pregnancy is unlikely, but reactivation of latent hhv-6 infection may be more common. no case of symptomatic hhv-6 prenatal infection has been reported. the significance of reactivation of hhv-6 in a pregnant woman and the production of infection and disease in the fetus and infant remains to be determined. primary infection in children occurs most often between 6 and 12 months of age, when maternally acquired passive antibodies against hhv-6 are waning. febrile illnesses in infants younger than 3 months of age have been described with hhv-6 infection, but infection before 3 months or after 3 years is uncommon. various studies involving serology and restriction enzyme analysis of hhv-6 isolates from mother/infant pairs support the idea that postnatal transmission and perhaps perinatal transmission from the mothers are common sources of infection. one study was unable to detect hhv-6 in breast milk by pcr analysis in 120 samples, although positive control samples seeded with hhv-6-infected cells did test positive. 119 given the limited occurrence of clinically significant disease and the absence of sequelae of hhv-6 infection in infants and children, the almost universal acquisition of infection in early childhood (with or without breastfeeding) and the absence of evidence that breast milk is a source of hhv-6 infection, breastfeeding can continue in women known to be seropositive for hhv-6. human herpesvirus 7 (hhv-7) is closely related to hhv-6 biologically. primary infection with hhv-7 occurs primarily in childhood, usually later in life than hhv-6 infection. the median age of infection is 26 months, with 75% of children becoming hhv-7 positive by 5 years of age. 63 seroprevalence of hhv-7 antibody has been reported to be 80% to 98% in adults, and passive antibody is present in almost all newborns. 306, 408 like hhv-6, hhv-7 infection can be associated with acute febrile illness, febrile seizures, and irritability, but in general it is a milder illness than with hhv-6 with fewer hospitalizations. virus excretion of hhv-7 occurs in saliva, and pcr testing of blood cells and saliva are frequently positive in individuals with past infection. 463 congenital infection of hhv-6 was detected via dna pcr testing in 57 of 5638 of cord blood samples (1%), but hhv-7 was not detected in any of 2129 cord blood specimens. 165 hhv-7 dna was detected by pcr in 3 of 29 breast milk mononuclear cell samples from 24 women who were serum positive for hhv-7 antibody. 137 in the same study, small differences were seen in the hhv-7 seropositive rates between breastfed infants and bottle-fed infants at 12 months of age (21.7% versus 20%), at 18 months of age (60% versus 48.1%), and at 24 months of age (77.3% versus 58.3%, respectively,). none of these differences were statistically significant. given that, in general, hhv-6 infection occurs earlier than hhv-7 infection in most infants and that hhv-6 is rarely found in breast milk, it seems unlikely that hhv-7 in breast milk is a common source of infection in infants and children. the infrequent occurrence of significant illness with hhv-7 infection, with the absence of sequelae except in patients who had transplantation surgery at older ages and the common occurrence of infection in childhood argue, that no reason to proscribe against breastfeeding for hhv-7 positive women exists. human papillomavirus (hpv) is a dna virus with at least 100 different types. these viruses cause warts, genital dysplasia, cervical carcinoma (types 6 and 11), and laryngeal papillomatosis. transmission occurs through direct contact and sexual contact. laryngeal papillomas are thought to result from acquiring the virus in passage through the birth canal. infection in pregnant women or during pregnancy does not lead to an increase in abortions or the risk for prematurity, and no evidence indicates intrauterine infection. hpv is one of the most common viruses in adults and one of the most commonly sexually transmitted infections. diagnosis is usually by histologic examination or dna detection. spontaneous resolution does occur, but therapy for persistent lesions or growths in anatomically problematic locations is appropriate. therapy can be with podophyllum preparations, trichloroacetic acid, cryotherapy, electrocautery, and laser surgery. interferon is being tested in the treatment of laryngeal papillomas, with mixed results. 109 prevention against transmission means limiting direct or sexual contact, but this may not be sufficient because lesions may not be evident and transmission may still occur. rintala et al 346 examined the occurrence of hpv dna in the oral and genital mucosa of infants during the first 3 years of life. hpv dna was identified in 12% to 21% of the oral scrape samples and in 4% to 15% of the genital scrape samples by pcr. oral hpv infection was acquired by 42% of children, cleared by 11%, and persisted in 10% of children; 37% of the children were never infected. they did not report on breast milk or breastfeeding in that study. the question of the source of the infection remains undetermined. the breast is a rare site of involvement. 110 hpv types 16 and 18 can immortalize normal breast epithelium in vitro. 441 hpv dna has been detected in breast milk in 10 of 223 (4.5%) of milk samples from 223 mothers, collected 3 days postpartum. 361 no attempt was made to correlate the presence of hpv dna in breast milk with the hpv status of an infant or to assess the "viral load" of hpv in breast milk or its presence over the course of lactation. a second study found dna of cutaneous and mucosal hpv types in 2 of 25 human milk samples and 1 of 10 colostrum samples. 64 no reports of hpv lesions of the breast or nipple and documented transmission to an infant secondary to breastfeeding are available. no increased risk for acquiring hpv from breast milk is apparent, and breastfeeding is acceptable. even in the rare occurrence of an hpv lesion of the nipple or breast, no data suggest that breastfeeding or the use of expressed breast milk is contraindicated. measles is another highly communicable childhood illness that can be more severe in neonates and adults. measles is an exanthematous febrile illness following a prodrome of malaise, coryza, conjunctivitis, cough, and often koplik spots in the mouth. the rash usually appears 10 to 14 days after exposure. complications can include pneumonitis, encephalitis, and bacterial superinfection. with the availability of vaccination, measles in pregnancy is rare (0.4 in 10,000 pregnancies), 148 although respiratory complications (primary viral pneumonitis, secondary bacterial pneumonia), hepatitis, or other secondary bacterial infections often lead to more severe disease in these situations. prenatal infection with measles may cause premature delivery without disrupting normal uterine development. no specific group of congenital malformations have been described in association with in utero measles infection, although teratogenic effects of measles infection in pregnant women may rarely manifest in the infants. perinatal measles includes transplacental infection when measles occurs in an infant in the first 10 days of life. infection from extrauterine exposure usually develops after 14 days of life. the severity of illness after suspected transplacental spread of virus to an infant varies from mild to severe and does not seem to vary with the antepartum or postpartum onset of rash in the mother. it is uncertain what role maternal antibodies play in the severity of an infant's disease. more severe disease seems to be associated with severe respiratory illness and bacterial infection. postnatal exposure leading to measles after 14 days of life is generally mild, probably because of passively acquired antibodies from the mother. severe measles in children younger than 1 year of age may occur because of declining passively acquired antibodies and complications of respiratory illness and rare cases of encephalitis. measles virus has not been identified in breast milk, whereas measles-specific antibodies have been documented. 1 infants exposed to mothers with documented measles while breastfeeding should be given immunoglobulin (ig) and isolated from the mother until 72 hours after the onset of rash, which is often only a short period after diagnosis of measles in the mother. the breast milk can be pumped and given to the infant because secretory iga begins to be secreted in breast milk within 48 hours of onset of the exanthem in the mother. table 13 -3 summarizes management of the hospitalized mother and infant with measles exposure or infection. 148 mumps is an acute transient benign illness with inflammation of the parotid gland and other salivary glands and often involves the pancreas, testicles, and meninges. mumps occurs infrequently in pregnant women (1 to 10 cases in 10,000 pregnancies) and is generally benign. mumps virus has been isolated from saliva, respiratory secretions, blood, testicular tissue, urine, csf in cases of meningeal involvement, and breast milk. the period of infectivity is believed to be between 7 days before and 9 days after the onset of parotitis, with the usual incubation period being 14 to 18 days. prenatal infection with the mumps virus causes an increase in the number of abortions when infection occurs in the first trimester. a small increase in the number of premature births was noted in one prospective study of maternal mumps infection. 383 no conclusive evidence suggests congenital malformations are associated with prenatal infection, not even with endocardial fibroelastosis, as originally reported in the 1960s. perinatal mumps (transplacentally or postnatally acquired) has rarely if ever been documented. natural mumps virus has been demonstrated to infect the placenta and infect the fetus, and live attenuated vaccine virus has been isolated from the placenta but not from fetal tissue in women vaccinated 10 days before induced abortion. antibodies to mumps do cross the placenta. postnatal mumps in the first year of life is typically benign. no epidemiologic data suggest that mumps infection is more or less common or severe in breastfed infants compared with formula-fed infants. although mumps virus has been identified in breast milk and mastitis is a rare complication of mumps in mature women, no evidence indicates that breast involvement occurs more frequently in lactating women. if mumps occurs in the mother breastfeeding can continue because exposure has already occurred throughout the 7 days before the development of symptoms in the mother and secretory iga in the milk may help to mitigate the symptoms in the infant. human parvovirus b19 causes a broad range of clinical manifestations, including asymptomatic infection (most frequent manifestation in all ages), erythema infectiosum (fifth disease), arthralgia and arthritis, red blood cell (rbc) aplasia (less often decreased white blood cells or platelets), chronic infection in immunodeficient individuals, and rarely myocarditis, vasculitis, or hemophagocytic syndrome. vertical transmission can lead to severe anemia and immune-mediated hydrops fetalis, which can be treated, if accurately diagnosed, by intrauterine transfusion. inflammation of the liver or cns can be seen in the infant, along with vasculitis. if the child is clinically well at birth, hidden or persistent abnormalities are rarely identified. no evidence indicates that parvovirus b19 causes an identified pattern of birth defects. postnatal transmission usually occurs person to person via contact with respiratory secretions, saliva, and rarely blood or urine. seroprevalence in children at 5 years of age is less than 5%, with the peak age of infection occurring during the schoolage years (5% to 40% of children infected). the majority of infections are asymptomatic or undiagnosed seroconversions. 417 severe disease, such as prolonged aplastic anemia, occurs in individuals with hemoglobinopathies or abnormal rbc maturation. attack rates have been estimated to be 17% to 30% in casual contacts but up to 50% among household contacts. in one study of 235 susceptible pregnant women, the annual seroconversion rate was 1.4%. 223 no reports of transmission to an infant through breastfeeding are available. excretion in breast milk has not been studied because of limitations in culturing techniques. rat parvovirus has been demonstrated in rat milk. the very low seroconversion rate in young children and the absence of chronic or frequent severe disease suggest that the risk for parvovirus infection via breast milk is not significant. the possibility of antibodies against parvovirus or other protective constituents in breast milk has not been studied. breastfeeding by a mother with parvovirus infection is acceptable. poliovirus infections (types 1, 2, and 3) cause a range of illness, with 90% to 95% subclinical, 4% to 8% abortive, and 1% to 2% manifest as paralytic poliomyelitis. a review by bates 29 from 1955 of 58 cases of poliomyelitis in infants younger than 1 month of age demonstrated paralysis or death in more than 70% and only one child without evidence of even transient paralysis. more than half the cases were ascribed to transmission from the mothers, although no mention was made of breastfeeding. breastfeeding rates at the time were approximately 25%. prenatal infection with polioviruses does cause an increased incidence of abortion. prematurity and stillbirth apparently occur more frequently in mothers who developed paralytic disease versus inapparent infection. 188 although individual reports of congenital malformations in association with maternal poliomyelitis exist, no epidemiologic data suggest that polioviruses are teratogenic. also, no evidence indicates that live attenuated vaccine poliovirus given during pregnancy is associated with congenital malformations. 89, 170 perinatal infection has been noted in several case reports of infants infected in utero several days before birth who had severe disease manifesting with neurologic manifestations (paralysis) but without fever, irritability, or vomiting. additional case reports of infection acquired postnatally demonstrate illness more consistent with poliomyelitis of childhood. these cases were more severe and involved paralysis, which may represent reporting bias. 89 no data are available concerning the presence of poliovirus in breast milk, although antibodies to poliovirus types 1, 2, and 3 have been documented. 270 in this era of increasing worldwide poliovirus vaccination, the likelihood of prenatal or perinatal poliovirus infection is decreasing. maternal susceptibility to poliovirus should be determined before conception and poliovirus vaccine offered to susceptible women. an analysis of the last great epidemic in italy in 1958 was done using a population-based case-control study. 336 in 114,000 births, 942 infants were reported with paralytic poliomyelitis. a group of matched control subjects was selected from infants admitted to the hospital at the same time. using the dichotomous variable of never breastfed and partially breastfed, 75 never-breastfed infants were among the cases and 88 among the control group. the authors determined an odds ratio of 4.2, with 95% confidence interval of 1.4 to 14, demonstrating that the risk for paralytic poliomyelitis was higher in infants never breastfed and lowest among those exclusively breastfed. because by the time the diagnosis of poliomyelitis is made in a breastfeeding mother, the exposure of the infant to poliovirus from maternal secretions has already occurred, and because the breast milk already contains antibodies that may be protective, no reason exists to interrupt breastfeeding. breastfeeding also does not interfere with successful immunization against poliomyelitis with oral or inactivated poliovirus vaccine. 71 the occurrence of human t-cell leukemia virus type i (htlv-i) is endemic in parts of southwestern japan, 66, 105, 207, 450 the caribbean, south america, 156 and sub-saharan africa. htlv-i is associated with adult t-cell leukemia/lymphoma and a chronic condition with progressive neuropathy. the progressive neuropathy is called htlv-i associated myelopathy or tropical spastic paraparesis. 136 other illnesses have been reported in association with htlv-i infection including dermatitis, uveitis, arthritis, sjögren syndrome in adults, and infective dermatitis and persistent lymphadenitis in children. transmission of htlv-i occurs most often through sexual contact, via blood or blood products, and via breast milk. infrequent transmission does occur in utero or at delivery and with casual or household contact. 291 seroprevalence generally increases with age and varies widely in different regions and in populations of different backgrounds. in some areas of japan, seropositivity can be as high as 12% to 16%, but in south america, africa, and some caribbean countries the rates are 2% to 6%. in latin america seropositive rates can be as high as 10% to 25% among female sex workers or attendees to std clinics. 156 in blood donors in europe, the seroprevalence of htlv-i has been reported at 0.001% to 0.03%. the seroprevalence in pregnant women in endemic areas of japan is as high as 4% to 5% and in nonendemic areas as low as 0.1% to 1.0%. htlv-1 is not a major disease in the united states. in studies from europe the seroprevalence in pregnant women has been noted to be up to 0.6%. these pregnant women were primarily of african or caribbean descent. 138 htlv-i antigen has been identified in breast milk of htlv-i positive mothers. 220 another report shows that basal mammary epithelial cells can be infected with htlv-i and can transfer infection to peripheral blood monocytes. 254 human milk from htlv-i positive mothers caused infection in marmosets. 221, 453 htlv-i infection clearly occurs via breastfeeding and a number of reports document an increased rate of transmission of htlv-i to breastfed infants compared with formula-fed infants.* ando et al 12, 13 in two separate reports demonstrated a parallel decline in antibodies against htlv-i in both formula-fed and breastfed infants to a nadir at approximately 1 year of age and a subsequent increase in antibodies from 1 to 2 years of age. the percentage of children seropositive at 1 year of age in the breastfed and formula-fed groups, respectively, was 3.0% and 0.6%, at 1.5 years of age it was 15.2% and 3.9%, and at 2 years of age it was 41.9% and 4.6%. a smaller group of children followed through 11 to 12 years of age demonstrated no newly infected children after 2 years of age and *references 9, 10, 12, 13, 178-180, 407. no loss of antibody in any child who was seropositive at 2 years of age. 12, 13 transmission of htlv-i infection via breastfeeding is also clearly associated with the duration of breastfeeding. 407, 409, 446, 447 it has been postulated that the persistence of passively acquired antibodies against htlv-i offers some protection through 6 months of life (table 13-4) . other factors relating to htlv-i transmission via breast milk have been proposed. yoshinaga et al 460 presented data on the htlv-i antigen producing capacity of peripheral blood and breast milk cells and showed an increased mother-to-child transmission rate when the mother' s blood and breast milk produced large numbers of antigen-producing cells in culture. 460 hisada et al 183 reported on 150 mothers and infants in jamaica, demonstrating that a higher maternal provirus level and a higher htlv-i antibody titer were independently associated with htlv-i transmission to the infant. ureta-vidal et al 421 reported an increased seropositivity rate in children of mothers with a high proviral load and elevated maternal htlv-i antibody titers. various interventions have been proposed to decrease htlv-i transmission via breastfeeding. complete avoidance of breastfeeding was shown to be an effective intervention by hino et al 180, 181 in large population of japanese in nagasaki. avoiding breastfeeding led to an 80% decrease in transmission. breastfeeding for a shorter duration is another effective alternative. ando et al 11 showed that freezing and thawing breast milk decreased the infectivity of htlv-i. sawada et al 363 demonstrated in a rabbit model that htlv-i immunoglobulin protected against htlv-i transmission via milk. it is reasonable to postulate that any measure that would decrease the maternal provirus load or increase the anti-htlv-i antibodies available to infants might decrease the risk for transmission. the overall prevalence of htlv-i infection during childhood is unknown because the majority of individuals do not manifest illness until much later in life. the timing of htlv-i infection in a breastfeeding population has been difficult to assess because of passively acquired antibodies from the mother and issues related to testing. furnia et al 139 in areas where the prevalence of htlv-i infection (in the united states, canada, or europe) is rare, the likelihood that a single test for antibody against htlv-i would be a false positive test is high compared with the number of true positive tests. repeat testing is warranted in many situations. 66 quantification of the antibody titer and the proviral load is appropriate in a situation when mother-to-child transmission is a concern. a greater risk for progression to disease in later life has not been shown for htlv-i infection through breast milk, but early-life infections are associated with the greatest risk for adult t-cell leukemia. 402 the mother and family should be informed about all these issues. if the risk for lack of breast milk is not too great and formula is readily available and culturally acceptable, then the proscription of breastfeeding, or at least a recommendation to limit the duration of breastfeeding to 6 months or less, is appropriate to limit the risk for htlv-i transmission to the infant. freezing and thawing breast milk before giving it to an infant might be another reasonable intervention to decrease the risk for transmission, although no controlled trials document the efficacy of such an intervention. neither ig nor antiviral agents against htlv-i are available at this time. human t-cell leukemia virus type ii (htlv-ii) is endemic in specific geographic locations, including africa, the americas, the caribbean, and japan. transmission is primarily through intravenous drug use, contaminated blood products, and breastfeeding. sexual transmission occurs but its overall contribution to the prevalence of htlv-ii in different populations remains uncertain. many studies have examined the presence of htlv-i and ii in blood products. pcr testing and selective antibody tests suggest that about half of the htlv seropositivity in blood donors is caused by htlv-ii. htlv-ii has been associated with two chronic neurologic disorders similar to those caused by htlv-i, tropical or spastic ataxia. 258 a connection between htlv-ii and glomerulonephritis, myelopathy, arthritis, t-hairy cell leukemia, and large granulocytic leukemia has been reported. mother-to-child transmission has been demonstrated in both breastfed and formula-fed infants. it appears that the rate of transmission is greater in breastfed infants.* htlv-ii has been detected in breast milk. 174 nyambi et al 304 reported that htlv-ii transmission did correlate with the duration of breastfeeding. the estimated rate of transmission was 20%. the time to seroconversion (after the initial loss of passively acquired maternal antibodies) for infected infants seemed to range between 1 and 3 years of age. 304 at this time avoidance of breastfeeding and limiting the duration of breastfeeding are the only two possible interventions with evidence of effectiveness for preventing htlv-ii mother-to-child transmission. 207 with the current understanding of retroviruses, it is appropriate in cases of documented htlv-ii maternal infection to recommend avoiding or limiting the duration of breastfeeding and provide alternative nutrition when financially practical and culturally acceptable. mothers should have confirmatory testing for htlv-ii and measurement of the proviral load. infants should be serially tested for antibodies to htlv-ii and have confirmatory testing if seropositive after 12 to 18 months of age. further investigation into the mechanisms of transmission via breast milk and possible interventions to prevent transmission should occur as they have for hiv-1 and htlv-i. human immunodeficiency virus type 1 (hiv-1) is transmitted through human milk. refraining from breastfeeding is a crucial aspect of preventing perinatal hiv infection in the united states and many other countries. the dilemma is the use of replacement feeding versus breastfeeding in countries where breastfeeding provides infants with significant protection from illness and death due to malnutrition or other infections. the question of the contribution of breastfeeding in mother-to-child hiv-1 transmission is not a trivial one when one considers the following: 3. the who estimates that 2.7 million people were newly infected with hiv-1 in 2007, with children younger than 14 years old making up 370,000 of that 2.7 million. (this number has declined due to increasing access to interventions to prevent mother-to-infant transmission. availability of antiretroviral therapy for prevention of mother-to-child hiv transmission in developing countries in 2007 was estimated to reach 33% of the mothers who needed it.) 419 4. breastfeeding contributes an estimated 10% to 20% increase in the overall mother-to-child transmission rates, over and above intrauterine and intrapartum transmission, when no specific interventions to prevent transmission via breastfeeding are utilized. 5. despite a dramatic increase in the number of people receiving antiretroviral therapy in developing countries (3 million), this represented only 31% of the individuals who needed treatment. 419 the evidence of hiv transmission via breastfeeding is irrefutable. multiple publications summarize the current evidence for hiv transmission via breastfeeding in the literature. 232, 341, 420 since 1985, case reports have documented hiv transmission via breast milk to children around the world. 182, 198, 249, 465 primary hiv infection in breastfeeding mothers, with the concomitant high viral load, is associated with a particularly high rate of hiv transmission via breast milk. palasanthiran et al 322 estimated that risk at 27%.large observational studies have demonstrated higher rates of hiv transmission in breastfed infants of mothers with chronic hiv infection compared with formula-fed infants. 43, 108, 124 a systematic analysis of published reports estimated the additional risk for perinatal hiv transmission due to breastfeeding to be 14% (95% confidence interval 7% to 22%). 117 more recently published cohort studies similarly attributed additional risk for hiv transmission due to breastfeeding at 4% to 22% over and above the risk from prenatal and intrapartum transmission. 38, 104, 121 laboratory reports demonstrate the presence of cell-free virus and cell-associated virus in breast milk as well as various immunologic factors that could block or limit infection.* a dose-response relationship has been observed, correlating the hiv viral load in human milk as well as a mother' s plasma viral load with an increased transmission risk for the breastfed infant. 335, 345, 351, 373 many of the potential risk factors associated with human milk transmission of hiv is higher the longer the duration of breastfeeding. 108, 251, 282, 290, 424 maternal characteristics related to transmission of hiv via human milk include younger maternal age, higher parity, lower cd4+ counts, higher plasma viral loads, and breast abnormalities (mastitis, abscess, or nipple lesions). characteristics of human milk that relate to a higher risk for transmission include higher viral load in the milk, lower concentrations of antiviral substances (lactoferrin, lysozyme), and lower concentrations of virus-specific cytotoxic t-lymphocytes, levels of various interleukins (il-7, il-15), 434, 435 secretory iga, and igm. mixed breastfeeding is also associated with a higher risk for hiv transmission compared with exclusive breastfeeding. 99, 100, 410 the measurable benefits of breast milk versus the relative risk for hiv transmission to the infant due to exclusive breastfeeding (with optimization of other factors to decrease hiv transmission) have been reported in a couple of studies. 97, 229 the measurable benefits of receiving breast milk versus the relative increased risk for hiv transmission will need to be determined in a prospective fashion in different locales. 247 a number of potential interventions to prevent breastfeeding transmission of hiv-1 can be utilized (box 13-3) . the simplest and most effective is the compete avoidance of human milk. this is a practical solution in places like the united states and other countries where replacement feeding as well as other strictly medical interventions are feasible and reasonable, and the risk for not providing breast milk to the infant is negligible. in resourcepoor situations, where the risk for other infections is high without the benefits of breast milk, exclusive breastfeeding is appropriate, with any other reasonable and culturally acceptable interventions to decrease hiv transmission via breast milk. potentially effective interventions include exclusive breastfeeding, early weaning versus breastfeeding for longer durations, education, and support to decrease the likelihood of mastitis or nipple lesions. 191 other possible interventions include treating a mother with antiretroviral therapy for her own health (cd4 counts less than 350) or prophylactically to decrease the human milk viral load, treating an infant prophylactically for a prolonged period of time (6 weeks to 6 months) to protect against transmission via breastfeeding, treating the milk itself to decrease the viral load (by pasteurization or other methods), 316, 318 treating acute conditions in mothers and infants (e.g., mastitis, breast lesions, infant candidiasis), and enhancing an infant' s own defenses via vitamins, immunization, or antiretroviral therapy. some of these may not be feasible in certain settings such as pasteurization or maternal antiretroviral therapy. others may not be culturally acceptable, such as treating expressed breast milk before giving it to an infant or even exclusive breastfeeding. significant data demonstrate the advantage of breastfeeding, even for hiv-infected or hivexposed infants. the complete avoidance of breastfeeding in certain situations may lead to increased risk for illness and death due to other reasons besides hiv transmission. 106 a study from kenya showed improved hiv-1-free survival rates in a formula-fed group of children born to hiv-positive mothers, but the breastfed and formula groups had similar mortality rates (24.4% versus 20.0%, respectively) and similar incidences of diarrhea and pneumonia in the first 2 years of life. 272 no difference in the two groups was seen in the prevalence of malnutrition, but the breastfed infants had better nutritional status in the first 6 months of life. arpadi et al 20 recommend additional nutritional interventions to complement breastfeeding in this population after 6 months of age. two reports from zambia document the benefit of exclusive breastfeeding for decreasing late hiv transmission and the lower mortality at 12 months in infants who had continued breastfeeding rather than had discontinued breastfeeding at 4 months of age. 229, 385 in malawi, hiv-infected and hiv-exposed infants who were breastfed (exclusive breastfeeding for 2 months and mixed feeding after that) had lower mortality at 24 months than those who were not breastfed. 405 a report from botswana examined breastfeeding plus infant zidovudine prophylaxis for 6 months versus formula feeding plus infant zidovudine for 1 month; this study showed a decreased risk for vertical transmission with formula feeding, but also increased cumulative mortality for the hiv-infected infants at 7 months of age who were in the formula-fed group. 411 a study from south africa examining the use of vitamin a also demonstrated less morbidity in hiv-infected children who were breastfed than not breastfed. 102 other abstract reports have shown increased morbidity in hiv-infected children due to diarrhea, gastroenteritis, and hospitalization after weaning from breastfeeding. 205, 226, 315, 413 exclusive breastfeeding in most areas of the world is essential to infant health and survival, even in the situation of maternal hiv infection. 97, 99, 100, 229 the duration of exclusive breastfeeding is crucial to decreasing the risk for hiv infection in infants versus the risk for malnutrition and other infections with early weaning. in the mashi study in botswana, thior et al 411 evaluated infants randomized to breastfeeding plus infant zidovudine for 6 months or formula feeding plus 1 month of infant zidovudine. the cumulative infant mortality was significantly higher at 7 months for the formula-fed group but at 18 months it was similar between the two groups. the breastfed infants were more likely to become hiv infected despite the 6 months of zidovudine prophylaxis. 411 becquet et al 33 analyzed data from cote d'ivoire for 2001 to 2005; 47% of the hivexposed infants were breastfed for a median of 4 months, and 53% were formula fed and observed for 2 years. no significant difference in the rate of hiv infection was seen in the two groups, and no significant difference between the two groups was seen for morbid events (diarrhea, acute respiratory infections or malnutrition) or hospitalization or death. the authors attributed these good outcomes to effective nutritional counseling and care, access to clean water, and the provision of a safe and continuous supply of breast milk substitute. 33 coovadia et al 97 studied exclusive breastfeeding in the first 6 months of life as an intervention in south africa. of the exclusively breastfed infants, 14.1% at 6 weeks of age and 19.5% at 6 months of age were hiv infected. breastfed infants who also were fed solids or formula milk were more likely to acquire infection than exclusively breastfed infants. the cumulative mortality at 3 months of age was markedly lower for exclusively breastfed infants (6.1%) versus 15.1% in the infants receiving mixed feedings. kuhn et al 230 examined the effects of early, abrupt weaning on hiv-free survival of 958 children in zambia. infants were randomly assigned to two different counseling programs that advised either abrupt weaning at 4 months or prolonged breastfeeding. in the weaning intervention group, 69% of mothers stopped breastfeeding by 5 months compared with a median duration of breastfeeding of 16 months in the control group. the study found no significant difference in hiv-free survival at • women and their health care providers need to be aware of the potential risk for transmission of hiv infection to infants during pregnancy and in the peripartum period and through breast milk. • documented, routine hiv education and routine testing with the consent of women seeking prenatal care are strongly recommended so that each woman knows her hiv status and the methods available both to prevent the acquisition and transmission of hiv and to determine whether breastfeeding is appropriate. • at delivery, education about hiv and testing with the consent of women whose hiv status during pregnancy is unknown are strongly recommended. knowledge of a woman' s hiv status assists in counseling on breastfeeding and helps each woman understand the benefits to herself and her infant of knowing her serostatus and the behaviors that would decrease the likelihood of acquisition and transmission of hiv. • women who are known to have hiv infections must be counseled not to breastfeed or provide their milk for the nutrition of their own or other' s infants. • in general, women who are known to be hiv seronegative should be encouraged to breastfeed. however, women who are hiv seronegative but at particularly high risk for seroconversion (e.g., injection drug users and sexual partners of known hiv-positive persons or active drug users) should be educated about hiv with an individualized recommendation concerning the appropriateness of breastfeeding. in addition, during the perinatal period, information should be provided on the potential risk for transmitting hiv through breast milk and about methods to reduce the risk for acquiring hiv infection. • each woman whose hiv status is unknown should be informed of the potential for hiv-infected women to transmit hiv during the peripartum period and through breast milk and the potential benefits to her and her infant of knowing her hiv status and how hiv is acquired and transmitted. the health care provider needs to make an individualized recommendation to assist the woman in deciding whether to breastfeed. 24 months in the two groups (83.9% versus 80.7%). children already infected by 4 months of age had a higher mortality if they were assigned to the early weaning group (73.6% versus 54.8%). additional analysis showed that in mothers with less severe hiv disease early weaning was clearly harmful to the infant. 231 arpadi et al 20 studied the growth of hiv-exposed, uninfected children who were exclusively breastfed for 4 months with rapid weaning to replacement foods or exclusively breastfed until 6 months and then continued breastfeeding with complementary foods. weight-for-age z scores dropped markedly in both groups from 4 to 15 months of age but less so in the continued breastfeeding group. length-for-age z score also dropped dramatically, but was not influenced by continued breastfeeding. even in this hiv-exposed, uninfected group of children, additional nutritional interventions are essential to complement breastfeeding beyond 6 months of age. 20 in recent years the discussion around preventing hiv transmission via breastfeeding and in the number of studies examining the important issues have increased. 98, 233, 283 the fact that intrapartum and perinatal transmission of hiv from mothers to infants has decreased markedly due to the increased utilization of antiretroviral therapy during pregnancy, delivery, and postnatally for prevention emphasizes the importance of now working harder to decrease breast milk transmission of hiv. in considering different possible interventions to decrease mother-infant hiv transmission, it is crucial to reemphasize the goals of optimizing maternal health and survival and optimizing infant health and survival at the same time. a laboratory report shows that mothers receiving highly active antiretroviral therapy (haart) while breastfeeding do have decreased whole breast milk hiv-1 viral loads (23/26 mothers had less than 50 copies/ml) compared with mothers who did not receive haart (9/25 with less than 50 copies/ml). however, the whole milk hiv-1 dna load (measured as "undetectable" at less than 10 copies/10 6 cells) was not significantly different in the haart (13 of 26 mothers)] and non-haart (15 of 23) groups. 378 hiv-1 dna is incorporated into cells found in breast milk. another group showed significantly lower hiv rna levels in the breast milk of women treated with nevirapine, zidovudine, and lamivudine compared with women not receiving antiretroviral therapy. 152 the use of maternal haart seems to decrease hiv-1 transmission via breastfeeding. one group working in mozambique, malawi, and tanzania working with mother-infants pairs receiving haart as prevention during pregnancy compared one cohort (809 mother-infant pairs) who received supplementary formula and water filters for the first 6 months of life with a second cohort (251 motherinfant pairs) breastfeeding exclusively and the mothers receiving haart for the first 6 months. the cumulative incidence rate of hiv infection at 6 months of age was 2.7% for the formula-fed infants and 2.2% for breastfed infants. through 6 months of age no apparent additional risk for late postnatal transmission of hiv was observed. 323 the petra study team working in tanzania, south africa, and uganda examined the efficacy of three shortcourse regimens of zidovudine and lamivudine in preventing early and late hiv transmission in this predominantly breastfeeding population. 332 there were four regimens: a, zidovudine and lamivudine starting at 36 weeks' gestation plus intrapartum medication and 7-days' postpartum treatment; b, same as a without the prepartum component; c, intrapartum zidovudine and lamivudine only; d, placebo. at week 6 the hiv transmission rates were 5.7% in group a, 8.9% in group b, 14.2% in group c, and 15.3% in group d. at 18 months the hiv infection rates were 15% in group a, 18% in group b, 20% in group c, and 22% in group d. although a measurable decrease in transmission at 6 weeks of age was observed, limited protection was seen at 18 months of age. an observational study from tanzania compared maternal haart for 6 months with exclusive breastfeeding and abrupt weaning at 5 to 6 months of age with a historical control of the same feeding schedule without the postnatal maternal haart. in the treatment group the cumulative hiv transmission was 4.1% at 6 weeks, 5.0% at 6 months, and 6.0% at 18 months of age. the cumulative hiv infection or death rate was 8.6% at 6 months and 13.6% at 18 months of age. the cumulative risk for hiv transmission was 1.1% between 6 and 18 months. the hiv transmission in this treatment group was half the transmission rate in the historical control group. 218 another study in sub-saharan africa with 6 months of maternal haart and exclusive breastfeeding for 6 months demonstrated 94% hiv-free survival at 12 months of age; the maternal and infant mortality rates for the treated mother-infant pairs were significantly lower than the country' s maternal and infant mortality rates. 264 antiretroviral therapy prophylaxis for infants is another investigated intervention to decrease hiv transmission via breastfeeding. in a study from cote d'ivoire comparing different groups over time, infants received zidovudine (zdv) alone as zdv prophylaxis, a single dose of nevirapine (nvp), and 7 days of zidovudine (zdv) as nvp/ zdv prophylaxis, or a single dose of nevirapine plus zidovudine and lamivudine (3tc) for 7 days as nvp/zdv/3tc prophylaxis. formula feeding (ff) was compared with exclusive shortened breastfeeding (esb) upto 4 months of age and prolonged breastfeeding (pb). the cumulative transmission rates at 18 months were 22.3% in 238 infants in the zdv + pb group, 15.9% in 169 infants in the nvp/zdv + esb group, 9.4%, in the 195 infants in the nvp/zdv +ff group, 6.8% in the 198 infants in the nvp/zdv/3tc + esb group, and 5.6% in the 126 infants in the nvp/zdv/3tc + ff group. 252 kumwenda et al 235 working in malawi demonstrated decreased hiv transmission with breastfeeding and two different infant prophylaxis regimens. at 9 months of age, they observed a 10.6% occurrence of hiv transmission for infants receiving a single dose of nevirapine plus 1 week of zidovudine compared with 5.2% in the group receiving a single dose of nevirapine plus 1 week of zidovudine plus 14 weeks of daily nevirapine, and 6.4% in the group receiving a single dose of nevirapine plus 1 week of zidovudine plus 14 weeks of nevirapine and zidovudine. 235 in the mitra study in tanzania in which the median time of breastfeeding was 18 weeks, the hiv transmission rate at 6 months in the infants who received zidovudine plus 3tc for 1 week plus 3tc alone for breastfeeding through 6 months of age was less than 50% of the transmission rate for those infants receiving only 1 week of zidovudine plus 3tc. 217 a summary of three trials in ethiopa, india, and uganda compared a single dose of nevirapine at birth for infants with 6 weeks of daily nevirapine in predominantly breastfed infants whose mothers were counselled regarding feeding per the who/ unicef guidelines. at 6 months 87 of 986 infants in the single-dose group and 62 of 901 in the extended-dose group were hiv infected, which was not statistically significant. the authors suggested that a longer course of infant antiretroviral prophylaxis might be more effective. 388 the potential effect of breastfeeding on the hivpositive mother needs to be adequately assessed in relation to the mother's health status. from uganda and zimbabwe mbizvo et al 271 reported no difference in the number of hospital admissions or mortality between hiv-positive and hiv-negative women during pregnancy. in the 2 years after delivery the hiv-positive women had higher hospital admission (approximately two times increased risk) and death rates (relative risk greater than 10) than hiv-negative women. 271 chilongozi et al 90 reported on 2292 hiv-positive mothers from four sub-saharan sites followed for 112 months. serious adverse events occurred in 166 women (7.2%); 42 deaths occurred in the hiv-positive women, and no deaths occurred in 331 hiv-negative women. 90 several studies have examined breastfeeding relative to mothers' health and reported conflicting results. the first study from kenya demonstrated a significantly higher mortality rate in breastfeeding mothers compared with a formula-feeding group in the 2 years after delivery. the hypothesized explanation offered by the authors for this difference was increased metabolic demands, greater weight loss, and nutritional depletion. 294 a second study from south africa showed an overall lower mortality rate in the two groups with no significant difference in mortality rate in the 10 months of observation. 101 kuhn et al 227 reported no difference in mortality at 12 months after delivery between 653 women randomly assigned to a short breastfeeding group (326 women, median breastfeeding duration 4 months, 21% still breastfeeding at 12 months) and a long breastfeeding group (327 women, 90% breastfeeding at 5 months, 72% breastfeeding at 12 months, median 15 months). the hiv-related mortality rates were high (4.9%), but not associated with prolonged lactation. 227 walson et al 433 followed 535 hiv-positive women for 1 to 2 years in kenya. the mortality risk was 1.9% at 1 year and 4.8% at 2 years of follow-up. although less than 10% of women reported a hospitalization during the 2 years, they experienced various common infections (pneumonia, diarrhea, tb, malaria, stds, urinary tract infections, mastitis). breastfeeding was a significant cofactor for diarrhea and mastitis but not for pneumonia, tb, or hospitalization. 433 in summary, breastfeeding of infants by hiv-positive mothers does lead to an increased risk for hiv infection in the infants. much remains to be understood about the mechanisms of hiv transmission via breast milk and the action and efficacy of different interventions to prevent such transmission. the complete avoidance of breastfeeding is a crucial component for the prevention of perinatal hiv infection in the united states and many other countries. in resource-poor settings, where breastfeeding is the norm and where it provides vital nutritional and infection protective benefits, the who, unicef, and the joint united nations programme on hiv/ aids (unaids) recommend education, counseling, and support for hiv-infected mothers so they can make an informed choice concerning infant feeding. mothers choosing to breastfeed should receive additional education, support, and medical care to minimize the risk for hiv transmission and to optimize their own health status during and after breastfeeding. mothers choosing to use replacement feedings should receive parallel education, support, and medical care for themselves and their infants to minimize the effect of the lack of breastfeeding. good evidence now shows that antiretroviral prophylactic regimens for mothers or infants while continuing breastfeeding does decrease postnatal hiv transmission. early weaning is associated with increased morbidity and mortality. further carefully controlled research is indicated to adequately assess the risks and benefits to infants and mothers of prolonged breastfeeding with antiretroviral prophylaxis for either or both mothers and infants. along with this, hiv testing rates must be improved at the same time as increased availability and access to antenatal care, hiv prevention services, and hiv medical care for everyone must be increased. the availability and free access to antiretroviral medications must also improve. the decision about infant feeding for hivpositive mothers remains a difficult one, but this is slowly changing with increasing options. the goals remain 100% hiv transmission prevention, optimal maternal health and survival, and long-term infant health and survival. human immunodeficiency virus type 2 (hiv-2) is an rna virus in the nononcogenic, cytopathic lentivirus genus of retroviruses. it is genetically closer to simian immunodeficiency virus than to hiv-1. the clinical disease associated with hiv-2 has similar symptoms to hiv-1 infection but progresses at a slower rate to severe immunosuppression. hiv-2 is endemic in western africa and parts of the caribbean and found infrequently in europe and north and south america. 190, 305 it is transmitted via sexual contact, blood, or blood products and from mother to child. routine testing for hiv-2 is recommended in blood banks. antibody tests used for hiv-1 are only 50% to 90% sensitive for detecting hiv-2. 65 specific testing for hiv-2 is appropriate whenever clinically or epidemiologically indicated. vertical transmission occurs infrequently. ekpini et al 121 followed a large cohort of west african mothers and infants: 138 hiv-1 positive women, 132 hiv-2 positive women, 69 women seropositive for both hiv-1 and 2, and 274 hiv seronegative women. a few cases of perinatal hiv-2 transmission occurred, but no case of late postnatal transmission was observed. 121 it is probable that hiv-2 transmission via breast milk is less common than with hiv-1, but insufficient data support that the risk for transmission is zero. mothers who test positive for hiv-2 should be tested for hiv-1, and guidelines for breastfeeding should follow those for hiv-1 until additional information is available. rabies virus produces a severe infection with progressive cns symptoms (anxiety, seizures, altered mental status) that ultimately proceeds to death; few reports of survival exist. rabies occurs worldwide except in australia, antarctica, and several island groups. in 1992 more than 36,000 cases of rabies were reported to the who, a number that is probably a marked underestimate of the actual cases. 67 between 1990 and 2003, 37 cases of human rabies were reported in the united states. 70, 78 postexposure prophylaxis is given to thousands of patients each year. rabies virus is endemic in various animal populations, including raccoons, skunks, foxes, and bats. because of aggressive immunization programs, rabies in domesticated dogs and cats in the united states is uncommon. the virus is found in the saliva and tears and nervous tissue of infected animals. transmission occurs by bites, licking, or simply contact of oral secretions with mucous membranes or nonintact skin. many cases of rabies in humans now lack a history of some obvious contact with a rabid animal. this may be a result of the long incubation period (generally 4 to 6 weeks, but can be up to 1 year, with reports of incubation periods of several years), a lack of symptoms early in an infectious animal, or airborne transmission from bats in enclosed environments (caves, laboratories, houses). person-to-person transmission via bites has not been documented, although it has occurred in corneal transplants. 44 rabies viremia has not been observed in the spread of the virus. no evidence exists indicating transmission through breast milk. in the case of maternal infection with rabies, many scenarios can occur before the onset of progressive, severe cns symptoms. the progression and severity of maternal illness can preclude breastfeeding, but separation of an infant from the mother is appropriate regardless of the mother' s status and method of infant feeding (especially to avoid contact with saliva and tears). breastfeeding should not continue when the mother has symptoms of rabies, and the infant should receive postexposure immunization and close observation. an infant may received expressed breast milk, but the expression must occur without possible contamination with saliva or tears from the mother. depending on the scenario, the nature of a mother' s illness, the possible exposure of an infant to the same source as the mother, and the exposure of a child to the mother, postexposure immunization of an infant may be appropriate. a more common scenario is a mother' s apparent exposure to rabies (without exposure for the infant), necessitating postexposure immunization of the mother with rabies vaccine. in the majority of cases, in the absence of maternal illness, breastfeeding can reasonably continue during the mother' s five-dose immunization series in 28 days. in a rare situation in which apparent exposure of mother and infant to rabies occurs together, postexposure treatment of both mother and infant should be instituted, and breastfeeding can continue. respiratory syncytial virus (rsv) is a common cause of respiratory illness in children and is relatively common in adults, usually producing milder upper respiratory tract infection in adults. no evidence indicates that rsv causes intrauterine infection, adversely affects the fetus, or causes abortion or prematurity. rsv does produce infection in neonates, causing asymptomatic infection, afebrile upper respiratory tract infection, bronchiolitis, pneumonia, and apnea. mortality rate can be high in neonates, especially in premature infants and ill full-term infants, particularly those with preexisting respiratory disease (hyaline membrane disease, bronchopulmonary dysplasia) or cardiac disease associated with pulmonary hypertension. rsv is believed to be transmitted via droplets or direct contact of the conjunctiva, nasal mucosa, or oropharynx with infected respiratory secretions. documentation of rsv infection is rarely made in adults, and spread from a mother or other household contacts probably occurs before a diagnosis can be made. therefore risk for rsv transmission from breast milk is probably insignificant compared with transmission via direct or droplet contact in families. in nurseries, however, it is appropriate to make a timely diagnosis of rsv infection in neonates to isolate infants from the others and prevent spread in the nursery. ribavirin is not recommended for routine use. it is infrequently used in patients with potentially life-threatening rsv infection. rsv infection should be suspected in any infant with rhinorrhea, nasal congestion, or unexplained apnea, especially in october through march in temperate climates. prophylaxis against rsv with rsv-specific immunoglobulin iv (rsv-igiv) during this season for infants at highest risk for severe disease is appropriate. debate surrounds the topic of the effect of passively acquired antibodies (in infants from mothers before birth) against rsv on the occurrence and severity of illness in neonates and infants. it appears that a higher level of neutralizing antibody against rsv in neonates decreases the risk for severe rsv disease. 153, 239 some controversy remains concerning the measurable benefit of breastfeeding for preventing serious rsv disease. 3, 54, 115 some studies have shown benefit and others no effect. controlling for possible confounding factors (e.g., smoking, crowded living conditions) in these studies has been difficult. at this point, no reason exists to stop breastfeeding with maternal rsv infection; a potential exists for benefit from nonspecific factors in breast milk against the rsv. infants with rsv infection should breastfeed unless their respiratory status precludes it. rotavirus infections usually result in diarrhea, accompanied by emesis and low-grade fever. in severe infections the clinical course can include dehydration, electrolyte abnormalities, and acidosis and can contribute to malnutrition in developing countries. generally, every child will have at least one episode of rotavirus infection by 5 years of age. 157 in developed countries, rotavirus is often associated with diarrhea requiring hospitalization in children younger than 2 years of age, but rarely associated with death. worldwide rotavirus is the leading cause of diarrhea-related deaths in children younger than 5 years old. estimates suggest that in children younger than 5 years old rotavirus infection leads to more than 100 million occurrences of diarrhea, 2 million hospital admissions, and 500,000 deaths each year. 157 fecal-oral transmission is the most common route, but fomites and respiratory spread may also occur. spread of infection occurs most often in homes with young children or in daycare centers and institutions. in hospitalized infants or mothers with rotavirus infection, contact precautions are indicated for the duration of the illness. no evidence indicates prenatal infection from rotavirus, but perinatal or postnatal infection from contact with the mother or others can occur. no case of transmission of rotavirus via breast milk has been documented. breast milk does contain antibodies to rotavirus for up to 2 years. human milk mucin has been demonstrated to inhibit rotavirus replication and prevent experimental gastroenteritis. 457 the mechanisms of rotavirus immunity are not well understood. they are thought to be multifactorial with cell-mediated immunity limiting severity and the course of infection, while humoral immunity protects against subsequent infections. innate and adaptive responses at the level of the mucosa are probably the most important. 134 exclusive breastfeeding may decrease the likelihood of severe rotavirus-related diarrhea by as much as 90%. 93, 377 although breastfeeding does not prevent infection with rotavirus, it seems to decrease the severity of rotavirus-induced illness in children younger than 2 years old. 93, 123, 184 at least one study suggested that this may represent simply the postponement of severe rotavirus infection until an older age. 93 one study suggested that protection against rotavirus rapidly declines upon discontinuation of breastfeeding. 356 this delay in rotavirus infection until the child is older may be beneficial in that the older child may be able to tolerate the infection or illness with a lower likelihood of becoming dehydrated or malnourished. continuing breastfeeding during an episode of rotavirus illness with or without vomiting is appropriate and often helpful to the infant. no reason to suspend breastfeeding by a mother infected with rotavirus is apparent. two rotavirus vaccines (rotateq and rotarix) have been licensed for use in more than 90 countries, but less than 20 countries have routine immunization programs. additional types of rotavirus vaccines are undergoing study in various countries, specifically examining the efficacy of the vaccines in low and medium income countries. 444 some of the explanations for the slow implementation of an effective vaccine globally include differences in protection with specific vaccines in high income countries compared with low or medium income countries, the unfortunate association with intussusception in the united states, the delayed recognition of the significant rotavirus-related morbidity and mortality, and the cost of the new vaccines. the question of variable efficacy of the specific rotavirus vaccines in developed and developing countries remains an important one. several trials are examining this issue and attempting to address factors such as maternal transplacentally transferred antibodies, breastfeeding practices (especially immediately before immunization with a live oral rotavirus vaccine), stomach acid, micronutrient malnutrition, interfering gut flora, and differences in the epidemiology of rotavirus in different locations. 327 evidence indicates that maternal immunization with rotavirus vaccine can increase both transplacental acquisition of antibodies and secretory iga in breast milk. 334 additionally, oral rotavirus vaccines have been able to stimulate a good serologic response in both formula-fed and breastfed infants, although the antigen titers may need to be modified to create an optimal response in all infants. 86 the actual protective effect of these vaccines in different situations and strategies will require measurement in ongoing prospective studies. congenital rubella infection has been well described, and the contributing variables to infection and severe disease have been elucidated. the primary intervention to prevent congenital rubella has been to establish the existence of maternal immunity to rubella before conception, including immunization with rubella vaccine and reimmunization if indicated. perinatal infection is not clinically significant. postnatal infection occurs infrequently in children younger than 1 year of age because of passively acquired maternal antibodies. the predominant age of infection is 5 to 14 years old, and more than half of those with infections are asymptomatic. postnatal rubella is a self-limited, mild viral infection associated with an evanescent rash, shotty adenopathy, and low-grade transient fever. it most often occurs in the late winter and spring. infants with congenital infection shed the virus for prolonged periods from various sites and may serve as a source of infection throughout the year. contact isolation is appropriate for suspected and proved congenital infection for at least 1 year, including exclusion from day care and avoidance of pregnant women, whereas postnatal rubella infection requires droplet precautions for 7 days after the onset of rash. rubella virus has been isolated from breast milk after natural infection (congenital or postnatal) and after immunization with live attenuated vaccine virus. both iga antibodies and immunoreactive cells against rubella have been identified in breast milk. breastfed infants can acquire vaccine virus infection via milk but are asymptomatic. because postpartum infection with this virus (natural or vaccine) is not associated with clinically significant illness, no reason exists to prevent breastfeeding after congenital infection, postpartum infection with this virus, or maternal immunization with rubella vaccine. severe acute respiratory syndrome (sars) is a term that could be applied to any acute serious respiratory illness caused by or associated with a variety of infections agents; since 2003, however, it has been linked with sars-associated coronavirus (sars-cov). in the global outbreak of 2002 to 2003, more than 8400 probable cases of sars and more than 800 deaths occurred. more than the actual number of affected individuals or its associated mortality rate (approximately 10% overall, and closer to 50% in persons older than 65 years of age), it was what we did not know about this new unusual illness, and the tremendous publicity surrounding it, that made sars such a sensation. we now know the cause of this illness, known as the sars-cov. sars-cov was shown not to be closely related to the previously characterized coronavirus groups. 265, 350 despite intense international collaboration to study the illness and the virus, many things are not known, such as the degree of infectiousness, the actual period of transmissibility, all the modes of transmission, how many people have an asymptomatic infection compared with those with symptoms or severe illnesses, how to make a rapid diagnosis of confirmed cases, and where it originated. at least 21 cases of probable sars in children have been described in the literature. 42, 187, 380, 387 in general, the illness in children is a mild, nonspecific respiratory illness, but in adolescents and adults it is more likely to progress to severe respiratory distress. it has been reported that children are less likely to transmit sars than adults. 187 the overall clinical course, the radiologic evolution, and the histologic findings of these illness are consistent with the host' s immune response playing a significant role in disease production. five infants were born to mothers with confirmed sars. the infants were born prematurely (26 to 37 weeks), presumably due to maternal illness. although two of the five infants had serious abdominal illnesses (other coronaviruses have been associated with reported outbreaks of necrotizing enterocolitis), the presence of sars-cov could not be demonstrated in any of these infants. 380 no evidence of vertical transmission of sars is available. the mode of feeding for any of the reported cases of young children with sars or the infants born to mothers with sars was not mentioned. as with other respiratory viruses predominantly transmitted by droplets, transmission via breast milk is an insignificant mode of transmission, if it occurs at all. the benefits of breastfeeding being what they are, mothers with sars should continue breastfeeding if they are able, or expressed breast milk can be given to an infant until the mother is able to breastfeed. in this era of worry about biologic terrorism, smallpox is an important concern. the concern for infants (breastfed or formula-fed) is direct contact with mothers or household members with smallpox. smallpox is highly contagious in the household setting due to person-to-person spread via droplet nuclei or aerosolization from the oropharynx and direct contact with the rash. additional potential exposures for infants include the release of a smallpox aerosol into the environment by terrorists, contact with a smallpox-contaminated space or the clothes of household members exposed to an aerosol, and infection via contact with a mother' s or a household member' s smallpox vaccination site. these risks are the same for breastfed and formulafed infants. no evidence for transmission of the smallpox virus via breast milk exists. a contact is defined as a person who has been in the same household or had face-to-face contact with a patient with smallpox after the onset of fever. patients do not transmit infection until after progression from the fever stage to the development of the rash. an exposed contact does not need to be isolated from others during the postcontact observation period (usually 17 days) until the person develops fever. temperature should be monitored daily in the exposed contact. personal contact and breastfeeding between mother and infant can continue until the onset of fever, when immediate isolation (at home) should begin. providing expressed breast milk for the infant of a mother with smallpox should be avoided because of the extensive nature of the smallpox rash and the possibility of contamination (from the rash) of the milk during the expression process. no literature documents transmission of the smallpox virus via expressed breast milk. the other issue for breastfeeding infants is the question of maternal vaccination with smallpox in a preexposure event vaccination program. children older than 1 year of age can be safely and reasonably vaccinated with smallpox in the face of a probable smallpox exposure. smallpox vaccination of infants younger than 1 year of age is contraindicated. breastfeeding is listed as a contraindication to vaccination in the preevent vaccination program. it is unknown whether vaccine virus or antibodies are present in breast milk. the risk for infection due to contact or aerosolization of virus from a mother' s smallpox vaccination site is the same for breastfed and formula-fed infants. the advisory committee on immunization practices also does not recommend preevent smallpox vaccination of children younger than 18 years old. 443 a report documents tertiary contact vaccinia in a breastfeeding infant. 140 a united states military person received a primary smallpox vaccination and developed a local reaction at the inoculation site. despite reportedly observing appropriate precautions, the individual' s wife developed vesicles on both areolae (secondary contact vaccinia). subsequently, the breastfeeding infant developed lesions on her philtrum, cheek, and tongue. both the mother and infant remained well and the infections resolved without therapy. culture and pcr testing confirmed vaccinia in both the mother' s and the infant' s lesions. the breast milk was not tested. 140 in a review from 1931 to 1981, sepkowitz 375 reported on 27 cases of secondary vaccinia in households. the cdc reported 30 suspected cases of secondary/tertiary vaccinia with 18 of those cases confirmed by culture or pcr. the 30 cases were related to 578,286 vaccinated military personnel. this is an incidence of 5.2 cases per 100,000 vac cinees and 7.4 cases per 100,000 primary vaccinees. 79 in a separate report on the civilian preevent smallpox vaccination program, 37,802 individuals were vaccinated between january and june 2003, and no cases of contact vaccinia were reported. 77 the risk for contact vaccinia is low. the risk is from close or intimate contact. in the above-mentioned case, the risk for the infant was contact with the mother' s breasts, the inadvertent site of her contact vaccinia. breastfed and formulafed infants are equally at risk from close contact in the household of a smallpox vaccinee or a case of secondary vaccinia, and separation from the individual is appropriate in both situations. if the breast of the nursing mother is not involved, expressed breast milk can be given to the infant. tt virus (ttv) is a recently identified virus found in a patient (tt) with posttransfusion hepatitis not associated with the other hepatitis-related viruses a through g. ttv has been described as an unenveloped, circular, single-stranded dna virus. 311 this virus is prevalent in healthy individuals, including healthy blood donors, and has been identified in patients with hepatitis. ttv dna has been detected in infants of ttv-positive and ttvnegative mothers. ohto et al 310 reported no ttv dna was detected in cord blood from 38 infants, and it was detected in only 1 of 14 samples taken at 1 month of age. they noted an increasing prevalence from 6 months (22%) to 2 years (33%), which they ascribed to acquisition via nonparenteral routes. in comparisons of the ttv dna in ttvpositive mothers and their ttv-positive infants, 6 of 13 showed high level nucleotide sequence similarity, and 7 of 13 differed by greater than 10%. 310 schröter et al 371 reported on ttv dna in breast milk examined retrospectively. notably, ttv dna was detected in 22 of 23 serum samples of infants at 1 week of age, who were born to 22 women viremic for ttv dna. twenty-four women who were negative for ttv dna gave birth to 24 children who were initially negative for ttv dna and remained negative throughout the observation period (mean 7.5 months, range 1 to 28 months). ttv dna was detected in 77% of breast milk samples from ttv viremic women and in none of the breast milk samples from ttv-negative women. no clinical or laboratory evidence of hepatitis was found in the 22 children who were observed to be ttv dna positive during the period of the study. 371 other authors have reported ttv in breast milk detected by pcr. they describe the absence of ttv dna in infants at 5 days and 3 months of age, and 4 of 10 infants were positive for ttv dna at 6 months of age, suggesting the late acquisition of infection via breastfeeding. 197 tt virus is transmitted in utero and is found in breast milk. no evidence of clinical hepatitis in infants related to ttv infection and no evidence for a late chronic hepatitis exist. given the current available information, no reason to proscribe breastfeeding by ttv-positive mothers is compelling. certainly more needs to be understood concerning the chronic nature of this infection and the possible pathogenesis of liver disease. no documented evidence indicates that women with breast cancer have rna of tumor virus in their milk. no correlation between rna-directed dna polymerase activity has been found in women with a family history of breast cancer. rna-directed dna polymerase activity, a reserve transcriptase, is a normal feature of the lactating breast. 91, 129, 352 epidemiologic data conflict with the suggestion that the tumor agent is transmitted through the breast milk. the incidence of breast cancer is low among groups who had nursed their infants, including lower economic groups, foreign-born groups, and those in sparsely populated areas. 262 the frequency of breast cancer in mothers and sisters of a woman with breast cancer is two to three times that expected by chance. this could be genetic or environmental. cancer actually is equally common on both sides of the family of an affected woman. if breast milk were the cause, it should be transmitted from mother to daughter. when mother-daughter incidence of cancer was studied, no relationship was found to breastfeeding. sarkar et al 360 reported that human milk, when incubated with mouse mammary tumor virus, caused degradation of the particular morphology and decreased infectivity and reverse transcriptase activity of the virions. they suggest that the significance of this destructive effect of human milk on mouse mammary tumor virus may account for the difficulty in isolating the putative human mammary tumor agent. sanner 359 showed that the inhibitory enzymes in milk can be removed by special sedimentation technique. he ascribes the discrepancies in isolating virus particles in human milk to these factors, which inhibit rna-directed dna po lymerase. the fear of cancer in breastfed female offspring of a woman with breast cancer does not justify avoiding breastfeeding. breastfed women have the same breast cancer experience as nonbreastfed women, and no increase is seen in benign tumors. daughters of breast cancer patients have an increased risk for developing benign and malignant tumors because of their heredity, not because of their breastfeeding history. 280, 287 unilateral breastfeeding (limited to the right breast) is a custom of tanka women of the fishing villages of hong kong. ing et al 194 investigated the question, "does the unsuckled breast have an altered risk for cancer?" they studied breast cancer data from 1958 to 1975. breast cancer occurred equally in the left and the right breasts. comparison of patients who had nursed unilaterally with nulliparous patients and with patients who had borne children but not breastfed indicated a highly significantly increased risk for cancer in the unsuckled breast. the authors conclude that in postmenopausal women who have breastfed unilaterally, the risk for cancer is significantly higher in the unsuckled breast. they thought that breastfeeding may help protect the suckled breast against cancer. 194 others 274 have suggested that tanka women are ethnically a separate people and that left-sided breast cancer may be related to their genetic pool and not to their breastfeeding habits. no mention has been made of other possible influences, such as the impact of their role as "fishermen" or any inherent trauma to the left breast. 274 in 1926, lane-claypon 240 stated that a breast that had never lactated was more liable to become cancerous. nulliparity and absence of breastfeeding had been considered important risk factors for breast cancer. macmahon et al 262 reported in 1970 that age at first full-term pregnancy was the compelling factor, and the younger the mother, the less the risk. in a collective review of the etiologic factors in cancer of the breast in humans, papaioannou 325 concludes, "genetic factors, viruses, hormones, psychogenic stress, diet, and other possible factors, probably in that order of importance, contribute to some extent to the development of cancer of the breast." 325 wing 449 concluded in her 1977 review on human milk and health that "in view of the complete absence of any studies showing a relationship between breastfeeding and increased risk of breast cancer, the presence of virus-like particles in breast milk should not be a contraindication to breastfeeding." henderson et al 173 gradually, studies have appeared challenging the dogma. brinton et al, 52 mctiernan and thomas, 276 and layde et al 245 showed the clearly protective effects of breastfeeding. another example is a study conducted to clarify whether lactation has a protective role against breast cancer in an asian people, regardless of confounding effects of age at first pregnancy, parity, and closely related factors. 458 in a hospital-based case-control study of 521 women without breast cancer, statistical adjustment for potential confounders and a likelihood ratio test for linear trend were done by unconditional logistic regression. total months of lactation regardless of parity was the discriminator. regardless of age of first pregnancy and parity, lactation had an independent protective effect against breast cancer in japanese women. 458 although breast cancer incidence is influenced by genetics, stress, hormones, and pregnancy, breastfeeding clearly has a protective effect. "there is a reduction in the risk of breast cancer among premenopausal women who have lactated. no reduction in the risk of breast cancer occurred among postmenopausal women with a history of lactation," according to newcombe et al, 299 reporting a multicenter study in 1993. varicella-zoster virus infection (varicella/chickenpox, zoster/shingles) is one of the most communicable diseases of humans, in a class with measles and smallpox. transmission is thought to occur via respiratory droplets and virus from vesicles. varicella in pregnancy is a rare event, although disease can be more severe with varicella pneumonia, and can be fatal. congenital varicella-zoster virus infection occurs infrequently, causing abortion, prematurity, and congenital malformations. a syndrome of malformations has been carefully described with congenital varicella-zoster virus infection, typically involving limb deformity, skin scarring, and nerve damage, including to the eye and brain. 148 perinatal infection can lead to severe infection in infants if maternal rash develops 5 days or less before delivery and within 2 days after delivery. illness in infants usually develops before 10 days of age and is believed to be more severe because of the lack of adequate transfer of antibody from the mother during this period and transplacental spread of virus to the fetus and infant during viremia in the mother. varicella in a mother occurring before 5 days before delivery allows sufficient formation and transplacental transfer of antibodies to the infant to ameliorate disease even if the infant is infected with varicella-zoster virus. mothers who develop varicella rash more than 2 days after delivery are less likely to transfer virus to the infant transplacentally; they pose a risk to the infants from postnatal exposure, which can be diminished by the administration of varicellazoster ig to the infant. postnatal transmission is believed to occur through aerosolized virus from skin lesions or the respiratory tract entering the susceptible infant's respiratory tract. airborne precautions are therefore appropriate in the hospital setting. infants infected with varicella-zoster virus in utero or in the perinatal period (younger than 1 month of age) are more likely to develop zoster (reactivation of latent varicella-zoster virus) during childhood or as young adults. postnatal varicella from nonmaternal exposure can occur but is generally mild when it develops after 3 weeks of age or when a mother has passed on antibodies against varicella-zoster virus via the placenta. severe postnatal varicella does occur in premature infants or infants of varicella-susceptible mothers. when a mother' s immune status relative to varicella-zoster virus is uncertain and measurement of antibodies to varicella-zoster virus in mother or infant cannot be performed promptly (less than 72 hours), administration of vzig 81 or ivig to the infant exposed to varicella or zoster in the postnatal period is indicated. ideally a mother' s varicella status should be known before pregnancy, when varicella virus vaccine could be given if indicated. varicella-zoster virus virus has not been cultured from milk, but varicella-zoster virus dna has been identified in breast milk. 459 antibody against varicella-zoster virus has also been found in breast milk. 270 breast milk from mothers who had received the varicella vaccine in the postpartum period was tested for varicella-zoster virus dna. varicella dna was not detected in any of the 217 breast milk samples from the 12 women, all of whom seroconverted after vaccination. 45 one case of suspected transfer of varicella-zoster virus to an infant via breastfeeding has been reported, but virus may have been transmitted by respiratory droplet or exposure to rash before the mother began antiviral therapy. 459 isolation of an infant from the mother with varicella and interruption of breastfeeding should occur only while the mother remains clinically infectious, regardless of the method of feeding. as soon as the infant has received varicella-zoster ig, expressed breast milk can be given to an infant if no skin lesions involve the breasts. persons with varicella rash are considered noninfectious when no new vesicles have appeared for 72 hours and all lesions have crusted, usually in 6 to 10 days. immunocompetent mothers who develop zoster can continue to breastfeed if the lesions do not involve the breast and can be covered because antibodies against varicella-zoster virus are provided to the infant via the placenta and breast milk and will diminish the severity of disease, even if not preventing it. conservative management in this scenario would include giving an infant varicella-zoster ig as well (see table 13 -5). 331 it is estimated that 150 to 300 asymptomatic cases of west nile infection occur for every 20 febrile illnesses and for every one case of meningoencephalitis associated with west nile virus. west nile fever is usually a mild illness of 3 to 6 days' duration. the symptoms are relatively nonspecific, including malaise, nausea, vomiting, headache, myalgia, lymphadenopathy, and rash. west nile disease is characterized by severe neurologic symptoms (e.g., meningitis, encephalitis, or acute flaccid paralysis, and occasionally optic neuritis, cranial nerve abnormalities, and seizures). children are infrequently sick with west nile virus infection and infants younger than 1 year of age have rarely been reported. 331 the case-fatality rate for 2003 in the united states was approximately 2.5%, but has been reported as high as 4% to 18% in hospitalized patients. the case-fatality rate for persons older than 70 years of age is considered to be higher, 15% to 29% among hospitalized patients in outbreaks in romania and israel. 331 the primary mechanism of transmission is via a mosquito bite. mosquitoes from the genus culex are primary vectors. the bird-mosquito-bird cycle serves to maintain and amplify the virus in the environment. humans and horses are incidental hosts. the pathogenesis of the infection is believed to occur via replication of the virus in the skin and lymph nodes, leading to a primary viremia that seeds secondary sites before a second viremia causes the infection of the cns and other affected organs. 59, 111 transmission has been reported in rare instances during pregnancy 7,73 via organ transplant 199 and percutaneously in laboratory workers. 75 a study of west nile virus infection in pregnancy documented four miscarriages, two elective abortions, and 72 live births. cord blood samples were tested in 55 infants and 54 of 55 were negative for anti-west nile virus igm. three infants had west nile virus infection, which could have been acquired congenitally. three of 7 infants who had congenital malformations might have been caused by maternal west nile virus infection based on timing in pregnancy, but no evidence of west nile virus etiology is conclusive. 312 west nile virus transmission occurs via blood and blood product transfusion, 186 and the incidence has been estimated to be as high as 21 per 10,000 donations during epidemics in specific cities. 40 no evidence of direct person-to-person transmission without the mosquito vector has been found. one case of possible west nile virus transmission via breastfeeding has been documented. 74 the mother acquired the virus via packed rbc transfusions after delivery. the second unit of blood she received was associated with other blood products from the same donation causing west nile infection in another transfusion recipient. eight days later the mother had a severe headache and was hospitalized with fever and a csf pleocytosis on day 12 after delivery. the mother' s csf was positive for west nile virus-specific igm antibody. the infant had been breastfed from birth through the second day of hospitalization of the mother. samples of breast milk were west nile virus-specific igg and igm positive on day 16 after delivery and west nile virus-specific igm positive on day 24. the same milk was west nile virus rna positive by pcr testing on day 16, but not on day 24 after delivery. the infant tested positive for west nile virus-specific igm in serum at day 25 of age, but remained well without fever. no clear-cut exposure to mosquitoes for the infant were reported. the cord blood and placenta were not available to be tested. igm antibodies can be found in low concentrations in breast milk, but this is not common or as efficient as the transfer of iga, secretory iga, or igg into breast milk. a review of west nile virus illness during the breastfeeding identified six occurrences of breastfeeding during maternal west nile virus illness. 177 five of the six infants had no illness or detectable antibodies to west nile virus in their blood. one infant developed a rash and was otherwise well after maternal west nile virus illness, but was not tested for west nile virus infection. two infants were identified who developed west nile virus illness while breastfeeding, but no preceding west nile virus infection was demonstrated in their mothers. two other breastfeeding infants developed west nile virus-specific antibodies after their mothers acquired west nile virus illness in the last week of pregnancy, but congenital infection could not be ruled out. live virus was not cultured from 45 samples of breast milk from mothers infected with west nile virus during pregnancy, but west nile virus rna was detected in two samples and 14 samples had igm antibodies to west nile virus. 177 the above data suggest that west nile virus infection through breastfeeding is rare. to date evidence of significant disease due to west nile virus infection in young breastfeeding children is lacking. at this time, no reason exists to proscribe breastfeeding in the case of maternal west nile virus infection if a mother is well enough to breastfeed. as with many other maternal viral illnesses, by the time the diagnosis is made in a mother, the infant may have already been exposed during maternal viremia and possible virolactia. the infant can and should continue to receive breast milk for the potential specific and nonspecific antiviral immunologic benefits. yellow fever virus is a flavivirus which is transmitted to humans by infected aedes and haemogogus mosquitos in tropical areas of south america and africa. large outbreaks occur when mosquitos in a populated area become infected from biting viremic humans infected with yellow fever virus. transmission from the mosquitos to other humans occurs after an incubation period in the mosquito of 8 days. direct person-to-person spread has not been reported. illness due to yellow fever virus usually begins after an incubation period of 3 to 6 days, with acute onset of headache, fever, chills, and myalgia. photophobia, back pain, anorexia, vomiting, and restlessness are other common symptoms. the individual is usually viremic for the first 4 days of illness until the fever and other symptoms diminish. liver dysfunction and even failure can develop as can myocardial dysfunction. cns infection is uncommon but symptoms can include seizures and coma. medical care should include intensive supportive care and fluid management. one case of congenital infection after immunization of a pregnant woman with the attenuated vaccine strain has been reported. one of 41 infants whose mothers had inadvertently received the yellow fever virus vaccine during pregnancy developed igm and elevated neutralizing antibodies against the yellow fever virus without any evidence of illness or abnormalities. 418 a more recent study 404 from brazil examined inadvertent yellow fever virus immunization during pregnancy during a mass vaccination campaign in 2000; 480 pregnant women received the yellow fever virus at a mean of 5.7 weeks' gestation, the majority of whom did not know their pregnancy status at the time. seroconversion occurred in 98.2% of the women after at least 6 weeks after vaccination. mild postvaccination illness (headache, fever, or myalgia) was reported by 19.6% of the 480 women. the frequency of malformations, miscarriages, stillbirths, and premature deliveries was similar to that found in the general population. at the 12-month follow-up point, 7% of the infants still demonstrated neutralizing antibodies against yellow fever virus, but after 12 months only one child was still seropositive. 404 transmission of the yellow fever vaccine virus through breastfeeding was recently reported from brazil. 85 the mother was immunized during a yellow fever epidemic in a nonendemic area in brazil; 15 days after delivering a healthy female infant (39 weeks' gestational age) the mother received the 17dd yellow fever vaccine, and 5 days later the mother reported headache, malaise, and low-grade fever that persisted for 2 days. the mother continued breastfeeding and did not seek medical care for herself. at 23 days of age the infant became irritable, developed fever, and refused to nurse. the infant developed seizures and subsequent evaluation of the infant demonstrated an abnormal csf and ct of the brain showed bilateral areas of diffuse low density suggestive of inflammation and consistent with encephalitis. yellow fever-specific igm antibodies were identified in the infant' s serum and csf. reverse-transcriptase polymerase chain reaction (rt-pcr) testing of the csf also demonstrated yellow fever virus rna identical to the 17dd yellow fever vaccine virus. breast milk and maternal serum were not tested for yellow fever virus. 85 yellow fever virus, wild or vaccine type, has not been identified in human breast milk, although another flavivirus, west nile virus, has been detected in milk from a few lactating women with west nile virus infection. 177 (see the section on west nile virus.) yellow fever vaccine-associated neurologic disease occurs at different rates in different age-groups, including 0.5 to 4.0 cases per 1000 infants younger than 6 months of age. 285 the 17d-derived yellow fever vaccines are contraindicated in infants younger than 6 months of age. since 2002, the advisory committee on immunization practices has recommended, based on theoretical risk, that yellow fever vaccine be avoided in nursing mothers, except when exposure in highrisk yellow fever endemic areas is likely to occur. 76 no case of transmission of yellow fever virus from an infected mother to her infant via breastfeeding or breast milk has been reported. published information on the severity of yellow fever virus infection in infants younger than 1 year of age, potential protection from passively acquired antibodies, or protection from breast milk is limited. no information on a differential risk in breastfed versus formula-fed infants is available. given the well documented method of transmission of yellow fever virus via mosquitos, and the lack of evidence of transmission via breast milk, it makes more sense to protect all infants against mosquito bites than to proscribe breastfeeding, even in the mother infected with yellow fever virus. continued breastfeeding or use of expressed breast milk will depend on a mother's health status and ability to maintain the milk supply while acutely ill. if another source of feeding is readily available then temporarily discarding expressed breast milk for at least 4 days of acute illness in the mother is a reasonable precaution. lyme disease, as with other human illnesses caused by spirochetes, especially syphilis, is characterized by a protean course and distinct phases (stages) of disease. lyme borreliosis was described in europe in the early twentieth century. since the 1970s, tremendous recognition, description, and investigation of lyme disease have occurred in the united states and europe. public concern surrounding this illness is dramatic. lyme disease is a multisystem disease characterized by involvement of the skin, heart, joints, and nervous system (peripheral and central). stages of disease are identified as early localized (erythema migrans, often accompanied by arthralgia, neck stiffness, fever, malaise, and headache), early disseminated (multiple erythema migrans lesions, cranial nerve palsies, meningitis, conjunctivitis, arthralgia, myalgia, headache, fatigue, and, rarely, myocarditis), and late disease (recurrent arthritis, encephalopathy, and neuropathy). the varied manifestations of disease may relate to the degree of spirochetemia, the extent of dissemination to specific tissues, and the host' s immunologic response. the diagnosis of lyme disease is often difficult in part because of the broad spectrum of presentations, inapparent exposure to the tick, and the lack of adequately standardized serologic tests. culture of the spirochete, borrelia burgdorferi, is not readily available. enzyme-linked immunosorbent assay (elisa), immunofluorescent assay, and immunoblot assay are the usual tests. pcr detection of spirochetal dna requires additional testing in clinical situations to clarify and standardize its utility. gardner 142 reviewed infection during pregnancy, summarizing a total of 46 adverse outcomes from 161 cases reported in the literature. the adverse outcomes included miscarriage and stillbirth (11% of cases), perinatal death (3%), congenital anomalies (15%), and both early-and late-onset progressive infection in the infants. silver 384 reviewed 11 published reports and concluded that lyme disease during pregnancy is uncommon, even in endemic areas. although the spirochete can be transmitted transplacentally, a significant immune response in the fetus is often lacking, and the association of lyme infection with congenital abnormalities is weak. 401, 448 little published information exists on whether b. burgdorferi can be transmitted via breast milk. one report showed the detection of b. burgdorferi dna by pcr in the breast milk of two lactating women with untreated erythema migrans, but no evidence of lyme disease or transmission of the spirochete in the one infant followed for 1 year. 369 no attempt to culture the spirochete was made, so it is not possible to determine if the detectable dna was from viable spirochetes or noninfectious fragments. in that same study of 56 women with untreated erythema migrans who had detectable b. burgdorferi dna in the urine, 32 still had detectable dna in the urine 15 to 30 days after starting treatment, but none had it 6 months after initiating therapy. ziska et al 466 reported on the management of nine cases of lyme disease in women associated with pregnancy; seven of the nine women were symptomatic at conception and six received antibiotics throughout pregnancy. follow-up of the infants showed no transmission of lyme disease, even in the seven infants who had been breastfed. 466 the lack of adequate information on transmission of b. burgdorferi via breast milk cannot be taken as proof that it is not occurring. if one extrapolates from data on syphilis and the treponema pallidum spirochete, it would be prudent to discuss the lack of information on the transmission of b. burgdorferi via breast milk with the mother or parents and to consider withholding breast milk at least until therapy for lyme disease has begun or been completed. if the infection occurred during pregnancy and treatment has already been completed, an infant can breastfeed. if infection occurs postpartum or the diagnosis is made postpartum, infant exposure may have already occurred. again, discussion with the mother or parents about withholding versus continuing breastfeeding is appropriate. after prenatal or postnatal exposure, an infant should be closely observed and empiric therapy considered if the infant develops a rash or symptoms suggestive of lyme borreliosis. treatment of mother and infant with ceftriaxone, penicillin, or amoxicillin is acceptable during breastfeeding relative to the infant' s exposure to these medications. doxycycline should not be administered for more than 14 days while continuing breastfeeding because of possible dental staining in the neonate. continued surveillance for viable organisms in breast milk and evidence of transmission through breastfeeding is recommended. a large body of information is available on various "lyme vaccines" used in dogs, but these vaccines are only partially protective and must be repeated yearly. preliminary information suggests that a vaccine for use in humans safely produces good serologic responses, but protective efficacy has not been demonstrated, and no information exists on its use during pregnancy or breastfeeding. syphilis is the classic example of a spirochetal infection that causes multisystem disease in various stages. both acquired syphilis and congenital syphilis are well-described entities. acquired syphilis is almost always transmitted through direct sexual contact with open lesions of the skin or mucous membranes of individuals infected with the spirochete, treponema pallidum. congenital syphilis occurs by infection across the placenta (placentitis) at any time during the pregnancy or by contact with the spirochete during passage through the birth canal. any stage of disease (primary, secondary, tertiary) in a mother can lead to infection of the fetus, but transmission in association with secondary syphilis approaches 100%. infection with primary syphilis during pregnancy, without treatment, leads to spontaneous abortion, stillbirth, or perinatal death in 40% of cases. similar to acquired syphilis, congenital syphilis manifests with moist lesions or secretions from rhinitis (snuffles), condylomata lata, or bullous lesions. these lesions and secretions contain numerous spirochetes and are therefore highly infectious. postnatal infection of an infant can occur through contact with open, moist lesions of the skin or mucous membranes of the mother or other infected individuals. if the mother or infant has potentially infectious lesions, isolation from each other and from other infants and mothers is recommended. if lesions are on the breasts or nipples, breastfeeding or using expressed milk is contraindicated until treatment is complete and the lesions have cleared. spirochetes are rarely identified in open lesions after more than 24 hours of appropriate treatment. penicillin remains the best therapy. evaluation of an infant with suspected syphilis should be based on the mother' s clinical and serologic status, history of adequate therapy in the mother, and the infant' s clinical status. histologic examination of the placenta and umbilical cord, serologic testing of the infant' s blood and csf, complete analysis of the csf, long bone and chest radiographs, liver function tests, and a complete blood cell count are all appropriate given the specific clinical situation. treatment of the infant should follow recommended protocols for suspected, probable, or proven syphilitic infection. 96 no evidence indicates transmission of syphilis via breast milk in the absence of a breast or nipple lesion. when a mother has no suspicious breast lesions, breastfeeding is acceptable as long as appropriate therapy for suspected or proven syphilis is begun in the mother and infant. giardiasis is a localized infection limited to the intestinal tract, causing diarrhea and malabsorption. immunocompetent individuals show no evidence of invasive infection, and no evidence exists that documents fetal infection from maternal infection during pregnancy. giardiasis is rare in children younger than 6 months of age, although neonatal infection from fecal contamination at birth has been described. 22 human milk has an in vivo protective effect against giardia lamblia infection, as documented by work from central africa, where the end of breastfeeding heralds the onset of giardia infection. 145 this has been reaffirmed in undeveloped countries around the world. the protective effect of breast milk has been identified in the milk of noninfected donors. 151 the antiparasitic effect does not result from specific antibodies but rather from lipase enzymatic activity. the lipase acts in the presence of bile salts to destroy the trophozoites as they emerge from their cysts in the gi tract. hernell et al 175 demonstrated that free fatty acids have a marked giardiacidal effect, which supports the conclusion that lipase activity releasing fatty acids is responsible for killing g. lamblia. g. lamblia have also been reported to appear in the mother' s milk, and the parasite has been transmitted to newborns via that route. the exact relationship of breastfeeding to transmission of g. lamblia and the effect on infants continue to be studied, even though symptomatic infection in breastfed infants is rare. 151 one report from the middle east suggests that even partial breastfeeding is protective against infection with intestinal parasites, including cryptosporidium and giardia lamblia. 41 breastfeeding by mothers with giardiasis is problematic mainly because of the medications used for therapy. metronidazole' s safety in infants has not been established, and little information is available on quinacrine hydrochloride and furazolidone in breast milk. paromomycin, a nonabsorbable aminoglycoside, is a reasonable alternative recommended for treatment of pregnant women. breastfeeding by a mother with symptomatic giardiasis is acceptable when consideration is given to the presence of the therapeutic agents in the breast milk. hookworm infection, most often caused by ancylostoma duodenale and necator americanus, is common in children younger than the age of 4 years, and there is at least one report on infantile hookworm disease from china. 374 this publication from the chinese literature reports hundreds of cases of infantile hookworm disease that include the common symptoms of bloody stools, melena, anorexia, listlessness, and edema. anemia, eosinophilia and even leukemoid reactions occur as part of the clinical pictures in young children. they also note at least 20 cases of hookworm diseases in newborn infants younger than 1 month of age. in the discussion of infantile hookworm infection, they note four routes of infection: direct contact with contaminated soil, "sand-stuffed" diapers, contaminated "washed/wet" diapers, and vertical equal to transmammary transmission or transplacental transmission. they postulated that infection of infants before 40 to 50 days of age would most likely be due to transplacental transmission and infection before environmental contact would most likely be due to transmammary transmission. ample evidence is available in veterinary medicine of transmammary spread of helminths. 302, 382 at least two reports suggest the possibility of transmammary transmission of hookworms in humans. setasuban et al 376 described the prevalence of necator americanus in 128 nursing mothers as 61% and identified n. americanus in breast milk in one case. nwosu 303 documented positive stool samples for hookworms in 33 of 316 neonates (10%) at 4 to 5 weeks of age in southern nigeria. the majority of neonatal infections were due to ancylostoma duodenale although necator americanus is more prevalent in that area of nigeria. examination of colostral milk did not demonstrate any hookworm larvae. 303 additional epidemiologic work is necessary to determine the potential significance of transmammary spread of helminths in humans, and more careful examination of breast milk as a source of hookworm infection is required before reasonable recommendation are possible. malaria is recognized as a major health problem in many countries. the effect of malaria infection on pregnant and lactating women and thus on the developing fetus, neonate, and growing infant can be significant. the four species of malaria, plasmodium vivax, p. ovale, p. malariae, and p. falciparum, vary in the specific aspects of the disease they produce. p. vivax exists throughout the world, but p. falciparum predominates in the tropics and is most problematic in its chloroquine-resistant form. malaria in the united states is most often seen in individuals traveling from areas where malaria is endemic. the parasite can exist in the blood for weeks, and infection with p. vivax and p. malariae can lead to relapses years later. transmission occurs through the bite of the anopheline mosquito and can occur via transfusion of blood products and transplacentally. congenital malaria is rare but seems to occur more often with p. vivax and p. falciparum. it usually presents in the first 7 days of life (range 1 day to 2 months). it may resemble neonatal sepsis, with fever, anemia, and splenomegaly occurring in the most neonates and hyperbilirubinemia and hepatomegaly in less than half. malaria in infants younger than 3 months of age generally manifests with less severe disease and death than in older children. possible explanations include the effect of less exposure to mosquitoes, passive antibody acquired from the mother, and the high level of fetal hemoglobin in infants at this age. 22 the variations in the infection rates in children younger 3 months of age during the wet and dry seasons support the idea that postnatal infection is more common than congenital infection. no evidence indicates that malaria is transmitted through breast milk. the greatest risk to infants is exposure to the anopheline mosquito infected with malaria. the main issues relative to malaria and breastfeeding are how to protect both mothers and infants effectively from mosquitoes and what drugs for treating malaria in mothers are appropriate during lactation. protection from mosquito bites includes screened-in living areas, mosquito nets while sleeping, protective clothing with or without repellents on the clothes, and community efforts to eradicate the mosquitoes. chloroquine, quinine, and tetracycline are acceptable during breastfeeding. sulfonamides should be avoided in the first month of an infant' s life, but pyrimethamine-sulfadoxine (fansidar) can be used later. mefloquine is not approved for infants or pregnant women. however, the milk/plasma ratio for mefloquine is less than 0.25, there is a large volume of distribution of the drug, high protein binding of the drug limits its presence in breast milk, and the relative importance of breastfeeding in areas where malaria is prevalent shifts the risk/benefit ratio in favor of treatment with mefloquine. the single dose recommended for treatment or the onceweekly dose for prevention allows for continued breastfeeding with discarding of the milk for short periods after a dose (1 to 6 hours). maternal plasma levels of primaquine range from 53 to 107 ng/ml, but no information is available on levels in human milk. primaquine is used in children, and once daily dosing in the mother would allow discarding milk with peak levels of drug. therefore breastfeeding during maternal malaria even with treatment is appropriate with specific medications. strongyloides stercoralis is a nematode (roundworm). most infections are asymptomatic, but clinically significant infection in humans can include larval skin invasion, tissue migration, intestinal invasion with abdominal pain and gi symptoms, and a loeffler-like syndrome due to migration to the lungs. immune-compromised individuals can develop dissemination of larvae systemically, causing various clinical symptoms. humans are the principal hosts, but other mammals can serve as reservoirs. infection via the skin by filariform larvae is the most common form of transmission; ingestion is an uncommon occurrence. transmammary transmission of strongyloides species has been described in dogs, ewes, and rats. 211, 302, 382 only one report of transmammary passage of strongyloides larvae in humans is available. in 76 infants younger than 200 days of age, 34% demonstrated the presence of strongyloides fuelleborni on stool examination. the clinical significance of this was not elucidated. strongyloides larvae was identified in only one sample of milk from 25 nursing mothers. 53 in the absence of an understanding of the clinical significance of strongyloides in the stools of young infants, given the lack of exclusion of the most common mechanism of transmission (through the skin) in the single report and the apparent infrequent evidence of these larvae in human milk, it is difficult to make any recommendations concerning breastfeeding and strongyloides. toxoplasmosis is one of the most common infections of humans throughout the world. the infective organism, toxoplasma gondii, is ubiquitous in nature. the prevalence of positive serologic test titers increases with age, indicating past exposure and infection. the cat is the definitive host, although infection occurs in most species of warmblooded animals. postnatal infection with toxoplasmosis is usually asymptomatic. symptomatic infection typically manifests with nonspecific symptoms, including fever, malaise, myalgia, sore throat, lymphadenopathy, rash, hepatosplenomegaly, and occasionally a mononucleosis-like illness. the illness usually resolves without treatment or significant complications. congenital infection or infection in an immunodeficient individual can be persistent and severe, causing significant morbidity and even death. although most infants with congenital infection are asymptomatic at birth, visual abnormalities, learning disabilities, and mental retardation can occur months or years later. the syndrome of congenital toxoplasmosis is clearly defined, with the most severe manifestations involving the cns, including hydrocephalus, cerebral calcifications, microcephaly, chorioretinitis, seizures, or simply isolated ocular involvement. the risk for fetal infection is related to the timing of primary maternal infection, although transmission can occur with preexisting maternal toxoplasmosis. 241 in the last months of pregnancy the protozoan is more readily transmitted to the fetus, but the infection is more likely to be subclinical. early in pregnancy the transmission to a fetus occurs less frequently but does result in severe disease. treatment of documented congenital infection is currently recommended, although duration and optimal regimen have not been determined, and reversal of preexisting sequelae generally does not occur. 343 prevention of infection in susceptible pregnant women is possible by avoiding exposure to cat feces or the organism in the soil. pregnant or lactating women should not change cat litter boxes, but if they must, it should be done daily and while wearing gloves. the oocyst is not infective for the first 24 to 48 hours after passage. mothers can avoid ingestion of the organism by fully cooking meats and carefully washing fruits, vegetables, and food preparation surfaces. 94 in various animal models, t. gondii has been transmitted through the milk to the suckling young. the organism has been isolated from colostrum as well. the newborn animals became asymptomatically infected when nursed by an infected mother whose colostrum contained t. gondii. only one report has identified t. gondii in human milk, and some question surrounds the reliability of that report. 241 transmission during breastfeeding in humans has not been demonstrated. breast milk may contain appropriate antibodies against t. gondii. given the benign nature of postnatal infection, the absence of documented transmission in human breast milk, and the potential antibodies in breast milk, no reason exists to proscribe breastfeeding by a mother known to be infected with toxoplasmosis. trichomonas vaginalis is a flagellated protozoan that can produce vaginitis (see chapter 16 for a discussion of vaginitis) but frequently causes asymptomatic infection in both men and women. the parasite is found in 10% to 25% of women in the childbearing years. it is transmitted predominantly by sexual intercourse, but it can be transmitted to the neonate by passage through the birth canal. this parasite often coexists with other stds, especially gonorrhea. infection during pregnancy or while taking oral contraceptives is more difficult to treat. some evidence suggests that infection with and growth of the parasite are enhanced by estrogens or their effect on the vaginal epithelium. no evidence indicates adverse effects on the fetus in association with maternal infection during pregnancy. occasionally female newborns have vaginal discharge during the first weeks of life caused by t. vaginalis. this is thought to be influenced by the effect of maternal estrogen on the infant' s vaginal epithelium and acquisition of the organism during passage through the birth canal. the organism does not seem to cause significant disease in a healthy infant. no documentation exists on transmission of t. vaginalis via breast milk. the difficulty encountered with maternal infection during lactation stems from metronidazole (flagyl), the drug of choice, being contraindicated for infants. case reports describe treatment of neonates with metronidazole without adverse effect. although topical agents containing povidone-iodine (betadine) or sodium lauryl sulfate (trichotine) can be effective when given as douches, creams, or suppositories, metronidazole remains the treatment of choice. the aap advises using metronidazole only with a physician' s discretion and considers its effect on a nursing infant unknown but possibly a concern. the potential concerns are metronidazole' s disulfiram-like effect in association with alcohol, tumorigenicity in animal studies, and leukopenia and neurologic side effects described in adults. on the other hand, metronidazole is given to children beyond the neonatal period to treat serious infections with various other parasites, such as entamoeba histolytica. the current recommendation for lactating women is to try local treatment first, and if these fail, then to try metronidazole. a 2-g single-dose treatment produces peak levels after 1 hour, and discarding expressed breast milk for the next 12 to 24 hours is recommended. if this treatment also fails, a 1-g twice-daily regimen for 7 days or a 2-g single daily dose for 3 to 5 days is recommended, with discarding of breast milk close to the dose and timing of feedings distant from the dose. infants who exclusively breastfeed are presumed at greater risk from exposure to metronidazole than those who are only partially breastfed. candida consists of multiple species. the most common species affecting humans include c. albicans as the dominant agent and c. tropicalis, c. krusei, and c. parapsilosis, as well as many other uncommon species. in general, candida exists as a commensal organism colonizing the oropharynx, gi tract, vagina, and skin without causing disease until some change disrupts the balance between the organism and the host. mild mucocutaneous infection is the most common illness, which can lead to vulvovaginitis, mastitis, or, uncommonly, oral mucositis in a mother, and thrush (oral candidiasis) and candidal diaper rash in an infant. invasive candidal infection occurs infrequently, usually when a person has other illness, impaired resistance to infection (hiv, diabetes mellitus, neutropenia; decreased cell-mediated immunity in premature infants or lbw or vlbw infants), or disrupted normal mucosal and skin barriers and has received antibiotics or corticosteroids. invasive disease can occur through local spread, and may occur more often in the genitourinary tract (urethra, bladder, ureters, kidneys), but usually develops in association with candidemia. the bladder and kidney are more frequently involved, but when dissemination occurs via candidemia, a careful search for other sites of infection should be made (e.g., retina, liver, spleen, lung, meninges). 279 transmission usually occurs from healthy individuals colonized with candida through direct contact with them or through contact with their oral or vaginal secretions. intrauterine infection can occur through ascending infection through the birth canal but is rare. no distinct syndrome of congenital candidal infection exists. most often an infant is infected in passing through the birth canal and remains colonized. postnatal transmission can occur through direct contact with caregivers. the mother and infant serve as an immediate source of recolonization for each other, especially during the direct contact of breastfeeding. for this reason, an infant and breastfeeding mother should be treated simultaneously when treating thrush, vulvovaginitis, diaper candidiasis, or mastitis. colonization with this organism usually occurs in the absence of any clinical evidence of infection. simultaneous treatment should occur even in the absence of any clinical evidence of candida infection or colonization in the apparently uninvolved individual of the breastfeeding dyad. no well-controlled clinical trials define the most appropriate or most effective method(s) of treatment for candidal infection in breastfeeding mother-infant pairs. the list of possible treatment products is extensive and includes many anecdotal and empirical regimens. in the face of this absence of data, brent 51 conducted a survey of members of the academy of breastfeeding medicine concerning the respondents' approach to diagnosis and treatment of thrush in the breastfeeding dyad. most of the respondents relied on the history and physical examination of the infant, but only a third rated the examination of the mother as very important in making a diagnosis. only 7% reported using laboratory testing to make the diagnosis. twentyone percent of the respondents reported using only oral nystatin for the infant when the mother was asymptomatic. almost half treated the infant and the mother with topical nystatin, and 13% used oral nystatin for the infant and oral fluconazole for the mother when the mother had breast pain. less than 5% used oral fluconazole for both infant and mother, and other therapies were used by about 15% of the respondents. for recurrence of persistence of the thrush, more respondents reported treating the mother or both the infant and mother with fluconazole, and almost a quarter reported using other therapies. considerable discussion of mammary candidosis/candidiasis, the clinical diagnosis of candidal involvement of the breast, the significance of pain with breastfeeding, and the presence or absence of candida albicans in milk samples is ongoing. 14, 133, 166 this topic will continue to be debated because additional prospective studies are necessary to clarify specific issues. data are inadequate to make specific recommendations about various clinical situations regarding candida and breastfeeding. clinical practice will vary with experience, especially for the more problematic clinical situations. some general guidelines follow. (see chapter 16 for a discussion of mastitis.) the treatment of mucocutaneous candidiasis should probably begin with a topical agent, such as nystatin, clotrimazole, miconazole, econazole, butaconazole, terconazole, or ciclopirox. treatment should continue for at least 2 weeks, even with obvious improvement in 1 or 2 days. failures most often result from inadequate therapy involving the frequency of application, careful washing and drying before application, or, in the case of diaper candidiasis, decreasing the contact of the skin with moisture. nystatin oral suspension is less effective for the treatment of oral candidiasis in infants, now compared with the past, supposedly due to increasing resistance. 154 gentian violet (diluted to 0.25% to 1.0%) applied to the breast or painted onto an infant' s mouth is being recommended more frequently. other topical preparations have been recommended for the mother' s breast including mupirocin, grapefruit seed extract, or mixtures of mupirocin, betamethasone ointments, and miconazole powder. controlled clinical trials for efficacy and toxicity are not available. when good adherence to the proposed regimen with topical agents fails, or when infant or mother are severely affected by pain and decreased breastfeeding, systemic therapy is appropriate. fluconazole and ketoconazole are the most commonly used systemic agents for oral or diaper candidiasis and vulvovaginitis or mastitis. fluconazole has a better side effect profile than ketoconazole, and more data are available concerning its safe use in children younger than 6 months of age and even neonates and premature infants. 87, 154, 209 fluconazole is not currently approved for use in infants younger than 6 months of age. for severe invasive infections in infants, amphotericin b with or without oral flucytosine, iv fluconazole, voriconazole or caspofungin are reasonable choices in different situations. use of itraconazole in infants has not been adequately studied to date. maternal use of fluconazole during breastfeeding is not contraindicated because only a small amount of medicine compared with the usual infant dose reaches the infant through breast milk. amphotericin or caspofungin therapy in mothers is also not contraindicated because these are both poorly absorbed from the gi tract. whenever a mother is treated for candidal mastitis or vulvovaginitis, the infant should be treated simultaneously, at least with nystatin oral suspension as the first choice of medication. any predisposing risk factors for candidal infection in mothers and infants should be reduced or eliminated to improve the chance of rapid, successful treatment and to decrease the likelihood of chronic or recurrent disease. for mothers, such interventions might include decreasing sugar consumption, stopping antibiotic use as soon as possible, and consuming some form of probiotic bacteria, such as acidophilus (in yogurt, milk, or pill form), to reestablish a normal colonizing bacterial flora. for infants, breastfeeding can enhance the growth of specific colonizing bacterial flora such as lactobacillus, which can successfully limit fungal growth. breastfeeding should continue with appropriate support and problem-solving with a professional who is knowledgeable about breastfeeding. hiv-1, hiv-2, htlv-i, and htlv-ii are the only infectious diseases that are considered absolute contraindications to breastfeeding in developed countries. when the primary route of transmission is via direct contact or respiratory droplets/particles, temporary separation of mother and infant may be appropriate (whether the infant is breastfed or formula fed), but expressed breast milk should be given to the infant for the organism-specific immunologic benefits in the mother' s milk. in most instances, by the time a specific diagnosis of infection is made for a mother, the infant has already been exposed to the organism and providing expressed breast milk to the infant should continue. (refer to appendix f for specific exceptions, such as lassa fever.) regarding antimicrobial therapy for mothers and continued breastfeeding, the majority of the medications commonly used in adults can be used to treat the same infection in infants. the additional amount of medication received by infants via breast milk is usually insignificant. in almost all instances, an appropriate antimicrobial agent for treating mothers that is also compatible with breastfeeding can be chosen. unless the risk to infants for transmission of an infectious agent via breast milk that leads to a clinically significant illness in the infants is documented, breastfeeding should continue. measles antibodies in the breast milk of nursing mothers spectrum of breast tuberculosis respiratory syncytial virus infection among young children with acute respiratory tract infection in iraq probable breast milk borne brucellosis in a young infant breast milk transmission of cytomegalovirus (cmv) infection congenital 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of circumcision in newborn outbreaks of community associated methicillin-resistant staphylococcus aureus brucellosis chez un nourrisson de 3 mois recovery of staphylococcal enterotoxin f from the breast milk of a woman with toxic-shock syndrome evidence for sexual and mother-to-child transmission of human t lymphotrophic virus type ii among guaymi indians transmission of cytomegalovirus to preterm infants through breast milk postnatally acquired cytomegalovirus infection via breast milk: effects on hearing and development in preterm infants pregnancy and lactation in relation to breast cancer risk morbidity among hiv-1-infected mothers in kenya: prevalence and correlates of illness during 2-year postpartum follow-up high concentrations of interleukin 15 in breast milk are associated with protection against postnatal hiv transmission low and undectable breast milk interleukin-7 concentrations are associated with reduced risk of postnatal hiv transmission lactation mastitis: a descriptive study of the experience breastfeeding does not pose any additional risk of immunoprophylaxis failure on infants of hbv carrier mothers recurrent neonatal group b streptococcal disease associated with infected breast milk transplacentally transferred maternal-infant antibodies to dengue virus vertical transmission of hepatitis a resulting in an outbreak in a neonatal intensive care unit immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 e6 or e7 perinatal transmission of hepatitis g virus (gb virus type c) and hepatitis c virus infections: a comparison advisory committee on immunization practices; healthcare infection control practices advisory committee: recommendations for using smallpox vaccine in a pre-event vaccination program global rotavirus surveillance: determining the need and measuring the impact of rotavirus vaccines recommendations for minimizing cmv exposure in breast milk fed very low birth weight (vlbw) preterm infants. available at www.breastfeeding. org/articals/cmvpremi.pdf maternal-infant transmission of htlv-i: frequency and time course of seroconversion. abstract w-53 mother-to-child transmission of human t-cell lymphotropic virus type i associated with prolonged breast-feeding maternal lyme disease and congenital malformations: a cord blood serosurvey in endemic and control areas human versus cow' s milk in infant nutrition and health human t-lymphocyte retroviruses working group on severe streptococcal infections: defining gas streptococcal toxic shock syndrome: rationale and consensus definition acquired cytomegalovirus infection of breast milk in infancy oral transmission of human t-cell leukemia virus type 1 into a common marmoset as an experimental model for milk-borne transmission evaluation of cytomegalovirus infections transmitted via breast milk in preterm infants with a real-time polymerase chain reaction assay sequelae of maternally derived cytomegalovirus infections in premature infants mother-to-infant transmission of hepatitis c virus human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis independent protective effect of lactation against breast cancer: a case-control study in japan a maternal factor for mother-to-child transmission: viral antigen-producing capacities in culture of peripheral blood and breast milk cells mother-to-infant transmission of hepatitis c virus infectious diseases of the fetus and newborn infant sensitive method for detection of human herpes viruses 6 and 7 in saliva collected in field studies survey of 34 pregnant women with hepatitis a and their neonates postnatal transmission of aids-associated retrovirus from mother to infant disseminated lyme disease and pregnancy. 9th annual international scientific conference on lyme disease and other tick-borne disorders a. siblings at home have active chickenpox when neonate and mother are ready for discharge from hospital.no no 1. mother: if she has a history of chickenpox, she may return home. without a history, she should be tested for varicellazoster virus antibody titer.* if test is positive, she may return home. if test is positive, she may return home. if test is negative, varicella-zoster ig † is administered and she is discharged home. 2. neonate: may be discharged home with mother if mother has history of varicella or is varicella-zoster virus-antibody positive. if mother is susceptible, administer varicella-zoster ig to infant and discharge home or place in protective isolation. b. mother has no history of chickenpox; exposed during period 6-20 days antepartum. ‡ ‡if exposure occurred less than 6 days antepartum, mother would not be potentially infectious until at least 72 hours postpartum. §considered noninfectious when no new vesicles have appeared for 72 hours and all lesions have crusted. ¶dosage of acyclovir for pregnant woman is 30 mg/kg/day; for seriously ill infant with varicella, 750 to 1500 mg/m 2 /day. elisa, enzyme-linked immunosorbent assay; fama, fluorescent antibody to membrane antigen; la, latex agglutination. key: cord-272052-8vvpm4tx authors: hartmann, katrin title: clinical aspects of feline immunodeficiency and feline leukemia virus infection date: 2011-10-15 journal: vet immunol immunopathol doi: 10.1016/j.vetimm.2011.06.003 sha: doc_id: 272052 cord_uid: 8vvpm4tx feline leukemia virus (felv) and feline immunodeficiency virus (fiv) are retroviruses with a global impact on the health of domestic cats. the two viruses differ in their potential to cause disease. fiv can cause an acquired immunodeficiency syndrome that increases the risk of developing opportunistic infections, neurological diseases, and tumors. in most naturally infected cats, however, fiv itself does not cause severe clinical signs, and fiv-infected cats may live many years without any health problems. felv is more pathogenic, and was long considered to be responsible for more clinical syndromes than any other agent in cats. felv can cause tumors (mainly lymphoma), bone marrow suppression syndromes (mainly anemia) and lead to secondary infectious diseases caused by suppressive effects of the virus on bone marrow and the immune system. today, felv is less important as a deadly infectious agent as in the last 20 years prevalence has been decreasing in most countries. feline immunodeficiency virus (fiv) and feline leukemia virus (felv) are among the most common infectious diseases of cats. belonging to the retroviral family, fiv is classified as a lentivirus, felv as a ␥-retrovirus. although fiv and felv are both retroviruses, they differ in their potential to cause disease. vaccines are available for both viruses; however, identification and segregation of infected cats remain the cornerstone for preventing new infections (levy et al., 2008) . in the united states, prevalence of both infections is about 2% in healthy cats and up to about 30% in high-risk or sick cats (o'connor et al., progression was variable, with death occurring in about 18% of infected cats within the first two years of observation (about five years after the estimated time of infection). an additional 18% developed increasingly severe disease, but more than 50% remained clinically asymptomatic during the two years (barr, 2000) . in a closed household of 26 cats, fiv infection did not adversely affect the cats' life expectancy (addie et al., 2000) . experimental fiv infection progresses through several stages, similar to hiv infection in people, including an acute phase, a clinically asymptomatic phase of variable duration, and a terminal phase sometimes named "feline acquired immunodeficiency syndrome" ("faids") (english, 1995; goto et al., 2000) . in experimental infection, an initial stage (acute phase) is sometimes noticed with usually transient and mild clinical signs, including fever, lethargy, signs of enteritis, stomatitis, dermatitis, conjunctivitis, respiratory tract disease, and generalized lymph node enlargement (obert and hoover, 2000) . the duration of the following asymptomatic phase varies but usually lasts many years. factors that influence the length of the asymptomatic stage include the pathogenicity of the infecting isolate (also depending on the fiv subtype), exposure to secondary pathogens, and the age of the cat at the time of infection (podell et al., 1997; pedersen et al., 2001) . in the last, symptomatic stage ("faids phase") of infection, clinical signs are a reflection of opportunistic infections, neoplasia, myelosuppression, and neurologic disease. in a survey study of 826 naturally fiv-infected cats examined at north american veterinary teaching hospitals, the most common disease syndromes were stomatitis, neoplasia (especially lymphoma and cutaneous squamous cell carcinoma), ocular inflammation (uveitis and chorioretinitis), anemia and leukopenia, opportunistic infections, renal insufficiency, lower urinary tract disease, and endocrinopathies such as hyperthyroidism and diabetes mellitus (levy, 2000a) . infections with many different "opportunistic" pathogens of viral, bacterial, protozoal, and fungal origin have been reported in fiv-infected cats. few studies, however, have compared the prevalence of most of these infections in fiv-infected and non-infected cats, and thus, their relevance as true secondary invaders is unclear. secondary infections are promoted by the immunosuppression caused be fiv through a progressive disruption of normal immune function. the most important immunologic abnormality demonstrated in experimental barlough et al., 1991; tompkins et al., 1991) as well as in natural (novotney et al., 1990; hoffmann-fezer et al., 1992) infection is a decrease in the number and relative proportion of cd4 + cells in the peripheral blood as well as in most primary lymphoid tissues (bull et al., 2003) . loss of cd4 + cells leads to inversion of the cd4/cd8 ratio. in addition, an increase in the proportion of cd8 + cells also contributes to the inversion hoffmann-fezer et al., 1992; willett et al., 1993) , in particular a population referred to as "cd8 + alpha-hi, beta-low cells" (shimojima et al., 1998 (shimojima et al., , 2003 phadke et al., 2006) , a subset of cd8 + cells that may contribute to suppression of viremia in fiv-infected cats. causes of cd4 + cell loss include decreased production secondary to bone marrow or thymic infection, lysis of infected cells induced by fiv itself (cytopathic effects), destruction of virus-infected cells by the immune system, or death by apoptosis (cell death that follows receipt of a membrane signal initiating a series of programmed intracellular events) (bishop et al., 1993; ohno et al., 1993 ohno et al., , 1994 johnson et al., 1996; guiot et al., 1997a,b; mizuno et al., 1997; mortola et al., 1998a,b; piedimonte et al., 1999; mizuno et al., 2001 mizuno et al., , 2003b tompkins et al., 2002) . the degree of apoptosis correlates inversely with cd4 + numbers and the cd4/cd8 ratio (holznagel et al., 1998) . fiv env proteins are capable of inducing apoptosis in mononuclear cells by a mechanism that requires cxcr4 binding . ultimately, loss of cd4 + cells impairs immune responses because cd4 + cells have critical roles in promoting and maintaining both humoral and cell-mediated immunity. a certain subset of cd4 + cells, termed "treg" (for t-regulatory cells), also seems to play an important role in fiv immunosuppression, and treg cells with suppressive activity have been documented during early (mexas et al., 2008) and chronic fiv infection (petty et al., 2008) . in fivinfected cats, increased activity of treg cells could thus play a role in suppressing immune responses to foreign antigens or pathogens. in addition, treg cells are themselves targets for fiv infection (joshi et al., 2005a,b; mexas et al., 2008) , and may serve as a fiv reservoir during the latent stage of infection capable of stimulating virus production . several other immunologic abnormalities can be found in fiv-infected cats. lymphocytes may loose the ability to proliferate in response to stimulation with mitogens or antigens, and priming of lymphocytes by immunogens may be impaired (hara et al., 1990; hosie and jarrett, 1990; taniguchi et al., 1990; barlough et al., 1991; taniguchi et al., 1991; torten et al., 1991; bishop et al., 1992a,b) . lymphocyte function may be reduced by altered expression of cell surface molecules, such as cd4, major histocompatibility complex ii antigens, or cytokines and cytokine receptors (willett et al., 1991; ohno et al., 1992; rideout et al., 1992; choi et al., 2000; mizuno et al., 2003a) , or through over-expression of abnormal molecules, such as receptors (nishimura et al., 2004) , leading to disrupted cytokines production or receptor function. impaired neutrophil adhesion and emigration in response to bacterial products have been described in fiv-infected cats (hanlon et al., 1993; kubes et al., 2003; heit et al., 2006) . natural killer cell activity may be diminished (zaccaro et al., 1995) or increased (zhao et al., 1995) in acutely or asymptomatically infected cats, respectively. changes in cytokine patterns include increased production of interferon (ifn)-␥, tumor necrosis factor (tnf)-␣, il-4, il-6, il-10, and il-12 (lerner et al., 1998; liang et al., 2000; orandle et al., 2000; ritchey et al., 2001) , but also differences in cytokine ratios (e.g., il-10/il-12 ratio) (dean et al., 1998; lehman et al., 2009) . another immunologic dysregulation observed in many fiv-infected cats is an excessive immune response leading to hypergammaglobulinemia flynn et al., 1994; gleich and hartmann, 2009 ). hypergammaglobulinemia reflects polyclonal b-cell stimulation and is a direct consequence of fiv infection, because healthy fiv-positive, specific pathogen-free (spf) cats also develop hypergammaglobulinemia (flynn et al., 1994) . in addition to increased igg, increased circulating immune complexes have been detected in fiv-infected cats (matsumoto et al., 1997) , potentially leading to immune complex deposition disorders, such as glomerulonephritis and uveitis. chronic ulcero-proliferative stomatitis is the most common syndrome found in cats naturally infected with fiv (affecting up to 50%). it characteristically originates in the pharyngeal area and spreads rostrally, especially along the maxillary teeth. lesions are often painful, and tooth loss is common. severe stomatitis can lead to anorexia and emaciation. histologically, the mucosa is invaded by plasma cells and lymphocytes, accompanied by variable degrees of neutrophilic and eosinophilic inflammation. the cause of this syndrome is uncertain, but the histologic findings suggest an immune response to chronic antigenic stimulation or immune dysregulation. circulating lymphocytes of cats with stomatitis have greater than normal expression of inflammatory cytokines (levy, 2000a) , further implicating immune activation in the pathogenesis of this condition. this type of stomatitis is not always correlated with fiv infection (quimby et al., 2008) , and is usually not seen in experimentally fiv-infected specific pathogen-free cats, suggesting that exposure to other infectious agents also plays a role (levy, 2000b) . concurrent feline calicivirus (fcv) infection is often identified in the oral cavity of these cats, and experimental and naturally occurring co-infection of fiv-infected cats with fcv results in more severe disease (tenorio et al., 1991; reubel et al., 1994) . neurologic signs have been described in both natural and experimental in acute as well as chronic fiv infections (dow et al., 1990 (dow et al., , 1992 podell et al., 1993; english et al., 1994; phillips et al., 1994; abramo et al., 1995) . about 5% of clinically affected fiv-infected cats have a neurological disease as a predominant clinical feature. neurologic disorders in fiv infection seem to be strain-dependent (power et al., 1998) . both central and peripheral neurologic manifestations are seen, comparable to the changes in hivinfected human beings. dementia in human patients with aids is often characterized by a slight decline in cognitive ability or behavior, changes that may be too subtle to recognize in cats. neurological abnormalities seen in naturally infected cats also tend to be more behavioral than motor. twitching movements of the face and tongue, psychotic behavior, compulsive roaming, dementia, loss of bladder and rectal control, and disturbed sleep patterns have been obeserved. other signs described include nystagmus, ataxia, seizures, and intention tremors gunn-moore et al., 1996; steigerwald et al., 1999) . abnormal forebrain electrical activity and abnormal visual and auditory-evoked potentials have also been documented in cats that appeared otherwise normal (podell et al., 1997 barr et al., 2000; phipps et al., 2000) . although the majority of fiv-infected cats do not show clinically observable neurologic signs, a much higher proportion of infected cats exhibit microscopic cns lesions. brain lesions may occur in the absence of massive infection, and abnormal neurological function has been documented in fiv-infected cats with only mild to moderate histological evidence of inflammation (barr, 2000) . pathologic findings include the presence of perivascular infiltrates of mononu-clear cells, diffuse gliosis, glial nodules, and white matter pallor. these lesions are usually located in the caudate nucleus, midbrain, and rostral brain stem (barr, 2000) . fiv enters the brain early following experimental infection, most likely via the blood-brain and blood-cerebrospinal fluid barriers. the exact mechanism of entry, and the factors that influence this entry, are not fully understood (fletcher et al., 2010) . tumor necrosis factor (tnf)-alpha seems to play an important role in lymphocyte migration through the blood-brain barrier (fletcher et al., 2010) . caused by the early brain infection, virus-induced cns lesions sometimes develop within two months of experimental infection (barr, 2000) . microglia and astrocytes are infected by fiv. the virus does not infect neurons. however, neuronal death has been associated with fiv infection; in particular, forebrain signs are often a result of direct neuronal injury from the virus. neurologic expression of fiv infection is highly strain-dependent. the fiv envelope seems to be a principal determinant of neuropathogenesis because the envelope, which is a domain of extensive molecular diversity, directly influences neuropathogenesis through the activation of select proteases (power et al., 2004) . however, while envelope proteins of some fiv strains can promote neuronal swelling and death, envelope proteins of other strains have negligible toxicity (bragg et al., 1999) . the exact mechanism of neuronal damage by fiv is unclear but may include neuronal apoptosis, effects on the neuron supportive functions of astroytes, toxic products released from infected microglia, or cytokines produced in response to viral infection. in vitro studies support the hypothesis that fiv infection may impair normal metabolism in cns cells, particularly astrocytes (barr, 2000) . documented abnormalities of astrocyte function include altered intercellular communication, abnormal glutathione reductase activity that could render cells more susceptible to oxidative injury, and alterations in mitochondrial membrane potential that disrupt energyproducing capacities of the cell (sellon, 1998) . astrocytes are by far the most common cell type of the brain and are important in maintaining cns neuronal mirovascular microenvironment. one of the most important functions of astrocyes is to regulate the level of extracellular glutamate, a major excitatory neurotransmitter that accumulates as a consequence of neuronal activity. excessive extracellular glutamate often results in neuronal toxicity and death. fiv infection of feline astrocytes can significantly inhibit their glutamate-scavenging ability, potentially resulting in neuronal damage (sellon, 1998 (sellon, , 2002 sellon and hartmann, 2006) . intracellular calcium levels seem to play a critical role, and changes in its levels may lead to neuronal dysfunction (meeker, 2007) . it has been shown that choroid plexus macrophages proliferate and release toxic factors in response to fiv (bragg et al., 2002) , and that these factors destabilize neuronal calcium homeostasis (bragg et al., 2002) . thus, choroid plexus macrophages may contribute to an inflammatory cascade in the brain that progresses independently of systemic and csf viral load. csf changes described in fiv-infected cats with neurologic disease include cellular pleocytosis and increases in concentrations of igg (dow et al., 1990) . viral rna may be detected in the csf of some cats and suggests parenchymal infection (ryan et al., 2003) . fiv-infected cats are about five times more likely to develop lymphoma or leukemia than non-infected cats and have a higher incidence of certain types of tumors (poli et al., 1994; callanan et al., 1996) . lymphomas (mostly b-cell lymphomas) (poli et al., 1994; terry et al., 1995; callanan et al., 1996; gabor et al., 2001) , leukemias, and a variety of other tumors have been described in association with fiv infection (fleming et al., 1991; hutson et al., 1991; buracco et al., 1992; callanan et al., 1992; poli et al., 1994; court et al., 1997; barr et al., 2000) , including squamous cell carcinoma, fibrosarcoma, and mast cell tumor. fiv provirus, however, is only occasionally detected in tumor cells (beatty et al., 1998a (beatty et al., ,b, 2002 wang et al., 2001) suggesting a more indirect role in lymphoma formation, such as decreased cell-mediated immune surveillance or chronic b-cell hyperplasia beatty et al., 1998b) . however, clonally integrated fiv dna was found in lymphoma cells from one cat that had been experimentally infected six years earlier (diehl and hoover, 1992; beatty et al., 1998a beatty et al., , 2002 indicating the possibility of an occasional direct oncogenic role of fiv in some animals. the prevalence of fiv infection in one cohort of cats with lymphoma was 50% (gabor et al., 2001) , much higher than the fiv prevalence in the population of cats without lymphomas, which is also supportive of a cause and effect relationship. beside causing a direct effect, fiv may alternatively increase cancer incidence by decreasing tumor immunosurveillance mechanisms, may promote tumor development through the immunostimulatory effects of replicating in lymphocytes, or may impair immunological control of felv infection and accelerate the proliferation of transformed lymphoid cells. felv is more pathogenic than fiv. historically, felv was considered to account for most disease-related deaths and to be responsible for more clinical syndromes than any other single agent in cats. it was proposed that approximately one-third of all tumor-related deaths in cats were caused by felv, and an even greater number of cats died of felv-related anemia and secondary infectious diseases caused by suppressive effects of the virus on bone marrow and the immune system. today, these statements have to be revised, as in recent years prevalence and consequently importance of felv as a pathogen in cats have been decreasing. still, if present in closed households with endemic feline coronavirus (fcov), felv, fiv, or all of these infections, felv infection has the greatest impact on mortality (addie et al., 2000) . the death rate of progressively felv-infected cats in multi-cat households is approximately 50% in two years and 80% in three years (cotter, 1998; levy, 2000c) , but much lower for cats kept strictly indoors in single-cat households. a large study in the united states compared the survival of more than 1000 felv-infected cats to more than 8000 age-and sex-matched uninfected control cats and found that in felv-infected cats median survival was 2.4 years compared to 6.0 years for control cats (levy et al., 2006) . clinical signs associated with felv infection are variable. although the virus was named after the contagious tumor that first garnered its attention, most infected cats are presented to the veterinarian not for tumors but for anemia or immunosuppression. of 8642 felv-infected cats examined at north american veterinary teaching hospitals, various co-infections (including fiv infection, fip, upper respiratory infection, hemotropic mycoplasmosis, and stomatitis) were the most frequent findings (15%), followed by anemia (11%), lymphoma (6%), leukopenia or thrombocytopenia (5%), and leukemia or myeloproliferative disease (4%) (cotter, 1991) . the exact mechanisms for the different clinical responses in progressively felvinfected cats are poorly understood. it is clear that the clinical course is determined by a combination of viral and host factors. some of these differences can be traced to properties of the virus itself, such as the subgroup that determines differences in the clinical picture (e.g., felv-b is primarily associated with tumors, felv-c is primarily associated with non-regenerative anemia). flynn et al. (2002) examined dominant host immune effector mechanisms responsible for the outcome of infection by using longitudinal changes in felv-specific cytotoxic t-lymphocytes (ctl). high levels of circulating felv-specific effector ctls appear before virusneutralizing antibodies in cats that have recovered from exposure to felv. in contrast, progressive infection with persistent viremia has been associated with a silencing of virus-specific humoral and cell-mediated immunity host effector mechanisms (flynn et al., 2002) . it is likely that the most important host factor that determines the clinical outcome of cats infected with felv is the age of the cat at the time of infection (hoover et al., 1976) . neonatal kittens develop marked thymic atrophy after infection ("fading kitten syndrome"), resulting in severe immunosuppression, wasting, and early death. as cats mature, they acquire a progressive resistance. when older cats become infected, they tend to have abortive or regressive infections or, if developing progressive infection, have at least milder signs and a more protracted period of apparent good health (levy, 2000c) . clinical signs associated with felv infection can be classified as tumors, immunosuppression, hematologic disorders, immune-mediated diseases, and other syndromes (including neuropathy, reproductive disorders, fading kitten syndrome). immunosuppression is caused by various mechanisms in felv-infected cats. it has been occasionally associated with un-integrated viral dna from replicationdefective viral variants (overbaugh et al., 1988) . these pathogenic immunosuppressive variants, such as felv-t, require a membrane-spanning receptor molecule (pit1) and a second co-receptor protein (felix) to infect t lymphocytes (lauring et al., 2002) . the latter protein is an endogenously expressed protein, which is similar to the felv receptor-binding protein of felv-b (barnett et al., 2003) . affected cats may develop thymic atrophy and depletion of lymph node paracortical zones following infection. lymphopenia and neutropenia are common. in addition, neutrophils of progressively infected cats have decreased chemotactic and phagocytic function compared with those of normal cats. in some cats, lymphopenia may be characterized by preferential loss of cd4 + helper t cells, resulting in an inverted cd4/cd8 ratio (like typically seen in fiv infection) (quackenbush et al., 1990; hoffmann-fezer et al., 1996) , but more commonly, substantial losses of helper cells and cytotoxic suppressor cells (cd8 + cells) occur (hoffmann-fezer et al., 1996) . many immune function tests of naturally felv-infected cats are abnormal, including decreased response to t-cell mitogens, prolonged allograft reaction, reduced immunoglobulin production, depressed neutrophil function, and complement depletion. il-2 and il-4 are decreased in some cats (linenberger and deng, 1999; levy, 2000c) , but felv does not appear to suppress il-1 production from infected macrophages. ifn-␥ may be deficient or increased. increased tnf-␣ has been observed in serum of infected cats and in infected cells in culture. each cytokine plays a vital role in the generation of a normal immune response, and the excess production of certain cytokines such as tnf-␣ can also cause illness. t-cells of felv-infected cats produce significantly lower levels of b-cell stimulatory factors than do those of normal cats (this defect becomes progressively more severe over time) (diehl and hoover, 1992) , but when b-cells of felv-infected cats are stimulated in vitro by uninfected t-cells, their function remains normal. in vaccination studies, felv-infected cats have not been able to mount an adequate immune response to vaccines, such as rabies. therefore, protection in a felv-infected cat after vaccination is not complete and comparable to that in a healthy cat, and more frequent vaccinations (e.g., every six months) have to be considered (lutz et al., 2009 ). in addition to immunosuppression, felv-infected cats can develop immune-mediated diseases caused by an overactive or dysregulated immune response to the virus. although humoral immunity to specific stimulation decreases, nonspecific increases of igg and igm have been noted. the loss of t-cell activity and the formation of antigen antibody complexes promote the immune dysregulation (pedersen, 1988) . immune-mediated diseases described in felv-infected cats include aiha (kohn et al., 2006) , glomerulonephritis (anderson and jarrett, 1971) , uveitis with immune complex deposition in iris and ciliary body (brightman et al., 1991) , and polyarthritis (pedersen, 1991) . chronic progressive polyarthritis can be triggered by felv; in about 20% of cats with polyarthritis, felv seems to be an associated agent (pedersen, 1991) . cats with glomerulonephritis have more circulating felv antigen that do other felv-infected cats. however, in a recent study felv-infected cats in general did not show significantly more commonly hypergammaglobulinemia in plasma electrophoretogram in contrast to fiv-infected cats (miro et al., 2007) , and hyperproteinemia is not a common problem in felv-infected cats (in contrast to fiv infection) (gleich and hartmann, 2009) . antigens that can lead to antigen antibody complex formation include not only whole virus particles but also free gp70, p27, or p15e proteins (day et al., 1980; tuomari et al., 1984) . hematopoietic disorders, particularly cytopenias caused by bone marrow suppression, are a common finding in felv-infected cats. hematologic disorders described in association with felv include anemia (non-regenerative or regenerative), persistent, transient, or cyclic neutropenia, platelet abnormalities (thrombocytopenia and platelet function abnormalities), aplastic anemia (pancytopenia), and panleukopenia-like syndrome. for the majority of pathogenic mechanisms in which felv causes bone marrow suppression, active virus replication is required. however, it has been demonstrated that in some felv antigen-negative cats, regressive felv infection without viremia may be responsible for bone marrow suppression. in a recent study including 37 cats with myelosuppression that had tested felv antigennegative in peripheral blood, 2/37 cats (5%) were found regressively infected with felv by bone marrow pcr (both had non-regenerative anemia) (stützer et al., 2010) . in these cats, felv provirus may interrupt or inactivate cellular genes in the infected cells, or regulatory features of viral dna may alter expression of neighboring genes. additionally, cell function of provirus-containing myelomonocytic progenitor and stromal fibroblasts that provide bone marrow microenvironment may be altered. alternatively, felv provirus may cause bone marrow disorders by inducing the expression of antigens on the cell surface, resulting in an immune-mediated destruction of the cell. anemia is a major non-neoplastic complication that occurs in a majority of symptomatic felv-infected cats (gleich and hartmann, 2009 ). anemia in felv-infected cats may have various causes. approximately 10% of felv-associated anemias are regenerative (shelton and linenberger, 1995) , most felv-associated anemias, however, are non-regenerative and caused by the bone marrow-suppressive effect of the virus resulting from primary infection of hematopoietic stem cells and infection of stroma cells that constitute the supporting environment for hematopoietic cells. in vitro exposure of normal feline bone marrow to some strains of felv causes suppression of erythrogenesis (cotter, 1998) . in addition to the direct effect of the virus on erythropoiesis, other factors can cause non-regenerative anemia in felv-infected cats (e.g., anemia of chronic disease promoted by high concentration of cytokines). felv infection may cause decreased platelet counts. it also may be responsible for platelet function deficits, and the lifespan of platelets is shortened in some felv-infected cats. thrombocytopenia (resulting in bleeding disorders) may occur secondary to decreased platelet production from felv-induced bone marrow suppression or leukemic infiltration. platelets harbor felv proteins as a result of infection, and megakaryocytes are frequent targets of progressive felv infection. immunemediated thrombocytopenia often accompanies imha in cats with underlying felv infection. felv infection also may cause decreased neutrophil or lymphocyte counts. neutropenia is common in felv-infected cats (brown and rogers, 2001 ) and generally occurs alone or in conjunction with other cytopenias. in some cases, myeloid hypoplasia of all granulocytic stages is observed, suggesting direct cytopathic infection on neutrophil precursors by felv. in some neutropenic felv-infected cats, an arrest in bone marrow maturation may occur at the myelocyte and metamyelocyte stages. immune-mediated mechanisms may alternatively be responsible in cases in which neutrophil counts recover with glucocorticoid treatment ("glucocorticoid-responsive neutropenia"). feline panleukopenia-like syndrome (fpls), also known as felv-associated enteritis (fae) or myeloblastopenia, consists of severe leukopenia (<3000 cells/l) with enteritis and destruction of intestinal crypt epithelium that mimics feline panleukopenia caused by feline panleukopenia virus (fpv) infection. however, fpv antigen has been demonstrated by ifa in intestinal sections of cats that died from this syndrome after being experimentally infected with felv (lutz et al., 1995) . fpv was also demonstrated by electron microscopy despite negative fpv antigen tests. it appears that this syndrome may actually not be caused by felv itself, as previously thought, but by co-infection with fpv. the syndromes also has been referred to as fae in cats with progressive felv infection because the clinical signs observed are usually gastronintestinal, including hemorrhagic diarrhea, vomiting, oral ulceration or gingivitis, anorexia, and weight loss (kipar et al., 2000 (kipar et al., , 2001 . it is still unclear whether all these syndromes have the same origin and are simply caused by co-infection with fpv (and even modified life fpv vaccines have been discussed) or if they are caused by felv itself (lutz et al., 1995) . hematopoietic neoplasia ("myeloprolifertaive disorders"), including leukemia, may also cause bone marrow suppression syndromes by replacing bone marrow cells. myelodysplastic syndrome (mds), characterized by peripheral blood cytopenias and dysplastic changes in the bone marrow, is a pre-stage of acute myeloic leukemia. it was found that changes of the ltr of felv (presence of three tandem direct 47-bp repeats in the upstream region of the enhancer (ure)) are strongly associated with the induction of mds (hisasue et al., 2009) . myelofibrosis, another bone marrow suppression syndrome characterized by abnormal proliferation of fibroblasts resulting from chronic stimulation of the bone marrow, such as chronic bone marrow activity from hyperplastic or neoplastic regeneration, may also be caused by felv. in severe cases, the entire endosteum within the medullary cavity can be obliterated. felv can cause different tumors in cats, most commonly lymphoma and leukemia, less commonly other hematopoietic tumors. the most important mechanism by which felv causes malignancy is by insertion of the felv genome into the cellular genome near a cellular oncogene (most commonly myc), resulting in activation and over-expression of that gene. these effects lead to uncontrolled proliferation of that cell (clone). a malignancy results in the absence of an appropriate immune response. felv-a may also incorporate the oncogene to form a recombinant virus (e.g., felv-b, fesv) containing cellular oncogene sequences that are then rearranged and activated. when they enter a new cell, these recombinant viruses are oncogenic. in a study of 119 cats with lymphomas, transduction or insertion of the myc locus had occurred in 38 cats (32%) (tsatsanis et al., 1994) . thus, felv-induced neoplasms are caused, at least in part, by somatically acquired insertional mutagenesis in which the integrated provirus may activate a proto-oncogene or disrupt a tumor suppressor gene. a recent study suggested that the u3-ltr region of felv transactivates cancer-related signaling pathways through production of a non-coding 104 base rna transcript that activates nf kappab (forman et al., 2009) . common integration sites for felv associated with lymphoma development have been identified in six loci: c-myc, flvi-1, flvi-2 (contains bmi-1), fit-1, pim-1, and flit-1. oncogenic association of the loci has been suggested because c-myc is known as a proto-oncogene, bmi-1 and pim-1 have been recognized as myc-collaborators, fit-1 appears to be closely linked to myb, and flit-1 insertion was shown to be associated with over-expression of cellular genes, e.g., activin-a receptor type ii-like 1 (acvrl1) (fujino et al., 2008) . flit-1 especially seems to play an important role in the development of thymic lymphomas and appears to represent a novel felv proviral common integration domain that may influence lymphomagenesis through insertional mutagenesis. among 35 felv-related tumors, five of 25 thymic lymphomas demonstrated proviral insertion within flit-1 locus, whereas none of four alimentary, five multicentric lymphomas, and one t-lymphoid leukemia examined had rearrangement in this region. expression of acvrl1 mrna was detected the two thymic lymphomas with flit-1 rearrangement, whereas normal thymuses and seven lymphoid tumors without flit-1 rearrangement had no detectable acvrl1 mrna expression (fujino et al., 2009) . the association between felv and lymphomas has been clearly established in several ways. first, these malignancies can be induced in kittens by experimental felv infection (rickard et al., 1969; hardy et al., 1973; jarrett et al., 1973) . second, cats naturally infected with felv have a higher risk of developing lymphoma than uninfected cats (hardy et al., 1973; essex et al., 1975) . third, most cats with lymphoma were -at least in earlier times when prevalence of felv was still higher -felv-positive in tests that detected infectious virus or felv antigens. previously, up to 80% of feline lymphomas and leukemias were reported to be felv related francis et al., 1977 francis et al., , 1979 hardy et al., 1980; reinacher, 1987; shelton et al., 1990; harrus et al., 2002) . however, since 1980, a dramatic reduction in the prevalence of viremia has been noted in cats with lymphoma (mauldin et al., 1995; moore et al., 1996; hartmann et al., 1998) . the decrease in prevalence of felv infection in cats with lymphoma or leukemia also indicates a shift in tumor causation in recent years. whereas 59% of all cats with lymphoma or leukemia were felv antigen-positive in one german study from 1980 to 1995, only 20% of the cats were felv antigen-positive in the years 1996 to 1999 in the same university veterinary clinic (hartmann et al., 1998) . a recent study in this area confirmed the low prevalence of felv antigenemia in cats with lymphoma; in the study, 16 of 77 cats (21%) were felv antigen-positive (stützer et al., 2011) . in a study in the netherlands, only four of 71 cats with lymphoma were felv-positive, although 22 of these cats had mediastinal lymphoma, which was previously highly associated with felv infection (teske et al., 2002) . today, a higher lymphoma incidence in older felv antigen-negative cats is observed. the major reason for the decreasing association of felv with lymphoma is the decreasing prevalence of felv infection in the overall cat population as a result of felv vaccination as well as testing and elimination programs. however, prevalence of lymphoma caused by felv may be higher than indicated by conventional antigen testing of blood. cats from felv cluster households had a 40-fold higher rate of development of felv-negative lymphoma than did those from the general population. felv-negative lymphomas have also occurred in laboratory cats known to have been infected previously with felv (rohn et al., 1994) . felv proviral dna was detected in lymphomas of older cats that tested negative for felv antigen, also suggesting that the virus may be associated with a larger proportion of lymphomas than previously thought. pcr detected proviral dna in formalin-fixed, paraffin-embedded tumor tissue in seven of 11 felv-negative cats with lymphoma (jackson et al., 1993) . however, other groups found evidence of provirus in only one of 22 (sheets et al., 1993) or in none of 61 felv antigen-negative lymphomas (hartmann et al., 1998) . the felv status of cats with lymphomas still varies, depending on the type and locations of tumors. lymphomas in felv antigen-positive cats are mainly of a t-cell origin; those in felv antigen-negative cats are mainly of a b-cell origin francis et al., 1979; hardy, 1981; neil et al., 1984) . this was confirmed in a recent study in which 8/28 (64.3%) felv antigen-negative cats with lymphoma had t-cell lymphoma, while none of the felv antigen-positive cats (0/8) had b-cell lymphoma (stützer et al., 2011) . a potential reason may be that felv transforms mature t cells and immature or prothymocytes, null cells, and possibly monocytes. transformation of mature b cells does not seem to occur, because feline lymphoma cell lines and primary tumors lack surface immunoglobulin expression (rojko et al., 1989) . the rare feline large granular lymphocyte (lgl) lymphoma, a morphologically distinct variant of feline lymphoma with grave prognosis, does not seem to be commonly associated with felv. in a study of 45 cats with lgl lymphoma, none of the cats was felv antigen-positive (krick et al., 2008) . similarly, low grade lymphomas are usually not associated with felv; in a study of 41 low grade lymphocytic lymphomas, none of the cats was felv antigen-positive (kiselow et al., 2008) . fibrosarcomas that are associated with felv are caused by fesv, a recombinant virus that develops de novo in felv-a-infected cats by recombination of the felv-a genome with cellular oncogenes (besmer et al., 1986) . through a process of genetic recombination, fesv acquires one of several oncogenes, such as fes, fms, or fgr (besmer et al., 1983) . as a result, fesv is an acutely transforming (tumorcausing) virus, leading to a polyclonal malignancy with multifocal tumors arising simultaneously after a short incubation period (mcdonald et al., 1976; pedersen et al., 1984) . with the decrease in felv prevalence, fesv also has become less common. fesv-induced fibrosarcomas are multicentric and usually occur in young cats. strains of fesv identified from naturally occurring tumors are defective and unable to replicate without the presence of felv-a as a helper virus that supplies proteins (such as those coded by the env gene) to fesv (rojko et al., 1994) . fibrosarcomas caused by fesv tend to grow rapidly, often with multiple cutaneous or subcutaneous nodules that are locally invasive and metastasize to the lung and other sites (pedersen et al., 1984) . solitary fibrosarcomas in older cats are not caused by fesv. these tumors are slower growing, locally invasive, slower metastasizing, and occasionally curable by excision combined with radiation and/or gene therapy. these tumors are usually injection site-associated sarcomas (isas) caused by the granulomatous inflammatory reaction at the injection site, commonly occurring after inoculation of adjuvant-containing vaccines. it has been demonstrated that neither fesv nor felv play any role in isas (ellis et al., 1996) . a number of other tumors have been found in felvinfected cats; some of them may have an association with felv, others have just been observed by chance simultaneously in an infected cat. iris melanomas, for example, are not associated with felv infections. an association has been hypothesized because of one study in which three of 18 eyes had positive test results for felv-fesv proviral dna (stiles et al., 1999) . however in a more recent study, immunohistochemical staining and pcr did not reveal felv or fesv in the ocular tissues of any cats with this disorder (cullen et al., 2002) . multiple osteochondromas (cartilaginous exostoses on flat bones of unknown pathogenesis) have been described in felv-infected cats. although histologically benign, they may cause significant morbidity if they occur in an area such as a vertebra and put pressure on the spinal cord or nerve roots (pool and carrig, 1972; lott-stolz, 1988) . in spontaneous feline olfactory neuroblastomas (aggressive, histologically inhomogenous tumors of the tasting and smelling epithelium of nose and pharynx with high metastasis rates), budding felv particles were found in the tumors and lymph node metastases, and felv dna was found in tumor tissue (schrenzel et al., 1990) . the exact role of felv in the genesis of these tumors is uncertain. cutaneous horns are a benign hyperplasia of keratinocytes that have been described in felv-infected cats (pedersen, 1991) , but again the role of felv is unclear. other syndromes directly caused by felv infection include felv-associated neuropathy, reproductive disorders, and fading kitten syndrome. although most neurologic signs seen in felv-infected cats are caused by lymphoma and lymphocytic infiltrations in brain or spinal cord leading to compression, in some cases no tumor is detectable with diagnostic imaging methods or in necropsy, and felv-induced neurotoxicity is suspected. anisocoria, mydriasis, central blindness, or horner's syndrome have been described in felv-infected cats without morphologic changes. in some regions (such as the southeastern united states), urinary incontinence caused by neuropathies in felv-infected cats has been described (carmichael et al., 2002) . direct neurotoxic effects of felv have been discussed as pathogenetic mechanisms. felv envelope glycoproteins may be able to produce increased intracellular free calcium leading to neuronal death (this has also been described in hiv-infected humans). a polypeptide of the felv envelope was found to cause dose-dependent neurotoxicity associated with alterations in intracellular calcium ion concentration, neuronal survival, and neurite outgrowth. the polypeptide from an felv-c strain was significantly more neurotoxic than the same peptide derived from an felv-a strain (fails et al., 1997; mitchell et al., 1997) . clinical signs in 16 cats with progressive felv infection and neurologic signs consisted of abnormal vocalization, hyperesthesia, and paresis pro-gressing to paralysis. some cats developed anisocoria or urinary incontinence during the course of their illness. affected cats developed gradually progressive neurologic dysfunction. microscopically, white-matter degeneration with dilation of myelin sheaths and swollen axons was identified in the spinal cord and brain stem of affected animals (carmichael et al., 2002) . immunohistochemical staining of affected tissues revealed consistent expression of felv p27 antigens in neurons, endothelial cells, and glial cells, and proviral dna was amplified from multiple sections of spinal cord (carmichael et al., 2002) . these findings suggest that in some felv-infected cats, the virus may directly affect cns cells cytopathically. felv-infected queens can transmit the virus transplacentally. reproductive failure in form of fetal resorption, abortion, and neonatal death is common if in utero felv infection occurs. the apparent infertility might actually be caused by early resorption of fetuses. abortions usually occur late in gestation, with expulsion of normal appearing fetuses. bacterial endometritis may accompany these abortions, particularly in cats with neutropenia (cotter, 1998) . kittens born to infected queens may become exposed to felv transplacentally, but heavy exposure also occurs at birth and throughout the nursing period. some kittens become immune, but most become progressively infected and die at an early age of the so-called fading kitten syndrome, characterized by failure to nurse, dehydration, hypothermia, thymic atrophy, and death within the first two weeks of life (levy, 2000c) . most knowledge about clinical aspects of fiv and felv infection as well as on the pathophysiology and immunological background of both infections derive from experimental studies. however, the relevance of these experimental data to the naturally occurring disease is unclear, and it remains an unanswered question on how or whether the experimental data can be applied to the clinical setting in the field. it is the impression of many clinicians that in most naturally infected cats, fiv does not cause a severe clinical syndrome. most clinical signs in fivinfected cats reflect secondary diseases such as infections and neoplasia to which fiv-infected cats are considered more susceptible. with proper care, fiv-infected cats can live many years and, in fact, may die at an old age from causes unrelated to their fiv infection. although felv causes more severe clinical syndromes, and despite the fact that progressive felv infection is associated with a decrease in life expectancy, many owners still elect to provide therapy for their felv-infected cats, and with proper treatment, felv-infected cats in single-cat households may also live for many years with good quality of life. as in fiv infection, diseases secondary to immunosuppression account for a large portion of the syndromes seen in felvinfected cats, and it is important to realize that many of these secondary diseases are treatable. many reports have been made of felv-infected cats having concurrent bacterial, viral, protozoal, and fungal infections, but few studies exist proving that these cats have a higher rate of infection than do felv-negative cats. thus, 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virus infection altered bone marrow dendritic cell cytokine production to toll-like receptor and cd40 ligation during chronic feline immunodeficiency virus infection fiv infection of il-2-dependent and -independent feline lymphocyte lines: host cells range distinctions and specific cytokine upregulation american association of feline practitioners' feline retrovirus management guidelines kirk's current veterinary therapy xiii small animal practice felv and non-neoplastic felv-related disease seroprevalence of feline leukemia virus and feline immunodeficiency virus infection among cats in north america and risk factors for seropositivity t cells overexpressing interferon-gamma and interleukin-10 are found in both the thymus and secondary lymphoid tissues of feline immunodeficiency virus-infected cats the effects of feline retroviruses on cytokine expression short original report. osteochondromatosis in the cat feline leukaemia. abcd guidelines on prevention and management panleukopenia-like syndrome of felv caused by co-infection with felv and feline panleukopenia virus serum concentration of circulating immune complexes in cats infected with feline immunodeficiency virus detected by immune adherence hemagglutination method chemotherapy in 132 cats with lymphoma: 1988-1994 characteristics of three strains of feline fibrosarcoma virus grown in cat and marmoset monkey cells feline immunodeficiency virus neuropathogenesis: from cats to calcium cd4+cd25+ regulatory t cells are infected and activated during acute fiv infection plasma electrophoretogram in feline immunodeficiency virus (fiv) and/or feline leukaemia virus (felv) infections felv envelope protein (gp70) variable region 5 causes alterations in calcium homeostasis and toxicity of neurons tnf-alpha-induced cell death in feline immunodeficiency virusinfected cells is mediated by the caspase cascade molecular cloning of feline tumour necrosis factor receptor type i (tnfr i) and expression of tnfr i and tnfr ii in lymphoid cells in cats quantitative analysis of fas and fas ligand mrnas in a feline t-lymphoid cell line after infection with feline immunodeficiency virus and primary peripheral blood mononuclear cells obtained from cats infected with the virus apoptosis enhanced by soluble factor produced in feline immunodeficiency virus infection a comparison of doxorubicin and cop for maintenance of remission in cats with lymphoma effect of interleukin-12 and interleukin-10 on the virus replication and apoptosis in t-cells infected with feline immunodeficiency virus inhibition of apoptosis and virus replication in feline immunodeficiency virus-infected cells by n-acetylcysteine and ascorbic acid transduction and rearrangement of the myc gene by feline leukaemia virus in naturally occurring t-cell leukaemias downmodulation of cd3epsilon expression in cd8alpha+beta− t cells of feline immunodeficiency virus-infected cats lymphocyte population changes in cats naturally infected with feline immunodeficiency virus report of the national felv/fiv awareness project relationship of lymphoid lesions to disease course in mucosal feline immunodeficiency virus type c infection apoptosis induced by tumor necrosis factor in cells chronically infected with feline immunodeficiency virus induction of apoptosis in a t lymphoblastoid cell line infected with feline immunodeficiency virus altered surface antigen expression on peripheral blood mononuclear cells in cats infected with feline immunodeficiency virus cd8+ thymic lymphocytes express reduced levels of cd8beta and increased interferon gamma in cats perinatally infected with the jsy3 molecular clone of feline immunodeficiency virus molecular cloning of a feline leukemia virus that induces fatal immunodeficiency disease in cats feline leukemia virus infection feline husbandry, disease and management in the multiple cat environment biological behavior of tumors and associated retroviremia in cats inoculated with snyder-theilen fibrosarcoma virus and the phenomenon of tumor recurrence after primary regression virulence differences between two field isolates of feline immunodeficiency virus (fiv-apetaluma and fiv-cpgammar) in young adult specific pathogen free cats transforming growth factor-beta/transforming growth factor-betarii signaling may regulate cd4+cd25+ t-regulatory cell homeostasis and suppressor function in feline aids lentivirus infection pathogenesis of a texas feline immunodeficiency virus isolate: an emerging subtype of clade b neurological abnormalities associated with feline immunodeficiency virus infection neurophysiologic and immunologic abnormalities associated with feline immunodeficiency virus molecular clone fiv-ppr dna inoculation protein degradation and apoptotic death in lymphocytes during fiv infection: activation of the ubiquitin-proteasome proteolytic system progressive encephalopathy associated with cd4/cd8 inversion in adult fivinfected cats frontal lobe neuronal injury correlates to altered function in fiv-infected cats aids-associated encephalopathy with experimental feline immunodeficiency virus infection malignant lymphoma associated with experimentally induced feline immunodeficiency virus infection multiple cartilagenous exostoses in a cat neurovirulence in feline immunodeficiency virusinfected neonatal cats is viral strain specific and dependent on systemic immune suppression comparative neurovirulence in lentiviral infections: the roles of viral molecular diversity and select proteases sleep patterns are disturbed in cats infected with feline immunodeficiency virus lymphocyte subset alterations and viral determinants of immunodeficiency disease induction by the feline leukemia virus felv-faids evaluation of the association of bartonella species, feline herpesvirus 1, feline calicivirus, feline leukemia virus and feline immunodeficiency virus with chronic feline gingivostomatitis infektionen mit dem felinen leukämie-virus (felv) effect of chronic feline immunodeficiency virus infection on experimental feline calicivirus-induced disease a transmissible virusinduced lymphocytic leukemia of the cat persistent upregulation of mhc class ii antigen expression on t-lymphocytes from cats experimentally infected with feline immunodeficiency virus constitutive expression of types 1 and 2 cytokines by alveolar macrophages from feline immunodeficiency virus-infected cats evolution of feline leukemia virus variant genomes with insertions, deletions, and defective envelope genes in infected cats with tumors feline lymphomas: immunological and cytochemical characterization dynamics of viral and proviral loads of feline immunodeficiency virus within the feline central nervous system during the acute phase following intravenous infection type c retroviral expression in spontaneous feline olfactory neuroblastomas feline immunodeficiency virus infection feline immunodeficiency virus infection recombinant feline leukemia virus genes detected in naturally occurring feline lymphosarcomas feline immunodeficiency virus and feline leukemia virus infections and their relationships to lymphoid malignancies in cats: a retrospective study (1968-1988) hematologic abnormalities associated with retroviral infections in the cat expansion of cd8alpha+beta− cells in cats infected with feline immunodeficiency virus phenotypic changes in cd8+ peripheral blood lymphocytes in cats infected with feline immunodeficiency virus effects of feline immunodeficiency virus on cognition and behavioral function in cats use of nested polymerase chain reaction (pcr) for detection of retroviruses from formalin-fixed, paraffin-embedded uveal melanomas in cats role of latent feline leukemia virus infection in nonregenerative cytopenias of cats incidence of persistent viraemia and latent feline leukaemia virus infection in cats with lymphoma altered mitogen response of peripheral blood lymphocytes in different stages of feline immunodeficiency virus infection humoral immune response to t cell dependent and independent antigens in cats infected with feline immunodeficiency virus chronic oral infections of cats and their relationship to persistent oral carriage of feline calici-, immunodeficiency, or leukemia viruses molecular analysis of tumours from feline immunodeficiency virus (fiv)-infected cats: an indirect role for fiv? chemotherapy with cyclophosphamide, vincristine, and prednisolone (cop) in cats with malignant lymphoma: new results with an old protocol feline immunodeficiency virus infection is characterized by b7+ctla4+ t cell apoptosis early events in the immunopathogenesis of feline retrovirus infections progressive immune dysfunction in cats experimentally infected with feline immunodeficiency virus genetic determinants of feline leukemia virus-induced lymphoid tumors: patterns of proviral insertion and gene rearrangement detection of circulating immune complexes by a clq/protein a-elisa during the preneoplastic stages of feline leukemia virus infection characterisation of lymphosarcomas in australian cats using polymerase chain reaction and immunohistochemical examination infection with feline immunodeficiency virus is followed by the rapid expansion of a cd8+ lymphocyte subset productive infection of t-helper lymphocytes with feline immunodeficiency virus is accompanied by reduced expression of cd4 defective natural killer cell cytotoxic activity in feline immunodeficiency virus-infected cats enhanced expression of novel cd57+cd8+ lak cells from cats infected with feline immunodeficiency virus key: cord-298905-c2uuvfm5 authors: horzinek, m. c. title: molecular pathogenesis of virus infections date: 1987 journal: experientia doi: 10.1007/bf01945522 sha: doc_id: 298905 cord_uid: c2uuvfm5 although a very wide range of viral diseases exists in vertebrates, certain generalizations can be made regarding pathogenetic pathways on the molecular level. the presentation will focus on interactions of virions and their components with target cells. using coronaviruses as examples the changes in virulence have been traced back to single mutational events; recombination, however, is likely to be an alternative mechanism by which virus-host interactions (e.g. the cell-, organor animal species-spectrum) can dramatically change. receptor molecules are essential for the early interactions during infection and some ot these have been identified. events in the target cell and the host organism are discussed, and wherever possible, aspects of virus evolution and cooperation between infectious agents are highlighted. with increasing knowledge about viral genes and their functions, in-depth study of the pathogenesis of virus diseases has become feasible and, in fact, fashionable. however, to write even a superficial review on this subject in general terms is not an easy task. i shall therefore discuss pathogenetic mechanisms using coronaviruses as examples the family of viruses we have studied in utrecht since 1978. i will allow for side-steps, however, when illustration is needed and when no pertinent studies have been done with coronaviruses. the interested reader may find more information on the molecular aspects of pathogenesis in the proceedings of a recent symposium devoted to this subject 3~ i should like to start by defining two terms which, in my opinion, are frequently used in a wrong context: pathogenicity and virulence. i shall designate a virus as pathogenic to describe the detrimental effect it may exert on a host species (e.g. rabies virus is pathogenic for man but apathogenic for frogs). in contrast, virulence refers to the disease-producing potential of a specific strain of a pathogenic virus (e.g. the flury low egg passage strain of rabies virus is avirulent for dogs but still virulent for kittens). epidemiologists have observed that diseases which emerge as new entities are only initially of a highly virulent nature (e.g. canine parvovirus enteritis in [ 978); the virulence then gradually lessens until symptomatology becomes slight or absent (e.g. classical swine fever today). in view of this tendency it is obvious that viruses which have reached a stage of commensalism are the 'fittest survivors' since their transmission is guaranteed by an unimpaired behavioral pattern of their hosts. upon closer examination, however, also viruses which have been labeled as innocuous to their hosts do induce pathology (e.g. lactic dehydrogenase virus of mice may cause an age-dependent polioencephalomyelitis23). the sudden emergence of new disease agents has lost much of its enigmatic character since some molecular mechanisms have become known. influenza virus owes its name to the florence epidemic in 1357 which was believed to have been caused by injluentia coeli (celestial influence); it is now one of the bestunderstood pathogens. coronaviruses cause respiratory, enteric and generalized infections and disease in animals and man. also neurologic symptoms have been observed, either under natural conditions e.g. in mouse hepatitis virus (mhv) type 4 infections, in the wake of an infection with feline infectious peritonitis virus (fipv) or as a laboratory artefact after intracerebral inoculation of some mhv mutants. the mhv-rodent model has been extensively studied to understand the mechanisms involved in virus induced chronic demyelination. coronaviruses enter the susceptible cell after adsorption to specific receptors by either membrane fusion or endocytosis -the decisive experiment still needs to be done. interaction of the viral surface protein -the club-shaped peplomer protruding from the envelope with a receptor molecule on the cytoplasmic membrane is a prerogative for this process. evidence has accumulated that already this earliest event during the infection process may determine its outcome. tropism for glial cells with resulting demyelination has been found in vivo and confirmed in vitro for the jhm strain of mhv type 4 21. evidence was presented that mutations in the gene coding for the peplomer glycoprotein result in attenuation of neurovirulence 4, 6. it was possible to protect mice by passive immunization using virus-neutralizing monoclonal antibodies. characterization of neutralization escape mutants obtained with the aid ofmonoclonal antibodies has shown that resistance at one epitope (e2b) resulted in changes also in another epitope (e2c) and vice versa. this simultaneous acquisition of resistance at multiple epitopes has been interpreted in terms of conformational changes in tile polypeptide chain triggered by a single point mutation 4. in addition, the mutants had lost the important biological property of infecting neurons. virus replication was found only in oligodendrocytes, the cell species responsible for providing the myelin sheaths for axons; wild-type mhv-4 infects both cell species. using the hepatotropic strain a59 of mhv, we have arrived at similar conclusions. whereas the wild type strain kills mice rapidly, the temperature-sensitive mutant ts342 is of an attenuated phenotype. it causes chronic demyelinating disease characterized by infection of oligodendrocytes and astrocytes, accompanied by widespread inflammatory reactions in the white matter. we were able to isolate three independent revertants of ts342 with biological properties indistinguishable from those of the wild type strain. again it appears that attenuation has resulted from single point mutations ~7,3s a single amino acid substitution in the surface glycoprotein has also been found responsible for the attenuation of neurovirulence in rabies virus 5. single stranded rna viruses have a predicted mutation rate as high as l0 -4 10 5. which has been experimentally confirmed for coronaviruses 4. with virus yields of about one hundred progeny infectious units per cell many mutants may be expected to arise during infection of an organism. apart from resulting in an attenuated phenotype for the individual, these mutants may display novel cell tropisms -also for cells of another animal species. when examining porcine, canine and feline coronaviruses belonging to the same antigenic cluster we found no quantitative nor qualitative differences at the level of the individual polypeptides ~~ however, changes in the host range may also be the consequence of recombinational events. recombinations have first been shown to occur in the mhv system 2~ but may be more common than anticipated. we have recently compared the nucleotide and derived amino acid sequences of porcine transmissible gastroenteritis virus (tgev) with those of feline infectious peritonitis virus (fipv) at the peplomer level. surprisingly, in an amino-terminal stretch of 274 residues, amino acid homology did not exceed 30% whereupon a sudden transition to a domain of 94% homology was found 11. one may speculate that tgev, which multiplies readily in carnivore cells, has recombined with an autochthonous feline coronavirus, thereby acquiring pathogenicity for the new host. in some virus-cell systems, cleavage of surface glycoproteins is a prerequisite for infectivity. in paramyxoviruses it is the fusion protein, in orthomyxoviruses the hemagglutinin protein which needs to be proteolytically processed. cells lacking the relevant enzymes produce noninfectious progeny in a single round of infection 16, 19. cell culture grown sendai virus did not cause lung pathology in mice unless it had been treated with trypsin before inoculation. mutants which possessed a fusion protein cleavable only by chymotrypsin did not produce pneumonia even when activated by this enzyme; only a single round of replication occurred in the organ which lacks the relevant enzyme 33 . these examples show that organ-specific enzymes may permit multiple rounds of infection resulting in organ pathology. however, the enzymes may also be contributed by co-infecting bacteria, as has been shown recently 34. when mice were exposed to the appropriate combination of strains of influenza virus and staphylococcus aureus, the animals came down with fatal disease whereas each of the agents inoculated alone caused no significant changes. in view of these findings the concept of %econdary' bacterial infections may need some adjustment, and antibiotic treatment of influenza pneumonia may not be so bizarre after all. also in some coronaviruses proteolytic cleavage of the surface glycoprotein by host cell proteases has been observed. fusion ensues in infected cells in vivo and in vitro and may be an important virulence determinant. trypsin treatment was shown to enhance plaque formation and cell fusion of an enteropathogenic bovine coronavirus 32. direct evidence for a correlation between cleavage and infectivity, however, has not been presented. the receptor molecules have been identified for several viruses and evidence is accumulating that they serve an essential function in the economy of the cell -otherwise they would probably have been lost from the membrane surface during evolution. identification of this function has been elusive, however, in most cases. the well-known genetic resistance of some strains of inbred mice to infection with mhv has been found to correlate with the absence of a virus-binding membrane molecule. this structure, probably a protein, was present on the membranes of cells from susceptible and semi-resistant mice and probably represents the mhv receptor 2. mouse-human cellular hybrids have been studied for localizing the receptor of picornaviruses on the human genome; the receptor for poliovirus was found to be coded by a gene on chromosome 1925 . from enzymatic digestion studies it appeared that picornaviruses do not all use the same receptor and that unrelated viruses may share the same receptor. binding of vesicular stomatitis virus (vsv) could be entirely inhibited by phosphatidylserine, a common component of cell membranes; this observation may explain the extremely wide host cell range of this rhabdovirus 31. rabies virus, another representative of this family, uses acetylcholine receptors for adsorption to neuromuscular junctions, which is compatible with the neurotropic nature of this pathogen 2~ in cell culture, however, rabies virus also infects non-neural cells, which indicates that it may utilize other receptors as well. monoclonal antibodies are now being widely employed for receptor identification. erythrocyte receptors have been studied in hemagglutinating viruses since red blood cells are an abundant source of plasma membrane; the significance of these receptors in relation to the virus-eclipsing structures is not clear, since erythrocytes do not play a role in the pathogenesis of most diseases (e.g. encephalomyocarditis of mice). complementarity between the three-dimensional shapes of the viral and the cellular structures is presumably the underlying principle of the interaction. in influenza virus the spikelike hemagglutinin glycoprotein is responsible for initial binding, and a cleft or pocket close to the top of the trimeric molecular aggregate serves as a receptacle for sialoligosaccharides on the cell surface. it is the receptor that generally permits a virus to enter a cell and if replication and amplification occurs, the cell is termed permissive; in an organism, the probability of a virus encountering a permissive cell is smallmost cell types are non-permissive for a given virus 3s. also, the receptor density on the surface of a permissive cell may vary with its cycle a6. for picornaviruses it has been shown that receptors indeed determine the host range24; for other viruses, the replication block must occur at a later stage. parainfluenzaviruses, for example, attach to neuraminic acid-containing receptors; since glycolipids and glycoproteins containing neuraminic acid abound in vertebrate cell membranes the adsorption/penetration process lacks the specificity required to explain the restrictions in host range and tissue tropism of paramyxoviruses 29. cooperation between infectious agents in the pathogenesis of a disease is of special interest in those cases where fulfillment of koch's postulates has been elusive. in addition to the proteolytic cleavage of the surface glycoprotein discussed above, examples can be given where cell surface receptors for one virus appear after infection with another virus; epstein-barr virus was proven to bind to and penetrate into cells lacking the homologous receptor after these cells had been infected with sendai virus 15. baby hamster kidney cells (bhk-21) which are normally refractory to infection with mhv-a59 became susceptible after infection with influenza virus. the (uncleaved) hemagglutinin was shown to provide the receptor on the apical cell surface which recognized the sialic acid residues on the superinfecting coronavirus 7. the disease produced by a virus is not necessarily the sum of the cytopathological events. also viruses non-cytopathogenic in tissue culture do produce disease. in many of these cases immunological mechanisms have been found responsible for the damage caused by the infection; some of these will be discussed later. the permissiveness, i.e., the degree to which a virus may enter a cell, replicate in it and emerge from it can be restricted at any point in the infectious cycle. because synthesis of viral components is entirely dependent upon cellular capacities, sharing of molecular functions has evolved. as with many parasite-host relationships, competition often results when shared resources are available in only limited amounts. after adsorption to the cell membrane, enveloped viruses may follow two pathways to have their genome enter the cytoplasmic compartment. in paramyxoviruses, for exampie, the viral envelope fuses with the cytoplasmic membrane and the contents of the former are released. alternatively, the virus may utilize the endocytotic pathway and may become internalized into endosomes which then are acidified by proton pumps. at a specific ph, fusion occurs again (between the viral envelope and the endosomal membrane) and the viral genome is released. this pathway is used by e.g. toga-, rhabdo-and orthomyxoviruses. in the latter, the hemagglutinin molecules undergo a conformational change which may bring the adjacent membranes into closer contact. the fatty acid chains covalently bound to fusogenic membrane proteins probably destabilize the membrane locally; indeed, deacylated hemagglutinin was found to be devoid of fusogenic activity. the detailed mechanism of fusion, however, is still unknown. a well-known feature following infection is the ability of many viruses to inhibit the synthesis of cellular macromolecules. the mechanism of this 'shut-off' has been extensively studied in vsv and recently reviewed ~4. vsv is a negative-stranded virus which contains a polymerase; upon infection, transcription starts and a 50-nucleotide leader rna is synthesized in addition to 5 mrna species. the leader rna, especially its central portion (bases 18-30) has been found responsible for the 'shut-off' of cellular rna synthesis. this sequence shows a high degree of homology with the lupus sm protein binding regions of small nuclear rnas. the earliest detectable effect of a vsv infection is the rapid and complete cessation of the prosessing and assembly of the small nuclear rnas, and it can be inferred that cellular macromolecular synthesis is inhibited and cell death ensues. it is interesting to note that ill-interferon production is not blocked by vsv infection. when vsv involved the ability to compromise rna processing, the host may have adapted to this intervention by evading the requirement for mrna splicing: the interferon gene lacks introns 36. the cytocidal effect of picornavirus infection has been pinpointed as being the inactivation of a cellular cap-binding protein which is essential for proper interaction between ribosomes and host mrna. poliovirus rna translation is independent of this protein 37. also in influenza virus infection cap structures are essential: these are cannibalized from host cell nuclear rna precursor molecules and used as primers for viral rna replication and synthesis 28. the cooperations between infectious agents as they may occur at the level of adsorption and penetration have been discussed above. also intracellular processes and their pathogenic consequences may be modified by coinfection. the best known example are the defective interfering (di) particles as they arise during e.g. high multiplicity passages of many viruses. di particles are mutants of the 'standard' virus which contain (sometimes extreme) deletions and sequence rearrangements. they usually interfere only homotypically (with the replication of the standard virus) at the level of macromolecular synthesis, probably by competition for a replication-relevant polypeptide whose supply in the cell is limited or for virion proteins. different populations of di particles can be generated in one system which may differ in biological properties. in the context of this review it is important to mention that restriction of multiplication of standard virus may result in the development of escape mutants resistant to interference. this virus in turn may generate its own di particles and the cycle repeats itself 9. it can be speculated that this mechanism grants an antibody-independent evolutionary advantage to the virus and maintains the labile equilibrium established in persistent infections. in cell culture, but also in experimental animal systems, there are cases where di particles modify an otherwise lethal infection and establish virus persistence (e.g., vsv, reo-, arena-and togaviruses in rodents). for example, adult mice were completely protected from the lethal effect of intranasally administered semliki forest virus, a togavirus, by di particles. the animals showed no clinical signs and cleared the standard virus from their brains in most instances; after several weeks, however, virus could still be isolated with appeared as virulent as the parental standard virus ~. it remains to be shown whether di particle-mediated long term virus persistence may give rise to chronic disease in old age. another form of cooperation was thought to be a speciality of plant viruses until recently. parasitic nucleic acids which are capable of very characteristically modulating the expres-sion of disease symptoms of a virus infection are named viral satellites. in spite of the absence of nucleotide sequence relationships between a viral satellite, its helper virus and its host, the trilateral interaction is highly specific ~3. it now appears that the hepatitis delta virus which causes an acute and severe form of hepatitis in humans must be classified as a satellite of hepatitis b virus. even more exciting is the observation that the circular rna of the satellite possesses sequence homologies with plant virus genomes which, together with the viroid character of the delta genome, have led to speculations about a possible plant origin of the animal virus 18. immunity-independent, genetic factors that determine the outcome of an infection have been identified, e.g. in flavivirus infections of mices; selection of genetically defined lines of chickens resistant to marek's disease, but not to the herpes virus infection, has been possible. the molecular interactions between viral gene products, either virion-borne or exhibited at the surface of an infected cell, and complementary structures from the host's immune system, are designed to thwart progression of the infection through the organism. in many cases, however, the immune reaction has been found to be detrimental for the host. autoimmune phenomena involving both the humoral and cellular limbs of the immune response have been identified in neurological conditions following infections with e.g. canine distemper virus3; invasion of brain tissue is supposed to cause changes in the molecular constitution of myelin and membrane components, making them recognizable as 'nonself'. another mechanism involves 'molecular mimicry' -the coincidental similarity between viral and host antigenic determinants, with antibodies recognizing both. such a crossreactivity was first reported between measles virus and myelin basic protein 36 and more examples have become known meanwhile. it remains to be shown whether the molecular similarity is indeed responsible for an autoimmune disease condition -the mere comparison of sequences is not sufficient to draw such a conclusion. the most complex form of autoimmune phenomena involves anti-idiotypic antibodies -those directed against the antigencombining site of an immunoglobulin molecule. these antibodies carry an 'internal image' of the original antigen. in the reovirus/mouse model, anti-idiotypic antibodies have been shown to bind to the viral receptor on the cytoplasmic membrane in a manner similar to virions 27. thus impairment of function or even cell injury may occur in tissues distant from those infected as a result of the antiviral immune response and its regulation through the idiotype/anti-idiotype network. of all coronavirus infections, feline peritonitis is probably the most enigmatic. in the ascitic fluid and in serum of diseased cats, high concentrations of igg are found, part of which is antibody formed in reaction to fipv. also extremely high-titering neutralizing antibody has no apparent protective value since more seropositive animals with higher titers are detected in catteries where fip had been a problem; high antibody titers are regarded as diagnostic and prognostic clues. the condition is fatal in the majority of cases. in experimentally infected cats antibody formation started 8-13 days before death and was accompanied by the appearance of circulating immune complexes followed by complement depletion ~2. it is our impression, however, that the immunological phenomena are directed also against host antigens. attempts to protect cats against this infection by different vaccine preparations have all been unsuccessful. sitespecific alteration of murine hepatitis virus type 4 peplomer glycoprotein e2 results in reduced neurovirulence characterization of an antigenic determinant of the g/ycoprotein that correlates with pathogenicity of rablies virus pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies cell surface influenza haemagglutinin can mediate infection by other animal viruses development of a virus-resistant inbred mouse strain for the study of innate resistance to arbo-b viruses reconstruction experiments demonstrating selective effects of defective interfering particles of mixed populations of vesicular stomatitis virus antigenic relationships among homologous structural polypeptides of porcine, feline and canine coronaviruses comparison of the peplomer protein sequences of porcine transmissible gastroenteritis virus (tgev) and feline infectious peritonitis virus (fipv) antibody, immune complexes, and complement activity fluctuations in kittens with experimentally induced feline infectious peritonitis viral satellites: parasitic nucleic acids capable of modulating disease expression interactions between small viral rnas of vesicular stomatitis virus and components of cellular gene expression sendal virus envelopes can mediate epstein-barr virus binding to and penetration into epstein-barr virus receptor-negative cells activation of influenza virus by trypsin treatment temperature sensitive mutants of mouse hepatitis virus strain a59: isolation, characterization and neuropathogenic properties the hepatitis delta virus possesses a circular rna enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide ls the acetylcholine receptor a rabies virus receptor? in vivo and in vitro models of demyelinating diseases: tropism of the jhm strain of murine hepatitis virus for cells of glial origin high frequency rna recombination of murine coronaviruses identification of lactate dehydrogenase-elevating virus as the etiological agent of genetically restricted, age-dependent polioencephalomyelitis of mice the mammalian cell-virus relationship. 1. attachment ofpoliovirus to cultivated cells of primate and non-primate origin human chromosome 19 carries a poliovirus receptor gene expression and modulation of virus receptors on lymphoid and myeloid cells: relationship to infectivity cell receptors for mammalian reovirus. 1. syngeneic monoclonal anti-idiotypic antibody identifies a celt surface receptor for reovirus transfer of 5'-terminal cap of globin mrna to influenza viral complementary rna during transcription in vitro molecular basis of virus disease molecular basis of virus disease inhibition of vsv binding and infectivity by phosphatidylserine: is phosphatidyl-serine a vsv-binding site? enhancement of plaque formation and cell fusion of an enteropathogenic coronavirus by trypsin treatment pneumotropism of sendal virus in relation to protease-mediated activation in mouse lungs. infect. lmmun synergistic role of staphylococcal proteases in the induction of influenza virus pathogenicity evidence for a unique human fibroblast interferon (ifn-fl i) chromosomal gene, devoid of intervening sequences purification of a factor that restores translation of vesicular stomatitis virus mrna in extracts form poliovirus infected hela cells restricted replication of mouse hepatitis virus a59 in primary mouse brain astrocytes correlates with reduced pathogenicity acknowledgment. i should like to thank willy spaan for helpful critical comments. key: cord-298131-zolwjl9u authors: xiao, shuqi; jia, jianyu; mo, delin; wang, qiwei; qin, limei; he, zuyong; zhao, xiao; huang, yuankai; li, anning; yu, jingwei; niu, yuna; liu, xiaohong; chen, yaosheng title: understanding prrsv infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing date: 2010-06-29 journal: plos one doi: 10.1371/journal.pone.0011377 sha: doc_id: 298131 cord_uid: zolwjl9u porcine reproductive and respiratory syndrome (prrs) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. prrs virus (prrsv) replicates mainly in porcine alveolar macrophages (pams) and dendritic cells (dcs) and develops persistent infections, antibody-dependent enhancement (ade), interstitial pneumonia and immunosuppression. but the molecular mechanisms of prrsv infection still are poorly understood. here we report on the first genome-wide host transcriptional responses to classical north american type prrsv (n-prrsv) strain ch 1a infection using solexa/illumina's digital gene expression (dge) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after n-prrsv infection and infection pathology. our results suggest that n-prrsv appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ade. upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during n-prrsv infection processes. n-prrsv-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. our systems analysis will benefit for better understanding the molecular pathogenesis of n-prrsv infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to prrs. porcine reproductive and respiratory syndrome (prrs), also called ''blue ear'' disease due to a typical, but not often observed hallmark of ''blue ears'', is widely accepted as being one of the most economically important diseases affecting swine industry. since its first appearance in the late 1980s in the us and europe, prrs has spread worldwide [1, 2, 3] . prrs is characterized with high mortality in piglets, reproductive failure (late-term abortions and stillbirths, premature farrowing, mummified pigs) in pregnant sows and respiratory disease (interstitial pneumonia, respiratory difficulties) in nursery and grower/finishing pigs, causing highly significant economic losses to the swine industry worldwide, resulting in .$ 560.32 million losses each year in the us alone [4] . the etiologic agent of prrs is prrs virus (prrsv), a small enveloped, linear, single, positive-stranded rna virus, which is a member of the family arteriviridae which includes lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus and enters in the newly established order delayed and their levels remain low, which can not eliminate effectively prrsv-infected cells [12, 13] . because of these features of prrsv infection, prrs has been one of the most challenging subjects of research in veterinary viral immunology [12] . regulation of immune responses and genetic resistance to infectious viral diseases is an area of concern for human and swine [14] . prrsv strongly modulates the host's immune responses, and changes the host's gene expression. studies showed that prrsv inhibits type i interferons (ifn-a/b, spi ifn), especially ifn-a [15] , and induces interleukin-10 (il10) [16, 17] . because the primary cellular target of prrsv is the porcine alveolar macrophages (pams) of the lung, several studies have analysed the immune responses of pams to prrsv infection. one group [18] used differential display reverse-transcription pcr to identify molecular genetic changes within prrsv-infected pams over a 24 h pi period. their results suggest that myxovirus resistance 1 (mx1) and ubiquitin specific proteases (usp) genes may play important roles in clinical disease during prrsv infection. notably, one recent paper on genome-wide transcriptional response of pams following infection with the lelystad prrsv strain (european type, eu prrsv) using affymetrix microarrays has been published during the preparation of our manuscript [19] . they found that the expression of beta interferon 1 (ifn-b) was strongly upregulated while the expression of il-10 and tnf-a was weakly upregulated. almost in the same time, the other group employed serial analysis of gene expression (sage) to examine the global expression of genes in vr-2332 prrsv strain (north american type, na prrsv)-infected pams. they identified over 400 unique tags with significantly altered expression levels [20] . in vitro studies will be useful for investigating how prrsv modifies genes expression in primary target cells, such as pams. however, many of the outstanding issues will be answered only in the context of prrsv-infected animals. hence, the characterization of host immune response under in vivo environment to prrsv is still an area in urgent need of investigation. lung pathogenesis is a major feature of prrsv infection. moreover, in addition to serving as a source of protein in the human diet, the pig is also an excellent biomedical model for humans because of the similarity in size and physiology, and in organ development and disease progression [14] . thus, understanding the host's immune response to prrsv infection is important not only for swine production but also for human consumption. however, to date, the immune response to prrsv in porcine lung has not been analyzed by transcriptome profiling. next generation high-throughput sequencing technology has been adapted for transcriptome analysis because of the inexpensive production of large volumes of sequence data [21, 22, 23, 24] . the technology developed by illumina (formerly solexa sequencing) [25] , which is also referred to as digital gene expression (dge) tag profiling, allows identification of millions of short rnas in a sample and of differentially expressed genes without the need for prior annotations. here we employed the illumina genome analyzer platform to perform a digital gene expression analysis of the porcine lung transcriptome response to n-prrsv infection, and used histopathology examination to analyze the pulmonary pathological changes of the infected-porcine lungs. the relationship between pulmonary gene expression profiles after n-prrsv infection and infection pathology was systematically analyzed. the comprehensive analysis of the global host response induced by n-prrsv suggested an inflammatory response, mediated by multiple inflammatory molecules early during infection that induced tissue injury, an immunosuppressive state, mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. our systems analysis will benefit for better understanding the molecular pathogenesis of n-prrsv infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to prrs. our study had been approved by animal care and use committee of guangdong province, china. all animal procedures were performed according to guidelines developed by the china council on animal care and protocol approved by animal care and use committee of guangdong province, china. nine conventionally-reared, healthy 6-week-old, crossbred weaned pigs (landrace6yorkshire) were selected from a highhealth commercial farm that has historically been free of all major pig diseases, such as prrsv, porcine circovirus type 2, classical swine fever virus, porcine parvovirus, pseudorabies virus, swine influenza virus and mycoplasma hyopneumoniae infections. all pigs were prrsv-seronegative determined by elisa (herdchek prrs 2xr; idexx laboratories) and absence of prrsv tested by rt-pcr. pigs were randomly assigned to two groups in the experiment and raised in isolation rooms. six pigs were inoculated with 6 ml viral suspension (4 ml intranasally and 2 ml intramuscularly) of classical north american type prrsv (n-prrsv) strain ch 1a, isolated from china in 1996, gifted by dr. zhang guihong, south china agricultural university) at a dose of 10 6.0 tcid50 ml 21 on day 0. three uninfected negative control (unc) pigs were treated similarly with an identical volume of dmem culture media from uninfected marc-145 cells 1 day prior to experimental infection, and were immediately necropsied. n-prrsv-inoculated pigs were clinically examined daily and rectal body temperatures were recorded from days 22 to 7 pi. viral reisolates were performed after the pigs were killed. the infected group showed positive, and the unc group was negative. tissue homogenates and serum were examined by n-prrsv-specific quantitative pcr (qpcr). the oligonucleotide primers used were nsp2f(59-gtgggtcggcaccagtt-39) and nsp2r , designed in the gene segment encoding for nsp2. the taqman probe, 59 fam-cacagttctacgcggtgcagg -tamra 39, was synthesized. three infected pigs randomly chosen were necropsied at each time point of 96 h pi and 168 h pi. lung samples were collected from unc group (c), three pigs at 96 h pi (n96), three pigs at 168 h pi (n168) and immediately frozen in liquid nitrogen for rna isolation or fixed in 10% neutralized buffered formalin for histological processing. lungs of unc and experimentally infected pigs were processed routinely for haematoxylin and eosin (h&e) and immunohistochemistry staining, as described previously [26] . total rna was extracted from frozen lungs using standard protocols (trizol) and then treated with dnase to remove potential genomic dna contamination according to the manufactures's protocols. rna integrity and concentration were evaluated by agilent 2100 bioanalyzer (agilent technologies). for rna library construction and deep sequencing, equal quantities of rna samples from three unc individual lungs were pooled, rna samples from the three infected pig lungs (n96) were pooled, and rna samples from the three infected individual lungs (n168) were pooled. approximately 6 mg of rna representing each group were submitted to solexa (now illumina inc.) for sequencing. sequence tag preparation was done with illumina's digital gene expression tag profiling kit according to the manufacturer's protocol. in brief, mrna was isolated from 6mg total rna by binding the mrna to a magnetic oligo bead. first-and secondstrand cdna were synthesized while the mrna was attached to the beads. the double stranded cdnas were digested with nlaiii to wash away all fragmens other than the 39 catg fragment attached to the oligo bead. then gex nlaiii adapter 1 was ligated at the site of nlaiii cleavage. in addition, gex nlaiii adapter 1 contains the sequence for the restriction enzyme mmei, subsequently, we applied the restriction enzyme mmei to create the 17 bp tag. the gex adapter 2 was ligated at the site of mmei cleavage. a pcr with 12 cycles was performed with two primers that anneal to the ends of the adapters to enrich the adapterligated cdna construct. the resulting 85 bp fragments were purified from 6% novex tbe page gel. subsequently, the purified cdna tags were sequenced on the illumina cluster station and genome analyzer. image recognition and base calling were performed using the illumina pipeline. all data is miame compliant. the raw data (tag sequences and counts) has been submitted to gene expression omnibus (geo) under series gse19970. for the raw data, we filtered adaptor tags, low quality tags and tags of copy number = 1 to get clean tags. subsequently, we classified the clean tags according their copy number in the library and show their percentage in the total clean tags and analyzed saturation of the library. the preprocessed database of all possible catg +17-nt tag sequences was created, using sus scrofa unigene (http://www.ncbi. nlm.nih.gov/unigene/ugorg.cgi?taxid = 9823, unigene build #35 sus scrofa, nov, 7th, 2008) from ncbi. for monitoring the mapping events on both strands, both the sense and the complementary antisense sequences were included in the data collection. information on the position of polyadenylation signals was also collected from the transcript dababase. then we aligned all clean tags to the reference sequences, and unambiguous tags were annotated. we counted the clean tag number corresponding to each gene. to compare the de of gene across samples (n96/c, n168/c, n168/n96), the number of raw clean tags in each library was normalized to tags per million (tpm) to obtain normalized gene expression level. de detection of gene or tag across samples was performed according to the previous description [27] . genes were deemed significantly differentially expressed with a p-value , 0.005, a false discovery rate (fdr) ,0.01 and an estimated absolute log2-fold change .0.5 in sequence counts across libraries. in order to verify the dge results, we used qpcr analysis. the rna samples used for the qpcr assays were both the same as for the dge experiments and independent rna extractions from biological replicates. qpcrs were done on the lightcycler480 (roche), with sybr-green detection (sybr primescript rt-pcr kit, takara biotechnology co., ltd.), according to the manufacture's instruction. each cdna was analyzed in triplicate, after which the average threshold cycle (ct) was calculated per sample. the relative expression levels were calculated with the 2 2ddct method. the results were normalized to the expression level of hprt1 and relative to the c sample. through browsing all health traits of pig quantitative trait locus (qtl) database (pigqtldb, http://www.animalgenome. org/qtldb/pig.html) by trait classes, we obtained mapping details of qtl on the corresponding pig chromosome. then pig affymetrix elements corresponding to health trait qtl regions were downloaded to an excel file. by matching the id of de genes to all genes in the qtl regions, we obtained de genes of the corresponding qtl region. pathway analysis was mainly based on the kyoto encyclopedia of genes and genomes (kegg) database. two-side fisher's exact test with a multiple testing and x2 test were used to classify the pathway category. the false discovery rate (fdr) was used to correct the p-value. we chose only pathway categories that had a p,0.05. within the significant category, the enrichment re was , where n f is the number of flagged proteins within the particular category, n is the total number of proteins within the same category, nf is the number of flagged proteins in the protein reference database list, n is the total number of proteins in the gene reference database list. stc is implemented entirely in java. the clustering algorithm first selects a set of distinct and representative temporal expression profiles. these model profiles are selected independent of the data. the clustering algorithm then assigns each gene passing the filtering criteria to the model profile that most closely matches the gene's expression profile as determined by the correlation coefficient. since the model profiles were selected independent of the data, the algorithm can then determine which profiles have a statistically significant higher numberthan genes assigned using a permutation test. this test determines an assignment of genes to model profiles using a large number of permutations of the time points. it then uses standard hypothesis testing to determine which model profiles have significantly more genes assigned under the true ordering of time points compared to the average number assigned to the model profile in the permutation runs. significant model profiles can either be analyzed independently, or grouped together based on similarity to form clusters of significant profiles [28, 29] . stc-go supports gene ontology enrichment analyses for sets of genes having the same significant temporal expression pattern. we select random samples of s a (s a is the number of genes assigned to the same model temporal expression profile r.) genes at each iteration and compute fisher's exact test p-values for the selected genes in all go biological categories [30] . the two-sided fisher's exact test p-value for a category reflects a test of the null hypothesis that the category is enriched in genes assigned to profile r with respect to what would have been expected by chance alone. to decide whether or not to follow up a category that appears enriched in these genes, we would know the statistical reliability of the apparent enrichment. to assess the significance of a particular category, we need to know the distribution of p-values that would occur by random chance. the percentage of false positives to be tolerated will generally depend on the relative costs of false positives and false negatives in whatever follow-up study is to be done. this way of framing the question leads us to specify the false discovery rate (fdr) for a set of categories, rather than significance level (p-value) for each category. with the significance at the 0.05 level, for a given category, the enrichment r e is given by r e~( i=m)=(s a =n) where i is the number of genes assigned to profile r within the go category of interest, m is the total number of genes within the go category of interest, and n is total number of unique genes in the gene reference database list. after n-prrsv infection, the affected pigs exhibited the following clinical symptoms within 3-7 days: fever of 40.08-40.8uc, depression, anorexia, rough hair coats, dyspnoea, reddening of skin, oedema of the eyelids, conjunctivitis, mild diarrhoea, shivering. those unc pigs did not show any obvious changes in body temperature and clinical signs. qpcr assay showed that n-prrsv virus was present in each of the 6 infected pigs. but n-prrsv nsp2 gene was not differentially expressed at 96 h pi and 168 h pi (table s1 ). histopathology examination of n-prrsv-affected pigs showed interstitial pneumonia in lungs with thickening of alveolar septa accompanied with infiltration of immune cells ( figure 1b ). most viral antigen was detected in alveolar cells and bronchiolar epithelial cells in lesions ( figure 1c ). to investigate the regulation of the host response to the n-prrsv virus, we considered the global gene expression profiles in lungs using solexa/illumina's dge system, a tag-based transcriptome sequencing method. we sequenced three porcine lung dge libraries from c, n96, n168 using massively parallel sequencing on the illumina platform. major characteristics of these three libraries were summarized table 1 . we obtained approximately 6.9 million total sequence tags per library with 515885 distinct tag sequences. prior to mapping these tag sequences to reference sequences, we filtered adaptor tags, low quality tags and tags of copy number = 1, producing approximately 6.6 million total clean sequence tags per library with 179589 distinct clean tag sequences. the c library had the highest number of both total sequence tags and distinct sequence tags; this was followed by the n168, n96 libraries. moreover, the c library had the highest ratio of number of distinct tags to total tags and the lowest percentage of distinct clean high copy number tags. the data showed that more genes were detected in the c library than other two libraries and more transcripts were expressed at lower levels in the c library. saturation analysis of capacity of libraries showed that new emerging distinct tags were gradually reduced with increasing of total sequence tags when the number of sequencing tags was big enough. when the number of sequencing tags reached 3 million, library capacity approached saturation ( figure s1 ). for tag mapping, we preprocessed one reference tag database that included 51670 sequences from sus scrofa unigene. to get the reference tags, we used nlaiii to digest all the samples and took all the catg+17 tags in the gene as the gene's reference tags, not only the 39 most one. we obtained 194664 total reference tag sequences with 172119 unambigous tag sequences. considering polymorphism across samples, tolerances were set to allow one mismatch in each alignment. by the criteria, 47.71%,51.04% of distinct clean tags mapped to the unigene virtual tag database, 39.42%,42.11% of the distinct clean tags mapped unambiguously to the unigene, and 52.29%,48.96% of the distinct clean tags didn't map to the unigene virtual tag database ( table 1 ). the occurrence of unknown tags was probably due to the incompleteness of pig genome sequencing. most solexa experimental tags matched to the 1st or 2nd 39 catg site in high-confidence transcripts ( figure s2 ). for solexa sequencing can distinguish between transcripts originating from both dna strands, employing the strand-specific nature of the sequencing tags obtained, we found evidence for bidirectional transcription in 6368 to 8271 of all detectable unigen clusters and 3889 to 4043 antisense-stand specific transcripts (table s2 ). by comparison, the ratio of sense to antisense strand of the transcripts was approximately 1.7:1 for all libraries. this suggests that in spite of the high number of antisense mapping events detected, the transcriptional regulation in the n-prrsv-induced immune response acts most strongly on the sense strand. to analyze the depth of transcriptome sampling in the dge libraries, we studied the rate of increase of the number of genes (sense+antisense strand) identified as the size of the corresponding library increases. when the library size reached one million, we could identify 45% and 30% all genes and genes identified by unambigous tags, respectively ( figure s3 ). at this time, library capacity approached saturation. to gain the global transcriptional changes in n-prrsv infected porcine lungs, we applied the method described previously [27] to identify de genes from the normalized dge data by pairwise comparisons between all differential time points (n96/c, n168/c, n168/n96) during infection. results showed that 5430 genes had p values ,0.005, false discovery rate (fdr) ,0.01 and estimated absolute log2-fold change .0.5 in at least one of the pairwise comparisons, which were declared to be differentially expressed during infection course (table s3) . to characterize the functional consequences of gene expression changes associated with infection with n-prrsv, we performed pathway analysis of de genes based on the kegg database by two-side fisher's exact test. we chose only significant pathway categories that had a p-value of ,0.05 and an fdr of ,0.05. as shown in figure s4 , the significant signaling pathways include cell adhesion molecules (cams), t cell receptor signaling pathway, antigen processing and presentation, toll-like receptor signaling pathway, biosynthesis of unsaturated fatty acids, pantothenate and coa biosynthesis, etc (table s4) . to validate de genes identified by solexa sequencing, we selected 8 genes for qpcr confirmation. the set included two down-regulated genes (epithelial chloride channel protein (aecc) and hyaluronan and proteoglycan link protein 1 (hapln1)) and six up-regulated genes (inflammatory response protein 6 (irg6), dead (asp-glu-ala-asp) box polypeptide 58 (ddx58), usp18, cxcl10, cytochrome p450 (cyp3a88), and cd209). data were presented as fold changes in gene expression normalized to the hprt1 gene and relative to the c sample. pearson's correlation coefficient (r) showed that both the dge and qpcr data (pooling samples) were highly correlated, for the genes modulated by n-prrsv had a high consistency and r values ranging from 0.781 (cyp3a88) to 0.997 (aecc) between the two methods ( figure 2 ). qpcr analysis (both pooling samples and independent rna extractions from biological replicates) confirmed the direction of change detected by dge analysis. this correlation indicated the reliability of dge results. qtl play a central role in linking genomic information with phenotypes. the ultimate goal of qtl studies is to identificate the actual gene(s) that are responsible for the phenotypic variation observed in a particular trait [31] . in the present paper, we mapped the de genes to pig qtl regions of health traits in pig qtldatabase (pigqtldb). our search found that 240 de genes were distributed in 18 different known qtl regions related to pig health traits ( figure s5 ; table s5 ). among the 240 de genes, 122 and 114 were located in qtl regions of the cd4-positive leukocytes and cd2-positive leukocytes, respectively; 53 were distributed in the qtl region of the band-formed neutrophils and cd8-positive leukocytes. immune responses against pathogens depend in part on the generation of fully differentiated 'killer' (or effector) and memory cd8 + t cell. effective priming and maintenance of cd8 + t cell responses to viral infection require 'help' from cd4 + t cells, the latter play also a critical role in programming cd8 + t cell memory development [32] . moreover, recent study showed that cd4 + t cells guide effector cytotoxic t lymphocytes (ctls) to virally infected tissues where they can destroy infected cells [32, 33] . in order to profile gene expression time series and search for the most probable set of clusters generating the observed time series, we used stc algorithm of gene expression dynamics, which explicitly took into account the dynamic nature of temporal gene expression profiles during clustering and identified the number of figure s6 ; table s3 ) with 4 (profile 1,6,0,7) significant cluster profiles which have significantly more genes assigned under the true ordering of time points compared to the average number assigned to the model profile in the permutation runs ( figure s6 ). then gene ontology (go) based on biological process (bp) enrichment analyses for sets of de genes having significant cluster profiles was performed by two-side fisher's exact test (table s6 and table s7 ; figures s7, s8 , s9 s10). we chose only significant go categories that had a p-value of ,0.05. the most prominently overrepresented go terms of significant cluster profile 1 (0,21,21) and profile 0 (0, 21, 22) , which are down-regulated genes, involved in regulation of lipid, cholesterol biosynthetic and metabolic process; regulation of skeletal muscle development, muscle cell differentiation; digestion; negative regulation of neuron apoptosis and neurological system process (table s6 ; figures s7 and s8) . the most prominently overrepresented go terms of significant cluster profile 6 (0,1,1) and profile 7 (0,1,2), which are up-regulated genes, included negative regulation of fibroblast proliferation; natural killer cell, macrophage, lymphocyte, mononuclear cell, leukocyte and t cell proliferation, differentiation and activation; complement activation, immune response, inflammatory response, defense response, and apoptosis; response to stimulus(stress); lipid and fatty acid metabolic process and oxidation; positive regulation of ubiquitinprotein ligase activity and protein proteolysis, protein targeting to mitochondrion (table s7 ; figure s9 and s10). these results are consistent with these genes and their associated processes playing important roles in n-prrsv replication and pathogenesis. viral infection of host leads to the initiation of antiviral innate immune responses, which results in the induction of expression of the type i interferons [34] . meanwhile, many viruses have also developed strategies to evade and subvert the immune response. as shown in figure 3a , transcripts of the ifn c was significantly induced in n-prrsv-infected pigs at days 4 through 7 pi, but short type i interferon (spi ifn) gene expression was suppressed, and interferon alpha 5 (ifna5) gene expression was markedly down-regulated. lipid rafts, lipid microdomains of the cell membrane enriched in sphingolipids, cholesterol and associated proteins, play critical roles in the life cycle of many viruses [35] . some viruses enhance their replication by modulating host cell lipid metabolism [36] . dge analysis of pigs infected with n-prrsv showed significant increase of transcript abundance in many genes involved in lipid metabolism, including those for apolipoprotein b48 receptor (apob48r), apolipoprotein-e (apoe), low density lipoprotein b (ldlb), phosphatidylinositol 3-kinase catalytic subunit type 3 (pik3c3) ( figure 3b ). perhaps n-prrsv alters hosts' lipid metabolism to create a lipid-rich intracellular environment to facilitate its own multiplication. moreover, we also observed that n-prrsv induced upregulation expression of anti-apoptotic genes in n-prrsv infected lungs, including myeloid cell leukemia sequence 1 (bcl2-related) (mcl1), nuclear factor kappa-b 1 (nfkb1), nfkb2, adrenomedullin (adm), and interleukin 10 (il10), and downregulation expression of pro-apoptotic genes, including bak protein (bak), (apoptosis-related protein 1) (apr1) ( figure 3d ) to inhibit apoptosis, which might prolong cell life and increase the yield of progeny virions. n-prrsv infection caused anorexia and subsequent slow growth. accordingly, we observed that transcript abundance of genes involved in digestion, such as gastric mucin (muc5ac) and cytochrome p450 (cyp39a1), was significantly decreased ( figure 3e ). simultaneously, transcript abundance of the genes associated with cell, muscle and cartilage development was markedly decreased ( figure 3f ). these genes include insulin-like growth factor binding protein 3 (igfbp3), collagen, type ii, alpha 1 isoform 2 (col2a1), connective tissue growth factor (ctgf), epidermal growth factor (egf). fever and heat shock fever is frequently the host's initial response to infection [37] . after viral infection, pathogen-associated molecular patterns (pamps) in viral proteins and nucleic acids were recognized by host pathogen-recognition receptors (prrs), such as toll-like receptors (tlrs), which trigger gene expression and synthesis of the il-1b precursor. active caspase-1 (casp1) cleaves the il-1b precursor into mature, bioactive il-1b, which is an inflammatory cytokine most responsible for fever [37, 38, 39] . as shown in figure 4a , transcript abundance of tlr1, 2, 4, 6, il-1b and casp1 was significantly increased in n-prrsv infected porcine lungs. moreover, transcript abundance of genes involved in the activation of casp1 and il-1b secretion including apoptosisassociated speck-like protein containing a card (asc), prostaglandin e synthase 2 (pge2) and phospholipase a2, group vii (pla2g7) was significantly increased ( figure 4a ). the expression of heat shock proteins (hsps), known as stress proteins, can be markedly upregulated by all cells under conditions of stress, such as increased temperature (fever) and viral infection [40] . transcript abundance for most of these heat shock genes, including 90-kda hsp (hsp90), hsp70, and heat shock protein beta-1 (hsp27) was significantly elevated in n-prrsv infected lungs relative to unc lungs ( figure 4b ). viral infection results in an inflammatory response, which is an essential component of the antiviral innate immune response [41] . after recognizing the pamps, either surface or intracellular prrs trigger intracellular signaling cascades that results in the activation of transcription factors, including nuclear factor-kb (nf-kb), interferon-regulatory factors (irfs), and signal transducer and activator of transcription (stats). as shown in figure 4a , transcripts of the toll-like prrs tlr1, tlr2, tlr4, tlr6, were significantly increased in n-prrsv-infected pigs at days 4 through 7 pi, but no change in tlr3 which specializes in the recognition of viral dsrna was detected. cytoplasmic prrs ( figure 5a ), retinoic-acid-inducible protein i (rig-i, ddx58) and melanoma differentiation-associated gene 5 (mda5), the two most relevant for defense against viruses, were expressed at a high level after n-prrsv infection. cell surface prrs such as cd14, md-2 protein (md2) and cd163 (which is probably involved in prrsv entry during uncoating [42] ) were likewise up-regulated expression after n-prrsv infection ( figure 5a ). after binding to n-prrsv viral pamps, prrs initiate intracellular signaling cascades that activate transcription factors, including irf1, irf7, irf9, but not irf3 and stat1, stat3, stat6 ( figure 5b ). activated transcription factors and stats in turn induce the transcription of specific sets of interferon-stimulated genes (isgs) [34, 43] , and expression of multiple inflammatory genes [44] , which induce a pro-inflammatory response and attract cells, such as neutrophils and macrophages, to sites of infection. accordingly, we observed significant increase of transcript abundance in many genes involved in isgs ( figure 5c ), pro-inflammatory cytokines (such as il1b, il8) ( figure 5d ), chemokines (ccl2, cxcl9, cxcl10) ( figure 5e ), adhesion molecules (vcam, icam1, sell), and other inflammatory molecules (such as mmp-2,) ( figure 5f ). moreover, immunoglobulin (such as igg2b, igg3) ( figure 5g ), three categories of fc receptors and mannose receptor c1 (mrc1) ( figure 5h ), and complement proteins ( figure 5i ) were also significantly induced in the n-prrsv-infected lungs. however, several complement inhibitors that possess inhibitory and/or decay-accelerating acitivity, such as decay-accelerating factor cd55, complement component 4 binding protein, alpha (c4bpa), c4bpb, were significantly repressed in the n-prrsvinfected lungs ( figure 5i ). cytotoxic t lymphocytes (ctls) detect cells infected with a virus and destroy them through perforin-mediated apoptosis [45] . cd8 + t cells activation require t cell receptors (tcrs) to recognize cognate antigenic peptides for presentation on mhc class i molecules displayed on the surface of antigen presenting cells (apcs) [32] . accordingly, we observed that transcript abundance of ubiquitin specific peptidase (usp) and ubiquitin enzyme ( figure 6a ), 16 proteasomes, and aminopeptidases ( figure 6b ) was significantly increased in n-prrsv-infected lungs. the transcript abundance of b2m, mhc class i antigen 2 (sla-2), sla-3, tap2, and chaperones (such as grp78) was markedly increased after infection with n-prrsv while the transcript abundance of sla-b was significantly decreased ( figure 6c ). in addition to recognization of cognate peptides presented by mhc class i molecules, cd8 + t cells activation needs also to receive 'costimulatory' signals and help by helper cd4 t + cells [33] . as shown in figure 6d and 6e, 8 cathepsins and 5 mhc class ii antigens were significantly induced in n-prrsv-infected lungs. the transcript abundance of costimulatory molecules (such as cd86, icos), cams, and tcrs/cd3 complex as well as co-receptor molecules (such as cd8a, table s3 for full gene names. doi:10.1371/journal.pone.0011377.g006 cd8b) was remarkably increased after infection with n-prrsv ( figure 6f and 6g) . activated ctls release perforin (pfr) and granzymes, which two effectors act collaboratively to induce apoptosis of target cells. as shown in figure 6h , prf1 and granzyme b (gzmb) transcript abundance was significantly elevated in n-prrsv infected lungs relative to unc lungs. in addition to cytotoxins released from ctls, the transcript abundance of other pro-apoptotic members ( figure 6i ), such as nfkbia, growth arrest and dna-damage-inducible protein alpha (gadd45a), bh3 interacting domain death agonist (bid), xiap-associated factor 1 (xaf1), cytochrome c (cycs), casp10, was also significantly increased after infection with n-prrsv, which can induce apoptosis of virus-infected cells. in addition, we also identified the upregulated expression of cytochrome b245 heavy chain (gp91-phox), a critical component of the membrane-bound oxidase of phagocytes (macrophages and neutrophils), and the downregulated expression of heme oxygenase 1 (hmox1) during n-prrsv infections, which might result in the oxidative stress response and subsequent oxidative damage of tissues ( figure 6j ). from the data presented in the paper, a model for the relationship between pulmonary gene expression profiles and infection pathology can be surmised in figure 7 , n-prrsv virus replicates and spreads by subverting host innate immune response and hijacking host lipid metabolism as well as inducing an antiapoptotic and anti-inflammatory state, as indicated by suppression expression of spi ifn, ifn-a, down-regulation expression of proapoptotic genes for bak, apr-1, sarp3, high levels expression of genes involved in lipid metabolism, such as apoe, ldlb, pik3c3, anti-apoptotic genes for mcl1, bcl2a1, chfr, adm, nfkb, il10, and anti-inflammatory molecule pge2 as well as cd163. infections of n-prrsv viruses resulted in fever and inflammatory response, as indicated by high expression of proinflammatory cytokines and chemokines, adhesion molecules, inflammatory enzymes and receptors, such as il-1b, il8, sell, icam, ccl2, cxcl9, cxcl10, b2m, proteasomes, cathepsins. this was compounded by cell death and elevated expression of nfkbia, xaf1, gadd45a, perforin, granzymes, and cytochrome c, coupled with increased ros-mediated oxidative stress, as indicated by by up-regulation expression of cytochrome b245. taken together, the n-prrsv infections may have resulted in an excessively immune and inflammatory response that contributed to tissue damage. infection of pigs with n-prrsv caused fever, dyspnoea, reddening of skin, oedema of the eyelids, conjunctivitis, depression, anorexia, mild diarrhoea. histopathology examination showed interstitial pneumonia in lungs with thickening of alveolar septa accompanied with infiltration of immune cells [46] (figure 1 ). although great efforts have been made by many researchers, the molecular basis of n-prrsv infection is largely unknown. here we report on the first genome-wide host transcriptional response to n-prrsv infection using solexa/illumina's digital gene expression (dge) system, a tag-based novel high-throughput transcriptome deep sequencing method. given the nature of the methodology of illumina's dge system, we have pooled biological replicates from three pigs for each group to make representative samples for deep sequencing analysis. we could reach a sequencing depth of 6.3-7.9 million tags per library (table 1) and found over 5000 genes to be differentially expressed during n-prrsv infection processes (table s3) . although others studies have also pooled biological replicates for library construction and deep sequencing [47, 48] , resulting in the lack of biological replicate, one may blur the impact of variations in pooling samples. because of the variations of pigs in response to prrsv infection, it is possible that one pig could significantly affect results without independent libraries. but we performed the qpcr validation both on the same pooled material that was used for deep sequencing and on independent rna extractions from each pig, which all confirmed the direction of change detected by dge analysis (figure 2 ). our dge analysis showed massive changes in the transcript abundace of known immune response genes and of genes that have been implicated in prrsv infection [18, 19, 20] . we also identified many interesting genes that had not been linked to prrsv infection in previous studies. for example, transcript abundace of lipid metabolism-related genes including apob48r, apoe, pik3c, was significantly increased during n-prrsv infection processes. alterations in lipid metabolism, perhaps including those with significant upregulation in this study, have been observed in response to infection by a range of viruses including sars-cov, hcv, influenza a virus, or dengue virus [35, 36, 49, 50] . although in vitro studies have investigated how prrsv modifying genes expression in pams [18, 19, 20] , many of the outstanding issues will be answered only in the context of prrsv-infected animals [51] . hence, we characterized the genome-wide transcriptome response to prrsv infection in porcine lung by deeping sequencing. but studies of transcript abundance in lung tissues have also their intrinsic limitations. for example, the transcriptome of lung tissues is actually a merging transcriptional responses of a wide range of cell types, some of which are infected, some of which are responding directly to the infectious process and others of which are bystanders. moreover, increased cellularity of tissues may be confused as biologically important increased transcript abundance. despite such limitations, our dge study offers a broad, system-wide window into molecular processes that regulate gene expression and also provides new leads for functional studies of candidate genes involved in host-virus interaction, as illustrated in this paper. the induction of expression of type i interferons (ifns; including ifn-a and ifn-b) is a well-known innate antiviral immune reaction in the virus-infected cells [52, 53] . however, n-prrsv infection suppressed spi ifn gene expression and decreased the transcript abundance of ifn-a ( figure 3a ). previous studies [19, 54, 55] , both in vitro and in vivo, have also showed that prrsv elicited only a minimal ifn-a production or even suppressed it's expression. the suppression of spi ifn, in particular of ifn-a, is probably a crucial step in the pathogenesis, because ifn-a has been shown to inhibit prrsv replication [11] . other viruses infection, such as the 1918 influenza virus [56] , hepatitis c virus (hcv) [57] , ebola virus [58] , also suppressed type i ifn gene expression which led to extensive viral replication and increased pathogenesis. irf3 plays an important role for type i ifn gene expression. the transcript abundance of irf3 was decreased intensively in n-prrsv-infected pigs by 168 h pi ( figure 4b ). one study [59] indicated that prrsv nsp1b inhibited irf3, and then down-regulated ifn-b gene expression. it is worth mentioning that the nsp1 of the influenza a can also suppress innate immunity by inhibiting irf3 activation, and subsequently disrupting the induction of a/b-interferon [60] . research has indicated that the expression of cd163, a prrsv receptor [61] , on macrophages in different microenvironments, in vivo, may determine the replication efficiency and subsequent pathogenecity of prrsv [62] . transcript abundance of cd163 was significantly increased after n-prrsv infection ( figure 5a ). the internalization of prrsv via cd163 in the target cells may induce the expression of il10, and in turn induce the expression of cd163 on neighboring undifferentiated monocytes and increased overall prrsv susceptibility [62] . moreover, infected pigs develop a strong and rapid humoral response but these initial antibodies do not confer protection and can even be harmful by mediating an ade [12] . these antibodies enhance prrsv viral replication by coating the virus and enhancing the internalization of viral particles into macrophages [12] . as shown in figure 5g and 5h, igg and fcc receptors were significantly induced during n-prrsv infection processes. interestingly, the presence of antibodies during feline enteric coronaviruses (fecvs) infection does not also provide sterilizing immunity, instead, these antibodies opsonize virus particles and facilitate their entry to monocytes and/or macrophages through fcc receptors, resulting in disease enhancement [51] . the activation of pro-inflammatory transcription factor nf-kb induces robust activation of the casp1 inflammasome and subsequent release of il-1b that cause fever and inflammation [63, 64, 65, 66] . accordingly, we identified upregulation expression of casp1, nf-kb, and il-1b genes during n-prrsv infection processes ( figure 4a ). nf-kb activation also enhanced the expression of matrix metalloproteinases (mmp2) and mmp9, two cytotoxic substances in prrsv-infected cells [11, 67] . similarly, transcript abundance of mmp2 and ngal (25 kda alpha-2-microglobulin-related subunit of mmp-9) was significantly increased in the lungs of n-prrsv-infected pigs ( figure 5f ). upregulation expression of mmps would likely facilitate infiltration of inflammatory cells and increase inflammation. upregulation expression of il8 (also known as cxcl8), which is an attractant for neutrophils and other polymorphonuclear leukocytes produced after acute infection, in prrsv-infected pams [68, 69] and lungs of n-prrsv-infected pigs ( figure 5d ), was observed. other chemokines such as ccl2 (also known as mcp1 ), cxcl9, cxcl10 (also known as ip10), which were significantly increased ( figure 5d ), may be also crucial for lymphocyte and macrophage infiltration into the sites of n-prrsv infection. ccl2, il8 and ip10 expression were upregulated during sars-cov [70, 71] , and murine coronavirus [72] infections process, which may recruit monocytes and/or macrophages to sites of infection and be a major cause of lung pathology. although the present study indicates that upregulation expression of pro-inflammatory molecules contributes to the pathogenesis of n-prrsv, increased transcript abundance of anti-inflammatory molecules, such as il10, pge2, was also detected in the study. upregulation of il10 gene expression was found previously in prrsv-infected porcine leukocytes, pams, dcs, and in vivo in prrsv infected pigs [16, 17, 19, 73] . perhaps an increase in pro-inflammatory molecules followed by increased anti-inflammatory molecules is the normal progression of events in inflammation [74] . the upregulation expression of il10 might skew the immune response away from a protective th1-cell response towards a non-protective th2-cell response, therefore impairing clearance of virus, which benefits viral infections [51] . upregulation expression of anti-inflammatory molecules and proinflammatory molecules occurring concurrently was also observed after sars-cov and fipv infection [75, 76] . antibodies might also contribute to immunopathogenesis through increasing the uptake of virus by macrophages, resulting in activation of these macrophages and secretion of pro-inflammatory cytokines and chemokines. antigen-antibody complexes might increase transcript abundance of complement ( figure 5i ), which leads to generation of the classical inflammatory response through the production of potent proinflammatory molecules [77] . furthermore, complement activation might also contribute to the development of pulmonary edema and oedema of the eyelids. further understanding the roles complement plays in the hostpathogen interactions may help to develop more effective pharmacological agents against infection. moreover, damage to the lungs of n-prrsv-infected pigs seems to occur directly by viral destruction of alveolar and bronchial epithelial cells and macrophages ( figure 1c) , as well as indirectly through production of immune mediators. activated ctls and nk cells release perforin (pfr) and granzymes, which two effectors act collaboratively to induce apoptosis of target cells. transcript abundance of pfr1 and granzymes increased in the lungs of n-prrsv infected pigs ( figure 6h ). pro-apoptotic molecules xaf1, bid, cyto c, casp10, aifm2, were significantly up-regulated after infection with n-prrsv, which may induce apoptosis of virus-infected cells ( figure 6i ). simultaneously, we also observed upregulation expression of anti-apoptotic genes in n-prrsv infected lungs, including bcl2a1, mcl1, chfr, nfkb, adm, il10 etc ( figure 3d ). upregulation expression of anti-apoptotic genes and pro-apoptotic genes occurring concurrently after n-prrsv infection seems in contradiction of each other. however, this may reflect a balance between apoptotic and anti-apoptotic mechanisms. perhaps prrsv actively induces an anti-apoptotic state to complete its virus replication cycle through delaying cell death while induces apoptosis of virus-infected cells after completion of virus replication to increase rate of spread of virus [19, 78, 79] . anti-apoptotic and pro-apoptotic activaties were also observed in prrsv-infected marc-145 cells, in which prrsv stimulated anti-apoptotic pathways early in infection while caused apoptosis of prrsv-infected cells late in infection [80, 81] . infection with n-prrsv also increased transcript abundance of nfkbia ( figure 6i ), an inhibitor of the tnf receptor activated transcription factor nf-kb. loss of nf-kb activity has been shown to increase the cytotoxic effects of tnf which resulted in increased cell death [82] . an increase of transcript abundance in proapoptotic genes might result in disruption of the mitochondria transmembrane potential, thereby inducing release of cyto c from mitochondrial membranes to induce apoptosis and secondary necrosis [83] . the production of ros, especially superoxide radicals, and the subsequent oxidative damage of cells and tissues are recognized as key contributors to the viral pathogenesis [82, 84] . ros-mediated oxidative stress might also be involved in inducing apoptosis by prrsv [81] . accordingly, we identified the remarkable upregulation of cytochrome b245 heavy chain (gp91-phox) ( figure 6j ), a critical component of the membrane-bound oxidase of phagocytes (macrophages and neutrophils), after infection with n-prrsv, that generated superoxide radicals that killed both infected and normal cells at sites of infection, which would further exacerbate the immunopathological response. infection of macrophages, monocytes and dcs that are essential for immune function, is likely to be a key component in n-prrsv-induced pathogenesis [19, 85, 86, 87] . apoptosis of infected cells causes immune suppression by two mechanism: apoptosis either induces a decrease in the numbers of immune cells that compromises both the innate and adaptive immune response in which they are unable to eradicate the primary infection, or impairs immunity by inducing immunosuppressive effects in the surviving cells [88] . for example, uptake of apoptotic cells by normal macrophages and dcs stimulates immune tolerance by inducing the release of anti-inflammatory cytokines, such as il10, and suppressing the release of pro-inflammatory cytokines [89] . histopathological analysis of the lymphnodes of pigs infection with n-prrsv revealed a profound depletion of immune cells compared with those of unc (data not shown). in summary, the data presented in this study suggest that n-prrsv appears to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ade. after infection of macrophages and possibly dcs, prr-pamp interactions triggered signaling cascades that increased the transcript abundance of multiple inflammatory molecules, including cytokines, chemokines, adhesion molecules and inflammatory enzymes that induce a proinflammatory response, activate and recruit immune cells, such as macrophages and neutrophils, to sites of infection for virus elimination and thereby produce the clinical symptoms of viral infection, such as fever, dyspnoea, interstitial pneumonia in lungs. further, antibodies and complement activation might exacerbate inflammatory response. n-prrsv might induce an immunosuppressive state, mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. figure s4 signaling pathways of de genes. pathway analysis was mainly based on the kegg database. a p-value of ,0.05 and an fdr of ,0.05 in the two-side fisher's exact test were selected as the significant criteria. the vertical axis is the pathway category and the horizontal axis is the log10(p value) of these significant pathways. found at: doi:10.1371/journal.pone.0011377.s004 (0.66 mb tif) figure s5 genes that distributed in the known pig qtls of health traits. the x axis represents the qtl symbol, and the y axis indicates the number of genes associated with health traits. see table s4 for full qtl names. figure s7 biological process go terms of profile 1. functional classification of the de genes was performed according to go biological processes. a p-value of ,0.05 in the two-side fisher's exact test were selected as the significant criteria. these de genes were sorted by the enrichment of go categories. the vertical axis is the go category and the horizontal axis is the enrichment of go. found at: doi:10.1371/journal.pone.0011377.s007 (1.53 mb tif) figure s8 biological process go terms of profile 0. functional classification of the de genes was performed according to go biological processes. a p-value of ,0.05 in the two-side fisher's exact test were selected as the significant criteria. these de genes were sorted by the enrichment of go categories. the vertical axis is the go category and the horizontal axis is the enrichment of go. found at: doi:10.1371/journal.pone.0011377.s008 (2.78 mb tif) figure s9 biological process go terms of profile 6. functional classification of the de genes was performed according to go biological processes. a p-value of ,0.05 in the two-side fisher's exact test were selected as the significant criteria. these de genes were sorted by the enrichment of go categories. the vertical axis is the go category and the horizontal axis is the enrichment of go. epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview isolation and identification of porcine reproductory and respiratory 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influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals ifn-alpha treatment enhances porcine arterivirus infection of monocytes via upregulation of the porcine arterivirus receptor sialoadhesin differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability apoptosis and caspases regulate death and inflammation in sepsis death-defying immunity: do apoptotic cells influence antigen processing and presentation? we thank the beijing genomics institute (bgi) shenzhen and genminix informatics ltd.,co for their providing us with technical assistance in dge and bioinformatics analysis. key: cord-298181-ypgb7uuc authors: hendaus, mohamed a title: why are children with bronchiolitis at risk of urinary tract infections? date: 2019-11-14 journal: risk manag healthc policy doi: 10.2147/rmhp.s222470 sha: doc_id: 298181 cord_uid: ypgb7uuc viral respiratory infections are frequently eliminated from human bodies without any sequelae. secondary serious bacterial infection (sbi) in children with acute bronchiolitis has been an apprehension expressed by health care providers. several published studies have shown an association between acute bronchiolitis and secondary bacterial infection, including urinary tract infections (uti). however, the proposed mechanism by which a virus can induce utis is not yet known. the aim of this commentary is to update the current evidence of risk of uti in children with bronchiolitis. we present several clinical studies related to the topic as well as a brief review of the potential pathophysiology of secondary infections that could present with viral respiratory illness. viral respiratory infections are frequently eliminated from human bodies without any sequelae. nevertheless, in some occasions viruses can evade the immune reaction of the airways, leading to austere respiratory diseases. 1 potent mechanical and immunosuppressive methods protect the lungs against external infections, but a solitary respiratory tract infection can change immunity and pathology. 2 secondary serious bacterial infection (sbi) in children with acute bronchiolitis has been an apprehension expressed by health care providers. 3 several published studies have shown an association between acute bronchiolitis and secondary bacterial infection, including urinary tract infections (uti). [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] however, the proposed mechanism by which a virus can induce utis is not yet known. in a review of the literature, the percentage of patients with fever with positive urine cultures ranged from 4.2% to 20.0% in infants <3 months of age and 0% to 7.4% in older children (3 to 36 months of age). 14 ralston et al 3 conducted a systematic review delineating the risk of occult sbi in young febrile infants presenting with either "clinical bronchiolitis" or "proven rsv infection". the review included 11 studies. [4] [5] [6] [7] [9] [10] [11] [12] [13] 15, 16 the rate of urinary tract infections in the 11 studies analyzed was 3.3% (95% confidence interval, 1.9-5.7%). the authors concluded the rate of urine cultures positive for bacteria was noteworthy, though asymptomatic bacteriuria may have muddled the results. recently, mcdaniel et al 17 conducted a systematic review and meta-analysis exploring the prevalence of uti in infants and young children with bronchiolitis when positive urinalysis (ua) results being incorporated into the uti definition. the investigators included 18 studies, [4] [5] [6] [7] 9, [11] [12] [13] 15, 16, [18] [19] [20] [21] [22] [23] [24] [25] seven of which had ua information. 4, 11, 16, 18, [20] [21] [22] the definition of positive ua varied among the studies. some considered positive ua as having more than 5 white blood cells per high-powered field, while others considered the ua positive by having bacteriuria, positive leukocyte esterase, or positive nitrite. the prevalence of uti in bronchiolitis in the 18 studies was 3.1% (95% ci, 1.8-4.6%). the authors further analyzed the data of the 7 studies where the presence of pyuria or nitrites was a diagnostic criterion to define uti and the prevalence of uti was 0.8% (95% ci, 0.3-1.4%). however, the above studies did not sub-categorize the prevalence of uti in bronchiolitis per specific respiratory virus as a trigger. hendaus et al 8 studied the prevalence of urinary tract infection in infants and children with bronchiolitis. the study included 835 pediatric patients with acute bronchiolitis. the mean (±sd) age at diagnosis was 3.47±2.99 months. there were 325 (39%) girls and 510 (61%) boys. participants were divided into three groups: group 1 comprised of children hospitalized with bronchiolitis and a positive diagnosis for respiratory syncytial virus (rsv) bronchiolitis; group 2 comprised of children hospitalized with clinical bronchiolitis with no virus detected; and group 3 comprised of children hospitalized with clinical with bronchiolitis and a positive diagnosis respiratory virus other than rsv. after applying inclusion and exclusion criteria, rsv [3.4%] ). the definition of uti was adopted from the american academy of pediatrics as clinicians should require both urinalysis results that suggest infection (pyuria and/or bacteriuria) and the presence of ≥50,000 colony-forming units (cfus) per milliliter of a uropathogen cultured from a urine specimen obtained through catheterization or suprapubic aspirate. 26 the overall prevalence of uti was 10%, and was most common in group 3 (13.4%) trailed by group 2 (9.7%), and was least common in group 1 (6%) (p=0.030). the most reasonable explanation of why the rate of uti was higher in the study conducted by hendaus et al 8 was because it was the first published study that sub-categorized the prevalence of uti in bronchiolitis per specific respiratory virus as a trigger. the epithelium is usually protected by a layer of mucus that functions as a border. [27] [28] [29] viruses can inflict impairment on host epithelial cells, and mammalian cells are susceptible to bacterial attachment during a viral sickness. 30 moreover, viruses can incapacitate the mucociliary clearance arrangement, causing increased attachment of bacteria to mucins and colonization. 31 viruses like the influenza and rsv might injure ciliated cells, causing ciliostasis, and hence worsening of mucociliary clearance. 30, 32, 33 furthermore, virus-induced cell demise weakens the mechanical elimination of the close pathogens and exhibits new receptors for bacterial adherence. 34 the respiratory virus-infected epithelia enable the attraction of inflammatory cells, including natural killer cells, neutrophils, macrophages, and eosinophils from the bloodstream into the infected area. 35 the epithelium identifies microorganisms through pattern recognition receptors, such as nucleotide-binding oligomerization domain (nod)-like receptors (nlrs), retinoic acid-inducible gene (rig)-like helicases, 36, 37 and toll-like receptors (tlrs). 29 nlrs and rig-like helicases activate innate immune reactions through cytosolic detection of viral and bacterial components, 37, 38 while tlrs are single, noncatalytic, membrane-spanning receptor proteins utilized by the innate immune system. 39 post-viral continued desensitization of lung sentinel cells to tlr signals might contribute to secondary bacterial infection. for instance, tlr4 and tlr5 pathways are modified after influenza virus infection, leading to decreased neutrophil attraction, hence resulting in increased attachment of bacteria. 38 several epithelial cells can also express the classical antiviral interferons (infs), especially ifn-α and ifnβ. 40, 41 the link between host cells and microorganisms during sickness prompts immune reaction that comprise the generation of pro-inflammatory molecules. in spite of their important role as a bactericidal, pro-inflammatory cytokines such as tnf-α produced in response to infection could be injurious to the host cells. 42 viruses can also have an impact in modulating many molecules such as intercellular adhesion molecule 1 (icam-1), carcinoembryonic antigen-related cellular adhesion 1 (ceacam-1), and platelet-activating factor receptor (paf-r), 43 resulting in a risk of bacterial adherence. 44 hendaus dovepress throughout a viral episode, tlr and rig-i-like receptor activation prompts fabrication of type i ifns, which can then boost the inflammatory response to tlr ligands including lipopolysaccharide (lps). 45, 46 interface between type i ifns and nod1/nod2 signalling results in bacterial recognition, and causes damaging effects in the virally infected host. 47 conclusions and recommendations the airway epithelium: soldier in the fight against respiratory viruses influenza virus lung infection protects from respiratory syncytial virus-induced immunopathology occult serious bacterial infection in infants younger than 60 to 90 days with bronchiolitis: 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host defense function of the airway epithelium in health and disease: clinical background effects of rhinovirus infection on the adherence of streptococcus pneumoniae to cultured human airway epithelial cells viral infection causes rapid sensitization to lipopolysaccharide: central role of ifn-alpha beta a role for ifn-alpha beta in virus infection-induced sensitization to endotoxin viral infection augments nod1/2 signaling to potentiate lethality publication of this manuscript has been funded by qatar national library. the author reports no conflicts of interest in this work. key: cord-283779-mudwcypl authors: lauretani, fulvio; ravazzoni, giulia; roberti, maria federica; longobucco, yari; adorni, elisa; grossi, margherita; de iorio, aurelio; la porta, umberto; fazio, chiara; gallini, elena; federici, raffaele; salvi, marco; ciarrocchi, erika; rossi, francesca; bergamin, marina; bussolati, giacomo; grieco, ilaria; broccoli, federica; zucchini, irene; ielo, giuseppe; morganti, simonetta; artoni, andrea; arisi, arianna; tagliaferri, sara; maggio, marcello title: assessment and treatment of older individuals with covid-19 multi-system disease: clinical and ethical implications date: 2020-05-11 journal: acta biomed doi: 10.23750/abm.v91i2.9629 sha: doc_id: 283779 cord_uid: mudwcypl covid-19 infection is a multisystem disease more frequent in older individuals, especially in those with multiple chronic diseases. this multimorbid and frail population requires attention and a personalized comprehensive assessment in order to avoid the occurrence of adverse outcomes. as other diseases, the covid-19 presentation in older patients is often atypical with less severe and unspecific symptoms. these subjects both at home and during hospitalization suffer isolation and the lack of support of caregivers. the geriatric care in covid-19 wards is often missing. the application of additional instruments would be necessary to facilitate and personalize the clinical approach, not only based on diseases but also on functional status. this narrative review starts from diagnostic evaluation, continues with adapted pharmacologic treatment and ends with the recovery phase targeting the nutrition and physical exercise. we developed a check-list of respiratory, gastro-intestinal and other less-specific symptoms, summarized in a table and easily to be filled-up by patients, nurses and general practitioners. as second step, we reported the clinical phases of this disease. far to be considered just viral infective and respiratory, this disease is also an inflammatory and thrombotic condition with frequent bacterial over-infection. we finally considered timing and selection of treatment, which depend on the disease phase, co-administration of other drugs and require the monitoring of renal, liver and cardiac function. this underlines the role of age not just as a limitation, but also an opportunity to increase the quality and the appropriateness of multidisciplinary and multidimensional intervention in this population. (www.actabiomedica.it) these countries differ in terms of percentages of population over 65, the most afflicted by infection, with italy reaching 23%. italy for instance has higher life expectancy than the majority of countries affected by covid-19 infection (83.4 overall vs 76.7 in china) (2) . these demographic differences could also explain the different outcomes between countries. italy has one of the highest covid-19 mortality (26,644 deaths) and case-fatality rate (7.2%), much higher than china (2.3%). interestingly, the case-fatality rate in italy and china are very similar for age-groups 0 to 69 years, but rates are higher in italy especially among those aged 80 years or older (52% of deaths and 20% of case fatality rate). this difference can be at least partially explained by the higher number of people aged 80 years or older (n=11563), age group having a very high fatality rate (22.7%) and not reported in china (2) . gender issue has been raised by scientists and epidemiologists with men experiencing higher prevalence (64.2 % in the last italian report) and severity (they die more and at earlier age) of covid-19 infection than women. many hypotheses have been formulated to explain this difference between two sexes. cov-id-19 virus can be localized in the testes, which are potential target of sars-cov-2 infection, and one of the reasons for the rapidly spreading disease. moreover, testosterone, the male hormone, has been shown to upregulate the expression of transmembrane protease, serine 2 (tmrpss2) which is an enzyme involved in the penetration of virus in the lung cells. age is accompanied by changes in immune competence and a higher prevalence of inflammation, socalled "inflammaging" (3) . the chronic increase in inflammatory cytokines, augmented by covid-19 infection, may explain the higher tendency for "the cascade leading to pulmonary fibrosis and insufficiency and activation of clotting" and poorer clinical prognosis, especially in multimorbid older persons (4) . multimorbidity defined by the concomitant presence of two or more chronic diseases, is highly prevalent in older persons, affecting more than 60% of people aged 65+ (5) . data collected in 21.551 sars-cov-2 italian patients who died from covid19 show that the mean number of diseases is 3.2 (median sd 3 ±1.9). seventy patients (3.7 % of sample) had no diseases, 273 (14.4%) 1 disease, 400 (21.2%) 2 diseases, and 1147 (68.7%) 3 or more (2) . cardio-renalrespiratory (heart failure, atrial fibrillation, chronic renal failure, copd), metabolic diseases (obesity and type 2 diabetes), active cancer during the last 5 years and dementia seem to be the clusters more associated with adverse clinical outcomes. as a consequence of multimorbidity, polypharmacy defined as the number of drugs reported at hospital admission and the potential drug-drug interactions require a careful evaluation in older covid-19 patients. the combination of antiviral and anti-inflammatory drugs (never tested before in these individuals) and the concomitant treatment for other chronic diseases, especially in subjects with smoking exposition or sarcopenic obesity, increase the risk of adverse drug effects. diarrhea, dehydration, acute kidney insufficiency and liver failure can frequently occur and need to be monitored (6) . diseases, drugs and the primum movens cov-id-19 are also associated with hyperactive delirium, especially in hospitalized patients with preexisting dementia and cognitive impairment (7) . this syndrome requires a multidisciplinary evaluation balancing cost/effectiveness of therapeutic treatment (sedation or precipitation of respiratory and cardiac failure) and opens a large window of ethical issues, especially in older patients (8) . as suggested by nice rapid guideline and the canadian frailty network, the assessment of all adults for frailty, irrespective of age and covid-19 status, is highly recommended especially at hospital admission (6) . as already reported for other diseases, the cov-id-19 clinical presentation in older patients is often atypical with less severe symptoms. these subjects both at home and during hospitalization also suffer the isolation and the lack of fundamental support of formal and informal caregivers required for their safety (9) . despite the peculiar aspects of older patients and the epidemiology of the phenomenon, the geriatric culture and care in covid-19 wards is often missing. their application together with additional instruments would be such necessary to facilitate and personalize the clinical approach, not only based on number of diseases but also on functional status of older patient (10) . this narrative review has the specific aim to address different aspects of covid-19 multi-system disease starting from diagnostic evaluation, continuing with innovative classification of phases and proposing sequential adapted pharmacological treatment. the document wants also focus on the recovery phase and ethical considerations regarding the risk of limited access of care and accelerated exitus in this vulnerable age-category. the most common symptoms of covid-19 disease in the adults are represented in table 1 . this table describes a check-list of more frequent symptoms in adults and would be a guide to orient patients and primary care physicians in assessing older patients with suspected covid-19 infection. the range of symptoms is similar for covid-19 and influenza infection, although the fraction with severe disease is different. for covid-19, actual data suggest that 80% of infections are mild or asymptomatic, 15% are severe infections, requiring oxygen and 5% are critical infections, requiring ventilation. these fractions of severe and critical infection would be higher than influenza infection (11, 12) . symptoms can be traditionally classified into two main groups, including respiratory and gastro-intestinal, and a third group of less organ specific. the quality and severity of symptoms can be different in older persons. the most common symptoms are fever (98%), cough (77%), dyspnea (63.5%) and muscle/joint soreness (13) . the rationale of symptoms distribution across organs is partially explained by the concentration of angiotensin-converting enzyme 2 (ace-2) virus receptors, which is particularly higher in the lung and lower in the gut. this can explain why less common symptoms include abdominal pain, vomiting and diarrhea and virus might be detected in stool samples although gastro-intestinal transmission remains to be demonstrated (14) . it has been also hypothesized that covid-19 virus can also alter central nervous system directly or alternatively disrupt the gut-barrier permeability and induces the gut-brain link via vagus nerve. this justifies the reduced sense of taste and smell, headache, dizziness and vertigo also observed in covid-19 patients (15) . elderly patients, especially with multiple chronic conditions, display less severe and atypical symptoms. the presence of mild symptoms is disproportionate to the severity of their illness (16) . they might be afebrile, without cough or sputum production, and show higher prevalence of muscle-joint pain, tachypnea, altered mental status or delirium, unexplained tachycardia and decrease in blood pressure (17) . atypical presentation may be due to several factors, including physiologic changes with age, comorbidities, and inability to provide an accurate history given the constant lack of caregivers during covid-19 hospitalization (18) . despite the presence of less severe and atypical symptoms, older patients have a significantly higher mortality. as nicely shown in an elegant retrospective study male sex, time from disease onset to hospitalization, abnormal kidney function, and elevated procalcitonin levels were all significant predictors of increased mortality (14) . swab and or lung ct scan. the diagnosis of covid-19 requires the combination of swab and radiologic features. the algorithm initially considers a swab performed with sterile cotton wool suitably rolled around the end of a glass or metal rod, and intended to be swiped on the surface of a natural pharynx and nose cavity. the main nasal swab tests examine the nasopharynx, where the back of the nose meets the top of the throat. this requires a trained hand to perform and some portion of the false negatives arises from improper procedures and poor compliance especially in older adults with acute confusion state (19) . the pharyngeal and nasal swab, once carried out (in some centers not even getting out of the car but with the prior authorization and appointment of the public health office of the local healthcare companies) is sent to an authorized laboratory where the presence of viral rna or genetic material of the virus is appreciated. in case of positivity, there is the certification that the subject has a covid-19 infection. but even if done correctly, the swab may produce a negative result. that is because as the disease progresses, the virus passes from the upper to the lower respiratory system. importantly, the swab test has a sensitivity of 60-70% and strictly depends on the timing of assessment. this means that in 30-40% of cases, even in the case of a negative buffer, the presence of the virus cannot be excluded. in these cases, the patient may be asked to try to cough up sputum -mucus from the lower lungs -or doctors may need to take a sample more invasively when a patient is under sedation. radiological findings are useful complements in the diagnosis covid-19 and in the management of one of its most common complications, pneumonia. the most common computed tomography (ct) findings of the covid-19 pneumonia are ground glass and/or consolidation, and mainly reflect the diffuse and bilateral alveolar damage and/or organizing interstitial pneumonia. it has also been reported a strong correlation between the severity of ct pulmonary findings and patients' outcome. hence, it has been suggested that chest ct could be used as a reliable diagnostic test in the emergency workup of covid-19, complementing pcr. a further confirmation of the covid-19 infection comes from a chest x-ray or even better from a high-resolution chest ct scan (hrct) which highlights the percentage of lungs and the number of lung lobes affected by the virus. the radiologist, using specific software, processes a visual score or score in percentage. the higher the visual score the greater the severity of the lung involvement of virus. visual scores at the time of admission to the hospital of more than 70% are usually associated with a bad prognosis and more than 50% identify a severe disease. another parameter assessed through the ct scan is the number of lung lobes affected by the infection, which can vary from 0/5 to 5/5. also in this case, the greater the number of lung lobes involved, the greater the severity of the ongoing lung involvement of virus. the diagnostic process is the first step of clinical assessment of the patient. interestingly the initial hypothesis that the covid-19 is just an infectious disease has been gradually abandoned. an intriguing recent theory suggests that there are different phases in the same disease ( figure 1 ). the viral disease is limited to phase 1, where an early infection (8 days of duration) predominates and the host fights to solve the infection. however, if the attempt fails, the activation of an exaggerated response is capable to damage different tissues and organs (kidney, liver, myocardium, brain). another interesting theory suggests an early endothelial cell damage induced by covid-19 as common mechanism of vascular impairment across different organs (20) . three other phases (mainly depicting the host response to virus) are even more important for the clinical course and the outcomes of the patients. more effective will be the host response to virus, more chances the individuals have to survive. together with clinical evaluation (for instance peripheral capillary oxygen saturation), the functional assessment should also guide clinicians in the admission to intensive care unit (icu), in selecting therapeutic choices, and in predicting clinical and functional responses. both uk and canadian frameworks suggest the usefulness of easy to use instruments such as the clinical frailty scale to assess frailty (10) . other additional tests include chair stand test (cst) which is one of the best and validated physical performance tests for older people, and it is reported to be associated with muscle strength of the lower leg. the cst is a simple and feasible physical performance test, even for evaluating older people with limited mobility. then, many representative cohort studies have demonstrated that the cst is a predictor of disability and falls in older people (21) . (figure 1 and table 2 ) phase 1. infectious-virological phase or early infection phase 1 (max duration 8 days). the virus is present in the upper airways and digestive tract and usually induces specific symptoms (dry cough, fever, fatigue with normal peripheral oxygen saturation, diarrhea, headache, conjunctivitis) in the adult individuals (13) . the body response produces immune (igm in phase 1, viral response predominates and respiratory and gastrointestinal symptoms can be treated at home with hydroxychloroquine and antivirals. in phase 2: pulmonary, fever and dyspnea worsen and rapid diagnosis by ct and hospitalization is required. in phase 3, pulmonary and hyperinflammatory, clinically represented by ards, corticosteroids and il-6 receptor antagonists should be started in sub-intensive wards. in phase 4, thrombotic, anticoagulant therapy should be introduced and admission to icu indicated. there is a transverse phase: bacterial over infection, typically characterized by high fever, increased white blood cells and procalcitonin, where broad-spectrum antibiotic therapy is the choice treatment. legend of figure . both responses, especially if supported by appropriate pharmacological treatment, translate into an infective resolution in 80% of cases. the different response in older patients and in different categories (fit, frail, disable) is an interesting topic to be investigated (12) . the phase 1 treatment includes drugs with mixed anti-viral and anti-inflammatory activity (hydroxychloroquine) and antibacterial drug with minimal anti-viral action (azithromycin) (14, 17) . these drugs act synergistically on heart rhythm and require, because of the frequent concomitant use and especially in subjects with previous cardiac disease, electrocardiogram (ekg) trace to monitor qtc interval (22) . low molecular weight heparin, in the presence of good renal and liver function, at prophylaxis doses is also suggested (23) . in older patients, these specific aspects require additional and careful evaluation given the inadequate formulas currently used to assess for instance renal function (24) . in about 80% of cases, the disease ends at this stage and can be managed at home. however, the onset and persistence of symptoms within 2-3 days requires an immediate communication to primary care physician (or general practitioner) for a timely diagnosis and therapy. pharmacological treatment must be accompanied by the adoption of home behavioral measures in order to avoid contagion of the other family members. if the fever is persistently higher ≥38°c, especially for more than 3-4 days, or if peripheral oxygen saturation drops below 95% and/or dyspnea increases, we should suspect an exaggerated inflammatory response and the extension to the lung and recommend the hospitalization. the typical serum biomarker picture of this phase could be represented by low wbc, crp and d-dimer mildly increased, normal troponin i hs levels ( table 2) . this phase usually occurs after 10 days on average from the onset of symptoms in which the virus migrates to the lower respiratory tract lung. characterizing symptoms lasting 5 days or longer, range from shortness of breath to severe dyspnea and fatigue. this phase can be characterized by low peripheral oxygen saturation (spo2 <95%). endothelial and initial cardiac damage are also possible (25) . at this stage, hospitalization in semi-intensive wards could be necessary. acute confusional state in older persons is frequently observed and sedative and palliative treatment are important and detrimental confounders. men experience more clinical complications than women. this different exposure can be explained by higher expression pattern of ace2 receptors in adult human testes at the level of single-cell transcriptome suggesting that this organ is a potential target of sars-cov-2 infection, and one of the reasons for the rapidly spreading disease (26) . the typical serum biomarker picture of this phase could be represented by normal wbc, further increase in crp and d-dimer levels, troponin i hs levels that require to be monitored for the potential involvement of myocardium and pericardium ( table 2) . phase 3. pulmonary-hyper-inflammatory phase 3, which is characterized by systemic symptoms with multi-organ involvement (ards sirs/ shock cardiac failure) (27, 28) . individualized treatment in this phase is required, considering for example corticosteroids (methylprednisolone 1 mg/kg/day or dexamethasone at 20 mg/day intravenously), human immunoglobulin, inhibitors of the il-6, il-2, and jak2 receptor. this phase requires hospitalization in icus or respiratory intensive care unit (29) . the typical biomarker picture of this phase could reproduce phase 2 ( table 2) . phase 4. vasculitic-thrombotic phase 4 (coexisting or immediately following the previous phase) consists of endothelial damage, local and diffuse thrombotic phenomena and pulmonary hypertension (30) . there is the rationale, especially in this phase, to support, at high dosages, and based on weight and renal function, the use of enoxaparin, very known also for its antiviral activity (31, 32) . the presence of pulmonary hypertension suggests also the potential usefulness of phosphodiesterase inhibitors releasing nitric oxide such as sildenafil (33) . the typical serum biomarker picture of this phase could be represented by normal wbc, very-high levels of d-dimer and troponin i hs levels that require to be monitored for the potential occurrence of thrombotic events in different organs ( table 2) . the separation of different phases of disease contributes to delineate a specific timing for starting appropriate pharmacological treatment and establishing setting (home and hospital wards) at increased intensity of care. in case of persistent fever, higher than 37.5°c for a time longer than 3 days and peripheral oxygen level lower than 95% after starting therapy, we should consider and proceed to hospitalization especially in multimorbid older patients with cardiac, respiratory diseases and diabetes. the use of antivirals is poorly supported by randomized controlled clinical trials performed only in adult patients (34) and should be limited to the initial phase of the disease. antivirals are poorly indicated during phase 2 (35), and not indicated at all during phases 3 and 4. vice-versa, the anti-inflammatory-immunosuppressive therapy, are contraindicated during phases 1 and 2 in which the organism/host is elaborating or implementing its defensive strategy. corticosteroids and other anti-inflammatory medications should be also carriedout, once having careful evaluated specific contraindications, during phases 3 and 4, where the combination anti-inflammatory/ anticoagulant therapy is suggested in case of significant increase of d-dimer and/or positive pulmonary ct with contrast. late phases are usually characterized by exaggerated phase response of the host which is harmful to the host and needs to be attenuated (28, 36) . this might be particularly detrimental in older patients where a chronic inflammatory status is often present. every single phase of the pathology is also influenced by the undergoing pharmacological treatment and related side effects. drug-drug interaction deserves particular attention especially in older persons with polypharmacy. all these medications may induce gastro-intestinal symptoms (especially diarrhea) and worsen kidney and liver function. the ekg at the basal entry should be carried out on regular basis to monitor the qtc interval and to exclude the potential myocardial and pericardial damage induced by the infectious process. treatment in this phase, usually lasting about 7-8 days, consists of drugs with anti-inflammatory activities. these drugs, such as chloroquine or hydroxychloroquine should be started as soon as possible (37) . however, their utilization is actually based on in vitro data (38, 39) and single open label non-randomized trial conducted in 36 patients with covid-19 (40) . antiviral drugs derive their use from trials verifying their effective treatment of other viruses including sars (severe acute respiratory syndrome-related coronavirus) and mers (middle east respiratory syndrome coronavirus). in particular, preliminary genomic studies on 2019-ncov showed that the sequence has similarities with the corresponding sars and mers enzymes, and this justifies why repurposing exiting sars and mers inhibitors for 2019-ncov (14) . although the use of many anti-viral drugs has been proposed, particular attention received lopinavir/ ritonavir and remdesivir. the first antiviral drug, lopinavir/ritonavir, has specific indication for treat hiv and was also utilized in the 2003 for sars. convincing evidence of its therapeutic effects on covid-19 is lacking. moreover, a recent randomized clinical trial found no different clinical effect compared to standard care on 2019-ncov infection. only in the modified intention-to-treat analysis, which excluded three pa-tients with early death, the between-group difference in the median time to clinical improvement (median, 15 days vs. 16 days) was significant, albeit modest (34) . another virally targeted agent is the remdesivir, a very promising drug, which is a drug currently being investigated as a potential covid-19 treatment through several clinical trials. in details, two phase iii randomized, placebo-controlled double-blind, multicenter trials were initiated in early february to investigate remdesivir in two different dosages 200 mg/day and 100 mg/day for 9 days with estimated complete results at the end of april 2020 (41) . finally, the favipiravir, an antiviral drug manufactured by japanese pharmaceutical company fujifilm toyama chemical, was approved for treatment of novel influenza on february 15, 2020 in china, and clinical trials testing this medication are undergoing. preliminary data from 80 patients indicated that favipiravir had more potent antiviral effect than lopinavir/ ritonavir and even with lower side-effects (42) . however, given that no current definitive specific treatment for covid-19 infection has been proved based on randomized clinical trial, who has now launched the solidarity trial to investigate four potential treatments: remdesivir, chloroquine/hydroxychloroquine; lopinavir and ritonavir; and lopinavir and ritonavir plus interferon-β. 1 the only limitation of this study is that will not be double blind, but it will include thousands of patients from several countries (43) . this phase normally is associated in the adults with the presence of persistent high fever. this symptom often requires admission to emergency department and hospitalization for the execution of pulmonary ct scan. this technique is the gold standard for the diagnosis of typical interstitial pneumonia. the most important observation of this infection phase is the rapid progression into pulmonary impairment with a rapid worsening hypoxia. therefore, patients who failed to standard oxygen therapy required an advanced oxygen/ventilatory. patients may also have increased work of breathing, demanding positive pressure breathing assistance, which could be guaranteed by non-invasive ventila-tion (including continuous positive airway pressure [cpap] or bi-level positive airway pressure [bipap]) in patients with hypoxemic respiratory failure. prone ventilation in patients with persistent severe hypoxic failure should be considered. finally, patients who are acutely deteriorating undergo intubation and mechanical ventilation. two thirds of patients who required critical care in the uk had mechanical ventilation within 24 hours of admission (44) . in this phase, the presence of elevated serum levels of inflammatory cytokines, such as il-6 could induce pulmonary damage or proliferative pulmonary phase. il-6 receptor antagonists (e.g., tocilizumab, sarilumab, siltuximab) can be used. in particular, the tocilizumab which is a monoclonal antibody that blocks the il-6 signalling pathway is currently used to treat rheumatoid arthritis. however, given the limited evidence on the safety or efficacy of the drug in clinical treatment of covid-19, the fda launched through a double blind, a randomised phase iii clinical trial as a treatment for severe covid-19 pneumonia with tocilizumab in combination with standard of care (44) . acute respiratory distress syndrome (ards) is an acute, diffuse, inflammatory form of lung injury related with high mortality. diagnostic criteria (berlin definition 2012) include non-cardiogenic respiratory failure, with respiratory symptoms, bilateral opacities on ct scan and presence of a moderate to severe impairment of oxygenation (45, 46) . the pao2/fio2 defines the severity of the ards (calculated data with a positive end-expiratory pressure (peep) or continuous positive airway pressure (cpap) ≥5 cm h2o) in the absence of cardiac failure or fluid overload. • mild ards -pao2/fio2 is >200 mmhg, but ≤300 mmhg. • moderate ards -pao2/fio2 is >100 mmhg, but ≤200 mmhg. • severe ards -the pao2/fio2 is ≤100 mmhg (47) . excessive inflammatory response is an essential characteristic of ards pathophysiology, with an increase of interleukin-1 beta (il-1β), interleukin-2 (il-2), il-6, interleukin-17 (il-17), interleukin-8 (il-8), tumor necrosis factor-α (tnf-α) and c-c motif chemokine ligand 2 (ccl2) (48) . it is known that in patients with ards, elevated plasma il-6 at baseline predict a poor survival (28) . also in covid-19 patients, higher il-6 levels are associated with an increased risk of hospitalization and other negative outcomes (49) . at this stage of the disease, patients typically show dyspnea, tachypnea, fever and tachycardia. they can also show severe, acute confusion (especially in older persons), respiratory distress and cyanosis. as lung dysfunction progresses, it is necessary to increase oxygen-therapy until non-invasive mechanical ventilation is required (50, 51) . the use of corticosteroids could be beneficial to modulate the excessive immune response, but their use is controversial. a recent study shows that the use of corticosteroids in ards reduced all-cause mortality and duration of mechanical ventilation, and increased ventilator-free days (52) . in this regard, we hypothesized that patients already taking corticosteroids for other diseases, such asthma, pulmonary fibrosis, rheumatologic diseases and without indication for bacterial over-infections, can take advantage from adequate dosages of corticosteroids. however, future clinical trials are required to verify these aspects. in this phase, convalescent plasma from patients who have recovered from viral infections can be used as a treatment. clinical trials to determine the safety and efficacy of convalescent plasma that contains antibodies to sars-cov-2 in patients with covid-19 have started. a small preliminary case-series of five critically ill patients reported clinical improvement after convalescent plasma transfusions (53) . another study of 10 patients with severe illness in china noted symptomatic improvement within 3 days. viral load was undetectable within 7 days in 70% of patients. no serious adverse reaction was noted. covid-19 and ards can evolve into thrombotic phenomena. prolonged inflammation is responsible for a pro-coagulation state, with activation of the endothelial vasoconstrictors and formation of lung micro thrombi, also found during autoptic examination (52, 54, 55) . intriguingly, sars-cov-2 can directly infect engineered human blood vessel organoids in vitro. very recent case-series in patients with cov-id-19 have demonstrated an endothelial cell involvement across vascular beds of different organs especially in those with preexisting thrombotic disease (32, 56) . for all these reasons, a vasculitic/thrombotic phase can be hypothesized during covid ards. clinically, episodes of intense dyspnea and respiratory distress may occur. fever can be resolved. the pro-coagulant state is characterized by an increase in the d-dimer, which must therefore be regularly analyzed (28) . in details, if d-dimer level, normally performed every three days, increase more than 6 times from admission to later check, this parameter represents a good index for identifying high-risk groups of venous thromboembolism and anticoagulant treatment, if not contraindicated, should be prescribed (57) . respiratory distress syndrome (ards) is a common complication of covid-19 infection. ozoline and colleagues demonstrated that in patients with ards higher plasma concentrations of tissue factor and plasminogen activator inhibitor-1 were present at day seven compared to non-ards (58) . the mechanisms contributing to this lung coagulopathy are localized tissue factor-mediated thrombin generation, and depression of bronchoalveolar plasminogen activator-mediated fibrinolysis, mediated by the pai-1 increase (59) . thus, treatment with heparin might be helpful in mitigating this pulmonary coagulopathy. moreover, adjunctive treatment with low-molecular-weight heparin (lmwh) within the initial seven-day onset of ards reduces the risk of 7-day mortality by 48% with a meaningful improvement of the pao2/fio2 ratio (31) . in the same study, the risk of 28-day mortality was reduced by 37% as well. in a report from a wuhan university hospital, heparin use was associated with lower mortality in patients with sepsis-induced-coagulopathy (sic) score ≥4 (40.0% vs 64.2%, p=0.029), but not in those with sic score <4 (29.0% vs 22.6%, p=0.419). in the same report patients with d-dimer > 3.0 ug/ml experienced a 20% mortality reduction after heparin treatment (32.8% vs 52.4%, p=0.017) (57) . another fascinating concept is the antiviral role of heparin which has been studied in experimental models. given its polyanionic nature, heparin can bind to several proteins and thus act as effective inhibitors of viral attachment (60). one example is in herpes simplex virus infections. heparin competes with the virus for host cell surface glycoproteins inhibiting the virus entrance in the cells. also, in zika virus infection, it prevents virus-induced cell death (61) . finally, the use of heparin at a concentration of 100 μg/ml halved the infection in an experimental model of cells injected with sputum from a patient with sars-associated cov pneumonia (62) . however, the clinical benefits in any of these viral infections are yet to be determined. moreover, heparin may also be helpful in microvascular dysfunction and this is of importance given the well-known role of endothelial dysfunction in the cardiac failure, another increasingly recognized complication of covid-19. finally, a recent document of the italian national drugs agency (63) advices to consider the use of lmwh in serious cases of covid-19 (defined by the presence of one of the following conditions: pao2/fio2 < 300, respiratory rate > 30/min and spo2< 93% at rest) when the d-dimer is markedly increased (4-6 fold) and the sic score is > 4 ( table 2 ) and myocardial infarction or other thrombotic events cannot be excluded. however, high rate of high incidence of venous thromboembolic events may occur in severe covid-19 patients, irrespective of anticoagulation (64) . all previous phases of the covid-infection can be complicated by the presence of bacterial over-infection. this condition should be suspected when specific serum biomarkers such as wbc and procalcitonin are pathologically elevated (table 2) (65) . in this case, specific antibiotic therapy should be promptly prescribed, even in accordance with suggested guidelines (66) . polypharmacy is one of the main characteristics in older subjects. there is an increased risk of adverse events in this specific age-group. although there are no food and drug administration (fda)-approved drugs to prevent or treat covid-19, nevertheless pre-liminary clinical research, based on in vitro-data, have suggested the use of pharmacologic agents as chloroquine or hydroxychloroquine, azithromycin, lopinavir/ritonavir and other anti-retrovirals (67) . some of these drugs may increase risk of qt prolongation, ventricular proarrhythmia and sudden cardiac death. some of the current covid-19 repurposed drugs have known risk of us food and drug administration adverse event reporting system (faers), long qt syndrome and torsade de points (tdp) and cardiac arrest for azithromycin, and hydroxychloroquine, and possible risk for lopinavir/ritonavir. in the prevention of qtc-prolongation, special attention should go to high-risk patients. age is one of the main determinants of this risk score which has been derived and validated by tisdale et al. (68) , for prediction of drug-associated qt prolongation among cardiac-care-unit-hospitalized patients. the application of this scale identifies maximum risk score of 21 and three different classes of risk, low (score ≤ 6 points), moderate (7-10 points) and high (≥ 11 points) ( table 3 ) (69) . the goal of qtc screening in this setting is not to identify patients whom are not candidates for therapy, but to identify those who are at increased risk for tdp in order that aggressive countermeasures may be implemented. 1. baseline a. discontinue and avoid all other non-critical qt prolonging agents. b. assess a baseline ecg, renal function, hepatic function, serum potassium and serum magnesium. c. when possible, have an experienced cardiologist/ electrophysiologist measure qtc, and seek pharmacist input in the setting of acute renal or hepatic failure. 2. relative contraindications (subject to modification based on potential benefits of therapy) a. history of long qt syndrome, or b. baseline qtc >500 msec (or >530-550 msec in patients with qrs greater than >120 msec) 3. ongoing monitoring, dose adjustment and drug discontinuation a. place on telemetry prior to start of therapy. b. monitor and optimize serum potassium daily. c. acquire an ecg 2-3 hours after the second dose of hydroxychloroquine, and daily thereafter. d. if qtc increases by >60 msec or absolute qtc >500 msec (or >530-550 msec if qrs >120 msec), discontinue azithromycin (if used) and/ or reduce dose of hydroxychloroquine and repeat ecg daily. e. if qtc remains increased >60 msec and/or absolute qtc >500 msec (or >530-550 msec if qrs >120 msec), reevaluate the risk/benefit of ongoing therapy, consider consultation with an electrophysiologist, and consider discontinuation of hydroxychloroquine (70) . during covid-19 infection adult and older patients may also experience a higher incidence of gastrointestinal symptoms including diarrhea. the ongoing treatment with antivirals and anti-inflammatory could worsen this symptomatology, increasing potassium and magnesium deficiency and amplifying the risk already described of cardiac events and arrhythmia. in older patients it is widely observed the chronic, not always appropriate, use of proton pump inhibitors (ppi). one year ppi treatment has been associated with increased risk of all-cause mortality (71) . authors suggest that magnesium deficiency, clostridium difficile infection and intestinal colonization with multidrug-resistant microorganisms might justify the link between inappropriate use of ppi and mortality (72) (73) (74) . interestingly their chronic use has been associated with malnutrition and functional decline (75, 76) , two main aspects to be assessed and monitored in older patients with covid-19 infection. older age and the presence of multimorbidity are almost invariably associated with impaired nutritional status and sarcopenia (77) . some studies have demonstrated that hospitalization and associated bed rest even for short time-period (20 days) promote detrimental reduction in muscle mass, strength and physical function, with altered aerobic exercise capacity (78, 79) . covid-19 also amplifies these symptoms if we consider that muscle pain and fatigue are frequent symptoms also in older persons. the bed rest and high inflammatory and hypercatabolic status following covid-19 infection can promote a further reduction in walking speed, stair ascent power and chair stand test. these functional parameters, as well as the loss of strength, may compromise the recovery of functional skills in the elderly and induce the loss of autonomy. although albumin and prealbumin circulating levels should not be considered as nutritional markers in patients with acute inflammatory response, studies have shown an association between low prealbumin levels and increased risk of respiratory failure with increased need for mechanical ventilation (80) . all infected patients at hospital admission, especially those at nutritional risk should undergo nutritional assessment and receive nutritional support as early as possible. there is evidence that nutritional derangements should be systematically and urgently managed in patients affected by covid-19, also considering that the immune response is weakened by inadequate nutrition. nutritional intervention should be complementary to pharmacological treatment and the presence of a standardized protocol would be extremely helpful. for example, in italy, a nutritional protocol has been developed and proposed by university of milan and pavia in lumbardy which is one of the main italian regions affected by the italian covid-19 crisis (81) . this is based on systematic supplementation of certain nutrients (e.g. vit. d, whey proteins and omega 3 fatty acids) with anabolic and anti-inflammatory activity, oligo-elements stimulating immune system and particularly indicated in this high systemic inflammaheart failure 3 one qtc-prolonging drug 3 * a cut-off ≥ 7 can be used to assess moderate-severe risk. modified by reference 69. tory and catabolic condition. obesity can be considered a specific type of malnutrition, where the excess of macronutrients intake could also be accompanied by micronutrients deficiency (82) . the centers for disease control and prevention considers those with bmi ≥ 40 kg/m 2 as being at risk for flu complications. during the 2009 h1n1 pandemic, obesity was recognized as an independent risk factor for complications from influenza (80) . it is now well accepted that obesity increases one's risk of being hospitalized with, and dying from, an influenza virus infection, and it can be considered a predictor for poor outcome during covid-19 infections (83) . it has been reported that the presence of obesity in a group of metabolic associated fatty liver disease (mafld) patients was associated with a ~ 6-fold increased risk of severe covid-19 illness (unadjusted-or 5.77, 95% ci 1.19-27.91, p=.029). given the high prevalence of obesity and overweight in european countries (30-70%), the challenge for virus pandemics is therefore to protect these subjects (84) . although the effects of covid-19 on patients with obesity have not yet been well-described, it is well known the impact of h1n1 influenza the care of patients with obesity and with severe obesity, due to its adverse effect on pulmonary function (85) . the increased morbidity associated with obesity in covid-19 infections may be explained by increased inflammatory cytokines, other important determinants of severity infection include basal hormone milieu, defective response of both innate and adaptive immune system and sedentariness. it has been suggested by recent evidences that a large obese population increases the chance of appearance of more virulent viral strain, prolongs the virus shedding throughout the total population and eventually may increase overall mortality rate of an influenza pandemic (86) . finally, some authors outlined a framework whereby adipose tissue may be as a reservoir for more extensive viral spread with increased shedding, immune activation and cytokine amplification (83) . even, there are no specific studies on nutrition management in covid-19 infection, espen promotes considerations based on the best of knowledge and clinical experience. first, patients at risk for poor outcomes and higher mortality following infection with sars-cov-2, namely older adults and multimorbid individuals, should be checked for malnutrition through screening and assessment. criteria can be used are the must criteria or, for hospitalized patients, the nrs-2002 criteria. recently it has been introduced the glim (global leadership initiative on malnutrition) criteria for malnutrition diagnosis. obese individuals should be screened and investigated according to the same criteria, as they are malnourished. in a recent review about potential interventions for novel coronavirus based on the chinese experience authors suggested that the nutritional status of each infected patient should be evaluated before the administration of general treatments (87) . subjects with malnutrition should optimize their nutritional status, ideally by diet counseling from an experienced professional. macronutrients intake proposed by espen are the following. energy needs can be assessed or predicted by equations or weight-based formulae such as: • 27 kcal per kg body weight and day; total energy expenditure for polymorbid patients aged >65 years; • 30 kcal per kg body weight and day; total energy expenditure for severely underweight polymorbid patients*; • 30 kcal per kg body weight and day; guiding value for energy intake in older persons, this value should be individually adjusted with regard to nutritional status, physical activity level, disease status and tolerance. *the target of 30 kcal/kg body weight in severely underweight patients should be cautiously and slowly achieved, as this is a population at high risk of refeeding syndrome. protein needs are usually estimated using formulae such as: • 1 g protein per kg body weight and day in older persons; the amount should be individually adjusted with regard to nutritional status, physical activity level, disease status and tolerance. • ≥ 1 g protein per kg body weight and day in polymorbid medical inpatients in order to prevent body weight loss, reduce the risk of complications and hospital readmission and improve functional outcome. fat and carbohydrate needs are adapted to the energy needs while considering an energy ratio from fat and carbohydrates between 30:70 (subjects with no respiratory deficiency) to 50:50 (ventilated patients) percent. also micronutrients, such as vitamins and minerals, should be ensured to potentially reduce disease negative impact, by supplementation and/or adequate provision. low levels or intakes of micronutrients such as vitamins a, e, b6 and b12, zn and se have been associated with adverse clinical outcomes during viral infections (86) . recently, a chinese review (88) proposed that also vitamin c, omega-3 polyunsaturated fatty acids, as well as selenium, zinc and iron should be considered in the assessment of micronutrients in covid-19 patients. oral nutritional supplements (ons) should be used whenever possible to meet patient's needs, when dietary counseling is not sufficient to reach nutritional goals. individuals infected with sars-cov-2 outside of the icu should therefore be treated to prevent or improve malnutrition. the oral route is always preferred when practicable. nutritional treatment should start early during hospitalization (within 24-48 h) and targets should be met gradually to prevent refeeding syndrome. ons provide energy-dense alternatives to regular meals and may be specifically enriched to meet targets in terms of protein as well as micronutrients (vitamins and trace elements). the daily estimated requirements of these nutrients should be regularly provided. nutritional treatment should continue after hospital discharge with ons and individualized nutritional plans; this is particularly important since preexisting nutritional risk factors continue to apply and acute disease and hospitalization are likely to worsen the risk or condition of malnutrition. according to espen statements, in multimorbid inpatients and in older persons with reasonable prognosis, when nutritional requirements cannot be met by the oral route, enteral nutrition (en) should be preferred to parenteral nutrition (pn), because of a lower risk of complications (related or not related to infectious). pn should not be started until all strategies to maximize en tolerance have been attempted. about the nutritional management of covid-19 patients admitted to intensive care units, espen guidelines on this specific topic are available giving suggestions on different stages of treatment according to patients' condition and respiration. infected patients not intubated who do not reach nutritional requirements by normal diet, first should be supplemented by ons, then en treatment can be considered. when limitations are present to en, pn can be prescribed. in covid-19 intubated and ventilated icu patients, enteral nutrition (en) should be started through a nasogastric tube; post-pyloric feeding should be performed in patients with gastric intolerance after prokinetic treatment or in patients at high-risk for aspiration; the prone position per se does not represent a limitation or contraindication for en. patients' energy expenditure can be derived from ventilator (vo2, oxygen consumption from pulmonary arterial catheter or vco2, carbon dioxide production), and energy is administered according to its value. hypocaloric nutrition (not exceeding 70% of ee) should be administered in the early phase of acute illness with increments up to 80 and 100% after day 3. regarding protein intake, 1.3 g/kg protein equivalents per day can be delivered progressively. in obese subjects, 1.3 g/ kg "adjusted body weight" protein equivalents per day is recommended. adjusted body weight is calculated as ideal body weight + (actual body weight -ideal body weight) * 0.33. after mechanical ventilation, patients may present swallowing difficulties and texture-adapted food can be considered after extubation. if swallowing is proven unsafe, en should be administered. in cases with a very high aspiration risk, post-pyloric en or, if not possible, temporary pn during swallowing training with removed naso-enteral tube can be performed. hydration status of patients should be considered and assessed after the acute and critical phases. high grade of inflammation and infectious status with long lasting fever period may cause dehydration which needs to be treated before discharge. furthermore, some patients with covid-19 show intestinal disease, thus nutritional and gastrointestinal function should be assessed for all patients. some authors suggest that nutritional support and application of prebiotics or probiotics should be suggested to regulate the balance of intestinal microbiota and reduce the risk of secondary infection due to bacterial translocation (89) . almost no information is available on metabolic and nutritional needs of icu survivors, and known nutritional practices reveal a poor nutritional performance during icu stay and after discharge. a few evidences showed that currently poor nutritional practices are adopted for older patients who leave the icu in the ward, and further research are needed to fill the gap. following hospital discharge, especially patients should comply with high-protein targets either by prolonged tube feeding or by enhanced high-protein oral nutrition (supplement) intake. further, nutritional and metabolic therapies such as anabolic/anti-catabolic agents in the recovery need urgent studies (90) . nutritional intervention should be combined (whether possible) with physical exercise in order to optimize its anabolic effect (91) . different phases and week programs could be also followed with the specific aim of recovering physical and motor skills (table 4) . phase 1. recover of orthostatism. once the acute phase has been resolved, the multidomain intervention should include exercise and target the recovery of orthostatic and motor skills. it would be important progressively increase the anti-gravity position starting from the sitting position on the bed with slow exercises and movements to be repeated several times a day, until the complete recovery of the upright position. phase 2. train balance and coordination of movements. following this first phase, static and dynamic balance exercise should be performed for improving balance impairment. holding on the back of a chair, stand on tiptoe and then return to the starting position, or keep the balance in monopodalic support. phase 3. regain muscle strength. low intensity muscle strengthening exercises might be useful for recovering strength and functional autonomy, improving stability, balance and reducing the risk of falls. for example, sitting back on a chair, slowly raise left leg until it is fully extended, pause for a breath, then slowly lower left leg back to the ground. this sequence should be repeated 10 times both sides. phase 4. start endurance training. aerobic exercise, like walking inside the house or stationary bike, can be started after the regaining of motor skills and strength, initially 10 minutes of activity then up to 20 minutes. maintenance: individual multicomponent exercise program. at the end of the total recovery, a multicomponent exercise program can include aerobic, resistance, balance, coordination and mobility training exercises (92) . twenty minutes of aerobic exercise every day and three days a week of resistance exercises at low and medium intensity should be the ideal choice for older people to enhance the protective role of physical activity (93) (94) (95) . the pathophysiology of the covid-19 infection especially in older adults requires a dynamic process with important clinical and ethical implications in the hospital and community care. now it is quite clear that the infection produces a systemic disease with different phases at increasing severity of symptoms. older patients infected by covid-19 often experience atypical and less severe symptoms in older persons, side-effects of the drugs and require specific nutritional and motor treatment for avoiding disability and death. by expanding the proposal of hasan k et al. (96) , we added to the already known infective, pulmonary and inflammatory, a potential iv phase for emphasizing the presence of a vascular-thrombotic process more frequent during the severe pulmonary disease. we also underlined the bacterial over-infection, which can be transversally present in all phases and requires the need of antibiotic treatment. as addressed by italian ethics committee it is ethically unacceptable, each selective care criterium based on «age, gender, condition and social role……and disability». these principles have been often ignored, especially in older covid-19 patients. examples reported from the sociologist giuseppe de rita and coming from uk or holland, describe that patients 70 year or older are invited to sign a declaration where they refuse to be cured if another younger patient requires the same treatment. a statement signed on march 23rd 2020 by the european geriatric medicine society (eugms) (97) suggests that advanced age should not by itself be a criterion for excluding patients from specialized hospital units and care. simplified models of comprehensive geriatric assessment and tailored interventions (including evaluation of frailty, hydration and nutritional with body mass index and cst, social and psychological support, management of polypharmacy) are mandatory to guide appropriate clinical approaches, especially if older subjects are really fit, without any cognitive and motoric dysfunction, and to improve the patient's quality of life (98) . these principles should be applied to every setting of care including community/primary care, hospital and nursing home placement. innovative organizing multidisciplinary models are especially important during the transition care and coronavirus outbreak, because older people might experience an understandable slowing down of physi-cal and mental capacities in the discharge from acute care with prolonged hospital stays and increased risk of iatrogenic consequences. all the necessary 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as therapy to fight against the mental and physical consequences of covid-19 quarantine: special focus in older people a tale of two pandemics: how will covid-19 and global trends in physical inactivity and sedentary behavior affect one another? considerations for obesity, vitamin d, and physical activity amidst the cov-id-19 pandemic physical activity for immunity protection: inoculating populations with healthy living medicine in preparation for the next pandemic covid-19 illness in native and immunosuppressed states: a clinical-therapeutic staging proposal state-ment of the eugms executive board on the covid-19 epidemic prevalence, incidence, and clinical impact of cognitive-motoric risk syndrome in europe, usa, and japan: facts and numbers update 2019 we deeply appreciated our patients and families for their patience and kindness and our nurse coordinator rosetta castellino and her staff for the excellent work and the strong support during covid 19 epidemic. each author declares that he or she has no commercial associations (e.g. consultancies, stock ownership, equity interest, patent/licensing arrangement etc.) that might pose a conflict of interest in connection with the submitted article key: cord-300965-ivczo1a7 authors: brown, m. m. title: don’t be the “fifth guy”: risk, responsibility, and the rhetoric of handwashing campaigns date: 2017-08-29 journal: j med humanit doi: 10.1007/s10912-017-9470-4 sha: doc_id: 300965 cord_uid: ivczo1a7 in recent years, outbreaks such as h1n1 have prompted heightened efforts to manage the risk of infection. these efforts often involve the endorsement of personal responsibility for infection risk, thus reinforcing an individualistic model of public health. some scholars—for example, peterson and lupton (1996)—term this model the “new public health.” in this essay, i describe how the focus on personal responsibility for infection risk shapes the promotion of hand hygiene and other forms of illness etiquette. my analysis underscores the use of constitutive and stigmatizing rhetoric to depict individual bodies, rather than environments, as prime sources of infection. common among workplaces, this rhetoric provides the impetus for encouraging individual behavior change as a hedge against infection risk. i argue, though, that the mandating of personal responsibility for infection risk galvanizes a culture of stigma and blame that may work against the aims of public health. signal that a pandemic [was] imminent.^so, the 100th day's headlining issues of economic recovery, job creation, and the wars in afghanistan and iraq came second to obama's discussion of h1n1, the pandemic strain of the influenza virus. obama opened his remarks, for example, by outlining the steps that his government had taken to protect the american people from the devastations of outbreak. 1 measures adopted to fend off h1n1 included carefully monitoring the spread of the novel strain and stockpiling medical supplies and drug treatments. on the advice of public health experts, the u.s. government had also considered closing public schools in response to suspected or confirmed cases. obama also urged parents and employers to develop contingency plans if the spread of h1n1 led to massive workplace and school closures. band finally,^obama continued, bi've asked every american to take the same steps you would to prevent any other flu: keep your hands washed, cover your mouth when you cough, stay home from work if you're sick, and keep your children home from school if they're sick.^adopting these various forms of illness etiquette, obama implied, demonstrated one's assumption of personal responsibility in response to the heightened risk of infection. this essay contributes to the ongoing examination among rhetoricians of health and medicine of constructions of responsibility and risk. 2 specifically, i explore the centrality of a rhetoric of personal responsibility to discursive efforts to manage infection risk. health humanities scholars and rhetoricians of health and medicine share a common concern for the ethical issues that arise from risk-management exercises undertaken in the name of public health. 3 practitioners, too, have a stake in more detailed understandings of the impact of risk discourse on the formation of health subjectivity. however, as keränen (2014b) explains in the health humanities reader, ba rhetorical perspective focuses on how specific symbolic patterns structure meaning and action^in health and public-health contexts (37). writing for this journal, for example, ding (2014b) stresses the economic and sociocultural effects of media portrayals of bat risk^populations during severe acute respiratory syndrome (sars). 4 my essay contributes to this scholarship with an account of how messaging that seeks to engage publics in outbreak management shapes their perceptions of responsibility and risk, not to mention of public health. as i argue, handwashing campaigns reinforce an individualistic model of public health, one premised to a significant extent on the necessity of behavior change rather than structural intervention. some scholars-for example, peterson and lupton (1996) -term this model the bnew public health.p ersonal responsibility serves essential functions in response to the threat of infection. however, the mandating of personal responsibility for infection risk has the potential to galvanize a culture of stigma and blame. too narrow a focus on personal responsibility may also diminish perceptions of the effectiveness of improved structural supports for those infected. during h1n1, for example, universal paid sick leave became a topic of debate, serving as a reminder of the need for an environment supportive of individual efforts to manage infection risk. in this essay, i characterize hand hygiene promotion as both a bconstitutive rhetoric^and a bstigmatizing rhetoric.^whereas a constitutive rhetoric encourages action through the cultivation of subjectivity, a stigmatizing rhetoric uses stigma to shape perceptions-also typically for the sake of influencing behavior change. i also describe how scholars of rhetoric of health and medicine have employed these two theories and explain their value to health humanities practitioners and scholars. i then examine the uses of constitutive and stigmatizing rhetoric in a u.s. state-level campaign to enforce bhygienic norms^(including, importantly, hand hygiene) within the workplace. my analysis reveals the centrality of stigma and blame to these efforts to encourage the assumption of personal responsibility. research on hand hygiene promotion finds that handwashing campaigns have a proven impact on health behaviors and thus, by extension, on health outcomes. so, why might those of us who have been exposed to these campaigns concern ourselves, perhaps unnecessarily, with their implications for our views of risk, responsibility, and public health? the reason i turn to in my conclusion is that the rhetorical means used to encourage personal responsibility may obscure perceptions of more effective approaches to the management of infection risk. handwashing campaigns also create opportunities to profit from and even exploit the stigma and blame that these texts associate with failures of personal responsibility. my goal, then, is mainly to explore the limits of personal responsibility-not just as an approach to infection risk, but more generally as a cornerstone of twenty-first-century public health. personal responsibility may be a cornerstone of public health, but hand hygiene promotion is an especially persuasive vehicle for popularizing an individualistic conception of infection risk. by bhand hygiene promotion,^i mean efforts to instruct a broad, lay public in hygiene practices typically used to reduce the transmission of disease-causing pathogens in hospitals and clinics. in this essay, i use bhandwashing campaigns^and bhand hygiene promotionî nterchangeably to describe the discursive encouragement of this habit. i also focus mainly on hand hygiene promotion within north america, where amid h1n1 handwashing campaigns and hand hygiene products alike became endemic. commenting on this trend in a new yorker essay, owen (2013) links the phenomenal success of gojo industry's blockbuster hand sanitizer, purell, to anxieties about infection risk. 5 today, hand sanitizer is a product category in its own right, and its popularity is sometimes regarded critically as both indicative of and responsible for a distinct shift in cultural perceptions of infection risk. 6 in my view, however, purell's unprecedented sales figures 7 are inextricably tied both to the increased promotion of hand hygiene in recent decades and to the ongoing individualization of public health. a drawback of undertaking a critique of hand hygiene is appearing to be against hand washing and other expressions of illness etiquette. hand hygiene is a vital form of infection control, and as such, it is also an ethical practice, particularly during an outbreak. rather than argue against hand hygiene, i explore the limits of hand hygiene promotion, as well as its implication in the deepening entrenchment of the new public health. in this respect, my essay draws its inspiration from the work of metzl, who in the introduction to his co-edited multidisciplinary anthology against health, writes that health is a bdesired state, but it is also a prescribed state and an ideological position^(2010, 2). the same argument applies to public health, which broadly speaking entails the strategic, organized effort to bpersuade a defined public to engage in behaviors that that will improve health or refrain from behaviors that are unhealthy^ (springston 2013, 713) . hand hygiene promotion especially invites further scrutiny because its prescriptive, ideological qualities far too often go unnoticed. hence, i focus my attention here on describing how handwashing campaigns benefit the overarching emphasis on personal responsibility for infection risk. an important precedent for my critique is plyushteva's analysis (2009) . 8 plyushteva examines the promotion of hand hygiene in developing countries, which she sees as having applications beyond the potential reduction high mortality rates due to infection. in fact, just as in north america, hand hygiene promotion directed at publics in developing countries aims to empower these publics to protect themselves from the risk of infection. since 2008, for example, global handwashing day has been celebrated annually on october 5. an initiative of the global public-private partnership for handwashing with soap (global ppphw), global handwashing day is bdedicated to increasing awareness and understanding about the importance of handwashing with soap as an effective and affordable way to prevent diseases and save lives.^the celebration also presents ban opportunity to . . . encourage people to wash their hands^-or, as explained in a global handwashing day press release, to inspire personal responsibility. 9 in developing countries, hand hygiene promotion's emphasis on personal responsibility may affect perceptions of entitlement to care. indeed, global handwashing day presents infection risk as managed not through the provision of clean water or proper sanitation but rather through the adoption of appropriate personal measures. underwritten by an array of corporate sponsors, global handwashing day also teaches people living in developing countries to become faithful consumers of hand sanitizer and soap, just like their counterparts in developed countries. current sponsors include colgate-palmolive, procter and gamble, and unilever, all companies with a massive stake in the global marketplace for personal hygiene products. (corporate sponsors may also have influenced the naming of the global public-private partnership for handwashing with soap. even the scholarship produced by the researchers working for this partnership typically includes this addendum.) global handwashing day's instruction in the consumption of personal hygiene products, too, has ties to the overarching emphasis on personal responsibility that defines the new public health. hand hygiene is promoted as a bdo-it-yourself vaccine,^a hedge against infection risk (apparently) even in settings in which infection risk often stems from poor sanitation and lack of access to clean water. 10 of course, regardless of context, hygiene habits have a proven impact on the transmission of disease-causing pathogens. hand hygiene limits the spread of diarrheal and respiratory diseases, which are among the leading causes of child mortality in developing countries. 11 children thus comprise a key audience for global handwashing day, which seeks to transform them into bchange agents^who have the capacity to bpositively influence other people's health behaviours^ (global ppphw 2015) . however, as plyushteva observes, global handwashing day's celebration of the life-saving power of individual behavior change potentially obscures understandings of the structural factors that shape infection risk. in developing countries, for example, the spread of disease stems from lack of access to clean water and adequate waste disposal and not mainly from a lack of agency per se. in tying infection risk to the bsuboptimal behaviour of the poor^(2009, 428), handwashing campaigns in developing countries exacerbate longstanding power imbalances, potentially reinforcing rather than removing obstacles to meaningful change. at the same time, hand hygiene promotion in this context expands the global marketplace for personal hygiene products, forging new opportunities to profit from the intractable problem of infectious disease. plyushteva's analysis is helpful to my own because she draws attention to hand hygiene promotion's insidiousness and stresses its consequent potential to serve a range of motivations. some of these motivations in fact conflict with the aims of public health, particularly in developing countries. bat first glance,^she argues, bthe cause of handwashing appears as apolitical and uncontroversial as can be^(428). so unproblematic is hand hygiene, and so important are efforts to promote it, that the very few criticisms of global handwashing day have largely been ignored. 12 for her part, plyushteva takes issue with the celebration's stigmatizing of people in developing countries bas traditional or backward, or, in a teleological view of development, pre-modern^(424). hand hygiene's buncontroversial façade^(420) also obscures the reality that individual behavior change is only ever a bpartial solution^(429) to the spread of disease. efforts to quell the spread of disease through behavior change also depend on the implementation of structural interventions-changes that create an environment supportive of personal responsibility. (i return to these limitations of personal responsibility in my conclusion.) i quote plyushteva at length because hers is the most recent scholarly critique of contemporary, globalized efforts to promote hand hygiene promotion. 13 her writing establishes a precedent for my critique of north american handwashing campaigns, which may also do more, politically and economically, than simply diminish the risk of infection. circulated within workplaces, schools, transit hubs, airports, community centers, groceries stores, and shopping malls, handwashing campaigns portray individual bodies, and body parts, as dangerous vectors of infectious disease. what makes these bodies, and parts, dangerous is both that they spread infection and because the disease-causing pathogens they transmit remain invisible to the individuals who transmit them. as a caption for a handwashing poster created by yale university's emergency management department in 2009, in response to h1n1, puts it, byou've got a mystery on your hands.t aking the form of pamphlets, posters, transit ads, web infographics, social media campaigns, and public service announcements, these texts caution that the power to prevent (and spread) infection is in our hands. sales figures for hand sanitizer alone illustrate the impressive new revenue streams generated by this individualization of infection risk. even in developed countries, where the assumption of personal responsibility is less likely to be impeded by structural issues, hand hygiene promotion may nevertheless skew perceptions of contextual or social determinants of infection risk. most notable among these factors may be the availability of sick leave or the effects on susceptibility of feelings of anxiety or stress. hand hygiene promotion invariably serves two distinct purposes. at one level, as exercises in risk communication, handwashing campaigns satisfy the obligation to inform publics about how to diminish the risk of infection. the most effective display of hand hygiene promotion's function as a form of risk communication may be the infographics, often posted in public restrooms, that illustrate the handwashing procedures practiced by healthcare professionals. these infographics teach handwashing methods, but they also serve to emphasize the need for personal responsibility in public settings. indeed, at another level, many handwashing campaigns often serve more expressly rhetorical goals. the most effective-and the most problematic-is the use of hand hygiene promotion to exacerbate a whole host of negative emotions, from anxiety, distrust, fear, and doubt to nausea and disgust. some of the most prominent voices behind the turn to hand hygiene promotion, particularly in developed countries, have emphatically defended the rhetorical utility of public health campaigns that inspire feelings of disgust. 14 in my close reading, i focus more on this latter function of handwashing campaign-that is, its use to foster emotional states that predispose audiences to the adoption of personal responsibility. hand hygiene promotion's alignment with an axiom of neoliberalism-the emphasis on personal responsibility-is also worthy of further examination. harvey describes the typical characteristics of the neoliberal state and explains, the bsocial safety net is reduced to a bare minimum in favour of a system that emphasizes personal responsibility. personal failure is generally attributed to personal failings, and the victim is all too often blamed^ (2005, 76) . harvey's account stresses the economic advantages of the neoliberal emphasis on personal responsibility. indeed, a neoliberal approach to infection risk has both shifted attention away from costlier programs of outbreak management and accorded private stakeholders unparalleled economic advantages. arguably, the main benefactors of personal responsibility for infection risk are the corporations that develop and distribute products in support of illness etiquette. yet the recent popularity of hand sanitizer does more than reflect the successful marketing of hand hygiene as an antidote to both uncertainty and infection. rather, this shift in consumptive patterns also illustrates the tremendous impact of handwashing campaigns on a risk-oriented subjectivity. alongside promoting a habit that may reduce the transmission of disease, handwashing texts heighten awareness of those who fail in their duty to limit the spread of infection. noncompliance with the dictates of hand hygiene promotion becomes grounds not merely for blame but also for suspicion about a person's moral worth. contemporary handwashing campaigns thus form a constitutive rhetoric, a mode of rhetorical appeal that calls into existence a shared collective identity. within hand hygiene promotion, the collectivity identity called into existence is that of the health citizen for whom participation in containing an outbreak is a personal responsibility. 15 white describes bconstitutive rhetoric^(a term that he coined) as bthe central art by which culture and community are established, maintained, and transformed^ (1985, 28) . scholars use constitutive rhetoric to explain the discursive formation of new social and political subjectivities. in demonstrating how some rhetorics discursively constitute the very subjects they address, many critics follow charland's model of constitutive rhetoric (1987) . 16 into white's theory, charland incorporates burke's notion of identification (1969) and althusser's idea of interpellation, or bhailing^(1971). as charland observes, constitutive appeals produce and reinforce new subject positions (1987) . by responding to these appeals, individuals affirm their membership in the community. constitutive rhetoric has been a useful analysis for health humanities scholars and practitioners. 17 anthropologist joseph dumit, for example, argues that strategies employed in pharmaceutical discourse create new opportunities for marketing drugs by constituting the individual as a body at risk of disease (2012). the strategic constitution of bodily risk, dumit argues, is essential to keeping americans on bdrugs for life.^scholars of rhetoric of health and medicine have employed constitutive rhetoric to critique the interpellation of headache patients as well as of patients as narrative subjects (segal 2005 ). derkatch has used constitutive rhetoric to account for the maintenance of professional boundaries in medicine (2012), whereas kopelson has shown in response to breast cancer, public health organizations mobilize citizens as consumer-activists (2013). majdik and platt describe the health subject constituted by the marketing campaign for a genetic testing product (2012). interpellation has also been a productive means for scholars to describe how public health officials shape perceptions of risk and responsibility in response to outbreak (briggs 2004; davis, stephenson, and flowers 2011) . hand hygiene promotion presents an opportunity to examine the constitutive functions of efforts to foster personal responsibility for infection risk. handwashing campaigns transform perceptions of responsibility for disease outbreaks. they do so by situating the risk of infection in individual bodies. the adoption of illness etiquette in response to hand hygiene promotion thus signals at least a partial acceptance of the new public health. 18 because it singles out the individual bodies-and individual body partsthat spread infection, hand hygiene promotion might be understood as both a constitutive rhetoric and a bstigmatizing rhetoric.^proposed by metzl in against health (2010) and premised on the writings of goffman ([1963] 1986), a stigmatizing rhetoric derives its conception of the bhealthy^from portrayals of the bunhealthy.^in other words, notions of poor health shore up understandings of good health. as metzl asserts, within a stigmatizing rhetoric, the baffirmation of one's own health depends on the constant recognition, and indeed the creation, of the spoiled health of others^ (2010, 5) . taking up metzl's refrain, some of the contributors to the multidisciplinary anthology, against health, critique the centrality of stigmatizing rhetoric to a neoliberal model of public health. lebesco, for example, argues that u.s. anti-obesity campaigns reinforce the valuing of bgood citizens [who] take care of their own health^ (2010, 78) at the expense of those classified as overweight or obese. handwashing campaigns potentially display such a stigmatizing rhetoric whenever their promotion of hand hygiene casts it as a prosocial behavior rather than as merely a method of infection control. according to these stigmatizing texts, the failure to observe hand hygiene has profound consequences in addition to the potential for infection. created by the florida department of health in response to h1n1, the bfifth guy^campaign illustrates the use of a constitutive, stigmatizing rhetoric to endorse the assumption of personal responsibility for infection risk. i chose this campaign both because of its focus on the workplace and because its messages about risk and responsibility later saw replication in other states (for example, by the michigan department of health). 19 the fifth guy campaign includes an interactive website that hosts a series of public service announcements (psas). together, these psas underscore the need for personal responsibility by dramatizing the tensions that arise when someone in the workplace ignores his duty to limit the spread of infection. underlying the fifth guy, as i argue, is the message that infection risk is exacerbated mainly by the failure to assume personal responsibility. my close reading of the fifth guy also reveals an emphasis on feelings of anxiety, fear, and even self-doubt. as a stigmatizing rhetoric, the fifth guy foregrounds these negative emotions to shore up the value of personal responsibility-in particular, its role in the maintenance of good health. the florida department of health's campaign employs the notion of the bfifth guy^to single out the person who ignores rather than assumes personal responsibility. (my references to the bfifth guy^describe the campaign, whereas discussions of the bfifth guy^refer to its main character.) the campaign has a basis in a study conducted by the american society for microbiology (asm), which found that four out of five people do wash their hands after using the restroom. in this campaign, the fifth guy is not only male but also young, able-bodied, and white. the fifth guy seeks to billustrate a simple point-most people respect certain hygienic norms.^those who do not observe these norms become bthat one person everyone whispers about.^within the campaign's configuration of personal responsibility, displays of illness etiquette are represented as much measures of moral worth as they are forms of infection control. the bfifth guy,^further, is portrayed as at risk of both sickness and social quarantine-exclusion from the group because he poses a threat to public health. to stress the value of personal responsibility, video public service announcements (psas) both televised and posted online exaggerate as deviant the fifth guy's violation ignorance of a workplace's bhygienic norms.^played by comedic actor ben spring, the fifth guy is, not surprisingly, central to the campaign's narrative of personal responsibility. two of the three psas showcase ben's tendency to come to work sick, for him, a point of pride, and for his coworkers, a source of disdain. ben also coughs and sneezes without covering his mouth and nose with his elbow. the videos bcougher^and bsick at home^dedicate considerable footage to shots of ben coughing into his hands, onto food in the lunchroom, during meetings, and in the faces of his fellow coworkers. ben is quite clearly ignorant of his body as potentially-and, in most instances, quite literally-a source of infection risk to the people around him. however, the fifth guy is used to emphasize the necessity of his coworker's efforts to compensate for his ignorance. bhow would i describe ben to you? the next black plague,b en's manager tells the camera in one psa: bthey're gonna say, 'how did it happen, was it rats?' no, it was ben over at amalgamated, responsible for the death of europe.^ben's violations of the dictates of illness etiquette make him an object of disgust within his workplace. more importantly, when illness arises within a workplace, his coworkers come to regard ben's body as its likeliest source. in the fifth guy, attention is paid to ben's body not as a site of sickness-or, put differently, a site of suffering-but as a site of infection risk. this situating of infection risk in individual bodies teaches the importance of avoidance of certain others as potentially (or, in ben's case, it seems, inherently) vectors of infectious disease. ben's coworkers leave the lunchroom when he enters, refuse to shake his hand or give him high fives, and send emails and issue prank calls urging him to go home. in other words, ben is to be avoided because he embodies the risk of infection in public. so, in avoiding ben, his coworkers assume personal responsibility for infection risk. ben's failures in this respect in turn imply that those who succumb to infection have only themselves to blame, perhaps because they, too, ignored the dictates of illness etiquette. avoidance and exclusion, however, are not the only strategies endorsed as both infectioncontrol measures and displays of personal responsibility. in the fifth guy, hand hygiene represents a hedge against infection risk and its absence a violation of the dictum of personal responsibility. bjust another day in the office^illustrates this dual function. in this psa ben's poor hand hygiene habits graphically come to life in the form of a urinal he carries around the office after leaving the restroom. in one scene, ben proudly places his urinal on a coworker's desk while asking for some paperwork. in other scenes, he dances along the office's corridors, embracing his urinal in a mock tango. depicting poor hand hygiene as a urinal makes some sense from the perspective of theories of fomite transmission of infection. these theories explain that, unless properly sanitized, inanimate materials or objects can become contaminated with infectious agents such as influenza virus. similarly, poor hand hygiene-or a lack of hand hygiene-increases the likelihood of the transmission pathogens, both from contaminated surfaces to individuals and between individuals as well. 20 yet, the goal of ben's urinal appears not to be to instruct the workers of florida in the problem of fomite transmission. instead, by emphasizing ignorance of illness etiquette as akin to intentionality, ben's out-ofplace urinal serves as an object lesson in hand hygiene as an expression of personal responsibility. ben is stigmatized-literally marked-to distinguish him from those who observe their obligation to illness etiquette. certainly, the fifth guy teaches hand hygiene as a display of personal responsibility. yet the campaign also reveals another expectation of the new public health, and that is the enforcement of individual behavior change among the non-compliant. frequently lacking the ability to confront him directly, ben's coworkers take advantage of the opportunity to make their concerns known to the camera. byes, i'd say he's a walking pandemic,^the receptionist comments just seconds after ben has left the restroom with his urinal-germs in tow. bquite frankly,^says the coworker whose desk has been sullied by ben's metaphorical urinal hands, bhe scares me.^acknowledging that it can be difficult to reproach our colleagues, bjust another day^ends with the words of a voiceover narrator: bfour out of five people wash their hands in the restroom. could someone talk to the fifth guy?^strategies for doing so appear on the page of the bfifth guy^website on which bjust another day^is posted. tips include emailing your coworker one of the campaign videos with the comment, bhey, sure glad you're nothing like this^or giving him or her ba new nickname like 'big loogie' or 'thunder cough'.^as these rather passive-aggressive strategies suggest, the assumption of personal responsibility for infection risk also involves participation in its enforcement. nevertheless, in using stigma to underscore personal responsibility, the fifth guy potentially both validates anxieties about infection risk and reassures that risk can always be managed. those who regularly encounter infection in the workplace or witness handhygiene violations in public restrooms may feel vindicated by the campaign's mockery of ben, the boffice superspreader.^after all, as the campaign implies, only careless people spread disease. with care, infection can invariably be avoided. the fifth guy's attributions of intentionality may be the campaign's most problematic feature and not simply because such attributions may be likely to exacerbate interpersonal conflicts within public settings. the use of a constitutive, stigmatizing rhetoric has consequences for shared perceptions of infection risk. it is to these perceptions that i now turn my attention. three configurations of infection risk emerge from the fifth guy's encouragement of personal responsibility. first, the most serious risk depicted throughout the campaign is exposure to ben, who is a bwalking pandemic,^possibly even the source of plague. in implying that infection risk is determined mainly by exposure to others, this configuration places undue emphasis on the need for hypervigilance in interpersonal interactions. in ben's story, the assumption of personal responsibility for infection risk takes the form of a kind of citizenepidemiology, with everyone working to root out sources of infection. yet shy of engaging in self-quarantine, most people exercise only limited control over their exposure to others. perhaps in recognition of this fact, the bfifth guy^instructs in subtle pressures that might be applied to those individuals determined to be the potential source of infection-for example, through stigma. second, infection risk is determined largely by one's ability to control and manage certain behaviors. conversely, failure to change habits increases our risk. different scenes from the fifth guy illustrate this formulation of infection risk. motivated by the threat of ben's behavior, his coworkers more than once demonstrate for the camera different practices for limiting infection risk. in displaying their compliance with illness etiquette to the camera rather than to ben, his colleagues indicate the necessity of habitual and bodily responses to the management of infection risk. a third assumption is underscored within the numerous texts that together form the campaign's overarching message about risk and responsibility. in the fifth guy, a lack of knowledge increases one's risk of infection. ben, who displays ignorance of his duty to manage risk, teaches that being knowledgeable reduces the risk of infection (not to mention the threat of expulsion from the group). other elements of the campaign reinforce this equation of knowledge with the assumption of responsibility for infection risk. visitors to the bfifth guy^website can, for example, take a quiz that tests their bhygiene iq.^their scores determine bwhich person^they are in the workplace drama of illness and infection. yet, as anyone who takes the quiz may quickly realize, it is only possible to either be the bfifth guy( ignorant) or not the fifth guy (not ignorant). users who select the incorrect answer to a series of five questions are also goaded to correct their mistakes by the message, bwrong. who are you, the fifth guy?^most of these wrong answers correspond with ben's behaviors in different scenes from the campaign psas. the didacticism of the campaign's testing of hygiene iq raises the question: what knowledge, exactly, do audiences gain through exposure to the bfifth guy^and campaigns like it? perhaps most importantly, the formulation of knowledge as a defense against infection risk teaches an individualistic approach to risk management. within this conception, the complex problem of emerging infectious diseases is most effectively resolved through personal transformations of our daily habits, not to mention of our relationships to one another. in the coming decades, it seems likely that the containment of outbreaks will depend more and more on a program of risk communication that teaches individuals how to protect themselves against infection. within the new public health, this focus on behavioral change is frequently regarded mainly as an alternative to the implementation of costlier, more comprehensive forms of protection, treatment, and care. problematically, however, this encouragement of the personal responsibility for infection risk ignores the influence of contextual and environmental factors. complex economic and social factors, from social support networks to gender, ethnicity, race, and culture, shape and determine the health of populations. instead, even those campaigns that single out the person who (like ben) does not adhere to the dictates of illness etiquette imply equality in our susceptibility to (or risk of) infection. despite its shortcomings, critics have only occasionally spoken out against the emphasis on personal responsibility for infection risk and the neoliberal model of public health it entails. shortly after president obama advised americans to help fend off a global pandemic by washing their hands, for example, cohen wrote a new york times column about the ethical dimensions of the 100th-day address (2009). was obama's counsel to americans to do their part by washing their hands and staying home from work bmerely good manners,^cohen wondered. or, should his comments instead be understood as a moral injunction, with serious implications for how the nation would cope with the outbreak? put simply, is hand hygiene a matter of etiquette-or is it a matter of ethics? while etiquette may bhave a trivial impact on others,^cohen deemed obama's h1n1 advisory a matter of ethics bbecause it concerns the effect of our actions on other people.ŵ ashing one's hands removes harmful, disease-causing pathogens, making the endorsement of the act an bethical imperative, meant to mitigate the harm we might do to others.^that hand hygiene has a personal benefit does not make the habit any more ethical-just more desirable, perhaps, because self-care for the most part overlaps with care of others. yet in defending hand hygiene as an ethical imperative, cohen claimed that even this commonsense health habit has its limits. a program of risk management that depends for its success on the assumption of personal responsibility may similarly be too limited an approach to the problem of infection. as cohen put it, the dictates of illness etiquette, although bfundamentally ethical, are not universally applicable.^efforts to mobilize citizens against infection risk require an environment supportive of their participation. adequate supports must exist to ensure that citizens can bdo the right thing.^to illustrate the limits of personal responsibility, cohen discussed the example of labour law: some employees, particularly low-wage workers, risk losing pay or even getting fired if they stay home from work to avoid infecting their coworkers. if we expect individuals to act ethically, we have a societal obligation to protect them when they do-for instance, by guaranteeing paid sick days to all. (2009) during h1n1, concern about the ability of individuals to behave according to the dictates of public health led to the introduction in the u.s. congress of a bill that would require most employers to provide workers sent home with infections such as influenza a minimum of five paid sick days. paid sick leave, supporters argued, could even be a benefit to the economy, since the policy could both increase productivity and reduce the spread of illness and infection around the workplace. i quote cohen's comments at length because he is one of few critics to publicly speak out about the ethical issues that arise from the increasing encouragement of personal responsibility for infection risk. (even owen [2013] , in describing in detail the brise of purell,^shies away from too staunch a critique of the implications of the turn to hand hygiene promotion.) despite the appeal of the argument that infection risk can be managed mainly through individual behavior change, most exercises in risk management depend for their success on an environment supportive of these changes. in implying that infection risk may be equally distributed across populations, handwashing campaigns exclude the insights of decades of research on the social determinants of health and diseases. in this context, rhetoricians of health and medicine and health humanities scholars contribute meaningful investigations of the rhetoric of personal responsibility and specifically of its emphasis on fear, anxiety, distrust, stigma, and blame. such analyses are sure to deepen conversations among scholars and practitioners about the long-term implications of a seemingly uncontroversial enterprise-the promotion of hand hygiene. as mentioned at this essay's outset, i do not wish to question hand hygiene's efficacy as a form of infection control. myriad studies report on the impact of hand washing on the risk of infection with the majority suggesting that the habit significantly limits the transmission of communicable diseases. 21 to abandon hand hygiene because of concerns about the rhetoric used to promote makes no sense. far from opposing handwashing campaigns, i have illuminated their broader implication in the ongoing individualization and responsibilization of public health, which is also in essence a neoliberalization of public health. hand hygiene promotion, as my analysis suggests, moralizes the spread of infection, making its publics more sensitive to their capacity to sicken, and be sickened, by others. in the context of outbreak, such a perception both potentially lessens expectations of various kinds of support, for example in the form of employment or health benefits. this perception also creates new opportunities for those who stand to profit from the negative emotions often highlighted in messaging about personal responsibility for infection risk. as the target of handwashing discourse, one might thus be wary of the implications of the turn to hand hygiene as a universal antidote to the crisis of emerging infectious diseases. despite its seemingly neutral objective as a form of risk communication, hand hygiene promotion galvanizes a culture of stigma, blame, and distrust in response to the threat of infection. to what extent might these effects in fact inhibit the need for cooperation in the face of a catastrophic outbreak? handwashing campaigns transform perceptions of infection risk, casting illness as a personal failing. this is not to say that infection is not partly a consequence of poor hand hygiene, but the reality is just that. poor hand hygiene is only a contributing factor and not the root cause of the heightened risk of outbreak. it may thus be time to consider alternatives, or complements, to a neoliberal model of public health. personal responsibility has its advantages-that much is clear-but a more expansive approach might better facilitate the cooperation, and compassion, that infectious-disease outbreaks demand. endnotes 1 for the full text of president obama's remarks, see btranscript: president obama's 100th-day press briefing^(2009). 2 see, for example, keränen (2011) ; angeli (2012); ding (2014a; 2014b) . 3 keränen also stresses a common interest in the formation of publics, not just through the bofficial texts of biomedicine,^but also through the practices they adopt in response to these texts (2014, 104). 4 wald (2008) in turn proposes the theory of the boutbreak narrative^to describe the influences of both media and popular culture on responses to infectious disease-in particular, those responses that generate stigma or discrimination. 5 in fact, for a decade prior to sars, purell languished in obscurity (owen 2013, 30) . 6 see sadler (2009) , which incorporates critiques of hand-sanitizer use from health historians jacalyn duffin and nancy tomes, both of whom regard the product's popularity as tied to anxieties about infection risk. 7 while worldwide sales figures vary from one source to another, a cnn story reports that shipments of hand sanitizer tripled during h1n1, from 1 million kilograms to 3 million kilograms (rooney 2009) . a more recent report (fottrell 2013) states that u.s. sales of hand sanitizer reached $300 million in 2009, and have since averaged nearly $200 million per year. 8 plyushteva's (2009) critique of hand hygiene promotion in developing countries documents only the latest stage in a longer arc of handwashing campaigns developed to generate sales for hand soap. vinikas (1989) , for example, chronicles the creation by soapmakers of the 1920s and 1930s of the cleaning institute, which worked to increase soap sales by inculcating schoolchildren into personal hygiene habits. see, also, vinikas (1995) , which illuminates the significance to modern advertising of early-twentieth-century efforts to promote personal hygiene. 9 for the full text of this press release, see royal society for the protection of nature, bglobal handwashing day observed in yoeseltse mss in samtse.1 0 the quotations in this paragraph derive from globalhandwashing.org, the website for the global ppphw. for a recent systematic review of the impact of hand hygiene promotion in developing countries, see whom plyushteva quotes, openly criticizes hand hygiene promotion in kerala as a poor substitute for structural interventions, such as the improvement of sanitation systems or provision of clean water another important historical precursor to plyushteva's critique is tomes, which documents the work of latenineteenth and early-twentieth century public health advocates to transform lay understandings of the spread of infection. tomes points out that bentrepreneurs and manufacturers curtis's (2011) review of the emerging body of scholarship on the use of public health discourse to trigger a disgust response in order to motivate individual behavior change my view of health citizenship derives mainly from petersen and lupton (1996), but it also has loose ties to rose and novas' (2005) notion of biological citizenship argues that before the 1980 provincial referendum the government of quebec sought support for quebec's separation from canada by constituting the province's inhabitants as ba distinct peuple.^by voting in support of separation a constitutive perspective is also consistent with foucault's theory of subjectivity formation. for a discussion of foucault's significance to health humanities, see petersen which instructs americans to bkeep calm and wash [their] hands,^implying that in washing their hands, citizens consent to their duty to cooperate in the event of an outbreak the fifth guy campaign has also been the subject of social-marketing case studies which is transmitted by fomites. among others, wald (2008) stresses the role of popular culture in circulating certain conceptions of outbreak-views of causality that overstate the role of the individual in triggering an outbreak. similarly, many of the essays in a 2002 special issue of american literary history discuss the longstanding influence of popular culture on understandings some of the most frequently-cited references to studies in support of hand hygiene appear on bshow me the science bideology and ideological state apparatuses.^in lenin and philosophy and other essays translated by ben brewster bmetaphors in the rhetoric of pandemic flu: electronic media coverage of h1n1 and swine flu btheorizing modernity conspiratorially: science, scale, and the political economy of public discourse in explanations of a cholera epidemic a rhetoric of motives health promotion materials. the u.s. centers for disease control and prevention bconstitutive rhetoric: the case of the 'peuple québécois bflu fighters.^the new york times bcompliant, complacent, or panicked? investigating the problematisation of the australian general public in pandemic influenza control global handwashing day. deb group ltd bdemarcating medicine's boundaries: constituting and categorizing in the rhetoric of a global epidemic: transcultural communication about sars. carbondale: southern illinois university press drugs for life: how pharmaceutical companies define our health btalk to the fifth guy bhand sanitizer spread faster than the flu.^marketwatch bhygiene and health: systematic review of handwashing practices worldwide and update of health effects bchildren as handwashing agents of change.^the global public-private partnership for handwashing with soap. accessed stigma: notes on the management of a spoiled identity bconcocting viral apocalypse: catastrophic risk and the production of bio(in)security 2014b. b'this weird, incurable disease': competing diagnoses in the rhetoric of morgellons.^in health humanities brisky appeals: recruiting to the environmental breast cancer movement in the age of 'pink fatigue bfat panic and the new morality.^in against health: how health became the new morality bselling certainty: genetic complexity and moral urgency in myriad genetics bintroduction: why against health?^in against health: how health became the new morality bhands across america: the rise of purell the new public health: health and self in the age of risk btalk to the fifth guy: a lesson in social marketing.ĉ ases in public bthis benevolent hand gives you soap: reflections on global handwashing day from an international development perspective.^journal of health management bpresident obama's 100th-day press briefing.^2009. the new york times. accessed bhand sanitizer in short supply as swine flu hits.^cnn money. accessed bbiological citizenship.^in global assemblages: technology, politics, and ethics as anthropological problems bglobal handwashing day observed in yoeseltse mss in samtse bdo you really need hand sanitizer?^cbc news health and the rhetoric of medicine bpublic health campaign.^in sage encyclopedia of public relations bhandwash or eyewash? selling soap in the name of public private partnerships.^india resource center blife in a time of germaphobia.^the globe and mail the gospel of germs: men, women, and the microbe in american life soft soap, hard sell: american hygiene in an age of advertisement contagious: cultures, carriers, and the outbreak narrative bcurrent who phase of pandemic alert for pandemic (h1n1).^the world health organization g l o b a l a l e r t a n d r e s p o n s e ( g a r ) . a c c e s s e d 2 9 a p r i l 2 0 0 9 key: cord-294568-12eyo13f authors: fernandes-matano, larissa; monroy-muñoz, irma eloísa; angeles-martínez, javier; sarquiz-martinez, brenda; palomec-nava, iliana donají; pardavé-alejandre, hector daniel; santos coy-arechavaleta, andrea; santacruz-tinoco, clara esperanza; gonzález-ibarra, joaquín; gonzález-bonilla, cesar raúl; muñoz-medina, josé esteban title: prevalence of non-influenza respiratory viruses in acute respiratory infection cases in mexico date: 2017-05-03 journal: plos one doi: 10.1371/journal.pone.0176298 sha: doc_id: 294568 cord_uid: 12eyo13f background: acute respiratory infections are the leading cause of morbidity and mortality worldwide. although a viral aetiological agent is estimated to be involved in up to 80% of cases, the majority of these agents have never been specifically identified. since 2009, diagnostic and surveillance efforts for influenza virus have been applied worldwide. however, insufficient epidemiological information is available for the many other respiratory viruses that can cause acute respiratory infections. methods: this study evaluated the presence of 14 non-influenza respiratory viruses in 872 pharyngeal exudate samples using rt-qpcr. all samples met the operational definition of a probable case of an influenza-like illness or severe acute respiratory infection and had a previous negative result for influenza by rt-qpcr. results: the presence of at least one non-influenza virus was observed in 312 samples (35.8%). the most frequent viruses were rhinovirus (rv; 33.0%), human respiratory syncytial virus (hrsv; 30.8%) and human metapneumovirus (hmpv; 10.6%). a total of 56 cases of co-infection (17.9%) caused by 2, 3, or 4 viruses were identified. approximately 62.5% of all positive cases were in children under 9 years of age. conclusion: in this study, we identified 13 non-influenza respiratory viruses that could occur in any season of the year. this study provides evidence for the prevalence and seasonality of a wide range of respiratory viruses that circulate in mexico and constitute a risk for the population. additionally, our data suggest that including these tests more widely in the diagnostic algorithm for influenza may reduce the use of unnecessary antibiotics, reduce the hospitalisation time, and enrich national epidemiological data with respect to the infections caused by these viruses. introduction acute respiratory infections (aris) represent the leading cause of morbidity and mortality worldwide [1] [2] , are the most common cause of outpatient care in adult patients [3] , and are responsible for 70% of hospitalisations due to respiratory diseases in child populations aged 1-4 years and up to 90% in infants under 1 year of age [4] . aris are a group of diseases with normally less than 15 days of evolution that are caused by different microorganisms. a viral aetiological agent is estimated to be present in up to 80% of cases [5] [6] [7] . these infections can occur in the upper and lower respiratory tract. upper aris may include one or more of the following conditions: rhinopharyngitis, pharyngoamygdalitis, sinusitis, and acute otitis media [8] [9] . lower aris include epiglottitis, laryngitis, laryngotracheobronchitis (croup), bronchitis, bronchiolitis, and pneumonia [9] . these infections are easily transmitted via coughing or sneezing. contagion occurs through the inhalation of aerosols and microdroplets that contain the causative agent. another important form of contagion is through direct contact of hands with objects contaminated with respiratory secretions from infected individuals, which can be self-inoculated into the nasal and buccal mucosae and/or into the ocular cavity [10] . a large amount of information is available concerning the timing and distribution of influenza viruses in the population following the reappearance of avian influenza a subtype h5n1 in 2003 and 2004 and the influenza a subtype h1n1 pandemic in 2009 [11] . influenza viruses are one of the main causative agents of aris worldwide; however, many other respiratory viruses for which insufficient epidemiological information is available can also cause aris. studies performed at the international level have frequently identified human respiratory syncytial virus (hrsv), human parainfluenza virus (hpiv), influenza virus (flu), human mastadenovirus (hmdv), rhinovirus (rv), and enterovirus (ev) and less frequently identified human metapneumovirus (hmpv), primate bocaparvovirus (pbpv), and human coronavirus (hcov) [12] . these viruses can serve as the causative agents for aris that occur outside of influenza season, when the rate of positivity can drop below 10%. although a large percentage of aris are caused by viral infections, the causative viruses have not been specifically identified in the majority of cases due to difficulties such as the high number of possible aetiological agents, similar symptoms among aris caused by different aetiological agents, the emergence of new viruses or new variants of previously described viruses, and the high cost of the detection tests [4] . thus, little information is available concerning the prevalence and seasonality of these viruses, mainly in undeveloped countries, where the possibilities of carrying out this type of study on a regular basis is unusual. the investigation of the causative agents of acute respiratory infections is important in order to lead the development of vaccines to target the most prevalent viruses and to reduce the unnecessary prescription of antibiotics and of antiviral oseltamivir phosphate. in addition, analysis like this can help to identify the age groups more susceptible to infection by each virus, in order to take the necessary actions for prevention and treatment. therefore, our aim was to determine the viral aetiology of aris in samples from patients who presented respiratory symptomatology but were negative for influenza by rt-qpcr testing. to evaluate the presence of noninfluenza respiratory viruses circulating in mexican population in the different seasons of the year, we analized every pharyngeal exudate sample received by the laboratorio central de epidemiología (lce) (central epidemiology laboratory) between the epidemiological week 40 of 2014 and the week 39 of 2015, according to the following criteria. all samples had a previous negative result for influenza by rt-qpcr and met one of the following operational case definitions: influenza-like illness (ili): a person of any age who presents a fever greater than or equal to 38˚c, a cough, and cephalalgia accompanied by one or more of the following symptoms: rhinorrhoea, coryza, arthralgias, myalgias, prostration, odynophagia, thoracic pain, abdominal pain, or nasal congestion. in patients under five years of age, irritability is considered a cardinal sign in place of cephalalgia. in patients older than 65 years, fever is not required as a cardinal symptom. severe acute respiratory infection (sari): a person of any age who presents difficulty breathing accompanied by a fever greater than or equal to 38˚c and a cough with one or more of the following symptoms: poor general condition, thoracic pain, polypnoea or acute respiratory distress syndrome (ards) or any death associated with ili or sari. a total of 872 samples were selected. (s1 table) total nucleic acid extraction the superscript™ iii platinum 1 one-step rt-qpcr system kit (invitrogen, carlsbad, califórnia, eua. catalog: 12574035) was used in a 7500 fast real-time pcr system (applied biosystems 1 , foster city, califórnia, eua) to amplify viral genetic material. primers and probes were used for each of the following viruses: hmpv, hrsv, hpiv 1-4, betacoronavirus 1 (βcov1), human coronavirus (hku1, 229e, nl63) (hcov), hmdv, rv, ev, and pbpv. the human rnase p (rp) gene was used as an internal control ( table 1 ). the viruses were evaluated in uniplex reactions with the following reaction mixture: 12.5 μl of 2x reaction mix, 0.5 μl of each primer and probe, 1 μl of enzyme, 5.5 μl of rnase-free water, and 5 μl of total dna or rna lyophilizates (amplirun1 vircell; granada-spain) were used as the positive controls for all evaluated viruses. all samples that presented amplification for any of the viral markers plus the internal control were considered positive results. all samples without amplification of the viral markers but with amplification of the internal control were considered negative results. samples that did not present amplification of the internal control were considered inadequate. descriptive statistics were used to analyse the prevalence of the viruses included in the study, with the percentages given with a 95% confidence interval. the chi-square test of homogeneity and independence and fisher's exact test were used to compare categorical variables (p-values <0.05 were considered significant). anova and student's t-test were used for hypothesis testing of the quantitative variables. the analyses were performed with the spss statistics version 24.0 software, and the graphics were generated with the statgraphics 1 centurion xvi.ii and microsoft 1 excel 1 2010 software. the information of the biological specimens used in the present study is not traceable to the patient data identity. all the samples were used in an anonymous way. the study was approved by the ethics committee and the research committee of the national committee of scientific research of the instituto mexicano del seguro social with the registration number r-2016-785-041. of the 872 samples analysed, 451 were from males (51.7%) and 421 were from females (48.3%). the average age was 41 years, with minimum and maximum extremes of 0 and 101 years, respectively. the population was divided into 4 age groups based on the age ranges included on the instituto mexicano del seguro social (imss; mexican social security institute) health cards. according to this stratification, the samples were distributed as follows: 265 samples corresponded to children 0-9 years of age (30.4%), 28 to young people 10-19 years of age (3.2%), 263 to adults 20-59 years of age (30.2%), and 316 to adults 60 years and older (36.2%). (table 2) . unintentionallya, the analysed samples were collected from patients from three geographical areas of the country as follows: the north zone (baja california, baja california sur, 25.9% of the samples (fig 1) . additionally, these data were analysed based on the age groups (s2 table and s1 file) . of the total samples analysed, 312 were positive for at least 1 virus (35.8%), with 47.1% from female patients (n = 147) and 52.9% from male patients (n = 165); there was no significant difference between the groups (p>0.05). of the total positive samples, rv had the highest number of detections (33.0%), followed by hrsv (30.8%), hmpv (10.6%), hmdv (9.6%), hpiv3 (8.7%), βcov1 (8.7%), ev (5.8%), and pbpv (4.5%). the other viruses presented percentages below 4%, although together they accounted for 10.3% of all positive cases. only hpiv2 was not detected (table 3 ) the 3 viruses most often identified in cases of co-infection were rv in 48.3% of the cases, followed by hrsv (35.7%) and hmdv (32.1%). interestingly, in the case of pbpv, despite it being the causal agent of only 19.6% of co-infections, these co-infections represented 78.6% of the pbpv cases detected. the behaviour of hmdv was similar, with 60% of all cases in which this virus was present involving a combination with other viruses. both of these cases represent a significant association (p<0.05) with co-infection (table 4) . the most commonly observed viral combination was hmdv + rv (16.1%), followed by hpiv3 + rv and hrsv + βcov1 (both 7.1%). although it occurred only 2 times, the co-infection with 4 viruses involved the same agents in both cases (hrsv + hmdv + βcov1 + pbpv). a comparison was performed between the days of hospitalization required by the patients with negative samples and those with samples positive for a single virus or in whom 2 or more viruses were detected. the analysis of this clinical data showed that the hospitalization was significantly higher in the negative samples than in the positive ones for a virus or with coinfections: 7.5, 5.1 and 4.5 days on average, respectively (p <0.05). we observed the same tendency with the average number of comorbidities and symptoms: 0.65 in the negative samples versus 0.35 in positive samples (p <0.05) and 7.7 in the negative samples against 7.4 in positive samples (p > 0.05), respectively. nevertheless, the number of clinical symptoms and comorbidities were greater in the aris positive for a single virus than in the patients with co-infections (p <0.05) ( table 5 ). the highest number of positive cases occurred in the group aged 0 to 9 years (62.5%) and the second highest in the group aged 60 years or older (20.5%), followed by the 20-to 59-year-old group (14.4%) and finally the 10-to 19-year-old age group (2.6%), which represented a significant difference (p<0.05). consistently, the 0-to 9-year-old age group comprised 91.1% of the co-infections. interestingly this group contained 92.9% of all pbpv infections, 86.7% of the hmdv infections, and 86.5% of the hrsv cases (fig 3) . when conducting the analysis of the overall behaviour of the viruses in the period covered by the study, we observed that the months with high and low positivity differed significantly table 4 . viruses participation in co-infections. (p<0.05). fig 4b-4e shows the monthly behaviour of each virus. the month of november presented the highest percentage of cases during the year ( fig 4a) . when the analysis was performed by season, we observed differences between the four seasons of the year (p<0.05). generally, summer had a significantly lower proportion of positive samples (24.5%; p<0.05), whereas fall had the highest proportion (44.9%; p<0.05). however, the individual trends of the viruses were not the same. for instance, spring (march 21 to june 20) accounted for the greatest proportions of rv, hmdv, hpiv4, and hpiv1 cases (29.5%, 38.5%, 46.8%, and 48.0%, respectively). in contrast, most hrsv (48.2%) and ev (72.6%) cases were detected in the fall (september 21 to december 20), and winter (december 21 to march 20) presented the highest prevalence of hmpv (47.3%) and βcov1 (60.5%). the other viruses (hcov 229e, hcov hku1, hpiv 3, pbpv, and hcov nl63) were detected throughout the year, with no seasonal trends observed (p>0.05). approximately 27 million ari cases occur annually in mexico [14] . these cases can be caused by a large variety of aetiological agents. however, the main purpose of epidemiological surveillance in the country is to detect the antigenic variations of influenza that are presented each season, which determine the changes in the vaccine composition. in 2013, the instituto de diagnóstico y referencia epidemiológicos (indre; institute of epidemiological diagnosis and reference) implemented a differential diagnosis of influenza that included 14 other respiratory viruses [15] . due to this, at this moment mexico does not have enough epidemiological information about the great diversity of viruses causing acute respiratory infection. this lack of information, makes physicians search only for influenza, it is therefore the most requested confirmatory test in the laboratory during the whole year, even when we are out of influenza season (season comprises from october to may), leading to useless negative influenza results in most of the cases, without identifying the real causal agent of ari in mexico. this problem have direct implications for each patient, as the clinician does not know the causal agent, the patient does not receive the suitable treatment. it also has implications that affect the whole society, because the ignorance of the circulation patterns and incidence of other respiratory viruses limit preventive actions by health institutions. according to data from the secretary of health, approximately 80% of the samples from patients with aris that are received for confirmation of influenza virus infection outside of flu season are negative for the different strains of this virus and remain without a defined aetiology. therefore, the objective of this study was to determine the viral aetiology of these infections and to analyse the behaviour of non-influenza respiratory viruses in the mexican population. beginning and ending with the 2015 influenza season, 872 samples collected over one year were evaluated to determine the presence of hmpv, hrsv, hpiv 1-4, βcov1, hcov, hmdv, rv, ev, and pbpv. these 14 respiratory viruses share symptoms with influenza but are rarely suspected or can be confused with influenza. in contrast to other studies in other countries investigating this issue, this study included samples corresponding to all age groups and regions of the country. therefore, the study population best represents the demographic distribution of aris in the country. in contrast to most of the prevalence studies of aris, in which all age groups are not normally analysed and data is limited to a single region of the country, in this study, nor the age of the patient, nor the region of the country from which the sample came were included as inclusion or exclusion criteria. therefore, the population analysed in this study represents better the demographic distribution of aris in our country. this type of study helps provide relevant data for the development of treatment and prevention strategies because the currently available antiviral agents and vaccines are primarily directed at influenza infection. however, the proportion of aris caused by different influenza viral agents is not negligible, with the detection range of non-influenza respiratory viruses spanning from 16.5 to 72.7% in studies conducted worldwide [4;16-20] , depending on factors such as the study design, study population, detection technique used, and period covered by the study. in our case, the prevalence of the analysed viruses was 35.8%, which was within the cited range. the most prevalent viruses were rv, hrsv, and hmpv, and the only virus that was not detected was hpiv2. this result was consistent with other studies in which type 2 parainfluenza virus had the lowest detection frequency [21-24]. however, there is evidence that its prevalence increases when hpiv-3 and hpiv-1 are reduced [25] [26] . consistent with our results, rv was identified as the most common virus in ari cases in several studies [17;20;22;27] in which the presence of influenza and other respiratory viruses from throat swabs is determined by molecular techniques of own design or using commercial panels. equal data were observed in a study of mexican children [28] . this virus is the major aetiological agent of the common cold [29], however, it may also be involved in serious aris [30] [31] [32] , which are primarily observed in patients with underlying comorbidities, such as asthma [33] [34] [35] or other chronic pulmonary diseases [36] , indicating a certain degree of opportunism. similar to rv, hrsv was identified in high proportions in other studies [4;37] , conducted in poland in which 380 individuals were analysed without age selection, and in mexico in which 383 children up to 5 years of age were studied. this trend is also observed in studies where a higher prevalence of influenza was observed [19;21;23;38] , and hrsv has been associated with the pathogenesis of asthma. more importantly, hrsv is the leading cause of child mortality caused by viruses [39] . the third most prevalent virus was hmpv. in mexico, the first study in which the presence of this virus was determined was published in 2005 [40] . subsequently, other mexican studies demonstrated its importance in the child population [16;41-42] . studies such as that of diaz et al. [16] demonstrated that hmpv was mostly associated with severe acute respiratory infections instead of mild and moderate infections. the importance of the differential diagnosis of other respiratory viruses in samples with negative influenza results becomes apparent when we observe the prevalence of the three main viruses identified in this study as well as their associations with severe cases and deaths, especially in the child population. co-infections represented 17.9% of the positive samples. this percentage ranged between 6.9 and 18.6% in other studies that also included various age groups [20;43-44] . however, much higher percentages have been found (above 30%) in study populations composed of children under 5 years of age [16;22-23;45] . our results confirmed that viral co-infection was common, especially in the child population because 91.1% of all co-infections occurred in the 0 to 9 year age group. this result could be attributed to slower viral elimination due to a still-developing immune system [46] . the virus most involved in co-infections was pbpv, as in 78.6% of the cases in which it was identified, it was found together with another virus. studies have reported identification percentages of this virus in conjunction with other agents ranging from 47.4 up to 90% [47] [48] . however, its prevalence in asymptomatic patients is also high (44% or 43%) [49] , which calls into question whether pbpv by itself is capable of generating disease [50] or if it only participates as a facilitator for another agent to infect the host. at present, cellular and animal models are still being developed for this virus [51] , therefore, the evidence needed to confirm this hypothesis does not yet exist. from a clinical perspective, whether the presence of a co-infection results in a more serious case or is a poor prognostic factor is a matter of controversy within the scientific community. although some studies have reported cases with these characteristics [52] [53] [54] , reports by other authors have suggested that co-infections are not synonymous with clinical differences or greater severity [55] [56] . in the study of martínez-roig et al. [57] , an inverse relationship was found between the number of viruses detected and the hospitalization time as well as the need for oxygen therapy. a similar relationship was observed in the study of canducci et al. [58] , where there was a greater hypoxia and lengthier hospitalization time for infections caused purely by hrsv compared to coinfections. in our study, the aris caused by a single virus also presented lengthier hospitalization times than aris caused by 2 or more viruses, although the differences were not statistically significant. notably, similar to the estimation of prevalence, differences in the reported ranges of and discrepancies in the severity associated with viral co-infections can be attributed to the detection techniques employed, the population, the time period of the study, and the viruses studied. because of the short period of time comprised by this study and the limited number of samples, it is not possible to state, but it can be suggested certain seasonal behaviors of some studied viruses. generally, the peak of respiratory infections occurs in the period between november and april in countries of the northern hemisphere [59] [60] . the influenza season is well known in mexico and worldwide; however, the seasons of other respiratory viruses are not well known. according to our results, the highest prevalence of these viruses in mexico appears to occur from october to march (autumn and winter), which coincides with the influenza season. detection was significantly higher in november of 2014; during this month, there was a marked decrease in the national mean temperature from 23.3 to 18.3˚c. according to cui et al. [61] , mean temperature is the key climatic parameter associated with the prevalence of many respiratory viruses because some viruses survive and/or replicate better at low temperatures [61] . on the other hand, viegas et al [62] proposed that mucus release by cilia was reduced when the temperature of the respiratory tract was lowered; consequently, the local innate immune response (neutrophil and natural killer (nk) cell migration) was also reduced, thereby promoting viral infection. climatic factors may also indirectly favour the transmission efficiency because low temperatures induce a change in social behaviour that favours interior overcrowding and increases the likelihood of close contact and transmission of infections [63] . for this reason, it is a common suggestion to avoid going to school or work when you are undergoing an infectious process. our results suggest that some viruses have a more marked seasonal trend than other viruses. in winter, the prevalences of hmpv, hpiv3, and bcov1 were higher than in the other seasons of the year. the circulation of hmpv in particular predominates in the colder seasons because a marked reduction in its prevalence is observed in the spring until it disappears completely in the summer. furthermore, in a study conducted in mexico city, in which 525 children between 1 and 15 years of age were analysed and in other study performed in china with 607 hospitalized patients was reported that the highest prevalence of hmpv occurs in the winter and that its seasonality overlaps with that of other viruses [19;43] . similar to hmpv, some authors agree that hrsv is a virus that circulates preferentially in colder seasons [19;43;64] . this study demonstrates that hrsv can cause aris in all seasons of the year but is consistent with other studies that report that its prevalence is highest during the fall and winter. conversely, rv seemed to be the virus best suited to the climatic conditions of the country because there were no significant differences in its prevalence in the different seasons. according to the results of a study conducted in china [61] , the optimal circulation temperature range of this virus is 15 to 25˚c, which is similar to the range of the mean mexican temperature and may justify its high prevalence in all months of the year. however, despite all of the information reported to date, factors affecting the circulation of different viruses remain unclear. annual research is needed to establish the seasonality of these viruses with more accuracy and precision. although influenza is one of the main causative agents of respiratory infections worldwide, it is of vital importance to determine the prevalence and timing of other causal agents. in this study, we identified 13 non-influenza respiratory viruses that occurred in any season of the year. this study provides evidence for the prevalence and seasonality of a wide range of respiratory viruses circulating in mexico that constitute a risk for the population. additionally, our data suggest that including these tests more widely in the diagnostic algorithm for influenza could reduce the use of unnecessary antibiotics, reduce the hospitalisation time, and enrich national epidemiological data regarding infections caused by these viruses. supporting information s1 human metapneumovirus in adults human metapneumovirus: review of an important respiratory pathogen reforzamiento pulmonar: su relación con la infección respiratoria aguda y la prescripción inadecuada de antibioticos non-influenza viruses in acute respiratory infections among young children. high prevalence of hmpv during the h1n1v.2009 pandemic in poland summary health statistics for u.s. children: national health interview survey viruses and bacteria in the etiology of the common cold bronchiolitis-associated hospitalizations among us children infecciones respiratorias agudas en menores de 5 años intervención educativa sibre infecciones respiratorias agudas 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children with viral respiratory infections? human bocavirus in chile: clinical characteristics and epidemiological profile in children with acute respiratory tract infections does respiratory virus coinfection increases the clinical severity of acute respiratory infection among children infected with respiratory syncytial virus? multipathogen infections in hospitalized children with acute respiratory infections human respiratory syncytial virus in children with lower respiratory tract infections or influenza-like illness and its co-infection characteristics with viruses and atypical bacteria in hangzhou viral coinfection in childhood respiratory tract infections two-year prospective study of single infections and co-infections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease health protection agency. respiratory pathogen circulation public health agency of canada. the respiratory virus detection surveillance viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china respiratory viruses seasonality in children under five years of age in seasonality and the dynamics of infectious diseases seasonal patterns of viral and bacterial infections among children hospitalized with community-acquired pneumonia in a tropical region we thank the san angel laboratory for their support for covering the costs of the publication of this manuscript. key: cord-272752-cobroc5h authors: brook, itzhak title: the challenges of treating tracheobronchitis in a laryngectomee due to nontypeable haemophilus influenzae: a case report date: 2018-08-20 journal: j med case rep doi: 10.1186/s13256-018-1764-2 sha: doc_id: 272752 cord_uid: cobroc5h background: laryngectomees run the risk of developing severe respiratory tract infections especially during the winter and when they do not wear a stoma cover. a case of severe tracheobronchitis in a laryngectomee is presented that illustrates the risks and difficulties encountered in managing this infection in a neck breather. case presentation: a 76-year-old caucasian man, a laryngectomee, presented with bacterial tracheobronchitis and conjunctivitis due to beta-lactamase-producing nontypeable haemophilus influenzae. he was febrile (38.9 °c; 102.0 f), and had repeated episodes of hypertension. he was treated with levofloxacin 500 mg/day, ciprofloxacin eye drops, acetaminophen, and guaifenesin. humidification of his trachea and the airway was sustained by insertions of saline into the stoma as well as breathing humidified air. the main challenge was to maintain the patency of his airway as the mucus was very dry and viscous and tended to stick to the walls of his trachea and the stoma. his condition improved within 7 days and he had a complete recovery. conclusions: maintaining the patency of the airway in laryngectomees who suffer from lower respiratory tract infection is of utmost importance as the mucus can be very dry and viscous and can stick to the walls of the trachea and the stoma. laryngectomees run a high risk of developing severe respiratory tract infections. following laryngectomy the tracheal epithelium becomes directly exposed to the relatively cold and dry ambient air entering the tracheostoma [1, 2] . this can cause: drying of the mucus, which makes it more viscous; reduction of ciliary activity that causes impaired mucociliary clearance [2] [3] [4] ; and tracheal epithelium damage (loss of ciliated cells, goblet cell hyperplasia, and excessive mucus production and metaplasia) [4] . severe pulmonary infections (tracheobronchitis and pneumonia) in laryngectomees are more frequent in the wintertime and the accompanying tracheal crusting often requires antibiotic treatment or even hospitalization [5] . tracheobronchitis in laryngectomees was described as a "suffocating" respiratory infection because of the difficulties in maintaining a patent airway in these patients [6, 7] . a case of severe tracheobronchitis in a laryngectomee is presented that illustrates the risks and difficulties encountered in managing this infection in a neck breather. a 76-year-old caucasian man who underwent laryngectomy 10 years earlier, presented with fever (38.9°c; 102.0°f), increased sputum production, and purulent conjunctivitis. these symptoms emerged gradually over a period of 48 hours. he noted increasing difficulty in coughing out his sputum that became brownish and viscous. he had been wearing a heat and moisture exchanger (hme) filter that covered his stoma and spoke through a tracheoesophageal voice prosthesis. the symptoms started a day after a very cold weather spell with temperatures of −7 to −1°c (19-31°f). he had to remove his hme on several occasions for extended periods of time to enable him to breathe when he walked outside his home. his past medical history included hypopharyngeal squamous cell carcinoma which was treated with intensity-modulated radiotherapy (imrt) 12 years earlier. a recurrence of the cancer 2 years later required laryngectomy. he had no signs of tumor recurrence since then. he also suffered from paroxysmal hypertension, diverticulitis, and migraines. he was vaccinated with the current influenza virus vaccine 3 month earlier. he had also received a pneumococcal polysaccharide vaccine (ppsv23) 2 years earlier. he was in mild respiratory distress especially when coughing. he had coughing spells and expectorated green-brown dry and viscous sputum. a physical examination revealed bilateral purulent conjunctivitis and auscultation of his lungs revealed coarse rhonchi and no crepitations. no lymphadenopathy was noted. the results of the rest of the physical and neurological examinations were within normal limits. a chest x-ray was normal. sputum and conjunctival culture grew heavy growth of beta-lactamase-producing nontypeable haemophilus influenzae (nthi) that was susceptible to levofloxacin and amoxicillin-clavulanate. a filmarray® respiratory panel 2 (rp2) polymerase chain reaction (pcr) system test did not detect 14 viruses (adenovirus, coronavirus hku1, coronavirus nl63, coronavirus 229e, coronavirus oc43, human rhinovirus/enterovirus, human metapneumovirus, influenza a, influenza b, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus) and four bacteria (bordetella pertussis, bordetella parapertussis, chlamydophila pneumoniae, mycoplasma pneumoniae). he was treated with orally administered levofloxacin 500 mg/day, ciprofloxacin eye drops, acetaminophen, and guaifenesin. humidification of his trachea and the airway was maintained by repeated insertions of 3-5 cc respiratory saline into the stoma at least once an hour and by breathing humidified air. the main challenge was to maintain a patent airway as the mucus was very dry and viscous and tended to stick to the walls of his trachea and the stoma. the mucus had to be repeatedly expectorated by vigorous coughing and by manual removal from the upper part of his trachea and stoma. he experienced repeated episodes of sustained elevated blood pressure (up to 210/110) and tachycardia (112/minute). this was managed by administration of clonidine 0.1 mg as needed (1-2/day). his fever started to decline 48 hours after antimicrobial therapy was started. the conjunctivitis improved within 36 hours. the sputum production declined and became less viscous over time, but persisted for 5 days. antimicrobial therapy was discontinued after 7 days. his condition improved and he had a complete recovery in 7 days. he was seen in the clinic every 2 months and showed no recurrence of his infection for the following 8 months. he received vaccination for h. influenzae b and prevnar 13® (pneumococcal conjugate vaccine; pcv13) 4 weeks after his recovery. this case report described the occurrence of a serious bacterial tracheobronchitis due to h. influenzae in a laryngectomee. it illustrates the difficulties in managing the airway access in laryngectomees because of the increased mucus production that can block them. it also highlights the need to recognize the potential of bacterial infection in this population. nthi is a known cause of community-acquired pneumonia in adults [8] and may be associated with severe disease and high mortality in higher risk populations. although bacteremia that complicates h. influenzae pneumonia occurs occasionally [9] , disseminated infections following nthi bacteremia are uncommon except in newborns and immunocompromised patients. h. influenzae was recovered from 6 of 27 (22%) tracheal aspirates of children with tracheostomy who had pneumonia [10] , and in 4 of 14 (29%) children with bacterial tracheitis [11] . the stoma in neck breathers allows the inhaled air to bypass the natural defenses (nasal hair and mucus membranes) of the upper airway that filter out dust and bacteria. wearing an hme that covers the stoma can provide important benefits [1, 2] . the hme filter serves as a stoma cover and creates a tight seal around the stoma. in addition to filtering dust and other larger airborne particles, hmes preserve some of the moisture and heat inside the respiratory tract and prevent their loss and adds resistance to the airflow [1, 2] . hme filters assist in restoring the temperature, moisture, and cleanliness of the inhaled air to the same condition as before laryngectomy. the number of bronchitis, tracheobronchitis, and pneumonia episodes as well as mortality due to these infections in non-hme users was found to be three times higher than in hme users [12] . laryngectomees especially those who do not wear an hme or have uncovered stoma are therefore at a higher risk for lower respiratory infections. the exposure of our patient to cold air without an hme most likely compromised his airway and led to the development of the infection. paroxysmal hypertension is a known complication following head and neck radiation, and can be attributed to damage to the carotid artery baroreceptors [13] . aggravation of this condition during our patient's illness further complicated his condition. treatment of tracheobronchitis in laryngectomees is more challenging and requires keeping the stoma open by manually removing accumulated mucus that can dry out and clog it, keeping the excessive sputum moist by breathing humidified air and inserting saline as needed, coughing out or suctioning accumulated sputum, keeping the patient well hydrated, and removing the hme during the illness or prior to coughing to prevent blocking it with the coughed out sputum. laryngectomees are at a higher risk of developing lower respiratory tract infections especially in the winter and when not wearing an hme. maintaining the patency of the airway is of utmost importance as the mucus can be very dry and viscous and can stick to the walls of the trachea and the stoma. the risk of acquiring these infections can be reduced by: getting vaccinated for respiratory pathogens that include streptococcus pneumoniae, h. influenzae, and the influenza viruses; washing hands before any stoma care; wearing an hme at all times; maintaining adequate respiratory tract humidification; and avoiding hypothermia or inhaling cold air. the author is the sole contributor to the report. the author read and approved the final manuscript. ethics approval and consent to participate ethics approval and consent to participate was obtained. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal. the author declares that he has no competing interests. pulmonary rehabilitation after total laryngectomy using a heat and moisture exchanger (hme) the laryngectomee guide. charleston: createspace publication mucociliary clearance and mucosal surface characteristics before and after total 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hypertension (pseudopheochromocytoma) key: cord-303320-3tjhisfg authors: petersen, eskild; chen, lin hwei title: reflections on travel-associated infections in europe date: 2015-01-31 journal: the lancet infectious diseases doi: 10.1016/s1473-3099(14)71055-2 sha: doc_id: 303320 cord_uid: 3tjhisfg nan as a reminder that although the term sepsis is a valuable clinical descriptor of a condition that almost every doctor will encounter, the catch-all nature of the term often conceals more than it reveals. determining the true incidence of sepsis and its underlying causes is important, not just to quantify the burden of disease and to ensure that adequate resources are provided to deal with it, but also because it informs priorities for clinical research. however, the use of registry data can have unexpected pitfalls. 3 factors such as ascertainment artefact, subtle changes in defi nitions, better and earlier diagnosis, and even reimbursement considerations can falsely seem to increase incidence in a population. 4 kaukonen and colleagues have published an analysis of trends in the incidence and mortality associated with sepsis in adults in australia and new zealand that carefully avoided these dangers. 5 they showed that, as for the data for children, incidence was rising but mortality was falling. but they also reported signifi cant diff erences in outcome depending on age and presence of comorbidities. the overall crude hospital mortality in patients with comorbidities was 33% compared with just 5% in patients younger than 44 years of age without other underlying complications. these data have important implications. imagine planning a phase 3 trial of a new intervention for sepsis in which the primary endpoint was hospital mortality. the calculation of the number of patients to enrol would be based on the anticipated eff ect size of the intervention and the mortality rate in the placebo group. if the study population contained a subgroup with a much lower than expected mortality, the calculations would be wrong (mortality in the placebo group would be lower) and a true eff ect of the intervention might be missed (a type 2 error). this possibility has concerned many people studying sepsis. furthermore, by using broad enrolment criteria, investigators might have included a very mixed population, only some of whom might be responsive to the intervention, thereby lowering the signal-to-noise ratio to a point where potentially valuable treatments have not been identifi ed. this possibility is the reason why accurate information about the incidence and severity of sepsis, be it in children or adults, is so important. it is also why researchers should be careful in describing exactly which patients they are talking about and in interpreting data. although sepsis is instantly recognisable (although sadly, not always as instantly as it should be), many investigators are in creasingly uneasy about using it as the basis of clinical trials. 6, 7 schlapbach and colleagues have provided valuable data showing that sepsis in children remains a signifi cant challenge, and discarding the term sepsis would not be wise. but paradoxically, meeting that challenge might mean that researchers no longer use sepsis as the starting point for clinical research in this diffi cult and frustrating fi eld. in the lancet infectious diseases, patricia schlagenhauf and colleagues 1 analyse travel-associated morbidity in 32 136 travellers presenting to european sites of the geosentinel network with a post-travel illness over the period 2008-12. knowledge about illness in travellers contributes to the understanding of disease epidemiology in our interconnected world. in addition to their own ill health, travellers serve as sentinels of infectious diseases; moreover, they can also transmit and disseminate infections. [2] [3] [4] travellers have shown these important roles in emerging infections such as sars (severe acute respiratory syndrome), dengue virus infection, chikungunya infection, and currently ebola virus disease, and also in antibiotic resistance. [3] [4] [5] [6] the geosentinel surveillance network, a collaboration between the international society of travel medicine and the us centers for disease control and prevention, has described signifi cant disease epidemiology in travellers, and has also identifi ed emerging infections through travellers such as sarcocystis on tioman island and the ongoing dengue outbreak in luanda, angola, in 2013. [7] [8] [9] [10] the strength of the geosentinel data, including the eurotravnet data analysed by schlagenhauf and colleagues, is the large number of reported cases from diff erent parts of the world. the weakness, however, is that they are mainly descriptive and variations in disease patterns over the years might be attributed to evolving travel patterns including changes in destinations and itineraries. thus, shifts in disease trends should be inter preted with caution because they might simply be related to changing exposure. moreover, some diseases (eg, dengue fever) also occur in countries not reporting to geosentinel, simply because they are not tourist destinations. even with these inherent shortcomings in the dataset, the fi ndings that tuberculosis transmission within europe remains a problem and that dengue transmission has occurred in europe provide new and very valuable insights into the transmission dynamics of these infections. the analysis of subgroups is an additional strength of the study showing reduced rates of malaria, hiv, and hepatitis type a in travellers who received pre-travel advice. the authors identifi ed travel to sub-saharan africa and asia to be signifi cantly associated with acquisition of travel-related illnesses; data for immigration travel also showed increased acquisition in travellers from africa. 1 interestingly, the trends over time showed an increased proportionate morbidity of vector-borne diseases, particularly malaria and dengue, from 2008 to 2012. further, the authors show the association between dengue virus and certain destinations and countries of origin, highlighting the popular destinations for various nationalities. previous analysis of dengue from the geosentinel network detected an increasing trend in travellers before national outbreak trends came to public recognition. 11 the detailed information provided by the authors might help national health authorities to formulate optimum advice for their travellers. the patterns of disease shown by the european sites relate to global disease outbreaks-eg, the predominance of infl uenza h1n1 pandemic among respiratory infections in 2009. 1,12 another relevant and timely example is chikungunya, which emerged in 2004 in the island countries in the indian ocean including comoros and reunion, and subsequently aff ected india, sri lanka, maldives, southeast asia, and more recently the americas and pacifi c islands. 10 the eurotravnet data parallel such developments in the localities aff ected, again showing the utility of travellers as sentinels of emerging infections. because chikungunya was fi rst documented in the caribbean in december, 2013, travellers with exposure in the americas would not have been included in this report by schlagenhauf and colleagues. importantly, the association of pre-travel consultation with signifi cantly reduced proportionate morbidity for plasmodium falciparum, hiv, and aids validates the goals of travel medicine practice. the fi nding that smaller proportions of travellers visiting friends or relatives obtained pre-travel advice compared with tourist travellers supports policies aimed at improving the provision of pre-travel interventions for travellers visiting friends or relatives. despite all its merits, a fi nal challenge remains. that is, the analysis cannot derive actual risk of acquiring each travel-related infection in view of its method of sampling through a consortium of travel and tropical medicine centres, which lacks a denominator-that is, the population at risk. nevertheless, this report on european travellers provides valuable information: an association of illness when travelling to sub-saharan africa and asia; the potential of acquiring endemic infections even when travelling to other parts of europe; an increased reporting of some vector-borne infections over time; the role of travellers as sentinels and disseminators of infections; and fi nally, the benefi ts of pre-travel health preparation. these fi ndings contribute to the creation of targeted pre-travel advice for particular diseases, populations, and destinations. men who have sex with men (msm) have a substantial burden of disease associated with human papillomavirus (hpv) infection, including anogenital warts, anal cancer, penile cancers, and oropharyngeal cancers. 1 however, the dynamics of hpv transmission in young msm are poorly understood. understanding of the natural history of hpv infection in men has become increasingly important for policy as more countries consider and adopt sex-neutral hpv vaccination programmes. in the lancet infectious diseases, huachun zou and colleagues 2 report high incidence of hpv infection in 200 australian adolescent and young msm aged 16-20 years (median age 19 years). over the 1 year follow-up period, they detected 48 incident defi nite hpv infections in the anus and ten incident defi nite hpv infections on the penis. defi nite incidence rate per 100 person-years for any anal hpv infection was 57 (95% ci 46-68), and 12 (6-21) for any penile hpv infection. the authors estimated per partner transmission through comparison with data from a study by goldstone and colleagues, 3 reporting higher probability from the penis to the anus than from the anus to the penis. the transmission estimates are the best we have for adolescent msm and the best data for this population that we will have any time soon. the data reported are best seen as rough estimates, albeit very good ones, in view of the many diff erences between the two samples. it will be important as others cite and use these data to keep some of their limitations in mind. first, estimates of partner incidence from goldstone and colleagues 3 are of a somewhat older sample of msm than those used by zhou and colleagues. 2 only half of the zou and colleagues' sample reported older sexual partners. furthermore, although the entire sample used by zou and colleagues was from australia, 2 only 15% of the goldstone sample was from australia, 3 and they had markedly lower prevalence of hpv infection compared with the rest of the study sample. second, the transmission estimates do not account for penile to penile transfer of hpv, such as through frottage. third, 4-6 years separate the collection of data in the two studies. these diff erences could bias estimates to be somewhat higher or lower than true rates. despite these and other potential limitations that zou and colleagues note, their data are novel and valuable. hpv transmission estimates are useful for many reasons, including to increase the precision of hpv vaccine cost-eff ectiveness models. 4, 5 zou and colleagues' fi ndings can inform policy decisions for the several countries that are debating routine provision of hpv vaccine to boys and men. hpv vaccination programmes that target young msm are appealing because they have higher risk for hpv-related disease than do other young men and are thus especially likely to receive benefi t from vaccination. 6 however, evidence suggests that risk-based vaccination strategies are not successful. for example, the usa abandoned risk-based vaccination when national eff orts to give hepatitis b vaccine to msm we declare no competing interests. travel-associated infection presenting in europe (2008-12): an analysis of eurotravnet longitudinal, surveillance data, and evaluation of the eff ect of the pre-travel consultation the traveller and emerging infections: sentinel, courier, transmitter the role of the traveler in emerging infections and magnitude of travel ndm-1 and the role of travel in its dissemination chikungunya: establishing a new home in the western hemisphere ebola virus disease outbreak-nigeria for the geosentinel surveillance network. spectrum of disease and relation to place of exposure among ill returned travelers for the geosentinel surveillance network. geosentinel surveillance of illness in returned travelers acute muscular sarcocystosis: an international investigation among ill travelers returning from tioman island detection on four continents of dengue fever cases related to an ongoing outbreak in luanda for the geosentinel surveillance network. seasonality, annual trends, and characteristics of dengue among ill returned travelers geneva: world health organization key: cord-274824-kaefedl1 authors: turski, waldemar a.; wnorowski, artur; turski, gabrielle n.; turski, christopher a.; turski, lechoslaw title: ahr and ido1 in pathogenesis of covid-19 and the “systemic ahr activation syndrome:” a translational review and therapeutic perspectives date: 2020-09-24 journal: restorative neurology and neuroscience doi: 10.3233/rnn-201042 sha: doc_id: 274824 cord_uid: kaefedl1 covid-19 is the acute illness caused by sars-cov-2 with initial clinical symptoms such as cough, fever, malaise, headache, and anosmia. after entry into cells, corona viruses (cov) activate aryl hydrocarbon receptors (ahrs) by an indoleamine 2,3-dioxygenase (ido1)-independent mechanism, bypassing the ido1-kynurenine-ahr pathway. the ido1-kynurenine-ahr signaling pathway is used by multiple viral, microbial and parasitic pathogens to activate ahrs and to establish infections. ahrs enhance their own activity through an ido1-ahr-ido1 positive feedback loop prolonging activation induced by pathogens. direct activation of ahrs by cov induces immediate and simultaneous up-regulation of diverse ahr-dependent downstream effectors, and this, in turn, results in a “systemic ahr activation syndrome” (saas) consisting of inflammation, thromboembolism, and fibrosis, culminating in multiple organ injuries, and death. activation of ahrs by cov may lead to diverse sets of phenotypic disease pictures depending on time after infection, overall state of health, hormonal balance, age, gender, comorbidities, but also diet and environmental factors modulating ahrs. we hypothesize that elimination of factors known to up-regulate ahrs, or implementation of measures known to down-regulate ahrs, should decrease severity of infection. although therapies selectively down-regulating both ahr and ido1 are currently lacking, medications in clinical use such as dexamethasone may down-regulate both ahr and ido1 genes, as calcitriol/vitamin d(3) may down-regulate the ahr gene, and tocopherol/vitamin e may down-regulate the ido1 gene. supplementation of calcitriol should therefore be subjected to epidemiological studies and tested in prospective trials for prevention of cov infections, as should tocopherol, whereas dexamethasone could be tried in interventional trials. because lack of physical exercise activates ahrs via the ido1-kynurenine-ahr signaling pathway increasing risk of infection, physical exercise should be encouraged during quarantines and stay-at-home orders during pandemic outbreaks. understanding which factors affect gene expression of both ahr and ido1 may help in designing therapies to prevent and treat humans suffering from covid-19. to trigger covid-19, sars-cov-2 enters cells via angiotensin-converting enzyme 2 (ace-2) by means of activating viral spike glycoproteins (sars-2-s) by transmembrane protease serine 2 (tmprss2) to engage them with the enzyme expressed in the surfactant secreting alveolar cells of the lung (letko, marzi, & munster, 2020; li et al., 2003) . how sars-cov-2 infection translates into systems pathology known as covid-19 remains unclear. however, infection with murine corona viruses activates the ahr in an ido1-independent manner, up-regulating expression of proviral tcdd-inducible-parp (tiparp) and modulating cytokines (grunewald, shaban, mackin, fehr, & perlman, 2020) . ahr is a nuclear receptor playing a critical role in immune and inflammatory processes and in modulating responses to the environment (veldhoen et al., 2008) . ahrs are kept in cytoplasm by chaperones hsp90 (heat shock protein 90) and xap (x-associated protein 2) and are transported into the nucleus after binding ligands to heterodimerize with arnt (aryl hydrocarbon receptor nuclear translocator), and to bind to dna to regulate gene expression. ido1 is a rate-limiting enzyme converting tryptophan to kynurenine, which is an endogenous ligand activating ahr. inflammatory factors, such as tumor necrosis factor-␣ (tnf␣) and interleukins activate ido1, whereas ahr also enhance their own activity through activation of a ido1-ahr-ido1 positive feedback loop prolonging the effects of ahr activation by other factors, including pathogens (larigot, jurice, dairou, & coumoul, 2018; neavin, liu, ray, & weinshilboum, 2018) . ahr can bind many diverse ligands including exogenous natural and synthetic, and endogenous chemicals (stockinger, di meglio, gialitakis, & duarte, 2014; stockinger, hirota, duarte, & veldhoen, 2011; wheeler, rothhammer, & quintana, 2017) . effects of clinically used drugs on gene expression mediated by ahr and ido1 in humans are hardly known. corona viruses (cov) activate ahr by an ido1-independent mechanism, bypassing the ido1kynurenine-ahr pathway, leading to mostly unrestricted up-regulation of downstream effectors such fig. 1 . ahr activation in cov-infected cells. cov activate ahr by means of ahr-activating ligand independent of ido1. upon activation ahr translocates to the nucleus to bind to genomic dna and to generate downstream effectors such as ahrr, cyps, tiparp, and cytokines. ido1 is induced by inflammatory factors, such as tnf␣ and interleukins 6 (il-6) and 1␤ (il-1␤). ahr also enhances its own activity through activation of ido1-ahr-ido1 positive feedback loop prolonging the effects of ahr activation by other pathways. activation of ido1 leads in immune cells to release of kynurenine, a tryptophan metabolite, which is an endogenous ligand activating ahr. exogenous ligands binding to ahr are dioxins such as tcdd. as pro viral factor tiparp, and to the modulation of cytokine gene expression, specifically, interleukin 1␤ (il-1␤), il-10, and tnf-␣ ( fig. 1) , which is consistent with the role for ahr activation in the host response to cov infection grunewald, shaban, mackin, fehr, & perlman, 2020; neavin, liu, ray, & weinshilboum, 2018) . this ability of cov allows other pathogens and comorbidities to enhance intensity of its infection by activating ahr via an ido1-dependent mechanism, also leading to inflammation. on the other hand, this ability to activate ahr by an ido1-independent mechanism prevents cov-induced infection from activating host immune-suppressive effects which depend on kynurenines to limit inflammation (jaronen, & quintana, 2014) . when cov infection persists, inflammation activates ido1 by massively releasing cytokines (chen et al., 2019; larigot, jurice, dairou, & coumoul, 2018; neavin, liu, ray, & weinshilboum, 2018) . this, in turn, perpetuates an already extensive viral activation of ahr by additional release of kynurenine. at that time self-limiting control mechanisms of the host immune response may derail due to intensity of ahr activation by multiple acting mechanisms, and a cytokine deluge may begin (fig. 2) . fig. 2 . activation of ahr signaling pathways contributing to pathogenesis of cov infections: a hypothesis. to induce inflammation (left upper panel), cov activate ahr by an ido1-independent mechanism resulting in up-regulation of downstream effectors such as tiparp (tcdd-inducible-parp), cytokines including interleukin 1␤ (il-1␤) and il-6, and tumor necrosis factor alpha (tnf-␣). characteristic clinical symptoms of early inflammation are congestion, fever and cough (left lower panel) complemented by fatigue, headache, anosmia, muscle aches, sore throat, nausea, diarrhea, and skin rashes. to induce thromboembolism (middle upper panel), another set of downstream effectors such as tf (tissue factor) and pai-1 (plasminogen activator inhibitor 1) need to be up-regulated. key clinical symptoms possibly indicating pulmonary, cardiovascular, or neurological complications are shortness of breath, chest pain, and confusion (middle lower panel) often accompanied by bluish lips and inability to wake. in addition, fibrosis with multiple organ injury develops when ahr activates cyp1a1/il-22 signaling pathway with stat3 (signal transducer and activator of transcription 3) promoting fibrogenesis and fibrosis (right upper panel). critical factors allowing early diagnosis are specific tissue biomarkers indicating organ damage such as troponin for the heart. in critically ill patients infected with cov multiple ahr signaling pathways may be activated simultaneously resulting in the systemic ahr activation syndrome (saas). critically ill patients infected with sars-cov-2 develop severe multisystem inflammatory syndrome consisting of respiratory distress, cardiovascular dysfunction, thrombosis, neurological manifestations, dysregulated inflammation, and fibrosis (leisman, deutschman, & legrand, 2020; oxley et al., 2020) . clinical experience is teaching us that individuals with sars-cov-2 infections are at high risk of fatal thromboembolism (wichmann et al., 2020) including the brain (oxley et al., 2020) . how sars-cov-2 infection can trigger thromboembolism also in young and formerly healthy humans is not understood. however, since cov activates ahr, it may also lead to up-regulation of downstream effectors such as tissue factor (tf) and plasminogen activator inhibitor 1 (pai-1) in endothelial cells, consistent with the role for ahr activation of ahr-tf/pai-1 axis as a part of the host response to cov infection (belghasem et al., 2019) . if the inflammatory vicious circle due to cytokine elevation persists and the ido1-kynurenine-ahr pathway becomes activated, the intensity of ahr activation may increase and thromboembolism may occur in different organs (fig. 2 ). individuals suffering from sars-cov-2 infections are also at high risk of multiple organ fibrosis including the lung, heart, kidney, and gastrointestinal tract organs (babapoor-farrokhran et al., 2020; ye, zhang, wang, huang, & song, 2020) . although ahr mainly contributes to metabolism of xenobiotics, it also regulates expression of genes involved in fibrogenesis (stevens, mezrich, & bradfield, 2009 ). after binding ligands, ahrs heterodimerize with arnt, and are transported into the nucleus to bind with xenobiotic response elements (xres) in promoter regions of the target genes including cyp1a1, cyp24a1, and il-22 (weidenbusch et al., 2017) . subsequently, il-22 binds to its receptor and after intermediate steps, phosphorylated stat3 (signal transducer and activator of transcription 3) acts as a transcription factor for ahr/il-22 signaling pathway (weidenbusch et al., 2017) and dysregulated fibrogenesis in multiple organs can begin, provided activation of ahrs persists (fig. 2) . most common adverse pregnancy outcome during the first two trimesters in women infected with cov is preterm birth (di mascio et al., 2020) . miscarriage, cesarean delivery, and perinatal death may occur more often, too (di mascio et al., 2020) . when infection manifests during the third trimester of pregnancy, such adverse outcomes may not occur (yan et al., 2020) . ahr is densely expressed in the human placenta and the ahr gene is up-regulated in placentas of women suffering from unexplained miscarriage (fan, su, yang, & zhao, 2017) . in that context, activation of placental ahrs by cov can trigger release of cytokines by placenta and fetus leading to activation of cyclooxygenase-2 (cox2) in trophoblasts and amniotic epithelial cells and release of prostaglandins such as pge2 and pgf2␣ (li et al., 2015) . we hypothesize that if the activation of ahrs during cov infection persists, activation of ahr-cox2/pg pathway may, in sensitive individuals, initiate myometrial contractions and lead to preterm birth (li et al., 2015) . protracted infection with sars-cov-2 may lead to injuries of multiple organs including the lung, heart, kidney, and liver (zheng, ma, zhang, & xie, 2020) . cov infected children can also develop such multiple organ injuries (cui et al., 2020) . during early infection, translocation of sars-cov-2/ace-2 complexes into the lung alveolar epithelial cells, or other organ epithelial cells, and massive intracellular viral replication (cava et al., 2020 ) may lead to cell death due to activation of ahrs leading to increased expression of cyp1 family genes such as cyp1a1, cyp1b1, and cyp1a2. this results in initiation of apoptosis via activation of caspases dependent pathways, and to ace-2 down-regulation, which after release of viruses from infected host cells offers a new entry establishing a multiple positive feedback loop accelerating infection (abassi, assady, khoury, & heyman, 2020) . during late phase of the disease, cytokine elevation related to persistent activation of ahrs by cov, can also induce tissue injury in diverse organs, and contribute to worsening of clinical manifestations and disease outcomes. cardiomyocytes exposed to tcdd, an ahr agonist, also respond with increasing cyp1a1 mrna and protein expression in an ahr dependent manner suggesting activation of the ahr/cyp1a1 signaling pathway (zhou et al., 2017) . therefore, we hypothesize, that the ahr/cyp1 signaling pathway may be activated by cov in organs undergoing tissue injuries during both early and late stages of disease, and that elevated cytokines such as il-6, il-8 or il-1ß (leisman, deutschman, & legrand, 2020 ) may activate diverse signal cascades causing and accelerating tissue injury. one such mechanism involves the notch-ahr-il22 pathway regulating tissue homeostasis (weidenbusch et al., 2017) . in macrophages, notch signaling enables production of il-6, which in turn amplifies notch-dependent signaling establishing another positive feedback loop accelerating synthesis of il-6 and perpetuating the notch-ahr-il-22 signaling pathway (weidenbusch et al., 2017) . if the inflammatory deluge due to cytokine elevation persists, multiple positive feedback loops become activated whereas the inhibitory feedback loops become gradually inactive. then the intensity of ahr activation may further increase and tissue damage may occur in different organs. in addition, ahr controls organ homeostasis by governing epithelial cell networks such as intestinal, alveolar, bronchial, or tubular, providing a barrier against toxins and pathogens, by supporting their integrity by means of preventing malignant transformation and allowing for their timely renewal (rothhammer, & quintana, 2019) . in intestine, activation of ahr by ido1 induced by oxazoles can suppress anti-inflammatory action of il-10 and lead to inflammation mediated by il-13 and ifn␥-producing killer t cells (iyer et al., 2018) . whether such a mechanism can be activated by viral activation of ido1 or ahr or both, and whether it may be functional in organs other than intestine, is not known. changes in smell and taste perceptions belong to the earliest symptoms indicating viral invasion of the nervous system by cov in humans (lechien et al., 2020; zubair et al., 2020) . however, olfactory sensory neurons do not express either ace-2 or related serine protease tmprss2 genes allowing sars-cov-2 passage through neuronal membranes (brann, tsukahara, weinreb, logan, & datta, 2020) . since cov including sars-cov-2 enter neurons and may induce brain injury in humans (zhou, zhang, wang, & gao, 2020) , and since in transgenic mice lacking ace-2, sars-cov-1 can enter the brain via olfactory bulbs and spread transsynaptically over many days towards interconnected cortical subfields, amygdala, hippocampus, thalamus, midbrain, periaqueductal gray, and pons causing neuronal death (netland et al., 2008) , we suspect an alternative non-ace-2 dependent entry mechanism allowing neuronal invasion by cov. when neuronal entry is established, sars-cov-2 will activate ahrs, as it does in non-neuronal cell populations, thereby increasing gene expression of the cyp1 family such as cyp1a1, cyp1b1, and cyp1a2 and initiate apoptosis via activation of caspases (rzemieniec et al., 2019) , resulting in progressive death of neurons and brain injury (zhou, zhang, wang, & gao, 2020) . it is intriguing that mechanisms involving activation of ahr in neurons leading to apoptotic brain injury have been confirmed in experimental models of stroke (cuartero et al., 2014) . the brain is complex compared to other organs thereby preventing neurotropic viruses from invading it all at once due to morphological organization and cellular diversity, long distances between different neuronal populations, and synaptic traps set between interconnected parts of it. as the silent and slow journey of cov through the brain towards the respiratory center continues, cell damage accumulating in initially affected neuronal populations can influence sensitivity of next targeted neuronal populations. since brain injury reported by netland et al. (2008) in sars-cov-1 infected mice was similar to that triggering spontaneous focal seizures in rats (cavalheiro et al., 1991) , we hypothesize that brain injury cumulating over time in infected individuals may lead to silent limbic seizures, or induce seizure threshold changes. it is intriguing, that even overt convulsions can occur in covid-19 patients (hepburn et al., 2020) and that careful analyses of cohorts of critically ill patients suffering from covid-19 are confirming such observations (roman et al., 2020), which, however, are still rare and require more attention during follow-up studies. brain stimulation studies in humans indicate that neuronal limbic/paralimbic networks, which are targeted by infection with cov in mice (netland et al. 2008) , and consist of amygdaloid and hippocampal neuronal networks, drive in humans ictal central apnea by affecting the periaqueductal gray (lacuey, hampson, harper, miller, & lhatoo, 2019) . the periaqueductal gray is responsible for modulation of pre-bötzinger complex neurons, which control the eupneic respiratory rhythm (subramanian, & holstege, 2013) . since periaqueductal gray is targeted by cov (netland et al., 2008) and it may also be affected by a dysfunctional limbic/paralimbic breathing modulation network in humans (lacuey, hampson, harper, miller, & lhatoo, 2019) , we hypothesize that in critically ill patients cov may reach the medulla and the pre-bötzinger complex, which contains respiration regulating cholinergic neurons (carey, dunn, & gaspari, 2013) , and contribute to their activation, apnea, and death. stimulation of the caudal part of the ventrolateral periaqueductal gray in rats inhibits both pre-inspiratory cells and the diaphragm leading to respiratory failure lacking dyspnea (subramanian & holstege, 2013) . if cov reach periaqueductal gray and enter cholinergic neurons in the pre-bötzinger complex, activation of ahr may also suppress activity of acetylcholinesterase (ache) in these neurons (xie et al., 2013) and induce stimulation of effector cells, eventually leading to inhibition of preinspiratory cells and apnea. early analyses indicate that exposure to air pollutants may complicate the course of covid-19 and lead to more severe and lethal outcomes (domingoa, & rovira, 2020) . atmospheric particulate matter air pollutants such as polycyclic aromatic hydrocarbons and dioxins, but also nitrogen oxide, ozone, and carbon monoxide have deleterious effects on the respiratory system in humans, and ahr is a target for several of them (o'driscoll, & mezrich, 2018) . individuals chronically exposed to such pollutants may be at higher risk of contracting viral infections since ahrs determine innate and adaptive immunity, and govern transcription of xenobiotic metabolizing enzymes such as cytochrome p450 with cyp1a1 and cyp1b1 (tsatsakis et al., 2020) . since ahr signaling is dependent on multiple proteins, activation of ahrs by urban dust, polycyclic aromatic hydrocarbons, or diesel exhaust may lead to up-regulation of multiple sets of downstream effectors promoting different pathologies and making humans more prone to pathogens including cov (tsatsakis et al., 2020) . another set of issues arises from clinical experience with sars-cov-2 indicating that severity of the disease in humans may vary from minimal to critically ill and disease duration from few days to many weeks. assuming that during clinically active covid-19 ahrs remain maximally activated poses the question of long-term sequelae of covid-19, which needs to be addressed by epidemiological studies in survivors. relevant clues guiding towards answers may arrive from epidemiological studies on human exposure to the agent orange contaminated with dioxin tcdd, which is an ahr agonist, pointing to increased prevalence of cancers, endocrine disorders, neurological disorders, pulmonary diseases, and liver cirrhosis (yi, hong, ohrr, & yi, 2014) . since cov persistently activate ahrs, this may lead to up-regulation of multiple sets of downstream effectors resulting in different pathologies (fig. 2) depending on time after infection, individuals overall state of health, comorbidities, and environmental factors affecting ahrs. we believe it is therefore appropriate to describe this disease as a systemic ahr activation syndrome (saas), which can manifest in an acute (current pandemic), and perhaps later, in a chronic form, in survivors. since cov initially bypass the common ido1kynurenine-ahr pathway, factors which activate ahr directly may facilitate infection. eliminating such factors may be critical for prophylaxis but also for therapy of cov infections. when cov enter the human body and symptoms are mild or disease is subclinical xu et al., 2020) , we hypothesize that cov activate ahr, but the ido1kynurenine-ahr pathway may be inactive (fig. 1 ). if so, the fate of viral infection may depend on the state of background ahr activation, which is determined by environmental pollutants, gender, age, diet, physical activity, and comorbidities. we hypothesize that as long as ahr remains activated due to cov infection and the symptoms are mild, eliminating factors known to up-regulate ahr, or implementing means known to down-regulate ahr should decrease severity of infection. when infection by cov has been fully established, and physicians care for patients suffering from severe symptoms oxley et al., 2020; xu et al., 2020) , we hypothesize that the ido1-kynurenine-ahr pathway is continuously activated due to protracted inflammation in addition to cov mediated activation of ahr. such a vicious circle can only be efficiently interrupted by simultaneous down-regulation of both ahr and ido1 (fig. 1) . a medication addressing activity of both ahr and ido1 simultaneously is dexamethasone (ott et al., 2015; vrzal et al., 2009 ), a glucocorticoid with a broad range of uses including asthma, chronic obstructive pulmonary disease, and rheumatoid arthritis. ahr was discovered as a receptor binding and mediating toxicity of the environmental dioxin tcdd (2,3,7,8-tetrachlorodibenzo-p-dioxin) (okey, 2007; opitz et al., 2011) . this initial observation may indicate that humans frequently exposed to environmental and other toxins activating ahr such as dioxins or pesticides may be more prone to, and at risk of, contracting infection due to cov more easily. surprisingly, calcitriol (vitamin d 3 ) in addition to its main mechanisms of action, may down-regulate expression of the ahr gene (takami, fujimaki, nishimura, & iwashima, 2015) . more surprisingly, deficiency of vitamin d 3 has indeed been reported to contribute to acute respiratory distress syndrome with fatalities increasing with age and comorbidities, both being associated with lower concentrations of vitamin d (grant et al., 2020) . the concentration of vitamin d decreases with age leaving the elderly particularly prone to viral infections including sars-cov-2, which is reflected in the pattern of covid-19 during the current pandemic. if confirmed in epidemiological studies, supplementation of vitamin d during winter should be recommended to all, and particularly in the elderly since vitamin d may down-regulate the ahr gene (takami, fujimaki, nishimura, & iwashima, 2015) , therefore decreasing susceptibility to viral infections. ido1 is a cytosolic, rate-limiting enzyme in the conversion of tryptophan, via kynurenine, which is a ligand of and activates ahr (jaronen, & quintana, 2014) , towards synthesis of nicotinamide (mellor, lemos, & huang, 2017) . activation of ido1 depresses t lymphocytes activation by starving them of tryptophan through its conversion to kynurenine (neavin, liu, ray, & weinshilboum, 2018) . ido1 gene transcription can be up-regulated by multiple viral, microbial and parasitic pathogens, cancers, or pregnancy (benavente et al., 2019; larigot, juricek, dairou, & coumoul, 2018; neavin, liu, ray, & weinshilboum, 2018) or it can be induced by the proinflammatory cytokine inf-␥ (neavin, liu, ray, & weinshilboum, 2018) limiting host immune response by means of generation of foxp3(forkhead box p3)expressing regulatory t cells to inhibit other cells of the immune system (larigot, juricek, dairou, & coumoul, 2018; neavin, liu, ray, & weinshilboum, 2018) . the most popular drug down-regulating ido1 is tocopherol/vitamin e (stapelberg et al., 2013) . vitamin e is an antioxidant that scavenges radicals, terminates oxidation of polyunsaturated fatty acids, and has multiple immunomodulatory actions such as on lymphocyte proliferation, prostaglandin e2 synthesis, affects gene transcription and translation, and it influences cell membrane and signal transduction (lee, & han, 2018) . it is intriguing that up to 60% of adults in the united states have too low vitamin e intake (<12 mg/d) (fulgoni, keast, bailey, & dwyer, 2011) . drawbacks of such a low intake are dysregulation of immune and inflammatory responses and decreased resistance to infections with elderly humans being hit the most. this is due to the fact that aging pathogen defense-related immune responses are weaker and inflammatory responses prolonged (meydani, lewis, & wu, 2018) . it is therefore conceivable, that insufficient supply with vitamin e may contribute to increased susceptibility in the elderly to infection with cov and may promote severity of covid-19. supplementation of vitamin e in elderly could reduce the risk of, and morbidity from, infections with cov. this has intuitively been recognized in great britain where covid-19 patients were supplemented with vitamins (calder, carr, gombart, & eggersdorfer, 2020) . in healthy conditions, tryptophan is metabolized to kynurenine by tryptophan-2,3-dioxygenase (tdo) in the liver and by ido1 in the brain and peripheral tissues such as small intestine, lungs, female genital tract or placenta (badawy, 2017; castro-portuguez, & sutphin, 2020; sedlmayr, 2007) . in addition, significant amount of kynurenine is metabolized by exercising muscles (agudelo et al., 2019; martin, azzolini, & lira ruas, 2020 ) and a small amount is used to activate ahr in response to minor inflammatory processes (badawy, 2017) . disrupting this healthy balance by immobility such as through stayat-home orders, may lead to an increase in kynurenine and its availability to activate ahr, therefore increasing an individual's risk to become prone to infection (agudelo, 2019; martin, azzolini, & lira ruas, 2020) . therefore, physical exercise during quarantine should be encouraged. in advanced disease, if liver function decreases and ceases ) and muscles are not stimulated due to immobilization in the hospital, ahr may become overwhelmed with kynurenines and activated via the ido1-kynurenine-ahr pathway. if such patients are subjected to intensive therapy in the icus, principles of care established to excel under regular conditions should perhaps be expanded by following biomarkers of tryptophan metabolism related to inflammation (badawy, & guillemin, 2019) . persistent activation of ahrs by cov may lead to up-regulation of multiple sets of downstream effectors governed by ahrs resulting in different phenotypic disease pictures depending on time after infection, individuals overall state of health, comorbidities, and environmental and other factors modulating ahrs. cov are perfect viruses invading hosts by avoiding collision with other pathogens because of activating the ahr when bypassing the ido1-kynurenine-ahr pathway. this pathway is employed by multiple, viral, microbial and parasitic pathogens to establish infection. this peculiarity enables massive upregulation of multiple ahr-dependent downstream effectors, whereas self-restricting mechanisms may remain at bay. therapies selectively down-regulating both ahr and ido1 are currently lacking and should be considered in the future by public and private innovation driven research organizations as therapies for viral infections. however, such therapies should be designed to leave physiological functions of both ahrs and ido1 intact. since dexamethasone may down-regulate both ahr and ido1 genes, in addition to its main mechanism of action, and is known to play a role in inflammatory diseases, its use in therapy of covid-19 should be studied in rigorous interventional trials. since calcitriol may down-regulate the ahr gene and is known to play a role in the spread of other viral infections, supplementation of calcitriol should be reviewed in epidemiological studies and tested in prospective trials for prevention of sars-cov-2 infections. supplementation of tocopherol should be reviewed in epidemiological studies and tested in prospective trials for prevention of sars-cov-2 infections in elderly since tocopherol may down-regulate ido1 gene and is known to play a role in response to viral infections and inflammation in aging. associations and conclusions revealed by our analyses should be considered with particular caution because we are reporting changes in the expression of genes only. such changes need to be translated into biological actions by multiple associated mechanisms, which have not been evaluated in our analyses. therefore, our hypotheses offer no information or confirmation of clinical therapeutic value of any discussed therapy in modulating of sars-cov-2 infection. before any conclusion can be drawn regarding a potential benefit of our recommendations regarding any medication in patients with covid-19 randomized controlled clinical trials and large observational population studies evaluating our concepts will be necessary. angiotensin-converting enzyme 2: an ally or a trojan horse? implications to sarscov-2-related cardiovascular complications skeletal muscle pgc-1␣1 reroutes kynurenine metabolism to increase energy efficiency and fatigue-resistance myocardial injury and covid-19: possible mechanisms kynurenine pathway of tryptophan metabolism: regulatory and functional aspects the plasma 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to reevaluate the correlation between long-term effects of anthropogenic pollutants on viral epidemic/pandemic events and prevalence the aryl hydrocarbon receptor links th17-cell mediated autoimmunity to environmental toxins dexamethasone controls aryl hydrocarbon receptor (ahr)-mediated cyp1a1 and cyp1a2 expression and activity in primary cultures of human hepatocytes gene expression profiling of the notch-ahr-il22 axis at homeostasis and in response to tissue injury control of immune-mediated pathology via the aryl hydrocarbon receptor autopsy findings and venous thromboembolism in patients with covid-19:a prospective cohort study ahr-mediated effects of dioxin on neuronal acetylcholinesterase expression in vitro pathological findings of covid-19 associated with acute respiratory distress syndrome coronavirus disease 2019 (covid-19) in pregnant women: a report based on 116 cases covid-19): a pictorial review agent orange exposure and disease prevalence in korean vietnam veterans: the korean veterans health study liver injury in covid-19: management and challenges covid-19 and the cardiovascular system mitochondrial activity and oxidative stress functions are influenced by the activation of ahr-induced cyp1a1 overexpression in cardiomyocytes sars-cov-2: underestimated damage to nervous system. travel medicine and infectious disease neuropathogenesis and neurologic manifestations of the coronaviruses in the age of coronavirus disease 2019 imbalanced host response to sars-cov-2 drives development of covid-19 genevestigator v3: a reference expression database for the meta-analysis of transcriptomes ontology-based meta-analysis of global collections of high-throughput public data ahr and ido1 in covid-19 query was limited to mrnaseq data from human lung tissues and cell lines. the results are marked with yellow background. it was revealed that significant up-regulation of ahr and ido1 occurred in the lung adenocarcinoma cell line calu-3 samples infected with sars-cov-2 (usa-wa1/2020 isolate) at a moi (multiplicity of infection) of 2 and harvested 24 h post-infection (hpi) versus mock-infected calu-3 cells. similar observation was made in human lung adenocarcinoma cell line a549 samples infected with respiratory syncytial virus (rsv) a2 strain (moi 2; 24 hpi) in comparison to mock control a p-value significance cutoff of 0.05 (without any multiple testing correction) and a minimum absolute fold-change cutoff of 1.2 were used by the software for identification of medications affecting expression of the genes of interest. then, grading of significant factors was conducted based on the following conditions: (1) total number of medication specific studies in which the gene was measured; (2) number of medication-specific studies in which the gene's expression was found to be significantly altered; (3) gene's degree of down-or up-regulation in comparison to all other genes with altered expression within each of the medication studies; (4) consistency of the gene's association across the medication studies. additional statistical criteria were applied, such as correction for multiple hypothesis testing, to finally rank the medications. the most significant result was assigned with a numerical score of 100, and the other medications' scores were normalized to the top-ranked result. analyses were performed separately for ahr and ido1 genes. only medications significantly downregulating both genes were plotted we conducted a gene expression data mining aimed to identify conditions under which ahr and ido1 undergo simultaneous upregulation. when the search was limited to human lung-derived samples, we confirmed that increased expression of ahr and ido1 genes occurred in response to viral infections, including sars-cov-2 and respiratory syncytial virus (rsv) (fig. 3a ). this was accompanied by elevation of proinflammatory gene expression, activation of the tf/pai-1 signaling pathway, and upregulation of cyp1a1 (fig. 3a) .in parallel, we investigated drugs in clinical use for propensity of affecting the expression of both ahr and ido1 genes by means of integrative analysis of bioinformatics data collected on human and non-human tissues to discover how the suspected key drivers of infection mediated by cov and perhaps by sars-cov-2, ahr and ido1, can be affected by medications already employed to treat other human diseases.surprisingly, there are licensed drugs possessing such dual antagonistic properties (fig. 3b ) such as rosiglitazone (antidiabetic), dexamethasone and betamethasone (glucocorticoids with broad range uses including asthma, chronic obstructive pulmonary disease and rheumatoid arthritis), infliximab (tnf-␣ inhibitor), isoniazid (antituberculotic), niacinamide (vitamin b 3 , dietary supplement), levonorgestrel (progestogen), promazine (neuroleptic), curcumin (herbal dietary supplement), lidocaine (local anesthetic and antiarrhythmic), and amitriptyline (antidepressant and anxiolytic). calcitriol (vitamin d 3 ) and fluticasone (steroid used for therapy of asthma and chronic obstructive pulmonary disease), preferentially down-regulated the ahr gene in the context of this analysis (fig. 3b) .no clinical data supporting use of any medications listed in fig. 3b in therapy or prevention of cov infections currently exist, nor is it known if any, how, and in which phase of the disease these medications can be recommended for use. rigorous clinical trials are necessary before any recommendation regarding future use of these medications in therapy of cov infections, including covid-19, can be made. key: cord-310371-pylrg91h authors: bishop, r.f.; kirkwood, c.d. title: enteric viruses date: 2008-07-30 journal: encyclopedia of virology doi: 10.1016/b978-012374410-4.00386-1 sha: doc_id: 310371 cord_uid: pylrg91h many viruses use the enteric tract as a route of entry to the human, animal, or avian host. the onset of acute enteritis is associated with infection by viruses that replicate at or near the site of entry into the intestinal mucosa, including caliciviruses, rotaviruses, adenoviruses, astroviruses, and coronaviruses. these ‘enteric’ viruses occur globally and share similar features. most are rna viruses that replicate in the cytoplasm of mature absorptive epithelial cells lining the villi of the small intestine, leading to inflammation and villus atrophy. vomiting and diarrhea can result in dehydration and death if untreated. despite abundant growth in vivo, they initially proved difficult or impossible to grow in vitro. most are genetically diverse, species specific, highly infectious within species and transmitted by the fecal–oral route. severe symptoms are most commonly associated with primary infections of young animals, and are followed by short-lived immunity. reinfections are common throughout life, but are often only mildly symptomatic. safe and effective vaccines have been developed to prevent severe rotavirus disease in young children. in addition to these enterotropic viruses, enteric disease can also result from spread to the intestine of hiv or cytomegaloviruses during the later stages of systemic disease in immunocompromised hosts. the intestinal tract, lined by replicating epithelial cells, bathed in nutrient fluids and maintained at optimal temperature provides an ideal milieu for growth of many viruses. 'enteric viruses' represent a wide spectrum of viral genera that invade and replicate in the mucosa of the intestinal tract, and that can be grouped as follows: . viruses causing localized inflammation at any level of the intestinal tract, predominantly in small intestinal mucosa, resulting in acute gastroenteritis, for example, rotaviruses, caliciviruses, adenoviruses, astroviruses; . viruses that multiply at any level of the intestinal tract, causing few enteric symptoms prior to producing clinical disease at a distant site, for example, measles virus, reoviruses (in mice) , enteroviruses (including polioviruses, coxsackieviruses, enteroviruses, hepatitis a and e); and . viruses that spread to the intestinal tract during the later stages of systemic disease, generally in an immunocompromised host, for example, human immunodeficiency virus (hiv), cytomegalovirus. this article focuses upon the first category of viruses that cause enteric disease associated with primary replication in the intestinal tract. acute gastroenteritis is one of the most common health problems worldwide. more than 700 million cases are estimated to occur annually in children less than 5 years of age, resulting in few deaths in developed countries, but more than 2 million deaths in developing countries. worldwide, a diverse group of viral, bacterial, and parasitic pathogens cause acute enteric symptoms including nausea, vomiting, abdominal pain, fever, and acute diarrhea. infections with viral agents, unlike those with bacterial or parasitic pathogens, cannot be treated with antibiotics, and many cannot be prevented by improvements in quality of drinking water, food, or sanitation. until the early 1970s most viral agents causing gastroenteritis in humans were largely unknown. studies using electron microscopy of intestinal contents resulted in the discovery of numerous viral enteropathogens now classified as caliciviruses, rotaviruses, astroviruses, or 'enteric' adenoviruses. caliciviruses are now recognized as the most important cause worldwide of outbreaks of viral gastroenteritis in humans of all age groups. rotaviruses are the single most important cause of life-threatening diarrhea in children <5 years old. astroviruses and adenoviruses also cause severe diarrhea in children. table 1 lists the characteristics of the major viruses associated with acute gastroenteritis. other viruses linked to gastroenteritis in humans include coronaviruses, toroviruses, picornaviruses, and picobirnaviruses. understanding many features of these 'enteric viruses' has been based on parallel studies of related viruses infecting animals. the family caliciviridae contain small rna viruses that cause enteric disease in a wide variety of hosts including cattle, pigs, rabbits, and humans. infections in other hosts, for example, sea lions, cats, and primates, appear to cause predominantly systemic and respiratory symptoms. caliciviruses are small nonenveloped viruses of 27-35 nm diameter ( figure 1) with a genome comprising a single-stranded positive-sense polyadenylated rna genome of 7400-7700 nucleotides (nt). typical calicivirus particles show cup-like hollows (calices) on the virus surface. some caliciviruses have an indistinct appearance described as 'feathery'. the 'norwalk agent' (now classified as a calicivirus) was identified in 1972 by kapikian and colleagues, using immune-electron microscopy (iem) to search for the causative agent of an 1968 outbreak of gastroenteritis in humans in norwalk, ohio, usa. many related viruses have been implicated as causes of gastroenteritis, and given names identifying the geographical location of the outbreak (e.g., marin county, snow mountain, hawaii, sapporo). classification of caliciviruses was hampered for many years by the inability to culture these viruses, and study their genetic and protein structure. caliciviruses causing enteric infections (in humans and other animals) are classified as belonging to the family caliciviridae, which is divided into four genera. the genus norovirus (nov), and genus sapovirus (sav) cause human and animal infections. other genera infect rabbits (lagovirus) or sealions, cats, and primates (vesivirus). the genome organization and reading frame usage differs between the four genera. for noroviruses, the genome contains three open reading frames (orfs). the largest (orf1) encodes a polyprotein which undergoes proteolytic cleavage to produce an ntpase, a 3c-like protease, and an rnadependent rna polymerase (rdrp). orf2 encodes the major capsid protein and orf3 a putative minor capsid protein. noroviruses are subdivided into five genogroups (gi-gv). genogroups gi, gii, and giv infect humans, giii infects cattle and gv infects mice. gi and gii contain at least 10 and 20 distinct genetic clusters, respectively. sapoviruses are divided into genogroups (gi-gv), of which gi, gii, giv, and gv infect humans. gi and gii are further divided into four and three genetic clusters. the inability to culture human caliciviruses delayed the introduction of diagnostic tests, resulting in an under-appreciation of the significance of these agents for many years. currently, over 20 different reverse transcription-polymerase chain reaction (rt-pcr) assays targeting regions on the rdrp gene and the capsid gene have been described and utilized in epidemiological studies. this large genetic diversity of human caliciviruses makes routine detection difficult. human nov are the leading causes of 'nonbacterial gastroenteritis' outbreaks in all age groups worldwide. outbreaks frequently occur in communities such as nursing homes, hospitals, schools, and cruise ships. no consistent seasonal variation has been observed. infection involves transmission via person-to-person contact or ingestion of contaminated food and water. epidemiological studies have identified caliciviruses in 60-95% of outbreaks in many countries. estimates of disease burden in usa suggest that caliciviruses are responsible annually for 23 million illnesses and 50 000 hospitalizations. strains of nov gii cluster 4 (gii-4) have been the most common type identified worldwide in the past 5 years (2001) (2002) (2003) (2004) (2005) in both adults and children. prevalence rates of calicivirus (predominantly nov) in young children admitted to hospital with acute gastroenteritis in many countries range from 3.5% to 20% annually. strains of sav, while playing a minor role overall, are more generally associated with childhood gastroenteritis than with disease in older children and adults. caliciviruses may be important enteric pathogens in patients with hereditary or acquired immunodeficiency. the genera norovirus and sapovirus are genetically diverse, and multiple strains co-circulate in human populations. individual dominant strains emerge every 2-5 years, and often have a global impact, such as the gii-4 strains identified in usa, europe, japan, and australia in 1995/ 1996 and again in 2004. nov recombinant strains, with polymerase and capsid genes derived from different ancestral clusters, have been identified in thailand and australia. repeated attempts to adapt nov and sav to growth in cell culture have failed. diagnostic techniques and analysis of antigenic variation rely predominantly on molecular biological techniques. cloning and expression of the major viral capsid protein (vp1) in baculovirus expression systems has led to the formation of virus-like particles (vlps) morphologically similar to native virus and their incorporation into enzyme immunoassay (eia) assays. pathogenesis of nov and sav infection is poorly understood as a result of the long-standing inability to adapt these viruses to cell culture, and the absence of a small animal model. symptomatic enteritis in human volunteers infected with 'norwalk agent' showed changes in jejunal biopsies (mucosal inflammation, absorptive cell abnormalities, villus shortening, and crypt hypertrophy) that persisted for at least 4 days after remission of clinical symptoms and reverted to normal after 2 weeks. no identifiable viral particles were detected by electron microscopy in any affected intestinal tissue. the recent demonstration that human noroviruses can infect and replicate in a three-dimensional cell culture model of human intestinal epithelium, should improve our understanding of the pathogenesis, and antigenic diversity of this important group of enteric viruses. these studies will also be enhanced by discovery of a norovirus that infects mice, and that replicates after transfection of cultured kidney cells. mechanisms of immunity to nov are unclear. infection results in formation of igg and igm serum antibody that are broadly reactive within, but not between, genogroups. the role of these antibodies in immune protection is unknown. infected individuals can develop short-term immunity to homologous viruses but the molecular diversity of nov circulating in communities makes it difficult to predict whether long-term immunity can develop. it is unclear why a proportion of exposed individuals remain uninfected during outbreaks. recent studies suggest that histo-blood group antigens and the secretor status may be genetic susceptibility markers for infection. at present the major control strategies for prevention of human calicivirus infection rely on prevention of contamination of food and water supplies. virus particles, later classified in the genus rotavirus, were first described in 1963 by adams and kraft as a cause of epidemic diarrhea in infant mice (edim). similar particles (ncdv) were recognized in 1969 by mebus and colleagues as a cause of severe diarrhea in newborn calves in nebraska, usa. neither virus was considered relevant as a causative agent of severe diarrhea in young children until 1973 when bishop, davidson, holmes, and ruck described a 'new virus' (later shown to be antigenically related to edim and ncdv) in duodenal biopsies and diarrheal feces from young children admitted to hospital in melbourne, australia with severe acute diarrhea. named because of their wheel-like appearance in negatively stained extracts examined by electron microscopy (rota ¼ latin for wheel) rotaviruses have since become established as causes of severe acute diarrhea in the young of many mammalian and avian species worldwide. rotavirus enteritis affects all children regardless of socioeconomic status, and results in over 600 000 deaths annually in young children in developing countries. rotaviruses are nonenveloped icosahedral viruses of 70 nm ( figure 2 ) diameter that belong to the genus rotavirus within the family reoviridae. the double-stranded rna genome is contained within a triple-layer of viral proteins (vps) comprising a core (vp1, vp2, vp3), an inner capsid (vp6), and an outer capsid (vp4, vp7). rotaviruses are classified into groups a to g based on serology of the vp6 protein. the majority of human and mammalian infections, due to group a viruses, are further classified into serotypes by antigenic differences on vp7 (g-serotypes) and into genotypes by genetic differences on vp4 (p-genotypes). to date there are at least 11 of 15 g-serotypes and 15 of 26 p-genotypes identified in humans. groups b and c have been identified infrequently in humans. groups d to g have been identified only in nonhuman mammalian or avian species. group a rotaviruses infect humans and other mammals repeatedly throughout life. most primary rotavirus infections in animals occur during the neonatal period. most primary rotavirus infections in humans occur during the first 24 months of life. children worldwide will experience at least one rotavirus infection by 5 years of age. in general, group a rotaviruses are species specific. however, rotavirus strains with gene segments of feline, bovine, or porcine origin have been isolated from children, suggesting the occurrence of cross-species infection in nature. cross-species infections can be established experimentally, and comprise one of the strategies for human vaccine development. the rotavirus genome consists of 11 segments of double-stranded rna that can be separated by polyacrylamide gel electrophoresis, allowing epidemiological studies mapping the genetic diversity of strains within and between serotypes. each gene segment encodes a separate protein, with the exception of gene segment 11 which encodes two proteins. reassortment of genes between human strains and human and animal strains occurs in vivo and in vitro. rotaviruses are transmitted from person to person by the fecal-oral route, or via aerosols. rotaviruses replicate in the cytoplasm of mature nonreplicating enterocytes lining the upper portions of the small intestinal villi, eventually causing cytolysis. profuse watery diarrhea results from a combination of mechanisms including malabsorption secondary to loss of enterocytes responsible for absorption and digestion, activation of the enteric nervous system, and stimulation of intestinal secretion by the rotavirus nonstructural protein nsp4. rotavirus antigenemia and viremia occur during the acute phase of severe primary rotavirus disease. as a result, complete rotavirus particles have been found in liver, lung, spleen, pancreas, thymus, and kidneys of experimental animals. it is not clear if the virus is replicating at these sites. clinical symptoms in children are strongly influenced by age, with severe often life-threatening diarrhea occurring after primary infection in young children, and in aged people in nursing homes. excretion of rotavirus particles in detectable numbers (by eia, rt/pcr) continues for 5-10 days, and occasionally up to 50 days. excretion can continue for months in immunodeficient children and animals. reinfections occur throughout life, and are usually asymptomatic or associated with mild symptoms. symptoms of primary infection require medical attention in 1:5 children, result in hospitalization in 1:65 children and death in 1:293 (almost all in young children in developing countries). treatment is based upon replacement of fluid and electrolyte loss, usually achieved by oral administration of fluids containing glucose and electrolytes. occasionally, delayed repair of the small intestinal mucosa is associated with disaccharide or monosaccharide malabsorption leading to malnutrition. primary rotavirus infection protects against severe symptomatic disease on reinfection, and is associated with humoral and cellular immune responses to individual rotavirus proteins. neutralizing antibody to vp4 and vp7 outer capsid proteins contribute to protection, possibly by interfering with viral replication and limiting the extent of intestinal damage. the role of immune responses to other proteins is uncertain. virus-specific cytotoxic tcells are not essential for protection. the importance of rotavirus disease worldwide and its contribution to childhood mortality in developing countries has resulted in strong initiatives, supported by the world health organization, to develop live oral rotavirus vaccines to be administered to infants before 3 months of age. two contrasting vaccines, a single attenuated g1p[8] human rotavirus and a pentavalent human-bovine g1-g4,p[8] reassortant vaccine, have been proved to be safe and effective in preventing severe rotavirus disease. both have the potential to radically change global childhood mortality and morbidity. adenoviruses were first detected in 1953 in cultured fragments of tonsillar and adenoidal tissue from children. they are nonenveloped icosahedral viruses approximately 80 nm in diameter (figure 3) . the genome is composed of double-stranded dna. the family adenoviridae comprises three genera: mastadenovirus (mammalian) classified into subgenera a-f representing more than 50 serotypes, aviadenovirus (birds), and a newly recognized genus atadenovirus identified in sheep and reptiles. most are readily cultivatable. adenovirus infections occur worldwide in many mammalian species, are species specific, usually associated with disease in the respiratory, urinary, and ocular systems, and are frequently shed in feces in the absence of any gastrointestinal symptoms. in 1975, flewett and colleagues in birmingham, uk, noticed the presence of large numbers of adenovirus particles in negatively stained extracts of diarrheal stools examined by em. these proved difficult to culture, were designated 'enteric' adenoviruses (ead), and are now classified as serotypes ead40 and ead41 within subgenus group f. cultivation of ead remains difficult. the most reliable growth has been achieved in human embryonic kidney cells (293 cells) immortalized by transfection with regions of ad5. ead40 and ead41 occur worldwide, causing severe acute enteritis in 5-15% of hospitalized young children. outbreaks occur at unpredictable intervals year-round with no seasonal prevalence. nosocomial epidemics occur in day-care nurseries and in hospital wards for children and adults. group a adenoviruses (serotypes 12, 18, 31) have also been implicated in epidemics, usually in older age groups. the adenovirus virion is composed of at least 10 different structural polypeptides and contains a linear 33-45 kbp dna. virus capsomers are arranged as hexons, the corners of which have antenna-like (fiber) projections presumed involved in cell attachment. the dna genomes of groups a-f are genetically diverse, and differences can be illustrated by analysis using genome restriction endonucleases. the heterogenous genome in groups a-f makes recombination between subgenera unlikely, with exception of groups a and f which show a close evolutionary relationship. diagnoses of ead infection rely on eia that detects the hexon antigen common to groups a-f, followed by determination of restriction enzyme patterns and/or reactions in eia incorporating neutralizing, monoclonal antibodies specific for ed40 and 41. ead replicate within the epithelial cells of the small intestine. group a adenoviruses have also been grown from mesenteric lymph nodes and appendices. the mechanisms causing diarrhea are not clear, but destruction of infected epithelial cells has a role. adenovirus diarrhea is more common in infants <12 months old than in older children, and can be protracted with a mean duration of 12 days. adenovirus diarrhea occurs in immunocompromised patients. nonseasonal epidemics of ead diarrhea occur in hospital wards, orphanages, and day-care nurseries. occasional fatal cases have been reported in children. evidence from animal models (with non-ead) suggests that viremia occurs, and can lead to infection of other tissues. the natural history of disease and development of immunity is unknown. astroviruses were first described in the uk in 1975 by madeley and cosgrove studying an outbreak of diarrhea in newborn babies in an obstetric hospital nursery. astroviruses are small, round nonenveloped plus-stranded rna viruses 28-30 nm diameter (figure 4) occasionally exhibiting virions with a superficial star shape. they are members of the genus astrovirus in the family astroviridae. they have been detected in humans (children and adults) and a range of mammalian (sheep, cattle, pigs, dogs, cats, and mice) and avian (turkeys, ducks) species, usually associated with diarrhea. there are currently eight serotypes of human astrovirus, designated hastv 1-8, based on reactivity with polyclonal antisera. hastv 1 is most common worldwide. prevalence rates as a cause of diarrhea vary from 2% to 16% (hospital-based studies), and 5% to 17% (community-based studies). most astrovirus infections have been recorded during colder months in temperate climates and year-round in tropical countries. a longitudinal study in mayan children in a poor community in mexico found a high prevalence (61%) of astrovirus infection in a birth cohort of 271 children followed for 3 years. infection occurred primarily in infants <12 months old, and showed a high rate of asymptomatic infection and prolonged shedding (2-17 weeks) in many infants. astrovirus infection has also been associated with persistent diarrhea (lasting for 14 days or more) in children in bangladesh. astroviruses are widespread in developed countries, causing outbreaks in day-care centers, hospitals, and nursing homes for the elderly. they are an important cause of enteritis in immunocompromised patients. the hast genome is a polyadenylated plus-stranded rna molecule of approximately 7 kbp. the genome contains two open reading frames (orfs), orf1a and -1b, code for nonstructural proteins and orf2 encodes for the capsid protein. genetic diversity in all serotypes exists, but no association has been shown between serotypes and ability to cause severe gastroenteritis. astrovirus diagnostic assays include commercially available eia kits, electron microscopy and rt-pcr detection and genotyping of diarrheal feces. acute astrovirus infection induces a mild watery diarrhea in young children that lasts for 2-3 days and may be associated with vomiting, fever, and anorexia. the lack of a small animal model has hampered studies of the mechanism of astrovirus-induced diarrhea. experimental models of astrovirus enteritis in turkeys and in gnotobiotic lambs show mild histopathological changes in the intestine (despite high mortality from severe osmotic diarrhea) together with viremia. experimental astrovirus infection in calves is asymptomatic, with viral replication apparently targeted to m cells. it is possible that none of these animal models illuminate pathogenesis of hastv infection in humans. cultivation of hastv was initially difficult, but can now regularly be achieved using a human colon cancer derived epithelial cell line (caco2 cells). members of the genera coronavirus and torovirus are enveloped plus-strand single-stranded rna viruses belonging to the family coronaviridae. electron microscopy shows them to be pleomorphic fringed particles 100-140 nm at maximum dimension ( figure 5 ). coronaviruses and toroviruses can be distinguished by differences in peplomer structure and reaction in iem using specific antisera. coronaviruses and toroviruses cause diarrhea, respiratory, and/or hepatic disease in many animal species, including cattle, mice, swine, cats, and dogs. in general, most of these viruses are species specific and disease is most severe in infant animals. transmission is fecal-oral and due to virus lability may require close contact. coronaviruses and toroviruses have been implicated in human diarrheal disease but there is still no consensus about their importance. similar particles have been seen frequently in children without diarrhea, particularly in children in developing countries. morphological similarities between these viruses and fragments of intestinal brush border make diagnosis difficult. several studies have implicated coronaviruses as causative agents of necrotizing enterocolitis outbreaks in newborn babies. toroviruses were first described as a cause of diarrhea by woode and colleagues in 1979 when breda virus was identified in a severe outbreak of neonatal calf diarrhea in usa. toroviruses are now also known to infect horses (berne virus) and swine. human infections were first described by flewett et al. in birmingham uk in 1984, but have rarely been reported since then. the pathogenesis of diarrhea has been studied in animal models using infection with coronavirus (tge) in piglets, and with breda viruses in calves. both replicate in epithelial cells of small intestine and descending colon causing diarrhea 24-72 h later. breda virus also replicates in crypt cells. many animal coronaviruses can be propagated in cell culture. isolation of human enteric coronaviruses is difficult and serological studies can be confounded by antibody resulting from repeated respiratory coronavirus infection. enteric infection can be confirmed in feces by detection of viral rna by rt-pcr, or iem using antibodies to the viral envelope glycoproteins. picornaviruses are 24-30 nm featureless spherical particles containing single-stranded positive-sense rna. they have been found in diarrheal feces from humans but their etiological role is often not clear. the first clear evidence implicating a picornavirus as an enteric pathogen identified aichi virus, as a cause of oyster-associated epidemics of gastroenteritis in japan in 1989. aichi viruses are now classified as a new genus kobuvirus within the family picornaviridae (kobu ¼ japanese for knob). isolation of aichi virus in vero cells has permitted development of eia and rt-pcr assays based on nucleotide sequence data. serological assays show seroconversion resulting from infection and a prevalence rate for antibody of 7.2% in japanese children aged 7 months to 4 years and rising to 80% in adults by age 35. the new genus parechovirus within the family picornavirideae contains at least one serotype (previously echovirus 22) that has been implicated as an enteric pathogen in humans. parvoviruses are small 22-26 nm single-stranded dna viruses comprising a genus parvovirus in the family parvoviridae. some animal parvoviruses have been clearly linked to enteritis including bovine, feline, mink, and canine strains. canine parvovirus infection emerged after 1977. this lethal neonatal enteric infection, accompanied by viremia and widespread systemic infection, shows a pathogenesis distinct from most enteropathogenic viruses. the virus infects and destroys crypt epithelial cells resulting in flat mucosa with fused and stunted villi. damage has been likened to that caused by radiation. other small viruses, resembling parvoviruses, have been seen by em in diarrheal feces in humans. evidence linking them to causation of disease is not convincing. they have often been present as dual infections with known enteric pathogens. in addition, their resemblance to some phages makes diagnosis uncertain. in the 1950s, the use of monkey kidney cell cultures for the growth of poliovirus revealed the presence of simian viruses and further study showed some of these to have properties consistent with enteroviruses. in parallel, investigations of viruses infecting domestic animals also revealed the presence of enteroviruses (and enteroviruslike particles) in pigs and cattle. enteroviruses have since been isolated from african buffalo, water buffalo, sheep, goat, deer, and impala and many have been shown to be related to bovine enterovirus isolates. in the past, the classification of enteroviruses was primarily based upon the physico-chemical properties of virions, growth in tissue-cultured cell lines, and by serotyping. in practice, this can be difficult with some isolates being poorly recognized by the reference antisera, or, the occurrence of (misleading) cross-reactivities between serotypes. the expansion of the sequence database together with more sensitive cloning/sequencing techniques have facilitated the elucidation of the genome structures of many picornaviruses. such analyses have replaced other techniques in the classification of picornaviruses, and this article discusses characteristics which are important for the classification of animal enteroviruses. bovine enteroviruses (bevs) are endemic in cattle in many regions of the world with infection typically enteroviruses of animals picobirnaviruses are 35-41 nm particles with a bi-or tri-segmented dsrna genome and have been detected in europe, south america, and australia. they are found significantly more often in patients with hiv-related diarrhea than those without diarrhea. their role in gastroenteritis in healthy individuals remains unknown diagnosis of noncultivable gastroenteritis viruses, the human caliciviruses foodborne viral gastroenteritis: challenges and opportunities gastroenteritis viruses. novartis foundation symposium viral gastroenteritis characterisation of torovirus from human faecal specimens immunity and correlates of protection for rotavirus vaccines new hope for defeating rotavirus comparative pathology of infection by novel diarrhoea viruses human sapovirus: genetic diversity, recombination and classification prevalence and genetic diversity of human astroviruses in mexican children with symptomatic and asymptomatic infections genetics of the rotaviruses pathogenesis of intestinal and systemic rotavirus infection norwalk and other human caliciviruses: molecular characterization, epidemiology and pathogenesis in vitro cell culture infectivity assay for human noroviruses the subgroup f adenoviruses application of a reverse transcription-pcr for identification and differentiation of aichi virus, a new member of the picornavirus family associated with gastroenteritis in humans key: cord-267023-w5ig7mrl authors: nori, priya; madaline, theresa; munjal, iona; bhar, shubha; guo, yi; seo, susan k.; porrovecchio, andrea; gancher, elizabeth; nosanchuk, joshua; pirofski, liise-anne; ostrowsky, belinda title: developing interactive antimicrobial stewardship and infection prevention curricula for diverse learners: a tailored approach date: 2017-07-20 journal: open forum infect dis doi: 10.1093/ofid/ofx117 sha: doc_id: 267023 cord_uid: w5ig7mrl background. to impart principles of antimicrobial stewardship (as) and infection prevention and control (ipc), we developed a curriculum tailored to the diverse aptitudes of learners at our medical center. methods. we integrated case-based modules, group learning activities, smartphone applications (apps), decision support tools, and prescription audit and feedback into curricula of the medical school, medicine residency program, infectious diseases (id) fellowship program, and hospital medicine program operations. interventions were implemented in 2012–2016 using a quasi-experimental before-and-after study design, and this was assessed using preand postintervention surveys or audit of antibiotic prescriptions. results. over 180 medical students participated in the as and ipc seminars. after smartphone app introduction, 69% reported using the app as their preferred source of antibiotic information. approximately 70% of students felt comfortable prescribing antibiotics for a known infection compared with 40% at baseline (p = .02), and approximately 83% were able to identify the appropriate personal protective equipment for specific scenarios. approximately 99% agreed that they have a role in promoting patient safety and preventing healthcare-associated infections as medical students. at 20 months, appropriateness of trainee antibiotic prescriptions increased by 20% (p < .01). almost all id fellows indicated that the as and ipc seminar was a vital training supplement. uptake of internist antibiotic recommendations using as decision support tools was approximately 70%. conclusions. all 5 interventions addressed learning objectives and knowledge gaps and are applicable across a range of environments. evaluating long-term impact of our curriculum is the focus of future study. in 2010, the infectious diseases society of america (idsa) noted that traditional infectious diseases (id) curricula were failing to stimulate active participation, recognize medical students as individual learners, or spark enthusiasm for id [1] . in 2011, abbo et al [2] surveyed faculty and residents at 2 teaching hospitals to assess knowledge, attitudes, and perceptions about antimicrobial use and resistance. the majority of respondents desired further education on antibiotics and agreed that better use of antibiotics would reduce resistance, but they did not feel responsible for antibiotic over prescribing. in a 2013 follow-up multicenter survey of fourth year medical students, 90% desired further education on antimicrobial prescribing, but only 40% were familiar with the role of antimicrobial stewardship (as) in promoting judicious antimicrobial use and preventing multidrug resistance [3, 4] . authors also noted that only 15% of students surveyed completed a clinical id rotation, an underused opportunity to augment id knowledge and explore a potential career path [3] . likewise, studies in medical student education reveal (1) poor knowledge and practices of infection prevention and (2) gaps in understanding of healthcare-associated infections (hais) due to lack of emphasis and suboptimal practices among supervising physicians [5] [6] [7] [8] . successful strategies in early medical education include active practice and feedback on proper hand hygiene, use of uv hand gel, with reinforcement of best practices in later years of training [7, 9] . however, there is no consensus on optimal techniques or implementation of an infection prevention curriculum. stewardship literature has established that education alone cannot sustain improvements in antimicrobial prescribing behaviors [10] . furthermore, new regulatory requirements will require concerted educational outreach to enable global improvements in antibiotic prescribing. president obama's 2014 national action plan for combating antibiotic resistant bacteria proposed a widespread implementation of antimicrobial stewardship programs (asp) to reduce antibiotic use by 20%-50% across all healthcare settings [11] . the centers for medicare and medicaid services and the joint commission proposed similar asp standards in 2016 [12, 13] . per ohl and luther [14] , the goal of stewardship education is not only to reduce total antibiotic use, but also to ensure that antibiotics are prescribed only when indicated, at the correct dose, route, and for the proper duration for each infection. in this study, we describe an integrated, multidisciplinary curriculum developed by the unified antimicrobial stewardship and infection prevention and control programs at the montefiore health system in bronx, new york. it consists of 5 educational strategies designed to bridge perceived learning gaps and lay the foundation for best practices in stewardship and infection prevention in medical students, postgraduate trainees, and mature clinicians. we conducted a series of diverse educational processes across distinct learning environments within our medical center. these were layered over time as an outgrowth of observed patterns of antibiotic prescribing and infection prevention behaviors across the spectrum of our learners. the majority used a quasi-experimental, before-and-after study design with preand postintervention knowledge assessment questions, surveys of learners, or chart review with post-antibiotic prescription audit as methods of evaluation. the χ 2 analysis was used to measure pre-and postintervention differences (microsoft excel). statistical significance was set at a p value of <.05 where applicable. montefiore medical center institutional review board approval was obtained where appropriate, and remaining studies were registered as quality improvement (qi) initiatives through the montefiore network performance group. the targeted learner, educational strategy, and method of assessment are detailed below. we developed 2 seminars integrated into the microbiology and infectious diseases course for second-year medical students at the albert einstein college of medicine, conducted annually in the medical school's state-of-the-art active learning studio. antimicrobial stewardship and infection prevention seminars were added to the existing curriculum to introduce students to patient-centered topics such as safety, quality, and antimicrobial resistance. attendance at both seminars, conducted over 2 hours each, was a course requirement. in the first seminar on appropriate antibiotic use, stewardship team members presented fundamental concepts of "bug-drug" matches, de-escalation, and use of the hospital antibiogram/ local susceptibility data using case-based multiple-choice questions as reinforcement. in small groups, they assembled a "toolkit" of core stewardship strategies to improve antibiotic use in mock clinical scenarios. in year 1 (2014), a printed antibiogram was provided. in years 2 and 3 (2015 and 2016), students downloaded a smart phone application (app) named, "appropriate use, " containing antibiograms and clinical practice guidelines developed by montefiore asp (supplementary figure 1 ). using an audience response system ([ars] turning technologies), students answered questions in 3 primary domains: (1) general antibiotic use, (2) principles of microbiology and testing, and (3) prescribing using the local antibiogram. students additionally participated in an anonymous, voluntary survey about their antibiotic overuse perceptions and preferred antibiotic resources. questions were adapted from a previously published study by abbo et al [3] . students were introduced to infection prevention bundles, personal protective equipment (ppe), and the transmission-based isolation precautions during a multidisciplinary patient safety seminar. in small groups, students identified appropriate precautions for mock patient scenarios, including clostridium difficile infection and tuberculosis, then practiced hand hygiene and proper donning and doffing of ppe. infection preventionists moderating the small group sessions used uv-reactive wash (glo germ) to identify areas of ongoing contamination and improve technique. they also worked with prevention bundles for hais such as catheter-associated bloodstream and urinary tract infections. a video describing the comprehensive patient safety seminar can be viewed at https://www.youtube.com/ watch?v=p8zybuoc8vy. at the conclusion of the patient safety seminar, students participated in an anonymous, voluntary survey evaluating the program. students' clinical microbiology/id final examination integrated questions on infection prevention drawn from the session. in 2012, we developed an id core curriculum for the medicine residency program consisting of recurring, 50-minute, case-based lectures on the recognition and management of typical inpatient infections (eg, community-acquired and healthcare-associated pneumonia, urinary tract infections, skin and soft tissue infections, etc). as an adjunct, we distributed a hospital antibiogram and a syndrome-based pocket antibiotic prescribing guide adapted from national guidelines and tailored to local microbiology (supplementary figure 2) . before the first lecture, we distributed an antibiotic pretest with 3 multiple-choice questions on common clinical scenarios designed to assess baseline prescribing knowledge. house staff inpatient antibiotic prescriptions were obtained from queries of the pharmacy's electronic database from november 2012 to september 2014. antibiotic regimens recommended by consulting services (ie, id, critical care medicine) were excluded from review, as were antiretroviral and antifungal prescriptions, because these often involved id comanagement. appropriateness of prescriptions was scored using 3 criteria. (1) were antibiotics indicated for a "true infection" (no alternative noninfectious primary diagnosis)? (2) did the "selected regimen" cover the infection and pathogens in question? (3) were antibiotic "dose" and "duration" appropriate? a senior medical resident and id attending physician independently reviewed each chart. antibiotic appropriateness by indication was assessed at 1 month preintervention and 1 month and 20 months postintervention. appropriateness of antibiotic dose and duration was assessed at 20 months postintervention. in 2013, the montefiore medical center and memorial sloan kettering cancer center jointly developed a free, half-day, intensive workshop on as and infection prevention and control (ipc) for id fellows. the first half-session consisted of 10 interactive ipc cases, including emerging infections, hospital outbreaks, hazardous exposures in laboratory personnel, among others. cases varied each year to include practical, everyday scenarios as well as relevant and timely global threats, such as ebola virus, middle east respiratory syndrome coronavirus, and zika virus. the second half-session addressed 5 challenging as scenarios, including drug shortages and conflict resolution with the "obstinate" prescriber. a premeeting survey evaluated fellows' existing participation in as and ipc. fellows answered multiple-choice questions using ars (turning technologies). solutions were discussed openly with a panel of experts from area hospitals (hospital epidemiologists, id pharmacists, and infection preventionists) and the new york city department of health. fianlly, fellows answered a postmeeting survey on perceptions, training needs, and the value of the workshop itself. in 2013, we developed an audit tool containing sepsis criteria, risk stratification for multidrug resistance, and diagnostic criteria for pneumonia, skin and soft tissue infections, and urinary tract infections for a smaller community hospital without fulltime asp (see supplementary figure 3 ). a 4-month qi initiative was designed to enable internal medicine faculty to serve as asp extensions (stewardship liaisons) to offer antibiotic recommendations to floor teams without providing direct patient care. initially, stewardship liaisons systematically screened all patients initiated on empiric antibiotics upon admission to 2 geriatric units using postprescription audit. liaisons used the audit tool and a validated antibiotic prescribing guide to assist chart review. cases were then discussed with asp, and recommendations on regimens, dose adjustments, or additional management were conveyed to the floor teams by the stewardship liaison in real-time, using one-to-one academic detailing and feedback. electronic medical record was reviewed for patient age, sex, nursing home residence, comorbidities, renal function, allergies, microbiology, imaging results, and length of hospitalization. acceptance rate of recommendations was determined from review of clinical notes and orders. unadjusted hospital length of stay was compared before and the after pilot. one hundred eighty-three students participated per year (2014-2016), and an average of 79 students answered the voluntary survey per year (43%). eighty percent of survey respondents believed that antibiotics are nationally overused. at least 90% believed that better use of antibiotics will reduce antibiotic resistance and that strong knowledge of antibiotics is important for success in medicine. forty percent admitted to taking antibiotics for a viral upper respiratory infection, and 91% reported that their friends have done the same. the appropriate use app was the preferred source of antibiotic information for 69% of students surveyed. two hundred twenty-two students downloaded the app over 2 years (108 downloads in 2015 and 114 downloads in 2016). pre-and post-app, there were no significant differences in the percentage of correct responses to questions on general antibiotic use (p = .64) or principles of microbiology and testing (p = .65). an increase in correct responses from 38% to 76% was observed for questions involving antibiogram use (p = .02) ( table 2 ). the app hits peaked at 330 in the immediate time frame after the february 2016 session. after app introduction, approximately 70% of students felt comfortable prescribing antibiotics for a known infection compared with 40% at baseline (p = .02) [15] . one hundred eighty-three students participated each year (2015 and 2016) and answered the preintervention survey, which revealed that only 23% of students felt either "very comfortable" or "somewhat comfortable" using ppe. only 17% of students recognized that central line insertion bundles reduce infection rates. only 14% were able to identify the appropriate ppe and isolation precautions for measles; however, 93% identified the appropriate hand hygiene associated with c difficile infection (soap and water instead of alcohol-based rubs). posttest results showed a significant improvement in ppe proficiency and knowledge of hai bundles (table 3) . on average, 70 students per year (38%) completed an anonymous survey evaluating the program. the majority of students rated the session as "effective" or "very effective" in achieving the learning objectives (85% and 97%, respectively, in 2015 and 2016) and answered exam questions about isolation, ppe, and hais correctly (range, 79%-97%). approximately 99% agreed that they have a role in promoting patient safety and preventing hais as medical students [16] . one hundred four internal medicine residents completed the antibiotic pretest, and the majority had 0-1 of 3 correct responses. one hundred fifty residents received the id core lectures with antibiogram and pocket prescribing card from november 2012 to september 2014, with an average of 3 lectures per resident per year. antibiotic orders of 104 unique prescribers were audited for appropriateness, with an average of 4 orders per resident. fifty-four percent of prescribers were interns, 28% were second-year residents, and 18% were thirdyear residents. a total of 425 patient charts were analyzed. antibiotics were indicated for a true infection in at least 80% of all cases reviewed (no alternative noninfectious diagnosis such as congestive heart failure, cardiac ischemia, or pulmonary embolism was encountered). preintervention appropriateness by indication was 60%, which improved to 70% at 1 month and 80% at 20 months postintervention (p = .049 and p < .01, respectively). at 20 months, appropriateness of antibiotic doses and durations was 92% and 86%, respectively (table 4) . a preand postintervention analysis by syndrome showed a statistically significant prescribing improvement only for urinary tract infections (57% preintervention, 86% at 20 months postintervention; p = .011) and respiratory infections (59% preintervention, 75% at 20 months postintervention; p < .01) [17] . syndrome-specific prescribing improvements for gastrointestinal, skin and soft tissue infections/osteomyelitis, and "other" infections (bloodstream, meningitis, c difficile, etc) were not statistically significant. on average, 25 id fellows attended the course annually, or >95% of second-or third-year id fellows, and 54% of all id fellows in the greater new york area. sixty-four percent of participants reported some formal as and ipc didactics and participation in regular as and ipc activities at their home institutions (ie, committee meetings or approval of restricted antibiotics). although 82% of participants expressed a professional interest in an ipc or as career after fellowship, more than half felt uncertain about possessing the skills to implement a stewardship program in future employment, and only half felt comfortable managing everyday ipc scenarios. more than 95% of participants agreed that the program was a valuable supplement to their id training and that case studies are an effective strategy for reinforcing as and ipc concepts. almost all participants desired additional training during fellowship. the primary critique each year was the limited time allotted for the workshop. in 2015, fellows from smaller programs with less as and ipc exposure also attended ( table 5 ). fellows from 12 of 16 local training programs have attended since the inaugural session in 2013 [18] . from august 2014 to november 2014, the asp/physician liaison collaborative team reviewed a total of 355 cases from 2 inpatient geriatric units. the mean age of patients was 73: 47% were male and 53% female. the most common syndromes reviewed were skin and soft tissue infections, osteomyelitis, pneumonia, urinary tract infections, and bloodstream infections. thirty-eight percent met sepsis criteria on admission, and 39% were residents of long-term care facilities. a variety of adjustments were recommended in 71.5% of reviewed cases, including dose reduction for diminished creatinine clearance, penicillin allergy clarification, optimization of gram-negative coverage based on local susceptibility patterns, and facilitation of id consultation. uptake of asp/physician liaison recommendations determined from postprescription audit was 69.5%. as a secondary outcome, we observed a 1-day reduction in average length of hospitalization compared with a similar time frame in 2013 (7.79 days [august 2013-november 2013] vs 6.69 days [august 2014-november 2014]) [19] . the idsa suggests real-time electronic question-answer sessions, peer instruction, and small group discussions as tools to enhance preclinical curricula in id [1] . these tools can effectively introduce as concepts to medical students, because studies indicate that only 65% of medicals schools address stewardship in their curricula [20, 21] . a recent collaborative study by macdougall et al [21] at the university of california san francisco suggests that early introduction of core as concepts to both pharmacy and medical students may foster appropriate antibiotic use as a shared responsibility of both professions. since its introduction in 2013, our as student seminar has evolved to better suit students' needs, and it has become a cornerstone of einstein's preclinical clinical microbiology and id curriculum. introduction of the appropriate use app resulted in a 30% increase in the percentage of students who felt more confident prescribing antibiotics for a given syndrome. likewise, introduction of an id core curriculum and tailored antibiotic guide for medical residents enabled a 20% increase in appropriate antibiotic prescriptions over time. to further leverage this success, we adapted our antibiotic guide to a smart phone app for all montefiore prescribers, which we launched during the centers for disease control's "get smart about antibiotics" campaign in november 2015. this "version 2.0" of the appropriate use app was downloaded onto over 200 devices and accessed over 1200 times within the first 6 months of introduction [22] . thus, similar tools, adapted across a range of learners, have proven successful and are popular throughout our institution. our experience also suggests that stewardship tools can aid experienced, non-id clinicians select appropriate antibiotic regimens for complex, elderly patients. attending physicians often serve in leadership roles in the medical unit, and they are charged with a myriad of responsibilities, including improving hospital throughput and use as well as teaching and evaluating trainees. challenges of outreach to the mature prescriber include (1) time constraints, (2) long-standing prescribing behaviors, (3) staff turnover, and (4) emphasis on individual patients over aggregate outcomes. education to improve prescribing at the senior level needs to respect existing attitudes, expectations, and knowledge, and maintain a collegial and collaborative atmosphere. our strategy has focused on recurring feedback sessions to prescriber groups and discrete detailing to individual prescribers. subtle reminders evoking the "public commitment" to responsible prescribing has been referred to as "antibiotic judo" [23] . our senior prescribers have also benefited from the prescribing tools initially developed for our students and trainees. our ipc session for second-year medical students enforced proper hand hygiene technique and introduced transmission-based isolation precautions and appropriate ppe at an early stage. many students requested additional training before the start of their clinical rotations, and they judged ours to be the most interactive session held in the education center in 2016. after the seminar, students felt much more comfortable identifying the appropriate ppe and isolation precautions for each scenario. our findings suggest that emphasis on ipc best practices in medical school promotes a culture of patient safety and lays the groundwork for sustained infection prevention behaviors as students mature into clinicians. the as and ipc interventions were implemented in different time frames and settings, which served to reduce potential confounding between learner groups. smartphone app was disseminated at the medical center only after it was introduced and studied in medical students. strategies presented herein share common themes and lessons learned (table 1) , but collective and individual limitations should be mentioned. we did not conduct an educational needs assessment before study implementation. interventions were designed based on our perceived educational needs extracted from daily interactions with learners. for example, we observed from >5000 stewardship pager interactions per year that 80%-90% of requests are for empiric antimicrobial regimens, often broad spectrum, or intended for patients with remote penicillin allergies, for whom more streamlined β-lactam regimens are more appropriate. quasi-experimental, before and after study design, particularly when applied to small sample sizes, has several limitations, including lack of random assignment and internal validity, generalizability of results, and robustness of conclusions on the effectiveness of educational techniques [24] . however, this methodology is common to published studies in stewardship and infection prevention. techniques described herein are best suited for adaptation at an academic medical center, because they have not been studied outside this setting. only 1 of the interventions was designed for and implemented in a community hospital. student learners were assessed using pre-and postintervention surveys and knowledge assessment questions, and thus the true impact on prescribing and infection prevention practices is not currently known. in the postgraduate residency curriculum, appropriateness of antibiotic dose and duration was not assessed at 1 month due to time constraints, and an antibiotic posttest was not administered due to a wide distribution of resident rotations across multiple sites throughout the bronx. in addition, a statistically significant improvement in syndrome-based antibiotic prescribing was not demonstrated in all categories due to insufficient sample sizes. in the id fellows' seminar, content of the program, mix of learners, and preand postassessment questions varied slightly each year, which may have affected survey results. although a majority of local id fellows attended our as and ipc workshop, the study was not powered to demonstrate statistically significant differences in pre-and postsurvey responses. past course attendees have not been surveyed to determine the role of as and ipc in their current careers and their perceived preparedness after completion of the workshop. a baseline audit of patient characteristics and antibiotic prescribing on the geriatric units was not conducted before stewardship implementation, and unadjusted length of stay was obtained from 2013 hospital discharge data only. reduced length of stay observed during the pilot was likely due to other factors in addition to asp intervention. fianlly, we have not yet studied the long-term impact of the curriculum as a whole or tracked individual learner progress on the path from medical student to mature clinician. the as and ipc curriculum development is a multidisciplinary endeavor of id specialists and clinical pharmacists, infection preventionists, medical school course directors, and residency and fellowship training program directors. we also suggest collaborating with other institutions to benefit from their particular expertise. to our knowledge, this is the first report describing a comprehensive as and ipc curriculum across a range of learners and environments. all strategies achieved intended short-term goals of addressing knowledge and training gaps, increasing confidence, and actively engaging participants. evaluating the long-term impact of individual strategies as part of an integrated curriculum such as ours should be the focus of future study. supplementary materials are available at open forum infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. commentary: idsa guidelines for improving the teaching of preclinical medical microbiology and infectious diseases faculty and resident physicians' attitudes, perceptions, and knowledge about antimicrobial use and resistance medical students' perceptions and knowledge about antimicrobial stewardship: how are we educating our future prescribers? implementing an antibiotic stewardship program: guidelines by the infectious diseases society of america and the society for healthcare epidemiology of america hand-hygiene behaviour, attitudes and beliefs in first year clinical medical students now please wash your hands': the handwashing behaviour of final mbbs candidates usage of ultraviolet test method for monitoring the efficacy of surgical hand rub technique among medical students critical gaps in knowledge of the epidemiology and pathophysiology of healthcare-associated infections hand hygiene in medical students: performance, education and knowledge infectious diseases society of america and the society for healthcare epidemiology of america guidelines for developing an institutional program to enhance antimicrobial stewardship new societal approaches to empowering antibiotic stewardship cms issues proposed rule that prohibits discrimination, reduces hospital-acquired conditions, and promotes antibiotic stewardship in hospitals health care provider education as a tool to enhance antibiotic stewardship practices is there an app for that? expanding the stewardship education armamentarium laying the foundation for better infection prevention and control practices through active learning in early medical education improving antimicrobial use starts with our trainees pooling nyc resources to educate fellows about antimicrobial stewardship and infection prevention and control engaging internists to champion antimicrobial stewardship on the wards a comprehensive survey of preclinical microbiology curricula among us medical schools an interprofessional curriculum on antimicrobial stewardship improves knowledge and attitudes toward appropriate antimicrobial use and collaboration is there an app for that 2.0: using an app to help house staff make more informed antimicrobial prescribing choices antibiotic judo working gently with prescriber psychology to overcome inappropriate use the use and interpretation of quasi-experimental studies in medical informatics we acknowledge dr. rosemarie conigliaro (montefiore internal medicine residency program director) for guidance and support for pursuits in medical education.financial support. our preclinical curriculum was supported by funding from the albert einstein college of medicine, grants for excellence in medical education program. dr. susan k. seo is supported in part through the nih/nci cancer center support grant p30 ca008748.potential conflict of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-295746-6e6itj3y authors: gu, young e.; park, ji y.; lee, mi k.; lim, in s. title: characteristics of human parainfluenza virus type 4 infection in hospitalized children in korea date: 2020-01-19 journal: pediatr int doi: 10.1111/ped.14049 sha: doc_id: 295746 cord_uid: 6e6itj3y background: the characteristics of human parainfluenza virus type 4 (hpiv4) infection are not thoroughly understood. we therefore clarified the characteristics of hpiv4 in korea. method: from january 2013 to december 2017, children admitted with respiratory tract infection at the department of pediatrics in chung‐ang university hospital were enrolled in the study. nasopharyngeal aspirate specimens were obtained from patients and tested for hpiv types by multiplex reverse transcription polymerase chain reaction. we retrospectively reviewed subject medical records, focusing on epidemiological and clinical characteristics. results: of the 12 423 npa specimens, 8,406 were positive by multiplex reverse transcription polymerase chain reaction for nine respiratory viruses, and 1,018 were positive for one of the four types of hpiv: 1,018 specimens led to the detection of 1,029 hpivs; 3ss (31.3%) were positive for hpiv1, 120 (11.7%) were positive for hpiv2, 356 (34.6%) were positive for hpiv3, and 231 (22.4%) were positive for hpiv4. of the hpiv‐positive patients, the mean age was 2.3 years (range, 0.1–12.7 years), 225 (97.4%) had no underlying disease, and 178 (77.1%) had a fever with a duration of 4.1 ± 2.3 days and a peak temperature of 39.0 ± 0.7 ℃. the most common diagnosis in hpiv4 infection was pneumonia (44.2%), followed by bronchiolitis (26.0%) and upper respiratory tract infection (24.3%). only 2.2% of patients were diagnosed with croup. although the most prevalent overall type of hpiv was hpiv3, hpiv4 generally caused acute respiratory tract infection in summer and early fall in an irregular annual pattern. conclusions: human parainfluenza virus type 4 is an important common pathogen of respiratory tract infections in pediatric patients in korea. hospital is the referral medical center and an 835-bed tertiary care university hospital in seoul, korea. nasopharyngeal aspirates were obtained at the time of admission. according to the results of the multiplex reverse transcription polymerase chain reaction (mrt-pcr) assay, a list of patients whose specimens were positive for hpiv types 1, 2, 3, or 4 was obtained. we retrospectively reviewed subject medical records between january 2013 and december 2017. we focused on the prevalence based on the date of collection, age, and diagnosis at admission for hpiv1, hpiv2, hpiv3, and hpiv4. furthermore, the clinical features and laboratory findings of hpiv4 were reviewed to identify their clinical characteristics. we defined the infection by only one type of hpiv as a single infection. the infection by one type of hpiv with other viruses or another type of hpiv was named the "coinfection group." collectively, all enrolled patients with either a single infection or coinfection were referred to as the "total infection group." we also classified hpiv-infected children into four groups: subjects aged <3 months, subjects aged between 3 and 24 months, subjects aged between 2 and 5 years, and subjects aged ≥5 years. this study was approved by the institutional review board of chung-ang university hospital (no. 1806-010-16186) and was exempt from informed consent approval from the ethical committee. viral dna or rna was extracted using the viral gene-spin kit (intron biotechnology, seoul, korea). extracted viral dna or rna was reverse transcribed and then analyzed using the anyplex ii rv16 detection (v1.1) assay (seegene, seoul, korea), which can detect respiratory viruses (adenovirus, rsv a/b, hmpv, influenza virus a/b, hpiv 1/2/3/4, rhinovirus, enterovirus, bocavirus, and coronavirus 229e/nl63/oc43), from january 2013 to april 2017, and the allplex tm respiratory panel 1, 2, 3 assay (seegene, seoul, korea), which can detect additional subtypes of influenza a (h1pdm09, h1 and h3), from may 2017 to december 2017. seegene viewer software was used to analyze the amplification results. anyplex ii rv16 detection (v1.1) assay gave the quantitative results as negative/1+/2+/3+ and allplex tm respiratory panel 1, 2, 3 assay gave those as ct value (ct ≤ 42; positive). we used two mrt-pcr panel kits from the same commercial company for separate study periods. however, evaluation of these panels revealed no significant difference in detecting hpiv4, 9 and both kits showed good sensitivity and specificity. 9, 10 statistical analysis statistical analysis was performed using spss software version 21.0 (ibm co., armonk, ny, usa). categorical variables were compared using the chi-square test and fisher's exact test. continuous variables were compared using t-test. the values of a ae b stood for mean ae standard deviation in continuous variables. p values under 0.05 were considered statistically significant. of the 12 423 npa specimens, 8,406 (67.7%) were positive for any respiratory viruses by mrt-pcr, and 1,018 (8.2%) were positive for more than one type of hpiv; 1,018 specimens yielded 1,029 hpivs; 322 (31.3%) were positive for hpiv1, 120 (11.7%) were positive for hpiv2, 356 (34.6%) were positive for hpiv3, and 231 (22.4%) were positive for hpiv4. of the total patients infected by hpiv4, 120 (51.9%) were male, the mean age at admission was 2.3 ae 1.8 years (range, 0.1-12.7 years), 225 (97.4%) children did not have any underlying disease and mean hospitalization duration was 4.9 ae 1.8 days. the coinfection rate of hpiv4 was the highest (58.9%); rates were 39.8% (128/322) for hpiv1, 41.7% (50/120) for hpiv2, and 46.6% (166/356) for hpiv3 (p = 0.000). the most common coinfecting virus in hpiv4 infections was rhinovirus (25.4%), followed by enterovirus (21.1%), adenovirus (18.3%), and bocavirus (14.6%). in the coinfection group, we obtained quantitative results from 133 specimens. in 2017, seven of eight specimens showed a lower ct value in hpiv4 than in other viruses. from 2013 to 2016, 23 of 125 (18.4%) specimens were in the same positive ranges (1+, 2+ or 3+) as other viruses and 26 of 125 (20.8%) specimens had greater positive ranges than other viruses. the group with a single hpiv4 infection did not differ from the coinfection group in age or in the presence of underlying diseases (p > 0.05). however, the single infection group required a 0.5-day longer hospital stay than the coinfection group required (p = 0.040), and patients in the group were more commonly female (p = 0.032) ( table 1) . when classifying hpiv-infected children into four age groups, the percentages of hpiv types were significantly different for each age group (p < 0.001). of the total number of hpivinfected children, 37 were aged under 3 months, 575 were from ages 3 months or older but were younger than 2 years, 346 were from ages 2 years or older but younger than 5 years, 60 were from ages 5 years or older. hpiv1 was shown to be present at similar percentages in each age group. however, hpiv2 showed a tendency to increase, and hpiv3 showed a tendency to decrease with aging ( fig. 1) . nonetheless, most hpiv-positive patients were infected with hpiv when they were 3 months or older but younger than 5 years (hpiv1 91.0%, hpiv2 83.3%, hpiv3 93.3%, and hpiv4 89.2%; p = 0.000). human parainfluenza virus type infections were more prevalent from april to august, although the seasonality differed by types of hpiv (fig. 2) . hpiv1 did not show any seasonal pattern; it occurred at any time during the year. hpiv2 occurred biennially from july to october. hpiv3 infections were prevalent from april to july every year. however, hpiv4 generally caused arti in summer and early fall in an irregular annual pattern. hpiv4 was not significantly detected in 2013 or 2016. in 2014 and 2015, the rate of hpiv4 detection was significantly higher at more than 15%. in 2017, the hpiv4 detection rate was typical at approximately 5% (fig. 3) . however, the most prevalent type of hpiv was different every year (p = 0.000). the most common type of hpiv was hpiv3 (49.6%) in 2013, hpiv1 (44.0%) in 2014, hpiv4 (36.5%) in 2015, hpiv1 (54.5%) in 2016, and hpiv3 (42.1%) in 2017. the percentages of diagnosis in each type of hpiv infections were not significantly different between the coinfection groups and the single infection groups (p = 0.900 in hpiv1, 0.142 in hpiv2, and 0.256 in hpiv3), except for hpiv4 (p = 0.001). in in hpiv4, bronchiolitis was significantly more common in the single infection group than in the coinfection group (p = 0.000, table 2 ). the prevalence of diagnosis differed by the type of hpiv in the single infection group (p = 0.000). in the single infection group, there were significant differences by type of hpiv in croup, bronchitis, and bronchiolitis (all p < 0.05). croup was more prevalent in hpiv1 and hpiv2 infections in the single infection group (p = 0.000). in hpiv3, bronchitis was more prevalent in the single infection group (p = 0.010). bronchiolitis due to hpiv4 was more common in the single infection group (p = 0.000). the most common diagnosis in hpiv4 infection was pneumonia (44.2%), followed by bronchiolitis (26.0%) and urti (24.3%) in the total infection group. lower respiratory tract infection diagnosis accounted for 75.8% of the total infection of hpiv4 and 82.1% of the single infection of hpiv4, which were the highest rates of lrti among hpiv types (p = 0.000). other types of hpiv were also shown to have high prevalence rates in lrtis (65.2% in hpiv1, 53.3% in hpiv2, and 69.4% in hpiv3; table 2 ). only 2.2% of hpiv4 infected patients, which was the lowest prevalence rate among hpiv types, were diagnosed with croup (p = 0.000). of the hpiv4-positive children, 77.1% (178/231) had a fever duration of 4.1 ae 2.3 days and a peak temperature of 39.0 ae 0.7°c. the most common symptom of hpiv4-infected patients was cough (82.3%), followed by sputum (61.0%), and rhinorrhea (60.2%). a small number of patients exhibited gastrointestinal symptoms, such as vomiting (9.1%) and diarrhea (7.4%). the most commonly detected physical sign was rale (36.8%) and wheezing (22.1%). laboratory findings for white blood cell counts (wbcs), absolute neutrophil counts (ancs), platelets, c-reactive protein (crp), aspartate aminotransferase (ast), and alanine aminotransferase (alt) were not significantly out of normal ranges. the clinical characteristics of single infection by hpiv4 were not significantly different from those of coinfection. wheezing was more frequently auscultated in the single infection group (p = 0.002). in laboratory findings, the anc of the coinfection group was higher and the platelet count lower than the equivalent counts in the single infection group (p = 0.028 and 0.008, respectively; table 1 ). human parainfluenza virus is one of the main pathogens of respiratory tract infection in children. 1-3 in our study, we found that 94.1% of hpiv types occurred arti under 5 years. hpiv1 and hpiv2 were prevalent in croup and hpiv1 and hpiv3 were prevalent in lrti. hpiv1 occurred at any time during the year; hpiv2 occurred biennially from july to october; and hpiv3 occurred from april to july every year. although hpiv4 has not been recognized as an important pathogen, we found that hpiv4 caused arti in summer and early fall in an irregular annual pattern, especially lrti with the highest prevalence. the seasonality of hpivs differs by region. in previous studies, hpiv1 infections are usually detected in the fall of odd-numbered years; hpiv2 infections do not exhibit a clear seasonal pattern but occur more commonly in autumn. 8, 11, 12 there are generally more cases of hpiv3 in spring and early summer each year. 5, 8, [11] [12] [13] however, hpiv4 usually occurs during late autumn and winter in temperate countries, 5, 11, 13 but in china, hpiv4 is more often observed during spring and summer. 14 the seasonal patterns of hpiv4 have not been well clarified because of the small number of samples and the small number of studies that have been conducted. 5, 8, 12 as a result, more studies in diverse countries are needed to understand the global characteristics of hpiv4. the korean centers for disease control and prevention (kcdc) observed respiratory pathogens from patients with arti as part of the korean influenza and respiratory surveillance system (kin-ress) project, beginning in may 2009. the results of a kcdc study about the types of hpivs were published in 2017. the authors could not determine the prevalence of hpiv4 in that study because they used the mrt-pcr panel without hpiv4. 15 studies of hpiv4 infection in korea are thus needed to understand the clinical characteristics and epidemiology in clinical areas. during our study period, the prevalence among hpiv types was highest in hpiv3, followed by hpiv1 and hpiv4. hpiv4 was the most frequent hpiv type in 2015 and the second most common type in 2014 (table 2 ). in many countries, hpiv2 is less frequently detected than hpiv1 or hpiv3. 16, 17 one study showed that hpiv4 was more frequently detected than hpiv2, suggesting that hpiv4 might be underestimated as a pathogen of arti. 18 in this study, the 5-year period prevalence of hpiv2 was lower than that of hpiv4 (1.9%, 231/12 423) and was observed to be 1.0% (120/12 423), which was the lowest detection rate among hpiv types. hpiv4 infection accounted for 22.4% (231/1,029) of total hpiv infections in the current study. this value shows that hpiv4 is more common than is believed. depending on the age group, the prevalence of hpiv types showed significant differences. as patients aged, hpiv2 became increasingly prevalent but the prevalence of hpiv3 decreased. we thought that tendency of each age group in hpiv2 and hpiv3 was influenced by the proportion of diagnosis (p = 0.000). hpiv2 was more prevalent in urti, and hpiv3 was more prevalent in lrti. we found some characteristics of hpiv4 infection. few children infected with hpiv4 developed croup in our study, as shown in previous studies. 11, 19 in the single-infection group with hpiv4, more children had wheezing and were diagnosed with bronchiolitis. most hpiv4 infections manifested as lrtis. the coinfection rate was also highest in hpiv4, as shown in a previous study. 19 in the present study, the most frequently coinfecting virus was rhinovirus, followed by enterovirus, adenovirus, and bocavirus. rhinoviruses occur year round except in winter. 20 rhinovirus is also frequently co-detected by mrt-pcr, 21 as is bocavirus. 22 furthermore, enterovirus occurs from summer to early fall, and adenovirus occurs from late summer to fall; these seasons overlap with hpiv4, according to the kinress project. we therefore believe that this overlap is the reason that these viruses were frequently co-detected with hpiv4 in this study. there are few studies on the epidemiology and characteristics of hpiv4 worldwide. as hpiv4 is considered a marginal respiratory pathogen, it is excluded from many commercial respiratory mrt-pcr panel kits, so many clinicians do not understand its features and have overlooked its clinical importance. thus, a strength of our study was that it included only korean data, which was from a 5-year period with a large group of enrolled children. however, this study also had some limitations. first, this study was performed in a single center for 5 years and thus does not reflect the general data from korea. furthermore, our center is a tertiary medical hospital, where many patients were referred with illness from a primary or secondary health care center. there might be a sampling bias that resulted in missing mild illness, such as urti. our pediatric department was visited by few children with immunodeficiencies, such as hemato-oncological diseases or immunodeficient diseases, so we could not explore the manifestation of hpiv4 in immunocompromised patients. second, this study was performed retrospectively, and all data were collected from medical charts, limiting the data available for analysis. although there are two subtypes of hpiv4 (4a and 4b), we could not distinguish between them. to overcome the limitations of the current study, further prospective nationwide multicenter studies are needed for a long period. in conclusion, hpiv4 is an important pathogen of respiratory tract infections in children. hpiv4 was found to be more common than previously thought and manifested with severe illnesses such as lrtis, especially bronchiolitis, in summer and early fall in an irregular annual pattern. we should therefore realize the impact of hpiv4 and the need to detect it in children with respiratory tract symptoms. none. the authors declare no conflict of interest. j.y.p. and m.k.l. contributed to the conception and design of this study; m.k.l. and y.e.g. collected data; y.e.g. performed the statistical analysis and drafted the manuscript; i.s.l. critically reviewed the manuscript and supervised the whole study process. all authors read and approved the final manuscript. © 2019 japan pediatric society clinical and genetic features of human metapneumovirus infection in children progress in the development of human parainfluenza virus vaccines epidemiology and clinical presentation of the four human parainfluenza virus types aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis use of laboratory and administrative data to understand the potential impact of human parainfluenza virus 4 on cases of bronchiolitis, croup, and pneumonia in alberta feigin and cherry's textbook of pediatric infectious diseases evaluation of allplex respiratory panel 1/2/3 multiplex real-time pcr assays for the detection of respiratory viruses with influenza a virus subtyping comparison of anyplex ii rv16 with the xtag respiratory viral panel and seeplex rv15 for detection of respiratory viruses epidemiology and clinical presentation of parainfluenza type 4 in children: a 3-year comparative study to parainfluenza types 1-3 estimates of parainfluenza virus-associated hospitalizations and cost among children aged less than 5 years in the united states epidemiological investigation and seroprevalence of human parainfluenza virus in mie prefecture in japan during 2009-2013 human parainfluenza virus type 4 infection in chinese children with lower respiratory tract infections: a comparison study global prevalence of the human parainfluenza virus. public health weekly rep characterization of acute respiratory infections among 340 infants in wuxi ten year retrospective evaluation of the seasonal distribution of agent viruses in childhood respiratory tract infections detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-pcr a prospective study of parainfluenza virus type 4 infections in children attending daycare picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season early detection of acute rhinovirus infections by a rapid reverse transcription-pcr assay prospective study of human bocavirus (hbov) infection in a pediatric university hospital in germany key: cord-271653-4q2olzx1 authors: libby, peter title: the heart in covid19: primary target or secondary bystander? date: 2020-04-10 journal: jacc basic transl sci doi: 10.1016/j.jacbts.2020.04.001 sha: doc_id: 271653 cord_uid: 4q2olzx1 summary: in the throes of the current covid-19 pandemic, interest has burgeoned in the cardiovascular complications of this virulent viral infection. as troponin, a biomarker of cardiac injury, often rises in hospitalized patients, its interpretation and actionability require careful consideration. fulminant myocarditis due to direct viral infection can certainly occur, but patients with increased oxygen demands due to tachycardia and fever, and reduced oxygen delivery due to hypotension and hypoxemia can cause myocardial injury indirectly. cytokines released during the acute infection can elicit activation of cells within pre-existing atherosclerotic lesions, augmenting thrombotic risk and risk of ischemic syndromes. moreover, microvascular activation by cytokines can cause not only myocardial injury but harm other organ systems commonly involved in covid-19 infections including the kidneys. dealing with the immense challenge of covid-19 disease, confronted with severely ill patients in dire straits with virtually no rigorous evidence base to guide our therapy, we must call upon our clinical skills and judgment. these touchstones can help guide us in selecting patients who might benefit from the advanced imaging and invasive procedures that present enormous logistical challenges in the current context. lacking a robust evidence base, pathophysiologic reasoning can help guide our choices of therapy for individual clinical scenarios. we must exercise caution and extreme humility, as often plausible interventions fail when tested rigorously. but act today we must, and understanding the multiplicity of mechanisms of myocardial injury in covid-19 infection will help us meet our mission unsupported by the comfort of strong data. in the throes of the current covid-19 pandemic, interest has burgeoned in the cardiovascular complications of this virulent viral infection. as troponin, a biomarker of cardiac injury, often rises in hospitalized patients, its interpretation and actionability require careful consideration. fulminant myocarditis due to direct viral infection can certainly occur, but patients with increased oxygen demands due to tachycardia and fever, and reduced oxygen delivery due to hypotension and hypoxemia can cause myocardial injury indirectly. cytokines released during the acute infection can elicit activation of cells within pre-existing atherosclerotic lesions, augmenting thrombotic risk and risk of ischemic syndromes. moreover, microvascular activation by cytokines can cause not only myocardial injury but harm other organ systems commonly involved in covid-19 infections including the kidneys. dealing with the immense challenge of covid-19 disease, confronted with severely ill patients in dire straits with virtually no rigorous evidence base to guide our therapy, we must call upon our clinical skills and judgment. these touchstones can help guide us in selecting patients who might benefit from the advanced imaging and invasive procedures that present enormous logistical challenges in the current context. lacking a robust evidence base, pathophysiologic reasoning can help guide our choices of therapy for individual clinical scenarios. we must exercise caution and extreme humility, as often plausible interventions fail when tested rigorously. but act today we must, and understanding the multiplicity of mechanisms of myocardial injury in covid-19 infection will help us meet our mission unsupported by the comfort of strong data. in the throes of the current pandemic, intense interest has burgeoned in cardiovascular involvement by the novel coronavirus known as covid-19. cardiologists as well as other practitioners who care for those with this virulent viral infection, and indeed the general public as to approach this question, we need to distinguish myocarditis due to infection of cardiac cells from myocardial ischemic injury. flow embarrassment to the heart muscle can result from lesions in epicardial coronary arteries or in the heart's microvasculature. cardiac ischemia can also arise from an imbalance between oxygen supply and demand, a type 2 acute coronary syndrome, a situation that can prevail in acute infections, particularly those that affect the lungs like covid19. several of these pathophysiologic pathways to myocardial ischemia may affect those without substantial or obstructive coronary artery atherosclerosis. hence, the distinction between these various mechanisms has important clinical consequences. the need for arduous imaging studies and invasive evaluation may vary considerably in these different scenarios, an issue of great import in acute care facilities stretched to or beyond their limits during a pandemic with a readily contagious and virulent infectious agent such as covid19. considering the pathophysiologic paths to cardiac injury can inform judgement regarding the necessity of transport of severely ill patients and the performance invasive procedures. the demographic most often affected by life-threatening and fatal covid-19 has a high prior probability of pre-existing atherosclerotic lesions: the elderly, evident male predominance, and those with pre-existing lung disease including that associated with cigarette smoking, a risk factor for atherosclerosis. remote infections such as the severe pneumonitis that too commonly complicates covid-19 can elicit an acute exacerbation of the chronic smoldering inflammation that characterizes coronary atherosclerotic lesions ( figure 2 ). the inflammatory cells at a site of regional infection such as the lungs in covid19 pneumonitis can produce cytokines such as interleukins (il)-1 and -6, and tumor necrosis factor, mediators that not only propagate local inflammation but can enter the systemic circulation. such circulating cytokines can stimulate macrophages within the plaque to augment local cytokine production and provoke an increase in tissue factor expression that renders lesions more thrombogenic. we have referred to this local response to systemic stimuli as an "echo" phenomenon. 4, 6 these same systemic cytokines can stimulate leukocyte adhesion molecule expression on the endothelial cells overlying established atheroma, boosting local recruitment of these inflammatory cells. these alterations in preexisting plaques can enhance their propensity to disrupt, be it by fibrous cap fissure or by superficial erosion, and provoke an acute coronary syndrome. even in individuals without pre-existing epicardial coronary artery disease, systemic cytokines released from sites of local infections such as in pneumonitis can affect intermural coronary vessels. they also can activate the microvascular endothelium, predisposing to vasomotor abnormalities, augmented thrombosis, reduced fibrinolysis, increased leukocyte adhesion, and other aspects of dysfunction of the microvessels of the coronary circulation. these effects wrought by distant infection can contribute to myocardial ischemia even in the absence of epicardial atherosclerosis, and could compound cardiac injury in those with flow limitation due to plaque in the larger coronary arteries. the cardinal signs of infection include fever and tachycardia, circumstances that increase the oxygen requirements of the myocardium (figure 3 ). hypoxemia produced by pneumonitis can decrease oxygen delivery to the myocardium. hypotension in sepsis and in cytokine storm can impair coronary perfusion. together these systemic effects of infection conspire to limit blood flow in the coronary arteries, and reduce oxygen supply while augmenting myocardial oxygen demand. these consequences of infection predispose to myocardial ischemia. they may aggravate the consequences of plaques that would not limit flow or provoke ischemia under usual conditions, and could produce ischemic injury even in those with little or no coronary artery atherosclerosis. at one end of the spectrum, a young individual with pristine coronary arteries might suffer severe myocardial injury due to a fulminant myocarditis caused by direct infection with covid-19 (figure 1, left) . at the other extreme, a person with advanced coronary atherosclerosis could suffer a type 1 or type 2 acute myocardial infarction without direct viral infection of cardiac cells (figure 1, right) . although we are early in our experience with this novel coronavirus disease, most patients affected by covid-19 encountered by cardiologists may have more secondary cardiac involvement then primary infective myocarditis. thus, many of our patients may fall into the zone between the two bookends depicted in figure 1 . it behooves us to consider the multiple mechanisms of cardiac injury in patients with covid-19 disease ( figure 4) . as in the bc (before covid) era, interpretation of rises in cardiac troponin requires consideration of the context of the clinical situation. not all rises in this biomarker of cardiac injury will result from coronary artery disease requiring invasive assessment or intervention. we urgently need randomized clinical trials to assess the value of interventions including anti-inflammatory therapies ranging from glucocorticoids to cytokine antagonism in addition to antiviral agents as outlined in the elegant exposition of atri et al. 3 dealing with the immense challenge of covid-19 disease, and confronted with severely ill patients in dire straits with virtually no rigorous evidence base to guide our therapy, we need to call upon our clinical skills and judgment. these touchstones can help guide us in selecting patients who might benefit from the advanced imaging and invasive procedures that present enormous logistical challenges in the current context. in the absence of a robust evidence base, we will also need to invoke pathophysiologic reasoning to guide our choices of therapy for each individual clinical scenario. we must do so with caution and extreme humility, recognizing how often plausible interventions fail when tested rigorously. but act today we must, and the overview of atri et al. and other recent compendia will help us meet our mission unsupported by the comfort of strong data. 3 coronavirus fulminant myocarditis treated with glucocorticoid and human immunoglobulin cardiac involvement in a patient with coronavirus disease 2019 (covid-19) covid-19 for the cardiologist: a state-of-the-art review of the virology, clinical epidemiology, cardiac and other clinical manifestations and potential therapeutic strategies. jacc: basic to translational science roles of infectious agents in atherosclerosis and restenosis: an assessment of the evidence and need for future research inflammation, immunity, and infection in atherothrombosis: jacc review topic of the week leukocytes link local and systemic inflammation in ischemic cardiovascular disease key: cord-273973-3uxg97tu authors: guenette, alexis; husain, shahid title: infectious complications following solid organ transplantation date: 2019-01-31 journal: critical care clinics doi: 10.1016/j.ccc.2018.08.004 sha: doc_id: 273973 cord_uid: 3uxg97tu infections in solid organ transplant recipients are complex and heterogeneous. this article reviews the clinical syndromes that will likely be encountered in the intensive care unit and helps to guide in the therapy and management of these patients. immunosuppression plays an integral role in solid organ transplant (sot) recipients because it increases graft survival; however, there are unintended consequences, such as infectious complications. one strategy aimed at assessing the functionality of the immune system consists of non-pathogen-specific immune monitoring, consisting of serum immunoglobulins, serum complement factors, peripheral blood lymphocyte subpopulations, soluble cd30, and iatp in cd4 1 t cells. 1 ideally, these would help to demonstrate one aspect of the overall "net state of immunosuppression." the "net state of immunosuppression" comprises all factors that may contribute to the risk of infection; this includes preexisting immune deficits, colonization with antimicrobial-resistant pathogens, immunosuppressive agents, acquired immunodeficiency, prior antimicrobial therapies, mucocutaneous barrier integrity, fluid collections, neutropenia, lymphopenia, and viral coinfections. 2 disclosure statement: a. guenette has no relationships with a commercial company that has a direct financial interest in subject matter or materials discussed in article or with a company making a competing product. s. husain has received grants from astellas, pfizer, and merck. a division of infectious disease, university health network, university of toronto, 585 university avenue, 11 vaccinations, surgical prophylaxis, universal prophylaxis, preemptive or presymptomatic therapy, targeted prophylaxis, and education avoidance are preventative strategies that have been implemented in sot recipients. trimethoprim/sulfamethoxazole prophylaxis is given in most institutions for 3 months to a lifetime to prevent pneumocystis pneumonia along with toxoplasma gondii, cyclospora cayetanensis, and many nocardia and listeria species. antiviral prophylaxis along with nucleic acid-based assays to prevent cytomegalovirus (cmv) and other herpesvirus infections has also transformed posttransplant care. 2 infections in sot recipients reflect the net balance between the recipient's epidemiologic exposures and immunosuppression. 2 alterations to the balance can be seen with antimicrobial prophylaxis, immunosuppression, and improved graft survival. 2 this balance is also affected during a period of graft rejection or intensification of immunosuppression (fig. 1 ). 2 in this article, the authors review infectious syndromes encountered in intensive care units among sot recipients. bloodstream infections (bsis) are associated with poor outcomes along with being the leading cause of mortality and morbidity in sot. [3] [4] [5] mortality as high as 24% 4,6,7 has been described, and in fact, once septic shock develops, mortality can reach 50%, 4, 8 although kalil and colleagues 9 demonstrated that there may be a decrease in mortality of transplant patients compared with nontransplant patients. it is thought that sot recipients do not necessarily clinically behave in the same manner due to underlying immunosuppression, and in fact, tend to present with organ failure and thrombocytopenia during sepsis. 10 universal risk factors for sepsis, regardless of transplanted organ, are cmv serology mismatch, particularly positive donor to negative recipient, cmv disease, which inherently demonstrates an immunomodulatory effect predisposing recipients to higher rates of bacterial and fungal sepsis, prolonged duration of graft cold ischemia, prolonged duration of surgical transplantation procedure, and requirement of large amounts of blood transfusion. 10, 11 management should consist of rapid initiation of intravenous antibiotics, rapid diagnosis, source control, aggressive search for pathologic condition that mimics severe sepsis, and reduction in immunosuppressive drugs. 10 nosocomial bsis are associated with an even increased risk of septic shock and failure of cure when compared with other bsis in sot patients. [3] [4] [5] 8, 12 grampositive bacteria are the most frequent source of bsis and are likely to be associated with intravascular catheters, especially in the nosocomial setting. 3, 4 however, with kidney transplant recipients (ktrs), gram-negative bacteria likely related to urinary tract infections (utis) are the primary source, regardless of the time period. the overall incidence of multidrug-resistant organisms (mdro) is increasing. mdro gram-negative organisms accounted for about 14% of isolates. 4 fluconazoleresistant candida spp accounted for up to 46% of cases of candidemia according to moreno and colleagues. 4, 13 vancomycin-resistant enterococci (vre) have become an emerging pathogen with studies documenting an incidence of up to 20.5% of nosocomial enterococcal spp bsis consistent with vre. these findings along with previous microbiological history and local antibiotic resistance patterns should be considered when determining empiric antimicrobial therapy. overall, there are limited studies on infective endocarditis (ie) in sold organ transplant recipients. the incidence of ie in a single center was 1%, with an estimated 171-fold higher incidence as compared with the general population, 14 with an overall mortality up to 57%. 15 there are limited data on the mode of infection and predisposing factors in sot recipients. underlying structural abnormalities may not appear to be a risk factor for ie as compared with the general population. 14, 15 according to paterson and colleagues, 15 50% of infections were due to aspergillus fumigatus or staphylococcus aureus, and 4% were due to viridans streptococci, which is in contrast to the general population. a combination of antibiotic therapy as described in infectious diseases society of america (idsa) guidelines, infective endocarditis in adults: diagnosis, antimicrobial therapy, and management of complications and surgical management, if warranted, is the current management. empiric management of suspected sepsis/bsi should include gram-positive coverage, in the presence of intravascular catheters, and broad gram-negative coverage. the choice of the antibiotic is dependent on the local epidemiology and previous microbiological data. empiric candida or antifungal coverage is not required, because the early initiation of antifungal has not been shown to improve the outcome in randomized controlled trials of mostly immunocompetent patients. 16, 17 bacterial pneumonia is the most common cause of lower respiratory tract infections. [18] [19] [20] [21] [22] [23] [24] according to giannella and colleagues, 25 community-acquired pneumonia (cap) was found in 40.7% and hospital-acquired pneumonia (hap) was found in 59.3% of sot recipients treated for pneumonia. in lung transplant recipients, bacterial pneumonia or bronchitis accounts for 32% to 63% of all infections with the incidence of bacterial pneumonia peaking in the first 4 to 8 postoperative weeks and then declines by the fourth month. perioperative antibiotics, which are focused on preoperative cultures from the recipient and donor, reduce the incidence of early bacterial pneumonia to less than 10%. 20, 21 regarding cardiac, hepatic, and renal transplants, the incidence of early bacterial pneumonia is 15%, 9%, and 4% to 6%, respectively, [18] [19] [20] 26, 27 with a mortality of 21% to 35% in liver and kidney transplant recipients. however, mortality between nosocomial and community-acquired infection was extreme at 58% compared with 8% 20-22 with mechanical ventilation and nosocomial infections at a higher increased risk for death. 18, [20] [21] [22] 24 in the initial perioperative infectious complications period, nosocomial pathogens, such as pseudomonas aeruginosa, escherichia coli, klebsiella species, acinetobacter species, and s aureus, including methicillinresistant s aureus (mrsa), should be considered in the immediate perioperative period; however, prolonged mechanical ventilation following transplant increases the risk for nosocomial pneumonia. [20] [21] [22] community-acquired pathogens, such as haemophilus influenzae, streptococcus pneumoniae, and legionella species, may be seen. with the implementation of trimethoprim-sulfamethoxazole prophylaxis, the incidence of nocardia pneumonia has decreased; however, it is still reported. 20, 28 empiric treatment should take into consideration previous microbiological data, local epidemiology, and recent clinical history with regards to empiric antibiotic coverage. respiratory viral infections are a significant cause of mortality and morbidity among transplant recipients, including influenza, respiratory syncytial virus, parainfluenza virus, rhinovirus, human metapneumovirus, and coronavirus. the seasonal pattern usually follows that of the general public. [29] [30] [31] disease can consist of mild congestion and rhinorrhea to more severe tracheobronchitis, bronchiolitis, and pneumonia. clinical manifestation can range from mild or atypical symptoms, including absence of fever, with lung transplant recipients presenting with a more severe clinical course and complications. 29 ,32 viral shedding is usually prolonged and seen even with the use of antivirals. 29, 32 transplant recipients are at higher risk of infectious complications, including fungal and bacterial pneumonia. respiratory viral infections appear to be a risk factor for both acute and chronic rejection, especially in lung transplant recipients. 29, [33] [34] [35] [36] diagnostic workup should consist of a nasopharyngeal swab, wash, or aspirate. if upper tract samples fail to document the cause or if there is clinical or radiological evidence of lower tract involvement, bronchoalveolar lavage (bal) should be considered. polymerase chain reaction (pcr)-based assays are commercially available with many centers adopting them because they are the most preferred mode of testing given the high sensitivity along with most allowing for simultaneously detecting a broad range of respiratory pathogens from a single sample. treatment, as is outlined in fig. 2 , includes supportive care and reduction in immunosuppression. adenovirus is a nonenveloped, lytic double-stranded dna virus that can be acquired de novo, through reactivation of a latent infection of the recipient, or from transplant organ. transmission occurs by respiratory route, person-to-person contact, or fecal-oral route. the true incidence among sot recipients is unknown, with most infections occurring within the first year after transplantation. 37 clinical manifestations can vary; however, when affecting lung transplant recipients, it can produce a range of clinical manifestations, including acute flulike illness, diffuse alveolar damage, or necrotizing pneumonia along with chronic changes, such as bronchiolitis obliterans, interstitial fibrosis, or bronchiectasis. [38] [39] [40] [41] viral culture, direct antigen detection, molecular methods, and histopathology are available for diagnosis with histopathologic evaluation, as the gold standard for the diagnosis of invasive disease. rapid antigen detection kits, in particular, immunofluorescence assays when processing respiratory specimens, are commercially available, which yield rapid and specific results. pcr, qualitative and quantitative, has emerged as a widely used tool for detection because it is highly sensitive and rapid. recovery of adenovirus from respiratory samples does not necessarily confirm disease because patients can shed asymptomatically for a prolonged period of time; therefore, it is essential to correlate with clinical findings along with detection of virus from other sites and histopathological findings. cidofovir has the best evidence to support its use in the treatment of adenoviral infections. brincidofovir, the lipid conjugate of cidofovir, has also demonstrated in vitro susceptibility and appears to be promising in vivo with regards to sot recipients; however, further studies are warranted. 42 cmv is a major pathogen in sot recipients, with the ability to cause end-organ disease. the immunomodulatory effects of cmv, impaired t-cell and phagocytic function, and cytokine dysregulation can lead to opportunistic infections, rejection, graft loss, and reduced survival. 43, 44 the transplant recipients who are at highest risk are seronegative recipients of seropositive organ, d1/rà, because they have no preexisting immunity, and seropositive recipients, dà/r1, are at intermediate risk. there is little difference between d1/r1 and dà/rà groups, with potentially worse outcomes in d1/r1 (fig. 3) . 43 clinical presentation consists of dyspnea, fever, and malaise with the identification of characteristic cmv cells in lung tissue. radiographic changes are nonspecific and include diffuse haziness, focal haziness, focal lobar consolidation, and no change. 20 diagnosis is made via cell culture viral isolation; however, this can be time consuming. therefore, the detection of cmv dna by pcr in the peripheral blood leukocytes, providing a sensitivity of 92% and specificity of 76% for cmv pneumonitis, along with the bal cmv dna pcr, is an alternative means to diagnosis. 20, 45 however, with regards to the bal findings, it would be imperative to differentiate between infections versus shedding; hence, the concomitant peripheral blood leukocyte pcr along with the clinical picture is necessary to help determine the diagnosis. treatment consists of ganciclovir or the oral alternative, valganciclovir. foscarnet and cidofovir are alternative options; however, they are primarily reserved when there is a concern for resistance or documented resistance because their side-effect profiles are less desirable. 20 cmv immunoglobulin in conjunction with treatment offers limited efficacy. 43 maribavir, brincidofovir, and letermovir are novel agents that may provide alternative options for treatment; however, further studies are warranted. candida is a frequent colonizer of lung transplant recipients, but less than 10% of patients colonized develop invasive disease. 20, 46 bronchial anastomotic infections may occur early in the postoperative setting, which can lead to anastomotic failure, parenchymal lung infection, and mediastinitis. [47] [48] [49] [50] [51] [52] artificial bronchial stents can serve as a potential site for infection. 47, 48 candida tracheobronchitis is based on visual inspection and histologic confirmation along with positive cultures from an appropriate specimen. 47 aspergillus species, a saprophytic organism, has higher rates of mortality, up to 54%, and an incidence of 3% in lung, 2.4% in heart, 2% in liver, and 0.2% in kidney transplant recipients. 47 infection may be due to reactivation or de novo infection following inhalation of the mold. renal failure, hemodialysis, repeated bacterial infections, leukopenia, cmv disease, high levels of immunosuppression, retransplantation, chronic exposure of the transplanted lung to the environment, and abnormal anatomic and physiologic function of the transplanted and, if still present, the native lung, airway ischemia, hypogammaglobulinemia, cystic fibrosis, and bronchial stent are all risk factors for invasive aspergillosis. 47, [53] [54] [55] [56] in lung transplant recipients, 20% to 40% are colonized with aspergillus with complicated infections affecting up to 13% of all patients. 20, [57] [58] [59] [60] colonization can also lead to bronchiolitis obliterans syndrome after lung transplantation. 47, 61 clinical manifestations can range from asymptomatic colonization to tracheobronchitis, invasive pulmonary aspergillosis, empyema, and disseminated disease, 47, 62 with symptoms including purulent sputum, fever, malaise, respiratory distress, and hemoptysis. 20, 63 aspergillus tracheobronchitis can cause airway obstruction, ulcerations, and pseudomembrane formation. 47 nonaspergillus mycelial fungi are also increasing with frequency, as high as 30%, and an overall mortality of 55% 20,64 ; however, zygomycosis (rhizopus and mucor species) and non-aspergillus hyalohyphomycosis (scedosporium apiospermum and fusarium species) have an even higher mortality of up to 100%. 20 endemic fungi, coccidioides immitis, histoplasma capsulatum, blastomyces dermatitidis, and cryptcoccus are additional pathogens that may need to be considered. radiological findings may demonstrate nodules, cavitary lesions, focal consolidation or patchy densities, wedge-shaped pleural-based lesions, air-filled bronchi with an intraluminal lesion, "air crescent" sign, and halo of decreased density. tissue invasion by fungal organisms is the gold standard for diagnosis of invasive fungal pneumonia. however, this may be difficult to obtain; therefore, international society for heart and lung transplantation developed guidelines, a 2010 working formulation for the standardization of definitions of infections in cardiothoracic transplant recipients, to assist in the diagnosis of not only fungal infections but also bacterial and viral infections. these definitions may also be applied to abdominal transplant recipients. treatment is as follows in fig. 4 ; however, be aware of drug-drug interactions with regards to immunosuppressive agents. it is important to note, however, that empiric management of mold infections in sot recipients is seldom necessary. the optimal approach is to pursue the diagnostic workup aggressively and treat accordingly even in lung transplant recipients. pneumocystis jirovecii, t gondii, mycobacterium tuberculosis, and nontuberculosis mycobacteria are other pathogens to also consider as possible causes of respiratory tract infections along with the other pathogens listed in fig. 5 . central nervous system (cns) infections can account for approximately 5% to 10% of cns lesions in transplant recipients. 20, 64 routine prophylaxis aimed at opportunistic infections along with a more conservative approach regarding immunosuppression has led to noticeable trends in infections in transplant recipients. for example, routine administration of trimethoprim-sulfamethoxazole for p jirovecii has likely contributed to the reduction in infections owing to t gondii, nocardia, and listeria monocytogenes along with acyclovir and valganciclovir attributing to the likely decline in herpesviridaerelated infections. 65, 66 clinical presentation will vary and may include fever, headache, meningismus, kernig and brudzinski signs, new-onset seizure, papilledema, altered sensorium, and/or focal neurologic deficits; however, because of their underlying immunosuppression, these may be subtle or absent. 65, 67 as listed in fig. 6 , possible causes range from community-acquired organisms, donor-derived infections, reactivation, to opportunistic pathogens. metastatic or direct lesions along with "pulmonary-brain" syndrome are also considerations in such organisms as cryptococcus, nocardia, aspergillus, zygomycetes, strongyloides, and toxoplasma; therefore, further investigations are warranted (eg, computed tomography of sinus and computed tomography of chest). 2, 67 evaluation should include neuroimaging along with lumbar puncture as soon as possible if no contraindication exists. cerebral spinal fluid examination should always include cell counts and differential, glucose, protein; routine smears; and cultures for bacteria, fungi, and mycobacteria. 65, 67 additional specialized testing, such as viral pcr, antigen or antibody, and 16s ribosomal rna, may be required depending on the clinical scenario. 65, 67 however, brain biopsy with appropriate staining may be the definitive diagnosis if findings are inconclusive. empiric treatment should cover common bacterial and viral pathogens; however, additional agents may be needed depending on the epidemiologic history. cholangitis is a common infection after liver transplant, and in fact, is the most common infection more than 1 year after liver transplant. 68 most cases occurred within 5 years and were associated with primary sclerosing cholangitis and rouxen-y anastomosis. 68 the most frequently identified bacteria are enterococcus spp and e coli 68, 69 ; however, other gram-negative bacilli and anaerobes should also be considered. 68, 69 bile leaks can occur in 2% to 25% of cases after liver transplantation, especially in living liver donor transplants. 70 clinical presentation varies with extent of the leak; however, symptoms can include abdominal pain, fever, or any sign of peritonitis. however, because of underlying immunosuppression, they can also be asymptomatic. 70 in these cases, elevations in serum bilirubin, fluctuations in cyclosporine, or bilious ascites should raise suspicion for a bile leak. 70 biliary strictures at the site of anastomosis can also present with fever, abdominal pain, but also jaundice and asymptomatic biochemical cholestasis. 70 bilomas can represent an additional source of infection. 41, 70 hepatic artery thrombosis, although more common in living donor liver transplants (ldlts), is uncommon with deceased donor liver transplant (ddlt) with an overall incidence up to 9% and can lead to complications, including hepatic abscesses, necrosis, sepsis, and graft loss. 71 vancomycin-resistant enterococcus faecium (vref) is of particular concern in liver transplant. pretransplant colonization increases rates of intra-abdominal and bsis after transplantation. 72, 73 in fact, hospital and intensive care unit stays are longer for patients with vref versus vancomycin-sensitive e faecium infections. 72 liver, pancreas, and intestinal recipients are at particular risk for fungal infections, most often caused by candida species. 41, 74, 75 regarding ddlt versus ldlt, there are variations with infectious complications. the rate of infection appears to be similar to ddlt; however, because of the more complex nature of the surgery, there are observable difference and specific concerns as detailed in box 1. [76] [77] [78] the clinical syndrome of hepatic dysfunction can range from mild elevated liver enzymes to hepatitis to fulminant hepatic failure. hepatic dysfunction can present in any sot recipient; however, of utmost concern would be liver transplant recipients. causes can range from infectious to noninfectious with noninfectious causes primarily an issue with liver transplant recipients regarding postoperative complications, recurrence of primary disease, drug-induced complications, and rejection. 79 the infectious causes listed in fig. 7 can range from donor-derived infections, postoperative complications, community-acquired organisms, reactivation, to opportunistic pathogens. evaluation should include imaging (eg, ultrasound, computed tomography, or mri) along with the appropriate infectious workup (eg, blood cultures, serum pcr, serology, antigen, and/or antibodies). if the diagnosis remains inconclusive, a liver biopsy may need to be pursued with appropriate staining obtained. diarrhea following transplantation is frequently observed and is estimated to occur in 22% to 52% of patients. [80] [81] [82] [83] [84] it can be associated with allograft loss and increased mortality. 80, 82, 85, 86 in fact, it results in 900,000 hospitalizations and 6000 deaths annually. 87, 88 the severity and cause of diarrhea can lead to hypovolemia and/or septic shock. diarrhea is a recognized side effect of some immunosuppressive agents; however, infectious causes should be considered based on the clinical picture. causes are detailed in fig. 8 . evaluation should include stool culture, ova and parasite and giardia antigen along with appropriate pcr, antigen testing, and/or special staining. imaging may include a computed tomography to evaluate for bowel wall edema along with colonic wall thickening and dilation. 87 esophagogastroduodenoscopy (egd) and/or colonoscopy along with biopsy may also be warranted. cmv colitis is diagnosed via histopathology obtained during a biopsy. serum pcr can be low to undetectable in this setting. if there is a clinical suspicion (eg, elevated serum pcr along with diarrhea, however the patient is unable to undergo colonoscopy), this may warrant empiric treatment. clostridium difficile infection (cdi) is among the most common health careassociated pathogen and is the most common cause of nosocomial infectious diarrhea. 89 the highest incidence of cdi in sot occurs within the first 3 months following the transplant and is likely related to frequent exposure to antimicrobials, health care settings, and immunosuppressants. 89 proton pump inhibitors (ppis) are known to be a risk for cdi and may still be used in the setting of mechanical ventilation, versus h2 blockers. 90 hospitalized patients who use ppis are twice as likely to develop cdi. 89, 91 fulminant colitis develops in up to 13% of sot recipients with cdi. 89, 92 relapsing disease is common, and protracted courses of therapy are often essential. 74, 93, 94 idsa and society for healthcare epidemiology of america recently updated the clinical practice guidelines for clostridium difficile infection in adults and children for 2017, and although this is for immunocompetent patients, these guidelines can be applied to sot recipients because there have been limited studies into the treatment of cdi in sot patients. the most common infectious complication in sot is uti, accounting for 45% to 72% of all infections, and 30% of all hospitalizations for sepsis in ktrs. 19, [95] [96] [97] [98] [99] most uti are seen within the first 6 months after transplant; however, it can occur any time after transplantation. 96, 100, 101 empirical treatment is imperative, as it has been demonstrated that inappropriate antibiotic therapy is associated with an increase in mortality. 100, 102, 103 to guide empirical therapy, local epidemiologic data, patient's microbiological history, and prior antibiotic use need to be taken into account. 95, 100 the most frequent organisms causing utis are gram-negative bacilli 100, 104 ; however, when a urinary catheter is involved, enterococci and s aureus should also be considered. the duration of treatment varies from 7-21 days depending on the clinical syndrome. recurrent uti, defined as 3 or more episodes of symptomatic utis over a 12month period or 2 in the previous 6 months, should prompt further investigation regarding anatomic and functional abnormalities along with behavioral modifications (eg, postcoital voiding) and may need a prolonged course of antibiotics, possibly 4 to 6 weeks. 95, 100, 101 asymptomatic bacteriuria should only be treated during the early postoperative period and up to 1 month after transplant in renal transplant patients. 100 the data on uti related to candida spp in sot are limited and mostly include ktr. in ktr, candida spp are the most frequently isolated fungal cause of uti. unfortunately, there are no clinical trials in the management of candida uti in sot. 100 candiduria is frequent, occurring in up to 11% of ktr; however, it is mostly asymptomatic. 95 asymptomatic candiduria is usually treated as there is concern regarding the allograft and potential for complications regarding the upper urinary tract; however, it should only be treated if the patient is neutropenic, or undergoing a urologic procedure. 95, 100 candiduria should be classified based on risk factors for disseminated candidiasis. urinary catheters should be removed or exchanged, and candiduria should be confirmed with a second, clean voided urine culture. disseminated candidiasis may be considered if clinical manifestations are consistent (eg, positive blood cultures, a second urine culture after removal or replacement of the urinary catheter, funduscopic examination, cultures from any other significant site, and kidney imaging). persistent candiduria along with infectious complications no indwelling catheters should prompt imaging of the kidneys and collecting system to exclude renal abscess, fungus balls, or other urologic abnormalities. treatment options can range from fluconazole treatment of choice, to echinocandins, amphotericin b, and flucytosine depending on the clinical situation and organism sensitivities with durations from at least 14 days for uti to 2 to 4 weeks for pyelonephritis (box 2). 100 sot recipients are a complex group of patients with diverse causes given the underlying immunosuppression. as with all infectious processes, rapid identification of the always consider previous microbiological data and local epidemiology with regards to empiric antibiotics if an intravascular catheter is involved, consider broad gram-positive cocci, including mrsa, coverage in addition to broad gram-negative coverage, including esbls and cres, if warranted empiric antifungal is not needed, unless there is a high index of suspicion always consider previous microbiological data and local epidemiology with regards to empiric antibiotics cap should include empiric coverage for atypicals along with community-associated organisms hap and vap should include broad gram-positive coverage, especially mrsa, along with broad gram-negative coverage, including esbls and cres if warranted influenza is the only virus with approved treatment, oseltamavir; therefore, this should be started empirically if there is a concern antifungals should not be started empirically, even in lung transplant recipients; however, fungal infections should be worked up thoroughly pathogen, source control, and adjustment of immunosuppression is the hallmark of treatment. there is a summary of the management of infections in sot recipients included in box 3. empiric therapy should consist of ceftriaxone and vancomycin ae acyclovir always consider previous microbiological data and local epidemiology with regards to empiric antibiotics if vre positive, will need to consider coverage using high-dose daptomycin or linezolid along with broad gram-negative coverage, including esbls and cres, if warranted regarding c difficile infections, oral vancomycin should be initiated empirically, if suspected, as first-line therapy always consider previous microbiological data along with local epidemiology with regards to empiric antibiotic decisions asymptomatic bacteriuria should only be treated in renal transplant patients during the first month posttransplantation antimicrobials should be tailored to the causative agent, with durations that generally range from 7 to 21 days depending on the clinical context fluconazole is the treatment of choice for cystitis and pyelonephritis if candida is the causative organism abbreviations: cre, carbapenem-resistant enterobacteriaceae; esbl, extended spectrum betalactamase inhibitors; vap, ventilator-associated pneumonia. clinical immune-monitoring strategies for predicting infection risk in solid organ transplantation from the classic concepts to modern practice epidemiology and risk factors for nosocomial bloodstream infections in solid organ transplants over a 10-year period bloodstream infections among transplant recipients: results of a nationwide 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recipients empirical fluconazole versus placebo for intensive care unit patients: a randomized trial empirical micafungin treatment and survival without invasive fungal infection in adults with icu-acquired sepsis, candida colonization, and multiple organ failure: the empiricus randomized clinical trial pulmonary infections in liver transplant recipients receiving tacrolimus. changing pattern of microbial etiologies infectious complications after kidney transplantation: current epidemiology and associated risk factors pneumonia infection in organ transplant recipients pneumonia in solid organ recipients: spectrum of pathogens in 217 episodes microbiologic features and outcome of pneumonia in transplanted patients infectious pulmonary complications in lung transplant recipients a standardized protocol for the treatment of severe pneumonia in kidney transplant recipients pneumonia in solid organ transplant recipients: a prospective multicenter study the diagnosis of pneumonia in renal transplant recipients using invasive and noninvasive procedures pulmonary complications in cardiac transplant recipients nocardiosis following solid organ transplantation: a single-centre experience rna respiratory viruses in solid organ transplantation respiratory viral infections in immunocompetent and immunocompromised persons clinical implications of respiratory virus infections in solid organ transplant recipients: a prospective study respiratory viral infections in transplant recipients respiratory viruses and chronic rejection in lung transplant recipients respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant a single-season prospective study of respiratory viral infections in lung transplant recipients respiratory viruses in transplant recipients: more than just a cold. clinical syndromes and infection prevention principles adenovirus in solid organ transplantation adenovirus pneumonia in lung transplant recipients adenovirus infection in the lung results in graft failure after lung transplantation assessment of adenovirus infection in adult lung transplant recipients using molecular surveillance ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus viral infections in solid organ transplant recipients: novel updates and a review of the classics late-onset cytomegalovirus disease as a significant complication in solid organ transplant recipients receiving antiviral prophylaxis: a call to heed the mounting evidence increased human cytomegalovirus (hcmv) dna load in peripheral blood leukocytes after lung transplantation correlates with hcmv pneumonitis infectious complications of lung transplantation. impact of cystic fibrosis invasive fungal infections in solid organ transplant recipients fungal infection in 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of risk factors aspergillus colonization of the lung allograft is a risk factor for bronchiolitis obliterans syndrome prevalence and outcome of invasive fungal infections in 1,963 thoracic organ transplant recipients: a multicenter retrospective study. italian study group of fungal infections in thoracic organ transplant recipients aspergillus infection in single and double lung transplant recipients opportunistic mycelial fungal infections in organ transplant recipients: emerging importance of non-aspergillus mycelial fungi cns infections in solid organ transplant recipients infections of the central nervous system in transplant recipients central nervous system syndromes in solid organ transplant recipients infectious complications more than 1 year after liver transplantation: a 3-decade nationwide experience the importance of late infections for the long-term outcome after liver transplantation biliary complications following liver transplantation etiology and management of hepatic 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recipients: mitos study gastrointestinal complications in renal transplant patients: a large, single-center experience diarrhoea following renal transplantation diarrhea in organ transplant recipients clinical practice. acute infectious diarrhea clostridium difficile infection in solid organ transplant patients histamine-2 receptor antagonists vs proton pump inhibitors on gastrointestinal tract hemorrhage and infectious complications in the intensive care unit strategies to prevent clostridium difficile infections in acute care hospitals fulminant clostridium difficile: an underappreciated and increasing cause of death and complications clostridium difficile infection in solid organ transplant recipients early and late onset clostridium difficileassociated colitis following liver transplantation urinary tract infections in solid organ transplantation urinary tract infections in renal transplant recipients hospitalizations for bacterial septicemia after renal transplantation in the united states infective complications in renal allograft recipients: epidemiology and outcome acute pyelonephritis represents a risk factor impairing long-term kidney graft function management of urinary tract infection in solid organ transplant recipients: consensus statement of the group for the study of infection in transplant recipients (gesitra) of the spanish society of infectious diseases and clinical microbiology (seimc) and the spanish network for research in infectious diseases (reipi) management of urinary tract infections and lymphocele in renal transplant recipients bloodstream infections caused by antibioticresistant gram-negative bacilli: risk factors for mortality and impact of inappropriate initial antimicrobial therapy on outcome mortality and delay in effective therapy associated with extended-spectrum beta-lactamase production in enterobacteriaceae bacteraemia: a systematic review and meta-analysis multidrug-resistant bacterial infection in solid organ transplant recipients key: cord-268830-8li6xhbu authors: kozak, robert; prost, karren; yip, lily; williams, victoria; leis, jerome a.; mubareka, samira title: severity of coronavirus respiratory tract infections in adults admitted to acute care in toronto, ontario date: 2020-03-29 journal: j clin virol doi: 10.1016/j.jcv.2020.104338 sha: doc_id: 268830 cord_uid: 8li6xhbu background: the world health organization has highlighted the need for improved surveillance and understanding of the health burden imposed by non-influenza rna respiratory viruses. human coronaviruses (covs) are a major cause of respiratory and gastrointestinal tract infections with associated morbidity and mortality. objectives: the objective of our study was to characterize the epidemiology of covs in our tertiary care centre, and identify clinical correlates of disease severity. study design: a cross-sectional study was performed of 226 patients admitted with confirmed cov respiratory tract infection between 2010 and 2016. variables consistent with a severe disease burden were evaluated including symptoms, length of stay, intensive care unit (icu) admission and mortality. results: covs represented 11.3% of all positive respiratory virus samples and oc43 was the most commonly identified cov. the majority of infections were community-associated while 21.6% were considered nosocomial. the average length of stay was 11.8 days with 17.3% of patients requiring icu admission and an all-cause mortality of 7%. in a multivariate model, female gender and smoking were associated with increased likelihood of admission to icu or death. conclusion: this study highlights the significant burden of covs and justifies the need for surveillance in the acute care setting. human coronaviruses (covs) are a significant cause of communityacquired respiratory tract infections. the symptoms associated with cov infection were first described over four decades ago [1] , and can range from relatively mild upper-to more severe lower-respiratory tract infections with increased severity in certain patient populations [1, 2] . it has been reported that immunocompromised patients, particularly hematopoietic cell transplant recipients, are at increased risk of lower respiratory tract infections, prolonged viral shedding and mortality, often comparable to what is seen with influenza virus [3, 4] . similar to other non-influenza respiratory viruses, covs are still relatively understudied despite being a common cause of hospital-and community-acquired respiratory infection [5, 6] . there is currently a paucity of canadian data on the burden of disease imparted by endemic covs, and their contribution to nosocomial respiratory virus outbreaks. this is likely due to the fact that laboratories may not routinely identify covs, and they are not generally reportable to public health agencies. thus, the world health organization (who) has highlighted the need for improved epidemiological surveillance and a better understanding of the health burden imposed by covs, as well as other non-influenza rna respiratory viruses [7] . four types of endemic covs are in current circulation, oc43, 229e, hku1, and nl63. recent findings demonstrate a seasonality for cov infections, with peak numbers being observed in the winter months [2] . however, this data is based on nationally-reported findings from the united states, and may not reflect local or national epidemiology in canada. moreover, the receptor-binding domain of the glycoprotein of 229e has undergone adaptation over the last 50 years [8] that ongoing viral evolution may influence which strains predominate from year to year. this is further supported by phylogenetic data examining oc43 isolates, which showed that the circulating genotypes in southeast asia changed over time [9] . a recent study in the midwestern usa reported frequent identification of hku1, whereas a separate study from china reported oc43 to be more prevalent [10, 11] . therefore, determining the regional prevalence is important to understand the burden of these infections. in acute care hospitals, much of the focus in diagnostics has been placed on influenza and respiratory syncytial virus (rsv) because of the severe infection and poor outcomes of hospitalized patients, yet the burden of cov in acute care is not well studied. most hospitals do not routinely test for cov resulting in gaps in our clinical and epidemiologic understanding of this virus. the predictors of severe infection are well known for cov associated with acute respiratory syndromes (eg. middle east respiratory syndrome cov, severe acute respiratory syndrome cov), yet few studies have identified these predictors for the more common four circulating cov strains such as oc43, 229e, hku1 and nl63 [12, 13] . the primary objective of this study was to describe the burden of cov among patients admitted to an acute care hospital in toronto, canada over a six-year period, and identify the predictors of severe disease. this cross-sectional study was performed at sunnybrook health sciences centre, a tertiary-care hospital with over 1300 total beds serving acutely ill and rehabilitating patients as well as long-term care residents. institutional ethics approval was obtained (reb#066-2017). the study participants included admitted patients ≥17 years of age who tested positive for a cov infection between january 1st 2010 and december 31st 2016. outpatients and residents of the affiliated longterm care facility were excluded. viral test results were obtained from nasopharyngeal (np), mid-turbinate (mt) swabs, and bronchoalveolar lavages (bals) tested as part of routine care for respiratory viruses using multiplex pcr (xtag rvp, xtag rvp fast v2 or rpp, luminex). viral targets in this assay included: influenza viruses a & b, rsv, adenovirus, rhinovirus/enterovirus, human metapneumovirus, parainfluenza viruses type 1-4 and coronavirus species oc43, 229e, nl63 and hku1. demographic and clinical data were obtained for all patients meeting inclusion criteria. cases were considered to be communityacquired if they were diagnosed within 72 h of admission and nosocomial if they were diagnosed ≥72 h after admission [14] . lower respiratory tract involvement was defined as radiographic evidence of acute disease, determined on review of radiology reports. dependent (outcome) variables were those associated with severity and burden of disease. these included: number of symptoms, presence or absence of fever, need for oxygen therapy or intubation, chest radiography changes, isolation of bacteria by conventional culture, admission to an intensive care unit (icu), number of days spent in the icu, antimicrobial and antiviral use, length of stay in hospital and death. independent variables included coronavirus strain (oc43 vs. non-oc43), gender, smoking status (not a smoker, previously a smoker, current smoker), and age. the age variable was converted into a categorical variable with three categories including: less than 60 years of age, patients between 61 and 80 years of age, and patients over 80 years of age. the chi-squared/fisher's exact test, kruskal wallis, mann-whitney and unpaired t-tests were used to assess the presence of statistically significant correlations between dependent and independent variables. statistically significant correlations were included in univariable logistic or non-parametric regression analyses to evaluate the predictive ability of the independent variable. a bivariate and a multivariate logistic regression analysis were also performed including the following variables: age, smoking status (current or previous smoker vs. non-smoker), viral strain (oc43 vs. non-oc43), nosocomial vs. community acquired infection, gender, and number of comorbidities (3 or more vs. less than 3). statistical analysis was performed using sas university edition (sas institute, cary, nc, usa). during the study period, 5038 samples were positive for a respiratory virus of which 11.3 % (n = 569) were positive for cov representing the third most frequently identified pathogen after influenza viruses and rhinoviruses/enteroviruses (fig. 1a) . it was noted that infections were identified year-round, but the peak number of cases occurred between november and february each year (data not shown). the number of cov infections increased between 2010 and 2016 ( fig. 1c) . from these samples, 226 patients met study inclusion criteria. amongst the covs, the most frequently identified strain was oc43, representing 50 % (n = 285) of covs, followed by 229e (22.3 %, n = 127), hku1 (13.9 %, n = 79) and nl63 (13.7 %, n = 78) (fig. 1b) . the age of patients spanned from 18 to 99 years old and the median age was 77 (table 1) . additionally, the distribution of cases was similar between males and females (44.7 % vs. 55.3 %). as shown in table 1 , comorbidities were common amongst our cohort, with vascular, cardiac and pulmonary comorbidities being the most frequently reported. only 3.1 % of patients were current smokers, and 12.8 % were former smokers. community-acquired infections accounted for 78.3 % (n = 177) of cases, and nosocomial infections accounted for 21.6 % (n = 49). symptoms included cough (48.6 %, n = 110), shortness of breath (sob) (37.1 %, n = 84), and fever (29.6 %, n = 67). furthermore, 81 % of patients had a chest x-ray performed within 24 h of presentation, and the majority of individuals (57.3 %, n = 130) demonstrated acute radiographic changes. hematology and biochemistry laboratory investigations indicated 30.9 % (n = 70) had elevated white blood cell counts. additionally, 38.9 % (n = 88) of patients had decreased lymphocytes counts (table 1) . bacterial co-infections were noted in 7.1 % of patients, and viral co-infections were detected in 3.9 % of patients, and in both groups no clear pathogen predominated (tables 1 & 2 ) . the average length of stay in hospital was 13 days (range 1−354 days), and 17.3 % required admission to the icu with a mean duration of 11.8 days (range 1-240 days). all-cause mortality was 7%. the predictor variables associated with severe disease outcomes are presented in table 3 . patients with oc43 had 2-fold odds of requiring o2 or intubation compared to non-oc43 strains, while no difference in mortality or icu admission was found based on cov strain. increased age was associated with increased numbers of symptoms, comorbidities, and radiographic changes. a bivariate analysis identified both this cross-sectional study spanning 6-years suggests that cov accounts for an important burden of respiratory infection, representing 1 out of 9 viral respiratory infections, with a propensity to cause lowerrespiratory tract infection and severe outcomes. notably, all-cause mortality and risk of icu admission were similar to rates reported for influenza and rsv [15, 16] . our findings indicate that cov is not a benign infection among those who are hospitalized, and is similar to available data elsewhere. garbino and colleagues found that 31 % of patients in their cohort were admitted to the icu, and noted an all-cause mortality of 10 %. lowerrespiratory tract infections (lrtis) are the fourth leading cause of mortality globally, and characterizing the epidemiology of respiratory viruses is a necessary first step to reducing the burden of disease [7] . predictors of severe outcome including need for icu admission or mechanical ventilation have been described for mers-cov, but there is a paucity of data on other covs [12, 17] . in our cohort smoking predicted icu admission and/or mortality, which is similar to what was reported in a prior study on patients infected with hku1 cov [18] the impact of gender on outcomes of cov, as determined by our multivariate analysis is in contrast with what is reported for mers cov. with other coronaviruses including mers-cov, there is often a predominance of male cases [19, 20] . however, females represented the majority of cov infections in our cohort, and our analysis indicated that female gender was associated with more severe outcome. this finding differs from what has been reported for sars-cov patients in singapore [21] , and merscov 17] where male gender was predictive of poor outcomes. interestingly, our bivariate analysis indicated that nosocomial acquisition was associated with poor prognosis. similar findings have been noted for infections with mers cov, where acquisition of the virus in the hospital was predictor of 72 h mortality in a multivariate analysis of cases in saudi arabia [13] . our findings indicate that there is heterogeneity in circulating strains, as oc43 and 229e were more prevalent than nl63 or hku1. previous studies have similarly shown oc43 and 229e account for up to approximately 30 % of common colds [22] , and our data indicate that approximately 70 % of coronavirus infections in our cohort were due to these two strains. more recent four-year prevalence data from military personnel in the usa revealed season-to-season variability where oc43 and 229e alternated as the most common strain identified [23] . other studies have highlighted prevalence of a particular strain, often showing variation between locations and patient populations (eg. transplant vs. non-transplant; inpatient vs. icu etc) [3, 10, 11, 23] . sequencing analysis by lau and colleagues of clinical isolates of oc43 suggested recombination may play a role in the generation of novel cov genotypes [24] . this highlights the importance of determining the local epidemiology, and suggests that genomic sequencing of isolates may be a necessary next step to investigate genetic changes over time. interestingly, there is increasing reports of both co-infections with multiple respiratory viruses and viral and bacterial pathogens, and data suggesting this may be associated with more severe disease [25] [26] [27] [28] . in our cohort bacterial co-infections were only identified in 7.9 % of individuals, and viral co-infections in 3.1 %; both of which are lower than what has been reported by other groups [28, 29] , and we did not have sufficient numbers to investigate any correlations with disease severity. however, this is an area where additional studies are needed. our study has several important limitations. we only included patients who presented to the hospital for acute care, thus representing a subset of the most ill patients with cov infection in the community. furthermore, it cannot be discounted that higher number of positives were seen during influenza season due to heightened testing of respiratory viruses during this time of year. importantly, our study did not include asymptomatic or subclinical cases or non-cov controls, which makes it challenging to identify determinants of severity relative to these other populations. moreover, as data emerges on the association of covs with central nervous system sequalae, future studies should investigate the incidence of strokes, and seizures in cases of cov infection [30] . the number of patients receiving extracorporeal membrane oxygenation should also be considered in subsequent studies. since our assay was not quantitative we are unable to determine the role of viral load in influencing disease severity, although it has been noted by others that viral load did not correlate with outcome [31] . furthermore, we did not include biomarkers associated with liver function. it has been reported that elevated alt was associated with adverse outcomes in patients infected with sars [32] , and further investigation is necessary to determine the prognostic value in other cov infections. finally, the relatively small numbers of cases of each strain (e.g. oc43, 229e, hku1, nl63) prevented separate analysis of any potential effects of individual strains on patient outcome. our study describes burden and risk factors associated with disease severity in patients infected with cov at a single urban healthcare centre. at present there is likely an under-reporting of cov infections in canadian hospitals, as many laboratories do not routinely test for these pathogens. collectively, this study highlights the significant burden of covs and justifies the need for surveillance in the acute care setting. credit author statement r.k. and s.m. were involved in the conceptualization and design of the study. r.k., k.p., l.y., v.w., j.a.l. were involved in data collection and analysis. k.p. and j.a.l. performed statistical analysis and interpretation. manuscript writing was performed by r.k., j.a.l. and s.m. and all authors participated in editing. all authors declare no conflicts of interest table 2 co-infections identified among 226 adult patients hospitalized with coronavirus respiratory tract infection. pathogen identified viral cytomegalovirus (n = 1), entero/rhinovirus (n = 3), human metapneumovirus (n = 2), parainfluenza virus 4 (n = 1), respiratory syncytial virus (n = 2) bacterial capnocytophage spp. (n = 1), coagulase-negative staphyloccocci (n = 4), escherchia coli (n = 2), haemophilus influenzae (n = 2), moraxella spp. (n = 3), streptococcus pneumoniae (n = 3), klebsiella pneumoniae (n = 1), pseudomonas aeruginosa (n = 1) table 3 association between severity of coronavirus infection and clinical factors based on univariate logistic regression analysis. independent variables included coronavirus strain (oc43 vs. non-oc43), gender, smoking status (not a smoker, previously a smoker, current smoker), and age. effects of a "new" human respiratory virus in volunteers human coronavirus circulation in the united states clinical significance of human coronavirus in bronchoalveolar lavage samples from hematopoietic cell transplant recipients and patients with hematologic malignancies prolonged shedding of human coronavirus in hematopoietic cell transplant recipients: risk factors and viral genome evolution viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study global epidemiology of non-influenza rna respiratory viruses: data gaps and a growing need for surveillance receptor-binding loops in alphacoronavirus adaptation and evolution identification and evolutionary dynamics of two novel human coronavirus oc43 genotypes associated with acute respiratory infections: phylogenetic, spatiotemporal and transmission network analyses epidemiology and clinical characteristics of human coronaviruses oc43, 229e, nl63, and hku1: a study of hospitalized children with acute respiratory tract infection in guangzhou, china, eur human coronavirus-hku1 infection among adults in mers transmission and risk factors: a systematic review the predictors of 3-and 30-day mortality in 660 mers-cov patients influenza surveillance-united states, 1992-93 and 1993-94 severe morbidity and mortality associated with respiratory syncytial virus versus influenza infection in hospitalized older adults clinical determinants of the severity of middle east respiratory syndrome (mers): a systematic review and meta-analysis clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia sex matters -a preliminary analysis of middle east respiratory syndrome in the republic of korea presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study sars in singapore-predictors of disease severity isolation of rhinoviruses and coronaviruses from 38 colds in adults species-specific clinical characteristics of human coronavirus infection among otherwise healthy adolescents and adults, influenza other respir molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination dual respiratory virus infections human coronavirus alone or in co-infection with rhinovirus c is a risk factor for severe respiratory disease and admission to the pediatric intensive care unit: a one-year study in southeast brazil epidemiology and microbiological investigations of communityacquired pneumonia in children admitted at the emergency department of a university hospital coronavirus hku1 and other coronavirus infections in hong kong detection of respiratory viruses and legionella spp. by realtime polymerase chain reaction in patients with community acquired pneumonia, scand human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method clinical significance of hepatic derangement in severe acute respiratory syndrome key: cord-277327-il8uaavn authors: couch, md, robert b.; englund, md, janet a. title: respiratory viral infections in immunocompetent and immunocompromised persons date: 1997-03-17 journal: am j med doi: 10.1016/s0002-9343(97)00003-x sha: doc_id: 277327 cord_uid: il8uaavn the acute respiratory illnesses are the most common type of acute illness in the united states today. the respiratory viruses—which include influenza viruses, parainfluenza viruses, respiratory syncytial virus (rsv), rhinoviruses, coronaviruses, and adenoviruses—cause the majority of these illnesses. some of these viruses cause illness throughout the year, whereas others are most common in winter. all population groups experience these infections and illnesses. as the number of elderly persons and those with underlying disease increases, awareness is growing that these common infections can have serious consequences. this has recently been emphasized for immunocompromised persons. at the m.d. anderson cancer center (mdacc), infection surveillance of mostly hospitalized adults with leukemia or a recent bone marrow transplant yielded a respiratory virus from 181 of 668 (27.1%) respiratory illness episodes. in descending order of frequency, infections with rsv, rhinoviruses, influenza viruses, parainfluenza viruses, and adenoviruses were detected in each of three surveillance years. high frequencies of nosocomial acquisition occurred, as has been noted in prior reports. similarly, persistence of infection and high frequencies of pneumonia and death among infected patients occurred, which have also been noted earlier. at mdacc, pneumonia occurred in 58–78% of infected patients, and 22–44% died. the role of the virus infection in many cases of pneumonia is uncertain, but death from pure viral pneumonia is well documented. a number of immune deficiencies in this patient population and options for control of these infections have been described that can, respectively, account for the medical problem and provide ways to approach prevention and treatment. a cute respiratory conditions are the most common acute conditions occurring among persons in the united states. 1 their incidence exceeds that of injuries, digestive conditions, and infective and parasitic conditions combined. infection with a respiratory virus is the most common cause of an acute respiratory condition. the variety and omnipresence of respiratory viruses along with their facility for spread among human populations ensure their occurrence as causes of infection and illness in all human populations. this includes persons of all ages, the healthy and the unhealthy, and the immunocompetent and the immunocompromised. the unusual severity of illnesses occurring among immunocompromised persons infected with a respiratory virus has been emphasized recently. this article is an overview of respiratory viral infections in both immunocompetent and immunocompromised persons. the respiratory viruses are listed in table i . all serotypes in each genus are significant causes of acute respiratory illness (ari), except for the adenoviruses, of which nine serotypes (1-7, 14, and 21) are important causes of ari. a large number of other viruses can cause ari or prominent respiratory symptoms; however, they are better known for other disease manifestations. these include many enteroviruses, measles virus, herpes simplex type 1 virus, and epstein-barr virus (the cause of infectious mononucleosis). in all, 20 distinct viruses plus ú100 different rhinoviruses cause ari in humans. this number of viruses-along with the facts that reinfection may occur, other viruses can produce ari, and a large portion of ari cases presumed to be viral cannot be ascribed to a specific virus using current technology-explains the high incidence of viral respiratory infections and illnesses in all human populations. the respiratory viruses exhibit a predilection for infecting the respiratory passages. localization of j the most common infections in developed countries j the cause of an extraordinary amount of morbidity, they are the most important contributor to loss of time from work or school j a major cause of severe disease, hospitalization, and death from infection, particularly among very young children and the elderly j a major predisposing cause of otitis media, sinusitis, and acute bacterial pneumonia j a major cause of acute respiratory insufficiency in persons with underlying lung disease (asthma, chronic obstructive pulmonary disease, etc.) j a major cause/contributor to severe disease and death among immunocompromised persons the infection induces symptom complexes that are well known to physicians: the common cold, pharyngitis, laryngitis, tracheobronchitis, laryngotracheobronchitis in young children (croup), bronchiolitis, and influenza. more than one symptom complex may occur, and upper respiratory illness, lower respiratory illness, or acute diffuse respiratory illness may be the appropriate designation. the respiratory viruses also cause pneumonia, particularly among infants and very young children, although most infections in immunocompetent persons do not lead to pneumonia. respiratory viral infections occur throughout the year, although they are more common in winter than in summer. they affect persons of all ages, but their incidence is higher among children than among adults. infection may be asymptomatic or induce a severe and lethal infection. a variety of complications of viral respiratory infection have been described; most notable are secondary bacterial infections causing otitis media, sinusitis, or pneumonia. in these cases, a respiratory virus infection impairs the defense mechanisms that keep these anatomic sites free from infection with bacteria. among the other described complications are post-infectious encephalitis, the guillain-barré syndrome, reyes syndrome after influenza in children, and disseminated intravascular coagulation with influenza. a summary of the major medical features of the acute viral respiratory infections appears in table ii . they are a major cause of morbidity in healthy populations; they are a major cause of severe disease, hospitalization, and death among the very young, the elderly, and persons with underlying disease, even if they are immunocompetent. severe disease is also common among immunocompromised persons with an acute respiratory viral infection, but the contribution of the virus to the severe disease and death in these populations is not always clear. results of surveillance of immunocompromised adult cancer patients at the m.d. anderson cancer center (mdacc) who were experiencing a respiratory illness are shown in table iii for a 3-year period. sampling was limited to hospitalized patients with leukemia and hospitalized or clinic patients who had recently received a bone marrow transplant. a combined nasal wash and throat swab specimen was tested for virus as described previously. 2 a total of 668 illness episodes were sampled for virus: 235 isolates were obtained from sampling of 223 illness episodes; ú1 virus was isolated for 12 episodes. thus, 33.4% of respiratory illness episodes in these mostly hospitalized patients yielded a virus. when herpes simplex virus and cytomegalovirus isolations were excluded, 181 (27.1%) episodes were virus positive; two episodes yielded two respiratory viruses. it should be emphasized that 27.1% represents a minimal estimate of the frequency of infection with a respiratory virus among these patients with an ari, since serology was not done and repeat sampling was minimal. the distribution of isolates according to type of respiratory virus is shown in table iv . as noted, each of the major respiratory viruses was detected in each year of sampling. the most commonly detected virus was rsv, followed closely by the picornaviruses. of picornaviruses that were further categorized, 90% were rhinoviruses and 10% were enteroviruses. influenza a or b virus was present each year, as were parainfluenza viruses; 92% of the parainfluenza viruses were type 3. 3 the adenovirus isolates have not been typed. the distribution of isolates by time of year is shown in figure 1 . excessive specimen contamination and limited sampling is the probable explanation for the absence of isolates during the summer of 1993. nevertheless, the overall pattern is for rhinoviruses, adenoviruses, and parainfluenza 3 virus to occur throughout the year, with periodic increases in frequency. the occurrence of rsv and influenza viruses was confined to the winter and spring, thus accounting for an overall winter increase. in summary, respiratory viral infections, which are highly prevalent among immunocompetent persons with a respiratory illness, are also highly prevalent among adult immunocompromised cancer patients experiencing a respiratory illness. the pattern of occurrences by time of year are similar to those described for these viruses among immunocompetent persons, except for the frequency of rsv infection. 4 this frequency is distinctly unusual for adult immunocompetent patients and may represent an increased susceptibility for this infection among immunocompromised patients (glezen wp, unpublished data, 1996). alternatively, increased occurrences of more severe disease that increases recognition of rsv infection or increased transmission for this virus in the social-environmental circumstance of the populations sampled might explain the finding. three major and relatively distinctive features of respiratory viral infections among immunocompromised patients are (1) high frequencies of nosocomial acquisition, (2) persistence of infection beyond the time periods reported for immunocompetent patients, and (3) high frequencies of pneumonia and death in association with the infection. published reports of frequencies of nosocomial infections among hospitalized patients are summarized in table v. 3,5 -11 infection frequencies for the different studies and the different viruses varied between 55% and 83%; there are no apparent differences in frequency for the different viruses. these infections will have been acquired from an infected person. this could be another ill patient, but most cases are probably acquired from exposure to an infected person with little or no illness who brings the virus into the hospital. hospital personnel, including doctors and nurses, have been identified as probable sources; visitors, particularly children who exhibit high infection frequencies, are an alternative source. a major contributing factor to these infection frequencies is the prolonged stay that is characteristic of hospitalized patients who are immunocompromised. reports of persistence of respiratory viral infection among immunocompromised children are shown in table vi ; no reports for adults were identified. 6,7,12 -17 the range and single-case durations are greater for immunocompromised children than for immunocompetent children in all reports. as noted, some children shed virus for months. although the durations vary, prolonged infection characterized all three virus infections. the frequencies of pneumonia and death associated with a documented infection among immunocompromised adult leukemia and bone marrow transplant patients hospitalized at mdacc are shown in table vii . 3,11,18 -26 pneumonia occurred in 58 -78% of infected persons, and 22 -44% died. although overall frequencies of pneumonia and death appear similar for each virus, analysis of patterns of occurrences suggested a greater severity for rsv infection after bone marrow transplantation or chemotherapy than for the other viruses. moreover, the apparent value of early aerosol ribavirin and intravenous immunoglobulin (iv ig) therapy for rsv reduced the overall frequencies for that virus. 8 the role of the virus infection in causing the pneumonia and death is uncertain in many of these patients, who frequently have multiple infections. however, histologic examination of lung tissue from many who died indicates that the virus infection is the primary pathogen in many cases. considerable effort has been expended in identifying the major immune correlates and mechanisms for defense against a respiratory virus infection. a variety of nonspecific defenses contribute to resistance to infection and recovery from infection. these defenses include the mucous layer covering the respiratory mucosa, fever, the inflammatory response, a and b interferon, and nonspecifically activated phagocytes. the primary defense against acquisition of a specific virus is antibody to that virus, whereas the primary mechanisms for recovery are both humoral (antibody) and cell-mediated immune responses. current information indicates that the primary immunoglobulin and antibody in the upper respiratory passage is polymeric iga that is formed locally in submucosal tissues in response to antigen and transported across epithelial cells into upper respiratory secretions. 23 with descent into the lower respiratory passage, the proportion of 7s igg in secretions increases, as does the proportion of 7s igg antibody. 24 this antibody is derived primarily from serum. thus, following infection, the major antibody protecting the nasopharynx is considered to be iga, whereas that protecting the lower respiratory passages is igg. both types of antibody can, however, be present at both sites, and both antibody types are desirable for optimal protection against a specific virus. once infection occurs, immune defenses are activated to eliminate the infection and thereby bring about recovery from illness. both a and b interferon act early in infection and are important nonspecific mechanisms for promoting elimination of infection. specific antibodies that are induced may contribute to virus elimination in a variety of ways, including aggregation for clearance, opsonization, and early lysis of infected cells; lysis may be complement mediated or represent antibody-dependent cell-mediated cytotoxicity (adcc). the major specific cell-mediated immune response promoting recovery is cd8 t lymphocyte-mediated cytotoxicity. this mechanism has been shown to be of major importance in myxovirus and paramyxovirus infections in animal models. 25, 26 a number of immune deficiencies have been described in immunocompromised persons that would be reflected in one or more deficiencies of the immune mediators summarized above. lum 27 has described deficiencies after bone marrow transplantation, a circumstance representing an extreme degree of immune deficiency. these patients exhibit b lymphocyte impairment manifested by a reduced ability of b cells to respond to stimulatory cytokines, such as il-4, reduced serum immunoglobulin levels, and depressed primary and secondary responses to antigens. deficiencies in t cell function are indicated by reduced cd4 lymphocyte and cd4/cd8 cell ratios, reduced t helper cell and increased suppressor cell activity, reduced proliferative responses, reduced delayed-type hypersensitivity responses, and reduced cd8 lymphocyte cytotoxic function. reduced neutrophil numbers and function are of major importance for the occurrence and control of bacterial infection; but, except for an ability to mediate adcc, no role for neutrophils in defense against these virus infections has been suggested. also accompanying the chemotherapy used in many transplant cases is the occurrence of mucositis with the loss of mucosal integrity and possibly iga antibody occurrence in the upper respiratory passages. 27 reduced mucosal integrity and local iga antibody would lead to increased susceptibility to upper respiratory infection, while reduced serum antibody levels would increase susceptibility to lower respiratory infection. although increased susceptibility to infection in immunocompromised patients has not been proven, kempe et al 28 reported an increased rate of influenza virus infection among immunocompromised children compared with an immunocompetent population. of interest is the finding that the increase was accounted for primarily by infection among those with preexisting serum antibody. a deficiency in new antibody synthesis induced by infection would lead to increased susceptibility to reinfection. reduced t lymphocyte numbers and function with reductions in t cell cytotoxic function would lead to reduced ability to clear virus, with a resulting increase in both magnitude and duration of infection. an increased magnitude of infection would lead to an increased likelihood of viral pneumonia as well as secondary bacterial or fungal pneumonias that occur because of impairment of antimicrobial defenses by both the immune deficiency and the viral infection. thus, the immune deficiencies described for immunocompromised persons can account for a high frequency of viral respiratory infections as well as an increased severity of illness and a significant risk of death. the described variability in frequency and severity of the infections among the various populations of immunocompromised persons should reflect the degree of immune deficiency. this appears to be the case, as persons who are less immunocompromised than others seem to have less of a problem with viral respiratory infections. [29] [30] [31] the three general categories of options for control of viral respiratory infections are (1) prevention of exposure, (2) provision of immunity, and (3) administration of antivirals. table viii lists many of the modalities that might be used. the high frequency of nosocomial acquisitions indicates that hospital infection control practices oriented toward preventing exposure would be rewarding. (early results of such practices at the mdacc are reported in the article by raad and colleagues in this supplement.) the only vaccine available for any of the respiratory viruses is inactivated influenza vaccine. although immune responsiveness is impaired in immunocompromised patients, any response should be beneficial. immunization of patients could be supplemented by immunization of contacts (as currently recommended by the committee on immunization practices of the centers for disease control) and immunization of donors for bone marrow transplant recipients, as transfer of b cell memory has been demonstrated. 32, 33 although interferon-a induces common cold-like symptoms when given intranasally for long periods, use for a shorter term or despite occurrence of side effects could be considered for prevention of rhinovirus, coronavirus, and rsv infections, for which it is effective. [34] [35] [36] enhancing immunity by passive immunization is currently done for some immunocompromised patients, but could be targeted toward a specific pathogen, such as was done for prevention of rsv infections in infants with underlying lung disease. 37 adoptive immunization with donor lymphocytes for bone marrow transplant patients has been used and reported to be effective for control of other viral infections. 38 antivirals are currently available for influenza a and rsv. amantadine and rimantadine are approved for prevention and treatment and could be superior to vaccine for prevention of influenza a in this population, although data to support this possibility are lacking. treatment should be short term, as resistance develops rapidly. 39 ribavirin by aerosol is approved for treatment of severe rsv disease in infants but may be effective in adults as well. 6,8,15,40 -43 controlled and uncontrolled studies have indicated value for ribavirin in treatment of influenza a and b and parainfluenza virus infections. 16,44 -47 comment it is becoming increasingly clear that the respiratory viruses commonly infect immunocompromised persons and that these infected persons may exhibit severe respiratory illnesses with high frequencies of pneumonia and death. although not well documented, there is surely a gradient of infection and illness severity that relates to the degree and type of impairment of immune function. it is not clear at present that impairment of immune function can lead to increased susceptibility to infection, although early data suggest this is true and described immune impairments would make this likely. on the other hand, there is little doubt that severe pure viral pneumonias and the characteristic prolonged infections that occur in immunocompromised patients are attributable to deficiencies in mechanisms of recovery. the severity of rsv infections in patients with extreme degrees of immunodeficiency is notable; an understanding of the immune basis for this severity could aid in designing immune approaches for prevention and treatment of these infections. similarly, such an understanding of the other respiratory viral infections could be of value in the care not only of immunocompromised persons but also of immunocompetent persons. most patients with pneumonia are infected or colonized with bacteria and fungi and at death yield such organisms from lung tissue; many, however, exhibit a histologic pattern of virus pneumonia only and yield evidence of a specific virus in tissues. it is not clear at present whether the most significant role of respiratory viral infection in the pneumonias is as a primary pathogen, a contributing pathogen, or a pathogen predisposing to secondary infection with bacteria or fungi that then cause pneumonia. the last of these -their role as predisposing pathogens -is considered to be the major role for these infections among immunocompetent patients who develop pneumonia. options are available for control of some of the respiratory viral infections, and efforts to recognize and apply these options are needed while other options are being identified and developed. perhaps the greatest need at present, however, is for practicing physicians to appreciate the role that respiratory viral infection can play as a cause of serious disease in immunocompromised patients. current estimates from the national health interview survey, united states efficacy of sequential annual vaccination with inactivated influenza virus vaccine respiratory disease due to parainfluenza virus in adult bone marrow transplant recipients mycoplasmal infections of the respiratory tract nosocomial transmission of respiratory syncytial virus in immunocompromised adults an outbreak of respiratory syncytial virus in a bone marrow transplant center respiratory syncytial virus infection in children with compromised immune function combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients influenza b in transplant patients influenza b virus infection in pediatric solid organ transplant recipients influenza a virus infections among hospitalized adult bone marrow transplant recipients. bone marrow transplantation medical progress: cellular response to respiratory viruses with particular reference to children with disorders of cellmediated immunity respiratory syncytial virus illnesses in human immunodeficiency, virus-infected and noninfected children. pediatr virus infections in children with acute lymphoblastic leukaemia treatment of respiratory viral infection in an immunodeficient infant with ribavirin aerosol ribavirin treatment of viral pneumonitis in severe combined immunodeficiency disease parainfluenza 3 virus and other common respiratory pathogens in children with human immunodeficiency virus infection respiratory syncytial virus (rsv) disease among hospitalized adult immunocompromised patients with leukemia community respiratory virus infections among hospitalized adult bone marrow transplant recipients epidemiology of influenza a virus infection in patients with acute or chronic leukemia influenza among hospitalized adult immunocompromised patients with leukemia community respiratory virus (crv) infections in hospitalized adult patients with leukemia host defenses at mucosal surfaces immunoglobulin a in secretions from the lower respiratory tract the specificity and function of t lymphocytes induced by influenza a viruses roles of ab and gd t cell subsets in viral immunity the kinetics of immune reconstitution after human marrow transplantation influenza in children with cancer viral pneumonia in recipients of solid organ transplants influenza a in immunocompromised patients orthomyxoviral and paramyxoviral infections in transplant patients bone marrow transplantation for malignant disease intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers prevention of natural colds by contact prophylaxis with intranasal alpha2-interferon the efficacy of intranasal interferon a-2a in respiratory syncytial virus infection in volunteers. antiviral research prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children donor leukocyte infusion as therapy of life-threatening adenoviral infections after t-cell-depleted bone marrow transplantation emergence and apparent transmission of rimantadine-resistant influenza a virus in families respiratory syncytial virus and parainfluenza virus infections in the immunocompromised host respiratory syncytial virus pneumonia in a cardiac transplant recipient case report: severe respiratory syncytial virus pneumonia in an adult renal transplant recipient: successful treatment with ribavirin successful therapy with ribavirin of late onset respiratory syncytial virus pneumonitis complicating allogeneic bone marrow transplantation ribavirin aerosol treatment of influenza parainfluenza virus respiratory infection after heart transplantation: successful treatment with ribavirin association of parainfluenza virus type 3 infection with allograft rejection in a liver transplant recipient nebulized ribavirin for influenza b viral pneumonia in a ventilated immunocompromised adult key: cord-269734-u43gt8fh authors: teijaro, j.r. title: pleiotropic roles of type 1 interferons in antiviral immune responses date: 2016-09-20 journal: adv immunol doi: 10.1016/bs.ai.2016.08.001 sha: doc_id: 269734 cord_uid: u43gt8fh since isaac's and lindenmann's seminal experiments over 50 years ago demonstrating a soluble factor generated from heat killed virus-stimulated chicken embryos could inhibit live influenza virus replication, the term interferon has been synonymous with inhibition of virus replication. while the antiviral properties of type 1 interferon (ifn-i) are undeniable, recent studies have reported expanding and somewhat unexpected roles of ifn-i signaling during both acute and persistent viral infections. ifn-i signaling can promote morbidity and mortality through induction of aberrant inflammatory responses and recruitment of inflammatory innate immune cell populations during acute respiratory viral infections. during persistent viral infection, ifn-i signaling promotes containment of early viral replication/dissemination, however, also initiates and maintains immune suppression, lymphoid tissue disorganization, and cd4 t cell dysfunction through modulation of multiple immune cell populations. finally, new data are emerging illuminating how specific ifn-i species regulate immune pathology and suppression during acute and persistent viral infections, respectively. systematic characterization of the cellular populations that produce ifn-i, how the timing of ifn-i induction and intricacies of subtype specific ifn-i signaling promote pathology or immune suppression during acute and persistent viral infections should inform the development of treatments and modalities to control viral associated pathologies. many viruses harbor viral proteins with specific functions geared toward preventing ifn-i production and/or signaling, highlighting the evolutionary selective pressure exerted by ifn-i during viral replication (devasthanam, 2014) . the absence of ifn-i signaling during acute virus infection in vivo increases virus replication, dissemination, and lethality during multiple viral infections in animal models. global deletion of ifnar1 results in enhanced mortality during vesicular stomatitis virus (vsv), vaccinia virus (vv), west nile virus (wnv), and lymphocytic choriomeningitis virus (lcmv) infections (muller et al., 1994) . moreover, infection of ifnar1 ko mice with acute lcmv armstrong (arm) (nakayama et al., 2010; zhou, cerny, fitzgerald, kurt-jones, & finberg, 2012) and treatment of arm-infected mice with an ifnar1 neutralizing antibody elevated viral loads and promoted virus persistence (teijaro et al., 2013; wilson et al., 2013) . dendritic cell-specific deletion of ifnar1 results in elevated virus replication and systemic persistence of the cw3 strain of murine norovirus (mnov) despite increased cell-mediated and humoral adaptive immune responses (nice et al., 2016) . ifn-i signaling has been shown to be essential for controlling wnv infection and restricting viral pathogenesis (sheehan, lazear, diamond, & schreiber, 2015) . mice deficient in ifnar1 signaling display increased susceptibility to wnv infection (pinto et al., 2014; samuel & diamond, 2005) . during infection with the coronavirus, mouse hepatitis virus (mhv-a59), the magnitude of the ifn-i and -ii responses directly correlated with viral loads (raaben, koerkamp, rottier, & de haan, 2009 ). moreover, ifn-i produced by plasmacytoid dendritic cells (pdcs) was essential to control virus replication and prevent mortality following mhv-a59 infection in mice (cervantes-barragan et al., 2007) . during experimental infection of mice and nonhuman primates with the lassa hemorrhagic fever virus, delayed or reduced induction of ifn-i and downstream gene signatures correlated with high viral loads and fatal outcome (baize et al., 2009; yun et al., 2012) . deletion of ifn-i related signaling pathways during respiratory virus infections in animal models results in diverse effects depending on the virus strain and genetic background (durbin et al., 2000; price, gaszewska-mastarlarz, & moskophidis, 2000) . in the context of respiratory viral infection, genetic deletion of stat1 reduced virus control, enhanced pathology, and mortality during sars-cov and influenza virus infection (durbin et al., 2000; frieman et al., 2010) . interestingly, stat1-deficient animals were highly susceptible to influenza virus infection, displaying elevated viral titers and increased pathology compared to stat1-sufficient mice. studies in mouse models of influenza virus have revealed conflicting evidence for the role of ifnar1 in controlling influenza virus replication, morbidity, and mortality. infection of ifnar1 à/à mice with the pr8 strain of influenza virus resulted in altered recruitment of ly6c hi vs ly6c int monocytes in the lung, translating into increased production of the neutrophil chemoattractant, kc (cxcl8), elevated numbers of neutrophils in the lung and increased morbidity and mortality (seo et al., 2011) . therefore, modulation of type 1 interferon signaling and production needs to be balanced to have enough to control virus infection but not promote excessive inflammation. the discrepancy between influenza pathogenicity in ifnar1 and stat1-deficient mice was later clarified when animals lacking both ifnar1/ifn-λ were unable to control influenza virus replication. this is further supported in humans where null mutations in the human interferon regulatory factor-7 gene results in reduced ifn-i and -iii production from myeloid dcs and pdcs and life-threatening seasonal influenza virus infection (ciancanelli et al., 2015) . exposure of bone marrow cells to ifn-i prior to their recruitment to lung endows these cells with an antiviral program that protects from virus infection after entry into the infected lung (hermesh, moltedo, moran, & lopez, 2010) . deletion of the ifn-β or ifnar1 genes in mice with a functional mx1 gene increased virus replication and reduced the ld 50 20-fold (koerner, kochs, kalinke, weiss, & staeheli, 2007) . infection of ifnar1-deficient mice with low dose mouse adapted h1n1 influenza viruses resulted in mortality, elevated viral loads, exacerbated lung pathology, and reduced numbers of il-10-producing cells as compared to ifnar1-sufficient controls (arimori et al., 2013) . moreover, exogenous administration of il-10 to ifnar1-deficient animals following influenza virus infection partially restored survival and ameliorated lung pathology. thus, ifn-i can be protective during influenza virus infection either through suppressing virus spread or prompting induction of immune-suppressive cytokines to reign in excessive inflammation. in addition to directly inhibiting virus propagation, ifn-i also has potent immune stimulatory functions which support the resolution of virus infection. ifn-i promotes upregulation of mhc-i expression in multiple cell lineages (lindahl, gresser, leary, & tovey, 1976a , 1976b , which is required for optimal t cell stimulation, differentiation, expansion, and killing of virus-infected cells. autocrine signaling of ifn-i on dendritic cells promotes their activation and t cell stimulatory capacity (montoya et al., 2002) . ifn-i signaling during virus infection promotes conversion of pdcs into myeloid derived dcs and impairs hematopoietic differentiation of bone marrow progenitors into dcs (sevilla, mcgavern, teng, kunz, & oldstone, 2004; zuniga, mcgavern, pruneda-paz, teng, & oldstone, 2004) . following exposure to ifn-i, metallophilic macrophages induce expression of the usp18 protein which prevents jak1 phosphorylation and inhibits ifn-i signaling in these cells. in turn, repression of ifn-i signaling allows for restricted virus replication in these macrophages, promoting the production of viral antigens which are recognized by b cells, the final result is the facilitation of antiviral antibody generation and enhanced virus control (honke et al., 2012) . ifn-i also exerts potent costimulatory effects directly on cd8 t cells, enhancing cd8 t cell proliferation upon ifnar1 signaling (curtsinger, valenzuela, agarwal, lins, & mescher, 2005; kolumam, thomas, thompson, sprent, & murali-krishna, 2005) . the timing of cd8 t cell exposure to ifn-i significantly influences the differentiation and magnitude of the response (welsh, bahl, marshall, & urban, 2012) . exposure of naïve cd8 t cells to apc and ifn-i prior to antigenic stimulation promotes the maintenance of a naïve phenotype with reduced proliferation despite production of effector cytokines. direct ifn-i signaling on naïve and memory t cells promotes rapid apoptosis, inhibits proliferation, and promotes early effector differentiation of memory cells upon exposure. blockade of ifn-i signaling during wnv infection has significant effects on t cell expansion, cytokine production, and differentiation when administered during the maturation phase of the t cell response, however, had no effect when given prior to infection (pinto et al., 2011) . moreover, low dose priming with the vv ankara strain had little effect on effector or memory t cell recall in ifnar1 à/à mice (volz, langenmayer, jany, kalinke, & sutter, 2014) . in addition to t cells, ifn-i signaling is known to be important for nk cell function. ifn-i signaling promotes nk cell cytolytic capacity and survival during acute viral infection (hwang et al., 2012; martinez, huang, & yang, 2008; nguyen et al., 2002) and was recently reported to protect antiviral cd8 t cells from nk cell lytic effects (crouse et al., 2014; xu et al., 2014) . reconstitution of ifnar1 à/à mice with ifnar1 +/+ nk cells restored early control of vv infection in vivo (martinez et al., 2008) , suggesting that nk cell intrinsic ifnar1 signaling is important for early control of vv replication. moreover, direct ifn-i signaling on nk cells was required to induce nk cell ifn-γ production during acute lcmv infection. early ifn-γr signaling was required for promoting initial virus control in the peritoneum (mack, kallal, demers, & biron, 2011) , suggesting that ifn-i signaling directly on nk cells promotes virus control during acute lcmv infection. ifn-i signaling during viral infection can also signal to regulatory t cells and subsequently alter their suppressive functions. it was recently demonstrated that ifnar1 signaling on foxp3 + tregs limits their suppressive function during acute lcmv infection, thus promoting virus control (srivastava, koch, pepper, & campbell, 2014) . deletion of ifnar1 on foxp3 + cells blunted virus-specific t cell responses and elevated virus loads. thus, ifn-i signaling on suppressive t cell populations temporarily suspends suppressive function and allows for optimal antiviral t cell responses during an ongoing viral infection. similar to effects on t cells, ifn-i signaling has both positive (le bon et al., 2001) and negative effects on antiviral b cell responses. the survival and maturation of immature b cells can be inhibited by ifn-i signaling (lin, dong, & cooper, 1998) . in contrast to immature b cells, ifn-i signaling promotes b cell activation, antibody production, and isotype switch following influenza, vsv, and wnv infection (coro, chang, & baumgarth, 2006; fink et al., 2006; purtha, chachu, virgin, & diamond, 2008; rau, dieter, luo, priest, & baumgarth, 2009 ). however, it was also reported that influenza virus-specific antibody levels were elevated at later time points following influenza virus challenge in ifnar1-deficient mice compared to ifnar1-sufficient controls (price et al., 2000) . during acute lcmv infection, blockade of ifn-i signaling in both wild-type and stat3-deficient mice enhanced t follicular helper cell (t fh ), germinal center b cell differentiation, and anti-lcmv antibody responses (ray et al., 2014) . elevated antibody responses during acute viral infections following ifnar1 blockade suggest that, in certain circumstances, ifn-i signaling can restrain optimal antiviral antibody responses. the correlation of an aggressive immune response and severe disease following influenza virus infection in humans and animal models has been discussed previously (la gruta, kedzierska, stambas, & doherty, 2007 ). an aggressive innate response, with elevated recruitment of inflammatory leukocytes to lung, likely contributed to the morbidity of the 1918 influenza infection (ahmed, oldstone, & palese, 2007; kobasa et al., 2007) . in fact, lung injury during infection of macaques with the 1918 h1n1 influenza virus strain directly correlated with early dysregulated inflammatory gene expression, including elevated ifn-i signatures (cilloniz et al., 2009; kobasa et al., 2007) . more recently, clinical studies on avian h5n1-infected humans documented a significant association between excessive early cytokine responses and immune cell recruitment as predictive of poor outcome (de jong et al., 2006 ). an aberrant cytokine/chemokine response was observed in patients with severe disease during the most recent h1n1 pandemic in 2009 (arankalle et al., 2010) . type i interferon signaling is well known to inhibit influenza virus replication and spread (garcia-sastre & biron, 2006) . the production of the ns1 protein, one of 11 viral proteins, acts to inhibit type 1 interferon production and signaling (hale, randall, ortin, & jackson, 2008) , suggesting that ifn-i signaling exerts substantial selection pressure on virus fitness. deletion or mutation of the ns1 gene results in significant increases in the levels of type 1 interferon in infected cells and significantly lower virus titers both in vitro and in vivo (garcia-sastre, egorov, et al., 1998; jiao et al., 2008; kochs, garcia-sastre, & martinez-sobrido, 2007) . despite strong evidence demonstrating extensive antiviral properties of ifn-i, several studies also suggest pathogenic roles for ifn-α during influenza virus infection. the production of several proinflammatory cytokines and chemokines is known to be amplified by ifn-i receptor signaling. in addition to protective effects of ifn-i signaling, pathogenic roles for ifn-i have been reported during influenza virus infection ( fig. 1a) . appearance of ifn-α in lavage fluid directly coincides with symptom onset during human experimental influenza virus infection (hayden et al., 1998) , suggesting that ifn-i signaling and pathological responses in humans temporally coincide. recently, it was paradoxically reported that deletion of ifnar1 or depletion of pdcs in svev129 mice inhibited pulmonary pathology and improved survival following lethal influenza virus challenge (davidson, crotta, mccabe, & wack, 2014) . reduced immune pathology and enhanced survival in mice deficient in ifn-i signaling transpired without significant increases in viral loads or impediment of eventual viral clearance (fig. 1b) . in contrast to deletion of ifn-i signaling, treatment of influenza virus-infected mice with ifnα resulted in enhanced morbidity and mortality; thus, ifn-i can promote pathological consequences during acute influenza virus infection. over the past 5 years, we identified that therapeutic administration of sphingosine 1 phosphate (s1p) analogs early during influenza virus infection in mice resulted in reduced morbidity and mortality . s1p is a lipid metabolite converted from ceramide precursors to sphingosine. fig. 1 ifn-i signaling enhances cytokine/chemokine amplification, innate immune cell recruitment, and immune pathology during respiratory viral infections. (a) viral infection in the lung with influenza or sars-cov promotes the induction of delayed ifn-i production which enhances cytokine/chemokine production, recruitment of nk cells, and neutrophils and inflammatory macrophage/monocytes all which contribute to lung immune-mediated pathology. (b) blockade or genetic deletion of ifnar1 blunts cytokine/chemokine amplification, inhibits recruitment of nk cells, neutrophils, and inflammatory macrophages/monocytes resulting in reduced immunopathology, and improved survival. treatment of mice with s1p1r agonists early during influenza virus infection suppresses ifn-i amplification from plasmacytoid dendritic cells which lowers ifn-i levels. the end result is blunting of cytokine/chemokine amplification, inhibition of nk cell, neutrophil, and inflammatory macrophage/monocyte recruitment into the lung, reduced immunopathology, and improved survival. the subsequent phosphorylation by sphingosine kinase 1 and 2 produces bioactive s1p in vivo where it acts on s1p-specific g-protein couples receptors (gpcrs) (chalfant & spiegel, 2005) . the levels of bioactive s1p are regulated through the actions of s1p phosphatases and lyases which dephosphorylate and degrade s1p, respectively. highest levels of s1p are found in the blood and lymph with significantly lower levels maintained in peripheral tissues (cyster, 2005) . s1p binds and signals through five gpcrs denoted as s1pr1-5 which couple to various g-protein signaling effectors. the expression of s1p receptors is heterogeneous, being found on both hematopoietic and nonhematopoietic lineages (im, 2010) . the functional coupling to multiple heterotrimeric g-proteins promote the diverse cellular functions associated with s1p receptor signaling. signaling through these five receptors is known to modulate multiple cellular processes including: cell adhesion, migration, survival, proliferation, endocytosis, barrier function, and cytokine production (rivera, proia, & olivera, 2008) . recently, we identified a novel regulatory function of s1pr1 signaling in blunting early cytokine amplification and innate immune cell recruitment following influenza virus infection (fig. 1b) . early administration of a promiscuous s1pr agonist, aal-r, or an s1p1r-selective agonist (cym-5442) significantly blunted production of multiple pro-inflammatory cytokines and chemokines following infection with either wsn or human pandemic h1n1 2009 influenza virus walsh et al., 2011) . further, both aal-r-and cym-5442-mediated reduction of early innate immune cell recruitment and cytokine/chemokine production correlated directly with reduced lung pathology and improved survival during h1n1 2009 influenza virus infection. while these s1pr agonists clearly inhibited innate immune responses, significant inhibition of activated t cell recruitment into the lung at various times post infection occurred in mouse adapted (marsolais et al., 2009 ) and human pathogenic strains of influenza virus . the above findings were extended using genetic and chemical tools to probe functions of the s1p1 receptor (s1p1 gfp knockin transgenic mice, s1p1 receptor agonists and antagonists), revealing that pulmonary endothelial cells modulate innate immune cell recruitment and cytokine/chemokine responses early following influenza virus infection . importantly, s1p1r agonist treatment blunted cytokine/chemokine production and innate immune cell recruitment in the lung independently of endosomal and cytosolic innate sensing pathways (teijaro, walsh, rice, rosen, & oldstone, 2014) . further, s1p1r signaling suppression of cytokine amplification was independent of multiple innate signaling adaptor pathways but required the myd88 adaptor for cytokine amplification following influenza virus challenge. immune cell infiltration and cytokine production were found to be distinct events, both orchestrated by signaling through the s1p1r. suppression of early innate immune responses through s1p1r signaling also reduced mortality during infection with human pathogenic strains (h1n1/2009 swine) of influenza virus in a ferret model, demonstrating that s1pr1-mediated blunting of influenza virus pathogenesis in mice could be extended to a model more closely resembling human disease. the link between s1pr1 and ifn-α amplification following influenza virus infection was striking. in fact, the absence of ifnar1 abolished cytokine amplification and the capacity of s1p1r agonists to further blunt cytokine/chemokine responses (teijaro et al., 2016 . to understand how s1pr1 signaling regulates ifn-α and cytokine amplification, we assessed the pulmonary cell subsets that produce ifn-α and cytokines/chemokines following influenza virus challenge. expression of s1p1r was quickly observed in purified pdcs; moreover, s1p1r agonists suppressed ifn-i induction/amplification from both mouse and human pdcs following influenza virus simulation (teijaro et al., 2016) . further mechanistic studies revealed that s1p1r agonist-mediated suppression was independent of gi/o signaling and required signaling through the s1p1r c-terminus. biochemically, s1p1r agonists accelerated the turnover of ifnar1 and promoted trafficking to lysosomes for degradation, abrogating stat1 phosphorylation, blunting the ifn-i autoamplification loop. the fact that ifn-i production/signaling can down modulate s1pr1 expression/activity indirectly through upregulation of cd69 which promotes internalization of s1pr1 in t cells is significant (shiow, 2006) and suggests that s1p1r and ifn-i signaling are closely linked and capable of counter regulating one another. an additional study also reported ifn-i modulation in pdcs via other s1prs (dillmann et al., 2016) , suggesting that this phenomenon could be more promiscuous than originally thought. similar to influenza virus infection, aberrant innate cytokine/chemokine responses and immune cell recruitment into lungs correlate with disease severity in human patients (huang et al., 2005) . ifn-i signaling during murine sars-cov infection appears to be dispensable for virus control while also potentiating immune pathology. however, the role ifn-i signaling plays in this pathology has only recently been systematically addressed. deletion of ifnar1 in mice does not mirror the enhanced viral loads or pathological consequences observed in stat1 à/à mice in sars-cov infection, suggesting an ifnar1-independent stat-1-dependent pathway is necessary for controlling sars-cov (frieman et al., 2010) . this study provocatively suggests that ifn-i signaling is dispensable for controlling sars-cov replication in vivo. recently, an important study was published where the authors further highlighted the importance of ifn-i signaling in respiratory virus pathology by reporting that delayed ifn-i induction and signaling during sars-cov infection in mice promoted the development and infiltration of inflammatory monocyte-macrophages into the lung, resulting in exacerbated lung pathology and lethal pneumonia (channappanavar et al., 2016) . attenuation of ifn-i signaling either through genetic deletion or through antibody neutralization of ifnar1 prevented inflammatory monocyte-macrophage infiltration into the lung, abrogated lung immune pathology, and resulted in mild clinical disease. importantly, genetic deletion or blockade of ifn-i signaling resulted in control of viral loads similar to control animals, reinforcing that ifn-i signaling is dispensable for control of sars-cov infection in vivo. one possibility is that in the absence of ifn-i signaling, induction of an ifn-iii (ifn-λ) antiviral program may effectively limit viral replication. the results found in this study were strikingly similar to those found in influenza virus-infected svev129 mice and suggest that strategic modulation of ifn-i signaling could ameliorate pathologies associated with severe respiratory virus infection. collectively, the studies above suggest that ifn-i signaling is essential to cytokine and chemokine amplification and innate immune cell recruitment and can promote excessive immunopathology during acute respiratory viral infections (fig. 1) . importantly, that ifn-i production and signaling can be blunted without enhancing virus propagation following acute respiratory viral infection suggests that this pathway can be modulated without compromising host antiviral responses. the correlation between blunting ifn-i signaling, lessened immune pathology, and improved survival during multiple respiratory viral infections highlight the need to mechanistically dissect how ifn-i promotes immune pathology during these infections. the role of ifn-i signaling in restraining chronic/persistent viral infection is well documented. inhibition of ifn-i signaling by antibody blockade of ifnar1 results in elevated virus replication early following lcmv cl13 infection and treatment of mice with ifn-i during the early stages of persistent lcmv infection promotes rapid virus control (wang et al., 2012) . mechanistically, ifn-i therapy increased expansion of virus-specific cd8 t cells and prevented t cell exhaustion; however, whether this was due to ifn-i-mediated immune stimulatory effects, lowering of antigen levels, or both was not systematically addressed. an additional study reported that deletion of the 2 0 -5 0 oligoadenylate synthetase-like 1 gene prior to lcmv cl13 infection facilitated sustained ifn-i production/signaling, promoted t cell expansion, reduced t cell exhaustion, and promoted rapid virus control (lee, park, jeong, kim, & ha, 2013) . similar to persistent lcmv infection, ifn-i administration can exert protective effects through slowing siv replication and disease progression if administered early following infection (sandler et al., 2014) and has shown some efficacy in patients with persistent hiv infection (asmuth et al., 2010; azzoni et al., 2013) . moreover, treatment with pegylated ifn-α in conjunction with the antiviral drug ribavirin was the standard of care for treating patients with chronic hepatitis c virus (hcv) infection until recently (heim, 2013; moreno-otero, 2005) . however, despite success in hcv therapy, the modest efficacy observed following ifn-α administration requires ribavirin and, even in combination, only a slim majority of patients respond. moreover, patients who fail to control hcv following ifn-i therapy were reported to express a higher ifn-i gene signature prior to treatment (sarasin-filipowicz et al., 2008) . similar trends were observed following ifn-i administration during hiv and siv infections, where ifn-i administration had only the modest effects if given during established persistent infection (asmuth et al., 2008; hubbard et al., 2012) . the reasons for the discrepancies observed in human persistent viral infections, where ifn-i therapy can promote control (50-60% of hcv patients) while in others (during established hiv infection) minimal benefit is observed, remain unknown. one could imagine a scenario where in some persistently infected hcv patients, elevated ifn-i signatures persist, and addition of pegylated ifn-α provides minimal benefit while patients with lower ifn-i signatures respond to the therapy. whether treatment with pegylated ifn-α earlier during infection (prior to sustained ifn-i signatures) would be beneficial would be interesting to discern. a similar profile appears to exist in persistent siv infection, where early administration of ifn-i promotes control of viral loads and pathogenesis, while later administration has modest effects on viral titers and disease outcome. during infection with a model gamma herpesvirus, mhv68, the lack of ifn-i signaling exacerbated virus replication, increased reactivation from latency, and resulted in enhanced morbidity and mortality (barton, lutzke, rochford, & virgin, 2005; dutia, allen, dyson, & nash, 1999) . taken together, ifn-i therapy may be beneficial during the early stages of persistent, latent chronic viral infection, or infections with lower ifn-i signatures; however, blocking ifn-i signaling either alone or in conjunction with antiviral or immune checkpoint therapies may prove more effective once virus persistence and elevated ifn-i signatures are established. however, the ultimate outcome will likely depend on the persistent virus studied, genetic susceptibilities of individuals, and subtype and timing of ifn-i species produced; all which require further investigation. moreover, given the undesirable side effects of ifn-i administration, ifn therapy can do as much harm as good during viral infection, highlighting the need for developing alternative approaches to treat persistent viral infections. during persistent viral infections, chronic immune activation, negative immune regulator expression, an elevated interferon signature, and lymphoid tissue destruction correlate with disease progression. elevated ifn-i signatures have been observed during lcmv infection in mice (hahm, trifilo, zuniga, & oldstone, 2005) and hiv and hcv infections in humans and nonhuman primates (bosinger et al., 2009; jacquelin et al., 2009; wieland et al., 2014) . chronic immune activation following hiv infection has been reported, and suppression of this hyperactivated state has been proposed as a potential strategy to alleviate hiv-associated pathologies (boasso, hardy, anderson, dolan, & shearer, 2008; d'ettorre, paiardini, ceccarelli, silvestri, & vullo, 2011) . disease following experimental siv infection in rhesus macaques correlates with elevated ifn-i production and inflammatory signatures (jacquelin et al., 2009; manches & bhardwaj, 2009 ). in contrast, siv infection in sooty mangabeys and african green monkeys, which develop modest pathology despite equivalent viral loads as macaques, correlate with reduced ifn-i and inflammatory gene signatures (bosinger et al., 2009) . similar correlations with respect to reduced immune activation exist in hiv-infected elite controllers, although whether reduced immune activation follows virus control is uncertain (deeks & walker, 2007; saez-cirion et al., 2007) . blockade of pd-1 signaling during chronic siv infection reduces hyperimmune activation and microbial translocation in rhesus macaques and lowers ifn-i signatures in the blood and colon (dyavar shetty et al., 2012) . moreover, an elevated interferon signature is observed in hcv-infected patients despite limited control of virus replication and development of liver pathology (guidotti & chisari, 2006; su et al., 2002; wieland et al., 2014) . in fact, hcv infection in culture blocks isg protein expression through activation of rna-dependent protein kinase (garaigorta & chisari, 2009) , creating a paradoxical ifn-i-dependent viral advantage. thus, ifn-i signaling pathways have the potential to aid viral fitness and promote pathology during persistent viral infection. these studies further highlight the viability of the ifn-i signaling system as a target to promote control of persistent viral infection. while the literature suggests a causative role for ifn-i in contributing to pathogenesis of persistent virus infections, definitive studies assessing how ifn-i neutralization affects the outcome of virus persistence were lacking until recently. two laboratories assessed the role ifn-i signaling plays during persistent infection using the lcmv clone-13 (cl13) strain of virus. during their investigation, they found that blockade of ifn-i signaling using an ifnar1 neutralizing antibody reduced immune system activation, decreased expression of negative immune regulatory molecules il-10 and pd-l1 and restored lymphoid architecture in mice persistently infected with lcmv (fig. 2) . importantly, blockade of ifnar1 both prior to and following established persistent lcmv infection promoted faster virus clearance and required an intact cd4 t cell compartment (teijaro et al., 2013; wilson et al., 2013) . blockade of ifn-i signaling significantly enhanced cd4 t cell differentiation into th1 effectors as well as increased t fh cell differentiation (osokine et al., 2014) . the above studies demonstrate for the first time a direct causal link between ifn-i signaling, immune activation, negative immune regulator expression, lymphoid tissue disorganization, and long-term virus persistence. more recently, it was reported that during cl13 infection, both type i and ii interferon promoted the induction and suppressive capacity of cd95 + cd39 + immune regulatory dcs (iregdcs), respectively (cunningham et al., 2016) . while ifn-γ promoted the differentiation of iregdcs from monocytes, ifn-i promoted the suppressive functions of iregdcs. genetic deletion of ifnar1 prevented the expression of pd-l1 and production of il-10 from iregdcs, relieving their suppressive capabilities. in addition to modulating the suppressive capacity of iregdcs, ifn-i signaling also limited their generation/expansion. during mnov infection, selective genetic deletion of ifnar1 in dcs increased expression of the cellular activation markers cd80, cd86, and mhcii, suggesting that direct ifn-i signaling on dcs may be responsible for restraining dc function in vivo (nice et al., 2016) . generation of elevated numbers of iregdcs was also observed during hiv and mycobacterium tuberculosis infections as well as cancer, suggesting that iregdc generation is common in immunosuppressive environments. the ifn-i-driven immune-suppressive state during persistent lcmv infection also inhibits macrophage function. a recent study found that mice infected with the persistent docile strain of lcmv have impaired humoral immune responses to a superinfecting vsv infection (honke et al., 2016) . the absence of virus replication in cd169 + macrophages was not due to antiviral cd8 t cell-mediated killing of cd169 + macrophages but instead the result of sustained ifn-i responses and an elevated ifn-i antiviral gene program. in turn, reduction in vsv replication and antigen production in cd169 + macrophages reduced antigen production in these cells which was essential for antiviral antibody generation. the existence of multiple ifn-i subspecies (14 ifn-α species in mice and 13 in humans in addition to ifn-β) suggests that either the ifn-i system requires redundancy to be effective or that individual ifn-i species evolved to execute specific functions. certainly, different ifn-α species and β display varying degrees of affinity for the ifnar1/2 receptor complex (ng, mendoza, garcia, & oldstone, 2016; thomas et al., 2011) , with ifn-β displaying the highest binding affinity. lcmv persistence was influenced more by ifn-β than ifn-α signaling as treatment of mice infected with lcmv cl13 with an ifn-β neutralizing antibody displayed accelerated virus clearance compared to a polyclonal ifn-α antibody which had minimal effects on virus control (ng et al., 2015) . ifn-β neutralization did not exacerbate early virus replication, improved lymphoid architecture, and enhanced virus-specific cd4 and cd8 t cell responses. however, while ifn-β neutralization clearly promoted faster virus clearance as compared to neutralization with a polyclonal ifn-α antibody, the contribution of ifn-α species not neutralized by the polyclonal antibody used was not investigated. nevertheless, neutralizing ifn-β may promote adaptive immune control of virus without significantly affecting virus replication and thus may represent a safer approach to promoting control of persistent virus infection in vivo. the dichotomy between ifn-α and β was further highlighted upon infection of new zealand black (nzb) mice with lcmv cl13. infection of nzb mice with cl13 resulted in early lethality that was found to be due to cd8 t cell-dependent thrombocytopenia and pulmonary endothelial cells loss (baccala et al., 2014) . interestingly, despite upregulation of pd-1/pd-l1 expression and il-10 production, t cell function remained intact. moreover, this enhanced pathology correlated with elevated ifn-i protein levels and gene signatures; however, unlike infection in c57bl/6j mice, the pathology required ifn-α signaling and was ifn-β independent. it was recently reported that ifn-β signaling required binding to ifnar1 but was independent of ifnar2. deletion of ifnar1 ameliorated lps-induced sepsis induction, while ifnar2 à/à mice were unaffected (de weerd et al., 2013) ; thus, it would be interesting to test how ifnar2 à/à nzb mice respond to cl13 infection. the above studies demonstrate that ifn-α and -β species can differentially modulate immune responses in various viral infections, highlighting the importance of future investigation into how different ifn-i subtypes modulate viral control and disease pathogenesis. several important questions still remain that provide exciting avenues for investigating the roles of ifn-i signaling during viral infection in the future. although ifn-i signaling can trigger various downstream effector pathways, how signaling via select ifn-i species dictate specific outcomes following viral infections remain incompletely understood. specifically, there is a great need to understand the roles individual ifn-i-α and -β subsets play in restraining viral replication or promoting immune inflammatory/suppressive programs in vivo. further, how ifn-i signaling in specific cellular subsets in vivo regulates immune pathological and immunesuppressive responses will be interesting to dissect. the ifnar1-floxed mouse strain which was generated recently will be instrumental in future studies to investigate this question. illuminating what cell types require ifn-i signaling in vivo should pave the way for generating a detailed understanding of the cellular and molecular mechanisms by which ifn-i signaling acts to promote immune pathology and suppression in acute and persistent viral infections. the capacity of ifn-i signaling to promote immune pathology during acute respiratory viral infection appears in animal models of both influenza and sars-cov infection. the necessity of ifn-i signaling to restrain viral spread during acute viral infection suggest that targeting the ifn-i signaling pathway may be ill advised. however, one wonders whether targeting specific ifn-i species to suppress detrimental inflammation can be achieved without compromising virus clearance during acute respiratory viral infections. moreover, the production of ifn-λ during respiratory viral infection may be sufficient to control viral loads while ifn-i signaling is inhibited. recent results in mouse models suggest this may be possible; however, further studies are needed. moreover, whether the effects observed in mice will translate to human respiratory viral infections is unknown and should be investigated with caution. in the context of the immune-suppressive programs elicited by ifn-i signaling during persistent virus infection, the recent demonstration that blockade of ifn-β enhanced virus control by inducing improved lymphoid architecture and enhanced virus-specific cd4 and cd8 t cell responses, suggest that targeting selective ifn-i species can redirect immune responses sufficiently to promote immune-mediated virus control. importantly, relief of the immune-suppressive environment in this case was not accompanied by elevated viral loads following treatment with ifn-β-neutralizing antibody, suggesting that more selective modulation of specific ifn-i species can allow for preservation of some antiviral functions. the mechanisms by which the different ifn-i species interact with the ifnar1 and ifnar2 receptors to induce differential downstream signaling suggests this pathway could be manipulated pharmacologically. it is interesting to postulate whether small molecules or biologics could be developed to block binding/signaling of specific ifn-i species (i.e., ifn-β or specific α-species). for example, could ifn-β signaling be selectively inhibited without altering ifn-α species engagement with the ifnar1/2 receptor complex during ongoing viral infection using a small molecule or antibody therapeutic? could a small molecule be designed to reverse aspects of the immune-suppressive environment and promote virus control without compromising virus replication? on the contrary, could selective ifn-i agonists be developed to increase ifn-i signaling in a productive way to lower viral loads and bring persistent/chronic viral infection under control? a similar question could be posited during acute viral infections where ifn-i signaling promotes aberrant inflammation and immune pathology. moreover, it would be interesting to investigate whether selective biological or pharmacological modulation of ifn-i signaling may translate to treat autoimmune disease states associated with elevated and sustained ifn-i signaling. however, any therapy that enhances or blocks ifn-i signaling will need to be approached carefully, given the delicate balancing act required for controlling virus replication while safely modulating immune responses. protective immunity and susceptibility to infectious diseases: lessons from the 1918 influenza pandemic role of host immune response and viral load in the differential outcome of pandemic h1n1 (2009) influenza virus infection 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doi: 10.1016/j.jhep.2020.09.031 sha: doc_id: 256508 cord_uid: ce59ovan an epidemic of acute respiratory syndrome (covid-19) started in humans in wuhan in 2019, and became a pandemic. groups from china identified and sequenced the virus responsible for covid-19, named sars-cov-2, and determined that it was a novel coronavirus (cov) that shared high sequence identity with batand pangolin-derived sars-like covs, suggesting a zoonotic origin. sars-cov-2 is a member of coronaviridae, a family of enveloped, positive-sense, single-stranded rna viruses that infect a broad range of vertebrates. the rapid release of the sequence of the virus has allowed the development of diagnostic tools (e.g., rt-pcr). additionally, serological tests can allow identification of persons who have been infected. in humans, covs tend to cause mild to moderate upper respiratory tract infections. the fatality rate is around 1-3% for infected persons. an acute respiratory distress syndrome (ards) likely due to an uncontrolled immune activation (“cytokine storm”) occurs in patients with severe disease and poor prognosis. risk factors for mortality include: advanced age, obesity, diabetes, hypertension and other comorbidities. drug repurposing has been used to rapidly identify potential treatment for covid-19, which could move quickly to phase-3. better knowledge of the virus, its enzymes, will be mandatory to develop more potent and specific direct-acting antiviral agents (daa). in the long term, a vaccine to prevent infection would be crucial; however even if successful it might not be available before 2021-22. to date, with the exception of intravenous remdesivir and dexamethasone, which have modest effects in moderate to severe covid-19, no strong clinical evidence supports the efficacy and safety of any other drugs against sars-cov-2. the aim of this review is to provide insights on the discovery of sars-cov-2, its virology, the diagnostic tools, and the ongoing drug discovery effort. the world health organization (who) announced on march 11 th 2020, that the outbreak of "coronavirus disease 2019" (covid19) , which initially started in asia, has become pandemic. on september 4 th 2020 the etiologic agent "severe acute respiratory syndrome (sars)-cov-2 has spread all over the world with a global estimation of around 26 million confirmed cases and around 865,000 deaths [1] . the rapid availability of the virus rna genome sequence was instrumental in the development of diagnostic tools and for the identification of experimental treatments. in this review, we will clarify aspects related to the discovery of sars-cov-2, virological features, diagnostic tools, complex pathogenesis, including a focus on the liver and gastro-intestinal lesions, and of course drug discovery. the causative agent of covid-19 is a novel coronavirus (cov) officially named sars-cov-2. it was named after sars-cov-1, because of a genomic homology [2] . coronaviruses are enveloped, large positive-sense single-stranded rna viruses (+ssrna), gathered in the coronaviridae family. covs can infect a broad range of vertebrates, including bats, birds, pangolins, snakes, mice, and humans. due to sequence similarities with ratg13 bat and pangolin cov strains, it is currently thought that sars-cov2 has a zoonotic origin and has secondary acquired human-to-human spreading capacity [3] . in particular, the acquisition of i) mutations in the receptor binding area, ii) a polybasic furin cleavage site (rrrar) at the junction of subdomain 1 and 2 of the spike protein and iii) a site of o-linked glycosylation in the same area, has enabled the virus to efficiently interact with high affinity (via its spike protein) with its bona fide cellular receptor (angiotensin-converting enzyme 2 (ace-2)) [4] , to become more virulent and pathogenic, while potentially evading immune response by o-glycan epitope masking [3] . where does the virus replicate? following replication and sub-genomic rna synthesis, the viral structural proteins, are translated and inserted into the endoplasmic reticulum (er). these proteins move along the secretory pathway into the er-golgi intermediate compartment. in infected cells, the cov rna-synthesizing machinery associates with modified er membranes that are transformed into the viral replication organelle; double-membrane vesicles appear to be the central hub for viral rna synthesis [5] . otherwise, the duration of sars-cov-2 is significantly longer in stool samples than in respiratory samples [6] . disrupted cell membrane [9] . viral particles were observed in the epithelial cells by electron microscopy suggesting that these lesions might be partially caused by a direct cytotoxicity. a better understanding of the functions/roles of viral proteins, as well as of the virus replication cycle, with a particular attention on host-cell/virus interactions, will allow the identification of novel or a better characterization of targets for antiviral agent development. the success of drug development for hepatitis c virus (hcv) has inspired scientists to achieve the same for other viruses [10] . entry process. many cell types could express ace2 and transmembrane serine protease 2 (tmprss2), the two cellular factors important for virus entry [11] , including nasal and lower airway epithelial cells (pneumocytes), lung resident immune cells, endothelial cells, as well as neurons, enterocytes, cardiomyocytes, hepatocytes, kidney cells [12] [13] [14] . but the presence of mrna in these cell-types is not sufficient. study of the expression at protein levels of these entry factors, as well as the demonstration of bona fide viral entry and active replication in all these cell-types is yet missing. interestingly, it was very recently shown that ace2 is an interferon-stimulated-gene (isg) [15] , meaning that the presence of ifns in the microenvironment of the virus replication site could further enhance the spreading of the virus. the molecular details of the entry process, involving the spike protein and host receptor/coreceptor, have already been studied [16] . polybasic furin cleavage site at the junction of subdomain 1 and 2 of the spike protein, may explain the large number of cell types that may be infected by the virus with the consequent diverse organ manifestations, including, possibly the thrombotic complications as a result of possible endothelial cell infection. this research will facilitate the identification of neutralizing antibodies or small molecules, which could target this step of the life cycle. covs encode several enzymes that are crucial for the replication of the virus and ideal target for antiviral development, amongst which two proteases/proteinases (plpro and 3clpro), the rdrp, a helicase, an mrna-cap-methyltransferase, and an exoribonuclease. these enzymes have been well studied for sars-cov, and thanks to the high homology between the two sars covs, we could expect functional similarities and the possible repurposing of drugs [3] [4] 11] . the rdrp (also identified as nsp12) bears the main enzymatic activity of the replicase complex. recent advance in antiviral research against hcv [10, 17] , has confirmed that rdrp are major target for very specific antiviral discovery. similarly to hcv, sars-cov2 genome is characterized by a positive-sense single-strand rna and share a similar replication cycle requiring a rdrp. this polymerase displays resembling catalytic mechanisms and key conserved amino acids in the active site. the rdrp 3d structure has been readily characterized [18] [19] . interestingly it has a large nterminal extension containing a kinase-like fold. the polymerase domain, like hcv, is composed of three subdomains; a fingers subdomain, a palm subdomain, and a thumb subdomain. moreover, j o u r n a l p r e -p r o o f also the 3-chymotrypsin-like protease (3clpro) is vital to virus replication and the 3clpro cleavage sites are highly conserved, so it could be a promising drug target [18] . plpro and 3clpro/mpro are essential enzymes for the proteolytic processing of cov replicase polyprotein; their activities are needed very early in the infection process to step-by-step release other viral enzymatic activities. they are also attractive targets for specific antiviral discovery. sars-cov1 and sars-cov2 share high amino-acid sequence identities for these two proteases and 3d structures are also available. moreover biochemical assays are also available for functional testing, at least for the sars-cov proteins [20] . plpro is a cysteine protease, encoded by nsp3, and involved in the release of nsp1 to 3, as well as in the regulation of host innate immunity functions to allow viral escape [20] . although the similarity between plpro of sars-covs is not very high, the catalytic domain, around the triad cyst-his-asp, is well conserved; therefore drug already in the pipeline for sars-cov1 might be repurposed. 3ctpro/mpro is encoded by nsp 5, forms a functional homodimer, utilizes a catalytic dyad cys-his, and is involved in the release of nsp4 to 16 from polyprotein. its activity is key for cov replication-cycle and its inhibition very efficient at stopping viral replication. due to the dimeric nature of this protease, not only catalytic inhibitors, but also allosteric ones can be developed increasing possibilities of success. moreover the very high similarity of 3ctpro/mpro sars-covs allow repurposing of drugs [20] . some specific antiviral screenings have been promptly started on 3ctpro of sars-cov-2 and several drug candidates already identified [21] [22] . beside virologic aspects of covid-19, it is also important to better understand immunologic ones, as well as their mutual amplification responsible for an increased pathogenesis in patients. if the virus can be studied in cell culture model, immunological aspects of covid-19 can only be studied either in relevant animal model or during clinical studies, using patient samples. it is now rather well established that in patients with poor outcome there is an uncontrolled "cytokine storm", featuring a local and systemic production of pro-inflammatory cytokines such as il-6, tnf-α and il-1β [23] [24] [25] [26] [27] . recently, it has been reported that ace2 is a human interferon-stimulated gene (isg) in vitro using airway epithelial cells and extend the findings to in vivo viral infections. these data suggest that sars-cov-2 could exploit species-specific interferon-driven upregulation of ace2, a tissue-protective mediator during lung injury, to enhance infection [28] . more studies are needed to clarify the origin of this massive and uncontrolled cytokine production. covid-19 tests can be grouped as nucleic acid, serological, antigen, and ancillary tests, all of which play distinct roles in hospital, point-of-care, or large-scale population testing [29] . methods for the detection of viral nucleic acid. pcr tests for sars-cov2 have been available since january 2020. rt-qpcr-based assays performed on respiratory specimens has j o u r n a l p r e -p r o o f emerged as the cornerstone of covid-19 diagnostic testing. the usa centers for disease control and prevention (cdc) has developed a widely used sars-cov2 rt-qpcr assay [30] . the kit contains pcr primer-probe sets for two regions of the viral nucleocapsid gene (n1 and n2), and for the human rnase-p gene to ensure the rna extraction was successful. this assay differs from the who's one, which target sars-cov2 rdrp and e genes [31] . to avoid potential cross-reaction with other endemic covs, as well as potential genetic drift, at least two molecular targets should be included in the assay. evolution and potential mutations in sars-cov-2 genome strengthens the need to continue optimizing the oligonucleotides by global updated sharing of sars-cov-2 genomes [32] . theoretical specificity of most rt-qpcr assays is 100% because the primer design is specific to the sars-cov-2 genome. occasional false positive results may occur due to technical errors or reagent contamination [33] . a cycle threshold (ct) value of rt-qpcr less than 40 is generally interpreted as positive when results are interpreted as qualitative [34] [35] . quantitative interpretation of ct as indicators of the copy number of sars-cov-2 rna in specimens needs an appropriate standard curve with adequate limit of detection [36] . a rigorous assessment of the diagnostic accuracy of the many newly introduced sars-cov-2 assays is hampered [37] [38] . the sensitivity of viral rna testing varies with timing of testing relative to exposure. a false positive result erroneously labels a person infected, with consequences including unnecessary quarantine and contact tracing. false negative results are more consequential, because infected persons may not be isolated and can infect others. one modeling study estimated that the probability of a falsenegative result in an affected patient decreases from 100% on day 1 to 67% on day 4 [40] . on the day of symptom onset, the median false-negative rate estimation was 38%. sample pooling strategy was suggested to offer a viable alternative to detect community transmission at a time when tests are in short supply globally [41] [42] [43] . one potential limitation of pool testing is that the false-negative rate may increase owing to dilution of positive samples. point-of-care pcr kits can shorten the turnaround time for screening and diagnosing patients with suspected sars-cov-2. these rapid tests typically have lower throughput and are generally more expensive than other tests. time efficient methods that do not require thermal cycling have been designed and are evaluated [44] . crispr-cas12/cas13-based assay are also currently in development for point-of-care use [45] [46] . nature of samples tested. the current diagnostic strategy to identify patients with covid-19 is to test samples taken from the respiratory tract to assess for the presence of sars-cov-2 specific nucleic acid targets [47] . a nasopharyngeal specimen is the preferred choice for testing, but oropharyngeal, mid-turbinate, or anterior nares samples are also acceptable [48] . a pharyngeal virus shedding was shown to be very high during the first week of symptoms [49] . infectious virus was readily isolated from throat-and lung-samples, but not from stool ones. serum and urine were usually negative for the presence of viral nucleic acid [50] [51] . the viral load in nasopharyngeal samples peaks within the first few days after symptom onset, before declining [48, [51] [52] . for nasopharyngeal specimen, samples should be obtained by using a flocked swab to enhance the j o u r n a l p r e -p r o o f collection and release of cellular material [53] [54] . samples taken from sputum, endotracheal aspirates, and bronchoalveolar may have greater sensitivity than upper respiratory tract specimens [50] . inadequate sample collection may result in a false-negative test. bronchoalveolar lavage fluid specimens had the highest positive rates of sars-cov2 rt-qpcr assay [50] . a single nasopharyngeal swab has become the preferred swab, as it is well tolerated and safe. saliva may also be an alternative specimen source that requires less personal protective equipment and fewer swabs, but requires further validation [55] [56] . serologic testing. if rt-qpcr-based molecular assays for detecting sars cov-2 in respiratory specimens remain the current reference standard for diagnosis, point-of care technologies, and serologic immunoassays have also rapidly emerged [57] [58] [59] . serologic tests that identify antibodies to sars-cov-2 from clinical specimens may be less complex than molecular tests [60] . as antibody responses to infection take days to weeks to be reliably detected [60] , their utility for diagnosing acute infections is limited [48] . serologic assays might be more relevant in surveying for asymptomatic infection or in scenarios in which patients present to medical care with late complications of disease, when rt-qpcr may be falsely negative [55, 61] . seroconversion in most cases of sars occurs during the second week of symptoms [49] . for sars-cov-2 infection, timing of seroconversion appears similar to or slightly earlier than in sars-cov-1 infection [62] . in a study of 285 patients with covid-19, 100% of patients were tested positive for antiviral igg within 19 days after symptom onset, with seroconversion for igg and igm occurring simultaneously or sequentially [61] . negative results would not exclude covid-19 infection, particularly among those with recent exposure to the virus. the viral spike protein is perceived as the clear candidate for inclusion in an immunoassay that detects whether antibodies are present [58; 63] . the other protein that appears to be important antigen for the development of serological assays is the n protein (structural component of the nucleocapsid). indeed, antibodies to this protein are frequently detected in covid-19 patient [64] [65] , suggesting that n protein may be one of the immunodominant antigens in the early diagnosis of covid-19 [60, [66] [67] . it is now established that sars-cov-2 pre-existing immune reactivity can exist in the general population. serum samples from patients with covid-19 showed some cross-reactivity for the sars-cov-1 nucleocapsid antigens [61, [68] [69] . a recent study detected sars-cov-2-reactive cd4+ t cells in 50% of unexposed individuals, suggesting cross reactive t cell recognition between circulating ''common cold'' coronaviruses and sars-cov-2 [66] . t cell reactivity was highest against proteins other than the coronavirus spike protein, but t cell reactivity was also detected against spike. several monoclonal antibodies have been described that target the s glycoprotein of sars-cov-2 from memory b cells of an individual who was infected with sars-cov-1 in 2003 [68] . one antibody (s309) potently neutralizes sars-cov-2 by engaging the receptor-binding domain of the s glycoprotein. enzyme-linked immunosorbent assays (elisa) and clia are common laboratory platforms that can measure antibody titers (igg and igm). a variation of these tests can use magnetic, proteincoated microparticles, known as a chemiluminescent microparticle immunoassay. being able to quantify antibodies will be important for identifying convalescent plasma donors with abundant titers and studying how the immune system responds to the virus. neutralizing antibodies (nabs) play important roles in virus clearance and have been considered as a key immune product for protection or treatment against viral diseases. in covid-19, transfusion of convalescent plasma or serum from recovered patients was also considered as a promising therapy [71] . the neutralization assay is a laboratory-based test that uses live virus and cell culture methods to determine if patient antibodies can prevent viral infection in vitro [72] . because immunofluorescence assay is a labor-intensive method, a substantial number of the new commercial covid-19 antibody tests developed as screening tests are not elisa-based. they are lateral flow immunoassays (lfia), which provide no quantitative information. these qualitative lfia represent typically small, portable rapid diagnostic tests (rdt), and can be used at point of care. conclusions on serologic testing. antibody testing is ramping up quickly, with a growing list of commercial kits and test protocols from academic researchers [57] . many questions will have to be answered. the first and most urgent is the validation of serologic tests. a recent meta-analysis showed wide range sensitivities from 66% in lfias to 98% in the clias [73] ; sensitivities were higher with increased time after symptom onset. the specificities are excellent (99%). assays must be optimized further, independently validated, and used in an algorithm format to achieve the highest possible accuracy for decision making [74] [75] . second, there is insufficient data of the magnitude and duration of antibody responses after infections. although data suggest that neutralizing titers correlate with severity of infection [61] , it remains elusive, whether this effect is caused by ongoing somatic hypermutation or ongoing production of highly potent antibodies that were initially generated. moreover, any documentation that limits individual freedoms on the basis of biology risks becoming a platform for restricting human rights [76] . physiopathology. several potential pathogenic mechanisms may be involved including coagulopathy, endothelial dysfunction, and excessive release of pro-inflammatory cytokines. the endothelial dysfunction caused by infection activates an excessive thrombin generation and inhibits fibrinolysis, which designates hypercoagulability [77] . lymphopenia is frequent in patients with covid-19 [78] . the cytokine release syndrome could have a major role in patients with severe covid-19 as in acute respiratory distress syndrome (ards) [79] . the pathological features of covid-19 related ards are diffuse alveolar damage with hyaline membrane formation with fibrin deposition and a few multinucleated enlarged cells [7] [8] . in patients who died from covid-19associated respiratory failure, the histologic pattern in the peripheral lung was diffuse alveolar j o u r n a l p r e -p r o o f damage with perivascular t-cell infiltration [9] . the lungs also showed distinctive vascular features, consisting of severe endothelial injury, but also widespread thrombosis with microangiopathy. alveolar capillary microthrombi were frequent, with a high amount of new vessel growth (intussusceptive angiogenesis). transmission by asymptomatic carriers. several findings are consistent with person-to-person transmission of this novel coronavirus in hospital and family settings . a case of sars-cov-2 infection acquired outside asia in which transmission appears to have occurred during the incubation period [81] . furthermore, in a previously reported family cluster, some of the family members had positive rt-qpcr results without any symptoms [47] . clinical characteristics. among 1,099 patients from china with laboratory-confirmed covid-19, 5.0% were admitted to the intensive care units (icu), 2.3% underwent invasive mechanical ventilation, and 1.4% died [78] . the most common symptoms were fever and cough. the median incubation period was 4 days. in another study including 191 patients, with 54 who died in hospital, half of the patients had a comorbidity, with hypertension being the most common, followed by diabetes and coronary heart disease [52] . in-hospital death was associated with older age, higher sequential organ failure assessment (sofa) score, and d-dimer greater than 1 μg/ml on admission. in another study of the 1,591 patients infected with sars-cov-2 admitted to icu in italy, the median age was 63 years and 82% were male [82] . among 1,300 patients with available respiratory support data, 99% needed respiratory support, including 88% who received mechanical ventilation and 11% who received non-invasive ventilation. finally, in this case series of critically ill patients admitted to icus, the majority were older men and icu mortality was 26%. moreover, data from previous coronavirus infections such as severe acute respiratory syndrome and middle east respiratory syndrome, as well as emerging data from the covid-19 pandemic, suggest there could be substantial fibrotic consequences following sars-cov-2 infection [83] . imaging findings. the hallmarks of covid-19 were bilateral and peripheral ground-glass and consolidative pulmonary opacities [84] . notably, 56% of patients with early disease had a normal ct. with a longer time after the onset of symptoms, ct findings were more frequent, including consolidation, bilateral and peripheral disease, greater total lung involvement, linear opacities, "crazy-paving" pattern and the "reverse halo" sign. bilateral lung involvement was observed in 28% in early phase and 88% in late phase of the disease. ct scans at the time of symptoms may increase diagnosis rate since rt-pcr sensitivity may be as low as 60% [85] . also, chest x-ray findings in covid-19 patients frequently showed bilateral lower zone consolidation [86] . coagulopathy disorders. sars-covid-19-induced infection can be associated with a coagulopathy, findings consistent with infection-induced inflammatory changes as observed in patients with disseminated intravascular coagulopathy (dic) [87] . the initial coagulopathy of covid-19 presents with elevation of d-dimer and fibrin/fibrinogen-degradation products. covid-19-j o u r n a l p r e -p r o o f associated coagulopathy should be managed as it would be for any critically ill patient, using thromboembolic prophylaxis and standard supportive care measures for those with sepsis-induced coagulopathy or dic. current data do not suggest the use of high anticoagulation doses [87] . among all the numerous clinical manifestations associated with covid-19 infection, we can recall cardiological lesions with acute myocardial injury [88] ; neurological lesions with encephalitis and myalgia [89] [90] , cutaneous manifestations with rash and urticaria [91] , and acute kidney injury [92] . (figure 3) . elevation of liver enzyme occurs in 5 to 50 % of patients. the pattern of liver injury is mainly at hepatocellular rather than cholestatic level [93] [94] , with hepatocyte degeneration, focal necrosis, capillary bile duct cholestasis and inflammation in the portal area, but interestingly sars-cov-2 cannot be detected in samples [95] . frequently, the severity of liver injury had been correlated to the severity of covid-19. the presence of underlying chronic liver diseases could render covid-19 patients at higher risk of severe liver injury, such as acute-on-chronic liver failure [96] , with data suggesting that nafld/madld could be an independent risk factors for severe covid-19 [97] [98] . the virus was found in stool samples in around 50% of patients with covid-19, with around 18% of them complaining of abdominal pain and diarrhea [99] . it was demonstrated that sars-cov2 is capable to productively replicate in ace2-positive enterocytes [12] . due to the abundance of the virus in the small intestine, liver cell exposure through the hepatic reticular system is expected. hepatic default immune status might play a critical role in covid-19 infection. indeed, it has been shown that in patients with mafld, the polarization status of macrophage might be skewed due to metabolic stimuli such as fatty acids and thus affecting host-inflammatory response to signals generated from the gut-liver axis [97] . in covid-19, the "cytokine storm" bears resemblance to sars caused by the sars-cov-1, where cytokine storm has been associated with disease [100] [101] [102] . on the other hand, direct cytopathic damage by sars-cov-2 is also possible as there are entry receptors ace-2 in cholangiocytes [103] . also, learning from sars experience, the use of antibiotics, antivirals, together with possible secondary bacterial infection, might lead to liver injury in covid-19 patients [104] . moreover, tocilizumab is evaluated for the treatment of covid-19 patients with serious lung damage and accompanying elevated blood levels of il-6 [105] . prophylactic nucleoside analogs against hepatitis b virus had been recommended for those hepatitis b surface antigen positive covid-19 patients planned for immunosuppressive therapy [102] . liver damage, which lead to drug withdrawal, has been reported in patients treated with remdesivir. accordingly, it is not recommended for those covid-19 patients with alt> 5 times uln or with liver decompensation to receive remdesivir [106] . lastly, hypoxia and shock induced by covid-19related complications may also cause hepatic ischemia [107] . to manage liver injury related to covid-19, several guidelines have been issued [100] [101] [102] 108] . suffered from gastrointestinal symptoms such as nausea or vomiting, diarrhea and anorexia [109] , with similar incidence among adults and children [110] . patients with gastrointestinal symptoms may require longer duration of hospitalization [78] [79] 111] . in some patients, gastrointestinal without respiratory symptoms might be the presenting clinical features [112] [113] . the underlying mechanism may be related to the abundant expression of ace2 mrna and receptor protein in the enterocytes [112] [113] . histological changes with the presence of plasma cell and lymphocytes infiltration in patients' lamina propria of enterocytes suggested an immune-mediated response [114] . the capability of sars-cov2 to infect enterocytes has also been demonstrated in human intestinal organoids [12] . one of the major concerns of enteric infection is whether fecal source can lead to fomite transmission, especially when infective aerosols are generated from the toilet plume. indeed, cluster of cases infected with covid-19, in analogy to "amoy garden" during the sars in 2003, has recently been suggested in hong kong [115] . in accordance to the surface stability study on plastic and different materials, sars-cov-2 could remain viable up to 72 hours [116] . in one study, in 20% of patients, sars-c0v2 rna remained positive in feces despite clearance in the respiratory specimen [114] . taking together, it is of great importance that the presence of sars-cov-2 in the stool need to be determined for the epidemiology control of covid-19. there is a major concern with the potential concomitant infection of sars-cov-2 with influenza or other respiratory diseases such a respiratory syncytial virus, or tuberculosis or even bacterial infections or mycoplasma. co-infection with sars-cov-2 and influenza a virus in a patient with pneumonia has been reported in china [117] . covid-19 might be underdiagnosed because of false-negative tests for upper respiratory specimens or co-infection with other respiratory viruses. prevention and transmission control measures. washing hands frequently, using mask and social distancing are important. china banned travel to and from wuhan city on 23 january 2020, and this shutdown was associated with the delayed arrival of covid-19 in other cities by approximately 3 days [118] . suspending intra-city public transport, closing entertainment venues and banning public gatherings were associated with reductions in case incidence. early on, the spatial distribution of covid-19 cases in china was explained well by human mobility data [119] . following the implementation of control measures, this correlation dropped and growth rates became negative in most locations. a contact-tracing application, which builds a memory of proximity contacts and immediately notifies contacts of positive cases could achieve epidemic control if used by enough people [120] . much like with influenza, antiviral drugs to be effective likely need to be started early in infection course. in turn, this represents a burden to identify drugs that are indeed effective against the virus in clinical trials. patients with early disease may benefit from antiviral agents to reduce viral load, patients with severe and late disease may benefit from anti-inflammatory j o u r n a l p r e -p r o o f drugs. furthermore, in the beginning of the disease, anti-inflammatory drugs might be harmful by increasing viral load. drug repurposing. drug repurposing (also called drug repositioning or reprofiling) is a strategy for identifying new uses for approved or investigational drugs that are outside the scope of the original medical indication [121] . this strategy offers various advantages over developing an entirely new drug, with a reduction risk of failure because safety has already been evaluated. but also the time frame and the cost can be reduced, because most of the preclinical testing and safety assessment have been done. an extensive repositioning activity of approved drugs has been embarked for the covid pandemic. a selection of drugs being tested for covid-19 are represented in table 1 . for example of large randomized ongoing trial the design of "solidarity", is provided in table 2 . has also been combined with azithromycin, an antibiotic. hcq would inhibit sars-cov-2 entry into cells. few data are coming from reports and small studies [122] . a systematic review on the efficacy and safety of hcq for the treatment of covid-19 concluded that there is currently no evidence from rcts to inform on hcq efficacy [123] . in a multicenter, open label, randomized controlled trial, 150 patients admitted to hospital with laboratory confirmed covid-19 were included in the intention to treat analysis (75 patients assigned to hcq plus standard of care (soc), 75 to soc [124] . there was no difference in term of efficacy between the 2 arms. adverse events were higher in hcq recipients than in non-recipients. lopinavir is an antiretroviral protease inhibitor used in combination with ritonavir in hiv therapy, which has shown some antiviral activity against sars-cov [125] . a randomized, controlled, openlabel trial involving hospitalized adult patients with confirmed sars-cov-2 infection and severe respiratory illness covid-19 was performed [126] . patients were randomly assigned to receive either lopinavir-ritonavir, in addition to soc, or soc alone. there were no differences between groups (virologic aspects, duration of disease, mortality), indicating that there is no benefit in hospitalized adult patients with severe covid-19. cell culture data suggest that this compound demonstrates activity with an ec 50 of 26.6 µm [127] . one wonders why it was selected for clinical trials with such a weak activity. evaluating in humans repurposed drugs that are essentially ineffective in culture against sars-cov-2 is being repeated over and over again wasting time and resources. remdesivir is a prodrug of a nucleotide analog that is intracellularly metabolized to an analogue of adenosine triphosphate that inhibits viral rna polymerases. remdesivir has broad-spectrum activity against members of several virus families, including filoviruses (e.g., ebola) and coronaviruses (e.g., sars-cov and mers-cov [128] . six large studies are ongoing ( table 3) . unfortunately remdesivir must be given intravenously for at least 5 days, although an aerosol formulation is being developed. were reported [129] . from the 53 patients whose data were analyzed, clinical improvement was observed in 36/53 patients (68%). a randomized, double-blind, placebo-controlled, multicenter trial was performed in china [106] . mortality at day 28 was similar between the two groups (14% died in the remdesivir group vs 13% in the placebo group). there was no difference in the two groups regarding clinical improvement and viral load decreased. this trial did not attain the predetermined sample size because the outbreak of covid-19 was brought under control in china, therefore, it is difficult to reach a definitive conclusion. gilead is conducting two randomized, open-label, multicenter, phase 3 clinical studies to evaluate the safety and efficacy of two dosing durations -5 days and 10 days -of remdesivir in adults diagnosed with covid-19 (the simple studies). the first simple study is involving hospitalized patients with confirmed sars-cov-2 infection, oxygen saturation of 94% or less while they were breathing ambient air, and radiologic evidence of pneumonia [130] . in the second simple study patients were randomized to receive open-label remdesivir for 5 or 10 days or soc alone. at day 11, a higher proportion of patients in the 5-day treatment group achieved improvement in clinical status versus the soc group, achieving statistical significance for a ≥ 1-point improvement in ordinal scale (p=0.026)(gilead press release). however, most clinician would have preferred to see a decrease in mortality on treatment. clearly another controlled study will have to be performed soon. among other antiviral drugs being tested for covid-19 we can quote arbidol [131] [132] , favipiravir [133] , famotidine [134] [135] , and camostat (tmprss2 inhibitor) [11] . dexamethasone. glucocorticoids may modulate inflammation-mediated lung injury and thereby reduce progression to respiratory failure and death. in a controlled, open-label trial of patients hospitalized with covid-19, patients were randomly assigned to receive oral or intravenous dexamethasone (6 mg once daily) for up to 10 days or to receive soc alone [136] . in the dexamethasone group, the incidence of death was lower than that in the soc group among patients receiving invasive mechanical ventilation (29.3% vs 41.4%) and among those receiving oxygen without invasive mechanical ventilation (23.3% vs 26.2%) but not among those who were receiving no respiratory support at randomization (17.8% vs 14.0%). in a recent trial involving patients with ards who were undergoing mechanical ventilation, mortality at 60 days was 15 percentage points lower among those receiving dexamethasone than among those receiving soc [137] . in the early phase of the infection, anti-inflammatory drugs may not be efficient (maybe harm-full) increasing viral load. viral shedding in sars-cov-2 appears to be higher early in the illness and declines thereafter [49, 54, 138] . the greater mortality benefit of dexamethasone in patients with covid-19 who are receiving respiratory support and among those recruited after the first week of their illness suggests that at that stage the disease may be j o u r n a l p r e -p r o o f dominated by inflammation, with active viral replication playing a secondary role. clearly a trial of the combination remdesivir and dexamethasone may yield interesting results. early triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin was safe and superior to lopinavir-ritonavir alone in alleviating symptoms and shortening the duration of viral shedding and hospital stay in patients with mild to moderate covid-19. future clinical study with interferon beta-1b as a backbone is warranted [139] . tocilizumab (actemra), also known as atlizumab, and sarilumab (kevzara) are both immunosuppressive drugs, mainly for the treatment of rheumatoid arthritis. they are both humanized monoclonal against the interleukin-6 receptor (il-6r), and are given by injection. clinical trials are ongoing. moreover other monoclonal antibodies or agents targeting other inflammatory cytokines (tnf-α, il-1β...) should be soon tested. kinase inhibitors. the janus activating kinase (jak)-signal transducer and activator of transcription (stat) pathway has been implicated as a key driver in many inflammatory diseases. with the onset of small molecule inhibitors that can selectively and specifically target key jaks involved in controlling downstream inflammation, exploration for their utility across a broad range of diseases has become a rapidly expanding field [139] [140] , including viral infections (eg. hiv; [141] [142] [143] ). baricitinib and ruxolitinib are two known jak inhibitors. recently, artificial intelligence enabled the identification of a group of approved drugs that could inhibit clathrin-mediated endocytosis and thereby inhibit viral infection of cells [144] [145] . the drug targets are members of the numbassociated kinase (nak) family. baricitinib was identified as a nak inhibitor, with a particularly high affinity for aak1, a pivotal regulator of clathrin-mediated endocytosis. this drug is also known to target jak and could have a dual action against the virus and inflammation [146] . the nih/niaid sponsored actt-2 study is still ongoing and compares remdesivir to remdesivir plus baricitinib in moderate to severe patients. in a small uncontrolled cohort of veterans affairs patients with moderate-severe covid-19, treatment with baricitinib plus hydroxychloroquine was associated with recovery in 11 of 15 patients [147] . two other kinase inhibitors, namely imatinib mesylate and dasatinib, could also be envisaged to treat covid-19 [148] . furthermore, ruxolitinib (another jak inhibitor-incyte) is being evaluated in a multicenter phase-ii clinical trial [149] . convalescent plasma. the immediate use of convalescent plasma provides a promising treatment. in a preliminary uncontrolled case series of 5 critically ill patients with covid-19 and ards, administration of convalescent plasma containing neutralizing antibody was followed by improvement in their clinical status [71] . the limited sample size of this study precludes a definitive statement about the efficacy of this treatment. vaccines are the most effective strategy for preventing infectious disease since they reduce morbidity and mortality, and they are more cost-effective than treatment. despite previous coronaviruses epidemics, there is still no approved vaccine for human coronaviruses [150] . we will have to improve our understanding and knowledge regarding immune response to sars-cov2. interestingly, in rhesus macaques, comparing the humoral and cellular immunity between primary infection and re-challenge revealed notably enhanced neutralizing antibody and immune responses [151] . these results suggest that primary sars-cov-2 exposure protects against subsequent reinfection in rhesus macaques. in human, in a large study of the icelandic population observed that humoral response did not decline within 4 months after infection, that 44% of persons who had been infected had not been diagnosed with pcr, and that the fatality rate was 0.3% [152] . we have also to recall that few cases of sars-cov-2 re-infection were reported. epidemiological, clinical, serological and genomic analyses confirmed that the patient had reinfection instead of persistent viral shedding from first infection [153] . this case lead to several open questions: how frequent is reinfection? are reinfections less severe than the first? will vaccine protect against reinfections? these results suggest sars-cov-2 may continue to circulate among the human populations despite herd immunity due to natural infection or vaccination. further studies of patients with re-infection will shed light on protective correlates important for vaccine design. regarding vaccine development, among the different strategies, we can recall the use of recombinant subunit vaccine, dna or mrna vaccine. subunit vaccines are believed to be highly safe because they are expected to induce the immune system without introducing infectious viruses [154] . a better knowledge of sars-cov-2 s or/and n protein organizations will be require to develop such vaccines. the sars-cov-2 spike (s) glycoprotein mediates host cell attachment and is required for viral entry; it is the primary vaccine target for many candidate sars-cov-2 vaccines. dna vaccines are based on direct injection of plasmids encoding the antigens, followed by a large range of immune responses. mrna-based vaccines contain mrnas encoding the antigens, which are translated at the host cellular machinery by vaccination [155] . mrna vaccines have advantages over conventional vaccines, by the absence of genome integration, the improved immune responses, the rapid development, and the production of multimeric antigens [155] [156] . preliminary report of an mrna vaccine against sars-cov-2 was published [157] . the candidate [158] . long-term assessment will be relevant given that natural history studies suggest that sars-cov may not generate long-lived antibody responses [159] . furthermore safety evaluation will be mandatory, since there have been concerns about the potential for vaccineassociated enhanced respiratory disease. of the three doses evaluated, the 100-μg dose elicits high neutralization responses and th1-skewed cd4 t cell responses, coupled with a reactogenicity profile that is more favorable than that of the higher dose. the results of two early phase covid-19 vaccine trials were reported, one at oxford university (uk), with support from astrazeneca [160] , and the second supported by cansino biologics in china [161] . both groups used an adenoviral vector, and both report the vaccine achieving humoral responses to the sars-cov-2 spike glycoprotein receptor binding domain by day 28 as well as tcell responses. both report local and systemic mild adverse events such as fever, fatigue, and injection site pain. in neither trial was a severe adverse event reported. although these preliminary data are encouraging sars-cov-2 is a novel pathogen in humans, and many of the technologies being used to build vaccines are relatively untested. there is still a long way to go and phase 3 trials of these vaccines will require thousands of subjects in order to confirm efficacy and safety. the rapid sequencing of the virus has allowed the development of diagnostic tools. table 4 summarize future directions. a test and trace programs will be essential. later, "test, trace and treat (t3)" programs will become mandatory once effective drugs would have been identified and safe therapies developed (figure 4) . several issues will be important to understand. it will be important to precise how transmissible and pathogenic is sars-cov-2 in the ongoing and future epidemic. furthermore, it will be important to improve diagnostic tools. ideally a single or combined test that provides virological and serological output would be ideal. in many countries, at the end of containment, strict recommended measures will be important to avoid new waves of contamination. however, few innovative treatment modalities have been discovered since the bulk of the effort to date has been focused on a vaccine. vaccines might not be enough to quell this pandemic. open-reading-frames (orfs), allowing structural, non-structural and accessory viral protein synthesis [87] . sars-cov2 is 29,903 base-long and contains 6 majors orfs, as well as additional accessory genes; the reference sequence is registered in genbank with id: mn908947.3 [1] . (a and b) up to 28 different polypeptides are potentially produced in fine from the different orfs and after polyprotein processing by viro-encoded proteases [87] . if the rna genome contained in virions can already serve, after cell entry, as a template for the synthesis of non-structural proteins, which are to achieve sars-cov-2 elimination there will be a need to improve protection, testing, treating and preventing strategies. a test and trace programs will be essential. later, test, trace and treat (t3) programs will become mandatory once effective and safe therapies are developed j o u r n a l p r e -p r o o f immunoassays: detection/quatification of seroconversion: patient igm and igg specific to sars-cov-2 spike or nucleocapsid proteins. -neutralization assay: quantitative information on antibodies able to inhibit virus growth ex vivo, -enzyme-linked immunosorbent assay (elisa): quantification of antibodies specific to the virus, -immunochromatography assay: qualitative lateral flow assay (rapid diagnostic test) : detection of antibodies specific to the virus. rapid diagnostic tests (rdt): -works with venous whole blood, serum, or plasma; -rapid test (15 min); no instrument required, -only qualitative: aid in the screening and diagnosis in combination with rt-pcr, -aid in risk stratification, and cohort study. who covid-19 situation report 181 a pneumonia outbreak associated with a new coronavirus of probable bat origin the proximal origin of sars angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a unifying structural and functional model of the coronavirus replication organelle: tracking down rna synthesis viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province, china pathological findings of covid-19 associated with acute respiratory distress syndrome a novel coronavirus from patients with pneumonia in china pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 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sars-cov-2 infection protects against rechallenge in rhesus macaques disappearance of antibodies to sarsassociated coronavirus after recovery safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial immunogenicity and safety of a recombinant adenovirus type-5-vectored covid-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial define mechanisms determining establishment of sars-cov-2 infection: characterize all steps of the virus replication cycle 2. define structure and function of the sars-cov-2 enzymes and their interractions 3. understand physiopathology and immune response 4. improve methods for study of the replication cycle and virus-host interactions to reveal new targets for therapeutic approaches develop and validate diagnostic tools improving sensibility and specificity (serology, rapid diagnostic test) supported in part by nih grant r01-ai-141327, the center for aids research/nih grant p30-ai-050409 and national science foundation grant 2032273 (to rfs).j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f key: cord-307813-elom30nx authors: yip, tsz-fung; selim, aisha sami mohammed; lian, ida; lee, suki man-yan title: advancements in host-based interventions for influenza treatment date: 2018-07-10 journal: front immunol doi: 10.3389/fimmu.2018.01547 sha: doc_id: 307813 cord_uid: elom30nx influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m2 ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m2 ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. keywords: host factors, influenza, cytokines, metabolism, immunomodulation introduction influenza remains a source of public health concern. influenza a virus (iav) has been the cause of historical noxious pandemics, such as the spanish flu 1918 h1n1, asian flu h2n2 1957, hong kong h3n2 flu 1968, and more recently the pandemic of h1n1 2009 (swine flu). influenza also causes seasonal epidemics and outbreaks with high morbidity and mortality rates such as the 2015 h1n1 outbreak in india (1, 2) . the error-prone nature of the viral rna polymerase (rdrp) and virus' capacity for genetic re-assortment (antigenic drift and shift) result in the viral components' susceptibility to mutations, allowing the viruses to evade the immune system and increases their resistance to control strategies. currently, influenza vaccination and two classes of antiviral drugs-m2 ion channel blockers (amantadine and rimantadine) and neuraminidase (na) inhibitor (oseltamivir, zanamivir, and peramivir)-and the novel treatment option using polymerase inhibitor (favipiravir) are considered as mainstays in influenza infection treatment and control. the use of influenza vaccinations remains challenging due to antigenic drifts and shifts, with seasonal variation of new circulating species. production of vaccine is time consuming with efficacy concerns, especially in the case of pandemic. variations in vaccine efficacy caused by age should be aware, with studies suggesting that vaccineconferred protection may not be optimal in certain age groups (3) . the disadvantages of using the conventional antiviral drugs have also been a concern. significant levels of resistance to both classes of drugs have been repeatedly reported (4, 5) . high level of resistance (up to 91%) to m2 blockers has been reported in h3n2 virus strain in american isolates (6) . resistance has also been reported in h5n1 virus (7) . iav resistance to na inhibitors has also become an increasingly prevalent concern, with the recent highly fatal outbreak of influenza a(h1n1)pdm09 in india 2015 associated with oseltamivir drug resistance (8, 9) . in addition, a large cluster of influenza a(h1n1)pdm09 viruses in japan was found to have increased oseltamivir and peramivir drug resistance (5) . there is an urgent need to search for alternative targets to treat influenza virus infections, including non-viral targets such as host cellular factors; which are promising as viruses rely on the host machinery for replication. while host immune response is intended to confer a degree of protection against the infection, an impaired or exaggerated host immune response could be detrimental-iav h5n1 and h7n9 virus infection was reported to exaggerate aberrant cytokine release, resulting in a cytokine storm that caused accelerated host death (10) (11) (12) . many recent studies have focused on the investigation of targeting host factors to control virus replication as well as modulate immune response, which we have previously evaluated (13) . in this review, we will discuss the latest studies (in the past 5 years) on the investigation of novel host-based approaches with potential for influenza treatment. the replication cycle of iav can be grossly divided into four different stages: (1) entry, (2) genome nuclear import, (3) replication and protein synthesis, and (4) genome nuclear export, apical transport, assembly, and budding. as an obligate intracellular pathogen, iavs are heavily dependent on host machinery for replication and propagation. to this extent, studies employing genome-wide rna interference (rnai) to screen for host factors involved in iav replication cycle have been performed (14, 15) and an increasing number of approaches targeting these host factors to control iav replication have been investigated. entry of iav into the host cell is divided into several steps (16, 17) . first, hemagglutinin (ha) on the surface of iav binds to the terminal α-sialic acid on the host cell receptor. this induces the internalization of the viral particle by clathrin-dependent, caveolin-, and clathrin-independent endocytosis (18) . macropinocytosis was revealed as an alternative entry pathway for iav (19) , which subsequently enters the canonical endocytic pathway (20, 21) . the vesicle-containing viral particle forms an early endosome (also known as sorting endosome), which matures into a late endosome as the endocytic pathway progresses. a gradual decrease in intraluminal ph from ph 6.5 to 5.0, mediated by v-atpase proton pump (22) , takes place as the endosome matures (23, 24) . this ph drop in the endosomal lumen induces a conformational change in ha, which is activated by proteolytic cleavage to generate ha1 and ha2 from precursor molecule ha0 (25, 26) . this conformational change triggers the fusion of the viral envelope with the endosomal membrane, releasing the viral genome into the cytoplasm. acidification of the endosome causes the subsequent acidi fication of viral lumen via the iav m2 proton channel (27) , which in turn promotes the dissociation of m1 layer from both the viral envelope (24) and the viral ribonucleoprotein (vrnp) complex (28) . interestingly, a sharp decrease in ph from neutral to an acidic ph of 5.0 as utilized by acid bypass has been observed to be sub-optimal for viral replication. it is hence proposed that a gradual decrease in endosomal ph is necessary for sequential reduction in viral stiffness, dissociation of m1 from the np in the vrnp complex, destabilization of m1 layer from the viral envelope, and the eventual conformational change of the ha for the release of viral genome and proteins to the cytoplasm from late endosome (24) . proteolytic cleavage of ha0 to ha1/ha2 is an important step in iav replication. this cleavage relocates ha2, converting previously uncleaved ha0 to a metastable conformation that induces membrane fusion at acidic ph (29) . inefficient cleavage and activation of ha leads to low infectivity (30) . as identified proteins encoded by the viral genome do not possess proteolytic properties, the virus is dependent on host protease for the cleavage of ha. this provides a potential target to control iav infection. ha is commonly cleaved by trypsin-like proteases at the single arginine residue at position 329. human airway epithelium serine proteases hat and tmprss2 were identified as the host factors for cleavage at this residue (31) . aprotinin, purified from bovine lung (32) , is a protease inhibitor with a long history of clinical use as an antifibrinolytic agent in cardiac surgery (33) . its potential as an anti-iav drug has been recognized for over a decade (34) and has been shown to reduce the infectivity of a broad spectrum of iav strains (34, 35) both in vitro (26) and in vivo (36) . once withdrawn from the western drug market due to its association with mortality (33), aprotinin has been approved as a locally administered, small-particle aerosol drug for the treatment of iav infection in russia (36) . however, side-effects associated with the systemic administration of aprotinin raises the need for an alternative protease inhibitor for use in treatment of iav infections. camostat, a serine protease inhibitor, was reported to demonstrate anti-iav potential in mice dating back to 1996 (37) , but little to no research has been conducted to develop it into an anti-iav treatment. it was revisited and proven to be one of the most efficient serine protease inhibitors for the inhibition of iav replication in primary human tracheal epithelial cells in vitro when tested compounds were used at similar molarities (35) . at present, camostat is widely administered for the treatment of liver fibrosis, chronic pancreatitis, and cancer (38, 39) , making it a highly promising candidate for drug repurposing. despite the lack of association between camostat and increased mortality (as with aprotinin), reports of camostat potentially inducing acute eosinophilic pneumonia (38) warrants the need for careful consideration and further research into the repositioning of drugs from the same class. highly pathogenic iav, such as the h5 and h7 subtypes, are reported to have ha cleavage sites rich in basic residues (30) . the polybasic nature of the cleavage sites provides multiple targets for a broad spectrum of proteases, including the more ubiquitously expressed intracellular proteases such as furin (40) . this increased protease spectrum could be utilized by these viruses for the activation of ha prior to viral budding, allowing for evasion of potential inhibition by exogenously administered serine protease inhibitors. furthermore, an in vivo study utilizing mice treated with a single protease inhibitor prior to infection with h7 virus bearing a polybasic cleavage site showed poor efficacy despite good results were obtained for infection with h1n1 virus bearing single cleavage site (41) , suggesting strain specificity in using serine protease inhibitors to treat iav infections. endosomal acidification is required for the release of iav genome (in the form of a vrnp complex) into the cytoplasm (24) . research has shown that an increase in endosomal ph during the early phases of infection could inhibit iav infection in vitro (42) , bringing to light the possibility of controlling iav infection through the prevention of endosomal acidification. the v-atpase inhibitor bafilomycin a1, when used at high concentrations (10-100 nm) has been proven to inhibit iav replication through the efficient suppression of v-atpase (43, 44) . however, prominent cytotoxicity to host cells was also observed at such concentrations (44) . interestingly, lower concentrations (0.1 nm) of bafilomycin a1 lack inhibitory effects on v-atpase attenuated iav replication due to disruption of endosomal trafficking. thus, bafilomycin a1 is suggested to exert its antiviral function via distinct mechanisms at differing concentrations. diphyllin, isolated from the plant cleistanthus collinus, is a natural compound able to induce a v-atpase inhibitory effect (45) . in contrast to bafilomycin a1, diphyllin is well-tolerated in vitro without inducing obvious cytotoxic effects (46) . most notably, diphyllin is found to effectively inhibit replication of viral strains resistant to amantadine and/or oseltamivir (46) . since drug resistance to these widely administered antivirals is of major public health concern (13), diphyllin is regarded as a promising antiviral against drug-resistant iav strains. the release of iav genomic material during replication requires the fusion of the endosomal membrane with the viral envelope. since cholesterol plays a major role in controlling the fluidity of the lipid bilayer in cells, it is hence suspected to have a role in the infection cycle of iav. interferon-induced transmembrane proteins (ifitms) are proteins expressed in many vertebrates (including humans) and are found on the plasma membrane, the membranes of early and late endosomes, as well as on lysosomes (47, 48) . while humans express ifitm1, ifitm2, ifitm3, ifitm5, and ifitm10, only ifitm 1, 2, and 3 are both immune-related as well as interferon (ifn)-inducible (48) , and have been observed to restrict the replication of different viruses, including iav (49) . studies suggest that ifitms limit viral infection by reducing membrane fluidity and hence restrict the hemifusion (the mixing of lipid bilayer without the release of viral content) of viral and endosomal membranes (50) , probably via the disruption of cholesterol homeostasis of late endosomes, where viral fusion and genome release conventionally take place (51) . a recent study using rnai also demonstrated that cholesterol homeostasis can be regulated via acid phosphatase 2 (acp2)-mediated niemann-pick c2 activity and impaired the membrane fusion of iav and influenza b virus (ibv) (52) , further suggesting the importance of controlling cholesterol homeostasis in the release of viral genome to cytoplasm. on the contrary, later studies suggest that ifitm3 exerts its antiviral activity in a cholesterol-independent manner, showing that an increase in cholesterol composition of late endosomal membranes fail to inhibit viral membrane fusion (53) . in addition, studies suggested the accumulation of cholesterol level in the late endosome does not inhibit the iav genome release into cytoplasm (54, 55) . with the modulation of cholesterol levels in host endosomal membrane as a mean to inhibit iav host cell entry is still under debate, further studies are required before clear conclusions can be drawn. by comparing the mirna profiles of the iav-permissive hek 293t cells and (less permissive) hela cells, mirna-33a has been identified as a negative regulator for iav infection via the inhibition of archain 1 (arcn1, also known as δ-copi) (56). arcn1 is a subunit of the copi complex that is required for intracellular trafficking and endosome function (57) , depletion of which has been reported to inhibit iav infection (14) . despite impaired iav internalization caused by arcn1 depletion via sirna (56, 58), it was not able to recapitulate through acute inhibition of copi complex by pharmaceutical means (58) . it is hypothesized that the long-term (lasting days) perturbation on arcn1 by rnai affected the general endosomal trafficking network, a phenomena which cannot be recapitulated by acute pharmaceutical inhibition to block iav infection (58) . the potential of targeting arcn1 for iav treatment deserves further investigation, despite the favorable results from rnai studies. nuclear import of vrnp complexes from the cytoplasm following fusion of the viral and the endosomal membrane is required for replication to take place (59) . an early study suggested that vrnp complexes could be transported to the periphery of the nucleus (60), while recent studies report that vrnp complexes utilize the importin-α-importin-β1 (impα-impβ1) system for nuclear import (59, 61) and lacking of importin-α7, in an importin-α7 knockout mouse model were found to be resistant to iav infection (62) . ivermectin has long been clinically administered for the treatment of parasitosis (63) , but has recently come to attention as a potential inhibitor of impα/β (64) . ivermectin inhibition of impα/β has shown to inhibit the replication of rna viruses such as dengue virus and hiv-1 (64) . ivermectin was recently tested for the inhibition of iav in vitro, with nuclear import of vrnp complex (of both wild-type and antiviral mxa escape mutant) efficiently inhibited (65) . given ivermectin's longstanding record of clinical applications and fda-approved status, repurposing of this drug for the treatment of iav should be considered, especially while under threat of pandemic iav outbreak. following the import of the vrnp complex into the nucleus of the host cell, rdrp uses the vrna as a template to synthesize mrna or crna. synthesized crna remains in the nucleus for new vrna generation, while mrna is exported out of the nucleus for translation. viral protein products are either transported to the cell surface via golgi (in case of ha and na) or imported back into the nucleus to bind with vrna, forming new vrnp complex (59) . numerous host factors are involved in this process and hence could be possible targets for therapeutic intervention. out of the eight genome segments of iav, the m and ns segments are well known for undergoing splicing to generate at least two different mrnas per individual segment (66, 67) . cdc2-like kinase 1 (clk1) is a kinase which regulates alternative splicing of pre-mrna (68) . inhibition of clk1 by the chemical tg003 or knockdown of clk1 is shown to cause a decrease in m2 mrna generation and disrupt downstream m2 protein expression, prominently reduced iav propagation (15) . clypearin and corilagin were both found to be potent anti-iav compounds, with a higher therapeutic index than tg003 in vitro (69) . clypearin is isolated from herbs used by chinese medicine practitioners for treating respiratory tract diseases during replication, viral mrna is exported from the nucleus to cytoplasm, where protein synthesis takes place. human rna polymerase ii activity is found to be correlated with iav replication through the inhibition of nuclear export of certain viral mrnas, such as m1 mrna (70) . cyclosporine a (csa) is a fda-approved drug with immunomodulatory functions (71) that has been found to have an anti-iav effect in both cyclophilin a (cypa)-dependent and -independent manners (72) . the cypa-dependent effect was found to correlate with nuclear export of vrnp complex (see targeting nuclear export complex). the cypa-independent effect caused inhibition of host rna polymerase ii. csa is a prospective drug candidate for treatment of iav infections with a relatively high barrier for development of intrinsic drug resistance, as opposed to commonly used antivirals (73) . nuclear rna export factor 1 (nxf1) is a host factor that has been identified to be involved in the nuclear export of iav mrna. the knockdown of nxf1 in hek 293t cells revealed prominent viral mrna nuclear retention in host cell nucleus (74) . protectin d1 (pd1), an endogenously produced lipid in the respiratory tract, has been identified to have potent anti-inflammatory and antiviral effects (75) . pd1 production was notably found to be reduced in the lungs of iav-infected mice. therapeutic administration of pd1 was shown to significantly reduce iav mrna expression, lower lung viral titer, as well as improve survival of iav-infected mice. mechanistic studies revealed attenuated cytoplasmic translocation of viral mrna with such treatment. a decrease in recruitment of viral transcripts to nxf1 was observed while nuclear export of host rna remained largely unaffected, suggesting a role of pd1 in regulating nxf1 in nuclear export of viral rna. natural pd1 expression in the human airway makes this an ideal candidate for novel therapeutics in the treatment of iav infection. the eukaryotic initiation factor-4a (eif4a) family plays an important role in protein translation (76, 77) . eif4a impairment has been proven to be related to antiviral activity in a broad spectrum of rna viruses in vitro (78) , with inhibition of iav mrna translation (79) . the eif4a inhibitors, silvestrol and pateamine a were demonstrated to arrest viral protein synthesis, thus blocking viral genome replication in vitro (80) . although both silvestrol and pateamine a caused high cytotoxicity at the concentration required effective for iav inhibition, drugs targeting mrna translation for various diseases have been approved by fda or are under active development (81) . as such, inhibition of iav infections by disrupting mrna translation may well be a therapeutic approach in the future. post-translational modifications during protein maturation ensure proper function of proteins, with proteins of iav no exception. nitazoxanide, a fda-licensed drug used to treat enteritis, was found to be effective in controlling iav infection by interfering with ha n-glycosylation as well as intracellular trafficking in host cell and eventually led to a reduction in viral budding (82) . despite the mechanism of nitazoxanide being presently unknown, its ability to inhibit replication of numerous viruses [iav, respiratory syncytial virus, coronavirus, hepatitis b virus, and many others (83) ] suggests that it may act on host machinery. the drug has also been proven in vitro to inhibit the propagation of many circulating strains of human iav, including those resistant to oseltamivir or zanamivir (84) . nitazoxanide has a high barrier of resistance to iav (83) and other viral strains resistance to neuraminidase inhibitors (85), making it a very promising therapeutic target for iav treatment. the drug is currently under phase iii clinical trials (83). in the later stage of viral replication, viral rnas of iav packed with rdrp and np (known as vrnp complexes) are exported from the nucleus (59), assembled (86) , and transported to the plasma membrane [apical in polarized cells (87) ] for budding. novel targets for influenza treatment frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1547 exportin 1 (xpo1, also known as crm1) is well known for its function in the nuclear export of protein (88) and rna, including viral rna (89) . similar to hiv (89, 90) , iav viral rna does not directly bind to xpo1 but is instead held together by several viral proteins. the viral nuclear export protein (nep, or previously known as ns2) and the vrnp complex have been proposed as the nuclear export complex (91) . cellular xpo1 has been proven to be crucial in the nuclear export of the vrnp complex, with early studies using leptomycin b (lmb), a potent xpo1 inhibitor, revealing that in vitro inhibition of xpo1 led to nuclear retention of vrnp complex (92, 93) . however, lmb was deemed unsuitable for development as a potential drug in the phase i clinical trial due to observed cytotoxic effects (94) . verdinexor (also known as kpt-355) is a new bioavailable selective inhibitor of xpo1. it has been shown to be effective against different strains of iavs both in vitro and in vivo as prophylactic and therapeutic treatments (95, 96) . it is worth mentioning that delayed administration of verdinexor at day 4 post-infection was still deemed beneficial, with reduced viral load in vivo (96) . this suggests a prolonged therapeutic time window when compared to the mainstay antiviral drugs such as oseltamivir, where recommended administration is at the early stage of infection (within 48 h of symptom onset) (97) . currently, verdinexor has passed the phase i clinical study trials, suggesting that it does not pose severe cytotoxic effects as lmb does. in addition, a recent report demonstrated that a new drug, dp2392-e10, which binds and inhibits the function of xpo1, can suppress iav replication in vitro (98) further strengthens the concept of iav intervention by targeting xpo1. viral m1 protein is crucial in assisting the nuclear export of vrnp complex. it was commonly suggested that m1 protein links vrnp complex to viral nuclear export protein nep which interacts with xpo1 for nuclear export (59) . thus, viral m1 protein may serve as a target to inhibit nuclear export of vrnp. as previously mentioned (see inhibition of mrna export), csa inhibits iav replication via both cypa-dependent and -independent mechanisms. a recent study using a transgenic mice overexpressing cypa showed greater resistance to iav challenge (99) . in the cypa-dependent mechanism, csa enhances the binding of cypa to m1 protein (72) , increases the self-association of m1, and hinders m1 nuclear import (100) . csa also promotes the cypa-dependent degradation of viral m1 protein (72, 101) . csa seems to be a promising drug to inhibit the nuclear export of vrnp complex by inhibiting viral m1 protein stability and function. recently, cd151, a tetraspanin (defined by four transmembrane domains with conserved residues) that is expressed abundantly in lungs and interacts with integrins has been implicated in the regulation of iav replication in vitro and in vivo (102) . knockdown of cd151 in primary human nasal epithelial cells resulted in the nuclear retention of host xpo1, viral np, nep, and m1 proteins, with an increased survival rate observed in iavinfected cd151 knockout mice. co-immunoprecipitation assays suggest that cd151 interacts with viral np, m1, and nep proteins (102) ; however, the exact domains involved in interaction and the mechanism of cd151 function in nuclear export remain unclear. given that a small molecule inhibitor for cd151 is now under development (103) , more data revealing the role of cd151 in iav infection and subsequent use in targeting cd151 as anti-iav therapy is anticipated. during iav infection, raf/mek/erk signaling cascade is activated, while the inhibition of mek by u0126, probably mediated via myosin (light chain) (104), a known motor protein, impairs the nuclear export of vrnp complexes (105) . suppressing iav replication by inhibition of raf/mek/erk signaling cascade has been illustrated both in vivo (106) and in vitro (105) . the replication of ibv (107) as well as borna disease virus (108) was shown to be inhibited by u0126, suggesting the versatility of this approach in controlling infection by different viruses. despite being effective when administered locally to lungs via aerosol, u0126 has little effect when administered orally (106) . another mek inhibitor, ci-1040 (also known as pd184352) was shown to have high potency against iav in vitro (106). ci-1040 has completed phase ii clinical trials as an anti-tumor drug, with the application of ci-1040 as a potential anti-iav drug candidate recently revisited. unlike u0126, ci-1040 is orally bioavailable and oral administration of ci-1040 at 48 h post-infection protected 60% of the iav-infected mice, while the oseltamivir-treated group experienced a 100% death rate (109) . oseltamivir is known to be effective only when administered in the early stages of iav infection. this suggests the potential use of ci-1040 as an agent used in iav treatment due to its potentially longer therapeutic time window than mainstay antivirals. formyl peptide receptor 2 (fpr2) located at the host cell surface was identified as an erk stimulator (110) . antagonizing fpr2 promoted the survival of iav-infected mice (110) . furthermore, fpr2 antagonists have been described to possess antiviral activity against not only iav but also ibv infection (111) , promoting the idea that antagonizing fpr2 to suppress raf/mek/erk signaling cascade could potentially be a novel approach for the treatment of a broad spectrum of influenza viruses. after the nuclear export of the vrnp complexes, host cell's intracellular transport mechanism is required to deliver vrnp complexes to the host plasma membrane for the assembly of viral rnas and proteins at the final stage of viral replication. among the various vesicular compartments found in a cell, the rab11a + endosomes are known to recycle endocytosed membrane proteins and lipids to the plasma membrane for membrane homeostasis (112) , a property utilized by many rna viruses, including iav (87, (113) (114) (115) . iav progeny virus production was found to be significantly reduced in rab11a + knockdown human cell lines (116) . furthermore, vrnp complex plasma membrane transport perturbation was observed in rab11a knockdown cells (114, 115) ; in cells expressing deletion mutant of rab11 family interacting proteins (87) ; as well as cells treated with chemicals to interfere microtubule (114) . direct interaction of vrnp complex with rab11a has also been verified (114, 115) , demonstrating the dependence of vrna complex transport on rab11a + vesicles and the microtubule network during viral replication. since rab11a proteins do not confer any mobile properties to the vesicle, molecular motors such as kinesins are required for the active transportation of vesicles through cytoskeletons. kif13a, a kinesin-3 family member, was recently identified as a molecular motor for plasma membrane transportation of vrnp-loaded rab11a + vesicles (117) . kif13a knockdown was found to reduce progeny virus production. overexpression of a mutant form of kif13a lacking in motor capacity resulted in disruption of the plasma membrane distribution of vrnp complex during later stages of infection. this data suggest that the apical transport of viral components via rab11a or kif13a could potentially serve as therapeutic targets against iav infection. further examination is merited. tubulin acetylation and deacetylation affects microtubule stability (118) . histone deacetylase 6 (hdac6) was found to deacetylate α-tubulin, one of the subunits of microtubule (119) . a study has demonstrated that hdac6 is involved in iav replication (120) . inhibition of hdac6 by tubacin or knockdown of hdac6 gene resulted in an increase of progeny virus production with vrnp complex redistributed toward the periphery of infected cells. in addition, transportation of ha to the plasma membrane for viral budding was also found to be inhibited by hdac6. this data suggests that activation of hdac6 by its stimulant could be a potential approach to anti-iav therapy, despite hdac6 stimulants still being under development. while several studies have suggested iav transmission between cells through apical membranes (121) and intercellular connections (122) , virus budding from cell membranes remains the major route for transmission of viruses to uninfected cells. na is responsible for the cleavage of sialic acid to prevent the interaction between ha and the host cell during viral budding. besides, viral na, viral ha, m1 as well as m2, are also suggested to play an important role in the initiation of the budding process (123, 124) . in section "controlling cholesterol homeostasis, " we discussed the involvement of host cholesterol in viral membrane fusion and viral genome release to cytoplasm. recent studies have demonstrated that host cholesterol may also play an important role in viral budding. it was demonstrated that overexpression of annexin a6 (anxa6), a phospholipid binding protein, could lead to a decrease in cholesterol levels within the golgi apparatus and plasma membrane (55), ultimately causing a reduction in egression of progeny virion from infected cells (54) . this reduction could be reversed by the addition of exogenous cholesterol (55) . similar to anxa6 overexpression, addition of a hydrophobic polyamine, u18666a, could reduce cholesterol level in plasma membr ane, also inhibited viral replication (55) . since iav is assumed to bud from lipid rafts (cholesterol-rich plasma membrane domains) (123) , it was demonstrated that anxa6 overexpression or u18666a treatment could hinder progeny virus production by lowering the cholesterol content in the plasma membrane. this hypothesis was strengthened through recent studies resolving the cholesterol-binding site of viral m2 protein, suggesting that iav m2 clustering (which provides membrane curvature for scission) is mediated by cholesterol (125) . a recent report utilizing two different fda-approved cholesterol-lowering drugs, gemfibrozil and lovastatin, stated that there was reduction in stability and infectivity of progeny virus compared to that replicating within cholesterol-sufficient host cells (126) . taken together, this data suggests that controlling cellular cholesterol content would be an effective alternative with drugs available for repurposing iav treatment. further in vivo works are needed to confirm this hypothesis. the gi-type g-protein coupled receptor α2-adrenergic receptors (α2-ars) have been recently identified as a key host factor involved in iav replication (127) . apical transport of the viral protein ha is inhibited by low intracellular camp level after stimulating the α2-ar-mediated signaling. in vitro stimulation of α2-ar by its agonist clonidine inhibits iav replication. therapeutic administration of clonidine reduced pulmonary edema and improved survival rate of iav-infected mice. development of a new antiviral targeting the α2-ar-mediated signaling seems promising and deserves further investigation. although targeting host factors for viral interventions generally provides a better resistance barrier, emergence of resistance may still arise (61) . therefore, combined use of interventions targeting both virus and host factors have been recommended to reduce opportunities for viral development of resistance. one such example would be the combined administration of na inhibitor (oseltamivir) alongside an anti-host factor [such as v-atpase inhibitor diphyllin (46) , ha maturation inhibitor nitazoxanide (85) , fpr2 antagonists (111) , and xpo1 inhibitor verdinexor (96) ]. while further direct assessment for the ease of emergence of escape mutants between single and combinatory use of drugs is required, the synergistic effects of a combined, multi-drug approach observed thus far highly suggest an increased effectiveness over a single-drug approach. table 1 summarizes novel host targets regulating iav replication. compared to rnai, small molecular chemicals remain the best choice as drug candidates due to their fast acting and easy-todeliver properties. although small molecular chemicals targeting certain host factors aforementioned have yet to be developed, their rnai-identified involvement in the iav replication cycle provide leads for the development of new iav interventions. the immune system aims to protect the host from infection and clear the pathogen once an infection occurs. in addition, the complex networks formed between the host physiology and the immune system co-operatively shape the disease outcome; modulations on the networks could alleviate disease severity in iav infections. the immunological responses elicited by iav infection has been reviewed in detail (128) (129) (130) . at the initial stage of iav infection, the respiratory epithelial cells are the primary target for infection. once the infection is initiated, the recognition of infection is accomplished via the detection of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) (see toll-like receptors), and lead to the expression and secretion of different cytokines and chemokines, such as il-6, il-8, tumor necrosis factor (tnf)-α, and ccl2 as well as type i and iii ifns. as sentinel cells, alveolar macrophages could also be infected, inducing cytokines and is the main source of type i ifns (128, 129) . type i ifns are known inducer for the upregulation of death receptor 5, which is the receptor for tnfrelated apoptosis-inducing ligand (trail), in lung pneumocytes (128) . il-8 and ccl2 produced by both epithelial cells and macrophages act as chemoattractants for neutrophils and monocytes, respectively. neutrophils are one of the earliest immune cells being recruited to the site of infection (131) with transmigration of neutrophils carry out by adhesion molecules, such as cd11a, cd11b, and cd18 (132) . in addition to the antiviral activity of neutrophil-released reactive oxygen species (ros), defensin and pentraxin (132) , uptaking iav by neutrophils could also help in controlling viral propagation as these cells do not support replication of iav (133) . besides controlling viral replication, neutrophils also play an important role in guiding the migration of iav-specific cd8 + t-cells in the infection site by secreting and leaving a trail of cxcl12 (134) . infiltrated monocytes will, however, differentiate into macrophages or dendritic cells (dcs). the monocytes-derived macrophages are reported to be a permissive host for iav production (135) , sustaining inflammation by producing cytokines in a magnitude larger than that of the resident alveolar macrophages. the monocyte-derived dc as well as the resident airway cd11c low b220 + plasmacytoid dc (pdc) and two types of conventional dcs (cd103 + cd11b low and cd103 − cd11b hi ) acquire the antigen of the invading pathogen through either direct infection or up-taking infected dead cells (129) . in the presence of type i ifns, dcs mature when encountering pamps from invading pathogen (129) . depending on the sub-cellular localization of the antigen, cytosolic and endosomal antigen will be loaded onto major histocompatibility complex (mhc) class i and ii molecules respectively (130) . once mature, dcs migrate from the infection site to the draining lymph nodes via the interaction of ccr7 and ccl19/ccl21 (130, 136) for antigen presentation via mhc class i and ii to naïve cd8 + and cd4 + t-cells, respectively (137) (138) (139) (140) . interestingly, monocytesderived dcs that engulfed the infected dead cells are poor antigen presenters for cd8 + t-cells and require the transfer of intact mhc class i/peptide complex to lymph node-resident cd8α + dcs which are the most efficient antigen-presenting cells to cd8 + t-cells (137) . in addition to antigen presentation, pdc are well known for their high ability in type i ifns production to limit viral propagation (141) . within the lymph node, naïve cd8 + t-cells are activated by the dcs, differentiate and clonal expand into cytotoxic t-lymphocytes (ctls) with the aid of various cytokines, including ifn-γ, il-12, type i ifns, and il2 (142, 143) , and the help from activated cd4 + t helper cells (144). differentiated ctls downregulate their lymph node homing receptor ccr7 and upregulate ccr4 and cxcr3 for the migration to the site of infection. within the site of infection, ctls control viral replication by targeting and inducing apoptosis of virus-infected cells via the secretion of perforin and granzymes as well as the ligation of death receptors on the infected cells by tnf, fas ligand, and trail. on the other hand, cd4 + t-cells are activated by the presentation of mhc class ii/ antigen complex by dcs, with co-stimulatory receptors such as cd28 expressed on the t-cells and the ligand for cd28 (cd80 and cd86) expressed on dcs playing an important role (144). activation of cd4 + t-cells lead to differentiation into different effector cells subsets, including the classical th1 and th2, and the more recently identified regulatory t cells, follicular t helper cells, th9, and th17 subsets (144). th1 cells regulate to the differentiation of ctls as mentioned whereas th2 cells contributes to the activation of b-cells through cd40l. within the pregerminal center of the lymph node, the follicular t helper cells interact with antigen-primed b-cells and promote their proliferation. antigen-primed b-cells differentiates into plasmablast and undergo antibody class-switching in the germinal center (145) . detailed functions of regulatory t cells, follicular t cells, th9, and th17 cells are discussed elsewhere (144, 145). plasmablasts enter the blood-stream, are recruited to the inflamed tissue, and terminally differentiate into plasma b cells which specialize in the production of antibody for pathogen neutralization, opsonization, and antibody-dependent cell-mediated cytotoxicity, etc. memory t-and b-cells are also developed during the maturation process, and has been discussed and reviewed elsewhere (146) (147) (148) (149) . a schematic diagram showing a summary of the immune response after iav infection has been illustrated in figure 1 . the yin and yang theory is always used to describe the importance in balancing the host immune response. in the light of this theory, the treatment strategy aims to suppress the overwhelming activation of the host immune response and in reverse to compensate any unfavorable suppression. although adaptive immune responses are important in viral clearance, the immediate innate immunity play an important role in the early control of an infection, and conversely, is a major factor for disease severity due to immunopathology. dysregulated immune responses caused by viral infections have been implicated in severe disease development (150, 151) , such as acute lung injury (ali). ali in its most severe form, known as acute respiratory distress syndrome (ards), is reported to be the most prevalent cause of mortality in iav-infected patients (152) . studies suggested that iav strains could be associated with either over-activating (human infection by avian h5n1 and h7n9) (153, 154) or suppressing (h1n1, h3n2) (155) immune response. recent history has seen the outbreak of iav pandemics of varying severity takes place at the cost of millions of lives. one such example would be the deadly spanish flu of 1918, which claimed the lives of 20-50 million of the 500 million people infected worldwide. the pathological examination of lung sections from mice infected with reconstituted 1918 iav virus revealed necrotizing bronchiolitis and severe alveolitis in tissue, with neutrophils observed as the predominant inflammatory cell type present (156) , suggesting neutrophil involvement in the pathogenesis of iav infection. the majority of immune cells in blood circulation are neutrophils; of which they are among the first innate immune cells recruited to the site of infection (131) . neutrophils characteristically control microbial infections by generating bactericidal (157) neutrophil extracellular traps (nets), consisting of granule proteins, histones, and decondensed chromatin (131) . both protective and destructive role of neutrophils in iav infections have been described. the contrasting role of neutrophils could be explained by factors such as viral strain and viral dose used in different experimental setup, etc. the protective role of neutrophils was observed when mice infected with a low, non-lethal dose of iav h3n2 strain hkx31 displayed neutrophil-mediated viral clearance via phagocytosis (132, 158) . depletion of neutrophils has found to enhance viral load in the iav-infected animals (158) . on the contrary, this protective nature is disputed due to the association of neutrophil-generated nets. extensive net formation was observed in mice infected with pr8, an iav strain highly pathogenic to mice (159) . histones and myeloperoxidase within the net induce cell death of lung epithelium and endothelium (157) , leading to the loss of integrity of the alveolarcapillary barrier, a characteristic of ali. yet, while histones have been shown to suppress iav replication in vitro (160) , in vivo study demonstrated that there was increase in lung inflammation and damage in iav-infected mice treated with histones (161) . interestingly, co-treatment of lethally infected mice with anti-histone antibody and oseltamivir resulted in an increase in animal survival when compared to infected mice groups treated solely with oseltamivir (161) . in agreement with the in vitro and in vivo data, it has been reported that net produced by cultured neutrophils from patient with h7n9 and severe h1n1 infection increased alveolar epithelial cell permeability (162) leading to ali. more importantly, plasma net level positively correlated with the disease severity index (including higher acute physiology and chronic health evaluation ii score) and multiple organ dysfunction syndrome (162) , further demonstrating the detrimental role of net in the pathogenesis of severe iav infections. studies have demonstrated the involvement of superoxide dismutase and myeloperoxidase in netosis, the formation of net (159) . the presence of anti-myeloperoxidase antibody as well as the superoxide dismutase inhibitor (detc) significantly reduced netosis. finally, tetrahydroisoquinolines (163) and a panpeptidylarginine deiminase (pad) inhibitor, named cl-amidine (164) have been suggested to inhibit netosis. despite it has been reported that during iav h1n1 infection, pad4 knockout mice displayed only slight improvement in weight loss and a slight prolonged but no end-point survival advantage was observed compared to wt mice (165) , based on the extensive findings presented above, targeting net to prevent ali in the severe case of iav infection, including the highly pathogenic avian iav, remain promising and may warrant further investigation. innate lymphoid cells are cells of lymphoid lineages that do not express antigen-specific b-or t-cell receptors (166) . similar to t-helper cells, they are classified into subsets by their ability to produce type 1 (th1), type 2 (th2), and type 3 (th17 and th22) cytokines. previous studies confirmed the involvement of ilcs of group 2 linage (ilc2) in iav infection and airway inflammation (166, 167) . on the positive side, during the recovery phase of iav infection, ilc2 expresses amphiregulin which promote airway epithelium repair (166, 168) , thus facilitating the recovery of the infected lung. on the other hand, in response to il-33 produced by macrophages, dcs, and nkt cells, ilc2 secretes il-5 and il-13 and induce airway hyper-responsiveness. recruitment of eosinophils by il-5 to the lung also mediates airway inflammation (166) . since eosinophilia is a characteristic of allergic asthma and influenza is a major cause for morbidity and mortality in asthma patients (166) , it will be of particular interest to investigate the role of ilc2 in iav infection, particularly in asthma patients. ilc1s have been initially described as immature nk cells residing in the liver and share many phenotypic similarities with nk cells (169) . it was recently appreciated that tissue-resident ilc1s other than the previously recognized nk cells are the major early source of the antiviral ifn-γ at the primary site of various viral infection, including iav (170) . interestingly, ifn-γ was found to suppress ilc2 activity and reduce il5 production which exacerbates disease severity during influenza a(h1n1) pdm09 infection (171) . this data may highlight a link between ilc1 and ilc2 and suggesting ilc1 can suppress ilc2 activity via ifn-γ production during iav infection. with ilcs finally identified, functions of these cells and their role in immune response to tumors and pathogen infections have been massively investigated in recent years. type i ifns, prostaglandin i2, corticosteroids, and testosterone have been reported to suppress ilc2 activity (172, 173) . in addition to il-33, the epithelial cytokines il-25, thymic stromal lymphopoietin, as well as the lipid mediator prostaglandin d2 were found to activate ilc2 (173) . the therapeutic potential of these ilc2 activators and suppressors is yet to be deduced. with more and more studies demonstrating the involvement of ilc in iav infection, the interplay between different ilc subtypes in iav infection would, therefore, be an interesting area to explore and modulate the ilc activity may be a future approach to combat iav infection. reactive oxygen species, generated by specialized enzymes such as nadph oxidases, are released during iav infection (174) . the nadph oxidase family consists of enzymes containing different catalytic subunit named nox1-5 and dual oxidase (duox) 1 and 2. ros have been reported to display both beneficial (limiting viral replication) and detrimental (promoting ali) effects in the course of iav infection. interestingly, the protective or destructive effect of ros is dependent on the enzyme of which the ros is generated (174) . dual oxidase1 and 2 are found to be host-protective (174, 175) . in vitro, ros generated by nuclear duox indirectly regulates the splicing of iav mrnas via the nuclear speckle-associated splicing complex (175) . in addition to altering viral mrna splicing, ros generated by doux2 has been attributed to the production of ifn-λ, an important anti-iav ifn. in response to iav infection, increased viral mrna replication was observed when duox2 was silenced in vitro (176) . increased viral replication was also observed in mice with doux silenced (175) , further depicting the protective role of doux in iav infection. unlike doux, nox2 activation could be harmful to host. iav infection was reported to induce nox2-dependent endosomal ros production (177) . ros could target the conserved cys98 on toll-like receptor (tlr) 7, and inhibit tlr7-mediated type i ifn expression during a mild iav h3n2 infection in vivo (177) . iav-infected mice treated with specific nox2 inhibitor, cholestanol-conjugated gp91ds-tat, were found to have reduction in endosomal ros production, restored tlr7 activity, and displayed a decreased viral load (177) . in addition to nox2, nox4dependent ros production has also been reported to activate mapk/erk signaling (178) , enhancing the export of vrnp complex, thus increasing viral replication (see targeting the raf/ mek/erk pathway). nox4 knockdown resulted in a reduction of viral replication in vitro (178) . targeting the different nadph oxidase isoforms, instead of scavenging ros should be considered as the therapeutic approach for iav infection, as doux-mediated ros production is beneficial (175, 176) , while nox2 and nox4 are harmful during iav infections (177, 178) . finally, ns1 (not to be confused with iav ns1 protein) has been demonstrated to be a nox inhibitor, which could inhibit the activity of nox1, nox2, and nox4. a study demonstrated that ns1 suppresses iav-induced nox2 and significantly inhibits iav virus replication (179) . besides cholestanolconjugated gp91ds-tat and ns1 aforementioned, apocynin, a phagocytic nox2 inhibitor as well as ros scavenger (180) (181) (182) , has been demonstrated to ameliorate hyper upregulation of cytokines induced by iav infection through socs1 and socs3 in vitro (154) and reduce peri-bronchial inflammation and viral titer in vivo (183) . interestingly, ebselen, another nox2 inhibitor and glutathione peroxidase mimetic, could reduce inflammatory status measured in bronchoalveolar lavage fluid (balf) of mice pre-exposed to cigarette smoke and subsequently infected with iav (184) . taken together, these reports highlight the potential use of nadph oxidases inhibitors and ros scavengers to treat iav infections. dysregulated cytokine production has been associated with the elevated mortality rate observed in severe iav infections (185, 186) . as such, the immunomodulation of cytokines are regarded as promising therapeutic tactics. recent advancements developed with this approach will be highlighted in the following section. tumor necrosis factor has two main functions during viral infection-it activates nf-κb, inducing the expression of cytokines responsible for the host immune response; and induces apoptosis through activation of a signaling cascade involving tradd, fadd, and caspase 3, 7, 8, and 10 (187) (188) (189) . tnf is known to be highly upregulated in iav-infected hosts, especially in hosts infected with highly pathogenic iav (153, 190) . however, it is both protective and counter-protective functions associated with tnf that makes it a target in the treatment of iav. the protective role of tnf is observed during infection by low pathogenic iav, where extrinsically derived tnf is responsible for attenuating tissue-damaging cd8 + t-cell response (191) . in addition to recruiting monocytic cells to the infection site, cd8 + t-cells response was observed to deteriorate lung pathology (192) and damage healthy, non-infected lung epithelial cells (193) upon iav infection. furthermore, tnf deficiency has been associated with an increased detection of il-15 and il-6 in balf (192) , which promote the survival of and proliferation of cd8 + t-cells (194, 195) and subsequent tissue damage. exacerbated lung pathology caused by the upregulation of the monocyte chemoattractant protein-1 was observed in tnf −/− mice infected with sub-lethal dose of iav (196) . in addition, decreased cd8 + t-cell contraction due to enhanced expression of the anti-apoptotic protein bcl-2 was observed in sub-lethally iav-infected tnfdeficient mice when compared to wt mice (192) . as a whole, there is substantial evidence supporting the protective role of tnf in iav infection. on the other hand, the correlation of tnf with pulmonary edema has been well-documented (197) . tnf has been observed to stimulate the expression of cxcl2 in alveolar epithelial cells in a transgenic mice model resembling extensive iav infection in lung tissue, causing alveolar damage, lung edema, and hemorrhage (198) . in addition to lung edema, tnf has also been reported to correlate with iav-associated encephalopathy (199, 200) . however, it is notable that despite iav-associated encephalopathy, direct invasion of the central nervous system is rare (201) , suggesting that iav-associated encephalopathy could instead be a result of peripheral infection. furthermore, tnf has been shown to increase the permeability of the blood-brain barrier (bbb) (202, 203) , contributing to neural damage (204) . these studies further support an anti-tnf approach as a potential therapy for severe iav infection. at present, etanercept, an anti-tnf drug administered in the treatment of rheumatoid arthritis, is the only tnf inhibitor (or even tnf directed treatment) tested for iav treatment. etanercept has been shown to protect against the in vivo lethal infection of mice with a highly virulent, mouse-adapted iav strain (205) , with observations made of an increased survival rate with decreased morbidity, expression of the proinflammatory cytokine il-6, lung injury, and edema (205) . the protective role of il-6 was demonstrated in mice challenged with sub-lethal iav infection. il-6-deficient mice displayed exacerbated pulmonary damage (206, 207) and lung injury due to an observed decline in the survival of alveolar type ii cells and alveolar epithelial cells (207) . iav suppresses the anti-apoptotic mcl-1 and bcl-xl expression, causing cell death of neutrophils which are critical in viral clearance (206) . addition of il-6 restored the expression of mcl-1 and bcl-xl in vitro and is considered as the underlying mechanism for the observed survival advantage of wt mice over il-6 knockout mice during mild iav infection. il-6 has also been shown to induce the proliferation of lung il-10 + regulatory t cells and il-27, which act to limit excessive proliferation of cd8 + t-cells and subsequent cd8 + -inflicted damage. this would hence prevent the tissue damage observed in lung immunopathology (208) . despite the apparent protective role of il-6, high levels of il-6 in serum or cerebrospinal fluid have been reported in severe neurologically complicated iav cases, with il-6 used as a marker for prognosis (199-201, 209, 210) . the role of il-6 in regulation of bbb permeability was reported (211) , with potentially detrimental neurological complications. as such, the suppression of hyper-induced il-6 as a form of therapy in severe iav infection should be considered. one such option is the anti-il6 antibodybased drug tocilizumab, which is currently administered clinically for the treatment of rheumatoid arthritis. however, study on the usage of this drug to treat hyper upregulation of il-6 due to severe iav infection has yet to be conducted. on the other hand, in a case of h1n1 virus-induced ards, the use of an extracorporeal cytokine hemoadsorption device to remove cytokines including tnf and il-6 from the bloodstream (212) has showed beneficial to the patient (213) . more research is required to confirm whether the removal or neutralization of il-6 could be a potential therapy for severe iav infections. the activation of cd8 + t-cell is crucial for viral clearance. it should, however, be tightly regulated to limit cd8 + t-cell inflicted host cell damage. il-6 mediates il-27 induction (208) . il-27 acts to suppress cd8 + t-cells and reduce morbidity through il-10 and regulatory t-cells (208) . much like other immunomodulatory approaches, the timing for applying il-27 should be carefully assessed. compared to placebo-treated iavinfected group, early administration of il-27 to iav-infected mice in fact led to poorer viral clearance, increased morbidity, and deteriorated lung histopathology, while il-27 administration during the recovery phase (5-10 days post-infection) accelerated recovery and improve lung immunopathology (214) . notably, il-27 could also suppress th17 responses and increases susceptibility to secondary s. aureus infection (215) . therefore, co-administration of antibiotics should be considered when utilizing il-27 as potential iav treatment. both type i and iii ifns have antiviral properties, with viruses counteract ifns to gain an advantage for their propagation. the iav viral protein ns1 inhibits the production of ifns by antagonizing irf-3, a key transcriptional factor for ifns. this prevents the processing of cellular pre-mrnas (including those for ifns) and directly interacts with retinoic acid-inducible gene (rig)-i receptors, which are critical in innate sensing, to suppress ifn production during infection (216, 217) . in addition to inhibiting ifn expression, the induction of socs3 inhibits ifns signaling by suppressing cytokine signaling has been documented (155) . the recognition of 5′ triphosphate on viral rna by rig-i receptor is shown to induce the expression of socs3, which in turn represses type i ifns expression (155) . due to ifns being a key contributor to antiviral immune response, an impairment of type i or iii ifn production may cause the escalation of otherwise mildly pathogenic iav infection into a life-threatening one (218) . while type i ifn has been demonstrated to inhibit iav replication in vitro (219) ; the in vivo administration of type i ifn in animal models only displayed effectiveness in a prophylactic capacity. a lowered viral titer was detected in the nasal wash of test animals. however, host susceptibility to iav infection remained unchanged (219) . notably, this protective effect is only conferred by an optimal dose of type i ifn of low to moderate amounts (10-100 units per mice daily); with higher dosages (1,000-10,000 units per mice daily) shown to increase morbidity (220) . in addition, clinical trials demonstrated that prophylac tic administration of type i ifn reduced disease severity and lowered susceptibility to iav in males and participants aged 50 or above (221) . despite relatively successful results seen in the prophylactic use of ifns, its therapeutic use is of greater clinical relevance. mice treated with type i ifn post-iav infection showed a successful reduction in lung iav titer but displayed increased morbidity and mortality in comparison to vehicle-treated mice (222) . a possible explanation for this phenomenon is the induction of excessive inflammatory response and trail-dr5-mediated epithelial cell death by type i ifn (223) , which accounts for the observed lung pathology in iav-infected animals treated with type i ifn (224) . in addition, downregulation of γδ t-cells by type i ifn has been correlated with increased susceptibility to secondary s. pneumoniae infection (225) , further arguing against the potential use of type i ifns for the treatment of iav infection. in comparison to type i ifns, the administration of type iii ifns may provide advantages in the control of iav replication (176, 222, 224) without the risk of previously reported type i ifns-mediated immunopathologic side-effects (222, 224, 226) . however, a recent study aiming to stimulate ifns signaling through the systematic administration of rig-i ligand post-iav infection demonstrated that type i, but not type iii ifns signaling is important in conferring protection during fatal iav infection in vivo (227) . though, this study did not measure the production of type i and iii ifns as well as any changes in viral load with respect to ifnar or ifnlr knockout. in addition, while human immune cells are not primary targets in iav infection, they could be susceptible to iav and become efficient host cells for virus replication. they are reported to possess a subpar response to type iii ifns (222) ; leading to the preliminary conclusion that solely using type iii ifn as treatment may not be feasible. as such, reports suggesting the use of type iii ifns over type i ifns as a front-line therapeutic agent to counter iav infections may require further investigation. the inhibition of cox-2 by selective inhibitors, nimesulide and celecoxib, was previously demonstrated to suppress the hyper upregulation of pro-inflammatory cytokines induced by highly pathogenic avian iav (228) (229) (230) . in addition, the use of zanamivir in tandem with a specific cox-2 inhibitor was shown to increase the survival rate of mice lethally infected with avian h7n9 iav, when compared to mice treated solely with zanamivir (229) . activated cox-2 regulates downstream prostaglandin production. one such example is pge2, a major type of prostaglandin recently demonstrated to play an important role during iav infection. pge2 was significantly upregulated in response to iav infection, leading to the inhibition of antiviral type i ifn production in macrophages and the subsequent increase in virus replication (231) . the use of chemicals ah6809 and gw627368x to antagonize pge2 downstream signaling molecules ep2 and ep4 respectively, was shown to induce antiviral type i ifn production. the in vivo treatment of mice lethally challenged iav with both ep2 and ep4 antagonists significantly improved the survival rate. a recent study demonstrated the ability of a modified tcm decoction to reduce peg2 production and subsequent morbidity in mice lethally challenged with iav. improved lung pathology was observed (232) . the long history of clinical tcm use supports the clinical feasibility of peg2 inhibition as an option to treat severe iav infections. pattern recognition receptors on host cells sense specific pamps present on the viral surface or generated during replication. prrs can be broadly divided into two classes by their function or location. when defined by location, prrs are classified into 3 groups-membrane-bound (tlrs and c-type lectin receptors), cytosolic (rig-i-like and nod-like receptors), and secreted (collectins and pentraxins) (233) . significant research has been conducted on prrs with regards to iav infection. tlrs and rig-i receptors have been extensively studied for their major roles in eliciting host immune responses (cytokine and ifn expression) during iav infection (234) (235) (236) . rig-i receptors have been investigated for their functional relevance to iav infection and targeting these receptors as a form of iav treatment has been extensively reviewed (237) (238) (239) . this section will cover recent research on tlrs and the targeting of different tlrs to treat iav infection. humans have been identified to express tlr1-10, while mice have been identified to express functional tlr1-9 as well as tlr11-13 (240) . most tlrs-with the exception of tlr3utilize myd88 as an adaptor protein during signal transduction. tlr3 utilizes trif as an adaptor. tlr4 is known for its ability to utilize either myd88 or trif, with the choice of adaptor dependent on its sub-cellular location (241) . different tlrs, such as tlr3, 7, and 8 (240) as well as tlr2, tlr4, and most recently tlr10 (235) , have been revealed to play a role in the orchestration of host immune responses contributing to iav pathogenesis. with tlr10 being an exception (242) (243) (244) , tlr activation largely causes the release of pro-inflammatory cytokines, with hypercytokinemia leading to ali as a major cause of mortality in severe iav infections. in addition to dysregulated cytokine release, excessive production of ros has been associated with ali development. in fact, lung injury during severe pulmonary infections, such as iav and sars, could be caused by oxidative stress (245) . iav infection activates nadph oxidase that subsequently produces oxidized papc, an endogenous phospholipid. the oxidized papc serves as an agonist for tlr4, activating a tlr4-trif-traf6-nf-κb signaling cascade to eventually trigger the release of il-6, ultimately inducing the onset of ali. in addition to oxidized papc, the induction of endogenous protein s100a9 upon intracellular prr ddx21 recognition of iav subsequently induces the activation of tlr4, further contributing to iav-induced mortality (246) . since tlr4 has been proven to be important in ali induction (and hence iav-related mortality), manipulating the stimulation and antagonism of tlr4 could potentially reduce the severity of iav infections. eritoran (e5564) is a specific tlr4 antagonist initially purposed for the treatment of sepsis, but a failed a phase iii clinical trial due to improved patient care in the placebo group prevented its eventual use in sepsis treatment (247) . in vivo administration of eritoran in mice lethally infected with iav resulted in improved clinical score, lung pathology results, and reduced viral titer. delayed administration of eritoran, at day 6 after infection beyond the recommended therapeutic time window (within 48 h after the first display of clinical symptom) for use of oseltamivir (248), also demonstrated a significant benefit to infected mice compared to non-treated group, suggesting a prolonged therapeutic time window for iav treatment when compared to mainstay antiviral drug treatment. a newer and structurally simpler specific tlr4 antagonist, fp7 (249), alongside a newly developed decoy peptide 2r9 that has been shown to disrupt tlr2, 4, 7, and 9 signaling via tirap, has been shown to protect mice from lethal iav infection (250) . these results support the potential use of tlr4 antagonism as a means to treat severe iav infection. the suppression of other tlr signaling pathways-such as blocking tlr2-mediated signaling through the use of an anti-tlr2 antibody, significantly protected against lethality when administered on day 2 and 4 post-iav infection (251) . a study also demonstrated that h5n1-infected tlr3 knockout mice had better survival than h5n1-infected wild-type mice, which is evident through the significantly faster regaining of body weight post-infection, lower viral titer in the lung, and fewer pathological changes in the lung (252) . an increasing number of tlr antagonists are now under development (253, 254) , alongside several other agents also shown to have effects on tlrs. polysaccharides isolated from r. isatidis, a traditional chinese medicinal herb used to treat iav infection, have recently been shown to inhibit pro-inflammatory cytokines such as il-6 and ccl-5 in vitro by down-regulating upstream tlr3 expression (255) . menk, an endogenous protein expressed in the adrenal medulla, was shown to both prophylactically and therapeutically increase the survival rate while reducing viral-caused lung pathology and viral titer in mice lethally challenged with iav (256) . this was determined to be caused by the downregulation of tlr7. these results suggest the potential of down-regulating tlr expression in the treatment of iav infection. the above-mentioned data suggest modulation of tlr signaling or expression as a promising approach in treating severe influenza disease and deserves immediate investigation. table 2 summarizes new immunomodulatory approaches to combat iav infections. it is well documented that patients with diabetes mellitus have a greater tendency to develop severe iav infection than healthy patients (257) . hyperglycemia increases susceptibility of the host to iav infection via viral uptake, through the promotion of v-atpase assembly (258) and immunosuppression (257) . in addition, viruses rely on host metabolism to perform essential functions during replication (259) (260) (261) (262) . these processes exert a large energy demand on the host within a very short period of time (263) ; energy of which is supplied by and is dependent on host metabolism. iav viruses have been reported to modify the metabolic state of the host. for example, increased c-myc-dependent glycolysis and glutaminolysis has been demonstrated in infected cells (264) . the changes in glucose and glutamine metabolism were reversed upon the addition of bez235, which inhibited the iav-mediated c-myc induction. administration of bez235 2 days prior to infection and up to 4 days post-infection was shown to decrease lung viral titer and improve the survival rate in iav-infected mice. small molecules such as clotrimazole and α-mangostin that target lipid metabolism have also been demonstrated to suppress iav replication in vitro (264) . in addition to being important for generating energy and biosynthesis, recent research demonstrates that cellular metabolism affects immune cell function. dysregulated immune responses observed in many diseases are associated with specific metabolic configurations. viruses, influenza inclusive (265) , were found to induce drastic alterations in metabolic levels and programs (263) . macrophages in infected hosts were observed to have marked differences in the krebs cycle, a key metabolic pathway. this is of significance due to the role of macrophages, which are immune cells critical in the pathogenesis of many inflammatory diseases (263, 265, 266) . in activated macrophages, succinate, a krebs cycle intermediate, was found to possess inflammatory signal. accumulation of succinate generates ros, leading to subsequent activation of hypoxia-inducible factor 1α and the induction of cytokines such as il-1β (267) . a recent study identified the ability of itaconate, another krebs cycle-derived metabolite, to block the production of inflammatory factors. this prevented inflammation, protecting mice from lethal levels of inflammation that can occur during infection (268) . this data suggest the critical roles of krebs cycle intermediates in regulating cytokine profiles and inflammation. metabolites generated by innate immune cells in distinct configurations could have different roles beyond that of bioenergetics, with functions in signaling regulation, transcription, and orchestrating innate immune responses. despite the lack of research conducted thus far on the application of immunometabolic approaches to influenza treatment, the prospect of manipulating immune responses by modulating immune cell metabolic state is promising. further research should focus on the identification of metabolites for modulation of immune cell function with substantial improvement of therapeutic strategies to treat iav disease. latest advancements in high-throughput technologies, e.g., meta bolomics is a useful approach to systematically investigate the changes of metabolic mechanisms during iav infections. identification of important metabolites involved during iav infection should be a new approach by modulating the host metabolism for interventions. multiple host-based intervention strategies against influenza have been developed or are under development. while approaches targeting host machinery required for virus replication seem to be promising thus far, additional research is needed to determine the effect of modulating host immune response on influenza treatment. this is increasingly important, since targeted host factors may play distinct roles in response to infection by different influenza viral strains (252) , making the management of influenza through solely targeting a single specific host factor is difficult. host-based interventions offer obvious advantages over conventional antivirals, such as a higher barrier to drug resistance (73, 83, 107) due to greater genetic stability of host factors than the mutation-prone nature of viral components. in addition, administration feasibility is a key factor to consider the usage of drugs. the mainstays of antivirals for iav infections, the na inhibitors, and m2 blockers, are recommended to be administered within 48 h of symptom onset for optimal antiviral activity. this short treatment window may not be fully fulfilled in a clinical setting. novel host-based interventions were reported to have therapeutic time windows longer than this conventional timeframe (96, 109, 214, 251) , even up to 6 days post-infection (248) , providing a clear clinical advantage over na inhibitors and m2 blockers. in addition, hypercytokinemia and ards could contribute to disease severity and mortality in instances of severe influenza infection, with virustargeting antivirals providing little to no alleviation of such complications. since host immune response is indispensable in host defense against invading pathogens, the use of immune-modulators to suppress detrimental effects while retaining beneficial protection of the host remains challenging. the timing and dosage of medication administration would be critical in determining the drug effectiveness in influenza treatment. targeting virus-induced metabolic changes to restore host normal metabolism may be a new direction to combat influenza disease. further research in the 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infection for therapeutic intervention metabolic effects of influenza virus infection in cultured animal cells: intra-and extracellular metabolite profiling krebs cycle rewired for macrophage and dendritic cell effector functions succinate is an inflammatory signal that induces il-1beta through hif-1alpha itaconate is an anti-inflammatory metabolite that activates nrf2 via alkylation of keap1 key: cord-268553-2o4k24og authors: lin, chun; chen, huanzhu; he, ping; li, yazhen; ke, changwen; jiao, xiaoyang title: etiology and characteristics of community-acquired pneumonia in an influenza epidemic period date: 2019-03-08 journal: comp immunol microbiol infect dis doi: 10.1016/j.cimid.2019.03.004 sha: doc_id: 268553 cord_uid: 2o4k24og purpose: the etiology of community-acquired pneumonia (cap) in hospital patients is often ambiguous due to the limited pathogen detection. lack of a microbiological diagnosis impairs precision treatment in cap. methods: specimens collected from the lower respiratory tract of 195 cap patients, viruses were measured by the single-plex real-time pcr assay and the conventional culture method was exploited for bacteria. results: among the 195 patients, there were 46 (23.59%) pure bacterial infections, 20 (10.26%) yeast infections, 32 (16.41%) pure viral infections, 8 (4.10%) viral-yeast co-infections, and 17 (8.72%) viral-bacterial co-infections. the two most abundant bacteria were acinetobacter baumannii and klebsiella pneumoniae, whereas the most common virus was influenza a. conclusions: non-influenza respiratory microorganisms frequently co-circulated during the epidemic peaks of influenza, which easily being ignored in cap therapy. in patients with bacterial and viral co-infections, identifying the etiologic agent is crucial for patient’s therapy. community-acquired pneumonia (cap) is a common disease and a significant cause of morbidity and mortality worldwide [1] . the severity of the disease can be affected by various factors including age, immune status of the host, and the single or mixed infection. the etiology of cap is essential because most respiratory illnesses lead to similar clinical presentations. currently, the ambiguous of etiology and lack of a microbiological diagnosis in cap impairs pathogen-directed antimicrobial therapy [2] . in the last decade, the long-term implications of respiratory viral infection among susceptible hosts have increasingly been recognized [3] . although approximately 200 million cases of viral cap occur every year, the incidence of viral pneumonia still has been underestimated. in hospital, bacteria remain the first consideration in pulmonary infections, and antibiotic therapy is still the cornerstone of the majority of cap. influenza virus infection (ivi) causes annual epidemics that result in estimated 1 billion infections, including 3-5 million severe cases and 250,000-500,000 mortality cases worldwide each year [4] . ivi can cause primary viral pneumonia, which may progress to a potentially fatal outcome [5] . it is difficult to distinguish the cause of cap induced by flu, other respiratory viruses or bacteria based solely on clinical symptoms, and the conventional lab biomarkers of viral and/or bacterial infections do not differ in influenza-positive compared with influenza-negative patients [6] . influenza can temporarily suppress host immune defenses, leading to bacterial complications [7] . secondary bacterial pneumonia is a frequent cause of excess mortality during influenza epidemics, but the epidemiology remains unclear [8] . hospital admissions during the 2009 influenza epidemic season showed a moderate to strong association between influenza and bacterial pneumonia, whereas the interaction is modest or non-existent during nonepidemic periods [9] . bacteria including streptococcus pneumoniae, hemophilia influenzae, and staphylococcus aureus, often interact with respiratory viruses (esp. the influenza virus), and shape the outcome of respiratory infection [10] . studies have shown the evidence of bacterial invasion is more than 90% in influenza-infected cases, and the high rates of co-infections exist in these patients, indicating that viral infection may enhance both susceptibility and severity of subsequent bacterial infection [8, 11, 12] . in vitro studies have shown that influenza infection enhanced susceptibility to pneumococcal pneumonia by about 100-fold in a week, but the interaction between influenza and bacteria is not limited in pneumococcal pneumonia [13] . in the hospital, viral testing among patients with respiratory symptoms is uncommon [14] , and the determination of the microbiological etiology is severely hampered in cap by the difficulty of obtaining specimens from the infected area (esp. from lower respiratory tract), samples could be easily contaminated by respiratory conditioned pathogens. at present, the epidemiological history of influenza-like illness (ili) and the incidence and clinical presentation of cap caused by viruses other than influenza during an influenza epidemic season were limited [15] [16] [17] . in this study, we report on the surveillance of respiratory microorganisms, and its laboratory biomarkers, in cap patients admitted to a hospital during the january to august 2016. the specific microbiome patterns, their clinical significance, and the antibacterial/antiviral treatment were analyzed in these patients simultaneous. we collected sputum specimens from cap patients who were admitted to the first affiliated hospital of shantou university medical college, shantou city, guangdong province in china from january to august 2016. pneumonia was diagnosed as an acute illness with fever, cough, or dyspnea / tachypnea, and at least one new focal chest sign, which was confirmed by finding lung shadowing on the chest radiographs that were likely to be new and without other obvious causes [18] . patients with cystic fibrosis or hiv infection were excluded from our study. the sputum specimens were collected and stored in a swab storage solution (copan, italy) and stored at −80°c. the nucleic acid was extracted using a beads viral dna/rna extraction kit (tianlong, china) and np968 nucleic acid extractor (tianlong, china). respiratory viruses were detected using an agpath-id™ one-step rt-pcr kit (applied biosystems, usa) and using a 7500 real-time pcr system (applied biosystems, usa). negativecontrols (depc-treated water) were included in each analytical procedure. fourteen respiratory viruses including influenza a, influenza b, parainfluenza viruses 1-3 (piv1-3), respiratory syncytial virus (rsv), coronaviruses 229e, oc43, hku1, and nl63, human metapneumovirus (mpv), human rhinovirus (hrv), bocavirus (bov), and adenovirus (adv) were measured. all the above procedures were followed the manufacturer's protocol. all sputum specimens were cultured according to china national standard protocols to detect respiratory bacteria and yeast. specimens were inoculated in blood agar, eosin methylene blue agar, and chocolate agar and incubated for 24-48 h at 37°c. colony identification was undertaken using a vitek 2 compact full automatic identification system (biomérieux, france data were analyzed by spss 19.0. categorical variables were summarized and compared by χ 2 test or fisher's exact test, the comparisons between the two groups were made by using the bonferroni test level adjustment method. continuous variables with normal distributions were used mean ± standard deviation(sd) to describe and compare using multiple variance of multiple samples. medians and interquartile ranges (iqr) were used to describe non-normal distribution of continuous variables, with comparisons based on the nonparametric kruskal-wallis h test for significance of the differences among more than two groups and the mann-whitney u test was used for significance of the differences between two groups. in table 1 , 195 patients included 130 males and 65 females. fifteen cases were children with a mean age of 0-14 yrs, and 180 cases were adults with a mean age of 60 (15-91 yrs). twenty patients (10.26%) previously had a pulmonary-associated disease, which included 6 cases of copd, 3 cases of tuberculosis, 3 cases of pulmonary heart disease, and 4 cases of emphysema. a total of 106 patients (54.36%) had at least 1 underlying disease, including high blood pressure, diabetes, heart disease, epilepsy, tumors and hepatitis. after admission, broad-spectrum antibiotics were prescribed to 113 patients (57.95%) as an empiric or definitive therapy, and 18 patients were given antiviral treatment. 39 patients had been given antiviral plus antibiotic treatment. the most frequently used antibiotic were quinolones, cephalosporins and β-lactams, and the most frequently used antiviral agents were oseltamivir, ribavirin, recombinant human interferon α-1b. unfortunately, 9 of the 195 patients died, all were adults (male: 6; female: 3), aged 55-91 yrs, with an average age of 69.89 yrs. a single-bacterial infection was found in 46 patients (23.59%), a single-yeast infection was found in 20 patients (10.26%) and a singleviral infection was found in 32 patients (16.41%). co-infections (virus + bacterium and virus+ yeast) was found in 25 patients (18.82%), and the pathogens could not be unidentified in 72 cases (36.92%) ( table 1) . bacterial infection was identified in 63 (32.31%) of the 195 patients. the two frequently detected bacteria were a. baumannii and k. pneumoniae, which accounting for 49.21% of the bacterial infection. in addition, there were 7 cases infected by p. aeruginosa and s. aureus, respectively, and 6 cases were e. coli (table 4 ). three cases had more than one bacterial infection, which was s. aureus plus k. ornithinolytica, a. baumannii plus p. aeruginosa and s. pneumoniae plus e. aerogenes, respectively. viral infection was identified in 57/195 (29.23%) of the patients. influenza a, influenza b, piv3 and rsv were the most common viruses among all cases, followed by 229e, mpv, oc43, and piv2 (table 4) . specifically, 8 patients had more than one virus, accounting for 4.10% of the virus-positive group. dual viral infection was observed in rsv coinfected with hrv, piv3 and hku1, respectively, piv2 co-infected with bov, hku1 and 229e, respectively, and influenza a co-infected with mpv and hrv, respectively. 25 patients had co-infections, including a single virus and a single bacterial/yeast co-infections in 21 cases, two viruses and a single bacterium /yeast co-infections in 4 cases. in these patients, bacterial/yeast co-infections with non-influenza viruses were more frequent than bacterial /yeast and influenza co-infections (table 5 ). fever, cough, and shortness of breath were the most common clinical signs observed in 50%, 30.43%, and 23.91% of the bacterial cases, in 75%, 78.13%, and 34.38% of the viral cases, in 60%, 35%, and 40% of the yeast cases, and 64%, 68% and 44% of the co-infections cases, respectively. the bacterial group was significantly different from the viral group and the co-infected group in cough (p＜0.0083), at the same time, the viral group and the yeast group in cough also has significant differences(p＜0.0083) ( table 2) . 4 patients with pure non-influenza infection and 4 patients with non-influenza and bacterium / yeast co-infection were admitted to the icu, and 3 patients died in the non-influenza co-infected with bacterium / yeast group (table 3) . in these patients, pharyngeal discomfort was only found in the pure influenza group. bacterium / yeast co-infected with non-influenza viruses were more common than bacterium/ yeast and influenza co-infections. wbc counts in the non-influenza groups were significantly higher than in the influenza groups, and the highest wbc counts were observed in the non-influenza and bacterium / yeast co-infected group. the plt count (p < 0.01) also had the similar trend. in the flu season, the cases of a. baumannii 's infection was greater than those in the non-flu season (15 cases vs. 1 case). it was noteworthy that bacteria such as h. influenzae, b. catarrhalis, s. maltophilia, e. cloacae, k. ornithinolytica, s. marcescens, s. haemolyticus, s. pneumonia were not detected in the non-flu season. the emergence of influenza viruses (flu a and flu b) was more common in the flu season, other virus like mpv and 229e were smaller than flu in the influenza season (table 4 ). pathogen-directed therapy avoids unnecessary antibiotic or antiviral use, facilitating more timely and hence more effective use of drugs, help prevent secondary spread of infection, all of which shorten hospital stays and have a major impact on patient management and disease prognosis [19] [20] [21] . accurate and rapid etiologic diagnosis is crucial to pathogen-directed therapy. to date, the gold standard for cap diagnosis is still based on the radiographic findings [22] . the radiological findings of multilobar infiltration or pleural effusion generally indicate greater severity [23] . although etiological studies have been performed, current microbial diagnostic tests frequently do not allow clinicians to rule out bacterial infection with certainty [24] . obtaining an qualified sample may be a challenge as specimens were easily contaminated by the bacteria located in upper airways [25] . for increasing the detection rate of pathogens, esp. for infection occurred in the lower respiratory tract, alveolar lavage fluid may be better in reflecting infectious status than the sputum. however, alveolar lavage fluids require semi-invasive surgery, which is certainly not routine in non-intubated patients, esp. to children [2] . under this condition, taking sputum as the sample is more practical. therefore, the relevant cap guidelines recommend sputum examinations for patients with moderate to severe cap. in this study, a high microbial yield can be achieved when real time pcr assays plus the conventional diagnostic methods, such as bacterial cultures [26, 27] . the single-plex real time table 2 the clinical symptoms of bacterium group, virus group, yeast group and co-infected group. pcr assay were exploited for detecting 14 respiratory viruses, which could improve diagnostic efficacy, particularly in diagnosing respiratory viral infections [28] . etiologic diagnosis in the cap patients reached 63.08%, we still had 36.92% cap patients without clearly microorganisms diagnose. for patients with comorbidities, the use of antibiotics prior to sampling may obscure a potential bacterial detection [29] . previous studies revealed that etiology remains unknown in approximately one-half of the cases [30, 31] , indicating that establishment of a precise pathogen diagnosis for cap patients is challenging. better diagnostic methods are warranted to distinguish viral from p＜0.01 when the influ group is compared with non-influ group ** p＜0.01 when the influ group is compared with non-influ + bacterium/yeast group; δ p＜0.01 when the non-influ group is compared with influ + bacterium/ yeast group; ▲▲ p＜0.01 when the influ + bacterium/yeast group is compared with non-influ + bacterium/ yeast group. the number of bacterial infections and viral infections in the influenza seasons and non-flu seasons in 2016. bacterial cap, which will clarify if these viral infections cause cap on their own or merely predispose the patient to bacterial co-infections [32] . differences in epidemiology make the knowledge of local etiology crucial for the appropriate choice of empirical antimicrobial treatment [28] . even there was a severe influenza outbreak during our study, other respiratory viral infections were more than influenza infection (63.16% vs. 36.84%). in all pathogens that cause cap, bacteria occupied the majority of pathogens, and the most common bacterium was a. baumannii. our results was not in keeping with the previous studies, in which the most frequently identified pathogens were burkholderia pseudomallei (29%), s. pneumoniae (20%), k. pneumoniae (19%) and h. influenza (11%), respectively [33] . although a. baumannii is an important pathogen of nosocomial infections, we often isolated it from infectious patients who is first admitted to the hospital, indicating that a. baumannii has a certain popularity in this area. most communityacquired cases of a. baumannii pneumonia are from tropical or subtropical countries in the asia-pacific region. in thailand, severe cap is caused by a. baumannii, and its mortality is 10 times higher than hospital-acquired a. baumannii pneumonia [33, 34] . in another aspect, most of our patients had underlying diseases, we could not exclude the possibility that patients had the bacterial infection in last hospital admission. the pathogen remains in the respiratory tract, causing clinical symptoms when the body's immunity is reduced. the outbreak of a. baumannii infection is also associated with multidrug resistance, and carbapenems have been considered to be a major factor in resistance to multidrug-resistant a. baumannii infection [34] . moreover, the mortality of carbapenem-resistant a. baumannii pneumonia patients is higher than that of carbapenem-sensitive a. baumannii pneumonia patients [35, 36] . s. pneumoniae and h. influenza, as co-pathogens, often seem to be part of a mixed infection (virus and bacterium) in adults with cap [26] , representing the most common combination with respiratory viruses [2] . the incidence of mixed infections was significant among patients admitted to the hospital with cap [37] . in our study, 16.41% of cap patients had single virus, and the co-infections (virus + bacterium and virus+ yeast) were found in 12.82% cap patients. the incidence of coinfections was in agreement with a previous study [38] . the most common bacterium co-detected with a virus was k. pneumoniae, as well as the yeast in our study, indicating that geographical variation exists in the prevalence of bacterium or yeast strains expressing factors that enable efficient disease potentiation during viral epidemics, which should be considered one explanation for regional differences in severity [8] . the diversity in etiology revealed that understanding the local etiology of cap is essential to inform clinical management decisions. respiratory viruses usually followed seasonal patterns of activity [39] , and epidemic influenza was noted in our studied area, which was active throughout the study period, and there was a high detecting rate of influenza a/influenza b was from january to may. during an influenza epidemic season, a high percentage of samples from cap patients contained at least one virus, and dual infections were very common [40] . the respiratory virus other than influenza accounted for 63.16% viral cap in our study, revealing that respiratory viruses can be cocirculating even during the highest epidemic peaks of influenza that showed a seasonal distribution. bacterial or fungal infections secondary to influenza viral infections were common [41] , though we could not exclude that the increasing co-infections cap was due to secondary viral infection. as the clinical signs and symptoms overlapped between influenza and non-influenza viruses, pathogen-directed therapy is difficult if only based on clinical symptoms. studies have shown that delayed antiviral treatment is associated with high risk of progression to severe disease required icu admission or prolonged hospital stay, and even causing death [42] . the microbiome's characterization and diversity are closely linked to the host status, and the host immune response may play a determining role in the infectious exacerbation of cap. the intrinsic complexity of the host-microbiome relationship currently limits the clinical indications of microbiome analysis [25] . diabetes and high blood pressure were the most common underlying disease, which were known to be associated with an increased risk of complications and death among critically ill patients [43] . detection of respiratory secretions by pcr method has provided a rapid and definitive diagnosis for the ilis in hospitalized adults [44] . unfortunately, it is not routine test in most clinic labs. appropriate empiric therapy is likely to be suboptimal without narrowing diagnostic possibilities to the most likely possibilities pending definitive pathogen identification [44] . effective, targeted therapy for cap requires an understanding of the heterogeneity of etiology and the individual host response to infection [45] . this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. this study was approved by the ethics committee of shantou university medical college and informed consents were obtained from the patients. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of 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diagnosis of pneumonia in epidemiological studies infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults diagnostic tests for agents of community-acquired pneumonia microbiome, biofilms, and pneumonia in the icu etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods community-acquired pneumonia in chile: the clinical relevance in the detection of viruses and atypical bacteria etiology of community-acquired pneumonia and diagnostic yields of microbiological methods: a 3-year prospective study in norway bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza a (h1n1) -united states microbial etiology of community-acquired pneumonia in the adult population of 4 municipalities in eastern finland british thoracic society standards of care c. bts guidelines for the management of community acquired pneumonia in adults respiratory viruses associated with community-acquired pneumonia in children: matched case-control study community-acquired acinetobacter baumannii pneumonia acinetobacter baumannii: evolution of a global pathogen pneumonia caused by extensive drug-resistant acinetobacter baumannii among hospitalized patients: genetic relationships, risk factors and mortality comparison of pneumonia-and non-pneumonia-related acinetobacter baumannii bacteremia: impact on empiric therapy and antibiotic resistance pneumococcal coinfection with human metapneumovirus british thoracic society standards of care c. british thoracic society guidelines for the management of community acquired pneumonia in childhood viral pneumonia the use of a multiplex real-time pcr assay for diagnosing acute respiratory viral infections in children attending an emergency unit critical illness from 2009 pandemic influenza a virus and bacterial coinfection in the united states predictors and outcomes of respiratory failure among hospitalized pneumonia patients with 2009 h1n1 influenza in taiwan early blood glucose control and mortality in critically ill patients in australia adult human metapneumonovirus (hmpv) pneumonia mimicking legionnaire's disease genomic landscape of the individual host response and outcomes in sepsis: a prospective cohort study this study was supported by the shantou science and technology project (grant numbers. 180709174010328).this study was made possible by the generous support of the american people through the united states agency for international development (usaid) emerging pandemic threats program-2 (predict-2) (cooperative agreement no. aid-oaa-a-14-00102). the contents were the responsibility of the authors and do not necessarily reflect the views of usaid or the united states government. key: cord-283545-vu8lt3w6 authors: brabb, thea; newsome, denise; burich, andrew; hanes, martha title: infectious diseases date: 2011-12-16 journal: the laboratory rabbit, guinea pig, hamster, and other rodents doi: 10.1016/b978-0-12-380920-9.00023-7 sha: doc_id: 283545 cord_uid: vu8lt3w6 although guinea pigs are sensitive and susceptible to the development of lesions from a wide range of viruses, bacteria, protozoa, and parasites, only a small number of organisms cause natural infection and only a portion of that group cause clinical disease. this chapter discusses naturally occurring diseases of guinea pigs, although some data from experimental infections have also been covered as they relate to the pathogenesis of the disease. the material presented includes background, etiology, epizootiology/pathogenesis, clinical manifestations, pathology, diagnosis, prevention, and therapy. the diseases are discussed in an alphabetical order based on the taxonomic groups to which the organisms belong and are independent of the order of perceived importance of the various diseases. the federation of european laboratory animal science associations recommends monitoring for guinea pig adenovirus, guinea pig cytomegalovirus, sendai virus, ectoparasites, endoparasites, e. cunniculi, and a variety of bacteria including bordetella bronchiseptica, chlamydia psittaci, corynebacterium kutscheri, dermatophytes, pasteurellaceae, salmonella, streptobacillus moniliformis, streptococcus, yersinia pseudotuberculosis, and clostridium piliforme. virus-associated necrotizing ronchopneumonia in guinea pigs is a spontaneous multifactorial disease that has low morbidity, high mortality, and a worldwide distribution. although guinea pigs are sensitive and susceptible to the development of lesions from a wide range of viruses, bacteria, protozoa, and parasites, only a small number of organisms cause natural infection and only a portion of that group cause clinical disease. the intent of this chapter is to discuss naturally occurring diseases of guinea pigs, although some data from experimental infections may be covered as they relate to the pathogenesis of the disease. the material is presented under the following headings: background, etiology, epizootiology/pathogenesis, clinical manifestations, pathology, diagnosis, prevention, and therapy. the diseases are discussed in an alphabetical order based on the taxonomic groups to which the organisms belong and are independent of the order of perceived importance of the various diseases. in laboratory colonies, routine surveillance of guinea pigs is conducted monthly, quarterly, or yearly as required. the federation of european laboratory animal science associations (felasa) recommendations include monitoring for guinea pig adenovirus, guinea pig cytomegalovirus, sendai virus, ectoparasites, endoparasites, e. cunniculi, and a variety of bacteria including bordetella bronchiseptica, chlamydia psittaci, corynebacterium kutscheri, dermatophytes, pasteurellaceae, salmonella spp., streptobacillus moniliformis, streptococcus spp., yersinia pseudotuberculosis, and clostridium piliforme. most laboratories also monitor for these additional viruses, simian virus-5 (sv5), pneumonia virus of mice (pvm), reovirus, lymphocytic choriomeningitis virus (lcmv), and parainfluenza virus-3 (piv-3). these viruses are routinely monitored both to minimize the risk of transference to mouse and rat colonies (pvm, reovirus, sendai virus, lcmv) and to identify infections that are zoonotic (lcmv, piv-3). the viral taxonomy used follows the recommendations of the viiith international committee on taxonomy of viruses (fauquet, 2005) , but well-established common names for viruses are used when appropriate. historically, pvm, parainfluenza viruses, cytomegalovirus, and reovirus have been commonly found in guinea pig colonies. from 1984 to 1988 serologic investigations of laboratory animal colonies originating from ten different european countries found seropositivity for four viral infections in guinea pig stocks; reovirus, pvm, sendai virus, and sv5 (see paramyxoviridae section for discussion of serological specificity of sendai virus and sv5) (kraft and meyer, 1990) . in a 2004 study guinea pigs reared conventionally were positive serologically for sendai virus, pvm, and reovirus (park et al., 2006) . adenoviridae: guinea pig adenovirus (gpadv) background virus-associated necrotizing bronchopneumonia in guinea pigs is a spontaneous multifactorial disease that has low morbidity, high mortality, and a worldwide distribution (percy and barthold, 2007) . after the first description in germany and experimental reproduction of the disease (kunstyr et al., 1984; naumann et al., 1981) , sporadic spontaneous cases were reported from the united states, australia, and switzerland (brennecke et al., 1983) . a 10-year endemic "in-house" occurrence of adenovirus-associated disease was reported in a british pharmaceutical research facility. in all described spontaneous cases, some additional stress factors were involved (pring-akerblom et al., 1997) . etiology gpadv is a member of the adenoviridae, genus mastadenovirus that contains canine and bovine adenoviruses as well (fauquet, 2005) . a portion of the hexon gene was cloned and sequenced and found to be eimeria 666 giardia duodenalis 667 klossiella cobayae 667 leishmania enrietti 668 toxoplasma gondii 668 tritrichomonas caviae 669 trypanosoma cruzi 669 prevention and therapy animals with gpadv are not appropriate for guinea pig pulmonary research projects. rederivation of infected colonies with aseptic hysterectomy or embryo transfer should eliminate the virus. in addition, there could be a concern that natural infection with gpadv would interfere with the use of adenovirus gene vectors. this was examined in a study of gpadv naturally infected guinea pigs that were used as recipients of a human adenoviral vector carrying the gene for green fluorescent protein (hankenson et al., 2010) . in this model system, no difference in transfection-efficiency was seen in guinea pigs naturally infected with gpadv or gpadv-free. three members of the family herpesviridae that specifically infect guinea pigs are catalogued in the eighth report of the international committee on the taxonomy of viruses (fauquet, 2005) ; caviid herpes virus 2 (commonly known as guinea pig cytomegalovirus and in some publications as caviid herpesvirus 1 (staczek, 1990) ), caviid herpes virus 1 (also known as guinea pig herpes-like virus or hsiung-kaplow herpesvirus, or in some publications as caviid herpesvirus 2 (staczek, 1990) ), and caviid herpes virus 3 (also known as guinea pig x virus) (fauquet, 2005) . in addition, equid herpes virus 1, a virus of horses, has been shown to cause severe disease in guinea pigs in one outbreak in a zoo (wohlsein et al., 2010) . of these viruses, guinea pig cytomegalovirus is the most commonly isolated herpes virus from guinea pigs and serological reactivity to this virus in colonies is not unusual. guinea pig cytomegalovirus is used extensively in research as an animal model for human cytomegalovirus. is consistently recovered from the blood and many other tissues including lung, spleen, and kidney during a 10-day period beginning at day 2 post-inoculation . viral clearance from the blood and most tissues occurs by 10 days post-infection, but virus remains consistently detectable in the urine and salivary gland during the chronic phase of infection (bia et al., 1979; hsiung et al., 1980) . less consistently, virus is found in splenic macrophages, b lymphocytes, kidneys, spleen, pancreas, and cervix of infected animals during this chronic phase. pregnancy can lead to more severe disease and changes in viral load following experimental gpcmv infection griffith and hsiung, 1980; griffith et al., 1983) . pregnant guinea pigs may develop interstitial pneumonia, splenomegaly, and necrosis of the liver, kidney, thymus, pancreas, and bone marrow (griffith et al., 1983) . the mortality rate for pregnant animals during an acute viremic episode following experimental infection is significantly greater than for non-pregnant females. although the fetus is susceptible to gpcmv infection at any time throughout gestation, transplacental transmission occurs most readily during the acute phase of maternal infection. the frequency of stillbirths and viral infection in neonates is highest in animals infected late in gestation. in neonates, brain lesions are observed and virus can be recovered from the salivary glands and, to a lesser extent, from the brain, lungs, pancreas, and liver of the neonates. in some cases, lesions are detected in the salivary glands of offspring up to 14 weeks postpartum. placental infection may be detected after maternal clearance of viremia and maintained despite the presence of significant levels of gpcmv neutralizing antibody. the site of infection is localized at the transitional zone between the capillarized labyrinth and the noncapillarized interlobrium of the placenta. whenever infection of the fetus is demonstrated, the placenta is invariably infected; however, infected placentas may be associated with uninfected fetuses. the status of maternal immunity before pregnancy can favorably affect the outcome of maternal or fetal infections with virulent gpcmv strains (staczek, 1990) . clinical manifestations natural infection with gpcmv causes a latent, persistent infection in the salivary gland generally without serious disease in animals in the vivarium (staczek, 1990; van hoosier and robinette, 1976) . however, the death of a sow and fetuses (motzel and wagner, 1989) and disseminated infection, including pneumonia, in guinea pigs that were not being manipulated experimentally have been reported (van hoosier et al., 1985) . illness is more likely when associated with pre-existent immune suppression or pregnancy (percy and barthold, 2007) . transient increase in mononuclear cells is seen as well as histopathological evidence of lymphoid hyperplasia of the spleen in both the t cell and b cell zones, although no changes were noted in the cervical and mesenteric lymph nodes (dowler et al., 1984) . intranuclear inclusions in tissues have not been noted in vivo, although they are evident in tissue culture cells (dowler et al., 1984; hsiung et al., 1971a hsiung et al., , 1971b van hoosier and robinette, 1976) . virus can be recovered from the lymph nodes and spleen of infected animals well after clinicopathological signs of infection end. serologically, this virus is distinct from gpcmv and gpxv and can be differentiated from these viruses both morphologically and serologically . prevention and therapy gphlv is not a clinically important disease of guinea pigs, but could be a complicating factor in research either through interaction with other viruses or cytopathological effects on cultured cells. herpesviridae (fauquet, 2005) . it was originally isolated from leukocytes of strain 2 guinea pigs that were free of gphlv and gpcmv . in one study of 112 hartley and strain 2 guinea pigs tested serologically for gpxv, 38% were found to be positive. inoculation of gpxv into hartley guinea pigs caused viremia which persisted for at least 3 weeks . virus could be recovered 5-6 months after inoculation suggesting a persistent infection. in this study, 50% of the animals died between 6 and 11 weeks postinoculation. hepatic necrosis was the only consistent lesion seen. gpxv is both morphologically and serologically distinct from gpcmv and gphlv. prevention and therapy natural disease from gpxv is unknown, but similar to gphlv, this virus may be a complicating factor in research. background and etiology ehv1 is an equine pathogen that causes a persistent infection in horses with a variety of clinical presentations including respiratory disease, abortion, neonatal death, and neurologic disease (reed and toribio, 2004) . ehv1 is a species in the genus varicellovirus of the alphaherpesvirinae subfamily (fauquet, 2005) . other members of this genus include the type species human herpesvirus 3 (varicella-zoster virus or chickenpox) and bovine herpesvirus 1 (infectious bovine rhinotracheitis virus). one recent report describes infection with ehv1 resulting in hindlimb paralysis, ataxia, abortion, or stillbirth in 18 of 80 guinea pigs at a european zoo (wohlsein et al., 2010) . in this outbreak, thomson gazelles (equus thomsoni) kept in the same building as the guinea pigs were first affected and suffered a short course of fatal neurologic disease. the source of the virus was unclear, although a similar strain of virus was isolated 6 months earlier from affected black bears (ursus americanus) and clinically unaffected onagers (equus hemionus kulan). in the second outbreak, a similar strain of virus was also recovered from clinically unaffected zebra (equius quagga boehmi) housed in the same building as the guinea pigs and gazelles. pathology lympho-histiocytic meningoencephalitis was seen predominantly in the olfactory bulb and the frontal cortical regions of the brain with neuronal and glial necrosis, gliosis, and intranuclear inclusion bodies (wohlsein et al., 2010) . ehv1 antigen was demonstrated in the neurons, neuronal processes and glial cells by immunohistochemistry and encapsulated herpes viral particles of 120-150 nm were detected by electron microscopy. diagnosis differentiation from other members of the herpesviridae is important for diagnosis. immunohistochemistry, virus isolation, pcr, and dna sequencing were used in this report to identify the virus involved (wohlsein et al., 2010) . prevention and therapy ehv1 infection of guinea pigs is an example of the severe disease seen when members of the alphaherpesvirinae infect unusual hosts similar to human herpes virus i (herpes simplex virus) which has been shown to infect guinea pigs experimentally (wohlsein et al., 2010) and has also been reported to cause naturally acquired clinical disease in rabbits (weissenbock et al., 1997) and chinchillas (wohlsein et al., 2002) . separation of species, control of fomites, and use of appropriate personal protective equipment can be used to prevent transmission of members of the alphaherpesvirinae subfamily of viruses to aberrant hosts such as guinea pigs. there has been one report of pox-like virus particles obtained from tissue culture samples of fibrovascular proliferations involving the rear limb of half of a colony of guinea pigs (hampton et al., 1968) . particles resembling pox viruses were observed by electron microscopy. arenaviridae: lymphocytic choriomeningitis virus (lcmv) background the first identified arenavirus, lcmv, was isolated in 1933 and shown to cause aseptic meningitis in humans and was named for its ability to cause lymphocytic choriomeningitis in mice and monkeys upon intracerebral injection (amman et al., 2007; charrel and de lamballerie, 2010; percy and barthold, 2007; van hoosier and robinette, 1976) . lcmv is a rodent-borne zoonotic arenavirus endemic in housemice (mus musculus) worldwide. infection by lcmv in people is known to cause acute central nervous system disease and congenital malformations. etiology the viral genus arenavirus includes 22 viral species, the type species is lcmv (fauquet, 2005) . like other arenaviruses, lcmv is a non-cytopathic, enveloped, single-stranded rna virus. epizootiology and pathogenesis lcmv is uncommon to rare in guinea pigs (fox, 2002; percy and barthold, 2007) . in may 2005, the centers for disease control and prevention reported a cluster of lcmv infections among four solid organ recipients in rhode island and massachusetts who received organs from a single apparently asymptomatic donor (centers for disease control and prevention [cdc], 2005a) . recipients became gravely ill shortly after transplantation; three subsequently died. lcmv was identified as the etiologic agent. viral sequences from the organ recipients were identical to those from a pet hamster acquired by the donor's household 17 days before organ donation. sequence and phylogenetic data provided strong support for the presence of the same lcmv lineage in hamsters and guinea pigs in the rhode island pet store and the ohio distribution center (amman et al., 2007; cdc, 2005b) . clinical manifestations clinical signs of lcmv are uncommon in the guinea pig, however meningitis with hind limb paralysis has been reported (van hoosier and robinette, 1976) . pathology lcmv in guinea pigs results in lesions involving the brain including lymphocytic infiltrates in meninges, choroid plexus, and ependyma (percy and barthold, 2007) . lymphocytic infiltrates are also seen in the liver, adrenal, and lungs. experimentally, neutrophilic destruction of the splenic red pulp, focal bone marrow necrosis, lymphopenia, and death occur following infection with the more virulent (we) strain (djavani et al., 1998) . diagnosis diagnosis is typically by pcr, ifa, elisa, or mfia (charrel and de lamballerie, 2010; fox, iii. guinea pigs 2002; percy and barthold, 2007) . detection of viral antigen in tissue or section via immunohistochemistry is also feasible. immunity to lcmv is primarily cellular, but virus-specific antibody is produced. prevention and therapy lymphocytic choriomeningitis is a zoonotic disease of concern with rodents kept as pets (pickering et al., 2008) . human infection occurs most commonly through exposure to secretions or excretions of infected animals. in addition, lcmv can contaminate transplantable tumor or cultured cell lines from mouse, hamster, and guinea pig, and viral stocks (ilar, 1991; percy and barthold, 2007) . in mice, lcmv has been shown to limit tumor induction by polyomavirus, mouse mammary tumor virus, and transplantable guinea pig leukemia, delay tumor rejection, and alter sensitivity to endotoxins (ilar, 1991) . natural infections would be expected to interfere with research studies involving enterohepatic, lymphoid, musculoskeletal, nervous, respiratory, and urinary systems. coronavirus-like particles were found in the feces of young guinea pigs that developed wasting, anorexia, and diarrhea with low morbidity and mortality (jaax et al., 1990) . at necropsy, there were large amounts of mucoid material throughout the small intestine. histologically, the primary lesion was acute to subacute necrotizing enteritis involving the distal ileum with blunting and fusion of the affected villi, necrosis and loss of enterocytes, and mucosal syncytial cells were evident. by electron microscopy, viral particles consistent with coronavirus were demonstrated in fecal matter from affected animals. coronavirus shedding was also noted in the feces of clinically normal guinea pigs (marshall and doultree, 1996) . the virus has not been isolated. paramyxoviridae is composed of pleomorphic, enveloped, linear, negative-sense single-stranded rna viruses occurring worldwide in animals and humans, often associated with subclinical infections of the respiratory tract (fauquet, 2005; simmons et al., 2002) . the family is split into two subfamilies, paramyxovirinae and pneumovirinae (fauquet, 2005) . in guinea pigs, three distinct members of the paramyxovirinae have been described; human parainfluenza virus 3 (including human parainfluenza virus 3, cavian parainfluenza virus 3, and guinea pig parainfluenza virus 3), sendai virus, and simian virus 5 (sv5). in addition, murine pneumonia virus, a member of the pneumovirinae, has been described in guinea pigs. (fauquet, 2005) . sequence analysis of a guineapig parainfluenza virus 3 isolated in 1998 from a colony of guinea pigs in japan indicates that it has 95.6-97.9% nucleotide identity to human parainfluenza 3 virus and 71.0-79.6% to bovine parainfluenza virus 3 suggesting this virus was introduced into guinea pigs from a human with human parainfluenza virus 3 (ohsawa et al., 1998) . a novel paramyxovirus (cavian parainfluenza virus 3) was isolated in 2002 from a colony of guinea pigs (simmons et al., 2002) . this virus has 94% nucleotide identity to guinea-pig parainfluenza virus 3 and human parainfluenza virus 3 and 80% nucleotide identity to bovine parainfluenza virus 3. epizootiology and pathogenesis seroconversion in endemic parainfluenza-3-positive breeding colony was studied in 2002 (blomqvist et al., 2002) and indicated that pups become infected from 2 weeks to 8 weeks of age. the virus did not persist as sentinels exposed to 4-5-month-old seropositive animals did not seroconvert. experimental infections indicate that seroconversion in naã¯ve animals occurs approximately 10 days following infection (graziano et al., 1989) . in an infection study guinea pigs inoculated with cavian parainfluenza virus 3, sendai virus, simian virus 5 (sv-5), murine pneumonia virus, or bovine parainfluenza virus developed no clinical signs of disease or lesions during the eightweek course of the study although they developed robust homologous or heterologous serologic responses to the majority of the antigens (simmons et al., 2002) . the serologic response, using an elisa, of those inoculated with sv-5 was modest or equivocal. in addition, sv-5 elisa resulted in the highest degree of non-specific reactivity among all of the paramyxovirus assays. in one study, no clinical signs or lesions were seen when guinea pigs were infected with cavian parainfluenza virus 3 (simmons et al., 2002) . in a separate study, guinea pigs were shown to be susceptible to human parainfluenza virus 3, developing peribronchiolar and interstitial lesions (clyde, 1980) . diagnosis identification of the virus via pcr and sequencing is ideal as cross-reactive serological results make interpretation of serology difficult (simmons et al., 2002) . it is apparent that human parainfluenza virus 3 can be transmitted to guinea pigs and infection-control measures such as the use of face masks, gloves, and laminar flow hoods to prevent transmission in laboratory settings is prudent. it isn't known whether human or cavian parainfluenza virus 3 can be transmitted to humans from guinea pigs. etiology sendai virus, also known as murine parainfluenza virus, is the type species in the genus respirovirus, which also contains the species human parainfluenza virus 3, bovine parainfluenza virus 3, and human parainfluenza virus 1 (fauquet, 2005) . numerous studies have reported seropositivity to sendai virus, including a publication in 1978 which describes seropositive reactions by hi or cf in seven out of 16 colonies tested over a 13-year period in the united states (parker et al., 1978) . in addition, there is one report of isolation of sendai virus from guinea pigs associated with mice (van hoosier and robinette, 1976) , although no clinical signs were reported. more recently, five of 15 european guinea pig colonies were seropositive to either sendai virus, human parainfluenza virus 3, or both (schoondermarkvan de ven et al., 2006) . inoculation of guinea pigs with sendai virus in one report resulted in no clinical signs of disease or lesions (simmons et al., 2002) . diagnosis as with the other paramyxoviridae, lack of specificity can result in false-positive results with routine serological testing (simmons et al., 2002) . in one study, sendai elisa-positive samples were seen in guinea pigs infected with pneumonia virus of mice and bovine parainfluenzavirus 3. guinea pigs were also positive by ifat in the case of bovine parainfluenza virus 3 infection. the importance of sendai virus infections in guinea pigs is unclear. however since it is a significant disease of mice housed in laboratory animal colonies, guinea pigs should be maintained free of sendai virus. in addition, sendai virus is used as a vector in research and natural infection or sero-reactivity may interfere with such uses (baker, 1998) . sv5 is a species of the genus rubulavirus which also contains mumps virus and human parainfluenza virus 4 (fauquet, 2005) . sv5 was initially isolated from simian kidney cell cultures and has also been shown to infect guinea pig cells in vitro (zakstelskaya et al., 1976) . serological reactivity to sv5 has been reported in guinea pig colonies (kraft and meyer, 1990) . seropositve guinea pigs show no clinical signs or lesions and inoculation with sv5 results in seroconversion, but not clinical signs of disease (simmons et al., 2002) . infection of guinea pigs with sendai virus, pneumonia virus of mice, or human parainfluenza virus 3 can result in positive sv5 elisa results which were negative by indirect fluorescent antibody test, indicating low specificity of the sv5 elisa (simmons et al., 2002) . etiology pvm is a member of the genus pneumovirus, species murine pneumonia virus (fauquet, 2005) . other members of the genus pneumovirus include the human and bovine respiratory syncytial viruses. the complete genomes of two strains of this virus were sequenced in 2005 (thorpe and easton, 2005) . pvm is most often subclinical in immunocompetent mice although in immunodeficient mice, it can cause chronic wasting with cyanosis and dyspnea (percy and barthold, 2007) . seropositive reactions to pvm have been seen historically in guinea pig colonies in animals without apparent disease (van hoosier and robinette, 1976) . inoculation of guinea pigs with a strain of pvm that caused mortality in immunocompetent and immunodeficient mice resulted in no clinical signs, although the guinea pigs seroconverted within 11 days after inoculation (griffith et al., 1997) . prevention and therapy transmission of pvm from guinea pigs to mice, or vice versa, appears to be possible and similar in this regard to sendai virus. in laboratory colonies, it is important to keep guinea pigs free of pvm in order to minimize potential transmission to mice. it is unclear whether laboratory guinea pigs may harbor a poliovirus which, in 1911, was described as the cause of a disease called guinea pig lameness (hansen et al., 1997; van hoosier and robinette, 1976) . a similar disease was described by rohrer in 1958 (van hoosier and robinette, 1976) . in these investigations, sera from affected guinea pigs reacted positively to theiler's murine encephalomyelitis virus (tmev) antigens. clinical signs involved a flaccid paralysis and weight loss with an incubation period of 9-23 days, and a duration of 1-2 weeks. affected animals had meningomyeloencephalitis. more recently, two pet shop guinea pigs were reported to suffer from rear limb paresis and generalized debilitation. the guinea pigs had extremely high titers against "poliovirus", while healthy guinea pigs from the same pet shop were negative (hansen et al., 1997) . the diseased guinea pigs recovered iii. guinea pigs fully after treatment with vitamin c in the drinking water. further testing of other guinea pig colonies by the same authors demonstrated antibody responses to tmev in 35 out of 152 laboratory guinea pig sera. positive results were found in two out of six breeding centers, and in three out of three experimental units, all of which purchased guinea pigs from one of the seropositive breeding colonies. guinea pig sera from some of the breeding units were also positive serologically for other infections; adenovirus, pvm, reovirus type 3, sendai virus, sv5 and encephalitozoon cuniculi. in experimental studies of susceptibility of laboratory animals to six strains of human poliovirus, newborn guinea pigs were the only species of laboratory animals in which multiplication of any of the viruses could not be detected (koroleva et al., 1975) . naturally occurring antibodies to reovirus have been detected in laboratory guinea pig colonies by ifa, elisa, and mfia (percy and barthold, 2007; van hoosier and robinette, 1976) . no clinical signs or lesions have been described. experimentally guinea pigs have been exposed to a reovirus associated with the sars coronavirus (liang et al., 2005) . background cavian leukemia was first described in 1954 by congdon and lorenz as being caused by a retrovirus, however herpes viral inclusions have also been seen in leukemic guinea pigs (jungeblut and opler, 1967; opler, 1969; percy and barthold, 2007; van hoosier and robinette, 1976) . etiology type c retrovirus particles have been seen in cases of cavian leukemia van hoosier and robinette, 1976 ). an endogenous guinea pig retrovirus was induced by bromodeoxyuridine (michalides et al., 1975; nayak, 1974) . molecular hybridization techniques were used to show that guinea pig virus nucleotide sequences are endogenous to both domestic (cavia porcellus) and indigenous (cavia aperea) guinea pigs, but cannot be detected in the dna of other mammals tested (dahlberg et al., 1980) . in 1982, a transplantable leukemia (ksl) of unknown etiology, that had the characteristics of a b cell tumor, was found in a female sewall-wright strain 2 guinea pig. ksl leukemia was also shown to be distinct from another guinea pig lymphatic leukemia (l2c) with respect to cell morphology, antigenicity, and in vivo growth rate (key, et al., 1983) . a c-type virus was reported in the urine of diabetic guinea pigs (lee et al., 1978) . clinical manifestations clinical signs of cavian leukemia include lethargy, pale mucous membranes, secondary infections, peripheral lymph node enlargement, and death (van hoosier and robinette, 1976) . leukocyte counts may vary from 25 000-250 000, with the preponderance of cells being lymphoblastic. peripheral lymphadenopathy is generally evident. pathology enlarged lymph nodes in the cervical, axillary, mediastinal, retroperitoneal, and inguinal areas are seen in cavian leukemia with splenomegaly and hepatomegaly (van hoosier and robinette, 1976) . there is a moderate infiltration of leukemic cells in all organs including the spleen, liver, bone marrow, lung, thymus, gastrointestinal associated lymphoid tissue (galt), heart, eyes, and adrenals (opler, 1969) . treatment and control there is no described treatment. rabies has been reported in a pet guinea pig associated with a bite from a raccoon (eidson et al., 2005) . most clostridia are large, spore-forming, grampositive, anaerobic rods. the one exception is c. piliforme which is gram negative and is discussed in the section covering gram-negative organisms below. two species of clostridia are discussed here, c. difficile and c. perfringens. background severe profuse diarrhea, associated with clostridium difficile in guinea pigs treated with antibiotics, is an important and preventable disease process. etiology clostridium difficile is most commonly associated with antibiotic-induced typhilitis in guinea pigs, although other organisms including escherichia coli, clostridium perfringens, c. sordelli, c. histolyticum, and bacillus pumilis have been implicated in different studies (boot et al., 1989; brophy and knoop, 1982; knoop, 1979; lowe et al., 1980; pakes, 1981, 1982; rehg et al., 1980; . c. difficile is a common anaerobic, spore-forming, fecal-borne, gram-positive, rod-shaped organism that generally produces both an enterotoxin (toxin a) and a cytotoxin (toxin b) that are crucial in the pathogenesis of disease (greenwood, 2007) . pathogenesis normal guinea pigs may carry low resident populations of c. difficile in the intestines and toxins can be identified in normal cecal contents (boot et al., 1989) . in one study, c. difficile was recovered by culture in one out of eight normal guinea pigs and 18 out of 34 guinea pigs with typhilitis. oral or parenteral antibiotic administration, including penicillin, ampicillin, streptomycin, clindamycin, lincomycin, spiramycin, aureomycin, erythromycin, and bacitracin, have all been implicated in causing antibiotic-associated typhilitis by permitting an overgrowth of primarily gram-negative organisms including toxinproducing clostridium such as c. difficile (young et al., 1987) . the enteric flora of guinea pigs is primarily grampositive and those antibiotics that target gram-positive organisms preferentially are of greatest risk to cause this disease (farrar and kent, 1965) . one study demonstrated that in the 24-48 hours post-administration of penicillin, there was a significant increase in gram-negative anaerobic and aerobic (coliforms predominantly) bacteria bringing the levels of coliform bacteria from less than 100 organisms per gram to 10 8 -10 9 organisms per gram. this shift in the normal gut flora allows bacteria such as c. difficile, which is generally nearly undetectable, to predominate (farrar and kent, 1965; fox, 2002; percy and barthold, 2007) . other gram-negative organisms, e. coli in particular, and other clostridium spp. also can contribute to disease. clostridium difficile (toxin)-associated typhilitis has also been diagnosed in guinea pigs not treated with antibiotics that had been rederived by caesarian section and were exposed to mice as a source of anaerobic flora (boot et al., 1989) . two years after rederivation, an outbreak of typhilitis was investigated. young guinea pigs 2-4 weeks old and a few adults were affected. clostridium difficile was isolated from half of the animals tested and the cytotoxin was identified in cecal contents from 95% of the affected animals. co-infection with c. perfringens and encephalitozoon cuniculi was present. the authors speculate that the anaerobic flora that the animals were exposed to was not sufficient to protect guinea pigs from c. difficile infection. this same flora has been shown to be unsuitable for rats and rabbits. clinical manifestations generally, affected guinea pigs have weight loss, rough hair coats, anorexia, dehydration, decreased activity, and hypothermia beginning 24-48 hours after antibiotic administration (farrar and kent, 1965; fox, 2002; knoop, 1979; . some animals have no stool, while others have diarrhea and acute death has been reported. pathology at necropsy, the cecum is often gasfilled and dilated with hemorrhage evident in the cecal mucosa. histopathologically, there is usually hyperplasia of the ileal mucosa as well as ulceration, edema, and degeneration of the cecal epithelium with leukocytic infiltration. diagnosis c. difficile can be isolated from cecal contents via anaerobic culture (boot et al., 1989; greenwood, 2007) . detection of toxin in cecal contents, tissue, or feces via an enzyme immunoassay, cytotoxin activity in cell culture, serum neutralization assay, or pcr are considered the gold standard, however, false positives and false negatives are not uncommon (greenwood, 2007; houser et al., 2010; songer, 1996) . prevention and therapy antibiotic-associated diarrhea is usually treated symptomatically. in humans, administration of metronidazole or vancomycin is standard (greenwood, 2007; rupnik et al., 2009; songer, 1996) . anecdotal information suggests that administration of yogurt or other lactobacillus-containing products in conjunction with antimicrobial agents will prevent or minimize the effects of antibiotic-associated enteritis. a recent study attempted to induce antibiotic-associated enteritis by a single subcutaneous injection of clindamycin . c. difficile and other enteric pathogens were not isolated, however, several guinea pigs that received clindamycin developed enteritis. in affected animals, administration of antibiotics with the oral lactobacillus preparation did not result in any clinical improvement over those animals receiving antibiotics alone. certain pcr ribotypes of c. difficile are found in both human and animal populations, suggesting that this can be a zoonotic disease, although most of these studies are concerned with c. difficile found in food animals rather than guinea pigs (rupnik et al., 2009) . clostridium perfringens is a ubiquitous gram-positive anaerobic spore-forming bacterium that is the primary cause of enterotoxemia in domestic animals. c. perfringens strains produce a variety of toxins, as many as 17 exotoxins, although four major toxins are associated with typing of the bacteria; alpha, beta, episilon, and iota. infections typically occur via contaminated food, water, bedding, or soil and primarily affect sheep, pigs, and cattle. confirmed c. perfringens infections have been reported in germ-free guinea pigs in 1970 and, more recently, in a conventionally housed guinea pig (feldman et al., 1997; madden et al., 1970; songer, 1996) . clinical signs seen in the conventionally housed guinea pig were lethargy and anorexia. at necropsy, there were fibrinous adhesions involving the small intestine and liver (feldman et al., 1997) . the ileum and cecum had ecchymotic hemorrhages. histopathology revealed acute, multifocal, severe, necrotizing, ulcerative typhlitis and ileitis as well as focal, severe, necrotizing, centrilobular vascular thrombosis of the liver with infarction and coagulative necrosis of the adjacent hepatocyes. pcr was used iii. guinea pigs to identify the toxins of c. perfringens both in the intestine and the liver. corynebacterium etiology corynebacterium spp., members of the family corynebacteriaceae, are gram-positive, non-sporeforming, non-motile, aerobic, pleomorphic rods with coccoid or club-shaped appearance that are catalase-positive and non-acid-fast (boone et al., 2001; greenwood, 2007) . pathology corynebacterium spp. can cause disease in guinea pigs, but are also part of the normal flora. specifically, they are commonly isolated from guinea pig conjunctiva. in one study, 27 out of 30 clinically normal pet guinea pigs grew corynebacterium spp. from conjunctival cultures (coster et al., 2008) . spontaneous disease of guinea pigs associated with corynebacterial infections has rarely been reported, but have involved numerous different species of corynebacterium. in one report, corynebacterium pyogenes was isolated from a guinea pig with septicemia (ganaway, 1976) . further research with that agent demonstrated it was pathogenic when administered ip to other guinea pigs, but not if it was given orally or intranasally. in another report, c. kutscheri was isolated from the lung of a guinea pig during an epizootic of group c, beta-hemolytic streptococcal disease. further research with this isolate was similar to c. pyogenes, in that intraperitoneal administration resulted in disease while intranasal administration did not. the organism was also given intravenously in this case and was rapidly fatal. in a retrospective study of urinary bladder calculi in guinea pigs, it was reported that corynebacterium renale was the most common isolate from urine and bladder samples (hawkins et al., 2009) . of the 44 cases submitted for bacterial culture, 77% were negative. the most common pure culture isolate was c. renale, which was also commonly found in mixed infections with staphylococcus and streptococcus spp. diagnosis diagnosis is made by recovering the organism using aerobic culture conditions, generally on blood agar plates with standard biochemical tests to differentiate corynebacterium from other species (greenwood, 2007) . pcr and sequencing has been used for further identification (greenwood, 2007; hawkins et al., 2009) . prevention and therapy many corynebacterium spp. have significant resistance to a number of antibiotics and this may be true of some organisms in guinea pigs as well (greenwood, 2007) . in the study isolating c. renale from the urine and bladder wall, many of those animals were already on antibiotics when those isolates were obtained (hawkins et al., 2009 ). in addition, the corynebacterium spp. recovered from the normal conjunctiva of guinea pigs, demonstrated some bacterial resistance with six out of nine isolates resistant to oxytetracycline, four out of nine resistant to polymyxin b, two out of nine resistant to bacitracin, and one out of nine resistant to erythromycin, gentamicin, amikacin, and ciprofloxacin (coster et al., 2008) . guinea pigs are highly susceptible to m. tuberculosis. natural infection has rarely been reported in the literature, but in those cases, the sources of the infections were believed to be from contact with human cases (van hoosier and robinette, 1976) . background markham and markham (1966) studied the prevalence of staphylococcal organisms in various animal species. a 42% incidence of coagulasepositive staphylococcus was noted in the nasal passages of guinea pigs. this was the greatest incidence of staphylococcal infection of any species tested, including humans. all of the guinea pigs cultured appeared clinically normal. etiology the staphylococcus genus, members of the staphylococcaceae family, are common inhabitants of the skin, oral cavity, respiratory system, and intestine, and cause suppurative lesions and septicemia in all species (songer and post, 2005) . these gram-positive cocci are coagulase-positive, catalase-positive, oxidasenegative, non-spore-forming and facultative anaerobic organisms (harkness and wagner, 1995; songer and post, 2005) . in guinea pigs, staphylococcus aureus has been isolated from cases of ulcerative pododermatitis and exfoliative dermatitis (percy and barthold, 2007) . pathogenesis despite the existence of speciesspecific serotypes and biotypes, human staphylococcus aureus phage types have been recovered from animals, and human-animal contact is one means of transmission of s. aureus (harkness and wagner, 1995) . additionally, direct contact with an infected animal or contaminated surface, as well as spread via aerosol can result in the dissemination of infection. ulcerative pododermatitis, or dermatitis of the footpad, is also known as "bumblefoot". trauma to the footpad from fighting, a sharp cage edge, or abrasive flooring creates a site of entry for bacteria (harkness and wagner, 1995) . obese guinea pigs, as well as any other natural or experimental cause that results in a sedentary lifestyle, can predispose an animal to bumblefoot, since perfusion is decreased and the pressure on the feet is increased (brown and donnelly, 2008; harkness and wagner, 1995) . exfoliative, or acute staphylococcal, dermatitis has also been associated with s. aureus in guinea pigs. strain 13 guinea pigs are reported with this condition more frequently than other strains, and mortality rates due to this condition are higher in young animals, especially if born to an affected dam that is too painful to nurse (ishihara, 1980; percy and barthold, 2007) . clinical manifestations swollen, erythemic paws and lameness are noted with bumblefoot (brown and donnelly, 2008) . paws will often have erosions or ulcerations on the palmar or plantar surfaces. osteoarthritis or osteomyelitis may result, and animals that develop such sequela have a poorer prognosis. alopecia and erythema of the ventral abdomen, leading to cracking and scabbing of the epidermis, are often observed with exfoliative dermatitis (fox, 2002; ishihara, 1980; percy and barthold, 2007) . in addition to its association with ulcerative pododermatitis and exfoliative dermatitis, pneumonia, mastitis, conjunctivitis, cheilitis, and osteoarthritis have been attributed to s. aureus infection in guinea pigs (fox, 2002) . systemic amyloidosis can develop secondary to chronic s. aureus infection (brown and donnelly, 2008; fox, 2002; percy and barthold, 2007) . pathology hypertrophy of the stratum corneum with a minimal inflammatory response is noted histologically in cases of dermatitis associated with s. aureus (percy and barthold, 2007) . diffuse cellulitis is the primary lesion associated with ulcerative pododermatitis (brown and donnelly, 2008) . in cases that progress to amyloidosis, amyloid may be noted in the liver, spleen, adrenal glands, and pancreatic islets (ganaway, 1976) . diagnosis stained impression smears of lesions can demonstrate clumps of gram-positive cocci (harkness and wagner, 1995) . s. aureus can often be cultured directly from affected tissues and from the upper respiratory tract and pharynx of affected animals (percy and barthold, 2007) . blood agar or selective mediums, such as mannitol salt or staph 110 agar, can be used for primary culture (songer and post, 2005) . s. aureus colonies often present with a golden pigmentation. prevention and therapy prevention of ulcerative pododermatitis and exfoliative dermatitis is easier than treatment. preventing cutaneous wounds by providing sturdy, smooth flooring to guinea pigs, offering non-abrasive feedstuff, and preventing obesity are highly effective in minimizing infection with s. aureus. pododermatitis and exfoliative dermatitis are not usually conditions conducive to lesion drainage and surgical removal (brown and donnelly, 2008) . lesions can be cleaned, but the possibility of causing greater trauma to the affected area must be considered. also, disinfectants that dry out the skin or agents that are cytotoxic to fibroblasts and reduce white blood cell viability and phagocytic efficiency, such as povidone-iodine or chlorhexidine, should be avoided. long-term antibiotic treatment is necessary, and enrofloxaxin or ciprofloxacin administered twice a day for 2-6 months has been reported in the treatment of ulcerative pododermatitis. use of an analgesic agent, especially a non-steroidal antiinflammatory drug such as meloxicam, is also recommended. low-level laser therapy, or phototherapy, has been reported as successful in treating bumblefoot by accelerating angiogenesis and stimulating vasodilatation, although such therapy can only be used after the infection is first controlled. two strains of streptococcus cause disease in guinea pigs. streptococcus pneumoniae can lead to severe pneumonia and streptococcus equi subsp. zooepidemicus is associated with the development of chronic lymphadenitis . infections due to s. equi subsp. zooepidemicus are more prevalent in guinea pigs than are pneumococcal infections. members of the streptococcus genus are part of the streptococcaceae family and consist of gram-positive, catalase-negative, facultative anaerobic bacteria that are non-spore-forming and non-motile (songer and post, 2005) . background streptococcus pneumoniae was first recognized in guinea pigs in europe in the late 1800s . homberger et al. (1945) are credited with the first report of this organism in guinea pigs in the united states; the animals were from a colony in massachusetts and presented with signs of respiratory infection. etiology streptococcus pneumoniae is an oval-to lancet-shaped, encapsulated coccus that presents in pairs (diplococcus) or short chains (fox, 2002; percy and barthold, 2007) . s. pneumoniae is an î±-hemolytic streptococcus that ferments trehalose and is optochin-sensitive (harkness and wagner, 1995; keyhani and naghshineh, 1974; songer and post, 2005) . additionally, it is not categorized according to one of the lancefield groupings (songer and post, 2005) . serovars of pneumococci are differentiated according to their capsular polysaccharide content, and serovars 4 and 19 are the two most often recovered from guinea pigs (fox, 2002; percy and barthold, 2007) . asymptomatic guinea pigs can carry pneumococci in the upper respiratory passages; the carrier rate for laboratory colonies can be as high as 50-55% (fox, 2002; percy and barthold, 2007) . while iii. guinea pigs pneumonia epidemics due to s. pneumoniae seldom occur in well-managed facilities, stressors which may predispose to infection include poor husbandry, pregnancy, sudden or prolonged changes in temperature, poor ventilation, shipping, inadequate nutrition, concurrent infections, and experimental procedures (nakagawa et al., 1986; percy and barthold, 2007) . transmission of s. pneumoniae is by aerosol or via direct contact with abraided skin or oral mucosa (fox, 2002; percy and barthold, 2007; wagner et al., 1976) . infection can also be passed at the time of parturition by an infected dam. abortions and stillbirths can accompany a high mortality rate during an epizootic outbreak in a guinea pig colony (percy and barthold, 2007) . in 1974, a spontaneous epizootic of respiratory infection involving 2400 guinea pigs was caused by s. pneumoniae type 19 (keyhani and naghshineh, 1974) . during this outbreak, 450 guinea pigs died within 33 days of infection, and a short course of listlessness and failure to thrive were the only noted clinical signs prior to death. in less acute cases of s. pneumoniae, guinea pigs display depression and lethargy, anorexia, and have ruffled fur. they may have a wet nose, nasal or ocular discharge (rhinitis, conjunctivitis), sneezing, coughing, dyspnea, or torticollis (head tilt due to otitis media) (fox, 2002; wagner et al., 1976) . wagner, owens, kusewitt, and corley reported that otitis media occurred in 177 of 1373 guinea pigs necropsied during a 6-year period in the 1970s . streptococcus pneumoniae was noted in 20% of these cases, and was the most common bacteria isolated. gupta et al. (1970) , reported mastitis caused by s. pneumoniae in two guinea pigs. pathology acute bronchopneumonia is most often associated with s. pneumoniae infections. fibrinous exudate, a polymorphonuclear cell infiltrate and thrombosis of the pulmonary vessels may be observed in the lung (fox, 2002; keyhani and naghshineh, 1974; percy and barthold, 2007) . other pyogenic processes noted in infected animals include pericarditis, peritonitis, otitis media, arthritis, endometritis, and suppurative meningitis (fox, 2002; percy and barthold, 2007) . additionally, splenitis, fibrinopurulent meningitis, metritis, and lymphadenitis with focal hepatic and ovarian abscessation have been reported (percy and barthold, 2007) . pneumococci are readily demonstrated on impression smears and in stained tissue sections . diagnosis culture of nasal washings is an antemortem means of diagnosing s. pneumoniae (homburger et al., 1945) , as is culture of the nasal passages (harkness and wagner, 1995) . post-mortem, direct smears and culture of inflammatory exudates can be performed. s. pneumoniae is a fastidious bacterium that grows best when incubated under 5-10% co 2 on blood agar or enrichment media (fox, 2002; percy and barthold, 2007) . serological diagnosis via elisa has been reported (matsubara et al., 1988) . prevention and therapy antibiotic treatment of a clinical animal usually results in reversion to a sub-clinical carrier state, so elimination of clinical animals is recommended whenever possible (fox, 2002) . chloramphenicol, trimethoprim-sulfa combinations, and tetracycline have been used during epizootic outbreaks (harkness and wagner, 1995; homburger et al., 1945; keyhani and naghshineh, 1974) . prevention should focus on good husbandry, adequate diet, early isolation of streptococcal carrier animals, and reduction of environmental stressors. the same s. pneumoniae serovars commonly detected in guinea pigs have been isolated from humans (songer and post, 2005) . s. pneumoniae can lead to respiratory and neurological disease in humans, especially in older and immune-compromised individuals. to date, interspecies transmission of s. pneumonia has not been reported (harkness and wagner, 1995; percy and barthold, 2007) . charles boxmeyer (1907) described his findings of epizootic lymphadentitis in over 3000 guinea pigs in 1907. cunningham (1929) reported on 20 guinea pigs from a colony of 150 that had "lumps," and from which "the same unusual type of mucoid hemolytic streptococcus was isolated". cunningham noted a 3:1 female to male ratio among the animals with chronic lymphadenitis. etiology streptococcus equi. subsp. zooepidemicus is a lancefield group c streptococcus that is betahemolytic, encapsulated, and ferments sorbitol (percy and barthold, 2007; songer and post, 2005) . s. equi subsp. zooepidemicus has a high degree of dna homology with s. equi subsp. equi. s. equi subsp. zooepidemicus has been recovered from human infections, while s. equi subsp. equi has not (songer and post, 2005) . pathogenesis guinea pigs can carry s. equi subsp. zooepidemicus asymptomatically in the nasopharynx and conjunctiva (percy and barthold, 2007) , but compared to s. pneumonia, clinical disease is more often noted in guinea pigs infected with s. equi subsp. zooepidemicus. as noted by cunningham, females are indeed more susceptible to disease than males (cunningham, 1929; fox, 2002; percy and barthold, 2007) . strain 2 guinea pigs also appear to be more sensitive than other strains (fox, 2002; percy and barthold, 2007; wagner et al., 1976) . transmission is via aerosol, as well as direct contact with abraded skin or oral mucosa (harkness and wagnerl 1995; fox, 2002; percy and barthold, 2007) . the organism can be transmitted via bite wounds and secondary to abrasion by rough foodstuff (ganaway, 1976) . while this is likely the route of entry for most natural infections, murphy et al. (1991) showed that the oral mucosa does not need to be abraded in order for entry of the bacterium. the female genital tract can be infected during farrowing (percy and barthold, 2007) . after initial entrance, the organism travels to the draining lymph node and replicates (harkness and wagner, 1995) . cervical lymphadenitis is commonly noted in guinea pigs when the point of entry for infection is via the conjunctiva or nasal mucosa (harkness and wagner, 1995) . clinical manifestations clinical disease associated with s. equi subsp. zooepidemicus is colloquially referred to as "lumps," due to the pyogenic nature of the bacteria and its propensity to travel to and replicate in the lymph nodes (fox, 2002; percy and barthold, 2007) . while the cerival lymph nodes are the most common site of s. equi subsp. zooepidemicus replication and abscessation, any lymph node, and practically any organ, can be affected (harkness and wagner, 1995) . a common presentation for infection with this organism is an animal that appears in good condition except for a swelling in the neck at the position of the cervical lymph node (fox, 2002) . the swelling is often soft to firm, pus-filled, non-fluctuant and freely moveable (percy and barthold, 2007) . cunningham's (1929) observations were that "in some cases, one or two nodes were greatly enlarged; in others, as in one animal with cervical node involvement, several plum-sized encapsulated abscesses formed a collar across the front and side of the neck". depending on what additional organs are affected, torticollis (if middle ear involvement), dyspnea and cyanosis or nasal and ocular discharge (if respiratory involvement), or hematuria and hemoglobinuria (if septic) may be noted (fox, 2002; harkness and wagner, 1995) . abortions, stillbirths and unexpected deaths are possible (fox, 2002) . during epizootic outbreaks, septicemia, acute pneumonia, and high mortality can occur (harkness and wagner, 1995) . pathology the most common finding at necropsy is that of an abscessed, encapsulated cervical lymph node (fox, 2002) . the node architecture is often destroyed by central necrosis and peripheral fibrosis, and a thick pustular exudate is present (cunningham, 1929; fox, 2002; percy and barthold, 2007) . smears of the exudate reveal numerous gram-positive cocci in short chains (cunningham, 1929) . a generalized lymphadenitis may be noted, as may retroorbital abcessation, otitis media, pneumonia, pleuritis, pericarditis, or hepatitis (cunningham, 1929; fox, 2002; percy and barthold, 2007) . diagnosis s. equi subsp. zooepidemicus can be cultured from affected tissues or heart blood (harkness and wagner, 1995; percy and barthold, 2007; quesenberry and carpenter, 2004; wagner et al., 1976) . culture requires blood agar, on which after 24 hours, a clear zone of hemolysis is observed (harkness and wagner, 1995) . trimming of teeth, elimination of sharp edges to feeders, and feeding nonabrasive feed may assist in prevention of s. equi subsp. zooepidemicus infection (fox, 2002) . infected animals should be isolated from the colony. surgical removal of abscess contents and capsule can be done with creation of drainage and lavage. antibiotic therapy is required. in epizootic cases, depopulation of affected animals and disinfection of the environment is advised. enzootic disease represents significant potential for research complications. disease-free replacement stock should be obtained and is available from commercial vendors. vaccines are typically type-specific and do not provide reliable protection (fox, 2002; mayora et al., 1978) . actinobacillus actinobacillus lignieresii and actinobacillus equuli have been noted in laboratory guinea pigs (songer and post, 2005) . the genus actinobacillus is a member of the pasteurellaceae family (boone et al., 2001) . these organisms are pleomorphic gram-negative rods that on gram stain, can have a "morse code" appearance (songer and post, 2005) . actinobacillus lignieresii and actinobacillus equuli were cultured from 36 guinea pigs, rats and mice during a 6-month period at the university of missouri research animal diagnostic and investigative laboratory (lentsch and wagner, 1980) . organisms grew on blood agar inoculated with swabs from the posterior nasopharynx, conjunctiva, and middle ear. conjunctivitis was noted in one animal and otitis media was noted in six. background tartakowsky is credited with one of the first accounts of bordetella infection in guinea pigs in the late 1880s (mccartney and olitsky, 1923; smith, 1913) . woolfrey and moody (1991) in their review of this organism stated "ferry, using the name bacillus bronchisepticus; mcgowan, using the term bacillus bronchicanis; and torrey and rahe, using ferry's epithet, independently observed the association of a small gram-negative coccobacillus with outbreaks of "canine distemper" and respiratory tract illness in laboratory animals such as the cat, rabbit, and guinea pig". in the early 1970s, b. bronchiseptica was still considered to be the primary cause of acute pneumonia in guinea pigs and while it is not often noted in modern laboratory facilities, it continues to be a major cause of respiratory disease in pet guinea pigs and this species is thought to be quite susceptible to infection (percy and barthold, 2007; rigby, 1976) . etiology bordetella bronchiseptica is a short, gramnegative rod or coccobacillus, that is aerobic, motile, and non-spore-forming (fox, 2002; percy and barthold, 2007) . b. bronchiseptica is oxidase, catalase, and urease positive, as well as positive for citrate utilization and nitrate reduction (songer and post, 2005; woolfrey and moody, 1991) . b. bronchiseptica is considered a commensal organism in the guinea pig, as well as many other mammalian species, including humans (fox, 2002; percy and barthold, 2007; woolfrey and moody, 1991) . the bordetella genus is a member of the alcaligenaceae family (songer and post, 2005) . epizootiology and pathogenesis b. bronchiseptica is carried by guinea pigs in the nasal cavity and trachea, and up to 20% of non-clinical animals in a colony can be carriers (ganaway, 1976) . stressful events, such as transport or pregnancy, may trigger an acute fatal pneumonia in carriers or animals with previously inapparent infections. transmission of b. bronchiseptica is via aerosol spread, direct contact, direct contact with respiratory secretions of infected animals, or fomites (fox, 2002; ganaway, 1976) . b. bronchiseptica adheres to ciliated respiratory epithelium and causes ciliary paralysis (fox, 2002) . pathogenic b. bronchiseptica produce adhesion and cytolytic toxins that lead to an inflammatory response, antiphagocytic activity, and dermo necrosis. while not explicitly described in the guinea pig, the impairment in mucociliary clearance caused by the binding of bordetella is known to reduce the clearance of additional respiratory pathogens in other animal species. the incubation period is 5-7 days (ganaway, 1976) . the highest levels of morbidity and mortality due to b. bronchiseptica have been noted in young guinea pigs, as well as strain 2 animals (fox, 2002; ganaway, 1976) . it is possible for infected animals to develop immunity and clear the organism. yoda et al. (1972) , noted that within 6-54 weeks of initial infection, 69 of 79 naturally infected guinea pigs completely recovered from infection. clinical manifestations infection with b. bronchiseptica is often subclinical in nature (fox, 2002) . it has been suggested that the clinical signs associated with bordetella in the past have actually been due to infections with a secondary invader, such as guinea pig adenovirus, which has not been recognized as an infectious agent of the guinea pig for as long as b. bronchiseptica. during an epizootic outbreak, death may follow respiratory disease and septicemica within a 24-72-hour period (fox, 2002) . abortion and stillbirths can occur in pregnant dams (percy and barthold, 2007) . in an enzootically infected colony, sporadic deaths may occur throughout the year, with highest levels during the winter months. inappetance, depression, nasal discharge, dyspnea, and cyanosis may also be observed (fox, 2002) . pathology at necropsy, cranioventral pulmonary consolidation is seen, and whole lobes or individual lobules will present as dark red to red-gray in color (ganaway, 1976; percy and barthold, 2007) . a bloodtinged froth is noted in the trachea. mucopurulent exudates may be noted in affected airways. pleuritis and otitis media may be observed. suppurative bronchopneumonia is noted histologically, with an influx of heterophils and mononuclear cells. uterine infections may result in dead fetuses or pyosalpinx. diagnosis b. bronchiseptica can be cultured on blood or macconkey's agar from nasal or other respiratory secretions (songer and post, 2005) . in cases of otitis media, b. bronchiseptica can be isolated from the middle ear, and in cases of metritis, from the uterus (fox, 2002; yoda et al., 1972) at 35-37â°c (songer and post, 2005) . smith and baskerville medium selects for b. bronchiseptica, and is another option for culture (songer and post, 2005) . elisa and ifa tests have been developed for the detection of guinea pig bordetella, and have been noted as equally successful in identifying infected animals as cultures of the respiratory tract (boot et al., 1993a; wullenweber and boot, 1994) . purchasing bordetellafree animals is the best means of control in laboratory colonies of guinea pigs. it is recommended that rabbits and guinea pigs not be co-housed since guinea pigs are highly susceptible to disease and rabbits are frequently sub-clinical carriers of b. bronchiseptica (charles river laboratories, 2011) . rats and other rodents can be additional sources of infection (rigby, 1976) . antibiotic treatment with fluroquinolones or trimethoprimsulfonamides may successfully resolve clinical signs, but elimination of infection is rare, with animals often reverting to the sub-clinical state. if infection is suspected in a colony, restocking, test and cull, or rederivation may be more beneficial than treatment (fox, 2002) . the efficacy of canine, porcine, human, and autogenous bordetella vaccines and bacterins has been evaluated by several individuals; reports suggest that these vaccines do not completely protect guinea pigs from infection, but a decrease in the incidence and severity of clinical disease has been noted in experimentally challenged animals (matherne et al., 1987; stephenson et al., 1989) . b. bronchiseptica apparently does not cause epidemic disease in man, but it can cause a whooping cough-like syndrome in immune-competent children or respiratory tract infections in immune-suppressed individuals (songer and post, 2005) . endocarditis, meningitis, and peritonitis have also been associated with b. bronchiseptica infections in humans. the ease of transmission between guinea pigs and humans in unknown. background and etiology spirochetes of the genus brachyspira (formerly serpulina) have been associated with diarrhea and colitis in a variety of animals including birds and mammals. brachyspira contains seven distinct species. of these, two species have been implicated in guinea pigs, either in natural infections or as an animal model; b. hyodysenteriae, the primary cause of swine dysentery, and b. pilosicoli, a zoonotic agent associated with disease in chickens, pigs, and humans. spirochetes have been associated with disease in guinea pigs in several publications, although purposeful infection to demonstrate causality has not been done and in many cases, identification of the species involved has also not been accomplished (duhamel, 2001) . guinea pigs have been used as an animal model for b. hyodysenteriae infection. two reports in the 1970s describing tyzzer's disease in guinea pigs also report observing spirochetes on histopathology (mcleod et al., 1977; zwicker et al., 1978) . the significance of the spirochete infections in the progression of the disease seen is unclear. a subsequent report describes a series of cases submitted for necropsy between 1992 and 1996 in belgium (vanrobaeys et al., 1998) . in 29 of 33 cases when spirochetes were identified, the presenting complaints were either sudden death or a short course of diarrhea followed by sudden death. flagellated parasites were seen in 12 animals and lesions consistent with vitamin c deficiency in six cases. gross necropsy of affected animals most commonly demonstrated liquid, occasionally mucoid or hemorrhagic large intestinal contents, and dilation of the cecum and colon. histologically, the cecum was characterized by hyperemia, mucosal infiltration of neutrophils, a mixed inflammatory infiltrate in the lamina propria, and occasionally, necrosis of crypt epithelium. lesions involving other organs were present in 30 of the 37 animals identified, suggesting multifactorial disease processes. in a report of a single pet guinea pig, clinical signs of tenesmus, tachycardia, pale mucous membranes, and dehydration were associated with acute rectal prolapse (helie et al., 2000) . histopathology identified superficial mucosal necrosis and ulceration and congestion with hemorrhage in the intussuscepted segment. although spirochetes and organisms consistent with eimeria spp. were seen in the rest of the intestinal tract, no other lesions were noted. b. pilosicoli was identified by pcr. diagnosis diagnosis can be made by identifying typical organisms on histopathology. pcr of intestinal contents is diagnostic (helie et al., 2000) . prevention and therapy treatment with ranidazole stopped the spread of the disease in some cases as described by vanrobaeys et al. (1998) , although cessation of treatment resulted in resumption of disease. as the majority of reports describe co-pathogens, controlling copathogens may be the most effective prevention. brucella spp. are zoonotic, gram-negative, strictly aerobic, intracellular, non-motile coccobacilli, which infect a variety of species, including guinea pigs (martirosyan et al., 2011; pappas, 2010; van hoosier and robinette, 1976) . natural infections in guinea pigs have been described likely associated with contaminated food, bedding, or other biological materials (van hoosier and robinette, 1976) . in these outbreaks, guinea pigs developed disease similar to that seen in other species including testicular and joint swelling as well as abscesses in the liver and pancreas. modern husbandry practices should limit transmission of brucella spp. to guinea pigs. campylobacter jejuni is a species of curved, rod-shaped, non-spore-forming, gram-negative, microaerophilic, bacteria that naturally colonize the digestive track of birds and numerous other species, including domestic farm animals, dogs, cats, various wildlife, and rodents (horrocks et al., 2009 ). it is a common cause of human gastroenteritis worldwide primarily from contamination of food. it was isolated from laboratory animal guinea pigs in one report where the prevalence of c. jejuni in a multispecies laboratory animal facility was investigated (meanger and marshall, 1989) . in this report, no clinical signs were seen in any of the animals involved and pathological analysis was not done. guinea pigs have been used as an animal model of c. jejuni infection (burrough et al., 2009; coid et al., 1987) . background and etiology guinea pig inclusion conjunctivitis was first described in 1964 associated with an experiment to examine the correlation between vitamin deficiency and the development of trachoma virus (murray, 1964) . the organism was identified as a type of chlamydia psittaci, a member of the psittacosis-lymphogranuloma-trachoma group, first thought to be protozoa, then viruses because they could pass through a 450-nm filter, and finally in 1966 recognized as bacteria (longbottom and coulter, 2003; moulder, 1966; songer and post, 2005) . as members of the family chlamydiaceae, they are iii. guinea pigs gram-negative, obligate intracellular pathogens that infect a variety of animals, including amoeba, birds, sheep, humans, and guinea pigs (greenwood, 2007) . recently they have been divided into two genera: chlamydia and chlamydophila. chlamydophila species include c. caviae as well as c. pneumoniae (humans, horses, koalas) and c. psittaci (birds) (everett et al., 1999; murray, 1964) . unlike most bacteria, the cell walls of chlamydiaceae do not have a peptidoglycan layer, but they do have a major outer-membrane protein, which is the determinant for serologic classification (greenwood, 2007; songer and post, 2005) . they also have a unique life cycle which consists of two forms; a small infectious elementary body which is more similar to a spore in that it is environmentally resistant and not metabolically active and a larger metabolically active, non-infectious reticulate body that is responsible for division within the host cell. epizootiology and pathogenesis generally, c. caviae infection is thought to be endemic in guinea pig colonies (percy and barthold, 2007) , although its detection in colonies has been variable. in a survey done in 1964 of five herds of guinea pigs, conjunctival staining revealed the organism in four out of five herds, although not in any guinea pigs less than 4 weeks of age (murray, 1964) . in 2006, two breeding facilities of conventional guinea pigs in korea were examined by conjunctival staining and no positive organisms were seen (park et al., 2006) . a survey of pet guinea pigs with normal conjunctiva, also revealed no positive staining (coster et al., 2008) , which may reflect the sensitivity of the test used or the actual prevalence of c. caviae. in a study using real-time pcr for diagnosis, c. caviae was detected in 12 out of 12 guinea pigs tested, four out of four rabbits, and one dog from samples obtained from a diagnostic laboratory (pantchev et al., 2010) . c. caviae in a second report was identified via pcr from conjunctival swabs from a rabbit, cat, and human that lived in the same household as infected guinea pigs (lutz-wohlgroth et al., 2006) . also utilizing pcr and dna sequencing, c. caviae was detected in foals with conjunctivitis, rhinitis, and coughing, indicating the host range of the species of chlamydiaceae may continue to be refined as new diagnostic tests are utilized (gaede et al., 2010) . one study identified acanthamoebae spp. via pcr from two guinea pigs with conjunctivitis that were also pcr positive for c. caviae (lutz-wohlgroth et al., 2006) . their role in the disease process is unclear. urogenital disease has been reported in natural infections with c. caviae and has been reproduced in experimental models (lutz-wohlgroth et al., 2006; mount et al., 1973) . in experimental infections, inoculation with c. caviae in the lower genital tract of female guinea pigs causes a self-limiting infection lasting 3-4 weeks. typically, the infection involves the epithelium of the cervix with very little inflammation. however, in 80% of the animals, the infection progresses to involve the oviducts, with half of the animals developing pathologic changes in the oviducts including inflammation and fibrosis rank and sanders, 1992) . infection can be transferred from infected male animals to females during sexual intercourse and vertically from dam to pups (padilla-carlin et al., 2008; miyairi et al., 2010) . in experimental infections, both cell-mediated and humoral immunity are important in recovery and resistance to re-infection (miyairi et al., 2010) . early infection is characterized by influx of polymorphonuclear cells while later in infection, lymphocytes predominate. t-cell depletion results in chronic infection and delayed clearance of the organisms. decreased antibody response also correlates with delayed clearance of the organisms (miyairi et al., 2010; rank et al., 1979) . clinical manifestations c. caviae has been primarily associated with conjunctivitis. guinea pigs have reddened, swollen conjunctiva with serous to purulent discharge, follicular hypertrophy, and pannus (deeb et al., 1989; lutz-wohlgroth et al., 2006; murray, 1964; padilla-carlin et al., 2008; strik et al., 2005) . generally the conjunctivitis resolves in 3-4 weeks. occasionally, conjunctivitis is accompanied by rhinitis. in one study, nasal discharge was present in 31 of 39 of the animals with ocular discharge (lutz-wohlgroth et al., 2006) . vaginal discharge was seen in 25% (ten of 39) of the animals although the organism could not be recovered by pcr from either the nose or the vagina. abortion was recorded in two animals. an early study also reported rhinitis, bronchopneumonia and abortions, although this study was complicated by infection with streptococcus pneumonia and bordetella bronchiseptica (schmeer et al., 1985) . pathology histological evaluation of the eyes and surrounding tissue reveals a mixed inflammatory response and congestion beneath the conjunctival epithelium (deeb et al., 1989; lutz-wohlgroth et al., 2006) . histological examination of the urogenital tract has primarily been described following experimental infection and consists of inflammation of the endometrium, mesosalpinx, and oviduct with a mixed inflammatory infiltrate and fibrosis rank and sanders, 1992) . diagnosis diagnosis is by cytology, pcr, culture, tissue assays, and serology (songer and post, 2005) . giemsa, gimenez, or macchiavello stains on cytologic preparations reveal inclusions in the cytoplasm. inclusions are a dark purple with giemsa and red with gimenez or macchiavello stains. cytology can be insensitive hence pcr can be used in addition (pantchev et al., 2010; strik et al., 2005) . in one study, 31 samples were tested via cytology and pcr (lutz-wohlgroth et al., 2006) . twenty-one were positive by pcr for c. caviae, however, no samples were positive by cytology. as c. caviae is an obligate intracellular pathogen, it cannot be cultured in cell-free media, but only on specific cell lines (songer and post, 2005) . this is an insensitive and difficult process that is not typically used for diagnosis. direct fluorescent antibody test and enzyme immunoassays can be used to detect antigens in cytology samples or tissues (percy and barthold, 2007; songer and post, 2005) . prevention and therapy generally, the disease is self-limiting and treatment is not required (percy and barthold, 2007; quesenberry and carpenter 2004) . no current vaccine prevents chlamydial infection, although vaccination studies have demonstrated a decrease in the intensity of the disease following vaccination (padilla-carlin et al., 2008) . recognition that zoonotic transmission is a possibility should guide appropriate personal protective equipment choices (lutz-wohlgroth et al., 2006) . citrobacter is a genus of gram-negative coliform bacteria of the family enterobacteriacae (boone et al., 2001) . in the 1960s and 1970s, a disease eventually named transmissible murine colonic hyperplasia caused by coliform bacteria of the genus citrobacter was recognized (mundy et al., 2005; percy and barthold, 2007; petty et al., 2010) . originally described as unusual biotypes of citrobacter freundii, these organisms were eventually separated into their own species, citrobacter rodentium, on the basis of dna sequencing studies (luperchio et al., 2000; petty et al., 2010) . whether the organisms that caused severe septicemia and death in guinea pigs in 1988 (ocholi et al., 1988) and other disease syndromes in hysterectomy-rederived guinea pigs (boot and walvoort, 1986) are c. freundii as originally indicated or c. rodentium is unclear. pathology there are two reports of disease associated with citrobacter spp. in guinea pigs (boot and walvoort, 1986; ocholi et al., 1988) . first, citrobacter spp. were isolated from germ-free guinea pigs that were exposed to defined flora from gnotobiotic mice (boot and walvoort, 1986) . citrobacter spp. were isolated from the middle ear, spleen, mammary gland, and kidney of some animals as well as the intestinal tract, suggesting an ability to cause varied disease in guinea pigs that are not populated with normal flora. second, an epizootic of citrobacter spp. septicemia in a large colony of conventional guinea pigs was reported (ocholi et al., 1988) . animals presented with diarrhea, dyspnea, and decreased appetite. mortality in weanlings and breeders was 115 of 1300 animals. gross necropsy revealed lung consolidation with pleural adhesions as well as enteritis and thickening of the intestinal wall. fibrinous pneumonia and septic thrombi in the capillaries of the lung, liver, and spleen were seen histopathologically. citrobacter spp. were consistently isolated in pure culture from the lung, liver, spleen, and intestine. diagnosis culture is the traditional method of detecting citrobacter spp. which grow well on normal media, are aerobic or facultative anaerobic, ferment glucose, produce catalase, but not oxidase, and are generally lactose-negative or later lactose-fermenting (greenwood, 2007) . however, pcr is more sensitive than bacterial culture for detection of c. rodentium in feces from mice (mckeel et al., 2002) . prevention and therapy c. freundii strains utilize a number of methods to encode resistance to ampicillin and cephalosporins (pfeifer et al., 2010) . enrofloxacin has been successful in treating mice infected with c. rodentium (maggio-price et al., 1998) . prevention would include eliminating contact with c. rodentium-infected mice and other rodents. clostridium piliforme (tyzzer's disease) background clostridium piliforme, first described in 1917 (tyzzer, 1917) , causes severe disease in many laboratory animals as well as other mammals (percy and barthold, 2007) . although rare in modern colonies, outbreaks in mice, rats, rabbits, and guinea pigs have been reported and c. piliforme is found in both wild (wobeser et al., 2009 ) and domestic mammals (borchers et al., 2006; ikegami et al., 1999) . while tyzzer's disease is not generally considered zoonotic, infection of an hivpositive patient has been reported (smith et al., 1996) . etiology clostridium piliforme (formerly bacillus piliformis) is a gram-negative, spore-forming, anaerobic rod that can survive for as long as five years in the environment (percy and barthold, 2007; songer and post, 2005) . pathogenesis c. piliforme is thought to be transmitted via fecal-oral transmission. in experimental models, oral inoculation was successful when other routes of inoculation did not result in disease (waggie et al., 1987) . vertical transmission has been reported in one case of hysterectomy-rederived guinea pigs (boot and walvoort, 1984) . in outbreaks of tyzzer's disease in other species, usually predisposing factors such as stress, overcrowding, poor sanitation, or immunemodulating treatments contribute to the development of disease (percy and barthold, 2007 (boot and walvoort, 1984; mcleod et al., 1977; zwicker et al., 1978) typically seen in weanlings. subclinical infections have been noted (boot and walvoort, 1984) . pathology lesions are usually found only in the gastrointestinal tract in young guinea pigs, although liver lesions have been reported in some animals (sparrow, 1978; waggie et al., 1987) . grossly, pinpoint gray foci occur on the ileum, cecum, and colon as well as enlarged, reddened mesenteric and colonic lymph nodes (mcleod et al., 1977; sparrow, 1978; waggie et al., 1987; zwicker et al., 1978) . white to tan pinpoint foci are also observed on the liver. microscopically, foci of necrosis occur in the mucosa of the ileum, cecum, and colon as well as foci of periportal coagulative hepatocellular necrosis when the liver is involved. clusters of intracellular organisms can be observed via silver stain or giemsa stain in affected enterocytes and hepatocytes. in two reports, affected animals have also had large numbers of spirochetes evident at necropsy (mcleod et al., 1977; zwicker et al., 1978) . diagnosis c. piliforme cannot be cultured using standard media, although it can be isolated by inoculation of cell lines, embryonated hen's eggs, and primary mouse or chick embryo cell cultures (niepceron and licoism, 2010; percy and barthold, 2007) . therefore, diagnosis typically relies on characteristic lesions and organisms observed on histopathology using a warthin-starry silver stain or giemsa stain (percy and barthold, 2007) . pcr has been used to detect c. piliforme in feces from rats (furukawa et al., 2002) . nested pcr was developed to increase the specificity of detection in tissues, better separating c. piliforme from other closely related organisms used conserved regions of 16s ribosomal rna (niepceron and licois, 2010). serology to detect antibodies has been used to monitor colonies for this organism (boot and walvoort, 1984; kraft and meyer, 1990) . treatment and control treatment of individual animals has not been reported. tyzzer's disease affects a wide variety of animals and immune-compromised people. prevention entails modern husbandry and sanitation practices, preventing contamination of food and bedding, and separation of species. escherichia coli are aerobic, straight gram-negative rods and are part of the normal intestinal flora of most vertebrates, including guinea pigs (crecelius and rettger, 1943; songer and post, 2005) . several pathotypes have been reported (e.g. epec-enteropathogenic strains), some which produce cytotoxins. diarrhea, rough hair coats, wasting and death have been reported to be associated with e. coli infection in conjunction with other environmental stressors (ganaway, 1976) . in addition, there is one report of a compilation of cases of mastitis in guinea pigs from a diagnostic lab over a four year period (kinkler et al., 1976) . of the 26 cases where cultures were taken, e. coli was found in 17 cases with the next most common isolate being klebsiella pneumonia in six cases. diagnosis may be obtained by culture of affected organs on macconkey agar and differentiation from other enteric gram-negative bacteria by a number of biochemical reactions such as indole production and utilization of certain sugars (greenwood, 2007) . background perkins (1901) described an epizootic outbreak in 25 laboratory guinea pigs that resulted in the death of all but two animals 18-24 hours after the onset of disease. a short bacillus, often paired, was isolated in the blood, spleen, liver, and peritoneal exudates. disease was reproduced in naã¯ve guinea pigs subsequently administered the bacterium by the intraperitoneal route. perkins' findings were similar to those of howard (1899), who inoculated dogs, rabbits, mice, pigeons, and guinea pigs with a bacillus organism isolated from human patients. etiology klebsiella pneumoniae is a gram-negative, rod-shaped bacillus from the genus klebsiella and family enterobacteriaceae (boone et al., 2001) . k. pneumoniae is facultatively anaerobic, oxidase-negative, and produces acid and gas from lactose. it is an enteric bacterium, noted in the intestinal tract of 5% of healthy humans (ganaway, 1976) . it can also reside in the skin and mouth. k. pneumoniae causes pneumonia in guinea pigs of both sexes and all ages, although there are few reports of natural infections of this animal species with this organism. clinical manifestations in the epizoonotic outbreak described by perkins, animals presented as anorexic and ungroomed, and their illness escalated in severity until, in some animals, a comatose condition ensued (perkins, 1901) . animals usually died 18-24 hours after the onset of clinical signs. in other epizootic outbreaks, dyspnea has been reported prior to reaching the comatose state (ganaway, 1976) . less severely infected animals may be able to recover from disease and develop immunity to re-infection (perkins, 1901) . otitis media and other signs consistent with a state of septicemia have been associated with clinical disease (fox, 2002; ganaway, 1976; kohn, 1974) . pathology seropurulent or serofibrinous peritonitis is noted at necropsy (fox, 2002; ganaway, 1976; perkins, 1901) . additionally, such exudates may be observed in the pleural cavity and pericardial sac. congestion of the liver, spleen, and kidney has been noted, as has hepatic coagulative necrosis and degeneration of the renal tubule cells. an acute, necrotizing bronchopneumonia may be observed. diagnosis diagnosis of k. pneumonia is usually via culture of the upper respiratory tract. the bacterium has been isolated from blood, spleen, liver, peritoneal exudate, and cerebrospinal fluid (fox, 2002; ganaway, 1976; perkins, 1901) . k. pneumoniae can be differentiated from k. oxytoca, another klebsiella spp. frequently recovered in animals, by the fact that it is indole-negative and does not grow at 10â°c, while k. oxytoca is indolepositive and does grow at 10â°c. antibiotic treatment for klebsiella infection should be based on sensitivity of culture isolates. ceftriaxone and cefodizime were found effective at clearing k. pneumoniae in experimentally infected guinea pigs (drago et al., 1999) , although generally, chloramphenicol, enrofloxacin, trimethoprimsulfa, and aminoglycoside drugs are considered the safest antibiotic choices for use in guinea pigs. background and etiology proliferative enteritis associated with campylobacter-like organisms has been reported in many species including pigs, hamsters, rats, rabbits, foals, nhps, ferrets, and deer (lawson and gebhart, 2000) . in the early 1990s, molecular techniques resulted in the identification of lawsonia intracellularis as the causative agent of many of these syndromes. since that time, diagnostics, including pcr, have enabled the diagnosis of l. intracellularis infection (collins et al., 2011) . pathology there have been two reports of disease in guinea pigs associated with campylobacter-like organisms, both prior to the identification of l. intracellularis. in 1981, one 3.5-month-old guinea pig, treated with steroids for 90 days as part of a study, displayed no clinical signs of disease but on necropsy had marked duodenal mucosal hyperplasia with intracellular warthin-starry stained argyrophilic bacteria in affected epithelial cells (elwell et al., 1981) . two cagemates of this guinea pig, that had the same steroid treatment, died at days 13 and 30. one had diarrhea for two days prior to death. histopathology revealed acute enteritis with mucosal erosions without mucosal hyperplasia in both animals. bacteria similar to that seen in the first case were found in the epithelial cells. the authors noted this organism was very similar to the organism associated with transmissible mucosal hyperplasia of hamsters (subsequently identified as l. intracellularis). in 1983, two dams and five young guinea pigs in a laboratory in japan developed diarrhea, anorexia, weight loss, and dehydration. two young animals died 5-7 days post-onset of clinical signs. grossly, there was thickened jejunum and ileum. histopathologically, there was hyperplasia and proliferation of the epithelial cells and dilation of the glands primarily in the ileum and the distal part of the jejunum. immature epithelial cells contained various numbers of intracytoplasmic, non-membrane-bound, curved organisms resembling campylobacter spp. bacteria. diagnosis characteristic histopathological lesions, immunohistochemistry, and pcr are diagnostic (collins et al., 2011; lawson and gebhart, 2000) . antibiotics have been used to treat a similar proliferative enteritis in swine with success (lawson and gebhart, 2000) . care should be taken to avoid penicillins and other antibiotics implicated in antibiotic-associated dysbacteriosis (percy and barthold, 2007) . background and etiology leptospira are tightly coiled, aerobic, gram-negative, flagellated spirochetes belonging to the genera leptospira, family leptospiraceae, phylum, spirochaetes (boone et al., 2001) . before 1989, the genus, leptospira, was divided into two species, l. interrogans (pathogenic) and l. biflexa (non-pathogenic) which each contained strains separated into numerous serovars (levett, 2001) . more recently, dna analysis has identified 20 species of leptospira and revisions of these taxonomic groups are ongoing (galloway and levett, 2010; levett, 2001) . leptospira are zoonotic organisms with a world-wide distribution that infect a variety of mammals, including mice, rats, carnivores, and ruminants (ko et al., 2009; levett, 2001; percy and barthold, 2007) . wild guinea pigs in south america (cavia porcellus apera), infected with leptospira spp., have been implicated as the natural reservoir responsible for transmitting the disease to domestic cattle (blood et al., 1963) . in domestic guinea pigs, there have been only a few reports of natural infection, although the guinea pig infection models have been used extensively in leptospira research (ko et al., 2009; van hoosier and robinette, 1976) . data from research models and infections in other species indicate that transmission occurs from direct contact with infected urine, soil, or water as leptospira are excreted in the urine (ko et al., 2009; lourdault et al., 2009 ). a report from 1937 describes a natural infection where one out of 12 guinea pigs died from leptospirosis (ganaway, 1976) . pathological findings include jaundice, petechial hemorrhages in the skin, fascia, and muscles, and ecchymosis in the lung. a more recent report of a case of leptospirosis in a 21-year-old man, described 20 out of 50 guinea pigs that died on his farm in germany. three of the remaining guinea pigs were tested and were serologically positive for leptospira serovar bratislava similar to the man. no necropsies of affected animals were described. diagnosis direct examination of infected tissue, urine, or blood with dark-field microscopy, immunoflourescence, or light microscopy with special stains is useful for diagnosis (greenwood, 2007; levett, 2001) . culture and pcr are additional diagnostic tools. growth in culture can be slow. generally, antibody detection is used to demonstrate previous infection. antimicrobial treatment of individual animals is generally successful in other species (pischke et al., 2010; van de maele et al., 2008) , although in guinea pigs, care must be taken to avoid antibiotic-associated dysbacteriosis (percy and barthold, 2007) . prevention includes preventing exposure to wild rodents and leptospira-infected food, bedding, soil, or water. background listeria monocytogenes was first discovered in 1926 by murray et al. (1926) , who isolated it from laboratory rabbits and guinea pigs. since that time, few cases have been reported in guinea pigs, although guinea pigs are very susceptible and have been used extensively to model the pathogenesis of listeriosis (cossart, 2007; disson et al., 2009; fox, 2002) . etiology listeria monocytogenes is a facultative intracellular, non-spore-forming, zoonotic, food, soil-, and silage-borne bacterium which causes disease in birds and many mammals. pathology in spontaneous infections of guinea pig colonies, l. monocytogenes infection has been correlated with acute death, reproductive disorders such as abortions and still births, as well as conjunctivitis (chukwu et al., 2006; colgin et al., 1995; fox, 2002) . in an outbreak associated with feeding infected greens to a colony of 80 guinea pigs, the mortality rate was reported to be 80-100% with lung and liver lesions predominating (chukwu et al., 2006) . conjunctivitis was reported in one outbreak affecting 11 out of 49 newly arrived guinea pigs (colgin et al., 1995) . l. monocytogenes was cultured from 10 of 12 specimens submitted. the animals presented with unilateral or bilateral serous to purulent discharge with hyperemic conjunctiva. histologically, there was corneal ulceration, stromal edema, and vascular proliferation. the conjunctiva contained an inflammatory infiltrate and the lacrimal gland had multifocal areas of inflammation and necrosis. intracytoplasmic gram-positive rods were seen in the conjunctival epithelium and no chlamydial organisms were identified on conjunctival scrapings or by histopathology. experimentally, guinea pigs have been shown to be susceptible to listeriainduced conjunctivitis. pasteurella multocida background pasteurellosis is rare in presentday guinea pig laboratory colonies. reports of epizootic outbreaks due to gram-negative coccobacilli were reported in europe in the late 1800s (wright, 1936) . etiology pasteurella multocida, a gram-negative, non-spore-forming and non-motile rod is a member of the pasteurella genus and the pasteurellaceae family (songer and post, 2005) . spp. are mucosal commensal organisms of the oropharynx and gastrointestinal tract of many species, including man (songer and post, 2005) . when stressed, the animal's immune system can be overwhelmed and, in guinea pigs, pneumonia may result. p. multocida can be transmitted to man via contact with infected saliva, which is likely to occur as a result of a bite wound or scratch. clinical manifestations wright detailed an outbreak of pasteurellosis that killed 600 guinea pigs over a 1-year period (wright, 1936) . clinical signs were not noted prior to death. p. multocida has been isolated from cases of conjunctivitis (percy and barthold, 2007) . pathology fibrinopurulent serositis is present in cases of guinea pig pasteurellosis (fox, 2002; ganaway, 1976; wright, 1936) . such serositis has been noted in the pericardium, pleura, and peritoneum. lung consolidation may also be observed (fox, 2002) . diagnosis pasteurella infection is diagnosed via culture. the bacteria do not reliably grow well on macconkey agar, therefore blood agar is routinely used for isolation (ganaway, 1976; songer and post, 2005) . plates should be incubated at 3-5% co 2 at 37â°c for 24-48 hours (songer and post, 2005) . in animal tissue or fluids, p. multocida appears as a bipolar rod after aniline dye staining, but in a smear taken from culture, it stains evenly (ganaway, 1976) . unlike p. pneumotropica, p. multocida is o-nitrophenyl-ã�-d-galactoside (onpg) and urease-negative (songer and post, 2005) . p. pneumotropica is also positive for the fermentation of mannose, while p. multocida is negative. an elisa-based assay has been developed to monitor antibodies to the sp group of pasteurellaceae, which is one of five serologically distinct groups of pasteurellaceae in guinea pigs (boot et al., 1995) . prevention and therapy antibiotic treatment of p. multocida should be based on culture sensitivities. antimicrobial therapy will eliminate clinical signs, but will not eliminate the organism from the colony (songer and post, 2005) . background scherago (1937) reported on a fatal epizootic septicemia in a colony of young guinea pigs received from a local breeder. animals died within 5-6 hours after showing clinical signs and "gram-negative, non-spore-forming encapsulated rods with rounded ends and straight sides appearing singly and occasionally in pairs end to end" were found on smears from the peritoneal cavities of all examined animals. the route of infection was not determined in this colony, but scherago did complete the required steps to fulfill koch's postulates and demonstrated that the infection was due to pseudomonas caviae. p. caviae has since been reclassified as aeromonas caviae, of the aeromonadaceae family. pseudomonas aeruginosa was noted as the cause of pulmonary botryomycosis in two guinea pigs (bostrom et al., 1969) . etiology p. aeruginosa, a member of the genus pseudomonas, is part of the pseudomonadaceae family (songer and post, 2005) . pseudomonas spp. are gramnegative rods that are aerobic, non-spore-forming, oxidase-positive, and non-fermentative. pseudomonas organisms thrive in aqueous environments (songer and post, 2005) . additionally, they are ubiquitous in soil, decaying organic matter, and vegetation. pseudomonas spp. are opportunistic pathogens of both humans and animals, yet, there are few reports of infections caused by these agents in guinea pigs (fox, 2002) . bostrum et al. (1969) , did not describe the clinical signs associated with the epizootic outbreak of pulmonary botryomycosis. otitis media, conjunctivitis, and an inflamed prostate gland are signs that have been associated with pseudomonas in other reports of guinea pig infection (fox, 2002) . pathology a focal, necrotizing bronchopneumonia, along with pulmonary consolidation has been described in cases of guinea pig pseudomonas infection (bostrom et al., 1969; fox, 2002; ganaway, 1976; percy and barthold, 2007) . bostrum noted atypical "sulfur granules", resembling the spherules of coccidioides immitis, in the lungs of affected animals (bostrom et al., 1969) . diagnosis p. aeruginosa can be cultured from infected tissues (songer and post, 2005) . p. aeruginosa grows well on macconkey agar and trypticase soy agar. additionally, cetrimide agar, a commercial formulation, selectively isolates p. aeruginosa. the best means of controlling pseudomonas is to reduce possible sources of infection, such as damp bedding or stagnant water. animal drinking water should be monitored for p. aeruginosa, and in some situations, water may be chlorinated to eliminate contamination (songer and post, 2005) . reducing stressors, such as overcrowding and concurrent infection, also aids in control. pseudomonas spp. have an inherent resistance to many antimicrobials. salmonella background some of the earliest reports of salmonellosis in guinea pigs date back to the 1800s (nelson and smith, 1927) . multiple outbreaks of natural infections in guinea pig colonies in the united states were documented throughout the early 1900s (holman, 1916; nelson and smith, 1927) . since this period of time, as laboratory standards have grown more stringent and general hygiene and sanitation have improved, the occurrence of salmonellosis in laboratory guinea pigs has declined greatly (fox, 2002; harkness and wagner, 1995; percy and barthold, 2007) . etiology members of the genus salmonella are gram-negative, non-spore-forming bacilli that are facultative anaerobes. salmonella other than s. typhi or s. paratyphi produce hydrogen sulfide, as well as acid and gas from glucose, but not lactose (ganaway, 1976; songer and post, 2005) . salmonella is a member of the enterobacteriaceae family. while multiple serovars have been recovered from guinea pigs, the most frequently isolated are s. typhimurium and s. enteritidis (fox, 2002; ganaway, 1976; harkness and wagner, 1995; quesenberry and carpenter, 2004; percy and barthold, 2007) . humans are susceptible to disease from the same serovars and phage types as guinea pigs (fish et al., 1968; percy and barthold, 2007; rigby, 1976 ). pathogenesis guinea pigs are extremely susceptible to salmonella (fox, 2002) . transmission of salmonella is primarily fecal-oral or via ingestion of contaminated feed (contaminated with feces of infected guinea pig or a wild rodent) (fox, 2002; harkness and wagner, 1995; percy and barthold, 2007; quesenberry and carpenter, 2004) . entry via the conjunctiva has also been reported in guinea pig epizootics (iijima et al., 1987; moore, 1957) . additionally, ingestion of contaminated blood or tissue can transmit salmonella (fox, 2002) . stressed animals, due to conditions such as weaning, parturition, old age or poor nutrition, are more susceptible to infection (quesenberry and carpenter, 2004) . recovered animals can develop an asymptomatic carrier state with intermittent shedding in the feces, making elimination of this bacterium from a colony difficult (fox, 2002; harkness and wagner, 1995; percy and barthold, 2007) . the incubation time for salmonella is 5-7 days and epizootic or enzootic patterns of infection are possible (fox, 2002; ganaway 1976) . salmonella occurs as peracute septicemia or acute, subacute, or chronic enteritis (songer and post, 2005) . acute disease is characterized by high morbidity and low to moderate mortality. clinical signs include depression, weakness, and fever. dehydration and electrolyte imbalance may be severe. infection spreads rapidly within a colony. conjunctivitis and abortions have also been noted (percy and barthold, 2007) . although weight loss is present, diarrhea may or may not occur in guinea pigs. in an epizootic outbreak, young guinea pigs may die suddenly without prior signs of illness. pathology in acute and peracute cases, there may be no lesions seen at necropsy (harkness and wagner, 1995; percy and barthold, 2007) . splenomegaly may be observed in subacute and chronic infections. minute white foci and/or yellowish-white nodules, up to several millimeters in diameter, may occur in the spleen, liver, or other lymph nodes. similar foci or nodules may also occur in the lung, pleura, peritoneum, and in the wall of the uterus. rupture of the nodules may result in a suppurative pleuritis, peritonitis, and/or pericarditis. gas may be observed in the small and large intestine, along with catarrhal enteritis (fish et al., 1968) . on histopathology, lesions support bacterial invasion, with subsequent necrosis and abscess formation (ganaway, 1976) . nodules are noted to be "typhoidlike" granulomas, with areas of central necrosis, surrounded by histiocytes. polymorphonuclear leukocytes and histiocytes are present in the peyer's patches of the ileum and jejunum. diagnosis salmonella can be isolated from blood, feces, and affected organs (most commonly from the spleen) (fox, 2002) . recovery requires special conditions: enrichment in a broth such as selenite-f or tetracycline and culture at 37â°c on macconkey's or brilliant green agar (fox, 2002; songer and post, 2005) . (harkness and wagner, 1995; rigby, 1976) . all fresh fruit and vegetables should be thoroughly washed and stored in airtight containers. since so many species of mammals are potential carriers, the exclusion of wild rodents, birds, and other vermin is essential. when increased colony mortality is apparent and a chronic case is recognized at necropsy, salmonella infection is likely widespread and well established in the colony. in situations such as this, it is reasonable to depopulate the colony, sanitize the premises, and restock with salmonella-free guinea pigs. treatment on an individual basis will include the appropriate antibiotics with fluid therapy. animals that become asymptomatic after treatment may become carriers, a problem with zoonotic potential. antimicrobial resistance is expanding; 90% of salmonella isolates from domestic animals demonstrate multi-drug resistance (songer and post, 2005) . background streptobacillus moniliformis is one of the causative agents of rat bite fever, a significant worldwide, zoonotic, systemic infection of humans associated with contact with rats (gaastra et al., 2009; khatchadourian et al., 2010) . it was described in early recorded history in india and was recognized in the us as early as 1839 as a syndrome that developed following contact with rats (gaastra et al., 2009) . s. moniliformis also causes haverhill fever, a systemic infection of those who eat food contaminated with rat urine or feces (abdulaziz et al., 2006; mcevoy et al., 1987; shanson et al., 1983) . etiology streptobacillus moniliformis, a facultative anaerobic, non-motile, non-capsulate, pleomorphic, gram-negative rod, is the type species of the genera, streptobacillus, family leptotrichiaceae within the phylum fusobacteria (greenwood, 2007; nolan et al., 2009) . its genome has been completely sequenced (nolan et al., 2009) . epizootiology and pathogenesis s. moniliformis is commonly found in the nasopharynx of wild and pet rats (gaastra et al., 2009; wullenweber, 1995) , although typically excluded from modern spf laboratory animal facilities (nicklas et al., 2002; pritchett-corning et al., 2009) . the organism has also been found in carnivores, calves, pigs, turkeys, and a variety of rodents including guinea pigs presumably following contact with infected rats (gaastra et al., 2009; wullenweber, 1995) . most commonly, s. moniliformis in guinea pigs has been associated with cervical lymphadenitis and other localized abscesses (aldred et al., 1974; fleming, 1976; percy and barthold, 2007; van hoosier and robinette, 1976; wullenweber, 1995) . severe necrotizing bronchopneumonia with pyogranuloma formation was reported in one guinea pig by kirchner et al. (1992) . no clinical signs of disease were noted in their report. diagnosis gram stain of smears can be suggestive of infection by demonstrating short bacilli in chains or long filaments followed by bacterial culture and isolation (greenwood, 2007; kirchner et al., 1992; wullenweber, 1995) although because of the fastidious nature of s. moniliformis, pcr is more commonly used for detection . if culture is attempted, samples must be cultured on a media containing serum, blood or ascitic fluid, such as loeffler's serum medium (greenwood, 2007) . cross-reactive sequences between leptotrichia spp. and s. moniliformis have been demonstrated and can result in false positives when pcr is used for diagnosis (boot et al., 2008; wouters et al., 2008) . sequence of the amplicon can differentiate these outcomes. as the entire sequence of s. moniliformis has recently been completed (nolan et al., 2009) , new primers for diagnosis may be forthcoming. antibodies to s. moniliformis can be demonstrated by elisa (boot et al., 1993b (boot et al., , 2006 . because the organism is not spread easily, outbreaks can be controlled by culling affected guinea pigs (van hoosier and robinette, 1976) . treatment of individual animals with antibiotics is effective in other species (gaastra et al., 2009; wullenweber, 1995) , although treatment should be approached with caution because of the zoonotic potential of this organism. prevention of contact with infected rats, other animals that contact wild or infected rats, or infected food and/or bedding should eliminate introduction of this organism (van hoosier and robinette, 1976) . yersinia pseudotuberculosis background ganaway states that "one of the earliest recognized bacterial disease of the guinea pig was caused by yersinia pseudotuberculosis" (ganaway, 1976) . in present day colonies of laboratory guinea pigs, natural outbreaks of pseudotuberculosis are rare (fox, 2002; percy and barthold, 2007) . etiology yersinia pseudotuberculosis, an ubiquitous enteropathogen, is a gram-negative, non-spore-forming, facultative anaerobic rod that is oxidase-negative and catalase-positive (ganaway, 1976) . it is non-hemolytic and produces both exotoxin and enzyme (fox, 2002) . growth is optimal at 20-30â°c (fox, 2002; ganaway, 1976) . the genus yersinia is part of the family enterobacteriaceae and, in addition to guinea pigs, it causes pseudotuberculosis in other rodents, cats, and turkeys (songer and post, 2005) . epizootiology and pathogenesis y. pseudotuberculosis can result in clinical disease or a sub-clinical carrier state. guinea pigs are very susceptible to infection with this bacterium (fox, 2002; ganaway, 1976) . y. pseudotuberculosis is shed in feces, therefore ingestion of food contaminated with feces from a carrier guinea pig, wild rodent, or bird can transmit the organism, as can aerosol spread, or entry via a bite wound or skin laceration (fox, 2002; ganaway, 1976; rigby, 1976) . dams can pass infection to their pups (fox, 2002) . orally absorbed organisms disseminate from the intestinal tract to the mesenteric lymph nodes. acute and chronic forms of disease have been described (ganaway, 1976; rigby, 1976 ). an acute septicemic form of infection can lead to mortality in 24-48 hours. a chronic infection that often lasts several weeks to months and ultimately results in death is the more classic pseudotuberculosis presentation. the classic clinical presentation associated with y. pseudotuberculosis is that of wasting and lymphadenitis, sometimes with accompanying diarrhea, and frequently resulting in death about a month after initial infection (obwolo, 1977; sebesteny, 1976) . the acute form of the disease can present with no clinical signs or consist of one to two days of coughing and respiratory distress prior to death (rigby, 1976) . pathology necropsy of animals chronically or subacutely infected with yersinia pseudotuberculosis reveals nodules throughout the body, including the regional lymph nodes, spleen, liver, lung, and bone marrow (fox, 2002; percy and barthold, 2007) . the grayish-white spherical nodules vary from miliary pinpoint to 2-3 cm in diameter and may contain creamy to caseous exudate. in acute cases, nodules are noted in the intestinal wall, especially at the location of the terminal ileum and cecum, and often with accompanying mucosal ulceration and acute enteritis (percy and barthold, 2007) . lung congestion may be seen in acute cases (rigby, 1976) . yersinia nodules often have a central area of necrosis with neutrophilic infiltration; foamy macrophages are often noted in the periphery (fox, 2002; ganaway, 1976 ). blood vessels within the nodule may contain bacterial emboli. in chronic lesions, fibroblasts and epithelial cells proliferate and nodules may become granulomatous, but do not calcify. diagnosis y. pseudotuberculosis can be cultured from abscesses (rigby, 1976) . direct smears may also iii. guinea pigs be beneficial. in acute septicemic cases, yersinia may be cultured from the blood (ganaway, 1976) . yersina spp. can be cultured on standard media and y. pseudotuberculosis can be differentiated from y. pestis, the causative agent of plague, by the fact that it is urease, and rhamnose-positive, while y. pestis is negative for both of these growth conditions (songer and post, 2005) . treatment and control euthanasia of infected animals is advised due to the fact that antibiotic treatment can result in a carrier state, which, due to its zoonotic potential, puts human handlers and staff at risk. a killed vaccine has been unsuccessful at protecting guinea pigs from infection, but a more recent live vaccine has produced promising results (quintard et al., 2010) . background dermatophytosis, also known as ringworm or tinea, indicates infection with pathogenic fungi in the phylum ascomycota that cause the bulk of superficial fungal infections in humans and animals (ameen, 2010; howard et al., 2003) . the fungi are keratinolytic and keratinophilic and infection is usually limited to cornified layers of the skin, hair, or nails (howard et al., 2003; weitzman and summerbell, 1995) . their importance as pathogens is clear in that the majority of dermatophytes infecting animals are zoonotic (chermette et al., 2008) . originally, the dermatophytes were organized into three anamorphic, or asexual, genera, microsporum, trichophyton, and epidermophyton. the primary structure associated with asexual reproduction is the conidium and classification into these genera was based on the morphology of the macroconidia in culture. subsequent studies revealed sexual reproduction in certain microsporum and trichophyton spp. that occurs by means of an ascospore found within an ascus. the dermatophyte sexual states (teleomorphs) are classified in the genus, arthroderma (weitzman and summerbell, 1995) . recent efforts at phylogenetic analysis and identification of dermatophytes have been achieved by sequencing 28s ribosomal dna (rdna) and internal transcribed spacer (its) regions flanking 5.8s rdna (drouot et al., 2009; frealle et al., 2007; symoens et al., 2011) . dermatophyte species can also be divided into three groups determined by their adaptation to a certain habitat. humanassociated dermatophytes are anthropophilic (e.g. trichophyton rubrum), those primarily found on animals and occasionally infecting humans are zoophilic (e.g. trichophyton mentagrophytes), and species persisting in the soil are termed geophilic (e.g. microsporum gypseum) (weitzman and summerbell, 1995) , however some overlap does occur. zoophilic and geophilic dermatophytes infecting humans cause a more intense inflammatory reaction than anthropophilic species (weitzman and summerbell, 1995) . etiology dermatophytosis in animals is most often caused by the anamorph species microsporum and trichophyton, however the infecting species can vary with the geographic region (bond, 2010) . of these genera, trichophyton mentagrophytes is the most common cause (aho, 1980; balsari et al., 1981; drouot et al., 2009; feuerman et al., 1975; lopez-martinez et al., 1984; mcaleer, 1980a mcaleer, , 1980b papini et al., 1997; pombier and kim, 1975; stenwig, 1985; vangeel et al., 2000) . t. mentagrophytes is a species complex with several variants including both anthropophilic and zoophilic pathogens. pollock (2003) considered t. mentagrophytes var mentagrophytes as the most common variant infecting rodents, however the specific variant is not always reported in the literature. the teleomorphs are known for the t. mentagrophytes complex and there are two, arthroderma benhamiae and a. vanbreuseghemii. the a. benhamiae genome has been sequenced (burmester et al., 2011) . the different variants and sexual states of the t. mentagrophytes complex coupled with more recent phylogenetic analyses presents confusion about proper nomenclature and species assignment (drouot et al., 2009; fumeaux et al., 2004) . infection with either m. canis or m. gypseum has also been reported in the guinea pig but less frequently (feuerman et al., 1975; papini et al., 1997) . guinea pigs, and other animals, are infected by direct exposure to an infected animal, contaminated environments, or fomites. arthroconidium produced by septation of hyphae are infectious and persist in the environment for months to years (bond, 2010; weitzman and summerbell, 1995) . hyphae invade the stratum corneum eventually colonizing the hair follicles. the sequenced genome of a. benhamiae revealed numerous predicted protease-encoding genes and their secretion was confirmed experimentally in a keratin digestion assay indicating an important role in invasion and pathogenesis (burmester et al., 2011) . young guinea pigs appear to be more susceptible to disease and other predisposing and susceptibility factors include pregnancy, high temperature and humidity, health status, and genetics (pollock, 2003; pombier and kim, 1975) . surveys of dermatophytosis in guinea pigs have been reported from around the world, with prevalence ranging from 1.3-56% (balsari et al., 1981; drouot et al., 2009; feuerman et al., 1975; lopez-martinez et al., 1984; papini et al., 1997; vangeel et al., 2000) . clinical manifestations infected guinea pigs are most often asymptomatic but can exhibit lesions typical of dermatophytosis. when present, lesions are most common on the head and appear to begin on the nose or muzzle and then can spread over the body to the trunk and limbs (figure 23 .2). circumscribed or irregular lesions with combinations of alopecia, scale, crust, erythema, and ulceration develop, with or without pruritis, and spontaneous remission is possible (drouot et al., 2009; mcaleer, 1980a; pollock, 2003; pombier and kim, 1975) . pustules may be associated with the lesions due to secondary bacterial infections. furthermore, in one outbreak of t. mentagrophytes, affected animals were in poor body condition and there was approximately 50% mortality in young guinea pigs (pombier and kim, 1975) . onychomycosis is rare in the guinea pig (drouot et al., 2009) . pathology microscopic analysis of affected epidermis and adnexa can include acanthosis, vascular congestion, dermal lymphocytic infiltration, folliculitis, perivascular, and interstitial dermatitis, and intra-epidermal pustules (chermette et al., 2008; pombier and kim, 1975) . diagnosis microscopy and culture are the primary means of diagnosing dermatophytosis. wood's lamp can be used as a screening tool for m. canis and infected hairs will fluoresce with an apple-green color. however, false negatives can occur (chermette et al., 2008) . hairs are plucked or the skin scraped from the periphery of an active lesion or from the whole lesion if there is no evidence of inflammation and samples placed in 10% potassium hydroxide to remove keratin. the samples are then examined for hair or skin invasion with hyphae and/or arthroconidia. to increase sensitivity, fluorescent microscopy with calcofluor white and congo red can be used (bond, 2010; robert and pihet, 2008) . geimsa staining will also identify arthroconidia. all the dermatophytes infecting guinea pigs will have ectothrix hair shaft involvement. prior to obtaining a skin or hair sample for culture, the area should be wiped with alcohol to reduce contamination. saprophytic fungi contamination of the fur may hinder isolation of dermatophytes (aho, 1983) . hair, skin, or crusts are collected using a sterile toothbrush, swab, forceps, scalpel blade, or surgical scrub brush and placed onto culture medium. sabouraud's dextrose agar supplemented with cyclohexamide and chloramphenicol is commonly used. dermatophyte test medium is available, however contamination can cause both false-positive and falsenegative results (robert and pihet, 2008) . if the animal is simply a mechanical carrier, results of repeated testing will vary (chermette et al., 2008) . biopsy samples can be stained with periodic acid-schiff or silver stains to detect arthroconidia and hyphae (figure 23 .3) (chermette et al., 2008) . as mentioned earlier, polymerase chain reaction (pcr) amplification and sequencing of its and 28s ribosomal dna (drouot et al., 2009; fumeaux et al., 2004; symoens et al., 2011) will identify isolates, and mating experiments can be performed (hironaga et al., 1981) . reducing exposure to infected animals and contaminated environments is critical in preventing dermatophytosis. assuring a wellmanaged facility with no overcrowding, parasite control, and proper temperature and humidity regulation are important preventive components (pollock, 2003; iii. guinea pigs pombier and kim, 1975) . infected animals should be removed, the environment thoroughly decontaminated and any items that cannot be disinfected should be discarded. animal care staff should wear proper personal protective equipment when handling potentially contaminated items or bedding since zoonosis is a concern (mcaleer, 1980a). dilute bleach, benzalkonium chloride, and glutaraldehyde are effective surface disinfectants (pollock, 2003) . in a laboratory setting, it is doubtful that animals will be treated for ringworm. readers are referred to other summaries of available topical and systemic treatments (pollock, 2003) . background the microsporidia (members of the phylum microspora) are a group of single-celled intracellular spore-forming eukaryotic parasites that infect a wide range of animals, from insects to mammals. the defining and unique feature of the phylum is the presence of a polar filament (tube) in the mature spore . the microsporidia also have one of the smallest genomes of any eukaryote and lack several key features, such as typical mitochondria. long thought to be protozoa, certain characteristics indicated that these organisms were fungi-like such as presence of a chitinous spore wall, genes for trehalose metabolism, and intranuclear division (brosson et al., 2006; dunn and smith, 2001) . recently, lee et al. (2008) concluded that microsporidia are true fungi related to zygomycetes. of the microsporidial species infecting humans, enterocytozoon bieneusi is the most common with lesser, but still significant, numbers of cases due to the encephalitozoon spp. the majority of patients are immunocompromised as a result of hiv infection, aids, or are organ transplant recipients. however, healthy immunecompetent persons are also susceptible to disease (didier and weiss, 2006; mathis et al., 2005) . animals are hosts for those common species strongly supporting their zoonotic potential. etiology the primary agent of microsporidiosis in guinea pigs is encephalitozoon cuniculi (syn. nosema). e. cuniculi was first reported in the rabbit as the cause of "infectious motor paralysis" (percy and barthold, 2007) and subsequently has been identified in a wide range of animal species . katinka et al. (2001) have sequenced the 11 chromosomes of the approximately 2.9-mb genome, highlighting the genome compaction and shortened protein sequences that reflect its intracellular location and host dependence. provide an informative summary of the mammalian microsporidian life cycle. environmentally resistant spores are shed in feces, urine, and mucus and then ingested or inhaled. transplacental transmission has been documented in the rabbit (baneux and pognan, 2003) and has been claimed in the guinea pig (boot et al., 1988) . predictably, primary sites of infection commonly include the small intestine, respiratory tract, and placenta. an unknown signal triggers the polar filament (tube) to be extruded from the spore and infectious sporoplasm is injected into the host cell cytoplasm. the sporoplasm then divides into meronts (merogony) that differentiate through intermediary forms before becoming mature spores (sporogony). for e. cuniculi and other encephalitozoon spp., developing spores are packaged in a parasitophorous vacuole. the host cell ruptures releasing spores that commonly disseminate to the kidney, liver, and brain. host protection from infection is primarily achieved by cell-mediated immunity with lesser contributions from the humoral immune system (khan et al., 2001) . from serologic surveys, prevalence of encephalitozoonosis in guinea pig colonies has ranged from 13%-63%. this variability may be due to differences in housing and husbandry for the particular colony and diagnostic methodology (boot et al., 1988; gannon, 1980) . clinical manifestations encephalitozoonosis is subclinical in the guinea pig (boot et al., 1988; gannon, 1980; illanes et al., 1993; moffatt and schiefer, 1973; wan et al., 1996) . pathology gross lesions of encephalitozoonosis in the guinea pig are not consistently found and when present have only been described in the kidney. infected animals may have pale kidneys and/or pitting of the renal cortex (gannon, 1980; moffatt and schiefer, 1973) . histologic lesions are found primarily in the brain and kidney. focal to multifocal granulomatous encephalitis may be evident in different regions of the brain, with or without associated necrosis, and perivascular and meningeal mononuclear cell infiltrate (moffatt and schiefer, 1973; wan et al., 1996) . organisms approximately 1-1.5 âµm in width î�³ 1.5-2.5 âµm in diameter (illanes et al., 1993; can be found within or adjacent to lesions or free in the tissue with no associated inflammation. in the kidney, interstitial nephritis and necrosis, fibrosis, perivascular cuffing and tubular ectasia have all been described (boot et al., 1988; gannon, 1980; moffatt and schiefer, 1973) . here, organisms may occasionally be visualized in renal epithelial cells and collecting tubule lumens. absence of typical lesions in the brain or kidney in a seropositive animal may be due to the multifocal nature of the lesions and insufficient sections being analyzed (gannon, 1980; wan et al., 1996) . other lesions reported include necrotic liver foci and interstitial pneumonia with perivascular and peribronchial lymphoid accumulation. diagnosis serology tests including carbon immunoassay, ifa, elisa, multiplex fluorescent immunoassay, and western blot have all been or currently are being used for colony monitoring (percy and barthold, 2007; . presence of typical histopathologic lesions and special stains confirm the infection. while indistinct with hematoxylin and eosin staining, spores are periodic acid-schiff, gram, and modified trichrome positive (figure 23.4) . chemofluorescent agents such as calcofluor white can also be used (garcia, 2002) and spores are birefringent (tiner, 1988) . pcr primers are available (baneux and pognan, 2003; ghosh and weiss, 2009 ) providing a fast and easily interpretable means of diagnosis and the genome for e. cuniculi has recently been sequenced providing further opportunities for novel molecular diagnostic strategies (katinka et al., 2001) . urine, brain, and kidney are optimal specimens for testing by pcr. electron microscopy is the gold standard for diagnostic confirmation and offers species identification which is particularly important for human infections (garcia, 2002) . prevention and therapy e. cuniculi-free guinea pigs are commercially available and therefore, protection from contaminated animals or environments is the primary means of prevention (gannon, 1980) . faced with contamination, eradication may be successful through serological analysis and euthanasia of seropositive animals (baker, 2007) coupled with environmental decontamination. e. cuniculi spores are environmentally stable for days to weeks depending on the temperature and humidity, but are susceptible to common disinfectants (jordan et al., 2006; waller, 1979) . vertical transmission is thought to occur in guinea pigs (boot et al., 1988) , so hysterectomy rederivation should be used with caution and offspring extensively tested. it is unlikely that laboratory guinea pigs would be treated for encephalitozoonosis. two of four laboratory-bred guinea pigs were found dead several days to two weeks after adoption (kunstyr et al., 1980) . diarrhea was noted in one animal prior to death and each had gross evidence of enteritis at necropsy which was confirmed histologically. the yeast candida pintolopesii, a normal inhabitant of the gut, was isolated from the small intestine, lung, and ascitic fluid of one animal. the authors hypothesized that a change in social structure, diet, and environment precipitated disease. cryptococcus neoformans is a basidiomycetous yeast causing serious infections of the lungs and central nervous system in immunocompromised persons (boekhout and guã©ho, 2003) . betty (1977) reported chronic, subclinical cryptococcal meningitis in laboratory-reared dunkin hartley guinea pigs with no evidence of generalized infection. the source of infection was not identified. van herck et al. (1988) described dermal cryptococcosis of an adult male pet guinea pig. histopathologic analysis of a large ulcerative lesion on the dorsal aspect of the nose showed extensive focal ulcerative dermatitis with infiltrating neutrophils, plasma cells, lymphocytes, and macrophages with edema and ovoid organisms. dermal and subcutaneous tissue also contained large numbers of these thick-walled organisms that were positive by periodic acid-schiff staining. enterocytozoon bieneusi is a significant cause of intestinal microsporidiosis in immunocompromised persons and has been identified in many mammalian and nonmammalian hosts (mathis et al., 2005) . microsporidian spores were found in the feces of a two-year-old male entered into a prospective study of pediatric enteric parasites in peru. to investigate the source of infection, stool was analyzed from animals in the household, including guinea pigs, chickens, dogs, and cats. seven of eight asymptomatic guinea pigs were positive for spores, whereas all the other animals were negative. polymerase chain reaction and sequence analysis confirmed that both the child and guinea pigs were infected with e. bieneusi and with the same genotype. furthermore, this specific genotype was found in guinea pigs from other unrelated iii. guinea pigs households suggesting that guinea pigs are the natural host and there is zoonotic potential for this organism. there is a single report from brazil of a naturally occurring histoplasmosis outbreak in laboratory guinea pigs (correa and pacheco, 1967) . clinical signs of disease in adults included progressive emaciation and hindlimb dysfunction before death. young animals exhibited a hunched back, ruffled fur, and conjunctivitis with discharge before dying at two months of age. gross and histologic lesions were numerous; however, for these authors a characteristic lesion of colitis with the wall expanded by numerous lymphocytes, macrophages, epithelioid and giant cells with basophilic organisms free or inside macrophages and culture results established the diagnosis. there may have been a link between this outbreak and histoplasmosis diagnosed in a cow on a farm from which the grass used to feed the guinea pigs was sourced. hortaea werneckii, the causative agent of tinea nigra in people, was diagnosed in a household guinea pig in japan (sharmin et al., 2002) . lesions included a focal area of ulceration and alopecia on the back and black pigmentation on the palmar aspect of the right forepaw. h. werneckii was detected by mycologic culture of the back lesion and dna sequencing of an amplified region of large subunit ribosomal dna. the authors hypothesized that the animal contracted the infection from the environment, but could not definitively rule out contamination with the fungus rather than a real infection. paecilomyces spp. are occasional opportunists in humans and animals (summerbell, 2003) . this fungus was identified during routine monitoring from a single conventionally housed laboratory guinea pig with no apparent clinical signs . paecilomyces spp. were most commonly identified in specific pathogenfree rats from one particular animal facility with the trachea and lungs being the most common site of infection in all animals analyzed. mild dermatitis was noted in a group of hairless mice; however, the vast majority of animals had no clinical or histologic evidence of disease. a hairless mutant arose in a closed colony of hartley guinea pigs. in addition to abnormal haircoat development, affected guinea pigs were smaller than their haired siblings and died early due to infections otherwise associated with an abnormal immune system such as systemic cytomegalovirus and balantidiasis, and pneumocystis pneumonia (reed and o'donoghue, 1979) . abnormal thymic and lymphoid follicle morphology and agammaglobulinemia confirmed the immunodeficiency. protozoa rarely cause disease in guinea pigs and intestinal protozoa are often considered part of the normal flora. in this review, the more frequently reported protozoa of domestic guinea pigs will be discussed. the reader is referred to parasites of laboratory animals (baker, 2007) and morphology and taxonomy of the intestinal protozoa of the guinea pig (nie, 1950) for a more detailed description of the protozoa of this species. the nomenclature used in this section is in accordance with the 2007 reference. amoebiasis etiology endolimax caviae and entamoeba caviae (syn. entamoeba cobayae) are the causative agents of amoebiasis in guinea pigs (levine, 1985) . nie (1950) reported the incidence of endolimax caviae to be 18% and the incidence of entamoeba caviae to be 14% in a colony of guinea pigs studied at the university of pennsylvania. pathogenesis endolimax caviae and entamoeba caviae are observed in the cecum of the guinea pig (nie, 1950) . both of these organisms are thought to feed upon the fecal material and microflora of the host intestine, without invasion of tissue or consumption of blood. transmission is via ingestion of cysts passed in the feces. clinical manifestations infection with either organism does not generally result in clinical signs (levine, 1985) . pathology entamoeba and endolimax are usually non-pathogenic (baker, 2007; levine, 1985) . diagnosis trophozoites can be observed in a smear of intestinal contents (baker, 2007; levine, 1985) . cysts are detected by zinc sulfate float. endolimax cavieae trophozoites are smaller than those of entamoeba caviae, with an average diameter of 1.6 âµm and 14.4 âµm, respectively (nie, 1950) . the cyst forms of either organism have rarely been observed, and little detail has been recorded on their appearances. prevention and therapy generally, treatment is not necessary for guinea pig amoebiasis. balantidia caviae background upon initial description, controversy developed over whether b. caviae was the same organism as b. coli in swine (vetterling, 1976) . morphological descriptions and infection studies have since proved that the two species are distinct. etiology balantidium caviae is the causative organism. pathogenesis b. caviae transmission is via ingestion of cysts in the feces (baker, 2007) . clinical manifestations if the mucosal barrier of the intestine is compromised, b. caviae can become invasive and result in enteritis; otherwise infection is non-pathogenic (baker, 2007) . pathology b. caviae may be observed in the walls of the cecum and colon, but penetration can occur postmortem, a consideration that should be made when diagnosing infection at the time of necropsy (baker, 2007; nie, 1950) . if invasion is ante-mortem, intestinal inflammation should be present; more often, though, b. caviae is considered a commensal protozoan. diagnosis in addition to diagnosing b. caviae in histologic sections of the cecum or colon, ciliated organisms with micro-and macro-nuclei can be observed in fresh fecal smears (baker, 2007; fox, 2002) . proper hygiene is most important in controlling b. caviae infection in guinea pigs (baker, 2007) . tetracyclines have been used to treat balantidiasis in other species. background c. wrairi is named after the walter reed army institute of research where the organism was first identified in a colony of laboratory guinea pigs (vetterling et al., 1971) . etiology cryptosporidium wrairi is the causative organism. pathogenesis the route of infection for c. wrairi is likely fecal-oral, with ingestion of oocysts in feces (baker, 2007; fox, 2002) . experimental infection has shown that the duration of infection for animals older than 16 weeks is usually short, lasting as little as 1-2 weeks, after which the organism is cleared (chrisp et al., 1990) . younger animals have a longer duration of infection. recovered animals appear to be refractory to subsequent infection. clinical manifestations clinical signs are more likely to be seen in young animals, with weight loss noted most commonly (baker, 2007; fox, 2002) . if infection is severe, anorexia, a potbellied appearance and a greasy hair coat may be observed with or without accompanying diarrhea (baker, 2007) . morbidity and mortality, especially in young animals, can reach levels of 50% during an outbreak (harkness and wagner, 1995; percy and barthold, 2007) . echinococcus coli has been associated with clinical cases of c. wrairi, but the significance of this infection is unknown (percy and barthold, 2007) . subclinical infections with c. wrairi are thought to be possible (baker, 2007; fox, 2002) . pathology erosion, hyperemia, and inflammation of the small intestine have been observed with c. wrairi infection (baker, 2007; chrisp et al., 1992; harkness and wagner, 1995) . edema of the lamina propria and hyperplasia of the crypt epithelium occurs. in chronic infections, villous atrophy and bridging, metaplasia of the mucosal epithelium, and lymphocyte infiltration of the lamina propria have been noted. developmental stages of c. wrairi can be seen throughout the intestine, with highest concentrations of the organism noted in the brush border of the ileum. diagnosis oocysts can be seen in fecal floats (gressler et al., 2010; percy and barthold, 2007) . kinyoun staining of fecal smears and microscopic examination of mucosal scrapings or histological sections are also diagnostic options. prevention and therapy prevention is the best means of control for c. wrairi. oocysts are resistant to many disinfectants or require high concentrations and/or long contact times before being rendered noninfectious. heating above 65â°c for greater than 5 minutes has been described to be successful for the destruction of cryptosporidium organisms, as has exposure to temperatures below 0â°c (baker, 2007; harkness and wagner, 1995) . successful treatment with sulfonamides has been reported, but the efficacy of this treatment has been questioned (fox, 2002; percy and barthold, 2007; sebesteny, 1976) . harkness suggests the "extra-label" use of high doses of the coccidiostat, decoquinate, to treat cryptosporidiosis (harkness and wagner, 1995) . while c. wrairi is not known to infect humans, the genus has a general lack of specificity and precautions should be taken to prevent zoonotic transmission (baker, 2007; harkness and wagner, 1995) . eimeria background reports of coccidia in guinea pigs date back to the late 1800s, although the observed organisms were initially thought to be a variety of rabbit coccidian (vetterling, 1976) . in the early 1920s, bugge and heinke showed that coccidia noted in the guinea pig were indeed distinct from those of the rabbit and in 1924 sheather named the guinea pig coccidia e. caviae. iii. guinea pigs etiology eimeria caviae is the causative organism. pathogenesis transmission of e. caviae is fecaloral, with ingestion of oocysts in feces (baker, 2007) . clinical manifestations clinical signs due to e. caviae are noted in severe infections and include diarrhea, anorexia and a rough hair coat (baker, 2007; ellis and wright, 1961; rigby, 1976) . the first appearance of diarrhea is often 10-11 days after initial infection (baker, 2007; elsheikha et al., 2009; percy and barthold, 2007) . watery, pasty, and hemorrhagic forms of diarrhea have all been reported (baker, 2007; elsheikha et al., 2009; fox, 2002; percy and barthold, 2007) . severe infections can result in death (baker, 2007; fox, 2002) . in mild infections or infections of older animals, diarrhea is likely to resolve in 4-7 days (baker, 2007; fox, 2002) , after which constipation may follow (fox, 2002) . stress, such as a diet change or transport, may exacerbate an otherwise nonpathogenic infection of e. caviae in the guinea pig (ellis and wright, 1961; elsheikha et al., 2009; rigby, 1976) . pathology the large intestine is most affected by e. caviae, with organisms most often settling in the proximal colon (baker, 2007; elsheikha et al., 2009) . hyperemia, edema, and petechial hemorrhage of the colonic mucosa may be seen (baker, 2007) . white or yellow plaques may be present in the colon or cecum. watery intestinal contents can contain blood (baker, 2007; percy and barthold, 2007) . dilated cystic crypts of liekberkuhn may be observed (baker, 2007) . developmental stages of e. caviae can be seen in intact epithelial cells or free in the intestinal lumen. enterocytes may slough and an infiltration of polymorphonuclear and mononuclear cells is possible (baker, 2007; percy and barthold, 2007) . microgametocytes and macrogametocytes may be present in the cecal and colonic mucosa (percy and barthold, 2007) . diagnosis oocysts are seen on fecal float (baker, 2007; fox, 2002; percy and barthold, 2007) . fox et al. (2002) recommend using a flotation medium of 1.33 specific gravity. the prepatent period of e. caviae is 11-12 days, yet diarrhea may occur before day 11; therefore, a fecal float performed at the beginning of diarrhea may result in a false-negative finding. multiple floats performed every 4 or 5 days for several weeks is recommended (vetterling, 1976) . organisms may also be identified on mucosal scrapings or histopathology sections (baker, 2007; fox, 2002; percy and barthold, 2007) . sulfonamides are recommended for treatment. minimizing stress and providing an adequate level of vitamin c help to prevent clinical infection. oocysts in the feces take 6 days to become infective, therefore regular cleaning of pans will help to minimize the spread of infection (rigby, 1976) . ammonia, followed by a thorough rinsing, has been used to clean cages and pans of infected animals (elsheikha et al., 2009 ). steam has also been used to clean enclosures. calhoon notes that eimeria-free colonies can be achieved via cesarean rederivation (calhoon and matthews, 1964) . etiology the causative agent of guinea pig giardiasis is giardia duodenalis, formally referred to as as g. caviae (baker, 2007; vetterling, 1976) . pathogenesis giardia duodenalis is a flagellate of guinea pigs that is transmitted by the fecal-oral route or via ingestion of contaminated food or water (baker, 2007) . clinical manifestations overt signs of infection, such as diarrhea, are rare, although some animals are severely infected and may present as weak and moribund (baker, 2007) . pathology the small intestine, primarily the duodenum, is colonized by trophozoites (baker, 2007) . colonization may result in mild inflammatory lesions, decreased villar height, and cystic enlargement of duodenal crypts. diagnosis diagnosis of g. duodenalis is via direct fecal smear, where trophozoites or cysts may be observed (baker, 2007) . only trophozoites, and not cysts, are found in diarrheic feces (vetterling, 1976) . shedding of g. duodenalis is intermittent, so unless multiple fecal smears are performed, there is a high likelihood of obtaining a falsenegative result. felasa recommends including giardia spp. on guinea pig breeding colony health monitoring reports (rehbinder et al., 1996) . prevention and therapy metronidazole and fenbendazole are treatment options for guinea pig giardiasis (baker, 2007) . infection can be prevented by thorough environmental sanitation using a quaternary ammonium product or sodium hypocholorite. hot temperatures will destroy cysts. cysts thrive in moist areas, so environments must be kept dry. the zoonotic potential of g. duodenalis of guinea pigs is unknown, but the possibility of transmission between guinea pigs and humans is suspected. klossiella cobayae background k. cobayae is also known as klassia caviae, as it was described and named simultaneously by two individuals, pearce and sangiori (respectively) working independently of each other (vetterling, 1976) . occurrence in modern laboratory guinea pigs is rare (percy and barthold, 2007) . etiology klossiella cobayae is the causative organism. pathogenesis k. cobayae is a parasite of the epithelial cells of the renal tubules, as well as the endothelial cells associated with glomerular capillaries and other organs, such as the spleen and lungs (griffiths, 1971; van andel et al., 1995; vetterling, 1976) . transmission is via ingestion of sporocysts in urine (baker, 2007) . (baker, 2007; sebesteny, 1976) . heavy infection can lead to an irregular surface and a gray mottled appearance of the kidney (baker, 2007; fox, 2002) . interstitial and perivascular lymphocytic and histiocytic infiltration may occur (baker, 2007) . a large number of interstitial fibroblasts may also be observed. diagnosis sporocysts are passed in the urine, yet observation of this stage in the urine is difficult (baker, 2007) . most often, diagnosis is made at the time of necropsy by identifying the different developmental stages of k. cobayae in glomerular capillaries or in the cytoplasm of epithelial cells lining the renal tubules (baker, 2007; percy and barthold, 2007) . felasa recommends including klossiella spp. as part of a health monitoring report for guinea pig breeding colonies (rehbinder et al., 1996) . prevention and therapy control of k. cobayae is best achieved through prevention. contamination of food and bedding with infective urine should be minimized (baker, 2007) . sulfonamides might be effective forms of treatment. background leishmania enrietti was first noted in laboratory guinea pigs in 1948 in brazil (medina, 1946) . according to the review by machado et al., this flagellate was next reported in 1967 in a guinea pig captured from the outskirts of curitiba (machado et al., 1994) . the sandfly, lutzomyia monticola, which is frequently noted on the trunks of curitibia pine trees, was proposed as a possible vector. etiology leishmania enrietti is the causative organism. pathogenesis little is known about the lifecycle of l. enrietta, but other members of the genus leishmania have indirect lifecycles and sandfly vectors (baker, 1998 (baker, , 2007 nie, 1950) . l. enrietta results in cutaneous infection. an infected guinea pig may present with a cutaneous nodule at the site of entry 1-2 weeks after infection (baker, 2007) . additionally, ulcers can develop on the feet, ears, nose, and genitalia of infected animals. ulcers will go through a series of pathologic changes, such as necrosis of surrounding macrophages 4-5 weeks after initial infection (especially in naive animals), followed by an infiltrate of giant cells, plasma cells, and lymphocytes at the periphery of the ulcer. by 7 weeks, giant cells are gone and fibroblasts appear, indicating resolution. resolution is usually complete by 10 weeks post-infection. hematogenous spread of the organism can occur and l. enrietti may be observed in the lymph nodes. diagnosis detection is via identification of amastigotes in lesion histology or via culture of the organism (baker, 2007) . control is achieved by elimination of vectors. treatment with meglumine antimoniate has been attempted, with inconsistent success (thomaz-soccol et al., 1996) . contact with possible sandfly vectors should be avoided. background this organism was first reported in brazil by carini and migliano (vetterling, 1976) . in early reports, t. gondii was mistaken for sarcocystis in the guinea pig (kean and grocott, 1945) . etiology toxoplasma gondii is the causative organism. pathogenesis cats are the definitive hosts of t. gondii (baker, 2007) . oocysts are released in cat feces and are ingested by guinea pigs which act as intermediate hosts for t. gondii. cysts can remain viable in a guinea pig for up to five years before ingestion by the definitive host. in addition to the guinea pig, a large number of mammalian and avian species are intermediate hosts to t. gondii, including humans. ingestion of contaminated biological material and transplacental transmission are alternative routes of transmission for the guinea pig (baker, 2007; percy and barthold, 2007) . clinical manifestations t. gondii infection in guinea pigs is often asymptomatic and, therefore, frequently goes undiagnosed (baker, 2007; griffiths, 1971; percy and barthold, 2007) . infection in pregnant sows can result in vulvar bleeding and abortion (fox, 2002) . in a case of spontaneous toxoplasma encephalitis in a guinea pig, spastic paralysis, opisthotonos, and loss of urethral and anal sphincter control were reported (markham, 1937) . pathology hepatitis, pneumonia, encephalitis, and uterine infection (possibly blood-filled), have been noted in infected animals, as have cysts in both the myocardium and central nervous system (baker, 2007; fox, 2002; percy and barthold, 2007) . in spontaneously infected animals that develop toxoplasma encephalitis, congested blood vessels of the meninges were noted and were surrounded by mononuclear leukocytes (markham, 1937) . diagnosis serology is often used for diagnosis in guinea pigs (baker, 2007) . cysts can be observed on histology. mice or hamsters can be inoculated with tissue homogenate from suspected positives and act as sentinels of infection. felasa recommends including toxoplasma gondii on guinea pig breeding colony health monitoring reports (rehbinder et al., 1996) . sulfadiazine and pyrimethamine are reported treatment options for clinical cases of t. gondii, yet the effectiveness of such treatments is questionable (baker, 2007; fox, 2002) . temperatures above 60â°c kill oocysts (baker, 2007) . strict sanitation and periodic colony monitoring are necessary for t. gondii control. background tritrichomonas caviae has also been referred to as trichomonas caviae and t. flagelliphora (tanabe, 1925) . etiology tritrichomonas caviae is one of the largest intestinal flagellates of the guinea pig, measuring an average of 16 î¼m long by 8.5 î¼m wide (baker, 2007) . no cyst stage is known. pathogenesis transmission of t. caviae is fecaloral (baker, 2007) . clinical manifestations tritrichomonas caviae is a non-pathogenic flagellate of guinea pigs (baker, 2007) . pathology t. caviae is most often noted in the cecum, although it has been observed in the duodenum, jejunum and lower ileum as well (baker, 2007) . although rare, tissue invasion and cecal or colonic ulceration are possible. diagnosis diagnosis of t. caviae is via direct fecal smear (tanabe, 1925) . prevention and therapy t. cavaie does not usually result in clinical signs or pertinent pathology and does not routinely require treatment (baker, 2007) . guinea pigs, among other mammals, are reservoirs for the flagellate t. cruzi (baker, 2007; vetterling, 1976) . reduviid bugs act as vectors, becoming infected after a blood meal from a positive animal. t. cruzi mature and reproduce within the vector. the bugs then go on to deposit t. cruzi-containing feces on humans during another blood meal. the organism enters human skin through a wound (potentially the bug bite) or contact with mucous membranes. chagas disease may result in humans. in the guinea pig, t. cruzi can encyst in multiple organs, including the skin and cardiac muscule. t. cruzi has been noted in domesticated guinea pigs of south america exposed to reduviid vectors. clinical and pathological signs are not described. the reader is once again directed to flynn's parasites of laboratory animals for further discussion on many of the parasites discussed below, including anatomical appearances and detailed life cycles (baker, 2007) . the nomenclature used in this section is also in accordance with this reference. note: the common names of arthropod organisms have been used in place of the name of the resulting condition (e.g. flies instead of myiasis). ctenocephalides felis has been noted on pet guinea pigs living in a household with cats and/or dogs. infected guinea pigs presented with pruritus, alopecia, dermal crusts, and anemia (white et al., 2003) . aqueous-based pyrethrin sprays have been recommended as the safest treatment option for such infection. nosopsyllus fasciatus, the northern rat flea, can also inhabit guinea pigs, although similarly to c. felis, infection of laboratory guinea pigs is rare (fox, 2002) . it has been stated that "fatal myiasis involving at least three species of flies, lucilia sericata, calliphora vicina and calliphora vomitoris, have been reported in guinea pigs" (baker, 2007) . eggs are deposited on the skin of a guinea pig by an adult fly, where larvae will develop and then feed on living or necrotic animal tissue (baker, 2007) . (baker, 2007) . g. porcelli, the most commonly observed of the three, is known as the "slender louse" due to its general appearance, especially when compared to g. ovalis or the "oval louse" (baker, 2007; harkness and wagner, 1995; kim et al., 2008) . several species of sucking lice (order anoplura) have been noted on wild guinea pigs in south america: pterophtirus alata and polyplax spinulosa (dittmar, 2002) . these later lice are listed in table 23 .1, but not discussed here in detail. pathogenesis biting lice attach to hair shafts and abrade the skin to ingest cutaneous fluids (fox, 2002; kim et al., 2008) . transmission is via direct contact (harkness and wagner, 1995; kim et al., 2008) . the life cycles of guinea pig lice are not specifically described, but in general, biting lice go through the following stages: egg, three nymphal stages, and adult (baker, 2007) . clinical manifestations g. ovalis, g. porcelli or t. hispidium infection of a guinea pig can be asymptomatic or may result in pruritus and a rough hair coat or alopecia (baker, 2007; coman et al., 2009; harkness and wagner, 1995; kim et al., 2008; percy and barthold, 2007; rigby, 1976) . scabs and crusts are sometimes noted secondary to scratching, especially around the ears (harkness and wagner, 1995) . pathology signs of louse infection are usually superficial. dermatitis may be noted grossly. diagnosis lice (adults or nits) can be seen on the haircoat of an infected animal, with or without the aid of a magnifying lens (figure 23 .6) (baker, 2007; griffiths, 1971 ). on a dead animal, lice will migrate to the warm tips of the hair as the skin cools (griffiths, 1971) . tape tests and flea combing can also be used to diagnose louse infestation (coman et al., 2009) . as stated previously, g. porcelli is slender in appearance (1.0-1.5 mm î�³ 0.3-0.4 mm), compared to g. ovalis (1.0-1.2 mm î�³ 0.5 mm) (figure 23 .7) (huerkamp et al., 1996) . t. hispidum's dimensions are similar to those of g. ovalis, yet can be differentiated by the fact that t. hispidum has five abdominal segments as compared to g. ovalis' eight. in reports of successful control of guinea pig louse infestations, both infected animals and the environment have been treated (harkness and wagner, 1995; kim et al., 2008) . systemic ivermectin (baker, 2007; white et al., 2003) , carbamate or pyrethrin-based powders (baker, 2007; griffiths, 1971) , romavermectin b1 (coman et al., 2009 ) and a combination of imidacloprid and moxidectin (kim et al., 2008) have been noted as effective drug choices for treating infected guinea pigs. hypochlorite bleach has been used for environmental decontamination (kim et al., 2008) . guinea pigs are susceptible to infection with several species of mites, some occurring more commonly than others. chirodiscoides caviae and trixacarus caviae are the most commonly noted infectious mites in guinea pigs, with demodex caviae, myocoptes musculinus, sarcoptes scabies and notoedres muris reported less frequently (baker, 2007; fox, 2002; white et al., 2003) . infection with the astigmatic mite, acarus farris, was described in a case report of two guinea pigs (linek and bourdeau, 2005) . psocoptes cuniculi has been described on a pet guinea pig in close contact with a pet rabbit (yeatts, 1994) . white et al. (2003) note cheyletiella parasitivorax as a cause of pruritus and scaling along the dorsum of guinea pigs. the more commonly noted mites, c. caviae and t. cavaie, will be discussed below. the first reports and illustrations of c. caviae were by stanley hirst in the early 1900s (hirst 1922 ). hirst noted this mite attached to the hairs of the back of the guinea pig. etiology chirodiscoides caviae is the causative organism (figure 23.8 ). pathogenesis c. caviae is a species-specific fur mite that tends to concentrate in the lumbar area and the lateral aspects of the hindquarters (harkness and wagner, 1995; percy and barthold, 2007; schonfelder et al., 2010) . c. caviae feed on the scales of hair shafts (hirsjarvi and phyala, 1995) and transmission is via direct contact with an infected animal (fox, 2002; schonfelder et al., 2010) or with infected cage debris or bedding (fox, 2002) . the life cycle of c. caviae has not been specifically described, although mites in general commonly go through egg, larval, nymphal, and adult stages (baker, 2007) . concurrent infection with lice is not uncommon (white et al., 2003) , and mixed infection with demodex caviae (detected via deep skin scrapping) has also been reported (schonfelder et al., 2010) . clinical manifestations infection with c. caviae can be asymptomatic or, if infestation is heavy, pruritis, alopecia, hyperemia, and dermal crusts may be seen (coman et al., 2009; fox, 2002; hirsjarvi and iii. guinea pigs phyala, 1995; lumeij and cremers, 1986; schonfelder et al., 2010) . anorexia may result secondary to grooming pruritic areas (schonfelder et al., 2010) . the appearance of c. caviae mites themselves is the major pathologic finding (fox, 2002) . mites and ova are located on the hair shafts of the guinea pigs, not burrowed into the skin. diagnosis mites may be seen with the unaided eye, hand lens or dissecting microscope. c. caviae can also be found on a superficial scrape (harkness and wagner, 1995; percy and barthold, 2007; schonfelder et al., 2010) . combing, hair plucking, and cellophane tape testing can aid in isolating mites for observation (white et al., 2003) . prevention and therapy many treatment options have been described in the literature for c. caviae. dilute ivermectin (diluted in aqua/propylene glycol) sprays have been successful, especially for treating large colonies of infected animals (baker, 2007; hirsjarvi and phyala, 1995; white et al., 2003) . selamectin, 12-15 mg/ kg, applied twice at 2-week intervals has also eliminated infection (baker, 2007; fox, 2002; schonfelder et al., 2010) . pyrethrin sprays and powders have been used to rid the environment of c. caviae (harkness and wagner, 1995; hirsjarvi and phyala, 1995) . 1% virkon s â® was used to treat an automatic watering system (hirsjarvi and phyala, 1995) . older successful treatments include the use of dichlorvos vapors (henderson, 1973) as well as immersion of shaved animals in 0.15% trichlorfon (lumeij and cremers, 1986 ) . background t. caviae was discovered in a colony of albino guinea pigs in the early 1970s by fain and was described as a disease very similar in appearance to sarcoptic mange in other animals (dorrestein and vanbronswijk, 1979; fain et al., 1972) . the first report of t. caviae in north america was in 1980 at davis, california (mcdonald and lavoipierre, 1980) . etiology trixacarus caviae is the causative organism (figure 23 .8). trixacarus caviae is a species-specific sarcoptic mite that most commonly affects guinea pigs that are 1-3 years of age (dorrestein and vanbronswijk, 1979) . if left untreated, the parasite load peaks 1 month post-infection, after which it slowly regresses (fuentealba and hanna, 1996) . the life cycle of t. caviae includes an egg, larval, two nymphal, and a final adult stage, all of which inhabit the host guinea pig (baker, 2007) . transmission is by direct contact, with nymphal or larval stages indicated in establishing new infestations (baker, 2007; rothwell et al., 1991) . within 72 hours, pups born to an infected guinea pig dam will show clinical signs consistent with t. caviae infestation (beck et al., 2007; kummel et al., 1980) . humans have been reported to develop transient pruritic, papulovesicular dermatitis after contact with infected animals, although mites appear to be incapable of persisting on human skin (fuentealba and hanna, 1996; kummel et al., 1980; mederle and indre, 2009) . t. caviae may be asymptomatic and result in non-clinical carrier animals, t. caviae has been reported as the most common and important cause of dermatitis in guinea pigs (dorrestein and vanbronswijk, 1979; fuentealba and hanna, 1996; percy and barthold, 2007; white et al., 2003) . severe pruritis and excoriation along with erythema and alopecia can accompany dermatitis in some animals (ackerman, 1987; beck et al., 2007; dorrestein and vanbronswijk, 1979) . in those guinea pigs that are clinical, grayish to yellow or white crusts may be present on the skin and can be dry or slightly greasy (ackerman, 1987; dorrestein and vanbronswijk, 1979) . secondary bacterial infections of infected skin areas are possible (fuentealba and hanna, 1996) . infection usually starts on the face and ears, spreading to the lumbar regions and lateral aspects of the legs (dorrestein and vanbronswijk, 1979; percy and barthold, 2007) . the intense pruritic response in some guinea pigs is reported to be due to an initial allergic reaction to mite antigen (fox, 2002) . extreme restlessness and emaciation have been noted in affected animals, especially in those that seemingly scratch uncontrollably (beck et al., 2007) . this constant scratching can lead to "fits" of muscular spasms or sporadic epileptiform seizures (ackerman, 1987; beck et al., 2007; dorrestein and vanbronswijk, 1979; kummel et al., 1980) caused by generalized pruritus-induced hyperesthesia (baker, 2007) . pathology orthokeratotic hyperkeratosis and acanthosis have been noted in animals showing clinical signs of t. caviae infection (ackerman, 1987; dorrestein and vanbronswijk, 1979; fuentealba and hanna, 1996; zenoble and greve, 1980) . a polymorphonuclear leucocytic infiltrate is present in the dermis (dorrestein and vanbronswijk, 1979; percy and barthold, 2007; rothwell et al., 1991; zenoble and greve, 1980) . adult mites may be noted in "tunnels" or burrows located in the hyperkeratotic areas (dorrestein and vanbronswijk, 1979; zenoble and greve, 1980) . such tunnels approach the epidermis perpendicularly, with some entering the mouth of hair follicles (zenoble and greve, 1980) . eggs may also be present in tunnels (dorrestein and vanbronswijk, 1979) . t. caviae mites have sharp spines on the cuticle of their dorsums (zenoble and greve, 1980) , which differentiates them from notoedres (ackerman, 1987) . the anus of the female t. caviae mite is located dorsally, while this structure is located terminally on s. scabiei (ackerman, 1987; baker, 2007; fuentealba and hanna, 1996) . diagnosis deep skin scrapes of crusted areas with 10% koh reveal t. caviae (dorrestein and vanbronswijk, 1979; fain et al., 1972; fuentealba and hanna, 1996; mederle and indre, 2009 ). zajac et al. (1980) report that live mites could be demonstrated for 5 days postmortem from deep skin scrapings taken from a guinea pig held at 4â°c after death. prevention and therapy treatment of both affected animals and the environment has been employed to rid guinea pig colonies of t. cavaie. multiple treatment regimens have been reported as successful over the years, including lindane baths (dorrestein and vanbronswijk, 1979; zajac et al., 1980) , weekly lime sulfur dips (ackerman, 1987; mcdonald and lavoipierre, 1980; zenoble and greve, 1980) , ivermectin injections (white et al., 2003) , and monthly spot-on treatment with 10% imidacloprid and 1% moxidectin (beck et al., 2007) . dilute lime sulfur can also be used to treat the environment (white et al., 2003) . valium has been used to control seizure-like activity related to t. cavaie infection by some, whereas others suggest that treating the mite infection alone eliminates this sequela to infection (beck et al., 2007; white et al., 2003) . according to harkness and wagner (1995) , tapeworms rarely infect guinea pigs. anoplocephala spp. and monoecocestus parcitesticulatus (table 23 .1) have been reported in the intestines of south american guinea pigs (baker, 2007; sardella and fugassa, 2009 ). etiology baylisascaris procyonis is the causative organism. pathogenesis guinea pigs are paratenic hosts of b. procyonis and contract infection by ingesting eggs in raccoon feces (baker, 2007) . once ingested, larvae will hatch and penetrate the small intestine of the guinea pig, migrate through the liver to the lungs and disseminate throughout the body via the circulatory system. larvae then encapsulate where they remain until ingested by a raccoon. clinical manifestations no signs are present in the guinea pig due to b. procyonis unless organisms migrate to the brain. migration can result in lethargy, head tilt and ataxia, which may progress to cachexia, stupor, hyperexcitability, lateral recumbency, torticollis, or opisthotonos (van andel et al., 1995) . pathology in cases were b. procyonis migrates to the brain, multifocal eosinophilic granulomatous inflammation and neutrophilic infiltration is seen, along with perivascular lymphoid cuffing and malacia (van andel et al., 1995) . eosinophilic granulomata have been noted in the lungs of some infected animals. diagnosis histology is diagnostic for b. procyonis (baker, 2007) . the baermann extraction technique can be used to extract organisms from the cerebral tissue of clinical animals (van andel et al., 1995) . prevention and therapy prevention, rather than treatment, is the most logical approach for b. procyonis control, as clinical signs are often not noted until the end stage of infection and diagnosis relies on histopathology. contamination of bedding or food with raccoon feces must be avoided. ova can be viable for years in soil and weeks to months in straw and are resistant to most disinfectants (fox, 2002) . removal of contaminated bedding and the autoclaving of caging may be required (baker, 2007) . humans, similarly to guinea pigs, can contract infection by ingesting raccoon feces, but transmission from guinea pigs to humans is not feasible (fox, 2002) . etiology paraspidodera uncinata is the causative organism. epizootiology and pathogenesis p. uncinata is the cecal worm of guinea pigs and the most common nematode infecting this species (griffiths, 1971) . the prevalence of p. uncinata in wild guinea pigs of south america was found to be 37%, while the prevalence was noted as 23.8% in a laboratory colony of guinea pigs (coman et al., 2009; dittmar, 2002) . transmission is via ingestion of an embyronated egg in feces (baker, 2007; fox, 2002) . eggs become infectious 3-5 days after they are initially shed (fox, 2002) . the life cycle is not well described (baker, 2007) . clinical manifestations infections are normally asymptomatic, but with heavy parasitic loads weight loss, diarrhea, and disability are possible (fox, 2002) . pathology infection can, and often does, occur without pathologic changes (baker, 2007; percy and iii. guinea pigs barthold, 2007; rigby, 1976) . if cecal worms migrate through the intestinal mucosa, a hemorrhagic typhlitis may be seen along with capillary ectasis in the submucosa (coman et al., 2009 ). diagnosis adult worms may be seen in the cecum at necropsy and eggs may be observed in feces ( figure 23 .9) (baker, 2007; rigby 1976) . prevention and therapy levimasole administered at 25 mg/kg, either orally or subcutaneously, has most often been reported as an effective treatment for p. uncinata in the guinea pig (baker, 2007; eliazian et al., 1975) . other reported treatment regimens include oral mebendazole at 50 mg/kg (baker, 2007) and romavermectin b1 given subcutaneously at a dose of 0.2 mg/kg twice at a three-week interval (coman et al., 2009) . etiology pelodera strongyloides is the causative organism. pathogenesis p. strongyloides organisms are usually found in damp soil or vegetation (white et al., 2003) . bedding can be a possible source of infection. clinical manifestations while typically nonpathogenic, if p. strongyloides invade hair follicles dermatitis can result (baker, 2007; white et al., 2003) . pathology organisms may be found in hair follicles of clinically infected animals. the observation of larvae in skin scrapings or biopsies signifies infection (white et al., 2003) . adults may be observed in the bedding. prevention and therapy cages should be kept dry, as organisms thrive in moist environments (white et al., 2003) . the bedding of any positive animal should be replaced. a 1-2% chlorhexadine bath has been recommended for infected animals as a means of preventing secondary infections. trichinellosis due to trichinella spiralis is possible in guinea pigs, but rare, especially in laboratory animals that seldom come in contact with infected material (raw or undercooked meat) (sebesteny, 1976) . enteritis could develop in infected animals if t. spiralis organisms migrate through the intestinal wall. fasciola background infection in present laboratoryhoused guinea pigs is rare, but when fasciola organisms are noted it is often in conjunction with recent diet supplementation of a leafy vegetable carrying encysted trematodes (baker, 2007; fox, 2002) . etiology two fasciola species are noted in guinea pigs, fasciola hepatica and fasciola gigantica (baker, 2007) . f. gigantica is slightly larger in size than f. hepatica, although f. gigantica rarely reach patency in guinea pigs. pathogenesis guinea pigs ingest encysted fasicola metacercariae on vegetation (baker, 2007; fox, 2002) . once ingested, metacercariae hatch in the small intestine and migrate through the wall to the peritoneal cavity and the liver. juvenile flukes develop in the liver and, once mature, burrow in the bile ducts. eggs are shed and enter the gut, where they pass in the feces. a snail intermediate host is necessary for maturation from cercariae to infectious metacercariae. metacercariae encyst on leafy vegetation. clinical manifestations emaciation and anemia have been associated with fasciola infection (sebesteny, 1976) . posterior paresis was noted in animals that developed cysts in the lumbar musculature after aberrant fluke migration (baker, 2007) . pathology hepatic congestion and hemorrhage, especially around portal vessels, central veins and sinusoids is possible (baker, 2007) . fibronecrotic tracks and granulomas may also be observed in the liver. other pathology due to fluke infection is often the result of aberrant migration, resulting in cysts in areas such as the kidney, the peritoneal cavity and the pelvic cavity. cysts often contain coffee-brown-colored material. diagnosis fasciola spp. can be observed on fecal flotation (baker, 2007) . prevention and therapy prevention is best achieved by limiting the amount of leafy vegetation supplemented in a guinea pig's diet; if supplemented, do not feed vegetation from fluke-endemic locations. fasciola in ruminants is treated with albendazole and clorsulon (baker, 2007) . haverhill fever with spine involvement trixacarus caviae infestation in a guinea pig studies on fungal flora in hair from domestic and laboratory animals suspected of dermatophytosis. i. dematophytes saprophytic fungi isolated from the hair of domestic and laboratory animals with suspected dermatophytosis the isolation of streptobacillus moniliformis from cervical abscesses of guinea-pigs epidemiology of superficial fungal infections pet rodents and fatal lymphocytic choriomeningitis in transplant patients flynn's parasites of laboratory animals natural pathogens of laboratory mice, rats, and rabbits and their effects on research dermatophytes in clinically healthy laboratory animals in utero transmission of encephalitozoon cuniculi strain type i in rabbits target tissues associated with genital infection of female guinea pigs by the chlamydial agent of guinea pig inclusion 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failure to thrive, and altered immunologic function in a colony of t-cell receptor transgenic mice: possible role of citrobacter rodentium spontaneous toxoplasma encephalitis in the guinea pig staphylococci in man and animals. distribution and characteristics of strains chronic excretion of coronaviruslike particles in laboratory guinea pigs an evolutionary strategy for a stealthy intracellular brucella pathogen efficacy of commercial vaccines for protecting guinea pigs against bordetella bronchiseptica pneumonia zoonotic potential of the microsporidia serodiagnosis of streptococcus pneumoniae infection in guinea pigs by an enzyme-linked immunosorbent assay prevention of cervical lymphadenitis in guinea pigs by vaccination an epizootic in laboratory guinea pigs due to trichophyton mentagrophytes keratinophilic fungi on four animal groups studies on the etiology of snuffles in stock rabbits: paranasal sinusitis, a factor in the interpretation of experimental results trixacarus caviae infestation in two guinea pigs outbreak of fever caused by streptobacillus moniliformis molecular, biological, and in vivo characterization of the guinea pig cytomegalovirus (cmv) homologs of the human cmv matrix proteins pp71 (ul82) and pp65 (ul83) comparison of an espb gene fecal polymerase chain reaction assay with bacteriologic isolation for detection of citrobacter rodentium infection in mice intestinal tyzzer's disease and spirochetosis in a guinea pig campylobacter jejuni infection within a laboratory animal production unit trixacarus caviae infestation in guinea pigs, case report. lucrari stiintifice medicina veterinara xlii estudos sobre leishmaniose: i. primeiros casos de leishmaniose espontã¢nea observados em cobã¡ios biochemical properties of the bromodeoxyuridine-induced guinea pig virus duration of untreated chlamydial genital infection and factors associated with clearance: review of animal studies microsporidiosis (encephalitozoonosis) in the guinea pig observations 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of rabbits toxicity of cecal filtrates from guinea pigs with penicillin-associated colitis natural infections of guinea-pigs conventional methods for the diagnosis of dermatophytosis haematological and pathological responses to experimental trixacarus caviae infection in guinea pigs clostridium difficile infection: new developments in epidemiology and pathogenesis paleoparasitological analysis of rodent coprolites in holocenic samples from patagonia an epizootic septicemia of young guinea pigs caused by pseudomonas caviae animal models of congenital cytomegalovirus infection: an overview of progress in the characterization of guinea pig cytomegalovirus (gpcmv) quantitative-competitive pcr monitoring of viral load following experimental guinea pig cytomegalovirus infection analysis of the nucleotide sequence of the guinea pig cytomegalovirus (gpcmv) genome course of chlamydia-induced inclusion conjunctivitis in the guinea pig in experimental animal husbandry concureent infesation of 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dermatophytes from domestic animals in norway efficacy of a commercial bacterin in protecting strain 13 guineapigs against bordetella bronchiseptica pneumonia conjunctival swab cytology from a guinea pig: it's elementary! ascomycetes, apergillus, fusarium, sporothrix, piedraia, and their relatives molecular analysis and mating behaviour of the trichophyton mentagrophytes species complex a study of trichomonas from the guinea-pig new isolation of leishmania enriettii muniz and medina, 1948 in paranastate, brazil, 50 years after the first description, and isoenzymatic polymorphism of the l. enriettii taxon genome sequence of the non-pathogenic strain 15 of pneumonia virus of mice and comparison with the genome of the pathogenic strain j3666 birefringent spores differentiate encephalitozoon and other microsporidia from coccidia a fatal disease of the japanese waltzing mouse caused by a spore-bearing bacillus (bacillus piliformis cerebrospinal larva migrans due to baylisascaris procyonis 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cuniculus) the dermatophytes dermatologic problems in guinea pigs tularemia, plague, yersiniosis, and tyzzer's disease in wild rodents and lagomorphs in canada: a review fatal epizootic equine herpesvirus 1 infections in new and unnatural hosts spontaneous human herpes virus type 1 infection in a chinchilla (chinchilla lanigera f. dom.) human infections associated with bordetella bronchiseptica dogs as vectors of streptobacillus moniliformis infection an epidemic of pasteurella infection in a guinea-pig stock streptobacillus moniliformis -a zoonotic pathogen. taxonomic considerations, host species, diagnosis, therapy, geographical distribution interlaboratory comparison of enzyme-linked immunosorbent assay (elisa) and indirect immunofluorescence (iif) for detection of bordetella bronchiseptica antibodies in guinea pigs rabbit mite infestation development of resistance to reinfection of bordetella bronchiseptica in guinea pigs recovered from natural infection an evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs mange caused by trixacarus caviae in guinea pigs persistent sv5 virus infection in continuous cell cultures sarcoptid mite infestation in a colony of guinea pigs naturally occurring tyzzer's disease and intestinal spirochetosis in guinea pigs key: cord-300815-1vy787md authors: fang, li-qun; sun, yu; zhao, guo-ping; liu, li-juan; jiang, zhe-jun; fan, zheng-wei; wang, jing-xue; ji, yang; ma, mai-juan; teng, juan; zhu, yan; yu, ping; li, kai; tian, ying-jie; cao, wu-chun title: travel-related infections in mainland china, 2014–16: an active surveillance study date: 2018-07-20 journal: lancet public health doi: 10.1016/s2468-2667(18)30127-0 sha: doc_id: 300815 cord_uid: 1vy787md background: transmission of infection through international travel is a growing health issue, and the frequency of imported infection is increasing in china. we aimed to quantify the total number of infections imported into mainland china by arriving travellers. methods: we actively surveyed arriving travellers at all 272 international entry–exit ports in mainland china. suspected cases were detected through fever screening, medical inspection, self-declaration, and reporting by on-board staff. participants completed a standardised questionnaire with questions about demographics, their travel itinerary (including detailed information about all countries or regions visited), and clinical manifestations. nasopharyngeal swabs, sputum samples, faecal samples, vomitus, blood, and serum were collected as appropriate for diagnoses. diagnosis was made by specific laboratory tests according to the national technical guidelines. infections were classified as respiratory, gastrointestinal, vector-borne, blood-transmitted and sex-transmitted, or mucocutaneous. we divided arriving travellers into two groups: travellers coming from countries other than china, and travellers coming from hong kong, macau, and taiwan. we integrated surveillance data for 2014–16, calculated incidences of travel-related infections, and compared the frequency of infections among subgroups. findings: between jan 1, 2014, and dec 31, 2016, 22 797 cases were identified among 805 993 392 arriving travellers—an overall incidence of 28·3 per million. 45 pathogens were detected in participants: 18 respiratory (19 662 cases), ten gastrointestinal (189 cases), seven vector-borne (831 cases), seven blood-transmitted and sex-transmitted (1531 cases), and three mucocutaneous (584 cases). both the overall number and incidence of infection were more than five times higher in 2016 than in 2014. case numbers and incidences also varied substantially by province, autonomous region, and municipality. overall, 17 643 (77%) infections were detected by fever screening, but 753 (49%) blood-transmitted and sex-transmitted infections were identified through medical inspection. 14 305 (73%) cases of respiratory infection and 96 (51%) of gastrointestinal infections were in tourists. tuberculosis, hepatitis a virus, vector-borne, and blood-transmitted and sex-transmitted infections were common among chinese labourers who worked abroad. dengue and malaria were most commonly diagnosed in travellers arriving from africa. 12 126 (93%) of the 12 985 cases arriving from hong kong, macau, or taiwan were respiratory infections. hand, foot, and mouth disease accounted for 2·90% of infections in travellers from hong kong, macau, or taiwan and 0·31% of infections in international travellers. interpretation: this report is the first to characterise the profile of travel-related infections among arriving travellers in mainland china. our findings should increase public awareness of the potential risk of imported infections, and help health-care providers to make evidence-based health recommendations to travellers. funding: the natural science foundation of china. the number of international travellers has more than doubled worldwide during the past two decades, from 527 million in 1995 to 1·186 billion in 2015. 1 this rapid increase in cross-border travelling has become the main driver of the global spread of infections, as exemplified by international transmission of severe acute respira tory syndrome, dengue, 2009 influenza a (h1n1), and zika virus. [2] [3] [4] [5] timely identification of infections among arriving travellers can help to alert the medical and public health communities of outbreak threats before they affect the general population of that country. surveillance of travel-related infections is important for global public health as international travel continues to increase worldwide. 6, 7 china has become the world's fourth most popular destination in terms of arrivals, with 57 million tourists in 2015, 1 and has inevitably been affected by travelrelated infections. malaria and dengue have been introduced by arriving travellers from countries where those diseases are endemic. [8] [9] [10] an outbreak of chikungunya virus caused by infected travellers has been recorded in main land china, 11 and zika virus, yellow fever, and rift valley fever have all been imported within the past 8 years. [12] [13] [14] [15] in response to the increasing risk of imported infections, the general administration of quality supervision, inspection and quarantine of the people's republic of china (aqsiq) obliged all air and surface ports to do active surveillance of infections among arriving travellers. systematic surveillance began in 2014 to reduce risk of autochthonous transmission and help to inform evidence-based advice for travellers. we integrated the data from all entry-exit ports in mainland china to characterise travel-related infections, define the demographic features of imported cases, identify risk groups and exposure countries or regions, and assess the effectiveness of surveillance for further improvement. we did an active surveillance study of all travel-related infections from jan 1, 2014, to dec 31, 2016 , at all 272 international entry-exit ports to mainland china, comprising 69 airports, 128 water (sea and river) ports, and 75 land (highway and railway) entry-exit stations (appendix p 2). we also gathered data for the total number of people arriving in each of the 22 provinces, five autonomous regions, and four municipalities of mainland china. all patient data were anonymised. because this study constituted public health surveillance rather than research in human beings, ethical approval from institutional review boards was not required. participants or their guardians provided written informed consent for collection of biological samples (appendix p 4). people who were suspected of having an infection but who did not consent to collection of samples were made known to local centres for disease control and prevention for potential further follow-up. active surveillance was done among all arriving travellers at a quarantine station before they passed through customs at each entry-exit port. suspected cases were detected through four approaches: fever screening, medical inspection, self-declaration, and reporting by on-board staff (appendix p 3). all people with suspected infections were quarantined according to who's international health regulations and the rules for the implementation of frontier health and quarantine law of the people's republic of china. participants completed a standardised questionnaire with questions about demographics, their travel itinerary (including detailed information about all countries or regions visited), and clinical manifestations. infections were diagnosed by laboratory testing at the international travel health-care centre of each provincial entry-exit inspection or the quarantine bureau, per the surveillance technical scheme developed by aqsiq. 16 quarantine officers at entry-exit ports suggested which tests should be done on the basis of clinical manifestations in each suspected patient. they collected nasopharyngeal swabs, sputum samples, faecal samples, vomitus, blood, or serum samples as appropriate for diagnoses. according to core capacity requirements for surveillance and response in who's international health regulations and the frontier health and quarantine law of china, aqsiq listed 28 required and 40 recommended infectious diseases for causative testing. 17, 18 diagnosed infections were classified as respiratory, gastrointestinal, vector-borne, blood-transmitted and sex-transmitted, or mucocutaneous according to standard clinical practice evidence before this study we searched pubmed and isi web of science with the terms "travel" and "infection" or "infectious disease", and "global spread" and "infection" or "infectious disease" for work published in any language between jan 1, 2000, and oct 31, 2017. we noted that previous studies mainly focused on surveillance of post-travel illness, which was usually based on clinician-based surveillance systems-eg, geosentinel, which tracks infectious diseases and other adverse health outcomes related to travel. several identified studies were about the epidemiological features of imported malaria or about febrile illnesses or hiv infection at one port of china. we did not find any studies of active surveillance for various infections among arriving travellers at all entry-exit ports throughout mainland china. to the best of our knowledge, our report is the first to characterise the spectrum of travel-related infections, and reveals variety in frequency of each infection by traveller type, exposure country or region, and arrival provinces of mainland china in 2014-16, representing the pattern of travel-associated infections during travel. our findings imply that health-care providers should make evidence-based health recommendations to travellers before travel, and do destination-specific medical assessments of arriving travellers when they are ill, based on our estimates of the incidence of infections. active surveillance at entry ports can identify imported cases with emerging or re-emerging infections to prevent or at least postpone local transmission. in addition to entry surveillance upon arrival, follow-up surveillance (especially contact tracing of highly communicable infections) is needed to better understand the whole profile of travel-related infections. overall, our findings should help to increase public health awareness about the potential risk of imported infections to mainland china. see online for appendix in china (appendix pp [5] [6] . patients with infections were informed of their diagnosis, and recommended for treatment. we also informed local centres for disease control and prevention of each case, and close contacts were informed for prevention and quarantine, if necessary, according to who's international health regulations and the frontier health and quarantine law of china. we excluded people without specific diagnoses, people with ambiguous itineraries, and those whose final diagnosis had been identified before travelling. we used epidata 3.0 to establish a structured database. each case was geo-referenced to a world map with arcgis (esri, redlands, ca, usa) according to the exposure location where patients might have been infected. the exposure location was defined as the country or region that the participant travelled from. for travellers who visited several destinations, exposure location was established according to their itinerary on the basis of incubation period or known patterns of endemicity. arriving travellers were classified into two groups: travellers coming from countries other than china, and travellers coming from hong kong, macau, and taiwan descriptive statistics were calculated for all variables. continuous variables were summarised as median and range. we estimated the annual incidence of each infection at national and provincial levels. proportions were calculated according to various categories. we constructed graphs to show distribution patterns of proportion among different subgroups, and created thematic maps according to entry province and exposure countries. the study funder had no role in study design; data collection, analysis, or interpretation; or writing of the report. w-cc and l-qf had full access to all data in the study, and had final responsibility for the decision to submit for publication. of 805 inner mongolia (6) heilongjiang (4) jilin (3) liaoning (6) 45 types of pathogens were detected in participants: 18 respiratory pathogens (19 662 cases), ten gastrointestinal pathogens (189 cases), seven vector-borne pathogens (831 cases), seven blood-transmitted and sex-transmitted pathogens (1531 cases), and three mucocutaneous pathogens (584 cases; figure 2a) . the most frequent respiratory infection was influenza virus, followed by rhinovirus ( figure 2a) . dengue was the most common vector-borne disease, hepatitis b virus infection was the most common blood-transmitted and sex-transmitted infection, and hand, foot, and mouth disease was the most common mucocutaneous infection ( figure 2a) . 16 237 (83%) respiratory infections, 76 (40%) gastrointestinal infections, 553 (67%) vector-borne infections, and 346 (85%) of the 406 cases of hand, foot, and mouth disease were detected by fever screening (figure 2b). 9812 (43%) of participants arrived from a different country (140 countries represented), whereas 12 985 (57%) came from hong kong, macau, or taiwan. the types and distribution of infection differed substantially between these two groups (figure 3). 12 126 (93%) of the 12 985 cases arriving from hong kong, macau, or taiwan were respiratory infections. influenza was the most common infection in both groups, accounting for 6738 (69%) cases in the international group and 10 749 (83%) cases in the hong kong, macau, and taiwan group ( figure 3) . however, hepatitis b and c virus infections, syphilis, hiv, malaria, and dengue were substantially more common in the international group, whereas respiratory infections with rhinovirus, para influenza virus and metapneumovirus, and hand, foot, and mouth disease were more common in travellers from hong kong, macau, and taiwan ( figure 3) . in the international travel group, infection was acquired in the western pacific region by 5153 (53%) people, in the south-east asia region by 2666 (27%), in the european taiwan region by 838 (9%), in the american region by 432 (4%), in the african region by 448 (5%), and in the eastern mediterranean region by 275 (3%). influenza accounted for more than two-thirds of cases among travellers who acquired infections in the western pacific, south-east asia, european, american, and eastern mediterranean regions (appendix p 7). people with gastrointestinal infections mainly travelled from countries in the south-east asia and western pacific regions ( figure 4b ). vector-borne diseases, including dengue and malaria, were predominantly diagnosed among travellers from countries in western and central africa and the south-east asia region (figure 4c). cases with mucocutaneous infections mostly come from countries in the western pacific region and eastern europe (figure 4). we noted seasonal patterns of travel-related infections, which differed between the international travellers (infection peak in november and december) and those who travelled from hong kong, macau, or taiwan (respiratory infections peak in february and june, mucocutaneous infection peak in may and july; appendix p 8). to our knowledge, ours is the first multicentre study of travel-related infections among travellers arriving in mainland china. we characterised the range of travel-related infections, and showed how they varied by traveller type, exposure country or region, and province of arrival to mainland china. our findings, which are based on surveillance data for 45 infections among 22 797 travellers who arrived at the 272 entry-exit ports to the mainland, are helpful for increasing public health awareness about the potential risk of imported infections. most previous studies about this topic in mainland china explored the epidemiological features of imported malaria, or febrile illnesses or hiv infection at a single entry-exit port. 8, 9, [19] [20] [21] [22] by comparison with geosentinel, a clinician-based global surveillance system that tracks infectious diseases and other adverse health outcomes related to travel, 23 we actively surveyed infections among travellers upon arrival in mainland china. this method enabled us not only to profile imported infections, identify risk groups, and formulate optimum advice for travellers (as done previously on the basis of data from geosentinel or its linked european surveillance network, eurotravnet), [24] [25] [26] but also to estimate the incidence of each infection by using the total number of arrivals as denominator. additionally, active surveillance of arriving travellers enables timely identification of imported infections, allowing for alerting of public health authorities of threats before autochthonous transmission. both case numbers and incidence of infection have increased over time among travellers arriving in mainland china (table) . this increase probably reflects the worldwide epidemic trend of infectious diseases. 27 improvements in diagnostic procedures and rapid tests might have also contributed to the increase. case numbers and incidence varied greatly between the 31 provinces, autonomous regions, and municipalities (figure 1), which implies that each province should optimise a unique strategy for surveillance of, and response to, specific infections. the diagnostics used in our study were based on the surveillance technical scheme developed by aqsiq, 16 whereby diagnosis was based on recommended specific laboratory tests. accordingly, we classified diagnosis according to the main transmission route of each infection: respiratory, gastrointestinal, vector-borne, blood-trans mitted and sex-transmitted, and mucocutaneous. over four-fifths of respiratory infections and about two-thirds of vector-borne and mucocutaneous infections were detected by fever screening (table), suggesting that fever screening is essential to identification of febrile patients. our findings also suggest that medical inspection should be sustained to identify people with mucocutaneous infections other than hand, foot, and mouth disease or blood-transmitted and sex-transmitted infections, which are often associated with obvious clinical manifestations such as rashes, vomiting, and jaundice (appendix pp 9-10). additionally, on-board staff should be encouraged to report patients they suspect of being infected for timely identification. chinese labourers (ie, chinese people who work overseas, mostly in the manufacturing, construction, forestry, fishing, transportation, and catering industries, although some are highly skilled workers) abroad seemed prone to tuberculosis, hepatitis a virus infection, vectorborne infections, and blood-transmitted and sextransmitted infections (figure 2c). pre-travel advice to labourers should focus on such public health threats. additionally, we suggest that on-site health education and primary health care should be provided to chinese labourers in the countries where they work. rubella, acute conjunctivitis, and herpes zoster virus infections were frequently detected in sailors (figure 2c), suggesting that close contact and working in humid environments are risk factors for transmission of these infections. although the infection profiles of international travellers and travellers from hong kong, macau, and taiwan differed substantially, influenza was the most common infection in both groups (figure 3). our findings show the epidemic pattern of influenza in different global regions, 28 and imply that vaccination should be considered if travelling to a region where influenza transmission is ongoing. 29 people with blood-transmitted and sextransmitted diseases were mostly male labourers and sailors (table). although the reasons for high risk in these populations were unclear, our study shows the sociodemographic characteristics of people with these diseases arriving in mainland china, which were significantly associated with travel-associated sexually transmitted infections in data gathered by geosentinel travel medicine clinics worldwide. 30 further research is required to investigate effective intervention measures. dengue and malaria were frequent among international travellers arriving from africa (appendix p 7). imported dengue has caused autochthonic transmission in new areas of china, europe, and the usa. [31] [32] [33] [34] in our study, we also noted the arrival of patients with zika virus, yellow fever virus, and rift valley fever to mainland china since february, 2016. [12] [13] [14] [15] governments and the public health community should be aware of these new threats to prevent possible local epidemics. in travellers from hong kong, macau, and taiwan, the incidence of hand, foot, and mouth disease was high (figure 3). we could not establish where these travellers were infected, because they usually travel frequently to and from mainland china. detailed data about itineraries should be collected to help to clarify the infection exposure site. the warm and humid climate in hong kong, macau, taiwan, and southern china could favour transmission of hand, foot, and mouth disease. fortunately, a vaccine against the ev71, the most prevalent virus strain, is commercially available and has a good protective effect. 35 we mapped travel-related infections globally to show differences in terms of exposure country and region ( figure 4) . health-care professionals should be aware of the specific risks to travellers, and should provide targeted pre-travel consultations. accordingly, countryspecific or region-specific vaccination and prophylaxis are strongly recommended to reduce the burden of travel-related infections. our study had several limitations. because our findings were based on surveillance of travellers when entering mainland china, most cases had clinical manifestations of disease upon arrival (appendix pp 9-10). patients who were infected but did not develop symptoms until after arrival were not included in our study. thus, the frequency of travel-related infections are underestimated. by contrast, for infections such as tuberculosis, hepatitis b and c virus infections, hiv, and syphilis, which have long latent or incubation periods, the location of acquisition is difficult to establish. for example, chinese citizens who travelled overseas and were diagnosed upon arrival back to mainland china could have acquired the infection before travelling. thus, the frequency of such infection in arriving travellers could have been overestimated. although we estimated the overall incidence of each infection, would could not calculate the numerical risk for travellers coming from a particular country or region because of the lack of denominator data. furthermore, we could not compare the demographic characteristics of the 22 797 people with an infection to those without an infection, because demographic data were not collected for all arriving travellers. in conclusion, our findings suggest that health-care providers should provide evidence-based health recommendations to travellers before travel and should do destination-specific medical assessments of arriving travellers who are ill. active surveillance at entry-exit ports can help with timely identification of people with emerging or re-emerging infections to prevent or at least postpone local transmission. 36, 37 in addition to entry surveillance, follow-up surveillance-especially contact tracing of highly communicable infections-will be necessary to understand the entire profile of travel-related infections. we declare no competing interests. world tourism organization. unwto tourism highlights transmission of the severe acute respiratory syndrome on aircraft global spread and persistence of dengue spread of a novel influenza a (h1n1) virus via global airline transportation potential for zika virus introduction and transmission in resource-limited countries in africa and the asia-pacific region: a modelling study approach to fever in the returning traveler surveillance for travel-related disease-geosentinel surveillance system epidemiologic features of overseas imported malaria in the people's republic of china plasmodium falciparum malaria importation from africa to china and its mortality: an analysis of driving factors the changing epidemiology of dengue in china, 1990-2014: a descriptive analysis of 25 years of nationwide surveillance data isolation, identification and genomic characterization of the asian lineage zika virus 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of exposure among ill returned travelers geosentinel surveillance of illness in returned travelers travel-associated infection presenting in europe (2008-12): an analysis of eurotravnet longitudinal, surveillance data, and evaluation of the effect of the pre-travel consultation who. emergencies: disease outbreaks global circulation patterns of seasonal influenza viruses vary with antigenic drift latitudinal patterns of travel among returned travelers with influenza: results from the geosentinel surveillance network, 1997-2007 travel-associated sexually transmitted infections: an observational cross-sectional study of the geosentinel surveillance database climate and the timing of imported cases as determinants of the dengue outbreak in guangzhou, 2014: evidence from a mathematical model autochthonous dengue fever in croatia first two autochthonous dengue virus infections in metropolitan france efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in china entry screening to delay local transmission of 2009 pandemic influenza a (h1n1) effectiveness of screening for ebola at airports we thank the medical inspection officers, on-board staff, and laboratory-test personnel who contributed to the detection, epidemiological investigation, and diagnosis of all suspected infections. key: cord-311908-sgdq6j6x authors: atkins, g. j.; mcquaid, s.; morris‐downes, m. m.; galbraith, s. e.; amor, s.; cosby, s. l.; sheahan, b. j. title: transient virus infection and multiple sclerosis date: 2000-09-28 journal: rev med virol doi: 10.1002/1099-1654(200009/10)10:5<291::aid-rmv278>3.0.co;2-u sha: doc_id: 311908 cord_uid: sgdq6j6x multiple sclerosis (ms) is a chronic, demyelinating disease of the cns in which autoimmunity to myelin plays a role in pathogenesis. the epidemiology of ms indicates that it may be triggered by a virus infection before the age of adolescence, but attempts to associate a specific virus with ms have produced equivocal results. many studies of the aetiology of ms have postulated that a persistent virus infection is involved, but transient virus infection may provide a plausible alternative mechanism that could explain many of the inconsistencies in ms research. the most studied animal model of ms is chronic relapsing experimental autoimmune encephalomyelitis (creae), which is induced in susceptible animals following injection of myelin components. while creae cannot provide information on the initiating factor for ms, it may mimic disease processes occurring after an initial trigger that may involve transient virus infection. the disease process may comprise separate triggering and relapse phases. the triggering phase may involve sensitisation to myelin antigens as a result of damage to oligodendrocytes or molecular mimicry. the relapse phase could be similar to creae, or alternatively relapses may be induced by further transient virus infections which may not involve infection of the cns, but which may involve the recrudescence of anti‐myelin autoimmunity. although current vaccines have a high degree of biosafety, it is suggested that the measles‐mumps‐rubella vaccine in particular could be modified to obviate any possibility of triggering anti‐myelin autoimmunity. copyright © 2000 john wiley & sons, ltd. the possible involvement of viruses in the aetiology of multiple sclerosis (ms) is a subject that has created much controversy. from epidemiological studies based on immigration data and the occurrence of clusters, it has been suggested that an environmental factor(s) triggers ms before the age of adolescence, while symptoms of the disease are not observed until years later. it is also apparent that there is a genetic susceptibility to ms, so a hypothesis for the aetiology of ms formulated on this evidence is that it is a disease triggered by an environmental factor in genetically susceptible individuals during childhood [1±8] . circumstantial evidence suggests that the environmental factor in ms could be a virus [7±14] . several human and animal virus infections are characterised by the cns demyelination that is also characteristic of ms (table 1) . these include subacute sclerosing panencephalitis (sspe), caused by a persistent measles virus infection, and human t cell lymphotropic virus-i (htlv-i)associated myelopathy, which is a slowly progressive neurological disease characterised by in¯ammatory in®ltrates and demyelination in the cns, and is caused by an exogenous retrovirus. many viruses have been implicated in ms itself [13, 14] (table 2 ), including measles virus and more recently ebv, [15] human herpesvirus 6 (hhv6) [16] and human endogenous retroviruses [17] . there is also evidence to suggest that some of the relapses which are characteristic of most ms cases are preceded by virus infections [18±20] . for example, a recent report demonstrated a signi®cantly higher exacerbation rate in ms patients following in¯uenza virus infection, suggesting that in¯uenza infection may trigger relapses [21] . evidence for the involvement of virus infection in ms also comes from several animal diseases in which persistent virus infection gives rise to demyelination [10,11,22±24] . while these studies point to a viral aetiology of ms, no virus has yet been de®nitely associated with this disease despite intensive effort. one possible explanation for the equivocal results obtained for identi®cation of viruses common uncharacterised infections transient associated with ms is that, while the disease is triggered by a virus infection, the subsequent course of the disease may not require virus persistence. it is probable that virus persistence does occur in some cases, but it may or may not be relevant to the progression of the disease. it is also possible that a number of different viruses, rather than one speci®c type, may act as a trigger for ms in susceptible individuals. another important factor that requires consideration is the heterogeneity in the detailed neuropathology and clinical disease progression in ms, suggesting that the disease may not have a single aetiology. it is possible that primarily progressive ms and relapsing remitting ms are two distinct disease entities [25] . ms may in fact be divided into at least ®ve disease subtypes on the basis of detailed analysis of neuropathology [26, 27] , and it is possible that only some of these may have viral involvement. the main assumption that has been made in considering the possible viral aetiology of ms is that a persistent infection by one virus is involved. here we review the evidence for transient virus infection, with a variety of agents, as an alternative to persistent infection in the aetiology of ms. in an attempt to understand the aetiology of ms, animal models have been used. the most studied autoimmune model of ms is experimental autoimmune encephalomyelitis (eae), which may be induced in a number of animal species by immunisation with myelin antigens or transfer of myelin-reactive class ii restricted cd4+ t lymphocytes [10] . the type of eae induced is dependent on the immunisation protocol, animal strain and antigen used. in some cases the protocol results in an acute monophasic disease and in others a chronic relapsing disease (creae) occurs. creae resembles ms because animals develop accumulating neurological de®cit following each disease phase. eae is characterised by in¯ammatory in®ltration of the cns associated with lesions of demyelination, and the topography of lesions within the cns is dependent on the nature and origin of the autoantigen [28] . the extent of demyelination varies with the protocol used but is more pronounced when myelin-speci®c antibodies are injected following transfer of mbp-speci®c t cells or at the onset of spinal cord homogenate-induced disease [29, 30] . the possible role of anti-myelin antibodies is supported by the ®ndings of myelin reactive antibodies in the blood of ms patients, and recently autoantibodies directed to myelin basic protein (mbp) and myelin oligodendrocyte protein (mog) peptides have been detected within lesions of demyelination in the cns of ms patients [31] . while antibodies may be involved in the development of demyelination, many other immunological mechanisms, including the release of in¯ammatory mediators of disease such as tnf-a and reactive oxygen species, or the action of cytotoxic t-lymphocytes, may play a role in the myelin damage [32] . the chronicity of the disease both in creae and possibly ms may be due to the perpetuation of autoimmune responses by determinant spreading. this process occurs as a result of myelin damage in the initial phase of disease, when new myelin antigens are released. the release of such antigens may subsequently induce new t-cell speci®cities, some of which may be pathogenic [33] . in contrast to eae are the virus-induced models, in which both natural and experimental infections of animals result in widespread demyelination and neurological de®cit. the naturally occurring diseases include canine distemper virus of dogs, and visna virus infection of sheep, both of which have been used to study the mechanism of virusinduced demyelination as models for ms. laboratory virus infections of mice and rats have also been used as models to understand the processes of virus-induced demyelination. these experimental models include murine coronavirus, where persistent virus infection induces an eae-like disease [22, 34] . myelin reactive t cells are also observed following experimental infection of rats with measles virus and some of these t cell lines are encephalitogenic, supporting the hypothesis that a virus trigger can lead to autoimmunity and myelin damage [35±37]. another virus model of ms is theiler's murine encephalomyelitis virus (tmev) infection of mice, which produces a chronic demyelinating disease associated with virus persistence [10, 11, 22, 23, 38] . autoimmune responses to myelin antigens are observed following infection with tmev, and while they may not play a major role in the initiation of demyelination, autoimmunity to myelin components probably contributes to lesion progression in chronically diseased animals [23] . an alternative model of virus-induced demyelination is semliki forest virus (sfv) infection of mice [39, 40] . sfv induces acute immune-mediated demyelination in mice, which is repaired and does not recur in most mouse strains. the initial immune-mediated demyelination induced by sfv infection may be due to targeting of sfv-infected oligodendrocytes by cytotoxic t-cells. it is clear, however, that t-cell responses to mbp [41±43] and to a range of encephalitogenic myelin epitopes (m. morris-downes and s. amor, unpublished data) are also induced. a mog peptide showing sequence similarity to the sfv e2 envelope protein, as well as the e2 peptide itself, has been shown to induce creae in mice, and it has been suggested that this molecular mimicry may contribute to sfv-induced demyelination [44] . in most mouse strains, for example in balb/c mice, myelin repair is complete by 3 months after avirulent sfv infection. however, in sjl mice, small lesions of demyelination persist up to a year after infection and some of these lesions show active in¯ammatory demyelination. long-term lesions do not correlate in individual mice with persistence of the virus genome, but do correlate with expression of the cytokines tnf-a or ifn-c [45, 46] . expression of these cytokines in the cns is undetectable in balb/c mice by 3 months after infection, but continues in most sjl mice beyond a year after infection [45] . sjl mice, which are susceptible to eae, appear to have a defect in innate suppression of immune responses, which is associated with secretion of lower levels of the regulatory cytokine tgf-b [41] . prior infection with sfv predisposes mice to eae, even in strains that are normally resistant to eae, and infection with sfv has been shown to exacerbate eae [42, 47, 48] . it is clear that the demyelination characteristic of sfv infection is immune-mediated and it appears that this may be triggered by virus infection of oligodendrocytes early in infection. such infection may induce damage to the infected cells and the subsequent release of myelin antigens by the induction of oligodendrocyte cell death [49] . sfv-infected oligodendrocytes could also be targeted by cd8+ t-lymphocytes [50, 51] or alternatively, uninfected oligodendrocytes and/or myelin in the vicinity of aggressive immune responses may be damaged by the action of cytokines such as tnf-a [43, 51] . either way, normally sequestered myelin antigens are likely to be presented to the immune system to induce autoimmunity to myelin. a possible mechanism whereby recrudescence of immunity in the cns could be triggered by a virus infection is suggested by a study on in¯uenza virus infection of mice. following an initial cns infection, subsequent infections led to the long-term persistence of activated cytotoxic t-lymphocytes in the brain parenchyma, long after the infection was cleared [52] . another study that does not involve virus infection, but may nonetheless provide information concerning the possible viral aetiology of ms, concerns the exacerbation of brain damage following eae induction. if a brain cryolesion is in¯icted on rats after induction of eae by injection of myelin components, eae lesions are enhanced [53] . this may be consistent with the proposal that the brain damage caused by virus infection could enhance eae in a manner that is not speci®c to the virus. evidence for the involvement of speci®c viruses in ms comes from serological studies and direct virus detection. it was found many years ago that antibody levels to several common viruses are elevated in ms patients, but it is not clear whether this is related to the cause of ms or an epiphenomenon. many viruses have been detected in cns autopsy tissue from ms patients [13±17] ( table 2 ); many such claims have either been retracted or have not been con®rmed by other workers. the inconsistent detection of viruses in cns autopsy tissue from ms patients is illustrated by a study in which brain samples from 8 cases of ms and 56 controls were examined by in situ hybridisation [54] . samples were examined for genomic rna sequences of measles virus, canine distemper virus, rubella virus and simian virus 5, all viruses that have been implicated in ms. positive hybridisation was detected in two of the ms cases for measles virus only, but was also detected in one of the controls. the latter was a neurological control that was a case of disseminated cmv infection, but which showed demyelination that is uncharacteristic of cmv infection [54] . in subsequent investigation, the number of ms cases found to be positive for measles virus rna has increased to 4 out of 14 cases examined (s. mcquaid and l. cosby, unpublished data). in another study, measles virus rna could not be detected by rt-pcr in peripheral blood leucocytes from ms patients [55] . measles, mumps and rubella virus rna has been shown to be absent from brain autopsy tissue from ms patients by rt-pcr [56] . these and other studies indicate that most cases of ms are not associated with persistent virus infection, at least with the viruses tested, but a minority of cases may be associated with the persistence of measles virus in the cns. more recently attention has focused on hhv6 [16] , ebv [57] and the ms-associated retroviruses [17] . while causal associations have yet to be de®nitely made with ms, it is possible that infection with a single virus alone is insuf®cient for the development of ms and dual infections such as retrovirus and ebv are required [15] . some positive evidence is available, however, linking transient virus infection with ms or exacerbations of ms. common virus infections, identi®ed by their symptoms rather than by virus detection, have been associated with exacerbations of ms [18, 20] . apart from post-vaccination encephalitis, rabies vaccination has been associated with a disease that is indistinguishable from ms at autopsy [12] , and it has been shown that the semple vaccine strain of rabies virus can induce anti-myelin autoimmunity [58] . recent studies have suggested that both in¯uenza infection and in¯uenza vaccination are associated with an increased frequency of exacerbations of ms [21] . like the evidence for viruses in ms, the data are controversial, since other studies have failed to ®nd an association between exacerbations of ms and in¯uenza vaccination [59] . a summary of possible mechanisms for the involvement of transient virus infections in stim-ulating anti-myelin autoimmunity is shown in figure 1 . it is possible that sensitisation to myelin antigens may follow an initial trigger involving a virus infection. subsequently, recrudescence of anti-myelin autoimmunity could occur during relapse phases induced by further transient virus infections. the anti-myelin autoimmunity may only produce a threshold of damage to myelin, resulting in clinical signs and symptoms, in a minority of patients with a genetic susceptibility to ms. this may involve an inability to suppress anti-myelin autoimmunity. the initial phase could result in damage to myelin and the release of normally sequestered myelin antigens, which would then prime the immune system to induce anti-myelin autoimmunity. in the case of a virus infection, this initial phase could involve damage to oligodendrocytes. such damage could be caused by direct infection of oligodendrocytes and the induction of cell an alternative mechanism of priming of the immune system to myelin antigens may be molecular mimicry. many bacterial and viral proteins share sequence and structural homologies with known autoantigens. several viruses share part of the sequence of myelin proteins in their genome [60, 61] , and a number of peptides based on these sequences activated myelin-speci®c t cell clones [61] . sequence similarity between mbp and hepatitis b virus exists, and this peptide sequence induced eae in rabbits [62] ; also, a rubella peptide shows sequence similarity to myelin proteolipid protein [63] . a mechanism similar to molecular mimicry may be the incorporation of self-antigens into the envelope of budding viruses leading to autosensitisation. if vesicular stomatitis virus, a model enveloped virus, is grown in mbp-expressing cell cultures it is highly ef®cient in triggering t-cell responses to mbp, both in vitro and in vivo [64] . thus, viruses could act as presenters of host antigen to the immune system. this initial triggering phase could be followed by a relapse phase in which reactivation of myelin-speci®c t-cells already present in the cns occurs, or alternatively myelin-reactive immunity is induced outside the cns and in®ltration of the brain parenchyma by lymphocytes and other components of the immune system occurs. in either case, such activation may be a result of a transient virus infection which compromises the blood±brain barrier and leads to further in¯ux of cells and soluble components [65, 66] . a possible mechanism for the recrudescence of anti-myelin autoimmunity, as well as those mentioned above for the triggering phase, may be the activation of myelin-reactive t-cells by viral superantigens. such activation relies on the preexistence of myelin reactive t cells but does not occur via the classical t-cell receptor : peptide : mhc interaction. instead it involves the polyclonal activation of t cells through the vb element of the t cell receptor. it has been demonstrated that eae relapses may be induced with staphylococcal superantigens, although injection of the same superantigens into naive mice failed to induce eae [67] . it is feasible that viruses may also act in this way, as has been shown for murine mammary tumour virus [68] . if the proposed triggering mechanism is correct, then de®nitive evidence for it could not be obtained from ms autopsy or biopsy tissue, because the myelin damage resulting in the triggering event probably occurred some time previously and only the consequences of this would be evident. also, if the damage were a result of virus infections, it may not be speci®c to any one virus, as at least in experimental infection many infections may give rise to a similar pathology, suggesting that any virus (including non-neurotropic viruses) that induces myelin sensitisation could act as a trigger. with regard to the activity of known human viruses in the induction of myelin damage, there is evidence that virus infections associated with cns demyelination can cause damage to oligodendrocytes. measles virus has been implicated in ms [54] , and virus infection of oligodendrocytes is seen in cases of sspe [69] , which is a rare human demyelinating disease caused by a persistent infection with measles virus (figure 2a) . similarly, progressive multifocal leucoencephalopathy (pml) is a rare human demyelinating disease caused by persistent infection with a human papovavirus. this virus has also been implicated in ms [70] , and virus infection of oligodendrocytes is seen in this disease (figure 2b) . in the sfv model, infection of oligodendrocytes occurs early in infection, and is followed by acute immune-mediated demyelination [71, 72] (figure 2e, f) . virus infection of oligodendrocytes by sfv is also seen in rat mixed glial cell cultures [73] (figure 2c, d) . the damage to oligodendrocytes induced by sfv infection may be due to induction of programmed cell death or apoptosis, since this is induced in cultured rat oligodendrocytes infected by sfv [74] . however, it has also been proposed that some mature oligodendrocytes in the animal survive sfv infection, and are capable of carrying out the remyelination observed later in the disease [75] . such virus-infected oligodendrocytes could be targeted by cd8+ t-lymphocytes [49] , which would also result in myelin damage. rubella virus, a togavirus like sfv, and another virus that has been implicated in ms [76] , shows tropism for oligodendrocytes in rat mixed glial cell cultures [77] . in canine distemper, which is a disease of dogs caused by a virus that has also been implicated in ms in the past [13] , damage to oligodendrocytes also occurs, although this does not appear to be associated with productive virus infection, which mainly occurs in astrocytes [78, 79] . lymphoproliferative responses to mbp have been detected in patients with encephalitis after measles virus, vzv or rubella virus infection, and after rabies vaccination [80] . in the case of measles virus, a t cell-mediated response to mbp was induced following intracerebral infection of rats; such sensitised t-cells showed no crossreactivity to measles virus, but could induce eae by adoptive transfer to naõ ève recipients. also, a normally non-encephalitogenic mbp peptide induced eae in measles virus-infected rats [37] . this is further evidence that virus infection of the cns can sensitise to subsequent autoimmune demyelination. the chronic immune-mediated demyelination that occurs in ms could be maintained by either of two mechanisms following sensitisation of the immune system to myelin antigens after the initial trigger. one mechanism could be similar to creae. another possibility is that the relapses characteristic of most cases of ms could be induced by further transient virus infections, which may not necessarily involve infection of the cns. the clinical evidence that transient virus infections could be implicated in relapses of ms is mentioned above; there is also indirect evidence that such a mechanism could operate. many virus infections lead to the production of pro-in¯ammatory cytokines such tnf-a or ifn-c. infection of sjl mice with sfv results in the expression of these cytokines in the cns for more than a year after the initial infection [45] . it is known that administration of ifn-c to ms patients results in exacerbation of the disease [81] . also, the demyelination in measles virus encephalitis cannot be associated with the presence of measles virus antigen in the cns at time of death, although it is not clear whether virus infection of the cns occurred earlier in the course of the disease [82] . it is possible that administration of ifn-b, which is a treatment currently used for ms with partial success [83] , could ameliorate the effects of some transient infections that may trigger exacerbations of ms. however, adminis-tration of ifn-b may also have an immunomodulatory effect [84] . it is possible that virus infection could induce secretion of pro-in¯ammatory cytokines that could penetrate the cns parenchyma from the blood and lead to the recrudescence of anti-myelin autoimmunity by reactivation of previously primed t-cells. it is also possible that such myelin-reactive t-cells could penetrate the brain parenchyma following damage to the blood±brain barrier caused by transient virus infection [66] , or following the secretion of pro-in¯ammatory cytokines and/or chemokines by cells within the cns (astrocytes and microglia as well as lymphocytes) [85] . if such a mechanism were to operate, it may not be virus-speci®c, but may be induced by many different transient infections. much of the evidence cited to support the idea that ms is associated with a speci®c persistent virus infection of the cns has been challenged, or shown to be incorrect. it is possible that there are still unknown viruses which infect the cns and which may contribute to ms pathogenesis. current candidates for persistent virus infection associated with the aetiology of ms include hhv6, ebv and human endogenous retroviruses. whether these viruses are indeed involved in the pathogenesis of at least some cases of ms is still unknown. however, ms may comprise a group of diseases of different aetiologies: persistent virus infection may represent one such aetiology, and the mechanisms triggered by transient infection described here may represent another. since the circumstantial evidence that ms is associated with a virus infection has not been refuted, it seems reasonable to consider the possibility that most cases of ms are not associated with a persistent virus infection, but that other mechanisms involving transient virus infection could operate. if ms is induced and/or exacerbated by transient virus infection, there are several corollaries that may explain the equivocal results obtained for the association of viruses with ms. firstly, it would not be possible in most cases to detect virus in autopsy tissue from ms patients, or in biopsy samples taken after the initial triggering phase. secondly, virus infections need not be speci®c, and it is possible that a range of viruses with common properties could be involved in either the triggering or maintenance phases. thirdly, it is probable that, if viruses are involved in triggering and/or maintaining ms, that these are common viruses that only have this effect in a minority of genetically susceptible individuals. if transient virus infection is involved in the triggering or maintenance of the antimyelin autoimmunity that is a risk factor for ms, the role of vaccination must be considered. of the common diseases for which vaccines are available, measles, mumps and rubella viruses have all been implicated in ms [9, 13, 14] . there is con¯icting evidence as to whether in¯uenza or hepatitis b vaccination can trigger cns in¯ammation [21, 59, 86, 87] . however, possible mechanisms of induction of cns in¯ammation may be different for hepatitis b and in¯uenza vaccines, which are non-replicative, compared with the measlesmumps-rubella (mmr) vaccine, which is a cocktail of three replicating attenuated viruses. wildtype measles, mumps and rubella viruses are all known to infect the cns, and the urabe am 9 mumps vaccine strain, now no longer used as a vaccine component, caused cns symptoms (aseptic meningitis) in a small minority of recipients [88±90] , showing that it is neurotropic. in developed countries, the mmr vaccine is routinely given to children to prevent disease caused by measles, mumps and rubella virus infections [89] . although it is clear that the incidence of ms has not been signi®cantly affected by the introduction of the vaccine, this could be explained if either the wild-type infections or the vaccine could trigger one type of ms in a minority of individuals. the mechanism by which vaccines may induce cns in¯ammation could be but one of a number of different mechanisms operating as an ms trigger. the hypothesis that vaccination could trigger ms is consistent with current knowledge of the epidemiology of ms, in particular the hypothesis deduced from studies of the epidemiology of ms, that it is a disease triggered by an environmental factor exerting its effect in a minority of genetically susceptible individuals before the age of adolescence. in the uk, rubella vaccination was introduced in 1970 for girls only but was extended to both sexes in 1988 when the mmr vaccine was introduced [89] . recently, peripheral neuropathy has been associated with rubella vaccination in which anti-mbp reactivity was prominent [91] . however, as has been suggested for in¯uenza [21] , vaccination may be less likely to trigger ms or ms exacerbations than the virulent infection. vaccination has been the most successful method of controlling virus diseases [92] , and current evidence indicates that the bene®ts of vaccination outweigh any disadvantages, for which there is no conclusive evidence at present. thus there is no justi®cation for the discontinuation of vaccination either in the general population or for ms patients. however, although current vaccines have a high degree of biosafety, a small risk associated with vaccination, which is much less than that associated with the wild-type infection, cannot be excluded. the evidence available at present does not indicate that vaccination can trigger ms and/or exacerbations of ms, but this possibility warrants further investigation of vaccines, particularly the mmr vaccine. in particular, the ability of vaccine strains to stimulate anti-myelin autoimmunity in a manner similar to wild-type virus strains, and as a combination vaccine, should be investigated. it is possible that the more rational design of vaccines, based on recombinant dna technology, could improve them and circumvent any possible problems which may be associated with current vaccines. it may be possible to replace live virus vaccines with engineered virus vaccines expressing only desired protective epitopes. the use of new genetically manipulated vaccines will create its own controversy, and the use of such vaccines can only be justi®ed if they are demonstrably more effective and have greater biosafety than conventional vaccines currently in use. of particular concern is a report that the plasmid backbone used to construct naked dna vaccines, which have been suggested as replacements for current vaccines [93] , has been shown to potentiate eae. the mechanism appears to be the induction of th1-promoting cytokines [94] . however, dna vaccines have poor immunogenicity and persist for long periods in the host tissue [95] (m. m. morris-downes and g. j. atkins, unpublished results). other types of prototype recombinant vaccines include naked rna vaccines and recombinant suicide particles based on the sfv genome [40, 96] . such vaccines stimulate immune responses more ef®ciently than naked dna vaccines [97] , and induce apoptosis which may lead to the removal of the vaccine from inoculated tissue [98] . this may lead to a more transient stimulation of pro-in¯ammatory cytokine synthesis than is possible either with conventional attenuated vaccines or naked dna vaccines. if the mechanism of induction of myelin damage by viruses were known, it may be possible to express only protective epitopes using such vaccine vectors. it may also be possible to omit known encephalitogenic sequences from such vaccines, and to circumvent any virus functions, such as a tropism for oligodendrocytes, which lead to myelin damage. multiple sclerosis and childhood infections serendipity and multiple sclerosis epidemiology is sporadic ms caused by an infection of adolescence and early childhood? a case±control study of birth order position further evidence in support of the hypothesis that one cause of multiple sclerosis is childhood infections the evolution of multiple sclerosis epidemiology epidemiologic evidence for multiple sclerosis as an infection multiple sclerosis, a disease acquired in childhood genetic factors in the pathogenesis of ms pathogenesis of multiple sclerosisðthe immune diathesis and the role of viruses animal model systems of ms pathogenesis of virus induced demyelination pathogenesis of multiple sclerosis. a critical reappraisal molecular biology of multiple sclerosis. russell wc viral etiology of multiple sclerosis: where does the truth lie? a putative new retrovirus associated with multiple sclerosis and the possible involvement of epstein±barr virus in this disease herpesvirus 6 might trigger multiple sclerosis moller larsen a. expression of sequence variants of endogenous retrovirus rgh in particle form in multiple sclerosis viral infections trigger multiple sclerosis relapsesða prospective seroepidemiological study acyclovir treatment of relapsing-remitting multiple sclerosis. a randomized, placebo-controlled, double-blind study clinical viral infections and multiple sclerosis effects of in¯uenza vaccination and in¯uenza illness on exacerbations in multiple sclerosis analysis of the molecular basis of neuropathogenesis of rna viruses in experimental animals: relevance for human disease? persistent infection with theiler's virus leads to cns autoimmunity via epitope spreading adoptive transfer of eae-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis primarily chronic progressive and relapsing/remitting multiple sclerosis: two immunologically distinct entities neuropathology in multiple sclerosis: new concepts distinct patterns of multiple sclerosis pathology indicates heterogeneity in pathogenesis experimental autoimmune encephalomyelitis: the antigen speci®city of t lymphocytes determines the topography of lesions in the central and peripheral nervous system augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies directed against a myelin/ oligodendrocyte glycoprotein anti-myelin antibodies modulate experimental allergic encephalomyelitis in biozzi abh mice identi®cation of auto-antibodies associated with myelin damage in multiple sclerosis multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system functional evidence for epitope spreading in the relapsing pathology of experimental autoimmune encephalomyelitis adoptive transfer of eae-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis characterization of measles virus-induced autoimmune reactions against myelin basic protein in lewis rats induction of autoimmune reactions to myelin basic protein in measles virus encephalitis in lewis rats synergistic interaction between measles virus infection and myelin basic protein peptide-speci®c t cells in the induction of experimental allergic encephalomyelitis in lewis rats the balance between persistent virus infection and immune cells determines demyelination semliki forest virus infection of mice: a model for the genetic and molecular analysis of viral pathogenicity the molecular pathogenesis of semliki forest virus: a model virus made useful immune responses, and autoimmune outcome, during virus infection of the central nervous system predisposition to eae induction in resistant mice by prior infection with semliki forest virus production and role of cytokines in the cns of mice with acute viral encephalomyelitis molecular mimicry between a viral peptide and a myelin oligodendrocyte glycoprotein peptide induces autoimmune demyelinating disease in mice long-term effects of semliki forest virus infection in the mouse central nervous system multiplication of virulent and demyelinating semliki forest virus in the mouse central nervous systemðconsequences in balb/c and sjl mice effect of viral infection on experimental allergic encephalomyelitis in mice facilitation of experimental allergic encephalomyelitis by irradiation and virus infection: role of in¯ammatory cells in vivo depletion of cd8+ t cells prevents lesions of demyelination in semliki forest virus infection role of immune responses in protection and pathogenesis during sfv infection characterization of the cellular and cytokine response in the central nervous system following semliki forest virus infection long-term persistence of activated cytotoxic t lymphocytes after viral infection of the central nervous system focal brain damage enhances experimental allergic encephalomyelitis in brain and spinal cord examination of eight cases of multiple sclerosis and 56 neurological and non-neurological controls for genome sequences of measles virus, canine distemper virus, simian virus 5 and rubella virus failure to detect measles virus rna by reverse transcription-polymerase chain reaction, in peripheral blood leucocytes of patients with multiple sclerosis absence of measles, mumps and rubella viral gene sequences from multiple sclerosis brain tissue by polymerase chain reaction increased risk of multiple sclerosis after late epstein-barr virus infection: a historical prospective study b-cell responses to myelin basic protein and its epitopes in autoimmune encephalomyelitis induced by semple rabies vaccine a multicenter, randomized, doubleblind placebo-controlled trial of in¯uenza immunization in multiple sclerosis sequence homology between certain viral proteins and proteins related to encephalomyelitis and neuritis molecular mimicry in t-cell mediated autoimmunity: viral peptides activate human t cell clones speci®c for myelin basic protein amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity multiple sclerosis and molecular mimicry autoimmunity caused by host cell protein-containing viruses breakdown of the blood±brain barrier precedes symptoms and other mri signs of new lesions in multiple sclerosis viral infection at the blood±brain barrier in multiple sclerosis:ðan ultrastructural study of tissues from a uk regional brain bank induction of relapsing paralysis in experimental autoimmune encephalomyelitis by bacterial superantigen reverse transcriptase-dependent and -independent phases of infection with mouse mammary tumor virus: implications for superantigen function the signi®cance of measles virus antigen and genome distribution in the cns in sspe for mechanisms of viral spread and demyelination polyomavirus models of brain infection and the pathogenesis of multiple sclerosis semliki forest virus neurovirulence mutants have altered cytopathogenicity for central nervous system cells oligodendrocyte infection and demyelination produced in mice by the m9 mutant of semliki forest virus pathogenicity of semliki forest virus for the rat central nervous system and primary rat neural cell culturesð possible implications for the pathogenesis of multiple sclerosis death mechanisms in cultured cells infected by semliki forest virus morphology of oligodendrocytes during demyelination in optic nerves of mice infected with semliki forest virus antibody response to rubella virus structural proteins in multiple sclerosis multiplication of rubella and measles viruses in primary rat neural cell cultures: relevance to a postulated triggering mechanism for multiple sclerosis oligodendroglial degeneration in distemper: apoptosis or necrosis? aspects of canine distemper and measles virus encephalomyelitis measles encephalomyelitisðclinical and immunologic studies multiple sclerosis and gamma interferon measles encephalomyelitis: lack of evidence of viral invasion of the central nervous system and quantitative study of the nature of demyelination interferon beta-1b is effective in relapsing remitting multiple sclerosis interferon beta in the treatment of multiple sclerosis. mechanism of action chemokines and chemokine receptors in cns pathology encephalitis after hepatitis b vaccinationðrecurrent disseminated encephalitis or ms? no increase in demyelinating diseases after hepatitis b vaccination complications of mumps vaccines immunisation against infectious disease mumps component in combined measles, mumps and rubella vaccines peripheral neuropathy associated with anti-myelin basic protein antibodies in a woman vaccinated with rubella virus vaccine defending vaccines from the enemy within dna immunization exacerbation of viral and autoimmune animal models for multiple sclerosis by bacterial dna dna vaccines with a kick vaccination with recombinant suicidal dna/rna recombinant semliki forest virus particles expressing louping ill virus antigens induce a better protective response than plasmid-based dna vaccines or an inactivated whole particle vaccine the semliki forest virus vector induces p53-independent apoptosis this review was greatly in¯uenced by a workshop on the infectious aetiology of multiple sclerosis held in brighton, uk in august 1999 by the us and uk multiple sclerosis societies; we are very grateful to the organisers. key: cord-293871-hzes7mwt authors: mcguinness, sarah l.; wu, henry m. title: pretravel considerations for non-vaccine-preventable travel infections date: 2018-11-26 journal: travel medicine doi: 10.1016/b978-0-323-54696-6.00007-0 sha: doc_id: 293871 cord_uid: hzes7mwt pretravel advice should be tailored to the individual following a thorough review of his or her itinerary, planned activities, and host characteristics. in addition to vaccinations and malaria chemoprophylaxis, a pretravel consultation should include advice on regionally endemic or emerging non–vaccine-preventable infections that can cause severe illness or chronic morbidity. these include mosquito-borne infections such as dengue, chikungunya, and zika, and regionally endemic severe respiratory infections such as middle east respiratory syndrome (mers) and some strains of avian influenza. zika virus is notable given its capacity for sexual transmission and association with congenital birth defects. preventive advice for other potentially relevant infections associated with specific exposures or activities (e.g., schistosomiasis and leptospirosis from freshwater exposure) should be provided where relevant. understanding the epidemiology and prevention of these infections is crucial to providing a comprehensive pretravel consultation. to provide optimal advice, travel health providers should be able to educate the traveler on preventive measures against key travel-related infections, including those for which no vaccine is available. tailoring this advice for an individual requires a thorough review of the travelers' itinerary and planned activities, consideration of the travelers' host characteristics, and a working knowledge of the epidemiology of relevant diseases. travelers play an important role in the global epidemiology of infectious diseases; therefore ensuring that travelers are aware of specific preventive measures not only protects the health of the individual but has the potential to protect the health of their communities. in this chapter, pretravel considerations for major non-vaccine-preventable infectious diseases are covered, including specific advice for dengue, chikungunya, zika, middle east respiratory syndrome coronavirus (mers-cov), and avian influenza. dengue virus (denv), chikungunya virus (chikv), and zika virus (zikv) are globally important mosquito-borne viruses spread via aedes aegypti and a. albopictus. the public health impact of these viruses has increased dramatically over the last 50 years, with epidemics of increasing size, geographic reach, and severity recorded. with factors such as population growth, urbanization, globalization, travel, and climate change facilitating increased transmission, travel medicine practitioners in temperate countries are increasingly likely to see returned travelers with these infections. furthermore, since the ranges of aedes vectors extend into temperate areas, infected returned travelers can precipitate outbreaks of these viruses in nonendemic regions. aedes mosquitoes are typically daytime biters and have a preference for the morning and late afternoon hours (crepuscular periods). 1 a. aegypti, the primary mosquito vector for dengue, chikungunya, and zika, is found in tropical, subtropical, and some temperate climates and has adapted to cohabit with humans in both urban and rural environments. 2 a. aegypti typically lays eggs in manmade or artificial containers in or around the home and can bite indoors. 2 a. albopictus (the asian tiger mosquito) can live in a broader temperature range and at cooler temperatures than a. aegypti and thus has a wider geographic distribution, extending into temperate regions. a. albopictus feeds on animals as well as humans, prefers natural habitats, usually bites outdoors, and is generally considered a less efficient vector of human disease than a. aegypti. 1 aedes mosquitoes can be found in temperate areas, including southern europe (a. albopictus), northern queensland in australia (a. aegypti), and southeastern regions of the united states (both species) 2,3 ( fig. 7.1 ). denv is a flavivirus that is the most common and arguably most important arbovirus globally. originating in africa, denv is now endemic in more than 100 countries across africa, southeast asia, the americas, the western pacific, and the eastern mediterranean regions. 4, 5 estimates suggest 390 million infections occur worldwide annually, with 70% of cases occurring in asia. 6 denv has four distinct serotypes (denv 1-4), with most endemic countries reporting circulation of all four serotypes. 7 primary infection provides lifelong serotype-specific protection but only short-lived cross-protection to other serotypes. 4, 8 broadly neutralizing antibodies are produced following a second dengue infection, and symptomatic disease is rarely seen with subsequent infections. 9 pretravel advice should be tailored to the individual following a thorough review of his or her itinerary, planned activities, and host characteristics. in addition to vaccinations and malaria chemoprophylaxis, a pretravel consultation should include advice on regionally endemic or emerging non-vaccine-preventable infections that can cause severe illness or chronic morbidity. these include mosquito-borne infections such as dengue, chikungunya, and zika, and regionally endemic severe respiratory infections such as middle east respiratory syndrome (mers) and some strains of avian influenza. zika virus is notable given its capacity for sexual transmission and association with congenital birth defects. preventive advice for other potentially relevant infections associated with specific exposures or activities (e.g., schistosomiasis and leptospirosis from freshwater exposure) should be provided where relevant. understanding the epidemiology and prevention of these infections is crucial to providing a comprehensive pretravel consultation. between nonhuman primates, small mammals, and aedes mosquitoes. 14 however, in outbreaks chikv can spread without the need for animal reservoirs. 14 among populations with no prior immunity, chikv outbreaks can be explosive, and attack rates as high as 70% have been documented. 15 introduction of chikv into asia occurred during or before the 1950s and led to outbreaks in india and southeast asia. 16 reemergence of chikv from africa in 2004 resulted in major outbreaks involving millions of people across the islands of the indian ocean in 2005 (including the comoros islands, la reunion, and mauritius) and india in 2005-2006. 16 furthermore, introduction of chikv to temperate areas in this period resulted in autochthonous transmission in italy and france. 17, 18 the first report of local transmission of chikv in the americas occurred in 2013 in saint martin, with subsequent spread to >40 countries and territories across north, central, and south america. 15 the unprecedented magnitude of chikv outbreaks in recent years is probably attributable to several factors including increased urbanization, global travel, and a series of adaptive mutations in the virus which have resulted in enhanced transmission by a. albopictus. 16 the incubation period of chikungunya is typically 2-4 days (range 1-14 days). 14 most chikungunya infections are symptomatic, with more than 85% of people with serologic evidence of infection reporting a history of symptoms. 16 chikungunya infection is characterized by sudden onset of fever and severe, potentially disabling arthralgia. notably, the name chikungunya is derived from a makonde word describing the bent posture that can be seen with severe arthralgia. 16 the arthralgia/ arthritis is usually symmetric and affects multiple joints, with fingers, wrists, ankles, elbows, toes, and knees the most often affected. 14 additional symptoms include headache, myalgia, conjunctivitis, and rash. the case fatality rate of chikungunya is <1%, but disease can be associated with significant acute and long-term morbidity secondary to debilitating polyarthralgia/arthritis. 15 although self-limiting in most individuals, some of those affected develop chronic joint pain that may last for months to years, with older people (>35-45 years) more predisposed. 14, 18 unlike dengue, in which nonsteroidal antiinflammatory drugs (nsaids) are contraindicated, antiinflammatory drugs are indicated for symptomatic management of chikungunya infections. zikv is a flavivirus that was first isolated in 1947 from a rhesus monkey in the zika forest of uganda, with the first human cases detected in uganda and tanzania in 1952. 19,19a following its discovery, the virus remained in relative obscurity for over 50 years, with only 14 cases reported until 2007, when an explosive outbreak infected approximately three-quarters of the population of yap, federated states of micronesia. 20 subsequent outbreaks occurred across the pacific islands from 2013 to 2016; spread of the virus to brazil in march 2015 preceded subsequent transmission throughout latin america, the caribbean, mexico, and florida and texas in the united states. 21, 22 as of february 2018, 86 countries, territories, or subnational areas have reported evidence of vectorborne zikv transmission. 23 unlike denv and chikv, there is now substantial evidence that direct person-to-person transmission of zikv is possible-both horizontally through sexual transmission, and vertically from the mother to the fetus during pregnancy. 22 transmission through blood transfusion has been reported, 22 as well as a single case report of transmission probably resulting from close contact with bodily fluids from an infected patient. 24 the incubation period is thought to be similar to other mosquito-borne flaviviruses 21 with an estimated range from 3-14 days. 25 male-to-female, male-to-male, and femaleto-male transmission to unprotected sexual contacts of returning following a short incubation period, with symptoms typically beginning 4-7 days (range 3-14 days) after exposure, dengue can present with a wide spectrum of illnesses, from asymptomatic infection to severe and fatal disease. 5 most infections are asymptomatic or subclinical; symptomatic infections occur in approximately one-third of cases. 4 patients who recover after a self-limited febrile illness, typically characterized by fever, headache, retroorbital pain, arthralgia, and myalgia, are classified as having dengue. 5 the small proportion who progress to capillary (plasma) leakage with or without bleeding, circulatory collapse, or severe end organ impairment are designated as having severe dengue. 5 epidemiologic risk factors for severe dengue include young age, secondary infection with a different serotype, and infection with a more virulent strain of virus. 4, 10 severe dengue occurs in approximately 1%-3% of dengue cases, with case fatality rates ranging from <1%-5%; the greatest burden of severe dengue occurs in children and infants in endemic countries. 11 dengue is a common diagnosis in travelers, accounting for >3% of presentations to geosentinel surveillance clinics. most infections are acquired in asia, followed by the americas, with only a small proportion acquired in africa. 12 the incidence of dengue infection in travelers ranges from 10.2-30 infections per 1000 person months, and varies according to travel destination, duration, and season of travel. 11 regionspecific peaks of travel-related dengue infections have been demonstrated for southeast asia (june and september), south central asia (october), and south america (march). 13 viraemic travelers can introduce dengue into new areas, with autochthonous transmission documented in the continental united states, europe, and australia. 11, 12 some travelers with dengue may require hospitalization or even evacuation. 10 studies of dengue in travelers have reported a dengue hemorrhagic fever prevalence of 0.9%-3%, though this is likely an overestimate as patients experiencing more severe symptoms are more likely to seek medical attention. 11 epidemiologic studies in endemic settings have shown that the risk of severe disease is significantly higher during a second denv infection than during a primary infection. 5 however, a lack of consensus exists regarding risk factors for severe disease in travelers. 11 results of one study in travelers suggest that severe dengue may occur at similar rates among cases with primary and secondary infections. 9 given that most dengue infections are asymptomatic, and that severe dengue in travelers is rare, travelers with a history of dengue infection need not avoid known dengue areas but rather should be advised to use the personal protection strategies outlined in box 7.1 to prevent subsequent infection. chikv is a mosquito-borne alphavirus first isolated in tanzania in 1952. 14 in africa, chikv exists in an enzootic sylvatic transmission cycle • wear an insect repellent containing an active ingredient such as deet or picaridin, particularly during daylight hours when the mosquitoes are most active. • wear long-sleeved shirts and long pants to help protect yourself from bites. light-colored clothes are best. • treat clothes and shoes with an insecticide such as permethrin or purchase pretreated clothing. • use mosquito coils, plug-in mosquito repellent devices, or insecticide surface sprays inside your accommodation; or stay in screened or air-conditioned accommodation. 29 testing may also be considered in asymptomatic potentially exposed pregnant women after considering risk of infection, patient preferences, and clinical judgment. 29 nonpregnant individuals and couples traveling to zika-affected areas should also be counseled on measures to prevent sexual transmission and congenital infection. due to the risk of prolonged viral shedding in semen, public health authorities advise men with risk of zika exposure to wait 3 months from the last possible exposure to zika (or after onset of symptoms following symptomatic infection) before attempting procreation. 22 most authorities advise women to wait 8 weeks from the last possible exposure to zika (or after onset of symptoms following symptomatic infection) before attempting to conceive 22 ; one exception is the who, which advises women to wait 6 months before attempting conception. 30 because many pregnancies are unplanned, all sexually active travelers and female partners of travelers should also practice measures to prevent congenital zika infection. readers are encouraged to review the most updated recommendations for prevention of sexual transmission of zikv and congenital zika infection from public health authorities including who and the us centers for disease control and prevention (cdc) ( the 2003 severe acute respiratory syndrome (sars) outbreak highlighted the potential for travelers to introduce novel respiratory infections into their home countries. more recently, concern has focused most on the emergence of middle eastern respiratory syndrome (mers) and certain strains of avian influenza. travelers has been reported with the former felt to be most prominent. sexual transmission from patients with both symptomatic and asymptomatic disease has been described. 22 currently it appears that zika can remain in semen longer than in other body fluids (including cervical mucus, vaginal fluids, urine, and blood). 26 in semen, zikv rna has been detected as long as 188 days after the onset of symptoms, and infectious virus has been cultured up to 69 days after symptom onset. 22 most zikv infections are asymptomatic, with serosurvey studies indicating that only 19% of those infected report clinical illness. 20 in symptomatic cases, illness is generally mild and self-limiting with symptoms including fever, rash, pruritus, arthralgia, myalgia, conjunctivitis, and headache. 21 zikv has been associated with neurologic complications including guillain-barré syndrome (gbs) and adverse fetal outcomes including congenital microcephaly. an association with gbs was first reported in 2013-2014 during the french polynesian outbreak. more than 20 countries have now reported an increased incidence of gbs and/or laboratory confirmation of a zikv infection among gbs cases. 23 in february 2016, following reports from brazil of microcephaly in babies whose mothers had been exposed to zika during pregnancy, the world health organization (who) declared that zika constituted a public health emergency of international concern (pheic). 19 microcephaly is one of several neurologic and musculoskeletal birth defects described in congenital infection; this constellation of findings is now known as congenital zika syndrome. 27 although a live attenuated tetravalent dengue vaccine (cyd-tdv; dengvaxia) has been registered in several countries, and several other dengue vaccine candidates are in clinical development, 8 at this time no dengue vaccine is licensed for travelers. likewise no vaccines are licensed for chikungunya or zika; therefore prevention of these viruses largely relies on personal protection strategies that limit contact between humans and aedes mosquitoes (see box 7.1 and chapter 6; insect protection) as well as avoidance of travel during peak transmission or outbreak periods. travelers returning to nonendemic areas with aedes mosquitos (see fig. 7 .1) should also be advised to avoid mosquito bites on their return to prevent local transmission. symptomatic travellers should seek medical evaluation immediately. infection with chikv is thought to result in lifelong protective immunity. 14 duration of zikv immunity following infection is currently unknown. in contrast, due to the multiple serotypes, individuals can be infected with dengue up to four times, and travelers with a history of infection should be educated about the potential risks of subsequent infections. due to the high prevalence of asymptomatic infections and the risk of sexual and vertical transmission, zika-specific preventative advice is important for those traveling to zika affected areas. pregnant women who do not reside in zika transmission risk areas should be advised not to travel to areas with risk; if travel cannot be avoided, advice to prevent mosquito bites and sexual transmission should be given. 28 measures to prevent sexual transmission include abstaining from sexual activity or use of condoms during sexual activity (including vaginal, anal, and oral sex, and sharing of sex toys) during the entire pregnancy. 28 pregnant women possibly exposed to zikv due to travel or sexual contact should discuss the potential exposure with their although no vaccines are available at this time for prevention of mers and avian influenza (h5n1 and h7n9) in travelers, providers should routinely review the most recent epidemiology of severe respiratory infections reported by authorities such as the who and cdc (see table 7 .1) and promote general hygiene and other preventative measures to travelers to these areas (box 7.2). while seasonal influenza vaccination does not protect against avian influenza or mers, vaccine-related prevention of seasonal influenza may reduce the chance of coinfections and overall risk of respiratory infections. travelers should also be advised to inform their health care providers of their travel history whenever seeking medical care for respiratory (and other illnesses) acquired during or soon after travel (see chapter 59). travel medicine providers should also be familiar with risk areas and specific preventive advice for other non-vaccine-preventable infectious diseases with regional distributions. some of the more important of these infections are presented in table 7 .2, along with specific preventive advice, such as insect bite avoidance (e.g., for prevention of african trypanosomiasis) or freshwater contact avoidance (e.g., for prevention of schistosomiasis or leptospirosis). mers is a respiratory infection caused by mers coronavirus (mers-cov). first described in 2012, 31 mers is an endemic infection in the arabian peninsula with epidemic potential in health care and travel settings. following an incubation period of 2-14 days, initial symptoms of mers are similar to many common viral respiratory infections and include fever, rhinorrhea, sore throat, and muscle aches. rapid progression to acute respiratory distress syndrome may follow, but mild and asymptomatic infections have also been described. 32, 33 nausea, vomiting, or diarrhea, and acute kidney injury can also occur. 32 risk factors for severe mers include age >50 years and comorbid conditions such as hypertension, diabetes, heart disease, end stage renal disease, chronic lung disease, cancer, or those receiving immunosuppressive therapy. 32 among confirmed cases reported to who up to july 2017, 35% have been fatal. 33a seroepidemiologic studies indicate that mers-cov circulates in dromedary camels in the middle east and africa, and direct contact with camels has been described in 33% of primary cases without known exposure to mers cases or health care settings. 34 the route of transmission from dromedaries to humans is unclear, but contact with infectious bodily secretions and fluids are suspect, and consumption of raw dromedary products has also raised some concern. 32 person-to-person transmission is primarily described in health care settings, although transmission among household close contacts has been described. 35 all cases of mers reported outside of the arabian peninsula have occurred in returned travelers, or as a result of secondary transmission from a patient with recent travel to the arabian peninsula, as was the case for the 2015 health care-associated outbreak in south korea that resulted in 186 cases and 36 deaths. 32, 36, 37 in this outbreak, delayed mers diagnoses and inadequate infection control precautions led to multiple generations of infections affecting other patients, visitors, and health care workers. 37, 38 the risk of traveler-initiated health care-associated outbreaks with mers mirrors the global experience of sars. like mers, the viral agent of sars is a coronavirus that emerged in southern china in 2002 and subsequently caused over 8000 infections and 774 deaths in more than 20 countries. 38, 39 the reservoir of sars coronavirus (sars-cov) is unknown, but some cases appear to have resulted from contact with animals used for human consumption such as civet cats. 40 although no cases of sars have been reported since 2004, the potential for reemergence is possible. 39 while seasonal influenza is a common infection among travelers, other influenza types including certain strains of avian influenza can also pose a risk to travelers. although avian influenza typically affects birds, human cases and outbreaks occur sporadically. avian influenza strains associated with severe respiratory infections with high mortality rates in humans include the highly pathogenic avian influenza a h5n1, 41 and more recently, novel avian influenza a h7n9. 42 although the majority of human infections caused by avian influenza are linked to direct contact with infected birds (primarily poultry), unsustained person-to-person transmission has been reported for h5n1 and h7n9. 41, 43 furthermore cocirculation of different influenza a viruses in humans and animals raises the concern of reassortment leading to new strains that spread more readily from person to person. 42 h5n1, first described in southern china in 1996, has since been documented in over 60 countries in asia and africa. 41 long-term sequelae of chikungunya virus disease: a systematic review zika: the origin and spread of a mosquito-borne virus zika virus: history of a newly emerging arbovirus zika virus outbreak on yap island, federated states of micronesia zika virus epidemiology, prevention, and potential future treatments of sexually transmitted zika virus infection zika virus classification tables fatal zika virus infection with secondary nonsexual transmission estimated incubation period for zika virus disease persistence of zika virus in body fluids-preliminary report congenital zika virus infection beyond neonatal microcephaly update: interim guidance for health care providers caring for pregnant women with possible zika virus exposure-united states (including us territories) world health organization. prevention of sexual transmission of zika virus-interim guidance update isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome asymptomatic mers-cov infection in humans possibly linked to infected dromedaries references the efficacy of repellents against aedes, anopheles, culex and ixodes spp.-a literature review the dengue vector aedes aegypti: what comes next the global distribution of the arbovirus vectors aedes aegypti and ae. albopictus the global distribution and burden of dengue global spread of dengue virus types: mapping the 70 year history dengue vaccine: who position paper a travel medicine view of dengue and dengue hemorrhagic fever geographic expansion of dengue: the impact of international travel dengue fever and international travel geosentinel surveillance network. geosentinel surveillance of illness in returned travelers geosentinel surveillance network. seasonality, annual trends, and characteristics of dengue among ill returned travelers chikungunya: a re-emerging virus epidemiology of chikungunya in the americas chikungunya virus infections infection with chikungunya virus in italy: an outbreak in a temperate region arrival of chikungunya virus in the new world: prospects for spread and impact on public health increase in human infections with avian influenza a(h7n9) virus during the fifth epidemic-china avian influenza a (h5n1) infection with respiratory failure and meningoencephalitis in a canadian traveller avian influenza a(h7n9) virus infection in 2 travelers returning from china to canada human african trypanosomiasis leishmaniasis in travelers: a literature review histoplasmosis infections worldwide: thinking outside of the ohio river valley leptospirosis: an emerging disease in travelers rickettsial infections in the tropics and in the traveler myiasis in travelers schistosomiasis in travelers and migrants multicenter geosentinel analysis of rickettsial diseases in international travelers mers-cov global summary and assessment of risk risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia transmission of mers-coronavirus in household contacts the clinical and virological features of the first imported case causing mers-cov outbreak in south korea preliminary epidemiological assessment of mers-cov outbreak in south korea ii, 21163 transmission characteristics of mers and sars in the healthcare setting: a comparative study who guidelines for the global surveillance of severe acute respiratory syndrome (sars) lessons from the severe acute respiratory syndrome outbreak in hong kong global epidemiology of avian influenza a h5n1 virus infection in humans, 1997-2015: a systematic review of individual case data epidemiology of avian influenza a h7n9 virus in human beings across five epidemics in mainland china key: cord-297834-me1ajoyb authors: schountz, tony; prescott, joseph title: hantavirus immunology of rodent reservoirs: current status and future directions date: 2014-03-14 journal: viruses doi: 10.3390/v6031317 sha: doc_id: 297834 cord_uid: me1ajoyb hantaviruses are hosted by rodents, insectivores and bats. several rodent-borne hantaviruses cause two diseases that share many features in humans, hemorrhagic fever with renal syndrome in eurasia or hantavirus cardiopulmonary syndrome in the americas. it is thought that the immune response plays a significant contributory role in these diseases. however, in reservoir hosts that have been closely examined, little or no pathology occurs and infection is persistent despite evidence of adaptive immune responses. because most hantavirus reservoirs are not model organisms, it is difficult to conduct meaningful experiments that might shed light on how the viruses evade sterilizing immune responses and why immunopathology does not occur. despite these limitations, recent advances in instrumentation and bioinformatics will have a dramatic impact on understanding reservoir host responses to hantaviruses by employing a systems biology approach to identify important pathways that mediate virus/reservoir relationships. hantaviruses (family bunyaviridae, genus hantavirus) are negative-stranded, trisegmented viruses that cause about 200,000 disease cases annually, with case fatality rates of 0.5%-40%, depending on the virus [1] . the viral gene segments encode four or five polypeptides. the large (l) segment encodes the rna-dependent rna polymerase (rdrp), the medium (m) segment encodes a precursor that is posttranslationally cleaved into gn and gc glycoproteins, and the small (s) segment encodes the nucleocapsid (n) protein. some hantaviruses encode a second nonstructural polypeptide (nss) downstream from the n start site in frame 2 [2, 3] . little is known about the immunomodulatory abilities of these proteins; however, there is evidence the n, gn and nss may alter host cellular responses during infection. more than 20 hantavirus species have been classified, and many more unclassified hantaviruses have been identified. they are hosted by several species of rodents (rodentia), insectivores (insectivora), and bats (chiroptera) [4] . little work has been conducted to understand hantavirus infections of insectivores or bats; however, much is known about the ecology of rodent-borne hantaviruses because of their impact on human health (table 1) . a central problem of hantavirus/reservoir host research is the lack of reagents and methods for experimentally examining the immune response. recent experimental work on the immunology of rodent reservoirs, summarized in an exceptional review by easterbrook and klein [5] , has begun to clarify these issues. the immune response is energetically expensive for wild animals, thus the findings of experimental studies will be critical for understanding the ecoimmunology of reservoir hosts of hantaviruses [6, 7] , and experiments using wild rodents in natural or semi-natural environments [8, 9] will be required to validate laboratory findings. rodentia is the largest mammalian order and is comprised of about 1,800 species [10] . only a few dozen species have been identified as susceptible hosts, and most hantaviruses are hosted by a single rodent species [11] . in each hantavirus/rodent reservoir relationship, infection results in two prominent features: no conspicuous pathology and persistent infection [12] [13] [14] . the earliest report of experimental infection of a reservoir host with its hantavirus was by lee et al. [15] that described infection of striped field mice (apodemus agrarius) with hantaan virus. inoculated mice developed chronic infection with transient viremia, and shed virus principally in urine, saliva and, to a lesser extent, feces, despite the production of neutralizing antibodies. currently, three laboratory infection systems have been developed to study hantavirus infections of reservoir hosts: seoul virus (seov) infection of the norway rat (rattus norvegicus), puumala virus (puuv) infection of the bank vole (myodes glareolus), and sin nombre virus (snv) infection of the deer mouse (peromyscus maniculatus) [12, 14, 16] . in humans, these viruses are etiologic agents of hemorrhagic fever with renal syndrome (hfrs; seov, puuv) and hantavirus cardiopulmonary syndrome (hcps; snv) [1]. these diseases share many pathologic similarities and because little damage to the endothelium occurs during infection, it is thought that the inflammatory immune response contributes to pathogenesis [17, 18] . because the norway rat is a model organism with many specific reagents, including monoclonal antibodies to immune markers, significant progress has been made toward understanding the reservoir host immune response to seov [19] [20] [21] . fewer methods and reagents are available for bank voles and deer mice. despite these limitations, emerging technologies will be useful for understanding why rodent reservoirs are infected without disease and why they are unable to clear infection. a significant limitation of hfrs research is that none of the hfrs-causing hantaviruses cause disease in animal models. however, two new world hantaviruses cause an hcps-like disease in syrian golden hamsters (mesocricetus auratus): andes virus (andv) and maporal virus (mapv) [22, 23] . andv causes most hcps cases in south america; however, no human cases of disease have been associated with mapv. andv is hosted by the long-tailed pygmy rice rat (oligoryzomys longicaudatus) and mapv is hosted by the delicate pygmy rice rat (oligoryzomys delicatus) [24, 25] . andv is considered an animal biosafety level-4 pathogen in most nations, whereas mapv is considered an absl-3 virus [26] . the natural route of transmission among reservoir rodents is thought to be principally through aerosols and/or biting [27] . however, experiments have been equivocal in clarifying transmission mechanisms. weanling bank voles caged with infected individuals lead to transmission as early as 14 days post exposure [14] . similar experiments examining snv transmission between deer mice have been less informative; however, in artificial enclosure experiments, transmission appeared to be facilitated by deer mice with higher amounts of viremia and wounding [8] and males likely play a dominant role in transmission in natural populations [9, 28, 29] . in bank voles, offspring of puuv-infected dams were less likely to be infected after exiting the nest because of protective maternal antibody [30] . although it is thought horizontal transmission is most important, work by hutchinson et al. [31] showed that vertical transmission occurred among cotton rats (sigmodon hispidus) infected with black creek canal virus, so it is possible that both routes may influence transmission at the population level. the route of transmission has important ramifications in terms of the host immune response where, presumably, a mucosal response occurs with aerosol transmission and a localized response at a bite site. experimental data have also shown that patterns of the expression of genes related to the immune response are different in infected males and females [32] , and it is likely these differences have important roles in hantavirus ecology. spillover to other rodent species also occurs [33] [34] [35] [36] , but it is unknown if the rodents remain infected. recent work has shown that deer mice are experimentally susceptible to andv; however, virus is cleared several weeks after infection [37] . a pronounced th2/tfh gene expression profile occurs, including il-4 pathway activation, that does not appear to be substantially activated in snv-infected deer mice [13, 38] . this system provides an opportunity to identify viral and reservoir host factors that are important for sterilizing immunity that clears infection. the principal target cells of infection in rodents (and humans) are the microvasculature endothelial cells of many tissues [39] . experimental intramuscular infection of deer mice with snv resulted in detectable virus in the lungs as few as two days later [13] . many organs appeared infected, although infection was limited to the vasculature within those tissues. two infectious outcomes occur in experimentally infected deer mice; a disseminated infection of three or more organs, or a restricted infection of the lungs and heart [40] . the relevance of these two patterns to transmission efficiency is unknown. the levels of viral rna vary dramatically in infected deer mice, with most having modest to moderate levels of rna at the peak of infection. however, some deer mice have significantly greater amounts of viral rna, suggesting these deer mice may produce substantially more virus than others, and it is possible they transmit virus more efficiently (e.g., "supershedders") [13] . this also occurs in semi-natural transmission experiments [9] and suggests certain individuals may have a prominent role in population-level transmission of hantaviruses. most serological assays for detecting antibody responses in hantavirus reservoirs use virus neutralization, elisa or strip immunoblotting [12, 29, [41] [42] [43] . while some of these assays are igg-specific, others use antiserum to whole igg, including the light chains. since light chains are shared by all immunoglobulins, these detection antibodies are not igg-specific. moreover, no assays are in place for detecting iga, which should be prominent in mucosal infections. igm assays have been problematic despite the availability of anti-igm capture antisera that are cross-reactive with igm from at least one hantavirus reservoir species [44] . some immunoglobulins have isotypes with specific effector activities, such as complement fixation or antibody-dependent cell cytotoxicity. laboratory house mice have four igg isotypes; igg1, igg2a, igg2b and igg3. it is likely that reservoirs also have immunoglobulin isotypes with distinct effector functions and which might predominate during hantavirus infections. these reagent deficiencies are a current obstacle for assessing antibody responses in rodent reservoir hosts. despite these limitations, many field studies have been conducted examining antibody responses in natural and semi-natural hantavirus infections of rodent reservoirs [34, [45] [46] [47] [48] [49] [50] [51] . in experimentally-infected deer mice, snv nucleocapsid-specific antibodies can be detected in serum as early as 10 days post infection, and neutralizing antibody can be detected after three weeks post infection [13] . similarly, experimentally-infected bank voles produce puuv-specific antibodies two to three weeks after inoculation [14] and rats experimentally infected with seov also produce igg within two weeks post inoculation [52] . the presence of igg in these naturally and experimentally infected reservoirs is an indicator of class switching and affinity maturation, events that are mediated by t cells [53] . thus, rodents mount adaptive t cell/b cell immune responses to their reservoir hantaviruses; however, it appears to be inadequate for virus clearance. while inflammatory signatures are present [13, 20, 54] , the magnitude of these signals appears to be modest relative to expression levels found in a syrian hamster pathology model of hcps [55] . it is noteworthy that immunization of rodent reservoirs with homologous nucleocapsid antigen or plasmids encoding the antigen protects from subsequent challenge [56, 57] , suggesting infection can be prevented in reservoir hosts. several hantavirus proteins have been implicated in modulation of the host cell's antiviral defenses ( table 2 ). the gn glycoproteins of pathogenic new world hantaviruses and seov possess an immunoreceptor tyrosine activation motif (itam) in the cytoplasmic tail that binds to fyn tyrosine kinase, and the itam may also interact with lyn, syk, and zap-70 kinases found in lymphocytes [58, 59] , although there is no evidence that lymphocytes are susceptible to hantaviruses. the itam may also promote polyubiquitination of the gn polypeptide to facilitate its degradation [60] ; however, it is unclear how it impacts the host response to infection. presumably, the itam interferes with the antiviral response of an infected cell since the motif is cytoplasmic. the gn protein may also alter the rig-i pathway that leads to irf3 phosphorylation and subsequent ifnβ expression [61] . the nucleocapsid may also antagonize the expression of ifnβ by binding to importin-α and interfering with nf-κb nuclear transport, which is required for ifnβ expression [62] [63] [64] . additionally, both caspase 3 and granzyme b are targets of the nucleocapsid of some hantaviruses [65] and both are essential components of ctl-mediated apoptosis. the lack of damage to the endothelium of infected rodent reservoirs suggests this may be an important mechanism of preventing viral clearance. the nucleocapsid of andv, but not other hantaviruses, also inhibits autophosphorylation and activation of tbk1, an enzyme that activates irf3 and nf-κb and induction of type i interferon gene expression [66] . recently, putative nonstructural nss sequences have been identified in some hantaviruses [3, 67] . this sequence is in an alternative reading frame of the nucleocapsid transcript. in other bunyaviruses, nss has anti-interferon activity [68] [69] [70] ; however, its role in hantavirus infections is less well characterized. importantly, these studies have been conducted with cells from nonreservoir hosts where they, presumably, are not optimized for manipulating the immune response in a manner that benefits the virus but without host pathology. future studies should examine the roles of these proteins in cells from reservoir hosts. the presence of high-titer igg antibodies during hantavirus infections of reservoir hosts indicates both t cell and b cell responses occur because t cells induce class switching and affinity maturation of antibodies produced by antigen-specific b cells. in experimental infections of rats with seov and deer mice with snv, early infection results in subtle inflammatory signatures, but a regulatory t cell (treg) response predominates at persistence (figure 1 ) [20, 54] . treg responses are critical for suppressing inflammation [71, 72] ; however, inflammation is a prominent feature of hantavirus disease in humans [73] and hamsters [55, 74] . in other viral diseases, the occurrence of a treg response is associated with persistent infection because these cells, while suppressing inflammation, also prevent virus clearance [75, 76] . for reservoirs of hantaviruses, the treg response may limit inflammatory immunopathology to an otherwise innocuous infection, but it may also impair virus clearance. how this relationship evolved is unknown, but considering the presence of hantavirus proteins with immunomodulatory activities, it suggests the viruses may manipulate the host response to favor persistence; a treg response may prevent sterilizing immunity, thus allow virus to remain in a population. it may also explain the ecoimmunology and ecology of hantavirus infections of reservoir hosts, thus studies assessing the energetics of inflammatory and anti-inflammatory immune responses should be performed. much of the work examining reservoir responses to hantaviruses has been conducted in rats infected with seov and deer mice infected with snv or andv. using cdna arrays, klein et al. [77] identified nearly 2000 genes that were differentially expressed in male and female rats infected with seov. many immune-associated transcription factors, proinflammatory, antiviral, t cell and ig family member genes were significantly higher in females, which may reduce transmission from females. in deer mice infected with snv, early expression signatures of a mixed th1/th2/treg response were present in virus-specific cd4 + t cells, including ifnγ, il4, il5 and tgfβ, before transitioning to a treg-like response at persistence [54] . the expression of immune response genes differed in the spleen, where signatures of inflammation occurred within two days, peaking by days 10 to 15 before subsiding, and lungs, where little immune gene expression occurred [13] . some deer mice produced nucleocapsid-specific igg by day 10 while others had igg around day 20 or later, and neutralizing antibody was not detected until after 3 weeks. . during acute infection, snv elicits a modest inflammatory response that initially limits, but does not clear, virus. within a few weeks, the response transitions to a regulatory response that may allow episodic recrudescence of virus that can be shed. in deer mice infected with snv, early expression signatures of a mixed th1/th2/treg response were present in virus-specific cd4 + t cells, including ifnγ, il4, il5 and tgfβ, before transitioning to a treg-like response at persistence [54] . the expression of immune response genes differed in the spleen, where signatures of inflammation occurred within two days, peaking by days 10 to 15 before subsiding, and lungs, where little immune gene expression occurred [13] . some deer mice produced nucleocapsid-specific igg by day 10 while others had igg around day 20 or later, and neutralizing antibody was not detected until after 3 weeks. assessment of cytotoxic t cell responses of reservoir hosts has not been reported. most assays that assess ctl functions require susceptible syngeneic target cells, which have been difficult to obtain with reservoir hosts. susceptible primary cell lines from reservoir hosts have been produced [37, 78] , but these are typically obtained from embryonic fibroblasts, thus matching of mhc alleles for use in ctl assays is difficult. many zoonotic viruses antagonize the innate immune response in human cells, and their pathogenic potential often correlates with their abilities to inhibit the innate response in vitro ( [79] [80] [81] [82] for review). pathogenic hantaviruses inhibit antiviral responses despite high levels of replication, whereas nonpathogenic viruses are efficiently recognized and elicit innate responses that limit replication [83, 84] . this antagonistic capacity must have evolved in the reservoir hosts of these hantaviruses because humans are dead-end hosts. to date, few studies have addressed the interactions between hantaviruses and their rodent hosts in vitro. this is partially due to the unavailability of cell lines, and the reagents and techniques to generate primary cell cultures from the various reservoirs of hantaviruses. the norway rat/seov system is the most tractable for studying virus/reservoir interactions. inoculation of rat lung-derived endothelial cells with seov resulted in virus replication, but little or no induction of cytokines or chemokines, suggesting seov can efficiently antagonize antiviral responses [21] . despite this, endothelial cells increased their expression of the protein pd-l1, which correlated with the ability of these cells to induce treg cell activity. in addition, antigen presenting cells isolated from norway rats and infected with seov in vitro were resistant to stimulation, suggesting that virus infection inhibits the normal signaling activities of these cells [85] . thus antagonism of the innate immune response likely allows for viral replication, while at the same time promotes an anti-inflammatory response that limits immunopathology. similar studies have been performed using bank vole cells infected with puuv [78] . embryonic fibroblasts inoculated with puuv did not express increased amounts of ifnβ or mx2, although non-related viruses were able to induce up-regulation of these genes. this suggests, as with seov, puuv efficiently antagonizes host innate responses in its natural reservoir. the syrian golden hamster develops an hcps-like disease when infected with andv or mapv [22, 23] . hamsters inoculated with andv mount an inflammatory response, as measured by elevated mrna encoding pro-inflammatory mediators, prior to succumbing to the disease [55] . infection also results in the activation of the adaptive immune response, characterized by antigen-specific proliferation of t cells and the generation of virus-specific antibodies [74, 86] . in contrast, snv, which is highly pathogenic in humans, replicates in hamsters, but does not cause disease and is cleared by the immune response [87] . passaging of snv in hamsters results in a virus that is able to replicate efficiently and cause a persistent infection similar to what is seen in the rodent reservoir, yet still does not cause disease [88] . examination of immune responses elicited by andv (pathogenic in hamsters) and snv (non-pathogenic in hamsters) showed that passaged snv evoked a stronger adaptive immune response than did andv; however, andv infection induced a much stronger innate immune response at late time points, despite both viruses replicating to similar levels. depletion of t cells did not alter the outcome of infection [74, 86] ; thus, these data suggest that, at least in the hamster model, the activation of the t cell-mediated immune response is not responsible for immunopathogenesis, and perhaps the innate immune response, either elicited from infected endothelial cells, macrophages and/or neutrophils, might contribute to disease. infectious diseases and the immune response are complex processes that are challenging to study, even with substantial reagent resources and mature methodologies. many difficulties exist for studying hantavirus infections of their reservoir hosts, including the lack of molecular and immunological reagents, methods for experimental investigation, and that few reservoir species have been colonized for laboratory use. in addition, pathogenic hantaviruses require bsl-3 and/or absl-4 containment, which presents logistical hurdles for examining the reservoir host/hantavirus relationship. despite these limitations, novel instrumentation, particularly those for transcriptome profiling (e.g., rna-seq) and metabolomics, and development of molecular and cellular methods, provide an opportunity to rapidly develop the tools necessary for examining reservoir host responses using a systems biology approach. the key feature of these new technologies is that they are species-independent in the data they generate, but they require significant computational and bioinformatics resources. infection triggers a cascading host response that is highly orchestrated by the vertebrate immune system. many genes are modulated (expressed or repressed) during the course of infection and identification of mrna and noncoding rnas can be used to identify the mechanisms that control, or fail to control, disease. moreover, some infectious diseases, including hantavirus disease, have substantial immunopathologic components. the quantitative assessment of the transcriptional landscape (patterns of gene expression) can be used to profile the host responses in infected and uninfected animals of the same species, or a disease model species to reservoir host species to identify mechanisms of susceptibility or resistance. rna-seq is one such method for profiling transcriptional landscapes [89] . the depth of coverage and costs of rna-seq have improved dramatically in the last few years, and it is likely to become less expensive. however, the computational resources necessary for using rna-seq for studying host responses is substantial, often requiring hundreds of gigabytes of ram and multicore, multiprocessor systems typically found in servers running a linux operating system [90] . this depth is often necessary to detect rnas that occur in extremely low abundance because their proteins are highly potent (e.g., cytokines). even then, it is possible that differentially expressed genes may not be detected and other, more sensitive methods, such as real-time pcr, may be required to validate pathway signatures. despite these requirements, bioinformatics tools for rna-seq are now widely available, many of which are free. a typical first step of differential gene expression profiling is the de novo assembly of all rna-seq samples from an experiment, which represents the totality of expressed genes from the experiment. there are several de novo assemblers available, including the trinity suite [90] and oases [91] . each of these packages has advantages and disadvantages, thus it is important to understand how each performs assemblies, particularly isoforms that may have specific activities. included in the trinity package is rsem [92] that estimates transcript abundances, including isoforms, in experimental samples by counting reads from replicates against the de novo assembly. an important feature of rsem is that it does not require an annotated genome; it determines the abundance of transcripts from the unannotated assembly, identifies differentially expressed transcripts, and provides a 95% credibility interval for each gene. additional tools, such as deseq and edger [93, 94] provide statistical evaluation of differential gene expression between samples and provide quantitative (higher/lower) and qualitative (on/off) data. the differentially expressed transcripts are subsequently identified by other means (e.g., blast) and can then be mapped to pathways (such as reactome or kegg) [95, 96] to visualize [97] where viruses may influence the host response and identify mechanistic targets of hantaviruses. in recent years, micrornas (mirna) have been identified that are important regulators of antiviral responses. activation of tlr/rig-i pathways leads to the expression of several mirnas [98] that likely are important in lymphocyte functions [99] . because hantavirus gn targets rig-i [61] , it is possible that these mirnas are dysregulated, which could provide the virus an advantage over the host cell response. while mirna expression has been evaluated in hantavirus-infected human endothelial cells, epithelial cells and macrophages [100, 101] , no work has been conducted to examine the role of mirnas in reservoir host cells infected with hantaviruses. considering the importance of mirna in host responses, it is likely they play an instrumental role in the immunological events leading to persistent infection of the reservoir hosts. the use of rna-seq can identify global mirna expression [102] and clarify their regulatory roles in infected reservoirs. metabolic products can provide substantial information about the interactions of viruses and infected host cells, and the how immune system responds to infection [103, 104] . the field of metabolomics is young but potentially informative for understanding hantavirus/reservoir host interactions. many viruses metabolically remodel the host cell to optimize infection. because metabolic products (e.g., carbohydrates, lipids, prostaglandins, etc.) are identical or highly similar between vertebrate species, this approach may help identify which enzymes, by virtue of their metabolic products, may be targeted by hantaviruses. while there are no reports of metabolic assessment of hantavirus infections, rift valley fever virus (rvfv) modulates the activity of adenosine 5' monophosphate-activated protein kinase (ampk) in infected cells. this enzyme is a regulator of several metabolic pathways, including enhancement of catabolic pathways such as autophagy and atp production, but it represses anabolic pathways, such as lipid biosynthesis. infection of cells by several viruses, including rvfv, results in activation of ampk and restriction of viral replication, suggesting an antiviral role for this enzyme [105] . other studies have revealed metabolic pathway targeting by viruses [106] [107] [108] , thus efforts to examine how hantaviruses may manipulate the metabolomes of infected cells could lead to the identification of therapeutic targets for treating hantavirus disease. the use of monoclonal antibodies has been particularly challenging for hantavirus/reservoir research. identification of cell surface markers could shed light on what cells respond during hantavirus infection of reservoir hosts. while cell surface antigens tend to be more divergent, intracellular proteins, such as antiviral proteins, tend to be more conserved, particularly phosphoepitopes. thus, it is likely that many available antibodies to house mouse (mus musculus) or norway rat antiviral proteins will be cross-reactive with orthologs from hantavirus rodent reservoir species. without information as to which proteins may be of interest, screening of antibodies is a daunting and expensive task since most likely will not be informative. however, combined with rna-seq and metabolomic data, it is likely that target proteins and pathways can be identified so that investigators can focus their efforts and resources. in addition, software tools can help predict whether antibodies may be cross-reactive to proteins from reservoir hosts [109] . the use of cytokines or neutralizing cytokine antibodies to perturb the host response during infection has identified many mechanisms that contribute to susceptibility or resistance. for example, administration of ifnγ to laboratory mice facilitates clearance of lcmv, whereas antibody that neutralizes ifnγ impairs ctl responses and clearance, thus leading to persistent infection [110, 111] . depletion of immune cell subsets with antibodies can also determine the roles of those cells during infection. this approach revealed a critical role for cd4 + t cells for sustaining ctl responses to lcmv [112] . other than the norway rat, an array of cytokines and antibodies for experimental manipulation of the host response of hantavirus reservoirs is substantially limited. thus, it is difficult to determine the mechanisms controlling the host response of reservoirs. some cytokines are broadly cross-reactive and can be used for studying reservoir responses. recombinant house mouse gm-csf and human il-2 stimulate deer mouse cells [113] , and it is likely that other commercially-available cytokines can also be used. identification of cross-reactive cytokines should be a high priority of the hantavirus community. with genome and transcriptome sequencing, many cytokine genes can be rapidly cloned into expression vectors that are codon-optimized for the expression system of choice using de novo synthesis services (e.g., geneart, life technologies). moreover, some antibodies may be cross-reactive with reservoir species' orthologs [114] . anti-mouse cd4 (clone gk1.5) and anti-rat cd8β (clone 341) antibodies can be used to deplete cd4 + and cd8 + t cells from hamsters, respectively [74, 86] , and they may also be useful for reservoir host studies to examine the roles of these cells. finally, it is difficult to assess ctl activity in reservoir hosts. most colonies are established with wild rodents that are highly polymorphic. this limits use of traditional ctl assays that require mhc class i-matched target cells. the generation of highly inbred strains is challenging and may result in alleles that do not represent the natural biology of hantavirus infection, thus it may not be desirable to generate highly inbred rodents. it is possible to establish mhc homozygotes with controlled breeding and screening of littermates for the same haplotypes. even then, the generation of susceptible cell lines can be problematic. while endothelial cell lines can be generated with retroviral transformation, it is possible the cells may have activated antiviral pathways that could alter in vitro ctl responses. the use of growth factors for expanding endothelial cells in culture may be more attractive. until methods are established for generating syngeneic, susceptible target cells, assessment of ctl responses in reservoir hosts will be difficult. the immunological relationships between hantaviruses and their rodent reservoir hosts are complex. infection typically leads to disseminated infection within a few days but without conspicuous signs of disease. expression of immune genes can be detected in as little as a few days that suggests innate and adaptive immune activation, but the magnitude of expression is substantially less than in hantavirus pathology models. the lack of reagents and methodologies for studying hantavirus reservoirs, most of which are not model organisms, presents a significant challenge. however, new technologies have recently emerged that are cost effective and species-independent, but which generate large amounts of data that require substantial computational and bioinformatics support for data reduction. with these tools, it should be possible to accelerate research to understand the relationships of hantaviruses and their reservoir hosts. work was provided by the department of microbiology, immunology and pathology, colorado state university (ts), nih grant ai054461 (ts), and intramural program of niaid, nih (jp). t.s. and j.p. reviewed the literature and wrote the manuscript. the authors declare no conflict of interest. hantaviruses: a global disease problem tula and puumala hantavirus nss orfs are functional and the products inhibit activation of the interferon-beta promoter the andes hantavirus nss protein is expressed from the viral small mrna by a leaky scanning mechanism phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents immunological mechanisms mediating hantavirus persistence in rodent reservoirs long-term patterns of immune investment by wild deer mice infected with sin nombre virus refining approaches and diversifying directions in ecoimmunology transmission ecology of sin nombre hantavirus in naturally infected north american deermouse populations in outdoor enclosures increased detection of sin nombre hantavirus rna in antibody-positive deer mice from montana, usa: evidence of male bias in rna viremia the delayed rise of present-day mammals a global perspective on hantavirus ecology, epidemiology, and disease experimental infection model for sin nombre hantavirus in the deer mouse (peromyscus maniculatus) kinetics of immune responses in deer mice experimentally infected with sin nombre virus experimental infection with puumala virus, the etiologic agent of nephropathia epidemica, in bank voles (clethrionomys glareolus) intraspecific transmission of hantaan virus, etiologic agent of korean hemorrhagic fever, in the rodent apodemus agrarius modes of seoul virus infections: persistency in newborn rats and transiency in adult rats high levels of cytokine-producing cells in the lung tissues of patients with fatal hantavirus pulmonary syndrome the pathogenesis of nephropathia epidemica: new knowledge and unanswered questions seoul virus enhances regulatory and reduces proinflammatory responses in male norway rats regulatory t cells enhance persistence of the zoonotic pathogen seoul virus in its reservoir host seoul virus-infected rat lung endothelial cells and alveolar macrophages differ in their ability to support virus replication and induce regulatory t cell phenotypes a lethal disease model for hantavirus pulmonary syndrome maporal viral infection in the syrian golden hamster: a model of hantavirus pulmonary syndrome the delicate pygmy rice rat (oligoryzomys delicatus) is the principal host of maporal virus (family bunyaviridae, genus hantavirus). vector borne zoonotic dis an outbreak of hantavirus pulmonary syndrome laboratory management of agents associated with hantavirus pulmonary syndrome: interim biosafety guidelines hantaviral infections of rodents: possible scenarios dynamics of puumala hantavirus infection in naturally infected bank voles (clethrinomys glareolus) epizootiology of sin nombre and el moro canyon hantaviruses, southeastern colorado maternal antibodies contribute to sex-based difference in hantavirus transmission dynamics transmission of black creek canal virus between cotton rats sex differences in the recognition of and innate antiviral responses to seoul virus in norway rats natural history of sin nombre virus in western colorado juquitiba-like hantavirus from 2 nonrelated rodent species rodent host specificity of european hantaviruses: evidence of puumala virus interspecific spillover dobrava-belgrade virus spillover infections experimental andes virus infection in deer mice: characteristics of infection and clearance in a heterologous rodent host expression profiling of lymph node cells from deer mice infected with andes virus hantavirus-induced immunity in rodent reservoirs and humans persistent sin nombre virus infection in the deer mouse (peromyscus maniculatus) model: sites of replication and strand-specific expression high prevalence of hantavirus antibodies in bank voles (clethrionomys glareolus) captured in the vicinity of households afflicted with nephropathia epidemica a survey of hantavirus antibody in small-mammal populations in selected united states national parks rapid field immunoassay for detecting antibody to sin nombre virus in deer mice development of an elisa to detect sin nombre virus-specific igm from deer mice (peromyscus maniculatus) serologic and genetic identification of peromyscus maniculatus as the primary rodent reservoir for a new hantavirus in the southwestern united states yellow pigmy rice rat (oligoryzomys flavescens) and hantavirus pulmonary syndrome in uruguay black creek canal virus infection in sigmodon hispidus in southern florida detection of antibody for the serodiagnosis of hantavirus infection in different rodent species genetic and serologic analysis of black creek canal virus and its association with human disease and sigmodon hispidus infection cocirculation of multiple hantaviruses in texas, with characterization of the small (s) genome of a previously undescribed virus of cotton rats (sigmodon hispidus) population, environmental, and community effects on local bank vole (myodes glareolus) puumala virus infection in an area with low human incidence. vector borne zoonotic dis social status does not predict responses to seoul virus infection or reproductive success among male norway rats t cells and follicular dendritic cells in germinal center b-cell formation and selection regulatory t cell-like responses in deer mice persistently infected with sin nombre virus pathogenesis and host response in syrian hamsters following intranasal infection with andes virus genetic vaccines protect against sin nombre hantavirus challenge in the deer mouse (peromyscus maniculatus) yeast-expressed puumala hantavirus nucleocapsid protein induces protection in a bank vole model hantavirus pulmonary syndrome-associated hantaviruses contain conserved and functional itam signaling elements identification of a functional itam-like sequence within g1 cytoplasmic tail of hantaan virus tyrosine residues direct the ubiquitination and degradation of the ny-1 hantavirus g1 cytoplasmic tail hantavirus gnt elements mediate traf3 binding and inhibit rig-i/tbk1 directed ifnbeta transcription by blocking irf3 phosphorylation modulation of apoptosis and immune signaling pathways by the hantaan virus nucleocapsid protein hantaan virus nucleocapsid protein binds to importin alpha proteins and inhibits tumor necrosis factor alpha-induced activation of nuclear factor kappa b inhibition of tnf-alpha-induced activation of nf-kappab by hantavirus nucleocapsid proteins hantavirus-infection confers resistance to cytotoxic lymphocyte-mediated apoptosis an innate immunity-regulating virulence determinant is uniquely encoded by the andes virus nucleocapsid protein gene tula hantavirus nss protein accumulates in the perinuclear area in infected and transfected cells la crosse bunyavirus nonstructural protein nss serves to suppress the type i interferon system of mammalian hosts nss protein of rift valley fever virus induces the specific degradation of the double-stranded rna-dependent protein kinase bunyamwera bunyavirus nonstructural protein nss counteracts the induction of alpha/beta interferon natural regulatory t cells in infectious disease regulatory t cells in virus infections hantavirus pulmonary syndrome. pathogenesis of an emerging infectious disease t cells are not required for pathogenesis in the syrian hamster model of hantavirus pulmonary syndrome the role of virus-induced regulatory t cells in immunopathology regulatory cells and infectious agents: detentes cordiale and contraire differential expression of immunoregulatory genes in male and female norway rats following infection with seoul virus a model system for in vitro studies of bank vole borne viruses sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon nipah and hendra virus interactions with the innate immune system antiviral escape strategies developed by bunyaviruses pathogenic for humans the pathogenesis of influenza virus infections: the contributions of virus and host factors pathogenic and nonpathogenic hantaviruses differentially regulate endothelial cell responses differential antiviral response of endothelial cells after infection with pathogenic and nonpathogenic hantaviruses seoul virus suppresses nf-kappab-mediated inflammatory responses of antigen presenting cells from norway rats the adaptive immune response does not influence hantavirus disease or persistence in the syrian hamster temporal analysis of andes virus and sin nombre virus infection of syrian hamsters hamster-adapted sin nombre virus causes disseminated infection and efficiently replicates in pulmonary endothelial cells without signs of disease field guide to next-generation dna sequencers de novo transcript sequence reconstruction from rna-seq using the trinity platform for reference generation and analysis oases: robust de novo rna-seq assembly across the dynamic range of expression levels accurate transcript quantification from rna-seq data with or without a reference genome differential expression analysis for sequence count data a bioconductor package for differential expression analysis of digital gene expression data the reactome pathway knowledgebase kegg: kyoto encyclopedia of genes and genomes cytoscape: a software environment for integrated models of biomolecular interaction networks micrornas in the regulation of tlr and rig-i pathways potential roles for short rnas in lymphocytes andes virus regulation of cellular micrornas contributes to hantavirus-induced endothelial cell permeability hantaviruses induce cell type-and viral species-specific host microrna expression signatures computational analysis of noncoding rnas lipids at the interface of virus-host interactions multifaceted roles for lipids in viral infection amp-activated kinase restricts rift valley fever virus infection by inhibiting fatty acid synthesis dengue virus infection perturbs lipid homeostasis in infected mosquito cells metabolic effects of influenza virus infection in cultured animal cells: intra-and extracellular metabolite profiling viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection modulation by gamma interferon of antiviral cell-mediated immune responses in vivo mechanism of recovery from acute virus infection. viii. treatment of lymphocytic choriomeningitis virus-infected mice with anti-interferon-gamma monoclonal antibody blocks generation of virus-specific cytotoxic t lymphocytes and virus elimination mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that lyt-2 + t lymphocytes mediate clearance of virus and regulate the antiviral antibody response generation of competent bone marrow-derived antigen presenting cells from the deer mouse (peromyscus maniculatus) discrimination of peromyscus maniculatus leukocytes by flow cytometry the authors thank brian hjelle, charles h. calisher, rushika perera and heinz feldmann for helpful comments, support and advice leading to the preparation of this manuscript. support for this key: cord-302833-6kntd89t authors: radonovich, lewis j.; bessesen, mary t.; cummings, derek a.; eagan, aaron; gaydos, charlotte; gibert, cynthia; gorse, geoffrey j.; nyquist, ann-christine; reich, nicholas g.; rodrigues-barradas, maria; savor-price, connie; shaffer, ronald e.; simberkoff, michael s.; perl, trish m. title: the respiratory protection effectiveness clinical trial (respect): a cluster-randomized comparison of respirator and medical mask effectiveness against respiratory infections in healthcare personnel date: 2016-06-02 journal: bmc infect dis doi: 10.1186/s12879-016-1494-2 sha: doc_id: 302833 cord_uid: 6kntd89t background: although n95 filtering facepiece respirators and medical masks are commonly used for protection against respiratory infections in healthcare settings, more clinical evidence is needed to understand the optimal settings and exposure circumstances for healthcare personnel to use these devices. a lack of clinically germane research has led to equivocal, and occasionally conflicting, healthcare respiratory protection recommendations from public health organizations, professional societies, and experts. methods: the respiratory protection effectiveness clinical trial (respect) is a prospective comparison of respiratory protective equipment to be conducted at multiple u.s. study sites. healthcare personnel who work in outpatient settings will be cluster-randomized to wear n95 respirators or medical masks for protection against infections during respiratory virus season. outcome measures will include laboratory-confirmed viral respiratory infections, acute respiratory illness, and influenza-like illness. participant exposures to patients, coworkers, and others with symptoms and signs of respiratory infection, both within and beyond the workplace, will be recorded in daily diaries. adherence to study protocols will be monitored by the study team. discussion: respect is designed to better understand the extent to which n95s and mms reduce clinical illness among healthcare personnel. a fully successful study would produce clinically relevant results that help clinician-leaders make reasoned decisions about protection of healthcare personnel against occupationally acquired respiratory infections and prevention of spread within healthcare systems. trial registration: the trial is registered at clinicaltrials.gov, number nct01249625 (11/29/2010). healthcare personnel (hcp) are exposed to respiratory pathogens in many clinical settings [1] . infected hcp may spread infection to their patients [2] [3] [4] [5] or coworkers [3] [4] [5] [6] , to family members [4, 7] , or to other community members [4, 8] . respiratory viral infections among healthcare workers can negatively impact delivery of healthcare services [9] [10] [11] . united states national guidelines call for modes of transmission to dictate infection control measures [3] . for most human respiratory viruses, the precise mode(s) of person-to-person transmission is incompletely understood [12, 13] . the predominant mode of transmission for some human respiratory pathogens, such as influenza virus, respiratory syncytial virus, and coronavirus is believed to be droplet transmission. airborne transmission plays a role with some human respiratory pathogens via small aerosol particles, often called droplet nuclei [3] . airborne transmission is the predominant mode of transmission for mycobacterium tuberculosis [3, 14] and recent evidence has suggested a larger role than previously thought for influenza a and b viruses [15, 16] . disposable respiratory protective devices (rpd) that fit tightly to the wearer's face, sometimes called airpurifying respirators or filtering facepiece respirators, are primarily designed to protect the wearer against infection spread by ill patients. n95 filtering facepiece respirators (commonly known as "n95 respirators") are one type of rpd capable, with proper facial fit and usage, of reducing inhalation of airborne particulates by a factor of 10 or greater [17] . medical masks (mm), typically called surgical masks in operative settings, are primarily devised to protect patients against infection spread by the wearer [18] . both types of devices also serve as a physical barrier keeping sprays and splashes of infectious materials and contaminated hands and objects away from oronasal region of wearer. although rpd and mm are capable of filtering particulates [19] , rpd are designed to filter smaller particulates that may remain airborne for long periods. a tight seal between the respirator and the wearer's face is designed to prevent leakage of particulates, a feature not provided by loose-fitting mm. the u.s. department of labor's occupational safety and health administration (osha) requires employers to ensure each hcp, who may be exposed to airbornetransmissible infections in the workplace, receives an rpd with an adequate respirator-to-face seal that is determined during a mandated annual "fit-test". however, evidence is inconclusive that rpd are better than mm at protecting hcps from respiratory infections in clinical settings [20] [21] [22] [23] [24] [25] , despite tight-fitting rpd produced by manufacturers, with higher levels of exposure reduction validated by numerous laboratory studies [19, [26] [27] [28] , and the use of a complete respiratory protection program (e.g., training, initial and annual fit test) as defined by osha to protect hcp. intuitively, rpd should better protect hcp against airborne infections than mm, but objective evidence has not validated this supposition. one possibility that may explain this discrepancy between expectations and observations is pragmatic: hcp, in general, do not tolerate n95 respirators as well as medical masks [29, 30] , perhaps prompting them to remove respirators more frequently and/or for longer periods, increasing the likelihood of exposure to infections. models have shown that 25 % or more non-wear time during exposure negates any significant differences in protective ability between types of rpds [17, 31] . given the difficulty with hcp adherence to guidelines [4] and general dissatisfaction [4, [32] [33] [34] with rpd, medical masks worn more consistently may provide similar levels of reduction in respiratory viral disease transmission as n95 respirators. this key gap in knowledge has contributed to discrepant clinical and public health recommendations about respiratory protection for hcp [35, 36] . needed are additional well-designed clinical trials conducted in patientcare settings during outbreaks of respiratory infections. the following is an abridged version of the full research protocol for the respiratory protection effectiveness clinical trial (respect). to compare the effectiveness of n95 respirators and medical masks at protecting hcp from acquiring viral respiratory illnesses in the workplace. null hypothesis: the incidence of laboratory confirmed influenza (primary), influenza-like illness (ili), acute respiratory illness (ari) and other respiratory infections will not be different between hcps who practice 2007 guidelines (medical masks) or 2009 guidelines (n95 respirators). alternative hypothesis: the incidence of laboratory confirmed influenza (primary), influenza-like illness (ili), acute respiratory illness (ari) and other respiratory infections will be different between hcps who practice the cdc's 2007 guidelines for influenza protection (medical masks) versus 2009 guidelines for influenza protection (n95 respirators). respect is a prospective comparison of respiratory protective equipment to be conducted at multiple, geographically distributed u.s. study sites. hcp who work in outpatient settings will be cluster-randomized to wear n95 respirators [37] or mm [38] for protection against infections during respiratory virus season, the "intervention" period. the null hypothesis assumes n95 and mm intervention groups will have no differences in outcomes, including (1) laboratory confirmed influenza or (2) influenza-like illness (ili), (3) acute respiratory illness (ari), and (4) laboratory confirmed respiratory illness (lcri). the alternative hypothesis asserts the incidence of at least one outcome would be different between intervention groups. because respiratory virus season varies year-to-year in onset, severity, and duration, multiple season-years of the study will be necessary to account for expected variance and optimally generalize the resulting knowledge. the beginning of each season's data collection will be independently determined for each study site using an epidemiologic predictive tool designed for respect to capture the largest possible number of respiratory infections. these data will be collected for twelve weeks each season. participant exposures to patients, coworkers, and others with symptoms and signs of respiratory infection, both within and beyond the workplace, will be recorded in daily diaries. adherence to study protocols will be measured by the study team at each site. since periodic changes in infection control guidance and practice may occur over the study years, participants will be expected to adhere to the most up-to-date guidance issued by the centers for disease control and prevention (cdc) and local policies at each study institution, at a minimum. for example, a participant randomized to the mm arm will be expected to don an n95 when participating in an aerosol-generating procedure, assuming no further changes in pertinent national guidance [39] . the study participants will be recruited from outpatient settings where patients are relatively likely to present with symptoms and signs of acute respiratory infection. participants will be eligible to enroll for multiple study seasons, yet each will be provided with informed consent and complete enrollment procedures prior to each study season. clinical study sites will be distributed geographically: participants will be cluster-randomized to one of the following n95 respirators or mm models, selected because they are commonly used in u.s. medical facilities, including the respect study sites. participants who participate in more than one of the study years will be cluster-randomized anew each year. n95 respirators: (1) precept 15320 or (2) kimberly clark technol fluidshield 47107. all subjects participating in the study will be required to pass an osha-accepted respirator fit test for the n95 respirator model(s) available at the study site. no fit testing of medical masks will be performed as these devices are not designed to be tight-fitting to the face and studies [19, 20] have shown that their fit capabilities are generally low. filter performance although medical masks are loose-fitting, they create a physical barrier that helps prevent splashes and sprays from reaching the wearer's mucous membranes. in addition to passage around the mask, some of the small particle aerosols are able to pass through the mask's filter media. therefore, in addition to rpds, filtration testing was done on medical masks prior to enrollment of subjects to ensure consistency between models across study locations. the filtration performance of the n95 respirators and medical mask models in the study were tested in a manner similar to that used by the national institute for occupational safety and health (niosh). devices were attached to a test fixture and placed in a tsi 8130 automated filter tester operated with an air flow rate of 85 liters per minute. the tsi 8130 uses a photometer to measure the flux of light scattering from aerosol particles. polydispersed particles (mass median diameter of~0.3 microns) were generated from a 2 % nacl solution and passed through each device being tested for 1 min. each test was repeated 3 times with a fresh n95 respirator or medical mask. to be certified as an n95 respirator, filter penetration needs to be less than 5 % (or 95 % efficient). as shown in table 1 , the average penetration percentages for the niosh certified n95 respirators were an order of magnitude lower than those of medical masks, which are not niosh certified. filter results between n95 respirator models and between medical mask models were comparable. filter airflow resistance was measured simultaneously using the tsi 8130. as filter airflow resistance increases, more energy expenditure is required for ventilation during device wear and the greater potential for perception of discomfort [40] . the medical mask models selected for this study have filter airflow resistance levels about half of that of the n95 respirators. however, one study [40] found that subjective and physiological responses were not different among subjects exercising while wearing devices purposely made with different filter airflow resistance levels (3 mm h 2 o, 6 mm h 2 o, and 9 mm h 2 o) in the range similar to those of the devices in this study (table 1) . participants will be instructed to don a new n95/mm with each patient interaction, every time a participant encounter occurs within 6 feet of a patient who has suspected or confirmed respiratory infection. hand hygiene will be recommended to all participants in accordance with cdc guidelines [41] and policies at each study institution. trained research assistants will observe participants during study periods to assess adherence to their assigned intervention arms and hand hygiene. a portable computer equipped with data recording software (handyaudit; toronto, canada) will be used to document adherence. participants will be expected to complete surveys about their attitudes and opinions concerning personal protective equipment before and after each seasonal study period. during the twelve week data collection period each year, participants will self-document (a) perceived occupational exposures to patients or coworkers who have symptoms or signs of respiratory infection, (b) perceived anterior nasal and pharyngeal swabs [42] [43] [44] [45] [floqswabs utm (99-08024), diagnostic hybrids; athens, oh] will be collected by research assistants when symptomatic with study defined respiratory symptoms, as well as two, randomized asymptomatic swabs during each seasonal study period. swabs will be collected when (a) participants self-report respiratory symptoms within a 24 h period, and again if participants remain symptomatic after 7 days; and (b) randomly, on all participants, twice during the active intervention period. the primary outcome measures will be the incidence of: laboratory-confirmed influenza (lci) a or b infection in participants, defined as a) detection of influenza virus by reverse-transcription polymerase chain reaction (rt-pcr) in an upper respiratory specimen swab collected within seven days of symptom onset, or b) influenza seroconversion defined as at least a 4-fold rise in hemagglutination inhibition antibody (hai) titers to influenza a or b virus from the pre-to post-season serological samples that is not deemed attributable to vaccine. the secondary outcome measures will be the incidence of: (1) acute respiratory illness (ari) defined as the occurrence of one sign or two symptoms ( the incidence rate ratios between participants randomly assigned to wear n95 respirators or medical masks will be estimated for each of the primary and secondary outcomes. investigators will be paired and provided with blinded information about clinical and laboratory data to determine if a participant meets a primary or secondary outcome. if the paired investigators do not agree, a principal investigator will adjudicate the outcome. assays will be performed at johns hopkins university. collected respiratory specimens will be stored at −80°c until analyzed using multiplex pcr (plex-id, abbott labs, chicago il). automated extraction of nucleic acid (na) from respiratory specimens will be performed utilizing nordiag's arrow instrument and the magna pure robotic system (roche indianapolis, in) per manufacturer instructions. each extraction run will include a quality control (natrol respiratory validation panel 3, zeptometrix inc., buffalo ny); runs with control failures will be repeated. purified na will be amplified via rt-pcr using a broad respiratory virus identification kit (plex-id rvs 3.0, abbott molecular, des plaines, il). desalting of rt-pcr product and electrospray mass spectrometry-based na analysis will be performed on the plex-id analyzer instrument. if funding is sufficient, samples will also be assayed by rt-pcr for bordetella pertussis, mycoplasma pneumoniae, and for chlamydophila pneumoniae. each study season, blood samples will be collected twice from each participant; one sample will be collected within two weeks of the beginning of the intervention period and a second within two weeks of the end of the intervention period. hemagglutination inhibition (hai) antibody assays will be performed on serum for influenza a and b virus strains, dependent on the antigens in each annual trivalent vaccine using standard methods [46, 47] . in brief, serial 2-fold dilutions of serum samples will be incubated with 8 hemagglutinin units of influenza antigen and a turkey red blood cell suspension. the serum hai antibody titer will be defined as the dilution factor of the highest serum dilution that completely inhibits agglutination of turkey red blood cells in the presence of type-specific hemagglutinin antigen. assays will be performed at the immunology core lab for the study at the va saint louis veterans affairs healthcare system. to optimize compliance and generalizability, a clusterrandomized design will be utilized. all participants working in the same clinical unit will be assigned to wear the same respiratory protective equipment (i.e., an n95 or mm) during patient interactions for the entire 12 week seasonal study period. clusters will be pairmatched within each study site based on the characteristics of each clinical cluster, including the (a) number of participants (b) occupational location, such as an emergency department, urgent care or primary care, (c) patient population served, such as children or adults, and (d) requirements for participants to wear additional protective equipment, such as goggles donned by dental hygienists. for each study season, the clinics in each matched pair will be randomly assigned to opposing study arms. for matched pairs participating in multiple study seasons, random sequences of arm assignments will ensure each is assigned to both study arms during the multi-year study. each study season, an individual not involved in the study implementation and data analyses will perform the randomization scheme for each study site, using a random number generator in microsoft excel. the principal investigators will be blinded to the randomization scheme prior to assignment. incidence rates of lci, ari, ili, and lcri among cluster-randomized participants will be compared. the relationships between incidence of clinically diagnosed and laboratory-confirmed illnesses will be analyzed with attention to potential confounders, such as participants' demographics, study arm compliance, attitudes and opinions about infection control, receipt of influenza vaccination, and infectious exposures within and beyond the workplace. standard statistics will describe baseline characteristics and follow-up measures, summarized by treatment arm and stratified by study site. to assess the primary outcome, a logistic regression model will be fit using a dichotomous variable to indicate whether a participant became infected with a respiratory pathogen. the odds of infection between the two treatment groups will be reported with a 95 % confidence interval. for secondary outcomes, poisson loglinear mixed effects regression models will assess the difference in seasonal respiratory infection rates between intervention groups. cluster-and individual-level random effects will be considered to account for clustered observations. additional covariates may be added to the models to adjust for confounding. participants will be encouraged to complete the study. those who withdraw from an intervention arm will be encouraged to complete follow-up laboratory specimen collection. an intent-to-treat analysis, in which all available data on all randomized participants are included, will be used for the primary comparison of interventions. a per-protocol secondary analysis will compare treatment effectiveness, accompanied by a planned sensitivity analysis that accounts for participants from whom researchers were not able to obtain a second serological sample. to detect a 25 % reduction (i.e., a relative risk of 0.75) in the incidence of laboratory confirmed influenza or laboratory confirmed respiratory illness among participants wearing an n95 respirator, compared to participants wearing a medical mask, respect will need to accumulate approximately 10,024 or 5104 personseasons of data over four seasons respectively. sample size calculations are based on several assumptions about the incidence rate and levels of withincluster correlation. the attack rate laboratory-confirmed influenza during a single study season is assumed to be 20 % among unvaccinated individuals in the medical mask group. we assume 65 % of our population will be administered a vaccine that is 65 % effective in preventing influenza infection. vaccine effectiveness at the higher end of published reports (86 % in health care workers) will lead to a reduction in the yearly attack rate to approximately 8.8 %, and effectiveness at the lower end of published reports (51 % in the general population) would lead to an increased yearly attack rate of approximately 13.4 %. importantly, the anticipated effect on the needed sample size of annual variations in influenza incidence is larger than the expected impact of variation in vaccine effectiveness. the respect study will need 157 independent clusters with a median size of 16 participants each to achieve 80 % power to detect a relative risk of 0.75 between n95 and surgical masks at preventing laboratory-confirmed influenza infection, with a type-i error rate of 0.05. the total number of individuals participating each season will need to be approximately 2506, with 10,024 total person-seasons accumulating over the multi-year study. for the secondary outcome of laboratory confirmed respiratory illness, the estimated total number of clinics will need to be 80, the total number of individuals participating each season will need to be 1276, and total person-seasons accumulated need to be 5104 (table 4) over the multi-year study. the sample size are made using the clusterpower software package for r [48] . power is estimated using an expected annual attack rate of 12 % {12 % = 0.35*0.2 + 0.65*(1-0.65)*0.2} [13] . this yearly attack rate translates into a 4-year attack rate of 39 % {39 % = 1-(1-(0.35*0.2 + 0.65*0.35*0.2)) 4 . accounting for correlation of outcomes within clusters by assuming the correlation coefficient is 0.1, leads to a design effect of 2.5. for scenarios representing the lower and higher ends of anticipated attack rates in the medical mask group, two quantities were calculated (a) the power to detect a relative-risk of 0.75 between the n95 group and the medical mask group and (b) the relative-risk that can be detected with 80 % power (table 5 ). for all of these calculations the two-sided type i error probability is 0.05. potential outcome analysis for laboratory-confirmed influenza some data on the primary outcome may be missing due to participants withdrawing from the study early and missing the second serological sample. to account for the unavoidable uncertainty posed by missing primary outcome data, due to participant withdrawal or loss of follow-up, a sensitivity analysis will be conducted that randomly assigns binary outcomes to participants who did not complete the study. a two-dimensional grid will be created that varies the influenza attack rates among participants who withdraw. withdrawal attack rates in both arms will be fixed between half and twice the observed attack rates, based on complete data. by varying these two parameters across the grid, and for each combination, the adjusted odds ratio will be calculated by averaging across n = 50 imputed datasets for each point on the grid. analysis of differential withdrawal the characteristics at the time of randomization for participants without complete follow-up will be examined. to assess the potential biases introduced by differential withdrawal among different n95 respirators, a comparison of withdrawal rates and time to withdrawal will be included as an ancillary analysis to the analyses of the primary and secondary outcomes. respect will be approved by the institutional review board at each participating study site and the centers for disease control and prevention, prior to study initiation. (an unabridged version of the respect protocol was approved by the intitutional review board at each study site and the centers for disease control and prevention). viral respiratory infections cause a wide range of illnesses, varying from mild to severe, in hcps who may spread infection to their patients, family members, and other community members. healthcare-associated infections cost $10b annually in u.s [49] . factors influencing transmission of respiratory infections in healthcare facilities include the population density of ill patients in healthcare settings, the types of exposures within healthcare settings, the administrative and physical structures of healthcare facilities, and intrinsic characteristics of virulence [3] . measures to prevent transmission within healthcare facilities include hcp vaccination, handhygiene, cleaning and disinfection of inanimate surfaces, pre-and post-exposure antiviral chemoprophylaxis, patient isolation, and personal protective equipment [3, 6] . respect is designed to better understand the extent to which ppe, specifically represented by differences in exposure reduction afforded by n95s and mms, reduces clinical illness among hcps. while it may seem that n95 respirators should better protect hcps than mm against airborne infections in the workplace, this notion has not been validated by objective clinical evidence. low tolerance to respirator wear among hcps may prompt more frequent or longer periods of removal, compared to mm, to an extent that the benefits of higher levels of filtration and lower levels of leakage around the facial seal afforded by respirators is offset or subjugated. key sources of variability in hcp health outcomes are difficult to control for, even in a rigorously designed clinical study such as respect. for example, the inability to prevent hcp community exposures to respiratory infections and the inherent year-to-year variation of viral respiratory infections provide a challenging setting in which to evaluate the effectiveness of personal protective equipment. while community-acquired infections may pose a significant source of exposure for hcps, this type of exposure, if occurring non-differentially between study arms, would bias the results from respect towards the null hypothesis. key reasons for choosing a cluster-randomized design are (a) to increase compliance by equipping all members of a healthcare team with the same equipment and (b) to capture indirect effects of the intervention at the cluster-level, such as herd immunity [50] . a fully successful study would produce clinically relevant results that help clinician-leaders make reasoned decisions about protection of hcps against occupationally acquired respiratory infections and prevention of spread within healthcare systems. abbreviations ari, acute respiratory illness; cdc, centers for disease control and prevention; dsmb, data safety monitoring board; hai, hemagglutination inhibition antibody; hcp, healthcare personnel; ili, influenza like illness; lcri, laboratory confirmed respiratory illness; mm, medical mask; n95, n95 respirator; niosh, national institute for occupational safety and health; osha, occupational safety and health administration; ppe, occupational protective equipment; respect, respiratory protection effectiveness clinical trial; rpd, respiratory protective devices; rt-pcr, reverse-transcriptase polymerase chain reaction; us, united states. occupationally acquired infections in health care workers. part i healthcare providers as sources of vaccine-preventable diseases guideline for isolation precautions: preventing transmission of infectious agents in health care settings preparing for and influenza pandemic: personal protective equipment for healthcare workers health care-associated infection outbreak investigations by the centers for disease control and prevention transmission of influenza: implications for control in health care settings risk of transmission of severe acute respiratory syndrome to household contacts by infected health care workers and patients surveillance network of catalonia spain tm. implication of health care personnel in measles transmission disruption of services in an internal medicine unit due to a nosocomial influenza outbreak the potential influence of common viral infections diagnosed during hospitalization among critically ill patients in the united states outbreak of pandemic 2009 influenza a/h1n1 infection in the hematology ward: fatal clinical outcome of hematopoietic stem cell transplant recipients and emergence of the h275y neuraminidase mutation epidemiology of viral respiratory infections how contagious are common respiratory tract infections? aerodynamics of droplet nuclei aerosol transmission is an important mode of influenza a virus spread modes of transmission of influenza b virus in households commentary: the use of respirators to reduce inhalation of airborne biological agents disposable surgical face masks for preventing surgical wound infection in clean surgery. the cochrane library surgical mask filter and fit performance surgical mask vs n95 respirator for preventing influenza among health care workers: a randomized trial a randomized clinical trial of three options for n95 respirators and medical masks in health workers efficacy of face masks and respirators in preventing upper respiratory tract bacterial colonization and co-infection in hospital healthcare workers n95 respirators or surgical masks to protect healthcare workers against respiratory infections: are we there yet? simulated workplace performance of n95 respirators respiratory protection for healthcare workers in the workplace against novel h1n1 influenza a: a letter report simulated workplace protection factors for half-facepiece respiratory protective devices performance of an n95 filtering facepiece particulate respirator and a surgical mask during human breathing: two pathways for particle penetration professional and home-made face masks reduce exposure to respiratory infections among the general population respirator tolerance in health care workers discomfort and exertion associated with prolonged wear of respiratory protection in a health care setting b95: a new respirator for health care personnel reusability of facemasks during an influenza pandemic: facing the flu health care workers' views about respirator use and features that should be included in the next generation of respirators differences in the compliance with hospital infection control practices during the 2009 influenza h1n1 pandemic in three countries institute of medicine committee on respiratory protection for healthcare workers in the workplace against novel h1n1 influenza a letter to president obama on federal ppe guidance interim recommendations for facemask and respirator use to reduce 2009 influenza a (h1n1) virus transmission guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings prevention strategies for seasonal influenza in healthcare settings impact of low filter resistances on subjective and physiological responses to filtering facepiece respirators hand hygiene task force: guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force comparison of combined nose-throat swabs with nasopharyngeal aspirates for detection of pandemic influenza a/h1n1 2009 virus by real-time reverse transcriptase pcr nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses comparative study of nasopharyngeal aspirate and nasal swab specimens for detection of influenza comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction diagnostic procedures for viral, rickettsial, and chlamydial infections concepts and procedures for laboratory based influenza surveillance. atlanta: world health organization collaborating centers for reference and research in influenza clusterpower: power calculations for cluster-randomized and cluster-randomized crossover trials health care-associated infections: a meta-analysis of costs and financial impact on the us health care system design and analysis issues in cluster-randomized trials of interventions against infectious diseases we wish to thank the members of the respect team (complete list to be provided prior to publication). respect is jointly funded by the u.s. all authors read and approved the final manuscript. all authors meet icmje guidelines. lr and tp conceived and designed the study, coordinated and supervised the study and drafted the manuscript. mb, ae, cn, mr, cs, and ms designed the study, coordinated and supervised the study and drafted the manuscript. dc designed the study, conceived and designed the epidemiologic and statistical analyses and drafted the manuscript. gg and cg designed the study, conceived and designed laboratory analyses and drafted the manuscript. nr designed the study, conceived and designed epidemiologic and statistical analyses, coordinated and supervised the study and drafted the manuscript. rs designed the study and drafted the manuscript. the authors declare that they have no competing interests. the findings and conclusions in this manuscript are the authors' own and do not necessarily represent the views of the national institute for occupational safety and health, the u.s. department of veterans affairs, or other affiliates. mention of product names does not imply endorsement. submit your next manuscript to biomed central and we will help you at every step: key: cord-302918-0nk7zyod authors: broor, s.; bharaj, p.; chahar, h. s. title: human metapneumovirus: a new respiratory pathogen date: 2008-11-01 journal: j biosci doi: 10.1007/s12038-008-0067-y sha: doc_id: 302918 cord_uid: 0nk7zyod human metapneumovirus is a recently recognized pathogen of acute respiratory tract infection (ari) in children as well as elderly and immunocompromised adults. the virus belongs to the family paramyxoviridae, sub family pneumovirinae and genus metapneumovirus. through genetic analysis it has been characterized into two groups a and b which are further divided into four sub-lineages. the virus is difficult to grow in tissue culture and hence reverse transcriptase-polymerase chain reaction (rt-pcr) for n and l gene is the method of choice for diagnosis. the virus has been seen in all countries with seasonal distribution in winter months for temperate and spring/summer for tropical countries. f gene is the most conserved among different lineages and efforts are underway to design recombination vaccine using f gene. acute respiratory tract infections (ari) are a leading cause of morbidity and mortality worldwide (denny and loda 1986; hijazi et al 1996) . of the 10 million deaths of children less than 5 years of age throughout the world 1·9 million children died from acute lower respiratory tract infections (alri) in the year 2000, 70% of them in sub-sahara africa and south asia (williams et al 2002) . although the incidence rates of ari are almost similar in both developed and developing countries, the mortality is higher in developing countries (shapiro 1998) . the risk of pneumonia is 3-6 times higher in children from developing countries (10-20%) as compared to developed countries (3-4%) (victora et al 1999) . approximately 0.5 million children die due to alri in india each year, accounting for one fourth of the 1.9 million global alri deaths (reddaiah and kapoor 1988; ahmad et al 2000; williams et al 2002) . all classes of micro organisms including viruses, bacteria and protozoa are capable of infecting the respiratory tract, but viruses and bacteria are the common pathogens. a variety of viruses, including, respiratory syncytial virus (rsv), infl uenza viruses, parainfl uenza viruses, adenoviruses, coronaviruses and picornaviruses, are associated with different respiratory syndromes in all age groups (grondahl et al 1999; weigl et al 2000) . however, despite extensive diagnostic testing, a substantial portion of respiratory tract infections still cannot be attributed to any known pathogens. in about a third cases of alri (liolios et al 2001) and a half of upper respiratory tract infections (uri) (nokso-koivisto et al 2002) no pathogen could be detected .these observations suggest that unknown pathogens may be responsible for a substantial proportion of respiratory tract disease. metapneumovirus is a recently discovered etiological agent of acute respiratory illness (ari). it was fi rst reported in 2001 from the netherlands from nasopharyngeal aspirates (taken over a 20 year period) in 28 hospitalized children and infants with ari having signs and symptoms similar to that of rsv infection (van den hoogen et al 2001) . the virus was found to be closely related to the avian pneumovirus, a member of the metapneumovirus genus, and was called human metapneumovirus (hmpv) . although the virus was not isolated until recently, antibodies to the virus have been shown to be present in all persons ≥ 8 years of age in serum samples collected since 1958 (van den hoogen et al 2001) . thus the virus is not a newly emerging pathogen and did not recently "jump" to the human population from an animal reservoir. genetic analysis of the virus showed that it is an rna virus of the paramyxoviridae family and pneumovirinae subfamily along with rsv. the avian pneumoviruses (apv) (formerly turkey rhinotracheitis virus) is highly related to hmpv and these two viruses have been separated by taxonomists into a separate genus, metapneumovirus (lamb et al 2000) . initial electron microscopic examination revealed that the viral isolate had morphology similar to paramyxoviruses. spherical enveloped particles have a mean diameter of ~200 nm. in addition, fi lamentous and pleomorphic particles are also present. the virion is surrounded by a lipid envelope derived from the plasma membrane of the host cell into which the three virus glycoproteins, the attachment (g), fusion (f), and small hydrophobic (sh) proteins, are inserted (collins and mottet 1993) . the rna genome associates with viral proteins to form the helical nucleocapsid (represented on the right and in the centre of the virion on the left in fi gure 1). the proteins consist of the nucleocapsid protein (n), the phosphoprotein (p), and the large polymerase (l) protein. the m2-1 transcriptional enhancer protein is also thought to be associated with the nucleocapsid. the nucleocapsid is surrounded by the matrix (m) protein, which forms a link between the nucleocapsid and the lipid membrane of the virus particle (easton et al 2004) . full length sequences of at least 4 hmpv genomes have been reported (van den hoogan et al 2002; biacchesi et al 2003; herfst et al 2004) . the negative sense non-segmented rna genome of the virus is about 13 kb in length (easton et al 2004) . the hmpv genome is predicted to encode 9 proteins in the order 3-n-pm-f-m2-sh-g-l-5 (the m2 gene is predicted to encode 2 proteins, m2-1 and m2-2, using overlapping open reading frames, as in rsv) (van den hoogan et al 2002) (fi gure 2). the genome also contains noncoding 3'leader which has the viral promoter, 5' trailer and intergenic regions, consistent with the organization of rsv . however there are some differences in the genome of apv/hmpv as compared to rsv, they lack the 2 nonstructural proteins ns1 and ns2 complete virion nucleocapsid located at the 3' end of rsv genomes. these proteins counteract host interferons; therefore the lack of these genes in the metapneumoviruses may have important implications for the relative pathogenicity of these viruses compared with rsv strains (collins et al 2001) . sequence identity between apv and hmpv open reading frames is 56% to 88% (van den hoogen et al 2002). although the function of each of the gene products has not been formally tested, it is likely that the function of the proteins can be predicted by comparison with other paramyxoviruses. the f (fusion), g (glycosylated) and sh (short hydrophobic) proteins are integral membrane proteins on the surface of infected cells and virion particles. the f protein appears to be a classic viral fusion protein, with a predicted nonfurin f1/f2 cleavage site near a hydrophobic fusion peptide and 2 heptad repeats in the extra cellular domain that facilitate membrane fusion. the predicted g protein of hmpv exhibits the basic features of a glycosylated type ii mucin-like protein but interestingly lacks the cluster of conserved cysteines sometimes termed the "cysteine noose" that is found in the rsv and apv g proteins. g gene of hmpv tends to be highly variable like rsv g gene which may be due to host immune selection pressure (crowe 2004) . the function of the sh proteins of both the viruses remains unknown. the n (nucleoprotein), p (phosphoprotein) and l (large, polymerase) proteins are replication proteins in the nucleocapsid, the m2-1 and m2-2 proteins are regulatory proteins and the m (matrix) protein may coordinate viral assembly of viral nucleocapsids with envelope proteins (crowe 2004) . the classifi cation and naming of serotypes/serogroups, genotypes, subgroups, strains, variants and isolates of hmpv is still evolving. based on genomic sequence and phylogenetic analysis, there are two major genotypes of hmpv, designated a and b (biacchesi et al 2003; van den hoogen et al 2003) . these analyses are based on sequencing of the n, m, f, g, or l gene, and the genotype groupings are concordant regardless of which gene is studied (bastein et al 2003; biacchesi et al 2003; boivin et al 2004; peret et al 2004) . full length sequences of genomes from viruses representing the 2 major subgroups show that the diversity between hmpv subgroup a and b sequences is greatest for the sh and g proteins (59 and 37% identity, respectively), biacchesi et al 2003; and is more than rsv subgroup a and b. hmpv f protein, which is predicted to be the principal target of protective antibodies, is more conserved in hmpv strains than rsv. the overall level of genome nucleotide sequence identity and aggregate proteome amino acid sequence identity between the two hmpv subgroups were 80 and 90%, respectively, similar to the respective values for rsv a and b groups (hamelin et al 2004) . based on the sequence analysis of two surface glycoprotein encoding genes namely f and g genes each of the major lineages are further subdivided into two genetic sub-lineages known as a1, a2 and b1, b2, the signifi cance of this diversity is unclear at this time van den hoogen et al 2004) (fi gure 3). in a recent report huck and colleagues have reported a new sub lineage within the a lineage. previously reported a2 lineage is found to consist of two clusters namely, a2a and a2b. among all the sub-lineages of hmpv the a2 sub-lineage shows the greatest diversity (huck et al 2006) . the genetic diversity in hmpv strains also affects antigenic diversity, but to what extent has not been resolved. studies with experimental infection of animals have suggested that the two lineages exhibited 48% antigenic relatedness based on reciprocal cross-neutralization assay with post-infection hamster sera (skiadopoulos et al 2004) . however cross protection studies in hamsters and nonhuman primates have shown that each strain provided a high level of resistance to reinfection with the homologous or heterologous strain, supporting the conclusion that the two hmpv genetic lineages are highly related antigenically and are not distinct antigenic subtypes or subgroups skiadopoulos et al 2004; van den hoogen et al 2004) . hmpv f protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection (macphail et al 2004; skiadopoulos et al 2004) . the extent of cross-protection in rodents cannot be extrapolated directly to the human situation because the animals are only semi permissive hosts for hmpv replication. although diffi cult to assess, the extent of cross-protection is important to estimate because vaccine developers will choose to develop either a monovalent or a bivalent vaccine formulation based on this factor. hmpv has been associated with ari in all age groups, with more severe diseases occurring in young children, elderly individuals and immunocompromised hosts. the virus causes a variety of clinical syndromes in children that are typical of the paramyxoviruses, including upper and lower respiratory tract illnesses. the clinical characteristics of hmpv infections are not distinctive, thus, differentiating it from other respiratory viruses on clinical grounds is not possible (stockton et al 2002) . a study conducted at vanderbilt university medical center on the association of the virus in a cohort of 2000 subjects of age 0-5 years, followed during a 25-year period revealed that hmpv was associated with common cold (complicated by otitis media in one third), bronchiolitis, pneumonia, croup and exacerbation of reactive airway disease (williams et al 2004) .the clinical profi le of illness caused by hmpv was similar to that caused by rsv. in outpatients with alri, hmpv was detected in 12% of cases, which was second only to rsv (williams et al 2004) . hoarseness has also been observed more frequently in hmpv infection as compared to rsv (falsey et al 2003) . further compared with rsv infections, the children who develop hmpv infection are somewhat older, and the severity of disease is usually somewhat less than rsv peiris et al 2003; van den hoogan et al 2003; viazov et al 2003) . human mpv infection is not restricted to the very young children but also occurs in adults and elderly subjects. in adults it usually causes fl u like illness and colds . in fragile elderly hmpv causes more severe disease than in healthy elderly or young adults (falsey et al 2003) . in a study among 10 elderly of > 65 years of age who had underlying illness, 4 developed pneumonitis and 2 died due to hmpv infection . immune status of infected subjects is also important in determining the severity of illness. hmpv infection has been reported in immunocompromised individuals. in 3 patients with acute lymphoblastic leukemia who had alri, hmpv was the sole pathogen detected and one of them died . hmpv infection has also been reported in hiv infected children but it is not clear if the severity of illness is more in this group (madhi et al 2003) . respiratory virus infections are often associated with recurrent wheezing in children and exacerbations of asthma in older patients. acute wheezing and asthma exacerbations have been associated with hmpv infection in some studies (jartti et al 2002; peiris et al 2003) but all studies have not shown this association (rawlinson et al 2003) . in some studies in children with asthma hmpv was found more frequently than rsv van den hoogen et al 2003) . a study by vicente et al (2006) suggested that hmpv genotype a might be more pathogenic than genotype b, causing greater clinical severity in children whereas no differences in disease severity associated with either genotype (agapov et al 2006) was observed in another study. because the seasonal distributions of hmpv and rsv overlap, the potential for dual infection exists. several studies have found hmpv -rsv co-infection rate of approximately 5-10% (esper et al 2004; viazov et al 2003; williams et al 2004; xepapadaki et al 2004) . however, greensill et al (2003) reported that 70% of rsv-infected children who required intensive care in liverpool, united kingdom, were co-infected with hmpv, suggesting that dual infection with rsv and hmpv may predispose for severe infection particularly in otherwise healthy children. in another study from the united kingdom, hmpv and rsv co-infection conferred an increased risk of admission to the pediatric intensive care unit (semple et al 2005) . however, such synergistic association has not been found in other, population-based and case-control studies of hospitalized children esper et al 2003; maggi et al 2003) . the possible synergistic interaction between hmpv and the severe acute respiratory syndrome (sars) coronavirus has been recently postulated during the 2003 sars outbreak in canada and hong kong as many individuals were found to have co-infection with hmpv (chan and li 2003; poutanen et al 2003) . on the other hand, such synergy between hmpv and sars was not confi rmed in experimental macaque models . in one case report, in an infant who had sars, fatal encephalitis was correlated with hmpv infection as hmpv rna was detected post-mortem in brain and lung tissue (schildgen et al 2005) . cases of severe hmpv infections in adults and reinfection in immunocompromised subjects suggest that, despite the universal infection in childhood, new infections can occur throughout life due to incompletely protective immune responses and/ or acquisition of new genotype. since severe disease is seen mainly in pediatric patients, it suggests that naturally acquired infection induces partial protection against the disease. however, it should be emphasized that there is no cross -protection among the virus strains. a recent report has described a child who suffered from two episodes of hmpv infection during a one month period, each caused by a different strain (vargas et al 2004) . the conventional model of attachment for the members of subfamily pneumovirinae including genus pneumovirus and metapneumovirus involves the interaction of viral g protein with a molecule or molecules on the host cell surface (levine et al1987) .while the precise nature of the cellular receptor and mechanism of entry has not been identifi ed, current evidence suggests that one or more cellular glycosaminoglycans or heparin-like molecules are involved in virus attachment and entry (feldman et al 2000) . following g-protein-mediated attachment, the f glycoprotein promotes ph-independent fusion between the cell membrane and the virus envelope. for the f protein to become functional, it has to be cleaved by cellular proteases, and the hydrophobic amino terminal region of the f1 component then promotes the fusion process, which introduces the internal components of the virion into the cytoplasm of the host cell, where the remainder of the infectious cycle takes place. recent experimental work using primates (chimpanzees, cynomolgus and rhesus macaques, african green monkeys) and small animals (hamsters, cotton rats, mice and ferrets) has been performed to characterize the pathogenesis associated with this viral infection; hmpv replicates to a various extent in the upper and lower respiratory tracts of these experimental animals, although clinical symptoms after intranasal challenge have only been observed in chimpanzees, cynomolgus macaques and balb/c mice so far (van den hoogen et al 2001; alvarez et al 2004; kuiken et al 2004; skiadopoulos et al 2004; . in balb/c mice and cotton rats, time course studies have indicated that peak viral titers are found around day 4-5 after infection and decrease thereafter alvarez et al (2004a) and , have demonstrated that hmpv may present an initial biphasic replication pattern in lungs of balb/c mice, with hmpv rna still detectable more than 180 days after infection. such persistence could be explained by an aberrant t helper cell type 2-like immune response, with impaired virus clearance after primary hmpv infection (alvarez and tripp 2005) . signifi cant pulmonary infl ammatory changes have been found in balb/c mice and cotton rats (alvarez et al 2004; . interestingly signifi cant infl ammatory changes were still present in the cotton rat animal model more than 21 days after viral challenge . increase in many cytokines and chemokines such as interleukin il-2, il-8, il-4, inf-γ, macrophage infl ammatory protein 1α and monocyte chemotactic protein has been observed in the lungs or bronchoalveolar lavage of both mice and cotton rats in response to hmpv challenge (alvarez et al 2004 . in humans, hmpv infection has also been associated with an increase of il-8 in upper respiratory tract secretions and with chronic infl ammatory changes of the airways, with presence of, intra alveolar foamy and hemosiderin-laden macrophages (laham et al 2004 , vargas et al 2004 . when compared with rsv, hmpv infections in humans seem to induce lower levels of infl ammatory cytokines such as il-12, tumor necrosis factor α, il-6 and il-1β (laham et al 2004) . thus far, no experimental human volunteer studies have been reported. since its initial description in 2001, hmpv has been isolated from individuals of all ages with ari, and has been identifi ed in every continent. hmpv infections have been documented in europe [united kingdom ( stockton et al 2002 ) , finland (jartti et al 2002 ) , italy (maggi et al 2003) , france (freymuth et al 2003) , germany ( viazov et al 2003) , spain (vicente et al 2003) , norway (dollner et al 2004) ]; america, canada , united states (esper et al 2003 , falsey et al 2003 , brazil (cuevas et al 2003) australia (nissen et al 2002) , asia [hongkong , japan (ebihara et al 2004) , korea (kim and lee 2005) thailand (samransamruajkit et al 2005) ] and africa [yemen, (al-sonboli et al 2005) , south africa (ludewick et al 2005) ]. in a preliminary study from pune in india hpmv was found in 5 of 26 children with ari (rao et al 2004) . in a study of children seen in a large referral hospital in delhi hmpv was detected in 12% of ari cases by reverse transcriptase-polymerase chain reaction (rt-pcr) (banerjee et al 2007) . role of hmpv in ari has been evaluated in many studies mostly using molecular methods. in young hospitalized children hmpv has been detected in 5-10% of ari cases. bastein et al 2003; bovin et al 2003; esper et al 2003 esper et al , 2004 mcintosh and mcadam 2004) . however in one study from italy the rates of hospitalizations due to hmpv infection varied from 7-43% (maggi et al 2003) . the rates of detection of hmpv in adults are usually lower than children with rates of about 3% in general community (stockton et al 2002) . it is interesting to note that detection rates of hmpv have generally been higher in retrospective studies than in prospective studies, an observation consistent with a degree of selection bias. this indicates that large prospective studies are needed in order to clarify the role of hmpv in various clinical conditions (hamelin et al 2004) . most hmpv infections occur in < 5 years of age, with children <2 years of age being most at risk for serious hmpv infections. in one study the peak was seen at 4-6 months of age (van den hoogen et al 2004). however, hmpv infections tend to occur in slightly older children compared to rsv van den hoogen et al 2003) . the activity of hmpv in temperate climates peaks between december and february (maggi et al 2003; van den hoogen et al 2003) where as in subtropics it peaks in springsummer . the peak of activity of hmpv at any given location often coincides with or follows the peak of rsv activity esper et al 2004) . outbreaks of hmpv are to be a local phenomenon unlike infl uenza virus, where two or three strains spread across the globe each year. strains of hmpv differ from community to community, and strains identifi ed in one location may be quite similar to strains identifi ed in other locations in different years. for example, the prototype strain identifi ed in the netherlands is genetically similar to strains identifi ed in australia; new haven, connecticut and quebec, canada in different years (esper et al 2004) . we in delhi also found that 2 lineages of hmpv circulated during the same season i.e. in 2005-06 a2b and b1 lineages were seen with a2b being the predominant genotype where as in 2006-07 b1 and b2 were found to co-circulate with predominance of b1 (broor s, et al, unpublished data) . although formal transmissions studied have not been carried out, transmission most likely is through large particle respiratory secretions and fomites as is true for rsv (human metapneumovirus 2006) . nosocomial transmission has been reported in hospital setting (pieris et al 2003). presence of hmpv antibodies in serum samples obtained in 1958 showed that the virus has been circulating for at least for the past 50 years in the netherlands (van den hoogen et al 2001). by the age of 5 years, >90% of individuals screened have evidence of hmpv infection. the seroprevalence of hmpv specifi c antibody in adults is nearly 100% (van den hoogen et al 2001, leung et al 2005) . the hmpv-specifi c antibodies in infants <3 months of age has been detected in >90% of the cases tested, indicating that maternally derived antibodies are present in young children (leung et al 2005) . whether these hmpv specifi c antibodies protects against infection or lessens the severity of illness is not known. hmpv replicates poorly in conventional cell culture and is relatively diffi cult to isolate. many strains show reliable cytopathic effect (cpe) in tertiary monkey kidney cell line/llcmk-2 cells . the cpe is quite variable, some strains show syncytia formation and other only produce rounding . cpe is usually observed after 10-21 days (mean 17 days). confi rmation of cpe is done by rt-pcr or indirect immunofl uorescence staining using hmpv specifi c antibodies. recent reports have shown that hmpv can replicate effi ciently in hep-2 cells but does not produce good cpe. van den hoogen et al (2004) reported that vero cell clone 118 is permissible for expression of virus from all four lineages and cpe is easier to observe. many laboratories are now using this cell line for routine virus isolation . in addition shell vial culture for rapid isolation of hmpv has also been described (reina et al 2007) . they showed that shell vial culture using commercial llcmk-2 cells should be a method for isolating hmpv from respiratory samples in pediatric population. direct immunofl uorescence assay (dfa) using virus specifi c antibodies is a rapid method to detect respiratory viruses and is commonly used in diagnostic laboratories. commercially available hmpv specifi c antibodies have been developed for direct immunofl uorescence assays, though this method may not be as sensitive as rt-pcr for the detection of hmpv (ebihara et al 2005 , landry et al 2005 , percivalle et al 2005 . because of the unavailability of rapid antigenic detection assays in the past and slow growth in tissue culture molecular methods have become the method of choice for the diagnosis of hmpv infection. cote et al (2003) reported that primers that bind regions of the n and l genes are highly sensitive for the detection of hmpv strains of both genotypes. however, it is reported that the p gene of hmpv is an ideal target for the molecular detection and genotyping as p gene provides the conserved region for designing rt-pcr primers and adequate variability to permit the accurate genotyping of the virus into 2 main lineages and 4 sublineages . real time pcr has also been described for hmpv which allows amplifi cation and quantitation of this pathogen in clinical samples. real-time rt-pcr is a powerful method to detect hmpv and other respiratory viruses. the assay has been developed to detect the n gene of all known hmpv lineages (maertzdorf et al 2004) and real time rt-pcr targeting the n and / or l gene is more sensitive, specifi c, and rapid method to detect this virus in clinical samples than conventional rt-pcr (mackay et al 2003) . nucleic acid sequence based amplifi cation assay (nasba) targeting the m gene has also been developed for detection of hmpv infection in respiratory specimens (dare et al 2007) . enzyme-linked immunosorbent assay (elisa) can also be used for hmpv serological testing using the hmpv-infected cells (falsey et al 2003 . recently, elisa methods using viral n or f protein expressed in prokaryotic or recombinant vesicular stomatitis virus (vsv) (leung et al 2005) and recombinant baculovirus system (liu et al 2007) have been developed to detect antibodies against hmpv. use of these assays in sero-epidemiologic studies might be helpful for detection of antibody response against hmpv infections thus increasing the understanding of the human immune responses to hmpv and permitting better understanding of the epidemiology of this virus. however, since infections with hmpv are universal, serologic testing for diagnosis can only help if four fold increases in antibody titers or seroconversion is demonstrated (prins and wolthers 2004) . the development of a safe and effective vaccine to protect against hmpv is a reasonable goal. several promising vaccine candidates have been tested in animal models. a live recombinant human parainfl uenza virus that contains the hmpv f gene has been shown to induce hmpv-specifi c antibodies and to protect experimental animals from hmpv challenge (skiadopoulos et al 2004) . a chimeric bovine/human parainfl uenza virus 3 expressing the hmpv f elicits neutralizing antibodies against both parainfl uenza virus and hmpv (tang et al 2005) . however, the results of these animal challenge studies should be interpreted cautiously. there are many limitations of the small-animal model for testing potential hmpv vaccines, not the least of which is that the pathogen is highly host restricted. the safety and effi cacy of a recombinant hmpv vaccine may be diffi cult to predict. chimeric hmpv/avian pneumovirus recombinant viruses in which the n or p gene of hmpv was replaced with the corresponding gene from avian pneumovirus are attenuated in african green monkeys (the p gene chimera is 10-to 1,000-fold more attenuated than the n chimera), though both are as immunogenic as wild-type hmpv (pham et al 2005) . this represents another strategy for the development of recombinant attenuated live hmpv vaccines. other than infl uenza virus, antiviral therapy for respiratory viruses has not shown tremendous potential. it is unknown what role the host's infl ammatory response plays during hmpv disease. nonetheless, antiviral compounds have been tested with hmpv. the antiviral activity of ribavirin to inhibit the replication of hmpv is equivalent to that observed with rsv (wyde et al 2003) . other compounds, such as nmso3, a sulphated sialyl lipid that has been shown to have potent antiviral activity against rsv in tissue culture cells, have been shown to have anti-hmpv activity in vitro (wyde et al 2004) . it is likely that an hmpv-neutralizing monoclonal antibody for prophylaxis of high-risk infants (similar to the anti-rsv f humanized monoclonal antibody currently used for prevention of severe rsv disease) will be developed and tested. the progress towards an effective antiviral strategy for hmpv is currently limited by the scant data on pathogenesis of the virus in the natural host. since the discovery of hmpv in 2001, the virus has been identifi ed worldwide. hmpv is a common respiratory pathogen, particularly in infants and young children. the virus is associated with both upper and lower respiratory tract infections and may be a trigger for asthma. there are two major genotypes of hmpv, whether these genotypes represent distinct serotypes re-mains controversial. the major challenges faced by the medical and scientifi c communities are: the understanding of the pathogenesis of hmpv disease and the development of a safe and effective vaccine to protect against infection and disease caused by this newly recognized respiratory virus. genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness the decline in child mortality: a reappraisal respiratory syncytial virus and human metapneumovirus in children with acute respiratory infections in yemen human 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membrane orientation and oligomerization of the small hydrophobic protein of human respiratory syncytial virus respiratory syncytial virus comparative evaluation of realtime pcr assays for detection of the human metapneumovirus human metapneumovirus as a major cause of human respiratory tract disease human metapneumovirus and respiratory syncytial virus, brazil ; emerg diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis acute respiratory infections are the leading cause of death in children in developing countries outbreak of human metapneumovirus infection in norwegian children nucleotide sequence of the genes encoding the matrix and small hydrophobic proteins of pneumonia virus of mice animal pneumovirus: molecular genetics and pathogenesis human metapneumovirus infection detection of human metapneumovirus antigens in nasopharyngeal secretions by an immunofl uorescent-antibody test human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children a 1-year experience with human metapneumovirus in children aged -5 years human metapneumovirus infections in young and elderly adults the fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate aetiology: koch's postulates fulfi lled for sars virus presence of the new human metapneumovirus in french children with bronchiolitis human metapneumovirus in severe respiratory syncytial virus bronchiolitis rapid identifi cation of nine micro-organisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-pcr: feasibility study human metapneumovirus: a new player among respiratory viruses development and validation of an enzyme-linked immunosorbent assay for human metapneumovirus serology based on a recombinant viral protein human metapneumovirus infection in adults with community-acquired pneumonia and exacerbation of chronic obstructive pulmonary disease recovery of human metapneumovirus genetic lineages a and b from cloned cdna laboratory diagnosis of acute lower respiratory tract viral infections in children metapneumovirus and acute wheezing in children human metapneumovirus-associated lower respiratory tract infections in korean infants and young children experimental human metapneumovirus infection of cynomolgus macaques (macaca fascicularis) results in virus replication in ciliated epithelial cells and pneumocytes with associated lesions throughout the respiratory tract differential production of infl ammatory cytokines in primary infection with human metapneumovirus and with other common respiratory viruses of infancy family paramyxoviridae detection of human metapneumovirus in clinical samples by immunofl uorescence staining of shell vial centrifugation cultures prepared from three different cell lines seroepidemiology of human metapneumovirus (hmpv) on the basis of a novel enzyme-linked immunosorbent assay utilizing hmpv fusion protein expressed in recombinant vesicular stomatitis virus demonstration that glycoprotein g is the attachment protein of respiratory syncytial virus comparison of a multiplex reverse transcription-pcr-enzyme hybridization assay with conventional viral culture and immunofl uorescence techniques for the detection of seven viral respiratory pathogens intracellular processing, glycosylation, and cell surface expression of human metapneumovirus attachment glycoprotein human metapneumovirus genetic variability molecular assays for detection of human metapneumovirus use of the p gene to genotype human metapneumovirus identifi es 4 viral subtypes identifi cation of small-animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hmpv) and implications for hmpv vaccine design human metapneumovirus-associated lower respiratory tract infections among hospitalized human immunodefi ciency virus type 1 (hiv-1)-infected and hiv-1-uninfected african infants real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages human metapneumovirus associated with respiratory tract infections in a 3 year study of nasal swabs from infants in italy human metapneumovirus -an important new respiratory virus viral etiology of frequently recurring respiratory tract infections in children children with respiratory disease associated with metapneumovirus in hong kong respiratory tract reinfections by the new human metapneumovirus in an immunocompromised child rapid detection of human metapneumovirus strains in nasopharyngeal aspirates and shell vial cultures by monoclonal antibodies characterization of human metapneumoviruses isolated from patients in north america sequence polymorphism of the predicted human metapneumovirus g glycoprotein chimeric recombinant human metapneumoviruses with the nucleoprotein or phosphoprotein open reading frame replaced by that of avian metapneumovirus exhibit improved growth in vitro and attenuation in vivo identifi cation of severe acute respiratory syndrome in canada human metapneumovirus: a new pathogen in children and adults first detection of human metapneumovirus in children with acute respiratory infection in india: a preliminary report asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection acute respiratory infections in rural underfi ves comparison of different cell lines and incubation times in the isolation by the shell vial culture of human metapneumovirus from pediatric respiratory samples human metapneumovirus in infants and young children in thailand with lower respiratory tract infections induction of acute otitis media by human metapneumovirus dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis epidemiology of acute respiratory infections the two major human metapneumovirus genetic lineages are highly related antigenically and the fusion (f) protein is a major contributor to this antigenic relatedness human metapneumovirus as a cause of community-acquired respiratory illness a host-range restricted parainfl uenza virus type 3 (piv3) expressing the human metapneumovirus (hmpv) fusion protein elicits protective immunity in african green monkeys prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients antigenic and genetic variability of human metapneumoviruses pathology of human metapneumovirus infection: insights into the pathogenesis of a newly identifi ed respiratory virus high prevalence of human metapneumovirus infection in young children and genetic heterogeneity of the viral isolates human metapneumovirus and community-acquired respiratory illness in children differences in clinical severity between genotype a and genotype b human metapneumovirus infection in children potential interventions for the prevention of childhood pneumonia in developing countries: improving nutrition epidemiological investigation of nine respiratory pathogens in hospitalized children in germany using multiplex reversetranscriptase polymerase chain reaction human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children estimates of world wide distribution of child deaths from acute respiratory infections comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by nmso3 in tissue culture assays human metapneumovirus as a causative agent of acute bronchiolitis in infants key: cord-309488-8guapzke authors: dodd, r. title: other emerging viral pathogens date: 2006-08-15 journal: isbt sci ser doi: 10.1111/j.1751-2824.2006.00043.x sha: doc_id: 309488 cord_uid: 8guapzke nan for many years, the greatest concerns about blood safety were focused upon viral infections, most notably hepatitides and retroviral diseases. as a result of experience with these infections, it was widely anticipated that future transfusiontransmissible infections would result from viruses causing persistent infection, most probably parenterally transmitted. however, until around the year 2000, most effort relating to emerging infections was directed towards parasitic diseases. attention was refocused on viral infections as a result of the outbreak of west nile virus (wnv) disease in the usa along with the recognition that it was transmissible by transfusion [1] . this paper will briefly review currently emerging viruses other than wnv, which have been shown to be, or which have the potential to be transmitted by transfusion. specifically omitted are cmv, ebv, hepatitis a virus and the b19 erythrovirus, which are demonstrably transmitted by transfusion, but which do not fit the current definition of emerging agents. it should be noted that most rna viruses and also hbv may mutate at a high frequency as a result of an absence of repair mechanisms during nucleic acid replication. this accounts for the large number of different strains and genotypes that are seen among these viruses, in addition to the pseudotypes that develop in individual infected patients. in the case of hbv, selection pressure can additionally lead to the appearance of so-called escape mutants that fail to express some of the antigens that are normally recognized by neutralizing antibodies [2] . there are a number of different clades of hiv that are broadly grouped as m, o and n. the latter two groups are genetically more distant and, in the case of group o, infection is not always detected by procedures that have been developed to detect antibodies to clade b which was originally most common in the usa and other developed countries [3] . the potential for continuing development of new clades and of recombinant viruses continues, and appropriate surveillance is necessary to ensure that sensitive tests are always available. in the usa, if a test is not validated for the detection of group o strains, donors must be deferred for a history of travel, sexual contact or residence in countries where these strains are found. similarly, there are six major genotypes of hcv which are distributed around the world in varying frequencies [4] . although they may differ somewhat in their pathogenesis and responsiveness to therapy, there do not seem to be the same difficulties in detection as have been seen with hiv [5] . this is also true of the eight genotypes of hbv [6] . however, in addition to hbv genotypes, there are also a number of mutations, some of which result in the absence of effective expression of all of the epitopes, particularly on the common 'a' antigen. tests constructed with a limited repertoire of monoclonal detection antibodies may be unable to detect some of these mutant strains. again, however, appropriate surveillance and continuous improvement of test kits usually overcomes these problems. severe acute respiratory syndrome (sars) is an example of the explosive emergence of an infection apparently new to humans. the disease, in common with most emerging infections, is a zoonosis and it is thought to have been transmitted to humans in south-east china through the preparation of exotic mammals for human consumption, late in 2002 [7, 8] . as its name suggests, the disease manifested primarily with respiratory symptoms, but in the more serious cases, was also systemic. it was extremely infectious by the respiratory route, and probably also by the faecal-oral route. as a result of rapid air transportation of infected individuals, it spread rapidly across the globe. between 1 november 2002 and 10 july 2003, the who reported 5910 symptomatic cases, plus another 2527 for which no dates were available. fortunately, this single outbreak was contained, and apart from a small handful of cases among laboratory workers, there does not appear to have been a recurrence of the disease. the infectious agent was rapidly identified by molecular techniques as a coronavirus, novel to humans, but endemic among certain mammalian species in south-eastern china [9, 10] . it is reasonable to ask why this outbreak of a new respiratory infection led to concern about blood safety. in addition, as an entirely new disease, nothing was known about it, so preventative measures were taken in accordance with the precautionary principle. the who issued recommendations to reduce the risk of transfusion transmission, as did the fda. these measures included deferral of donors with a history of the disease or contact with a patient and those with a history of travel from an affected area. even relatively casual travel exposure (such as travel through the airport in toronto) led to temporary deferral, at least in the usa. subsequent to the development of these precautions, it became apparent that there could be viraemia during symptomatic disease, but it is still unknown whether there is a presymptomatic viraemia. additionally, epidemiologic studies showed that there were asymptomatic infections, as judged by the prevalence of antibodies to the sars coronavirus [11] . again, however, it is unknown whether such asymptomatic infection is accompanied by viraemia. there were no recorded cases of transfusiontransmission of sars during the outbreak, although it is unclear whether they would actually have been recognized if they did occur. the human herpes virus 8 is the most recently recognized herpes virus to infect humans [12] . it is known to be the causative agent of kaposi's sarcoma, both classical and human immunodeficiency virus-associated, and is the likely causative agent of several other rare disorders, including primary effusion lymphoma and multicentric castleman's disease [13] . it is naturally transmitted through saliva or sexual contact and has been transmitted through organ transplantation. in some cases, recipients of infected organ transplants have subsequently developed kaposi's sarcoma [14] [15] [16] . because of the pathogenic potential of this virus and because of the evident potential for blood-borne transmission, there has been a continuing undercurrent of concern about hhv-8 among those involved with blood safety. it is important to note, however, that hhv-8 is not a new agent: rather, it is newly described. presumably, blood from hhv-8-infected individuals has been transfused for many years, yet there is no extant evidence of an association between transfusion and kaposi's sarcoma or other hhv-8-associated disease states. evidence for transmissibility of hhv-8 by transfusion has been equivocal at best and there continues to be a somewhat anxious wait for the definitive answer to this key question. in 1997, blackbourn and colleagues [17] reported on the detection of hhv-8 dna in the blood of a seropositive blood donor; based on evidence of in vitro passage of the virus to allogeneic cells, the authors expressed concern about the potential for transmission by transfusion. in the same year, however, operskalski and colleagues [18] used the tss repository to examine samples from 10 recipients of blood from 14 donors who were subsequently shown to be hhv-8-seropositive. none of the recipients showed any evidence of infection. similarly, negative data on recipients of seropositive blood units have been reported from jamaica [19] . in contrast, cannon and associates [20] demonstrated an increased prevalence of hhv-8 antibodies among female injection drug users-a result that was interpreted as evidence favouring blood-borne transmission. other studies generated similar results. finally, there have been reports of an increased prevalence of hhv-8 antibodies among some, but not all, groups of blood recipients [21, 22] . it is perhaps worth pointing out that there is, as yet, no clear gold standard for hhv-8 antibodies and that the performance characteristics of available tests differ considerably and do not seem to correlate well with the presence of viral dna [23, 24] . false-positive reactions have also been noted. dollard and colleagues [25] reported on a study of patient samples from the fact study, which was a major effort designed to determine the frequency of transfusion-associated infections in a large cohort of cardiac surgery patients from 1986 to 1990. among the 284 patients who were initially seronegative, 2 had unequivocal increases in hhv-8 antibody titres 6 months postoperatively. this translated to a risk of 0·082% per unit transfused. although there were no infections among a control group of 75 non-transfused individuals, the difference was not significant. the overall frequency of existing positive test results in the study population was quite high, at 11·3%, although, in a different study, the donor prevalence was found to be 2·4%. as with other studies, there is no clear donorrecipient linkage or real proof that transfusion-associated transmission had occurred. linked donor samples were not available. the study adds to the weight of circumstantial evidence, however, that hhv-8 may be transmissible via transfusion. more convincing evidence of transmissibility has been developed in a study in uganda, and was recently presented in preliminary form. given the variable behaviour characteristics of tests for hhv-8 antibodies, and the absence of clear evidence that dna testing would be effective, it is unclear how interventions for this infection will be implemented, if needed. it is, however, quite likely that leucoreduction will be able to effect a substantial, if not complete, abrogation of infectivity. dengue is caused by a mosquito-borne flavivirus which is endemic in most of the tropics. it has the potential for emergence or re-emergence in areas where the vector mosquitoes exist, including the south-eastern usa. by way of example, an outbreak of dengue has occurred in tropical queensland, australia; it was thought to have been introduced by infected travellers. although infection with dengue virus is usually acute, the example of wnv shows that this is not necessarily a barrier to transmission by transfusion. however, given the very high incidence of dengue, it is perhaps surprising that more cases of transfusion-transmitted disease have not been reported. in fact, there has been one well-documented case reported in hong kong, plus a potential case in a bone marrow transplant recipient in puerto rico. it seems likely that the infectious viraemic period must be very short, and/or that cases are not readily recognized as transfusion-transmitted in an epidemic environment. a complication of epidemic dengue for transfusionists is the fact that, because there are four circulating strains of the virus, patients may suffer a second infection. in such cases, an even more serious disease, dengue haemorrhagic fever may result [26] . this disease state often requires platelet transfusions to correct bleeding, thus further stressing transfusion services. investigations are currently under way to determine the extent of dengue viraemia among blood donors in epidemic areas, such as brazil and puerto rico. this information will help to establish the potential risk of transmission and indicate the extent to which a testing strategy would be effective. there is considerable current concern about the possibility of an influenza pandemic. this concern is based upon the historical fact that there have been periodic pandemics associated with the circulation of new strains of the virus in humans and the current outbreak of the h5n1 strain of avian influenza, which causes high mortality when it does infect humans [27] . in addition, other strains of avian influenza viruses are also circulating. while there is no evidence that h5n1 will (or will not) result in a human pandemic, its presence has triggered significant levels of pandemic preparedness. as with the sars virus, there is evidence that h5n1 will result in viraemia during symptomatic infection, but it is unknown whether there is a presymptomatic or asymptomatic viraemia. thus, it is unclear whether there will be a risk to blood safety during an influenza pandemic. consequently, it is unclear whether donors would need to be deferred after disease, or after contact with infected individuals. there do not appear to be any documented cases of transfusion transmission of influenza during normal periodic outbreaks, but it is unclear whether such a transmission would actually be recognized. it is also unclear how an intravenously inoculated infection would be manifested. what is clear is that an influenza pandemic would have a profound effect upon the ability of the medical system to provide an adequate supply of blood in the face of widespread sickness among donors and blood centre staff. additionally, it is not entirely clear how a pandemic would impact the needs for blood. hepatitis e virus (hev) causes an epidemic form of hepatitis that is self-limited [28] . the disease is somewhat similar to hepatitis a, although it is much more severe in pregnancy. transmission is by the faecal-oral route and is most often water-borne. the virus is related to the calicivirus group and, as such, is a non-enveloped virus that has an rna genome. although there is some evidence (based largely on seroprevalence studies) that hev is present in the usa, it is found predominantly in tropical countries. indeed, most cases identified in the usa appear to have resulted from infections that occurred in countries where hev is endemic. hev infection is self-limited and acute and it has generally been thought that there is little risk of transmission by transfusion. until recently, no cases of transfusion-associated hev infection of disease have been reported [29] . however, a number of recent cases of such transmission have been reported from non-endemic areas, emphasizing that acute infections are also transmissible by transfusion, as long as there is an asymptomatic viraemic phase [30, 31] . this has, of course, been abundantly illustrated by the example of west nile virus in the usa [1] . there has been a continuing search for additional hepatitis viruses and, over the past few years, two different viruses or virus groups have been identified in this context. the first of such putative hepatitis viruses was identified by two separate groups in the late 1990s. in one case, scientists at abbott laboratories looked for genomic sequences related to those of an existing isolate known as the gb virus (gbv), which had previously been associated with hepatitis in a physician. three viruses were identified, one of which (termed gbv-c) was found among a number of human sources [32] . working in parallel, but using a different approach, scientists at gene-laboratories isolated viral rna sequences and characterized a virus they termed hepatitis g virus (hgv) [33] . it is generally accepted that these two isolates were, in fact, representatives of essentially the same virus group, which is now known as hgv. hgv, like hcv, appears to be closely related to the flavivirus group. it is found among a relatively high proportion of the normal population, as exemplified by blood donors. its presence has been demonstrated both by seroprevalence studies, in which the frequency of antibodies is 3% to 15%, and more interestingly by detection of viral rna in the plasma of 1% to 3% of normal subjects [34] . perhaps not surprisingly, the virus is readily transmissible by transfusion and is found at high prevalence among individuals who have undergone multiple transfusions. however, it has not proved possible to demonstrate that infection with hgv is associated with hepatitis or even with signs of mild liver disease, such as elevated alt levels. indeed, hgv appears to be a virus that is currently in search of a disease. the term 'hepatitis' in its name may be a misnomer, attributable only to the fact that the virus was found in association with hepatitis in the first place. it is also important to recognise that the worldwide distribution of hgv clearly shows that it is not a new virus but, rather, one that has coexisted with humans for many centuries. there have, however, been some intriguing observations that clearly suggest that hgv/gbv-c may have an impact on the course of hiv disease. for example, studies have shown lower mortality among co-infected individuals relative to those with hiv only [35, 36] . the mechanism for this effect is unclear, but may be due to the effects of infection on the levels of a number of chemokines [37] . curiously, another two viruses, ttv and sen-v, were separately identified among individuals with hepatitis and were also shown to be poorly, if at all, associated with hepatitis. these viruses were also found to cause prolonged viraemia and, in some cases, turned out to be present in up to 90% of the population. like hgv, they were readily transmitted by transfusion. both viruses were thought to be representatives of the circovirus group: small, non-enveloped viruses with a circular dna genome. this group of viruses had not previously been described among humans. workers in japan used representational difference analysis to isolate dna sequences from three patients with unexplained post-transfusion hepatitis. the sequence was established as viral, and the virus was named ttv, reflecting the initials of the patient from whom it was isolated [38] . a considerable amount of research has revealed some of the key features of this agent [39] . it is a small virus with a covalently closed, circular dna genome of around 3800 bases. the virus is thought to be non-enveloped and is most closely related to the circoviruses, which are responsible for a number of diseases in plants and a handful of mammalian or avian disease states. the classification of ttv is currently incomplete, and proposals have been made to place it in at least two genera. what has become apparent is that ttv is a member of an extremely diverse group of viruses, as demonstrated by considerable variation in genomic sequence. epidemiologic studies have confirmed that ttv is a widely distributed virus and have clearly established that it is transmissible by transfusion. interestingly, it also appears to be transmitted by the vertical, faecal-oral, and, perhaps, other routes. a key issue is the clinical significance of this group of viruses. although the original source of the virus and its apparent association with alt elevations implied that ttv was indeed a hepatitis virus, this identification no longer seems tenable. indeed, there are far more infections without alt elevations than with such evidence of liver disease. even in clear, transfusion-associated transmission of ttv, the recipients did not manifest alt elevations in any pattern that could be associated with the infection. thus, at this stage, there is little evidence that this virus expresses any pathogenic potential. however, it is certainly too early to conclude that this entire group of viruses is without any clinical significance. after the recognition of ttv, the search for hepatitis viruses continued, and primi and colleagues [39, 40] used degenerate primers derived from ttv to probe samples from selected patients. an isolate was identified and named sen-v (after the initials of the source patient, an hiv-infected injection drug user). eventually, at least eight different strains were isolated, termed a through h. the sen group has been shown to be one branch of the ttv group, seeming to share its epidemiologic characteristics. although two of the strains (d and h) have been associated with evidence of transfusion-associated hepatitis, a causal relationship has not been established [40, 41] . over the past few years, there have been a number of unexpected cases of transmission of infectious agents through organ transplantation. in some cases, such transmissions could imply that the agents may be transmissible by blood, which is certainly true of trypanosoma cruzi , a protozoan parasite that has been involved in at least three clusters of transplantassociated transmission. two viruses have been similarly involved, although it must be acknowledged that neither are currently considered to be emerging pathogens. there have been two well-documented cases of transmission of rabies by organ transplantation [42, 43] , plus one cluster of transmissions of lymphocytic choriomeningitis virus (lcmv) [44] . rabies virus infection is not thought to result in viraemia and it was considered likely that the transmission resulted from infection of, and via the sympathetic innervation of the transplanted organs. on the other hand, lcmv may generate high levels of viraemia in its normal rodent hosts and viraemia was likely in the organ donor, who was shown to have acquired the infection from a pet hamster. there have, however, been no reports of transfusion transmission of either rabies virus or lcmv. at this time, no specific interventions are recommended for blood donors who may have been exposed to either infection. concern has been expressed about the potential for transmission of simian foamy virus (sfv), a retrovirus. it has been found to infect a small number of animal handlers and it has been theorized that such individuals might be able to transmit the virus through transfusion of their blood. however, foamy viruses seem to be apathogenic, both in their natural hosts and in humans. the underlying concern is that viruses that jump species may turn out to be highly pathogenic in the new host. additionally, as discussed above, many retroviruses are prone to frequent mutation, so that pathogenic strains could emerge. the issue has been discussed by regulatory authorities in the usa, and to date, not action has been taken. however, in canada, animal handlers who have frequent contact with primates are deferred. one report details look-back to recipients of blood from an animal handler infected with sfv, but no recipient tested had any evidence of infection with the virus [45] . at the time of writing, there has been an unexpected outbreak of mumps in the midwestern states of the usa and previously in the uk. although there have been no recorded cases of transmission of mumps virus by transfusion, it is known that there is a viraemic phase and modest precautions to reduce the theoretical risk of transmission have been recommended by the aabb in the usa. in the developed world, the major transfusion-transmitted agents (with the exception of syphilis) have traditionally been viral. interestingly, the general expectation at the end of the 20th century was that the next threat to blood safety would turn out to be a persistent, sexually transmitted virus. however, this was not the case. the unexpected emergence of wnv and its transmissibility by transfusion has taught us to broaden our thinking around the concept of transfusionassociated infection. nevertheless there are relatively few emerging viral infections that have shown any evidence of transmission by this route. there are certainly numerous viruses that have the potential for such transmission and continued vigilance is necessary. there are, however, no clear patterns that will define appropriate target patient groups for surveillance and we will have to continue to rely upon haemovigilance systems and astute clinicians to identify cases of emerging viral infections among blood recipients. careful and appropriate use of the precautionary principle should also help to maintain the safety of the blood supply. transmission of west nile virus through blood transfusion in the united states in 2002 molecular virology of hepatitis b virus significance of hiv-1 genetic subtypes hepatitis c virus 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novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology molecular properties, biology, and clinical implications of tt virus, a recently identified widespread infectious agent of humans sen virus infection and its relationship to transfusion-associated hepatitis genomic and molecular evolutionary analysis of a newly identified infectious agent (sen virus) and its relationship to the tt virus family investigation of rabies infections in organ donor and transplant recipients -alabama transmission of rabies virus from an organ donor to four transplant recipients update. interim guidance for minimizing risk for human lymphocytic choriomeningitis virus infection associated with pet rodents simian foamy virus infection in a blood donor key: cord-288238-36hiiw91 authors: keshavarz, mohsen; solaymani-mohammadi, farid; namdari, haideh; arjeini, yaser; mousavi, mohammad javad; rezaei, farhad title: metabolic host response and therapeutic approaches to influenza infection date: 2020-03-05 journal: cell mol biol lett doi: 10.1186/s11658-020-00211-2 sha: doc_id: 288238 cord_uid: 36hiiw91 based on available metabolomic studies, influenza infection affects a variety of cellular metabolic pathways to ensure an optimal environment for its replication and production of viral particles. following infection, glucose uptake and aerobic glycolysis increase in infected cells continually, which results in higher glucose consumption. the pentose phosphate shunt, as another glucose-consuming pathway, is enhanced by influenza infection to help produce more nucleotides, especially atp. regarding lipid species, following infection, levels of triglycerides, phospholipids, and several lipid derivatives undergo perturbations, some of which are associated with inflammatory responses. also, mitochondrial fatty acid β-oxidation decreases significantly simultaneously with an increase in biosynthesis of fatty acids and membrane lipids. moreover, essential amino acids are demonstrated to decline in infected tissues due to the production of large amounts of viral and cellular proteins. immune responses against influenza infection, on the other hand, could significantly affect metabolic pathways. mainly, interferon (ifn) production following viral infection affects cell function via alteration in amino acid synthesis, membrane composition, and lipid metabolism. understanding metabolic alterations required for influenza virus replication has revealed novel therapeutic methods based on targeted inhibition of these cellular metabolic pathways. influenza virus infection (ivi) is one of the most common infectious agents, capable of infecting a variety of avian and mammalian species. the virus is responsible for seasonal epidemics, leading to 3-5 million severe infections and 250,000-500,000 deaths annually [1, 2] . despite the annual vaccination program, the high mortality rate caused by influenza infection and its various complications, including chronic lung disease, cardiac disease, asthma, and metabolic disorders, is yet to be adequately addressed [3] [4] [5] . in 1956, eagle et al. first indicated that the addition of glucose to hela cell medium could promote the generation of poliovirus progeny [6] . results of a published study showed that the replication of the influenza virus depends on host cellular metabolism such that metabolites including nucleic acids, proteins, glycoproteins, and lipids are crucially required for the life cycle of the influenza virus [7] . recent research on a mouse model showed that influenza infection could affect more than 100 metabolite markers in serum, lung, and bronchoalveolar lavage fluid [8] . acquiring the required materials from the host cell to self-replicate, the virus can disrupt biochemical processes such as glycolysis, fatty acid (fa) synthesis, and glutamine pathways [9] . it is of particular importance for scientists to broaden their horizon on the metabolic changes during influenza infection, which in turn paves the way for preventing life-threatening consequences. influenza infection actively provokes the pro-oxidant condition in the host cell to facilitate viral proliferation and pathogenesis. increased expression of influenza m2 protein can activate protein kinase c and increase reactive oxygen species (ros) production [10] . on the other hand, pb1-f2 decreases superoxide anion dismutase 1 (sod1) expression and consequently disrupts the ros scavenging process [11] . in people with influenza infection, increased levels of dna, lipid, and protein oxidation products are found in plasma and urine [12] [13] [14] . also, increased levels of ros and inducible nitric oxide synthase (inos) have been observed as markers of oxidative stress in the lungs of people who died due to pandemic influenza infection [15] . ros-producing enzymes induced by influenza infection mainly include nadph oxidase (nox) and xanthine oxidase, upregulation of which causes the impaired defensive function of antioxidants [16] . an increase in ros production, along with impaired antioxidant function, ultimately leads to a profound change in redox homeostasis of the cell [16] [17] [18] [19] . nox2 is a phagocytic enzyme that is involved in the production of ros induced by influenza virus [19] [20] [21] [22] , and impaired nox2 expression results in a lack of increased rns and ros production following influenza infection [20] . xanthine oxidase is also an ros-producing enzyme that is induced by influenza infection [23, 24] , and its inhibition can hinder ros increase in the cell. on the other hand, increased expression of sod1 reduces influenza virus titers within the cell [25] . it is also reported that influenza infection significantly increases ros production by inducing nox4, and the proliferation of this virus in lung epithelial cells is dependent on redox-sensitive pathways activated by nox4-derived ros [16] . glutathione (gsh) is a vital antioxidant in the cell, and its cellular content is inversely related to influenza virus replication in the cell [26, 27] . it is indicated that higher levels of gsh, antioxidant enzymes such as glutathione peroxidase, and the anti-apoptotic protein bcl-2 in the lungs of female mice result in superior resistance of these mice to influenza infection. in contrast, male mice are more susceptible to this infection due to higher expression of nox4. this difference is due primarily to the higher ability of female mice to maintain cellular redox homeostasis [28] . amatore et al. also showed that an increase in gsh content in organs by affecting gsh-dependent antiviral pathways strengthens the immune system, in particular th1 cell response, and decreases viral replication [29] . however, gsh depletion results in a deviation of the response towards th2 cells [30] . furthermore, oxidative stress following infection can induce the transcription factor nf-kb, which subsequently leads to increased levels of inflammatory cytokines, including interleukin (il)-1β, il-6, ifn, and tnf [31, 32] . ifns are one of these cytokines that trigger and affect t cell metabolism via mediating glucose uptake, glycolysis, and lipid synthesis. ifn can also exert its function on metabolic changes by producing several mediators including indoleamine-2,3-dioxygenase (ido) and nitric oxide (no), both of which appear to have either an inducible or an inhibitory role in viral replication [33] . since tryptophan is critical for t cell proliferation, depletion of this amino acid by ido suppresses the immune system through the stimulation of t regulatory cells. no, on the other hand, inhibits viral replication via changes in the structure of viral proteins [34] . in this review, we first discuss the metabolic abnormalities during influenza infection and then shed light on the role of immunometabolites that regulate cellular metabolism. the following sections summarize recent evidence about the novel therapeutic approaches that target metabolic pathways in influenza infection. viruses take advantage of various cellular mechanisms to replicate efficiently. vital metabolic pathways of host cells are one of the most widely used mechanisms targeted by viruses, resulting in considerable changes. in this context, studies have revealed that various human viruses, such as cytomegalovirus [35] [36] [37] , rubella [38, 39] , dengue [40] , mumps [41] , poliovirus [42, 43] and reovirus [44] can strongly affect host cell glycolysis, lipid metabolism, and glutaminolysis. furthermore, a review by sanchez and lagunoff delineated the activation of these metabolic processes by several viruses [45] . as will be discussed in this section, influenza as a highly pathogenic human virus interferes tremendously with the host metabolic cycles and thereby forces them to produce viral particles more efficiently. the metabolism and concentration of glucose in the cell play a cardinal role in the homeostasis of cellular metabolic procedures [46] . shortly after the onset of ivi, the rate of glucose uptake by infected cells increases continually, and the subsequently enhanced glycolysis results in higher glucose consumption, production of viral particles [47, 48] , and extracellular concentration of lactate. the relationship between glycolysis and ivi has shown that influenza infection at a higher multiplicity of infection (moi) raises the glycolytic activity of the cells [49] . in a study by kohio and adamson, a dose-specific increase in influenza infection was associated with higher glucose levels, whereas the treatment of cells with glycolysis inhibitors remarkably suppressed the viral replication. however, the viral infection could be retriggered by adding atp to the cell environment. this study revealed that enhancing vacuolar-type atpase (a proton pump essential for influenza uncoating) via increasing glucose metabolism and, as a result, higher available atp resources, augments the virus infection [50] . the observations, as mentioned above, reveal a significant increase in atp and glucose consumption within cells following influenza infection and also highlight the dependence of the influenza virus on the glycolysis pathway for energy production. the viral replication has the highest use of atp during influenza infection, releasing large quantities of energy in the form of heat. this process can increase the temperature of infected cells by 4-5°c. as viral proliferation increases, the cellular atp level drops sharply, resulting in reduced potential and stability of the mitochondrial membrane [51] . based on available results, patients with metabolic disorders can develop more severe influenza infection compared to healthy hosts. there have been several studies showing that diabetes can increase the risk of influenza infection, the severity of the disease, and the fatal consequences of this infection [52] [53] [54] [55] . about 90% of patients with type 2 diabetes are overweight, and obesity is a significant risk factor for severe influenza infection [56] . this reinforcing effect of diabetes on influenza may be due to the inhibitory effect of hyperglycemia on the immune system [57] [58] [59] . it has been shown that this hyperglycemia-associated immunosuppression and susceptibility to influenza infection can be alleviated by insulin administration and diabetes control [60] . on the other hand, the pentose phosphate pathway (ppp), as another glucose-consuming pathway reported to be enhanced by ivi [61] , contributes to a higher yield of nucleotides and atp for viral replication [62] . significant up-regulation of the ppp key enzymes in influenza-infected cells, including glucose 6-phosphate dehydrogenase (g6pd) and 6-phosphogluconate dehydrogenase (6pgd), was reported by janke et al. [63] . g6pd enzyme is also responsible for the generation of nadph [64] , a critical component of fatty acid biosynthesis. the level of g6pd activity specifies the ability of the cell to clear the accumulated ros. cells with an average g6pd level can retain the appropriate gsh/gssg ratio and keep the ros production at a tolerably low level, indicating that g6pd activity has an inverse correlation with cellular ros level [65] . disruption of redox balance has been shown to contribute to replication and virulence of several viruses [66] [67] [68] , and g6pd deficiency can cause this disruption. despite the results reported by janke et al., g6pd activity seems to have an inverse relation with some other respiratory viral infections. for example, in an in vitro study, after infection with human coronavirus (hcov) 229e, the production of viral particles in g6pd-deficient or g6pd-knockdown cells was higher than in healthy cells, and this was correlated with increased oxidant production [67] . molecular mechanisms through which the virus can control the metabolic pathways have been thoroughly identified. smallwood et al. have shown that an increase in glucose uptake, glycolysis, and glutaminolysis following influenza infection may be related to the loss of pi3k/akt/mtor pathway homeostasis and subsequent increase in c-myc expression in the infected cells [9] . regarding the available results, the mechanistic target of rapamycin complex 1 (mtorc1) and mtorc2 signaling can be activated by a variety of influenza virus proteins. the viral hemagglutinin (ha) protein, along with virus replication, can upregulate pdpk1-mediated phosphorylation and activate akt, which is required for induction of the mtorc1 signaling pathway by the influenza virus. on the other hand, influenza m2 protein is capable of down-regulation of the mtorc1 inhibitor redd1, thereby enhancing the mtorc1 activation [69] . mtorc1 signaling, in turn, promotes c-myc expression at the translational level [70] . additionally, the ns1 protein can effectively promote the activity of mtorc2, which, in turn, upregulates c-myc through foxo inhibition [71] . moreover, akt-dependent inactivation of foxos can increase glycolysis [72, 73] by removing the suppressive force of c-myc [74] [75] [76] . myc enhances glycolysis by upregulating expression of the glucose transporter glut1, glycolytic genes, and lactate dehydrogenase (ldh), as the converter of pyruvate to lactate [77, 78] . mtorc1 also mediates upregulation of hypoxia-inducible factor-1α (hif-1α), a factor that increases the expression of various genes [79] , including several glycolytic enzymes, glucose transporters, and ldh [80] [81] [82] . akt is able to promote the expression and membrane localization of glut1 as well as the function of phosphofructokinase [83, 84] . furthermore, akt is demonstrated to activate srebp in an mtorc1-dependent manner [85] and to upregulate srebp by enhancing the stability of its processed form [86] . srebp1 is shown to be required for the mtorc1induced increase in the expression of g6pd, which is the rate-limiting enzyme of the ppp oxidative branch [79] . in addition to the above-mentioned metabolic pathways, the influenza virus exhibits disruptive effects on some other metabolic processes, which gives rise to metabolic disorders and atp crisis. previous studies have found that the influenza infection increases the cellular synthesis of fatty acids [87] , with some of their derivatives, including eicosanoids [88] , and these molecules are natural endogenous ligands and stimulators of peroxisome proliferation-activated receptors (ppars) [88] [89] [90] . ppars are a group of nuclear receptor proteins that act as transcription factors and regulate the expression of different genes [91] involved in cellular differentiation and metabolism of carbohydrates, lipids, and proteins [92] . furthermore, all types of ppars discovered so far are able to suppress the activity of the pyruvate dehydrogenase (pdh) enzyme (known as a catalyzer of the oxidative decarboxylation of pyruvate leading to acetyl-coa production) in various organs through the upregulation of pyruvate dehydrogenase kinase (pdk)-4 [93] . in this respect, extremely low pdh enzyme activity has been found after influenza infection in vitro. enhanced pdk4-mediated inhibition of pdh has been found in the lung tissue of influenza-infected mice. this enzyme inhibition contributes to an erroneous process, which causes significant disruption of glucose oxidation, cellular respiration, and lipid metabolism ( fig. 1) [7, 94] . following infection, levels of phospholipids and several lipid derivatives undergo perturbations. several lines of evidence have shown that in obese mice due to abnormalities in the fig. 1 metabolic changes caused by influenza infection and related mechanisms. several anabolic and catabolic processes can be affected: higher glucose uptake and metabolism in glycolysis and pentose phosphate pathways, higher nucleotide catabolism, increase in biosynthesis of fatty acids including arachidonic acid, the precursor of proinflammatory lipids, and also enhanced glutaminolysis and protein synthesis. activation of mtorc1&2 signaling and downstream factors by influenza infection may have an essential role in the upregulation of these metabolic processes. in addition, high atp consumption and reduced β-oxidation, as well as glucose oxidation by influenza infection, contribute to the atp crisis and hence influenza-related multi-organ failure [96] . this influenza-mediated elevation of aa, consistent with inflammation, has also been reported in the same study [97] . the accumulation of the above-mentioned proinflammatory lipids in the cell leads to the promoted synthesis of eicosanoids and inflammatory mediators, thus exacerbating post-infection necrosis, inflammation, and tissue damage in the lungs [96] . in a prospective cohort study on influenza-infected subjects, lipid inflammatory mediators in serum samples of patients were mainly aa-derived oxylipins, including txb2, 15-deoxy-12,14-pgd2, 20-hete, 5,6-dhet, 5-oxoete, lte4, and 12-hpete. although all of these metabolites were shown to be elevated shortly after the infection, 5,6-dhet and 5-oxoete levels remained considerably high up to 4 weeks postinfection, indicating a constant pulmonary inflammation [98] . the pulmonary surfactant system, which is involved in suppressing influenza infection in the respiratory tracts [99] , can be disrupted due to significant influenza-induced changes in the abundance of different types of pc and pe species as the major components of surfactants [96, 100] . tanner et al. proposed a principal correlation between influenza replication and choline lipids metabolism. they found an ivi-mediated reduction in ester-linked pc species as well as an increased level of sphingomyelin (sm) [101] , probably connected with expending cellular choline stores for sm synthesis. this led to an increase in sm species within the infected cell. sm and short-chain fatty acid-containing ether-linked pc (epc) species were found in higher amounts in both infected cells and virions and therefore appeared to be involved in viral morphogenesis. on the other hand, long-chain fatty acidcontaining epc was increased in infected cells while having low levels in the structure of the virion, highlighting the role of this phospholipid in replication of the virus [101] . evidently, higher production of these complex lipids in the cell will require increased biosynthesis of fatty acids. in this regard, the results of a study revealed that influenza infection could induce fatty acid biosynthesis and cholesterol metabolism in human lung basal epithelial tumor cells [87] . since srebps are thoroughly identified as stimulators of expression of many genes involved in lipid and sterol biosynthesis, including fatty acid synthase [102] [103] [104] , their upregulation by the influenza virus (through induction of mtorc1 signaling, as discussed earlier) may logically explain the increased rate of lipogenesis [105] . a coincidence between increased fatty acid synthesis and a decline in fatty acid β-oxidation has been found during influenza infection, which is attributed to a variety of mechanisms directly or indirectly related to viral replication. for instance, the sharp increase of proinflammatory cytokines during influenza infection [106] causes decreased hepatic fatty acid β-oxidation both in vitro and in vivo [107, 108] , most likely through excessive nitric oxide and other related free radicals [109] . in addition, increased temperature of cells during infection (which could be the result of virus replication and fever) causes heat stress which in turn can considerably downregulate carnitine palmitoyltransferase ii (cpt ii) activity and reduce the β-oxidation and atp levels in fibroblasts of influenza-associated encephalopathy patients and healthy volunteers [110] . a study on influenza-infected mice demonstrated a significant depression in long hepatic chain fatty acid β-oxidation at both the mrna and protein level, as several β-oxidation essential enzymes were reduced by > 50% [97] . a significant decrease in mitochondrial fatty acid β-oxidation simultaneously with increased biosynthesis of fatty acids and membrane lipids may reflect the fact that the virus stores structural lipids to produce more infectious particles. in addition to impaired metabolism in mitochondria, influenza infection induces severe peroxisomal lipid metabolism disorders, which can be inferred from abnormal levels of several specific long-chain fatty acids [101] (fig. 1) . the aforementioned metabolic processes are not the only pathways affected by influenza virus infection. this virus has the ability to induce higher consumption rates of glutamine during glutaminolysis, which can be attributed to transient c-myc overexpression [9] . myc acts to regulate glutamine uptake and its utilization in the cell [111] . it has been demonstrated that catalytic activity of glutaminase, as the key enzyme in glutaminolysis, greatly increases following the infection [63] . moreover, essential amino acids, especially tryptophan, are other materials whose quantities have been shown to decline in infected tissues [112] . mtorc1 can up-regulate protein synthesis through several downstream factors [113] . thus, induction of mtorc1 signaling by the influenza virus leads to higher usage of essential amino acid storages for concurrent production of large amounts of viral and cellular proteins. infection of influenza virus can also alter the cellular level and metabolism of purines and pyrimidines [8, 98, 100] , and is associated with both increased activities of nucleotide catabolism core enzymes including adenosine deaminase (ada) and xanthine oxidase (xo) and elevated levels of inosine, hypoxanthine, xanthine, and uric acid in serum and bronchoalveolar lavage fluid. enhanced catabolic degradation of nucleotides and their metabolites can facilitate the production of superoxide and contribute to the pathogenesis of influenza infection [23] . interferons are well-known cytokines with a powerful capability of altering the cellular functions following viral infections. these alterations affect protein synthesis, composition of the membrane, cellular proliferation, and nutritional status [34] . interferon stimulated genes (isgs) are the effector components whose transcription could be induced by type i ifns and ifnγ [114, 115] . studies have recently underscored the general effect of ifns on the energy metabolism of cells, mostly by promoting glycolysis. for instance, ifnβ has been shown to induce the glucose uptake of embryonic fibroblasts in a pi3/akt-dependent manner, thereby increasing atp production [116] . it has also been demonstrated that type i ifn can stimulate oxygen consumption in a range of cells, including conventional dendritic cells (dcs), keratinocytes, and memory t cells [117] . indeed, the high yield of atp and mitochondrial fitness guarantee the host cell's need for energy in plasmacytoid dcs (pdcs) and non-hematopoietic cells following challenges with viral pathogens [118] . these studies emphasized the mediatory effect of type i ifn on glycolysis induction via ifnar1, tyk2, and stat1. it has also been shown that influenza infection stimulates pdcs to enhance their glycolysis and develop a warburg-like remodeling of energy metabolism. this enhanced glycolysis leads to higher ifn production and, consequently, more potent antiviral activity [118] . ifnγ induces metabolic reprogramming of m1 macrophages as a rapid increase in aerobic glycolysis, followed by a reduction in oxidative phosphorylation. this metabolic reprogramming maintains cell viability and the inflammatory response while reducing dependence on mitochondrial oxidative metabolism. excessive production of pro-inflammatory cytokines and chemokines in human monocytes/macrophages can be blocked by inhibition of aerobic glycolysis [119] . also, activation of macrophages by ifnγ induces expression of the atp-citrate lyase enzyme (acly), and blockage of acly activity reduces the production of ros and nitric oxide [120] . there is a strong consensus that influenza replication is crucially dependent on fatty acids [97] , which makes it a fascinating target for therapeutic modalities [45] . thus, the ability of ifn to channel the fas from biosynthesis to catabolism via fatty acid oxidation (fao) is currently known as a promising antiviral strategy in pdcs [117] , which requires further research for more elucidation. several lines of current evidence have revealed the antiviral activity of type i ifn to be exerted through hampering glucosederived cholesterol and fatty acid synthesis [121, 122] . sterol regulatory binding protein 2 (srebp2), along with srebp1, is known as the leading transcription factor which orchestrates the biosynthesis pathway of sterol, whose inhibited transcription and expression can be strongly mediated by ifns via ifnar1 [123] (fig. 2) . [124] . this recognition can lead to the induction of an inflammatory response that, in turn, controls the replication and spread of the virus [31] . h5n1, h7n7, and h7n9 were correlated with increased transcription of the cytokine response in mice. severe infection with h7n9, h7n7, h5n1, or 1918 virus can lead to upregulation of inflammatory cytokine genes along with downregulation of lipid metabolism and coagulation genes [125] . this uncontrolled proinflammatory response accompanied by an inadequate anti-inflammatory response is referred to as the cytokine storm [31] . monocytes/macrophages, neutrophils, and lung epithelial cells have useful roles in the cytokine storm developed by influenza infection [126] . severe cytokine storm, with greater levels of interferons and tumor necrosis factors, has been recognized in patients hospitalized due to influenza infection [127] . such influenza-induced cytokine storms, together with viral virulence, can develop severe lung injury in patients [128, 129] . it is believed that the level of the cytokine storm is directly associated with the severity of the disease caused by influenza infection [130, 131] . some specific polymorphisms in immune system genes have determinative roles in the outcome of influenza infection. our previous studies have shown a relationship between cytokine gene polymorphisms and severity of the influenza disease. several cytokines were evaluated after influenza a/h3n2 virus infection, among which il-17 rs2275913 gg and ag, gg and gt of il-10 (rs1800872) and il-28 (rs8099917) genotype tt polymorphisms were associated with increased risk of influenza infection. in contrast, il-1β (rs16944) (gg) and il-28 (rs8099917) gg and tg genotypes were associated with reduced risk of infection [132] . in another study, an association between il-1β rs16944 and il-17 rs2275913 genotypes and severe influenza disease was found while il-10 rs1800872 and il-28 rs8099917 polymorphisms were not associated with influenza disease. also, lacking an a allele in il-17 rs2275913 could increase the risk of influenza a (h1n1) infection [2] . such polymorphisms in immune system genes may be associated with some metabolic changes and, in turn, may reinforce the metabolic disorders following influenza infection. however, additional studies are needed in this field to confirm or reject this opinion. ifns are known to be capable of depletion of polyamines to limit virus replication. polyamines are small ornithine-derived polycationic molecules which encompass three molecules: putrescine, spermidine, and spermine. spermidine and spermine depletion is one of the compelling mechanisms through which ifns produce their antiviral effects on the replication of rna viruses. mechanistically speaking, polyamines appear to play a pivotal role in the processes of rna transcription and protein translation of viruses, making them a promising target to combat viral infections [133] . l-tryptophan is one of the nine essential amino acids with a remarkable role in immunosuppression and tolerance and is also essential in protein, kynurenine, and serotonin synthesis [122] . ido is an intracellular enzyme that induces production of kynurenine from l-tryptophan, thereby acting to deplete tryptophan and modulate the immune system following viral infections [134] . having two ifn-stimulated response elements and three ifnγ-activated sites in the promoter of ido, ifnγ acts as the most powerful inducer of ido1 expression [135] . ido has also been shown to be expressed during influenza infection [136] . dendritic cells, macrophages, and epithelial cells can express ido [137, 138] , and since the primary target for replication of influenza is primarily found to be respiratory epithelial cells, understanding the role of ido during influenza infection is of particular importance. there exists a coincidence of peak ido1 and ifn-k expression during influenza infection. also, mouse lung airways considerably express ifn type i and iii following infection with influenza [139] . these findings emphasize that there is upregulated expression and enhanced function of ido during influenza infection, which is found to be induced by ifn-i. moreover, ifn-i is thought to signal the adjacent cells via ifn-ir and stimulate them to produce ido [140] . nonetheless, the ifn-mediated ido induction during influenza infection generally has undesirable consequences and establishes immune tolerance [136] . indeed, an inhibitory effect of tryptophan depletion on t cell responses has been confirmed. also, ido induces kynurenine derivation from tryptophan, leading to stimulation of regulatory t cells [141] . nowadays, ido is hypothesized to be part of the "metabolic, immune regulation," which plays a protective role in immune responses and inhibits the overreaction of these responses against influenza infection. a pleiotropic role has been attributed to ido during infections, which gives rise to the opposing outcomes (fig. 3) [142] . research on the role of ido in influenza infection has been mainly focused on the murine models of influenza infection, emphasizing the increased ido activity and its maximum expression correlated with increased lymphocyte numbers in the respiratory tract [143] . nitric oxide (no) is a gaseous free radical with accessible vasodilatory and microbicidal functions [144] . however, the antiviral effect of no has also been documented, leading to reduced viral load and more efficient clearance of infection [145] . nitrosylation of viral molecules has been offered as an antiviral mechanism employed by no [146] . also, no synthase-mediated generation of no leads to the depletion of l-arginine, thereby reducing the level of polyamines. thus, ifn-induced nos2 represents antiviral activities, and this, in turn, may exhibit another mechanism through which ifns hinder viral infections such as influenza [34] . despite existing data regarding the antiviral activity of no, many studies have considered no as a double-edged sword with both pathogenic and viricidal effects. the role of no in the pathogenesis of pneumonia caused by influenza virus infection has been described in mice. the ifnγ response induces greatly increased levels of inos in the lungs of infected mice, leading to the production of a significant amount of no and peroxynitrite species, which are among the most important pathogenic factors in influenza virus-induced pneumonia in mice [147] . uetani et al. also observed overexpression of the inos gene in human airway epithelial cells induced by influenza a virus infection [148] . in addition, no produced by phagocytic cells has antiviral activity that is simultaneous with nonspecific damage of host cells and viral pathogenesis [149] . in a survey by nin et al. on pandemic a/h1n1 influenza infection, all cases showed increased levels of inos protein, tyrosine nitration, and oxygen free radicals, indicating the production of peroxynitrite. their results revealed the involvement of oxidative and nitrative stress in the pathogenesis of h1n1 influenza virus-induced acute respiratory distress syndrome (ards) [15] . influenza-induced cytokines such as ifnγ stimulate no release from human airway epithelial cells [150] [151] [152] . as mentioned previously, the influenza infection induces upregulation of hif-1α. interestingly hif-1α-knockout macrophages show decreased expression of inos after ifnγ stimulation [153] , indicating the possible involvement of hif-1α in influenza pathogenesis. it has been shown that infection of h5n1 and 1918 viruses induces higher levels of no in mice compared to the seasonal h1n1 virus and, as a result, they develop more intense pathogenic outcomes, while mice with inos deficiency showed reduced morbidity, mortality, and cytokine production in the lungs following h5n1 and 1918 virus infection. also, systemic administration of nos inhibitor could postpone weight loss and death among 1918 virus-infected mice [154] . in another survey, the delivery of no to influenza-infected mice could not improve the lung infection and survival of mice, indicating that no administration was not a suitable treatment strategy for influenza although this was probably due to the difficulty of determining concentrations of no that are both viricidal and safe in host airways [155] . also, it is reported that no released from s-nitroso-n-acetylpenicillamine (snap) reduces the replication of influenza virus in a dose-dependent manner. the production of no in airway epithelial cells can lead to antiviral rather than harmful effects following influenza infection provided that its production is precisely controlled [156] . since the influenza virus affects about 20% of the world population annually, preventive and therapeutic approaches require much closer attention. therapeutic drugs for influenza infection fall into three groups: 1) neuraminidase inhibitors (zanamivir, oseltamivir, laninamivir, peramivir), 2) m2 inhibitors (rimantadine and amantadine), and 3) polymerase inhibitors (favipiravir) [157, 158] . antiviral drug resistance has recently emerged as a global problem which can bring about a remarkable financial and social burden [159] . therefore, further research is urgently needed to develop novel and promising antiviral drugs. many of the metabolic pathways in influenza infections are increasingly changing, dampening of which appears to hamper the virus replication. one of the newly developed strategies aiming to hinder influenza infection is targeting metabolic pathways and restoration of hemostasis in cells ( table 1 ). the pi3k/mtor signaling pathway has been shown to play a pivotal role in a variety of cellular pathways, including proliferation and nutrient uptake, and its activation increases the glucose uptake through the up-regulation of cell surface glucose transporter [163] . bez235 alters glucose metabolism via blockage of the pi3k/mtor pathway, and some clinical trials are underway to assess this strategy in cancer therapy (smith et al., 2012). on the other hand, several lines of evidence have demonstrated that sirna targets the pi3k-akt-mtor pathway, thereby warding off influenza infection [164] . in a new study by smallwood et al., it was found that although bez235 did not interfere with the early stages of the infection, it could finally reduce the viral progeny and result in prolonged survival in mice challenged by the influenza virus. indeed, bez235 induced hemostasis in the pi3k/mtor pathway via phosphorylation of p85 and 4e-bp1 and through reconstitution of metabolic status, which was already altered by the virus [9] . it has been found that there is an elevated level of pdk4 in lung, liver, and heart during influenza infection, while the levels of atp and pdh, a key enzyme in the regulation of glucose, lipid and atp levels in human cells, are shown to be reduced [156] . furthermore, dichloroacetate (dca) is a pyruvate dehydrogenase kinase inhibitor with anti-tumor activity in a variety of carcinomas. studies have also indicated that diisopropylamine dichloroacetate (dada) could ameliorate the metabolism of hepatocytes in chronic liver disease [165] . in a study by yamane et al. attempting to evaluate the effect of dada on influenza-infected mice (pr8), oral administration of dada was found to not only restore the activity of pdh and atp in affected organs but also suppress cytokine storm and viral replication [94] . sterols are intermediate metabolites that play an essential role in a broad spectrum of biological pathways, including inflammation. research has shown that interferon production following the immune response in viral infection regulates sterol production paths. blanc et al. revealed that sterol metabolism pathway regulators such as simvastatin, zometa (zoledronic acid), and fpt inhibitor iii could effectively hinder h5n1 influenza replication and cytokine production, which makes them promising therapeutic candidates in acute patients [121, 161] . on the other hand, as mentioned earlier, srebps are transcription factors that have a critical role in the process of lipogenesis. studies have shown that these factors can play a variety of roles, such as energy supply and post-translational protein modification, as well as in the propagation of various groups of viruses such as influenza viruses. a study has shown that the am580 compound, which is a retinoid derivative, inhibits srebp-linked pathways, and it has antiviral activity against influenza a and coronavirus in vitro and in vivo [105] . concerning the fact that mitochondria and glycolysis are two sources of energy production, they play vital roles in the regulation of innate immunity responses. during the immune system response, and especially the cytokine storm, following influenza infection, atp synthesis in the mitochondria decreases, leading to weakened innate immune responses (dengbing yao). studies have revealed that traditional herbal medicines have an important role in improving influenza-like symptoms in infected patients. results of a study demonstrated that pre-treatment of infected cells with hochuekkito (a traditional japanese herbal medicine) could activate both mitochondrial and glycolytic energy metabolism and thereby intensify symptoms [160] . also, the effects of traditional chinese medicine (modified jiu wei qiang huo) on h1n1 infected mice were evaluated in another study. the results showed that this herbal medicine could ameliorate weight loss and inflammatory mediators in infected mice through the regulation of amino acid, fatty acid, and arachidonic acid pathways [162] . based on recent studies, influenza virus infection can interfere with cellular metabolic pathways either directly or indirectly via stimulation of immune system mediators. through enhancing the activity of the mtorc1 complex, the influenza virus strengthens several metabolic pathways, including glycolysis, glutaminolysis, pentose phosphate, and fatty acid synthesis, to provide more atp and structural materials for viral replication. on the other hand, β-oxidation suppression following viral infection can help to supply essential fatty acids for the synthesis of structural lipids. however, exhausting cellular atp resources due to virus replication, as well as an increase in pro-inflammatory lipid synthesis, will ultimately lead to irreversible cell damage. innate immune responses following influenza infection play a crucial role in metabolic alterations. ifn is one of these mediators that acts on several metabolites such as ido and no and thereby affects lipid and amino acid pathways. since the drug resistance in influenza infection is a global concern, research on designing novel therapeutic modalities to tackle pandemics is of particular importance. thus, a clear understanding of the metabolic alterations during influenza infection would be tremendously helpful for 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infection in vivo characterization of an indoleamine 2,3-dioxygenase-like protein found in humans and mice ido expression by dendritic cells: tolerance and tryptophan catabolism indoleamine 2,3 dioxygenase and metabolic control of immune responses induction and role of indoleamine 2,3 dioxygenase in mouse models of influenza a virus infection nitric oxide synthase in innate and adaptive immunity: an update does nitric oxide play a critical role in viral infections? role of nitric oxide in immune responses against viruses: beyond microbicidal activity pathogenesis of influenza virusinduced pneumonia: involvement of both nitric oxide and oxygen radicals central role of doublestranded rna-activated protein kinase in microbial induction of nitric oxide synthase nitric oxide-induced nitrative stress involved in microbial pathogenesis constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo concurrent production of interleukin-2, interleukin-10, and gamma interferon in the regional lymph nodes of mice with influenza pneumonia differential activation and antagonistic function of hif-α isoforms in macrophages are essential for no homeostasis inducible nitric oxide contributes to viral pathogenesis following highly pathogenic influenza virus infection in mice inhaled nitric oxide therapy fails to improve outcome in experimental severe influenza inhibition of influenza virus replication by nitric oxide emergence of multidrug-resistant influenza a(h1n1)pdm09 virus variants in an immunocompromised child treated with oseltamivir and zanamivir affinity of rimantadine enantiomers against influenza a/m2 protein revisited influenza virus drug resistance: a time-sampled population genetics perspective japanese herbal medicine, restores metabolic homeostasis between mitochondrial and glycolytic pathways impaired by influenza a virus infection modulation of sterol biosynthesis regulates viral replication and cytokine production in influenza a virus infected human alveolar epithelial cells modified jiu wei qiang huo decoction improves dysfunctional metabolomics in influenza a pneumonia-infected mice proliferation, survival and metabolism: the role of pi3k/akt/mtor signalling in pluripotency and cell fate determination inhibition of influenza a virus replication by antagonism of a pi3k-akt-mtor pathway member identified by gene-trap insertional mutagenesis superior anti-tumor efficacy of diisopropylamine dichloroacetate compared with dichloroacetate in a subcutaneous transplantation breast tumor model key: cord-300793-tuq8z6gm authors: weiss, robin a; mcmichael, anthony j title: social and environmental risk factors in the emergence of infectious diseases date: 2004 journal: nat med doi: 10.1038/nm1150 sha: doc_id: 300793 cord_uid: tuq8z6gm fifty years ago, the age-old scourge of infectious disease was receding in the developed world in response to improved public health measures, while the advent of antibiotics, better vaccines, insecticides and improved surveillance held the promise of eradicating residual problems. by the late twentieth century, however, an increase in the emergence and re-emergence of infectious diseases was evident in many parts of the world. this upturn looms as the fourth major transition in human–microbe relationships since the advent of agriculture around 10,000 years ago. about 30 new diseases have been identified, including legionnaires' disease, human immunodeficiency virus (hiv)/acquired immune deficiency syndrome (aids), hepatitis c, bovine spongiform encephalopathy (bse)/variant creutzfeldt-jakob disease (vcjd), nipah virus, several viral hemorrhagic fevers and, most recently, severe acute respiratory syndrome (sars) and avian influenza. the emergence of these diseases, and resurgence of old ones like tuberculosis and cholera, reflects various changes in human ecology: rural-to-urban migration resulting in high-density peri-urban slums; increasing long-distance mobility and trade; the social disruption of war and conflict; changes in personal behavior; and, increasingly, human-induced global changes, including widespread forest clearance and climate change. political ignorance, denial and obduracy (as with hiv/aids) further compound the risks. the use and misuse of medical technology also pose risks, such as drug-resistant microbes and contaminated equipment or biological medicines. a better understanding of the evolving social dynamics of emerging infectious diseases ought to help us to anticipate and hopefully ameliorate current and future risks. popular writing on emerging infectious diseases resounds with dire warnings about the threat of modern 'plagues' and losing the 'war against microbes.' this adversarial language obscures the fact that most of the microbial world is either neutral toward, or supportive of, human well-being and survival. indeed, we would not survive long without commensal microbes such as the beneficial strains of escherichia coli in our gut. that aside, the study of emerging infections is more than a passing fad. the recent rate of identification of such infections, the impact of the sars outbreak, the devastation caused by aids, and the ever-present threat of a new influenza pandemic indicate that we cannot control our disease destiny. nor are emerging infections unique to humans; the irish potato famine in 1845 and the english foot-and-mouth disease epidemic in 2001 underscore the consequences for human societies of disease emergence in crops and livestock. emerging infectious diseases in humans comprise the following: first, established diseases undergoing increased incidence or geographic spread, for example, tuberculosis and dengue fever; second, newly discovered infections causing known diseases, for example, hepatitis c and helicobacter pylori; and third, newly emerged diseases, for example, hiv/aids and sars. this perspective will discuss the human ecology of both the (apparently) new and re-emerging diseases. interest in infectious disease has itself recently re-emerged. in 1972, burnet and white commented, "the most likely forecast about the future of infectious disease is that it will be very dull. there may be some wholly unexpected emergence of a new and dangerous infectious disease, but nothing of the sort has marked the past fifty years" 1 . today, we may criticize the short-sightedness of our mentors' generation, yet in demographic terms they were essentially correct because the proportion of deaths from infectious disease has fallen throughout the twentieth century 2,3 (fig. 1) . humankind currently faces neither apocalyptic extinction nor even a population reduction such as occurred in europe during the black death of the fourteenth century. rather, overpopulation in relation to environmental resources remains a more pressing problem in many developing countries, where poor economic and social conditions go hand-in-hand with infectious disease. in industrializing countries during the nineteenth century, a major reduction in enteric infections was achieved by separating drinking water from sewage-an environmental change that probably saved more lives than all the twentieth century vaccines and antibiotics together. today, however, the growth of shanty towns without sanitation around the megalopolis cities of asia, africa and south america is recreating similar conditions, and in the past 40 years cholera has made a remarkable re-emergence through its longest ever (seventh) pandemic 4 . in most countries, life expectancy has risen over the past 50 years 5 (fig. 2) . the most important exception is those regions where hiv infection is rife. moreover, during the past 15 years, falling living standards in some african countries and the breakdown of public health infrastructure in ex-soviet nations has aided the re-emergence of transmissible diseases like tuberculosis 4,6 . further, severe outbreaks such as the 1918-1919 influenza a pandemic temporarily reversed the decline of deaths caused by infectious disease. the 50 million estimated deaths from that pandemic 7 represented about 2% of the global population at that time, and is twice as many as the cumulative aids mortality of the past 20 years. the next influenza pandemic may be just around the corner, and may spread even faster 8 , if access to appropriate vaccines and drug treatment is not available 9, 10 . for other newly emerging infections that make headlines, such as sars, ebola or vcjd, it is important to keep a sense of demographic proportion. placing these emerging infections on a 'richter' scale of human mortality (box 1) shows that they elicit scarcely detectable minor tremors in numbers of fatalities -despite the fear they invoke. we do not know, however, which one might leap to the top of the scale like hiv has done; indeed, it may be a completely unknown agent, as the sars coronavirus was two years ago. a major challenge is to predict which infection presages the next big quake, hopefully allowing preventive action. like any other animal or plant species, humans have been prone to infection by pathogens throughout their evolutionary history. such ancient infections by helminth and protozoan parasites, bacteria, fungi and viruses are endemic, eliciting a range of effects from a heavy burden of disease (e.g., malaria) to being essentially commensal in immunocompetent hosts (e.g., most types of herpesvirus and papilloma virus). other infections depend on an animal reservoir for their maintenance; their infection of humans may be pathogenic, but it has little part in the evolving ecology of the microbe or parasite. an estimated 61% of the 1,415 species of infectious organisms known to be pathogenic in humans are transmitted by animals 11 , for which the human represents a dead-end host. occasionally, however, a zoonotic infection adapts to human-to-human transmission and diversifies away from its animal origin. epidemic diseases are generally caused by infections that are directly transmissible between humans. hiv is a recent example of a long line of human infections initiated by a switch of host species, stretching back to the origins of measles and smallpox. free-living microbes may also find a human niche that suits their lifestyle, such as the lung for legionella pneumophila and the gut for vibrio cholerae. legionnaires' disease, first recognized in philadelphia in 1976, is the environmental equivalent of a zoonosis. it is seldom passed directly from person to person but it was human ingenuity in designing warm, aerated, humid 'artificial lungs' called air-conditioning systems that allowed the microbe to proliferate and become an opportunistic colonizer of the human lung. cholera, which was unknown beyond the ganges delta before it spread widely in asia and the middle east during the period 1815-1825, at around that time horizontally acquired a toxin gene and other factors in a genetic package that helped it to colonize the gut; the resultant diarrhea aids dispersal of the microbe 12, 13 . human society has undergone a series of major transitions that has affected our pattern of infectious disease acquisition and dissemination 4 . these transitions illustrate the interrelationship between environmental, social and behavioral influences on the emergence and subsequent spread of infectious disease. some infections were acquired when our australopithecine ancestors left their arboreal habitat to live in the savannah. this ecological change included exposure to new species of mosquito and tick as vectors for infection. after the emergence of homo sapiens, the eventual migration of neolithic hunter-gatherers out of africa 50,000 to 100,000 years ago exposed them to new infections in distant regions. the first major transition of prehistoric/early historic times gave rise to the epidemic, or 'crowd,' infections. this change must have started in the millennia following the advent of agriculture-from around 10,000 years ago-as agriculturally based society developed larger, denser populations. the domestication of livestock and the rich dividends available in human settlements to other animals (e.g., rodents, dogs and various insects) provided further opportunities for pathogens to move between species. sometimes such a pathogen (or a mutant strain thereof) would have been well suited to humans as a new host species, and, if human numbers were adequate, could therefore persist indefinitely as a human infection. thus, measles emerged about 7,000 years ago, probably from rindepest of cattle, and diverged to become an exclusively human infection when population size and density became sufficient to maintain the virus without an animal reservoir. similarly, smallpox became epidemic about 4,000 years ago, possibly evolving from camelpox, its closest phylogenetic relative. the next two transitions were primarily to do with great extensions in the spread of infectious diseases, entering distant populations as 'new infections.' thus, the second historical transition occurred in classical times as large eurasian civilizations came into commercial and military contact. they inadvertently exchanged their pools of infections, and vectors such as rats and fleas, across the mediterranean basin, the middle east, india and china. the plague of athens in 430 bce during the peloponnesian war vividly described by thucidides may represent the first report of typhus. this rickettsial infection is transmitted from rats to humans and thence among louse-ridden humans. typhus frequently accompanies human conflict and deprivation, as seen in a recent outbreak among rwandan refugees in burundi 14 . the justinian plague of 542 ce devastated the eastern mediterranean region and probably extended as far as china 15 like the black death 800 years later (and both are attributable to yersinia pestis 16 ). the third historical transition accompanied the era of worldwide exploration and colonization by europeans from circa 1500 ce onward. a contemporary account by one of hernan cortes' fellow conquistadors, bernal diaz, recalls that they might well have failed to overthrow the mighty aztec empire had they not been aided by a raging epidemic. this was possibly a combination of smallpox and measles, both wholly unknown to the new world population. curiously, the columbian exchange was unidirectional regarding infectious diseases; the one contentious possible exception being syphilis. the new world is believed to have had substantially fewer human zoonotic infections 15, 17 , and vector-borne infections like chagas' disease did not travel in the absence of an appropriate vector. two centuries later, captain cook unwittingly repeated the decimation of indigenous peoples through syphilis, measles and tuberculosis in many of the pacific islands, whereas lord jeffrey amherst deliberately attempted to spread smallpox among 'hostile' native americans, one of the better documented cases of germ warfare 18 . the transmission dynamics of infections in naive populations is markedly different from those in which the majority of adults are immune 19 . onboard the beagle, charles darwin observed with his customary acuity, "wherever the values are approximate global death rates for the year 2003, taken from the world health organization (who) and other sources. hbv and hcv, hepatitis b and c viruses; rsv, respiratory syncytial virus; hpv, human papilloma viruses; vcjd, variant creutzfeldt-jakob disease. two major, novel causes of mortality top the list: cigarette smoking and hiv infection; they emerged in the twentieth century and continue to increase in many developing countries. among the chronic and re-emerging infections, malaria and tuberculosis are near the top, so it becomes apparent why there is a need for the global fund for malaria, tuberculosis and aids. accidental injuries, particularly road deaths, continue to rise, with 85% occurring in developing countries 54 . although 2003 was the year of the sars outbreak 52,55 , less than 1,000 people actually died as a result of sars coronavirus infection despite the collateral damage to daily life, psychological wellbeing and economic activity in the affected cities. this richter scale represents a snapshot in time. twenty years ago, hiv was three logs further down the scale, whereas polio was three logs higher. fifty years ago, malaria was finally eradicated from europe, where it had formerly been widespread, including in england (shakespeare's 'ague'). bacterial respiratory diseases used to have a more important role in human mortality and, despite concern over multi-resistance to antibiotics 40, 56 , the situation is considerably better than in the era before the advent of antibiotics. common bacterial infections of childhood, such as diphtheria and whooping cough, have become rarities in the developed world, largely through vaccination. viral diseases have similarly been reduced. thanks to effective immunization policies of the who, smallpox was eradicated in 1977; polio and measles viruses, which have no animal reservoir, may soon be eliminated in the same way. the european has trod, death seems to pursue the aboriginal …most of the diseases have been introduced by ships and what renders this fact remarkable is that there might be no appearance of the disease among the crew which conveyed this destructive importation." today we are living through the fourth historical transition of globalization. urbanization, dense and usually impoverished peri-urban settlements, social upheaval, air travel, long-distance trade, technological developments, land clearance and climate change all influence the risks of infectious disease emergence and spread. although some of the apparent increase in infectious disease may be attributable to better diagnostic methods and surveillance, there seems little doubt that more incidents are occurring, and have the potential to spread more widely than 50 years ago, as outbreaks and spread of infections like nipah virus and sars would not have passed unnoticed. as humans encroach further into previously uncultivated environments, new contacts between wild fauna and humans and their livestock increases the risk of cross-species infection 20 . this process will only diminish as wild species become rarer and eventually endangered, like the great apes today. an example of such contact followed the establishment of piggeries close to the tropical forest in northern malaysia, where, in 1998, the nipah virus first crossed over from fruit bats (flying foxes, pteropus spp.) to pigs and thence to pig farmers 21 . destruction of natural forest has also encouraged fruit bats to relocate nearer human habitation, like the large colony in the botanic gardens in the heart of sydney. indeed, in 1997, hendra, a related paramyxovirus of australian fruit bats 22 , fatally infected a veterinarian examining a sick horse. rodents continue to be sources of re-emerging infections, as witnessed in the 1990s with hantaviruses in the united states. rodentborne hantavirus is prevalent in agricultural systems in south america and east asia, in arid grasslands in north america and elsewhere. in mid-1993, an unexpected outbreak of acute, sometimes fatal, respiratory disease occurred in humans in the southwestern united states 23 . this 'hantavirus pulmonary syndrome' was caused by a previously unrecognized virus, maintained primarily within the native deermouse, and transmitted through excreta. the 1991-1992 el niño event, with unseasonal heavy summer rains and a proliferation of piñon nuts, hugely amplified local rodent populations which led to the 1993 outbreak 23, 24 . in south america, there have been several outbreaks of hantavirus and arenavirus infections linked to forest clearance and the growth of rodent populations in the new grasslands 4 . habitat destruction is not the only cause of increased human infection, however. dengue virus is extending its range and prevalence because its mosquito vector breeds rapidly in the urban environment 25 . in the united states, nature conservation and increased woodland in the eastern states has led to the emergence of lyme disease. this disease is caused by a tick-borne spirochete and the presence of tick-infested deer near suburban homes leads to ticks residing on bushes adjacent to baseball diamonds and gardens. intensification of production of meat and meat products has led to new infections 26 . most notorious is vcjd in the uk arising from consumption of contaminated food products of cattle affected by bse 27 . bse, or 'mad cow disease,' emerged in british cattle in 1986 because of industrialized cannibalism, whereby rendered neural tissue and bone meal from slaughtered cattle were recycled into cattle feed, as well as into pies and hamburgers for human consumption 28 . originally, infectious prions from scrapie in sheep were the suspected source, but it now seems more likely that it arose from a bovine with sporadic prion disease. the extent of the human epidemic remains unclear. although natural transmission is unsustainable (r 0 < 1 in both cattle and humans), there are concerns that vcjd might be transmissible through blood transfusions 29 . without effective diagnostic tests for presymptomatic vcjd infection, this situation is extremely unfortunate. other recently emergent food-borne infections include e. coli o157:h7, which is harmless to cattle but toxic to humans, and salmonella enteriditis in chickens. better hygiene in abattoirs, butchers and domestic kitchens can greatly reduce the incidence of infection. in theory, closed and intensive farming of a single species should reduce the risk of cross-species infection (fig. 3) . but it also allows large-scale epidemics to emerge, as seen recently for avian influenza strains in southeast asia and the netherlands 8, 30 . ancient dietary taboos, such as those of hindus, muslims and jews regarding pork as unclean, doubtless had their roots in protection from infectious disease. today, an increasing demand for consumption of exotic and wild animals raises new risks of infectious diseases such as sars (box 2). changing patterns of human behavior and ecology affect two distinct steps in the emergence of new infectious disease. the first is an increased opportunity for animal-to-human infection to occur owing to greater exposure, which may be necessary but not sufficient to lead to the emergence of a new human infection. the second step is the opportunity for onward transmission once a person has become infected. for each novel epidemic, such as the 1918-1919 influenza pandemic or aids, there are probably thousands of failed transfers. some infections simply do not take in the new host. innate hostspecific restrictions on viral replication have recently become evident for primate lentiviruses 31 , which may explain why certain species that harbor simian immunodeficiency virus, but not others more commonly in contact with humans, gave rise to hiv-1 and hiv-2. even in the case of hiv-1, only one pedigree of three independent chimpanzee-tohuman crossover events 32 has given rise to the aids pandemic, whereas the other two smolder as poorly transmissible infections. fatal pathogenesis is not necessarily coupled with infectiousness 12 , which is evident for h5n1 avian influenza in humans 9 . but genetic reassortment between avian and human influenza viruses could easily give rise to a new, rapidly spreading strain 8 . a poorly infectious pathogen may not spread at all from the index case, as is usual with rabies, or may only infect close contacts and soon peter out, as seen with lassa fever and ebola virus. sars nearly became self-sustaining but was brought under control. some of the most insidious infections are those with long, silent incubation periods during which the person is infectious. these emerge surreptitiously so that when the new disease is eventually recognized, as aids was in 1981, the infection has already spread far beyond control. like the ships of centuries past, the speed of modern air travel works wonders for the dispersal of infectious diseases. sars was eventually constrained by quarantine and strict adherence to infection control guidelines in hospitals, but not before it quickly traveled from guangzhong to hong kong and on to toronto. if ebola broke out in a city with a busy international airport, it might also travel across continents in a similar manner. brockmann 33 has modeled how rapidly such infections can move once they reach a major airport hub; closing the hubs becomes an immediate imperative. we cannot be sure what the initial vector was for the arrival of west nile virus into north america in 1999: a migratory bird blown off course, an infected human with a valid air ticket or a stowaway mosquito on a similar flight. whatever the means of entry and early colonization of crows in new york, it has taken less than four years to reach the pacific coast 25 . thus, west nile virus has found a new reservoir in american birds, just as yellow fever virus reached new world primates 350 years earlier. microbes frequently capitalize on situations of ecological, biological and social disturbance. biologically weakened and vulnerable populations-especially if also socially disordered and living in circumstances of privation, unhygienic conditions and close contact-are susceptible to microbial colonization. the severity of the bubonic plague (black death) in mid-fourteenth-century europe seems to have reflected the nutritional and impoverishment consequences of several preceding decades of unusually cold and wet weather with crop failures compounding the incipient destabilization of the hierarchical feudal system. many of the rapid and marked changes in human social ecology in recent decades have altered the probabilities of infectious disease emergence and transmission. these changes include increases in population size and density, urbanization, persistent poverty (especially in the expanding peri-urban slums), the increased number and movement of political, economic and environmental refugees, conflict and warfare. political ignorance, denial and obduracy often compound the risk of infectious disease transmission-as has been tragically observed with hiv/aids in parts of africa, where widespread poverty, a culture of female disempowerment and political instability further "if there is any conceivable way a germ can travel from one species to another, some microbe will find it," wrote william mcneill in his classic text plagues and peoples 15 . for millennia, small farmsteads accommodated mixed species living closely with humans-goats, pigs, cattle, ducks, geese, chickens and perhaps a water buffalo or a donkey-and exchanged infections. when species are raised separately but are sold together, the opportunity for cross-infection moves from the farm to the marketplace. the 1997 outbreak of avian influenza in hong kong occurred in mixed markets, where live chickens, quail and ducks were stacked together in close quarters with humans. the h5n1 virus that emerged may have been derived by recombination between those of different avian hosts 8 . after 1997, mixed species were separated into different areas of the markets. but this year's h5n1 virus is spreading among intensively reared chickens across southeast asia. the increasing predilection for meat of exotic species has exacerbated the risk of exposure to infections not previously encountered, and this situation probably triggered the sars epidemic 55 . although we are still not sure of the natural reservoir species of sars coronavirus, the live markets and restaurants in guangzhong sold small carnivores, and several species of civet cat, racoon dog and ferret badger captured in china, laos, vietnam and thailand, were brought into close proximity 57 . clearly, some of the palm civet cats were infected with sars-related viruses, but it is less clear whether they represent the original source species. there is a danger in incriminating the wrong species; if the true reservoir resides in the rodent prey of these carnivores, then culling the predators may be counterproductive. stopping the exotic meat trade altogether would seem to be a simple solution to prevent the reappearance of sars, but once the taste for it has been established, that may prove no more practical than attempting to prohibit the tobacco trade. in africa, bushmeat also poses a serious problem for emerging infectious diseases, as well as for nature conservation. sick animals may be more easily captured. for example, 21 human deaths owing to ebola virus infection ensued from the butchering of a single chimpanzee 58 . hiv has crossed from chimpanzees to humans on at least three occasions, and a higher number of zoonotic events from sooty mangabeys are indicated for hiv-2 (ref. 32). whether these cross-species infections arose from butchering the animals or from keeping them as pets is unknown, but a recent survey of primate hunters in africa showed that they are susceptible, like handlers of primates in captivity, to infection (though not disease) from foamy retroviruses 59 . the escalating intercontinental trade in exotic pets can lead to unexpected infectious disease outbreaks. the united states has only recently imposed more stringent regulations and quarantine following cases of monkeypox in humans and in prairie dogs introduced by rodents imported from africa as pets 60 . exacerbate the problem 34, 35 . but we have little understanding of why the prevalence of hiv infection varies so greatly between cities in sub-saharan africa 36 . the urban environment has only recently become the dominant human habitat. urbanism typically leads to a breakdown in traditional family and social structures, and entails greater personal mobility and extended and changeable social networks. these features, along with access to modern contraception, have facilitated a diversity of sexual contacts and, hence, the spread of sexually transmitted diseases 37 . this risk is further amplified by the growth in sex tourism in today's internationally mobile world, which capitalizes on the desperation and ignorance of poverty, combined with exploitative behaviors, in developing countries. more generally, cities often function as highways for 'microbial traffic' 38 . rapid urbanization boosts certain well established infectious diseases, such as childhood pneumonia, diarrhea, tuberculosis and dengue, and facilitates dissemination of various 'emerging' diseases-as occurred for sars in the high-rise housing of hong kong. crowded and dilapidated public housing can potentiate infectious disease transmission through drug abuse and sexually transmitted infections 39 . technological advances in medicine and public health can also inadvertently promote the emergence and spread of infectious disease. it has become commonplace to quip that you go to the hospital at the peril of acquiring an intractable nosocomial infection such as methicillin-resistant staphylococcus aureus 40 , and such infections killed around 40 times as many people as sars did in 2003 (box 1). multidrug-resistant tuberculosis has also become a major problem, and, paradoxically, regions with health programs that reduced wildtype tuberculosis strains can develop into 'hot zones' for multidrugresistant tuberculosis 41 . by far the most effective medical vector of infectious disease has been the syringe and needle. drucker et al. 42 have charted the massive increase in the use of injecting equipment over the past 100 years. individuals with hemophilia treated with pooled clotting factors became almost universally infected with hepatitis b and c viruses before diagnostic screening tests were developed. over 20% of such affected individuals also became infected with hiv 43 , and more recently, transmission of west nile virus by blood transfusion and by organ transplantation has been reported 44, 45 . the use of contaminated needles among intravenous drug users has had similar consequences. infectious diseases have also been amplified by the use of nonsterile medical injections in developing countries 42 . egypt has the highest prevalence of hepatitis c infection in the world because of the use and reuse of syringes and needles in an earlier public health campaign to reduce bilharzia by medication given by injection. the transmission of cjd through contaminated surgical instruments is another example of iatrogenic spread of infection 29 . biological medicines produced from animal-cell substrates present an inherent potential hazard for introducing new infections. great care must be taken to ensure that live attenuated vaccines grown in animal cells or eggs are devoid of pathogens ; for example, several early batches of live and inactivated polio vaccine unwittingly contained live sv40 virus, a polyoma virus of macaques. after sv40 was discovered in 1960, polio vaccine production shifted to virus propagation in primary kidney cells of african green monkeys. these cultures were free of sv40 but possibly contained sivagm, a relative of hiv that fortunately does not infect humans 31 . the irony of the sv40 story is that the united states food and drug administration prohibited the use of well known, permanent cell lines demonstrably free of adventitious infectious agents, for fear that such immortalized cells might exert oncogenic properties on the vaccine. there is no epidemiological evidence of increased tumor incidence in those populations who are known to have received sv40-contaminated polio vaccine. but there have been a number of recent claims of an association of sv40 dna sequences in a variety of human malignancies 46 , although these findings remain controversial 47 . the ultimate medical means of introducing animal viruses into humans is xenotransplantation. the implantation of animal cells or tissues into immunosuppressed individuals seems to be a perfectly designed way to encourage cross-species infection. it is astonishing that trials were started without much thought about the consequences for potentially emerging pathogens, for example, porcine retroviruses 48 . the generation of genetically modified knockout or transgenic animals to prevent hyperacute rejection of donor tissues may exacerbate the infection hazard 49, 50 . happily, there is no evidence so far of retrovirus infection in individuals who were exposed to living pig cells 50 , and clinical xenotransplantation is now stringently regulated; so it seems all the more extraordinary that cellular therapies with fetal lamb cells and extracts continue to be practiced with impunity in alternative medicine clinics in europe and the far east. novel infectious diseases can emerge in any part of the world at any time. hiv and ebola came out of africa, avian influenza and sars from china, nipah virus from malaysia, bse/vcjd from the uk and hantavirus pulmonary syndrome from the americas. it is difficult to predict what new disease will come next or where it will appear, but changing ecological conditions and novel human-animal contacts will be useful clues as to which horizons require scanning with most scrutiny. we must expect the unexpected. as a codicil, another factor that needs to be taken into account is the potential impact of the hiv pandemic on the emergence of other infectious diseases 51 . we already know that persons with aids act as 'superspreaders' of tuberculosis, and we can only speculate what course the sars outbreak might have taken had someone incubating the disease flown to durban rather than toronto 52 . people with aids may persistently harbor infections that would otherwise be transient, and this could hamper the eradication of measles and polio. multivalent pneumococcus vaccines are ineffective in hiv-infected people with cd4 + lymphocyte levels below 200/µl, whereas live 'attenuated' vaccines such as vaccinia can cause virulent disease in the immunocompromised host. immunodeficient persons living at high density could also be the seed-bed for microorganisms that are initially ill adapted to human infection to evolve into transmissible human pathogens. thus, an infection from a zoonotic or environmental source-for example, the mycobacterium avium intracellulare complex-could conceivably emerge as the tuberculosis of the twenty-first century, although direct transmission between individuals with aids of such opportunistic infections have not been documented so far. we shall give girolamo frascatoro the last word on emerging and re-emerging infectious diseases by quoting from his treatise de contagione, published almost 450 years ago, "there will come yet other new and unusual ailments in the course of time. and this disease [syphilis] will pass way, but it later will be born again and be seen by our descendents." we are grateful to m. e. chamberland, h. w. jaffe and s. leff for critically reading the manuscript. natural history of infectious disease human frontiers, environments and disease. past patterns, uncertain futures the challenge of emerging and re-emerging infectious diseases environmental and social influences on emerging infectious diseases: past, present and future mortality trends and setbacks: global convergence or divergence? betrayal of trust: the collapse of global public health (hyperion updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic molecular constraints to interspecies transmission of viral pathogens influenza: old and new threats antivirals and antiviral strategies risk factors for human disease emergence virulence and pathogenesis the cambridge world history of human disease jail fever (epidemic 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emergence of human pathogens mortality before and after hiv infection in the complete uk population of haemophiliacs transmission of west nile virus through blood transfusion in the united states in 2002 transmission of west nile virus from an organ donor to four transplant recipients cell and molecular biology of simian virus 40: implications for human infections and disease sv40 in human cancers-an endless tale? infection of human cells by an endogenous retrovirus of pigs transgenic pigs and virus adaptation reduced sensitivity to human serum inactivation of enveloped viruses produced by pig cells transgenic for human cd55 or deficient for the galactosyl-α(1-3) galactosyl epitope gulliver's travels in hivland what have we learnt from sars? mission now possible for aids fund research on preventing road traffic injuries in developing countries is needed severe acute respiratory syndrome antimicrobial resistance worldwide: causes, challenges and responses animal origins of sars coronavirus: possible links with the international trade in small carnivores zoonoses and haemorrhagic fever. in safety of biological products prepared from mammalian cell culture naturally acquired simian retrovirus infections in central african hunters human monkeypox: an emerging zoonosis the authors declare that they have no competing financial interests. key: cord-269181-1h3wbhq4 authors: perelmutter, l.; potvin, l.; phipps, p. title: immunoglobulin e response during viral infections date: 1979-08-31 journal: journal of allergy and clinical immunology doi: 10.1016/0091-6749(79)90046-0 sha: doc_id: 269181 cord_uid: 1h3wbhq4 abstract one hundred and three patients (90 nonatopics and 13 atopics) with respiratory infections to various viral agents were studied retrospectively with respect to ige immunoglobulin levels during acute (1 to 7 days) and convalescent (8 to 30 days) phases of infection. it was found that 59% of patients had a decrease of 20% or more in ige level, 27% remained the same, and only 14% showed a rise of 20% or more from the acute to the convalescent phases of infection. ige levels decreased up to 3 to 4 wk after symptoms and the degree of decrease was more apparent for the nonatopics who had higher ige levels in their acute phase of infection. less dramatic decrease in ige was observed for the 13 atopics studied. the changes in ige levels during the viral infectious period are discussed in terms of possible cellular mechanisms that may control ige immunoglobulin. one hundred and three patients (90 nonatopics and 13 atopics) with respiratory i!fections to curious viral ugents were studied retrospectively with respect to ige immunoglobulin levels during acute (i to 7 days) and convalescent (8 to 30 days) phases of infection. it was found that 59% of patients had a decrease of 20% or more in ige level, 27% remained the same, and only 14% showed a rise of 20% or more from the acute to the convulescent phases of infection. ige levels decreased up to 3 to 4 wk after symptoms and the degree of decrease was more apparent for the nonatopics who had higher ige levels in their acute phase of infection. less dramatic decrease in ige was observed for the 13 atopics studied. the changes in ige levels during the viral infectious period are discussed in terms of possible cellular mechanisms that may control ige immunoglobulin. clinical studies dating back almost 40 yr have suggested that upper respiratory tract infections are associated with bronchial asthma and can precipitate or potentiate attacks of bronchial asthma', ' and wheezing in asthmatic patients. 33 4 other studies have shown that killed influenza vaccine can increase bronchial sensitivity to drugs such as methacholine.5 by viral isolations and serologic techniques, a variety of viruses including respiratory syncytial virus, parainfluenza virus, corona virus, and rhinovirus have been identified in patients with asthma. in addition, ida et al6 found an enhancement of ige-mediated histamine release from human basophils and ultravioletinactivated herpes simplex virus-1, influenza a, and adenovirus-1. from the recent results of frick et a1.,7 a possible virus infection association was found with the onset of allergic sensitization in infants. other investigations have shown both increased83 g and decreasedlo> l1 levels of ige in association with viral infections. the purpose of the present study was to examine more closely the relationship between viral infections and ige levels. for this purpose, a retrospective study was performed on a population of patients with upper respiratory symptoms of viral origin. all patients were followed clinically, screened for viral and bacterial infections, and ige and rast determinations were performed during the course of their acute and convalescent phases of infection. the records of the regional virus laboratory were inspected and a list prepared of patients showing a rise 24fold in antibody level (by complement-fixation test)12 against one of the common respiratory agents, between the first and second specimens of paired sera. from this list 103 individuals were chosen (from november, 1976, to march, 1977) on the basis of having been diagnosed as having an acute upper and/or lower respiratory infection, the onset of which was 8 or fewer days before submission of the first blood specimen. individuals with chronic viral infections and/or bacterial infections were eliminated from the study. the antigens in the test were as follows: influenza a (soluble), influenza b (soluble), parainfluenza, type i. parainfluenza, type ii, parainfluenza, type iii, adenovims, respiratory syncytial virus, herpes simplex virus, psittacosis group, and myoplasma pneumoniae. in every case, the clinical condition was sufficiently severe to warrant hospitalization of the patient. most patients manifested bronchitis, tonsillitis or pharyngitis. their ages ranged from 6 mo to 60 yr. about half were under the age of 15 yr and the rest between the ages of 16 and 60 yr. clinical histories were taken, particularly noting the patients' infections (recent and past) and atopic state. all patients were screened for allergies using a radioallergosorbent test piscataway, n. j.). all samples were examined in duplicate. in our hands, a 20% variation was found for ige levels over the range used in this study considering both kit-to-kit variation as well as variation with the same kit. all kits were standardized not only with the standards provided with the prist kit but also with several sera whose ige levels were over the working range. each pair of acute and convalescent sera were always run with the same kit. a change of 20% or more in ige level was considered significant. the phadabas rast for ige antibodies was determined for acute phase samples for all patients against the more common allergens found in the ottawa area, such as tree (maple, birch, oak, elm), grass (sweet vernal, meadow fescue, rye grass, timothy), weed (short ragweed, western ragweed, wormwood, mugwort, russian thistle), mold (a/rernaria, asprrgillus, cladosporiutn), animal (cat, dog), food (egg, milk, fish), and house dust allergens. an individual was considered positive to rast if a 2+ reaction was found for one or more allergens. fig. 1 . it was found that 59% of patients had a decrease in ige level of 20% or more, 27% remained the same, and only 14% showed a rise of 20% or more for this immunoglobulin. fig. 2 shows a scattergram summarizing the ige levels of the nonatopic population as a function of time after onset of infection. for each patient, acute and convalescent ige values were plotted. fig. 3 shows a similar scattergram for the atopic patients. for the nonatopics (fig. 2) , 2 populations of patients emerged, one segregating at ige levels greater than 60 u/ml (nonatopic i) and the other, below this value (nonatopic ii). figs. 4 and 5 show the average ige value for a given time after onset of infection for the nonatopic and atopic groups respectively. decreases in ige levels were observed for the high and low nonatopic groups as well as for the atopic group of patients. it can be seen (figs. 4 and 5) that decreases in ige occurred from the time of onset of symptoms up to 3 wk after symptoms, after which the ige values leveled off. table i shows the geometric mean values of ige for nonatopic groups i and ii as well as the atopic group during the acute and convalescent phases of infection. it can be seen that ige levels decreased for all 3 groups but more dramatically for nonatopic i. in the present study, it was found that naturally occurring viral respiratory infections modulated serum ige levels in both nonatopic as well as atopic individuals. these results confirm those of previous decreaselo. l1 in ige associated with viral infections. in this study, ige levels decreased up to 3 to 4 wk after symptoms. on the other hand, the ige levels viral agent to precipitate the asthmatic condition. for some individuals, the viral agent may be capable of "turning off" the formation of ige immunoglobulin in sufficient time to prevent the development of the disease state. the degree to which this will occur will depend, in part, on the cellular mechanisms that control the formation of ige immunoglobulins. it is possible that during viral infection t helper cells are stimulated during the acute phase of infection for both atopics and nonatopics to produce ige immunoglobulin and ige antibody to the infectious agent. the viruses may also activate t suppressor cells for the nonatopics during the convalescent phase to decrease ige levels. for atopics, there is increasing evidence'" that there is a genetically determined defect in some or all of the t lymphocytes which makes them more vulnerable to the inhibitory action of cyclic adenosine monophosphate (camp). this substance is considered to be inhibitory, particularly for suppressor t cells. thus, it might be expected that during the convalescent phase of viral infection for atopics a de-upper respiratory tract infections due to virus are associated with the onset of bronchial asthma.'-l it is tempting to speculate that ige antibodies to the infectious agent react in the respiratory tract with the 130 perelmutter et al. feet in the t suppressor cells may lead to continual hyperproduction of ige and hence bronchial asthma. the role of allergy in viral respiratory tract infections viruses as precipitants of asthmatic attacks in children the association of viral and mycoplasma infections with recurrence of wheezing in the asthmatic child the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children increased response of asthmatic subjects to methacholine after influenza vaccine enhancement of ige-mediated histamine release from human basophils by viruses: role of interferon virus infection association with onset of allergic sensitization in infants peacock lb: an evaluation of quantitative serum immunoglobulins determinations in clinical practice a study on the ige levels of military recruits and association with hla antigens viral infections and ige levels signification des valeurs basses d'ige scriques. les immunoglobuline e. symposium ige diagnostic procedures for viral and rickettsial infections serum ige levels in healthy children quantified by a sandwich technique (prist) ige in cytomegalovirus mononucleosis ige response in heterophil-positive infectious mononucleosis ige responses during immunization with influenza antigens specific ige antibodies to ebv in infectious mononucleosis. presented at the third international congress of immunology strannegard 0, strannegard i-l: t lymphocyte numbers and function in human ige-mediated allergy key: cord-272955-kkkrkgg1 authors: belsy, acosta; odalys, valdés; alexander, piñón; clara, savón; angel, goyenechea; grehete, gonzalez; guelsys, gonzalez; luis, sarmiento; pedro, más; guadalupe, guzmán maría; alina, llop; pilar, perez breña ma; inmaculada, casas title: molecular characterization of adenoviral infections in cuba: report of an unusual association of species d adenoviruses with different clinical syndromes date: 2009-03-12 journal: arch virol doi: 10.1007/s00705-009-0338-4 sha: doc_id: 272955 cord_uid: kkkrkgg1 adenoviruses are common pathogens that are responsible for a wide variety of infectious syndromes. the objectives of this study were to identify and characterize members of different adenovirus species at the molecular level and to describe the correlation between viruses and clinical syndromes during a period of 4 years. between 2002 and 2006, 45 of 512 respiratory specimens (8%) from patients with acute respiratory tract infection tested positive for adenovirus. four adenovirus isolates from samples sent for enterovirus isolation were also analyzed. this research identified 49 confirmed cases of human adenovirus infection by pcr and/or viral culture. the most common diagnosis was upper respiratory infection (44%). human adenovirus d was the major species found (59%), followed by human adenovirus c (36%) and human adenovirus b (4%). human adenovirus 5 was the major serotype found producing bronchiolitis, followed by human adenovirus 6. in patients with upper respiratory infection, the major serotype found was human adenovirus 17. viruses of the species human adenovirus d were identified in seven (77%) cases of acute febrile syndrome. four isolates from clinical materials obtained from patients with encephalitis, acute flaccid paralysis and meningoencephalitis were identified as belonging to the species human adenovirus d. our data demonstrate a surprising result about the identification of an unusual association of viruses of the species human adenovirus d with different clinical syndromes. this observation could be evaluated as a possible indicator of the emergence of a novel strain but further studies are required. human adenoviruses (hadvs) are members of the family adenoviridae, whose members infect hosts across a broad spectrum of vertebrates. there are 51 serotypes of hadv in the genus mastadenovirus, distributed in six species, a-f (formerly subgroups or subgenera) on the basis of their physicochemical, biological and genetic properties. a new serotype isolated recently (52) has been proposed to represent a new species g [21] . human adenoviruses are associated with sporadic infection, and community and institutional outbreaks. these viruses cause a variety of clinical manifestations, such as conjunctivitis, pneumonia, gastroenteritis, and hemorrhagic cystitis. some serotypes can occasionally infect tissues of the central nervous system (cns) and cause aseptic meningitis, meningoencephalitis, and encephalitis. they can cause especially severe disease in infants, young children, immunocompromised persons, and transplant recipients [2, 5, 14, 15, 25, 30, 31] . serosurveys suggest that virtually all people are exposed to hadv during childhood [22, 35] . they can remain in an asymptomatic carrier state until at least young adulthood [18] , and virus may be actively shed long after symptomatic infection [16] . over the past decades, neutralization tests, elisa, and virus isolation had been used for the detection and identification of adenovirus serotypes. however, these methods are relatively complicated, labour-intensive, and timeconsuming, and they have low sensitivity. [26, 39, 42] . these disadvantages have limited their use. amplification of the viral genome by pcr has been introduced as a convenient and powerful alternative for molecular diagnosis. additionally, genome amplification allows further characterization of the adenovirus serotype by sequence analysis [1, 7, 34] . the objectives of this study were the identification and molecular characterization of different hadv isolates and to describe the correlation between viruses and clinical syndromes during a period of 4 years. between october 2002 and september 2006, 512 respiratory specimens (nasopharyngeal swabs and pharyngeal washes) from patients with acute respiratory tract infection (arti) were sent specifically to the national reference laboratory of respiratory viruses for testing respiratory viruses, including influenza virus a, b and c, human respiratory syncitial virus (hrsv), human parainfluenza virus 1-4, hadv, human coronavirus, human rhinovirus and human metapneumovirus (hmpv). routine virological testing for respiratory pathogens was performed using a combination of direct immunofluorescence, isolation in cell culture and pcr assays [9, 10, 27] . in addition, four samples (three stools and one cerebrospinal fluid) from the enterovirus (ev) laboratory with previous viral isolation without identification were analyzed. nasopharyngeal swabs and pharyngeal washes were collected in 3 ml of virus transport medium (mem, gibco-brl, life technologies, paisley, scotland; penicillin 200 u/ml, and streptomycin 200 lg/ml, biowhittaker, ma; mycostatin 200 u/ml, sigma; bovine serum albumin 0.25%, merck, darmstadt, germany) the specimens were frozen and stored at -70°c until the analysis was carried out. a human embryonic fibroblast cell line was used for primary isolation of ev. tubes with 80% confluent monolayers were inoculated with 0.2 ml of homogenized samples. cells were fed with 2 ml of 2% fetal calf serum in basal medium eagle and visualized for cytopathic effect (cpe) twice a week. when a cpe that was not typical of ev was observed, the monolayer was scraped and tested for adenovirus antigen by immunofluorescence with a specific monoclonal antibody (chemicon, temecula, ca). total viral rna/dna from 200-ll aliquots from clinical samples or supernatant of infected cell culture was extracted using the guanidinium thiocyanate method as described previously by casas et al. [6] . the lysis buffer included 100 copies of the cloned, amplified product of the internal control described by coiras et al. [9] . it was used for checking the extraction process, the amplification efficiency, and the presence of inhibitors in the clinical specimens. after processing, the dried pellet was resuspended in 15 ll of rnase-free sterile water. negative controls consisting of rnase-free sterile water (sigma) were treated following the same procedure. for each assay, known positive controls, derived from infected viral cells, were added. in all cases, nucleic acid extracts (5 ll) were examined for influenza virus a, b and c, hadv, hrsv a and b [9] , human parainfluenza virus 1-4, coronaviruses, rhinoviruses, and ev [10] , using nrt-pcr. in addition, a multiplex npcr assay for human herpesvirus was used in the case of a patient with encephalitis [40] . the protocols employed have been published previously and used for clinical diagnosis. three different biosafety cabinets were used for master mix preparation, sample handling and primary reaction product handling. different laboratory wear and coats were used for each cabinet. amplicon detection was done in a different room. specimens that were positive for hadv were processed by two independent nested reactions with 20 pmol of adhex2f, nt 20485 to 20503; (5 0 cccittyaaccacc accg3 0 ) and adhex1r, nt 20836 to 20854; (5 0 katg gggtaragcatgtt3 0 ) or 20 pmol of adhex1f, nt 20380 to 20400; (5 0 caacacctaygastacatgaa3 0 ) and adhex2r, nt 20632 to 20652; (5 0 acatccttbc kgaagttcca3 0 ) degenerate primers as described previously [7] . these nested primer pairs were designed to bind inside the hexon protein coding region of the adenovirus genome [1] . the pcr products were purified with qiaquick pcr purification kit (qiagen) according to the manufacturers's protocol and were sequenced in both directions using the primer pairs described above. sequences were obtained using an automatic dna sequencer (abi prism 3700; applied biosystems) and a big dye terminator cycle sequencing kit version 3.1 (applied biosystems). the fragment size sequenced corresponded to 122 amino acids, from amino acid position 540-662 of the hexon protein. this fragment is located at the external surface l2 of the hexon protein monomer but does not overlap with the hrv7 domain [11] . the nucleotide sequences obtained were first analysed using the chromas software (version 1.3). the forward and reverse sequences were combined using bioedit and were compared and aligned with the corresponding previously published sequences of prototype viruses available from genbank, using the clustal x (version 1.83) program. specimens that yielded identity scores c90%, were considered to be good genotype matches. phylogenetic trees were inferred using programs from the mega package (version 4) and reconstructed using the neighbourjoining method. the evolutionary distances were estimated using kimura's two-parameter method [24] . the statistical significance of a particular tree topology was evaluated by 1,000 replicates of boostrap re-sampling. the criteria for serotype assignation were the shortest distance from a prototype strain, as deduced from the phylogenetic tree ( fig. 1) , and the highest similarity value obtained after sequence comparisons. the genbank accession numbers of the nucleotide sequences presented in this study are: eu179755-eu179763, eu179765, eu179768-eu179785 and eu439569-eu439589. in the present report, the nested pcr method used was able to detect different hadvs in clinical samples and supernatant culture with a sensitive internal control system to assure the quality of reaction conditions in each individual tube. in this investigation, 49 confirmed cases of hadv infection were identified by pcr and/or viral culture ( table 1) . the monthly collection of clinical samples is shown in table 1 . human adenovirus was detected throughout the year; however, 38 samples (77%) were collected between june and september. two significant outbreaks of hadv infection were identified, one in july of 2005 and the other in september of 2006. more than half of the positive samples (53%) were from children younger than 5 years of age, and 50% of them were under 6 months of age. thirty-two percent were from patients between 5 and 20 years, and 14% were from adults between 21 and 64 years of age. no difference in gender distribution was observed. the most common diagnosis was upper respiratory infection (44%). medical records documented the following symptoms at initial presentation: nasal congestion (98%), sore throat (83%) and cough (78%). all patients were febrile. fourteen (28%) patients had bronchiolitis. acute febrile syndrome (18%) was defined as the concurrence of fever, cold symptoms, headache, weakness, vomiting and diarrhoea, a common but non-specific symptom. in these cases, the results of diagnostic assays for the detection of other potentially pathogenic enteric microorganisms were negative. an interesting finding was the detection of hadv in a patient 64 years of age who was admitted with a diagnosis of encephalitis. no human herpesvirus, influenza virus, ev or flavivirus genomes were found in the cerebrospinal fluid of this patient. human adenovirus dna was detected in the supernatant of a cell culture infected with viruses obtained from fecal specimens taken from a patient with acute flaccid paralysis (afp), as well as in two cases of meningoencephalitis. overall, 73% of hadv positive patients were hospitalized. partial hexon nucleotide sequences for 49 different hadv types were obtained. a blast search in the genbank database for all of the amplicon sequences determined in the present study was done. a 100% agreement with existing genbank sequences for serotypes 1, 3, 5, and 6 was obtained. an agreement ranging from 99 to 100% was obtained for prototype strains of human adenovirus d. an alignment of the hexon gene sequences was computed by using sequences from representative serotypes of species b (hadv 3), c (hadvs 1, 5, and 6), and d (hadvs 9, 10, 13,15,17, 19, 23, 25, 28, 30, 32, 33, 37-39, 43, 44, and 47-49) . in order to evaluate the phylogenetic relationships between individual prototype viruses and positive cases of hadv infection by pcr and/or viral culture, phylogenetic trees were constructed using a phylogeny reconstruction algorithm (the neighbour-joining method) and a nucleotide substitution method . phylogenetic and sequence similarity analysis permitted accurate species classification and serotype identification except for species d hadv, for which serotypes could not be clearly defined. the resulting phylogenetic tree showed three different clusters, represented by the species b, c and d. the bootstrap values ( fig. 1) were, in general, quite high, and especially at nodes between species. however, these values were less good for some nodes within species d. the assignment of a serotype to a clinical isolate was done by calculating the shortest distance between the isolate and a prototype strain. a total of 31 (63%) hadvs were serotyped, and typing results yielded some interesting observations. the major species found was hadv-d, with 29 clinical cases (59%), followed by hadv-c, with 18 cases (36%), and hadv-b, with two cases (4%). in bronchiolitis cases, hadv-5 (71%) was the major serotype, followed by hadv-6 (14%) and single cases of hadv-1 and hadv-d. in the group of patients with upper respiratory infection, the major serotype found was hadv-17 (77%). hadv-5 (22%) was the second-most common serotype, and five specimens were typed as hadv-d; one of them recovered in 2003 and four in 2005. lastly, two specimens recovered in 2003 were hadv-3. hadv-d was the species identified in nine (100%) cases detected during an atypical outbreak of acute febrile syndrome in july of 2005. four isolates from clinical materials obtained from patients with severe disease such as encephalitis, afp and meningoencephalitis were identified as hadv-d. hadvs are categorized by species (hadv-a, hadv-b , hadv-c, hadv-d, hadv-e and hadv-f), and further by serotype (ad1-ad51), and cause a wide spectrum of illnesses in both children and adults, including those involving the cns. a new recently isolated serotype (52) has been proposed to belong to a new species g [3, 13, 21] . more than half of the positive samples (53%) were from children younger than 5 years of age, and 50 % of them were under 6 months of age. the highest incidence of adenoviral infection occurs in children from 6 months to 5 years of age, although outbreaks have been noted when susceptible hosts, such as military recruits, adolescents, visitors to summer camps, and sometimes nursing home residents, congregate together. explanations for the relative lack of illness in the youngest or in older individuals include the presence of transplacentally acquired maternal antibody in young infants and the development of neutralizing antibody to the most common adenoviral strains in the majority of children older than 5 years old. the age distribution of hadv in developing countries appears to be similar to that in developed countries [8] . the results in this study showed that adenovirus infections were more common in children younger than 5 years of age, and 50% of them were under 6 months of age. most patients were hospitalized (73%); nevertheless, none of these had severe respiratory infections. in cuba, infants are considered special patients by pediatricians. this report also describes the seasonal variation of adenovirus infections in cuba. respiratory viral agents usually have characteristic seasonal patterns in temperate and tropical climates. in temperate climates, the majority of isolations of respiratory viruses are in the winter. in central america, there are few studies published about the epidemiological characteristics of adenoviruses. the cuban island is located in the caribbean sea at the entrance of the gulf of mexico. the climate of cuba is semitropical, and two seasons are generally recognized: a rainy season from may to october and dry season from november to april. cuba is often hit by hurricanes from june to november, resulting in great economic loss and temporarily interrupted sanitary conditions. the average minimum temperature is 21°c (70°f), and the average maximum is 27°c (81°f). the warmest month is july with 30°c (86°f). in this study, adenovirus was detected throughout the year; however, two significant outbreaks of hadv infection were identified, the first in july of 2005, when hurricane dennis hit severely the western part of cuba, and the second in september of 2006. we believe that the effect of temperature and rainfall appears to be a determinant of the timing of those outbreaks. figure 1 displays a phylogenetic tree built from an alignment of the nucleotide sequences of the amplicons obtained from clinical samples and each serotype prototype. as shown in fig. 1 , each serotype is clearly distinct. serotypes belonging to the same species cluster together. in cuba, human adenovirus circulates in the pediatric population throughout the year. furthermore, members of adenovirus species c of are commonly involved in respiratory diseases in the pediatric population. two previous studies have been reported on the associations between specific clinical syndromes and hadv serotypes. the analysis of the occurrence of hadv-c revealed that hadv-1 and hadv-6 were the predominant serotypes in children with acute respiratory diseases, and hadv-37 was the most prevalent one associated with conjunctivitis [36, 41] . human adenovirus respiratory infections have usually been associated with species b, c, and e [8] . hadv-c includes hadv-1, hadv-2, hadv-5, and hadv-6. these serotypes are commonly associated with febrile respiratory illness in children and are noted to be endemic in certain regions or in epidemics [32] . the significant proportion of infection with hadv-1, hadv-3, hadv-5 and hadv-6 among patients with respiratory infection is consistent with previous reports [7, 20, 23, 32] . however, we detected hadv-d in a child with bronchiolitis, and in 15 cases of upper respiratory infection. hadv-d are rarely isolated in respiratory illness surveys, and strong causal correlations are generally lacking. nevertheless, this association was previously described by casas et al. [7] . on the other hand, hadv-d was identified in a group of patients with acute febrile syndrome and in four isolates from clinical materials obtained from patients with encephalitis, afp and meningoencephalitis. sporadic cases or small outbreaks of neurological disease following adenovirus infection are well documented [12, 17, 25, 38] . the high prevalence of infection with hadv-d in previously healthy patients is not consistent with previous reports. this association was unexpected, and a detailed analysis of this sample set will be published in a separate report. human adenovirus is unique among the common respiratory viruses in that it can spread to organs other than the respiratory system, resulting in conjunctivitis, gastroenteritis, acute hemorrhagic cystitis, and meningoencephalitis. the liver, spleen, pancreas, kidney, or heart may also be involved in disseminated infections, both in previously healthy people and in immunocompromised patients (e.g., infants, patients with aids, transplant recipients) [33] . hadv-d has been frequently associated with severe adenovirus infection in these patients. in our study, we reviewed the medical records and did not find that the patients suffered from aids or another type of immunodeficiency. however, we thought that is important to take into account that the highest percentage of hadv-d was identified in children. the identification of such a large number of hadv-d infections is extremely interesting and a major and unique finding. we recognize that the limited sequence data analyzed (370 nt in a relatively conserved region of the hexon) for typing did not generate enough results. the sequencing of the hypervariable regions of the hexon gene (hvr7 alone or hvr1-6) and fiber gene of select strains will certainly provide a solid basis for confirming the species/serotype identification [29, 37, 43] . we used the method previously published by casas et al. [7] , to facilitate sequencing and typing of hadvs using the hexon protein coding region of the hadv genome. this method was extensively validated using clinical samples and prototype strains, and we also noted unusual associations between specific serotypes and clinical presentation. the primers and pcr conditions have also been extensively validated. some years ago, adenovirus infection was considered to have little consequence. however, much has changed since these early epidemiological studies were conducted. in contrast to the modest number of hadvs recognized 35 years ago, 51 unique serotypes are now recognized, and a new serotype isolated recently (52) has been proposed to belong to a new species g. different serotypes have been found to have different tissue tropisms that correlate with different clinical manifestations of infection. partial epidemiological investigations have revealed that, among some specific serotypes, multiple genetic variants exist that often have quite different geographical distributions and associated virulence [19, 21, 28, 30] . our study expands the range of hadv serotypes that have been reported elsewhere in association with major clinical syndromes. further independent testing is needed to verify these associations. however, future studies of hadv should not exclude these rare serotypes, and they should be kept in mind. unfortunately, because adenoviruses can be asymptomatically shed for prolonged periods of time, recovery of hadv from the upper respiratory tract or stool samples by culture does not confirm it as the cause of a specific disease. accordingly, recovery of hadv should always prompt an effort to identify any additional or alternate potential explanations for symptoms present at the time of a positive viral culture [4, 16] . additional research is needed for the development of better rapid methods to detect and to quantify hadv in various body fluids (blood, stool, and throat). finally, our findings, combined with the existence of several reports concerning the association of hadv with different syndromes confirm that members of the family adenoviridae, mainly the serotypes belonging to hadv-c are common causal agents of respiratory infection. in contrast, serotypes of hadv-d could be involved in the aetiology of acute respiratory infection and neurological disorders in previously healthy people. however, this observation needs to be confirmed in a larger study. our identification of hadv-d associated with different syndromes may signify the emergence of new genomic variants that have the potential to spread globally. further studies of such typing with other molecular typing methods are necessary. these reports show that surveillance, serotyping and molecular characterization methods need to be improved in order to identify emerging adenovirus variants. authors wish to thank all of the physicians who provided samples and clinical data. we also acknowledge all technicians and researchers of the virology deparment of the instituto de medicina tropical pedro kourí, la habana, cuba, who collaborated with this investigation. thanks to dr enrique tabarés, from universidad autónoma de madrid, spain, for his help. rapid and sensitive diagnosis of human adenovirus infections by a generic polymerase chain reaction outcome and clinical course of 100 patients with adenovirus infection following bone marrow transplantation family adenoviridae infections in 18, 000 infants and children in a controlled study of respiratory tract disease. ii. variation in adenovirus infections by year and season intravenous cidofovir therapy for disseminated adenovirus in a pediatric liver transplant recipient new method for the extraction of viral rna anddna from cerebrospinal fluid for use in the polymerase chain reaction molecular identification of adenoviruses in clinical samples by analysing a partial hexon genomic region molecular and clinical characteristics of adenoviral infections in taiwanese 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encephalopathy associated with adenovirus infection serotyping of adenoviruses on conjunctival scrapings by pcr and sequence analysis detection and typing of human herpesviruses by multiplex polymerase chain reaction the incidence of adenoviruses in viral conjunctivitis characterization of fastidious adenovirus type 40 and 41 by dna restriction analysis and by neutralizing monoclonal antibodies species-specific identification of human adenoviruses by a multiplex pcr assay acknowledgment this work was supported in part by the cuban national program of surveillance and control of acute respiratory infection. the nucleotide sequence was performed in the instituto de salud carlos iii, madrid, spain, and was funded by the proposal mpy-1177/06 entitled caracterización molecular de hadv detectados en muestras clínicas de pacientes con infección respiratoria. the key: cord-103630-nt3ogyzl authors: deal, c. l.; thauland, t. j.; signer, r.; nelson, s. f.; undiagnosed diseases network,; lee, h.; butte, m. j. title: recurrent respiratory viral diseases and chronic sequelae due to dominant negative ifih1 date: 2020-07-06 journal: nan doi: 10.1101/2020.07.01.20105379 sha: doc_id: 103630 cord_uid: nt3ogyzl viral respiratory infections are the most common childhood infection worldwide. however, even common pathogens can have significant consequences in the context of patients with primary immunodeficiency diseases. more than half or viral infections annually are due to rhinovirus/enterovirus strains. most clinical manifestations of viral infection are mild. however 3% of cases result in hospitalization in patients who have no other known risk factors. these patients may have an inborn error of immunity, a genetic susceptibility to viral infections. here we present the case of an adult male who suffered respiratory viral infections his whole life and developed chronic, inflammatory damage to sinuses and lungs as a consequence. genomic sequencing identified compound heterozygous variants in the ifih1 gene, encoding the protein melanoma differentiation association protein 5 (mda5), a rig-i-like cytoplasmic sensor of rna intracellular infections. we show a dominant negative effect on these variants on the level of interferon-induced expression of mda5 protein. this work supports that loss-of-function variants in ifih1 affect the sensing of viral infections. underlying genomic variants may dictate the point at which recurrent, respiratory viral infections leave commonplace experience and incur lasting damage. viral respiratory infections are the most common childhood infection worldwide. however, even prevalent pathogens can have significant consequences in patients with primary immunodeficiency diseases (pid). more than half of viral infections annually are due to rhinovirus/enterovirus strains. most clinical manifestations of viral infections are mild. however, 3% of cases result in hospitalization in patients who have no other known risk factors (1) . these patients may have a genetic susceptibility to viral infections. interferon-induced helicase c domain-containing protein 1 (ifih1) is a gene encoding melanoma differentiation association protein 5 (mda5), a rig-i-like cytoplasmic sensor of long double-stranded rna, which is key in innate immune recognition of rna viruses. on binding viral dsrna, mda5 triggers signaling molecules ips-1, irf3 and irf7 resulting in transcription of type 1 interferons (2) . autosomal dominant gain-of-function variants in ifih1 are known to cause aicardi-goutières syndrome (3) . other variants have been found to predispose to autoimmune conditions like type-1 diabetes, vitiligo, autoimmune thyroiditis, and sle (4) . furthermore, biallelic loss of function variants in ifih1 result in severe infections due to common viral illnesses (2, 3) , and one study suggested a possible dominant negative role for heterozygous loss-of-function variants (1) . patients with loss-of-function variants in ifih1 are susceptible to common viral pathogens, particularly human rhinovirus. importantly, patients with these variants have a normal evaluation of their circulating immune cells, because the defect does not affect the generation or function of adaptive immune cells like t and b cells. however, patients carrying these variants are at risk of severe clinical manifestations due to viral infections as well as bacterial superinfections. understanding who is susceptible can allow for better preventative care for these individuals. a 57-year-old male was referred to the undiagnosed diseases network for chronic pansinusitis. throughout childhood he suffered numerous viral upper respiratory infections that were often accompanied by wheezing. in his late 20s he developed wheezing episodes with shortness of breath that were refractory to all treatments other than oral corticosteroids. in his 30s and 40s these episodes became more frequent, increasing from two episodes annually to five episodes annually. in his 50s he developed anosmia and was found to have nasal polyps. he underwent 3 sinus surgeries for polyposis and sinusitis as well as tympanostomy tubes bilaterally for bilateral ear pain. after surgical intervention, he continued to suffer from pansinusitis ( figure 1a) . he did not suffer from fungal infections, skin infections, invasive bacterial infections, or chronic viral infections such as warts, cmv, or ebv. he had eosinophilic asthma but normal total ige. he had negative skin prick tests to aeroallergens and undetectable serum-specific ige to aeroallergens. he tolerates aspirin. his chest ct showed evidence of atelectasis and bronchiectasis ( figure 1b) . immune evaluation revealed mildly low total igg of 577 mg/dl due to mildly low igg1 and igg3 subclasses. he had normal lymphocyte subsets, normal pneumococcal titers after immunization with ppv23 (pneumovax), and protective tetanus and diphtheria titers long after immunization (table 1) . he had a history of laparoscopic nissen fundoplication for gerd. he was found to have an ascending aortic aneurysm, and family history was only remarkable for cardiac disease in mother, sister and brother. he recently developed signs of peripheral neuropathy. a part of his evaluation included ophthalmology screen which revealed ocular albinism ( figure s1 ). all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july 6, 2020. . https://doi.org/10.1101/2020.07.01.20105379 doi: medrxiv preprint whole exome sequencing revealed compound heterozygous variants in ifih1 resulting in nm_022168:c.1879g>t np_071451.2:p.e627* (rs35744605) and c.1066c>a p.p356t (rs150317197) (5-8) ( figure 1c) . the global minor allele frequency of the first variant in the population is roughly 0.003 and the second is 0.0006, which each fall below the population maximum allele frequency and so could be causative variants (9) . we pursued these variants knowing that a dominant negative variant could play a role in pathogenesis. the second variant has been biochemically studied in isolation, expresses well, and showed a near-normal ability to drive ifn-β in response to poly-i:c stimulation (3). the first variant, on the other hand, was ascribed to act in a dominant negative fashion when cooverexpressed in 293t cells with wt mda5 protein (1), but this effect was not shown in primary cells. thus, we sought to determine the amount of mda5 in our patients' cells. since mda5 expression is responsive to interferon α (ifn-α) stimulation, pbmcs from patient and control were stimulated overnight with ifn-α. cells were lysed and examined by western blot using a monoclonal antibody targeted to a region more c-terminal than the missense variant and more n-terminal than the truncation variant. in this way, the expression of both the truncated and mutated forms of the protein could potentially be detectable. we saw none of the truncated form of the protein in primary cells. the patient's mda5 levels were lower by over half at baseline as compared to control, when normalized to gapdh. an increase in expression of mda5 was seen after stimulation with ifn-α in both the subject and a control ( figure 1d and figure s2 ). in each condition, the relative amount of mda5 expression was ~20% of the control, well below the 50% one would expect if the protein amount were determined by the missense variant alone. these results show that the nonsense variant disproportionately and dominantly reduces the expression or stability of the protein bearing the missense variant. there have been few case reports of ifih1 loss-of-function in children, and no cases yet reported in adults. therefore, the complete clinical phenotype still remains poorly understood. patients described thus far have a narrow spectrum of severe and recurrent respiratory viral infections with bacterial superinfections, but no other major viral infections (ebv, warts) (3). prior cases report normal immune evaluation including responses to vaccines, with perhaps the exception of minor abnormalities in igg and igg subclasses, as seen here. our patient was incidentally found to have ocular albinism and ascending aortic aneurysm. it is unlikely these findings are related to his ifih1 variants, but are pertinent findings in a poorly described phenotype. the nonsense mutation in the middle of the protein eliminates the c-terminal regulatory domain that is essential for recognizing viral dsrna. this results in a disruption of overall protein stability by the truncated variant (1). therefore, in our patient, these variants resulted in decreased mda5 expression. our case thus supports the previous demonstration of a potential dominant negative mechanism in this disease. the ifih1 gene tolerates a higher frequency of loss-of-function variants than would be expected for a rare disease. evolutionary pressures on genes required for survival should, over time, "purify" away loss-of-function variants to low frequencies. on the other hand, loss-offunction variants of ifih1 carry protection from autoimmunity (8,10); conversely, gain-offunction variants with improved viral sensing predispose patients to autoimmunity (4). thus, there may be greater tolerance to loss-of-function mutations than is seen in other pid genes, a potential example of "heterozygote advantage." this reason may explain the relatively high frequency of nonsense variants in the population (0.3% for the nonsense variant, with an allele all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july 6, 2020. . https://doi.org/10.1101/2020.07.01.20105379 doi: medrxiv preprint frequency highest in european populations per gnomad). it is the delicate balance between an efficient immune response and autoimmunity that balances the selective pressures on this gene. the phenotype of "susceptibility" to respiratory viral infections that have almost universal prevalence raises an important question about when it would be pragmatically useful to actually proceed with an immunological workup for individuals suffering from viral infections. we suggest that all patients with recurrent or severe, respiratory viral infections, including covid-19, should be evaluated, especially those with evidence of irreversible inflammation and scarring (bronchiectasis, polyp disease). if this subject's inborn error of immunity had been discovered earlier in life, some of his permanent complications may have been preventable. in summary, we present the oldest case of an individual suffering from compound heterozygous ifih1 variants resulting in a phenotype of recurrent viral infections, nasal polyposis and chronic pansinusitis due to bacterial and fungal organisms, marked chronic eosinophilia, severe asthma with ground glass opacities and bronchiectasis, as well as neuropathy. human subjects: the patient enrolled in this study provided written informed consent. all methods in this study were approved by the national human genome research institute (nhgri) central institutional review board. antibodies: anti-cd3ε af647 (hit3a), cd19 bv421 (hib19), cd56 af647 (5.1h11), cd14 bv421 (hcd14), cd11c af488 (3.9), and human trustain fcx were from biolegend. anti-mda-5 (d74e4) was from cell signaling technology. anti-gapdh (g-9) was from santa cruz biotechnology. fluorescently labeled anti-mouse and anti-rabbit secondary antibodies were from li-cor biosciences. facs analysis: pbmcs were isolated from whole blood by density centrifugation. fc receptors were blocked, and samples were stained on ice for 20 min followed by three washes. data were collected on a cytek dxp instrument and analyzed with flowjo (treestar). western blots: pbmcs were stimulated overnight with the indicated concentrations of ifn-α (cell signaling technology) in media comprising rpmi plus 10% fbs, 1x pen/strep, 10 mm hepes, 1 mm sodium pyruvate, and 55 μm 2-mercaptoethanol. cell pellets were lysed with ripa buffer supplemented with halt protease inhibitors (thermofisher), and insoluble material was removed by centrifugation. reduced lysates were separated by sds-page and transferred to nitrocellulose membranes. membranes were blocked with pbs-tween containing 5% bsa and probed with primary and secondary antibodies. data were collected on a sapphire biomolecular imager (azure biosystems) and analyzed with the 'gels' plugin in imagej. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july 6, 2020. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july 6, 2020. . severe viral respiratory infections in children with ifih1 loss-of-function mutations recurrent and prolonged infections in a child with a homozygous ifih1 nonsense mutation recurrent rhinovirus infections in a child with inherited mda5 deficiency the a946t variant of the rna sensor ifih1 mediates an interferon program that limits viral infection but increases the risk for autoimmunity identification of loss of function mutations in human genes encoding rig-i and mda5: implications for resistance to type i diabetes loss-of-function mutations e627x and i923v of ifih1 are associated with lower poly(i:c)-induced interferon-β production in peripheral blood mononuclear cells of type 1 diabetes patients reduced expression of ifih1 is protective for type 1 diabetes rare variants of ifih1, a gene implicated in antiviral responses the mutational constraint spectrum quantified from variation in 141,456 humans analysis of predicted loss-of-function variants in uk biobank identifies variants protective for disease the authors declare no competing interests. key: cord-275997-4ibeidyw authors: goldrick, barbara a. title: the practice of infection control and applied epidemiology: a historical perspective date: 2005-10-31 journal: am j infect control doi: 10.1016/j.ajic.2005.04.250 sha: doc_id: 275997 cord_uid: 4ibeidyw the united states health care system and patient populations have changed substantially over the past several decades. the practice of infection control also has evolved since the landmark study on the efficacy of nosocomial infection control project, and infection control professionals (icps) must continue to develop the knowledge and skills necessary to practice infection prevention and control. practice analyses of infection control conducted between 1982 and 2001 were analyzed to determine changes in practice. these data reflect a 145% increase in infection control activities over a 20-year period. however, resources for infection control and prevention have not kept pace with this increased activity. in addition, the current trend toward mandatory reporting of health care-associated infections (hais) among several states will add more tasks for icps with limited resources, at the risk of spending less time on prevention and control activities. in keeping with its philosophy of quality health care and responsible public reporting, the association of professionals in infection control and epidemiology, inc, continues to explore the issue of mandatory reporting of hais. the practice of infection control and applied epidemiology: a historical perspective barbara a. goldrick, rn, phd, mph, cic chatham, massachusetts the united states health care system and patient populations have changed substantially over the past several decades. the practice of infection control also has evolved since the landmark study on the efficacy of nosocomial infection control project, and infection control professionals (icps) must continue to develop the knowledge and skills necessary to practice infection prevention and control. practice analyses of infection control conducted between 1982 and 2001 were analyzed to determine changes in practice. these data reflect a 145% increase in infection control activities over a 20-year period. however, resources for infection control and prevention have not kept pace with this increased activity. in addition, the current trend toward mandatory reporting of health care-associated infections (hais) among several states will add more tasks for icps with limited resources, at the risk of spending less time on prevention and control activities. in keeping with its philosophy of quality health care and responsible public reporting, the association of professionals in infection control and epidemiology, inc, continues to explore the issue of mandatory reporting of hais. (am j infect control 2005;33:493-500.) the major infection control movement emerged in the united states in the 1970s. and by the mid-1970s, thousands of hospitals throughout the country had infection surveillance and control programs (iscps) in place. however, it was not until the landmark study on the efficacy of nosocomial infection control (senic), which was conducted by the centers for disease control (cdc) in the mid-1970s, that the link between iscps and the reduction of nosocomial infections (nis) in acute care facilities was established. 1 the senic demonstrated that effective iscps were associated with a 32% reduction in nis. 2 the senic project found that, prior to 1970, very few hospitals in the united states had an infection control professional (icp), but, by 1977, 80% of the hospitals had at least 1 icp. 3 in the early 1970s, the cdc recommended that hospitals have at least 1 full-time equivalent (fte) icp for every 250 beds 4 ; however, the senic found that only 28% of the icps surveyed worked full-time specifically on infection control activities, and the amount of time was related to hospital size, with an overall average of 24 hours per week. 3 the extensive 1976 revisions to the joint commission on accreditation of hospitals (jcah) guidelines for infection control, which formally recommended a position responsible for surveillance, added further impetus to include icp positions to hospital infection control programs. 5 until the senic, there were no published studies that assessed the dimensions of infection control practice; therefore, the senic project included a study of the role of the icp. nearly all (94%) of icps surveyed in 1976-1977 were registered nurses (rns), with two thirds of these having a diploma in nursing, one fourth a baccalaureate degree, and 8% an associate degree. the senic also identified 5 practice activities among icps: surveillance, policy development, training, epidemic investigations, and consulting 3 (table 1) . however, there were no certification standards for the icp at the time of the senic project. the association for practitioners in infection control, inc. (apic), a multidisciplinary organization, was established in 1972 to meet the education and practice needs of icps in the united states. the name was changed to the association for professionals in infection control and epidemiology, inc, in 1994 to recognize the organization's maturation and evolution into the broader context of health care delivery in the united states. 6 apic's sister organization, community and hospital infection control association-canada (chica-canada), was incorporated in 1976 for icps practicing in canada. 7 the apic developed 8 educational standards for infection control practice in 1980, which included epidemiology; microbiology; infectious diseases; sterilization, disinfection, and sanitation; patient care practices; education; management and communication; and employee health. 8 these standards were consolidated into the apic curriculum for infection control practice, which was published in 1983. 9 the first infection control practice analysis (pa) was conducted in 1982 at the request of the certification board of infection control (cbic), which was established in 1981. 10 the first pa survey collected demographic data and data from a task inventory rating scale, which consisted of 99 task statements, categorized according to the 8 educational standards of apic. 8 a randomized stratified sample of 600 icps who would receive the pa questionnaire was deliberately skewed toward larger hospitals (.100 beds) because the pa committee felt that these hospitals would most likely employ icps who performed the full range of infection control activities. based on inclusion/exclusion criteria, a total of 317 icp respondents' data were used for analysis to determine key tasks for infection control practice. 11 most (88%) of respondents in the 1982 pa survey were rns who worked in community hospitals (78%) with .200 beds (73%). all but 4% of the respondents' hospitals were accredited by the jcah. 12 although the majority of the respondents were full-time employees, 17% spent less than 20 hours/week in infection control, and many held multiple positions within the hospital; however, 54% of the icps surveyed indicated that infection control was their primary responsibility. the majority (72%) of respondents had been in infection control practice between 2 and 10 years, and nearly all (96%) had attended educational programs in infection control. sixty tasks (activities) related to the 8 areas/dimensions of infection control practice were identified as relevant in the 1982 pa 11 (table 1 ). the 1982 pa was the basis for the first infection control certification examination, which was offered in the united states in 1983. 11 it also defined and described the scope of infection control practice for the first time and established a baseline for measuring progress and changes in practice. 11 recertification for icps is required every 5 years; therefore, the cbic repeats the pa every 5 years. 10 by the 1980s, infection control practice had expanded beyond the acute care setting. infection control practice also had changed considerably. new infectious diseases such as acquired immunodeficiency syndrome had emerged, prospective payment systems for hospitals were in place, and the joint commission on accreditation of health care organizations (jcaho; formerly jcah) instituted outcome measures as part of the accreditation process. these changes required new or enhanced skills for the icp. therefore, in 1987, 5 years after the first infection control pa, a study was conducted to update and revalidate the original (1982) pa to ensure that the content of the certification examination was a valid representation of infection control practice. a modified delphi technique was used between 2 panels of icps and further validated by a third panel of subject matter experts for a total of 29 icps. there was a high level of agreement between respondents on the 1982 pa, the 1987 icps, and the expert panel, providing content validity for the certification examination based on current infection control practice. eight new tasks (activities) were identified for a total of 67 tasks in 8 areas (dimensions) of infection control practice (table 1) . these additional tasks ''reflect[ed] an expanding role of the icp as a planner and manager.'' 13 the cbic conducted its second pa survey among icps in 1992, which included a random sample of canadian icps for the first time. the original 1982 pa list was used with modifications to reflect current infection control practice and terminology. a total of 577 responses were available for analysis. the majority (87%) of respondents were rns; 9% held an associate degree; 44% held a baccalaureate degree; and 24% held an advanced degree. 14 the majority of respondents had 9 years or more in infection control practice (56%), worked in hospitals with greater than 200 beds (64%) less than 40 hours a week (52%), and were certified in infection control (72%). ninety-five tasks identified in the 1992 pa were organized into 5 major practice dimensions describing the responsibilities of icps in the united states and canada: infectious process, surveillance/epidemiologic investigation, transmission of infection, management and communication, and education 14 (table 1) ; however, new tasks were added, and outdated tasks were eliminated. 14 each pa builds on those conducted previously and is an important component of ensuring content validity that reflects current infection control practice. this process of content validation involves systematic collection of information that describes behaviors and activities (tasks) performed by occupants of the practice in question. 15 because of the numerous changes in health care delivery, which affected the scope and practice of infection control since the 1992 pa, a more contemporary analysis of infection control practice was needed in the 1996 pa survey conducted by the cbic. the application of decision rules ensured that the resulting certification examination accurately reflected infection control practice in the united states and canada, regardless of the region or size of facility. a representative sample across all health care settings was selected from the apic and the chica-canada icps to receive the 1996 pa questionnaire. 16 a return of 1530 responses significantly exceeded the n 5 1067 required by power analysis. as in past pa surveys, the majority (85%) of respondents in the 1996 survey were rns. thirteen percent of respondents held an associate degree; 41% held a baccalaureate degree; and 22% had an advanced degree. 16 the majority of respondents in the 1996 pa survey worked in acute care settings (65%), worked in facilities with greater than 100 beds (68%), and worked less than 40 hours per week in infection control (62%). nineteen percent of the respondents worked in long-term care (14%), mental health facilities (3%), and rehabilitation centers (2%). thirty-five percent of the respondents had 10 or more years of experience in infection control practice, but less than half (48%) were certified in infection control. 16 the completed inventory on the 1996 pa resulted in127 tasks organized into 5 major practice areas, 16 as listed in table 1 . the apic/chica-canada infection control and epidemiology: professional and practice standards were published in 1999. 17 the document has the following 2 sections: (1) professional standards that the icp is expected to meet or exceed and (2) practice standards that the icp is capable of meeting, regardless of applicability to the specific practice setting. these professional and practice standards were synthesized into the most current pa survey, which was conducted by the cbic in 2001, to ensure that the resulting content outline was consistent with current standards of infection control practice. the primary source for the 2001 pa survey questionnaire was the 1997 infection control professional task list. 16 the target population for the survey consisted of icps in the united states and canada who met the eligibility requirements for taking the certification examination. a final sample of 1306 responses were available for analysis in the 2001 pa survey, which significantly exceeded the n 5 1067 required by power analysis. therefore, results from this sample could be generalized to icps in the united states and canada. in the continuing process of serving the international infection control community, the survey instrument also was distributed to a selected sample of international icps. 18 table 1 outlines the 6 major areas of infection control practice identified in the 2001 pa. two changes in the major areas of practice were made. these included a category that incorporates both education and research. research had not been considered in previous pas; however, because there were only 5 tasks associated with the ''research'' category, it was combined with the ''education'' category, and the category was renamed ''education and research.'' several new tasks associated with employee health also were identified. as a result, all aspects of employee health were separated into a new category: ''infection control aspects of employee health,'' which had previously been included in the category that addressed preventing and controlling the transmission of infectious agents. 18 table 1 indicates that 147 tasks related to the 6 areas of infection control practice were identified in the 2001 pa. the majority of respondents in the 2001 pa survey was rns (80%), held a baccalaureate degree or advanced degree (73%), and was certified in infection control (84%). more than half (55%) of the respondents had been in infection control practice more than 10 years. most of the respondents (56%) worked in an acute care hospital with $200 beds (51%) with #1 full-time equivalent icp (59%), and nearly half (49%) worked 40 or more hours a week. eight percent of the respondents worked in long-term care (compared with 14% in 1996), 4% worked in public health, and 2% worked in mental health facilities. 18 the 2001 pa reflected changes in the practice of infection prevention/control and applied epidemiology at that time and identified the responsibilities of the icp both in the united states and in canada. 18 however, several new infection control activities have been identified since 2001. to remain current, the cbic will conduct its next pa in 2006, which will include tasks related to changes in infection control practice since 2001. table 1 illustrates that the practice of infection control evolved significantly between 1976 and 2001. figure 1 shows the increase in infection control tasks from 60 to 147 between 1982 and 2001, a 145% increase over a 20-year period, with little or no additional resources allotted to infection control programs during that time. although the 2001 pa survey 18 was undertaken early in 2001, long before the september 11, 2001 , attacks on the united states and the department of homeland security was formed, the cdc developed a plan to upgrade the nation's public health infrastructure to respond to acts of biological terrorism. 19 in 1999, the apic and the cdc developed a bioterrorism readiness plan ''. to serve as a tool for [icps] and health care epidemiologists to guide the development of practical and realistic response plans for their institutions in preparation for a real or suspected bioterrorism attack.'' 20 as a result, many icps across the united states developed policies and procedures based on the apic and the cdc recommendations to prepare their health care facilities to deal with bioterrorism, and, although bioterrorism was not a separate infection control practice category listed in the 2001 pa, 18 the tasks related to attacks of biologic warfare are another layer in the practice of infection control, which deals with the prevention and control of the transmission of infectious agents (table 1 ). since the september 11 attacks, there has been a substantial increase in icps involvement in emergency preparedness and management for bioterrorist attacks and emerging infections. for example, the recent emergence of severe acute respiratory syndrome (sars) and the threat of a new influenza pandemic are further examples of the need for due diligence in infection control. the main lesson learned from the sars outbreak was that it was contained through the conscientious application of enhanced infection control measures at the national and local levels. these same measures will defeat sars should it reemerge. control of an emerging infection requires swift action by health care providers and an adequate public health infrastructure. 21 as change in the health care system continues, infection control practice also must evolve. icps are instrumental in ensuring that their health care facilities comply with new regulations and guidelines, which include education of health care workers on proper hand hygiene, prevention of needlestick injuries, isolation techniques, and appropriate use of personal protective equipment, and are prepared for emerging and reemerging infectious diseases. in addition, changes in the us health care infrastructure occurred during the past 20 years when many hospitals merged or became part of multihospital health systems that included health care facilities across the continum of care. these changes in the organization and delivery of health care services prompted an expansion of the role of many icps to include not only the responsibility for infection control programs in acute care and/or long-term care facilities but the added responsibility for nonacute health services such as freestanding surgery centers, medical and dental clinics, rehabilitation units, child and adult day care centers, and home care. however, icps report that resources for the infection control staff have remained static despite the need to respond to emerging infections and implementation of new regulations and guidelines. emergence of multidrug-resistant microorganisms in all health care facilities also has necessitated increased icp activity. 22 o'boyle et al used a delphi method to study infection control tasks in addition to those found in the 1996 pa, along with additional responsibilities. 22 a panel of experts (n 5 32) from 20 states, who represented icps in acute care, long-term care, and community settings, identified 14 new tasks added to the 127 listed in the 1996 pa. the delphi panel also estimated the percentage of time used in the major infection control practice domains. 22 the activity with the greatest average estimated time was surveillance (27%), followed by education (16%), prevention (14%), and communication (14%). the activity with the least average estimated time was control measures (8%). lack of adequate resources was seen as influencing icps' ability to perform tasks across all infection control functions. 22 the panelists in the study determined that a ratio of 0.8 to 1.0 icp for every 100 occupied acute care beds was adequate infection control staffing. 22 despite the additional responsibilities involved in the practice of infection control, the ratio of 1 icp for every 250 acute care beds has continued in many health care facilities in the united states since the 1976 senic project. 2 however, the ratio of icps per 250 beds was a recommendation that was not evidence based. two recent reports recognized that the complexity of the current practices of icps has changed considerably since the senic and that the old ratio of 1 icp per 250 beds is no longer adequate. the authors also recommend that infection control programs be based on the scope of the infection control program rather than bed size. 23, 24 in the past year alone, 5 new guidelines for infection control practice were published. icps must be aware of these guidelines and implement those recommendations that are strongly evidence based. 25 the cdc recently released draft documents of revised isolation procedures and tuberculosis prevention guidelines. when the final documents are published, icps must implement the new recommendations. in addition, revised jcaho infection control standards, which went into effect in january 2005, include an emergency management plan (standard ic.6.10) that requires health care facilities to respond to epidemics and infections that may require expansion of patient care over extended periods of time. 26 the first report, which addressed the ratio of icps per number of beds since the senic project, was published in the canadian journal of infection control in the summer of 2001. 27 a canadian infection control alliance, consisting of infection control experts, was asked to reach consensus on the key components and resources needed to support effective infection prevention and control programs across the health care continuum: acute care settings, long-term care facilities, and community and home care settings. the alliance recommended 3 full-time equivalent icps for every 500 beds in acute care settings and 1 full-time equivalent icp for every 150 to 250 beds in long-term care facilities. the projected needs for icps in each of these settings was determined based on the expertise represented by members of the alliance. 27 22 and the canadian infection control alliance study 27 were conducted before the september 11 attacks on the united states and therefore do not reflect current infection control practice, which now includes emerging infections and bioterrorism preparedness. one state, new jersey, recently recognized the importance of infection control programs and published revised hospital licensure regulations that mandate an icp ratio of 1 full-time icp per 200 adjusted-occupied beds as a minimum standard. the adjusted-occupied bed calculation takes into account patient days, outpatient factors, and case mix. also mandated was certification by the certification board in infection control and epidemiology for all icps within 5 years of beginning infection control practice. 30 the 1999 landmark institute of medicine (iom) report on medical errors identified nosocomial infection surveillance as a model for voluntary patient safety reporting systems. 31 the national nosocomial infection surveillance (nnis) system, created by the cdc in 1970 to establish a national nosocomial infections database, is the nation's largest and oldest performance measurement system devoted to hospital-acquired infections. 32 the nnis system started with 62 hospitals in 1970, and, by 2000, approximately 315 hospitals were participating in the nnis system. participation in the nnis system is voluntary and involves only acute care facilities in the united states. surveillance data are collected uniformly by trained icps using standardized protocols that target inpatients at high risk of infection and are reported routinely to the cdc at which they are aggregated into a national database. participating hospitals are assured by law that the cdc will not provide any information that would identify any individual or institution and that the data received will be held in strict confidence. 32 icps in the nnis system collect data for selected ''surveillance components'': adult and pediatric intensive care units, high-risk nursery, and surgical patients, using standard cdc definitions that include both clinical and laboratory criteria. nnis data provide benchmarks to guide hospitals within and outside the nnis system to improve efforts aimed at reducing infection rates. nnis reports are published in the biomedical literature on a regular basis, with the latest providing data from january 1992 through june 2004. 33 the infrastructure of the nnis system offers a national resource on which to build improved voluntary patient safety monitoring efforts, as outlined in the 1999 iom report. 34 other than the nnis system, there currently is no national standardized method for collecting hospital infection data. in addition, because each hospital monitors those infections and procedures that are most risky for their specific patient populations, all hospitals do not monitor the same infections. nonetheless, by early 2005, 5 states-pennsylvania, missouri, illinois, virginia, and florida-had new regulations, which mandate that icps report hais to state agencies; however, each state's requirements differ. for example, the missouri regulations call for the ''methodologies and systems for data collection established by the federal centers for disease control and prevention national nosocomial infection surveillance system, or its successor .'' 35 the florida bill, on the other hand, would allow patients to request and obtain information about hospital infection rates. 36 mandatory reporting of hais is currently pending in at least 30 other states. 6 legislation for mandatory reporting of hais in california was vetoed by governor schwarzenegger, making the following case: 37 ''. infection control programs have considerable merit and are currently in effect. the department of health services and the joint commission on accreditation of health care organizations scrutinize hospital infection control programs and the national quality initiative is expected to more than double the number of quality indicators tracked by may 2005. this calls into question the need of a new program to address this issue .. the absence of data auditing and review by impartial clinical experts may call into question the quality and ultimate validity of the data on hospital-acquired infections ..'' the healthcare infection control practices advisory committee (hicpac) (formerly the hospital infection control practices advisory committee), which is authorized under the public health service act, advises the secretary, department of health and human services (dhhs) and the cdc regarding the practice of infection control and strategies for surveillance, prevention, and control of hais, antimicrobial resistance, and related events in settings in which health care is provided. the committee also advises the cdc on periodic updating of existing guidelines, development of new guidelines, and other policy statements regarding the prevention of hais. 38 in response to the recent states' legislation regarding mandatory public reporting of hais, hicpac released a document, guidance on public reporting of healthcare-associated infections, which includes recommendations for use by policy makers and organizations that are tasked to design and implement public reporting systems for hais. however, based on an extensive review of the scientific literature, hicpac found no conclusive evidence for or against public reporting of hais as a method to prevent or control their occurrence in health care settings. 39 the apic supports the right of consumers and purchasers of health care to expect quality health care and responsible public reporting of performance indicators. 6 in its 1998 position paper entitled ''release of nosocomial infection data,'' 40 the apic outlined specific guidelines for interhospital comparison of hai surveillance data. these included (1) trained icps who use standardized protocols for data collection, (2) maintenance of a continued level of surveillance over time, (3) consistent use of valid case definitions for identifying infections, (4) appropriate use of denominator data and time periods for rate-based data, and (4) risk stratification to control for different levels of illness among patients. 40 in keeping with its philosophy, mandatory reporting of hais has become a high priority issue for the association. on march 14, 2005 , the apic released its ''position on mandatory public reporting of healthcare-associated infections.'' 6 the apic continues to explore this issue with other stakeholders and to identify and develop informational resources to assist its members at the local level. 6 the united states health care system and patient populations have changed substantially over the past several decades. the practice of infection control also has evolved, and icps must continue to develop the knowledge and skills necessary to practice infection prevention and control as changes in health care, standards, guidelines, and regulations evolve. practice analyses of infection control conducted between 1982 and 2001 reflect an increase in infection control activities from 60 to 147 tasks in 6 areas of infection control practice, a 145% increase over a 20-year period. however, resources have not kept pace with the increase in infection control activities. in addition, the recent trend toward public reporting of hais will add more tasks for icps with limited resources, at the risk of spending less time on prevention and control activities. in keeping with its philosophy of quality health care and responsible public reporting, the apic continues to explore this issue. the emergence of infection surveillance and control programs in us hospitals: an assessment, 1976 the efficacy of infection surveillance and control programs in preventing nosocomial infections in us hospitals the infection control nurse in us hospitals, 1976-1977: characteristics of the position and its occupant a hospital program for control of nosocomial infections joint commission on accreditation of hospitals (jcah) association for professionals in infection control and epidemiology (apic) community and hospital infection control association (chica)-canada educational standards of the association for practitioners in infection control the apic curriculum for infection control practice. volumes i and ii certification board of infection control and epidemiology (cbic) part ii: tasks, knowledge, and abilities for practice a task analysis of infection control practitioners, 1982. part i: methodology and demography validating the certification process for infection control practice job analysis 1992: infection control practitioner practice analysis: building the foundation for validity job analysis 1996: infection control practitioner apic/chica-canada infection control and epidemiology: professional and practice standards practice analysis for infection control and epidemiology in the new millennium biological and chemical terrorism: strategic plan for preparedness and response bioterrorism readiness plan: a template for healthcare facilities public health grand rounds staffing requirements for infection control programs in us health care facilities: delphi project requirements for infrastructure and essential activities of infection control and epidemiology in out-of-hospital settings: a consensus panel report requirements for infrastructure and essential activities of infection control and epidemiology in hospitals: a consensus panel report. society for healthcare epidemiology of america the state of the science of health care epidemiology, infection control, and patient safety critical access hospitals surveillance, prevention and control of infection development of a resource model for infection prevention and control programs (ipcps) in acute, long term, and home care settings: conference proceedings of the infection prevention and control alliance development of a resource model for infection prevention and control programs (ipcps) in acute, long term, and home care settings: conference proceedings of the infection prevention and control alliance canadian report: icp needs across the health care continuum institute of medicine (iom) national nosocomial infections surveillance (nnis) system report, data summary from characteristics of hospitals and infection control professionals participating in the national nosocomial infections surveillance system section 11 consumer union healthcare infection control practice advisory committee (hicpac) healthcare infection control practice advisory committee (hicpac) association for professionals in infection control and epidemiology (apic) the next report from the cdc containing comparative healthcare-associated infection rates, which will include data from the national nosocomial infections surveillance (nnis) system and the first full year of data from the new national healthcare safety network (nhsn), will be published in june 2006. although nnis hospitals began collecting data under the new nhsn protocols beginning in january 2005, they have not been able to report these data to cdc yet, because of unforeseen complexities encountered in launching the web-based reporting tool. until the new report is available, the nnis report issued in october 2004 (published in december 2004) may be used for comparison purposes. key: cord-286337-qk90xb3a authors: hanada, shigeo; pirzadeh, mina; carver, kyle y.; deng, jane c. title: respiratory viral infection-induced microbiome alterations and secondary bacterial pneumonia date: 2018-11-16 journal: front immunol doi: 10.3389/fimmu.2018.02640 sha: doc_id: 286337 cord_uid: qk90xb3a influenza and other respiratory viral infections are the most common type of acute respiratory infection. viral infections predispose patients to secondary bacterial infections, which often have a more severe clinical course. the mechanisms underlying post-viral bacterial infections are complex, and include multifactorial processes mediated by interactions between viruses, bacteria, and the host immune system. studies over the past 15 years have demonstrated that unique microbial communities reside on the mucosal surfaces of the gastrointestinal tract and the respiratory tract, which have both direct and indirect effects on host defense against viral infections. in addition, antiviral immune responses induced by acute respiratory infections such as influenza are associated with changes in microbial composition and function (“dysbiosis”) in the respiratory and gastrointestinal tract, which in turn may alter subsequent immune function against secondary bacterial infection or alter the dynamics of inter-microbial interactions, thereby enhancing the proliferation of potentially pathogenic bacterial species. in this review, we summarize the literature on the interactions between host microbial communities and host defense, and how influenza, and other acute respiratory viral infections disrupt these interactions, thereby contributing to the pathogenesis of secondary bacterial infections. influenza and bacterial pneumonia are the leading cause of morbidity and mortality from infectious diseases worldwide. influenza and other respiratory viral infections predispose patients to secondary bacterial super-infections, which are frequently associated with a more severe clinical course. it is estimated that the so-called "spanish flu" pandemic of h1n1 influenza a virus from 1918 to 1919 resulted in more than 50 million deaths, with many caused by bacterial superinfection leading to secondary pneumonia (1) (2) (3) (4) (5) (6) (7) . even in the antibiotic era, over half of patients with severe infections in the 1957 h2n2 and 1968 h3n2 pandemics had bacterial complications (8) (9) (10) . bacterial co-infection was also detected in ∼30% of cases in the 2009 h1n1 pandemic, with high mortality rates despite administration of appropriate antibiotics (11) (12) (13) (14) (15) (16) (17) (18) . thus, it is evident that a better understanding of the pathogenesis of secondary bacterial pneumonia following viral infections is needed in order to make therapeutic strides for this devastating complication. the mechanisms of post-viral bacterial infection are complex, comprising multifactorial processes mediated by interactions between viruses, bacteria, and the host immune system. the pathogenesis of super-infection has been attributed to direct mucosal/epithelial damage by influenza virus, increased bacterial colonization of the upper and lower respiratory tracts (urt and lrt, respectively), and dysregulation of immune responses, which all lead to increased susceptibility to secondary bacterial infections. however, emerging evidence suggests that our microbial communities residing on our mucosal surfaces likely shape the rigor of our immune responses and shape the ecological relationships between host and pathogens. over the past 10 years, intense interest has focused on examining how the microbial communities which inhabit our bodies-which some consider to be a separate "organ system" given the sheer physical bulk, number of genes, and metabolic activities-govern the balance between health and susceptibility to diseases, including infections. this raises the possibility that disruptions in the normal microbial communities by an acute viral infection might contribute to the development of post-viral bacterial pneumonia. the recent development of culture-independent methods of microbial identification has enabled the study of microbial communities on mucosal surfaces of the human body, referred to as "microbiota." the microbial communities of mammalian hosts are diverse, comprised of bacterial, viruses, archaea, parasites, and fungi. the human microbiome project (hmp) and other similar large-scale sequencing projects worldwide have characterized the distinct microbial communities that have adapted to the unique environmental niches within our bodies, such as the gut, skin, airways, genitourinary tract, and oral cavity. the gut microbiome, in particular, has been shown to play an integral role in shaping the immune system starting early in life, with continued influence on priming the nature and robustness of immune responses throughout one's lifetime. the respiratory tract also harbors distinct communities of microbes, with multiple discrete ecological niches (e.g., nasal cavity, oropharynx, upper airways) that vary in terms of temperature, ph, oxygen tension, mucus production, and other factors. the effects of viral infections on both the gut and respiratory microbiome have recently undergone examination. surprisingly, influenza infection has been found to result in significant changes in the gut microbiome, despite the lack of detectable virions in the gi tract. by comparison, the effects of viral infection on the respiratory microbiome appear to be relatively modest, but detectable. while the effects of these alterations on risk of secondary bacterial pneumonia have not been studied, potential mechanisms by which these changes might modulate susceptibility to secondary bacterial infections include alterations in the nature and magnitude of the immune response in the host (microbiome on host effects) and facilitating growth of pathogens in the absence of normal commensals (inter-microbial effects). in this article, we review the current understanding of how alterations in the microbiome following viral infection might alter host immune responses and increase susceptibility to secondary bacterial infections. although the term "microbiome" encompasses all microbial communities, there is currently a paucity of studies on how the mycobiome (fungal microbiome) and the virome (viral microbiome) affect host defense against respiratory infections and vice-versa; thus, this review will focus on the bacterial microbiome literature. of the niches in the body, the gut microbial community has been the most intensively studied, with over 20,000 publications to date. while the virome and mycobiome (fungi) are also being analyzed, the bulk of the literature has focused on the bacterial component of the microbiome, and thus most of our understanding of the relation of the gut microbiome to host immunity and pathogenesis of chronic diseases comes largely from studies of the bacterial community. during health, the human gut bacterial community is diverse, with each individual harboring over 100 trillion bacteria, comprised of over 150 different species. the gastrointestinal microbiota is dominated by firmicutes (e.g., lactobacillus, bacillus, and clostridium) and bacteroidetes (e.g., bacteroides), with lower abundances of proteobacteria (e.g., escherichia) and actinobacteria (e.g., bifidobacterium) (19, 20) . wild-living mice exhibit more diverse microbiomes, with significant abundance of proteobacteria as well as firmicutes and bacteroidetes (21) . the gut microbiome, in addition to its metabolic functions in the host, plays an integral role in the development, instruction, and priming of the immune system. germ-free (gf) mice (which lack microbiota) have markedly underdeveloped gut-associated lymphoid tissues, decreased number and smaller-sized peyer's patches and mesenteric lymph nodes, and defects in antibody production, compared to specific pathogen free (spf) mice. not surprisingly, germ-free animals exhibit increased susceptibility to multiple types of infections, including viruses, bacteria, and parasites (22) (23) (24) (25) (26) (27) . however, compared to free-living mice or laboratory animals exposed to gut flora from wild mice, spf animals have a more limited microbial community and are also more susceptible to inflammatory diseases, with a reduced immune repertoire including deficits in memory responses (21, 28, 29) . although an extensive discussion of the healthy gut microbiome and its impact on host immunity is beyond the scope of this review, we will highlight a few important aspects of how the intestinal bacterial community microbiome maintains a healthy host immune environment. first, bacterial metabolites generated by gut commensals contribute to the maintenance of intact epithelial integrity, regulatory t-cell development, and a relatively anti-inflammatory immune state. in particular, short-chain fatty acids (scfas) such as acetate, propionate, and butyrate are fermentation products of dietary fiber and carbohydrates by large intestinal bacteria (30) . in addition to being a major energy source for intestinal epithelial cells, scfas promote the development of naive cd4 + t cells into regulatory t cells (31, 32) , induce "tolerogenic" dendritic cells in the intestinal mucosa (33), and limit autoimmity (34, 35) . at the same time, microbial metabolites are integral for promoting immune responses in the gut against pathogens, including inducing secretion of il-18 (36) and defensins (37, 38) . thus, the products of microbiome metabolism are integral to the appropriate regulation of mucosal barrier integrity and immune homeostasis. in addition, specific members of the bacterial community have been shown to foster the proper maturation and development of the immune system. while this is still an area undergoing intense investigation, one notable example is the discovery that segmented filamentous bacteria are critical promoters of intestinal mucosal iga production (39, 40) and th17 cell induction (41, 42) . dysbiosis, or an imbalance in the normal composition of the microbiome, is associated with a variety of chronic diseases, many of which are characterized by chronic inflammation or abnormal metabolism, including inflammatory bowel disease, cardiovascular disease, and diabetes. thus, fostering appropriate levels of diversity and composition of the gut microbial community is critical for promoting health and immune homeostasis. during health, the composition of the microbiome is governed by a number of selective pressures unique to each anatomic niche, including temperature, nutrient availability, ph, oxygen tension, and the local immune environment. shortterm perturbations in the gut microenvironment caused by illness, antibiotic usage, or dietary changes (e.g., starvation) can alter the gut microbiome and subsequently lead to transient alterations in immune responses. thus, investigating whether influenza and other respiratory viruses alter the gastrointestinal microbiome could have mechanistic implications for viralmediated suppression of antibacterial immune responses. although the composition of the gastrointestinal microbiome is largely influenced by dietary patterns, respiratory viral infections could also contribute, along with other stress inducers such as broad-spectrum antibiotics exposure and chronic inflammation. using animal models of pulmonary infections by influenza and respiratory syncytial virus (rsv), multiple groups have shown that the gut microbiome is clearly impacted by respiratory viral infections, despite the lack of detectable respiratory virus in the gut (43) (44) (45) (46) (47) . in a murine model of influenza infection, the investigators found that although the total numbers of bacteria in the gut did not decrease, there was a reduction in the quantities of segmented filamentous bacteria (sfb) and lactobacillus/lactococcus, accompanied by increases in enterobacteriaceae. interestingly, although sfb have previously been shown to induce th17 cells (41, 48) , flu-infected mice had increased il-17a levels and numbers of th17 cells in the small intestine and colon, which appeared to contribute to intestinal injury (43) . in this study, antibiotic treatment prior to influenza infection ameliorated the degree of intestinal injury, but not lung injury, suggesting that gut dysbiosis contributed to local but not systemic inflammation. other groups have similarly reported increased proteobacteria (the phylum of which enterobacteriaceae are members) (44, 45) , decreased firmicutes (which include sfb, lactobacillus and lactococcus species), and increased bacteroidetes (47) following infection by flu or rsvs but not after administration of live attenuated influenza vaccine (laiv), indicating that live viral infection is required for these changes (47) . the increase in proteobacteria appears to be mediated by type i interferons (ifns) (18) , which not only depleted anaerobic bacteria but also increased susceptibility to secondary salmonella colitis. however, caloric restriction also figure 1 | shifts in the mouse gut microbiome in the setting of influenza infection. during an acute respiratory viral infection, changes in the bacterial composition of the gut microbiome can be observed despite the absence of detectable virus in the gastrointestinal compartment. this suggests that systemic immune signals, physiologic changes (e.g., weight loss), and other still unknown factors are disrupting the normal ecology of the gut, thereby leading to dysbiosis. however, the majority of these studies have been conducted in laboratory animals housed under spf conditions. it remains to be determined whether human patients and mammalian hosts with more diverse baseline gut microbiota (i.e., mice in the wild), exhibit similar qualitative or quantitative changes. results in increased relative abundance of proteobacteria and increased bacteroidetes to firmicutes ratio, raising the possibility that decreased oral intake during influenza may contribute to changes in the microbiome (45, 47, 49, 50) . it has also been shown that influenza infection alters intestinal microbiota composition through type ii ifn produced by lung-derived t cells recruited to the intestine (43) . thus, changes in the gut microbiome appear to result not from direct viral effects but from systemic inflammatory signals that travel from the lung and trigger local inflammatory responses in the gut (figure 1 ). interactions between respiratory tract infections and the gut microbiome are bidirectional. while respiratory viral infections can change the gut microbiome, the gut microbiome also shapes the adaptive immune responses against respiratory pathogens. mice pretreated with an antibiotic cocktail showed increased morbidity and mortality during influenza infection (51, 52) . the severity of infection was associated with reductions in dendritic cell migration rate and the number of local t cells. mice given a 4 week oral course of broad-spectrum antibiotics before respiratory viral infection mounted an attenuated anti-pr8 antibody response, were incapable of inducing cd4 + t cell-mediated ifn-γ response to pr8 antigen, and had fewer influenza-specific cd8 + t cells (51, 52) . these mice also had higher viral titers in their lungs (51) . germ-free mice and antibiotic-treated mice also exhibit impaired antibody responses to seasonal influenza vaccination, which was restored by oral administration of flagellated e. coli, demonstrating a dependence on tlr5-mediated sensing of the host microbiota (53) . the gut microbiome is essential for priming innate immune responses against pulmonary infections as well. during viral infections, the degree of macrophage response to respiratory viruses depends on the presence of gut microbes. macrophages from animals treated with antibiotics exhibited defective responses to type i and ii ifns and impaired capacity to limit viral replication, suggesting that intestinal microbiota provide immune stimulation that establishes an "activation threshold" for innate antiviral immune responses (52) . a comparison of c57bl/6 mice from the jackson laboratory (which lack sfb in the stool) and taconic biosciences (which are sfb positive) revealed that sfb-deficient animals have increased lung bacterial burdens and more severe pneumonia when challenged with methicillin-resistant staphylococcus aureus (mrsa) (54) , which was associated with decreased il-17-mediated responses in the lung. another study using broad-spectrum antibiotic treatment followed by intranasal administration of s. pneumoniae in mice demonstrated that microbiome depletion led to decreased survival, increased lung bacterial burden, and increased systemic dissemination of bacteria (55) . antibiotic-pretreated animals displayed altered cytokine profiles in the lung compared to untreated controls following s. pneumoniae infection, including significantly decreased tnf-α levels at 6 and 24 h after infection. additionally, in the microbiota-depleted group, alveolar macrophages and blood neutrophils exhibited decreased phagocytic activity, and decreased inflammatory cytokine production following ex vivo stimulation by toll-like receptor (tlr) ligands such as lipoteichoic acid (lta) (55) . these effects might be mediated in part by decreased nod1 sensing of meso-dap (diaminopimelic acid)-containing peptidoglycan found in gut microbiota, which previously was shown to be essential for priming innate immune responses to s. pneumoniae (56) . thus, antibiotic-induced disruptions in the normal gut microbial community alter multiple aspects of normal host defense against acute respiratory pathogens (figure 2 ). collectively, the studies above suggest that modulation of the gastrointestinal tract microbiome plays an important role in acute respiratory infections, but precisely how the microbiome should be manipulated to promote appropriate immune responses during acute respiratory infections is unclear. currently, clinical studies have shown that although probiotics do not influence the incidence of respiratory tract infection, they do reduce the severity of symptoms and duration of the illness (57, 58) . pinpointing which members of the gut microbial community are essential for proper immune priming is challenging, but necessary for guiding further microbiome-based therapies. clostridium orbiscindens, a member of the human gut microbiome, has been found to produce desaminotyrosine (dat) from metabolism of flavonoids and amino acids. antibiotic-treated mice exhibited markedly decreased fecal and serum dat levels, which was associated with attenuated type i ifn responses to influenza infection and increased mortality (59) . thus, identification of dat-producing microbiota might serve as a modality for priming type i ifn responses against viral infections. another group demonstrated that oral administration of lactobacillus plantarum enhanced the type i ifn response and lowered viral titers in the lungs in a murine model of influenza infection (60) . other lactobacillus strains are known to enhance tnf-α and ifnγ production by nasal lymphocytes upon influenza infection (61) . oral administration of a probiotic cocktail containing lactobacillus restored the immune response and enhanced the activation of signaling pathways associated with recognition of single-stranded rna virus (62) . an alternative approach to administering probiotics is to alter the local metabolic environment to regulate immune responses. a recent report demonstrated that animals fed a high fiber diet had increased generation of scfas, leading to enhanced antiviral cd8 + t cell immune responses and attenuated neutrophil-mediated lung injury during influenza infection, resulting in improved survival (63) . thus, one strategy for decreasing the incidence of post-viral bacterial infections is to limit the severity of the primary viral infection. however, activation of antiviral immune responses, including type i and type ii ifns, have been associated with increased susceptibility to secondary bacterial pneumonia (64, 65) . thus, another strategy is to enhance immune responses against common bacterial causes of pneumonia. one group re-colonized antibiotic-treated or germ-free mice with groups of cultivatable commensal bacteria, and found that administration of lactobacillus reuteri, enterococcus faecalis, lactobacillus crispatus, and clostridium orbiscindens, which are strong stimulators of nod2 (i.e., cytosolic receptor for muramyl dipeptide, which is found in cell walls of certain bacteria), are able to protect against bacterial pneumonia by enhancing gm-csf production (66) . whether viral-induced changes in the gut microbiome is associated with immune defects that promote secondary bacterial pneumonia, or whether the impaired antibacterial defenses observed in virally-infected hosts can be restored by augmenting certain components of the microbiome are important areas to be investigated. the microbiome of the respiratory tract has also been investigated in the context of viral infections. its role in the development of secondary bacterial pneumonia following influenza and other acute respiratory viral infections is unclear. the respiratory tract is the main site of continuous contact with frontiers in immunology | www.frontiersin.org figure 2 | effects of antibiotic pre-treatment on immune responses to influenza, streptococcus pneumoniae, and lipoteichoic acid (lta). the effects of the gut microbiome on immune responses to respiratory pathogens have been investigated by administration of oral antibiotics to generate alterations in the gut flora, followed by acute infection, and analyzing host immune responses compared to non-antibiotic-pretreated animals. multiple aspects of innate and adaptive immune responses are altered in antibiotic treated animals, including decreased antibody production, decreased phagocytic activity, and decreased inflammatory cytokine production by innate immune cells (e.g., alveolar and peritoneal macrophages) following ex vivo stimulation with tlr ligands. exogenous microbes. as is the case with the gut, immunity at the mucosal interface of the respiratory tract is a constant balance of tolerance of commensal and non-invasive microbes and immune activation against pathogens. the urt and lrt have similar microbial community compositions, although microbe densities are much higher in the former in healthy hosts. several factors are known to influence airway microbiome composition including infection history, age, genetics, and structural lung disease. the urt is an interconnected system consisting of the anterior nares, nasal cavity, nasopharynx, sinuses, eustachian tube, middle ear cavity, oral cavity, oropharynx, and larynx, each of which serve as distinct niches with their own microbial communities. in healthy adults, bacteria present in the nasal cavity are typically those associated with skin, predominantly members of the actinobacteria (e.g., corynebacterium spp., propionibacterium spp.), followed by firmicutes (e.g., staphylococcus spp.), and proteobacteria (67) (68) (69) . the oropharynx contains members of firmicutes, proteobacteria, and bacteroidetes, including streptococcus, neisseria, haemophilus, and lachnospira spp. (68, 70, 71) . skin and oral cavity lineages are represented in the nasopharynxe.g., streptococcus, staphylococcus, corynebacterium, and prevotella (70, 72, 73) . a limited number of pathogens including streptococcus pneumoniae, neisseria meningitides, and haemophilus influenzae are commensal bacteria of the urt. in healthy individuals, the microbial community richness (i.e., the total number of bacterial taxa) is lower in the lrt than that in the urt (70, (74) (75) (76) . contrary to dogma that normal healthy lungs are a sterile environment, a distinct, and somewhat dynamic lung microbiome can be identified using sequencing technology, with microaspiration serving as the primary route of microbial immigration from the urt to the lrt (76, 77) . the major phyla in healthy lungs are bacteroidetes and firmicutes, which mainly include prevotella, veillonella, and streptococcus (78) (79) (80) . individuals with chronic airway diseases (e.g., cystic fibrosis, copd) have increased bacterial populations in the lungs (77) and differences in the relative abundance of certain species (81) . impaired airway clearance due to intrinsic or extrinsic factors leads to the proliferation of bacterial species that can exploit this growth opportunity (82) . how respiratory viral infection affects the diversity of microbial communities and whether viral-induced dysbiosis influences immune functions is being examined. nonetheless, bacterial colonization of the urt is generally considered as the first step in the development of invasive bacterial infections (83, 84) , including secondary bacterial infections following respiratory viral infection. bacterial abundance, species diversity, and factors that shape the immune response to subsequent infections are discussed in greater detail below. respiratory viruses enter the human body through the urt and are the most common type of acute infections of the respiratory tract. one possible mechanism by which influenza and other viral infections might predispose infected hosts to secondary bacterial pneumonia is by altering the microbial composition of the upper respiratory tract, fostering enhanced growth of pathogens, and facilitating the subsequent entry of large bacterial loads into the lrt (85) . this section will examine recent literature on how acute respiratory viral infections have changed the urt microbiome. given the effects of viruses on enhancing bacterial adherence to the epithelium (86) (87) (88) , it is perhaps not surprising that multiple studies of human subjects as well as in animal models have shown that viral infections are associated with increased colonization by potentially pathogenic bacteria (known as "pathobionts"). a comparative analysis using qpcr to detect specific bacteria in adult patients with or without influenza a infection showed that staphylococcus aureus, s. pneumoniae, and h. influenzae were present in 12, 24, and 32% of infected patients, respectively as compared to 5, 11, and 10% of uninfected patients (89) . in experimental in vitro models, viral infections increase the colonization rates of various bacteria in the urt (90-95), including s. pneumoniae and h. influenzae (86) (87) (88) . in children, influenza is associated with a 15-fold increase in nasopharyngeal titer of s. pneumoniae (96) . animal models have similarly confirmed that viral infection, particularly influenza, increases bacterial colonization rates in the urt, enhancing the risk of secondary bacterial infections (97) (98) (99) . higher pneumococcal colonization density has been linked to respiratory virus coinfection and invasive pneumococcal pneumonia, after adjusting for age and sex (85) . another case-control study comparing nasopharyngeal bacteria with and without pneumonia also found an association between nasopharyngeal load of s. pneumoniaebut not of h. influenza and m. catarrhalis-and viral coinfection and pneumonia (96) . in addition, viral infections potentially may enhance transmission of bacteria. in a study of mice colonized with s. pneumoniae and then infected with influenza a virus 3 days after, s. pneumoniae transmission occurred only when all mice were infected with influenza and was blocked by an influenza-neutralizing antibody (95) . however, while specific bacteria might gain a competitive advantage during viral infections, this does not universally translate to all bacterial taxa. a recent study of subjects with and without respiratory viral infections demonstrated lower overall bacterial reads from nasopharyngeal samples in virally-infected subjects compared with uninfected controls (100) . the relationship between acute viral infections and bacterial colonization appears to be bidirectional. bacterial carriage or their ligands can increase or decrease viral infectivity rate, thereby positively or negatively influencing the subsequent host immune response to viral infection. viral replication in the respiratory tract can be enhanced by exposure to s. pneumoniae (101) . patients harboring s. pneumoniae are more likely to experience subsequent acute respiratory illness episodes than those without colonization (102) . in addition, bacteria present in the airways can modulate host responses against viral infection. the presence of a nasopharyngeal commensal protected mice against rsv-induced airway hyperresponsiveness. rsv-infected mice who underwent antibiotic-mediated depletion of streptococcus viridans in the nasopharynx exhibited increases in number of inflammatory lymphocytes and airway hyperresponsiveness, and decreases in regulatory t cell number and transforming growth factor-β production (103) . others have shown that colonization of the urt with s. aureus drastically reduced influenza-induced acute lung injury and mortality in mice by recruiting a c-c chemokine receptor type 2 + cluster of differentiation (cd)11b + monocyte subset to the lungs and inducing an m2 macrophage phenotype (104) . with the availability of next-generation 16s rrna sequencing, microbiome-based studies have attempted to discern global patterns of change in the bacterial community of each anatomic niche during viral infections, such as changes in diversity. diversity can be assessed using a variety of indices, such as total number of unique species of the microbiome (i.e., richness) or other measures that account for both richness and the evenness of relative abundance of the members of the community (e.g., shannon index). results from microbiome analyses have not demonstrated consistent changes in diversity when comparing virally infected subjects with healthy controls. this is not surprising given the variability of the subjects sampled, differences in type and severity of viral infections, type and timing of sample collection, and analysis methodology. in some studies, increased bacterial diversity appeared to be associated with influenza severity. a french study of children admitted to the hospital with influenza revealed increased diversity of the nasopharyngeal microflora with increased influenza severity (105) . children with severe influenza showed decreased relative abundance of s. aureus and increased abundance of prevotella, streptobacillus, porphyromonas, granulicatella, veillonella, fusobacterium, and haemophilus. a recent chinese study in patients with h7n9 avian influenza demonstrated significantly increased diversity in the oropharyngeal microbiome of h7n9-infected patients compared to healthy controls, particularly h7n9 patients with secondary bacterial pneumonia (106) . conversely, a french study of nasopharyngeal samples and a south korean study of oropharyngeal samples from patients with acute respiratory viral infections both displayed decreases in diversity indices during viral infections compared to healthy controls (71, 100) . both studies included subjects ranging from infants to adults >80 years of age, limiting conclusions about age-related effects. longitudinal studies conducted in healthy volunteers who underwent experimental self-innoculation with rhinovirus also failed to demonstrate significant changes in diversity of the urt microbiome, while administration of laiv vaccine to healthy adults led to increases in diversity measures following viral challenge (73, 107) . thus, unlike other diseases where decreased diversity is considered deleterious to the host, the effects of viral infections on diversity per se are variable and not presently considered a good indicator of risk for complications, including secondary bacterial pneumonias. microbiome sequencing studies also enable investigators to identify changes in abundance among multiple bacterial taxa simultaneously, beyond just what can be cultured individually. this allows investigators to determine what groups of bacteria are changing in unison during viral infection and which are existing in competition with one another. this information may have implications for the development of probiotic therapies (as discussed below). a recent metagenomics-based study in france reported enrichment of s. aureus, s. pneumoniae, h. influenzae, moraxella catarrhalis and klebsiella pneumoniae in nasopharyngeal samples of subjects with confirmed respiratory viral infections compared to healthy controls (100) . an examination of the oropharyngeal microbiome of pneumonia patients with and without 2009 influenza a h1n1 pandemic viral infection showed that firmicutes (which include staphylococcus and streptococcus spp.) and proteobacteria (mainly pseudomonas amygdali, p. fluorescens, pseudomonas sp. uk4, acinetobacter baumanii and a. junii)-were significantly enriched in patients with influenza (108) . another study of patients with 2009 pandemic h1n1 influenza infection revealed that the predominant phyla of the upper respiratory tract (nasal and nasopharyngeal samples) in patients harboring pandemic h1n1 were actinobacteria, firmicutes, and proteobacteria although normal controls were not included; however, the authors suggested that flu is associated with an expansion of proteobacteria (109) which is generally less abundant in healthy hosts. these findings are supported by another group who found that moraxella and enterobacter spp. (which are classified as proteobacteria) were the most highly represented bacteria in nasopharyngeal samples obtained from patients with pandemic h1n1 influenza (110) . however, these studies demonstrated that there was considerable inter-subject variability, highlighting the need for longitudinal studies to decipher changes following viral infection. investigators have also sought to determine whether specific viruses are consistently linked to enrichment of certain bacterial taxa. in the nasopharyngeal compartment of aboriginal and non-aboriginal children in australia, positive associations were detected between hrv and s. pneumoniae, h. influenza, and moraxella catarrhalis carriage as well as between adenovirus and m. catarrhalis (111) . another study examining the presence of 20 respiratory viruses by pcr panel and prevalence of bacterial carriage in the nasopharynx of children found a strong positive association between s. aureus colonization and influenza virus (112). moreover, s. pneumoniae colonization was positively associated with the presence of hrv and enteroviruses; h. influenzae was positively associated with hrv and rsv; and m. catarrhalis colonization was positively associated with coronaviruses and adenoviruses. a 16s rrna sequencing-based study conducted in infants with acute rsv or hrv respiratory infections reported that infants with rsv had significantly higher abundance of staphylococcus spp. compared to hrv-infected infants (113) . an analysis of the urt bacterial content of 57 healthy asymptomatic individuals and 59 patients with influenza virus, parainfluenza, hrv, rsv, coronavirus, adenovirus, or metapneumovirus by culture-independent pyrosequencing revealed six distinct bacterial profiles-i.e., streptococcus + prevotella + veillonella, streptococcus + haemophilus + neisseria, streptococcus, moraxella, haemophilus, and klebsiella. these profiles, however, were not associated with virus type but were linked to the age of subjects (71) . given that many human studies are cross-sectional in nature, it remains unclear whether post-viral bacterial pneumonias might be the result of viral infections enhancing bacterial colonization or acquisition, colonizing bacteria influencing host susceptibility to respiratory viral infections, or a combination of both. another complicating factor particularly in cross-sectional studies examining the microbiome during viral infections is that the groups are not well-controlled and the sample numbers are relatively small considering the number of variables that could affect the respiratory tract microbiome-such as age, gender, oral hygiene and nose-picking habits, healthcare-based employment status, smoking status, medication use, exposure to small children, etc. the underlying type of viral infection, sampling timepoint after onset of infection, severity of infection, and concomittant antimicrobial usage are other confounding factors. this may underlie the highly variable and sometimes discrepant observations from microbiome studies in patients with viral infections. there have been few clinical studies comparing baseline pre-and post-infection microbiomes in otherwise healthy individuals with acute viral infections due to the difficulty of sampling before infection. however, the relatively few studies available provide insights into the dynamicity and stability of bacteria colonization patterns over time, and whether and how perturbations brought on by acute viral infections alter these patterns. in healthy children, the major phyla among nasopharyngeal microbiotas are proteobacteria, firmicutes, bacteroidetes, actinobacteria, and fusobacteria, with moraxella, haemophilus, streptococcus, flavobacteria, dolosigranulum, corynebacterium, and neisseria as predominant genera. changes in nasopharyngeal microbiome diversity were observed across seasons, with a predominance of proteobacteria and fusobacteria in fall-winter and bacteroidetes and firmicutes in spring; these differences were independent of recent antibiotics and viral co-infection (114) . however, another analysis of two nasopharyngeal washes collected 5.5-6.5 months apart from 40 children and adolescents with asthma showed no significant differences in nasopharyngeal microbiome diversity across seasons, although mean relative abundances of haemophilus, moraxella, staphylococcus, and corynebacterium varied significantly between summer and fall samples and between age groups. moreover, in 87.5% of patients, operational taxonomic units (otus) in patients varied significantly between time points (115) . an investigation of the frequency and seasonal variation in bacterial and viral load in asymptomatic healthcare professionals during the winter and summer months showed that of the 100 subjects tested during the winter, 34 were colonized with at least one bacterial species and 11 tested positive for at least one virus. the most frequently detected pathogens were methicillinresistant staphylococcus aureus (mrsa), m. catarrhalis, and coronavirus. in contrast, of the 100 subjects tested during the summer, 37 harbored at least one bacterium (mainly mrsa and k. pneumoniae) and four tested positive for one virus (116) . several larger scale surveillance studies of mainly pediatric populations have examined the natural temporal patterns in bacterial colonization during viral infections. one clinical investigation assessed the presence and density of s. pneumoniae, h. influenzae, and m. catarrhalis in the nasopharynx of children during urt infection and in the healthy state, and reported that the proportion of children colonized with these bacteria was higher during infection than during asymptomatic surveillance visits. mean density of all bacterial species was significantly higher at each visit when a virus was detected. interestingly, the percentage of colonized children and bacterial density were also higher at asymptomatic visits in which virus was detected than at those in which virus was not detected (117) . another study of 31 families with small children using longitudinal nasal swab sampling demonstrated that rhinovirus infection was associated with increased acquisition of s. pneumoniae from the community as well as increased transmission of s. pneumoniae within the family (118) . other groups have examined the effects of experimental innoculation of hrv into the urt (nares) (figure 3) . these studies reported no significant changes in total read counts or of the main phyla (e.g., actinobacteria, firmicutes, and proteobacteria) over time in nasopharyngeal samples (73) or throat swabs (119) . in the oropharyngeal compartment, rhinovirus infection was associated with a strong trend toward transient increases in the relative abundances of h. parainfluenzae, neisseria subflava and a weak trend toward an increase in s. aureus (119) . by 60 days, abundance of these bacteria had returned to baseline. nasopharyngeal sampling showed completely opposite results, with decreased relative abundance of haemophilus and neisseria spp., but an increase in the normal nasal commensal, propionibacterium, in subjects following hrv infection (73) . no differences in staphylococcus were observed. however, the number of subjects were small in both studies, limiting the power to detect changes over time. nasopharyngeal microbiota composition has been shown to be altered by influenza vaccination (figure 3) . administration of live attenuated influenza vaccine (laiv), which is nasally instilled, to healthy children increased the nasal colonization density of s. pneumoniae in subjects who harbored this bacterium at the time of vaccination, and transiently increased rates of colonization by h. influenza (120) . in healthy adult volunteers, it was demonstrated that intranasal laiv administration induced an increase in the diversity of the nasopharyngeal microbiome, figure 3 | changes in the human upper respiratory tract microbiome following viral exposure. given that bacterial pneumonia frequently arises as a result of aspirated bacterial pathogens, a potential mechanism by which viral infections might increase the risk of secondary bacterial infections is through increased colonization of the upper respiratory tract by bacterial pathogens. in human subjects, live attenuated influenza vaccine (laiv) and human rhinovirus (hrv) have been shown to disrupt the local host bacterial community, with increased relative abundance of potential pathogens (or pathobionts), such as staphylococcal and neisseria species. the major changes in the upper respiratory tract microbiome are highlighted here. as well as relative abundances of staphylococcus and bacteroides (107). these changes were not observed in subjects given saline nasal spray. in a mouse model, bacterial density in the nasopharynx after laiv administration was increased as much as 100,000 times compared to influenza-naive hosts, and the duration of carriage of s. pneumoniae or s. aureus was also increased 2 to 5-fold (121) . however, systemic vaccination can also alter the urt microbiome. a longitudinal study of healthy subjects found a significant association between the presence of lactobacillus helveticus, prevotella melaninogenica, streptococcus infantis, veillonella dispar, and bacteroides ovatus and influenzaspecific h1 and h3 iga antibody response (122) . thus, it is remarkable that a relatively mild viral stimulus such as flu vaccine can lead to detectable changes in the urt microbiome. although the data are still preliminary, animal studies have suggested that antiviral immune activation contributes to changes in the urt microbiome and facilitate colonization by potential pathogens, such as s. aureus. in a mouse model of s. aureus nasal colonization, the absence of type i ifn receptor was associated with decreased persistence of bacteria (107) . type iii ifn, which is also induced during influenza infections, led to changes in the nasal microbiome, including increased numbers of culturable bacteria. increased upper respiratory tract persistence of s. aureus as well as increased risk of s. aureus pneumonia was observed in flu-infected wildtype mice compared to mice lacking the type iii ifn receptor (123) . currently, however, is it unclear to what extent viral-induced changes in the urt microbiome alter subsequent immune responses against secondary bacterial infections. compared to studies of the urt microbiome, studies of the lrt microbiome following viral infections are relatively scarce due to the difficulty of obtaining uncontaminated samples from the lung. samples of convenience, such as sputum, suffer from oral contamination, but bronchoscopic samples are invasive and expensive to obtain on a regular basis. moreover, it is unclear whether outside of patients with chronic lung disease (e.g., copd), the lung microbial burden is of sufficient magnitude to exert robust effects on immune responses and risk of secondary bacterial infection during viral infection. data from a mouse model of influenza infection seem to indicate that flu infection has only a modest effect on bacterial counts, diversity and composition of the lung microbiome (46) . in subjects with chronic obstructive pulmonary disease (copd) after hrv infection but not in healthy individuals, there was an increase in bacterial burden and growth of bacteria present at baseline, particularly h. influenzae (124) . the researchers observed that the growth of bacteria seemed to arise from the existing community. s. pneumoniae intranasally inoculated into mice pre-infected with influenza virus first colonized the nose, followed by the trachea and lungs several days later with purulent inflammation. however, this effect was not observed in uninfected animals. this suggests that pneumococcal infection may sequentially develop from the urt to the lrt in influenza virus-infected subjects (97) . thus, it is possible that some individuals with influenza infection might develop changes in their lung microbiome as a result of changes in their urt microbial communities. respiratory viruses not only alter the bacterial community in the urt, but also promote bacterial colonization of the lrt by a variety of mechanisms that impair bacterial clearance. first, mucus production in the respiratory tract is increased to facilitate viral clearance during infections. however, excessive mucus production can lead to airway obstruction by impeding mucociliary clearance (125) . second, viral infections can also reduce ciliary beat frequency and the number of ciliated cells, disrupt the coordinated movement of cilia, and impede the repair of respiratory epithelial cells, further leading to reduced mucociliary clearance (126, 127) . third, respiratory viral infections impair innate immune responses against bacteria (128) (129) (130) . innate immune cells including macrophages and neutrophils are recruited to the lung by cytokines and chemokines for phagocytosis and bactericidal activity. prior viral infections dysregulate both alveolar macrophages (64, (131) (132) (133) (134) (135) (136) and neutrophils (65, 128, 129) , thereby inhibiting bactericidal activity. thus, with multiple aspects of pulmonary host defense impaired, it would not be entirely surprising if a subset of influenza infected patients developed secondary bacterial pneumonia as a result of being unable to clear aspirated pathobionts from the urt. in addition to enabling us to determine what is present during states of health, large-scale sequencing-based microbiome analyses have also revealed who is not present during disease. it has long been appreciated that mechanisms have evolved in bacteria that confer competitive advantages, permitting them to survive in an otherwise inhospitable host environment. however, interspecies competition also maintains homeostasis of the microbial community, either through their abilities to capture scare resources (e.g., iron), or targeted killing of other bacteria (e.g., bacteriocins), preventing one microbe from dominating the community. thus, it is possible that the immune response incited by acute viral infections, changes in the host epithelial surface caused by the virus, or the virus itself might lead to elimination of a host commensal that is responsible for keeping pathobionts in check. for example, s. epidermidis and propionibacterium acnes abundance in the nares has been shown to be negatively associated with s. aureus carriage (67) . understanding these interactions may create new avenues for therapeutic interventions aimed at reducing colonization by pathogenic bacteria during influenza epidemics or pandemics. one group of commensals that has been examined for its role in inhibiting nasal carriage by s. aureus and s. pneumoniae is corynebacterium spp. an early study in japan reported on the effects of introducing a corynebacterium strain into the nares of healthy adult hospital workers who were persistent carriers of s. aureus, with successful eradication in 71% of subjects (137). the mechanism appeared to be bacteriocin-independent. in comparison, s. epidermidis implantation did not have an effect. whether the s. epidermidis strain used expressed the serine protease esp, which inhibits biofilm formation by s. aureus and nasal colonization (138) , is unknown. subsequent studies by another group reported that c. pseudodiphtheriticum inhibited s. aureus growth, whereas c. accolens and s. aureus appeared to support each other's growth (139) . conversely, other investigators observed that corynebacterium spp. were enriched in children who were not nasally colonized with pneumococcus, and demonstrated that c. accolens inhibit s. pneumoniae growth in vitro by expressing a lipase that releases free fatty acids from skin surface triacylglycerols, which inhibit pneumococcal growth. thus, painstaking identification and mechanistic interrogation of interspecies competition between commensals might lead to novel insights as to how viral infections might confer competitive advantage to pathobionts, and how to exploit natural strategies employed by commensals to restore homeostasis to the host microbial niche. interestingly, a recent preclinical study using a murine model of rsv and s. pneumoniae superinfection employed nasal priming by a c. pseudodiphtheriticum strain to augment host defense against the viral infection, which enhanced clearance of secondary bacterial challenge and reduced lung injury measures (140) . finally, direct effects of the infecting virus on bacteria that comprise the microbiome may facilitate the transition from pathobiont to pathogen. a metagenomic analysis showed that ph1n1-associated airway microbiotas were enriched in genes associated with cell motility, transcriptional regulation, metabolism, and response to chemotaxis compared to the same bacteria in non-infected patients (108) . these data imply that influenza infection perturbs the respiratory microbiome, leading to the production of secondary metabolites including immune-modulating molecules. viruses have also been found to impair bacterial biofilm formation and disrupt existing biofilm (141) (142) (143) (144) . influenza has been shown to affect the s. pneumoniae transcriptome in terms of downregulating expression of genes associated with the colonizer state and upregulations of bacteriocins (142) . thus, direct effects of viruses on bacterial transcriptional patterns might be a mechanism by which colonizing bacteria acquire invasive potential, thereby leading to bacterial superinfections. there are several areas that must be addressed by future respiratory microbiome research. first, it is necessary to standardize protocols used to analyze the respiratory microbiome, including sampling, processing, and bioinformatics methodologies. for example, sputum may be an appropriate material for investigations of respiratory diseases since it contains components of the lrt and can be obtained easily. however, more reliable information on the lrt requires invasive samples such as bal or protected specimen brush frontiers in immunology | www.frontiersin.org or bronchial/lung biopsies. second, most studies are limited to experiments conducted in animal models. even in human studies, most analyses have been performed in a small number of patients and have described bacterial communities in the urt. the role of microbial communities outside of the lungs including gut, sinus, and skin should be considered in the context of airway diseases. third, most studies on the microbiome have focused on the bacterial component, and have largely omitted fungi and viruses. the role of viruses-including the vast number of phages that infect bacteria-and fungi in respiratory diseases cannot be examined through 16s rrna gene analyses, and there are no studies describing the composition and role of the respiratory virome due to the difficulty of comprehensive analyses for viruses. fourth, it is not sufficient to study microbial communities based on species composition; a functional characterization through transcriptome and proteasome analyses is necessary to understand mechanistic role of microbiome on outcomes of infection. finally, mucosal microbiome manipulations by vaccines, antibiotics, and probiotics in the gastrointestinal and respiratory tract niches represent novel approaches for the prevention, treatment, and management of acute and chronic lung diseases. however, given that antibiotic therapy could affect commensal bacteria and hasten the emergence of drug-resistant bacteria, more research is needed on the long-term effects of this therapy. animal models should be developed to study the influence of the urt and lrt microbiomes on immune responses to respiratory viral infections; only then will it be possible to consider the clinical application of microbiome modulation strategies. respiratory viral infections can initiate a cascade of host immune responses that alter microbial growth conditions in the urt, lrt, and the gut (supplemental table 1 ). activation of influenzainduced antiviral interferon pathways can lead to inadequate innate immune cell responses during host defense against secondary bacterial infections, resulting in the proliferation of potentially pathogenic bacterial species. concomitant changes in the gut microbiome caused by the initial viral infection may also alter immune cell priming against secondary bacterial challenge, although this has not been examined to date. although the picture is incomplete, recent microbiome literature provides additional insights into the pathogenesis of dysregulated immune responses following acute viral infections, that may promote the development of secondary bacterial pneumonias (figure 4) . clarifying the differences and dynamics of respiratory microbiota in healthy subjects and chronic lung diseases during acute respiratory viral infections can elucidate pathogenesis of viralbacterial interactions and provide a basis for developing novel approaches for the prevention, treatment, or management of acute respiratory infection and exacerbation of chronic lung diseases. sh and jd co-wrote the manuscript. sh, jd, and mp designed the figures and table. kc and mp edited and provided critical revisions of the manuscript. all authors approve the final version and agree to be accountable for the content of the manuscript. jd is supported by a research grant from the national institutes of health (grant no. r01hl108949). the views 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microbiol host physiologic changes induced by influenza a virus lead to staphylococcus aureus biofilm dispersion and transition from asymptomatic colonization to invasive disease dynamic changes in the streptococcus pneumoniae transcriptome during transition from biofilm formation to invasive disease upon influenza a virus infection interkingdom signaling induces streptococcus pneumoniae biofilm dispersion and transition from asymptomatic colonization to disease streptococcus pneumoniae modulates staphylococcus aureus biofilm dispersion and the transition from colonization to invasive disease key: cord-310055-9qj8d2f7 authors: gerace, elisabetta; lo presti, vincenzo di marco; biondo, carmelo title: cryptosporidium infection: epidemiology, pathogenesis, and differential diagnosis date: 2019-10-22 journal: eur j microbiol immunol (bp) doi: 10.1556/1886.2019.00019 sha: doc_id: 310055 cord_uid: 9qj8d2f7 cryptosporidium is a protozoan that infects a wide variety of vertebrates, including humans, causing acute gastroenteritis. the disease manifests with abdominal pain and diarrhea similar to that of choleric infection. in the immunocompromised hosts, the parasite causes prolonged infections that can also be fatal. for this reason, cryptosporidiosis is considered one of riskiest opportunistic infections for patients with acquired immunodeficiency syndrome. the best way to control the infection in these patients is setting up sensitive and specific diagnostic tests for epidemiological surveillance and morbidity reduction. here, we summarized the general aspects of cryptosporidium infection focusing on available diagnostic tools used for the diagnosis of cryptosporidiosis. molecular methods currently available for its detection and progress in the development of new diagnostics for cryptosporidiosis are also discussed. cryptosporidiosis is a worldwide infection caused by the protozoan cryptosporidium, a parasite that infects many species of vertebrates, including humans, causing acute gastroenteritis, abdominal pain, and diarrhea [1] . cryptosporidiosis is transmitted primarily through the fecal-oral route, i.e., by ingesting viable oocysts of animal and/or human origin, emitted with feces that contaminated food or water [2, 3] . although the waterborne transmission of infectious pathogen is well documented, neither the natural reservoir nor the exact infection route of cryptosporidia is well-known [4] . cryptosporidium was first discovered by tyzzer in 1907 [5] , but for a longtime, it was thought to be a non-pathogenic parasite. only since 1976, it was recognized as an opportunistic pathogenic parasite, when 2 human cases of cryptosporidiosis were reported to be associated with diarrhea [6, 7] . however, cryptosporidium remained unknown as a significant human pathogen of acute diarrhea disease until 1982, when it was recognized as a causative agent of self-limiting diarrhea for the general population and a life-threatening disease for immunocompromised persons such as those receiving immunosuppressive agents and acquired immunodeficiency syndrome (aids) patients [4, 8, 9] . the parasite completes its life cycle within a single host (monoxen cycle), alternating asexual and sexual reproduction [3, 5] . although more than 30 species have been included in the genus cryptosporidium, only 2 species, namely, cryptosporidium parvum and cryptosporidium hominis, commonly infect humans [10] [11] [12] . c. parvum is responsible for most cattle infections, and consequently, it is considered to be responsible for most zoonotic infections in humans [10] . cryptosporidium spp. exhibit little host specificity, and different members of this genus have been reported to infect multiple hosts, such as mammals, marsupials, birds, reptiles, and fish [13, 14] . the intracellular protozoan parasite cryptosporidium is a human and veterinary pathogena member of the phylum apicomplexa that include other pathogens such as plasmodium spp., eimeria spp., neospora, babesia, and theileria [15] . however, in contrast to other parasitic protozoa of this phylum, such as toxoplasma gondii or plasmodium falciparum, the species of the genus cryptosporidium cannot be cultivated in vitro [16] . since there is no vaccine commercially available to prevent cryptosporidium infection, and these parasites have certain characteristics that make them highly contagious (i.e., survival in the environment for a long time and resistance to chlorine-based disinfectants), the only way to avoid the spreading of the parasite to other people is the introduction of preventive measures to control the transmission of the germs that are shed in feces [12] . these precautions are especially important for people with weakened immune systems [17] . infection is initiated by ingestion of oocysts, each of which contains 4 sporozoites that hatch at the intestinal level, releasing infectious sporozoites [3, 5] . after excystation, these sporozoites are ingested into a modified host membrane separated from the cytoplasm by a dense layer; then, the location of parasites within the host is not intracellular but extracytoplasmic [18] . within the parasitophorous vacuole, the parasite undergoes asexual or schizogony reproduction, leading to the production of 8 merozoites within a type i meront [19] . the merozoites can invade the neighboring epithelial cells and propagate the infection to other sites of the intestines. during this stage, the merozoites can undergo 2 distinct replicative cycles: an asexual stage characterized by multiplication of merozoites (type i meront) and production of thin-walled oocysts that autoinfect the host and/or a sexual stage with formation of type ii meront, which, after differentiation in microgametocytes and macrogametocytes, will unite to form the zygote [3] . the diploid zygote, through a process called sporogony, will form 4 sporozoites within thick or thin-walled oocysts. the thick-walled oocyst, protected by a resistant wall, after releasing in the feces is shed into the environment, ready to infect a new individual ( figure 1 ) [19, 20] . although the number of worldwide reported cases of cryptosporidiosis in the last years is increased with a number of 3 cases per 100,000 population, numerous indicators (i.e., clinical symptoms) indicate that the frequency of infection is likely to be 100-fold higher than the number of reported cases [21] . the prevalence of cryptosporidium infection is significantly lower in industrialized countries compared to developing countries since, in the latter, there are many people that still lack a basic level of drinking water and sanitation [22, 23] . in developing countries, cryptosporidiosis is rarely reported in immunocompetent persons, while this infection causes approximately 10-15% of cases of severe diarrheal illness mostly in malnourished children less than five years old [22, 23] . cryptosporidium spp. have been isolated worldwide, and outbreaks of cryptosporidiosis associated with swimming pool or contaminated drinking water have been reported in several countries [24, 25] . although there are more than 30 species of cryptosporidium, only few species like c. hominis, c. parvum, c. meleagridis, c. felis, and c. canis are commonly found in people [10, 26, 27] . among these species, c. hominis and c. parvum are most frequently found in intestinal infections in humans, although they differ in host range, with the former that infects exclusively humans, while the latter has an infectious cycle involving humans and ruminants, and thereby, usually c. parvum infects people who have close contact with large numbers of animals [28] [29] [30] . although the transmission from animals is possible, it is very uncommon, and cryptosporidium infection transmitted from sheep, horses, goats, and rodents has been rarely reported [12, 30, 31] . c. canis and c. felis, the dog-and the cat-adapted species, respectively, have been reported to cause infection in individuals without being sick at all [21, 30] . however, human infections with cryptosporidium species from pets are very rare [10, 14] . in immunocompetent persons, cryptosporidium infection usually produces a bout of watery diarrhea, although the infection in some persons may not lead to the symptoms [4, 23, 32] . the disease is likely underestimated, since the diarrhea usually resolves without any treatment [19] . although even people who do not have direct contact with animals may be infected, those who have direct contact with infected animals (particularly calves) or swallow pool water or drink untreated water are at a higher risk of contracting cryptosporidiosis [22, 24, 25] . cryptosporidium infections are also more common in individuals who are in poor health or who have weakened immune systems (e.g., human immunodeficiency virus (hiv)/ aids, cancer, and transplant patients) [19, [33] [34] [35] . between the parasites that caused about 1 million deaths every year, cryptosporidiosis resulted in over 50,000 deaths [36, 37] . moreover, cryptosporidium is one of the most important protozoan pathogen that cause waterborne outbreaks worldwide [19, 32, 38] . cryptosporidium lives in the intestines of the infected individuals and animals in the form of oocysts, which will be released in the feces [19] . after infection, the parasite have been released into the feces from an infected human or animal. in the gastrointestinal tract, the oocysts are broken releasing 4 sporozoites (a) that invade the mucosa to establish a cycle of repeated endogenous reinfection (endogenous autoinfection). the trophozoites remain in the apical portion of the cell (b) and undergo asexual division (merogony) to form merozoites (c). the merozoites released from type i meronts enter adjacent host cells to form type ii meronts. type ii meronts do not recycle but enter host cells to form the sexual stages (d). the type ii meronts form merozoites that differentiate in sexual reproductive microgamonts (male) and macrogamonts (female) (e). after fertilization, the zygote develops into resistant thick-walled oocysts (f) that are excreted from the host in the fecal material. however, some oocysts are thin-walled (g) and excyst within the same host in a process of autoinfection (h) alters the function of the intestinal barrier, increasing its permeability, absorption, and secretion of fluid and electrolytes, and thereby, the severity, persistence, and outcome of the infection depend on the degree of the immunocompromised status [39, 40] . the oocysts are very resistant to chlorine, chloramines, and chlorine dioxide, which are commonly used in methods of water system disinfection, and remain vital for infection in the environment for a long time [21] . humans become infected with cryptosporidium by touching anything that has come in contact with contaminated feces, although the most common mode of transmission is represented by ingestion of oocysts in contaminated food and water or air [21, 40] . recent studies indicate that cryptosporidiosis may be transmitted by inhalation of aerosolized droplets via respiratory secretions or by coughing, in addition to the well-documented fecal-oral transmission [41] . pulmonary infections also have been reported [41, 42] . immunocompromised hosts are more susceptible to infection than people with a healthy immune system, and in the subjects with hiv/aids, the parasite often causes a chronic, prolonged form of a disease, which is difficult to treat and can even result in death [37] . in these patients, fever and malabsorption are common, and the parasite can cause inflammatory disease of the biliary tree leading to biliary tract obstruction, sclerosing cholangitis, papillary stenosis, and pancreatitis [33, 37] . for this reason, cryptosporidiosis is considered one of the riskiest opportunistic infections for patients with acquired immune deficiency syndrome [37] . the diagnosis of cryptosporidiosis is usually made by microscopic detection of the parasite oocysts, oocyst antigens, or oocyst dna in stool samples. since the most common symptom of cryptosporidiosis is a watery diarrhea, the differential diagnosis for cryptosporidium includes bacterial, viral, and parasitic enteric pathogens associated with acute diarrhea such as rotaviruses, coronaviruses, escherichia coli, and salmonella spp. [43, 44] . however, gastrointestinal disorders may also have noninfectious causes, such as inflammatory bowel disease in humans [45] . diagnosis of cryptosporidiosis is usually made by microscopically identifying the presence of oocysts of 4 to 6 μm in diameter in the stool of the infected subjects [44, 46] . however, since the detection of cryptosporidium oocysts can be difficult, three fecal samples collected on separate days should be microscopically examined for detection of oocysts prior to exclude a cryptosporidium infection in subjects with severe diarrhea [44, 45] . in addition, for detection of oocysts in stool, sample must be concentrated using the formalin-ether sedimentation method prior to microscopic examination [47] . the oocysts of cryptosporidium can be also observed by acid-fast (modified ziehl-neelsen method) or phenol-auramine staining on unconcentrated fecal smears, where the oocysts stain red and bright yellow, respectively [44, 45] . however, much attention should be given to this staining since the oocysts may also appear as "ghost" cells [48] . in addition, although the oocysts of cryptosporidium are half the size of those of cyclospora cayetanensis (about 4-5 μm in diameter vs. 9-10 μm in diameter), another coccidian protozoan parasite that infects the intestine of humans causing acute diarrhea, much attention should be given when evaluating stool samples since the oocysts of both parasites are autofluorescent and acid-fast ( figure 2 ) [46, 47] . in addition, although c. cayetanensis has a life cycle similar to cryptosporidium, its oocysts are unsporulated and not infective when shed in the feces, and thereby, direct fecal-oral transmission cannot occur [46] . although routine diagnosis of cryptosporidiosis is generally made by the microscopic identification of oocysts in fecal smears, this method, despite being easy to use and low cost, unfortunately has a low sensitivity (≤ 30%). moreover, accurate diagnosis of cryptosporidiosis using this technique is largely dependent on the experience of the microscopist. sensitivity can be improved by performing modified acidfast stain, a staining generally performed if there are structures suspicious for cryptosporidium, which has been reported to be associated with a sensitivity of 55%. however, these methods cannot distinguish between cryptosporidium species [49, 50] . in addition to the above described methods, watery or mushy stools can be examined for the laboratory diagnosis of cryptosporidiosis using different techniques such as the enzyme-linked immunosorbent assay (elisa) and immunochromatographic test, which have good sensitivity and specificity for detection of cryptosporidium antigens [51] [52] [53] . although the commercial kits have a range of sensitivities and specificities higher than that of the microscopic methods (ranging from 58 to 95%), previous studies have shown that these antigen/antibody-based detection methods are also ineffective if the burden of this parasite in the patients is below the minimum threshold [53] . in addition, these methods are more expensive than polymerase chain reaction (pcr), which is now accepted in most laboratories as the gold standard for the detection of this parasite in the stool. previous studies have shown that compared to pcr, microscopy, elisa, and immunochromatographic test (ict) are less convenient in terms of cost, sensitivity, and specificity and also are more time consuming [54] [55] [56] . although there have been important advances in diagnostic tools (i.e., the availability of multiplex pcr assays for the detection of intestinal protozoa), the accessibility to this molecular technique is limited in some labs and totally absent in others. in addition, the expense and requirement for technical expertise have limited their use particularly in high-prevalence regions such as developing countries. despite the global prevalence and the high impact of cryptosporidiosis, principally in immunocompromised patients, major deficiencies exist in the current control programs, especially in terms of available diagnostic tools. in addition, most common diagnostic tests tend to misdiagnose the disease in endemic areas. in particular, microscopic techniques are the most widely used method for detection of cryptosporidia in stool samples, and the diagnostic accuracy of these methods is largely dependent on the experience of the microscopist. since early diagnosis is the best way to fight the infection, there is a need to develop molecular techniques that are sensitive, specific, easy to perform, cost-effective, and highthroughput. this work was supported by fondo per il finanziamento delle attività base di ricerca (ffbra2017) cryptosporidiosis: epidemiology and impact cryptosporidiosis: current trends and challenges cryptosporidiosis: biology, pathogenesis and disease morbidity, mortality, and long-term consequences associated with diarrhoea from cryptosporidium infection in children younger than 5 years: a metaanalyses study a hundred-year retrospective on cryptosporidiosis overwhelming watery diarrhea associated with a cryptosporidium in an immunosuppressed patient acute enterocolitis in a human being infected with the protozoan cryptosporidium cryptosporidium spp. and cryptosporidiosis epidemiological observations on cryptosporidiosis and molecular characterization of cryptosporidium spp. in sheep and goats in kuwait cryptosporidium species in humans and animals: current understanding and research needs assessing the impact of environmental exposures and 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cryptosporidium, microsporidia and isospora infection in hiv-infected people: a global systematic review and meta-analysis. parasites vectors parasitic infection among hiv/aids patients at bela-bela clinic, limpopo province, south africa with special reference to cryptosporidium cryptosporidium infection in solid organ transplantation burden of disease from cryptosporidiosis widespread occurrence of cryptosporidium infections in patients with hiv/aids: epidemiology, clinical feature, diagnosis, and therapy post-infection symptoms following two large waterborne outbreaks of cryptosporidium hominis in northern sweden cryptosporidium parvum disrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins intestinal and pulmonary infection by cryptosporidium parvum in two patients with hiv laboratory diagnosis of cryptosporidiosis laboratory diagnosis of cryptosporidiosis comparative diagnostic techniques for cryptosporidium infection comparison of current methods used to detect cryptosporidium oocysts in stools differences in the detection of cryptosporidium and isospora (cystoisospora) oocysts according to the fecal concentration or staining method used in a clinical laboratory detection of intestinal protozoa in the clinical laboratory evaluation of four commercial rapid immunochromatographic assays for detection of cryptosporidium antigens in stool samples: a blind multicenter trial multisite performance evaluation of an enzyme-linked immunosorbent assay for detection of giardia, cryptosporidium, and entamoeba histolytica antigens in human stool evaluation of an immunoassay-based algorithm for screening and identification of giardia and cryptosporidium antigens in human faecal specimens from saudi arabia evaluation of the roche lightmix gastro parasites multiplex pcr assay detecting giardia duodenalis, entamoeba histolytica, cryptosporidia, dientamoeba fragilis, and blastocystis hominis is real-time pcr-based diagnosis similar in performance to routine parasitological examination for the identification of giardia intestinalis, cryptosporidium parvum/cryptosporidium hominis and entamoeba histolytica from stool samples? evaluation of a new commercial multiplex pcr assay and literature review comparison of three commercial multiplex pcr assays for the diagnosis of intestinal protozoa all authors conceived the original idea, cb wrote the manuscript with support from eg and vml. the authors declare no conflict of interest. key: cord-286574-t9z2ynt5 authors: nan title: speaker presentations date: 2017-09-30 journal: international journal of antimicrobial agents doi: 10.1016/s0924-8579(17)30340-0 sha: doc_id: 286574 cord_uid: t9z2ynt5 nan increased mortality rates in patients infected with major resistant pathogens are as follows; • mrsa: mrsa can cause almost 2-fold higher mortality (or 1.93) compared with mssa strains according to a meta-analysis (cosgrove et al. 2003) . mrsa kills more american people every year (∼19,000) than emphysema, hiv/aids, parkinson's disease and homicide combined (klevens et al. 2007 ). • esbl-producing gram-negative bacilli: a meta-analysis showed that crude rrs were significantly higher mortality in esbl-associated bacteremia ( pooled rr 1.85) (schwaber et al. 2007 ). • carbapenem-resistant k.pneumoniae: in patients with carbapenem-resistant k.pneumoniae bacteremia, the crude mortality rate was 71.9%, while it was 21.9% in control subjects (borer et al. 2009 ). a mortality risk ratio was 3.3 for patients with carbapenem-resistant k. pneumoniae bacteremia. increased mortality and morbidity in resistant infections is due to treatment failure of antibiotic therapy which is associated with bacterial fitness, greater severity of underlying illness, delays in initiating effective therapy and lack of effective therapy (friedman et al. 2016) . due to mismatch between choice of empirical antibiotics and in vitro susceptibility test results, administration of effective antibiotics was delayed by 6-times in the case of antibiotic-resistant e.coli and k.pneumoniae infections compared with susceptible strains (72 hours vs 11 hours) (lautenbach et al. 2001) . also, patients infected with resistant strains are more frequently associated with more severe underlying illness requiring longer hospitalization. for example, patients with cre infections are more likely to be a transplant recipient, require mechanical ventilation, a prolonged hospitalization, icu stays, or use of central venous catheters (borer et al. 2009 ). patients infected with resistant strains are more likely to develop complications and long-term sequelae. for example, mrsa infection caused higher incidence of complications by 69% compared with mssa infection. the most frequent complications are a progression of the local infection (rr 3.25) and pneumonia (rr 2.28) (cecchini et al. 2015) . once amr emerges in the hospital, resistant pathogens can cause additional cases of bacterial infections that affect more patients. emergence of amr in major human pathogens also affects safety and efficacy of surgical procedures, cancer chemotherapy, organ transplantation, and intensive care. therefore, amr is not just an infectious disease issue, but rather an issue in surgery, cancer care, organ transplantation and whole health system. amr can also negatively affect the daily hospital activities such as total closure of an affected ward or unit or cancellation of elective surgery. economic impact of amr is difficult to quantify, which consists of direct costs associated with use of more expensive antibiotics, special equipments, longer hospital stay, and isolation procedures etc., and indirect costs mainly due to loss of productivity of the patients. according to the world bank report to estimate the amr impact on global gdp in -2050 (world bank, 2016 , in the optimistic "low-amr" scenario, global economic output is projected to be 1% lower by 2030 ($1 trillion) and 1.1% lower by 2050 than in the base case. in the pessimistic "high-amr impact" scenario, global economic output would be 3.2% lower in 2030 ($3.4 trillion) and 3.8% lower by 2050 than in the base case. it means that in the "high-amr" scenario, economic damage will be greater than that in 2008-2009 global financial crisis which caused 3.6% drop in the global gdp. economic impact of amr differs by countries with different economic level. low-income and lower middle-income countries would have greater impact of economic loss (5.6% loss of gdp) due to amr compared with high-income countries (3.1% loss of gdp), particularly in the "high-amr" scenario. global economy is negatively affected by amr in various aspects such as international trade, livestock production, and healthcare expenditures. by 2050, the volume of global real exports may decrease by 1.1% in the "low-amr" scenario and by 3.8% in the "high-amr" scenario. in the "high-amr" scenario, healthcare expenditures in 2050 would be as 25% higher than the baseline values for low-income countries, 15% higher for middle-income countries, and 6% higher for high-income countries. the additional expenditures in 2050 would be $1.2 trillion annually in the "high-amr" scenario. in the us, cdc estimated the cost of amr as a total of $55 billion per year : $20 billion in excess for direct healthcare costs and $35 billion for indirect societal costs due to loss of productivity (cdc, 2013) . in europe, the overall economic burden of amr was estimated to be at least 1.5 billion euros; 60 % for direct costs and 40% for indirect costs (ecdc/emea, 2009 ). data on the economic impact of amr in major pathogens showed that healthcare costs significantly increase in the treatment of infections caused by antibiotic-resistant strains (maragakis et al. 2008) ; mrsa bacteremia (us$ 6,916), mrsa surgical site infection (us$ 13, 901) , vre infection (us$ 12, 766) , and esbl or kpc-producing e.coli or klebsiella infection (1.7fold increase). amr is obviously one the most serious and urgent issues with devastating impact on clinical medicine, economic growth and societal system and function. g20 leaders' declaration which was announced in july 2017 also underlined the importance of combating amr through international collaboration. for more effective and robust implementation of global action plan to combat amr, appropriate evaluation of impact of amr should be performed, particularly in developing world which would have greater damage due to amr. school of public health, the university of hong kong, china the development, large-scale production and widespread use of antimicrobial drugs in the twentieth century was a turning point in human history, resulting in dramatic improvements in medical care and reduction of deaths. over time, however, rising levels of antimicrobial resistance (amr) among a wide range of pathogens has placed such gains at risk of being lost. reducing amr is now a top-level global public health priority. in principle, the major high-level goals consist of optimizing the use of such drugs in health and agriculture and minimizing environmental contamination; sustaining the development of new classes of antimicrobials drugs and other medicines and making them affordable and accessible to all who need them; and much more effective application of infection control and prevention principles. for decades, technical solutions have been the primary approach used for addressing amr. more recently, fao, oie and who in combination with like-mined champions have embarked upon a more political and broader "one health" approach to increase awareness and engagement beyond scientific and medical groups. this change is the basis for the 2015 global action for amr, the 2016 high level meeting on amr held at the un general assembly and attention to amr by groups such as the g20. while such results have been instrumental in broadening the awareness and attention paid to amr, it is now critical to adopt concrete and focused activities to consolidate and build upon these gains. the fundamental building blocks will be proposed and discussed. murdoch university, australia methicillin-resistant staphylococcus aureus (mrsa) was initially a healthcare associated pathogen limited to distinct lineages often associated with multi-drug resistance. however, in the late 1980s mrsa began to emerge in the community. in the beginning, this was mostly confined to closed communities, i.e. australian aboriginals, but around the late 1990s community-associated mrsa (ca-mrsa) emerged worldwide in the general population. the ca-mrsa clones evolved independently of the ha-mrsa clones, and typically, possessed type iv or v sccmec elements, were non multi-drug resistant, and were positive for the panton valentine leucocidin (pvl) toxin. whilst regionally many unique ca-mrsa clones have emerged, only a few clones predominate within a region, and even fewer clones have a global distribution. the reasons for the variation in the prevalence and dominance of ca-mrsa clones between different geographical niches remains unclear. although the epidemiology of ca-mrsa varies considerably, four major global ca-mrsa clones have been described: st8-mrsa-iv (usa300) and st30-mrsa-iv (south west pacific clone) which and are considered pandemic; st80-mrsa-iv (european clone) which predominates in europe, africa and the middle east; and st59-mrsa-iv/v t (taiwan/asia pacific clone) in asia. however, using whole genome sequencing analysis, at least two of the clones have different clades that have evolved independently of each other. in clonal complex 8 nine phylogenetic clades with at least eight independent events of methicillin-resistance acquisition have been identified. subsequently, it has been hypothesised usa300 did not evolve from the healthcare-associated st8-iv usa500 clone, the historic ca-mrsa from western australia, or the pvl-positive mssa clone from trinidad and tobago or western africa, but most likely from pvl-positive mssa circulating within the usa. the ancestor of the usa300 clade emerged in central europe in the mid-19 th century and exported to north america in the early 20 th century. once in north america the clone progressively acquired the usa300 characteristic genetic specifications, diversified and spread globally including africa. within the usa300 clade two closely related st8-iv clones are recognised: usa300, primarily isolated in north america (st8-mrsa-iva, spa t008 luks-pv/lukf-pv, acme positive, msra-mediated macrolide resistance genes) and the usa300 latin american variant, primarily isolated in south america (st8-mrsa-ivc, spa t008, luks-pv/lukf-pv, acme negative, tetk, copper and mercury resistance genes). similarly, st59 mrsa is also not a single clone but consists of two major clades, one originating in the usa and the other in east asia. furthermore, two distinct subclades within the east asia clade have been identified: pvlnegative st59-mrsa-iv (asia pacific clone) and the multi-resistant pvl positive st59-mrsa-v t (taiwan clone) which possesses two distinct ccrc genes. recently three newly described pvl-positive ca-mrsa clones have been reported in multiple countries: st93-mrsa-iv (queensland ca-mrsa) from australia, and st772-mrsa-v t (bengal bay) and st22-mrsa-iv from the indian subcontinent. st22-mrsa-iv, first reported in india in 2010, is genetically distinct from the healthcare associated st22-mrsa-iv (emrsa-15) having acquired different sccmec types and sub types. of concern, st772-mrsa-v t and st22-mrsa-iv are multi-resistant hyper-virulent ca-mrsa that have recently been associated with nosocomial transmission. the co-emergence of multiple ca-mrsa lineages that have risen independently in most parts of the world has been striking. to date no single genetic or epidemiological factor has been identified that accounts for the extraordinary success of some genetically distinct ca-mrsa clones. over time, some ca-mrsa clones will replace ha-mrsa clones, which will have significant clinical and public health implications. along the way they will become increasing antibiotic resistant making the distinctions between ha-mrsa and ca-mrsa blurred and antimicrobial treatment difficult. methicillin-resistant staphylococcus aureus (mrsa) has been a major cause of healthcare-associated infections with significant morbidity and mortality, and it has become one of the most important nosocomial pathogens in many countries worldwide since 1980s. in particular, several mrsa clones have been successful in spreading and causing infections in the hospitalized patients. the prevalence rates of mrsa in s. aureus isolates from the hospitalized patients have become high enough to spread into the community. interestingly, during the past two decades, the emergence of community-associated (ca-) mrsa clones has greatly affected the global epidemiology of s. aureus infection. circulating ca-mrsa clone varies according to the region, and it includes st8 (usa300), st80, st30, st59, st72, st772 and st22 which mostly belong to sccmec type iv or v. as a result, the proportion of mrsa in community-acquired s. aureus infections has been significantly increasing in many countries. furthermore, many reports have shown that ca-mrsa clones have replaced traditional hospital-endemic mrsa clones. such an influx of dominant ca-mrsa clones into the hospitals has been making the 'search and destroy' policy which have contributed to maintain low mrsa rates in some countries difficult to maintain. in addition, there has been concern that healthcareassociated infections such as surgical site infection caused by ca-mrsa may increase. in this talk, an update on ca-mrsa epidemiology in healthcare-associated infection and concerns on infection prevention and control will be discussed. the newly emerging middle east respiratory syndrome coronavirus (mers-cov) causes a severe respiratory infection with a high mortality rate (∼35%), and has been a global threat due to continuous outbreaks in the arabian peninsula and international spread by infected travelers since 2012. from may to july 2015, a large outbreak initiated by an infected traveler from the arabian peninsula swept south korea, and resulted in 186 confirmed cases with 38 deaths (case-fatality rate: 20.4%). here, we show the rapid emergence and spreading of a mutant mers-cov with reduced affinity to the human receptor, cd26, during the korean outbreak. we isolated thirteen new viral genomes from fourteen infected patients treated at a hospital and found that twelve of them possess a point mutation in the receptor-binding domain (rbd) of viral spike (s) protein. we also analyzed clinical data and specimens from fourteen mers patients treated in a hospital who collectively represent a wide spectrum of disease severity, ranging from mild febrile illness to fatal pneumonia. comparative and kinetic analyses revealed that high viral loads, weak antibody responses, and lymphopenia accompanying thrombocytopenia were associated with disease mortality, whereas persistent and gradual increases in lymphocyte responses might be required for effective immunity against mers-cov infection. the correlation of the virological and immunological responses with disease severity and mortality, as well as their responses to current antiviral therapy, may have prognostic significance during the early phase of mers. severe fever with thrombocytopenia syndrome (sfts) was discovered as an infectious disease caused by a novel bunyavirus in china in 2011 (n engl j med, 2011 . the causative agent for sfts is named sfts virus (sftsv), which is a tick-borne virus and belongs to the family buniyaviridae, genus phlebovirus. the virus infection causes generalized infections with a high case fatality rate. in late 2012, it was discovered that sfts was also endemic to japan (takahashi t, et al., jid, 2014) . sfts is endemic to china, south korea, and japan. since the discovery of sfts endemic to japan in january 2013, approximately 240 patients with sfts have been reported to the national institute of infectious diseases. the case fatality is about 25%. most of the patients were aged over 40's. the pathophysiology of sfts has been studied through the pathological examination of sfts patients, who died. through the pathological studies on sfts, sftsv is present in the lymph node tissues. there are two pathological types, sftsv-positive lymph node-localized type and sftsv-positive lymph node-generalized types. all the patients, in whom bone marrow aspiration test was performed, showed hemophagocytic syndrome, indicating that cytokine storm plays an important role in pathogenesis of sfts. most fatal sfts patients showed symptoms of hemorrhage and deterioration in consciousness. one of the patients showed a symptom of bloody vomit. real time imaging of the stomach by endoscopic examination revealed the presence of ulcerative lesions with whoozing hemorrhage (kaneyuki s, et al., jjid, 2017) . the pathophysiological features behind the high case fatality rate in sfts are hemophagocytosis, hemorrhagic tendencies due to thrombocytopenia, disseminated intravascular coagulation, and ulcerative lesions appeared in the gastrointestinal tracts, and multi-organ failure. the efficacy of antiviral agent, favipiravir, which was developed by dr. furuta y and his colleagues (toyama chemical co., ltd, japan), in the treatment of sfts was evaluated using mice lacking the type i interferon alpha receptor (ifnar −/− ) (tani h, et al., msphere, 2016) . favipiravir inhibited replication of sftsv in vero cells by 5 log units, with a 50% inhibitory concentration (ic 50 ) and ic 90 of 6.0 and 22 µm, respectively. intraperitoneal or oral administration of favipiravir for 5 days to ifnar−/− mice infected with lethal sftsv significantly improved survival rates (100% survival) without causing body weight loss and reduced the viral load in the serum. although ribavirin also inhibited sftsv replication, it was quite less effective than favipiravir both in vitro and in vivo. a time-of-drug-addition study revealed that therapeutic favipiravir treatment of sftsv infection in ifnar−/− mice was effective. these results suggest that favipiravir might be a promising candidate as antiviral drug against sfts. the study suggested that favipiravir is a candidate drug for the treatment of sfts. in the presentation, i will present the overview of the epidemiology and pathophysiology evaluated through pathological autopsy, and the development strategy of antiviral drug therapy for sfts. this disease, sfts, continues to be endemic to china, korea, and japan for the future. we can not escape from the risk being infected with sftsv. on the other hand, there is a potential that favipiravir has a therapeutic effect in the treatment of sfts. safe and efficacious vaccine against sfts should be developed. furthermore, i will discuss the issues to be addressed to reduce the sfts disease burden. the challenges posed by infectious diseases in the 21 st century are forever growing more complex and numerous. there are no magic bullets or simple solutions that we can employ in our battle against the array of pathogens that every day attack humans, animals and plants. but there is much that we can do to mitigate the changing landscape of infectious diseases threats. this presentation will expand upon dr. osterholm's previous plenary session lecture. it will go into detail describing the major steps we can and must take to tip the balance between the growing threat of infectious diseases and our ability to anticipate these threats and prevent and control them before they can result in increased morbidity and mortality. the polymyxins, colistin ( polymyxin e) and polymyxin b, are effective antibiotics against most multidrug-resistant (mdr) gram-negatives and are currently considered as the last-line drugs for treating severe bacterial infections. polymyxin resistance among gram-negatives has increased gradually for the last few years, and knowledge of its multifaceted resistance mechanisms is expanding. typically, colistin resistance is due to chromosomally mediated modulation of two-component regulatory systems leading to modification of lipid a that resulted in reduced affinity to polymyxins. clones of polymyxin-resistant gram-negatives have spread in some hospitals, but have not significantly affected the use of polymyxins. these resistance genes are generally not transmissible between bacteria and so have not disseminated widely. however, a newly discovered plasmid-mediated polymyxin resistance gene mcr-1 has been detected in many countries in different geographical regions of the world since its first report in 2016. therefore, it seems inevitable that plasmid-mediated transfer of polymyxin resistance will seriously reduce the lifespan of the polymyxins as the backbone of regimens against infections due to mdr gram-negatives. this review provides an update on epidemiology and mechanisms of polymyxin resistance among different commonly encountered mdr gram-negative bacilli. some unresolved questions with respect to polymyxin resistance are also discussed. one of the few remaining options for the infectious disease caused by multiple-drug resistant gram-negative bacilli is colistin. accordingly, emerging mobile colistin resistance mcr-1 for phosphoethanolamine transferase became a death threat to public health. various enterobacteriaceae carrying mcr-1-plasmids from human beings, animals, and environments were reported in asia, europe, africa, and north and south america. in south korea, by the retrospective screening for the animal-oriented escherichia coli strains, the mcr-1 positive isolates have been identified. to evaluate mcr-1-positive clinical strains, a total of 9396 enterobacteriaceae isolates collected between 2010 and 2015 were screened and, finally, three strains, two escherichia coli belonging to either st1011 or st101 and one enterobacter aerogenes, harboring the gene were identified. all possessed inci2 mcr-1-plasmids and colistin mics were 4 g/ml in e. coli strains and 32 g/ml in e. aerogenes. the bla ctx-m-55 gene for extended-spectrum beta-lactamase was co-carried in the mcr-1-plasmid of e. aerogenes and extra plasmids carrying bla ctx-m-55 , bla ctx-m-27 , and bla ndm-9 were co-harbored by e. coli strains. one of the e. coli strains produced complete conjugal machinery presenting the best efficiency of plasmid transfer while e. aerogenes having a truncated prepilin peptidase pilu resulting in complete loss of conjugal activity. the plasmid in e. coli transferred efficiently to e. coli recipients compared to k. pneumoniae and to enterobacter cloacae probably due to the species-specificity allowed by rearranged shufflon. previously identified 11 mcr-1-positive e. coli strains of animal-origin from south korea also possessed inci2 mcr-1-plasmid and the non-self-transferable mcr-1-plasmid in e. aerogenes was closely associated to those, while the self-transferable mcr-1-plasmid in e. coli strains were alike to each other. the plasmid carrying the gene, or the bacterial host harboring the plasmid, has come from animals as the previous reports illustrated. better stewardship for the proper usage of antimicrobials as well as a collaborating surveillance study as a concept of one-health is needed. what to use in the clinical practice? yohei doi md, phd university of pittsburgh, usa while colistin resistance in enterobacteriaceae due to the plasmid-mediated mcr genes has drawn much attention, the threat of colistin resistance that impacts patient care most currently exists in extensively drug-resistant (xdr) bacteria which have acquired colistin resistance through chromosomal mutations in species such as klebsiella pneumoniae, pseudomonas aeruginosa and acinetobacter baumannii. unlike the mcr mechanisms that readily spread horizontally via plasmids, colistin resistance in these xdr species usually arises through selective pressure in patients upon treatment with colistin, though outbreaks have also been reported on occasions. development of colistin resistance in these strains means few or even no remaining active agents. however, patients who are affected by these bacteria are medically complex, heterogenous, and difficult to enroll into clinical trials for many reasons. consequently, clinical data associating colistin resistance, antimicrobial therapy given and patient outcome are scarce and retrospective in nature for the most part. for k. pneumoniae, limited data suggest that mortality of infection from colistin-resistant strains may be lower when treated with gentamicin-containing regimens than those without gentamicin, but this approach is only applicable when the infecting strain is susceptible to this agent. fortunately, the approval of ceftazidime-avibactam has ameliorated concerns over colistin resistance in k. pneumoniae at least for the time being, as therapy with ceftazidime-avibactam appears to improve clinical outcome of infected patients over those treated with colistin-based regimens. interestingly, gastrointestinal decolonization with gentamicin may reduce infections and mortality in those known to be colonized with colistin-resistant k. pneumoniae, but the recent spread of mcr genes in humans brings into question the long-term viability of this approach. there are even less clinical data regarding the treatment of infections caused by colistin-resistant p. aeruginosa and a. baumannii. in vitro data variably support colistin-based combinations with partner agents including carbapenems, rifampicin, fosfomycin, and even gram-positive agents like vancomycin in the case of a. baumannii, but without accompanying robust clinical data. there are several novel agents with activity against colistin-resistant strains in late-stage clinical development. they include novel beta-lactam-beta-lactamase inhibitor combinations, a siderophore cephalosporin, an aminoglycoside and tetracyclines. most of them target carbapenem-resistant enterobacteriaceae, while some are also active against p. aeruginosa and a. baumannii. these new agents are bound to change the paradigm for the treatment of infections caused by colistin-resistant gram-negatives, but uncertainties are still likely to remain, including which agent to use, how to optimize dosing in to maximize efficacy and minimize toxicity as well as potential for development of resistance, and whether use of more than one agent would still be needed. analyte health, usa in the united states, telehealth represents a rapid, convenient, and cost-effective solution for non-life threatening conditions, including acute respiratory infections, urinary tract infection (uti), and sexually transmitted infections (stis). over 1.25 billion outpatient "bricks and mortar" visits occur annually in the us, and it is estimated that one third of these visits (417 million) can be handled through telehealth. furthermore, one third of this subgroup of consults (140 million) are related to infectious disease causes. until recently, the provision of diagnostic testing, in conjunction with telemedicine physician consults, was difficult due to diverse geo-locations of patients, physicians and testing laboratories. for example, in the virtual world of telehealth, a patient can be located in california, with the physician licensed to practice in california located three time zones away in new york. linkage of the physician and patient is now easily accomplished by electronic means (e.g., video conferencing, phone calls, text messaging). however, obtaining specimens from the patient and then transporting the specimen to the appropriate testing laboratory has been a challenge, such that most telemedicine consults are performed without diagnostic testing and empiric treatment is provided. this approach, especially with acute respiratory infection, could result in overuse or underuse of antibiotics. this lecture will describe new approaches to diagnostic testing for infectious diseases in the telehealth ecosystem. included is a description of the infrastructure requirements required and/or recently developed to accommodate specimen acquisition and testing through aggregation ("uber"-ization), of both physicians and laboratory patient service centers, provision of "round the clock" mobile collection (in-home collection) services and self-collection and self-testing. also, the cost-savings of these approaches, in contrast to traditional bricks and mortar approaches will be discussed. rapid molecular, point of care testing instruments are now available for the accurate and complete (no confirmation testing required) testing for group a streptococcus for throat swab specimens, and influenza and rsv for nasopharyngeal swab specimens; future testing capabilities include sexually transmitted diseases like neisseria gonorrhoeae, chlamydia trachomatis and human papilloma virus (hpv). these new devices will accommodate the next inflection point in virtual medicine: point of patient testing, including testing the patients in their homes. the world bank estimates that epidemics will cost $6 trillion dollars in the 21 st centuryroughly $60 billion per year. the $6 billion cost of the ebola epidemic was its economic impact (in countries that can least afford it); however the impact of ebola on families, social customs, and health infrastructure will have long term consequences. in addition, funding for epidemic diseases waxes and wanes with the passage of the epidemic, and as a consequence longer term preventive solutions, such as vaccines, have little means for progressing. in 2016 several nations and charitable foundations launched a new enterprise known as cepi, the coalition for epidemic preparedness innovations. cepi will fund the development of vaccine candidates for diseases of epidemic potential, the initial candidates are mers, nipah and lassa fever. we will review product development activities in these diseases and also detail attempts by international organizations to try to develop a framework around responses to public health emergencies of international concern. carbapenemases, from kpc to ndm to oxa-48: diverse enzymes in diverse clones david m. livermore norwich medical school, university of east anglia, norwich nr4 7tj, uk carbapenemase-producing enterobacteriaceae present a growing problem. the predominant enzymes are members of the kpc, ndm, imp, vim and oxa-48 families. rarer types include imi, sme and fri enzymes, also some members of the ges family. kpc, imi, sme, fri and ges enzymes belong to class a; ndm, imp and vim are class b metallo carbapenemases, whilst oxa-48 and its relatives, e.g. oxa-181, belong to class d. this diversity of enzymes is reflected in a diversity of properties. kpc carbapenemases hydrolyse all widely available β-lactams and are inhibited by avibactam and vaborbactam. by contrast the metallo types evade avibactam and vaborbactam and attack all β-lactams except monobactams, which are often compromised by co-produced esbl and ampc enzymes. oxa-48, which is inhibited by avibactam but not vaborbactam, has little activity against oxyimino-cephalosporins but, again, is often accompanied by cephalosporin-hydrolysing esbls. the origins of most clinically-important carbapenemases are obscure; the exception is oxa-48, which is a chromosomal escape from shewenella spp. it is assumed that other carbapenemase genes similarly escaped from unknown environmental organisms. escape often entails gene mobilisation by insertion sequences, followed by capture by transposons and plasmids, which then facilitate dissemination. bla kpc , regardless of geographic origin, is generally carried by tn4401, often within pkpqil plasmids, whilst bla oxa-48 is often encoded by tn1999-related elements, carried on a small range of plasmids. bla oxa-181 differs only slightly from bla oxa-48 but appears to be a separate escape from shewenella, being associated with an isecp1 element on tn2013, a quite different transposon; moreover bla oxa-181 is epidemiologically linked to india, whereas classical bla oxa-48 links to turkey and the middle east. bla imp and bla vim metallo-carbapenemases occur as cassettes within class i integrons. bla ndm is generally linked to isaba125, carried by a wide diversity of different plasmids and may have arisen as a chimera between apha6, encoding its first six amino acids but otherwise deleted, and a pre-existing metallocarbapenemase gene of unknown origin. the plasmids encoding oxa-48-like enzymes and all the major metallo types (i.e. imp, ndm and vim) spread among strains and species. these enzymes all have a predilection for klebsiella pneumoniae, but frequently occur also in escherichia coli, enterobacter and other enterobacteriaceae species. individual producer strains cause local outbreaks but no single strain has become nationally or globally widespread. rather, when producers became prevalent, the common pattern is one of small clusters within a wider epidemiology of plasmid transfer among strains. by contrast, kpc carbapenemases (carried by tn4401 transposons within pkpqil plasmids) are strongly associated with k. pneumoniae st258. this lineage, along with its variants (e.g. st512), has become internationally widespread and is responsible for the major expansion of 'carbapenemase-producing enterobacteriaceae' in e.g. greece, italy, brazil and israel. only in the last of these countries has it been brought under control, achieved by a determined and centrally mandated infection control effort. not all kpc problems are linked to st258. in manchester, england, the problem is more akin to that with other carbapenemase types, entailing plasmid transfer among strains and species. the plasmids responsible are pkpqil variants with large substitutions resulting in the replacement of the partitioning and replication functions, potentially explaining their spread. the diversity of carbapenemase types and hosts exacerbates the problem of resistance. self-evidently, it harder to devise new pharmaceuticals that inhibit or evade multiple carbapenemase families than to develop those that inhibit or evade single or closely related enzyme types. what is more, experience suggests that it is harder to mobilise infection control staff and efforts against 'plasmid epidemics,' as with most carbapenemases, than against single strains epidemics. carbapenem-resistant enterobacteriaceae (cre) has been increasingly reported worldwide in the past 10 years, which represents a serious threat to public health. invasive cre infections are associated with high mortality, and cre has the potential to spread widely. carbapenem resistance in enterobacteriaceae can mainly result from two different mechanisms. some cre, which possess either ampc or extended-spectrum β-lactamase (esbl) with concomitant porin mutations, can render the organism non-susceptible to carbapenems. more importantly, some cre may result from production of carbapenemases that break down carbapenems. carbapenemases belong to heterogenous group of β-lactamases: molecular class a ( penicillinases), class b (metalloenzymes), and class d (oxacillinases). in 2011, clsi and eucast updated the carbapenem clinical breakpoints for enterobacteriaceae, in order to better predict treatment outcomes and to provide therapeutic alternatives for carbapenem-resistant bacteria. these breakpoints were based on patient response, pharmacokinetic/pharmacodynamic information, and in vitro minimal inhibitory concentration data. with the new lower breakpoints, routine testing for carbapenemase in clinical laboratory became not required for patient care. however, by these updated breakpoints, not all carbapenemase producing cre were non-susceptible to carbapenems. using the updated breakpoints, a study identified that occasional isolates of klebsiella pneumoniae with kpc or vim carbapenemases were susceptible to one or more carbapenem. as of now, few clinical data are available to support that mic is more important than presence of carbapenemase when predicting carbapenem treatment outcome. the different breakpoints for imipenem and meropenem in clsi and eucast may need evaluation for optimal patient care as well as epidemiologic study. carbapenemase detection and characterization are recommended for public health and infection control. currently, there are a variety of the phenotypic assays such as modified hodge test, the carba np test, modified carbapenemase inactivation method, and molecular assays. none of the currently described phenotypic test can detect all carbapenemases. molecular tests are often used as the golden standard, but they have inherent limitations such as missing novel variants and/or previously undescribed enzymes. cre continue to present challenges related to both treatment decisions by physicians and detection methods applied at laboratories. continued efforts to improve detection methods, by making them rapid, sensitive, and unbiased for specific carbapenemase, will hopefully allow for better patient care and prevention of further cre dissemination. ami neuberger, m.d. infections caused by carbapenem-resistant enterobacteriacea (cre) are more difficult to treat both because our current arsenal of antibiotics is limited and because some of the available antibiotics are less efficacious or have not been rigorously studied. the presentation will provide a brief overview of the future of treatment of cre infections. there are a number of controversies regarding the modes of administration of antibotics for cre infections: high versus lower dose, continuous or prolonged versus intermittent administration, duration of antibiotic treatment, and mono versus dual therapy. current evidence supports prolonged or continuous administration of β-lactam antibiotics. new randomized controlled trials of mono versus dual antimicrobial therapy, and short versus long duration of treatment for gram-negative bacteremia are ongoing, with results expected to be available in 2018. older antibiotics, such as colistin, aminoglycosides, fosfomycin, and tigecycline are increasingly used together with newly approved drugs such as ceftazidime-avibactam. some new antibiotics in advanced stages of development ( phase 2 or 3 trials) will be reviewed. these drugs will include combinations of carbapenems or aztreonam with β-lactamses inhibitors, new combinations of cephalosporins with β-lactamses inhibitors, cefiderecola novel cephalosporin, plazomicinan aminoglycoside, and eravacyclinea new fluorocycline. the advantages and disadvantages of these drugs will be discussed. novel approaches to antibiotic development include the use of antibacterials produced from previously "non-culturable" bacteria, and development of new entry mechanisms of antibiotics into gram-negative bacilli. treatment of cre infections is likely to undergo rapid changes in the upcoming years, but any such change will have to be accompanied by comprehensive infection-control programs, antibiotic stewardship programs in hospitals and in the community, and judicious use of new antibiotics. one health approaches, 'one hdealth, one medicine', have been globally recognized to control zoonotic diseases. world organization of animal health (oie) has reported 60% of human pathogens are animal origin and more than 75% of emerging animal diseases are zoonoses. this means collaboration and cooperation between animal and human medicine together can only solve the problem. recent huge outbreaks of highly pathogenic avian influenza (hpai) and middle east respiratory syndrome (mers) in korea have been more pay attention to implement one health approaches in practice. we experienced several hpai epidemics past ten years and the mers in 2015 and had to bear huge damages. one health becomes a key approach to control zoonotic diseases systemically and effectively. a 'one health' approach to minimize the antimicrobial resistance in humans and animals need collaboration among the responsibility of all three parts; human health, animal health and environmental health-communities. surveillance of antimicrobial usage and resistance provides important data for the identification of resistance problems and contributing factors for the development and spread of resistance at a national and local level. through the painful korean experience of these zoonotic diseases and global challenge to amr brings us to establish the effective preventive method and early diagnosis as critical control strategies. prevention and control of infections is essential in fighting antimicrobial resistance. thus, to minimize infections in animal and human and to decrease the volume of antimicrobials used, collaborative efforts should be implemented to improve animal and human health. one health activities on nipah in bangladesh: a high risk country for zoonotic disease spillover to man epidemiological investigations in bangladesh implicated consumption of date palm sap as the major route of transmission of the virus from bats to humans. in bangladesh date palm sap is collected from date palm trees for consumption either as fresh or as fermented beverage. the sap is collected in clay pots attached to the top portion of the tree, where the tree has been denuded of bar, so that the sap can ooze overnight into the collection pots. outbreaks of disease typically occur in the winter months, the interval in which date palm sap is collected. as well, case-control studies have identified consumption of date palm sap beverage as a risk factor at the individual level. videos using infrared cameras have documented that fruit bats visit date palm trees at night and contaminate the collection pots by licking the oozing sap and by urinating in the collection pots. ethnographic studies of affected populations has enabled development of a behavior change intervention to reduce consumption of date palm sap. in addition, bamboo skirts placed around the denuded bark area of the date palm trees and collection pots have been developed to obstruct contamination of collected date palm sap by bat saliva and urine. of great concern, epidemiological studies in bangladesh have also identified person-to-person transmission of the virus between patients and persons who are in close contact with patient secretions, often in hospital settings. a study of nipah outbreaks between 2001 and 2007 attributed 51% of all cases to person-to-person transmission, though only 7% of patients were assessed as having transmitted their infection onward via this route. importantly, handwashing seemed to be effective in reducing person-to-person transmission. in aggregate, these studies illustrate the power of multidisciplinary collaborations under the rubric of one health, and, in view of the documented person-to-person transmission in bangladesh and the potential for emergence of new genetic variants of nipah that are capable of sustained human-to-human transmission, underscore the need for continued surveillance and control efforts, including development of effective vaccines. mycoplasma pneumoniae (mp) is one of the most common causes of community-acquired pneumonia in children and young adults. emerging resistance to macrolides among mp is of great concern since a macrolide-resistant mp strain was first reported in 1997, most notably in japan, china, and korea. although macrolides are recommended for the first-line treatment for mp pneumonia, the efficacy of macrolides in the treatment of m. pneumoniae infection remains unclear. in addition, with the increase in macrolide resistance, concerns about the efficacy of macrolides for the treatment of mp pneumonia in children have been raised. given the versatile features of mp pneumonia, which are determined by the patient's age, the immunologic response of the host, and extrapulmonary manifestations, a more comprehensive approach must be established to analyze the clinical outcome of mp pneumonia according to the presence of macrolide resistance. initially, treatment of mp pneumonia with antimicrobials was supported by a randomized trial of 290 marine recruits that showed a shortening of fever duration, alleviation of cough, and improvement of chest x-rays. a recent systematic review that evaluated the effect of treating mp pneumonia demonstrated that there was no significant clinical benefit of antimicrobial therapy in children with mp pneumonia. although much is not known about clinical impact of macrolide resistance on the severity of mp pneumonia, macrolide resistance alone does not seem to explain the severity of mp pneumonia. some studies have reported that patients infected with macrolide-resistant strains had more febrile days and a longer duration of persistent cough than those infected with macrolide-susceptible strains, suggesting poorer response to macrolide treatment in macrolide-resistant strains. the results highlight the need for well-designed prospective studies to assess a therapeutic benefit from macrolides and an adjunctive therapeutic strategy in the treatment of children with macrolide-resistant mp pneumonia. mayo clinic, usa mycoplasma pneumoniae (mp) can cause upper and lower respiratory tract infections in children and adolescents, with communityacquired pneumonia (cap) comprising the major burden of the disease. mp infections are generally mild and self-limiting. some patients, of any age, may develop severe and fulminant infection with pulmonary complications and/or extrapulmonary manifestations that may affect almost every organ. this presentation will focus on pulmonary infections due to mp. it is estimated that 3-10% of children with mp respiratory infection develop cap and less than 5% are severe enough to require hospitalization, although this may change with increasing prevalence of mrmp. children with mp cap present with a longer duration of fever compared with children with cap due to other organisms. complicating the diagnosis of mp has been the insensitivity of testing as the organism does not grow well in culture, the inability to perform reliable and sequential serologic testing or pcr testing; the co-existence of mp with other pathogens; and asymptomatic carriage of mp in 21-56% of patients. pathogenic effects on the respiratory tract may be direct by active infection, indirect by infection-induced immune mechanisms, or both. the immunopathology is poorly understood, in particular the role of cell-mediated immunity (cmi). studies have demonstrated increased concentrations in il-8 and il-18 in acute phase serum and pleural fluid samples and inf gamma, il-6 and ip-10 in patients with mrmp and that severity of cap correlated positively with the size of cutaneous induration following intradermal injection of mp antigens. it's been postulated that cytoadherence of mp to the respiratory epithelium initiates an immune response with progression to an excessive inflammatory response and a vigorous cmi response leading to pulmonary injury and severe clinical illness. macrolides have been and remain the drugs of choice for treatment of mp infections in children. alternative drugs such as tetracyclines and fluoroquinolones are not first line due to age-related adverse effects. macrolide resistant m. pneumoniae (mrmp) have been reported as early as the 1970s in japan with rates now greater than 90% in areas of japan and china. korea reported mrmp in 2010. mrmp in north america was reported in 2008 with a prevalence now of approximately 13%. europe has reported rates of 2-26% and israel as high as 30%. many countries have unreported rates likely due to the lack of susceptibility testing. the mechanism of resistance is genetic with point mutations in a few positions of the domain v of the peptides transferase loop of the 23s rrna where macrolides bind the 50s rrna subunit. mp pneumonia was more prevalent throughout korea in 2011 and caused more severe clinical features than at any other time, which led to an unpublished observation that it may be associated with mrmp. despite the increasing prevalence of resistance, macrolides remain the drugs of choice for treatment of mp infections in children, but require antimicrobial stewardship. with increasing clinical severity of mp pneumonia and growing evidence for the role of an overreactive host defense, the role of corticosteroids has been investigated. in a study of 12 healthy patients followed retrospectively all had either severe pneumonia (high fever, respiratory distress or initial lobar pneumonic consolidation with or without pleural effusion) or refractory pneumonia ( prolonged fever of >7 days or persistent consolidation of more than one lobe of the lung despite appropriate antimicrobial therapy), were diagnosed serologically, had no viral co-infection, received appropriate antimicrobials (macrolide or beta-lactam) and methylprednisolone at 30 mg/kg/day × 3 days. all improved. although mrmp may cause severe refractory mp, susceptibility testing was not performed in the above study. most often, mp causes a benign respiratory illness that may involve both upper and lower respiratory tracts. but mp has the potential to cause severe disease. the emergence of mrmp has led to severe mp pneumonia. but macrolide resistance is only one potential cause of severe disease. an exuberant host inflammatory response with release of cytokines and, perhaps mediated largely by cmi, appears to play a role in some children and adolescents. in the latter situation, corticosteroids may prove beneficial but more research needs to be done. over the last three decades, many studies have investigated the best approaches to promote hand hygiene practices and improve hand hygiene indicators, in particular healthcare workers' (hcws) compliance. early studies on hand hygiene improvement in healthcare were focused on single interventions promoting the importance of handwashing and introducing the use of antimicrobial soaps. in 1994, our group in geneva conducted the first large-scale epidemiological research on hand hygiene, identifying major risk factors for noncompliance and demonstrating the critical role of alcohol-based handrubs (abhrs) as major system change to replace handwashing with soap and water, while included in a multimodal strategy to change hcw behavior. the strategy combines easy access to abhr ( proven to bypass the time constraint on hcws), hcw education, performance monitoring and feedback, reminders in the workplace and institutional safety climate. the strategy (referenced as the "geneva model of hand hygiene promotion) was proven successful, and hand hygiene improvement was associated with significant reduction in healthcare-associated infections (hais) and spread of multi-resistant organisms. between 2000 and 2006, the "geneva model of hand hygiene promotion" was replicated in single, as well as multiple healthcare institutions, at local, regional and national level, with success. since 2005, the world health organization (who) mandated our group to lead the first global patient safety challenge (clean care is safer care) with the main objectives to raise awareness about hai worldwide, mobilize nations toward ipc activities and promote best ipc practices, in particular hand hygiene. the recent meta-analysis by luangasanatip et al. demonstrates the critical role of the who multimodal approach in successful hand hygiene promotion. nevertheless, several knowledge gaps in hand hygiene monitoring and efficacy remain. in my lecture i will focus on the following topic areas related to key questions in the hand hygiene research agenda: • studies on direct and indirect monitoring of hand hygiene compliance, including new devices for observation and feedback; • studies of the influence of i) handrubbing duration; ii) volume of abhr used, and iii) the optimal sequence of the handrubbing steps within the "how to handrub" 6-step technique, in the reduction of bacterial counts on hcws hands; • studies on the burden of disease and implementation of infection prevention and control strategies worldwide; • studies of the possible role of patient participation and empowerment in hand hygiene promotion. hepatitis c virus (hcv) infection is one of the most common chronic liver disease. globally, it was estimated that in 2005, more than 185 million people had hcv antibodies ( prevalence of 2.8%). most patients infected with hcv acquired the disease through intravenous drug use or blood transfusion, the latter of which has become rare since routine testing of the blood supply for hcv began in 1990. nosocomial transmission of hcv has been documented in several health care settings. where there are stringent infection control protocols to prevent transmission, particularly through unsafe medication injection practices, nosocomial transmission has still been reported, generally because of breaches in protocol. rare sources of transmission of hcv include contaminated equipment used during the performance of procedures and other breakdowns of infection control procedures or aseptic techniques leading to person-to-person transmission. screening for hepatitis c virus (hcv) infection is an important component of successful control of hcv for the infected individual and for public health purposes. diagnostic tests for hepatitis c virus (hcv) are serologic assays that detect antibodies to hepatitis c, and molecular assays that detect or quantify hcv rna, other investigations such as genotype testing, serum fibrosis panels and liver biopsy may help to predict the response to treatment and prognosis. most cases of acute hcv infection are anicteric and asymptomatic, with fewer than 25% being clinically apparent. fulminant hepatitis c is rare. of those who go on to have chronic infection, a substantial proportion will develop cirrhosis, and a subset of those develop hepatocellular carcinoma. active surveillance based on mdr gram-negative pathogens? anucha apisarnthanarak md active surveillance is one of the common strategies employed to control mdr-gram negative pathogens. although listed in the guideline, the true value of active surveillance for mdr gram-negative pathogens has never been demonstrated. several considerations should be made when infection preventionists implement active surveillance as part of control measures. these include the specific reservoir of each specific mdr gram-negative pathogens, resource availability for isolation precaution, the turn-around time of active surveillance culture, whether institution is able to implement other infection prevention strategies together with active surveillance culture. in this session, i will address the pro-and con-for active surveillance culture for mdr gram-negative pathogens. application of active surveillance culture in resource-limited settings will also be discussed. also, rates of catheter associated urinary tract infection (cauti) and c-line associated blood stream infection (clbsi) significantly decreased from 1.85 to 0.88 ( per 1000 catheter-days, f = 10.14, p < 0.0001) and from 3.40 to 2.20 ( per 1,000 catheter-days, f = 14.17, p < 0.0001). in subgroup analysis, rates of vap, cauti and clbsi were significantly decreased regardless of organizational and institutional characteristics of icus. in summary, all of the da-hais have shown a significant reduction in the last 10 years, however v-ur has year-wise significantly increased trend for past 10-years, also uc-ur and cl-ur have not decreased trend significantly. we need effort to make reduction of device utilization ratios and associated infections. invasive fungal infections (ifi) are primary causes of mortality, and rapid and accurate diagnostic laboratory tests are required to improve patient outcomes. although histopathologic examinations of tissues can detect ifis, tissue morphology alone is insufficient to distinguish between aspergillus and other fungi, including fusarium, scedosporium, or mucorales. therefore, species identification using culture and non-culture methods is necessary. recently, assays targeting ribosomal dna have been used to identify infectious fungi in tissues. notably, analyses using frozen tissues were superior to those using formalin-fixed paraffin-embedded tissues. because pan-fungal primers hybridized dna from other eukaryotes, the pan-fungal approach was limited in tissues that primarily contained human dna and little fungal dna. diagnostic tests that rely on fungal cultures are common for ifis. recently, however, dna sequencing and matrix-assisted laser desorption/ ionization time-of-fight mass spectrometry (maldi-tof ms) have been used to rapidly and accurately identify fungal pathogens recovered from cultures. sequence-based identifications have been particularly useful for identifying cryptic species that were misidentified by microscopic analyses or were identified only to the complex level. some cryptic species are resistant to azole antifungal agents, and molecular methods have been developed to detect azole-and echinocandin-resistant candida species as well as azole-resistant aspergillus species. blood cultures are the gold standard for diagnosing candidemia; however, cultures require 1-3 days of growth followed by an additional 1-2 days for identification and antifungal susceptibility testing. such timeframes lead to delays in treatment initiation. additionally, up to onethird of patients with invasive candidiasis fail to test positive in blood cultures. pcr and the 1,3-β-d-glucan (bdg) assay are more sensitive than blood cultures for patients with deep-seated candidiasis. bdg is a cell wall component in candida and other fungal species, except mucorales and cryptococcus, and is included in the eortic/msg revised diagnostic criteria for invasive fungal diseases. however, bdg assays are expensive and labor-intensive, making them unavailable in most developing countries. the bdg assay is highly sensitive but lacks specificity; thus, it is typically suitable only for ruling out the causes of candidiasis. serum galactomannan (gm) is a cell wall component of aspergillus, and can be detected in serum using a commercial test (platelia™ aspergillus eia; biorad, usa). the serum gm test is considered aspergillus-specific, and multiple studies demonstrated high sensitivities (∼70%) in sera from patients with hematological malignancies or allogeneic hematopoietic stem cell transplantations. however, gm sensitivity is low in non-neutropenic patients and recipients of solid organ transplants. additionally, the gm test is associated with poor predictive values in patients receiving mold-active antifungal prophylaxis. however, the detection of gm in bal fluid is high (>70%) even among patients receiving mold-active antifungal therapies. multiple studies have shown that pcr was more sensitive than culture methods at detecting aspergillus in blood and respiratory fluids. multiple pcr assays are commercially available for the detection of aspergillus spp.; however, none is fda-approved. moreover, aspergillusspecific pcr is not recommended for clinical use as only few assays are standardized and validated. the t2candida assay (t2 biosystems, usa) was recently introduced for the detection of five common candida species from whole-blood samples. the t2candida assay is the only fda-approved diagnostic test that offers rapid diagnosis (3-5 hours) and specific organism identification with detection limits of 1 cfu/ml. the development of additional diagnostic tests will facilitate the early diagnosis and management of ifis in high-risk patients. the newest treatment strategies for candidemia thomas f. patterson professor of medicine, ut health san antonio, san antonio, texas usa candidemia is an important cause of morbidity and mortality especially in hospitalized and immunocompromised patients. despite advances in antifungal therapy, the number antifungal drugs and drug classes for treating these serious infectious remains limited. management is further complicated by the fact that it remains difficult to establish an accurate diagnosis which is compounded by the need for early therapy. fluconazole remains a useful antifungal agent especially for follow-on therapy after stabilization of infection but resistance is high for some species including c. glabrata. thus, the echinocandins have become recommended as primary therapy in most patients. however, development of echinocandin drug resistant strains has been reported worldwide. the development of drug resistance has often been tied to antifungal drug use, particularly for some species like candida glabrata. while multidrug resistance is uncommon, increasing reports of multidrug resistance to the azoles, echinocandins and polyenes have occurred in several candida species, most notably candida glabrata and more recently candida auris. new agents are under development for candidemia, including echinocandins improved pharmacokinetic profiles and those available for oral use. in addition, other agents with new targets of action are also undergoing preclinical development. diagnosis of infection and detection of antifungal resistance is critical to the successful management of patients with these infections. new therapies are aimed at improving outcomes in candidemia. epidemiology and management of mucormycosis and invasive aspergillosis: are there lessons to be learned? centre for infectious diseases and microbiology laboratory services, icpmr, westmead hospital, university of sydney, new south wales, australia the epidemiology of aspergillus and mucorales infections may be changing. although invasive aspergillosis (ia) remains a substantive cause of morbidity in patients with neutropenia, hematologic malignancy and organ transplants, there is an expansion in the spectrum of at risk patients. patients in the intensive care unit (icu), with chronic lung disease, hiv/aids and those on immunomodulating drugs are amongst relatively understudied populations. diagnostic difficulties including the interpretation of aspergillus cultures contribute to this limitation. other than the expansion in host risk groups, is the shift in etiology of ia, with cryptic or uncommon species emergent. in addition, azoleresistant a. fumigatus infections pose an increasing dilemma in regions of europe and asia (<5-30% resistance rates). in general, voriconazole is recommended for the primary treatment of ia regardless of site of infection. other azoles, the echinocandins or amphotericin b compounds may be used as second line or salvage therapy. primary combination therapy is not routinely advised. surgical derbridement or resection is an important adjunct where feasible. in the presence of azole resistance, either liposomal amphotericin b (l-amb) or a voriconazole-echinocandin combination is preferred. in vitro susceptibility testing is recommended for all ia cases. isavuconazole has good activity against aspergillus and new antifungal drugs with anti-aspergillus activity are in development. many cases of mucormycosis affect hosts with malignancy and stem cell transplantation, but organ transplantation, diabetes mellitus and iron overload are also important underlying conditions although the use of certain calcineurin inhibitors may be associated with lower risk. emerging risks include underlying rheumatological/autoimmune conditions. trauma-related cases may also be increasing and outbreaks of infection following natural disasters and iatrogenic exposure described. pathogen epidemiology varies with region with emergence of uncommon genera such as apophysomyces. despite best practice management and reversal of risks, mortality is up to 80%. treatment is based on case series and expert opinion. surgical debridement/resection combined with antifungals improves outcomes. initial treatment with l-amb is preferred agent although optimal dosage is uncertain. isavuconazole appears to be as efficacious as l-amb. combination polyene-echinocandin can be considered for extensive disease or salvage therapy, with posaconazole typically employed as step-down therapy. treatment algorithms may change with wider availability of iv posaconazole; other new drugs are in the pipeline. endemic fungal infection in the asia-pacific region (in the context of altered immunity) national university health system, singapore less is known about the incidence and characterization of deep mycoses in the asia pacific region. geoclimatic conditions, population demographics and health resource accessibility vary within the respective asian countries and also differ from west. invasive fungal diseases (ifd) encountered in the region will be discussed with specific highlights on the unique challenges faced pertaining to host immune susceptibility and in the management of these diseases. candidemia and candidiasis constitute the highest proportion of the ifds. in particular, the contribution by candida tropicalis in tropical asia pacific is not to be overlooked. candida tropicalis infection, linked to more severe disease, is seen in patients with weakened immunity such as hematological malignancies and in neonates. the host immune factors recently identified as predisposing to oro-esophageal candidiasis will also be discussed. chronic (cavitatory) pulmonary aspergillosis (cpa) in asia is often a sequelae to old tuberculosis as well as aspergillosis in the critically ill is under-recognized. the immune interplay between the ubiquitous mold and the host defense during severe illness will be explored. cryptococcosis and penicilliosis have traditionally been seen in patients infected with the human immunodeficiency virus (hiv). while cryptococcus infections have been recognized to occur beyond the context of hiv and even in apparently immunocompetent subjects, it is only recently that the immune susceptibility of such patients to cryptococcus are being dissected. similarly the notable predilection of talaormyces marneffei (and other rare fungi) in the asia pacific region is being linked to other non-hiv-attributed host immune defects including a novel disease trait seemingly distinct to asians. pre-exposure prophylaxis: from science to implementation rossana a. ditangco md prep for hiv refers to the preventive strategy of taking a medical agent prior to hiv exposure. this method specifically involves the oral intake of an antiretroviral drug (arv) by hiv-negative individuals who are at high risk of acquiring the virus in order to prevent hiv infection. recent studies have demonstrated safety and efficacy of dual antiretroviral (arv) oral pre-exposure prophylaxis (prep) in preventing sexual and parenteral transmission of hiv infection. the iprex study, a randomized controlled efficacy trial in men who have sex with men (msm) and a small contingent of male-to-female transgender women (tgw) is particularly relevant to asia and the pacific. in this study, emticitrabine/tenofovir disoproxil fumarate (ftc/tdf or truvada ® ) safely achieved a 44% per-protocol reduction in new hiv infections. however, risk reduction was 92% in ftc/tdf recipients having detectable study-drug blood levels, indicative of much greater efficacy associated with increased adherence. subsequent pharmacologic modeling and follow-up studies demonstrated daily use of these agents not being required for achieving optimal protection, declining on a gradual scale associated with decreased frequency of use . this research showed a reduction of hiv infection risk of 99% for 7 doses, 96% for 4 doses and 76% for 2 doses of ftc/tdf per week. the protective efficacy of non-daily dosing (89% risk reduction) was subsequently confirmed in a placebo-controlled trial of intermittent (on-demand) ftc/tdf prep among msm in france and canada. mathematical modeling using iprex findings demonstrated substantial reductions in new hiv infection associated with modest prep program coverage in msm, while increased prep adherence was shown to have the largest population level preventive impact with greatest cost-effectiveness . prep implementation has been shown feasible in open-label extensions and project sites with research and program capacity. despite its excellent safety and efficacy profile, the proof of implementation of prep in resource-limited settings outside of these situations still needs to be delivered. applications of prep in resource limited situations must be grounded in the reality of existing health systems and the interface between community-level primary care clinic-and hospital-based services. a number of unknowns remain with respect to the delivery of hiv prep for msm/tgw in these environments. among others, access, uptake and adherence, effectiveness and behavioral and social impact effects are not described. taking part in a prep project or using arv drugs may disclose same-sex behavior and hiv risk. these in turn may provoke negative reactions or undue pressures in the social, professional and family environment. participants may see certain services or privileges being revoked or denied. in a worst case scenario, disclosure may lead to denial or termination of health or life-insurance, rental agreements, employment or promotion. these negative repercussions constitute violations of individual rights and may adversely impact project participation and prep access, uptake and adherence. of particular concern are poor adherence and increases in sexual risk behavior, the latter potentially accelerating sexually transmitted infections (sti) followed by increased hiv acquisition and transmission in the non-prep serviced portion of at risk population. at present countries are at varying stages of introducing daily oral prep using tdf/ftc, which is now recommended by who as an option for people at substantial risk of hiv. some countries have national programs; others have smaller-scale programs; others aren't offering prep at all. the benefit of prep as a prevention strategy goes beyond its clinical efficacy. prep programs could increase hiv testing uptake and contact with health care provider for hiv counseling and education services and early diagnosis and treatment of sexually transmitted infection. scientific evidence of efficacy and safety of prep, mathematical models for cost effectiveness and potential elimination of new hiv infection and projected benefit beyond clinical effectiveness provide rationale for making prep available as additional layer of hiv prevention strategy. center for aids research, kumamoto university, kumamoto, japan despite the significant reduction in morbidity and mortality following combination antiretroviral therapy (art) there is emerging evidence that people with successfully treated hiv infection by art age prematurely, leading to progressive multi-organ disease referred as comorbidities. one of the major factors involved in this pathogenic process is residual viral replication by persistently infected cells surviving in vivo and subsequent chronic inflammation. in contrast to the current art that only targets viral replication neutralizing or nonneutralizing antibodies against the envelope proteins of hiv-1 have been reported to have an adcc (antibody-mediated cellular cytotoxicity) activity that eliminate hiv-1 infected cells in vitro. the discovery of potent and broadly neutralizing antibodies (bnabs) against hiv has made passive immunization a potential strategy for the prevention and treatment of hiv infection. the bnabs 3bnc117 and vrc01 targeting the hiv cd4-binding site has been tested in treatment naïve patients and on treatment patients for the delay in plasma viral rebound after the discontinuation of art. monoclonal antibody 10-1074 targets the v3 glycan supersite was used at the highest dose of 30 mg/kg and showed a rapid decline in viremia. however, virologic analyses revealed the emergence of multiple independent neutralization resistant mutants in every case. we conducted a phase 1b clinical trial of passive transfer of neutralizing monoclonal antibody kd-247, which is reactive against the tip of the v3 region. eligible subjects were randomized to receive one of the 3 doses of kd-247 and the treatment was found safe and well tolerated. we observed significant decrease in hiv-rna in the 16 mg/kg cohort of kd-247 with two in six cases who achieved >1 log reduction of hiv-rna. long-term suppression of viral load was observed for one patient despite significant decrease in plasma concentration of kd-247, suggesting effects of antibody other than neutralization. we previously reported that the neutralization escape mutants to kd-247 became sensitive to chemokine cc receptor (ccr)5 inhibitors such as maraviroc or cenicriviroc. conversely, resistance mutants to ccr5 inhibitors became sensitive to several neutralizing antibodies including kd-247. furthermore, a series of in vitro experiments suggested synergistic effects of the combination of kd-247 and ccr5 antagonists including maraviroc (figure) . these results taken together, suggest that the combination of neutralizing antibodies with ccr5-inhibitors would be a promising candidate of intensification therapy added onto the current suppressive art aiming toward functional cure of the disease. programmable nucleasesincluding zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens) and rna-guided engineered nucleases (rgens) derived from the bacterial clustered regularly interspaced short palindromic repeat (crispr)-cas (crisprassociated) systemenable targeted genetic modifications in cultured cells, as well as in whole animals and plants. the value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific dna cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. however, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. knowledge of nucleasespecific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications. group cpf1 is a recently reported effector endonuclease protein of the class 2 crispr-cas system. cpf1 has several differences from cas9: cleavage with 5' overhangs, shorter guide rna, and a longer distance between the seed sequence and cleavage site, which could provide potential advantages for some cases of genome editing such as nonhomologous end joining-based gene insertion and efficient genome editing using homology-directed repair. however, limited information is available about cpf1 activity profiles in mammalian cells, precluding its wide use for genome editing. furthermore, both selection of highly efficient guide rnas and collection of big data for determination of parameters of rna-programmable nucleases are currently laborious and costly due to lack of a reliable high-throughput approach to determine rnaprogrammable nuclease activity in mammalian cells. here, we performed en masse evaluation of guide rna and cpf1 activity using synthetic target sequences and deep sequencing. using this in vivo high-throughput approach, we determined on-and off-target activity profiles and protospacer adjacent motif (pam) sequences of cpf1. we found that sequence features of high activity ascpf1 guide rnas are distinct from those of spcas9 and that the pam of as and lbcpf1 in mammalian cells is tttv, rather than tttn, which was previously determined using an in vitro system. evaluation of off-target activity showed that cpf1 target sequences can be divided into three regions: a 6 base pair (bp) seed, a 13 bp trunk, and a 6 bp promiscuous region. these results should serve as a useful guide to select cpf1 as a genome editing tool. more importantly, our in vivo high-throughput evaluation system will greatly facilitate both the selection of efficient and precise guide rnas and the generation of big data for the development of advanced prediction programs for on-and off-target activities. tasp works if an infected person knows his/her hiv status. a diagnosis should have been made as early as possible, and treatment offered promptly, so that the time gap between infection and viral suppression can be narrowed. around the world, late diagnosis is still common. early diagnosis often hinges not just on easy access to voluntary testing, freedom from stigma, but also the availability of screening. selftesting and the submission of specimens (urine, saliva, dried blood spots) for laboratory testing are some solutions, albeit the existence of technical problems, and the need for establishing linkage to care. periodic testing campaigns could impact hiv epidemiology, as illustrated in our modelling study parametrized by hong kong's surveillance data. for patients enrolled in treatment programs, optimization of regimen is crucial to ensure lifelong maintenance of haart without adverse reactions. in the developed world, integrase inhibitor is now used in first-line regimens, which has the capacity of reducing viral load to undetectable range within weeks. their access and that of single tablet regimens, are however limited in places where most hiv patients are. for some antiretrovirals, for example abacavir and efavirenz, knowledge of the host genotype (hla-b5701 and cyp2b6-516gt respectively) could minimize the occurrence of adverse reactions which might otherwise compromise adherence. notably adherence is the key to lifelong maintenance of virus suppression, and there is no simple solution to guarantee adherence over years, or tens of years. whereas all efforts could be made to promote testing and improve treatment coverage, the underdiagnosed interval is often where the achilles heel is. except for a small number of cases presenting as acute infection, the seroconversion time is often unknown. using modified back calculation methods to estimate the seroconversion year of over 3,000 patients in hong kong, we found that the interquartile range of their underdiagnosed intervals was 1-4 years with a range between 0 and 10 years. the lengths of undiagnosed intervals were associated with age and the routes of hiv transmission. obviously people with low perceived risk of hiv infection had longer undiagnosed interval and were more likely to be late for diagnosis. from mathematical modelling, increase of undiagnosed intervals would offset infections averted by tasp even if good coverage and high adherence were assumed. apparently, tasp is a necessary yet insufficient intervention to achieve hiv elimination. tasp targets people infected with hiv, whereas interventions for non-infected individuals would contribute to the reduction of the size of populations requiring tasp. one good example of the latter is pre-exposure prophylaxis, but it's only accessible to handfuls of people at risk around the world. effective protocols are needed, and risk compensation is a new concern. let's not forget promotion of protected sex, provision of methadone maintenance (and other harm reduction measures) for injection drug users, and community-level behavioral interventions. they continue to be powerful components of the armamentarium of measures to combat hiv, complementing tasp to achieve hiv elimination. stephan harbarth health care-associated infection (hcai) is a major global issue in patient safety. it affects hundreds of millions of people worldwide, complicates the delivery of patient care, contributes to patient deaths and disability, promotes resistance to antibiotics, and generates additional expenditure to that already incurred by the patients' underlying disease. indeed, hcai is a growing international problem. patients are becoming more susceptible to infections because of more serious underlying illnesses. poor compliance with hand hygiene by health care staff, increased recourse to invasive medical devices, and care of the critically ill as well as lack of access to safe water and unclean instruments and environmental surfaces all play a role. the environment of patient care is also important. factors such as understaffing, high bed occupancy, and increased patient transfers all create new risks of infection. the first global patient safety challenge created a worldwide focus on reducing hcai as a vital element of the safety of patient care. the last two years provided important and clinically relevant research data for prevention of hcai in different patient populations. my presentation will summarise the results of clinical trials and systematic reviews for the reduction of various types of hcai, and will discuss them in the context of the current relevant scientific and clinical background. in particular, i will discuss recent data on the epidemiology and prevention of nosocomial infections in intensive care units, present new approaches to prevention of surgical site infections as well as catheter-related bloodstream infections, describe recent advances in hand hygiene research and attempt to briefly summarise specific challenges related to the management of infections caused by multidrugresistant microorganisms. overall, hcais remain one of the key challenges of hospital care and significantly contributes to morbidity and mortality. papers published in the last 2 years remind us that further reductions of hcai rates are possibleoften with the help of simple and rather inexpensive interventions. rapid and accurate diagnosis is critical for the effective treatment of life threatening infections, such as bloodstream, respiratory tract and complicated urinary tract infections (utis). these clinical syndromes are difficult to diagnose due to complex aetiology and challenging clinical sample types (e.g. blood, sputum). current culture based diagnosis often has sub-optimal specificity and sensitivity and is too slow to impact on patient management. shotgun metagenomics sequencing has the potential to change the way we diagnose infection, combining rapidity with comprehensiveness beyond that of current methods. real-time nanopore sequencing technology provides the rapid turnaround necessary for infectious diseases diagnostics applications at point-of-care. there are challenges in applying sequencing to infection diagnosis, however, including high human:pathogen nucleic acid ratios, low pathogen numbers and low quality nucleic acid, depending on the disease and the clinical sample type. it is, therefore, vital to carefully design, develop, and optimise diagnostics pipelines before attempting to apply them to clinical samples. i will describe how we develop our minion based infectious diseases diagnostics pipelines with examples from our ongoing research on pneumonia, sepsis and utis. protein synthesis enzymes as primary defense system against infection aminoacyl-trna synthetases (arss) are essential protein synthesis enzymes, making a covalent linkage of their cognate amino acids to trnas. since the catalytic activities of arss are always required for the viability of organisms, they are constitutively expressed and ubiquitously present. due to these features, arss are the first to be exposed to broad spectrum of stresses and challenges. in fact, they have shown diverse roles as rapid responding signal mediators beyond protein synthesis. here we show our recent findings on arss as primary defense system against bacterial and viral infections. for instance, tryptophanyl-trna synthetase (wrs), is rapidly secreted out from monocytes upon bacterial infection and primes innate immune responses. the secretion was far more rapid than the induction of innate immune system. in another case, glutamyl-prolyl-trna synthetase (eprs) plays a unique role in viral clearance. eprs blocks pcbp2mediated ubiquitination of mavs (mitochondrial antiviral signaling protein), leading to the inhibition of viral replication. based on these findings, other arss are expected to play unique roles against infection, thereby collectively serve as a primary shielding system. victor lim international medical university, kuala lumpur, malaysia the world is facing a crisis in antimicrobial resistance (amr). it has been projected that if nothing is done 10 million people will die from antimicrobial-resistant infections annually by 2050 and the economic cost will also be correspondingly high at one hundred trillion us dollars. the overuse of antimicrobial agents is a major driver for the emergence of resistance. improving antibiotic stewardship is therefore crucial in meeting the challenges posed by amr. to do so would require recognition of the problem by all stakeholders at all levels and the political will to solve. adequate planning and provision of resources (including legislation) are essential. most importantly however, it would require a behaviour change among all stakeholders. there is increasing recognition that providing evidence from health research while necessary is insufficient for the delivery of optimal health care. there is a need to translate knowledge into action. the field of knowledge translation (kt) is the scientific study of methods for closing the knowledge-to-practice gap, and of the barriers and facilitators inherent in this process and such methods must be employed if we are to enjoy any success in antibiotic stewardship. the esrc working group on amr report 2014 stated that although amr involves biological processes, the context which determines the operation of these biological mechanisms is shaped by social, cultural, political, and economic processes. 1 behavioural science is a multidisciplinary study of human (and animal) behavior and encompasses the disciplines of psychology, anthropology, sociology and economics. the contribution of behavioural science in improving antibiotic stewardship is crucial but has only been recognized lately. 2 antibiotic prescribing is a complex behaviour, carried out by an informed individual often making a subjectively rational choice. a recent report by the department of health and public health england summarised the evidence on behavioural change and antibiotic prescribing in healthcare settings. it pointed out the lack of underpinning psychological theory and behavioural science in most of the studies which have been done so far. 3 as a result interventions may not be effective in changing behavior. to change behavior we need to understand the drivers of behavior. drivers of prescriber behavior may be intrinsic or extrinsic. intrinsic factors include an altruistic motivation to do one's best for the patient, ignorance, duration of practice, the tendency to yield to patient demands, fear (both of progression of disease and losing the patient), and a lack of understanding of the resistance problem. extrinsic factors would include patient demands, the influence of the pharmaceutical industry, diagnostic uncertainty, patient comorbidities and social class, workload and time pressures, role modelling by senior colleagues and financial incentives. interventions to improve antibiotic stewardship had included prescriber education and training, the issuance of antibiotic guidelines, the use of electronic decision support systems, audit and feedback, rapid and near patient testing to reduce diagnostic uncertainty, restriction strategies (pharmacy and laboratory) as well as financial strategies. to increase awareness of the problem of amr among the public social marketing strategies have been employed including the use of the mass media. although a variety of interventions have been employed over the last 5 decades; their overall effectiveness is subject to question. results have been variable. what works in one setting may not work in another as behavior change is a complex process and influenced by social, economic, ethnic and cultural beliefs. many studies are cross-sectional in nature and the sustainability of the intervention is not measured. to design any strategy or intervention there is a need to understand both prescriber and patient/public behavior and the factors that drive it using methodologies that are established in the behavioural sciences. any intervention has to be specific to the local context and the target population. a combination of strategies may be necessary to modify behavior for desired outcomes. department of infectious diseases, institute of infectious diseases and epidemiology, tan tock seng hospital, singapore antimicrobial resistance in hospitals is characterized by widespread dissemination of multidrug-resistant and extensively drug resistant bacteria, including methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum beta-lactamase and carbapenemase producing enterobacteriaceae, and pandrug-resistant acinetobacter baumannii. a combination of enhanced infection control and antimicrobial stewardship is often recommended to combat antimicrobial resistance in hospitals. multiple strategies are recommended by professional societies for antimicrobial stewardship, with differing levels of evidence and effectiveness. several recent well conducted systematic reviews and meta-analyses have shown that antimicrobial stewardship is effective in reducing broadspectrum antibiotic use and antimicrobial resistance without increasing adverse clinical outcomes. restrictive strategies produce more immediate effects but persuasive strategies are associated with more sustained impact. in addition to commonly adopted strategies of pre-authorization and prospective review and feedback, a greater variety of antimicrobial stewardship interventions are now recommended. in particular, evidence-based clinical care paths for common infections are associated with improved mortality. however antimicrobial use in hospitals is part of the greater context of antimicrobial use and resistance within the one health continuum. increasingly national and regional antimicrobial stewardship efforts must move outside of hospitals. damps: neutrophil dysfunction in experimental sepsis and post-septic complications related to immunosuppression jaroslaw zmijewski university of alabama at birmingham, usa damps: neutrophil dysfunction in experimental sepsis and post-septic complications related to immunosuppression bone using a murine model of polymicrobial septic peritonitis, we demonstrated that treatment with anti-hmgb1 ab significantly diminished sepsis-induced dysfunction of neutrophil nadph oxidase activity. importantly, confirmatory experiments revealed that blocking hmgb1 prevents neutrophils dysfunction. in summary, these results suggest that hmgb1 accumulation in the late phase of sepsis plays a specific role in the development of immunosuppression and specifically affects neutrophil-dependent antibacterial function in sepsis survivors. epidemiology of extensively-resistant gram-negative in mainland china hui wang peking university people's hospital, pr china multidrug resistance in gram-negative bacteria, especially carbapenem-resistant enterobacteriaceae (cre), is a critical public health threat in china advances in next-generation sequencing (ngs) platforms and microbial bioinformatics have positioned ngs to play an increasing role in clinical microbiology laboratories. next-generation sequencing is being applied to microbial isolates as well as directly to clinical specimens benchtop sequencers suitable for use in clinical microbiology laboratories are now available. sequences may be generated with various approaches (e.g., paired-end, mate-pair) and either aligned against a reference strain or assembled de novo with subsequent analytic strategies including single nucleotide polymorphism (snp) analysis and core genome multilocus sequence typing (cgmlst), among others. these approaches impact turnaround time, cost, technical difficulty, and accuracy. how results compare to those of historical methods not only does it allow for pathogen identification, but gene content information can also be used for resistance prediction, typing, and assessment for other relevant genes, such as those encoding virulence factors. our experience applying this approach to the diagnosis of prosthetic joint infection will be presented we found that loss of parkin, ubiquitin ligase implicated in autophagy and mitophagy occurs several hours after pro-inflammatory engagement in macrophages and lungs of mice subjected to intratracheal instillation of endotoxin. parkin dissipation was also accompanied with diminished activity in ampactivated protein kinase (ampk), a major sensor and metabolic regulator of immune homeostasis. we hypothesize that ampk activation will overcome loss in parkin-mediated autophagy and thus, diminish severity of ali. we found that ampk activators metformin or aicar did not recover the amounts of parkin. however, ampk activation promoted autophagy and improved bacterial clearance. in summary, our results show that parkin deficiency increased macrophage pro-inflammatory activation and the severity of ali mayo clinic, usa infectious diseases diagnostic testing is currently in a revolution vis-à-vis delivering new technologies. a myriad of new technologies are in use or under development, including matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (maldi-tof ms), rapid multiplex (i.e., panel) nucleic acid amplification tests (naats), point-of-care microbiology naats, and rapid phenotypic bacterial susceptibility testing, to name a few. these technologies provide new tools to combat antimicrobial resistance, which is especially important in an era of rising resistance. today, there is increased use of unneeded broad spectrum therapy because of the need to address the possibility of resistance until such a time as results of conventional diagnostic tests are available. this situation can potentially be ameliorated by more rapid diagnostics. in addition, over-prescription of antibacterial agents in general can potentially be addressed by rapid tests which exclude the need to prescribe antibacterial agents in the first place. an overview of new rapid diagnostics for infectious diseases that can potentially help combat antimicrobial resistance will be presented.ideal new diagnostics are more rapid, less expensive, and more accurate than existing diagnostics. unfortunately, not all new diagnostics meet all these criteria, with cost often being high. to address this situation, outcomes studies evaluating new diagnostics have assumed an important role. outcomes that should be addressed include antibiotic and further test avoidance, patient outcomes (e.g., length of stay, morbidity, mortality), patient and provider satisfaction, and infection transmission. results of outcomes studies then need to be used to inform the development of practice guidelines for use of these tests (which may vary from practice-to-practice) and also to inform ideal reconfiguration/development of tests by diagnostics companies. our experience with a recent randomized controlled clinical trial evaluating a rapid multiplex naat for testing positive blood culture bottles will be presented.it is an exciting time for diagnostics, with new technologies and opportunities for studies to determine how best to use these technologies in patient care. div. infectious diseases, dept. internal medicine, the catholic university of korea, koreasepsis is a significant syndrome in medicine and one of a major contributing cause of death world-widely.prompt recognition and aggressive management is the key element of improving survival from this brutal disease. unfortunately, sepsis is very complex immunologic event according to etiology and individuals' characteristics. as a result, a degree of organ dysfunction result from sepsis, an effect of protocolized treatment, and outcome could not be same.resuscitation with fluid and vasopressors is an initial and essential treatment of severe sepsis and septic shock. surviving sepsis campaign (ssc) continues to revise the treatment guideline and the initial steps for treatment are converged to use crystalloid fluid and use norepinephrine to whom do not respond to fluid therapy. in 2001, rivers et al suggested protocolized quantitative resuscitation method, otherwise known as early goal-directed therapy (egdt). this method was attractive in hospital settings because of clear notification of parameters, target, and drugs. the uncertainty of the effect of the individual component of bundle approach in treating severe sepsis/septic shock has been debated and egdt has been challenged following the failure to show a mortality reduction in subsequent large multicenter randomized controlled trials. effective fluid resuscitation means an effective restoration of tissue perfusion. although mean arterial pressure (map) can be a representative marker of tissue perfusion, central venous pressure (cvp) and serum lactic acid do not. as you already know, we do not have precise parameters of individual patient's hemodynamic status and also evaluating an effect of therapy we do. what is the proper volume of resuscitating fluid and how to monitor are also important issues we should have interest? although the crystalloid fluid is recommended as an initial therapy of resuscitation, the benefit, and harm of colloidal fluid, and alternative fluid is still evaluating.vasopressor therapy is needed to who do not respond fluid resuscitation. but there are many unresolved questions such as target map, optimal timing, and duration. norepinephrine has been recommended since the first ssc guideline, because more potent and effective at reversing hypotension, and less adverse events compared to dopamine. epinephrine and vasopressin are regarded as next-line or partner drug with norepinephrine. the effect of other vasopressors such as angiotensin ii and endothelin-1 are evaluating.medical experience clearly says that fluid resuscitation and vasopressor are very essential and initial element to improve the survival of the sepsis and septic shock patients. but we do not exactly know how to do accurately and properly. i will briefly present the current consensus and controversies about fluid resuscitation, monitoring of patient's hemodynamics, and use of vasopressors in septic patients in this lecture. the antimicrobial stewardship program (asp) is recognized as the most important tool for proper use of antibiotics. however, in order to properly implement asp, systematic training and tools are needed for medical staff prescribing antibiotics.basic education consists of face-to-face training for medical staffs. however, face-to-face education alone is difficult to maximize its effectiveness. when medical staffs prescribe, a variety of tools are being used to prescribe antibiotics properly.the most basic program is the antibiotic prescription restriction program. this program is a system that antibiotics which can only be used for certain resistant strains in hospitals are used with the permission of an antibiotic manager (usually an infectious diseases specialist).most hospitals in korea with infectious diseases specialists have this system. the infectious disease division of hallym universuty kangnam sacred heart hospital have a list of restricted antibiotics that contain vancomycin, linezolid, carbapenem, piperacillin/tazobactam, tigecycline, liposomal amphotericin b, echinocandin, voriconazole as restricted antibiotics.since the 2000s, antibiotic prescription programs have been introduced in korea and used in various hospitals. the purpose of this program is to recommend appropriate antibiotics according to the impression at the time of admission to the patient. when the causative bacteria are cultured, they help to prescribe appropriate antimicrobial agents according to the cultures. advanced programs automatically adjust the antibiotic dose according to the renal function. the number of antibiotic doses is also automatically recommended. when antibiotics need to be mixed with other solutions, it is automatically recommended that the solutions be mixed.currently, these antibiotic prescription programs have begun to apply the learning function of artificial intelligence and recommend antibiotics that frequently used depending on the name of diagnosis. if you are using antibiotics which could be expected to be drug interactions, you can also show alarms via pop-ups.in 2016, the korean society for chemotherapy developed a mobile app to help prescribe antibiotics. from 2017 to 2018, the korean society of chemotherapy and korea cdc (center for disease control and prevention) are conducting this project together. the antibiotic program is a collaborative project with hospital and clinic antibiotic prescription programs.this lecture will discuss antibiotic prescription programs used in korea and discuss the developmental direction of this program. key: cord-276907-b855tj7x authors: giersing, birgitte k.; vekemans, johan; nava, samantha; kaslow, david c.; moorthy, vasee title: report from the world health organization’s third product development for vaccines advisory committee (pdvac) meeting, geneva, 8–10th june 2016 date: 2019-11-28 journal: vaccine doi: 10.1016/j.vaccine.2016.10.090 sha: doc_id: 276907 cord_uid: b855tj7x abstract the third meeting of who’s product development for vaccines advisory committee (pdvac) was held in june 2016, with a remit to revisit the pathogen areas for which significant progress has occurred since recommendations from the 2015 meeting, as well as to consider new advances in the development of vaccines against other pathogens. since the previous meeting, significant progress has been made with regulatory approvals of the first malaria and dengue vaccines, and the first phase iii trials of a respiratory syncytial virus (rsv) vaccine candidate has started in the elderly and pregnant women. in addition, pdvac has also supported vaccine development efforts against important emerging pathogens, including middle eastern coronavirus (mers cov) and zika virus. trials of hiv and tuberculosis vaccine candidates are steadily progressing towards pivotal data points, and the leading norovirus vaccine candidate has entered a phase iib efficacy study. who’s immunization, vaccine and biologicals (ivb) department is actively working in several pathogen areas on the recommendation of pdvac, as well as continuing horizon scanning for advances in the development of vaccines that may benefit low and middle income countries (lmics), such as the recent licensure of the enterovirus 71 (ev71) vaccine in china. following on from discussions with who’s strategic advisory group of experts (sage) on immunization, pdvac will also look beyond licensure and consider data needs for vaccine recommendation and implementation to reduce the delay between vaccine approval and vaccine impact. who's pdvac was established by the department of immunization, vaccines and biologicals (ivb) in 2014, following a review of who's process for strategic priority setting for vaccines. the need for a group to advise who specifically on vaccine product development was highlighted, to accelerate vaccine availability and ensure accessibility of vaccines to low and middle income countries (lmics). pdvac's remit is to advise on the product development strategy of vaccine candidates at phase ii of clinical evaluation or earlier, and to report its proceedings to the who's principal committee on immunization policy recommendations: the strategic advisory group of experts on immunization (sage). the pdvac committee has a critical role in assessing the evolving vaccine development landscape and in helping to define where and how who can be most impactful, according to three criteria: likelihood of a product emerging from the pipeline, as defined by probability of technical and regulatory success, and the extent of awareness, activity and investment in a given area, a clear role for who with perceived added value for engagement in the pathogen area. typically, who engages in a pathogen area by working with a broad set of key vaccine development stakeholders to develop consensus on pivotal clinical trial design, vaccine roadmaps, or guidance documents on desired vaccine properties, referred to as preferred product characteristics (ppcs). ppcs define who preferences for the properties of vaccines to be used in lmics that are 5-10 years from licensure, and inform target product profiles in use by manufacturers and funders for vaccines. pdvac also encourages developers to be aware of the process and requirements for who prequalification (pq). who prequalification is a service to unicef and other un agencies that purchase vaccines once they have been licensed, to determine the acceptability, in principle, of vaccines from different sources for supply to these agencies. it aims to ensure that diagnostics, medicines, vaccines and immunizationrelated equipment and devices for high burden diseases meet global standards of quality, safety and efficacy, and are appropriate for use in lmics contexts in order to optimize the potential benefit of these interventions [1] . the third pdvac meeting was held in geneva from 8-10th june 2016. dr. jean-marie okwo-bele, director of ivb, opened proceedings with a synopsis of the significant milestones in vaccine development in the nine months since the previous meeting in september 2015: the first dengue and malaria vaccines have been licensed or achieved the equivalent of licensure, respectively, the first rsv vaccine candidate has entered phase iii studies in the elderly and pregnant women, the most advanced hiv vaccine candidate has met its endpoints in the interim analysis of a phase ii study, and preparations to commence an efficacy study are underway, who convened the mers-coronavirus r&d community, and a phase i clinical study is now underway (nct02670187), ebola virus vaccines are under review and have progressed to the point of consideration for licensure in record time, there are co-ordinated efforts to develop a zika virus vaccine as expeditiously as possible. a pdvac working group has overseen the development of a zika virus vaccine target product profile (tpp), and developed regulatory considerations towards phase i and emergency use authorization. in addition to these significant advances in vaccine development, the uk government published in may 2016 the report on 'tackling drug-resistant infections globally' that it commissioned in collaboration with the welcome trust [2] . the report highlights the urgent need to reduce reliance on currently available antimicrobials, without which today's 700,000 deaths per year from drug resistant microbes is forecasted to increase to 10 million, by 2050. the cost in terms of lost global production due to infections that are not controllable due to antimicrobial resistance (amr) is estimated to be $100 trillion by 2050 if no action is taken [2] . the development of vaccines against pathogens that are currently controlled by antimicrobials has become an imperative, as they have the potential to reduce the prevalence and spread of drug resis-tance, as well as to reduce the use of antimicrobials more broadly [3] . the decade for vaccines' global vaccine action plan (gvap) mid-term review, required an assessment of progress against objectives since its inception in 2011, and strategic planning to achieve the stated targets within the remaining 5 years. part of pdvac's remit is to review the vaccine development pipeline and consider the priority activities for ivb, within this context. during the remaining timeframe of gvap, a number of vaccines could reach licensure, and who needs to ensure early engagement with policy makers regarding potential vaccine implementation, as well as alignment with gavi's vaccine investment strategy. to facilitate information sharing, and tracking of progress within the global vaccine development community, the who has established and maintains an online 'vaccine pipeline tracker' in which information regarding all current clinical studies in several different pathogen areas can be found [4] . in addition, landscape analyses for 25 pathogens from the 2015 meeting have been collated within a special issue of the journal 'vaccine' and all are available through open access [5] . these documents are authored by independent subject matter experts and review the status of vaccine candidate development, as well as assessing possible pathways to regulatory approval. pdvac reports progress on the global vaccine development pipeline to who's strategic advisory group of experts (sage) on immunization. at the meeting in april 2016, advances in the development of interventions (vaccines and monoclonal antibodies) for respiratory syncytial virus (rsv) were presented for information. the reports from the october 2015 and april 2016 sage meetings are available online [6, 7] . much of the discussion focused on the need to better understand the key factors for early for implementation, as well as safety and efficacy data to support the assessment of a vaccine for policy recommendation. as emerging vaccines are likely to require new vaccination platforms, such as maternal immunization, or visits outside of the current vaccination schedule, such as for the recently licensed malaria vaccine rts,s, cost-effectiveness data informing their optimal use and potential impact must be generated in line with conventional clinical data required for regulatory approval, to minimise the delay between vaccine licensure and uptake [8] . the goals of this third pdvac meeting were to revisit the pathogen areas where there has been significant progress to report since recommendations from the 2015 meeting, as well as to: review status of vaccine development in 7 new pathogen areas where there has been significant vaccine development progress, or where there is significant disease burden but r&d has stalled, refine the workplan and strategic directions for ivb in specific pathogen areas, identify cross-cutting issues that accelerate vaccine development or prepare for policy decisions, where appropriate, consider how to better align pdvac's vaccine development activities and strategies with other areas of research, to inform the vaccine development community regarding steps to be considered beyond vaccine licensure, and who processes for vaccine policy recommendation. the gvap is a 10-year strategic framework derived from the decade of vaccines collaboration [9] to prevent millions of deaths by 2020 through more equitable access to existing vaccines for people in all communities. within this framework is a specific objective that supports research and development of innovations that will maximise the benefits of immunization, with indicators for progress towards development of hiv, malaria, tuberculosis and influenza vaccines. the gvap has just completed its midterm review stage, and following the 2015 recommendation from sage [10] , the 2016 gvap assessment will highlight advances made in these areas. these four pathogen areas are standing agenda items for discussion at pdvac. in 2014, mycobacterium tuberculosis (mtb) killed 1.5 million people (0.4 million of whom were co-infected with hiv) and is now the world's most deadly infectious disease [11] . approximately 480,000 cases/annum are multi-drug resistant (mdr) or extensively drug resistant (xdr) and some strains are untreatable. in 2014, six million new cases of mtb were reported to who, fewer than two-thirds (63%) of the 9.6 million people estimated to have contracted the disease. this means that 37% of new cases were not detected or reported. a vaccine is imperative to achieving the end tb goals [12] , particularly through reaching the population who are undiagnosed and continue to transmit disease. as such, the tb vaccine development community has turned its focus to the development of vaccines targeted to adolescents and adults as the age-groups with highest burden of active disease and the source of mtb transmission. modelling studies suggest that prevention of pulmonary disease in this population from primary infection and from reinfection or reactivation of existing infections is the most effective strategy to prevent mtb infection and disease in infants and children [13] . the most advanced vaccine candidates are targeting this indication, including current neonatal bcg replacement candidate vaccines that are also undergoing evaluation as a booster in later life. several of these candidates are in proof-of-concept clinical studies and are approaching key endpoints through prevention of infection or disease, or prevention of disease due to reinfection in this these target populations in the next 12-24 months [14] . with this in mind, pdvac recommended that who prioritize and facilitate consensus building with respect to the development of strategic goal(s) and ppc(s) for vaccines targeted to adolescents and adults, in the first instance. there are several candidates and platforms in the pipeline that target this goal in this population, as well as other important target populations [4] . pdvac acknowledged the significant need for development for these vaccines in parallel, as well as continued efforts to understand the biological mechanism of disease to support the immunological rationalization of candidates. the pox-protein public private partnership (p5) consisting of sanofi, glaxosmithkline (gsk), bill & melinda gates foundation, the us military hiv research program (mhrp), and the hiv vaccine trials network (hvtn) have been collaborating with the us national institutes of allergy and infectious disease (niaid) to optimize and assess the efficacy of the alvac/heterologous prime boost approach, following the demonstration of partial efficacy in the rv144 trial in thailand [15] . the interim data from a phase i/ ii study (hvtn 100) met its humoral and cellular immunological 'go' criteria, exceeding the rv144 responses against sub-saharan clade c antigens. extrapolation of these responses to those observed with rv144, suggest that the optimised vaccine could offer at least 50% protection following a 12 month booster. based on these data, a randomised placebo controlled phase iib/iii efficacy trial (hvtn702) enrolling 5400 subjects was initiated in late 2016 in south africa, and will evaluate alvac (clade c) prime/ bivalent recombinant gp120 protein with mf59 adjuvant as a heterologous boost, as well as the effect of a booster at 12 months [16] . futility analyses will be undertaken early in the 2 year followup period. correlate of protection studies and assessment of crossreactivity to other regional clades are included in the study design. discussions with the south african medicines control council (mcc) are ongoing, and licensure in south africa could be as early as 2021. other vaccine candidates are in development, including janssen's heterologous prime boost approach with ad26/gp140, currently undergoing dose regimen selection in phase i/iia trials. antibody-mediated prevention using broadly neutralizing, potent monoclonal antibody (bnmabs) approaches are also undergoing phase i/iia clinical evaluation. the niaid/vaccine research centre's vrc01 broadly neutralising mab is the most advanced candidate which has been shown to neutralise cd4 binding of 90% of viral isolates. hvtn 703/hptn 081 and hvtn 704/hptn 085 are phase iib studies to evaluate the efficacy of vrc01 in reducing acquisition of hiv-1 infection in high risk populations in the americas and sub-saharan africa, and started enrolment in 2016. if shown to be effective, administration of vrc01 could be positioned as a long-acting supplement to increase effectiveness of anti-retrovirals. pdvac commended the advances in hiv vaccine development, and requested to be kept informed about progress with hvtn702. currently, there are no known intentions for global studies with the p5 candidate vaccine, or to seek who prequalification. pdvac encourages the p5 partners and the south african hiv vaccine development community to keep who fully informed about progress with the trial. concerns were expressed regarding the lack of follow-on studies in thailand, given that the initial landmark rv144 trial was performed there. despite the substantial reduction over the last 15 years (over 50% for global malaria mortality in children aged <5 years), mainly due to greater investments in malaria control, the who estimates there were 214 million malaria cases in 2015, 88% of which were in africa. of the 438,000 people who died from the disease in 2015, 90% reside in africa [17] . given the increase in multi-drug and insecticide resistance, there remains an urgent need for a vaccine to combat malaria. as reported in the 2015 pdvac meeting summary [18] , the european medicine's agency (ema) provided a positive scientific opinion, indicating a favorable assessment of the risk-benefit balance of rts,s/as01 from a regulatory perspective. in october 2015, two advisory bodies to who, namely sage and the malaria policy advisory committee (mpac), recommended pilot implementation studies of the 4-dose schedule of the rts,s/as01 vaccine in 3-5 distinct epidemiological settings in sub-saharan africa, at sub-national level, covering moderate-to-high transmission settings, with three doses administered to children between 5 and 9 months of age, followed by a fourth dose 15-18 months later. the intent of these pilot studies is to assess: the feasibility of providing all four doses of rts,s to the target age group through existing health services; the impact of rts,s on child mortality; whether there are any safety issues, particularly evidence of any causal relationships between rts,s administration and either meningitis or cerebral malaria (both signalled in the phase iii trials), whether introduction of the vaccine impacts positively or negatively on existing country immunization programs and on the use of currently recommended malaria control measures. in 2013, the malaria vaccine technology roadmap was updated to include licensure of vaccines targeting plasmodium falciparum and plasmodium vivax by 2030, with protective efficacy of at least 75% against clinical malaria, and that reduce transmission of the parasite and thereby substantially reduce the incidence of human malaria parasite infection [19] . the vaccine candidate pipeline is robust, and includes novel antigens and platforms [4] . second generation vaccines are expected to provide higher protection than rts,s in the longer term. optimised tools are needed to measure incremental improvements and predict potential cost effectiveness of new candidates. the development of controlled human malaria infection (chmi) models, efforts to harmonize elements of clinical trial design and standardization of various assays continue. pdvac stressed the importance of the development of 2nd generation malaria vaccines in parallel to the pilot implementation program for rts,s, and proposed that the current version of the vaccine roadmap be updated, potentially in 2018, in light of the rts,s pilot implementation. in 2015, pdvac noted that development of universal influenza vaccines will be challenging and protracted, particularly due to the lack of a regulatory pathway for novel antigens that operate through induction of t-cell immunity. rather, pdvac recommended that there be a focus on the definition of, and the collection of data to support implementation of 'improved' seasonal flu vaccines that would offer more immediate impact in lmics. pdvac advised who to develop strategic public health goals and ppcs for improved seasonal influenza vaccines, and to provide guidance on data requirements that would be needed to establish improved performance of such vaccines. a working group has been established, and has proposed a draft statement of unmet public health need: 'safe and well-tolerated influenza vaccines that are effective at preventing severe influenza illness, that provide protection beyond a single year, and that are programmatically suitable for use, are needed for low-and middleincome countries.' draft 5-and 10-year strategic goals for development of influenza vaccines that induce broader and more durable protection against severe illness caused by influenza a strains have been developed. these strategic goals and the draft ppc for nextgeneration influenza vaccines were presented at the upcoming eighth who meeting on development of influenza vaccines [20] . pdvac reaffirmed the value of ppcs based on the two different approaches. there is a public health need to develop improved performance of currently available seasonal vaccines to offer protection over multiple seasons, and against drifted strains, with a view to generating shorter timelines to achieving availability and access in lmics. as part of this effort, it will be necessary to define the criteria needed to demonstrate clinical benefit, and additional data requirements to support policy recommendations. efforts to develop 'universal' vaccines that target conserved antigens, or conserved components of antigens, should continue in parallel, with a focus on identifying correlates of protection to support a regulatory pathway for this novel class of vaccines. diarrheal disease remains the second leading cause of death in children under 5 years of age. although mortality has declined over the past four decades, morbidity has not declined significantly, despite improvements in water and sanitation and benefits from oral rehydration therapy. there are nearly 2.7 billion cases of diarrheal disease every year, many with acute and chronic effects such as growth stunting and cognitive impairment. these long term sequelae significantly impact quality of life and economic potential, and are estimated to affect one-fifth of children globally. in 2015, pdvac recommended that who expand its remit to include support for enteric vaccine development, particularly against enterotoxigenic escherichia coli (etec) and shigella. one of the main objectives of the planned who engagement in this area will be to ensure that the design of the phase iii efficacy study, including definition of primary/secondary endpoints and long-term follow up, and the data generated, will be relevant to support a policy recommendation from sage. another key objective is to develop a who preferred product characteristics document which outlines who preferences, including considerations for development towards a potential combined vaccine. several vaccines are in development, with two etec candidates and seven shigella candidates currently in clinical studies. for etec, the most advanced vaccine is etvax adjuvanted with dmlt, which is being developed for both a pediatric and traveller's indication. a phase i/ii dose escalation, age de-escalation study in children is currently ongoing in bangladesh, with intent to further age deescalate into 6 week-old infants in late 2016. in parallel, a phase iib study in travellers is planned to begin in 2017. based on an encouraging phase iib immunization and challenge study and additional positive protection studies in non-human primates (nhps), an adhesin-based subunit etec vaccine (fta) is moving forward with an accelerated clinical program designed to move a complete multi-valent vaccine into descending age field trials in 2020. the most advanced shigella candidate is trivalent shigella killed whole cell (tswc) composed of formalin-inactivated s. flexneri 2a, s. flexneri 3a, and s. sonnei, expected to offer coverage across about 80% of isolates. a phase i study has been completed and a challenge trial with s. flexneri 2a prototype will begin in 2017, followed by a study that will assess co-administration with etvax. both etvax and tswc are being developed for oral administration. other promising shigella vaccines in early stage clinical testing include two live attenuated vaccines, wrrs1 and shigetec. wrrs1 is in a descending age study in bangladesh, while shigetec, which is a combination shigella-etec combination vaccine, will begin a phase i study in early 2017. three subunit approaches for shigella are also in phase i/ii studies; the prototype s. flexneri 2a bioconjugate vaccine (flexyn2a), invaplex and the generalized module for membrane antigens (gmma). one of the critical strategic issues is whether to prioritize the licensure and approval of an etec vaccine, or to focus on the development of a combination with shigella that will likely delay the timeline to vaccine availability. epidemiologic data suggest that both intra-and inter-country disease heterogeneity is likely to exist and this may drive vaccine preferences, and presentation optimization. these data are critical to inform decision-making by country policymakers. for this reason, development of who derived preferred product characteristics for etec and shigella vaccines, alone and in combination is needed. in april 2016, plos released a collection on 'the global burden of norovirus & prospects for vaccine development' [21] , which includes the most current estimates on global norovirus disease burden of over 200,000 deaths in low resource countries, and a global economic burden of more than $60 billion [22] . recent molecular analyses of samples from the community based longitudinal birth cohort mal-ed study suggest that norovirus is the most common diarrheal pathogen in the first year of life, and the second most common in the second year of life. there are 5 vaccine candidates in development, including three strategies to develop a combination vaccine against other enteric pathogens. however, only one candidate, which is composed of two vlps based on the gi.i and gii.4 norovirus genotypes, has entered clinical studies, a phase iib study began recently [23] . the advent of cell culture methods for norovirus will facilitate many advancements, including the optimization of a neutralization assay and enable the assessment of antisera against this vaccine to block binding of a diverse genotypes. in addition, in response to the 2015 pdvac recommendation to consider incorporating norovirus surveillance within the who global rotavirus surveillance network, a survey of the capability and capacity at representative global sites has been performed to support a pilot study proposal. the recently published epidemiology and burden of disease data indicate that norovirus fulfils the pdvac criterion of unmet public health for a vaccine in lmics. however, the ability of the candidates in the pipeline to offer protection over the range of circulating and emerging viral genotypes, and therefore the duration of protection of these vaccines, is currently unknown. it is conceivable that the vaccine will need to be periodically re-formulated, to include emerging genotypes. in addition to infants as a priority target population, adults and particularly the elderly are at risk, requiring the potential need for two vaccine formulations and/or presentations. fortunately, at the current time, development of a norovirus vaccine that may offer efficacy in the context of low and middle income countries is proceeding with investment from the private sector, however an assessment of vaccine programmatic suitability and applicability to prequalification is needed, prior to phase iii trials to ensure the vaccine is appropriate for use in lmics, assuming it is demonstrated to offer coverage over circulating genotypes within lmics. rotavirus is the leading cause of severe diarrhea among all children below 5 years of age worldwide, causing 20-40% of severe diarrheal hospitalisations, and is associated with significant mortality, with the latest mortality estimates at 215,000 deaths in 2013 [24] . the introduction of the live-attenuated oral rotavirus vaccines, rotateq and rotarix, in 2008 has had significant direct and indirect impact in countries where they are in use, including saving lives and reducing hospitalizations. however, in gavieligible and lmic countries in asia and africa the vaccine effectiveness is lower, with protective efficacy observed from 40 to 70% against severe rotavirus diarrhea over the first year of life. waning of protection has also been observed in these settings, with lower protection rates (25-50%) in the second year of life. in comparison, in high-income countries protection is higher (70-90%) and persists into the second year of life. thus, despite the enormous success of the live oral rotavirus vaccines, several challenges and issues remain such as the lower protection in gavi-eligible and lmic countries in africa and asia, together with the high cost of available vaccines. despite an overall acceptable safety profile, the intussusception rate seems to be slightly increased by vaccination (occurrence 1-3/100 000 oral rotavirus vaccine recipients) in high income countries. several new oral, live-attenuated vaccines, composed of alternative strains, are in mid-to late-stage clinical development. the current who guidance document for the quality, safety and efficacy of oral live attenuated rotavirus vaccines [25] would be applicable for these next generation oral, live-attenuated vaccines. of these new oral rotavirus vaccines, rotavac 20c (developed by bbil) is the only vaccine currently licensed for use in children, having been approved for use in india in 2014. this vaccine is available on the private market in india and staged roll out in public health system is planned in four states in india. another live rotavirus vaccine is being evaluated in a randomised placebo controlled trials in india (nct02133690) and in niger (nct02145000). efforts are underway to develop non-replicating rotavirus vaccines (nrrv) as second generation rotavirus vaccines, which may avoid the risk of intusseseption. the most advanced candidate is p2-vp8 ⁄ , a trivalent truncated vp8 ⁄ of rotavirus genotypes p [8] , p [4] and p [6] , currently in phase ii clinical testing with a parenteral route of administration (nct02646891). for both nrrvs and additional oral, live-attenuated vaccines in development, pdvac encouraged the rationalization of target product profiles for these new candidates, to clearly articulate the distinguishing/advantageous features over the existing vaccines, i.e. cost, safety, efficacy in lmic, stability, breath of protection, etc. the potential for any of these vaccine candidates to be included in combination with other emerging enteric vaccines will clearly be advantageous and should be encouraged and explorations of combination with ipv could be considered. clostridium difficile is the leading cause of healthcare-associated diarrhoeal disease in the high-income countries, and is strongly associated with increasing age and frailty, immunodeficiency and in particular, modification of the normal flora through antibiotic use. the results of infection range from asymptomatic carriage through mild infection to severe diarrheal disease, with complications including pseudomembraneous colitis and toxic megacolon. in the us alone, it is believed to have caused approximately 0.5 million infections and 29,000 deaths in 2012 [26] . current interventions include antibiotic treatments, but their use can trigger relapse on withdrawal. data on the burden of disease in lmics is lacking, however hospital based studies in india, thailand and south korea suggest that the c. difficile infection is widespread, and global (douce, manuscript in preparation). there is a correlation between toxin neutralising antibody in human serum and disease protection; antibodies against toxin a are associated with protection against acute diarrhea, whilst immune responses to toxin b appear to be effective against severe disease and relapse. toxin-mediated disease is recapitulated in the syrian golden hamster, which is the standard preclinical model for demonstration of proof of concept. currently there are three vaccines in clinical development. a toxoid vaccine candidate (containing toxins a and b) recently completed a phase ii study in healthy adults and demonstrated induction of high levels of neutralizing antibodies [27] and a phase iii study has been initiated. a genetically modified, detoxified whole cell vaccine has also completed phase ii, alhough results have not yet been reported. in phase i, the vaccine was shown to be safe and induced toxin-specific neutralizing antibodies that were sustained for 12 months [28] . the third candidate is an adjuvanted recombinant protein encoding binding domains of both toxins, and the results of a phase i trial has been reported [29] , and a phase ii study has been completed. passive immunity by administration of a monoclonal antibody is also in phase iii evaluation (nct01513239 and nct01241552). pdvac agreed that the role for who in facilitating c. difficile vaccine development is not clear given the lack of data regarding the disease burden in lmics. however, it would be useful to understand the potential effectiveness of a vaccine in low resource contexts, and pdvac raised the possibility of testing existing samples from the gems and mal-ed studies for the presence of c. difficile. in addition, it would be helpful to assess the impact that these vaccines many have on reducing the use, and cost of antibiotics, and to consider this in the value proposition for vaccine decision-making. h. pylori is a highly motile, gram-negative bacterium that infects the mucus layer lining the stomach. infection typically occurs in childhood, although symptoms and clinical disease develop in only a minority of infected individuals during their lifetime. h. pylori is associated with gastritis, which causes several pathologies including gastric peptic and duodenal ulcer disease. most significantly, long term infection can result in gastric adenocarcinoma (ga) in later years of life; 65-90% of ga cases are due to h. pylori infection. ga is the 3rd leading cause of death due to cancer, globally (723,000 deaths in 2012, 8.8% of all cancers) [30] . the global prevalence of h. pylori is believed to be approximately 50% with the highest mortality rates in east asia and eastern europe. the route of transmission is poorly characterised but the oraloral route appears to be a common mechanism, as well as vertical transmission from mother to child. if untreated, most h. pylori infections are sustained for life, and 15% of those infected are thought to develop an associated pathology. if diagnosed, h. pylori infections are currently treatable with combination antimicrobial therapies. however antibiotic resistance is increasing, with 20% of patients in some countries currently failing first treatment and 5% failing two rounds of therapy. antimicrobial treatment offers no protection against reinfection. the choice of indication for an h. pylori vaccine is challenging: a prophylactic vaccine would likely need to be given to children in the first few years of life (to reach the maximum number of the target group while uninfected) but would need to offer long term protection to demonstrate clinical benefit against ga. an effective therapeutic vaccine however could be given at almost any age and would ideally be given by the 4th decade of life, prior to the peak of ga development which typically occurs from 50 years of age. the most advanced candidate is a urease toxin fusion approach and has completed phase iii trials in children, in china, and demonstrated 71.8% efficacy against natural acquisition of infection [31] . however, protection appeared to wane to 55% over 2-3 years and next steps for this vaccine are not clear. several other candidates are in preclinical development with one close to phase i studies. pdvac concluded that the burden of h. pylori is significant, and that a vaccine that is able to protect against infection, with sufficiently long duration of protection, would be of public health benefit. therapeutic candidates are currently too upstream in development for there to be a role for pdvac. maternal immunization is increasingly considered as a strategy to prevent maternal and/or neonatal disease. this approach has been proven to protect against maternal and neonatal tetanus and has been in place for decades. who recommends influenza and pertussis vaccination of pregnant women to prevent disease in mothers and newborns, respectively. however, for the first time there are now vaccines in development, specifically indicated for immunization of pregnant women as the target population. respiratory syncytial virus vaccines are most advanced in this area followed by group b streptococcal vaccines. since the 2015 pdvac meeting, a special journal issue dedicated to the issues regarding the maternal immunization vaccination strategy has been published and a great deal of work is underway to strengthen the maternal immunization platform [32] . due to the advanced stage of rsv vaccine and monoclonal antibody development, rsv was presented to sage for information in april 2016. rsv causes 33.8 million episodes of lower respiratory infection (lri) annually in children and approximately 200,000 deaths, 99% of which are in lmics [33] . recently updated estimates for rsv acute and severe lri (community based and hospitalized) disease and deaths will be published by the rsv global epidemiology network (rsv-gen) in early 2017. in addition, the pneumonia etiology research for child health (perch) study will present and publish results on the etiology of severe and very severe pneumonia in hospitalized infants and children in 9 sites in africa and asia. preliminary data analyses indicate that rsv was the leading pathogen in infants with severe pneumonia in this study. there are four rsv intervention strategies currently in development: (1) maternal immunization to enable passive transfer of maternal antibodies to the foetus in utero, (2) birth or early infant passive immunization with a long-acting monoclonal antibody, (3) active pediatric immunization and (4) vaccination of the elderly. the most advanced maternal immunization candidate begun phase iii efficacy testing in late 2015 following the demonstration of induction of palivizumab-competing antibodies (measured by elisa) in women of childbearing age (pmid: 26259809) and pregnant women. this efficacy trial has a group sequential design and will enroll 5000-8255 participants in a randomised placebo controlled trial across multiple sites in both the northern and southern hemispheres, and is expected to take 2-4 years to complete. monoclonal antibody development for the prevention of rsv in pediatrics is the next most advanced, with an extended half-life candidate (medi8897) that has been shown to be more potent in vitro than the currently licensed palivizumab. one dose may offer protection for up to 6 months. a phase iib clinical study in infants born at 29-35 week gestation is planned, and the fda recently granted fast-track designation for this product. since the palivizumab patent recently expired, who in collaboration with the university of utrecht will develop a 'biosimilar' of palivizumab and reduce costs for lmic markets through high yield production and a novel financing plan [34] . the estimated price is $us 250 per child for the full 5 month dose series and the first market authorization is expected in late 2017. pediatric rsv vaccine candidates are the least advanced, however two adenovirus-based approaches have entered the clinic since the last pdvac meeting. a chimp adenovirus (chad) candidate is currently in phase i testing in adults, to be followed by age de-escalation into seropositive, and ultimately seronegative infants. ad26 is also being evaluated as a heterologous primeboost regimen, currently in phase i testing in adults. a number of pediatric vaccine candidates developed by the laboratory of infectious diseases, nih are in phase i trials in infants and children. of note, a vaccine containing a deletion of the m2-2 gene showed evidence of diminished replication, enhanced immunogenicity, and asymptomatic 'boosting' (anamnestic response) following naturally acquired rsv infection (pmid: 26537255). two vaccine candidates are in clinical development for the elderly with a post-fusion f-based adjuvanted nanoparticle in phase iii efficacy testing, with data expected in early 2017. pdvac fully supported the following sage recommendations and called for who and partners to develop plans to support global policy-making for rsv maternal immunization as well as passive immunization with long-acting mab, following licensure. particular areas of emphasis include: (1) rsv surveillance to determine seasonality and age-stratified rsv disease burden and community morbidity and mortality, especially in africa and south-east asia (2) assessment of the long term effects of rsv interventions and the potential impact of vaccination on reducing recurrent wheeze, which, if demonstrated, would substantially increase the costeffectiveness and impact of rsv preventive interventions (3) generation of cost-effectiveness and impact data. sage also emphasized the need for strengthening of the maternal immunization platform in collaboration with the influenza, tetanus and pertussis vaccine communities, along with preparations for potential country introductions of rsv vaccine. there is an urgent need to establish a who prequalification pathway for monoclonal antibodies, which does not currently exist. as a rsv vaccine or extended half-life monoclonal ab may become available in the next 5 years, it will also be imperative to initiate early discussions with financing bodies, and to align with the gavi vaccine investment strategy (vis) to avoid delay in achieving the potential major public health impact of rsv immunization if recommended for use by who. globally, gbs remains the leading cause of sepsis and meningitis in young infants, with its greatest burden in the first 90 days of life. intrapartum antibiotic prophylaxis (iap) for women at risk of transmitting gbs to their newborns has been effective in reducing the young infant gbs disease burden in many high income countries, but iap uptake is limited and difficult to implement in lmics. immunization of pregnant women with a gbs vaccine represents an alternative pathway to protecting newborns and young infants from gbs disease, through prevention of gbs colonization and transplacental antibody transfer to the fetus in utero. pdvac prioritized gbs in 2015 and encouraged who to engage on developing guidance on the development pathway for gbs vaccines, including development of a ppc guidance document and a vaccine roadmap. in april 2016, who convened its first consultation on gbs vaccine development [35] . the focus was on gbs maternal immunization development programs targeting lmic with the ultimate goal of reducing global newborn and young infant deaths. the major knowledge gaps about the disease burden characterization were identified. recent data suggesting that gbs is an under-reported cause of stillbirth may have profound implications on the estimate of the global public health impact of a future gbs vaccine. the relationship between gbs colonization and prematurity should also be clarified. disease surveillance in hic also suggest an important residual unmet medical need, despite implementation of iap. two major pharmaceutical companies are currently developing a multivalent polysaccharide conjugate vaccine, based on the available evidence of an association between trans-placental maternalfoetal transfer of antibodies targeting polysaccharides of the gbs envelope, acquired as a consequence of natural exposure, and a reduced risk of invasive infant disease. a vaccine incorporating five of the eleven described gbs serotypes is predicted to cover over 95% of the global circulating serotypes, but the risk of serotype replacement is unknown. an alternative approach is targeting surface expressed proteins, in an attempt to confer broad protection across all serotypes. epidemiological studies evaluating the role of maternal antibodies acquired following natural exposure will determine whether a protective threshold at birth can serve as an acceptable vaccine-induced correlate of protection. until additional epidemiological and immunological data are available, estimating vaccine efficacy against invasive gbs disease in neonates and young infants in a double blind placebo-controlled vaccine trial remains the gold standard for generating the evidence required to determine potential public health impact and inform policy decision-making. pdvac endorsed the consensus-based prioritization of future activities including the development of a ppc and vaccine development technology roadmap. efforts should be made to raise awareness of the burden of gbs disease and potential public health value of a gbs vaccine, particularly in countries that lack local epidemiological data. as with rsv, efforts must be made to leverage and strengthen the maternal immunization platform by alignment with other vaccines that are administered in pregnancy, including the brighton collaboration's considerations for safety monitoring through the global alignment of immunization safety assessment in pregnancy (gaia) [36] . antimicrobial-resistant infections currently claim at least 50,000 lives each year across europe and the us alone, but amr affects many hundreds of thousands in other areas of the world [2] . in 15 european countries, more than 10% of bloodstream staphylococcus aureus infections are caused by methicillin-resistant strains (mrsa), with several of these countries seeing resistance rates closer to 50%. emerging resistance to treatments for other diseases, such as tb, malaria and hiv, have enormous impacts in lower-income settings, and by 2050, the death toll due to amr infections in africa is predicted to be approx. 4,000,000 per year. as mentioned above, each year almost 0.5 million cases of drugresistant tb are reported, and these are extremely costly to treat; an mdr case costs 8-15-fold more to treat than drug a sensitive case, while an xdr case is 25-32-fold more expensive [37] . the who estimates that approximately $8 billion per year is required to support tb care and control efforts in lmics. this is significantly more than the current investment in tb vaccine development programs. the o'neill review on antimicrobial resistance estimated that by 2050 drug-resistant infections could be claiming 10 million lives per year and at an economic cost to the global gdp in excess of $100 trillion. at the sixty-eighth world health assembly in may 2015, a global action plan to tackle antimicrobial resistance, including antibiotic resistance, was adopted [38] . its goal is to ensure continuity of successful treatment and prevention of infectious diseases with effective and safe medicines -including vaccines -that are quality-assured, used in a responsible way, and accessible to all who need them. the amr global action plan (gap) is based on 10 work streams ranging from national plans, stewardship of antibiotics, encouraging r&d through developing new business plans and assessing environmental drivers. one work stream focuses on vaccines to prevent amr. the who gap workstream on vaccines to prevent amr is based on three complementary approaches: increasing the use of existing vaccines; developing vaccines against high burden diseases currently treated systematically with antibiotics; and prioritizing the development of vaccines for diseases where antibiotic resistance is significant. these three approaches, and the challenges associated with implementing them are summarised below: a). increasing use of existing vaccines: while it is logical that increasing the use of existing vaccines would reduce infections and result in reduced use of antibiotics, it is not always clear which vaccines, in which populations, would have the greatest impact on reducing antibiotic use and potentially amr, and should therefore be prioritized. for example, it has been shown that the use of pcv-7 in children results in a roughly 50% reduction in antibiotic-resistant strains of s. pneumonia, and the use of pcv-10 reduces outpatient antibiotic purchase, leading to the suggestion that global pediatric coverage with pcv could prevent 11 million days of antibiotic use in children annually. however, it is thought that the bulk of pneumonia, and antibiotic use for s. pneumonia infections, is in older adults. this would suggest that demonstrating efficacy of pneumonia vaccines to reduce antibiotic use in older adults and an expanded use of these vaccines in that population group where very few countries have a vaccination policy may have a substantial impact on reducing amr. other existing vaccines which could impact antibiotic use include pertussis, haemophilus influenza, neisseria meningitides, typhoid, as well as influenza which, although not directly susceptible to antibiotic treatment, does result in bacterial super-infections and accounts for up to 30% of excess (winter-related) antibiotic prescriptions in some countries. in order for a rational evidence-based policy on expanding the use of existing vaccines a prioritization exercise needs to be performed, taking into account the disease burden in different populations, the antibiotic use associated with that disease burden, and an evaluation of how many days of antibiotic use would be avoided with each dose of vaccine administered. this exercise is particularly challenging since in most of the world antibiotics are taken in response to a symptom, rather than an identified infection. this means that, for example, preventing salmonella typhi-induced infection with vaccines may have minimal impact on antibiotic use for severe diarrhea. the prioritization exercise therefore needs to consider not only the disease burden, but the symptom burden and the proportion of that burden due to the vaccine-preventable infection. b). developing vaccines for diseases that are consistently treated with antibiotics, where amr is not currently an issue, but where the vaccine could reduce antibiotic use. one such example is group a streptococcus (gas). while gas is not directly associated with antibiotic resistance (there is little evidence of resistance to date) it has a high disease burden and is a source of extensive antibiotic use. in addition, it is thought that vaccine development is feasible. however to date there has been no significant effort from industry, possibly because of the weak market assessment since it can be treated with antibiotics, and such treatment is cheap. however the indirect costs from such antibiotic use, increased environmental exposure to antibiotics and expansion of amr have not been considered. taking these costs into account may contribute to the value proposition for developing and using such a vaccine. other such candidates could include group b streptococcus, and m. catarhalis and non-typeable haemophilus influenza, both responsible for otitis media which is another source of significant antibiotic prescription. while including potential impact on reduction of antibiotic use, prioritization of these candidate vaccines also needs to consider technical feasibility, whether the antibiotic use is appropriate, and whether alternative non-antibiotic approaches may make vaccine use less attractive. for example: while there are over 700 million cases of otitis media per year in under -5 year olds, for which antibiotic treatment is usually prescribed which could justify development of a vaccine, most otitis media resolves and new guidelines recommend limiting antibiotic treatment. another example is urinary tract infections which are frequent in elderly patients and a cause of significant antibiotic use, yet there is little supporting evidence that these infections could be effectively reduced by vaccination. a prioritization exercise is therefore required for vaccines that are considered technically feasible, takes into account the potential amr impact, and therefore could contribute to the cost effectiveness of the vaccine if they were developed. to achieve this, the evaluation of impact on amr is recommended to be included in the review of vaccine conducted by pdvac. c). the third and most challenging approach is developing vaccines against pathogens that are frequently antibiotic resistant and becoming increasingly difficult to treat, the so-called eskape pathogens [39] . this list includes staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumonia, as well as clostridium difficile and tuberculosis. there are numerous challenges in this approach. the first is that although infection with these may result in significant morbidity, the current global disease burden of many of these infections remains relatively low so prophylactic vaccination of the entire population would not be cost-effective. tuberculosis is however an example with significant disease burden and rapidly expanding multi-drug resistance. secondly many of these infections are associated with ageing, where immune decline may make immune interventions poorly effective, or are associated with penetrative medical interventions where the time available to induce a protective immune response may be insufficient. and finally, despite significant efforts to make effective vaccines against some of these has so far proven to be difficult. of these, tuberculosis appears to have the greatest global burden and public health impact, and tb vaccines are also the subject of extensive research. the amr gap activity in this area, to be conducted by ivr is firstly to promote tb vaccine research and development through facilitation of preferred product characteristics and the establishment of a roadmap for vaccine use, and highlighting the impact that the vaccine will have on antibiotic use and antibiotic resistance. additional activities involve monitoring the state of development of vaccines against the pathogens that are becoming antibiotic resistance, and facilitating their development. gas is a ubiquitous human pathogen that causes a broad disease spectrum, from mild to severe, the most serious of which is rheumatic heart disease (rhd). rhd affects approximately 30 million people globally, of whom 1 million experience heart failure and an estimated 300,000 die. gas is also a major cause of invasive disease, with a case fatality rate of 10-15% in high income countries, and as high as 38% in lmics [40] . on the milder end of the spectrum, gas causes approx. 615 million cases of pharyngitis per year, resulting in 60-70% of cases being treated with broad spectrum antibiotics, rather than penicillin (9% of cases), to which gas is universally susceptible. this extensive use of unnecessary and inappropriate antibiotics increases the likelihood of amr emergence against antibiotics that are used to treat a range of pathogens. previous human challenge studies, as well as preclinical animal models suggest that it is feasible to develop a vaccine against gas, and since the previous pdvac meeting, phase i studies for one candidate has been initiated in adults, and two additional candidates are expected to enter phase i studies in the next 12 months. despite this encouraging progress, significant debate remains as to the appropriate indication and optimal clinical endpoints, and the regulatory pathway for a vaccine to prevent or reduce rhd is unclear. in addition, there is a perception that increased prescription of penicillin would be an equally as effective and a significantly more cost effective method of reducing conditions that result from gas infection. these issues are likely major stumbling blocks in incentivising investment in gas vaccine development. gas has been prioritized by pdvac previously, with a recommendation to develop a business case for both a global market, and also specifically for lmics which would focus on prevention of severe outcomes in resource poor settings. despite significant effort, it has been very difficult to engage stakeholders in this activity. on the recommendation of pdvac, who convened a consultation in december 2016 to examine the value proposition for gas vaccines, considering its potential impact across both high income and lower income settings -including the consideration of how current antibiotic treatment practices may increase amr, as well as to investigate the perceived regulatory obstacles. s. aureus is a bacterium that is found as both an asymptomatic colonizer of the skin and nares of human hosts, as well as a frequent cause of human disease. it causes a spectrum of clinical manifestations of varying severity, and is the most commonly isolated pathogen from skin and soft-tissue infections, septic arthritis, pneumonia, endovascular infections, osteomyelitis, catheter/other foreign-body infections, septicaemia, and toxic shock syndrome. methicillin-resistant s. aureus (mrsa) has been documented to be emerging at a rapid and increasing rate since the antibiotic was first introduced in 1959, and hospital-associated mrsa (ha-mrsa) clones are now recognized to be the leading cause of nosocomial infections both in the united states and around the world, in high income as well as lmics. the emergence of communityassociated mrsa (ca-mrsa) in the past several decades is of concern, as is the emergence of highly resistant vancomycin-resistant s. aureus (vrsa). to date, active and passive immunization approaches have been based on increasing the concentration of opsonic antibodies to single surface antigens, and all have failed to demonstrate protection. antigenic variation, the multiple invasion pathways and lack of a surrogate of protection all present significant obstacles to vaccine development. following the failure of single antigen vaccine approaches, most development efforts are now focused on multiple antigens, and a number of candidates are in preclinical development. one multi antigen approach, comprised of 4 antigens including two capsule polysaccharides, clumping factor a and a manganese transport protein, is the most advanced [41] . current efforts are also focused on further characterizing the immunopathology and immunity of s. aureus infections to identify new antigenic targets, and developing more representative preclinical models in which opsonising and/or neutralising immune responses are measured. to date, none of the vaccine candidates in development have contemplated target populations or indications that are prevalent in lmics. focus has been on development of a vaccine that will protect against life-threatening s. aureus infections in high income countries, but it is hoped that such a vaccine would also protect against all s. aureus infections including more commonly encountered skin and soft tissue infections, and therefore be applicable in lmic contexts. since the 2015 pdvac meeting, a new global health sector strategy on sexually transmitted infections has been developed for 2016-2021 and adopted by who member states at the 69th world health assembly. within this strategic framework, sti vaccine development was highlighted as key need for future sti control [42] . in addition, the global roadmap for vaccines against stis has been updated and included in the who special issue on pipeline vaccines published in vaccine [43] . currently, the only sti vaccine candidates that are undergoing or approaching clinical development are against herpes simplex virus (hsv) and chlamydia trachomatis, and as such discussion was limited to these pathogens. hsv is the leading cause of genital ulcer disease, and a particular concern for lmics as it increases both acquisition and transmission of hiv infection. hsv type 2 and type 1 disease burden estimates were recently updated [44, 45] , and it is estimated more than half a billion people live with genital hsv infection, worldwide. pdvac previously recommended that improved global estimates of neonatal herpes burden be generated, and assessment of available data has recently been completed with preliminary estimates of >14,000 new cases globally, an incidence rate of approx. 10/100,000 births, which is concerning because of a case fatality rate of 60% (looker, submitted) . the incidence is likely to be under-estimated in lmics where hsv infection rates are highest and poor healthcare infrastructure means that neonatal herpes cases are likely to be undetected, but primary data are lacking. ongoing evaluation of hsv infection as part of the child health and mortality prevention surveillance (champs) network will help to address this burden gap. at the 2015 meeting, the advance of therapeutic vaccine candidates for hsv-2 was highlighted, and the role of these types of vaccines in modulating the interaction between hsv and hiv acquisition was discussed as an important consideration for these vaccines in lmics. in consideration of this, and with who support, a systematic review/meta-analysis of hsv-2 and risk of hiv acquisition including 54 studies will inform modelling of the potential impact of an hsv-2 vaccine on hiv incidence, and is expected to be published in late 2016. a review of biological mechanisms of hsv-hiv interaction and implications for vaccine development has also been drafted. the pipeline for therapeutic vaccines remains robust, with 5 candidates in clinical development, the most advanced of which now has data demonstrating significant reductions in hsv2 shedding (55%) and days with genital lesions (60%) over 12 months [46] . in response to these positive data, niaid has formed an hsv working group to propose desired characteristics for therapeutic and prophylactic vaccines for hsv, including indication, priority target populations, clinical trial endpoints, and safety and efficacy criteria. this document could form the foundation for a who consultative process to generate a guidance document on preferred product characteristics (ppc). pdvac encouraged who to actively collaborate and support development of ppcs for hsv vaccines. chlamydia trachomatis is a gram-negative bacterium that can infect genital, ocular and lung epithelium. it includes three sets of serovars: -serovars ab, b, ba, or c -cause ocular trachoma, which can lead to blindness -serovars d-k -cause sexually transmitted infection resulting in urethritis, cervicitis, pelvic inflammatory disease (pid) (and associated infertility, ectopic pregnancy, and chronic pelvic pain), neonatal pneumonia, and neonatal conjunctivitis -serovars l1, l2 and l3 -cause lymphogranuloma venereum c. trachomatis can ascend to the upper genital tract and cause pelvic inflammatory disease (pid), which can in turn lead to long-term sequelae including tubal factor infertility, ectopic pregnancy, and chronic pelvic pain. other adverse outcomes of chlamydia include preterm birth, neonatal conjunctivitis and pneumonia, and increased hiv risk. currently management is through screening programs in some high income countries that are not feasible in resource constrained settings, where most cases are likely never diagnosed. who estimates that there were 131 million new cases of chlamydia in 2012 [47] with most cases among adolescents and young adults. the global burden of chlamydia-associated pid, infertility and other sequelae has not been well characterised and estimates of the proportion of infertility presumed to be associated with genital infection (e.g., have a fallopian tube etiology) in africa are outdated [48] , but are thought to be approx. 65-85% in women seeking fertility care. there are several vaccine candidates currently in preclinical development, with a subunit vaccine based on the chlamydial major outer membrane protein (momp) and live-attenuated (plasmid-deficient) approaches being the most advanced. the momp candidate entered phase i clinical testing in late 2016, and a phase i study with the live attenuated candidate will commence in 2017. the intended goal of a chlamydia vaccine is to decrease upper genital tract sequelae, however pid is challenging to use as clinical endpoint as it is difficult to definitively diagnose and the causes of pid are multi-factorial (typically the result of c. trachomatis in 1/3 of cases). the chlamydia vaccine community is seeking guidance and consensus building on clinical endpoints for clinical studies, including evaluating the potential role of biomarkers, radiologic, and other measures of upper tract ascension, infection, inflammation and damage. improved global burden of disease data and vaccine impact modelling on long term sequelae are also needed to define the investment case for these vaccines. pdvac commended the progress towards the first vaccine study against chlamydia since the 1960s and look forward to discussing the path ahead once the early clinical data are available. this section refers to vaccines that have been licensed, or are approaching licensure in some areas of the world, but are currently limited in their use outside any single who region. in some instances, the vaccines may have the potential of offering broader public health impact by expanding approval and use in other geographical regions, and pdvac is seeking to understand the perspective in this regard. ev71 is one of the most common causes of hand-foot-andmouth disease (hfmd). sporadic ev71 outbreaks have occurred globally since it was first isolated in 1969, but from the late 1990s a series of large hfmd epidemics caused by ev71 have been reported in the asia-pacific region. in china alone, 7.2 million cases were reported between 2008 and 2012, of which 2457 (0.03%) were fatal [49] . children less than 5 years of age have the highest risk of disease, and although infection is unusually mild and selflimiting, severe infections can result in neurological and cardiopulmonary complications, and death. several ev71 vaccine candidates are in development, and it was stated at the meeting that the chinese national regulatory authority has licensed two ev71 vaccines, with another in progress. the first licensed vaccine was developed by the institute of medical biology, chinese academy of medical science, and has been approved for use to prevent ev71 disease in 6-71 month olds, based on a phase iii study that demonstrated 95% efficacy over 12 month [50] . sinovac has also licensed in activated vaccine, with supportive phase iii data in 6-35 month olds [51] . beijing vigoo is in the process of licensing its inactivated ev71 vaccines in china (nct01508247). all three vaccines are adjuvanted with aluminum hydroxide. given that ev71 outbreaks occur in other areas of the world (recently reported in spain [52]), further discussions are warranted in the international health community about how to assess the role of the chinese vaccines during outbreaks outside china. in the wake of the 2014-15 ebola outbreak, various strategies were proposed to avoid such crises from reoccurring. key to improving r&d preparedness and response is determining which pathogens are likely to be the greatest threat, creating consensus with respect to product development strategies and coordinating global funding for complementary r&d efforts going forward. to tackle these questions, and at the request of its 194 member states, who convened a broad global coalition to develop the r&d blueprint [53] as a sustainable platform for accelerated r&d, with two complementary objectives: to develop (and implement) a roadmap for r&d preparedness for known priority pathogens, and to enable roll-out of an emergency r&d response as early and as efficiently as possible the main approaches underpinning the improvement of preparedness within the r&d blueprint include: as reported in the 2015 meeting summary, a consultation to initiate work towards a mers cov roadmap was held in december 2015 with aims of defining the key basic and applied research activities, identifying the priority technologies and capacities to support vaccine development, and finally understanding the financing/procurement opportunities. following this meeting, a draft roadmap was developed and underwent public consultation prior to finalisation and publication [54] . it is well accepted that the extraordinary rate of ebola virus vaccine development was as a result of unprecedented collaboration and co-ordination of global vaccine r&d activities, and the availability of a number of candidate vaccines that could enter clinical phase evaluation [55] . in the face of another pheic so soon following ebola virus disease (evd) outbreak, the global vaccine community is rallying, and reflecting on lessons learned from the experience in west africa only 2 years ago. at the time of responding to the evd emergency, the availability of well-characterised pre-clinical models and robust data was essential for the comparative evaluation and selection of candidates to move into clinical studies. novel recombinant viral vector platforms, in combination with recombination proteins have been validated by evd experience and it could be argued are now less risky for development of vaccine against future pathogens, but manufacturing feasibility and scale-up capabilities still need to be confirmed for the most novel platforms. critically, sustainable public sector push and pull investment mechanisms beyond the initial emergency response phase need to be created, to incentivise manufacturers to engage in the long term commitment to developing and licensing vaccines that may only be used in outbreak or emergency scenarios. pdvac noted that a target product profile for a second generation ebola virus vaccine is under development that will likely cover ebola zaire, ebola sudan, and marburg filoviruses, and will need to demonstrate longer duration of protection. this tpp will provide guidance about whos preferences and minimally acceptable criteria for vaccines in this area. during the discussion it was clarified that who tpps include minimally acceptable criteria, whereas preferred product characteristics specify only preferences. the status of zika virus epidemiology and the understanding of its pathogenesis and associated sequelae are evolving so rapidly that publications on these issues are almost immediately out of date. pdvac's role has been to oversee a working group that has developed a target product profile (tpp) for use in an emergency, or future outbreak scenario. the tpp was made available for public consultation, after which subject matter experts, global regulators, developers and manufacturers were convened to discuss the regulatory considerations for developing a vaccine with the characteristics described in the tpp. the finalised tpp and position paper are publically available [57] . in addition to reviewing the status of vaccine development against pathogens, pdvac considered a number of cross-cutting issues that could better integrate and therefore facilitate product development efforts for vaccines and other interventions. in addition to the significant morbidity and mortality that drives the development of vaccines against pathogens for which vaccines are currently not available, the who estimates that there are approximately 1.5 million deaths per year in children under 5 from vaccine preventable diseases [58, 59] . one of the reasons for this striking immunization gap is the cost and logistical challenges of delivery of these vaccines, over and above the cost of their manufacture. the remit of who's immunization practices advisory committee (ipac) is to provide strategic advice on immunization practices, tools, and technologies intended to improve the delivery of immunization programs at the country level. it oversees the recently formed delivery technologies working group (dtwg) composed of public health organizations, funders and procurement agencies as well as vaccine developers to evaluate r&d in novel delivery technologies and devices, for example the microarray patch, and compact, pre-filled auto-disable injection technologies (cpad). of particular focus for this group is the development and evaluation of a framework to analyze high-level trade-offs between important variables such as development, procurement and supply chain costs, coverage, efficacy, and safety in order to facilitate investment decisions by product developers, vaccine manufacturers, global policy makers, in-country decision makers and procurement agencies. this framework is referred to as total systems effectiveness (tse). the intent of this delivery technology working group is to offer a platform for discussion and guidance regarding vaccine preferences for lmics, early on in development, so that ultimately the vaccine is suitable for programmatic use. the dtwg reports directly to ipac, but has potential overlap with activities that are overseen by pdvac, particularly in consideration of second generation vaccines or new vaccines that may be developed with an alternative presentation to that of a needle and syringe. pdvac was supportive of the dtwg and encouraged continued communication between vaccine development and device/delivery technology development to identify potential opportunities for novel combination product development. there are several pathogen areas where mab are being developed as vaccine-like interventions, as their single dose regimen and long half-life render them amenable for lmics contexts, where they could offer significant public health benefit. candidates for rsv and rabies are approaching licensure within the next 5 years, and a who procedure for who prequalification is urgently needed to avoid delay implementation. this gap has been recognized and will be addressed. the scope of pdvac overlaps with several other research agendas such as gvap, amr, new delivery technologies and development and consolidation of maternal immunization platforms. the pdvac research agenda needs to be clearly communicated, and pdvac and ivb will strive to be well-informed of efforts in other research areas, to help shape and align strategy where appropriate. future pdvac meetings will consider these potential overlaps in more detail, as well as how pdvac and ivb can facilitate development of integrated product development approaches. since its inception in 2014, pdvac has reviewed the pipeline and vaccine development status of 33 different pathogens. pdvac will continue to review new pathogen areas as candidates progress into clinical studies, providing that who engagement will likely facilitate the use of vaccines to reduce disease burden in lmics. one such pathogen is cytomegalovirus (cmv), which is a leading cause of congenital infections worldwide, resulting in 17-20% of infants developing permanent sequelae including hearing loss and neurodevelopmental disabilities. there are little data on cmv infection in lmics, but a recent systemic review suggests that birth prevalence ranges are higher in lmics than in europe and north america [60] , and several clinical trials of vaccine candidates are ongoing. as such, an assessment of cmv vaccine development will be undertaken by pdvac 2017. with rsv, tb, hiv and enteric candidates approaching pivotal data points, understanding what data are needed to support earlier policy implementation and outcomes will be key, as well as understanding the potential impact of vaccines within the broader control strategy -including diagnostics and other preventatives -for these pathogens. vaccine impact modelling, and understanding the composite set of cost drivers through to vaccine delivery will be important. interaction with who's ipac and pq teams will increase going forward, to strengthen the link between product development and programmatic requirements. under the recommendation of pdvac, who will seek to broaden its role to support development of value propositions for vaccines against pathogens for which there is a poorly defined business case, for example gas and hsv. raising awareness of lmic disease burden and requirements/procedures for access to lmics markets may help to incentivise financing development of these vaccines. of key consideration may be the potential for these vaccines to reduce the emergence of amr, and pdvac recommended that this be considered as a criterion in future landscape analyses and ppc guidance documents. pdvac and who will continue to align activities with the priorities within the who r&d blueprint. pdvac is aware that several other organizations which are responsible for emergency preparedness have been through a process to prioritize their r&d agendas, with some commonality and some complementarity to the pathogens listed in the who blueprint. in this arena, pdvac will continue its horizon scanning role, and will advocate for commitment to product development of vaccines for emerging diseases to progress through robust preclinical proof of concept to generation of phase i data, as a minimum. pdvac strongly recommends the collaboration with other groups to co-ordinate advocacy and funding for vaccine development to prepare for the inevitable future emergencies. procedure for assessing the acceptability, in principle, of vaccines for purchase by united nations agencies tackling drug resistant infections globally: final report and recommendation how can vaccines contribute to solving the antimicrobial resistance problem? who vaccine pipeline tracker moorthy vasee s, who product development for vaccines advisory committee (pdvac) pipeline analyses for 25 pathogens mind the gap: jumping from vaccine licensure to routine use global vaccine action plan (gvap) impact and cost-effectiveness of new tuberculosis vaccines in low-and middle-income countries status of vaccine research and development of vaccines for tuberculosis vaccination with alvac and aidsvax to prevent hiv-1 infection in thailand report from the world health organization's product development for vaccines advisory committee (pdvac) meeting who global consultation on universal influenza vaccines the global burden of norovirus & prospects for vaccine development the vast and varied global burden of norovirus: prospects for prevention and control global, regional, and national estimates of rotavirus mortality in children <5 years of age who guidance document for the quality, safety and efficacy of oral live attenuated rotavirus vaccines current status of clostridium difficile infection epidemiology defining the optimal formulation and schedule of a candidate toxoid vaccine against clostridium difficile infection: a randomized phase 2 clinical trial a phase 1, placebo-controlled, randomized study of the safety, tolerability, and immunogenicity of a clostridium difficile vaccine administered with or without aluminum hydroxide in healthy adults safety, immunogenicity and dose response of vla84, a new vaccine candidate against clostridium difficile, in healthy volunteers efficacy, safety, and immunogenicity of an oral recombinant helicobacter pylori vaccine in children in china: a randomised, double-blind, placebo-controlled, phase 3 trial brewinski isaacs and a. sobanjo-ter meulen advancing maternal immunization programs through research in low and medium income countries global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis moorthy vasee s. who consultation on group b streptococcus vaccine development: report from a meeting tb vaccine research & development: a business case for investment clinical relevance of the eskape pathogens invasive group a streptococcus infection among children development of a multicomponent staphylococcus aureus vaccine designed to counter multiple bacterial virulence factors the global roadmap for advancing development of vaccines against sexually transmitted infections: update and next steps global estimates of prevalent and incident herpes simplex virus type 2 infections in 2012 global and regional estimates of prevalent and incident herpes simplex virus type 1 infections in 2012 global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting the pill, chlamydia and pid hand, foot, and mouth disease in china, 2008-12: an epidemiological study an inactivated enterovirus 71 vaccine in healthy children efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in china a roadmap for mers-cov research and product development: report from a world health organization consultation on a path to accelerate access to ebola vaccines: the who's research and development efforts during the 2014-2016 ebola epidemic in west africa meeting report: who consultation on considerations for regulatory expectations of zika virus vaccines for use during an emergency global, regional, and national causes of child mortality in 2008: a systematic analysis systematic review of the birth prevalence of congenital cytomegalovirus infection in developing countries we gratefully acknowledge the 2016 pathogen specific landscape analyses that were prepared by the following individuals: this report contains the collective views of an international group of experts, and does not necessarily represent the decisions or the stated policy of the world health organization, nor of the u. s. government.bsg is an employee of the u.s. government. this work was prepared as part of his official duties. title 17 u.s.c. §105 provides that 'copyright protection under this title is not available for any work of the united states government'. key: cord-299255-wnf8fozk authors: chan, m.y.; smith, m.a. title: infections in pregnancy date: 2017-11-27 journal: comprehensive toxicology doi: 10.1016/b978-0-12-801238-3.64293-9 sha: doc_id: 299255 cord_uid: wnf8fozk infections during pregnancy may affect a developing fetus. if left untreated, these infections can lead to the death of the mother, fetus, or neonate and other adverse sequelae. there are many factors that impact infection during pregnancy, such as the immune system changes during pregnancy, hormonal flux, stress, and the microbiome. we review some of the outcomes of infection during pregnancy, such as preterm birth, chorioamnionitis, meningitis, hydrocephaly, developmental delays, microcephaly, and sepsis. transmission routes are discussed regarding how a pregnant woman may pass her infection to her fetus. this is followed by examples of infection during pregnancy: bacterial, viral, parasitic, and fungal infections. there are many known organisms that are capable of producing similar congenital defects during pregnancy; however, whether these infections share common mechanisms of action is yet to be determined. to protect the health of pregnant women and their offspring, additional research is needed to understand how these intrauterine infections adversely affect pregnancies and/or neonates in order to develop prevention strategies and treatments. . intrauterine infection, comprehensive toxicology, 2nd edn, vol. 12, pp. 239-258, isbn 978-0-08-046884-6, 10.1016/b978-0-08-046884-6.01523-2. preterm birth babies born before 37 completed weeks of gestation. sepsis a life-threatening condition where a whole body inflammatory response is activated due to an infection. sequelae a pathological condition resulting from a prior disease or injury. stillbirth a baby born dead after 24 completed weeks of pregnancy. torch an acronym for toxoplasmosis, other, rubella, cytomegalovirus (cmv), and herpes infections that are some of the most common infections associated with congenital anomalies. vertical transmission when an infection is transmitted directly from the mother to an embryo, fetus, or baby during pregnancy or childbirth. in the 1940s, serious birth defects in infants born to women infected with rubella during their first trimester provided the first evidence that adverse pregnancy outcomes could result from maternal infection. it has become well recognized that infections during pregnancy, in addition to affecting the mother, may affect or be passed to the developing fetus. infections are caused by bacteria, viruses, or parasites that have invaded body tissues or have released toxins disrupting normal functions and eliciting an inflammatory response to help combat the microorganism. infections, or the inflammatory responses to an infection, can result in adverse effects including preterm delivery, birth defects, developmental delays, or stillbirths. the main focus of this article is to explore the variety of pathogens that can lead to adverse effects to the fetus or neonate. there are various factors that can impact infection and susceptibility during pregnancy, some of these will be examined in detail, focusing on new data and information. databases searched for this information included pubmed, google scholar, and web of science focusing on publications from 2007 to 2016. since the publication of the previous comprehensive toxicology 2nd edition, new sections included in this article discuss the transmission of intrauterine infections; the factors impacting infection; examples of classic and new pathogens capable of causing intrauterine infections; and an updated table of additional selected pathogens detailing the exposures, symptoms, and outcomes of infection. bacteria, viruses, and other organisms are able to be passed from mother to child, a phenomenon known as transmission. the specific symptoms of transmitted infections depend on the individual pathogen and the stage of pregnancy at the time of infection. transmission occurs in the womb during pregnancy or during birth. pathogens can enter the uterus and infect the fetus through several routesdthe most common routes are vertical transmission, such as hematogenous route where pathogens gain entry to the fetus by passing through the placenta, and ascending transmission, such as pathogens traveling up the reproductive tract of the pregnant woman. other less common routes are passage from the abdominal cavity and through the fallopian tubes, invasive medical procedures, or the birthing process (richardson et al., 2010) . the main routes of intrauterine infection during pregnancy are vertical transmissions. a vertically transmitted infection is caused by a pathogen transmitted directly from the mother to an embryo, fetus, or baby during pregnancy or childbirth; this type of transmission is also known as mother-to-child transmission. vertical transmission during the perinatal period can be subcategorized as a perinatal infection. vertical transmission generally refers to transplacental transmission. the pathogen reaches the placenta through the mother's blood (hematogenous transmission). these pathogens must have the ability to overcome the placental barriersdsyncytiotrophoblast interface, decidual-trophoblast interface, and physical obstacles (robbins and bakardjiev, 2012) . pathogens may overcome these barriers through a damaged syncytiotrophoblast interface; invasion of the uterine-trophoblast interface directly or cell-to-cell transport (e.g., listeria monocytogenes); or immune cells may allow for barrier crossing (transport of human immunodeficiency virus mediated by leukocytes) (robbins and bakardjiev, 2012; lagaye et al., 2001) . when the placenta is infected, this may allow for the pathogen to invade the fetus. ascending infections occur when infectious pathogens residing in the external genitalia of the mother access the amniotic sac. upon infection, the amniotic sac may become compromised and rupture. once ruptured, the pathogens may inoculate the amniotic fluid, spread as a biofilm over the exposed amnion, and invade choriodecidual tissue planes (kim et al., 2009; redline, 2012) . the fetus can become infected by aspirating the microorganism to the lungs, ingesting the pathogens, or by the pathogen penetrating the ear canal. see section on chorioamnionitis for details of post amniotic fluid infection. susceptibility of infection in women differs due to natural variation; however, a number of other factors also influence this susceptibility. for intrauterine infections, there are some common factors that impact susceptibility to infection. epidemiologic studies have shown that pregnant women have an increased incidence of contracting a variety of infections like influenza, varicella, measles, severe acute respiratory syndrome, tuberculosis, listeriosis, pneumocystis, toxoplasmosis, and malaria (sappenfield et al., 2013; riley et al., 2011; jamieson et al., 2006) . the severity of infection has been suggested to vary at different stages of pregnancy, likely from the unique immunological alterations that occur during different stages of pregnancy (faucette et al., 2015) . to accommodate the genetic differences between the mother and the fetus and to prevent allogenic rejection of the fetus, the maternal immune system shifts toward a t-helper 2 bias (jamieson et al., 2006) . maternal hormone levels change during pregnancy, and interplay between the sex hormones and immune system may alter the efficiency of the maternal immune system. these changes are thought to contribute to an increase in susceptibility toward certain infectious diseases which may change the severity of illness the mother experiences and fetal mortality rates. pregnant women may be considered immunosuppressed, though not in the traditional sense of the word. a fetus derives half of its genetic material from its father; thus, the fetus is essentially a foreign tissue in the maternal uterine environment. the fetus' susceptibility to rejection from its mother's immune system has been compared to an organ transplant (jamieson et al., 2006) . evidence suggests that the maternal immune system tolerates fetal antigens by suppressing cell-mediated immunity while retaining normal humoral immunity and response (jamieson et al., 2006) . though only occurring locally at the maternal-fetal interface, this suppression prevents the rejection of the fetal tissue and has been suggested that it may affect maternal response to infection (jamieson et al., 2006; erlebacher, 2013) . the exact immunological tolerance is not completely understood. during pregnancy, there is a systematic shift from t-helper 1 (th1) to t-helper 2 (th2) cell-mediated responses in the mother. in a th1-dominated response, the th1-type t lymphocytes and proinflammatory cytokines amplify cell-mediated immunity allowing for recognition of the body's own cells that engulf the pathogen and express pathogen-related antigens on their surfaces (jamieson et al., 2006; corr et al., 2007) . when shifting to the th2 cell-mediated response, the th2-stimulating cytokines dominate and suppress the th1 t cell responses locally allowing for adequate humoral immune response while the cell-mediated immunity is compromised (thaxton and sharma, 2010) . hormonal changes during pregnancy promote this shift from th1 to th2. hormones can contribute to a modified immune response by altering the functions of immune cells and can have effects on the outcome of infection during pregnancy (robinson and klein, 2012) . during pregnancy, steroid sex hormone levels change drastically and are considerably higher than at any other time during a woman's life, particularly pregnancy-associated hormones including estradiol, estriol, progesterone, corticosteroids, and prolactin (robinson and klein, 2012) . the interplay between sex hormones and the immune system is complex and often contradictorydestradiol can enhance innate immunity in cellmediated and humoral adaptive immune responses, yet progesterone can suppress the maternal immune response and alter helper t cell responses (kourtis et al., 2014; robinson and klein, 2012) . research is needed to fully understand the changes in hormones and the immune system during pregnancy and the interplay between the two systems. pregnancy is a stressful change in a woman's body. additional stressors beyond that of a pregnancy impact not only the health of the mother but also the development of the fetus. stress can either enhance or suppress the immune system and thereby change the mother's susceptibility and severity to certain infections. mechanisms linking maternal stress to infant development are complex. stressors can enhance or suppress immune function, which can change susceptibility to infections. the duration of stress impacts the health of the individual. suppressed immunity can occur by decreasing immune cell number and function or increasing active immunosuppressive mechanisms. maternal stress may be mediated through biological and behavioral mechanisms like the neuroendocrine pathway, which can result in the activation of the maternal-placental-fetal endocrine systems or an immune/inflammatory pathway, where maternal stress may modify the characteristics of systemic and local immunity by increasing susceptibility of the intrauterine and fetal environments to the immune inflammatory processes (wadhwa et al., 2001) . it is likely that the placenta plays a vital part in mechanisms linking maternal stress and infant development. traditionally, intrauterine infections were thought to have originated from pathogens in the vaginal tract that ascend into the intrauterine environment. the placenta was considered sterile during gestation and the presence of any bacteria in clinical cultures was a diagnostic for intrauterine infection (hillier et al., 1988) . however, the presence of placental or membrane bacteria has been observed in the absence of histological infection and recognition that the presence of bacteria does not always have the expected corresponding clinical outcomes when observed (stout et al., 2013; leviton et al., 2010; han et al., 2009; buhimschi et al., 2009; steel et al., 2005; mysorekar and cao, 2014) . of all the cells in the human body, only 10% are human somatic and germ cells; the remaining 90% are made of microflora (savage, 1977) . the microflora found in and on humans are composed of diverse niche communities of microbial species varying by site or organ. as a whole, all these communities make up the microbiome. with the initiatives set by the national institutes of health with the human microbiome project in 2007, the microbiome has been found to be increasingly integral with the healthy functions of a human (turnbaugh et al., 2007; peterson et al., 2009) . human-microflora interactions occur across body sites and have roles in metabolism, immunity, development, and behavior of the host (o'hara and shanahan, 2006; cabreiro and gems, 2013) . the communities consist of a balance of commensal bacteria and beneficial bacteria that help humans with normal functions. when the balance is upset, it can result in disease and unfavorable alterations; under certain conditions, an outgrowth of potential pathogenic bacteria, a decrease in the number of beneficial bacteria, or an imbalance of commensal microflora may contribute to the pathogenesis of disorders which may affect the susceptibility rate for infectionsdincluding intrauterine infections (round and mazmanian, 2009; hill and artis, 2010; honda and littman, 2012; o'hara and shanahan, 2006) . infection with a pathogen leads to a change in the normal microbiome which can have an effect on the intrauterine system (hacquard and schadt, 2015) . for instance, a decrease in the diversity in the placental microbiome has been correlated with lower birth weight in human infants (zheng et al., 2015) . many of the diverse groups of oral cavity bacteria have been found in the intrauterine environment and associated with adverse pregnancy outcomes suggesting that the oral cavity may act as a reservoir for infection (mysorekar and cao, 2014; fardini et al., 2010; han, 2011) . these bacteria are capable of hematogenous transmission, similar to the transient bacteremia possible during periodontal infections (fardini et al., 2010; han, 2011; offenbacher et al., 1999) . for example, maternal oral infections, like acute gingival infection and chronic periodontal infections, are multifactorial disorders where microbial dental biofilms are considered the primary agent initiating inflammation (chambrone et al., 2011; offenbacher et al., 2006; xiong et al., 2006) . although inflammation is typically restricted to local periodontal tissues, the pathogens can stimulate bacteremia and translocate to distant tissues (champagne et al., 2000; xiong et al., 2006) . microorganisms have the ability to transfer from one location to another, as noted by studies on periodontal disease and its impact on pregnancy. as we continue to discover the complex roles and interactions between the human body and the microbiome that inhabits the body, it is likely that correlations between the health of the intrauterine microbiome and the susceptibility to intrauterine infection will be discovered. once infected, there are a multitude of adverse pregnancy outcomesdpreterm births, chorioamnionitis, infections of the central nervous system (cns), and sepsisdwhich are detailed in the following section (richardson et al., 2010) . premature births, or preterm births, are defined as births that occur prior to 37 completed weeks of gestation, and they are associated with 5%-18% of pregnancies (liu et al., 2012) . premature births are the leading cause of neonatal morbidity and mortality in the developed and developing world (goldenberg et al., 2000) . infants who are born prematurely have a higher risk of serious disability or mortality than their full-term counterparts, with those infants born before the 30th week of pregnancy being especially at risk of prenatal mortality and morbidity. potential problems for premature babies are an increased risk of short-term complications due to immaturity of organ systems, neurodevelopmental disorders, intellectual disabilities, vision/hearing impairments, or even death (cdc, 2015e; romero et al., 2014) . intrauterine infection is a frequent and important mechanism that may contribute to nearly 40% of preterm births (goldenberg et al., 2008; agrawal and hirsch, 2012; romero et al., 2007) . however, this number may be higher because many infections are likely to be subclinical and the pathogenesis is not detected due to the lack of sensitivity of conventional culture techniques (racicot et al., 2013) . outcomes of infection in a pregnant host are dependent upon various factors including gestational stage of fetus, previous exposure and immunity of the mother, variable immunity among individuals, the placenta's ability to protect the fetus from infection, and the developing immune system in the fetus (richardson et al., 2010) . there are different pathways by which preterm birth from bacterial infection are known to occur. two major events that are required for bacteria to induce preterm labor are (1) bacterial colonization in gestational tissues or the fetus and/or (2) bacterial counts reaching a threshold that elicits maternal immune inflammatory response that will induce preterm labor (romero et al., 2002; richardson et al., 2010) . preterm labor is more frequent when a fetal inflammatory response is triggered as diagnosed by an increase in il-1 and il-6 and a decrease in il-10 (romero et al., 2007) . compared to term infants, preterm births have a higher incidence rate of temperature instability, respiratory distress, apnoea, hypocalcaemia, seizures, jaundice, kernicterus, feeding difficulties, periventricular leukomalacia, and hospitalizations (saigal and doyle, 2008) . effects of preterm birth can manifest later in childhood (huddy et al., 2001) . studies on low birth weight have shown that most of these infants showed sequelae like cognitive defects, academic underachievement, grade failures, and the need for increased remedial assistance during mid-childhood and adolescence continuing during life (saigal and doyle, 2008) . there is still much about the pathogenesis and mechanisms of preterm birth that is not understood and animal models, such as sheep, have/are being utilized to gain knowledge that could lead to methods and treatments that can improve neonatal outcomes (racicot et al., 2013; goldenberg et al., 2008) . the condition in which the membranes that surround the fetusdthe chorion and amnion, and the amniotic fluiddare infected by bacteria is commonly referred to as "chorioamnionitis." chorioamnionitis complications are associated with significant and/or long-term adverse outcomes for the mother (postpartum infections and sepsis) and/or the infant (stillbirth, premature birth, neonatal sepsis, chronic lung disease, and brain injury) (tita and andrews, 2010) . it has been estimated that one of every three preterm neonates is born to a mother with intraamniotic infection, identified with either cultivation or molecular microbiologic techniques (berger et al., 2009) . it is important to also note that microbial colonization of chorioamniotic membranes does not always result in a fetal or maternal inflammatory response (romero et al., 2007) . chorioamnionitis is typically associated with bacteria that have low virulence and enter the uterus by ascending from the lower reproductive tract; it is rare that transmission is spread hematogenously (one notable exception: listeria monocytogenes) (richardson et al., 2010; tita and andrews, 2010) . the presence of organisms in the amniotic cavity elicits an intense inflammatory response (racicot et al., 2013) . the inflammatory response may intensify and progress into fetal inflammatory response syndrome which predisposes preterm neonates to shortand long-term consequences (goldenberg et al., 2008) . a maternal fever is an important criterion for the diagnosis of chorioamnionitis. the presence of maternal fever of greater than 38 c (100.4 f) and at least two of the following criteria: maternal leukocytosis (greater than 15,000 cells/mm 3 ), maternal tachycardia (greater than 100 beats/min), fetal tachycardia (greater than 160 beats/min), uterine tenderness, and/or foul odor of the amniotic fluid. these thresholds are associated with higher rates of neonatal and maternal morbidity (polin and committee on fetus and newborn, 2012) . additional details of the mechanisms of chorioamnionitis can be found in richardson, pollak et al. (2010) and in tita and andrews (2010). adverse effects to the cns is a common result of intrauterine infections in neonates. transversal of the blood brain barrier (bbb) must occur for a pathogen to access the cns (polin and harris, 2001) . the bbb secretes cerebrospinal fluid (csf) while physically separating the brain from the intravascular department. the purpose of the bbb is to control the flow of molecules and ions that enter and leave the brain in order to maintain a neutral environment (kim, 2003; richardson et al., 2010) . the extent of effect depends on the maturity of the developing brain, timing of infection, or amount of pathogens (kim, 2014) . there are an estimated six cases of bacterial meningitis per 100,000 people worldwide (ninds/nih, 2015b); however, there is little known about bacterial meningitis in the fetus (ninds/nih, 2015b). generally, pregnancy has not been associated with an increased risk of bacterial meningitis with the exception if the cause is due to streptococcus pneumoniae, listeria monocytogenes, or cryptococcus neoformans (baldwin and roos, 2011; adriani et al., 2012) . clinical effects of meningitis are sepsis and increased intracranial pressure; chronic changes can occur such as hydrocephalus, encephalomalacia, or subdural effusion (kim, 2014) . neonatal meningitis is the most common and serious bacterial infection during the neonatal period, with the most common pathogens being enterobacteria, e.coli, and group b streptococcus (kim, 2014) . hydrocephaly is a condition where excess csf accumulates in the brain: it is also known as hydrocephalus or "water on the brain" (ninds/nih, 2015a). the excessive accumulations cause an abnormal expansion of the brain's ventricles producing pressure on the brain (ninds/nih, 2015a). due to the malleability of an infant's skull, the added pressure on the brain may result in an enlarged head size for the infant as the skull widens to relieve pressure (richardson et al., 2010) . causes of hydrocephalus are still not well understood; possible causes in the fetus are infections, genetic defects, developmental disorders, meningitis, premature birth complications, or traumatic head injury (richardson et al., 2010 ; ninds/nih, 2015a). microcephaly is a medical condition present at birth or developed within the first few years of life wherein the circumference of the head is smaller than normal because the brain has either not developed properly or growth has stopped (ninds/nih, 2015c). it is most often caused by genetic abnormalities during the early months of fetal development, but it may be induced if the mother is infected or affected by pathogens, drugs or alcohol, or certain toxic chemicals during pregnancy (ninds/nih, 2015c). recently, pregnant women infected with the zika virus had a high incidence of babies born with microcephaly. see sections examples of infection during pregnancy, viral infections, and subsection zika virus, for examples and a discussion. the result of having microcephaly varies. babies born with microcephaly will have smaller than normal heads that fail to grow through infancy. microcephaly can be an isolated condition or occur in combination with other major birth defects (cdc, 2016b) . depending on the severity of microcephaly, problems can range from mild to severe and are often lifelong, such as impaired cognitive development, developmental delay, facial distortions, dwarfism or short stature, hyperactivity, seizures, difficulties with coordination and balance, feeding problems, hearing loss, vision problems, and other brain or neurological abnormalities (ninds/ nih, 2015c; cdc, 2016b). severe microcephaly can also be life-threatening (cdc, 2016b) . yet some will develop normally and their head will grow bigger especially if they are otherwise growing and developing normally (ninds/nih, 2015c). infections during pregnancy may result in developmental delays for the infant. pathogens that are able to interact with the cns have capabilities of causing morbidities in fetuses and premature infants. some of these impairments may not manifest at birth or in the neonatal period but later in life. many neonates are able to survive major insults, like infection, without any evidence of impairment due to the plasticity of the developing brain and improvements in medical care; yet, in others, these insults can cause varying degrees of long-term neurodevelopmental impairment such as cerebral palsy, with half of those continuing to develop cognitive and behavioral deficits (mwaniki et al., 2012; stoll et al., 2004) . the exact mechanisms whereby pathogens lead to developmental delays are not yet known. molecular events such as the release of inflammatory cytokines by brain cells during infection may have a significant role in the brain injury (dammann and leviton, 1997) . for instance, ascending intrauterine infections have been suggested to significantly increase the risk of fetal brain damage due to the initiation of fetal inflammatory response syndrome (berger and soder, 2015; dammann and leviton, 1997; de vries, 2009 ). yet, whether there are exposure-specific or pathogen-specific patterns by which cytokines act in response to inflammation during infection is unknown. sepsis is a systematic syndrome that occurs in response to infection of the blood and stems from an additional medical condition, like chorioamnionitis or a cns infection (nigms/nih, 2015) . during sepsis, intravenous coagulation may occur, blocking blood flow to vital organs and creating hypoxic and ischemic conditions. mortality in cases of sepsis is due to the failure of multiple organsdtypically, a single organ fails leading to the dysfunction of other organ systems. there is early and late onset of neonatal sepsis. early onset is typically a consequence of fetal infection in utero or during parturition within the first week of life. sepsis can begin in utero when the fetus inhales or swallows infected amniotic fluid; during parturition, sepsis is capable of developing within the hours or days after birth when colonized skin or mucosal surfaces are compromised (polin and committee on fetus and newborn, 2012) . maternal treatment with intrapartum intravenous antimicrobial agents is the only intervention proven to reduce the incidence of early-onset neonatal sepsis (cdc, 2010b; polin and committee on fetus and newborn, 2012). late-onset sepsis can result from colonization during birth via vertical transmission of a colonized mother's anorectal and vaginal areas; case reports have suggested possible horizontal transmission in the postnatal period through breast milk (berardi et al., 2013; polin and committee on fetus and newborn, 2012) . depending on the pathogen and the neonatologists' discretion, a full course of antimicrobials may be used to treat the neonate (rubin et al., 2002) . vancomycin has been a frequent choice of empiric antimicrobial treatment of neonates with suspected late-onset sepsis (rubin et al., 2002) . chorioamnionitis is a major risk factor for neonatal sepsis and increased risk of early-onset sepsis (polin and committee on fetus and newborn, 2012; wolfs et al., 2012) . additionally, pathogens associated with neonatal meningitis have the ability to induce neonatal sepsis. as mentioned previously, neonates that survive a cns infection may experience developmental delays and impairment. a study showed that the degree of impairment was more likely to be severe in septic preterm neonates than in nonseptic neonates (mwaniki et al., 2012) . infections that are acquired in utero or during birth are a significant cause of fetal and neonatal mortality which can contribute to early and later childhood morbidity. organisms shown to cause these congenital conditions are included in the torch complex. the torch acronym represents toxoplasma gondii, other infections, rubella, cytomegalovirus, and herpes viruses as a concept emphasizing that these infections have been shown to cause stillbirths, perinatal morbidity, and 2%-3% of all congenital anomalies (neu et al., 2015; stegmann and carey, 2002) . these conditions are far from the only kinds that can cause congenital conditions. specific examples of types of pathogens that affect pregnancy and the developing fetus are listed in table 1 . to explore some of the mechanisms unique to pathogens (bacterial, viral, parasitic, and fungal), a few examples are elaborated in this section. listeriosis is predominantly a foodborne disease associated with the consumption of contaminated foodsdprimarily in dairy products. populations most susceptible to l. monocytogenes infection are people with a compromised immune system, older people, pregnant women, and neonates. these populations account for at least 90% of reported infections (cdc, 2013a) . pregnant women are about 10 times more susceptible to l. monocytogenes infection than healthy, nonpregnant adults; about one in seven (14%) cases of listeriosis occur during pregnancy (cdc, 2013a; 2013c) . infection during pregnancy can cause spontaneous preterm labor, fetal loss, and illness or death in newborn infants (cdc, 2013c; richardson et al., 2010) . symptoms of infection may include gastrointestinal symptoms, influenza-like illness with fever, headache, myalgia, and/or backache, but 29% of the infection cases are asymptomatic (mylonakis et al., 2002; jackson et al., 2010) . animal studies on rhesus monkeys (smith et al., 2003) , guinea pigs (williams, 2006) , and gerbils (roulo et al., 2014) have shown a relationship between increasing bacterial load and adverse pregnancy outcomes. a fetus becomes infected when l. monocytogenes crosses the placenta, which has been suggested to be a reservoir for reinfection (bakardjiev et al., 2006; de luca et al., 2015) . neonatal infection occurs in 68% of the cases of maternal infection; 68% of the cases may make a full recovery, but in 12.7% of cases, there may be long-term sequelae (mylonakis et al., 2002) . infection may lead to severe health conditions such as septicemia, pneumonia, or meningitis and in about 25% of cases, the occurrence of preterm delivery with birth weights lower than normal or stillbirth may occur (mylonakis et al., 2002) . septicemia is the main symptom that neonates display with a 15%-50% mortality rate. other symptoms include respiratory problems, pneumonia, and the formation of minute abscesses in bodily organs (farber and peterkin, 1991) . a neonate can develop late-onset listeriosis through natural birth if the mother's vaginal tract has been colonized by l. monocytogenes (cdc, 2013c) . bacteremia can be caused by ascending infection, transplacental passage of the organism, or by inhalation of amniotic fluid (becroft et al., 1971; de luca et al., 2015) . second-and third-trimester infections can result in premature delivery followed by neonatal illness or preterm delivery of a stillborn (farber and peterkin, 1991) . for additional discussion of mechanisms, see richardson, pollak et al. (2010) . streptococcus are classified by their hemolytic properties with two species known to affect pregnancydalpha-hemolytic streptococci (group a strep) and beta-hemolytic streptococci (group b strep). we will be detailing examples from group a strep and group b strep in the following section because of their potential impact on pregnancy. a group a streptococci, streptococcus pneumoniae or pneumococcus, is a significant human bacterium. it is part of the normal microbiome in the upper respired tract, but opportunistic, if conditions allow. it is one of the most common causes of severe pneumonia and can cause pneumococcal meningitis, which is the most common form of meningitis and the most serious (ninds/nih, 2015b; cdc, 2015d) . coexisting disease in the mother increases the risk of contracting pneumonia in pregnancy. a pregnancy complicated with pneumonia can have adverse fetal effects; 44% of women experienced preterm labor and of the preterm births, 36% had respiratory failure (munn et al., 1999) . pneumonia in pregnancy can result in inflammation and edema of alveoli, restricting the number available for oxygen transport. fetal oxygen delivery decreases when the maternal oxygen saturation falls to < 90% (goodnight, 2005) . birth weights were significantly lower at delivery during pregnancies complicated by pneumonia (goodnight, 2005) . invasive group b streptococci (group b strep) or streptococcus agalactiae is the leading infectious cause of maternal chorioamnionitis, puerperal endometritis, and early-onset neonatal sepsis (phares et al., 2008; cdc, 2010b) . it causes invasive disease primarily in infants, pregnant/postpartum women, and older adults, with the highest incidence among young infants (phares et al., 2008) . there are two main types of group b strep diseasedearly-onset disease and late-onset disease. early-onset disease occurs during the first week of life and late-onset disease occurs from the first week through the first three months of life. during early-onset disease, group b strep commonly causes sepsis, pneumonia, and, sometimes, meningitis (cdc, 2014) . for late-onset disease, the same illnesses are found as in the early onset, but meningitis is more common during this type of disease (cdc, 2014) . the primary risk factor for neonatal infection transmission is colonization of the mother's genital tract through direct exposure during birth, or in fetuses through ascension into the amniotic fluid then into the placenta. group b strep can also cause miscarriages, stillbirths, and preterm deliveries (cdc, 2014) . the exact mechanisms are generally unknown (cdc, 2014). campylobacter are found ubiquitously in the environment and considered normal intestinal flora in most mammals and birds. the consumption of raw or undercooked poultry or crosscontamination of other foods is the most common source of campylobacteriosis in industrialized nations, responsible for more than 90% of all sporadic cases in humans (dasti, 2010; allos, 2001) . more than 90% of all human campylobacter infections are caused by campylobacter jejuni (c. jejuni) and campylobacter coli, with increasing rates of infection reported during the summer months. infants are one of the two most infected populations, the second group are patients between 15 and 39 years (dasti, 2010) . c. jejuni, campylobacter fetus (c. fetus), and other strains have caused stillbirths/abortions in pregnant women (smith, 2002) . c. jejuni and c. fetus are two strains known to cause illness in pregnant/postpartum women and their fetuses/newborn infants. though most cases have no complications, enteritis caused by c. jejuni infection can result in stillbirth or premature labor (allos, 2001) . transmission of the pathogen to offspring can occur either transplacentally or through vaginal delivery (smith, 2002; richardson et al., 2010) . additional details into the mechanisms of campylobacter intrauterine infection and the impacts on the fetusdsuch as stillbirthdmay be found in the second edition of comprehensive toxicology, volume 8, intrauterine infections (richardson et al., 2010) . rubella was the first virus recognized to result in birth defects. there are a number of viruses that can adversely affect the developing fetus (table 1) . viruses act through different mechanisms of action; there are some common features of viral infections that adversely affect pregnancy. transmission occurs vertically from the mother to the fetus. if the placenta is not infected, the virus does not appear to spread to the fetus. various factors influence the likelihood of the virus infecting the fetus such as infection state of the mother and gestational age of the fetus. outcomes of viral infections are generally spontaneous abortion, stillbirth, prematurity, and organ pathogenesis. in the following section are provided examples of viruses, additional examples may be found in table 1 . cytomegalovirus (cmv) is a herpes virus that infects people of all ages. once infected, the virus stays within a person's body for life. in the united states, it is estimated that 50%-80% of adults are infected with cmv by the age of 40 (cdc, 2010a). most healthy children and adults infected with cmv exhibit no symptoms; those that do exhibit symptoms tend to be like other illnessesdfever, sore throat, fatigue, and swollen glands. cmv can cause serious disease in pregnant women and their babies, people who care for infants and children, and the immunocompromised. it is the most common viral infection in the developing fetus; approximately 1 in 150 children is born with congenital cmv infection. transmission of this virus is generally through direct contact with body fluids. cmv can be transmitted from a pregnant woman to her fetus during pregnancy by the virus crossing the placenta and infecting the fetus' blood. congenital cmv infection in developed countries occurs between 0.3% and 2.45% of all live births (peckham, 1990; alford et al., 1990; bonalumi et al., 2011) . most infants born with congenital cmv infection (90%) appear healthy at birth and 80% of the healthy appearing infants never develop symptoms or disabilities. health problems or disabilities may appear 2 or more years after birth or never (cdc, 2010a). fetal damage from cmv infection can include growth retardation and cns abnormalities (kimberlin et al., 2003) . additional details regarding the mechanisms of cmv may be found in the second edition of comprehensive toxicology, volume 8, intrauterine infections (richardson et al., 2010) . influenza viruses are a group of rna viruses and are the leading cause of serious wintertime respiratory morbidity worldwide. studies about the effects of influenza-related illness during pregnancy have shown an impact on the health of pregnant women. specific changes in a woman's immune system, heart, and lungs during pregnancy increase the susceptibility to severe illness, hospitalizations, and possibly death. there is an increased chance for serious problems for unborn babies, including premature labor and delivery (mcneil et al., 2011) . there are three distinct genera of influenza viruses: a, b, and c. for this example, we will be looking at the outcomes specific to the subtype of influenza adthe 2009 h1n1 influenza a virus. women hospitalized with h1n1 infection were estimated to have a three times increased risk of delivering preterm (yates et al., 2010) . emergency cesarean deliveries were frequently reported as urgent or emergent in one study regarding the 2009 h1n1 influenza (haberg et al., 2013) . the unusually high cesarean section delivery is a likely effect of deteriorating maternal condition that impacts the overall fetal status (mendez-figueroa et al., 2011) . infants of ill mothers can test positive for h1n1 influenza, but it is an unusual occurrence. this raises the possibility of vertical transmission, but additional studies are needed on the impacts of maternal infection with influenza on neonatal outcomes. long-term follow-up studies on infants born to women with influenza infection should be performed to fully understand the impacts on overall outcomes when evaluating influenza in pregnancy. vaccines are available and recommended to pregnant women; see centers for disease control and prevention (2015) for recommendations (cdc, 2015b). hepatitis refers to the inflammation of the liver. though inflammation can be caused by various means, most commonly it is due to a virus. there are five main types of hepatitis virusesda, b, c, d, and e. hepatitis a and e are generally caused by the intake of contaminated food or water; hepatitis b, c, and d typically occur as parenteral contact with infected bodily fluids. although hepatitis e has a high vertical transmission rate (50%), there is still limited data on complications (ornoy and tenenbaum, 2006; kumar et al., 2004) . for this example, we concentrate on hepatitis b due to the effects it can cause on the pregnancy, to the fetus, and potential teratogenic effects of drugs used to prevent infection. maternal hepatitis b virus (hbv) infection has not been shown to increase major congenital defects in offspring. reports of a higher incidence of low birth weight and prematurity have been noted during acute infection compared to the general population (jonas, 2009 ). the higher incidence may be attributed to the changes in the maternal immune system contributing to a suppressed immune response against hbv. a high frequency of hbv perinatal transmission occurs in up to 90% of infants born to women positive for hbv infection. hbv is able to cross the placental barrier but presumed to be the cause of a minority of infections for those mothers not immunized (jonas, 2009) . transmission of the hbv can occur prenatally when the virus enters the placenta through maternal blood and crosses the placental barrier gaining access to the fetus or during birth when the newborn is exposed to infected cervical secretions. increased likelihood of intrauterine transmission has been associated with the level of hbv dna in the mother. prevention of hbv transmission can be performed by identifying the hepatitis b surface antigen in pregnant women and providing hepatitis b immune globulin and hepatitis b vaccine to the infants within 12 h of birth (cdc, 2015f). the virus was first identified in africa in 1947 and was named after the zika forest in uganda, where it was first isolated (dick, 1953) . zika virus is a single-stranded rna virus spread to people when they are bitten by an infected aedes species mosquito (arbovirus). it is related to other flaviviruses including dengue and chikungunya (triunfol, 2016) . transmission is typically through exposure to infected blood but transmission can result from vertical transmission from a pregnant woman to her fetus, sexual transmission, laboratory exposure, and potentially through blood and organ transplant (musso et al., 2015; foy et al., 2011; cdc, 2016a) . many zika infections are asymptomatic; common symptoms of zika are fever, rash, joint pain, conjunctivitis, muscle pain, and headache which may appear 3-7 days after infection, last several days to a week, and are generally mild (cdc, 2015g; schuler-faccini and rasmussen, 2016) . severe disease is uncommon; fatalities are rare (cdc, 2015g); however, guillain-barre syndrome has been reported in patients following suspected zika virus infection (oehler et al., 2014) . the virus usually remains in the blood of an infected person for a week and can persist longer in other secretions such as semen. once a person has been infected, they are likely to be protected from future infections schuler-faccini and rasmussen, 2016) . the centers for disease prevention and control (cdc) acknowledges the link between zika virus infection in pregnant women and subsequent birth defectsdspontaneous abortion and fetal demise schuler-faccini and rasmussen, 2016) . a pregnant woman already infected with zika can pass the virus to her fetus during pregnancy or around the time of birth . brain defects linked with zika in the fetus include decreased total brain tissue with resulting microcephaly, calcium deposits in the brain indicating brain damage, excess fluid in the brain cavities and surrounding the brain, absent or poorly formed brain structures, and abnormal eye development cdc, 2016b; schuler-faccini and rasmussen, 2016) . zika can be sexually transmitted and can be passed from an infected partner before their symptoms start, while they exhibit symptoms, and after their symptoms end (who, 2016a; schuler-faccini and rasmussen, 2016) . the zika virus can remain in semen (2-10 weeks) longer than in any other body fluids, including vaginal fluids, urine, and blood (foy et al., 2011; higgs, 2016; oster, 2016; musso et al., 2015) . brazil's ministry of health made an announcement as of december 2015 for ".women in the northeast of the country not to get pregnant for the foreseeable future" (npr, 2015) . in jan. 2016, the cdc issued a travel notice alert level 2 regarding the zika virus in south america, central america, mexico, the caribbean, and puerto rico. the world health organization has declared a public health emergency of international concern on feb. 1, 2016 (who, 2016b . since 2015, 71 countries and territories reported evidence of vector-borne zika virus transmission (who, 2016a). as of summer 2016, the united states reported 43 locally acquired mosquito-borne cases all from the state of florida (cdc, 2016a). parasitic infections affect pregnant women worldwide and directly or indirectly lead to a variety of adverse fetal effects. these effects include intrauterine retardation of growth, congenital malformations, and fetal loss. depending on when the infection takes place and the stage of pregnancy the fetus is in, severe fetal consequences may occur during development or be asymptomatic until later in the pregnancy. malaria is a protozoan disease transmitted by female mosquitoes of the genus anopheles. the parasites multiply in the host's liver and subsequently infect the red blood cells causing symptoms of fever, headache, and vomiting; if not treated, the infection can become life-threatening by disrupting the blood supply to vital organs. in 2015, there were an estimated 214 million malaria cases and 43,800 malaria deaths; young children, pregnant women, and those nonimmune travelers are the most at risk when infected (who, 2014) . in areas with high transmission rates, more than 70% of malaria deaths occur in the age group of children under the age of five (who, 2015b). pregnant women have increased susceptibility to p. falciparum malaria. malaria during pregnancy increases the risk of maternal and fetal anemia, stillbirth, spontaneous abortion, low birth weight, and neonatal death (who, 2015a). the effects of low birth weight have an odds ratio of two to seven times higher effects when it is the woman's first pregnancy compared to women who have had multiple pregnancies prior (desai et al., 2007; white et al., 2014) . in malaria-endemic countries, pregnant women are at increased risk of developing severe p. falciparum malaria with a very high mortality rate, approximately 50% (white et al., 2014) . maternal hiv infection also predisposes pregnant women to malaria, congenital malaria, and exacerbates reductions in birth weight (steketee et al., 2001) . the risk of infant death is high if maternal malaria occurs late in pregnancy (bardaji et al., 2011; white et al., 2014) . congenital malaria occurs in roughly 5% of neonates but clears spontaneously in 62% of cases (falade et al., 2007) . the condition is typically defined as the presence of asexual forms of malaria parasites in the peripheral blood within the first 7 days of life but frequently reported with the presence of the parasites in cord blood (menendez and mayor, 2007) . p. falciparum contributes to 8%-14% of low birth weight deliveries (white et al., 2014) . with increased malaria prevention (early diagnosis and treatment) and control measures, the malaria mortality rates have fallen globally among all age groups. there is, however, a concern of p. falciparum resistance in some countries to artemisinin, one of the core compounds in world health organization recommended treatments for uncomplicated malaria (who, 2015b). toxoplasmosis is caused by the protozoan parasite toxoplasma gondii (t. gondii) that infects most species of warm-blooded animals. it has a complex life cycle. sexual reproduction can only occur in the digestive tract of felidae hosts (cats). asexual reproduction can occur in any warm-blooded vertebrate. humans can be infected by eating undercooked meat of animals harboring tissue cysts, consuming contaminated food or drinking water, contact with contaminated environmental samples, blood transfusion or organ transplant, or by vertical transmission. in humans, those infected are generally asymptomatic; when symptoms occur, it is usually mild and flu-like lasting for weeks to months and until the cycle returns to a dormant state (cdc, 2013b). the parasites form tissue cysts and may remain throughout the life of the host (cdc, 2013b). if a woman has contracted toxoplasmosis before becoming pregnant, the fetus will be protected due to the mother's developed immunity (woudwyk et al., 2012) . so maternal-fetal transmission only occurs when a pregnant woman is newly infected with toxoplasma. global estimates of maternal-fetal transmission are approximately 20%-33% of newly infected pregnant women (carlier et al., 2012) . although transmission rates during the first trimester of pregnancy are low (below 6%), when it does occur the damage to the fetus is the most severe at this time (carlier et al., 2012) . the second and third trimesters' transmission rates increase to 22%-40% and 58%-72%, respectively; the highest proportions of transmission occur in the last few weeks before birth (carlier et al., 2012) . transmission may result in a miscarriage, stillbirth, or a child with congenital toxoplasmosis. infants infected before birth may be asymptomatic at first but may develop later in life with potential vision impairment/loss, mental disability, and seizures (cdc, 2013b) . treatment is available for pregnant women, newborns, and infants involving sulphonamides or spiramycin, but due to the parasites residing within cells in a less active phase, it is difficult for medication to completely eliminate the cysts (richardson et al., 2010; cdc, 2013b) . fungi are ubiquitous, found outdoors, on many inside surfaces, and on human skin. most fungi are harmless, but some types can be harmful to health. anyone can be affected by fungal diseases (cdc, 2015c). serious fungal infections are uncommon during pregnancy; in some cases, infection may occur with higher frequency during pregnancy, potentially increasing maternal mortality and cause fetal loss or prematurity (ostrosky-zeichner and rex, 2010 ). an extreme outcome of a fungal infection is the spread of the fungus through the blood to the spinal cord leading to fungal meningitis. candida is a genus of yeasts and is the most common cause of infections worldwide (manolakaki et al., 2010) . there are over 150 species. many of the species are harmless commensals or endosymbionts under normal conditions; when conditions are sufficient, they can invade and cause disease. candida albicans (c. albicans) and candida parapsilosis (c. parapsilosis) are the two most likely species to cause candidiasis and candidemia. up to 40% of pregnant women may have vaginal colonization with candida spp. (goldenberg et al., 2000; digiulio, 2012) . candida spp. have been isolated from amniotic fluid of spontaneous preterm birth mothers (maneenil et al., 2015) . cases studied have shown intraamniotic infection can cause fetal death or fetal candidiasis with impaired cns outcomes in humans (marelli et al., 1996; benjamin et al., 2006; darmstadt et al., 2000; siriratsivawong et al., 2014) . in neonatal candidiasis, c. albicans and c. parapsilosis are the most commonly isolated species and cause the majority of fungal infections in very low birth weight infants. though c. albicans is not specifically associated with the risk of preterm delivery, there has been some evidence that suggests the elimination of the yeast reduces the risk of a late miscarriage and preterm birth (maneenil et al., 2015) . c. albicans is usually vertically transmitted, whereas c. parapsilosis is mostly transmitted by contact other than parentchild (waggoner-fountain et al., 1996; reef et al., 1998; chapman and faix, 2000; abi-said et al., 1997) . pathogenesis likely involves ascension of the infection from maternal candida infection then leading to amniotic fluid infection and chorioamnionitis/funisitis (siriratsivawong et al., 2014) . approximately 9%-10% of nosocomial blood infections in neonates are fungal infections; 85.6% of these infections are caused by candida spp. (blaszkowska and goralska, 2014) . the mortality rate is the second highest ranging from 40% to 70% (blaszkowska and goralska, 2014) . at risk neonates can develop candidiasis, a dermatitis caused by candida spp. the dermatitis usually resolves on its own in neonates born full-term; however, the condition may advance to candidemia in very low birth weight infants possibly leading to death. some of the risk factors for dermatitis are vaginal birth, extreme prematurity, low birth weight, hyperglycemia, and steroid administration. congenital cutaneous candidiasis can present in a variety of symptomsddiffuse skin eruption in the absence of systemic illness to severe systemic disease leading to stillbirth or early neonatal death (darmstadt et al., 2000) . cutaneous eruptions occur in 82% of cases and are typically detected within 24 h of birth (darmstadt et al., 2000) . diagnosis of congenital candidiasis may be delayed due to the similar appearance to more common rashes in newborns which increases risk for adverse sequelae (siriratsivawong et al., 2014) . adhesion is a major virulence factor for this fungus. candida spp. has the capability of forming biofilm which allows it to adhere to skin, mucus layers, and surfaces of tubes/catheters that may be used on neonates during hospitalization. there is evidence that candida spp. has the ability to form biofilms on intrauterine devices (iud). due to iud application directly affecting the vaginal ecosystem, there may be a need for the consideration of candida spp. internal exposure for those women planning to get pregnant after removing the iud (caliskan et al., 2011) . since the 1940s' outbreak of rubella, it has become well recognized that infections during pregnancy can affect the mother and fetus. for most women, pregnancy is a normal and healthy state. but there are factors that may make pregnant women more susceptible to infection. changes in the immune system and the altering effects of hormones affect the immune system's susceptibility and responses to infection. stress is common during pregnancy, but high levels may increase the risk of certain problems with delivering a healthy neonate by influencing the already altered immune functions. the diversity in the human microbiome has been correlated with the health of humans; as we learn more about the interconnecting relations between the microbiome and our health, promoting specific diversity may be able to protect the mother and fetus during pregnancy in the future. infection has long been established as the leading cause of preterm births which has its own risks such as the infection of the cns or low birth weight, which can allow the neonate to be more susceptible to infections postpartum. chorioamnionitis indicates infection of the intrauterine environment and is a major risk factor for neonatal sepsis, which can cause those neonates to commonly present with multiple insults or lead to a stillbirth. stillbirths are an end to a pregnancy when the conditions in the mother are unable to keep the fetus alive. intrauterine infections are a diverse group of organisms that can cause a range of adverse outcomes in pregnancy. the torch complex is a reminder of organisms that can produce congenital defects when a woman is infected during pregnancy. the mechanisms of some infections are currently unknown and additional research is needed to understand how these pathogens cause adverse outcomes in pregnancy. those that are understood may be preventable; others, like the zika virus, are emerging and are only now being studied as outbreaks occur. with the ever globalizing world, the risk of being exposed or contracting these infections rises as organisms can travel across continents in mere hours. environmental factors such as changes in climate may allow for some organisms to move into more populated areas, exposing more individuals. the understanding of intrauterine infections is vital to ensure the health of all future mothers and their offspring. many factors influence the susceptibility of becoming infected. contracting an intrauterine infection has health impacts on the mother and fetus. the fetus's development is a major concern after being exposed to an intrauterine infection but predicting the outcome for any individual is difficult with our current knowledge. there are many known organisms that are capable of producing congenital defects during pregnancy. however, there are new emerging organisms that have yet to be studied. additional research to understand the mechanisms and risks associated with these organisms is critical for the health of all potential mothers and their offspring. see also: 5.06. maternally mediated developmental toxicity. 5.08. epigenetics and the developmental origins of health and disease. the epidemiology of hematogenous candidiasis caused by different candida species bacterial meningitis in pregnancy: report of six cases and 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development cytomegalovirus infection in pregnancy: review of the literature hepatitis b in pregnancy fetal inflammatory response in women with proteomic biomarkers characteristic of intra-amniotic inflammation and preterm birth worms need microbes too: microbiota, health and aging in caenorhabditis elegans in vitro biofilm formation and relationship with antifungal resistance of candida spp. isolated from vaginal and intrauterine device string samples of women with vaginal complaints congenital parasitic infections: a review cytomegalovirus (cmv) and congenital cmv infection prevention of perinatal group b streptococcal disease people at risk [online vital signs: listeria illnesses, deaths, and outbreaksdunited states candidiasis [online flu vaccine safety and pregnancy fungal diseases homepage | cdc pneumococcal disease preterm birth viral hepatitis [online zika virus case counts in the us | zika virus | cdc facts about microcephaly evidence grade associating periodontitis to preterm 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who. who (2016a) who | zika virus and complications ihr 2005) emergency committee on zika virus and observed increase in neurological disorders and neonatal malformations dose response, infectivity and stillbirths in pregnant guinea pigs inoculated with listeria monocytogenes inflammation-induced immune suppression of the fetus: a potential link between chorioamnionitis and postnatal early onset sepsis study of the uterine local immune response in a murine model of embryonic death due to tritrichomonas foetus periodontal disease and adverse pregnancy outcomes: a systematic review influenza a/h1n1v in pregnancy: an investigation of the characteristics and management of affected women and the relationship to pregnancy outcomes for mother and infant the placental microbiome varies in association with low birth weight in full-term neonates key: cord-273019-hbpfz8rt authors: glingston, r. sahaya; deb, rachayeeta; kumar, sachin; nagotu, shirisha title: organelle dynamics and viral infections: at cross roads date: 2018-06-25 journal: microbes infect doi: 10.1016/j.micinf.2018.06.002 sha: doc_id: 273019 cord_uid: hbpfz8rt viruses are obligate intracellular parasites of the host cells. a commonly accepted view is the requirement of internal membranous structures for various aspects of viral life cycle. organelles enable favourable intracellular environment for several viruses. however, studies reporting organelle dynamics upon viral infections are scant. in this review, we aim to summarize and highlight modulations caused to various organelles upon viral infection or expression of its proteins. a unique feature of eukaryotic cells is the presence of distinct membrane bound structures called organelles. this subcompartmentalization of eukaryotic cells is very essential for its optimum function. organelles achieve this due to the presence of a unique set of proteins and distinctive lipid composition that determines their function. they are dynamic and interact with the surrounding organelles to regulate their biogenesis and function [1, 2] . association of organelle dysfunction with human diseases/ disorders is reported and widely acknowledged [3] . it is interesting that not only organelles are required for proper functioning of cells, but are also required for the successful infection of a virus [4, 5] . it is well established that viruses develop alternate strategies to survive in the cells. the steps of the viral life cycle include entry, translation, replication, assembly and egress [6] . moreover, viruses have developed remarkable ways to complete their life cycle by targeting specific cell organelles and processes. organelles like mitochondria, er, peroxisomes play an important role in innate immunity and host defence [7] . recently lipid droplets have also been reported to be essential for innate response against viral infection [8] . the viral life cycle is also a dynamic process that leads to the extensive host cellular reorganization. characterization of this spatiotemporal reorganization is a key step in order to understand the molecular mechanism of viral infection. localization of the viral proteins to an appropriate sub-cellular compartment is part of the strategy to hijack the host machinery and necessary pathways leading to establishment of an infection [9, 10] . with the advent of several modern microscopy and proteomics methods, it is now clear that the localization and function of many host proteins are altered due to viral infections. not only the host proteins but the intracellular localization of the viral proteins and their interactions with the host proteins are also extensively studied using these methods [10] . however, currently our understanding of organelle dynamics during viral infections is still at its infancy. current review briefly summarizes our knowledge of the various cell organelles/compartments following virus infection. this topic at the interjection of virology and cell biology represents an emerging research area of molecular investigations in virology. presence of nucleus that houses the genome is what distinguishes eukaryotic cells from prokaryotes (fig. 1 ). it is a double membrane organelle and is the site for gene regulation and transcription. nuclear envelope comprising of nuclear pores is the barrier between the nuclear content and the rest of the cell. nucleolus is the region where ribosomal subunit assembly takes place in the nucleus. sub nuclear structures comprising of several proteins which regulate many cellular processes like apoptosis, dna damage, etc are called pml-nbs. many dna and rna viruses depend on the host nuclear proteins for their replication [11] . several strategies are used by viruses in order to deliver its genome to the host cells [12] . usually, when cells undergo mitosis, there is a temporary disassembly of the nuclear envelope (ne) which allows some viruses to enter into the nucleus [13] . entry and integration of murine leukemia virus (mlv) into host nucleus depends on the ne breakdown during mitosis [14] . various human immunodeficiency virus (hiv) preintegration complex proteins, such as the matrix [15] , vpr [16] , integrase [17, 18] were found to contain nuclear localization signal (nls) in their genome sequence. this nls interacts with the nuclear transport receptors, which transports the viral proteins through the nuclear pore complex (npc). similarly, the influenza virus nucleoprotein (np) present in the viral ribonucleoprotein complex (vrnp) contains nls1, nls2 and nls3, which helps in its binding to cellular importins resulting in transportation of the vrnp to the nucleus [19e21] . in another strategy, the capsid of certain viruses gets attached to the cytoplasmic side of the host npc either directly or with the help of importins. this interaction acts as a signal for capsid disassembly followed by entry of the viral genome along with their proteins through the npc into the nucleus [12] . studies on the herpes simplex virus-1 (hsv-1) infection on vero, bhk-21 and ptk 2 cells reported transportation of viral tegument-capsid by dynein to the cytoplasmic side of npc [22, 23] . the hsv-1 capsid binds to npc with the help of importin b and subsequently releases dna into the nucleus through the npc [24] . similarly, the capsid protein of adenovirus (ad) on binding with the nuclear transport protein nup214 attaches to the cytoplasmic side of npc and releases the genetic material into the nucleus [25] . some small viruses such as hepatitis b virus (hbv), baculovirus, etc were found to cross the npc and release their genome at the nuclear side of npc or within the nucleus [12] . the capsid protein of hbv was reported to interact with npc via the nuclear receptor nup153 in xenopus laevis oocytes [26] . furthermore, this binding depends on importin a, b and phosphorylated core protein present on its capsid [27] . upon infection, hbv capsid enters the nucleus through the npc and subsequently releases its genome into the nucleoplasm [28] . similarly, capsid proteins of simian virus 40 (sv40) were found to enter the nucleus through npc [29] . it has been reported that the disruption of the ne and nuclear lamina temporarily aid the nuclear entry of viruses such as parvoviruses [30] . disruption of ne in xenopus oocytes and both ne and nuclear lamina in mouse fibroblast cells upon parvovirus infection and minute virus of mice (mvm) was reported using electron microscopy analysis [30, 31] . presumably, viruses evolved different mechanisms to enter nucleus in order to facilitate their replication cycle and survivability inside the host cells. after the successful entry into the nucleus, both dna and rna viruses exploit various nuclear components such as nucleolus, promyelocytic leukaemia nuclear body (pln) and/or nuclear proteins in order to facilitate their replication (fig. 2) [11] . nucleolin, fibrillarin and b23 (nucleophosmin) are examples of nucleolar proteins reported essential for various functions like post transcriptional process, ribosome assembly, etc [32] . these multifunctional host nucleolar proteins were reported to be incorporated into the replication and translation complex of many viruses [11] . many dna viruses like hsv and ad were reported to be involved in relocalization or disruption of the host nucleolar proteins [11] . for instance, the transfection of the recombinant ul24 protein of hsv-1, tagged with an n-terminal hemagglutinin in vero cells resulted in the redistribution of nucleolin and b23 [33, 34] . another nucleolar protein fibrillarin was also found to be redistributed in spots throughout the nucleus but in an ul24 independent manner [33] . the core protein of ad was found to be associated with the nucleolus in hela cells [35] . further studies showed that this association results in the relocation of the nucleolin to the cytoplasm of the infected cells [36] . the ad-induced relocation of nucleolin was proposed to be a strategy used by virus to suppress its activity [36] . additionally, infection of hela cells with ad resulted in inhibition of synthesis and maturation of rrna [37] . nucleolar localization of the viral capsid and rna binding proteins of many rna viruses has been reported [11] . for example, the encephalomyocarditis viral (emcv) proteins 2a and 3bcd and human rhinovirus hrv 3c protease were found to localize to the nucleoli resulting in inhibition of cellular rna transcription [38, 39] . it has been reported that the expression of tat and rev proteins of hiv-1 in cos7 cells resulted in their accumulation in dense fibrillar component in the nucleolus [40] . moreover, rev protein was reported to be involved in deforming the nucleolar architecture [40] . similarly, the viral n protein was found to be localized to the nucleolus in addition to the cytoplasm upon infection of coronavirus resulting in delayed cell cycle to facilitate viral assembly [41] . poliovirus (pv) or rhinoviruses interact with nucleolin and blocks the nuclear import leading to accumulation of nucleolin in the cytoplasm of the infected cells [42] . the accumulated nucleolin interacts with the internal ribosome entry site (ires) element present in the upstream 5 0 end of the viral genome to stimulate its translation [43] . on the other hand, these changes result in the shutdown of cellular transcription leading to the downregulation of the host defence mechanism [42] . infection of human respiratory syncytial virus (hrsv) in a549 cells resulted in depletion of nucleolin [44] . although, both dna and rna viruses behave differently towards acquiring the nuclear niche however the goal being to establish control over cellular transcription and favour its genome over the host. pml nb is another subnucleolar component, which contains proteins such as pml and sp100. these proteins are induced by interferons and hence pml nb can act as a target for viruses to escape the antiviral signalling response [45] . redistribution of the pml nb has been reported upon infection of hsv-1 to bhk-21 cells [46] . similarly, ebna lp protein of epstein-barr virus (ebv) was found to displace sp100 from pml nbs in burkitt's lymphoma and hep2 cells [47] . the viral e4-orf3 protein induced reorganization of pml nbs into elongated track like structures upon infection of cv1 cells with human ad5 [48] . later, it was found that this reorganization was responsible for the downregulation of the host antiviral response [49] . another example is the infection of human foreskin fibroblasts (hffs) with human cytomegalovirus (hcmv) resulting in the accumulation of pp71 at pml nbs in the infected cells [50] . further studies showed that pp71 was responsible for the proteasomal degradation of pml-nb protein daxx, which is important for inducing intrinsic immune response against hcmv infection [50] . some rna viruses also result in the redistribution and degradation of host pml nbs in order to neutralize the antiviral response. the ring finger protein z of lymphocytic choriomeningitis virus interacts with pml protein and leads to the redistribution of pml-nbs from the nucleolus to the cytoplasm [51] . the expression of 3c protease of emcv in cho cells was reported to target pml-nbs and promote its degradation in proteasome and by sumo-dependent mechanism [52] . interestingly, cho cells infected with rabies virus were reported to contain enlarged pml nbs [53] . disruption of the nucleocytoplasmic trafficking in the host cells is another major alteration caused due to viral infections ( fig. 2 ) [54] . two conserved proteins pul50 and pul53 of hcmv were reported to remodel the nuclear lamina of helfs (primary human lung fibroblasts) for the budding of virions [55] . interaction between human papillomavirus (hpv) and host mitotic chromosomes has been documented [56] . infection of porcine bone marrow cells with african swine fever virus (afsv) resulted in fragmentation of the nucleus [57] . the organelle found in continuation with the nucleus in a cell is the er (fig. 1) . protein synthesis and folding (rough er), lipid synthesis, calcium regulation (smooth er), etc are some of the important functions of the er. multiple structural domains of the er are reported which enable it to serve as a site for various important functions in a cell. several morphological alterations of er such as vesicle formation, invagination of the membrane, zippered appearance, etc have been reported upon viral infection in different cell lines (fig. 2) . the large malleable surface of the er aids viruses to form protective compartments to set up their replication machinery. these compartments known as viroplasm not only concentrate viral and host proteins required for viral genome replication but also protect the viral genome from cellular nucleases [58] . in order to construct these compartments, viruses alter host's fatty acid metabolism, induce rearrangement of the membrane constituents and also recruit cellular machinery to produce proteins essential for its replication [59, 60] . infection of vaccinia virus (vacv) in bs-c-1, hela and rk-13 cells leads to the formation of vacv membranes derived from the er membrane [61] . in case of dengue virus (denv-2), the genomic rna was observed to localize over the rough er in c6/36 aedes albopictus cells [62] . denv infection in human hepatoma 7 (huh7) cells leads to invagination of er membrane resulting in the formation of spherules or vesicles containing double stranded rna [63] . hela cells and cos-1 cells on infection with pv 1 cause formation of single membrane tubules which later mature into double membrane vesicles (dmvs) [64, 65] . similarly, severe acute respiratory syndrome corona virus (sars-cov) induced formation of er derived dmvs upon infection of vero cells [66, 67] . zippered appearance of er was observed upon infection of infectious bronchitis virus (ibv) on mammalian, avian and tracheal epithelial cells [68] . studies with the plant viruses like the potato virus x (pvx) reported tgbp2 protein induced reorganization of the er and appearance of vesicular structures in tobacco plants [69] . effect of the viral protein expression on sterol biosynthesis apart from the alterations in er morphology have also been reported [59, 70] . several viral proteins need to be glycosylated at their n-terminal to ensure proper folding and for the incorporation into virions [71] . hence, a common phenomenon observed upon viral infection is interference with the host post translational machinery and competition with the cellular proteins to undergo these modifications [72] . for example, protein porf2 of hepatitis e virus (hev) gets glycosylated in the er upon its expression in cos-1 cells and huh7 mammalian cells [73, 74] . this increased biosynthetic load is proposed to increase in accumulation of malformed proteins resulting in er stress (fig. 2) . er gets relieved from this stress by inducing a pathway called unfolded protein response (upr) pathway [72] . upr serves to enhance the cell's degradation ability in order to establish er homeostasis [72] . transport of misfolded, misassembled proteins from the er to the cytosol and clearance by the ubiquitin proteasome system takes place via the er associated degradation (erad) pathway [72] . however, some viruses use erad pathway for their advantage by co-opting erad to disassemble and gain access to the cytosol of the host cell [75] . upon sv40 infection of cv-1, hela and 3t6 cells, induction of erad factors in turn induce er membrane reorganization into distinct subdomains called foci and the accumulation of sv40 in these foci was observed [76] . later, sv40 particles travel across the er membrane to reach the cytosol [76] . many enveloped and non-enveloped viruses that exploit er functions like upr, erad to facilitate their replication have been discussed elsewhere [71] . in addition to the above listed mechanisms, er mediated nlinked glycosylation plays a very important role in the survival of the viruses. it has been very elegantly demonstrated that the biogenesis of influenza could modulate the host immune response [77] . similarly, the role of n-glycans in the host immunity against hiv has been documented [77] . these studies corroborate to a common point where the level of glycosylation in the viral surface glycoproteins could alter their antigenicity. mitochondria are double membrane bound organelles that comprise their own genome ( fig. 1 ). they are involved in various functions like fatty acid metabolism, apoptosis, calcium homeostasis, etc. considered evolutionarily the oldest organelle of a eukaryotic cell, they are indispensable as power house of the cell. mitochondrial morphology is maintained by a series of interlinked events namely mitochondrial fusion, fission and mitophagy [78] . mitochondrial fusion helps in exchanging matrix metabolites and intact mitochondrial dna while mitochondrial fission helps in sorting impaired mitochondria from the healthy population which are further eliminated by a process called mitophagy [79] . all the above dynamic events are altered upon viral infection in order to facilitate its replication (fig. 2) [80] . for example, it was reported that hbv infection in the cell induces mitochondrial fission followed by mitophagy in order to attenuate apoptosis [81] . on the other hand, expression of orf-9b protein of sars-cov virus promoted mitochondrial fusion in hek293 cells [82] . upregulation of mitophagy and degradation of the mitochondrial antiviral signalling protein (mavs) in order to attenuate the antiviral immune response in non-small cell lung cancer (nsclc) cells was reported upon measles virus infection [83] . the expression of matrix protein (m) of human parainfluenza virus type 3 (hpiv3) in hek293t and hela cells was reported to induce mitophagy resulting in the suppression of type1 interferon response [84] . hbv induces mitophagosome formation which upon fusion with lysosomes leads to mitophagy and prevents apoptosis, thus facilitating persistent infection in the huh7 cells [81] . similarly, newcastle disease virus (ndv) was reported to induce mitophagy, which promotes ndv replication by preventing caspase dependent apoptosis in human non-small cell lung cancer a549 cells [85] . viruses can alter the intracellular distribution of mitochondria either to prevent the release of mediators of apoptosis or to meet their energy requirement during replication by concentrating them near their viral factories [86] . hbv x protein induces the perinuclear clustering of mitochondria in huh7 cells [87] and afsv leads to the transport of mitochondria to viral assembly sites in vero cells [88] . mitochondrial dna codes for proteins essential for respiratory functions of a cell. certain viruses evade the mitochondria associated antiviral response by damaging the host cell mitochondrial dna, which is essential for synthesizing enzymes for optimum mitochondrial function [86] . for example, mitochondrial dna degradation is induced in mammalian cells upon expression of ul12.5, an amino-terminally truncated ul12 isoform of hsv-1 [89] . raji cell lines infected with hepatitis c virus (hcv) also exhibit mitochondrial dna depletion [90] . maintenance of mitochondrial/cellular ca 2ã¾ homeostasis is vital for various cellular functions. many viruses are involved in altering the mitochondrial calcium homeostasis in order to meet their needs during replication [86] . hcmv upon infection causes calcium influx into mitochondria from er [91] . on the other hand, expression of 2b protein of coxsackievirus resulted in reduced signalling of ca 2ã¾ between the er-golgi and the mitochondria in hela cells resulting in suppression of apoptosis [92] . rotavirus was also reported to alter the calcium homeostasis in the host cell throughout its life cycle [93] . mitochondria are the major source of ros in a cell and a balance between ros production and scavenging is crucial for optimum functioning of the cells. upon viral infection mitochondria undergo oxidative stress and result in an increased production of ros which in turn reduces virus replication [86] . interestingly, ros is also involved in activating many host cellular pathways favourable for viral replication and pathogenesis. several viruses like hiv, hcv, adv, ebv, etc result in increased oxidative stress upon infection [86] . on the other hand, both increase and decrease of oxidative stress is employed as a survival strategy by hbv [94, 95] . many viral proteins reach mitochondria through the mitochondria-associated membrane [96] a sub-compartment of the er or are targeted directly from the cytosol and result in an altered mitochondrial permeability transition pore (mptp) [97] . mptp is also responsible for the maintenance of mmp and provides energy for atp synthesis. altering mptp leads to passive swelling, outer membrane rupture, osmotic water flux, and release of proapoptotic factors leading to cell death [98] . in general, increased mmp induces apoptosis, while decreased mmp prevents apoptosis [86] . viruses are proposed to decrease mmp to prevent cell death in order to promote their replication. however, in later stages, they may trigger an increase in mmp to release the progeny virions by apoptosis [86] . the m11l protein of myxoma pox virus prevents the loss of mmp in cos-7 monkey kidney cells, hela cells, thp-1 human monocytes and jurkat t lymphocytes [99] . on the other side, the r protein of hiv-1 induces the loss of mmp in cem-c7 and jurkat cells and results in apoptosis [100] . viruses alter the host mitochondrial metabolic pathways in order to maintain cellular energy homeostasis essential to ensure efficient replication and to avoid mitochondrial antiviral response particularly in case of slow replicating virus [101] . some viruses modulate the normal cells to increase aerobic glycolysis and use glucose biosynthetically, which helps especially enveloped viruses to increase their available pool of fatty acids, lipids and nucleotides during their replication [102, 103] the feline leukemia virus infection on lung fibroblasts (flf-3) resulted in a 30e40% increase in glucose uptake and lactic acid production [104] . an increase in the lactic acid production upon infection of the chick embryo cells with rous sarcoma virus was reported [105] . hcmv upon infection of hffs, human retinal pigment epithelial cells (arpe19), human embryonic lung fibroblasts (mrc5) and vero cells enhances glycolytic flux and directs the supply of carbon from glucose to tca cycle. this helps to facilitate fatty acid biosynthesis while hsv-1 upon infection of same cell lines directs the central carbon metabolism in order to induce the production of pyrimidine nucleotide components [106] . hcv core protein suppresses mitochondrial complex 1 activity and impaired function of electron transport cycle leading to ros accumulation in hepatoma cells of transgenic mice [107] . mitochondrial involvement in virus survival is quite relevant to the fact that viruses require a source of energy to favour the active processes involved in their life cycle. peroxisomes are single membrane bound dynamic organelles required for b-oxidation of fatty acids and ros metabolism (fig. 1) . they have developed diverse functions which are organism and environment dependent. they are unique with respect to proliferation, as they can increase in number by both growth and division of pre-existing organelles and formation of new organelles from the er. many viruses or viral proteins are reported to localize to peroxisomes and/or exploit their functions to facilitate their replication in the host cells [108] . presence of a peroxisome targeting signal (pts) in the protein sequence is reported to be essential for targeting proteins into the peroxisomes [109] . however, it was also proposed that viral proteins without pts may get associated with host peroxisomal proteins in the cytosol which then ferry the viral protein into the peroxisomes in a piggyback fashion [108] . for instance, hcmv encodes a protein called vmia reported to be localized to peroxisomes in hffs and hepg2 cells [110] . the vmia interaction with the host pex19 protein aids the viral protein localization to the peroxisomes. fragmentation of peroxisomes upon vmia expression in hepg2 was also reported [110] . in addition, peroxisomal localization of two cymbidium ring spot viral proteins p33 and p92 was reported in yeast [111] . a role for peroxisomes in the replication of tbsv has also been reported. the viral replication protein p33 interacts with the host peroxisomal protein pex19 for its targeting to the peroxisomal membrane and subsequent replication [112, 113] . the host hsp70 protein was reported to promote the localization of the viral replication proteins to the peroxisomes in yeast cells [114] . n-terminal protease n pro of pestivirus is another example of a viral protein that is associated with peroxisomes and facilitates its survival and replication [115] . some members of the family tombusviridae are involved in remodelling the peroxisomal membrane resulting in the formation of vesicular structures called "profoundly modified peroxisomes" or "peroxisome derived multivesicular bodies" [108] . for example, the infection of cymbidium ring spot virus resulted in the formation of small vesicles at the periphery of the peroxisomes in plant leaf tissues [116] . at the later stage of infection, the entire peroxisomal matrix is occupied by these vesicles [116] . similarly, the cucumber necrosis virus (cnv) induces the formation of peroxisomal vesicles in which the viral rna replication occurs in yeast cells [117] . studies revealed upregulation of peroxisomal biogenesis in endothelial cells upon latent infection of kaposi's sarcoma associated herpes virus [118] . it was also reported that proteins involved in peroxisomal lipid metabolism were essential for the survival of latently infected human dermal microvascular endothelial cells and lymphatic endothelial cells [118] . interestingly, hiv infection reduces the number of peroxisomes in infected cells due to upregulation in the levels of micrornas that inhibit production of peroxisome biogenesis factors [119] . it has been reported that the west nile virus (wnv) and denv infection on a549 and hek293t cells result in the degradation of peroxisomes [120] . the capsid proteins of both denv and wnv were reported to interact with the peroxisomal protein pex19 required for peroxisome biogenesis. degradation and redistribution of pex19 from perinuclear region to juxtanuclear region was reported in the infected cells. in addition, reduced level of the antioxidant enzyme catalase was observed in the infected cells. these alterations resulted in an impairment of early antiviral signalling of the peroxisomes. expression of the pts containing vp4 protein of the rotavirus, in ma104 cell lines resulted in peroxisomal localization [121] . the role for such a localization was speculated to utilize the peroxisomal lipid metabolism for the supply of cholesterol for lipid raft synthesis, required for the viral replication. peroxisomal b-oxidation metabolism leads to the production of myristoyl-coa by shortening the fatty acids chain which could be exported to the cytosol for nmyristoylation of the viral proteins vp2 and vp6 of rotavirus [108] . studies on hiv suggested an interaction between viral nef protein and the peroxisomal enzyme thio-esterase using yeast two hybrid system [122] . further studies reported an increase in the enzymatic activity of human acyl-coa thio-esterase 3 on binding with the nef protein [123] . the increased activity of the peroxisomal enzyme was speculated to contribute in alteration of the subcellular morphology and in downregulation of the host antiviral response [108] . extensive modifications of proteins for proper functioning and targeting in a cell takes place at the golgi apparatus. other functions of golgi inevitable for the cell are carbohydrate synthesis and lipid transport. the unique stacked structural organization of the golgi is essential for these functions (fig. 1) . the golgi apparatus is composed of three regions namely cis golgi network (cgn), medial and trans golgi network (tgn). various viruses and viral proteins have been identified to localize to these golgi regions during their life cycle. for example, hela cells upon infection with adeno-associated virus type 5 (aav-5) led to an accumulation of the viral particles in the tgn and in the golginetwork associated coated vesicles [124] . in sars-cov, the transmembrane domain of the orf7b protein contains the retention signal required for accumulation of the protein in the cgn and tgn [125] . reports suggest bunyaviruses assemble in the golgi as a result of its retention signal in the glycoprotein (gn) of the virus [82, 126] . fragmentation of golgi body is a common phenomenon that occurs as a result of various viral infections (fig. 2) . orf virus (a parapox virus) causes disruption and fragmentation of golgi in vero cells. this structural modification affects the late vesicular export machinery and results in downregulation of the host immune response [127] . similar phenomenon was also reported upon expression of 3c protease of foot-and-mouth disease virus in vero cells [128] . infection of hrv1a on wi-38, 293t, and h1-hela cells was reported to induce fragmentation of golgi body, rearrangement of the golgi membranes into vesicles which are utilized as sites of rna replication [129] . another incidence of the virus induced golgi body fragmentation was observed upon infection of sars-cov that resulted in death of vero cells [130] . many rna viruses were also found to alter the integrity of golgi complex of the host cells. for example, the expression of n-terminal non-structural protein of norwalk virus in crandell-rees feline kidney (crfk) and hela cells co-localizes to golgi complex and induces its disassembly into discrete aggregates [131] . another example is the pv infection on vero cells which results in complete disruption of the cgn into fragments scattered throughout the cytoplasm. the expression of pv protein 2b in vero, normal rat kidney (nrk) and cos-7 cells also resulted in golgi bodies disassembly [132] . similarly, expression of protein 3a of avian encephalomyelitis virus (aev) in chick embryo brain (ceb) cells and cos-7 cells resulted in depletion of golgi stacks and in severe disassembly of golgi bodies [133] . an interesting alteration in the morphology of golgi apparatus was observed upon infection of turnip mosaic virus (tumv) on n. benthamiana plant. tumv infection was observed to induce amalgamation of golgi apparatus, er and chloroplast [134] . mccoy mouse fibroblast cells led to accumulation of golgin-97 a tgn resident protein in the viral factories [135] . similarly, 3a protein of some picornaviruses were also found to interact with a golgi apparatus resident protein namely golgi adaptor protein acyl coenzyme a (acyl-coa) binding domain protein 3 (acbd3/gpc60), acts as an adaptor to recruit phosphatidylinositol 4-kinase class iii beta (pi4kiii) in infected cells [136] . evidence shows that the tomato spotted wilt virus (tswv) glycoprotein gn localizes to golgi membranes and induces deformation of the membranes into pseudo-circular and pleomorphic structures in tobacco plant cells [137] . upon infection of hela cells, rabbit kidney cells and mouse monocytes-macrophages cell lines with vacv, viral progeny becomes enwrapped in the membrane derived from tgn cisterna to form the enveloped virus [138] . several viral proteins localize to the golgi body and mature by undergoing post translational modifications such as glycosylation, phosphorylation, etc. rubella virus was reported to undergo a golgi dependent maturation upon infection of bhk-21 and vero cells [139] . the inner tegument protein pul37 of the dna virus hsv was identified to be responsible in directing the viral capsids to the tgn in order to undergo secondary envelopment in different cell lines [140] . the glycoproteins of bunyamwera virus undergo primary maturation by modifying their sugar composition in the tgn upon infection of the bhk-21 and vero cells [141] . lipid droplets are single membrane bound organelles with a lipid core that primarily consists of neutral lipids like triacylglycerols (fig. 1) . they are essential for lipid storage and metabolism in a cell. these stored lipids can be used to generate and maintain energy homeostasis and hence they are central to the cellular function. lipid droplets are dynamic intracellular organelles which are required for storing lipids in a cell. they play a major role in energy homeostasis and membrane trafficking [142] . many rna viruses exploit this energy storing capacity of lipid droplets to facilitate their replication [142] . upon expression, various viral proteins are reported to localize to the lipid droplets [143] . for example, upon hcv infection, the hcv ns5a protein localizes to the surface of the lipid droplets with the help of a host factor diacylglycerol acyltransferase 1 (dgat1) in huh7 and hek293t cells [143] . another study reported that hcv core 3a protein is involved in downregulating the expression of phosphoinositide 3-kinase (pi3k)/phosphatase and tensin (pten) which in turn induces the accumulation of enlarged lipid droplets in human huh7 and hepg2 cells [144] . in another example, denv c protein was reported to bind and interact with perilipin 3 a protein present on the surface of the lipid droplets in hepg2 cell lines [145] . a similar localization of denv c protein was also observed upon infection of denv2 in bhk-21, hepg2, and c6/36 ht mosquito cells of a. albopictus [146] . additionally, the denv c protein localization on lipid droplets was also found to be essential for the denv2 replication [146] . denv induces autophagy and results in a reduction in lipid droplet area in huh7.5 cells, a subline derived from huh7 cells [147, 148] . further analysis of the infected cells showed reduced levels of triglycerides in the lipid droplets and suggests that atp generation by b-oxidation of fatty acids is essential for robust replication of viral rna [147, 148] . another study reports that rotavirus recruits lipid droplets into their viroplasm compartments upon infection of ma104, caco-2, bsc-1, and cos-7 cell lines [149] . confocal microscopy studies reported that two ld-associated proteins namely perilipin a and adrp colocalize with rotaviral proteins present in the viroplasm [149] . overexpression of the hbv x (hbx) protein was found to induce lipid accumulation in hepatic cells [150, 151] . na and colleagues found that hbx induces a pathway that involves the expression of liver x receptor and its associated genes result in accumulation of lipid droplets in hepg2 cell lines [152] . lysosomes are single membrane-bound organelles which house enzymes involved in the degradation of various extracellular and intracellular macromolecules such as proteins, pathogens, etc through phagocytic or autophagic pathway. plant and fungal vacuoles have similar degradative and storage functions like the lysosomes (fig. 1) . the specialized acidic lumen is the unique feature of these organelles which is needed to keep the enzymes active. viruses utilize lysosomal enzymes in order to facilitate their replication and release within the host cell [153] . it has been suggested that lysosomal enzymes are involved at different stages of vacv and mouse hepatitis virus replication [153] . they proposed a possibility for viruses to recruit lysosomal enzymes inside phagosomes in order to uncoat and release their genome in the host cell. a possible role for the lysosomal enzymes in enhancement of glycolysis in viral infected cells was also proposed. recent studies reported the accumulation of hav progeny in the lysosome of the host hepg2 cells. the maturation of these viral particles was reported to be catalysed by the lysosomal protease [154] . sv40 infection in bsc-1 and 3t3 cell lines resulted in swelling up of lysosomes followed by the release of lysosomal enzyme into the cytoplasm [155] . since lysosomes play a major role in the host's antiviral response, they are likely to become a target for certain viruses. the x protein of the hbv leads to inhibition of lysosomal acidification leading to loss of functioning [156] . however, the lysosome's ability to fuse with autophagosomes was found to remain unaffected. this virus induced accumulation of immature lysosomes resulting in the suppression of autophagic degradation was followed by development of hbv-associated hepatocellular carcinoma [156] . deglycosylation of the lysosome-associated membrane proteins by neuraminidase (na) of h5n1 influenza virus resulted in destruction of lysosomes. this was followed by cell death due to the release of hydrolytic lysosomal enzymes to the cytoplasm [157] . shubin and colleagues studied the expression of 3c protease of hav in a549 and human lung epidermoid carcinoma (calu-1) cell lines and found that hav 3c protease induces the development of non-acidic cytoplasmic vacuoles which originate from several types of lysosomal/endosomal organelles [158] . similarly several viruses like hiv, adenovirus, pv have been reported to cause lysosomal rupture [159] . tobacco plant when infected with cucumber mosaic virus (cmv), the viral replicase complex that constitutes the replicaseassociated protein cmv1a and rna dependent rna polymerase protein cmv2a was reported to localize on the vacuolar membrane [160] . singapore grouper iridovirus (sgiv) infection on grouper embryonic cells (gecs) from the brown-spotted grouper epinephelus tauvina results in the formation of a large intracellular vacuole for viral accumulation. later, the virus recruits the host cytosolic membrane-bending proteins in order to induce tubulation of vacuolar membrane [161] . the individual vacuoles fuse together to form a large vacuole which in turn fuses with the cell membrane. these events aid in the release of virions [161] . semliki forest virus (sfv) targets the vacuolar atpase in order to cause acidification of vacuoles resulting in low intra luminal ph [162] . upon infection with the sfv the endocytic vacuoles with low ph trigger membrane fusion essential for the viral pathogenesis in bhk-21 cells [163] . vacuolar proton atpase activity leading to low intra endosomal ph was reported to be required for the entry of reovirus into the host cells [164] . acidification of vacuoles by cellular v-atpase in human dermal fibroblast cells upon infection of hcmv was reported to be required for the formation of the specialized compartment for virion assembly [165] . apart from the above discussed well defined organelles of a cell, viruses also modify and hijack various vesicular structures and protein complexes of the host cell to ensure efficient infection. we discuss these essential compartments/structures in this section. endosomes are required for internalization of extracellular material into the cells and lead them to lysosomes for degradation or recycle back to the plasma membrane. multi vesicular bodies are vesicular compartment of the endocytic pathway and contain intraluminal vesicles. autophagosomes are double membrane vesicular structures that sequester the cytoplasm containing proteins, organelles, etc and direct them to degradation (fig. 1) . efficient cellular entry of most viruses is via endocytic pathway that comprises of various endosomes. several viruses like influenza, sfv, vsv, sv40, ebola, etc use different endocytic pathways like clathrin, caveoli or micropinocytosis to gain entry into the cells [6,166e168] . viruses modify or induce the formation of vesicles like endosomes in order to facilitate their multiplication inside the host cells [169] . sfv modifies the endosomal and lysosomal membrane of the infected bhk-21 cells for the construction of its replication site [170] . further the endosomes and lysosomes were reported to fuse together resulting in the formation of cytoplasmic vacuoles [170] . sv40 was reported to trigger the formation of endocytic vacuoles that migrate and fuse with the nuclear membrane upon infection of cv-1 cells leading to the migration of virions into the infected cell nucleus [171, 172] . endosomal sorting complexes required for transport (escrt) catalyse the process of invagination of endosomal membrane and result in the formation of multiple vesicular bodies (mvb) in eukaryotic cells [173] . mvb act as an intermediate for transporting ubiquitinated or misfolded protein to lysosomes [173] . mvb are also reported to play an active role in endolysosomal transport and budding of the virus. several rna and dna viruses hijack the escrt machinery in order to facilitate their release from the infected cells [169] . the matrix protein vp40 recruits tsg101 protein and escrt-1 complex constituting of vps28, vps37b and vps4 in order to direct the ebola virus into multivesicular bodies that assists in their budding [174] . hoffmann and colleagues reported that upon infection of hepatoma derived cell lines by the dna virus hbv, cellular a-taxilin acts as an adaptor for the binding of large hbv surface antigen with escrt components. this aids in recruiting the escrt machinery for the release of hbv-dna containing particles [175] . various rna viruses alter autophagosomes and induce or suppress autophagy in order to complete their life cycle or to escape from the host antiviral response [176] . it has been reported that the coxsackie b3 infection triggers the formation of autophagosomes in hela and hek293 cells [177] . prevention of lysosomal fusion with autophagosomes also enhanced coxsackie virus replication in the host cells. wild type mouse embryonic fibroblast (mef) and huh7 cells also exhibit enhanced autophagosome formation upon denv 2 infection [178] . the viruses modulating the phenomenon of cellular autophagy for their advantage have been reviewed elsewhere [176] . proteasome is a complex of proteases that selectively degrades intracellular proteins. this complex is tightly regulated and identifies polyubiquitinated proteins for degradation process. recent studies identified that viruses hijack ups in several ways to enhance their infectivity [179] . this is achieved by degradation of host proteins of ups, enhancing the function of viral proteins by modifications, hampering the modifications of signalling molecules of innate immunity, etc [179] . virus induced ubiquitination and subsequent proteasomal degradation of p53 as a strategy is employed by dna viruses such as hpv, adv, etc [180] . viral protein x of hiv-2 was reported to be responsible for the ubiquitination and proteasomal degradation of the host protein samhd1 that inhibits hiv infection [181] . an interesting strategy by denv was reported where the viral protein ns5 stimulates proteasomal degradation of stat2 and blocks type 1 ifn signalling and thus evades the host immune mechanisms [182] . similarly selective degradation of ns3 (non-structural protein) of the zika virus via proteasome mediated pathway was recently reported [183] . this proteasome dependant degradation of the viral protein was proposed as a strategy for the host antiviral mechanism. a role for ups has also been reported in plant viral infections. components of rna silencing such as argonaute 1 are targeted to proteasomal degradation by viruses like potato virus x, enamovirus, etc for efficient infection [184, 185] . this review summarizes the modifications that organelles encounter upon viral infection in a cell. understanding organelle dynamics under various conditions is a fundamental question that has attracted researchers for a long time. the importance of organelle dynamics and function is highlighted by many examples of diseases/disorders where it is affected. alterations in organelles such as shape, content, dynamics and eventually the function as a result of viral infections is observed. not only viruses but pathogenic bacteria have also been reported to alter organelles for their survival and infection. as emphasized in this paper, recent studies have shown that many viruses encode proteins that are targeted to various cellular organelles and control their functions. certainly there exists a close relationship between organelle dynamics and viral infections but thorough characterization will highlight their relevance to pathogenesis. advanced methods in microscopy and proteomics have enabled such characterization and the molecular details of virus-host interactions and viral replication in host cells is now understood in detail for few viruses. it is important to determine how various organelle proteins are temporally and spatially regulated upon viral infections leading to altered functions. organelles not as independent entities but a role for inter-organelle communication/inter-organelle cross talk in a cell for optimum functioning is now unequivocally accepted. this is another very interesting aspect to be explored to enhance our understanding of the virus-host interaction mechanisms to enable design of new antiviral strategies. our understanding on the organelle-virus interaction has been rapidly increasing with the advent of new molecular biology tools and advance imaging techniques. although we know the basic modus operandi of the viruses, there might be a novel virus that behaves different than the existing dogma with respect to host cellular architecture. the authors declare to no conflict of interest. ground control to major tom: mitochondria-nucleus communication the upsides and downsides of organelle interconnectivity an organelle-specific protein landscape identifies novel diseases and molecular mechanisms hiv-1 and the host cell: an intimate association virulence and pathogenesis virus entry by endocytosis signaling organelles of the innate immune system lipid droplet density alters the early innate immune response to viral infection virus factories: associations of cell organelles for viral replication and morphogenesis exploring and exploiting proteome organization during viral infection nuclear remodelling during viral infections how viruses access the nucleus the road to chromatin -nuclear entry of retroviruses integration of murine leukemia virus dna depends on mitosis two nuclear localization signals in the hiv-1 matrix protein regulate nuclear import of the hiv-1 pre-integration complex characterization of hiv-1 vpr nuclear import: analysis of signals and pathways hiv-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway integrase interacts with nucleoporin nup153 to mediate the nuclear import of human immunodeficiency virus type 1 nuclear import and export of influenza virus nucleoprotein application of bioinformatics-coupled experimental analysis reveals a new transport-competent nuclear localization signal in the nucleoprotein of influenza a virus strain importin alpha nuclear localization signal binding sites for stat1, stat2, and influenza a virus nucleoprotein function of dynein and dynactin in herpes simplex virus capsid transport microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro import of adenovirus dna involves the nuclear pore complex receptor can/nup214 and histone h1 nucleoporin 153 arrests the nuclear import of hepatitis b virus capsids in the nuclear basket phosphorylationdependent binding of hepatitis b virus core particles to the nuclear pore complex nuclear import of hepatitis b virus capsids and release of the viral genome role of nuclear pore complex in simian virus 40 nuclear targeting pushing the envelope: microinjection of minute virus of mice into xenopus oocytes causes damage to the nuclear envelope parvoviral nuclear import: bypassing the host nuclear-transport machinery nucleoli: composition, function, and dynamics involvement of ul24 in herpes-simplex-virus-1-induced dispersal of nucleolin involvement of the ul24 protein in herpes simplex virus 1-induced dispersal of b23 and in nuclear egress adenovirus core protein v is delivered by the invading virus to the nucleus of the infected cell and later in infection is associated with nucleoli adenovirus protein v induces redistribution of nucleolin and b23 from nucleolus to cytoplasm effects of adenovirus infection on rrna synthesis and maturation in hela cells encephalomyocarditis virus (emcv) proteins 2a and 3bcd localize to nuclei and inhibit cellular mrna transcription but not rrna transcription rhinovirus 3c protease precursors 3cd and 3cd' localize to the nuclei of infected cells the post-transcriptional regulator rev of hiv: implications for its interaction with the nucleolar protein b23 localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus nucleolin stimulates viral internal ribosome entry site-mediated translation quantitative proteomic analysis of a549 cells infected with human respiratory syncytial virus ifn enhance expression of sp100, an autoantigen in primary biliary cirrhosis hsv-1 ie protein vmw110 causes redistribution of pml mediation of epstein-barr virus ebna-lp transcriptional coactivation by sp100 adenovirus replication is coupled with the dynamic properties of the pml nuclear structure adenovirus e4 orf3 protein inhibits the interferon-mediated antiviral response inactivating a cellular intrinsic immune defense mediated by daxx is the mechanism through which the human cytomegalovirus pp71 protein stimulates viral immediate-early gene expression two ring finger proteins, the oncoprotein pml and the arenavirus z protein, colocalize with the nuclear fraction of the ribosomal p proteins sumoylation promotes pml degradation during encephalomyocarditis virus infection rabies virus p and small p products interact directly with pml and reorganize pml nuclear bodies viral interactions with the nuclear transport machinery: discovering and disrupting pathways remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pul50 and pul53 papillomavirus interaction with cellular chromatin phenotypic and cytologic studies of lymphoid cells and monocytes in primary culture of porcine bone marrow during infection of african swine fever virus wrapping membranes around plant virus infection inhibition of sterol biosynthesis reduces tombusvirus replication in yeast and plants endoplasmic reticulum: the favorite intracellular niche for viral replication and assembly direct formation of vaccinia virus membranes from the endoplasmic reticulum in the absence of the newly characterized l2-interacting protein a30.5 intracellular localisation of dengue-2 rna in mosquito cell culture using electron microscopic in situ hybridisation composition and three-dimensional architecture of the dengue virus replication and assembly sites cellular origin and ultrastructure of membranes induced during poliovirus infection remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagylike origin for virus-induced vesicles ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes the potato virus x tgbp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection tombusviruses upregulate phospholipid biosynthesis via interaction between p33 replication protein and yeast lipid sensor proteins during virus replication in yeast opportunistic intruders: how viruses orchestrate er functions to infect cells arms race between enveloped viruses and the host erad machinery mutational analysis of glycosylation, membrane translocation, and cell surface expression of the hepatitis e virus orf2 protein cytoplasmic localization of the orf2 protein of hepatitis e virus is dependent on its ability to undergo retrotranslocation from the endoplasmic reticulum the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis bap31 and bip are essential for dislocation of sv40 from the endoplasmic reticulum to the cytosol global sitespecific n-glycosylation analysis of hiv envelope glycoprotein the interplay between mitochondrial dynamics and mitophagy mitochondrial fusion, fission, and mitochondrial toxicity mitochondrial dynamics and viral infections: a close nexus hepatitis b virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis sarscoronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome mitophagy in viral infections the matrix protein of human parainfluenza virus type 3 induces mitophagy that suppresses interferon responses mitophagy promotes replication of oncolytic newcastle disease virus by blocking intrinsic apoptosis in lung cancer cells viruses as modulators of mitochondrial functions hepatitis b virus x protein induces perinuclear mitochondrial clustering in microtubule-and dyneindependent manners migration of mitochondria to viral assembly sites in african swine fever virus-infected cells herpes simplex virus eliminates host mitochondrial dna hepatitis c virus triggers mitochondrial permeability transition with production of reactive oxygen species, leading to dna damage and stat3 activation human cytomegalovirus pul37x1 induces the release of endoplasmic reticulum calcium stores the coxsackievirus 2b protein suppresses apoptotic host cell responses by manipulating intracellular ca2ã¾ homeostasis role of ca2ã¾in the replication and pathogenesis of rotavirus and other viral infections human hepatitis b virus-x protein alters mitochondrial function and physiology in human liver cells hbx sensitizes cells to oxidative stress-induced apoptosis by accelerating the loss of mcl-1 protein via caspase-3 cascade influence of cytosolic and mitochondrial ca2ã¾, atp, mitochondrial membrane potential, and calpain activity on the mechanism of neuron death induced by 3-nitropropionic acid viral product trafficking to mitochondria, mechanisms and roles in pathogenesis a pore way to die: the role of mitochondria in reperfusion injury and cardioprotection the myxoma poxvirus protein, m11l, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore the hiv-1 viral protein r induces apoptosis via a direct effect on the mitochondrial permeability transition pore a renewed focus on the interplay between viruses and mitochondrial metabolism systemslevel metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy dynamics of the cellular metabolome during human cytomegalovirus infection glycolysis during early infection of feline and human cells with feline leukemia virus glycolysis in chick embryo cell cultures transformed by rous sarcoma virus divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production viruses exploiting peroxisomes import of peroxisomal matrix and membrane proteins peroxisomes are platforms for cytomegalovirus' evasion from the cellular immune response expression of the cymbidium ringspot virus 33-kilodalton protein in saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal localization of the tomato bushy stunt virus replication protein p33 reveals a peroxisome-to-endoplasmic reticulum sorting pathway the host pex19p plays a role in peroxisomal localization of tombusvirus replication proteins a key role for heat shock protein 70 in the localization and insertion of tombusvirus replication proteins to intracellular membranes the pestivirus n terminal protease n(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of irf3 by n(pro.) the fine structure of cymbidium ringspot virus infections in host tissues. iii. role of peroxisomes in the genesis of multivesicular bodies the role of the p33:p33/p92 interaction domain in rna replication and intracellular localization of p33 and p92 proteins of cucumber necrosis tombusvirus integrated systems biology analysis of kshv latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism micro-rnas upregulated during hiv infection target peroxisome biogenesis factors: implications for virus biology, disease mechanisms and neuropathology flavivirus infection impairs peroxisome biogenesis and early antiviral signaling identification of a type 1 peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle binding of hiv-1 nef to a novel thioesterase enzyme correlates with nef-mediated cd4 down-regulation a novel acyl-coa thioesterase enhances its enzymatic activity by direct binding with hiv nef endocytosis of adeno-associated virus type 5 leads to accumulation of virus particles in the golgi compartment the transmembrane domain of the severe acute respiratory syndrome coronavirus orf7b protein is necessary and sufficient for its retention in the golgi complex a signal for golgi retention in the bunyavirus g1 glycoprotein orf virus interferes with mhc class i surface expression by targeting vesicular transport and golgi foot-and-mouth disease virus 3c protease induces fragmentation of the golgi compartment and blocks intra-golgi transport fragmentation of the golgi apparatus provides replication membranes for human rhinovirus 1a the open reading frame 3a protein of severe acute respiratory syndrome-associated coronavirus promotes membrane rearrangement and cell death norwalk virus n-terminal nonstructural protein is associated with disassembly of the golgi complex in transfected cells poliovirus infection and expression of the poliovirus protein 2b provoke the disassembly of the golgi complex, the organelle target for the antipoliovirus drug ro-090179 membrane-association properties of avian encephalomyelitis virus protein 3a impact on the endoplasmic reticulum and golgi apparatus of turnip mosaic virus infection a trans-golgi network resident protein, golgin-97, accumulates in viral factories and incorporates into virions during poxvirus infection the 3a protein from multiple picornaviruses utilizes the golgi adaptor protein acbd3 to recruit pi4kiiibeta tomato spotted wilt virus glycoproteins induce the formation of endoplasmic reticulum-and golgi-derived pleomorphic membrane structures in plant cells assembly of vaccinia virus: the second wrapping cisterna is derived from the trans golgi network structural maturation of rubella virus in the golgi complex inner tegument protein pul37 of herpes simplex virus type 1 is involved in directing capsids to the trans-golgi network for envelopment key golgi factors for structural and functional maturation of bunyamwera virus emerging role of lipid droplets in host/pathogen interactions lipid droplets and viral infections down-regulation of phosphatase and tensin homolog by hepatitis c virus core 3a in hepatocytes triggers the formation of large lipid droplets dengue virus capsid protein binding to hepatic lipid droplets (ld) is potassium ion dependent and is mediated by ld surface proteins dengue virus capsid protein usurps lipid droplets for viral particle formation dengue virus-induced autophagy regulates lipid metabolism dengue virus and autophagy rotaviruses associate with cellular lipid droplet components to replicate in viroplasms, and compounds disrupting or blocking lipid droplets inhibit viroplasm formation and viral replication hepatitis b virus x protein induces lipogenic transcription factor srebp1 and fatty acid synthase through the activation of nuclear receptor lxralpha hepatitis b virus x protein induces hepatic steatosis via transcriptional activation of srebp1 and ppargamma liver x receptor mediates hepatitis b virus x protein-induced lipogenesis in hepatitis b virusassociated hepatocellular carcinoma activation of lysosomal enzymes in virus-infected cells and its possible relationship to cytopathic effects lysosomes serve as a platform for hepatitis a virus particle maturation and nonlytic release lysosomal changes in lytic and nonlytic infections with the simian vacuolating virus (sv40) hepatitis b virus x protein inhibits autophagic degradation by impairing lysosomal maturation neuraminidase of influenza a virus binds lysosome-associated membrane proteins directly and induces lysosome rupture protease 3c of hepatitis a virus induces vacuolization of lysosomal/endosomal organelles and caspase-independent cell death lysosomal cell death at a glance a zinc finger protein tsip1 controls cucumber mosaic virus infection by interacting with the replication complex on vacuolar membranes of the tobacco plant visualization of assembly intermediates and budding vacuoles of singapore grouper iridovirus in grouper embryonic cells involvement of the vacuolar h(ã¾)-atpase in animal virus entry membrane and protein interactions of a soluble form of the semliki forest virus fusion protein the entry of reovirus into l cells is dependent on vacuolar proton-atpase activity cellular v-atpase is required for virion assembly compartment formation in human cytomegalovirus infection endocytosis via caveolae endocytosis of simian virus 40 into the endoplasmic reticulum cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes membrane dynamics associated with viral infection biogenesis of the semliki forest virus rna replication complex fusion of sv40-induced endocytotic vacuoles with the nuclear membrane interaction of endocytotic vacuoles with the inner nuclear membrane in simian virus 40 entry into cv-1 cell nucleus escrt complexes and the biogenesis of multivesicular bodies involvement of vacuolar protein sorting pathway in ebola virus release independent of tsg101 interaction identification of alpha-taxilin as an essential factor for the life cycle of hepatitis b virus divergent roles of autophagy in virus infection autophagosome supports coxsackievirus b3 replication in host cells autophagic machinery activated by dengue virus enhances virus replication irhom2 is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting identification of three functions of the adenovirus e4orf6 protein that mediate p53 degradation by the e4orf6-e1b55k complex molecular determinants for recognition of divergent samhd1 proteins by the lentiviral accessory protein vpx ns5 of dengue virus mediates stat2 binding and degradation viperin restricts zika virus and tick-borne encephalitis virus replication by targeting ns3 for proteasomal degradation the silencing suppressor p25 of potato virus x interacts with argonaute1 and mediates its degradation through the proteasome pathway the enamovirus p0 protein is a silencing suppressor which inhibits local and systemic rna silencing through ago1 degradation we sincerely apologize to our colleagues for any research study or review publication not being cited in this review. this is only due to space limitation. research in the laboratory of sn is supported by grants from science and engineering research board ( key: cord-256282-vqus7vlg authors: cox, michael j; loman, nicholas; bogaert, debby; o'grady, justin title: co-infections: potentially lethal and unexplored in covid-19 date: 2020-04-24 journal: the lancet. microbe doi: 10.1016/s2666-5247(20)30009-4 sha: doc_id: 256282 cord_uid: vqus7vlg nan respiratory viral infections predispose patients to co-infections and these lead to increased disease severity and mortality. most fatalities in the 1918 influenza outbreak were due to subsequent bacterial infection, particularly with streptococcus pneumoniae. 1 poor outcomes in the 2009 h1n1 influenza pandemic were also associated with bacterial co-infections, although few studies captured these data. 2 despite the proven importance of co-infections in the severity of respiratory diseases, they are understudied during large outbreaks of respiratory infections. zhou and colleagues 3 showed that in the current coronavirus disease 2019 (covid-19) pandemic, 50% of patients with covid-19 who have died had secondary bacterial infections, and chen and colleagues 4 have recorded both bacterial and fungal co-infections. although 71% of the admitted patients with covid-19 received antibiotic drugs, no information is available on the antimicrobial sensitivities of the organisms that were identified, or on the type and duration of antimicrobial treatment. chronic obstructive pulmonary disease (copd) is a risk factor for severe covid-19 disease and many patients with copd will have underlying chronic bacterial infections before severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection, but this important information is not being reported. more data on co-infections are urgently required to establish their importance in covid-19 severity and mortality. diagnosing co-infections is complex. the organism itself might be carried by the patient before the viral infection, might be part of an underlying chronic infection, or might be picked up nosocomially. in the uk, national institute for health and care excellence (nice) treatment guidance for severe acquired pneumonia is broadspectrum antibiotic co-amoxiclav plus a macrolide to cover atypical organisms. currently, antibiotic use is high (74·5%) among patients with covid-19 who are admitted to intensive care units, rendering culture-based microbiological testing less sensitive. patients with covid-19 are kept on invasive mechanical ventilation for a long time (mean 9·1 days [sd 5·5]), increasing chances of hospital and ventilator acquired infections. hence, early diagnosis of co-infection is required, preferably using methods capable of detecting a broad range of potential pathogens and antimicrobial resistances, with subsequent monitoring for infection development. to accurately diagnose and study co-infection in covid-19, patients should be recruited on admission to intensive care units and sampled longitudinally throughout the disease course using cultureindependent techniques capable of identifying complex mixed infections without previous target selection, such as whole-genome metagenomics. 5 such a study would provide valuable surveillance data on the pathogens causing co-infections and antimicrobial resistance in the intensive care setting, thereby helping inform antibiotic prescribing policy. rapid characterisation of coinfection is essential in the management and treatment of the most severe covid-19 cases, could help to save lives, and will improve antimicrobial stewardship throughout the course of the pandemic. predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness the role of pneumonia and secondary bacterial infection in fatal and serious outcomes of pandemic influenza a(h1n1)pdm09 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection for further discussion about co-infection and covid-19 see key: cord-270294-g95skuik authors: johnstone, jennie; majumdar, sumit r.; fox, julie d.; marrie, thomas j. title: viral infection in adults hospitalized with community-acquired pneumonia prevalence, pathogens, and presentation date: 2008-12-31 journal: chest doi: 10.1378/chest.08-0888 sha: doc_id: 270294 cord_uid: g95skuik background the potential role of respiratory viruses in the natural history of community-acquired pneumonia (cap) in adults has not been well described since the advent of nucleic amplification tests (nats). methods from 2004 to 2006, adults with cap who were admitted to five hospitals were prospectively enrolled in the study, and clinical data, cultures, serology, and nasopharyngeal swabs were obtained. nats from swabs were tested for influenza, human metapneumovirus (hmpv), respiratory syncytial virus (rsv), rhinovirus, parainfluenza virus 1–4, coronaviruses (oc43, 229e, and nl63), and adenovirus. results a total of 193 patients were included; the median age was 71 years, 51% of patients were male, and 47% of patients had severe cap. overall, 75 patients (39%) had a pathogen identified. of these pathogens, 29 were viruses (15%), 38 were bacteria (20%), 8 were mixed (4%), and the rest were “unknown.” influenza (n = 7), hmpv (n = 7), and rsv (n = 5) accounted for most viral infections; other infections included rhinovirus (n = 4), parainfluenza (n = 3), coronavirus (n = 4), and adenovirus (n = 2). streptococcus pneumoniae was the most common bacterial infection (37%). compared with bacterial infection, patients with viral infection were older (76 vs 64 years, respectively; p = 0.01), were more likely to have cardiac disease (66% vs 32%, respectively; p = 0.006), and were more frail (eg, 48% with limited ambulation vs 21% of bacterial infections; p = 0.02). there were few clinically meaningful differences in presentation and no differences in outcomes according to the presence or absence of viral infection. conclusions viral infections are common in adults with pneumonia. easily transmissible viruses such as influenza, hmpv, and rsv were the most common, raising concerns about infection control. routine testing for respiratory viruses may be warranted for adults who have been hospitalized with pneumonia. community-acquired pneumonia (cap) is one of the most clinically important diseases in adults, affecting 5 to 20 per 1,000 adults per year. 1 of these, at least 20 to 40% will require hospitalization for the treatment of their pneumonia.f cap management guidelines 3 have been influenced by older cap etiology studies," which helped to direct empiric therapeutic antimicrobial choices for therapy against bacterial pathogens such as streptococcus pneumoniae, haemophilus influenzae, and "atypical" bacwww.chestjoumal.org teria, including chlamydophila pneumoniae, mycoplasma pneumoniae, and legionella pneumophila. although cap guidelines 3 acknowledge respiratory viruses as a "cause" of pneumonia, few recommendations are made regarding management, largely due to the paucity of data regarding prevalence, clinical presentation, and outcomes. furthermore, viral etiology studies in pneumonia are difficult to interpret as noninvasive viral detection methods are often considered to be only markers of infection rather than the cause of pneumonia." clearly, much better knowledge of the potential role of respiratory viruses present in patients with pneumonia is needed. most published studies's? of respiratory viruses have relied on tests with relatively poor sensitivity such as serology and direct fluorescent antigen (dfa) tests. such tests are limited in the sample type to which they can be applied and are not suitable for a broad range of respiratory viruses. more recently, the introduction of highly sensitive nucleic acid amplification tests (nats) has dramatically improved our ability to detect multiple viral pathogens such as influenza, respiratory syncytial virus (rsv), rhinovirus, parainfluenza, and adenovirus. such tests can be undertaken using a small single sample of respiratory secretions with results available with rapid turnaround times. 7 -12 in addition, these tests have allowed us to detect emerging respiratory viruses such as human metapneumovirus (hmpv) and coronaviruses, viruses that are difficult to grow in cell culture. [13] [14] [15] to date, there have been few studies, 5, 7, [9] [10] [11] 16 ,17 reported in patients with pneumonia using nats to detect viral infection, and these studies have either not included clinical data 7.9.11 or have not tested for all potentially important respiratory viruses in a comprehensive manner.lv-!? better knowledge of the role of infection with respiratory viruses in adults with pneumonia may lead to better management. thus, we performed a prospective study in consecutive adults who had "from the department of medicine (drs, johnstone, majumdar, and marne), faculty of medicine and dentistry, university of alberta, edmonton, ab, canada; and the department of microbiology (dr. fox), provincial laboratory for public health, calgary, ab, canada. dr. majumdar was supported by the alberta heritage foundation for medical research (health scholar) and the canadian institutes of health research (new investigator). this project was funded in part by an establishment grant (to dr. marrie) from the alberta heritage foundation for medical research. funding sources had no role in study design, data collection, data analysis or interpretation, or writing of the report. all authors participated in the study conception, design, analysis, interpretation of results, and revision of the manuscript, and approved the final version of the manuscript. dr. johnstone drafted the initial manuscript. dr. marrie acquired the data, obtained funding for the study, and will act as guarantor, the authors have reported to the accp that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article, been admitted to the hospital with cap, and sought to describe their pathogens, clinical presentation, and outcomes. from january 2004 to january 2006, consecutive adults (~18 years of age) who had been admitted to five hospitals in edmonton, ab, canada, with cap were enrolled in a prospective study of pneumonia, patients were excluded from the study if they had received antibioties or been hospitalized within the prior 2 weeks, were unable or unwilling to provide informed consent, or had the following conditions: immunocompromised (ie, had received > 10 mg of prednisone per day for > 1 month, other immunosuppressives, had cancer with reeent chemotherapy, or had hiv with a cd4 count of < 250 cells/u.l), tuberculosis; bronchiectasis, cystic fibrosis; or pregnancy. all patients gave written informed consent, and the health research ethics board of the university of alberta approved the study. we did not record data on patients who were unable to provide consent or who did not meet the enrollment eriteria. pneumonia was defined as an acute lower respiratory tract illness with two or more of the following symptoms or signs: cough; productive cough; fever; chills; dyspnea; pleuritic chest pain; crackles, and bronchial breathing plus an opacity or inflltrate seen on a chest radiograph that was interpreted as pneumonia by the treating physician. to characterize the severity of the pneumonia itself, we calculated the pneumonia severity index (psi) using the methods of fine et al. is clinical, radiographlc, and laboratory data and short-term outcomes were collected by a trained research nurse; the nurse was masked to microbiology results at the time of data collection. patients were followed up throughout their hospital stay until discharge, routine blood culture, sputum specimens, nasopharyngeal (np) swabs, and serum samples were processed for each patient according to the study protocol. np swabs that were submitted for the detection of viral pathogens first underwent d fa testing for influenza a and b, rsv, and parainfluenza virus 1-3 (imagen; dakocytomation ltd; ely, uk), in addition, expanded testing of np samples was undertaken for a range of respiratory pathogens by nats using extraction and amplification methods that have been described previously'! i briefly. nats were designed to amplify and detect influenza a and b, hmpv, rsv, rhinovirus, parainfluenza 1-4, coronaviruses (oc43, 229e, and nie3), and adenoviruses. all the nats utilized in this study have been published, and the assay parameters evaluated. 1 1.i 2. 19 .2o laboratory validation of these assays confirmed a limit of detection of :s 100 copies (cloned target or synthetic rna) or one or fewer tissue culture infectious dose of 50% (for culturable viruses). the specificity of all assays was confirmed using samples and spiked materials 'containing high loads of alternative respiratory pathogens. (further details on viral nats are available from j.d.f. on request [also see references 11, 12, 19, and 20] .) bacterial infections were identified using standard laboratory protocols, acute and convalescent serum samples were collected on the day of hospital admission and were repeated 4 to 6 weeks later, serum samples were tested for the presence of c pneu-numiae and chlamydia psittaci igm and igg by a mieroimmunofluorescence assay," m pneumoniae igm enzyme immunoassay (platelia, biorad; hercules, ca), 22 coxiellabumetii phase i and phase ii titers by indirect immunofluorescence.w and l pneumophila titers by indirect immunofluorescence.s-m pneunumiae and l pneumophila were also tested using nats, and were validated as above but with a limit of detection of :s; 100 copies (cloned target or synthetic rna) or :s; 1 cfu. 11 a diabtj10sis of respiratory viral infection was made if a virus was detected by nat or dfa and a coexisting bacterial pathogen was not identified. a diagnosis of bacterial infection was made if a viral pathogen was not detected, and the following criteria were met: (1) isolation of a respiratory pathogen from purulent sputum (defined as an adequate quality sputum sample with > 25 leukocytes and < 10 epithelial cells per x 100 magnification field) or blood culture 1,2,,; (2) a fourfold rise in igg titers for c pneumoniae (> 1:32) and c psittaci (> 1:32)1; (3) a single increased igm titer for m pneutnoniae (> 1:64) or c pneunwniae (> 1:16)1; (4) an antibody titer of> 1:1,024to l pneumophila in a serum specimen obtained during either the acute or convalescent phase', (5) a fourfold rise in antibody titer to> 1:128 or a fourfold rise in antibodies to c bumetii', (6) a single titer of > 1:128 to a phase ii c bumetii antigen'. or (7) the detection of m pneutnoniae or l pneumophila by nat.1i a mixed infection was defined as the presence of both respiratory virus and bacteria, as defined above, last, if no pathogens were detected, based on the tests used in the study protocol, we classified this as "unknown." patient characteristics and outcomes according to pathogen were compared using x 2 test, fisher exact test, student t test, or mann-whitney u test, as appropriate. although we present data for .ji pathogen categories, our primary analyses compare viral infection to bacterial infection. the few viral cases (n = 29) in our sample precluded attempts at multivariable analyses. all data were analyzed using a statistical software package (spss, version 15.0; spss inc; chicago, il). three hundred patients were enrolled into the study, and 193 patients (64%) had evaluable np swabs. the reasons for nonevaluable np specimens included insufficient sample (n = 68) and missed collection (n = 39). because the primary purpose of the study was to evaluate for the presence of viral pathogens, we excluded those patients without np swabs. there were essentially no differences between those with evaluable np swabs and those without for either clinical characteristics or outcomes, with the following exceptions: impaired functional status (35% vs 21%, respectively; p = 0.02); lobar pneumonia seen on a chest radiograph (72% vs .57%, respectively; p = 0.009); and median length of stay (7 vs 6 days, respectively; p = 0.02). we considered the 193 patients with evaluable np swabs to be our final study sample. sputum and blood cultures were requested for all patients, but these were not performed in some patients due to their inability to produce a sputum specimen (n = 106), their refusal of a blood draw (n = 61), or death (n = 5). convalescent serum samples were not obtained in 153 patients because they did not return for follow-up blood work (n = 146) or died (n = 7). overall, the median age of patients was 71 years (interquartile range [iqr], 58 to 80 years), 51% were male, and 47% had severe cap (psi class 4 or 5). in total, 75 patients (39%) had a respiratory pathogen identified, whereas pathogens were classified as "unknown" in 118 patients (61%). of those patients with a pathogen identified, 29 (39%) had a viral infection, 38 (51%) had a bacterial infection, and 8 (11%) had a mixed viral and bacterial infection. of the 29 patients with a viral infection, the most common organisms were influenza a (n = 3), influenza b (n = 4), hmpv (n = 7), and rsv (n = 5). other organisms included coronavirus (n = 4), rhinovirus (n = 4), parainfluenza (n = 3), and adenovirus (n = 2). three patients had two viruses detected. of 29 patients with viral infections, 18 (62%) had influenza, hmpv, or rsv as a cause (table 1) . of the 38 patients with bacterial infection, 14 infections (37%) were caused by s pneumoniae and 9 infections (24%) were caused by common atypical pathogens (table 1) . patients with viral infections were older than those without viral infections (median age, 76 vs 64 years, respectively; p = 0.01), were more likely to have underlying cardiac disease (66% vs 32%, respectively; p = 0.006), and tended to be more frail (eg, 48% had severely limited ambulation vs 21% of those with bacterial pneumonia; p = 0.02). other differences included the presence of chest pain, which was far less common in those patients with a viral infection than in those with a bacterial infection (7% vs 37%, respectively; p = 0.004) [ table 2 ]. in terms of laboratory findings, those with viral, infections were far more likely to have a normal leukocyte count than those without viral infection (74% vs 14% leukocytes, respectively; p < 0.001). all cases of viral infection occurred between the months of october and may, with one exception (one episode of rhinovirus infection occurred in july), whereas bacterial infections occurred year round (fig 1) . although the importance of s pneurrwniae and atypical bacterial pathogens is well understood in patients with cap, in this prospective cohort study we have now demonstrated the significant potential contribution of respiratory viruses in patients presenting with pneumonia. indeed, fully one sixth of all cases (15%) in this cohort of adults who were hospitalized with pneumonia had a respiratory virus identified; alternatively, more than one third of those there were no significant differences in outcomes according to the pathogens identified. specifically, there were no differences in median length of hospital stay (patients with viral infection, 7 days [iqr, 6 to 10 days]; patients with bacterial infection, patients (39%) with a pathogen identified had a respiratory viral infection. influenza, hmpv, and rsv comprised almost two thirds of all cases of viral infection and, in our study, were acquired during the influenza season. there were some differences in presentation between those patients with a viral infection and those without, including the following: older age; presence of cardiac disease (but in the near absence of chest pain on presentation); and greater frailty. of note, patients with a viral infection were far more likely than patients with bacterial pneumonia to have a normal leukocyte count. despite these apparent differences, given that the majority of our cohort never had a respiratory pathogen identified, it is obviously very difficult to distinguish the presence or absence of viral infection in patients with pneumonia. this is further borne out by the fact that outcomes were virtually identical irrespective of the pathogens involved. adult cap etiology studies-" conducted prior to the use of nats estimated viral involvement in 0.3 to 30% of all cap cases. testing was generally based on serologic conversion or positive dfa test results for influenza a or b; rsv; parainfluenza virus 1,2, and 3; or adenovirus. in our cohort, viral infection without evidence of bacterial coinfection was detected 15% of the time, which falls within the commonly reported range. 26 a study by marcos et al.!? which used nats, reported a similar prevalence of viral infection in spain. however, the study by marcos et al lo differed from ours in several noteworthy ways, as follows: they did not test for hmpv; and they included immunocompromised patients in their study. our results also differ from those of jennings et al,5 as follows: they documented a viral infection 29% of the time in their cohort of adults with cap, but, surprisingly, more than a third of infections were attributed to rhinovirus, and almost one fifth were mixed infections. the impact of rhinoviruses in our study may be underestimated as data 27 have indicated that the picornavirus family of viruses is much more variable than originally thought. it is extremely difficult to design and validate assays to pick up all divergent rhinoviruses, and the original assay design that we utilized in this study would not identify all those that have been reported.f this is an inherent limitation in the type of study undertaken; as we identify more novel respiratory pathogens and variants, it is inevitable that some will have been missed. most older etiology studies-" have reported influenza infection in patients with pneumonia 4 to 19% of the time, followed by rsv. influenza was the most common virus identified in our study, affecting 4% of patients; however, we found hmpv to occur as commonly as influenza, and more frequently than rsv. this important finding has not been widely documented as most respiratory virus studies 7 ,10,29,3o have not included testing for hmpv, largely due to the difficulty in its identification in the past. to our knowledge, only two previous etiology studiess-!? used nats for detecting hmpv. one study.!? which was restricted to copd patients with pneumonia, found hmpv as a pathogen in 4.1% of cases; another study" from new zealand found no cases of hmpv. the strengths of this study include its prospective nature and the thorough collection of data from a cohort of consecutive patients who had been admitted to the hospital with cap. there are also several limitations to the study. first and foremost, despite our best efforts and a detailed study protocol, a number of bacterial investigations iie, blood culture, sputum culture, and convalescent serum specimens) www.chestjournal.org were missed, thereby potentially underestimating the number of cases of bacterial pneumonia and (potentially) underestimating the number of mixed infections. the number of missed bacterial investigations may be the reason for our 61% rate of unknown infections, although our rate of recovery is similar to other studies 22 ,3 1--34 that have reported 47 to 60% unknown infections, second, we excluded patients without evaluable np swabs from our analyses. not obtaining specimens for conducting a nat was a study protocol violation in 13% of patients (39 of 300 patients). we speculate that either np swabs were not collected when patients transitioned from the emergency department to the wards, or that there was a miscommunication with the reference laboratory regarding when or where to send the study-related swabs, that said, there were few important clinical differences between patients with and without evaluable np swabs, with two excep-chest/134/6/ december, 2008 tions. those patients without evaluable np swabs were more likely to have lobar pneumonia, which, according to our data, would bias the results toward bacterial infection. those patients without evaluable np swabs were also more likely to be functionally impaired, which would bias the results toward viral infection. third, there is potential for both falsepositive and false-negative np results, although testing with nats has been reported to have excellent sensitivity and speciflcity.p as noted above, sequence divergence for the rhinoviruses (and, potentially for the other virus groups) may also have led to some underestimation of the number of viral infections. fourth, we detected viruses in the upper respiratory tract using np specimens, which does not necessarily equate with the causation of pneumonia. however, the purpose of this study was to describe the potential role of respiratory viral infection in those patients with pneumonia; a study describing "confirmed" viral pneumonia would require lung tissue samples from all enrolled patients. last, our overall sample size might be considered small by some, and our cohort was drawn from only one health region in canada, which might limit the generalizability of the results to some degree. in our study, patients with pneumonia and respiratory viral infection were older and more frail than 1146 those without evidence of viral infection. differentiating between patients with viral infection and those without based on clinical findings and routine laboratory test results remains a challenge. indeed, although we were unable to perform a multivariable logistic regression analysis due to the small sample size, it seems unlikely that any constellation of symptoms, signs, and routine laboratory findings will ever reliably differentiate between the presence or absence of a virus. 3 , 1o,29 ,30 current guidelines:] recommend empiric antibiotic therapy targeted against common bacterial pathogens for patients who are admitted to the hospital with pneumonia. how to manage patients with pneumonia and a respiratory viral infection, without a documented coexisting bacterial pathogen, is far less clear. future research, similar to that found in the pediatrics literature, is needed to help answer whether empiric therapv with antibiotics can be discontinued in this clinic~1 seenario.f' perhaps most importantly, the inability to identify patients with a respiratory virus without comprehensive respiratory viral testing is a concern from the perspective of infection control. the presence of a respiratory viral infection can result in nosocomial outbreaks. for instance, outbreaks due to influenza have been well documented.s" and cases of rsv and hmpv nosocomial transmission are increasingly recognized. 21i , 36.37 the nosocomial spread of respiratory viruses among adults poses the biggest threat to immunocompromised patients, including frail elderly patients: 3 ,31i-40 the current infection control guidelines recommend placing patients with a suspected respiratory viral infection in private rooms or cohorting them with patients with the same viral infection as a way to prevent transmission." given the 15% prevalence of viral infection in adults in our study, and the indistinguishable presentation from typical bacterial pneumonia, our results suggest routine isolation (with droplet and contact precautions) of all adults with pneumonia, from the time of hospital admission until respiratory viral infection is ruled out, should be considered to help prevent the nosocomial transmission of respiratory viruses. this suggested approach should become logistically feasible when the turnaround time for nat results is < 24 h and as the price of testing with nats decreases over time. this will be facilitated by emerging commercial viral identification assays that are both accurate and relatively inexpensive.p conclusion infections with respiratory viruses are common in patients who are hospitalized with pneumonia, com-prising 39% of all identified pathogens and 15%of all patients in our study. influenza, hmpv, and rsv were the most common respiratory viruses identified. in patients presenting with pneumonia, it remains difficult to differentiate patients with viral infection from those without viral infection. our findings suggest that routine testing for common respiratory viruses may be warranted for all adults hospitalized with pneumonia. community-acquired pneumonia: clinical features and outcomes bts guidelines for the management of community acquired pneumonia in adults infectious diseases society of america!american thoracic society consensus guidelines on the management of communityacquired pneumonia in adults microbiological profile of community-acquired pneumonia in adults over the last 20 years incidence and characteristics of viral community acquired pneumonia in adults lack of sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults improved diagnosis of the etiology of community acquired pneumonia with real-time polymerase chain reaction polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and b viruses, respiratory syncytial virus and parainfluenza viruses 1, 2, 3 and 4 the role of viruses in tbe aetiology of community acquired pneumonia in adults enhanced identification of viral and atypical bacterial pathogens in lower respiratory tract samples with nucleic acid amplification tests nucleic acid amplification tests for detection of respiratory viruses newly identified respiratory viruses a newly discovered human pneumovirus isolated from young children with respiratory tract disease a previously undescribed coronavirus associated with respiratory disease in humans comparison of multiplex reverse transcription pcr enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens human metapneumovirus infection in adults with community acquired pneumonia and exacerbation of chronic obstructive pulmonary disease a prediction rule to identify low-risk patients with community-acquired pneumonia diagnosis and epidemiological studies of human metapneumovirus using real-time pcr detection of a broad range of human adenoviruses in respiratory tract samples with a sensitive multiplex real-time pcr assay the microimmunofluorescence test for chlamydia pneumoniae infection: technique and interpretation evaluation of four commercial immunoglobulin g and immunoglobulin m specific enzyme immunoassays for diagnosis of mycoplasnw pneumoniae infections detection and persistence of specific igm antibody to coxiella bumetii by enzymelinked immunosorbent assay: a comparison with immunofluorescence and complement fixation tests reactivity of serum from patients with suspected legionellosis against 29 antigens of legionellaceae and legionella-like organisms by indirect immunofluorescence assay patients admitted to hospital with suspeeted pneumonia and normal chest radiographs: epidemiology, microbiology, and outcomes community acquired viral pneumonia masstag polymerasechain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during viral pneumonia in older adults etiology of communityacquired pneumonia in hospitalized patients in chile viral communityacquired pneumonia in nonimmunocompromised adults viral lower respiratory traet infection in the elderly: a prospective inhospital study pneumonia: etiology. epidemiology, and outcomes at a teaching hospital in argentina etiology of severe pneumonia in the very elderly microbial etiology of acute pneumonia in hospitalized patients impact of the rapid diagnosis of influenza on physician decision-making and patient management in the pediatric emergency department: results of a randomized, prospective, controlled trial an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility a summer outbreak of human metapneumovirus infection in a long-term care facility viral infections in immunocompromised patients: what's new with respiratory viruses? a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections brief communication: fatal human metapneumovirus infection in stem-cell transplant recipients guidelines for preventing health-care-associated pneumonia nucleic acid amplification tests for detection and analysis of respiratory viruses: the future for diagnostics? acknowledgment: thanks to carol mangan, our research nurse, for her invaluable contribution, and to the provincial laboratory for their comprehensive laboratory help. thanks also go to sipi garg and meagan rosenthal for help with preparing the database for analyses. key: cord-253256-909chgl0 authors: bajwa, sukhminder jit singh; sarna, rashi; bawa, chashamjot; mehdiratta, lalit title: peri-operative and critical care concerns in coronavirus pandemic date: 2020-03-28 journal: indian j anaesth doi: 10.4103/ija.ija_272_20 sha: doc_id: 253256 cord_uid: 909chgl0 world health organization (who) declared novel coronavirus outbreak a “pandemic” on march 11(th), 2020. india has already reached stage 2 (local transmission) and the indian government, in collaboration with the indian council of medical research (icmr), is taking all necessary steps to halt the community transmission(stage 3). anaesthesiologists and intensivists around the globe are making untiring efforts akin to soldiers at the final frontier during war. all efforts pertaining to adequate staffing, personal protective equipment (ppe) and strict adherence to hand hygiene measures are being stressed upon to prevent in-hospital transmission. in this article, all outbreak response measures including triaging, preparation of isolation rooms, decontamination and disinfection protocols as well as fundamental principles of critical care and anaesthetic management in covid-19 cases is being discussed. all the recommendations have been derived from the past experiences of sars (severe acute respiratory syndrome) and mers (middle east respiratory syndrome) outbreak as well as upcoming guidelines from the international health fraternity and indian health services. corona virus has created havoc amongst people of all nationalities with disturbingly rising count of affected people worldwide. a large database study of 72,314 affected chinese nationals has given a preliminary case-fatality rate of 2.3% with 81% experiencing mild flu symptoms, while rest of the 14% having severe symptoms and 5% being critically ill; death rate increasing exponentially with severity. [1] although the fatality has till now been reported lower than previous outbreaks of ebola, sarsandmersit is expected to rise if ipc (infection prevention and control) policies are not laid down and followed religiously. an estimated maximum likelihood (ml) of reproductive number of covid-19 infection was calculated to be 2.28 with a serial interval of 7 days which can only be curtailed by strict quarantine measures. [2] as anaesthesiologists and intensivists, our prime involvement in resuscitation, airway management, peri-surgical as well as critical care of such cases cannot be undermined. the need of the hour therefore is to update ourselves with clinicopathological spectrum of the deadly disease and provide evidence based medical services to all hospitalized cases with suspected covid-19 infection. all the recommendations below have been influenced by the past experiences of corona virus outbreaks and desire improved measures for personal protection of health care workers and consequent prevention of nosocomial transmission of infection. a systematic literature search was made using search engines including pubmed, google and google scholar with the use of the following single-text words and combinations: anaesthesia, corona virus, covid-19, intensivists from the year 2000 to 2020. the references of relevant articles were cross-checked and the articles containing all these keywords were thoroughly checked for imbibing the review. the data was also checked with national and international databases from internet and various newspapers. indian health authorities have submitted a total record of 283 lab-confirmed cases-2449 indians, 39 foreign nationalsand22 cured and discharged post-quarantine till the time of writing this article i.e., 21 th march, 2020. [3] the first three cases in india were students returning from china. they were successfully isolated in kerala and declared negative.till date,4 deaths have been reported from our country as a result of this deadly virus with old age (more than 60 years), hypertension and male gender being stated as independent risk factors. [4] the real number of patients carrying the virus is very difficult to establish as many people are avoiding the tests and many are not disclosing at all. the real danger to us as intensivists and anaesthesiologists is from these patients who have not been tested despite being potentially positive for harboring the virus. in this scenario, it is prudent to follow the national and international guidelines and treat any suspected patient as positive and to take necessary precautions during anaesthesia and critical care of such patients. screeningfor suspect cases n-cov is a positive sense, single-stranded rna corona virus belonging to sub-group sarbecovirus which closely resembles two bat-derived sars-like corona viruses but distant from sars-cov and mers-cov. preliminary phylogenetic analysis suggests bats as the original host of this virus with human to human transmission confirmed via droplet, contact and fomites and average incubation period of 2-10 days. [5] since a vast majority of indian institutions have anaesthesiologists as in charge of the emergency triaging and intensive care units, it becomes essential that our fellow colleagues should be aware of every minute details of this deadly pandemic. the following terminology has been standardized for isolation of a suspect case [ table 1 ]. although wuhan is considered the epicenter of outbreak, the increased number of cases being reported worldwide hasabolished the diagnostic link with ban on all tourist visas to india with effect from 13 th march 2020. for tracing contacts and categorizing risk of procuring infection, refer to table 2 . while the high and medium-risk contacts are advised immediate isolation with controlled local and air travel in medical transport vehicles with laboratory evaluation guided by symptomatology, low risk contacts are advised to avoid prolonged public exposure and if symptomatic get lab tests done when deemed appropriate. however, as anaesthesiologists and intensivists, the biggest challenge we will face in coming times is from the asymptomatic patients oncestage3 of the disease is crossed. it becomes imperative that during all these times, we should see only emergency and critical patients with an emphasis on personal safety as well as taking all the universal precautions against corona virus. for any emergency surgery in such patients, planning of anaesthesia becomes very crucial while taking all the necessary precautions. planning for anaesthesiaand surgery in covid-suspect cases anaesthesiologists and intensivists are the soldiers at the final frontier of corona disaster as all patients landing up in the hospital with severe respiratory distress will be primarily seen under their domain. while applying all precautionary measures pertaining to health care workers treating covid-infected individuals, necessary critical illness support needs to be provided using the current knowledge about the pathophysiology and management of covid infection. some patients with mild-moderate symptoms can also be encountered by the anaesthesiologists while being posted for surgeries. patients with mild symptoms resembling the symptomatology of other similar flu like illnesses are the real threat as chances of precautionary lapses are high in such cases. further,accidental chances of getting infection becomes highest when the positive patients without any symptomatology are being treated like normal patients. elective surgeries in these patients can be postponed till the resolution of symptoms while an emergency surgery undertaken with due risk consent as this is entirely a new clinical scenario for anaesthesia and surgery. indian journal of anaesthesia | volume 64 | issue 4 | april 2020 all suspect patients posted for surgery must be kept in isolation room till their shifting to or (operating room). for the operative procedure, negative pressure isolation rooms with minimum 12 air exchanges per hour. [7] temporary conversion of otherwise positive pressure ors into negative pressure with independent provision for air conditioning and humidification should be preferred. the negative pressure can be created by reducing the amount of air volume entering the inlet duct, while maintaining the air volume exiting through the other duct. two adjacent ors with one being utilized for the procedure and other as anteroom for the staff and doctors to wear and dispose of all ppes is ideal. the biggest challenge comes from the diversity of operation theater infrastructure set ups in our country which makes it difficultfor the implementation of universal guidelines meant for such outbreaks. therefore, such surgeries should be undertaken only in select centers with all the facilities. the policies for such clinical interventions have to be formulated by the appropriate health authorities at central governmentlevelwhich can be implemented uniformly across the nation. during such emergency surgeries, the following protocols should be followed religiously. this must be done in the isolation itself where the patient is kept. various personal protective equipment have been described for preventing aerosol and droplet transmission from patient to the anaesthesiologist. this includes-gown and shoe cover long length, full sleeved gown and shoe covers which are water resistant provide protection from any spillage of oral, nasal secretions or vomitus. it is essential for all suspect patients to wear surgical masks to prevent any droplet transmission while coughing or yawning. a plastic face shield to cover front as well as side of face, n-95 masks or pcm 2000 or powered air purifying respirators for mouth and nose, eye shields and head caps for peri-orbital mucous membranes and hair respectively must be worn to protect any transfer of virus emitted in patient's secretions to anaesthesiologist's body. all these protective equipment must be checked for proper-fit to prevent any uncovered zone. double-gloving is advised to all health care workers providing care to such patients to prevent any cross-contamination. once any procedure is done, the outer glove should be used to remove gown, shoe cover and face shield while the inner glove removed as soon as possible afterwards and disposed of with all other equipment in double-zip lock plastic container. any evidence of soiling of the glove also reprimands its removal and replacement with fresh pair of gloves. hand washing all the 9 steps of hand-washingwith alcohol based hand rubs must be adhered to strictly before wearing gloves for any procedure, before and after touching any contaminated area, anaesthesia trolley, airway cart or other instruments, and after removing gloves. protocol of keeping alcohol based hand wash at the entrance of every ward and icu helps in better compliance of health care workers.all ppes after exposure should be locked in a double zip lock plastic bag and discarded in a touch-free disposal. [8] after taking due risk for the procedure, along with nil-per-oral instructions of 2 hours for clear fluids, it is important to counsel the patient's relatives regarding need for strict isolation as well as their own testing. transportation to operation theatre during transportation, covid-19 infected patients must wear a surgical mask and be transported to the negative pressure or in an evacuated passageway and elevator preferably covered by plastic disposable material to be disposed of thereafter. all the ppes must be worn by the transporting nurse and doctor. these patients are highly anxious especially at seeing the level of protection worn by the team of doctors which gives them an impression of being doomed. adequate premedication in the form of benzodiazepines like midazolam and alprazolam or opioids like fentanylshould be prescribed before shifting to or. all ventilated patients must be intubated and transferred via special bain's circuit connected with the tube via hepa filter. the position of filter must be between the endotracheal tube connector and y-limb to prevent any aerosol transfer from the patient to portable ventilator and vice-versa. [8] hfnc or bi-pap therapy should be avoided due to increased generation of aerosols. [9] the patient must be suctioned by closed suction apparatus before transfer and given intravenous sedation and muscle relaxation to prevent any coughing and movement which might lead to disconnection of tube and dispersion of droplets. [10] adequate training regarding transport of such patients and mock drills should be done for proper implementation. after preparation of all drugs in disposable syringes, arranging for face mask, airway, endotracheal tube, stillete, bougie, laryngoscope blade, circuit, soda lime, etc. all in disposable form, the patient is taken in the or and monitors attached. as all the health care workers have to cover themselves from head to toe, there is decreased heat loss and sweating. this can be mitigated by using a lower temperature setting of 20-22 degree celsius in the operating room. [8] the leads for ecg, nibp, spo2 and etco2 should be single use or covered in sterile plastic cover to be discarded after use. in case of predicted difficult intubation, disposable blade and c-mac video laryngoscope should be kept standby while avoiding decision to conduct awakefibreoptic intubation due to potential of coughing during the procedure. [11] intravenous cannulation must be done with sterile drape and gloves to prevent any spillage. it is recommended to administer anti-emetics or prokinetics in every case as prophylaxis for vomiting. [12] the patient must be preoxygenated with 100% o2 for 3 minutes using the disposable circuit and hepa filter barrier for denitrogenation.it is suggested to use total intravenous anaesthesia in place of inhalational due to lack of isposablevaporizers and cost concern. rsi (rapid sequence induction) is the procedure of choice for intubation to abolish need for mask ventilation and regurgitation. after administering appropriate inductionagent in the form of propofol or thiopentone and muscle relaxant such as succinylcholine or rocuronium in desired doses, intubation is done by the most experienced anaesthesiologist in minimum possible time and confirmed using capnography. in cases of severely decreased po2/fio2 ratio, modified rsi can be done with low pressure ventilation before intubation. [13] it is important to reemphasize the use of "double-glove" technique during intubation. it is suggested to use total intravenous anaesthesia in place of inhalational for maintenance of anesthesia due to lack of disposable vaporizers and cost concern. the maintenance of anaesthesia can be done using disposable 50ml syringes and pmo lines. the depth of anaesthesiashould be maintained at all times to prevent any bucking in between the procedure. the setting of ventilatory parameters should be in accordance with the lung protective strategies with optimal peep and pplat<35 mmhg to achieve an spo2 >90% and admissible hypercapnia. [14] in case devices like point-of-care ultrasound (usg) have been used during the procedure, the machine and the probe along with the wire must be covered with a plastic sheath which is removed and discarded after use. any invasive procedures like epidurals or putting a central venous line should be done under usg guidance to prevent chances of failure. after the surgery, the decision to extubate should only be taken ideally in asymptomatic patients and threshold for keeping the patient intubated kept low in order to avoid any procedure with assumed dispersion of infective material in the environment. [15] in case of decision to extubate, the bucking on the tube should be avoided by "deep plane of extubation" along with anti-aspiration prophylaxis. after the patient is transported back to isolation, or should be kept closed with adequate air exchanges before allowing for subsequent patient care. all the ppes and materials used by the operating team and anaesthesiologists must be removed and sent for disposal. the left-over drugs and plastic sheets must be discarded. the pregnant, elderly and patients with co-morbid conditions have been implicated much more than general healthy population in terms of risk of infection and severity. considerations for all these high-risk populations are similar to those advocated during sars and mers outbreak. the individuals must be vaccinated against influenza and pneumonia causing bacteria to prevent secondary lrtis. [15] the patients with cardiovascular disorders are advised guideline-directed statins and diuretics to prevent potential risk of acute coronary syndrome and heart failure. other co-morbidities must be managed with respective treatment guidelines aggressively to improve outcome. in pregnant females, there is an additional risk of premature rupture of membranes, preterm birth, low birth weight and fetal distress in case of confirmed infection especially in the third trimester. as a precautionary measure, pregnant females should refrain from unnecessary travel and public transport together with practicing good personal hygiene. hence, any confirmed case coming in pregnant state must be screened for fetal well-being and advised betamethasone therapy for fetal lung maturity if anticipating pre-term delivery. the timing of delivery is entirely dependent on the condition of the mother and the fetus, wherein stable cases can be delivered at term vaginally, while critically ill mothers delivered earliest possible. as no shedding of virus has been found in the amniotic fluid, vaginal delivery can be performed but for early cord clamping and maternal separation for 2 weeks postnatally to prevent direct contact transmission. [16] till date, no guidelines have been issued regarding the anesthetic management of infected pregnant patients for caesarean section. however,spinal anaesthesia have been reported to be safe in one patient undergoing emergency c-section in wuhan. [17] there is no advisory issued against breastfeeding, but direct suckling from mother's breast is not advocated. rather expression of mother's milk and palade feeding is suitable. after prompt diagnosis based on aforementioned criterion, case must be reported by the provisional public health authorities to the national body within 24 hours in their own jurisdiction and transferred to negative pressure isolation cabin in the icu. as most of the icus are not equipped with negative and positive pressure regulations in india, an alternative approach is using hepa-carbon-photo-catalysis air purification systems as alternate means of source control. [14] the financial considerations for current icu models running in india to meet international standards in terms of quality assurance and workforce is humongous which can only be met by public-private partnership in health sector and generous gdp devoted to health. the clinical spectrum of covid-19 suspected patients can range from mild flu like symptoms to those mimicking community acquired pneumonia. in very severe cases, patients can present as ards (acute respiratory distress syndrome) with refractory hypoxemia requiring mechanical ventilation. the history taking, sampling for detection of all viral, bacterial pathogens involved in upper respiratory tract infection in the form of oropharyngeal or nasopharyngeal swabs and lower respiratory tract by sputum, endotracheal aspirate, or broncho-alveolar lavage must be sent for rt-pcr or nucleic acid detection. [14] throat swab to be collected for all virological examination should be stored in three-shelled receptacle for avoiding any spillage. more than one specimen is found to yield better results. viral rna has also been found in stools, urine and serum of infected individuals, hence can be used as alternate specimens. [14] the nasopharyngeal specimen and suction tip specimen must be sent for other bacteriological examination to rule out any secondary infection. decreased lymphocyte count, cd4 and cd8 cells along with increased crp, esr, il-6 and normal pct have been most consistent findings with this viral etiology. [18] abg analysis, liver, cardiac and kidney functions must be monitored for overall assessment of severity of infection. the radiological findings reported in these cases range from diffuse segmental or sub-segmental ground-glass opacities signifying interstitial oedema seemingly like "paving-stone" with extensive exudation into alveolar cavities leading to patchy areas of consolidation. the recent reports confirm more sensitivity of ct-scan than rt-pcr (98% versus 71%) for diagnosis of covid infection. [19] supportive therapy in the form of supplemental oxygen and antipyretics should be immediately started in patients with sari. liberal fluid administration should be avoided for risk of worsening oxygenation and periodic hemodynamic assessment used to guide goal-directed therapy.along with it, adequate nutritional support with balanced proportions of proteins, carbohydrates, vitamins and minerals boosts immunity to fight the infection. empirical antimicrobials must be given within one hour based on the clinical diagnosis, local epidemiology and susceptibility data to cover all likely pathogens causing community acquired pneumonia even if suspected to have covid. [20] a typical coverage would involve injection ceftriaxone 2gm i/v bd, azithromycin/levofloxacin 500mg od and doxycycline 100mg bd.de-escalation must be practiced after microbiological evidence.neuraminidase inhibitor like oseltemavir can be initiated for influenza when there is local circulation or other risk factors. systemic corticosteroids have controversial role in reducing mortality with viral pneumonia or ards. however, in rapidly progressing severe diseases, they provide symptomatic relief and faster resolution of pulmonary lesions, so advocated by many. [21] [22] [23] a landscape of therapeutic agents are being questioned and tested for use against 2019-cov with priority research on remdesivir and lopinavir/ritonavir + interferon beta. dgca body in india has put a nod on "restricted use" of chloroquine phosphate 500mg bd, lopinavir/ritonavir (400 mg/100 mg) for treating the virus. [24, 25] the samples must be repeated at least every 2-4 days for viral clearance until there are two consecutive negative results 24 hours apart in a clinically recovered patient. recognition of patients in respiratory distress not responsive to oxygen therapy must be swift and anaesthesiologiststrained in endotracheal intubation and all necessary equipment must be kept standby if need arises. post intubation by rsi, lung protective strategies involving use of lower tidal volumes (4-8 ml/kg predicted body weight), high peep and lower inspiratory pressures (plateau pressure <30 cmh 2 o) for meeting the ph goal of 7.30-7.45 have been postulated to prevent volutrauma, barotraumas, atelectrauma and biotrauma. [26, 27] deep sedation in the form of midazolam, propofol or fentanyl infusions are recommended to curb patient's respiratory drive and prevent dyssynchrony. the few indications of continuous neuromuscular blockade in the setting of severe ards has been agreed upon like ventilator dyssynchrony, inability to achieve target tidal volumes or refractory hypoxemia/hypercapnia. [28] in fulminating cases, prone ventilation for 12-18 hours per day and use of ino(inhaled nitric oxide) for bronchodilatation is recommended, failing which ecmo (extra corporeal membrane oxygenation) is the only modality for survival. it should only be offered in expert centres with a sufficient case volume to maintain expertise and that can apply the ipc measures required for covid patients. along with mechanical ventilation, periodic hemodynamic assessment by static procedures like passive leg raising and dynamic procedures like serial stroke volume measurements, variations in pulse pressure, inferior vena cava size, or stroke volume changes with intrathoracic pressure during mechanical ventilation must be done for guiding fluid therapy and achieving optimal response. in resuscitation of adults with septic shock, 30 ml/kg of isotonic crystalloid like ringer lactate must be administered in the first 3 hours for volume repletion. [29] if map target is not fulfilled on fluid loading and signs of volume overload appear, discontinuation of fluid administration is advised. vasopressors should be then initiated, norepinephrine being the first-line in adult patients. epinephrine or vasopressin can be added if first line drug fails. all these therapies must be instituted timely to prevent multi-organ dysfunction which carries grave prognosis. [30, 31] a term called "protected code blue "had come into existence during sars outbreak which emphasize on the use of paprs rather than n-95 masks and specialized ppes during resuscitation owing to the dynamic nature of the procedure with high risk of airborne transmission. the need for resuscitation should always be anticipated to allow for preparation of isolation room, disposable resuscitation packages instead of trolley and formation of four members team with designated roles. all the team members should wear ppes checked by infection control coach and then enter the isolation bringing the defibrillator and packages along with. [31] along with standard code blue protocol, each action should be considered in the lines of risk of aerosol transmission versus benefit to the patient. [32] future research and perspective beyond all these general measures, research is underway in different phases of completion would soon elaborate on candidate vaccines and therapeutic agents like hydroxychloroquine against covid. presently numerous false alarms are being raised from one city to another and from one country to another but nothing concrete or any definite vaccine has been developed till date. the laws of nature will definitely take their own course but till then human beings will be the worst sufferers. this is neither the first challenge nor the final one which humanshave to face when the natural ecological milieu is disturbed. at present best prophylaxis is conveyed by one sentence being used globally "stay home, stay safe" apart from taking other recommended precautions. financial support and sponsorship nil. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention serial interval of novel coronavirus (covid-19) infections all information related to novel coronavirus host susceptibility to severe covid-19 and establishment of a host risk score: findings of 487 cases outside wuhan transmission dynamics and evolutionary history of 2019-ncov coronavirus disease 2019 (covid-19) situation report -55 air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient perioperative care provider's considerations in managing patients with the covid-19 infections cross-contamination via anesthesia equipment? practical recommendations for critical care and anesthesiology teams caring for novel coronavirus (2019-ncov) patients outbreak of a new coronavirus: what anaesthetists should know staff safety during emergency airway management for covid-19 in hong kong 2020 features, evaluation and treatment coronavirus (covid-19) for the zhongnan hospital of wuhan university novel coronavirus management and research team, evidence-based medicine chapter of china international exchange and promotive association for medical and health care (cpam). a rapid advice guideline for the diagnosis and treatment of 2019-novel coronavirus (2019-ncov) infected pneumonia (standard version) acc clinical bulletin cardiac implications of novel wuhan coronavirus (covid-19) american college of cardiology (acc) novel corona virus disease (covid-19) in pregnancy: what clinical recommendations to follow? emergency caesarean delivery in a patient with confirmed coronavirus disease 2019 under spinal anesthesia a family cluster of pneumonia associated with 2019 novel coronavirus indicating person to person transmission: a study of a family cluster correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases care for critically ill patients with covid-19 clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected: interim guidance 2020 use of glucocorticoid in the treatment of severe acute respiratory syndrome cases clinical analysis of 190 cases of outbreak of atypical pneumonia in guangzhou in spring the possibility of using lopinave/litonawe(lpv/r) as treatment of novel coronavirus 2019-ncov pneumonia: a quick systemic review based on earlier coronavirus clinical studies ministry of health & family welfare, directorate general of health services(emr division), government of india. guidelines on clinical management of covid-19 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: adescriptive study formal guidelines: management of acute respiratory distress syndrome preparing for the most critically ill patients with covid-19: the potential role of extracorporeal membrane oxygenation managing sepsis and septic shock. current guidelines and definitions endothelial biomarkers in human sepsis: pathogenesis and prognosis for pathological findings of covid-19 associated with acute respiratory distress syndrome mount sinai hospital critical care unit. sars resources. protected code blue isa family benevolent fund" • isa encourages members to join family benevolent fund of indian society of anaesthesiologists (isa-fbf) to help our colleagues' and our own families when they face the testing moments of their life. • become an isafbf member, not for you, but to help our colleague's families by donating rs.300/-per year /death. • to become an isafbf member kindly visit our website isafbf.com or contact your city branch/state/president/secretary • contact for details & application forms: dr. sugu varghese there are no conflicts of interest. key: cord-292871-vgposxom authors: falsey, ann r.; mccann, robert m.; hall, william j.; criddle, mary m.; formica, maria a.; wycoff, dennis; kolassa, john e. title: the “common cold” in frail older persons: impact of rhinovirus and coronavirus in a senior daycare center date: 2015-04-27 journal: j am geriatr soc doi: 10.1111/j.1532-5415.1997.tb01474.x sha: doc_id: 292871 cord_uid: vgposxom objective: to evaluate the incidence and impact of rhino‐virus and coronavirus infections in older persons attending daycare. design: prospective descriptive study. setting: three senior daycare centers in rochester, new york. patients: frail older persons and staff members of the daycare centers who developed signs or symptoms of an acute respiratory illness measurements: demographic, medical, and physical findings were recorded on subjects at baseline and during respiratory illness. nasopharyngeal specimens for viral culture as well as acute and convalescent sera for coronavirus 229e enzyme immunoassay (eia) were obtained for all illnesses. results: during the 44 months of study, 352 older persons experienced 522 illnesses. thirty‐five (7%) of 522 cultures were positive for rhinovirus and 37 (8%) of 451 serologies were positive for coronavirus 229e infection. the clinical syndromes associated with rhinovirus and coronavirus infection were similar and characterized by nasal congestion, cough, and constitutional symptoms. no patient died or was hospitalized, but approximately 50% had evidence of lower respiratory tract involvement. the average illness lasted 14 days. during the same period, 113 staff developed 338 respiratory illnesses. eight percent were identified as coronavirus and 9% as rhinovirus. cough, sputum production, and constitutional symptoms were significantly more common among older persons. conclusions: rhinovirus and coronavirus 229e are common causes of moderately debilitating acute respiratory illnesses among older persons attending daycare. objective: to evaluate the incidence and impact of rhinovirus and coronavirus infections in older persons attending daycare. design: prospective descriptive study. settlng: three senior daycare centers in rochester, new york. patients: frail older persons and staff members of the daycare centers who developed signs or symptoms of an acute respiratory illness measurements: demographic, medical, and physical findings were recorded on subjects at baseline and during respiratory illness. nasopharyngeal specimens for viral culture as well as acute and convalescent sera for coronavirus 229e enzyme immunoassay (eia) were obtained for all illnesses. results: during the 44 months of study, 352 older persons experienced 522 illnesses. thirty-five (7%) of 522 cultures were positive for rhinovirus and 37 (8%) of 451 serologies were positive for coronavirus 229e infection. the clinical syndromes associated with rhinovirus and coronavirus infection were similar and characterized by nasal congestion, cough, and constitutional symptoms. n o patient died or was hospitalized, but approximately 50% had evidence of lower respiratory tract involvement. the average illness lasted 14 days. during the same period, 113 staff developed 338 respiratory illnesses. eight percent were identified as coronavirus and 9% as rhinovirus. cough, sputum production, and constitutional symptoms were significantly more common among older persons. conclusions: rhinovirus and coronavirus 229e are common causes of moderately debilitating acute respiratory illnesses among older persons attending daycare. j am geriatr soc 45:706-711,1997. r morbidity and mortality in older persons. viruses, such as influenza and respiratory syncytial virus (rsv), have been shown to be the cause of serious disease in this age group. ' less is known about the impact of rhinoviruses and coronaviruses, the causative agents in the majority of "common colds.'" approximately 20 to 40% of upper respiratory tract infections (uris) in adults are caused by rhinoviruses, and 5 to 15% are caused by coronaviruses. more than 100 serotypes of rhinoviruses and two major serotypes of coronavirus (229e and oc43) have been ider~tified.~-~ reinfections with both viruses occur throughout life, in part because of multiple serotypes and incomplete immunity.'-' while these infections lead to significant time lost from work and school, they generally do not result in serious disease in children or young adults."i2 rates of acute respiratory tract infections diminish with advancing age, most likely as a result of less frequent exposure^.'^ however, frail older persons attending adult daycare centers may represent a special population at increased risk of infection and at risk for complications from these common infection^.'^ the purpose of this study was to evaluate prospectively the prevalence and clinical features of rhinovirus and coronavirus 229e infections in frail older persons attending senior daycare. volunteers were recruited from three sites of a senior daycare program in rochester, new york. these facilities allow frail older persons who are nursing home-eligible by new york state medicaid standards to be maintained at home by providing comprehensive medical and social services. all attendees of the daycare program were recruited to take part in the study. daycare participants were scheduled to attend the center, on average, 3.5 days per week, with a minimum of once a week. ill subjects were encouraged to attend the program for evaluation by center physicians. in addition, all staff members with direct contact with older participants were also recruited for the surveillance study. informed consent was obtained from volunteers upon entrance into the daycare program. if volunteers were unable to provide informed consent, consent was obtained from their legal guardians. baseline information, including medical history and demographics, were obtained from daycare participant's medical records. subjects were examined at base-line, and arterial oxygenation saturation (sao,) was measured percutaneously by pulse oximetry (ohmeda-biox iv-boulder, co). baseline serum samples were collected from daycare participants and staff members. surveillance for acute respiratory illnesses took place between january 30, 1992, and october 2, 1995. possible respiratory illnesses among daycare workers or attendees were reported by the daycare staff to the project nurse for evaluation. an acute respiratory illness was defined as nasal congestion, sore throat, new or increased cough, wheezing, sputum production, or respiratory difficulty with or without fever. illness evaluations consisted of a directed history and physical exam, mcasurement of sao,, and nasopharyngeal swab for viral culture. subjects were evaluated each day they attended the center until symptoms resolved. end of illness was defined as resolution of symptoms and physical findings. if participants were well upon return to the center after an absence of several days, they were questioned as to when symptoms resolved. staff members who became ill answered brief questionnaires and had nasopharyngeal cultures taken. four-week convalescent sera were obtained from as many staff and participants as possible. viral cultures and sera were not collected from asymptomatic individuals. nasopharyngeal swabs were performed by gently rubbing the posterior nasal turbinates and posterior pharynx with cotton tip swabs. swabs were placed in veal infusion broth, transported to the laboratory on ice, and inoculated onto wi-38 cell cultures (biowhittaker, walkersville, md) within 6 hours of collection. tubes were incubated at 33°c on roller drums and observed for 10 days for cytopathic effect (cpe). rhinovirus infection was identified by typical cpe and confirmed by acid lability testing. serologic evidence of coronavirus infection was defined as a greater than 4 rise in coronavirus-specific igc as measured by enzyme immunoassay (eia). coronavirus antigens were prepared by expanding coronavirus 229e virus in wi-38 cells. at the point of visible cpe, monolayers were scraped, and cellular material was pelleted in a sorvall at 500 g for 15 minutes. pellets were resuspended in 0.5% np40, and suspensions were sonicated every 15 minutes for 1 hour. eia plates were prepared by coating 229e antigen to immulon round bottom plates in bicarbonate buffer and stored at 4°c overnight. control plates were prepared by using uninfected wi-38 cell lysates prepared by the above procedure. acute and convalescent sera was added in serial 2-fold dilutions from 1:400 to 1:102,400 in duplicate to control and antigen plates. serum igc was detected with alkaline phosphatase conjugated goat a human igg followed by substrate. coronavirus titer was defined as the highest titer with an optical density (o.d.) 2 0.100 and at least twice the o.d. of the control plate. as part of an ongoing surveillance project for respiratory infections in the daycare centers, all nasal specimens were cultured for other viruses (influenza, rsv, parainfluenza, enteroviruses), and all sera were tested by eia for influenza a and b, parainfluenza, and rsv infection. details of these methodologies and the results of this project will be presented in a future publication. during the 44 months of study, 352 older daycare attendees were enrolled and participated in the surveillance project for a mean of 43.6 2 33.7 months. all illnesses were evaluated with viral cultures, and 451 of 522 specimens (86%) had acute and convalescent sera available for coronavirus eia. both coronavirus 229b and rhinovirus infections were identified in approximately 7% of all illnesses. viral cultures were positive for rhinovirus in 35/522 (6.8%) specimens and coronavirus serology was positive in 37/451 (8.0%) paired sera tested. sixty-one subjects experienced 72 separate infections. fifty persons had a single infection, and 11 subjects had multiple infections. three subjects had two different rhinovirus infections, two subjects had two episodes of coronavirus infection, and six people had one rhinovirus and one coronavirus infection each during the study period. six individuals had evidence of concurrent mixed viral infcctions. four persons had 4-fold rises in both rsv and coronavirus titers. two subjects, one with coronavirus infection and one with rhinovirus, had serologic evidence of parainfluenza infection. thus, in 32 illnesses, coronavirus 229e was the sole pathogen identified, and in 34 illnesses, rhinovirus was the only organism found. the clinical features associated with illnesses were analyzed only in cases where either coronavirus or rhinovirus was the only agent identified. coronavirus 229e infections were identified most commonly during the winter and early spring whereas rhinovirus activity was sporadic but tended to be more frequent in the summer and fall ( figure 1 ). interestingly, when coronavirus was circulating, rhinovirus activity nearly ceased. while no clear outbreaks of infection occurred at any daycare center, periods of viral activity typically involved small clusters of three to six older persons and several staff members. clusters of rhinovirus or coronavirus infections were not infrequently preceded by an ill staff member. in the spring of 1995, a 4-week period of increased coronavirus activity at one center, involving seven people, was preceded by 2 days with a coronavirus-infected staff member. the mean age of the 61 subjects who experienced illnesses was 78.5 ? 7.1 years old. the demographics and clinical characteristics of the group that became infected were reflective of the group as a whole except that diabetes was less common in the infected group (1 1 vs 27%, p = .01) ( table 1 ). approximately two-thirds of subjects had underlying cardiac disease, and 21 yo had chronic pulmonary disease. in addition, there were no significant differences between the group infected with rhinovirus compared with the group infected with coronavirus. the clinical syndromes produced by coronavirus 229b and rhinovirus were nearly identical ( table 2 ). most illnesses were characterized by nasal congestion, cough, and constitutional symptoms. low-grade fever was not uncommon, but temperature of 101°f or greater occurred in only three subjects, two with coronavirus and one with rhinovirus. although subjects generally recovered without significant sequelae, illnesses lasted, on average, 14 days. approximately 50% of illnesses were associated with evidence of lower respiratory tract involvement as defined by the presence of sputum production, shortness of breath, new wheezing and/or new rales on exam. thirty-six percent complained of feeling short of breath. twenty-two percent had wheezing, and 46% had rales found on auscultation of the chest. notably, few subjects had wheezing (3%) or rales (13%) on baseline examinations. although mean sao, measurements for the group dropped only a modest amount from 95.3 ? 1.9 at baseline to 94.2 ? 2.0, p = .003 when ill, seven individuals had a greater than 4-point drop in sao, during illness. one individual with coronavirus had a fall in sao, from a baseline of 95% to 89% while ill. four illnesses were evaluated with chest roentgenograms, of which three were normal and one showed congestive heart failure. nine percent of subjects with rhinovirus and 16% with coronavirus received bronchodilators. antibiotics were frequently prescribed in both groups ( table 3) . one individual with rhinovirus infection was hospitalized and treated for congestive heart failure with complete recovery. no deaths occurred. of note, the six illnesses associated with rsv or parainfluenza were not significantly different from those with rhinovirus or coronavirus alone. when individuals with underlying cardiac or pulmonary disease were compared with those without, no significant difference in the severity of rhinovirus or coronavirus infections was noted. wheezing was found to be equally prevalent in those with chronic lung disease as in those without pulmonary problems (23 vs 22%). subjects who developed wheezing during their illnesses were symptomatic slightly longer (15.3 5 6.6 days vs 12.4 2 5.9, p = .095) and received antibiotics much more frequently (86% vs 33%, p < .001) than those who had no evidence of bronchospasm. during the same time period, 113 staff members developed 338 respiratory illnesses ( figure 1b) cough, sputum production, and constitutional symptoms were significantly more common among older persons. twenty percent of daycarc staff missed work secondary to rhinovirus or coronavirus illnesses. this study represents the largest series to date of prospectively identified cases of coronavirus 229e and rhinovirus infections in older persons. our data show that both viruses are common in the daycare setting, and although infections did not generally result in serious complications, many were clinically significant with prolonged symptoms and evidence of lower respiratory tract involvement. relatively little information has been reported on the impact of these common viruses in frail older populations. in a study of acute respiratory infections in nursing home patients by nicholson et al., 12 persons were identified as having coronavirus infection by eia, and three had evidence of lower respiratory in~olvement.'~ in the same study, 11 individuals were found to have rhinovirus infection, one of whom had lower respiratory tract disease. in our previous study of 14 nursing home residents with rhinovirus infections, all illnesses were mild, with only 21 % complaining of sputum production and 14% noting shortness of breath. no patient was hospitalized or died.16 in contrast, in a recent report by wald et al. describing an outbreak of rhinovirus infection that affected 35 institutionalized older persons, a high percentage (66%) of subjects had lower respiratory tract symptoms, and 52% had new abnormalities on lung exam.i7 persons with underlying lung disease had more severe illnesses, with two individuals requiring hospitalization, one radiographically documented pneumonia, and one death secondary to respiratory failure. although all participants of the present study recovered without serious sequelae in contrast to our previous study in the nursing home, these subjects were more seriously ill. similar to the study by wald and colleagues, our current subjects frequently had evidence of lower respiratory tract involvement with new auscultatory findings, symptoms of dyspnea, and a drop in arterial oxygen saturation. additionally, subjects were ill for approximately 2 wecks compared with the usual 2 to 4 days of illness in the young healthy adult. it is also note worthy that, during this era of increasing antimicrobial resistance, antibiotics were prescribed during 50% of illnesses caused by rhinoviruses and coronaviruses. rhinovirus and coronavirus infections have only rarely been found to be the cause of pneumonia in adults, even in severely immunocompromised patient^.^.^.'".'^ however, both viruses have been implicated as a precipitating factor in exacerbations of asthma and copd.''-2z consistent with the published literature, no older subjects in our study had evidence of invasive disease or pneumonia. this contrasts sharply with infection with influenza, rsv, or parainfluenza in older persons where rates of pneumonia can be high and excess mortality rates have been n~t e d . ' * '~-~~ the relatively milder illnesses associated with rhinoviruses and coronaviruses likely reflect the biological characteristics of these viruses. rhinovirus replicates poorly at core body temperature of 37°c and appears to produce symptoms via chemical mediators rather than direct viral invasion.26 although less well studied because of fastidious growth requirements, coronaviruses also do not appear to cause significant damage to respiratory epithelium. the lower respiratory signs of wheezing and rales without evidence of pneumonia in our patients suggest that these viruses cause disease in older persons by aggravating preexisting congestive heart failure or inducing bronchospasm. the incidence of rhinovirus and coronavirus 229e infections in the daycare centers was found to be nearly identical. since we tested only for one of the two most common serotypes of coronavirus infection and the incidence of 229e and oc43 are roughly equivalent, it is possible that the number of illnesses attributable to coronavirus may actually have been double what was reported. the small intermittent clusters of infections in staff and participants at each center suggest that these viruses were introduced into the centers from outside sources. however, once introduced, some element of nosocomial spread is also likely because of close contact between staff and older persons. the daycare policy that encourages participants to attend daycare so they can receive medical attention from the on-site physicians may have influenced the overall incidence of infection in the daycare. however, this concern must be balanced with the need to provide medical care to this very debilitated group of older persons. the daycare center requires that employees with febrile illnesses and/or uncontrolled respiratory symptoms stay out of work until symptoms resolve. however, many of the common respiratory viruses do not cause fever or severe symptoms in young healthy persons, and, therefore, most healthcare workers suffering from upper respiratory illnesses do not miss work. because many individuals, both staff and participants, will be experiencing 'colds' and be in close contact throughout the winter months, good infection control practices in daycare centers are critical. most respiratory viruses, with the exception of influenza viruses, require relatively close contact for transmission.'' rhinoviruses can be transmitted either by fomites and autoinoculation or by aerosol ~p r e a d . '~.~~ although less information is available about the transmission of coronavirus, it is likely they are also spread by fomites and close contact.' many authorities in the field of pediatrics feel that control of respiratory infections in children's daycare centers is nearly impossible because of the nature of young children's activities.30 however, in senior daycare centers, the outlook for infection control may be more hopeful. since transmission of these agents is caused, in part, by fomites, careful handwashing may interrupt ~pread.'~ in addition, architectural design of centers with attention to square feet per resident and adequate ventilation may be important for future control of respiratory infection^.^' in summary, coronavirus 229 and rhinoviruses were found to be common causes of acute respiratory illnesses among the staff and participants of a senior daycare program. although illnesses were not as severe as those associated with other viral pathogens such as influenza and rsv, older subjects were moderately debilitated by these infections. attention should be paid to basic infection control principles to limit spread of these common viruses. respiratory syncytial virus or influenza? lancet isolation of rhinoviruses and coronaviruses rhinovirus infections in tecumseh, michi-1993 gan: frequency of illness and number of serotypes vitro and in volunteers: evidence of heterogeneity among 229e-related strains the time course of the immune response to experimental coronavirus infection of man characteristics of illness and antibody response the behavior of recent isolates of human respiratory coronavirus in rhinovirus infections in rhinovirus infections in an industrial population. 1. the occurrence of illness virologic studies of acute respiratory disease in young adults coronavirus infection in working adults seroepidemiologic survey of coronavirus 2298 infections in a population of children rhinoviruses in seattle families, 1975-1979 acute respiratory illness in an american community: the tecumseh study acute respiratory tract infections in daycare centers for older persons viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings a rhinovirus outbreak among residents of a long-term care facility principles and practices of infectious diseases impact of respiratory virus infection in patients with chronic chest disease association of viral and mycoplasma pneumoniae infections with acute respiratory illness in patients with chronic obstructive pulmonary diseases viruses as precipitants of asthmatic attacks in children respiratory syncytial virus and influenza a infections in the hospitalized elderly parainfluenza outbreaks in extended-care facilities -united states excess pneumonia and influenza hospitalization in the us due 26 epidemiology and control of nosocomial virus infec hand-to-hand transmission of to infmenza epidcmics, 1970-78 726. home architecture and influenza-a attack rates the authors thank the staff and attendees of ils for their participation, christine brower for data management, and joanne prives for transcription assistance. key: cord-268133-obwo7741 authors: ponce, josé burgos; tjioe, kellen cristine title: overlapping findings or oral manifestations in new sars‐cov‐2 infection? date: 2020-06-10 journal: oral dis doi: 10.1111/odi.13478 sha: doc_id: 268133 cord_uid: obwo7741 we have read the short communication “oral vesiculobullous lesions associated with sars‐cov‐2 infection” (martin carreras‐presas, amaro sanchez, lopez‐sanchez, jane‐salas, & somacarrera perez, 2020) by dr. martín carreras‐presas et al. with great interest. we congratulate the team for contributing to the knowledge about this devastating infection in such challenging times. however, we raise some concerns that must be addressed. we have read the short communication "oral vesiculobullous lesions associated with sars-cov-2 infection" (martin carreras-presas, amaro sanchez, lopez-sanchez, jane-salas, & somacarrera perez, 2020) by dr. martín carreras-presas et al. with great interest. we congratulate the team for contributing to the knowledge about this devastating infection in such challenging times. however, we raise some concerns that must be addressed. first, the diagnosis of covid-19 is difficult in mild cases once its signals and symptoms may resemble other infections. the non-confirmed status of sars-cov-2 infection in the two first cases is worrisome once the oral lesions the patients presented are compatible with other frequent diseases. second, we detected several weaknesses in the diagnostic process as follows. in the first case, the authors state the lesions "resemble recurrent herpetic stomatitis, however, it was the first time the patient had them". the primary infection by herpes simplex virus (hsv) may be subclinical (crimi et al., 2019) ; thus, this not a reason to rule out hsv infection. moreover, the other signals and symptoms the patient presented -compatible with an infection -may have led to an immunosuppressed state and potentially contributed to the reactivation of hsv. in the second case, the patient presented the classical clinical appearance of varicella zoster virus recurrent infection: multiple vesicles unilaterally spread at the route of the maxillary branch of the trigeminal nerve. in this case, the authors stated that "the patient did not have any previous history of herpetic infection". however, zoster is the secondary infection of the varicella zoster virus. and, again, these first two patients did not have the diagnosis of covid-19 confirmed. in the third case, the patient developed oral lesions several days after hospital discharge and multiple drug therapy. since some of the drugs are associated with oral manifestations (israr et al., 2011) , we raised the possibility that these lesions are more related to the drugs or immunosuppression driven by the drugs rather than to covid-19. even traumatic injuries should be considered in the differential diagnosis. third, the authors affirmed that "further studies need to be carried out in order to determine whether oral manifestations are common in patients affected by sars-cov-2 infection or if the emotional distress of the situation itself could trigger such lesions". we add that the so-called "oral this article is protected by copyright. all rights reserved manifestations" might actually be other overlapping diseases. and we strongly agree that further studies -or more detailed examination of the patients? -should be performed. finally, the lesions the patients presented might be closely associated with distress (crimi et al., 2019) . in such extraordinary conditions the patients were -possibility of having covid-19, social isolation, potential loss of family or friends, and detriment in economic conditions -the mental status must be considered. we understand the eagerness to understand how covid-19 can impact the oral cavity balance but very preliminary cases should be carefully analyzed before its publication. in times when the information is rapidly spread and often misinterpreted, we must provide accurate and complete data about the cases and findings we are broadcasting. herpes virus, oral clinical signs and qol: systematic review of recent data the hiv protease inhibitor lopinavir/ritonavir (kaletra) alters the growth, differentiation and proliferation of primary gingival epithelium oral vesiculobullous lesions associated with sars-cov-2 infection key: cord-267973-uvz7kavu authors: do, lien anh ha; bryant, juliet e.; tran, anh tuan; nguyen, bach hue; tran, thi thu loan; tran, quynh huong; vo, quoc bao; tran dac, nguyen anh; trinh, hong nhien; nguyen, thi thanh hai; le binh, bao tinh; le, khanh; nguyen, minh tien; thai, quang tung; vo, thanh vu; ngo, ngoc quang minh; dang, thi kim huyen; cao, ngoc huong; tran, thu van; ho, lu viet; farrar, jeremy; de jong, menno; van doorn, h. rogier title: respiratory syncytial virus and other viral infections among children under two years old in southern vietnam 2009-2010: clinical characteristics and disease severity date: 2016-08-08 journal: plos one doi: 10.1371/journal.pone.0160606 sha: doc_id: 267973 cord_uid: uvz7kavu background: despite a high burden of respiratory syncytial virus (rsv) infections among children, data on demographic and clinical characteristics of rsv are scarce in low and middle income countries. this study aims to describe the viral etiologies, the demographic, epidemiological, and clinical characteristics of children under two years of age who were hospitalized with a lower respiratory tract infections (lrti), focusing on rsv (prevalence, seasonality, subgroups, viral load) and its association with disease severity. methods: a prospective study among children under two years of age, hospitalized with lrti was conducted in two referral pediatric hospitals in ho chi minh city, vietnam, from may 2009 to december 2010. socio-demographic, clinical data and nasopharyngeal swabs were collected on enrolment and discharge. multiplex real-time rt-pcr (13 viruses) and quantitative rsv rt-pcr were used to identify viral pathogens, rsv load and subgroups. results: among 632 cases, 48% were rsv positive. rsv infections occurred at younger age than three other leading viral infections i.e rhinovirus (rv), metapneumovirus (mpv), parainfluenza virus (piv-3) and were significantly more frequent in the first 6 months of life. clinical severity score of rsv infection was significantly higher than piv-3 but not for rv or mpv. in multivariate analysis, rv infection was significantly associated with severity while rsv infection was not. among rsv infections, neither viral load nor viral co-infections were significantly associated with severity. young age and having fever at admission were significantly associated with both rsv and lrti severity. a shift in rsv subgroup predominance was observed during two consecutive rainy seasons but was not associated with severity. conclusion: we report etiologies, the epidemiological and clinical characteristics of lrti among hospitalized children under two years of age and risk factors of rsv and lrti severity. respiratory syncytial virus (rsv) is the leading cause of lower respiratory tract infections (lrtis) in young children. 50% of children are infected by rsv during their first year of life, and by 3 years of age, 100% have experienced at least one infection [1] . previous studies [2] [3] [4] have shown the importance of rsv in hospitalized children in vietnam. hospital records from the two main paediatric referral centers in ho chi minh city show that 77% of hospitalized children with lrti are under 2 years old (unpublished data). in addition, severe disease from rsv infection seems to exclusively occur in this population [3, 5] . however, information on detailed clinical, epidemiological features and virological characteristics of rsv infections (e.g. disease burden, demographics, seasonal variations of rsv and other viral infections, circulating genotypes and subgroups, viral load) or on the frequency / impact of other respiratory viruses among vietnamese children under two years old are limited [6] . here, we aimed to describe the viral etiologies and the demographic, epidemiological, and clinical characteristics of children under two years of age who were hospitalized with a lrti, focusing on rsv (prevalence, seasonality, subgroups, viral load) and its association with disease severity. the study was conducted from may 2009 to december 2010 (to cover two rsv seasons, that normally coincide with the rainy season) at children's hospital 1 (ch1) and children's hospital 2 (ch2), the two largest pediatric referral centers for southern vietnam, located in ho chi minh city. children from the respiratory wards (rw), emergency care units (ecu) and intensive care units (icu) were eligible for inclusion in the study. patients admitted to the rw are typically those that initially present to the outpatient clinics and were subsequently indicated for admission, while those admitted to ecu or icu typically presented with more severe symptoms. inclusion criteria were age between 1 month to 2 years, cough, a clinical diagnosis of lrti based on who criteria [7] , and onset of symptoms 4 days prior to hospital admission. exclusion criteria were patients with known non-respiratory or non-infectious respiratory diseases such as asthma. were collected on the day of enrolment (day 1) and on day 7 or discharge (if patients were discharged before day 7). edta blood were used for whole blood cells counting, and liver enzyme measurement by ch1 and ch2 laboratory. swabs were placed in viral transport medium [8] and kept at 4°c for a maximum of 24h, then aliquoted and stored at -80°c until further processing for molecular diagnostics [9, 10] . the study was approved by the institutional review board of children's hospitals 1 and 2, the scientific and ethics committee of the hospital for tropical diseases, ho chi minh city, vietnam and by the oxford university tropical research ethics committee (oxtrec), oxford, uk. written informed consent was obtained from parents or legal guardians of children prior to enrolment into the study. rna extraction from nasopharyngeal swabs was done as described previously [11] . rna was analyzed by multiplex real-time rt-pcr on a roche light cycler 480 ii thermocycler (roche diagnostics, penzberg, germany) [9, 10] and rsv locked nucleic acid (lna) real-time rt-pcr (lna assay) on a dna engine peltier thermocycler and chromo 4 real-time pcr system detector (bio-rad, hercules (ca), usa) [11] . the multiplex real-time pcr detects 13 other human respiratory viruses: influenza virus a (flu a); influenza virus b (flu b); adenovirus (adv), enterovirus (env), human metapneumovirus (mpv), human coronaviruses (cov-229e, oc43, hku1, sars cov & nl63), human rhinovirus (rv a, b and c), parainfluenza virus 1, 2 and 3, 4 (piv1, 2, 3, 4), parechovirus (pev) and human bocavirus (bov) [9, 10] . the lna assay assesses viral load (log10 copies/ml) of rsv subgroups a and b (rsv a, rsv b) [11] . for every rsv positive patient, the second sample (at discharge or day 7) was also assessed by lna assay. definitions. severe disease on admission was defined as having an oxygen saturation of spo2<92 or requiring supplemental oxygen or ventilatory support (by nasal cannula, nasal continuous positive airway pressure (ncpap), mask cpap or mechanical ventilation/intubation) or clinical cyanosis. a clinical severity score (adapted from [12] [13] [14] [15] ) was introduced to grade the clinical status of patients at enrolment (table 1) . only patients from whom all components in the score table were available were given a clinical severity score. long hospitalization was defined as longer than 7 days. statistical analysis. associations between categorical and continuous variables were analyzed using the mann-whitney-u test or kruskal-wallis test for continuous variables, and the fisher exact test for dichotomous variables. spearman's rank correlation coefficient was used to assess a general monotonic trend between two continuous variables. a simple linear regression model was used to measure linear associations between rsv viral load and age or day of illness or number of leucocytes in blood. multivariable logistic regression analysis was performed to assess risk factors for severe disease or long hospitalization. the following variables were considered for the models: age, sex, premature birth, previous hospitalization for respiratory illness, other household members sick at home, living with smoker(s), number of members at home, fever, rsv infection (rsv positivity, viral load, and subgroup) and rv infection (rv positivity as single infection or co-infection with rsv). the models' predictive ability was investigated by calculating the area under the receiver operating characteristic (roc) curve of the model (auc), i.e the higher the auc the better prediction power the model has. the hosmer-lemeshow goodness-of-fit test was done to assess model adequacy. the hosmer-lemeshow test of goodness-of-fit tests the null hypothesis that there is no difference between the observed and predicted values of the response variables. therefore, when the test is not significant (p>0.05) the null hypothesis cannot be rejected and this means that the model fits the data well. risk factors for disease severity or long hospitalization were further assessed using odds ratios (or) and 95% confidence intervals (95% cis). all statistical tests were performed as two-tailed tests at 5% significance in either r version 2.13.1 (r foundation for statistical computing, vienna, austria) or intercooled stata version 9.2 (college station, tx, usa). a total of 632 children aged 1-24 months (median 7, iqr 4-12 months) were enrolled into the study between may 2009 and december 2010. 10/632 (2%) patients were admitted to icu, 36/ 632 (6%) to the ecu and 586/632 (92%) to the respiratory ward. the monthly distribution of children hospitalized for lrti and enrolled in this study during the study period are shown in fig 1. diagnoses on admission were based on clinical symptoms, routine laboratory tests and chest radiography; physicians were unaware of viral diagnostic laboratory results. 438/632 (69%) patients were diagnosed with bronchiolitis; 164/632 (26%) with pneumonia, 22/632 (3%) with combined bronchiolitis and pneumonia, 3/632 (0.4%) with laryngotracheitis, and 5/ 632 (0.8%) with undifferentiated lrti. in addition to respiratory symptoms, 21/632 (3%) patients had diarrhoea on admission, and 4/632(0.6%) had congenital heart disease (ventricular or atrial septum defects). only 18/598 (3%) had blood culture test which was part of standard clinical care at hospitals and only 5/18 were positive (1 staphylococcus aureus, 1 streptococcus pneumoniae, 1 haemophilus influenzae, 1 pseudomonas aeruginosa, 1 klebsiella spp). 535/632 (85%) children received antibiotics, 100% children with clinical diagnosis of pneumonia received antibiotics. high alt** 1 high ast** 1 discharge information was available for 596/632 (94%) cases: 363/596 (61%) of patients fully recovered; 226/596 (38%) had incomplete recovery at the time of discharge; 4/596 (1%) went home without permission (mostly due to economic reasons, patients could not pay hospitalization fee) or formal hospital discharge; and 3 patients (1%) died in the hospital. one fatal case (8 months old) was diagnosed with septicemia and severe pneumonia (s. pneumoniae was recovered from blood culture; no respiratory viruses were detected in nasopharyngeal swabs) and died on the second day of admission. the two other fatal cases were 6 and 3 months old and did not have any severe indications on admission but deteriorated quickly after 5 days of hospitalization. no organisms were recovered from blood culture and virology results yielded a single rv infection and a triple infection (rv, piv-3 and bov), respectively. viral etiologies were identified in the vast majority (91%, 574/632) of patients; single viral infections accounted for 375/632 (59%) of cases and co-infections with multiple viruses were found in 199/632 (31%) ( table 2) . rsv was the most frequently detected: overall (302/632, 48%) and in single infections, while rv was most frequently detected in co-infections (table 2) . a significantly higher proportion of rv, env, bov, adv, piv-2, piv-3, cov and pev were detected among co-infections when compared to the single infections (table 2 ). for mpv, rv, piv-2, piv-3 and piv-4, the relative viral load in single infections was significantly higher than in co-infections (table 2) . rsv-rv co-infection was the most common double infection, identified in 147/632 (23%) cases. triple infections were identified in 47/632 (7%) and in 5/632 cases, 4 different viruses were detected (rsv-rv-piv3-cov, env-rv-cov-bov, adv-env-rv-piv2; rsv-adv-env-rv). (2) median log copies/ml rsva (iqr) 7.5 (6.7-8.2) 7.7 (6.7-8.1) 7.4 (6.3-8.2) 0.2 (1) rsv b, n(%) 139 (22) 93 (25) 46 (23) 0.7 (2) median log copies/ml rsvb (iqr) 7.6 (6.9-8.2) 7.7 (7.1-8.2) 7.5 (6.6-8.0) 0.2 (1) flu, n(%) median cp-value flu b (iqr) 31 (29-33) 32 (29) (30) (31) (32) (33) 30 (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) 0.8 (1) adv, n(%) 37 (6) 3 (1) 34 (17) 0.001 (2) median mpv, n(%) 30 (5) 17 (5) 13 (7) 0.3 (2) median (1) piv-3, n(%) 47 (7) 18 (5) (1) pev, n(%) 12 (2) 2 (1) 10 (5) 0.001 (2) median cp-value pev (iqr) 28 (26) (27) (28) (29) 28 (26) (27) (28) (29) (30) (31) 28 (27) (28) (29) 0.7 (1) bov, n(%) 42 (7) 7 (2) 35 (18) 0.001 (2) median cp-value bov (iqr) 29 (25-33) 23 (22) (23) (24) (25) (26) (27) (28) (29) (30) 30 (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) 0.1 (1) p-value was calculated between single infections and co-infections groups, (1) mann-whitney test, in the univariate analysis, lrti severity was associated with younger age (median age in months [iqr] = 5 [3] [4] [5] [6] [7] [8] [9] [10] in severe cases versus 7 [4] [5] [6] [7] [8] [9] [10] [11] [12] in non-severe cases, mann-whitney test pvalue = 0.001 -s1 fig) , presence of fever (72%, 76/106 in severe cases versus 47%, 247/526 in non-severe cases, fisher exact p-value = 0.001), living in a household with a high number of people (median household members [iqr] = 5 [4] [5] [6] in severe cases versus 4 [3] [4] [5] in nonsevere cases, mann-whitney test p-value = 0.03), or having other household members sick at home (50%, 52/103 in severe cases versus 37%, 193/528 in non-severe cases, fisher exact pvalue = 0.001). co-infections were more frequent among the non-severe cases, and the clinical severity score was inversely correlated to the number of viruses detected (spearman cor = -0.09, p = 0.04). among the single infections, viral load was not associated with severity. for rv, single infections were significantly more common (21%, 22/106) in severe cases versus in non-severe cases (12%, 65/526) (fisher's exact test p-value = 0.02). the relationship between rv and severity was also observed in increased odds ratios for elevated (worse) clinical score (or = 1.86, 95% ci (1.08-3.17), p = 0.02). similar relationships were not observed for rsv single infection cases. in multivariate analyses, significant predictors of severity within the entire study population were younger age, presence of fever, and rv single infection ( table 3 ). the hosmerlemeshow goodness-of-fit test result was χ 2 = 5.94, p-value = 0.65) and the auc for the prediction model was 0.74. similarly, predictors of long hospitalization were younger age, presence of fever, and previous hospitalization with respiratory illness were significant ( table 3 ). the hosmer-lemeshow goodness-of-fit test result was χ 2 = 7.38, p-value = 0.5) and the area under the roc curve for the prediction model of long hospitalization was 0.66. rsv infection was not correlated to disease severity, or to duration of hospitalization (table 3) . rsv infections occurred at a younger age than the 3 other leading single viral infections, i.e rv, mpv, piv-3 (tables 4 and 5) , and were significantly more frequent in the first 6 months of life (fisher exact test p-value = 0.001). in contrast, none of the other viruses exhibited significant age group distributions (tables 4 and 5). a significant inverse correlation was observed between rsv load at enrolment and patient age. indeed, on average rsv viral loads decreased by 0.03 log copies/ml per month increase in age (t = -2.37, p-value = 0.02). a higher clinical severity score was found only among rsv single infection cases compared to those of piv-3 single infection cases (mann-withney test p-value = 0.02, table 5 ), but not in any of the other pair-wise comparisons. while rsv single infection had a significantly shorter duration of hospitalization, a higher prevalence of elevated ast and a lower leucocyte count than rv single infection cases (table 5 , mann-withney test p-value = 0.02, 0.02 and 0.001, respectively). we also observed a significantly lower number of leucocytes in rsv single infection versus those in rsv co-infection (mann-withney p-value = 0.001), and an inverse (2) 12 (71) 0.9 (2) 10 (56) 0.3 (2) median number of household members (iqr) 4 (4-6) 4 (4-6) 0.7 (1) 4 (3) (4) (5) 0.3 (1) 4 (3-6) 0.4 (1) living with smokers, n (%) 102/196 (52) 51/86 (59) 0.3 (2) 5 (29) 0.07 (2) 12 (67) 0.2 (2) medical story (2) 16 (94) 0.07 (2) 13 (72) 0.8 (2) premature birth, n(%) 20/199 (10) 14/85 (16) 0.1 (2) 0/16 (0) n.a 1/17 (6) 0.5 (2) daycare, n (%) 25/197 (13) 10/84 (12) 0.9 (2) 2/16 (13) 1.0 (2) 5/18 (28) 0.08 (2) previous hospitalization with respiratory illness, n (%) 22 (11) 23 (26) 0.001 (2) 2 (12) 0.9 (2) 3 (17) 0.5 (2) other household members sick at home, n (%) 76/197 (39) 36/85 (42) 0.6 (2) 5 (29) 0.5 (2) 5 (28) 0.4 (2) clinical characteristics fast breathing, n(%) 166 (83) 75 (86) 0.4 (2) 14 (82) 1.0 (2) 14 (78) 0.6 (2) cyanosis, n(%) 6 (3) chest indrawings, n(%) 193 (96) 81 (94) 0.3 (2) 15 (88) 0.1 (2) 15 (83) 0.01 (2) stridor, n(%) wheezing, n(%) 192 (96) 76 (88) 0.01 (2) 16 (94) 0.8 (2) 17 (94) 0.8 (2) fever (>37.5°c), n(%) 112 (56) 50 (57) 0.8 (2) 7 (41) 0.2 (2) 6 (33) 0.07 (2) fever ( 38.5°c), n(%) 46 (23) 13 (15) 0.1 (2) 5 (29) 0.5 (2) 2 (11) 0.2 (2) rash, n(%) 2 (1) 3 (3) 0.1 (2) 0 (0) n.a 1 (6) 0.1 (2) runny nose, n(%) 181 (91) 70 (81) 0.03 (2) 15 (88) 0.8 (2) 17 (94) 0.5 (2) low spo 2 , n(%) 19/140 (14) 6/60 (10) 0.5 (2) 1/14 (7) 0.5 (2) 1/17 (6) 0.4 (2) median of duration of hospitalization (iqr) 6 (5-7) 6 (5-8) 0.02 (1) 6 (4-7) 0.9 (1) 6 (5-7) 0.9 (1) duration of hospitalization 7 days, n(%) 77 (38) 43 (49) 0.08 (2) 7 (41) 0.8 (2) 7 (39) 1.0 (2) high alt, n(%) 54 (27) 16 (18) 0.1 (2) 2 (12) 0.2 (2) 3 (17) 0.3 (2) high ast, n(%) 44 (22) 9 (10) 0.02 (2) 4 (23) 0.9 (2) 3 (17) 0.6 (2) median of number of white cells in blood (k/mm 3 ) (iqr) 10 (8-14) 13 (11) (12) (13) (14) (15) (16) (17) 0.001 (1) 11 (9-13) 0.8 (1) 11 (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) 0.6 (1) severe cases, n(%) 36 (18) 22 (25) 0.2 (2) 3 (18) 1.0 (2) 3 (17) 1.0 (2) median of clinical score (iqr) 6 (4-7) (n = 186) 5 (4-7) (n = 80) 0.5 (1) 5 (4-6) 0.4 (1) 4.5 (4-6) 0.02 (1) diagnosis bronchiolitis, n(%) 162 (81) 65 (75) 0.3 (2) 10 (59) 0.06 (2) 9 (50) 0.005 (2) treatments antimicrobial agents, n(%) 169 (84) 62 (71) 0.01 (2) 17 (100) 0.07 (2) 17 (94) 0.2 (2) corticosteroids, n(%) 9 (4) 6 (7) 0.4 (2) 1 (6) 0.8 (2) bronchodilators, n(%) 168 (84) 67 (77) 0.2 (2) 9 (52) 0.002 (2) 10 (56) 0.004 (2) supplemental oxygen, n(%) 31 (15) 21 (24) 0.08 (2) 2 (12) 0.7 (2) 2 (11) 0.6 (2) outcomes full recovery, n(%) 119/195 (61) 46/81 (57) 0.5 (2) 11 (73) 0.8 (2) 11 (65) 0.9 (2) severe cases, n(%) 36 (18) 22 (25) 0.2 (2) 3 (18) 1.0 (2) 3 (17) 0.9 (2) death, n(%) p-values were based on comparison with children having rsv single infections. (1) mann-withney test, (2) fisher's exact test, n.a: not applicable correlation between rsv load in nasopharyngeal swabs and leucocyte count in blood collected at enrolment (coeff = -0.56, 95%ci = -0.98 to -0.14, p-value = 0.009). among 302 rsv positive cases, 156/302 (52%) were rsv a, 139/302 (46%) were rsv b, and 7/302 (2%) were both rsv a and b. there was no difference in proportion of a and b subgroups between single and co-infections, and no difference in viral load among subgroups at enrolment (table 2) . strong seasonal variations of rsv prevalence with peaks during the rainy season from may to october were observed over the two seasons of the study. rsv b was dominant during the first season and rsv a during the second season (fig 2) . overall case numbers for mpv and influenza were insufficient to detect patterns of seasonality. the most frequently detected viruses in rsv co-infection were rv, env and adv (s1 table) . in the univariate analysis, the number of viruses co-detected with rsv or the rsv viral load was not associated with rsv severity, or with clinical severity score (cor = 0.06, p = 0.1 and cor = -0.3, p = 0.2, respectively). rsv patients with long duration of hospitalization had a significantly higher rsv viral load than others (median of rsv load (iqr) 8 (7-8) versus 7 (7) (8) mann-withney test p-value = 0.004, respectively). no associations with rsv subgroups and severity or duration of hospitalization were found. in multivariate analyses for the rsv-infected population, the logistic regression analysis of ten selected predictors identified age (or = 0.88, 95%ci:0.81-0.95, p-value = 0.001), fever (or = 4.80, 95%ci:2.26-10.19, p-value = 0.001), and having another household member sick at home (or = 2.04, 95%ci: 1.03-4.02, p-value = 0.04) as independent variables associated with disease severity in rsv-infected children ( table 3 ). the hosmer-lemeshow goodnessof-fit test result was χ 2 = 5.73, p-value = 0.68) and the area under the roc curve for the prediction model was 0.76. for risk factors of long hospitalization among rsv patients, only fever (or = 2.67, 95%ci: 1.59-4.48, p-value = 0.001) was a significant independent predictor ( table 3 ). the hosmer-lemeshow goodness-of-fit test result was χ 2 = 9.35, p-value = 0.31 and the auc for the prediction model of long duration hospitalization was 0.67. again, rsv subgroups or rsv viral load were not associated with disease severity or long hospitalization in multivariate analyses. over the last decade, multiplex molecular diagnostics have revolutionized the diagnostics of respiratory infections and greatly expanded the available data on viral etiologies and coinfection [6, [16] [17] [18] [19] [20] [21] [22] . here, using a previously described multiplex assay to detect 13 different respiratory viruses, viruses were identified in 91% of enrolled patients, and viral co-infection was found in 31%. the high prevalence of viral infections among lrti cases, the predominance of rsv and rv infections, and the co-infection rates between rsv and rv, env and adv in our study were similar to other published work in comparable populations [21, 23] . in these previous studies, among hospitalized children, the proportion of viral causes ranged from 93-97%; rsv and rv were the leading causes, ranging from 64 to 73% and from 30-34%, respectively [21, 23] . our study confirms the importance of rsv infection in children under two as shown by many other studies [17, 21, [23] [24] [25] and is the first study to examine the demographic, clinical and virological characteristics of rsv infections in south vietnam. our results confirm and extend previous observations regarding associations of rsv infections with young age compared to the 3 other leading viral infections (rhinovirus (rv), metapneumovirus, parainfluenza virus (piv-3), wheezing, runny nose, leucopenia (among rsv single versus rsv co-infection or rv single infection) and risk factors (premature birth) [17, 21, [23] [24] [25] [26] . in addition, we observed a significant negative correlation between rsv load in nasopharyngeal swabs and leucocyte count in blood collected at enrolment (coeff = -0.56, 95%ci = -0.98 to -0.14, pvalue = 0.009). these findings are consistent with previous reports [26, 27] and it could be hypothesized that leukocytes in rsv infection are being recruited at specific sites away from the circulating blood [28, 29] . one of our aims was to determine risk factors for severity and long hospitalization among all study patients and among rsv-infected patients. we observed that rsv load at enrolment was significantly related to long hospitalization in univariate analysis, but this was not confirmed in the multivariate analysis; and rsv infection, rsv viral load, rsv subgroups and rsv slope (i.e rsv load dynamic between two time points: admission and discharge, it was calculated by dividing the difference of viral load at admission and at discharge by the number of days between these two time points-data not shown), did not correlate to disease severity or long hospitalization. only young age and fever were independent predictors for disease severity in both populations (study population and rsv population). reports about the relation between rsv infection [30, 31] or rsv load [12, 26, 32, 33] or rsv subgroups [34] [35] [36] [37] [38] and disease severity have been contradicting. marguet et al. reported that rsv infection caused more severe disease than rv infection among children under 1 year of age. in a study by tran et al. conducted in children hospital 2, ho chi minh city, targeting hospitalized children under 15 years, rsv positive children had significantly higher clinical severity scores compared to rsvnegatives. however, this comparison was based on univariate analysis without considering other confounding factors such as age [6] . papadopoulos et al. found, among children less than 18 months, that the presence of rv increases the risk for severe disease significantly, by approximately five-fold. rsv infection was also correlated with severity, however, this did not reach significance [30, 31] . rsv loads at the second collection had a significant negative correlation (correlation = -0.33, p-value = 0.009) with day of illness, while this correlation was not found for the enrolment samples which were collected on any day during the first days of illness (s2 fig). this rsv load trend during rsv infection was also observed in other studies [39] and confirmed the study samples were collected on the early day of rsv illness. thus far, observations regarding the relation between viral co-infections and disease severity have been contradictory and biased by different study designs and viral diagnostic tools used [24, 40] . similar to other studies, we observed a significantly lower proportion of co-infections in severe cases compared to non-severe cases [21, 41] . however, among rsv infected cases, we did not observe significant differences in disease severity between single and co-infected cases. in addition, there were significant differences between rsv-rv coinfections (3/45, 6.6%) versus rsv-env (9/27, 75%)-fisher's exact p-value = 0.006 but no significant versus rsv-adv co-infections (2/18, 11%). interestingly, and in agreement with papadopoulos et al. [31] , although rv was detected mostly as a coinfection, rv single infection was identified as an independent risk factor of severe disease. this finding should be interpreted with caution because the role of rv in the pathogenesis of severe lrti remains a topic of debate [30, 31, [42] [43] [44] [45] [46] [47] . cross-reactivity in the pcr detection of env and rv using 5'utr primers is a known problem, particularly for ev-d68 which has a rhinoviral 5' utr sequence, and this complicates ascertainment of env and rv diagnoses [48] . despite the fact that the study population from our current study and our previous study [3] and from other studies [2, 6] consisted of hospitalized children and is therefore not representative of vietnamese children in general, a consistent rsv seasonal pattern with peaks during the rainy season (between may and october) was observed from 2005 to 2010. the clear shift of rsv subgroups was not observed during the period 2005 to 2007 [3] but we observed this in our current study and tran et al. also confirmed the dominance of rsv a during the same period [6] . in many studies, rsv has been reported as a highly seasonal infection and rsv outbreaks are frequently associated with the rainy season in areas with tropical and subtropical climate [5, 49] . possible explanations for this include meteorological effects such as humidity and uvb radiation on the environmental stability of rsv viruses. the stability of rsv in aerosols was shown to increase with higher humidity [50] . moreover, population behaviors as staying indoors during cold or rainy seasons may facilitate transmission of rsv. from this cohort, we have recently reported analysis of rsv whole genomes [51] and are currently analysing host expression profiles of blood and nasopharyngeal swabs at two time points (manuscript in preparation). there are a number of limitations to our study. firstly, few bacterial diagnostic results were obtained from patients. the role of bacteria as a cause of lrti or as cause of superinfection, especially in rsv and influenza virus infection [52] , is important for antibiotic intervention strategies. lower airway secretions (sputum for example) are considered the optimal specimen type for detecting bacterial (co-) infection, yet are often difficult to obtain from young children, and can only be readily obtained from intubated children. secondly, only a limited number of patients were enrolled as compared to the total number of lrti hospitalizations in two hospitals during the study period (632/45134, 1%) although the number of enrolled patients followed a similar pattern as the total number of patients (fig 1) . in summary, our study has contributed detailed clinical and virological data on rsv and other viruses in respiratory infections among children under 2 years old, the most vulnerable age group, in a lower middle income setting in asia: vietnam. the data on seasonality of viruses are crucial for health care management, such as 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hyperreponsiveness through regulation by complement c3a and tachykinins role of rhinovirus in hospitalized infants with respiratory tract infections in spain correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients rhinovirus associated with severe lower respiratory tract infections in children respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants role of real-time reverse transcription polymerase chain reaction for detection of respiratory viruses in critically ill children with respiratory disease: is it time for a change in algorithm? pediatr crit care med equal virulence of rhinovirus and respiratory syncytial virus in infants hospitalized for lower respiratory tract infection viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china the epidemiology of respiratory syncytial virus lower respiratory tract infections in children less than 5 years of age in indonesia the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus direct whole-genome deepsequencing of human respiratory syncytial virus a and b from vietnamese children identifies distinct patterns of inter-and intra-host evolution increased susceptibility to bacterial superinfection as a consequence of innate antiviral responses we acknowledge study nurses hoang thi minh tu, nguyen thi hong ngoc, nguyen viet truong, nguyen thi ngoc ha, nguyen thi thanh nha, le thi kim loan, ho huynh nhu, tran thi tuyet nhung, huynh thi phuong thao, for sample collection; staff in data management at oucru for entering data; vo nhi ha and nguyen thi thanh thuy and the clinical trials unit at oucru and children's hospital 1 and 2 for coordinating the trial.this study was funded by the wellcome trust of great britain (077078/z/05/z). the funder had no role in study design, data collection or analysis, the decision to publish, or preparation of the manuscript. wrote the paper: lahd jeb hrvd mdj. key: cord-259194-9zllvfqb authors: cupples, sandra a. title: transplant infectious disease: implications for critical care nurses date: 2011-11-02 journal: crit care nurs clin north am doi: 10.1016/j.ccell.2011.08.001 sha: doc_id: 259194 cord_uid: 9zllvfqb infection is an important issue for critical care nurses as they care for patients throughout all phases of the transplant continuum: potential organ donors, transplant candidates, and transplant recipients. this article has reviewed salient issues relative to infections in each of these patient populations, including patients with vads, and has highlighted key points pertaining to bacterial, viral, and fungal infections. while they are on the transplant waiting list. urinary tract infections are common in kidney and pancreas transplant candidates. kidney transplant candidates are also at risk for infections in the native kidneys and occult abscesses. liver transplant candidates may have intra-abdominal infections or aspiration pneumonia. pneumonia is also common in the heart and lung candidate populations. hospitalized candidates are at risk for catheter-or device-related infections, such as those associated with dialysis access devices or ventricular assist devices (vads). as of august 2011, there were more than 3100 candidates on the heart transplant waiting list in the united states. 2 to date, only 949 heart transplant procedures have been performed in the united states in 2011. thus the demand for donor hearts far exceeds the supply. 3 vads were developed to augment circulation in patients with end-stage heart disease. these devices have been approved by the food and drug administration for three purposes: to bridge patients to heart transplantation, to bridge patients to recovery of their native myocardial function, and to provide permanent support for patients who are not deemed to be suitable heart transplant candidates ("life-time" or "destination" therapy). 4 vads can support the right or left ventricle or both. they stabilize the patient's condition by increasing cardiac output, improving perfusion to vital organs, and restoring mobility. 5, 6 these devices are typically implanted through a median sternotomy incision and placed in a pre-peritoneal or intra-abdominal pocket. the major components of a vad are inflow and outflow cannulae, unidirectional valves, a polyurethane chamber (for pulsatile devices), and a pump or rotor. the device is connected to an external power source through a driveline that exits through the abdominal wall (fig. 1) . 7, 8 vads contain biomaterials and, unfortunately, none of these materials are biologically inert. 9 therefore, events that occur at the host-implant interface can trigger aberrant immune activation. when the patient's blood comes in contact with the foreign vad surface, t cells can become activated and initiate a defective proliferative response and subsequent activation-induced cell death. as a result, the patient's immune system is impaired and the patient may be more susceptible to infection. 4, 5, 7, 9 infection is a common complication of vad therapy. 6 vad-related infections may delay or prevent transplantation altogether and they are a major cause of morbidity and mortality in lifetime therapy patients. 10 -12 the most recent international society for heart and lung/mechanical circulatory support device registry data indicate that infection occurred in 32.5% of the 655 vad patients enrolled in this database and that patients with vad infections had a 7.9% mortality rate. 13 device-related infection rates reported in the literature have ranged between 13% and 80%. 5 potential infection sites include the surgical site or any component of the vad (driveline, device pocket, or pump membrane). driveline infections are the most common; however, more than half of all infections involve several device sites simultaneously. 7 vad infections may remain localized or become systemic. if the infection spreads to multiple sites, serious complications such as bloodstream infections, bacteremia, sepsis, and endocarditis can ensue. 7 device-, patient-, and mechanical-related factors can contribute to vad infections. device-related factors include the exposure of percutaneous drivelines to pathogens and the vad cavities and pockets that can harbor pathogens. these microorganisms can cause blood flow through the pump to become turbulent; this in turn enables the pathogens to adhere to the surface of the device. 4 patient-related risk factors for infection include older age, poor nutritional status, indwelling catheters, prolonged intubation, postoperative bleeding, blood transfusions, multiorgan dysfunction, comorbidities such as diabetes mellitus and obesity, prolonged hospitalization before vad implantation, and surgical reexploration. 4, 6, 7 mechanical trauma to the driveline exit site is frequently associated with late-onset (ͼ30 days after implantation) infections. driveline trauma occurs when, for example, the controller or battery pack is dropped or when the driveline is snared on an object. these accidents result in shearing or torsion injuries that can lead to infection. 12 device-related infections can occur at any time; however, the majority develop between 2 weeks and 2 months of implantation. 7 gram-positive pathogens, particularly staphylococcus species, cause most infections. 14 these organisms are able to form a protective biofilm that blunts the host immune response and enables them to attach to and grow on inanimate surfaces. 8 fungal and gram-negative bacilli, such as pseudomonas aeruginosa and the enterobacter and klebsiella species, are other causative agents; these particular pathogens are associated with poorer outcomes. 4, 7 the administration of broad-spectrum antibiotics often leads to the development of fungal infections. 7 the clinical manifestations of vad-related infections are varied. presentation may be subtle or acute. if a device-related infection is suspected, the patient must have a thorough evaluation that includes a comprehensive physical examination and extensive work-up including blood cultures with gram stains. if possible, cultures should be obtained before initiation of antimicrobial therapy. 7 other sources of infection, such as pneumonia, urinary tract or catheter-related infections, must be investigated appropriately. additional diagnostic tests are site specific. for example, ultrasound is used to evaluate suspected pump pocket infections; transesophageal echocardiograms are useful in the setting of vad-related endocarditis. table 1 lists the typical signs and symptoms of devicerelated infections and potential treatment options. the evidence regarding the impact of device-related infection and posttransplant outcomes is mixed. some studies have indicated that these infections do not reduce 1-year 15 or overall 7, 16 survival. other studies have found that serious device-related infections can persist into the posttransplant period 7, 17 and are associated with decreased early 11 and long-term 17 posttransplant survival. although assist devices are often associated with infection, the benefits of this life-saving therapy are thought to outweigh the infection risk. 7 the major clinical implications for pre-and postoperative nursing care are listed in table 2 . infections can be transmitted via the allograft itself. 18 a donor-derived disease transmission is defined as "any disease present in the organ donor that is or has the potential to be transmitted to at least one of the recipients." 19(p234) donor-derived infectious diseases are rare. unexpected transmissions, that is, those that were either unrecognized in the donor or for which the donor was not screened, occur in fewer than 1% of all solid organ transplantation procedures. 19 although rare, these infections cause significant morbidity and mortality. factors that promote infection in potential organ donors include the use of medical devices and the treatment of patients in certain units that have high rates of bacterial contamination. 20 it is important to note that treatment of donor infections itself can further increase the potential donor's risk of iatrogenic infection, for example, via the insertion of intravascular catheters for antimicrobial therapy, the administration of immunomodulating medications such as corticosteroids, and prolonged hospitalization. 21 for a number of reasons, infections in potential organ donors may be difficult to diagnose: • the donor may not have the clinical manifestations of infection due to insufficient numbers or virulence of pathogens. • hemorrhage or aggressive fluid resuscitation may dilute both organisms and serologic infection markers such that they are undetectable by conventional laboratory tests. • the donor may not mount a fever response because brain death causes loss of temperature control and poikilothermia (a phenomenon whereby body temperature decreases to that of the environment). • the donor's white blood cell count may be already elevated due to trauma, tissue inflammation, or medications such as corticosteroids. 21 as a consequence, the diagnosis of infection may rely on culture and urinalysis reports, polymerase chain reaction (pcr) and nucleic acid testing results, characteristics of sputum, and changes in chest radiographs and computed tomography (ct) scans. 21 potential organ donors undergo a rigorous infectious disease evaluation. organ procurement and transplantation network (optn) policies mandate that potential donors must be screened for the following pathogens: human immunodeficiency 22 potential heart donors are screened for toxoplasmosis. many donors are also screened for nosocomial infections such as methicillinresistant staphylcoccus aureus or vancomycin-resistant enterocci. because infection can be transmitted via transfusions, serologic testing is typically performed both before and after a potential donor receives blood products. in addition, family members are questioned about the potential donor's infection risk, including prior infection exposure, history, and immunizations; travel to endemic areas; and risky behaviors such as intravenous drug abuse. table 3 displays donor organ acceptance and exclusion criteria based on the results of infectious disease screening. the acceptance of organs from donors with known infections with or exposure to hiv, hepatitis, or other viruses remains controversial. 21 given that the number of transplant candidates on the waiting list far exceeds the number of available organs, strategies to expand the donor pool include accepting donors with certain infections, higher-risk serological profiles, and social histories suggestive of prior exposure to bloodborne infections as well as donors who may be more at risk for transmitting infections (eg, older donors and donors with long icu stays). 20 informed consent effective treatment of bacterial infections in potential organ donors can result in successful transplantation. 21 box 1 displays important principles of antibiotic selection and administration for potential organ donors. the organ procurement organization's (opo's) coordinator has major responsibilities regarding the prevention and treatment of infections and reporting known infections to transplant centers that could potentially receive organs from infected donors. infections that must be reported to the transplant center are listed in box 2. moreover, all antimicrobial agents that are given to the potential donor must be documented and reported to each transplant center that receives an organ from that donor. 21 when a transplant center is informed that one of its organ recipients is confirmed positive for or has died from a potential donor-derived transmissible disease, that center must notify, within one working day, the opo that procured that organ. the opo must then notify the optn. these reports are forwarded to unos and uploaded to the disease transmission advisory committee's (dtac's) secure website. 1. select a bactericidal antibiotic over a bacteriostatic antibiotic. 2. use medication that will most directly target the identified bacteria to prevent the removal of harmless bacteria, the promotion of selective overgrowth of fungi, resistant organisms, or abnormal bacterial strains (eg, clostridium difficile), and the development of gene mutations and highly resistant organisms. 3. substitute directed antibiotic for broad-spectrum agent once sensitivities are available. 9. consider antibiotics specifically effective against anaerobic bacteria in the setting of facial injuries, pulmonary aspiration, or contaminated wounds from an injury scene. 10. administer antibiotics intravenously to maximize bioavailability. 11. adjust antibiotic doses in the setting of renal failure, hepatic failure, and older donor age. dtac data indicate that, between 2005 and 2007, there were 80 donors with reported possible donor-derived infectious disease transmission, 30 recipients with confirmed (proven, probable, or possible) donor-derived infections, and 14 recipient deaths attributed to donor-derived infections. these deaths were due to hepatitis c, tuberculosis, hiv, chagas disease, bacteremias, candidemia, strongyloides, and lymphocytic choriomeningitis virus. 18 there are three major factors that determine a transplant recipient's risk of infection. these include the patient's epidemiological exposure, either in the box 2 infections that must be reported to the transplant center 35 known conditions that may be transmitted by the donor organ must be communicated to the transplant center. these may include, but are not limited to, the following: hospital or in the community; the patient's current antimicrobial regimen, if any; and the patient's net state of immunosuppression, which is defined as "the combined effect of all of the factors that determine the patient's susceptibility to infection." 24(p138), 25 the net state of immunosuppression includes the patient's current immunosuppressive regimen (number and strength of antirejection agents), as well as any of the following concurrent factors: infection with an immunomodulating virus (eg, cmv or ebv); metabolic or autoimmune disorders (eg, diabetes mellitus); neutropenia or lymphopenia; disruption of mucocutaneous barriers; and surgical sequelae (eg, fluid collections). 25 approximately 80% of all transplant recipients have at least one significant infection during the first posttransplant year. 26 the three major groups of posttransplant pathogens are bacteria, viruses, and fungi (fig. 2) . bacterial infections are the most common, 26 followed by viral and fungal infections. bacterial infections frequently occur at the transplant site. bacterial pneumonias are common among all types of solid organ transplant recipients. nosocomial pathogens of particular concern include clostridium difficile, vancomycin-resistant enterococcus, methicillin-resistant staphylococcus aureus, and extended-spectrum ␤-lactamase gram-negative bacilli. common organ-specific bacterial infections and associated risk factors are listed in table 4 . most posttransplant viral infections are caused by two groups of pathogens: the herpes viruses (cmv, ebv, hsv 1 and 2, and varicella zoster) and the hepatitis viruses. viral infections are particularly deleterious because they have both direct and indirect effects. the direct effect is the clinical syndrome caused by the virus itself, such as cmv pneumonia or hepatitis. indirect effects include potential injury to the allograft, rejection, oncogenesis, and the virus's ability to alter the net state of immunosuppression, thereby increasing the patient's susceptibility to other opportunistic infections. the herpes viruses are characterized by latency. this means that once the virus is present, the patient will harbor the viral genome for life. immunosuppression, particularly augmented immunotherapy in the setting of rejection, can trigger replication of latent herpes viruses. 24 cytomegalovirus is the most important pathogen that affects transplant recipients. there is a bidirectional relationship between cmv and rejection. cmv can trigger rejection and the inflammatory effects of rejection and rejection therapy can increase cmv viral replication. the allograft is more likely to be affected by a cmv infection than a native organ. thus, liver transplant recipients with cmv infections are prone to develop vanishing bile duct syndrome, heart transplants recipients are at risk for coronary artery vasculopathy, lung transplant recipients are at risk for bronchiolitis obliterans, and so forth. the most common types of cmv disease are hepatitis, pneumonitis, and gastroenteritis. with regard to cmv serostatus, the risk of developing a posttransplant cmv infection is highest in cmv-seronegative recipients who receive an allograft from a cmv-seropositive donor and lowest in cmv-seronegative recipients who receive an allograft from a cmv-seronegative donor. recipients who receive potent antirejection therapy such as antithymocyte globulin are also at increased risk for developing a cmv infection. a concurrent critical illness can lead to the reactivation of a latent cmv infection; this is thought to be associated with proinflammatory cytokines and subsequent downregulation of the immune system. agents used to prevent or treat cmv infection include ganciclovir, valganciclovir, acyclovir, and cmv immune globulin. foscarnet is often used to treat ganciclovirresistant organisms. because cmv can be transmitted through blood transfusions, cmv-seronegative transplant candidates and recipients should receive cmv-negative, leukocyte-poor, or filtered blood products. 1, 24 given that most adults are ebv-seropositive, most posttransplant ebv infections in adults are reactivated from latent pretransplant infections. however, ebv-seronegative recipients can acquire an ebv infection through blood transfusions or community exposure. the incidence of posttransplant ebv infections is highest in multiorgan and intestinal transplant recipients followed, in decreasing order, by kidney-pancreas, lung, heart, liver, and kidney recipients. intravenous ganciclovir has been used as preemptive therapy for patients at high risk for ebv infections, for example, patients receiving antilymphocyte antibody therapy for rejection. the clinical sequelae of ebv infection range from a relatively mild mononucleosis-like syndrome to posttransplant lymphoproliferative disease (ptld). treatment options for mononucleosis include acyclovir. ptld is a set of syndromes that ranges from a benign, self-limiting polyclonal proliferation of b cells to an aggressive, malignant, monoclonal lymphoma. risk factors for ptld include pretransplant ebv-negative serostatus, primary ebv infection, high ebv viral load, cmv serostatus mismatch (recipient is cmv negative and donor is cmv positive), cmv disease, potent rejection treatment, and type of allograft. the incidence of ptld is highest in intestinal transplant recipients. treatment options for ptld range from antiviral agents (acyclovir, ganciclovir) and decreased immunosuppression for the benign polyclonal form to chemotherapy, radiation, resection, and decreased immunosuppression for malignant monoclonal lymphoma. although invasive fungal infections have the lowest incidence of all infections, they are associated with the highest morbidity and mortality rates. 27 risk factors for fungal infections include the use of high-dose corticosteroids and broad-spectrum antibiotics, rejection that requires increased immunosuppression, allograft dysfunction, and a simultaneous infection with an immunomodulating virus such as cmv. 24 two genera, aspergillus and candida, cause the vast majority of posttransplant fungal infections. together, these two pathogens account for more than 80% of invasive fungal infections. these infections typically present during the first month posttransplant, 27 but they can occur at any time. the most common fungal infection that involves the respiratory tract is invasive aspergillosis, which may affect approximately 30% of solid organ transplant recipients. 28 other portals of entry include the gastrointestinal tract and the skin. the risk of disseminated candidiasis is highest in neutropenic patients with central venous catheters who have received broadspectrum antibiotics and who have had prolonged icu stays. 29 liver transplant recipients are at highest risk for invasive candidiasis, followed, in decreasing order, by pancreas, lung, heart-lung, kidney, and heart transplant recipients. 29 pediatric transplant recipients are often at higher risk for posttransplant infections for a number of reasons, including: • lack of immunity to common pathogens such as cmv and ebv • incomplete immunizations • increased technical difficulty and prolonged transplant operative time due to pretransplant palliative surgeries • inability to close the abdomen or chest due to placement of a large allograft into a small child • social behavior of children in densely populated day care and school settings. 30 acute mediastinitis can develop after the implantation of mechanical circulatory assist devices or after heart, lung, and heart-lung transplantation. the risk of posttransplant mediastinitis is higher if the patient had a mechanical circulatory assist device or a total artificial heart as a bridge to transplantation. there are preoperative, intraoperative, and postoperative risk factors for mediastinitis. examples of preoperative risk factors include diabetes mellitus, prior sternotomy, renal failure requiring dialysis, prolonged hospitalization before the transplant surgery, and obesity. the risk of developing mediastinitis is more than double in patients with a body mass index greater than 30. intraoperative risk factors include blood transfusions and prolonged cardiopulmonary bypass, aortic cross-clamp, and operative times. examples of postoperative risk factors include surgical reexploration, prolonged icu stay, prolonged mechanical ventilation (ͼ24 -48 hours), having a tracheostomy, cardiopulmonary resuscitation, poor perioperative and postoperative glucose control, and low posttransplant cardiac output. 31 the major etiologic pathogens associated with mediastinitis include, in decreasing order, gram-positive cocci (staphylococcus aureus, staphylococcus epidermidis, enterococcus spp., streptococcus spp.), gram-negative bacilli (escherichia coli, enterobacter spp., klebsiella spp., proteus spp., other enterobacteriaceae, and pseudomonas spp.), and fungi (candida albicans). 31 the initial clinical manifestations of mediastinitis may be subtle: mild chest pain, and edema or erythema along the sternal incision. 24 the most common presenting symptom is fever; it may be associated with localized infection, erythema, cellulitis, purulent drainage, pleuritic-like pain, and sternal instability. diagnostic studies include ct scans, cultures, and laboratory tests. laboratory findings include elevations in the white blood cell count, c reactive protein, and procalcitonin. the latter test is particularly useful in distinguishing between rejection and infection. once mediastinitis is diagnosed, treatment should be initiated promptly. therapeutic options include surgical drainage/débridement, wound irrigation, tailored parenteral antimicrobial agents, and nutritional support. 31 in transplant recipients, central nervous system (cns) infections are among the most deleterious because they can be difficult to diagnose and treat. diagnosis is often challenging because presenting symptoms, such as mental status changes, seizures, focal neurologic deficits, and headache, may be blunted by immunosuppressive therapy. moreover, the neurotoxic effects of antibiotics, antiviral agents, and immunosuppressants themselves may make diagnosis even more complicated. 32, 33 the first step in diagnosing a suspected cns infection is a neuroimaging study to establish the presence, location, potential etiology, and characteristics of any lesion(s). magnetic resonance imaging studies of the brain or spinal cord or both are typically preferred to ct scans. neuroimaging studies are useful in determining if the infection is focal or nonfocal and if it involves the meninges. other diagnostic tests include cerebrospinal fluid analyses, electroencephalograms, viral polymerase chain reaction tests, cultures, and serologic tests. brain biopsies are rarely done, except in the setting of posttransplant lymphoproliferative disease and brain abscesses. the posttransplant interval, patient-specific risk factors, and the timing and evolution of clinical manifestations also help to inform the diagnosis. 32 cns infections may be caused by fungi, viruses, or bacteria. fungal infections carry the highest mortality rate-90% or higher-of all pathogens. 32 most brain abscesses are associated with aspergillus. these abscesses tend to occur early in the posttransplant period, particularly in recipients who have multiple risk factors for infection such as surgical reexploration, dependence on mechanical ventilation or dialysis, or retransplantation. 33 unlike meningitis in immunocompetent patients, posttransplant meningitis is typically caused by fungi. in this setting, the patient often develops a systemic infection that subsequently spreads to the cns. viral cns infections may be associated with the reactivation of a latent virus, such as jc virus-induced progressive multifocal leukoencephalopathy, or they may be caused by a new exposure to a pathogen such as west nile virus. other pathogens commonly associated with posttransplant encephalitis include the herpes simplex virus, varicella zoster virus, ebv, and cmv. bacterial cns infections are more frequently caused by listeria and nocardia rather than more common bacterial pathogens. 32 due to the severity of cns infections in transplant recipients, an infectious disease consult, coupled with prompt diagnosis and treatment, is imperative. empiric, broad-spectrum antimicrobial agents are typically administered until the causative organism is identified. 32 immunosuppressive agents blunt the inflammatory response to infection; however, in most cases, transplant recipients with infections will have an increase in temperature. some infections, however, tend to occur in the absence of fever. these include pneumocystis pneumonia, focal fungal lung infections, and cryptococcal meningitis. 29 patients with a persistent fever greater than 38°c or acute pulmonary infiltrates or both are typically hospitalized for an infection workup. fevers of unknown origin are most commonly associated with cmv or ebv viral syndromes. it is important to note that fevers in transplant recipients may also be caused by drug reactions (particularly antilymphocyte therapy), pulmonary emboli, deep vein thrombosis, and rejection. rejection-induced fever typically occurs in lung transplant recipients. it occurs less commonly in kidney and liver transplant recipients and rarely in heart recipients. 24, 29 given that other human beings are the most frequent source of infection in the patient's environment, it is essential that nurses prevent the nosocomial transmission of respiratory viruses and the transmission of organisms through contaminated hands or inanimate objects. 29 in addition, it is important for critical care nurses to: • follow standard precautions • use aseptic techniques with vascular and urinary catheters • ensure that ventilator circuits, catheters, and dressings are changed per protocol • inspect all percutaneous catheter sites for signs of infection • maintain closed systems for urinary and suction catheters • keep the head of the bed elevated to decrease the risk of aspiration. • restrict access to patients by visitors and staff with colds or other contagious illnesses • avoid transporting transplant recipients through areas of hospital construction • promptly recognize and report the clinical manifestations of infections • obtain and report diagnostic test results in a timely manner • administer and document antimicrobial agents in a timely manner • assess and report the vaccination status of transplant candidates and recipients • know the cmv, ebv, and other pertinent serostatuses of the donor and recipient. 21, 29 infection is an important issue for critical care nurses as they care for patients throughout all phases of the transplant continuum: potential organ donors, transplant candidates, and transplant recipients. this article has reviewed salient issues relative to infections in each of these patient populations, including patients with vads, and has highlighted key points pertaining to bacterial, viral, and fungal infections. approach to the immunocompromised host with infection in the intensive care unit organ procurement and transplantation network organ procurement and transplantation network. transplants by donor type complications in patients with ventricular assist devices nonvalvular cardiovascular device-related infections ventricular assist devices in the adult ventricular assist device-related infections device-related infections: a review immunobiologic consequences of assist devices for the randomized evaluation of mechanical assistance for the treatment of congestive heart failure (rematch) study group. long-term mechanical left ventricular assistance for end-stage heart failure left ventricular assist device-related infection: treatment and outcome late-onset driveline infections: the achilles' heel of prolonged left ventricular assist device support mechanical circulatory support device database of the international society for heart and lung transplantation: third annual report -2005 infection in ventricular assist devices: prevention and treatment effect of left ventricular assist device infection on post-transplant outcomes infections during left ventricular assist device support do not affect posttransplant outcomes lvad bloodstream infections: therapeutic rationale for transplantation after lvad infection donor-derived disease transmission events in the united states: data reviewed by the optn/unos disease transmission advisory committee the epidemiology and prevention of donor-derived infections donor infection and transmission to recipient of a solid allograft bacterial infection during adult donor care available at: http:// optn.transplant.hrsa.gov/policiesandbylaws2 the ast infection disease community of practice. screening of donor and recipient prior to organ transplantation transplant complications: infectious diseases infection in solid organ transplant recipients bacterial infections in the early period after liver transplantation: etiological agents and their susceptibility clinical aspects of invasive candidiasis in solid organ transplant recipients new therapies for fungal pneumonia risk factors and approaches to infections in transplant recipients intestine transplantation mandell, douglas and bennett's principles and practice of infectious diseases neurologic manifestations of transplant complications infections of the central nervous system in transplant recipients drive-line exit site infection in a patient with axial flow pump support: successful management using vacuum-assisted therapy lung and heart-lung transplantation liver transplantation liver transplantation pancreas and kidney-pancreas transplantation pancreas and simultaneous pancreas-kidney transplantation key: cord-283826-lgyc3sro authors: stiehm, e. richard; orange, jordan s.; ballow, mark; lehman, heather title: therapeutic use of immunoglobulins date: 2010-11-05 journal: adv pediatr doi: 10.1016/j.yapd.2010.08.005 sha: doc_id: 283826 cord_uid: lgyc3sro nan medical science and thereby placed in the hands of the physician a victorious weapon against illness and death.' ' since then antibodies in multiple forms (animal and human serums, immune globulins and monoclonal antibodies) have been developed, primarily for prevention of infectious diseases, and less commonly for their treatment. these antibodies are presented in table 1 . this section reviews their uses, with an emphasis on their value in the treatment of human infections, as summarized in table 2 . antibody works by several mechanisms. it can neutralize viruses and bacterial toxins, lyse bacteria with the aid of complement, prevent the spread of microbes to adjacent cells or along nerve roots, coat bacteria for opsonization by phagocytes, block microbial attachment by saturating microbial receptors, and facilitate lysis of infected cells by binding them to cytotoxic cells with an fc receptor. antibody is particularly valuable in bacterial diseases associated with toxin production because much of the tissue damage results from action of the toxin; these can be neutralized rapidly by antibody before antibiotics kill the bacterium. anthrax (bacillus anthracis) anthrax is a rare but serious infection, predominantly of ruminant animals, caused by an aerobic gram-positive rod [1] . humans are infected through the skin (cutaneous anthrax), by ingestion (gastrointestinal anthrax), or by inhalation of anthrax spores (inhalational anthrax) [1] . the last often results from prolonged exposure to animal hides or carcasses or infected soil, and rarely by deliberate spore exposure in the bioterrorism setting. after inhalation the spores are ingested by alveolar macrophages and transported to regional nodes, where the spores germinate and release potent exotoxins. these toxins damage cell membranes, increase capillary permeability, cause pulmonary damage, and lead to shock and cardiovascular collapse. a vaccine is available for individuals at high risk for exposure and for the military. before the antibiotic era and as early as 1903, anthrax antitoxin (usually equine) was used in therapy [2] . an antitoxin is of value in a bioterrorism attack, both before and after exposure. the us government is collecting plasma from immunized donors to develop a human high-titer igiv [3] . a human monoclonal antibody is being tested in animals and humans [4] . diphtheria (corynebacterium diphtheriae). many of the adverse effects of diphtheria result from the action of its potent toxin on the heart, central nervous system, and other organs [5] . thus the prompt use of antitoxin is indicated, in addition to antibiotics [6] . the dose used depends on the localization and severity of infection, ranging from 20,000 units for mild infection of short duration to 120,000 units for severe illness with neck edema. the equine antitoxin is given intravenously, so must be preceded by skin testing for hypersensitivity and possible desensitization. the antitoxin is available through the us centers for disease control (cdc). a smaller dose of antitoxin can be used in asymptomatic, exposed, susceptible individuals. before the availability of diphtheria vaccine, antitoxins were given to health care workers caring for patients with diphtheria [7] . tetanus (clostridium tetani). equine antitoxin for the treatment of tetanus was initiated by von behring in the 1890s for toxin neutralization. extensive studies have been carried out to determine the optimal dose of antitoxin and the possible benefit of intrathecal antitoxin, particularly in tetanus neonatorum, a common problem in developing countries [8] . since the 1960s a human tetanus immune globulin (tig) has been available, but in some areas of the world equine antitoxin is still used. tig is given to unimmunized or incompletely immunized patients who sustain contaminated or deep puncture wounds [8] . the recommended dose of tig is 250 iu, along with initiation of active immunization. if tig is unavailable, human ivig can also be used; it contains variable titers of tetanus antitoxin but a minimal dose of 200-400 mg/kg is suggested for tetanus prophylaxis [8, 9] . clostridium difficile gastroenteritis. clostridium difficile infection of the gastrointestinal tract is usually associated with antibiotic-associated diarrhea, often with pseudomembranous colitis and sometimes toxic megacolon [10] toxic strains of clostridium difficile release 2 distinct toxins, both of which have potent cytotoxic and inflammatory properties [11] . infection generally leads to an antibody response to the toxin, and most individuals older than 2 years have such antibodies. high levels of these antibodies acquired after colonization may result in the asymptomatic carrier state [12] . some patients with symptomatic infection, many of whom are immunodeficient or immunosuppressed, develop antibiotic-resistant diarrhea; many have low or absent igg antibodies to toxin a. such patients may respond to ivig given 300 to 500 mg/kg every 1 to 3 weeks [13] . such therapy increases antitoxin levels, controls the diarrhea, and prevents relapses [14, 15] . controlled trials have not been performed. botulism (clostridium botulinum). botulism is a severe paralytic poisoning resulting for the ingestion or absorption of neurotoxin or spores of clostridium botulinum. several variants are recognized: food poisoning from ingestion of contaminated canned food, wound botulism from a contaminated soft-tissue infection, inhalational botulism among individuals working with the toxin or in a bioterrorist event, infantile botulism (see next section), and adult-type infant botulism in adults with preexisting gastrointestinal disease [16] [17] [18] . in the last 2 types, ingested spores multiply in the gastrointestinal tract to elaborate toxin; the absorbed toxin results in a paralytic disorder. a few cases of botulism have been associated with use of botulism toxin for cosmetic use [19, 20] . an heptavalent fab fragment equine antitoxin (hbat) to types a, b, c, d, e, f and g is available in the united states through the cdc [21, 22] . sensitivity testing must be conducted before their use. antitoxin to all 3 types is given unless the toxin type is known. additional doses may be needed in severe wound botulism. antitoxin can also by used prophylactically in individuals known to have ingested contaminated food. it is not used for infantile botulism. infantile botulism (clostridium botulinum). this severe paralytic disorder of infants results from the ingestion of clostridium botulinum spores in baby formulae or food, resulting in slow onset of constipation, abdominal bloating, poor feeding, and respiratory paralysis [22] . such infants must be hospitalized for prolonged periods for tube feeding and respiratory support, often for as long as 6 to 9 months. human iv botulism immune globulin is available for treatment of infantile botulism [23] . despite its high cost ($50,000 per vial) it is cost-effective because of the shortened hospital stay needed. gas gangrene (clostridium perfringens). there is no antitoxin for gas gangrene. respiratory infections with streptoccocci, streptococcus pneumonia, haemophilus influenzae, and neisseria meningitides are reduced in immunodeficient patients receiving immunoglobulin therapy. these patients include young infants with poor antibody responses to polysaccharide antigens, patients infected with the human immunodeficiency virus (hiv), and patients with primary antibody immunodeficiencies. before antibiotics, immune serum or animal serum was used as therapy for severe bacterial infection [24, 25] . other studies suggest that a large dose of ivig decreases the frequency of otitis in patients with recurrent otitis and normal immunity [26] . thus regular use of ivig in antibody-deficient patients in doses of 400 to 600 mg/kg every 3 to 4 weeks or an equivalent amount given subcutaneously decreases the frequency and severity of otitis and other respiratory tract infections [27, 28] . circulating antibody may play a role in the prevention and treatment of invasive group a streptococcal infection [29] . newborns with transplacental antibody and patients on ivig rarely develop streptococcal illnesses. equine antitoxin was used with some success in the treatment of erysipelas and scarlet fever in the 1920s and 1930s [30] . a preventive vaccine against the streptococcal m protein has been contemplated but is not yet unavailable. treatment with ivig, in addition to antibiotics, is probably beneficial [25, 31] . streptococcal pyrogenic exotoxins types a, b, and c and mitogenic factor elaborated by certain strains of streptococci may be responsible for these complications. these exotoxins are potent superantigens that activate certain t lymphocytes directly, leading to synthesis and/or release of multiple cytokines with resultant shock, fever, and organ failure. ivig contains neutralizing antibodies to these antigens of varying titers from batch to batch [32] . despite this variability ivig is recommended, in addition to antibiotics, in the management of these infections, not only to neutralize pyrogenic toxins but to dampen cytokine storm and release [33] . controlled trials are unavailable but case reports and large series compared with historical controls are encouraging [34] . large doses of ivig are recommended (eg, 1-2 g/kg over several days). staphylococcal infections are ubiquitous and of varying severity, ranging from superficial skin infections to deep-seated cellulitis, osteomyelitis, and overwhelming shock [35, 36] . these severe infections occur when the organism is resistant to antibiotics or is a strain associated with toxin production. one well-recognized syndrome is toxic shock associated with tampon use in menstruating women [36] . this syndrome results from release of the toxic shock syndrome toxin-1, a potent superantigen that initiates the release of multiple cytokines and a clinical picture of rapidly progressive fever, shock, and organ failure. most authorities recommend a high dose of ivig to neutralize the toxin and dampen cytokine storm [35, 37] . a second situation in which ivig may be of value is in neonatal staphylococcal infection, usually coagulase-negative staphylococcus epidermidis. this is the most common cause of sepsis in premature infants and is aggravated in part by the use of catheters and central lines [38, 39] . one controlled study indicated that ivig was of value in decreasing the incidence of this infection [40] . other studies were not confirmatory, possibly because of differences in titer for the protective antibodies [39] . immunoglobulin is also used in the treatment of antibiotic-resistant staphylococcal infection. older studies from waisbren [41] and current studies from russia suggest clinical benefit [42] . animal studies support such a combined approach [43] . newborns, particularly premature newborns with birth weight less than 2000 g are potential candidates for immunoglobulin therapy in view of the frequency and severity of infections. all newborns have low levels of igm and iga, and, if premature, a deficiency of transplacental maternal igg, the deficiency of which is proportional to the degree of immaturity [44] . premature infants also have defects in antibody synthesis, complement levels, opsonic activity, neutrophil mobilization and killing, and cellular immune responses [44] . accordingly several studies sought to determine the value of igiv in the prevention or early treatment of infection in premature infants. these studies differ in terms of entry criteria, immunoglobulin dose and duration, and end points (eg, type and severity of infection, survival). meta-analyses of prospective, randomized, placebo-controlled prevention studies suggest a slight reduction (3%) in the frequency of sepsis but no difference in mortality, length of nursery stay, or other complications of prematurity [45] [46] [47] . by contrast meta-analysis of 6 controlled studies for the treatment of proven sepsis, involving 262 premature infants, showed that igiv therapy reduced mortality from 20% to 11%, a significant difference [48] . there was a suggestive benefit for infants with suspected sepsis also. infants with neutropenia may particularly benefit. because a common cause of neonatal sepsis is staphylococcus epidermidis, a hyperimmune staphylococcal ivig may be of particular benefit in the prevention of neonatal sepsis. two recent studies of igiv from either immunized donors (altastaph) [49] or selected donors with high titers to a fibrinogen-binding protein (veronate) [50] did not show a significantly decreased incidence of infection. studies of monoclonal antibodies to staphylococcal antigens are in progress. thus the 1990 national institutes of health consensus statement that igiv should not be given routinely to infants of low birth weight but that it may be of value in selected premature newborns with proven or suspected infection remains valid [51] . patients undergoing severe stress associated with trauma, extensive surgery, or intensive care have profound exposure to and susceptibility to infection, usually as a result of enteric gram-negative infections [52, 53] . monoclonal antibodies, igm-enriched igiv, and regular igiv have been studied in these situations with inconclusive results [5] . laupland and colleagues [54] reviewed 14 randomized trials of igiv and found suggestive benefit in terms of length of stay in the intensive care unit (icu) and mortality. similar studies in pediatric patients in the icu have not been performed. despite the lack of controlled trials, igiv is often used in critically ill patients, particularly neutropenic patients, because of possible benefit and rare side effects. although many viral diseases are prevented by immunoglobulin, just a few are amenable to antibody therapy, as presented in table 2 . this section focuses on some viral diseases in which antibodies can be used in therapy. although smallpox (variola) has been eradicated from the world since 1977, immunization with live vaccinia virus (cowpox virus) is still used by the military and by certain laboratory personnel working with vaccinia [5] . further, smallpox is a potential bioterrorism weapon so a supply of vaccinia immune globulin (vig) is being stockpiled by the us government for complications of smallpox vaccine and for a response to biological warfare. kempe [55] used immune globulin from vaccinated individuals (vig) to prevent the spread in a 1953 outbreak of smallpox in madras, india. he also showed that vig could be used to treat the not infrequent complications of smallpox vaccine including vaccinia eczematum, generalized vaccinia, autoinoculation, and prevention of spread to high-risk individuals exposed to a recently vaccinated individual. vig, both for iv and intramuscular (im) use, is prepared from vaccinated donors and is commercially available. the usual dose is 100 mg/kg [56] . parvovirus is a dna virus that causes fifth disease (slapped cheek syndrome, a common exanthem of childhood that usually provides lifelong immunity to subsequent exposure [57] ). parvovirus infects erythroid progenitors (its receptor is the common red cell p antigen) to cause red cell aplasia in patients with congenital or acquired immunodeficiencies including hiv, immunosuppressed organ transplant recipients, and patients with sickle cell disease [57] [58] [59] . igiv contains neutralizing antibody to parvovirus such that prolonged highdose therapy can eradicate the infection. parvovirus infection during pregnancy can also cause fetal hydrops [60] . arthritis and chronic fatigue syndrome are uncommon manifestations of chronic parvovirus infections [60, 61] . the ivig dose needed to eradicate parvovirus in not established but is large (1-2 g/kg) and should be repeated until the virus is eradicated as indicated by serum polymerase chain reaction analysis [62, 63] . antibodies to cytomegalovirus (cmv) either in the form of hyperimmune iv cmv immune globulin (cmvig-cytogam) or regular igiv have been used for more than a decade to prevent cmv infection in recipients of bone marrow and solid organ transplant [64] . cmvig is prepared from donors with high anti-cmv titers but regular igiv also contains cmv antibodies at lower titers. testing of donor and recipient for cmv infection, the use of cmv antibodynegative blood donors, and the use of antiviral drugs have greatly reduced the indications for cmv antibody [65] . cmvig is still used in heart and heartlung transplants (along with antivirals) if either the donor or the recipient is cmv-seropositive [66] . cmvig is also of suggestive benefit in severe cmv pneumonitis along with antiviral treatment [67] . cmvig may also be of value for in utero cmv infection; infusions of cmvig were given intraperitoneally at 28 and 29 weeks to a cmv-infected fetus, with possible benefit [68] . nigro and colleagues [69] gave 31 pregnant women with primary cmv infection cmvig during pregnancy; some women received additional cmvig into the amniotic sac or umbilical cord. only one woman gave birth to an infant with cmv infection compared with cmv infection in 7 of 14 infants of control women who did not receive antibody therapy. these data are encouraging but are not from well-controlled studies. thus the use of cmvig in recipients of organ transplant, severe cmv infections, or in utero cmv infections is unproved but of suggestive therapeutic benefit. transplacental maternal antibody has a proven preventive effect in herpes simplex virus (hsv) infection in the newborn period: mothers with a reactivated herpex infection (ie, preexisting infection) during delivery are 10-fold less likely to transmit hsv to their newborn infants during vaginal delivery than are mothers with primary hsv infection acquired during late pregnancy [70] . masci and colleagues [71] used ivig to prevent recurrent genital hsv infection with suggestive benefit. the value of hsv monoclonal antibody or ivig is being evaluated for treatment of disseminated neonatal disease. epstein-barr virus (ebv) antibodies are present in variable titers in ivig, particularly in cmvig, because donors with high titers of cmv often have high titers of ebv. a few patients with posttransplant ebv-induced lymphoproliferative syndrome or hepatitis have been treated successfully with a combination of igiv or cmvig, antiviral therapy and interferon-a [72] [73] [74] . similar results have been achieved in ebv infection in x-linked lymphoproliferative syndrome: such patients have a hereditary predisposition to overwhelming ebv infection [75] . varicella-zoster immune globulin (vzig), available since 1978, is prepared from plasma with high titers to vz virus [76] . the commercial product vari-zig is used for the prevention or modification of susceptible high-risk immunodeficient or immunosuppressed children exposed to chickenpox or shingles. it is also used in susceptible women during late pregnancy, newborn infants whose mother develops chickenpox perinatally, and exposed premature infants of less than 28 weeks' gestation. it is not of benefit in established chickenpox or zoster infection [77] . encephalomyelitis. before poliovirus vaccine was introduced, immunoglobulin was used in the prevention of poliomyelitis [78] . immunodeficient individuals are susceptible to chronic enteroviral encephalitis, usually echovirus or coxsackievirus or less commonly, attenuated poliovirus vaccine strains [79] [80] [81] [82] . regular doses of igiv given to antibody-deficient patients have markedly reduced the frequency of enterovirus encephalitis in these patients. attenuated poliovirus has been replaced in many countries by inactivated (salk) vaccine. high-dose ivig (sufficient to increase the serum igg levels to 1000 mg/ml) has been used successfully in immunodeficient patients with enteroviral encephalomyelitis [80] [81] [82] [83] . some patients have been given intrathecal infusions [80, 81] . not all ivig-treated patients are cured: some may have viral strains for which the ivig has no neutralizing antibody. for these instances typing of the cerebrospinal fluid and treatment with selective ivig units with antibodies to the infecting serotype may be necessary. antiviral therapy with pleconoril has also been used [83] . neonatal enteroviral infection. severe and sometimes fatal disseminated enterovirus infection can develop in neonates [84] [85] [86] . high-dose ivig has been used in such infants with suggested benefit in decreasing the severity of the illness [84] . maternal plasma may also be used in the likelihood that the mother has antibody to the organism involved [85] . ivig has also been used to prevent spread to unaffected infants in a nursery [86] . unless the titer in the ivig is known, large doses are recommended. an increasingly important use of hyperimmune hepatitis b immune globulin (hbig) is to prevent hepatitis b recurrence in hepatitis b-seropositive recipients of liver transplant, many of whom are transplanted because of complications of hepatitis b [87, 88] . hepatitis b reoccurs in half of the patients in 3 years [89] . such recurrences can be reduced significantly by giving large doses of hbig for a prolonged period beginning at the time of transplantation and continuing indefinitely after transplantation [89] . antiviral agents such as lamivudine are also given simultaneously. the dose of hbig after transplantation is varied so as to maintain a continuous serum anti-hbs titer. hepatitis b vaccine can also be given to induce active immunity. the 2 types of hbig available include the 16% igim used for prophylaxis in newborns of hepatic b-positive mothers and for unimmunized exposed susceptibles and a 5% hbig for iv use in liver transplantation. the use of the latter adds a considerable cost to liver transplantation. the university of california at los angeles medical center spends $500,000 per year on hbig, nearly all for the liver transplant program. a hyperimmune hepatitis c immune globulin for hepatitis c liver transplantation is also under study. monoclonal antibodies to hepatitis b and c are under development. west nile fever. west nile fever, caused by the west nile virus, is common in many tropical regions where culex mosquitoes are endemic. it has spread to europe and the united states, and can also be transmitted by infected blood and organ transplantation. several case reports and animal studies suggest that ivig prepared from seropositive donors modifies the severity and mortality [90, 91] . ebola. ebola virus, a filivirus, causes severe and often fatal hemorrhagic fever in tropical africa. there is no effective antiviral agent. goat hyperimmune serum protected guinea pigs from experimental infection if given within 72 hours of exposure. this product was used for emergency prophylaxis in 4 patients exposed by a laboratory accident. only one developed mild infection [92] . equine serum has protected monkeys against low-dose virus challenge but not high-dose virus challenge [93] . blood from convalescing patients has also been used with promising results [94] . other animal antisera have been developed, as have monoclonal antibodies. tick-borne encephalitis. tick-borne encephalitis caused by a flavivirus is endemic in central europe. a vaccine is available as is a hyperimmune immune globulin. a combination has been also used [95, 96] . argentine hemorrhagic fever. argentine hemorrhagic fever caused by the junin virus has a high mortality from vascular or neurologic complications. maiztegui and colleagues [97] found that immune plasma given before the ninth day of illness reduced mortality to 1% among 91 patients given immune plasma compared with 16.5% mortality among 97 patient given normal plasma. severe acute respiratory distress syndrome. convalescent plasma and ivig have been used in the treatment of severe acute respiratory distress syndrome caused by a corona virus. studies were inconclusive [98] . antibody is a time-honored way to prevent viral infection after exposure, and has a crucial role in the treatment of bacterial diseases associated with toxin production. it is also of value in prevention of certain viral infections as well as in the treatment of parvovius, enterovirus infection, and certain regional viral infections. polyclonal immunoglobulin, now used in scores of diverse disorders [99] [100] [101] , was first used in the prevention of infectious diseases. in 1952, ogden bruton [102] reported a child with agammaglobulinemia and initiated the first use of repeat injections of immunoglobulin as replacement therapy. in his report c-globulin fractionated from human plasma was administered subcutaneously to an 8-year-old boy who had no known c-globulin in a serum protein electorophoresis. this child had multiple infections, including 19 episodes of septicemia, which were ameliorated by chronic treatment with the immunoglobulin. this experience represented the dawn of immunoglobulin therapy for primary immunodeficiency and defined its use in a disease for which no therapeutic alterative was available. since then, the study of primary immunodeficiency has expanded markedly. there are now more than 140 distinct diagnoses, most of which have defects of humoral immunity [103] . approximately 1 in 2000 people are living with a primary immunodeficiency in the united states, of whom greater than 50% have an antibody deficiency potentially requiring immunoglobulin replacement therapy [104] . other primary immunodeficiency registries confirm that greater than 50% have an antibody deficiency [105] [106] [107] [108] . treatment with immunoglobulin remains the best therapeutic option for most of these patients. characteristics of antibody immunodeficiencies appropriate for replacement therapy are presented in table 3 . the clearest indications for immunoglobulin therapy are those associated with an absence of b cells (category i). these patients are unable to make antibodies or immunoglobulin i. examples include agammaglobulinemia and certain types of severe combined immunodeficiency. several gene defects may be responsible for these illnesses [109] , but all need immunoglobulin replacement therapy. the next category (ii) of patients needing immunoglobulin are those who have b cells but cannot make igg and generate specific igg antibodies. because igg represents the major defense of humoral immunity against infection, these patients also require immunoglobulin replacement therapy. this diagnostic category includes the hyper igm syndrome (higm) and common variable immunodeficiency (cvid). higm is caused by several specific gene mutations [110] , but most cvid cases have no identifiable genetic lesions [111] . diagnosis can be made by either identifying a specific gene mutation, or by defining the quantitative and qualitative deficit of igg [112] . as in patients in category i, continuous and uninterrupted replacement therapy with immunoglobulin is warranted. if the diagnosis is confirmed molecularly, immunoglobulin therapy must be continued. in a few cases, it may be clinically appropriate to stop immunoglobulin therapy once during a lifetime to determine if the defect is fixed [1] . this strategy should not be repeated if the single trial indicates a persistent deficit. if a trial off immunoglobulin therapy is considered, this should be performed in late spring or summer, when respiratory infections are less prevalent. a third diagnostic category (iii) of antibody deficiencies is those associated with qualitative defects in humoral immunity [113] . these patients have b cells and produce normal quantities of igg but the quality of igg is diminished. these individuals are unable to respond appropriately to specific antigenic challenges such as vaccinations or infections. this category includes those with specific antibody deficiency with normal immunoglobulins [113] and certain patients with nemo (nf-kappa;-b essential modulator) deficiency [114, 115] . diagnosis is made after documentation of an ineffective vaccination response, a failed humoral response to an infection, or a specific molecular/ genetic diagnosis linked to this category [112] . a fourth category (iv) includes patients with lower than expected levels of igg but who are able to mount effective antibody responses. this category forms a subset of individuals referred to as having ''isolated hypogammaglobulinemia'' when only the igg level is low. although hypogammaglobulinemia can be a component of many immunologic defects, in isolated hypogammaglobulinemia antibody quality is adequate, with normal responses to vaccination or infection. because the normal age-specific ranges of igg define the lower limit at the 2.5th percentile, one of 40 individuals has low levels of igg. the question becomes, when there is no deficit of antibody quality, is isolated hypogammaglobulinemia clinically a problem? it is also important to discern when hypogammaglobulinemia represents a primary versus a secondary problem with increased loss of igg. examples of the latter include draining chylothorax [116] or intestinal lymphangiectasia [117] . in these individuals, the hypogammaglobulinemia is less likely to cause a problem because antibody synthesis is intact and often accelerated. in patients with primary hypogammaglobulinemia, the level of igg that is associated with a definitive risk for infection is not defined, especially when antibody quality is intact [112] . some insurance companies recommend replacement therapy for patients who have an igg level less than 400 mg/dl and a history of recurrent infection. although that situation may be reasonable, questions still exist about how to manage the patient recognized as having primary hypogammaglobulinemia with low igg levels (ie, <150) but no history of infection. diagnostic examples include transient hypogammaglobulinemia of infancy (thi) [118] [119] [120] or otherwise unexplained primary hypogammaglobulinemia [121] . the former diagnosis is established in retrospect, as the igg level normalizes with age. thus, in select cases of thi immunoglobulin replacement may be considered as a temporizing measure. however, primary hypogammaglobulinemia remains a difficult diagnostic and therapeutic dilemma. other patients (a fifth diagnostic category [v]) have a deficiency of one of the 3 major igg subclasses, igg1, igg2, or igg3. igg4 deficiency is common and should not be considered an abnormality [99] . although a deficiency of one of the major igg subclasses indicates some immunologic deviation, most of these patients have a normal total igg level, intact responses to specific antigens, and are not candidates for immunoglobulin replacement therapy. those with impaired antibody specificity do not fall in to this category, but into the third category. however, even without impairment in antibody quality, immunoglobulin replacement in some patients in a deficiency subclass does reduce the incidence of infections [122, 123] . nevertheless, most insurers in the united states have additional criteria for justifying therapy in patients with igg in deficiency subclasses. a final diagnostic category is patients with recurrent infection who do not have hypogammaglobulinemia subclass deficiency or deficits of antibody quality. thus, they have infectious susceptibility without evidence of identifiable immune abnormality. the infectious burden in these individuals can be high and most certainly has an explanation, so nonhumoral diagnoses should be aggressively sought. there are also patients an explanation of whose infectious susceptibility presently evades clinical science. immunoglobulin replacement therapy has been considered in these individuals under certain circumstances. immunoglobulin preparations for antibody immunodeficiencies although bruton [102] gave immunoglobulin to his patient by the subcutaneous (sc) route, subsequent patients until 1970 received immunoglobulin by weekly im injections [124] . this strategy was necessary because the immunoglobulin preparations were not purified to the degree required for iv administration. in the early 1970s, immunoglobulin preparations with low quantities of immunoglobulin aggregates were developed for iv administration. ivig and igiv have numerous advantages, including achieving high peak and trough igg levels and convenient monthly dosing regimens. although limited studies have compared ivig with imig, the iv route has become the preferred route of immunoglobulin administration worldwide [125] . seven ivig preparations are currently approved by the us food and drug administration (fda) for replacement therapy in primary immunodeficiency (table 4 ). each has been studied in a licensing trial in patients with primary immunodeficiency and found to be safe and effective. the primary end point in most of these clinical trials has been the prevention of serious bacterial infection compared with the expected frequency of such infections before diagnosis [126] . the rate of infection can be surprisingly high, as shown by bruton's [102] first patient mentioned earlier. the early diagnosis and treatment of primary immunodeficiency with immunoglobulin products has reduced morbidity and mortality and considerable savings of health care expenditures [127, 128] . all ivig products are purified from human plasma pools under strict manufacturing guidelines. although each manufacturer has its own process there are more similarities than differences in the various methods. all processes remove non-igg impurities and igg aggregates and add stabilizers to prevent in vitro aggregate formation. despite these efforts, adverse reactions during ivig administration are not uncommon [129] . all immunoglobulin manufacturers have robust measures to screen donors and to inactivate blood-borne pathogens; the safety of immunoglobulin preparations in the last decade has been superb [130] . there are subtle differences among different ivig products from different companies; several companies have more than one product on the market [131] . this situation can lead to confusion about which ivig to administer to which patient. in general, most ivig products are tolerated by most patients. the characteristics of the individual ivig preparations, as outlined in table 2 , may help in selecting the best product for each patient. they differ as to concentration, stabilizers, sugar content, iga content, sodium content, and osmality. the volume of individual vials, storage requirements, need to reconstitute a lyophilized product before use, local availability, and price are also variable. many patients may tolerate one product more effectively than another. thus, when a patient tolerates a particular immunoglobulin product it is advisable to continue with that product whenever possible [99] . three other preparations of immunoglobulin are approved by the us fda (table 5) . one is approved for im administration and two for sc administration. few patients receive their immunoglobulin by the im route. the sc administration of immunoglobulin resurfaced in 1980 in the united states [132] . subcutaneous immunoglobulin (scig) is usually given in the abdominal wall or thigh with a thin bored needle and an infusion pump, delivered over several hours. although its initial use in the united states was limited, the sc route gained popularity in europe; extensive clinical experience indicated that it was equivalent to ivig therapy [133, 134] . a crossover trial with ivig and a us fda licensing trial showed that scig was equivalent to ivig in preventing infection in primary immunodeficiency [135, 136] . scig has advantages and disadvantages compared with ivig therapy (many related to patient preferences) and these have been reviewed extensively [137] [138] [139] . one advantage of scig over ivig is the markedly decreased incidence of systemic reactions [134, 135] . another is eliminating the need for iv access or indwelling iv access devices. the most serious disadvantage is the need for more frequent administration (at least weekly) to administer sufficient immunoglobulin [140] . another disadvantage is less frequent physician encounters because most scig infusions are given at home by caretakers or home infusion companies. the dose and frequency of immunoglobulin therapy is a complex topic and draws on both evidence-and experience-based sources. these recommendations are presented in a several reviews and consensus statements [99, 112, 138, 139, 141] . the recommendations include starting doses of 400 to 600 mg/kg/mo. after several months this dose can be altered depending on the trough level and the clinical response. patients vary as to their requirement to maintain reasonable resistance to infection [141, 142] . scig is typically used after the patient has been on ivig for several months. the weekly scig dose is usually one-fourth of the previous monthly ivig dose. some immunoglobulin-naive patients are started on immunoglobulin therapy with scig so the number of initial doses may need to be increased. the amount of scig given at a single site for an adult is usually 20 ml of the 16% solution (ie, 3.2 g). more than one site can be used simultaneously to deliver the target dose. this procedure has been facilitated by the availability of special tubing, needle sets, catheters, and pumps. infusion site reactions are not uncommon but are rarely severe [136] . ivig is usually administered monthly and scig is usually administered weekly, but other schedules are often used. these schedules include shorter or longer intervals between infusions of ivig to achieve a satisfactory clinical response. scig can be given biweekly, or divided into more frequent injections, even small daily doses. the latter is generally self-administered at home, well tolerated, and preferred by some patients because of the small daily dose needed [143, 144] . trough levels of immunoglobulin achieved must be considered. several studies have correlated resistance to infection with specific igg trough levels. targeting a specific trough level may be feasible for patients with agammaglobulinemia who have a profound deficiency of igg [126] but more difficult for other antibody deficiencies [145] [146] [147] . in agammaglobulinemia, a trough level of 500 mg/dl is a minimally acceptable level and 800 mg/dl a more desirable trough level [99, 126] . these recommendations may not be appropriate in other disorders in which baseline igg levels and antibody titers are variable; in these cases the clinical response must be considered. polyclonal immunoglobulin is essential therapy for the primary antibody immunodeficiency diseases. the different disorders in which immunoglobulin therapy are used are reviewed. several immunoglobulin products are available for their treatment; they have similar therapeutic properties but there are individual differences among the available products. immunoglobulin can be given either intravenously (ivig) or subcutaneously (scig). dosage, frequency of infusions, achieved trough levels, and advantages and disadvantages of ivig and scig are discussed. the early 1980s witnessed an increase in the use of ivig as an immunomodulator for inflammatory and autoimmune disorders. more than 70% of the ivig prescribed is for patients with autoimmune and inflammatory diseases, despite the fact that ivig is approved for just a handful of indications (box 1). in the late 1990s, this situation led to an ivig shortage, compromising those patients who depend on igg replacement therapy to correct their underlying antibody deficiency. in 2006, the american academy of allergy, asthma and immunology's committee on primary immunodeficiency evaluated the use of ivig for multiple disorders. the strength of the evidence for a beneficial effect and the basis for this recommendation were classified (box 2). this section reviews the use of ivig for the autoimmune and inflammatory conditions in this report (box 3), in the context of a review of the mechanisms of action of ivig in these conditions. the multiple effects of ivig on the innate and adaptive immune system are illustrated in fig. 1 . historical note: ivig in immune thrombocytopenic purpura the first use of ivig for an autoimmune process was in children with immune thrombocytopenic purpura (itp). imbach and colleagues [148] observed that antibody-deficient patients receiving ivig who also had itp had a marked increase in platelet count after ivig infusions. subsequently, these investigators examined the therapeutic effects of ivig in children with a primary diagnosis of itp; they used high-dose ivig (400 mg/kg) for 4 consecutive days. the investigators reported a dramatic increase in platelet count within hours of the administration of ivig. in some patients, the increase in platelet count was sustained; in others, repeat ivig treatments were necessary. box 3 presents the indications for ivig in autoimmune cytopenias as well its likely benefit. several hypotheses have been proposed to explain the rapid increase in platelet count (or other antibody-coated cells) after ivig administration. the most accepted hypothesis is that high-dose ivig induces an fc receptor blockade of reticuloendothelial cells in the liver and spleen, preventing them from removing antibody-sensitized cells. debre and colleagues [149] provided evidence for this hypothesis when they infused fcc fragments in children with itp, and showed an increase in platelet count after the infusion. the fc receptor blockade theory may account for the rapid increase in platelet count after the ivig infusion, but not for the longterm benefits of ivig. thus additional mechanisms have been sought. one such mechanism, supported by animal studies, is that ivig stimulates inhibitory fccriib receptors found on a variety of cell types including b cells that in turn inhibit antibody and immune function [150] . samuelsson and colleagues [151] showed in a mouse model of itp that ivig suppresses or inhibits antiplatelet antibody production through this fccriib receptor. subsequently ravetch and colleagues [152, 153] identified distinct motifs in the ivig that have a propensity to engage and activate the fccriib inhibitor receptor that inhibits antibody synthesis. these distinct properties were attributed to the carbohydrate moiety in the ivig molecule, representing about 5% of the total igg molecule. more than 30 different covalently attached carbohydrate glycans in the igg molecule have been identified. glycosylation of the igg is essential for binding to all fcc receptors. the important glycan moiety in the igg molecule is attached to the asparagine (asn 297 ) in the second domain of the constant region of the igg molecule. using a k/bxn serum-induced arthritis model in mice, kaneko [152] showed that igg at 1 g/kg inhibited the inflammatory arthritic process. deglycosylated or neuraminidase-treated ivigs were unable to inhibit this inflammation. kaneko then showed that ivig enriched for the sialylated glycan moiety had comparable inhibitory effects on the inflammatory process at one-tenth of the dosage used with intact ivig. this investigator showed that this inhibitory activity resided in the igg fc fragment, and was dependent on fccriib expression on effector macrophages. anthony and colleagues [154] have engineered a recombinant/sialylated human igg1 fc protein that had the same immune modulating activity as native ivig. these investigators showed that the action of sialylated fc in the rheumatoid arthritis mouse model is mediated through the interaction of sialylated fc with the sign-r1 receptor on macrophages [154] . the investigators propose that the interaction between sialylated fc and sign-r1 produces an antiinflammatory state that upregulates inhibitory fccriib receptors on effector cells, making these cells more resistant to triggering by immune complexes. they suggest that dc-sign, the human homolog of sign-r1, has a comparable role for the antiinflammatory effects of igg fc fragments. another mechanism proposed by yu and lennon [155] suggested that the administration of high-dose ivig augments the catabolism of endogenous serum igg. igg catabolism occurs through a process by which the igg molecule binds to a specialized fc receptor found on endothelial cells (eg, fcrn), which protects the igg molecule from normal catabolism and its removal from the plasma. this process accounts for the long serum igg half-life (21 days). high-dose ivig saturates the fcrn receptor, resulting in the accelerated catabolism of autoantibodies [156] [157] . hansen and balthasar [157] have supporting data in a rat model of immune thrombocytopenia using monoclonal antibodies. the uses of ivig in several autoimmune inflammatory neuropathies are presented in box 3. the fda has recently approved the use of ivig in chronic inflammatory demyelinating polyneuropathy. this table also shows the evidence-based efficacy of ivig in rheumatic disorders. aside from the mechanisms involving the fccriib inhibitory receptor and the accelerated catabolism of autoimmune antibodies through the fcrn receptor, it has also been proposed that the administration of ivig can regulate autoreactive b cells by restoring the idiotypic-antiidiotypic network. other autoimmune diseases may be associated with a deficiency of these antiidiotypic antibodies, which are believed to regulate the production and activity of these autoantibodies (box 4). kazatchkine and colleagues [158] showed that f(ab 9 ) 2 fragments prepared from ivig could bind to several autoantibodies (eg, antifactor viii, antithyroglobulin, anti-dna, antiintrinsic factor, neutrophil cytoplasmic antigens), and thus lead to increased catabolism of these autoimmune antibodies and prevent them from inducing tissue injury [159] . these investigators postulated that ivig may work, at least in part, in certain autoimmune diseases by neutralizing the functional activity of various autoantibodies or inhibiting their binding to their respective autoantigens [160] . another mechanism by which ivig may benefit autoimmune disease is by preventing the uptake of complement on target tissues. berger and colleagues [161] showed that high concentrations of igg inhibit the uptake of c3 on antibodysensitized erythrocytes. thus, any inflammatory or autoimmune process that involves a c3b-or c4b-dependent process could be modulated by ivig therapy. this situation is best exemplified in patients with dermatomyositis in whom the disease is mediated by activation of c3 and deposition of the membrane attack complex on the endomysial capillaries [162] . treatment with ivig inhibits complement-induced inflammation by decreasing complement deposition on the endomysial capillaries of muscle tissues [163, 164] . this mechanism of ivig is relevant not only in dermatomyositis but also in guillain-barrã© syndrome and myasthenia gravis [165, 166] . as shown in box 3, ivig is used in many other inflammatory diseases. however, the evidence-based data for several of these diseases are not so strong as some of the autoimmune disorders discussed earlier. nevertheless, one inflammatory disease in which ivig may be beneficial is toxic epidermal necrolysis or stevens-johnson syndrome. patients with toxic epidermal necrolysis have high levels of serum-soluble fas ligand that bind to fas receptors on keratinocytes to induce apoptosis (cell death). viard and colleagues [167] showed that the anti-fas antibodies in ivig block the interaction of fas ligand with fas receptors on the keratinocytes, preventing destruction of the epithelium. ivig contains antibodies to several cell-surface molecules [168] including antibodies to a 10-peptide sequence containing the (arg-gly-asp) motif that is expressed on cell surfaces and matrix proteins that are part of the integrin adhesion system. ivig inhibits the adhesion of b cells to fibronectin and inhibits platelet aggregation [169] . turhan and colleagues [170] and chang and colleagues [171] investigated the effect of ivig on a mouse model of sickle cell acute vasoocclusive crisis, in which the adhesion of sickled red blood cells to leukocytes causes the vasoocclusive disease. in this model, high-dose ivig given after the onset of a crisis resulted in improved blood flow and prolonged survival. these investigators showed that ivig reverses acute vasoocclusive crisis in sickle cell mice by inhibiting neutrophil adhesion to the capillary endothelial cells. the various mechanisms of the antiinflammatory and immunomodulatory properties of ivig are reviewed. the first use of ivig was in the treatment of immune thrombocytopenia, presumably because of fc receptor blockade. other mechanisms are reviewed as well as the evidence for the value of ivig in multiple disorders. ivig may have yet undiscovered immunomodulating properties on both the innate and adaptive immune systems. future advances will include a better understanding of its mechanisms of action and modification of the igg molecule to enhance its immunomodulating properties. idiopathic thrombocytopenic purpura (ia-a) might provide benefit autoimmune neutropenia autoimmune hemolytic anemia (iii-d) posttransfusion purpura (iii-d) inflammatory neuropathies definitely beneficial: guillain-barrã© syndrome (ia-a) chronic inflammatory demyelinating polyneuropathy multifocal motor neuropathy (ia-a) probably beneficial: myasthenia gravis (ib-iia-b) eaton myasthenic syndrome (ib-a) igm antimyelin-associated glycoprotein paraprotein-associated peripheral neuropathy (ib-a) stiff man syndrome (ib-a) might provide benefit: relapsing-remitting multiple sclerosis (ia-a) intractable childhood seizures rasmussen syndrome (iib-b) acute disseminated encephalomyelitis (iii-c) lumbosacral or brachial plexitis human t-lymphotropic virus-1-associated myelopathy (iii-c) postinfectious cerebellar ataxia acute idiopathic dysautonomia (iii-d) unlikely to be beneficial: demyelinating neuropathy associated with monoclonal igm (ib-a) amyotrophic lateral sclerosis (iii-c) poems syndrome paraneoplastic neuropathies (iii-c) rheumatologic and organ-specific autoimmune diseases definitely beneficial: graves ophthalmopathy (ib-a) probably beneficial: autoimmune uveitis (iia-b) might provide benefit: severe rheumatoid arthritis autoimmune diabetes mellitus (iib-b) vasculitides and antineutrophil antibody syndromes systemic lupus erythematosus (iii-d) unlikely to be beneficial: antiphospholipid antibody syndrome toxic epidermal necrolysis/ stevens-johnson syndrome (iia-b) might provide benefit: steroid-dependent asthma (ib-a) prevention of acute humoral rejection in renal transplants (ib-a) treatment of acute humoral rejection in renal transplants pediatric autoimmune neuropsychiatric disorder associated with streptococcus (pandas) (iib-b) subset of women (repeat second-trimester loss) with spontaneous recurrent abortions unlikely to be beneficial: nonsteroid-dependent asthma (ib-a) prevention of chronic gvhd after bone marrow transplantation (ib-a) chronic fatigue syndrome atopic dermatitis (iia-b) use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology principles and practice of infectious diseases studies on anthrax: clinical report of ten human cases hhs to buy 20,000 courses of anthrax antitoxin. available at: cidrap.umn raxibacumab or the treatment of inhalational anthrax philadelphia: saunders/elsevier report of the committee on infectious diseases history of the american pediatric society 1887-1965 report of the committee on infectious diseases anti-tetanus toxoid antibodies in intravenous gamma globulin: an alternative to tetanus immune globulin treatment of clostridium difficile infection clostridium difficile-associated diarrhea and colitis: clinical manifestations, diagnosis, and treatment asymptomatic carriage of clostridium difficile and serum levels of igg antibody against toxin intravenous immunoglobulin for the treatment of severe, refractory, and recurrent clostridium difficile diarrhea treatment with intravenously administered gamma globulin of chronic relapsing colitis induced by clostridium difficile toxin intravenous immunoglobulin therapy for severe clostridium difficile colitis botulinum toxin as a biological weapon: medical and public health management syndrome of infant botulism handbook for epidemiologists, clinicians and laboratory workers botulism in 4 adults following cosmetic injections with an unlicensed, highly concentrated botulinum preparation severe botulism after focal injection of botulinum toxin investigational heptavalent botulinum antitoxin (hbat) to replace licensed botulinum antitoxin ab and investigational botulinum antitoxin e botulism and infant botulism human botulism immune globulin for the treatment of infant botulism treatment of haemophilus influenzae infection and of meningococcic and pneumococcic meningitis return to the past: the case for antibody-based therapies in infectious diseases respiratory syncytial virus-enriched globulin for the prevention of acute otitis media in high risk children prophylactic intravenous immunoglobulin in hivinfected children with cd4 ã¾ counts of 0.20 ã� 10 9 /l or more: effect on viral, opportunistic, and bacterial infections national institute of child health and human development (nichhd) intravenous immunoglobulin study group. intravenous immune globulin for the prevention of bacterial infections in children with symptomatic human immunodeficiency virus infection passive antibody therapies: progress and continuing challenges antitoxin versus no antitoxin in scarlet fever adjunctive treatment of streptococcal toxic shock syndrome using intravenous immunoglobulin: case report and review different preparations of intravenous immunoglobulin vary in their efficacy to neutralize streptococcal superantigens: implications for treatment of streptococcal toxic shock syndrome clinical usefulness of intravenous human immunoglobulins in invasive group a streptococcal infections: case report and review intravenous immunoglobulin for streptococcal toxic shock syndrome-a comparative observational study report of the committee on infectious diseases vaginal tampon model for toxic shock syndrome philadelphia: saunders/elsevier opsonic antibodies to staphylococcus epidermidis: in vitro and in vivo studies using human intravenous immune globulin the role of intravenous immunoglobulin for the prevention and treatment of neonatal sepsis intravenous immune globulin for the prevention of nosocomial infection in low-birth-weight neonates the treatment of bacterial infections with the combination of antibiotics and gamma globulin immunotherapy against antibiotic-resistant bacteria: the russian experience with an antistaphyloccocal hyperimmune plasma and immunoglobulin synergism between human gamma globulin and chloramphenicol in the treatment of experimental bacterial infections the physiologic immunodeficiency of immaturity meta-analyses of the effectiveness of intravenous immune globulin for prevention and treatment of neonatal sepsis administration of intravenous immunoglobulins for prophylaxis or treatment of infection in preterm infants: meta-analysis intravenous immunoglobulin for preventing infection in preterm and/ or low birth-weight infants (cochrane review). the cochrane library intravenous immunoglobulin for suspected or subsequently proven infection in neonates a blinded, randomized, multicenter study of an intravenous staphylococcus aureus immune globulin multicenter study to assess safety and efficacy of inh-a21, a donor-selected human staphylococcal immunoglobulin, for prevention of nosocomial infections in very low birth weight infants nih consensus development conference: diseases, doses, recommendations for intravenous immunoglobulin. hlb newsletter polyvalent immunoglobulins for prophylaxis of bacterial infections in patients following multiple trauma the secondary immunodeficiencies polyclonal intravenous immunoglobulin for the treatment of severe sepsis and septic shock in critically ill adults: a systematic review and meta-analysis studies on smallpox and complications of smallpox vaccination smallpox (variola) doling r, editors. mandell, douglas and bennett's principles and practice of infectious diseases parvovirus b19 infection: aplastic crisis, erythema infectiosum and idiopathic thrombocytopenic purpura parvovirus infection and its treatment report of the committee on infectious diseases persistent parvovirus-associated chronic fatigue treated with high dose intravenous immunoglobulin persistent b19 parvovirus infection in patients infected with human immunodeficiency virus type 1 (hiv-1): a treatable cause of anemia with aids chronic pure red cell aplasia caused by parvovirus b19 in aids: use of intravenous immunoglobulin: a report of eight patients cytomegalovirus infection in kidney transplantation: prophylaxis and management clinical practice guidelines: prevention of cytomegalovirus disease after renal transplantation comparison of combined prophylaxis of cytomegalovirus hyperimmune globulin plus ganciclovir versus cytomegalovirus hyperimmune globulin alone in high-risk heart transplant recipients immunotherapy of cmv infections intraperitoneal administration of cytomegalovirus hyperimmunoglobulin to the cytomegalovirus-infected fetus passive immunization during pregnancy for congenital cytomegalovirus infection report of the committee on infectious diseases intravenous immunoglobulins suppress the recurrences of genital herpes simplex virus: a clinical and immunological study successful treatment of epstein-barr virus infection with ganciclovir and cytomegalovirus hyperimmune globulin following kidney transplantation treatment with ganciclovir and ig for acute epstein-barr virus infection after allogeneic bone marrow transplantation the effect of intravenous immunoglobulin and interferonalpha on epstein-barr virus-induced lymphoproliferative disorder in a liver transplant recipient immune-mediated hematologic and oncologic disorders, including epstein-barr virus infection passive immunization against varicella zoster infections varicella-zoster infections experimental studies on passive immunization against poliomyelitis: i. protection with human gamma globulin against intramuscular inoculation and combined passive and active immunization chronic enteroviral meningoencephalitis in agammaglobulinemic patients intraventricular gamma-globulin for the management of enterovirus encephalitis successful treatment of echovirus meningoencephalitis in sex-linked agammaglobulinaemia by intrathecal and intravenous injection of high titer gammaglobulin chronic enteroviral meningoencephalitis, in agammaglobulinemia: case report and literature review enteroviral meningitis: natural history and outcome of pleconaril therapy neonatal enterovirus infection: virology, serology, and effects of intravenous immune globulin investigation of treatment failure in neonatal echovirus 7 infection use of normal immunoglobulin in an echovirus 11 outbreak in a special-care baby unit prophylaxis in liver transplant recipients using a fixed dosing schedule of hepatitis b immunoglobulin liver transplantation in hbsag-positive hbv-dnanegative cirrhotics: immunoprophylaxis and long term outcome hbv-infection in liver transplantation in hbsag positive patients: experience with long-term immunoprophylaxis hyperimmune gammaglobulin for the treatment of west nile virus encephalitis using high titer west nile intravenous immunoglobulin from selected israeli donors for treatment of west nile virus infection preparation and use of hyperimmune serum for prophylaxis and therapy of ebola virus infections evaluation of immune globulin and recombinant interferon-a2b for treatment of experimental ebola virus infections treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients tick-borne encephalitis vaccination against tick-borne encephalitis (tbe): influence of simultaneous application of tbe immunoglobulin on seroconversion and rate of adverse events efficacy of immune plasma in treatment of argentine haemorrhagic fever and association between treatment and a late neurological syndrome sars: systematic review of treatment effects use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology intravenous immunoglobulin utilization in the canadian atlantic provinces: a report of the atlantic collaborative intravenous immune globulin utilization working group prescribing intravenous immunoglobulin: summary of department of health guidelines primary immunodeficiency diseases: an update from the international union of immunological societies primary immunodeficiency diseases classification committee population prevalence of diagnosed primary immunodeficiency diseases in the united states primary immunodeficiency diseases in latin america: the second report of the lagid registry primary immunodeficiency diseases in australia and new zealand primary immunodeficiency syndromes in italy: a report of the national register in children and adults primary immunodeficiency diseases in norway primary b cell immunodeficiencies: comparisons and contrasts defects of class-switch recombination common variable immunodeficiency: an update on etiology and management practice parameter for the diagnosis and management of primary immunodeficiency natural history of selective antibody deficiency to bacterial polysaccharide antigens in children hypomorphic nuclear factor-kappab essential modulator mutation database and reconstitution system identifies phenotypic and immunologic diversity human nuclear factor kappa b essential modulator mutation can result in immunodeficiency without ectodermal dysplasia acute chylothorax in children: selective retention of memory t cells and natural killer cells intestinal lymphangiectasia: a protein-losing enteropathy with hypogammaglobulinemia, lymphocytopenia and impaired homograft rejection the outcome of patients with hypogammaglobulinemia in infancy and early childhood impaired specific antibody response and increased b-cell population in transient hypogammaglobulinemia of infancy infants presenting with recurrent infections and low immunoglobulins: characteristics and analysis of normalization therapy for patients with recurrent infections and low serum igg3 levels efficacy of intravenous gammaglobulin for immunoglobulin g subclass and/or antibody deficiency in adults the gamma globulins. iv. therapeutic uses of gamma globulin efficacy of intravenous immunoglobulin in primary humoral immunodeficiency disease early and prolonged intravenous immunoglobulin replacement therapy in childhood agammaglobulinemia: a retrospective survey of 31 patients impact of a physician education and patient awareness campaign on the diagnosis and management of primary immunodeficiencies pharmacoeconomics of immunoglobulins in primary immunodeficiency adverse reactions and pathogen safety of intravenous immunoglobulin european surveillance of immunoglobulin safety-results of initial survey of 1243 patients with primary immunodeficiencies in 16 countries differences between igiv products: impact on clinical outcome immunoglobulin replacement therapy by slow subcutaneous infusion subcutaneous immunoglobulin replacement in patients with primary antibody deficiencies: safety and costs home treatment of hypogammaglobulinaemia with subcutaneous gammaglobulin by rapid infusion the comparison of the efficacy and safety of intravenous versus subcutaneous immunoglobulin replacement therapy safety and efficacy of self-administered subcutaneous immunoglobulin in patients with primary immunodeficiency diseases subcutaneous immunoglobulin replacement in primary immunodeficiencies subcutaneous administration of igg subcutaneous immunoglobulin replacement therapy for primary antibody deficiency: advancements into the 21st century pharmacokinetics of immunoglobulin administered via intravenous of subcutaneous routes individualizing the dose of intravenous immune serum globulin for therapy of patients with primary humoral immunodeficiency biologic igg level in primary immunodeficiency disease: the igg level that protects against recurrent infection replacement igg therapy and self-therapy at home improve the health-related quality of life in patients with primary antibody deficiencies quality of life and health-care resource utilization among children with primary immunodeficiency receiving home treatment with subcutaneous human immunoglobulin the effect of two different dosages of intravenous immunoglobulin on the incidence of recurrent infections in patients with primary hypogammaglobulinemia. a randomized, double-blind, multicenter crossover trial comparison of the efficacy of igiv-c, 10% (caprylate/chromatography) and igiv-sd, 10% as replacement therapy in primary immune deficiency. a randomized double-blind trial efficacy of intravenous immunoglobulin in the prevention of pneumonia in patients with common variable immunodeficiency high dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura in childhood infusion of fc gamma fragments for treatment of children with acute immune thrombocytopenic purpura fcg receptor as regulators of immune responses anti-inflammatory activity of ivig mediated through the inhibitory fc receptor anti-inflammatory activity of immunoglobulin g resulting from fc sialylation recapitulation of ivig anti-inflammatory activity with a recombinant igg fc identification of a receptor required for the anti-inflammatory activity of ivig mechanism of intravenous immune globulin therapy in antibody-mediated autoimmune diseases accelerated autoantibody clearance by intravenous immunoglobulin therapy: studies in experimental models to determine the magnitude and time course of the effect effects of intravenous immunoglobulin on platelet count and antiplatelet antibody disposition in a rat model of immune thrombycytopenia v region-mediated selection of autoreactive repertoires by intravenous immunoglobulin (ivig) the concept of anti-idiotypic regulation of selected autoimmune diseases by intravenous immunoglobulin anti-idiotypic suppression of autoantibodies to factor viii (antihaemophilic factor) by high-dose intravenous gammaglobulin intravenous and standard immune serum globulin preparations interfere with uptake of 125i-c3 onto sensitized erythrocytes and inhibit hemolytic complement activity modulation of complement-mediated immune damage by intravenous immune globulin intravenous immune globulin for dermatomyositis controlled studies with high-dose intravenous immunoglobulin in the treatment of dermatomyositis, inclusion body myositis, and polymyositis increased in vitro uptake of the complement c3b in the serum of patients with guillain-barrã© syndrome, myasthenia gravis and dermatomyositis intravenous immune globulin therapy for neurologic diseases inhibition of toxic epidermal necrolysis by blockade of cd95 with human intravenous immunoglobulin intravenous immunoglobulin: an update on the clinical use and mechanisms of action inhibition of cell adhesion by antibodies to arg-gly-asp (rgd) in normal immunoglobulin for therapeutic use intravenous immune globulin prevents venular vasoocclusion in sickle cell mice by inhibiting leukocyte adhesion and the interactions between sickle erythrocytes and adherent leukocytes intravenous immunoglobulins reverse acute vaso-occlusive crises in sickle cell mice through rapid inhibition of neutrophil adhesion key: cord-279255-v861kk0i authors: dhama, kuldeep; khan, sharun; tiwari, ruchi; sircar, shubhankar; bhat, sudipta; malik, yashpal singh; singh, karam pal; chaicumpa, wanpen; bonilla-aldana, d. katterine; rodriguez-morales, alfonso j. title: coronavirus disease 2019–covid-19 date: 2020-06-24 journal: clin microbiol rev doi: 10.1128/cmr.00028-20 sha: doc_id: 279255 cord_uid: v861kk0i in recent decades, several new diseases have emerged in different geographical areas, with pathogens including ebola virus, zika virus, nipah virus, and coronaviruses (covs). recently, a new type of viral infection emerged in wuhan city, china, and initial genomic sequencing data of this virus do not match with previously sequenced covs, suggesting a novel cov strain (2019-ncov), which has now been termed severe acute respiratory syndrome cov-2 (sars-cov-2). although coronavirus disease 2019 (covid-19) is suspected to originate from an animal host (zoonotic origin) followed by human-to-human transmission, the possibility of other routes should not be ruled out. compared to diseases caused by previously known human covs, covid-19 shows less severe pathogenesis but higher transmission competence, as is evident from the continuously increasing number of confirmed cases globally. compared to other emerging viruses, such as ebola virus, avian h7n9, sars-cov, and middle east respiratory syndrome coronavirus (mers-cov), sars-cov-2 has shown relatively low pathogenicity and moderate transmissibility. codon usage studies suggest that this novel virus has been transferred from an animal source, such as bats. early diagnosis by real-time pcr and next-generation sequencing has facilitated the identification of the pathogen at an early stage. since no antiviral drug or vaccine exists to treat or prevent sars-cov-2, potential therapeutic strategies that are currently being evaluated predominantly stem from previous experience with treating sars-cov, mers-cov, and other emerging viral diseases. in this review, we address epidemiological, diagnostic, clinical, and therapeutic aspects, including perspectives of vaccines and preventive measures that have already been globally recommended to counter this pandemic virus. o ver the past 2 decades, coronaviruses (covs) have been associated with significant disease outbreaks in east asia and the middle east. the severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) began to emerge in 2002 and 2012, respectively. recently, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), causing coronavirus disease 2019 (covid19) , emerged in late 2019, and it has posed a global health threat, causing an ongoing pandemic in many countries and territories (1) . health workers worldwide are currently making efforts to control further disease outbreaks caused by the novel cov (originally named 2019-ncov), which was first identified in wuhan city, hubei province, china, on 12 december 2019. on 11 february 2020, the world health organization (who) announced the official designation for the current cov-associated disease to be covid-19, caused by sars-cov-2. the primary cluster of patients was found to be connected with the huanan south china seafood market in wuhan (2) . covs belong to the family coronaviridae (subfamily coronavirinae), the members of which infect a broad range of hosts, producing symptoms and diseases ranging from the common cold to severe and ultimately fatal illnesses, such as sars, mers, and, presently, covid-19. sars-cov-2 is considered one of the seven members of the cov family that infect humans (3) , and it belongs to the same lineage of covs that causes sars; however, this novel virus is genetically distinct. until 2020, six covs were known to infect humans, including human cov 229e (hcov-229e), hcov-nl63, hcov-oc43, hcov-hku1, sars-cov, and mers-cov. although sars-cov and mers-cov have resulted in outbreaks with high mortality, others remain associated with mild upper-respiratory-tract illnesses (4) . newly evolved covs pose a high threat to global public health. the current emergence of covid-19 is the third cov outbreak in humans over the past 2 decades (5) . it is no coincidence that fan et al. predicted potential sars-or mers-like cov outbreaks in china following pathogen transmission from bats (6) . covid-19 emerged in china and spread rapidly throughout the country and, subsequently, to other countries. due to the severity of this outbreak and the potential of spreading on an international scale, the who declared a global health emergency on 31 january 2020; subsequently, on 11 march 2020, they declared it a pandemic situation. at present, we are not in a position to effectively treat covid-19, since neither approved vaccines nor specific antiviral drugs for treating human cov infections are available (7) (8) (9) . most nations are currently making efforts to prevent the further spreading of this potentially deadly virus by implementing preventive and control strategies. in domestic animals, infections with covs are associated with a broad spectrum of furthermore, it acts as a critical factor for tissue tropism and the determination of host range (45) . notably, s protein is one of the vital immunodominant proteins of covs capable of inducing host immune responses (45) . the ectodomains in all covs s proteins have similar domain organizations, divided into two subunits, s1 and s2 (43) . the first one, s1, helps in host receptor binding, while the second one, s2, accounts for fusion. the former (s1) is further divided into two subdomains, namely, the n-terminal domain (ntd) and c-terminal domain (ctd). both of these subdomains act as receptorbinding domains, interacting efficiently with various host receptors (45) . the s1 ctd contains the receptor-binding motif (rbm). in each coronavirus spike protein, the trimeric s1 locates itself on top of the trimeric s2 stalk (45) . recently, structural analyses of the s proteins of covid-19 have revealed 27 amino acid substitutions within a 1,273-amino-acid stretch (16) . six substitutions are located in the rbd (amino acids 357 to 528), while four substitutions are in the rbm at the ctd of the s1 domain (16) . of note, no amino acid change is seen in the rbm, which binds directly to the angiotensinconverting enzyme-2 (ace2) receptor in sars-cov (16, 46) . at present, the main emphasis is knowing how many differences would be required to change the host tropism. sequence comparison revealed 17 nonsynonymous changes between the early sequence of sars-cov-2 and the later isolates of sars-cov. the changes were found scattered over the genome of the virus, with nine substitutions in orf1ab, orf8 (4 substitutions), the spike gene (3 substitutions) , and orf7a (single substitution) (4) . notably, the same nonsynonymous changes were found in a familial cluster, indicating that the viral evolution happened during person-to-person transmission (4, 47) . such adaptive evolution events are frequent and constitute a constantly ongoing process once the virus spreads among new hosts (47) . even though no functional changes occur in the virus associated with this adaptive evolution, close monitoring of the viral mutations that occur during subsequent human-to-human transmission is warranted. the m protein is the most abundant viral protein present in the virion particle, giving a definite shape to the viral envelope (48) . it binds to the nucleocapsid and acts as a central organizer of coronavirus assembly (49) . coronavirus m proteins are highly diverse in amino acid contents but maintain overall structural similarity within different genera (50) . the m protein has three transmembrane domains, flanked by a short amino terminus outside the virion and a long carboxy terminus inside the virion (50) . overall, the viral scaffold is maintained by m-m interaction. of note, the m protein of sars-cov-2 does not have an amino acid substitution compared to that of sars-cov (16) . the coronavirus e protein is the most enigmatic and smallest of the major structural proteins (51) . it plays a multifunctional role in the pathogenesis, assembly, and release of the virus (52) . it is a small integral membrane polypeptide that acts as a viroporin (ion channel) (53) . the inactivation or absence of this protein is related to the altered virulence of coronaviruses due to changes in morphology and tropism (54) . the e protein consists of three domains, namely, a short hydrophilic amino terminal, a large hydrophobic transmembrane domain, and an efficient c-terminal domain (51) . the sars-cov-2 e protein reveals a similar amino acid constitution without any substitution (16) . the n protein of coronavirus is multipurpose. among several functions, it plays a role in complex formation with the viral genome, facilitates m protein interaction needed during virion assembly, and enhances the transcription efficiency of the virus (55, 56) . it contains three highly conserved and distinct domains, namely, an ntd, an rna-binding domain or a linker region (lkr), and a ctd (57) . the ntd binds with the 3= end of the viral genome, perhaps via electrostatic interactions, and is highly diverged both in length and sequence (58) . the charged lkr is serine and arginine rich and is also known as the sr (serine and arginine) domain (59) . the lkr is capable of direct interaction with in vitro rna interaction and is responsible for cell signaling (60, 61) . it also modulates the antiviral response of the host by working as an antagonist for interferon (ifn) and rna interference (62) . compared to that of sars-cov, the n protein of sars-cov-2 possess five amino acid mutations, where two are in the intrinsically dispersed region (idr; positions 25 and 26) , one each in the ntd (position 103), lkr (position 217), and ctd (position 334) (16) . besides the important structural proteins, the sars-cov-2 genome contains 15 nsps, nsp1 to nsp10 and nsp12 to nsp16, and 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14) (16) . all these proteins play a specific role in viral replication (27) . unlike the accessory proteins of sars-cov, sars-cov-2 does not contain 8a protein and has a longer 8b and shorter 3b protein (16) . the nsp7, nsp13, envelope, matrix, and p6 and 8b accessory proteins have not been detected with any amino acid substitutions compared to the sequences of other coronaviruses (16) . the virus structure of sars-cov-2 is depicted in fig. 2 . sequence percent similarity analysis. we assessed the nucleotide percent similarity using the megalign software program, where the similarity between the novel sars-cov-2 isolates was in the range of 99.4% to 100%. among the other serbecovirus cov sequences, the novel sars-cov-2 sequences revealed the highest similarity to bat-sl-cov, with nucleotide percent identity ranges between 88.12 and 89.65%. meanwhile, earlier reported sars-covs showed 70.6 to 74.9% similarity to sars-cov-2 at the nucleotide level. further, the nucleotide percent similarity was 55.4%, 45.5% to 47.9%, 46 .2% to 46.6%, and 45.0% to 46.3% to the other four subgenera, namely, hibecovirus, nobecovirus, merbecovirus, and embecovirus, respectively. the percent similarity index of current outbreak isolates indicates a close relationship between sars-cov-2 isolates and bat-sl-cov, indicating a common origin. however, particular pieces of evidence based on further complete genomic analysis of current isolates are necessary to draw any conclusions, although it was ascertained that the current novel sars-cov-2 isolates belong to the subgenus sarbecovirus in the diverse range of betacoronaviruses. their possible ancestor was hypothesized to be from bat cov strains, wherein bats might have played a crucial role in harboring this class of viruses. splitstree phylogeny analysis. in the unrooted phylogenetic tree of different betacoronaviruses based on the s protein, virus sequences from different subgenera grouped into separate clusters. sars-cov-2 sequences from wuhan and other countries exhibited a close relationship and appeared in a single cluster (fig. 1 ). the covs from the subgenus sarbecovirus appeared jointly in splitstree and divided into three subclusters, namely, sars-cov-2, bat-sars-like-cov (bat-sl-cov), and sars-cov (fig. 1) . in the case of other subgenera, like merbecovirus, all of the sequences grouped clinical microbiology reviews than italy. a john hopkins university web platform has provided daily updates on the basic epidemiology of the covid-19 outbreak (https://gisanddata.maps.arcgis.com/ apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6) (238) . covid-19 has also been confirmed on a cruise ship, named diamond princess, quarantined in japanese waters (port of yokohama), as well as on other cruise ships around the world (239) (fig. 3) . the significant events of the sars-cov-2/covid-19 virus outbreak occurring since 8 december 2019 are presented as a timeline in fig. 5 . at the beginning, china experienced the majority of the burden associated with covid-19 in the form of disease morbidity and mortality (65), but over time the covid-19 menace moved to europe, particularly italy and spain, and now the united states has the highest number of confirmed cases and deaths. the covid-19 outbreak has also been associated with severe economic impacts globally due to the sudden interruption of global trade and supply chains that forced multinational companies to make decisions that led to significant economic losses (66) . the recent increase in the number of confirmed critically ill patients with covid-19 has already surpassed the intensive care supplies, limiting intensive care services to only a small portion of critically ill patients (67) . this might also have contributed to the increased case fatality rate observed in the covid-19 outbreak. the novel coronavirus was identified within 1 month (28 days) of the outbreak. this is impressively fast compared to the time taken to identify sars-cov reported in foshan, guangdong province, china (125 days) (68) . immediately after the confirmation of viral etiology, the chinese virologists rapidly released the genomic sequence of sars-cov-2, which played a crucial role in controlling the spread of this newly emerged novel coronavirus to other parts of the world (69) . the possible origin of sars-cov-2 and the first mode of disease transmission are not yet identified (70) . analysis of the initial cluster of infections suggests that the infected individuals had a common exposure point, a seafood market in wuhan, hubei province, china (fig. 6 ). the restaurants of this market are well-known for providing different types of wild animals for human consumption (71) . the huanan south china seafood market also sells live animals, such as poultry, bats, snakes, and marmots (72) . this might be the point where zoonotic (animal-to-human) transmission occurred (71) . although sars-cov-2 is alleged to have originated from an animal host (zoonotic origin) with further humanto-human transmission (fig. 6 ), the likelihood of foodborne transmission should be ruled out with further investigations, since it is a latent possibility (1). additionally, other clinical microbiology reviews potential and expected routes would be associated with transmission, as in other respiratory viruses, by direct contact, such as shaking contaminated hands, or by direct contact with contaminated surfaces (fig. 6) . still, whether blood transfusion and organ transplantation (276) , as well as transplacental and perinatal routes, are possible routes for sars-cov-2 transmission needs to be determined (fig. 6 ). from experience with several outbreaks associated with known emerging viruses, higher pathogenicity of a virus is often associated with lower transmissibility. compared to emerging viruses like ebola virus, avian h7n9, sars-cov, and mers-cov, sars-cov-2 has relatively lower pathogenicity and moderate transmissibility (15) . the risk of death among individuals infected with covid-19 was calculated using the infection fatality risk (ifr). the ifr was found to be in the range of 0.3% to 0.6%, which is comparable to that of a previous asian influenza pandemic (1957 to 1958) (73, 277) . notably, the reanalysis of the covid-19 pandemic curve from the initial cluster of cases pointed to considerable human-to-human transmission. it is opined that the exposure history of sars-cov-2 at the wuhan seafood market originated from humanto-human transmission rather than animal-to-human transmission (74) ; however, in light of the zoonotic spillover in covid-19, is too early to fully endorse this idea (1). following the initial infection, human-to-human transmission has been observed with a preliminary reproduction number (r 0 ) estimate of 1.4 to 2.5 (70, 75) , and recently it is estimated to be 2.24 to 3.58 (76) . in another study, the average reproductive number of covid-19 was found to be 3.28, which is significantly higher than the initial who estimate of 1.4 to 2.5 (77) . it is too early to obtain the exact r 0 value, since there is a possibility of bias due to insufficient data. the higher r 0 value is indicative of the more significant potential of sars-cov-2 transmission in a susceptible population. this is not the first time where the culinary practices of china have been blamed for the origin of novel coronavirus infection in humans. previously, the animals present in the liveanimal market were identified to be the intermediate hosts of the sars outbreak in china (78) . several wildlife species were found to harbor potentially evolving coronavirus strains that can overcome the species barrier (79) . one of the main principles of chinese food culture is that live-slaughtered animals are considered more nutritious (5) . after 4 months of struggle that lasted from december 2019 to march 2020, the covid-19 situation now seems under control in china. the wet animal markets have reopened, and people have started buying bats, dogs, cats, birds, scorpions, badgers, rabbits, pangolins (scaly anteaters), minks, soup from palm civet, ostriches, hamsters, snapping turtles, ducks, fish, siamese crocodiles, and other animal meats without any fear of covid-19. the chinese government is encouraging people to feel they can return to normalcy. however, this could be a risk, as it has been mentioned in advisories that people should avoid contact with live-dead animals as much as possible, as sars-cov-2 has shown zoonotic spillover. additionally, we cannot rule out the possibility of new mutations in the same virus being closely related to contact with both animals and humans at the market (284) . in january 2020, china imposed a temporary ban on the sale of live-dead animals in wet markets. however, now hundreds of such wet markets have been reopened without optimizing standard food safety and sanitation practices (286) . with china being the most populated country in the world and due to its domestic and international food exportation policies, the whole world is now facing the menace of covid-19, including china itself. wet markets of live-dead animals do not maintain strict food hygienic practices. fresh blood splashes are present everywhere, on the floor and tabletops, and such food customs could encourage many pathogens to adapt, mutate, and jump the species barrier. as a result, the whole world is suffering from novel sars-cov-2, with more than 4,170,424 cases and 287,399 deaths across the globe. there is an urgent need for a rational international campaign against the unhealthy food practices of china to encourage the sellers to increase hygienic food practices or close the crude live-dead animal wet markets. there is a need to modify food policies at national and international levels to avoid further life threats and clinical microbiology reviews economic consequences from any emerging or reemerging pandemic due to close animal-human interaction (285) . even though individuals of all ages and sexes are susceptible to covid-19, older people with an underlying chronic disease are more likely to become severely infected (80) . recently, individuals with asymptomatic infection were also found to act as a source of infection to susceptible individuals (81) . both the asymptomatic and symptomatic patients secrete similar viral loads, which indicates that the transmission capacity of asymptomatic or minimally symptomatic patients is very high. thus, sars-cov-2 transmission can happen early in the course of infection (82) . atypical clinical manifestations have also been reported in covid-19 in which the only reporting symptom was fatigue. such patients may lack respiratory signs, such as fever, cough, and sputum (83) . hence, the clinicians must be on the look-out for the possible occurrence of atypical clinical manifestations to avoid the possibility of missed diagnosis. the early transmission ability of sars-cov-2 was found to be similar to or slightly higher than that of sars-cov, reflecting that it could be controlled despite moderate to high transmissibility (84) . increasing reports of sars-cov-2 in sewage and wastewater warrants the need for further investigation due to the possibility of fecal-oral transmission. sars-cov-2 present in environmental compartments such as soil and water will finally end up in the wastewater and sewage sludge of treatment plants (328) . therefore, we have to reevaluate the current wastewater and sewage sludge treatment procedures and introduce advanced techniques that are specific and effective against sars-cov-2. since there is active shedding of sars-cov-2 in the stool, the prevalence of infections in a large population can be studied using wastewater-based epidemiology. recently, reverse transcription-quantitative pcr (rt-qpcr) was used to enumerate the copies of sars-cov-2 rna concentrated from wastewater collected from a wastewater treatment plant (327) . the calculated viral rna copy numbers determine the number of infected individuals. the increasing reports of virus shedding via the fecal route warrants the introduction of negative fecal viral nucleic acid test results as one of the additional discharge criteria in laboratory-confirmed cases of covid-19 (326) . the covid-19 pandemic does not have any novel factors, other than the genetically unique pathogen and a further possible reservoir. the cause and the likely future outcome are just repetitions of our previous interactions with fatal coronaviruses. the only difference is the time of occurrence and the genetic distinctness of the pathogen involved. mutations on the rbd of covs facilitated their capability of infecting newer hosts, thereby expanding their reach to all corners of the world (85) . this is a potential threat to the health of both animals and humans. advanced studies using bayesian phylogeographic reconstruction identified the most probable origin of sars-cov-2 as the bat sars-like coronavirus, circulating in the rhinolophus bat family (86) . phylogenetic analysis of 10 whole-genome sequences of sars-cov-2 showed that they are related to two covs of bat origin, namely, bat-sl-covzc45 and bat-sl-covzxc21, which were reported during 2018 in china (17) . it was reported that sars-cov-2 had been confirmed to use ace2 as an entry receptor while exhibiting an rbd similar to that of sars-cov (17, 87, 254, 255) . several countries have provided recommendations to their people traveling to china (88, 89) . compared to the previous coronavirus outbreaks caused by sars-cov and mers-cov, the efficiency of sars-cov-2 human-to-human transmission was thought to be less. this assumption was based on the finding that health workers were affected less than they were in previous outbreaks of fatal coronaviruses (2) . superspreading events are considered the main culprit for the extensive transmission of sars and mers (90, 91) . almost half of the mers-cov cases reported in saudi arabia are of secondary origin that occurred through contact with infected asymptomatic or symptomatic individuals through human-tohuman transmission (92) . the occurrence of superspreading events in the covid-19 outbreak cannot be ruled out until its possibility is evaluated. like sars and mers, covid-19 can also infect the lower respiratory tract, with milder symptoms (27) . the basic reproduction number of covid-19 has been found to be in the range of 2.8 to 3.3 based on real-time reports and 3.2 to 3.9 based on predicted infected cases (84) . coronavirus infection in humans is commonly associated with mild to severe respiratory diseases, with high fever, severe inflammation, cough, and internal organ dysfunction that can even lead to death (92) . most of the identified coronaviruses cause the common cold in humans. however, this changed when sars-cov was identified, paving the way for severe forms of the disease in humans (22) . our previous experience with the outbreaks of other coronaviruses, like sars and mers, suggests that the mode of transmission in covid-19 as mainly human-to-human transmission via direct contact, droplets, and fomites (25) . recent studies have demonstrated that the virus could remain viable for hours in aerosols and up to days on surfaces; thus, aerosol and fomite contamination could play potent roles in the transmission of sars-cov-2 (257) . the immune response against coronavirus is vital to control and get rid of the infection. however, maladjusted immune responses may contribute to the immunopathology of the disease, resulting in impairment of pulmonary gas exchange. understanding the interaction between covs and host innate immune systems could enlighten our understanding of the lung inflammation associated with this infection (24) . sars is a viral respiratory disease caused by a formerly unrecognized animal cov that originated from the wet markets in southern china after adapting to the human host, thereby enabling transmission between humans (90) . the sars outbreak reported in 2002 to 2003 had 8,098 confirmed cases with 774 total deaths (9.6%) (93) . the outbreak severely affected the asia pacific region, especially mainland china (94) . even though the case fatality rate (cfr) of sars-cov-2 (covid-19) is lower than that of sars-cov, there exists a severe concern linked to this outbreak due to its epidemiological similarity to influenza viruses (95, 279) . this can fail the public health system, resulting in a pandemic (96) . mers is another respiratory disease that was first reported in saudi arabia during the year 2012. the disease was found to have a cfr of around 35% (97) . the analysis of available data sets suggests that the incubation period of sars-cov-2, sars-cov, and mers-cov is in almost the same range. the longest predicted incubation time of sars-cov-2 is 14 days. hence, suspected individuals are isolated for 14 days to avoid the risk of further spread (98) . even though a high similarity has been reported between the genome sequence of the new coronavirus (sars-cov-2) and sars-like covs, the comparative analysis recognized a furin-like cleavage site in the sars-cov-2 s protein that is missing from other sars-like covs (99) . the furin-like cleavage site is expected to play a role in the life cycle of the virus and disease pathogenicity and might even act as a therapeutic target for furin inhibitors. the highly contagious nature of sars-cov-2 compared to that of its predecessors might be the result of a stabilizing mutation that occurred in the endosome-associated-protein-like domain of nsp2 protein. similarly, the destabilizing mutation near the phosphatase domain of nsp3 proteins in sars-cov-2 could indicate a potential mechanism that differentiates it from other covs (100) . even though the cfr reported for covid-19 is meager compared to those of the previous sars and mers outbreaks, it has caused more deaths than sars and mers combined (101) . possibly related to the viral pathogenesis is the recent finding of an 832-nucleotide (nt) deletion in orf8, which appears to reduce the replicative fitness of the virus and leads to attenuated phenotypes of sars-cov-2 (256) . coronavirus is the most prominent example of a virus that has crossed the species barrier twice from wild animals to humans during sars and mers outbreaks (79, 102) . the possibility of crossing the species barrier for the third time has also been suspected in the case of sars-cov-2 (covid19) . bats are recognized as a possible natural reservoir host of both sars-cov and mers-cov infection. in contrast, the possible intermediary host is the palm civet for sars-cov and the dromedary camel for mers-cov infection (102) . bats are considered the ancestral hosts for both sars and mers (103) . bats are also considered the reservoir host of human coronaviruses like clinical microbiology reviews hcov-229e and hcov-nl63 (104) . in the case of covid-19, there are two possibilities for primary transmission: it can be transmitted either through intermediate hosts, similar to that of sars and mers, or directly from bats (103) . the emergence paradigm put forward in the sars outbreak suggests that sars-cov originated from bats (reservoir host) and later jumped to civets (intermediate host) and incorporated changes within the receptor-binding domain (rbd) to improve binding to civet ace2. this civetadapted virus, during their subsequent exposure to humans at live markets, promoted further adaptations that resulted in the epidemic strain (104) . transmission can also occur directly from the reservoir host to humans without rbd adaptations. the bat coronavirus that is currently in circulation maintains specific "poised" spike proteins that facilitate human infection without the requirement of any mutations or adaptations (105) . altogether, different species of bats carry a massive number of coronaviruses around the world (106) . the high plasticity in receptor usage, along with the feasibility of adaptive mutation and recombination, may result in frequent interspecies transmission of coronavirus from bats to animals and humans (106) . the pathogenesis of most bat coronaviruses is unknown, as most of these viruses are not isolated and studied (4) . hedgehog coronavirus hku31, a betacoronavirus, has been identified from amur hedgehogs in china. studies show that hedgehogs are the reservoir of betacoronavirus, and there is evidence of recombination (107) . the current scientific evidence available on mers infection suggests that the significant reservoir host, as well as the animal source of mers infection in humans, is the dromedary camels (97) . the infected dromedary camels may not show any visible signs of infection, making it challenging to identify animals actively excreting mers-cov that has the potential to infect humans. however, they may shed mers-cov through milk, urine, feces, and nasal and eye discharge and can also be found in the raw organs (108) . in a study conducted to evaluate the susceptibility of animal species to mers-cov infection, llamas and pigs were found to be susceptible, indicating the possibility of mers-cov circulation in animal species other than dromedary camels (109) . following the outbreak of sars in china, sars-cov-like viruses were isolated from himalayan palm civets (paguma larvata) and raccoon dogs (nyctereutes procyonoides) found in a live-animal market in guangdong, china. the animal isolates obtained from the live-animal market retained a 29-nucleotide sequence that was not present in most of the human isolates (78) . these findings were critical in identifying the possibility of interspecies transmission in sars-cov. the higher diversity and prevalence of bat coronaviruses in this region compared to those in previous reports indicate a host/ pathogen coevolution. sars-like coronaviruses also have been found circulating in the chinese horseshoe bat (rhinolophus sinicus) populations. the in vitro and in vivo studies carried out on the isolated virus confirmed that there is a potential risk for the reemergence of sars-cov infection from the viruses that are currently circulating in the bat population (105) . the disease caused by sars-cov-2 is also named severe specific contagious pneumonia (sscp), wuhan pneumonia, and, recently, covid-19 (110) . compared to sars-cov, sars-cov-2 has less severe pathogenesis but has superior transmission capability, as evidenced by the rapidly increasing number of covid-19 cases (111) . the incubation period of sars-cov-2 in familial clusters was found to be 3 to 6 days (112) . the mean incubation period of covid-19 was found to be 6.4 days, ranging from 2.1 to 11.1 days (113) . among an early affected group of 425 patients, 59 years was the median age, of which more males were affected (114) . similar to sars and mers, the severity of this ncov is high in age groups above 50 years (2, 115) . symptoms of covid-19 include fever, cough, myalgia or fatigue, and, less commonly, headache, hemoptysis, and diarrhea (116, 282) . compared to the sars-cov-2-infected patients in wuhan during the initial stages of the outbreak, only mild symptoms were noticed in those patients that are infected by human-to-human transmission (14) . the initial trends suggested that the mortality associated with covid-19 was less than that of previous outbreaks of sars (101) . the updates obtained from countries like china, japan, thailand, and south korea indicated that the covid-19 patients had relatively mild manifestations compared to those with sars and mers (4). regardless of the coronavirus type, immune cells, like mast cells, that are present in the submucosa of the respiratory tract and nasal cavity are considered the primary barrier against this virus (92) . advanced in-depth analysis of the genome has identified 380 amino acid substitutions between the amino acid sequences of sars-cov-2 and the sars/sarslike coronaviruses. these differences in the amino acid sequences might have contributed to the difference in the pathogenic divergence of sars-cov-2 (16) . further research is required to evaluate the possible differences in tropism, pathogenesis, and transmission of this novel agent associated with this change in the amino acid sequence. with the current outbreak of covid-19, there is an expectancy of a significant increase in the number of published studies about this emerging coronavirus, as occurred with sars and mers (117) . sars-cov-2 invades the lung parenchyma, resulting in severe interstitial inflammation of the lungs. this is evident on computed tomography (ct) images as ground-glass opacity in the lungs. this lesion initially involves a single lobe but later expands to multiple lung lobes (118) . the histological assessment of lung biopsy samples obtained from covid-19-infected patients revealed diffuse alveolar damage, cellular fibromyxoid exudates, hyaline membrane formation, and desquamation of pneumocytes, indicative of acute respiratory distress syndrome (119) . it was also found that the sars-cov-2infected patients often have lymphocytopenia with or without leukocyte abnormalities. the degree of lymphocytopenia gives an idea about disease prognosis, as it is found to be positively correlated with disease severity (118) . pregnant women are considered to have a higher risk of getting infected by covid-19. the coronaviruses can cause adverse outcomes for the fetus, such as intrauterine growth restriction, spontaneous abortion, preterm delivery, and perinatal death. nevertheless, the possibility of intrauterine maternal-fetal transmission (vertical transmission) of covs is low and was not seen during either the sars-or mers-cov outbreak (120) . however, there has been concern regarding the impact of sars-cov-2/covid-19 on pregnancy. researchers have mentioned the probability of in utero transmission of novel sars-cov-2 from covid-19-infected mothers to their neonates in china based upon the rise in igm and igg antibody levels and cytokine values in the blood obtained from newborn infants immediately postbirth; however, rt-pcr failed to confirm the presence of sars-cov-2 genetic material in the infants (283) . recent studies show that at least in some cases, preterm delivery and its consequences are associated with the virus. nonetheless, some cases have raised doubts for the likelihood of vertical transmission (240) (241) (242) (243) . covid-19 infection was associated with pneumonia, and some developed acute respiratory distress syndrome (ards). the blood biochemistry indexes, such as albumin, lactate dehydrogenase, c-reactive protein, lymphocytes (percent), and neutrophils (percent) give an idea about the disease severity in covid-19 infection (121) . during covid-19, patients may present leukocytosis, leukopenia with lymphopenia (244), hypoalbuminemia, and an increase of lactate dehydrogenase, aspartate transaminase, alanine aminotransferase, bilirubin, and, especially, d-dimer (244) . middle-aged and elderly patients with primary chronic diseases, especially high blood pressure and diabetes, were found to be more susceptible to respiratory failure and, therefore, had poorer prognoses. providing respiratory support at early stages improved the disease prognosis and facilitated recovery (18) . the ards in covid-19 is due to the occurrence of cytokine storms that results in exaggerated immune response, immune regulatory network imbalance, and, finally, multiple-organ failure (122) . in addition to the exaggerated inflammatory response seen in patients with covid-19 pneumonia, the bile duct epithelial cell-derived hepatocytes upregulate ace2 expression in liver tissue by compensatory proliferation that might result in hepatic tissue injury (123) . coronavirus can cause disease in several species of domestic and wild animals, as well as humans (23) . the different animal species that are infected with cov include horses, camels, cattle, swine, dogs, cats, rodents, birds, ferrets, minks, bats, rabbits, snakes, and various other wild animals (20, 30, 79, 93, 124, 125, 287) . coronavirus infection is linked to different kinds of clinical manifestations, varying from enteritis in cows and pigs, upper respiratory disease in chickens, and fatal respiratory infections in humans (30) . among the cov genera, alphacoronavirus and betacoronavirus infect mammals, while gammacoronavirus and deltacoronavirus mainly infect birds, fishes, and, sometimes, mammals (27, 29, 106) . several novel coronaviruses that come under the genus deltacoronavirus have been discovered in the past from birds, like wigeon coronavirus hku20, bulbul coronavirus hku11, munia coronavirus hku13, white-eye coronavirus hku16, night-heron coronavirus hku19, and common moorhen coronavirus hku21, as well as from pigs (porcine coronavirus hku15) (6, 29) . transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), and porcine hemagglutinating encephalomyelitis virus (phev) are some of the coronaviruses of swine. among them, tgev and pedv are responsible for causing severe gastroenteritis in young piglets with noteworthy morbidity and mortality. infection with phev also causes enteric infection but can cause encephalitis due to its ability to infect the nervous system (30) . bovine coronaviruses (bocovs) are known to infect several domestic and wild ruminants (126) . bocov inflicts neonatal calf diarrhea in adult cattle, leading to bloody diarrhea (winter dysentery) and respiratory disease complex (shipping fever) in cattle of all age groups (126) . bocov-like viruses have been noted in humans, suggesting its zoonotic potential as well (127) . feline enteric and feline infectious peritonitis (fip) viruses are the two major feline covs (128) , where feline covs can affect the gastrointestinal tract, abdominal cavity (peritonitis), respiratory tract, and central nervous system (128) . canines are also affected by covs that fall under different genera, namely, canine enteric coronavirus in alphacoronavirus and canine respiratory coronavirus in betacoronavirus, affecting the enteric and respiratory tract, respectively (129, 130) . ibv, under gammacoronavirus, causes diseases of respiratory, urinary, and reproductive systems, with substantial economic losses in chickens (131, 132) . in small laboratory animals, mouse hepatitis virus, rat sialodacryoadenitis coronavirus, and guinea pig and rabbit coronaviruses are the major covs associated with disease manifestations like enteritis, hepatitis, and respiratory infections (10, 133) . swine acute diarrhea syndrome coronavirus (sads-cov) was first identified in suckling piglets having severe enteritis and belongs to the genus alphacoronavirus (106) . the outbreak was associated with considerable scale mortality of piglets (24,693 deaths) across four farms in china (134) . the virus isolated from the piglets was almost identical to and had 95% genomic similarity with horseshoe bat (rhinolophus species) coronavirus hku2, suggesting a bat origin of the pig virus (106, 134, 135) . it is also imperative to note that the sads-cov outbreak started in guangdong province, near the location of the sars pandemic origin (134) . before this outbreak, pigs were not known to be infected with bat-origin coronaviruses. this indicates that the bat-origin coronavirus jumped to pig by breaking the species barrier. the next step of this jump might not end well, since pigs are considered the mixing vessel for influenza a viruses due to their ability to be infected by both human and avian influenza a viruses (136) . similarly, they may act as the mixing vessel for coronaviruses, since they are in frequent contact with both humans and multiple wildlife species. additionally, pigs are also found to be susceptible to infection with human sars-cov and mers-cov, making this scenario a nightmare (109, 137) . it is only a matter of time before another zoonotic coronavirus results in an epidemic by jumping the so-called species barrier (287) . the host spectrum of coronavirus increased when a novel coronavirus, namely, sw1, was recognized in the liver tissue of a captive beluga whale (delphinapterus leucas) (138) . in recent decades, several novel coronaviruses were identified from different animal species. bats can harbor these viruses without manifesting any clinical disease but are persistently infected (30) . they are the only mammals with the capacity for self-powered flight, which enables them to migrate long distances, unlike land mammals. bats are distributed worldwide and also account for about a fifth of all mammalian species (6) . this makes them the ideal reservoir host for many viral agents and also the source of novel coronaviruses that have yet to be identified. it has become a necessity to study the diversity of coronavirus in the bat population to prevent future outbreaks that could jeopardize livestock and public health. the repeated outbreaks caused by bat-origin coronaviruses calls for the development of efficient molecular surveillance strategies for studying betacoronavirus among animals (12) , especially in the rhinolophus bat family (86) . chinese bats have high commercial value, since they are used in traditional chinese medicine (tcm). therefore, the handling of bats for trading purposes poses a considerable risk of transmitting zoonotic cov epidemics (139) . due to the possible role played by farm and wild animals in sars-cov-2 infection, the who, in their novel coronavirus (covid-19) situation report, recommended the avoidance of unprotected contact with both farm and wild animals (25) . the live-animal markets, like the one in guangdong, china, provides a setting for animal coronaviruses to amplify and to be transmitted to new hosts, like humans (78) . such markets can be considered a critical place for the origin of novel zoonotic diseases and have enormous public health significance in the event of an outbreak. bats are the reservoirs for several viruses; hence, the role of bats in the present outbreak cannot be ruled out (140) . in a qualitative study conducted for evaluating the zoonotic risk factors among rural communities of southern china, the frequent human-animal interactions along with the low levels of environmental biosecurity were identified as significant risks for the emergence of zoonotic disease in local communities (141, 142) . the comprehensive sequence analysis of the sars-cov-2 rna genome identified that the cov from wuhan is a recombinant virus of the bat coronavirus and another coronavirus of unknown origin. the recombination was found to have happened within the viral spike glycoprotein, which recognizes the cell surface receptor. further analysis of the genome based on codon usage identified the snake as the most probable animal reservoir of sars-cov-2 (143) . contrary to these findings, another genome analysis proposed that the genome of sars-cov-2 is 96% identical to bat coronavirus, reflecting its origin from bats (63) . the involvement of bat-derived materials in causing the current outbreak cannot be ruled out. high risk is involved in the production of bat-derived materials for tcm practices involving the handling of wild bats. the use of bats for tcm practices will remain a severe risk for the occurrence of zoonotic coronavirus epidemics in the future (139) . furthermore, the pangolins are an endangered species of animals that harbor a wide variety of viruses, including coronaviruses (144) . the coronavirus isolated from malayan pangolins (manis javanica) showed a very high amino acid identity with covid-19 at e (100%), m (98.2%), n (96.7%), and s genes (90.4%). the rbd of s protein in cov isolated from pangolin was almost identical (one amino acid difference) to that of sars-cov-2. a comparison of the genomes suggests recombination between pangolin-cov-like viruses with the bat-cov-ratg13-like virus. all this suggests the potential of pangolins to act as the intermediate host of sars-cov-2 (145) . human-wildlife interactions, which are increasing in the context of climate change (142) , are further considered high risk and responsible for the emergence of sars-cov. covid-19 is also suspected of having a similar mode of origin. hence, to prevent the occurrence of another zoonotic spillover (1), exhaustive coordinated efforts are needed to identify the high-risk pathogens harbored by wild animal populations, conducting surveillance among the people who are susceptible to zoonotic spillover events (12) , and to improve the biosecurity measures associated with the wildlife trade (146) . the serological surveillance studies conducted in people living in proximity to bat caves had earlier identified the serological confirmation of sars-related covs in humans. people clinical microbiology reviews living at the wildlife-human interface, mainly in rural china, are regularly exposed to sars-related covs (147) . these findings will not have any significance until a significant outbreak occurs due to a virus-like sars-cov-2. there is a steady increase in the reports of covid-19 in companion and wild animals around the world. further studies are required to evaluate the potential of animals (especially companion animals) to serve as an efficient reservoir host that can further alter the dynamics of human-to-human transmission (330) . to date, two pet dogs (hong kong) and four pet cats (one each from belgium and hong kong, two from the united states) have tested positive for sars-cov-2 (335) . the world organization for animal health (oie) has confirmed the diagnosis of covid-19 in both dogs and cats due to human-to-animal transmission (331) . the similarity observed in the gene sequence of sars-cov-2 from an infected pet owner and his dog further confirms the occurrence of human-to-animal transmission (333) . even though asymptomatic, feline species should be considered a potential transmission route from animals to humans (326) . however, currently, there are no reports of sars-cov-2 transmission from felines to human beings. based on the current evidence, we can conclude that cats are susceptible to sars-cov-2 and can get infected by human beings. however, evidence of cat-to-human transmission is lacking and requires further studies (332) . rather than waiting for firmer evidence on animal-to-human transmission, necessary preventive measures are advised, as well as following social distancing practices among companion animals of different households (331) . one of the leading veterinary diagnostic companies, idexx, has conducted large-scale testing for covid-19 in specimens collected from dogs and cats. however, none of the tests turned out to be positive (334) . in a study conducted to investigate the potential of different animal species to act as the intermediate host of sars-cov-2, it was found that both ferrets and cats can be infected via experimental inoculation of the virus. in addition, infected cats efficiently transmitted the disease to naive cats (329) . sars-cov-2 infection and subsequent transmission in ferrets were found to recapitulate the clinical aspects of covid-19 in humans. the infected ferrets also shed virus via multiple routes, such as saliva, nasal washes, feces, and urine, postinfection, making them an ideal animal model for studying disease transmission (337) . experimental inoculation was also done in other animal species and found that the dogs have low susceptibility, while the chickens, ducks, and pigs are not at all susceptible to sars-cov-2 (329) . similarly, the national veterinary services laboratories of the usda have reported covid-19 in tigers and lions that exhibited respiratory signs like dry cough and wheezing. the zoo animals are suspected to have been infected by an asymptomatic zookeeper (335) . the total number of covid-19-positive cases in human beings is increasing at a high rate, thereby creating ideal conditions for viral spillover to other species, such as pigs. the evidence obtained from sars-cov suggests that pigs can get infected with sars-cov-2 (336). however, experimental inoculation with sars-cov-2 failed to infect pigs (329) . further studies are required to identify the possible animal reservoirs of sars-cov-2 and the seasonal variation in the circulation of these viruses in the animal population. research collaboration between human and animal health sectors is becoming a necessity to evaluate and identify the possible risk factors of transmission between animals and humans. such cooperation will help to devise efficient strategies for the management of emerging zoonotic diseases (12) . rna tests can confirm the diagnosis of sars-cov-2 (covid-19) cases with real-time rt-pcr or next-generation sequencing (148, 149, 245, 246) . at present, nucleic acid detection techniques, like rt-pcr, are considered an effective method for confirming the diagnosis in clinical cases of covid-19 (148) . several companies across the world are currently focusing on developing and marketing sars-cov-2-specific nucleic acid detection kits. multiple laboratories are also developing their own in-house rt-pcr. one of them is the sars-cov-2 nucleic acid detection kit produced by shuoshi biotechnology (double fluorescence pcr method) (150) . up to 30 march 2020, the u.s. food and drug administration (fda) had granted 22 in vitro diagnostics emergency use authorizations (euas), including for the rt-pcr diagnostic panel for the universal detection of sars-like betacoronaviruses and specific detection of sars-cov-2, developed by the u.s. cdc (table 1) (258, 259) . recently, 95 full-length genomic sequences of saras-cov-2 strains available in the national center for biotechnology information and gisaid databases were subjected to multiple-sequence alignment and phylogenetic analyses for studying variations in the viral genome (260) . all the viral strains revealed high homology of 99.99% (99.91% to 100%) at the nucleotide level and 99.99% (99.79% to 100%) at the amino acid level. overall variation was found to be low in orf regions, with 13 variation sites recognized in 1a, 1b, s, 3a, m, 8, and n regions. mutation rates of 30.53% (29/95) and 29.47% (28/95) were observed at nt 28144 (orf8) and nt 8782 (orf1a) positions, respectively. owing to such selective mutations, a few specific regions of sars-cov-2 should not be considered for designing primers and probes. the sars-cov-2 reference sequence could pave the way to study molecular biology and pathobiology, along with developing diagnostics and appropriate prevention and control strategies for countering sars-cov-2 (260) . nucleic acids of sars-cov-2 can be detected from samples (64) such as bronchoalveolar lavage fluid, sputum, nasal swabs, fiber bronchoscope brush biopsy specimen, pharyngeal swabs, feces, blood, and urine, with different levels of diagnostic performance (table 2) (80, 245, 246) . the viral loads of sars-cov-2 were measured using n-gene-specific quantitative rt-pcr in throat swab and sputum samples collected from covid-19-infected individuals. the results indicated that the viral load peaked at around 5 to 6 days following the onset of symptoms, and it ranged from 10 4 to 10 7 copies/ml during this time (151) . in another study, the viral load was found to be higher in the nasal swabs than the throat swabs obtained from covid-19 symptomatic patients (82) . although initially it was thought that viral load would be associated with poor outcomes, some case reports have shown asymptomatic individuals with high viral loads (247) . recently, the viral load in nasal and throat swabs of 17 symptomatic patients was determined, and higher viral loads were recorded soon after the onset of symptoms, particularly in the nose compared to the throat. the pattern of viral nucleic the results of the studies related to sars-cov-2 viral loads reflect active replication of this virus in the upper respiratory tract and prolonged viral shedding after symptoms disappear, including via stool. thus, the current case definition needs to be updated along with a reassessment of the strategies to be adopted for restraining the sars-cov-2 outbreak spread (248) . in some cases, the viral load studies of sars-cov-2 have also been useful to recommend precautionary measures when handling specific samples, e.g., feces. in a recent survey from 17 confirmed cases of sars-cov-2 infection with available data (representing days 0 to 13 after onset), stool samples from nine cases (53%; days 0 to 11 after onset) were positive on rt-pcr analysis. although the viral loads were lower than those of respiratory samples (range, 550 copies per ml to 1.21 ϫ 10 5 copies per ml), this has essential biosafety implications (151) . the samples from 18 sars-cov-2-positive patients in singapore who had traveled from wuhan to singapore showed the presence of viral rna in stool and whole blood but not in urine by real-time rt-pcr (288) . further, novel sars-cov-2 infections have been detected in a variety of clinical specimens, like bronchoalveolar lavage fluid, sputum, nasal swabs, fibrobronchoscope brush biopsy specimens, pharyngeal swabs, feces, and blood (246) . the presence of sars-cov-2 in fecal samples has posed grave public health concerns. in addition to the direct transmission mainly occurring via droplets of sneezing and coughing, other routes, such as fecal excretion and environmental and fomite contamination, are contributing to sars-cov-2 transmission and spread (249) (250) (251) (252) . fecal excretion has also been documented for sars-cov and mers-cov, along with the potential to stay viable in situations aiding fecal-oral transmission. thus, sars-cov-2 has every possibility to be transmitted through this mode. fecal-oral transmission of sars-cov-2, particularly in regions having low standards of hygiene and poor sanitation, may have grave consequences with regard to the high spread of this virus. ethanol and disinfectants containing chlorine or bleach are effective against coronaviruses (249) (250) (251) (252) . appropriate precautions need to be followed strictly while handling the stools of patients infected with sars-cov-2. biowaste materials and sewage from hospitals must be adequately disinfected, treated, and disposed of properly. the significance of frequent and good hand hygiene and sanitation practices needs to be given due emphasis (249) (250) (251) (252) . future explorative research needs to be conducted with regard to the fecal-oral transmission of sars-cov-2, along with focusing on environmental investigations to find out if this virus could stay viable in situations and atmospheres facilitating such potent routes of transmission. the correlation of fecal concentrations of viral rna with disease severity needs to be determined, along with assessing the gastrointestinal symptoms and the possibility of fecal sars-cov-2 rna detection during the covid-19 incubation period or convalescence phases of the disease (249) (250) (251) (252) . the lower respiratory tract sampling techniques, like bronchoalveolar lavage fluid aspirate, are considered the ideal clinical materials, rather than the throat swab, due to their higher positive rate on the nucleic acid test (148) . the diagnosis of covid-19 can be made by using upper-respiratory-tract specimens collected using nasopharyngeal and oropharyngeal swabs. however, these techniques are associated with unnecessary risks to health care workers due to close contact with patients (152) . similarly, a single patient with a high viral load was reported to contaminate an entire endoscopy room by shedding the virus, which may remain viable for at least 3 days and is considered a great risk for uninfected patients and health care workers (289) . recently, it was found that the anal swabs gave more positive results than oral swabs in the later stages of infection (153) . hence, clinicians have to be cautious while discharging any covid-19infected patient based on negative oral swab test results due to the possibility of fecal-oral transmission. even though the viral loads in stool samples were found to be less than those of respiratory samples, strict precautionary measures have to be followed while handling stool samples of covid-19 suspected or infected patients (151) . children infected with sars-cov-2 experience only a mild form of illness and recover immediately after treatment. it was recently found that stool samples of sars-cov-2-infected children that gave negative throat swab results were positive within ten days of negative results. this could result in the fecal-oral transmission of sars-cov-2 infections, especially in children (290) . hence, to prevent the fecal-oral transmission of sars-cov-2, infected covid-19 patients should only be considered negative when they test negative for sars-cov-2 in the stool sample. a suspected case of covid-19 infection is said to be confirmed if the respiratory tract aspirate or blood samples test positive for sars-cov-2 nucleic acid using rt-pcr or by the identification of sars-cov-2 genetic sequence in respiratory tract aspirate or blood samples (80) . the patient will be confirmed as cured when two subsequent oral swab results are negative (153) . recently, the live virus was detected in the selfcollected saliva of patients infected with covid-19. these findings were confirmative of using saliva as a noninvasive specimen for the diagnosis of covid-19 infection in suspected individuals (152) . it has also been observed that the initial screening of covid-19 patients infected with rt-pcr may give negative results even if they have chest ct findings that are suggestive of infection. hence, for the accurate diagnosis of covid-19, a combination of repeated swab tests using rt-pcr and ct scanning is required to prevent the possibility of false-negative results during disease screening (154) . rt-pcr is the most widely used test for diagnosing covid-19. however, it has some significant limitations from the clinical perspective, since it will not give any clarity regarding disease progression. droplet digital pcr (ddpcr) can be used for the quantification of viral load in the samples obtained from lower respiratory tracts. hence, based on the viral load, we can quickly evaluate the progression of infection (291) . in addition to all of the above findings, sequencing and phylogenetics are critical in the correct identification and confirmation of the causative viral agent and useful to establish relationships with previous isolates and sequences, as well as to know, especially during an epidemic, the nucleotide and amino acid mutations and the molecular divergence. the rapid development and implementation of diagnostic tests against emerging novel diseases like covid-19 pose significant challenges due to the lack of resources and logistical limitations associated with an outbreak (155) . sars-cov-2 infection can also be confirmed by isolation and culturing. the human airway epithelial cell culture was found to be useful in isolating sars-cov-2 (3). the efficient control of an outbreak depends on the rapid diagnosis of the disease. recently, in response to the covid-19 outbreak, 1-step quantitative realtime reverse transcription-pcr assays were developed that detect the orf1b and n regions of the sars-cov-2 genome (156) . that assay was found to achieve the rapid detection of sars-cov-2. nucleic acid-based assays offer high accuracy in the diagnosis of sars-cov-2, but the current rate of spread limits its use due to the lack of diagnostic assay kits. this will further result in the extensive transmission of covid-19, since only a portion of suspected cases can be diagnosed. in such situations, conventional serological assays, like enzyme-linked immunosorbent assay (elisa), that are specific to covid-19 igm and igg antibodies can be used as a high-throughput alternative (149) . at present, there is no diagnostic kit available for detecting the sars-cov-2 antibody (150) . the specific antibody profiles of covid-19 patients were analyzed, and it was found that the igm level lasted more than 1 month, indicating a prolonged stage of virus replication in sars-cov-2-infected patients. the igg levels were found to increase only in the later stages of the disease. these findings indicate that the specific antibody profiles of sars-cov-2 and sars-cov were similar (325) . these findings can be utilized for the development of specific diagnostic tests against covid-19 and can be used for rapid screening. even though diagnostic test kits are already available that can detect the genetic sequences of sars-cov-2 (95), their availability is a concern, as the number of covid-19 cases is skyrocketing (155, 157) . a major problem associated with this diagnostic kit is that it works only when the test subject has an active infection, limiting its use to the earlier stages of infection. several laboratories around the world are currently developing antibody-based diagnostic tests against sars-cov-2 (157). chest ct is an ideal diagnostic tool for identifying viral pneumonia. the sensitivity of chest ct is far superior to that of x-ray screening. the chest ct findings associated with covid-19-infected patients include characteristic patchy infiltration that later progresses to ground-glass opacities (158) . early manifestations of covid-19 pneumonia might not be evident in x-ray chest radiography. in such situations, a chest ct examination can be performed, as it is considered highly specific for covid-19 pneumonia (118) . those patients having covid-19 pneumonia will exhibit the typical ground-glass opacity in their chest ct images (154) . the patients infected with covid-19 had elevated plasma angiotensin 2 levels. the level of angiotensin 2 was found to be linearly associated with viral load and lung injury, indicating its potential as a diagnostic biomarker (121) . the chest ct imaging abnormalities associated with covid-19 pneumonia have also been observed even in asymptomatic patients. these abnormalities progress from the initial focal unilateral to diffuse bilateral ground-glass opacities and will further progress to or coexist with lung consolidation changes within 1 to 3 weeks (159). the role played by radiologists in the current scenario is very important. radiologists can help in the early diagnosis of lung abnormalities associated with covid-19 pneumonia. they can also help in the evaluation of disease severity, identifying its progression to acute respiratory distress syndrome and the presence of secondary bacterial infections (160) . even though chest ct is considered an essential diagnostic tool for covid-19, the extensive use of ct for screening purposes in the suspected individuals might be associated with a disproportionate risk-benefit ratio due to increased radiation exposure as well as increased risk of cross-infection. hence, the use of ct for early diagnosis of sars-cov-2 infection in high-risk groups should be done with great caution (292) . more recently, other advanced diagnostics have been designed and developed for the detection of sars-cov-2 (345, 347, (350) (351) (352) . a reverse transcriptional loopmediated isothermal amplification (rt-lamp), namely, ilaco, has been developed for rapid and colorimetric detection of this virus (354) . rt-lamp serves as a simple, rapid, and sensitive diagnostic method that does not require sophisticated equipment or skilled personnel (349) . an interactive web-based dashboard for tracking sars-cov-2 in a real-time mode has been designed (238) . a smartphone-integrated home-based point-of-care testing (poct) tool, a paper-based poct combined with lamp, is a useful point-of-care diagnostic (353) . an abbott id now covid-19 molecular poct-based test, using isothermal nucleic acid amplification technology, has been designed as a pointof-care test for very rapid detection of sars-cov-2 in just 5 min (344) . a crispr-based sherlock (specific high-sensitivity enzymatic reporter unlocking) diagnostic for rapid detection of sars-cov-2 without the requirement of specialized instrumentation has been reported to be very useful in the clinical diagnosis of covid-19 (360) . a crispr-cas12-based lateral flow assay also has been developed for rapid detection of sars-cov-2 (346) . artificial intelligence, by means of a three-dimensional deep-learning model, has been developed for sensitive and specific diagnosis of covid-19 via ct images (332) . tracking and mapping of the rising incidence rates, disease outbreaks, community spread, clustered transmission events, hot spots, and superspreader potential of sars-cov-2/covid warrant full exploitation of real-time disease mapping by employing geographical information systems (gis), such as the gis software kosmo 3.1, web-based real-time tools and dashboards, apps, and advances in information technology (356-359). researchers have also developed a few prediction tools/models, such as the prediction model risk of bias assessment tool (probast) and critical appraisal and data extraction for systematic reviews of prediction modeling studies (charms), which could aid in assessing the possibility of getting infection and estimating the prognosis in patients; however, such models may suffer from bias issues and, hence, cannot be considered completely trustworthy, which necessitates the development of new and reliable predictors (360) . recently emerged viruses, such as zika, ebola, and nipah viruses, and their grave threats to humans have begun a race in exploring the designing and developing of advanced vaccines, prophylactics, therapeutics, and drug regimens to counter emerging viruses (161) (162) (163) 280) . several attempts are being made to design and develop vaccines for cov infection, mostly by targeting the spike glycoprotein. nevertheless, owing to extensive diversity in antigenic variants, cross-protection rendered by the vaccines is significantly limited, even within the strains of a phylogenetic subcluster (104) . due to the lack of effective antiviral therapy and vaccines in the present scenario, we need to depend solely on implementing effective infection control measures to lessen the risk of possible nosocomial transmission (68) . recently, the receptor for sars-cov-2 was established as the human angiotensin-converting enzyme 2 (hace2), and the virus was found to enter the host cell mainly through endocytosis. it was also found that the major components that have a critical role in viral entry include pikfyve, tpc2, and cathepsin l. these findings are critical, since the components described above might act as candidates for vaccines or therapeutic drugs against sars-cov-2 (293) . the majority of the treatment options and strategies that are being evaluated for sars-cov-2 (covid-19) have been taken from our previous experiences in treating sars-cov, mers-cov, and other emerging viral diseases. several therapeutic and preventive strategies, including vaccines, immunotherapeutics, and antiviral drugs, have been exploited against the previous cov outbreaks (sars-cov and mers-cov) (8, 104, (164) (165) (166) (167) . these valuable options have already been evaluated for their potency, efficacy, and safety, along with several other types of current research that will fuel our search for ideal therapeutic agents against covid-19 (7, 9, 19, 21, 36) . the primary cause of the unavailability of approved and commercial vaccines, drugs, and therapeutics to counter the earlier sars-cov and mers-cov seems to owe to the lesser attention of the biomedicine and pharmaceutical companies, as these two covs did not cause much havoc, global threat, and panic like those posed by the sars-cov-2 pandemic (19) . moreover, for such outbreak situations, the requirement for vaccines and therapeutics/drugs exists only for a limited period, until the outbreak is controlled. the proportion of the human population infected with sars-cov and mers-cov was also much lower across the globe, failing to attract drug and vaccine manufacturers and clinical microbiology reviews producers. therefore, by the time an effective drug or vaccine is designed against such disease outbreaks, the virus would have been controlled by adopting appropriate and strict prevention and control measures, and patients for clinical trials will not be available. the newly developed drugs cannot be marketed due to the lack of end users. the s protein plays a significant role in the induction of protective immunity against sars-cov by mediating t-cell responses and neutralizing antibody production (168) . in the past few decades, we have seen several attempts to develop a vaccine against human coronaviruses by using s protein as the target (168, 169) . however, the developed vaccines have minimal application, even among closely related strains of the virus, due to a lack of cross-protection. that is mainly because of the extensive diversity existing among the different antigenic variants of the virus (104) . the contributions of the structural proteins, like spike (s), matrix (m), small envelope (e), and nucleocapsid (n) proteins, of sars-cov to induce protective immunity has been evaluated by expressing them in a recombinant parainfluenza virus type 3 vector (bhpiv3). of note, the result was conclusive that the expression of m, e, or n proteins without the presence of s protein would not confer any noticeable protection, with the absence of detectable serum sars-cov-neutralizing antibodies (170) . antigenic determinant sites present over s and n structural proteins of sars-cov-2 can be explored as suitable vaccine candidates (294) . in the asian population, s, e, m, and n proteins of sars-cov-2 are being targeted for developing subunit vaccines against covid-19 (295) . the identification of the immunodominant region among the subunits and domains of s protein is critical for developing an effective vaccine against the coronavirus. the c-terminal domain of the s1 subunit is considered the immunodominant region of the porcine deltacoronavirus s protein (171) . similarly, further investigations are needed to determine the immunodominant regions of sars-cov-2 for facilitating vaccine development. however, our previous attempts to develop a universal vaccine that is effective for both sars-cov and mers-cov based on t-cell epitope similarity pointed out the possibility of cross-reactivity among coronaviruses (172) . that can be made possible by selected potential vaccine targets that are common to both viruses. sars-cov-2 has been reported to be closely related to sars-cov (173, 174) . hence, knowledge and understanding of s protein-based vaccine development in sars-cov will help to identify potential s protein vaccine candidates in sars-cov-2. therefore, vaccine strategies based on the whole s protein, s protein subunits, or specific potential epitopes of s protein appear to be the most promising vaccine candidates against coronaviruses. the rbd of the s1 subunit of s protein has a superior capacity to induce neutralizing antibodies. this property of the rbd can be utilized for designing potential sars-cov vaccines either by using rbd-containing recombinant proteins or recombinant vectors that encode rbd (175) . hence, the superior genetic similarity existing between sars-cov-2 and sars-cov can be utilized to repurpose vaccines that have proven in vitro efficacy against sars-cov to be utilized for sars-cov-2. the possibility of cross-protection in covid-19 was evaluated by comparing the s protein sequences of sars-cov-2 with that of sars-cov. the comparative analysis confirmed that the variable residues were found concentrated on the s1 subunit of s protein, an important vaccine target of the virus (150) . hence, the possibility of sars-cov-specific neutralizing antibodies providing cross-protection to covid-19 might be lower. further genetic analysis is required between sars-cov-2 and different strains of sars-cov and sarslike (sl) covs to evaluate the possibility of repurposed vaccines against covid-19. this strategy will be helpful in the scenario of an outbreak, since much time can be saved, because preliminary evaluation, including in vitro studies, already would be completed for such vaccine candidates. multiepitope subunit vaccines can be considered a promising preventive strategy against the ongoing covid-19 pandemic. in silico and advanced immunoinformatic tools can be used to develop multiepitope subunit vaccines. the vaccines that are engineered by this technique can be further evaluated using docking studies and, if found effective, then can be further evaluated in animal models (365) . identifying epitopes that have the potential to become a vaccine candidate is critical to developing an effective vaccine against covid-19. the immunoinformatics approach has been used for recognizing essential epitopes of cytotoxic t lymphocytes and b cells from the surface glycoprotein of sars-cov-2. recently, a few epitopes have been recognized from the sars-cov-2 surface glycoprotein. the selected epitopes explored targeting molecular dynamic simulations, evaluating their interaction with corresponding major histocompatibility complex class i molecules. they potentially induce immune responses (176) . the recombinant vaccine can be designed by using rabies virus (rv) as a viral vector. rv can be made to express mers-cov s1 protein on its surface so that an immune response is induced against mers-cov. the rv vector-based vaccines against mers-cov can induce faster antibody response as well as higher degrees of cellular immunity than the gram-positive enhancer matrix (gem) particle vector-based vaccine. however, the latter can induce a very high antibody response at lower doses (167) . hence, the degree of humoral and cellular immune responses produced by such vaccines depends upon the vector used. dual vaccines have been getting more popular recently. among them, the rabies virus-based vectored vaccine platform is used to develop vaccines against emerging infectious diseases. the dual vaccine developed from inactivated rabies virus particles that express the mers-cov s1 domain of s protein was found to induce immune responses for both mers-cov and rabies virus. the vaccinated mice were found to be completely protected from challenge with mers-cov (169) . the intranasal administration of the recombinant adenovirus-based vaccine in balb/c mice was found to induce long-lasting neutralizing immunity against mers spike pseudotyped virus, characterized by the induction of systemic igg, secretory iga, and lung-resident memory t-cell responses (177) . immunoinformatics methods have been employed for the genomewide screening of potential vaccine targets among the different immunogens of mers-cov (178) . the n protein and the potential b-cell epitopes of mers-cov e protein have been suggested as immunoprotective targets inducing both t-cell and neutralizing antibody responses (178, 179) . the collaborative effort of the researchers of rocky mountain laboratories and oxford university is designing a chimpanzee adenovirus-vectored vaccine to counter covid-19 (180) . the coalition for epidemic preparedness innovations (cepi) has initiated three programs to design sars-cov-2 vaccines (181) . cepi has a collaborative project with inovio for designing a mers-cov dna vaccine that could potentiate effective immunity. cepi and the university of queensland are designing a molecular clamp vaccine platform for mers-cov and other pathogens, which could assist in the easier identification of antigens by the immune system (181) . cepi has also funded moderna to develop a vaccine for covid-19 in partnership with the vaccine research center (vrc) of the national institute of allergy and infectious diseases (niaid), part of the national institutes of health (nih) (182) . by employing mrna vaccine platform technology, a vaccine candidate expressing sars-cov-2 spike protein is likely to go through clinical testing in the coming months (180 (298) . the process of vaccine development usually takes approximately ten years, in the case of inactivated or live attenuated vaccines, since it involves the generation of long-term efficacy data. however, this was brought down to 5 years during the ebola emergency for viral vector vaccines. in the urgency associated with the covid-19 outbreaks, we expect a vaccine by the end of this year (343) . the development of an effective vaccine against covid-19 with high speed and precision is the combined result of advancements in computational biology, gene synthesis, protein engineering, and the invention of advanced manufacturing platforms (342) . the recurring nature of the coronavirus outbreaks calls for the development of a pan-coronavirus vaccine that can produce cross-reactive antibodies. however, the success of such a vaccine relies greatly on its ability to provide protection not only against present versions of the virus but also the ones that are likely to emerge in the future. this can be achieved by identifying antibodies that can recognize relatively conserved epitopes that are maintained as such even after the occurrence of considerable variations (362) . even though several vaccine clinical trials are being conducted around the world, pregnant women have been completely excluded from these studies. pregnant women are highly vulnerable to emerging diseases such as covid-19 due to alterations in the immune system and other physiological systems that are associated with pregnancy. therefore, in the event of successful vaccine development, pregnant women will not get access to the vaccines (361) . hence, it is recommended that pregnant women be included in the ongoing vaccine trials, since successful vaccination in pregnancy will protect the mother, fetus, and newborn. the heterologous immune effects induced by bacillus calmette guérin (bcg) vaccination is a promising strategy for controlling the covid-19 pandemic and requires further investigations. bcg is a widely used vaccine against tuberculosis in high-risk regions. it is derived from a live attenuated strain of mycobacterium bovis. at present, three new clinical trials have been registered to evaluate the protective role of bcg vaccination against sars-cov-2 (363) . recently, a cohort study was conducted to evaluate the impact of childhood bcg vaccination in covid-19 pcr positivity rates. however, childhood bcg vaccination was found to be associated with a rate of covid-19-positive test results similar to that of the nonvaccinated group (364) . further studies are required to analyze whether bcg vaccination in childhood can induce protective effects against covid-19 in adulthood. population genetic studies conducted on 103 genomes identified that the sars-cov-2 virus has evolved into two major types, l and s. among the two types, l type is expected to be the most prevalent (ϳ70%), followed by the s type (ϳ30%) (366) . this finding has a significant impact on our race to develop an ideal vaccine, since the vaccine candidate has to target both strains to be considered effective. at present, the genetic differences between the l and s types are very small and may not affect the immune response. however, we can expect further genetic variations in the coming days that could lead to the emergence of new strains (367) . there is no currently licensed specific antiviral treatment for mers-and sars-cov infections, and the main focus in clinical settings remains on lessening clinical signs and providing supportive care (183) (184) (185) (186) . effective drugs to manage covid-19 patients include remdesivir, lopinavir/ritonavir alone or in a blend with interferon beta, convalescent plasma, and monoclonal antibodies (mabs); however, efficacy and safety issues of these drugs require additional clinical trials (187, 281) . a controlled trial of ritonavirboosted lopinavir and interferon alpha 2b treatment was performed on covid-19 hospitalized patients (chictr2000029308) (188) . in addition, the use of hydroxychloroquine and tocilizumab for their potential role in modulating inflammatory responses in the lungs and antiviral effect has been proposed and discussed in many research articles. still, no fool-proof clinical trials have been published (194, 196, 197, (261) (262) (263) (264) (265) (266) (267) (268) (269) (270) (271) (272) . recently, a clinical trial conducted on adult patients suffering from severe covid-19 revealed no benefit of lopinavir-ritonavir treatment over standard care (273) . the efforts to control sars-cov-2 infection utilize defined strategies as followed against mers and sars, along with adopting and strengthening a few precautionary measures owing to the unknown nature of this novel virus (36, 189) . presently, the main course of treatment for severely affected sars-cov-2 patients admitted to hospitals includes mechanical ventilation, intensive care unit (icu) admittance, and symptomatic and supportive therapies. additionally, rna synthesis inhibitors (lamivudine and tenofovir disoproxil fumarate), remdesivir, neuraminidase inhibitors, peptide (ek1), antiinflammatory drugs, abidol, and chinese traditional medicine (lianhuaqingwen and shufengjiedu capsules) could aid in covid-19 treatment. however, further clinical trials are being carried out concerning their safety and efficacy (7) . it might require months to a year(s) to design and develop effective drugs, therapeutics, and vaccines against covid-19, with adequate evaluation and approval from regulatory bodies and moving to the bulk production of many millions of doses at commercial levels to meet the timely demand of mass populations across the globe (9) . continuous efforts are also warranted to identify and assess viable drugs and immunotherapeutic regimens that revealed proven potency in combating other viral agents similar to sars-cov-2. covid-19 patients showing severe signs are treated symptomatically along with oxygen therapy. in such cases where the patients progress toward respiratory failure and become refractory to oxygen therapy, mechanical ventilation is necessitated. the covid-19-induced septic shock can be managed by providing adequate hemodynamic support (299) . several classes of drugs are currently being evaluated for their potential therapeutic action against sars-cov-2. therapeutic agents that have anti-sars-cov-2 activity can be broadly classified into three categories: drugs that block virus entry into the host cell, drugs that block viral replication as well as its survival within the host cell, and drugs that attenuate the exaggerated host immune response (300) . an inflammatory cytokine storm is commonly seen in critically ill covid-19 patients. hence, they may benefit from the use of timely anti-inflammation treatment. anti-inflammatory therapy using drugs like glucocorticoids, cytokine inhibitors, jak inhibitors, and chloroquine/hydroxychloroquine should be done only after analyzing the risk/benefit ratio in covid-19 patients (301). there have not been any studies concerning the application of nonsteroidal anti-inflammatory drugs (nsaid) to covid-19-infected patients. however, reasonable pieces of evidence are available that link nsaid uses with the occurrence of respiratory and cardiovascular adverse effects. hence, as a cautionary approach, it is better to recommend the use of nsaids as the first-line option for managing covid-19 symptoms (302) . the use of corticosteroids in covid-19 patients is still a matter of controversy and requires further systematic clinical studies. the guidelines that were put forward to manage critically ill adults suggest the use of systemic corticosteroids in mechanically ventilated adults with ards (303) . the generalized use of corticosteroids is not indicated in covid-19, since there are some concerns associated with the use of corticosteroids in viral pneumonia. stem cell therapy using mesenchymal stem cells (mscs) is another hopeful strategy that can be used in clinical cases of covid-19 owing to its potential immunomodulatory capacity. it may have a beneficial role in attenuating the cytokine storm that is observed in severe cases of sars-cov-2 infection, thereby reducing mortality. among the different types of mscs, expanded umbilical cord mscs can be considered a potential therapeutic agent that requires further validation for managing critically ill covid-19 patients (304) . repurposed broad-spectrum antiviral drugs having proven uses against other viral pathogens can be employed for sars-cov-2-infected patients. these possess benefits of easy accessibility and recognized pharmacokinetic and pharmacodynamic activities, stability, doses, and side effects (9) . repurposed drugs have been studied for treating cov infections, like lopinavir/ritonavir, and interferon-1␤ revealed in vitro anti-mers-cov action. the in vivo experiment carried out in the nonhuman primate model of clinical microbiology reviews common marmosets treated with lopinavir/ritonavir and interferon beta showed superior protective results in treated animals than in the untreated ones (190) . a combination of these drugs is being evaluated to treat mers in humans (miracle trial) (191) . these two protease inhibitors (lopinavir and ritonavir), in combination with ribavirin, gave encouraging clinical outcomes in sars patients, suggesting their therapeutic values (165) . however, in the current scenario, due to the lack of specific therapeutic agents against sars-cov-2, hospitalized patients confirmed for the disease are given supportive care, like oxygen and fluid therapy, along with antibiotic therapy for managing secondary bacterial infections (192) . patients with novel coronavirus or covid-19 pneumonia who are mechanically ventilated often require sedatives, analgesics, and even muscle relaxation drugs to prevent ventilator-related lung injury associated with human-machine incoordination (122) . the result obtained from a clinical study of four patients infected with covid-19 claimed that combination therapy using lopinavir/ritonavir, arbidol, and shufeng jiedu capsules (traditional chinese medicine) was found to be effective in managing covid-19 pneumonia (193) . it is difficult to evaluate the therapeutic potential of a drug or a combination of drugs for managing a disease based on such a limited sample size. before choosing the ideal therapeutic agent for the management of covid-19, randomized clinical control studies should be performed with a sufficient study population. several classes of routinely used antiviral drugs, like oseltamivir (neuraminidase inhibitor), acyclovir, ganciclovir, and ribavirin, do not have any effect on covid-19 and, hence, are not recommended (187) . oseltamivir, a neuraminidase inhibitor, has been explored in chinese hospitals for treating suspected covid-19 cases, although proven efficacy against sars-cov-2 is still lacking for this drug (7) . the in vitro antiviral potential of fad-approved drugs, viz., ribavirin, penciclovir, nitazoxanide, nafamostat, and chloroquine, tested in comparison to remdesivir and favipiravir (broad-spectrum antiviral drugs) revealed remdesivir and chloroquine to be highly effective against sars-cov-2 infection in vitro (194) . ribavirin, penciclovir, and favipiravir might not possess noteworthy in vivo antiviral actions for sars-cov-2, since higher concentrations of these nucleoside analogs are needed in vitro to lessen the viral infection. both remdesivir and chloroquine are being used in humans to treat other diseases, and such safer drugs can be explored for assessing their effectiveness in covid-19 patients. several therapeutic agents, such as lopinavir/ritonavir, chloroquine, and hydroxychloroquine, have been proposed for the clinical management of covid-19 (299) . a molecular docking study, conducted in the rna-dependent rna polymerase (rdrp) of sars-cov-2 using different commercially available antipolymerase drugs, identified that drugs such as ribavirin, remdesivir, galidesivir, tenofovir, and sofosbuvir bind rdrp tightly, indicating their vast potential to be used against covid-19 (305). a broadspectrum antiviral drug that was developed in the united states, tilorone dihydrochloride (tilorone), was previously found to possess potent antiviral activity against mers, marburg, ebola, and chikungunya viruses (306) . even though it had broad-spectrum activity, it was neglected for an extended period. tilorone is another antiviral drug that might have activity against sars-cov-2. remdesivir, a novel nucleotide analog prodrug, was developed for treating ebola virus disease (evd), and it was also found to inhibit the replication of sars-cov and mers-cov in primary human airway epithelial cell culture systems (195) . recently, in vitro study has proven that remdesivir has better antiviral activity than lopinavir and ritonavir. further, in vivo studies conducted in mice also identified that treatment with remdesivir improved pulmonary function and reduced viral loads and lung pathology both in prophylactic and therapeutic regimens compared to lopinavir/ritonavir-ifn-␥ treatment in mers-cov infection (8) . remdesivir also inhibits a diverse range of coronaviruses, including circulating human cov, zoonotic bat cov, and prepandemic zoonotic cov (195) . remdesivir is also considered the only therapeutic drug that significantly reduces pulmonary pathology (8) . all these findings indicate that remde-sivir has to be further evaluated for its efficacy in the treatment of covid-19 infection in humans. the broad-spectrum activity exhibited by remdesivir will help control the spread of disease in the event of a new coronavirus outbreak. chloroquine is an antimalarial drug known to possess antiviral activity due to its ability to block virus-cell fusion by raising the endosomal ph necessary for fusion. it also interferes with virus-receptor binding by interfering with the terminal glycosylation of sars-cov cellular receptors, such as ace2 (196) . in a recent multicenter clinical trial that was conducted in china, chloroquine phosphate was found to exhibit both efficacy and safety in the therapeutic management of sars-cov-2-associated pneumonia (197) . this drug is already included in the treatment guidelines issued by the national health commission of the people's republic of china. the preliminary clinical trials using hydroxychloroquine, another aminoquinoline drug, gave promising results. the covid-19 patients received 600 mg of hydroxychloroquine daily along with azithromycin as a single-arm protocol. this protocol was found to be associated with a noteworthy reduction in viral load. finally, it resulted in a complete cure (271) ; however, the study comprised a small population and, hence, the possibility of misinterpretation could arise. however, in another case study, the authors raised concerns over the efficacy of hydroxychloroquine-azithromycin in the treatment of covid-19 patients, since no observable effect was seen when they were used. in some cases, the treatment was discontinued due to the prolongation of the qt interval (307) . hence, further randomized clinical trials are required before concluding this matter. recently, another fda-approved drug, ivermectin, was reported to inhibit the in vitro replication of sars-cov-2. the findings from this study indicate that a single treatment of this drug was able to induce an ϳ5,000-fold reduction in the viral rna at 48 h in cell culture. (308) . one of the main disadvantages that limit the clinical utility of ivermectin is its potential to cause cytotoxicity. however, altering the vehicles used in the formulations, the pharmacokinetic properties can be modified, thereby having significant control over the systemic concentration of ivermectin (338) . based on the pharmacokinetic simulation, it was also found that ivermectin may have limited therapeutic utility in managing covid-19, since the inhibitory concentration that has to be achieved for effective anti-sars-cov-2 activity is far higher than the maximum plasma concentration achieved by administering the approved dose (340) . however, ivermectin, being a host-directed agent, exhibits antiviral activity by targeting a critical cellular process of the mammalian cell. therefore, the administration of ivermectin, even at lower doses, will reduce the viral load at a minor level. this slight decrease will provide a great advantage to the immune system for mounting a large-scale antiviral response against sars-cov-2 (341). further, a combination of ivermectin and hydroxychloroquine might have a synergistic effect, since ivermectin reduces viral replication, while hydroxychloroquine inhibits the entry of the virus in the host cell (339) . further, in vivo studies and randomized clinical control trials are required to understand the mechanism as well as the clinical utility of this promising drug. nafamostat is a potent inhibitor of mers-cov that acts by preventing membrane fusion. nevertheless, it does not have any sort of inhibitory action against sars-cov-2 infection (194) . recently, several newly synthesized halogenated triazole compounds were evaluated, using fluorescence resonance energy transfer (fret)-based helicase assays, for their ability to inhibit helicase activity. among the evaluated compounds, 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-iodophenyl) hydrazinyl]-4h-1,2,4-triazole-3-thiol and 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-chlorophenyl) hydrazinyl]-4h-1,2,4-triazole-3-thiol were found to be the most potent. these compounds were used for in silico studies, and molecular docking was accomplished into the active binding site of mers-cov helicase nsp13 (21) . further studies are required for evaluating the therapeutic potential of these newly identified compounds in the management of covid-19 infection. monoclonal antibodies (mabs) may be helpful in the intervention of disease in clinical microbiology reviews cov-exposed individuals. patients recovering from sars showed robust neutralizing antibodies against this cov infection (164) . a set of mabs aimed at the mers-cov s protein-specific domains, comprising six specific epitope groups interacting with receptor-binding, membrane fusion, and sialic acid-binding sites, make up crucial entry tasks of s protein (198, 199) . passive immunization employing weaker and strongly neutralizing antibodies provided considerable protection in mice against a mers-cov lethal challenge. such antibodies may play a crucial role in enhancing protective humoral responses against the emerging covs by aiming appropriate epitopes and functions of the s protein. the cross-neutralization ability of sars-cov rbd-specific neutralizing mabs considerably relies on the resemblance between their rbds; therefore, sars-cov rbd-specific antibodies could cross-neutralized sl covs, i.e., bat-sl-cov strain wiv1 (rbd with eight amino acid differences from sars-cov) but not bat-sl-cov strain shc014 (24 amino acid differences) (200) . appropriate rbd-specific mabs can be recognized by a relative analysis of rbd of sars-cov-2 to that of sars-cov, and cross-neutralizing sars-cov rbd-specific mabs could be explored for their effectiveness against covid-19 and further need to be assessed clinically. the u.s. biotechnology company regeneron is attempting to recognize potent and specific mabs to combat covid-19. an ideal therapeutic option suggested for sars-cov-2 (covid-19) is the combination therapy comprised of mabs and the drug remdesivir (covid-19) (201) . the sars-cov-specific human mab cr3022 is found to bind with sars-cov-2 rbd, indicating its potential as a therapeutic agent in the management of covid-19. it can be used alone or in combination with other effective neutralizing antibodies for the treatment and prevention of covid-19 (202) . furthermore, sars-cov-specific neutralizing antibodies, like m396 and cr3014, failed to bind the s protein of sars-cov-2, indicating that a particular level of similarity is mandatory between the rbds of sars-cov and sars-cov-2 for the cross-reactivity to occur. further assessment is necessary before confirming the effectiveness of such combination therapy. in addition, to prevent further community and nosocomial spread of covid-19, the postprocedure risk management program should not be neglected (309) . development of broad-spectrum inhibitors against the human coronaviral pathogens will help to facilitate clinical trials on the effectiveness of such inhibitors against endemic and emerging coronaviruses (203) . a promising animal study revealed the protective effect of passive immunotherapy with immune serum from mers-immune camels on mice infected with mers-cov (204) . passive immunotherapy using convalescent plasma is another strategy that can be used for treating covid-19-infected, critically ill patients (205) . the exploration of fully human antibodies (human single-chain antibodies; huscfvs) or humanized nanobodies (single-domain antibodies; sdab, vh/vhh) could aid in blocking virus replication, as these agents can traverse the virus-infected cell membranes (transbodies) and can interfere with the biological characteristics of the replicating virus proteins. such examples include transbodies to the influenza virus, hepatitis c virus, ebola virus, and dengue virus (206) . producing similar transbodies against intracellular proteins of coronaviruses, such as papain-like proteases (plpro), cysteinelike protease (3clpro), or other nsps, which are essential for replication and transcription of the virus, might formulate a practical move forward for a safer and potent passive immunization approach for virus-exposed persons and rendering therapy to infected patients. in a case study on five grimly sick patients having symptoms of severe pneumonia due to covid-19, convalescent plasma administration was found to be helpful in patients recovering successfully. the convalescent plasma containing a sars-cov-2specific elisa (serum) antibody titer higher than 1:1,000 and neutralizing antibody titer more significant than 40 was collected from the recovered patients and used for plasma transfusion twice in a volume of 200 to 250 ml on the day of collection (310) . at present, treatment for sepsis and ards mainly involves antimicrobial therapy, source control, and supportive care. hence, the use of therapeutic plasma exchange can be considered an option in managing such severe conditions. further randomized trials can be designed to investigate its efficacy (311) . potent therapeutics to combat sars-cov-2 infection include virus binding molecules, molecules or inhibitors targeting particular enzymes implicated in replication and transcription process of the virus, helicase inhibitors, vital viral proteases and proteins, protease inhibitors of host cells, endocytosis inhibitors, short interfering rna (sirna), neutralizing antibodies, mabs against the host receptor, mabs interfering with the s1 rbd, antiviral peptide aimed at s2, and natural drugs/medicines (7, 166, 186) . the s protein acts as the critical target for developing cov antivirals, like inhibitors of s protein and s cleavage, neutralizing antibodies, rbd-ace2 blockers, sirnas, blockers of the fusion core, and proteases (168) . all of these therapeutic approaches have revealed both in vitro and in vivo anti-cov potential. although in vitro research carried out with these therapeutics showed efficacy, most need appropriate support from randomized animal or human trials. therefore, they might be of limited applicability and require trials against sars-cov-2 to gain practical usefulness. the binding of sars-cov-2 with ace2 leads to the exacerbation of pneumonia as a consequence of the imbalance in the reninangiotensin system (ras). the virus-induced pulmonary inflammatory responses may be reduced by the administration of ace inhibitors (acei) and angiotensin type-1 receptor (at1r) (207) . several investigations have suggested the use of small-molecule inhibitors for the potential control of sars-cov infections. drugs of the fda-approved compound library were screened to identify four small-molecule inhibitors of mers-cov (chlorpromazine, chloroquine, loperamide, and lopinavir) that inhibited viral replication. these compounds also hinder sars-cov and human covs (208) . therapeutic strategies involving the use of specific antibodies or compounds that neutralize cytokines and their receptors will help to restrain the host inflammatory responses. such drugs acting specifically in the respiratory tract will help to reduce virus-triggered immune pathologies in covid-19 (209) . the later stages of coronavirus-induced inflammatory cascades are characterized by the release of proinflammatory interleukin-1 (il-1) family members, such as il-1 and il-33. hence, there exists a possibility that the inflammation associated with coronavirus can be inhibited by utilizing anti-inflammatory cytokines that belong to the il-1 family (92) . it has also been suggested that the actin protein is the host factor that is involved in cell entry and pathogenesis of sars-cov-2. hence, those drugs that modulate the biological activity of this protein, like ibuprofen, might have some therapeutic application in managing the disease (174). the plasma angiotensin 2 level was found to be markedly elevated in covid-19 infection and was correlated with viral load and lung injury. hence, drugs that block angiotensin receptors may have potential for treating covid-19 infection (121) . a scientist from germany, named rolf hilgenfeld, has been working on the identification of drugs for the treatment of coronaviral infection since the time of the first sars outbreak (19) . the sars-cov s2 subunit has a significant function in mediating virus fusion that provides entry into the host cell. heptad repeat 1 (hr1) and heptad repeat 2 (hr2) can interact and form a six-helix bundle that brings the viral and cellular membranes in close proximity, facilitating its fusion. the sequence alignment study conducted between covid-19 and sars-cov identified that the s2 subunits are highly conserved in these covs. the hr1 and hr2 domains showed 92.6% and 100% overall identity, respectively (210) . from these findings, we can confirm the significance of covid-19 hr1 and hr2 and their vital role in host cell entry. hence, fusion inhibitors target the hr1 domain of s protein, thereby preventing viral fusion and entry into the host cell. this is another potential therapeutic strategy that can be used in the management of covid-19. other than the specific therapy directed against covid-19, general treatments play a vital role in the enhancement of host immune responses against the viral agent. inadequate nutrition is linked to the weakening of the host immune response, clinical microbiology reviews making the individual more susceptible. the role played by nutrition in disease susceptibility should be measured by evaluating the nutritional status of patients with covid-19 (205) . for evaluating the potential of vaccines and therapeutics against covs, including sars-cov, mers-covs, and the presently emerging sars-cov-2, suitable animal models that can mimic the clinical disease are needed (211, 212) . various animal models were assessed for sars-and mers-covs, such as mice, guinea pigs, golden syrian hamsters, ferrets, rabbits, nonhuman primates like rhesus macaques and marmosets, and cats (185, (213) (214) (215) (216) (217) (218) . the specificity of the virus to hace2 (receptor of sars-cov) was found to be a significant barrier in developing animal models. consequently, a sars-cov transgenic mouse model has been developed by inserting the hace2 gene into the mouse genome (219) . the inability of mers-cov to replicate in the respiratory tracts of animals (mice, hamsters, and ferrets) is another limiting factor. however, with genetic engineering, a 288-330 ϩ/ϩ mers-cov genetically modified mouse model was developed and now is in use for the assessment of novel drugs and vaccines against mers-cov (220). in the past, small animals (mice or hamsters) have been targeted for being closer to a humanized structure, such as mouse dpp4 altered with human dpp4 (hdpp4), hdpp4-transduced mice, and hdpp4-tg mice (transgenic for expressing hdpp4) for mers-cov infection (221) . the crispr-cas9 gene-editing tool has been used for inserting genomic alterations in mice, making them susceptible to mers-cov infection (222) . efforts are under way to recognize suitable animal models for sars-cov2/covid-19, identify the receptor affinity of this virus, study pathology in experimental animal models, and explore virus-specific immune responses and protection studies, which together would increase the pace of efforts being made for developing potent vaccines and drugs to counter this emerging virus. cell lines, such as monkey epithelial cell lines (llc-mk2 and vero-b4), goat lung cells, alpaca kidney cells, dromedary umbilical cord cells, and advanced ex vivo three-dimensional tracheobronchial tissue, have been explored to study human covs (mers-cov) (223, 224) . vero and huh-7 cells (human liver cancer cells) have been used for isolating sars-cov-2 (194) . recently, an experimental study with rhesus monkeys as animal models revealed the absence of any viral loads in nasopharyngeal and anal swabs, and no viral replication was recorded in the primary tissues at a time interval of 5 days post-reinfection in reexposed monkeys (274) . the subsequent virological, radiological, and pathological observations indicated that the monkeys with reexposure had no recurrence of covid-19, like the sars-cov-2-infected monkeys without rechallenge. these findings suggest that primary infection with sars-cov-2 could protect from later exposures to the virus, which could help in defining disease prognosis and crucial inferences for designing and developing potent vaccines against covid-19 (274). in contrast to their response to the 2002 sars outbreak, china has shown immense political openness in reporting the covid-19 outbreak promptly. they have also performed rapid sequencing of covid-19 at multiple levels and shared the findings globally within days of identifying the novel virus (225) . the move made by china opened a new chapter in global health security and diplomacy. even though complete lockdown was declared following the covid-19 outbreak in wuhan, the large-scale movement of people has resulted in a radiating spread of infections in the surrounding provinces as well as to several other countries. large-scale screening programs might help us to control the spread of this virus. however, this is both challenging as well as time-consuming due to the present extent of infection (226) . the current scenario demands effective implementation of vigorous prevention and control strategies owing to the prospect of covid-19 for nosocomial infections (68) . follow-ups of infected patients by telephone on day 7 and day 14 are advised to avoid any further unintentional spread or nosocomial transmission (312) . the availability of public data sets provided by independent analytical teams will act as robust evidence that would guide us in designing interventions against the covid-19 outbreak. newspaper reports and social media can be used to analyze and reconstruct the progression of an outbreak. they can help us to obtain detailed patient-level data in the early stages of an outbreak (227) . immediate travel restrictions imposed by several countries might have contributed significantly to preventing the spread of sars-cov-2 globally (89, 228) . following the outbreak, a temporary ban was imposed on the wildlife trade, keeping in mind the possible role played by wild animal species in the origin of sars-cov-2/covid-19 (147) . making a permanent and bold decision on the trade of wild animal species is necessary to prevent the possibility of virus spread and initiation of an outbreak due to zoonotic spillover (1) . personal protective equipment (ppe), like face masks, will help to prevent the spread of respiratory infections like covid-19. face masks not only protect from infectious aerosols but also prevent the transmission of disease to other susceptible individuals while traveling through public transport systems (313) . another critical practice that can reduce the transmission of respiratory diseases is the maintenance of hand hygiene. however, the efficacy of this practice in reducing the transmission of respiratory viruses like sars-cov-2 is much dependent upon the size of droplets produced. hand hygiene will reduce disease transmission only if the virus is transmitted through the formation of large droplets (314) . hence, it is better not to overemphasize that hand hygiene will prevent the transmission of sars-cov-2, since it may produce a false sense of safety among the general public that further contributes to the spread of covid-19. even though airborne spread has not been reported in sars-cov-2 infection, transmission can occur through droplets and fomites, especially when there is close, unprotected contact between infected and susceptible individuals. hence, hand hygiene is equally as important as the use of appropriate ppe, like face masks, to break the transmission cycle of the virus; both hand hygiene and face masks help to lessen the risk of covid-19 transmission (315) . medical staff are in the group of individuals most at risk of getting covid-19 infection. this is because they are exposed directly to infected patients. hence, proper training must be given to all hospital staff on methods of prevention and protection so that they become competent enough to protect themselves and others from this deadly disease (316) . as a preventive measure, health care workers caring for infected patients should take extreme precautions against both contact and airborne transmission. they should use ppe such as face masks (n95 or ffp3), eye protection (goggles), gowns, and gloves to nullify the risk of infection (299) . the human-to-human transmission reported in sars-cov-2 infection occurs mainly through droplet or direct contact. due to this finding, frontline health care workers should follow stringent infection control and preventive measures, such as the use of ppe, to prevent infection (110) . the mental health of the medical/health workers who are involved in the covid-19 outbreak is of great importance, because the strain on their mental well-being will affect their attention, concentration, and decision-making capacity. hence, for control of the covid-19 outbreak, rapid steps should be taken to protect the mental health of medical workers (229) . since the living mammals sold in the wet market are suspected to be the intermediate host of sars-cov-2, there is a need for strengthening the regulatory mechanism for wild animal trade (13) . the total number of covid-19 confirmed cases is on a continuous rise and the cure rate is relatively low, making disease control very difficult to achieve. the chinese government is making continuous efforts to contain the disease by taking emergency control and prevention measures. they have already built a hospital for patients affected by this virus and are currently building several more for accommodating the continuously increasing infected population (230) . the effective control of sars-cov-2/covid-19 requires high-level interventions like intensive contact tracing, as well as the quarantine of people with suspected infection and the isolation of infected individuals. the implementation of rigorous control and preventive measures together might control the r 0 number and reduce the transmission risk (228) . clinical microbiology reviews considering the zoonotic links associated with sars-cov-2, the one health approach may play a vital role in the prevention and control measures being followed to restrain this pandemic virus (317) (318) (319) . the substantial importation of covid-19 presymptomatic cases from wuhan has resulted in independent, self-sustaining outbreaks across major cities both within the country and across the globe. the majority of chinese cities are now facing localized outbreaks of covid-19 (231) . hence, deploying efficient public health interventions might help to cut the spread of this virus globally. the occurrence of covid-19 infection on several cruise ships gave us a preliminary idea regarding the transmission pattern of the disease. cruise ships act as a closed environment and provide an ideal setting for the occurrence of respiratory disease outbreaks. such a situation poses a significant threat to travelers, since people from different countries are on board, which favors the introduction of the pathogen (320). although nearly 30 cruise ships from different countries have been found harboring covid-19 infection, the major cruise ships that were involved in the covid-19 outbreaks are the diamond princess, grand princess, celebrity apex, and ruby princess. the number of confirmed covid-19 cases around the world is on the rise. the success of preventive measures put forward by every country is mainly dependent upon their ability to anticipate the approaching waves of patients. this will help to properly prepare the health care workers and increase the intensive care unit (icu) capacity (321) . instead of entirely relying on lockdown protocols, countries should focus mainly on alternative intervention strategies, such as large-scale testing, contract tracing, and localized quarantine of suspected cases for limiting the spread of this pandemic virus. such intervention strategies will be useful either at the beginning of the pandemic or after lockdown relaxation (322) . lockdown should be imposed only to slow down disease progression among the population so that the health care system is not overloaded. the reproduction number (r 0 ) of covid-19 infection was earlier estimated to be in the range of 1.4 to 2.5 (70); recently, it was estimated to be 2.24 to 3.58 (76) . compared to its coronavirus predecessors, covid-19 has an r 0 value that is greater than that of mers (r 0 ͻ 1) (108) but less than that of sars (r 0 value of 2 to 5) (93) . still, to prevent further spread of disease at mass gatherings, functions remain canceled in the affected cities, and persons are asked to work from home (232) . hence, it is a relief that the current outbreak of covid-19 infection can be brought under control with the adoption of strategic preventive and control measures along with the early isolation of subsequent cases in the coming days. studies also report that since air traffic between china and african countries increased many times over in the decade after the sars outbreak, african countries need to be vigilant to prevent the spread of novel coronavirus in africa (225) . due to fear of virus spread, wuhan city was completely shut down (233) . the immediate control of the ongoing covid-19 outbreaks appears a mammoth task, especially for developing countries, due to their inability to allocate quarantine stations that could screen infected individuals' movements (234) . such underdeveloped countries should divert their resources and energy to enforcing the primary level of preventive measures, like controlling the entry of individuals from china or countries where the disease has flared up, isolating the infected individuals, and quarantining individuals with suspected infection. most of the sub-saharan african countries have a fragile health system that can be crippled in the event of an outbreak. effective management of covid-19 would be difficult for low-income countries due to their inability to respond rapidly due to the lack of an efficient health care system (65) . controlling the imported cases is critical in preventing the spread of covid-19 to other countries that have not reported the disease until now. the possibility of an imported case of covid-19 leading to sustained human-to-human transmission was estimated to be 0.41. this can be reduced to a value of 0.012 by decreasing the mean time from the onset of symptoms to hospitalization and can only be made possible by using intense disease surveillance systems (235) . the silent importations of infected individuals (before the manifestation of clinical signs) also contributed significantly to the spread of disease across the major cities of the world. even though the travel ban was implemented in wuhan (89) , infected persons who traveled out of the city just before the imposition of the ban might have remained undetected and resulted in local outbreaks (236) . emerging novel diseases like covid-19 are difficult to contain within the country of origin, since globalization has led to a world without borders. hence, international collaboration plays a vital role in preventing the further spread of this virus across the globe (237) . we also predict the possibility of another outbreak, as predicted by fan et al. (6) . indeed, the present outbreak caused by sars-cov-2 (covid-19) was expected. similar to previous outbreaks, the current outbreak also will be contained shortly. however, the real issue is how we are planning to counter the next zoonotic cov epidemic that is likely to occur within the next 5 to 10 years or even sooner (fig. 7) . several years after the global sars epidemic, the current sars-cov-2/covid-19 pandemic has served as a reminder of how novel pathogens can rapidly emerge and spread through the human population and eventually cause severe public health crises. further research should be conducted to establish animal models for sars-cov-2 to investigate replication, transmission dynamics, and pathogenesis in humans. this may help develop and evaluate potential therapeutic strategies against zoonotic cov epidemics. present trends suggest the occurrence of future outbreaks of covs due to changes in the climate, and ecological conditions may be associated with humananimal contact. live-animal markets, such as the huanan south china seafood market, represent ideal conditions for interspecies contact of wildlife with domestic birds, pigs, and mammals, which substantially increases the probability of interspecies transmission of cov infections and could result in high risks to humans due to adaptive genetic recombination in these viruses (323) (324) (325) . the covid-19-associated symptoms are fever, cough, expectoration, headache, and myalgia or fatigue. individuals with asymptomatic and atypical clinical manifestations were also identified recently, further adding to the complexity of disease transmission dynamics. atypical clinical manifestations may only express symptoms such as fatigue instead of respiratory signs such as fever, cough, and sputum. in such cases, the clinician must be vigilant for the possible occurrence of asymptomatic and atypical clinical manifestations to avoid the possibility of missed diagnoses. the present outbreak caused by sars-cov-2 was, indeed, expected. similar to clinical microbiology reviews previous outbreaks, the current pandemic also will be contained shortly. however, the real question is, how are we planning to counter the next zoonotic cov epidemic that is likely to occur within the next 5 to 10 years or perhaps sooner? our knowledge of most of the bat covs is scarce, as these viruses have not been isolated and studied, and extensive studies on such viruses are typically only conducted when they are associated with specific disease outbreaks. the next step following the control of the covid-19 outbreak in china should be focused on screening, identification, isolation, and characterization of covs present in wildlife species of china, particularly in bats. both in vitro and in vivo studies (using suitable animal models) should be conducted to evaluate the risk of future epidemics. presently, licensed antiviral drugs or vaccines against sars-cov, mers-cov, and sars-cov-2 are lacking. however, 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origin and continuing evolution of sars-cov-2 covid-19: the race for a vaccine all authors substantially contributed to the conception, design, analysis, and interpretation of data and checking and approving the final version of the manuscript, and we agree to be accountable for its contents.this compilation is a review article written, analyzed, and designed by its authors and required no substantial funding to be developed.all authors declare that there are no existing commercial or financial relationships that could, in any way, lead to a potential conflict of interest. with 25 years of research and teaching experience in the areas of microbiology, immunology, virology, public health, medicine, and biomedicine as an eminent researcher, he has developed several diagnostics, vaccines, immunomodulatory modules, and hypotheses to counter infectious diseases of animals, poultry, and public health concerns. he has to his credit 600 publications, 6 books, and 65 book chapters. dr. dhama has been recognized as an extremely productive researcher in the journal nature. he has been honored with 50 best paper awards and other recognitions. he is an naas (national academy of agricultural science, india) associate and has worked as nodal officer, wto, and member, wildlife health specialist group (iucn). he is actively serving as editor-in-chief, co-eic, editor, and member, editorial board, of nearly 20 scientific journals. his google scholar h-index is 47 and scopus h-index is 31. sharun khan, m.v.sc., is currently working as a researcher in the stem cell laboratory, division of surgery, icar-indian veterinary research institute, izatnagar, india. his area of interest is regenerative medicine with a focus on understanding cell biology and molecular pathways involved in the maintenance and differentiation of stem cells originating from different tissues. he has particular interest and knowledge in the fields of veterinary medicine, pharmacology, infectious diseases of animals, wildlife diseases, diagnosis and therapy of animal diseases, nutrition, and biomedicine. with excellent academic records, he has received awards and recognitions (fellowships and scholarships) and participated in national and international workshops, training programs, and courses. he has a keen interest in learning excellent scientific writing skills and has published 30 papers, including in international journals of repute. he is highly enthusiastic about gaining knowledge of advancements in educational and scientific research areas.ruchi tiwari is currently working as assistant professor in the department of veterinary microbiology, college of veterinary sciences, duvasu, mathura, india. she is currently pursuing her ph.d. (hons) degree from duvasu. with an excellent academic record and 10 years of research and teaching experience, she has expertise in the field of diagnosis, prevention, and control of important livestock/poultry diseases/pathogens having public health significance, along with particular reference to veterinary microbiology, immunology, ethnoveterinary medicine, alternative and complementary therapies, and bacteriophage therapy. dr. tiwari has published 150 research/review articles and 5 book chapters. she has been honored with the young scientist award, best paper awards (10) key: cord-263927-hnsyas9q authors: peci, adriana; winter, anne‐luise; gubbay, jonathan b.; skowronski, danuta m.; balogun, elizabeth i.; de lima, cedric; crowcroft, natasha s.; rebbapragada, anu title: community‐acquired respiratory viruses and co‐infection among patients of ontario sentinel practices, april 2009 to february 2010 date: 2012-08-09 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2012.00418.x sha: doc_id: 263927 cord_uid: hnsyas9q please cite this paper as: peci et al. (2012) community‐acquired respiratory viruses and co‐infection among patients of ontario sentinel practices, april 2009 to february 2010. influenza and other respiratory viruses 7(4), 559–566. background respiratory viruses are known to cocirculate but this has not been described in detail during an influenza pandemic. objectives to describe respiratory viruses, including co‐infection and associated attributes such as age, sex or comorbidity, in patients presenting with influenza‐like illness to a community sentinel network, during the pandemic a(h1n1)pdm09 in ontario, canada. methods respiratory samples and epidemiologic details were collected from 1018 patients with influenza‐like illness as part of respiratory virus surveillance and a multiprovincial case–control study of influenza vaccine effectiveness. results at least one virus was detected in 668 (65·6%) of 1018 samples; 512 (50·3%) had single infections and 156 (15·3%) co‐infections. of single infections, the most common viruses were influenza a in 304 (59·4%) samples of which 275 (90·5%) were influenza a(h1n1)pdm09, and enterovirus/rhinovirus in 149 (29·1%) samples. the most common co‐infections were influenza a and respiratory syncytial virus b, and influenza a and enterovirus/rhinovirus. in multinomial logistic regression analyses adjusted for age, sex, comorbidity, and timeliness of sample collection, single infection was less often detected in the elderly and co‐infection more often in patients <30 years of age. co‐infection, but not single infection, was more likely detected in patients who had a sample collected within 2 days of symptom onset as compared to 3–7 days. conclusions respiratory viral co‐infections are commonly detected when using molecular techniques. early sample collection increases likelihood of detection of co‐infection. further studies are needed to better understand the clinical significance of viral co‐infection. a novel influenza virus, a(h1n1)pdm09 emerged in april 2009 and spread rapidly, primarily through human-tohuman transmission. several million people were infected globally. 1 an important feature of this virus was that it mostly affected younger people with 60% of patients under 18 years of age, suggesting possible pre-existing immunity in the elderly due to previous exposure to antigenically related influenza strains. 2, 3 the assumption made in most pandemic plans before 2009 was that the pandemic virus would be the dominant circulating respiratory virus. 4 few studies performed extensive respiratory testing beyond influenza during the pandemic, and fewer still focused on community cases. casalegno et al. documented cocirculation and co-infection of a(h1n1)pdm09 with rhinovirus during the pandemic. 5 watanabe et al. found a wide range of etiologic agents were identified among respiratory samples that were influenza negative, highlighting the need to diagnose other viral organisms that can co-circulate with influenza. 6 louie et al. investigated samples from laboratory-confirmed fatal a(h1n1)pdm09 cases during the pandemic and bacterial pathogens were identified in 22 of 77 samples. 7 prior to the 2009 pandemic, respiratory viral co-infection was reported in 7-27% of respiratory samples submitted for viral diagnosis. [8] [9] [10] [11] [12] [13] higher proportions of influenza a, respiratory syncytial virus (rsv), parainfluenza viruses, and rhinovirus, compared with other circulating viruses have been detected in patients with co-infections. [14] [15] [16] [17] [18] co-infection has not been fully explored due to limitations of several studies. some studies focused on younger age groups, hospitalized patients or deceased individuals, which does not represent the general population. [8] [9] [10] 12, 15, 19, 20 others have utilized a small sample size or limited their focus to certain viral pathogens, underestimating the role of other viruses in co-infection. 9, 15, 16, 20, 21 this study enrolled community patients presenting with (ili) to a community sentinel network, during the influenza pandemic a(h1n1)pdm09 in ontario, canada and documented the profile of respiratory viruses causing ili symptoms. this study aimed to describe respiratory viruses including co-infections and host-associated attributes such as age, sex, and comorbidity. data were collected as part of a multiprovincial case-control sentinel network study that has been described elsewhere. 21 the sentinel network included 117 sentinels across the province of ontario (with a population of 13ae4 million) who volunteered to participate in the study. it was anticipated that each sentinel would submit an average of 1-2 samples per week from their clinical practice during the study period, april 21, 2009 to february 25, 2010 . this period was chosen to span the full pandemic in ontario. eligible patients were ontario residents, who presented to a sentinel's office with influenza-like illness (ili) within seven days of symptom onset; number and selection of eligible patients was at the sentinel's discretion. ili was defined as acute onset of fever and cough and one or more of the following: sore throat, myalgia, arthralgia, headache or prostration. standard information was collected including date of birth, sex, chronic conditions, symptom onset, and sample collection date. the main outcome was the number of respiratory viruses detected per sample. samples were categorized as negative, single infection or co-infection when no virus, one virus, or at least two viruses were detected, respectively. age was determined as age at symptom onset and categorized as 0-4, 5-14, 15-29, 30-54, 55-64, and 65+years. time to sample collection was calculated as the difference between sample collection and symptom onset dates and categorized as less than or equal to twodays or 3-7 days. chronic condition was defined as heart ⁄ lung ⁄ renal ⁄ metabolic ⁄ blood ⁄ immune conditions or conditions that compromise the management of respiratory secretions and increase risk of aspiration and categorized as yes ⁄ no. this study was approved by the university of toronto's ethics board and all patients gave verbal consent to participate. a nasal or nasopharyngeal sample was collected from each patient using starswabò multitrans collection swab and transported at 4°c for testing at public health ontario laboratory (phol)-toronto; in this study, each sample represents one patient. viral rna was extracted directly from samples using nuclisens ò easymag ò (biomérieux, inc., marcy l'etoile, france). samples were tested for influenza a and influenza b by influenza real-time reverse transcriptase (rrt)-pcr and also for influenza a, influenza b, enterovirus ⁄ rhinovirus, rsv, parainfluenza, adenovirus, coronaviruses, and metapneumovirus by a commercial multiplex pcr method [luminex respiratory viral panel (luminex molecular diagnostics, toronto, on, canada) or seeplex rv (seegene usa, rockville, md, usa)]. in the event of discrepant results between the two methods, positive results for influenza a by either method were considered positive. rrt-pcr was used for subtyping of influenza a samples; all influenza a specimens were subtyped, but not all attempts were successful. statistical analyses were performed using stata software version 10.0 (statacorp, college station, tx, usa). descriptive analyses were conducted to derive the proportion of single, co-infection and no infection as well as describe patient characteristics using chi-square. crude and adjusted multinomial logistic regression were employed to evaluate any association of single, co-infection and noinfection with patient characteristics including age, sex, chronic condition, and time to sample collection. odds ratios (or) with 95% confidence intervals (ci) were calculated. a total of 1018 respiratory samples from 1018 patients with influenza-like illness were included in this study after excluding 102 (9ae1%) samples that did not meet study inclusion criteria (figure 1 ). at least one respiratory virus was detected in 668 (65ae6%) of the samples. of the 831 detected viruses, influenza a was the most frequent accounting for 452 (54ae4%) followed by enterovirus ⁄ rhinovirus 194 (23ae3%) and rsv 120 (14ae4%) ( table 1) . of 452 influenza a viruses, 408 (90ae3%) were a(h1n1)pdm09, two (0ae4%) were h3, and 42 (9ae3%) could not be subtyped presumably due to low viral load. peaks in detection for influenza a occurred in june and october 2010, for enterovirus ⁄ rhinovirus in september 2010, and for rsv in october 2010 ( figure 2 ). a single virus was detected in 512 (50ae3%) samples. of these, 304 (59ae4%) were influenza a and 208 (40ae6%) were other respiratory viruses, the most common being enterovirus ⁄ rhinovirus, detected in 149 (29ae1%) samples (table 2) . peaks for single infection occurred in june and september 2009, which were mainly due to the increase in influenza a and enterovirus ⁄ rhinovirus, respectively ( figure 3 ). viral co-infection was detected in 156 (15ae3%) of the samples of which 149 (95ae5%) were dual infections and seven (4ae5%) triple infections. one hundred and fortyeight (94ae9%) of the co-infections were combination of a(h1n1)pdm09 and another respiratory virus and eight (5ae1%) were non-influenza combinations. the most common co-infections were influenza a ⁄ rsv b and influenza a ⁄ enterovirus ⁄ rhinovirus, responsible for 64ae1% and 21ae8% of co-infections, respectively ( table 3 ).the highest proportion of co-infection was detected in october, corresponding with peak activity of influenza a and rsv ( figure 3 ). the median age of patients in the study was 23 years with a range of 3 months-96 years of age ( table 4 ). the highest proportion of single and co-infections was observed in children 5-14 and 0-4 years of age, respectively. the proportion of those with no infection detected steadily increased with age, peaking at the elderly, aged 65 years and over ( figure 4 ). females were overrepresented, comprising 599 (58ae8%) of the patients included in this study. patients with no-infection, single infection, and co-infection did not differ with regards to sex. two hundred and nineteen (21ae6%) patients had a chronic condition. of these, 37ae4% had no virus detected, 44ae8% had single infections, and 17ae8% had co-infections, whereas among the 795 participants without comorbidities, the distribution was 33ae6%, 51ae8%, and 14ae6%, respectively; however, that was not statistically significant ( table 4 ). the median number of days from symptom onset to sample collection was two with a range of 0-7 days. five hundred and eighty-two (57ae2%) and 436 (42ae8%) samples were collected within 2 days and 3-7 days, respectively. of the 582 samples collected within 2 days of onset, 32ae9% had no virus detected, 48ae5% had single infections, and 18ae6% had co-infections, whereas among those collected within 3-7 days, the distribution was 36ae3%, 52ae8%, and 11%, which was statistically significant. in crude and adjusted multinomial logistic regression, patients with single and co-infections were compared to those with no infection. compared to the elderly, patients under 65 years of age were more likely to have a single infection; the highest likelihood was observed in children 5-14 years of age (table 5) . patients under 30 years of age were more likely to have co-infections compared with patients 65 and over; this was most evident in the 0-4 age group. presence of a chronic condition did not increase the likelihood of single infection but increased the likelihood of co-infection; this did not achieve significance. co-infection was more likely detected in patients who had samples collected within 2 days as compared to 3-7 days; this did not apply for those with single infections. there was no sex difference. in this study, 66% of samples tested during the 2009 pandemic in ontario had at least one virus detected and 15% had co-infections. these findings are consistent with reports from other studies with the range of co-infection reported from 7-27%. [8] [9] [10] [11] [12] [13] however, positivity and co-infection rates vary widely between studies. there are various reasons for this finding: firstly, detection methods differ notably between studies, which impacts sensitivity, specificity and other technical parameters; secondly, viruses targeted differ from one study to another as does the study population. 21 this study was conducted during the influenza pandemic a(h1n1)pdm09 which was associated with an increased number of samples submitted and high detection of figure 2 ). this demonstrates the importance of monitoring circulating respiratory viruses when advising clinicians to prescribe antivirals empirically during a pandemic. 2 (0-7) 2 (0-7) 2 (0-7) 2 (0-7) *p-value <0ae05 is considered significant. **time to specimen collection = collection date-symptom onset date. despite the higher prevalence of enterovirus ⁄ rhinovirus (23ae4%) than rsv (14ae5%), co-infection with a(h1n1) pdm09/rsv was more common than a(h1n1)pdm09 ⁄ enterovirus ⁄ rhinovirus, accounting for 64ae1% and 22ae8% of the co-infections, respectively. this may reflect the younger age of patients infected by a(h1n1)pdm09, who were therefore also at greater risk of rsv. in addition, rsv cocirculated with a(h1n1)pdm09 more than enterovirus ⁄ rhinovirus, which peaked before the second wave (figure 2 ). there may also be preferential interactions among certain pathogens; viral interactions were not assessed in this study. 23 when other respiratory samples positive for enterovirus ⁄ rhinovirus were further evaluated at our laboratory, they all were confirmed as rhinovirus, not enterovirus. 24 single infection was more commonly detected in those less than 65 years of age. it is known that respiratory infections are more common in children for several reasons, including an immature immune system, lack of preexisting immunity particularly to new emerging viruses, and greater viral exposure opportunities. 11, 18, 25 younger patients shed higher levels of virus when infected and also may be brought for medical care earlier than older patients, facilitating detection in these groups. 26 in addition, lower detection of single and co-infection in elderly may be explained by pre-existing immunity to a(h1n1)pdm09 and other respiratory viruses. 2 co-infection was more common in persons less than 30 years of age as compared to older adults. these data are congruent with findings from a previously published study where co-infection was more likely in younger than older adults. 18 the combined effect of predominance of a(h1n1)pdm09 and the greater likelihood of infection with other respiratory viruses among younger ages likely explains our age-related findings of co-infection during the pandemic, which may not be generalizable to a typical influenza season. the presence of comorbidities did not increase the likelihood of having a single infection but increased the likelihood of co-infections; this did not achieve statistical significance. patients with chronic conditions are at higher risk of severe disease and consequently may be more likely to seek medical care. 26 selection bias is unlikely to influence these results as the proportion of patients with comorbidities was similar (21ae5%) to that in ontario's population (20ae3%). 27 sample collection within 2 days of symptom onset was found to independently increase the likelihood of detecting a viral co-infection but not single infection. long et al. reported an inverse relationship between duration of symptoms and viral detection rate due to greater viral shedding earlier in the disease process. 11 this study was designed to examine circulating viruses and co-infection. the presence of more than two viruses in the same sample may not always indicate clinical infection. as viruses may be detected in asymptomatic patients, it is impossible to determine which viruses caused symptoms. previous studies suggest co-infection may manifest higher disease severity, which may shorten the time to medical care and viral detection; disease severity was not assessed in the current study. 19, 28 as viral-bacterial co-infections also occurred during the pandemic, it will be interesting for further studies to investigate their characteristics and impact on disease severity. 28, 29 in summary, a(h1n1)pdm09 was frequently detected among community patients with ili. however, other respiratory viruses cocirculated with a(h1n1)pdm09 during the pandemic, reinforcing the need to test for other viral agents even during a pandemic to appropriately guide clinical treatment decisions. viral diagnosis, primarily a(h1n1)pdm09, was made more often in patients less than 65 years of age. viral co-infection was commonly detected in this study and was most likely detected in individuals less than 30 years of age. earlier sample collection improves the detection of viral co-infections. understanding the contribution of other circulating respiratory pathogens during a pandemic may lead to improved individual diagnosis and recommendations for community-based clinicians, and more effective prevention and treatment of respiratory infections, including use of influenza antivirals. novel swine-origin influenza a (h1n1) virus in humans a (h1n1) influenza virus pandemic: a review complications of seasonal and pandemic influenza who checklist for influenza pandemic preparedness planning 2005. department of communicable disease surveillance and response global influenza programme. available online: http: ⁄ ⁄ a (h1n1)v, rvs: the race for hivernal pandemics respiratory virus infections among hospitalized patients with suspected influenza a h1n1 2009 virus during the first pandemic wave in brazil bacterial coinfections in lung tissue specimens from fatal cases of the association of newly identified respiratory viruses with lowers respiratory tract infections in korean children association of rhinovirus infection with increased disease severity in acute bronchiolitis single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-y response prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens. duration of symptoms significantly affects detection rate frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction multipathogen infections in hospitalized children with acute respiratory infections development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay low mortality rates related to respiratory virus infections after bone marrow transplant incidence of common respiratory viral infections related to climate factors in hospitalized children in hong kong respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology prevalence of human respiratory viruses in adults with acute respiratory tract infections in beijing correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection impact of respiratory virus infections on persons with chronic underlying conditions human metapneumovirus and respiratory syncytial virus in hospitalized danish children with acute respiratory tract infection association between the 2008-09 seasonal influenza vaccine and pandemic h1n1 illness during spring-summer 2009: four observational studies from canada evidence from multiplex molecular assays for complex multipathogen interaction in acute respiratory infections rhinovirus outbreaks in long-term care facilities viruses in communityacquired pneumonia in children aged less than 3 years old; high rate of viral confection correlation of pandemic (h1n1) 2009 viral load with disease severity and prolonged viral shedding in children self-reported ph1n1 influenza vaccination coverage for ontario streptococcus pneumonia coinfection is correlated with the severity of h1n1 pandemic influenza invasive group a streptococcal infection concurrent with 2009 h1n1 influenza authors wish to acknowledge the staff of molecular diagnostics and virus detection departments at public health ontario laboratory-toronto, the ontario and national sentinel influenza vaccine effectiveness study and the college of family physicians of canada funded by the canadian institute of health research. dms was principal investigator on a clinical trial of pediatric influenza vaccine dose response for which influenza vaccine was provided free by sanofi pasteur. jbg has received research grants from glaxosmithkline inc. and hoffman-la roche ltd to study antiviral resistance in influenza. key: cord-264408-vk4lt83x authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: animal models of human viral diseases date: 2017-06-23 journal: animal models for the study of human disease doi: 10.1016/b978-0-12-809468-6.00033-4 sha: doc_id: 264408 cord_uid: vk4lt83x as the threat of exposure to emerging and reemerging viruses within a naïve population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. recent outbreaks of middle east respiratory syndrome corona virus, ebola virus, chikungunya virus, and zika virus illustrate the emerging threats that are encountered. by utilizing animal models in this endeavor, the host response to viruses can be studied in a more complex and integrated context to identify novel drug targets, and assess the efficacy and safety of new products rapidly. this is especially true in the advent and implementation of the fda animal rule. although no one animal model is able to recapitulate all aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to consider prior to an animal study are route of viral exposure, species of animal, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand disease progression, pathogenesis, and immunologic responses to viral infections in humans. furthermore, to test vaccines and medical countermeasures, animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease course. a good animal model of viral infection should allow assay of many parameters of infection, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most studies in small animal models when possible, and studies in nhps to fill in the remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen free (spf), transgenic and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated to humans. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables collection of larger or more frequent blood samples, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains, thus greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic ferrets are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically, the closest species to humans, thus disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns pertaining to experimentation on nhps along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure the fewest number of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, depending on the pathogen, the route of exposure and the dose should mimic the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on viruses for each family that are of the greatest concern for public health worldwide. norovirus, the genus of which norwalk is the prototypic member, is the most common cause of gastroenteritis in the united states (hall et al., 2013) . there are five distinct genogroups (gi-gv) and numerous strains of norwalk virus, including the particularly significant human pathogens gi.1 norwalk virus, gii.2 snow mountain virus, and gii.1 hawaii virus. in developing countries, norwalk virus, also known as "winter vomiting virus," is responsible for approximately 200,000 deaths annually (patel et al., 2008) . a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants (chen et al., 2009; lutgehetmann et al., 2012; turcios-ruiz et al., 2008) . symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation is normally 1-3 days, with symptoms persisting for 2-3 days (koopmans and duizer, 2004) . viral shedding can range from 6 to 55 days in healthy individuals (atmar et al., 2014) . however, longer illness duration can be indicative of immunocompromised status, with the elderly and young having a prolonged state of shedding (harris et al., 2008; rockx et al., 2002) . interestingly, individuals vary greatly in susceptibility to norovirus infection depending on their fucosyl transferase 2 (fut2) allele functionality and histoblood group antigen status, with type a and o individuals susceptible and types ab and b resistant (hutson et al., 2005) . transmission occurs predominately through the oralfecal route with contaminated food and water being a major vector (atmar and estes, 2001; becker et al., 2000; koopmans and duizer, 2004) . vomiting results in airborne dissemination of the virus with areas of 7.8 m 2 being contaminated and subsequent transmission from oral deposition of airborne particles or contact with contaminated fomites, which can remain contaminated for up to 42 days (makison booth, 2014; tung-thompson et al., 2015) . each vomiting event in a classroom setting elevates the risk of norovirus illness among elementary students with proximity correlating with attack rates (evans et al., 2002; marks et al., 2003) . viral titers in emesis and fecal suspensions are as high as 1.2 × 10 7 and 1.6 × 10 11 ges (genomic equivalent copies per milliliter), respectively and the 50% infectious dose is 1320 ges (atmar et al., 2014) . therefore, outbreaks can be extremely difficult to contain. therapeutic intervention consists of rehydration therapy and antiemetic medication (bucardo et al., 2008; moe et al., 2001) . no approved vaccine or therapeutic is available, and development has been challenging given that immunity is short-lived after infection, new strains rapidly evolve and the correlates of protection are not completely understood (chen et al., 2013) . however, one promising strategy utilized a virus-like particle (vlp)-based vaccine that protected or reduced infection by almost 50% in human volunteers (aliabadi et al., 2015; atmar et al., 2011) . given the relatively benign disease in adults, experimental challenge has been carried out on human volunteers (ball et al., 1999; tacket et al., 2000) . viral titers are determined by shedding in feces and sera with histopathology changes monitored by biopsies particularly of the duodenum. the ph of emesis samples collected containing virus is consistent with viral replication in the small intestine with reflux to the stomach (kirby et al., 2016) . additionally, norwalk virus has been shown to bind to duodenal tissue (chan et al., 2011) . however, this type of research is technically difficult and expensive, and thus other models have been developed. a major hindrance to basic research into this pathogen is the lack of permissive cell culture systems or animal models for norwalk virus. nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus are monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity is measured exclusively by assay of viral shedding (rockx et al., 2005) . incidentally, more viruses were needed to create an infection when challenging by the oral route than by the intravenous (iv) route (purcell et al., 2002) . chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route (bok et al., 2011) . although none of the animals developed disease symptoms, viral shedding within the feces was observed within 2-5 days postinfection and lasted anywhere from 17 days to 6 weeks. viremia never occurred and no histopathological changes were detected. the amount and duration of viral shedding was in-line with what is observed upon human infection. as such, chimeric chimpanzee-human antinorovirus neutralizing antibodies have been explored as a possible therapeutic strategy (chen et al., 2013) . a recently identified calicivirus of rhesus origin, named tulane virus, has been used as a surrogate model of infection. unlike norwalk virus, tulane virus can be cultured in cells. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever 2 days postinfection. viral shedding was detected for 8 days. the immune system produced antibodies that dropped in concentration within 38 days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease (sestak et al., 2012) . a murine norovirus has been identified and is closely related to human norwalk virus (karst et al., 2003) . however, clinically the virus presents a different disease. the murine norovirus model does not include observable gastrointestinal clinical signs, possibly in part because rodents lack a vomiting reflex. additionally, mice infected with norovirus develop a persistent infection in contrast to human disease (hsu et al., 2006 (hsu et al., , 2007 khan et al., 2009) . porcine enteric caliciviruses can induce diarrheal disease in young pigs, and an asymptomatic infection in adults (wang et al., 2006 . gnotobiotic pigs can successfully be infected with a passaged clinical norovirus isolate by the oral route. diarrheal disease developed in 74% of the animals and virus was detected in the stool of 44% of the animals. no major histopathological changes or viral persistence was noted (cheetham et al., 2006) . calves are naturally infected with bovine noroviruses (scipioni et al., 2008) . experimental challenge of calves by oral inoculation with a bovine isolate resulted in diarrheal disease 14-16 h postinfection. recovery of virus was achieved after 53.5 and 67 h postinfection (otto et al., 2011) . eastern equine encephalitis virus (eeev), western equine encephalitis virus (weev), and venezuelan equine encephalitis virus (veev) present with near synonymous symptoms. the majorities of human cases are asymptomatic, but can present as a flu-like illness progressing to central nervous system (cns) involvement to include seizures and paralysis. mortality rates vary among the virus, with the highest reported for eev at 36%-75% followed by weev and lastly veev at less than 1% (ayers et al., 1994; griffin, 2007; steele and twenhafel, 2010) . there are currently no licensed vaccines or therapies but a recent phase 1 clinical trial of a veev dna vaccine resulted in veev-neutralizing antibody responses in 100% of the subjects (hannaman et al., 2016) . mouse models have been developed for numerous routes of infection including cutaneous, intranasal (in), intracranial (ic), and aerosol. eeev susceptibility in mouse models is correlated with age, with younger mice being more susceptible than adults. importantly, eeev pathogenesis is dependent on route of infection with delayed progression upon subcutaneous (subq) exposure (honnold et al., 2015) . newborn mice display neuronal damage with rapid disease progression, resulting in death (murphy and whitfield, 1970) . similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, while inoculation via the subq route causes a pantropic infection eventually resulting in encephalitis (liu et al., 1970; morgan, 1941) . a general drawback to the usage of the mouse model is the lack of vascular involvement during the disease course (liu et al., 1970) . after subq inoculation with weev, suckling mice started to show signs of disease by 24 h and died within 48 h (aguilar, 1970) . the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on day 4-5 postinfection. mice were severely ill by day 8 and appeared hunched and dehydrated. death occurred between days 7 and 14 with brain and mesodermal tissues, such as heart, lungs, liver, and kidney involvement (aguilar, 1970; monath et al., 1978) . intracerebral and in routes of infection resulted in a fatal disease that was highly dependent on dose while intradermal (id) and subq inoculations caused only 50% fatality in mice regardless of the amount of virus (liu et al., 1970) . comparing susceptibility of inbred and outbred strains revealed that cd-1, balb/c, a/j, and c57bl6 mice were all highly susceptible to experimental infection via subq inoculation when challenged prior to 10 weeks old with cns involvement and lethality (blakely et al., 2015) . subq/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans (macdonald and johnston, 2000) . virus begins to replicate in the draining lymph nodes at 4 h postinoculation. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis (charles et al., 1995; steele et al., 1998) . aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death (charles et al., 1995; steele and twenhafel, 2010; steele et al., 1998) . in challenge of c3h/hen mice with high dose veev caused high morbidity and mortality (julander et al., 2008b) . viral titers in brain peaked on day 4 postchallenge and remained elevated until animals succumbed on day 9-10 postchallenge. protein cytokine array performed on brains of infected mice showed elevated il-1a, il-1b, il-6, il-12, mcp-1, ifnγ, mip-1a, and rantes levels. this model was used successfully to test antivirals against veev (julander et al., 2008a) . additionally, a veev vaccine inactivated with 1,5-iodonaphthyl azide v3526 protects against both footpad and aerosol challenge with virulent veev in a mouse model (gupta et al., 2016) . guinea pigs and hamsters have also been developed as animal models for eeev studies (paessler et al., 2004; roy et al., 2009) . guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed in brain lesions in the experimentally challenged animals (roy et al., 2009) . subq inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis, and death. in addition, parenchyma necrosis were observed in the liver and lymphoid organs (paessler et al., 2004) . harlan sprague-dawley hamsters develop viremia and progress to respiratory, gastrointestinal, and nervous system involvement when inoculated via subq route. vasculitis and encephalitis were both evident in this model, which mirrors the human disease clinical spectrum (paessler et al., 2004) . weev is highly infectious to guinea pigs and has been utilized for prophylactic screening (sidwell and smee, 2003) . studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases (zlotnik et al., 1972) . cns involvement was evident with intracerebral, intraperitoneal (ip), and id inoculations (zlotnik et al., 1972) . ip inoculation of weev is fatal in guinea pigs regardless of amount of virus inoculum, with the animals exhibiting signs of illness on day 3-4, followed by death on day 5-9 (nalca, unpublished results) . id, im, or iv inoculations of eeev in nhps cause disease, but does not reliably result in neurological symptoms (dupuy and reed, 2012) . intracerebral infection of eeev produces nervous system disease and fatality in monkeys (nathanson et al., 1969) . the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in nhps when they are inoculated by the peripheral route (wyckoff, 1939) . in and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but are less drastic than intracerebral injections (wyckoff, 1939) . the aerosol route of delivery will result in uniformly lethal disease in cynomolgus macaques (reed et al., 2007) . in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed and nhps became moribund and were euthanized between 5-9 days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals (steele and twenhafel, 2010) . similar clinical signs and pathology were observed when common marmosets were infected with eeev by the in route (adams et al., 2008) . both aerosol and in nhp models had similar disease progression and pathology as seen in human disease. very limited studies have been performed with nhps. reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model could be useful for testing of vaccines and therapeutics against weev (reed et al., 2005) . veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at 12-72 h after infection and lasted less than 12 h. secondary fever generally began on day 5 and lasted 3-4 days (gleiser et al., 1961) . veev-infected nhps exhibited mild symptoms, such as anorexia, irritability, diarrhea, and tremors. leukopenia was common in animals exhibiting fever (monath et al., 1974) . supporting the leukopenia, microscopic changes in lymphatic tissues, such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, and focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection (gleiser et al., 1961) . the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day 6 postinfection during the secondary febrile episode. similar to these observations, when cynomolgus macaques were exposed to aerosolized veev, fever, viremia, lymphopenia, and clinical signs of encephalitis were observed but the nhps did not succumb to disease (reed et al., 2004) . a common marmoset model was utilized for comparison studies of south america (sa) and north america (na) strains of eeev (adams et al., 2008) . previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected in with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized 4-5 days after the challenge. although sa strain-infected animals developed viremia, they remained asymptomatic and survived until the end of study. chikungunya virus (chikv) is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics centered within africa and southeast asia (griffin, 2007) . the virus is transmitted by aedes aegypti and aedes albopictus mosquitoes. given the widespread endemicity of aedes mosquitoes, chikv has the potential to spread to previously unaffected areas. this is typified by the emergence of disease reported for the first time in 2005 in the islands of south-west indian ocean, including the french la reunion island, and the appearance in central italy in 2007 (charrel et al., 2007; rezza et al., 2007) . the incubation period following a mosquito bite is 2-5 days, leading to a self-limiting acute phase that lasts 3-4 days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported (powers and logue, 2007) . individuals typically develop a stooped posture due to the pain. for approximately 12% of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of 3 and 18 years old have an increased risk of developing neurological manifestations (arpino et al., 2009) . there is currently no approved vaccine or antiviral. wild-type c57bl/6 adult mice are not permissive to chikv infection by id inoculation. however, it was demonstrated that neonatal mice were susceptible and severity was dependent upon age at infection. six-dayold mice developed paralysis by day 6, and all succumbed by day 12, whereas 50% of 9-day-old mice were able to recover from infection. by 12 days, mice were no longer permissive to disease. symptomatic mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications (couderc et al., 2008; ziegler et al., 2008) . an adult mouse model has been developed by injection of the ventral side of the footpad of c57bl/6j mice. viremia lasted 4-5 days accompanied with foot swelling and noted inflammation of the musculoskeletal tissue morrison et al., 2011) . adult ifnα/βr knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within 3 days. this model was useful in identifying the viral cellular tropism for fibroblasts (couderc et al., 2008) . icr and cd-1 mice can also be utilized as a disease model. neonatal mice inoculated subq with a passaged clinical isolate of chikv developed lethargy, loss of balance, and difficulty walking. mortality was low, 17 and 8% for newborn cd-1 and icr mice, respectively. the remaining mice fully recovered within 6 weeks after infection (ziegler et al., 2008) . a drawback of both the ifnα/βr and cd-1 mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised (teo et al., 2012) . a chronic infection model was developed using recombinant activating gene 1 (rag1 −/− ) knockout mice. in this study, mice inoculated via the footpad lost weight in comparison to the control group. both footpad and subq injected mice developed viremia 5-6 days postinfection, which was detectable up to 28 days postinfection. inflammation was evident in the brain, liver, and lung of the subq inoculated animals at 28-56 days postinfection. despite minimal footpad swelling on day 2 postinfection, on day 14 there was severe muscle damage noted at necropsy, which resolved by day 28 (seymour et al., 2015) . golden hamsters serve as another option for small animal modeling. although hamsters do not appear to develop overt clinical symptoms following subq inoculation, viremia developed in the majority of animals within 1 day postinfection with clearance following from day 3 to 4. histologically, inflammation was noted at the skeletal muscle, fascia, and tendon sheaths of numerous limbs. this study was limited in the number of animals utilized, and more work is needed to further develop the hamster model (bosco-lauth et al., 2015) . nhp models of disease include adult, aged, and pregnant rhesus macaques in addition to cynomolgus macaques (broeckel et al., 2015) . differing routes of infection (subq, iv, and im) have been successfully administered, although there is not a clear understanding of the role that route of transmission plays in subsequent pathogenesis and clinical symptoms. typically, viremia is observed 4-5 days postinfection with a correlation between infectious titer and time to viremia observed in cynomolgus but not rhesus (labadie et al., 2010; messaoudi et al., 2013) . fever began at 1-2 days postinfection and persisted for 2-7 days and 3-7 days in cynomolgus and rhesus, respectively and coincided with rash (chen et al., 2010; labadie et al., 2010; messaoudi et al., 2013) . overall blood chemistries changed in conjunction with initiation of viremia, and returned to baseline 10-15 days postexposure (chen et al., 2010) . cns involvement has been difficult to reproduce in nhp models, although it was reported that high inoculum in cynomolgus did result in meningoencephalitis (labadie et al., 2010) . the nhp models have been utilized to conduct efficacy testing on novel vaccines and therapeutics (broeckel et al., 2015) . dengue virus (denv) is transmitted via the mosquito vectors a. aegypti and a. albopictus (moore and mitchell, 1997) . given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to denv. this results in approximately 50 million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia (gubler, 2002) . it is estimated that there are 20,000 deaths each year due to dengue hemorrhagic fever (dhf) (guzman and kouri, 2002) . there are four distinct serotypes of denv, numbered 1-4, which are capable of causing a wide clinical spectrum that ranges from asymptomatic to severe with the development of dhf (world health organization, 1997) . incubation can range from 3 to 14 days, with the average being 4-7 days. the virus targets dendritic cells and macrophages following a mosquito bite (balsitis et al., 2009) . typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure (world health organization, 1997) . thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis (gregory et al., 2010) . severe disease has a higher propensity to occur upon secondary infection with a different denv serotype (thein et al., 1997) . this is hypothesized to occur due to antibody dependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in order to further progress toward an effective drug or vaccine, small human cohort studies have taken place. however, to provide statistically relevant results, testing must progress in an animal model. in developing an animal model, it is important to note that mosquitoes typically deposit 10 4 -10 6 pfu, and is considered the optimal range during experimental challenge . denv does not naturally replicate effectively in rodent cells, creating the need for mouse-adapted strains, engineered mouse lines, and a variety of inoculation routes to overcome the initial barrier. several laboratory mouse strains including a/j, balb/c, and c57bl/6 are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis (huang et al., 2000; paes et al., 2005; shresta et al., 2004) . a higher dose of an adapted denv strain induced dhf symptoms in both balb/c and c57bl/6 souza et al., 2009) . this model can also yield asymptomatic infections. a mouse-adapted strain of denv 2 introduced into ag129 mice developed vascular leak syndrome similar to the severe disease seen in humans (shresta et al., 2006) . passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased and animals died due to vascular leak syndrome (balsitis et al., 2010) . another mouse-adapted strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability (souza et al., 2009) . ic injection of suckling mice with denv leads to death by paralysis and encephalitis, which is rare in human infection (lee et al., 2015; parida et al., 2002; zhao et al., 2014a) . immunocompromised mice have also been used to gain an understanding of the pathogenesis of denv. the most well-defined model is ag129 which is deficient in ifnα/β and γ receptors and can recapitulate dhf/dss if a mouse-adapted strain is utilized yauch et al., 2009) . scid mice engrafted with human tumor cells develop paralysis upon infection, and thus are not useful for pathogenesis studies (blaney et al., 2002; lin et al., 1998) . df symptoms developed after infection in nod/scid/il2rγko mice engrafted with cd34 + human progenitor cells (mota and rico-hesse, 2011) . rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and labadapted strain of denv 2 (kuruvilla et al., 2007) . a passaged clinical isolate of denv 3 was used to create a model in immunocompetent adult mice. ip injection in c57bl/6j and balb/c caused lethality by day 6-7 postinfection in a dose dependent manner. the first indication of infection was weight loss beginning on day 4 followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, ast and alt, were also observed. viremia was established by day 5. this model mimicked the characteristic symptoms observed in human dhf/dss cases (costa et al., 2012) . vascular leakage was also observed when c57bl/6 were inoculated with denv 2 (st john et al., 2013) . a murine model was developed that utilized infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of denv 2. infected mosquitoes then fed upon the mouse footpad to allow for transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the number of mosquitoes it was exposed to, with 4-5 being optimal. detectable viral rna was in line with historical human infection data. severe thrombocytopenia developed on day 14. this model is notable in that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus (cox et al., 2012) . nhp models have used a subq inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection (marchette et al., 1973) . the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes (marchette et al., 1980) . throughout these preliminary studies, no clinical disease was detected. in order to circumvent this, a higher dose of denv was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day 3 and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed (onlamoon et al., 2010) . a robust antibody response was observed in multiple studies (marchette et al., 1973; onlamoon et al., 2010) . marmosets also mirror human dengue infection, developing fever, leukopenia, and thrombocytopenia following subq inoculation (omatsu et al., 2011 (omatsu et al., , 2012 . nhps are able to produce antibodies similar to those observed during the course of human infection, making them advantageous in studying ade. sequential infection led to a cross-reactive antibody response which has been demonstrated in both humans and mice (midgley et al., 2011) . this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was 3-to 100-fold higher than previously reported, however, no clinical signs were apparent (goncalvez et al., 2007) . the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in 1937 uganda in (smithburn et al., 1940 . after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early 1950s (melnick et al., 1951; taylor et al., 1953) and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia (hayes, 1989) . the virus first reached the western hemisphere in the summer of 1999, during an outbreak involving humans, horses, and birds in the new york city metropolitan area (centers for disease control and prevention, 1999; lanciotti et al., 1999) . since 1999, the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from 3 to 6 days (monath and tsai, 2002) . the mortality rate following neuroinvasive disease ranges from 4% to 11% (asnis et al., 2000; hayes, 1989; hubalek and halouzka, 1999; komar, 2000) . the most severe complications are commonly seen in the elderly, with reported case fatality rates from 4% to 11%. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e., asymptomatic, fever, encephalitis) (pogodina et al., 1983) . thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subq inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia (lieberman et al., 2009; pletnev et al., 2003) . this is mirrored in new zealand white rabbits in that they only develop fever and low levels of viremia following inoculation via footpad (suen et al., 2015) . id inoculation of both marmosets and rhesus macaques did not yield any clinical signs of disease including fever. viremia was detected in both nhp species, but marmosets developed a higher titer for a greater duration than rhesus (verstrepen et al., 2014) . wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and ip routes, resulting in encephalitis and 100% mortality. id route pathogenesis studies indicated that langerhans dendritic cells are the initial viral replication sites in the skin (brown et al., 2007; johnston et al., 1996) . the infected langerhans cells then migrate to lymph nodes and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases (byrne et al., 2001; cunha et al., 2000; diamond et al., 2003; fratkin et al., 2004) . the swiss mouse strain was inoculated ip in order to screen a variety of viral lineages to assess differences in pathogenesis (bingham et al., 2014) . tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared asymptomatic during the first 5 days, became lethargic at approximately day 6, and developed neurologic symptoms between days 7 and 10 . many of the severely affected animals died 7-14 days after infection. viremia was detected in the hamsters within 24 h after infection and persisted for 5-6 days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the previously mentioned monkey experiments by pogodina et al. (1983) , persistent wnv infection was found in the brains of hamsters. zika virus recently came to the forefront of public health concerns with the outbreak in brazil at the end of 2015. the clinical disease spectrum is highly variable with reports of a flu-like illness accompanied by rash, guillan-barre syndrome, and microcephaly in newborns (ramos da silva and gao, 2016) . to date, a correlation between gestational age at which exposure to the virus occurs and severity of microcephaly is not fully understood (brasil et al., 2016) . however, a recent study of pregnant women in columbia found that infection with zika virus during the third trimester was not associated with any obvious structural abnormalities of the fetus (pacheco et al., 2016) . transmission of the virus occurs via the bite from an infected a. aegypti or a. albopictus (ramos da silva and gao, 2016) . other reported routes of exposure include sexual transmission and blood transfusion (cunha et al., 2016; d'ortenzio et al., 2016; hills et al., 2016; mccarthy, 2016) . the emergence of this virus with no approved vaccine or therapy, and few diagnostic options demonstrates the utility of well-characterized animal model development. it was first demonstrated in 1956 that experimentally infected mosquitoes could be used to transmit the virus to mice and nhps (boorman and porterfield, 1956) . a129 mice were susceptible to nonadapted zika virus infection following subq inoculation of the limbs. mice began to lose weight 3 days postinfection and met euthanasia criteria by day 6. microscopic lesions within the brain were noted upon necropsy. in conjunction, viral rna was detected in the blood, brain, ovary, spleen, and liver of the infected mice. wild-type 129sv/ev mice were also challenged with no observable clinical disease. however, viral rna was detected at day 3 postinfection in the blood, ovary and spleen, and then remained at detectable levels in the ovaries and spleen on day 7 (dowall et al., 2016) . footpad inoculation of the virus leads to a fatal disease in ag129 mice by day 7 postinoculation with significant histopathological changes in the brain noted at necropsy (aliota et al., 2016) . ag129 mice were also observed to develop neurologic disease by day 6 postexposure (rossi et al., 2016) . immunocompetent mice are resistant to infection via the subq route (rossi et al., 2016) . recently, a mouse model was identified to verify vertical transmission of the virus. pregnant c57 mice were injected either ip or in utero into the lateral ventricle of the fetal brain. ip inoculation induced transient viremia in the pregnant mice on day 1. viral rna was detected in five out of nine placentas on day 3 postinfection. the virus was able to infect the radial glia cells in the fetal brain and leads to a reduction in the cortical neural progenitors . viral exposure via cerebroventricular space/ lateral ventricle of the fetal brain exhibited small brain size at day 5 postexposure in addition to cortical thinning (cugola et al., 2016; li et al., 2016a) . ifnar1 −/− pregnant mice exposed to the virus had nonviable fetuses. in the same study, wild-type mice were given an anti-ifnar antibody prior to and during infection resulting in detectable virus in the fetal head with mild intrauterine growth restriction (miner et al., 2016) . all of these murine studies will further study of the pathogenesis of vertical transmission and the resulting neurological disorders in conjunction with screening novel countermeasures. nhp studies are currently ongoing for animal model development. numerous viruses from the coronavirus (cov) family exist that infect a wide range of animals. six species have been identified that can infect humans. two of these are alpha coronavirues: hcov-229e and hcov-nl63. four are beta coronavirueses: hcov-oc43, hcov-hku1, hcov-sars, and mers-cov. hcov-229e and hcov-oc43 were first detected in the 1960s from the nasal passages of humans with the "common cold" (gaunt et al., 2010) . hcov-nl63, which was first isolated in 2004, causes upper and lower respiratory infections of varying intensity and has been continuously circulating among humans (van der hoek et al., 2006) . hcov-hku1, first isolated in 2002, has been identified more sporadically but also causes respiratory infections (lau et al., 2006) . a significant portion of common cold infections in humans are caused by coronaviruses. in 2002 and 2012, two human coronaviruses, sars-cov and mers-cov, emerged that caused a great deal of alarm since these infections have resulted in nearly 10 and 40% fatality, respectively (assiri et al., 2013; peiris et al., 2004) . the etiologic agent of severe acute respiratory syndrome (sars), sars-cov, emerged in 2002 as it spread throughout 32 countries in a period of 6 months, with 8437 confirmed infections and 813 deaths (roberts and subbarao, 2006; world health organization, 2003) . no additional cases of community acquired sars-cov infection have been reported since 2004. the natural reservoir of sars-cov is the horseshoe bat and the palm civet is an intermediate host (lau et al., 2005) . the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to 6 days and can be detected in stool, suggesting other modes of transmission are also possible (pearson et al., 2003; rabenau et al., 2005; rota et al., 2003) . sars-cov infection has a 10% case fatality with the majority of cases in people over the age of 15 (peiris et al., 2003; wang et al., 2004) . after an incubation period of 2-10 days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough (drosten et al., 2003) . in some sars cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within 2 weeks and coincides with the induction of high levels of neutralizing antibodies (tan et al., 2004) . in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses (chen and subbarao, 2007; perlman and dandekar, 2005) . infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection (perlman and dandekar, 2005) . virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues (cheng et al., 2004) . sars-cov can replicate in many species, including: dogs, cats, pigs, mice, rats, ferrets, foxes, and monkeys (roper and rehm, 2009) . no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (∼10%), viral replication, and pathology (roberts et al., 2008) . in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models utilize mice, hamsters, ferrets, and nhps. mouse models of sars-cov typically are inoculated by the in route under light anesthesia (roberts et al., 2005) . young, 6-to 8-week-old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinate, with a peak on day 2 and clearance by day 5 postexposure (mcauliffe et al., 2004) . there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis) . in sars-cov infection of c57bl/6 (b6), also yields reduced weight gain and viral k. viral disease replication in the lungs, with a peak on day 3 and clearance by day 9 (glass et al., 2004) . in contrast, balb/c mice 13-14 months of age show weight loss, hunched posture, dehydration, and ruffled fur on day 3-6 postexposure (bisht et al., 2004) . interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. 129s mice and b6 mice show outcomes to sars-cov infection similar to those observed for balb/c mice but have lower titers and less prolonged disease. while the aged mouse model is more frequently used then young mice, it is more difficult to obtain large numbers of mice older than 1 year (table 33 .1). a number of immunocompromised knockout mouse models of in sars-cov infection have also been developed. 129svev mice infected with sars-cov by the in route develop bronchiolitis, with peribronchiolar inflammatory infiltrates and interstitial inflammation in adjacent alveolar septae . viral replication and disease in these mice resolves by day 14 postexposure. beige, cd1 −/− , and rag1 −/− mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs (glass et al., 2004) . stat1 ko mice infected in with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths . the stat1 ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. angiotensin converting enzyme 2 (ace2) and cd209l were identified as cellular receptors for sars-cov, with affinity for the spike (s) protein of the virus (jeffers et al., 2004) . the variations in the ace2 sequence across animal species could partially explain the differences in infection severity (li et al., 2016b; sutton and subbarao, 2015) . since mice in particular have a greater number of sequence differences in ace2, transgenic mice were created that express human ace2 (mccray et al., 2007; netland et al., 2008; yang et al., 2007) . unlike other murine models of sars-cov, mice expressing hace2 had up to 100% mortality, with severity correlating to the level of hace2 expression (tseng et al., 2007) . with high levels of hace2 expression, mice developed a severe lung and brain infection. however, cns k. viral disease infection is only rarely observed in humans infected with sars-cov. syrian golden hamsters (strain lvg) are also susceptible to in exposure of sars-cov. after the administration of 10 3 tcid 50, along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis (roberts et al., 2005) . there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. limited mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred (roberts et al., 2008) . damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal (it) inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge (skowronski et al., 2005) . clinical disease has been observed in several studies excluding one, perhaps due to characteristics of the inoculating virus (kobinger et al., 2007) . sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day 4 postexposure as well as mild alveolar damage in 5%-10% of the lungs, occasionally accompanied by severe pathology within the lungs (martina et al., 2003; ter meulen et al., 2004) . with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or it routes generally results in a very mild infection that resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness but others have rash, lethargy, and respiratory signs and pathology martina et al., 2003; mcauliffe et al., 2004; rowe et al., 2004) . rhesus have little to no disease and only have mild findings upon histopathological analysis (rowe et al., 2004) . agms infected with sars-cov have no overt clinical signs but dad and pneumonitis has been documented (mcauliffe et al., 2004) . viral replication has been detected for up to 10 days in the lungs of agms; however, the infection resolves, and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were involved in the sars-cov outbreak. it and in inoculation of civets with sars-cov results in lethargy, decreased aggressiveness, fever, diarrhea, and conjunctivitis . leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. squirrel monkeys, mustached tamarinds, and common marmosets have not been susceptible to sars-cov infection (greenough et al., 2005; roberts et al., 2008) . vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine, and have included live-attenuated, killed, dna and viral-vectored vaccine strategies (cavanagh, 2003) . an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease (perlman and dandekar, 2005; weiss and scott, 1981) . as such, immune mice had th2type immunopathology upon sars-cov challenge (tseng et al., 2012) . severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported (weingartl et al., 2004) . additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance (clay et al., 2012) . mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. mouse-adapted sars with uniform lethality was developed from 15 serial passages in the lungs of young balb/c mice (mccray et al., 2007; roberts et al., 2007; rockx et al., 2007) . middle east respiratory syndrome (mers-cov) emerged in saudi arabia and is associated with fever, severe lower respiratory tract infection, and oftentimes renal failure (al-tawfiq et al., 2016; omrani et al., 2015) . mers patients can also occasionally manifest with neurological symptoms. mers-cov infection has a high fatality rate. infections in humans can also be asymptomatic. as of october 2015, there were 1589 confirmed cases and 567 deaths (li et al., 2016b) . bats serve as the likely natural reservoir since virus with 100% nucleotide identity to the index case was isolated from egyptian tomb bats (memish et al., 2013) . spread to humans likely comes from infected dromedary camels (adney et al., 2014; azhar et al., 2014) . the host range for mers-cov is dependent on the binding of the viral s protein to the host receptor, which is human dipeptidyl peptidase four (hdpp4), also known as cd26 (raj et al., 2013) . the expression and distribution of dpp4 in the human respiratory tract has recently been well characterized (meyerholz et al., 2016) . interestingly, dpp4 expression is preferentially localized to alveolar regions, perhaps explaining why mers predominantly manifests as an infection of the lower respiratory tract. humans with preexisting pulmonary disease have increased dpp4 expression in alveolar epithelia. small animals typically used for viral disease research, such as mice, hamsters, guinea pigs, and ferrets are naturally nonpermissive to mers-cov infection due to a low binding efficiency of the viral s protein to the host dpp4 (sutton and subbarao, 2015). in contrast the rhesus macaque and common marmoset have complete homology to human dpp4, allowing productive mers-cov infection to occur falzarano et al., 2014; munster et al., 2013; yao et al., 2014) . new zealand white rabbits can be infected with mers-cov, and virus was isolated from the upper respiratory tract, but there were no clinical symptoms or significant histopathological changes (haagmans et al., 2015) . due to the lack of strong binding affinity of the mers-cov s protein to the murine dpp4 receptor, wildtype mice are not susceptible to mers-cov infection. as such, several approaches have been used to create susceptible murine animal models of mers-cov infection by inducing the expression of hdpp4. one approach utilized an adenovirus vector expressing hdpp4 to transduce mice (zhao et al., 2014b) . these mice developed pneumonia but survived mers-cov infection. in mers-cov infection of mice with global expression of hdpp4 resulted in id 50 and ld 50 values of <1 and 10 tcid 50 , respectively (tao et al., 2016) . thus, mers-cov infection of these transgenic mice can be either sublethal or uniformly lethal depending on the dose. inflammatory infiltrates were found in the lungs and brain stems of mice with some focal infiltrates in the liver as well. another strategy uses transgenic mice expressing hdpp4 under either a surfactant protein c or cytokeratin 10 promoter (li et al., 2016b) . in mers-cov infection in these mice resulted in a uniformly lethal disease characterized by alveolar edema and microvascular thrombosis and mononuclear clear cell infiltration in the lungs. the brain stem was also impacted by the infection. dpp4 expression with an ubiquitously expressing promoter from cytomegalovirus also had a uniformly lethal infection with predominant lung and brain involvement, but numerous other tissues were also impacted and contained virus (agrawal et al., 2015) . common marmosets infected with 5.2 × 10 6 tcid 50 (emc-2012) mers-cov by the combined in, oral, ocular, and it routes capitulate the severe disease in human infections (falzarano et al., 2014) . the animals manifested moderate to severe clinical disease, with interstitial infiltration of both lower lung lobes. two of nine animals became moribund between days 4 and 6. viral rna was detected in nasal and throat swabs, various organs, and in the blood of some animals, indicating a systemic infection. histologically, animals showed evidence of acute bronchial interstitial pneumonia as well as other pathological defects. infection of rhesus macaques with mers-cov results in a mild clinical disease characterized by a transient lung infection with pneumonia. rhesus macaques were inoculated with at least 10 7 tcid 50 (emc-2012) mers-cov either by the it route or a combined in, it, oral, and ocular inoculation . the result was a mild respiratory illness including nasal swelling and a short fever with all animals surviving. viral rna was recovered from nasal swab samples and replicating virus was found in lung tissue . mild pathological lesions were found only in the lungs. radiographic imaging of the lungs revealed interstitial infiltrates, which are signs of pneumonia (yao et al., 2014) . interestingly, mer-cov infection is more severe in marmosets compared to rhesus macaques (falzarano et al., 2014) . this is despite the finding that both species have complete homology with humans within the dpp4 domain that interacts with the viral s protein. other host factors influencing disease severity have not yet been identified. transgenic mouse models expressing hdpp4 are ideal for initial development and screening of mers-cov countermeasures, and marmosets can be used for final selection and characterization. filoviridae consists of three genera, ebolavirus and marburgvirus, and a newly discovered group, cuevavirus (kuhn, 2008) . it is thought that various species of bats are the natural host reservoir for these viruses that have lethality rates from 40% to 82% in humans. there is evidence that the egyptian rousette bat (rousettus aegyptiacus) is the natural reservoir for marburgviruses but may not be for ebolaviruses (jones et al., 2015) . marburg virus (marv) first emerged in 1967 in germany when laboratory workers contracted the virus from agms (chlorocebus aerthiops) that were shipped from uganda. ebolaviruses sudan and zaire (sudv and ebov) caused nearly simultaneous outbreaks in 1976 in what is now the democratic republic of congo (drc). the most recent outbreak of ebov in west africa was by far the largest with over 28,000 suspected, probable and confirmed cases and over 11,000 deaths. bundibugyo virus (bdbv) first emerged in 2007 in bundibugyo, uganda with 56 confirmed cases . two other ebolaviruses are known: taï forest (tafv) (previously named cote d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. filovirus disease in humans is a characterized by aberrant innate immunity and a number of clinical symptoms: fever, nausea, vomiting, k. viral disease arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, and anorexia as well as numerous others (mehedi et al., 2011; wauquier et al., 2010) . approximately 10% of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.) (table 33 .2). natural transmission in an epidemic is through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, im, ip, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures . since filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. one issue to take note of in animal model development of filovirus infection is the impact of particle to plaque-forming unit (pfu) ratios on lethality, wherein it is possible that increasing the dose could actually decrease infectivity due to an immunogenic effect produced by inactive virions in the stock. additionally, the plaque assay used to measure live virions in a stock may greatly underestimate the true quantity of infectious virions in a preparation (alfson et al., 2015; smither et al., 2013a) . immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type 1 interferon response (bray, 2001) . however, wild-type inbred mice are susceptible to filovirus that has been mouse adapted (ma) by serial passage in mice (bray et al., 1999) . marv angola was particularly resistant to adaptation, but after 24 serial passages in scid mice, infection caused severe disease in balb/c and c57bl/6 mice when administered in or ip (qiu et al., 2014) . these mice had pathology with some similarities to infection in humans including lymphopenia, thrombocytopenia, liver damage, and viremia. balb/c mice, which are the strain of choice for ip inoculation of ma-ebov, are not susceptible by the aerosol route (bray et al., 1999; zumbrun et al., 2012a) . for aerosol infection of immunocompetent mice, a panel of bxd (balb/c x dba) recombinant inbred strains were screened and one strain, bxd34, was particularly susceptible to airborne ma-ebov, with 100% lethality to low or high doses (approximately 100 or 1000 pfu) ( zumbrun et al., 2012a) . these mice developed weight loss of greater than 15% and succumbed to infection between days 7 and 8 postexposure. the aerosol infection model utilizes a whole-body exposure chamber to expose mice aged 6-8 weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately 1.6 µm and a geometric standard deviation (gsd) of approximately 2.0 for 10 min. another approach uses immunodeficient mouse strains, such as scid, stat1 ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by ip or aerosol routes (bray, 2001; lever et al., 2012; zumbrun et al., 2012a) . mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection (bray et al., 1999) . liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps (zumbrun et al., 2012a) . while most mouse studies have used ma-ebov or ebov, an ip mouse-adapted marv model is also available (warfield et al., 2007 (warfield et al., , 2009 ). ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds (panchal et al., 2012) . recently, a model was created using immunodeficient nsg [nonobese diabetic (nod)/scid/il-2 receptor chain knockout] mice with transplanted human hematopoietic stem cells from umbilical cord blood. these mice were susceptible to lethal wt (nonadapted) ebov by ip and in exposure (ludtke et al., 2015) . the transplanted mice had all of the cellular components of a fully functional adaptive human immune system and upon ebov (brannan et al., 2015; lever et al., 2012) . interestingly, inoculation of infa/br −/− mice with tafv and restv does not result in clinical signs. yet another strategy uses knockout mice lacking possible receptors for filovirus entry, such as niemann-pick c1 and c2 (npc1 and npc2). npc2 (−/−) mice were fully susceptible to infection with ebov but npc1 (−/−) mice were completely resistant (herbert et al., 2015) . hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models (gowen and holbrook, 2008; herbert et al., 2015) . an ip ma-ebov infection model has been developed in syrian hamsters gowen and holbrook, 2008; herbert et al., 2015; tsuda et al., 2011) . this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, utilizes male 5-to 6-week-old syrian hamsters which are infected with 100 ld 50 of ma-ebov. virus is present in tissues and blood collected on day 4 and all animals succumbed to the disease by day 6. infected hamsters had severe coagulopathy and uncontrolled host immune responses, similar to what is observed in primates. (ebihara et al., 2010) guinea pig models of filovirus infection have been developed for ip and aerosol routes using guinea pigadapted ebov (gp-ebov) and marv (gp-marv) (choi et al., 2012; connolly et al., 1999; twenhafel et al., 2015; zumbrun et al., 2012c) . guinea pig models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. a comparison of ip infection of outbred guinea pigs with guinea pig-adapted marv angola and marv ravn revealed similar pathogenesis (cross et al., 2015) . infection with either strain resulted in features of the disease that are similar to what is seen in human and nhp infection, such as viremia, fever, coagulopathy, lymphopenia, elevated liver enzymes (alt and ast), thrombocytopenia, and splenic, gastrointestinal and hepatic lesions. gp-marv-ravn had a delayed disease progression relative to gp-marv-ang. hartley guinea pigs exposed to aerosolized gp-ebov develop lethal interstitial pneumonia. this is in contrast to subq infection of guinea pigs, aerosol ebov challenge of nhps, and natural human infection (twenhafel et al., 2015) . both subq and aerosol exposure of guinea pigs to gp-ebov resulted in only mild lesions in the liver and spleen. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses (100 or 1000 pfu target doses) but lethality drops with low (less than 1000 pfu) presented doses of airborne gp-marv and more protracted disease is seen in some animals (our unpublished observations) (zumbrun et al., 2012c) . weight loss of between 15% and 25% is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day 5, occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day 6 postexposure. blood chemistries (i.e., alt, ast, alkp, and bun) indicating problems with liver and kidney function are also altered late in the disease course. transmission of ebov has been documented from swine to nhps via the respiratory tract (kobinger et al., 2011) . as such, guinea pigs have been used to establish transmission models (wong et al., 2015a,b) . nonexposed guinea pigs were placed in the cages with infected guinea pigs 1 day postexposure to gp-ebov. guinea pigs challenged intanasally were more likely to transmit virus to naive cagemates than those that were exposed by the ip route. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans (dye et al., 2012) . old world primates have been primarily used for development of ip, im, and aerosol models of filovirus infection ( twenhafel et al., 2013) . uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, agms (alves et al., 2010; carrion et al., 2011; davis et al., 1997; hensley et al., 2011a; reed et al., 2007; zumbrun et al., 2012b) . low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. ebov-makona, the strain responsible for the recent large outbreak in west africa, was compared to the "prototype" 1976 ebov strain (marzi et al., 2015) . the disease in cynos was similar for both viruses, but disease progression was delayed for ebov-makona. this delay as well as the lower fatality rate in the 2014 epidemic compared to the 1976 outbreak suggest that ebov-makona is less virulent. the large number of cases in the 2014-15 ebov outbreak brought to light previously underappreciated eye pathology and ocular viral persistence in survivors. while survivors of nhp filovirus infection are infrequent, necrotizing scleritis, conjunctivitis, and other ocular pathology has been observed in ebov-infected animals (alves et al., 2016) . prominent features of the filovirus infections in nhps are onset of fever by day 5 postexposure, viremia, lymphopenia, tachycardia, azotemia, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. petechial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species (zumbrun et al., 2012b) . immunological parameters have been evaluated and t, b, and natural killer cells are greatly diminished as the infection progresses (fernando et al., 2015) . a cytokine storm occurs with rises in ifnγ, tnf, il-6, and ccl2 (fernando et al., 2015) . however, there is also evidence from transcriptional profiling of circulating immune cells that the early immune response is skewed toward a th2 response (connor et al., 2015) . strikingly, animals surviving challenge may have a delay in the production of inflammatory cytokines and chemokines (martins et al., 2015) . clinical disease parameters may have a slightly delayed onset in aerosol models. dyspnea late in infection is a prominent feature of disease after aerosol exposure (zumbrun et al., 2012b) . aerosol filovirus infection of nhps results in early infection of respiratory lymphoid tissues, dendritic cells, alveolar macrophages, blood monocytes, and fibroblastic reticular cells followed by spread to regional lymph nodes then multiple organs (ewers et al., 2016; twenhafel et al., 2013) . a number of pronounced pathology findings include multifocal hepatic necrosis and fibrin accumulation, particularly within the liver and the spleen. for aerosolized marv infection of rhesus, the most significant pathology included destruction of the tracheobronchial and mediastinal lymph nodes (ewers et al., 2016) . lymphocytolysis and lymphoid depletion are also observed (alves et al., 2010) . multilead, surgically implanted telemetry devices are useful in continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection (zumbrun et al., 2012b) . the most recently developed telemetry devices can also aid in plethysmography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. standardized filovirus-infected nhp euthanasia criteria have also been developed to enhance reproducibility for studies that evaluate therapeutic and vaccine countermeasures (warren et al., 2014) . filovirus infection of common marmosets (callithrix jacchus) is also a viable model to study the disease course. respiratory infection of marmosets with marv results in a lethal infection with fever, hemorrhaging, transient rash, disseminated viral infection, increases in liver function enzymes, coagulopathy, hepatitis, and histological lesions particularly in the kidney and liver (smither et al., 2013b) . marmosets are similarly susceptible to infection with ebovkikwit (smither et al., 2015) . thus, ebov or marv infection of marmosets produces features of the disease that are very similar to that of other nhps and humans. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate (rockx et al., 2012) . outbreaks due to nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra virus outbreaks have yet to be reported outside of australia (luby et al., 2009a,b) . hendra was the first member of the genus identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from bat to human. nipah has the distinction of transmission among, although the exact route is unknown (homaira et al., 2010) . the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized as needed for successful transmission (fogarty et al., 2008) . both viruses have a tropism for the neurological and respiratory tracts. the incubation period for hendra virus is 7-17 days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting (hanna et al., 2006) . disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure (playford et al., 2010) . nipah virus has an incubation period of 4 days to 2 weeks (goh et al., 2000) . much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within 24-48 h (lo and rota, 2008). survivors of infection typically make a full recovery; however, 22% suffer permanent sequelae, including persistent convulsions (tan and chua, 2008) . at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because documentation of human cases is at terminal stages. the best small animal model is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died within 4-17 days postinfection. the progression of disease and timeline is highly dependent on dose and route of infection. in inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas ip resulted in tremors and paralysis within 24 h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, with the brain having the highest titer. this model is used for vaccination and passive protection studies (guillaume et al., 2009; rockx et al., 2011; wong et al., 2003) . the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge (torres-velez et al., 2008; williamson et al., 2001) . inoculation with hendra virus via the subq route leads to a generalized vascular disease with 20% mortality. clinical signs were apparent 7-16 days postinfection with death occurring within 2 days of cns involvement. higher inoculum has been associated with development of encephalitis and cns lesions. id and in injection does not lead to disease, although the animals are able to seroconvert upon challenge. the inoculum source does not affect clinical progression. nipah virus challenge only causes disease upon ip injection and results in weight loss and transient fever for 5-7 days. virus was shed through urine and was present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder (hooper et al., 1997) . ferrets infected with hendra or nipah virus display the same clinical disease as seen in the hamster model and human cases (bossart et al., 2009; pallister et al., 2011) . upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within 6-9 days including fever, coughing, and dyspnea. lesions do develop in the ferret's brains, but to a lesser degree than seen in humans. cats have also been utilized as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is 4-8 days and leads to respiratory and neurological symptoms (mungall et al., 2007; johnston et al., 2015; westbury et al., 1996) . this model has proven useful for vaccine efficacy studies. squirrel and agms are representative of the nhp models. for squirrel monkeys, nipah virus is introduced by either the in or iv route and subsequently leads to clinical signs similar to humans, although in challenge results in milder disease. upon challenge, only 50% of animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge (marianneau et al., 2010) . agms are very consistent model of both viruses. it inoculation of the viruses results in 100% mortality, and death within 8.5 and 9-12 days postinfection for hendra and nipah viruses, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis rockx et al., 2010) . the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue, and nipah virus can be located in urine . pigs develop a respiratory disease upon infection with both nipah and hendra viruses (berhane et al., 2008; li et al., 2010; middleton et al., 2002) . oral inoculation does not produce a clinical disease, but subq injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as 4 days postinfection. nipah virus can also be transmitted between pigs. nipah virus was able to induce neurological symptoms in 20% of the pigs, even though virus was present in all neurological tissues regardless of symptoms (weingartl et al., 2005) . within the pig model, it appeared that nipah virus had a greater tropism for the respiratory tract, while hendra for the neurological system. horses are also able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra viruses. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within 2 days (marsh et al., 2011; williamson et al., 1998) . animals died within 4 days postexposure and have interstitial pneumonia with necrosis of alveoli (murray et al., 1995a,b) . virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but are nonpermissive to infection (westbury et al., 1995; wong et al., 2003) . suckling balb/c mice succumb to infection if the virus is inoculated intracranially (mungall et al., 2006) . in exposure with nipah does not induce a clinical disease; however, there is evidence of a subclinical infection in the lungs following euthanasia of the mice (dups et al., 2014) . in addition, a human lung xenograph model in nsg mice demonstrated that the human lung is highly susceptible to nipah viral replication and damage (valbuena et al., 2014) . embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within 4-5 days postinfection (tanimura et al., 2006) . annually, respiratory syncytial virus (rsv) is responsible for the lower respiratory tract infections of 33 million children under the age of 5, which in turn results in 3 million hospitalizations and approximately 200,000 deaths (nair et al., 2010) . within the united states, hospital costs alone amount to over 600 million dollars per year (paramore et al., 2004) . outbreaks are common in the winter (yusuf et al., 2007) . the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and spreads to the lower respiratory tract. incubation for the virus is 2-8 days. rsv is highly virulent leading to very few asymptomatic infections (collins and graham, 2008) . disease manifestations are highly dependent upon the age of the individual. rsv infections in neonates produce nonspecific symptoms including overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting rsv at this age results in an increased chance of developing childhood asthma (wu et al., 2008) . young children develop recurrent wheezing while adults have exacerbation of previously existing respiratory conditions (falsey et al., 2005) . common clinical symptoms are runny nose, sneezing, and coughing accompanied by fever. mortality rates from rsv in hospitalized children are 1%-3% with the greatest burden of disease seen in 3-4 month olds (ruuskanen and ogra, 1993). hematopoietic stem cell transplant patients, solid organ transplant patients, and copd patients are particularly vulnerable to rsv infection and have mortality rates between 7.3% and 13.3% upon infection (anderson et al., 2016) . although there are almost 60 rsv vaccine candidates which are in preclinical and clinical phases, there is no licensed vaccine available and ribavirin usage is not recommended for routine treatment (american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis, 2006; higgins et al., 2016; kim and chang, 2016) . animal models of rsv were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated rsv (fi-rsv) vaccine. this vaccine induced severe respiratory illness in infants whom received the vaccine and were subsequently infected with live virus (kim et al., 1969) . mice can be used to model rsv infection, although a very high in inoculation is needed to achieve clinical symptoms (jafri et al., 2004; stark et al., 2002) . strain choice is crucial to reproducing a physiological relevant response (stokes et al., 2011). age does not affect primary disease manifestations (graham et al., 1988) . however, it does play a role in later sequelae showing increased airway hyperreactivity . primary rsv infection produces increased breathing with airway obstruction (jafri et al., 2004; van schaik et al., 1998) . virus was detected as early as day 3 and reached maximum titer at day 6 postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. a humanized mouse model was recently developed by in inoculation. the challenged mice experienced weight loss and demonstrated a humoral and cellular immune response to the infection (sharma et al., 2016) . cotton rats are useful given that rsv is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after in inoculation (boukhvalova et al., 2009; niewiesk and prince, 2002) . viral replication is 50-to 1000-fold greater in the cotton rat model than mouse model (wyde et al., 1993) . the cotton rats develop mild to moderate bronchiolitis or pneumonia (grieves et al., 2015; prince et al., 1999) . although age does not appear to factor into clinical outcome, it has been reported that older cotton rats tend to take longer to achieve viral clearance. viral loads peak by the 5th day, dropping to below the levels of detection by day 8. the histopathology of the lungs appears similar to that of humans after infection (piazza et al., 1993) . this model has limited use in modeling the human immune response to infection as challenge with the virus induces a th2 response in cotton rats, whereas humans tend to have a response skewed toward th1 (culley et al., 2002; dakhama et al., 2005; ripple et al., 2010) . fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally with rsv via in inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is therefore useful in studying mucosal immunity during infection (gitiban et al., 2005) . ferrets infected by it were found to have detectable rsv in throat swabs up to day 7 postinfection, and positive qpcr up to day 10. immunocompromised ferrets were observed to have higher viral loads accompanied with detectable viral replication in the upper respiratory tract (stittelaar et al., 2016) . chimpanzees are permissive to replication and clinical symptoms of rsv including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms or high levels of viral replication (belshe et al., 1977) . bonnet monkeys were developed an inflammatory response by day 7 with viral rna detected in both bronchial and alveolar cells (simoes et al., 1999) . the chimpanzee model has been proven useful for vaccine studies (hancock et al., 2000; teng et al., 2000) . sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine rsv (meyerholz et al., 2004) . lambs are also susceptible to human respiratory syncytial infection (olivier et al., 2009; sow et al., 2011) . when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after 6 days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within 14 days. rsv replication peaked at 6 days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features with humans (plopper et al., 1983; scheerlinck et al., 2008) . the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; 16 ha and 9 na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within 10 days (taubenberger and morens, 2008) . the rate of infection is estimated at 10% in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately 40,000 people in the united states die each year from seasonal influenza (dushoff et al., 2006) . thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing (braun et al., 2007b) . influenza virus replicates in the upper and lower airways, peaking at approximately 48-h postexposure. infection can be more severe in infants and children under the age of 22, people over the age of 65, or immunocompromised individuals where viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis (barnard, 2009; glezen, 1982) . pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza virus itself, accounting for ∼27% of all influenza associated fatalities (alonso et al., 2003; ison and lee, 2010; speshock et al., 2007) . death, often due to ards can occur as early as 2 days after onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation (taubenberger and morens, 2008) . the h5n1 avian strain of influenza, has lethality rates of around ∼50% (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus the potential for global spread is a major concern (matrosovich et al., 2004; wang et al., 2016) . h7n9 is another avian influenza a strain that infected more than 130 people and was implicated in 37 deaths. approximately 75% of infected people had a known exposure to birds. there is no evidence of sustained spread between humans but these viruses are of great concern for their pandemic potential (zhang et al., 2013) . the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. (2012) . swine are not frequently utilized but are also a potentially useful model for influenza research since they share many similarities to human anatomy, genetics, susceptibility, and pathogenesis (rajao and vincent, 2015). lethality rates can vary with virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, super infection, and sepsis, severe with ards, and neurologic manifestations (barnard, 2009) . also, models can utilize seasonal or avian strains and have been developed to study transmission, important for understanding the potential for more lethal strains, such as h5n1 for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines (gilbert and mcleay, 2008; hagenaars et al., 2008; ortigoza et al., 2012) . inoculation is by the in route, utilizing approximately 60 µl of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with a mmad of <5 µl. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c57bl/6j mice. males and females have equivalent disease but influenza is generally more infectious in younger 2-to 4-week-old (8-10 g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. most inbred laboratory mice lack the mxa gene which is an important part of human innate immune response to influenza infection. the mouse homolog to mxa, mx1 is defective in most inbred mouse strains (staeheli and haller, 1987) . mice with the knocked-in mx1 gene have a 1000-fold higher ld-50 for an influenza a strain (pr8) than wildtype background c57bl/6 mice (grimm et al., 2007) . weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs in influenza infected mice. for more virulent strains, mice may require euthanasia as early as 48 h postexposure, but most mortality occurs from 5 to 12 days postexposure accompanied by decreases in rectal temperature (sidwell and smee, 2000). pulse oximeter readings and measurement of blood gases for oxygen saturation are also used to determine the impact of influenza infection on respiratory function (sidwell et al., 1992) . virus can be isolated from bronchial lavage (bal) fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild to moderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha-1-acidglycoprotein and lung weight also frequently occur. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa 2,3 predominates over sa 2,6 which is why h1, h2, and h3 subtypes usually need to be adapted to mice and h5n1, h2, h6, and h7 viruses do not require adaptation (o'donnell and subbarao, 2011). to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. h5n1 and the 1918 pandemic influenza virus can cause lethal infection in mice without adaptation (gao et al., 1999; taubenberger, 2006) . h5n1 infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia ( dybing et al., 2000; gubareva et al., 1998; lu et al., 1999) . as in humans, the virulence of h5n1 is attributable to damage caused by an overactive host immune response. additionally, mice infected with the 1918 h1n1 influenza virus produce severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia (barnard et al., 2007) . reassortment influenza viruses of the 2009 h1n1 virus and a low-pathogenicity avian h7n3 virus can also induce disease in mice without adaptation . in superinfection models, a sublethal dose of influenza is given to mice followed 7 days later by in inoculation of a sublethal dose of a bacterial strain, such as s. pneumoniae or s. pyogenes (chaussee et al., 2011) . morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to 2 weeks. at least one transmission model has also been developed in mice. with h2n2 influenza, transmission rates of up to 60% among cagemates can be achieved after infection by the aerosol route and cocaging after 24 h (schulman, 1968). rats (f344 and sd) inoculated with rat-adapted h3n2 developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes (daniels et al., 2003) . additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets (lowen et al., 2006) . cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies (eichelberger et al., 2004) . cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted (eichelberger et al., 2006; ottolini et al., 2005) . nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at 24 h postexposure. virus was cleared from the lung by day 3 and from the nares by day 66, but animals had bronchial and alveolar damage, and pneumonia for up to 3 weeks. there is also a s. aureus superinfection model in cotton rats (braun et al., 2007a) . coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans (lambkin et al., 2004; maines et al., 2012; mclaren and butchko, 1978) . like humans, ferrets exclusively express neu5ac, which acts as a receptor for influenza a virus, a feature likely contributing to the susceptibility of ferrets to human-adapted influenza a virus strains (ng et al., 2014) . the glycomic characterization of ferret respiratory tract tissues demonstrated some similarities and some differences to humans in terms of the potential glycan binding sites for the influenza virus (jia et al., 2014) . ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa 2,6 and sa 2,3) within the airways similar to that of humans (van riel et al., 2007) . influenza was first isolated from ferrets infected in with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments (huber et al., 2008; lambkin et al., 2004; maines et al., 2012) . when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by in inoculation of 0.25-0.5 ml containing approximately 10 4 -10 6 egg id 50 dropwise to each nostril. however, a larger inoculum volume of 1.0 ml has also been explored as being more appropriate, yielding more severe and consistent respiratory tract pathology, likely because the larger inoculum is more widely distributed in the lower respiratory tract (moore et al., 2014) . video tracking to assign values to activity levels in ferrets can aid ferret studies, eliminating the need for collection of subjective and arbitrary clinical scores (oh et al., 2015) . viral replication in the upper respiratory tract is typically measured by nasal washes, but virus can also be measured in bronchoalveolar lavage fluid using a noninvasive technique (lee et al., 2014) . influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts 3-5 days for mild to moderately virulent strains (maher and destefano, 2004) . ferrets are more susceptible to influenza a than influenza b strains and are also susceptible to avian influenza h5n1 strains without adaptation (zitzow et al., 2002) . however, the localized immune responses within the respiratory tract of ferrets infected with influenza a and b have been characterized and are similar (carolan et al., 2015) . virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirrors that seen in humans (stark et al., 2013) . nonadapted h1n1, h2n2, and h3n2 have mild to moderate virulence in ferrets. the sequencing of the ferret genome has allowed for the characterization of the ferret host response using rnaseq analysis . distinct signatures were obtained depending on the particular influenza strain to inoculate the ferrets. also helpful is the sequencing and characterization of the influenza ferret infectome during different stages of the infection in naïve or immune ferrets (leon et al., 2013) . since influenza infection is particularly devastating to the elderly population, an aged ferret model of h1n1 influenza infection was developed (paquette et al., 2014) . features associated with increased clinical disease are weakened hemagglutinin antibody generation and attenuated th1 responses. pregnant and breastfeeding women and infants are also susceptible to more severe illness from influenza virus. to study this dynamic, a breastfeeding mother-infant ferret influenza infection model was created (paquette et al., 2015) . notably, the mammary gland itself harbored virus and transcript analysis showed downregulation of milk production genes. in support of the development of therapies, the ferret influenza model for pharmacokinetic/pharmacodynamics studies of antiviral drugs as also been developed (reddy et al., 2015) . critical to this model is ensuring pronounced clinical signs and robust viral replication upon influenza infection. strains of low virulence have predominant replication in the nasal turbinates of ferrets. clinical signs and other disease indicators in ferrets are similar to that of humans with mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia (svitek et al., 2008) . replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract of ferrets correlates with severe disease and lethal outcome. h5n1-infected ferrets develop severe lethargy, greater interferon response, transient lymphopenia, and replication in respiratory tract, brain, and other organs (peng et al., 2012; zitzow et al., 2002) . immunocompromised humans have influenza illness of greater duration and complications. immunocompromised ferrets infected with influenza similarly had prolonged virus shedding (van der vries et al., 2013) . interestingly, antiviral resistance emerged in both humans and ferrets with immunocompromised status infected with influenza. alveolar macrophage depleted of ferrets infected with 2009 pandemic h1n1 influenza also had a more severe disease with greater viral replication in the lungs and greater induction of inflammatory chemokines (kim et al., 2013) . a superinfection model resembling that of mice has been developed by in instillation of influenza in 6-to 8-week-old ferrets followed by in inoculation of s. pneumonia 5 days later (peltola et al., 2006) . this typically resulted in otitis media, sinusitis, and pneumonia. transmission models in ferrets have recently met with worldwide media attention and controversy with regard to the study of h5n1 (enserink, 2013; fouchier et al., 2012; herfst et al., 2012; oh et al., 2014) . in general, some subtypes, such as the 2009 h1n1, can transmit easily through aerosol and respiratory droplets (munster et al., 2009) . of concern, h7n9 isolated from humans was more pathogenic and readily transmissible between ferrets by larger respiratory droplets and smaller particle aerosols (kreijtz et al., 2013; richard et al., 2013; zhang et al., 2013) . h5n1 became transmissible by adopting just four mutations, spreading between ferrets in separate cages (imai et al., 2012) . transmission occurs more readily at the height of pyrexia, but for the 2009 h1n1 in particular, can occur before fever is detected (roberts et al., 2012) . ferret-to-ferret transmission of a mouseadapted influenza b virus has also been demonstrated (kim et al., 2015) . since ferrets can be expensive and cumbersome, influenza infection has been characterized and a transmission model developed in the guinea pig; however, this is a newer model with infrequent utilization thus far (lowen et al., 2014) . old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models which are deficient for h5n1 vaccine studies because there is a lack of correlation with hemagglutination inhibition (murphy et al., 1980) . of old world primates, cynomolgus macaque (macaca fascicularis) is most frequently utilized for studies of vaccines and antiviral drug therapies (stittelaar et al., 2008) . h5n1 and h1n1 1918 infection of cynos is very similar to humans (rimmelzwaan et al., 2001) . cynos develop fever and ards upon in inoculation of h5n1 with necrotizing bronchial interstitial pneumonia . nhps are challenged by multiple routes k. viral disease (ocular, nasal, and tracheal) simultaneously 1 × 10 6 pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h5n1 results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the 1918 h1n1 caused a very high mortality rate due to an aberrant immune response and ards and had more than 50% lethality (humans only had a 1%-3% lethality) (kobasa et al., 2007) . ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains, such as h3n2 (o'donnell and subbarao, 2011) . influenza-infected rhesus macaques represent a mild disease model for vaccine and therapeutic efficacy studies (baas et al., 2006) . host microarray and qrt-pcr proved useful for analysis of infected lung tissues. other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates, such as squirrel and cebus monkeys (baskin et al., 2009) . domestic pig models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h5n1 (isoda et al., 2006; lipatov et al., 2008) . while pigs infected with influenza may have fever, anorexia, and respiratory signs, such as dyspnea and cough, mortality is rare (van der laan et al., 2008) . size and space requirements make this animal difficult to work with, although the development of minipig models may provide an easier to use alternative. cat and dog influenza models have primarily been utilized to study their susceptibility to h5n1 with the thought that these animals could act as sentinels or could serve to transmit the virus to humans (giese et al., 2008; rimmelzwaan et al., 2006) . these models are not generally used to better understand the disease in humans or for testing vaccines or antivirals. rift valley fever virus (rvfv) causes epizootics and human epidemics in africa. rvfv mainly infects livestock, such as sheep, cattle, goats, etc. after 2-4 days incubation period, animals show signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers (balkhy and memish, 2003) . mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans (abu-elyazeed et al., 1996; mundel and gear, 1951) . after 2-to 6-day-incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever (ikegami and makino, 2011; laughlin et al., 1979; peters and linthicum, 1994) . depending on the severity of the disease when the symptoms start, 10%-20% of the hospitalized patients might die in 3-6 days or 12-17 days after the disease onset (ikegami and makino, 2011) . hepatic failure, renal failure or dic, and encephalitis are demonstrated within patients during postmortem examination. live domestic animals especially sheep and goats were used to develop animal models of rvfv (weingartl et al., 2014) . this study indicated that goats were more resistant to the disease compared to sheep. the viremia in goats was lower and had a shorter duration with only some animals developing fever. the susceptibility is influenced by route of infection, breed of animals, the rvfv strain, and growth conditions as well as the passage history. therefore, it might be difficult to establish an animal model with domestic ruminants. mice are one of the most susceptible animal species to rvfv infection. several mouse models including balb/c, ifnar −/− , mbt/pas, 129 and c57bl/6 were exposed to rvfv via parental or aerosol routes of infection (ross et al., 2012) . subq or ip routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice (mims, 1956; smith et al., 2010) . mice start to show signs of decreased activity and ruffled fur by day 2-3 postexposure. immediately following these signs, they become lethargic and generally die 3-6 days postexposure. ocular disease or the hemorrhagic form of the disease has not been observed in mouse models so far (ikegami and makino, 2011) . increased viremia and tissue tropism were reported in mice with (smith et al., 2010) increased liver enzymes and lymphopenia observed in sick mice. aerosolized rvfv causes faster and more severe neuropathy in mice compared to the parental route (dodd et al., 2014; reed et al., 2014) . the liver is a target organ following aerosol exposure and liver failure results in fatality. rats and gerbils are also susceptible to rvfv infection. the rat's susceptibility is dependent on the rat strain utilized for the challenge model and route of exposure. there is also noted age dependence in the susceptibility of rats. while wistar-furth and brown norway strains, and young rats are highly susceptible to rvfv infection, fisher 344, buffalo and lewis strains, and old rats demonstrated resistance to infection via subq route of infection (findlay and howard, 1952; peters and slone, 1982) . similar pathologic changes, such as liver damage and encephalopathy were observed in both rats and mice. the recent study by bales et al. (2012) showed that aerosolized rvfv caused similar disease outcome in wistar-furth and aci rats while lewis rats developed fatal encephalitis which was much more severe than the subq route of infection. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils (anderson et al., 1988) . similar to the rat model, the susceptibility of gerbils was also dependent on age. natural history studies with syrian hamsters indicated that the liver was the target organ with highly elevated alt levels and viral titers (scharton et al., 2015) . lethargy, ruffled fur, and hunched posture were observed in hamsters by day 2 post-subq inoculation and the disease was uniformly lethal by day 2-3 postexposure. this model has been successfully used to test antivirals against rvfv (scharton et al., 2015) . studies thus far showed that rvfv does not cause uniform lethality in a nhp model. ip, in, iv, and aerosol routes have been utilized to develop nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort . monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to humans, such as pathological changes in the liver and hemorrhagic disease. there was no ocular involvement in this model. smith et al. compared iv, in and subq routes of infection in common marmosets and rhesus macaques (peng et al., 2012) . marmosets were more susceptible to rvfv infection than rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subq routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of in challenged marmosets. a recent study by hartman et al. (2014) demonstrated that aerosolized rvfv only caused mild fever in cynomolgus macaques and rhesus macaques, while agms and marmosets had encephalitis and succumbed to disease between days 9 and 11 postexposure. in contrast to other lethal models, the brain was the target organ in agms and marmosets. although no change was observed in ast levels, alp levels were increased in marmosets. little or no change was observed in hepatic enzyme levels in agms. lack of information regarding human disease concerning the aerosol route of exposure makes it difficult to evaluate these animal models. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, appears to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans (ergonul and whitehouse, 2007) . incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts 1-9 days. during the prehemorragic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients (mardani and keshtkar-jahromi, 2007; swanepoel et al., 1989) . over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. (fagbami et al., 1975; nalca and whitehouse, 2007; shepherd et al., 1989; smirnova, 1979) . until recently, the only animal that manifests disease is the newborn mouse. infant mice ip infected with cchfv resulted in fatality around day 8 postinfection (tignor and hanham, 1993) . pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day 6) and the brain (day 7). the recent studies utilizing knockout adult mice were successful to develop a lethal small animal model for cchfv infection (bente et al., 2010; bereczky et al., 2010) . bente et al. infected stat1 knockout mice by the ip route. in this model, after the signs of fever, leukopenia, thrombocytopenia, viremia, elevated liver enzymes and proinflammatory cytokines, mice were moribund and succumbed to disease in 3-5 days postexposure. the second model was developed by using interferon alpha/beta (ifnα/β) receptor knockout mice (ifnar −/− ) (bereczky et al., 2010) . similar observations were made in this model as in the stat1 knockout mouse model. animals were moribund and died 2-4 days after exposure with high viremia levels in liver and spleen. characterization studies with ifnar −/− mice challenged with different routes (ip, in, im, and subq) showed that cchfv causes acute disease with high viral loads, pathology in liver and lymphoid tissues, increased proinflammatory response, severe thrombocytopenia, coagulopathy, and death, all of which are characteristics of human disease . proinflammatory cytokines and chemokines, such as g-csf, ifnγ, cxc-cl10, ccl2 increased dramatically day 3 postchallenge and gm-csf, il-1a, il-1b, il-2, il-6, il-12p70, il-13, il-17, cxcl1, ccl3, ccl5, and tnf-α concentrations were extremely elevated at the time of death/euthanasia. this model is also utilized to test therapeutics, such as ribavirin, arbidol, and t-705 (favipiravir) successfully (oestereich et al., 2014) . experimental vaccines developed for cchf were evaluated in this model provided protection compare to unvaccinated mice (buttigieg et al., 2014; canakoglu et al., 2015, p. 725) . thus, the ifnar −/− mouse model would be a good choice to test medical countermeasures against cchfv, although they have an impaired ifn and immune response phenotype. other laboratory animals, including nhps, show little or no sign of infection or disease when infected with cchfv (nalca and whitehouse, 2007) . butenko et al. utilized agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day 4 postinfection, the animals did not show signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. fagbami et al. (1975) infected two patas monkeys (erythrocebus patas) and one guinea baboon (papio papio) with cchfv. whereas all three animals had low-level viremia between days 1 and 5 after inoculation, only the baboon serum had neutralizing antibody activity on day 137 postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia (rabinovich et al., 1972) and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least 3 months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes (blagoveshchenskaya et al., 1975 (blagoveshchenskaya et al., ). shepherd et al. (1989 infected 11 species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. whereas scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (gerbilliscus brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and mastomys coucha), and syrian hamsters were negative for virus. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed onset of viremia earlier than those infected by the subq or ip routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather rodents (schmaljohn and nichol, 2007) . rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by inhalation of infected rodent saliva, feces, and urine (xu et al., 1985) . human infections can normally be traced to a rural setting with activities, such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention (lednicky, 2003) . the viruses have a tropism for endothelial cells within the microvasculature of the lungs (zaki et al., 1995) . there are two distinct clinical diseases that infection can yield: hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses (nichol, 2001) . hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul (lednicky, 2003) . incubation lasts 2-3 weeks and presents as flu-like in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops which can further progress to shock in approximately 15% patients. overall mortality rate is 7%. infection with dobrava and hantaan viruses are typically linked to development of severe disease. hps was first diagnosed in 1993 within southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus (snv) (centers for disease control and prevention, 1993) . this virus is still the leading cause within north america of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay (padula et al., 2000; stephen et al., 1994) . the first report of hps in maine was recently documented (centers for disease control and prevention, 1993). andes virus (andv) was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans (wells et al., 1997) . the fulminant disease is more lethal than that observed of hfrs with a mortality rate of 40%. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation (peters and khan, 2002) . incubation typically occurs 14-17 days following exposure (young et al., 2000) . unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within 48 h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted (hallin et al., 1996) . syrian golden hamsters are the most widely utilized small animal model for hantavirus infection. hamsters inoculated im with a passaged andes viral strain died within 11 days postinfection. clinical signs did not appear until 24 h prior to death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged snv isolate. no hamsters developed any symptoms during the course of observation. however, an antibody response to the virus that was not dose dependent was determined via elisa. hamsters infected with andv have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day 8 and lasted up to 12 days postinfection. infection of hamsters with andv yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver . normally, snv does not cause a disease in hamsters (wahl-jensen et al., 2007) . but a recent study showed that immunosuppression with dexamethasone and cyclophosphamide in combination causes lethal disease with snv in hamsters (brocato et al., 2014) . the disease was very similar to the disease caused by andv in hamsters. lethal disease can be induced in newborn mice, but does not recapitulate the clinical symptoms observed in human disease (kim and mckee, 1985) . the disease outcome is very much dependent on the age of the mice. younger mice are much more susceptible to virus than the adult mice. adult mice exposed to hanta virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease (seto et al., 2012; wichmann et al., 2002) . knockout mice lacking ifnα/β are highly susceptible to hanta virus infection (muller et al., 1994) . in a study of panel of laboratory strains of mice, c57bl/6 mice were most susceptible to a passaged hanta viral strain injected ip. animals progressed to neurological manifestation including paralyses and convulsions, and succumbed to infection within 24-36 h postinfection. clinical disease was markedly different from that observed in human cases (wichmann et al., 2002) . in a recent study, 2-weekold icr mice was exposed to htnv strain aa57 via the subq route (seto et al., 2012) . mice started to show signs of disease by day 11 postinoculation. piloerection, trembling, hunching, loss of body weight, labored breathing, and severe respiratory disease were observed in mice. studies to develop nhp models were not successful until recently. nhps have been challenged with new world hantaviruses; however, no clinical signs were reported (hooper et al., 2006; mcelroy et al., 2002) . cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease (klingstrom et al., 2002; sironen et al., 2008) . challenge with andv to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within 5 days postinfection. four of six aerosol exposed monkeys and 8 of 11 iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. in a recent study, rhesus macaques were inoculated by the intramuscular route with snv passaged only in deer mice (safronetz et al., 2014) . characteristics of hps disease including rapid onset of respiratory distress, severe pulmonary edema, thrombocytopenia, and leukocytosis were observed in this promising model. viremia was observed 4-10 days prior to respiratory signs of the disease that were observed on days 14-16 postinoculation. with all aspects, this animal model would be very useful to test medical countermeasures against hanta virus. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all of these viruses share common clinical manifestations (mccormick and fisher-hoch, 2002) . lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april (curtis, 2006) . this virus is responsible for 100,000-500,000 infections per year, leading to approximately 5000 deaths (khan et al., 2008) . outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases have sprung up in germany, netherlands, united kingdom, and the united states due to transmission to travelers on commercial airlines (amorosa et al., 2010) . transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex (curtis, 2006) . humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease occurs in 20% of individuals. the incubation period is from 5 to 21 days and initial onset is characterized by flu-like illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease that is permanent for a third of those affected. the overall fatality is about 1%; however, of those admitted to the hospital it is between 15% and 25%. there is no approved vaccine and besides supportive measures, ribavirin is effective only if started within 7 days (mccormick et al., 1986a,b) . the primary animal model used to study lassa fever is the rhesus macaque (jahrling et al., 1980) . aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus mirrored more closely the disease course and histopathology observed in human infection (danes et al., 1963) . iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt (lukashevich et al., 2003) . disease was dose dependent with iv, intramuscular, and subq inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be utilized, and mimic a more natural route of infection (peters et al., 1987) . within this model, the nhp becomes viremic after 4-6 days. clinical manifestations were present by day 7 and death typically occurred within 10-14 days (lukashevich et al., 2004; rodas et al., 2004) . intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns (hensley et al., 2011b) . this pathogenicity is seen in select cases of human lassa fever (cummins et al., 1992; gunther et al., 2001) . a marmoset model has recently been defined utilizing a subq injection of lassa fever virus. virus was initially detected by day 8 and viremia achieved by day 14. liver enzymes were elevated and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases (carrion et al., 2007) . mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is dependent on the mhc background, age of the animal, and inoculation route (salvato et al., 2005) . stat1 knockout mice inoculated ip with both lethal and nonlethal lassa virus strains develop hearing loss accompanied by damage to the inner ear hair cells and auditory nerve (yun et al., 2015) . guinea pig inbred strain 13 was highly susceptible to lassa virus infection. the outbred hartley strain was less susceptible, and thus strain 13 has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus (jahrling et al., 1982) . infection with pichinde virus passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death (lucia et al., 1990; qian et al., 1994) . the guinea pig is an excellent model given that it not only results in similar disease pattern, viral distribution, histopathology, and immune response to humans (connolly et al., 1993; katz and starr, 1990) . infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig models. the virus was injected ip resulting in lethargy and anorexia within 6-7 days. virus was first detected at 3 days, and reached maximum titers within 5 days. neurological symptoms began to appear at the same time, and all animals died by day 9. pneumonitis, pulmonary hemorrhage, and edema were also present (sbrana et al., 2006) . these results were recapitulated with a nonadapted pichinde virus (buchmeier and rawls, 1977; gowen et al., 2005; smee et al., 1993) . the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects 0.6% of the world's population. a greater proportion of infections and deaths occur in subsaharan africa. worldwide, there are approximately 1.8 million deaths per year with over 260,000 being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv-1 and hiv-2, with hiv-2 having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv-1 (de cock et al., 2011) . hiv targets t-helper cells (cd4+), macrophages, dendritic cells (fields et al., 2007) . acute infection occurs 2-4 weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd8+ t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd4+ t-cells decline to less than 200 cells/µl; thus cell-mediated immunity becomes impaired and the person is more susceptible to opportunistic infections as well as certain cancers. hiv has a narrow host range likely because the virus is unable to antagonize and evade effector molecules of the interferon response (thippeshappa et al., 2012) . humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of an intact and functional human innate and adaptive immune responses (berges and rowan, 2011) . the scidhu hiv infection model has proven useful, particularly in screening antivirals and therapeutics (denton et al., 2008; melkus et al., 2006) . a number of different humanized mouse models have been developed for the study of hiv, including rag1 −/− γc −/− , rag2 −/− γc −/− , nod/scidγc −/− (hnog), nod/scidγc −/− (hnsg), nod/scid blt, and nod/scidγc −/− (hnsg) blt (karpel et al., 2015; li et al., 2015; shimizu et al., 2015) . cd34+ human stem cells derived from umbilical cord blood or fetal liver are used for humanization (baenziger et al., 2006; watanabe et al., 2007) . hiv-1 infection by ip injection can be successful with as little as 5% peripheral blood engraftment (berges et al., 2006) . vaginal and rectal transmission models have been developed in blt scid hu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv-1 viremia occurs within approximately 7 days postinoculation . in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans (brainard et al., 2009) . importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hivinfected humanized mice and at least some mechanisms of pathogenesis that occur in hiv-infected humans, also occur in the hiv-infected humanized mouse models (baenziger et al., 2006; neff et al., 2011) . the advantage of these models is that these mice are susceptible to hiv infection and thus the impact of drugs on the intended viral targets can be tested. one caveat is that while mice have a "common mucosal immune system," humans do not, due to differences in the distribution of addressins (holmgren and czerkinsky, 2005) . thus, murine mucosal immune responses to hiv do not reflect those of humans. another strategy uses a human cd4-and human ccr5-expressing transgenic luciferase reporter mouse to study hiv-1 pseudovirus entry (gruell et al., 2013) . hiv-1 transgenic (tg) rats are also used to study hiv related pathology, immunopathogenesis, and neuropathology (lentz et al., 2014; reid et al., 2001) . the clinical signs include skin lesions, wasting, respiratory difficulty, and neurological signs. brain volume decreases have been documented and the hiv-1 tg rat is thus used as a model of neuropathology in particular. there are a number of important nhp models for human hiv infection (hessell and haigwood, 2015) . an adaptation of hiv-1 was obtained by four passages in pigtailed macaques transiently depleted of cd8(+) cells during acute infection (hatziioannou et al., 2014) . the resulting disease has several similarities to aids in humans, such as depletion of cd4(+) t-cells (kimata, 2014) . simian immunodeficiency virus (siv) infection of macaques has been widely used as a platform for modeling hiv infection of humans (demberg and robert-guroff, 2015; walker et al., 2015) . importantly, nhps have similar, pharmacokinetics, metabolism, mucosal tcell homing receptors, and vascular addressins to those of humans. thus, while the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero or through breast milk (keele et al., 2009; miller et al., 2005; stone et al., 2009) . there are also macaque models to study the emergence and clinical implications of hiv drug resistance (van rompay et al., 2002) . these models most routinely utilize rhesus macaques (macaca mulatta), cynomolgus macaques (m. fasicularis), and pigtailed macaques (macaca nemestrina). all ages are used, depending on the needs of the study. for instance, use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently employed. female pigtailed macaques have been used to investigate the effect of the menstrual cycle on hiv susceptibility (vishwanathan et al., 2015) . studies are performed in bsl-2 animal laboratories and nhps must be simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied (ebenezer et al., 2012) . exposure in model systems is typically through a single high-dose challenge. iv infection of rhesus macaques with 100 tcid 50 of the highly pathogenic siv/ deltab670 induces aids in most macaques within 5-17 months (mean of 11 months) (fuller et al., 2012) . peak viremia occurs around week 4. aids in such models is often defined as cd4+ t-cells that have dropped to less than 50% of the baseline values. alternatively, repeated low dose challenges are often utilized, depending on the requirements of the model (henning et al., 2014; moldt et al., 2012; reynolds et al., 2012) . since nhps infected with hiv do not develop an infection with a clinical disease course similar to humans, siv or siv/hiv-1 laboratory-engineered chimeric viruses (shivs) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease which progresses to aids, and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv-1 and is more closely related to hiv-2. however, the polymerase region of siv is 60% homologous to that of hiv-1 and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors, thus hiv-1 rt is usually put into siv for such studies (uberla et al., 1995) . sivmac239 is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt, or integrase inhibition (witvrouw et al., 2004) . nhps infected with sivmac239 have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd4+ t-cell depletion. additionally, sivmac239 utilizes the cxcr5 chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry (veazey et al., 2003) . nhps infected with shiv strains, may not develop aids, but these models are useful in testing vaccine efficacy . for example, rt-shivs and env-shivs are useful for testing and evaluation of drugs that may target the envelope or rt, respectively (uberla et al., 1995) . one disadvantage of the highly virulent env-shiv (shiv-89.6 p), is that it uses the cxcr4 coreceptor. of note, env-shivs that do use the cxcr5 coreceptor are less virulent; viremia develops then resolves without further disease progression (humbert et al., 2008) . simian-tropic (st) hiv-1 contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for 3 months, followed by clearance (haigwood, 2009) . a number of routes are utilized for siv or shiv infection of nhps, with iv inoculation the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) 1 month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase susceptibility to infection by atraumatic vaginal instillation (burton et al., 2011) . upon vaginal instillation of 500 tcid 50 of shiv-162p3, peak viremia was seen around 12 days postexposure with greater than 10 7 copies/ml and dropping thereafter to a constant level of 10 4 rna copies/ml at 60 days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac251 (barouch et al., 2012) . an example of an intrarectal model utilized juvenile (2-year-old) pigtailed macaques, challenged intrarectally with 10 4 tcid 50s of siv mne027 to study the pathogenesis related to the virulence factor, vpx (belshan et al., 2012) . here, viremia peaked at approximately 10 days with more than 10 8 copies/ml. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistent high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of haart therapy on virus and t-cells in the male genital tract, adult (3-to 4-year-old) male cynomolgus macaques were intravenously inoculated with 50 aid50s of sivmac251 and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization (moreau et al., 2012) . pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course (abel, 2009) . importantly, mother-to-infant transmission models have also been developed (jayaraman et al., 2004) . pregnant female pigtailed macaques were infected during the second trimester with 100 mid 50 shiv-sf162p3 by the iv route. four of nine infants were infected, one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission as well as the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating, and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and trim alleles), differences between challenge strains, and challenge routes (letvin et al., 2011) . nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the long-term clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade (van rompay et al., 2008) . unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study which used an adenovirus-based vaccine approach (buchbinder et al., 2008) . vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv exposure is more difficult to protect than mucosal exposure and is used as a "worst case scenario." however, efficacy at one mucosal route is usually comparable to other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect (bosch and de sanjose, 2002) . there are over 100 human papillomaviruses, with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately one third of papillomaviruses are transmitted sexually. of these, virulent subtypes, such as hpv-16, hpv-18, hpv-31, hpv-33, and hpv-45 place individuals at high risk for cervical and other cancers. up to 35% of head and neck cancers are caused by hpv-16, particularly oropharyngeal cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no wild-type animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist which use animal papillomaviruses in their natural host or a very closely related species (borzacchiello et al., 2009; brandsma, 1994; campo, 2002) . these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines (rabenau et al., 2005) . wild-type inbred mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, orthotopically transplanted or transgenic, but they are often used to look at immunogenicity of vaccines (jagu et al., 2011; oosterhuis et al., 2011) . transgenic mice used for hpv animal modeling typically express the viral oncogenes e5, e6, e7, or the entire early region of hpv-16 from the keratin 14 promoter which is only active in the basal cells of the mouse epithelium (chow, 2015) . cancers in these models develop upon extended estrogen exposure (maufort et al., 2010; ocadiz-delgado et al., 2009; stelzer et al., 2010; thomas et al., 2011) . transgenic mice with constitutively active wnt/b-catenin signaling in cervical epithelial cells expressing the hpb oncoprotein e7 develop invasive cervical squamous carcinomas (bulut and uren, 2015) . the tumors occur within 6 months approximately 94% of the time. another model uses c57bl/6 mice expressing the hpv16-e7 transgene which are then treated topically with 7,12-dimethylbenz(a)anthracene (dmba) (de azambuja et al., 2014) . these mice developed benign and malignant cutaneous lesions. cervical cancers can also be induced in human cervical cancer xenografts transplanted onto the flanks of athymic mice and serially transplanted thereafter ( hiroshima et al., 2015; siolas and hannon, 2013) . a wild-type immunocompetent rodent model uses m. coucha, which is naturally infected with mastomys natalensis papillomavirus (mnpv) (vinzon et al., 2014) . mnpv induces papillomas, keratoacanthomas, and squamous cell carcinomas and provides a means to study vaccination in an immunocompetent small animal model. wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which is a very closely related species ( breitburd et al., 1997) . in this model, papillomas can range from cutaneous squamous cell carcinomas on one end of spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) causes florid warty lesions in mucosa of the oral cavity within 4-8 weeks postexposure in experimental settings (johnston et al., 2005) . the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv-6 and hpv-11 make this a useful model (nicholls et al., 1999) . these lesions typically spontaneously regress 4-8 weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged 10 weeks to 2 years, with no history of copv, are typically used for these studies. infection is achieved by application of a 10 µl droplet of virus extract to multiple 0.5 cm 2 scarified areas within the mucosa of the upper lip of anesthetized beagles (nicholls et al., 2001) . some investigators have raised concerns that dogs are not a suitable model for high-risk hpv-induced oral cancer (staff, 2015) . bovine papillomavirus (bpv) has a wider host range than most papillomaviruses, infecting the fibroblasts cells of numerous ungulates (campo, 2002) . bpv-4 infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases, such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, rhesus papillomavirus (rhpv), a sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques is very similar to hpv-16 and is associated with the development of cervical cancer ( ostrow et al., 1990; wood et al., 2007) . monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including k. viral disease squirrels (charatan, 2003; khodakevich et al., 1986) . mpxv was discovered during the pox-like disease outbreak among laboratory monkeys (mostly cynomolgus and rhesus macaques) in denmark in 1958. no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until 1970 in zaire (the present drc) with continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of 2003, a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and western hemisphere. among 72 reported cases, 37 human cases were laboratory confirmed during an outbreak (nalca et al., 2005; sejvar et al., 2004) . it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-to-person spread occurs by large respiratory droplets or direct contact (jeézek and fenner, 1988) . most of the clinical features of human monkeypox are very similar to those of ordinary smallpox (breman and arita, 1980) . after a 7-to 21-dayincubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash (di giulio and eckburg, 2004; jeézek and fenner, 1988) . a typical maculopapular rash follows the prodromal period, generally lasting 1-3 days. the average size of the skin lesions are 0.5-1 cm and the progress of lesions follows the order: macules, papules, vesicles, pustules, umblication then scab, and desquamation and lasts typically 2-4 weeks. the fatality rate is 10% among the unvaccinated population and death generally occurs during the 2nd week of the disease (jeézek and fenner, 1988; nalca et al., 2005) . mpxv is highly pathogenic for a variety of laboratory animals and many animal models have been developed by using different species and different routes of exposure (table 33 .3). due to unavailability of variola virus (smallpox) to develop animal models and similar disease manifestations in humans that are similar, mpxv is one of the pox viruses that are utilized very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat1-deficient c57bl/6 mouse, icr mouse, prairie dogs, african dormice, ground squirrels, and gambian pouched rats are highly susceptible to mpxv by different exposure routes (americo et al., 2010; falendysz et al., 2015; hutson et al., 2009; osorio et al., 2009; sbrana et al., 2007; schultz et al., 2009; sergeev et al., 2016; stabenow et al., 2010; tesh et al., 2004; xiao et al., 2005) . cast/eij mice, one of the 38 inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose dependent mortality after in exposure to mpxv. studies with ip route of challenge indicated a 50fold higher susceptibility to mpxv when compared to in route (americo et al., 2010) . scid-balb/c mice were also susceptible to the ip challenge route and the disease resulted in mortality on day 9 postinfection (osorio et al., 2009) . similarly, c57bl/6 stat1 −/− mice were infected in with mpxv and the infection resulted in weight loss and mortality 10 days postexposure. recently sergeev et al. (2016) showed that in challenge of icr mice with mpxv resulted in purulent conjunctivitis, blepharitis, and ruffled fur in these mice although there was no death. the mouse models mentioned here are very promising for screening therapeutics against poxviruses but testing in additional models will be required for advanced development. high doses of the mpxv by ip or in routes caused 100% mortality in 6 days postexposure and 8 days postexposure, respectively, in ground squirrels (tesh et al., 2004) . the disease progressed very quickly and most of the animals were lethargic and moribund by day 5 postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv strains in ground squirrels by the subq route resulted in systemic disease and mortality in 6-11 days postexposure. the disease resembles hemorrhagic smallpox with nosebleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. another study by sergeev et al. (2017) showed that in challenge with mpxv caused fever, lymphadenitis, and skin rash in ground squirrels 7-9 days postexposure. mortality was observed in 40% of the animals 13-22 days postexposure (sergeev et al., 2017) . since mpxv was transmitted by infected prairie dogs in the us outbreak, this animal model has been more thoroughly studied and utilized to test therapeutics and vaccines compared to other small animal models ( hutson et al., 2009; keckler et al., 2011; smith et al., 2011; xiao et al., 2005) . studies using in, ip, and id routes of exposure showed that mpxv was highly infectious to prairie dogs, ip infection with the west african mpxv strain caused a more severe disease and 100% mortality than challenge by the in route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to ip route, the in route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in ip-infected animals (xiao et al., 2005) . hutson et al. (2009) african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and most importantly generalized pox lesions. therefore, this model has the utility for testing therapeutics and vaccines against pox viruses. furthermore, mpxv challenged prairie dogs were used to perform in vivo bioluminescent imaging (bli) studies (falendysz et al., 2015) . bli studies showed real time spread of virus in prairie dogs as well as potential routes for shedding and transmission. the african dormouse is susceptible to mpxv by a footpad injection or in routes (schultz et al., 2009) . mice had decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of 200 and 2000 pfu provided 100% mortality with a mean time to death of 8 days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and lung hemorrhage were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. in a recent study, comparison of the disease pathogenesis was performed by using live bioluminescence imaging in the cast/eij mouse and african dormouse challenged with low dose of mpxv (earl et al., 2015) . following in challenge, mpxv dissemination occurred through the blood or lymphatic system in dormice compared to dissemination that was through the nasal cavity and lungs in cast/eij mice. the disease course was much faster in cast/eij mice (earl et al., 2015) . considering the limited availability of prairie dogs, ground squirrels and african dormice, lack of reagents specific for these species, and not having commercial sources of these species, these small animal models are as attractive for further characterization and vaccine, and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal model for mpxv (edghill-smith et al., 2005; johnson et al., 2011; nalca et al., 2010; stittelaar et al., 2006; zaucha et al., 2001) . during our studies using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day 6 postexposure (nalca et al., 2010) . complete blood count and clinical chemistries showed abnormalities similar to human monkeypox cases with leukocytosis and thrombocytopenia (huhn et al., 2005) . whole blood and throat swabs had viral loads peak around day 10, and in survivors, gradually decrease until day 28 postexposure. since doses of 4 × 10 4 pfu, 1 × 10 5 pfu, or 1 × 10 6 pfu resulted in lethality for 70% of the animals, whereas a dose of 4 × 10 5 pfu resulted in 85% lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, animals succumbed to disease before developing pox lesions. with the low challenge dose, pox lesions were observed but they were few in comparison to the iv model. a recent study also evaluated the cytokine levels in aerosol challenged animals. (tree et al., 2015) . tree et al. (2015) showed that ifnγ, il-1rα, and il-6 increased dramatically on day 8 postexposure the day that death was most likely to occur, and viral dna was detected in most of the tissues. these results support the idea of a cytokine storm causing mortality in monkeypox disease. mpxv causes dose dependent disease in nhps when given by the iv route (johnson et al., 2011) . studies showed that a 1 × 10 7 pfu iv challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an it infection model skips the upper respiratory system and deposits virus into the trachea, delivering the virus directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation. fibrinonecrotic bronchopneumonia was described in animals that received 10 7 pfu of mpxv intratracheally (stittelaar et al., 2006) . although a similar challenge dose of it mpxv infection resulted in a similar viremia in nhps to the aerosol route of infection, the timing of the first peak was delayed by 5 days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately 100-fold lower. this suggests that local replication is more prominent after aerosol delivery compared to the it route. an intrabronchial route of exposure resulted in pneumonia in nhps (johnson et al., 2011) . delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and it infection models, the number of pox lesions was much less than in the iv infection model. a major downside of the iv, it, and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus to the blood stream or to the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting the subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human varv infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b virus (hbv) is one of the most common infections worldwide with over 400 million people chronically infected and 316,000 cases per year of liver cancer due to infection (lee, 1997) . the virus can naturally infect both humans and chimpanzees (guha et al., 2004) . hbv is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture (lai et al., 2003) . the age at which one is infected dictates the risk of developing chronic disease (hyams, 1995) . acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms of hbv infection last for a few weeks before resolving (ganem and prince, 2004) . after this acute phase, lifetime immunity is achieved (wright and lau, 1993) . of those infected, less than 5% will develop the chronic form of the disease. chronicity is the most serious outcome of the disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is 100 times more likely to develop in a chronically infected individual than a noncarrier (beasley, 1988) . the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepatitis virus suggest that x protein may be responsible (spandau and lee, 1988). many individuals are asymptomatic until complications emerge related to chronic hbv carriage. chimpanzees have a unique strain that circulates within the population (hu et al., 2000; . it was found that 3%-6% of all wild-caught animals from africa are positive for hbv antigen ( lander et al., 1972) . natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease (prince, 1972) . to date, chimpanzees are the only reliable method to ensure that plasma vaccines are free from infectious particles (prince and brotman, 2001 ). this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially ideal for these studies given that their immune response to infection directly mirrors humans (nayersina et al., 1993) . recent regulations by the national institute of health (nih) and restrictions to use great apes as animal models forced researches to find alternate models for hbv infection. other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hbv, none develops hepatic lesions or liver damage as noted by monitoring of liver enzymes (pillot, 1990 ). mice are not permissible to infection, and thus numerous transgenic and humanized lines that express hbv proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease (guha et al., 2004) . recently, the entire genome of hbv was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection (huang et al., 2012) . another model that has been developed is hydrodynamic injection of hbv genomes in the liver of mice (liu et al., 1999; yang et al., 2002) . although this model is very stressful to mice and has liver toxicity, it is successfully used to evaluate antivirals against hbv (mccaffrey et al., 2003) . liver chimeric mouse models are an additional set of surrogate models for hbv infection (dandri and lutgehetmann, 2014) . in these models human hepatocytes are integrated into the murine liver parenchyma (allweiss and dandri, 2016) . this model might be used to test antivirals as well as to study the molecular biology of hbv infection. hbv can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses (mason et al., 1982) . the woodchuck hepatitis virus induces hepatocellular carcinoma (summers et al., 1978) . within a population, 65%-75% of all neonatal woodchucks are susceptible to chronic infection (cote et al., 2000) . a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within 3 years of the initial infection in woodchucks, whereas human carcinogenesis takes much longer (gerin et al., 1989) . the acute infection strongly resembles what occurs during the course of human disease. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage (tennant, 2001) . challenge with virus in neonates leads to a chronic infection while adults only develop the acute phase of disease (buendia, 1992) . a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. the marmot himalayan develops an acute hepatitis with a productive infection (lucifora et al., 2010) . hepatitis d virus (hdv) is dependent upon hbv to undergo replication and successful infection in its human host (gerin, 2001) . there are two modes of infection possible between the viruses: coinfection where a person is simultaneously infected or superinfection in which a chronic carrier of hbv is subsequently infected with hdv (purcell et al., 1987) . coinfection leads to a similar disease as seen with hbv alone; however, superinfection can result in chronic hdv infection and severe liver damage (guilhot et al., 1994) . both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d (ponzetto et al., 1991) . a recently published report demonstrated the use of a humanized chimeric upa mouse to study interactions between the two viruses and drug testing (lutgehetmann et al., 2012) new models ranging from nhps to small animals and representing the disease characteristics in humans are necessary to study viral and host factors that drive disease pathogenesis and evaluate medical countermeasures. the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus is a low passage clinical isolate thus animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that occurs in natural disease. in order to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the aforementioned characteristics cannot always be satisfied; however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for licensure to satisfy fda "animal rule." this rule applies to situations in which vaccine and therapeutic efficacy cannot safely or ethically be tested in humans; thus licensure will come only after preclinical tests are performed in animal models. many fields in virology are moving toward standardized models that can be used across institutions to test vaccines and therapeutics. a current example of such an effort is within the filovirus community, where animal models, euthanasia criteria, assays, and virus strains are in the process of being standardized. the hope is that these efforts will enable results of efficacy tests on medical countermeasures compared across institutions. this chapter has summarized the best models available for each of the viruses described. the rhesus macaque pediatric siv infection model-a valuable tool in understanding infant hiv-1 pathogenesis and for designing pediatric hiv-1 prevention strategies prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the 1993 outbreak in egypt common marmosets (callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels. emerg generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pathological changes in brain and other target organs of infant and weanling mice after infection with nonneuroadapted western equine encephalitis virus particle-to-pfu ratio of ebola virus influences disease course and survival in cynomolgus macaques progress toward norovirus vaccines: considerations for further development and implementation in potential target populations characterization of lethal zika virus infection in ag129 mice experimental in vitro and in vivo models for the study of human hepatitis b virus infection a model of meningococcal bacteremia after respiratory superinfection in influenza a virus-infected mice middle east respiratory syndrome coronavirus: current situation and travel-associated concerns aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques necrotizing scleritis, conjunctivitis, and other pathologic findings in the left eye and brain of an ebola virus-infected rhesus macaque (macaca mulatta) with apparent recovery and a delayed time of death 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health organization, geneva. world health organization clinical aspects of hepatitis b virus infection civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates evidence of a causal role of winter virus infection during infancy in early childhood asthma vertical transmission of zika virus targeting the radial glial cells affects cortex development of offspring mice the antiviral activity of sp-303, a natural polyphenolic polymer, against respiratory syncytial and parainfluenza type 3 viruses in cotton rats experimental infection of prairie dogs with monkeypox virus. emerg epidemiological studies of hemorrhagic fever with renal syndrome: analysis of risk factors and mode of transmission hydrodynamic injection of viral dna: a mouse model of acute hepatitis b virus infection mice transgenic for human angiotensin-converting enzyme 2 provide a model for sars coronavirus infection an animal model of mers produced by infection of rhesus macaques with mers coronavirus a protective role for dengue virus-specific cd8+ t cells the incubation period of hantavirus pulmonary syndrome animal model of sensorineural hearing loss associated with lassa virus infection the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus. epidemiol hantavirus pulmonary syndrome. pathogenesis of an emerging infectious disease the pathology of experimental aerosolized monkeypox virus infection in cynomolgus monkeys (macaca fascicularis) hiv-1 infection and pathogenesis in a novel humanized mouse model h7n9 influenza viruses are transmissible in ferrets by respiratory droplet induction of neutralizing antibodies against four serotypes of dengue viruses by mixbiediii, a tetravalent dengue vaccine rapid generation of a mouse model for middle east respiratory syndrome an animal model for studying the pathogenesis of chikungunya virus infection pathogenesis of avian influenza a (h5n1) viruses in ferrets lethal crimean-congo hemorrhagic fever virus infection in interferon alpha/beta receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy the pathogenesis of western equine encephalitis virus (w.e.e.) in adult hamsters with special reference to the long and short term effects on the c.n.s. of the attenuated clone 15 variant development of a murine model for aerosolized filovirus infection using a panel of bxd recombinant inbred mice a characterization of aerosolized sudan ebolavirus infection in african green monkeys, cynomolgus macaques, and rhesus macaques characterization of disease and pathogenesis following airborne exposure of guinea pigs to filoviruses manuscripts in preparation opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the us army. key: cord-271172-y48dovux authors: potter, christopher william title: chapter 25 respiratory tract viruses date: 1998-12-31 journal: principles of medical biology doi: 10.1016/s1569-2582(97)80009-8 sha: doc_id: 271172 cord_uid: y48dovux summary respiratory tract infections are among the commonest of illnesses, and most individuals will experience two to five infections during each year of their lives. the illnesses vary from relatively mild common colds caused by rhinoviruses and coronaviruses, to severe bronchiolitis and pneumonia caused by adenoviruses and influenza viruses and respiratory syncytial virus (rsv) in infants: the former is associated with little morbidity and no mortality, while influenza is responsible annually for between 1 and 25 thousand deaths per 50 million population. over 140 viruses cause respiratory tract infections, with the added complications of influenza viruses where new antigenic variants are recognized almost annually; and immunity to infection by one virus strain offers little or no protection to infection by others. knowledge of the mechanisms of spread of respiratory viruses is largely understood and has helped in infection control; however, the clinical signs and symptoms of infection tend not to be diagnostic of the causative agent; and although vaccines have been developed for the more serious infections such as influenza and some adenovirus infection, none are available for other important infections. treatment is largely symptomatic, but the compounds ribovirin for rsv infection and amantadine for influenza virus infection have been shown to be effective. much remains to be discovered before more effective measures can be implemented to limit the enormous costs incurred by these infections. the number of viruses involved is large, and the spectrum of illness complex: in the present chapter, the viruses are described, together with the features of the epidemiology, pathogenesis, clinical disease, and treatment. virus infections of the respiratory tract affect all people in all places at all times throughout life. over 140 viruses are involved: the average child under the age of five years or adult in the developed countries will experience four to five and two to four infections each year, respectively; and although many infections are relatively mild, some are life-threatening. douglas and edelstein (1988) have said that, "about three hundred million cases of acute respiratory disease occur in the united states each year, accounting for about one hundred and fifty million visits to physicians. the cost of these illnesses exceeds one billion dollars exclusive of time lost for work": this statistic, adjusted for population, can be directed at any country. the size of the problem demands much of a general physician's time, while the importance has focused the attention of epidemiologists, microbiologists, pharmaceutical companies and many others. the viruses which cause respiratory tract infections in man include both rna and dna viruses in six families: this does not include many viruses which can cause respiratory symptoms, but where the principal tissue or organ of infection is other than the respiratory tract. these families are listed in table 1 , together with data on the finer classification into sub-families, genera, or sub-genera, the number of distinct types of virus in each group and the number of these that cause human respiratory infections. thus, there are six families encapsulating 21 sub-divisions and over 240 viruses, of which over 140 cause respiratory tract infections in man, while the remainder cause other types of infection or infections in other species. the exact numbers are complicated by the rhinoviruses where more than 100 serotypes are known, but many more probably exist, and by influenza a and b virus which exhibit continuing genetic change producing new antigenic variants in most years. the respiratory tract can be considered to be stratified horizontally into levels, beginning with the nasal passages and descending sequentially to the throat, trachea, bronchi, bronchioles, and the alveoli: it is convenient to divide the respiratory tract in this manner, since many viruses have a predilection for infection of a particular level, and these associations are shown in table 2 . thus, the common cold, limited to the nasal passages, is caused commonly by rhinoviruses and virology. rhinoviruses belong to the family picornaviridae and are distinguished from other members of this family by a higher particle density, acid lability which limits intestinal infection, relative heat stability, resistance to a variety of detergents and survival for several hours or even days outside the body. virus particles contain an internal rna which is single-stranded with positive polarity surrounded by a capsid of 60 capsomeres arranged in icosahedral symmetry: particles are approximately 30 nm in diameter. rhinoviruses replicate in human cells and less well in the cells of other primates: the conditions for growth are a relatively low ph of 7.0 to 7.2, a low temperature of 33 °c and aeration of cell cultures; and under these conditions cytopathic effects can be observed. the growth cycle takes some 11 to 17 hours; and each infected cell yields 10-200 virus particles. cross-neutralization tests indicate over 100 distinct serological types, but some sharing of epitopes does exist (hamparian et al., 1987) . epidemiology. the frequency of respiratory infections is 2 to 4 each year for adults, with a higher incidence in children: most are conmion colds of which some 30% are due to the rhinoviruses, and a number of serotypes can circulate concurrently with no evidence of cross-immunity. infections tend to be most common in spring and autumn, and are spread by large and small droplets of nasal secretions from infected persons, by direct spread from nasal secretions via hand to hand contact and auto-inoculation of nasal or conjunctival mucosa or via inanimate objects: infection can be initiated by very little virus, and the viruses can survive for long periods outside the body. peak titers appear in nasal secretions 1 to 3 days after the onset of symptoms, and virus persists for 5 to 7 days; however, virus secretion can continue for 2 to 3 weeks. a serum antibody response is seen in 30 to 90% of affected patients and a local iga response in 70 to 100%: immunity lasts 2 to 4 years, but the multiplicity of circulating serotypes gives litde or no protection against clinical disease (gwaltney, 1982) . pathogenesis. the pathogenesis of rhinovirus infection is not understood: viruses grow to high titer in the nasal epithelium, but biopsies of antigen-positive nasal mucosa cells show litde pathological change. infection causes progressive loss of ciliated epithelial cells, an inflammatory cell infiltrate, edema, hyperemia and a seromucinous exudation (hendley, 1983) . virus replication is predominantly in the nasal mucosa: other cell types such as in the nares and throat are relatively resistant. clinical features. after an incubation period of 1 to 4 days during which virus titers increase, symptoms of nasal obstruction and discharge, sneezing and coughing occur; a sore throat with erythema and headache may be experienced, but fever and systemic symptoms are usually absent. the symptoms usually improve after 2 to 3 days; but symptoms may last for 7 days, and in 25% of cases for 14 days. complications include sinusitis and otitis media; rhinoviruses have been isolated from the middle ear, but more often a bacterium has been found in conjunction with virus. small particle infection can cause tracheobronchitis and bronchiolitis, and exacerbations of asthma and chronic bronchitis are commonly precipitated by rhinovirus infection. diagnosis. rhinoviruses can be recovered from infected persons by inoculating nasal secretions onto monkey or human cells and incubating these in a roller drum at 33 °c to maximize the cytopathic effects; this appears 2 to 6 days after infection (al-nakib and tyrrell, 1988) . isolates are serotyped using a battery of neutralizing antisera in various combinations, but this is seldom carried out except for research purposes; and the lack of any specific treatment means that laboratory diagnosis is seldom attempted. treatment. there is no specific treatment for rhinovirus infections, and the treatments recommended are to relieve symptoms: nasal decongestants can be used to relieve obstruction; gargles will relieve the sore throat; and analgesics can be used if necessary. if the cough is severe, suppressants such as codeine or dextromethorphan can be given. the multiplicity of antigenic types makes vaccine development impractical, and volunteer studies have shown that inactivated vaccines have litde protective effect. locally applied interferon a suppresses virus replication, but long-term use is cytotoxic causing irritation, bleeding, and ulceration in some patients. virology. coronaviruses, within the family coronaviridae are divided into four antigenic groups containing a total of 13 serotypes of which two are infectious agents of man. the singular structure and method of replication distinguish these viruses. virus particles are 80 to 160 nm in diameter, and contain a large singlestranded rna with positive polarity and a nuclear protein coiled internally to form the nucleocapsid. this core is surrounded by a lipid bilayer into which are inserted three distinct glycoproteins which radiate from the surface: these are a receptor binding glycoprotein which initiates cell fusion and induces neutrahzing antibody production; a glycoprotein with combined neuraminidase and hemagglutinating activity; and a glycoprotein which binds to the internal nucleocapsid and effects virus budding. coronaviruses can be cultivated in organ cultures of human embryonic trachea or less successfully in tissue cultures of human cells. the serotypes are distinguished by neutralization tests, or more commonly by immunofluorescence or elisa tests. epidemiology. coronavirus infections usually occur in the winter and early spring months, with epidemics every 2 to 3 years: the two serotypes causing human infection tend to be exclusive, and 5 to 30% of common colds are due to these viruses. infection is spread by small particle aerosols; many infections are asymptomatic, and may not induce an immune response, but clinical infections invariably induce detectable antibody; however, antibody titers may not be sustained, and reinfection with the same serotype can occur within months (isaacs et al., 1983) . almost 100% of adults have detectable antibody to both serotypes. pathogenesis and clinical disease. these features are similar to those described for rhinoviruses; however, the incubation period is usually longer at 2 to 5 days, the illness persists for 2 to 18 days, some 25% of patients exhibit a mild fever but throat infection and cough is less frequent. complications of infection are unusual, but isolated cases of pneumonia are recorded; and these viruses cause exacerbations of asthma and chronic bronchitis. the pathogenesis of infection is little understood, but the evidence suggests a slow and progressive destruction to the ciliated epithelium. diagnosis and treatment. diagnosis of coronavirus infection is both difficult and unsatisfactory: organ cultures are the most sensitive for virus isolation but are rarely available, and tissue culture systems are relatively insensitive. direct demonstration of virus in cells in respiratory secretions by immunofluorescence or elisa are used in some laboratories, but most diagnoses are made retrospectively by demonstrating a significant rise of antibody following infection (schieble and kapikian, 1979) . as for rhinovirus infection, treatment is symptomatic: no antiviral drugs have been developed and no vaccine is available for coronavirus infections. experimentally, a-interferon has been shown to ameliorate symptoms and reduce virus replication. some 50% of common colds, characterized as an afebrile illness localized to the nasal epithelium and presenting with nasal obstruction and discharge, sneezing and coughing are caused by rhinoviruses and coronaviruses; the remainder are caused by a number of other viruses including echoviruses, coxsackie a and b viruses. respiratory syncytial virus, influenza c and parainfluenza viruses ( table 2) . as these infections are relatively mild and a large number of agents are involved, exact laboratory diagnosis is seldom undertaken except for research purposes, and there are no specific treatments. virology. there are 47 serotypes of human adenoviruses in six subgenera in the genus mastadenovirus in the family adenoviridae: eight serotypes in three subgenera cause respiratory infection in man (table 2 ). virus particles contain single-stranded dna surrounded by at least 11 polypeptides. the outer capsid is made up of 252 capsomeres arranged in an isocosahedral of 20 triangles with 12 vortices giving a particle diameter of 60 to 80 nm: 240 capsomeres are hexons with six neighbors and are composed of a single protein with common epitopes for all serotypes; and the remaining 12 capsomeres are pentons with five neighbors, each composed of a base with a fiber-like projection radiating outwards and are serotype specific. human adenoviruses only replicate sequentially in human cells. virus particles attach to cell receptors via the penton fiber and then to other cell receptors by the hexons: following entry, cellular dna and protein synthesis are inhibited; replication takes place in the cell nucleus; newly-formed pentons are toxic to cells; and virus is released after cell death. the division of human adenoviruses into six subgenera is based on the sequence homology of the dna genome, dna fragment analysis following restriction enzyme treatment, oncogenicity for newborn hamsters, the molecular mass of the internal proteins, length of fibers and precent g + c in the viral dna. the division into serotypes is based on neutralization tests which show no cross-reactivity. this classification is important, since it is consistent with the association of the viruses with various human infections (wadell, 1984) . epidemiology. adenoviruses cause both sporadic and epidemic infections: five per cent of acute respiratory infections and ten per cent of febrile infections of children, together with 3 and 7% of these infections in adults, are caused by these viruses; and some 10% of all pneumonias in children are due to adenovirus infection. transmission is by respiratory droplets, whilst in children fecal/oral transmission is important: fecal excretion can continue for weeks and months after acute infection. by the age of 10 years, 50% of children have antibody to serotypes 1,2, and 5; antibody to other serotypes is less common, but in total the results reflect the importance of these viruses in human infection. pathogenesis. adenoviruses, spread by droplet infection, impinge on epithelial cells in the pharynx or in the lower respiratory tract to enter and kill cells by a combination of inhibition of cellular metabolism, virus replication and the toxic effects of the penton: the results are extensive desquamation of affected areas, causing sore throat, necrotizing bronchitis, bronchiolitis and interstitial pneumonia. in lymphoid cells, infection causes hypertrophy: affected cells can harbor latent virus for months, years or throughout life; and although 50% of excised tonsils and adenoids contain latent virus, there is no evidence that this can exacerbate, except possibly in pertussis syndrome. clinical disease. adenoviruses cause some 5% of all acute respiratory disease (ard) seen in children aged five years or less (brandt et al., 1969) . these are predominantly due to serotypes 1,2 and 5 (subgenera b); and the frequency of these infections in children means adult infections are unusual. spread is by droplet infection: after an incubation period of 2 to 4 days, symptoms begin with pharyngitis, cough, nasal congestion and coryza, whilst fever, exudative tonsillitis, malaise, headache and myalgia are often seen. infection tends to progress over a 2 to 3 day period, and resolves in 5 to 8 days. infection may extend to cause laryngotracheobronchitis, bronchitis and pneumonia, the last being particularly important in young children and the cause of some deaths. sporadic infections of both children and adults are also caused by adenovirus types 3 and 7 (subgenera c): these present as above, but laryngotracheobronchitis and pneumonia are more commonly seen, with 40 to 50% showing radiological evidence of pneumonia; and with conjunctivitis, when the infection is known as pharyngo-conjunctival fever, and that occasionally is seen as epidemics among children and in families. the respiratory tract complications are particularly severe in immunocompromised patients. probably due to overcrowding and fatigue, adenovirus types 3,4 and 7 cause epidemic ard in military recruits: some 80% of recruits can become infected, 20 to 40% require hospitalization for lower respiratory tract infection and pneumonia, and deaths are recorded. adenoviruses have been frequendy isolated from patients with whooping cough syndrome in conjunction with bordetella pertussis', this is usually reported as adenovirus type 5. however, there is little evidence that adenoviruses can produce this syndrome alone: the presence of adenovirus may be due to reactivation from tonsillar tissue by bordetella pertussis to complicate the infective process. diagnosis. adenoviruses are present in throat swabs, throat or nasal washings or feces of infected persons. virus growth is best in tissue culture of human embryonic kidney, but hela and kb cells are a more available alternative. growth is recognized by a characteristic cytopathic effect; however, this may take days or weeks to develop, but virus can be demonstrated early in infected cells by immunofluorescence with specific antibody. isolates can be identified as adenoviruses with antisera to the common epitopes of the hexon using complement fixation, immunofluorescence or elisa tests; and specific serotypes identified by neutralization tests. there are no effective antiviral agents for the treatment of adenovirus infection, and treatment is limited to the relief of symptoms. in addition, there is no adenovirus vaccine; however, the problem of ard in military recruits is of such importance that a vaccine has been developed: live adenovirus types 4 and 7, the main viruses responsible for ard, are given orally as a coated enteric preparation; this establishes an intestinal infection, induces an immune reaction and, in bypassing the respiratory tract, does not cause symptoms (tarafuji etal, 1979) . virology. epstein-barr virus (eb v) is a member of the family herpetoviridae, and the only member of the sub-family gammaherpesvirinae to cause infection in humans: the virus is distinguished from other herpetoviridae in replicating or establishing a latent infection in p-lymphocytes, and the potential for promoting tumorgenicity of these cells. the virus is relatively large at 150 to 200 nm diameter: particles are composed sequentially of linear double-stranded dna and an internal core of proteins; a capsid of 162 capsomeres arranged in an icosahedral form; a protein tegument; and a trilaminar envelope derived from the cellular membrane into which are inserted spikes of several virus-specific glycoproteins. infection is primarily of the epithelial cells of the oropharynx, and via the complement receptor cd21: virus infection from these cells spreads to p-lymphocytes where a few cells undergo lytic infection while the majority support a latent infection that leads to cell proliferation. the mechanism for this latency is not fully understood, but is believed to be due to the absence or low-level of host cell transcriptional factors that are essential for virus replication: the result is a persistent, life-long p-lymphocyte infection. epidemiology serological studies from most developed countries have indicated that eb v infection is common, and 80 to 90% of adults have been infected: in developing countries, infection occurs earlier and is more common. once infected, a subject will remain a virus excretor for months, years and probably life, with continuous virus production from a variable number of p-lymphocytes in the oral cavity. infection is accompanied by an intense immune response; and it is the balance between virus production and immune status which determines the extent of virus secretion at any one time. the immune response is not sufficient to resolve the illness, as infected lymphocytes appear to be resistant to cytotoxic t cells and there is a down-regulation of hla antigen expression. the immortalization of p-lymphocytes is polyclonal and leads to cell proliferation; this can be further exaggerated in immunosuppressed patients, such as hiv or malaria infected persons, which in turn can lead to a chromosome translocation where the c-myc oncogene comes adjacent to a strong promoter: the outcome is a malignant cell transformation giving rise to burkitt's lymphoma (lenair and bornkamm, 1987) . pathogenesis. infection is from saliva of previously infected subjects: since virus production is low, transmission requires close contact, and peaks of infection are seen at ages 1-6 and 14-20 years corresponding to the ages of early and adolescent intimacy. the exact site of infection remains unknown, but waldeyer's ring, rich in lymphocytes, epithelial cells or the salivary glands are suggested by various authors. virus-infected (3-lymphocytes are disseminated throughout the body via the blood stream, and antigen-positive cells can be detected in most organs and tissues. infection is accompanied by an intense immune reaction to virus and to the proliferating p cells which includes virus-specific antibody, heterophile antibody, autoantibodies and rheumatoid factor (robinson and stevens, 1984) ; indeed, the clinical disease is due to the nature and the intensity of the immune responses. the changing pattern of the immune state regulates virus secretion, and the infectivity of the patient for others. clinical features. following contact with infected saliva, there is an incubation period of 30-45 days followed by a short prodromal illness of headache, malaise and fatigue: after this, the definitive symptoms of glandular fever occur. most patients complain of a sore throat with hyperemia and hyperplasia of the lymphoid tissue; exhibit an exudate over the pharynx; have a fever which lasts for some 10 days; and have cervical or general lymphadenopathy: fever and malaise can persist for weeks or months; secondary infections are common; blood dyscrasias occur; splenic enlargement is common, but rarely leads to rupture; and a mild hepatomegaly is seen in 5 to 10% of patients associated with a transient jaundice. mild rubilliform skin rashes can occur; however, ampicillin causes a maculopapular rash and is not used in patients with sore throats. oral cavity obstruction due to massive enlargement of tonsils, adenoids and epiglottis may require emergency treatment. fatalities are recorded, but are usually associated with immunocompromised patients. diagnosis. the symptoms of glandular fever usually alert the physician who can confirm his suspicions by a number of laboratory tests. the virus is difficult to cultivate: in vitro growth is only seen in lymphocytes, but only a few cells support virus replication; however, virus protein or dna can be demonstrated in infected cells by western blotdng or dna hybridizafion tests, respectively. serological tests include the demonstration of serum igm antibody to virus capsid proteins using an elisa test, or the detecuon of the heterophile antibody response using the paul bunnell test. atypical monocytes can form up to 20% of the peripheral blood leucocytes. treatment and control. both interferon and acyclovir have been shown to diminish virus secretion during treatment, but relapses occur when treatment is stopped: more importantly, neither treatment significantly ameliorates symptoms, and are therefore not recommended. in the absence of specific therapy, treatment is supportive: the sore throat can be treated with analgesics; and some suggest corticosteroids limit the duration of illness, possibly by the effect on the immune response. the importance of ebv in initiating burkitt's lymphoma in african children where the incidence is 1:5000, has focused attention on developing an ebv vaccine; such vaccines are undergoing clinical trials at the present time. although febrile sore throats are caused by adenovirus and ebv infection, a large number of other viruses can produce the same clinical symptoms; these include influenza a, b, and c, numerous serotypes of coxsackie and echoviruses and parainfluenza viruses. collectively, these latter viruses are responsible for more than half the febrile sore throats which occur; and collectively, viruses are responsible for over 90% of the febrile sore throats caused by infectious agents. virology. influenza viruses are the only viruses of the genus orthomyxovirus in the family orthomyxoviridae, affecting man, birds, horses, pigs, and other species. the viruses are approximately spherical with a diameter of 80 to 120 nm. each virus particle consists of single-stranded rna of negative polarity segmented into eight fragments of varying size. the rna is closely associated with a nuclear protein (np) and the polymerase enzyme complex to form a helical structure: the np takes one of three antigenic forms which allows influenza viruses to be classified into types a, b, and c. surrounding a nuclear protein is the matrix or membrane protein, and this in turn is surrounded by a lipid bilayer. inserted into the bilayer, and radiating from the surface, are two virus glycoproteins. the most numerous glycoprotein is the hemagglutinin (ha) which is the receptor binding component; the classification of influenza types into subtypes is based principally on the different antigenic forms of the ha (hi, h2, h3) molecule. the second glycoprotein is the neuraminidase (na) which is important in both cell infection and in facilitating the release of newly formed virus from the surface of infected cells; the na is antigenically variable (n,,n2) and this variation is used in the subtype classification of the viruses. influenza viruses grow in human and monkey cells and in the amniotic and allantoic cavity of embryonated hens' eggs. absorbed virus is uncoated and the virus rna together with the polymerase enzyme complex pass to the cell nucleus. replication, which is dependent on cell rna synthesis, produces virus components which pass to the cell membrane for assembly, and are then budded from the cell surface: the complete cycle takes 8 to 10 hours. epidemiology. influenza holds a unique position among the viruses causing respiratory tract infection, since it commonly and dramatically causes local outbreaks or widespread epidemics, and these occur in most parts of the world and in some countries in most years. epidemics occur suddenly and without warning, and the number of people infected range from few hundreds to hundreds of thousands: although short-lived, epidemics can infect up to 70% of a population, with clinical disease occurring in 50% of infected subjects, and deaths due direcdy or indirectly to influenza number from 1000 to 25,000 per 50 million persons per year in developed countries. the importance of this infection in causing morbidity and mortality is reflected in the enormous scientific effort made to understand the virus, the nature of the disease and to devise methods for control. when influenza virus isolates are cross-referenced to patients, time and place, several patterns are seen. firstly, most pandemics and widespread epidemics are caused by influenza a viruses; influenza b viruses are associated with self-limiting epidemics which occur in families or small communities; and influenza c virus is associated with sporadic infections, mainly among young children. influenza a exhibits the greatest antigenic diversity; influenza b exhibits some variation; and influenza c is relatively stable. secondly, the recorded patterns of influenza a epidemics during the past century exhibit two phenomena. every 10 to 15 years since records began in 1890, influenza virus has undergone major antigenic changes in the ha molecule, termed antigenic shift: the emergence of these new subtypes has resulted in the pandemics seen in the years 1933 (hi), 1947(h1), 1957(h2) and 1968(h3); and caused the epidemics which followed until the next new subtype emerged. these subtypes are distinct, and immunity to one provides no protection against infection by others. the origin of new subtypes cannot be by simple mutation from previously existing strains, since many genetic changes occur and intermediary strains are not found. two theories for the origin of new subtypes are advanced. firstly, two influenza viruses, one of human origin and the other probably of avian origin, infect the same cell: due to the segmental nature of the virus genomes, reassortant virus is easily produced combining the properties to infect man and the ha glycoprotein of the non-human strain. the new subtypes can now cause pandemic infection in populations with no previous immunity. an alternative theory is that the various subtypes circulate sequentially over a period of 70 to 80 years, since on two occasions antibody to new serotypes has been detected in sera from elderly people years prior to the emergence of that new subtype to cause pandemic infection: where the viruses survive between times is unknown. between the times of antigenic shift, epidemics occur in most years and the strains which cause them exhibit antigenic drift; these strains belong to the same subtype, but do not cross-react completely, and infection by one strain does not induce solid immunity to later emerging strains. this sequential accumulation of mutations arises naturally, and is selected by antigenic pressure in the immune and partially-immune population. in addition to antigenic drift and shift, viruses isolated from different places at the same time, and even viruses from different individuals in the same epidemic, can exhibit antigenic differences; this is known as inter-and intra-epidemic variation, and both underline the difficulty in matching vaccine virus to epidemic strains, and contributes to the disappointing low levels of immunity induced by inactivated influenza vaccines. the degree of crossprotection is directly related to the degree of cross-reaction of the virus ha, and this is shown in table 3 . influenza b viruses exhibit antigenic drift but not antigenic shift. pathogenesis. the pathogenesis of influenza has not been agreed among researchers, and many features are not understood. virological investigations indicate that infection is from virus inhaled as droplets on to the epithelial cells of both the upper and lower respiratory airway. histological studies of nasal exudate and tracheal biopsies indicate that the major site of infection is the ciliated columnar epithelial cells which become progressively rounded and swollen, and exhibit vacuolation with loss of ciliation; the progression usually begins in the tracheal bronchial epithelium and then ascends. the result is the widespread destruction of the ciliated epithelium down to the basement membrane which itself is not affected; the lesions become increasingly permeable with polymorphonuclear infiltration and edema. because of the generalized symptoms of uncomplicated influenza, viremic spread has been suspected; however, there is no conclusive evidence that viremia occurs. in contrast, virus infections have been associated with ecg and eeg changes; some unconfirmed observations of virus antigen in brain and heart tissue have been published; and infection can be associated with viral encephalitis, particularly among children. these findings suggest dissemination of either virus or virus products from the respiratory tract; virus is known to grow in leucocytes and the release of pyrogens or cytokines offers an explanation for some systemic symptoms. clinical disease. the symptoms of influenza tend to be constant regardless of the subtype or strain of virus; however, the clinical features of influenza in young children may vary from that of adults, with croup a more common symptom in children and sore throats more common in adults. droplet infection is followed by an incubation period of approximately 24 to 96 hours: the onset of illness is usually abrupt. symptoms include fever, headache, photophobia, shivering, dry cough, malaise, aching of muscles and a dry, ticking throat which can lead to the voice becoming husky or lost. the eyes are often watery, burning and painful on movement. fever is usually continuous, and typically lasts some 3 days: in a percentage of patients, a second rise in temperature may occur, usually smaller than the first, which gives the infection a biphasic fever curve. the cough may persist for several days; the nose can be blocked or show a purulent discharge; and myalgia is most severe in leg muscles, but also may involve the other extremities. acute illness usually resolves within 7 days, but patients frequently complain of feeling listless for weeks, and depression is a common residual complaint. infections caused by influenza b resemble closely those caused by influenza a; in contrast, influenza c is usually a mild upper respiratory tract infection. the complications of infection include tracheobronchitis and bronchiolitis: these patients exhibit a productive cough and chest tightness, and crepitations are commonly heard but the lungs are usually radiologically clear. these complications are most conmionly seen in patients with obstructive bronchitis and in older people, and death from influenza can result in such patients. pneumonia in patients with influenza virus infection can be primary or secondary. in viral pneumonia, patients developed a persistent fever and leucocytosis, dyspnea, hypoxia, and cyanosis; this follows the acute symptoms described above. sputum specimens show no bacterial cause, and a proportion of these patients die of diffuse hemorrhagic pneumonia. more commonly, pneumonia following influenza is due to secondary bacterial infection, principally with staphylococcus aureus, but also with streptococcus pneumoniae, hemophilus influenzae and other bacterial species: this complication is a major cause of death among elderly people and those with underlying disease such as congestive heart failure and chronic bronchitis. in addition, patients with diabetes, renal disease, alcoholism and those who are pregnant also have an increased susceptibility to secondary bronchopneumonia. influenza is also associated with myalgia, a common feature of acute disease, but clinical myositis and myoglobinuria can occur: the symptoms develop after the onset of respiratory infection, when muscles become painful and tender, but without neurological symptoms. an important complication of influenza infection is the syndrome known as reye's syndrome characterized by encephalopathy and fatty liver degeneration; this is chiefly seen at age 8 to 15 years, and among those hospitalized the mortality can be as high as 50%. the association of reye's syndrome following infection by influenza a or b or other viruses has been fully demonstrated, but the pathogenesis remains obscure (carey et al., 1976) : researchers have highlighted the association of virus infection with treatment of fever with high concentrations of aspirin, and for this reason aspirin should not be given to patients in this age group. more conjectural is the association of influenza infection in pregnancy with congenital abnormality; this is not justified with our current knowledge. further complications reported are ketoacidosis in diabetic patients, acute viral encephalitis in children, guillain-barre syndrome, sudden infant death syndrome and toxic shock syndrome resulting from the dual association of staphylococcus aureus and influenza infection. diagnosis. influenza viruses can be recovered from throat washings or swabs by inoculating tissue cultures of kidney tissue from rhesus monkeys, chicks, and a variety of other species. after incubation, newly produced virus can be detected in supernatant fluids by the ability to agglutinate erythrocytes (hemagglutination), or the adherence of erythrocytes to virus particles assembled on the cell surface (hemadsorption). influenza virus can also be cultured in the amniotic cavity of embyronated eggs: after incubation, high titers of virus are found in the amniotic fluid, and are detected by hemagglutination. the viruses are recognized as influenza a, b, or c by complement fixation tests using extracts of infected cells containing high concentrations of np antigen, and type-specific antisera. further identification of influenza isolates into sub-types and strains is dependent upon antigenic differences in the ha. this is determined by hemagglutination inhibition (hi) tests against antisera raised in experimental animals against a range of virus subtypes and strains: the titer of each antisera against homologous virus is known prior to testing, and the pattern of hi titers found against an unknown influenza virus determines the strain and type. however, this is a highly specialized typing system which is the responsibility of who reference laboratories who constantly type new isolated viruses in a worldwide endeavor to detect new virus variants as they arise. proof of influenza infection can also be obtained by demonstrating a rise in specific complement fixating or hi antibodies in sera collected early after the onset of symptoms and 14 to 21 days later. control and treatment. the constant, almost annual antigenic changes seen in influenza virus a and to a lesser extent influenza b means that vaccines need to be developed for each new epidemic strain. at present, vaccines are produced by inoculating virus into embryonated eggs, purifying and inactivating the resultant virus growth to give a whole virus, disrupted virus or virus subunit (ha and na) vaccines (potter, 1982) . to date, no live attenuated virus vaccines are available. inactivated vaccines produce few reactions, but most are mild and ephemeral; induce serum antibody in the majority of subjects, but immunity in only 60 to 90% of vaccinees. due to the severity and fatalities from influenza, vaccine is offered annually to at-risk patients: these include persons aged 65 yrs of age, patients with a history of chronic chest or heart disease, and patients with asthma, renal dysfunction and metabolic disorders. in some years, such as when a new subtype is recognized, key personnel in industry and social services should be offered vaccine. at present, the treatment of influenza is symptomatic: patients are advised to remain in bed for 2 to 3 days until the acute symptoms subside; symptoms of headache and fever are treated with paracetamol; codeine linctus can relieve the cough; insomnia may be treated by barbiturates or promethazine; and antibiotics are indicated when chest complications are present or suspected. the use of prophylactic antibiotics in patients with chronic chest disease is common, but not recommended. the compound amantadine, and the analog rimantidine, are active against influenza: an oral dose of 100 mg per day given to people in contact with influenza decreases the chance of infection by some 70%, and given to patients with clinical disease can reduce both the length and severity of disease (dolin et al., 1976) . virology. parainfluenza viruses of the genus paramyxovirus in the family paramyxoviridae are distinguished by the size and shape of the nucleocapsid, biochemical similarity, antigenic cross-reactivity and the presence of a surface glycoprotein with combined hemagglutination and neuraminidase activity. virus particles consist of single, non-segmented, negative-strand rna, and three internal proteins surrounded by a lipid bilayer with a fourth protein, into which are inserted the hemagglutinin/neuraminidase molecule and a fusion protein which both radiate from the surface of the virion particle. the complete virion has a diameter of 150-200 nm. the viruses replicate in primary human and monkey cells with assembly of new virus particles taking place in the cytoplasm and release by budding: the effect on the cells is lytic with cytopathic effect and occasionally syncytia formation. viruses can be detected by hemadsorption of guinea-pig erythrocytes to infected cells through virion particles budding through the cell membrane. four serotypes cause respiratory infection in man, and are individually recognized by various tests including hemagglutination inhibition, hemadsorption inhibition and neutralization tests. epidemiology. parainfluenza viruses types 1, 2, and 3 are the major cause of tracheobronchitis and croup in young children; type 3 is frequently associated with pneumonia; and type 4 causes mild upper respiratory tract infections. infection is by droplets and requires only a small dose of virus; virus from infected persons is shed for 3 to 10 days, but in some cases can continue for 3 to 4 weeks. infection by all types is worldwide with peak numbers occurring in the winter months: epidemics are frequently recorded, and reinfection common (chapman et al., 1981) . over 50% have antibodies to one or more of these viruses by age 2 years, and over 75% by age 4 years. pathogenesis. mild infections are mostly of the nose and throat with minimal involvement of the lower respiratory tract; more extensive infection by types 1 and 2 involves the larynx, trachea, and bronchi with pneumonia occurring in some 15 to 20% of patients; and type 3 infection causing a higher incidence of bronchiolitis and pneumonia. virus replication has a lytic effect on the epithelial cells, whilst in the trachea and bronchi infection causes excess mucus production leading to atelectasis and pneumonia. infection induces an ige antibody response in serious cases which in turn initiates histamine release: it is thought that these responses are important in the pathogenesis of infection. clinical features. following an incubation period of 2 to 4 days, primary infection in children is seen as a rhinitis and pharyngitis with erythema: some evidence of bronchitis is commonly seen with hoarseness, cough with croup and bronchitis with rhonchi. fever is recorded and lasts 2-3 days. in more severe cases, infection extends to produce a heightened fever, a laryngotracheobronchitis with a barking cough and croup which lasts for 48 to 72 hours; symptoms may worsen to cause air hunger and cyanosis, sternal and intercostal retractions, airway obstruction and glottic and subglottic narrowing (parrott et al., 1962) : if pneumonia develops, the cough is productive, and radiological examination may show interstitial and perihylar infiltration. diagnosis. viruses present in throat washings can be cultivated on monkey kidney cells: after replication, virus particles can be demonstrated on the surface of infected cells by hemadsorption. alternatively, virus can be detected directly by immunofluorescence tests on respiratory secretions (ray and minnich, 1987) . diagnosis based on serological tests is less satisfactory: primary infection induces a type-specific antibody response detectable by hemagglutination inhibition, complement fixation, or neutralization tests, but subsequent infections induce a heterotypic response. control and treatment. much research has been carried out on the development of a parainfluenza virus vaccine but none is available at the present time: inactivated vaccines induce serum antibodies, but only partial immunity. treatment is symptomatic: children may be nursed in plastic tents supplied with cool moistened oxygen for 2 or 3 days to relieve respiratory symptoms; severe obstruction may require endotracheal intubation or tracheostomy; and accumulative and excessive tracheobronchial secretion may require bronchoscopy aspiration. antibiotics are used where investigations indicate secondary bacterial infection. the use of corticosteroids is contentious, but aerosolized preparations of the antiviral compound ribavirin may be valuable. some 50% of cases of laryngotracheitis and croup in children under age three years are due to parainfluenza virus infection; the remaining cases are due to influenza a and b, respiratory syncytial virus (rsv) and various serotypes of coxsackie and echoviruses. the severity of infection indicates laboratory investigation; thus, the contribution of these latter agents to the syndrome is well-documented. acute bronchitis, or more commonly tracheo-bronchitis since contiguous respiratory compartments are usually involved, has been associated with adenovirus type 3, 4 and 7 infection in both children and adults, but other serotypes have been identified. among the other infections causing bronchitis are rhinoviruses and rsv virus in children, and measles and influenza a virus infections in children and adults: these infections may precede secondary bacterial infection. exacerbation of chronic bronchitis is frequently associated with virus infections; these include a wide range of viruses, but are most commonly caused by rsv, rhinoviruses, and parainfluenza viruses. virology. respiratory syncytial virus (rsv) belongs to the genus pneumovirus within the family paramyxoviridae; and is distinguished by the form of a nucleocapsid, replication entirely in the cell cytoplasm and the absence of hemagglutinin and neuraminidase glycoproteins. the virus particle is structurally similar to that of other members of the family consisting of a single, non-segmented, negative strand of rna and three internal proteins surrounded by a lipid bilayer with two associated proteins: inserted into the outer aspect of the lipid bilayer are spikes of an attachment protein and a fusion protein. the diameter of the virion is 120 to 300 nm. rsv replicates in the cytoplasm of a range of human and animal cells: cell death is principally the result of cell fusion, and the formation of multinucleate syncytia indicates the presence of virus and gives the virus its name. antigenic variants are known, and this has resulted in recognition of two subgroups. epidemiology. rsv causes annual epidemics in the winter months in most countries which are indicated by an abrupt rise in the number of pediatric admissions to hospital (glezen and denny, 1973) . infection is spread by large droplets and therefore require close contact; by hand from nasal and conjunctival secretions; or via inanimate objects and self-inoculation: the virus is highly infectious, and 50% of children are infected by 1 year of life, and all children by age 2 years, with recurrent infection common. the virus causes 75% of all bronchiolitis and 25% of all pneumonia cases seen in children under 1 year of age: the mortality rate is 0.5 to 2.5% with most in children with underlying heart or respiratory disease. infection is essentially an upper respiratory tract infection in children aged less than 6 weeks or over 6 months; however, between these age limits 30% of infections involve the lower respiratory tract. recovery is accompanied by serum antibody and a cell-mediated immune response which protects against subsequent lower respiratory tract infection; the local iga antibody response is ephemeral, allowing further upper respiratory tract infections in later life. pathogenesis. after an incubation period of 3 to 6 days, infection begins in the nasopharynx with virus titers reaching a maximum at 2 to 3 days, declining between 3 to 6 days but can be detected in some patients for 3 weeks. spread to the lower respiratory tract is by cell-to-cell interaction in the respiratory epithelium and via aspirates. cell infection is cytopathic following cell fusion, causing inflammation and necrosis with associated plugging of the airways; but other factors are involved in the disease which are not fully defined: these include immunopathology due to antibody production, the formation of antigen-antibody complexes, delayed hypersensitivity reactions, an exaggerated cytotoxic t-cell response and an ige response as described for parainfluenza virus infection (welliver et al., 1984) . clinical disease. in children aged less than 6 weeks or greater than 6 months infection is usually seen as an upper respiratory tract (urt) infection with rhinitis, a mild fever, sneezing, and wheezing; some 40% of children exhibit a lower respiratory tract involvement with tachypnea, rales, and rhonchi. more severe lower respiratory tract (lrt) infection may occur, but this is more common at age 6 to 24 weeks: following the mainly urt infection, patients develop a bronchiolitis with dyspnea, severe tachypnea, and intercostal and substernal retraction; and in most severe cases an added pneumonia occurs with hypoxia and cyanosis. radiological appearances vary from normal to that of a bacterial pneumonia, but clinical severity is not mirrored by the radiological changes. the infection lasts 6 to 12 days with patients showing improvement after 3 to 4 days, but in severe cases symptoms may persist for several weeks. diagnosis. aspirates or nasal secretions contain virus that can be detected by inoculating tissue cultures which show syncytia formation following virus replication, or by direct tests for virus antigen using immunofluorescence tests. infection induces a rise in serum antibody detected by complement fixation or neutralization tests. treatment. patients with lrt infection commonly require hospitalization for supportive therapy: reduction of fever and hydration is commonly adequate, but in more severe cases oxygen may be required to assist breathing; mechanical removal of respiratory secretions may be necessary, and blood gases should be monitored. the infection responds to treatment with the antiviral compound ribovirin, and administration of this compound as a small particle aerosol has proven effective (hall et al., 1983) . no effective vaccine has been developed despite 20 years of dedicated research. studies have shown that 50 to 90% of bronchiolitis cases are caused by rsv; and characterized by necrosis and sloughing of the bronchiolar epithelial leading to the plugging of small airways, obstruction and atelectasis (hall et al., 1986) . however, other viruses more appropriately associated with other compartments of the respiratory tract, can produce the same pathological changes and clinical symptoms. thus, bronchiolitis has been associated with infections by influenza viruses a and b, and adenoviruses; and in young children with parainfluenza virus infection: these virus infections are described under separate headings. pneumonia, characterized by radiological changes, physical signs and pathology, is uncommonly related to infection by any virus; however, three reservations should be admitted. firstly, cases of acute pneumonia due to adenovirus and influenza viruses, although unusual, are well-documented, and fatalities have been recorded following these infections. secondly, severe infection by viruses in higher compartments of the respiratory tract, can extend to cause pneumonia: these include adenovirus and influenza viruses again, and rsv and parainfluenza viruses in young children. thirdly, primary pneumonia is a rare presentation by measles, chicken pox (varicella/zoster) and cytomegalovirus (cmv) infection: although unusual in normal subjects these are more commonly seen in immunocompromised persons, where the infection can be devastating. pneumonia in the immunocompromised by measles, chicken pox or cmv is usually an extension of typical infection to involve the lungs, but can present without a rash or with an atypical rash in patients with no history of infection. patients develop cough and chest pains, and more seriously dyspnea and cyanosis; x-rays may show evidence of viral pneumonia with atypical, patchy consolidation; and deaths are recorded in 40% or more of immunocompromised patients. pathologically, the alveoli contain edema fluid with macrophages, but few polymorphonuclear cells; and typically and diagnostically, giant multinuclear cells. the viruses can be grown from the bronchial secretions; however, since these are rapidly progressing infections and suggested treatments are available for two of these viruses, quicker methods such as the direct demonstration of virus by immunofluorescence or elis a tests on cells in secretions are needed. there is no treatment for measles virus pneumonia; acyclovir or ganciclovir are reported to be of value in the treatment of cmv; and acyclovir will prevent pneumonia in chicken pox patients, but has no proven value in treating established pneumonia. respiratory tract infections are among the commonest of illnesses, and most individuals will experience two to five infections during each year of their lives. the illnesses vary from relatively mild common colds caused by rhinoviruses and coronaviruses, to severe bronchiolitis and pneumonia caused by adenoviruses and influenza viruses and respiratory syncytial virus (rsv) in infants: the former is associated with little morbidity and no mortality, while influenza is responsible annually for between 1 and 25 thousand deaths per 50 million population. over 140 viruses cause respiratory tract infections, with the added complications of influenza viruses where new antigenic variants are recognized almost annually; and immunity to infection by one virus strain offers little or no protection to infection by others. knowledge of the mechanisms of spread of respiratory viruses is largely understood and has helped in infection control; however, the clinical signs and symptoms of infection tend not to be diagnostic of the causative agent; and although vaccines have been developed for the more serious infections such as influenza and some adenovirus infection, none are available for other important infections. treatment is largely symptomatic, but the compounds ribovirin for rsv infection and amantadine for influenza virus infection have been shown to be effective. much remains to be discovered before more effective measures can be implemented to limit the enormous costs incurred by these infections. the number of viruses involved is large, and the spectrum of illness complex: in the present chapter, the viruses are described, together with the features of the epidemiology, pathogenesis, clinical disease, and treatment. common cold viruses-rhinoviruses infections in 18,000 infants and children in a controlled study of respiratory tract disease. i. adenovirus pathogenicity in relation to serological type and illness syndrome the epidemiology of tracheobronchitis in pediatric practice a nationwide outbreak of reye's syndrome. amer a controlled trial of amantadine and rimantadine in the prophylaxis of influenza a infection epidemiology of acute lower respiratory disease in children rhinoviruses. in: viral infections of humans: epidemiology and control aerosalised ribavirin treatment of infants with respiratory syncytial virus infection respiratory syncytial viral infection in children with compromised immune function a collaborative report: rhinoviruses-extension of the numbering system from 89-l(x) rhinovirus colds: immunology and pathogenesis epidemiology of coronavirus respiratory infections burkitt's lymphoma, a human cancer model for the study of the multistep development of cancer: proposal for a new scenario inactivated influenza virus vaccine efficiency of immunofluorescence for rapid diagnosis of common respiratory viruses production of autoantibodies to cellular antigens by human b cells transformed by epstein-barr virus coronaviruses. in: diagnostic procedures for viral, rickettsial and chlamydial infections simultaneous administration of live, enteric-coated adenovirus 4, 7 and 21 vaccines: safety and immunogenicity molecular epidemiology of human adenoviruses defective regulation of immune responses in respiratory syncytial virus infection rhinoviruses in seattle families 1975-1979 the tecumseh study of respiratory disease vi. frequency of the relationship between outbreaks of coronavirus infection world-wide epidemiology of human adenovirus infections influenza seminars in respiratory infections parainfluenza virus bronchiolitis: epidemiology and pathology respiratory syncytial virus epidemiology of respiratory syncytial virus infection in washington d.c. ill composite analyses of eleven consecutive yearly epidemics respiratory syncytial virus: brief review key: cord-260267-nau9kayk authors: ren, lili; gonzalez, richard; xie, zhengde; xiong, zhaohui; liu, chunyan; xiang, zichun; xiao, yan; li, yongjun; zhou, hongli; li, jianguo; yang, qingqing; zhang, jing; chen, lan; wang, wei; vernet, guy; paranhos-baccalà, gláucia; shen, kunling; wang, jianwei title: human parainfluenza virus type 4 infection in chinese children with lower respiratory tract infections: a comparison study date: 2011-06-01 journal: j clin virol doi: 10.1016/j.jcv.2011.05.001 sha: doc_id: 260267 cord_uid: nau9kayk background: human parainfluenza viruses (hpivs) are a leading cause of acute respiratory tract infections (artis). although hpiv-4 has been associated with mild artis for years, recent investigations have also associated hpiv-4 infection with severe respiratory syndromes and with outbreaks of artis in children. objectives: to characterize the role of hpiv-4 and its clinical features in children with acute lower respiratory tract infections (alrtis) in beijing, china. study design: nasopharyngeal aspirates were collected from 2009 hospitalized children with alrtis between march 2007 and april 2010. rt-pcr and pcr analyses were used to identify hpiv types and other known respiratory viruses. results: hpivs were detected in 246 (12.2%) patients, of whom 25 (10.2%) were positive for hpiv-4, 11 (4.5%) for hpiv-2, 51 (20.7%) for hpiv-1, 151 (61.4%) for hpiv-3, and 8 (3.3%) were co-detected with different types of hpivs. like hpiv-3, hpiv-4 was detected in spring, summer, and late fall over the study period. seasonal incidence varied for hpiv-1 and -2. the median patient age was 20 months for hpiv-4 infections and 7–11 months for hpiv-1, -2, and -3 infections, but the clinical manifestations did not differ significantly between hpiv-1, -2, -3, and -4 infections. moreover, co-detection of hpiv-4 (44%) with other respiratory viruses was lower than that of hpiv-1 (62.7%), hpiv-2 (63.6%), and hpiv-3 (72.7%). conclusions: hpiv-4 plays an important role in chinese paediatric alrtis. the epidemiological and clinical characteristics reported here improve our understanding of the pathogenesis associated with hpiv-4. background: human parainfluenza viruses (hpivs) are a leading cause of acute respiratory tract infections (artis). although hpiv-4 has been associated with mild artis for years, recent investigations have also associated hpiv-4 infection with severe respiratory syndromes and with outbreaks of artis in children. objectives: to characterize the role of hpiv-4 and its clinical features in children with acute lower respiratory tract infections (alrtis) in beijing, china. study design: nasopharyngeal aspirates were collected from 2009 hospitalized children with alrtis between march 2007 and april 2010. rt-pcr and pcr analyses were used to identify hpiv types and other known respiratory viruses. results: hpivs were detected in 246 (12.2%) patients, of whom 25 (10.2%) were positive for hpiv-4, 11 (4.5%) for hpiv-2, 51 (20.7%) for hpiv-1, 151 (61.4%) for hpiv-3, and 8 (3.3%) were co-detected with different types of hpivs. like hpiv-3, hpiv-4 was detected in spring, summer, and late fall over the study period. seasonal incidence varied for hpiv-1 and -2. the median patient age was 20 months for hpiv-4 infections and 7-11 months for hpiv-1, -2, and -3 infections, but the clinical manifestations did not differ significantly between hpiv-1, -2, -3, and -4 infections. moreover, co-detection of hpiv-4 (44%) with other respiratory viruses was lower than that of hpiv-1 (62.7%), hpiv-2 (63.6%), and hpiv-3 (72.7%). conclusions: hpiv-4 plays an important role in chinese paediatric alrtis. the epidemiological and clinical characteristics reported here improve our understanding of the pathogenesis associated with hpiv-4. © 2011 elsevier b.v. all rights reserved. human parainfluenza viruses (hpivs) are a leading cause of acute respiratory tract infections (artis). 1-3 hpivs account for 6.8% of hospitalizations due to fever and/or acute respiratory illnesses in children younger than 5 years of age. [1] [2] [3] [4] [5] [6] [7] [8] [9] four types of hpivs, 1 through 4, have been identified. studies of hpivs have focused primarily on hpiv-1 to -3, and detection methods include mainly cell culture and immunofluorescence. as the recovery rate of hpiv-4 in cell culture is inherently low and most clinical laboratories do not screen for hpiv-4, 10,11 the role of hpiv-4 in artis is unclear and may be underestimated. recent studies indicate that hpiv-4 is associated with respiratory infections including bronchitis and pneumonia. [10] [11] [12] [13] [14] however, the prevalence and clinical characteristics of hpiv-4 in chinese paediatric patients with acute lower respiratory tract infections (alrtis) have not been addressed fully. to characterize the role and clinical features of hpiv-4 in children with alrtis in beijing, china. nasopharyngeal aspirates (npa) were collected from children with alrtis, upon admission to beijing children's hospital between march 2007 and april 2010. no repeat samples or lower respiratory tract samples were collected. to exclude typical bacterial infections, physicians enrolled patients with body temperature ≥38 • c, respiratory symptoms and normal or low leukocyte count. similar numbers of samples were collected during each season over the study period, except in august 2009 when only eight samples were collected. nucleic acids were extracted from npa using nuclisens easymag tm (biomérieux, france). nested rt-pcr was used to detect hpivs, as described previously. 3, 5 invariant ␤-actin gene was used as an internal control for efficient extraction and amplification of viral nucleic acids. analytic sensitivity of rt-pcr was 1-10 copies for hpiv-2 and -4 and 10-100 copies for hpiv-1 and -3. to avoid contamination, the pcr process (including master mixture preparation for pcr, nucleic acid extraction, reaction installation and electrophoresis) was performed in different rooms. strict controls were used during the process of nucleic acid extraction and pcr analysis to monitor contamination. all pcr products were verified by sequencing. each specimen was also screened for other respiratory viruses, as described elsewhere. 3,15,16 distribution frequencies of hpivs were compared using pearson's chi square test or fisher's exact test. continuous variables for population parameters such as age, maximum temperature and duration of fever, laboratory investigations and other parameters were compared using one-way analysis of variance. p values <0.05 were statistically significant. a total of 2009 patients (1278 males and 731 females), 0.5 month to 16 years old (mean 2.5 years, median nine months), were enrolled in this study. hpiv rna was detected in 246 (12.2%) patients, from 1 month to 13 years old (mean 1.5 years; median nine months). the prevalence of hpiv was not significantly different between males (13.3%) and females (10.3%). twenty-five (10.2%) patients were positive for hpiv-4, 11 (4.5%) for hpiv-2, 51 (20.7%) for hpiv-1 and 151 (61.4%) for hpiv-3. the detection rate of hpivs varied significantly between age groups (x 2 = 57.680, p < 0.001). compared to hpiv-1 to -3, hpiv-4 was detected at a higher rate in 36-72 months-old patients and at a lower rate in 6-36 months-old patients ( table 1 ). the median age of hpiv-4 cases (20 months) was higher than that of hpiv-1 (11 months), hpiv-2 (10 months), and hpiv-3 cases (7 months). the seasonal distribution of hpivs fluctuated (fig. 1) 2008-2009, with varying seasonal incidence. hpiv-2 appeared sporadically during the study period (fig. 1 ). of the hpiv-positive patients, 63.8% were diagnosed with pneumonia, 15% with bronchopneumonia, 13% with bronchitis and 8.1% with peribronchitis or other conditions. the maximum body temperature was similar for patients with hpiv-4 and patients with other hpiv infections. congenital heart disease was reported in one (4%) patient with hpiv-4, four (3.9%) with hpiv-1, eight (5.3%) with hpiv-3, and in two (25%) co-infected with hpiv-4 and hpiv-1 or hpiv-4 and hpiv-3. the frequency of symptoms did not differ significantly between patients with different types of hpiv. chills, diarrhoea and vomiting were observed most frequently in patients with hpiv-2 ( table 1 ). the percentage of neutrophilic granulocytes, mean lymphocyte count or percentage of lymphocytes was similar between patients infected by different hpiv types. overall, the clinical manifestation of hpiv-4 infection resembles that of other hpiv types. co-infection was detected in 167 (67.9%) of the hpiv-positive cases, including eight (3.3%) co-detected with different types of hpivs and 159 (66.8%) co-detected with other respiratory viruses. co-detection rates of all hpiv types were statistically different ( table 2 ). hpiv-4 had the lowest co-detection rate, which was significantly lower than that of hpiv-3 (x 2 = 7.854, p = 0.005; table 2 ). respiratory syncitial virus (rsv) (65 cases) and rhinovirus (64 cases) were co-detected with hpivs most often ( table 2) . while co-infections of hpivs with rsv occurred mainly in spring and winter, those for rhinovirus occurred year round, corresponding to the seasonality of these viruses. clinical characteristics did not differ significantly between patients with single hpiv infections and those with co-infections. we present the first detailed study of hpiv-4 infections in chinese children with alrtis. in our study, we found that hpiv-3 cases are most prevalent, as previously reported, 2 followed by hpiv-1, then hpiv-4, and hpiv-2 cases. clinical manifestations did not differ significantly between hpiv-1, -2, -3, and -4 infections, but the median age of patients with hpiv-4 was higher than that for patients with any of the other hpiv types. in temperate climates, hpiv-1 and -2 infections occur annually during late fall and early winter, 1,2 whereas hpiv-4 infections occur biennially during late fall and winter, 2, 11, 14, 17 and hpiv-3 infections occur mainly during late spring and summer. 2 the seasonal distributions of hpiv-1, -3, and -4 reported here differ from those of previous reports. these discrepancies may be attributed to different geographical regions and study years. however, as the number of positive cases is limited in our study, large-scale investigation of hpiv incidence over a broader geographical range and longer time period is needed to better understand the seasonal patterns of hpivs. it is particularly interesting that hpiv-4 was more prevalent during spring and summer in 2007 and 2008 than in 2009 and 2010. we speculate that this finding is associated with the duration of protective immunities. if the protective immunity lasts long, the interval of epidemics could be longer. however, this hypothesis requires testing by immunological studies. to investigate the rate and role of co-infection, we screened each specimen for hpivs and other common respiratory viruses. most hpiv-positive patients (159, 66.8%) were co-infected with other viruses, but the co-detection rate was lowest in patients infected with hpiv-4. future investigations may want to consider screening for additional pathogens, such as bacteria, and using samples from different geographic locations to provide insight into the clinical significance of co-infections. overall, our results confirm that hpiv-4 is an important cause of severe symptoms associated with paediatric alrtis, which should be screened for routinely. [13] [14] [15] [16] parainfluenza viruses epidemiological features of parainfluenza virus infections: laboratory surveillance in england and wales, 1975-1997 prevalence of human respiratory viruses in adults with acute respiratory tract infections in beijing parainfluenza virus infection of young children: estimates of the populationbased burden of hospitalization simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays adult croup: a rare but more severe condition a longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion pediatric hospitalizations for croup (laryngotracheobronchitis): biennial increases associated with human parainfluenza virus 1 epidemics epidemiology of respiratory infections in young children: insights from the new vaccine surveillance network human parainfluenza virus 4 outbreak and the role of diagnostic tests clinical and molecular epidemiology of human parainfluenza virus 4 infections in hong kong: subtype 4b as common as subtype 4a infections due to parainfluenza virus type 4 in children parainfluenza virus type 4 infections in pediatric patients human parainfluenza type 4 infections wu and ki polyomavirus present in the respiratory tract of children, but not in immunocompetent adults human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-pcr we thank the clinicians of beijing children's hospital for assisting with sample collections. this study was supported by grants from the national major the authors report no conflicts of interest. key: cord-300314-fbppvt75 authors: bikov, andras; boots, agnes; bjerg, anders; jacinto, tiago; olland, anne; skoczyński, szymon title: 13th ers lung science conference. the most important take home messages: news from the underground date: 2015-06-17 journal: breathe (sheff) doi: 10.1183/20734735.04015 sha: doc_id: 300314 cord_uid: fbppvt75 the 13th ers lung science conference (lsc) was organised to bring academics together from all over the world to present and discuss the latest developments regarding lung infection and immunity. the conference took place in breathtaking estoril, portugal; however, it wasn’t the beautiful surroundings that were our main motivation to attend, but instead the scientific merit of the conference and the chance to create new scientific collaborations. the scientific programme [1] was packed with the most up-to-date content in the field of lung infection and immunity and included some of the top researchers within this exciting area. moreover, the convenient size of the lsc offered the opportunity to renew and intensify friendships and collaborations. in particular, for researchers at the start of their career, this is a great feature and we therefore warmly recommend the lsc to ers juniors members! the 13th ers lung science conference (lsc) was organised to bring academics together from all over the world to present and discuss the latest developments regarding lung infection and immunity. the conference took place in breathtaking estoril, portugal; however, it wasn't the beautiful surroundings that were our main motivation to attend, but instead the scientific merit of the conference and the chance to create new scientific collaborations. the scientific programme [1] was packed with the most up-to-date content in the field of lung infection and immunity and included some of the top researchers within this exciting area. moreover, the convenient size of the lsc offered the opportunity to renew and intensify friendships and collaborations. in particular, for researchers at the start of their career, this is a great feature and we therefore warmly recommend the lsc to ers juniors members! during the first session, the complex mechanisms of immunity and host defense were discussed. the role of dendritic cells in sensing viral infections was explained as well as the three recognition pathways these cells use to sense pathogen components: toll like receptors (tlrs), dexd/h-box helicases and c-type lectin [2, 3] . interestingly, one very specific c-type lectin, i.e. dngr-1, does not directly sense pathogens but instead recognises f-actin exposed by necrotic cells [4] . understanding the receptors and signalling pathways regulating dendritic cell activation opens a wide area of research regarding the regulation of the immune system with respect to other causes of cell necrosis, such as immunotherapy of cancer or treatment of idiopathic pulmonary fibrosis (ipf). indeed, although little is known about the exact triggers initiating ipf, it is now recognised that infectious exacerbations are a determining factor in the progression and worsening of this disease [5] and that bacterial load can be regarded as an independent predictor of disease progression. an increase in the ipf-lung bacterial load might be due to several potentially pathogenic bacteria and might be linked to polymorphisms in the gene muc5b and to abnormalties in macrophage function [6] . it was concluded that immunity is not only a defense mechanism but can also become a therapeutic target that can be either upregulated to fight diseases such as cancer or downregulate to combat auto-activating diseases. another research area emphasised the importance of host microbiome interactions in future development of chronic lung diseases. germ-free mice models were presented that highlighted the clinical importance of early-life multi-bacterial stimuli in developing pulmonary immunity. in these models, a variety of cellular mediators including dendritic cells and t lymphocytes play a role in decreasing the future likelihood of developing atopic asthma, and similar close interactions between host and microbiome are present in humans [7] . another important aspect in terms of future possible respiratory pathogen prophylaxis, was the elucidation of bacterial sensing therein. recognition of living bacteria, as opposed to dead ones, through so-called viability-associated pamps (vita-pamps) induces a stronger immune response via a t1 helper cell response cascade [8] . the last presentation of the session highlighted the evidence for increased de novo hyaluronan synthesis in a murine influenza infection model. it was shown that by reducing hyaluronan content, the disease severity and time to recovery were decreased, thereby giving hope for future specific treatment targets [9] . the session devoted to the inflammasome highlighted not only the important differences between the four known bona fide inflammasomes but also the role of the epithelium inflammasome in fighting infections induced by intra-cellular pathogens [10, 11] . the four well-described human inflammasomes comprise nlrp1, nlrp3, nlrc4 and aim2 that share their dependence on caspase-1 activation for cytokine production and that are all so-called pattern-recognition receptors capable of sensing molecules excreted by or indicating the presence of invading pathogens [12, 13] . nlrc4 combats bacterial pathogens by recognising their flagellin and rod proteins in a tlr5-independent manner whereas nlrp3 offers protection against intracellular pathogens by sensing bacterial toxins, microbial rna or extracellular atp [14] . examples of such intracellular pathogens, inducing respectively inflammation or infection, are porphyromonas gingivalis and chlamydia pneumoniae. indeed, infecting cells or mice with these pathogens elucidates the importance of a functional epithelium inflammasome in combatting both inflammation and infection [15, 16] . interestingly, all the discussed pulmonary and intestinal models describe a rather general signalling pathway for the activation of both nlrp3 and nlrp 4, beginning with a potassium efflux and resulting in a caspase-1-induced interleukin-1β production needed to recruit anti-inflammatory chemokines and neutrophils [12, 14] . although the exact mechanism underlying the activation of the inflammasome by the observed potassium efflux is not yet known, it has been suggested that the production of reactive oxygen species might also be involved [17] . the early evening session on friday featured talks by mike catchpole from the european center for disease prevention and control (ecdc; stockholm, sweden) and virologist bart haagmans (rotterdam, the netherlands) on recent infectious outbreaks. according to dr catchpole, the ever-increasing urbanisation, multinational and trans-continental travel and trade together with an aging population puts the world of today at greater danger of pandemics than ever before. less (in)famous than sars and ebola, the recent spread of the west nile virus into southeastern europe was highlighted as an effect of climate change, which favour its mosquito vector [18] . in the meantime, advances in technology and knowledge present promising countermeasures. the genomes of pathogens can today be rapidly sequenced for the development of treatments and vaccines, but still limiting the spread of disease by exit screening of travellers remains a fundamental method. it as a means of disease surveillance has yielded mixed results [19, 20] , and detection of new outbreaks to date relies on the vigilance of local clinicians. dr haagmans' talk exemplified this. the meticulous testing and sequencing of the first case in saudi arabia of a novel coronavirus infection that had high morbidity and mortality ultimately led to several additional, previously unrecognised cases being identified. the similarity with the dreaded sars outbreak led to the idea of an animal virus reservoir, and dr haagmans told the fascinating story of how the disease was ultimately traced back to racing dromedaries [21] . the alertness of the audience despite the intense day demonstrated the strength of such sessions, where clinical and epidemiological perspectives combine to fertilise mechanistic research. novel data on lung infections, particularly on the involvement of pulmonary macrophages herein, were presented during the saturday morning session. these cells have pluripotent functions in host defense and scott buddinger added new data about their role in pulmonary fibrosis [22] . the migration of bone marrow-derived macrophages to lung tissue is increased in lung fibrosis and regulated by caspase 8. subsequently, the balance between bone marrow-derived and tissueresident macrophages is skewed toward the former fraction. christin becker presented a novel pathway which is involved in influenza virus-induced epithelial dysfunction. influenza-infected macrophages start producing interferon and tumour necrosis factor-related apoptosis-inducing ligand (trail) which decrease the expression of epithelial na-k atpase. viruses also lead to delayed activation of airway macrophages as shown by tracy hussell. interestingly, airway macrophages of copd patients respond slower to infectious agents compared with similar cells from healthy individuals. this delayed response is also augmented by hyaluronic acid accumulation in the airways. in summary, all three presentations highlighted potential new therapeutic targets for drug development. the young investigator session on saturday featured the top five communications competing for the william macnee award, named after the distinguished professor and former ers president. the importance and impact of the lsc was once again shown, given the high scientific quality of these five presentations. the work presented included the demonstration of, in a rhinovirus infection scenario, the requirement of cell contact between neutrophils and monocytes for preformed mediators to be released and reduce cytokine mrna and protein levels moreover, it was shown that bacterial burden was not increased following an experimental rhinovirus infection in either healthy or asthmatics individuals. also, a secretory leukocyte protease inhibitor variant (slpi-a16g) was proposed as a promising tool for anti-inflammatory treatment in lungs with cystic fibrosis. the dysregulation of regulatory t cells function by camp-responsive element binding protein and upstream micrornas was also a topic of this session, as it was demonstrated that this might contribute to chronic inflammatory diseases. the final communication, and also the winner of the award, was presented by rena brauer from the cedars-sinai medical center (los angeles, ca, usa). she concluded that syndecan-1, a cell surface heparin proteoglycan with multiple roles in healthy and pathological conditions, is cytoprotective to the cells of the lung epithelium and attenuates lung injury during influenza virus infection. congratulations to all the presenting authors and all the research teams that participated in this great session! one of the most important and successful sessions was the ers junior members and fellows networking session co-organised by our colleagues anne olland and agnes boots. during this session, presentations regarding different career opportunities within the field of pulmonary research and clinical care were given by maria belvisi and lynne murray. additionally, liz elvidge talked about the possibilities and pitfalls when preparing your resume and job interview. the session was concluded with a discussion with the audience during which a lot of attending juniors used the chance provided to ask advice regarding their career paths. since this mixture of expertise and young passion is off great importance to the ers and jmc, it has already been decided that a similar initiative will take place during the next lsc. during the sunday morning session we heard three very exciting presentations on alveolar macrophage and neutrophil function in the lungs. venizelos papayannopoulos showed data that neutrophils, in addition to phagocytosis and antibacterial granulae, are also capable of releasing webbing structures called neutrophil extracellular traps (nets). in addition, neutrophils seem to implement different strategies selectively depending on the microbe size, e.g. they react differently to fungi in their hyphae or spore forms [23] . ricardo jose highlighted in his presentation the role of protease-activated receptor 1 (par1) in streptococcus pneumonia induced infection. par1 was shown to lead to an exaggerated inflammatory response, involving cytokines such as il-1β, cxcl1, ccl2 and ccl7. its antagonist, a molecule called sch530348, significantly reduced neutrophil recruitment without affecting macrophage number. finally, jahar bhattacharya presented results on macrophage-epithelial communication, recently published in nature [24] . it was shown that sessile alveolar macrophages form gap junctions with alveolar epithelium via connexin 43. knocking out this junction leads to increased neutrophil influx, suggesting a regulatory role in host defense. particularly, hours after bacterium-induced neutrophil migration, the connection between alveolar macrophage and epithelial cells is essential to suppress neutrophil involvement. to conclude, we are convinced that the lsc offers an excellent opportunity to boost young respiratory researchers' scientific careers and to provide all means to establish interesting networks and friendships for life. therefore, we will attend the next lsc and we hope to see even more juniors there in future years! antiviral immunity via rig-i-mediated recognition of rna bearing 5′-diphosphates syk signaling in dendritic cells orchestrates innate resistance to systemic fungal infection f-actin is an evolutionarily conserved damage-associated molecular pattern recognized by dngr-1, a receptor for dead cells the role of bacteria in the pathogenesis and progression of idiopathic pulmonary fibrosis genome-wide association study identifies multiple susceptibility loci for pulmonary fibrosis host-microbe interactions in lung disease detection of prokaryotic mrna signifies microbial viability and promotes immunity accumulation of hyaluronan in the lumen of the airways enhances the pathology of respiratory viral infection sensing and reacting to microbes through the inflammasomes inflammasomes bridge signaling between pathogen identification and the immune response nod-like receptors in lung diseases the naip/nlrc4 inflammasomes caspase-1 dependent il-1β secretion is critical for host defense in a mouse model of chlamydia pneumoniae lung infection atp-dependent activation of an inflammasome in primary gingival epithelial cells infected by porhyromonas gingivalis reactive oxygen species at the crossroads of inflammasome and inflammation comparison of five influenza surveillance systems during the 2009 pandemic and their association with media attention big data. the parable of google flu: traps in big data analysis middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation west african ebola epidemic after one year -slowing but not yet under control alveolar macrophages: plasticity in a tissue-specific context neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity key: cord-284386-emh9feb3 authors: chatterjee, saptarshi; sarkar, apurba; chatterjee, swarnajit; karmakar, mintu; paul, raja title: studying the progress of covid-19 outbreak in india using sird model date: 2020-06-23 journal: indian j phys proc indian assoc cultiv sci (2004) doi: 10.1007/s12648-020-01766-8 sha: doc_id: 284386 cord_uid: emh9feb3 we explore a standard epidemiological model, known as the sird model, to study the covid-19 infection in india, and a few other countries around the world. we use (a) the stable cumulative infection of various countries and (b) the number of infection versus the tests carried out to evaluate the model. the time-dependent infection rate is set in the model to obtain the best fit with the available data. the model is simulated aiming to project the probable features of the infection in india, various indian states, and other countries. india imposed an early lockdown to contain the infection that can be treated by its healthcare system. we find that with the current infection rate and containment measures, the total active infection in india would be maximum at the end of june or beginning of july 2020. with proper containment measures in the infected zones and social distancing, the infection is expected to fall considerably from august. if the containment measures are relaxed before the arrival of the peak infection, more people from the susceptible population will fall sick as the infection is expected to see a threefold rise at the peak. if the relaxation is given a month after the peak infection, a second peak with a moderate infection will follow. however, a gradual relaxation of the lockdown started well ahead of the peak infection, leads to a nearly twofold increase of the peak infection with no second peak. the model is further extended to incorporate the infection arising from the population showing no symptoms. the preliminary finding suggests that random testing needs to be carried out within the asymptomatic population to contain the spread of the disease. our model provides a semi-quantitative overview of the progression of covid-19 in india, with model projections reasonably replicating the current progress. the projection of the model is highly sensitive to the choice of the parameters and the available data. electronic supplementary material: the online version of this article (10.1007/s12648-020-01766-8) contains supplementary material, which is available to authorized users. in the post-ww-2 era, the world probably has not witnessed such catastrophic morbidity and the looming threat of severe economic challenges caused by the worldwide outbreak of the disease covid-19 caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the detection of the disease in the human host was first reported in wuhan, china, on december 31, 2019 as a cluster of cases of pneumonia. as the highly contagious disease transmitted rapidly all over the globe, the outbreak was declared as a pandemic by the who on march 11, 2020. tackling the outspread of the disease is found to be very challenging across the world for the following reasons: (a) conventional flu-like symptoms in human carriers and (b) human-to-human transmission via asymptomatic human hosts and (c) the absence of a proper clinical doctrine (e.g., vaccines, drugs, concrete ideas about the immunological response, etc.). extensive testing and the imposition of containment measures to maintain social distancing turn out to be the effective remedies to prevent disease transmission at the current stage of the epidemic at several places. to evaluate the impact of these preventive measures on infection spread, recovery, death tolls, and various other associated factors, mathematical models become useful in predicting realistic, quantitative estimates. a preliminary analysis suggests that the classic mean-field susceptible-infected-recovery-death (sird) model by kermack and mckendrick [1, 2] , can be used to obtain a quantitative picture of the epidemic [3] [4] [5] [6] [7] [8] . in this article, implementing the sird model, we report the temporal progress of covid-19 transmission in india, various indian states and compare it with some other countries around the world. a similar model used by fernandez-villaverde et al [5] provides a detailed overview of the pandemic situation in the usa and many other countries. india implemented a nation-wide lockdown from march 25, 2020. on the day of the announcement of nation-wide lockdown, india had about 657 corona positive cases, while the first covid-19 positive was detected on january 30, 2020. the socioeconomic constraints in the indian context alludes that: (a) 'too-prolonged' lockdown is difficult to sustain; (b) the sole imposition of containment measures without a manifold increase in testing capacity is a futile endeavor; (c) if the implementation of the lockdown measures is lenient, containment of the spread is highly improbable. henceforth, the feasible solution for limiting the spread lies in carefully balancing various key epidemiological factors. that is where the importance of the current model predictions becomes relevant. this study further highlights the effect of lockdown on the disease spread and predictions about the variability in the infection peak upon the severity of the containment measures (and/or the lack of it). the model predicts that, in india, the height of the peak infection decreases with stricter lockdown, but at the cost of 'time' (position of the peak shifts to a later month). thus, with a large susceptible, the infection will stay for a long time if existing infections are not quarantined immediately or no proper medicine/vaccine is employed. the key is to quarantine the infection in small pockets while in lockdown and prevent inter-pocket transmission. the model further underlines that in the highly contagious zones ('red' zones where covid-19 positive cases continue to grow), if the lockdown is extended and enforced with proper quarantine measures, the new infections will gradually plummet down flattening the covid-19 curve at a much faster rate. our study also explores the plausibility of universality in the spread of the covid-19 outbreak amongst different countries [6] and compares the situation in india with few other countries (e.g., germany, south korea, usa, spain) in the relevant time window (february-april). due to the simplicity of the sird model, we found it difficult to fit the observed patterns of the pandemic using the available data. the real data for analysis in india's context is collected from the repository with an interactive interface hosted at https://www.covid19india.org. the data for other countries are taken from the repository with an interactive interface hosted at https://www.worldometers.info/coronavirus. the purpose of this article is not to make any quantitative prediction that should be used to design policies, but for the research purpose only. we employ the standard sird model where the population n is divided into sub-population of susceptible (s), infected (i), recovered (r) and dead (d) for all times t. thus, the following set of mean-field differential equations governs the temporal dynamics of the population of susceptible (s), infected (i), recovered (r), dead (d) and describes a comprehensive picture of the sird epidemic evolution: drðtþ dt ¼ ci ð3þ here, b, c, d are the parameters determining the characteristics of infection, recovery and deaths respectively (fig. 1a ). note that, in the current scenario i represents the population of symptomatic infection. when a susceptible person interacts with an infectious person, the susceptible become infected at a rate bsi=n. large variability is observed in the rate c that an infected individual is no longer infectious or equivalently has recovered in this simplified model. literature [9] [10] [11] suggests that, on the average, infectiousness appears to start from 2 to 3 days before the symptoms are visible. the infectiousness increases to its peak before the arrival of the symptoms and remains for about 7-9 days after the peak infection. thus, an infected individual remains infectious for about 12 days on the average and then recover. in our preliminary analysis, we set the recovery rate c $ 1=12, which however does not give the best fit for all the cases we studied. in essence, the numerical values of the model parameters are obtained from the best fit. initial values (time t ¼ 0 days) of the number of infected, recovered and deaths ði 0 ; r 0 ; d 0 þ, are chosen from real data. the choice for the initial number of susceptible (s 0 ) is quite difficult. in the absence of antibody, the entire population can be susceptible to the covid-19 pandemic. nevertheless, the geographical, social, and economic characteristics of a region (and various other demographic factors) can substantially influence this number. we used two different approaches to get an estimate of s 0 . first, we study the data for the large countries where the cumulative positive cases have reached closer to a plateau. though, the infected population at the plateau can be determined only when the epidemic is over. dividing this number by the total population of the country gives a fraction that appears to be of the order for 10 à3 for germany, usa, spain, italy, and 10 à4 and 10 à5 for south korea and china, respectively. thus, an estimate of the susceptible may be obtained by multiplying the population of a country by this fraction. the number of susceptible obtained in this way, however, indicates a lower bound as many individuals with mild or no symptoms go unreported. another possibility to estimate the fraction would be to test the number of positive cases by the number of tests carried out. this number would be an upper bound since there are many regions within a country that remains completely isolated and the populations in such pockets would not be susceptible. the ratio between the number of positive cases and the total number of tests for different countries are given in the following; the fraction is 0.159 for the usa, 0.016 for south korea (as per data up to may 7), 0.1072 for spain, 0.063 for germany (as per data up to may 10). conventionally, in epidemiological modeling s 0 $ n. in our simulation, we have reasonably varied s 0 within this range to obtain the best fit with real data in a case by case manner (i.e., for india, few indian states and other countries). with the formulation of the model, comes the quantitative estimate of the speed at which the disease spreads across a population. in other words, from the deterministic sird model, the objective is to assess how fast a human carrier would infect people belonging to the population of susceptible. the quantity that determines the transmission speed of the pandemic is the effective reproduction number or replacement number (r e ) [12] . often the basic reproduction number r 0 , defined as the average number of secondary infections that occur when an infectious person (primary or source of infection) is placed into a susceptible population, is used in the epidemiological models. r 0 can be estimated from the very early stage of the infection when the infectious person mixes freely with the susceptible population. estimating r 0 is often challenging due to lack of unbiased data as all secondary infections cannot be determined exactly; especially for covid-19, where asymptomatic cases are hardly identified (fig. 1b) . the effective reproduction number (r e ), which we used in this study, evaluates the mean number of new infections (infected from the susceptible pool) directly transmitted/induced by a typical infected person and can vary over the entire duration of the infection (fig. 1c) . in the sird model, r e can be represented as b=ðc þ dþ. from the best fit of the data, we find that c [ [ d, yielding r e $ b=c. if r e [ 1, the disease starts spreading in a population infecting more and more people, but spreading does not occur if r e falls below 1. it is easy to notice that longer a person remains infectious (i.e. 1=c days), can give rise to very large r e even if the number of infectious interactions per day (i.e., b) is small. containment measures in terms of social distancing and lockdown have been implemented world-wide to mitigate the transmission speed of the outbreak. we implemented the effect of lockdown in the model by modifying the infection rate and obtained the best-fit. we chose the following functional form of time-dependent infection ratẽ bðtþ where it gradually decreases after the containment measures are enforced [5, 6] . before lockdown, the infection rate is b which is constant. when the lockdown is imposed on day s (counted from the initial time point t ¼ 0 or day 0 as chosen in the simulation), the time-dependent infection ratebðtþ diminishes with every progressing day which is assumed to vary exponentially in the following manner [6] : here, f 2 ½0; 1 is the infection parameter (or interaction parameter) and t is the delay in the number of days before the effect of lockdown is visible in the propagation of infection. without lockdown f ¼ 1, referring to rapid infection while f ¼ 0 means that infection is contained (e.g., no interaction between infected and susceptible population, hence no transmission). f 2 ½0; 1 reflects the asymptotic mitigation of the infection ratebðtþ, when containment measures are imposed. lower the value of f, stricter is the containment measures (or the manifestation of the same) [5, 6] . here,bðtþ has an initial value b which is constant.bðtþ diminishes over time and reaches a value fb as containment measures continue. essentially, the initial value of b determines the characteristic properties of the disease which depends on the effective interaction of people in a region, social behavior, density of population, etc. the terminal value fb reflects the effect of the containment and how the social distancing is being maintained. in the current model setup,bðtþ is meant to account for the changes in the behavior of infection spread due to social distancing and containment measures. this is an external parameter that is expected to decay with time to a smaller value fb when physical contact is avoided. taking a cue from the previous studies [5, 6] , the functional form ofbðtþ is constructed to account for the apparent changes on the infection growth due to containment measures and social distancing. t controls the effective speed at which the slow down in the disease transmission occurs due to the enforced containment measures. the model simulation, data analysis, and plotting are carried out in python. the analysis of the covid-19 data, using the deterministic compartmental sird model, sheds light on the primary characteristics of the temporal evolution of the pandemic. relevant parameter values chosen for the india and few indian states are listed in table s1 -s2. the best-fit parameters chosen for foreign countries are listed in table s3 . we carried out the sird model analysis on covid-19 progression in india's context (and few other countries) with realistic variations in following parameters: rates of infection (b), recovery (c) and deaths (d), the initial number of susceptible (s 0 ) and the effective reproduction number (r e ). detailed results are described in the following and illustrated in figs. 2, 3, 4, 5, 6, 7, 8, 9 and 10, fig. s1 -s4. in a nutshell, we start with the initial susceptible population (s 0 ) varied within the range $ 1-3 million, keeping the effective reproduction number r e fixed at $ 4.0, and show how the model prediction fits with the indian data without a lockdown, the location of the infection peak and the relative deviation from the real data (fig. 2a) . the best fit is obtained by tuning the rates of infection (b), recovery (c), and deaths (d) keeping s 0 constrained in the mentioned range. then, we incorporate the effect of containmentmeasures/lockdown in the functional form of time-depen-dentbðtþ and show how the effect of the containmentmeasures has altered the location and the height of the infection peak (fig. 2b) . next, we explore how the variability in the effective reproduction number r e influences the infection peak (figs. 3, 4) . furthermore, we analyze the covid-19 progression in few indian states e.g., kerala, maharashtra, delhi, and west bengal (figs the first covid-19 positive human host was reported in india on january 30, 2020. the exponential growth of the number of infections, from 30th january onward, reached a number 657 on march 25, 2020, the day on which india imposed a nation-wide lockdown (fig. s1a ). using the sird model, we first explored what could have happened, if the containment measures had not been undertaken. as mentioned earlier, we chose the factor $ 10 à3 (obtained in case of germany and few other countries by dividing the cumulative population at infection peak by the actual population of the country) and multiplied it with the indian population of $ 10 9 to estimate the lower bound of the susceptible population (s 0 ). it turns out that it would be a 'good' estimation to have a 'working' s 0 in the range $ 10 6 . with susceptible population s 0 varied in the range $ 1-3 million (for fixed r e $ 4:0), the peak of the infection occurs in the first half of may (fig. 2a) . as expected, the peak height (infected population at the peak) increases with increasing s 0 . for an initial susceptible pool of s 0 $ 10 6 , the peak reaches a height of 0.4 million, whereas the peak jumps to $ 1.2 million for s 0 $ 3 million (fig. 2a) . the total death toll is estimated to reach about 30,000-100,000 for s 0 in range $ 1-3 million, during july-august, 2020 (fig. 2a) . next, we introduced the effect of containment-measures in the infection ratebðtþ (eqs. 5-6). numerical analysis is carried out to investigate whether the progression of the outbreak is mitigated after the lockdown is imposed. fig. 4 best fit of the infection and death curves with the real data freely varying the effective r e which appears to be $ 7.4 in the early stage. the color shades enveloping the curves denote variation in susceptible population within a range of $ 0.8-1:2 â 10 6 . the real data considered for fitting are from march 2, 2020. relevant parameters for the analysis are b ¼ 0:257 day à1 , c ¼ 0:0315 day à1 , d ¼ 0:0032 day à1 , s ¼ 29 days, t ¼ 7 days, f ¼ 0:375 3.2. effect of containment measures: how well is india doing? from the real data, it appears that the infection rate begins to reduce, 15-20 days after the national lockdown is implemented (fig. 2a, inset) . we further observed that the growth curve for the infected population displays a straightening feature during the lockdown time frame. this is expected to be observed if containment measures are initiated; the unhindered exponential growth before the lockdown slows down due to the effect of containment measures during the lockdown. while slowing down and deviating from the exponential trajectory, the infection growth curve (time progression of the infected population size) acquires a distinctive straightening feature until the very recent surge (fig. 2a , inset). next, adding the lockdown effects into the picture, we fit the theoretically obtained infection growth curve with the real data. the best fit with the current set of parameters demonstrates that, due to the effect of the present lockdown, the infection peak dwarfs down to about 0.10 million from about 0.40 million in 'without lockdown' scenario (dashed curve, fig. 2b and inset). the infection time evolution of the population of infected and dead for maharashtra, (c) if lockdown was not imposed, (d) due to effect of lockdown peak is projected to reach a peak at the end of june, tentatively (fig. 2b, 2b , inset). the estimated death toll also reduces substantially compared to the earlier scenario without containment measures. however, the model also shows that the situation can be improved further. the infection growth curve can be dwarfed down further if the lockdown is extended and reinforced stringently in covid-19 prone zones. in that case, the infection growth curve noticeably flattens with the infection peak reduced further. as mentioned earlier, s 0 is a very crucial parameter in governing the position and the height of the infection peak. in the following, we summarize how the variations in the size of the susceptible population s 0 influence the infection growth curve. keeping r e fixed at $ 4.0, we varied the size of the total susceptible population within a range of 1-3 million (fig. 2a, b) . the model analysis shows that the larger the size of the susceptible population, the higher the infection peak (fig. 2) . moreover, for the larger size of the susceptible population, attainment of the infection peak is delayed with the infection peak shifted to a later time zone (fig. 2b, 2b, inset) . these characteristic features are consistent in both without and with lockdown scenarios. thus, it is evident that the key to containing the outspread lies in keeping s 0 small. this is feasible only when interactions between a demographic region with the recent occurrence of infections and a region with no 'latest' the next question that crops up is what happens to the magnitude of effective reproduction number (r e ) when containment measures are put in place. we discuss in the following, how the effective r e changes with time during the lockdown (fig. 3 ). we start with r e in range 3:5 r e 4:4 in the beginning. as the lockdown is implemented, less number of people interact. therefore, the effective infection ratebðtþ starts decreasing over time. how much the reduction would be forbðtþ in longer time regime, is determined by the factor f in eq. (5). the reducedbðtþ settles at a value fb due to the containment effects. thus, if the recovery rate c is fixed, the r e will diminish and reach a valueb ðtþ c $ fb c . the decrease in r e due to the effect of lockdown is evident in fig. 3 where the effective r e reduces to $ 40% of its initial value (before lockdown). the smeared color shades enveloping the dashed lines label the variations in r e within the mentioned range. as expected, the higher the value of r e , the taller the infection peak. this feature is consistent both in presence and absence of lockdown (fig. 3, inset) . next, we investigate, whether the value of r e , extracted from the best fit with real data, is unique (of course with marginal variation) or the variation is non-marginal. instead of fixing r e in the beginning, we varied the rates of infection (b), recovery (c), and deaths (d) without any restriction on the resulting value of r e . we aimed to verify whether the best fit of real data with the theoretical curves (infection, recovery, and death) can be obtained for a set of (b; c; d), other than the already chosen values in figs. 2 and 3, with no apparent constraint on the values of r e . we find that, for a fixed size of the susceptible population of about 0.8-1.2 million, the real data can still be fitted with the theoretical curves, even if the r e is large (r e $ 7:4, fig. 4 ). similar to figs. 2 and 3, the active infection cases deviate from the theoretical infection curve without lockdown, as the enforced lockdown effectively slows down the progression of infection (fig. 4, inset) . note here, that in comparison with figs. 2 and 3, the location and height of the infection peaks change (both in the cases of without lockdown and with lockdown), as effective r e is increased $ twofold (fig. 4) . consistent with the definition of r e , we observe that greater the value of r e , larger the size of the infected population (compare fig. 2b , r e $ 4:0; and fig. 4 , r e $ 7:4). it is also noteworthy to mention that here the recovery rate c = 0.0315 day à1 corresponds to about $ 31 days compared to 12 days as discussed earlier. prolonged infectiousness leads to the rise in the r e and consequently the total number of infected people. from the above observations, we connote that the exactness of r e can be ascertained, when we have more data points in the time evolution of the infected, recovered, and dead population. the current model setup may not be able to precisely pinpoint the exact 'real' r e . in india, the first covid-19 positive case was reported in indian state kerala on january 30, 2020, and now almost half of the active covid-19 positive cases are from another indian state maharashtra. in this note, we explore the covid-19 progression in these indian states along with delhi and west bengal and compare the features of pandemic progression with each other (figs. 5, 6, s1b). after the first case being detected in kerala on january 30, the second and third cases were reported on february 2-3. after february 3, there was no new case detected in kerala till march 7. the previous three cases were all recovered within february 20. the 'second-wave' of infections started from march 8. from march 8 onwards, there was a rapid upsurge of infections. however, about 2 weeks after the national lockdown is imposed, kerala reached its infection peak. it is evident from fig. 5a , b that the downfall of the infection is rapid, as the infection curve moved past its peak. if the lockdown was not enforced, the infection was projected to occur around mid-may. but, fig. 5b alludes that kerala implemented the containment measures so well that the infection peak occurred early at a much lower height (fig. 5a, b) . the model analysis further projects that due to the effect of lockdown, r e reduces to $ 18% of its initial value during the upsurge of infections before lockdown. the reduced value of r e is \ 1, which means kerala is on the way to become a covid-19 free state soon if the trend continues. in maharashtra, the first case was detected on march 9. the total infected population is yet to attain its peak. the projected infection peak would occur around the end of may or early june if the present trend continues and containment measures remain enforced in places (fig. 5c, d) . similarly, in delhi and west bengal, the infection growth curves are yet to attain their respective peaks (fig. 6a-d) . the first cases in these states were reported on march 2 and march 17, respectively. the peaks are projected to be reached at the end of june and mid-july for delhi and west bengal respectively, if the enforced lockdown remains deployed and the current trend continues (fig. 6b, d) . it is important to note that, in indian states, maharashtra, delhi and west bengal, the estimated r e plummets down to value [ 1:0, even after staying months under lockdown. among the indian states we analyzed, kerala turns out to be the only exception where the effective r e reduces to a value \1, meaning that further 'out-of-bound' spreading is unlikely to occur there if the current trend is followed. it is evident from fig. 7a -d and s1a that both germany and south korea have moved past the infection peak. the infected population is decreasing day by day in those countries. the best fit with real data is obtained for the initial r e $ 3:0 and $ 4.5 for germany and south korea, respectively. however, as the containment measures were undertaken in those countries, the effective transmission (or r e ) reduced to $ 20-25% from the initial values for the respective countries (fig. 7b, d) . this observation, suggests that the counter-measures to fight the pandemic (e.g., containment measures, social distancing, quarantining, testing, etc.), undertaken in these countries, were reasonably successful in repressing the outspread. moreover, the reduction of r e to 20-25 % of its initial values, rescales the r e for the respective countries to a value \ 1 which alludes that new infections are declining and any more 'out-of-bound' infection growth is unlikely to occur if the current trend is followed. we analyzed covid-19 progression data for two more countries: usa and spain (fig. 8a-d) . the usa is approaching the infection peak and will reach its peak shortly if the current trend continues (fig. 8b) . however, contrary to the usa, spain has already passed the infection peak and the infected population is decreasing gradually (fig. 8d) . spain imposed a nation-wide lockdown on march 14. model analysis (fitting parameter optimization) suggests that, due to the effect of lockdown, r e for spain reduced to 25 % of its initial value. but in the usa, the reduction in r e is only $ 42% implying that the implementation of local containment was not that stringent. fig. 10 representative schematic illustrating the aftermath of slowly relaxing (phasing out) of the local containment measures vs rapid lifting of the same. the boxes bordered in black depict the containment zones. rapid lifting of the local containment paves the way for inter-mingling between regions with no positive cases (green) and regions with positive cases found lately (red). this, in turn, results in rapid transmission of the disease across zones (all zones becoming red), rendering the purpose of preceding lockdown futile. on the contrary, initially, the partial lifting of local containment only in the green zones bars the import of transmissions from red zones. when a red zone becomes green, the local containment can be lifted from that region. due to the effect of this gradual lifting, the red zones diminish over time, with 'greens' taking over the 'reds' (color figure online) contrary to the infection curves for germany, south korea, spain (figs. 7, 8) , india is yet to reach the infection peak (the usa is about to reach the peak.). the following remarks briefly summarize where india stands compared to these countries. 1. the effective containment during the present lockdown in india indicates that the infected population might reach its peak at the end of june (fig. 2) whereas germany, south korea, and spain have already moved past the peak and daily new infections are decreasing (figs. 7, 8 ). 2. since india has a large population, the infection is expected to stay for a longer duration. germany, south korea, and spain might have the advantage of a smaller population of the susceptible. we allude that the higher the actual population of a country, the higher would be the effective size of the susceptible pool for that country while making the previous statement. the key is to contain the infections in small zones and prevent transmission between infectious and non-infectious zones. 3. the growth of the infected population in germany, south korea, and spain were greater than that experienced in india which gave india an additional advantage of 'buying precious time'. slow growth rate alludes to a smaller peak value at the zenith of the infection. however, as mentioned earlier, the height of the peak is subjected to the effective size of the initial susceptible pool (s 0 ). for better clarity and wider accessibility to general readers, we discuss and summarize the important observations from our study in q&a format in the following: to investigate the universality in the covid-19 outspread across different countries, we looked into iterative time lag maps for the cumulative confirmed infected (c = i?r?d), recovered (r), and dead (d) population [6] . using the iterative maps, we try to extract the correlation between a population on the day n and day n þ 1. from the recurrence plots (population count on n th day vs population count on ðn þ 1þ th day) in fig. s2 , we observe that the real data for all the cases follow the same power law of the following kind: f ðxþ ¼ ax b . the factor and exponent a and b are similar for all the countries considered in the plots. this finding indicates that there exists an underlying universality in the outspread of the pandemic across various countries. to check the predictiveness of the model, we compared the projected outcome with the real data accumulated over the last couple of weeks since our initial submission of the manuscript. we plotted the data and the model prediction in fig. s3a -s3b (similar to figs. 2b and 9d respectively) without altering the parameters. one can notice that, so far, the real data is bounded by the model predicted curves. nevertheless, the sird model is a drastically simplified approach to thoroughly understand the dynamics of covid-19 progression. from the available information, it is now becoming apparent that a susceptible person goes through a latent period of 2-3 days after coming in contact with an infected individual. subsequently, the person remains infectious for several days ( $ 5 days). the infectious individual may or may not develop symptoms. the current model does not incorporate any of these details and hence fitting is imperfect. moreover, data used to fit with the model also vary between different locations leading to uncertain predictions. a compartmental model with multiple species may be useful to study the dynamics of the sub-population [13] . the model used here to analyze the covid-19 progression is the well-known sird model. this is a standard epidemiological model with three characteristic parameters for infection, recovery, and death. the model estimates the number of infections within a closed (conserved) population of susceptible bearing the risk of contagion. note that this is 'not' a covid-19 specific model by construction. for years, people have used this model to study several outbreaks all across the globe [14] . in our study, the word 'covid-19' enters into the picture only through the best fitting of the 'real data', that determines the instantaneous rates. the biological and clinical nitty-gritty of covid-19 is beyond the scope of the model. the limitations of this current model prescription are given in the following: (a) the mean-field sird equations do not include the spatial variation in the population density. by construction, there is no spatial degree of freedom in the standard sird model. the term bsi in eq. (1) alludes that all the infected individuals are equally likely to interact with susceptibles and transmit the disease [14] . however, in a realistic scenario, this is unlikely to occur. in the current indian context, an individual living in a remote himalayan village is far less likely to encounter an infected person compared to an individual living near marine drive, mumbai. (b) the model assumes homogeneous exposure and response to the disease [14] . there are various key factors like different demographic features, ethnicity, lifestyle, socioeconomic strata of a sub-population that can induce variability in the exposure to the infection. (c) the model assumes that the contagiousness of an infected person remains constant throughout before his/her recovery and a person is capable of transmitting the disease right after s/he became infected which may not be realistic in the present context. (d) naturally, an increase in the number of tests would also increase the number of infections as more and more undetected infections will be detected. the availability of hospital beds, critical care facilities would also influence the recovery rate in reality. the simplistic model cannot account for all these factors. there are also a few ''what if'' paradigms that cannot be assessed via this model unless further compartments and rate parameters are added upon. few ''what if'' scenarios are: (a) what if the sars-cov-2 immunity is not permanent? [15] ; (b) what if there is seasonal variation in transmission rates of the disease? [15] ; (c) what if the vaccine becomes available within a certain period? and many more. in essence, to address each of these aspects, we need to formulate elaborate models brick by brick in a context-dependent manner. the current working handle of the sird model marks the preliminary footstep along that direction. we investigated the sensitivity of the model to parameter variations, focusing in particular on the parameters that change the rates of infection, recovery (b; c) and most importantly the effective reproduction number or replacement number (r e ) within a feasible range to see the effect the model prediction. the lockdown stringency, characterized by the time-dependentbðtþ, f, was varied to get an estimate of the infected population. corresponding data are shown in each figure by the shaded envelope around the mean curves. the model appears to be sensitive to the variation in the value of f and s 0 when compared with the real data. increasing (decreasing) the value s 0 , f, and r e rapidly increases (decreases) the population of total infection and death mostly around the peak and alters the position of the peak infection. these parameters can be decreased by enforcing local containment and social-distancing measures. the standard sird model does not contain any spatial degree of freedom. the mean-field nature of the current approach excludes the topological dependence of the model predictions. hence, the spatially explicit modeling incorporating infection hotspots and disease transmission from the hotspots to the rest of the places would yield a more realistic reconstruction of the scenario. a compartmentalized sird model simulated on a virtual network of all indian states where the network connectivity manifests the transmission spread from one state to another would be worthwhile. the spatial topology of the underlying network should also include details like the geographical topology of a region, human mobility, connectivity by transport system, health care facilities, etc. the detailed phenomenological reconstruction on a lattice-based model would shed light on the topological dependence of the predictions, robustly. a similar spatially explicit study using the seir model shows how the disease is transmitted from initial foci/local pockets of infections to entire italy [16] . we plan to adopt a similar approach in the indian context as a worthy future endeavor. we reiterate that the predictions based on the sird model are simple mean-field predictions. given the public interest on this pandemic, we put it as a disclaimer that, including topological effects may quantitatively alter the picture of the covid-19 progression in indian context up to a reasonable degree. a common perception of flu and other infectious diseases is that an infected individual spreads infection when symptoms appear. in the case of seasonal flu, infection mostly occurs when a person has symptoms [17] . however, as we understand from the literature survey, an individual with covid-19 would be contagious before developing symptoms. the incubation period for covid-19 is $ 5 days, and maximum infectiousness appears to be 2-3 days before the symptoms appear. thus infection spread by an individual is maximum before he/she becomes sick [10, 18] . due to limitations of the testing procedure, diagnosis takes about 5 days after symptoms are visible, i.e., 10 days from the day of infection. clearly, on the average, an infected individual is beyond the peak of maximum infectiousness after this time. thus, a reduced rate of infection demands early diagnosis and isolation of positive patients. this means that a covid-19 patient needs to be identified in the pre-symptomatic stage as evidence suggests the infectiousness of the patient before developing symptoms which is extremely challenging (effectively, rt-pcr needs to be carried out for every individual who might have come in contact with the patient). the epidemic becomes even more complex due to a majority of the infected individual who develops mild or no symptoms [18, 19] . therefore, even with isolating/ quarantining, all the infected covid-19 would not be eliminated for two reasons: a) normally an individual would be tested after symptoms appear which is when he/ she has passed the peak of the contagiousness, b) asymptomatically infected person, in general, are not tested but he/she is also contagious like the symptomatic individual. 4.1.6. gradual relaxation of the containment versus extended lockdown? we have investigated the possible effect of relaxing the containment measures at three different time points for india ( fig. 9a-d) . we find that if the containment would have been relaxed in the middle of may, i.e. the before the projected peak infection is reached at the end of june, the infected population would rise rapidly to a great extent (fig. 9a) . the peak height reduces, if the containment measures are relaxed, when the infection is close to the peak, a time point around the 3rd week of june (fig. 9b) . however, if the relaxation occurs a month after the peak infection, a second peak arrives which is lower than the first infection-peak (fig. 9c) . a third possibility is to gradually relax the containment measures after may 17. the model shows that in this case the original peak does not shift its position but becomes twofold higher than before (fig. 9d) . a gradual relaxation could be carried out in steps: (a) first, identify all the sensitive (red) and safe (green) zones having positive and no cases respectively. smaller the size of such zones, easier they can be managed by the administration, and necessary supplies can be arranged. it is important to seal the boundary of the red zones. (b) test for new cases carrying symptoms and randomly test a few having no symptoms. (c) dissolve the boundary between red and neighboring green zones once the red zone does not report a case for 2 weeks. this process will increase the size of the green zone where more and more people can communicate and business can restart. successively extending the relaxation from the local neighborhood to the cities, districts, states, the containment measures can be relaxed across the country. nevertheless, social distancing is mandatory even after the containment is officially lifted as there might be many undetected cases that can trigger the spread of the disease again. a schematic diagram in fig. 10 summarizes the above-mentioned steps of relaxation and the consequential aftermaths, pictorially. in a nutshell, 'too-early' lifting of containment measures, long before the infection reached its peak, makes the purpose of lockdown 'null-and-void'. the reduced infection ratebðtþ again starts increasing yielding a larger r e promoting the outspread. it is imperative to note that there are certain differences in the concepts and implications of lockdown, containment measures, and social distancing. in the indian context, ideally, lockdown implies that containment measures are enforced in every nook and corner of the country. however, the intervention of containment measures can be applied locally. for example, in principle, the nation-wide lockdown can be lifted, but containment measures can remain in action in places that are identified as infection hotspots. hence, the word 'lockdown' refers to the restrictions (social, economical) applied over a very large region (e.g., a state or the whole country) whereas the 'containment measures' refers to implementing the same disciplines locally as well as universally. social distancing, on the other hand, is rather a personalized affair. people can maintain social distancing even when a lockdown is not in place. in an ideal scenario, if all individuals within a closed community maintain social distancing without 'lockdown', new infections are unlikely to occur. in that note, our model projects that, with containment measures and social distancing in effect, the number of active infection (noncumulative) would be about 80,000-140,000 at the peak which is expected to appear sometime toward the end of june (fig. 3) . nevertheless, to reduce the economic impacts, it is essential to relax the strict containment measures applied across the country. through our model, we checked several scenarios for applying the relaxation and estimated the evolution of the infection. if the containment measures were suddenly relaxed after may 17, the peak infection would have increased sharply to 0.3-0.4 million (fig. 9a) . it is imperative to mention that, this number is estimated without altering the susceptible population which is about 10 à3 times the actual population observed for many large countries. the remaining population is considered to be shielded from the infection due to containment and demographic segregation. note that, due to containment (and social distancing), the size of the susceptible population is kept at a value of only several million while the total population of india is about 1.35 billion. upon lifting the containment measures, the effective susceptible population should also increase. in this simplistic model, since the attributes are based on a closed population, no additional increment in the number of susceptibles upon the lifting of containment measures is considered. in the event of unrestricted mixing of the population of the whole country, the peak infection might see a 10 3 fold rise which would be challenging for any health care system to deal with. thus, social distancing measures must remain in place unless the infectious population is contained or drastically reduced. in a nutshell, the four scenarios depicted in fig. 9a(fig. 9a) . but, in reality, in the indian context, we observed that the lockdown was still in place after may 17. thus, fig. 9a remains as a ' what could have been' scenario. note that when we say 'lockdown', we assume that both the containment measures and social distancing norms are enforced/followed. now the question is, do any of these plots (fig. 9a-d) corroborate with the present situation (real data as of may 29)? in india, 4th phase of lockdown was enforced (rather extended) from may 18 onward. however, during this phase, various relaxation measures were also given. in that note, the closest theoretical consideration would be the scenario depicted in fig. 9d . to analyze the situation, we compare two scenarios side by side from a more recent perspective (evolved when the manuscript was under revision): time evolution of infection curves (a) with lockdown (containment measures ? social distancing in place) as depicted in fig. 2b; (b) containment measures gradually relaxed as depicted in fig. 9d . in figs. 2b and 9d, the real data was up to may 7. in fig. s3a-s3b , we re-plotted the real data up to may 29, with the theoretical curves of figs. 2b and 9d. the real data up to may 29, in both fig. s3a -s3b, reasonably falls within the range enveloped by the theoretical curves (with marginal deviation). the range of the active infection peak in fig. s3a , is about 0.1-0.3 million occurring between the end of june to mid-july whereas in fig. s3b , the infection peak is predicted to be in the range of 0.2-0.3 million also occurring at the end of june. thus, from fig. s3a -s3b combined, we gather that the active infection peak may be above 0.1 million and likely to be in a range of 0.2-0.3 million. according to this analysis, the peak is likely to occur during the end of june and mid-july. however, this is merely a model prediction; the dynamics, in reality, depends on a myriad of factors which are beyond the scope of this simple model. although the nation is under lockdown, it is observed that the number of positive cases is still growing at large. a distinct feature of this growth is the local resurgence of infections. as gleaned from various news reports, even after several days with a few new cases, suddenly, there had been jumps in the covid-19 positive cases in quite a few places. in other words, 'lull' 'green' zones are, all of a sudden, turning into 'red' zones. we discuss a few plausible factors behind the resurgence: (a) cross-country reverse migration: due to the lockdown, a large population of migrant workers reeling at the bottom of the economic barrel got stranded in different places without much subsistence. these people started returning to their homes taking desperate measures. during this migration, humanto-human transmission of covid-19 might have occurred to a great extent due to a lack of social distancing adding fuel to the 'resurgence' of infection. (b) lack of 'test, trace and contain': interestingly, an important aspect of covid-19 is the number of patients who do not develop any symptoms (fig. 1b) . in india, primarily the testing capacity was devoted to the persons showing typical symptoms of covid-19. the asymptomatic pool largely remained unnoticed at the initial stages of infection outgrowth which probably contributed to the resurgence of infections. moreover, it is not sufficient to only isolate the positive cases but to trace all those people who came in contact with the individual tested positive and find the source of infection. this is known as 'contact tracing'. if the source of the infection is not traceable, this could indicate an insufficient testing or asymptomatically positive source. extensive use of the app-based modern technology may become useful to trace contacts, however, often at the cost of privacy. in india, where a large portion of the population has no 'digital footprint', contact tracing becomes even harder. south korea flattened the infection curve with extensive testing and other mentioned measures. in india, a similar endeavor of a magnitude proportional to its humongous population seems extremely challenging. with the limited capacity and huge population, randomized testing, at least in the infectious neighborhood, is an immediate solution to detect and isolate the asymptomatic individuals. in both the above-mentioned scenarios, the majority of the infection spreading is likely to be spearheaded by the asymptomatic human hosts who remain undetected due to a lack of randomized testing and come in social contact with others. this means that they would be infecting healthy people unknowingly. according to the who and the indian council of medical research (icmr), as much as 80% of the infected individual can be asymptomatic. thus, all the symptomatic cases reported so far contribute to only about 20% of the total infection. going by the reported number of cumulative infections 61,356 as on may 9, almost all of which are symptomatic, this would correspond to about 245,424 people who also had the virus but did not show any symptoms. together, about 306,780 people have actually been infected so far in india carrying symptoms or no symptoms. therefore, the number of people in the country who are still susceptible to the infection is still in the order of billion. one can realize that, with so many active infections, extensive mixing of the countrywide population soon after the lockdown is over (after 17 may) would cause a huge surge in the total number of infections which is nearly impossible to manage by any health care system. to estimate the asymptomatic population from the model, we rewrite the equations as follows: di a ðtþ dt ¼ sðbi s þ b 0 i a þ=n à ða þ m 0 þi a ; di s ðtþ dt ¼ ai a à ðm þ dþi s ; drðtþ dt ¼ m 0 i a þ mi s ; ddðtþ dt ¼ di s here, the total infectious population is segregated into two compartments: (a) symptomatic i s and (b) asymptomatic or mildly symptomatic i a population. a susceptible person can be infected upon contact with a symptomatic or asymptomatic individual with rates b; b 0 respectively. the infected individual can remain asymptomatic or mildly symptomatic and transit into a symptomatic state with rate a. the asymptomatic and symptomatic persons can recover at rates m 0 and m respectively. for a symptomatic individual, death occurs with rate d. for simplicity, we assumed no death for asymptomatic population. we find that with an s 0 in range $ 4-6 million and r e $ 4:0, new model data (fig. s4 ) matches with the previously plotted population of symptomatic infection (see fig. 2 ). the asymptomatic infection peak appears to be about 4 fold larger than the symptomatic infection peak. thus the current lockdown can only be relaxed in the presence of extensive testing of symptomatic and asymptomatic population and contact tracing. it is noteworthy to mention that the total number of cases reported in all over india as well as in various indian states are negligible compared to the total population of the country and states respectively. besides, the severity of the infection with symptoms is relatively less in india than in the usa and other large european countries. whether it is due to the effect of hot and humid weather of india or other meteorological parameters such as high uv index, future research would be able to evaluate. the model predicts the infection peak for india at the end of june or the first half of july (fig. 2) assuming that the social distancing measures will remain in place. the model shows that as the size of the susceptible population increases, the infection peak shifts to a later date (fig. 2b, inset) . thus, if the human mobility between regions increases, that would lead to an effective expansion in the number of susceptibles. this increase in susceptible population would not only lead to a surge in infections but also delay the occurrence of the infection peak by a few weeks. lately, we have been observing an uprise in the new covid-19 positive cases daily. this may be attributed to the transmission of the pathogen via asymptomatic carriers and reverse migration of migrant workers from one province to another within india. whether asymptomatic transmission in tandem with elevated human mobility plays a crucial role in the recent infection surge is a topic of our ongoing work [20] . our results also exemplify that, the best fit of the real data can be obtained for different r e values with the difference being non-marginal (figs. 3, 4) . this may be a limitation of the current model setup to zero in on the exactness of characteristic r e corresponding to the outspread. as mentioned earlier, a spatially explicit model considering a network of indian provinces connected by human mobility, domestic travel from one place to another, and corresponding disease transmission graphs may lead to deeper understandings of the dynamics of the ongoing pandemic in india. epidemic modelling: an introduction publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we thank santanu bhattacharya, heiko rieger, soumitra sengupta, jayanta kumar bhattacharjee, deb shankar ray, sankar prasad bhattacharyya, parongama sen for exciting discussions and valuable suggestions. sa.c. and a.s. were supported by a fellowship from the university grants commission (ugc), india. sw.c. thanks indian association for the cultivation of science, kolkata for financial support. m.k. was supported by a fellowship from csir, india. r.p. thanks iacs for support and grant no. emr/2017/001346 of serb, dst, india for the computational facility. key: cord-292367-ocbsmmt6 authors: el-masri, maher m.; oldfield, margaret title: exploring the influence of enforcing infection control directives on the risk of developing healthcare associated infections in the intensive care unit: a retrospective study date: 2012-02-29 journal: intensive and critical care nursing doi: 10.1016/j.iccn.2011.10.003 sha: doc_id: 292367 cord_uid: ocbsmmt6 summary background although strict adherence to infection control strategies is recognised as the simplest and most cost effective method to prevent the spread of healthcare associated infections (hais), measurement of the direct impact that such adherence may have on the risk of developing such infections has always been a challenge. purpose the purpose of this study was to compare the risk of hais before and during the sars outbreak. such comparison is intended to provide a surrogate measure of the influence that strict enforcement of infection control strategies during the sars outbreak may have had on the risk of hais. methods a retrospective chart review was conducted on the medical records of 400 intensive care patients who were admitted to the icu three months before and during the 2003 sars outbreak. results the rate of hais was higher in the pre-sars period than the sars period. specifically, 61.7% of all reported infections were diagnosed in the pre-sars period. the rate of hais in the pre-sars period was 14.5% as opposed to 9% during the sars period. adjusted logistic regression analysis suggested that the odds of hais were 2.2 times higher in the pre-sars period as compared to the sars period (or =2.2; 95%ci =1.08–4.49). conclusion our findings suggest that strict enforcement of infection control strategies may have a positive impact on the efforts to minimise the risk of hais. these findings carry a clinical significance that shall not be ignored with regard to our overall efforts to minimise the risk of developing hais in the icu. infection control guidelines in health care settings are designed to protect healthcare providers (hcps) and patients from the risk of infections. although hcps are expected to demonstrate full adherence with these guidelines, the literature suggests that adherence with infection control guidelines are less than optimal. for instance, several studies have shown that hand hygiene compliance amongst hcps ranges from 10 to 60% (arenas et al., 2005; korniewicz and el-masri, 2010; lankford et al., 2003; larson et al., 2005; saba et al., 2005; whitby and mclaws, 2004) . these statistics are especially concerning in light of the costly consequences of healthcare associated infections (hais) to patients and the health care system. hais, previously known as nosocomial or hospital acquired infections, are defined in a patient if the infection was not present at the time of hospital admission and does not develop within the first 48 hours post admission. it is estimated that 250,000 hospitalised canadians (one out of nine admissions) develop hais annually, of whom 8000-12,000 die as a result of these infections (canadian foundation of infectious diseases, 2008) . further, the additional economic cost of treating hais to the canadian healthcare system is estimated at $12,000-35,000 per patient (canadian foundation of infectious diseases, 2008) . over the last two decades, a plethora of studies have examined the risk factors of hais. however, the impact of adherence or lack of, with the guidelines of infection control was almost never reported in these studies. this is because it is very difficult to reliably measure and/or observe infection control practises amongst hcps on a continuous basis. yet, failure to account for infection control practises threatens the validity of our understanding of hais. although a number of studies (camins and fraser, 2005; won et al., 2004) examined the association between the implementation of hand hygiene promotion programmes and hais, infection control guidelines are far more encompassing than hand hygiene. to date, there is little evidence concerning the impact of strict enforcement of infection control guidelines on the rate of hais. during the outbreak of the severe acute respiratory syndrome (sars) in the spring of 2003, all canadian hospitals in the province of ontario imposed exceptionally strict enforcement of infection control strategies that were mandated by a series of directives from the ministry of health and long-term care (mohltc) (ministry of health and long-term care, 2003b) . these directives were continuously updated during the sars outbreak, but their most important points are summarised in appendix a, which provides a comparison between the before and during sars practises. whilst restrictions and enforcement of infection control strategies were implemented across all hospital units, they were especially tighter in intensive care units (icu) due to the vulnerability of icu patients and the tendency of sars to progress into a critical respiratory disease. the strict enforcement of, and adherence to the infection control guidelines at canadian hospitals during the sars outbreak presents a rare opportunity to examine the impact of such enforcement on the risk of hais amongst icu patients. therefore, the purpose of this study was to retrospectively compare the risk of developing hais in the icu before and during the sars outbreak. the intent of such comparison is to provide a surrogate measure of the influence that strict enforcement of infection control guidelines might have had on the risk of developing hais. a retrospective chart review was conducted on the medical records of 400 patients who were admitted to the intensive care unit of a community-based hospital in southwestern ontario. the reviewed charts were randomly selected and divided into two equal groups of 200 each, representing the three months that immediately preceded the sars (i.e., pre-sars) and the three months of the sars outbreak (march 15-june 15, 2003) . infection control practises/strategies during these two periods are outlined in appendix a. this sample of 400 charts was deemed sufficient to detect an absolute risk reduction of 10% in the risk of developing hais during the sars period, using an alpha of 0.05 and assuming 80% power. the inclusion criteria for the study required that patients be free from pre-existing hais upon icu admission and that they had an icu length of stay of 48 hours or greater. a patient was considered a positive hai case if he/she had a confirmed diagnosis of infection (types are outlined in table 1 ) that did not exist at the time of icu admission. diagnoses of hai's at the time of admission of our sample were made according to the cdc criteria for the definition of hais as outlined in garner et al. (1988) . for the purpose of statistical adjustment for potential confounding relationships, data were also collected on other commonly reported risk factors of hai (table 2 ). data were analysed using the predictive analysis software (pasw), version 18. basic descriptive statistics such as general frequencies of nominal variables, and means and standard errors of continuous variables were performed on the total sample and between the two groups (i.e., hai versus no hai). chi square comparisons were performed to compare hais between the pre-sars and sars periods. chi-square and t-test comparisons were also performed to compare the demographic and prognostic factors between those who had hais and those who did not. then, multivariate logistic regression analysis was performed to identify the independent (i.e. adjusted) association between the period (pre-sars versus sars) and the risk of developing hai, whilst adjusting for other demographic and prognostic variables. variables were included in the regression model based on a liberal criterion (i.e., p ≤ 0.25) concerning the association that each of these variables had with hais in the univariate analysis (hosmer and lemshow, 2000) . this liberal p value was chosen to avoid unnecessary deletion of potentially significant independent predictors from the regression model. a 95% confidence interval ( 95 ci) that did not include 1.0 was set as the criterion to establish significance. the total number of hais was 47 (11.8%), which were distributed across a number of specific infections as outlined in table 1 . our findings show that the rate of hais was higher in the pre-sars period than the sars period. specifically, the findings suggest that 61.7% of all reported infections were diagnosed in the pre-sars period. the findings further suggest that the rate of infection in the pre-sars period was 14.5% as opposed to 9% during the sars period. table 2 presents the univariate comparisons between the infected and non-infected patients on each of the study variables (i.e., ''each of the risk factors for hai's''). according to the variable inclusion criteria, all variables in table 2 were entered into the logistic regression model except for comorbidities and age; each of which had a p value that was ≥0.25. our findings suggested that the frequency of pneumonia, bacteraemia and urinary tract infections were all lower during the sars period than the pre-sars period. however, the rate of surgical site infections was higher during the sars period than the pre-sars period. interestingly, the most noticeable change in the frequency of infections happened in pneumonia (31.8% versus 14.9%) and urinary tract infections (12.8% versus 2.1%). it is difficult to explain the exact cause of this trend. however, pneumonia, bacteraemia and urinary tract infections are hais that are often associated with the use of invasive devices such as endotracheal tubes or invasive mechanical ventilation and suctioning in pneumonia, central venous catheters and chest tubes in bacteremia and urinary catheters in urinary tract infections. given that a number of mohltc's directives were specific to respiratory and high risk procedures (ministry of health and long-term care, 2003b) , it is possible that this trend of risk reduction is attributed in part to the strict enforcement of the guidelines outlined in those directives. it is unclear why the risk of surgical site infections and other infections were higher in the sars period. however, it is possible that these two findings were the result of chance due to the very small number of patients who acquired these infections in our sample. our findings show that, after adjusting for commonly reported risk factors of hais such as use of mechanical ventilation, use of central venous catheters and use of chest tubes; the odds of developing hais were 2.2 times higher in the pre-sars period than the odds of hais in the sars period. this adjusted finding suggests an independent association between the risk of developing hais and the time period (i.e., pre-sars versus sars). although we did not directly measure the impact of enforcing infection control strategies and vigilance of healthcare workers on the risk of hai between the two periods, this finding could be possibly explained in light of these strategies and the other changes that were implemented during the sars outbreak. this is because enforcement of infection control strategies through a series of specific directives from the mohltc was the main event that distinguished the change in practice between the two periods. additionally, our pre-sars data was obtained from the three months that immediately preceded the sars period whereby no change in practice other than those implemented to counter the sars outbreak took place. our findings support the argument that enforcing strict infection control strategies is associated with reduction in the risk of developing hais. for instance, won et al. (2004) reported that promoting a hand hygiene programme resulted in increase in hand hygiene from 43% to 80% and reduction of hais from 15.13 to 10.69 per 1000 patient-days. nonetheless, won et al. (2004) did not impose hand hygiene practises and did not report adjusted relationships between hand hygiene and hais. interestingly, our findings suggested that factors such as use of mechanical ventilation, central venous catheters and chest tubes remained significant predictors of hais in the adjusted analysis. these findings are consistent with those reported by other authors who suggested that the use of mechanical ventilation (bochicchio et al., 2008; magnason et al., 2008) , chest tubes (el-masri et al., 2004; oldfield et al., 2009) and central venous catheters (el-masri et al., 2004; garnacho-montero et al., 2008) were significant predictors of hais. however, within the context of this study, these findings suggest that despite enforcement of strict infection control strategies, the use of invasive devices poses additional risk of hais to icu patients. thus, it is important that invasive devices be used and handled properly so that their associated risk is minimised. it is also important that hcps pay equal attention to adhering to proper infection control practises and maintain proper disinfection and/or care of invasive devices. that includes limiting the use and duration of such devices based on patient specific indications and good understanding of proper practice guidelines concerning the use of such devices. the findings of our study could be used to highlight the importance of enforcing strict infection control strategies for the reduction of the risk of developing hais. given the significant financial and health costs that are associated with hais (burke, 2003) , it is imperative that health care agencies consider enforcing infection control policies at all times and not only during crises such as the sars outbreak. we realise that the measures imposed during the sars outbreak were exceptionally strict and included measures that may not be attainable or realistic for normal times. however, we believe that a reasonable enforcement of infection control policies is warranted. the current rate of 10-60% compliance (korniewicz and el-masri, 2010; maskerine and loeb, 2006) with hand hygiene guidelines and the incidence of hais in one of every nine canadian hospital admissions (cfid, 2008) are unacceptable and must be addressed. for instance, the literature suggests that hcps report higher compliance rate with hand hygiene after procedures than before procedures (korniewicz and el-masri, 2010; saba et al., 2005) , which may suggest that hcps are more likely to sanitise their hands out of fear for their own health than that of their patients. although we did not measure hcps adherence to hand hygiene in our study, anecdotal data suggest that hcps were strictly complying with infection control guidelines (including hand hygiene) due to institutional enforcement of these guidelines and internal motivation that was driven by fear for one's own health. therefore, we recommend that strategies to increase compliance with infection control guidelines do not only focus on enforcement, but also address the issue of internal enticement and motivation of hcps. it is encouraging to learn that in the time since the sars outbreak, health care facilities are making significant strides to encourage appropriate adherence with proper infection control. in many acute care facilities, hcps are required to complete an annual infection control learning programme. in ontario, alcohol-based hand rub must be within an arm's length of each patient's bed, to remove one more obstacle to performing appropriate hand hygiene. gloves are located within reach at almost every location in the unit. adherence with hand hygiene at the ''four needed moments'' is now regularly audited and publicly posted in nursing areas alongside the rates of common hais. although these efforts help to hold hcps accountable with regards to their adherence with infection control guidelines, it is important that they be coupled with strategies that create a sense of personal responsibility amongst hcps so that they are constantly mindful of the seriousness of breaching these guidelines. in conclusion, our findings, although use comparison of two time periods as a surrogate measure of adherence with infection control guidelines, imply that enforcement of such guidelines may have a positive impact on the efforts to minimise the risk of hais. whilst this study utilised a rare natural occurrence (i.e., the sars outbreak) in which infection control strategies were enforced throughout the healthcare system to explore the impact of enforcing infection control guidelines on hais, it is important that its findings be interpreted with caution due to its retrospective nature and the fact that it did not directly measure the association between enforcing infection control guidelines and hais. instead, it used the experience of policy enforcement that took place during the sars outbreak as a surrogate measure of adherence with proper infection control practises. in addition, patients in the two time periods may have had different baseline characteristics and/or different exposure times. further, our infection rates were reported based on an older version of the cdc criteria for the definition of healthcare associated infections, which however were not significantly changed in the 2008 updated criteria (horan et al., 2008) . despite these limitations, the findings of this study carry a clinical significance with regard to the influence that strict enforcement of hand hygiene guidelines may have on the risk of developing hais. finally, it is important to mention that other extreme measures such as the cancellation of all casual visitations, outpatient services and elective surgeries took place during the sars outbreak period. such measures may have been a contributing factor to our results. therefore, we recommend that these results be interpreted with caution within the context of this research. a multicentric survey of the practice of hand hygiene in haemodialysis units: factors affecting compliance blood product transfusion and ventilator-associated pneumonia in trauma patients infection control -a problem for patient safety reducing the risk of health care-associated infections by complying with cdc hand hygiene guidelines canadian foundation of infectious diseases. healthcare-associated infections predictors of nosocomial bloodstream infections among critically ill adult trauma patients risk factors and prognosis of catheter-related bloodstream infection in critically ill patients: a multicenter study cdc definitions for nosocomial infections cdc/nhsn surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting applied logistic regression exploring the factors associated with hand hygiene compliance of nurses during routine clinical practice influence of role models and hospital design on hand hygiene of healthcare workers hand hygiene behavior in a pediatric emergency department and a pediatric intensive care unit: comparison of use of 2 dispenser systems risk factors and outcome in icu-acquired infections ministry of health and long-term care. health update: severe acute respiratory syndrome severe acute respiratory syndrome (sars) archive: notices and directives examining the association between chest tube-related factors and the risk of developing healthcare-associated infections in the icu of a community hospital: a retrospective case-control study hand hygiene compliance in a hematology unit handwashing in healthcare workers: accessibility of sink location does not improve compliance handwashing program for the prevention of nosocomial infections in a neonatal intensive care unit summary of infection control practises before and during the sars outbreak. pre sars during sars key: cord-264569-q8nq2gbz authors: grünberg, k.; sterk, p. j. title: rhinovirus infections: induction and modulation of airways inflammation in asthma date: 2001-12-24 journal: clin exp allergy doi: 10.1046/j.1365-2222.1999.00011.x sha: doc_id: 264569 cord_uid: q8nq2gbz there is renewed interest in the role of respiratory virus infections in the pathogenesis of asthma and in the development of exacerbations in pre‐existing disease. this is due to the availability of new molecular and experimental tools. circumstantial evidence points towards a potentially causative role as well as to possibly protective effects of certain respiratory viruses in the cause of allergic asthma during early childhood. in addition, it now has become clear that exacerbations of asthma, in children as well as adults, are mostly associated with respiratory virus infections, with a predominant role of the common cold virus: rhinovirus. careful human in vitro and in vivo experiments have shown that rhinovirus can potentially stimulate bronchial epithelial cells to produce pro‐inflammatory chemokines and cytokines, may activate cholinergic‐ or noncholinergic nerves, increase epithelial‐derived nitric oxide synthesis, upregulate local icam‐1 expression, and can lead to nonspecific t‐cell responses and/or virus‐specific t‐cell proliferation. experimental rhinovirus infections in patients with asthma demonstrate features of exacerbation, such as lower airway symptoms, variable airways obstruction, and bronchial hyperresponsiveness, the latter being associated with eosinophil counts and eosinophilic cationic protein levels in induced sputum. this suggests that multiple cellular pathways can be involved in rhinovirus‐induced asthma exacerbations. it is still unknown whether these mechanisms are a distinguishing characteristic of asthma. because of the limited effects of inhaled steroids during asthma exacerbations, new therapeutic interventions need to be developed based on the increasing pathophysiological knowledge about the role of viruses in asthma. considering the rapid progress in the present research on the pathogenesis, pathophysiology, and immunopathology of asthma and chronic obstructive pulmonary disease (copd), it is remarkable that the understanding of the potential involvement of respiratory virus infections in these diseases is far from settled. this seems to be due to the only recent availability of data from (i) epidemiological studies using modern virological techniques, and (ii) experimental studies in animals as well as in man. these approaches are increasing our knowledge on the potential role of respiratory viruses in the onset of asthma and copd as well as in inducing exacerbations in pre-existing disease. in general, the information on the complex involvement of respiratory virus infections in the pathogenesis of asthma is increasing. it shows circumstantial evidence of a broad spectrum of effects: between a potentially causative or facilitatory role [1, 2] and an inhibitory or protective influence [3, 4] , probably depending on the type and age of infection. with regard to the importance of respiratory virus infections in exacerbations of pre-existing asthma, research during the past decade has provided clear positive evidence, from both epidemiological and experimental studies [5, 6] . this is less clear in copd. latent virus infections have been implicated in its pathogenesis [7] , but this still needs confirmation. furthermore, there are only very recent data on a potentially (limited?) role of respiratory viruses in causing exacerbations of pre-existing copd [8] . it is likely that respiratory viruses can have pro-inflammatory and immunomodulatory effects within the airways [9] . this has most extensively been studied for respiratory syncytial virus (rsv) [10, 11] and for human rhinovirus [5, 6, 12] . this review will focus on the latter, and will particularly discuss the association between the pro-inflammatory and pathophysiological effects of rhinoviruses in patients with asthma. there is little doubt that rhinoviruses are important as a trigger of asthma exacerbations. several clinical and epidemiological studies have described a close temporal association of respiratory virus infections with exacerbations in patients with asthma [13] . respiratory viruses can be identified in 10%-44% of the asthma exacerbations in adults [14, 15] , whilst in children identification rates vary from 26% to 83% [16] [17] [18] [19] . the use of the sensitive polymerase chain reaction technique to detect rhinovirus and coronavirus in the two most recent studies has resulted in the highest identification rates so far [15, 19] . among the various respiratory viruses identified, rhinovirus predominates in most of these studies, accounting for about 50% of the detected viruses [15] [16] [17] [18] [19] . the incidence of rhinovirus infections may even be higher in asthmatic patients as compared with nonasthmatic subjects [13, 20] , although rhinovirus shedding in the absence of cold symptoms does not seem to be associated with clinical worsening of asthma [17] . taken together, these findings indicate that rhinovirus infections may have a causal role in exacerbations of asthma. preliminary data confirm that this also occurs in exacerbations of copd, but to a lesser degree [8] . the observation that common colds are important in inducing exacerbations in asthma, offers the perspective of experimental studies that can be safely performed in humans in vivo [21] . indeed, experimental infections have been shown to be a useful tool for investigating the effects of rhinovirus infections in allergic disease or asthma [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] . such a model allows careful patient selection and monitoring, and intensive assessment of the rhinovirusinduced effects, under controlled circumstances and timing. thus, the effects of a rhinovirus infection can be assessed at the level of asthma symptoms, changes in use of asthma medication, lung function, and airway hyperresponsiveness, as well as the underlying airway pathology. so far, experimental rhinovirus infections in patients with asthma and/or atopic rhinitis have not been shown to induce a significant change in lung function, e.g. fev 1 , when measured during laboratory visits [22, [24] [25] [26] 28, 29, 31, 35] . this has been considered to be reassuring in terms of patient-safety of the experimental model; however, recent results from our laboratory are demonstrating that frequent home-recordings of fev 1 (three times daily) do decrease in atopic asthmatic patients in the acute phase of an experimental rv16 infection [27] . furthermore, the maximal decrease in fev 1 in the acute phase of the infection, expressed as percentage of the recent personal best, correlated significantly with the observed increase in airways hyperresponsiveness [27] . this suggests that there is transient worsening of airways obstruction after rhinovirus infection in asthma, which may improve spontaneously during the day, or as a consequence of repeated deepbreath manoeuvres as are being performed in the lung function laboratory. this points at either increased sensitivity to bronchoconstrictive stimuli and/or a reduced bronchodilating effect of a deep breath [36] . the effects of experimental rhinovirus infection on airway responsiveness vary among different studies, using different rhinovirus serotypes. lemanske et al. and gern et al. have demonstrated an enhanced hypersensitivity to histamine and allergen challenge after experimental rhinovirus 16 (rv16) infection in nonasthmatic patients with atopic rhinitis [24, 34] , which was significantly different from the lack of response in normals [34] . others, however, have not observed such an effect when using rhinovirus 39 [35] . in asthmatic subjects, halperin et al. has found increased hypersensitivity to histamine in only four of 22 subjects after experimental rhinovirus (serotype 39 and hh strain) infection [22] . more recently, in our laboratory cheung et al. have shown that rv16 increases asthma symptoms, coinciding an increase in the maximal bronchoconstrictive response to methacholine up to 15 days after infection [25] , pointing towards the development of excessive airway narrowing due to a rhinovirus infection. in a similar design, we have shown a significant increase in airway sensitivity to histamine in asthmatic subjects after rv16 infection, which was most pronounced in those subjects who had severe cold symptoms [28] . taken together, these data indicate that patients with asthma and/or atopic rhinitis may suffer more prominent pathophysiological consequences from a rhinovirus infection as compared with nonatopic nonasthmatic subjects, suggesting an interactive effect of virus-induced airways inflammation with features of the underlying disease, such as altered airway geometry [37, 38] and/or pre-existing allergic sensitization/ inflammation [39] . experimental animal studies have provided strong evidence of the induction of airways inflammation by respiratory viruses, as is apparent from epithelial shedding and morphological changes after, for example, parainfluenza infection in guinea pigs [40] . it has not, however, been resolved to what extent such induced inflammation contributes to the accompanying physiological changes. initial findings suggested a major role of inflammatory cells, such as t cells, basophils, and their mediators [41] . and indeed, virusinduced airway hyperresponsiveness could be transferred by bronchoalveolar lavage cells recovered after viral infection in guinea pigs [42] . however, more recent results obtained by using antibodies against pro-inflammatory cytokines, such as il-5, are indicative of (partly) independent induction of airways hyperresponsiveness and eosinophilic airways inflammation by respiratory viruses in guinea pigs in vivo [43] . apparently, neurogenic mechanisms can also be responsible for virus-induced hyperresponsiveness in animals. interestingly, this seems to be mainly due to impaired inhibitory mechanisms [44] . such impaired neurogenic protection might arise from reduced production or activity of the neuropeptide degrading enzyme neutral endopeptidasen [45] , or to dysfunction of inhibitory cholinergic m 2receptors [46] . in addition, there is evidence that virusinduced airways hyperresponsiveness in guinea pigs can be caused by impaired synthesis of nitric oxide (no) [47] . this is potentially important, since it has recently been shown that rhinovirus can induce no-synthase expression in human primary bronchial epithelial cells in vitro [48] , and that such no can reduce rhinovirus replication and the induced inflammatory cytokine release by epithelial cells [49] . interestingly, our latest data in asthmatics in vivo are in keeping with such a protective mechanism by no after rhinovirus infection (fig. 1 ) [50] , but this hypothesis remains to be tested using appropriate pharmacological blocking agents in asthmatics in vivo. what do we know about the inflammatory response to a rhinovirus infection in asthmatic and/or atopic rhinitis patients? it is likely that epithelial cells are playing an active and major role in this. in vitro, there is evidence that infection with rhinovirus in pulmonary epithelial cells and fibroblasts induces de novo synthesis of pro-inflammatory cytokines, such as gm-csf, il-11, and il-6, and the chemokines il-8 and rantes [51] [52] [53] [54] [55] by activating transcription factors such as nf-kb [53, 56, 57] . such epithelial chemokine release appears to be prolonged until 5 days after in vitro infection [58] . indeed, during common colds in vivo, these mediators and others were found to be elevated in nasal secretions [28, 57, [59] [60] [61] [62] [63] [64] [65] [66] [67] as well as in sputum (il-8, il-6) [30, 68] . in view of the chemotactic properties of the abovementioned chemokines on lymphocytes, basophils, neutrophils, and (primed) eosinophils, one can postulate that such chemokines drive the secondary recruitment of inflammatory cells [69] . indeed, a rhinovirus infection is known to lead to infiltration of leucocytes, particularly neutrophils and mononuclear cells, into nasal secretions [68, [70] [71] [72] rather than the nasal mucosa [59, 70, 73] . both in normals and asthmatic subjects this occurs in conjunction with an increase in the number of neutrophils in peripheral blood, whereas the number of circulating lymphocytes falls in the acute phase of infection [25, 26, 28, 74] . these effects, as well as the decrease in pc 20 in asthmatic subjects, are significantly related to the severity of the cold, as reflected by the cold score [28, 74] and the increase in il-8 in nasal lavage [28] , underlining the relationship between the severity of the cold and the pathophysiological consequences [17, 28] . in contrast to the findings in the nose, the only available study so far on the effects of a common cold on intrapulmonary airways inflammation showed that in the bronchial biopsies the numbers of t lymphocytes in the submucosa and eosinophils in the epithelium were elevated both in normal and/or in atopic asthmatic subjects following rv16 infection [26] . interestingly, the numbers of eosinophils remained elevated up to 6 weeks after infection in the six asthmatic subjects [26] . this is in keeping with the increased levels of eosinophilic cationic protein (ecp) in sputum that we have observed after experimental rv16 infection in asthmatics, which correlated significantly with the increase in airway sensitivity to histamine (fig. 2) [30] . one could speculate that local production of eosinophil chemotactic chemokines contributes to virus-induced eosinophilic airways inflammation in asthma. [75] [76] [77] [78] , and (ii) as the major rhinovirus receptor [79, 80] . it appears that icam-1 expression is up-regulated upon stimulation with rhinovirus in pulmonary epithelial cell lines in vitro [55, 56] . this has now been confirmed in vivo in a recent study from our laboratory demonstrating that experimental rhinovirus colds in asthmatics increase epithelial icam-1 expression in bronchial biopsy specimens [81] . hence, up-regulation of icam-1 by secondarily released cytokines such as il-1b and tnf-a, may facilitate rhinovirus infection in susceptible cell types [52, 55, 82] . icam-1-binding to its ligands [77] , lymphocyte function associated antigen (lfa-1, cd11a/cd18), and macrophage-1 antigen (mac-1, cd11b/cd18), is involved in the process of leucocyte adherence and migration, and in addition, may provide costimulatory signals for cd4 cell and lymphokine-activated killer cell activation, t-cell mediated cytotoxicity, and t-cell dependent b-cell activation. it has been postulated that cytokines such as tnfa, il-1b, ifn-g, and il-4 may alter the expression, function, or configuration of icam-1, thereby specifically promoting (tnfa, ifn-g, il-4) or impeding (il-6, il-1) lymphocyte adhesion to, for example, cytokine-pretreated fibroblasts or primary tracheal epithelial cells [52, 83, 84] . rhinovirus binding, however, does not seem to be affected by such cytokine-induced functional changes [52, 83, 84] , suggesting that virus-induced pro-inflammatory cytokines may facilitate infection, whilst promoting or inhibiting the interaction between epithelial cells and lymphocytes. what are the immunological changes following rhinovirus infection? incubation of either infectious or inactivated rhinovirus particles with a mixture of antigen presenting cells (apc; such as monocytes and also eosinophils [78] ) and lymphocytes can induce a proliferative response to rhinovirus [85] , while concomitantly hampering the monocytes-induced, icam-1mediated, specific t-cell proliferation and cytotoxicity to other 68 k. grünberg antigens [86] . this indicates that binding of rhinovirus to icam-1 may interfere with icam-1/lfa-1 interaction [86] . indeed, the icam-1 binding sites for lfa-1 and rhinovirus overlap partially [87] . natural killer cell activation and cytotoxicity, which is less dependent on icam-1/lfa-1 interaction, was not shown to be affected, or even increased after in vitro rhinovirus inoculation [86, 88, 89] . rhinovirus does not replicate inside monocytes and airway macrophages [90] ; however, the uptake of infectious rhinovirus particles (either mediated or not mediated by icam-1) by apcs may increase nonspecific t cells activation [91] . this is reflected by the increased expression of the activation marker cd69, but not cd25 on the cell membrane [91] , and spontaneous [85] or mitogen-induced secretion of il-2 and ifn-g in normal subjects [88, 91] . in addition, from 3 weeks after infection onward, antigenspecific lymphocyte proliferation could be demonstrated [88, 92, 93] , indicating a cell-mediated immune response to the infection [85] . such cd4 þ cells may be specific to either serotype-restricted or serotype-shared viral epitopes [85, 94, 95] . a recent report suggests that eosinophils, when expressing icam-1, may function as apcs, in that they induce t-cell proliferation when incubated with rhinovirus [78] . this is associated with an increase in cd18 expression (an icam-1 ligand), thus providing evidence for a possible positive feedback loop in rhinovirus-induced eosinophil activation within the airways. in a comparative study, using in vivo experimental rv39 infection, patients with allergic rhinitis appeared to have lower numbers of t-helper cells (either activated or not activated) and rv39-induced peripheral blood mononuclear cell proliferation as compared with normals [93] , while only in the rhinitis patients was an increase in proliferation to rv39 noted as early as during the acute phase of the infection. furthermore, the decrease in nk-activity was less marked in the rhinitis patients as compared with the normals [93] . this suggests that there may be diseaserelated differences in cell-mediated immunity, although it is still unclear as to how such differences might be interpreted in terms of mechanisms of rhinovirus-induced exacerbations of asthma. is rhinovirus actually present within the lower airways? in the nasopharynx, rhinovirus replicates in the adenoid tissue and the epithelium [96] , and can readily be detected in tissue culture or by reverse transcriptase-polymerase chain reaction [97] . evidence of rhinovirus infection of the intrapulmonary airways is more difficult to obtain. although rhinoviruses can occasionally be cultured from sputum [98] , tracheal brushings [31] , and bronchoalveolar lavage (bal) fluid [33, 99] , possible nasopharyngeal contamination precludes definite conclusions as to virus infection of the lower airways. the use of reverse transcriptase-polymerase chain reaction on bal cells, rather than bal fluid, has increased the likelihood of detecting lower airways infection, and has led to a detection rate of 80%, while the detection rate in nasal lavage fluid in the same subjects was 100% after experimental rv16 infection [100] . in view of the difficulties in detecting rhinoviruses in the lungs so far, and the relative fastidiousness of rhinoviruses for culture condition (particularly the relatively low optimal culture temperature of 33 њc), a high grade infection of the lower airways may not be very likely. one could speculate, however, that host factors such as increased icam-1 expression in the nasal epithelium [101] might increase the susceptibility of asthmatic and/or atopic patients to symptomatic rhinovirus colds [20] , even after repeated infection with the same serotype [23, 28, 102] . similarly, increased expression of icam-1 in the lungs [56, 75] could promote lower airways infection, and its pathophysiological consequences. apparently, in situ techniques to detect the viral genome [103] [104] [105] will need to be applied in order to conclusively assess viral contamination and/or infection within the lower airways. preliminary results using in situ hybridization on bronchial biopsy specimens obtained from normals and asthmatics after experimental infection may indeed be suggestive of the possibility that rhinovirus can infect the lower airways [106] ; however, confirmation of these results has to be awaited. there is little doubt that respiratory virus infections can have pro-inflammatory effects within the airways. whilst it is still unresolved as to whether this contributes to the onset of asthma, there is convincing evidence that rhinovirus infections in particular are associated with exacerbations of the disease. rhinovirus infection is able to activate bronchial epithelial cells, leading to the release of proinflammatory cytokines (chemokines) as well as of modulating substances (such as no). this can be accompanied by virus-specific t-cell proliferation weeks after infection, at early stages preceded by nonspecific t-cell responses that could contribute to virus-induced airways inflammation. by up-regulating icam-1 expression on, for example, epithelial cells and eosinophils, there is the possibility of positive feedback loops between rhinovirus infection and the inflammatory response. some of these events can be observed following experimental rhinovirus infection in patients in vivo. these are accompanied by clinical symptoms, airways obstruction, and worsened airway hyperresponsiveness. the latter is associated with changes in eosinophil counts and levels of ecp in induced sputum. this suggests, but does not prove, that eosinophilic inflammation can be involved in rhinovirus-induced airways inflammation and the associated exacerbations of asthma. in general, the clinical, physiological, and cellular responses to experimental rhinovirus infections in patients with asthma are relatively mild. this underlines the safety of this procedure, but may not adequately mimic the events occurring after a natural common cold. it can be postulated that this requires more complex experimental models, such as those with a pre-existing flare-up of allergic inflammation with, for example, up-regulated icam-1 [107] . this might be obtained by a design in which rhinovirus inoculation follows repeated low-dose allergen exposure [108, 109] . this may mimic the course of events that occur during spontaneous exacerbations more accurately [110] . it is hoped that such experiments will bring us a therapy for virus-induced acute exacerbations of asthma that is more successful than the ones currently available [111, 112] . rsv and chronic asthma persistent adenoviral infection and chronic airway obstruction in children role of viral infections in the inception of asthma and allergies during childhood: could they be protective? measles and atopy 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cytokines and rhinovirus-16 nitric oxide inhibits rhinovirus-induced cytokine production and viral replication in a human respiratory epithelial cell line relationship between exhaled nitric oxide and airway hyperresponsiveness following experimental rhinovirus infection in asthmatic subjects rhinoviruses induce prolonged interleukin-8 release, increases mrna and promotor activation in pulmonary epithelial and peripheral blood mononuclear cells infection of a human respiratory epithelial cell line with rhinovirus. induction of cytokine release and modulation of susceptibility to infection by cytokine exposure rhinovirus stimulation of interleukin-6 in vivo and in vitro. evidence for nuclear factor kb-dependent transcriptional activation rhinovirus 16 (rv16) stimulates production and release of the chemokines il-8 and rantes in subcultures of human primary bronchial epithelial cells rhinovirus infection of primary cultures of human tracheal epithelium: role of icam-1 and il-1b 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virus-specific t cells the major human rhinovirus receptor is icam-1 a cell adhesion molecule, icam-1, is the major surface receptor for rhinoviruses experimental rhinovirus (rv)-16 infection enhances icam-1 expression in bronchial mucosal biopsies of mildly asthmatic subjects, regardless of inhaled steroid treatment internalization of human rhinovirus 14 into hela and icam-1-transfected bhk cells binding of human rhinovirus and t cells to intercellular adhesion molecule-1 on human fibroblasts. discordance between effects of il-1 and ifn-gamma regulation of icam-1 expression and function in human dermal fibroblasts by il-4 characterization of the t cell response to human rhinovirus in children: implications for understanding the immunopathology of the common cold rhinovirus inhibits antigen-specific t cell proliferation through an intercellular adhesion molecule-1 dependant mechanism the arrangement of the immunoglobulin-like domains of icam-1 and the binding sites for lfa-1 and rhinovirus peripheral blood mononuclear cell interleukin-2 and interferon-gamma production, cytotoxicity, and antigen-stimulated blastogenesis during experimental rhinovirus infection rhinovirus induces natural killer-like cytotoxic cells and interferon alpha in mononuclear leukocytes rhinovirus enters but does not replicate inside monocytes and airway macrophages rhinovirus produces nonspecific activation of lymphocytes through a monocyte-dependent mechanism specific mononuclear cell response to rhinovirus effect of rhinovirus 39 infection on cellular immune parameters in allergic and nonallergic subjects proliferative responses of t cells primed against human rhinovirus to other rhinovirus serotypes rhinovirusspecific t cells recognize both shared and serotype-restricted viral epitopes sites of rhinovirus recovery after point inoculation of the upper airway use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms role of viruses and bacteria in acute wheezy bronchitis in childhood: a study of sputum rhinovirus as a lower respiratory tract pathogen in infants detection of rhinovirus rna in lower airway cells during experimentally induced infection allergen-specific challenge induces intercellular adhesion molecule 1 (icam-1 or cd54) on nasal epithelial cells in allergic subjects prechallenge antibodies: moderators of infection rate, signs, and symptoms in adults experimentally challenged with rhinovirus type 39 detection of rhinovirus rna in nasal epithelial cells by in situ hybridization localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization detection of rhinovirus infection of the nasal mucosa by oligonucleotide in situ hybridization localisation of rhinovirus in the bronchial epithelium of experimentallyinfected human volunteers bronchial biopsy evidence for leukocyte infiltration and upregulation of leukocyte-endothelial cell adhesion molecules 6 hours after local allergen challenge of sensitized asthmatic airways repeated low dose allergen exposure: a new investigational model of asthma as a persistent disease? repeated aerosol exposure to small doses of allergen. a model of chronic allergic asthma the relationship between upper respiratory infections and hospital admissions for asthma: a time-trend analysis effect of inhaled corticosteroids on episodes of wheezing associated with viral infection in school age children: a randomised double blind placebo controlled trial intermittent treatment with inhaled steroids for deterioration of asthma due to upper respiratory tract infection key: cord-274643-vjb2yt93 authors: kang, g. title: viral diarrhea date: 2008-08-26 journal: international encyclopedia of public health doi: 10.1016/b978-012373960-5.00571-2 sha: doc_id: 274643 cord_uid: vjb2yt93 viral gastroenteritis is among the most common illnesses affecting humans and has greatest impact at the extremes of age. the spectrum of disease can range from asymptomatic infections to severe disease with dehydration. intensive investigation of enteric infections in the past three decades has made it increasingly clear that viruses cause a significant proportion of enteric illnesses worldwide. in contrast to bacterial pathogens, enteric viruses cannot multiply outside their host; hence, the original inoculum into the common source determines infectivity. prevention of contamination of food and water will help control primary cases, whereas careful nursing and handwashing prevent secondary cases. acute gastroenteritis is among the most common illnesses affecting humans and has greatest impact at the extremes of age, severely affecting children and the elderly. the spectrum of disease can range from asymptomatic infections to severe disease with dehydration, which can be fatal. diarrheal disease continues to be a major cause of mortality in young children, particularly in developing countries. prior to 1972, the etiology of most episodes of gastroenteritis was unknown, and cases were attributed to a multitude of causes, including teething, weaning, diet, old age, drugs, and malnutrition, as well as infections. intensive investigation of enteric infections in the past three decades has resulted in the discovery of many new viral agents filling in the 'diagnostic gap' in diarrheal disease. with the identification of the norwalk virus, rotavirus, astroviruses, enteric adenoviruses, and other caliciviruses in the 1970s and subsequently, it has become increasingly clear that viruses cause a significant proportion of the enteric illnesses that did not earlier have a defined etiology. with improvements in sanitation and hygiene, and better standards of living, the proportion of diarrheal disease attributed to bacteria has decreased, resulting in an increase in the proportion of cases associated with viral infections. developments of new assays to identify viruses have also resulted in the ability to identify the viral etiology of episodes and epidemics of gastroenteritis. approximately 5 billion episodes of diarrhea occur worldwide annually, with virtually all children infected with the most common agents by the age of 3 years. widespread use of oral rehydration therapy in the past two decades has resulted in a significant decrease in mortality due to diarrhea, by about 50%, to 2.2 million annual deaths, occurring mainly in developing countries. infections with gastroenteritis viruses differ from bacterial enteric infections in that they affect children in both developing and developed countries, suggesting that they may also be transmitted by means unrelated to contaminated food or water. although feco-oral spread is the major route of transmission for all enteric viruses, transmission through contact, fomites, and a respiratory route has been suggested based on the recovery of these viruses from inanimate objects during outbreaks. the four distinct patterns of viral gastroenteritisendemic childhood diarrhea, outbreaks in closed communities, other food-or waterborne outbreaks among wider communities, and viral gastroenteritis in immunocompromised patients -reflect the differences in the pathogens, transmission, and host response. these have a direct bearing on strategies for prevention and control ( table 1) . the highest rates of viral gastroenteritis occur between 3 and 24 months of age. protection in early infancy is believed to be mediated by maternal antibodies, followed by acquisition of protective immunity through repeated exposure in early childhood. this pattern is seen with all viral enteropathogens. however, for the caliciviruses and astroviruses, immunity is not long-lasting, suggesting waning of immunity or lack of cross-protection between different viral strains. childhood diarrhea is best exemplified by the group a rotaviruses, but a similar pattern of infection and illness is seen with enteric adenoviruses, astroviruses, and sapoviruses. these agents infect children during the first few years of life, with first infections being symptomatic and protecting against subsequent disease. disease is caused by a limited number of specific serotypes and incidence decreases with increasing age. group a rotaviruses are the main cause of severe diarrhea in children under 5 years of age, and cause more than 130 million episodes per year throughout the world, and approximately 600 000 deaths annually. reports from europe, australia, and the united states indicate that rotavirus may be responsible for 20-60% of cases of gastroenteritis requiring hospitalization. recently, from asia, it has been estimated that 45% of gastroenteritis requiring admission is due to rotavirus, a higher percentage than previously recorded. of the 'non-group a' rotaviruses, group b rotavirus has been identified in epidemic outbreaks of severe diarrhea in adults in china and in symptomatic infections in children. outbreaks of diarrhea due to group c rotavirus have been identified in asia, europe, and south and north america, but are not common. human caliciviruses, consisting mainly of noroviruses and sapoviruses, are associated mainly with milder cases of gastroenteritis in children, causing greater than 20% of diarrheal disease in children in the community. in many studies, noroviruses are the second most common cause of gastroenteritis in children, following rotaviruses. enteric adenoviruses cause 10% of diarrheal disease in reports from developed countries and have a variable incidence of 2% to 30% depending on the region in developing countries. astroviruses were found in approximately 1% of cases, when electron microscopy was employed for detection, but with the availability of a commercial enzyme immunoassay and molecular techniques, the percentage of detection has increased in hospital and community settings to 5% to 10%. outbreaks in closed or semi-closed communities such as old-age homes, cruise ships, and hospitals are mainly due to caliciviruses. norovirus infections are a significant cause of outbreaks in adults in nursing homes and residential care facilities and can lead to an increased need for hospital care and increased mortality. nosocomial outbreaks occurring in hospitals have required the closure of wards in order to control infections. outbreaks due to noroviruses and to mixed viral infections have been reported among military personnel. outbreaks of norovirus gastroenteritis are also being recognized and are occurring with increasing frequency on cruise ships. attack rates as high as 30% have been observed among cruise ship passengers, and repeated outbreaks have continued even after cleaning and disinfection protocols were instituted on successive voyages. in addition to infections in adult patients, viral agents of gastroenteritis are an important cause of nosocomial infection in pediatric units. between 20% and 50% of cases of gastroenteritis caused by rotavirus in hospitals are considered to be of nosocomial origin, and nosocomial viral enteric infections have been documented in up to 6% of children admitted for more than 72 hours in both developed and developing countries. infections in older individuals are usually due to caliciviruses, although adenovirus infections have been documented. the prevalence and transmission of nosocomial infection may be explained by asymptomatic patients who excrete the virus and the relative resistance of these viruses to normal disinfectants. the microbiological contamination of food and water is a significant global problem. it is estimated that there are approximately 1.5 billion cases and over 3 million deaths worldwide annually. the microorganisms associated with about 50% of the foodborne disease outbreaks still go unrecognized, particularly those occurring in developing countries. the apparent failure to confirm a viral etiology in such outbreaks has been due largely to the lack of available tests, unavailability of food or water specimens, and the failure to report outbreaks of mild gastrointestinal disease. all of these factors have resulted in a drastic underestimate of the true scope and importance of foodor waterborne viral infection. the most common types of food-and waterborne viral disease are infectious hepatitis due to hepatitis a virus and acute viral gastroenteritis associated with the human caliciviruses. noroviruses, transmitted by the fecal-oral and the aerosol routes, are the most common cause of outbreaks of nonbacterial gastroenteritis in industrialized countries, but data from developing countries are lacking. noroviruses are responsible for an estimated 67% of all illnesses caused by known foodborne pathogens and for 96% of nonbacterial gastroenteritis in the united states. many outbreaks can be associated with the consumption of primarily or secondarily contaminated foods. shellfish and fruit implicated in outbreaks have been shown to be contaminated at the site where these foods are harvested or produced, whereas other foods, such as salads, cold foods, and sandwiches, have caused outbreaks after being contaminated by food handlers at the site of food preparation. shellfish, in particular oysters and clams that are raw or insufficiently cooked, is associated with noroviral outbreaks, frequently occurring because these shellfish filter contaminated seawater to feed and hence result in a concentration of virus. foodborne outbreaks due to rotaviruses, parvoviruses, and astroviruses are also occasionally reported. water is also a common source of outbreaks and may include water from municipal supplies, wells, recreational lakes, swimming pools, and ice machines. rotaviruses, caliciviruses, and some adenoviruses are important causes of waterborne disease outbreaks. post-recovery and secondary transmission are a particular concern in infections due to these agents. the main viral causes of severe gastroenteritis in immunosuppressed patients are cytomegalovirus (cmv) and epstein-barr virus (ebv), which mainly affect patients with aids and transplant recipients. cmv is a frequent pathogen in diarrhea associated with aids with cd4 counts under 100 cells/mm 3 . other viruses that produce hiv-associated gastroenteritis include astrovirus, picobirnavirus, calicivirus, and adenovirus. there is evidence of gastroenteritis due to astrovirus and adenovirus in both child and adult bone marrow transplant recipients. caliciviruses are now being increasingly recognized as a cause of chronic diarrhea in patients undergoing transplants. toroviruses have been found in association with diarrhea in immunocompromised children. criteria to define a virus as an etiologic agent of gastroenteritis include (1) the identification of the virus more frequently in study participants with diarrhea than in controls, (2) the demonstration of an immune response to the specific virus, and (3) the demonstration that the beginning and end of the illness correspond to the onset and termination of virus shedding, respectively. so far, these include human caliciviruses, rotaviruses, astroviruses, and the enteric adenoviruses. in immunocompromised patients, gastrointestinal cmv and ebv infections also cause significant morbidity. coronaviruses, toroviruses, the aichi virus, and picobirnaviruses have also been found to be associated with diarrhea in some studies, but definitive data are not yet available. similarly, in conditions such as hiv, it has been difficult to obtain definitive data on the role of enteric viruses in the causation of symptoms. the study of rotaviruses, enteric adenoviruses, and astroviruses has been facilitated greatly by the ability to propagate these viruses in cell culture, which has allowed the production of reagents for use in diagnostic studies, a better understanding of factors correlated with immunity to infection, and the elucidation of each virus's life cycle. although human caliciviruses have defied numerous attempts to propagate them in cell culture to date, recent developments in their study by using molecular biology techniques have increased our ability to diagnose and study infections due to these agents (see figure 1 ). rotaviruses are double-stranded rna viruses constituting a genus within the family reoviridae. the mature virus particles are triple layered, approximately 70 nm in diameter, and possess icosahedral symmetry. the rotavirus genome consists of 11 segments of doublestranded rna, which code for 6 structural viral proteins and 6 nonstructural proteins. of the nonstructural proteins, nsp4 is of particular interest, as it has enterotoxin-like activity and can induce diarrhea in mice. the classification of rotavirus into 7 different groups (a-g) is based on the antigenic specificity of the vp6 capsid proteins. of the 7 groups, only groups a, b, and c are known to infect humans. severe, life-threatening disease in children worldwide is caused predominantly by group a rotaviruses. variability in the genes encoding the two outer capsid proteins vp7 and vp4 forms the basis of the current strain typing of group a rotaviruses into g and p genotypes, respectively. all known g serotypes correspond with genotypes; more p genotypes than serotypes have been identified. the rapid evolution of rotaviruses by a variety of mechanisms provides one of the major challenges in epidemiological studies. these mechanisms include genetic drift, where an accumulation of point mutations generates genetic lineages leading to the emergence of antibody escape mutants, and genetic shift through gene reassortment during dual infection of a single cell. hence methods of virus typing need to be regularly monitored and updated to identify emerging novel strains of epidemiological importance. rotaviruses induce a clinical illness characterized by vomiting, diarrhea, abdominal discomfort, fever, and dehydration (or a combination of some of these symptoms) that occurs primarily in infants and young children and may lead to hospitalization for rehydration therapy. fever and vomiting frequently precede the onset of diarrhea. milder gastroenteric illnesses that do not require hospitalization are common. the highest attack rate is usually among infants and young children 6 to 24 months old. neonatal infections are largely asymptomatic. deaths from rotavirus gastroenteritis may occur from dehydration and electrolyte imbalance. the severity of diarrhea is measured by the vesikari score which includes duration and severity of diarrhea and vomiting, associated fever, and degree of dehydration. in older children and adults, rotavirus gastroenteritis occurs infrequently, although subclinical infections are common. rotaviruses also induce chronic symptomatic diarrhea in immunodeficient children. rotavirus infections can be severe and sometimes fatal in individuals of any age who are immunosuppressed for bone marrow transplantation. rotavirus infections have also been associated with necrotizing enterocolitis and hemorrhagic gastroenteritis in neonates in special-care units. recently, rotavirus antigenemia has been described early in infection in children requiring hospitalization and rna has been extracted from serum of antigenemic children and cerebrospinal fluid (csf) of children with seizures, but the clinical significance of these findings requires further investigation. rotaviruses infect the mature enterocytes on the tips of small intestinal villi, leading to villous atrophy with secondary hyperplasia of the crypts. it has been proposed that cellular damage is secondary to villous ischemia. the mechanism that induces the production of diarrhea is not well understood, although it appears to be mediated by the relative decrease of villous epithelium absorption in relation to the secretory capacity of the crypt cells, as well as the possible action of nsp4, the viral enterotoxin that has been shown to cause secretory diarrhea in rodents. there is a loss of intestinal permeability to macromolecules such as lactose, secondary to a decrease in disaccharidase in the intestine. the enteric nervous system is stimulated by this virus, leading to intestinal water and electrolyte secretion. the immunologic mechanisms responsible for protection against infection by rotavirus are still not well known. older children and adults usually have asymptomatic or mild infection unless an overwhelming infectious dose is delivered. several studies have shown that local intestinal immunity produced in response to infection protects against subsequent severe episodes of diarrhea. studies in children indicate that the antibody response in primary infection is homotypic with subsequent infections producing a broadening of the immune response. laboratory procedures for diagnosis of rotavirus include electron microscopy (em), passive latex agglutination assays (la), electropherotyping using polyacylamide gel electrophoresis (page), enzyme-linked immunosorbent assays (elisa), and reverse transcription-polymerase chain reaction (rt-pcr). in recent years, elisa has become the method of choice for screening. early studies on strain surveillance identified rotavirus serotypes using neutralization assays. monoclonal antibodies to specific serotypes were used. new methods have greatly improved data on circulating rotavirus strains and include multiplex rt-pcr based genotyping based on vp7 and vp4 genes, hybridization assays, and nucleotide sequencing. the term 'calicivirus' is derived from the latin calyx, meaning cup or goblet, and refers to the cup-shaped depressions visible by em. these cuplike depressions are more prominent in some strains, particularly the sapoviruses, leading to the characteristic star of david appearance from which caliciviruses get their name. caliciviruses (family caliciviridae) are a group of nonenveloped, icosahedral viruses with a single-stranded, positive-sense rna genome. the genome is 7.5 to 7.7 kilobases in length and has three open reading frames (orfs). noroviruses can be genetically classified into five different genogroups (gi, gii, giii, giv, and gv) which can be further divided into different genetic groups or genotypes. genogroup ii, the most prevalent human genogroup, presently contains 17 genotypes. genogroups i, ii, and iv infect humans, whereas genogroup iii is associated with bovine infections and genogroup v has recently been isolated in mice. noroviruses were named after the places where the outbreaks occurred. recently a numeric classification system has been proposed based on numbering genogroups with roman numerals and genotypes with numbers. for example, the genogroup ii norovirus, lordsdale virus, is a member of genotype 4, and therefore classified as a gii.4 norovirus. gii.4 viruses account for the majority of adult outbreaks of gastroenteritis and often sweep across the globe. sapoviruses, previously called the classical caliciviruses, based on their morphology, have 8 human genotypes in 5 genogroups, and have a similar system of strain designation as noroviruses. the incubation period for caliciviral infections is short, about 24 to 48 hours, and the mean duration of illness is 12 to 60 hours. nausea is prominent, with vomiting, nonbloody diarrhea, and abdominal cramps occurring in most cases. these symptoms are experienced by all age groups, but diarrhea is relatively more prevalent among adults, whereas a higher proportion of children experience vomiting. from 25% to 50% of affected persons also report headache, fever, chills, and myalgias. adults have died during illness caused by noroviruses, presumably from electrolyte imbalance. late sequelae have not been reported, but the elderly often report persistence of constitutional symptoms for up to several weeks. routes of transmission that have been documented include water, food (particularly shellfish and salads), aerosol, fomites, and person-to-person contact. infectivity can last for as long as 4 days after resolution of symptoms. presymptomatic shedding has been suspected on epidemiologic grounds but has not been proved in volunteer studies. other than a murine norovirus, noroviruses have not been grown in culture, making studies of pathogenetic mechanisms difficult. in studies carried out on volunteers, infection by calicivirus produces an expansion of the villi of the proximal small intestine. the epithelial cells remain intact with a shortening of the microvilli. the mechanism by which diarrhea is produced is unknown. in volunteer studies, infection by the norwalk virus induces a specific igg, iga, and igm serum antibody response, even in persons with preexisting antibodies. after norovirus infection, immunity appears to last for a few months but there is little or no evidence of long-term protection. volunteer studies conducted in the 1970s also suggest that some people are resistant to norwalk virus challenge. recently two host factors have been identified that may contribute to this resistance to infection. in volunteer studies homozygous recessives for the a (1,2) fucosyltransferases gene (fut2), who do not express h type-1 oligosaccharide were resistant to infection with norwalk virus (nonsecretors). there is also evidence to suggest that different norovirus strains bind to different blood group antigens. data on sapovirus infections and immune responses are not yet available. electron microscopy was initially used for identification of these viruses and continues to be used by many laboratories to screen stools for potential viral pathogens. this method is insensitive compared with molecular detection assays. currently, rt-pcr assays are the most common approach for establishing a diagnosis of norovirus infection. virus-specific primers are used to amplify conserved regions of the genome, usually in the polymerase or capsid genes. no single primer pair can detect all norovirus or sapovirus strains because of the high sequence diversity, but in most geographic regions, more than 90% of currently circulating strains can be detected using separate primer pairs for genogroups i and ii noroviruses and sapoviruses. antigen-detection elisa assays for noroviruses have been established in the last decade, but the first assays had a very narrow reactivity. more broadly reactive assays have been developed using monoclonal antibodies that recognize cross-reactive epitopes or multiple monoclonal antibodies, but are not widely used. serologic assays also have been developed to detect immune responses to infecting norovirus strains, but are used more in epidemiological studies than for diagnosis in individual patients. human astrovirus is the prototype of the astroviridae, a family of nonenveloped positive-sense rna viruses, measuring 38-41 nm. by direct em, astroviruses recovered from stool display a distinctive surface starlike appearance. the genome of astrovirus consists of positive-sense, single-stranded rna, 6.8 kb in length, organized in three orfs. all serotypes have at least three capsid proteins, p1, p2, and p3, with the p2 protein carrying the groupreactive epitopes and the p3 protein specifying serotype. astroviruses are classified into serotypes based on the reactivity of the capsid proteins with polyclonal sera and monoclonal antibodies. astroviruses can also be classified into genotypes on the basis of the nucleotide sequence of a 348-bp region of the orf2, and there is a good correlation with the serotypes. there are eight established genotypes. phylogenetic analyses have shown that it is common to find multiple astrovirus strains circulating in one region during a given period of time, and that there are also variations in the prevalent type with time, suggesting either a genetic shift or an introduction of new strains. serotype 1 is predominant in most studies, followed by 2, 3, 4, and 5. serotypes 6, 7, and 8 are rarely detected. clinically, these viruses cause similar symptoms to caliciviruses. like rotaviruses, astrovirus infections occur throughout the year with peaks in the winter months. infections have been shown to occur mainly in childhood. other studies showed that most of the cases of infection are detected in children under 5 years of age with the majority of the children being under 1 year of age. outbreaks of astrovirus infection involving children and elderly patients have been described and prolonged excretion documented in immunosuppressed, immunodeficient, and aids patients. significantly higher seroprevalence rates of astrovirus have been reported in adults exposed to contaminated water compared with a control group. the pathogenesis of the disease induced by astrovirus has not yet been established, although it has been suggested that viral replication occurs in intestinal tissue. in animal studies, atrophy of the intestinal villi is observed, as well as inflammatory infiltrates in the lamina propria leading to osmotic diarrhea. symptomatic astrovirus infection occurs mainly in small children and the elderly, which suggests both an acquisition of antibodies with increasing exposure and a reduction in antibodies with advancing age. studies in adult volunteers indicate that people with detectable levels of antibodies do not develop the illness, although epidemiological observations suggest that human astrovirus infections do not induce heterotypic immunity, as an episode of astrovirus diarrhea is not associated with a reduced incidence of a subsequent episode. em is an insensitive technique, because a high concentration of viral particles is required for detection and the typical five-or six-pointed star morphology is seen in less than 10% of particles. enzyme immunoassays have been developed including streptavidin-biotin assays for increased sensitivity of detection, and are used in most diagnostic laboratories. for epidemiological research, recently astrovirus-specific rt-pcr has been the screening method of choice. while some investigators have used highly sensitive primers targeted to conserved genomic regions coding for the nonstructural proteins and untranslated regions, others prefer to use primers from the capsid coding region which can be less sensitive but provide typing information. all adenovirus particles are nonenveloped, 60 to 90 nm diameter, with icosahedral symmetry easily visible in the electron microscope by negative staining, and are composed of 252 capsomers: 240 hexons and 12 pentons bearing fibers at the vertices of an icosahedron. the genome is linear, nonsegmented, double-stranded dna of 30 to 38 kbp. based on their immunologic properties, oncogenicity in rodents, genome, and morphology, adenoviruses are classified into six subgroups a through f with 51 serotypes. serotypes predominantly associated with human infections include h-40 and h-41, which belong to subgenus f, and occasionally h-31 in subgenus a. adenoviruses are widely recognized causes of respiratory, ocular, and genitourinary infections. however, serotypes 40 and 41 (previously called fastidious enteric adenoviruses) primarily affect the gut, contributing to 5% to 20% of hospitalizations for childhood diarrhea in developed countries. enteric adenoviruses have also been identified in pediatric gastroenteritis in developing countries. peak incidence is among children under 2 years of age, but older children and adults may be infected, with or without symptoms. incubation is between 3 and 10 days, with illness lasting 1 week or longer, longer than for other enteric viral pathogens. diarrhea is more prominent than vomiting or fever, and respiratory symptoms are often present. the lesions produced by serotypes 40 and 41 in the enterocytes lead to atrophy of the villi and compensatory hyperplasia in the crypts, with subsequent malabsorption and loss of fluids. a neutralizing antibody response made in response to infection results in control of disease and protection from reinfection with the same serotype. asymptomatic virus excretion can continue for prolonged periods even after an antibody response is documented in acute infection. although adenoviruses can be grown in culture, little data are available on the pathogenetic mechanisms of these agents of viral gastroenteritis. traditionally, adenoviruses have been detected and typed by em, virus culture, and neutralization assays. these assays are time-consuming, and more rapid serological assays including immunofluorescence, enzyme immunoassays, and latex agglutination have been developed. the rapid assays are useful in the diagnostic laboratory, but do not generally distinguish between serotypes. pcrbased techniques are more sensitive and relatively rapid, but have been shown to give discrepant results when compared with serotyping by neutralization. torovirus is a genus within the coronaviridae family, and toroviruses are known causes of diarrhea among cattle. these viruses have an envelope of 100-140 nm, with a helicoidal capsid and a single-stranded positive-sense rna genome. torovirus was detected for the first time in human gastroenteritis in 1984. they are associated with persistent and acute diarrhea in children, and may represent an important cause of nosocomial diarrhea. coronaviruses are well-established causes of diarrhea in animals and respiratory disease in humans. these viruses are between 60 and 220 nm, with helicoidal symmetry, a spiculated envelope which gives them the appearance of a crown, and a genome with positive-sense single-stranded rna. they have been identified in the stool of persons with gastroenteritis (usually children under 2 years of age), but human controls have been found to shed them with higher frequency, raising doubt about their etiologic role in human diarrhea. picobirnaviruses are small viruses, without an envelope, 30-40 nm in diameter, with an icosahedral capsid and a genome made up of two or three segments of bicatenary rna. reports from brazil documented human cases of diarrhea caused by picobirnavirus, which had been thought to be a cause of diarrhea only in animals. the importance of this pathogen is unknown, but it has been found in association with hiv and cryptosporidium-infected individuals. aichi virus, in the genus kobuvirus, was first recognized in 1989 in oyster-associated nonbacterial gastroenteritis in humans. aichi virus appears to morphologically resemble astroviruses when examined by em. recently aichi virus was isolated from pakistani children and from japanese travelers with gastroenteritis returning from tours of south-east asian countries. twenty-three percent of children under 2 years of age with gastroenteritis of unknown etiology were antigenpositive for pestivirus, compared with 3% of controls, in a study on an american indian reservation. no further studies have been done on the role of this agent in gastroenteritis. parvovirus-like particles have been identified by em in stool specimens of both well and ill persons in britain. the relationship of these particles to disease is unclear, but they have been associated with shellfish-related outbreaks of gastroenteritis. enteroviruses cause a wide spectrum of disease, in which gastroenteritis plays a minor role. although the entry of polio, coxsackie, echo, or other enteroviruses through the gut may cause incidental mild diarrheal symptoms, the spread of the virus through the bloodstream to other organs (e.g., central nervous system, heart, pleura, pancreatic islets) produces major disease manifestations. reports have linked some enteroviruses to illnesses in which diarrhea was the sole symptom; nevertheless, an outbreak or case of gastroenteritis should not be attributed to an enterovirus merely because it was isolated in the stool of an affected person. treatment of viral gastroenteritis is symptomatic, and its aim is to prevent or treat the dehydration secondary to the disease. dehydration is assessed using blood pressure, pulse, heart rate, skin turgor, fontanelle depression, mucous membranes, eyes, extremities, mental status and activity, urine output, and thirst. assessment of dehydration, particularly in children in community studies or by field workers, relies on lethargy, restlessness, appearance of eyes, skin turgor, and feeding/thirst. fluid and metabolic imbalances must be assessed and corrected. the most important factor predicting adverse outcome of viral gastroenteritis is delay in fluid and electrolyte therapy. clinically significant dehydration can occur within 6 hours of onset of illness, especially during primary infection in children. malnutrition, malignancy, and immunodeficient states predispose to a more severe episode of illness or unremitting diarrhea that can persist until the underlying condition is corrected. oral rehydration therapy is recommended for preventing and treating early dehydration and continued replacement therapy for ongoing losses. intravenous therapy is required in severe dehydration, shock, and decreased consciousness. in children, age-appropriate diet should be continued during oral rehydration and following intravenous rehydration. anti-emetics, anti-diarrheal agents, and antibiotics should not be given to children, although anti-emetics and anti-diarrheals may be used in adults. studies have shown that antirotavirus immunoglobulin as bovine hyperimmune colostrum or human milk may decrease the frequency and duration of rotavirus diarrhea. probiotics such as lactobacillus casei gg and saccharomyces boulardii reduce the frequency and duration of diarrhea. racecadotril, an enkephalinase inhibitor, has been shown in some studies to be useful in treating rotaviral diarrhea in children. zinc supplements have been suggested to reduce severity and duration of illness. preventive measures can limit the number of episodes of viral gastroenteritis both within the home and in institutions. diaper-changing areas should be separate from food preparation areas. diapers should be disposed of directly in the changing area and should be placed in closed bags. hands should be washed after contact with soiled diapers and clothing. interruption of transmission of the infection is extremely important, especially in hospitals and centers that care for small children. therefore, it is necessary to reinforce hygiene measures, such as handwashing, and clean all surfaces with suitable disinfectants. as viruses do not replicate outside a host, decreasing the potential inoculum is key to preventing further infection of susceptible hosts. further preventive measures in childcare facilities and hospitals include isolation and cohorting of ill children. asymptomatic infections probably also play an important role in the spread of infection. studies with vaccines against group a rotavirus began in 1982. the first vaccine developed was the tetravalent human-rhesus reassortant vaccine, which induces protection against the four main rotavirus serotypes, g1-g4. efficacy studies showed a reduction in the appearance of severe gastroenteritis caused by rotavirus in vaccinated children, and the vaccine was approved in the united states in 1998. however, the detection of an increase in the risk of intussusception after vaccination led to its suspension. in 2006, two licensed vaccines became commercially available. these are rotateq, a live oral attenuated pentavalent vaccine from merck which is recommended by the cdc advisory committee on immunization practices to be given at 2, 4, and 6 months with the last dose administered no later than 32 weeks, and rotarix from glaxosmithkline, a human strain derived monovalent live oral vaccine given in two doses at 2 and 4 months. rotateq is licensed in the united states and rotarix in many countries in europe and south america. these vaccines need to be evaluated in settings where there is marked diversity of rotaviruses and infection occurs at a younger age, as in developing countries. owing to the high cost of these vaccines, vaccine manufacturers in developing countries have initiated the process of formulating and testing vaccines based on local strains, which may address the issues of cost and heterotypic immunity in settings where different viruses circulate. dna-based and virus-like particle vaccines may also provide an alternate method of prevention in the future. studies on virus-like particle-based vaccines against norwalk virus have also been initiated with capsid proteins expressed in plants inducing an antibody response in experimental models. however, these potential vaccines are still in preclinical development. in summary, viral gastroenteritis is a major cause of morbidity in developed countries and mortality in developing countries. hygiene is the first preventive step in viral gastroenteritis and rehydration is the key to management of clinical illness. the range of organisms that can cause gastroenteritis is immense and vaccines will be important in reducing the impact of childhood gastroenteritis. also: adenoviruses; arboviruses; enteroviruses foodborne illnesses: overview infectious diarrhea in developed and developing countries viral gastroenteritis: perspectives in medical virology foodborne viruses: an emerging problem enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools gastroenteritis viruses: an overview probiotics for children: use in diarrhea human astrovirus diagnosis and typing: current and future prospects norovirus disease: changing epidemiology and host susceptibility factors viral infections of the gastrointestinal tract probiotic therapy of intestinal inflammation and infections clinical trials of rotavirus vaccines in europe -national institute of diabetes and digestive and kidney diseases s h wedner and d a ross, london school of hygiene and tropical medicine, london, uk 2008 elsevier inc. all rights reserved. micronutrient deficiencies -most notably vitamin a, zinc, iodine, and iron deficiencies -are a major public health problem globally, with low-income countries in africa and asia carrying the highest burden of disease. young children and pregnant and breastfeeding women are the main groups affected, because their relative requirements for micronutrients are higher and thus the impact of deficiency is more severe than in other population subgroups. micronutrient deficiencies have also been described as hidden malnutrition, as subnormal levels of key: cord-167157-z0lvcb3z authors: wang, xiubin bruce; ma, chaolun title: controlling the hidden growth of covid-19 date: 2020-05-19 journal: nan doi: nan sha: doc_id: 167157 cord_uid: z0lvcb3z the covid-19 pandemic has plagued the world for months. the u.s. has taken measures to counter it. on a daily basis, newly confirmed cases have been reported. in the early days, these numbers showed an increasing trend. recently, the numbers have been generally flattened out. this report tries to estimate the hidden number of currently alive infections in the population by using the confirmed cases. a major result indicates an existing infections estimate at about 10-50 times the daily confirmed new cases, with the stringent social distancing policy tipping to the upper end of this range. it clarifies the relationship between the infection rate and the test rate to put the epidemic under control, which says that the test rate shall keep up at the same pace as infection rate to prevent an outbreak. this relationship is meaningful in the wake of business re-opening in the u.s. and the world. the report also reveals the connections of all the measures taken to the epidemic spread. a stratified sampling method is proposed to add to the current tool kits of epidemic control. again, this report is a summary of some straight observations and thoughts, not through a thorough study backed with field data. the results appear obvious and suitable for general education to interested policymakers and the public. the outbreak of the cornonavirus covid-19 is an unfortunate incident in the 21st century that suddenly plagued numerous countries. it has brought much of the world economy to a halt. countries quickly motioned to put it under control. it is an anxious, agonizing process checking the newly confirmed cases of infection and death each day for such an extended, long time. people have observed italy, spain, and other countries after china that have been inflicted by this pandemic. now the u.s. became the seemingly world epicenter with a total confirmed infections exceeding one and half million [1] . as people watch on the daily spread of this pandemic, few know when this pandemic will be put to an end or whether it will spiral out of control again, although pundits worldwide have publicized many findings and predicts. what the general public observe each day are newly confirmed cases from day to day as well as the cumulative totals. the trend of the newly confirmed cases each day was increasing in the early days and is now pretty flattened out overall under the prevalent shelter-in-place policy [2] . what does this trend mean ? we try to answer the question and also examine a condition under which a stable, flat trend is sustainable. such an examination appears especially meaningful as the u.s. and the world approach to massive business reopening. additionally, in a later summary of all measures taken as of now, this report tries to clarify how they each take effect in the control of this pandemic in connection to the condition we identify here. this report proposes a sampling method for testing the communities. it does not discuss the feasibility of the proposed measures in terms of its technical and fiscal constraints. this report did not result from an intensive study. it does not contain field data but only reveals some inherent structural relationships. in an attempt to understand the driving factors of the epidemic spread, our perspective is different from most mainstream literature of epidemiology. the literature in epidemiology such as [3, 4, 5] generally simulate the interactive process of epidemic transmissive behaviors in a closed region by also considering population migration in and out in order to duplicate the transmissive process. the simulation realizes the mechanism expressed in an array of partial differential equations [6, 7] , in which the transmission rate is affected by the percentage of the infected in a nonlinear manner. similar to the literature in epidemiology such as [8, 9] , we try to understand and gauge the hidden world of the infected population, including the latent and active ones that are not detected yet. however, our method to estimate the undetected number of currently latent and active infections is based on the publicized numbers of the daily confirmed cases without having to resort to the epidemic process simulation. our result is simpler and easier to understand and use. note that this note treats the latent and active as in one undetected group with a comprehensive infection rate. we allow a different aggregate infection rate for each day. our weakness is that this report takes infection rate and detection rate as given parameters. this report does not study their specific values. current literature report that the total infected population could be six to ten times the cumulative total confirmed [9] . our finding shows that the total currently latent and active cases can be six to sixty times the incremental daily confirmed total in today's situation that the daily confirmations seem on a flat trend. this findings implies the total infections from 1 to about 10 times the total confirmed infections. one may also gauge the growth or declination of the infections in light of a necessary and sufficient condition for epidemic control that we have found in this report. in the remainder of this report, we will detail our discussion and derivation of the result. first, we describe the problem in a technical term that we believe characterizes the epidemic process well. table 1 tabulates the notation in the report. we first introduce the epidemic problem. problem statement there is a group of infected people on day one. out of them, n 1 is confirmed and quarantined. on day i, each infected case that is not confirmed and quarantined, referred to as hidden cases, grows at a daily rate of r i into more cases. at the end of each day i, hidden cases each have a probability p i being detected and put into quarantine while the undetected cases continue the infection the next day. the growth rate of r i is the net growth rate, which is the difference between the new infections deducted by the recovered and dead. day by day over a period of time, newly confirmed cases are reported by the number n 1 , n 2 , ..., and n n .the study is to use the daily confirmed numbers of infections to estimate the hidden infections and their trend of growth or declination. the goal is to propose measures for the epidemic control. clearly, there are a number of infections hidden out in the communities undetected. note that the hidden cases here are referred to as undocumented in [9] . the number of hidden cases is the primary interest here. be aware that the growth rate r i here is different from the transmission rate in the conventional epidemiology literature [9] , where the transmission rate is the number of infections that a currently active one may incur per day during the short span of active period. we assume that the confirmed cases have been perfectly quarantined and have lost their ability of further infection. one may consider their remnant infectious ability, even if under quarantine, is implicitly incorporated into the infectious capability of the hidden cases by allowing for a slightly higher rate r i . we simplify the process from the perspective of someone outside epidemiology by assuming each infected case has an equal average growth rate, which may not be accurate. one may take this growth rate as an aggregate measure of all the hidden cases as a whole. note we include the latent also in the hidden cases. for covid-19, some reports declare that the latent cases may also be infectious. the net infection (or net growth) rate is random. it is reasonable to assume a constant average rate with fluctuations (e.g., random outcomes) between days for a period over which the test means, ability, and policy do not change significantly. we assume a detection rate p i on day i to represent a percentage of the infections that are confirmed and quarantined on the day. the detection rate has to do with many measures, such as contact tracing of newly confirmed cases. the detected cases are the confirmed cases that the public observes in the daily report. each day, the reported cases are the tip of an iceberg, which public opinions and public policies are generally based upon. let's show the growth process of the infected population in the hindsight. assume the epidemic moves forward according to the numbered days, day 1, 2, ..., n, n+1,.... again, there are n 1 new cases confirmed on day 1. clearly, there holds p n = n 1 where n ≥ 2 as on table 2 . on table 2 , infected population is the number of infected people including those detected and quarantined on the day, the latent and the active but undetected, excluding the confirmed/quarantined on prior days. the theoretically detected new shows the analytical relationship of the daily confirmed cases to the total infected population. observed new is the daily confirmed cases in the public report. the following result becomes obvious. if we use ∆ n as the theoretically detected new on day n, clearly one can reach the following result, combining equations (1) and (2), we get a major result as illustrated in the following equation. again, p i is an outcome of a random variable at a constant (average) rate during a stable epidemic period. to simplify, one may simply assume that p n and p n+1 just represent their expected value. within two consecutive days, if there are no dramatic changes to the sampling methods and regulating policies for detecting the infected cases, one may assume p n ≈ p n+1 , which explains the approximation in equation (4) above. observation 1 when the newly confirmed cases stay flat from day n to day n + 1, the total infected cases on the two consecutive days may be deemed to roughly stay flat. again, the total infected population on day i mentioned in observation 1 excludes the detected and quarantined on prior days. observation 1 is trivial in the sense that an equal number of newly confirmed cases and an equal probability of detection naturally allude to an equal size of infected populations on two days. the above discussion also implies: our interest is in estimating the total infected population at the end of a day, the vast majority of whom are the undetected, active and latent infections. let take a look at a special case, which is roughly true in the u.s. today, where n n+1 nn ≈ 1.0. in this case, we have (1 − p n )(1 + r n ) ≈ 1.0, on which much the discussion ensued is based. we summarize the basic result here. the sufficient and necessary condition to keep an epidemic from growing is to satisfy the following condition: when (6) is in equality, the epidemic reaches an equilibrium, meaning the total hidden plus newly confirmed remain a stable population. as a special case, at low infection rates such as during shelter-in-place, the required detection rate of the infected is roughly equal to the infection rate in order to control the total infected population from growth. one knows, with social distancing and shelter-in-place orders, (1 + r n ) remains very low and yet above 1.0 as one may assume safely for the nation. then one can roughly estimate the detection probability p n needed. proposition 1 is intuitive and obvious. if (1 + r n ) is very close to 1.0 from values above 1.0, one may say that p n is close to zero from values also above zero when a stable trend of daily confirmed cases is observed. if p n is very close to zero, in a conservative estimation, say p n ≈ 0.01, the total hidden infected population would be about 100 times larger than the latest average daily confirmed amount. the closer (1+r n ) is to 1.0, the closer p n is to zero, and the larger the total infected population compared with the stable daily confirmed cases. note that in our simple discussion here, it is impossible to separate the effect of r n with p n in the growth (1 + r n )(1 − p n ). therefore, we have no certainty about the values of p n and r n respectively. researchers can only conduct additional studies in order to have separate estimates of the two variables. for public information, there are several scenarios about the guess at the total hidden cases, ranging from the most conservative to the most aggressive. note that these guesses of the infected total on the day (excluding prior confirmed) is under the circumstance of having flattened new cases each day. • conservative in this case, we assume a growth rate r n to be as reasonably large as possible. in a scenario in which each patient infects 2.5 others during an active period of 14 days, the daily infection rate appears to be 2.5 14 = 0.18 1 , meaning r n = 1.18. in this case, if the total new confirmation remains relatively stable, implying (1 − p n )(1 + r n ) ≈ 1.0, p n ≈ 0.1515, the total active cases of infection on that particular day would be nn pn ≈ 6.6n n . • moderate in the case that the infection rate, under fairly strict social distancing and shelter-in-place practices, has dropped to a third of that under the normal unrestricted social life as above, where (1 + r n ) ≈ 1.039, (1 − p n )(1 + r n ) ≈ 1.0 reveals p n ≈ 0.038, implying nn pn = 26.64n n is the total infected on day n. • aggressive this scenario is one that practices very stringent social distancing and shelter-in-place orders, let's assume that the infection rate is controlled to a tenth of the rate for the unrestricted case, meaning (1 + r n ) ≈ 1.018. in this scenario, if the total new confirmations each day remains flat (hypothetically), the equation (1 − p n )(1 + r n ) ≈ 1.0 alludes to p n ≈ 0.018. this means that the total infections in the population on day n is nn pn ≈ 56.5n n . the above example scenarios, all assuming the epidemic is under control by having the total newly confirmed cases flat over a period of days, indicate a large number of currently active and latent infections in the population ranging from 6 to 50/60 times of the daily confirmations. today, the daily confirmation within the u.s. has been in the range of twenty to thirty thousand cases. our simple result suggests that the total current infections in the communities, excluding the quarantined, probably would be in the range of 0.5 to 1.0 million. in the case that schools and businesses are reopened in the near future, where people reasonably practice social distancing and follow other published preventive guidelines, a new situation between the conservative and moderate scenarios listed above, if the new infection has flattened out or has reached a new equilibrium, the total hidden infections alive on the day would reasonably be in a range of 10-20 times the daily confirmed cases. of course, the daily confirmed cases after reopening is expected to be higher than under shelter-in-place. with reopening, the infection rate r n would be larger than when the shelter-in-place was enforced, probably by a few hundred percentage. the growth rate likely will be kr where k ≥ 2 if we use r for the growth rate during shelter-in-place. in order to have the total infected population non-increasing to form a new equilibrium, in light of equation (4), the newly confirmed cases shall not increase from day to day based on the average trend, which roughly means n n+1 nn ≈ 1.0. note that under shelter-in-place, one roughly has r i ≈ p i on day i, which corresponds to an equilibrium condition (1 + r) × (1 − p) ≈ 1.0, where p represents the stable detection rate during shelter-in-place. under business reopening, (1+r n )(1−p n ) = (1+kr)×(1−mp) is the new infection growth rate, where mp is the new detection rate required to control the infected population from growing, and m ≥ 1.0. if we have m ≈ k, we would have (1+r n )(1−p n ) = (1+kr)×(1−mp) ≈ (1+r)×(1−p) ≤ 1.0. the above logic assumes p n and r n are both small and that p n r n ≈ 0. to summarize, the following result appears to hold by itself. proposition 2 in a switch from shelter-in-place to business re-opening, to prevent the infected population from growing, if the infection rate becomes k times larger, the probability of having the infected to be detected shall be about k times larger accordingly. what does this larger detection probability mean? with social distancing and shelter-in-place, a low infection rate allows an equilibrium with a low detection rate, which is a balance between the new infections (recovery and death are considered a negative increase) and the ones quarantined. with business re-opening, the infection rate would probably spike, say by k times, if the proportion of the population being checked or sampled (or an equivalent of it) remains unchanged as under shelter-in-place, the infected population would explode. the rate of explosion, in light of the discussion at the beginning of this subsection, would be (1 − p n )(1 + r n ) ≈ 1 + (k − 1)r. there will be no equilibrium between the newly infected and the newly detected if the detection rate falls behind the infection rate. the practical meaning of this observation may be recapped as follows. we do not have a specific value for the daily infection rate r n before and after the business re-opening, which needs to be specially studied by professionals. with business re-opening, if the chance of detecting an infected is not keeping pace precisely with the infection rate increase, the infected population will keep growing. worthy of a note is that the detection rate here is the probability of a random infection in the community to be tested based on some mechanism (such as social contact tracing, body temperature check at some key locations, random sampling in the communities, etc.). this report is not in a complete agreement with some media reports such as [14] , which advocates for an absolute increased total number of tests. we think the sample rate or its equivalent is the factor effective to epidemic control. it's about the percentage of the people who shall be tested and quarantined, not the absolute number. again, stringent social contact tracing is an effective means to raise the percentage of infections to be quarantined. in this section, we briefly summarize the current strategies in dealing with covid-19. the previous section makes it clear that to put the epidemic under control, one must have inequality (6) satisfied. at the very minimum, (6) shall be an equality. all the measures so far undertaken by governments and other entities fall into two categories: controlling the infection rate and controlling the detection rate. • containing the infection rate the measures in this category include: shelterin-place, specially social distancing, quarantine policy, all means of disinfection or sanitation, medical treatment, etc. immunization as the final solution may be considered as a special means, • improving the detection rate this category includes social contact tracing, population sampling, test kits of improved accuracy, additional means such as quick antibody test, body temperature measurement as a pre-screening means for virus test, etc. it appears that much more can be done at the front of increasing detection rate. the difficulty is in the fact that to identify an infected resembles a witch hunt due to the randomness or uncertainty. google and apple are reported working together to develop social media applications for (voluntary) tracing social contact and for alert of potential danger of infection, which serves both purposes of reducing infection and increasing detection rates [15, 16] . contact tracing of an infected person remains a valid means. if contact tracing is effective, it equivalently increases the detection rate and decreases the infection rate simultaneously. stringent contact tracing is a proven means in other countries such as south korea. as mentioned shortly above, the public body temperature measurement through automatic machines when the public pass through key locations may prove to be an extremely beneficial means to greatly increase the detection rate of the infected. here, we propose a sampling method as an additional but not a substitute means to the existing measures. in light of condition (6), a sample rate that is equal to the infection rate, or other means such as very stringent social contact tracing that achieve an equivalent effect, would work. we mean here for a random sampling of the public. this random sampling may also be coupled with enhanced contact tracing of the community or social groups of newly confirmed infections. we propose a stratified sampling by prioritizing the high-risk groups. there are statistics in the u.s. to show different infection rates for population strata according to ethnicity, age, or other criteria ([1] [2] ). if the sampling cost is too high, a low-cost screening process may be first implemented such as a temperature check. temperature check at major traffic locations such as airports and subway stations is an effective pre-screening means. we propose a sample rate proportionate to the infection percentage of the strata weighted by the varying costs, one of the costs being life-threat. obviously, the aged groups, especially those with complications, have a more severe consequence from the infection. to understand the necessity of sampling in public, one might consider such a question: would social contact tracing alone meet the needs of control without sampling of the population? vast deployment of body temperature measurement machines is an example of large population sampling. there may be a debate here. but in our view, periodic sampling to a certain extent, preferably on a daily basis, is necessary to an enhanced social contact tracing. consider social networking in the context of covid-19. figure 1 illustrates this general idea. this social network (via proximity connection) contains the most effective entities in terms of spreading the virus. an individual may be an entity shown as a string that connects multiple dots (or nodes). an entity may be a household in which the household members are considered fully communicated internally in terms of virus infection. a string accounting for a household (e.g. the connected links of the network) may be connected to many other nodes such as shopping malls, schools, plants, etc. there are also nodes that many entities traverse. nodes and strings all have varying attributes that contribute to the virus spread to different degrees. schools, including k-12 and college education when face-to-face classes are resumed may be considered high-risk nodes. families of active members exposed to close contact with a large number of others are also considered as strings with large weight. the goal of dealing with this network is to identify the nodes and strings that may effectively circumvent the spread of covid-19. although much needs to be studied in future through the academic community regarding covid-19 spread, much can now be done by governments and people in their responsible positions. these include: identify critical locations with most social connections, identify entities that connect most critical locations (e.g. household with multiple members working at shopping malls, factories, attending schools simultaneously). a major job of the governments and administrations would be to implement measures that impede or this is a preliminary discussion of covid-19 spread in the process of test and quarantine. the main focus is about the general conditions needed to put the epidemic under control, a situation in which the total infected population size does not grow or even shrink. the results are intuitive and straightforward. this document may, therefore, serve as an educational material to the public or the government officials in positions relevant to disease control. the major finding is that the effective detection rate should keep up in pace with the infection rate such that (1 + r)(1 − p) ≤ 1.0. this finding implies that the detection rate under business re-opening shall keep pace with the expectedly spiking infection rate. this discussion has touched on the means in two categories, detection and infection rates control, respectively. enhanced social tracing may be an effective means of increasing the detection rate. the detection rate here is the probability of a random infection in a community to be selected and tested for infection random sampling based on strata is also proposed, which emphasizes the strata likelihood and also strata severity of infection. a principle is that the sample rate shall be consistent with the strata infection rate weighted by infection consequence. note that direct sampling for infection test may turn out to be too costly. therefore, vast deployment of automatic body temperature measurement machines at key locations with large volume of traffic, especially traffic that risk transmitting virus among regions or communities is necessary as a pre-screening step of social sampling. another interesting finding is that when the daily confirmed cases remain relatively stable a trend, an equilibrium has likely formed between infection and detection. this means (1 + r)(1 − p) ≈ 1.0. under social distancing and shelter-in-place, the infection rate is low, and obviously, the detection rate p is low as well. one may estimate the total alive infections hidden out in the communities of up to 50 times the daily confirmations. note that the same day total alive infections of 50 times the daily confirmation is equivalent to a total cumulative infections of about 10 times the cumulative daily infections. this estimate may serve as an alert to the policymakers when they prepare for business reopening. those undetected infections would incur a higher infection rate during business re-opening and require a higher detection rate accordingly. the needed higher detection rate would come from a heightened contact tracing system, which is desirably coupled with new community sampling practice. in fact, the infection rate and detection rate are both random numbers from day to day. therefore, the ratio between confirmed cases on two consecutive days may fluctuate around 1.0, not stably equal to a constant 1.0. the infection and detection rates may be taken as the expected values and be used for the trend analysis. if a model fully considers the randomness of the two rates, a reliable condition to control the epidemic is likely to be (1 + r)(1 − p) < 1.0 − ǫ, where ǫ is a small positive number representing a little room to allow for the randomness and errors in the process. without further extensive studies, the two parameters used here, r and p, would not be known exactly. however, the relationship and the qualitative meaning of the results are sound. this discussion was driven by interest, it did not involve extensive, data rich effort which are often backed by projects. therefore, the limitations of this report are multitude. we hope this report may bring its audience to an operations research perspective for this epidemic control. coronavirus disease 2019 (covid-19): cases in the u covid-19 united state cases the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study interventions to mitigate early spread of sars-cov-2 in singapore: a modelling study pattern of early human-to-human transmission of wuhan real-time forecasts of the covid-19 epidemic in china from estimating and simulating a sird model of covid-19 for many countries, states, and cities. no. w27128 why is it difficult to accurately predict the covid-19 epidemic? substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov-2) incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia pandemics explained: hghi and npr publish new state testing targets coronavirus phone tracing by apple and google could help america reopen. usa today, gannett satellite information network apple and google have a clever way of encouraging people to install contact-tracing apps for covid-19. the verge, the verge using social network analysis to assess communications and develop networking tools among climate change professionals across the pacific islands region this study is a self-motivated effort with partial support from a e.b. snead '25 career development professor i fund through the zachry department of civil and environmental engineering at texas a&m university. errors and mistakes are solely the authors'. key: cord-278682-s4gfbsqy authors: chan, w-m; liu, d t l; chan, p k s; chong, k k l; yuen, k s c; chiu, t y h; tam, b s m; ng, j s k; lam, d s c title: precautions in ophthalmic practice in a hospital with a major acute sars outbreak: an experience from hong kong date: 2005-04-29 journal: eye (lond) doi: 10.1038/sj.eye.6701885 sha: doc_id: 278682 cord_uid: s4gfbsqy many new infectious diseases in humans have been derived from animal sources in the past 20 years. some are highly contagious and fatal. vaccination may not be available and antiviral drugs are not effective enough. infectious control is important in clinical medicine and in ophthalmology. severe acute respiratory syndrome (sars), as an example, is a highly contagious respiratory disease that has recently been reported in asia, north america, and europe. within a matter of weeks, the outbreak has evolved to become a global health threat and more than 30 countries have been afflicted with a novel coronavirus strain (sars-cov) that is the aetiologic agent of sars. the primary route of transmission of sars appears involving close person-to-person contact through droplets. ophthalmologists may be particularly susceptible to the infection as routine ophthalmic examinations like direct ophthalmoscopy and slit-lamp examination are usually performed in a setting that has close doctor–patient contact. being the ophthalmology department of the only hospital in the world that has just gone through the largest outbreak of sars, we would like to share our strategy, measures, and experiences of preventing contracting or spreading of sars infection as an infection control model. sars is one of the many viruses against which personnel will need protecting in an ophthalmic setting. the experiences attained and the measures established might also apply to other infectious conditions spreading by droplets such as the avian influenza with h5n1. severe acute respiratory syndrome (sars) is the 'modern plague' of the new era for its infectivity, novelty, casualty, and high fatality. 1, 2 hong kong, guangdong province, some other provinces of china, singapore, taiwan, vietnam, canada, and the whole world are now under unprecedented threats from this deadly infectious disease. [1] [2] [3] [4] [5] [6] [7] [8] the culprit virulent agent is believed to be a novel coronavirus strain (sars-cov) that is not closely related to any of the previously characterized coronavirus, according to the world health organization (who) and local infection control experts. 4, 5 the awful impact of this disease can be reflected by the alarming health-care statistics recorded since the massive outbreak in hong kong on march 12, 2003 . the accumulative number of sars infection by october 15, 2003 was 1755 with a total number of deaths of 299 and among them eight of the deceased were doctors, nurses, and health-care assistants. 7 sars has unveiled many virgin health-care issues that have not caught the attention of the general public in the past and the whole healthcare system in hong kong has been significantly percolated and bombarded by sars in terms of changes in daily practices and everyday routines. ophthalmology is no exception. the ophthalmologist, as one of the frontline health-care providers, has to inevitably face these new challenges and the ever-changing expectation upon our profession from patients and the community. several clinical problems are particularly pertaining to the ophthalmic practice. firstly, what we can be certain right now is that this novel coronavirus-associated atypical pneumonia is extremely infectious as reflected by the high prevalence of infected health-care workers. by october 15, 2003 , 386 of 1755 patients (21.9%) were health-care workers of hospitals/clinics or medical students. most of them had a history of close contact with known or finally known sars patients. 7 it is still unclear whether ophthalmologists are at increased risk of infection in examining a proven or suspected sars patient with short working distance, especially in performing direct ophthalmoscopy and slit-lamp examination. secondly, the route of transmission is still enigmatic and the relative infective roles of different body fluids are still under active study. 1, 2 droplets and fomites are established main routes of transmission. stool and urine have also been implicated as the possible media in harboring the infective agent. [1] [2] [3] tears and eye discharges are body secretions that may be equally or potentially more hazardous to health-care personnel, particularly ophthalmologists. in fact, tears have been reported by who to be one of the body fluids conveying the novel sars-cov, although the infectivity or clinical significance is still not known for the time being. 8 the use of different instruments, which are in direct contact with patients' conjunctival mucosal surface may serve as possible routes and sources for disease transmission. the ultimate infectivity of the tears secretion and ocular discharge from sars patients may bring impacts on not only the daily ophthalmic practice but also the universal infection control measures practiced by general public and health-care workers. being the ophthalmology department of the only hospital in the world that has just gone through the largest outbreak of sars, we would like to share our strategy, measures and experience of preventing sars infection. in early march 2003, shortly after the sars outbreak within a medical ward in the prince of wales hospital, hong kong, a 39-year-old lupus woman admitted under eye ward for elective operation of wound revision of an enucleated eye developed a high fever of 39.21c on postoperative day 1 after an uneventful operation. supplementary oxygen was required to alleviate the shortness of breath and oxygen desaturation. chest radiography showed bilateral lung parenchymal infiltrates without consolidation. respiratory physician recognized her condition as a probable case of sars that required intensive care unit admission with ventilatory support. at 3 days prior to the present admission, she had been seen by two of our colleagues in the outpatient consultation room. immediate notification of the infection control unit of the hospital was performed. the sars command centre of the department of health of hong kong and the hospital authority consisting of infection disease clinicians, microbiologists, and epidemiologists were notified as well. in accordance with the infection surveillance protocol, the clinic doctors and nurses, who had a close contact with the patients, were required to have compulsory blood check for cell counts and chest radiography despite the fact that they did not exhibit any symptoms on initial assessment. the investigations were normal but they still have to be put on home quarantine for 7 days. subsequently, the policy was further geared up to 10 days home quarantine for those health-care workers having unprotected contacts of similar setting. the diagnosis of the patient was finally revised and confirmed to be lupus pneumonitis with systemic flare-up. she succumbed on day 11 after admission. with such an experience, we have consolidated both the infection control and containment measures with respect to in-patient and outpatient ophthalmic services. the level of infection control and precautions could be specifically targeted according to the level of regional epidemic, different clinical settings, and scenarios as follows. the eye clinic is one of the most crowded areas, with many patients. suboptimal infection control strategies in the clinic may inadvertently help the spread of sars infection and jeopardize the whole community infection containment policy. we have used modified who cases classification schemes in handling our eye patients in the clinic. three categories of patients are recognized based on a triage system run by experienced nurses. they are the general, suspect, and probable categories. [3] [4] [5] general categories in attending all ophthalmic outpatients, irrespective of their sars status, staff are encouraged to follow the universal precaution measures consisting of wearing surgical/n95 respirator mask, wearing gloves in handling blood, body fluids, secreta and excreta, wearing water-repellant or water-resistant gowns and goggles (visor) during splashes or sprays generating procedures such as measuring noncontact tonometry (nct) ( table 1 ). the most important of all is frequent hand washing, preferably with the use of chlorhexidine alcoholic handrub (0.5-1.0% chlorhexidine in 80% ethyl alcohol, or a similar commercial product, such as hexol s ) in the prevention of crossinfection. the gloves, if used for high-risk procedures, must be changed and hand hygiene must be performed in between each contact with different patients. staff are strongly advised not to touch their face-shield, eye protective wear, mask, head, and neck region before a thorough and complete hand washing procedure. visitors are restricted in the hospital or clinic complexes unless under exceptional circumstances such as the escort of demented or visually impaired patients or parents of small children. 3 suspect and probable categories for known cases of sars inpatients, the outpatient attendance is deemed contraindicated because of significant risk of crossinfection. any ophthalmic consultation should be completed within the quarantine ward instead. if an outpatient presented with overt sars symptoms and signs, immediate attention from the respiratory physicians and infection control officers should be summoned. the ophthalmic appointment should be deferred indefinitely until the status of infection is ascertained. for patients presenting with fever, contact history of sars patients within 10 days, or recovered sars patients shortly discharged from hospital, we have adopted a special outpatient follow-up strategy based on initial epidemiological and microbiological evidences that the main mode of transmission of sars is by droplets, direct contact with patient's secretions, and subsequent inoculation into mucous membranes. 3 infection control measures accentuating droplet precautions, with the application of barrier apparels and environmental cleaning as well as universal precautions, being the pivot of success in containing the infection, are of paramount importance. the personal protective equipment (ppe) with disposable caps, n95 respirator, goggles, face-shields, gloves, top and pants, and long protective gowns with high neck and long cuffed sleeves should be worn all the time when attending to these patients (table 1) . under circumstances such as between cases and immediately following a high-risk procedure, all health-care workers have to abide by the decontamination process consisting of removal of all potentially contaminated protective wear in proper sequence and putting on clean protective wear. once again, hand washing is strongly advised after removal of gloves and before attending another patient in order to minimize risk of crossinfection. it is very advisable that the suspect category patients be seen during the last time slot at which most of the patients of the general category have left the clinic already. a specifically designated consultation room with separate set of examining instruments and independent ventilatory system is highly desirable. the ventilatory system is preferably of exhaust fan type with filter, generating a negative room pressure with lamellar downward airflow. during the ophthalmic examination, patients themselves are required to put on the n95 respirator mask as well. self-adhesive surgical plastic sheets that are commonly used during cataract surgery wear gloves. discard gloves, wash or alcohol-rub the hands and then put on new gloves in-between case wear glove in high-risk procedure general categories: for all patients attending the ophthalmic outpatients in which the sars status is not certain. suspect and probable categories: for any known or suspect cases of sars from the history, symptoms or signs. high-risk procedures in ophthalmology: include those with short-distance contact, prolonged contacted, and involving splashes or sprays generating actions. direct ophthalmoscopic examination, irrigation and probing, nasal endoscopic examination, laser photocoagulation, measurement by noncontact tonometry, ophthalmic surgeries are some examples. can be applied directly but lightly over the face and the nostril areas, leaving behind the eyes for examination. this innovative infection control measure specific for eye examination can help preventing any splashing of droplets from inadvertent coughing or deep breathing. in sars, sars-cov could be transmitted through the contaminated hands and instruments in addition to droplets. the strong survival ability of sars-cov over surfaces of commonly used materials in domestic and working environments have been quantitatively proved by in vitro models. 9 therefore, the disinfection of the ophthalmic instruments as well as the environmental surfaces in frequent contact with patients are deemed as one of the key infection containment measures. susceptibility of human coronaviruses 229e and oc43 to chemical disinfection has been demonstrated by biological viral titration assay, 10, 11 whereas similar in vitro data concerning the efficacy of different hospital disinfectant products (us environmental protection agency (epa) registered disinfectants) against this novel coronavirus (sars-cov) is currently lacking. 12 nevertheless, we can still make use of the available chemical germicides that could inactivate related viruses with similar physical and biochemical properties as the possible sars agents. the adopted disinfection protocols in our ophthalmic practice are in line with the recommendation by the centers for disease control & prevention 13 and based on the scientific evidences provided by apic guidelines. 14 ophthalmic instruments especially those in direct contact with patients' mucosal membranes like the goldmann applanation prism tip and goldmann contact lens require intermediate level of disinfection. 14 they should be immediately and separately disinfected with 2% alkaline glutaraldehyde (cidex s ) for 20 min at room temperature or 6% hydrogen peroxide after use. 14 frequent (at least daily) disinfection of the apparatus (slit lamp, binocular indirect ophthalmoscope, etc) with hypochlorite (naocl) solution (1000 ppm, 1 in 50 dilution of 5.25 % household bleach, for 2 min) is mandatory. 14 the examination table and slit lamp will be cleaned and disinfected thoroughly before another patient is seen in that environment. surfaces that cannot be easily cleaned, such as keyboards of desktop computers, should be covered within plastic sheeting, wiped between cases, and changed at the end of the day. if the plastic wraps are grossly soiled with patient's tears or ocular secretion, they should be immediately removed and replaced. daily cleaning and disinfection of the environmental surfaces (eg, door knobs, phone, lavatory facilities) that have been frequently touched by patients and health-care workers are specifically targeted, in accordance with the recommendations by the cdc. 12, 13 unless it is otherwise indicated, the consultation waiting time and overall consultation time ought to be maintained at a safe minimum. after all, the outpatient attendance should be judiciously kept at a low level so as to curtail the risk of crossinfection. the different wards in hospital have been stratified into 'high risk' and 'all other areas'. high-risk areas refer to intensive care unit (icu), sars ward, infection triage or cohort fever ward, accident and emergency department, admission or observation wards. eye the eye ward belongs to the category of 'all other areas'. all patients admitted to the eye ward will be screened for symptoms and signs of sars by the house eye surgeon or resident (figure 1 ). any positive findings like fever, recent contact history or travel history to epidemic area will call for an admission to a centralized observation ward or infection triage ward instead. patients will be observed for at least 24 h and will be examined by the respiratory physician in addition to radiological investigation of the chest (chest radiography or computerized tomography thorax). suspect or probable cases will be transferred to another infection quarantine ward for further observation and management. once again, universal infection control and containment measures are practiced at all times. the ppe with the same level as 'suspect & probable categories of sars' in opd should be adopted (table 1) . ophthalmic consultation from other wards or specialties every consultation is screened by an ophthalmologist and the degree of urgency of the consultation is determined. for nonurgent consultations, appropriate outpatient appointment will be granted accordingly, provided patients can be discharged from hospital for at least 10 days before the appointment. in case of ophthalmic emergency or semiurgent cases (acute primary or secondary glaucoma, penetrating ocular trauma, alkali chemical injuries and the like), patients will be seen accordingly in their original wards. transfer of in-patients to the eye outpatient department or to the eye ward for the purpose of examination is regarded as absolutely contraindicated for the sake of preventing cross infection. we have arranged our eye colleagues to examine the patients within their original wards by a portable slit lamp, tonopen, and binocular indirect ophthalmoscope. proper and thorough disinfection of the instruments will be carried out by eye ward nursing staff after each consultation. in dealing with suspect or probable sars patients in other wards, ophthalmologists should maintain a high index of caution with the full standard barrier apparels or protective attire in carrying out short-range procedures or examinations. nonurgent ophthalmic operations like cataract operations, eyelid surgeries and squint extraocular muscle surgeries, and nonurgent ophthalmic interventions performed in outpatients such as barrier laser, pan-retinal photocoagulation, yag : nd laser capsulotomy, probing and syringing, incision and curettage will be performed selectively and proper precautions ought to be taken. ocular emergencies such as macula-on rheugmatogenous retinal detachment, ruptured eyeball and intraocular foreign body will be dealt with by arranging an emergency operation. the operating theater is regarded as a high-risk area for sars infection and universal precaution measures with barrier apparels (n95 and surgical mask, goggles, plastic apron, operating theater gown, and gloves) will be strictly practiced. given the facts that the sars-cov is highly infectious, and the observation that none of the staff and patients has been infected within the department of ophthalmology, in both in-patient and outpatient areas throughout this massive outbreak, it is an encouraging and vindicating efficacy of the precautions adopted. we, as ophthalmologists, had also safely performed several clinical studies involving direct patient contacts, close examinations and performance procedures of such as conjunctival scrapping, during the sars attack. 15, 16 universal precautions during the sars period are the basic measures. in a case-control study among 254 hong kong health-care workers with documented exposure to sars patients, none of the 69 staff reporting use of four infection control measures, namely mask, gloves, gowns, and hand washing, was infected. 17 on the contrary, at least one of the four measures had been omitted (p ¼ 0.0224) among all 13 infected staff. in a comparison between infected and noninfected cohorts, health-care personnel practicing hand washing (p ¼ 0.011) and personal protective equipments (wearing masks and gowns) were significantly associated with fewer infections. these data provide, at least to a certain extent, scientific rationale for our infection control precautions. ophthalmologists will inevitably get into contact with the tears and sometimes conjunctival mucosa of patients and unlike other body fluid or secreta, 1,2 the infectivity of tears from sars patients remains elusive in spite of preliminary studies. 15, 18 in the face of these uncertain infective risks and inadvertent close contact with patients, ophthalmologists may need to maintain extra vigilance with regard to infection control. in real life, the ophthalmic practices in the midst of the sars outbreak have been changed. the ophthalmologists in hong kong have abandoned the direct ophthalmoscopic examination in view of its short working distance. in ultrahigh risk patients proven to have sars, safer and easily accessible investigative tools like fundus photography might be an alternative to conventional fundus examination. the knowledge obtained from the sars outbreak in our hospital has been remarkable. it demonstrates the significance of infection control in clinical practice. strict infection control and containment at the appropriate level according to the infectivity, route of transmission, morbidity, and fatality caused by the suspected organism is important in preventing crossinfection. conclusions sars infection has swept through the continents and caused hundreds and thousands of mortality and morbidity. the cumulative numbers of probable sars worldwide by sept 26, 2003 was 8098, in which 1707 (21%) were health-care workers. a total of 774 patients died from the disease with a fatality ratio of 9.6%. 19 it was first recognized as a global threat in mid-march 2003, but was successfully contained in less than 4 months with international cooperation. on 5 july 2003, who reported that the last human chain of transmission of sars had been broken. 20 the epidemic, however, is not yet over. firstly, sporadic confirmed and probable sars-cov infection have been reported and it is important to note that the source of infection in the most recent case is still unknown. 21 secondly, even with the aid of molecular epidemiological techniques such as genetic fingerprinting, the natural reservoir, host, vector and media of transmission of sars-cov, is still enigmatic to mankind. 22, 23 owing to lack of a clear understanding of the epidemiological characteristics, a complete eradication of the sars-cov is remote and the world anticipates ongoing new cases. thirdly, mutation analysis has demonstrated continuous evolutional change of the genetic constitutions of sars-cov, which may encourage emergence of more virulent strains in a community without immunity against this pathogen. 24 with an interference of the human-animal ecology, more new virulent species are imminent, sars-cov infection is an example, and avian-flu is another. countries that were not involved during the heat of the outbreak have recently stepped up this national sars preventive measures. 25 in the postoutbreak period, all health-care workers, including ophthalmologists, should remain vigilant for the recurrence of sars and always incorporate risk-based infection control measures in care provision. sars is one of the many viruses against which personnel will need protection in an ophthalmic setting. the experiences we have learnt also apply to other highly infectious disease, which can be spread by means of droplets. 26 avian influenza a (h5n1) is an example. 27 it is a viral infection that can affect humans and cause serious disease and death. 27, 28 there is still no solid evidence of human-to-human transmission in most of the outbreaks involving human cases. 27 however, a recent clustering of cases in a family, probably resulting from person-to-person transmission during unprotected exposure to the critically ill index patient was recorded. 28 these findings indicate that inefficient transmission is possible and reinforce the importance of infection control precautions among health-care workers. 29 if h5 viruses do persist, they will likely continue to evolve, or reassort with existing human influenza viruses, to become more virulent and more easily transmitted subtypes from person-to-person. 26, 30 we must be well prepared for any h5n1 outbreaks as it is difficult to eradicate the existing virus in nature with the potential reservoirs in wild birds. 30 strict adoption of the regularly revised infection control guidelines in accordance to the updated clinical, epidemiological, and laboratory information is the only way to prevent further large-scale hospital outbreak or crossinfection. a major outbreak of severe acute respiratory syndrome in hong kong a cluster of cases of severe acute respiratory syndrome in hong kong hospital authority guideline on infection control of severe acute respiratory syndrome (sars) world health organization. case definitions for surveillance of severe acute respiratory syndrome (sars) severe acute respiratory syndrome (sars): multi-country outbreak-update 56 what everyone should know about severe acute respiratory syndrome (sars) latest figures on severe acute respiratory syndrome update 27 f one month into the global sars outbreak: status of the outbreak and lessons for the immediate future stability of sars coronavirus in human specimens and environment and its sensitivity to heating and uv irradiation survival of human coronaviruses 229e and oc43 in suspension and after drying on surfaces: a possible source of hospital-acquired infections chemical disinfection of non-porous inanimate surfaces experimentally contaminated with 4 human pathogenic viruses center for disease control and prevention. interim recommendations for cleaning and disinfection of the sars patient environment centers for disease control & prevention. supplement i: infection control in healthcare, home, and community settings apic guideline of selection and use of disinfectants tears and conjunctival scrapings for coronavirus (sars-cov) in patients with sars ocular screening in severe acute respiratory syndrome effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) the severe acute respiratory syndrome coronavirus in tears world health organization. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) world health organization. alert, verification and public health management of sars in the post-outbreak period new case of laboratoryconfirmed sars in guangdong, china -update 5 molecular epidemiology of the novel coronavirus that causes severe acute respiratory syndrome isolation and characterization of viruses related to the sars coronavirus from animals in southern china comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection japan steps up sars plans outbreak of avian influenza a (h5n1) virus infection in hong kong in 1997 probable person-toperson transmission of avian influenza a (h5n1) antibody response in individuals infected with avian influenza a (h5n1) viruses and detection of anti-h5 antibody among household and social contacts influenza a (h5n1) in hong kong: an overview key: cord-269627-mx1mjdqc authors: thiry, etienne; addie, diane; belák, sándor; boucraut-baralon, corine; egberink, herman; frymus, tadeusz; gruffydd-jones, tim; hartmann, katrin; hosie, margaret j.; lloret, albert; lutz, hans; marsilio, fulvio; pennisi, maria grazia; radford, alan d.; truyen, uwe; horzinek, marian c. title: feline herpesvirus infection. abcd guidelines on prevention and management date: 2009-07-31 journal: journal of feline medicine & surgery doi: 10.1016/j.jfms.2009.05.003 sha: doc_id: 269627 cord_uid: mx1mjdqc abstract overview feline viral rhinotracheitis, caused by feline herpesvirus (fhv), is an upper respiratory tract disease that is often associated with feline calicivirus and bacteria. in most cats, fhv remains latent after recovery, and they become lifelong virus carriers. stress or corticosteroid treatment may lead to virus reactivation and shedding in oronasal and conjunctival secretions. infection sick cats shed fhv in oral, nasal and conjunctival secretions; shedding may last for 3 weeks. infection requires direct contact with a shedding cat. disease signs feline herpesvirus infections cause acute rhinitis and conjunctivitis, usually accompanied by fever, depression and anorexia. affected cats may also develop typical ulcerative, dendritic keratitis. diagnosis samples consist of conjunctival, corneal or oropharyngeal swabs, corneal scrapings or biopsies. it is not recommended that cats recently vaccinated with a modified-live virus vaccine are sampled. positive pcr results should be interpreted with caution, as they may be produced by low-level shedding or viral latency. disease management ‘tender loving care’ from the owner, supportive therapy and good nursing are essential. anorexic cats should be fed blended, highly palatable food – warmed up if required. mucolytic drugs (eg, bromhexine) or nebulisation with saline may offer relief. broad-spectrum antibiotics should be given to prevent secondary bacterial infections. topical antiviral drugs may be used for the treatment of acute fhv ocular disease. the virus is labile and susceptible to most disinfectants, antiseptics and detergents. vaccination recommendations two injections, at 9 and 12 weeks of age, are recommended, with a first booster 1 year later. boosters should be given annually to at-risk cats. for cats in low-risk situations (eg, indoor-only cats), 3-yearly intervals suffice. cats that have recovered from fhv-associated disease are usually not protected for life against further disease episodes; vaccination of recovered cats is therefore recommended. shedding. kittens may therefore acquire the virus very early on. the outcome of the infection depends on the level of maternally derived antibodies (mda) they possess. when high levels are present, kittens are protected against disease and develop subclinical infection leading to latency; in the absence of sufficient mda, they may develop clinical signs. 4 in healthy small populations, the prevalence of viral shedding may be lower than 1%, and in large populations, especially when clinical signs are present, it may reach 20%. [5] [6] [7] this low prevalence probably reflects the intermittent nature of viral shedding during latency. in shelters, risks are higher: with 4% of shedders entering the shelter, 50% of cats may excrete the virus 1 week later. 8 the virus enters the cat's body via the nasal, oral or conjunctival routes. it causes a lytic infection of the nasal epithelium with spread to the conjunctivas, pharynx, trachea, bronchi and bronchioles. lesions are characterised by multifocal epithelial necrosis with neutrophil infiltration and inflammation. a transient viraemia associated with mononuclear cells has been observed exceptionally in neonates or hypothermic kittens, as fhv replication occurs preferentially at lower temperatures. 2 viral excretion starts 24 h after infection and lasts for 1-3 weeks. acute disease resolves within 10-14 days. some animals may develop chronic lesions in the upper respiratory tract and ocular tissues. the virus spreads along the sensory nerves and reaches neurons, particularly in the trigeminal ganglia, which are the main sites of latency. almost all infected cats become lifelong carriers. there is no easy diagnostic method to recognise latency, because the viral genome persists in the nucleus of the infected neurons without replication. reactivation with virus shedding can be induced experimentally by glucocorticoid treatment in approximately 70% of cats. other reactivating stressors include lactation (40%) and moving into a new environment (18%). 4, 8, 9 some adult cats may develop lesions at the time of viral reactivation. disease as a consequence of reactivation is referred to as 'recrudescence'. conjunctivitis may be associated with corneal ulcers, which may develop into chron-ic sequestra. stromal keratitis is a secondary immune-mediated reaction due to the presence of virus in the epithelium or stroma. damage to the nasal turbinates in acute disease predisposes some cats to chronic rhinitis. 2 passive immunity acquired via colostrum maternally derived antibodies protect kittens against disease during the first weeks of life, but in general levels are low in fhv infections. antibody may persist for up to 10 weeks, but in some studies about 25% of the kittens became mda-negative at only 6 weeks of age. 10, 11 active immune response natural fhv infection does not result in solid immunity as seen, for example, in feline panleukopenia virus infections. in general, the immune response protects against disease, but not against infection, and mild clinical signs have been observed following reinfection only 150 days after primary infection. virus neutralising antibody (vna) titres are often low and rise slowly -they may still be absent 40 days after infection. 12 it is likely that vna neutralises incoming virus during acute infection, and contributes to antibody-dependent cellular cytotoxicity and antibody-induced complement lysis. 13 as with other alphaherpesviruses, cellmediated immunity plays a very important role in protection, since vaccinated cats without detectable antibody are not necessarily susceptible to disease. by contrast, seroconversion has been shown to correlate with protection against virulent fhv challenge. 14 in these cases, antibodies may serve as an indicator of cellular immune responses, since t lymphocytes are required for the maintenance of b lymphocyte function. since fhv is a pathogen of the respiratory tract, mucosal cellular and humoral responses are significant. 15 although a correlation exists between fhv antibodies and protection against clinical signs, there is no test available to predict protection in individual cats. latent chronic infection is the typical outcome of an acute fhv infection, and intermittent reactivation gives rise to viral shedding in oronasal and conjunctival secretions. in general, the immune response protects against disease, but not against infection, and mild clinical signs have been observed following reinfection only 150 days after primary infection. feline herpesvirus infection typically causes acute upper respiratory and ocular disease (table 1) , which can be particularly severe in young kittens. erosion and ulceration of mucosal surfaces, rhinitis and conjunctivitis are common; occasionally, corneal dendritic ulcers are seen, which are considered pathognomonic (fig 1) . 16 typical clinical signs include fever, depression, anorexia, serous or serosanguineous ocular and/or nasal discharge, conjunctival hyperaemia, sneezing and, less frequently, salivation and coughing (fig 2) . secondary bacterial infection is common and secretions then become purulent (fig 3) . in particularly susceptible kittens, primary pneumonia and a viraemic state have been identified that can produce severe generalised signs and eventually death (fig 4) . 1 less frequently, oral and skin ulcers, dermatitis and neurological signs are observed. 1, 17 abortion is rare and, in contrast to other herpesvirus infections, not a direct consequence of viral replication. after virus reactivation and recrudescence, some cats may show acute cytolytic disease, as described above; others progress to chronic ocular immune-mediated disease. experimental evidence suggests that stromal keratitis with corneal oedema, inflammatory cell infiltrates, vascularisation and eventually blindness are the result of this pathogenic mechanism. 16 corneal sequestra and eosinophilic keratitis have been linked to the presence of fhv in the cornea and/or blood, but some affected cats have been found to be virus-negative. 18 viral dna has also been detected in the aqueous humour of a larger proportion of cats suffering from uveitis, as compared with 19 chronic rhinosinusitis, a frequent cause of sneezing and nasal discharge, has been associated with fhv infection. viral dna is detected in some affected cats, but is also found in control animals. 20 the virus does not replicate, suggesting that chronic rhinosinusitis is initiated by fhv infection and perpetuated by immune-mediated mechanisms. inflammation and remodelling then lead to the permanent destruction of nasal turbinates and bone, complicated by secondary bacterial infection. 21 often, fhv infection occurs in combination with feline calicivirus (fcv) and/or chlamydophila felis, bordetella bronchiseptica, mycoplasma species, staphylococcus species or escherichia coli infection, causing a multi-agent respiratory syndrome. 1 the preferred method for virus detection in biological samples is pcr. virus isolation is still a valid method for detecting infectious fhv, but is more time consuming. the sensitivity and specificity of the tests differ between laboratories because there is no standardisation. conventional pcr, nested pcr and real-time pcr are now routinely used by diagnostic laboratories to detect fhv dna in conjunctival, corneal or oropharyngeal swabs, corneal scrapings, aqueous humour, corneal sequestra, blood or biopsies. [22] [23] [24] most primers are based on the highly conserved thymidine kinase gene. molecular methods seem more sensitive than virus isolation or indirect immunofluorescence [ebm grade i]. 22, 25 since minute amounts of viral nucleic acids are detectable by pcr, they may or may not be associated with disease. positive test results should therefore be interpreted with caution. pcr may even detect viral dna in scrapings of the cornea and/or tonsils in non-productive infections. 19 consequently, its diagnostic value may be poor, depending also on the samples analysed (corneal scrapings and biopsies are more frequently positive than conjunctival ones) and the population tested (shelter cats are more likely to test positive than household pets). furthermore, pcr detects fhv dna in modified-live virus vaccines, though it is unknown whether vaccinal strains are detected in recently vaccinated animals and, if so, for how long. 26 a positive pcr result may represent lowlevel shedding or viral latency, and does not necessarily link fhv with the observed clinical signs, although it may predict future recurrence of signs. however, when quantitative real-time pcr is used, the virus concentration measured may provide additional information: high viral loads in nasal secretion or tears suggest active replication and fhv involvement in the clinical signs [ebm grade ii]. 23 if low copy numbers are detected in corneal scrapings, this would indicate a latent infection. virus isolation -that is, growing fhv in cell culture -is the traditional alternative to pcr. it is less sensitive than pcr but reveals viable virus, not just its dna. it also allows the simultaneous detection of fcv. in primary fhv infections, the virus is readily isolated from conjunctival, nasal and pharyngeal swabs or scrapings, or from postmortem lung samples. when the cause of disease has to be identified in chronic infections, virus isolation is more difficult. asymptomatic carriers can be detected by virus isolation, but both positive and negative predictive values of virus isolation are low. 9, 19 samples must be collected before fluorescein or rose bengal stain has been used on the patient. 27 samples should be sent swiftly or under refrigeration to the laboratory. for these logistical reasons, virus isolation is not used routinely for the diagnosis of fhv infection, despite its sensitivity in cases of acute disease. feline herpesvirus-specific proteins can be detected by immunofluorescent antibody (ifa) assay on conjunctival or corneal smears or biopsies. as with virus isolation, fluorescein instillation should be avoided before sampling, as this may give false-positive results. in chronic infections especially, ifa assay is less sensitive than virus isolation or pcr. 25 for the clinician, pcr diagnosis is more jfms clinical practice 551 convenient, because fluorescein can be used and samples can be mailed at ambient temperature. 16 it also allows for simultaneous detection of other feline respiratory and ocular pathogens, especially c felis and, less reliably, fcv. 24, 28 antibody detection antibodies to fhv can be detected in serum, aqueous humour and cerebrospinal fluid by serum neutralisation assay or elisa. 11, 19 owing to natural infection and vaccination, seroprevalence is high in cats, and the presence of antibodies does not correlate with disease and active infection [ebm grade i]. 19 moreover, serology does not distinguish between infected and vaccinated animals. neutralising antibodies appear 20-30 days after primary infection, and titres may be low, both in cases of acute and chronic disease. serology, therefore, is of only limited value in the diagnosis of fhv infection. 16 restoration of fluids, electrolytes and acid-base balance (eg, replacement of potassium and bicarbonate losses due to salivation and reduced food intake), preferably by intravenous administration, is required in cats with severe clinical signs. food intake is extremely important. many cats will not eat because of loss of their sense of smell or ulcers in the oral cavity. food should be highly palatable and may be blended and warmed up to increase the flavour. appetite stimulants (eg, cyproheptadine) may be used. if the cat does not eat for more than 3 days, a feeding tube should be placed. to prevent secondary bacterial infections, broad-spectrum antibiotics that achieve good penetration into the respiratory tract should be given in all acute cases. nasal discharge should be wiped away using saline and a local ointment. mucolytic drugs (eg, bromhexine) may be helpful. eye drops or ointments can be administered several times a day. nebulisation with saline can be used to combat dehydration of the airways. vitamins are given, though their value is unclear. antiviral drugs recommended for the treatment of acute fhv ocular disease are listed in table 2 . other drugs have been proposed for the treatment of fhv ocular infections, including bromovinyldeoxyuridine, cidofovir, famciclovir, hpma (n-[2-hydroxy propyl] methacryla mide), penciclovir, ribavirin, valaciclovir, vidarabine, foscarnet and lactoferrin. 37 the efficacy of these drugs is not supported by published data although recent data demonstrate the efficacy of topical ocular application of cidofovir on primary ocular fhv. 29 feline herpesvirus infection is common and may induce severe, and at times fatal, disease. the abcd therefore considers fhv to be a core vaccine component and recommends that all cats are vaccinated (see box on page 553). feline herpesvirus is a particular problem in cat shelters, and management measures to limit or contain the infection are as important as vaccination. where incoming cats are mixed with residents, high infection rates ensue. as a rule, therefore, newcomers should be quarantined for 2 weeks and kept individually -unless they are from the same household. shelter design and management should aim to avoid crosscontamination, and new cats should be vaccinated as soon as possible. if there is a 552 jfms clinical practice particularly high risk (eg, a recent rhinotracheitis episode), a modified-live virus vaccine is preferable, as it provides earlier protection. if acute respiratory disease is noted, laboratory identification of the agent with differentiation between fhv and fcv can be useful in designing appropriate preventive measures. in breeding catteries, fhv can cause major problems. the infection surfaces most often in young kittens before weaning, typically at around 4-8 weeks of age, as mda wane. the virus source is often the mother whose latent infection (carrier state) has been reactivated following the stress of kittening and lactation. clinical signs can be severe and frequently involve all kittens in the litter. mortality can occur, and some recovered kittens are left with chronic rhinitis. vaccination of the queen will not prevent this problem, because it will not prevent her from becoming a carrier. however, if she has a high antibody titre, the kittens will benefit from mda in the colostrum, which should provide protection for the first weeks of life. booster vaccinations of queens may therefore be indicated and should be given before mating. vaccination during pregnancy may be considered only as an exception. feline herpesvirus vaccines are not licensed for use in pregnant cats and an inactivated product may be preferable in these cases. breeding management plays a crucial role in vaccination protects against disease, but not necessarily against infection. however, it can reduce virus excretion upon infection. 2 all fhv vaccines currently marketed are divalent products and include fcv components (in some countries) or, more commonly, cocktails of other antigens. both modified-live and inactivated parenteral vaccines are available. subunit fhv vaccines and modified-live intranasal vaccines are no longer available in europe. there is no reason to choose any particular vaccine over another for routine vaccination, particularly as they are all based on the same single fhv serotype. modified-live vaccines retain some virulence and may induce clinical signs if administered incorrectly (eg, by accidental aerosolisation or spillage on the skin). post-vaccination serology is of limited value for predicting protection. methodological issues complicate titre comparisons, and cats that have not seroconverted have nevertheless been found to be protected. 14,30 following exposure to field virus, vaccinated cats usually show an anamnestic response. the abcd recommends that all kittens are vaccinated against fhv. maternal immunity can interfere with the response, and the primary course is therefore usually started at around 9 weeks of age, although some vaccines are licensed for earlier use. kittens should receive a second vaccination 2-4 weeks later, at around 12 weeks of age. this protocol has been developed to ensure optimal protection. in assessing currently available scientific evidence, the abcd recommends that boosters are given at annual intervals to protect individual cats against fhv field infection. an informed decision should be taken on the basis of a risk-benefit analysis; annual boosters are particularly important for cats in high-risk situations or environments. however, for cats in lowrisk situations (eg, indoor-only cats without contact with other cats), 3-yearly intervals are recommended. experimental studies and serological data from the field indicate that immunity against fhv lasts longer than 1 year in most vaccinated cats [ebm grade ii]. 38 however, for many cats this is not the case. in the field, most cats tested have either had titres against fcv and fpv, or have shown an anamnestic response after booster, but about 30% of the population had no detectable antibody against fhv and about 20% failed to show an anamnestic response after booster vaccinations. 14,30 assessment of the duration of immunity is complicated: vaccination does not provide complete protection even shortly after vaccination, and the degree of protection decreases with time. if booster vaccinations have lapsed, a single injection suffices if the interval since the last vaccination is less than 3 years; if it is more than 3 years, two vaccinations are recommended. boosters using fhv products from another manufacturer are acceptable. cats that have recovered from feline viral rhinotracheitis may not be protected against new disease episodes. because the cause of the clinical signs will not have been identified, and the cat may experience infections with other respiratory tract pathogens, vaccination of recovered cats is also recommended. the abcd considers vaccines that protect against fhv infections as being core. in breeding catteries, the virus source is often the mother whose latent infection (carrier state) has been reactivated following the stress of kittening and lactation. controlling fhv in catteries. queens should kitten in isolation, and their litters should not mix with those of other cats until they have been fully vaccinated. early vaccination should be considered for litters from queens that have had infected litters previously. the earliest age for which fhv vaccines are licensed is 6 weeks, but vaccination from around 4 weeks of age may be considered (kittens are already immunocompetent at that age), with repeated injections every 2 weeks until the normal primary vaccination course is started. vaccines cannot immunise animals with a compromised immune function, such as those with systemic disease, virus-induced immunodeficiency, nutritional deficits or combined genetic immunodeficiency, those receiving immunosuppressive drug therapy, or experiencing prolonged stress. although such patients should preferably be shielded from exposure to pathogens, this may be unattainable, and hence vaccination is considered. based on safety considerations, inactivated preparations are recommended in this situation. infectious disease of the dog and cat feline herpesvirus feline herpesvirus type 1 (feline rhinotracheitis virus) transmission of feline viral rhinotracheitis isolation of feline respiratory viruses from clinically healthy cats at uk cat shows a study of feline upper respiratory tract disease with reference to prevalence and risk factors for infection with feline calicivirus and feline herpesvirus factors associated with upper respiratory tract disease caused by feline herpes virus, feline calicivirus, chlamydophila felis and bordetella bronchiseptica in cats: experience from 218 european catteries common virus infections in cats, before and after being placed in shelters, with emphasis on feline enteric coronavirus experimental induction of feline viral rhinotracheitis (fvr) virus re-excretion in fvr-recovered cats vaccination against feline viral rhinotracheitis in kittens with maternally derived feline viral rhinotracheitis antibodies enzyme-linked immunosorbent assay for detection of feline herpesvirus 1 igg in serum, aqueous humour, and cerebrospinal fluid the dose response of cats to experimental infection with feline viral rhinotracheitis virus observations on recovery mechanisms from feline viral rhinotracheitis use of serologic tests to predict resistance to feline herpesvirus 1, feline calicivirus, and feline parvovirus infection in cats effects of a single dose of an intranasal feline herpesvirus 1, calicivirus, and panleukopenia vaccine on clinical signs and virus shedding after challenge with virulent feline herpesvirus 1 update on pathogenesis, diagnosis, and treatment of feline herpesvirus type 1 ulcerative facial and nasal dermatitis and stomatitis in cats associated with feline herpesvirus-1 detection of feline herpesvirus 1 dna in corneas of cats with eosinophilic keratitis or corneal sequestration evaluation of serologic and viral detection methods for diagnosing feline herpesvirus-1 infection in cats with acute respiratory tract or chronic ocular disease investigation of nasal disease in the cat -a retrospective study of 77 cases assessment of infectious organisms associated with chronic rhinosinusitis in cats high sensitivity polymerase chain reaction assay for active and latent feline herpesvirus-1 infections in domestic cats quantification of feline herpesvirus 1 dna in ocular fluid samples of clinically diseased cats by real-time taqman pcr duplex polymerase chain reaction assay to screen for feline herpesvirus-1 and chlamydophila spp. in mucosal swabs from cats comparison of pcr, virus isolation, and indirect fluorescent antibody staining in the detection of naturally occurring feline herpesvirus infections relative sensitivity of polymerase chain reaction assays used for detection of feline herpesvirus type 1 dna in clinical samples and commercial vaccines survival of equine herpesvirus-4, feline herpesvirus-1, and feline calicivirus in multidose ophthalmic solutions detection of chlamydophila felis and feline herpes virus by multiplex real-time pcr analysis effect of topical ophthalmic application of cidofovir on experimentally induced primary ocular feline herpesvirus-1 infection in cats duration of serologic response to three viral antigens in cats update on the diagnosis and management of feline herpesvirus-1 infection a cat with herpetic keratitis (primary stage of infection) treated with feline omega interferon efficacy of oral supplementation with l-lysine in cats latently infected with feline herpesvirus in vitro comparison of antiviral drugs against feline herpesvirus 1 efficacy of topical aciclovir for the treatment of feline herpetic keratitis: results of a prospective clinical trial and data from in vitro investigations synergistic antiviral activities of acyclovir and recombinant human leukocyte (alpha) interferon on feline herpesvirus replication treatment of feline herpesvirus-1 associated disease in cats with famciclovir and related drugs long-term immunity in cats vaccinated with an inactivated trivalent vaccine the european advisory board on cat diseases (abcd) is indebted to dr karin de lange for her judicious assistance in organising this special issue, her efforts at coordination, and her friendly deadline-keeping. the tireless editorial assistance of christina espert-sanchez is gratefully acknowledged. the groundwork for this series of guidelines would not have been possible without financial support from merial. the abcd particularly appreciates the support of dr jean-christophe thibault, who respected the team's insistence on scientific independence. key: cord-303490-rixuuytu authors: pazos, michael a.; kraus, thomas a.; muñoz-fontela, césar; moran, thomas m. title: estrogen mediates innate and adaptive immune alterations to influenza infection in pregnant mice date: 2012-07-05 journal: plos one doi: 10.1371/journal.pone.0040502 sha: doc_id: 303490 cord_uid: rixuuytu pregnancy is a leading risk factor for severe complications during an influenza virus infection. women infected during their second and third trimesters are at increased risk for severe cardiopulmonary complications, premature delivery, and death. here, we establish a murine model of aerosolized influenza infection during pregnancy. we find significantly altered innate antiviral responses in pregnant mice, including decreased levels of ifn-β, il-1α, and ifn-γ at early time points of infection. we also find reduced cytotoxic t cell activity and delayed viral clearance. we further demonstrate that pregnancy levels of the estrogen 17-β-estradiol are able to induce key anti-inflammatory phenotypes in immune responses to the virus independently of other hormones or pregnancy-related stressors. we conclude that elevated estrogen levels result in an attenuated anti-viral immune response, and that pregnancy-associated morbidities occur in the context of this anti-inflammatory phenotype. the immunological implications of pregnancy have motivated immunologists for more than half a century [1] . various mechanisms have been proposed to explain how rejection of an antigenically foreign fetus is prevented, from the absence of classical mhc class i molecules [2] , to regulatory t cell expansion [3] and specific suppression of paternally-derived antigens [4, 5] . pregnancy is also associated with reduced pathology and alleviation of symptoms in inflammatory autoimmune diseases such as multiple sclerosis [6] and rheumatoid arthritis [7] . the profound immunological changes in these disease states during pregnancy suggests that there exists a broad impact on immune regulation that is not specific to fetal antigens. empirical evidence suggests that pregnant women fair poorly in response to certain pathogen challenges, including listeria monocytogenes [8] , malaria [9] , sars coronavirus [10, 11] , varicella zoster [12] , rhinovirus [13] and influenza virus [14] , among others. of these, the increased risk of severe influenza infection is of particular public health concern. pregnancy has been an acknowledged risk factor for severe complications from influenza virus infection for nearly a century [15] , and has been observed during every major influenza pandemic including the pandemics of 1918 [15] , 1957 [16] , 1968 [17] , and 2009 [18] [19] [20] . implications of a severe influenza infection during pregnancy are life threatening for mother and child. significantly increased risks of cardiopulmonary events have been reported [14] , and mortality for the mother can be as high as 45% [21] . moreover, consequences to the fetus range from behavioral alterations and low birthweight, to preterm birth and spontaneous abortion [21] [22] [23] [24] . significant effort has been put into determining the safety and efficacy of vaccination during pregnancy [25, 26] , as well as the efficacy of neuraminidase inhibitors [20, 27] , but a deeper understanding of the changes in maternal immunity during pregnancy are needed in order to devise adequate therapeutic strategies. many clinical studies have been performed to empirically define the increased severity of influenza infections during pregnancy [14, 18, 21] . for safety and ethical reasons, these studies are conducted in a setting without viral challenge, and rely on observations made only after the onset of symptoms. although earlier work has established that the increased mortality to influenza infection during pregnancy can be observed in mouse models [28] [29] [30] [31] , recent work has primarily focused on characterization of associated morbidities in lethal infections with little insight into how immunity is impacted early in infection. increased morbidity due to influenza infection is typically associated with the third trimester of pregnancy, and correlates well with the highest levels of circulating estrogen. estrogens have long been known to possess potent immunomodulatory effects in various models of disease [32] [33] [34] , and have been proposed for therapeutic use in some clinical situations [35] . although pregnancy levels of estrogen have been associated with reduced levels of immune-mediated morbidity, severe morbidity is a hallmark of influenza infection during pregnancy. we sought to reconcile these observations and determine what role high levels of estrogen play in influenza infections during pregnancy. we have developed a model of influenza infection in pregnant mice and documented the major changes in the anti-viral response. moreover, using implantable hormone pellets, we have determined that estrogen alone is able to recapitulate many key alterations observed in pregnancy. estrogen treatment leads to early reductions in cytokine production, in particular type-i interferon (ifn), impacting downstream activation of adaptive immunity. additionally, estrogen treatment compromises effective antigen-specific cytotoxicity. ultimately these deficiencies result in significantly delayed viral clearance in pregnant animals, highlighting the presence of an anti-inflammatory mechanism in the context of enhanced morbidity. to determine the response to influenza virus infection during pregnancy, we challenged mice at gestational day 10.5 (e10.5) and non-pregnant controls with aerosolized pr8 virus in a controlled chamber. peak viral titers were reached in both sets of mice by 3 day post-infection (dpi) ( figure 1a) , as described previously [36] . by 7 dpi, non-pregnant mice showed significantly lower pulmonary viral titers, suggesting they were able to clear virus more efficiently than their pregnant counterparts. weight gain resulting from pregnancy outpaced any potential weight loss incurred during infection, although pregnant infected mice did lose weight compared to mock controls. this weight loss was delayed by approximately 2 days when compared to the weight loss of non-pregnant controls, suggesting that immuneassociated morbidities were delayed in pregnant animals ( figure 1b ). significant morbidity was readily evident in measures of gestational health. the number of fetuses per mother were not effected by infection in our model ( figure 1c ), but fetuses from influenza-infected mice failed to achieve the weight of their counterparts from mock-infected mothers. by e18.5 they were nearly 35% below the weight of controls ( figure 1d ). the length of gestation was also significantly impacted by infection with influenza virus. infected pregnant mice undergo labor much earlier than their mock-infected controls ( figure 1e ). all mock infected pregnant mice continued gestation beyond the bounds of our experiments, while infected mice universally delivered by e19. pups delivered by influenza-infected mothers were therefore not only premature, but additionally underweight for their gestational age. these results reinforce the evidence that the viability of the pregnancy is negatively impacted by infection with influenza virus, with a significant burden borne by the fetus, as is seen in clinical situations [23, 37] . as most clinical incidents of influenza infection are non-lethal, we wanted to model a viral infection with a milder pathology. pr8 is extremely virulent, and useful for establishing morbidities, but is 100% lethal in wild-type mice. as a non-lethal infection model, we made use of the reassortant mouse-adapted virus x31 (h3n2). infection of pregnant animals with this virus leads to full recovery, but with a significantly more pronounced delay in viral clearance ( figure 1f ). these data suggest that despite significant morbidity associated with fetal gestation, pregnant mice have an impaired ability to control influenza virus infection in both lethal and nonlethal models. given the well documented effects of sex hormones on immune modulation [38, 39] , as well as the increased attention estradiol has received in modulating disease during pregnancy [35, 40] , we decided to investigate the contribution of pregnancy-level 17-bestradiol independently of other hormonal and immunological modulations. we made use of a biodegradable pellet that was implanted beneath the skin and release 17-b-estradiol (e2). serum levels of 17-b-estradiol were found to be at third trimester levels [38] through the experiment ( figure 2a ). as we did with the pregnant animals, we tracked weight loss in estradiol-treated mice using a virulent pr8-derived virus (figure 2b ). initial weight loss was nearly identical before stabilizing in e2-pelleted mice, while placebo-pelleted mice continued to lose weight throughout the latter part of infection. these observations are compatible with our results in pregnant mice, where we found more significant weight loss in non-pregnant controls. viral growth and clearance in e2-pelleted mice mirrored pregnancy extremely well ( figure 2c ). using x31 virus to analyze viral clearance, we found peak titers were achieved in both groups by 3dpi. virus was cleared in the placebo-pelleted mice by 7dpi, but clearance was delayed in e2-pelleted mice. taken together, these data support the hypothesis that e2 is at least partially responsible for the delayed viral clearance found in pregnant animals. one of the critical early signaling events during an influenza infection is the release of type-i interferon (ifn). we have previously demonstrated that e2 was able to significantly impair type-i interferon signaling in human dendritic cells [41] . in order to extend these observations, we measured transcription of ifnb and interferon-stimulated genes (isgs) by quantitative real-time pcr on rna collected from whole lung during infection with x31. we observed a significant early impairment in this pathway in both pregnant and estrogen-treated mice ( figure 3a , 3b). the deficit was not only in ifnb mrna expression, but also activation of downstream isgs, suggesting a significant, biologically relevant impairment in ifn protein expression. we have previously shown that interferon signaling plays an important role in tissues outside the of lung [42] . antiviral instruction of peripheral leukocytes is important for effective control of viral infection and a feature of a robust immune response. using the spleen as a tissue that is not in direct contact with virus, we detected an impairment of isg upregulation in the periphery ( figure 3c ). this data suggests that cytokine-mediated early immune activation is inefficient in pregnant animals. to eliminate the possibility that estrogen treatment interferes with interferon signaling pathways in addition to interferon induction, we treated splenocytes directly with murine ifnb ( figure 3d ). we found e2 to have no effect on ifn signaling. in order to further dissect the mechanism of altered immune responses in pregnancy, we collected whole lung digests throughout x31 infection for analysis by elisa. at 3dpi, we observed several key early cytokines were under expressed in pregnant mice ( figure 4a ), including il-1a, il-1b, and tnf-a. we observed reduced ifn-c expression early in infection, as well as decreased expression of chemokines such as mcp-1 and kc. it is important to note that at this time point we do not detect any difference in viral loads. we repeated the experiment using our estrogen model and observed a similar modulation in many of the same cytokines ( figure 4b ). ifnc, kc, and tnf-a all were under expressed in the lungs of influenza infected e2 mice compared to placebo controls. these results suggest that both models establish a significant anti-inflammatory environment at critical early time points in infection. given the significant changes observed in early cytokine and chemokine expression, we investigated whether these changes lead to variations in migratory kinetics of early immune mediators. we did not observe any significant differences in the absolute number of cells infiltrating lung tissue during infection with x31 in pregnant ( figure 5a ) or e2-treated mice (data not shown). we found no differences in populations of natural killer cells, neutrophils, or cd11c + mhcii + cells that represent a mixture of activated exudate macrophages, dendritic cells and infiltrated monocyte-derived cells. in order to determine whether emigration from the lung was impaired, we investigated the number of dendritic cells in the draining mediastinal lymph node at key time points of infection and found no significant differences between pregnant mice and their non-pregnant controls ( figure 5b ), or between e2-pelleted mice and their placebo controls (data not shown). we next analyzed whether the function of cells migrating into the mediastinal lymph nodes was impaired. we found strongly reduced levels of cd86 expression on cd11c+ cells at critical early time points after infection in both pregnancy ( figure 5c ) and estrogen-treatment ( figure 5d ), suggesting a deficiency in proper dc maturation. the maturation state of dcs at these time points is a critical junction bridging innate and adaptive immunity. the upregulation of cd86 expression, as well as other markers of activation, is extremely sensitive to ifn signaling [43] [44] [45] . the adaptive immune response is critical for efficient clearance of viral infection. of particular importance is the activity of cytotoxic cd8 t cells [46] . in order to determine whether functional deficiencies in adaptive immunity may account for delayed viral clearance, we made use of an in vivo cytotoxicity assay. in this assay, animals are infected with pn-1, an influenza virus bearing the model antigen siinfekl. cytotoxic t cells are endogenously generated towards this peptide during infection. at 8dpi, two populations of target splenocytes are adoptively transferred. each differentially labeled with a fluorescent dye and incubated with either siinfekl or an irrelevant peptide. specific lysis of the antigen-bearing population is then calculated by comparing the relative proportion of the two target populations 16-18h after adoptive transfer ( figure 6a ). we observed significant reductions in antigen-specific cytotoxicity in both pregnant ( figure 6b ) and estrogen-treated ( figure 6c ) animals. these differences represent delays in the ability of pregnant and e2-treated mice to clear antigen specific targets. we next used intracellular cytokine staining techniques to determine the effect of estrogen on granzyme b production by t cells during x31 infection. granzyme b is a serine protease that serves an important role in cd8 t cell cytotoxicity. despite the fact that more virus is present in e2-pelleted mice at 7dpi, we found significant reductions in granzyme b production by intracellular cytokine staining ( figure 6d ). in order to determine whether cytokine signaling in cd8 t cells was more broadly affected, we also measured ifn-c production, a common measure of t cell activation. ifn-c levels were equivalent in both placebo and estrogen treatment ( figure 6e ). these results suggest that while we observe strongly reduced granzyme b levels and impaired cytotoxicity, lung-migrating cd8 t cells were capable of secreting equivalent levels of ifnc. we next investigated the antigen-specific proliferative response. animals were again infected with the pn-1 virus and adoptively transferred with cfse-labeled ot-i t cells, which are a transgenic cytotoxic t cells specific for the siinfekl antigen. at 4dpi, we analyzed draining lymph nodes for cfse dilution and found fewer cd8 t cells proliferating in estrogen treated mice ( figure 6f , 6g). those cells that did proliferate underwent fewer divisions than their placebo-treated counterparts ( figure 6h ). these results are consistent with our findings of immature dendritic cells in the lymph node at early time points. in addition to delays in proliferation, migration of total and influenza-specific cd8 t cells to the infected lung was delayed in x31 infection ( figure 6i ). these observations correlate well with the observed delays in viral clearance. similarly, at 7dpi, antigen specific cd8 t cells in the draining lymph node continue to be reduced in estrogen-treated animals ( figure 6j ). the total number of cd8 t cells is not significant reduced in the draining lymph node. this likely reflects the large number of t cells present in a lymphoid tissue. in order to distinguish between the effects of estrogen on delayed t cell activation by antigen presenting cells and the effects independent of antigen presentation, we used a single-shot model of e2-treatment. a single 100mg dose of e2 was administered to infected mice 6h prior to adoptive transfer of target cells. this dose was sufficient to partially replicate the defect in cytotoxicity observed in e2 mice ( figure 6k) . therefore, e2 impairs t cell cytotoxicity after dc stimulation has occurred. taken together, these data suggest two mechanisms of cytotoxic t cell deficiency. the first involving delayed proliferation and migration of antigenspecific cytotoxic t cells to the lung. the second involves a functional effect of estrogen treatment that occurs after activation of t cells and can be replicated in a short period of time after treatment. establishing a model of influenza infection during pregnancy in order to determine mechanism and intervention strategies is of primary importance. the 2009 h1n1 pandemic outbreak reestablished the risk of severe morbidity and increased mortality that influenza exposure presents to pregnant women, especially in the second and third trimesters [18] [19] [20] . significantly elevated mortality has been reported in mouse models [28] [29] [30] [31] . using an aerosol model of infection, we were able to detect quantifiable and clinically relevant increases in morbidity independent of lethality. notably, fetal pups were found to be significantly underweight, and were delivered prematurely, consistent with clinical observations [23, 37] . in addition to these measures of morbidity, we also observed evidence of an anti-inflammatory phenotype in pregnant mice. viral clearance was delayed in pregnancy, using both lethal and non-lethal infections. additionally, weight loss in pregnant mice was attenuated and delayed. altered pathology in response to influenza virus infection during pregnancy is likely mediated by multiple factors including changes to sex hormones and increased cardiopulmonary demands [47] . we focused on the impact of pregnancy-levels of estrogen for its well-defined impacts on immune function. estrogen is a particu-larly interesting immune modulator as it has been described as having bimodal effects on immunity [38] . circulating levels of estrogen in female mice have been associated with stimulating immune function in general, including exacerbating influenza virus pathogenesis [48] . this accounts for the relative susceptibility to influenza virus infection in non-pregnant females compared to males. this effect can be abolished by gonadectomy. gonadallyintact females were used in this study in order to directly compare pathogenesis in pregnancy or high-dose estrogen-treatment to healthy wild-type females. high levels of estrogen, on the other hand, have been associated with an anti-inflammatory phenotype, including in the context of influenza a virus infection [48] , and cannot be directly linked to the susceptibility of pregnant females to high morbidity. high-dose estrogen treatment has been used to alleviate symptoms in models of rheumatoid arthritis [34, 49] and multiple sclerosis [32, 33, 50] in response to clinical observations showing a reduction of symptoms in pregnant women with these diseases. excessive immune responses mediate much of the pathology associated with influenza virus [51, 52] . pregnancy-specific morbidities have also been associated with inflammation including premature parturition [53] [54] [55] . anti-inflammatory treatment can prevent loss of pregnancy. blocking tnf-a or administering antiinflammatory cytokines such as il-10 [56] have been shown to reduce morbidity. additionally, the placenta has been shown to employ negative regulatory mechanisms aimed at reducing inflammatory cytokine signaling [57] . given the well described anti-inflammatory phenotype of estrogen, and specifically the high while other groups have reported on increased morbidity to influenza infection during pregnancy, anti-inflammatory mechanisms in the context of infection have not been extensively characterized as they have been in the context of disease [6, 7] and of fetal rejection [3, 5] . because of a focus on morbidity, mouse models of influenza virus infection during pregnancy often rely on a high dose intranasal inoculum of pathogenic virus strains such as the 2009 pandemic h1n1 strain. using these models, morbidities such as elevated mortality and low fetal birth weight have been observed [30, 31] . clinical observations of preterm delivery have not been reported in a mouse model, and was not observed in one study [30] , despite increased cytokines levels. additionally, proinflammatory cytokine phenotypes have been reported in these models, and delays in viral clearance are often not supported, although these reports often focus on events late in infection or reflect divergent viral kinetics. we were concerned that models using highly pathogenic intranasal inoculation may not adequately model clinical pre-sentations of influenza virus infection and obscure relevant phenotypes. aerosolized infection closely models clinical disease [58, 59] and was able to replicate clinical observations of influenza infection during pregnancy. we focused on a early cytokine events within the first 24h of the onset of immunity in our model [36] . these inflammatory events have broad impacts on the outcome of immunity, and serve as the best indicators early inflammation. by making use of a model of infection using pregnancy-levels of 17-b-estradiol, we were able to measure its impact on immunity independently of other pregnancy-associated variables. in addition to demonstrating a similar delay in viral clearance, estrogentreated animals were also protected from the weight loss experienced by placebo controls. it is not clear how estrogen may protect mice from weight loss associated with influenza infection, but a likely mechanism may be decreased cytokineassociated cachexia. in both pregnant and estrogen-treated animals, early cytokine responses were reduced. estrogens have been linked to modulation of cytokine signaling, such as through antagonism of nf-kb pathway [38, 60] . sources often disagree as to the degree and direction of these changes depending on the concentration of estrogen, model organism, and method of stimulation [38, 39, 61] . indeed, we saw a strong trend towards elevated levels of the chemokine kc in the steady state of pregnancy, followed by significant deficit in kc expression upon infection. this complex regulation highlights the elaborate mechanisms involved in estrogen signaling [62] . pregnancy presents additional layers of complexity in cytokine regulation. several hormones circulating in pregnancy are immunologically active, including progesterone and prolactin [39, 63] . we did not observe identical regulation of all cytokines in pregnancy and estrogen-treatment, and some of these other hormones may account for some of this variation. the overlap remains significant, and both models demonstrate defects in early immune responses. over-expression of several regulated cytokines including il-1a, il-1b, tnf-a, and ifn-c have been associated with morbidities in influenza infection [52] , and spontaneous pregnancy loss [56, 64] . the ability of estrogen to suppress cytokine expression has been proposed as protective in influenza virus pathogenesis [48] . indeed, it may be that estrogen plays a protective role during pregnancy by muting early cytokine responses that would otherwise threaten fetal development [22, 54] . enhanced morbidities experienced during pregnancy may rely on the failure to clear virus following an impaired immune response. prolonged infection may result in a late cytokine response triggering premature delivery and significant morbidity. delayed weight loss in pregnant animals supports the hypothesis of a late development inducing pathology. alternatively, prolonged infection may lead to significant lung damage. others have reported that delayed lung repair during pregnancy leads significant cardiopulmonary demands during pregnancy, and may contribute to morbidity [31] . type-i ifns play a critical role in early immune responses to influenza, and impairment of this pathway during pregnancy implicates a significant alteration in the response to virus. transcription of ifn-b is a prototypical marker of early type-i interferon expression, and was found to be significantly reduced in both of our models. we did not find any evidence that estrogen treatment interfered with downstream signaling pathways, suggesting that this effect is mediated by reduced type-i interferon expression. isg levels were reduced not only in the lung, but additionally in the periphery. this is an important observation, as interferon signaling in the periphery is necessary for efficient antiviral immunity [42] . this reduction is well conserved in human models of both e2 treatment and pregnancy [13, 41] . the mechanism of interaction between estrogen and type-i ifn induction pathways is not currently understood, but could lead to significant insights. after the onset of inflammation, pregnant and e2-treated animals were able to control early virus titers as well as wild-type counterparts, despite reduced levels of interferon. influenza virus ns1 is a powerful inhibitor of inflammation and functions to counteract early checks on viral growth [36] , likely minimizing the impact of interferon deficiency at the earliest time points. interferon signaling also plays an important role in the activation and maturation of antigen-bearing dendritic cells [65, 66] , and serves as a bridge between innate and adaptive immunity [44, 45] . pregnancy-levels of estrogen were able to interfere with proper dendritic cell maturation. the reduced levels of the co-stimulatory molecule cd86 is particularly relevant for its role in cd8 t cell activation [67] . the primary goal of dendritic cell maturation is the efficient initiation of adaptive immunity, and in particular initiating proliferation of antigen-specific cd8 t cells [46] . as expected, we observed significant estrogen-associated delays in cd8 t cell proliferation in the lymph node at 4dpi, and significantly reduced numbers of antigen-specific cytotoxic t cells in the lung and lymph node at 7dpi. these numbers eventually recovered by 9dpi, correlating nicely with the kinetics of viral clearance in this model. because the observed phenotype results in delays in cd8 t cell recruitment to the lung, the timing of this observation is critical. observations made late in infection may not reflect this phenotype and may explain why others have failed to note significant differences in the numbers of cytotoxic t cells in similar models [31] . dysregulation of interferon function can be linked to delays in appropriate activation of cytotoxic cd8 t cells, and delays in viral clearance [68] . in addition to experiencing delays in proliferation and migration, these t cells are functionally compromised. cytotoxicity was decreased in an in vivo cytotoxicity assay, and granzyme b levels were strongly reduced in the lungs of infected mice. interestingly, ifn-c expression in cd8 t cells was unchanged, suggesting that these cells that reach the lung were at least partially activated, even while functionally compromised. a single dose of estrogen administered immediately prior to encountering target cells was sufficient to impair cytotoxic function, suggesting that this effect is not entirely mediated by inefficient dendritic cell stimulation. estrogen signaling pathways have been associated with lysosome biogenesis in cytotoxic t cells, and may represent a relevant mechanism [69] . notably, two mechanisms of impaired adaptive immunity can be observed. the first involves delayed proliferation in the draining lymph nodes and can account for delayed migration to the lung. this mechanism is likely the product of weak early interferon signaling events and impaired antigen presentation. those cells that do proliferate and are activated are additionally functionally compromised. this second mechanism is independent of antigen presentation and is at least partially responsible for a deficiencies in cytotoxic function. this mechanism has a rapid onset and can be observed within hours of estrogen treatment. this mechanism likely accounts for the observed decreases in granzyme b production that do not effect ifn-c levels in the same t cells. although pregnancy is a complex immunological state, it is clear that estrogen plays a key role. initially, estrogen reduces early cytokine responses and may function to protect from cytokineassociated morbidities. early recruitment of leukocytes is equivalent and early viral titers are controlled equally well in pregnant and wild type mice. the key deficit appears to be in priming of dendritic cells that migrate from the lungs to lymph nodes to initiate t cell expansion. this disruption in interferon signaling leads to suboptimal cd8 t cell activation and delayed viral clearance. estrogen also appears to impair cd8 t cell cytotoxicity independently of dendritic cell activity. it is not currently clear how these anti-inflammatory effects impact the viability of pregnancy, particularly fetal development and premature delivery, but evidence suggests it may play a protective role. deficits in viral clearance and prolonged exposure to infection may be act as stressors that trigger delayed morbidities despite the anti-inflammatory effects of estrogen. it is also not clear whether prolonged infection is of significant impact on morbidity absent pregnancy, as would be the case in hormone treatment. these are key questions that require further study and the establishment of novel models. there is overwhelming clinical value to understanding the mechanisms and contributions of sex hormones to immune modulation during pregnancy. animal models, such as those described in this report, allow for controlled infection conditions and detailed observation of early events during infection. understanding the mechanisms and immunological outcomes of pregnancy, particularly the role of steroid hormones, may lead to the development of therapeutic options for protection of women, their children, and patients receiving hormone replacement therapy. establishing the tenets of hormone mediated immune modulation may provide testable hypotheses for treatment of autoimmune diseases such as multiple sclerosis. mice c57bl/6 wild type, transgenic ot-i and syngeneic pregnant mice were purchased from jackson laboratories (bar harbor, ma). timed pregnant mice were delivered at e8.5 and allowed two days to rest before commencing the experiment. nonpregnant mice were age matched to timed pregnant mice. wildtype mice were implanted with placebo or estrogen pellets purchased from innovative research of america. estrogen pellets contained 35mg of 17-b-estradiol and were designed for 21-day release. surgery was performed 4-5 days prior to infection. mice were anesthetized with avertin (tribromoethanol, acros organics) and shaved behind the right ear. the pellet was implanted via small incision between the shoulder and ear and secured with a surgical clip. mice were monitored for signs of infection. for experiments using a single dose of estradiol, mice received a single 100mg subcutaneous injection of water soluble 17-b-estradiol (sigma) six hours prior to receiving target cells for in vivo cytotoxicity assay. the animals were housed in specific pathogenfree conditions. influenza virus strains a/pr/8/1934 (h1n1) (pr8), recombinant pr8-oti (h1n1) (pn-1), and a/x-31(h3n2) (x31) were propagated in 10-day-old embryonated eggs. virus titers were determined by tissue culture infectivity assay (tcid50) as previously described [70] . briefly, lungs were extracted and homogenized in pbs-gelatin (0.1%) and frozen in dry ice-ethanol for storage at 280uc. presence of the virus was detected by infecting mdck cells at 1:10 dilutions for 72h in the presence of trypsin. presence of virus was measure by testing for the presence of hemagglutination of chicken red blood cells. the limit of detection for this assay is approximately 8610 2 tcid50 per lung. all mice were infected using an inhalation exposure system a42x (glass-col, usa). virus was diluted in pbs to obtain a solution of 10 7.9 tcid50 in12ml. this solution was placed in a glass nebulizer and aerosolized for a total exposure of 30 min. this leads to 100% infection rate as described previously [36, 71] . we estimate between 10 and 100 infectious particles are passively inhaled during this period [72] . mock infected animals are exposed to aerosolized pbs. lungs or spleen at indicated time points were homogenized in 3ml of trizol reagent (invitrogen). rna was isolated as indicated by the manufacturer, and converted to cdna by rt-pcr using the transcriptor first strand cdna synthesis system (roche). qpcr reactions were performed using a lightcycler 480 ii (roche). all reactions were normalized to three housekeeping genes: a-tubulin, rps-11, and b-actin, as previously described [73] . the following primers were used in this study: b-actin -forward: aggtgacagcattgcttctg, reverse: gctgcctcaacacctcaac; a-tubulin lungs were perfused with cold pbs containing 0.5mm edta and immediately ground in hank's buffered saline solution containing 0.5mm edta and 0.5% fbs using the gentlemacs tissue dissociator (miltenyi biotec). mediastinal lymph nodes were mechanically disrupted. both tissues were then incubated with 0.25mg/ml collagenase (liberase type iii, roche) and 8000u/ml dnase i (invitrogen) for 20 min at 37uc. collagenase was inactivated with sterile hbss containing 2% fbs and the suspension was passed through a 70mm strainer. suspensions were then treated with red blood cell lysis buffer (bd biosciences), and resuspended in hbss containing 10mg/ml fc-receptor block (bd biosciences). total cell counts were then attained by hemocytometer. cells were then stained with antibodies for multiple surface antigens: cd8 (53-6.7), cd3 (17a2), cd11c (hl3), mhcii (m5/ 114.15.2), cd86 (gl-1), nk1.1 (pk136), ly6g (1a8). for intracellular cytokine staining, total lung was digested and a single cell suspension was incubated with brefeldin a-containing golgiplug (bd biosciences) for six hours, according to manufacturer's instructions. following staining of extracellular antigens, intracellular cytokine staining was performed using the cytofix/ cytoperm system (bd biosciences). intracellular antigens granzyme b (gb11), and ifn-c (xmg1.2) were stained following permeabilization. antibodies were purchased from bd bioscience, ebiosciences, and biolegend. kb/siinfekl pentamer was purchased from proimmune. influenza np-specific tetramers were kindly provided by dr. david woodland (trudeau institute). samples were acquired using cytomic fc500 coulter station (beckman coulter) and analyzed using flow jo software (treestar corp). whole lung was homogenized in pbs-gelatin (0.1%). cytokine concentration was measured by bead-based multiplex elisa (millipore) using a luminex 200 (luminex corporation). serum was used for detection of circulating 17-b-estradiol using a competitive estradiol elisa (cayman chemicals). mice were infected as described with pn-1 virus or mock infected with pbs. at 8dpi, 1-2.5610 6 target cells were adoptively transferred. splenocytes from a congenic naive donor mouse were used as target cells, and injected intravenously as a 1:1 ratio of two populations. one population was incubated with 100mg/ml of the h2-k b -restricted ova peptide, siinfekl, and labeled with 2.5mm cfse. the second population was incubated with irrelevant peptide and labeled with 0.25mm cfse. 16-18h posttransfer mice were sacrificed. spleens of recipient mice were disrupted, and suspended in red blood cell lysis buffer. total spleen was analyzed by flow cytometry for the relative proportion of the cfse-labeled cells. specific killing was calculated as previously described [74] . briefly, a normalization value (n) was calculated as (% low cfse/% high cfse) in mock infected animals for each treatment group. percent specific cytotoxicity was then calculated for each infected mouse as follows: (% low cfse x n -% high cfse)/(% low cfse x n) x 100. spleens from ot-i mice were mechanically disrupted and passed through a 70mm strainer. cells were suspended in red blood cell lysis buffer (bd biosciences), washed and suspended in hbss containing 0.5% fbs and 10mg/ml fc-receptor block (bd biosciences). cells were then incubated with a cocktail of biotinlabeled antibodies including: cd19 (1d3), b220 (ra3-6b2), mhcii (m5/114.15.2), gr-1 (rb6-8c5), nk1.1 (pk136), cd11b (m1/70), ter119 (ter119), and cd4 (gk1.5). cell suspensions were then washed and incubated with anti-biotin magnetic beads and passed over a macs magnetic column (miltenyi biotec). flow through was collected and verified by flow cytometry to have a purity of greater than 80%. for the cfse dilution assay, these cells were stained with 2.5mm cfse. 1610 6 -2.5610 6 labeled cd8 t cells were intravenously injected into recipient mice on the day of infection. at 4dpi, mice were sacrificed and lymph nodes were collected for analysis. results are expressed as mean +/2 standard deviation. data sets with multiple groups were analyzed by 1-way anova followed by newman-keuls multiple comparison test. weight loss was evaluated using a two-way anova. single time point analyses were evaluated using unpaired two-tailed student's t test. p 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pregnancy and post-partum periods estrogen receptors: how do they signal and what are their targets prolactin and autoimmunity cytokines and neonates interferon-beta pretreatment of conventional and plasmacytoid human dendritic cells enhances their activation by influenza virus antiviral-activated dendritic cells: a paracrine-induced response state cd86 has sustained costimulatory effects on cd8 t cells serves as a host antiviral factor that enhances innate and adaptive immune responses to influenza a virus the tumor-associated antigen ebag9 negatively regulates the cytolytic capacity of mouse cd8+ t cells a mouse model for immunization with ex vivo virus-infected dendritic cells influenza virusinduced dendritic cell maturation is associated with the induction of strong t cell immunity to a coadministered, normally nonimmunogenic protein experimental transmission of influenza virus infection in mice. i. the period of transmissibility a novel role for viral-defective interfering particles in enhancing dendritic cell maturation cutting edge: rapid in vivo ctl activity by polyoma virus-specific effector and memory cd8+ t cells we thank dr. david woodland for providing influenza np-specific tetramers, and olga herrera for technical assistance. key: cord-278816-l92lkj69 authors: brouard, j.; vabret, a.; bach, n.; toutain, f.; duhamel, j. f.; freymuth, f. title: prise en charge des pathologies respiratoires à adénovirus chez l’enfant immunocompétent à propos d’une étude rétrospective de 116 enfants hospitalisés date: 2004-05-31 journal: antibiotiques doi: 10.1016/s1294-5501(04)94248-3 sha: doc_id: 278816 cord_uid: l92lkj69 résumé les adénovirus sont une cause commune d’atteinte respiratoire ; bien que dépendant du sérotype, ils peuvent également être la cause d’atteintes extrarespiratoires. le diagnostic positif peut en être difficile. les résultats cliniques chez 116 enfants hospitalisés en raison d’une infection adénovirale ont été repris rétrospectivement. chez 71 enfants, le diagnostic virologique a été obtenu par immunofluorescence directe sur les aspirations nasales, 71 par culture virale de ces mêmes prélèvements. le tableau clinique de l’infection adénovirale est caractérisé par une fièvre élevée (moyenne 39°1c) et prolongée (durée moyenne 4,3 jours). l’atteinte des voies aériennes supérieures (rhinopharyngite, angine, otite) et des voies aériennes inférieures (bronchite, bronchiolite, pneumopathie) sont les plus fréquentes. douze enfants ont présenté des convulsions hyperpyrétiques, 6 avaient une méningite lymphocytaire. les examens complémentaires ont objectivé des valeurs allant de la normalité à celles évocatrices d’infection bactérienne. cinquante-neuf enfants furent adressés pour fièvre résistante à une antibiothérapie. les symptômes de l’atteinte respiratoire dues aux infections adénovirales s’étendent de la rhinite à la pneumopathie et la bronchiolite. les adénovirus peuvent entraîner des séquelles graves même chez l’enfant sain. les recherches sur les mécanismes moléculaires de l’infection virale sur les voies aériennes amèneront d’importantes voies de réflexion sur la nature des processus inflammatoires participant à l’asthme et à la bronchite chronique obstructive. la plupart des infections sont modérées et ne nécessitent qu’un traitement symptomatique. il n’existe pas actuellement de traitement antiviral efficace pour les infections adénovirales graves. le diagnostic virologique rapide par l’étude des sécrétions nasopharyngées est d’une grande utilité clinique. abstract adenoviruses most commonly cause respiratory illness; however, depending on the infecting serotype, they may also cause various other diseases. diagnosis may be difficult to achieve. the clinical findings for 116 children hospitalised with adenoviral infection were studied retrospectively. in 71 children, the diagnosis was based on detection of adenovirus antigen in the nasopharyngeal specimens and in 71 children on viral culture. the clinical picture of adenoviral infection was characterised by high-grade (mean 39°1c) and prolonged fever (mean duration 4,3 days). upper respiratory and lower respiratory symptoms were the most common infections. twelve had been admitted to the hospital due to febrile convulsions, 6 had meningitis. laboratory findings varied from normal values to values seen in bacterial infections. thus it was difficult to distinguish adenoviral disease from a bacterial disease. fifty-nine children were referred to the hospital due to infection unresponsive to antimicrobial therapy. symptoms of respiratory infection caused by adenovirus may range from the common cold syndrome to pneumonia, croup and bronchiolitis. adenoviruses can be responsible for severe consequences, even in previously healthy children. studies of the molecular mechanisms of viral infections of the airways could provide important insights into the nature of the inflammatory process involved in asthma and chronic obstructive pulmonary disease. most infections are mild and require no therapy or only symptomatic treatment. there are at present time no recognised antiviral agents that are effective in treating serious adenovirus disease. the rapid detection of adenovirus antigen in nasopharygeal specimens proved to have a great clinical value in the diagnosis. prise en charge des pathologies respiratoires à adénovirus chez l'enfant immunocompétent j. brouard, a. vabret, n. bach, f. toutain, j.f. duhamel, f. freymuth les adénovirus sont une cause commune d'atteinte respiratoire ; bien que dépendant du sérotype, ils peuvent également être la cause d'atteintes extrarespiratoires. le diagnostic positif peut en être difficile. les résultats cliniques chez 116 enfants hospitalisés en raison d'une infection adénovirale ont été repris rétrospectivement. chez 71 enfants, le diagnostic virologique a été obtenu par immunofluorescence directe sur les aspirations nasales, 71 par culture virale de ces mêmes prélèvements. le tableau clinique de l'infection adénovirale est caractérisé par une fièvre élevée (moyenne 39 °1c) et prolongée (durée moyenne 4,3 jours). l'atteinte des voies aériennes supérieures (rhinopharyngite, angine, otite) et des voies aériennes inférieures (bronchite, bronchiolite, pneumopathie) sont les plus fréquentes. douze enfants ont présenté des convulsions hyperpyrétiques, 6 avaient une méningite lymphocytaire. les examens complémentaires ont objectivé des valeurs allant de la normalité à celles évocatrices d'infection bactérienne. cinquante-neuf enfants furent adressés pour fièvre résistante à une antibiothérapie. les symptômes de l'atteinte respiratoire dues aux infections adénovirales s'étendent de la rhinite à la pneumopathie et la bronchiolite. les adénovirus peuvent entraîner des séquelles graves même chez l'enfant sain. les recherches sur les mécanismes moléculaires de l'infection virale sur les voies aériennes amèneront d'importantes voies de réflexion sur la nature des processus inflammatoires participant à l'asthme et à la bronchite chronique obstructive. la plupart des infections sont modérées et ne nécessitent qu'un traitement symptomatique. il n'existe pas actuellement de traitement antiviral efficace pour les infections adénovirales graves. le diagnostic virologique rapide par l'étude des sécrétions nasopharyngées est d'une grande utilité clinique. adenoviruses most commonly cause respiratory illness; however, depending on the infecting serotype, they may also cause various other diseases. diagnosis may be difficult to achieve. the clinical findings for 116 children hospitalised with adenoviral infection were studied retrospectively. in 71 children, the diagnosis was based on detection of adenovirus antigen in the nasopharyngeal specimens and in 71 children on viral culture. the clinical picture of adenoviral infection was characterised by high-grade (mean 39 °1c) and prolonged fever (mean duration 4,3 days). upper respiratory and lower respiratory symptoms were the most common infections. twelve had been admitted to the hospital due to febrile convulsions, 6 had meningitis. laboratory findings varied from normal values to values seen in bacterial infections. thus it was difficult to distinguish adenoviral disease from a bacterial disease. fifty-nine children were referred to the hospital due to infection unresponsive to antimicrobial therapy. plus de 200 virus antigéniquement distincts, appartenant à 8 genres différents, atteignent en général tout l'arbre respiratoire. leur diagnostic est souvent retardé par l'aspect peu spécifique de leur expression. les adénovirus (adv) humains comprennent 51 sérotypes, ils sont ubiquitaires et responsables d'un large éventail de syndrômes cliniques. quelques sérotypes sont particulièrement en cause dans la gravité de la maladie en phase aiguë même chez l'enfant immunocompétent. la littérature est relativement importante sur les atteintes adénovirales du sujet fragilisé, immunodéprimé ou porteur d'une cardiopathie. elle est par contre plus restreinte pour les enfants a priori sains : or chez eux des altérations bronchiolaires et bronchiques peuvent être à l'origine de lésions définitives parfois d'expression retardée. à partir d'une étude rétrospective personnelle cet article se focalise sur la pathologie respiratoire à adv chez l'enfant immunocompétent. les pneumopathies virales se définissent par l'existence d'une atteinte parenchymateuse : elles ne représentent qu'une faible part des infections respiratoires basses, environ 5 %. l'épidémiologie de quelques repères épidémiologiques syncytial (vrs), 10 à 15 % par les adénovirus (adv), 5 à 10 % par les virus parainfluenza (vpi), 5 % par les virus influenza [2] . la biologie moléculaire amène des modifications de l'épidémiologie descriptive en raison de sa plus grande sensibilité [3] . ces techniques permettent également la mise en évidence de co-infection virale [4] . les atteintes respiratoires à adv les plus fréquentes sont essentiellement endémiques, observées régulièrement d'octobre à mai et liées aux sérotypes 1, 2, 5, et 6. il s'agit de rhinopharyngites, de conjonctivites, de syndrômes apc (adénopharyngo-conjonctivites), de bronchites et de pneumonies, rarement de détresse respiratoire d'évolution sévère. cependant d'autres sérotypes évoluent seulement sous forme d'épidémies, plutôt hivernales, irrégulières dans le temps, au sein de collectivités de nourrissons, parfois d'enfants plus agés : ils sont parfois marqués par l'apparition de formes graves pour certains sérotypes (3, 5, 6, 7) . un aspect particulier est représenté par les épidémies d'atteintes respiratoires aiguës chez les recrues militaires, bien décrites aux usa, dont les principaux sérotypes sont le 4 et le 7 [5] . la prédominance de quelques sérotypes a été soulignée dans certaines zones géographiques [6, 7] . la circulation du sousgroupe b avec le génotype 7 h a été documentée lors de bronchiolites et de pneumopathies sévères en argentine [8] . ce même type a provoqué de redoutables infections nosocomiales avec une létalité approchant 30 % et la présence de séquelles chroniques chez 20 % des survivants [7, 8] . récemment ce sous-type a émergé au japon [9] . il est peut-être important d'être vigilant sur cette épidémiologie car cela pourrait annoncer un glissement du génotype circulant et les adv 7 comptent pour environ 20 % de tous les adv rapportés par l'oms [10] . caractères des adv : les adv sont des virus à capside nue très résistants dans le milieu extérieur ; il sont présents dans les voies aériennes et dans les selles des sujets infectés. les modes de contamination sont donc soit directs, se faisant par les sécrétions respiratoires, soit indirects par la contamination d'objets ou d'aliments responsables de transmissions nosocomiales. le portage d'adv latents est possible, la contamination peut s'effectuer par des sujets asymptomatiques. une étude a été récemment menée par notre équipe [11] . cent seize enfants manifestations respiratoires aiguës ches issues de la culture (63/71) a révélé que 6 sérotypes représentent 85 % des isolements : ce sont les sérotypes 1 (12 %), 2 (40 %), 3 (10 %), 5 (12 %), 6 (3 %), 7 (8 %). donc dans cette cohorte près d'un enfant sur cinq présente un sérotype potentiellent grave. une coinfection virale respiratoire est présente 14 fois (6 vrs, 3 vpi, 2 rhinovirus, 3 coronavirus), digestive 12 fois (9 rotavirus, 3 entérovirus). le motif principal ou le motif associé pour l'admission hospitalière a été le suivant : 52 % fièvre réfractaire à une antibiothérapie ambulatoire (n = 59), 23 % toux persistante de plus de trois jours (26), 12 % malaise ou convulsion (14), 9 % troubles digestifs (10), 3 % otite moyenne aiguë (3) et une cystite hémorragique. lorsqu'elle était présente, la fièvre était élevée (médiane 39 °1) et prolongée (moyenne 4,3 jours). la durée de l'hospitalisation a été en moyenne de 5,1 jours (+/-4,7 j). au cours de l'hospitalisation les diagnostics cliniques isolés ou associés ont été : atteinte des voies aériennes supérieures chez 86 % des enfants, atteinte respiratoire basse chez 76 %, gastro-entérites aiguës chez 30 %. douze enfants ont présenté des convulsions, parmi eux 6 avaient une méningite lymphocytaire mais l'identification virale dans le lcr est restée négative. le plus souvent les examens paracliniques peuvent orienter faussement vers une infection bactérienne, par l'élévation modérée des polynucléaires neutrophiles (moyenne = 10 100/mm 3 ) et de la c-reactive protein (moyenne = 32,8 mg/l), par la mise en évidence d'une bactérie potentiellement pathogène dans 17 des 23 examens de crachats effectués : l'orientation erronée a aussi été liée à la présence d'anomalies radiologiques au sein de 60 % des clichés thoraciques pratiqués. aucune différence significative n'a été mise en évidence entre sérotypes et signes cliniques, gravité (oxygénodépendance), durée de l'hospitalisation. douze enfants ont été réhospitalisés pour atteinte respiratoire, mais les enquêtes virologiques sont restées vaines, aucun de ceux-ci n'avaient initialement un sérotype à risque. les affections respiratoires aiguës furent à l'origine de la découverte des adv et restent la manifestation la plus connue symptoms of respiratory infection caused by adenovirus may range from the common cold syndrome to pneumonia, croup and bronchiolitis. adenoviruses can be responsible for severe consequences, even in previously healthy children. studies of the molecular mechanisms of viral infections of the airways could provide important insights into the nature of the inflammatory process involved in asthma and chronic obstructive pulmonary disease. most infections are mild and require no therapy or only symptomatic treatment. there are at present time no recognised antiviral agents that are effective in treating serious adenovirus disease. the rapid detection of adenovirus antigen in nasopharygeal specimens proved to have a great clinical value in the diagnosis. de l'infection chez l'enfant. la plupart d'entre elles restent modérées et focales, elles ne peuvent être cliniquement distinguées des infections dues à d'autres virus [1] . le spectre clinique peut être : une pharyngite, une angine, une otite moyenne aiguë, une laryngite, une bronchiolite, une pneumopathie. chez les sujets hospitalisés la fièvre est habituelle (94 %), prolongée, en moyenne : 5,4 jours (extrêmes de 2 à 13 jours) [12] . en l'absence d'enquête étiologique spécifique, il est impossible cliniquement ou biologiquement de distinguer une atteinte respiratoire secondaire à un adv d'une autre cause infectieuse [12, 13] . les pharyngites et les angines constituent un syndrome clinique fréquent, et les adv peuvent être identifiés chez 15 à 20 % des enfants porteurs d'une pharyngite isolée. elle peut être exsudative et souvent fébrile. la plupart des cas sont dus aux types 1, 2, 3 et 5 [12] . il est difficile de distinguer une amygdalite bactérienne d'une amygdalite virale. l'infection à adv reste une cause fréquente d'angines ou d'otites moyennes aiguës résistantes à l'antibiothérapie, fréquemment adressées alors aux admissions hospitalières [12, 13] . les fièvres pharyngo-conjonctivales sont parmi les manifestations cliniques les plus typiques et fréquentes de l'infection à adv [12, 14] particulièrement avec les sous-types 3 et 7. elles associent des signes non spécifique : un syndrome pseudogrippal (fièvre, malaise, myalgies), une toux, une congestion nasale, une angine érythémateuse, des adénopathies cervicales, à une conjonctivite uni ou bilatérale. les adv du sous-groupe c (adv 1, 2, 5, 6) rendent compte d'environ 5 à 10 % des infections respiratoires de l'enfant de moins de 6 ans. l'adv est une cause peu fréquente de laryngite. les atteintes par les adv peuvent parfois s'exprimer par une bronchiolite mais également par une maladie plus sévère. formes graves d'infections à adv : les pneumopathies à adv intéressent principalement le nourrisson, l'enfant en bas-âge, le sujet immunodéprimé, le transplanté rénal, médullaire ou pulmonaire et le patient porteur d'une cardiopathie ou d'une atteinte respiratoire chronique [15] [16] [17] . en dehors de ce contexte ces infections respiratoires sont le plus souvent bénignes, mais par-fois cette symptomatologie peut être marquée même chez l'enfant sain [18] . les sérotypes 3, 7 et 21 en particulier peuvent être responsables des atteintes respiratoires les plus graves [8, 19] . le début est alors brutal avec fièvre élevée, la symptomatologie pulmonaire comporte une toux rebelle, une dyspnée avec tachypnée et tirage, des râles bronchiques et parfois alvéolaires. les signes radiologiques sont souvent évocateurs, associant des infiltrats, une atteinte interstitielle et des adénopathies hilaires ; les épanchements pleuraux ont été décrits dans 15 à 25 % des cas [16, 20] . de telles atteintes ont une mortalité d'environ 10 %. après une régression initiale elles font place à une forme plus chronique de bronchiolite définissant la bronchiolite oblitérante. c'est un syndrome relativement rare caractérisé cliniquement par une pneumonie ou une bronchiolite sévère, avec une évolution chron i q u e c o n d u i s a n t s o u v e n t à d e s séquelles permanentes [21, 22] . l'histologie du tissu pulmonaire révèle alors des lésions nécrotiques des bronches et de l'épithélium bronchiolaire avec atteinte des glandes bronchiques. une atteinte pulmonaire persistante après une pneumopathie adénovirale à adv 7 est rapportée chez un tiers à deux tiers des sujets [23] . les lésions les plus graves sont représentées par : les dilatations des bronches, la bronchiolite oblitérante, le poumon clair unilatéral (syndrome de mcleod) ou, rarement, la fibrose pulmonaire [24] . infections disséminées : les pneumonies adénovirales s'accompagnent parfois d'une infection disséminée redoutable avec atteintes cardiaques (myocardite aiguë, péricardite aiguë), hépatiques, pancréatiques, rénales (glomérulonéphrite aiguë), et quelques signes encéphalitiques s'y associent souvent. la létalité est alors élevée, de l'ordre de 30 % [8] . absorbé à la surface des cellules de l'épithélium respiratoire, l'adv pénètre dans les cellules et s'y réplique. l'atteinte de l'ensemble de l'arbre respiratoire se réalise surtout de proche en proche, la plupart des infections restent locales dans les voies respiratoires supérieures voire asymptomatiques. plus rarement l'atteinte tissulaire se fait par voie lymphatique et sanguine. les lésions tissulaires résultent soit directement de l'infection par cytotoxicité, soit indirectement par la réponse inflammatoire et immunitaire. cette dernière est essentielle. les séquelles pulmonaires graves surviennent avec une particulière fréquence dans certaines populations homogènes, chez qui un facteur génétique pourrait intervenir mais le sous-type adénoviral garde un rôle essentiel [25, 26] . le site préférentiel de réplication des adv est l'épithélium respiratoire, la réplication semblant limitée au tissu lymphoïde, qui représente par contre un lieu essentiel de persistance virale [27] . les mécanismes pathogéniques de l'infection à adv sont peu connus. l'épithélium respiratoire est modérément atteint (vacuolisation, atteinte de l'épithélium cilié), sans réelle cytolyse mais avec des infiltrats péri-bronchiolaires et péri-vasculaires à prédominance lymphocytaire (t cytotoxique) ainsi que péri-alvéolaires (lymphocytes, macrophages, polynucléaires neutrophiles). il a été démontré que le facteur alpha de nécrose tumorale (tnf-α), l'interleukine (il)-1 et l'il-6 sont élaborés durant les deux à trois premiers jours de l'infection mais seul le tnf-α a un rôle majeur dans la phase précoce de la pathogénie. chez l'enfant une élévation des igg contenant des complexes immuns circulants spécifiques ou des valeurs sériques élevées d'il-6, d'il-8 et de tnf-α sont associées significativement aux adénoviroses graves [28] . à long terme l'infection à vrs est plutôt corrélée avec l'apparition d'un asthme [29] alors que l'atteinte adénovirale, surtout pour le 7 h, est corrélée avec des séquelles pulmonaires lourdes, dilatations des bronches et poumon hyperclair unilatéral. une équipe a étudié les différences de réponses immunitaires entre ces deux virus chez l'enfant. les cellules mononuclées périphériques (pbmc) produisent davantage d'il-10 lorsqu'elles ont été infectées par le vrs versus l'adv, alors qu'elles sécrètent plus d'interféron (ifn)-γ lorsqu'elles ont été infectées par l'adv versus le vrs. le ratio il-10/ifn-γ est significativement plus bas lors des infections par l'adv, ce qui suggère que l'adv induit une réponse de type th1 [30] . l'étude des mécanismes moléculaires par les mutants thermosensibles d'adv 5, par mutation des gènes des adn-binding protein ou de l'adn polymérase, révèle que la synthèse des protéines structurales et des virions ne sont pas nécessaires au développement des lésions pulmonaires. les pneumopathies à adv sont certainement plus liées à l'expression de certains gènes de ce virus qu'à la réplication virale [31, 32] . la réplication de l'adv se déroule en deux étapes successives : phase précoce survenant avant la réplication de l'adn génomique et phase tardive débutant en même temps que cette dernière. les gènes précoces sont lus dès le début de l'infection (e1a, e1b, e2a, e2b, e3, e4). le gène e1a est le premier transcrit et les protéines correspondantes contrôlent la transcription des autres gènes précoces. les protéines des gènes e1 et e3 jouent un rôle important dans l'établissement de l'infection lymphocytaire latente à adv et l'échappement des adv aux réponses inflammatoires et immunitaires de l'hôte. ces protéines agissent en inhibant les deux actions antivirales du tnf-α, sur le blocage de la réplication virale et la mort des cellules infectées. l'adn adénoviral peut persister plusieurs années dans le poumon après l'infection aiguë. la protéine adénovirale e1a (early region 1a) est exprimée dans le poumon et est capable d'assurer une dysrégulation majeure de l'expression des cytokines, avec une augmentation de l'expression de l'arn messager de la molécule d'adhésion icam-1, de la cytokine inflammatoire il-8 et ceci implique le facteur de transcription nf-κb [33] . les infections subaiguës des voies respiratoires par les adv pourraient être impliquées dans la genèse de certaines formes de bronchopathies chroniques obstructives chez l'enfant et chez l'adulte. macek et al. ont rapporté une étude chez des enfants ayant développé un asthme corticorésistant après une bronchiolite [34] . dans des biopsies bronchiques par bronchoscopie, il a été révélé dans la majorité de ces cas la présence d'une protéine adénovirale de la capside, et l'isolement d'un adv sur culture de cellules a été positive dans certains de ces échantillons. hegele et al. trouvent de plus grandes quantités d'adn adénoviral chez les patients souffrant de bronchopathie chronique obstructive, suggérant le rôle possible de l'adv dans cette maladie [35] . les observations in vivo et in vitro qui démontrent que l'infection adénovirale latente puisse être associée avec la persistance d'une inflammation à bas bruit dans le parenchyme pulmonaire périphérique, suggèrent un lien causal dans l'installation de la bronchite chronique obstructive [36] . parallèlement l'inflammation induite par la fumée de tabac est amplifiée chez les sujets emphysèmateux par l'expression latente de la protéine adénovirale e1a exprimée par les cellules épithéliales alvéolaires [37] . récemment, une étude rapporte l'implication possible de l'adv dans le développement de la dysplasie bronchopulmonaire du prématuré [38] . les aléas du diagnostic étiologique d'une pneumonie chez le jeune enfant conduit quasi systématiquement à l'indication de l'antibiothérapie en raison des risques potentiels de l'abstention thérapeutique [39] : celle-ci pourra être interrompue en fonction des résultats des examens complémentaires. la disponibilité des outils de diagnostic virologique de lecture rapide, fiables et réalisables au lit du malade, actuellement possible pour le vrs et la grippe, pourrait dans l'avenir modifier cette attitude [40] . l'abstention d'une antibiothérapie pourrait en effet être justifiée comptetenu des enquêtes épidémiologiques, mais à condition que soit assuré un suivi clinique rapproché et rigoureux. la bronchite aiguë n'associe par définition aucune atteinte du parenchyme pulmonaire. plus encore que les pneumopathies, l'origine virale prédomine, et l'abstention de toute prescription d'antibiotiques est justifiée en l'absence de risque vital de laisser une bactérie se développer. les critères d'hospitalisation lors d'une pneumopathie supposée virale sont communs avec ceux des infections bactériennes. en l'absence de score validé en pédiatrie, ils se fondent sur l'âge (inférieur à 6 mois), l'aspect général (syn-drome toxi-infectieux), une mauvaise tolérance respiratoire (tachypnée, signes de lutte respiratoire), l'existence d'une hypoxie estimée par la détection de la saturation en oxygène par l'oxymétrie de pouls. d'autres indicateurs sont à retenir : les difficultés à s'alimenter (dyspnée lors de la prise des biberons chez le nourrisson), une aggravation rapide de la maladie, les risques liés au terrain (cardiopathie, bronchopathie chronique, déficits immunitaires…), des conditions socio-économiques précaires ou les difficultés d'accès aux soins. en raison de leur persistance durant une à plusieurs semaines dans les sécrétions nasopharyngées des sujets infectés et de leur capacité de survie de plusieurs heures sur des surfaces inertes, les virus à tropisme respiratoire peuvent se transmettre plus particulièrement aux nourrissons immunologiquement naïfs [41] . il est essentiel de limiter ce risque au cours des hospitalisations, notamment dans les services d'immunodéprimés. la meilleure mesure de prévention reste le lavage des mains. il est recommandé le lavage régulier des surfaces susceptibles d'être contaminées (jouets, table à langer, outre le stéthoscope, otoscope etc) [42] . l'hydratation, le contrôle thermique, l'oxygénothérapie, la kinésithérapie respiratoire restent la base de la prise en charge des infections bronchopulmonaires virales [43] . le retentissement sur l'alimentation est directement fonction de l'intensité de la détresse respiratoire (dyspnée, essoufflement) ou secondaire aux vomissements provoqués par la toux. les risques de déshydratation sont présents chez le nourrisson et une perfusion initiale semble préférable à la mise en place d'une nutrition entérale à débit continu lors de la phase aiguë [44] . l'oxygénothérapie sera initiée lors d'une hypoxémie ; l'oxygène doit être humidifié et administré par lunettes nasales ; son débit sera adapté afin d'obtenir une saturation pulsée en oxygène transcutanée proche de 95 %. c'est une réelle prescription médicale dont les modalités doivent être clairement définies [45] . l'intensité de la détresse respiratoire, l'hypercapnie, conduit parfois à la réalisation d'une intubation endotrachéale avant mise sous ventilation mécanique. la kinésithérapie respiratoire nous semble justifiée lors de la phase sécrétante, sa technique exige une compétence obtenue par une grande pratique pédiatrique. l'accélération du flux expiratoire avec expectoration provoquée est la technique la plus utilisée. peu de références étayent l'efficacité de cet acte en dehors de la mucoviscidose [46] . malgré une prescription très large tant ambulatoire qu'hospitalière, il faut bien reconnaître la faiblesse des preuves cliniques concernant les mucomodificateurs [47] . la toux est un phénomène réflexe protégeant les voies respiratoires basses dont le respect doit être la première attitude. comme pour les mucomodificateurs, peu de preuves existent quant à l'efficacité des antitussifs [48] . en cas de persistance de la toux, le diagnostic d'infection bronchopulmonaire virale devra être révisé. l'inflammation bronchopulmonaire est particulièrement intense lors d'une infection virale, et elle a un double visage : d'une part, il s'agit d'une réaction normale de l'hôte contre l'infection, d'autre part l'amplification de cette réaction peut conduire à la constitution de lésions cicatricielles. peu d'études pédiatriques randomisées sont disponibles, à l'exception de quelques références concernant la corticothérapie inhalée lors des sifflements associés aux infections virales [49] mais dont on connaît les relations étroites avec l'asthme [50] . des vaccins inactivés et vivants expérimentaux ont été développés aux usa pour prévenir les épidémies de pneumonie par les sérotypes 4 et 7 chez les recrues militaires mais n'ont pas été utilisés chez l'enfant, et certaines interrogations sur un éventuel potentiel oncogène exigent de futures recherches [51] . ces vaccins ne sont pas disponibles en france. on possède peu d'informations sur l'éventuelle efficacité de l'utilisation préventive d'immunoglobulines intraveineuses. elle semble être une aide comme traitement adjuvant lors des infections disséminées du nouveau-né ou de l'immunodéprimé. chez le sujet immunodéprimé les infections adénovirales résultent de la réactivation d'infections latentes du tissu lymphoïde (adv 1,2,5) ou du rein ( adv 11, 34, 35) . la ribavirine semble avoir une efficacité en aérosol et surtout par voie intraveineuse. les protocoles thérapeutiques ne sont pas fixés [52, 53] . quelques essais d'utilisation réussis ont porté sur le cidofovir (hpmc ou cdv) parentéral [54] . les données chez l'enfant sont encore très fragmentaires. aucune thérapeutique n'a fait ses preuves après le diagnostic de bronchiolite constrictive. les seuls arguments expérimentaux concernent la corticothérapie si celle-ci pouvait être prescrite la veille de l'agression virale [55] . l'absence d'effet clinique des corticoïdes par voie systémique ou inhalée pourrait être due à une prescription trop tardive, les anomalies anatomiques étant fixées. les bronchodilatateurs inhalés sont également le plus souvent inefficaces [34] . on ne peut que limiter les rechutes infectieuses, qui aggravent la situation respiratoire, grâce à des mesures de prévention. le diagnostic d'infection respiratoire à adv repose sur la mise en évidence directe du virus ou de ses constituants dans les voies aériennes. la détection de cellules spécifiquement infectées par immunofluorescence signe l'atteinte virale de l'épithélium. mais cette méthode manque de sensibilité. les méthodes d'amplification moléculaire permettent une amélioration de la détection virale mais la détection de séquences d'adv ne traduit pas toujours une infection virale active, sachant cependant, que celle-ci n'est pas indispensable pour initier et entretenir une inflammation persistante des voies respiratoires inférieures. bilan de 3 480 aspirations nasales réalisées chez l'enfant en une période de six ans viral etiology in acute lower respiratory infections in children from a closed community diagnostic moléculaire des infections virales respiratoires communautaires les co-infections virales lors des bronchiolites du nourrisson immunocompétent strain variation in adenovirus serotypes 4 and 7a causing acute respiratory disease analysis of 15 different genome types of adenovirus type 7 isolated on five continents adenovirus type 7 associated with severe and fatal acute lower respiratory infections in argentina children adenovirus type 7 h respiratory infections : a report of 29 cases of acute lower respiratory disease molecular and serological characterization of adenovirus genome type 7 h isolated in japan worldwide epidemiology of human adenovirus infections pathologie respiratoire de l'enfant immunocompétent à adénovirus : étude de 116 sujets hospitalisés adenoviral diseases in children : a study of 105 hospital cases comparison of clinical characteristics of adenovirus and non-adenovirus pneumonia in children adenovirus infections in young children adenovirus infection in the immunocompromised patient viral pneumonia adenovirus pneumonia in lung transplant recepients severe adenovirus bronchiolitis in children adenovirus 7a: a community-acquired outbreak in a children's hospital adenovirus pneumonia adenovirus type 7b in children's hospital lung function in infants with chronic pulmonary disease after severe adenoviral illness chronic lung damage caused by adenovirus type 7 : a ten-year follow-up study factors predisposing to abnormal pulmonary function after adenovirus 7 pneumonia bronchiolitis obliterans, bronchiectasis, and other sequelae of adenovirus type 21 infection in young children pneumopathie sévère à adénovirus type 7 chez un adulte immunocompétent group c adenovirus dna sequences in human lymphoid cells cytokines in adenoviral diseases in children: association of interleukine-6, interleukine-8 and tumor necrosis factor alpha levels with clinical outcome respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years differential effects of respiratory syncytial virus and adenovirus on mononuclear cell cytokine responses the molecular basis of adenovirus pathogenesis les pneumonies à adénovirus identification of glucocorticoid-and adenovirus e1a-regulated genes in lung epithelial cells by differential display persistent adenoviral infection and chronic airway obstruction in children mechanisms of airway narrowing and hyperresponsiveness in viral respiratory tract infections role of latent viral infections in chronic obstructive pulmonary disease and asthma amplification of inflammation in emphysema and its association with latent adenoviral infection detection of microorganisms in the tracheal aspirates of preterm infants by polymerase chain reaction: association of adenovirus infection with bronchopulmonary dysplasia current management of community-acquired pneumonia in children: an algorithmic guideline recommendation rationalised prescribing for community acquired pneumonia: a closed loop audit nosocomial adenovirus infection in a paediatric respiratory unit l'isolement en pédiatrie les traitements non antibiotiques des pneumopathies communautaires de l'enfant why block a small hole abc of oxygen. acute oxygen therapy does chest physical therapies work? mucomodificateurs et anti-tussifs systematic review of randomised controlled trials of over the counter cough medecines for acute cough in adults effect of inhaled corticosteroids on episodes of wheezing associated with viral infection in school age children: randomised double blind placebo controlled trial rôle des infections virales et des infections à chlamydia pneumoniae et à mycoplasma pneumoniae au cours de l'asthme du nourrisson et du jeune enfant. à propos d'une étude épidémiologique chez 118 enfants vaccines for control of respiratory disease caused by adenoviruses intravenous ribavirin therapy in a neonate with disseminated adenovirus infection undergoing extracorporeal membrane oxygenation: pharmacokinetics and clearance by hemofiltration intravenous ribavirin treatment for severe adenovirus disease in immunocompromised children successful treatment of adenovirus disease with intravenous cidofovir in an unrelated stemcell transplant recipient obliterative bronchiolitis in children key: cord-283792-g7wyu8pc authors: hiltunen, raimo; josling, peter d.; james, mike h. title: preventing airborne infection with an intranasal cellulose powder formulation (nasaleze travel®) date: 2007 journal: adv ther doi: 10.1007/bf02877720 sha: doc_id: 283792 cord_uid: g7wyu8pc a total of 52 volunteers were recruited to take part in a dual-centered, randomized, blinded study so investigators could determine whether the level of airborne infection could be significantly reduced in patients randomly assigned to treatment with either nasaleze® cellulose extract alone or a combination of nasaleze cellulose and powdered garlic extract (pge). one puff into each nostril was recommended, and volunteers who developed an infection while traveling were told to use at least 3 puffs per nostril until symptoms were reduced. this study took place over an 8-wk period across finland and the united kingdom between november 2006 and march 2007. volunteers were instructed to use a 5-point scale to assess their health and to record infectious episodes and symptoms in a daily diary. the activetreatment group (nasaleze cellulose with pce) experienced significantly fewer infections than the control group (20 vs 57; p<.001) and far fewer days on which an infection was obviously present (126 d in the active group vs 240 d in the control group; p<.05). consequently, volunteers in the active group were less likely to pick up an airborne infection when pce was added to this novel cellulose extract. volunteers in the control group were much more likely to report more than 1 infectious episode over the treatment period or to endure longer periods of infection. the investigators concluded that the combination nasaleze travel formulation significantly reduced the number of airborne infections to which volunteers were exposed while traveling. the common cold is the world's most widespread viral infection; most adults develop approximately 2 to 5 colds per year, irrespective of where they live. more than 200 different viruses are known to cause symptoms of the common cold. some, such as rhinoviruses, seldom produce serious illness. others, such as parainfluenza and respiratory syncytial virus, generally produce mild infection in adults but can precipitate severe lower respiratory tract infection in young children. rhinoviruses (from the greek rhin, meaning "nose") cause an estimated 30% to 35% of all adult cold infections and are most active in early fall, spring, and summer. more than 110 distinct rhinovirus types have been identified. these agents grow best at temperatures of about 91°f-the temperature inside the human nose. scientists believe that coronaviruses cause a large percentage of all adult colds. these viruses bring on colds primarily in the winter and early spring. of the more than 30 types of coronaviruses that have been identified, 3 or 4 are known to infect humans. the importance of coronaviruses as a cause of colds is difficult to assess because, in contrast to rhinoviruses, they are difficult to grow in the laboratory. approximately 10% to 15% of adult cold infections are caused by viruses that are also responsible for other, more severe illnesses; these include adenoviruses, coxsackie viruses, echoviruses, orthomyxoviruses (including influenza a and b viruses, which cause flu), paramyxoviruses (including several parainfluenza viruses), respiratory syncytial virus, and enteroviruses. the causes of 30% to 50% of adult colds that are presumed to be viral in origin remain unidentified. the same viruses that produce colds in adults appear to cause colds in children. the relative importance of various viruses in pediatric colds, however, is unclear because it is difficult to isolate the precise cause of symptoms in studies of children with colds. in cases of airborne infection, the initial entry point in a human being is the nasal cavity. touching the skin or environmental surfaces, such as telephones and stair rails, that have cold germs on them and then touching the eyes or nose and inhaling drops of mucus full of cold germs from the air are the most common methods of transmission. unfortunately, airborne infections are commonplace all year round nowadays; although the chance of picking up an infection in the summer months is only 1 in 4 compared with the chance in winter, special factors may increase this risk. long-haul jet flights appear to pose a particular risk in that at no other time are humans likely to be squeezed as tightly together with 400 potential sources of common cold infection. the chances are that any number of passengers will have the tendency to spread an airborne infection in the confined space of a jetliner, making this an ideal environment for transmission of airborne disease. experiments on exposing uninfected volunteers to others with common cold infections have shown that the chances of catching a cold are directly related to the number of hours of exposure to infection. hence, one is much more likely to get a cold on a long-haul flight to the united states than on a short hop to europe. current lifestyles often demand air conditioning, which may contribute to infection. although the lining of the nose is covered by a thin layer of mucus that protects against infection, air conditioners unfortunately extract moisture from the air; therefore, they may cause some drying of the protective mucous blanket in the nose and predispose to infection. this feature is one that the active test compound nasaleze travel ® (cellulose and powdered garlic extract [pge] combination; nasaleze limited, isle of man, united kingdom) may improve significantly, simply because of the way it works. cold air may also help viruses establish a hold in the nose because they reproduce better in a cold environment. traveling by public transportation can significantly increase the risk of viral infection. although individuals may have already been exposed to current common cold viruses at home, they are likely to encounter new viruses to which they have no immunity as they circulate in public. travelers could actually be responsible for introducing new viruses into a foreign country if they have an active infection when they arrive for a holiday or a business meeting. with modern jet travel, viruses are spread rapidly, which is why influenza spreads so quickly around the world during an epidemic. unfortunately, because of the extensive number of airborne infections, reinfection is common. published literature on the activity of garlic extracts (among others) against viral infection is sparse. 1,2 it has been reported, however, that during an influenza epidemic, the former soviet union imported more than 500 tons of garlic cloves for acute treatment. 3 among the viruses thought to be sensitive to garlic extracts are human cytomegalovirus, human rhinovirus type 2, herpes simplex types 1 and 2, and influenza b. many consumers already take natural remedies such as echinacea, vitamin c, zinc, and garlic supplements for preventive purposes and report an absence of infection with colds and symptoms associated with viral replication. 4 cellulose powder is used as a thickener in many liquid nasal sprays and is generally regarded as safe for consumption. the unique proprietary grade of micronized cellulose used in this study (nasaleze ® ) is administered with a patented device that ensures that a suitable amount of material will be drawn from the container and delivered into the nose. compared with liquid nasal sprays, which require preservatives, powdered cellulose inhibits bacterial and viral growth to a limited extent. although it is not a medicine, this formulation is classified as a medical device that is safe to use throughout the year. this powdered cellulose product addresses the cause of allergic reactions, rather than the symptoms, because it works as a facial mask in preventing inhaled pollen, dirt, and allergens from reaching the lungs. in a healthy individual, the nose and the nasal tract positively extract these materials from inhaled air as it passes through on its way to serve the lungs. this filtration effect is created by very low peripheral air pressure (generated by the internal shape of the nasal tract) as the air goes through the system. this linear, low air pressure area positively attracts entrained allergens and at the same time attaches this material onto the mucous membranes (figs 1 and 2) . 5 mucus has the ability to rapidly adsorb and render harmless any particle that touches its surface. spent mucus is eventually disposed of via the digestive tract. thus, clean, allergen-free air is supplied to the lungs. a unique feature of the cellulose powder formulation described herein is that it turns into a gel on contact with the moisture that is always present in the nasal cavity. this gel is similar to normal mucus and helps to maintain delivery of a supply of clean air to the lungs. this survey was designed to determine whether the addition of a simple pge to nasaleze powdered cellulose would enhance the capability of this formulation to trap airborne infections, disarm them, and remove them safely into the stomach during normal mucociliary clearance. a randomized, blinded study design was applied in 2 countries-finland and the united kingdom-to test whether the addition of pge would increase the potential for preventing airborne infection among individuals traveling locally and nationally during the winter, when airborne infections are at their peak. after completion of recruitment through advertisements in daily newspapers in london and helsinki, 52 participants were selected. all volunteers kept a diary of their general well-being for 8 wk on a 5-point scale as they traveled to and from work or completed various other trips across the united kingdom or finland (and, in some cases, internationally) ( table 1) . if an infection occurred, volunteers noted the number and variety of symptoms, the day recovery began, and the day they felt completely better. the volunteers were separated into 2 groups of 26 participants each and were matched for age. the average age in each group was 38 y, and all participants were actively employed. volunteers who were already taking a garlic supplement were excluded, as were those who reported that they were housebound. a simple random number generator assigned volunteers to the active or the control group, and all were instructed to take 1 sniff in each nostril every day, according to the manufacturer's recommendation; if an infection developed, participants were instructed to take up to 3 sniffs per nostril on each day that the infection was present, so it could be determined whether the infectious period was reduced in either group. randomization codes were kept secure at the herbal research centre and were not broken until all diaries had been returned. volunteers were contacted several times during the study period to ensure that they were complying with the dosage regimen and that diary entries were made daily. participants in finland were also encouraged to record their daily diary scores online using a secure web site. after the diaries had been returned, the number of infections experienced by volunteers was counted. active infection was defined as a score of 3 or less that lasted for 4 days in succession. the duration of symptoms was expressed as the number of days on which a score of 3, 2, or 1 was recorded; average recovery time ended with a score of 4 or 5 taken across all recorded infections. the number of volunteers in each group who did not experience a single airborne infection throughout the study period was recorded. the total number of infectious episodes, the average length of symptoms in days, and the number of days on which the subject was challenged by an active infection were subjected to calculations of standard deviation, sample variance, and standard error of the difference of the means. data were analyzed by means of student t test, so that a probability coefficient could be attained that would allow for the calculated number of degrees of freedom. no participants withdrew from the study; therefore, an intention-to-treat analysis was performed on all completed diaries. at the end of the 56-day study, 57 major infections had been recorded in the control group, but the active group had recorded a total of only 20 infections. this result is highly significant (p<.001) in favor of the addition of pge to nasaleze cellulose powder as a preventive agent for airborne infections acquired during travel ( table 2 ). the control group reported 12 serious cases in which infection lasted for 7 days; the active group reported only 6 such cases. similarly, the number of days on which active infection was present, warranting a recorded score of 3 or less, was 240 in the control group; in the active group, this value was 126 days. this result is also highly significant (p<.05). during this study, 11 volunteers who were taking the control compound experienced multiple infectious episodes; only 2 volunteers who were taking active treatment had this experience, which suggests that this was indeed a preventive option (p<.05 level of significance). details of the statistical analysis indicate that the sample variance and the standard deviation were low, and that although most members of the 2 groups were female volunteers, they were well matched statistically with a standard error for the difference of the means of just 0.76 for the number of active airborne infections reported by each group, so that probability with a student t test was p<.01. significance dropped to p<.05 for the number of volunteers with multiple infectious episodes and the total number of reported days with an active infection. those with a serious infection that lasted 7 days and the number of participants who did not report an infection at all did not reach significance, but clear differences between groups were noted. the investigators believed that the nasaleze travel combination treatment product proved superior to the nasaleze cellulose extract treatment alone. volunteers were also asked to record in their diaries any other concerns that they had during the study, such as comments about the acceptability of taking the product, adverse effects, tastes, or other reasons that might warrant discontinuation of treatment. generally, the product was extremely well tolerated in both groups, although in the active group, several volunteers (n=3) wrote that they could easily taste the pge; however, this did not stop them from completing treatment. in this pilot investigation, 2 inert cellulose powder formulations, both dosed intranasally through a novel, patented delivery system, were compared in a randomized, blinded study, so the investigators could determine which formulation provided the best protection against various airborne infections. the volunteers were encouraged to go about their normal daily lives while traveling within local and national boundaries. some volunteers even ventured out internationally, so this was a genuinely fair assessment of the relative dangers of picking up an airborne infection during the winter, and of assessing how this might be prevented. the results clearly favored the nasaleze travel combination formulation. they indicate that a significant reduction in the number of airborne infectious pathogens picked up by volunteers was seen in this group as opposed to outcomes in the nasaleze cellulose powder alone group. examination of volunteer diaries clearly showed that the control group suffered much more than the active group in terms of the number and duration of infectious episodes. thus, it is concluded that the addition of a potentially antiviral compound, in this case, a pge, can significantly reduce the number of infectious challenges that people meet when they travel. the results also suggest that infection and reinfection may be effectively prevented through its daily use throughout the year, with reduced sick days leading to an enormous potential savings to national economies. this product clearly exhibits excellent antiviral activity, confirming that nasaleze cellulose extract combined with pge is an excellent carrier that allows any number of agents to be spread throughout the nasal cavity for an extended period of time. this novel property clearly warrants further investigation to determine the nature and method of its viral destruction when administered intranasally. in: garlic: the science and therapeutic application of allium sativum l. and related species a double blind placebo controlled evaluation of a garlic supplement in preventing the common cold use of cellulose powder in the treatment of seasonal allergic rhinitis a double blind, placebo controlled trial of inert cellulose powder for the relief of symptoms of hay fever in adults the authors would like to thank all volunteers in the united kingdom and in finland; charlotte duxbury for her excellent administrative assistance; and nasaleze international limited for supplying the test materials. key: cord-257399-p6of5fno authors: gentry, chris a; humphrey, mary beth; thind, sharanjeet k; hendrickson, sage c; kurdgelashvili, george; williams, riley j title: long-term hydroxychloroquine use in patients with rheumatic conditions and development of sars-cov-2 infection: a retrospective cohort study date: 2020-09-21 journal: lancet rheumatol doi: 10.1016/s2665-9913(20)30305-2 sha: doc_id: 257399 cord_uid: p6of5fno background: hydroxychloroquine is one of several agents being evaluated in the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. we aimed to examine whether patients with rheumatological conditions receiving chronic hydroxychloroquine therapy are at less risk of developing sars-cov-2 infection than those not receiving hydroxychloroquine. methods: this retrospective cohort study included de-identified information of all veterans in the us veterans health administration clinical administrative database aged 18 years or older with rheumatoid arthritis, systemic lupus erythematosus, or associated rheumatological conditions (based on international classification of diseases, 10th edition, diagnostic codes) who were alive on march 1, 2020. a propensity score was calculated for each patient, and each patient who was receiving hydroxychloroquine was matched to two patients who were not receiving hydroxychloroquine (controls). the primary endpoint was the proportion of patients with pcr-confirmed sars-cov-2 infection among those receiving chronic hydroxychloroquine versus the propensity-matched patients not receiving chronic hydroxychloroquine between march 1 and june 30, 2020. secondary outcomes were hospital admission associated with sars-cov-2 infection; intensive care requirement associated with sars-cov-2 infection; mortality associated with sars-cov-2 infection; and overall rates of any hospital admission and mortality (ie, all cause). multivariate logistic regression analysis was done to determine independent variables for the development of active sars-cov-2 infection. findings: between march 1 and june 30, 2020, 10 703 patients receiving hydroxychloroquine and 21 406 patients not receiving hydroxychloroquine were included in the primary analysis. the incidence of active sars-cov-2 infections during the study period did not differ between patients receiving hydroxychloroquine and patients not receiving hydroxychloroquine (31 [0·3%] of 10 703 vs 78 [0·4%] of 21 406; odds ratio 0·79, 95% ci 0·52–1·20, p=0·27). there were no significant differences in secondary outcomes between the two groups in patients who developed active sars-cov-2 infection. for all patients in the study, overall mortality was lower in the hydroxychloroquine group than in the group of patients who did not receive hydroxychloroquine (odds ratio 0·70, 95% ci 0·55–0·89, p=0·0031). in multivariate logistic regression analysis, receipt of hydroxychloroquine was not associated with the development of active sars-cov-2 infection (odds ratio 0·79, 95% ci 0·51–1·42). interpretation: hydroxychloroquine was not associated with a preventive effect against sars-cov-2 infection in a large group of patients with rheumatological conditions. funding: none. the emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in wuhan (hubei province, china) in late 2019 sparked a global pandemic that, towards the end of august, 2020, had resulted in close to 25 million known cases with almost 800 000 attributed deaths. [1] [2] [3] the pandemic reached the usa in january, 2020, and by the end of august more than 180 000 deaths had been recorded among 5·8 million cases. 4 as the research community launched several efforts to develop vaccine candidates, various potential pharmacotherapy options began to be explored on the basis of previous in-vitro and in-vivo studies of existing coronaviruses and on the few early in-vitro studies on sars-cov-2. 5, 6 in addition to antiviral agents, some atypical drugs, such as the antimalarial drugs chloroquine and hydroxychloroquine, demonstrated in-vitro activity. in the usa, chloroquine was not on the market, but hydroxy chloroquine had been available for several decades, principally used for treatment of patients with rheuma toid arthritis, systemic lupus erythematosus, and other associ ated autoimmune disorders. 7, 8 the us health-care community began to focus on hydroxy chloroquine and its potential role in the treatment of sars-cov-2 infection, and the federal government and lay media were soon to follow. there was sufficient interest in hydroxychloroquine for health-care institutions to increase procurement of the drug, causing shortages in the supply chain that threatened the continued avail ability of the drug relied upon by millions of patients with rheumatological conditions. 9 unfortunately, reports began to surface questioning whether patients with active sars-cov-2 infection received benefit from hydrox ychloroquine, and safety concerns arose that focused on its propensity to induce prolonged qt arrhythmias. [10] [11] [12] these data led the us food and drug administration to revoke the emergency use authorisation for hydroxy chloro quine and chloroquine to treat active sars-cov-2 infection on june 15, 2020, only 11 weeks after initial issuance. 13 a small trial reported preliminary evidence of short-term post-exposure prophylaxis of hydroxychloroquine among 821 household and occupational contacts of patients with newly diagnosed sars-cov-2 infection. 14 the trial failed to demonstrate a difference between hydroxy chloroquine and placebo among all participants or among the 20 laboratory-confirmed cases of infection, although the authors noted that a marginal benefit of hydroxychloroquine could not be ruled out. randomised trials designed to evaluate interventions to prevent active infection have a unique set of challenges, including sparse event rates over time, which results in the need for substantial time and effort to achieve the power necessary to detect an effect. the results of large prevention trials will probably remain unavailable for several months. however, an alternative approach of compiling observa tional data from large clinical administrative databases might be useful to more rapidly identify preventive effects of an intervention. we aimed to examine whether patients with rheuma tological conditions receiving chronic hydroxy chloroquine therapy are at less risk of developing sars-cov-2 infection compared with a propensity-matched group of patients not receiving hydroxychloroquine. in this retrospective cohort study, de-identified information was obtained from across all us veterans affairs medical centers (vamcs) for eligible patients aged 18 years or older throughout the veterans health administration (vha). the vha is the largest inte grated health-care system in the usa, providing care in 1255 health-care facilities, including 130 health-care centres and 1074 outpatient sites, serving 9 million enrolled veterans each year. 15 a central clinical and adminis trative relational database, the corporate data warehouse, maintains all information from the vha's comprehensive electronic medical record sys tem and is accessible to vha clinical researchers following a rigorous approval process. the patient cohort consisted of all veterans in the vha system who were alive as of march 1, 2020, who had international classification of diseases (10th edition; icd-10) diagnostic code entries for rheuma toid arthritis, systemic lupus erythematosus, and associ ated rheumatologi cal conditions recorded from vha encounters between oct 1, 2016, and march 1, 2020 (appendix p 1). data were collected to determine the following: evidence of receipt of hydroxychloroquine to the equivalent of at least four 90-day supplies since april 1, 2019, and a medication possession ratio calculation of 80% or more from july 1, 2019, to june 30, 2020, with the most recent receipt within a timeframe to include the date of march 1, 2020; 16 baseline demographic data as of march 1, 2020, to determine age, race, sex, and any tobacco use; all icd-10 codes from oct 1, 2016, to march 1, 2020, to determine the presence of chronic comorbidities; labora tory variables to assess organ dysfunction and to char acterise classification and progression of the patient's rheumatological condition from april 1, 2019, to june 30, 2020, including c-reactive protein, erythrocyte sedi mentation rate, white blood cell count, haemo globin, haematocrit, platelet count, blood urea nitrogen, serum creatinine, aspartate amino transferase, alanine amino trans ferase, lactate dehydro genase, and alkaline phos phatase; out patient prescriptions for methotrexate, leflu no mide, sulfasa lazine, tofacitinib, prednisone, angiotensin-convert ing enzyme 2 inhibitors, angiotensin ii receptor blockers, and zinc, vitamin d, and vitamin c preparations where availability included the date of march 1, 2020; and outpatient prescriptions or infusion evidence before this study we searched pubmed, up to may 1, 2020, for published clinical trials assessing the effect of hydroxychloroquine to prevent laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. the search terms used were "covid-19", "sars-cov-2", and "hydroxychloroquine". we identified a lack of well powered studies to determine the preventive effect of hydroxychloroquine on the development of laboratory-confirmed sars-cov-2 infection. our study is the first to examine a large nationwide population of individuals with rheumatological conditions receiving long-term hydroxychloroquine with high adherence rates, comparing the population to a propensity-matched population not receiving hydroxychloroquine. the primary endpoint, the development of laboratory-confirmed sars-cov-2 infection, was not significantly different between the two propensity-matched cohorts. the findings of this study expand the knowledge base on the role of hydroxychloroquine in sars-cov-2 infection, supporting preliminary data from smaller studies suggesting that hydroxychloroquine might not be an effective agent to prevent sars-cov-2 infection. see online for appendix clinic orders for adalimumab, certolizumab, etanercept, golimumab, infliximab, abatacept, rituximab, belimumab, or tocilizumab where last dose administered would remain active (based on frequency given) up to the date of march 1, 2020. all univariate variables were assessed for their association with the use of hydroxychloroquine. those univariate variables with a standardised mean difference of more than 0·10 were entered into a nominal multivari ate logistic regression model to determine indepen dent variables associated with the use of hydroxychloro quine. this model computed a propensity formula, and a propensity score was calculated for each participant. each patient who was receiving hydroxychloroquine was matched to two patients who were not receiving hydroxy chloroquine (controls) with the next-nearest propensity score to the patient receiving hydroxychloro quine, stratified by the vamc and rural or urban status, sorted by area zip code. the resultant propensity population was assessed with the following data collection for data points between march 1 and june 30, 2020: hospital admission dates and discharge dates; ward locations associated with hospital admission for any reason; discharge diagnostic icd-10 codes associated with each admission; emergency or urgent care clinic encounters; any influenza tests done at the individual facilities; any pcr test results for sars-cov-2; and dates of death if applicable. this study was approved by the university of oklahoma health sciences center institutional review board and the oklahoma city va healthcare system research and development committee. the primary endpoint was the proportion of pcrconfirmed sars-cov-2 infection among those receiv ing chronic hydroxychloroquine versus the propensity-matched patients not receiving chronic hydroxy chloro quine between march 1 and june 30, 2020. secondary endpoints comparing patients receiving hydroxy chloroquine with those not receiving hydroxychloro quine within the same time period were: hospital admission associated with sars-cov-2 infection; intensive care requirement associated with sars-cov-2 infection; mortality associated with sars-cov-2 infection; and overall rates of any hospital admis sion and mortality for both propensity-matched groups. we did a univariate analysis to determine variables associated with the development of sars-cov-2 infection, including receipt of chronic hydroxychloroquine. variables with a p value of 0·05 or less were entered into a multivariate logistic regression model to determine variables independently associated with the development of sars-cov-2 infection; receipt of chronic hydroxychloroquine was included in the multivariate model regardless of p value. for all tests and analyses except where specified, the a-priori level of significance was set at a p value of 0·05 or less. standardised mean difference measurements were con sidered well balanced if they were less than 0·25. categorical variables were assessed using χ² test and fisher's exact test where appropriate. wilcoxon rank sum test was used to assess continuous vari ables. univariate and multivariate analyses were done as described. all analyses were done with jmp/sas statistical software (version 12). there was no funding source for this study. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. an icd-10 code for any rheumatological-associated condition was found for 194 900 patients. of these patients, 18 516 were excluded because they died before march 1, 2020. 100 639 patients were excluded because of the presence of only a non-specific icd-10 diagnostic code indicating arthritis otherwise unspecified, arthropathy, several univariate variables were found to be associated with the selection of hydroxychloroquine at a significant level (appendix pp 2-4). the resultant multivariate logistic regression model derived from these variables resulted in a good fit, and odds ratios (ors) and 95% cis for variables found to be independently associated with hydroxychloroquine selection are presented in table 1. the baseline characteristics of 10 703 patients who received hydroxychloroquine compared with 21 406 patients who did not receive hydroxychloroquine were largely similar in the propensity-matched analysis, although some small numerical differences were significant, including younger mean age in those receiving hydroxychloroquine (64·8 years [sd 12·9]) compared with those not receiving hydroxychloroquine (65·4 years [13·3], p<0·0001; table 2). the incidence of active sars-cov-2 infections during the study period did not differ between the two groups (31 [0·3%] of 10 703 vs 78 [0·4%] of 21 406; or 0·79, 95% ci 0·52-1·20, p=0·27; table 3), resulting in an overall rate of infection of 3·39 cases per 1000 patients. the figure shows no difference in time to positive sars-cov-2 pcr test between groups (p=0·27), with day 0 indicating march 1, 2020. sars-cov-2-related secondary outcomes showed no significant differ ence between the two groups among patients who developed active sars-cov-2 infection. overall hospital admission did not differ between the groups; however, overall mor tality was lower in patients receiving hydroxychloroquine than in data are n (%) or mean (sd) unless otherwise stated. csdmard=conventional synthetic disease-modifying antirheumatic drug. *csdmards include hydroxychloroquine, methotrexate, lefunomide, and sulfasalazine; other csdmard refers to agents other than hydroxychloroquine. univariate variables associated with the development of sars-cov-2 infection are presented in the appendix (pp 5-7). the resultant multivariate logistic regression model showed that the following variables were independently associated with sars-cov-2 infection: presence of polyarthritis (or 4·01, 95% ci 1·76-7·89), non-white race (1·65, 1·08-2·50), urban residence (1·78, 1·14-2·89), receipt of vitamin c (3·31, 1·37-6·83), receipt of an angiotensin-converting enzyme 2 inhibitor (1·74, 1·08-2·72), elevated erythrocyte sedimentation rate (1·69, 1·08-2·59), and baseline c-reactive protein greater than 10 μg/ml (2·14, 1·21-3·63). receipt of hydroxy chloro quine was placed into the model but was not independently associated with sars-cov-2 infection (or 0·79, 95% ci 0·51-1·42). our study examined a large nationwide population of patients with rheumatological conditions to determine whether chronic hydroxychloroquine might protect against the development of sars-cov-2 infection. in this study, the proportion of patients with laboratory-confirmed sars-cov-2 infection did not differ between people with rheumatological conditions who received hydroxychloroquine and those who did not, suggesting that chronic hydroxychloroquine might not have a role in the prevention of sars-cov-2 infection. the overall rate of infection of 3·39 cases per 1000 patients in this study appears to be relatively close to the rate of infection in the vha healthcare system, as 18 560 infections had been diagnosed-out of a total of close to 9 million enrolled veterans-by june 29, 2020. 17 although there were no differences between groups in infection-related secondary out comes among patients who developed active sars-cov-2 infection, overall mortality was lower in patients receiving hydroxychloroquine. given that our study's primary purpose was to investigate the association between a drug and prevention of a specific infection, we cannot make con clusions about the observed difference in overall mortality. the high adherence to hydroxychloroquine might result in prolonged survival in patients with sys temic lupus erythematosus and rheuma toid arthritis, 8 and individ uals in the hydroxychloroquine were slightly younger than those in the control group. reports of hydroxychloroquine's in-vitro activity against sars-cov-2 led to rapid inclusion of the drug in clinical studies and to clinical use in patients with active infection. 6, 18 by contrast, other early investigations sug gested that hydroxychloroquine might lead to a delay in mounting an initial antiviral response and increase the initial viral load. [19] [20] [21] one of the first open-label studies by gautret and colleagues 22 showed that patients infected with sars-cov-2 who received hydroxychloroquine and azithromycin had a higher frequency of nasal clearance of the virus compared with patients who did not receive the drug combination. a follow-up study by the same authors that did not include a control group showed rapid trans formation to negative pcr test for sars-cov-2 in patients receiving hydroxychloroquine and azithromycin. 23 both studies were limited because of the study design, small sample sizes, and questionable exclusion of some patients from data analysis. despite the scarcity of substantial evidence, use of hydroxychloroquine with and without azithromycin was rapidly and widely incorporated into treatment protocols for sars-cov2 infection in the usa and globally in early march, 2020. rheumatology associ ations such as the european league against rheumatism and the american college of rheumatology raised con cerns over possible hydroxychloroquine shortages, noting its effectiveness to treat joint pain, autoimmune rashes, and autoimmune thrombotic events, to prevent lupus flares, and to potentially prolong survival in patients with systemic lupus erythematosus and rheumatoid arthritis. 24 more recently, results of studies evaluating hydroxy chloroquine for the treatment of active sars-cov-2 infection have been inconclusive. a meta-analysis includ ing three studies did not show earlier or higher frequency of viral clearance in patents receiving hydroxychloro quine, and showed a two times increase in death in these patients. 25 in addition, a large observational study of 1376 patients in new york city (ny, usa) reported no signifi cant association between hydroxychloroquine use and a combined endpoint of intubation or death (hazard ratio 1·04, 95% ci 0·82-1·32). 10 effective antiviral pharmacological intervention could fill an important gap to prevent sars-cov-2 infection and is likely to play an important part even after effective vaccines become available. so far, there have been no reports of studies evaluating the preventive effects of pharmacological agents other than hydroxy chloroquine against sars-cov-2 infection. a 5-day high-dose course of hydroxychloroquine given to 821 household and occupational contacts of sars-cov-2-infected individuals as post-exposure prophylaxis failed to show a difference between hydroxychloroquine and placebo in the development of compatible symptoms of disease (49 [11·8%] of 414 individuals vs 58 [14·3%] of 407 individuals, p=0·35). 14 however, only 20 participants developed laboratoryconfirmed sars-cov-2 infection in the study (11 [2·7%] of 414 participants in the hydroxy chloroquine group vs nine [2·2%] of 407 in the placebo group, p=0·82). the trial was halted prematurely for futility before the a-priori level of power could be reached. 14 another study indirectly investigated usage of hydroxy chloroquine and colchicine in 1317 individuals who tested positive for sars-cov-2 infection in israel compared with individuals who tested negative. 26 in that study, five of six baseline variables were not balanced between individuals testing positive versus those testing negative, there was no baseline comparison of patients receiving or not receiving hydroxychloroquine, and there was no analysis of adherence among the patients deemed to be receiving hydroxychloroquine (ie, those who had one prescription dispensed between january, 2020, and the patient's first sars-cov-2 positive or negative test). the study showed no difference in the proportion of patients receiving hydroxychloroquine who had a positive test versus those who only had a negative test; however, only three (0·23%) patients in the infected group had received a hydroxychloroquine prescription. studies evaluating prolonged hydroxychloroquine use for prevention of sars-cov-2 infection might provide more useful evidence than short post-exposure regimens. hydroxychloroquine has a long terminal half-life of approximately 45 days and a large volume of distribution. 7 these pharmacokinetic characteristics result in substantial drug accumulation in plasma and tissues over time. our study takes advantage of a setting in which a specific group of patients has been receiving chronic hydroxy chloroquine over several months to years as a novel virus emerges among the population, setting up an ideal premise to test the hypothesis that hydroxychloroquine might be effective in preventing sars-cov-2 infection. the standard limitations of a non-randomised, observational retrospective study using a clinical administrative database apply to our study. however, a rigorous multivariate regression-derived, propensity-matching process was used to produce two comparable groups. march 1, 2020, was our baseline event date, just days before the first known entry of sars-cov-2 infection into the vha. to gather baseline data, we collected all chronic comorbidity data for the 4 preceding years, and we collected laboratory data from 1 year previously (using the most recent value for each) for comprehensiveness. women comprised only 24% of the study population; however, this proportion is much higher than that in most studies done using the vha, as only approximately 5-10% of the enrolled veterans are female. adherence to hydroxychloroquine was mea sured by a 12-month history of prescriptions filled to produce a medication possession ratio. this method does not ensure that patients are taking the medication appropriately, but our strict threshold of including only those with a medication possession ratio of 0·8 or above improves the chances that our population was adherent. no similar measure of adherence was undertaken to evaluate other medications that the patient was receiving, although each medication was documented to have a dispense date with supply that included march 1, 2020. if one extrapolates the high level of adherence of the included patients using hydroxychloroquine to other areas such as ove rall medi cation adherence or healthy lifestyle choices (eg, exercise and diet), a claim could be made that this high level of adherence might create an imbal ance between the included patients using hydroxy chloroquine and those not using hydroxychloroquine. any perceived imbalance could be corrected with appropriate multivariate analyses. as an alternative, investigators could choose to include patients with poor adherence to hydroxychloroquine (34% of patients receiving hydroxychloroquine were excluded in our study for poor adherence) to assess the endpoint of prevention of sars-cov-2 infection; this approach would invite much-deserved criticism, however, of diluting any actual preventive effect of hydroxy chloroquine. the primary endpoint was the development of sars-cov-2 infection during the initial 4-month period of the pandemic as recorded in the us vha system. although we present a time-to-event description in the figure, our study was not designed to provide sophisticated analysis of time-to-event data. many institutions' policies regarding sars-cov-2 pcr testing have depended on test supply availability and prevalence in a particular area; therefore, many people suspected of having sars-cov-2 infection might not have been tested, particularly early in march. thus, given that our outcome measures relied on the objective parameter of a positive sars-cov-2 pcr test, some patients with active infection might not have been included. it is also possible that veterans were tested and treated outside the vha, so we might have not been able to find these patients using the corporate data warehouse database. however, the vha system is used primarily for the majority of the health-care needs of its enrolled veterans, so its electronic database is quite comprehensive for each veteran's healthcare data. to control for the variabilities in testing practices and area prevalence, each propensity-matched patient not on hydroxychloroquine was matched to a patient on hydroxy chloroquine enrolled in the same vamc and by rural or urban residence, sorted by area zip code. the devastation of the sars-cov-2 pandemic and the absence of an available vaccine make it imperative that the research community prioritises pharmacological treatment or prevention strategies effectively and efficiently. the use of observational data drawn rapidly from large clinical administrative databases might be an effective strategy to identify promising agents for further research or to identify agents that might not merit more effort. our data adds to the expanding knowledge base that suggests that hydroxychloroquine might not be an effective agent in the battle against sars-cov-2. cag was responsible for the concept of the study. cag and skt contributed to drafting of the article and critical revisions. cag, skt, rjw, mbh, gk, and sch contributed to the study design, data analysis, and data interpretation, and approved the final version of the article. we declare no competing interests. the vha corporate data warehouse protects information of veterans within the electronic database and does not generally allow sharing of data to individuals or entities outside the vha. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges review of the 2019 novel coronavirus (sars-cov-2) based on current evidence covid-19) weekly epidemiologic update an interactive web-based dashboard to track covid-19 in real time remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology american college of rheumatology guideline for the treatment of rheumatoid arthritis hydroxychloroquine or chloroquine for treatment or prophylaxis of covid-19: a living systematic review observational study of hydroxychloroquine in hospitalized patients with covid-19 clinical efficacy of hydroxychloroquine in patients with covid-19 pneumonia who require oxygen: observational comparative study using routine care data association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid-19 in new york state fda cautions against use of hydroxychloroquine or chloroquine for covid-19 outside of the hospital setting or a clinical trial due to risk of heart rhythm problems a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid-19 department of veterans affairs statistics at a glance standardizing terminology and definitions of medication adherence and persistence in research employing electronic databases us department of veterans affairs. department of veterans affairs covid-19 national summary hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro initial viral load and the outcomes of sars effects of hydroxychloroquine on immune activation and disease progression among hiv-infected patients not receiving antiretroviral therapy: a randomized controlled trial selective regulation of cytokine secretion by hydroxychloroquine: inhibition of interleukin 1 alpha (il-1-alpha) and il-6 in human monocytes and t cells hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study use of hydroxychloroquine and chloroquine during the covid-19 pandemic: what every clinician should know hydroxychloroquine in patients with covid-19: a systematic review and meta-analysis continuous hydroxychloroquine or colchicine therapy does not prevent infection with sars-cov-2: insights from a large healthcare database analysis no external funding was received for this study. this material is the result of work supported with the resources and use of facilities at the veterans affairs health care system (oklahoma city, ok, usa) and the vha corporate data warehouse. key: cord-261150-cdo7y3ob authors: fthenakis, g. c.; mavrogianni, v. s.; gallidis, e.; papadopoulos, e. title: interactions between parasitic infections and reproductive efficiency in sheep date: 2015-02-28 journal: veterinary parasitology doi: 10.1016/j.vetpar.2014.12.017 sha: doc_id: 261150 cord_uid: cdo7y3ob abstract this review article summarises the many reports in the literature, confirming that, in sheep, parasitic infections can adversely affect reproductive efficiency; examples, which refer to all parts of the reproductive cycle of sheep, are as follows: trichostrongylosis in ewe-lambs (which can lead to delayed attainment of puberty), myiosis of the prepuce (which can cause impediment of mating), chorioptic mange or trypanosomosis in rams (which can lead to testicular degeneration or azoospermia, respectively), trypanosomosis or sarcoptic mange in pre-conceptual ewes (which can lead to poor conception rates or reduced number of ovulations, respectively), toxoplasmosis or neosporosis in pregnant ewes (which are causes of abortion), trichostrongylosis or trematode infections in lactating ewes (which can cause reduction of milk yield and can be a risk factor for mastitis, respectively), cryptosporidiosis in newborn lambs (which can be a cause of deaths), coccidiosis in growing pre-weaned lambs (which can cause suboptimal growth rate). in other cases, the reproductive status of the animal can influence the parasitic infection; examples are as follows: the increase in faecal parasitic output during the peri-parturient period (as a consequence of the peri-parturient relaxation of immunity), the heavier trichostrongylid infections of twin lambs compared to lambs from single parities (as a consequence of developmental origin issues in twin lambs). all the above examples support the idea of presence of interactions between parasitic infections and reproductive efficiency in sheep. in sheep, 'reproductive efficiency' includes the ability of ewes to ovulate, be mated, conceive with semen from fertile rams, carry foetuses to term and, finally, lamb live-born lambs, which will be weaned in the appropriate time at an optimal bodyweight. the term implies efficient conception through active gametes, uninterrupted pregnancy, normal delivery of the newborn(s) ('eutocia'), unimpaired lactation within this frame, trichostrongylid gastrointestinal infections currently are among the major challenges in sheep health management, due to the widespread anthelmintic resistance in many parts of the world torres-acosta et al., 2012) , which increases potential adverse effects in health and welfare of animals. the present paper reviews and discusses interactions between parasitic infections and reproductive efficiency in sheep. the article focuses only on direct effects of parasites on the reproductive efficiency of sheep. nevertheless, it is noteworthy that many arthropods (flies, midges, ticks, etc.) can transmit various infective agents, which may adversely affect the reproductive efficiency of sheep. examples of such agents include anaplasma phagocytophilum (stuen and longbottom, 2011) , as well as various viruses, e.g., the bluetongue virus (osburn, 2007) , the rift valley fever virus (bath, 2007) and the schmallenberg virus (lievaart-peterson et al., 2012) , which have a foetopathic and/or abortifacient effect. in this article, the topics are organised in a pattern according to the reproductive cycle of small ruminants. puberty is the end point of a series of events affecting the development of the 'hypothalamo-pituitary-gonadal' axis leading to reproductive competence. from a practical point of view, puberty for females is the age at which the animal can support pregnancy to term. puberty is a complex mechanism involving primarily the reactivation of the gonadotropin-releasing hormone (gnrh) secretory system, affected by various factors (senger, 2003; ebling, 2005) . among those, energy-deprived animals have been found to have a delayed puberty that has been attributed to a lesser frequency of gnrh pulses and accordingly of luteinizing hormone (lh) pulses i'anson et al., 1997; polkowska et al., 2003) . the age at which ewe-lambs are mated, is crucial for productivity of a flock, since cost is high for maintaining unbred animals in a flock. management of ewe-lambs for enhanced reproductive performance requires increased energy availability. this can be achieved by either increasing energy intake by the animal (i.e., availability of high-energy ration or increased quantity of feed) or by reducing its energy drains (e.g., anthelmintic treatment) . possibly therefore, parasitic infections can have an effect in attainment of puberty through interaction with energy intake as discussed herebelow. there is evidence that parasitic infections, specifically gastrointestinal trichostrongylid or trypanosoma congolense (osaer et al., 1999) infections, can adversely affect onset of puberty and age at first lambing by depressing weight gain of affected animals. administration of a long-acting nematocide anthelmintic, which effectively protected treated ewe-lambs for up to 85 days post-ram introduction, allowed treated animals to exhibit their full reproductive potential during that time . treated ewe-lambs showed earlier reproductive activity, as expressed by short 'interval to first mating after ram introduction' and increased 'cycling rate'; this resulted in significantly younger 'age at first mating' . anthelmintic-treated ewe-lambs reached heavier bodyweight, which is a determinant for puberty in sheep ; thus, they were mated and conceived earlier than untreated controls. there are also similar findings in cattle, showing that nematode-infected heifers reach puberty with a delay compared to uninfected animals (díaz-torga et al., 2001) . genital myiosis caused by various dipteran insects, can lead to vulvar oedema and subcutaneous fistulae around the vulva in ewes and to difficult/partial exteriorising of penis and markedly thickened prepuce with subcutaneous fistulae along the tissue in rams (fragkou et al., 2013) . these lesions can impede mating. development of myiosis lesions takes place during the summer months (wall, 2012) . at para-mediterranean areas and other locations of similar (north hemisphere) or of respective (south hemisphere) geographical latitude, summer months coincide with the sheep reproductive season; at more northern (north hemisphere) or more southern (south hemisphere) latitudes, they precede that period (abecia et al., 2011 (abecia et al., , 2012 . tick predilection for the genital organs (vulvar mucosa, scrotum) of sheep has been reported (fourie and kok, 1995; mbuh et al., 2008) and may be responsible for local nuisance and/or inflammatory reaction, which may also adversely affect mating. one may also suggest that ectoparasitic infections with intense pruritus (e.g., heavy lice infestation, mange) could lead to reduced mating activity of rams, as these animals would be busy scratching rather than being sexually active. ridler et al. (2012) have proposed that parasitic diseases can affect the scrotal circumference of rams and that parasite control is important for keeping rams sound for breeding. scrotal circumference in rams has been associated with their reproductive performance (kafi et al., 2004; gouletsou and fthenakis, 2010) . however, gaglio et al. (2010) have not identified a significant effect of gastrointestinal trichostrongylid infection to semen parametres in rams. (rhodes, 1975 (rhodes, , 1976 has presented evidence that extensive chorioptic mange in the scrotum of rams led to reduced quality of their semen, due to seminal degeneration, as a consequence of long-standing increased intra-scrotal temperature due to the inflammation; semen quality was restored after cure of the skin lesions. lopes et al. (2009) have identified that toxoplasma gondii-infected rams have produced smaller volumes of ejaculate than healthy animals, whilst sangare et al. (2010) have reported azoospermia in tr. congolense-infected rams. finally, sarasa et al. (2011) have reported that sarcoptic mange can cause reduction of the testicular mass in capra pyrenaica (a wild small ruminant); these findings may have implications for sheep, in which species sarcoptic mange is a significant health problem (doukas et al., 2007) . moreover, parasitic agents may be transmitted through the semen of rams. t. gondii shedding has been identified in the semen of rams (de moraes et al., 2010a,b) and the possibility for transmission of the parasite to ewes during mating has been described (de moraes et al., 2010a,b) depending on the tachyzoite content of semen. in a recent study, dna of neospora caninum was detected in the semen of experimentally infected rams (syed-hussain et al., 2013) , although none of the ewes mated with those rams developed the disease. no relevant studies are available regarding besnoitia infection of rams. nevertheless, experimental work performed in bucks has indicated that besnoitia caprae can affect their genital system, with cysts of the protozoon identified in the testicular parenchyma and the scrotum (oryan et al., 2011) . other clinical studies have indicated that besnoitia besnoiti can affect the testicular parenchyma and scrotum of bulls (sekoni et al., 1992; dubey et al., 2013) , leading to reduced libido and suboptimal semen quality, although in subsequent studies the protozoon could not be detected in the semen of affected bulls (esteban-gil et al., 2014) . in general, there is still little published evidence to corroborate an adverse association between parasitism and testicular function (which is reflected in semen quality), although available results point out to that direction. possibly, parasitic infections can directly affect semen quality of rams and, hence, influence conception rates in a flock. alternatively, changes in testicular function can lead to reduced production of testosterone, which is a determinant of a ram's sexual behaviour and social ranking within a flock (parkinson, 1996) ; this may lead to changes in social interactions and behaviours during mating period. all the above can affect conception rates in a flock, especially if the ram:ewe ratio is at the lower acceptable level or if reproductive management techniques are applied in the flock. this is an area where further work will elucidate mechanisms and potential interactions between parasitism and reproduction. the period immediately before and around conception is a potentially vulnerable period. during that period, adverse developmental origin of the foetus might initiate, as the result of reduced availability of nutrients to the female animal (fleming et al., 2012) . the significance of increased energy available to ewes in the pre-conception period has been recognised for a long time (clark, 1934; walkden-brown et al., 1999; dobson et al., 2012) . increased energy is provided to ewes by means of modifying the nutritional regime in the period before ewes would be put with rams for mating. at the start of the mating season, ewes should have a body-condition score of '3' to '3.5' on the five-point scale (lovatt, 2010) . 'flushing' consists of administration of an additional quantity of concentrate feed mixture, on top of the ration administered to cover maintenance requirements of the animals and should commence at least 35 days before start of the mating period; that interval is equivalent to the length of two full oestrous cycles of sheep. in animals with appropriate body-condition score, this increased energy feeding aims to producing higher ovulation numbers, leading to greater number of lambs born per ewe. in animals at lower body condition score, there is a benefit to other reproductive parametres (e.g., earlier start of the annual reproductive activity, improved cycling and lambing rate), but no significant improvement in their fecundity (heasman et al., 1998; fthenakis et al., 2012) . the converse is also true. poor body condition of ewes is associated with reduced fertility, characterised by delayed oestrus development and reduced 'cycling rate' in a flock. ovulation rates decrease as body condition score of the animals deteriorates (dobson et al., 2012) , whilst embryo mortality at the early stages of pregnancy is higher in undernourished animals (gunn and doney, 1975) . in general, decreased energy availability around the peri-conceptional period, depresses reproductive performance of extensively (hill farming research organisation, 1979) or intensively (orskov, 1982) managed sheep and leads to reduced cyclic activity, reduced ovulation rates and suboptimal ova survival, as well as increased risk of early embryonic deaths (gunn, 1967) . decreased energy availability at the post-conception period leads to compensatory changes in the gravid uterus later in pregnancy, e.g., increased vascular density of the placentomes (zhu et al., 2007) , which in turn can lead to higher risk of foetal infection with parasitic abortifacient agents (section 6.1), especially in multiple pregnancies; health of the lamb(s) after birth is also adversely affected (fleming et al., 2012) . lambs from multiple parities were found to be more heavily infected with trichostrongylids than lambs from single parities (hayward et al., 2010) . twin foetuses develop a between-them competition for nutrients, are enveloped by a smaller placenta than single foetuses and live in a restricted physical space . the findings of hayward et al. (2010) are in line with the 'developmental origins of health and disease' concept (formerly known as foetal programming or the barker hypothesis), which implicates early in utero development and the maternal environment experienced during that period as being of significance to development of disease in adulthood (fleming et al., 2012) . the most significant energy-drain of clinically healthy sheep is parasitism by gastrointestinal helminths. the effects of these parasites in reducing energy availability for their hosts have been well documented (coop and kyriazakis, 1999) . these authors have proposed that gastrointestinal nematodes can reduce nutrient availability to the host, through a reduction in voluntary feed intake and/or a reduction in the efficiency of absorbed nutrients; the relative contribution of each of these two mechanisms depend on the species of parasite and its location in the gastrointestinal tract (coop and kyriazakis, 1999) . with regard to trematode infections, reduced feed conversion efficiency, present even in low burden infections (hawkins and morris, 1978) , as well as depressed appetite and feed intake, also occurring in these helminthoses (taylor et al., 2007; rojo-vázquez et al., 2012) , contribute to decreased energy availability for affected sheep. it thus becomes evident that parasitism by the above helminths may affect reproductive efficiency in ewes during the peri-conception period, mostly through the decreased energy availability for the animals. the precise adverse effects can vary depending on the level of parasitism and the general condition of the animal. it is also noteworthy that reduced feed intake occurring in such cases, would reduce the benefits of supplementation with high-energy feed before mating ('flushing'), as animals would not benefit from all energy provided. similar results have been published in cases of t. congolense infection (osaer et al., 1998) . affected animals showed lower rates of conception and pregnancy and had lower concentrations of progesterone, which is a major factor in establishment of pregnancy, with a function to synchronise development of the maternal endometrium with intrauterine arrival of the embryo (wilmut and sales, 1981; lawson and cahill, 1983) . as an association between luteal phase progesterone blood concentrations and embryo survival has been suggested (noakes, 1996) , perhaps the reduced progesterone levels may be responsible for the conception problems in affected ewes. fthenakis et al. (2001) reported that sarcoptes scabieiinfested ewes had fewer ovulations than uninfested animals and attributed that to nutrient availability at the pre-conception period. nevertheless, when progesterone and equine chorionic gonadotrophin were administered exogenously, no differences were evident between infested and healthy animals. this indirectly suggests reduced hormone concentrations in the parasitised animals during the peri-conceptional period, which lends further support to the idea of sarcoptic mange influencing embryo implantation and survival. in sheep, there are no reports directly associating quality of female gametes (ova) with parasitism. nevertheless, some relevant results have been published in cattle. tritrichomonas foetus has been reported to damage oocytes (benchimol et al., 2007) . also, n. caninum has been detected in bovine follicles during assisted reproduction manipulations (silva et al., 2012) , as well as in bovine foetuses (marques et al., 2011) , although there are reports suggesting that embryo transfer is a safe method to avoid vertical transmission of these protozoon (landmann et al., 2002; moskwa et al., 2008) . these implications should be borne in mind, as assisted reproductive technologies for sheep are developing and being applied in larger numbers of animals (amiridis and cseh, 2012) . in view of the above, one should consider the strategic administration of anthelmintic drugs before the start of the mating period, with a view to improve reproductive efficiency. in fact, mavrogianni et al. (2011) have reported that ewes given a broad-spectrum long-acting anthelmintic before the start of the mating period had a higher 'cycling rate' reflecting better functionality of the genital system of the treated animals and higher 'total lambs born per ewe' and 'liveborn lambs per ewe' indicating increased number of ovulations during the peri-conceptional period. in dairytype production systems, anthelmintic administration at the pre-conception period (which coincides with the end of a lactation period) would also contribute to maintaining a longer persistency of lactation (another energy-dependent function), although drug withdrawal periods (athanasiou et al., 2009 ) should be taken into account when designing strategic treatments. finally, anthelmintic administration before the mating season has the added advantage of avoiding the inadverted administration of albendazole or netobimin, broad-spectrum anthelmintics with confirmed embryotoxic properties (delatour et al., 1981; navarro et al., 1998) , to ewes at the first stage of pregnancy. as sheep have a seasonal pattern of reproductive activity, depending on the geographical latitude, the pre-mating period would also differ according to location. hence, administration of anthelmintics at that period would have differing effects from a parasitological viewpoint, resulting from the difference in season. this should be taken into account when strategic treatments are carried out at the pre-mating period. furthermore, one should have in mind the possibility of promoting anthelmintic resistance that way and should consider an appropriate cost-benefit analysis. one should always take into account that in para-mediterranean areas and locations of similar latitude (in the north hemisphere), as well as in locations of respective latitude in the south hemisphere, reproductive activity of sheep would start at the beginning of the summer (hence, anthelmintic administration should be planned for late spring). moreover, in more northern (north hemisphere) or more southern (south hemisphere) areas, reproductive activity of sheep would start in the autumn (hence, anthelmintic administration should be planned for late summer). foetopathies and abortions are significant problems in pregnant ewes and major sources of financial losses in sheep flocks (menzies, 2012) . various protozoa can cause abortion in ewes. the principal problem by parasites in pregnant ewes is toxoplasmosis, caused by the intracellular protozoon t. gondii, which is a confirmed abortifacient agent (buxton and rodger, 2007) . ewes often become infected through consumption of oocyst-contaminated concentrate feed, given to the animals as an energy-supplement (buxton and rodger, 2007) . if infection takes place before the 61st-80th day of gestation, embryonic death occurs, followed by absorption or expulsion of small embryos, rarely being noticed. if infection takes place later, up to the 110th-115th day of gestation, then abortion takes place. finally, if infection takes place after that, congenitally infected lambs are born. in embryos, the organism causes coagulative necrosis in the placental cotyledons, as well as microglial foci, representing an immune response (buxton and rodger, 2007) . toxoplasmosis has been well-studied around the world, with many scientific references describing all aspects of the disease (buxton and rodger, 2007; dubey, 2009) . neosporosis, caused by the protozoon n. caninum, is an emerging reproductive problem in ewes; many facets of the disease still remain unclear (dubey and schares, 2011) . in the initial literature, the infection had not been always associated with abortion (otter et al., 1997; chanton-greutmann et al., 2002) , despite evidence showing the abortifacient role of the organism in experimental infections (buxton et al., , 2001 . progressively, however, reports from various parts of the world have associated the parasite with abortion in ewes (masala et al., 2007; spilovská et al., 2009; howe et al., 2012; moreno et al., 2012) , although frequency and clinical significance of the problem require further elucidation (dubey and schares, 2011) . the organism establishes itself in the maternal caruncular septum, before crossing to the foetal placental villi. a direct foetopathic effect of the organism has been described to be the cause of abortion (innes, 2007; gibney et al., 2008) , although further studies are necessary to fully clarify the pathogenesis of the infection. other protozoa that have been reported with an abortifacient role, include sarcocystis ovicanis (s. tenella), s. arieticanis, trypanosoma brucei subsp. brucei, t. congolense, t. vivax and theileria spp. (buxton, 1998; heckeroth and tenter, 1998; bawa et al., 2000; nagore et al., 2004; batista et al., 2009 ). the relaxation of acquired immunity to parasites around lambing and its consequences have been well documented (armour, 1980; gibbs, 1986; barger, 1993) . this is manifested with a rise in faecal parasitic output and had initially, for nematode infections, been associated with increased prolactin concentrations. fleming, 1993 fleming, , 1996 investigated the potential role of prolactin and prostaglandin f 2␣ (two hormones, the concentrations of which increase at the end of pregnancy); they found that total egg production by haemonchus contortus in infected sheep increased after administration of prolactin, but not after 'administration of prostaglandin f 2␣ , to those animals. prolactin is a peptide hormone, responsible for initiating and sustaining lactation in ewes (castro et al., 2011) . it acts in a cytokine-like manner and as an important regulator of the immune system (rovensky et al., 1991) . blood concentrations of prolactin in pregnant ewes start to increase from the 115th-135th day of gestation (banchero et al., 2006) . beasley et al. (2010a,b) reported that, in ewes experimentally infected with trichostrongylus colubriformis, the rise in faecal egg counts at the end of pregnancy had been preceded by a decrease in the immunological competence of the ewes; this was shown by reduced numbers of circulating eosinophils and by decreased total antibody and igg 1 titres. the changes coincided with increased prolactin concentrations at the end of pregnancy, but, nevertheless, the authors considered that they were unrelated to hormonal effects; this confirmed a similar earlier hypothesis by coop et al. (1990) . as significant differences have been reported in blood concentrations of prolactin according to season (gomez brunet and lopez sebastian, 1991) , the peri-parturient rise in faecal parasitic output should have differed in accord with time of the year. according to coop and kyriazakis (1999) , this relaxation of immunity and the ensuing increase in faecal egg counts appear to have a nutrition-based background. under the conditions of high metabolic demand, which occur at the end of pregnancy, susceptibility of ewes to gastrointestinal parasites is increased (kahn et al., 2003) . finally, coop and kyriazakis (1999) proposed a nutritional, rather than an endocrinological, involvement in the relaxation of immunity during that period and presented the following arguments: (a) grade of immunity expression in pregnant ewes is consistent with the reproductive effort, i.e., the number of foetuses borne, (b) termination of pregnancy leads to abrupt restoration of immunity and (c) nutritional management of pregnant ewes can alter the time of first observation of the relaxation. beasley et al. (2012) reported that feeding ewes a low quality diet resulted in a peri-parturient rise in faecal parasitic output starting 24 days before lambing and increasing substantially thereafter, whilst in ewes fed a high quality diet there was only a short rise of small magnitude; these findings lend further support to the above theory. a peri-parturient increase of oocyst/cyst output has also been recorded in cryptosporidium (xiao et al., 1994; ortega-mora et al., 1999) and giardia (xiao et al., 1994) infections of pregnant ewes. in both cases, the authors recorded an increase of oocyst/cyst numbers in faeces of pregnant ewes, as well as an increase in the number of ewes, which excreted oocysts/cysts in their faeces. perhaps, a combination of all above factors may determine the whole process. the parasites can also play a role in increasing the metabolic pressure in pregnant ewes. the increased parasitic output during the pre-partum period has significant consequences for the epidemiology of the respective diseases. lambs are born in a contaminated environment and, thus, are exposed to the infective forms of the various parasites at a very young age. in sheep, pregnancy is a metabolically demanding period. during the first 100 days of pregnancy, there is a slow foetal growth (blanchart and sauvant, 1974; economides and louca, 1981) ; during the second month of pregnancy, when foetal attachment has been established and placental growth has been completed, foetus(es) can acquire up to 15-25% of their future birth bodyweight; finally, at the last stage of pregnancy, the ovine foetus(es) can develop rapidly, to acquire up to 75-80% of their future birth bodyweight . hence, energy requirements of pregnant ewes increase, as the end of pregnancy is approaching. in the final month of gestation, protein requirements of pregnant ewes also increase, due to foetal requirements and the need to prepare colostrum in the mammary glands . the situation may be aggravated in cases of heavy parasitic infections, as parasites increase the energy requirements of their hosts (coop et al., 1977; dakkak, 1990) , as well as protein synthesis by the host, and consequently protein requirements, due to tissue invasion and damage (solomons, 1993) . recently, papadopoulos et al. (2013) have shown that trematode infections (fasciola hepatica and dicrocoelium dendriticum) in pregnant adult ewes led to increased ␤hydroxybutyrate concentrations in blood, thus indicating a potential association between trematode infections and pregnancy toxaemia. the authors attributed that on the direct effects that trematodes exert on the liver of affected sheep, as well as on the general energy drain effects of parasitism on the pregnant animals; they suggested that in flocks where many risk factors for pregnancy toxaemia would accumulate (e.g., suboptimal feeding), synergistic effect of those, coupled with trematode infection, could lead to clinical cases of pregnancy toxaemia. valderrábano et al. (2006) took the opposite approach and reported that increased feeding allowance during pregnancy resulted in improved response of pregnant sheep against h. contortus infection. the findings support the idea that response of pregnant ewes to parasitic infections during pregnancy may be enhanced by increased nutrition planes in the earlier stages (valderrabano and uriarte, 2003) . potential metabolic problems caused by parasitic infections are expressed, ultimately, in the birth bodyweight of lambs born. osaer et al. (1999) have reported that lambs born from t. congolense-infected ewes were of smaller bodyweight than those born from healthy animals. moreover, gatongi et al. (1997) and fthenakis et al. (2005) have administered a nematocidal treatment to ewes at the end of pregnancy and found that birthweight of lambs from treated ewes was higher than that of lambs from untreated animals. albendazole and the respective pro-benzimidazole, netobimin, have a confirmed teratogenic effect to sheep embryos, causing skeletal, renal and/or vascular malformations (navarro et al., 1998) , when administered to ewes during the first stage of gestation. active metabolites of these drugs can cross the placenta and reach the foetal blood circulation (capece et al., 2002) . often, the affected foetuses are absorbed or expelled, so ewes will be seen as barren animals at the end of the lambing season. consequently, if there is a need for administration of these drugs, they should be given before start of the mating period, as pre-conception administration of the drug does not appear to cause a foetopathic effect during the subsequent pregnancy (teruel et al., 2011) . otherwise, anthelmintic drugs with no foetopathic effects must be used. the precaution should extend to later stages of the breeding season, if rams remain with ewes for a long period of time, as there is always the possibility for some ewes to have been mated later in the season . levamisole has also been reported to potentially cause abortion if administered in late pregnancy (braun, 1997) , hence, it should better be avoided at that period. tissue lesions caused by genital myiosis may result to development of connective tissue in the vaginal and vulvar regions; these lesions can cause dystocia at lambing, due to possible foetomaternal disproportion, as a result of the lesions narrowing the birth canal (arthur and bee, 1996) . also, presence of connective tissue can lead to difficulties in dilatation of the birth canal, which may also lead to dystocia. dystocia may also occur in cases of births of malformed embryos, formed consequently to albendazole/netobimin administration at the first stage of pregnancy (section 6.4). finally, leontides et al. (2000) have postulated that d. dendriticum-infection may act as a risk factor for retention of foetal membranes in ewes during the subsequent lambing; they attributed the effect to the reduced energy availability of parasitised ewes, which may affect leucocyte function of ewes, a determinant of placental retention (azawi, 2008) , that way potentially associating the parasitic infection with the increased incidence of the reproductive disorder in ewes. 8.1. peri-parturient rise in faecal parasitic output: post-partum period the increase in faecal parasitic output ('peri-parturient egg rise') continues after lambing and contributes to lambs for acquiring the infective forms of the various parasites at a young age. beasley et al. (2012) found that increased parasitic output from infected ewes was evident for up to 1.5 months after lambing, but, again, suggested that an association with endocrinological factors was unlikely (bar, possibly, a potential role for cortisol), concluding that some other factor(s) would be contributing to the relaxation of immunity to nematodes and the consequent increase in faecal parasitic output during the post-partum period (beasley et al., 2012) . suarez et al. (2009) and cruz-rojo et al. (2012) have documented that gastrointestinal nematode parasitism can cause 10-15% reduction in milk yield of affected ewes, as well as shorter persistency of lactation. anthelmintic treatment has also been found to increase milk production (rinaldi et al., 2007) . more specifically, fthenakis et al. (2005) and cringoli et al. (2009) have reported that administration of an anthelmintic with a long persistent activity at the final stage of pregnancy, resulted in a significant (up to 40%) increase in total milk production throughout the subsequent lactation period. finally, fthenakis et al. (2000) have identified a lower milk production in ewes with sarcoptic mange: up to 18% compared to healthy animals. the situation regarding potential effects on milk composition is not clear. cruz-rojo et al. (2012) have described that milk from ewes with gastrointestinal nematode parasitism had lower fat and total protein content during the last stage of lactation, but sechi et al. (2010) have not found a significant effect of parasitism on milk composition. it is clear that parasitism leads to reduction in milk production of affected animals. the above studies have been carried out in dairy breeds, with a view to estimate milk production and financial effects of parasitism for dairy farmers. results of direct studies of potential milk yield reduction due to parasitism, in the growth of lambs of the affected ewes are not available and can only inferred from the above reports. reduced milk yield of ewes leads to suboptimal growth rate of lambs (fthenakis and jones, 1990) and, during the neonatal period, even to increased death rate of lambs (dwyer, 2008; brozos et al., 2011) . in this context, it is noteworthy the report by juste jordán and garcía pérez (1991) , who found that adverse effects of parasitism in milk yield were more pronounced at the final stage of lactation, when, however, there is little dependence of lambs on maternal milk, as they consume solid feed. the nutritive value of milk is also dependent upon its composition. nevertheless, a variety of factors can influence milk composition (e.g., nutrition, genetics, stage of lactation, mammary health), which may be difficult to control in order to test potential adverse effects of parasitism; that may explain the unclear results among the respective studies. the major defence mechanism against bacteria invading into the mammary gland is phagocytosis (craven and williams, 1985) . the process is regulated through a variety of factors, among them energy resources of the host (greenberg and grinstein, 2002; stuart and ezekowitz, 2005) , which may indicate a potential adverse role for parasites. in two recent publications, mavrogianni et al. (2012 mavrogianni et al. ( , 2014 have shown the effects of gastrointestinal parasitic infections to development of mastitis in ewes. in a field study, trematode infections (f. hepatica and d. dendriticum) in lactating multiparous ewes have led to increased incidence of clinical or subclinical mastitis during the first two weeks post-partum (mavrogianni et al., 2014) . in an experimental study, deposition of mannheimia haemolytica into the teat duct of trichostrongylid-infected ewes resulted to development of clinical mastitis, whilst healthy controls developed only subclinical disease after challenge . the above studies were the first to confirm that parasitic infections predispose ewes to mastitis, both diseases being significant health and welfare problems in sheep flocks. it is interesting to note that in one of these studies (mavrogianni et al., 2014) , the association between trematode infection and mastitis was shown in the immediately post-partum period, when relaxation of immunity (sections 6.2 and 8.1) would be present. in the other one of the above papers , the authors have presented evidence of impaired local defence mechanisms in the mammary glands of parasitised ewes, which might explain pathways for the association observed. during infections with the various abortifacient parasitic agents, foetuses may survive depending upon their age at infection (section 6.1). in such cases, weak lambs, usually congenitally infected with the respective agent, may be born. such lambs may die soon after birth, from attacks of predators or from hypothermia, as often they are unable to stand up on time, suck and respond to external stimuli (wilsmore, 1984) . a significant and well documented (de graaf et al., 1999; fayer, 2004; taylor et al., 2007; shahiduzzaman and daugschies, 2013) health problem in newborn and young lambs is cryptosporidiosis. the disease is an infectious enteritis that causes high morbidity and mortality of affected animals. it is caused by the enteric protozoa cryptosporidium spp., which can affect newborns alone or in mixed infection with escherichia coli or viruses affecting the intestinal tract (e.g., rotavirus, coronavirus) (chatzopoulos et al., 2013) . cryptosporidium spp. are located at the microvilli in the intestine of affected lambs and impair intestinal function. the disease causes suboptimal growth rate and often death of affected animals, leading to heavy economic losses in the sheep industry. lambs which survive infection at a young age, remain asymptomatic carriers and shed oocysts, contributing to increased environmental contamination and infection of newborn lambs. giardia spp. is an intestinal protozoon affecting lambs at the end of the neonatal period (o'handley and olson, 2006) . infection is often asymptomatic, although it can cause diarrhoea, which becomes severe and life-threatening in cases of co-infection with other enteric pathogens (andrew thompson et al., 2008) . specific works in lambs have not been reported. in calves, infection with giardia duodenalis can lead to loss of the epithelial barrier function, villus atrophy and crypt hyperplasia in the small intestine, changes which may result in clinical disease usually characterised by intermittent diarrhoea (ruest et al., 1997) . in any case, the lesions would cause malabsorption, leading to suboptimal weight gain, reduced feed-efficiency and ill-thriftiness of the affected animals (olson et al., 1995; buret, 2007; sweeny et al., 2012) . in general, the significance of this infection has not yet been fully elucidated and further studies are needed. eimeria spp. are well-described enteric protozoa (taylor et al., 2007; andrews, 2011; taylor, 2012) , which can affect growing lambs from the age of 20 days onwards, causing coccidiosis. up to 11 different species have been reported to affect lambs; e. crandallis and e. ovinoidalis are considered to be the most pathogenic, perhaps because they cause extensive damage in both the small and the large intestine. infections usually remain inapparent, although affected lambs may not be thriving as expected. watery, haemorrhagic diarrhoea can occur and may result to death, if the infection is not properly managed. the onset of intense infection of lambs with gastrointestinal helminths (tapeworms and nematodes) coincides with the start of consumption of solid feed by these animals, specifically grazing. tapeworms (moniezia spp., avitellina spp., stilesia spp., thysaniezia spp.) are regarded of little pathogenic significance, only in heavy infections causing suboptimal growth rate and possibly clinical signs (taylor et al., 2007) . the effects of gastrointestinal nematode parasitism on the growth of unweaned lambs have been well documented. these parasites are of importance in mutton-type production systems, where lambs remain with their dams for over 100 days of age (sargison, 2009) . in contrast to that, in dairy-type production systems, lambs are weaned at a younger age (gelasakis et al., 2010) and, usually, are taken for slaughter; hence, chances for building up a significant production-limiting parasitic burden are minimal. gastrointestinal nematode infections cause significant growth retardation or delay in age of slaughter, which have been documented repeatedly in the older to the more recent scientific literature (coop et al., 1984; sweeny et al., 2012) . the significant financial losses associated with the growth retardation of unweaned lambs have led to the need for frequent anthelmintic treatments in these animals, which, in turn, have led to development of widespread anthelmintic resistance in sheep flocks around the world torres-acosta et al., 2012) . currently, many strategies for anthelmintic treatment of lambs in mutton-type production systems have been advocated, with emphasis given to strategic administration of the drugs (sargison, 2011 (sargison, , 2012 . the review has covered many aspects of interaction between parasitic infections and reproduction in sheep. in the majority of cases, parasitic infections lead to reduced reproductive efficiency, although there are a few cases where the reproductive stage of the animal influences the parasitic infection. further collaboration of parasitologists with obstetricians, endocrinologists and immunologists will contribute to deeper investigations into these topics and the elucidation of potential relationships, in order to improve health, welfare and production of sheep. pharmaceutical control of reproduction in sheep and goats hormonal control of reproduction in small ruminants assisted reproductive technologies in the reproductive management of small ruminants reproductive health management of sheep and goats the public health and clinical significance of giardia and cryptosporidium in domestic animals some aspects of coccidiosis in sheep and goats epidemiology of helminth disease in farm animals maternal dystocia: treatment proposals for withdrawal period of sheep milk for 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immunity to the gastrointestinal nematode trichostrongylus colubriformis in merino ewes tritrichomonas foetus damages bovine oocytes in vitro consequences of pregnancy on the evolution of lactation non-infectious prenatal pregnancy loss in the doe treatment and control of peri-parturient metabolic diseases: pregnancy toxemia, hypocalcemia, hypomagnesemia mechanisms of epithelial dysfunction in giardiasis protozoan infections (toxoplasma gondii, neospora caninum and sarcocystis spp.) in sheep and goats: recent advances the pathogenesis of experimental neosporosis in pregnant sheep toxoplasmosis and neosporosis immunity to experimental neosporosis in pregnant sheep placental transfer of albendazole sulphoxide enantiomers in sheep management effects on colostrogenesis in small ruminants: a review abortion in small ruminants in switzerland: investigations during two lambing seasons (1996-1998) with special regard to chlamydial abortions rotavirus infections in domestic animals studies of reproduction in sheep. 1. the ovulation rate of the ewe as affected by the plane of nutrition effect of anthelmintic treatment on the productivity of lambs infected with the intestinal nematode nutrition-parasite interaction teladorsagia circumcincta egg output at the outset of natural and induced lactation in ewes the effect of a daily intake of ostertagia circumcincta larvae on body weight, food intake and concentration of serum constituents in sheep defences of the bovine mammary gland against infection and prospects for their enhancement the effect of moxidectin 0.1% vs ivermectin 0.08% on milk production in sheep naturally infected by gastrointestinal nematodes effect of infection with teladorsagia circumcincta on milk production and composition in assaf dairy sheep. vet. parasitol gastrointestinal strongylosis and malabsorption of nutrients a review of the importance of cryptosporidiosis in farm animals experimental infection by toxoplasma gondii using contaminated semen containing different doses of tachyzoites in sheep characterization of reproductive disorders in ewes given an intrauterine dose of toxoplasma gondii tachyzoites during the intrauterine insemination albendazole: a comparison of relay embryotoxicity with embryotoxicity of individual metabolites metabolic cues for puberty onset in free grazing holstein heifers naturally infected with nematodes effects of stress on reproduction in ewes ovine sarcoptic mange: clinical features and economic impact toxoplasmosis in sheep-the last 20 years neosporosis in animals-the last five years development of early tissue cysts and associated pathology of besnoitia besnoiti in a naturally infected bull (bos taurus) in south africa the welfare of the neonatal lamb the neuroendocrine timing of puberty the effects of the quantity and quality of feed on the performance of pregnant and lactating goats no detection of besnoitia besnoiti dna in the semen of chronically infected bulls cryptosporidium: a water-borne zoonotic parasite acute or chronic administration of prolactin alters ovine infections of haemonchus contortus effects of exogenous reproductive hormones on haemonchus contortus populations in lambs nutrition of females during the peri-conceptional period and effects on foetal programming and health of offspring attachment preferences of hyalomma truncatum and hyalomma marginatum rufipes ticks (acari: ixodidae) on two sheep breeds effect of restricted nutrition on puberty in the lamb: patterns of tonic luteinizing hormone (lh) secretion and competency of the lh surge system internal and external determinants of the timing of puberty in the female observations in ovine myiosis in greece, with special reference to clinical findings and treatment of genital myiosis health management of ewes during pregnancy the effect of experimentally induced subclinical mastitis on milk yield of ewes and on the growth of lambs effects of sarcoptic mange on the reproductive performance of ewes and transmission of sarcoptes scabiei to newborn lambs effects of three anthelmintic regimes on milk yield of ewes and growth of lambs efficacy of moxidectin against sarcoptic mange and effects on milk yield of ewes and growth of lambs influence of gastrointestinal trichostrongylidosis on ram fertility effects of three nematode anthelmintic treatment regimes on flock performance of sheep and goats under extensive management in semi-arid kenya farm conditions and production methods in chios sheep flocks hypobiosis and the periparturient rise in sheep the extent of parasite-associated necrosis in the placenta and foetal tissues of cattle following neospora caninum infection in early and late gestation correlates with foetal death effect of season on plasma concentrations of prolactin and cortisol in pregnant, non-pregnant and lactating ewes clinical evaluation of reproductive ability of rams phagocytosis and innate immunity dietary energy and ovulation rate in sheep the interaction of nutrition and body condition at mating on ovulation rate and early embryo mortality in scottish blackface ewes depression of productivity in sheep infected with fasciola hepatica maternal effects and early-life performance are associated with parasite resistance across life in free-living soay sheep influence of restricted maternal nutrition in early to mid gestation on placental and fetal development at term in sheep comparison of immunological and molecular methods for the diagnosis of infections with pathogenic sarcocystis species in sheep science and hill farming hfro 1954-1979 potential involvement of neospora caninum in naturally occurring ovine abortions in new zealand the host-parasite relationship in pregnant cattle infected with neospora caninum effect of treatment with netobimin on milk production of sheep regional differences in the distribution of gonadotropin-releasing hormone cells between rapidly growing and growth-restricted prepubertal female sheep seasonal variation in semen characteristics, scrotal circumference and libido of persian karakul enhancing immunity to nematode parasites in single-bearing merino ewes through nutrition and genetic selection confirmation of the prevention of vertical transmission of neospora caninum in cattle by the use of embryo transfer modification of the embryo-maternal relationship in ewes by progesterone treatment early in the oestrous cycle a matched case-control study of factors associated with retention of fetal membranes in dairy ewes in southern greece schmallenberg virus infection in small ruminants-first review of the situation and prospects in northern europe aspects of toxoplasma infection on the reproductive system of experimentally infected rams (ovis aries) clinical examination of sheep neospora caninum: evaluation of vertical transmission in slaughtered beef cows (bos indicus) administration of a long-acting antiparasitic to pre-pubertal ewe-lambs in greece results in earlier reproductive activity and improved reproductive performance pre-existing gastrointestinal trichostrongylosis predisposes ewes to clinical mastitis after experimental mammary infection trematode infections in pregnant ewes can predispose to mastitis during the subsequent lactation period vaccination programs for reproductive disorders of small ruminants parasites of sheep and goats and their prevalence in bokova, a rural area of buea sub division occurrence of neospora caninum and toxoplasma gondii infections in ovine and caprine abortions studies on neospora caninum dna detection in the oocytes and embryos collected from infected cows identification, genetic diversity and prevalence of theileria and babesia species in a sheep population from northern spain anthelmintic induced congenital malformations in sheep embryos using netobimin infertility in the cow: general considerations, anatomical, functional and management causes giardiasis and cryptosporidiosis in ruminants effects of giardiasis on production in a domestic ruminant (lamb) very intensive systems role of adult sheep in transmission of infection by cryptosporidium parvum to lambs: confirmation of periparturient rise histopathologic and ultrastructural studies on experimental caprine besnoitiosis effects of trypanosoma congolense and nutritional supplements on establishment and outcome of pregnancy in trypanotolerant djallonké ewes effects of trypanosoma congolense infection and diet on puberty, age at first lambing and haematology changes in djallonké ewe lambs bluetongue results of a survey to determine whether neospora is a significant cause of ovine abortion in england and wales update on parasitic diseases of sheep anthelmintic resistance in sheep in europe: a selected review potential association between trematode infections and development of pregnancy toxaemia in sheep normal reproduction in male animals the effect of dietary protein restriction on the secretion of lh and fsh in pre-pubertal female lambs seminal degeneration associated with chorioptic mange of the scrotum of rams the effect of extensive chorioptic mange of the scrotum on reproductive function of the ram ram and buck management dairy goat production and the importance of gastrointestinal strongyle parasitism update on trematode infections in sheep evidence for immunomodulatory properties of prolactin in selected in vitro and in vivo situations morphological changes in the jejunum of calves naturally infected with giardia spp and cryptosporidium spp influence of an experimental trypanosoma congolense infection on the reproductive function of djallonke and sahelian rams in subhumid zone negative effect of the arthropod parasite, sarcoptes scabiei, on testes mass in iberian ibex, capra pyrenaica pharmaceutical control of endoparasitic helminth infections in sheep pharmaceutical treatments of gastrointestinal nematode infections of sheep-future of anthelmintic drugs effects of anthelmintic treatment on milk production in sarda dairy ewes naturally infected by gastrointestinal nematodes loss of libido and terminal sterility in a friesian bull naturally infected with besnoitia besnoiti in northern nigeria. a case report puberty therapy and prevention of cryptosporidiosis in animals increased incidence of dna amplification in follicular than in uterine and blood samples indicates possible tropism of neospora caninum to the ovarian follicle pathways to the impairment of human nutritional status by gastrointestinal pathogens the first finding of neospora caninum and the occurrence of other abortifacient agents in sheep in slovakia phagocytosis: elegant complexity treatment and control of chlamydial and rickettsial infections in sheep and goats epidemiology and effects of gastrointestinal nematode infection on milk productions of dairy ewes impacts of naturally acquired protozoa and strongylid nematode infections on growth and faecal attributes in lambs transmission of neospora caninum to sheep by transfer through semen emerging parasitic diseases of sheep veterinary parasitology albendazole sulphoxide administered prior to mating and its relation with fertilization and mouse embryo development anthelmintic resistance in sheep farms: update of the situation in the american continent management of pre-pubertal small ruminants: physiological basis and clinical approach effect of nutritional status and fat reserves on the periparturient immune response to haemonchus contortus infection in sheep effect of nutrition in early pregnancy on the periparturient relaxation of immunity to gastro-intestinal parasitism in prolific ewes role of male-female interaction in regulating reproduction in sheep and goats ovine cutaneous myiasis: effects on production and control effect of an asynchronous environment on embryonic development in sheep perinatal mortality in sheep with special reference to ovine enzootic abortion. the royal veterinary college periparturient rise in the excretion of giardia sp. cysts and cryptosporidium parvum oocysts as a source of infection for lambs periconceptional nutrient restriction in the ewe alters mapk/erk1/2 and pi3k/akt growth signaling pathways and vascularity in the placentome the authors have nothing to disclose. key: cord-270940-acwkh6ed authors: kallio-kokko, hannimari; uzcategui, nathalie; vapalahti, olli; vaheri, antti title: viral zoonoses in europe date: 2005-06-29 journal: fems microbiol rev doi: 10.1016/j.femsre.2005.04.012 sha: doc_id: 270940 cord_uid: acwkh6ed a number of new virus infections have emerged or re-emerged during the past 15 years. some viruses are spreading to new areas along with climate and environmental changes. the majority of these infections are transmitted from animals to humans, and thus called zoonoses. zoonotic viruses are, as compared to human-only viruses, much more difficult to eradicate. infections by several of these viruses may lead to high mortality and also attract attention because they are potential bioweapons. this review will focus on zoonotic virus infections occurring in europe. during the past 15 years a number of new virus infections have emerged or re-emerged. most of them, such as sin nombre and andes hantaviruses, sars coronavirus, avian influenza, nipah and hendra viruses, have appeared in subtropical or tropical regions. dengue is spreading to new areas and west nile virus has reached the new world. infections by several of these viruses may lead to high mortality and also attract attention because they are potential bioweapons. some viruses such as tick-borne encephalitis virus are spreading to new areas along with climate and environmental changes. most of these infections are zoonoses and clearly viruses shared by animals and humans are, unlike human-only viruses, much more difficult to eradicate. here, we review zoonotic virus infections occurring in europe. the infections like lassa fever and dengue that are imported to europe but are not indigenous to european nature will not be discussed in detail in the review. we have divided the virus infections into two categories, those that are transmitted to humans directly from vertebrate animals (like rodents, foxes, bats and birds) and those that are primarily transmitted by arthropods (mosquitoes, ticks, sandflies). the latter class is formed by arboviruses but notably they have vertebrate hosts in nature. hantaviruses are enveloped viruses with a tri-segmented negative-stranded genome and belong to the family bunyaviridae [1, 2] . the 6.4 kb l (large) segment rna encodes the 250 kda rna polymerase, the 3.6 kb m (medium) segment the two glycoproteins 68-76 kda gn and 52-58 kda gc, -formerly known as g1 and g2; and the 1.7 kb s (small) segment the 50-54 kda nucleocapsid protein (n) ( table 1 , fig. 1 ). in addition, the s segment of some hantaviruses has another open reading frame named ns but its product or function remains to be discovered. viral messenger rnas of the members of the bunyaviridae are not polyadenylated and are truncated relative to the genome rnas at the 3 0 termini. messenger rnas have 5 0 -methylated caps and 10-18 nontemplated nucleotides which are derived from host cell mrnas. the termini of all three segments are conserved and complementary to each other, a feature that has assisted in cloning and discovery of new hantaviruses. unlike most other bunyaviridae, hantaviruses are not arthropod-borne (arboviruses), but are rodent-borne, roboviruses. each hantavirus is primarily carried by a distinct rodent/insectivore species although a few host switches seem to have occurred during the tens of millions of years of their co-evolution with their carrier animals [3] . we now know that the genetic diversity of hantaviruses is generated partly by (i) genetic drift (accumulation of point mutations and insertions/deletions) leading to quasispecies [4, 5] , (ii) genetic shifts (reassortments of genome fragments within the same virus genotype/species), and (iii) according to recent findings [6, 7] , by homologous recombination, a mechanism not previously observed for negative-strand rna viruses. hantaviruses, which cause hemorrhagic fevers with renal syndrome (hfrs) in eurasia and hantavirus cardiopulmonary syndrome (hcps) in the americas, are prime examples of emerging and re-emerging infectious agents. like most of these infections hantaviral diseases are zoonoses. with the exception of the south-american andes virus, which can be transmitted directly from human to human, hantavirus infections are thought to be transmitted to humans primarily from aerosols of rodent excreta. only some [9, 10] . topografov hantavirus isolated from siberian lemmings (lemmus sibiricus) has not been detected in north european lemmings (lemmus lemmus) although it can grow in them [11] . infections in rodents are mainly asymptomatic and persistent. hantavirus infections are quite common in europe [8] (table 3) , puumala virus is common in northern europe, european russia and parts of central-western europe (fig. 2) . dobrava virus is found mainly in the balkans (fig. 2) . saaremaa virus has been detected in eastern and central europe but its epidemiology is not well defined (fig. 2) . apart from laboratory infections [12, 13] seoul virus has been detected in wild rats, only in france [14] . it is also apparent that many parts of europe, such as britain, poland and byelorussia, remain ''white'' on the european hantavirus map [8] . this means either that hfrs is rare or nonexistent in these regions or is not widely recognized and diagnosed by the biomedical community. in northern europe hfrs as well as the carrier rodents exhibit peaks in 3-4 year cycles [15] while in central europe the hfrs incidence follows the fluctuations of ''mast years'', i.e. the availability of beech and oak seeds for the hantavirus-carrying rodents. in central europe hfrs peaks in the summer while in northern europe most cases occur in late autumn and early winter, from november to january. risk factors to catch hantavirus infections and hfrs include professions such as forestry, farming, and military, or activities such as camping, and the use of summer cottages. males are more likely to be exposed than females [15, 16] . puumala and dobrava viruses both cause hfrs but the infections differ considerably in severity [17] : both are characterized by acute-onset fever, headache, abdominal pains, backache, temporary renal insufficiency -first oliguria, proteinuria and increase in serum creatinine and then polyuria -and thrombocytopenia but the extent of hemorrhages (hematuria, petecchiae, internal hemorrhages), requirement for dialysis treatment, hypotension and mortality are much higher in [19] . this is important since the infection is so common in many areas of europe [8] (table 3 , fig. 2 ). in addition, in some patients puumala virus infection may invade the pituitary gland and lead to mortality or at least hypophyseal insufficiency requiring hormone-replacement therapy [20] . the pathogenesis of hfrs is poorly understood [17, 21] . however, it is known that b3 integrins can mediate the entry of pathogenic hantaviruses [22] and that hantaviruses can regulate apoptosis [23] [24] [25] [26] 28] . also there is evidence [17, 21] that increased capillary permeability is an essential component in the pathogenesis of both hfrs and hcps, although different target tissues, kidneys and lungs are affected in the two diseases. hfrs patients show locally increased levels of tnf-a in the plasma and kidneys [27, 28] and high levels of urinary secretion of the proinflammatory cytokine il-6 [29] . studies with a monkey model mimicking human puumala virus infection [30] may assist in elucidating the mechanism of pathogenesis. of the four structural proteins, both in humoral and cellular immunity, the n protein appears to be the principal immunogen [31] . cytotoxic t-lymphocyte (ctl) responses are seen [32] and may be important both for protective immunity and pathogenesis of hantavirus infections [21] . the diagnosis of acute hfrs is primarily based on serology, since viral rna cannot be regularly detected in the blood or urine of patients [33, 34] . both immunofluorescence tests and enzyme immunoassays are widely used for detection of specific igm or low-avidity igg antibodies, characteristic of acute infection [35] [36] [37] . in addition, immunochromatographic 5-min igm-antibody tests [38, 39] have been developed. vaccines against hantavirus infections have been used for years in china and korea, but not in europe or the americas [40] . no specific therapy is used in europe, although both ribavirin and interferon-a have been successfully used in trials in china [41, 42] . a major problem is that at the time hfrs patients are hospitalized, virus replication is already disappearing. members of the genus lyssavirus within the family rhabdoviridae are bullet-shaped, enveloped viruses approximately 60 nm in diameter and 200 nm in length. the 12 kb non-segmented negative-strand genome encodes five proteins (starting from the 3 0 end): the 58-62 kda nucleoprotein (n), the 35-40 kda phosphoprotein (p), the 22-25 kda matrix protein (m), the trimeric 65-80 kda glycoprotein (g) and the 190 kda polymerase protein (l) ( table 1 , fig. 1 ). proteins are separately transcribed in cascade by a special mechanism from a single 3 0 -end promoter which results in a decreasing transcription and expression gradient for proteins encoded from the 3 0 to 5 0 end. the major antigen with neutralizing epitopes and pathogenetic determinants is the glycoprotein, which is responsible for receptor recognition and membrane fusion. after endocytosis the viral envelope fuses with endosomal membranes provoking the release of the internal viral nucleocapsid in the cytoplasm where transcription and replication takes place. the helically wound nucleocapsid results from the intimate association of the nucleocapsid protein and the rna genome. it serves as template for the polymerase (l protein and p cofactor) for transcription and replication. it buds to intracytoplamic membranes in infected neurons, but on plasma membranes in salivary gland epithelial cells. the lyssaviruses currently consist of 7 established genotypes, or lineages, of rabies(-like) viruses [56] of which the classical rabies virus, found throughout the world and associated with terrestrial mammalian hosts and american bats, forms genotype 1 ( table 2 , fig. 3 ). other lineages, or genotypes, are found in bats, of which mokola and lagos bat viruses seem to be less pathogenic, and towards which the vaccines based on the classical genotype 1 rabies virus are not protective [43a,43b] . in addition to the 7 genotypes, new bat lyssavirus genotypes have been recently found, e.g. from russia [43c]. during the past 100 years or more in europe, classical rabies virus has made two major host shifts, firstly from the dog to red foxes, and then to racoon dogs brought from east asia to be raised for fur; this species then became widely established in the wild (fig. 3) . phylogenetic data also suggest that westand southward spread of rabies virus occurred during the last century [44a] . although in europe the most important carriers of classical rabies virus are foxes and racoon dogs, the virus can be transmitted to secondary hosts such as domestic animals (dog, cat, cattle, horse, sheep) or e.g. deer -practically any mammal species could be a potential carrier. bats are a special case; lyssaviruses are maintained in bats, even in the absence of classical rabies in carnivores; thus in countries where classical rabies has been eliminated, bat rabies has become the dominating or only source of rabies virus and retains the potential for host-switching into the carnivore reservoir which constitutes a more direct threat for public health [44b]. as many bats are protected species, the detection or ''absence'' of bat rabies is dependent also on the intensity of screening efforts [45, 46] . as of the beginning of 2004, the following countries in europe were declared ''rabies-free'' by who (meaning no indigenous cases occurred during the last two years): belgium, cyprus, finland, greece, iceland, ireland, italy, luxembourg, norway, portugal and sweden (fig. 3) . in 2003, rabies virus was detected in europe in 7095 wild animals (excluding bats), 3951 domestic animals; 33 bat and 6 human rabies cases were diagnosed (see table 4 ). the countries where rabies was circulating include (in diminishing order of cases) russia, ukraine, lithuania, belarus, latvia, estonia, croatia, poland, slovakia, serbia-montenegro and turkey with hundred(s) to thousands of (animal) cases, romania, bosnia-herzegovina, germany, moldova and bulgaria with dozens of cases, and individual cases were registered (some imported and not affecting the rabies-free status) in slovenia, the netherlands, denmark, france, albania, finland, austria and switzerland ( members of 6 out of the 7 lyssavirus genotypes (except lagos bat virus, i.e. genotype 2) have caused rabies disease in man. the infection is inevitably fatal in humans or other mammals unless immune intervention (vaccination and administration of antibodies) is used. transmission may occur though the bite of an animal delivering the virus deep in to striated muscle or connective tissue, but infection may also occur after abrasion of the skin or licking of mucous membranes. the bite of a bat with small teeth may go unnoticed; on the other hand the bat lyssaviruses may infect human skin more easily than has been recognised. the incubation period is 20-60 days, but has been shown in some cases to be months, even years. the classical picture of rabies includes prodromal illness with fever and non-specific symptoms, as well as itching and local paresthesia. this is followed by neurological signs, consisting of either encephalitic ''furious rabies'' or paralytic ''dumb rabies''. in the former, episodic hyperactivity and excitation of the cns manifest as, e.g. hydrophobia, hypersalivation and convulsions. the patient finally develops paralysis, coma and cardiorespiratory failure. in the paralytic form, no excitation is seen, but paralytic disease develops to coma and death [45,52a,52b] . after entry of the virus to the body, the virus must gain access to peripheral nerves and to be transported towards the cns. rabies virus components are attached either directly or by encapsulated vesicles to the dynein motor carrying the ''cargo'' along the axonal microtubular system towards the cell stroma, approximately at a speed of 25 mm/day [45, 53, 54] . it is transmitted after replication on neuronal membranes transsynaptically to adjacent neurons and finally invades the cns where it first disturbs the limbic system associated with the excitation, and later neocortex, with little histopathological changes in neurons. centrifugal spread of the virus from the cns to many tissues through somatic and autonomic nerves also occurs, where the salivary glands are especially important for the spread of the virus to the next victim. immunological responses do not occur before cns involvement. all the viruses in phylogroup i are also pathogenic to mice by i.m. injection, and can cross-neutralize each other. this has practical implications, as fortunately the vaccine strains (of genotype 1) also appear to protect against the ebvl viruses [55] . a pathogenic determinant common to phylogroup i viruses seems to be amino acid r333 in the glycoprotein [56] ; substitution of this amino acid also abolishes the retrograde transport [57] . when symptoms develop, no cure is available. however, after exposure to a bite or scratch of a potentially rabid animal, rabies must and can effectively be prevented by post-exposure prophylaxis, originally developed by louis pasteur. the treatment includes in a non-vaccinated person (a) washing (with water and detergent) and disinfecting the wound to minimize the amount of cell-free virus (this alone can increase survival 50%), (b) starting a post-exposure vaccine regimen which includes (in europe) five doses of cell-culture derived vaccine intramuscularly into the deltoid muscle on days 0, 3, 7, 14, 28 and (c) in case of severe or deep injury additional passive anti-rabies immunoglobulin, which should be administered principally to the wound area. if the person was pre-vaccinated or the animal can be caught and studied for the presence of rabies, the protocol can be adjusted accordingly [45,52a] . clinical suspicion of rabies in a case of encephalitis of unknown origin is the starting point. ante mortem diagnostics can be achieved most easily with rt-pcr from saliva. in addition, rabies antigen can be detected in brain or nuchal skin biopsies, and in some cases antibodies in serum or csf may be found. post-mortem diagnostics is most rapid with antigen detection or rt-pcr from the brain, virus isolation is also possible. in addition to post-exposure prophylaxis, vaccines in humans can be used for pre-exposure prophylaxis (3 doses) in risk groups (veterinarians, wildlife workers, travellers to endemic areas; especially small children who may be unable to explain a potential exposure to a rabid animal) [45,52a,58] . the primary method for control of rabies is vaccination of dogs (and cats). this practice was established at the beginning of the last century in most of europe, but has not yet been achieved in many developing countries, where annually 60 000 people still die of rabies. in most of europe (excluding eastern europe) rabies has been eradicated from terrestrial wildlife species (carnivores) by aerial distribution of vaccine baits across the countryside. the vaccines used to protect wildlife species include attenuated vaccines as well as a recombinant vaccinia virus carrying the rabies virus glycoprotein (the latter has in one case caused a skin infection in man). although rabies in terrestrial animals can be controlled efficiently, eradication from the bat reservoir is not currently feasible. arenaviruses are the only members of the rna virus family arenaviridae. these viruses are enveloped, lipid solvent-sensitive, pleomorphic particles with a mean diameter of 120 nm (ranging between 50 and 300 nm). host cell-derived ribosomes are present in the virions, and give the virus particles a ''sandy'' appearance under the electron microscope, hence the name arenavirus (arena: sand in latin). the virion structure of arenaviruses is quite simple, virus particles contain two rna segments (s and l) linked to nucleocapsid proteins and viral polymerase molecules, and these nucleocapsidpolymerase complexes are surrounded by a lipid envelope into which two glycoproteins (g1 and g2) are linked protruding on the outside of the virion. arenaviral rna segments have an ambisense coding arrangement, the s segment (3.5 kb) encoding a 63-kda nucleocapsid protein (n) in the viral complementary sequence, and in the viral-sense 5 0 -end sequence a 75-kda glycoprotein precursor (gpc) which is posttranslationally cleaved to two glycosylated proteins 34-44 kda g1 and g2; and the l segment (7.2 kb) encoding a 180-kda viral polymerase (l) in the viral complementary sequence, and in the viral-sense 5 0 -end of the sequence a 10-14 kda ring finger z protein, which has been shown to have a role in arenavirus budding [59a] (table 1 , fig. 1 ). initiation of transcription may involve cap-snatching, although the transcription mechanism is not yet fully elucidated. arenaviruses include 23 viruses all carried by different rodent hosts (except tacaribe virus which has been isolated only from fruit bats) [59b]. arenaviruses are capable of causing chronic infections in their rodent hosts, and infectious virus is present in the blood and is also secreted into body fluids (saliva, urine, semen), which is presumably the route of transmission to humans. the appearance and incidence of arenaviral infections are closely associated with the distribution of the rodent host species and the rodent population dynamics. arenaviruses have co-evolved with their specific host species during millions of years, and have been divided into old world and new world groups first on a serological bass, and later into evolutionary lineages (new world group) using genetic analysis [60a,60b,60c]. both groups contain viruses that are included in the category a pathogen list (defined by cdc, usa), which means that the propagation of these agents is allowed only in biosafety level 4 laboratories. these highly pathogenic arenaviruses include the south american junin, machupo, guanarito, and sabia viruses from the new world group, and the african lassa virus from the old world group. all these viruses can cause hemorrhagic fevers in humans, and are considered potential bioterrorism agents being thus included in biohazard preparedness programs. in europe, these viruses occur only rarely as imported cases. the only arenavirus endemic in europe is lymphocytic choriomeningitis virus (lcmv), shown to circulate in mus musculus populations (table 2) , and associated with pet hamster derived epidemics [59b,61a]. relatively few epidemiological data are available concerning the actual distribution of lcmv in europe, but in addition to serological evidence from mus sp. material from spain [61b], antibodies against lcmv have been detected in rodent species other than mus musculus (our unpublished data), which indicates that other yet unknown arenaviruses may circulate in europe. at least ten arenaviruses have been reported to be able to cause disease in humans. as mentioned above, five arenaviruses (lassa, junin, machupo, guanarito, and sabia viruses) are even capable of causing a lifethreatening viral hemorrhagic fever [62a,62b] . none of these five viruses are endemic in europe, but a few imported lassa virus infections have been diagnosed in germany and great britain during the last few years with no secondary infections detected [63] [64] [65] . increased travelling increases also the risk for transmission of exotic arenaviruses to non-endemic areas such as europe. lcmv is thus far the only known endemic arenavirus in europe [62b]. in humans lcmv infections are mostly either asymptomatic or influenza-like diseases. in some cases aseptic meningitis or meningoencephalomyelitis is seen. lcmv is also capable of causing congenital infections manifested by hydrocephalus, microcephalus, chorioretinitis, and mental or psychomotor retardation [66,67a,67b] . lcmv infections are rarely fatal for humans. the diagnosis of arenaviral infections is based on serology and/or direct detection of the virus [68] . for serodiagnosis methods using immunofluorescence assay (ifa) as well as enzyme immunoassay (eia) have been described. either a four-fold rise in igg antibody titers or presence of igm antibodies is considered indicative of acute infection. the antibodies that appear first in the acute phase of infection are directed against the nucleocapsid protein; neutralizing antibodies against the glycoproteins appear later in the convalescent phase (if at all). this means that typing of the causative agent is difficult on a serological basis at the early stage of infection, and is actually possible only in the convalescent phase due to the slow rise of virus typespecific neutralizing antibodies. for direct detection of the virus, antigen detection assays are useful in the early diagnosis of lassa fever especially. also reverse transcriptase (rt)-pcr tests have been developed to detect arenaviral rna in patient samples [69a,69b,70a,70b,70c]. virus isolation attempts can also be successful. the supportive treatment of arenaviral infections includes ensuring the fluid, electrolyte and osmotic balance. in severe hemorrhagic fever-cases early diagnosis is important because the use of ribavirin has been found effective if the treatment commences within the first six days after onset of symptoms [71] . immune plasma containing neutralizing antibodies has also been useful in some cases. for prevention of arenaviral diseases, several attempts to develop vaccines have been made [72a] . one vaccine, candid 1, has been successfully used in the prevention of argentine hemorrhagic fever caused by junin virus with a clear reduction in the number of infections observed in humans [72b,73]. the genus orthopoxvirus in the family poxviridae consists of large 220-450 nm brick-shaped viruses, with a double-stranded dna genome (160-220 kb) ( table 1) , that are serologically cross-reactive and -protective. the middle part of the genome is very conserved among orthopoxviruses encoding structural proteins and replication machinery whereas the ends are more variable comprising genes involved with host-specificity and counteracting the immune response [74] . replication of orthopoxviruses occurs in the cytoplasm and includes translation of early mrnas (such as dna polymerases and immune defense molecules), dna replication, translation of intermediate mrnas (for late transcription factors), and late mrnas encoding structural proteins and late enzymes, respectively. altogether, e.g. the cowpox virus genome encodes nearly 200 open reading frames. intracellular non-enveloped virions are first formed, comprising the majority of the infectious viral progeny; some particles develop in er/golgi into enveloped, either cell-attached or extracellular viral particles, often motile due to attached actin tails [75] . homologous recombination occurs readily between orthopoxvirus sequences which has raised some concerns about the use of vaccinia virus-based vaccines in wild animals [76] . some orthopoxviruses are host-specific, whereas some are more promiscuous but have a distinct reservoir. their nomenclature may be misleading; e.g. monkeypox is not carried by monkeys, neither is cowpox carried by cows. smallpox or variola virus, now eradicated and historically the cause of one of the most feared human diseases, was specific to man. many other orthopoxviruses circulate in wildlife species and are often zoonotic. examples include the vaccinia virus, the modern smallpox vaccine, the origins of which are unclear but was originally described as cowpox by edward jenner [77] in late 18th century england, and which later has also re-escaped to nature in other parts of the world [78] ; monkeypox virus, pathogenic to primates, including humans, causing a smallpox-like disease with secondary transmission and with a likely reservoir in small rodents in central africa; cowpox virus, which is the main orthopoxvirus in europe and may be transmitted to man either directly from rodents or from a secondary carrier, typically cat. cowpox virus has been detected in western eurasia and in europe, voles of clethrionomys and microtus species and apodemus mice are the main reservoir hosts [79] ( table 2) . shedding of the virus from rodents is apparently transient. infection of cattle is rare; domestic cats relatively frequently present with clinical disease, but infection of zoo animals, e.g. elephants, has also been reported [80] . following the cessation of smallpox vaccination, more than 25 years ago, the number of humans susceptible to cowpox has increased. more than 60 human cowpox cases have been reported in the literature since 1969 [81, 82] . all age groups may acquire cowpox, but most cases have been in girls under 12 years of age, who have had a cat or e.g. a field mouse as a pet. infection probably occurs through abrasions in the skin; persons with atopic eczema are more prone to the infection. [81] [82] [83] [84] . the incubation period is 5-7 days, after which papules develop into lesions 1-3 cm in diameter which proceed through pustular, ulceral and eschar stages over a period of a about two weeks. they may be painful and vary in number, size and severity. local lymphadenopathy, pyrexia and nausea may occur; secondary bacterial infections are common. typically, solitary lesions are found, located mainly in fingers, hands or face (e.g. eyelid) [81, 85] . in 6-8 weeks the lesions heal gradually, some residual scars may remain. in some cases severe generalized skin infection occurs [83] , especially in atopic and in immunocompromised individuals and may in extreme cases lead to death [86] . cidofovir (a phosphorylated nucleoside analog of cytosine) may have potential as an antiviral against cowpox virus [87] . man-to-man transmission of cowpox virus (unlike for monkeypox) has not been reported. it is usually possible to detect orthopoxvirus particles directly from the skin lesions by electron microscopy. the virus can also be readily isolated in e.g. vero cells or chorioallantoic membrane of chicken embryos from the lesions and subsequently characterised [80] . several sensitive pcr approaches have been described, some related to the bioterrorism (smallpox) preparedness [88] [89] [90] ; in each case further typing at the species level is needed. in addition, during acute cowpox infection, igm antibodies and low-avidity igg antibodies have been detected [83] . following the cessation of smallpox vaccination approximately 30 years ago, the younger age groups are the most susceptible population, both to smallpox and to cowpox, which is more closely related to vaccinia virus. recent estimates indicate as low as 40% protection levels among europeans. for instance in finland, in the age group over 50, everybody had orthopoxvirus antibodies as measured by immunofluorescence assay. the seroprevalence decreased gradually towards younger age groups reflecting the gradual cessation of smallpox vaccination, with the last vaccinations in finland occurring in 1977 [83] . smallpox was finally declared to be eradicated from the world in 1980 [91] after which few people in europe have received the vaccine. in addition to wild rodents carrying cowpox, import of exotic pets may also pose a risk for orthropoxvirus transmission, as was seen in a recent outbreak of monkeypox virus in the usa [92] . orthomyxoviruses are enveloped, negative-strand rna viruses with 6-8 genome segments, of which avian influenza, (i.e. influenza a) viruses may cause severe disease in domestic poultry and cause zoonotic infections. the influenza viruses in wild aquatic birds are the source of these epidemics in chickens as well as providing a gene pool for reassortants with human influenza a viruses which then may become established in humanto-human transmission resulting in influenza pandemics. in addition, influenza a viruses are known pathogens of pigs, horses, mink, seals and whales [93, 94] . influenza a virus is 80-120 nm in diameter and has 8 genome segments varying in size from 0.89 to 2.3 kb. the three largest segments 1-3 encode the polymerase subunits pa (83 kda), pb1 (87 kda) and pb2 (84 kda), respectively; whereas the segments 4-6 each encode one viral protein, namely the 63 kda hemagglutinin (ha), the 56 kda nucleoprotein (np), and the 50 kda neuraminidase (na), respectively (table 1 , fig. 1 ). the two smallest segments, 7 and 8, encode each two proteins, the 28 kda matrix protein (m1) and the 11 kda membrane protein (m2), and the 27 kda ns1 and the 14 kda ns2 proteins, respectively (table 1 , fig. 1 ). in common with the bunyaviridae, the genomic rna is packed in the nucleoprotein which carries polymerase subunits, and the 5 0 and 3 0 -ends are conserved and complementary to each other and thus able to form panhandle structures in which the promoter regions reside. ''cap-snatching'' from cellular mrnas and an oligo-u motif are used to create viral mrnas starting with a cap structure and ending in a poly-a region. the virus enters the cells after binding to the sialic acid receptors by endocytosis, which is followed by acidification of the endocytic vesicle and, mediated by the ion channel forming m2 protein, of the interior of the virus, which leads then to fusion of the viral and endosomal membrane and release of the viral nucleocapsids to the cytoplasm, respectively. however, untypically for an rna virus, the replication, transcription and nucleprotein assembly occur in the nucleus. the envelope proteins are processed to the plasma membrane, where budding of the virions finally occurs. the envelope proteins are also the most important antigenic determinants. the homotrimeric hemagglutinin (ha), defines the ''h type'' which is responsible for the binding to the sialic-acid containing host cell receptors and membrane fusion properties. the neuraminidase (na) defines the ''n type'', and cleaves terminal sialic acid residues from glycoconjugates enabling the virus to reach target cells in the mucin-rich epithelium and facilitating release of the virus from the cells [93, 94] . the catalytic site of the neuraminidase is a target for antivirals oseltamivir and zanamivir, and the m2 protein is a target for amantadine and rimantadine. to become active, the hemagglutinin protein needs to be cleaved by trypsin-like proteases found in respiratory and gastrointestinal epithelia. human-adapted influenza viruses replicate in the respiratory tract, whereas in avians the virus replicates primarily in the gut. when transmitted to and within poultry, the normal hemagglutinin of influenza a virus h5 or h7 of wild aquatic birds of the ''low pathogenic type (lpai) may be mutated. accumulation of basic residues at the cleavage site makes the ha cleavable by most proteases of cells, such as furin, and results in ''highly-pathogenic avian influenza'' (hpai) virus able to replicate in most tissues killing rapidly up to 100% of chickens [93] [94] [95] . hpai, also known as ''fowl plague'' was first described in 1878 and the virus was first isolated in 1901 suggesting that humans have been directly exposed to avian influenza a viruses for centuries [96] . influenza a virus gene pools reside in wild aquatic birds where at least 15 ha types and 9 na types are found, as compared to 3 ha (h1-h3) and 2 na (n1, n2) types circulating in man. also, the genes in aquatic birds are in evolutionary stasis without undergoing changes due to selective pressures. influenza a viruses in man are under constant selective pressure imposed by population immunity causing the hemagglutinin of influenza a virus to change its antigenic properties by accumulation of mutations (genetic drift). however, through double infection and reassortment (genetic shift), novel (e.g. hemagglutinin) genes from aquatic birds may become established in human influenza viruses giving rise to pandemics due to lack of adaptation to and immunity in, humans. previously, it was thought that the different receptor specificity -favoring different side chains of sialic acid -of human and avian influenza viruses would make direct transmission of avian viruses to humans unlikely, but both could be expected to infect swine, which carry receptors for both. thus pigs are considered to be potential reservoirs for generating new influenza virus variants. it has only recently been discovered that viruses considered unique to avian species may also infect man, although this may involve change in receptor specificities [97] . in 1996 conjunctivitis in uk caused by avian influenza viruses (and previously, by seal influenza a viruses) was reported [98, 99] (table 2) . direct zoonotic transmission of avian influenza viruses to man resulting in human respiratory illness, was not known to occur or had not been diagnosed before the outbreak of h5n1 avian influenza virus in hong kong in 1997 where 6/18 patients died of lower respiratory tract infection [100] . after this, in europe, an outbreak of h7n1 hpai avian influenza was encountered in italy (1999) (2000) without reported transmission to humans [101] . in 2003, a major h7n7 hpai avian influenza outbreak occurred in the netherlands [102, 103] . during this epidemic, veterinarians and people who culled infected poultry were at greatest risk of infection. it was noted by active surveillance that human h7 infections (and simultaneous h3 infections) were occurring, and consequently prophylactic treatment with oseltamivir was started. the h7 virus was confirmed to be transmitted to 85 humans of which the majority had conjunctivitis, seven had an influenza-like illness; one veterinarian (who did not receive prophylactic oseltamivir) died. his symptoms started with high fever and headache two days after visiting a farm with infected chickens. one week later, he was admitted to hospital with pneumonia, where his status deteriorated to multi-organ failure, with death due to respiratory insufficiency 2 weeks after onset of symptoms. in addition, in three cases household primary contacts were shown to have the disease by human-to-human transmission [102] . both na inhibitors, zanamivir and oseltamivir inhibited virus obtained from humans during this outbreak and a significant difference was found in avian influenza virus detection between oseltamivir users (1/38) and those who had not taken prophylactic medication (5/52) [102] . outbreaks of avian influenza have continued to occur in other parts of the world, especially the devastating ongoing h5n1 epidemic in south-east asia since late 2003 with as of june 2005 a total of 54 deaths/107 cases in viet nam, thailand and cambodia. the epidemic has had a dramatic impact on poultry farming and industry with million birds dying or being destroyed to reduce virus dispersal. this ongoing epidemic clearly has ''pandemic potential''. avian influenza viruses may be detected by virus isolation (in cell culture or embryonated eggs) or rt-pcr which may be targeted at the specific ha subtype or may be generic to influenza a viruses (e.g. targeting the matrix protein gene) [104] . also, serological tests such as. hemagglutination inhibition may be used. typing may be based on serological methods (such as hemagglutination inhibition), specific primers/probes or sequencing. many commercial influenza a antigen tests detect the nucleoprotein or na activity and should be applicable to the avian viruses, although this is poorly studied and documented. furthermore, it should be noted that rt-pcr from throat swabs of the lethal case in the netherlands were negative [102] . avian influenza, or ''fowl plague'' outbreaks usually arise following contact with wild aquatic birds, such as mallards and ducks, in which the virus replicates as a rule without causing symptoms. the virus that is excreted in the gut can typically be isolated (or detected by rt-pcr) from cloacal swabs. hinshaw et al. [105] studied over nine thousand birds and detected 30% prevalence in young and 11% prevalence in adult birds. influenza virus is readily transmitted in cold environments and is stable for 126-207 days at +17â°c, or in wet faeces (as shown for h5n1 virus) for at least 4 days at 25â°c and more than 40 days at +4â°c [106, 107] . the main preventive and control measures include proofing the chicken breeding facilities against wild birds. raising chickens and turkeys in the open is a risk, minimizing secondary spread of outbreaks by stamping out the infected poultry, followed by cleaning, disinfection and controlling movements of humans and animals, trade embargoes and reporting the outbreaks (''fowl plague'' is in the top priority ''list a'' of the international animal health code of the office international des epizooties). selling poultry live, a common practice in south-east asia, is a definite risk factor. vaccination of poultry has been used as an additional control measure but may lead to undetected shedding and transmission of mutant virus selected under the pressure imposed by the vaccine [94] . to prevent further transmission to humans, rapid measures are needed due to the short incubation period. the dutch experience suggests that results can be achieved by personal protection (e.g. protective eye glasses, masks) for all workers who screen and cull poultry, vaccination with regular inactivated influenza virus vaccine and prophylactic oseltamivir for those handling potentially infected poultry, to be continued for 2 days after last exposure [102] . for humans, specific vaccines containing h5 and h7 antigens would be welcome. the only known zoonotic agent of the togaviridae family to cause human disease in europe is the mosquito-borne sindbis virus (sinv), in the genus alphavirus. sinv is distributed throughout the old world and australia and causes rashes and polyarthritis outbreaks in northern europe, similar to chikungunya, oã�nyong-nyong virus and ross river virus in far east asia, africa and australia, respectively, whereas other alphaviruses causing encephalitic infections in man (venezuelan, western and eastern equine encephalitis virus) are found in the new world. alphaviruses are enveloped, positive-strand rna viruses with an 11 kb genome. the non-structural proteins (nsp1-4) are encoded from the 5 0 -terminus of the genome, and a separate subgenomic 26s rna from the 3 0 -end is used as a messenger for the structural proteins: the 30-33 kda capsid protein (c), and the 45-58 kda envelope glycoproteins (e1 and e2) [94] (table 1 , fig. 1 ). sinv was first isolated in the nile delta in the 1950s from a pool of mosquitoes without knowledge of any disease association. it is now known to be the causative agent of mosquito-borne epidemic polyarthritis with accompanying rashes and when described in northern europe it was given the names ockelbo disease, pogosta disease, or karelian fever when found in sweden, finland and russia, respectively. most infections occur during august-september, and larger outbreaks tend to appear with a seven-year interval (e.g. in finland, 1282 serologically verified cases occurred in 1995, 600 cases in 2002; and in sweden 50-65 cases are reported during peak years) [108, 109] , with a peak incidence (56%) in 50-year old females. the association of sinv with pogosta disease was first discovered in the early 1960s and it is believed that the virus may have been distributed throughout northern europe around this time. this conclusion is based on the evidence that thousands of human and bird sera collected during the early 1960s in finland, were negative for sinv antibodies, whereas in the 1990s 2-5% of the population were sinvantibody positive [108] . antibodies to sinv without polyarthritis outbreaks have been recorded in italy, romania, greece and the former yugoslavia [109] . however, human disease due to sinv has been recorded in south africa, from where it may have originated. sinv was isolated from mosquitoes in sweden, norway and russia in the early 1980s [109] [110] [111] and sinv antibodies are found in wild tetraonic and migratory birds, most commonly (in sweden) from passeriformes such as redwing (turdus iliacus), fieldfare (turdus pilaris), blue tit (parus caeluleus), chaffinch (frinigilla coelebs), songthrush (turdus philomelos); thrushes have been suggested to be a major candidate as an amplifying host [109, 112] . in addition, antibodies are commonly found in galliformes [109, 108] . phylogenetically, sinv strains are similar in northern europe and south africa (in the north to south dimension), but differ considerably from strains circulating in asia and australia, where sinv-associated rash-arthritis is not recorded. this is consistent with the notion of a north-south dispersal of sinv strains by migratory birds [113] . several bird species can be infected experimentally [114] . it is evident that sinv cycles between birds and ornithophilic (culex and culiseta) mosquitoes (table 2) . however, it may spill over to other hosts (evidence from many vertebrates from a frog to a bear) and vectors (including ticks and aedes mosquitoes) [109] . recently, during an outbreak in finland in 2002, the causative agent of pogosta disease was isolated for the first time in europe from skin biopsies and a blood sample of patients [115] ; the virus strains were most closely related to sinv strains isolated from mosquitoes in sweden and russia 20 years previously. the incubation period for the disease is about one week and the onset is accompanied by arthritis/arthralgia and itchy rash as the dominant symptoms, and also fatigue, mild fever, headache and muscle pain. hematological laboratory parameters are within the normal range and levels of c-reactive protein (crp) are not elevated. the rash is usually located on the trunk and thighs and lasts for a couple of days. one third to a half of patients, suffer from joint pains for more than 12 months [116, 117] . usually several joints are affected, the most common being the ankle, wrists, knee, and finger joints (50% or more of patients), as well as hip, shoulder and elbow joints [118,119a,119b ]. diagnosis is based on serology using enzyme immunoassay, immunofluorescence assay or hemagglutinationinhibition, and detection of seroconversion or a 4-fold rise in titre between two samples, or positive igm in a single sample. the first sample is usually taken during the first week after onset of illness, another sample is required to diagnose or exclude sinv infection; igm antibodies are detectable until approximately 6 days post-onset, and igg antibodies can be detected approximately 10 days after onset of illness [119b,120] . in some cases persisting igm antibody can be detected years after infection [117] . for research purposes, the virus can be detected by 121] or isolated from skin biopsies [115] . no specific preventive measures are available, apart from avoiding mosquito bites. flaviviruses comprise a diverse group of pathogens that have been traditionally classified as arthropodborne viruses. they are linear positive single-stranded rna viruses with a monopartite genome (10-11 kb) that encodes 3 structural proteins: the 13 kda capsid protein (c), the 51-59 kda major envelope protein (e) and the 8.5 kda glycoprotein m (in mature virions); and 7 non-structural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b and ns5) (table 1, fig. 1 ). noncoding or untranslated regions flank the infectious rna genome. when seen in the electron microscope flaviviruses appear as uniform spherical particles, 40-60 nm in diameter. the virus particles consist of a lipid envelope that has a surface covered by protrusions that contain envelope (e) and membrane (m) structural proteins, organized as dimers. this envelope surrounds an isometric capsid protein of approximately 30 nm in diameter [122] [123] [124] . there are currently about 70 members in the family flaviviridae [125] which have been found infecting a wide variety of organisms including mammals, arthropods, avian and amphibians. many of these viruses are major pathogens of humans, domestic and farmed animals as well as wildlife species. with the possible exception of the dengue viruses, the flaviviruses are zoonotic, depending almost entirely for their existence on wildlife vertebrate and in many cases, invertebrate species. the type species of the genus is yellow fever virus (yfv), hence the term ''flavi'' from the latin word flavus, which in turn describes the yellowish color of the skin in yellow fever infections [126] . the classification, and serological and phylogenetic studies of flaviviruses reflect the importance of the vector on the biology and evolution of this genus. there are essentially three groups of flaviviruses: tick-borne, mosquito-borne and non-vectored flaviviruses, although this grouping is, to some extent, arbitrary since some mosquito-borne viruses are also transmitted by ticks and vice versa [127] (table 2 ). phylogenetic analysis also shows very strong correlations between genetic relationships, epidemiology and ecology of these viruses [128, 129] . some flaviviruses are responsible for a significant proportion of the morbidity and mortality that is registered annually worldwide. they cause epidemic outbreaks that involve encephalitis and/or haemorrhagic fever, often fatal and involving millions of humans or in some case birds or mammals. important flaviviruses affecting humans are the dengue viruses, yellow fever virus, west nile virus (wnv), tick-borne encephalitis virus (tbev), japanese encephalitis virus, saint louis encephalitis virus, and murray valley encephalitis virus among others. dengue virus alone causes more than 50-100 million cases worldwide each year and some 2500 million people are now at risk from dengue infections [130] . the most important flavivirus in europe is tbev, which is endemic in many european countries, and also in russia, ( table 5 , fig. 4 ) northern china and northern japan [126,131a] . it affects thousands of people annually and has a significant impact on public health. the virus is transmitted to humans mainly through a tick bite, however, the infection has also been reported to occur by drinking unpasteurised goat milk from viraemic animals [131b,131c]. the virus is maintained in nature in a cycle involving ticks and wild vertebrate hosts and also by transovarial and transstadial transmission in its vector [126, 132] . serological evidence and viral isolations, as well as sequence similarities have suggested that migratory birds could also play a role in the transmission of tbev from central europe to scandinavian countries; moreover, endemic areas of tbev are regions of high migratory bird activity [133] . tbev is classified taxonomically into three subtypes: european subtype, far eastern subtype, and siberian subtype. the first subtype is transmitted mainly by ixodes ricinus, and the last two by ixodes persulcatus [134, 135] . the distribution of tbev is well coordinated with the distribution of its vector, furthermore, different genotypes are located in distinct geographical areas and associated with specific vector hosts. recent data have shown the co-circulation of all three subtypes of tbev in the same geographical region, specifically in latvia, where the two vector ixodes species habitats meet [136] . however, the endemic region in europe is patchy and covers only part of the geographical range of e.g. ixodes ricinus. there has been an increase of the incidence of tbev in many of its endemic areas but not in austria where a countrywide successful vaccination campaign was established reducing the disease incidence to lower levels [131c,131d,137a]. tbev affects principally the nervous system and can cause several clinical features of different severity table 5 tick-borne enchephalitis viral infections in europe per country through time year 1990 year 1995 year 2000 year 2002 austria 89 109 60 60 belarus -a 66 23 18 croatia 23 59 18 30 czech r. 193 744 719 647 denmark --3 1 estonia 37 175 272 90 finland 9 23 41 38 france 2 6 0 2 germany -226 133 including meningitis, meningoencephalitis, meningoencephalomyelitis and meningoradiculoneuritis. hospitalization varies from days to months and in some cases years of treatment and rehabilitation are necessary as sequelae occur in approximately 1/3 of the patients. the incubation period of tbe is between 7 and 14 days. the disease is characteristically biphasic. the first phase (day 2-4 of onset of symptoms) is viraemic and can be either asymptomatic or fever, malaise, headache, anorexia, nausea, and muscle pains may be present. the second phase occurs in up to 30% of the patients after about 8 days lag period (approximately 21 days after the tick-bite) as a neurological disease of which about 0.5% has a fatal outcome [137b,137c,137d]. the neurological symptoms and severity vary: the clinical picture includes meningitis (in about half of the patients), meningoencephalitis, meningoencephalomyelitis and meningoradiculoneuritis. hospitalization varies between days and months and in some cases years of treatment and rehabilitation are necessary in case of e.g. paresis. altogether, neuropsychiatric sequelae occur in approximately 1/3 of the patients. louping ill virus (liv) is endemic in ireland, in the northern region of great britain and in norway (fig. 4) affecting principally sheep with a disease that is known as ovine encephalomyelitis, infectious encephalomyelitis of sheep, or trembling-ill. liv is transmitted to sheep by the tick vector ixodes ricinus. the natural life cycle of liv resembles that of tbev, it can be sustained in the natural environment through non-systemic transmission of virus between ticks cofeeding on rodents and other wild animals, which in turn infect grazing animals such as sheep and goat as a zoonotic disease. louping ill virus has also been observed in a bird-tick-bird cycle involving the red grouse (lagopus lagopus scoticus), ptarmigan bird species and the ixodes tick [138] . infection with liv in sheep is characterised by a biphasic fever, depression, ataxia, muscular in-coordination, tremors, posterior paralysis, coma, and death. there is evidence for infection by liv in other domestic species and wildlife, i.e. cattle, horses, pigs, dogs, deer, shrews, wood-mice, voles, and hares [139,140a] . it is generally believed that the majority of avian infections occur through a tick bite, however laboratory experiments and field observations have demonstrated that the red grouse can also become infected by feeding on infected ticks indicating that the vector bite is not the only route of infection [140b] . humans are also susceptible to infection with liv. however, the majority of the cases reported are accidentally acquired infections in laboratory workers. the second most frequent infection results from handling infected animal carcasses. infection with liv in humans can cause a neurological disease resembling the clinical picture observed for tbev infections, i.e. biphasic encephalitis, influenza-type illness, fever, articular pain, meningitis, myagia and poliomyelitis-like illness [141] . other possible transmission routes for liv infection to humans include drinking contaminated milk from goat or sheep in an acute phase of infection [142] , tick-bite, exposure to infective material, or through skin abrasions or wounds. antigenic and phylogenetic studies have shown that liv is most closely related to strains of the western european subtype of tbev and it is estimated to have emerged from this lineage approximately 800 years ago [143] . west nile virus (wnv) was first discovered in 1937 in uganda [144] . it occurs throughout africa, the middle east, europe, russia, india and indonesia and was recently introduced into north america (new york) [126, 145] . in the old world wnv is primarily considered to be an african virus which annually disperses northwards out of africa when birds migrate to europe, the middle east and asia [145] . in humans, the majority of wnv infections cause a nonsymptomatic or mild febrile illness; however some infections can cause encephalitis and in most severe cases can lead to death, particularly in elderly patients. the incubation period is between 3 and 15 days after a mosquito bite (http://www.cdc.gov/ncidod/ dvbid/westnile/wnv_factsheet.htm). west nile virus is an illustrative example of the human impact on the dispersal and evolution of flaviviruses. the virus appeared for the first time in the usa, in new york in 1999 causing sixty-two confirmed human infections and seven deaths [146] . the virus successfully over-wintered and during the next years dispersed widely throughout north america and now more recently also to central america and the caribbean. in north america the virus infects a very wide range of mosquito and animal species. the exact mechanism of introduction into north america is not known [146-148a] . to date, more than 14 000 human cases and 586 deaths from wnv have been reported in the united sates of america (http:// www.cdc.gov/ncidod/dvbid/westnile). in europe, outbreaks caused by wnv have been recorded since the early 1950s, especially in mediterranean countries, romania and southern russia (fig. 4) . larger outbreaks (over 800 cases) have been reported since the mid 1990s in urban settings, especially a large outbreak in bucharest, romania in 1996 [145a,145b,145c] . in southern russia, specifically in volgograd, astrakhan and krasnodar regions, wnv has caused large epidemics of approximately 1000 human cases, with 4% mortality rate of reported cases. molecular epidemiological studies have shown that the latest large outbreaks in volgograd were caused by strains genetically similar to that of romania-1996 , kenya-1998 and newyork-1999 reflecting the widespread distribution capacity of these epidemic viral strains [149b,151b]. the incidence of wnv in europe is largely unknown. phylogenetic studies have showed that wnv has diverged into two main lineages, which form internal clusters or clades [149c,150a,150b,150c]. more recently, a novel virus (rabensburg virus), antigenically and genetically closely related to wnv was isolated from a culex pipens mosquito pool in czech republic. this new virus could represent a third lineage of wnv, however more studies are needed to confirm this hypothesis or to determine whether or not this could represent a new member of the flaviviridae family [151a] . usutu virus (usuv) was first isolated from a mosquito in africa in 1959 [148c]. it was characterised serologically and classified within the japanese encephalitis virus serocomplex. usutu virus had rarely been isolated since then, with only one reported human case and being present only in two regions of africa. however, in the late summer of 2001, usuv was found in vienna in austria during a study of an avian epidemic characterized by fatal encephalitis that was thought to be caused by wnv. the virus caused die-off among the bird population, especially blackbirds, in vienna (fig. 4) and when isolated from birds and mosquitoes it showed 97% genetic identity to the african usuv [148b]. this is the first time usuv has been observed outside africa and also the first time it has been associated with fatal disease in animals. the virus re-appeared in the late summer of 2002 in the same region of vienna [149a] . most recently, in 2003 the virus has been isolated from humans (associated with rash in one patient) and from birds in austria and evidence exists as to the virus being able to overwinter establishing itself in this geographical area (nowotny, n. second european congress of virology, eurovirology 2004). as mentioned above the distribution of flaviviruses is worldwide, and we have described the zoonotic flaviviruses endemic to europe. however, it is important to note the increasing incidence of imported flaviviruses totaling over 500 cases mainly due to changes in human behavior, such as increased travel to non-european destinations (http://www.eurosurveillance.org). the dengue viruses (denv) are a world-wide public health problem. they are transmitted to man by mosquitoes, particularly aedes aegypti and cause a wide range of different clinical outcomes [151c,152a] . it is estimated that 100 million cases of dengue fever (df) and 250 000 of dengue hemorrhagic fever (dhf) cases occur annually [126] . in a study based on the epidemiology and clinical course of 294 travellers with symptoms of dengue infection it was found that the incidence of df among european travellers is underestimated, since diagnostic procedures, in general, for febrile patients often do not include tests for tropical arthropod-borne diseases, such as dengue virus [152b] . there have also been some imported cases of yfv to europe mainly by tourists travelling from endemic areas. however, the incidence of yfv as an imported disease is much lower than that of dengue infections (http://www.eurosurveillance.org) presumably because of the different nature of these diseases, denv having spread worldwide throughout the tropics and yfv occurring only in its endemic regions in africa and south america [152c]. traditionally, diagnosis of flaviviruses in europe has been restricted to detecting tbev infections through serological assays; igm-capture enzyme immunoassay, hemagglutination inhibition-, and immunofluorescence assay. igm antibodies in serum, and in some cases in cerebrospinal fluid during the neurological disease usually provide the diagnosis. rt-pcr is rarely positive at the second phase of the disease [153a]. because of the close antigenic relatedness of flaviviruses, laboratory findings can easily lead to misdiagnosis [153b]. in a study conducted in hungary, tbev-positive serum panels were tested retrospectively against wnv and some sera were found to show higher titres to wnv than to tbev when compared in serological assays (e. ferenczi, personal communication). as mentioned above, a study including travellers returning to europe from tropical destinations proved that many cases remain undetected. there is a need to improve the diagnosis for flaviviruses in order to determine the true incidence and prevalence of these infectious diseases in europe, and to study the ecology of imported viruses to determine which viruses have been able to establish themselves in the continent and which are still continuously being imported from africa or asia. meanwhile anti-vector campaigns and vaccination for travellers to endemic areas are the only measures to prevent and control these infectious diseases [146] . there are effective vaccines for tbev and yfv but a protective immunogen does not exist for denv or wnv. tbev vaccines from two commercial manufacturers (baxter and chiron) are available and widely used, especially in austria and germany, both are based on formalin-inactivated virions, three injections are needed for full protection. booster immunizations are recommended every 3-5 years for people living or spending holidays in endemic areas. 3.3. nairoviruses: crimean-congo hemorrhagic fever virus the genus nairovirus (family bunyaviridae) is composed of 34 predominantly tick-borne viruses that have been divided into seven serogroups [154] including several associated with severe human and livestock diseases (especially crimean-congo hemorrhagic fever virus (cchfv) and nairobi sheep disease virus). of the pathogenic viruses only cchfv causes significant human morbidity and mortality and is found in europe. like other members of the bunyaviridae family nairoviruses possess a tripartite single-stranded rna genome of negative polarity consisting of large (l), medium (m) and small (s) segments. the 12 kb l segment encodes an rna-dependent rna polymerase (deduced size 448 kda in cchfv), the 4.9 kb m segment encodes the precursor for the two envelope glycoproteins gn (75 kda in cchfv) and gc (37 kda in cchfv), and the 1.7 kb s segment the viral nucleocapsid n (50 kda) ( table 1 , fig. 1 ). like with other members of the bunyaviridae viral messenger rnas (mrna) are not polyadenylated and are truncated relative to the genome rnas at the 3 0 termini. messenger rnas have 5 0 -methylated caps and 10-18 nontemplated nucleotides which are derived from host cell mrnas. the nairovirus l segment encoding the rna polymerase is conspicuously large, almost twice the size of many other members within the family bunyaviridae, and may thus provide other functions such as viral helicase and a papain-like cysteine protease activity predicted from the nucleotide sequence [155, 156] . viral protein analysis has suggested that gn and gc may be derived from 85 and 140 kda precursors, of which the latter contains an n-terminal region with a highly o-glycosylated mucin-like domain [157] . the cchfv nucleocapsid protein colocalizes and interacts with human mxa protein; this interaction may explain the antiviral effect of interferons on cchfv [158] . similar to the hantavirus-rodent association, the high genetic variation of viruses of the genus nairovirus reflects the diversity of their predominant tick hosts [159] . within cchfv isolates, comparison between m and s segment phylogenetic groupings suggests that reassortment events have occurred in some virus lineages [160] . reverse genetics, recently established for cchfv [161] , provides a unique opportunity to study the biology of nairoviruses and tailor optimal therapeutic and prophylactic measures against cchfv infections. cchfv is transmitted most efficiently by hyalomma ticks, followed by rhipicephalus, dermatocentor spp and many other species of ixodid (hard) and some argasid (soft) ticks (table 2) . among ticks cchfv is capable of transmitting infection both transovarially and transstadially. the life cycle of cchfv also includes a tick-vertebrate host cycle involving both wild and domestic animals. the virus or antibodies against it have been detected in rodents, hares, hedgehogs and some birds but human infections seem to be principally from contacts with livestock (mainly cattle, sheep, goats) including in africa farmed ostriches and less often ticks [162, 163] . thus cchfv is more commonly seen in persons exposed to blood and tissues of infected animals during occupational activities, such as farming, herding, veterinary examination and abattoir work. nosocomial infection is common and often results in small outbreaks. yet, outdoor and recreational activities also represent a risk factor. in addition, horizontal transmission of cchfv from mother to child may occur [164] . the global distribution of cchf follows closely that of hyalomma spp. in the middle east, asia, africa and southeast europe [162, 163] . sequence analysis of cchfv s rna segments gives patterns following links between different geographic locations, in some cases suggesting links originating from trade in livestock and long-distance carriage of virus or infected ticks during bird migration [165] . in europe, cchf occurs in the balkan peninsula (albania, bulgaria, greece, turkey, yugoslavia) but also in southern russia, hungary, france and portugal [162, 166, 167] . the symptoms and signs of crimean-congo hemorrhagic fever are similar to those of other viral hemorrhagic fevers [162, 163] . the incubation period is generally short ranging usually from 2 to 9 days. there is typically a very sudden onset of illness with fever, rigors, chills, intense headache and backache or leg pains, myalgia, nausea, and vomiting. patients may also present with photophobia, somnolence and menigism with confusion or aggression. hemorrhages in the form of petecchiae, ecchymoses, epistaxis, melena and bleeding from various organs usually begin a few days later. tachycardia is common and lymphadenopathy is seen occasionally. the case-fatality rate is 20-35%. the pathology consists of hemorrhagic and necrotic lesions in various organs as well as fibrin deposits. the methods used for detection of cchfv infection in humans or livestock include indirect immunofluorescence on virus-infected or n-expressing transfected cells. both igm and igg antibodies reactive for cchf appear within a week in patients [162, 168] . alternatively a recombinant nucleocapsid protein-based enzyme immunoassay [169] [170] [171] may be used. rapid detection and quantification of cchfv rna is possible by real-time reverse transcription pcr [172] . ribavirin is clearly effective in treatment of crimean-congo hemorrhagic fever [173, 174] but supportive treatment (blood, thrombocytes, coagulation factors) is equally important. immune plasma from recovered patients has also been used to treat patients but not in controlled studies with a follow-up of virus-neutralizing activities [162] . the most effective method of preventing infections is to take measure to avoid exposure to ticks (protective clothing, tick repellent, frequent body searches to remove ticks). inactivated virus prepared in mouse brains has been used on a limited scale as a vaccine in southeastern europe and the former ussr but the sporadic and unpredictable occurrence of the disease renders it difficult to identify target populations and has slowed development of a safe and modern vaccine [162] . orthobunyaviruses belong to the family bunyaviridae. these enveloped viruses have a three-segmented negative-strand rna genome. the 0.97 kb s segment encodes the 19-25 kda nucleocapsid protein (n) and a 10-13 kda nonstructural protein (ns); the 4.5 kb m segment encodes the polyprotein precursor of two glycoproteins, g1 and g2 (108-210 kda and 29-41 kda, respectively), and a nonstructural protein nsm (15) (16) (17) (18) ; and the 6.9 kb l segment encodes the rna polymerase (260 kda) ( table 1 , fig. 1 ). viral messenger rnas are not polyadenylated and are truncated relative to the genome rnas at the 3 0 termini. messenger rnas have 5 0 -methylated caps and some nontemplated nucleotides which are derived from host cell mrnas. orthobunyaviruses are transmitted to humans by a variety of mosquito species, and in europe two known human pathogenic representatives of the genus circulate, tahyna virus and inkoo virus (mostly transmitted by aedes spp.) [109, 175, 176] (table 2 ). these viruses belong to the california serogroup, and their incidence is associated with the distribution of the carrier mosquitoes. the natural cycle of these viruses involve also mammal hosts. tahyna virus circulates throughout most of europe, whereas inkoo virus is mainly reported from northern europe [109,177a] . tahyna virus causes an influenza-like disease which may also present as a meningitis. the infection caused by inkoo virus is mostly sub-clinical or a mild disease, although encephalitis has been reported. the most typical symptoms associated with encephalitis or meningitis caused by californian serogroup viruses include fever, headache, nausea, vomiting, convulsions and confusion. in europe the seroprevalence for californian serogroup viruses varies from 1% to 69%, depending on the geographic area [109] . in finland the seroprevalence against inkoo virus varies from 2% to 6% (ã� land islands) to 61-69% (finnish lapland) [109, putkuri et al, unpublished results] . in disease-endemic areas the seropositivity of the population increases with age [177b]. the diagnosis of orthobunyaviruses is based on serology, either as a rise in igg-antibody titers, or the presence of igm antibodies. also rt-pcr methods are under development to detect viral rna in cerebrospinal fluid samples of patients with encephalitis. phleboviruses are one of the five groups within the family bunyaviridae. they have a negative singlestranded rna tripartite genome, namely: l (6.4 kb), m (3.2-4.2 kb) and s (1.7-1.9 kb). the l segment encodes a single protein of 240 kda (rna polymerase) in the complementary sense. the m segment encodes a protein precursor of 130 kda in the complementary sense which is cleaved into two glycoproteins g1 (56 kda) and g2 (59 kda). the s segment has an ambisense coding arrangement, and it encodes the nucleocapsid protein (n) of 27 kda in the complementary sense and a non-structural protein (nss) of 30 kda in the virion sense [178, 179] (table 1, fig. 1 ). viral messenger rnas (mrna) are not polyadenylated and are truncated relative to the genome rnas at the 3 0 termini. mrnas have 5 0 -methylated caps and some nontemplated nucleotides which are derived from host cell mrnas. when seen under the electron microscope the virions are spherical and enveloped and approximately 80-110 nm in diameter. glycoprotein projections of 5-10 nm are also seen embedded in the membrane. there are between 200 and 1500 spikes per virion [178, 179] . phleboviruses are arthropod-borne viruses and are transmitted mainly by phlebotomine sandflies, hence the derivation of its name ''phlebo'' (table 2) . however, mosquitoes and ticks can also transmit phleboviruses. phleboviruses are distributed throughout most of the world but have not been reported in australia [180] . the first documented record of a disease resembling those caused by phleboviruses dates back to the 1900s in the mediterranean countries of europe [178] . however, the first virus isolation occurred for rift valley fever virus in 1930 in kenya [181] . retrospective serological studies have indicated that outbreaks caused by phleboviruses have occurred since 1912 in the sub-saharan africa region [182] . phleboviruses are major causal agents of encephalitis in humans, especially among children, in mediterranean countries. they also have an important impact in livestock especially in african endemic areas. infection of livestock is characterised by a high rate of abortions and pathologies associated with hemorrhagic illness (leukopenia, thrombocytopenia, fibrin thrombi, intravascular coagulopathy, etc.) [183] [184] [185] . the distribution of the disease follows that of its vector. however the natural history of phleboviruses is largely unknown and for many species the amplifying host has not been identified [185] [186] [187] . in general, phlebovirus infections cause a 2-4 days illness characterised by an incubation period of 2-6 days followed by fever, general malaise, headache, photophobia, and back and joint pain [188] . the illness occurs during the summer months when the activity of phlebotomine flies is high [178] . there are three recognised members of the genus phlebovirus, associated with disease in humans. rift valley fever virus (rvfv), which is the type species of the genus and is transmitted by mosquitoes, causing an influenza-like disease that affects domestic animals and humans. the majority of human infections are asymptomatic; however, in 0.5% of cases it can cause severe haemorrhagic fever, encephalitis and death. the geographical distribution of rvfv includes africa, egypt, saudi arabia and yemen and occurs as epizootics involving aedes spp. mosquitoes and domestic animals [180] . toscana virus (tsv) causes a mild influenza-like disease that can occasionally develop into an acute neurological disease, such as meningitis or meningoencephalitis in italy and possibly other mediterranean countries. a similar virus (granada virus) was recently isolated in spain [193] . tsv is the number one cause of encephalitis among children in italy. the distribution of the virus follows that of its vector, phlebotomus perniciosus, found in italy, spain, portugal, and cyprus. human cases have also been reported from southern france and greece. the virus replicates in the sandfly vector population; however, experimental evidence suggests that an amplifying vertebrate host is needed to sustain the virus in the arthropod population [189] [190] [191] [192] [193] [194] . sandfly fever virus (sfv) sicilian and naples viruses can cause a mild-influenza like disease. the distribution of sfv also follows that of its vector, phlebotomus papatasii, found in the mediterranean basin extending to the middle east and arabian peninsula, the caucasus mountains, pakistan and india [185] [186] [187] . diagnostics of phlebovirus is performed mainly by igm-capture eia and ifa. the virus can be isolated using intracranial inoculation in suckling mice or using a susceptible tissue culture cell line (vero, llc-mk2, bhk-21) [178,195a] . toscana virus and naples sandfly fever virus group together and are distinct antigenically and phylogenetically from sicilian sandfly fever virus [195b] . prevention of infections by phleboviruses varies depending on the type species. immunising livestock with the formalin-inactivated or live-attenuated vaccines can prevent rvfv by avoiding epizootics and hence human infections [178, 196] . in the case of tsv and sfv, prevention is limited to controlling the vector population through insecticides and repellents [197, 198] . what are the reasons for the appearance of all these emerging and re-emerging viruses? have changes taken place in the viruses themselves? apparently not. the principal reasons are changes in the environment and human activity that have created new attacks with nature. factors such as climate change, increasing intensity of agriculture, the building of dams and introduction of other irrigation measures which generate new breeding sites for mosquitoes, crises and wars which bring rodents and arthropods closer to humans, population growth and especially in suburban slums, and global air traffic are important factors generating such new contacts, or may affect the ecological cycles of these viruses. many epidemics may be unexpected outcomes of such changes. moreover, the new molecular biology techniques facilitate detection of new viral agents. many prevention and control measures, such as the anti-aedes campaigns, have been abandoned in tropical countries. the yellow-fever vaccination campaigns have 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virus from sandflies and humans during the same season in aurangabad district, maharashtra state phlebotomus fever in egypt: isolation of phlebotomus fever viruses from phlebotomus papatasi the genus phlebovirus and its vectors clinical and serologic responses of volunteers infected with phlebotomus fever virus (sicilian type) phlebotomustransmitted toscana virus infections of the central nervous system: a seven-year experience in tuscany. scand toscana virus infections of the central nervous system in children: a report of 14 cases evidence of toscana virus infections without central nervous system involvement: a serological study high prevalence rates of antibody to three sandfly fever viruses (sicilian, naples, and toscana) among cypriots infections due to sandfly fever virus serotype toscana in spain central nervous system involvement during infection by phlebovirus toscana of residents in natural foci in central italy (1977-1988) distinction between bunyaviridae genera by surface structure and comparison with hantaan virus using negative stain electron microscopy phylogenetic relationships among members of the genus phlebovirus (bunyaviridae) based on partial m segment sequence analyses climate and satellite indicators to forecast rift valley fever epidemics in kenya evaluation of a new rift valley fever vaccine: safety and immunogenicity trials immunization against rift valley fever virus. studies of the immunogenicity of lyophilized formalin-inactivated vaccine zoonotic transmission of hepatitis e virus from deer to human beings key: cord-257392-u6jy6w1m authors: zhao, yanfeng; ben, haijing; qu, su; zhou, xinwen; yan, liang; xu, bin; zhou, shuangcheng; lou, qiang; ye, rong; zhou, tianlun; yang, pengyuan; qu, di title: proteomic analysis of primary duck hepatocytes infected with duck hepatitis b virus date: 2010-06-07 journal: proteome sci doi: 10.1186/1477-5956-8-28 sha: doc_id: 257392 cord_uid: u6jy6w1m background: hepatitis b virus (hbv) is a major cause of liver infection in human. because of the lack of an appropriate cell culture system for supporting hbv infection efficiently, the cellular and molecular mechanisms of hepadnavirus infection remain incompletely understood. duck heptatitis b virus (dhbv) can naturally infect primary duck hepatocytes (pdhs) that provide valuable model systems for studying hepadnavirus infection in vitro. in this report, we explored global changes in cellular protein expression in dhbv infected pdhs by two-dimension gel electrophoresis (2-de) combined with maldi-tof/tof tandem mass spectrometry (ms/ms). results: the effects of hepadnavirus infection on hepatocytes were investigated in dhbv infected pdhs by the 2-de analysis. proteomic profile of pdhs infected with dhbv were analyzed at 24, 72 and 120 h post-infection by comparing with uninfected pdhs, and 75 differentially expressed protein spots were revealed by 2-de analysis. among the selected protein spots, 51 spots were identified corresponding to 42 proteins by ms/ms analysis; most of them were matched to orthologous proteins of gallus gallus, anas platyrhynchos or other avian species, including alpha-enolase, lamin a, aconitase 2, cofilin-2 and annexin a2, etc. the down-regulated expression of beta-actin and annexin a2 was confirmed by western blot analysis, and potential roles of some differentially expressed proteins in the virus-infected cells have been discussed. conclusions: differentially expressed proteins of dhbv infected pdhs revealed by 2-de, are involved in carbohydrate metabolism, amino acid metabolism, stress responses and cytoskeleton processes etc, providing the insight to understanding of interactions between hepadnavirus and hepatocytes and molecular mechanisms of hepadnavirus pathogenesis. the hbv, prototype of the hepadnaviridae family, is a noncytopathic hepatotropic dna virus replicating via reverse transcription [1] . more than 350 million individuals are hbv carriers worldwide and over one-third of them develop serious liver diseases such as chronic hepatitis, cirrhosis and primary hepatocellular carcinoma [2] . major obstacles in hbv research have been the inability of the virus to infect cells in vitro and lack of adequate animal models for hbv infection, though primary human hepatocytes and heparg cell line have been used to study hbv infection [3] . human primary hepatocytes and heparg cells can support hbv life cycle, but have limitations in accessibility, reproducibility and low level of hbv replication, and a large amount of input virus was needed to infect low proportion of cells [4] [5] [6] . dhbv and woodchuck hepatitis b virus (whbv) are classified into the family of hepadnaviridae. thus for hepadnavirus infection primary hepatocytes of ducks (dhbv) and woodchucks (whbv) are still considered as suitable models for investigating the viral replication and pathogenesis [7, 8] . the development of proteomic methods has enabled us to investigate the changes of cellular protein expression at a global scale to reveal virus-host interactions [9] [10] [11] [12] . the effect of hepadnavirus replication on the host cells, such as the carcinoma derived hepatocyte lines transfected with the hbv genome, heparg cell lines or hbv trans-genic mice, have been investigated by using 2-de analysis [13] [14] [15] . in the present study, we intend to utilize the dhbv-pdhs system to explore global protein expression changes during hepadnavirus infection by 2-de. a total of 75 differentially expressed protein spots were revealed by 2-de between dhbv infected and uninfected pdhs, and 51 protein spots have been identified by ms/ms analysis. differential expression of beta-actin and annexin a2 was confirmed by western blot analysis, and potential roles of some differentially expressed proteins in the viral infection have been discussed. pdhs isolated from the same liver of dhbv-negative cherry valley duckling were infected with dhbv at multiplicity of infection of 30 (moi, based on dhbv dna genome equivalents) and cultured for 12, 24, 72 and 120 h in l15 medium supplemented with 5% fetal bovine serum (fbs). the efficiency of dhbv infection in pdhs was determined by indirect immunofluorescence using anti-dhbv pres monoclonal antibody. pdhs inoculated with phosphate-buffered saline (pbs, ph 7.2) as a control. at 12 h and 24 h after infection, only a few cells showed fluorescence (data not shown), and at 72 h post-infection about 30% of cells expressed viral large surface antigen, indicating that cells were infected with dhbv, showed in figure 1 . dhbv dna in pdhs was analyzed by southern blot hybridization using an alpha-32 p-dctp labeled dhbv-specific probe, and dhbv in the supernatant was detected by real time polymerase chain reaction (pcr). single stranded forms of intracellular viral dna and dhbv copy number increasing in the supernatant indicated the replication of dhbv 72 h and 120 h post-infection (additional file 1 and 2). differentially expressed proteins between dhbv infected and uninfected pdhs at 24, 72 and 120 h post-infection were analyzed using the 2-de. the gels were stained by a modified silver staining method compatible with mass spectrometry (ms) analysis and processed for image analysis. on the 2-de gels (ph 3-10 nl, 24 cm), about 1150~1350 protein spots were detected. compared with the parallel uninfected pdhs, 91 differentially expressed protein spots were revealed by 2-de (p-values less than 0.05 with at least a 1.5-fold difference in percentage of the volume), shown in figure 2 and additional file 3 (see also additional file 4), and a total of 75 differentially expressed non-redundant protein spots were analyzed by ms/ms. differentially expressed protein spots between dhbv infected and uninfected pdhs, were excised, digested ingel with trypsin and determined by ms/ms. among 75 differentially expressed protein spots, 51 protein spots were identified corresponding to 42 proteins (table 1 and additional file 5). with a mascot cutoff score of 72 (pvalue less than 0.05), 51 spots were identified, and 37 spots were matched to orthologous proteins of avian species (26 protein spots to gallus gallus, 4 spots to anas platyrhynchos and 7 spots to other avian species), listed in table 1 . some of the differentially expressed protein spots such as annexin a2, beta-actin, lamin a, destrin, aconitase 2 and mn superoxide dismutase were illustrated in enlarged formats (figure 3 ), and representative mass spectrum of annexin a2 (spot 49) analyzed by maldi-tof/tof ms was shown in figure 4 . isoforms of annexin a2, alpha-enolase, lamin a, glyceraldehyde-3phosphate dehydrogenase (gapdh), heat shock protein 70 (hsp70) and elongation factor 2 have been identified. for example, protein spot 49 (mw 31 kda and pi of 5.45) and spot 50 (mw 10 kda and pi of 5.78) down-regulated in dhbv infected pdhs were both identified as annexin a2, and up-regulated protein spot 26 (mw 65 kda and pi of 6.61) and spot 27 (mw 66 kda and pi of 6.4) were matched to lamin a (theoretical mw 73.1 kda and pi of 6.5) showed in figure 2 . biological functions of the differentially expressed proteins in the dhbv-infected pdhs, were analyzed according to the gene ontology criteria and classified into carbohydrate metabolism (29%), amino acid metabolism (14%), cytoskeletal/structural protein (24%), stress response (18%) and other functions (16%), as shown in table 1 . the roles of selected differentially expressed proteins reported in viral infections were showed in table 2 . expression levels of annexin a2, beta-actin, hsp70, destrin, and lamin a were validated by western blot analysis to confirm the dynamic alterations of protein expression during dhbv infection. equal amounts (30 μg) of cell lysates of dhbv-infected and uninfected pdhs at 12, 24, 72 and 120 h post-infection were separated by sds-page. duck beta-actin and annexin a2 expression were detected down-regulated in the dhbv-infected pdhs at 12-120 h post-infection with mouse anti-beta-actin and anti-duck-annexin a2 as primary antibodies ( figure 5a ), that were consistent with the protein expression pattern revealed by the 2-de analysis. duck hsp70, destrin, and lamin a were not detected by western blot analysis with the rabbit anti-human or anti-mouse hsp70 polyclonal antibodies, rabbit anti-human destrin polyclonal antibody and rabbit anti-human lamin a polyclonal antibody. the same amount protein of each sample were applied to a parallel sds-page gel and stained with coomassie brilliant blue ( figure 5b ). hbv infection remains a public health problem worldwide. because the lack of appropriate cell lines that can support hbv infection efficiently, the cellular and molecular mechanisms of hepadnavirus infection remain incompletely understood. the hepadnavirus animal infection models such as ducks (dhbv) and woodchucks (whbv) have been used to investigate the viral replication, pathogenesis or hepadnavirus-associated hepatocellular carcinoma. dhbv-pdhs model is a valuable model of hepadnavirus infection with high reproducibility and efficiency [16] . in the present study, global changes in cellular protein expression in dhbv-infected pdhs were explored by 2-de combined with ms/ms. among the 75 differentially expressed protein spots, 51 spots have been identified by ms/ms corresponding to 42 proteins, in which 30 spots were matched to orthologous proteins of gallus gallus or anas platyrhynchos, 7 spots to other avian species, and 14 spots to non-avian species, while mass spectra of the other 24 protein spots did not match to any proteins in the current databases, possibly due to the incomplete genome sequence of anas platyrhynchos or low abundance of those protein spots. in previously studies, tong performed a proteomic analysis comparing hepg2 with hepg2.2.15 in which hbv genome integrated into cellular chromosome [13] , and narayan revealed 19 differentially regulated features in heparg cells by 2-de [14] . hepg2.2.15 is a hbv replication cell model but not an infection model, while the human hepatoma heparg cells are susceptible to hbv, but 10~20% of cells can be infected regardless of the amount of virus used (moi > 200) [4, 6] . in previous studies, it has been showed that at moi of 30, about 50%~60% pdhs can be reproducibly infected with dhbv [17] . some of differentially expressed proteins identified in the present study, such as alpha-enolase, lamin a, gapdh and cofilin2 have not yet been reported in hepadnavirus proteomic analysis. viruses depend on host cell metabolism for their replication. elucidation of the pathways/processes involving in the viral life cycle will help to understand the mechanisms of viral infection. in the 2-de analysis, the identified differentially expressed proteins were classified into carbohydrate metabolism, amino acid metabolism, cytoskeletal/structural protein, stress response and other functions according to the gene ontology criteria. some of differentially expressed proteins identified in the present study have been reported playing roles in viral infections, as shown in table 2 . in dhbv infected pdhs, the expression of some carbohydrate metabolic enzymes, such as phosphoglycerate kinase 1, triosephosphate isomerase, phosphoglycerate mutase 1 etc, was up-regulated. the differentially expressed proteins involving in carbohydrate metabolism, suggests perturbed energy metabolism in dhbv infections. hepatitis c virus (hcv) infection reprograms the cellular metabolisms to favor glucose fermentation and glycolytic intermediates toward the metabolite synthesis that supports the viral life cycle [18] . in lymphocytic choriomeningitis virus infection, there was a significant increasing in transcripts promoting gluconeogenesis for viral mediate synthesis, and a decreasing in transcripts promoting glycogenolysis in the early stage of infection [19] . however, gapdh and alpha-enolase, key enzymes involving in glycolysis and gluconeogenesis, are decreased in dhbv infected pdhs. gapdh and alphaenolase have been found associating with the cell membrane and in secreted viral particles of influenza virus, lentiviral vector etc [20, 21] . gapdh may phosphorylate the hbv core protein, and binds to the pres1 region of the hbv envelope antigen and posttranscriptional regulatory element in regulating expression of surface antigen, suggesting that gapdh plays an important role in the life-cycle of hbv infection [22] [23] [24] . the host cellular carbohydrate metabolism affected by dhbv infection may benefit viral replication. alterations of cytoskeleton networks were found in many viral infections [25] [26] [27] . hepadnavirus needs to manipulate and utilize the host cytoskeleton to promote viral infection like many viruses, although the mechanism is still unclear [28] . in dhbv infected pdhs, the microfilament-associated proteins, beta-actin and cofilin-2 were down-regulated, and three microfilament-associated proteins such as transgelin, destrin, and collapsin response mediator protein-2b were up-regulated. actin plays an active role in maturation of the viruses [29, 30] . many viruses require actin for viral entry and establishment of figure 2 . b) accession no. is the mascot result of maldi-tof/tof searched from the ncbinr database. c) protein score was from maldi-tof/tof identification. the proteins that had a statistically significant score great than 72 (p < 0.05) were considered identified. d) theoretical/experimental molecular mass. e) theoretical/experimental molecular pi. f) not applicable, because the spots on the gels were too weak or non-detectable. g) a represents the spot on one of the gels was detectable, n represents the spot on one of the gels was too weak to detect. [31] [32] [33] [34] . however, the actin cortex beneath the plasma membrane can also be an obstacle for virus entry or budding [35] . it has been reported that dhbv entry depends on both intact microtubules and their dynamic turnover but not actin cytoskeleton [28] . therefore the role of actin in dhbv replication is required to further investigation. lamin a is key structural components of the nuclear lamina and lamins, involving in dna replication and gene expression, as well as presenting a natural barrier against most dna viruses such as human cytomegalovirus (hcmv), kaposi's sarcoma-associated herpesvirus, herpes simplex virus (hsv) 1 and epstein-barr virus [36] . lamin a/c is phosphorylated in hsv-infected cells supporting a role in regulating virus capsid nuclear egress [37, 38] . infection of epstein-barr virus induced disassembly of the nuclear lamina and redistribution of nuclear lamin for the nuclear egress [39] . the expression of lamin a with different isoforms, were up-regulated in dhbv infected pdhs, suggesting that lamin a may play a role in dhbv replication. in dhbv infected pdhs, up-regulated expressions of amino acid metabolism enzymes, catalyzing interconversion of glutamate, histidine, and proline (glutamate dehydrogenase 1, urocanate hydratase, delta-1-pyrroline-5-carboxylate dehydrogenase, the orthologs in human referred to protein 16, 19 and 20, 22 in table 1 ), indicate that glutamine metabolism is enhanced. switching the anaplerotic substrate from glucose to glutamine to accommodate the biosynthetic and energetic needs of the viral infection and to allow glucose to be used biosynthetically was reported in hcmv infection [40] . hcvinfected cells exhibit increased levels of the enzymes catalyzing glutamine flux to replenish metabolic intermediates through the latter half of the citric acid cycle providing substrates for atp production [18] . thus simi-lar mechanism of glutamine metabolism may be at work in dhbv infection. stress response associated proteins including endoplasmic reticulum stress associated proteins such as hsp70, and chaperonin containing t-complex polypeptide 1 (tcp1) and oxidative stress associated proteins such as antioxidant enzymes mn superoxide dismutase and peroxiredoxin-3 (similar to antioxidant protein isoform 2) were found to be up-regulated post dhbv infection. hsp70 assists folding of many newly synthesized polypeptides, and refolding of the proteins misfolded [41, 42] . hsp70 can enhance flock house virus replication [43, 44] . hsp70 and hsp90 participate in dengue virus entry as a receptor complex [45] . moreover, hbv p protein activation in vitro is fundamentally dependent on heat shock protein 70 family hsc70/hsp40 [46] . in hbv-replicating hepad38 cell, expressions of heat shock proteins (hsp70 and hsp90) and mn superoxide dismutase increase, after hbv replication induced by tetracycline [47] . in humanized transgenic mice, inhibition of hbv replication results in suppression of mn superoxide dismutase expression in hepatocytes [47, 48] . it suggests that oxidative stress can be induced by hepadnavirus replication as epstein-barr virus [49] . annexin a2, belongs to a family of calcium-dependent, phospholipid binding proteins, is involved in many biological processes, such as the ca 2+ dependent exocytosis, calcium transport and cell proliferation. it participates in viral infection, including assisting in the assembly of hiv in monocyte-derived macrophages [50] , as a cellular cofactor supporting hiv-1 infection [51] , enhancing cytomegalovirus binding and membrane fusion [52] and supporting the replication of influenza viruses by mediating activation of plasminogen [53] . it has been reported that hbv polymerase activity was inhibited by interacted with s100a10, a protein binding to annexin a2 [54] . in hepg2.2.15 compared with hepg2, annexin a2 was revealed down-regulated [55] , which was consistent with our observation in dhbv-pdhs model and confirmed by western blot analysis. it indicated that annexin a2 may involve in hepadnavirus infection and warrants further investigation. beta-actin and gapdh are usually referred as the internal standards for detections of rna transcription and protein expression of genes. however, those proteins were found to be down-regulated post dhbv infection by 2-de analysis. recently, accumulated evidence showed that in hbv-related hepatocellular carcinoma or viral infections, beta-actin and gapdh are unsuitable controls in quantitative mrna expression or western blot analysis due to variations in expression [56] [57] [58] [59] [60] , though there are controversial observations [61] . these findings therefore highlight the importance of re-evaluating the housekeeping genes whose expressions may be affected by hepadnavirus infection. in summary, the present study explored global changes in cellular protein expression of hepadnavirus infection by 2-de analysis, using a natural dhbv-pdhs infection system. forty-two differentially expressed proteins in dhbv infected pdhs have been identified by ms/ms. most of them involve in carbohydrate metabolism, amino acid metabolism, stress responses and cytoskeleton processes including alpha-enolase, beta-actin, lamin a and annexin a2. it suggests that those proteins may play important roles in hepadnavirus infection. differential expressions of annexin a2 and beta-actin were confirmed by western blot analysis. further investigation of the roles of the differentially expressed cellular proteins will help to understand cellular and molecular mechanisms of hepadnavirus infection. cherry valley ducks (anas platyrhynchos) were purchased from breeding center of shanghai institute of veterinary medical sciences, china. animal protocols were approved by the ethics committee of fudan university. three-day-old ducklings with no congenital dhbv infection detected by pcr (sence: 5'-ctcactttgtg-gatctcattg-3', antisense: 5'-atcggatagtcg-ggttgg-3'), were used for pdhs cultures. duck hepatocytes were isolated with in situ liver perfusion method with modifications. after the duck was anesthetized with approximately 0.3 ml of 0.75% pentobarbital sodium, the liver was perfused via portal vein with prewarmed liver perfusion medium (gibco laboratories), then the inferior vena cava was cut to effuse the buffer liquid when the liver was engorged. at first, perfusion was maintained at 15 to 20 ml per minute with 100 ml of liver perfusion medium until the liver became blanch, then the liver became soft followed by 50 ml digestion buffer with 1 μg/ml of collagenase type iv (sigma) in l15 medium (gibco). after the perfusion, the gallbladder was removed and hepatocytes were dispersed in l-15 medium. hepatocytes were filtered through sterilized gauze, centrifuged at 40 g for 4 min, and washed three times with hepatocyte wash medium (gibco). then 4 × 10 6 hepatocytes were seeded onto 100-mm-diameter dish and incubated in l-15 medium containing 5% fbs (gibco laboratories), 15 mm hepes, 100 u penicillin per liter, 100 mg of streptomycin per liter, 1 mg of insulin (sigma) per liter and 10 -5 m hydrocortisone-hemisuccinate (sigma) at 37°c. the medium was changed every day. infectious dhbv were produced by lmh-d2 cell line which carries a stably integrated dhbv dimer and constitutively secretes dhbv virions (generous gift of william mason, fox chase cancer center, usa) [62] . dhbv particles were obtained from lmh-d2 cells by ultracentrifugation, and the virus pellet was suspended in pbs with 10% glycerol. dhbv was quantified using real time pcr as a titer of 2 × 10 9 copies per milliliter. pdhs cultured 16 h after plating were infected with purified dhbv viral particles at moi of 30, and incubated at 37°c overnight. then, they were washed with pbs three times and cultured for 12, 24, 72 and 120 h in l15 medium supplemented with 5% fbs. the efficiency of dhbv infection in pdhs was determined by indirect immunofluorescence and southern blot hybridization. monolayers of pdhs grown on glass coverslips were fixed directly by adding 4% polyoxymethylene at room temperature for 20 min, washed twice with pbs and preincubated with 3% bovine serum albumin for 30 min. after incubation with a 1:100 dilution of monoclonal mouse anti-dhbv pres (generous gift of john c. pugh and william mason, fox chase cancer center, usa) at 37°c for 60 min, the cells were washed three times with pbs, subsequently incubated with a 1:200 dilution of fitc-conjugated sheep anti-mouse igg (gghl-90f, immunology consultants laboratory) at 37°c for 30 min. cell nucleus were stained with 1 μg/ml 4',6'-diamidino-2-phenylindole (dapi, sigma) and mounted in 50% glycerol in pbs. efficiency of dhbv infection was observed by the confocal laser scanning microscope (leica). to detect dhbv replication, intrac-ellular dna was extracted from dhbv infected or uninfected pdhs. forty micrograms of dna from each sample was separated on a 1.5% agarose gel, and analyzed by southern blot hybridization with an alpha-32 p-dctp labeled dhbv-specific probe for detection as described previously [63] . the dhbv-infected and control (uninfected) pdhs at 24, 72, and 120 h post-infection were washed three times with ice-cold pbs before harvesting and stored at -80°c. pdhs for dhbv infection or the uninfected controls were from the same duck in order to avoid individual differences. approximately 2 × 10 7 cells were lysed in 1 ml lysis buffer (7 m urea, 2 m thiourea, 2% (w/v) chaps, 50 mm dithiothreitol (dtt), 2% (v/v) ph 3-10 nonlinear immobilized ph gradient (ipg) buffer (amersham biosciences) containing 1% protease inhibitor cocktail (roche) and 1 mm pmsf (sigma)), then sonicated on ice for 12 cycles, each consisting of 5 s pulse and 10 s pause. after centrifugation at 20,000 g at 4°c for 1 h, the supernatants of lysates were divided into aliquots and the protein concentrations were determined by the bradford assay. then, aliquots were stored at -80°c for further analysis. the 2-de gels were performed using 24-cm ipg strips (ph 3-10, nonlinear, ge healthcare) in ettan ipgphor isoelectric focusing system (amersham biosciences) plus ettan-dalt six system (amersham biosciences) according to the manufacturer's instructions. to compensate the variability of gel electrophoresis, at least three replicate gels were performed for each group. in the first dimensional isoelectric focusing (ief), 120 μg proteins of each sample were diluted to 450 μl with rehydration buffer containing 8 m urea, 2% (w/v) chaps, 50 mm dtt, 0.5% (v/v) ampholyte (ph 3-10, nonlinear, amersham biosciences), and ipg strips were allowed to rehydrate in the above solution under mineral oil. ief was performed as follow: 30 v for 6 h (active rehydration); 60 v for 6 h (active rehydration); 500 v for 2 h, rapid; 1,000 v for 2 h, rapid; 4,000 v for 2 h, linear; linear ramping to 8,000 v for 2 h, and finally 8,000 v for about 7 h with a total of 64 kvh at 20°c. then the ipg strips were incubated in equilibration buffer (75 mm tris-hcl (ph 8.8), 6 m urea, 29.3% (v/v) glycerol, 2% (w/v) sds and 0.002% (w/v) bromophenol blue) containing 2% (w/v) dtt for 15 min with gentle agitation, followed by incubation in the same equilibration buffer supplemented with 2.5% (w/v) iodoacetamide for 15 min at room temperature. the second dimension sds-page was performed on 1 mm thick 12.5% polyacrilamide vertical gels in ettan-dalt six system using 5 w/gel for 30 min, and followed by 12 w/gel at 10°c until the bromophenol blue dye front reached the end of the gels. the gels were stained by a modified silver staining method compatible with ms analysis [64] and scanned at 300 dpi (dots/inch) using imagescanner (umax, amersham biosciences). images were captured and analyzed by imagemaster 2d platinum 6.0 software (amersham biosciences). the percentage of the volume of the spots representing a certain protein was determined in comparison with the total proteins present in the 2-de gel. to select differentially expressed protein spots, quantitative analysis was performed using the student's t-test to compare the percentage volumes of spots between dhbv infected and uninfected groups at three time points. the differentially expressed protein spots with p-values less than 0.05 were considered as significant differences, and at least 1.5 fold difference in percentage of the volume for each spot was set as a threshold. these protein spots were selected and subjected to in-gel tryptic digestion and identification by ms. the differentially expressed protein spots were manually excised from the sliver-stained gels (each gel of 120 μg protein) and placed into a 96-well microplate. the gel pieces were destained with a solution of 15 mm potassium ferricyanide and 50 mm sodium thiosulfate (1:1) at room temperature for 10 min, then washed twice with deionized water, each for 30 min, and dehydrated in 80 μl of acetonitrile (acn) twice. then the samples were swollen in a digestion buffer containing 25 mm nh 4 hco 3 and 12.5 ng/μl trypsin (promega) at 4°c after 30 min incubation, and incubated at 37°c for more than 12 h. the peptide mixtures from the gel were extracted twice using 0.1% trifluoroacetic/50% acn at room temperature, re-suspended with 0.7 μl matrix solution (α-cyano-4-hydroxy-cinnamic acid (sigma) in 0.1% trifluoroacetic, 50% acn), allowed to dry in air under the protection of n2. the peptide mixtures from samples were analyzed by 4700 maldi-tof/tof proteomics analyzer (applied biosystems). the uv laser was operated at a 200 hz repetition rate with wavelength of 355 nm. the accelerated voltage was operated at 20 kv. myoglobin digested by trypsin was used to calibrate the mass instrument with internal calibration mode. all acquired spectra of samples were processed using 4700 series explore software (applied biosystems) in a default mode. the parent mass peaks with mass range 700-3200 da and minimum s/n 20 were picked out for tandem tof/tof analysis. combined ms and ms/ms spectra were submitted to mas-cot (v2.1, matrix science) by gps explorer software (v3.6, applied biosystems) and searched with the following parameters: ncbinr database (release date: 2009.11), taxonomy of bony vertebrates or viruses, trypsin digest with one missing cleavage, none fixed modifications, ms tolerance of 100 ppm, ms/ms tolerance of 0.6 da and possible oxidation of methionine. known contaminant ions (human keratin and tryptic autodigest peptides, etc.) were excluded. mascot protein scores (based on ms and ms/ms spectra) with greater than 72 were considered statistically significant (p < 0.05). the individual ms/ms spectrum with statistically significant (confidence interval >95%) and best ion score (based on ms/ ms spectra) was accepted. to eliminate the redundancy of proteins that appeared in the database under different names and accession numbers, the protein belonging to the species anas platyrhynchos or with the highest protein score (top rank) was singled out. duck cdna of annexin a2 was amplified by reverse transcription-pcr with the primers designed according to the sequence of the chicken annexin a2 (genbank accession no. gi|45382533, sense: 5'-ccgctcgaggtc-ctctccaccacacaggt-3' and antisense: 5'-ccg ctcgaggtcctctccaccacacaggt-3'). the fulllength of duck annexin a2 amplified from pdhs, was cloned into prokaryotic expression plasmid pet-28a (novagen). recombinant annexin a2 with c-terminal fusion his-tag was induced by iptg, and purified by ni-nta affinity chromatography (qiagen). balb/c mice were immunized by the purified recombinant duck annexin a2 with freund's complete adjuvant (sigma). the serum was collected 2 weeks following the final injection and the levels of anti-duck-annexin a2 antibody titers from immunized mice were determined by western blot. differential expression of duck beta-actin, annexin a2, hsp70, destrin and lamin a were confirmed by western blot analysis. the primary antibodies for detection were as follow: a monoclonal antibody against beta-actin (sigma), rabbit anti-human lamin a polyclonal antibody (santa cruz biotechnology and proteintech), rabbit antihuman hsp70 polyclonal antibody (santa cruz biotechnology), rabbit hsp70 polyclonal antibody (bios), rabbit anti-human destrin polyclonal antibody (protein-teck) and mouse anti-duck-annexin a2 polyclonal antibody prepared in our laboratory described as above. thirty microgram proteins from each sample were separated in 12% sds-page gels and transferred to pvdf membranes using the transfer system (biorad). the blots were blocked with 5% nonfat milk for 2 h at room temperature and incubated at 4°c overnight with 1:200-1:500 dilution of primary antibody. the blots were then washed four times with pbs containing 0.1% tween-20, and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (santa cruz biotechnology) 1 hour at room temperature. after washed four times with pbs containing 0.1% tween-20, the bands were developed with ecl detection reagent (pierce). the same amount protein of each sample was applied to a parallel sds-page gel and stained with coomassie brilliant blue. hepatitis b virus immunopathogenesis hepatitis b virus infection--natural history and clinical consequences in vitro experimental infection of primary human hepatocytes with hepatitis b virus infection of a human hepatoma cell line by hepatitis b virus initiation of hepatitis b virus genome replication and production of infectious virus following delivery in hepg2 cells by novel recombinant baculovirus vector persistence of the hepatitis b virus covalently closed circular dna in heparg human hepatocyte-like cells age-related differences in amplification of covalently closed circular dna at early times after duck hepatitis b virus infection of ducks treatment of chronic viral hepatitis in woodchucks by prolonged intrahepatic expression of interleukin-12 proteome analysis of cultivar-specific deregulations of oryza sativa indica and o. sativa japonica cellular suspensions undergoing rice yellow mottle virus infection identification of epstein-barr virus (ebv) nuclear antigen 2 (ebna2) target proteins by proteome analysis: activation of ebna2 in conditionally immortalized b cells reflects early events after infection of primary b cells by ebv proteomic analysis of tobacco mosaic virus-infected tomato (lycopersicon esculentum m.) fruits and detection of viral coat protein identification of cellular proteins modified in response to african swine fever virus infection by proteomics proteomic analysis of cellular protein alterations using a hepatitis b virus-producing cellular model proteomic analysis of heparg cells: a novel cell line that supports hepatitis b virus infection proteomic analysis of hepatitis b surface antigen positive transgenic mouse liver and decrease of cyclophilin a duck hepatitis b virus: an invaluable model system for hbv infection infection and uptake of duck hepatitis b virus by duck hepatocytes maintained in the presence of dimethyl sulfoxide temporal proteome and lipidome profiles reveal hepatitis c virus-associated reprogramming of hepatocellular metabolism and bioenergetics gene expression in primate liver during viral hemorrhagic fever cellular proteins in influenza virus particles quantitative proteomic analysis of lentiviral vectors using 2-de identification of glyceraldehyde-3-phosphate dehydrogenase as a cellular protein that binds to the hepatitis b virus posttranscriptional regulatory element phosphorylation of the hepatitis b virus core protein by glyceraldehyde-3-phosphate dehydrogenase protein kinase activity protein kinase and nostimulated adp-ribosyltransferase activities associated with glyceraldehyde-3-phosphate dehydrogenase isolated from human liver almeras l: identification of cellular proteome modifications in response to west nile virus infection proteomics analysis of host cells infected with infectious bursal disease virus quantitative analysis of severe acute respiratory syndrome (sars)-associated coronavirus-infected cells using proteomic approaches: implications for cellular responses to virus infection itinerary of hepatitis b viruses: delineation of restriction points critical for infectious entry actin in transcription and transcription regulation actin-based motility of vaccinia virus local actin polymerization and dynamin recruitment in sv40-induced internalization of caveolae cellular motility driven by assembly and disassembly of actin filaments adenovirus endocytosis requires actin cytoskeleton reorganization mediated by rho family gtpases establishment of a functional human immunodeficiency virus type 1 (hiv-1) reverse transcription complex involves the cytoskeleton sfv infection in cho cells: cell-type specific restrictions to productive virus entry at the cell surface escape of herpesviruses from the nucleus us3 of herpes simplex virus type 1 encodes a promiscuous protein kinase that phosphorylates and alters localization of lamin a/c in infected cells effects of lamin a/c, lamin b1, and viral us3 kinase activity on viral infectivity, virion egress, and the targeting of herpes simplex virus u(l)34-encoded protein to the inner nuclear membrane epstein-barr virus bglf4 kinase induces disassembly of the nuclear lamina to facilitate virion production glutamine metabolism is essential for human cytomegalovirus infection molecular chaperones in the cytosol: from nascent chain to folded protein hsp70 chaperones: cellular functions and molecular mechanism proteomics analysis of the tombusvirus replicase: hsp70 molecular chaperone is associated with the replicase and enhances viral rna replication the cellular chaperone heat shock protein 90 facilitates flock house virus rna replication in drosophila cells heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells efficient hsp90-independent in vitro activation by hsc70 and hsp40 of duck hepatitis b virus reverse transcriptase, an assumed hsp90 client protein hepatitis b virus replication causes oxidative stress in hepad38 liver cells betulinic acid-mediated inhibitory effect on hepatitis b virus by suppression of manganese superoxide dismutase expression epstein-barr virus induces an oxidative stress during the early stages of infection in b lymphocytes, epithelial, and lymphoblastoid cell lines annexin 2: a novel human immunodeficiency virus type 1 gag binding protein involved in replication in monocyte-derived macrophages secretory leukocyte protease inhibitor binds to annexin ii, a cofactor for macrophage hiv-1 infection annexin ii enhances cytomegalovirus binding and fusion to phospholipid membranes annexin ii incorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin association of hepatitis b virus polymerase with promyelocytic leukemia nuclear bodies mediated by the s100 family protein p11 proteome responses to stable hepatitis b virus transfection and following interferon alpha treatment in human liver cell line hepg2 suitable reference genes for real-time pcr in human hbv-related hepatocellular carcinoma with different clinical prognoses selection of reference genes for real-time pcr in human hepatocellular carcinoma tissues validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative rt-pcr determination of suitable housekeeping genes for normalisation of quantitative real time pcr analysis of cells infected with human immunodeficiency virus and herpes viruses reference gene selection for quantitative real-time pcr analysis in virus infected cells: sars corona virus, yellow fever virus, human herpesvirus-6, camelpox virus and cytomegalovirus infections a protein-based set of reference markers for liver tissues and hepatocellular carcinoma efficient duck hepatitis b virus production by an avian liver tumor cell line rapid resolution of duck hepatitis b virus infections occurs after massive hepatocellular involvement pharmacia a: a modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry proteomic analysis of primary duck hepatocytes infected with duck hepatitis b virus proteome science we would like to thank professor alastair i. h. murchie for careful reading and correcting the english of the revised manuscript. this work was supported by national natural science foundation of china (30670092, j0730860) and the program of ministry of science and technology of china (2010dfa32100, the authors declare that they have no competing interests.authors' contributions dq was responsible for the conception and design of the study. yz were responsible for pdhs culture, virus infection and sample preparation. hb and sq carried out the 2-de gels experiments, image analysis and excised the protein spots. xz performed the mass spectrometric analyses. yz and ly confirmed the differential expression by western blot. yz and hb carried out the analysis and interpretation of data. dq, yz and hb wrote the manuscript. bx, sz, ql, ry, tz, py have been involved in drafting the manuscript or revising it critically for important content. all authors read and approved the final manuscript. key: cord-263744-zrzwhu0j authors: lin, sheng-wen; shen, ching-fen; cheng, chao-min title: potential trends of point-of-care diagnostics—the next generation of the laboratory diagnosis date: 2020-09-30 journal: diagnostics (basel) doi: 10.3390/diagnostics10100774 sha: doc_id: 263744 cord_uid: zrzwhu0j with the current worldwide outbreak of covid-19, developing rapid, effective, and convenient detection tools has become imperative [...]. current diagnostic methods for examining male infertility primarily rely on microscope-based examination and computer-assisted semen analysis (casa) systems to process and examine human semen [1] . although standard methods could provide a holistic approach for determining sperm quality, such approaches require bulky and costly microscopes, equipment, and well-trained technicians. moreover, these requirements are complicated, expensive, often costing hundreds of us dollars, and time intensive. they also limit accessibility to semen analysis in resource-limited areas and push the process into the category of non-routine health examinations. to meet the need for a fast, convenient, and easy-to-operate semen analysis tool, biochemical analysis might be a suitable diagnostic approach. a novel, extremely inexpensive, and easy-to-use alternative to conventional methodology can be found in the form of a paper-based diagnostic device capable of detecting total motile sperm concentration (tmsc) based on the mitochondrial activity of motile spermatozoa [2] (figure 1 ). this tool can be used to distinguish low tmsc semen samples from those with normal tmsc levels. the activity and function of mitochondria are key to sperm function and play a key role in many sperm functions including basic motility, acrosome reaction, and final fertilization [3] . moreover, the activity of succinate dehydrogenase (sdh), one of the mitochondrial respiratory chain enzymes, was found to be highly correlated with sperm quality, including sperm concentration, motility, and vitality [4] . this paper-based diagnostic device is used to assess mitochondrial activity of sperm based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt). mtt is a yellow tetrazolium salt that can be reduced by sdh, in the mitochondria of metabolically active cells, to purple-colored formazan. the amount of formazan is proportional to the metabolic activity of living cells, suggesting that mtt can be used as a powerful tool for providing basic information about sperm quality. preliminary result in the clinic or at bedside. results could be used to expedite follow-up care protocols. similar biochemical analysis tools can be developed to examine urine, tears, sweat, etc. these simple and easy-to-use diagnostic kits will be able to help clinicians and individuals obtain results more quickly and effectively. bacterial and viral infections are the most common reasons children receive medical care in clinics or small hospitals. as patients with bacterial and viral infections exhibit similar symptoms, symptom-based tests or a medical history review are often insufficient for distinguishing between bacterial and viral infections, even among experienced doctors. although treatment options for bacterial and viral infections differ substantially, children are often treated with empirical antibiotics because of the lack of rapid and accurate testing. this can lead to delayed diagnosis and antibiotic abuse, increasing the incidence of adverse events due to antibiotics, antibiotic resistance, and other potential confounding issues. current biochemical analyses based on biomarkers, such as procalcitonin (pct) and c-reactive protein (crp) methods, can provide some clues for the differential diagnosis of bacterial and viral infections, but their low sensitivity and specificity means that they cannot be relied upon in and of themselves for treatment. moreover, bacteria-virus interactions and their relationship to biofilm formation make pathogen diagnosis more challenging. both of these issues underscore the need for the development and use of poc diagnostics using host biomarkers to distinguish between bacterial and viral infections in pediatric patients. such an approach allows for more careful selection of appropriate therapies. we have discussed the use of several biomarkers, including tnf (tumor necrosis factor)-related apoptosis-inducing ligand (trail), interferon γ-induced protein 10 (ip-10) and family with sequence similarity 89 member a (fam89a) to analyze the urine of children. we preliminarily found a significant difference in the concentration of ip-10 among children with viral infections compared to children with bacterial infections (figure 2 ). from our preliminary data, it can be known that ip-10 compared with image detection, liquid biopsy could be more convenient and practical. it is not limited by location, instrument, time, or personnel. although liquid biopsy is relatively less likely to provide accurate results compared to image detection, it may be used to help doctors obtain a rapid, preliminary result in the clinic or at bedside. results could be used to expedite follow-up care protocols. similar biochemical analysis tools can be developed to examine urine, tears, sweat, etc. these simple and easy-to-use diagnostic kits will be able to help clinicians and individuals obtain results more quickly and effectively. bacterial and viral infections are the most common reasons children receive medical care in clinics or small hospitals. as patients with bacterial and viral infections exhibit similar symptoms, symptom-based tests or a medical history review are often insufficient for distinguishing between bacterial and viral infections, even among experienced doctors. although treatment options for bacterial and viral infections differ substantially, children are often treated with empirical antibiotics because of the lack of rapid and accurate testing. this can lead to delayed diagnosis and antibiotic abuse, increasing the incidence of adverse events due to antibiotics, antibiotic resistance, and other potential confounding issues. current biochemical analyses based on biomarkers, such as procalcitonin (pct) and c-reactive protein (crp) methods, can provide some clues for the differential diagnosis of bacterial and viral infections, but their low sensitivity and specificity means that they cannot be relied upon in and of themselves for treatment. moreover, bacteria-virus interactions and their relationship to biofilm formation make pathogen diagnosis more challenging. both of these issues underscore the need for the development and use of poc diagnostics using host biomarkers to distinguish between bacterial diagnostics 2020, 10, 774 3 of 4 and viral infections in pediatric patients. such an approach allows for more careful selection of appropriate therapies. we have discussed the use of several biomarkers, including tnf (tumor necrosis factor)-related apoptosis-inducing ligand (trail), interferon γ-induced protein 10 (ip-10) and family with sequence similarity 89 member a (fam89a) to analyze the urine of children. we preliminarily found a significant difference in the concentration of ip-10 among children with viral infections compared to children with bacterial infections (figure 2 ). from our preliminary data, it can be known that ip-10 in urine could be used to differentiate the infection mode, viral or bacterial, in children, and these data can potentially be used to guide clinical treatment, once its sensitivity and specificity in urine are defined. in urine could be used to differentiate the infection mode, viral or bacterial, in children, and these data can potentially be used to guide clinical treatment, once its sensitivity and specificity in urine are defined. the development and discovery of new biomarkers to gauge health status is ongoing. several biomarkers have recently demonstrated potential value for use as bacterial infection markers, including il-27, cd35, cd64, and presepsin [5] [6] [7] . while most studies have only detected biomarkers in blood samples, non-invasive samples such as nasopharyngeal aspirates, saliva, or urine may also be used to detect infection. non-invasive sample analysis provides several advantages; it is painless, enables self-collection, and offers superior safety for both patient and doctor. however, the use of non-invasive samples, such as urine and saliva, is usually limited by fluctuations in sample flow. this directly affects the sensitivity and specificity of detection. if this problem can be overcome, analysis of biomarkers within non-invasive samples could become a valuable approach for diagnosis and subsequent treatment of infants and children and will facilitate the development of easy-to-use poc. as the number of confirmed cases of covid-19 increases rapidly around the world, we know that conventional testing may become insufficient. we need more efficient, rapid diagnostic methods to maintain a sustainable medical ecosystem. in the future, we expect that testing by in non-invasive samples and the development of new biomarkers will lead to more reliable and convenient testing methods that will reduce transport requirements, the risk of infection transmission, stress on healthcare systems, and healthcare costs for both individuals and governments. the development and discovery of new biomarkers to gauge health status is ongoing. several biomarkers have recently demonstrated potential value for use as bacterial infection markers, including il-27, cd35, cd64, and presepsin [5] [6] [7] . while most studies have only detected biomarkers in blood samples, non-invasive samples such as nasopharyngeal aspirates, saliva, or urine may also be used to detect infection. non-invasive sample analysis provides several advantages; it is painless, enables self-collection, and offers superior safety for both patient and doctor. however, the use of non-invasive samples, such as urine and saliva, is usually limited by fluctuations in sample flow. this directly affects the sensitivity and specificity of detection. if this problem can be overcome, analysis of biomarkers within non-invasive samples could become a valuable approach for diagnosis and subsequent treatment of infants and children and will facilitate the development of easy-to-use poc. as the number of confirmed cases of covid-19 increases rapidly around the world, we know that conventional testing may become insufficient. we need more efficient, rapid diagnostic methods to maintain a sustainable medical ecosystem. in the future, we expect that testing by in non-invasive samples and the development of new biomarkers will lead to more reliable and convenient testing methods that will reduce transport requirements, the risk of infection transmission, stress on healthcare systems, and healthcare costs for both individuals and governments. world health organization. who laboratory manual for the examination and processing of human semen world health organization. who laboratory manual for the examination and processing of human semen point-of-care semen analysis of patients with infertility via smartphone and colorimetric paper-based diagnostic device the sperm mitochondrion: organelle of many functions seminal quality correlates with mitochondrial functionality presepsin values as markers of severity of sepsis interleukin-27: a novel biomarker in predicting bacterial infection among the critically ill combined use of biomarkers for distinguishing between bacterial and viral etiologies in pediatric lower respiratory tract infections the authors declare no conflict of interest. key: cord-285148-bch7814v authors: singanayagam, aran; joshi, priya v; mallia, patrick; johnston, sebastian l title: viruses exacerbating chronic pulmonary disease: the role of immune modulation date: 2012-03-15 journal: bmc med doi: 10.1186/1741-7015-10-27 sha: doc_id: 285148 cord_uid: bch7814v chronic pulmonary diseases are a major cause of morbidity and mortality and their impact is expected to increase in the future. respiratory viruses are the most common cause of acute respiratory infections and it is increasingly recognized that respiratory viruses are a major cause of acute exacerbations of chronic pulmonary diseases such as asthma, chronic obstructive pulmonary disease and cystic fibrosis. there is now increasing evidence that the host response to virus infection is dysregulated in these diseases and a better understanding of the mechanisms of abnormal immune responses has the potential to lead to the development of new therapies for virus-induced exacerbations. the aim of this article is to review the current knowledge regarding the role of viruses and immune modulation in chronic pulmonary diseases and discuss avenues for future research and therapeutic implications. chronic diseases are the leading cause of death worldwide and the third most common group of chronic diseases are chronic pulmonary diseases that account for an estimated four million deaths annually [1] . the most prevalent diseases of the respiratory tract are chronic obstructive pulmonary disease (copd), asthma, tuberculosis and lung cancer, and the most common genetic disease is cystic fibrosis (cf). copd is estimated to be the fourth leading cause of mortality by 2030 [2] and an estimated 300 million people suffer from asthma. copd, asthma and cf are all chronic inflammatory conditions but their etiology and pathogenesis differ markedly. copd and asthma are believed to be caused by exposure to relevant environmental agents (mainly cigarette smoke and aeroallergens, respectively) in patients with a susceptible genetic background, whereas cf is caused by mutations in the cf transmembrane regulator gene. the typical clinical course of these conditions is of chronic symptoms that are punctuated by periods of increased symptoms termed 'acute exacerbations'. acute exacerbations are now recognized to be significant events in the course of the disease and have enormous implications for patients, their caregivers and for healthcare providers. exacerbations accelerate disease progression, impair quality of life, cause significant morbidity for patients and are the major cause of mortality. in addition they are the major drivers of excess healthcare costs as they often result in unscheduled healthcare visits, treatment costs and above all hospitalizations. therefore, preventing exacerbations is a major therapeutic goal in all three diseases and one that has not been achieved with currently available treatments. despite the differences between copd, asthma and cf, all three have in common that respiratory virus infections are a major trigger of acute exacerbations. an important mechanism underlying this may be impaired host immune responses to virus infection and a better understanding of these mechanisms has the potential to lead to the development of new therapies that may be beneficial in different chronic pulmonary diseases. the aim of this article is to review the current knowledge regarding the role of viruses and host immune responses in asthma, copd and cf, and discuss avenues for future research and therapeutic interventions. although this article primarily focuses on acute exacerbations of chronic respiratory diseases, virus infection has also been implicated in the induction of asthma. asthma is strongly related to a genetic predisposition to develop allergic reactions to aeroallergens. however, not all individuals with atopy develop asthma and, therefore, it has been proposed that other environmental factors may act as 'triggers' to the development of asthma in genetically susceptible individuals. one such factor that has attracted much research interest is respiratory virus infections, in particular infection with respiratory syncytial virus (rsv). in the majority of cases rsv causes a self-limiting upper respiratory tract infection, but in infants under the age of one year it can cause a more serious infection of the lower respiratory tract -bronchiolitis -and studies have linked rsv bronchiolitis with an increased frequency of subsequent wheezing and asthma [3] . recently, it has been reported that rhinovirus (rv) infection is also related to the development of asthma [4] . however, these studies are unable to ascertain the direction of the relationship between viral infections and asthma, that is, whether infections cause asthma or infections occur more frequently in individuals predisposed to asthma. recent evidence has emerged supporting the later hypothesis. a study using data on hospitalization due to rsv infection for all twins born in denmark between 1994 and 2000 found that rsv hospitalization and asthma were positively associated but that a model in which asthma 'causes' rsv hospitalization fitted the data significantly better than a model in which rsv hospitalization 'causes' asthma [5] . a study of the temporal relationship between sensitization to aeroallergens and viral wheeze showed that allergic sensitization led to an increased risk of wheezing illnesses but viral wheeze did not lead to increased risk of subsequent allergic sensitization [6] . therefore, the link between asthma and virus infection may be due to genetically determined alterations in airway or immune responses that predispose infants both to infection and asthma, rather than virus infections causing asthma [7] . this will be discussed later in light of recent developments regarding innate immune responses in asthma but it is clear that the relationship between respiratory virus infections and the induction of asthma is complex and requires further study. asthma is the most common chronic respiratory disease affecting up to 10% of adults and 30% of children in the western world [8] . the global initiative for asthma (gina) defines asthma as 'a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. the chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. these episodes are usually associated with widespread, but variable, airflow obstruction within the lung that is often reversible either spontaneously or with treatment'. this definition refers to the key physiological marker of asthma -reversible airflow obstruction, and the key pathological characteristic -airways inflammation. the characteristic pattern of inflammation of allergic diseases and also in asthma involves eosinophils, mast cells and t helper 2 lymphocytes (th2) and a wide range of inflammatory mediators. asthma exacerbations are episodes characterized by progressive increase in shortness of breath, cough, wheezing and chest tightness, or some combination of these, and increased airflow obstruction that is manifested by reductions in measurements of lung function such as peak expiratory flow (pef). acute exacerbations are a common occurrence in asthma and the social and economic burden of asthma exacerbations is substantial, due to both the direct costs of healthcare utilization and the indirect costs associated with lost productivity. current therapies for asthma consist of bronchodilator and antiinflammatory medications, the mainstay of which are inhaled β 2 -agonists and inhaled corticosteroids, respectively. these are highly effective in relieving symptoms and reduce exacerbations by approximately 50% in clinical trials [9] . however, in 'real life' surveys of asthmatics a significant proportion of patients continue to experience acute exacerbations despite therapy and, therefore, prevention/treatment of exacerbations remains a major unmet clinical need in asthma [10] [11] [12] . it has long been recognized that viral respiratory tract infections are triggers for exacerbations of asthma in both adults and children but early studies reported low detection rates of viruses in asthma exacerbations casting doubt on this association. the development of highly sensitive and specific molecular diagnostic techniques using polymerase chain reaction (pcr) technology led to a reappraisal of the role of virus infections in asthma. studies using pcr detected viruses in approximately 80% to 85% of asthma exacerbations in school-aged children and 60% to 80% of exacerbations in adults. although respiratory virus infection can be detected in stable asthma patients detection rates are consistently lower than in exacerbated patients [13, 14] . therefore, these studies suggest that the majority of asthma exacerbations are associated with respiratory virus infections and that the low detection rates in earlier studies were a consequence of diagnostic methods with a low sensitivity. the most common viruses detected in these studies were rv. rvs are members of the picornaviridae family and are the most common cause for the common cold in both children and adults. more than 100 serotypes exist. virus typing classified rvs into rv-a and rv-b groups based on susceptibility to anti-viral drugs and on genetic sequence similarity. more recently a newly identified group termed rv-c has been identified based purely on sequencing data [15] . other respiratory viruses have been detected in subjects with asthma exacerbations including influenza, rsv, coronaviruses, human metapneumoviruses, parainfluenza viruses (piv) and adenoviruses. however, in a recent study in children the only virus type significantly associated with asthma exacerbations was rv [16] . the risk of exacerbation following virus infection is influenced by other factors such as allergy and environmental pollution. allergen sensitization, exposure to sensitizing allergens, and respiratory virus infection act in a synergistic manner to significantly increase the risk of hospitalization with acute asthma in both adults [17] and children [18] . the presence of high ambient levels of nitric oxide(no) is also associated with an increased risk of exacerbation following rv infection [19] . following discovery of the role of rv in asthma exacerbations research attention has focused on the mechanisms of susceptibility to virus infection in asthmatics. rv infection in healthy individuals results in a predominantly upper respiratory symptom syndrome ('common cold'), whereas in asthmatics infection results in lower respiratory symptoms and airflow obstruction ('acute exacerbation'). a study of co-habiting partners discordant for the presence of asthma demonstrated that asthmatics do not have a higher frequency of rv infections but have more severe lower respiratory symptoms and changes in airway physiology [20] . similar results have been reported in experimental rv infection studies in asthmatics and nonasthmatic control subjects [21] . therefore, it would appear that the consequences of virus infection in asthmatics are more severe than in non-asthmatics. understanding the mechanisms underlying increased disease severity is crucial to developing new strategies to treat virus-induced exacerbations. most research into virus-induced asthma exacerbations has focused on rv as these are the most common viruses detected in asthma exacerbations and well-characterized models of rv infection exist both in vitro and in vivo. rvs primarily enter and replicate in epithelial cells in the respiratory tract and trigger a cascade of immune and inflammatory responses. following viral entry into a cell, uncoating of the virus leads to the release of viral rna that is recognized by pattern recognition receptors including toll-like receptors (tlr)-3, -7 and -8, and the cytosolic rna helicases, retinoic acid inducible gene i (rig-i) and melanoma differentiation-associated protein-5 (mda-5) [22, 23] . the interactions between ligand and receptor trigger signaling cascades ultimately resulting in the activation of transcription factors such as interferon regulatory factor (irf)-3 and-7, nuclear factor-b (nf-b) and activating transcription factor 2 (atf2). these activated transcription factors translocate to the nucleus and induce transcription of the type i interferons (ifn-α and -β) and pro-inflammatory cytokines including interleukin (il)-8/cxcl8, il-6, epithelial-derived neutrophilactivating peptide 78 (ena-78/cxcl5) and ifn-γinduced protein 10 kda (ip-10/cxcl10) [24] [25] [26] [27] [28] . ifn-α and -β have both a direct antiviral effect through inhibition of viral replication in cells and an indirect effect through stimulation of innate and adaptive immune responses. the direct antiviral activity of type i ifns is mediated by various mechanisms including blocking viral entry into cells, control of viral transcription, cleavage of rna and blocking translation. these effects are mediated through the up-regulation of interferon stimulated genes (isgs) and the production of antiviral proteins. the indirect antiviral effect is mediated through induction of natural killer cell cytotoxicity [29] , up-regulation of the expression of major histocompatibility complex 1 (mhc-1) on cells and up-regulation of co-stimulatory molecules on antigen-presenting cells. therefore, a robust interferon response is central to effective antiviral responses and resolution of virus infections. recently a novel class of interferons termed type iii interferons, or interferonlambda (ifn-λ) has been described. the type iii ifns consist of ifn-λ1, 2, 3 (respectively, il-29, il-28a and il-28b) [30] . the ifn-λs utilize a different receptor than ifn-α/β but appear to have functional similarities, however much more is known about the mechanism of action of ifn-α/β. the pro-inflammatory mediators and cytokines induced by rv infection lead to chemoattraction of inflammatory cells such as neutrophils, lymphocytes and eosinophils. this inflammatory response contributes to virus clearance but is also responsible for the pathology induced by rv infections. the balance between antiviral and inflammatory responses following virus infection is likely to determine the clinical outcome of the infection. an effective antiviral response rapidly controls viral replication with a minimal inflammatory response and limited clinical illness. if antiviral responses are inadequate this is likely to result in uncontrolled viral replication, greater inflammatory response and more severe clinical illness ( figure 1 ). the evidence that clinical illness following virus infection is more severe in asthmatics has stimulated research into possible dysregulation of antiviral and inflammatory responses in asthmatics. in 2005 wark et al. examined the kinetics of virus replication in bronchial epithelial cells obtained from asthmatics and healthy volunteers and reported that viral replication is increased in cells from asthmatics compared to non-asthmatic subjects [31] . this was the first report indicating that the innate immune response to virus infection may be impaired in asthma. furthermore, the authors demonstrated that production of ifnβ was impaired in asthmatics and administration of exogenous ifn-β resulted in restoration of a normal antiviral response, and this was confirmed in a subsequent study [32] . deficient ifn-β production by bronchial epithelial cells [33] , as well as deficient ifn-α production by peripheral blood mononuclear cells [34] [35] [36] and dendritic cells [37] has also been reported in asthma. our group has also shown that ifn-α and ifn-β production by alveolar macrophages is impaired in asthmatics (manuscript submitted). furthermore, deficient ifn-λ production by epithelial cells and alveolar macrophages in asthmatics has also been reported and related to clinical outcomes following experimental rv [38] . however, other groups have not reported deficient ifn induction in epithelial cells from asthmatics [39, 40] . in experimental rv infections virus loads were higher and virus shedding prolonged in asthmatics but this was not statistically significant [21, 41] . therefore, although interferon deficiency is an exciting new mechanism underlying increased severity of virus infection in asthma it has not been conclusively demonstrated to occur in all asthmatic subjects studied. the studies in question were small with different experimental conditions such as cell culture techniques and virus dose. it is also possible that interferon deficiency occurs in some asthmatics only and it may also be related to disease severity, disease control or degree of atopy. further studies with more subjects and careful patient selection and characterization are required to provide answers to these ongoing research questions. up-regulation of interferonstimulated genes if interferon production in response to virus infection is impaired in asthmatics what are the possible molecular mechanisms underlying this? the discovery that ifn-α, ifn-β and ifn-λ are all deficient suggests that it is not a genetic defect as ifn-α and ifn-β are on different genetic loci than ifn-λ. a key family of proteins regulating both interferon production and allergic inflammation are the suppressor of cytokine signaling family (socs), and one member of this family, socs1, is a potent negative regulator of type i and type ii interferons and of th2 inflammation [42, 43] . socs1 is induced by type ii cytokines such as il-13 [44] and, therefore, persistent th2 inflammation may result in chronic up-regulation of socs1 and impaired interferon responses, but this hypothesis requires further investigation. in vitro infection of airway epithelial cells with rv induces the production of inflammatory mediators and this has also been reported in vivo in both experimental and naturally-acquired viral infections. chemokines and cytokines such as il-8, il-6 and regulated on activation, normal t-cell expressed and secreted (rantes) have been detected during virus infections in asthmatic patients [45] [46] [47] [48] [49] . however it remains unclear whether the inflammatory response following virus infection differs quantitatively or qualitatively in asthmatics. one experimental rv infection study reported increased nasal lavage levels of il-8 and il-1β in asthmatics [46] but not in control subjects; however, another study reported no differences in il-6, il-8, il-11 and granulocyte-monocyte-colony stimulating factor (gm-csf) levels in either nasal lavage or sputum between asthmatics and nonasthmatics [45] . increased sputum levels of il-10 but not rantes or il-8 have been reported in asthmatics [48] . these conflicting results highlight the need for further studies evaluating the inflammatory profile (preferably in the lower airway) in well-characterized patients and nonasthmatic controls following virus infection. many of the inflammatory mediators produced are chemoattractants and, therefore, following virus infection inflammatory cells are recruited to the lungs. a number of different inflammatory cells have been identified in both naturally-occurring and experimental virus infections in asthma. although stable asthma is characterized by eosinophilic inflammation, a number of studies have identified neutrophils as the key inflammatory cell in virus-induced asthma exacerbations [21, [50] [51] [52] . neutrophils release bioactive mediators such as the protease neutrophil elastase that have effects such as stimulation of mucous production and, therefore, are likely key contributors to the pathogenesis of asthma exacerbations. another key cell involved in immune and inflammatory responses in the lungs is the macrophage. there is evidence that rvs can infect macrophages and that in asthmatics macrophage responses to virus infection are altered. our group has reported that rvs infect macrophages and induce tnf-α production [53] and that production of the cytokine il-15, that plays a key role in linking innate and adaptive antiviral immune responses and promoting t cell anti-viral immune responses, is impaired in asthmatics [53] . as described previously, macrophage production of ifns in response to virus infection is also impaired in asthma [38] . therefore, there is evidence of impaired antiviral responses in asthmatics in macrophages as well as respiratory epithelial cells. increased lymphocyte numbers in bronchoalveolar lavage (bal) and bronchial biopsies in experimental rv infection in asthmatics has been reported, with increases in cd4+, cd8+ and nk cells [21, 54] . abnormalities of the acquired immune system in stable asthma have been well described with skewing of acquired immune responses towards a th2 profile. as robust antiviral responses require an adequate th1 response it is possible that in diseases such as asthma with predominant th2 cells antiviral immunity is impaired. impaired levels of the th1 cytokines il-12, -15, -18 and ifn-γ have all been reported in asthma [21, [55] [56] [57] . in human experimental rv infection lower respiratory symptoms, bronchial hyperreactivity, reductions in blood total and cd8+ lymphocytes and virus load are related to deficient ifn-γ, il-12 or il-15 responses and to augmented il-4, il-5, and il-13 responses [21, 55] . sputum ifn-γ/il-5 messenger rna ratio following virus infection is inversely related to both peak cold symptoms and the time to viral clearance [58] . therefore, augmented th2 and deficient th1 immune responses are associated with greater clinical illness following rv in asthma. the identification of impaired innate immunity in asthma suggests a possible mechanism not only for virus-induced asthma exacerbations but also for the link between respiratory virus infections and the subsequent development of asthma. it is possible that infants who will develop asthma in later life have impaired immune responses from birth and, therefore, are more likely to develop more severe disease manifestations (for example, bronchiolitis) following respiratory virus infection. most studies to date have focused on the role of the acquired immune system and identified reduced ifn-γ production as a significant risk factor both for subsequent wheezing illness and allergic sensitization [59] [60] [61] . no studies have investigated innate immune responses in infants prior to the development of symptomatic asthma but impaired ifn-α production has been reported in older children with atopic asthma [35] . in conclusion there is evidence that both innate and acquired immune responses in asthmatics are impaired and this may be a key mechanism underlying virusinduced asthma exacerbations and the link between virus infections and the subsequent development of asthma in infants. further studies are needed to determine whether these deficiencies are common to all asthmatics, whether they represent a specific asthma phenotype and how they relate to conventional measures of asthma control. another important research question is whether new interventions targeting the interferon pathways can prevent asthma exacerbations and even potentially prevent the development of asthma in infants. chronic obstructive pulmonary disease (copd) is the most common chronic respiratory condition in adults. the global initiative for obstructive lung disease (gold), a collaboration between the world health organization and the national heart lung and blood institute, defines copd as 'a preventable and treatable disease with some significant extrapulmonary effects that may contribute to the severity in individual patients. its pulmonary component is characterized by airflow limitation that is not fully reversible. the airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lung to noxious inhaled particles or gases' [62] . the main etiological agents linked with copd are cigarette smoking and biomass exposure and the inflammatory response consists of neutrophils, macrophages and cd8+ t cells and, therefore, differs from the allergic inflammation seen in asthma. pulmonary inflammation is further amplified by oxidative stress and excess proteases released by inflammatory cells recruited to the lung. as in asthma, acute exacerbations are a common occurrence in copd and become more frequent as the disease progresses [63] . exacerbations are a major cause of morbidity, mortality and healthcare costs and accelerate decline in lung function [64] and quality of life [65] in copd patients. historically, bacterial infections have been considered the predominant infectious etiology, however epidemiological data showing a greater frequency of exacerbations in the winter months [66] and frequent coryzal symptoms preceding exacerbations suggest a causal role for viruses [67] . older studies using cell culture and serologic diagnostic tests detected viral infection in only approximately 10% to 20% of exacerbations [68, 69] . however, these diagnostic methods have low sensitivity for virus detection especially for rvs that are the most common cause of upper respiratory tract infections. more recent studies using modern pcr-based techniques have allowed a re-evaluation of the importance of viruses in copd exacerbations and these studies have shown the presence of viruses in 47% to 56% of exacerbations [70] [71] [72] [73] . a recent systematic review evaluated weighted mean prevalence of respiratory viruses detected by pcr in patients with acute exacerbations of copd. eight studies were included with an overall prevalence of 34.1%, with picornaviruses including rvs being the most frequently detected pathogen, followed by influenza, parainfluenza, rsv and adenoviruses [74] . although these studies have higher detection rates they are likely to have underestimated the role of viral infections in copd exacerbation as they evaluated patients at the time of presentation to healthcare services which often occurs considerably later than the onset of exacerbation and by which time virus may no longer be detectable. although viruses are frequently detected in copd exacerbations, their presence during exacerbations does not prove a definite causative role. experimental infection using rv provides a novel tool for investigating relationships between virus infection and exacerbations. such studies have been previously conducted in asthma and yielded important insights into the mechanisms linking virus infection to exacerbations in asthma. a recent study from our group reported the first experimental rv infection study in copd [75] . copd patients and nonobstructed controls were infected with rv with sequential measurement of symptoms, lung function, inflammatory markers and virus load. following rv infection, copd subjects developed symptomatic colds followed by the typical lower respiratory symptoms of an acute exacerbation. symptoms were accompanied by objective evidence of airflow limitation and airways inflammation and inflammatory markers correlated with virus load. virus was detected in airway samples prior to the onset of symptoms and viral clearance was followed by symptom resolution and return of inflammatory markers to baseline levels. therefore, this study directly links respiratory virus infection to lower respiratory symptoms, airflow obstruction and airways inflammation in copd and provides novel evidence supporting a causative role for rv infection in copd exacerbations. much less is known regarding mechanisms of virusinduced exacerbations in copd compared to asthma. in the experimental infection study, symptoms, airflow obstruction and airways inflammation were more severe in the copd subjects compared to non-obstructed controls [75] . therefore, as is the case in asthma it would appear that clinical illness following rv infection is more severe in copd subjects, but the mechanisms underlying this are poorly understood. copd exacerbations are associated with increased levels of inflammatory mediators including tumor necrosis factor-alpha (tnf-α) [76] , il-8 [76, 77] , il-6 [78] , and leukotriene b4 [79] and inflammatory cells such as neutrophils [70, 77] and eosinophils [70] . however, few studies have examined the inflammatory response specific to virus-induced exacerbations. virus infection has been associated with high levels of il-6 [80, 81] and ip-10 [82, 83] and papi et al. reported that elevated sputum eosinophils were only seen in exacerbations in which a virus was present [70] . others have reported that the presence of rv is not associated with significant airway inflammation [84] and that only exacerbations associated with purulent sputum (presumed bacterial infection) are associated with airways inflammation [79] . from the data available no clear conclusions can be drawn regarding the inflammatory response to virus infection in copd and there are no studies comparing the effects of naturally-occurring virus infections in copd patients and non-copd controls. there is evidence from animal models that the inflammatory response to virus infection may be exaggerated in copd. in a mouse model of copd utilizing intranasal administration of lipopolysaccharide and elastase, infection with rv resulted in increased levels of tnf-α and il-13 compared to control mice [85] . this was accompanied by increased airway hyper-responsiveness and increased mucus production. similarly, in the human copd rv challenge study, increased levels of il-8 and neutrophil elastase were reported in copd subjects when compared to non-obstructed controls [75] . these studies suggest that copd is associated with an exaggerated inflammatory response to viral infection and this may explain the increased severity and duration of symptoms seen in these patients. in vitro studies have shown that cigarette smoke impairs release of ifn-β and ifn-α [86] . bal cells from copd patients infected ex vivo with rv demonstrated deficient induction of ifn-β with similar trends for deficient induction of ifns-α and -λ, associated with deficiency of the interferon stimulated gene cxcl10 [75] . similar findings have been reported in a mouse model where persistence of rv, increased airways inflammation and deficient induction of ifns-α, -β and -γ were reported in copd mice compared to controls [85] . however in vitro rv infection of epithelial cells from copd patients resulted in higher virus load and increased inflammatory mediators, but no differences in interferon production compared to cells from control subjects [87] . further studies examining the role of interferon deficiency in viral exacerbations are required as this may lead to potential future therapeutic application of interferon therapy in reducing exacerbation severity in copd. rvs bind to cells via intercellular adhesion molecule-1 (icam-1, major group rvs) or members of the low-density lipoprotein receptor family (minor group rvs). icam-1 is upregulated on the bronchial epithelium of patients with copd [87, 88] and, therefore, it is possible that increased icam-1 levels may permit greater virus binding and increased viral entry into epithelial cells in copd patients. the majority of studies have detected viruses at a greater frequency during acute exacerbations compared to the stable state. one study indicated that rsv is detected in nasal lavage at a similar frequency of around 25% in the stable state and during exacerbations [67] . this was followed by a similar study reporting detection of rsv in about 30% of sputum samples, with detection being related to greater airway inflammation and to a faster decline in lung function [89] . however, other studies have not reported increased rsv detection in stable copd [70, 71] . a study comparing virus loads between infants with acute respiratory infections and adult copd patients found that virus loads were 2000-fold higher in the infants, suggesting low-grade virus infection in copd [90] . the disparity between these findings is likely to be due to a combination of factors including differing sensitivity of the pcr techniques used, differences in severity of copd patients included or differences in the populations studied [91] . latent infection by adenovirus has also been proposed to be involved in the pathogenesis of copd. lung tissue from copd patients has been demonstrated to carry more group c adenoviral dna than matched nonobstructed smokers [92] . latent adenoviral infection in combination with cigarette smoke exposure in a guinea pig model caused an increase in lung volumes, airspace volume and reduced surface to volume ratio compared to smoke exposure alone [93] . additionally, adenovirus detection has been shown to be similar in exacerbated and stable copd patients [94] . some authors have postulated that the presence of rsv and adenovirus in stable copd may contribute to the pathogenesis of the disease as there are some common pathologic features between respiratory viral infection and copd including a predominance of cd8+ t lymphocytes. however, this remains a largely unproven hypothesis. cystic fibrosis (cf) is an autosomal recessive disease caused by mutations in the gene for the cystic fibrosis transmembrane regulator (cftr) protein. defective cftr function leads to abnormal transport of chloride and sodium across the pulmonary epithelium, resulting in viscous secretions in the lungs, recurrent bacterial infections and progressive loss of lung function. pulmonary involvement is the most common manifestation of the disease and respiratory failure the most common cause of death. respiratory infections are the leading cause of morbidity, decline in lung function and hospitalizations due to acute exacerbations. the major cause of infectious complications in cf has always been considered to be bacterial infection, with pseudomonas aeruginosa the most common organism detected. there has been relatively little research on the role of virus infections in cf but recent studies have suggested that viruses have a significant impact on the cf patient. the role of respiratory viruses in cf exacerbations is likely to have been under-appreciated in the past because older studies investigated only one virus type and the detection methods used were not sufficiently sensitive. newer pcr techniques have helped to improve detection and it is now becoming clear that viruses are implicated in exacerbations in cf. previous studies using serology, culture and immunoflourescence detected viruses in 10% to 28% of exacerbations in cf patients [95] [96] [97] [98] . in contrast, studies using pcr for virus detection have reported detection rates of 50% to 60% [99] [100] [101] . a number of different viruses have been detected in cf patients with the most common being rvs, influenza and rsv. the incidence of viral infections in children with cf is not elevated in comparison to healthy children but the severity of clinical illness associated with infection is greater [102] . viral infections are associated with deterioration in lung function and more severe clinical illness indicating that they contribute to disease progression thus demonstrating the clinical importance of research within this field [100, 103] . the mechanisms of viral-induced cf exacerbations and increased clinical illness are poorly understood with conflicting results from published studies. some authors have reported increased production of pro-inflammatory cytokines and chemokines by epithelial cells obtained from cf patients compared to healthy controls [102, 104] . however, others have failed to detect any differences in cytokine production between cf and normal cells [105, 106] . these differences may be due to different viruses used (rv, rsv, piv) and differences in cell culture techniques, but it remains unclear whether the cf epithelium is intrinsically pro-inflammatory in response to virus infection. another mechanism that has been postulated is a deficiency in antiviral innate immune responses in cf cells. increased replication following piv infection of cf cells has been reported and this was corrected by administration of ifnα [102] . ifn responses were not impaired but induction of nitric oxide synthase 2 (nos2) was impaired in cf. nos2 is required for production of no that has potent antiviral effects and, therefore, impaired no synthesis may be one mechanism of impaired antiviral host responses in cf. our group has reported reduced ifn-β and ifn-λ production and reduced isgs in cf epithelial cells [107] and, therefore, ifn deficiency may be relevant to cf as well as in asthma and copd. holtzman has proposed that 'hypersusceptibility' to virus infection, via defective interferon pathways, is a unifying pathway in asthma, copd and now cf [108] . both bacterial and virus infections are common in cf and copd and, therefore, co-infections are likely to be common. there is now increasing evidence that both viral and bacterial infections can modulate host immune responses and increase susceptibility to subsequent infection. there is abundant evidence from both human studies and animal models that influenza infection impairs antibacterial immunity and this can result in secondary bacterial pneumonia [109, 110] . however, much less is known regarding the effect of other respiratory viruses, such as rvs, on susceptibility to bacterial infection. in vitro studies have reported that rv infection increases bacterial adhesion to epithelial cells [111] [112] [113] and impairs macrophage immune responses to bacterial products [114] . we have found that experimental rv infection in copd is followed by secondary bacterial infection in 60% of patients and this is related to deficiency of the antimicrobial peptides elafin and secretory leukoprotease inhibitor (slpi) (submitted manuscript). there are also studies indicating that virus-bacteria interactions influence host immune responses in cf. chattoraj et al. reported that rv infection of cf cells liberates planktonic bacteria from biofilm [115] . planktonic bacteria express virulence factors and stimulate inflammatory responses more readily compared to biofilm bacteria and this was manifested by increased cytokine responses. evidence is also emerging that bacterial infection can increase susceptibility to viral infection. infection of epithelial cells by haemophilus influenzae (a common organism in copd) increases susceptibility to infection by rv, possibly by up-regulation of icam-1 [116] . cf cells infected with mucoid p.aeruginosa and then with rv produced less ifn and viral loads were higher compared to cells infected with the rv alone [117] . this effect was not seen in normal epithelial cells infected with pseudomonas and was related to the inhibition of akt phosphorylation and irf-3 activation -both prerequisites for the ifn response to rv infection. it is widely acknowledged that the main infectious cause of asthma exacerbations is virus infection and it is believed that bacteria play only a minor role. however, a recent study using culture-independent molecular methods for bacterial detection reported that the bacterial flora in the airways of asthmatics is closer to that of copd patients than of non-asthmatics [118] . the role of bacteria in asthma exacerbations needs to be revisited as virus-bacterial interactions may play a role in the pathogenesis of asthma exacerbations. this is a fertile area for further research. our knowledge of the interactions between respiratory viruses and bacteria, and how these influence host immune responses in pulmonary diseases, is still at an early stage. further research is required to understand better these complex relationships and to explore the implications they may have for the development of new therapies. there is now convincing data implicating respiratory viruses as a major cause of acute exacerbations in asthma, copd and cf. in all these conditions there is evidence that host immune responses to virus infection are impaired, but whether this occurs through a common mechanism, or whether mechanisms differ between the different diseases is unclear. further research is needed to elucidate the exact mechanisms of increased susceptibility to virus infection in pulmonary diseases, the interactions between viruses and bacteria and how these impact on host immune responses. a better understanding of these mechanisms has the potential to lead to the development of novel therapies that will reduce the impact of acute exacerbations in chronic pulmonary diseases. list of abbreviations atf: activating transcription factor; bal: bronchoalveolar lavage; cf: cystic fibrosis; cftr: cystic fibrosis transmembrane regulator; copd: chronic obstructive pulmonary disease; ena-78: epithelial-derived neutrophilactivating peptide 78; icam-1: intercellular adhesion molecule; ifn-α: interferon-alpha; ifn-β: interferon-beta; ifn-λ: interferon-lambda; ifn-γ: interferon-gamma; il: interleukin; ip-10: ifn-γ-induced protein-10; irf: interferon regulatory factor; isg: interferon stimulated genes; mda-5: melanoma differentiation-associated protein-5; nf-κb: nuclear factor-kappa b; no: nitric oxide; nos2: nitric oxide synthase 2; pcr: polymerase chain reaction; pef: peak expiratory flow; piv: parainfluenza virus; rantes: regulated on activation: normal t-cell expressed and secreted; rig-i: retinoic acid inducible gene i; rsv: respiratory syncytial virus; rv: rhinovirus; slpi: secretory leukoprotease inhibitor; socs: suppressor of cytokine signaling family; th1/2: t helper 1/2; tlr: toll-like receptors; 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enhances various bacterial adhesions to nasal epithelial cells simultaneously effects of rhinovirus infection on the adherence of streptococcus pneumoniae to cultured human airway epithelial cells rhinovirus disrupts the barrier function of polarized airway epithelial cells rhinovirus exposure impairs immune responses to bacterial products in human alveolar macrophages rhinovirus infection liberates planktonic bacteria from biofilm and increases chemokine responses in cystic fibrosis airway epithelial cells hershenson mb: h. influenzae potentiates airway epithelial cell responses to rhinovirus by increasing icam-1 and tlr3 expression pseudomonas aeruginosa suppresses interferon response to rhinovirus infection in cystic fibrosis but not in normal bronchial epithelial cells disordered microbial communities in asthmatic airways all the authors contributed equally to writing the manuscript. all authors read and approved the final manuscript. pre-publication history key: cord-309138-44qpk2vf authors: khanna, kanika; kohli, sukhmeen kaur; kaur, ravdeep; bhardwaj, abhay; bhardwaj, vinay; ohri, puja; sharma, anket; ahmad, ajaz; bhardwaj, renu; ahmad, parvaiz title: herbal immune-boosters: substantial warriors of pandemic covid-19 battle date: 2020-10-03 journal: phytomedicine doi: 10.1016/j.phymed.2020.153361 sha: doc_id: 309138 cord_uid: 44qpk2vf current scenario depicts that world has been clenched by covid-19 pandemic. inevitably, public health and safety measures could be undertaken in order to dwindle the infection threat and mortality. moreover, to overcome the global menace and drawing out world from moribund stage, there is an exigency for social distancing and quarantines. since december, 2019, coronavirus, sars-cov-2 (covid-19) have came into existence and up till now world is still in the state of shock.at this point of time, covid-19 has entered perilous phase, creating havoc among individuals, and this has been directly implied due to enhanced globalisation and ability of the virus to acclimatize at all conditions. the unabated transmission is due to lack of drugs, vaccines and therapeutics against this viral outbreak. but research is still underway to formulate the vaccines or drugs by this means, as scientific communities are continuously working to unravel the pharmacologically active compounds that might offer a new insight for curbing infections and pandemics. therefore, the topical covid-19 situation highlights an immediate need for effective therapeutics against sars-cov-2. towards this effort, the present review discusses the vital concepts related to covid-19, in terms of its origin, transmission, clinical aspects and diagnosis. however, here, we have formulated the novel concept hitherto, ancient means of traditional medicines or herbal plants to beat this pandemic. pandemic diseases are of global concern in the present era, to cause gigantic morbidity and transience, regardless of, extensive medical facilities. particularly, anti-viral therapies have been fraught because of surfacing of mutants competent enough to subdue the drugs targeting disease that has created panic all over. by this review, we suggest that herbal or medicinal plant formulations could be essential alternative strategy, a step ahead to battle these awful viruses. zhang and holmes, 2020). moreover, sars-cov-2 just like sars-cov utilizes the same ace2 receptors to infect its hosts (guo et al., 2020) . thus, the sites where ace2 protein is mainly expressed are the potential target sites for sars-cov-2 respectively. these regions are belong to type ii alveolar cells of the lungs and enterocytes of the small intestine (hamming et al., 2004; zheng, 2020) . nevertheless, there are some remarkable biological differences between the sars-cov-2 and the other beta-covs, which probably make it more infectious. consequently, the epidemiological dynamics of sars-cov-2 is different from previous human-cov outbreaks having striking local and global spread (zhang and holmes, 2020; zheng, 2020). although, sars-cov-2 shows greater human-to-human transmission efficiency, its crude fatality rate (0.25% to 5%) is comparatively far less than that of sars-cov which is approx. 10%. furthermore, sars-cov-2 has r 0 (basic reproduction number) of 4.7 to 6.6. this highly contagious nature of sars-cov-2 is supported by the fact that its spike (s) protein possess 10 to 20 time's greater affinity for ace2 receptors than sars-cov (zheng, 2020) . s-protein is the surface glycoprotein that assists the virus in the attachment to the host cells through its receptor-binding domain (rbd). s-protein has several domains, one of the sections termed as ectodomain has two subunits, s1 and s2, which form a crown-like structure around the virus (vellingiri et al., 2020) . besides, s-protein of sars-cov-2 contains a furin-like cleavage site at the s1-s2 junction, missing in other members of its sister clade. this additional cleavage site might also be responsible for greater pathogenicity of sars-cov-2 as it also occurs in highly infectious form of influenza virus but lacking in less pathogenic ones (coutard et al., 2020; zhang and holmes, 2020).3. origin and the natural reservoir for both the sars-cov and mers-cov were bats; however, these viruses infect humans through an intermediate host. palm civets are supposed to be the intermediate host of sars-cov while dromedary camels are of mers-cov (yin and wunderink, 2018) .unfortunately, the exact zoonotic origin of sars-cov-2 is still elusive. since sars-cov-2 shares 88% nucleotide homology with two sars-like cov found in bats (bat-sl-covzc45 and bat-sl-covzxc21) and 96% with ratg13 virus found in horseshoe bat (rhinolophus affinis), bats are considered to be its natural host (lu et al., 2020b; zhou et al., 2020b) . despite the 96% nucleotide similarity, the rbd of both the viruses varies significantly (mackenzie and smith, 2020) . however, due to the ecological separation of the bats from the humans and the requirement of some necessary mutations in the virus genome to cross the species barrier, sars-cov-2 likely has one or more mammalian intermediate hosts for efficient animal-to-human transmission (zhang and holmes, 2020) . analyses of interactions between rbd in sars-cov-2 and ace2 receptors in different hosts indicate that pangolins, snakes, and turtles may be the immediate host of sars-cov-2 (liu et al., 2020) (fig. 1) . recent phylogenetic studies on the genome of pangolin-cov found from dead malayan pangolins, which are illegally imported in china revealed that its genome is about 91% identical to sars-cov-2 and around 90% to ratg13 (bat-cov). besides, five of the six amino acids (contact residues) on rbd, vital for binding to ace2 receptors on hosts are consistent between sars-cov-2 and pangolin-cov, but only four between pangolin-cov and ratg13 and only one out of six between sars-cov and sars-cov-2 (zhang et al., person-to-person transmission is primarily reported in families, communities, and hospitals (guo et al., 2020) . droplet transmission is considered as the main route of person-to-person transmission (han et al., 2020) . the infection also spreads through direct contact and via fomite exposure i.e., direct contact to eyes, nose, and mouth after touching surfaces and objects contaminated by an infected individual morawskaa and cao, 2020) . in general, human-covs can remain infectious on some material surfaces for even 9 days (kampf et al., 2020) . besides, sars-cov-2 also spreads through contact with asymptomatic carriers . since sars-cov-2 infection is spreading at an unprecedented pace, airborne transmission too warrants meticulous determination (morawskaa and cao, 2020). in addition, sars-cov-2 was found in a stool sample, gastrointestinal tissue even urine and saliva of an infected person, suggests the possibility of intestinal and fecal-oral transmission (xiao et al., 2020; guan et al., 2020) . although no case of mother to child transmission has been reported yet, infection in two newborns of wuhan, china raises the question for vertical transmission (han et al., 2020) . apart from this, there are recent studies that reveal that there is a possibility of sexual transmission of covid-19 infection. there are an array of evidences for this transmission such as through faecal-oral route via gastrointestinal infection . this is most probably due to ace2 that enable virus entry and ace2 mrna is overexpressed in gastro-intestinal system as revealed through immunofluorescent analysis of rectal epithelial cells (hindson, 2020). moreover, rna analysis of sars-cov-2 also revealed that the viral particles can infect these cells therefore, it is quite predictable that sexual intercourse could be the possible way of contagion. there have been cases when person shows negative results on nasopharyngeal swabs, while, positive on rectal swabs, depicting that sexual transmission might be the possibility . henceforth, the physicians as well as doctors in particular recommend a strong message to discourage sexual practices if in any case of covid-19 infection. hence, comprehensive possible means of sars-cov-2 transmission warrants further study. suggests this novel sars-cov-2 affects elderly people with coexisting other medical complications more seriously. these results are consistent with previous studies conducted on severely ill patients which also advocates high mortality rate in elderly people with acute respiratory distress syndrome (ards) and comorbidities such as hypertension, diabetes, chronic obstructive lung disease and coronary heart disease zhou et al., 2020a) . the most common complications associated with sars-cov-2 infection are sepsis, respiratory and heart failure, ards, and septic shock (zhou et al., 2020a) . even though children of all ages face risk of sars-cov infection, the serious progression of infection and morbidity is rare in children and adolescents relative to the adults (lu et al., 2020a). sars-cov-2 infection is often categorized into three stages: first, asymptomatic phase; second, non-severe symptomatic phase; and third, severe respiratory symptomatic phase (shi et al., 2020) . usually, a small number of patient's progress to the severe stage and develop ards and/or multiorgan failure (cao et al., 2020). host's immune responses initiate as soon as sars-cov-2 binds to ace2 receptors and releases viral rna for replication. both the innate and adaptive immune response could be triggered in response to the sars-cov-2 infection (cao et al., 2020). however, immune responses are different between severely and moderately infected persons. in a blood sample of symptomatic hospitalised patients with mild to moderate sars-cov-2 infection before resolution of symptoms, immunological changes such as increase in the number of activated cd4+ helper t cells and cd8+ killer t cells, follicular helper t (tfh) cells, antibodysecreting cells (ascs) and antibodies particularly igg (immunoglobulin g)and igm (immunoglobulin m) were detected (thevarajan, 2020) . on the other hand, in severely infected patients, lymphocytopenia is a common denominator with substantial fall in numbers of natural killer cells, b cells, cd3+ t cells, cd4+ helper t cells, cd8+ killer t cells along with the increase in neutrophil-to-lymphocyte ratio (nlr) and c-reactive protein levels. additionally, in comparison to the non-severe patients, pro-inflammatory cytokines and chemokines such as tumor necrosis factor (tnf)-α, interleukin (il)-2, il-6, il-7, il-8, il-10, granulocyte-colony stimulating factor (gcsf), monocytechemoattractant protein 1 (mcp1) and macrophage inflammatory protein 1-alpha (mip1-alpha) are often reported to be elevated in serum levels of critically ill patients (huang et al., 2020; qin et al., 2020; wang et al., 2020a) . the elevated ratio of nlr, which is a biomarker of systemic inflammatory response syndrome, points to the devastated inflammatory state of icu patients (salciccioli et al., 2015) . moreover, uncontrolled levels of cytokines and chemokines cause over-active inflammatory responses or cytokine storm. this hyperactive immune response along with impaired adaptive immune response may trigger pulmonary injury, ards, viral sepsis and organ failure like complications, and eventually death in some cases (prompetchara et al., 2020) . the traditional chinese medicines and ayurveda since the vedic period (1500-500 bce) provides globally with potential remedies to lessen the severity of the illnesses caused by the traditional medicines have been in general disregarded in the novel research and expansion of contemporary drugs due to the fact that their translational ability is commonly i.e. curumin, is identified to block cytokine release, specifically interleukin-1, interleukin-6, pro-inflammatory cytokines and tumor necrosis factor-α and is directed to be consumed with milk (omara et al., 2010) . inhibition of the cytokine discharge is one of the prime clinical development associated with experimental modules of flu and other infectious diseases and have also been compared to covid-19 where similar cytokine storm play an imperative role in transience (sordillo et al., 2015) . moreover, ayush has recommended certain preventive and medicinal plants for prevention and prophylactic of covid-19 including warm extracts of tinospora cordifolia (advised for chronic fever), andrograhis paniculata (advised for fever and cold), cydonia oblonga, zizyphus jujube and cordia myxa (enhancing antioxidant, immune-modulatory, anti-allergic, smooth muscle relaxant, anti-influenza activity) and arsenicum album 30 (found effective against sars-cov-2, immune-modulator). the symptomatic management of covid-19 was suggested to be acquired from agastya haritaki the polysaccharides are a structurally multifaceted class of biomolecules that have diverse therefore, these biocompatible compounds were suggested to be employed in coating materials of various sanitary items for covid-19 deterrence. the naturally occurring products and phytomedicines are coming to the fore all around the (sengupta, 2020 ). according to their study, 'aurantiamide', an active metabolite from piper aurantiacum has chymotrypsin like-proteases like activities that is a ratified agent for inhibiting coronavirus. in addition to this, it also encompassespiperol, eugenol, catechol, caryophyllene, etragol, chavibetol, betlol, quercetin etc. that also grows body's self-defense mechanism either through reception of viruses or in waning off the viral load within the infected hosts (sengupta, 2020) .alongside, studies have been depicted the role of herbal drug gene-eden-vir/novirin against many noxious viruses and this drug is mainly comprised of quercetin, green tea, cinnamon, licorice and selenium (polansky and lori, 2020). they found that it disrupts the viral entry, infection, replication, viral proteases, viral quasi-speciesand triggers the immunity, thereby, can be evidently utilised against sars-cov-2 respectively (polansky and lori, 2020). interestingly, the role of tulsi for scientific evidence against covid-19 has also been elucidated (goothy et al., 2020). as, it is well known herbal plant for antiviral effects in inhibiting many deadly viruses like vaccinia, dengue, hepatitis, encephalitis etc. by enhancing their survival and defense ability. moreover, it also reinstate the physiological functions of body through its phenolic and antioxidative property that in turns shields the body from toxic substances (shivananjappa and joshi, 2012) . the most possible mechanism underlying its immunity boosters lies in triggering humoral and cellular immunity responses (vaghasiya et al., 2010) . apart from this, modulatory actions of gaba pathway also encompass their multi-modal therapeutic properties, therefore, we can conjecture that it could be efficient in cure of covid-19. nevertheless, the role of persian herbs have also been untangled against coronavirus covid-19 pandemic is a huge catastrophe that has caused devastating effects however, the biological activities can also be improved by ingesting high levels of naturally existing polyphenols that not only elicit immunity against viruses but they are also comprised of receptors that identifies and permit their cellular uptake for activating signaling cascade are also hindered by smoke.therefore, these steps can reboot the immune system and reinforce the inner forces to battle against severe viral infections covid-19 respectively. the most momentous weapon against any kind of viral infection is a strong immune system. vitamin a is an essential fat-soluble vitamin which has a major involvement in regulating vision, growth and maturity as well as protection of the mucosal and epithelium had the highest inhibitory potential against sars-cov-2, followed by galangal, sappan wood and curcuma species and it was further suggested that they might have antiviral potency against covid-19. additionally, different plant based sources of vitamins include, mushrooms, carrots, broccoli, almonds, citrus, guava, amla, avocados etc. fig. 3 describes the role of vitamins (a, c, e and d) in combating covid-19. zinc is one of the indispensible elements which has significant role in modulation of growth and development and the regulation of the immunomodulatory responses in addition to this, another trace element that has been reported to have wide range of pleiotropic effects ranges from anti-inflammatory to antioxidative is selenium (se) (rayman, 2012) . comparatively, the lower concentrations of se are related to enhancement in the jeopardy of mortality, meager immune responses and cognitive reduction, whereas the higher doses are affirmed to show antiviral activity (rayman, 2012) . supplementation of se results in elevation in concentration of se in the plasma, up-regulation of lymphocyte phospholipid levels and activity of gpox in the cell, subsequently leading elevation in cellular immune response. the humoral response was shown to be un-affected (broome et al., 2004) . similar reports of hasty clearance of poliovirus were also reported by se supplementation (ivory et al., 2017). various plants namely, beans, nuts, peanuts, grains, mushrooms, cereals, lentils, garlic, grains, etc. have been wide sources of minerals and must be included in diet. nutraceuticals are the products which are argued to provide physiological assistance and protection against varied persistent diseases. wide range of nutraceutical products have been isolated herbal products, dietary supplements, isolated nutrients, genetically engineered foods and processed cereals, beverages and soups (kalra, 2003) . certain nutraceuticals have been shown earlier to enhance the immune function. mccarty and dinicolantonio (2020) elucidated that, a few nutraceuticals were able to lower the degree of symptoms and provide respite to the patients infected from coronavirus and influenza. another significant product is probiotic, they are defined as live micro-organisms which bestow varied health benefits such as improvement in gastrointestinal activity (sanders, 2008) . they also induce certain specific immune responses by augmenting antibody synthesis (kanauchi et al., 2018) . a report by paks belong to mammalian kinases family also known as rac/cdc42-activated kinases that have been came into existence since decades. amid, these classes pak1 is most prominent category of "pathogenic kinases" that can cause a plethora of diseases like cancer, inflammation, malaria, dengue, immuno-suppression and many antiviral diseases, if behave abnormally (maruta, 2020) . strikingly, pak1-blockers (naturally existing) such as caffeic acid, its esters, bee-product propolis have been observed to inhibit rac that directly activates pak1 (xu et al., 2005) . however, previous studies reported that chloroquine a drug active against malaria also suppressed the incidence of sars/coronavirus infection, but its mode of action still remains unknown (vinecent, 2005) . later, it was reported that this drug induced cdk inhibitor (p21) to inhibit pak1 activity (maruta, 2014) . in line of forging argument, a recent study depicted that tumor-repressed phosphatase pten, mainly involved in impeding pak1 also inhibited the coronavirus-curbed llc2-linked fibrosis (lu, 2020). moreover, llc2 expression has been observed to correlate with ace2, a coronavirus receptor and ck2/ras-pak1-ap1 signaling cascade (chen et al., 2010) . henceforth, through all these assumptions we can deduce the pak1-reliance of corona pathogenesis and a suggestion of using pak1-blockers for treating pandemic covid-19 outbreak (fig. 4) inevitably, the conventional and recently accepted product propolis, obtained from bees have been known since ancient times. it is formerly obtained from honey-bee extract via honey-bees feeding the buds of poplar and willow trees, thereby, mixing their saliva into it to form hexagonal bee-hives in order to protect their larval species against many pathogenic organisms. this product synthesised is called as propolis and is regarded as herbal medicine with anti-viral properties. in contemporary world, propolis is identified in possessing anticancer characteristics due to the presence of an ingredient cape (caffeic acid phenyl ester) involved in down-regulation of rac for pak1 inhibition respectively (xu et al., 2005) . although, the anticancer constituents in propolis varies from product to product according to harvesting property of bee extracts. for instance, the anti-cancer element in brazilian propolis is artepillin c (arc), while in sub-tropical regions of taiwan and okinawa, polyphenols namely, nymphaeols act as direct inhibitors of pak1 .in view of the fact that, familiar thing among all categories is that they are comprised of pak1-blockers. ever since, has been elucidated that, pak1 tends to cause cancers, viral diseases like hiv, hepatitis, pappiloma, influenza, ebola, sars and corona virus along with immune system suppression of hosts, henceforth, propolis would be quintessential in blocking covid/coronavirus curbed fibrosis in respiratory tract and boosting the immunity of an individual (maruta, 2014) . the effectiveness of propolis also depends on the product, its chemical nature and action. to expedite, cape-containing propolis named 'bio 30', commercialised by new zealand is found to be highly active (maruta, 2014) . the optimal doses are 1 ml/10 kg of the body weight. regardless of this pre-formed formulation in the market, it could not be used against covid-19 eruption, due to paucity of its stock in market place. moreover, the available stocks are being predominantly used for treating patients with deadly cancers and prolonged treatment of genetic brain disorder, nf tumers (neuro fibromatosis type 1, 2) (maruta and he, 2020). in addition to this, the cell membrane permeability of caffeic acid and arc is relatively weak, owing to its -cooh moiety. nonetheless, this feature could be surmounted by triggering the cell membrane permeability via conversion their esters into 1, 2, 3-triazolyl esters that are thousand times more powerful than arc or caffeine esters (maruta and ahn, 2017) . along with this, melatonin hormone (pineal gland hormone) is a serotonin and possess anti-melanogenic activity that is a primitive factor as melanogenesis is directly correlated to pak1 . further, it also owns similarity with other anti-pak1 actions like anti-cancer, antiviral, antiinflammatory, immuno-boosters etc. therefore, it is pertinent to mention here that melatonin, a commonly available sleeping pill could be utilized against coronaviral infections. currently, many investigations are being carried out to high-light the significance of melatonin as another therapeutic resort or covid-19 adjuvant (zhang et al., 2020b) . furthermore, glucocorticoid hormone 'ciclesonide' is another choice to cure different inflammatory disorders and it has been widely commercialised under the brand name of alvesco. it has been officially approved since 1990 and been widely associated with treating adults as well as children. the molecular mechanism forming the basis for anti-inflammatory action is based on the potential of pak1-blockers that lays the foundation for its pathway. majorly, it works according to two basic reasons, firstly, inflammation is not possible without pak1 and apart from this, a herbal formulation mainly triterpenes or steroid named 'triptolide' derived from thunder god vine also inhibited rac followed by blocking pak1 route (maruta and ahn, 2017) . this medicine was previously treating the patients infected with dengue virus in restricting the proliferation of virus in lungs by blocking pak1 signaling cascade (liou et al., 2008) . conversely, due to its lower water solubility, few years back, its -oh group was phosphorylated to enhance its solubility (patil et al., 2015) . resultantly, phosphotase-receptive pro-drug of triptolide 'minnelide' is currently under clinical trials to cure covid-19 infections as well. another compound ivermectin has been screened from bacteria but with some lethal effects. interestingly, these ill-effects were removed later by chemically re-designing the compound. (hirokawa et al., 2005) . therefore, possessing all these features it was approved to cure rare cancer disease cutaneous lymphoma and commercialised as 'isodax' in market. however, research is being conducted with the belief that it could be most probably utilized for covid-19 therapy. it is need of an hour to clinically test various pak1-blockers against blocking virus replication to develop anti-covid-19 therapy for patients suffering worldwide. strata of mankind has been marred with the onset of the unexpected pandemics, leading to the atrocious effects on their entire communities. considering themselves invincible to conquer the world, they have now become the cocoons of their houses against covid-19 storm. unfortunately, sars-cov-2 has led the global population on their toes due to its highly infectious nature and susceptibility towards humans in causing higher mortalities among them. we can also introspect that about a lot many cases of covid-19 have been undetected and by looking into the ongoing trends, the cases are doing to inflate in the coming future. this would ultimately affect the world economy, specifically within developing and under-developing nations. besides, who has advised the world to take preventive measures, owing to which government has facilitated nationwide shutdowns, selfquarantine, physical distancing to ensure public safety. thus far, the prevailing drugs and vaccines for antiviral diseases have been used as clinical trials to fight against coronavirus. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influencethe work reported in this paper. vitamin d supplementation improves sustained virologic response in chronic hepatitis c (genotype 1)-naïve patients zinc supplementation promotes a a review on antiviral activity of the 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(nigella sativa) and its constituent thymoquinone as an antidote or a protective agent against natural or chemical toxicities methanol and aqueous extracts of ocimum kilimandscharicum (karpuratulasi) inhibits hiv-1 reverse transcriptase in vitro breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 betulinic acid: a new cytotoxic compound against malignant head and neck cancer cells investigation into sars-cov-2 resistance of compounds in garlic essential oil characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia antioxidant activity tests on novel triterpenoids from salvia macrochlamys long-term high copper intake: effects on indexes of copper status, antioxidant status, and immune function in young men structural basis of sars-cov-2 3clpro and anti-covid-19 drug discovery from medicinal plants revealing the potency of citrus and galangal constituents to halt sars-cov-2 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anti-inflammatory high-cbd cannabis sativa extracts modulate ace2 expression in covid-19 gateway tissues anti-influenza agents from plants and traditional chinese medicine adaptogens: herbs for strength. stam. stress rel contribution of selected vitamins and trace elements to immune function situationreport-88. world health organization who guidelines on safety monitoring of herbal medicines in pharmacovigilance systems evidence for gastrointestinal infection of sars-cov-2 downregulation of rac1 activation by caffeic acid in aortic smooth muscle cells characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study traditional chinese medicine in the treatment of patients infected with 2019-new coronavirus (sars-cov-2): a review and perspective herbs for viral respiratory infections herbal extracts as antiviral agents water extract of licorice had anti-viral activity against human respiratory syncytial virus in human respiratory tract cell lines mers, sars and other coronaviruses as causes of pneumonia identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 the traditional medicine and modern medicine from natural products covid-19: melatonin as a potential adjuvant treatment probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak a genomic perspective on the origin and emergence of sars-cov-2 sars-cov-2: an emerging coronavirus that causes a global threat clinical course and risk factors for mortality of adult in patients with covid-19 in wuhan, china: a retrospective cohort study a pneumonia outbreak associated with a new coronavirus of probable bat origin none list of tables: table 1 . herbal formulations as possible therapeutics against covid-19 infection mechanism of action therapeutic property references anti-inflammatory action by via modulation of gene expression of ace2 enzyme, serine protease tmprss2, protein pre-requisite for sars-cov2 invasion into host cells.adjunct therapy and utilised as mouthwash and throat gargle products clinically and home use owing to their potential to decrease viral entry via the oral mucosa. 15. highest ace2 enzyme inhibition, anti-inflammatory activity, modulated -gene expression for tnf-production in macrophages.bioactive compounds could be used for drug formulations. 17. ginkgolic acids impeded dna and protein synthesis by binding towards host cell receptors to activate cellsignaling pathways for arresting cell cycle as an inhibitory action. key: cord-269975-1ebmq7t8 authors: duplantier, allen j.; shurtleff, amy c.; miller, cheryl; chiang, chih-yuan; panchal, rekha g.; sunay, melek title: combating biothreat pathogens: ongoing efforts for countermeasure development and unique challenges date: 2020-05-27 journal: drug discovery targeting drug-resistant bacteria doi: 10.1016/b978-0-12-818480-6.00007-2 sha: doc_id: 269975 cord_uid: 1ebmq7t8 research to discover and develop antibacterial and antiviral drugs with potent activity against pathogens of biothreat concern presents unique methodological and process-driven challenges. herein, we review laboratory approaches for finding new antibodies, antibiotics, and antiviral molecules for pathogens of biothreat concern. using high-throughput screening techniques, molecules that directly inhibit a pathogen’s entry, replication, or growth can be identified. alternatively, molecules that target host proteins can be interesting targets for development when countering biothreat pathogens, due to the modulation of the host immune response or targeting proteins that interfere with the pathways required by the pathogen for replication. monoclonal and cocktail antibody therapies approved by the food and drug administration for countering anthrax and under development for treatment of ebola virus infection are discussed. a comprehensive tabular review of current in vitro, in vivo, pharmacokinetic and efficacy datasets has been presented for biothreat pathogens of greatest concern. finally, clinical trials and animal rule or traditional drug approval pathways are also reviewed. opinions; interpretations; conclusions; and recommendations are those of the authors and are not necessarily endorsed by the us army. the concept of bioterrorism and the intentional release of biothreat agents for purposes of harm to human and agricultural interests stimulates discussion of some unanswerable questions. questions ranging from protection of a nation's security to military defense tactics, all point to the gravity of the problem for which scientists are working together in many areas of study such as the development of novel medical countermeasures to combat lethal infections, the prevention of the spread of disease in the general populace, and design of field-worthy diagnostic tools. a biothreat organism is generally thought to be one causing severe or lethal disease or has potential to induce panic over the prospect of infection therewith; one with high pathogenicity and/or contagious infectivity; one with strong environmental stability or probable transmission as an aerosol; one with ease of large-scale production for far-reaching dissemination; and one that can be controlled for directing the release to only the intended target rather than accidental harm to the perpetrator [1] . improved preparedness for intentional release of bacteria, viruses, and toxins will not only protect military positions and strategies but will also increase ability to combat disease in naturally occurring epidemics of diseases caused by some of these organisms. the priorities for the development of medical countermeasures against these organisms have been defined through international discussions [2] [3] [4] . currently classified as tier 1 select agents are those pathogens of grave concern, whereas other useful classification categories are in use by us government entities such as national institute of allergy and infectious diseases and the us centers for disease control and prevention (cdc) list, denoting the pathogens as category a, b, and c agents [5] . various biothreat pathogens addressed in this chapter are grouped by general category and disease associated therewith (table 7 .1). there are many organisms on the cdc list; consequently, not all of them are addressed in this chapter [4] . the authors of this chapter have endeavored to provide a comprehensive survey of the literature and described the development hemorrhagic fever a crimean-congo hemorrhagic fever virus hemorrhagic fever a, b, c denote additional categorization into category a, b, and c pathogens, per niaid [5] . a tier 1 agents of human pathogenicity are presented [4] . two more tier 1 agents are rinderpest virus and foot-and-mouth disease virus, which are of agricultural concern (not covered in this chapter). b important non-tier 1 agents for which countermeasures are described in this chapter [4, 5] . see www.selectagents.gov for a comprehensive list of non-tier 1 select agents and toxins.sars, severe acute respiratory syndrome; mers, middle east respiratory syndrome-related coronavirus. of medical countermeasures against high-priority bacterial and viral biothreat agents where the most progress has been made, and/or the most novel ground has been broken. bacteria cause disease in humans by invading tissue, altering the host immune response, and/or producing toxins or virulence factors. many of the bacteria described here are difficult to treat clinically. the potential bacterial threat agents that pose the greatest risk to national security are ones that can be easily disseminated and result in high morbidity and mortality rates. the former soviet union is known to have weaponized at least 30 viral and bacterial agents, including several vaccine or drug-resistant strains [6] . each agent has unique properties that present both a distinct threat and challenge for detection, prevention, and control. bacillus anthracis and clostridium botulinum are gram-positive bacterial agents of grave biothreat concern. b. anthracis is a spore-forming bacterium that causes cutaneous, respiratory, or intestinal forms of anthrax disease, which is an acute, rapidly progressing infection in any form. the b. anthracis spores are highly stable both in the environment and in the exposed individuals and can be easily disseminated via the aerosol route, thus making it a dangerous bacterium [7] . the anthrax attacks in 2001 caused widespread panic, damage, disease, and death, which increased national awareness to the threat of bioterrorism. the bacterium produces a lethal toxin that disrupts the host innate responses during the early stages of infection and ultimately leads to septicemia and death of the host (fig. 7.1a) . antibiotic treatment requires a lengthy dosing regimen and is effective only if it is initiated during the early stage of the infection. two monoclonal antibody (mab)-based anthrax antitoxin therapeutics [abthrax (raxibacumab) and anthim (obiltoxaximab)] have been approved by the us food and drug administration (fda) and included in the strategic national stockpile for treating inhalational anthrax [8] . biothrax, the only licensed anthrax vaccine, is indicated for preexposure prophylaxis of disease in persons at high risk of exposure and postexposure prophylaxis of disease following suspected or confirmed b. anthracis exposure [8] . botulinum neurotoxin (bont), produced by c. botulinum, is extremely potent, lethal, and easy to produce, transport, and misuse. the toxin itself is the select agent, but the clostridium organism, as an isolate capable of producing the toxin, is also classified as a tier 1 select agent. there are seven serotypically distinct bonts (serotypes a-g) and they act by blocking neurotransmitter release and thereby preventing transmission of nerve impulses, which can lead to botulism, hallmarks of which are paralysis and respiratory arrest [9] (fig. 7.1b) . current treatment is limited to botulism immune globulin intravenous, human-derived antibotulism toxin antibodies for the treatment of infant botulism types a and b, and botulism antitoxin heptavalent (a-g), a mixture of immune globulin fragments developed from equine plasma for the symptomatic treatment of adult and pediatric botulism. the us army has developed a similar antitoxin based on equine neutralizing antibodies that is effective against a number of serotypes, but there is a limited supply and risk of horse serum sensitivity. an investigational vaccine also exists, but it offers limited protection and painful side effects [10] . many of the bacterial agents of biothreat concern are intracellular gram-negative organisms. intracellular bacteria are particularly difficult to treat because the intracellular niche protects bacteria from the innate or adaptive immune surveillance. these bacteria can enter host cells through phagocytosis, and to prevent their destruction in the endocytic pathways, intracellular bacteria have adapted to survive in a host lysosome and replicate within the acidic endolysosomal compartment (e.g., coxiella burnetii). another intracellular bacteria brucella spp. can traffic from a mature lysosome to endoplasmic reticulum-derived compartments, while bacteria such as burkholderia mallei, burkholderia pseudomallei, francisella tularensis, and yersinia pestis can prevent acidification and maturation of the phagosome and escape to the cytosol, where they can replicate and then disseminate to neighboring cells [12] [13] [14] . one characteristic feature of the b. mallei and b. pseudomallei intracellular life cycle is the fusion of infected mononuclear cells, forming multinucleated giant cells (mngcs). although the role of b. pseudomallei-induced mngcs is unclear, it is believed that cell fusion facilitates localized dissemination of the bacteria [15, 16] (fig. 7.1c) . brucella spp. are nonmotile bacteria that cause brucellosis, a world-wide chronic debilitating disease in both humans and animals. although not typically fatal, brucella spp. are stable and infectious as aerosols and can lead to sterility and abortions [17] . the nonmotile bacillus b. mallei is the causative agent of glanders that usually infects equids but is highly infectious to humans at low doses, producing septicemia, severe pulmonary infection, and chronic inflammation of the skin and eyes. b. mallei can be easily aerosolized, and even with antibiotic treatment there are high mortality rates [18] . the motile bacterium b. pseudomallei, the causative agent of melioidosis, is a close relative to b. mallei and can lead to severe illness in humans, such as pulmonary infection and septic shock. b. pseudomallei is an environmental saprophyte that is naturally resistant to many antibiotics [19] . q-fever is caused by direct contact with the nonmotile bacterium c. burnetii that was previously weaponized because of its ease of aerosolization, its environmental stability, and its ability to infect animals or humans with a single bacterium [20] . q-fever is not typically lethal but can be incapacitating, causing fever and difficulty breathing, and antibiotic therapy is not always effective, thus leading to persistent infections. f. tularensis is the causative agent of tularemia and is highly infectious, resulting in an acute, rapidly progressing local or systemic infection [21] . y. pestis, the causative agent of plague, is a nonmotile bacterium that can be disseminated by aerosol, transmitted from person-to-person, and is characterized by a severe clinical disease course with potentially high case-fatality rates. there is a limited window for effective treatment against plague, since the resulting respiratory and circulatory collapse from septic shock is usually fatal [22] . there are a great number of viruses on the list of select agents and toxins, and some of these can only be handled in the laboratory at the highest biocontainment level (biosafety level 4). the causative agents of some of the most lethal hemorrhagic fever infections are filoviruses, paramyxoviruses, and arenaviruses. filoviruses such as ebola virus (ebov) and marburg virus infect humans and nonhuman primates (nhps) and have caused large outbreaks in recent years. these viruses are likely transmitted in nature by fruit bats and are spread from person to person via contact with body fluids or fomites [23] . marburg virus was reportedly weaponized through activities carried out by the former soviet union [24] . because of the large scale of recent ebov outbreaks, this virus may have become available to nefarious people through access to corpses and contaminated clinical waste. two more viruses transmitted in nature at least in part by fruit bats are the nipah and hendra paramyxoviruses that belong to the henipaviridae family and cause severe neurological and/or respiratory diseases in humans [25] . these viruses can infect many domestic and agricultural animal species and are frequently transmitted between humans via droplets or fomites, leading to concerns of new human outbreaks in areas of malaysia, bangladesh, india, and australia where case-fatality rates range from 40% to 100% [26] . arenaviruses, specifically lassa virus (lasv) and junin virus (junv), machupo, and other south american viruses are transmitted not by bats but by peridomestic rodent species [27] . none of the filoviruses or henipaviruses has any fda-approved therapeutics or vaccines available for prevention or treatment of human disease, and while ribavirin is sometimes used to treat lassa fever, it is not a terribly effective drug against this viral infection [28] . variola virus (varv) is the causative agent of smallpox, a human viral disease for which tecovirimat was recently approved as a therapeutic by the fda, a successful therapeutic development story [29] . this pathogen has been eradicated since 1979 through successful vaccine campaigns [30] , but because the vaccine is no longer administered in most countries, populations may be susceptible in the event that varv or an intentionally modified or related poxvirus with similar virulence factors and similar human lethality is resurrected [31] . arthropod-transmitted alphaviruses and bunyaviruses are also biothreat concerns. for the alphaviruses specifically, the venezuelan encephalitis viruses (veev), eastern encephalitis viruses, and western equine encephalitis viruses belonging to the family togaviridae are found in the americas and cause equine disease [32, 33] . these new world alphaviruses cause encephalitis-like symptoms, are stable in the environment, grow to high titers easily in cell culture, are highly infectious by aerosol, and affect humans with incapacitating neurological disease, sometimes with high morbidity rates [33] . bunyaviruses such as rift valley fever virus and the related tick-transmitted nairovirus causing crimean-congo hemorrhagic fever also cause human diseases with high morbidity and mortality rates. some investigational new drug vaccines exist for these agents. these viruses replicate quickly in humans and cause rapid disease; therefore the timing for therapeutic intervention is short, making treatment postinfection very challenging. the lack of approved therapeutics available to combat biothreats may be in part attributed to the unique challenges for the discovery and development process of evaluating drugs that target select agents. foremost is the implementation of high-throughput screening (hts) efforts for the discovery of new compounds against authentic or wildtype biothreat bacterial and viral pathogens [34] . specifically, the requirement of work to be performed in high-level biocontainment laboratories (bsl3 or bsl4) is a major limiting factor since laboratories with these capabilities are not widely available. in addition, highly trained personnel that can handle infectious agents use robotic instruments and adhere to operational, engineering, and government regulations are a critical requirement for working with biothreat agents [34] . in the united states, strict guidelines have been instated for generating government-approved methods and processes for inactivation of pathogens before plates/samples can be brought out of biocontainment suites for further experimentation, and to track the inactivated material [35] . other challenges that need to be considered include the prevention of pathogen aerosolization while handling screening plates in biocontainment laboratories and ensuring that inactivation chemicals and methods are compatible with downstream procedures. new therapeutics effective against both natural and engineered resistant forms of bacterium are vital to the biodefense armory. screening for novel antimicrobials is traditionally done by scoring for growth inhibition in vitro, using the standard clinical & laboratory standards institute guidelines. this generally involves performing a dose-response assay in a multiwell plate format and monitoring growth in the absence or presence of the test compounds. the compound concentration that shows no visible growth is considered the minimum inhibitory concentration (mic). over the years, this approach has led to the discovery of only a limited number of novel antimicrobial compounds and resistance has already been generated against most of the antibiotics used in the clinic. one disadvantage to this approach is the inability to identify potent immunomodulatory compounds against intracellular pathogens that require the host for replication. new approaches to understanding bacterial pathogenesis have enabled researchers to elucidate mechanisms that could be targeted to control and clear infection in lieu of simply targeting in vitro bacterial viability. targeting the host under in vivo-like conditions (e.g., in cell culture or animal models) will be a key feature of study design to combatting intracellular pathogens that require the host for invasion and replication and will likely identify new host-directed therapeutics. the development of host-directed therapeutic (hdt) strategy relies on an understanding of the interactions between pathogens and their hosts and appropriate tools and hts assays to screen and identify therapeutics. technological progress in assay miniaturization has emerged from a combination of advanced robotic systems, high-throughput microscopy, automated image analysis, and data analysis using powerful bioinformatics tools, and this has led to the development of high-content imaging (hci), allowing for large-scale quantification of multiple cellular phenotypes at the system level. such phenotypic screening platforms rely on physiologically relevant host cell types that are permissive to pathogen infection and have the potential to identify compounds that modulate relevant biological processes in an unbiased, target, and mechanism-agnostic fashion. this cell-based approach has the added advantage that compounds that have greater mammalian cell membrane permeability, reduced cellular toxicity, and target the host proteins will be readily identified in the context of their desirable function in cells. pharmacologically active compounds can be selected that inhibit the uptake or intracellular replication of the bacterium or disrupt the host-pathogen interactions. the general workflow for high-throughput, imagebased phenotypic screening approach to identify hdts is outlined in fig. 7 .2 [36, 37] . using bacterial antigen-specific antibody to detect bacteria, this method can quantitate the number of intracellular or cell-associated bacteria and the effect of the compounds in reducing the bacterial number (% inhibition of bacterial infection), and cellular toxicity (based on loss in cell number). alternatively, one can use hci to quantitate the morphological changes of mngcs based on nuclei number and mngc size/area and use this phenotype to screen and identify compounds that prevent bacterial spread [38] . to overcome the problem of multidrug resistant bacteria, there is a growing focus on identifying small molecules that target drug resistant mechanisms or virulence factors, or agents that prevent/disrupt biofilm formation. virulence factors, such as secretion systems in gram negative bacterial pathogens, are promising therapeutic targets. specifically, the type 3 secretion system (t3ss) present in y. pestis is responsible for injecting effectors that target the cytoskeleton and proinflammatory signaling pathways. a number of techniques have been used to screen and identify potential t3ss inhibitors that can be adapted for biothreat pathogens. these include an enzyme-linked immunosorbent assay (elisa)-based detection of proteins secreted from enteropathogenic escherichia coli (epec), inhibition of sheep erythrocyte lysis by epec, inhibition of induction of a yope luciferase fusion in yersinia pseudotuberculosis, and a pseudomonas aeruginosa cell-based bioluminescent reporter screen [40] [41] [42] [43] . using a high-throughput luminescence screening assay, three compounds were identified that inhibit y. pestis t3ss-mediated cytotoxicity that relieves the growth inhibition associated with in vitro activation of t3ss [44] . another promising approach to disarm the bacteria is to prevent/disrupt biofilms, a barrier produced by bacteria to protect itself from the aggressive host environment. small molecule therapeutics that specifically disrupt or prevent the biofilm formation could be used in combination with antibiotics. the common method to quantitate biofilms is a colorimetric-based assay that utilizes a crystal violet dye to stain the biofilms and subsequent extraction of the dye using organic solvents or detergents [45] followed by absorbance measurement. to improve sensitivity, robustness, and throughput, a fluorescent-dye-based assay was developed, wherein the biofilms are stained with fm1-43 fluorescent dye and fluorescence signal is measured following organic extraction of the dye [46] . screening of a small molecule library in this assay identified rifabutin and ethavarine, as potential inhibitors of b. pseudomallei (bp82) and acinetobacter baumannii biofilm production, respectively, without directly affecting the bacterial growth. 2 phenotypic screening using high-throughput hci. cells susceptible to the pathogen of interest are seeded in hci plates. next day, cells are pretreated with appropriate concentration of the compounds and then infected with the pathogen of interest for optimal time wherein 70%-80% infection results. the infected plates are then submerged in 10% formalin for 24 h to inactivate the pathogen and to fix the cells. immunofluorescence staining is then performed, using a primary antibody specific to a pathogen antigen, and an appropriate fluorescence-labeled secondary antibody. dyes such as cell mask red and hoechst are added to detect the cell cytoplasm and nuclei, respectively. automated image acquisition and analysis is performed and data are analyzed using columbus software to quantitate the percentage of inhibition of pathogen infection and loss in cell number that represents cellular toxicity, in the presence of the compound [39] . hci, high-content imaging. there is a possibility that therapeutics targeting the virulence factors or other drug resistance mechanisms may not be effective by themselves and will need to be evaluated in combination with antibiotics to treat multiple drug resistance (mdr) infections. thus screening experiments designed to find combination therapies are warranted. to determine the synergy of two drugs (antibiotic and nonantibiotic), conventional checkerboard assays are set up wherein the two drugs are tested in combination at varying concentrations and the mic of each drug either alone or in combination is then used to calculate the fractional inhibitory concentration [47] . similarly, in the case of the biofilm assay, testing a biofilm disruptor and an antibiotic together at varying concentrations will help one to assess the effectiveness of combination therapies. unlike most bacteria, viruses require the mammalian host for replication. the virus life cycle can be divided into distinct stages that include the entry, uncoating, replication, genome packaging, assembly, maturation, and budding. various cell-based and in vitro biochemical assays have been developed to study virus life cycles as well as to screen and identify antivirals [48] . the conventional plaque-forming assay used to evaluate antivirals is time-consuming, not amenable for hts, and not very robust. alternatively, in the absence of more sophisticated instruments or technologies, a virus-induced cytopathic effect can be used as an endpoint to test antivirals. with advances in imaging instruments and informatics, a cell-based hci platform ( fig. 7 .2) that uses viral antigen-specific antibodies to detect and quantitate the viral infection is now a general approach to identify compounds that inhibit viral infection [39, 49, 50] . however, this approach will not provide information on which steps in the viral life cycle the inhibitors are disrupting. to help one to deconvolute the mechanism of action of identified hits ( fig. 7. 3), cells pretreated with an inhibitor prior to virus exposure can potentially identify compounds that inhibit viral entry, while treatment of cells after exposure (i.e., after the entry step) would identify compounds that inhibit intracellular replication and/or viral spread. assays utilizing recombinant noninfectious viruses have been generated to screen and identify inhibitors that target different stages of the viral life cycle [48] (fig. 7. 3). pseudotyped virion assays are well suited as safe alternatives for hts, since bsl3 and bsl4 wild-type pathogens are not required to complete the screens. these assays are based on viral vectors that harbor glycoproteins (gps) of different enveloped viruses and a reporter gene such as green fluorescent protein (gfp) or luciferase flanked by packaging signals, are used to generate chimeric replication-deficient viruses, and then used to screen and identify entry inhibitors. this approach has successfully identified entry inhibitors for lassa, ebola, and nipah viruses [51] [52] [53] [54] . cell fusion assays, including cell-cell or cell-virus fusions, have been developed to screen and identify hiv-1 fusion inhibitors, but to date no such assays have been developed for biothreat viruses [55] . reverse genetic systems or minigenome assays have proven to be valuable models to study rna virus replication and transcription. this model system is used to screen and identify antivirals [56, 57] . replication competent minigenome systems wherein some of the viral open reading frame is replaced with a reporter gene (gfp or luciferase) and the cdna copy cloned into a plasmid is cotransfected into mammalian cells with individual plasmids each containing a viral ribonucleoprotein (rnp). the target genes in the expression vectors are under the control of either a mammalian rna polymerase i or ii or t7 rna polymerase (which will require transfection of a plasmid containing the t7 rna polymerase) promoters. following transcription, the resulting viral rna is complexed with the rnp components and there is subsequent replication of the virus genome and expression of the reporter protein. minigenome systems have been developed for several biothreat pathogens, including filoviruses, arenaviruses, and bunyaviruses, and have been used to screen and identify small molecule inhibitors of filovirus and arenavirus replication [56] [57] [58] [59] [60] . to study the viral assembly and budding, another surrogate model that can be used is based on virus-like particles (vlps) that are mimics of viral protein assemblies made by reconstituting the viral recombinant structural proteins. vlps are noninfectious as they do not contain any viral genome, but are intrinsically immunogenic, and hence are being extensively investigated as potential vaccine candidates [61] . in the case of the ebov vlp-based assay, cotransfection of plasmids encoding the viral gp and the matrix protein (vp40) results in spontaneous formation of filamentous vlps that are released into the medium and can be quantitated by elisa [62] ; thus this model can be useful in drug discovery research [63] . to identify inhibitors of viral genome replication, in vitro biochemical assays targeting viral enzymes such as polymerases, methyltransferases, helicases, as well as viral and host proteases such as cathepsins or kinases have been developed. a number of antivirals that have been approved by the fda target either the dna or rna polymerases. incorporation of radioactive nucleotide either to a dna oligonucleotide by dna polymerase [64] or to a homopolymeric rna as a template by rna polymerase are common methods to determine polymerase activity [65] . a recent study reported the use of fluorescent dye to detect the double-stranded rna and the feasibility of developing this assay to screen and identify inhibitors of zika virus polymerase activity [66] . the host lysosomal protease cathepsin l (cat l) is necessary for the processing and cleavage of the gp of enveloped viruses, so that the virus can fuse with the host cell membrane and gain entry into the host. thus cat l has been regarded as an ideal target for drug discovery. a fluorescence resonance energy transfer (fret)-based cat l enzymatic assay was developed, wherein peptides derived from gps of viruses such as ebola, nipah, hendra, and severe acute respiratory syndrome and middle east respiratory syndrome coronavirus and containing cat l cleavage site were chemically conjugated with a quencher 5-carboxytetramethylrhodamine at the n-terminus and 5-carboxyfluorescein fluorophore at the c-terminus [67] . the intact peptides exhibited minimal to no fluorescence, but following cleavage of the peptide by cat l, there was an increase in fluorescence intensity. screening of a chemical library in this assay identified small molecules that selectively inhibited cat l-mediated cleavage of multiple viral peptides over host proneuropeptide y [67] . viral proteases are also good drug targets as they play a vital role in viral replication. for example, the ns2b-ns3 protease is highly conserved among the flaviviruses and a fret-based enzymatic assay using a synthetic peptide substrate [68] was developed to identify west nile virus protease inhibitors [69] . functional genomic screening using gene-trapping, crispr's gene editing, or rna interference (rnai) technologies has been applied to identify host factors that are required for replication or involved in pathogenesis of several biothreat viral and bacterial agents and are summarized in table 7 .2. the activities of several identified host factors can be perturbed by small molecules and thus serve as potential therapeutic platforms. for example, it was demonstrated that the novel host factor inositol-requiring enzyme 1α is required for brucella infection in mammalian cells [70] . reducing the levels of either the retromer cargo-adapter complex or retromer-associated sorting nexins abrogated c. burnetii replication [71] . multiple host kinases such as camp-dependent protein kinase, protein kinase b, and protein kinase c all play a role during c. burnetii infections [72, 73] . zhou et al. [74] identified tnfrsf9 and serpini1 that may promote activated macrophages in controlling f. tularensis replication. akimana et al. [13] showed that f. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and that phosphoinositide metabolism is essential for cytosolic proliferation of f. tularensis. connor et al. [14] revealed that 71 host proteins are required for intracellular survival of y. pestis. of particular, interest was the enrichment for genes involved in endosome recycling. using the gene trapping approach, carette et al. [76] first identified several host factors that are required for ebov infection. these include a cholesterol transporter niemann-pick c1 factor involved in the fusion of endosomes and lysosomes (homotypic fusion and protein sorting complex), biogenesis of endosomes (pikfyve), lysosomes (bloc1s1, bloc1s2), and targeting of luminal cargo to the endocytic pathway [76] . many of these hits reoccurred in several crispr and small interfering rna (sirna)/shrna screenings [77, 78, 81] . in addition, lysosomal protein (bri3) and a gtpase involved in the regulation of vesicle trafficking (rab39b), pi3k, calcium/ calmodulin kinase-related network, and de novo pyrimidine synthesis pathway are essential for ebov replication and transcription [78] [79] [80] . the application of rnai screening has been utilized for other viral pathogens such as henipavirus, junv, poxvirus, vaccinia virus, and veev [75, [82] [83] [84] [85] [86] . it is demonstrated that catalytic activity of fibrillarin, the enzymatic subunit of the snornp complex that is responsible for catalyzing the transfer of a methyl donor from a bound cofactor s-adenosyl methionine to ribose sugars of the target pre-rrna, is required for henipavirus infection [82] . voltage-gated calcium channel (vgcc) subunits were shown to be important in junv-cell fusion and entry into cells. gabapentin, an fda approved anticonvulsant drug against α 2 δ 2 subunitcontaining vgccs, inhibited replication of the vaccine strain of junv in mice [75] . other sirna-based screens against v. virus identified that amp-activated protein kinase (ampk) promotes viral entry through the control of actin dynamics, and knockdown of nuclear pore protein (nup62) arrests virion morphogenesis [83] [84] [85] . lastly, an sirna screen identified trafficking host factors that modulate veev infection [86] . an alternative approach to gain an in-depth understanding of host-pathogen interactions during infection is to construct a protein-protein interaction network between host protein and bacterial virulence factors. using a yeast two-hybrid (y2h) library, memiševic´ et al. [16] identified a molecular network that governs b. mallei infection. similarly, a y2h study conducted by yang et al. [87] showed the involvement of focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, toll-like receptor (tlr), and mapk signaling pathways during y. pestis infection. to complement the y2h study, reverse-phase protein microarray analysis was used to interrogate changes in protein expression and posttranslational modification. this further revealed the roles of ampk-α1, src, and gsk3β in regulating b. mallei and b. pseudomallei infection [88] , and thus, as viable host targets for countermeasure development. prior to the initiation of medical countermeasure development against specific pathogens, a target product profile (tpp) is needed to define the required features of potential drug candidates (e.g., route of administration, prophylactic vs therapeutic, trigger to treat, and onset of action requirements). once a tpp is in place, a screening funnel is drafted that sets laboratory criteria and defines clear go/no-go decision points that are needed to progress countermeasures from discovery through preclinical development and into human clinical trials. irrespective of the types of assays used for countermeasure screening, compounds identified as having significant inhibition in primary screens are validated in subsequent dose-response experiments to determine the half maximal effective concentration (ec 50 ) and cytotoxic concentration (cc 50 ). potent compounds that have an adequate selectivity index (e.g., >10) that is defined as a ratio of cc 50 / ec 50 , are then often tested in orthogonal assays in appropriate cells/tissues to better understand or validate the antipathogen activity. ideally, compounds are further optimized for potency, selectivity, physicochemical, and pharmacokinetic (pk) properties and safety prior to in vivo evaluation to assess efficacy in appropriate animal models of infection ( fig. 7.4) . many of the therapeutics that are in different stages of either preclinical or clinical development for select biothreat pathogens include small molecule antivirals (tables 7.3 and 7.4), antibody (or antibody cocktails) against viruses or bacteria/virulence factors (table 7 .5), and combination drug therapy (table 7 .6). the increased use of antivirals and antibiotics has set the stage for rapid adaptation mechanisms that microbes can use to counteract them. the development of antimicrobial resistance is one of the biggest public health threats and hence alternative approaches to treat infectious diseases are urgently needed. table 7 .7 lists the resistance mechanisms identified in each biothreat bacterial pathogen and provides references for targets of resistance. since stand-alone antibiotics may not be sufficient to overcome resistance and/or completely clear some biothreat bacterial infections, we have also included encouraging data on host directed therapeutics, and combination therapy. hdt is an emerging approach in the field of antiinfectives discovery. the strategies behind hdt can include modulation of host immune responses, or interference/manipulation/targeting of host-cell factors that are required for pathogen replication [202] . for example, in a potential bioterror scenario, where the identity of the etiological agent causing the disease is unknown, stimulation of innate immunity may be particularly useful as induced immune responses are often capable of providing protection against a broad range of pathogens. although no fda-approved hdt therapies are yet available for treating infectious diseases, we have summarized in this section the antimicrobial primary screening of small molecule chemical libraries in the phenotypic hci assay will identify compounds that inhibit pathogen infection as well as those that may contribute to cellular toxicity. generally, hits that show ≥50% infection inhibition and ≤20% loss in cell number are then subjected to hit triage or in silico filtering wherein compounds with optimal physicochemical properties such as solubility, lipinski's rule of 5, metabolism are selected for potency testing in the phenotypic screening assays. compounds that exhibit an ec 50 ≤ 1 µm and si, which is a ratio of cc 50 /ec 50 > 10, are then further optimized through iterative cycles of synthesis, testing in cell-based and orthogonal assays and in in vitro admet studies to improve potency and physicochemical properties. if the target of the hit molecule is identified then a target-based screen is performed and the hits identified are optimized through the iterative structureactivity relationship cycle. the lead series candidates are then evaluated in vivo for their pharmacokinetic properties and then for efficacy in appropriate challenge models of infection. admet, absorption, distribution, metabolism, excretion, and toxicity; hci, high-content imaging; si, selectivity index. antibiotics aigiv combination therapy with antibiotics and aigiv is more effective than antibiotics alone in a rabbit model of inhalational anthrax and improved survival compared to the antibiotic treatment alone [155] ciprofloxacin clindamycin treatment of rabbits with systemic anthrax with clindamycin and ciprofloxacin had improved efficacy compared to monotherapy and could be used to prevent relapse of infection [156] ciprofloxacin combination therapy for anthrax, including antiprotective antigen (pa igg) antibodies and ciprofloxacin in a rodent anthrax model increased survival significantly compared to ciprofloxacin treatment alone [157] levofloxacin raxibacumab combination therapy with raxibacumab, an igg1 monoclonal antibody that binds protective antigen, and the antibiotic levofloxacin provides protection in rabbits late in the disease course [158] oligochlorophen antibiotics targeting cytoskeletal proteins such as ftsz with oligochlorophen analogs is a promising new treatment method that has a 10-fold lower development of resistance compared to antibiotics used for anthrax treatment in humans [159] penicillin, meropenem, or rifampin linezolid treatment of antibiotic-resistant inhalation anthrax with linezolid and penicillin, meropenem, or rifampin had the inhibitory effect on mean lethal factor levels compared to the control groups and successfully treated fluoroquinolone-resistant b. anthracis infection [160] rifampin clindamycin combination therapy for anthrax with rifampin and clindamycin was shown to be synergistic in vitro [161] brucella spp. rifampin successful combination therapies used to treat pulmonary brucellosis in humans is doxycycline and rifampin for 6 weeks [162] burkholderia mallei antibiotic heat-killed vaccine combination of an antibiotic moxifloxacin, azithromycin, or sulfamethoxazole-trimethoprim, and vaccination using heat-killed b. mallei can protect balb/c mice from lethal glanders infection, potentially by stimulating immune responses, such as gamma interferon, which acts synergistically with antibiotic therapy to inhibit bacterial growth [163] enrofloxacin, trimethoprim, and sulfadiazine doxycycline successful 12-week combination treatment of parenteral administration of enrofloxacin and trimethoprim with sulfadiazine followed by oral administration of doxycycline eliminated b. mallei from glanderous horses during an outbreak [164] burkholderia pseudomallei combination therapy with farnesol a sesquiterpene alcohol that damages biofilm matrix and interferes with cell wall and peptidoglycan biosynthesis, facilitates antimicrobial penetration, and reduces the minimum biofilm eradication concentration for ceftazidime, amoxicillin, doxycycline, and sulfamethoxazole-trimethoprim in vitro [165, 166] ceftazidime avibactam avibactam restores susceptibility to ceftazidime for genetically diverse extremely drug resistant isolates of burkholderia from cystic fibrosis patients by binding pena and the combination treatment significantly improved survival of larvae infected with the drug resistant isolates [166] ceftazidime ifn-γ interferon gamma-induced reactive oxygen species with ceftazidime leads to synergistic killing of intracellular b. pseudomallei and markedly increases the effectiveness of antimicrobial therapy for the treatment of b. pseudomallei infection in mice [167, 168] clostridium botulinum bont serotypes and subtypes differences present a significant challenge for creating monoclonal antibody treatments for neutralization, by diversifying the v-regions of mabs and selecting cross reactivity, a combination treatment of three antibodies neutralized bont/f1, f2, f4, and f7 in mice and was 150 times more potent than equine antitoxin [169] coxiella burnetii doxycycline chloroquine combination therapy of doxycycline and hydroxychloroquine combination shortened the duration of therapy and reduced the number of relapses in patients with q fever endocarditis. and a case of q fever endocarditis with biological prosthetic aortic valve and aortic homograft was successfully treated with doxycycline and chloroquine combination therapy [170, 171] francisella tularensis cytochalasin b, ly294002, wortmannin, nocodazole, mg132, and xva143 inhibitors reduce f. tularensis update and reduce inflammatory cytokine production and can be used in combination with antibiotics to improve survival of infected mice [172] gentamicin membrane antigen immunization postexposure immunization with membrane protein fraction antigens and treatment with low-dose gentamicin increased survival of mice and significantly reduced bacterial burdens in the liver and spleen [173] potential of several small molecule immunomodulators and host cell factors that have been investigated to date. immunomodulators directly target the host rather than the pathogen (fig. 7.5 ). this is accomplished by targeting pattern recognition receptors, such as tlrs that are present on innate immune cells in the host to detect features of microbes known as pathogenassociated molecular patterns. since immunomodulators target host immune cells, they are an attractive candidate for use against bacterial agents as they are unlikely to result in the development of antibiotic resistance even after repeated use. in particular, the threat of an intentional release of a highly virulent bacterial pathogen that is either intrinsically resistant to antibiotics, or has been weaponized via the introduction of antibiotic resistance, makes immunomodulation an attractive complementary or alternative strategy to directly targeting bacterial biothreat agents. for example, a synthetic tlr9 agonist, 5'-c-phosphate-g-3' oligodeoxynucleotide (cpg odn), appears to be able to stimulate protective immunity against intracellular bacterial infection and/or eliminate chronic infections. indeed, studies in mice have demonstrated that the innate immune defenses activated by cpg odns protect against lethal challenge with b. anthracis, b. mallei, and combination therapy, including antibiotics with an efflux pump inhibitor, would be a novel mechanism to restore the efficacy of the antibiotic in resistant strains of y. pestis [174] antibody therapy corticosteroid the addition of antiinflammatory methylprednisolone, a corticosteroid, in combination with antibody therapy correlates with improved mouse survival, with reduction in neutrophil and matrix metalloproteinase 9 in the tissue, and the mitigation of tissue damage [175] ciprofloxacin l-97-1 a novel postexposure medical countermeasure l-97-1, an a 1 adenosine receptor antagonist blocks lps-induced activation of immunomodulatory cytotoxic substance accumulation to prevent acute lung injury, and in combination with ciprofloxacin improves survival of rats following infection with y. pestis [176] aigiv, anthrax immune globulin intravenous; ifn, interferon; lps, lipopolysaccharide; pa, protective antigen. aminoglycosides rnd-type efflux pumps, s-adenosyl-l-methioninedependent methyltransferase, amrr [186, 187] β lactams pena a , nlpd1, dacc, flgn, sch, tr70_0856, tr70_1911, ftsi, amrr, bper, bpet, spot, trna, rrna, proteins with unknown function, sers seryl-trna synthetase, and rnd efflux pump amrab-opra, and bpeef-oprc [186, [188] [189] [190] [191] [192] macrolides amrab-opra efflux pump [193] quinolones amrab-opra efflux pump, bpeab-oprb efflux pump [194] sulfamethoxazole/ trimethoprim rnd bpeef-oprc efflux pump, lysr-type regulator bpet bpes, ptr1, fola, amrr tetr-type regulator, amrab-opra, metf [186, 195] quinolones gyra a [196] tetracyclines putative protein secretion targets, biosynthesis of pantothenate and coenzyme a, aspartate biosynthesis, dna replication [197] francisella tularensis β lactams blab1 [198] chloramphenicol 23s rrna, the l4 and l22 ribosomal proteins, and overexpression of efflux pumps [199] quinolones gyra a and gyrb [200] f. tularensis or their surrogates [203] . similarly, human monocyte-derived macrophages treated with poly(i:c), a synthetic tlr3 agonist, showed significantly reduced intracellular f. tularensis [both schu 4 and lvs (live vaccine strains)] replication. mice administered with poly(i:c) before or after schu 4 or lvs infection showed reduced bacterial burden in the lungs and prolonged survival. mice treated with poly(i:c), challenged with f. tularensis, and then treated with levofloxacin showed 100% survival relative to no survival in animals receiving levofloxacin alone [204] . in addition to targeting innate immune cell receptors, there is a growing interest in modulating autophagy as an immunotherapeutic intervention. autophagy is a dynamic process that targets cellular cytoplasmic contents for lysosomal degradation. more specifically, xenophagy is a type of selective autophagy that specifically targets intracellular pathogens to lysosomes, retracing their replication and survival [205] . the use of autophagy inducer rapamycin, decreased the survival of b. pseudomallei in vitro [206] . however, several bacteria exploit autophagic machinery as part of their intracellular life cycles (i.e., brucella abortus, c. burnetii, and f. tularensis). therefore infection may be exacerbated by the induction of autophagy (fig. 7.1c ) [205] . research to further understand the balance between infective and protective cellular targets in the autophagy pathway may enhance its utilization as a therapeutic target. hts of fda-approved drugs is another approach to identifying compounds that were previously approved for other disease indications but may have the potential to be repurposed as antiinfectives. trifluoperazine (an antipsychotic), amoxapine (an antidepressant), and doxapram (a breathing stimulant) mitigated fatal y. pestis infection in a pneumonic plague murine model [207] . at 48 h postinfection, these drugs provided animals with up to 100% protection against challenge with bubonic or pneumonic plague agents when administered in combination with levofloxacin [208] . multiple fda-approved drugs targeting g-protein coupled receptors and calcium fluxes inhibited c. burnetii and b. abortus, whereas drugs targeting cholesterol traffic attenuated c. burnetii [209] . similarly, increasing evidence suggested statin, a 3-hydroxy-3-methylglutaryl-coenzyme-a reductase inhibitor, possesses antibacterial activity by the inhibition of sterols, prenylation, and isoprenoids (c. burnetii), the inhibition of antiinflammatory cytokines (y. pestis), and the modulation of phagosome maturation (c. burnetii) [210] . it was demonstrated that a low dose of gleevec, an anticancer drug inhibiting abl1, c-kit, and related protein tyrosine kinases, can increase the number of myeloid cells in the bone marrow, blood, and spleen and enhance antimicrobial responses in a mouse model of f. tularensis infection [211] . in the case of viruses, small molecule targeting of innate immune receptors has also shown efficacy in several relevant viral models of infection. for example, treatment with poly(ic:lc) has also been protective against ebov infection in nhps [212] . prophylactic pulmonary administration of tlr7 ligand (tmx201) significantly protected mice from lethal infection with veev [213] . tlr3 and tlr9 agonists have also been shown to improve the efficacy of postexposure therapeutics against smallpox [214] . sometimes modulation of host pathophysiological responses can be evaluated as a target. hemorrhagic fever virus pathophysiology includes the stimulation of procoagulant pathways and increased permeability of the vascular endothelium; therefore these processes are being evaluated as possible targets for therapeutic intervention. this could be accomplished by utilization of an anticoagulant, such as recombinant nematode anticoagulant protein c2 (rnapc2) that blocks initiation of the extrinsic coagulation pathway by inhibiting the tissue factor-factor viia complex [215, 216] . rnapc2 has been shown to be highly protective in macaques infected with a lethal dose of ebola zaire virus, when treatment was initiated 1 day post viral challenge [216, 217] . whereas hdt targets the host directly, antibody therapy is the passive process of activating the immune system to respond to microbial threats. sources of antibodies can include individuals that survive infection or have received a prophylactic vaccine against a microbe. alternatively, antibodies can also be generated ex vivo using cell culture. historically, antibody-based serum or plasma therapy has been widely used to treat a variety of infectious diseases. limitations for clinical use arise however from the polyclonal nature of serum antibodies, resulting in lot-to-lot variation, approaches for determination of correct dose levels and regimens, and a risk for allergic reactions and transmission of transfusion-borne diseases. in general, limited clinical applications for antibody therapy existed until the development of technology that allowed the production of mabs through the use of hybridomas [218] . hybridomas allow for the production of homogenous antibodies with the same specificity of a single immunoglobulin class and isotype. further advancements made it possible to humanize or generate fully human mabs. research advancements in the past 10-15 years have resulted in numerous mab-based therapies that have been approved for inflammatory and neoplastic diseases. infectious diseases have not been included in approved treatments. although many mab products targeting infectious diseases are in different stages of development, to date, one mab-based product, synagis (palivizumab), is currently approved for use in infectious diseases (rsv) [219] , while two mabs abthrax (raxibacumab) and anthim (obiltoxaximab) have been approved under the fda's animal efficacy rule for treatment of inhalation anthrax [8] . for treatment of ebola infection, the single mab mab114 and zmapp, a cocktail of three "humanized" mabs, have advanced in product development and are being tested for efficacy in the ongoing ebola outbreak in the democratic republic of the congo (nct03719586, www.clinicaltrials.gov). it is clear that mabs offer a highly specific, potent, and generally safe platform for antimicrobials and may be a useful alternative to immune plasma. it is imperative to find appropriate niches in infectious diseases, specifically those caused by biothreat agents, where new antibody-based treatments could prove to be efficacious [220] . table 7 .5 summarizes key research in antibody therapy across different bacterial and viral families of some current biothreat agents. the utilization of mab therapy for the prophylactic or therapeutic treatment of biothreat agents varies depending on the agent. in all cases however, the challenge for the development of effective therapeutic antibodies against viruses is the viruses' heterogeneity and mutability. a related problem is the low binding affinity of cross-reactive antibodies that are capable of neutralizing a variety of primary isolates. finally, the cost of large-scale production of mabs is a limiting factor for continued use. a solution to the challenges with viral mutagenicity may be found in the identification of potent new mabs that target highly conserved viral structures, which are critical for virus entry into cells. alternatively, utilization of combination therapy, whereby, a cocktail of several mabs may be used or mabs may be combined with other drugs, such as antiviral compounds, may overcome mutagenicity issues. these areas of research will continue to be a major focus of biothreat agent therapeutic research [221] . for countermeasures against lethal viral infections (i.e., category a), table 7 .3 lists reported studies in either mice or nhps that have shown significant benefits to survival in challenge models. the table also includes in vitro potencies, viral strains, specific animal species, dosing regimens, routes of administration, pks, and benefits to survivaldata necessary for the reader to relate in vitro potency to in vivo efficacy, assess/interpret results, and make comparisons. the corresponding chemical structures are provided in fig. 7 .6. table 7 .4 displays the status/results of clinical trials for therapeutics used for the treatment of infections caused by ebov and lasv. noteworthy, most of these clinical trials were underpowered without appropriate controls and hence results may be speculative. combination therapies are an excellent approach to improve treatment outcomes, shorten treatment duration, and overcome microbial resistance mechanisms caused by biothreat pathogens. combination therapy may incorporate antibiotics or antivirals with hdt or antibody therapy at rationally designed treatment schedule. in this way the usage of multiple treatment modalities can synergize to optimize the mechanism of action of biothreat-targeted therapies. table 7 .6 includes combination therapies that have been used to treat each biothreat bacteria. in the case of viral infections, while combination therapy has been used for treatment of patients with human immunodeficiency virus (e.g., combination of nucleoside, nonnucleoside, protease, and/or host-targeted inhibitors) or chronic hepatitis c virus infection (e.g., combination of polymerase and rna-binding protein ns5a inhibitors), to date there are no reported studies for biothreat viral agents. several β lactam antibiotic drugs have been able to overcome deactivation when delivered in combination with inhibitors that target extended-spectrum β lactamases (enzymes that are overexpressed in the mdr pathogens, inactivate the β lactam antibiotic by cleaving the β lactam ring and thus one of the major contributors of antibiotic resistance). the β lactam/β lactamase inhibitor combination drugs that have been fda-approved include augmentin xr (amoxicillin/clavulanate combination), unasyn (ampicillin/sublbactam combination), and zosyn (piperacillin/tazobactam combination). in the case of biothreat bacteria, the combination of ceftolozone and tazobactam exhibited increased in vitro susceptibility to a variety of clinical, environmental, and animal strains of b. pseudomallei, but to date it has not been evaluated in in vivo efficacy studies [222] . challenges to developing countermeasures against biothreat agents are many, but some of the unique and key challenges are pk differences in healthy versus infected subjects, mapping the biodistribution of the countermeasure to the biodistribution of the pathogen, and limited opportunities to run randomized, controlled clinical trials. preclinical studies typically require a pk study in healthy animals to guide dose selection prior to testing a countermeasure in an animal model of infection. in that regard, it is critical to understand what cells and tissues the pathogen is infecting over time so that countermeasures can be properly designed to reach infected tissue. for example, countermeasures against pathogens causing encephalitis require drug to reach the central nervous system (cns). in contrast, ebov was found to infect lymph nodes, spleen, and liver in nhps 2-3 days following viral challenge, and by days 5-6 the virus was detected throughout the body (fig. 7.7 ) [217] . thus if one was designing a countermeasure against ebov infection, it would likely require a wide tissue distribution in order to be effective. to complicate things further, infected animals often have altered metabolizing enzymes (e.g., cytochrome p450s) [223] , tissues, and barriers (e.g., blood-brain barrier) making drug exposure difficult to predict. running pk experiments in the presence of infection would eliminate many of these variables, but this is seldom done for countermeasures to biothreat agents, since it requires running these experiments in biocontainment labs. because biothreat pathogens cause infrequent human cases and outbreaks in generally remote areas of the world, planning a traditional human clinical trial with large numbers of participants is not feasible. even when the west african ebola outbreak of more than 28,600 cases was unfolding in 2014-2016, and now that there is a large outbreak unfolding in the democratic republic of the congo, the amount of clinical efficacy data that have been collected for ebov therapeutics is quite limited. the limitations are due to difficulty of performing clinical research in a remote outbreak setting where cultural, geographical, and political barriers may hinder or halt trial planning [225] . the bulk of the efficacy data for ebola published in the literature has been garnered through animal studies. to enable product development for viral, bacterial pathogens as well as for chemical, toxin, and radiological threat agents for which outbreaks or cases are sparse, the fda issued an animal rule, codified 21 cfr 314.600 in 2002, that proposes to permit consideration of product development and efficacy data obtained from animal studies for drug licensing, in lieu of human clinical trials when such trials would be unfeasible or unethical [226] . since introducing the animal rule in 2002, the fda has approved more than a dozen products, including several therapeutics for anthrax, plague, botulinum toxin, and smallpox [8, 29] . the animal rule does not provide an expedited pathway to fda approval for drugs and can certainly be more challenging than traditional drug development pathways. the developer must compile a significant body of data to prove efficacy of the drug against the target therapeutic indication. first in 2009, and updated now into a formal document published in 2015 [226] , the fda has released guidance for industry describing critical data elements required for animal efficacy studies for drug approval under the animal rule: (1) the pathophysiology of disease and the mechanism of action by which the drug prevents or ameliorates disease must be reasonably well understood; (2) it is desired that the efficacy must be demonstrated in two animal species, although multiple studies in one species can be acceptable if the animal model is sufficiently well characterized and accurately predicts the human response; (3) the animal study end point must be clearly related to the desired human efficacy end point, such as enhancement of survival; and (4) pk and pharmacodynamics data must be generated in the animal studies to allow selection of an effective dose in humans. under the animal rule, efficacy studies are expected to demonstrate that drug effectiveness in animals reliably indicates efficacy in humans. thus while traditional human clinical efficacy studies require demonstration that the therapy is effective, the animal rule imposes an additional burden on investigators to establish a drug candidate's mode of action in at least one animal model that reproduces accurate human disease pathology. further, the animal rule outlines considerations for the development of the model(s), to include the use of an isolate of the etiologic agent that was known to cause human disease (e.g., agent was isolated from a fatal human case if it is a lethal disease, such as ebola) [227] . there is also a requirement that the infection model using the chosen pathogen strain must present the same or similar pathophysiology as the human disease. definitive animal model efficacy evaluations should be performed only after careful model development studies have been performed and accepted by the regulators. these studies are known as natural history studies and are carefully designed to investigate and describe the course of the disease in the animal species, through clinical, serological, and histopathological evaluations, to compare the features of the disease in the model to the features of disease in human cases. it is important to consider the route of pathogen exposure (nasal, oral, and aerosol routes) to the animal because this will model the natural or unnatural modes of exposure predicted for humans, where a biorelease would constitute an unnatural exposure. a dose of challenge agent that is thought to be predictive of the human exposure level in a biorelease scenario should be used to develop the model, and that dose should be well characterized and reproducible by a quantitative measure. the route of drug delivery, dose administration timing, and treatment regimen in response to a biorelease scenario must also be considered when designing the animal model studies for a drug under development for such an indication. it is possible that a biorelease scenario would not be immediately known, and a period of time might pass before people begin to develop symptoms. studies evaluating the cutoff time for drug to still be effective, and what are the triggers for treatment should be investigated in the animal model. animal rule pivotal efficacy studies are essentially performed in place of traditional phase 3 clinical studies, so they must be done in the containment laboratory under a quality system [226] . use of fda good laboratory practice or other comparable quality system with high levels of documentation and data integrity is paramount, so data packages can withstand regulatory review and audit [228] . the studies must be designed so that the program will collect the same results and conclusions one would expect from a well-designed traditional phase 3 trial, but in addition a pivotal animal efficacy study must describe a mechanism of action for the treatment modality to prevent or block disease or tissue infection and damage [227] . the pathologic mechanism needs to be consistent and well understood across both the human and animal models, such as the mechanism of pathogen entry into the target host cell, toxicological mechanism of lethal factors, or germination of spores and dissemination of bacterial infection in target cells and tissues, all of which may be mechanisms the drug under study is known to block; this must be proven in the animal model. products developed under the animal rule are subject to postmarketing or field studies when the product is actually used in the scenarios for which it was developed, and this is required to verify a product's clinical benefit [8] . part of the approval process is a requirement to have postmarketing study plans in place, for quick execution should an event occur in which the drug would be field tested. approval may also come with restrictions for off-label use, distribution, or access. actual use will also come with requirements to inform patients of the conditions under which the drug was approved by virtue of only animal efficacy data, making them informed consumers as to the risks of possible nonefficacy or unknown effects in cases of human disease. with the advancement of systems and synthetic biology and the ease of genetic modification, biothreats are becoming more complex and there is a growing need for novel treatments that can have broad-spectrum activity against new, remerging, and engineered pathogens. developing novel countermeasures that can effectively treat and prevent massive casualties is an ongoing challenge that remains a central priority for future research. the development of novel therapies relies on an improved understanding of the host-pathogen interactions. key virulence factors have been identified and targeted for potential treatment options, including biofilm and t3ss inhibitors for bacterial infections, and viral entry or polymerase inhibitors for viral infections. combining hts with systems biology provides a robust, coordinated approach to identifying therapeutic targets. since stand-alone antibiotics or antivirals may not be sufficient to overcome resistant or engineered biothreat infections, a focus on combination therapy, antibodies, and hdts is the key countermeasure. although many challenges are faced when developing novel therapies for biothreat pathogens and no fda-approved hdts are yet available for treatment, there is potential for novel small molecule host-targeted immunomodulators to be developed. screening of fda-approved drugs is a powerful approach to possibly repurpose drugs for new disease indication and/or identify compounds that are safe and effective in humans, which can also have antibacterial or antiviral capabilities. three fda-approved drugs have shown potential in mice against pneumonic plague [207] . serious challenges still remain with the prevalence of antibiotic resistance that jeopardizes the effectiveness of our current treatment options for bacterial threats. in addition, the complex intracellular life cycle of many biothreat pathogens requires therapeutics that can penetrate the host cell. there are limited fda-approved viral countermeasures for prevention and treatment. none of the filoviruses or henipaviruses has approved therapeutics or vaccines available for human disease. some vaccines exist for new world alphaviruses, but no current therapeutics are effective for treatment after infection. focused efforts using hts to develop novel, effective, and broad-spectrum medical countermeasures will provide a robust response capability 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are early and sustained targets of infection experimental drugs poised for use in ebola outbreak us depart of health and human services, center for drug evaluationproduct development under the animal rule: guidance for industry establishing efficacy of human products using animals: the us food and drug administration's "animal rule chapter 48-bioresearch monitoring inspection of nonclinical laboratories conducting animal rule specific studies key: cord-262524-ununcin0 authors: bankhead, armand; mancini, emiliano; sims, amy c.; baric, ralph s.; mcweeney, shannon; sloot, peter m.a. title: a simulation framework to investigate in vitro viral infection dynamics date: 2011-12-31 journal: procedia computer science doi: 10.1016/j.procs.2011.04.195 sha: doc_id: 262524 cord_uid: ununcin0 abstract virus infection is a complex biological phenomenon for which in vitro experiments provide a uniquely concise view where data is often obtained from a single population of cells, under controlled environmental conditions. nonetheless, data interpretation and real understanding of viral dynamics is still hampered by the sheer complexity of the various intertwined spatio-temporal processes. in this paper we present a tool to address these issues: a cellular automata model describing critical aspects of in vitro viral infections taking into account spatial characteristics of virus spreading within a culture well. the aim of the model is to understand the key mechanisms of sars-cov infection dynamics during the first 24hours post infection. we interrogate the model using a latin hypercube sensitivity analysis to identify which mechanisms are critical to the observed infection of host cells and the release of measured virus particles. in the past ten years there has been a growing interest in modeling viral infections for the study and characterization of host infection dynamics. early mathematical models were typically based on ordinary differential equations (odes) and focused on extracting key parameters of the infections dynamics [1, 2] . later on, interest moved to investigating how spatial relations affected the system dynamics, thus moving to cellular automata (ca) models [3, 4, 5] . more recently the attention has shifted to viral respiratory diseases due to the increasing danger of pandemics such as the "spanish flu" that caused over 50 million deaths worldwide in 1918. near pandemics such as severe acute respiratory syndrome (sars), resulting in 8,096 infected cases and a 9.6% death rate, have provided unfortunate reminders their continued health risk and significant economic impact [6] . new research projects are being funded to investigate the different scales of threat, from the epidemiological spread of a virus in a population to its early phases of the infection in a cell culture. among the papers published on this topic, in particular on the influenza virus [7, 8, 9] , there are bocharov's ode model [10] and beauchemin's ca model [3, 4, 11] . both models open access under cc by-nc-nd license. described the dynamics of the influenza infection in the upper epithelial areas of the lungs and were validated by clinical data. although they take into account the effect of the immune system on the dynamics of the disease, describing the infection until its final outcome (more than a week after the first infection), none of them analyzed which mechanisms where critical in the dynamics of the first and second round of infection. to understand the key mechanisms of viral respiratory diseases, we focus instead on early phases of viral infection by using in vitro experiments observing lung cells up to 24 hours post infection (pi). the in vitro experiments provide measurements of virus titer, spatial characteristics of cell growth and, through green fluorescent protein (gfp) imaging, of the infection spread. our computational model focuses on simulating the early stages of a viral infection in a population of cells plated on a culture well. the choice of a ca model was natural since the in vitro infections being studied use host lung cancer cell lines that form a fixed mono-layer in which spatially dependent aspects of infection may be present [12, 13] . we developed this computational model using the multi-agent system visualization (masyv) platform [3] . in contrast to previous models, we explicitly focus on the dynamics of virus spread on a population of cells, supported by experimental data from an in vitro model system. we also explicitly model the infectious viral particles as discrete entities, whereas in previous models the infection of cells followed simple ca rules depending on the states of neighboring cells. these viral particles are released by infected cells according to a specific function based on time post infection, and move over the well with a random walk algorithm. this representation allows us to model the mechanisms of virus spread in an environment where the virus is not confined and can also infect cells not adjacent to the infected ones. in section 2 we describe the model design and its main features. we also describe the sars infection experimental data used to parameterize the model. in section 3 we present a sensitivity analysis that identifies the critical mechanisms characterizing the early phases of the infection. we also show that the model can explain the experimentally observed virus titer data and allows a deeper understanding of the infection dynamics in the in vitro experiments. the computational model is built using beauchemin's masyv platform. the software consists of a server providing i/o and supervisory services to the various client modules where the simulation is actually coded. our choice to use masyv was partially driven by flexible and powerful graphical visualization routines that facilitate comparison to images provided by the experimental collaborators. masyv has a c-based api and is open source allowing finalized custom models to be easily shared. we discuss novel contributions and differences from the previous modules. the original module details are presented in beauchemin et al. [3] . our model reproduces a viral infection on a population of cells plated on a culture well. in our client we consider, as the target of the viral infection, calu-3 cells that are a human airway epithelial cell line derived from human lung cancer. we model these host cells using a 130x130-site ca model where each site represents either one calu-3 cell or an empty space. at the beginning of the simulation each lattice site is initialized and labelled with "uninfected" or "empty" states as described below in section 2.2. uninfected cells are initially stochastically infected with virus through a first round of infection at the beginning of the simulation, described in section 2.3, and once infected progress through the following states: containing: initial infection state representing viral entry and hijacking of host cell mechanisms necessary for viral replication. expressing: cell is actively producing and assembling virus capsids and genomes internally, but has not begun releasing virion. infectious: assembled virion is being released from the host cell according to the release function (section 2.4) by examining the experimental viral titer data shown in figure 1 we derived temporal delay of the state transition between containing and infectious. the ~ log 10 3 viral titer measurements at time points 0, 4 and 7 hours post infection are residual virus left over from the initial infection after washing. we set the delay for release of new infectious viral particles to 7 hours to represent the jump in observed virus titer between 7 and 12 hours. cells release virus particles according to a viral release function, described below. virus particles are entities that diffuse from one lattice point to another according to a random walk algorithm (section 2.4). these free-floating virus particles stochastically infect uninfected cells in a second round of infection. the ca lattice is therefore like a tissue of immobile cells with infective virions moving over it. we represent only infectious viral particles to relate their count in the model with the viral titer measured in terms of plaque forming unit per ml (pfu/ml). a pfu is a measure of the number of viral particles capable of forming plaques per unit of volume, thus only infectious particles are counted in the viral titer. the ca lattice is updated synchronously and the boundary conditions for both the epithelial and viral particles are periodic, i.e. epithelial cells and viral particles can grow or move outside the boundary of the virtual well. we use a honeycomb neighbourhood: each lattice site is considered adjacent to six sites and cells can replicate only if a neighbouring site is empty or if it contains a dead cell. only uninfected cells are allowed to replicate. when cells on the lattice are initialized all uninfected cells are stochastically assigned a time_to_death value between 1 and an arbitrary cell_lifespan variable. cell_lifespan was chosen to be large enough such that 5%, a minority of the cells, die naturally within the simulated 24 hours. cell_lifespan is not affected by infection as in vitro experimental observations showed no increase in cell death due to infection within 24 hours. in the laboratory before an in vitro infection is performed, 10 6 cells are first plated in each of the 6 culture wells present in a dish, and given time to settle down on the well surface to create a cell mono-layer, before the virus is applied to the cell culture. in the plating process, a suspension of cells is plated onto the surface of the well that adhere to the plastic in small clumps or as single cells forming "islands" that are homogeneously distributed throughout the well. cells located on the inside of these islands (surrounded by other cells) do not replicate due to contact inhibition whereas cells on the outer edges of each island replicate until they are completely surrounded by other cells. as a result each island continues to grow until there is no adjacent empty space on the well surface. to represent this behaviour we used a set of images ( figure 2 ) that shows how cells are distributed on the culture well just before the infection is performed. the number of cells forming each isolated island was counted for each image obtaining a distribution of island sizes with an average of 116 cells per island and a standard deviation of 74 cells per island. this data was used to generate randomly positioned clusters of cells on the grid, each with a size extracted from the measured distribution. islands are placed iteratively until the simulated confluency matches the experimentally measured confluency. a cell line doubling time is defined as the time required for the population of cells to double in number and we used a doubling time of 48 hours for calu-3 cells (ardizzoni lab, personal communication). cell density directly affects local cell growth since cells completely surrounded by other cells are not able to replicate. for this reason in our model the initial spatial distribution of uninfected cells affects the overall cell growth on the virtual culture well. in order to obtain a correct cell growth in our model we used a parameter called division_time that measures the time necessary to a uninfected cell to duplicate. we adjusted division_time to match the simulated doubling time with the experimentally-derived one. this was accomplished by analyzing cell growth of uninfected cells over 48 hours as a function of time for different values of the parameter. this led us to fix the value of division_time to 10 hours. cells in the culture well are initially infected using a multiplicity of infection (moi) of 2 (see section 2.7.4). the moi is calculated as the ratio of infectious viral particles to the number of calu-3 target cells. by definition the proportion of infected cells is given by the poisson distribution that describes the infection process [14] . using the experimental moi would theoretically give us the exact percentage of infected cells at the beginning of the experiment but even though the number of particles can be measured with good accuracy not all the viral particles used in this first infection process are infective and the estimate is derived using veroe6 cells. this leads to the definition of an effective moi, that is the moi given by the number of particles able to truly infect the calu-3 host cells. initially viral particles infect a proportion of the plated calu-3 cells before being washed away. we use the resulting proportion of infected cells estimated through the standard moi definition as a starting point of our simulation. after cells are initialized on the lattice, they are assigned an infected state according to equation 2, which describes the probability of at least one viral particle entering a cell. in the equations below, n represents the number of virus particles and moi is the multiplicity of infection used in the experiment. equation 2, the probability of a cell being infected by 1 or more virus particles, is derived from equation 1, the probability of being infected by a single virus particle, commonly used to describe viral infection as a poisson process [14] . although the expected moi is experimentally known, as mentioned in the "experimental data" section it is often over-estimated. we add the free parameter infectiousness for two purposes: 1) to scale moi over-estimation and 2) predict the number of initially infected cells when this data is not available. in the sensitivity analysis we discuss the importance of this parameter. moi n * e moi n! (1) after cells transition to an infectious state virus particles are released with a probability described by a sigmoidal function shown in figure 4 . the probability that one or more infectious viral particles are released in each time step is given by v(t) (equation 3 ). in this equation t represents time since the cell was infected and parameters a, b, and c are derived by fitting an experimentally-derived viral release curve produced by ka-wai et al. [15] . since we had no direct measure of the amount of virions per cell released by sars infected cells we rescaled the sigmoidal function with the free parameter vir_release. when v(t) > 1 an infectious cell will release one or more virions with a probability of 1. decimal digits less than one are treated as a probability for an additional virus particle to be released. infectious virions diffuse in the virtual well according to a simple random walk: for each virion in the virtual well at each time step of the simulation we perform a number of random walk steps given by the free parameter num_diff_steps. each viral particle has an equal chance to move to one of the six neighboring lattice sites and at each time step performs num_diff_steps movements on the lattice. the need of using a free parameter for the diffusion of virions was given by the lack of data regarding the virion diffusion coefficient on the well with specific conditions of the media used to cover the cell culture. viral titer measures only infectious particles present in the media using plaque forming units (pfu) per ml. for this reason we model only infectious particles [14] . in addition it is more computationally efficient to release and diffuse only the infectious viral particles. once released, virus particles may infect cells with an uninfected state located at the same lattice site. we assume each viral particle independently infects a nearby cell with a probability following the binomial distribution. for each lattice site the probability p second round (equation 4) is used to calculate whether or not a given uninfected cell is infected by n viral particles given n total local virus particles. p bp represents the probability of a virus-receptor binding event leading to a cell's infection by a single viral particle during a given model time step. we refer to the free parameter, p bp , as binding_prob below. once a cell is successfully infected, n viral particles are removed from the local virtual media located at the cell's lattice site. for these experiments we used a virus derived from our severe acute respiratory syndrome coronavirus (sars-cov) wild type infectious clone in which we engineered the green fluorescent protein (gfp) in place of open reading frame 7a/b. sars-cov gfp stock titers were calculated using standard plaque tittering methods. briefly, confluent monolayers of veroe6 cells in 6 well plates were infected with serial 1:10 dilutions (usually 10e-1 to 10e-6) of stock virus for 1 hour at 37 o c. the monolayers were covered with a solution of 0.8% low melting point agarose (seachem), 1x minimal essential media high glucose (mem, invitrogen), 10% fetal clone ii (hyclone), and 1x antibiotic/antimycotic (invitrogen), which solidifies trapping the virus to allow cell to cell viral spread but not release of virions into the media. plates were incubated at 37 o c for 36 hours, stained with neutral red for 2 to 5 hours, the stain removed and the plaques (holes in the monolayer generated when viruses kill the host cells) are visualized and counted on a light box. stock titers were calculated as plaque forming units (pfu) per ml. calu-3 cells were plated at a density of 1x10 6 per well in 6 well plates in mem containing 10% fetal clone ii and 1x antibiotic/antimycotic. cells were incubated at 37 o c at 5% co 2 levels for 2 days prior to infection with a media change 24 hours post plating. sars-cov gfp stocks (7.5x10 7 ) were used to infect each well at a multiplicity of infection of 2 or the addition of 2 infectious virions per cell in each well. at each time point, 100ul of media was harvested from each well to titer via plaque assay. (number of cells per well assumed to be 2x10 6 at 2 days post plating.) images shown in figure 2 were taken using phase contrast microscopy images were taken at standard exposure times at 10x magnification. at the indicated times post infection, images of the infected and mock-infected calu-3 cells were taken in living cells real time using a fluorescent light to excite gfp [16] at each time point, three to five fields of sars-gfp infected cells were assessed using imagej (http://rsbweb.nih.gov/ij/). in imagej, we gated the light signal to generate spots in each cell and used the spot counting algorithm determine the total number of cells per field. gfp positive cells were then counted and averaged. for each candidate parameter set, î¸, a simulation fitness (eq. 5) was calculated based on a comparison of two experimental measurements: virus titer and proportion infected cells. both components of simulation fitness, f(î¸), are normalized by a maximum error (me) term to balance their contribution. f titer = log 10 vt exp(t) log 10 vtsim(t) 2 (6) randomized weight bootstrapping (1000 iterations) was used to determine me titer and the derivation of me gfp is described below. equation 6 shows f titer is the sum of squares difference between experimentally derived virus titer, vt exp , (figure 1 ) and scaled virus titer produced by the simulation, vt sim . only the last three time points (7, 12, 24 hours) are compared because earlier time point titer values were skewed by residual virus. simulation output, vt sim , is scaled by î± to account for differences in the number of simulated cells versus the number of in vitro cells. using a lattice size of 130x130 an average of 7,636 total starting simulated cells were produced. the virus titer was scaled by a factor of 10 6 /7,636 because the experimental virus titer was produced from a population of 10 6 in vitro cells. as described in the experimental data section 2.7, in vitro gfp measurements were used to quantify the proportion of infected cells at 12 hours post infection. this additional biological information was used to constrain our model's parameter space. the f gfp portion of simulation fitness (equation 7) is the absolute value of the difference between simulated proportion of infected cells i sim to experimentally measured proportion infected, i exp . since i exp was measured to be .11 (on average 11 of 100 cells are infected) at 12 hours, a maximum error, me gfp , of .89 was used to normalize f gfp between zero and one. to assess the importance of model free parameters to simulation outcome, a latin hypercube sampling (lhs) sensitivity analysis was performed [17, 18] . this two-tiered sampling method was implemented by dividing each free parameter's range into 1000 intervals. intervals for each parameter were shuffled without replacement and then randomly sampled using a uniform distribution resulting in 1000 samplings of the parameter space. all possible probability parameter values were considered for infectiousness and binding_prob between 0 and 1. maximum values for num_diff_steps and vir_release were arbitrarily chosen (12 and 30 respectively). a spearman rank correlation was then used to measure statistical dependence, rho, between free parameter and single run simulation fitness. the following are free parameters (described above) contained in our model and assessed using lhs: we also examined how free parameters affect simulation output with time. using lhs sampling, we tested relations between free parameters and two simulation outputs: virus titer and % of infected cells. figure 6 (a) shows that infectiousness and vir_release are significant over 24 hours of simulated infection and that their correlations start to diverge at ~12 hours. again, binding_prob and num_diff_steps do not show a significant correlation with virus titer. however, parameters necessary for the second round of infection do impact the proportion of infected cells shown in figure 6 (b) where we see an increase in binding_prob and vir_release correlation between 7 and 12 hours, corresponding to a decrease in the infectiousness correlation. this decrease can be attributed to the second round of infection in which a greater population of cells has been infected. the simultaneous increase in correlation of binding_prob and vir_release indicates that additional rounds of infection are playing a significant role in viral spread. although simplistic compared to in vivo model systems, the interpretation of in vitro experiments is still confounded by biological complexity and disparate data types. explanatory models are critical for understanding and hypothesis generation. this agent-based modeling framework may be used to investigate first and second round of infection mechanisms using free parameters able to be tuned to allow the model to incorporate disparate types of experimental data. we also take into account spatial aspects of infection, including biases in culture well cell growth and diffusion of infectious virions. virus titer data and gfp infectivity data from a sars infection of calu-3 cells is used as an example to illustrate the model's capacity to interpret experimentally derived data. lhs sensitivity analysis indicates that a small population of cells is initially infected and that additional rounds of infection are responsible for virus titer measurements. a significant relationship between infectiousness and both the simulation fitness and simulation outputs (virus titer and proportion infected cells) indicates the importance of this parameter on the resulting infection dynamics. this result also demonstrates the importance of the accurate cell and infectious virus particle counts for the moi calculation. finally our model highlights the importance of intracellular processes leading to virus release. one possible future step is to include additional detail regarding intracellular processes of virus replication and move to a multi-scale spatio-temporal model. future work is planned to incorporate microarray data and make predictions regarding host response and expression in order to examine connections between infection state and signaling in the immune response. simulated annealing has been used to identify free parameters that fit the described model to virus titer data and may be used to predict the number of infected cells or other un-measured data types to support experimental modeling efforts. these off-line predictions could then be used to interpolate a single-cell function representing host response postinfection. we also plan to train the model with data from multiple virus strains to investigate how virus population and host response dynamics differ. finally, we also plan to investigate the effects of initial spatial distribution of infected cells on viral pathogenesis for multiple virus strains. all authors must sign the transfer of copyright agreement before the article can be published. this transfer agreement enables elsevier to protect the copyrighted material for the authors, but does not relinquish the authors' proprietary rights. the copyright transfer covers the exclusive rights to reproduce and distribute the article, including reprints, photographic reproductions, microfilm or any other reproductions of similar nature and translations. authors are responsible for obtaining from the copyright holder permission to reproduce any figures for which copyright exists. hiv-1 dynamics in vivo: virion clearance rate, infected cell lifespan, and viral generation time modelling viral and immune system dynamics a simple cellular automaton model for influenza a viral infections probing the effects of the well-mixed assumption on viral infection dynamics structured model of influenza virus replication in mdck cells sars cases with onset of illness from 1 kinetics of influenza a virus infection in humans antiviral resistance and the control of pandemic influenza: the roles of stochasticity, evolution and model details modeling the viral dynamics of influenza a virus infection mathematical model of antiviral immune response iii. influenza a virus infection modeling influenza viral dynamics in tissue. artificial immune systems multi-scale modelling in computational biomedicine modeling dynamic systems with cellular automata, chapter 21 role of atp in influenza virus budding severe acute respiratory syndrome coronavirus infection of human ciliated airway epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs a methodology for performing global uncertainty and sensitivity analysis in systems biology critique of and limitations on the use of expert judgements in accident consequence uncertainty analysis this work was made possible by funding from the national institute of allergy and infectious diseases, nih, department of health and human services contract # hhsn272200800060c thanks to casey long for his work in plating calu-3 cells for sars-cov infection experiments. also thanks to dr.andrea cavazzoni of the unit of experimental oncology (university of parma) for the useful discussions. key: cord-284322-synuzaxm authors: borel, nicole; dumrese, claudia; ziegler, urs; schifferli, andrea; kaiser, carmen; pospischil, andreas title: mixed infections with chlamydia and porcine epidemic diarrhea virus a new in vitro model of chlamydial persistence date: 2010-07-27 journal: bmc microbiol doi: 10.1186/1471-2180-10-201 sha: doc_id: 284322 cord_uid: synuzaxm background: chlamydiae induce persistent infections, which have been associated with a wide range of chronic diseases in humans and animals. mixed infections with chlamydia and porcine epidemic diarrhea virus (pedv) may result in generation of persistent chlamydial infections. to test this hypothesis, an in vitro model of dual infection with cell culture-adapted pedv and chlamydia abortus or chlamydia pecorum in vero cells was established. results: infected cultures were investigated by immunofluorescence (if), transmission electron microscopy (tem) and re-infection experiments. by if, chlamydia-infected cells showed normal inclusions after 39 hpi. dual infections with chlamydia abortus revealed a heterogenous mix of inclusion types including small inclusions consisting of aberrant bodies (abs), medium-sized inclusions consisting of abs and reticulate bodies and normal inclusions. only aberrant inclusions were observable in dual infection experiments with chlamydia pecorum and pedv. tem examinations of mixed infections with chlamydia abortus and chlamydia pecorum revealed aberrant chlamydial inclusions containing reticulate-like, pleomorphic abs, which were up to 2 μm in diameter. no re-differentiation into elementary bodies (ebs) was detected. in re-infection experiments, co-infected cells produced fewer ebs than monoinfected cells. conclusions: in the present study we confirm that pedv co-infection alters the developmental cycle of member species of the family chlamydiaceae, in a similar manner to other well-described persistence induction methods. interestingly, this effect appears to be partially species-specific as chlamydia pecorum appears more sensitive to pedv co-infection than chlamydia abortus, as evidenced by tem and if observations of a homogenous population of aberrant inclusions in pedv chlamydia pecorum co-infections. chlamydiae are implicated in a wide variety of diseases in both animals and humans. although acute infections in animal chlamydioses are the most commonly reported, chronic chlamydial infections are also associated with a variety of diseases in humans and animals. these latter infections are characterized by inflammation and scarring resulting in significant damage of the host. a causative role in chronic diseases requires that chlamydiae persist within infected tissue for extended periods of time. current theories, based primarily on in vitro data, suggest that chlamydial persistence, and the resulting chronic inflammation, is linked to morphological and metabolic conversion of the actively replicating and intracellular reticulate body (rb) into an alternative, non-replicative form known as an aberrant body (ab) [1] . in vitro, alterations of the normal developmental cycle of chlamydia trachomatis and chlamydia pneumoniae can be induced by interferon-γ (ifn-γ), tumor necrosis factor-α (tnf-α) and penicillin g exposure as well as amino acid or iron deprivation and monocyte infection [2, 3] . to date, in vitro models for animal pathogens, chlamydia abortus and chlamydia pecorum have not been described although both organisms are associated with chronic disease in koalas and small ruminants [1] . in pigs, several chlamydial species, including chlamydia abortus, chlamydia psittaci, chlamydia pecorum and chlamydia suis, have been implicated in a variety of disease conditions including conjunctivitis, pneumonia, pericarditis, polyserositis, arthritis, abortion and infertility [4] . in the gastrointestinal tract, chlamydiae appear to be highly prevalent but only occasionally cause enteritis. they have been found in the intestine of diarrheic and healthy pigs and could be demonstrated in mixed enteric infections [5] [6] [7] . pospischil and wood [7] first described an association between chlamydiaceae and lesions in the intestinal tract of pigs and assumed a synergistic effect in co-existence with salmonella typhimurium. further, mixed infections with eimeria scabra, cryptosporidia, and porcine epidemic diarrhea virus (pedv) have been described in the past. pedv, a member of the family coronaviridae, is a well-known cause of diarrhea in pigs. after the identification of pedv in 1978 by pensaert and debouck [8] , more than a decade passed before the virus could be adapted for propagation in cell cultures. examination of infected vero cell cultures by direct immunofluorescence revealed single cells with granular cytoplasmic fluorescence as well as formation of syncytia with up to 50-100 nuclei or more. typical features of syncytial cells were growth, fusion and detachment from cell layers after they had reached a certain size [9] . biomolecular studies revealed major genomic differences between cell culture-adapted (ca)-pedv and wild type virus [10, 11] . cell culture model of co-infection with ca-pedv and chlamydia has been established recently [12] to investigate the interaction of ca-pedv and chlamydiaceae in mixed infections and to detect possible synergistic or additive effects of possible significance in clinical enteric disease in pigs. in that study, abnormally large chlamydial forms were observed in dually infected cell layers by immunofluorescence suggesting that ca-pedv co-infection might alter the chlamydial developmental cycle in a manner similar to that observed during persistent infections. to confirm these initial observations, we established a cell culture model of mixed infections with chlamydia and a cell culture-adapted porcine epidemic diarrhea virus (ca-pedv) and hypothesized that this would result in the generation of persistent chlamydial forms. this data demonstrates that ca-pedv co-infection, indeed, alters the developmental cycle of chlamydia pecorum and chlamydia abortus in a similar manner to other inducers of chlamydial persistence. vero cells can be co-infected with chlamydia and ca-pedv immunofluorescence (if) labeling was used to investigate the morphologic differences of chlamydia between monoinfected and dually infected monolayers using chlamydia and ca-pedv specific antibodies. control and mock-infected cells did not stain with either antibody. ca-pedv monoinfected cells showed brilliant and distinct, red cytoplasmic fluorescence. syncytia were characterized by accumulation of nuclei in the center or the periphery of the multi-nucleated cells and moderate to bright, fine-granular, cytoplasmic ca-pedv labeling (figure 1b) . syncytia were categorized into small (2-15 nuclei), medium (16-30 nuclei) and large (more than 30 nuclei). in single infection experiments, syncytia at 24 h post infection were mostly large with fewer medium sized syncytia observed (data not shown). numbers of syncytia in ca-pedv single and dual infections were counted on the whole coverslip and mean values were determined. no difference of viral syncytia numbers for ca-pedv monoinfection and dual infection with chlamydia abortus were seen (data not shown). in contrast, numbers of viral synyctia in dual infections with chlamydia pecorum were diminished compared to the respective ca-pedv single infections (table 1) . if microscopy of chlamydial single infections revealed intracytoplasmic, mainly round to ovoid, sharply outlined inclusions with brilliant, green fluorescence. chlamydia abortus and chlamydia pecorum infected cells had one to five, finely granular (consisting mainly of ebs) inclusion(s) per cell at 39 h post infection ( figure figure 1 morphology of chlamydia pecorum mono-and coinfection with pedv. a) vero cells were infected with chlamydia pecorum 1 moi for 39 h, with subsequent pedv inoculation and labelled with an anti-chlamydia antibody (green); b) double infected monolayer were labelled for ca-pedv in red, chlamydia in green and dna in blue; c) chlamydia pecorum mono-infected vero cells labelled with an anti-chlamydia antibody (green) and dna staining (blue); d) inclusion size was measured as described and the frequency of chlamydial inclusions assembled into sizes of 50 μm 2 area groups depicted. the difference between mono and double infected monolayers was statistically analyzed using students t-test. the groups were significantly different with p = 0.0044. 1c &2a). in general, chlamydial inclusions were smaller and had more variable forms in chlamydia pecorum than in chlamydia abortus single infections. infectivity was almost 100% and a moderate number of free ebs could be observed. compared to single infections, the size and shape of chlamydial inclusions in pedv co-infections was highly variable. in chlamydia abortus co-infection experiments, three types of inclusions were observed: (i) small inclusions consisting of 1-10 aberrant bodies (abs), (ii) medium-sized inclusions consisting of abs and reticulate bodies (rbs), and (iii) large (normal) inclusions consisting of ebs as seen in the single infection experiments (figure 2b ). in contrast, dual infections with ca-pedv and chlamydia pecorum resulted in the exclusive production of aberrant inclusions containing between 2-50 abs. chlamydial inclusions in viral syncytia grew even larger than in non-viral infected vero cells. overall, no normal chlamydial inclusions were observed (figure 1a &1b) . image analysis was used to compare inclusion size in single chlamydiae-infected vero cells with the inclusion size in vero monolayers that subsequently underwent ca-pedv virus infection. to this end, inclusion size was determined in μm 2 and all inclusions were assembled into groups covering 50 μm 2 and multiples of this area. the average frequency of chlamydia pecorum inclusions between 100 μm 2 and 400 μm 2 was significantly reduced when cells were subsequently infected with ca-pedv. in other words, chlamydia pecorum inclusions vero cells were infected with chlamydia abortus with subsequent pedv inoculation and stained as with an anti-chlamydia antibody and dapi; c) frequency of inclusions with various sizes was calculated and mono and double infected cells were compared according to the inclusion size. the difference between mono and double infected monolayers was statistically analyzed using students t-test. the groups were significantly different with p = 0.0132. a numbers of syncytia for ca-pedv monoinfection and dual infection with chlamydia pecorum were counted on the whole coverslip. b vero cells were mock infected (mock), c. pecorum infected, ca-pedv infected (ca-pedv) and chlamydia pecorum/ca-pedv co-infected as described. were highly significant smaller in ca-pedv dual infections than in those infections without the addition of virus ( figure 1d ) as analyzed by t-test (p = 0.0044). the additional changes observed in the shape of all inclusions growing in virus-infected monolayers indicated the induction of chlamydia pecorum persistence, since the finely dispersed staining reverted to grape-like structures (figure 1a &1b ). the changes of chlamydial inclusion size by subsequent virus addition to chlamydia abortus are different to those we observed in the chlamydia pecorum dual infection experiments. the frequency of inclusions observed between a size range of 0-200 μm 2 was significantly (p = 0.0132) reduced under virus infection but the amount of medium sized and big inclusions 300 -700 μm 2 was increased ( figure 2c ). the morphology of chlamydia abortus inclusions was also found to differ in the population when co-infected with ca-pedv. smaller inclusions were generally observed in aberrant shapes compared to larger inclusions, which appeared similar to normal actively growing inclusions showing finely dispersed staining (figure 2b ). this effect might be due to an incomplete induction of persistence of chlamydia abortus when cells were ca-pedv coinfected. co-infection with ca-pedv induced ultrastructural morphological changes in chlamydia abortus and chlamydia pecorum persistent forms of chlamydia trachomatis and chlamydia pneumoniae are well described by their characteristic electron microscopic appearance [2, 13, 14] . thus, chlamydial ultrastructure in single and co-infected cells was compared by transmission electron microscopy (tem). at 24 h after viral infection, viral-induced syncytia containing vacuoles filled with viral particles were present in ca-pedv-monoinfected and dual infections. the viral particles showed the typical coronavirus morphology with a diameter between 50 to 130 nm (data not shown). at 39 h after chlamydial infection, large intracytoplasmic chlamydial inclusions in single infected cells could be observed in vero cells infected with chlamydia abortus or chlamydia pecorum. the inclusions observed contained variable numbers of morphologically normal rbs and ebs and were generally located near the host cell nucleus, often surrounded by mitochondria (figure 3a and 3b) . tem examinations of mixed infections (ca-pedv and chlamydia abortus or chlamydia pecorum) revealed aberrant chlamydial inclusions containing fewer bacteria than typical inclusions and were located in viral syncytia or single cells without viral infection. aberrant inclusions consisted of reticulate-like, pleomorphic, aberrant bodies (abs), which were in general larger in diameter (up to 2 μm) than typical reticulate bodies (rbs), with a sparse densitometric appearance and no re-differentiation into elementary bodies (ebs). as already observed in if investigations, three types of inclusions were present in dual infections with ca-pedv and chlamydia abortus (figure 3c ), whereas dual infections with ca-pedv and chlamydia pecorum resulted in the exclusive production of aberrant inclusions consisting of 2-50 abs (figure 3d ). neither chlamydial inclusions nor ca-pedv virions were visible in mock-infected cells. previous studies have demonstrated that chlamydial persistent forms are non-infectious [2] . reduced number or even a lack of ebs in co-infected cells in tem suggested arrested chlamydial developmental cycle with halted maturation from rb to eb. to ascertain the effect of ca-pedv inhibition of chlamydial eb production, the yield of infective chlamydial progeny was determined after 40 h of re-infection in three independent experiments for chlamydia abortus (figure 4a ) and for chlamydia pecorum (figure 4b ). neither mock nor ca-pedv monoinfected cells produced detectable infectious ebs, whereas chlamydia abortus and chlamydia pecorum single infections cells produced abundant ebs. coinfected cells produced fewer infectious ebs than nonviral infected cells, demonstrating that production of infectious chlamydial progeny was essentially diminished by ca-pedv-co-infection. eradication of infectious eb production was almost complete in chlamydia pecorum double infection, analyzed by reinfection experiments and found to be statistically different as analyzed by ttest (p = 0.0145) (figure 4b ). in chlamydia abortus reinfection analysis, several ebs could still be observed in spite of the co-infection with ca-pedv (figure 4a this data is consistent with the observations from our if and ultrastructural analysis. chlamydial co-infection does alter ca-pedv infection depending on the chlamydial species but does not alter viral ultrastructure to determine whether chlamydial pre-infection altered subsequent viral infection, numbers of syncytia and ca-pedv-infected cells from single and co-infected monolayers of three unrelated experiments were counted. mock-infected and chlamydia only infected cells produced no virions. the difference between virus-infected cells and co-infection with chlamydia abortus was minimal. the number of syncytia detected were within the same range (data not shown) indicating that chlamydial co-infection with chlamydia abortus does not alter ca-pedv infection or the development of syncytia. in contrast, numbers of syncytia in co-infection with chlamydia pecorum were reduced compared to single ca-pedv infection (table 1) . overall numbers of single viral infected cells were low in both single and co-infection experiments, and no significant difference between the two chlamydial species was obvious (data not shown). viral morphology was also studied by tem. in ca-pedv single and co-infected cells, viral particles were unaltered indicating that chlamydial co-infection did not induce any changes in viral ultrastructural morphology. while a previous study [12] primarily investigated the interaction of ca-pedv and chlamydiaceae in mixed infections to detect possible synergistic or additive effects of these two pathogens, questions remained about whether viral infection could potentially induce the persistent chlamydial phenotype. enlarged chlamydial inclusions were described in that study in the ca-pedv co-infection model with chlamydia abortus and chlamydia pecorum but no further ultrastructural analysis has been subsequently performed. in this study, in vitro models of chlamydia abortus and chlamydia pecorum persistence were established using co-infection with ca-pedv. several experimental methods were used to demonstrate the characteristic features of chlamydial persistence, including altered ultrastructural morphology and decreased production of infectious ebs. our results demonstrated that ca-pedv-co-infection alters the chlamydial developmental cycle similarly to other inducers of chlamydial persistence. a similar co-infection model has been recently described by deka et al. (2006) [15] . in that study, it was shown that chlamydia trachomatis enters a viable but non-cultivable, persistent state with herpes simplex virus type 2 (hsv-2) co-infected host cells. in contrast, a similar study investigating a coinfection model with chlamydia trachomatis and genital mycoplasmas, mycoplasma genitalium and mycoplasma hominis, did not change the morphology of chlamydial rbs, indicating that co-infection of these two microorganisms is likely to be independent and not related to the onset of chlamydial persistence [16] . in the study by deka et al. (2006) [15] , hela monolayers were first infected with chlamydia trachomatis and 24 h later with hsv-2. in our study, the optimal experimental protocol for co-infection procedure was developed, based on our own earlier study [12] , and optimization experiments performed as a part of the current study (data not shown). to this end, vero monolayers were first infected with chlamydia and later with ca-pedv, thus the suspected inducer of persistence would be introduced after chlamydial infection and differentiation into rbs. simultaneous infection of chlamydia and ca-pedv has been performed earlier [12] , but did not result in persistent infection in our preliminary experiments (data not shown) and was not considered further as interference of chlamydial infection and concurrent viral uptake could have influenced the results. viral infection and subsequent development of syncytia was not affected by co-infection with chlamydia abortus as demonstrated by unaltered numbers of syncytia observed in the coinfection experiments. in contrast, viral syncytia formation was dramatically decreased in vero cells double infected with ca-pedv and chlamydia pecorum. if chlamydia pecorum infection might induce a down regulation of the host pedv receptor needed for syncytium formation at 14-15 hours post-chlamydial infection, this could produce a reduction in syncytium formation without reducing viral entry or replication -the possible persistence inducer mechanism. interestingly, chlamydial persistence was more prominent in co-infection with chlamydia pecorum than with chlamydia abortus, indicating possible species-specific differences. limited reports are available for in vitro models of chlamydial persistence from non-chlamydia trachomatis and chlamydia pneumoniae strains. kaltenboeck and storz (1992) [17] suggested that strain 1710s of chlamydia pecorum is highly nutrient dependent and this could elicit aberrant forms. indeed, aberrant forms of this strain were significantly present in our study. previously, only limited data have been published on persistent infection of l cells with an ovine abortion strain of chlamydia psittaci (current classification: chlamydia abortus) [18] . it should be noted, that in the latter study, chlamydial persistence was not demonstrated using the characteristic features now associated with the morphology of persistent chlamydial infections. detailed description of electron microscopic observations on the effects of penicillin on the morphology of chlamydia psittaci cal10 in l cells showing aberrant chlamydial stages were published by matsumoto and manire [13] . the different occurence of persistent forms in coinfection with chlamydia abortus and chlamydia pecorum, respectively, has not been described before. differences between persistence behaviour are already known (reviewed by hogan et al., 2004) [1] not only between different chlamydial species but also between different serovars and strains of chlamydia pneumoniae and chlamydia trachomatis, respectively. the fact that chlamydia pecorum strain 1710s is an original swine isolate whereas chlamydia abortus strain s26/3 originates from a sheep abortion and, thus, from another animal species could have an impact but needs further investigation. the mechanism by which ca-pedv interferes with chlamydial developmental cycle and chlamydial persistence is still unclear. it is known that vero cells, a monkey kidney epithelial cell line, is deficient for interferon production [19] ; thus, this cytokine group well known to be capable of inducing in vitro persistence in chlamydia pneumoniae [1] , cannot be relevant for our co-infection persistence model. co-infection experiments with ca-pedv are best performed with vero cells, as they have been shown to be permissive for viral replication in contrast to other cell lines such as pd5, pk 15, and hrt18 cell lines [9] . specific measurements of primate cytokines in our co-infection model are planned in the future to elucidate the mechanism leading to chlamydial persistence. the herpes simplex virus (hsv) co-induced chlamydia trachomatis persistence model [15] has been recently been shown not to be mediated by any known persistence inducer or anti-chlamydial pathway recently [20, 21] . instead, it was hypothesized by the authors that hsv-2 attachment and/or entry into the host cell is sufficient for stimulating chlamydial persistence, suggesting a potential novel host signaling pathway could be responsible for inducing chlamydial persistence. a very recent publication by the same group showed that hsv replication is not necessary for persistence induction and that chlamydial activity could be recovered after coinfection with uv-inactivated hsv-2. finally, it was concluded that the interaction of hsv glycoprotein d with the host cell surface is crucial to trigger chlamydial persistence [22] . female genital tract infection often has a complex etiology, where chlamydia trachomatis is present together with one or more genital agents. epidemiological and clinical studies have shown that double infection with hsv-2 and chlamydia trachomatis occurs in vivo; thus, the in vitro model described by deka et al. (2006) [15] represents a realistic situation in human medicine. similarities exist to the in vitro model established in this study as simultaneous intestinal infection with different pathogens is possible in swine in vivo. a recent study [23] documented the occurrence of aberrant chlamydial bodies in vivo in intestinal tissues of pigs. in this study, aberrant bodies of chlamydia suis were demonstrated and characterized in the gut of pigs experimentally infected with salmonella typhimurium by transmission electron microscopy. it was concluded by pospischil et al. [23] that aberrant bodies occur in vivo in pigs and that the gnotobiotic pig model might be suitable for the study of chlamydial persistence in vivo. available intestinal tissues from experimentally infected gnotobiotic piglets (single infection and coinfection with chlamydia and ca-pedv, respectively) will be investigated in the future with the aim of further characterization of abs in vivo. although chronic chlamydial diseases in animals are of economic impact, the pig model may also reveal the important link between persistence in vitro and in vivo and, thus, help to elucidate mechanisms of chronic human chlamydial infections in the future. the present study reports a new persistence model of chlamydia in co-infection with porcine epidemic diarrhea virus (pedv). pedv-co-infection altered the chlamydial developmental cycle similarly to other known inducers of chlamydial persistence. this new animal model could provide the important link between persistence in vitro and in vivo and, thus, would help to elucidate mechanisms of chronic human chlamydial infections in the future. growth medium (gm) for normal cell propagation was minimal essential medium (mem) with earle's salts, 25 mm hepes, without l-glutamine (gibco, invitrogen, carlsbad, ca) and supplemented with 10% fetal calf serum (fcs) (bioconcept, allschwil, switzerland), 4 mm glutamax-i (200 mm, gibco) and 0.2 mg/ml gentamycin (50 mg/ml, gibco). gm without gentamycin was used for the propagation of cells for infection experiments. infection medium was prepared as gm but without gentamycin and fcs, and was used for the infection and for the 24 h incubation period after the infection with ca-pedv, respectively. incubation medium was prepared as gm without gentamycin, freshly supplemented with 1 μg/ml cycloheximide (sigma, buchs sg, switzerland), and used after an infection for estimation of the chlamydial titer (ifu determination). vero 76 cells (african green monkey kidney cells, crl 1587 american type culture collection) were seeded on round plastic coverslips (13 mm diameter, bibby sterilin, stone, uk) and cultured in gm without gentamycin at 37°c until they reached confluence. before inoculation, the cells were washed once with phosphate buffered saline (pbs). two different chlamydial strains of chlamydiaceae were used in this study: chlamydia abortus s26/3 (ovine abortion strain, kindly donated by dr. g.e. jones, moredun research institute, edinburgh, gb) and chlamydia pecorum 1710s (intestinal swine isolate, kindly provided by prof. j. storz, baton rouge, louisiana, la, usa). for initial culturing, chlamydial strains were cultured in embryonated chicken eggs, and yolk sac material was harvested, diluted 1:2 in sucrose-phosphate-glutamate (spg) medium and stored at -80°c. yolk sac-derived chlamydiae were then propagated in hep-2 cell (atcc ccl-23) monolayers and elementary bodies (ebs) were harvested and purified by disruption of hep-2 cell monolayers with a cell scraper, sonication and centrifugation over a renografin density gradient as described elsewhere [24] . eb suspensions were stored in sucrosephosphate-glutamic acid buffer at -80°c, after which viable titers were established using standard methods. moi of 1 was used for chlamydial monoinfection and mixed infection, respectively. ca-pedv strain cv777 (kindly provided by prof. dr. m. ackermann, institute of virology, university of zurich) was propagated as previously described [9] . the virus (10 5,5 tcid 50 /ml) was used undiluted (1 ml 10 5,5 tcid 50 /ml). vero cells, an african green monkey kidney cell line (atcc crl 1587), were used for all infection experiments. they were propagated in gm without gentamycin at 37°c in an atmosphere of 5% co 2 . vero cells were divided into four groups: for mock infection, chlamydial infection, ca-pedv infection, and both chlamydia and ca-pedv double infection. host cells were infected with a moi of 1 for chlamydia and an infective dose of 1 × 10 5,5 tcid 50 /ml for ca-pedv, respectively. for ca-pedv monoinfections and negative controls, infection medium was used. all co-infection experiments were done three times and monoinfections with chlamydia and ca-pedv were performed simultaneously. the optimal experimental protocol (adding the virus several hours after chlamydial infection) for co-infection procedure was developed before (data not shown). for dual infections, cell monolayers were first infected with chlamydia at a moi of 1. all coverslips were centrifuged at 1000 × g for 1 h at 25°c. timepoint 0 (t 0 ) was defined after centrifugation and supernatant was replaced subsequently by incubation medium. infected monolayers were then incubated for 14 h at 37°c (t 0 -t 14 ). all cell layers for dual infections or ca-pedv monoinfection were exposed to a ca-pedv suspension (1 × 10 5,5 tcid 50 ), the samples were centrifuged again for 1000 × g for 1 h at 25°c and incubated for 24 h at 37°c. after this incubation period, all monolayers were fixed and further investigated by indirect immunofluorescence and transmission electron microscopy. re-infection experiments were performed to compare the production of infectious chlamydial elementary bodies (ebs) between monoinfections and mixed infections. for indirect immunofluorescence analyses, infected cells were fixed in absolute methanol (-20°c) for 10 min. and if labeling of cell cultures was performed immediately after fixation. for viral antigen detection, a mouse monoclonal antibody against the m protein of pedv (mcab 204, kindly provided by prof. dr. m. ackermann, institute of virology, university of zurich), diluted 1:4 in pbs supplemented with bsa, and an alexa fluor 594-conjugated secondary antibody (goat anti-mouse, 1:500, molecular probes, eugene, usa) were used. chlamydial inclusions were labeled with a chlamydiaceae family-specific mouse monoclonal antibody directed against the chlamydial lipopolysaccharide (mlps; clone aci-p, progen, heidelberg, germany) and a secondary alexa fluor 488-conjugated secondary antibody (goat anti-mouse, 1:500, molecular probes). dna was labeled with 1 μg/ml 4', 6-diamidin-2'-phenylindoldihydrochlorid (dapi, molecular probes). all staining procedures were conducted at room temperature. antibody incubations were carried out for 1 h (primary antibodies) or 45 min (secondary antibodies), respectively, with three washes of pbs following fixation, between and after applications of the different antibodies. dually infected cell layers were stained using sequential double immunofluorescence labeling. uninfected vero cells were used as a negative control. coverslips were mounted with immumount (shandon, pittsburgh, usa) on glass slides and investigated using a leica fluorescence microscope. coverslips from all experimental conditions were fixed in 2.5% glutaraldehyde (electron microscopy sciences, ft. washington, usa) for 1-2 h, and processed by routine methods for embedding in epoxy resin (fluka). appropriate areas for ultrastructural investigation were selected using semithin sections (1 μm) stained with toluidine blue (fluka, buchs sg, switzerland). ultrathin sections (80 nm) were mounted on gold grids (merck eurolab ag, dietlikon, switzerland), contrasted with uranyl acetate dihydrate (fluka) and lead citrate (lead nitrate and tri-natrium dihydrate; merck eurolab ag) and investigated in a philips cm10 electron microscope. at 39 h after chlamydial infection, monolayers were scraped into 1 ml of cold infection medium, pelleted and resuspended in 1 ml of fresh medium. infected host cells were lysed by sonication and centrifuged (500 g for 5 min) to remove pellet cell debris. supernatants were centrifuged once (4,000 g for 60 min). final eb pellets were resuspended in 200 μl of spg and used to infect vero cells plated on glass coverslips in duplicate in dilution series. all coverslips were centrifuged at 1000 × g for 1 h at 25°c. after centrifugation, the vero cells were refed with medium containing 1 μg/ml cycloheximide and subsequently incubated for 40 h at 37°c. fixation and staining of chlamydia, ca-pedv and dna was performed as described above. the number of inclusions in 20 random microscopic fields per sample was determined using a leica fluorescence microscope at a magnification of 200 ×. duplicate coverslips were counted and the counts were averaged. the number of inclusion-forming units (ifu) in the indiluted inoculum was then calculated and expressed as ifu per 10 6 cells as described by deka et al., 2006 [15] . from duplicate samples of three independent experiments uniform random sampled images were acquired using a widefield microscope (leica lx, leica microsystems mannheim, germany). cells and inclusions were automatically detected according to size, shape and intensity and counted using imaris (bitplane ag, zürich switzerland). chlamydial persistence: beyond the biphasic paradigm persistent chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis morphologic and antigenic characterization of interferon gamma-mediated persistent chlamydia trachomatis infection in vitro prevalence of intestinal chlamydial infection in pigs in the midwest, as determined by immunoperoxidase staining intestinal chlamydia in finishing pigs intestinal chlamydia in pigs a new coronavirus-like particle associated with diarrhea in swine propagation of the virus of porcine epidemic diarrhea in cell culture sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf pedv leader sequence and junction sites mixed infections in vitro with different chlamydiaceae strains and a cell culture adapted porcine epidemic diarrhea virus electron microscopic observations on the effects of penicillin on the morphology of chlamydia psittaci chlamydia pneumoniae expresses genes required for dna replication but not cytokinesis during persistent infection of hep-2 cells chlamydia trachomatis enters a viable but non-cultivable (persistent) state within herpes simplex virus type 2 (hsv-2) co-infected host cells chlamydia trachomatis and genital mycoplasmas in the co-infection model -in vitro study biological properties and genetic analysis of the omp a locus in chlamydiae isolated from swine persistent infection of l cells with an ovine abortion strain of chlamydia psittaci characterization of the interferon regulatory factor 3-mediated antiviral response in a cell line deficient for ifn production an early event in the herpes simplex virus type-2 replication cycle is sufficient to induce chlamydia trachomatis persistence herpes simplex virus co-infection-induced chlamydia trachomatis persistence is not mediated by any known persistence inducer or anti-chlamydial pathway interaction of hsv-2 glycoprotein d with the host cell surface is sufficient to induce chlamydia trachomatis persistence aberrant chlamydial developmental stages in the gastrointestinal tract of pigs spontaneously and experimentally infected with chlamydia suis purification on renografin density gradients of chlamydia trachomatis grown in the yolk sac of eggs submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors would like to thank lisbeth nufer of the laboratory staff at the institute of veterinary pathology, zurich, for her excellent technical assistance. we would also like to thank dr. monika engels and eva loepfe, institute of virology (head: prof. m. ackermann), vetsuisse faculty, university of zurich for providing the porcine epidemic diarrhea virus. we thank dr. adam polkinghorne, queensland university of technology, brisbane, australia, for manuscript editing. this work was supported by a grant from the university of zurich (forschungskredit).author details 1 institute of veterinary pathology, vetsuisse faculty, university of zurich, zurich, switzerland. 2 center for microscopy and image analysis, university of zurich, zurich, switzerland. authors' contributions nb conceived of the study, planned the experiments, and drafted the manuscript. cd and uz performed the imaging and statistical analyses. as and ck carried out the cell culture experiments including immunofluorescence and transmission electron microscopy. ap participated in the design and coordination of the study and helped to draft the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-257644-9f30s0gy authors: mu, xingjiang; pridgeon, julia w.; klesius, phillip h. title: comparative transcriptional analysis reveals distinct expression patterns of channel catfish genes after the first infection and re-infection with aeromonas hydrophila date: 2013-09-12 journal: fish shellfish immunol doi: 10.1016/j.fsi.2013.08.027 sha: doc_id: 257644 cord_uid: 9f30s0gy to determine whether transcriptional levels of channel catfish (ictalurus punctatus) genes are differentially regulated between a first infection with aeromonas hydrophila and a re-infection, suppression subtractive hybridization (ssh) was performed in this study using anterior kidney cdna after the re-infection as tester. of the 96 clones isolated from the ssh library, 28 unique expressed sequence tags (ests) were obtained, of which eight were confirmed to be slightly but significantly (p < 0.05) more up-regulated by the re-infection at 6 h post infection (hpi). expression kinetics studies at 3, 6, 12, 24, and 48 hpi revealed that the eight ests were significantly (p = 0.016) more up-regulated by the first infection, with a major peak at 3 hpi. a total of 96 genes reported in literature to be up-regulated by bacterial infections were selected and subjected to expression analysis at 3 hpi. of the 96 selected genes, 19 were found to be significantly (p < 0.05) induced by a. hydrophila after the first infection and the re-infection. the 19 genes belonged to the following five main categories: 1) toll-like receptor (tlr2, tlr3, tlr5, tlr21); 2) antimicrobial peptide (nk-lysin type 1, nk-lysin type 2, nk-lysin type 3, cathepsin d, transferrin, hepcidin); 3) cytokine or chemokine (interleukin-1β, interleukin-10, tumor necrosis factor α, chemokine cxcl-10); 4) signaling proteins (cadherin egf lag seven-pass g-type receptor 1, very large inducible gtpase 1, arginine deiminase type 2, lymphokine-activated killer t-cell originated protein kinase); 5) lysozyme (lysozyme c). overall, the total 27 genes (8 ests plus the 19 selected genes) were significantly (p < 0.001) more induced by the first infection. peaked expression of lysozyme c and serum lysozyme activity after the first infection were seen at 24 hpi, whereas that after the re-infection were seen at 12 hpi, suggesting that both innate and adaptive immunity were involved in the defense against the re-infection of a. hydrophila. aeromonas hydrophila is a causative agent of motile aeromonad septicaemia (mas) [1, 2] . swelling of tissues, dropsy, red sores, necrosis, ulceration, and hemorrhagic septicemia are typical symptoms of mas [3, 4] . many fish species can be affected by mas, including tilapia [5, 6] , catfish [7, 8] , goldfish [9, 10] , common carp [11, 12] , and eel [13] . as an opportunistic pathogen, a. hydrophila caused outbreaks in fish farms with high mortality rates [14e16] . in west alabama, mas disease outbreak caused by a. hydrophila in 2009 alone has led to an estimated loss of more than 3 million pounds of food size channel catfish [17] . virulence studies have revealed that al09-71, a 2009 west alabama isolate of a. hydrophila used in this study, is highly virulent to channel catfish, killing fish within 24 h post exposure [18] . both innate and adaptive immunity play important roles in the defense of fish against bacterial infections [19] . immunity of fish to bacterial infections is largely mediated by cellular immune responses with humoral antibodies having a secondary role [20, 21] . it was reported that live attenuated a. hydrophila induced the upregulation of multiple immune genes in channel catfish [22] . in addition, infection with a. hydrophila induced peak up-regulation of several genes at 6 h post injection (hpi) [22] . however, it was unknown whether gene expression levels in channel catfish after re-infection with a. hydrophila were induced higher compared to that by the first infection. therefore, the objectives of this study were to: 1) identify up-regulated genes in channel catfish after a second infection of a. hydrophila compared to a single infection at 6 hpi by suppression subtractive hybridization (ssh); 2) compare the gene expression patterns of at different time (0, 3, 6, 12, 24, and 48 hpi) after the first infection and the re-infection to determine the peak response time; and 3) screen genes that were reported in literature and identify genes up-regulated by the first infection and the re-infection at the peak response time. the al09-71 isolate of a. hydrophila was obtained from diseased channel catfish in 2009 from west alabama. the isolate has been confirmed to be a. hydrophila through biochemical and molecular identification [17] . bacterial cultures were grown in tryptic soy broth (tsb) (fisher scientific, pittsburgh, pa) for 24 h at 28 c. channel catfish (26.2 ae 3.3 g) were obtained from stocks maintained at usda-ars, aquatic animal health research laboratory (auburn, al, usa). all fish were maintained in dechlorinated water in 340 l tanks. prior to experiments, fish were acclimated in flow-through 57-l aquaria supplied with w0.5 l h à1 de-chlorinated water for 14 days. experimental fish were confirmed to be culture-negative for bacterial infection by culturing posterior kidney tissues from representative groups of fish on tryptic soy agar plates. a 12:12 h light:dark period was maintained and supplemental aeration was supplied by air stones. mean dissolved oxygen was w5.6 mg l à1 at water temperature w27 c, with ph w7.1 and hardness w100 mg l à1 . fish were fed w3% body weight daily with commercial dry fish food. prior to the experimental infection, fish were moved to 57-l flow through aquaria and acclimated for 14 days. the sub-lethal infection dose of a. hydrophila al09-71 given to fish was 1 â 10 4 colony forming unit per fish (cfu/fish) based on previous challenge result (ld 50 ¼ 1 â 10 5 cfu/fish [17] ). prior to injection, thirty untreated fish were used to collect samples at 0 h. the remaining fish were divided into three groups (150 fish/group): 1) control group [intraperitoneally (ip) injection with 100 ml tsb]; 2) innate group (ip injection with a. hydrophila al09-71 at 1 â 10 4 cfu/fish); 3) adaptive group (ip injection with a. hydrophila al09-71 at 1 â 10 4 cfu/fish followed by a second injection with a. hydrophila al09-71 at 2 â 10 4 cfu/fish at 28 days post first injection). anterior kidney samples were collected from 30 fish at 0, 3, 6, 12, 24, 48 hpi and 10 tissues were pooled together as one sample (10 fish/pool, 3 pools per time point). all anterior kidney tissues were flash frozen on dry ice immediately after sampling and stored at à80 c until rna extraction. total rna was isolated from anterior kidney tissues using trizol reagent (invitrogen, carlsbad, ca) following the manufacturer's protocol. all rnas were treated with dnase provided by the dnafree kit (ambion, austin, tx) to eliminate any dna in the rna sample. rna samples were quantified on a nanodrop nd-1000 spectrophotometer (nanodrop technologies, rockland, de). the first strand cdnas used for quantitative pcr were synthesized using 2 mg of total rna, amv reverse transcriptase, and oligo-dt primer provided by the cloned amv first strand cdna synthesis kit (invitrogen, carlsbad, ca). for subtractive library construction, total rnas were extracted from pooled anterior kidney samples at 6 hpi after the first infection or the re-infection of a. hydrophila al09-71. the 6 hpi time point was chosen based on previous research results on gene upregulation in channel catfish in response to a. hydrophila [22] . cdnas were then synthesized using pcr-select cdna subtraction kit (clontech, palo alto, ca). two-step subtractive hybridizations were performed according to procedures described previously [22] . briefly, two primary hybridization reactions (a and b) were formed by adding excess amounts of first infection cdna (driver) to second infection cdna (tester) samples at a 50:1 ratio. the samples were denatured for 2 min at 98 c and allowed to anneal for 8 h at 68 c. the remaining single-stranded, adaptor-ligated tester cdnas were substantially enriched in each hybridization reaction for overexpressed sequences because non-target cdnas present in the tester and driver could form hybrids. after filling in the adapter ends with dna polymerase, over-expressed sequences (tester cdna) had different annealing sites on their 3 0 -and 5 0 -ends. the molecules were then subjected to suppression subtraction pcr. the pcr products were then cloned into pgem-t easy vector (promega, madison, wi). plasmids were transformed into one shot ò top10 competent cells (invitrogen, carlsbad, ca). transformed cells were plated on luriaebertani (lb) plates containing ampicillin (100 mg/ ml) and x-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) (40 mg/ml). from the library, a total of 96 colonies were subsequently picked to grow overnight in lysogeny broth (lb) in the presence of ampicillin (100 mg/ml) at 37 c and 235 rpm in innovaô 4000 incubator shaker (new brunswick scientific, edison, nj). overnight cultures were then sent to usda-ars msa genomics laboratory in stoneville, ms for plasmid dna extraction and dna sequencing with an abi 3730 genetic analyzer (applied biosystems, foster city, ca). raw sequence base calling and trimming was conducted at the msa genomics laboratory by using phred with a cut-off score of q20. vector and adaptor sequences were manually trimmed. trimmed cdna sequences were then analyzed using the national center for biotechnology information (ncbi) blast program to search for sequence homologies. sequencing results of different clones were used to design genespecific primers (supplementary table 1 ) by using the primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www. cgi). quantitative pcr (qpcr) was performed using applied biosystems 7500 real-time pcr system (applied biosystems, foster city, ca). for each cdna sample, channel catfish 18s ribosomal rna primers were included as an internal control to normalize the variation in cdna amount as published previously [22] . all qpcr was performed using platinum ò sybr ò green qpcr supermix-udg with rox (invitrogen, carlsbad, ca) in a total volume of 12.5 ml. the qpcr mixture consisted of 1 ml of cdna (input rna of 10 ng), 0.5 ml of 5 mm gene-specific forward primer, 0.5 ml of 5 mm gene-specific reverse primer and 10.5 ml of 1 â sybr green supermix. the qpcr thermal cycling parameters were 50 c for 2 min, 95 c for 10 min followed by 40 cycle of 95 c for 15 s and 60 c for 1 min. all qpcr was run in duplicate for each pooled cdna sample (10 kidney samples per pool; three pools were analyzed). the fluorescence intensities of the control and treatment products for each gene, as measured by cycle threshold (c t ) values, were compared and converted to fold differences by the relative quantification method [23] using the relative expression software tool 384 v. 1 (rest) and assuming 100% efficiencies. expression differences between control and treatment groups were assessed for statistical significance using a randomization test in the rest software. the mrna expression levels of all samples were normalized to the levels of 18s ribosomal rna gene in the same sample. expression levels of 18s were constant between all samples (<0.30 change in c t ). each primer set amplified a single product as indicated by a single peak present for each gene during melting curve analysis. the relative transcriptional levels of different genes were determined by subtracting the cycle threshold (c t ) of the sample by that of the 18s rrna, the calibrator or internal control, as per the formula: dc t ¼ c t (sample) à c t (calibrator). the relative expression level of a specific gene at different time points were compared to that of fish at time 0 using formula 2 àddct where ddc t ¼ dc t (time point x) à dc t (time point 0) as described previously [22] . to search for more channel catfish genes that might be differentially induced by a re-infection of a. hydrophila compared to that by a first infection, a total of 96 channel catfish genes reported in literature were screened in this study, including the following: 1) 43 genes induced by edwardsiella ictaluri [24] ; 2) 28 genes upregulated by flavobacterium columnare [25] ; 3) 5 toll-like receptors (tlrs) up-regulated by e. ictaluri [26] ; 4) 20 genes upregulated by a. hydrophila [22] . primers for these 96 genes were purchased from sigmaealdrich (st. louis, mo). lysozyme activity was measured using published procedures [27, 28] with slight modifications. briefly, lyophilized micrococcus lysodeikticus (sigmaealdrich) at a concentration of 0.2 mg/ml in sodium acetate buffer (0.1 m; ph 6.0) was used as a substrate. serum (20 ml/well in duplicate) from ten fish/group at different time points after injected with tsb or a. hydrophila were placed in a 96-well plate containing 250 ml of m. lysodeikticus cell suspension per well. hen egg white (hew) lysozyme was used as an external standard. the absorbance values of 0 min and 20 min post incubation (30 c) were measured at 450 nm. the rate of reduction in absorbance of samples was converted to lysozyme concentration (mg/ml) using a standard curve of hew lysozyme. the results were expressed as mg/ml equivalent of hew lysozyme. the effect of serum on cell proliferation was performed using published procedures [29, 30] with slight modifications. serum samples collected at 28 days post first or second injection of a. hydrophila was used in this study. briefly, logarithmic phase a. hydrophila al09-71 bacterial cultures were diluted in tryptic soy broth to make a final concentration of 1.0 â 10 6 cfu/ml based on published procedures [29, 30] . the assay mixture contained 10 ml serum and 50 ml diluted bacterial culture. heat-inactivated (70 c, 30 min) serum samples at 28 days post first or second injection of a. hydrophila were used as negative controls. plates were incubated at 28 c for 2 h. the number of viable cells in each well was determined by celltiter 96 ò aq ueous non-radioactive cell proliferation inner salt] (mts) and an electron coupling reagent phenazine methosulfate (pms) was added to each well. the mts was then bioreduced by viable cells into a formazan product. the absorbance of the formazan product was measured at 490 nm using a biorad 680 microplate reader (biorad, hercules, ca) at 0, 5, 15, 30, 60, and 180 min post mts addition. relative increases in od values were calculated using the following formula: increased od value ¼ od value after incubation à od value at 0 min of incubation. all statistical analyses were performed using sigmastat 3.5 software (systat software, inc, point richmond, ca). differences in expression levels of gene at one time point, lysozyme activity, and inhibitory effect on cell proliferation were analyzed with student ttest and the significance level was defined as p < 0.05. statistical differences of expression levels of up-regulated genes at different time points after first infection compared to that after the second infection were analyzed with mannewhitney u significance test, a well-known non-parametric statistical hypothesis test to assess whether one of two samples of independent observations tends to have larger values than the other. a total of 96 clones were obtained from the subtractive library using the secondary infected fish as tester and primary infected fish as driver. all 96 clones were subjected to sequencing. of the 96 clones, 94 contained inserts. blastn analysis revealed that 28 unique expressed sequence tags (ests) ( table 1) were obtained from the 94 inserts. the insert sizes of the 28 unique ests ranged from 124 bp to 1054 bp, with average size of 452 bp (table 1) . eighteen and seven of the 28 sequences shared high identities with channel catfish (ictalurus punctatus) and zebra fish (danio rerio), respectively ( table 1 ). the 28 ests listed in table 1 were deposited in genbank dbest under accession numbers jk993536 to jk993563. of the 28 unique ests, 8 were significantly (p < 0.05) more upregulated by the second exposure to a. hydrophila compared to that by the first exposure at 6 hpi (fig. 1) . of the 8 ests, two (a10: xbai element 5 and b04: xbai element 7) were up-regulated greater than 6-fold by the second infection (fig. 1) . the other six ests that were also significantly more up-regulated by the second infection were: 1) a08: tlr20-1; 2) c04: inward rectifier potassium channel 13; 3) c09: immunoglobulin heavy chain gene locus; 4) c11: prolactin; 5) c12: nadh dehydrogenase subunit 2; 6) e05: reverse transcriptase-like protein (fig. 1 ). expression kinetics studies at 3, 6, 12, 24, and 48 hpi revealed that the 8 ests were significantly (p ¼ 0.016) more up-regulated by the first infection, with a major peak at 3 hpi (fig. 2) . the significantly (p < 0.05) higher expression levels of tlr20-1 induced by first infection compared to that by second infection peaked at 3 hpi, followed by a decreased peak at 24 hpi ( fig. 2a) . similar expression pattern was observed for xbai element 5 (fig. 2b) , xbai element 7 (fig. 2c) , inward rectifier potassium channel 13 (fig. 2d) , immunoglobulin heavy chain locus gene (fig. 2e) , and reverse transcriptase-like protein (fig. 2h ). significantly (p < 0.05) higher expression of prolactin (fig. 2f ) and nadh dehydrogenase subunit 2 (fig. 2g ) induced by first infection compared to that by second infection with the peak at 3 hpi were also observed. based on expression kinetics study results of the 8 ests identified by ssh, the time point of 3 hpi was chosen to screen the 96 selected genes. of the 96 genes selected from literature, 19 were found to be significantly (p < 0.05) induced by a. hydrophila at 3 hpi compared to that by tsb, regardless whether it was the first infection or the re-infection. the 19 genes belonged to the following five main categories: 1) toll-like receptor (tlr2, tlr3, tlr5, tlr21); 2) antimicrobial peptides (amps) (nk-lysin type 1, nk-lysin type 2, nk-lysin type 3, cathepsin d, transferrin, hepcidin); 3) cytokine or chemokine (interleukin-1b/il-1b, interleukin-10/il-10, tumor necrosis factor a/tnfa, chemokine cxcl-10); 4) signaling proteins (cadherin egf lag seven-pass g-type receptor 1, very large inducible gtpase 1, arginine deiminase type 2, lymphokine-activated killer t-cell originated protein kinase); 5) lysozyme (lysozyme c). overall, the expression of the four tlrs identified by the screen at various time points was significantly (p ¼ 0.005) more induced by the first infection compared to that by the second infection. the expression of tlr2 (fig. 3a) after injection with a. hydrophila once and twice both peaked at 3 hpi, with no statistical difference (p > 0.05) between the two. at 6 hpi, tlr2 was significantly (p < 0.05) more induced by the second infection compared to that by the first infection (fig. 3a) . at 12 and 24 hpi, tlr2 was significantly (p < 0.05) more induced by the first infection compared to that by the second infection (fig. 3a) . similar pattern was also observed for tlr3 (fig. 3b) and tlr21 (fig. 3d) . however, the expression pattern of tlr5 (fig. 3c) after the first infection of a. hydrophila was not statistically different from that after the second infection. the significantly (p < 0.05) higher expression level of tlr5 in fish injected with a. hydrophila compared to that with tsb peaked at 3 hpi (fig. 3c) . overall, the expression of the six amps identified by the screen at various time points was significantly (p < 0.001) more induced by the first infection compared to that by the second infection. of the 6 amps identified by the screen, the expression levels of transferrin (fig. 4a ) and hepcidin (fig. 4b) induced by a. hydrophila were the highest (>50 fold). the expression of transferrin had a major peak at 12 hpi, regardless whether it was a first infection or a second infection (fig. 4a) . the expression of hepcidin had peaks at 3 and 12 hpi, with significantly (p < 0.05) higher expression at 12 hpi induced by first infection (fig. 4b) . the expression of nklysin type 3 was highly and significantly (p < 0.05) induced by the first infection compared to that by the second infection at 24 hpi (fig. 4c) . similar pattern was also observed for nk-lysin type 1 (fig. 4d) and nk-lysin type 2 (fig. 4e) . the expression of cathepsin d after infection had peak at 12 hpi, with a significantly (p < 0.05) higher expression level induced by the first infection compared to that by the second infection (fig. 4f) . overall, the expression of the four cytokine or chemokine identified by the screen at various time points was significantly (p < 0.001) more induced by the first infection compared to that by the second infection. of the four cytokine or chemokine, the expression level of il-10 at 12 hpi induced by the first infection was the highest (up to 80 fold) (fig. 5a) . the expression of il-10 had a peak at 3 hpi, regardless whether it was a first infection or a second infection (fig. 5a ). its peaked expression at 12 hpi induced by the first infection was significantly (p < 0.05) higher than that by the second infection (fig. 5a) . the expression pattern of il-1b induced by the first infection was similar to that by the second infection (fig. 5b) . however, the expression levels of il-1b was induced significantly (p < 0.05) higher by the first infection compared to that by the second infection (fig. 5b) . chemokine cxcl-10 was induced the most at 24 hpi by the first infection (fig. 5c) , which was significantly (p < 0.05) higher than that by the second infection. the expression of tnfa was induced the most at 12 hpi by the first infection, which was significantly higher than that by the second infection (fig. 5d ). overall, the expression of the four signaling proteins identified by the screen at various time points was significantly (p < 0.001) more induced by the first infection compared to that by the second infection. the expression of cadherin egf lag seven-pass g-type receptor 1 peaked at both 3 and 24 hpi after the first infection, with significantly higher expression induced by the first infection compared to that by the second infection (fig. 6a) . similar pattern was observed for arginine deiminase type ii (fig. 6b) . the expression of the very large inducible gtpase 1 peaked at both 3 and 12 hpi after the first infection, with significantly (p < 0.05) higher expression induced by the first infection compared to that by the second infection (fig. 6c) . the expression of the lymphokine-activated killer t-cell originated protein kinase also peaked at both 3 and 12 hpi after the infection, regardless whether the infection time was once or twice (fig. 6d) . however, its highest expression was observed at 12 hpi after the first infection (fig. 6d ). the expression of lysozyme c identified by the screen at various time points was significantly (p ¼ 0.026) more induced by the first infection compared to that by the second infection. its expression after the first and second infection peaked at 24 and 12 hpi, respectively (fig. 7) , with significantly (p < 0.05) higher expression induced by the first infection at both time points. serum lysozyme activity at various time points after the first infection was significantly (p ¼ 0.005) higher than after the second infection (fig. 8) . its activity peaked at 24 hpi after the first infection, whereas its activity peaked at 12 hpi after the second infection. the serum lysozyme activity at 24 hpi after the first infection was significantly (p < 0.05) higher that that after the second infection (fig. 8) . at 28 days post infection, serum of channel catfish infected with a. hydrophila exhibited significantly (p < 0.05) higher inhibitory effect on the cell proliferation of a. hydrophila compared to the serum of tsb treated control fish (fig. 9a) , regardless whether the fish was infected once or twice. serum of tsb treated control fish also significantly (p < 0.05) inhibited the cell proliferation of a. hydrophila (fig. 9a) . when serum samples of fish were heatinactivated, no inhibitory effect on cell proliferation was observed (fig. 9b ). using ssh technique, 28 unique ests were identified from a total of 96 clones, of which 8 were confirmed to be slightly but significantly up-regulated by the second infection at 6 hpi compared to that by the first infection. of the eight ests, two xba elements (xbai element 5 and xbai element 7) were up-regulated greater than 6-fold by the second infection. in channel catfish, xba elements were a/t-rich tandem repetitive non-coding sequences [31] . non-coding rna sequences play important roles in regulating transcription of protein-coding genes [32] , including immunity-related genes [33] . transcriptome analysis of the host response to severe acute respiratory syndrome coronavirus (sars-cov) infection across four founder mouse strains has revealed differential expression of 500 ncrnas during infection [34] , indicating that differential expression of ncrnas is widespread in response to infection. taken together, these results suggest that the overexpression of ncrnas such as xbai elements might play important roles in immunity. expression kinetics studies at 3, 6, 12, 24, and 48 hpi revealed that the 8 ests were significantly more up-regulated by the first infection, with a major peak at 3 hpi. therefore, 3 hpi was chosen to screen the 96 selected genes. of the 96 genes selected from literature, 19 were found to be significantly induced by a. hydrophila at 3 hpi compared to that by tsb, regardless the infection time (first infection or second infection). the fact that ssh failed to identify these genes could be due to the following reasons. firstly, we used cdnas from the second infection as tester in an attempt to identify genes that are more up-regulated in adaptive response, which might have contributed to the failed discovery of other genes. secondary, we chose 6 hpi instead of 3 hpi as the time point for ssh which might have contributed to the failed discovery of the known immunity-related genes identified by the screen. nonetheless, ssh identified differentially expressed genes that were previously reported to be up-regulated by bacteria infections, including tlr20 and nadh dehydrogenase, both of which were reported to be upregulated in channel catfish by edwardsiella icatluri infection [24, 26] . in addition, ssh also identified genes that were previously not reported to be induced by infection in channel catfish, such as xbai elements, prolactin, and inward rectifier potassium channel. prolactin, a peptide hormone that shares many properties with cytokines [35] , plays an important role in immunity by inducing the expression of the genes encoding the major phagocyte nadph oxidase components and ros production in fish macrophages via the jak2/stat/irf-1 signaling pathway [36, 37] . macrophages have two types of potassium channels: inwardly rectifying potassium channel and voltage-gated potassium channel [38, 39] . potassium channel activity is required for the induction of nitric oxide and respiratory burst response in activated macrophages in goldfish (carassius auratus) [40] . of the 96 genes selected from literature, 19 were found to be significantly induced by a. hydrophila at 3 hpi, including four tlrs. expression kinetics study revealed that the expression pattern of tlr2, tlr3, or tlr21 after the second infection was different from that after the firs infection. however, tlr5 exhibited similar expression patterns after the first and the second infections, with significant peaked up-regulation at 3 hpi. tlrs are evolutionarily conserved receptors that function in innate immunity through recognition of the conserved pathogen-associated molecular patterns (pamps) of an invading pathogen and eliciting inflammatory and immune responses [41] . the best characterized ligand that tlrs recognize include: (1) lipoproteins by tlr2; (2) dsrna by tlr3; (3) lipopolysaccharide (lps) by tlr4; and (4) bacterial flagellins by tlr5 [42] . the recognition of pamps by tlrs will trigger an intracellular signaling cascade which activates signaling molecules such as cytokines, chemokines, interferons (ifns), and costimulatory molecules that aid in the development of the immune response [43] . in addition to significant up-regulation of tlr5 at 3 hpi, four signaling proteins were also found to be significantly up-regulated at 3 hpi after the first infection or the second infection, including cadherin egf lag seven-pass g-type receptor 1, very large inducible gtpase 1, arginine deiminase, and lymphokine-activated killer t-cell originated protein kinase. at 3 hpi, the expression of the four cytokine or chemokine also peaked, although the expression of il-1b and chemokine cxcl-10 after the first infection were the highest. when the expression levels of the total 27 genes at each time point after the first infection were compared to each other, the expression levels of the two xbai elements were up-regulated the most (w300 fold). at 6 hpi, the expression levels of transferrin and il-1b were the highest. at 12 hpi, the expression levels of il-10 and chemokine cxcl-10 were the highest. at 24 hpi, the expression levels of xbai element 5 and xbai element 7 were the highest. at 48 hpi, transferrin had the highest expression. il-1b is an important early response pro-inflammatory cytokine that mediates immune regulation in both innate and adaptive immunity [44] . when il-1b was over-expressed in common carp (cyprinus carpio), the macrophage functions such as production of superoxide anion and phagocytosis were significantly stimulated [45] . il-10 is reported to be an anti-inflammatory cytokine that plays a crucial role in the regulation of inflammation by down-regulating expression of other cytokines such as il-1b [46] . in consistent with previous report, we also observed the lowest expression level (<10 fold) of il-1b at 12 hpi when il-10 was expressed the highest (w60 fold). significant up-regulation of hepcidin and transferrin has been reported in channel catfish after infection with e. ictaluri or a. hydrophila [22, 30] . in consistent with previous reports, this study also revealed that hepcidin and transferrin were induced by infection with a. hydrophila. taken together, these results suggest that transcriptional regulation of immune genes plays and important role in the immunity of channel catfish against bacterial infections. when the expression levels of the 27 genes at different time points after the first infection were compared to that after the second infection, significantly higher expression levels of these genes were induced by the first infection. lower transcriptional level of genes in rainbow trout (oncorhynchus mykiss) re-infected with yersinia ruckeri o1 at 35 days post the first infection has been previously reported [47] . taken together, these results suggest that adaptive immunity might play an important role in the defense against the re-infection. significantly higher bactericidal activity of serum samples at 28 days post infection with a. hydrophila was observed in this study when compared to that of fish serum treated only with tsb. in addition, peaked expression and activity of lysozyme after the first infection was at 24 hpi, whereas that after the second infection was at 12 hpi, further suggesting that adaptive immunity in fish must have developed, therefore enabling faster production of lysozyme c to kill the bacteria. in summary, 96 clones were isolated from the ssh library. of the 96 clones, 28 ests were obtained, of which 8 were confirmed to be slightly but significantly more up-regulated by the twice exposure at 6 hpi. expression kinetics studies at 3, 6, 12, 24, and 48 hpi revealed that the 8 ests were significantly more up-regulated by the first infection, with a major peak at 3 hpi. of 96 genes that were reported to be up-regulated by bacterial infections in literature, 19 were found to be significantly induced by a. hydrophila at 3 hpi. the 19 genes belonged to the following five main categories: 1) toll-like receptor (tlr2, tlr3, tlr5, tlr21); 2) antimicrobial peptide (nklysin type 1, nk-lysin type 2, nk-lysin type 3, cathepsin d, transferrin, hepcidin); 3) cytokine or chemokine (interleukin-1b, interleukin-10, tumor necrosis factor a, chemokine cxcl-10); 4) signaling proteins (cadherin egf lag seven-pass g-type receptor 1, very large inducible gtpase 1, arginine deiminase type 2, lymphokine-activated killer t-cell originated protein kinase); 5) lysozyme (lysozyme c). significantly (p < 0.05) higher bactericidal activity of channel catfish serum at 28 days post the first infection compared to the fish serum treated with tryptic soy broth was observed in this study. peaked expression of lysozyme c and peaked lysozyme activity after the first infection was at 24 hpi, whereas that after the re-infection was at 12 hpi. taken together, our results suggest that both innate and adaptive immunity were involved in the defense of catfish against the re-infection of a. hydrophila. we thank drs. dunhua zhang (usda-ars) and mediha aksoy (tuskegee university) for critical reviews of the manuscript. we thank beth peterman (usda-ars) for her excellent technical hematological and biochemical parameters in common carp, cyprinus carpio, following herbal treatment for aeromonas hydrophila infection immune response of gilthead seabream (sparus aurata) following experimental infection with aeromonas hydrophila aeromonas hydrophila septicaemia of indian major carps in some commercial fish farms of west godavari district virulence and histopathology of aeromonas hydrophila (sah 93) in experimentally infected tilapia, oreochromis mossambicus (l.) antagonism of aeromonas hydrophila by propolis and its effect on the performance of nile tilapia endosulfan increases seric interleukin-2 like (il-2l) factor and immunoglobulin m (igm) of nile tilapia (oreochromis niloticus) challenged with aeromona hydrophila role of 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relative quantification in real-time rt-pcr identification and expression profile of multiple genes in the anterior kidney of channel catfish induced by modified live edwardsiella ictaluri vaccination identification and expression profile of multiple genes in channel catfish fry ten minutes after modified live flavobacterium columnare vaccination expression profiles of tolllike receptors in anterior kidney of channel catfish, ictalurus punctatus (rafinesque), acutely infected by edwardsiella ictaluri on the variation in the catalytic activity of lysozyme in fishes enzyme characterisation and gene expression profiling of atlantic salmon chicken-and goose-type lysozymes cloning and expression of a hepcidin gene from a marine fish (pseudosciaena crocea) and the antimicrobial activity of its synthetic peptide expression profiles of seven channel catfish antimicrobial peptides in response to edwardsiella ictaluri infection characterization of an a/t-rich family of sequences from channel catfish (ictalurus punctatus) epigenetic regulation by long noncoding rnas transcription of il17 and il17f is controlled by conserved noncoding sequence 2 unique signatures of long noncoding rna expression in response to virus infection and altered innate immune signaling prolactin, growth hormone, and insulin-like growth factor-i in the immune system induction of genes encoding nadph oxidase components and activation of ifn regulatory factor-1 by prolactin in fish macrophages prolactin-releasing peptide is a potent mediator of the innate immune response in leukocytes from salmo salar kv1.3 potassium channels in human alveolar macrophages differential voltage-dependent kþ channel responses during proliferation and activation in macrophages induction of nitric oxide and respiratory burst response in activated goldfish macrophages requires potassium channel activity the toll receptor family and microbial recognition divergent toll-like receptors in catfish evolution of interleukin-1beta the immune responses of common carp, cyprinus carpio l., injected with carp interleukin-1beta gene interleukin 10 (il-10) inhibits human lymphocyte interferon gammaproduction by suppressing natural killer cell stimulatory factor/il-12 synthesis in accessory cells innate immune response in rainbow trout (oncorhynchus mykiss) against primary and secondary infections with yersinia ruckeri o1 catfish genetics research unit) for their excellent sequencing work. we thank beth peterman (usda-ars) for her excellent technical support. we also thank the management team of the aquatic animal health research unit for daily care and management of the fish. this study was supported by the usda/ars cris project #6420-32000-024-00d. the use of trade, firm, or corporate names in this publication is for the information and convenience of the reader. such use does not constitute an official endorsement or approval by the united states department of agriculture or the agricultural research service of any product or service to the exclusion of others that may be suitable. supplementary material related to this article can be found at http://dx.doi.org/10.1016/j.fsi.2013.08.027. key: cord-103342-stqj3ue5 authors: prakash, meher k; kaushal, shaurya; bhattacharya, soumyadeep; chandran, akshay; kumar, aloke; ansumali, santosh title: a minimal and adaptive prediction strategy for critical resource planning in a pandemic date: 2020-04-10 journal: nan doi: 10.1101/2020.04.08.20057414 sha: doc_id: 103342 cord_uid: stqj3ue5 current epidemiological models can in principle model the temporal evolution of a pandemic. however, any such model will rely on parameters that are unknown, which in practice are estimated using stochastic and poorly measured quantities. as a result, an early prediction of the long-term evolution of a pandemic will quickly lose relevance, while a late model will be too late to be useful for disaster management. unless a model is designed to be adaptive, it is bound either to lose relevance over time, or lose trust and thus not have a second-chance for retraining. we propose a strategy for estimating the number of infections and the number of deaths, that does away with time-series modeling, and instead makes use of a 'phase portrait approach'. we demonstrate that, with this approach, there is a universality to the evolution of the disease across countries, that can then be usedto make reliable predictions. these same models can also be used to plan the requirements for critical resources during the pandemic. the approach is designed for simplicity of interpretation, and adaptivity over time. using our model, we predict the number of infections and deaths in italy and new york state, based on an adaptive algorithm which uses early available data, and show that our predictions closely match the actual outcomes. we also carry out a similar exercise for india, where in addition to projecting the number of infections and deaths, we also project the expected range of critical resource requirements for hospitalizations in a location. the world health organization (who) has declared covid-19, a disease caused by the novel coronavirus (sars-cov-2), as a pandemic. covid-19 is causing infections and deaths globally on a scale that has not been seen in this century. the virus made a zoonotic transition into humans and then continued person to person transfer. 1 analysis of the 1918 spanish flu suggests a need for timely action from the governments. 2, 3 with no vaccines or treatment options, prevention via social distancing and city or nation-wide lockdowns have become the catchword for containing the spread of infections. these restrictions on the movement of people of course have adverse effects on the economies. there have been several efforts to model the spread of the pandemic [4] [5] [6] to understand the gravity of the situation that one is facing 7 as well as to suggest travel restrictions 8, 9 and to guide policy measures 10-12 on the extent of testing or the implementation of the lockdowns. further, the unexpected surge in patients requiring hospitalizations and intensive care is leading to the collapse of health care systems globally. gearing up for providing the health care supplies including critical equipment such as ventilators requires an accurate estimate of the number of infections. but the key information that is required for this modelling, which is the number of infected people at any time, is in itself marred by uncertainties. there has been an acute shortage of testing kits required for extensive screening. as a result, although symptomatic patients may have been traced or tested, a significant fraction of the global infections are believed to have been through asymptomatic patients. 13 although a pandemic such as covid-19 appears as a once-in-a-century event, the 21st century has already seen a few others which drew very close: sars in 2003, swine flu in 2009, mers in 2015. thus, having tools to quickly plan for the critical resource requirements is important. the nature of transmissible diseases such as covid-19 is that the number of infected people grows exponentially. the leaders of many countries have made a sudden switch from complacency to active mitigation policies, as if they were caught by surprise while expecting linear trends. the nature of the numerics associated with the long-term pandemic trajectory evolution is that the errors in estimation also increase exponentially, no matter how detailed the model is. an early prediction of a pandemic that forecasts the evolution for many months will accrue errors very quickly, and a model that is developed much later in the evolution will only be relevant for post facto analyses, and not for policy decisions or disaster management. 2 therefore, the models should be designed to be adaptive, such that their projections are constantly corrected. they should also be simple enough to be interpretable by the various stakeholders (with differing abilities to understand the detailed models) involved in the management of the pandemic. further, due to the differences in the allowed freedom of movement or the rigour of policy implementation in various states or provinces in different countries, the predictions should be recalculated. the focus of this work is to set up a framework that can be useful to estimate the need for critical resources such as hospital beds and ventilators, the need for which changes with time and with the region (province, state or district) of interest. inter alia, we also make predictions based on early data for italy and new york state, update them, and demonstrate good agreement between the subsequent evolution of the pandemic and our predictions. our approach can be summarized as follows: the covid-19 data from most countries suggests that, especially in the growing phase of the pandemic, the number of active cases and the number of hospitalizations are both proportional to the total number of infections: approximately around 70-90 % and 20-30%, respectively. this conclusion is arrived at by eliminating "time" as a variable, and focusing only on the above-cited ratios. moreover, it is quite easy to update our estimates on a weekly basis. a simple law that can capture the spread of infections is where α is the rate of transmission, i is the number of infected people, r is the number of people who recover from infection and p is the total population. further details can be added by noting that i is the sum of symptomatic and asymptomatic patients, and that α differs for the two groups and across communities depending on their social contact structure or policies of various degrees of isolation. however, this is not done here. when the infections are in a growing phase, the data suggests that i r, which in turn suggests that i can be used as a proxy for the number of active infections. coming to the focus of this work, we sugguse that the number of active infections can be the basis for critical resource planning during the evolution of the pandemic. also, regardless of the scale of the pandemic, it remains manageable only when p i. this allows one to approximate eq. 1 as where r is the effective rate of the spread of infection. the parameter r depends on the infection under consideration. during the early days of the pandemic, when detailed biological understanding of the disease spread is still elusive, it can be a challenge to estimate the parameter r accurately. one approach is to estimate i is an integer-valued variable, and further, its values would be small at the outset. moreover an analysis of the raw time series data from several countries reveals that there is no universal value for r across countries. this is illustrated in figure: 1a. even the cumulative value of i does not reveal any universal trend (see figure: 1b). therefore it is necessary to adopt a different approach. our approach is to eliminate time as a variable. by doing so, we found three universal trends, one partly expected and the others not so immediately apparent: 1. the infection rate is proportional to the number of current infections ( figure: 2a). in principle this is expected from eq 2. in practice this finding is interesting because the rates from all countries are comparable, especially during the early days of the spread of infection before any policy changes are implemented. this also underscores the fact that the biology of the disease is similar from one country to the other, without further mutations in the virus or differences in immunity levels of the different populations. this commonality across the data from different countries has been previously highlighted by many. 14 2. interestingly, there is also another universal pattern across the countries, where the rate of deaths caused by the infection is proportional to the number of deaths ( figure:2b) . further, the fluctuations in this data are lesser in extent compared to that in figure: 2a, a feature that we will exploit later. it is not immediately apparent why the rate of deaths should be proportional to the cumulative deaths. one possible explanantion is that the number of deaths depends directly on the number of infections. 3. the cumulative number of deaths at any time is directly related to the number of overall infections(figure:3a). considering the wide variation across countries in the duration of the hospitalization and critical care, it is not apparent why this should be so. however, the similarity in the data from across the countries is 4 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.08.20057414 doi: medrxiv preprint as noted earlier, it is not easy to scale up the number of test kits and to arrange tests in large populations. thus the official count of the number of infected individuals at any given time is far from being very precise. an alternative way of estimating the number of infections is via monitoring of the the number of deaths. the key idea is that the fraction of infected people who may die are most likely to be in hospitals and thus a fraction of the reported infection by the state. while this fraction might depend on country and quality of testing, it is more likely that an infected person who may die due to other health complications will be in hospital. to do this, we incorporate a scaling factor to account for the extensiveness of the testing. it is believed that, to date, the most extensive screening tests have been performed by south korea, detecting most of the symptomatic and asymptomatic infected individuals. thus, using the data from south korea as a reference standard, the deaths versus infections curve has been readjusted as seen in figure:3a cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . figure 3 : using the i-d plot for estimating number of infections. these plots depict the universal relation between i and d, barring a corrective factor. this uncertainty factor that was calculated from the covid-19 infection data of south korea as a reference can be used for estimating infections when the number of deaths are known. β = corrected infections reported infections was assumed to be 1 for south korea. at any time in the evolution of the pandemic shown on this i versus d graph, using the known number of deaths in that country, the number of infections were rescaled to match with the infections in south korea and this factor was used as β. the figure illustrates the procedure used for β when deaths are slightly lower than 10, equal to 10 and 20. of a higher median age (for example in italy) are infected compared to individuals of a slightly lower median age (for example in germany). however, remembering that the objective is to plan for the resources needed for the individuals who are currently infected, the lower death rate is likely to predict the number of infections on the safer side as far as resource planning is concerned. the estimate of infections from these two different approaches will be used to set the limits on the number of infected individuals, and the resources required. the preceding subsection addressed the difficulties in measuring i, the number of infections. in this subsection we discuss the estimation of r, the rate at which the infection spreads. a typical way to estimate r is to fit the time series of i(t) by an exponential. as pointed out earlier, when i is small, fitting a time series to an integervalued (and possibly noisy) variable is problematic. instead, we use the slope of di/dt versus i ( figure:2a) to estimate r. instead of applying eq 2 exactly with all the attendant uncertainties in i and r, we use a piecewise strategy. a simple discretization of this in time steps of ∆t gives i(t + ∆t) = i(t) + r t,t+∆t · i(t). it was seen from the covid-19 infection data from several countries that as the number of daily new infections fluctuates considerably, the trends from the data can be captured only by following the data over a few days. we work with a ∆t = 7 days, since inferring r from the data drawn over a shorter duration or making a prediction more fine-grained than this do not seem to be relevant from a practical point of view. the multiplicative factors r t,t+∆t estimated week by week, using the universal patterns observed in figure: 2b, are given in supplementary table 1 . we make two predictions for i(t): one using the reported infections from the country, and another using the reported deaths. the scaling factor β is re-calibrated once a week in an adaptive fashion, using the data until that week. in essence, the exponential function of time has been replaced by a geometric series for each week. the results from adaptively predicting the number of covid-19 related deaths from italy are illustrated in 6 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.08.20057414 doi: medrxiv preprint 7 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.08.20057414 doi: medrxiv preprint reported infections (ny) predictions using infections predictions using deaths a b figure 5 : adaptive predictions for new york state. the adaptive predictions were performed for the data from new york state, just as described in figure: 4. a lockdown can cause two changes in our model. first, depending on the levels of restrictions on movement such as social distancing, r t,t+∆t will reduce or even drop to zero. the r t,t+∆t in the weeks following important restrictions on movement from different countries was also used as a lower bound on how fast the new infections can decrease. second, when a country imposes a lockdown by imposing restrictions on international or interstate movement, the different physically separated regions will follow their own independent growth trajectory. the weekly growth factor to be applied to each specific state or province will depend upon the number of infected in the province at the time of the decision, with some regions lagging behind the others by weeks. one thing we learnt from covid-19 is that each country or province, before it becomes a transmission vector for other places, has a lead of several weeks or months over those places. the lead of course depends on the extent of people travelling between these places. a disease that was once confined to a specific region, diffuses locally and jumps to distant locations almost like a jump-diffusion process. there is a self-replication phenomenon at different scales, wherein the universalities in the patterns between the nations and at a national level, now are reflected between the individual states or provinces. this observation can be used by the governments of these provinces to plan for the worst-case scenario. each pandemic is different in its ability to persist, infect, and diffuse through the population. while the medical knowledge and critical equipment required may differ from the earlier pandemics, as long as the universalities discussed in this work are seen in the data, one can certainly learn from other countries, states or provinces that experienced the same pandemic a few weeks or months earlier. 8 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint table 1 ). 15 table 1 lists the projected number of ventilators required in each state as well as the gross national estimate for a period of four weeks beginning from april 7th, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. in this work we aimed to provide tools for critical resource planning in the wake of a pandemic. we do this by introducing three attributes that are not common in epidemiological modeling: we used the number of deaths as an additional and surrogate marker for the otherwise uncertain number of infections. given the data is stochastic, and with several unknowns, we focused on making weekly predictions based on a geometric series with piecewise adaptive ratio. adapting the geometric ratio week by week serves two objectives: (i) smoothing out the daily fluctuations in data, and (ii) bridging the gap between predictions made using early data, and current data. the simplicity of the formalism, and its data-aware design permit us to make adaptive predictions for the next few weeks, starting at any time. although covid-19 is a rare pandemic, the quick, simple and adaptive principles laid out here, embracing the limitations of testing and drawing on the universalities of the progression, should be relevant for any pandemic spreading through person to person contact. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.08.20057414 doi: medrxiv preprint a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster the effect of public health measures on the 1918 influenza pandemic in us cities public health interventions and epidemic intensity during the 1918 influenza pandemic real-time forecasts of the covid-19 epidemic in china from early dynamics of transmission and control of covid-19: a mathematical modelling study an interactive web-based dashboard to track covid-19 in real time real-time tentative assessment of the epidemiological characteristics of novel coronavirus infections in wuhan, china impact of international travel and border control measures on the global spread of the novel 2019 coronavirus outbreak the effect of travel restrictions on the spread of the 2019 novel coronavirus on fast multishot epidemic interventions for post lock-down mitigation: implications for simple covid-19 models age-structured impact of social distancing on the covid-19 epidemic in india substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) countries test tactics in war against covid-19 the authors declare no competing interests. key: cord-267115-6jqdi417 authors: giobbe, giovanni giuseppe; bonfante, francesco; zambaiti, elisa; gagliano, onelia; jones, brendan c.; luni, camilla; laterza, cecilia; perin, silvia; stuart, hannah t.; pagliari, matteo; bortolami, alessio; mazzetto, eva; manfredi, anna; colantuono, chiara; di filippo, lucio; pellegata, alessandro; li, vivian sze wing; eaton, simon; thapar, nikhil; cacchiarelli, davide; elvassore, nicola; de coppi, paolo title: sars-cov-2 infection and replication in human fetal and pediatric gastric organoids date: 2020-06-24 journal: biorxiv doi: 10.1101/2020.06.24.167049 sha: doc_id: 267115 cord_uid: 6jqdi417 coronavirus disease 2019 (covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection is a global public health emergency. covid-19 typically manifests as a respiratory illness but an increasing number of clinical reports describe gastrointestinal (gi) symptoms. this is particularly true in children in whom gi symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. by contrast, fetuses seem to be rarely affected by covid-19, although the virus has been detected in placentas of affected women. these observations raise the question of whether the virus can infect and replicate within the stomach once ingested. moreover, it is not yet clear whether active replication of sars-cov-2 is possible in the stomach of children or in fetuses at different developmental stages. here we show the novel derivation of fetal gastric organoids from 8-21 post-conception week (pcw) fetuses, and from pediatric biopsies, to be used as an in vitro model for sars-cov-2 gastric infection. gastric organoids recapitulate human stomach with linear increase of gastric mucin 5ac along developmental stages, and expression of gastric markers pepsinogen, somatostatin, gastrin and chromogranin a. in order to investigate sars-cov-2 infection with minimal perturbation and under steady-state conditions, we induced a reversed polarity in the gastric organoids (rp-gos) in suspension. in this condition of exposed apical polarity, the virus can easily access viral receptor angiotensin-converting enzyme 2 (ace2). the pediatric rp-gos are fully susceptible to infection with sars-cov-2, where viral nucleoprotein is expressed in cells undergoing programmed cell death, while the efficiency of infection is significantly lower in fetal organoids. the rp-gos derived from pediatric patients show sustained robust viral replication of sars-cov-2, compared with organoids derived from fetal stomachs. transcriptomic analysis shows a moderate innate antiviral response and the lack of differentially expressed genes belonging to the interferon family. collectively, we established the first expandable human gastric organoid culture across fetal developmental stages, and we support the hypothesis that fetal tissue seems to be less susceptible to sars-cov-2 infection, especially in early stages of development. however, the virus can efficiently infect gastric epithelium in pediatric patients, suggesting that the stomach might have an active role in fecal-oral transmission of sars-cov-2. severe acute respiratory syndrome coronavirus 2 is responsible for a pandemic that has proven catastrophic, due to the lack of immunity in the human population and the range of pathological features associated with infection, including severe and often life-threatening respiratory syndromes causing major health, social and economic consequences. the virus has been shown to infect respiratory epithelial cells and to spread mainly via the respiratory tract 1 . as governments and international health agencies seek effective policies to minimise infections, maintain health care delivery, and eventually ease movement restrictions, understanding the pathogenesis and the various mechanisms for transmission is of the utmost importance. it is well established that adults are more likely than children to develop symptoms upon sars-cov-2 infection, but little is known on the role of children in transmission of the disease. a growing body of literature suggests that replication at the level of the gastrointestinal (gi) tract not only occurs in a large proportion of confirmed cases 2 , but it also extends the overall duration of shedding, after viral clearance from the respiratory tract has occurred 3 . interestingly, infected children have been shown to be particularly prone to develop gi symptoms which can be moderate-to-severe, leading to intensive care unit (icu) admission and mimicking, in some cases, symptoms of appendicitis 4 . additionally, sars-cov-2 was detected by means of electron microscopy in stool samples, elevated concentrations of sars-cov-2 rna were detected in air samples collected in patients' toilet areas 5 and rectal swabs from mildly symptomatic pediatric patients persistently tested positive, even after viral clearance from the upper respiratory tract had occurred 6 . this evidence, together with the recent demonstration of a high receptor density at the level of the oral cavity and tongue 7 , raises important questions about the likelihood of fecal-oral transmission and whether therapeutic interventions to reduce gastrointestinal infection will play a role in the control of the disease. however, a recent report of 244 consecutive covid-19 positive children from wuhan did not find any difference in fecal nucleic acid rt-pcr between children with or without gi symptoms, suggesting that rt-pcr detection of the virus was not due to gut infection but coming instead from the respiratory tract from swallowed sputum 8 . defining the role of the gi tract in sars-cov-2 infection may also help understand the risks of vertical transmission during gestation since amniotic fluid is swallowed by the fetus during gestation and viral contamination has been isolated from the placenta of an affected woman 9 . while samples from affected mothers have so far failed to prove that amniotic fluid, cord blood, and breast milk contain sars-cov-2 10 , very limited data are available at this stage for pregnant women with covid-19, and even fewer data are available on intrauterine vertical transmission 11 . while severe symptoms and death have been recorded in infants as young as 10 months of age 12 , very few cases of pathology in neonates have been associated with infection 13 and when 6 newborns from infected mothers were screened for sars-cov-2, they tested negative for the virus 10 . however, this limited cohort cannot exclude the possibility of infection of the fetuses. reliable human in vitro gi model systems that faithfully reproduce infection dynamics and disease mechanisms will prove key to advance our understanding of sars-cov-2 replication and pathology in the gi tract. little information is available with respect to the distribution of the viral receptor angiotensinconverting enzyme 2 (ace2) at the level of the gi tract of humans 14 . in particular, we lack fundamental information regarding which region of the gi system is the target of replication and primarily associates with the prolonged shedding of sars-cov-2 in both pediatric and adult patients. organoids have attracted great attention, enabling in vitro disease modeling and providing an ideal tool for studying infectious pathogens, particularly of the gi tract 15 . recent studies have demonstrated how sars-cov-2 can efficiently infect human intestinal enteroids 16, 17 , providing evidence in support of the hypothesis that sees sars-cov-2 as a fecaloral transmissible pathogen. however, it remains to be elucidated whether access to the duodenum depends on passive transport of infected oral fluids across the stomach, or on active viral replication in the gastric mucosa. human gastric organoids derived from adult patients and induced pluripotent stem cells have proved to be instrumental for the generation of reliable in vitro models for the characterization of infectious agents 18, 19 . organoid derivation from human fetal organs has been shown for the intestine 20 , liver 21 and pancreas 22 . here, we describe the novel derivation of proliferative progenitors from human fetal stomach and their expansion in vitro as enterospheres. furthermore, we provide insight into the ability of sars-cov-2 to infect an organoid-based model of the gastric mucosa at both fetal and pediatric ages. this work aims to unravel the susceptibility of the stomach to sars-cov-2 infection through the development of an innovative expandable in vitro model that faithfully reproduces the gastric microenvironment. a deeper understanding of the susceptibility of the human stomach to sars-cov-2 infection and replication could lay the foundations for the development of therapeutic options to reduce gastrointestinal infection. organoids are organized three dimensional structures that can be grown from isolated stem cells found in adult and fetal tissues. in order to derive a novel in vitro gastric model of fetal origin, we firstly characterized the tissues isolated from human fetuses and compared them to gastric mucosa obtained from pediatric patients undergoing surgery (fig. 1a) . developing stomach structures are shown in fig. 1b from carnegie stage (cs) 23 (corresponding to mid-week 8) to post conception week (pcw) 21. gastric crypts start to invaginate between pcw 11 and pcw 12 and form a clearly defined crypt at around pcw 20 (fig. 1c) . we characterized the appearance of gastric markers during fetal development. mucin 5ac positive pit mucous cells were evident at pcw 11, while pepsinogen c (marking chief cells) started to emerge at around pcw 20 (fig. 1d) . mucin 6, a gland mucous cell marker, was constitutively expressed from early week 8 (cs 20), together with enteroendocrine cells marked by chromogranin a that were present from mid-week 8 (cs 23) (fig. 1e) . we then defined three distinct groups of gastric epithelial tissues based on gland maturity: 1) early fetal stomachs from pcw 8 to pcw 15; 2) late fetal stomachs from pcw 17 to pcw 21; 3) pediatric stomachs. real time quantitative pcr (qpcr) was performed on gastric tissues obtained from these three groups to examine the gene expression changes of stem cell and differentiated cell markers. a significant correlation between developmental stage and mrna expression was observed for axin2, mucin 5ac (muc5ac), pepsinogen a5 (pga5), with a similar trend for chromogranin a (chga) and atpase h+/k+ transporting subunit beta (atp4b) (fig. 1f) . on the other hand, expression of leucine-rich repeat-containing g-protein coupled receptor 5 (lgr5) and somatostatin (sst) were significantly higher in the late fetal stomachs. following gastric tissue characterization, we efficiently extracted glandular crypts from fetal and pediatric stomachs utilizing chelating buffers and mechanical stress. to improve compatibility with subsequent clinical application of this organoid system, isolated fetal cells were expanded in a chemically defined medium, without the use of animal serum or conditioned media. each gastric cytokine, based on previous work 19 was screened and selectively removed from the organoids split to single cells and grown for 10 days to allow clonal organoid formation. while r-spondin 1,wnt-3a and noggin withdrawal led to more differentiated morphology, chir99021 (gsk-3 inhibitor) proved to be essential in the formation of fetal gastric organoids starting from single cells (fig. 2a) . no difference was found among fetal and pediatric organoid growth in the medium. we then performed isolation of several gastric organoid lines (supplementary table 1 ). the isolation protocol proved to be highly efficient and we obtained a biobank composed of 5 lines of early fetal stage (from cs23 to pcw 11), 6 lines of late fetal stage (from pcw 18 to pcw 21), and 4 lines of pediatric stage organoids (from 4 months-to 11 years-old). expanding organoids were stained for the epithelial marker ezrin (ezr) and luminal polarized f-actin fig. 2c . muc5ac was present on the luminal side of the organoids of all stages, with a relatively lower expression in the early pcw 11 (fig. 2c ). organoids were expanded and counted for several months, showing higher rate of expansion for earlier fetal stages (fig. 2d) . no plateau was reached in any of the curves even after several months, showing the possibility to obtain stable fetal gastric organoid lines ( supplementary fig. 1 ). after weekly passaging for more than 10 weeks, we further characterized the organoid lines to evaluate genomic stability. single nucleotide polymorphism (snp) arrays on early fetal, late fetal and pediatric organoids showed no chromosomal duplications, no large deletions, nor other karyotype aberrations, demonstrating the organoids are genetically stable after prolonged in vitro culture (fig. 2e) . real time pcr was performed on organoids grouped in early fetal (cs 23 to pcw 11), late fetal (pcw 18 to pcw 20) and pediatric. stem cell crypt markers lgr5 and axin2 were expressed in these organoids, indicating the presence of proliferating cells. muc5ac showed a pattern of increased expression along differentiation comparable to the tissue of origin in fig. 1f . the expression patterns of muc6 and sst were also comparable to the tissue of origin. on the other hand, chga showed an inverted pattern of expression, while transcript expression of proton pump transporter atp4b, responsible for gastric acid secretion, was lost in the organoid model (fig. 2f) . next, we characterized the transcriptomics of gastric epithelial tissues and gastric-derived organoids, at three developmental stages. rna-seq was performed on the three groups of early fetal, late fetal and pediatric samples. principal component analysis (pca) showed smaller heterogeneity in the organoid groups derived at different stages of fetal and pediatric development with respect to the primary tissues analyzed at the same stages, which may also include some heterogeneity from the surrounding cells as a result of the isolation procedure (fig. 3a) . when pca was performed including only organoid samples, the overall variability due to the different developmental stage was comparable to that between biological replicates within the same group ( supplementary fig. 2a ). this analysis suggests that transcriptional differences related to the developmental stage of the tissue of origin could be more subtle than those captured at pca level. we then analyzed the expression of typical gastric markers in organoids derived from tissues at different stages 23 . the only differentially expressed gene (deg) was muc5ac, which was more highly expressed in organoids from tissues at later developmental stages (fig. 3b) , confirming the qpcr results above (fig. 2f) . we did not observe processes of "intestinalization" of the organoids in culture, as cdx2 expression was negligible ( supplementary fig. 2c ). consistent with the qpcr results in fig. 2f , expression of atp4a and atp4b proton transporters were not detectable in the rna-seq, confirming the absence of the parietal cells in the organoids ( supplementary fig. 2d ). on the other hand, most putative genes identifying gastric crypt stem cells (sox9, olfm4, procr, mki67, tacstd2) were expressed at all developmental stages ( supplementary fig. 2e ). rna-seq analysis on the gastric primary tissues showed a significant increase in transcript levels of the functional markers along the developmental stage (fig. 3c) , confirming that the temporal trend shown by pca (fig. 3a) is related to specific gastric developmental stages. when we performed hierarchical clustering analyses of the previously reported genes representing the six stomach cellular subtypes 23 , most of these genes from the analysis were not degs (fig. 3d) . indeed, these six cell types are known to be all co-present at different stages of embryo development from pcw 7 to 25 23 . furthermore, we clustered degs between pair of conditions to reproduce a pseudo-temporal profile between the three developmental stages considered (fig. 3e) . we highlighted in fig. 3f the results of a pathway enrichment analysis from selected clusters that displayed gastric-related functions. full results are reported in (supplementary file 1). in order to validate both fetal and pediatric gastric organoids as functional in vitro models of sars-cov-2 infection and replication, we optimized the culture condition for viral infection in a 3d system (fig. 4a) . standard organoids of endodermal organs have a luminal polarity facing the internal portion of the structure, with an apical (inner) f-actin and zonula occludens-1 (zo-1), and basal (external) lamina marked by b-4 integrin (b4-int) (fig. 4b) . such inner polarity might be an obstacle to an efficient viral infection in vitro, given that the apical side is luminal. in addition, matrigel might impede efficient diffusion of the virus, thus affecting the likelihood of establishing an infection, and subsequent detection and quantification of the viral progeny released from the infected organoids. to maximize the efficiency of infection and the effective quantification of viral progeny released, we reverted the polarity of the gastric organoids 24 to expose the apical side of the cells on the outer side. organoids were removed from the surrounding extracellular matrix and cultured in suspension for 3 days, resulting in the exposure of the apical f-actin on the outer side, accompanied with muc5ac secretion externally (fig. 4c) . conversely, zo-1 and b4-int expression was inverted compared to standard organoids in fig. 4b . full 3d deconvolution images of reversed organoids are shown in supplementary fig. 3a . we then analyzed the absolute expression of ace2 and transmembrane protease serine 2 (tmprss2) sars-cov-2 receptors in our gastric models to evaluate the sars-cov-2 infection potential. rna-seq data analysis showed that expression of ace2 was significantly lower in early fetal stomachs compared to the pediatric ones, while late fetal samples' higher variability prevents drawing a final conclusion. on the other hand, tmprss2 mrna expression was consistently high throughout the stomach samples ( fig. 4d) . we further performed rna sequencing on rp-gos to evaluate the transcriptional changes. pca analysis showed similar clustering among the different stages between rp-gos ( supplementary fig. 3b ) and normal polarity organoids ( supplementary fig. 2a) . a comparable pattern of expression for ace2 and tmprss2 was observed also in rp-gos, with ace2 significantly higher expressed in pediatric organoids (fig. 4e ). protein expression of ace2 was further confirmed by immunofluorescence staining in all the rp-gos derived at pcw11, pcw 20 and pediatric stages (fig. 4f) . to investigate the susceptibility of organoids to sars-cov-2 infection, we selected a sars-cov-2 isolate obtained from the pharyngeal swab of a 14-year-old pediatric patient. purity of the isolate was confirmed by means of molecular testing comprising an extended panel of bacterial and viral respiratory agents. reversed-polarity gastric organoids derived at pcw11, pcw 20 and pediatric stages and normalpolarity organoids from the same stages were infected by trained virologists in a biosafety level 3 (bsl3) laboratory. after a 2-hour infection, the organoids were cultured up to 96 hours in suspension and checked for structural integrity and viability by visual examination on a daily basis (fig. 5a) . vero e6 cells were used as a susceptible substrate for sars-cov-2, to validate the infection in vitro (supplementary fig. 4a ). in next, we performed rna-seq analysis on non-infected and infected organoids samples at each developmental stage. interestingly, we identified significantly degs in samples from pcw 20 (19 degs) and pediatric organoids (13 degs), but not in pcw11 samples (fig. 6a ). all the degs in both developmental stages were up-regulated after the infection, among which 10 genes (cmpk2, ddx58, dhx58, herc5, ifi44, ifit2, ifit3, irf7, mx1, rsad2) were in common. we further performed an analysis to find the degs associated with the infection irrespective of the developmental stage, identifying a further 8 degs (bst2, eif2ak2, herc6, ifi44l, lamp3, slc1a3, stat1, usp18), for an overall number of degs equal to 30. intriguingly, approximately 63% of the degs identified in this study as respondent to the infection were previously found to be degs in a literature survey of transcriptomic data on sars-cov, where 38 genes were identified as degs at the intersection of at least 9 studies (fig. 6b ). among the common degs, some were first responders of the infection process, like the ddx58 and ifih1 encoding the viral rna sensors rig-i and mda5 respectively, and their regulators, such as dhx58; others more downstream players of the response, such as oas2 that is activated by detection of dsrna to inhibit viral replication, ifit2 that inhibits the expression of viral mrnas, and bst2 that limits viral secretion. all the 30 degs identified in this study, except slc1a3, showed an up-regulation in response to the infection in samples from pcw 20 and the pediatric patient (fig. 6c) . ifi44l showed the highest fold change both in pcw 20 and in pediatric samples. this gene was previously found to be a marker of viral infection compared with to bacterial infection 25 and more recently described as a negative modulator of innate immune responses induced after virus infections 26 . type i, ii and iii interferon (ifn) transcripts were not differentially expressed between non-infected and infected rp-gos. we then performed an enrichment analysis within the reactome database to understand the functional implications of the degs up-regulation after the infection in pcw20 and pediatric samples (fig. 6d ). interestingly, the majority of degs fell within pathways associated with the innate response to viral infection, particularly those involved in the regulation of type i ifn alpha/beta by cytoplasmic patternrecognition receptors (prrs) such as rig-i and mda5, and the expression of ifn stimulated genes (isgs). moreover, to capture more subtle differences between non-infected and infected samples that do not emerge in the deg analysis, we performed a quantitative set analysis for gene expression (qusage) 27 within the gene ontology database (supplementary file 2). categories related to the ifn response to the infection were identified in all three sample groups (pcw11, pcw20, and pediatric), other categories were reported for completeness, but require further studies to understand their relevance. reliable in vitro models capable of reproducing complex in vivo systems are becoming increasingly important in life sciences and play a crucial role in the investigation of emerging pathogens of devastating sanitary and economic impact like sars-cov-2. in the context of the covid-19 pandemic, it is still unclear how gastrointestinal virus replication might affect the clinical outcome of infection, the development of immunity and the transmission dynamics in the population. while it has been shown that sars-cov-2 is frequently detected in rectal samples of affected children and adults, it remains to be determined if the virus is able to produce a primary infection throughout the entire gi tract, or if its presence could be related in part to a passive transport of contaminated sputum coming from the upper respiratory tract. most importantly, the ability of sars-cov-2 to persist in the gi tract after respiratory clearance, has not yet been fully elucidated in terms of viral infectivity, possibly impairing important public health and policy measures for the control of the disease. these concerns are particularly relevant in children who appear on average to suffer a less severe respiratory illness compared to adults, despite recording more prominent gi symptoms with clinical pictures mimicking appendicitis 28 , a hyperinflammatory shock syndrome (paediatric multisystem inflammatory syndrome -temporally associated with sars-cov-2, pims-ts) 29 , or acting as relatively asymptomatic carriers of the virus. those risks have prompted clinical guidelines recommending the avoidance of aerosol producing procedures, including upper gi endoscopies, in children with confirmed or suspected covid-19 for the safety of frontline clinical staff and other patients. susceptibility of the different portions of the gi tract to sars-cov-2 infection has not been fully characterized and due to a paucity of autopsy reports targeting the gastric compartment 2 , the capacity of sars-cov-2 to infect the gastric mucosa is still unclear. two recent studies show that the sars-cov-2 receptor angiotensin converting enzyme 2 (ace2) is highly expressed on differentiated enterocytes and that intestinal organoids derived from the small intestine can be easily infected by sars-cov-2 16, 17 . interestingly, intestinal organoids derived from both human and horseshoe bats are fully susceptible to sars-cov-2 infection and sustain robust viral replication. although vertical transmission of sars-cov-2 seems to be anecdotal, it is still unclear if this lack of infection relates to the inability of the virus to migrate through the placenta 30 , to the low susceptibility of the fetal cells to infection, or simply on low viremic loads. whereas human fetal intestinal organoids have already been reported 20 , a reliable 3d culture in vitro model of human gastric mucosa at different developmental stages has been challenging to achieve. in this study, we describe successful derivation of human gastric organoids from both fetal and pediatric samples and we demonstrate that gastric cells are susceptible to sars-cov-2 infection. furthermore, we describe how a reversed-polarity organoid model can help expose the apical domains, in direct contact with the surrounding microenvironment, so that pathogens can easily access surface receptors on the cells. past studies showed laborious pathogen infection in human gastric organoids by microinjection of helicobacter pylori solution into the lumen of each organoid 18, 19 . other studies showed disruption of 3d organoid organization in favor of a 2d monolayer culture to overcome inner polarity problems in h. pylori infection 31 . recently, in sars-cov-2 infection studies, intestinal organoid 3d structures were sheared to expose the apical viral receptors and then reaggregated in ecm hydrogel droplets 16, 17 . in these studies, infection of organoids upon shearing and embedding was attained, as proved by the immunofluorescent staining of viral antigens and detection of viral rna. however, release of the infectious progeny in the culture supernatants differed greatly, ranging from positive titers of around 10 1-2 tissue culture infectious dose 50% (tcid50)/ml 16 to 10 4-5 tcid50/ml 17 . such discrepancy could depend on multiple factors, but we believe that the laborious nature of this approach might increase inter-operator and inter-laboratory variability, resulting in the generation of less-reproducible data. to overcome these complications, we decided to prevent the system perturbation and infect human gastric organoids under steady-state conditions. taking advantage of a polarity reversion study 24 , we generated fetal and pediatric cultures of rp-gos in suspension. in this condition of exposed apical polarity and absence of surrounding matrigel, we could infect organoids and readily titrate the infectivity of the progeny virus, recording infection level comparable to those shown by zhou et al. 17 , taking into account an ffu-to-tcid50 conversion factor of 0.28 (data not shown, from previous validation of assays). interestingly, when we infected gastric organoids through shearing and re-embedding in matrigel, infection was achieved but failed to detect virus in the supernatants, indicating this approach as suboptimal for our purposes. we demonstrated that the rp-gos are fully susceptible to sars-cov-2 infection, with an efficiency of replication that correlates directly with the developmental stage of origin. quantification of gene transcripts coding for the viral receptors ace2 and tmprss2 suggested that the observed levels of replication are not dependent on difference in the density of receptors, given their statistically comparable expression across the three stages. nonetheless, variation in the protein-to-mrna ratio across organoids of different developmental stage should be taken into account and receptivity investigated in future work. immunofluorescence staining for the nucleocapsid indicated a clear cytosolic localization of this protein that in some cells was associated with the presence of the cleaved caspase 3, confirming the occurrence of apoptosis in the gi compartment 16 . apoptosis in infected gi mucosal cells might account at least in part for the frequent abdominal pain, vomit and diarrhea described in covid-19 patients 32 , in particular in pediatric populations. apoptosis is one of the key mechanisms of cells to restrict viral infections by destruction of the cellular machinery indispensable for virus replication; on the other hand, selected viruses have evolved diverse adaptative strategies to control this phenomenon in their favor 33 . to this respect, sars-cov was shown to replicate in vitro to high titers in cells undergoing apoptosis and to low titers in cells where cytopathic effect was limited and a persistent infection was established 34 . interestingly, induction of apoptosis for sars-cov was proved to be caused by a nuclear localization of the nucleocapsid protein that in turn resulted in its cleavage by caspases 6 and 3. the precise mechanism underpinning nucleocapsid cleaving, apoptosis and the replication efficiency of sars-cov remains unexplored. we reckon that similar mechanistic studies are of great interest to decipher the pathology of sars-cov-2 in the gi system and its implications on virus shedding and transmissibility. rp-go of late fetal and pediatric age infected with sars-cov-2 shared a transcriptional footprint surprisingly similar to those described for infected human small intestine enteroids 16, 17 in which type i ifn genes were either poorly expressed or undetectable, despite enterocytes and gastric cells displaying moderate levels of isgs primarily involved in the recognition of viral rna. moreover, our transcriptional data are in considerable agreement with clinical and experimental profiles derived from covid-19 patients, infected normal human bronchial epithelial cells and in vivo studies in ferrets 35 that highlighted a negligible expression of genes of the ifn family but a robust expression of chemokines and isgs. our data provide novel evidence in support of the hypothesis that pathogenesis of covid-19 is at least in part dependent on a reduced innate antiviral response and an unbalanced cytokine production. nevertheless, in our model chemokines were not differentially expressed, whereas in small intestine organoids, zhou et al 17 reported degs coding both chemokine receptors and ligands. since we conducted a bulk rna-seq analysis and the number of infected cells in our organoids were still a minority, we speculate that many processes specific of infected cells, most likely did not reach a statistically significance level and might have gone undetected, hence imposing a cautionary approach in our interpretation of data. interestingly, a large overlap of degs with previous transcriptomic studies of sars-cov infection was found, including the peculiar feature of a limited/absent type i ifn induction and the recruitment of a subset of cytoplasmic prrs. similarly to sars-cov, in which orf3b and orf6 are the main antagonists of ifn, a recent study currently under peerrevision 36 indicates the sars-cov-2 orf3b protein as a potent ifn inhibitor, supporting ours and the published transcriptomic data herein discussed. our gastric organoid system offers a unique tool to characterize the replication of viruses and some of the associated pathological consequences of infection. this innovative model could represent an in vitro scalable platform for the development and testing of antiviral drug candidates targeting the gi system. a deeper understanding of the pathogenic mechanism underpinning the viral colonization of the gi system will potentially expand the available therapeutic options for the inhibition and preventing of gi infection, in an attempt to suppress viral shedding and halt spreading of the disease. the clinical importance of our findings relates to the worrisome phenomenon of prolonged shedding of sars-cov-2 from the gi tract and calls for further research to assess the risk of vertical transmission in infected women. defining the susceptible age and the target anatomical sites will prove of crucial importance for the implementation of sensitive and sustainable diagnostic screening for the identification of contagious asymptomatic patients. human fetal stomachs were dissected from tissue obtained immediately after termination of pregnancy from 8 to 21 pcw (post conception week), in compliance with the bioethics legislation in the uk. human pediatric gastric surgical biopsies were collected after informed consent, in compliance with all relevant ethical regulations for work with human participants, following the guidelines of the licenses 08nd13 and 18ds02. fetal stomachs and pediatric biopsies were collected in ice-cold sterile phosphate buffered solution (pbs -sigma-aldrich) and processed within a few hours of collection. gastric crypt stem cells were isolated from specimens following a well-established dissociation protocol 19 . briefly, fetal stomachs where cut open longitudinally along the lesser curvature, while @ 0.5 cm 2 pediatric biopsies where processed as they were obtained. specimens were cold-washed in a plate with chelating buffer (sterile milli-q water (merck millipore) with 5.6 mmol/l na2hpo4, 8.0 mmol/l kh2po4, 96.2 mmol/l nacl, 1.6 mmol/l kcl, 43.4 mmol/l sucrose, 54.9 mmol/l d-sorbitol, 0.5 mmol/l dl-dithiothreitol, ph 7, all from sigma-aldrich). mucus was removed with a glass coverslip and mucosa was stripped from muscle layer. tissue was cut in small pieces, transferred in a 15 ml tube in new chelating buffer and pipetted repeatedly. supernatant was discarded and 10 ml of 10 mm edta was added and incubated for 10 min at room temperature. edta was discarded and mucosa pieces were washed in ice cold pbs with ca 2+ /mg 2+ (sigma-aldrich). tissue was transferred to a new 10 cm plate on ice and pressure was applied on top with a sterile 3.5 cm plate, to release the crypts from the mucosa. table 2 . cell were passaged every 6-8 days. to passage the organoids, matrigel droplets were disrupted by pipetting in the well and transferred to tubes on ice. cells were washed with 10 ml of cold basal admem+++ and centrifuged at 200 g at 4â°c. (first method) for single cell dissociation, supernatant was discarded, and the pellet resuspended in 1 ml of tryple (thermo fisher) and incubated for 5 min. after incubation organoids were disaggregated by pipetting, and 10 ml of ice-cold admem+++ was added to dilute and inhibit tryple. (second method) for standard organoid passage during expansion, the organoid pellet was resuspended in 1.5 ml of ice-cold admem+++ and organoids were manually disrupted by narrowed (flamed) glass pipette pre-coated in bsa 1% in pbs, to avoid adhesion to the glass. cells were washed, pelleted and supernatant discarded. almost-dry pellets of disaggregated organoids (or single cells) were thoroughly resuspended in cold liquid matrigel, aliquoted in 30 âµl droplets in pre-warmed multi-well plates and incubated at 37â°c for 20 min to form a gel. rho-kinase inhibitor (tocris) was added to single cell dissociated organoids. medium was added and changed every 3 days. fully grown gastric organoids at day 7 after single cell disaggregation were removed from surrounding extracellular matrix using a modified published protocol 24 . matrigel was dissolved with 60 min treatment of the droplets with cell recovery solution (corning) at 4â°c. organoids were retrieved from the plates using 1% bsa-coated cut-end tips and transferred to 1% bsa-coated 15 ml tubes. cells were extensively washed with ice-cold pbs and centrifuged at 200 g for 5 min at 4â°c. supernatant was discarded, the pellet was resuspended in complete medium and transferred to non-tissue culture treated low-adhesive multiwell plates (pre-coated in 1% bsa). organoids were cultured in suspension for 3 days to allow reversion of polarity, before use in infection experiments. for rna isolation from stomach tissues, i) pediatric stomach biopsies consisted of only mucosal layer from surgical samples; ii) late fetal stomachs were cut open and mucosal layer was isolated; iii) early fetal stomachs were processed with no layer isolation, given the small size of the samples. mucus was removed from all the samples with a glass coverslip to prevent rna loss during the isolation protocol, and tissues washed in ice-cold pbs. then the tissues were finely cut with a scalpel on a petri-dish on ice and transferred to 1.5 ml tubes. recovery solution (corning) at 4â°c. cells were then washed in ice cold pbs to remove matrix leftovers that could interfere with rna isolation. organoids were centrifuged at 200 g at 4â°c and supernatant discarded. dry pellets of tissues and organoids were lysed with rlt buffer (qiagen). rna was isolated with rneasy mini kit (qiagen) following manufacturer's instructions. total rna was quantified using the qubit 2.0 fluorimetric assay (thermo fisher scientific). rna reverse transcription was performed using the high-capacity cdna reverse transcription kit (thermo fisher), according to the manufacturer's instructions. reverse transcription was done using the t100 thermal cycler (bio-rad). the qrt-pcr was performed with taqman gene expression assay probes (thermo fisher) according to the manufacturer's instructions. the following probes (all from thermo fisher) were used: gapdh (glyceraldehyde 3-phosphate dehydrogenase), lgr5 (leucine-rich repeat-containing gprotein coupled receptor 5), axin2 (axin-like protein), muc5ac (mucin 5ac), muc6 (mucin 6), pga5 (pepsinogen a5), sst (somatostatin), gast (gastrin), chga (chromogranin a), atp4b (atpase h+/k+ transporting subunit beta). reactions were performed on step one plus real-time pcr system (applied biosystems) and results were analyzed with stepone (version 2.3) software (life technologies). gapdh expression was used to normalize ct values for gene expression, and data were shown as relative fold change to controls (early fetal stage), using â��â��ct method, and presented as mean â± sem. for rna-seq data of original tissues and organoids with spontaneous polarity, total rna (100 ng) from each sample was prepared using quantseq 3' mrna-seq library prep kit (lexogen gmbh) according to manufacturer's instructions. the amplified fragmented cdna of 300 bp in size were sequenced in singleend mode using the nova seq 6000 (illumina) with a read length of 100 bp. illumina novaseq base call (bcl) files were converted into fastq files through bcl2fastq (version v2.20.0.422) following software guide. sequence reads were trimmed using bbduk software (bbmap suite 37.31), following software guide, to remove adapter sequences, poly-a tails and low-quality end bases (regions with average quality below 6). alignment was performed with star 2.6.0a 37 on hg38 reference assembly obtained from cellranger website (ensembl 93), following online site guide. the expression levels of genes were determined with htseq-count 0.9.1 by using cellranger pre-build genes annotations (ensembl assembly 93). all transcripts having <1 cpm in less than 4 samples and percentage of multimap alignment reads > 20% simultaneously were filtered out. for rna-seq data of non-infected and infected rp-gos, a total of 600 pg of rna was used as input for the synthesis of cdna with the smart-seq v4 ultra low input rna kit for sequencing (takara bio usa, mountain view, ca, usa). manufacturer suggested protocol was followed, with minor modifications. 75 pg of dna generated with smart-seq v4 kit were used for preparation of library with nextera xt dna library preparation kit (illumina inc., san diego, ca, usa), following suggested protocol. libraries were sequenced in pair-end mode using a nova seq 6000 sequencing system on an sp, 100 cycles flow cell (illumina inc., san diego, ca, usa). illumina novaseq base call (bcl) files were converted into fastq files through bcl2fastq (version v2.20.0.422) following software guide. alignment was performed with star 2.6.0a34 on hg38 reference assembly obtained from the gencode website (primary assembly v. 32). transcripts estimated counts were determined with rsem 1.3.0 38 by using the gencode v.32 genes annotations. all genes having <1 cpm in less than 2 replicates of the same condition were filtered out. differentially expressed genes (degs) were computed with edger 39 , using a mixed criterion based on p-value, after false discovery rate (fdr) correction by benjamini-hochberg method, lower than 0.05 and absolute log2(fold change) higher than log2(1.5). this analysis was paired between non-infected and infected samples derived from the same original sample. for rna-seq data of organoids with spontaneous polarity, degs were clustered according to a flat, increasing, or decreasing profile according to the differential expression analysis between pairs of time points. principal component analysis was performed by singular value decomposition (svd) on log2(cpm+1) data, after centering, using matlab r2017a (the mathworks). degs over-representation analysis of gene ontology (go) and reactome categories was performed using cluego (version 2.5.4) 40 . reactome hierarchy was visualized using cluego within cytoscape 41 . hierarchical clustering of degs was performed on median-centered log2(cpm+1) data in matlab, using euclidean distance and complete linkage. log-normalized expression data were analyzed by the quantitative set analysis for gene expression (qusage) 27 bioconductor package. vero e6 cells (atccâ® crl 1586â�¢) were maintained in dulbecco's modified eagle's medium (dmem, thermo fisher) supplemented with 10% fetal calf serum (fcs), penicillin (100 u/ml) and streptomycin (100u/ml) (all from thermo fisher) at 37â°c in a humidified 5% co2 incubator. the sars-cov-2 isolate was obtained from a nasopharyngeal swab collected from a 14-year-old boy in italy. briefly, the swab viral transport medium was filtered through a 0.22 âµm filter, serially diluted and incubated onto a confluent layer of vero e6 cells, for 5 days. to ensure purity of the viral isolate, the supernatant of the highest dilution in which cytopathic effect was visible was tested for the presence of 21 human respiratory pathogens including sars-cov-2, using the qiastat-dx respiratory sars-cov-2 panel (qiagen). viral stocks were produced infecting at a multiplicity of infection (moi) of 1 vero e6 cells cultured in dmem supplemented with 2% fcs, penicillin (100 u/ml) and streptomycin (100u/ml) and incubating the cells for 48 hours. supernatants were collected when 80% cells exhibited cytopathic effect and cleared by low-speed centrifugation before being stored at -80â°c. all infections in this paper were performed using the third culture passage of the original isolate. intact organoids were embedded in two 3 âµl-drops of matrigel per well, in 24-well plates. embedded organoids were washed once in dmem and infected at a moi of 0.5 by incubation with 250 âµl of an expansion medium viral suspension for 2 hours. after removal of the inoculum, organoids were washed twice with a dmem solution and 400 âµl of complete medium were added to each well to maintain the culture at 37 â°c with 5% co2. reversed-polarity organoids were infected at a moi of 1 by incubation with 250 âµl of an expansion medium viral suspension for 2 hours. after infection, organoids were washed twice in dmem to remove unbound virus. rp-gos were dispersed in a 400 âµl expansion medium at 37 â°c with 5% co2. for all organoid cultures 50 âµl of supernatant were harvested at 0, 24, 48, and 72 hours post infection. an equal volume of expansion medium replaced the sampled supernatant at each collection time. an extra sample at 96 hours post infection was collected for the rp-gos. samples were stored at -80â°c before titration through the ffa. supernatants of organoid cultures and aliquots of viral stocks were serially diluted and incubated on confluent monolayers of vero e6 cells, in 96-well plates, for 1 hour. culture medium formulation was the same used for virus propagation. after infection, the inoculum was removed and an overlay of mem, 2% fbs, penicillin (100 u/ml) and streptomycin (100u/ml) and 0.8% carboxy methyl cellulose was added. after 27 hours, the overlay medium was removed and cells were fixed in a 4% paraformaldehyde (pfa) phosphate buffered solution (pbs), for 30 minutes at 4â°c. upon removal, cells were permeabilized by incubation with a 0.5 % triton x-100 solution for 10 minutes. immunostaining of infected cells was performed by incubation of the j2 anti-dsrna monoclonal antibody (1:10,000; scicons) for 1 hour, followed by 1-hour incubation with peroxidase-labeled goat anti-mouse antibodies (1:1000; dako) and a 7 min incubation with the true blueâ�¢ (kpl) peroxidase substrate. solution of 1% bovine serum albumin and 0.05% tween-80 in pbs was used for the preparation of working dilutions of immuno-reagents. after each antibody incubation, cells were washed 4 times through a 5 min incubation with a 0.05% tween-80 pbs solution. focus forming units (ffu) were counted after acquisition of pictures at a high resolution of 4800 x 9400dpi, on a flatbed scanner. human gastric tissues were fixed in 4% paraformaldehyde (pfa -sigma-aldrich) for 2 hours and embedded in paraffin wax, then cut at 7 âµm on a microtome. hematoxylin and eosin (h&e) tissue slides were stained according to manufacturer's instructions with hematoxylin and eosin (h&e) (thermo fisher). immunostaining was performed by blocking and permeabilizing the tissue slides with pbs + triton x-100 0.1% with bsa 0.5%. organoid whole-mounts were blocked and permeabilized with pbs + triton x-100 0.5% with bsa 1% for 2 h at room temperature in rotation. primary antibodies were incubated in blocking buffer for 24h at 4â°c in rotation and extensively washed in pbs + triton x-100 0.1%. secondary antibodies were incubated overnight at 4â°c in rotation and extensively washed. slides were mounted in mounting medium, while floating organoids were moved to a glass-bottomed petri dish and blocked with a coverslip on top. the full list of primary and secondary antibodies is presented in supplementary table 3. organoids were imaged in bright field using a zeiss axio observer a1. immunofluorescence images of whole-mount staining and sections were acquired on a confocal microscope zeiss lsm 710. infected organoid immunofluorescence images were acquired on a leica tcs sp5. statistical analyses were performed using the following software: matlab (v. r2017a) for pca, pie plot, bar plot, hierarchical clustering with proteomic and rna-seq data. graphpad prism mac (v. 6.0h) was used with all other graphs and charts. 42 . c) hierarchical clustering of deg genes included in (b), data were median-centered for each pair of non-infected and infected conditions. d) results from an enrichment analysis within reactome database of degs highlighted in (a). symbol size is proportional to number of genes. p-value < 10 -5 . white symbols were not enriched and were added to highlight the hierarchy between categories within reactome structure. virological assessment of hospitalized patients with covid-2019 evidence for gastrointestinal infection of sars-cov-2 prolonged presence of sars-cov-2 viral rna in faecal samples gastrointestinal features in children with covid-19: an observation of varied presentation in eight children aerodynamic analysis of sars-cov-2 in two wuhan hospitals 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in vitro model of human development and disease sars-cov-2 productively infects human gut enterocytes. science (80-. ) infection of bat and human intestinal organoids by sars-cov-2 modelling human development and disease in pluripotent stem-cell-derived gastric organoids in vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury long-term expansion of functional mouse and human hepatocytes as 3d organoids extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture tracing the temporal-spatial transcriptome landscapes of the human fetal digestive tract using single-cell rna-sequencing controlling epithelial polarity: a human enteroid model for host-pathogen interactions a qpcr expression assay of ifi44l gene differentiates viral from bacterial infections in febrile children novel functions of ifi44l as a feedback regulator of host antiviral responses quantitative set analysis for gene expression: a method to quantify gene set differential expression including gene-gene correlations clinical characteristics of 58 children with a pediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 hyperinflammatory shock in children during covid-19 pandemic does the human placenta express the canonical cell entry mediators for sars-cov-2? a novel human gastric primary cell culture system for modelling helicobacter pylori infection in vitro review article: gastrointestinal features in covid-19 and the possibility of faecal transmission caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis cell type-specific cleavage of nucleocapsid protein by effector caspases during sars coronavirus infection imbalanced host response to sars-cov-2 drives development of covid-19 sars-cov-2 orf3b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant sequence analysis star: ultrafast universal rna-seq aligner rsem: accurate transcript quantification from rna-seq data with or without a reference genome edger: a bioconductor package for differential expression analysis of digital gene expression data cluego: a cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks cytoscape: a software environment for integrated models of biomolecular interaction networks interferon-induced transmembrane protein (ifitm3) is upregulated explicitly in sars-cov-2 infected lung epithelial cells dc is founder, shareholder, and consultant of next generation diagnostic srl. all the other authors of the study declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. the authors declare that all data supporting the findings of this study are available within the article, its supplementary information, attached files, and online deposited data (gastric rna-seq data: gse_xxxx, it will be deposited during revision), or from the authors upon reasonable request. key: cord-263811-w0983x19 authors: decaro, nicola; martella, vito; buonavoglia, canio title: canine adenoviruses and herpesvirus date: 2008-05-22 journal: vet clin north am small anim pract doi: 10.1016/j.cvsm.2008.02.006 sha: doc_id: 263811 cord_uid: w0983x19 canine adenoviruses (cavs) and canine herpesvirus (chv) are pathogens of dogs that have been known for several decades. the two distinct types of cavs, type 1 and type 2, are responsible for infectious canine hepatitis and infectious tracheobronchitis, respectively. in the present article, the currently available literature on cavs and chv is reviewed, providing a meaningful update on the epidemiologic, pathogenetic, clinical, diagnostic, and prophylactic aspects of the infections caused by these important pathogens. hybridization [19] . they also exhibit different hemagglutination patterns and cell tropism. cav-1 recognizes the vascular endothelial cells and hepatic and renal parenchymal cells as targets for viral replication, whereas cav-2 replicates efficiently in the respiratory tract and, to a limited extent, in the intestinal epithelia [20] [21] [22] . infection by cavs has been described worldwide in several mammalian species. dogs, red foxes, wolves, and coyotes are highly susceptible to cav infection [3] . the overall prevalence of antibodies to cavs in european red foxes (vulpes vulpes) in australia was 23.2%, with marked geographic, seasonal, and age differences [23] , whereas the prevalence of antibody was 97% in island foxes (urocyon littoralis) in the channel islands, california [24] . antibodies to cavs were also detected in free-ranging terrestrial carnivores and marine mammals in alaska and canada, including black bears (ursus americanus), fishers (martes pennanti), polar bears (ursus maritimus), wolves (canis lupus), walruses (odobenus rosmarus), and steller sea lions (eumetopias jubatus) [25, 26] . recently, a fatal cav-1 infection has been reported in a eurasian river otter (lutra lutra) [27] . canine ich is a systemic disease described in canidae and ursidae. cav-1 replication in vascular endothelial cells and hepatocytes produces acute necrohemorrhagic hepatitis, and the disease is more severe in young animals [28, 29] . transmission occurs through animal-to-animal contact or indirectly through exposure to infectious saliva, feces, urine, or respiratory secretions. cav-1 is shed in urine up to 6 to 9 months after infection [30] . the incubation period in dogs is 4 to 6 days after ingestion of infectious material and 6 to 9 days after direct contact with infected dogs [31] . the mortality rate is 10% to 30% [32] . coinfections with canine coronavirus (ccov) [33, 34] , canine distemper virus (cdv) [34] [35] [36] [37] , or canine parvovirus [34] can exacerbate the disease, increasing the mortality rates. fever (>40 c) is the earliest clinical sign and displays a biphasic course. after the first febrile peak (1-2 days), some dogs recover from the infection. dogs displaying a second peak of hyperthermia frequently undergo a more severe form of ich. commonly observed symptoms are depression, loss of appetite, increased heart rate, hyperventilation, vomiting, and diarrhea. abdominal pain and distention can occur as a result of accumulation of serosanguineous or hemorrhagic fluid and enlargement of the liver. frequently, hemorrhagic diathesis is observed with epistaxis, congestion, or hemorrhage of the mucous membranes and skin. respiratory distress can also be observed as a consequence of laryngitis, tracheitis, and, less frequently, pneumonia. neurologic signs (hypersalivation, ataxia, and seizures) are rare in dogs and are associated with vascular damage in the central nervous system (cns) [28, 38] . corneal opacity (''blue eye''; fig. 1 ) and interstitial nephritis may occur 1 to 3 weeks after recovery because of deposition of immune complexes [39] [40] [41] . hematologic findings include leukopenia (<2000 cells/ll of blood; mainly attributable to a decrease in neutrophil count), increase in the serum transaminases (only in the severe forms of disease) [42] , and coagulation disorders associated with disseminated intravascular coagulation (dic; thrombocytopenia, altered platelet formation, and prolonged prothrombin time) [43] . proteinuria (albuminuria) can easily reach values greater than 50 mg/dl because of immunomediated glomerulonephritis [29] . at necropsy, the dogs that die during the acute phase of the disease often appear in good nutritional state. external examination can reveal ecchymoses and petechial hemorrhages, whereas the abdominal cavity contains abundant clear or serosanguineous fluid. the liver is enlarged, yellowish brown, congested, and spotted with small rounding areas of necrosis; the gallbladder appears thickened, edematous, and grayish or bluish white opaque in color (fig. 2) . edema of the gallbladder wall is a constant finding. congestion and hemorrhagic lesions are observed in the spleen, lymph nodes ( fig. 3) , thymus, pancreas, and kidneys. lungs show patchy areas of consolidation because of bronchopneumonia. hemorrhagic enteritis can also be observed (fig. 4) [3, 28] . histologic changes in the liver are characterized by centrolobular necrosis, along with neutrophilic and mononuclear cell infiltration and intranuclear inclusions in the kupffer's cells and hepatocytes. multifocal areas of congestion, hemorrhage, and leukocyte infiltration can be observed in several organs, mainly in the liver and kidneys, because of vascular damage and inflammation. interstitial nephritis and iridocyclitis with corneal edema are also present in dogs recovering from ich [44] . the route of infection by cav-2 is oronasal. respiratory signs are consistent with damage of bronchial epithelial cells. cav-2 infections rarely result in overt clinical signs, however, despite the presence of extensive lung lesions. clinical signs typical of itb are observed when cav-2 infection is complicated by other viral or bacterial pathogens of dogs, including canine parainfluenza 3 virus [45] , cdv [46] [47] [48] , bordetella bronchiseptica [49] , mycoplasmas [50, 51] , and streptococcus equi subsp. zooepidemicus [52] [53] [54] . in addition, other viruses with tropism for the respiratory tract have been recently identified and associated with itb-like forms in dogs, such as influenza a virus [54, 55] , a pantropic variant of ccov [56] , and the canine respiratory coronavirus (crcov) [57, 58] . chv and mammalian reoviruses have rarely been reported from dogs with itb and likely do not play a major role in the disease complex [59, 60] . itb (kennel cough) is an acute and highly contagious respiratory disease of dogs affecting the larynx, trachea, bronchi, and, occasionally, lower respiratory tract [61] . kennel cough is typically a complex of diseases caused by viral pathogens (eg, cavs, chv, canine parainfluenza virus, reoviruses) in association with bacteria, mainly b bronchiseptica and mycoplasma spp. most frequently, a dry hacking cough is observed as a consequence of an uncomplicated, self-limiting, and primarily viral infection of the trachea and bronchi. in complicated forms, which are more common in pups and immunocompromised dogs, secondary bacterial infections and involvement of pulmonary tissue overlap the viral infection. cough is usually associated with mucoid discharges. the condition may progress to bronchopneumonia and, in the most severe instances, death [61] . usually, cns involvement is not seen, although death in pups with neurologic disease associated with cav-2 infection has been reported [62] . at postmortem examination, red areas of consolidation can be observed in the lungs, especially in the complicated forms. histologically, necrotizing bronchitis and bronchiolitis obliterans may be observed. infection of type 2 alveolar cells is associated with interstitial pneumonia and the presence of viral inclusion bodies in their nuclei [63] [64] [65] [66] [67] [68] . hematologic findings (eg, leukopenia, prolonged blood clotting, increased activities of alanine aminotransferase [alt] and aspartate aminotransferase [ast]) may be indicative of cav-1 infection, although the increase of transaminases is commonly observed only in severely affected or moribund dogs. postmortem findings and histopathologic changes are highly suggestive of cav-1 infection. confirmation of a diagnosis of ich is obtained by virus isolation on permissive cell lines, such as madin darby canine kidney (mdck) cells. a polymerase chain reaction (pcr) protocol has recently been developed for molecular diagnosis [69] . ocular swabs, feces, and urine can be collected in vivo for virus isolation and pcr. postmortem samples can be withdrawn from the kidney, lung, and lymphoid tissues. the liver is rich in arginase, which inhibits viral growth in cell cultures [70] , but it represents the most important organ for histopathologic examination [28, 29] . viral growth in cells is revealed by rounding cells that form clusters and detach from the monolayers [34] . immunofluorescence (if) can detect viral antigens in infected cell cultures and in acetone-fixed tissue sections or smears. viral replication can also be demonstrated by detection of nuclear inclusion bodies in the cells after hematoxylin-eosin staining. neither virus isolation nor if is able to distinguish between the two adenovirus types. because cav-2 can also be detected in the internal organs and feces of vaccinated or acutely infected dogs [46] and cav-1 is also frequently isolated from respiratory secretions, trachea, and lungs, distinction between cav-1 and cav-2 necessarily deserves laboratory examination. restriction fragment length polymorphism analysis on viral genomes using the endonucleases psti and hpaii generates differential patterns [17, 18] . detection and differentiation of cav-1 and cav-2 by pcr with a single primer pair are also possible [69] . although cavs agglutinate erythrocytes of several species, hemagglutination is not used in routine diagnosis [71] . because most dogs are vaccinated and cav-2 infection is frequent in dogs, serology has low diagnostic relevance [21, 39] . treatment of ich is primarily symptomatic and supportive. dehydration and dic require administration of fluids, plasma, or whole-blood transfusions and anticoagulants. hyperammonemia attributable to hepatic and renal damages can be corrected by oral administration of nonabsorbable antibiotics and lactulose and by oral or parenteral administration of potassium and urinary acidificants (ascorbic acid). supportive therapy may facilitate the clinical recovery of infected dogs, provided that there is time for hepatocellular regeneration [29] . uncomplicated forms of cav-2-associated itb can be treated with glucocorticoids, antitussives, and bronchodilators as cough suppressants. aerosol therapy can be effective in dogs displaying excessive accumulation of tracheal and bronchial secretions. antimicrobial therapy is recommended in the complicated forms and when the lower respiratory tract seems to be involved [29] . use of vaccines has greatly reduced the burden of ich in canine populations. initial attempts were made with cav-1 inactivated vaccines, which require repeated inoculations [72] . cav-1-based modified-live virus (mlv) vaccines proved to be highly effective but were associated with interstitial nephritis and corneal opacity [22] . administration of cav-1 in conjunction with cdv vaccines was also associated with postvaccinal encephalitis [73] . because cav-1 and cav-2 are able to confer cross-protection, the current vaccines contain mlv cav-2, which is not able to induce renal or ocular damage. the cav-2 attenuated strain toronto a26/61 is contained in most vaccine formulations [22, 74] . in the absence of maternally derived antibodies (mdas), a single dose administered subcutaneously or intramuscularly is protective against ich and itb. because of the possible interference of mdas, however, the vaccination schedule requires administration of at least two vaccine doses at a 3-to 4-week interval, starting when pups are 8 to 10 weeks old. intranasal administration of an mlv cav-2 vaccine has been proposed to overcome mda interference, but it may be associated with the onset of mild respiratory disease [29] . vaccination is usually repeated yearly, although after administration of two doses of cav-2 vaccine, immunity seems to persist for more than 3 years [75, 76] . although extensive vaccination has greatly reduced the incidence of cav infections, re-emergence of ich has been described in italy, likely as the result of parallel trading of pups with uncertain sanitary status from eastern european countries [34] . at the moment, there are few data on the molecular epidemiology of cavs, but it is commonly accepted that vaccine breaks occur rarely with cav vaccines, because the viruses are genetically stable. accordingly, cav infection in vaccinated dogs has been associated with mda interference in the early life of the pups rather than with emergence of variants genetically distant from the prototype strains contained in cav-2 vaccines [34] . cause chv was first described in the mid-1960s as the causative agent of a fatal septicemic disease of puppies [77] . chv is included in the alphaherpesvirinae subfamily, herpesviridae family [78] . the virus is sensitive to lipid solvents, is readily inactivated at temperatures greater than 40 c, and is rapidly inactivated by common disinfectants. chv seems to be a monotypic virus, as defined by antigenic comparison of various isolates [77, 79] . the genome structure of chv resembles that of other members of the alphaherpesvirus subfamily [80] [81] [82] [83] . southern blot hybridization and sequence analysis of various genes have shown a close genetic relatedness to feline herpesvirus (fhv-1), to phocid herpesvirus 1, and to the equid herpesviruses 1 and 4 [84] [85] [86] . the host range of chv is restricted to dogs [87] . antibodies to chv have been detected in sera of european red foxes (v vulpes) in australia [23] and germany [88] , however, and in sera of north american river otters (lontra canadensis) from new york [89] , whereas a chv-like virus has been isolated from captive coyote pups [90] . the virus seems to be present worldwide in domestic and wild dogs. serologic surveys have shown a relatively high prevalence of chv in household and colony-bred dogs. the prevalence of antibodies in dogs was 88% in england, 45.8% in belgium, and 39.3% in the netherlands [91] [92] [93] . serologic studies in italy have revealed a high prevalence in kenneled dogs (27.9%), whereas the prevalence was lower in pets (3.1%) [94] . in the united states, fulton and colleagues [95] studied the prevalence of antibodies against chv in washington and found only a 6% seroprevalence. transmission occurs by direct contact with oronasal or genital secretions, because chv is quickly inactivated in the environment. the age of the pups at the time of infection is critical for the outcome of the disease. infection of susceptible puppies at 1 to 2 weeks of age may be associated with fatal generalized necrotizing and hemorrhagic disease, whereas infection of pups older than 2 weeks of age and adult dogs is often asymptomatic [77] . infection in older dogs seems to be restricted to the upper respiratory tract [96] . also, chv has been identified in corneal swabs of adult dogs with corneal ulcerations [97] . transplacental transmission of chv and fetal death may also occur [98] , and chv infection is suspected in dogs with fertility disorders. the high susceptibility of newborn pups to fatal acute chv-induced disease is likely related to the fact that pups have low and poorly regulated body temperature and chv growth is optimal at lower than normal body temperature [99] . neonatal mortality chv infection is generally fatal in neonatal pups lacking maternally derived immunity. death of 1-to 4-week-old pups is most common. neonatal pups may be infected during passage through the birth canal or by contact with oronasal secretions of other dogs. the duration of illness in newborn pups is 1 to 3 days. signs include vocalization, anorexia, dyspnea, abdominal pain, incoordination, and soft feces, whereas the rectal temperature is not elevated and may be low. serous or hemorrhagic nasal discharge and petechial hemorrhage on the mucous membranes may also be observed. in pups less than 1 week of age at the time of infection, chv replicates in the nasal mucosa, pharynx, and tonsils before spreading by means of the blood (in macrophages) to the liver, kidneys, lymphatic tissues, lungs, and cns. the incubation period is approximately 6 to 10 days. death in affected litters usually occurs over a period of a few days to a week. litter mortality can reach a peak of 100%. in pups older than 2 to 3 weeks of age at the time of infection, chv infection is generally asymptomatic, although cns signs, including blindness and deafness, have been described [100] . reproductive disorders chv can cause occasional in utero infections that result in death of the fetus or pup shortly after birth [77, 98] . pregnant dogs infected at midgestation or later may abort weak or stillborn pups. pups may seem normal at parturition but die within a few days of birth. the infected dams develop protective immunity, and chv-related diseases are not observed in subsequent litters because maternally derived immunity protects the pups during the first week of life when they are most susceptible. primary genital infections in susceptible adult animals may be associated with lymphofollicular lesions and vaginal hyperemia (fig. 5) . male animals may have similar lesions over the base of the penis and the prepuce. respiratory disease chv has been detected in dogs with itb [101] , but its role remains controversial. experimental infection has been shown to cause mild clinical symptoms of rhinitis and pharyngitis [96] or tracheobronchitis [102] . experimental infection by the intravenous route in adult foxes resulted in fever, lethargy, and respiratory signs, although peroral infection did not [103] . a long-term survey in a population of dogs in a shelter has demonstrated chv in 9.6% of lung and 12.8% of tracheal samples. chv infections occurred later than other viral infections. chv was detected more frequently at weeks 3 and 4 after a dog's introduction in the kennel, whereas crcov and canine parainfluenza were detected more frequently within the first and second weeks, respectively. interestingly, chv infection was apparently related to more severe respiratory signs [53] . in a 1-year study in training centers for working dogs, however, seroconversion to chv seemed to be more frequent in dogs infected with crcov [104] , suggesting virus reactivation after disease-induced stress. after symptomatic and asymptomatic infections, dogs remain latently infected and virus may be excreted at unpredictable intervals over periods of several months or years. reactivation of latent virus may be provoked by environmental or social stress or, experimentally, by immunosuppressive drugs (corticosteroids) or antilymphocyte serum. latent virus persists in the trigeminal ganglia and other sites, such as lumbosacral ganglia, tonsils, and parotid salivary glands [3, [105] [106] [107] . latently infected dogs represent a source of infection for susceptible animals, and this is of particular concern in breeding dogs that can ensure chv transmission through genital secretions. multifocal areas of necrosis and hemorrhage may be observed in most organs, including the lungs, liver, brain, and intestine, with the kidneys being the most classic organ affected. circumscribed areas of hemorrhage and necrosis on a pale gray cortex give the organs a spotted appearance (fig. 6) . lymph nodes and spleens appear enlarged. meningoencephalitis also is common. necrosis in the placenta is observed in infected pregnant animals. fetal lesions are similar to those seen in affected puppies. diagnosis of chv infection may be achieved by isolation of the virus on permissive cell lines. the virus can be adapted for growth on canine primary or secondary kidney or testicular cells and in canine cell lines. growth is optimal at 34 c to 35 c, with diminished virus yields at temperatures higher than 36 c. in cell cultures, virus growth is revealed by formation of typical clusters of rounded cells that tend to detach, and for certain isolates, by formation of syncytia with type a intranuclear inclusions. pcr assays are available, significantly increasing diagnostic reliability and sensitivity [107] . serologic screenings to evaluate the neutralizing antibodies may be useful to investigate the presence of chv in kennels. because chv growth is optimal at temperatures lower than 36 c [99] , attempts were made to influence the evolution of chv-induced disease in experimentally infected pups. experimentally infected newborn pups reared at elevated temperatures that raised their body temperature to 38.5 c to 39.5 c survived chv infection but presented with permanent neurologic damage [108] . likewise, residual neurologic damage may be observed in infected dogs treated with antiviral drugs, such as vidarabine. accordingly, neither artificial temperature nor vidarabine may be applied for the therapy of chv. an inactivated subunit vaccine is available commercially in europe. the vaccine should be administered to bitches during heat or the initial stages of pregnancy and again at the sixth to seventh week of gestation. a temperature-resistant mutant of chv attenuated through serial cell passages has been proposed as an mlv vaccine [109] , but its safety and efficacy have not been evaluated and such a vaccine is not available commercially. cav infections have been satisfactorily controlled in the past decades as a consequence of the vaccination programs adopted in all developed countries. nevertheless, there are some concerns about the possible introduction of infected dogs from areas of uncertain epidemiologic conditions, in which both cav types are widespread as a result of the lack of systematic canine immunization [34] . cav vaccines have been proved to be safe and effective for prevention of ich and itb, conferring protection against more recent cav strains, albeit prepared with old cav-2 strains [28, 110] . conversely, chv is still circulating in canine populations worldwide, mainly in shelters and breeding kennels. active immunization is recommended in pregnant bitches to prevent fatal infections in newborn pups [111] . when the mdas decrease, however, pups born to vaccinated bitches become susceptible and, along with unvaccinated dogs, maintain chv infection. it is unclear whether vaccination prevents chv infection and virus shedding through secretions. in addition, control of the infection is hindered by the fact that chv is often associated with asymptomatic infections, and the real prevalence of chv infection is likely underestimated [87] . the intensification of surveillance activity using new diagnostic techniques and molecular analysis tools may help to investigate the epidemiology of cav and chv infections more thoroughly and to plan adequate measures of control. epizootic fox encephalitis. i. general description a comparison of certain intranuclear inclusions found in the livers of dogs without history of infection with intranuclear inclusions characteristic of the action of filtrable viruses infectious diseases of the dog and cat. philadelphia: wb saunders co propagation of infectious canine hepatitis virus in tissue culture cultivation and modification of infectious canine hepatitis virus 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virions during replication of canine herpesvirus at elevated temperature herpesvirus canis: aspects of pathogenesis and immune response studies of respiratory disease in random-source laboratory dogs: viral infections in unconditioned dogs experimental production of canine tracheobronchitis (kennel cough) with canine herpesvirus isolated from naturally infected dogs experimental infection of european red foxes (vulpes vulpes) with canine herpesvirus investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations detection of canine herpesvirus dna in the ganglionic neurons and the lymph node lymphocytes of latently infected dogs virus reactivation in bitches with a medical history of herpesvirus infection detection of canine herpesvirus 1 in a wide range of tissues using the polymerase chain reaction temperature as a factor of resistance of young puppies to canine herpesvirus small-plaque variant of canine of canine herpesvirus with reduced pathogenicity for newborn pups guidelines for the vaccination of dogs and cats. compiled by the vaccination guidelines group (vgg) of the world small animal veterinary association (wsava) protection of puppies against canine herpesvirus by vaccination of the dams key: cord-255623-qdpdsye9 authors: pham, hien t.; nguyen, phuc t. t.; tran, sinh t.; phung, thuy t. b. title: clinical and pathogenic characteristics of lower respiratory tract infection treated at the vietnam national children's hospital date: 2020-03-11 journal: can j infect dis med microbiol doi: 10.1155/2020/7931950 sha: doc_id: 255623 cord_uid: qdpdsye9 lower respiratory tract infections are commonly caused by viruses and cause significant morbidity and mortality among children. early identification of the pathological agent causing these infections is essential to avoid unnecessary antibiotic use and improve patient management. multiplex pcr techniques were recently developed to detect multiple viral pathogens using a single pcr reaction. in this study, we identify viral pathogens in children with respiratory infections. we collected 194 nasopharyngeal aspirates from infants (2–24 months old) with lower respiratory tract infections treated at the vietnam national children's hospital between november 2014 and june 2015 and assessed the presence of 16 virus types and subtypes by multiplex pcr using the xtag respiratory viral panel (rvp) assay. overall, 73.7% of the samples were positive for at least one virus, and 24.2% corresponded to infections with multiple viruses. the most common viruses were respiratory syncytial virus and enterovirus/rhinovirus. these viruses were more frequent among younger patients (2–5 months old) and caused symptoms similar to those of bronchiolitis and pneumonia. the most common clinical manifestation caused by respiratory tract infection was bronchiolitis. elevated neutrophils levels were associated with adenovirus infection. our results showed that the xtag respiratory viral panel (rvp) can effectively detect multiple viruses causing respiratory infections in children and that the nasopharyngeal aspirates are a good sample choice to detect respiratory viruses in children. applying this approach in the clinical setting would improve patient management and allow early diagnosis, thus avoiding the unnecessary use of antibiotics. acute respiratory infections (aris) are common among children worldwide. although they have similar incidence rate in developed and developing countries, the mortality rate is higher in developing countries [1] [2] [3] . aris are responsible for approximately 20% of all deaths in children under 5 years in 2006; about 70% of these deaths occur in sub-saharan africa and the southern regions of asia. viruses are the main causative agents of respiratory infection, but bacteria, fungi, and parasites can cause them as well. e leading ari-causing viruses are rhinovirus and respiratory syncytial virus (rsv) [4] , with rsv affecting mainly infants under 1 year of age [5, 6] . viruses primarily infect and replicate in the airway epithelium, causing injuries in the proximal (conducting) and distal airways (alveoli and parenchyma), where gas exchange occurs. viral infection can have multiple clinical manifestations, such as pneumonia, defined as an inflammation of the lung parenchyma. is condition is often associated with visible changes on chest x-rays, ctscanning, or gallium scanning and with abnormalities in alveolar gas exchange. e presentations of viral pneumonia vary considerably depending on the age and immunological competence of the host, as well as the viral pathogen. viral pneumonia is an important cause of morbidity and mortality in immune compromised individuals and children [7] . e clinical presentation depends on the specific causative agent but typically includes fever and lower respiratory tract symptoms, such as tachypnoea, nonproductive cough, wheeze, and increased breath sound [7, 8] . inappropriate antibiotic use during acute respiratory infection has led to an increase in antimicrobial resistance [9] . erefore, early detection of the pathogen causing respiratory infection may prevent antibiotic issue and help improve patient management [10] . however, the lack of rapid, affordable, sensitive, and specific diagnostic tools is one of the main reasons for inefficient diagnosis. pcr has revolutionized the field of infectious disease diagnosis. multiplex pcr, a variant that uses several pairs of primers to amplify more than one target sequence in a single tube, was developed to detect different viruses in a single sample [11] . previous studies showed that multiplex assays using a limited amount of sample had similar performance to that of traditional methods when used for clinical diagnosis [12] . additionally, they have the benefit of detecting a large number of viruses in a short time. e xtag respiratory viral panel fast (rvp fast) is a qualitative nucleic acid multiplex test that simultaneously detects and identifies the presence of nucleic acids from multiple respiratory viruses in a single tube. it works with samples such as nasopharyngeal swabs, nasal aspirates, nasopharyngeal aspirates, and bronchoalveolar lavages from individuals suspected of respiratory tract infections. e xtag rvp fast test has shown superior sensitivity when compared to single real-time pcr and conventional pcr assays [11, 13, 14] . in the present study, we used the xtag rvp fast assay to identify the viruses causing ri in children and the relationship between specific viruses and clinical outcome. is cross-sectional study was held between november 2014 and june 2015. we enrolled 194 pediatric infants (2-24 months old) who had lower respiratory tract infections and were treated at the vietnam national children's hospital. we collected nasopharyngeal aspirates from each participant. patients were divided into 3 age groups. e first group included infants between 2 and 5 months (n � 71) who were breastfed and likely received passive immunity from their mothers. e second group included infants between 6 and 11 months old (n � 66) who spent most of their time at home. e third groups included infants between 12 and 24 months old (n � 57) who attended different nurseries. is study was approved by the vnch ethics committee (approval number: 521b/nch-rich). written informed consent was obtained from the parents or legal guardians of the children enrolled in the study. we collected 2 ml of nasopharyngeal aspirate from each participant and stored the samples at −70°c until analysis. npa collection was performed by trained nurses. e catheter was inserted into the nostril to a depth of 5 to 7 cm and drawn back while applying gentle suction with a syringe. npa sample (200 μl) from the patient was subjected to total nucleic extraction after addition of internal control bacteriophage ms2 (20 μl) using the magna pure lc total nucleic acid isolation kit (roche diagnostics, germany) on magna pure lc 2.0 platform, following the manufacturer's instructions [15] . e method is based on magnetic-bead technology. e procedure included cellular destruction, nucleic acid binding on beads, and washing steps to remove cellular and purified nucleic acid elution. extracted nucleic acids were eluted in 50 µl of elution buffer and stored at −80°c for rvp fast assay. cluding subtypes h1n1 (1977), h1n1pdm09, h3n2, respiratory syncytial virus a and b (rsva, rsvb), enteroviruses including rhinoviruses (ev/rhi), human parainfluenza viruses 1-4 (piv1-4), human metapneumovirus (hmpv), adenovirus (adv), human coronavirus nl63 (hcov nl63), hcov hku1, hcov 229e, hcov oc43, and human bocavirus (hbov). e assay was performed according to the manufacturer's instructions. assay performance was controlled using bacteriophage lambda included in every run. e assay comprised two steps: a multiplex pcr amplification step and hybridization step. 2.6. multiplex rt-pcr. pcr amplification was performed on the applied biosystems gene amp 9700 pcr (applied biosystems, usa) with the following program: preheating at 50°c for 20 min; template denaturation at 95°c for 15 min; 34 cycles of 95°c for 30 s, 59°c for 30 s, and 72°c for 30 s; and final extension at 72°c for 2 min and kept at 4°c until ready tousle. e hybridization reaction was performed on luminex 100/200 system (lmd, toronto, canada). e hybridization assay includes 20 µl xtag rvp fast bead mix, 2 µl amplified nucleic acid, and 75 µl streptavidin, r-phycoerythrin conjugate (sa-pe) reporter. all reagents were incubated at 45°c for 20 minutes. signal acquisition was done on a luminex 100/200 instrument. data were analyzed and reported by xtag data analysis software (tdas). e infections were classified as pneumonia, bronchiolitis, or bronchitis based on clinical findings and chest x-rays (cxr). pneumonia was defined as ari characterized by infiltrates on the cxr. bronchiolitis was defined as ari in children under 2 years who presented wheezing and hyperaeration and atelectasis or peribronchial thickening in the cxr. bronchitis was defined as ari in children over 2 years old who presented wheezing, hyperaeration, and peribronchial thickening on the cxr. analysis. statistical analysis was performed using fisher's exact test. p < 0.05 was considered significant (spss 13). e xtag rvp fast assay system detected one or more respiratory viruses in 73.7% (143/194) of the samples (table 1) . rsv was the most common virus (51%, 73/143), followed by enterovirus/rhinovirus (ev/rhi), parainfluenza (piv), adenovirus (adv), influenza a/b, and human bocavirus (hbov). overall, 96 samples were positive for a single virus, 37 for 2 viruses, 9 for 3 viruses, and 1 for 4 viruses. groups. rsv and ev/rhi were detected more frequently in children under 6 months (figure 1 ). for other viruses, there was no difference among age groups. children with viral infection in the upper respiratory tract showed symptoms such as runny nose, cough, and hoarseness. some of them also present lower respiratory tract symptoms such as wheezing, severe cough, breathlessness, and respiratory distress, which may be due to bronchiolitis or pneumonia. we divided the patients according to three clinical manifestations: pneumonia, bronchiolitis, and bronchitis, and investigated whether the detected viruses were associated with a specific clinical manifestation. our analysis showed that in most cases, rsv infections induced bronchiolitis (n � 45), followed by pneumonia (n � 22, p � 0.004) and bronchitis (n � 6, p � 0.0015). ev/rhi infections more often induced pneumonia or bronchiolitis (n � 19 for both) instead of bronchitis (n � 6, p � 0.03) (figure 2 ). other total positive specimens 143 73 62 28 15 6 9 3 3 single infection 96 44 28 12 3 3 3 1 1 double infection 37 22 26 11 7 3 2 1 1 triple infection 9 6 7 in the respiratory syncytial virus-(rsv-) infected group, the prevalence of pneumonia symptoms was significantly lower than that of bronchiolitis symptoms and significantly higher than that of bronchitis symptoms. in the enterovirus (ev)/rhinovirus (rhi) infected group, the prevalence of pneumonia symptoms was significantly higher than that of bronchitis symptoms. other viruses showed a similar prevalence of each manifestation. * p < 0.05. viruses showed a similar prevalence of each clinical manifestation. virus infections. all respiratory viruses may cause symptoms such as nasal congestion, runny nose, wheezing, and cough. we found no significant association between the viruses and a specific symptom. we studied whether the presence of specific viral agents was associated with clinical parameters such as respiratory rate (breaths/min), heart rate (beats/min), and peripheral capillary oxygen saturation (spo 2 , %) or with blood test parameters such as white blood count, neutrophil count, or c-reactive protein (crp). elevated neutrophils were associated with adenovirus infection (or � 1.4, p � 0.006, figure 3 ). ere were no other noticeable associations. crp was slightly higher in the adenovirus and enterovirus/rhinovirus infection groups than in the negative group (p � 0.29 and 0.32, respectively). neutrophils were significantly higher in the adenovirus infection group (48.7%) than in the negative group (35.6%, p � 0.0058). in this study, we calculated the frequency of different respiratory viruses present in children with lower respiratory tract infection at the vietnam national children's hospital. e high proportion of viruses detected in pediatric ari patients agreed with previous studies done in vietnam [5] . with the severity of children with respiratory infections, while those viruses were detected rarely in the north of vietnam due to the difference of the location and climate between the south and north of vietnam. [16] . rsv and ev/ rhi were the most frequent viruses, which was similar to the findings of a study done in southern vietnam [6] . rsv, rhinoviruses, influenza viruses, parainfluenza viruses, enteroviruses, coronaviruses, and certain strains of adenovirus are the leading causes of viral respiratory infections in children. e nasal or respiratory secretions from children with viral respiratory tract infections contain more viruses than those from infected adults. e increased output of viruses, together with an overall reduced attention to hygiene, makes children more likely to spread their infection to others. when viruses invade the cells of the respiratory tract, they trigger inflammation and mucus production, which causes nasal congestion, runny nose, scratchy throat, and cough. e small airways of young children can be significantly narrowed by inflammation and mucus, making breathing difficult. airway problems are most common in infections caused by parainfluenza viruses, rsv, and human metapneumovirus [1, 8, 17] . bronchiolitis occurs predominantly in the first year of life and with decreasing frequency in the second and third years. it is characterized by inflammatory obstruction of the small airways and hyperinflation of the lungs and typically presents along with breathing problems and wheezing. rsv is the primary causative agent of bronchiolitis worldwide, causing between 70 or 80 percent of aris during the high season [18] [19] [20] . rsv was the only virus associated with bronchiolitis in this study. neutrophils are immune cells that are present in many lung diseases associated with acute respiratory distress syndrome (ards) and may contribute to acute lung injury. neutrophils are poorly studied with respect to viral infection. we observed an association between elevated neutrophil count and adenovirus infection, which might indicate an association between neutrophil count and damage to the alveolar epithelium. finding biomarkers to diagnose specific viral infections is essential to improve patient care [21] . crp is an acute phase protein synthesized by the liver in response to il-6 increase, which is used as a biomarker of inflammation [22] and to distinguish between bacterial and viral infections. it is not well known if crp levels differ between different viral respiratory infections. in this study, we found no significant association between crp and a specific virus. bronchiolitis was the most common clinical characteristic of lower respiratory tract infection at the vietnam national children's hospital. xtag rvp fast can effectively detect different virus from specimens with low viral loads. e assay should be applied in the clinic for the screening of multiple respiratory viral infections. e data used to support the findings of this study are included within the article. is study has been approved by the vnch ethics committee (approval number: 521b/nch-rich). written informed consent was obtained from all patients enrolled in the study or their legal guardians. e authors declare that there are no conflicts of interest regarding the publication of this article. e changing face of pediatric respiratory tract infections: how human metapneumovirus and human bocavirus fit into the overall etiology of respiratory tract infections in young children respiratory viral infections and host responses; insights from genomics multiplex pcr: optimization and application in diagnostic virology fatal respiratory infections associated with rhinovirus outbreak clinical and epidemiological characteristics of acute respiratory virus infections in vietnamese children human rhinovirus infections in hospitalized children: clinical, epidemiological and virological features comparison of the luminex xtag respiratory viral panel with xtag respiratory viral panel fast for diagnosis of respiratory virus infections mini-symposium: microbiological diagnostic procedures in respiratory infection respiratory virus infection access to a polymerase chain reaction assay method targeting 13 respiratory viruses can reduce antibiotics: a randomised, controlled trial diagnostic errors that lead to inappropriate antimicrobial use multiplex pcr and emerging technologies for the detection of respiratory pathogens assessment of the usefulness of multiplex real-time pcr tests in the diagnostic and therapeutic process of pneumonia in hospitalized children: a single-center experience comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital comparison of the luminex xtag respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections fully automated nucleic acid extraction: magna pure lc human bocavirus in children with acute respiratory infections in vietnam cellular immunity and lung injury in respiratory virus infection respiratory syncytial virus infection pathogen screening and prognostic factors in children with severe ards of pulmonary origin respiratory syncytial virus infections: recent prospects for control association between c-reactive protein and metabolic syndrome in korean adults c-reactive protein: ligands, receptors and role in inflammation key: cord-023817-39r3a4fd authors: singh, namita; burpee, tyler title: rotavirus and noroand caliciviruses date: 2012 journal: textbook of clinical pediatrics doi: 10.1007/978-3-642-02202-9_120 sha: doc_id: 23817 cord_uid: 39r3a4fd nan rotavirus is the most common cause of severe diarrhea in infants and children of developed and developing countries worldwide. globally, rotavirus gastroenteritis causes the death of more than half a million children younger than 5 years of age. this illness creates a disease burden to virtually all societies around the world. rotaviruses are included in genus rotavirus, part of the reoviridae family. negative contrast electron microscopy reveals the viral particles take on a wheel-like appearance in feces, leading to the prefix ''rota.' ' rotavirus particles are large in size (1,000 å ), nonenveloped, and have three concentric layers of proteins surrounding a viral genome. the genome is comprised of 11 segments of double-stranded rna, a characteristic that allows reassortment during natural infection to yield new strains. these segments encode six structural viral proteins (vps) that form virus particles and six nonstructural proteins (nsps). the nsps are synthesized in infected cells and interact with host proteins to influence pathogenesis and the immune response to infection. the rotavirus outer capsid shell is made of the protein vp7. spike-like projections protrude through the capsid shell and are formed by the glycoprotein vp4. the threelayered capsid renders it stable and resistant to the acidic ph in the stomach and to the digestive enzymes in the small intestine. this eases the fecal-oral transmission and delivery of the virus into the small intestine, where rotavirus causes pathological changes in structure and function. rotaviruses can be classified into groups a-e, according to antigenic groups on the major capsid antigen, vp6. only groups a, b, and c have been shown to infect humans, with group a causing the preponderance of human rotaviral gastrointestinal disease. rotaviruses are further classified into g and p types based on the identification of antigens on the outer capsid proteins vp7 and vp4. most severe infections in young children are caused by serotypes g1-4. in general, the more densely populated countries show the most complex patterns of serotype prevalence. during the last two decades, g1 infections appear to have predominated globally. worldwide, approximately 40% of hospitalizations for diarrhea in children younger than 5 years of age are attributable to rotavirus infection. the virus is identified in the stool in 10-40% of children admitted for acute diarrhea in developing countries and 35-50% in developed countries. over 525,000 children younger than 5 years of age die annually from rotavirus, with more than 85% of these deaths occurring in african and asian nations. in the united states, prior to the vaccine's introduction, rotavirus infection accounted for 400,000 doctor visits, 200,000 emergency room visits, 50,000 hospitalizations, and 20-60 deaths per year, with costs amounting to $1 billion yearly. see > fig. 120 .1: rotavirus disease burden. virtually all children have been infected with rotavirus by the age of 5, with various degrees of severity. it is common to have progressively less severe subsequent rotavirus infections as each causes a boost in mucosal immunity. serious rotavirus infections occur most often in children 4-24 months of age. neonatal infection is often asymptomatic in healthy, full-term infants, presumably due to passive immunity from transplacental and breastmilk antibodies. adults are rarely severely affected, but roughly 20% of adult household contacts of an infected infant may develop symptomatic disease. rotavirus gastroenteritis has a seasonal variation pattern. in the united states and other countries with a temperate climate, infections predominate during the winter months, with annual epidemics occurring between december and june. regional variations also exist within particular climates or countries. for example, the united states rotavirus season starts in the southwest in the fall and ends in the northeast in the spring. in europe, the season begins in the southern region and spreads north over the fall to spring months. rotavirus infections fluctuate less in the tropics, though a recent systematic review of 26 studies from tropical areas concluded that infections were more prominent in the coolest and driest months of the year. rotavirus is primarily transmitted person-to-person via the fecal-oral route. in developing regions, it may also be spread via fecally contaminated water. infected individuals usually shed large quantities of virus, up to 10^10 particles per gram of stool. as few as ten viral particles may cause infection in rotavirus-naïve patients. viral shedding may occur up to 2 days prior to the initiation of symptoms and continues for an average of 4 days, though shedding has been reported for up to 21 days in immunocompetent patients. immunocompromised patients may excrete the virus for even longer periods. the virus may survive at least 4 h on hands, days to weeks on environmental surfaces, and up to weeks in drinking water. transmission is increased in settings such as child day-care centers and family homes, where diaper changing has been identified to be the highest-risk activity. additionally, rotavirus has been found on toys, faucets, hand-washing areas, and even food preparation areas, indicating that fomites may play a role in viral transmission. the high infectivity, presymptomatic shedding, and prolonged environmental life span are all important factors in the transmission of rotavirus. the transmission from animals to humans is rare. there is evidence, however, that animal rotaviruses can infect humans via direct transmission of the virus or by contributing one or more rna segments to reassortants with human strains. the respiratory spread of rotavirus via aerosolized particles has also been suggested. rotavirus has been detected in respiratory secretions in a small number of patients, and cases of pneumonia have been described. rotavirus rna from air samples taken from rooms of hospitalized children with rotavirus gastroenteritis indicate that airborne spread may be a route of transmission of rotavirus, especially in hospital and day-care settings. rotavirus has its most profound effects in the gastrointestinal tract, yet systemic infection has been reported. rna and proteins of rotavirus have frequently been detected in the blood of infected children, as well as the liver, heart, lung, and central nervous system. rotavirus particles are ingested orally. in the small intestine, the vp4 spikes on the capsid surface may be proteolyzed by the enzyme trypsin. this cleavage causes a conformational change in the spike structure, which then exposes other attachment sites on the surface glycoprotein. the virus is then attached to host receptors on enterocytes, and the entry process begins. the outer capsid shell is removed, and double-layered particles are inserted into the cytoplasm. these particles produce capped viral messenger rnas (mrnas) that are then displaced from the transcribing particles into the cell cytoplasm. there, they are then translated into proteins and replicated to new genomic rna. this viral replication process is unique and occurs in viroplasms, electron-dense structures near the cell nucleus, and endoplasmic reticulum. newly assembled particles with newly replicated dsrna bud into the endoplasmic reticulum from the viroplasm and are equipped with outer capsid proteins. fully developed virus particles are released from enterocytes via cell lysis or delivery of particles to the apical plasma membrane of cells. rotavirus primarily invades mature enterocyte villi, sparing the intestinal crypts. with enterocyte destruction, fluid moves into the intestinal lumen, resulting in a net loss of fluid and electrolytes in stool. malabsorption occurs secondary to the loss of absorptive enterocytes, the decreased synthesis of lactase and other digestive enzymes, and the alteration of tight junctions between enterocytes that leads to paracellular leakage. the rotavirus toxin, nsp4, is a nonstructural glycoprotein that may contribute to the pathophysiology by inducing a secretory component to the diarrhea. this viral enterotoxin is reported to increase intracellular calcium and activate cellular chloride channels, in turn increasing the secretion of chloride and consequently water into the lumen. nsp4 has also been found to activate the secretory reflexes of the enteric nervous system (ens), further contributing to diarrhea. microischemia of villi, impaired polar transport of sucrase-isomaltase to the membrane surface, neuronally mediated intestinal hypersecretion, and altered intestinal motility are other possible mechanisms of rotavirus injury. histologically, rotavirus is associated with a wide spectrum of changes, ranging from virtually normal mucosa or mild enterocyte vacuolization and loss to more significant villous blunting and crypt hyperplasia. the degree of inflammation is usually milder than that caused by other intestinal pathogens. there appears to be no direct correlation between histological findings and disease symptoms. after a 2-7 day incubation period, symptoms often start abruptly with vomiting, followed by watery diarrhea. this is a noninflammatory process, with blood and white cells typically absent from the stool. in about one-third of patients, fever of over 38.9 c is reported. other clinical features of acute rotaviral infection include anorexia and lethargy, with abdominal cramping being less common. the diarrhea can range from mild to severe, with resultant dehydration, shock, electrolyte imbalance, and even death. severe, dehydrating rotavirus infection occurs mostly in children age 3-35 months. rotavirus is generally a selflimited virus, with vomiting settling within 24-48 h and diarrhea in 2-7 days. some studies have noted respiratory symptoms and otitis media in up to half of patients with rotavirus infection. intussusception in rotavirus infection may be caused by a disturbance in the motility of the gastrointestinal tract during an acute infection, as opposed to a typical lead point. lipopolysaccharides may slow intestinal motility through the induction of various inflammatory agents, such as prostaglandins, cytokines, and nitric oxide. the center for disease control and prevention (cdc) defines a confirmed case of rotavirus gastroenteritis as diarrhea (3 or more loose stools within 24 h) or vomiting (1 or more episodes in a 24 h period) in a child with a positive stool detection of rotavirus by a standard assay, such as an enzyme immunoassay (eia). confirmation of rotavirus infection is necessary for reliable surveillance and can be clinically useful to avoid inappropriate antimicrobial use. the most widely used method to detect rotavirus antigen in stool is eia. these assays typically detect the rotavirus group antigen present on vp6. many eia kits are commercially available, providing rapid, inexpensive results with 90-100% sensitivity. strains may be further characterized by additional enzyme immunoassay or reverse transcriptase polymerase chain reaction (rt-pcr). research centers use other techniques for detection and surveillance of rotavirus, including electron microscopy, rt-pcr, nucleic acid hybridization, sequence analysis, and culture. these techniques are more labor intensive and provide little additional clinical utility. the non-bloody, watery diarrhea of rotavirus gastroenteritis is clinically indistinguishable from that caused by other enteric viruses, including norovirus and other caliciviruses, enteric adenovirus, and astrovirus. that said, rotavirus associated acute diarrhea may be more severe and more frequently associated with fever and vomiting. the presence of blood or leukocytes in the stool should suggest alternative diagnoses, including bacterial etiologies such as salmonella, shigella, yersinia, campylobacter, and escherichia coli. noninfectious etiologies, including intussusception, must also be considered in the infant with this presentation. protozoal infection, particularly entamoeba histolytica, giardia lamblia, and cryptosporidium parvum, should also be included in the differential diagnosis. as mentioned, rotavirus infection is most frequently selflimited, with cessation of vomiting within 2 days and diarrhea within 7 days. severe, dehydrating infection occurs more often in young children from age 3-35 months. malnutrition is known to increase the severity of infection, with delayed small intestinal recovery and altered inflammatory responses. acute complications include dehydration, sodium imbalance, and possible seizures. reye syndrome, encephalitis, rectal bleeding, and intussusception have all been associated with rotavirus, but evidence showing a causative effect is lacking. rotavirus gastroenteritis is generally a self-limited illness, and treatment is largely supportive. initial management should focus on the identification and correction of any underlying fluid and electrolyte imbalance. the assessment of dehydration and the use of oral rehydration therapy are critical and are reviewed elsewhere in this text (> chap. 187, ''acute gastroenteritis in infants and children''). undernourished children are particularly at risk of severe and/or persistent symptoms, and great care must be taken to encourage early resumption of normal feeding, typically including breastfeeding. there is no role for antibiotics in the treatment of rotavirus gastroenteritis. the use of antiemetics and antidiarrheals is generally avoided. please see > chap. 187, ''acute gastroenteritis in infants and children'' for further details. the oral administration of a probiotic, lactobacillus gg, is effective in both reducing viral shedding and shortening symptom duration by roughly 1 day. this improvement is most notable when the probiotic is given early in the course of the illness and seems most prominent in young children. the mechanism may be due to an enhancement of the immune response against the virus. orally administered human immune globulin, as an investigational therapy in immunocompromised patients, was found to shorten the course of diarrhea and decrease viral excretion. further investigation is required, and the cost-benefit ratio may not justify usage of this therapy on a wide-scale basis. rotavirus attack rates are similar between developed and developing regions, suggesting that improved sanitation is unlikely to play a significant part in disease prevention. the high infectivity of the virus makes control measures difficult. rotaviruses are relatively resistant to chemical disinfectants used widely in hospitals. effective agents include chlorhexidine gluconate and quaternary ammonium compounds with high alcohol content (70%). hand washing with plain soap is often ineffective against rotavirus and may even spread the virus over a larger area of the hands. the use of a waterless, alcohol-based handcleaning agent before and after patient contact is recommended. breastfeeding plays a protective role against acquiring rotavirus. this may be due to the presence of anti-rotaviral secretory iga and trypsin inhibitors in breast milk. breast-fed infants also excrete fewer viruses than formula-fed infants. based upon the significant morbidity and mortality of childhood rotaviral infection worldwide, great attention has been focused upon the development of a successful vaccine. since 1983, multiple candidate vaccines have been tried. initially, vaccines based upon the use of animal rotavirus strains that are not pathogenic in humans failed to provide sufficient clinical protection. rotashield (wyeth-lederle vaccines, philadelphia, pa), a tetravalent simian/human reassortant rotavirus vaccine, was licensed by the fda in 1998, based upon data showing an 80-100% protection against dehydrating rotavirus diarrhea. within 9 months of its release, the cdc's vaccine adverse event reporting system (vaers) reported 15 cases of intussusception in infants who had received the vaccine. subsequent case control and retrospective cohort studies verified a temporal association. the relative risk for intussusception within 2 weeks following the first vaccine dose exceeded 20 in both studies, prompting the vaccine's withdrawal from the market. two subsequent oral rotavirus vaccines are now licensed in many nations around the world, including the united states. these vaccines have led to the decline of rotaviral infection and mortality worldwide. rotarix™ (glaxosmithkline biologicals, rixensart, belgium, 2006) is based on a live attenuated human rotavirus strain, g1p. rotateq™ (merck & co., inc., whitehouse station, nj, usa, 2008) is a pentavalent human-bovine reassortant, with a low rate of replication in the human gastrointestinal tract and a low rate of fecal shedding. large phase iii clinical trials have demonstrated that both rotarix™ and rotateq™ are well tolerated. they have also been demonstrated to be immunogenic and highly efficacious, preventing 74-87% of all cases of rotavirus gastroenteritis and greater than 85% of those associated with severe diarrhea. in africa and asia, where more than 85% of rotavirus associated deaths occur, rotateq™ vaccination reduced cases of severe rotavirus diarrhea by greater than 50% during the first year of life when the disease burden and mortality is greatest. the world health organization's strategic advisory group of experts declared that rotavirus vaccines should be included in national immunization programs worldwide, particularly in nations with high diarrheal fatalities. diarrhea-associated deaths in these developing countries could be reduced by 25%. the rotateq™ vaccine is recommended to be given as a three-dose series to infants between the ages of 6-32 weeks. the rotarix™ vaccine is recommended to be administered as a two-dose series at the ages of 2-4 months. details of the vaccine schedules are provided in > table 120 .1. contraindications to the rotavirus vaccine include a previous severe life-threatening allergic reaction to any components of the vaccine and some immunocompromised states. data from the cdc's vaers has shown that the observed rate of intussusception in rotateq™-vaccinated children is not higher than the age-adjusted background rate of intussusception. similarly, studies show no increased risk of kawasaki syndrome with the administration of rotateq™ vaccine. since the introduction of these vaccines in 2006, the incidence of rotavirus diarrhea in infants has dramatically decreased. one study noted that from 1986 to 2006, nearly 20% of hospitalized gastroenteritis patients younger than 5 years of age tested positive for rotavirus in the stool. in the three seasons after vaccine introduction (2007) (2008) (2009) , the percentage dropped to 12.4%, 9.6%, and 6.4%, resulting in a decline of 66% by the study's termination. furthermore, the rotavirus season has been found to be shortened and delayed. see > fig. 120 .2: hospitalizations in children due to laboratory-confirmed rotavirus gastroenteritis. newer approaches, such as non-replicating virus-like particle (vlp) vaccines, are presently being evaluated. meanwhile, the current rotavirus vaccines remain a success in decreasing the morbidity and mortality from this global health disease. norwalk virus carries historical import as the first confirmed viral etiology for human gastroenteritis when it was identified by electron microscopy in stools from a severe outbreak of diarrhea in norwalk, ohio in 1972. subsequently, similar appearing viruses were often called ''norwalk-like viruses.'' recent reclassifications now define the family as caliciviridae, which are 20-40 nm, non-enveloped, single-stranded rna viruses. within this family are four distinct genera. the norovirus genus accounts for roughly 95% of calicivirus-associated gastroenteritis and is discussed here in greater detail. the other three genera (sapovirus, lagovirus, and vesivirus) are less clinically relevant. human norovirus strains are classified into several distinct genogroups and subgroups. genogroup ii has been identified as the most common strain infecting humans worldwide. a new pandemic strain emerges every 2 -4 years. noroviruses are the most common cause of nonbacterial gastroenteritis outbreaks worldwide. infections may occur year round, though more cases are reported during winter months. both children and adults can be affected. the center for disease control (cdc) estimates that noroviruses cause 21 million cases of gastrointestinal illness annually in the united states, accounting for half of all foodborne disease outbreaks. norovirus infection leads to an estimated 70,000 hospitalizations and 500 deaths annually in the united states. there, only rotavirus leads to more hospitalizations for gastroenteritis in children. in developing countries, norovirus is also a common etiology for diarrhea. in india and peru, 15% and 31%, respectively, of stool samples in hospitalized pediatric gastroenteritis patients tested positive for norovirus via pcr ( > table 120 .2). . not to be given after 8 months of age rotavirus and noro-and caliciviruses norovirus outbreaks frequently occur in closed environments, such as cruise ships, camps, nursing homes, or schools. typically, the outbreaks originate from direct contamination by an infected food handler. food contaminated at its source, such as oysters from contaminated water, has also been described. as the viruses are highly contagious, outbreaks can be explosive, with potentially thousands of people being infected in a short period of time. norovirus is typically transmitted via the fecal-oral route or through the ingestion of contaminated food or water. additionally, vomitus has been shown to contain infectious particles. as few as 10-100 virions are required for infection. once exposed, roughly 30% of individuals may shed the virus prior to the onset of symptoms. viral shedding then peaks 1-3 days after illness develops and may persist for up to 3 weeks. the virus is stable from freezing temperatures up to 60 c. all of these factors contribute to the ease of spread and the potential for large outbreaks. the incubation period ranges from 12 -48 h. disease onset is then quite rapid, with vomiting and non-bloody, watery diarrhea. fever occurs in roughly 40% of cases, and other constitutional symptoms such as headache, myalgias, and chills are common. in one-third of patients, the illness is often mild and short-lived, with 85% of patients experiencing less than 3 days of vomiting and diarrhea. the risk for dehydration requiring hospitalization is greatest in children less than 5 years of age and in adults 65 years and older. in the infected host, the proximal duodenum demonstrates villous broadening and blunting, crypt-cell hyperplasia, cytoplasmic vacuolization, and inflammatory cell infiltration into the lamina propria. histologically, the stomach and colon are spared. intestinal brush border enzymes are diminished during acute infection, with resultant carbohydrate malabsorption. mild steatorrhea is also noted. the prominent nausea and vomiting associated with norovirus gastroenteritis may relate to delayed gastric emptying, which has been documented in symptomatic adults. the preponderance of vomiting and the high attack rate across all age groups are characteristic features of calicivirus gastroenteritis. norovirus outbreaks can be distinguished from those caused by preformed toxins by the slightly longer incubation period (12-48 h vs. 2-6 h for toxins) and the emergence of secondary cases in household contacts. in general, calicivirus infection tends to have a milder, less dehydrating course than rotavirus. that said, the illness is not reliably clinically distinguishable from that caused by other enteric viruses, including rotavirus, enteric coronavirus, enteric adenovirus, and astrovirus. human noroviruses cannot be cultured. reverse transcriptase polymerase chain reaction (rt-pcr) can be used to detect viral rna from stool or emesis samples, in addition to environmental swabs from food or water, in special circumstances. the technique is challenging as human caliciviruses are genetically diverse. consequently, several sets of primers must be used to confirm infection. real-time quantitative rt-pcr assays have increased both the sensitivity and specificity of this diagnostic modality. rt-pcr detection is available in some public health and research laboratories but is not readily commercially available in most areas. enzyme immune assays (eias) for norovirus detection have been approved for commercial use in some countries, though the poor sensitivity of current assays (<50%) limits their use in sporadic cases. in outbreak situations, however, they can be used to rapidly identify norovirus as the causative agent. in many situations, microbiologic confirmation of a suspected norovirus outbreak is not possible. the ''kaplan criteria'' were developed in 1982 to distinguish outbreaks caused by norovirus from those caused by bacterial etiologies. the criteria (vomiting in greater than 50% of affected persons, a mean illness duration of 12-60 h, a mean incubation period of 24-48 h, and no bacterial pathogen identified in stool culture) are highly specific (99%) and moderately sensitive (68%) in this regard. as roughly 30% of norovirus-induced outbreaks will not satisfy all four criteria, it is still important to consider this virus in the appropriate clinical setting. until norovirus diagnostic tests become widely available, the application of these criteria may be the most useful diagnostic aid in identifying food-borne gastroenteritis outbreaks due to norovirus. as noted above, calicivirus gastroenteritis is, in general, fairly mild and self-limited. immunocompromised hosts, infants, and the elderly are at the highest risk for protracted illness and more severe dehydration. reports have arisen suggesting an association of norovirus with necrotizing enterocolitis in newborns, benign seizures in infants, and inflammatory bowel disease exacerbations in pediatric patient. further studies are required to investigate these possible links. there is no specific treatment available. as with other causes of viral gastroenteritis, supportive care and attention to fluid and electrolyte balance is crucial. please see > chap. 187, ''acute gastroenteritis in infants and children'' for additional details. during an outbreak, preventing secondary spread is important in halting further progression. enforcing personal hygiene, using contact precautions, decontaminating environmental surfaces, and using an rotavirus and noro-and caliciviruses alcohol-based hand sanitizer have all been found to decrease the spread of infection. people with diarrhea due to norovirus refrain should refrain from the use of recreational water venues, such as pools and lakes, for at least 2 week following the resolution of symptoms. the development of effective preventative measures is of great interest, given the significant socioeconomic burden of large, prolonged outbreaks. in contrast to rotavirus, humans do not acquire long-term immunity with norovirus infection, making vaccine development challenging. a virus-like particle vaccine is currently being evaluated. acute infectious nonbacterial gastroenteritis: intestinal histopathology. histologic and enzymatic alterations during illness produced by the norwalk agent in man detection by pcr of eight groups of enteric pathogens in 4, 627 faecal samples: re-examination of the english case-control infectious intestinal disease study (1993-1996) diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses immune response of children who develop persistent diarrhea following rotavirus infection real-time surveillance to assess risk of intussusception and other adverse events after pentavalent, bovine-derived rotavirus vaccine natural history of human rotavirus infection discovery of rotavirus: implications for child health rotavirus antigenaemia and viraemia: a common event? rotavirus: to the gut and beyond! norovirus infection as a cause of diarrhea-associated benign infantile seizures asymptomatic endemic rotavirus infections in the newborn the zoonotic potential of rotavirus reduction in acute gastroenteritis hospitalizations among us children after introduction of rotavirus vaccine: analysis of hospital discharge data from 18 us states vpd surveillance manual transmission of rotavirus and other enteric pathogens in the home. pediatric infect disease j (the healthy home summit: the significance of cleanliness and disinfection in the home and its link to infection control) detection of rotavirus rna in hospital air samples by polymerase chain reaction (pcr) * 828 update on rotavirus trends and the importance of surveillance intussusception: 354 cases in 10 years norovirus gastroenteritis rotaviruses: from pathogenesis to vaccination probiotics for children: use in diarrhea oral bacterial therapy reduces the duration of symptoms and of viral excretion in children with mild diarrhea protection against neonatal rotavirus infection by breast milk antibodies and trypsin inhibitors gastrointestinal norovirus infection associated with exacerbation of inflammatory bowel disease intussusception, infection, and immunization: summary of a workshop on rotavirus progress in understanding norovirus epidemiology seasonality of rotavirus 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and trends, using capture-recapture methods norwalk virus: how infectious is it reevaluation of epidemiological criteria for identifying outbreaks of acute gastroenteritis due to norovirus: united states outbreak of necrotizing enterocolitis caused by norovirus in a neonatal intensive care unit safety and efficacy of a pentavalent human-bovine (wc3) reassortant rotavirus vaccine evaluation of two commercial enzyme immunoassays for the detection of norovirus in faecal samples from hospitalised children with sporadic acute gastroenteritis rotavirus incidence and genotype distribution before and after national rotavirus vaccine introduction in belgium a functional nsp4 enterotoxin peptide secreted from rotavirus-infected cells protein-energy malnutrition delays small-intestinal recovery in neonatal pigs infected with rotavirus key: cord-272596-yxvg8357 authors: wu, jian jun; jin, yu; lin, na; xie, zhi ping; yu, jie mei; li, jin song; cao, chang qing; yuan, xin hui; song, jin rong; zhang, jing; zhao, yang; gao, xiao qian; duan, zhao jun title: detection of human bocavirus in children with acute respiratory tract infections in lanzhou and nanjing, china date: 2014-11-30 journal: biomedical and environmental sciences doi: 10.3967/bes2014.110 sha: doc_id: 272596 cord_uid: yxvg8357 abstract objective the aim of this study was to explore the prevalent characteristics of hbov1 and its co-infection. methods pcr was used to detect hbov1-dna (hbov1) and other viruses. a multivariate logistic regression model was used to explore possibility of co-detected for related viruses. results the positivity rates in nanjing and lanzhou were 9.38% (74/789) and 11.62% (161/1386), respectively (p<0.05). the hbov1 positive group was younger than negative group (p<0.05). seasonal differences were noted, with a higher frequency of infection in december and july. hbov1-positive children [72.34% (169/235)] were co-infected with other respiratory viruses. multifactorial analysis showed no correlations between hbov1 and the clinical classification, region, gender, age, or treatment as an outpatient or in a hospital. correlations were identified between hbov1 infections with adv (or=1.53, 95% ci 1.03-2.28), rsv (or=0.71, 95% ci 0.52-0.98), and ifva (or=1.77, 95% ci 1.00-3.13). conclusions presence of hbov1 in nasopharyngeal aspirates did not correlate with region or gender, although the prevalence of hbov1 was higher in younger children. there were no correlations between hbov1 and other variables, except for the season and adv, rsv, or ifva infections. introduction espiratory infections are often the result of combined bacterial and viral infections [1] , and severe acute respiratory infections are the leading cause of mortality among children worldwide [2] . the traditional viruses known to cause respiratory tract infections include adenovirus (adv), influenza virus (ifv) a and b, human rhinoviruses (hrv), and respiratory syncytial virus (rsv). new viruses have been discovered in recent years, including human metapneumovirus (hmpv) [3] , the coronaviruses hcov-nl63 [4] , and hcov-hku1 [5] [6] , as well as human bocavirus (hbov) which was the first virus identified by molecular virus screening in 2005 [7] . more than two-thirds of childhood pneumonia cases are associated with viral infections [8] [9] . reports of hbov in respiratory tract infections have increased rapidly in numerous countries and regions throughout the world [10] [11] [12] [13] [14] [15] [16] [17] , with hbovs detected in 1.5%-19% of respiratory tract secretions and 0.8%-9.1% of fecal samples [12] [13] . thus, increasing evidence suggests that hbov1 is an important cause of respiratory tract infections [10] [11] [12] 14] . hbov2 [18] , hbov3 [19] , and hbov4 [20] were isolated recently in stool samples from patients with diarrhea. all four hbov genotypes can be detected in stool of patients with acute gastroenteritis, while hbov1 and hbov2 were reported in respiratory tract samples [12] . however, pathogen testing of respiratory infected patient sputum found the presence of other viruses and bacteria in addition to hbov1. thus, it remains to be clarified whether high-rates of hbov co-infection in the respiratory tract lead to disease, or represents only a concomitant viral infection. there is also a lack of large-scale hbov studies determining whether there are ethnic and regional differences in hbov detection rates, since the incidence and clinical presentation of hbov vary widely. furthermore, the epidemiological characteristics of hbov1 and its relationships with other viruses need to be elucidated. from november 2007 to july 2011, nasopharyngeal aspirates (npas) were obtained from 2175 children <173 months of age with acute respiratory tract infection. all cases of children with acute respiratory tract infections were included in our study, data were collected using a standardized questionnaire, diagnostic criteria based on practical pediatrics (seventh edition), including: acute upper respiratory tract infections (including acute laryngitis, herpes angina, acute tonsillitis), acute bronchitis (including asthmatic bronchitis), pneumonia (including bronchiolitis), and acute exacerbation of bronchial asthma. inclusion criteria: (1) patients with the 'acute respiratory infection' diagnostic criteria characteristics from inpatient or outpatient, (2) patients or legal guardian signed an informed consent form, (3) age of patients were <15 years, (4) the course of disease of patients were within 48 h. exclusion criteria: (1) the patients did not meet the diagnostic criteria, (2) although within 48 h, the patients had received anti-viral or anti-inflammatory drug treatment, (3) patients associated with cardiovascular, liver, kidney, hematopoietic system, or other serious diseases, (4) the sample of patients could not be completely collected. of these samples, 1386 were from the first hospital of lanzhou university (lanzhou, china) and 789 were from nanjing children's hospital, medical school of nanjing university (nanjing, china). acute respiratory tract infections were classified according to who definitions [21] ; children with symptoms of acute upper respiratory tract infection (urti) and acute lower respiratory tract infection (lrti) were diagnosed in the presence of characteristic manifestations of rhinitis, pharyngitis, tracheitis, bronchitis, pneumonia, and severe pneumonia. the clinical diagnostic classification (i-iv) was based on previous studies [22] . it was assessed at the time of enrollment and categorized as follows. i grade: very mild (upper respiratory tract symptoms/signs only); ii grade: mild (lower respiratory tract symptoms/signs +/2 upper respiratory tract symptoms/signs but not needing hospital admission); iii grade: moderate (lower respiratory tract symptoms/signs +/2 upper respiratory tract symptoms/signs, needing hospital admission but with oxygen saturations in air >93% on pulse oximetry); iv grade: severe (lower respiratory tract symptoms/signs +/2 upper respiratory tract symptoms/signs, needing hospital admission and oxygen with saturations in air <93%). informed consent was obtained from the parents of all children who provided specimens and the study protocol was approved by the ethics committee of each hospital. demographic and clinical data were recorded from all patients and specimens were stored at -80 °c for further analysis. viral rna and dna were extracted from the npas by using a qiaamp® viral rna mini kit (qiagen, hilden, germany), which extracts viral rna and dna simultaneously according to the manufacturer's instructions. the nested pcr assay with primers (forward1 5'-a ggtaaaacaaatattgcaaa ggccat agtc-3' and reverse1 5'-tgggagttctctccgtccgtat c-3', forward2 5'-agggtttg t ctttaacgattgc agac aac-3', and reverse2 5'-tatacacagagt cgtcagcac tatgag-3') was performed using ex-taq dna polymerase (takara, beijing, china) to amplify a 455-bp fragment targeting a portion of the ns1 genes, as reported previously [23] . the amplicons from a second-round of pcr were analyzed by 1.5% agarose gel electrophoresis and sequenced with the big-dye terminator cycle sequencing kit and the automated abi prism 310 genetic analyzer (qiagen, beijing, china). sequences were edited using the dna star software package. blast (www.ncbi. nlm.nih.gov) was used to confirm gene identities in the national center for biotechnology information (ncbi) databases. all samples were tested for 11 pathogens using standard reverse transcription pcr. these pathogens included ifva, ifvb, hmpv, rsv, hrv, piv types 1-3, hcov-nl63, and hku1 [7, [24] [25] [26] . a different pcr method was used to detect adv [27] . all sample data were done using epidata analysis. categorical data and the median ages of children in different groups were compared using the chi-square (χ 2 ) test and mann-whitney test, respectively. binary logistic regression analysis and independent variables were defined by region (lanzhou=1, nanjing=2), gender (male=1, female=2), age (months), season (1-12), disease classification (i-iv), and inpatient/outpatient (inpatient=1, outpatient=2). the 11 other respiratory viruses were also defined as independent variables (positive=1, negative=0). hbov1 infection (positive=1, negative=0) was the dependent variable in multivariate analysis. eigenvalues and condition indices were used to assess multi-colinearity in the regression models. all tests were two-tailed and p≤0.05 was considered statistically significant. all analyses were performed using spss version 13.0. the 2175 children with acute respiratory infections in this study were aged 9 d to 173 months (the median age was 26 months). the majority of patients (74.11%) were aged >24 months. the ratio of males to females was 2.0:1.0 and that of inpatients to outpatients was 6.35:1. the case ratio of lanzhou to nanjing was 1.76:1.0. respiratory tract infection disease classifications at grade iii-iv accounted for 66.73% of cases. hbov1-dna was detected by nested pcr in 235 specimens, an overall frequency of 10.80%. hbov1 positive rates were 11.62% (161/1386) in lanzhou and 9.38% (74/789) in nanjing (χ 2 =2.61, p>0.05). the ratio of males to females with hbov1-dna was 1.83:1 but the hbov1 positive rate did not differ based on gender or region ( table 1 ). the positivity rates were 6.97% (53/760) for the <6 months old group, 14.55% (112/770) for the 7-24 months old group, 12.30% (53/431) for the 25-60 months old group, 13.64% (9/66) for the 61-72 months old group, 5.41% (8/148) for the >72 months old group (figure 1 ). hbov1-dna was detected in every month, but detection peaked in december (13.75%), followed by 13.16% in july, 12.70% in february, 12.50% in april, and 11.29% in may (figure 2 ). the seasonal distribution of hbov1-dna differed significantly (χ 2 =19.97, p<0.05) in the positive and negative groups. while our study used highly sensitive and low error probability nested pcr to screen hbov1, hbov1 detection rates varies widely. since our techniques result in highly precise and sensitive detection of hbov1 infection, environmental or socio-economic factors may potentiate different patient risk factors. previous studies had shown that the peak of hbov1 infection is in the summer [12, 14] or spring [37, 39] . in our study however, the peak seasons of hbov1 infection occurred in february, april, july, and december during the 48 months collecting period. thus, the seasonal frequency of hbov1 infection should be further investigated as it appears to be more common in the colder seasons [37] . regardless, the seasonal peaks of hbov1 infection have differed among counties and climate regions. previous studies have determined that hbov1 has a high co-infection rate with other respiratory viruses [12] [13] , but its pathogenic role was still debatable due to high frequency of co-infection. hbov1 may exist in the respiratory tracts as a bystander without causality to a patient's current symptoms [13] . additionally, hbov1 may have an important role in acute respiratory infections among children [43] . in patients with respiratory complaints, hbov1 can be found alone or, more often, in combination with other viruses known to cause respiratory complaints [44] . hbov1 may be a factitive with other respiratory viruses for the observed symptions, suggested by the findings that symptomatic children had higher hbov1 viral load than asymptomatic children [41] . in our study, 72.34% of hbov1-positive patients had co-infection with 10 different respiratory viruses, which is similar to previous reports [12] [13] [14] [46] [47] [48] . hbov1 co-infection has been reported with rsv [8] [9] 14, [45] [46] [47] [48] , ifva [12, 14] , and adv [8] [9] in children and adults with respiratory tract infections, and also with hmpv infections in children with community-acquired pneumonia [49] . thus, the reasons underlying the high co-infection rate of hbov1 need further study and leads to questions regarding hbov1 pathogenicity [12] . recent studies based on epidemiological evidence show that hbov1 was likely to be a major contributor to acute respiratory tract disease, particularly in infants and chlidren [13] . hbov1 co-infections with other viruses have been associated with greater disease burden (i.e., more hospitalizations and loss of school days) in children with respiratory tract infection than in those with hbov infection alone. many factors might have an impact on hbov1 infection and a larger study seemed to corroborate a synergistic relationship between hbov1 and the pathogenesis of respiratory syndromes such as influenza-like illnesses, bronchiolitis, and pneumonia [47] . however, our regression model showed that hbov1-positivity was highly correlated with season, rsv, adv, and ifva (p<0.05). hbov1 belongs to the parvoviridae family with viral head-to-tail genome sequences that use typical rolling circle replication [50] [51] . hbov1 may be triggered by helper viruses, although it had been hypothesized that picornaviruses may be a factor that promotes clinical illnesses [52] . our analysis suggested a possible synergistic effect between hbov1 and adv and ifva (or>1). the results should be confirmed by further clinical and experimental data. nevertheless, given that the or value for rsv infection in multivariate analysis was <1, it was estimated that related to the wide distribution of rsv. unfortunately, the analysis results did not show a link between age and hbov1, which may be due to the fact that infants and young children were the primary participants in our study. this connection requires further investigation before any interactions can be confirmed. deficiencies still existed in our manuscript because some patients or legal guardians did not agree to take part in our study. as such, some cases were missed, so potential sample selection bias were not absolutely avoided. however, our large sample size likely weakened any potential sample bias. samples of cases that were excluded were not collected in this study, so the frequency of infection in exclusive samples of cases needs to be revealed by more studies. in addition, distribution of hbov1 positive infections in children without respiratory tract infections remains crucial to be explored for revealing relationships between hbov1 in the health and the diseased. in summary, we examined hbov1 in children with respiratory infections but require further information to elucidate its pathogenic role. our understanding of this virus would be greatly enhanced by large-scale molecular epidemiological studies of pathogens in different regions. none of the authors has a conflict of interest. zd and yj designed the study. cc, xy, js, jz, yz, and xg collected the samples. jw, jl, zx, and xg performed the pcr tests. jw and nl analyzed the data. jw, jy, and nl wrote and finalized the manuscript. all authors read 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journal: j nutr health aging doi: 10.1007/s12603-020-1512-3 sha: doc_id: 34436 cord_uid: yhb8m1si objective: poor dietary habits are considered to be the second-leading risk factors for mortality and disability-adjusted life-years (dalys) in the world. dietary patterns are different based on cultural, environmental, technological, and economic factors. nutritional deficiencies of energy, protein, and specific micronutrients have been shown to contribute to depressed immune function and increased susceptibility to infections. we aimed to explore the relation of dietary factors with global infection and mortality rates of covid-19 in this study. design: in the current ecological study, the countries that had national dietary data from the global dietary databases of the united nations and coronavirus disease statistics from the world health organization (who) were included. the countries that had coronavirus disease statistics from the who were consecutively checked for the recent data of the dietary factors. setting: world. participants: 158 countries across the world. measurements: infection and mortality rates of covid-19; dietary factors. results: the median crude infection and mortality rates by covid-19 were 87.78 (iqr: 468.03) and 0.0015 (iqr: 0.0059), respectively. the two highest percentage of the crude infection rate were between 0 and 500 (75.9%) and 500–1000 (8.9%) per one million persons. the regression analysis showed that the crude infection rate has been increased by raising consuming fruits (beta: 0.237; p=0.006) and calcium (beta: 0.286; p=0.007) and was decreased with rising consuming beans and legumes (beta: −0.145; p=0.038). the analysis showed that the crude mortality rate was increased by raising consuming sugar-sweetened beverages (beta: 0.340; p<0.001). whereas, the crude mortality rate by covid-19 has been decreased by increasing fruits consuming (beta: −0.226; p=0.047) and beans and legumes (beta: −0.176; p=0.046). conclusion: the present study showed the higher intake of fruits and sugar-sweetened beverages had a positive effect on infection and mortally rates by covid-19, respectively. in contrast, the higher intake of beans and legumes had a negative effect on both increasing infection and mortality rates. poor dietary habits are considered to be the second-leading risk factors for mortality and disability-adjusted life-years (dalys) in the world. the poor dietary habits are responsible for 10.3 million deaths and 229.1 million dalys in 2016 (1) . for example, the following dietary habits are among the leading risk factors for early death and disability in european countries. the habits are low intakes of whole grains, fruit and vegetables, and nuts and seeds, and high intakes of alcohol and sodium. the western dietary habits are consuming diet processed, high in red and processed meat, diets with high in sugar-sweetened beverages, and low in milk. these kinds of dietary habits are regarded to be a rising health concern. dietary patterns are different based on cultural, environmental, technological, and economic factors. however, the dietary patterns are becoming similar due to increasing living standards and growing globalization of the food sector (2, 3) . mertens et al. (4) explored the dietary intakes in four different european counters using individual-level dietary intake in adults in nationally-representative surveys of denmark, france, czech republic, and italy. they reported a higher intake of fruits and vegetables and lower intakes of sweetened beverages and alcohol in italy. while individuals in denmark and the czech republic had a higher intake of vegetables. a comparison of population subgroups within countries shows that there is a difference in the dietary preferences, beliefs, and practices for particular consumer groups. for example, highly-educated persons and women have a higher intake of fish, nuts, and seeds along with lower intake of red and processed meats (5) . the individual-level reported dietary data of the countries could be used as a useful tool to make a connection between health and environment with foods as their common denominator (1) . a recent review study reported that the detailed assessment of patients for the dietary and nutritional risks along with medical, lifestyle, and environmental factors with suitable risk management strategies make the sensible way to deal with the covid-19 (6) . the diet and nutrition have a variance impact on the immune system competence. in addition, they determine the risk and severity of the infections. the relation between diet, nutrition, infection, and immunity is bidirectional (7) . the macro-, micronutrients, and phytonutrients in diet, such as fruits and colorful vegetables improve healthy immune responses. the microand phytonutrients provide the antioxidants and the antiinflammatory nutrients, like beta-carotene, vitamin c, vitamin e, and polyphenolic compounds resulting in modulating the immune functions (8, 9) . nutritional deficiencies of energy, protein, and specific micronutrients have been shown to contribute to depressed immune function and increased susceptibility to infections. the sufficient intake of iron, zinc, and vitamins a, e, b6, and b12 is vital for the overall maintenance of immune function (10) . the new epidemics of coronavirus disease 2019 (covid-19) has become a pandemic to the world currently. we make a hypothesis that geographical variation in dietary factors could have a role in infection and mortality rates of covid-19 in the world. therefore, we aimed to explore the relation of dietary factors with global infection and mortality rates of covis-19 in this study. in the current ecological study, the countries that had national dietary data from the global dietary databases of the united nations (11) and coronavirus disease statistics from the world health organization (who) were included (12) . the countries that had coronavirus disease statistics from the who were consecutively checked for the recent data of the dietary factors. the countries/states met eligibility criteria for this investigation if they had the statistics from the who coronavirus disease (covid-19) situation dashboard from the website of the world health organization by 24 april 2020 (12) . the following countries were excluded from the analysis due to not having the statistics of the covid-19; comoros, north korea, kiribati, lesotho, malawi, marshall islands, micronesia, nauru, palau, samoa, sao tome, and principe, solomon islands, south sudan, tajikistan, tonga, turkmenistan, tuvalu, vanuatu, and yemen. the following countries were excluded from the study due to not having data on the national dietary factors on the website of the global dietary database (11) the populations of the countries were extracted from the united nations statistics division (13) . the estimated populations of the year 2018 were considered for the countries. some of the countries had not the population for the year 2018. therefore, the authors checked for the years 2015, 2016, and 2017. accordingly, the population of 2017 was used for the following country; algeria. the population of 2015 was considered for the following countries: lybia; sierra leone. the population of 2016 was extracted for the following countries: mali; mauritania; papua new guinea; sudan and 2017 for the following countries; bhutan; bosni; burkina faso; fiji; guyana; niger; nigeria; pakistan; uae. the populations of the following countries were not available for the 2015-2019 period. therefore, the population of the following countries was not included in this study based on the eligibility criteria. these countries were the central african republic; djibouti; djibouti; dominica; gabon; kosovo; lebanon; liberia; moldova; russia; saint kitts and nevis; syria; somalia, the democratic republic of the cong. finally, 158 countries/states were included in this study. the general characteristics of the countries were presented in median (interquartile range [iqr], mean (std. deviation), and number (percentage). the confirmed and dead cases were presented in median and interquartile range due to the nonnormal distribution of the data. the normality of the outcomes was examined in drawing a histogram and box plot. the number of confirmed cases was divided by the total population of a country multiplied by 1000,000 to obtain the infection rate of covid-19 per one million persons. the number of dead cases was divided by the total number of confirmed cases and divided by total population multiplied by 1000,000 to obtain the mortality rate/1000,000 persons.. the infection and mortality rates were determined in a median and interquartile range following dealing with the potential outliers. the upper limit values were considered for the extremely higher limit values in the infection and mortality rates. the crude infection rate was categorized into the following groups; 0-500; 500-1000; 1000-1500; 1500-2000; 2000-2500; and > 2500 per one million person. the infection and mortality rates were transformed through the ln technique to obtain a normally distributed histogram. no ethical aspect was applicable to this study. the median crude infection rate by covid-19 was 87.78 (iqr: 468.03) ranged between 0.00 and 24034.51 per 1000,000 persons. the median mortality rate by covid-19 was 0.0015 (iqr: 0.0059) ranged between 0.00 and 1.3728 per 1000,000 persons. the two highest percentage of the crude infection rate were between 0 and 500 (75.9%) and 500-1000 (8.9%) per one million persons ( table 1) . the study showed the crude infection rate was raised with increasing consuming fruits (r=0.416; p<0.001), unprocessed red meats (r=0.457; p<0.001), fruit juices (r=0.390; p<0.001), total protein (r=0.275; p<0.001), calcium (r=0.550; p<0.001), potassium (r=0.371, p<0.001), and total milk (r=0.450; p<0.001). regarding crude mortality rate per 1000,000 persons; the study showed that crude mortality rate was raised with increasing consuming unprocessed red meats (r=0.234; p=0.007), sugar sweetened beverages (r=0.224; p=0.010), fruit juices (r=0.272; p=0.002), calcium (r=0.288; p=0.001), and total milk (r=0.230; p=0.008). however, the mortality rate was decreased following increasing consuming non-starchy vegetables (r=-0.196; p=0.023), see table 2 . the regression analysis showed that the crude infection rate has been increased by raising consuming fruits (beta: 0.237; p=0.006) and calcium (beta: 0.286; p=0.007). however, the infection rate was decreased with rising consuming beans and legumes (beta: -0.145; p=0.038), table 3 . the effect of dietary factors on the crude mortality rate by covid-19 was examined in the regression analysis. the analysis showed that the crude mortality rate was increased by raising consuming sugar-sweetened beverages (beta: 0.340; p<0.001). whereas, the crude mortality rate by covid-19 has been decreased by increasing fruits consuming (beta: -0.226; p=0.047) and beans and legumes (beta: -0.176; p=0.046), as presented in table 4 . the comparison of dietary factors in countries with different infection rates was examined in table 5 and fig 2. the study showed that the countries with higher infection rates between 1500 and above had a higher intake of fruits (p=0.002), fruit juices (p<0.001), calcium (p<0.001), potassium (p<0.001), and total milk (p<0.001). however, these countries had a lower intake of unprocessed red meats (p<0.001) and total protein (p=0.013). the aim of the food-based dietary guidelines is to maintain the general health of the population and prevent non-communicable diseases (14) . most of the food-based dietary guidelines recommend intake of whole grains, fruit and vegetables, low-fat dairy and fish, and low intake of red and processed meat, sugar-sweetened food products, alcohol, and salt (15) . the present study showed that the crude infection rate by covid-19 has been increased by raising consuming fruits, calcium and decreased with increasing consuming beans and legumes. regarding the mortality rate, the analysis showed that the crude mortality rate was increased by raising consuming sugar-sweetened beverages and decreased by increasing fruits consuming and beans and legumes. the anti-inflammatory strategies inside foods, nutrients, or (16, 17) since the coronavirus has serious inflammatory consequences for acute pneumonia in persons (18) . the human coronavirus infections cause mild to severe diseases, systemic inflammation, high fever, cough, and acute respiratory tract infection and dysfunction in internal organs leading to death. this virus is classified as a ribonucleic acid (rna) virus. the virus has a genome that often escapes the innate immune system, particularly if it is malfunctioning (19) . entering coronavirus into the organism activates innate immunity, which intervenes in the first instance to engulf the invader. the severity of the diseases locates within the ability of innate immune cells to stem viral infection (20) . the virus has less ability to replicate itself and induce the pathological state in the case of the stronger innate immune system. when the immune system is suppressed by the virus, the body activates the adaptive immunity. the coronavirus enables to produce viral enzymes and proteases. these enzymes and proteases can damage the immunity and inhibit the signaling pathways of type i interferon (ifn) along with the nuclear factor-κb, facilitating innate immune evasion (21) . apart from the age-related micronutrient inadequacy, the nutritional status of a person has a role in the developing risk of sars-cov-ii infection, the clinical course, and the disease outcomes. hence, the maintenance of host macro-and micronutrient status is considered to be a crucial preventive measure for covid-19 (6) . the coronavirus infection is primarily attacked by immune cells, however, the virus has developed viral proteins overtime that counteracts with the innate immune system (22) . some of the viral proteins antagonize interferon (inf) and stimulate inflammatory proteins, such as il-1 family member cytokines (23) . the inflammatory state and pathogenesis of the disease are escalated after abnormal production of cytokines as shown in sars (24) . our hypothesis is that the higher intake of fruits makes the persons at further risk of infection by the covid-19. despite fruits and vegetables have anti-inflammatory and antioxidant factors and have an important role in enhancing the immune system responses (25) . but higher intake of these micronutrients makes a barrier in improving the human immune system or response to the pathogens due to the role of the fruits with a high glycemic index. our study showed that beans and legumes have a positive role in reducing the infection rate by the covid-19. the human body requires the substates in the plant proteins to improve or respond to the vial pathogens because of the human body unable to produce these substrates (26) . therefore, the body needs these substates to protect the organs against the coronavirus. we assume that the immune body system unable to recognize the virus at the early times. comparison of dietary factors in countries with different infection rates therefore, the available proteins are essential for the body to make a response to the pathogen. the beans and legumes have been effective to reduce the rate of mortality by the covid-19 as well. the role of age in the suppression of the immune system must not be overlooked. the population of the countries with a higher infection rate is older compared to the counters with a low infection rate (27) . for example, france and italy compared to iraq and saudi arabia. the available evidence indicates that adults aged 60 years and older and patients with preexisting medical conditions are more likely to have sever-even deadly-coronavirus infection that other population groups (28) . therefore, we can make the further hypothesis that the aged population of the countries with high infection rates has been the main factor in the low immune system. the impacts of aging on the immune system can reflect at multiple levels. the levels are decreased production of b and t cells in bone marrow and thymus and diminished functions of mature lymphocytes in secondary lymphoid tissues. so, the elderly persons do not respond to immune challenges as robustly as the young individual (29) . the higher intake of fruits and vegetables may not be beneficial to enhance the immune system in aged populations. diet alone may be insufficient and tailored micronutrient supplementation based on specific age-related needs necessary (7) . many micronutrients are required for immune-competence, especially vitamin a, c, d, e, bs, iron, selenium, and zinc. moreover, the dietary pattern is essential to maintain the nutritional status of an individual. however, the diet alone could not be adequate in certain metabolic and lifestyle conditions, such as elderlies, co-existing medical conditions, cigarette smoking, or occupational exposure to environmental toxins (7) . the fruits have several vitamins and minerals. the fruits at a ground level may not be quite suitable to make the final judgment. the older persons over the age of 60-65 experience some immune dysregulation with less ability to respond to immune challenges and response to pathogens, antigens, and mitogens decreases (30) . the decrease in the number of circulating lymphocytes and loss of immune cells are characteristics of the immune system in older people (31) . moreover, the older peoples have reduced the production of t cells in the involved thymus and consequently diminished function of mature lymphocytes in secondary lymphoid tissues (29) . the lifetime of exposure to antigens and to several sources of oxidative stress cause dysregulation in the immune system that makes them at further risk of infections than other age groups (31) . the role of fruits in enhancing immunity, such as micronutrients is in exhibiting pleiotropic roles in supporting immune function. the vitamins and minerals support to develop and maintain the physical barriers, produce and activate antimicrobial proteins (32) . some other mechanisms of micronutrients are supporting the growth, differentiation, and motility/chemotaxis of innate cells; phagocytic and killing activities of neutrophils and macrophages, and promotion of and recovery from inflammation (e.g. cytokine production and antioxidant activity (32) . the potential mechanisms of the fruits may back to the antiviral immune induction, the modulation of immunoregulatory defense, induction of autophagy and apoptosis, genetic or epigenetic regulation (33) . stimulation of defensins and cathelicidins may reduce the replication of the virus and raise the levels of anti-inflammatory cytokines, and reducing levels of pro-inflammatory cytokines (34) . here our hypothesis is that a higher intake of fruits suppresses the role of stimulation of defensins and cathelicidins. the common denominator that reflects the role of nutrition and dietary recommendations against viral infections; including covid-19 is the relation between diet and immunity (35) . this is why we made our hypothesis based on the immunological effects of a higher intake of fruits in patients with covid-19 by taking into account the patients' ages. the evidence highlights that diet has an important effect on the immune system and disease vulnerability of peoples. the role of nutrients or nutrient combinations back to their effects on the immune system through the cell activation, modification in the production of signaling molecules, and gene expression (36) . the relation of fruits and beans and legumes on crude mortality rate is weak (p=0.047 and p=0.046, respectively) in contrast with the strong relation of sugar-sweetened beverages (ssbs) (p<0.001). the possible role of sugar-sweetened beverages on infection rate may back to its role in weight gain and the risk of obesity. a review study of observational and clinical trials showed that a higher intake of ssbs raised the risk of weight gain and obesity (37) . the evidence has been confirmed elsewhere (38, 39) . accordingly, maccioni et al. (40) recruited 1455 individuals aged 18-70 in a cross-sectional study on airway infection in germany. the study reported that obese persons have a consistently higher frequency of upper and lower respiratory tract infections (rtis). the evidence has been reported elsewhere (41, 42) . obesity is responsible for the dysregulation of the immune system through mediation in different immune, metabolic, and thrombogenic responses (43) . the higher intake of ssbs has been reported in high-income countries (44) . the effect of higher calcium intake on raising infection rates could be due to the effect of calcium on the risk of some other chronic diseases rather than its direct effect. a meta-analysis showed the increased incidence of myocardial infarction in persons who consume higher levels of calcium with a pooled relative risk of 1.27, 95% confidence interval 1.01 to 1.59, p=0.038 (45) . in addition, calcium has been reported as a trigger for ischemic cell death (46) . the daily recommended allowance/intake of the dietary factors are different across the countries. it is required to mention that food intake varies markedly based on the sociodemographic factors; like age gender, and educational level. we did not make stratification the results of the study based on the socio-demographic aspects since the who has not published the covid-19 confirmed cases according to age, gender, and educational level. besides, the cross-country caparison of individual-level dietary data is challenged by the dietary surveys performed with various survey characteristics and data collection methods with a possible influence in the comparison of the results. however, we used the fao dietary data that represent the nationally representative sample of all age-sex, and educational level categories. the present study showed the higher intake of fruits and sugar-sweetened beverages had a positive effect on infection and mortally rates by covid-19, respectively. in contrast, the higher intake of beans and legumes had a negative effect on both increasing infection and mortality rates. the possible reason for the role of fruits and sugar-sweetened beverages on infection and mortally rates back to the indirect effect of weight gain and obesity and the role of age. the authors do not declare any conflicts of ineptest. global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2016: a systematic analysis for the global burden of disease study importance of government policies and other influences in transforming global diets global panel on agriculture and food systems for nutrition: food systems and diets: facing the challenges of the 21st century geographic and socioeconomic diversity of food and nutrient intakes: a comparison of four european countries meatless days" or "less but better"? exploring strategies to adapt western meat consumption to health and sustainability challenges 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cardiovascular events: meta-analysis calcium in ischemic cell death key: cord-022399-66mzbynu authors: hopkins, graham; pearson, richard title: basic microbiology date: 2009-05-15 journal: ophthalmic drugs doi: 10.1016/b978-0-7506-8864-2.50005-2 sha: doc_id: 22399 cord_uid: 66mzbynu nan • patients with 'red eye' • contact lens solutions and the claims made for them by manufacturers • preventive measures following contact tonometry and foreign body removal • the constituents of eye drops and the maintenance of sterility. the science of microbiology covers organisms invisible to the naked eye. microorganisms include protozoa, fungi, bacteria, rickettsia, chlamydia and viruses. protozoa and fungi are the only microorganisms that have eukaryotic cells similar in structure to those of higher organisms. such cells have inclusions like nuclei and an endoplasmic reticulum. fungi and protozoa can be either parasitic or free-living. bacteria are simpler cells (prokaryotic cells) but some species are capable of an independent existence in simple environments. however, many bacteria are parasitic or saprophytic and live on the tissues of living or dead organisms. rickettsia and chlamydiae lack many of the structures found in more complicated cells and are thus obligate intracellular parasites. viruses are the simplest microorganisms and can multiply only by utilizing a host cell's biochemical systems. of the above, the bacteria have received most attention. bacteria are important because of their ubiquity -that is, their ability to infect and multiply in varied environments -and the ability of many types of bacteria to cause disease -their pathogenicity. to reduce problems caused by bacteria, it is important to understand something of their structure, growth, environmental and metabolic requirements, classification, relationship with disease and the particular problems they can cause to the eye. the cytoplasm of bacterial (prokaryotic) cells ( fig. 3.1) is notable for the absence of the discrete structures normally found in eukaryotic cells ( fig. 3 enzymes are instead located on the cell membrane. there is also no endoplasmic reticulum or golgi apparatus; the ribosomes are found free in the cytoplasm. there is no nucleus and no nuclear membrane, and when the cell divides there is no mitosis. genetic material is carried on a single strand of dna, unlike the genetic material of eukaryotic cells. which is organized into chromosomes. some bacteria contain additional dna molecules called plasmids, which are not essential for the organism's life but can confer on it very special properties such as resistance to antibiotics. plasmids are most commonly found in gramnegative bacteria and can spread from one cell to another. surrounding the bacterial cell cytoplasm is a thin, selectively permeable, elastic, lipoprotein membrane -the plasma membrane. it is the site of action of many of the bacterial enzymes and controls entry of substances into the cell. being elastic, the cell membrane does not determine the shape of the bacterial cell. this is the function of the cell wall, a rigid, permeable structure principally composed of a substance called murein. because of the osmotic pressure of the cytoplasm, the cell membrane is usually pushed hard against the inside of the cell wall by a pressure of up to 20 atmospheres. the cell wall is relatively thick, especially in gram-positive bacteria, and is chemically unrelated to the cell walls of higher plants. in some bacteria, the cell wall is surrounded by a layer of extracellular material referred to as the capsule (if it is closely associated with the cell wall) or slime layer (if the relationship is looser). this is a poorly organized layer of large molecules such as polysaccharides or polypeptides. the effect of this layer is to impede the ingress of substances (useful and harmful) into the cell wall. the result is that the cells tend to grow and divide at a slower rate but are more resistant to antibacterial chemicals, viruses (bacteriophages), phagocytes and other adverse agents. this layer might also inhibit antibody formation against the bacteria, thereby rendering the bacteria more harmful to the body. additionally, it can act to form an adhesion between the bacterial cell and a host cell, which also increases its virulence. one of the best known bacteria to form a capsule is mycobacterium tuberculosum, the causative organism of tuberculosis. flagella are found on the outside of some types of bacteria; the number of flagella per cell is constant for each species. flagella are long, filamentous structures containing a contractile protein, flagellin, which is similar to muscle myosin. the presence of flagella normally confers the ability for motion that, it is assumed, allows the bacterium to migrate to more favourable environments. some bacteria have the ability to move without possessing flagella. these are the spiral forms, which move by twisting the whole body. also projecting from the surface of some bacteria are fimbriae, formed of protein, which instead of facilitating motion act to hold the bacterial cell to a host cell or another bacterial cell. the fimbriae are very specific for the molecule to which they attach and fimbriated bacteria are found to be more virulent than those that lack these appendages. bacteria are ubiquitous and can exist in many environments that are far too hostile for the cells of higher organisms. more fastidious bacteria have requirements closer to those of the internal environment of animals and hence are more likely to be parasitic and pathogenic. nutritional all organisms have a requirement for carbon, hydrogen, oxygen and requirements nitrogen. as hydrogen and oxygen can be obtained from water, it is the requirement for the other elements -nitrogen and carbon -that is most critical. some bacterial species can obtain their nutrient requirements from inorganic sources. they obtain energy from other sources, e.g. bacterial chlorophyll allows some bacteria to use sunlight as a source of energy and synthesize simple organic compounds. others can utilize inorganic nitrogen, providing they are supplied with an organic source of carbon. such organisms are found in soil and are responsible for maintaining its fertility. yet others require both organic carbon and nitrogen to survive. pathogenic bacteria need other complicated growth factors and minerals. oxygen requirements although oxygen can be obtained from water, some types of bacteria need atmospheric oxygen and cannot exist without it. these bacteria are termed obligate aerobes. others are the exact opposite and cannot exist in the presence of oxygen, requiring anaerobic situations (obligate anaerobes). the majority, however, are facultative anaerobes, which means they can exist in either the absence or presence of oxygen. physical conditions different bacteria can exist at both high and low extremes of both ph and temperature. pathogens prefer the medium state of ph 7 and 37°c. acidophilic bacteria prefer a low ph, whereas basophiles like a high one. thermophilic bacteria grow best at between 55 and 80°c, whereas the spores of bacillus stearothermophilus can withstand boiling. some organisms can exist at very low temperatures. growth bacteria reproduce by binary fission. the cell divides and two equal daughter cells are formed. as there is no nucleus there is no mitosis. the time between a daughter cell being formed and itself dividing to form two new cells is called the generation time and varies greatly between species. it also varies with environmental conditions and the supply of nutrients. some bacteria multiply very quickly and divide every 20 minutes. others, like m. tuberculosus, take hours or even days. when a new sterile environment with finite limits is colonized, the bacterial cell population goes through four phases (fig. 3.3 ). • lag phase: the original innoculum remains dormant and no increase in number is seen. • log phase: there is an exponential growth in the number of organisms and the logarithm of the number of cells is directly proportional to time. it is during the early stages of this phase that the bacterial population is most susceptible to antibacterial agents. • stationary phase: the number of viable organisms remains constant because the number of new organisms is equal to the number dying. this phase can be brought about by a depletion of essential nutrients, a change in the oxygen level or an accumulation of metabolites which regulate the growth. • decline phase: the number of viable organisms declines. sporulation certain bacteria can produce endospores. these compact masses, which have a very resistant coat, are formed inside the vegetative cell. once formed, the rest of the cell disintegrates, releasing the spore. spores can withstand adverse environments that would be lethal to the vegetative cell. when the conditions are right, the spore germinates into one vegetative cell (sporulation is not a form of multiplication). once a pure colony of an organism has been isolated by successive culturing, it is often necessary to find out which organism is present. not only genus and species require elucidation but also the particular strain. to elicit this information the following techniques can be used: • microscopy and staining • differential media and biochemical tests • serological testing • bacteriophage typing. microscopy and gram's stain divides bacteria into gram-positive and gram-negative differential staining bacteria. bacteria are fixed onto a microscopy slide and stained with a dark purple stain. the slide is then covered with an iodine solution that acts as a mordant, i.e. fixes the stain onto the organisms. the next step is the decolourizing process, in which the slide is treated with a solvent. a counterstain completes the process and the slide is viewed under the microscope. if the organism has resisted decolourization it is termed gram positive and will appear purple under the microscope. if the original stain has been lost, the colour of the counterstain will show through and the organism will be deemed gram negative. this is a fundamental method of classifying bacteria. other differential stains have been used. for example, acid-fast staining, in which the organisms are subjected to a decolourizing process using acid. specific stains can be used to show the presence of sporeforming bacteria. examination under the microscope not only gives information about the organisms' staining characteristics but, of course, about the shape. basically, bacteria can be spherical (cocci), rod-shaped (bacilli) or spiral. cocci can be divided according to their form of aggregation. some bacteria appear in just one direction and form chains (streptococci) whereas others give the appearance of a bunch of grapes (staphylococci). however, the appearance of aggregations under the microscope can sometimes be deceptive and other tests are necessary to differentiate between streptococci and staphylococci. differential media and special media that support the growth of some types of bacteria and biochemical tests not others can be useful in bacterial typing. other tests examine the organism's ability to break down hydrogen peroxide, to liquefy protein and to ferment certain sugars. media containing blood are useful in differentiating streptococci. serological testing bacteria possess many potentially antigenic substances and one of the body's defences against bacterial invasion is to produce antibodies to these antigens. these antibodies are specific to the antigens and this specificity can assist in the determination not only of the genus and species but also the strain of bacteria present. bacteriophage typing bacteriophages are viruses that attack bacteria. they invade the bacterial cell just like any other host cell. once inside they combine with the bacterial dna and change the genetic material. this effect can be destructive and the whole cell is taken over, producing new phage particles. bacteriophages are species specific to the bacterium they invade. viruses ( fig. 3 .4) are much smaller than bacteria (18-300 nm). whereas all known bacteria will be trapped by a 0.22 μm filter (sterilizing filter), many viruses will pass through, hence the term 'filtrable viruses'. viruses can infect any form of higher organism and are usually divided into animal viruses, plant viruses and bacteriophages. viruses consist of either rna or dna (never both) surrounded by a layer of protein (capsid) or a membrane (referred to as the envelope). the outer covering of the virus particle plays a vital role in the initial infection of the cell and the spread of a virus within the host. the components of the covering layer are very antigenic and are involved in the host's immune response. capsids are rigid structures and tend to be very protective against adverse environments such as desiccation and detergents. such viruses tend to retain their infectivity on fomites and will withstand the adverse conditions in the gut, i.e. low ph and the presence of proteases. envelopes are less protective and can be disrupted by acids, detergents and drying out. thus these virus particles must remain in aqueous solution to survive and must be transmitted by droplet infection, blood or other body fluids; they will not survive the gut. the nucleic acid may be single strand or double strand. viruses contain few if any enzymes and are entirely reliant on the host cell to bring about replication; they are obligate intracellular parasites. they vary greatly in size and in the number of genes they carry (from three to several hundred). virus reproduction does not take place by binary fission but, generally, by the following pattern: • the virus particle (virion) becomes attached to the surface of the cell. specific receptors are usually involved, which results in viral preference for certain cells within the host. for example, the aids (hiv) virus binds to cd4 receptors, which are found on t cells. • the virus particle passes into the cell. • the virus particle becomes uncoated, releasing the viral genetic material into the host cytoplasm if it is rna. the genetic material of dna viruses must be delivered into the nucleus. • the viral genetic material induces the cell to produce different macromolecules that are essential for the production of new viral particles. • assembly of new virus particles takes place within the host cell; these are then released. the release can bring about disruption of the host cell. the new virus particles are available to infect new cells. the classification of viruses is more difficult than that of bacteria. many criteria are used in classifying viruses. • morphology • nucleic acid type • immunological properties • transmission methods • host and cell tropisms • symptomatology and pathology. the following are the main groups of animal viruses: these organisms are more complex than viruses but less complex than bacteria. they can multiply only in susceptible cells and, unlike viruses, contain both dna and rna. they multiply by binary fission and are susceptible to certain antibiotics. chlamydiae exist in two forms: (1) as an elementary body (300 nm) that can exist outside the host body and is the infectious unit; and (2) as a reticulate body (1000 nm), which exists only inside the host cell and is not infectious. chlamydiae attack mucous membranes and inhibit host cell protein synthesis. they rely on the host to provide atp, which they cannot generate. they synthesize their own nucleic acids and proteins. there are two species, chlamydia trachomatis (which causes trachoma, inclusion conjunctivitis) and chlamydia psittaci (which causes psittacosis). as causative organisms of disease, fungi are less important than bacteria and viruses. of the tens of thousands of species of fungus, probably only about 100 are pathogenic. some of these are capable of producing very severe infections (e.g. filamentous keratitis), others result in more trivial infections (e.g. athlete's foot). fungi can colonize non-living structures and lead to spoilation, e.g. hydrogel contact lenses. fungi are composed of eukaryotic cells with the normal inclusions, e.g. a nucleus and mitochondria. they can be divided into four groups according to their structure: • moulds: these grow as a mycelium, which is composed of filamentous multicellular structures called hyphae. the mycelium is divided into the vegetative mycelium, which grows into the substrate and assimilates nutrients, and the aerial mycelium, which produces the spores either asexually by budding or sexually by the fusion of two cells. • yeasts: these fungi occur as single cells and reproduce by budding. • yeast-like fungi: both hyphae and yeast cells exist together. • dimorphic fungi: these fungi exist either as yeasts or as filaments. if they are grown on artificial media they appear as hyphae; when they inhabit a living host they occur as yeast cells. protozoal parasites are best known for their ability to colonize and, in some cases, parasitize the alimentary tract, particularly in hosts in tropical countries. however, protozoal parasites are capable of living in other parts of the body and of causing serious pathological conditions. the most important of these are the various forms of amoebae, which are primitive acellular organisms with a simple life cycle. they exist in two forms: • the active trophozite, which moves around its environment using pseudopodia (the cytoplasm extends in one direction and the rest of the organism is drawn along to follow it) • the dormant cyst, which -like the spores of bacteria -is more resistant than the active trophozite to unfavourable environments. reproduction is either by simple binary fission of the active trophozite or the formation of multinucleated cysts. some amoebae are free living whereas others, such as entamoeba coli and entamoeba gingivalis, inhabit parts of the alimentary tract as commensals. others, such as entamoeba hystolytica (the causative organism of ameobic dysentery), are potent pathogens. naegleria fowleri causes a rapidly fatal (4 to 5 days) meningoencephalitis. acanthamoeba is an important pathogen in causing keratitis, especially in wearers of hydrogel contact lenses (see chapter 16). humans play host to a large number of bacteria that normally do no harm and, to varying degrees, contribute to the body's well-being. for example, bacteria live on the dead surface of the skin and prevent other more potentially dangerous organisms from occupying the site. bacteria that inhabit the gut provide the body with the vitamin k that is essential for the production of prothrombin, in exchange for nutrients. these commensal organisms only maintain this mutually advantageous relationship providing they remain in their proper place. one tissue's commensal is another tissue's pathogen. it is not always the microorganism itself that causes the harm, but substances that the organism produces. some bacteria live and multiply in food, producing toxins as they do so. when the food is ingested, the toxins produce adverse effects. clostridium botulinum is such an organism and the toxin it produces, botulinum toxin, is fatal in minute quantities. staphylococcus aureus can also bring about food intoxication. most microorganisms, however, cause disease by acting as parasites on the body, gaining access by a variety of routes: • direct contact: this normally means sexual contact; the disease is classed as a venereal disease. this method of transmission favours the very fastidious bacteria that exist only with difficulty outside the human body. treponema pallidum (which causes syphilis) is so fastidious that it has never been grown on lifeless media. • indirect transmission: infection is passed from one person to another by an inanimate object (called a fomite), e.g. bedclothes, used dressings. • dust-borne infection: dust contains discarded human cells and dried water droplets. these are likely to carry bacteria, especially spores. • droplet infection: bacteria are present in the fine spray that is exhaled with forced expirations such as coughing and sneezing. • water-borne infection: water is an excellent medium for transmitting infection. public health and sanitation prevent this until some disaster interrupts the supply of clean drinking water. • insect-borne infection: biting and sucking insects can carry organisms from an infected host and transfer them to a new one. • maternal transmission: infections can be passed from mother to child, either while the child is still in the womb or during birth (e.g. ophthalmia neonatorum). the eye is at risk from infection by opportunistic and invasive organisms via a variety of routes. in addition to congenital ocular infections, microorganisms can gain access as a result of: • direct contact, e.g. herpes simplex • airborne infections • insect-borne infections, e.g. trachoma • migration of bacteria from the nasopharynx • metastatic infection from other loci in the body • trauma, especially penetrating injuries • infected contact lenses • infected eyedrops and lotions • infected instruments. the lids, cornea and conjunctiva, being the most exposed parts of the eye, are at most risk from infection. infections of these tissues are far more common than those of deeper tissues but they are also less serious. the eye is covered with tears, which contain a number of antimicrobial agents to reduce the incidence of infections. the result is a fairly low level of microbial contamination in the fornices. tears contain immunoglobulins a, g and m (iga, igg and igm; reim 1983) in different proportions to those found in plasma, suggesting that their origin is secretory rather than just the result of filtration. tears contain two other agents with marked antibacterial properties: lysozyme and beta-lysin. lysozyme is an enzyme capable of dissolving the cell wall of bacteria, especially gram-positive bacteria. the level of lysozyme decreases with age and is reduced in patients with dry eye syndrome (mackie & seal 1976) . beta-lysin acts principally on the cell membrane (ford et al 1976) and works in concert with lysozyme. as the cell membrane is the site of action of the bacterial enzymes, the effect is quite marked. beta-lysin is also present in aqueous humour. staphylococci some staphylococci are normal inhabitants of the skin and mucous membranes, whereas other species are capable of producing conditions such as boils, abscesses and even a fatal septicaemia. others can cause food poisoning by the liberation of an enterotoxin. resistance to certain antibiotics develops easily; the term 'hospital staph' used to be applied to some resistant forms -the modern term is mrsa (methicillin-resistant staphylococcus aureus). staphylococci are differentiated from streptococci by the presence of an enzyme that breaks down hydrogen peroxide (catalase). pathogenic staphylococci possess coagulase, which clots blood plasma. in the eye, staphylococci can cause infections of the lids, lacrimal apparatus, conjunctiva and cornea (davis et al 1978) . infections of the lash follicle can result in the formation of a stye (hordeolum). staphylococci can also produce acute or chronic blepharitis, which is sometimes associated with acute conjunctivitis (brook 1980; brook et al 1979; brown 1978) . staphylococci were also found to be present in a large number of cases of ophthalmia neonatorum in one study (jarvis et al 1987) . the routine use of prophylactic eye ointments to prevent ophthalmia neonatorum has led to the emergence of resistant strains. hedberg et al (1990) reported an outbreak of erythromycin-resistant staphylococcal conjunctivitis in a newborn nursery in which erythromycin eye ointment was used as a prophylactic agent. following septicaemia, staphylococci have caused endophthalmitis (bloomfield et al 1978) . because staph. aureus is so common, it is often employed in the efficiency testing of preservative systems. staphylococcus epidermis is normally considered to be a commensal and is a normal inhabitant of the skin. unlike staph. aureus, it produces white colonies. maske et al (1986) found a higher than normal incidence in a group of patients with bacterial corneal ulcers. it has been suggested that staph. epidermis releases a toxin to cause some of the signs of blepharitis and keratopathy (mcgill et al 1982) . streptococci streptococci lack the enzyme catalase and are characterized by their ability to cause haemolysis complete haemolysis is brought about by beta-haemolytic streptococci; the haemolysis produced by alphahaemolytic species is incomplete and leads to the formation of a green pigment. there are also non-haemolytic streptococci. streptococci can produce local and general infections. one of the most common local infections of beta-haemolytic streptococci is the streptococcal sore throat, which in young children can extend into the middle ear to cause otitis media. on the skin they can cause impetigo. betahaemolytic streptococci infections give rise to puerperal fever, wound sepsis and endocarditis. it is fortunate that penicillin continues to be effective against many strains of streptococci. in the eye, streptococci can cause conjunctivitis, dacryoadenitis, dacryocystitis and blepharitis (brook 1980) . jones et al (1988) reported corneal ulcers, endophthalmitis, conjunctivitis and dacryocystitis resulting from streptococcal infections. the ability of streptococci to cause sightthreatening infections is of concern because many strains are not susceptible to gentamicin, an antibiotic often chosen to treat such infections. the neisseriae is a group of gram-negative bacteria that include the normal flora of the respiratory system and the pathogens which cause meningitis (neisseria meningitidis), and gonorrhoea (neisseria gonorrhoeae). in the eye, neisseriae species can infect the lids, lacrimal apparatus and conjunctiva and n. gonorrhoea is best known as the one-time principal cause of ophthalmia neonatorum, an infection that occurs as the infant passes down the birth canal. the disease becomes manifest between the second and fifth day after birth, when the lids become swollen and there is a bilateral purulent discharge. the lids are tightly closed and difficult to open and the acute phase lasts for 4-6 weeks. the condition is treated with topical and systemic antibacterials. if treatment is not carried out, the cornea can become involved and the eye lost. however, other causative organisms, such as chlamydia species, and other causes (paradoxically, the overenthusiastic use of silver nitrate) are more important today (jarvis et al 1987) . corynebacterium corynebacteria are non-motile gram-positive rods; they do not form diphtheriae spores. some species are normally resident in the human respiratory tract. corynebacterium diphtheriae, when infected with the appropriate bacteriophage, produces a powerful exotoxin that causes diphtheria. this disease results in the growth of a membrane across the throat, leading to suffocation. it can similarly affect the eyelids, with the appearance of such membranes on the inner surface of the lids. the conjunctiva can become involved in the same way. diphtheroids have been isolated in a proportion of infected conjunctivae (brooke et al 1979; brown 1978 ). clostridium species the clostridia are a group of obligate anaerobes notorious for their pathogenicity. in particular, they include clostridium botulinum, which, when it infects food, produces botulinum toxin. although botulinum toxin ingestion is potentially fatal, this substance has been used to paralyse the antagonist muscles in cases of paralytic strabismus (elston & lee 1985) and other ocular disorders (alpar 1987 ). clostridium tetani is a possible infectant of deep wounds and prophylaxis against the effects of its toxin is routine. other clostridia, such as clostridium perfringens, clostridium welchii and clostridium oedematiens cause gangrene. gas gangrene of the lids has been reported (crock et al 1985) . this is by far the biggest group of pathogens, most of which are facultative anaerobes. pseudomonas pseudomonas aeruginosa is perhaps the most notorious of bacteria for aeruginosa causing ocular problems and is normally found in small numbers in the gut and on the skin. it is a common contaminant of water and has been cultured from jacuzzis (brett 1985) . its numbers are kept in check by the presence of other organisms but as it is resistant to many antibiotics it can gain dominance if the surrounding organisms are suppressed. ps. aeruginosa produces a bluish green colour when grown on media. it has a characteristic odour and is pyogenic, the presence of green pus suggesting the presence of a pseudomonal infection. ps. aeruginosa is an opportunistic organism and is normally kept at bay by the body's defence mechanisms. if these are breached, a serious infection often results. ps. aeruginosa can infect burns, especially if of large area, and can also gain hold in patients who are immunecompromised. ps. aeruginosa is an extremely versatile organism in that it can metabolize fluorescein and hydroxybenzoates as carbon sources for energy, which means that it can survive in conditions that are alien to most other organisms. the organism is susceptible to antibiotics such as gentamicin and polymixin. in the eye, ps. aeruginosa can produce meibomitis, conjunctivitis and corneal ulcers and is one of the causes of ophthalmia neonatorum (cole et al 1980) . should access be gained to the sterile interior of the eye, then panophthalmitis might result and, indeed, has been responsible for causing more than one serious case of hospital acquired disease leading to the loss of an eye (crompton 1978) . ps. aeruginosa is an important test organism for contact lens solutions and eyedrop preservative systems, not only for its virulence when an infection is established but also because of its biochemical versatility, which sometimes makes it difficult to eradicate. haemophilus species these small, aerobic organisms get their name from their requirement for enriched media containing blood for culturing in vitro. they include certain important human pathogenic organisms. haemophilus influenzae is a secondary invader, which helps to produce some of the symptoms of influenza and can produce inflammation in most parts of the respiratory tract. bordetella pertussis is another member of this group that affects the respiratory system, causing whooping cough, which is transmitted by airborne infections from one person to another. b. pertussis cannot exist for long periods outside the body. similarly, haemophilus ducreyi is so fastidious in its requirements that it can only be transmitted sexually and, consequently, is the causative organism of chancroid, a form of venereal disease. h. influenzae and h. ducreyi can infect ocular tissues. two members of this group, however, are particularly noted for their ability to cause conjunctivitis: haemophilus aegyptius (haemophilus conjunctivitidis, koch-weeks bacillus) is often the cause of acute epidemic conjunctivitis, especially in school children and moraxella lacunata (morax-axenfeld bacillus) is another well-known causative organism of conjunctivitis. the most important members of the herpes group as far as the eye is concerned are herpes zoster, herpes simplex and cytomegalovirus. herpes zoster (varicella) virus results in chickenpox in children. this is a mild, highly contagious disease characterized by a vesicular rash. the disease leaves the patient with a continuing immunity to the disease. in the adult, a reactivation of the virus leads to shingles, in which an area of the body becomes covered with a painful rash. evidently the virus is stored in a sensory ganglion, the shingles attack being caused by a migration of the virus along the nerve root. when the nerve affected is the ophthalmic division of the trigeminal nerve, the area served by it exhibits signs, i.e. the eye, the orbit and surrounding areas. this is known as herpes zoster ophthalmicus, in which the cornea becomes inflamed and oedematous, and sensitivity may be impaired permanently. secondary infection can occur, leading to ulceration and scarring. herpes simplex can be differentiated into types i and ii. type ii is associated with genital herpes and neonatal herpes. transplacental infection with type ii virus has led to the development of neonatal cataract (cibis & burge 1971) . type i causes cold sores, inflammation of the oral cavity, encephalitis and dendritic ulcers. dendritic ulcers are so called because of their branching pattern. as the ulcer extends it might lose this appearance and become amoeboid or geographic. the patient complains of pain, photophobia, blurring of vision and a watery discharge (unlike that from bacterial conjunctivitis). in the early stages, infection affects only the epithelium, later progressing to the superficial stroma. the cornea becomes oedematous and there is further loss of stroma and possible vascularization. the condition is treated with intense local antiviral therapy. herpes simplex can also produce a keratoconjunctivitis similar to that caused by adenovirus 8 (darougar et al 1978) (see below). the third virus in this group is cytomegalovirus, which normally inhabits the female reproductive tract giving rise to congenital infections. congenital infections can give rise to chorioretinitis, optic atrophy and cataract. adenovirus 8 adenovirus 8 gives rise to epidemic keratoconjunctivitis sometimes called 'eye hospital eye' because of its possible transmission by contaminated instruments. the infection produces severe acute conjunctivitis, which can spread, leading to keratitis. marked discomfort can last for months. adenovirus was the cause of 8% of cases of acute conjunctivitis in one study (wishart et al 1984) . pox viruses this group of viruses includes smallpox and cowpox. a relatively uncommon skin condition affecting young adults and childrenmolluscum contagiosum -is caused by a pox virus. transparent nodules (2-3 mm in diameter) appear on the skin of the arm, legs, back and face, with possible involvement of the lid margins and conjunctiva. this condition is sometimes seen in patients with aids and aids-related complex (arc). the best known member of this group is rubella (german measles), which can be passed from mother to baby in the uterus, leading to congenital defects in 30% of the children of mothers suffering rubella in the first trimester of pregnancy. particularly affected are the heart, ears and eyes. ophthalmic defects lead to microphthalmia, cataracts and congenital glaucoma. retroviruses the human immunodeficiency virus (hiv) is present in many of the body fluids of affected individuals, including tears. although there has been no recorded case of transmission via infected contact lenses, it has become a point of concern of contact lens practitioners. the virus has also been recovered from contact lenses worn by patients with aids and arc (aids-related complex; tervo et al 1986) . aids can have certain ocular manifestations, partially because the patients are, from the very nature of the disease, more likely to develop opportunistic infections such as cytomegalovirus retinitis. conjunctival kaposi's sarcoma is another ocular complication (kanski 1987) . this dimorphic opportunistic fungus is normally found in the mucous membranes of the mouth, vagina, gut and eye (liotet et al 1980) . it causes oral thrush in newborn infants and terminally ill patients. in the eye it can cause corneal ulcers, conjunctivitis and severe uveitis. aspergillus niger this fungus, which is not dimorphic, grows in the form of mycelia. often found in vegetable matter, it can cause bronchial problems; it can also produce severe local infection in the eye, especially after injudicious use of local corticosteroids, which tend to mask the clinical signs of the infection, allowing it to get a stronger foothold. a case of aspergillus panophthalmitis has been reported in a patient after excision of a pterygium who received beta radiation treatment (margo et al 1988) . aspergillus niger, which also can be found in the healthy eye (liotet et al 1980) , can infect hydrogel contact lenses and destroy them. other species of aspergillus have been implicated in contact lens contamination. for example, yamaguchi (1984) reported growth on a contact lens of aspergillus flavus and filppi et al (1973) found that aspergillus fumagatus penetrated hydrogel contact lenses. aspergillus species are not the only ones to infect contact lenses. yamamoto et al (1979) found cephalosporium acremonium growing on a contact lens worn for the treatment of metaherpetic keratitis. trachomatis) endemic in many areas. where it is endemic, it affects over 90% of the population. associated with poor living conditions, this organism is passed on by insects and contaminated objects such as bedclothes (fomites). it is sometimes a resident of the female genital tract (barton et al 1985) and can produce a form of ophthalmia neonatorum (markham 1979) . the incidence of chlamydial ophthalmia neonatorum varies. in one study in america, 1.4% of all newborn babies acquired chlamydial conjunctivitis (schacter et al 1979) and similar findings were reported in sweden (persson et al 1983) and wolverhampton, uk (preece et al 1989) . the last report concluded that screening for chlamydial antigen was not justified as the condition could be easily treated with oral erythromycin. the condition starts as a mild inflammation of the conjunctiva, with the development of small follicles, which become larger. the cornea is invaded and vascularized, resulting in pannus, which can lead to severe scarring and contraction, which causes deformity of the eyelids. symblepharon and trichiasis are also seen. in temperate climates, the organism results in inclusion conjunctivitis. acanthamoebae like naegleria can infect the brain, producing a form of amoebic encephalitis (ma et al 1990) . amoebic keratitis has been reported as a result of infection with acanthamoebae species (moore et al 1986) . the patients in the report were myopes corrected with hydrogel contact lenses who had used salt tablets dissolved in distilled water during disinfection procedures. this infection has so far proved to be difficult to treat. many 'agents' have the ability to kill or inactivate microorganisms. within this broad term we encompass the body's defence mechanisms, i.e. the white blood cells and circulating antibodies of the blood, the gastric hydrochloric acid and the lyzosyme and beta-lysin of tears; other microorganisms, such as bacteriophages, must also be considered as antimicrobial agents. here we are concerned with three basic groups: • physical agents capable of rendering objects and chemicals free of contamination • antimicrobial preservatives that are incorporated into solutions to maintain sterility • chemotherapeutic agents used either to treat or prevent an infection in the body. it is important to define certain terms that are relevant to this subject and are sometimes used incorrectly: • sterilization: the killing or removal of all viable organisms (including bacterial spores) from an object or pharmaceutical product by the use of chemical or physical agents. • disinfection: a lesser process than sterilization by which the capacity of an object to cause infection is removed. a disinfected product might not be sterile. • antisepsis: a similar degree of decontamination as disinfection but referring to solutions and chemicals that are safe to apply to surfaces of the body. • chemotherapeutic agents: described as bactericidal or bacteriostatic; the former are actually capable of killing the bacteria (although not necessarily bacterial spores) whereas bacteriostatic agents prevent bacteria from growing and rely on the body's own defence mechanism to get rid of the organisms. all physical agents are forms of energy and the antimicrobial action is dependent on supplying sufficient to cause disruption to the cell. bacteria and other microorganisms are far more resistant to adverse situations than animal cells and can withstand environments that would be quickly lethal to us. the effect of antimicrobial agents follows a firstorder reaction in which the log number of survivors is inversely proportional to the time. the time for 1 log cycle reduction, i.e. the time taken for 90% of the bacteria to be killed, is called the d value or decimal death time, this value reducing as the antimicrobial effect of the antimicrobial increases. heat heat is one of the best-known disinfecting and sterilizing agents. it is used for sterilizing solutions (providing that the substances are thermostable), dressings and some instruments. the effectiveness of heat is increased by the presence of water, especially if the ph is raised, the use of moist heat bringing hydrolysis to bear on the organisms as well as pyrolysis. temperatures of around 60°c will kill most viruses, as well as the vegetative cells of pathogenic bacteria and fungi, whereas boiling brings about the demise of spores of pathogenic bacteria. however, there are organisms whose spores will withstand boiling for long periods. therefore, to obtain sterility without compromising the product, autoclaves are used. these work on the 'pressure cooker' principle by heating the product in steam (not air) to a defined temperature and specified time, which is usually 121°c for 15 minutes. the use of dry heat is far less efficient and temperatures of up to 160°c for 1 hour are needed to kill spores. disinfection, as opposed to sterilization, can be brought about by boiling for 10-15 minutes. temperatures below boiling can reduce the number of microorganisms present and are used for materials that cannot withstand heating at high temperatures; milk is pasteurized at 60-70°c, for example. the temperature inside a soft lens storage subjected to heating by steam will not reach boiling but the temperature attained (95°c) is very bactericidal. thermal disinfection of contact lenses is discussed in chapter 11. freezing freezing cultures of bacteria will markedly reduce the number of bacteria, as a proportion will be damaged by the formation of ice crystals. however, the rest will survive in a dormant state even at temperatures as low as that of liquid nitrogen. indeed, this process is used to store cultures of bacteria. ionizing radiation this technique is used for disposable plastic items and for paper products such as fluorescein-impregnated strips. all types of radiation are lethal to microorganisms (alpha, beta and gamma rays). usually it is gamma rays that are used at a dosage of 2.5-3.5 mrad. ultraviolet radiation light is only bactericidal at low wavelengths (240-280 nm -the uvc region) and at this level does not penetrate well, making it suitable only for surface and aerial disinfection. filtration solutions of thermolabile drugs can be sterilized by passing the solution through a 0.22 μm filter, which retains all bacteria (the smallest bacterium is about 0-5 μm). the filters, which are sterilized before use, will not remove viral contamination. ultrasonics sound will kill bacteria but high power inputs are required. ultrasonic cleaners with antibacterial cleaners have been used on contact lenses (see chapter 11). a whole range of substances is incorporated into products to prevent the growth of microorganisms. these are used in foods and drinks and cosmetics but are most important in multidose sterile pharmaceutical solutions to ensure that the product is protected from microbial attack while it is in use. these compounds are selected for their ability to kill or inhibit the growth of microorganisms, particularly bacteria and fungi. the rate of kill represented by the d value depends on the concentration of the preservative, but is not always a simple inverse relationship, i.e. with the d value inversely proportional to the concentration of the preservative compound. with some compounds the effect is exponential, with a reduction of concentration to half of the original leading to an increase in d value of a factor of 2 8 or 256. such compounds are thus quickly inactivated by dilution. these agents can damage human cells and it is necessary to use them in as low a concentration as possible to reduce toxicity, the final concentration therefore representing a compromise between safety and efficacy. to achieve greater efficacy without increasing the toxic effects, mixtures of preservatives are often used. many such agents have been used in the past; the following are those in common use. benzalkonium chloride benzalkonium chloride (bak) has a detergent action that disrupts the cell membrane; it is by far the most commonly used preservative for eye drops. benzalkonium chloride has a disruptive effect on the tear film when used in concentrations of 0.01% and greater (wilson et al 1975) . it is often found combined with ethylene diamine tetra-acetic acid sodium edentate (edta), which is a chelating agent that combines with divalent ions (normally calcium) to form a non-ionizable complex. edta has a slight antibacterial action of its own but is principally used to enhance the bactericidal action of benzalkonium chloride. they produce mercury ions that react with sulphydryl groups of essential enzymes. they are slower in action against certain organisms but are less quickly inactivated by dilution than other compounds. unlike benzalkonium chloride, mercury compounds are not potentiated by the addition of edta (richards & reary 1972) . in fact, morton (1985) found that edta actually reduces the antimicrobial efficacy of thiomersal. significant penetration of mercury-containing compounds into the aqueous humour following their use has been recorded by winder et al (1980) . these compounds have been demonstrated to have cytotoxic effects that are time and concentration dependent (takahashi 1982) but that are less than those of benzalkonium chloride (gasset et al 1974) , and their use in many contact lens solutions has led to the increasing incidence of allergic reactions (gold 1983 ). chlorhexidine gluconate chlorhexidine is a useful alternative to benzalkonium chloride and is used when the latter is incompatible with the active ingredient. it is very toxic to the corneal endothelium in concentrations of 20 μg/ml and, if the epithelium is perfused, the result is a sloughing of the cells without corneal swelling (green et al 1980) . oxidizing agents strong oxidizing agents are very bactericidal. probably the best known is hydrogen peroxide, which kills most vegetative forms at a concentration of 3-6%; stronger concentrations ( > 10%) will dispose of spores. the halogens are also strong oxidizing agents and part of the action of iodine-and chlorine-based disinfectant systems is the oxidation of essential enzymes. although iodine in simple solution is still occasionally used it is most often complexed with another compound to form an iodophor. iodine can be complexed with poly-vinylpyrrolidine to form povidone iodine. other compounds other compounds that have been used include chlorbutol, cetrimide, phenylethanol, hydroxybenzoates and chlorocresol. the treatment of infections has evolved somewhat since the treatment of syphilis with mercurial compounds. developments have led to the introduction of agents that are more effective against the infecting organism and less toxic to the host. anti-infectives tend to be specific against groups of organisms, e.g. antibacterials and antifungals, although there is some overlap. certain antibacterials are effective against chlamydiae. the mode of action of antibacterial agents varies greatly. to produce their desired effect without producing a toxic reaction from the patient they must interfere with some specific function important to the parasitizing cells. these include: inhibition of the many of the common antibiotics produce their activity by interfering formation of with the formation of cell walls. penicillin and the other beta-lactam the cell wall antibiotics, such as the cephalosporins, work in this manner, as do vancomycin and bacitracin. the antibiotic molecules combine with enzymes responsible for the synthesis of the cell wall, preventing new wall being laid down and the destruction of existing material. in the normal bacterial cell the membrane is pushed against the cell wall by osmotic pressure. without this constraint water balance is not maintained and cell death occurs. these antibiotics are most effective against populations which are actively growing as this is the stage of maximum cell wall production. such bacteria are normally bactericidal. the cell membrane of the prokaryotic cell is even more important than the cell membrane that of the eukaryotic cell because the former lacks mitochondria and so respiratory membranes are located on the membrane. any disruption of the membrane, as well as interfering with the transport of substances into and out of the cell, will have an effect on cell respiration. inhibition of protein many of the more modern antibiotic groups can be found under this synthesis heading -the tetracyclines, aminoglycosides, macrolides chloramphenicol and clindamycin. their site of action is the ribosomes, where they either bind directly or prevent the binding of trna. many of these antibiotics are bacteriostatic. the most important group in this section are the quinolones, of which acid synthesis new members are constantly introduced. also included are rifampicin, used in the modern treatment of tuberculosis, and metronidazole, often used in the dental treatment of anaerobic infections. rifampicin inhibits the formation of rna whereas the quinolones interfere the development of dna. the best known antimetabolites are the sulphonamides, which interfere with uptake of para-amino-benzoic acid (paba), which prevents the synthesis of folic acid. trimethroprim is taken up by an enzyme necessary for the ultilization of folic acid. interference with the metabolism of folic acid inhibits the production of new genetic material. the administration of antimicrobial agents for the treatment of infection requires the achievement of adequate levels of the antimicrobial agent as quickly as possible. for many antibiotics, this level is referred to as the minimum inhibitory concentration (mic), which is the concentration that prevents visible growth after a 24-hour incubation period. unless the route of administration can achieve a level substantially higher than the mic for the invading organism, it is unlikely that successful treatment of the infection will be achieved. for bactericidal agents, the figure quoted is minimum bactericidal concentration (mbc), which is the concentration that kills 99.99% of the bacterial cells. the higher the concentration of the agent, the greater will be the effect on the organism. failure to achieve these levels can lead to the development of resistant strains. in any population there will be a proportion of organisms that are resistant to the agent. in the presence of the agent these will be selected and will form the majority of the population. some bacteria develop resistance by altering their metabolic pathways to avoid those with which the antimicrobial interacts. other bacteria produce enzymes capable of destroying the chemical, e.g. penicillinase, which is an enzyme produced by certain strains of staphylococci and is capable of breaking down penicillin. although there has been no recorded case of a patient contracting aids from a contaminated contact lens, this condition has highlighted the necessity for good practice hygiene. the aids virus is not the only organism (opportunist or invasive) that could be transmitted in an optometrist's practice and simple disinfection procedures should be employed in order to protect both practitioner and patient. the first consideration is one of cleanliness, as clean objects and surfaces are easier to disinfect and will remain uncontaminated for longer. normal contaminants will harbour bacteria, protect them from antibacterial agents, provide them with nutrients and inactivate disinfectants. jacobs (1986) has laid down simple infection control guidelines for optometrists and contact lens practitioners. basically, anything that can be boiled without adversely affecting its performance should be, e.g. bowls, soft contact lenses. items of equipment that will touch the eye should be swabbed with 70% alcohol, e.g. tonometer heads, chin rests, trial frames. working surfaces should be treated with 1% sodium hypochlorite solution, which is effective against bacteria and viruses. at levels as low as 500 ppm (0.05%) this solution destroys herpes simplex, adenovirus 8 and enterovirus 70 within 10 minutes (naginton et al 1983) . such procedures will protect the patient more than will a prophylactic eye drop. in the interests of self-protection, the practitioner should have no open cut uncovered but the added precaution of wearing gloves is only necessary for high-risk patients, e.g. patients who are hiv positive. botulinum toxin and its uses in the treatment of ocular disorders detection of chlamydia trachomatis in the vaginal vault of women who have had hysterectomies endophthalmitis following staphylococcal sepsis in renal failure patients pseudomonas aeruginosa and whirlpools anaerobic and aerobic bacterial flora of acute conjunctivitis in children anaerobic and aerobic bacteriology of acute conjunctivitis the conjunctival flora of nursing home patients and staff herpes simplex virus induced cataracts pseudomonas ophthalmia neonatorum: a cause of blindness gas gangrene infection of the eye and orbit medical ethics and hospital-acquired disease acute follicular conjunctivitis and keratoconjunctivitis references due to herpes simplex virus in london paralytic strabismus: the role of botulinum toxin penetration of hydrophilic contact lenses by aspergillus fumagatus identification of a nonlysozymal bacteriocidal factor (betalysin) in human tears and aqueous humor cytotoxicity of ophthalmic preservatives the war on thiomersal chlorhexidine effects on corneal epithelium and endothelium outbreak of erythromycin resistant staphylococcal conjunctivitis in a new born nursery infection control guidelines for optometrists and contact lens practitioners asbell p a 1987 ophthalmia neonatorum. study of a decade of experience at the mount sinai hospital ocular manifestation of aids conjunctival flora of healthy people quantitative tear lysozyme essay in units of activity per microlitre aspergillus panophthalmitis complicating treatment of pterygium genital tract to eye infection -tissue culture of chlamydia trachomatis management of bacterial corneal ulcers pathophysiology of bacterial infection in the external eye radial keratoneuritis as a presenting sign in acanthamoeba keratins edta reduces the antimicrobial efficacy of thiomersal tonometer disinfection and viruses neonatal chlamydial eye infection: an epidemiological and clinical study chlamydia trachomatis infection in infants. a prospective study normal and pathological components of tears and conjunctival secretion changes in antibacterial activity of thiomersal and pmn on autoclaving with certain adjuvants prospective study of chlamydial infection in neonates cytotoxicity of mercurial preservatives in cell culture recovery of htlv-iii from contact lenses effect of benzalkonium chloride on the stability of the precorneal tear film in rabbit and man penetration of mercury from ophthalmic preservatives into the human eye prevalence of acute conjunctivitis caused by chlamydia adenovirus, and herpes simplex virus in an ophthalmic casualty department herpetic keratitis and corneal destruction treacher collins prize essay. inflammatory diseases of the outer eye fungus growth on soft contact lenses with different water contents fungal invasion of a therapeutic soft contact lens and cornea key: cord-264916-c4n0kyog authors: zimmerman, keith; kearns, fiona; tzekov, radouil title: natural protection of ocular surface from viral infections – a hypothesis date: 2020-07-09 journal: med hypotheses doi: 10.1016/j.mehy.2020.110082 sha: doc_id: 264916 cord_uid: c4n0kyog a pandemic outbreak of a viral respiratory infection (covid-19) caused by a coronavirus (sars-cov-2) prompted a multitude of research focused on various aspects of this disease. one of the interesting aspects of the clinical manifestation of the infection is an accompanying ocular surface viral infection, viral conjunctivitis. although occasional reports of viral conjunctivitis caused by this and the related sars-cov virus (causing the sars outbreak in the early 2000s) are available, the prevalence of this complication among infected people appears low (∼1%). this is surprising, considering the recent discovery of the presence of viral receptors (ace2 and tmprss2) in ocular surface tissue. the discrepancy between the theoretically expected high rate of concurrence of viral ocular surface inflammation and the observed relatively low occurrence can be explained by several factors. in this work, we discuss the significance of natural protective factors related to anatomical and physiological properties of the eyes and preventing the deposition of large number of virus-loaded particles on the ocular surface. specifically, we advance the hypothesis that the standing potential of the eye plays an important role in repelling aerosol particles (microdroplets) from the surface of the eye and discuss factors associated with this hypothesis, possible ways to test it and its implications in terms of prevention of ocular infections. ocular surface infections caused by viruses are a considerable public health problem worldwide. for example, an analysis of the 1985 national ambulatory medical care survey found that ~1% of all primary care office visits in the united states were related to conjunctivitis [1] . if one assumes that the proportion remained the same, which would translate to ~2.3m visits in 2019. similar proportion has been reported for uk [2] . infectious conjunctivitis 1 is more prevalent than the other types (e.g. allergic, chemical, etc.) and viral conjunctivitis is estimated to be the most common cause of infectious conjunctivitis, at up to 80% of all cases [3] . of all different types of viruses as candidates of causing infectious conjunctivitis, adenoviruses have been found to be the most common pathogen, in up to 90% of all cases [4] . however, it has to be kept in mind that exhaustive testing related to the causation of conjunctivitis is not always possible, which may introduce bias in reported results. apart from adenoviruses, other types of viruses reported to be isolated from the conjunctiva and implicated in the development of conjunctivitis are herpes simplex virus, enterovirus 70 and coxsackie a24 variant virus [5] . somewhat surprisingly, the majority of the viruses responsible for respiratory viral infections are not a major cause of infectious conjunctivitis [6] . thus, even the adenovirus causing the majority of infectious conjunctivitis cases is subtype d, while strains that cause respiratory infections are subtypes b, c and e and they rarely cause conjunctivitis ( table 1 in [6] ). similarly, only one of the influenza viruses subtypes (h7, causing avian influenza), is a significant cause of conjunctivitis, with ~80% of the cases with this infection presenting with conjunctivitis [7] , while the much more common subtypes causing human infection with influenza virus subtypes that are more commonly associated with respiratory illness are an infrequent cause of the disease [6] . recently, the worldwide attention has been focused on a viral infection caused by a coronavirus, resulting in a global pandemic. this infection is most often associated with respiratory symptoms and its major complication is overwhelmingly a disease of the lower respiratory tract, a bilateral viral pneumonia [8] and, therefore, can be classified as a respiratory viral infection. it is caused by a coronavirus (sars-cov-2) [9] , while the disease has been named covid-19 [10] . this virus belongs to the subfamily of coronaviruses, a term proposed in 1968 to describe a group of enveloped, positive-sense, single-stranded rna viruses with similar form, including a characteristic appearance of the envelope glycoproteins in electron microscopy observation, recalling the solar corona [11] . this proposal was accepted and currently this type of viruses are classified as subfamily orthocoronavirinae (of family coronaviridae) and contains 4 major groups (genera): alpha-, beta-, gamma-and deltacoronaviruses, a total of 25 species, the majority of which are found in animals, but not humans [12] . the viruses found either exclusively or not in humans typically cause respiratory tract illnesses [13] . seven types of coronaviruses are known to cause disease in humans. the two types that were discovered in the 1960s, hcov-oc43 (a betacoronavirus) and hcov229e (an alphacoronavirus), both causing mostly mild upper respiratory tract illnesses in adults. they are still in circulation, together with three types identified in the 2000s: hcov-nl63, hcov-hku1 (both alphacoronaviruses) and mers-cov (a betacoronavirus), the first two casing mild upper and lower respiratory tract infections in adults, while the third one causing more severe infections. another virus, sars-cov (a betacoronavirus), identified also in the early 2000s, causing severe acute respiratory syndrome (sars), is no longer circulating in humans after strict and coordinated public health measures [13] . the new virus, sars-cov-2, has closest similarity (40-90% identity) to the first sars-cov virus [14] and shows similar clinical manifestations, although with a lower mortality rate [15] . generally, coronaviruses infecting humans have been rarely associated with ocular surface infections [6] . only rare reports of conjunctivitis have been associated with hcov-nl63 [16] and no reports have been presented for sars-cov [17] . whether and to what extent these types of viruses can be spread through ocular surface exposure remains a subject of debate and uncertainty. for example, the sars-cov virus was detected in the conjunctiva from 3 probable cases (out of 36) in one study [18] , but not in two other studies in tears and conjunctival scraping samples [19, 20] , triggering questions about the mechanism and details of identification of the virus in ocular tissues and fluids [21, 22] . two recent studies by xia et al. [23] and wu et al. [24] , showed also a low rate of virus detection in conjunctiva (4%). a recent meta-analysis including 1,167 patients, indicates that frequency of conjunctivitis associated with sars-cov-2 infection (covid-19) was generally low: ~1.1% (3% in severe and 0.7% nonsevere covid-19 patients) [25] . on the other hand, two recent reports showed expression of established sars-cov [26] and sars-cov-2 [27] receptors, the angiotensin-converting enzyme 2 (ace2) and transmembrane protease serine subtype 2 (tmprss2) in human conjunctiva, limbus and cornea [28, 29] . this finding indicates that ocular surface cells including in the conjunctiva could be susceptible to the virus and theoretically serve as a point of entry for the viral infection. therefore, a question arises: if the receptors for the virus are present in ocular surface tissues, why the incidence of ocular surface infection is so low and isolating the virus from ocular surfaces presents such a challenge? our hypothesis is the standing potential of the eye interacts with microdroplets carrying the virus and prevents -either partially or in whole -microdroplets from landing and attaching to the ocular surface. such an interaction would greatly reduce the probability of virus presence on, and in turn reduce infections in, ocular surface tissues. the fact the eye is electrically charged was first demonstrated in an animal eye by du bois-reymond in 1849 [30] and re-discovered by frithiof holmgren in 1865 in a fish eye [31]. the first rigorous confirmation of the standing potential of the eye as a corneo-retinal potential in humans was provided by mowerer et al. in 1935 [32] . in the 1960s, detailed investigation of the magnitude of the standing potential under standardized light-adapted conditions showed that it is on average ~+0.7 mv (range 0.25 to 1.1 mv), with the cornea having a positive charge compared to the back of the eye [33] and it was shown that this potential is relatively stable on an hourly, daily and weekly basis, but shows some diurnal variation. it has to be noted that these measurements were made indirectly with electrodes placed on both sides of the eyeball, at the outer and inner canthus of the eye, and, therefore, the true electrical potential in front of the cornea is likely to be higher (+5 mv), which was suspected since the 1940s based on animal studies and confirmed in humans in the 1970s with more direct laboratory measurements [34] . it was estimated further that if the corneal potentials were measured relative to the skin on the forehead or cheek, the potential difference would be even higher at +10 to +15 mv [35] . this potential could be further enhanced by electrostatic charges on the eyelid skin. it is well-documented that human oily skin can become highly positively electrostatically charged, as it is usually listed near the top of the triboelectric series [36] . bioaerosols generated by human exhalation are considered a possible route for sars-cov-2 spread [37] [38] [39] [40] . in support of this possibility, previous studies testing of air samples showed the presence of another coronavirus (mers-cov) have found the virus in 4 of 7 air samples [41] . studies of the sars-cov virus also suggested airborne transmission [42] and infective droplet inhalation [43] , although some uncertainty remains [44] . influenza virus rna was also recovered from air sampled in a hospital [45] . finally, a recent study identified seasonal human coronaviruses, influenza viruses and rhinoviruses in exhaled breath and coughs of patients with respiratory disease [46] . currently, it is generally accepted that bioaerosols as an infectious disease transmission medium should be divided into three groups based on aerodynamic droplet diameter: small particles (less than 10 µm), that can remain airborne, larger droplets (more than 20 µm), which settle relatively quickly to the ground and an intermediate size (10-20 µm) may either settle or remain airborne [47] . apart from the consensus built around this reasonable classification, there is a great deal of variation within the range and distribution of particle size reported by different studies, which likely depends largely on the methodology used to count and classify the particles. thus, the following discussion of this topic should not be treated as a comprehensive analysis of this subject, but rather as one specific perspective on the reported results in view of the matter discussed here. the measurement of the size of exhaled particles in a 2011 study showed that more than 82% of all exhaled particles from three healthy and 16 human rhinovirus (hrv)-infected subjects were within 0.3-0.5 µm diameter range, placing it firmly in the small droplets category [48] . given that the average diameter of the sars-cov-2 virus is ~0.12 µm [49, 50] and assuming that the maximal viral concentration per droplet should be an occupancy of ~30% of the whole surface of the droplet 2 , the maximal viral load per particle can be estimated between 8 and 21 virions/droplet 3 . although this appears like a small load per droplet, the same study found that the droplet production varied dramatically between subjects, with some subjects (4 out of 17) producing ~3,500 particles/liter (range ~1,000 to ~7,000), equivalent to ~28,000 droplets/min, with the potential to spread more than 500,000 viral particles/min via airborne particles. in contrast, the rest of the subjects produced on average ~7.4 particles/liter (equivalent to ~52 droplets/min with some participants generating 0 droplets), thus, supporting the hypothesis that some people could be "high spreaders" of the viral infection. it should be emphasized that this study estimated the droplet size and production under regular breathing. the result is likely different when sneezing. a study estimating the droplet sizes from sneezing found almost exclusively large droplets with diameter larger than 50 µm (and up to 1,000 µm) [51] . for comparison, cough in healthy volunteers appeared to generate particles with less than 1 µm in diameter [52] . some studies suggest that large droplets only originate from the oral cavity [53] . although studies with coronavirus are not available, one study measured influenza virus in cough samples and found that 35% of the influenza rna was isolated from particles >4 µm diameter, while 23% of influenza rna was isolated from particles 1 to 4 µm diameter, and 42% in particles <1 µm [54] . high spreaders for the influenza virus were confirmed in another study [55] . the size and viral load have potential importance for the probability of landing on ocular surfaces. thus, larger droplets, loaded with more viral particles are expected to have poorer aerodynamic characteristics and be more susceptible to gravity forces and quicker landing. given lower airflow speed under normal breathing conditions, the main type of viral airborne spread seems to be small droplets, which can stay afloat for many minutes to hours, but carry relatively little viral load per droplet. as the intensity of exhaled airflow increases, as in loud talking, singing, coughing and sneezing, the droplet formation becomes more and more dominated by larger and larger droplets with high viral load per droplet and high initial velocity, but short airborne time. water. although it is assumed that bioaerosol contains some amount of water, the exact water content of bioaerosol microdroplets is difficult to determine and probably varies a lot, depending on the relative humidity and temperature of ambient air. most physiological models of human breathing assume 100% relative humidity and 36-37° c in the air of the lower respiratory tract, in accordance with experimental data [56, 57] . with expiration (tidal breathing and room temperature air), some of this water content is reabsorbed in the upper respiratory tract, where the temperature is assumed to be ~32°c below the glottis [57] and vary in the naso-pharyngeal cavity, from ~25°c in the nasal vestibule, to ~30°c in the internal nasal valve area and ~32°c in the middle turbinate [58, 59] , with some differences (~2°c) between the air and nasal mucosa, allowing for an efficient water reabsorption during exhaling, but also providing favorable conditions for condensation and droplet formation. these values change with change in ambient air temperature, as inhalation of cold air lowers the temperature in both the upper [60] and lower respiratory tract [57] , thus further improving the conditions for water condensation during exhalation. once the bioaerosol leaves the human body, several factors will affect the water content, probably the most important one being change in droplet size, influenced by the rate of evaporation [61] , interaction with each other (coalescence or fragmentation [62] ) and relative position in relation to the center of airflow [63] . these effects are complex and still insufficiently understood in the case of exhaled aerosol. organic components. the main organic component of exhaled human air is an airway lining fluid component, which is highly diluted in water, with a consensus estimate for dilution between 2,000 to 10,000 times [64, 65] . the airway lining fluid likely contains various types of lipids and electrolytes. thus, a recent study identified 75 glycerophospholipids, 13 sphingolipids, 5 sterol lipids and 12 neutral glycerolipids in samples from healthy volunteers [66] , with a large variation in composition between subjects, underscoring the complexity of organic content of exhaled air condensate. within this context, another phenomenon should be pointed out, namely the effect of temperature and relative humidity. two aspects should be mentioned. first, natural evaporation from the lungs depends on temperature and humidity -at relatively low temperature (~2° c), water loss depends very little on relative humidity (2.0-2.2 ml h 2 o per l/breath/min), but at higher temperature (~27° c), the range increases dramatically (0.5-2.1 ml h 2 o per l/breath/min) with much less water loss in more humid air [67] . the second aspect to be mentioned is the water loss at low relative humidity, which is substantial [68] . both factors would apply to most modern air-conditioned indoor environments, where the temperature is kept typically in the range of 20-23° c and humidity at ~40%. that electrical potential value does not seem very strong as a generator of an electrostatic repelling force (coulombic force), but it has to be kept in mind that the volume of space to be protected from particles in front of the cornea is not large, particles need only be deflected less than 1 cm in front of the corneal surface, as the thickness of the eyelid margins is only about 2 mm [69] . considering that ocular surface area exposed to air is typically 1.25-1.75 cm 2 [70] [71] [72] (although it can reach intermittently up to 3 cm 2 , depending on the visual task), the "protected volume of air" 4 in front of the cornea is probably less than 0.4 cm 3 and does not exceed 0.75 cm 3 . furthermore, human eyelashes act as passive dust controlling system and reduce evaporation and particle deposition up to 50% [73] , further facilitating the defense against foreign particles (including microdroplets). what is the effect of blinking on the airflow within the "protected volume of air" is currently unknown, however, it can be speculated that it would create some change in low-velocity airflow, as it was estimated that blinking increases corneal temperature by 1.3° c [74] and this could create convection air microcurrents away from the corneal surface. the speed and direction of local airflow can be further modified by artificial objects in eye vicinity. for example, it is highly likely that even regular eyeglasses can restrict the airflow around the eyes, although we are not aware of a quantitative evaluation of this phenomenon. another factor to be considered under the scenario of direct, close human-to-human communication is that the majority of microdroplets would not reach the eye. a recent study using a 3d-printed realistic face and airway model, with an outlet of a particle jet centered at the nose of the model at a distance of 20 cm, found that more 80% of the generated aerosol particles with an initial velocity of 0.94 m/s was deposited on the lips rather than on the eyes [75] . electrostatic force 4 the "protected volume of air" in this context can be defined as the volume of air enclosed in the space between the upper and lower eyelids when the eyelids are open, and the gaze is directed forward in primary position. electrostatic (coulombic) forces can play considerable part in aerosol deposition [76] [77] [78] [79] , especially for particle size range 0.01 µm to 5 µm [80] , which, as discussed below is the most important range of water microdroplet particles carrying viruses. additionally, it was shown that even relatively low voltage (12v) ionizer device effectively captured airborne transmitted calicivirus, rotavirus and influenza virus and prevented airborne transmitted influenza a between animals [81] . however, it needs to be emphasized that, according to hoque (2010) : "no specific distribution has been identified in the literature to describe charge distribution in bioaerosols" [82] , and, thus further work is needed to clarify the role of electrical charge of bioaerosols for deposition on human tissue in the real world. to the best of our knowledge, direct measurements of the electrical charge of bioaerosols generated by human exhalation have not been published to date. therefore, we would advance some theoretical considerations supported by some indirect experimental data to explore what would be the more likely overall change of the droplets comprising the bioaerosol generated by exhalation and will focus on bioaerosol containing mostly coronaviruses. one such consideration is that the ph of exhaled breath condensate is slightly alkaline, e.g. in normative database from 404 subjects, the mean ph was 7.83, and the median ph was 8.0 [83] and similar results were obtained in another report and a meta-analysis [84, 85] . however, the role of bioaerosol de-aeration with co 2 -free gas is unclear. therefore, it is possible that the net charge of bioaerosol droplets generated by breathing and other activities is positive. if this would be the case, it would explain the low probability of airborne particles to land in sufficient numbers to the ocular surfaces and remain there for long enough time to attach to receptors and cause inflammation. of note, the overall isoelectric point of coronaviruses has not been reported in the literature. most of the factors discussed above would apply to relatively similar indoor controlled environments, including air conditioning, central heating, etc., with relatively slow airflow and limited temperature and humidity range. the production rate, spread and infective potential of bioaerosol from exhaled air would be very different in outdoor environments. it is likely that the outdoor infectious potential of bioaerosol would be much more dependent on environmental factors, such as temperature, humidity, wind speed and direction, air ionization, solar irradiance, etc. currently, most people in industrialized nations spend most of their time indoors. a large (n= 9,196), 2-year probability-based telephone survey in the us (1992) (1993) (1994) found that the respondents reported spending an average of ~87% of their time in enclosed buildings and ~7.6% outdoors, confirming similar findings from earlier surveys in industrialized countries [86] . however, the exact proportion of infectious events for viral respiratory diseases occurring in indoor environments vs. outdoors is unknown and it is likely to be different for different types of viruses. this hypothesis could be tested by measuring the electrical charge of bioaerosol generated by normal breathing in healthy subjects and in patients with viral infections caused by different viruses, causing respiratory infections or with suspected aerosol transmission pathway. an established reliable isoelectric point for all studied viruses would be very helpful in modeling the relationship between virion electrical charge and droplet electrical charge. the discrepancy between the theoretically expected high rate of concurrence of viral ocular surface inflammation and its observed relatively low occurrence in covid-19 is surprising. natural protective factors related to anatomical and physiological properties of the eyes could prevent the deposition of large number of virus-loaded particles on the ocular surface and play a protective role. it is possible that the standing potential of the eye plays an important role in repelling aerosol particles (microdroplets) near the surface of the eye and serve as major contributing factor in securing either complete protection or fast elimination of certain type of bioaerosols containing viruses. a comparison of eye problems in primary care and ophthalmology practices management of ophthalmic disease in general practice conjunctivitis: a systematic review of diagnosis and treatment acute conjunctivitis: truth and misconceptions a twenty-one year surveillance of adenoviral conjunctivitis in sapporo ocular tropism of respiratory viruses past, present, and possible future human infection with influenza virus a subtype h7 clinical characteristics of coronavirus disease 2019 in china sars-cov-2 is an appropriate name for the new coronavirus editorial -differences and similarities between severe acute respiratory syndrome (sars)-coronavirus (cov) and sars-cov-2. would a rose by another name smell as sweet? human coronaviruses: what do they cause a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 comparison of confirmed covid-19 with sars and mers cases -clinical characteristics, laboratory findings, radiographic signs and outcomes: a systematic review and meta-analysis human coronavirus nl63 the severe acute respiratory syndrome the severe acute respiratory syndrome coronavirus in tears tears and conjunctival scrapings for coronavirus in patients with sars ocular screening in severe acute respiratory syndrome sars virus in tears? lancet infect dis the severe acute respiratory syndrome coronavirus in tears evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov-2 infection characteristics of ocular findings of patients with coronavirus disease conjunctivitis and covid-19: a meta-analysis efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor expression of sars-cov-2 receptor ace2 and tmprss2 in human primary conjunctival and pterygium cell lines and in mouse cornea ace2 and tmprss2 are expressed on the human ocular surface, suggesting susceptibility to sars-cov-2 infection untersuchingen uber die thierische elektrizitat. reimer holmgren f (1865) method att objective effekten av ljusintryck pa retina the corneo-retinal potential difference as the basis of the galvanometric method of recoirding eye movements changes in the electro-oculogram potential level the directly recorded standing potential of the human eye ocular artifacts in recording eegs and event-related potentials. ii: source dipoles and source components transmission of 2019-ncov infection from an asymptomatic contact in germany rapid expert consultation on the possibility of bioaerosol spread of sars-cov-2 for the covid-19 pandemic aerosol and surface transmission potential of sars-cov-2 singapore novel coronavirus outbreak research t (2020) detection of air and surface contamination by sars-cov-2 in hospital rooms of infected patients consideration of the aerosol transmission for covid-19 and public health extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards evidence of airborne transmission of the severe acute respiratory syndrome virus viral infections in workers in hospital and research laboratory settings: a comparative review of infection modes and respective biosafety aspects airborne transmission of communicable infection--the elusive pathway influenza virus rna recovered from droplets and droplet nuclei emitted by adults in an acute care setting respiratory virus shedding in exhaled breath and efficacy of face masks recognition of aerosol transmission of infectious agents: a commentary origin of exhaled breath particles from healthy and human rhinovirus-infected subjects properties of coronavirus and sars-cov-2 viral architecture of sars-cov-2 with post-fusion spike revealed by characterizations of particle size distribution of the droplets exhaled by sneeze cough aerosol in healthy participants: fundamental knowledge to optimize droplet-spread infectious respiratory disease management airborne spread of infectious agents in the indoor environment measurements of airborne influenza virus in aerosol particles from human coughs exposure to influenza virus aerosols during routine patient care physiology humidification: basic concepts in: esquinas am (ed) humidification in the intensive care unit thermal mapping of the airways in humans temperature profile in the nasal cavity simultaneous in vivo measurements of intranasal air and mucosal temperature nasal mucosal temperature after exposure to cold, dry air and hot, humid air kinetics and evaporation of water drops in air interaction of water droplets in air flow at different degrees of flow turbulence evaporation and movement of fine water droplets influenced by initial diameter and relative humidity exhaled breath condensate: methodological recommendations and unresolved questions exhaled breath condensate: an overview lipid phenotyping of lung epithelial lining fluid in healthy human volunteers water lost through breathing dynamics of airborne influenza a viruses indoors and dependence on humidity basic conceptsdiseases and disorders of the orbit and ocular adnexa e-book ocular surface-area as an informative index of visual ergonomics the exposed ocular surface and its relationship to spontaneous eyeblink rate in elderly caucasians digital photography as a novel technique of measuring ocular surface dimensions eyelashes divert airflow to protect the eye mathematical model of thermal effects of blinking in human eye measuring the administered dose of particles on the facial mucosa of a realistic human model aerosol wall losses in electrically charged chambers combined effects of electrophoresis and thermophoresis on particle deposition onto flat surfaces an eulerian model for particle deposition under electrostatic and turbulent conditions direct numerical-simulation of particle entrainment in turbulent channel flow particle deposition in a nearly developed turbulent duct flow with electrophoresis ionizing air affects influenza virus infectivity and prevents airborne-transmission development of computational fluid dynamics based multiple linear and neural network metamodels for bioaerosol fate and normative data for ph of exhaled breath condensate exhaled breath condensate ph in adult croatian population without respiratory disorders: how healthy a population should be to provide normative data? a review of the usefulness of non-invasive exhaled breath condensate ph analysis for diseases diagnosis with elements of meta-analysis: an update from 2012 the national human activity pattern survey (nhaps): a resource for assessing exposure to environmental pollutants no conflict of interest. key: cord-277539-xt2nt11e authors: kochhar, anuraj singh; bhasin, ritasha; kochhar, gulsheen kaur; dadlani, himanshu; thakkar, balvinder; singh, gurkeerat title: dentistry during and after covid-19 pandemic: pediatric considerations date: 2020 journal: int j clin pediatr dent doi: 10.5005/jp-journals-10005-1782 sha: doc_id: 277539 cord_uid: xt2nt11e this article is a rumination on the outbreak of the dreaded coronavirus disease-2019 (covid-19) pandemic which has engulfed both the developed and the developing countries, thereby causing widespread global public health concerns and threats to human lives. although countries have made varied efforts, the pestilence is escalating due to the high infectivity. it is highly likely that dental professionals in upcoming days will come across covid-19 patients and sars-cov-2 carriers, and hence must ensure a tactful handling of such patients to prevent its nosocomial spread. despite the avalanche of information that has exploded in relation to this rapidly spreading disease, there is a lack of consolidated information to guide dentists regarding clinical management including precautions to take materials to use and postprocedure care, during and after the covid-19 pandemic. available sources of information have been analyzed, while relying on peer-reviewed reports followed by information available from the most respected authoritative sources, such as who, centers for disease control and prevention (cdc), and ada. this review aims to provide a comprehensive summary from the available literature on covid-19, its insinuation in dentistry, recommendations that have been published, and the actual in-practice implications, so a plan can be formulated and adapted to the circumstances of each dental practice during the pandemic and the times to follow. how to cite this article: kochhar as, bhasin r, kochhar gk, et al. dentistry during and after covid-19 pandemic: pediatric considerations. int j clin pediatr dent 2020;13(4):399–406. coronavirus disease-2019 (covid-19) pandemic presented as a black swan event, later spreading its tentacles into almost all spheres of life. the carcinogenic consequences have made it the need of the hour that dental healthcare professionals understand the mortiferous effects of this dreaded disease in order to prevent its malignant spread. the sars outbreak was the first readily communicable infectious disease that the world faced in the 21st century. it was presumed not to be the last such contagion. 1 the covid-19 outbreak that was the first reported in the area of wuhan, china, drastically developed into a public health quandary. 2 who has declared a global emergency with millions of people presently in quarantine, self-isolation, or lockdown. 3 constant diligent efforts to curb the effects of the pandemic are being carried out but the situation is still escalating owing to the high transmissibility of the virus leading to a community spread. it is speculated that dental professionals will come across covid-19 patients and sars-cov-2 carriers, and hence must ensure a careful handling of such patients to prevent its nosocomial spread. despite the avalanche of data that is available in relation to this disease, there is confusion due to a lack of consolidated information to guide dentists. the purpose of this review is to provide a comprehensive summary from the available literature on covid-19, its insinuation in dentistry, recommendations that have been published, and the actual in-practice implications, so a plan of measures can be formulated and adapted according to the circumstances of each dental practice during the pandemic and the times to follow. initially, covid-19 started as a zoonotic infection, but soon human transmission started through nosocomial routes and later viruses were found in respiratory droplets as well. 4 this causes an alarming concern to dentists as they deal with the orofacial region. also, of concern is the presymptomatic transmissibility for covid-19, during the incubation period up to 14 days, with the mean of 5-6 days which has been documented by aggressive reconnaissance of clusters of confirmed patients. [5] [6] [7] this is buttressed by information, via contact-tracing efforts that few individuals can test positive for the disease 1-3 days before the development of symptoms. 8 it is noteworthy that infectious droplets or aerosols are still a prerequisite for transmission. asymptomatic transmission has also been reported similar to presymptomatic, by measures of contact tracing, wherein an individual test positive for covid-19, however, presents without any symptoms. 3, 6, 7, 9 owing to the transmission by presymptomatic and asymptomatic patients, the high r 0 (basic reproductive number/ infectious agent's epidemic potential) for sars-cov-2 which ranges between 1.4 and 6.5, with an average of 3.28, might be justified. 10, 11 even in those who are symptomatic, several people with covid-19 express only faint clinical presentation, such as cough, 12 however, this is a cause of concern in dentistry, because, there is an increased exposure risk if performing or being present for an aerosol-generating medical procedure (agmp). aerosolgenerating medical procedure comprises tracheal intubation, extubation, cardiopulmonary resuscitation, nebulizer treatment, sputum induction, noninvasive ventilation, tracheostomy, manual ventilation prior to intubation, and bronchoscopy. 12 however, surprisingly, there is no mention of dental procedures. 13 fomites and feco-oral are other possible transmission routes that require attention. recent updates suggest the possible routes of entry to be via the exposure of mucous membranes of nose, mouth, and eyes, 10, 14 and raise the concern for hand hygiene by the dental team as well as patients. another matter of concern is air conditioner airflow direction, which was implicated as a means of transmission in guangzhou, china and was published by centers for disease control and prevention (cdc). 15 knowledge of signs and symptoms of covid-19 is important for the dental practitioners. an elaborate history must be taken and should include questions regarding any flu-like symptoms, ranging from cough, shortness of breath, fever, and diarrhea, trouble breathing persistent pain or pressure in the chest. mildly symptomatic patients also report alterations in smell or taste in sars-cov-2 infections which often were the first apparent symptoms. questions regarding the same by the dentist can also prove to be beneficial in the initial diagnosis by the dentist, but results must be interpreted with caution. 16 detailed medical history is imperative as presence of comorbidities (obesity, diabetes, hypertension, cardiovascular disease, pulmonary disease, and renal disease) have been found to be risk factors for the disease. 17, 18 diagnostic tests sars-cov-2 tests may either detect the virus itself [viral ribonucleic acid (rna)]/antigens or detect the serological antibodies produced in response. 19 real-time pcr test which mainly makes use of a nasopharyngeal swab is still the confirmatory test. 20, 21 rapid testing and monitoring which is hot off the fire, detects igm, iga, igg, or total antibodies, 19 and can be used by the dental practitioners for in-clinic screening of patients, with the results being available within a short span of time. 22 of the many rapid test kits that have been developed recently, as on april 24, 2020, 45 have been approved by fda 23 (fig. 1) . however, dentists should be cautioned, as early in the disease a single nasopharyngeal swab for pcr test is only 70% sensitive. 24 on the contrary, although the rapid test infers expedited diagnosis of covid-19, false-negatives are a bigger problem which cannot be overlooked. hoffman et al., stated that the precision of the test was 94.1% for igm and 98% for igg, considering rt-pcr positive cases as true positives. further they outlined that although a sensitivity of 69 and 93.1% for igm and igg was observed, the assay showed an overall specificity of 100 and 99.2% for igm and igg, respectively. 25 similar results were obtained by the researchers at cleveland clinic who in an unpublished report stated a false-negative rate of 14.8%. 26 hence, one must be forewarned that a negative test alone should not be used as the sole basis for ruling out covid-19 infections and making patient management decisions, instead should be supplemented with history, clinical signs and symptoms, radiological findings, and epidemiological information. 27 to et al., found the presence of the novel coronavirus in selfcollected saliva specimens of 91.7% patients which might be a viable source for diagnosis. 28 also, a study by wyllie et al. showed that the collection of saliva can be self-administered, more sensitive, and viable as compared to nasopharyngeal swabs and could make accurate sars-cov-2 testing a possibility, be it at home or in a dental setting with lower risk to healthcare workers. 29 another valuable tool that can be used as an adjunct in the dental clinics is pulse oximeter, which is fast, easy, noninvasive, and harmless, to detect silent hypoxia, in covid-19 patients. this has become a recent topic for consideration but requires further studies to be validated. 30 prophylactic hydroxychloroquine or chloroquine for asymptomatic healthcare professionals while treating covid-19 patients has been advocated by the indian council of medical research (icmr). the advised prophylaxis with hydroxychloroquine (400 mg twice on day 1, followed by 400 mg once a week) is a matter of concern as it can cause an undue optimistic perception of a drug whose efficacy is not verified for the particular use, the advantages unseen, and the undesirable consequences, including potentially lethal cardiac arrhythmia in certain individuals, overlooked. [31] [32] [33] also, prophylaxis could last for the entire duration of the pandemic, and in countries where malaria is highly prevalent, appropriate resistance monitoring of plasmodium species is needed. 34 a chemoprophylaxis advice without reliable evidence might be questionable. 30 hence, risks vs benefits should be evaluated, a thorough medical assessment and physicians' approval for the same must be taken. sound knowledge of the spread of sars-cov-2 is required to prevent its transmission in the dental practice. though numerous routes of infections exist in the dental scenario, aerosols are one of the predominant routes for transmission of pathogens including sars-cov-2, therefore stringent infection control measures are imperative. [35] [36] [37] [38] thus, for patient screening and infection control, specific proposals and strategies for dental healthcare practice are critical. dentists must abide by the most recent recommendations from international, federal and local public health authorities. before a patient comes to the clinic, a telephonic appointment must be made with the dentist. aggressive screening of patients, attendants, dental practitioners, and the staff should be performed for covid-19. patients must be enquired about any close contact with a known covid-19 patient or symptoms including fever, dry cough, and shortness of breath over the phone and where possible, video call. it can help screen suspected covid-19 patients and also identify whether the patient requires in-office treatment. 2 another recommended method by the cdc is an online bot nicknamed clara which can act as a screening aid 39 (fig. 2) . *positive pcr confirms infection. antibody test not required **patient should be treated with caution in accordance with most updated cdc guidelines, as no information still available about infectivity during convalescence 3 ***treatment to be carried out after cdc guidelines fulfilled 3 proposed clinic contingency plan *this is a contingency plan outline, and can be used as a skeleton to formulate a customised plan for a dental practice while following the most updated national and international guidelines **sensitivity and accuracy of the test must be checked prior to use and interpretation though teledentistry has now become an integral part of care, some patients may not be comfortable with it. studies have reported that telehealth presented an impediment to traditional healthcare and many patients were discontented with the same, 40,41 when their primary anticipations were not met. 42 also, while minor signs and symptoms may seem unimportant to the patient, these assumptions of aggressive telescreening are relying heavily on patients' information or lack thereof. dentists should make the patient comfortable, gain their confidence, and encourage them to report even the mildest of symptoms, which they might usually ignore. dental treatment of any suspected or confirmed covid-19 patient ideally must be deferred for 2 weeks from the time of exposure. 35 analgesics and/or antibiotics can be considered as therapeutic agents in certain cases. during the peak of the pandemic dental treatments should be categorized according to the severity as well as, the extent of invasiveness and risk of the procedure, especially when the federal and local public health authorities have deferred elective treatment. treatment should be considered after risk vs benefit evaluation 43 keeping in mind the possibility of disease progression, as reversible pulpitis might progress to irreversible, irreversible to apical periodontitis, and further on. there might be consequent tooth loss as well. 44 dentistry might see a splurge of patients once life resumes its "normalcy" with patients presenting with dental problems of much more severity and poor prognosis. if a patient requires in-person visit, temperature needs to be checked at the point of entry itself, preferably with a noncontact thermometer, 3 followed by a questionnaire and rapid test if available. to avoid transmission, magazines, toys, and other unnecessary items must be removed from the clinic and appointments should be staggered. 3 in pediatric dental setup, only one parent should accompany the child. alcohol-based hand rub (abhr) should be available at appropriate locations in the waiting area to help improve hand hygiene by children, parents, and staff. if treatment needs to be performed, informed consent 2 must be obtained and the operatory must be prepared for the same. treatment of any suspected covid-19 patient if required, must preferably utilize a negative pressure/airborne infection isolation room (aiir). 2 furthermore, a portable high-efficiency particulate air (hepa) filter with negative ion generator may be considered, 45 which are available mostly in hospital based settings only. 46 in a recent study, after sars-cov-2 application on copper and only cardboard, no live viable sars-cov-2 was observed after 4 and 24 hours, respectively. however, viable virus was more stable and discerned up to 72 hours on stainless steel and plastic. 47 this recent ground-breaking research about the aerosol and surface viability of the virus might provide a beneficial implication for dentistry as well. cardboard use as barriers and the use of coppercoated instruments instead of stainless steel may be considered as a substitute and further research is recommended for the same. 47 who recommended abhr formulations with 80% ethanol or 75% 2-propanol, have been assessed against sars-cov and mers-cov, and were found to be effective. benzalkonium chloride, however, has less efficiency than either of the alcohols, against coronaviruses. 35, 38 due to the current situation, healthcare organizations that grapple with paucity of abhr can consider local production of formulations as described by the fda policy for compounding of certain alcohol-based hand sanitizer products. 3 few studies illustrate that poor compliance with hand hygiene practices remains a challenge for infection prevention and control (ipac) among practitioners all over the world who mistake gloving as a substitute for hand washing. 48, 49 alcohol-based hand rub use should be encouraged between all patients, they should be provided in appropriate areas close to the health workers, and additionally hand-washing promoted when hands are visibly soiled. use of custom-fit n95 respirator, eye protection, face shield, and overgown during agmp on confirmed or suspected covid-19 patients have been propounded. 35, 37, 38 long-sleeved gloves with double-gloving technique, eyewear including side shields or fullface shields, and hair covers/hoods are recommended. 35 the use of powered air-purifying respirator (papr) in invasive treatments is suggested by few. 13, 50 donning a surgical mask or n95 is predicated on the size and dispersion of the respiratory secretions, and the dimension of droplets known to be contagious for a specific pathogen, rather than the size of the virus itself. 51 although wearing double gloves, overgown, and face/eye protection that have been overemphasized during agmp along with papr in invasive treatments, 13,50 but the reduced dexterity owing to the double layers of gloves, diminished visibility by flexible face shields, and back ache due to paprs may result in poor compliance. 52 thus, rather than donning everything, the most appropriate personal protective equipment (ppe) to be used in covid-19 patients is contingent on the clinical procedure being performed. association for the advancement of medical instrumentation (aami)-level 2 gown, surgical mask with face shield, and single gloves are advisable for caretakers who are not involved in high-risk agmp. 53 for healthcare personnel present in the operatory during an agmp, aami level-2 gown, n95 mask, eyeshield, head cover, and single gloves are advised; and practitioners performing agmp, should deploy aami level-3 gown, neck cover, and two pairs of gloves. 53 the coated fabric that has to be used must pass iso 16603 and iso 22612 testing for the usage of ppe kit. 54 another concern is contamination during doffing but abiding by doffing recommendations by cdc may lead to reduced crosscontamination, in comparison with a lack of guidance. 55 though this has not been paid heed to, nitrile gloves show better penetration resistance to viral load, when compared to latex. 56 when comparing latex and nitrile gloves in terms of barrier performance, both were superior to vinyl gloves. 57 sars-cov-2 has been found to be susceptible to oxidation, hence, pre-procedural rinse incorporating oxidative agents like 1% hydrogen peroxide or 0.2% povidone iodine is advocated to reduce viral load, though substantial evidence is not available. 38 spraying local anesthetics must be avoided due to the propensity to disseminate the virus in aerosols. 35, 38 however, if treatment is to be performed under ga, lidocaine should be used before endotracheal intubation as coughing can produce aerosols. 58 furthermore, ultrasonic scalers, three-way syringes, and high-speed handpieces must be obviated. 2 if indispensable, antiretraction handpieces 35 or electric friction grip handpieces 59 must be used to prevent aspiration and expulsion of debris and fluids. 35 suction, low or high volume can reduce aerosol production. 41, 42 if restorative or endodontic treatment is necessary, chemomechanical caries removal, such as carisolv and papain gel, is a viable alternative. 35, 60 also, silver diamine fluoride and gic can be used to restore teeth to prevent disease progression 61, 62 (table 1) . the reduction in bacteria-laden aerosols with rubber dam has been recommended for aerosol-generating procedures by peng et al. 35 studies by samaranayake et al. have suggested 78% reduction by and 70-88% reported by cochran et al. 63 hence, caution needs to be observed despite its use. extraoral radiography must be preferred over intraoral to reduce saliva production. 2, 35, 38 in case intraoral digital films are needed, supplemental barrier protection with finger 64 aerosols are the foremost reason for panic, owing to cross infection through it. during this pandemic, high-volume evacuators (hves) may offer a promising solution by controlling aerosol particles before they leave the mouth and can reduce 90-98% of aerosols, regardless of source. 35, 37, 65 air-cleaning systems also used for the same purpose significantly reduce the potentially hazardous aerosols created as shown by early studies; but still there is a dearth of published evidence for the same. 66 following every dental procedure, rigorous disinfection of all the surrounding surfaces should be carried out. therefore, there should be a time lapse of at least an hour between subsequent appointments to perform thorough decontamination. this has also been advocated by ti and colleagues. 52 moreover, there may be a backflow of pathogens with the use of handpieces in the water tubes of the dental chair. owing to this, purging should be appropriately done. all sterilizable instruments must be timely cleaned, disinfected, and sterilized. all disposables should be considered highly infected medical waste and discarded appropriately. 27, 58 the potency of human coronaviruses on inanimate objects is up to 9 days at room temperature. although data on the transmissibility of sars-cov-2 from contaminated surfaces to hands were not found, 5-second contact time has been documented to transmit 31.6% of influenza a virus to hands. 67, 68 ethanol at concentrations between 62% and 71%, 0.1-0.5% sodium hypochlorite, and 2% glutaraldehyde reduced coronavirus infectivity within 1 minute exposure time. a comparable effect is expected against the sars-cov-2 and these agents should be used for appropriate surface disinfection. 67 hydrogen peroxide vaporizer can also be used to decontaminate the operatory. 3, 52 various advisories and specifications have been put forward by the governing authorities. despite these recommendations, absolute infection control measures sometimes require constant reiteration. 49 therefore, the administration of the dental healthcare practice must make sure and evolve infection prevention and occupational health programs. written infection prevention policies and procedures including standard operating protocols for the services provided by the facility should be formulated and maintained. provision of supplies necessary for hand hygiene products in easily accessible areas should be ensured. definitive infection prevention education and training must be made available. the complete team must be provided with appropriate ppe for the procedure to be performed and their role in the same, to prevent droplet and contact infections. dental healthcare settings must institute procedural protocols for donning and doffing ppe. prior to the treatment, the dental team must identify and review • extra-oral • if intra-oral with double barrier and finger cots *above the age of 5 years **thorough evaluation and clinical signs and symptoms to be noted, risk vs benefit evaluation and individualistic approach required. this table has been created using information from suri et al., 3 alharbi et al., 43 peng et al., 35 and meng et al. 37 the in-procedural protocols, and plan ahead for triage, pre-procedure operatory preparation, minimizing aerosols during procedure and postprocedure disinfection. routine evaluation and reevaluation of the infection prevention program, including adherence to infection prevention practices should be established. • in these unforeseen times, events are unfolding rapidly. hence, all dental practitioners should be abreast with the latest news and guidelines in accordance with state, federal, national, and international bodies. • especially in pediatric dentistry, children should be made to feel comfortable with the new ppe and protocols in the dental practice setting in order to reduce fear and increase cooperation. • a customized approach should be taken by practices to safeguard patients, patients' families, and dental healthcare personnel during and after this pandemic. • although currently catastrophic, even after the critical peak of the outbreak has been contained, stringent ipac protocols need to be updated, followed, and reviewed. severe acute respiratory syndrome and dentistry: a retrospective view coronavirus disease 19 (covid-19): implications for clinical dental care clinical orthodontic management during the covid-19 pandemic can we contain the covid-19 outbreak with the same measures as for sars? pattern of early human-to-human transmission of wuhan anesthetic management of patients with covid 19 infections during emergency 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when a covid-19 patient needs an operation: operating room preparation and guidance simulation as a tool for assessing and evolving your current personal protective equipment: lessons learned during the coronavirus disease (covid-19) pandemic clothing for protection against infectious agents -test method for resistance to dry microbial penetration personal protective equipment for preventing highly infectious diseases due to exposure to contaminated body fluids in healthcare staff testing for viral penetration of non-latex surgical and examination gloves: a comparison of three methods in-use barrier integrity of gloves: latex and nitrile superior to vinyl recommendations for anesthesia in patients suspected of coronavirus 2019-ncov infection high speed handpieces an evaluation of different caries removal techniques in primary teeth: a comparitive clinical study topical silver diamine fluoride for managing dental caries in children and adults oral health management of children during the epidemic period of coronavirus disease 2019 the efficacy of the rubber dam as a barrier to the spread of microorganisms during dental treatment assessing the effectiveness of direct digital radiography barrier sheaths and finger cots contaminated dental aerosols a pilot study of bioaerosol reduction using an air cleaning system during dental procedures persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents survival of influenza viruses on environmental surfaces key: cord-281417-z6k30y1m authors: waggoner, stephen n; reighard, seth d; gyurova, ivayla e; cranert, stacey a; mahl, sarah e; karmele, erik p; mcnally, jonathan p; moran, michael t; brooks, taylor r; yaqoob, fazeela; rydyznski, carolyn e title: roles of natural killer cells in antiviral immunity date: 2016-02-29 journal: current opinion in virology doi: 10.1016/j.coviro.2015.10.008 sha: doc_id: 281417 cord_uid: z6k30y1m natural killer (nk) cells are important in immune defense against virus infections. this is predominantly considered a function of rapid, innate nk-cell killing of virus-infected cells. however, nk cells also prime other immune cells through the release of interferon gamma (ifn-γ) and other cytokines. additionally, nk cells share features with long-lived adaptive immune cells and can impact disease pathogenesis through the inhibition of adaptive immune responses by virus-specific t and b cells. the relative contributions of these diverse and conflicting functions of nk cells in humans are poorly defined and likely context-dependent, thereby complicating the development of therapeutic interventions. here we focus on the contributions of nk cells to disease in diverse virus infections germane to human health. stephen n waggoner 1,2,3,4 , seth d reighard 1,2,3 , ivayla e gyurova 1,4 , stacey a cranert 1 , sarah e mahl 1 , erik p karmele 1 , jonathan p mcnally 1 , michael t moran 1,2 , taylor r brooks 1 , fazeela yaqoob 1,2 and carolyn e rydyznski 1,2 natural killer (nk) cells are important in immune defense against virus infections. this is predominantly considered a function of rapid, innate nk-cell killing of virus-infected cells. however, nk cells also prime other immune cells through the release of interferon gamma (ifn-g) and other cytokines. additionally, nk cells share features with long-lived adaptive immune cells and can impact disease pathogenesis through the inhibition of adaptive immune responses by virus-specific t and b cells. the relative contributions of these diverse and conflicting functions of nk cells in humans are poorly defined and likely context-dependent, thereby complicating the development of therapeutic interventions. here we focus on the contributions of nk cells to disease in diverse virus infections germane to human health. the prevention and control of virus infections involves a complex interplay between diverse cell types of the innate and adaptive immune systems. natural killer (nk) cells are a type of innate lymphoid cell (ilc) that unquestionably play an important role in immune defense against infection in both mice and humans. the contribution of nk cells to cytolytic killing of virusinfected cells is well-established and prominently featured in immunology textbooks. likewise, the importance of early and potent production of pro-inflammatory cytokines like interferon gamma (ifn-g) by nk cells is widely accepted. more recently, there is increasing evidence that nk cells play a key regulatory role in shaping adaptive immune responses to control infection [1] . in this capacity, nk cells have been shown to kill both antigen-presenting cells [2,3] and virus-specific t cells [4,5 ,6,7 ,8 ,9,10] , and can produce anti-inflammatory cytokines like interleukin-10 (il-10) to suppress immunity [11] [12] [13] . nk cells can also play a beneficial regulatory role in stimulating adaptive immunity [14] . finally, a series of recent intriguing studies have questioned the 'innate' nature of nk cells by advancing the concept of long-lived memory nk cells that can contribute to viral control during latent infections or following re-infection [15] [16] [17] . in general, while the significance of nk cells in host defense against virus infection is clear, the relative contributions of their diverse and often conflicting functions (figure 1) to antiviral immunity is poorly defined in humans. therefore, it is difficult to determine whether nk cell activity is beneficial or detrimental during vaccination [18] , and whether strategies to cure chronic infection should aim to enhance or subvert nk cells. this uncertainty is almost undoubtedly compounded by the context-dependence of nk cell activity in different virus infections. in order to complement more in-depth summaries of the regulatory [1], antiviral [19] , and memory functions [20] of nk cells, this review focuses on highlighting what is presently known about the potential involvement of nk cells in different types of virus infections relevant to human disease. herpesviridae: since 1989, it has been clear that rare individuals genetically deficient in nk cells or the functional activity of nk cells display heightened susceptibility to severe diseases conferred by infection with herpesviruses [21 ], including cytomegalovirus (cmv), varicella zoster virus (vzv), epstein-barr virus (ebv), and herpes simplex virus (hsv). this is paralleled by the increased susceptibility of mice lacking nk cells or particular nk-cell receptors to murine cmv (mcmv) infection [22, 23] , wherein infected cells are targets of nk-cell cytolytic attack and the antiviral effects of ifn-g [24] . notably, several herpesviruses encode genes that promote evasion of nk-cell antiviral function [25] . together, these data provide compelling evidence for the importance of the direct antiviral functions of nk cells during herpesvirus infection. in this context, the mcmv model was used to reveal the long-lived nature of nk cells with features of adaptive immune cells [15] . infection of susceptible mice triggered the clonal expansion of nk cells expressing the mcmvspecific activating receptor, ly49h. following contraction, a small population of these cells persisted long-term in mice and demonstrated enhanced recall function against infection compared to unprimed ly49h-expressing nk cells. in humans, an analogous population of cd94/nkg2c-expressing nk cells characterized by epigenetic changes in the ifng locus [26] and other immune loci [27 ,28 ] becomes prominent after hcmv infection [29, 30] . together, these results suggest nk cells have evolved to recognize and control herpesvirus infections in a sustained fashion that leaves a phenotypical and functional imprint on the nk cell repertoire in infected individuals. despite the clear importance of nk cells in immune defense against herpesviruses, several groups have uncovered regulatory functions of nk cells in these infections. removal of nk cells enhanced antiviral t cells responses during mcmv infection [31] , which has been attributed to crosstalk between nk cells and antigen-presenting cells like dendritic cells [2,32-34] as well as production of il-10 by nk cells [11] . additionally, there is some speculation that severe t cell-mediated pathology in the absence of cytotoxic function in hemophagocytic lymphohistiocytosis patients, who suffer severe pathology during uncontrolled virus infections, arises as a consequence of both loss of cytotoxic-mediated elimination of virus infected cells and nk cell-mediated cytotoxic regulation of adaptive immunity [35] . nk cell subversion of antiviral t cells also appeared to be important in preventing development of autoimmune inflammatory conditions associated with persistent herpesvirus infections [36 ] . however, it is unclear to what extent these regulatory functions of nk cells contribute to the antiviral responses against herpesvirus in humans, wherein absence of nk cells is associated with loss of viral control [21 ] . a recently developed model of ebv infection of humanized mice, in which nk cells prevented mononucleosis-like disease by targeting infected cells [37] , may be useful in trying to parse out the relative contributions of nk cell functions to human disease. papovaviridae: the condition of nk cell deficiency in humans is also associated with a loss of control of human papillomavirus (hpv) infection [38] , suggesting that this virus may demonstrate herpesvirus-like susceptibility to nk cell-mediated antiviral function. in addition, the virus-like particles of hpv in vaccines aimed at preventing hpv-induced cancers are potent stimulants of human nk cell activity and crosstalk with dendritic cells [39]. this is not surprising given the vital role of nk cells in antitumor immunity and the propensity of hpv to trigger carcinogenesis. thus, hpv may represent a useful model to examine the induction and function of virus-specific memory nk cells in humans. polyomaviridae: a microrna encoded by two human polyoma viruses, jc and bk, targets the transcripts of a ligand for the activating nk cell receptor, nkg2d, in order to prevent nk cell-mediated lysis of infected cells [40] . similarly, mouse models of polyomavirus infection have revealed a role for nk cells in preventing virus-induced tumor development [41] that is subverted when virus-induced inflammation curtails the expression of a ligand for nkg2d [42] . together, these studies establish that nk cells are important players in immune defense against tumor-promoting dna viruses via elimination of either transformed cells during these infections. poxviridae: nk cells were discovered shortly before the eradication of smallpox, the major poxvirus contributing to human disease. therefore, little is known about the contributions of nk cells to antiviral immunity. nk cells have the potential to (a) recognize and kill virus-infected cells or release antiviral pro-inflammatory cytokines that can inhibit virus replication. these activities can be protective, but can also contribute to (b) pathological damage of host tissues. inflammation and viral antigens can also trigger the development of (c) long-lived memory nk cells that may protect against reinfection or prevent viral reactivation from latency. by contrast, (d) nk cell promotion or inhibition of adaptive immune cells (e.g. t and b cells) or other innate cells (e.g. dendritic cells) can shape the overall immune response against the virus which can have consequences for (e) viral control, disease pathogenesis, and infection outcome. role of nk cells in smallpox pathogenesis. however, ectromelia virus provides a mouse model of smallpox and vaccinia virus is similar enough to smallpox that it served as the active component for vaccination and facilitated global smallpox eradication. in each of these viral infections, nk cells have been shown to play a crucial early role in viral control that involves ifn-g and the cytolytic protein, perforin [22, [43] [44] [45] . moreover, both viruses encode proteins that interfere with nk cell function [46-48]. more recently, memory nk cells that can mediate protection against re-infection were shown to be induced following vaccinia virus administration in mice [17] . thus, like other dna viruses, poxviruses appear to be susceptible to the antiviral effects of nk cells and drive the development of memory nk cells that may provide lasting protection. adenoviridae: similar to other dna viruses, there is evidence that human adenoviruses evade nk cell antiviral functions by sequestering or preventing up-regulation of ligands of activating nk-cells receptors on infected cells [49, 50] . nevertheless, nk cells play a critical role in eliminating adenoviral vectors in the liver [51-53], which may be beneficial during natural adenovirus infection but can also inhibit the efficacy of adenovirus-mediated gene transfer. hepadnaviridae: hepatitis b virus (hbv) infection can result in various infectious pathologies ranging from acute to chronic infections associated with liver disease. dysfunction of nk cells in infected individuals, driven predominately by heightened expression levels of il-10 and tgf-b has been associated with failure to control hbv replication and chronic infection [54,55], consistent with a potential direct antiviral role of nk cells against hbv. however, nk cells can also kill virus-specific cd8t cells in a trail-dependent manner in chronically infected individuals [5 ], thereby suppressing the ability of antiviral t cells to control infection. moreover, the killing of infected hepatocytes by nk cells also contributes to liver damage and development of disease, whereas nk-cell killing of activated stellate cells in hbv-infected individuals may prevent the development of fibrosis [56,57]. these reports suggest nk cells play both beneficial and detrimental roles during hbv infection but the overall contribution of these functions to disease severity remains unclear. arenaviridae: by contrast to the vital role of nk cells in control of herpesvirus infection, nk cells do not suppress replication of lymphocytic choriomeningitis virus (lcmv) in mice [22] , even in the absence of adaptive immune cells [58] . for this reason, the lcmv model was a valuable tool for uncovering the functional contribution of nk cell regulatory function to disease pathogenesis without a confounding contribution of nk cells to direct viral control. nk cells are potently activated by the inflammatory cytokine milieu (e.g. type i ifn) during lcmv infection, resulting in suppression of virus-specific cd4 and cd8t cell responses as well as antigenpresenting cell function [4,9,59,60,61 ]. ironically, type i ifn is also critical for protecting antiviral t cells from nk-cell mediated cytolysis [7 ,8 ] . importantly, nk cell inhibition of antiviral t and b cells could prevent fatal immunopathology [4] and facilitate viral persistence during chronic lcmv infection [4,9,60,61 ]. similar regulatory functions of nk cells during acute lcmv infection contributed to diminished virus-specific memory t-cell and b-cell responses [62 ] . whether nk cells play a similar role in determining disease outcome during infections of humans with lassa, machupo, and other arenaviruses remains to be explored. there is some in vitro evidence that lassa-virus infected apcs stimulate nk cell cytolytic function, which is inhibited by viral nucleoprotein as a viral evasion strategy [63] . flaviviridae: a number of studies have shown that establishment and maintenance of chronic hepatitis c virus (hcv) infection is associated with nk cell dysfunction [57], consistent with an important antiviral function of these cells against this major human pathogen. similar to hbv infection, chronic hcv-induced nk cell activity has the potential to potentiate or limit liver damage and fibrosis depending on the interactions between nk cells and hepatocytes, stellate cells, or other leukocytes. by contrast to chronic hbv infection, depletion of nk cells did not appear to enhance the responses of antiviral cd8t cells [5 ], suggesting immunoregulatory functions of nk cells may be less pronounced in this type of chronic viral infection. the relevance of nk cells to pathogenesis of a number of vector-borne flaviviruses, including dengue virus, west nile virus, and yellow fever virus, has been reported. notably, the yellow fever vaccine 17d (yf-17d) is typically a highly efficacious vaccine, but a recent study found reduced vaccine efficacy in a cohort of vaccine recipients that was associated with an inflamed innate compartment that included highly activated nk cells [64 ] . the high frequency of activated nk cells correlated with poor vaccine responses, including weaker induction of neutralizing antibodies, which suggests a potential role for regulatory nk cells in controlling responses to the yf-17d vaccine or even natural yellow fever virus infection. alternatively, it remains possible that these activated nk cells with an exhausted-like phenotype may be poor candidates for the induction of memory nk cells, which may be critical in immune defense against this virus. preliminary studies in mouse models of dengue and west nile virus infections largely support the concept of direct antiviral functions of nk cells against these pathogens, which is bolstered by reports of viral strategies to evade nk cell mediated attack [65, 66] . orthomyxoviridae: there are conflicting reports concerning the role of nk cells in pathogenesis of influenza a virus infection. most of the data from animal models suggests that nk cells may be directly antiviral [67] , able to recognize and kill virus-infected cells through interactions with influenza hemagglutinin and the receptor, nkp46 [68, 69] . this has been extended to the idea that influenza vaccines stimulate nk cell memory that may be beneficial during subsequent viral infection [70] . by contrast, one report asserted that nk cells suppress influenza-specific t cells and modulate antiviral defense in a regulatory capacity [71] . how these disparate functions fit with the apparent contribution of human nk cells to immunity during influenza infection remains to be determined. paramyxoviridae: respiratory syncytial virus (rsv) is a life-threatening pediatric pathogen associated with severe acute lung pathology. in a mouse model of rsv infection, nk cells made ifn-g that contributed both to the lung disease [72] and to the failure of adaptive immunity to control infection [73] . these limited results suggest that nk cells may not only be dispensable for control of rsv infection, but may in fact be undesirable in rsv immunity in order to limit pathology and optimize adaptive anti-rsv immune responses. although there are few studies of nk cells in ebola virus infection in humans, the repertoire of host nk cell receptors, or killer immunoglobulin-like receptors (kirs), has been linked to fatal outcome of ebola [74] . this may relate to the potential of human nk cells to recognize and kill ebola virus-infected dcs in vitro [75] . in fact, immunization of mice with ebola virus-like particles could protect against lethal ebola virus challenge in an nk cell-dependent and perforindependent manner [76,77], highlighting a possible direct antiviral role for nk cells against this important human pathogen as well as the potential value in vaccine-induced generation of ebola-specific memory nk cells. retroviridae: the role of nk cells in retrovirus infection has been widely studied and yet it remains unclear due to the overall complexity in understanding the correlates of protection against these viruses [19] . in the case of infection with human t-cell leukemia virus type 1 (htlv-1), the sum of available evidence suggests that nk cells are not involved in control of htlv-1 infection or disease outcome [78] . by contrast, genetic studies have revealed an association between the presence of nk cell receptors (e.g. kirs) and slower progression toward aids disease [79] . this is most likely due to nk cell-mediated killing of infected cells, since the protective kir alleles are associated with enhanced nk cell cytolysis in vitro [80] and hiv appears to mutate in order to escape nk cell-mediated kir-facilitated immune pressure [81] . hiv also exerts multiple effects on infected cells in order to subvert nk cell-mediated killing [82] [83] [84] [85] . nevertheless, the activation of nk cells or the lack of certain inhibitory nk cell receptors has been positively correlated with aids progression [86] , suggesting that these cells may promote disease while trying to combat the infection. other studies have highlighted an inverse relationship between nk cells and antiviral t cells [87] , which suggests an element of nk-cell regulation of adaptive immunity at play in the determination of pathogenesis of hiv infection. in fact, nk-cell mediated suppression of adaptive immunity has been observed at distinct stages of friend retrovirus infection of mice, whereas other time points reveal nk cell control of retrovirus replication that was recently shown to be suppressed by regulatory t cells [88, 89] . these studies highlight the potentially complex relationship between nk cells and virus in disease pathogenesis. there has been extensive analysis of changes in nk cell phenotype in function in nonhuman primate models of simian immunodeficiency virus (siv) infection [90] . although nk cells appear capable of antiviral activity against siv, the distribution, phenotype, and functionality are compromised in chronic siv infection [91] [92] [93] . a recent and very exciting study not only revealed the presence of functional siv antigen-specific nk cells present in infected macaques, but highlighted the potential to stimulate virus-specific memory nk cells in macaques through adenoviral vector-mediated vaccination [94 ] . in combination with the realization that hiv-specific memory nk cells could be generated in conventional mice [16] and that distinct memory-like populations of nk cells are present in the blood of hiv-infected but seronegative individuals [95] , these studies highlight the incredible potential of developing means to elicit retrovirus-specific memory nk cell responses through immunization that may prevent infection. rhabdoviridae and togaviridae: although vesicular stomatitis virus (vsv) is a human pathogen, its current importance to human health is in the use of vsv as an oncolytic anti-tumor treatment [96] . as antitumor effector cells, nk cells and vsv would appear to be on the same team against tumors. however, the direct antiviral functions of nk cells against vsv and other oncolytic viruses can limit the antitumor functionality via rapid clearance the virus itself [97, 98] . thus, there may be cases in humans where reduced durability of vaccine or therapeutic viral vectors may be a detrimental consequence of nk cell antiviral functions. by contrast, there is a clear beneficial role for nk cells in therapy with oncolytic togaviruses, like sindbis, which stimulate both direct antitumor functions of the nk cells as well as the positive feedback stimulation of other arms of the immune response via nk cell-derived ifn-g [99] . bunyaviridae: in an analogous manner to hcmv, infection of humans with hantavirus stimulates a rapid and sustained expansion of nkg2c-expressing nk cells [100] . it remains unclear whether this expanded population of nk cells is contributing to viral control, or causing tissue damage associated with hantavirus hemorrhagic fever. il-15 rather than virally encoded factors appears to be the driving force in this nk cell expansion, suggesting there may not be specific nk cell recognition of hantavirus via an nk cell receptor as was the case for mcmv. nonetheless, this may represent another instance of the induction of long-lived nk cells with features of adaptive immune cells. picornaviridae and coronaviridae: depletion of nk cells or low levels of nk cell cytolytic function is associated with increased virus replication and more severe disease during infections with coxsackie virus, encephalomyocarditis virus, and theiler's murine encephalitis virus [101] [102] [103] . nk cells have been similarly implicated in direct inhibition of virus replication and stimulation of liver damage during mouse hepatitis virus (mhv) infection [104, 105] . whether nk cells also play a direct antiviral role in human infections with picornaviruses (coxsackie) or coronaviruses (sars) is not known. in conclusion, there are clear instances in humans where nk cells play an important role in combatting virus infection, most notably against dna viruses. this function can be detrimental when nk cells cause immunopathology or when nk cells are too effective at eliminating viral vectors in oncotherapy and gene therapy trials. there are also defined instances of long-lived memory nk cells that may contribute to immunity and health in ways that are not currently apparent. importantly, the regulatory function of human nk cells cannot be overlooked in the interpretation of experimental results and evaluation of factors contributing to disease. however, current clinical practices must largely favor strategies to stimulate or induce only the antiviral functions of nk cells. the value of activating nk cells in chronic infection has been realized during therapeutic vaccination of sivinfected macaques [106 ] , restoration of hiv-specific t cell responses in human hiv infection [107 ] , ribavirin/ interferon therapy of chronic hepatitis c virus infection in human patients [108] , il-15-based potentiation of anti-hiv immune responses in humanized mice [109] , and probiotic enhancement of control of influenza virus infection in mice [110] . nevertheless, there are also reports that subversion of nk cell function can enhance adaptive immune responses that facilitate better control of virus replication in chronic infection [5 ,36,111 ] . moreover, attempts to subvert the regulatory function in order to enhance adaptive immunity may have detrimental consequences for nk cell-mediated host resistance against latent herpesviruses and other viral pathogens present in most healthy adults. continued evaluation of the specific context-dependent roles of nk cells in human virus infection will be necessary to guide attempts to modulate nk cells in therapy or prevention of infection. waggoner sn, daniels ka, welsh rm: therapeutic depletion of natural killer cells controls persistent infection. j virol 2014, 88:1953-1960 . in a mouse model of chronic infeciton, the authors demonstate that there could be beneficial effects for viral control associated with removal of regulatory nk cells and associated enhancement of antiviral t cell responses. systemic but not local infections elicit immunosuppressive il-10 production by natural killer cells increased natural cytotoxicity receptor expression and relevant il-10 production in nk cells from chronically infected viremic hcv patients nk-cell-mediated killing of target cells triggers robust antigen-specific t-cell-mediated and humoral responses adaptive immune features of natural killer cells critical role for the chemokine receptor cxcr6 in nk cellmediated antigen-specific memory of haptens and viruses generation of cellular immune memory and b-cell immunity is impaired by natural killer cells in mouse models of acute infection, the authors discovery that suppressive functions of nk cells limit the induction and memory t and b cells, in part by impairing the germinal center response and undermining the development of neutralizing antibodies the exonuclease domain of lassa virus nucleoprotein is involved in antigenpresenting-cell-mediated nk cell responses in volunteer yellow fever vaccine recipients in africa and europe, the authors demonstrate that an active immune environment, including activated and exhausted nk cells, is deterimental to vaccine-induced t and b cell responses. this is a clear extension of discoveries in mouse models to the human population, which reveals the potential for identification of individuals who will be poor vaccine responders and highlights the potential for role of natural killer and gamma-delta t cells in west nile virus infection control of acute dengue virus infection by natural killer cells in vivo treatment of mice and hamsters with antibodies to asialo gm1 increases morbidity and mortality to pulmonary influenza infection elucidating the mechanisms of influenza virus recognition by ncr1 lethal influenza infection in the absence of the natural killer cell receptor gene ncr1 influenza vaccine induces intracellular immune memory of human nk cells nk cells regulate cd8+ t cell priming and dendritic cell migration during influenza a infection by ifngamma and perforin-dependent mechanisms natural killer cells are involved in acute lung immune injury caused by respiratory syncytial virus infection neonatal antibody responses are attenuated by interferongamma produced by nk and t cells during rsv infection association of kir2ds1 and kir2ds3 with fatal outcome in ebola virus infection nkp30-dependent cytolysis of filovirusinfected human dendritic cells htlv-1: persistence and pathogenesis hla/kir restraint of hiv: surviving the fittest differential natural killer cell-mediated inhibition of hiv-1 replication based on distinct kir/hla subtypes hiv-1 adaptation to nk-cell-mediated immune pressure degranulation of natural killer cells following interaction with hiv-1-infected cells is hindered by downmodulation of ntb-a by vpu the human immunodeficiency virus type 1 nef and vpu proteins downregulate the natural killer cell-activating ligand pvr mhc class i chain-related protein a shedding in chronic hiv-1 infection is associated with profound nk cell dysfunction differential microrna regulation of hla-c expression and its association with hiv control innate partnership of hla-b and kir3dl1 subtypes against hiv-1 impact of protective killer inhibitory receptor/human leukocyte antigen genotypes on natural killer cell and t-cell function in hiv-1-infected controllers distinct roles of nk cells in viral immunity during different phases of acute friend retrovirus infection activated regulatory t cells suppress effector nk cell responses by an il-2-mediated mechanism during an acute retroviral infection in vivo administration of a jak3 inhibitor to chronically siv infected rhesus macaques leads to nk cell depletion associated with transient modest increase in viral loads accumulation of cytotoxic cd16+ nk cells in simian immunodeficiency virusinfected lymph nodes associated with in situ differentiation and functional anergy suppression of a natural killer cell response by simian immunodeficiency virus peptides progression to aids in siv-infected rhesus macaques is associated with distinct kir and mhc class i polymorphisms and nk cell dysfunction antigen-specific nk cell memory in rhesus macaques the authors made the important discovery that siv and shiv infection of macaques triggers the generation of functional viral antigen-specific nk cells. importantly, siv-specific memory nk cells were also induced in macaques following immunization with an adenoviral vector. these nk cells appeared to mediate antigen-specific effector functions via the nkg2 family of receptors distinct natural killer cells in hiv-exposed seronegative subjects with effector cytotoxic cd56(dim) and cd56(bright) cells and memory-like cd57(+)nkg2c(+)cd56(dim) cells vesicular stomatitis virus as an oncolytic vector enhanced oncolytic potency of vesicular stomatitis virus through vector-mediated inhibition of nk and nkt cells nk cells impede glioblastoma virotherapy through nkp30 and nkp46 natural cytotoxicity receptors activation of cytotoxic and regulatory functions of nk cells by sindbis viral vectors rapid expansion and long-term persistence of elevated nk cell numbers in humans infected with hantavirus murine natural killer cells limit coxsackievirus b3 replication sex-dependent, early cytokine production by nk-like spleen cells following infection with the d variant of encephalomyocarditis virus (emcv-d) role of natural killer cells as immune effectors in encephalitis and demyelination induced by theiler's virus increased killing of liver nk cells by fas/fas ligand and nkg2d/nkg2d ligand contributes to hepatocyte necrosis in virus-induced liver failure natural cytotoxicity against mouse hepatitis virus-infected cells ii. a cytotoxic effector cell with a b lymphocyte phenotype therapeutic envelope vaccination in combination with antiretroviral therapy temporarily rescues siv-specific cd4(+) t-cell-dependent natural killer cell effector responses in chronically infected rhesus macaques cd4+ t-cell help enhances nk cell function following therapeutic hiv-1 vaccination the authors show that therapies that restore virusspecific t cells have the capability of enhacing antiviral nk cell responses ribavirin improves the ifn-gamma response of natural killer cells to ifn-based therapy of hepatitis c virus infection in vivo activation of human nk cells by treatment with an interleukin-15 superagonist potently inhibits acute in vivo hiv-1 infection in humanized mice the authors are supported by national institutes of health grants da038017 and ai118179, the cincinnati children's research foundation, the alpha omega alpha honor medical society, the albert j. ryan foundation, the cancerfree kids pediatric cancer research alliance, and the ellison medical foundation. our apologies to colleagues whose work could not be cited due to space limitations. key: cord-266963-belin2jq authors: cowling, benjamin j; leung, gabriel m title: epidemiological research priorities for public health control of the ongoing global novel coronavirus (2019-ncov) outbreak date: 2020-02-13 journal: euro surveill doi: 10.2807/1560-7917.es.2020.25.6.2000110 sha: doc_id: 266963 cord_uid: belin2jq nan it is now 6 weeks since chinese health authorities announced the discovery of a novel coronavirus (2019-ncov) [1] causing a cluster of pneumonia cases in wuhan, the major transport hub of central china. the earliest human infections had occurred by early december 2019, and a large wet market in central wuhan was linked to most, but not all, of the initial cases [2] . while evidence from the initial outbreak investigations seemed to suggest that 2019-ncov could not easily spread between humans [3] , it is now very clear that infections have been spreading from person to person [2] . we recently estimated that more than 75,000 infections may have occurred in wuhan as at 25 january 2020 [4] , and increasing numbers of infections continue to be detected in other cities in mainland china and around the world. a number of important characteristics of 2019-ncov infection have already been identified, but in order to calibrate public health responses we need improved information on transmission dynamics, severity of the disease, immunity, and the impact of control and mitigation measures that have been applied to date. infections with 2019-ncov can spread from person to person, and in the earliest phase of the outbreak the basic reproductive number was estimated to be around 2.2, assuming a mean serial interval of 7.5 days [2] . the serial interval was not precisely estimated, and a potentially shorter mean serial interval would have corresponded to a slightly lower basic reproductive number. control measures and changes in population behaviour later in january should have reduced the effective reproductive number. however, it is too early to estimate whether the effective reproductive number has been reduced to below the critical threshold of 1 because cases currently being detected and reported would have mostly been infected in mid-to late-january. average delays between infection and illness onset have been estimated at around 5-6 days, with an upper limit of around 11-14 days [2, 5] , and delays from illness onset to laboratory confirmation added a further 10 days on average [2] . chains of transmission have now been reported in a number of locations outside of mainland china. within the coming days or weeks it will become clear whether sustained local transmission has been occurring in other cities outside of hubei province in china, or in other countries. if sustained transmission does occur in other locations, it would be valuable to determine whether there is variation in transmissibility by location, for example because of different behaviours or control measures, or because of different environmental conditions. to address the latter, virus survival studies can be done in the laboratory to confirm whether there are preferred ranges of temperature or humidity for 2019-ncov transmission to occur. in an analysis of the first 425 confirmed cases of infection, 73% of cases with illness onset between 12 and 22 january reported no exposure to either a wet market or another person with symptoms of a respiratory illness [2] . the lack of reported exposure to another ill person could be attributed to lack of awareness or recall bias, but china's health minister publicly warned that pre-symptomatic transmission could be occurring [6] . determining the extent to which asymptomatic or pre-symptomatic transmission might be occurring is an urgent priority, because it has direct implications for public health and hospital infection control. data on viral shedding dynamics could help in assessing duration of infectiousness. for severe acute respiratory syndrome-related coronavirus (sars-cov), infectivity peaked at around 10 days after illness onset [7] , consistent with the peak in viral load at around that time [8] . this allowed control of the sars epidemic through prompt detection of cases and strict isolation. for influenza virus infections, virus shedding is highest on the day of illness onset and relatively higher from shortly before symptom onset until a few days after onset [9] . to date, transmission patterns of 2019-ncov appear more similar to influenza, with contagiousness occurring around the time of symptom onset, rather than sars. transmission of respiratory viruses generally happens through large respiratory droplets, but some respiratory viruses can spread through fine particle aerosols [10] , and indirect transmission via fomites can also play a role. coronaviruses can also infect the human gastrointestinal tract [11, 12] , and faecal-oral transmission might also play a role in this instance. the sars-cov superspreading event at amoy gardens where more than 300 cases were infected was attributed to faecal-oral, then airborne, spread through pressure differentials between contaminated effluent pipes, bathroom floor drains and flushing toilets [13] . the first large identifiable superspreading event during the present 2019-ncov outbreak has apparently taken place on the diamond princess cruise liner quarantined off the coast of yokohama, japan, with at least 130 passengers tested positive for 2019-ncov as at 10 february 2020 [14] . identifying which modes are important for 2019-ncov transmission would inform the importance of personal protective measures such as face masks (and specifically which types) and hand hygiene. the first human infections were identified through a surveillance system for pneumonia of unknown aetiology, and all of the earliest infections therefore had modelling studies incorporating healthcare capacity and processes pneumonia. it is well established that some infections can be severe, particularly in older adults with underlying medical conditions [15, 16] , but based on the generally mild clinical presentation of 2019-ncov cases detected outside china, it appears that there could be many more mild infections than severe infections. determining the spectrum of clinical manifestations of 2019-ncov infections is perhaps the most urgent research priority, because it determines the strength of public health response required. if the seriousness of infection is similar to the 1918/19 spanish influenza, and therefore at the upper end of severity scales in influenza pandemic plans, the same responses would be warranted for 2019-ncov as for the most severe influenza pandemics. if, however, the seriousness of infection is similar to seasonal influenza, especially during milder seasons, mitigation measures could be tuned accordingly. beyond a robust assessment of overall severity, it is also important to determine high risk groups. infections would likely be more severe in older adults, obese individuals or those with underlying medical conditions, but there have not yet been reports of severity of infections in pregnant women, and very few cases have been reported in children [2] . those under 18 years are a critical group to study in order to tease out the relative roles of susceptibility vs severity as possible underlying causes for the very rare recorded instances of infection in this age group. are children protected from infection or do they not fall ill after infection? if they are naturally immune, which is unlikely, we should understand why; otherwise, even if they do not show symptoms, it is important to know if they shed the virus. obviously, the question about virus shedding of those being infected but asymptomatic leads to the crucial question of infectivity. answers to these questions are especially pertinent as basis for decisions on school closure as a social distancing intervention, which can be hugely disruptive not only for students but also because of its knock-on effect for child care and parental duties. very few children have been confirmed 2019-ncov cases so far but that does not necessarily mean that they are less susceptible or that they could not be latent carriers. serosurveys in affected locations could inform this, in addition to truly assessing the clinical severity spectrum. another question on susceptibility is regarding whether 2019-ncov infection confers neutralising immunity, usually but not always, indicated by the presence of neutralising antibodies in convalescent sera. some experts already questioned whether the 2019-ncov may behave similarly to mers-cov in cases exhibiting mild symptoms without eliciting neutralising antibodies [17] . a separate question pertains to the possibility of antibody-dependent enhancement of infection or of disease [18, 19] . if either of these were to be relevant, the transmission dynamics could become more complex. a wide range of control measures can be considered to contain or mitigate an emerging infection such as 2019-ncov. internationally, the past week has seen an increasing number of countries issue travel advisories or outright entry bans on persons from hubei province or china as a whole, as well as substantial cuts in flights to and from affected areas out of commercial considerations. evaluation of these mobility restrictions can confirm their potential effectiveness in delaying local epidemics [20] , and can also inform when as well as how to lift these restrictions. if and when local transmission begins in a particular location, a variety of community mitigation measures can be implemented by health authorities to reduce transmission and thus reduce the growth rate of an epidemic, reduce the height of the epidemic peak and the peak demand on healthcare services, as well as reduce the total number of infected persons [21] . a number of social distancing measures have already been implemented in chinese cities in the past few weeks including school and workplace closures. it should now be an urgent priority to quantify the effects of these measures and specifically whether they can reduce the effective reproductive number below 1, because this will guide the response strategies in other locations. during the 1918/19 influenza pandemic, cities in the united states, which implemented the most aggressive and sustained community measures were the most successful ones in mitigating the impact of that pandemic [22] . similarly to international travel interventions, local social distancing measures should be assessed for their impact and when they could be safely discontinued, albeit in a coordinated and deliberate manner across china such that recrudescence in the epidemic curve is minimised. mobile telephony global positioning system (gps) data and location services data from social media providers such as baidu and tencent in china could become the first occasion when these data inform outbreak control in real time. at the individual level, surgical face masks have often been a particularly visible image from affected cities in china. face masks are essential components of personal protective equipment in healthcare settings, and should be recommended for ill persons in the community or for those who care for ill persons. however, there is now a shortage of supply of masks in china and elsewhere, and debates are ongoing about their protective value for uninfected persons in the general community. the table summarises research gaps to guide the public health response identified. in conclusion, there are a number of urgent research priorities to inform the public health response to the global spread of 2019-ncov infections. establishing robust estimates of the clinical severity of infections is probably the most pressing, because flattening out the surge in hospital admissions would be essential if there is a danger of hospitals becoming overwhelmed with patients who require inpatient care, not only for those infected with 2019-ncov but also for urgent acute care of patients with other conditions including those scheduled for procedures and operations. in addressing the research gaps identified here, there is a need for strong collaboration of a competent corps of epidemiological scientists and public health workers who have the flexibility to cope with the surge capacity required, as well as support from laboratories that can deliver on the ever rising demand for diagnostic tests for 2019-ncov and related sequelae. the readiness survey by reusken et al. in this issue of eurosurveillance testifies to the rapid response and capabilities of laboratories across europe should the outbreak originating in wuhan reach this continent [23] . in the medium term, we look towards the identification of efficacious pharmaceutical agents to prevent and treat what may likely become an endemic infection globally. beyond the first year, one interesting possibility in the longer term, perhaps borne of wishful hope, is that after the first few epidemic waves, the subsequent endemic re-infections could be of milder severity. particularly if children are being infected and are developing immunity hereafter, 2019-ncov could optimistically 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chughtai, abrar ahmad; khan, wasiq title: use of personal protective equipment to protect against respiratory infections in pakistan: a systematic review date: 2020-03-04 journal: j infect public health doi: 10.1016/j.jiph.2020.02.032 sha: doc_id: 305146 cord_uid: iprzeigk like other low-income countries, limited data are available on the use of personal protective equipment (ppe) in pakistan. we conducted a systematic review of studies on ppe use for respiratory infections in healthcare settings in pakistan. medline, embase and goggle scholar were searched for clinical, epidemiological and laboratory-based studies in english, and 13 studies were included; all were observational/cross-sectional studies. the studies examined ppe use in hospital (n = 7), dental (n = 4) or laboratory (n = 2) settings. policies and practices on ppe use were inconsistent. face masks and gloves were the most commonly used ppe to protect from respiratory and other infections. ppe was not available in many facilities and its use was limited to high-risk situations. compliance with ppe use was low among healthcare workers, and reuse of ppe was reported. clear policies on the use of ppe and available ppe are needed to avoid inappropriate practices that could result in the spread of infection. large, multimethod studies are recommended on ppe use to inform national infection-control guidelines. healthcare workers are at the frontline when treating infectious disease cases and at high risk of acquiring influenza and other respiratory infections [1] [2] [3] . several outbreaks of new infectious diseases have occurred in recent decades, such as the outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in 2002-2003 [4] , influenza pandemic (h1n1) in 2009 [5] , middle east respiratory syndrome coronavirus (mers-cov) in 2012 [6] and ebola virus diseases in 2014-2016 [7] . many healthcare workers were infected and died during these outbreaks because of a lack of infection control [4, 8, 9, 10] various infection control strategies are used to protect healthcare workers from respiratory and other infections in healthcare settings [11, 12] . these strategies can be broadly classified as administrative control measures, environmental control measures and the use of personal protective equipment (ppe). administrative control measures include developing policies and procedures, implementing triage protocols and providing health education and trainings. environmental control measures includes ensuring proper ventilation, establishing airborne infection isolation and negative pressure rooms, developing systems for cleaning and waste disposal. [13, 14] . ppe is commonly used in healthcare settings as standard or transmission based precaution to protect healthcare workers from infections and to prevent further spread to patients around them [14, 15] . ppe is generally ranked lowest in the infection control hierarchy due to less effectiveness compared to other control measures and high expenditure in the long run. therefore, most infection control guidelines recommend using ppe together with other administrative and environmental control measures. however, ppe is important during the early stage of an outbreak or a pandemic when drugs, a vaccine and other control measures are not available, or access is limited. commonly used ppe to protect from respiratory infectionsare; face masks, respirators, gloves, and goggles or face shields [16] . face masks (or medical masks) and respirators are the most commonly used ppe to protect from influenza and other respiratory infection in healthcare settings. however, these two products are not the same. face masks are not designed for respiratory protection and are used to avoid respiratory droplet and spray of body fluids on the face. they are also used by sick patients to prevent spread of pathogens to others (referred to as "source control"), or by surgeons in the operating theatre to maintain a sterile operating field. face masks are not fit to the face and have [17] . respirators are designed for respiratory protection and are used to protect from respiratory aerosols [18] . a properly fitted respirator provides better protection again respiratory infections than a face mask. gloves are used to protect hands from blood and body fluids, including respiratory secretions. goggles and face shields are used to prevent transfer of respiratory pathogens into the eyes from contaminated hands and other sources. gowns, coveralls, surgical hoods and shoe covers can also be used where procedures on infectious patients generate aerosols or when a new respiratory virus has emerged [18] . there is an ongoing debate about the selection and use of various types of ppe in healthcare settings. this is mainly because of a lack of high quality studies on the use of ppe. most studies are observational and on the use of masks and/or respirators [18] . to date, only five randomized clinical trials have been conducted on use of ppe in hospital settings and all were on face masks/respirators [18] . moreover, most studies on ppe use were conducted in high/middle income countries and currently there are limited data from lowincome countries where the burden of infectious diseases is high. it is therefore important to examine the use of ppe in low resource countries to inform infection control policies. pakistan has a population of about 200 million. as a low-income country, its gross domestic product is low, as is its expenditure on health [19] . the country has one of highest rates of infant and maternal mortality in the south asia region. infectious diseases are still among the main causes of death, particularly in young children. health and surveillance systems are generally weak and limited data are available on infection prevention and control strategies. the aim of this study was to examine the use of ppe for respiratory infections in healthcare settings in pakistan. a systematic review was conducted using the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines. we searched for studies on the electronic databases medline and embase using selected key words. a combination of keywords were used including: 'face mask' or 'mask' or 'medical mask' or 'surgical mask' or 'cloth mask' or 'respirator' or 'gloves' or 'gowns' or 'coverall' or 'surgical cap/hood' or 'shoe/boot covers' or 'goggles' or 'face shield' or 'eye protection' and ' respiratory infection' or 'respiratory tract infection' or 'respiratory diseases', 'outbreaks' or 'infectious disease' or 'influenza' or 'pandemic influenza' or 'flu' or 'tuberculosis' or 'pneumonia' and 'pakistan' or 'punjab' or 'sindh' or 'balochistan' or 'khyber pakhtunkhwa'. we used an open date strategy up to december 2017. we anticipated that studies published in local journals might not be indexed on the medline or embase, therefore, an additional search was made on google scholar using the same keywords. we set a limit of 20 results per page on google scholar and first three pages were reviewed for each keyword search. after the initial search; we reviewed titles and abstracts and selected studies for full text review (fig. 1) . clinical, epidemiological and laboratory-based studies conducted in any part of pakistan and published in english were included in the review. the focus of this systematic review was on the use of ppe for prevention of respiratory infections. therefore, we only included those studies which examined the use of facemask and/or respirator in healthcare settings, with or without other ppe. we only included those studies where ppe was discussed for respiratory infections. studies where ppe was examined for general infection control were also included, given respiratory protective equipment (face masks and/or respirators) was mentioned. we excluded studies on the use of ppe only for bloodborne infections. conference abstracts and poster presentations were also excluded. a total of 137 studies were found in the initial search. after reviewing titles and abstracts, 46 studies were selected for full text review. finally, 13 articles were included in this review (table 1 ) [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] . we only found observational/cross-sectional studies on the use of ppe for infectious diseases in healthcare settings in pakistan. in all studies data were collected through questionnaires or interviews. no clinical trials or laboratory-based studies on the use of ppe in such settings were found. seven studies examined the use of ppe in hospital [20] [21] [22] [23] 25] and among those, two examined the ppe perceptions among medical students [24] or pharmacy students [32] . two studies were conducted in the laboratory settings [26, 27] while, four in dental settings [28] [29] [30] [31] two studies focused on the use of ppe for influenza [24, 32] , two were for tuberculosis [20, 25] and nine studies were on multiple respiratory diseases, including influenza [21, 22] or general infections [23, [26] [27] [28] [29] [30] [31] . only two studies examined the use of ppe alone [21, 22] , while other studies examined other infection control practices as well [20, [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] . guidelines and standard operating procedures on ppe do not exist in most of the hospitals [23] or laboratories [26, 27] in pakistan. two studies examined the guidelines and current practices on the use of face masks/respirators for influenza, tuberculosis and sars in pakistan [21, 22] . recommendations on the use of masks were reported to be inconsistent and different types of product were recommended and used in various healthcare settings [21, 22] . face masks were the most commonly used ppe to protect from respiratory infections in most hospitals in pakistan. medical masks were generally used to protect from influenza, tuberculosis and other respiratory infections, while the use of respirators was limited to high-risk situations [21, 22] . in a cross-sectional survey among final-year pharmacy students in seven universities of karachi, about 60% of 443 participants highlighted the need to cover the nose or mouth to protect from influenza and about 57% highlighted the use of face masks, gloves and other ppe [32] . laboratory coat and gloves were the most commonly used ppe in the laboratories in pakistan while face masks and eye covers were rarely used [26, 27] . a survey of dentists working in various settings (dental colleges, hospitals and private clinics) showed that face masks and gloves were also commonly used ppe [29] . the use of ppe was also reported to be low among health workers. according to a hospital-based survey, face masks are not provided to patients with tuberculosis and respirators are not provided to the healthcare workers [23] . another survey showed that 25% of participants used ppe for patients with suspected tuberculosis and 56% used ppe for patients with confirmed tuberculosis [25] . a study in a ward for patients with multidrug-resistant tuberculosis reported that 65% of the healthcare workers used n95 respirators and 58% were provided with a mask [20] . a study on 253 biosafety level (bsl) 2 laboratory workers showed that ppe was not used by about half of the staff (46.2%) [27] . a countrywide survey showed that almost one third (31.9%) of bsl-2 laboratory workers did not routinely use ppe [26] . both gloves and laboratory coats were used by only 26.7% of the personnel, while a laboratory coat or gloves alone were used by 30.4% and 8.1%, respectively. less than 1% of all the respondents across pakistan reported using eye covers [26] . in a survey of medical students during pandemic (h1n1) 2009, 75% said that they would use a face mask to protect from infection. students with less risk perception were more hesitant to use face masks [24] . the use of face masks was common in dental practice and according to various surveys, 68-100% of dentists wear masks during dental procedures [28] [29] [30] [31] . across all the studies in dental settings, more than 90% also used gloves. among the ppe, face masks were considered the most bothersome to use by wearers. reuse of ppe was also reported in many studies, mainly because of unavailability of ppe and lack of training. gowns are shared among the healthcare workers in hospital many times [23] . two surveys in dental clinics showed that more than half of the dentist reuse masks during routine work [28, 29] . the availability of ppe was generally low in all healthcare settings [21, 22, 28] and varied according to the type [23] ; gloves and masks were available while gowns and n95 respirators were not available in several wards [23] . a shortage of ppe was also reported during sars and pandemic (h1n1) 2009 [21, 22] . a lack of training was a common issue reported and most healthcare workers were not trained in the use of ppe. most of the studies (11/13) discussed other infection control practices as well, in addition to the use of ppe. other non-standard infection control practices included reuse of syringes, improper waste disposal, a lack of hand hygiene practices, non-isolation of infectious cases and low influenza vaccination among healthcare workers. we reviewed the use of ppe in various healthcare settings in pakistan. a lack of guidelines and standard operating procedures, inconsistent policies and practices, low compliance, and non-availability and reuse of ppe were the main issues highlighted in this study. evidence is lacking on the use of ppe in hospitals and other healthcare settings in pakistan and most studies are of low quality. clinical studies should be conducted to examine the effectiveness of ppe and improve the compliance. reuse of ppe may increase the risk of self-contamination to the wearer and this practice should be discontinued. there is a need to improve the availability of ppe and healthcare workers should be trained. ppe is generally considered lowest in the infection control hierarchy and is generally recommended in combination with other control measures. other infection control practices in such settings should also be examined. different types of ppe are used by healthcare workers in pakistan, which reflects a lack of standard policies and guidelines. the different policies and practices may be because of the different recommendations by the world health organization (who) and the united states (us) centers for disease control and prevention (cdc) [33, 34] . debate continues about the selection and use ppe for different infections, for example, face masks versus respirators, gowns versus coveralls, face shields versus goggles [7, 18, 34, 35] . selection of ppe mainly depends on mode of transmission, however, several individual and organizational factors also contribute the selection and use of ppe, such as risk perception, presence of adverse events, pre-existing medical illness, availability and cost [7] . respiratory infections are generally transmitted through contact, droplet and/or airborne routes. gloves should be used to protect from infections transmitted through contact (e.g. respiratory syncytial virus and adenovirus), face masks should be used for droplet infections (e.g. influenza and coronavirus) and a respirator should be used to protect form airborne infection (e.g. tuberculosis and measles). however, infection transmission is rarely by only one route and most infections are transmitted by more than one route [36] . for example, influenza and sars primarily transmit through droplet and contact routes, but airborne transmission has also been reported [37, 38] . similarly, ebola primarily transmits through direct contact with blood and body fluids [39] , but animal studies have shown that airborne transmission is also possible [7] . the risk of transmission further increases during aerosol-generating and other high-risk procedures [40, 41] . moreover, uncertainty exists about how pathogens transmit during outbreaks and pandemics [14, 34, 42, 43] . therefore, superior ppe should be used where the mode of transmission is uncertain, the case-fatality rate is high and pharmaceutical interventions are not available [7] . infection control guidelines in pakistan need to be updated urgently to reflect these recommendations. given that mers cov is circulating in the eastern mediterranean region (emr), policies and practices on the use of ppe in other countries of the region should also be examined. our study also reported low availability of ppe in hospital, dental and laboratory settings in pakistan. the availability of ppe is a challenge, not only in low-resource counties, but also in highincome countries, particularly during outbreaks and pandemics when the use of ppe greatly increases [46, 47] . this may result in non-standard practices such as reuse and extended use of ppe. shortages of ppe were even reported in many high-income countries during the 2009 influenza h1n1 pandemic and staff had to use various alternatives [46] [47] [48] . the availability of ppe is important to ensure proper use and compliance. low use of ppe among laboratory workers in pakistan may be due to non-availability and a lack of resources. for example, ppe use was relatively higher in laboratory workers in punjab, which is an affluent province, than other provinces [26] . moreover ppe use was reported more in the private sector in pakistan than the public sector which has fewer resources [27] . proper use of ppe depends on several factors such as availability, knowledge, training, risk perception and comfort [7, 44, 45] . this study showed the compliance with the use of ppe was generally low among healthcare workers and was mainly due to unavailability of ppe, discomfort and a lack of training. while the use ppe depends on many factors, a greater perception of risk was positively associated with compliance [24] . continuous use of face masks and respirators may have psychological and physiological effects on the wearer and result in more adverse events [49] [50] [51] . compliance with the use of face masks has been shown to be based on the nature of the disease, infectiousness of patients and the performance of high-risk procedures [52] . previous studies have tested the precede (predisposing, reinforcing and enabling) framework to examine healthcare workers' compliance with universal precautions [53] . the results showed that reinforcing factors, such as availability of ppe and less job hindrance, and enabling factors, such as safety climate and regular feedback, were significant predictors of compliance with ppe [53] . in addition, the health belief model [54] was also used to examine the compliance and use of face mask during the sars outbreak [55, 56] . perceived susceptibility (vulnerability to acquiring sars and close contact with case), perceived benefits (that face masks can prevent infection) and cues to action (someone asked them to use face masks) were significant predictors of protective behaviour and use of face masks [55] . our study showed that most healthcare workers were not trained on the use of ppe in pakistan. the risk of infection can be reduced with proper training and availability of policies and standard operating procedures [57] . however, regular monitoring is also required to make sure that healthcare workers are using ppe according to the protocols. a study in the us reported many deviations from the protocols even though all healthcare workers were trained [15] . this may result in self-contamination to the wearers and the spread of infection to others [58] . training programmes should be arranged for newly recruited staff and then annual refresher courses should be provided. our study had some limitations. the initial search was made on medline and embase but very few studies were retrieved because many papers are not indexed on these databases. therefore, we also searched google scholar but we only reviewed the first 3 pages after each search so some studies could have been missed. however, we checked the references lists of the relevant studies and could not find any other studies. our search was up to 2017 and studies in 2018 were not included. we only considered ppe in this study and did not examine other infection control practices. the use of ppe is generally recommended with other administrative and environmental control measures. the selection and use of ppe vary according to the type of healthcare worker and working environment. face masks and gloves were the most commonly used ppe to protect from respiratory and other infections. overall, compliance with the use of ppe was low, and non-availability and reuse of ppe were reported. most studies were observational and large-scale prospective studies are needed to collect more evidence about the use of ppe in healthcare settings in pakistan. no funding sources. aac tested the filtration of mask samples by 3 min in another study; 3m products were not used in this study. wk declares none. as this was a systematic review of published data, ethics approval was not required. aac devised the structure and topic areas for this review and made the initial search. wk and aac reviewed titles and abstracts and selected studies for full text review. aac prepared the first draft of manuscript and both authors contributed equally to the final manuscript. influenza and rhinovirus infections among health-care workers nosocomial transmission of measles among healthcare workers tuberculosis among health care workers emergencies preparedness, 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self-contamination during doffing of personal protective equipment key: cord-276758-k2imddzr authors: siegel, jane d.; rhinehart, emily; jackson, marguerite; chiarello, linda title: 2007 guideline for isolation precautions: preventing transmission of infectious agents in health care settings date: 2007-12-07 journal: am j infect control doi: 10.1016/j.ajic.2007.10.007 sha: doc_id: 276758 cord_uid: k2imddzr nan . clinical syndromes or conditions warranting additional empiric transmission-based precautions pending confirmation of diagnosis table 3 . infection control considerations for highpriority (cdc category a) diseases that may result from bioterrorist attacks or are considered bioterrorist threats table 4 . recommendations for application of standard precautions for the care of all patients in all health care settings table 5 . components of a protective environment 1. the transition of health care delivery from primarily acute care hospitals to other health care settings (eg, home care, ambulatory care, freestanding specialty care sites, long-term care) created a need for recommendations that can be applied in all health care settings using common principles of infection control practice, yet can be modified to reflect setting-specific needs. accordingly, the revised guideline addresses the spectrum of health care delivery settings. furthermore, the term ''nosocomial infections'' is replaced by ''health care-associated infections'' (hais), to reflect the changing patterns in health care delivery and difficulty in determining the geographic site of exposure to an infectious agent and/ or acquisition of infection. 2. the emergence of new pathogens (eg, severe acute respiratory syndrome coronavirus [sars-cov] associated with sars avian influenza in humans), renewed concern for evolving known pathogens (eg, clostridium difficile, noroviruses, communityassociated methicillin-resistant staphylococcus aureus [ca-mrsa]), development of new therapies (eg, gene therapy), and increasing concern for the threat of bioweapons attacks, necessitates addressing a broader scope of issues than in previous isolation guidelines. 3. the successful experience with standard precautions, first recommended in the 1996 guideline, has led to a reaffirmation of this approach as the foundation for preventing transmission of infectious agents in all health care settings. new additions to the recommendations for standard precautions are respiratory hygiene/cough etiquette and safe injection practices, including the use of a mask when performing certain highrisk, prolonged procedures involving spinal canal punctures (eg, myelography, epidural anesthesia). the need for a recommendation for respiratory hygiene/cough etiquette grew out of observations during the sars outbreaks, when failure to implement simple source control measures with patients, visitors, and health care workers (hcws) with respiratory symptoms may have contributed to sars-cov transmission. the recommended practices have a strong evidence base. the continued occurrence of outbreaks of hepatitis b and hepatitis c viruses in ambulatory settings indicated a need to reiterate safe injection practice recommendations as part of standard precautions. the addition of a mask for certain spinal injections grew from recent evidence of an associated risk for developing meningitis caused by respiratory flora. 4. the accumulated evidence that environmental controls decrease the risk of life-threatening fungal infections in the most severely immunocompromised patients (ie, those undergoing allogeneic hematopoietic stem cell transplantation [hsct] ) led to the update on the components of the protective environment (pe). 5. evidence that organizational characteristics (eg, nurse staffing levels and composition, establishment of a safety culture) influence hcws' adherence to recommended infection control practices, and thus are important factors in preventing transmission of infectious agents, led to a new emphasis and recommendations for administrative involvement in the development and support of infection control programs. 6. continued increase in the incidence of hais caused by multidrug-resistant organisms (mdros) in all health care settings and the expanded body of knowledge concerning prevention of transmission of mdros created a need for more specific recommendations for surveillance and control of these pathogens that would be practical and effective in various types of health care settings. this document is intended for use by infection control staff, health care epidemiologists, health care administrators, nurses, other health care providers, and persons responsible for developing, implementing, and evaluating infection control programs for health care settings across the continuum of care. the reader is referred to other guidelines and websites for more detailed information and for recommendations concerning specialized infection control problems. part i reviews the relevant scientific literature that supports the recommended prevention and control practices. as in the 1996 guideline, the modes and factors that influence transmission risks are described in detail. new to the section on transmission are discussions of bioaerosols and of how droplet and airborne transmission may contribute to infection transmission. this became a concern during the sars outbreaks of 2003, when transmission associated with aerosol-generating procedures was observed. also new is a definition of ''epidemiologically important organisms'' that was developed to assist in the identification of clusters of infections that require investigation (ie multidrug-resistant organisms, c difficile). several other pathogens of special infection control interest (ie, norovirus, sars, centers for disease control and prevention [cdc] category a bioterrorist agents, prions, monkeypox, and the hemorrhagic fever viruses) also are discussed, to present new information and infection control lessons learned from experience with these agents. this section of the guideline also presents information on infection risks associated with specific health care settings and patient populations. part ii updates information on the basic principles of hand hygiene, barrier precautions, safe work practices, and isolation practices that were included in previous guidelines. however, new to this guideline is important information on health care system components that influence transmission risks, including those components under the influence of health care administrators. an important administrative priority that is described is the need for appropriate infection control staffing to meet the ever-expanding role of infection control professionals in the complex modern health care system. evidence presented also demonstrates another administrative concern: the importance of nurse staffing levels, including ensuring numbers of appropriately trained nurses in intensive care units (icus) for preventing hais. the role of the clinical microbiology laboratory in supporting infection control is described, to emphasize the need for this service in health care facilities. other factors that influence transmission risks are discussed, including the adherence of hcws to recommended infection control practices, organizational safety culture or climate, and education and training. discussed for the first time in an isolation guideline is surveillance of health care-associated infections. the information presented will be useful to new infection control professionals as well as persons involved in designing or responding to state programs for public reporting of hai rates. part iii describes each of the categories of precautions developed by the health care infection control practices advisory committee (hicpac) and the cdc and provides guidance for their application in various health care settings. the categories of transmission-based precautions are unchanged from those in the 1996 guideline: contact, droplet, and airborne. one important change is the recommendation to don the indicated personal protective equipment (ppe-gowns, gloves, mask) on entry into the patient's room for patients who are on contact and/or droplet precautions, because the nature of the interaction with the patient cannot be predicted with certainty, and contaminated environmental surfaces are important sources for transmission of pathogens. in addition, the pe for patients undergoing allogeneic hsct, described in previous guidelines, has been updated. five tables summarize important information. table 1 provides a summary of the evolution of this document. table 2 gives guidance on using empiric isolation precautions according to a clinical syndrome. table 3 summarizes infection control recommendations for cdc category a agents of bioterrorism. table 4 lists the components of standard precautions and recommendations for their application, and table 5 lists components of the pe. a glossary of definitions used in this guideline also is provided. new to this edition of the guideline is a figure showing the recommended sequence for donning and removing ppe used for isolation precautions to optimize safety and prevent self-contamination during removal. appendix a provides an updated alphabetical list of most infectious agents and clinical conditions for which isolation precautions are recommended. a preamble to the appendix provides a rationale for recommending the use of 1 or more transmission-based precautions in addition to standard precautions, based on a review of the literature and evidence demonstrating a real or potential risk for person-to-person transmission in health care settings. the type and duration of recommended precautions are presented, with additional comments concerning the use of adjunctive measures or other relevant considerations to prevent transmission of the specific agent. relevant citations are included. new to this guideline is a comprehensive review and detailed recommendations for prevention of transmission of mdros. this portion of the guideline was published electronically in october 2006 and updated in november 2006 (siegel jd, rhinehart e, jackson m, chiarello l and hicpac. management of multidrug-resistant organisms in health care settings, 2006; available from http://www.cdc.gov/ ncidod/dhqp/pdf/ar/mdroguideline2006.pdf), and is considered a part of the guideline for isolation precautions. this section provides a detailed review of the complex topic of mdro control in health care settings and is intended to provide a context for evaluation of mdro at individual health care settings. a rationale and institutional requirements for developing an effective mdro control program are summarized. although the focus of this guideline is on measures to prevent transmission of mdros in health care settings, information concerning the judicious use of antimicrobial agents also is presented, because such practices are intricately related to the size of the reservoir of mdros, which in turn influences transmission (eg, colonization pressure). two tables summarize recommended prevention and control practices using 7 categories of interventions to control mdros: administrative measures, education of hcws, judicious antimicrobial use, surveillance, infection control precautions, environmental measures, and decolonization. recommendations for each category apply to and are adapted for the various health care settings. with the increasing incidence and prevalence of mdros, all health care facilities must prioritize effective control of mdro transmission. facilities should identify prevalent mdros at the facility, implement control measures, assess the effectiveness of control programs, and demonstrate decreasing mdro rates. a set of intensified mdro prevention interventions is to be added if the incidence of transmission of a target mdro is not decreasing despite implementation of basic mdro infection control measures, and when the first case of an epidemiologically important mdro is identified within a health care facility. this updated guideline responds to changes in health care delivery and addresses new concerns about transmission of infectious agents to patients and hcws in the united states and infection control. the primary objective of the guideline is to improve the safety of the nation's health care delivery system by reducing the rates of hais. instruct symptomatic persons to cover mouth/nose when sneezing/ coughing; use tissues and dispose in no-touch receptacle; observe hand hygiene after soiling of hands with respiratory secretions; wear surgical mask if tolerated or maintain spatial separation, .3 feet if possible. *during aerosol-generating procedures on patients with suspected or proven infections transmitted by respiratory aerosols (eg, severe acute respiratory syndrome), wear a fittested n95 or higher respirator in addition to gloves, gown, and face/eye protection. -proper construction of windows, doors, and intake and exhaust ports -ceilings: smooth, free of fissures, open joints, crevices -walls sealed above and below the ceiling -if leakage detected, locate source and make necessary repairs d ventilation to maintain $12 air changes/hour d directed air flow; air supply and exhaust grills located so that clean, filtered air enters from one side of the room, flows across the patient's bed, and exits on opposite side of the room d positive room air pressure in relation to the corridor; pressure differential of .2.5 pa (0.01-inch water gauge) d air flow patterns monitored and recorded daily using visual methods (eg, flutter strips, smoke tubes) or a hand-held pressure gauge d self-closing door on all room exits d back-up ventilation equipment (eg, portable units for fans or filters) maintained for emergency provision of ventilation requirements for pe areas, with immediate steps taken to restore the fixed ventilation system d for patients who require both a pe and an airborne infection isolation room (aiir), use an anteroom to ensure proper air balance relationships and provide independent exhaust of contaminated air to the outside, or place a hepa filter in the exhaust duct. (2) reaffirm standard precautions as the foundation for preventing transmission during patient care in all health care settings; (3) reaffirm the importance of implementing transmission-based precautions based on the clinical presentation or syndrome and likely pathogens until the infectious etiology has been determined ( table 2) ; and (4) provide epidemiologically sound and, whenever possible, evidence-based recommendations. this guideline is designed for use by individuals who are charged with administering infection control programs in hospitals and other health care settings. the information also will be useful for other hcws, health care administrators, and anyone needing information about infection control measures to prevent transmission of infectious agents. commonly used abbreviations are provided, and terms used in the guideline are defined in the glossary. medline and pubmed were used to search for relevant studies published in english, focusing on those published since 1996. much of the evidence cited for preventing transmission of infectious agents in health care settings is derived from studies that used ''quasiexperimental designs,'' also referred to as nonrandomized preintervention and postintervention study designs. 2 although these types of studies can provide valuable information regarding the effectiveness of various interventions, several factors decrease the certainty of attributing improved outcome to a specific intervention. these include: difficulties in controlling for important confounding variables, the use of multiple interventions during an outbreak, and results that are explained by the statistical principle of regression to the mean (eg, improvement over time without any intervention). 3 observational studies remain relevant and have been used to evaluate infection control interventions. 4, 5 the quality of studies, consistency of results, and correlation with results from randomized controlled trials, when available, were considered during the literature review and assignment of evidencebased categories (see part iv: recommendations) to the recommendations in this guideline. several authors have summarized properties to consider when evaluating studies for the purpose of determining whether the results should change practice or in designing new studies. 2,6,7 this guideline contains 4 changes in terminology from the 1996 guideline: 1. the term ''nosocomial infection'' is retained to refer only to infections acquired in hospitals. the term ''health care-associated infection'' (hai) is used to refer to infections associated with health care delivery in any setting (eg, hospitals, long-term care facilities, ambulatory settings, home care). this term reflects the inability to determine with certainty where the pathogen was acquired, because patients may be colonized with or exposed to potential pathogens outside of the health care setting before receiving health care, or may develop infections caused by those pathogens when exposed to the conditions associated with delivery of health care. in addition, patients frequently move among the various settings within the health care system. 8 of infectious agents, a susceptible host with a portal of entry receptive to the agent, and a mode of transmission for the agent. this section describes the interrelationship of these elements in the epidemiology of hais. i.b.1. sources of infectious agents. infectious agents transmitted during health care derive primarily from human sources but inanimate environmental sources also are implicated in transmission. human reservoirs include patients, [20] [21] [22] [23] [24] [25] [26] [27] [28] hcws, 17, [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] and household members and other visitors. [40] [41] [42] [43] [44] [45] such source individuals may have active infections, may be in the asymptomatic and/or incubation period of an infectious disease, or may be transiently or chronically colonized with pathogenic microorganisms, particularly in the respiratory and gastrointestinal tracts. other sources of hais are the endogenous flora of patients (eg, bacteria residing in the respiratory or gastrointestinal tract). [46] [47] [48] [49] [50] [51] [52] [53] [54] i.b.2. susceptible hosts. infection is the result of a complex interrelationship between a potential host and an infectious agent. most of the factors that influence infection and the occurrence and severity of disease are related to the host. however, characteristics of the host-agent interaction as it relates to pathogenicity, virulence, and antigenicity also are important, as are the infectious dose, mechanisms of disease production, and route of exposure. 55 there is a spectrum of possible outcomes after exposure to an infectious agent. some persons exposed to pathogenic microorganisms never develop symptomatic disease, whereas others become severely ill and even die. some individuals are prone to becoming transiently or permanently colonized but remain asymptomatic. still others progress from colonization to symptomatic disease either immediately after exposure or after a period of asymptomatic colonization. the immune state at the time of exposure to an infectious agent, interaction between pathogens, and virulence factors intrinsic to the agent are important predictors of an individual's outcome. host factors such as extremes of age and underlying disease (eg, diabetes 56,57 , human immunodeficiency virus/acquired immune deficiency syndrome [hiv/ aids], 58,59 malignancy, and transplantation 18, 60, 61 ) can increase susceptibility to infection, as can various medications that alter the normal flora (eg, antimicrobial agents, gastric acid suppressors, corticosteroids, antirejection drugs, antineoplastic agents, immunosuppressive drugs). surgical procedures and radiation therapy impair defenses of the skin and other involved organ systems. indwelling devices, such as urinary catheters, endotracheal tubes, central venous and arterial catheters, [62] [63] [64] and synthetic implants, facilitate development of hais by allowing potential pathogens to bypass local defenses that ordinarily would impede their invasion and by providing surfaces for development of biofilms that may facilitate adherence of microorganisms and protect from antimicrobial activity. 65 some infections associated with invasive procedures result from transmission within the health care facility; others arise from the patient's endogenous flora. 46 clothing, uniforms, laboratory coats, or isolation gowns used as ppe may become contaminated with potential pathogens after care of a patient colonized or infected with an infectious agent, (eg, mrsa, 88 vancomycin-resistant enterococci [vre], 89 and c difficile 90 ). although contaminated clothing has not been implicated directly in transmission, the potential exists for soiled garments to transfer infectious agents to successive patients. i.b.3.b. droplet transmission. droplet transmission is technically a form of contact transmission; some infectious agents transmitted by the droplet route also may be transmitted by direct and indirect contact routes. however, in contrast to contact transmission, respiratory droplets carrying infectious pathogens transmit infection when they travel directly from the respiratory tract of the infectious individual to susceptible mucosal surfaces of the recipient, generally over short distances, necessitating facial protection. respiratory droplets are generated when an infected person coughs, sneezes, or talks 91, 92 or during such procedures as suctioning, endotracheal intubation, [93] [94] [95] [96] cough induction by chest physiotherapy, 97 and cardiopulmonary resuscitation. 98, 99 evidence for droplet transmission comes from epidemiologic studies of disease outbreaks, [100] [101] [102] [103] from experimental studies, 104 and from information on aerosol dynamics. 91, 105 studies have shown that the nasal mucosa, conjunctivae, and, less frequently, the mouth are susceptible portals of entry for respiratory viruses. 106 the maximum distance for droplet transmission is currently unresolved; pathogens transmitted by the droplet route have not been transmitted through the air over long distances, in contrast to the airborne pathogens discussed below. historically, the area of defined risk has been a distance of , 3 feet around the patient, based on epidemiologic and simulated studies of selected infections. 103, 104 using this distance for donning masks has been effective in preventing transmission of infectious agents through the droplet route. however, experimental studies with smallpox 107,108 and investigations during the global sars outbreaks of 2003 101 suggest that droplets from patients with these 2 infections could reach persons located 6 feet or more from their source. it is likely that the distance that droplets travel depends on the velocity and mechanism by which respiratory droplets are propelled from the source, the density of respiratory secretions, environmental factors (eg, temperature, humidity), and the pathogen's ability to maintain infectivity over that distance. 105 thus, a distance of , 3 feet around the patient is best considered an example of what is meant by ''a short distance from a patient'' and should not be used as the sole criterion for determining when a mask should be donned to protect from droplet exposure. based on these considerations, it may be prudent to don a mask when within 6 to 10 feet of the patient or on entry into the patient's room, especially when exposure to emerging or highly virulent pathogens is likely. more studies are needed to gain more insight into droplet transmission under various circumstances. droplet size is another variable under investigation. droplets traditionally have been defined as being . 5 mm in size. droplet nuclei (ie, particles arising from desiccation of suspended droplets) have been associated with airborne transmission and defined as , 5 mm in size, 105 a reflection of the pathogenesis of pulmonary tuberculosis that is not generalizeable to other organisms. observations of particle dynamics have demonstrated that a range of droplet sizes, including those of diameter $ 30 mm, can remain suspended in the air. 109 the behavior of droplets and droplet nuclei affect recommendations for preventing transmission. whereas fine airborne particles containing pathogens that are able to remain infective may transmit infections over long distances, requiring aiir to prevent its dissemination within a facility; organisms transmitted by the droplet route do not remain infective over long distances and thus do not require special air handling and ventilation. examples of infectious agents transmitted through the droplet route include b pertussis, 110 influenza virus, 23 adenovirus, 111 rhinovirus, 104 mycoplasma pneumoniae, 112 sars-cov, 21, 96, 113 group a streptococcus, 114 and neisseria meningitides. 95, 103, 115 although rsv may be transmitted by the droplet route, direct contact with infected respiratory secretions is the most important determinant of transmission and consistent adherence to standard precautions plus contact precautions prevents transmission in health care settings. 24, 116, 117 rarely, pathogens that are not transmitted routinely by the droplet route are dispersed into the air over short distances. for example, although s aureus is transmitted most frequently by the contact route, viral upper respiratory tract infection has been associated with increased dispersal of s aureus from the nose into the air for a distance of 4 feet under both outbreak and experimental conditions; this is known as the ''cloud baby'' and ''cloud adult'' phenomenon. [118] [119] [120] i.b.3.c. airborne transmission. airborne transmission occurs by dissemination of either airborne droplet nuclei or small particles in the respirable size range containing infectious agents that remain infective over time and distance (eg, spores of aspergillus spp and m tuberculosis). microorganisms carried in this manner may be dispersed over long distances by air currents and may be inhaled by susceptible individuals who have not had face-to-face contact with (or even been in the same room with) the infectious individual. [121] [122] [123] [124] preventing the spread of pathogens that are transmitted by the airborne route requires the use of special air handling and ventilation systems (eg, aiirs) to contain and then safely remove the infectious agent. 11, 12 infectious agents to which this applies include m tuberculosis, 124-127 rubeola virus (measles), 122 and varicella-zoster virus (chickenpox). 123 in addition, published data suggest the possibility that variola virus (smallpox) may be transmitted over long distances through the air under unusual circumstances, and aiirs are recommended for this agent as well; however, droplet and contact routes are the more frequent routes of transmission for smallpox. 108, 128, 129 in addition to aiirs, respiratory protection with a national institute for occupational safety and health (niosh)-certified n95 or higher-level respirator is recommended for hcws entering the aiir, to prevent acquisition of airborne infectious agents such as m tuberculosis. 12 for certain other respiratory infectious agents, such as influenza 130, 131 and rhinovirus, 104 and even some gastrointestinal viruses (eg, norovirus 132 and rotavirus 133 ) , there is some evidence that the pathogen may be transmitted through small-particle aerosols under natural and experimental conditions. such transmission has occurred over distances . 3 feet but within a defined air space (eg, patient room), suggesting that it is unlikely that these agents remain viable on air currents that travel long distances. aiirs are not routinely required to prevent transmission of these agents. additional issues concerning small-particle aerosol transmission of agents that are most frequently transmitted by the droplet route are discussed below. although sars-cov is transmitted primarily by contact and/or droplet routes, airborne transmission over a limited distance (eg, within a room) has been suggested, although not proven. [134] [135] [136] [137] [138] [139] [140] [141] this is true of other infectious agents as well, such as influenza virus 130 and noroviruses. 132, 142, 143 influenza viruses are transmitted primarily by close contact with respiratory droplets, 23, 102 and acquisition by hcws has been prevented by droplet precautions, even when positive-pressure rooms were used in one center. 144 however, inhalational transmission could not be excluded in an outbreak of influenza in the passengers and crew of an aircraft. 130 observations of a protective effect of ultraviolet light in preventing influenza among patients with tuberculosis during the influenza pandemic of 1957-1958 have been used to suggest airborne transmission. 145, 146 in contrast to the strict interpretation of an airborne route for transmission (ie, long distances beyond the patient room environment), short-distance transmission by small-particle aerosols generated under specific circumstances (eg, during endotracheal intubation) to persons in the immediate area near the patient also has been demonstrated. aerosolized particles , 100 mm in diameter can remain suspended in air when room air current velocities exceed the terminal settling velocities of the particles. 109 sars-cov transmission has been associated with endotracheal intubation, noninvasive positive pressure ventilation, and cardiopulmonary resuscitation. 93, 94, 96, 98, 141 although the most frequent routes of transmission of noroviruses are contact and foodborne and waterborne routes, several reports suggest that noroviruses also may be transmitted through aerosolization of infectious particles from vomitus or fecal material. 142, 143, 147, 148 it is hypothesized that the aerosolized particles are inhaled and subsequently swallowed. roy this conceptual framework can explain rare occurrences of airborne transmission of agents that are transmitted most frequently by other routes (eg, smallpox, sars, influenza, noroviruses). concerns about unknown or possible routes of transmission of agents associated with severe disease and no known treatment often result in the adoption of overextreme prevention strategies, and recommended precautions may change as the epidemiology of an emerging infection becomes more well defined and controversial issues are resolved. i.b.3.d.ii. transmission from the environment. some airborne infectious agents are derived from the environment and do not usually involve person-to-person transmission; for example, anthrax spores present in a finely milled powdered preparation can be aerosolized from contaminated environmental surfaces and inhaled into the respiratory tract. 150, 151 spores of environmental fungi (eg, aspergillus spp) are ubiquitous in the environment and may cause disease in immunocompromised patients who inhale aerosolized spores (through, eg, construction dust). 152, 153 as a rule, neither of these organisms is subsequently transmitted from infected patients; however, there is 1 well-documented report of person-to-person transmission of aspergillus sp in the icu setting that was most likely due to the aerosolization of spores during wound debridement. 154 the pe involves isolation practices designed to decrease the risk of exposure to environmental fungal agents in allogeneic hsct patients. 11, 14, 15, [155] [156] [157] [158] environmental sources of respiratory pathogens (eg, legionella) transmitted to humans through a common aerosol source is distinct from direct patient-to-patient transmission. i.b.3.e. other sources of infection. sources of infection transmission other than infectious individuals include those associated with common environmental sources or vehicles (eg, contaminated food, water, or medications, such as intravenous fluids). although aspergillus spp have been recovered from hospital water systems, 159 the role of water as a reservoir for immunosuppressed patients remains unclear. vectorborne transmission of infectious agents from mosquitoes, flies, rats, and other vermin also can occur in health care settings. prevention of vectorborne transmission is not addressed in this document. this section discusses several infectious agents with important infection control implications that either were not discussed extensively in previous isolation s80 vol. 35 no. 10 supplement 2 guidelines or have emerged only recently. included are epidemiologically important organisms (eg, c difficile), agents of bioterrorism, prions, sars-cov, monkeypox, noroviruses, and the hemorrhagic fever viruses (hfvs). experience with these agents has broadened the understanding of modes of transmission and effective preventive measures. these agents are included for information purposes and, for some (ie, sars-cov, monkeypox), to highlight the lessons that have been learned about preparedness planning and responding effectively to new infectious agents. i.c.1. epidemiologically important organisms. under defined conditions, any infectious agent transmitted in a health care setting may become targeted for control because it is epidemiologically important. c difficile is specifically discussed below because of its current prevalence and seriousness in us health care facilities. in determining what constitutes an ''epidemiologically important organism,'' the following criteria apply: d a propensity for transmission within health care facilities based on published reports and the occurrence of temporal or geographic clusters of more than 2 patients, (eg, c difficile, norovirus, rsv, influenza, rotavirus, enterobacter spp, serratia spp, group a streptococcus). a single case of health care-associated invasive disease caused by certain pathogens (eg, group a streptococcus postoperatively, 160 in a burn unit, 161 or in a ltcf; 162 legionella spp, 14, 163 aspergillus spp 164 ) is generally considered a trigger for investigation and enhanced control measures because of the risk of additional cases and the severity of illness associated with these infections. i.c.1.a. clostridium difficile. c difficile is a sporeforming gram-positive anaerobic bacillus that was first isolated from stools of neonates in 1935 165 and identified as the most frequent causative agent of antibioticassociated diarrhea and pseudomembranous colitis in 1977. 166 this pathogen is a major cause of health care-associated diarrhea and has been responsible for many large outbreaks in health care settings that have proven extremely difficult to control. important factors contributing to health care-associated outbreaks include environmental contamination, persistence of spores for prolonged periods, resistance of spores to routinely used disinfectants and antiseptics, hand carriage by hcws to other patients, and exposure of patients to frequent courses of antimicrobial agents. 167 antimicrobials most frequently associated with increased risk of c difficile include third-generation cephalosporins, clindamycin, vancomycin, and fluoroquinolones. since 2001, outbreaks and sporadic cases of c difficile with increased morbidity and mortality have occurred in several us states, canada, england, and the netherlands. [168] [169] [170] [171] [172] the same strain of c difficile has been implicated in all of these outbreaks; 173 this strain, toxinotype iii, north american pulsedfield gel electrophoresis (pfge) type 1, and polymerase chain reaction (pcr)-ribotype 027 (nap1/027), has been found to hyperproduce toxin a (a 16-fold increase) and toxin b (a 23-fold increase) compared with isolates from 12 other pfge types. a recent survey of us infectious disease physicians found that 40% of the respondents perceived recent increases in the incidence and severity of c difficile disease. 174 standardization of testing methodology and surveillance definitions is needed for accurate comparisons of trends in rates among hospitals. 175 it is hypothesized that the incidence of disease and apparent heightened transmissibility of this new strain may be due, at least in part, to the greater production of toxins a and b, increasing the severity of diarrhea and producing more environmental contamination. considering the greater morbidity, mortality, length of stay, and costs associated with c difficile disease in both acute care and long-term care facilities, control of this pathogen is becoming increasingly important. prevention of transmission focuses on syndromic application of contact precautions for patients with diarrhea, accurate identification of affected patients, environmental measures (eg, rigorous cleaning of patient rooms), and consistent hand hygiene. using soap and water rather than alcohol-based handrubs for mechanical removal of spores from hands and using a bleachcontaining disinfectant (5000 ppm) for environmental disinfection may be valuable in cases of transmission in health care facilities. appendix a provides for recommendations. i.c.1.b. multidrug-resistant organisms. in general, mdros are defined as microorganisms-predominantly bacteria-that are resistant to 1 or more classes of antimicrobial agents. 176 although the names of certain mdros suggest resistance to only a single agent (eg, mrsa, vre), these pathogens are usually resistant to all but a few commercially available antimicrobial agents. this latter feature defines mdros that are considered to be epidemiologically important and deserve special attention in health care facilities. 177 other mdros of current concern include multidrug-resistant streptococcus pneumoniae, which is resistant to penicillin and other broad-spectrum agents such as macrolides and fluroquinolones, multidrug-resistant gram-negative bacilli (mdr-gnb), especially those producing esbls; and strains of s aureus that are intermediate or resistant to vancomycin (ie, visa and vrsa). mdros are transmitted by the same routes as antimicrobial susceptible infectious agents. patient-to-patient transmission in health care settings, usually via hands of hcws, has been a major factor accounting for the increase in mdro incidence and prevalence, especially for mrsa and vre in acute care facilities. [199] [200] [201] preventing the emergence and transmission of these pathogens requires a comprehensive approach that includes administrative involvement and measures (eg, nurse staffing, communication systems, performance improvement processes to ensure adherence to recommended infection control measures), education and training of medical and other hcws, judicious antibiotic use, comprehensive surveillance for targeted mdros, application of infection control precautions during patient care, environmental measures (eg, cleaning and disinfection of the patient care environment and equipment, dedicated single-patient use of noncritical equipment), and decolonization therapy when appropriate. the prevention and control of mdros is a national priority, one that requires that all health care facilities and agencies assume responsibility and participate in community-wide control programs. 176, 177 a detailed discussion of this topic and recommendations for prevention published in 2006 is available at http:// www.cdc.gov/ncidod/dhqp/pdf/ar/mdroguideline2006. pdf. i.c.2. agents of bioterrorism. the cdc has designated the agents that cause anthrax, smallpox, plague, tularemia, viral hemorrhagic fevers, and botulism as category a (high priority), because these agents can be easily disseminated environmentally and/or transmitted from person to person, can cause high mortality and have the potential for major public health impact, might cause public panic and social disruption, and necessitate special action for public health preparedness. 202 general information relevant to infection control in health care settings for category a agents of bioterrorism is summarized in table 3 . (see http:// www.bt.cdc.gov for additional, updated category a agent information as well as information concerning category b and c agents of bioterrorism and updates.) category b and c agents are important but are not as readily disseminated and cause less morbidity and mortality than category a agents. health care facilities confront a different set of issues when dealing with a suspected bioterrorism event compared with other communicable diseases. an understanding of the epidemiology, modes of transmission, and clinical course of each disease, as well as carefully drafted plans that specify an approach and relevant websites and other resources for disease-specific guidance to health care, administrative, and support personnel, are essential for responding to and managing a bioterrorism event. infection control issues to be addressed include (1) identifying persons who may be exposed or infected; (2) preventing transmission among patients, hcws, and visitors; (3) providing treatment, chemoprophylaxis, or vaccine to potentially large numbers of people; (4) protecting the environment, including the logistical aspects of securing sufficient numbers of aiirs or designating areas for patient cohorts when an insufficient number of aiirs is available; (5) providing adequate quantities of appropriate ppe; and (6) identifying appropriate staff to care for potentially infectious patients (eg, vaccinated hcws for care of patients with smallpox). the response is likely to differ for exposures resulting from an intentional release compared with a naturally occurring disease because of the large number of persons that can be exposed at the same time and possible differences in pathogenicity. various sources offer guidance for the management of persons exposed to the most likely agents of bioterrorism. federal agency websites (eg, http://www. usamriid.army.mil/publications/index.html and http:// www.bt.cdc.gov) and state and county health department websites should be consulted for the most upto-date information. sources of information on specific agents include anthrax, 203 smallpox, [204] [205] [206] plague, 207,208 botulinum toxin, 209 tularemia, 210 and hemorrhagic fever viruses. 211, 212 i.c.2.a. pre-event administration of smallpox (vaccinia) vaccine to health care workers. vaccination of hcwsl in preparation for a possible smallpox exposure has important infection control implications. [213] [214] [215] these include the need for meticulous screening for vaccine contraindications in persons at increased risk for adverse vaccinia events; containment and monitoring of the vaccination site to prevent transmission in the health care setting and at home; and management of patients with vaccinia-related adverse events. 216, 217 the pre-event us smallpox vaccination program of 2003 is an example of the effectiveness of carefully developed recommendations for both screening potential vaccinees for contraindications and vaccination site care and monitoring. between december 2002 and february 2005, approximately 760,000 individuals were vaccinated in the department of defense and 40,000 in the civilian or public health populations, including approximately 70,000 who worked in health care settings. no cases of eczema vaccinatum, progressive vaccinia, fetal vaccinia, or contact transfer of vaccinia were reported in health care settings or in military workplaces. 218, 219 outside the health care setting, there were 53 cases of contact transfer from military vaccinees to close personal contacts (eg, bed partners or contacts during participation in sports such as wrestling 220 ). all contact transfers were from individuals who were not following recommendations to cover their vaccination sites. vaccinia virus was confirmed by culture or pcr in 30 cases, 2 of which resulted from tertiary transfer. all recipients, including 1 breast-fed infant, recovered without complications. subsequent studies using viral culture and pcr techniques have confirmed the effectiveness of semipermeable dressings to contain vaccinia. [221] [222] [223] [224] this experience emphasizes the importance of ensuring that newly vaccinated hcws adhere to recommended vaccination site care, especially those caring for high-risk patients. recommendations for pre-event smallpox vaccination of hcws and vacciniarelated infection control recommendations are published in the morbidity and mortality weekly report, 216, 225 with updates posted on the cdc's bioterrorism website. 205 i.c.3. prions. creutzfeldt-jakob disease (cjd) is a rapidly progressive, degenerative neurologic disorder of humans, with an incidence in the united states of approximately 1 person/million population/year. 226, 227 cjd is believed to be caused by a transmissible proteinaceous infectious agent known as a prion. infectious prions are isoforms of a host-encoded glycoprotein known as the prion protein. the incubation period (ie, time between exposure and and onset of symptoms) varies from 2 years to many decades. however, death typically occurs within 1 year of the onset of symptoms. approximately 85% of cjd cases occur sporadically with no known environmental source of infection, and 10% of cases are familial. iatrogenic transmission has occurred, with most cases resulting from treatment with human cadaver pituitary-derived growth hormone or gonadotropin, 228, 229 from implantation of contaminated human dura mater grafts, 230 or from corneal transplants. 231 transmission has been linked to the use of contaminated neurosurgical instruments or stereotactic electroencephalogram electrodes. [232] [233] [234] [235] prion diseases in animals include scrapie in sheep and goats, bovine spongiform encephalopathy (bse, or ''mad cow disease'') in cattle, and chronic wasting disease in deer and elk. 236 bse, first recognized in the united kingdom in 1986, was associated with a major epidemic among cattle that had consumed contaminated meat and bone meal. the possible transmission of bse to humans causing variant cjd (vcjd) was first described in 1996 and was subsequently found to be associated with consumption of bse-contaminated cattle products primarily in the united kingdom. there is strong epidemiologic and laboratory evidence for a causal association between the causative agent of bse and vcjd. 237 although most cases of vcjd have been reported from the united kingdom, a few cases also have been reported from europe, japan, canada, and the united states. most persons affected with vcjd worldwide lived in or visited the united kingdom during the years of a large outbreak of bse (1980) (1981) (1982) (1983) (1984) (1985) (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) and may have consumed contaminated cattle products during that time (see http://www.cdc.gov/ncidod/ diseases/cjd/cjd.htm). although there has been no indigenously acquired vcjd in the united states, the sporadic occurrence of bse in cattle in north america has heightened awareness of the possibility that such infections could occur and have led to increased surveillance activities. updated information may be found at http://www.cdc.gov/ncidod/diseases/cjd/cjd.htm. the public health impact of prion diseases has been reviewed previously. 238 vcjd in humans has different clinical and pathologic characteristics than sporadic or classic cjd, 239 including (1) younger median age at death (28 [range, 16 to 48] vs 68 years), (2) longer median duration of illness (14 months vs 4 to 6 months), (3) increased frequency of sensory symptoms and early psychiatric symptoms with delayed onset of frank neurologic signs; and (4) detection of prions in tonsillar and other lymphoid tissues, not present in sporadic cjd. 240 similar to sporadic cjd, there have been no reported cases of direct human-tohuman transmission of vcjd by casual or environmental contact, droplet, or airborne routes. ongoing blood safety surveillance in the united states has not detected sporadic cjd transmission through blood transfusion; 241-243 however, bloodborne transmission of vcjd is believed to have occurred in 2 patients in the uited kingdom. 244, 245 the following fda websites provide information on steps currently being taken in the united states to protect the blood supply from cjd and vcjd: http://www.fda.gov/cber/gdlns/cjdvcjd.htm and http:// www.fda.gov/cber/gdlns/cjdvcjdq&a.htm. standard precautions are used when caring for patients with suspected or confirmed cjd or vcjd. however, special precautions are recommended for tissue handling in the histology laboratory and for conducting an autopsy, embalming, and coming into contact with a body that has undergone autopsy. 246 recommendations for reprocessing surgical instruments to prevent transmission of cjd in health care settings have been published by the world health organization (who) and are currently under review at the cdc. questions may arise concerning notification of patients potentially exposed to cjd or vcjd through contaminated instruments and blood products from patients with cjd or vcjd or at risk of having vcjd. the risk of transmission associated with such exposures is believed to be extremely low but may vary based on the specific circumstance. therefore, consultation on appropriate options is advised. the united kingdom has developed several documents that clinicians and patients in the united states may find useful (see http://www.hpa.org.uk/infections/topics_az/cjd/ information_documents.htm). i.c.4. severe acute respiratory syndrome. sars is a newly discovered respiratory disease that emerged in china late in 2002 and spread to several countries. 135, 140 in particular, mainland china, hong kong, hanoi, singapore, and toronto have been significantly affected. sars is caused by sars-cov, a previously unrecognized member of the coronavirus family. 247, 248 the incubation period from exposure to the onset of symptoms is typically 2 to 7 days, but can be as long as 10 days and in rare cases even longer. 249 the illness is initially difficult to distinguish from other common respiratory infections. signs and symptoms usually include fever above 38.08c and chills and rigors, sometimes accompanied by headache, myalgia, and mild to severe respiratory symptoms. a radiographic profile of atypical pneumonia is an important clinical indicator of possible sars. compared with adults, children are affected less frequently, have milder disease, and are less likely to transmit sars-cov. 135, [249] [250] [251] the overall case fatality rate is approximately 6%; underlying disease and advanced age increase the risk of mortality (see http://www.who.int/csr/sarsarchive/2003_05_07a/en/). outbreaks in health care settings, with transmission to large numbers of hcws and patients, haa been a striking feature of sars; undiagnosed infectious patients and visitors have been important initiators of these outbreaks. 21, [252] [253] [254] the relative contribution of potential modes of transmission is not known precisely. there is ample evidence for droplet and contact transmission; 96, 101, 113 however, opportunistic airborne transmission cannot be excluded. 101, [135] [136] [137] [138] [139] 149, 254 for example, exposure to aerosol-generating procedures (eg, endotracheal intubation, suctioning) has been associated with transmission of infection to large numbers of hcws outside of the united states. 93, 94, 96, 98, 253 therefore, aerosolization of small infectious particles generated during these and other similar procedures could be a risk factor for transmission to others within a multibed room or shared airspace. a review of the infection control literature generated from the sars outbreaks of 2003 concluded that the greatest risk of transmission is to those who have close contact, are not properly trained in use of protective infection control procedures, and do not consistently use ppe, and that n95 or higher-level respirators may offer additional protection to those exposed to aerosol-generating procedures and high-risk activities. 255, 256 organizational and individual factors that affect adherence to infection control practices for sars also were identified. 256 control of sars requires a coordinated, dynamic response by multiple disciplines in a health care setting. 9 early detection of cases is accomplished by screening persons with symptoms of a respiratory infection for history of travel to areas experiencing community transmission or contact with sars patients, followed by implementation of respiratory hygiene/cough etiquette (ie, placing a mask over the patient's nose and mouth) and physical separation from other patients in common waiting areas. the precise combination of precautions to protect hcws has not yet been determined. at the time of this publication, the cdc recommends standard precautions, with emphasis on the use of hand hygiene; contact precautions, with emphasis on environmental cleaning due to the detection of sars-cov rna by pcr on surfaces in rooms occupied by sars patients; 138, 254, 257 and airborne precautions, including use of fit-tested niosh-approved n95 or higher-level respirators and eye protection. 258 in hong kong, the use of droplet and contact precautions, including the use of a mask but not a respirator, was effective in protecting hcws. 113 however, in toronto, consistent use of an n95 respirator was found to be slightly more protective than a mask. 93 it is noteworthy that no transmission of sars-cov to public hospital workers occurred in vietnam despite inconsistent use of infection control measures, including use of ppe, which suggests other factors (eg, severity of disease, frequency of high-risk procedures or events, environmental features) may influence opportunities for transmission. 259 sars-cov also has been transmitted in the laboratory setting through breaches in recommended laboratory practices. research laboratories in which sars-cov was under investigation were the source of most cases reported after the first series of outbreaks in the winter and spring of 2003. 260 lessons learned from the sars outbreaks are useful in devising plans to respond to future public health crises, such as pandemic influenza and bioterrorism events. surveillance for cases among patients and hcws, ensuring availability of adequate supplies and staffing, and limiting access to health care facilities were important factors in the response to sars. 9 guidance for infection control precautions in various settings is available at http://www.cdc.gov/ncidod/sars. i.c.5. monkeypox. monkeypox is a rare viral disease found mostly in the rain forest countries of central and west africa. the disease is caused by an orthopoxvirus that is similar in appearance to smallpox but causes a milder disease. the only recognized outbreak of human monkeypox in the united states was detected in june 2003, after several people became ill after contact with sick pet prairie dogs. infection in the prairie dogs was subsequently traced to their contact with a shipment of animals from africa, including giant gambian rats. 262 this outbreak demonstrates the importance of recognition and prompt reporting of unusual disease presentations by clinicians to enable prompt identification of the etiology, as well as the potential of epizootic diseases to spread from animal reservoirs to humans through personal and occupational exposure. 263 only limited data on transmission of monkeypox are available. transmission from infected animals and humans is believed to occur primarily through direct contact with lesions and respiratory secretions; airborne transmission from animals to humans is unlikely but cannot be excluded, and may have occurred in veterinary practices (eg, during administration of nebulized medications to ill prairie dogs 264 ). in humans, 4 instances of monkeypox transmission in hospitals have been reported in africa among children, usually related to sharing the same ward or bed. 265, 266 additional recent literature documents transmission of congo basin monkeypox in a hospital compound for an extended number of generations. 267 there has been no evidence of airborne or any other person-to-person transmission of monkeypox in the united states, and no new cases of monkeypox have been identified since the outbreak in june 2003. 268 the outbreak strain is a clade of monkeypox distinct from the congo basin clade and may have different epidemiologic properties (including human-to-human transmission potential) from monkeypox strains of the congo basin; 269 this awaits further study. smallpox vaccine is 85% protective against congo basin monkeypox. 270 because there is an associated case fatality rate of , 10%, administration of smallpox vaccine within 4 days to individuals who have had direct exposure to patients or animals with monkeypox is a reasonable policy. 271 for the most current information on monkeypox, see http://www.cdc.gov/ncidod/mon keypox/clinicians.htm. i.c.6. noroviruses. noroviruses, formerly referred to as norwalk-like viruses, are members of the caliciviridae family. these agents are transmitted via contaminated food or water and from person to person, causing explosive outbreaks of gastrointestinal disease. 272 environmental contamination also has been documented as a contributing factor in ongoing transmission during outbreaks. 273, 274 although noroviruses cannot be propagated in cell culture, dna detection by molecular diagnostic techniques has brought a greater appreciation of their role in outbreaks of gastrointestinal disease. 275 reported outbreaks in hospitals, 132 148 and large crowded shelters established for hurricane evacuees 285 has demonstrated their highly contagious nature, their potentially disruptive impact in health care facilities and the community, and the difficulty of controlling outbreaks in settings in which people share common facilites and space. of note, there is nearly a 5-fold increase in the risk to patients in outbreaks when a patient is the index case compared with exposure of patients during outbreaks when a staff member is the index case. 286 the average incubation period for gastroenteritis caused by noroviruses is 12 to 48 hours, and the clinical course lasts 12 to 60 hours. 272 illness is characterized by acute onset of nausea, vomiting, abdominal cramps, and/or diarrhea. the disease is largely self-limited; rarely, death due to severe dehydration can occur, particularly in elderly persons with debilitating health conditions. the epidemiology of norovirus outbreaks shows that even though primary cases may result from exposure to a fecally contaminated food or water, secondary and tertiary cases often result from person-to-person transmission facilitated by contamination of fomites 272, 287 and dissemination of infectious particles, especially during the process of vomiting. 132, 142, 143, 147, 148, 272, 278, 279 widespread, persistent, and inapparent contamination of the environment and fomites can make outbreaks extremely difficult to control. 147, 274, 283 these clinical observations and the detection of norovirus dna on horizontal surfaces 5 feet above the level that might be touched normally suggest that under certain circumstances, aerosolized particles may travel distances beyond 3 feet. 147 it is hypothesized that infectious particles may be aerosolized from vomitus, inhaled, and swallowed. in addition, individuals who are responsible for cleaning the environment may be at increased risk of infection. development of disease and transmission may be facilitated by the low infectious dose (ie, , 100 viral particles) 288 and the resistance of these viruses to the usual cleaning and disinfection agents (ie, they may survive , 10 ppm chlorine). [289] [290] [291] an alternate phenolic agent that was shown to be effective against feline calicivirus was used for environmental cleaning in one outbreak. 275, 292 there are insufficient data to determine the efficacy of alcohol-based hand rubs against noroviruses when the hands are not visibly soiled. 293 absence of disease in certain individuals during an outbreak may be explained by protection from infection conferred by the b histo-blood group antigen. 294 consultation on outbreaks of gastroenteritis is available through the cdc's division of viral and rickettsial diseases. 295 i.c.7. hemorrhagic fever viruses. hfv is a mixed group of viruses that cause serious disease with high fever, skin rash, bleeding diathesis, and, in some cases, high mortality; the resulting disease is referred to as viral hemorrhagic fever (vhf). among the more commonly known hfvs are ebola and marburg viruses (filoviridae), lassa virus (arenaviridae), crimean-congo hemorrhagic fever and rift valley fever virus (bunyaviridae), and dengue and yellow fever viruses (flaviviridae). 212, 296 these viruses are transmitted to humans through contact with infected animals or via arthropod vectors. although none of these viruses is endemic in the united states, outbreaks in affected countries provide potential opportunities for importation by infected humans and animals. furthermore, there is a concern that some of these agents could be used as bioweapons. 212 person-to-person transmission has been documented for ebola, marburg, lassa, and crimean-congo hfvs. in resource-limited health care settings, transmission of these agents to hcws, patients, and visitors has been described and in some outbreaks has accounted for a large proportion of cases. [297] [298] [299] transmission within households also has been documented in individuals who had direct contact with ill persons or their body fluids, but not in those who did not have such contact. 300 evidence concerning the transmission of hfvs has been summarized previously. 212, 301 person-to-person transmission is associated primarily with direct blood and body fluid contact. percutaneous exposure to contaminated blood carries a particularly high risk for transmission and increased mortality. 302, 303 the finding of large numbers of ebola viral particles in the skin and the lumina of sweat glands has raised concerns that transmission could occur from direct contact with intact skin, although epidemiologic evidence to support this is lacking. 304 postmortem handling of infected bodies is an important risk for transmission. 300, 305, 306 in rare situations, cases in which the mode of transmission was unexplained among individuals with no known direct contact have led to speculation that airborne transmission could have occurred. 297 however, airborne transmission of naturally occurring hfvs in humans has not been documented. a study of airplane passengers exposed to an in-flight index case of lassa fever found no transmission to any passengers. 307 in the laboratory setting, animals have been infected experimentally with marburg or ebola virus through direct inoculation of the nose, mouth, and/or conjunctiva 308,309 and by using mechanically generated viruscontaining aerosols. 310, 311 transmission of ebola virus among laboratory primates in an animal facility has been described. 312 the secondarily infected animals were in individual cages separated by approximately 3 meters. although the possibility of airborne transmission was suggested, the investigators were not able to exclude droplet or indirect contact transmission in this incidental observation. guidance on infection control precautions for hvfs transmitted person-to-person have been published by the cdc 1,211 and by the johns hopkins center for civilian biodefense strategies. 212 the most recent recommendations at the time of publication of this document were posted on the cdc website on may 19, 2005. 313 inconsistencies among the various recommendations have raised questions about the appropriate precautions to use in us hospitals. in less developed countries, outbreaks of hfvs have been controlled with basic hygiene, barrier precautions, safe injection practices, and safe burial practices. 298, 305 the preponderance of evidence on hfv transmission indicates that standard, contact, and droplet precautions with eye protection are effective in protecting hcws and visitors coming in contact with an infected patient. single gloves are adequate for routine patient care; doublegloving is advised during invasive procedures (eg, surgery) that pose an increased risk of blood exposure. routine eye protection (ie goggles or face shield) is particularly important. fluid-resistant gowns should be worn for all patient contact. airborne precautions are not required for routine patient care; however, use of aiirs is prudent when procedures that could generate infectious aerosols are performed (eg, endotracheal intubation, bronchoscopy, suctioning, autopsy procedures involving oscillating saws). n95 or higher-level respirators may provide added protection for individuals in a room during aerosol-generating procedures ( table 3 , appendix a). when a patient with a syndrome consistent with hemorrhagic fever also has a history of travel to an endemic area, precautions are initiated on presentation and then modified as more information is obtained ( table 2) . patients with hemorrhagic fever syndrome in the setting of a suspected bioweapons attack should be managed using airborne precautions, including aiirs, because the epidemiology of a potentially weaponized hemorrhagic fever virus is unpredictable. numerous factors influence differences in transmission risks among the various health care settings. these factors include the population characteristics (eg, increased susceptibility to infections, type and prevalence of indwelling devices), intensity of care, exposure to environmental sources, length of stay, and frequency of interaction between patients/residents with each other and with hcws. these factors, as well as organizational priorities, goals, and resources, influence how different health care settings adapt transmission prevention guidelines to meet their specific needs. 314, 315 infection control management decisions are informed by data regarding institutional experience/epidemiology; trends in community and institutional hais; local, regional, and national epidemiology; and emerging infectious disease threats. i.d.1. hospitals. infection transmission risks are present in all hospital settings. however, certain hospital settings and patient populations have unique conditions that predispose patients to infection and merit special mention. these are often sentinel sites for the emergence of new transmission risks that may be unique to that setting or present opportunities for transmission to other settings in the hospital. i.d.1.a. intensive care units. intensive care units (icus) serve patients who are immunocompromised by disease state and/or by treatment modalities, as well as patients with major trauma, respiratory failure, and other life-threatening conditions (eg, myocardial infarction, congestive heart failure, overdose, stroke, gastrointestinal bleeding, renal failure, hepatic failure, multiorgan system failure, and extremes of age). although icus account for a relatively small proportion of hospitalized patients, infections acquired in these units account for . 20% of all hais. 316 in the national nosocomial infection surveillance (nnis) system, 26.6% of hais were reported from icu and high-risk nursery (neonatal icu [nicu]) patients in 2002 (nnis, unpublished data). this patient population has increased susceptibility to colonization and infection, especially with mdros and candida spp, 317,318 because of underlying diseases and conditions, the invasive medical devices and technology used in their care (eg central venous catheters and other intravascular devices, mechanical ventilators, extracorporeal membrane oxygenation, hemodialysis/filtration, pacemakers, implantable left-ventricular assist devices), the frequency of contact with hcws, prolonged lengths of stay, and prolonged exposure to antimicrobial agents. [319] [320] [321] [322] [323] [324] [325] [326] [327] [328] [329] [330] furthermore, adverse patient outcomes in this setting are more severe and are associated with a higher mortality. 331 outbreaks associated with various bacterial, fungal, and viral pathogens due to common-source and person-to-person transmissions are frequent in adult icus and pediatric icus (picus). 31, [332] [333] [334] [335] [336] [337] i.d.1.b. burn units. burn wounds can provide optimal conditions for colonization, infection, and transmission of pathogens; infection acquired by burn patients is a frequent cause of morbidity and mortality. 319, 338, 339 the risk of invasive burn wound infection is particularly high in patients with a burn injury involving . 30% of the total body surface area (tbsa). 340, 341 infections occurring in patients with burn injuries involving , 30% of the tbsa are usually associated with the use of invasive devices. mssa, mrsa, enterococci (including vre), gram-negative bacteria, and candida spp are prevalent pathogens in burn infections, 53, 339, [342] [343] [344] [345] [346] [347] [348] [349] and outbreaks of these organisms have been reported. [350] [351] [352] [353] shifts over time in the predominance of pathogens causing infections in burn patients often lead to changes in burn care practices. 342, [354] [355] [356] [357] burn wound infections caused by aspergillus spp or other environmental molds may result from exposure to supplies contaminated during construction 358 or to dust generated during construction or other environmental disruption. 359 hydrotherapy equipment is an important environmental reservoir of gram-negative organisms. its use in burn care is discouraged based on demonstrated associations between the use of contaminated hydrotherapy equipment and infections. burn wound infections and colonization, as well as bloodstream infections, caused by multidrug-resistant p aeruginosa, 360 acinetobacter baumannii, 361 and mrsa 351 have been associated with hydrotherapy; thus, excision of burn wounds in operating rooms is the preferred approach. advances in burn care (specifically, early excision and grafting of the burn wound, use of topical antimicrobial agents, and institution of early enteral feeding) have led to decreased infectious complications. other advances have included prophylactic antimicrobial use, selective digestive decontamination, and use of antimicrobial-coated catheters; however, few epidemiologic studies and no efficacy studies have been performed to investigate the relative benefit of these measures. 356 there is no consensus on the most effective infection control practices to prevent transmission of infections to and from patients with serious burns (eg, single-bed rooms, 357 laminar flow, 362 and high-efficiency particulate air [hepa] filtration, 359 or maintaining burn patients in a separate unit with no exposure to patients or equipment from other units 363 ). there also is controversy regarding the need for and type of barrier precautions in the routine care of burn patients. one retrospective study demonstrated the efficacy and cost-effectiveness of a simplified barrier isolation protocol for wound colonization, emphasizing handwashing and use of gloves, caps, masks, and impermeable plastic aprons (rather than isolation gowns) for direct patient contact. 364 however, to date no studies have determined the most effective combination of infection control precautions for use in burn settings. prospective studies in this area are needed. i.d.1.c. pediatrics. studies of the epidemiology of hais in children have identified unique infection control issues in this population. 63, 64, [365] [366] [367] [368] [369] pediatric icu patients and the lowest birth weight babies in the nicu monitored in the nnis system have had high rates of central venous catheter-associated bloodstream infections. 64 38, 377 ) . close physical contact between hcws and infants and young children (eg. cuddling, feeding, playing, changing soiled diapers, and cleaning copious uncontrolled respiratory secretions) provides abundant opportunities for transmission of infectious material. such practices and behaviors as congregation of children in play areas where toys and bodily secretions are easily shared and rooming-in of family members with pediatric patients can further increase the risk of transmission. pathogenic bacteria have been recovered from toys used by hospitalized patients; 378 contaminated bath toys were implicated in an outbreak of multidrug-resistant p. aeruginosa on a pediatric oncology unit. 80 in addition, several patient factors increase the likelihood that infection will result from exposure to pathogens in health care settings (eg, immaturity of the neonatal immune system, lack of previous natural infection and resulting immunity, prevalence of patients with congenital or acquired immune deficiencies, congenital anatomic anomalies, and use of life-saving invasive devices in nicus and picus). 63 there are theoretical concerns that infection risk will increase in association with innovative practices used in the nicu for the purpose of improving developmental outcomes, such factors include cobedding 379 and kangaroo care, 380 which may increase opportunity for skin-to-skin exposure of multiple gestation infants to each other and to their mothers, respectively; although the risk of infection actually may be reduced among infants receiving kangaroo care. 381 children who attend child care centers 382, 383 and pediatric rehabilitation units 384 may increase the overall burden of antimicrobial resistance by contributing to the reservoir of ca-mrsa. [385] [386] [387] [388] [389] [390] patients in chronic care facilities may have increased rates of colonization with resistant garm-negative bacilli and may be sources of introduction of resistant organisms to acute care settings. 50 i.d.2. nonacute health care settings. health care is provided in various settings outside of hospitals, including long-term care facilities (ltcfs) (eg nursing homes), homes for the developmentally disabled, behavioral health service settings, rehabilitation centers, and hospices. 391 in addition, health care may be provided in non-health care settings, such as workplaces with occupational health clinics, adult day care centers, assisted-living facilities, homeless shelters, jails and prisons, school clinics, and infirmaries. each of these settings has unique circumstances and population risks that must be considered when designing and implementing an infection control program. several of the most common settings and their particular challenges are discussed below. although this guideline does not address each setting, the principles and strategies provided herein may be adapted and applied as appropriate. i.d.2.a. long-term care. the designation ltcf applies to a diverse group of residential settings, ranging from institutions for the developmentally disabled to nursing homes for the elderly and pediatric chronic care facilities. [392] [393] [394] nursing homes for the elderly predominate numerically and frequently represent longterm care as a group of facilities. approximately 1.8 million americans reside in the nation's 16,500 nursing homes. 395 estimates of hai rates of 1.8 to 13.5 per 1000 resident-care days have been reported, with a range of 3 to 7 per 1000 resident-care days in the more rigorous studies. [396] [397] [398] [399] [400] the infrastructure described in the department of veterans affairs' nursing home care units is a promising example for the development of a nationwide hai surveillance system for ltcfs. 401 lctfs are different from other health care settings in that elderly patients at increased risk for infection are brought together in one setting and remain in the facility for extended periods; for most residents, it is their home. an atmosphere of community is fostered, and residents share common eating and living areas and participate in various facility-sponsored activities. 402, 403 because able residents interact freely with each other, controlling infection transmission in this setting can be challenging. 404 a residents who is colonized or infected with certain microorganisms are in some cases restricted to his or her room. however, because of the psychosocial risks associated with such restriction, balancing psychosocial needs with infection control needs is important in the ltcf setting. 405 274, 277, 278, 280 ) and bacteria, including group a streptococcus, 162 , b pertussis, 414 nonsusceptible s pneumoniae, 197, 198 other mdros, and c difficile 415 ). these pathogens can lead to substantial morbidity and mortality, as well as increased medical costs; prompt detection and implementation of effective control measures are needed. risk factors for infection are prevalent among ltcf residents. 394, 416, 417 age-related declines in immunity may affect the response to immunizations for influenza and other infectious agents and increase the susceptibility to tuberculosis. immobility, incontinence, dysphagia, underlying chronic diseases, poor functional status, and age-related skin changes increase susceptibility to urinary, respiratory, and cutaneous and soft tissue infections, whereas malnutrition can impair wound healing. [418] [419] [420] [421] [422] medications (eg, drugs that affect level of consciousness, immune function, gastric acid secretions, and normal flora, including antimicrobial therapy) and invasive devices (eg, urinary catheters and feeding tubes) heighten the susceptibility to infection and colonization in ltcf residents. [423] [424] [425] finally, limited functional status and total dependence on hcws for activities of daily living have been identified as independent risk factors for infection 400, 416, 426 and for colonization with mrsa 427,428 and esbl-producing klebsiella pneumoniae. 429 several position papers and review articles provide guidance on various aspects of infection control and antimicrobial resistance in ltcfs. [405] [406] [407] [430] [431] [432] [433] [434] [435] the centers for medicare and medicaid services has established regulations for the prevention of infection in ltcfs. 436 because residents of ltcfs are hospitalized frequently, they can transfer pathogens between ltcfs and health care facilities in which they receive care. 8, [437] [438] [439] [440] this also is true for pediatric long-term care populations. pediatric chronic care facilities have been associated with the importation of extendedspectrum cephalosporin-resistant, gram-negative bacilli into a picu. 50 children from pediatric rehabilitation units may contribute to the reservoir of community-associated mrsa. 384, [388] [389] [390] i.d.2.b. ambulatory care. over the past decade, health care delivery in the united states has shifted from the acute, inpatient hospital to various ambulatory and community-based settings, including the home. ambulatory care is provided in hospital-based outpatient clinics, nonhospital-based clinics and physicians' offices, public health clinics, free-standing dialysis centers, ambulatory surgical centers, urgent care centers, and other setting. in 2000, there were 83 million visits to hospital outpatient clinics and more than 823 million visits to physicians' offices; 441 ambulatory care now accounts for most patient encounters with the health care system. 442 adapting transmission prevention guidelines to these settings is challenging, because patients remain in common areas for prolonged periods waiting to be seen by a health care provider or awaiting admission to the hospital, examination or treatment rooms are turned around quickly with limited cleaning, and infectious patients may not be recognized immediately. furthermore, immunocompromised patients often receive chemotherapy in infusion rooms, where they stay for extended periods along with other types of patients. little data exist on the risk of hais in ambulatory care settings, with the exception of hemodialysis centers. 18, 443, 444 transmission of infections in outpatient settings has been reviewed in 3 studies. [445] [446] [447] goodman and solomon 445 summarized 53 clusters of infections associated with the outpatient setting between 1961 and 1990. overall, 29 clusters were associated with common source transmission from contaminated solutions or equipment, 14 were associated with person-to-person transmission from or involving hcws, and 10 were associated with airborne or droplet transmission among patients and health care workers. transmission of bloodborne pathogens (ie, hbv, hcv, and, rarely, hiv) in outbreaks, sometimes involving hundreds of patients, continues to occur in ambulatory settings. these outbreaks often are related to common source exposures, usually a contaminated medical device, multidose vial, or intravenous solution. 82, [448] [449] [450] [451] [452] in all cases, transmission has been attributed to failure to adhere to fundamental infection control principles, including safe injection practices and aseptic technique. this subject has been reviewed, and recommended infection control and safe injection practices have been summarized. 453 airborne transmission of m tuberculosis and measles in ambulatory settings, most often emergency departments, has been reported. 34, 127, 445, 447, [454] [455] [456] measles virus was transmitted in physicians' offices and other outpatient settings during an era when immunization rates were low and measles outbreaks in the community were occurring regularly. 34, 122, 457 rubella has been transmitted in the outpatient obstetric setting; 33 there are no published reports of varicella transmission in the outpatient setting. in the ophthalmology setting, adenovirus type 8 epidemic keratoconjunctivitis has been transmitted through incompletely disinfected ophthalmology equipment and/or from hcws to patients, presumably by contaminated hands. 17, 445, 447, [458] [459] [460] [461] preventing transmission in outpatient settings necessitates screening for potentially infectious symptomatic and asymptomatic individuals, especially those at possible risk for transmitting airborne infectious agents (eg, m tuberculosis, varicella-zoster virus, rubeola [measles]), at the start of the initial patient encounter. on identification of a potentially infectious patient, implementation of prevention measures, including prompt separation of potentially infectious patients and implementation of appropriate control measures (eg, respiratory hygiene/cough etiquette and transmission-based precautions) can decrease transmission risks. 9, 12 transmission of mrsa and vre in outpatient settings has not been reported, but the association of ca-mrsa in hcws working in an outpatient hiv clinic with environmental ca-mrsa contamination in that clinic suggests the possibility of transmission in that setting. 462 patient-to-patient transmission of burkholderia spp and p aeruginosa in outpatient clinics for adults and children with cystic fibrosis has been confirmed. 463, 464 i.d.2.c. home care. home care in the united states is delivered by more than 20,000 provider agencies, including home health agencies, hospices, durable medical equipment providers, home infusion therapy services, and personal care and support services providers. home care is provided to patients of all ages with both acute and chronic conditions. the scope of services ranges from assistance with activities of daily living and physical and occupational therapy to the care of wounds, infusion therapy, and chronic ambulatory peritoneal dialysis. the incidence of infection in home care patients, other than that associated with infusion therapy, has not been well studied. [465] [466] [467] [468] [469] [470] however, data collection and calculation of infection rates have been done for central venous catheter-associated bloodstream infections in patients receiving home infusion therapy [469] [470] [471] [472] [473] and for the risk of blood contact through percutaneous or mucosal exposures, demonstrating that surveillance can be performed in this setting. 474 draft definitions for home care-associated infections have been developed. 475 transmission risks during home care are presumed to be minimal. the main transmission risks to home care patients are from an infectious home care provider or contaminated equipment; a provider also can be exposed to an infectious patient during home visits. because home care involves patient care by a limited number of personnel in settings without multiple patients or shared equipment, the potential reservoir of pathogens is reduced. infections of home care providers that could pose a risk to home care patients include infections transmitted by the airborne or droplet routes (eg, chickenpox, tuberculosis, influenza), skin infestations (eg, scabies 69 and lice), and infections transmitted by direct or indirect contact (eg, impetigo). there are no published data on indirect transmission of mdros from one home care patient to another, although this is theoretically possible if contaminated equipment is transported from an infected or colonized patient and used on another patient. of note, investigations of the first case of visa in home care 186 and the first 2 reported cases of vrsa 178, 180, 181, 183 found no evidence of transmission of visa or vrsa to other home care recipients. home health care also may contribute to antimicrobial resistance; a review of outpatient vancomycin use found that 39% of recipients did not receive prescribed antibiotics according to recommended guidelines. 476 although most home care agencies implement policies and procedures aimed at preventing transmission of organisms, the current approach is based on the adaptation of the 1996 guideline for isolation precautions in hospitals, 1 as well as other professional guidance. 477, 478 this issue has proven very challenging to the home care industry, and practice has been inconsistent and frequently not evidence-based. for example, many home health agencies continue to observe ''nursing bag technique,'' a practice that prescribes the use of barriers between the nursing bag and environmental surfaces in the home. 479 although the home environment may not always appear clean, the use of barriers between 2 noncritical surfaces has been questioned. 480, 481 opportunites exist to conduct research in home care related to infection transmission risks. 482 i.d.2.d. other sites of health care delivery. facilities that are not primarily health care settings but in which health care is delivered include clinics in correctional facilities and shelters. both of these settings can have suboptimal features, such as crowded conditions and poor ventilation. economically disadvantaged individuals who may have chronic illnesses and health care problems related to alcoholism, injected drug use, poor nutrition, and/or inadequate shelter often receive their primary health care at such sites. 483 infectious diseases of special concern for transmission include tuberculosis, scabies, respiratory infections (eg, n meningitides, s pneumoniae), sexually transmitted and bloodborne diseases (eg, hiv, hbv, hcv, syphilis, gonorrhea), hepatitis a virus, diarrheal agents such as norovirus, and foodborne diseases. 285, [484] [485] [486] [487] a high index of suspicion for tuberculosis and ca-mrsa in these populations is needed; outbreaks in these settings or among the populations they serve have been reported. [488] [489] [490] [491] [492] [493] [494] [495] [496] patient encounters in these types of facilities provide an opportunity to deliver recommended immunizations and screen for m tuberculosis infection, along with diagnosing and treating acute illnesses. 497 recommended infection control measures in these nontraditional areas designated for health care delivery are the same as for other ambulatory care settings. therefore, these settings must be equipped to observe standard precautions and, when indicated, transmission-based precautions. as new treatments emerge for complex diseases, unique infection control challenges associated with special patient populations must be addressed. i.e.1. immunocompromised patients. patients who have congenital primary immune deficiencies or acquired disease (eg. treatment-induced immune deficiencies) are at increased risk for numerous types of infections while receiving health care; these patients may be located throughout the health care facility. the specific immune system defects determine the types of infections most likely to be acquired (eg, viral infections are associated with t cell defects, and fungal and bacterial infections occur in patients who are neutropenic). as a general group, immunocompromised patients can be cared for in the same environment as other patients; however, it is always advisable to minimize exposure to other patients with transmissible infections, such as influenza and other respiratory viruses. 498, 499 the use of more intense chemotherapy regimens for treatment of childhood leukemia may be associated with prolonged periods of neutropenia and suppression of other components of the immune system, extending the period of infection risk and raising the concern that additional precautions may be indicated for select groups. 500, 501 with the application of newer and more intense immunosuppressive therapies for various medical conditions (eg, rheumatologic disease, 502, 503 inflammatory bowel disease 504 ), immunosuppressed patients are likely to be more widely distributed throughout a health care facility rather than localized to single patient units (eg, hematologyoncology). guidelines for preventing infections in certain groups of immunocompromised patients have been published previously. 15, 505, 506 published data provide evidence to support placing patients undergoing allogeneic hsct in a pe. 15, 157, 158 in addition, guidelines have been developed that address the special requirements of these immunocompromised patients, including use of antimicrobial prophylaxis and engineering controls to create a pe for the prevention of infections caused by aspergillus spp and other environmental fungi. 11, 14, 15 as more intense chemotherapy regimens associated with prolonged periods of neutropenia or graft-versus-host disease are implemented, the period of risk and duration of environmental protection may need to be prolonged beyond the traditional 100 days. 507 i.e.2. cystic fibrosis patients. patients with cystic fibrosis (cf) require special consideration when developing infection control guidelines. compared with other patients, cf patients require additional protection to prevent transmission from contaminated respiratory therapy equipment. [508] [509] [510] [511] [512] such infectious agents as b cepacia complex and p aeruginosa. 463, 464, 513, 514 have unique clinical and prognostic significance. in cf patients, b cepacia infection has been associated with increased morbidity and mortality, [515] [516] [517] whereas delayed acquisition of chronic p aeruginosa infection may be associated with an improved long-term clinical outcome. 518, 519 person-to-person transmission of b cepacia complex has been demonstrated among children 516 and adults 520 with cf in health care settings 463, 521 and from various social contacts, 522 most notably attendance at camps for patients with cf 523 and among siblings with cf. 524 successful infection control measures used to prevent transmission of respiratory secretions include segregation of cf patients from each other in ambulatory and hospital settings (including use of private rooms with separate showers), environmental decontamination of surfaces and equipment contaminated with respiratory secretions, elimination of group chest physiotherapy sessions, and disbanding of cf camps. 97, 525 the cystic fibrosis foundation has published a consensus document with evidence-based recommendations for infection control practices in cf patients. 20 i.f. new therapies associated with potentially transmissible infectious agents i.f.1. gene therapy. gene therapy has has been attempted using various viral vectors, including nonreplicating retroviruses, adenoviruses, adeno-associated viruses, and replication-competent strains of poxviruses. unexpected adverse events have restricted the prevalence of gene therapy protocols. the infectious hazards of gene therapy are theoretical at this time but require meticulous surveillance due to the possible occurrence of in vivo recombination and the subsequent emergence of a transmissible genetically altered pathogen. the greatest concern attends the use of replication-competent viruses, especially vaccinia. to date, no reports have described transmission of a vector virus from a gene therapy recipient to another individual, but surveillance is ongoing. recommendations for monitoring infection control issues throughout the course of gene therapy trials have been published. [526] [527] [528] i.f.2. infections transmitted through blood, organs, and other tissues. the potential hazard of transmitting infectious pathogens through biologic products is a small but ever-present risk, despite donor screening. reported infections transmitted by transfusion or transplantation include west nile virus infection, 529 cytomegalovirus infection, 530 cjd, 230 hepatitis c, 531 infections with clostridium spp 532 and group a streptococcus, 533 malaria, 534 babesiosis, 535 chagas disease, 536 lymphocytic choriomeningitis, 537 and rabies. 538, 539 therefore, it is important to consider receipt of biologic products when evaluating patients for potential sources of infection. i.f.3. xenotransplantation. transplantation of nonhuman cells, tissues, and organs into humans potentially exposes patients to zoonotic pathogens. transmission of known zoonotic infections (eg, trichinosis from porcine tissue) is of concern. also of concern is the possibility that transplantation of nonhuman cells, tissues, or organs may transmit previously unknown zoonotic infections (xenozoonoses) to immunosuppressed human recipients. potential infections that potentially could accompany transplantation of porcine organs have been described previously. 540 guidelines from the us public health service address many infectious diseases and infection control issues that surround the developing field of xenotransplantation; 541 549 policies and procedures that explain how standard precautions and transmission-based precautions are applied, including systems used to identify and communicate information on patients with potentially transmissible infectious agents, are essential to ensure the success of these measures. these policies and procedures may vary according to the characteristics of the organization. a key administrative measure is the provision of fiscal and human resources for maintaining infection control and occupational health programs that are responsive to emerging needs. specific components include bedside nurse 550 and infection prevention and control professional (icp) staffing levels, 551 inclusion of icps in facility construction and design decisions, 11 clinical microbiology laboratory support, 552, 553 adequate supplies and equipment including facility ventilation systems, 11 adherence monitoring, 554 assessment and correction of system failures that contribute to transmission, 555, 556 and provision of feedback to hcws and senior administrators. 433, 547, 548, 557 the positive influence of institutional leadership has been demonstrated repeatedly in studies of hcws' adherence to recommended hand hygiene practices. 176, 177, 433, 547, 548, [558] [559] [560] [561] [562] [563] health care administrators' involvement in the infection control processes can improve their awareness of the rationale and resource requirements for following recommended infection control practices. several administrative factors may affect the transmission of infectious agents in health care settings, including the institutional culture, individual hcw behavior, and the work environment. each of these areas is suitable for performance improvement monitoring and incorporation into the organization's patient safety goals. 542, 543, 545, 564 ii.a.1.a. scope of work and staffing needs for infection control professionals. the effectiveness of infection surveillance and control programs in preventing nosocomial infections in ust hospitals was assessed by the cdc through the study on the efficacy of nosocomial infection control (senic project) conducted between 1970 and 1976. 565 in a representative sample of us general hospitals, those with a trained infection control physician or microbiologist involved in an infection control program and at least 1 infection control nurse per 250 beds were associated with a 32% lower rate of the 4 infections studied (cvc-associated bloodstream infections, ventilator-associated pneumonias, catheter-related urinary tract infections, and surgical site infections). since the publication of that landmark study, responsibilities of icps have expanded commensurate with the growing complexity of the health care system, the patient populations served, and the increasing numbers of medical procedures and devices used in all types of health care settings. the scope of work of icps was first assessed in 1982 566-568 by the certification board of infection control, and has been reassessed every 5 years since that time. 557, [569] [570] [571] the findings of these analyses have been used to develop and update the infection control certification examination, which was first offered in 1983. with each new survey, it becomes increasingly apparent that the role of the icp is growing in complexity and scope beyond traditional infection control activities in acute care hospitals. activities currently assigned to icps in response to emerging challenges include (1) surveillance and infection prevention at facilities other than acute care hospitals (eg, ambulatory clinics, day surgery centers, ltcfs, rehabilitation centers, home care); (2) oversight of employee health services related to infection prevention (eg, assessment of risk and administration of recommended treatment after exposure to infectious agents, tuberculosis screening, influenza vaccination, respiratory protection fit testing, and administration of other vaccines as indicated, such as smallpox vaccine in 2003); (3) preparedness planning for annual influenza outbreaks, pandemic influenza, sars, and bioweapons attacks; (4) adherence monitoring for selected infection control practices; (5) oversight of risk assessment and implementation of prevention measures associated with construction and renovation; (6) prevention of transmission of mdros; (7) evaluation of new medical products that could be associated with increased infection risk (eg, intravenous infusion materials); (8) communication with the public, facility staff, and state and local health departments concerning infection control-related issues; and (9) participation in local and multicenter research projects. 433, 548, 551, 557, 572, 573 none of the certification board of infection control job analyses addressed specific staffing requirements for the identified tasks, although the surveys did include information about hours worked; the 2001 survey included the number of icps assigned to the responding facilities. 557 there is agreement in the literature that a ratio of 1 icp per 250 acute care beds is no longer adequate to meet current infection control needs; a delphi project that assessed staffing needs of infection control programs in the 21 st century concluded that a ratio of 0.8 to 1.0 icp per 100 occupied acute care beds is an appropriate staffing level. 551 a survey of participants in the nnis system found an average daily patient census of 115 per icp. 315 results of other studies have been similar: 3 per 500 beds for large acute care hospitals, 1 per 150 to 250 beds in ltcfs, and 1.56 per 250 in small rural hospitals. 572, 574 the foregoing demonstrates that infection control staffing no longer can be based on patient census alone, but rather must be determined by the scope of the program, characteristics of the patient population, complexity of the health care system, tools available to assist personnel to perform essential tasks (eg, electronic tracking and laboratory support for surveillance), and unique or urgent needs of the institution and community. 551 furthermore, appropriate training is required to optimize the quality of work performed. 557, 571, 575 ii.a.1.a.i. infection control nurse liaison. designating a bedside nurse on a patient care unit as an infection control liaison or ''link nurse'' is reported to be an effective adjunct to enhance infection control at the unit level. [576] [577] [578] [579] [580] [581] such individuals receive training in basic infection control and have frequent communication with icps, but maintain their primary role as bedside caregiver on their units. the infection control nurse liaison increases the awareness of infection control at the unit level. he or she is especially effective in implementating new policies or control interventions because of the rapport with individuals on the unit, an understanding of unit-specific challenges, and ability to promote strategies that are most likely to be successful in that unit. this position is an adjunct to, not a replacement for, fully trained icps. furthermore, the infection control liaison nurses should not be counted when considering icp staffing. there is increasing evidence that the level of bedside nurse staffing influences the quality of patient care. 582, 583 adequate nursing staff makes it more likely that infection control practices, including hand hygiene, standard precautions, and transmission-based precautions, will be given appropriate attention and applied correctly and consistently. 551 a national multicenter study reported strong and consistent inverse relationships between nurse staffing and 5 adverse outcomes in medical patients, 2 of which were hais (urinary tract infections and pneumonia). 582 the association of nursing staff shortages with increased rates of hai has been demonstrated in several outbreaks in hospitals and ltcfs, and with increased transmission of hepatitis c virus in dialysis units. 22, 417, 550, [584] [585] [586] [587] [588] [589] [590] [591] [592] [593] [594] [595] [596] in most cases, when staffing was improved as part of a comprehensive control intervention, the outbreak ended or the hai rate declined. in 2 studies, 589,595 the composition of the nursing staff (''pool'' or ''float'' vs regular staff nurses) influenced the rate of primary bloodstream infections, with an increased infection rate occurring when the proportion of regular nurses decreased and that of pool nurses increased. ii.a.1.c. clinical microbiology laboratory support. the critical role of the clinical microbiology laboratory in infection control and health care epidemiology has been well described 552, 553, [597] [598] [599] and is supported by the infectious disease society of america's policy statement on the consolidation of clinical microbiology laboratories published in 2001. 552 the clinical microbiology laboratory contributes to preventing transmission of infectious diseases in health care settings by promptly detecting and reporting epidemiologically important organisms, identifying emerging patterns of antimicrobial resistance, and assessing the effectiveness of recommended precautions to limit transmission during outbreaks. 597 outbreaks of infections may be recognized first by laboratorians. 161 health care organizations need to ensure the availability of the recommended scope and quality of laboratory services, a sufficient number of appropriately trained laboratory staff members, and systems to promptly communicate epidemiologically important results to those who will take action (eg, providers of clinical care, infection control staff, health care epidemiologists, and infectious disease consultants). 600 as concerns about emerging pathogens and bioterrorism grow, the role of the clinical microbiology laboratory assumes ever-greater importance. for health care organizations that outsource microbiology laboratory services (eg, ambulatory care, home care, ltcfs, smaller acute care hospitals), it is important to specify by contract the types of services (eg, periodic institution-specific aggregate susceptibility reports) required to support infection control. several key functions of the clinical microbiology laboratory are relevant to this guideline: ii.a.2. institutional safety culture and organizational characteristics. safety culture (or safety climate) refers to a work environment in which a shared commitment to safety on the part of management and the workforce is understood and maintained. 558, 619, 620 the authors of the institute of medicine's report titled to err is human 542 acknowledged that causes of medical error are multifaceted but emphasized the pivotal role of system failures and the benefits of a safety culture. a safety culture is created through (1) the actions that management takes to improve patient and worker safety, (2) worker participation in safety planning, (3) the availability of appropriate ppe, (4) the influence of group norms regarding acceptable safety practices, and (5) the organization's socialization process for new personnel. safety and patient outcomes can be enhanced by improving or creating organizational characteristics within patient care units, as demonstrated by studies of surgical icus. 621, 622 each of these factors has a direct bearing on adherence to transmission prevention recommendations. 256 measurement of an institution's culture of safety is useful in designing improvements in health care. 623, 624 several hospitalbased studies have linked measures of safety culture with both employee adherence to safe practices and reduced exposures to blood and body fluids. [625] [626] [627] [628] [629] [630] [631] one study of hand hygiene practices concluded that improved adherence requires integration of infection control into the organization's safety culture. 560 several hospitals that are part of the veterans administration health care system have taken specific steps toward improving the safety culture, including error-reporting mechanisms, root cause analyses of identified problems, safety incentives, and employee education. [632] [633] [634] ii.a.3. adherence of health care workers to recommended guidelines. hcws' adherence to recommended infection control practices decreases the transmission of infectious agents in health care settings. 116, 561, [635] [636] [637] [638] [639] several observational studies have shown limited adherence to recommended practices by hcws. 558, [639] [640] [641] [642] [643] [644] [645] [646] [647] [648] [649] [650] [651] [652] [653] [654] [655] [656] observed adherence to universal precautions ranged from 43% to 89%. 640, 641, 648, 650, 651 the degree of adherence often depended on the specific practice that was assessed and, for glove use, the circumstance in which the practice was applied. observed rates of appropriate glove use has ranged from a low of 15% 644 to a high of 82%. 649 however, 92% and 98% adherence with glove use have been reported during arterial blood gas collection and resuscitation, respectively, procedures in which considerable blood contact may occur. 642, 655 differences in observed adherence have been reported among occupational groups in the same health care facility 640 and between experienced and nonexperienced professionals. 644 in surveys of hcws, self-reported adherence was generally higher than actual adherence found in observational studies. furthermore, where an observational component was included with a self-reported survey, self-perceived adherence was often greater than observed adherence. 656 among nurses and physicians, increasing years of experience is a negative predictor of adherence. 644, 650 education to improve adherence is the primary intervention that has been studied. whereas positive changes in knowledge and attitude have been demonstrated, 639, 657 no or only limited accompanying changes in behavior often have been found. 641, 643 self-reported adherence is higher in groups that received an educational intervention. 629, 658 in one study, educational interventions that incorporated videotaping and performance feedback were successful in improving adherence during the study period, but the long-term effect of such interventions is not known. 653 the use of videotaping also served to identify system problems (eg, communication and access to ppe) that otherwise may not have been recognized. interest is growing in the use of engineering controls and facility design concepts for improving adherence. whereas the introduction of automated sinks was found to have a negative impact on consistent adherence to handwashing in one study, 659 the use of electronic monitoring and voice prompts to remind hcws to perform hand hygiene and improving accessibility to hand hygiene products increased adherence and contributed to a decrease in hais in another study. 660 more information is needed regarding ways in which technology might improve adherence. improving adherence to infection control practices requires a multifaceted approach that incorporates continuous assessment of both the individual and the work environment. 558, 560 using several behavioral theories, kretzer and larson concluded that a single intervention (eg, a handwashing campaign or putting up new posters about transmission precautions) likely would be ineffective in improving hcws adherence. 661 improvement requires the organizational leadership to make prevention an institutional priority and integrate infection control practices into the organization's safety culture. 560 a recent review of the literature concluded that variations in organizational factors (eg, safety climate, policies and procedures, education and training) and individual factors (eg, knowledge, perceptions of risk, past experience) were determinants of adherence to infection control guidelines for protection against sars and other respiratory pathogens. 256 surveillance is an essential tool for case finding of single patients or clusters of patients who are infected or colonized with epidemiologically important organisms (eg, susceptible bacteria such as s aureus, s pyogenes [group a streptococcus] or enterobacter-klebsiella spp; mrsa, vre, and other mdros; c difficile; rsv; influenza virus) for which transmission-based precautions may be required. surveillance is defined as the ongoing systematic collection, analysis, interpretation, and dissemination of data regarding a health-related event for use in public health action to reduce morbidity and mortality and to improve health. 662 the work of ignaz semmelweis delineating the role of person-toperson transmission in puerperal sepsis is the earliest example of the use of surveillance data to reduce transmission of infectious agents. 663 surveillance of both process measures and the infection rates to which they are linked is important in evaluating the effectiveness of infection prevention efforts and identifying indications for change. 554, [664] [665] [666] [667] the study on the efficacy of nosocomial infection control (senic) found that different combinations of infection control practices resulted in reduced rates of nosocomial surgical site infections, pneumonia, urinary tract infections, and bacteremia in acute care hospitals; 565 however, surveillance was the only component essential for reducing all 4 types of hais. although a similar study has not been conducted in other health care settings, a role for surveillance and the need for novel strategies in ltcfs 397, 433, 668, 669 and in home care [469] [470] [471] [472] have been described. the essential elements of a surveillance system are (1) standardized definitions, (2) identification of patient populations at risk for infection, (3) statistical analysis (eg, risk adjustment, calculation of rates using appropriate denominators, trend analysis using such methods as statistical process control charts), and (4) feedback of results to the primary caregivers. [670] [671] [672] [673] [674] [675] data gathered through surveillance of high-risk populations, device use, procedures, and facility locations (eg, icus) are useful in detecting transmission trends. [670] [671] [672] identification of clusters of infections should be followed by a systematic epidemiologic investigation to determine commonalities in persons, places, and time and to guide implementation of interventions and evaluation of the effectiveness of those interventions. targeted surveillance based on the highest-risk areas or patients has been preferred over facility-wide surveillance for the most effective use of resources. 672, 675 however, for certain epidemiologically important organisms, surveillance may need to be facility-wide. surveillance methods will continue to evolve as health care delivery systems change 391, 676 and user-friendly electronic tools for electronic tracking and trend analysis become more widely available. 673, 677, 678 individuals with experience in health care epidemiology and infection control should be involved in selecting software packages for data aggregation and analysis, to ensure that the need for efficient and accurate hai surveillance will be met. effective surveillance is increasingly important as legislation requiring public reporting of hai rates is passed and states work to develop effective systems to support such legislation. 679 the education and training of hcws is a prerequisite for ensuring that policies and procedures for standard and transmission-based precautions are understood and practiced. understanding the scientific rationale for the precautions will allow hcws to apply procedures correctly, as well as to safely modify precautions based on changing requirements, resources, or health care settings. 14,654,680-687 one study found that the likelihood of hcws developing sars was strongly associated with less than 2 hours of infection control training and poor understanding of infection control procedures. 688 education regarding the important role of vaccines (eg, influenza, measles, varicella, pertussis, pneumococcal) in protecting hcws, their patients, and family members can help improve vaccination rates. [689] [690] [691] [692] education on the principles and practices for preventing transmission of infectious agents should begin during training in the health professions and be provided to anyone who has an opportunity for contact with patients or medical equipment (eg, nursing and medical staff; therapists and technicians, including respiratory, physical, occupational, radiology, and cardiology personnel; phlebotomists; housekeeping and maintenance staff; and students). in health care facilities, education and training on standard and transmission-based precautions are typically provided at the time of orientation and should be repeated as necessary to maintain competency; updated education and training are necessary when policies and procedures are revised or when a special circumstance occurs, such as an outbreak that requires modification of current practice or adoption of new recommendations. education and training materials and methods appropriate to the hcw's level of responsibility, individual learning habits, and language needs can improve the learning experience. 657, [693] [694] [695] [696] [697] [698] [699] [700] [701] education programs for hcws have been associated with sustained improvement in adherence to best practices and a related decrease in device-associated hais in teaching and nonteaching settings 638, 702 and in medical and surgical icus (coopersmith, 2002 #2563) . several studies have shown that in addition to targeted education to improve specific practices, periodic assessment and feedback of the hcw's knowledge and adherence to recommended practices are necessary to achieve the desired changes and identify continuing education needs. 561, [703] [704] [705] [706] [707] the effectiveness of this approach for isolation practices has been demonstrated in the control of rsv. 116, 683 patients, family members, and visitors can be partners in preventing transmission of infections in health care settings. 9,42,708-710 information on standard precautions, especially hand hygiene, respiratory hygiene/cough etiquette, vaccination (especially against influenza), and other routine infection prevention strategies, may be incorporated into patient information materials provided on admission to the health care facility. additional information on transmission-based precautions is best provided when these precautions are initiated. fact sheets, pamphlets, and other printed material may include information on the rationale for the additional precautions, risks to household members, room assignment for transmission-based precautions purposes, explanation of the use of ppe by hcws, and directions for use of such equipment by family members and visitors. such information may be particularly helpful in the home environment, where household members often have the primary responsibility for adherence to recommended infection control practices. hcws must be available and prepared to explain this material and answer questions as needed. hand hygiene has been frequently cited as the single most important practice to reduce the transmission of infectious agents in health care settings 558, 711, 712 and is an essential element of standard precautions. the term ''hand hygiene'' includes both handwashing with either plain or antiseptic-containing soap and water and the use of alcohol-based products (gels, rinses, foams) that do not require water. in the absence of visible soiling of hands, approved alcohol-based products for hand disinfection are preferred over antimicrobial or plain soap and water because of their superior microbiocidal activity, reduced drying of the skin, and convenience. 558 have been associated with a sustained decrease in the incidence of mrsa and vre infections primarily in icus. 560, 561, [713] [714] [715] [716] the scientific rationale, indications, methods, and products for hand hygiene have been summarized in previous publications. 558, 716 the effectiveness of hand hygiene can be reduced by the type and length of fingernails. 558, 717, 718 individuals wearing artificial nails have been shown to harbor more pathogenic organisms, especially gram-negative bacilli and yeasts, on the nails and in the subungual area compared with individuals with native nails. 719, 720 in 2002, the cdc/hicpac recommended (category ia) that artificial fingernails and extenders not be worn by hcws who have contact with high-risk patients (eg, those in icus and operating rooms), due to the association with outbreaks of gram-negative bacillus and candidal infections as confirmed by molecular typing of isolates. 30, 31, 558, [721] [722] [723] [724] the need to restrict the wearing of artificial fingernails by all hcws who provide direct patient care and those who have contact with other high-risk groups (eg, oncology and cystic fibrosis patients) has not been studied but has been recommended by some experts. 20 currently, such decisions are at the discretion of an individual facility's infection control program. there is less evidence indicating that jewelry affects the quality of hand hygiene. although hand contamination with potential pathogens is increased with ring-wearing, 558, 725 no studies have related this practice to hcw-to-patient transmission of pathogens. ppe refers to various barriers and respirators used alone or in combination to protect mucous membranes, airways, skin, and clothing from contact with infectious agents. the choice of ppe is based on the nature of the patient interaction and/or the likely mode(s) of transmission. specific guidance on the use of ppe is provided in part iii of this guideline. a suggested procedure for donning and removing ppe aimed at preventing skin or clothing contamination is presented in figure 1 . designated containers for used disposable or reusable ppe should be placed in a location convenient to the site of removal, to facilitate disposal and containment of contaminated materials. hand hygiene is always the final step after removing and disposing of ppe. the following sections highlight the primary uses of and criteria for selecting this equipment. ii.e.1. gloves. gloves are used to prevent contamination of hcw hands when (1) anticipating direct contact with blood or body fluids, mucous membranes, nonintact skin and other potentially infectious material; (2) having direct contact with patients who are colonized or infected with pathogens transmitted by the contact route (eg, vre, mrsa, rsv 558, 726, 727 ); or (3) handling or touching visibly or potentially contaminated patient care equipment and environmental surfaces. 72, 73, 558 gloves can protect both patients and hcws from exposure to infectious material that may be carried on hands. 73 the extent to which gloves will protect hcws from transmission of bloodborne pathogens (eg, hiv, hbv, hcv) after a needlestick or other puncture that penetrates the glove barrier has not yet been determined. although gloves may reduce the volume of blood on the external surface of a sharp by 46% to 86%, 728 the residual blood in the lumen of a hollow-bore needle would not be affected; therefore, the effect on transmission risk is unknown. gloves manufactured for health care purposes are subject to fda evaluation and clearance. 729 nonsterile disposable medical gloves made of various materials (eg, latex, vinyl, nitrile) are available for routine patient care. 730 the selection of glove type for nonsurgical use is based on various factors, including the task to be performed, anticipated contact with chemicals and chemotherapeutic agents, latex sensitivity, sizing, and facility policies for creating a latex-free environment. 17, [731] [732] [733] for contact with blood and body fluids during nonsurgical patient care, a single pair of gloves generally provides adequate barrier protection. 733 however, there is considerable variability among gloves; both the quality of the manufacturing process and type of material influence their barrier effectiveness. 734 whereas there is little difference in the barrier properties of unused intact gloves, 735 studies have shown repeatedly that vinyl gloves have higher failure rates than latex or nitrile gloves when tested under simulated and actual clinical conditions. 730, [734] [735] [736] [737] for this reason, either latex or nitrile gloves are preferable for clinical procedures that require manual dexterity or will involve more than brief patient contact. a facility may need to stock gloves in several sizes. heavier, reusable utility gloves are indicated for non-patient care activities, such as handling or cleaning contaminated equipment or surfaces. 11, 14, 738 during patient care, transmission of infectious organisms can be reduced by adhering to the principles of working from ''clean'' to ''dirty'' and confining or limiting contamination to those surfaces directly needed for patient care. it may be necessary to change gloves during the care of a single patient to prevent cross-contamination of body sites. 558, 739 it also may be necessary to change gloves if the patient interaction also involves touching portable computer keyboards or other mobile equipment transported from room to room. discarding gloves between patients is necessary to prevent transmission of infectious material. gloves must not be washed for subsequent reuse, because microorganisms cannot be removed reliably from glove surfaces, and continued glove integrity cannot be ensured. furthermore, glove reuse has been associated with transmission of mrsa and gram-negative bacilli. [740] [741] [742] when gloves are worn in combination with other ppe, they are put on last. gloves that fit snugly around the wrist are preferred for use with an isolation gown, because they will cover the gown cuff and provide a more reliable continuous barrier for the arms, wrists, and hands. proper glove removal will prevent hand contamination (fig 1) . hand hygiene after glove removal further ensures that the hands will not carry potentially infectious material that might have penetrated through unrecognized tears or that could have contaminated the hands during glove removal. 558, 727, 740 ii.e.2. isolation gowns. isolation gowns are used as specified by standard and transmission-based precautions to protect the hcw's arms and exposed body areas and prevent contamination of clothing with blood, body fluids, and other potentially infectious material. 24, 88, 261, [743] [744] [745] the need for and the type of isolation gown selected is based on the nature of the patient interaction, including the anticipated degree of contact with infectious material and potential for blood and body fluid penetration of the barrier. the wearing of isolation gowns and other protective apparel is mandated by the occupational safety and health administration's (osha) bloodborne pathogens standard. 738 clinical and laboratory coats or jackets worn over personal clothing for comfort and/or purposes of identity are not considered ppe. when applying standard precautions, an isolation gown is worn only if contact with blood or body fluid is anticipated. however, when contact precautions are used (ie, to prevent transmission of an infectious agent that is not interrupted by standard precautions alone and is associated with environmental contamination), donning of both gown and gloves on room entry is indicated, to prevent unintentional contact with contaminated environmental surfaces. 54, 72, 73, 88 the routine donning of isolation gowns on entry into an icu or other high-risk area does not prevent or influence potential colonization or infection of patients in those areas, however. 364, [746] [747] [748] [749] isolation gowns are always worn in combination with gloves, and with other ppe when indicated. gowns are usually the first piece of ppe to be donned. full coverage of the arms and body front, from neck to the mid-thigh or below, will ensure protection of clothing and exposed upper body areas. several gown sizes should be available in a health care facility to ensure appropriate coverage for staff members. isolation gowns should be removed before leaving the patient care area to prevent possible contamination of the environment outside the patient's room. isolation gowns should be removed in a manner that prevents contamination of clothing or skin (fig 1) ; the outer, ''contaminated'' side of the gown is turned inward and rolled into a bundle, and then discarded into a designated container for waste or linen to contain contamination. ii.e.3. face protection: masks, goggles, and face shields. ii.e.3.a. masks. masks are used for 3 primary purposes in health care settings: (1) placed on hcws to protect them from contact with infectious material from patients (eg, respiratory secretions and sprays of blood or body fluids), consistent with standard precautions and droplet precautions; (2) placed on hcws engaged in procedures requiring sterile technique, to protect patients from exposure to infectious agents carried in the hcw's mouth or nose; and (3) placed on coughing patients to limit potential dissemination of infectious respiratory secretions from the patient to others (ie, respiratory hygiene/cough etiquette). masks may be used in combination with goggles to protect the mouth, nose, and eyes, or, alternatively, a face shield may be used instead of a mask and goggles to provide more complete protection for the face, as discussed below. masks should not be confused with particulate respirators used to prevent inhalation of small particles that may contain infectious agents transmitted through the airborne route, as described below. the mucous membranes of the mouth, nose, and eyes are susceptible portals of entry for infectious agents; other skin surfaces also may be portals if skin integrity is compromised (by, eg, acne, dermatitis). 66, [750] [751] [752] [753] therefore, use of ppe to protect these body sites is an important component of standard precautions. the protective effect of masks for exposed hcws has been demonstrated previously. 93, 113, 754, 755 procedures that generate splashes or sprays of blood, body fluids, secretions, or excretions (eg, endotracheal suctioning, bronchoscopy, invasive vascular procedures) require either a face shield (disposable or reusable) or a mask and goggles. [93] [94] [95] [96] 113, 115, 261, 738, 756 the wearing of masks, eye protection, and face shields in specified circumstances when blood or body fluid exposure is likely is mandated by osha's bloodborne pathogens standard. 738 appropriate ppe should be selected based on the anticipated level of exposure. two mask types are available for use in health care settings: surgical masks that are cleared by the fda and required to have fluid-resistant properties, and procedure or isolation masks. 757,#2688 to date, no studies comparing mask types to determine whether one mask type provides better protection than another have been published. because procedure/isolation masks are not regulated by the fda, they may be more variable in terms of quality and performance than surgical masks. masks come in various shapes (eg, molded and nonmolded), sizes, filtration efficiency, and method of attachment (eg, ties, elastic, ear loops). health care facilities may find that different types of masks are needed to meet individual hcw needs. ii.e.3.b. goggles and face shields. guidance on eye protection for infection control has been published. 758 the eye protection chosen for specific work situations (eg, goggles or face shield) depends on the circumstances of exposure, other ppe used, and personal vision needs. personal eyeglasses and contact lenses are not considered adequate eye protection (see http://www.cdc.gov/ niosh/topics/eye/eye-infectious.html). niosh guidelines specify that eye protection must be comfortable, allow for sufficient peripheral vision, and adjustable to ensure a secure fit. a health care facility may need to provide several different types, styles, and sizes of eye protection equipment. indirectly vented goggles with a manufacturer's antifog coating may provide the most reliable practical eye protection from splashes, sprays, and respiratory droplets from multiple angles. newer styles of goggles may provide better indirect airflow properties to reduce fogging, as well as better peripheral vision and more size options for fitting goggles to different workers. many styles of goggles fit adequately over prescription glasses with minimal gaps. although effective as eye protection, goggles do not provide splash or spray protection to other parts of the face. the role of goggles in addition to a mask in preventing exposure to infectious agents transmitted through respiratory droplets has been studied only for rsv. reports published in the mid-1980s demonstrated that eye protection reduced occupational transmission of rsv. 759, 760 whether this was due to the prevention hand-eye contact or the prevention of respiratory droplet-eye contact has not been determined. however, subsequent studies demonstrated that rsv transmission is effectively prevented by adherence to standard precautions plus contact precautions and that routine use of goggles is not necessary for this virus. 24, 116, 117, 683, 761 it is important to remind hcws that even if droplet precautions are not recommended for a specific respiratory tract pathogen, protection for the eyes, nose, and mouth using a mask and goggles or a face shield alone is necessary when a splash or spray of any respiratory secretions or other body fluids is likely to occur, as defined in standard precautions. disposable or nondisposable face shields may be used as an alternative to goggles. 758 compared with goggles, a face shield can provide protection to other facial areas besides the eyes. face shields extending from the chin to crown provide better face and eye protection from splashes and sprays; face shields that wrap around the sides may reduce splashes around the edge of the shield. removal of a face shield, goggles, and mask can be performed safely after gloves have been removed and hand hygiene performed. the ties, earpieces, and/or headband used to secure the equipment to the head are considered ''clean'' and thus safe to touch with bare hands. the front of a mask, goggles, and face shield are considered contaminated (fig 1) . ii.e.4. respiratory protection. the subject of respiratory protection as it applies to preventing transmission of airborne infectious agents, including the need for and frequency of fit testing is under scientific review and was the subject of a 2004 cdc workshop. 762 respiratory protection currently requires the use of a respirator with n95 or higher-level filtration to prevent inhalation of infectious particles. information about respirators and respiratory protection programs is summarized in the guideline for preventing transmission of mycobacterium tuberculosis in health care settings. 12 respiratory protection is broadly regulated by osha under the general industry standard for respiratory protection (29 cfr 1910 .134), 763 which requires that us employers in all employment settings implement a program to protect employees from inhalation of toxic materials. osha program components include medical clearance to wear a respirator; provision and use of appropriate respirators, including fit-tested niosh-certified n95 and higher-level particulate filtering respirators; education on respirator use, and periodic reevaluation of the respiratory protection program. when selecting particulate respirators, models with inherently good fit characteristics (ie, those expected to provide protection factors of $ 10% to 95% of wearers) are preferred and theoretically could preclude the need for fit testing. 764, 765 issues pertaining to respiratory protection remain the subject of ongoing debate. information on various types of respirators is available at http://www.cdc.gov/niosh/ npptl/respirators/respsars.html and in several previously published studies. 764, 766, 767 a user-seal check (formerly called a ''fit check'') should be performed by the wearer of a respirator each time that the respirator is donned, to minimize air leakage around the face piece. 768 the optimal frequency of fit testing has not been determined; retesting may be indicated if there is a change in wearer's facial features, onset of a medical condition that would affect respiratory function in the wearer, or a change in the model or size of the respirator that was initially assigned. 12 respiratory protection was first recommended for protection of us hcws from exposure to m tuberculosis in 1989. that recommendation has been maintained in 2 successive revisions of the guidelines for prevention of transmission of tuberculosis in hospitals and other health care settings. 12, 126 the incremental benefit from respirator use, in addition to administrative and engineering controls (ie, aiirs, early recognition of patients likely to have tuberculosis and prompt placement in an aiir, and maintenance of a patient with suspected tuberculosis in an aiir until no longer infectious), for preventing transmission of airborne infectious agents (eg, m tuberculosis) remains undetermined. although some studies have demonstrated effective prevention of m tuberculosis transmission in hospitals in which surgical masks instead of respirators were used in conjunction with other administrative and engineering controls. 636, 769, 770 the cdc currently recommends n95 or higher-level respirators for personnel exposed to patients with suspected or confirmed tuberculosis. currently, this recommendation also holds for other diseases that could be transmitted through the airborne route, including sars 261 and smallpox, 108,129,771 until inhalational transmission is better defined or health care-specific ppe more suitable for preventing infection is developed. wearing of respirators is also currently recommended during the performance of aerosol-generating procedures (eg, intubation, bronchoscopy, suctioning) in patients with sars-cov infection, avian influenza, and pandemic influenza (see appendix a). although airborne precautions are recommended for preventing airborne transmission of measles and varicella-zoster viruses, no data are available on which to base a recommendation for respiratory protection to protect susceptible personnel against these 2 infections. transmission of varicella-zoster virus has been prevented among pediatric patients using negativepressure isolation alone. 772 whether respiratory protection (ie, wearing a particulate respirator) will enhance protection from these viruses has not yet been studied. because most hcws have natural or acquired immunity to these viruses, only immune personnel generally care for patients with these infections. [773] [774] [775] [776] although there is no evidence suggesting that masks are not adequate to protect hcws in these settings, for purposes of consistency and simplicity, or because of difficulties in ascertaining immunity, some facilities may require the use of respirators for entry into all aiirs, regardless of the specific infectious agent present. procedures for safe removal of respirators are provided in figure 1 . in some health care settings, particulate respirators used to provide care for patients with m tuberculosis are reused by the same hcw. this is an acceptable practice providing that the respirator is not damaged or soiled, the fit is not compromised by a change in shape, and the respirator has not been contaminated with blood or body fluids. no data are available on which to base a recommendation regarding the length of time that a respirator may be safely reused. sharps-related injuries. injuries due to needles and other sharps have been associated with transmission of hbv, hcv, and hiv to hcws. 777, 778 the prevention of sharps injuries has always been an essential element of universal precautions and is now an aspect of standard precautions. 1, 779 these include measures to handle needles and other sharp devices in a manner that will prevent injury to the user and to others who may encounter the device during or after a procedure. these measures apply to routine patient care and do not address the prevention of sharps injuries and other blood exposures during surgical and other invasive procedures addressed elsewhere. [780] [781] [782] [783] [784] since 1991, when osha first issued its bloodborne pathogens standard to protect hcws from blood exposure, the focus of regulatory and legislative activity has been on implementing a hierarchy of control measures. this has included focusing attention on removing sharps hazards through the development and use of engineering controls. the federal needlestick safety and prevention act, signed into law in november 2000, authorized osha's revision of its bloodborne pathogens standard to more explicitly require the use of safety-engineered sharps devices. 785 the cdc has provided guidance on sharps injury prevention, 786,787 including guidelines for the design, implementation and evaluation of a comprehensive sharps injury prevention program. 788 ii.f.2. prevention of mucous membrane contact. exposure of mucous membranes of the eyes, nose, and mouth to blood and body fluids has been associated with the transmission of bloodborne viruses and other infectious agents to hcws. 66, 751, 753, 778 the prevention of mucous membrane exposures has always been an element of universal precautions and is now an element of standard precautions for routine patient care 1, 752 and is subject to osha bloodborne pathogen regulations. safe work practices, in addition to wearing ppe, are designed to protect mucous membranes and nonintact skin from contact with potentially infectious material. these include keeping contaminated gloved and ungloved hands from touching the mouth, nose, eyes, or face and positioning patients to direct sprays and splatter away from the caregiver's face. careful placement of ppe before patient contact will help avoid the need to make adjustments to ppe and prevent possible face or mucous membrane contamination during use. in areas where the need for resuscitation is unpredictable, mouthpieces, pocket resuscitation masks with 1-way valves, and other ventilation devices provide an alternative to mouth-to-mouth resuscitation, preventing exposure of the caregiver's nose and mouth to oral and respiratory fluids during the procedure. ii.f.2.a. precautions during aerosol-generating procedures. the performance of procedures that can generate small-particle aerosols (aerosol-generating procedures), such as bronchoscopy, endotracheal intubation, and open suctioning of the respiratory tract, have been associated with transmission of infectious agents to hcws, including m tuberculosis, 789 sars-cov, 93, 94, 98 and n meningitidis. 95 protection of the eyes, nose, and mouth, in addition to gown and gloves, is recommended during performance of these procedures in accordance with standard precautions. the use of a particulate respirator is recommended during aerosol-generating procedures when the aerosol is likely to contain m tuberculosis, sars-cov, or avian or pandemic influenza viruses. ii.g.1. hospitals and long-term care facilities. options for patient placement include single-patient rooms, 2-patient rooms, and multibed wards. of these, single-patient rooms are preferred when transmission of an infectious agent is of concern. although some studies have failed to demonstrate the efficacy of single-patient rooms in preventing hais, 790 other published studies, including one commissioned by the aia and the facility guidelines institute, have documented a beneficial relationship between private rooms and reduced infectious and noninfectious adverse patient outcomes. 791, 792 the aia notes that private rooms are the trend in hospital planning and design. however, most hospitals and ltcfs have multibed rooms and must consider many competing priorities when determining the appropriate room placement for patients (eg, reason for admission; patient characteristics, such as age, gender, and mental status; staffing needs; family requests; psychosocial factors; reimbursement concerns). in the absence of obvious infectious diseases that require specified airborne infection isolation rooms (eg, tuberculosis, sars, chickenpox), the risk of transmission of infectious agents is not always considered when making placement decisions. when only a limited number of single-patient rooms is available, it is prudent to prioritize room assignments for those patients with conditions that facilitate transmission of infectious material to other patients (eg, draining wounds, stool incontinence, uncontained secretions) and those at increased risk of acquisition and adverse outcomes resulting from hais (due to, eg, immunosuppression, open wounds, indwelling catheters, anticipated prolonged length of stay, total dependence on hcws for activities of daily living). 15, 24, 43, 429, 793, 794 single-patient rooms are always indicated for patients placed on airborne precautions in a pe and are preferred for patients requiring contact or droplet precautions. 23, 24, 409, 434, 795, 796 during a suspected or proven outbreak caused by a pathogen whose reservoir is the gastrointestinal tract, the use of single-patient rooms with private bathrooms limits opportunities for transmission, especially when the colonized or infected patient has poor personal hygiene habits or fecal incontinence, or cannot be expected to assist in maintaining procedures that prevent transmission of microorganisms (eg, infants, children, and patients with altered mental status or developmental delay). in the absence of continued transmission, it is not necessary to provide a private bathroom for patients colonized or infected with enteric pathogens as long as personal hygiene practices and standard precautions (especially hand hygiene and appropriate environmental cleaning) are maintained. assignment of a dedicated commode to a patient, and cleaning and disinfecting fixtures and equipment that may have fecal contamination (eg, bathrooms, commodes, 797 scales used for weighing diapers) and the adjacent surfaces with appropriate agents may be especially important when a single-patient room cannot be assigned, because environmental contamination with intestinal tract pathogens is likely from both continent and incontinent patients. 54, 798 the results of several studies that investigated the benefit of a single-patient room in preventing transmission of c difficile were inconclusive. 167, [799] [800] [801] some studies have shown that being in the same room with a colonized or infected patient is not necessarily a risk factor for transmission; 790,802-804 however, for children, the risk of health care-associated diarrhea is increased with the increased number of patients per room. 805 these findings demonstrate that patient factors are important determinants of infection transmission risks. the need for a single-patient room and/or private bathroom for any patient is best determined on a case-by-case basis. cohorting is the practice of grouping together patients who are colonized or infected with the same organism to confine their care to a single area and prevent contact with other patients. cohorts are created based on clinical diagnosis, microbiologic confirmation (when available), epidemiology, and mode of transmission of the infectious agent. avoiding placing severely immunosuppressed patients in rooms with other patients is generally preferred. cohorting has been extensively used for managing outbreaks of mdros, including mrsa, 22 813 rotavirus, 814 and sars. 815 modeling studies provide additional support for cohorting patients to control outbreaks; 816-818 however, cohorting often is implemented only after routine infection control measures have failed to control an outbreak. assigning or cohorting hcws to care only for patients infected or colonized with a single target pathogen limits further transmission of the target pathogen to uninfected patients, 739, 818 but is difficult to achieve in the face of current staffing shortages in hospitals 582 and residential health care sites. [819] [820] [821] however, cohorting of hcws may be beneficial when transmission continues after implementing routine infection control measures and creating patient cohorts. during periods when rsv, human metapneumovirus, 822 parainfluenza, influenza, other respiratory viruses, 823 and rotavirus are circulating in the community, cohorting based on the presenting clinical syndrome is often a priority in facilities that care for infants and young children. 824 for example, during the respiratory virus season, infants may be cohorted based solely on the clinical diagnosis of bronchiolitis, due to the logistical difficulties and costs associated with requiring microbiologic confirmation before room placement and the predominance of rsv during most of the season. however, when available, single-patient rooms are always preferred, because a common clinical presentation (eg, bronchiolitis), can be caused by more than 1 infectious agent. 822, 823, 825 furthermore, the inability of infants and children to contain body fluids, and the close physical contact associated with their care, increases the risk of infection transmission for patients and personnel in this setting. 24, 794 ii.g.2. ambulatory care settings. patients actively infected with or incubating transmissible infectious diseases are frequently seen in ambulatory settings (eg, outpatient clinics, physicians' offices, emergency departments) and potentially expose hcws and other patients, family members, and visitors. 21, 34, 127, 135, 142, 826 in response to the global outbreak of sars in 2003 and in preparation for pandemic influenza, hcws working in outpatient settings are urged to implement source containment measures (eg, asking coughing patients to wear a surgical mask or cover coughing with tissues) to prevent transmission of respiratory infections, beginning at the initial patient encounter, 9,261,827 as described in section iii.a.1.a. signs can be posted at the facility's entrance or at the reception or registration desk requesting that the patient or individuals accompanying the patient promptly inform the receptionist of any symptoms of respiratory infection (eg, cough, flulike illness, increased production of respiratory secretions). the presence of diarrhea, skin rash, or known or suspected exposure to a transmissible disease (eg, measles, pertussis, chickenpox, tuberculosis) also could be added. prompt placement of a potentially infectious patient in an examination room limits the number of exposed individuals in the common waiting area. in waiting areas, maintaining a distance between symptomatic and nonsymptomatic patients (eg, . 3 feet), in addition to source control measures, may limit exposures. however, infections transmitted through the airborne route (eg, m tuberculosis, measles, chickenpox) require additional precautions. 12, 125, 828 patients suspected of having such an infection can wear a surgical mask for source containment, if tolerated, and should be placed in an examination room (preferably an aiir) as soon as possible. if this is not possible, then having the patient wear a mask and segregating the patient from other patients in the waiting area will reduce the risk of exposing others. because the person(s) accompanying the patient also may be infectious, application of the same infection control precautions may be extended to these persons if they are symptomatic. 21, 251, 829 family members accompanying children admitted with suspected m tuberculosis have been found to have unsuspected pulmonary tuberculosis with cavitary lesions, even when asymptomatic. 42, 830 patients with underlying conditions that increase their susceptibility to infection (eg, immunocompromised status 43, 44 or cystic fibrosis 20 ) require special efforts to protect them from exposure to infected patients in common waiting areas. informing the receptionist of their infection risk on arrival allows appropriate steps to further protect these patients from infection. in some cystic fibrosis clinics, to avoid exposure to other patients who could be colonized with b cepacia, patients have been given beepers on registration so that they may leave the area and receive notification to return when an examination room becomes available. 831 ii.g.3. home care. in home care, patient placement concerns focus on protecting others in the home from exposure to an infectious household member. for individuals who are especially vulnerable to adverse outcomes associated with certain infections, it may be beneficial to either remove them from the home or segregate them within the home. persons who are not part of the household may need to be prohibited from visiting during the period of infectivity. for example, in a situation where a patient with pulmonary tuberculosis is contagious and being cared for at home, very young children (age under 4 years) 832 and immunocompromised persons who have not yet been infected should be removed or excluded from the household. during the sars outbreak of 2003, segregation of infected persons during the communicable phase of the illness was found to be beneficial in preventing household transmission. 249, 833 several principles guide the transport of patients requiring transmission-based precautions. in the inpatient and residential settings, these include the following: 1. limiting transport of such patients to essential purposes, such as diagnostic and therapeutic procedures that cannot be performed in the patient's room. 2. when transport is necessary, applying appropriate barriers on the patient (eg, mask, gown, wrapping in sheets or use of impervious dressings to cover the affected areas) when infectious skin lesions or drainage are present, consistent with the route and risk of transmission. 3. notifying hcws in the receiving area of the patient's impending arrival and of the necessary precautions to prevent transmission. 4. for patients being transported outside the facility, informing the receiving facility and the medi-van or emergency vehicle personnel in advance about the type of transmission-based precautions being used. for tuberculosis, additional precautions may be needed in a small shared air space, such as in an ambulance. 12 cleaning and disinfecting noncritical surfaces in patient care areas is an aspect of standard precautions. in general, these procedures do not need to be changed for patients on transmission-based precautions. the cleaning and disinfection of all patient care areas is important for frequently touched surfaces, especially those closest to the patient, which are most likely to be contaminated (eg, bedrails, bedside tables, commodes, doorknobs, sinks, surfaces and equipment in close proximity to the patient). 11, 72, 73, 834 the frequency or intensity of cleaning may need to be changed, based on the patient's level of hygiene and the degree of environmental contamination and for certain infectious agents with reservoirs in the intestinal tract. 54 this may be particularly important in ltcfs and pediatric facilities, where patients with stool and urine incontinence are encountered more frequently. in addition, increased frequency of cleaning may be needed in a pe to minimize dust accumulation. 11 special recommendations for cleaning and disinfecting environmental surfaces in dialysis centers have been published previously. 18 in all health care settings, administrative, staffing, and scheduling activities should prioritize the proper cleaning and disinfection of surfaces that could be implicated in transmission. during a suspected or proven outbreak in which an environmental reservoir is suspected, routine cleaning procedures should be reviewed, and the need for additional trained cleaning staff should be assessed. adherence should be monitored and reinforced to promote consistent and correct cleaning. us environmental protection agency-registered disinfectants or detergents/disinfectants that best meet the overall needs of the health care facility for routine cleaning and disinfection should be selected. 11, 835 in general, use of the existing facility detergent/disinfectant according to the manufacturer's recommendations for amount, dilution, and contact time is sufficient to remove pathogens from surfaces of rooms where colonized or infected individuals were housed. this includes those pathogens that are resistant to multiple classes of antimicrobial agents (eg, c difficile, vre, mrsa, mdr-gnb 11, 24, 88, 434, 745, 795, 836 ). most often, environmental reservoirs of pathogens during outbreaks are related to a failure to follow recommended procedures for cleaning and disinfection, rather than to the specific cleaning and disinfectant agents used. [837] [838] [839] [840] certain pathogens (eg, rotavirus, noroviruses, c difficile) may be resistant to some routinely used hospital disinfectants. 274, 291, [841] [842] [843] [844] [845] [846] the role of specific disinfectants in limiting transmission of rotavirus has been demonstrated experimentally. 841 also, because c difficile may display increased levels of spore production when exposed to non-chlorine-based cleaning agents, and because these spores are more resistant than vegetative cells to commonly used surface disinfectants, some investigators have recommended the use of a 1:10 dilution of 5.25% sodium hypochlorite (household bleach) and water for routine environmental disinfection of rooms of patients with c difficile when there is continued transmission. 843, 847 one study found an association between the use of a hypochlorite solution and decreased rates of c difficile infections. 846 the need to change disinfectants based on the presence of these organisms can be determined in consultation with the infection control committee. 11, 846, 847 detailed recommendations for disinfection and sterilization of surfaces and medical equipment that have been in contact with prion-containing tissue or high risk body fluids, and for cleaning of blood and body substance spills, are available in the guidelines for environmental infection control in health care facilities 11 and in the guideline for disinfection and sterilization. 847 medical equipment and instruments/devices must be cleaned and maintained according to the manufacturers' instructions to prevent patient-to-patient transmission of infectious agents. 86, 87, 324, 848 cleaning to remove organic material always must precede highlevel disinfection and sterilization of critical and semicritical instruments and devices, because residual proteinacous material reduces the effectiveness of the disinfection and sterilization processes. 835, 847 noncritical equipment, such as commodes, intravenous pumps, and ventilators, must be thoroughly cleaned and disinfected before being used on another patient. all such equipment and devices should be handled in a manner that will prevent hcw and environmental contact with potentially infectious material. it is important to include computers and personal digital assistants used in patient care in policies for cleaning and disinfection of noncritical items. the literature on contamination of computers with pathogens has been summarized, 849 and 2 reports have linked computer contamination to colonization and infections in patients. 850, 851 although keyboard covers and washable keyboards that can be easily disinfected are available, the infection control benefit of these items and their optimal management have not yet been determined. in all health care settings, providing patients who are on transmission-based precautions with dedicated noncritical medical equipment (eg, stethoscope, blood pressure cuff, electronic thermometer) has proven beneficial for preventing transmission. 74, 89, 739, 852, 853 when this is not possible, disinfection of this equipment after each use is recommended. other previously published guidelines should be consulted for detailed guidance in developing specific protocols for cleaning and reprocessing medical equipment and patient care items in both routine and special circumstances. 11, 14, 18, 20, 739, 835, 847 in home care, it is preferable to remove visible blood or body fluids from durable medical equipment before it leaves the home. equipment can be cleaned onsite using a detergent/disinfectant and, when possible, should be placed in a plastic bag for transport to the reprocessing location. 20,738 although soiled textiles, including bedding, towels, and patient or resident clothing, may be contaminated with pathogenic microorganisms, the risk of disease transmission is negligible if these textiles are handled, transported, and laundered in a safe manner. 11, 854, 855 key principles for handling soiled laundry are (1) avoiding shaking the items or handling them in any way that may aerosolize infectious agents, (2) avoiding contact of one's body and personal clothing with the soiled items being handled, and (3) containing soiled items in a laundry bag or designated bin. if a laundry chute is used, it must be maintained to minimize dispersion of aerosols from contaminated items. 11 methods of handling, transporting, and laundering soiled textiles are determined by organizational policy and any applicable regulations; 738 guidance is provided in the guidelines for environmental infection control in health care facilities. 11 rather than rigid rules and regulations, hygienic and common sense storage and processing of clean textiles is recommended. 11, 856 when laundering is done outside of a health care facility, the clean items must be packaged or completely covered and placed in an enclosed space during transport to prevent contamination with outside air or construction dust that could contain infectious fungal spores that pose a risk for immunocompromised patients. 11 institutions are required to launder garments used as ppe and uniforms visibly soiled with blood or infective material. 738 little data exist on the safety of home laundering of hcw uniforms, but no increase in infection rates was observed in the one published study, 857 and no pathogens were recovered from home-or hospital-laundered scrubs in another study. 858 in the home, textiles and laundry from patients with potentially transmissible infectious pathogens do not require special handling or separate laundering and may be washed with warm water and detergent. 11, 857, 858 the management of solid waste emanating from the health care environment is subject to federal and state regulations for medical and nonmedical waste. 859, 860 no additional precautions are needed for nonmedical solid waste removed from rooms of patients on transmission-based precautions. solid waste may be contained in a single bag of sufficient strength. 861 the combination of hot water and detergents used in dishwashers is sufficient to decontaminate dishware and eating utensils. therefore, no special precautions are needed for dishware (eg, dishes, glasses, cups) or eating utensils. reusable dishware and utensils may be used for patients requiring transmission-based precautions. in the home and other communal settings, eating utensils and drinking vessels should not be shared, consistent with principles of good personal hygiene and to help prevent transmission of respiratory viruses, herpes simplex virus, and infectious agents that infect the gastrointestinal tract and are transmitted by the fecal/oral route (eg, hepatitis a virus, noroviruses). if adequate resources for cleaning utensils and dishes are not available, then disposable products may be used. important adjunctive measures that are not considered primary components of programs to prevent transmission of infectious agents but nonetheless improve the effectiveness of such programs include (1) antimicrobial management programs, (2) postexposure chemoprophylaxis with antiviral or antibacterial agents, (3) vaccines used both for pre-exposure and postexposure prevention, and (4) screening and restricting visitors with signs of transmissible infections. detailed discussion of judicious use of antimicrobial agents is beyond the scope of this document; however, this topic has been addressed in a previous cdc guideline (http://www.cdc.gov/ncidod/dhqp/pdf/ar/ mdroguideline2006.pdf). ii.n.1. chemoprophylaxis. antimicrobial agents and topical antiseptics may be used to prevent infection and potential outbreaks of selected agents. infections for which postexposure chemoprophylaxis is recommended under defined conditions include b pertussis, 17,862 n meningitides, 863 b anthracis after environmental exposure to aeosolizable material, 864 influenza virus, 610 hiv, 865 and group a streptococcus. 160 orally administered antimicrobials also may be used under defined circumstances for mrsa decolonization of patients or hcws. 866 another form of chemoprophylaxis involves the use of topical antiseptic agents. for example, triple dye is routinely used on the umbilical cords of term newborns to reduce the risk of colonization, skin infections, and omphalitis caused by s aureus, including mrsa, and group a streptococcus. 867, 868 extension of the use of triple dye to low birth weight infants in a nicu was one component of a program that controlled a long-standing mrsa outbreak. 22 topical antiseptics (eg, mupirocin) also are used for decolonization of hcws or selected patients colonized with mrsa, as discussed in the mdro guideline 866, [869] [870] [871] [872] ii.n.2. immunoprophylaxis. certain immunizations recommended for susceptible hcws have decreased the risk of infection and the potential for transmission in health care facilities. 17, 873 the osha mandate requiring employers to offer hbv vaccination to hcws has played a substantial role in the sharp decline in incidence of occupational hbv infection. 777, 874 the routine administration of varicella vaccine to hcws has decreased the need to place susceptible hcws on administrative leave after exposure to patients with varicella. 774 in addition, reports of health care-associated transmission of rubella in obstetric clinics 33, 875 and measles in acute care settings 34 demonstrate the importance of immunization of susceptible hcws against childhood diseases. many states have requirements for vaccination of hcws for measles and rubella in the absence of evidence of immunity. annual influenza vaccine campaigns targeted at patients and hcws in ltcfs and acute care settings have been instrumental in preventing or limiting institutional outbreaks; consequently, increasing attention is being directed toward improving influenza vaccination rates in hcws. 35, 610, 689, [876] [877] [878] transmission of b pertussis in health care facilities has been associated with large and costly outbreaks that include both hcws and patients. 17, 36, 41, 100, 682, 826, 879, 880 hcws in close contact with infants with pertussis are at particularly high risk because of waning immunity and, until 2005, the absence of a vaccine appropriate for adults. but 2 acellular pertussis vaccines were licensed in the united states in 2005, 1 for use in individuals age 11 to 18 years and the other for use in those age 10 to 64 years. 881 current advisory committee on immunization practices provisional recommendations include immunization of adolescents and adults, especially those in contact with infants under age 12 months and hcws with direct patient contact. 882, 883 immunization of children and adults will help prevent the introduction of vaccine-preventable diseases into health care settings. the recommended immunization schedule for children is published annually in the january issues of the morbidity and mortality weekly report, with interim updates as needed. 884, 885 an adult immunization schedule also is available for healthy adults and those with special immunization needs due to high-risk medical conditions. 886 some vaccines are also used for postexposure prophylaxis of susceptible individuals, including varicella, 887 influenza, 610 hepatitis b, 777 and smallpox 225 vaccines. 17, 873 in the future, administration of a newly developed s aureus conjugate vaccine (still under investigation) to selected patients may provide a novel method of preventing health care-associated s aureus (including mrsa) infections in high-risk groups (eg, hemodialysis patients and candidates for selected surgical procedures). 888, 889 immune globulin preparations also are used for postexposure prophylaxis of certain infectious agents under specified circumstances (eg, varicella-zoster virus, hbv, rabies, measles and hepatitis a virus 17, 832, 873 ). the rsv monoclonal antibody preparation palivizumab may have contributed to controlling a nosocomial outbreak of rsv in one nicu, but there is insufficient evidence to support a routine recommendation for its use in this setting. 890 ii.n. 3 24, 43, 44, 372 and sars 21, [252] [253] [254] . effective methods for visitor screening in health care settings have not yet been studied, however. visitor screening is especially important during community outbreaks of infectious diseases and for high-risk patient units. sibling visits are often encouraged in birthing centers, postpartum rooms, pediatric inpatient units, picus, and residential settings for children; in hospital settings, a child visitor should visit only his or her own sibling. screening of visiting siblings and other children before they are allowed into clinical areas is necessary to prevent the introduction of childhood illnesses and common respiratory infections. screening may be passive, through the use of signs to alert family members and visitors with signs and symptoms of communicable diseases not to enter clinical areas. more active screening may include the completion of a screening tool or questionnaire to elicit information related to recent exposures or current symptoms. this information is reviewed by the facility staff, after which the visitor is either permitted to visit or is excluded. 832 family and household members visiting pediatric patients with pertussis and tuberculosis may need to be screened for a history of exposure, as well as signs and symptoms of current infection. potentially infectious visitors are excluded until they receive appropriate medical screening, diagnosis, or treatment. if exclusion is not considered to be in the best interest of the patient or family (ie, primary family members of critically or terminally ill patients), then the symptomatic visitor must wear a mask while in the health care facility and remain in the patient's room, avoiding exposure to others, especially in public waiting areas and the cafeteria. visitor screening is used consistently on hsct units. 15, 43 however, considering the experience during the 2003 sars outbreaks and the potential for pandemic influenza, developing effective visitor screening systems will be beneficial. 9 education concerning respiratory hygiene/cough etiquette is a useful adjunct to visitor screening. ii.n.3.b. use of barrier precautions by visitors. the use of gowns, gloves, and masks by visitors in health care settings has not been addressed specifically in the scientific literature. some studies included the use of gowns and gloves by visitors in the control of mdros but did not perform a separate analysis to determine whether their use by visitors had a measurable impact. [892] [893] [894] family members or visitors who are providing care to or otherwise are in very close contact with the patient (eg, feeding, holding) may also have contact with other patients and could contribute to transmission in the absence of effective barrier precautions. specific recommendations may vary by facility or by unit and should be determined by the specific level of interaction. there are 2 tiers of hicpac/cdc precautions to prevent transmission of infectious agents, standard precautions and transmission-based precautions. standard precautions are intended to be applied to the care of all patients in all health care settings, regardless of the suspected or confirmed presence of an infectious agent. implementation of standard precautions constitutes the primary strategy for the prevention of health care-associated transmission of infectious agents among patients and hcws. transmission-based precautions are for patients who are known or suspected to be infected or colonized with infectious agents, including certain epidemiologically important pathogens, which require additional control measures to effectively prevent transmission. because the infecting agent often is not known at the time of admission to a health care facility, transmission-based precautions are used empirically, according to the clinical syndrome and the likely etiologic agents at the time, and then modified when the pathogen is identified or a transmissible infectious etiology is ruled out. examples of this syndromic approach are presented in table 2 . the hicpac/cdc guidelines also include recommendations for creating a protective environment for allogeneic hsct patients. the specific elements of standard and transmission-based precautions are discussed in part ii of this guideline. in part iii, the circumstances in which standard precautions, transmission-based precautions, and a protective environment are applied are discussed. tables 4 and 5 summarize the key elements of these sets of precautions standard precautions combine the major features of universal precautions 779, 895 and body substance isolation 639 and are based on the principle that all blood, body fluids, secretions, excretions except sweat, nonintact skin, and mucous membranes may contain transmissible infectious agents. standard precautions include a group of infection prevention practices that apply to all patients, regardless of suspected or confirmed infection status, in any setting in which health care is delivered (table 4 ). these include hand hygiene; use of gloves, gown, mask, eye protection, or face shield, depending on the anticipated exposure; and safe injection practices. also, equipment or items in the patient environment likely to have been contaminated with infectious body fluids must be handled in a manner to prevent transmission of infectious agents (eg, wear gloves for direct contact, contain heavily soiled equipment, properly clean and disinfect or sterilize reusable equipment before use on another patient). the application of standard precautions during patient care is determined by the nature of the hcw-patient interaction and the extent of anticipated blood, body fluid, or pathogen exposure. for some interactions (eg, performing venipuncture), only gloves may be needed; during other interactions (eg, intubation), use of gloves, gown, and face shield or mask and goggles is necessary. education and training on the principles and rationale for recommended practices are critical elements of standard precautions because they facilitate appropriate decision-making and promote adherence when hcws are faced with new circumstances. 654, [680] [681] [682] [683] [684] [685] an example of the importance of the use of standard precautions is intubation, especially under emergency circumstances when infectious agents may not be suspected, but later are identified (eg, sars-cov, n meningitides). the application of standard precautions is described below and summarized in table 4 . guidance on donning and removing gloves, gowns and other ppe is presented in figure 1 . standard precautions are also intended to protect patients by ensuring that hcws do not carry infectious agents to patients on their hands or via equipment used during patient care. 21, 254, 896 the strategy proposed has been termed respiratory hygiene/cough etiquette 9, 827 and is intended to be incorporated into infection control practices as a new component of standard precautions. the strategy is targeted at patients and accompanying family members and friends with undiagnosed transmissible respiratory infections, and applies to any person with signs of illness including cough, congestion, rhinorrhea, or increased production of respiratory secretions when entering a health care facility. 40, 41, 43 the term cough etiquette is derived from recommended source control measures for m tuberculosis. 12, 126 the elements of respiratory hygiene/cough etiquette include (1) education of health care facility staff, patients, and visitors; (2) posted signs, in language(s) appropriate to the population served, with instructions to patients and accompanying family members or friends; (3) source control measures (eg, covering the mouth/nose with a tissue when coughing and prompt disposal of used tissues, using surgical masks on the coughing person when tolerated and appropriate); (4) hand hygiene after contact with respiratory secretions; and (5) spatial separation, ideally .3 feet, of persons with respiratory infections in common waiting areas when possible. covering sneezes and coughs and placing masks on coughing patients are proven means of source containment that prevent infected persons from dispersing respiratory secretions into the air. 107, 145, 897, 898 masking may be difficult in some settings, (eg, pediatrics), in which case the emphasis by necessity may be on cough etiquette. 899 physical proximity of , 3 feet has been associated with an increased risk for transmission of infections through the droplet route (eg, n meningitidis 103 and group a streptococcus 114 ) and thus supports the practice of distancing infected persons from others who are not infected. the effectiveness of good hygiene practices, especially hand hygiene, in preventing transmission of viruses and reducing the incidence of respiratory infections both within and outside [900] [901] [902] health care settings is summarized in several reviews. 558, 716, 903 these measures should be effective in decreasing the risk of transmission of pathogens contained in large respiratory droplets (eg, influenza virus, 23 adenovirus, 111 b pertussis, 826 and m pneumoniae 112 ). although fever will be present in many respiratory infections, patients with pertussis and mild upper respiratory tract infections are often afebrile. therefore, the absence of fever does not always exclude a respiratory infection. patients who have asthma, allergic rhinitis, or chronic obstructive lung disease also may be coughing and sneezing. although these patients often are not infectious, cough etiquette measures are prudent. hcws are advised to observe droplet precautions (ie, wear a mask) and hand hygiene when examining and caring for patients with signs and symptoms of a respiratory infection. hcws who have a respiratory infection are advised to avoid direct patient contact, especially with high-risk patients. if this is not possible, then a mask should be worn while providing patient care. iii.a.1.b. safe injection practices. the investigation of 4 large outbreaks of hbv and hcv among patients in ambulatory care facilities in the united states identified a need to define and reinforce safe injection practices. 452 the 4 outbreaks occurred in a private medical practice, a pain clinic, an endoscopy clinic, and a hematology/oncology clinic. the primary breaches in infection control practice that contributed to these outbreaks were reinsertion of used needles into a multiple-dose vial or solution container (eg, saline bag) and use of a single needle/syringe to administer intravenous medication to multiple patients. in 1 of these outbreaks, preparation of medications in the same workspace where used needle/syringes were dismantled also may have been a contributing factor. these and other outbreaks of viral hepatitis could have been prevented by adherence to basic principles of aseptic technique for the preparation and administration of parenteral medications. 452, 453 these include the use of a sterile, single-use, disposable needle and syringe for each injection given and prevention of contamination of injection equipment and medication. whenever possible, use of single-dose vials is preferred over multiple-dose vials, especially when medications will be administered to multiple patients. outbreaks related to unsafe injection practices indicate that some hcws are unaware of, do not understand, or do not adhere to basic principles of infection control and aseptic technique. a survey of us health care workers who provide medication through injection found that 1% to 3% reused the same needle and/or syringe on multiple patients. 904 among the deficiencies identified in recent outbreaks were a lack of oversight of personnel and failure to follow up on reported breaches in infection control practices in ambulatory settings. therefore, to ensure that all hcws understand and adhere to recommended practices, principles of infection control and aseptic technique need to be reinforced in training programs and incorporated into institutional polices that are monitored for adherence. 453 iii.a.1.c. infection control practices for special lumbar puncture procedures. in 2004, the cdc investigated 8 cases of postmyelography meningitis that either were reported to the cdc or identified through a survey of the emerging infections network of the infectious disease society of america. blood and/or cerebrospinal fluid of all 8 cases yielded streptococcal species consistent with oropharyngeal flora and there were changes in the csf indices and clinical status indicative of bacterial meningitis. equipment and products used during these procedures (eg, contrast media) were excluded as probable sources of contamination. procedural details available for 7 cases determined that antiseptic skin preparations and sterile gloves had been used. however, none of the clinicians wore a face mask, giving rise to the speculation that droplet transmission of oralpharyngeal flora was the most likely explanation for these infections. bacterial meningitis after myelography and other spinal procedures (eg, lumbar puncture, spinal and epidural anesthesia, intrathecal chemotherapy) has been reported previously. [905] [906] [907] [908] [909] [910] [911] [912] [913] [914] as a result, the question of whether face masks should be worn to prevent droplet spread of oral flora during spinal procedures (eg, myelography, lumbar puncture, spinal anesthesia) has been debated. 915, 916 face masks are effective in limiting the dispersal of oropharyngeal droplets 917 and are recommended for the placement of central venous catheters. 918 in october 2005, hicpac reviewed the evidence and concluded that there is sufficient experience to warrant the additional protection of a face mask for the individual placing a catheter or injecting material into the spinal or epidural space. there are 3 categories of transmission-based precautions: contact precautions, droplet precautions, and airborne precautions. transmission-based precautions are used when the route(s) of transmission is (are) not completely interrupted using standard precautions alone. for some diseases that have multiple routes of transmission (eg, sars), more than 1 transmission-based precautions category may be used. when used either singly or in combination, they are always used in addition to standard precautions. see appendix a for recommended precautions for specific infections. when transmission-based precautions are indicated, efforts must be made to counteract possible adverse effects on patients (ie, anxiety, depression and other mood disturbances, 919-921 perceptions of stigma, 922 reduced contact with clinical staff, [923] [924] [925] and increases in preventable adverse events 564 ) to improve acceptance by the patients and adherence by hcws. iii.b.1. contact precautions. contact precautions are intended to prevent transmission of infectious agents, including epidemiologically important microorganisms, which are spread by direct or indirect contact with the patient or the patient's environment as described in section i.b.3.a. the specific agents and circumstance for which contact precautions are indicated are found in appendix a. the application of contact precautions for patients infected or colonized with mdros is described in the 2006 hicpac/cdc mdro guideline. 926 contact precautions also apply where the presence of excessive wound drainage, fecal incontinence, or other discharges from the body suggest an increased potential for extensive environmental contamination and risk of transmission. a single-patient room is preferred for patients who require contact precautions. when a single-patient room is not available, consultation with infection control personnel is recommended to assess the various risks associated with other patient placement options (eg, cohorting, keeping the patient with an existing roommate). in multipatient rooms, $ 3 feet spatial separation between beds is advised to reduce the opportunities for inadvertent sharing of items between the infected/colonized patient and other patients. hcws caring for patients on contact precautions wear a gown and gloves for all interactions that may involve contact with the patient or potentially contaminated areas in the patient's environment. donning ppe on room entry and discarding before exiting the patient room is done to contain pathogens, especially those that have been implicated in transmission through environmental contamination (eg, vre, c difficile, noroviruses and other intestinal tract pathogens, rsv). 54, 72, 73, 78, 273, 274, 739 iii.b.2. droplet precautions. droplet precautions are intended to prevent transmission of pathogens spread through close respiratory or mucous membrane contact with respiratory secretions as described in section i.b.3.b. because these pathogens do not remain infectious over long distances in a health care facility, special air handling and ventilation are not required to prevent droplet transmission. infectious agents for which droplet precautions are indicated are listed in appendix a and include b pertussis, influenza virus, adenovirus, rhinovirus, n meningitides, and group a streptococcus (for the first 24 hours of antimicrobial therapy). a single-patient room is preferred for patients who require droplet precautions. when a single-patient room is not available, consultation with infection control personnel is recommended to assess the various risks associated with other patient placement options (eg, cohorting, keeping the patient with an existing roommate). spatial separation of $ 3 feet and drawing the curtain between patient beds is especially important for patients in multibed rooms with infections transmitted by the droplet route. hcws wear a mask (a respirator is not necessary) for close contact with infectious patient; the mask is generally donned on room entry. patients on droplet precautions who must be transported outside of the room should wear a mask if tolerated and follow respiratory hygiene/cough etiquette. iii.b.3. airborne precautions. airborne precautions prevent transmission of infectious agents that remain infectious over long distances when suspended in the air (eg, rubeola virus [measles], varicella virus [chickenpox], m tuberculosis, and possibly sars-cov), as described in section i.b.3.c and appendix a. the preferred placement for patients who require airborne precautions is in an aiir, a single-patient room equipped with special air handling and ventilation capacity that meet the aia/facility guidelines institute standards for aiirs (ie, monitored negative pressure relative to the surrounding area; 12 air exchanges per hour for new construction and renovation and 6 air exchanges per hour for existing facilities; air exhausted directly to the outside or recirculated through hepa filtration before return). 12, 13 some states require the availability of such rooms in hospitals, emergency departments, and nursing homes that care for patients with m tuberculosis. a respiratory protection program that includes education about use of respirators, fit testing, and user seal checks is required in any facility with aiirs. in settings where airborne precautions cannot be implemented due to limited engineering resources (eg, physician offices), masking the patient, placing the patient in a private room (eg, office examination room) with the door closed, and providing n95 or higher-level respirators or masks if respirators are not available for hcws will reduce the likelihood of airborne transmission until the patient is either transferred to a facility with an aiir or returned to the home environment, as deemed medically appropriate. hcws caring for patients on airborne precautions wear a mask or respirator, depending on the disease-specific recommendations (see section ii.e.4, table 2 , and appendix a), that is donned before room entry. whenever possible, nonimmune hcws should not care for patients with vaccine-preventable airborne diseases (eg, measles, chickenpox, smallpox). diagnosis of many infections requires laboratory confirmation. because laboratory tests, especially those that depend on culture techniques, often require 2 or more days for completion, transmission-based precautions must be implemented while test results are pending, based on the clinical presentation and likely pathogens. use of appropriate transmission-based precautions at the time a patient develops symptoms or signs of transmissible infection, or arrives at a health care facility for care, reduces transmission opportunities. although it is not possible to identify prospectively all patients needing transmission-based precautions, certain clinical syndromes and conditions carry a sufficiently high risk to warrant their use empirically while confirmatory tests are pending (see table 2 ). icps are encouraged to modify or adapt this table according to local conditions. transmission-based precautions remain in effect for limited periods (ie, while the risk for transmission of the infectious agent persists or for the duration of the illness (see appendix a). for most infectious diseases, this duration reflects known patterns of persistence and shedding of infectious agents associated with the natural history of the infectious process and its treatment. for some diseases (eg, pharyngeal or cutaneous diphtheria, rsv), transmission-based precautions remain in effect until culture or antigen-detection test results document eradication of the pathogen and, for rsv, symptomatic disease is resolved. for other diseases (eg, m tuberculosis), state laws and regulations and health care facility policies may dictate the duration of precautions. 12 in immunocompromised patients, viral shedding can persist for prolonged periods of time (many weeks to months) and transmission to others may occur during that time; therefore, the duration of contact and/or droplet precautions may be prolonged for many weeks. 499, [927] [928] [929] [930] [931] [932] the duration of contact precautions for patients who are colonized or infected with mdros remains undefined. mrsa is the only mdro for which effective decolonization regimens are available. 866 however, carriers of mrsa who have negative nasal cultures after a course of systemic or topical therapy may resume shedding mrsa in the weeks after therapy. 933, 934 although early guidelines for vre suggested discontinuation of contact precautions after 3 stool cultures obtained at weekly intervals proved negative, 739 subsequent experiences have indicated that such screening may fail to detect colonization that can persist for . 1 year. 27, [935] [936] [937] likewise, available data indicate that colonization with vre, mrsa, 938 and possibly mdr-gnb can persist for many months, especially in the presence of severe underlying disease, invasive devices, and recurrent courses of antimicrobial agents. it may be prudent to assume that mdro carriers are colonized permanently and manage them accordingly. alternatively, an interval free of hospitalizations, antimicrobial therapy, and invasive devices (eg, 6 or 12 months) before reculturing patients to document clearance of carriage may be used. determination of the best strategy awaits the results of additional studies. see the 2006 hicpac/cdc mdro guideline 926 for a discussion of possible criteria to discontinue contact precautions for patients colonized or infected with mdros. although transmission-based precautions generally apply in all health care settings, exceptions exist. for example, in home care, aiirs are not available. furthermore, family members already exposed to diseases such as varicella and tuberculosis would not use masks or respiratory protection, but visiting hcws would need to use such protection. similarly, management of patients colonized or infected with mdros may necessitate contact precautions in acute care hospitals and in some ltcfs when there is continued transmission, but the risk of transmission in ambulatory care and home care has not been defined. consistent use of standard precautions may suffice in these settings, but more information is needed. a pe is designed for allogeneic hsct patients to minimize fungal spore counts in the air and reduce the risk of invasive environmental fungal infections (see table 5 for specifications). 11, [13] [14] [15] the need for such controls has been demonstrated in studies of aspergillosis outbreaks associated with construction. 11, 14, 15, 157, 158 as defined by the aia 13 and presented in detail in the cdc's 2003 guideline for environmental infection control in health care facilities, 11,860 air quality for hsct patients is improved through a combination of environmental controls that include (1) hepa filtration of incoming air, (2) directed room air flow, (3) positive room air pressure relative to the corridor, (4) well-sealed rooms (including sealed walls, floors, ceilings, windows, electrical outlets) to prevent flow of air from the outside, (5) ventilation to provide $ 12 air changes per hour, (6) strategies to minimize dust (eg, scrubbable surfaces rather than upholstery 939 and carpet, 940 and routinely cleaning crevices and sprinkler heads), and (7) prohibiting dried and fresh flowers and potted plants in the rooms of hsct patients. the latter is based on molecular typing studies that have found indistinguishable strains of aspergillus terreus in patients with hematologic malignancies and in potted plants in the vicinity of the patients. [941] [942] [943] the desired quality of air may be achieved without incurring the inconvenience or expense of laminar airflow. 15, 157 to prevent inhalation of fungal spores during periods when construction, renovation, or other dust-generating activities that may be ongoing in and around the health care facility, it has been recommended that severely immunocompromised patients wear a high-efficiency respiratory protection device (eg, an n95 respirator) when they leave the pe. 11, 14, 944 the use of masks or respirators by hsct patients when they are outside of the pe for prevention of environmental fungal infections in the absence of construction has not been evaluated. a pe does not include the use of barrier precautions beyond those indicated for standard precuations and transmission-based precautions. no published reports support the benefit of placing patients undergoing solid organ transplantation or other immunocompromised patients in a pe. these recommendations are designed to prevent transmission of infectious agents among patients and hcws in all settings where health care is delivered. as in other cdc/hicpac guidelines, each recommendation is categorized on the basis of existing scientific data, theoretical rationale, applicability, and, when possible, economic impact. the cdc/hicpac system for categorizing recommendations is as follows: category ia. strongly recommended for implementation and strongly supported by well-designed experimental, clinical, or epidemiologic studies. category ib. strongly recommended for implementation and supported by some experimental, clinical, or epidemiologic studies and a strong theoretical rationale. category ic. required for implementation, as mandated by federal and/or state regulation or standard. category ii. suggested for implementation and supported by suggestive clinical or epidemiologic studies or a theoretical rationale. no recommendation; unresolved issue. practices for which insufficient evidence or no consensus regarding efficacy exists. health care organization administrators should ensure the implementation of recommendations specified in this section. agents into the objectives of the organization's patient and occupational safety programs. 542 assume that every person is potentially infected or colonized with an organism that could be transmitted in the health care setting and apply the following infection control practices during the delivery of health care. iv.a.1. during the delivery of health care, avoid unnecessary touching of surfaces in close proximity to the patient to prevent both contamination of clean hands from environmental surfaces and transmission of pathogens from contaminated hands to surfaces. 72 airborne precautions does not need to wear a mask or respirator during transport if the patient is wearing a mask and infectious skin lesions are covered. category ii v.d.6. exposure management immunize or provide the appropriate immune globulin to susceptible persons as soon as possible after unprotected contact (ie, exposure) to a patient with measles, varicella, or smallpox: category ia d administer measles vaccine to exposed susceptible persons within 72 hours after the exposure or administer immune globulin within 6 days of the exposure event for high-risk persons in whom vaccine is contraindicated. 17,1030-1033 d administer varicella vaccine to exposed susceptible persons within 120 hours after the exposure or administer varicella immune globulin (vzig or an alternative product), when available, within 96 hours for high-risk persons in whom vaccine is contraindicated (eg, immunocompromised patients, pregnant women, newborns whose mother's varicella onset was , 5 days before or within 48 hours after delivery). 887,1033-1035 d administer smallpox vaccine to exposed susceptible persons within 4 days after exposure. 108 vi. protective environment (see table 4 airborne infection isolation room (aiir). formerly known as a negative-pressure isolation room, an aiir is a single-occupancy patient care room used to isolate persons with a suspected or confirmed airborne infectious disease. environmental factors are controlled in aiirs to minimize the transmission of infectious agents that are usually transmitted from person to person by droplet nuclei associated with coughing or aerosolization of contaminated fluids. aiirs should provide negative pressure in the room (so that air flows under the door gap into the room), an air flow rate of 6 to 12 air changes per hour (ach) (6 ach for existing structures, 12 ach for new construction or renovation), and direct exhaust of air from the room to the outside of the building or recirculation of air through a highefficiency particulate air filter before returning to circulation. ( ambulatory care setting. a facility that provides health care to patients who do not remain overnight; examples include hospital-based outpatient clinics, non-hospital-based clinics and physician offices, urgent care centers, surgicenters, free-standing dialysis centers, public health clinics, imaging centers, ambulatory behavioral health and substance abuse clinics, physical therapy and rehabilitation centers, and dental practices. bioaerosol. an airborne dispersion of particles containing whole or parts of biological entities, including bacteria, viruses, dust mites, fungal hyphae, and fungal spores. such aerosols usually consist of a mixture of monodispersed and aggregate cells, spores, or viruses carried by other materials, such as respiratory secretions and/or inert particles. infectious bioaerosols (ie, those containing biological agents capable of causing an infectious disease) can be generated from human sources (eg, expulsion from the respiratory tract during coughing, sneezing, talking, singing, suctioning, or wound irrigation), wet environmental sources (eg, high-volume air consitioning and cooling tower water with legionella) or dry sources (eg, construction dust with spores produced by aspergillus spp). bioaerosols include large respiratory droplets and small droplet nuclei (cole ec. ajic 1998; 26: 453-64) . caregiver.. any person who is not an employee of an organization, is not paid, and provides or assists in providing health care to a patient (eg, family member, friend) and acquire technical training as needed based on the tasks that must be performed. cohorting. in the context of this guideline, this term applies to the practice of grouping patients infected or colonized with the same infectious agent together to confine their care to one area and prevent contact with susceptible patients (cohorting patients). during outbreaks, health care personnel may be assigned to a cohort of patients to further limit opportunities for transmission (cohorting staff). colonization. proliferation of microorganisms on or within body sites without detectable host immune response, cellular damage, or clinical expression. the presence of a microorganism within a host may occur with varying durations but may become a source of potential transmission. in many instances, colonization and carriage are synonymous. droplet nuclei. microscopic particles , 5 mm in size that are the residue of evaporated droplets and are produced when a person coughs, sneezes, shouts, or sings. these particles can remain suspended in the air for prolonged periods and can be carried on normal air currents in a room or beyond, to adjacent spaces or areas receiving exhaust air. engineering controls. removal or isolation of a workplace hazard through technology. an airborne infection isolation room, a protective environment, engineered sharps injury prevention device, and a sharps container are examples of engineering controls. epidemiologically important pathogen. an infectious agent that has one or more of the following characteristics: (1) readily transmissible, (2) a proclivity toward causing outbreaks, (3) possible association with a severe outcome, and (4) difficult to treat. examples include acinetobacter spp, aspergillus spp, burkholderia cepacia, clostridium difficile, klebsiella or enterobacter spp, extended-spectrum beta-lactamaseproducing gram-negative bacilli, methicillin-resistant staphylococcus aureus, pseudomonas aeruginosa, vancomycin-resistant enterococci, vancomycin-resistant staphylococcus aureus, influenza virus, respiratory syncytial virus, rotavirus, severe acute respiratory syndrome coronavirus, noroviruses, and the hemorrhagic fever viruses. hand hygiene. a general term that applies to any one of the following: (1) handwashing with plain (nonantimicrobial) soap and water, (2) antiseptic handwashing (soap containing antiseptic agents and water), (3) antiseptic handrub (waterless antiseptic product, most often alcohol-based, rubbed on all surfaces of hands), or (4) surgical hand antisepsis (antiseptic handwash or antiseptic handrub performed preoperatively by surgical personnel to eliminate transient hand flora and reduce resident hand flora). 558 health care-associated infection (hai). an infection that develops in a patient who is cared for in any setting where health care is delivered (eg, acute care hospital, chronic care facility, ambulatory clinic, dialysis center, surgicenter, home) and is related to receiving health care (ie, was not incubating or present at the time health care was provided). in ambulatory and home settings, hai refers to any infection that is associated with a medical or surgical intervention. because the geographic location of infection acquisition is often uncertain, the preferred term is considered to be health care-associated rather than health care-acquired. healthcare epidemiologist. a person whose primary training is medical (md, do) and/or masters-or doctorate-level epidemiology who has received advanced training in health care epidemiology. typically these professionals direct or provide consultation to an infection control program in a hospital, long-term care facility, or health care delivery system (also see infection control professional). health care personnel, health care worker (hcw). any paid or unpaid person who works in a health care setting (eg, any person who has professional or technical training in a health care-related field and provides patient care in a health care setting or any person who provides services that support the delivery of health care such as dietary, housekeeping, engineering, maintenance personnel). hematopoietic stem cell transplantation (hsct). any transplantation of blood-or bone marrow-derived hematopoietic stem cells, regardless of donor type (eg, allogeneic or autologous) or cell source (eg, bone marrow, peripheral blood, or placental/umbilical cord blood), associated with periods of severe immunosuppression that vary with the source of the cells, the intensity of chemotherapy required, and the presence of graft versus host disease (mmwr 2000; 49: rr-10). high-efficiency particulate air (hepa) filter. an air filter that removes .99.97% of particles . 0.3 mm (the most penetrating particle size) at a specified flow rate of air. hepa filters may be integrated into the central air handling systems, installed at the point of use above the ceiling of a room, or used as portable units (mmwr 2003; 52: rr-10). home care. a wide range of medical, nursing, rehabilitation, hospice, and social services delivered to patients in their place of residence (eg, private residence, senior living center, assisted living facility). home health care services include care provided by home health aides and skilled nurses, respiratory therapists, dieticians, physicians, chaplains, and volunteers; provision of durable medical equipment; home infusion therapy; and physical, speech, and occupational therapy. immunocompromised patient. a patient whose immune mechanisms are deficient because of a congenital or acquired immunologic disorder (eg, human immunodeficiency virus infection, congenital immune deficiency syndromes), chronic diseases such as diabetes mellitus, cancer, emphysema, or cardiac failure, intensive care unit care, malnutrition, and immunosuppressive therapy of another disease process [eg, radiation, cytotoxic chemotherapy, anti-graft rejection medication, corticosteroids, monoclonal antibodies directed against a specific component of the immune system]). the type of infections for which an immunocompromised patient has increased susceptibility is determined by the severity of immunosuppression and the specific component(s) of the immune system that is affected. patients undergoing allogeneic hematopoietic stem cell transplantation and those with chronic graft versus host disease are considered the most vulnerable to health care-associated infections. immunocompromised states also make it more difficult to diagnose certain infections (eg, tuberculosis) and are associated with more severe clinical disease states than persons with the same infection and a normal immune system. infection. the transmission of microorganisms into a host after evading or overcoming defense mechanisms, resulting in the organism's proliferation and invasion within host tissue(s). host responses to infection may include clinical symptoms or may be subclinical, with manifestations of disease mediated by direct organisms pathogenesis and/or a function of cell-mediated or antibody responses that result in the destruction of host tissues. infection control and prevention professional (icp). a person whose primary training is in either nursing, medical technology, microbiology, or epidemiology and who has acquired specialized training in infection control. responsibilities may include collection, analysis, and feedback of infection data and trends to health care providers; consultation on infection risk assessment, prevention, and control strategies; performance of education and training activities; implementation of evidence-based infection control practices or those mandated by regulatory and licensing agencies; application of epidemiologic principles to improve patient outcomes; participation in planning renovation and construction projects (eg, to ensure appropriate containment of construction dust); evaluation of new products or procedures on patient outcomes; oversight of employee health services related to infection prevention; implementation of preparedness plans; communication within the health care setting, with local and state health departments, and with the community at large concerning infection control issues; and participation in research. certification in infection control is available through the certification board of infection control and epidemiology. infection control and prevention program. a multidisciplinary program that includes a group of activities to ensure that recommended practices for the prevention of health care-associated infections are implemented and followed by health care workers, making the health care setting safe from infection for patients and health care personnel. the joint commission on accreditation of healthcare organizations requires the following 5 components of an infection control program for accreditation: (1) surveillance: monitoring patients and health care personnel for acquisition of infection and/or colonization; (2) investigation: identification and analysis of infection problems or undesirable trends; (3) prevention: implementation of measures to prevent transmission of infectious agents and to reduce risks for device-and procedure-related infections; (4) control: evaluation and management of outbreaks; and (5) reporting: provision of information to external agencies as required by state and federal laws and regulations (see http://www.jcaho.org). the infection control program staff has the ultimate authority to determine infection control policies for a health care organization with the approval of the organization's governing body. long-term care facility (ltcf). a residential or outpatient facility designed to meet the biopsychosocial needs of persons with sustained self-care deficits. these include skilled nursing facilities, chronic disease hospitals, nursing homes, foster and group homes, institutions for the developmentally disabled, residential care facilities, assisted living facilities, retirement homes, adult day health care facilities, rehabilitation centers, and long-term psychiatric hospitals. mask. a term that applies collectively to items used to cover the nose and mouth and includes both procedure masks and surgical masks (see http://www.fda. gov/cdrh/ode/guidance/094.html#4). multidrug-resistant organism (mdro). in general, a bacterium (excluding mycobacterium tuberculosis) that is resistant to 1 or more classes of antimicrobial agents and usually is resistant to all but 1 or 2 commercially available antimicrobial agents (eg, methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum beta-lactamase-producing or intrinsically resistant gram-negative bacilli). 176 nosocomial infection. derived from 2 greek words, ''nosos'' (disease) and ''komeion'' (to take care of), refers to any infection that develops during or as a result of an admission to an acute care facility (hospital) and was not incubating at the time of admission. personal protective equipment (ppe). a variety of barriers used alone or in combination to protect mucous membranes, skin, and clothing from contact with infectious agents. ppe includes gloves, masks, respirators, goggles, face shields, and gowns. procedure mask. a covering for the nose and mouth that is intended for use in general patient care situations. these masks generally attach to the face with ear loops rather than ties or elastic. unlike surgical masks, procedure masks are not regulated by the food and drug administration. protective environment. a specialized patient care area, usually in a hospital, with a positive air flow relative to the corridor (ie, air flows from the room to the outside adjacent space). the combination of high-efficiency particulate air filtration, high numbers (.12) of air changes per hour, and minimal leakage of air into the room creates an environment that can safely accommodate patients with a severely compromised immune system (eg, those who have received allogeneic hemopoietic stem cell transplantation) and decrease the risk of exposure to spores produced by environmental fungi. other components include use of scrubbable surfaces instead of materials such as upholstery or carpeting, cleaning to prevent dust accumulation, and prohibition of fresh flowers or potted plants. quasi-experimental study. a study undertaken to evaluate interventions but do not use randomization as part of the study design. these studies are also referred to as nonrandomized, pre-/postintervention study designs. these studies aim to demonstrate causality between an intervention and an outcome but cannot achieve the level of confidence concerning an attributable benefit obtained through a randomized controlled trial. in hospitals and public health settings, randomized control trials often cannot be implemented due to ethical, practical, and urgency reasons; therefore, quasi-experimental design studies are commonly used. however, even if an intervention appears to be effective statistically, the question can be raised as to the possibility of alternative explanations for the result. such a study design is used when it is not logistically feasible or ethically possible to conduct a randomized controlled trial, (eg, during outbreaks). within the classification of quasi-experimental study designs, there is a hierarchy of design features that may contribute to validity of results (harris et al. cid 2004 :38: 1586 . residential care setting. a facility in which people live, minimal medical care is delivered, and the psychosocial needs of the residents are provided for. respirator. a personal protective device worn by health care personnel over the nose and mouth to protect them from acquiring airborne infectious diseases due to inhalation of infectious airborne particles , 5 mm in size. these include infectious droplet nuclei from patients with mycobacterium tuberculosis, variola virus [smallpox], or severe acute respiratory syndrome and dust particles that contain infectious particles, such as spores of environmental fungi (eg, aspergillus spp). the centers for disease control and prevention's national institute for occupational safety and health (niosh) certifies respirators used in health care settings (see http://www.cdc.gov/niosh/topics/respirators/). the n95 disposable particulate, air-purifying respirator is the type used most commonly by health care personnel. other respirators used include n-99 and n-100 particulate respirators, powered air-purifying respirators with high-efficiency filters, and nonpowered fullfacepiece elastomeric negative pressure respirators. a listing of niosh-approved respirators can be found at http://www.cdc.gov/niosh/npptl/respirators/disp_part/ particlist.html. respirators must be used in conjunction with a complete respiratory protection program, as required by the occupational safety and health administration, which includes fit testing, training, proper selection of respirators, medical clearance, and respirator maintenance. respiratory hygiene/cough etiquette. a combination of measures designed to minimize the transmission of respiratory pathogens through droplet or airborne routes in health care settings. the components of respiratory hygiene/cough etiquette are (1) covering the mouth and nose during coughing and sneezing, (2) using tissues to contain respiratory secretions with prompt disposal into a no-touch receptacle, (3) offering a surgical mask to persons who are coughing to decrease contamination of the surrounding environment, and (4) turning the head away from others and maintaining spatial separation (ideally .3 feet) when coughing. these measures are targeted to all patients with symptoms of respiratory infection and their accompanying family members or friends beginning at the point of initial encounter with a health care setting (eg, reception/triage in emergency departments, ambulatory clinics, health care provider offices). 126 (srinivasin a iche 2004; 25: 1020; http://www.cdc.gov/flu/ professionals/infectioncontrol/resphygiene.htm). safety culture. shared perceptions of workers and management regarding the level of safety in the work environment. a hospital safety climate includes the following organizational components: (1) senior management support for safety programs, (2) absence of workplace barriers to safe work practices, (3) cleanliness and orderliness of the worksite, (4) minimal conflict and good communication among staff members, (5) frequent safety-related feedback/training by supervisors, and (6) availability of ppe and engineering controls. 618 source control. the process of containing an infectious agent either at the portal of exit from the body or within a confined space. the term is applied most frequently to containment of infectious agents transmitted by the respiratory route but could apply to other routes of transmission, (eg, a draining wound, vesicular or bullous skin lesions). respiratory hygiene/cough etiquette that encourages individuals to ''cover your cough'' and/or wear a mask is a source control measure. the use of enclosing devices for local exhaust ventilation (eg, booths for sputum induction or administration of aerosolized medication) is another example of source control. standard precautions. a group of infection prevention practices that apply to all patients, regardless of suspected or confirmed diagnosis or presumed infection status. standard precautions represents a combination and expansion of universal precautions 778 and body substance isolation. 1109 standard precautions are based on the principle that all blood, body fluids, secretions, excretions except sweat, nonintact skin, and mucous membranes may contain transmissible infectious agents. standard precautions include hand hygiene and, depending on the anticipated exposure, use of gloves, gown, mask, eye protection, or face shield. in addition, equipment or items in the patient environment likely to have been contaminated with infectious fluids must be handled in a manner to prevent transmission of infectious agents (eg, wear gloves for handling, contain heavily soiled equipment, properly clean and disinfect or sterilize reusable equipment before use on another patient). surgical mask. a device worn over the mouth and nose by operating room personnel during surgical procedures to protect both surgical patients and operating room personnel from transfer of microorganisms and body fluids. surgical masks also are used to protect health care personnel from contact with large infectious droplets (. 5 mm in size). according to draft guidance issued by the food and drug administration on may 15, 2003 , surgical masks are evaluated using standardized testing procedures for fluid resistance, bacterial filtration efficiency, differential pressure (air exchange), and flammability to mitigate the risks to health associated with the use of surgical masks. these specifications apply to any masks that are labeled surgical, laser, isolation, or dental or medical procedure (http://www.fda.gov/cdrh/ode/guidance/094.html#4). surgical masks do not protect against inhalation of small particles or droplet nuclei and should not be confused with particulate respirators that are recommended for protection against selected airborne infectious agents (eg, mycobacterium tuberculosis). other species s use contact precautions for diapered or incontinent persons for the duration of illness or to control institutional outbreaks. giardia lamblia s use contact precautions for diapered or incontinent persons for the duration of illness or to control institutional outbreaks. noroviruses s use contact precautions for diapered or incontinent persons for the duration of illness or to control institutional outbreaks. persons who clean areas heavily contaminated with feces or vomitus may benefit from wearing masks, because virus can be aerosolized from these body substances; 142,147,148 ensure consistent environmental cleaning and disinfection with focus on restrooms even when apparently unsoiled. 272, 1060 hypochlorite solutions may be required when there is continued transmission. [289] [290] [291] alcohol is less active, but there is no evidence that alcohol antiseptic handrubs are not effective for hand decontamination. 293 cohorting of affected patients to separate airs paces and toilet facilities may help interrupt transmission during outbreaks. rotavirus c di ensure consistent environmental cleaning and disinfection and frequent removal of soiled diapers. prolonged shedding may occur in both immunocompetent and immunocompromised children and the elderly. 930 also for asymptomatic, exposed infants delivered vaginally or by c-section and if mother has active infection and membranes have been ruptured for more than 4 to 6 hours until infant surface cultures obtained at 24 to 36 hours of age negative after 48 hours of incubation. 1065 susceptible hcws should not enter room if immune caregivers are available; no recommendation for face protection of immune hcws; no recommendation for type of protection (ie, surgical mask or respirator) for susceptible hcws. in an immunocompromised host with varicella pneumonia, prolong the duration of precautions for duration of illness. postexposure prophylaxis: provide postexposure vaccine as soon as possible but within 120 hours; for susceptible exposed persons for whom vaccine is contraindicated (immunocompromised persons, pregnant women, newborns whose mother's varicella onset is # 5 days before delivery or within 48 hours after delivery) provide vzig, when available, within 96 hours; if unavailable, use ivig. provide airborne precautions for exposed susceptible persons and exclude exposed susceptible health care workers beginning 8 days after first exposure until 21 days after last exposure or 28 if received vzig, regardless of postexposure vaccination. 1032 variola (see smallpox) vibrio parahaemolyticus (see gastroenteritis) vincent's angina (trench mouth) s viral hemorrhagic fevers due to lassa, ebola, marburg, crimean-congo fever viruses s, d, c di single-patient room preferred. emphasize: use of sharps safety devices and safe work practices, hand hygiene; barrier protection against blood and body fluids on entry into room (single gloves and fluid-resistant or impermeable gown, face/eye protection with masks, goggles or face shields), and appropriate waste handling. use n95 or higher-level respirator when performing aerosol-generating procedures. largest viral load in final stages of illness when hemorrhage may occur; additional ppe, including double gloves, leg and shoe coverings may be used, especially in resource-limited settings where options for cleaning and laundry are limited. notify public health officials immediately if ebola is suspected. 212, 313, 738, 770 also see table 3 *type of precautions: a, airborne precautions; c, contact; d, droplet; s, standard; when a, c, and d are specified, also use s. y duration of precautions: cn, until off antimicrobial treatment and culture-negative; di, duration of illness (with wound lesions, di means until wounds stop draining); 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methicillin-resistant staphylococcus aureus contamination bacterial contamination on the surface of hospital linen chutes designing linen chutes to reduce spread of infectious organisms iatrogenic contamination of multidose vials in simulated use: a reassessment of current patient injection technique a large outbreak of hepatitis b virus infections associated with frequent injections at a physician's office a large nosocomial outbreak of hepatitis c and hepatitis b among patients receiving pain remediation treatments patient-to-patient transmission of hepatitis c virus through the use of multidose vials during general anesthesia an outbreak of hepatitis c virus infections among outpatients at a hematology/oncology clinic streptococcal meningitis following myelogram procedures a prospective study to determine whether cover gowns in addition to gloves decrease nosocomial transmission of vancomycin-resistant enterococci in an intensive care unit parainfluenza virus infections after hematopoietic stem cell transplantation: risk factors, response to antiviral therapy, and effect on transplant outcome parainfluenza virus 3 infection after stem cell transplant: relevance to outcome of rapid diagnosis and ribavirin treatment serial observations of chronic rotavirus infection in an immunodeficient child an outbreak of imipenem-resistant acinetobacter baumannii in critically ill surgical patients epidemiology of methicillin-resistant staphylococcus aureus at a university hospital in the canary islands nosocomial acquisition of methicillin-resistant staphylococcus aureus during an outbreak of severe acute respiratory syndrome increase in methicillin-resistant staphylococcus aureus acquisition rate and change in pathogen pattern associated with an outbreak of severe acute respiratory syndrome an outbreak of mupirocin-resistant staphylococcus aureus on a dermatology ward associated with an environmental reservoir risk of secondary meningococcal disease in health-care workers an outbreak of measles at an international sporting event with airborne transmission in a domed stadium an outbreak of airborne nosocomial varicella herpes zoster causing varicella (chickenpox) in hospital employees: cost of a casual attitude identification of factors that disrupt negative air pressurization of respiratory isolation rooms an evaluation of hospital special ventilation room pressures nosocomial transmission of tuberculosis associated with a draining abscess an outbreak of tuberculosis among hospital personnel caring for a patient with a skin ulcer secondary measles vaccine failure in healthcare workers exposed to infected patients a cluster of primary varicella cases among healthcare workers with false-positive varicella zoster virus titers airborne transmission of nosocomial varicella from localized zoster zoster-causing varicella: current dangers of contagion without isolation detection of aerosolized varicella-zoster virus dna in patients with localized herpes zoster measles vaccination after exposure to natural measles use of live measles virus vaccine to abort an expected outbreak of measles within a closed population measles, mumps, and rubella vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of mumps: recommendations of the advisory committee on immunization practices (acip) general recommendations on immunization: recommendations of the advisory committee on immunization practices (acip) postexposure effectiveness of varicella vaccine postexposure varicella vaccination in siblings of children with active varicella centers for disease control and preverntion. vaccinia (smallpox) vaccine: recommendations of the advisory committee on immunization practices (acip) smallpox vaccination: a review. part i: background, vaccination technique, normal vaccination and revaccination, and expected normal reactions smallpox in tripolitania, 1946: an epidemiological and clinical study of 500 cases, including trials of penicillin treatment ventilation for protection of immune-compromised patients efficacy of portable filtration units in reducing aerosolized particles in the size range of mycobacterium tuberculosis dolin r, editors. mandell, douglas and bennett's principles and practice of infectious diseases control of communicable diseases manual outbreak of amebiasis in a family in the netherlands parasitic disease control in a residential facility for the mentally retarded: failure of selected isolation procedures west nile virus: epidemiology, clinical presentation, diagnosis, and prevention person-to-person transmission of brucella melitensis isolation of brucella melitensis from human sperm prevention of laboratoryacquired brucellosis chlamydia pneumoniae as a new source of infectious outbreaks in nursing homes an epidemic of infections due to chlamydia pneumoniae in military conscripts an outbreak of surgical wound infections due to clostridium perfringens acquisition of coccidioidomycosis at necropsy by inhalation of coccidioidal endospores donor-related coccidioidomycosis in organ transplant recipients centers for disease control and prevention. acute hemorrhagic conjunctivitis outbreak caused by coxsackievirus a24 outbreak of adenovirus type 30 in a neonatal intensive care unit an outbreak of epidemic keratoconjunctivtis in a pediatric unit due to adenovirus type 8 a large outbreak of epidemic keratoconjunctivitis: problems in controlling nosocomial spread nosocomial transmission of cryptococcosis cryptococcal endophthalmitis after corneal transplantation probable transmission of norovirus on an airplane centers for disease control and prevention. prevention of hepatitis a through active or passive immunization: recommendations of the advisory committee on immunization practices (acip) hepatitis a outbreak in a neonatal intensive care unit: risk factors for transmission and evidence of prolonged viral excretion among preterm infants excretion of hepatitis a virus in the stools of hospitalized hepatitis patients hospital outbreak of hepatitis e herpes simplex virus infections neonatal herpes infection: diagnosis, treatment and prevention human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children listeria moncytogenes cross-contamination in a nursery neonatal listeriosis due to cross-infection confirmed by isoenzyme typing and dna fingerprinting outbreak of neonatal listeriosis associated with mineral oil neonatal cross-infection with listeria monocytogenes nosocomial malaria and saline flush plasmodium falciparum malaria transmitted in hospital through heparin locks nosocomial malaria from contamination of a multidose heparin container with blood hospital-acquired malaria transmitted by contaminated gloves clustering of necrotizing enterocolitis: interruption by infection-control measures how contagious is necrotizing enterocolitis? an outbreak of rotavirus-associated neonatal necrotizing enterocolitis increased risk of illness among nursery staff caring for neonates with necrotizing enterocolitis outbreak of adenovirus 35 pneumonia among adult residents and staff of a chronic care psychiatric facility nosocomial adenovirus infection: molecular epidemiology of an outbreak a recent outbreak of adenovirus type 7 infection in a chronic inpatient facility for the severely handicapped an outbreak of multidrugresistant pneumococcal pneumonia and bacteremia among unvaccinated nursing home residents human-to-human transmission of rabies virus by corneal transplant human rabies prevention, united states, 1999: recommendations of the advisory committee on immunization practices (acip) rhinovirus and the lower respiratory tract concurrent outbreaks of rhinovirus and respiratory syncytial virus in an intensive care nursery: epidemiology and associated risk factors rhinovirus infection associated with serious lower respiratory illness in patients with bronchopulmonary dysplasia nosocomial ringworm in a neonatal intensive care unit: a nurse and her cat nosocomial transmission of trichophyton tonsurans tinea corporis in a rehabilitation hospital molecular epidemiology of staphylococcal scalded skin syndrome in premature infants an outbreak of fatal nosocomial infections due to group a streptococcus on a medical ward an outbreak of group a streptococcal infection among health care workers clusters of invasive group a streptococcal infections in family, hospital, and nursing home settings isolation techniques for use in hospitals us government printing office rethinking the role of isolation practices in the prevention of nosocomial infections the authors and hicpac gratefully acknowledge dr larry strausbaugh for his many contributions and valued guidance in the preparation of this guideline. the mode(s) and risk of transmission for each specific disease agent listed in this appendix were reviewed. principle sources consulted for the development of disease-specific recommendations for the appendix included infectious disease manuals and textbooks. 831, 1039, 1040 the published literature was searched for evidence of person-to-person transmission in health care and non-health care settings with a focus on reported outbreaks that would assist in developing recommendations for all settings where health care is delivered. the following criteria were used to assign transmission-based precautions categories: d a transmission-based precautions category was assigned if there was strong evidence for person-to-person transmission via droplet, contact, or airborne routes in health care or non-health care settings and/or if patient factors (eg, diapered infants, diarrhea, draining wounds) increased the risk of transmission. d transmission-based precautions category assignments reflect the predominant mode(s) of transmission. d if there was no evidence for person-to-person transmission by droplet, contact or airborne routes, then standard precautions were assigned. d if there was a low risk for person-to-person transmission and no evidence of health care-associated transmission, then standard precautions were assigned. d standard precautions were assigned for bloodborne pathogens (eg, hbv, hcv, hiv) in accordance with cdc recommendations for universal precautions issued in 1988. 778 subsequent experience has confirmed the efficacy of standard precautions to prevent exposure to infected blood and body fluid. 776, 777, 863 additional information relevant to use of precautions was added in the comments column to assist the caregiver in decision-making. citations were added as needed to support a change in or provide additional evidence for recommendations for a specific disease and for new infectious agents (eg, sars-cov, avian influenza) that have been added to appendix a. the reader may refer to more detailed discussion concerning modes of transmission and emerging pathogens in the background text and for mdro control in the mdro guideline. key: cord-270091-sqrh8ylt authors: cohen, pascal; guillevin, loïc title: vascularites associées aux infections virales date: 2004-11-30 journal: la presse médicale doi: 10.1016/s0755-4982(04)98936-1 sha: doc_id: 270091 cord_uid: sqrh8ylt résumé des virus, causes de vascularites si la plupart des vascularites systémiques sont de cause inconnue, la responsabilité d’une infection virale a été démontrée de façon formelle pour certaines d’entre elles, un traitement spécifique pouvant les guérir définitivement. chaque virus incriminé rend compte d’un type particulier de vascularite. infection par le virus de l’hépatite b (vhb) elle est la cause de la périartérite noueuse dans 36 à 50 % des cas. la symptomatologie apparaît de façon aiguë, en général dans les mois suivant l’infection; elle est comparable à celle observée en l’absence d’infection par vhb. cryoglobulinémies liées au virus de l’hépatite c (vhc) les manifestations cliniques sont celles d’une vascularite systémique avec un tropisme plus particulier pour la peau (atteinte le plus souvent inaugurale et presque constante), les nerfs périphériques, les glomérules. elles surviennent assez tardivement au décours de l’infection. vascularites associées à l’infection par le vih (virus de l’immunodéficience humaine) il existe un fort tropisme pour le système nerveux périphérique (multinévrites) et central. au cours de l’infection aiguë par le parvovirus b19 des lésions de vascularite ont été parfois signalées après la phase virémique, se limitant généralement à une ou plusieurs poussées de pupura vasculaire prédominant aux membres inférieurs. après infection par le virus varicelle-zona une vascularite se développe parfois, sous la forme d’un déficit neurologique central (déficit moteur avec ou sans aphasie environ un mois après un zona ophtalmologique), d’une atteinte de la rétine, plus rarement de la peau ou des reins. vascularites associées à l’infection par le cytomégalovirus (cmv) survenant essentiellement chez le sujet immunodéprimé, les vascularites après infection par le cmv sont diffuses intéressant surtout le tube digestif, en particulier le côlon, le système nerveux central et la peau. une complication rare de l’infection à htlv1 (human t-cell lymphoma virus) une vascularite rétinienne prenant volontiers la forme d’une rétinite nécrosante est souvent associée à une paraparésie spasmodique. stratégie thérapeutique pour de nombreuses vascularites d’origine virale, les traitements corticoïdes et immunosuppresseurs ne sont indiqués qu’en seconde intention en cas d’échec des antiviraux et de l’association d’antiviraux et d’échanges plasmatiques. summary viruses, the cause of vasculitis although the majority of systemic vasculitis are of unknown causes, the responsibility of a viral infection has been formally demonstrated in some of them and specific treatment can permanently cure them. each virus incriminated accounts for a particular type of vasculitis. hepatitis b viral infection (hbv) is the cause of polyarteritis nodosa in 36 to 50% of cases. the onset of the symptomatology is acute, usually within a few months following the infection; it is comparable to that observed in the absence of hbv infection. cryoglobulinemia related to the hepatitis c virus (hcv) the clinical manifestations are those of systemic vasculitis with particular tropism for the skin (involvement generally inaugural and almost constant), peripheral nerves and the glomerula. they occur fairly late during the infection. vasculitis associated with hiv infection there is strong tropism for the peripheral (multi-neuritis) and central nervous system. during acute parvovirus b19 infection vasculitis lesions have occasionally been reported following the viremic phase, generally limited to one or several flares of vascular purpura predominating on the lower limbs. following varicella-herpes zoster infection vasculitis occasionally develops in the form of a central neurological deficiency (locomotor deficiency with or without aphasia around one month after an ophthalmologic herpes zoster) or involving the retina or, more rarely, the skin or the kidneys. vasculitis associated with cytomegaloviral infection predominantly observed in immunodepressed patients, vasculitis after cmv infection is diffuse and basically involving the digestive tube, notably the colon, the central nervous system and the skin. a rare complication of an htlv1 infection vasculitis of the retina often in the form of necrotic retinitis is often associated with spasmodic paraparessia. therapeutic strategy for many vasculitis of viral origin, corticosteroid and immunosuppressive treatments are only indicated in second intention following failure with antiviral agents and the combination of antivirals and plasma exchanges. des virus, causes de vascularites si la plupart des vascularites systémiques sont de cause inconnue, la responsabilité d'une infection virale a été démontrée de façon formelle pour certaines d'entre elles, un traitement spécifique pouvant les guérir définitivement. chaque virus incriminé rend compte d'un type particulier de vascularite. infection par le virus de l'hépatite b (vhb) elle est la cause de la périartérite noueuse dans 36 à 50 % des cas. la symptomatologie apparaît de façon aiguë, en général dans les mois suivant l'infection; elle est comparable à celle observée en l'absence d'infection par vhb. cryoglobulinémies liées au virus de l'hépatite c (vhc) les manifestations cliniques sont celles d'une vascularite systémique avec un tropisme plus particulier pour la peau (atteinte le plus souvent inaugurale et presque constante), les nerfs périphériques, les glomérules. elles surviennent assez tardivement au décours de l'infection. vascularites associées à l'infection par le vih (virus de l'immunodéficience humaine) il existe un fort tropisme pour le système nerveux périphérique (multinévrites) et central. au cours de l'infection aiguë par le parvovirus b19 des lésions de vascularite ont été parfois signalées après la phase virémique, se limitant généralement à une ou plusieurs poussées de pupura vasculaire prédominant aux membres inférieurs. après infection par le virus varicelle-zona une vascularite se développe parfois, sous la forme d'un déficit neurologique central (déficit moteur avec ou sans aphasie environ un mois après un zona ophtalmologique), d'une atteinte de la rétine, plus rarement de la peau ou des reins. survenant essentiellement chez le sujet immunodéprimé, les vascularites après infection par le cmv sont diffuses intéressant surtout le tube digestif, en particulier le côlon, le système nerveux central et la peau. l es causes des maladies systémiques sont généralement inconnues et l'implication d'agents infectieux est plus souvent suspectée que démontrée.les vascularites systémiques ne font pas exception à la règle et la plupart d'entre elles sont de cause inconnue. c'est notamment le cas des vascularites associées aux anticorps anti-cytoplasme des polynucléaires neutrophiles (anca),mais aussi du purpura rhumatoïde 1 et de la plupart des vascularites quel que soit le calibre ou le mécanisme de l'atteinte vasculaire. à l'inverse, c'est au cours des vascularites que l'on a impliqué formellement la responsabilité d'infections virales [2] [3] [4] et que l'on a montré qu'un traitement spécifique pouvait les guérir définitivement. nous analyserons les principales vascularites nécrosantes virales et les traitements adaptés à leur prise en charge. nous n'aborderons pas les artérites à cellules géantes qui font l'objet d'un autre développement 5 . de nombreux virus ont été décrits comme pouvant être responsables de vascularites, le plus souvent de façon anecdotique. nous avons résumé dans le tableau 1 les divers virus impliqués. dans de nombreux cas, sans nier l'imputabilité du virus, peu d'observations identiques ont été rapportées. de nombreuses observations de vascularites ont été décrites chez l'animal. de très nombreux virus ont été impliqués : adénovirus 6 , porcine reproductive and respiratory syndrome virus 7 , herpes simplex virus chez la souris 8 , htlv (human t-cell lymphoma virus) chez le rat 9 , cytomégalovirus chez le rat 10 , coronavirus chez la souris 11 ,etc.certaines de ces artérites sont proches de maladies humaines, telle l'artérite nécrosante du cheval due au virus de l'artérite équine qui ressemble à la périartérite noueuse (pan) 12 au cours de l'infection par le vih (virus de l'immunodéficience humaine), de nombreuses manifestations rhumatologiques ont été décrites. les vascularites représentent moins de 1 % de ces manifestations 46, 47 . elles ont été rapportées à tout stade de l'infection. certaines d'entre elles sont de toute évidence reliées à une infection opportuniste sur terrain immunodéprimé comme les infections à cmv (cytomégalovirus) ou à vzv (virus varicelle zona) [48] [49] [50] . ailleurs, elles sont associées à un traitement médicamenteux [51] [52] [53] [54] [55] . due au vhb est une forme typique de pan. la symptomatologie apparaît de façon aiguë et, le plus souvent, dans les mois suivant l'infection par le vhb. dans la plupart des cas, il est toutefois difficile d'identifier la date précise du contage. nous avons observé que, lorsque le contage était daté, la pan survenait 4 mois plus tard. l'hépatite virale est généralement absente ou se limite à une élévation des transaminases qui est au double ou au triple de la normale. dans notre série de 110 patients, nous avons eu 3 malades avec une pan survenant en phase aiguë de l'hépatite (série personnelle,non publiée).nous avons aussi observé que la probabilité d'obtenir une séroconversion hbe/anti-hbe était plus élevée lorsque l'infection était récente. les signes cliniques sont exposés dans le tableau 2. l'atteinte rénale est une néphropathie vasculaire d'origine ischémique. elle s'accompagne volontiers d'une hypertension artérielle maligne 72 . tous les symptômes sont comparables à ceux que l'on observe dans les pan sans vhb. parmi ceux-ci, les signes digestifs sont fréquents et souvent sévères.les patients se plaignent de douleurs abdominales persistantes, de perforation ou d'hémorragie. parmi les examens complémentaires, on retiendra : une biologie montrant des signes non spécifiques d'inflammation ; une sérologie virale témoignant de la réplication du vhb avec la positivité de l'adn viral qui s'ajoute à la présence des antigènes hbs et hbe, de l'anti-hbc, sans anticorps anti-hbe ni anti-hbs.une cryoglobulinémie est rarement présente. les anca sont constamment absents, comme au cours de toute pan. l'artériographie digestive et rénale, lorsqu'elle est réalisée, montre dans plus de la moitié des cas des anévrysmes et des sténoses siégeant sur l'ensemble des branches de l'aorte abdominale et sur les artères de calibre inférieur (figures 1 et 2). au niveau rénal on peut aussi observer des infarctus parenchymateux (figure 3). lorsqu'ils sont nombreux,ils sont la cause d'une insuffisance rénale avec hypertension artérielle sévère. l'évolution de la pan due au vhb est habituellement favorable si le traitement est adapté. la mortalité initiale n'est toutefois pas exceptionnelle et les complications digestives survenant durant les premières semaines de la maladie rendent compte de la majorité des causes précoces de décès. le taux de rechute est faible, n'excédant pas 8 % à 5 ans 2 . la survie à 5 ans est de 70 % (non publié).le pronostic peut être calculé dès le diagnostic en appliquant le five factor score 73 .les paramètres de mauvais pronostic sont l'atteinte rénale (2 points), l'atteinte digestive sévère (1 point) ;l'atteinte cardiaque (1 point) et l'atteinte du système nerveux central (1 point). selon que le malade a 0, 1 ou 2 points, la mortalité à 5 ans s'étend de 12 à 50 %. . il s'agit d'accidents vasculaires ischémiques uniques ou multiples. la vascularite est soit prouvée histologiquement sur biopsie ou autopsie, soit indirectement visible en angiographie qui peut montrer de façon diffuse des altérations de calibre des artères intracérébrales. d'autres observations plus anecdotiques ont été rapportées comme des vascularites granulomateuses ou des atteintes macroanévrysmales multiples 106 . de nombreux autres tableaux cliniques de vascularite ont été rapportés comme un purpura vasculaire au cours d'une séroconversion 107 , des vascularites nécrosantes cutanées pures 108, 109 , une érythrocyanose des doigts 110 ou une ischémie digitale 65, 111 ; ce polymorphisme clinique souligne la difficulté d'affirmer le rôle possible du vih. il faut toujours garder à l'esprit la possibilité de vascularites immuno-allergiques, en particulier aux médicaments antiviraux 104, 112 . enfin, le lien avec un syndrome de churg et strauss ou une maladie de behçet paraît plus fortuit que vraisemblable 113 . . les manifestations cliniques sont celles habituellement observées au cours de la pan. toutefois,la fréquence de cette étiologie au sein de l'ensemble des pan reste faible 135, 136 . les mêmes remarques peuvent s'appliquer à la maladie de wegener. les études utilisant la détection du virus par pcr confirment l'authenticité de l'association mais celle-ci ne représente qu'une minorité des cas 136, 137 . chez l'enfant comme chez l'adulte, l'infection par le vzv est parfois compliquée d'une vascularite qui s'exprime sous la forme d'un déficit neurologique central, d'une atteinte de la rétine,plus rarement de la peau ou des reins [145] [146] [147] [148] [149] [150] [151] [152] [153] [154] [155] . l'atteinte neurologique centrale est assez caractéristique et stéréotypée : un mois environ après un zona localisé au visage, en particulier à l'oeil, survient un déficit moteur, avec ou sans aphasie, d'installation soudaine 145 , des symptomatologies beaucoup plus sévères ont été rapportées et correspondent à la dissémination de l'infection. ce sont de véritables encéphalopathies subaiguës, voire des encéphalomyélites ou encore des myélites avec de multiples déficits. il s'agit alors d'une vascularite des petits vaisseaux diffuse à l'encéphale.des tableaux défi-citaires moins sévères d'angéite granulomateuse des vaisseaux leptoméningés ont aussi été décrits. la rétinite nécrosante est une complication rare de l'infection par le vzv [160] [161] [162] . elle est uni, voire bilatérale, et se traduit par une baisse rapide de l'acuité visuelle. elle correspond à une vascularite des petits vaisseaux choroïdiens et rétiniens. la preuve virologique est obtenue soit par immunofluorescence, soit par pcr dans le liquide intraoculaire ou sur le matériel biopsique. le virus de la varicelle a aussi été cherché par technique pcr sur des biopsies d'artérite à cellules géantes de l'adulte, avec des résultats variables : tantôt présent dans 26 %, tantôt dans aucune des biopsies.il est donc peu probable que le vzv soit impliqué dans cette affection 163, 164 . les vascularites associées à l'infection par le cmv touchent essentiellement les sujets immunodéprimés, en particulier lorsque l'immunité cellulaire est altérée comme chez les patients porteurs du vih. la vascularite est diffuse et intéresse surtout le tube digestif, en particulier le côlon, le système nerveux central (vaisseaux méningés et extraduraux dont ceux de la moelle) et la peau [165] [166] [167] . la vascularite pourrait être la conséquence directe de l'infection endothéliale mais d'autres facteurs immunologiques semblent intervenir, car la seule présence du cmv dans les cellules endothéliales ne suffit pas à établir un lien étiologique. il est d'ailleurs nécessaire d'obtenir la lésion caractéristique d'une image d'inclusions à cmv en "oeil de hibou" dans les cellules endothéliales au sein du foyer de vascularite. l'atteinte digestive peut aller d'une simple anorexie à des douleurs abdominales avec fièvre, diarrhée et quelquefois hémorragie digestive voire perforation ; la laparotomie est souvent nécessaire et la vascularite est mise en évidence sur la pièce opératoire ou autopsique [167] [168] [169] [170] [171] . 170, 174 . l'atteinte cutanée serait péjorative parce qu'elle pourrait témoigner de la dissémination de l'infection 165 chez des patients particulièrement immunodéprimés. les lésions ne sont pas spécifiques : purpura vasculaire qui prend volontiers un aspect nécrotique, nodules et vésicules ou encore éruptions cutanées maculo-papuleuses. par contre, chez les sujets immunocompétents, elle semble exceptionnelle 177 . de rares observations de pan ont été décrites au cours de la primoinfection par le cmv 178, 179 . dans un travail portant sur 11 patients suivis pour une pan, la recherche de cmv par méthode pcr n'était positive l'infection à htlv1 est rarement compliquée de vascularite ;cette dernière a un tropisme neurologique central 181 192, 193 qui fut le premier à décrire la responsabilité liée à l'hépatite b au cours de la périartérite noueuse 194 , nous décidâmes d'inclure dans un protocole prospectif tous les patients atteints de pan hbv+ et de traiter les patients par une combinaison d'antiviraux et d'échanges plasmatiques 195 . initialement, les patients recevaient une courte corticothérapie intensive, ne dépassant pas 2 semaines et les malades, préalablement traités par corticoïdes, arrêtaient ce traitement dans le même délai. l'arrêt brutal de la corticothérapie et des immunosuppresseurs précédemment prescrits avait pour but de renforcer l'action antivirale dont l'action s'exerçait à un moment de réplication maximale. les échanges plasmatiques, en éliminant les immuns complexes circulants, ont été prescrits systématiquement chez tous les patients.trois à quatre échanges par semaine durant 3 à 4 semaines sont nécessaires. ensuite, les échanges plasmatiques sont espacés puis interrompus lorsque la pan est en rémission,c'està-dire au bout de 2 mois environ. nous avons toujours effectué des échanges plasmatiques par centrifugation ou par filtration avec un soluté de substitution majoritairement constitué d'albumine.dans la mesure où nous n'avons pas observé d'insuffisance hépatocellulaire chez nos patients,il n'a pas été nécessaire d'utiliser du plasma frais congelé en substitution. les antiviraux permettent de diminuer, puis d'arrêter la réplication du virus de l'hépatite b.au fil des années, plusieurs types d'antiviraux ont été successivement testés. dans notre première série prospective 195 , l'antiviral était la vidarabine que nous prescrivions par voie intraveineuse, à raison de 15 mg/kg/j pendant une semaine, puis 7,5 mg/kg/j durant 2 semaines.ce traitement était efficace mais, en raison de sa toxicité neurologique et hématologique, il n'est aujourd'hui plus commercialisé sous forme intraveineuse. la forme intramusculaire a donné des résultats superposables avec une tolérance elle-même médiocre. nous avons utilisé cette stratégie chez 33 patients, dont 24 nouvellement diagnostiqués et 9 ne répondant pas à une corticothérapie et aux immunosuppresseurs.la guérison a été obtenue dans les trois quarts des cas et une séroconversion hbe/anti-hbe dans la moitié des cas. la séroconversion hbs/anti-hbs n'était obtenue que dans 18 % des cas. l'interféron alpha est devenu,ensuite, le traitement de référence des hépatites virales b et a donc pris la place de la vidarabine. la stratégie thérapeutique n'a pas été modifiée.la dose de l'interféron était de . on peut espérer que les nouveaux traitements antiviraux ou leur combinaison améliorent encore les résultats. on peut aussi observer une dissociation entre l'évolution de la vascularite et celle de l'infection virale. la vascularite s'améliore souvent rapidement et certains patients guéris-sent définitivement en 3 semaines. habituellement l'amélioration se produit en une dizaine de jours et elle est complète au bout de 2 mois environ. lorsque nous utilisions la vidarabine,nous observions une augmentation des transaminases au moment de la séroconversion. il s'agit là d'une réponse immunologique normale, reflétant la capacité du patient à restaurer sa réponse immunitaire.toutefois, cette élévation des transaminases peut être très marquée et, à une reprise, nous avons observé une hépatite fulminante mortelle survenant au moment de la séroconversion. lorsque nous avons substitué l'interféron à la vidarabine,la réponse a été très différente avec une diminution progressive des transaminases,si elles étaient élevées au début de la maladie, et il n'y a pas eu d'effet rebond à l'arrêt du traitement. des résultats superposables ont été obtenus avec la lamivudine. une fois le traitement interrompu, le clinicien se trouve confronté à 2 situations différentes : • la périartérite noueuse est guérie et il y a eu séroconversion. a priori, il n'y aura plus de rechute. il n'est donc pas nécessaire de proposer un autre traitement sinon celui des séquelles de la périartérite noueuse (traitement anti-hypertenseur par exemple) ; • le malade a guéri de sa périartérite noueuse mais a toujours une réplication virale.on entre alors dans le cadre du traitement des hépatites chroniques persistantes et, indépendamment de la pan, d'autres stratégies thérapeutiques antivirales peuvent être proposées. le risque de rechute de la pan demeure, mais reste très faible. il est important de distinguer traitement purement symptomatique des manifestations de la vascularite et trai-tement étiologique antiviral c. les manifestations cliniques justifient bien souvent le recours aux corticoïdes et parfois aux immunosuppresseurs.toutefois, aucune étude prospective ne précise la place respective de ces traitements. il faut aussi garder à l'esprit la présence d'une infection virale sous-jacente, limiter ainsi la posologie et la durée des corticoïdes, réserver aux manifestations rebelles ou particulièrement menaçantes l'utilisation des immunosuppresseurs. l'indication des échanges plasmatiques (qui permettent d'épurer de façon rapide et massive de grandes quantités de cryoglobuline et de complexes immuns circulants) n'est pas unanime 198-204 ; ils semblent utiles dans les atteintes glomérulaires avec insuffisance rénale [198] [199] [200] [201] . l'éradication virale c est au centre du traitement de la maladie. le recul est encore insuffisant pour affirmer que l'obtention d'une éradication virale c suffit à contrôler la maladie [206] [207] [208] .les études cliniques portant sur le traitement antiviral au cours des cryoglobulinémies sont rares 24, 74, [209] [210] [211] [212] [213] [214] [215] [216] . la première étude thérapeutique contrôlée 209 chez les sujets immunodéprimés, cette complication justifie un traitement antiviral le plus précoce pos-sible par acyclovir en perfusions intraveineuses. la place éventuelle de la corticothérapie ne peut être précisée. il est toutefois difficile de la proposer chez ces patients déjà très immunodéprimés. malgré une thérapeutique précoce, le pronostic demeure sombre ; la majorité des observations rapportées est autopsique 148, 149, 155, 157, 158, 160 . le traitement de l'atteinte neurologique centrale qui fait suite à un zona céphalique est plus difficile à codifier car il n'est pas rare d'observer une récupération complète spontanée ; de nombreux auteurs soulignent l'intérêt du traitement antiviral par aciclovir, avec ou sans corticothérapie. les réponses cliniques sont parfois spectaculaires, même sur terrain immunodéprimé comme le vih, mais des séquelles neurologiques sont possibles 147, 148 . le terrain immunodéprimé fait toute la gravité de cette complication et impose un traitement antiviral le plus précocement possible par ganciclovir ou foscarnet par voie veineuse. l'association à une corticothérapie ne paraît pas logique en raison de l'immunodépression. malgré un traitement antiviral rapide, la mortalité demeure élevée. plus qu'ailleurs, il est probablement nécessaire de limiter le recours aux thérapeutiques immunosuppressives chez les patients infectés par le vih,et d'instituer un traitement antirétroviral puissant 222 . cependant, l'expérience clinique, certes limitée, montre qu'il est possible de traiter ces patients par les corticoïdes,et ce même de façon prolongée sur plusieurs mois 62, 63, 223, 224 . en effet, les observations rapportées ne font pas état de complications systémiques, notamment infectieuses. d'autres auteurs ont même traité par cyclophosphamide sans complication infectieuse opportuniste 225 . dans l'expérience de gisselbrecht et al. 65 , le traitement symptomatique des manifestations cliniques a été privilégié : très brève corticothérapie limitée à une semaine relayée par les échanges plasmatiques. cette méthode est fondée sur l'épuration rapide et massive d'immuns complexes, supposés être impliqués dans l'infection par le vih. les résultats obtenus sur 6 patients ont été particulièrement bons et ce volet thérapeutique symptomatique a été 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of refractory symptomatic hepatitis c virus related mixed cryoglobulinemia with ribavirin and interferon alpha interferon alpha-2a therapy in cryoglobulinemia associated with hepatitis c virus mixed cryoglobulinemia in chronic hepatitis c infection. a clinicopathologic analysis of 10 cases and review of recent literature effects of two different alpha-interferon regimens on clinical and virological findings in mixed cryoglobulinemia treatment of mixed cryoglobulinemia with recombinant interferon alpha and adjuvant therapies effects of long-term course of alpha interferon in patients with chronic hepatitis c associated to mixed cryoglobulinemia affections extra-hépatiques certainement liées au virus de l'hépatite c response to interferon alpha treatment and disappearance of cryoglobulinaemia in patients infected by hepatitis c virus intravenous interferon alpha treatment of mixed cryoglobulinemia associated with chronic hepatitis c virus infection randomized trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis c virus. international hepatitis interventional therapy group interferon alpha 2b alone or in combination with ribavirin as initial treatment for chron,ic hepatitis c. hepatitis interventional therapy group prolonged complete remission after 2-chlorodeoxyadenosine therapy in a patient with refractory essential mixed cryoglobulinemia fludarabine treatment of cryoglobulinemic glomerulonephritis rituximab for the treatment of type ii mixed cryoglobulinemia the spectrum and treatment of virus-associated vasculitides retroviral associated vasculitis of the nervous system frosted branch angiitis in a child with hiv infection biopsyproven cerebral vasculitis associated with cocaine abuse key: cord-284195-qarz4o2z authors: ansumali, santosh; prakash, meher k title: a very flat peak: exponential growth phase of covid-19 is mostly followed by a prolonged linear growth phase, not an immediate saturation date: 2020-04-11 journal: nan doi: 10.1101/2020.04.07.20055772 sha: doc_id: 284195 cord_uid: qarz4o2z when actively taking measures to control an epidemic, an important indicator of success is crossing the "peak" of daily new infections. the peak is a positive sign which marks the end of the exponential phase of infection spread and a transition into a phase that is a manageable. most countries or provinces with similar but independent growth trajectories had taken drastic measures for containing the covid-19 pandemic and are eagerly waiting to cross the peak. however, the data after many weeks of strict measures suggests that most provinces instead enter a phase where the infections are in a linear growth. while the transition out of an exponential phase is relieving, the roughly constant number of daily new infections differ widely, range from around 50 in singapore to around 2000 just in lombardy (italy), and 7600 in spain. the daily new infection rate of a region seems to depend heavily on the time point in the exponential evolution when the restrictive measures were adopted, rather than on the population of the region. it is not easy to point the critical source of these persistent infections. we attempt to interpret this data using a simple model of newer infections mediated by asymptomatic patients, which underscores the importance of actively identifying any potential leakages in the quarantine. given the novelty of the virus, it is hard to predict too far into the future and one needs to be observant to see if a plan b is needed as a second round of interventions. so far, the peak achieved by most countries with the first round of intervention is extremely flat. sars spread in around 29 countries, infecting around 8,096 individuals globally [1] . quarantine strategies were implemented by several governments, and they were effective in reducing the number of newer infections [2] . the number of casualties from other epidemics such as swine flu in 2009, mers in 2015 were also similarly contained. however, the infections caused by the novel coronavirus (covid-19) continue to increase. the world health organization has declared covid-19 as a pandemic, the first one in the 21 st century [3] . since so far there is no known treatment or vaccine, after weighing the damages to the lives versus that to the economy, most governments across the world have implemented the nonpharmaceutical interventions such as strict measures of social distancing, or even lockdowns and curfews, guided by the historic response to the . for an emerging pandemic such as covid-19, a first natural scientific impulse is to model it via standard epidemiological models [4] [5] such as the susceptible-infected-recovered (sir) model to predict how rapidly the infections can spread without an intervention or how quickly a lockdown program may be planned [6] . governments and public health modelers are interested in understanding the effectiveness of various strategies [7] [8] [9] [10] starting from a complete lockdown, or a reduced social contact [11] and a subsequent release of restrictions in a phased manner. many such models have already been developed to model covid-19, and have been guided by the past intuitions from modeling how the epidemic spread declines with changes in season or with active containment strategies, and the success of china which after 2 months of lockdown reports no new covid-19 infection cases consistently for more than 3 weeks. in the time of a pandemic, an important question asked on daily basis by public and policy makers is when the pandemic is going to 'cross the peak'. crossing the peak signifies that one may expect fewer cases of infection, compared to the previous day. it sends positive signals of pandemic containment to the people as well as to the economy and other aspects of the social life. it also indicates the time for lowering the guard is not too far. as such, a few weeks after these strict measures, and noting the reported success of china, governments of various provinces and countries are waiting for the new daily infections to cross over the peak. because of the drastic measures, the number of daily new cases are no longer increasing in many places. however, it is worrying that they do not have clear signatures of a downward trend either. in this context, we perform a detailed analysis of the nature of this peak, and whether it has been achieved. as it turns out, most provinces and countries that implemented containment are no longer in an exponential growth phase, but rather enter a new, and possibly unexpected, linear growth phase which we discuss here. in the early stage of infection spread, each infected individual becomes a vector for transmission the infection. thus, rate of increase infections can be captured by a simple model, di/dt=r.(i-r).(p-i), eq (1) where i is the number of infected individuals, r is the number of recovered individuals, p is the total population and rate of transmission is r. specifically, with the current variant of covid-19, where the median recovery time is 10-15 days, the exponential increase in i is always much faster than the slow growth of r. one can see from the data in the growth phase of the pandemic spread in any country that typically r < 10-15% of i, and i << p. in principle, one can also consider detailed models such as the sir model, or even detailed agent-based models assuming a more realistic social contact structure. however, the simple phenomenological model di/dt=a.i eq (2) does capture the growth of i. the number of infections in a well diffused society, community or a province would thus grow as i(t)=i0 exp(at), where i0 is the number infections at t=0. however, the transmission across countries or less-frequented provinces, occurs much through a jump-diffusion process, with only occasional jumps over these boundaries, and a diffusion within the region. as a consequence, the trajectory of a country with two epicenters can be thought as two independent weakly interacting subsystems which leads to emergence of a multi-exponential i(t)= i0,1 exp(a1t)+ i0,2 exp(a2t) with the i0,1 and i0,2 being the number of infections in these two decoupled regions at t=0, and a1, a2 the rates in these two regions which may or may not be same depending on mobility in the cities, any other restrictions imposed by the local governors. for administrative reasons, one may be interested in following the trajectory of the world, or of a specific country. but depending upon the lag between these multiple hotspots, the exponential nature of the growth gets masked. thus, for detailed studies, one needs to unmask this data by decoupling these multiple exponentials and focusing on the individual provinces, which are possibly separated from other provinces by travel restrictions. after decoupling, it is clear that the different regions very similar exponential growth curves. decoupled data show a shift from exponential to linear growth. we first illustrate the qualitative change in the spread of infection using the data from the number of infected cases in lombardy [13] . as figure 1 shows, about a week after the lockdown, the growth in lombardy transitioned from an exponential to a linear growth. in figure 2 , we study the growth in south korea, singapore, saudi arabia, switzerland, spain, [14] germany and two of its states (bayern, baden) and another italian province venice. it is apparent from the data that the later part of the data from all these countries shows a clearly linear trend. even the number of deaths recorded on a daily basis also show the same linear trend. clearly a transition from the exponential regime is a relief. and this transition happens around 10 days from the day of the restrictive measure, possibly coinciding with the distribution of the incubation time. however, the slope of these curves, which indicate the number of new daily infections is very different for the different regions. south korea's response by contact tracing and extensive testing has been widely praised. although the number of daily new cases has crossed a peak there are still an average of 100 cases every day from the 12 th of march till the 3 rd of april. while 100 new cases may be a manageable number in terms of resource allocation, several other countries or provinces are in a linear growth regime with much larger daily new cases: switzerland (980), italy (≈4200), germany (≈5600), spain (≈7600) despite the containment measures. to date, other than china which continues to report nearly zero new infected cases every day for the past few weeks, all other countries are either in an exponential phase or a linear growth phase. the daily new infected cases for the provinces and countries we analyzed in figures 1, 2 are given in table 1 . the dependence of the daily new cases on various factors such as the extent of testing, the population of the region, etc was studied in figure 3 . a strong correlation was observed with the number of cumulative infections in the region, and the number of daily infections at the time when the containment measures were taken. which seems to suggest that covid-19 infections at this point are held in a pause. looking back, if the quarantine or lockdown decision is taken later in time the average number of daily new cases would have been significantly higher. of course, the same message applies for the future before relaxing the social restrictions that have helped contain the spread of the covid-19. there is now enough evidence that one main difference of covid-19 has been the high rate of transmission by the asymptomatic individuals . in an attempt to model the observed transition from an exponential to a linear growth phase, we resort to simple rate equations by including a, which is the number of asymptomatic patients. l is the infection rate via asymptomatic individuals, d reflects the natural rate of reduction in the numbers of asymptomatic patients post-incubation period, b is the rate at which asymptotic individuals transmit to other individuals, r is the rate at which by performing tests one reduces the a by moving them to a quarantine. c is the rate of increase due to non-human sources such as aerosols or contact surfaces. we perform the simple analysis with µ=0, assuming the recovery rate is much slower than the rate of infection. a median hospitalization time of 3 weeks from the data does support this assumption, as in the exponential phase the increase in the number of infections in these 3 weeks is much higher. however, the following analysis should remain valid even if µ≠0. in figure 4 , we illustrate a simulation of these differential equations to show the transition to linear regime, the nature of the peak, constant rate of daily fluctuations and the reduction in the number of the daily infection rate with r. in eq. 3a, the dependence of di/dt on i is the main reason for the exponential growth. a quarantine of infected individuals removes this dependence with a=0, and turns the behavior to one of linear growth. the number of asymptomatic individuals in principle will reduce to zero after a strict implementation of quarantine, followed by the decay rate of infection in the individuals. however, assuming our model is realistic, a sustained increase in infections appears to be possible only through a leakage in the quarantine program. the lowest rate of spread of i will be in the steady state da/dt=0, when where g' is the growth in a, due to a leakage from i. since the measures are already in place, this will be a weak coupling. as long as a≠0, the cumulative number of infected individuals will continue to increase until most or all of the susceptible individuals are infected (eq.3a), which is quite undesirable considering the scale of devastation that has already been caused to even the developed countries by roughly 1 in 500 infections. according to this model, and not too far from common sense, a significantly high rate of testing r and keeping a check on the potential leakage from infected individuals even under a quarantine condition can reduce the number of asymptomatic individuals. in this work, we note by studying the covid-19 infection data from several countries which implemented quarantine that the exponential growth phase ends, but it is followed by a linear growth phase. a deviation from an exponential growth phase is a relief to the population, and a sign of success of the containment measures. the significance of this is that there are no new infections caused by an individual who is understood be infected. however, via indirect route or secondary effects there is a still a constant rise in the cumulative number of infections, in many places with a very high daily rate. the peak is flat. the unmoderated peak is understood at the population level using established sir models, when most or all of the susceptible individuals develop infections and immunity. in the first week of april, the number of global covid-19 infections reached 1 million. the hospital resources, health care personal, economies are already overwhelmed by the pandemic, when as little as 1 in 500 are infected in many developed countries. so, at this point in this work we do not attempt to project the dates when a much higher fraction of the society is infected, or evaluate the consequences of such mass scale infection, which may also come with several other assumptions such as the reduction of the virulence upon spreading, etc. further, the definition of who is susceptible is not yet clear. in the initial months of covid-19 infection, people over an age of 65, and with comorbidities were considered highly susceptible. however, although at a much lower rate, one begins to hear about healthy individuals in their 20s or 30s succumbing to covid-19. instead, we focus on the peak that is achievable by active interventions, such as the ones sars, mers had seen. a peak explained in a lay language is the time when for the first time the number of new infections are lower than the previous day crossed. however, with stochastic fluctuations on a daily basis, this statement needs to be interpreted by observing consistent trends over a few days. however, the number of daily covid-19 infections in many places has been roughly constant, at least for 3 to 4 weeks, after containment measures. even if eventually the new infections do decline because of any reason, it must be understood that the high number of daily infections present a strange situation of having chronic and acute severity simultaneously for at least many weeks, if not longer. however, if instead of following the trends in the daily fluctuations, if one fits a sigmod function to the cumulative infections it can lead to confusing interpretation. extrapolating the slowing exponential trend in the early days following the quarantine, the sigmoid will predict a peak. however, this peak will shift when the same fit is repeated in the days later, with a dominant linear component, as in reality a linear graph does not have a peak. we explore the possibility that the constant rate of new infections is a false signal from the constant rate of testing. if it is indeed the case, the screening tests are showing infections of people not just asymptomatic, but asymptomatic and not contagious. if there is a way of distinguishing the latter, it must be clarified to reduce the global panic levels. however, prima facie this possibility can be refuted because although the countries such as south korea and singapore on the one extreme are performing extensive screening tests (43 and 70 tests on average for every detected infection), countries such as switzerland are performing tests only when there are significant symptoms, or pre-existing vulnerabilities, and of course for health care personnel. thus, the new infections arising purely as an artefact of over testing does not seem like a possibility. so far, other than china, most countries have shown only a transition from an exponential to a linear growth phase. the hope is that eventually the linearity will fade out with an exponential decay of the numbers of asymptomatic and infected individuals or at least reduce in intensity as it did in south korea, which showed a shift from a constant daily new infections of 600 to daily new infections of 100 (the second linear regime in figure 2 ). the second linear regime from south korea thus presents an interesting case study. south korea had ramped up its testing capacity from about 1000 per day in early february to around 10,000 per day from february 25 till at least early april. whether the reduced number of daily infections a few weeks after this ramp up is a consequence of the tests or of any contact tracing that allowed them to test and isolate the large number of asymptomatic individuals or purely depends on the decay time of the infection in the asymptomatic individuals which was has so far been underestimated to be around 10 days needs to be understood with an in-depth analysis of the policies and implementation, which we could not perform even after parsing through the information that is publicly available. it is clear that the availability or implementation of newer resources such as massimmunizations, therapeutic interventions or even the chance that the sars-cov-2 reduces in lethality due to mutations or seasonal variations were not considered in our analyses. several other models have made the predictions of the peak of the infection at the population level. when any of these possibilities arise, it is possible to adapt those models to predict the peak for a newer country or region which still did not implement those interventions. however, the reality today is different and none of these options at our disposal. we instead focus on how this scenario is evolving, based on the real data rather than assumptions. whether the number of daily new infections continue at the constant but very high daily rate . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . or decline, should be tracked from the real data rather than making a prediction based on past experiences from other infections. if this hope does not get the support of the data, each country depending upon its current overload of active infections and health care resources needs to have a "plan b". presently the only two interventions available are testing and isolation. both of these are of course qualitative in nature. how extensively to perform the tests, how restrictive and privacy-limiting should the isolation be, has been interpreted differently by different countries. in summary, we ask three questions: has the implementation of these strategies been successful? the answer we find is that it is. from an exponential behavior, most countries transitioned to a linear behavior showing that the infected individuals are not causing new infections. and this happened roughly around 10 days after the implementation of the social distancing or quarantine policies. while this is a moment of triumph and many may be wondering when is a good time to lower the guard and relax the restrictions, we ask the second question: if the goal of reducing the number of daily infections below a manageable level been achieved. from the data after 3 to 4 weeks of strict measures by many countries, it has not yet reached this level. this happens because instead of a decline in the number of daily cases, they saturate, and that too at very high values (7600 in spain, 5600 in germany, etc). we then raise the third question for an open interpretation, not to be restricted by the limited understanding of the authors: why this linear regime persists for so many weeks, and would it reduce in intensity naturally or require a newer intervention such as extensive testing or prevention of unexpected leakages in the system of isolation and quarantine through health care workers, essential services or any other means we cannot imagine today. if there are indeed such leakages, these are not the ones that can be predicted by following the overall number of infections of states or countries as was done in this work, but rather by taking a detailed audit, by tracking the need and rigor of implementation in each industry and segment of the society. we analyze the data using a simple model that seems to suggest that if the linearity persists, this may be due to leakages in the quarantine system, and can be partly compensated by increasing the rate of testing. theoretically, it is possible that the measures adopted by china were much more stringent compared to other countries which allowed a reduction in the new infections. however, given the gravity of the situation, we present our observation, analysis and model in all its humility, for an open interpretation. the aim of this work is mainly to point to existence of this new, and at least for us, linear growth phase with a very high number of daily new infections, which brings a very hard to manage mix of acute and chronic societal burden. given the gravity of the situation the world is facing, the data of the linear phase needs an open interpretation by all the experts. with all good intentions, we wish the slope of this linearity to be reduced in a few weeks, and the linearity shown in this work is not relevant in longer term. however, the data is not currently in favor of such wishes. thus, to understand if the existing interventions are working one needs to detach oneself from the notion of a peak followed by a decline or even the linear table 1 . the data up to 1 st of april was used in these analyses (the data from germany and spain is from the 3 rd of april). the legends in the figures suggest that the number of days for which the growth continued in a linear regime. south korea and singapore have two distinct linear regimes, and the duration in both are indicated. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . the slope from the linear regime was compared with several factors to see if any factor could potentially help interpret the linear regime. it is understood that all these factors are not entirely independent but some are connected. the cumulative infections and the number of daily cases around the time when the transition occurred are the most correlated. the number of tests performed per single detection is poorly correlated, and the other variables such as population of the region do not appear correlated. as much as the linear regime suggests the end of the exponential growth phase, a correlation of the daily cases with the average number of infections at the time of transition seems to suggest that the growth is only maintained in a "pause", frozen at the state where the quarantines are implemented. 3) were simulated to see if the observations can be captured. the simulation was performed with parameters such as a=0.1, and the transmission via asymptomatic is individuals is much lesser with l=0.01. at this growth stage of the pandemic since the data from most countries show a recovery rate of around 10%, we performed the simulation with µ=0. the asymptomatic (blue), and recorded infections (red) with two different testing rates r=0.2 and 1.2 initiated after the lockdown period (30 on axis which represents the days) are shown in panels a and b. as expected a transition from an exponential to linearity is observed. panel c shows the daily new infections when r=1.2. since the eq.(3) is stochastic, we added an incubation period drawn randomly from a beta-distribution with a mean of 10 days. for this choice of parameters which were chosen to qualitatively emulate the peak observed in south korea, a a low level of daily new infections persist. however, by varying r, it was seen that these average number of these daily new cases decreases with the r and increases with the time of lockdown (data not shown). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . along with other relevant information for each country or province. *estimated from the german national testing as of 1 april 2020. § the infection data from south korea shows two different linear regimes. the first one which immediately follows the end of the exponential regime is considered. ¶ singapore also showed two linear regimes, a very short linear regime early on, followed by what appeared to be an exponential phase. however, since the absolute numbers are very low and noisy during the first linear regime in this work, the second linear regime was considered. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.07.20055772 doi: medrxiv preprint summary of probable sars cases with onset of illness from 1 modelling strategies for controlling sars outbreaks who director-general's opening remarks at the media briefing on covid-19-11 real-time tentative assessment of the epidemiological characteristics of novel coronavirus infections in wuhan, china, as at 22 impact of non-pharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand age-structured impact of social distancing on the covid-19 epidemic in india countries test tactics in 'war' against covid-19 the simulations driving the world's response to covid-19 the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) data from the official repository of protezione civile an interactive web-based dashboard to track covid-19 in real time key: cord-283588-j27q53oz authors: gebicki, jerzy; wieczorkowska, marzena title: covid-19 infection: mitohormetic concept of immune response date: 2020-07-14 journal: cell death discov doi: 10.1038/s41420-020-00297-9 sha: doc_id: 283588 cord_uid: j27q53oz nan peroxidase 1 knockout (gpx1−/−) mice was much higher than that of wild-type mice. these findings demonstrate that, contrary to much current thinking, early intervention targeting nlrp3 inflammasome activity can induce timely and efficient activation of the innate immune response during acute infection. clearly, this observation can be linked directly to the mitohormetic concept. 1-mna, previously regarded as a useless metabolite of na excreted with urine, has been shown to possess significant anti-inflammatory properties 5 . the pharmacological properties of 1-mna are quite numerous, and have been documented for many diseases and disorders 6 . the mitohormetic concept of anti-inflammatory activity by 1-mna is presented in fig. 1 . as aox expression is particularly high in respiratory tissues, it may be expected that there would be significant 1-mna anti-inflammatory activity in the airways as well. indeed, the excretion of 1-mna with urine has been found to be significantly reduced in respiratory syncytial virus (rsv) infection 7 . it has been suggested that the weakened ability to fend off inflammation during rsv infection is likely due to lower levels of 1-mna 7 . taking all of the above into consideration, the use of vitamin b3 to prevent inflammation damage associated with covid-19 seems rational 1 . however, a better effect is likely to be achieved by direct application of 1-mna. the lower levels of 1-mna observed in some airway diseases, including viral infections, may further suggest that 1-mna plays an important physiological role in regulation of the innate immune response. conflict of interest j.g. is co-inventor of the patents protecting the pharmacological use of 1-mna. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. received: 25 may 2020 revised: 13 june 2020 accepted: 3 july 2020 covid-19 infection: the perspectives on immune responses role of sirtuins in lifespan regulation is linked to methylation of nicotinamide widespread cellular distribution of aldehyde oxidase in human tissues found by immunohistochemistry staining protective effect of inflammasome activation by hydrogen peroxide in a mouse model of septic shock 1-methylnicotinamide: a potent anti-inflammatory agent of vitamin origin 1-methylnicotinamide (mna), a primary metabolite of nicotinamide, exerts anti-thrombotic activity mediated by a cyclooxygenase-2/ prostacyclin pathway using urine metabolomics to understand the pathogenesis of infant respiratory syncytial virus (rsv) infection and its role in childhood wheezing key: cord-255781-55zrmgxq authors: bergman, scott j.; ferguson, mckenzie c.; santanello, cathy title: interferons as therapeutic agents for infectious diseases date: 2011-12-31 journal: infectious disease clinics of north america doi: 10.1016/j.idc.2011.07.008 sha: doc_id: 255781 cord_uid: 55zrmgxq this article explains the rationale for development of interferons as therapeutic agents, and describes commercial products available today. it also provides a summary of studies that have been performed with interferons for use as exogenous biological response modifiers in viral infections. overall, the best data exist for treatment of viral hepatitis b and c, for which interferons are a cornerstone of therapy. although infections with human papillomavirus and common cold viruses sometimes respond favorably to interferons, their outcomes are far from ideal. finally, the role of interferons as vaccine adjuvants is still being explored but could be promising. replication in vitro at concentrations as low as pg/ml-the development of ifns as clinically useful drugs has been largely disappointing. this fact can be attributed partly to their short half-life in vivo and their extensive side effects. in fact, many symptoms of viral infections such as influenza can be blamed on endogenous ifn release. the adverse effects prevalent at therapeutic doses include fever, myalgia, and headache, dubbed "flulike symptoms," along with bone marrow suppression leading to leukocytopenia and thrombocytopenia, plus central nervous system manifestations including depression. 1 ifns have been studied for the treatment or prevention of herpes zoster, herpes simplex, and cytomegalovirus infections, but the successful development of acyclovir and ganciclovir gave clinicians safer and more effective alternatives for dealing with these viruses. 5, 6 ifns can also be used in the treatment of multiple sclerosis and certain cancers, but this article reviews the therapeutic applications of ifns for infectious diseases, focusing on viral infections. ifns are not absorbed orally because of their large amino acid sequence, which is susceptible to the proteolytic enzymes in the digestive tract. however, ifn-a is readily absorbed after both intramuscular and subcutaneous injection. 7 this rapid absorption combined with a short half-life means that frequent injections are needed to maintain adequate concentrations in the body. both commercially available ifn-a products in the united states have now been chemically attached to polyethylene glycol (peg) to enhance their half-life and make once-weekly dosing possible. this coupling not only makes administration easier, but also reduces side effects by having a predictably lower peak concentration of the exogenous cytokine. both pegylated inf-a2a (pegasys) and ifn-a2b (peg-intron) are obtained from escherichia coli by recombinant methods. these agents consist of naturally occurring small proteins with molecular weights of 15,000 to 27,600 da. 3 each is considered a first-line option for the treatment of chronic hepatitis c virus (hcv) infection in combination with ribavirin. more details on this use and others are described later in this article. along with the list of additional indications approved by the food and drug administration shown in table 1 , ifn-a was shown to be an effective treatment for the symptoms of an aggressive case of chronic active epstein-barr virus, but did not eliminate infection entirely. 8 therefore, additional studies would need to be performed before recommendation for this use. human leukocyte derived ifn-an3 (alferon n) injection contains a spectrum of a ifns, and is only approved for the treatment of refractory or recurring condylomata acuminata in adult patients. a low-dose oral version is in development for use in the treatment and prevention of influenza. 9 both versions have been studied against human immunodeficiency virus (hiv)-1 infection, but with little success. 10,11 ifn alfacon-1 (infergen) is considered the synthetic "consensus interferon" because it contains a nonnatural sequence of ifn-a amino acids all chosen for the highest activity against viral hepatitis. to date, no pegylated formulation of this product has been brought to market. all the a ifns include a black-box warning in their prescribing information about how their use .may cause or aggravate fatal or life-threatening neuropsychiatric, autoimmune, ischemic and infectious disorders. patients should be monitored closely with periodic clinical and laboratory evaluations. therapy should be withdrawn in patients with persistently severe or worsening signs and symptoms related to side effects. in many, but not all cases, these resolve after stopping therapy. 12, 13 ifn-b1a (avonex or rebif) and ifn-b1b (betaseron) are recombinant proteins with 166 and 165 amino acids, respectively. these b ifns have antiviral and immunomodulatory properties too, but their use at this time is limited to treatment of multiple sclerosis, not infections. ifn-g1b (actimmune) injection is used regularly for the prevention of infections in patients with chronic granulomatous disease along with antibacterials and antifungals. 14 its mechanism of action for this purpose is not entirely known, but long-term studies show a definite benefit. 15 ifn-g can also be used as a salvage therapy for mycobacterial infections, but is not routinely used for treatment of this or other infections. 16 topical imiquimod 5% (aldara) and 3.75% (zyclara) creams do not have inherent antiviral activity alone, but instead induce ifn-a, ifn-b, and ifn-g plus tumor necrosis factor (tnf)-a through toll-like receptors (tlrs). local application to external genital and perianal warts results in an immunomodulatory response that stimulates cytokines, which have antiviral action and cause a reduction in both viral load and wart size. 3 chronic infection with hepatitis b virus (hbv) and hcv affects over 400 million people worldwide. [17] [18] [19] chronic viral hepatitis is a leading cause of cirrhosis, liver transplantation, and hepatocellular carcinoma. with the development of a vaccination series for hepatitis b in the mid-1980s, along with increased public education and awareness, acute infection rates of both hbv and hcv in the united states have declined steadily. 19 hbv is a double-stranded dna virus whereas hcv is a single-stranded rna virus, both of which are capable of significant morbidity and mortality in chronic infection. the exact mechanisms of hepatic injury from hbv and hcv infection are not completely understood. because asymptomatic carriers with normal liver transaminases exist, it is likely multiple immune-mediated mechanisms result in hepatocyte damage as opposed to the virus itself being directly cytotoxic. following acute viral infection, the innate immune response initiates formation of nk cells, followed by virus-specific cd4 1 t cells and cd8 1 cytotoxic t lymphocytes. nk cells stimulate production of ifn-a/b and promote cellular clearance of viral proteins through disruption of the replication process. following successful clearance, either spontaneously or by treatment with ifn, peripheral cytotoxic t lymphocytes and cd4 1 t-cell response persists. 20 chronic infection is likely a result of failed innate and adaptive immunity. specifically, chronic infection with hcv has been associated with impaired t-cell and nk-cell response. [21] [22] [23] [24] genetic factors also likely influence progression of disease and predisposition to adverse effects. 25 although an abundance of research has investigated the immune response in relation to chronic viral hepatitis, many areas of uncertainty still exist. standard ifn-a, the first approved ifn for viral hepatitis, lacked several desirable pharmacokinetic properties. the addition of peg created an ifn that has a slower rate of absorption, reduced elimination, and a longer half-life, necessitating less frequent dosing and fewer adverse effects. furthermore, the peg moiety results in reduced immunogenicity and sterically hinders the antigenic binding site. 26, 27 although pegylated ifn has replaced standard ifn-a in treatment of chronic hbv and hcv, as many as 40% to 50% of patients still fail to respond to treatment. successful response depends on many factors including but not limited to viral genotype, viral load, and degree of liver fibrosis. 25 chronic hepatitis b and c are treated similarly with peginterferon (pegifn); however, only pegifn-a2a is fda-approved in the united states for treatment of hbv. both pegifn products are administered as subcutaneous injections once weekly for durations up to 48 weeks, dependent on viral genotype and early viral response for treatment of hcv. pegifn-a2b is dosed based on body weight (1.5 mg/kg once weekly) whereas pegifn-a2a is a fixed dose (180 mg/wk). ribavirin is used in combination with pegifn for treatment of hcv. the exact mechanism of action of ribavirin as an adjunctive antiviral agent in hcv is not completely understood. 12, 13 some studies have proposed ribavirin to act as an ifn-stimulated gene inducer to improve second-phase viral decline. 28 protease inhibitors (boceprevir and telaprevir) are recently approved adjunctive oral agents for the treatment of chronic hcv with pegifn and ribavirin. to date, all studies of protease inhibitors have been conducted in patients with hcv genotype 1, and have shown an increase in sustained virologic response (svr) rates particularly for patients previously unresponsive to ifn therapy. [29] [30] [31] [32] [33] [34] [35] the use of ifn for the treatment of chronic hbv and hcv has represented a mainstay of treatment for several decades. the specific mechanisms behind the antiviral effects of ifn for hepatitis are complex. ifn-stimulated genes are induced by ifn and disrupt viral replication. hundreds of ifn-stimulated genes are thought to exist. viperin, isg20 and protein kinase r (pkr) are just a few of the most commonly cited. it is also highly possible that ifn-stimulated genes work synergistically to produce antiviral activities. 36, 37 a lack of pkr can lead to an environment conducive to hcv replication, though it may not be a good predictor of exogenous ifn response. the study of ifn-stimulated genes and their role in determining who responds to ifn therapy has been evaluated in several studies. 25, 36, 38 additional studies of ifnstimulated gene expression are needed to clarify which are directly involved in successful viral response, in what capacity they affect response, and whether pharmacotherapy directed at induction of ifn-stimulated genes can help improve treatment response. chronic hbv infection can be successfully treated with ifn monotherapy. 39 loss of viral dna and antibody formation are successful outcomes associated with ifn treatment. the mechanism of ifn antiviral activity varies depending on hepatitis be antigen (hbeag)-positive or hbeag-negative disease. in hbeag-positive patients, an immune response is stimulated by ifn whereas in hbeag-negative disease, ifn acts directly as an antiviral. 5 hbeag-negative disease tends to be more difficult to treat, and is associated with a longer duration of disease and a higher likelihood of complications such as cirrhosis. 20 several oral nonnucleoside reverse transcriptase inhibitors are also available for treatment of hbv (entecavir, tenofovir, adefovir, lamivudine, and telbivudine). although ifn is still considered a first-line alternative and provides the advantage of defined treatment duration rather than potentially lifelong administration, these oral agents are often used in therapy because of their ease of use and reduced number of side effects associated with treatment. the ability of hcv to evade the host immune response has produced a complex rna virus capable of lingering infection, ultimately resulting in opportunities for increased risk of transmission and complications from advanced liver disease. much of the research regarding the use of ifn for chronic viral hepatitis has focused on use in hcv. following treatment with ifn, a decline in hcv rna occurs over several phases. a rapid inhibition of rna production within the first 1 to 2 days of treatment is followed by a second, slower phase associated with clearance of infected cells. 23, 40, 41 the interferons for infectious disease immune response to endogenous ifn produced by innate immunity and that administered exogenously can differ in terms of antiviral activities based on the phase of viral decline. studies have shown that response to ifn-based treatment for hcv may be affected by differences in ifn signaling and induction. it is likely that hcv has mechanisms to avoid recognition by the innate immune response, and as such inhibits the ability of hcv-infected cells to generate ifn. 25, 38, 42 early studies conducted in nonresponders to current therapy showed wide genetic diversity, with many showing no common traits to predict nonresponse to ifn therapy. 25, 38, 43 however, in 2009 several major studies were published associating a singlenucleotide polymorphism (snp) just upstream from interleukin-28b gene (il28b) with ifn response in patients with hcv genotype 1. [44] [45] [46] [47] [48] additional evidence points to the fact that the il28b polymorphism is also linked to spontaneous clearance of hcv. 48, 49 the il28b variant encodes for ifn-l3, a type iii ifn belonging to the interleukin (il)-10 superfamily, which function in a manner similar to type i ifns, resulting in ifn-stimulated gene induction. [49] [50] [51] the genome-wide association study conducted by ge and colleagues 45 evaluated more than 1600 treatment-naïve hcv genotype 1 patients, the majority of whom originated from the ideal study. results from logistic regression showed that the il28b polymorphism was a stronger predictor of svr than baseline viral load, ethnicity, or degree of fibrosis. further research in this area is needed to clearly identify a future role for genotype testing and further clarify whether it may influence response to therapy in other hcv genotypes. a multicenter, randomized, controlled study by mangia and colleagues 52 analyzed 268 caucasian patients with hcv genotype 2 (n 5 213) and 3 (n 5 55). out of 61% of patients who achieved rapid virologic response (rvr), il28b genotype was not associated with svr, whereas in those patients who did not achieve rvr a significant difference in svr was noted based on il28b genotype. at this time genotype testing for il28b is not routinely recommended for all hcv patients planning to undergo treatment, but it may be in the future. if done, it should not be used as the only factor when choosing a treatment strategy. 49 the complexity of viral defense mechanisms and subsequent effect on the host response has led not only to development of chronic infections but also to a lack of a viable vaccine. hcv viral polymerase lacks a proofreading capability, creating a more diverse target for vaccine development. 18 additional challenges include the lack of a suitable animal model to mimic a human environment and medium for viral growth. 18 one of the major limitations to ifn therapy is adverse effects. malaise, gastrointestinal effects, neuropsychiatric effects, neutropenia, and anemia can all limit the effectiveness of treatment by necessitating dosage reductions or treatment discontinuation. for newer ifn therapies to be successful, they must induce an antiviral response while at the same time limiting adverse effects. albinterferon is a new ifn therapy currently in development for the treatment of chronic hcv. this product is a combination of ifn-a2b fused to recombinant human albumin. one of the advantages with this product is that it only requires once or twice monthly dosing. 53 not much is known at this time about the immunomodulating effects of albinterferon in hcv. it has been shown to have similar svr and adverse event rates to traditional pegifn when used in combination with ribavirin. [54] [55] [56] research into ifn-l as an agent to treat hcv has also been initiated. it is hypothesized that l ifns may be associated with less adverse effects than ifn-a because ifn-l receptors are primarily found in hepatocytes. 49, 57 specifically, research into new investigational pharmacotherapy in the form of pegylated il-29 (ifn-l1) in patients with hcv genotype 1 who relapsed following traditional treatment with peg-ifn-a and ribavirin appears promising. 50, 57 both ifn-l1 and ifn-l3 share a common receptor and have a similar sequence identity. 57 a 4-week, open-label study conducted in 56 patients with chronic hcv genotype 1 was designed to assess pegifn-l1 in combination with ribavirin. 57 it was a dose escalation study conducted in 3 parts. parts 1 and 2 evaluated patients who relapsed following treatment with ifn-a, and part 3 included treatment-naïve patients. in part 1, pegifn-l monotherapy (1.5 mg/kg or 3 mg/kg) was administered subcutaneously every 2 weeks or weekly. in parts 2 and 3, a range of pegifn-l dosages (0.5 mg/kg, 0.75 mg/kg, 1.5 mg/kg, or 2.25 mg/kg) were administered weekly in combination with ribavirin twice daily (1000 mg if weight <75 kg and 1200 mg if weight !75 kg). the primary outcomes were safety and tolerability. pharmacokinetics and viral load reduction were evaluated as secondary end points. commonly reported adverse effects with pegifn-l included fatigue (29%), nausea (12%), myalgia (11%), and headache (9%). most adverse events were mild or moderate in severity. four patients (7%) experienced treatment-related toxicity and required doses to be withheld. one patient experienced grade 3 thrombocytopenic purpura and another patient had elevated alanine aminotransferase, aspartate aminotransferase, and bilirubin levels. both events were considered to be related to treatment with pegifn-l. aminotransferase elevations occurred most often in patients who received high-dose (3 mg/kg) pegifn-l monotherapy. no clinically relevant decreases in absolute neutrophil count occurred. also, hemoglobin values remained consistent with known effects in patients who received ribavirin therapy. viral activity decreased in the majority of patients who relapsed with previous treatment, with 23 of 24 patients achieving at least a greater than 2-log reduction in hcv rna. six of 7 treatment-naïve patients achieved a similar reduction in viral load and 2 achieved undetectable hcv rna levels. kinetic data showed a linear relationship between dose and exposure independent of body weight, which may prompt future research to evaluate a fixed dose of pegifn-l. 57 larger, longer, controlled, and blinded studies of ifn-l as a viable treatment option in hcv are needed to define its place in therapy and benefits over existing ifn therapy. studies in other hcv genotypes are also needed. in addition, with the advent of protease inhibitors, more research will be necessary to evaluate how direct antivirals and il28b genotyping interact in guiding treatment decisions. adjunctive therapy with agents that induce or restore ifn-stimulated gene expression has recently been evaluated in patients with hcv. s-adenosylmethionine (same) given orally was evaluated in an open-label study in 24 patients with chronic hcv, genotype 1 who were considered nonresponders to previous ifn and ribavirin treatment. 41 same was administered at a dose of 800 mg twice daily in combination with pegifn-a2a (180 mg/kg weekly) and weight-based ribavirin (1000 mg if weight <75 kg and 1200 mg if weight !75 kg). the primary outcome was change in firstphase and second-phase viral decline. treatment response and ifn-stimulated gene expression were also evaluated after up to 72 weeks of treatment. results showed significant improvement in second-phase viral decline assessed at 2 weeks. svr was also evaluated; however, this study was not powered to detect differences in virologic response rates. furthermore, at the time of publication not all patients had reached 24 weeks post treatment, so the full effects on svr were not fully known. the addition of same showed greater induction of ifn-stimulated genes, including viperin, myxovirus resistance protein, and isg15, compared with control. adverse effects noted with same were mild and mostly related to gastrointestinal upset, likely as a result of lactose in the tablet preparation. 41 additional research is aimed at investigating structure-activity relationships, and preliminary pharmacokinetic studies on oral ifn inducers that act on tlrs in the treatment of hcv. 58 upper respiratory tract infection in the form of "the common cold" can be caused by a variety of viruses including rhinovirus, coronavirus, influenza, parainfluenza, respiratory syncytial virus, adenovirus, coxsackie, and echovirus families among others. 59 symptoms may include rhinorrhea, nasal obstruction, cough, fever, and sore throat. the disease is usually mild and self-limited, but several trials have addressed treatment or prevention of the common cold with therapeutic agents. ifns were once one of the most popular prospects for this purpose, but the minor benefit that was derived from them was counteracted by the adverse effects inflicted. 60 an early double-blind trial with ifn-a2b intranasal drops did demonstrate that with use for several days before experimentally induced rhinovirus infection, common cold symptoms were significantly fewer in study participants compared with placebo-drop users. 61 administration of the drops 4 times daily was superior to a higher dose given once daily at preventing infection. short-term use was well tolerated, but obviously it is not realistic for everyone to use intranasal drops 4 times daily throughout the entire cold season. in an attempt to prevent natural infection during the period of increased acute respiratory tract virus activity, a twice-daily nasal spray was studied in volunteers over 28 days. 62 there was a significant decrease in the number of rhinovirus infections noted, but not in any other types of viral respiratory tract infections including parainfluenza. adverse events with the ifn formulation were common in this placebocontrolled trial. during the first week alone, 20% of participants receiving ifn spray reported nosebleeds. this number increased to 41% by the end of the study. providing ifn prophylaxis for family members of those infected with common cold viruses is a more targeted approach to therapy. several studies have addressed the usefulness of ifn nasal sprays in this scenario. seven days of use did significantly reduce rhinovirus infections in 2 different trials when compared with placebo for both individuals (7.9% vs 15.5%) and their families (3.3% vs 33.3%, both p<.05), but not in 2 other studies when lower doses were given for a shorter 5-day course. [63] [64] [65] [66] overall, the intranasal dose of ifn needed to protect against upper respiratory tract infection appears to cause significant unwanted effects. 67 infection with coronavirus and respiratory syncytial virus has also been an object of investigation for ifn-a2b nasal sprays, but with little success. 68, 69 a study of intranasal human lymphoblastoid ifn-an1 (wellferon) suggested lower prophylactic activity for influenza than it did for rhinovirus. 70 because results of prophylactic trials with ifns for common cold viruses were not favorable, use in the treatment of infection seemed a logical application for this biological response modifier. although some benefit was originally seen with twice-daily ifn-a2b intranasal drops for treatment of experimentally induced rhinovirus, 71 no advantage was clear when an intranasal spray was used once daily for 5 days to treat natural infection. 72 increased rates of blood in the mucus were again noted for participants receiving the intervention, and the ifn group experienced more secondary complications requiring prescription of antibiotics. the investigators concluded that intranasal ifn was ineffective for treating the common cold and was associated with clinically significant side effects. similar trials with ifn-b-serine and ifn-g formulations, although initially positive, have shown equally disappointing clinical results. [73] [74] [75] [76] even though the prospects of further study on ifns for upper respiratory tract infection appear limited, one modern trial did demonstrate an added benefit of intranasal ifn-a2b in combination with an antihistamine (chlorpheniramine) and nonsteroidal anti-inflammatory drug (ibuprofen) at reducing common cold symptoms, showing that at least one group is still interested in studying the topic. 77 investigators have also recently begun research on an alternative therapeutic approach for rhinovirus infections using the ifn and tnf-a inducer, imiquimod. application of this intranasal cream in primates has shown promising results in terms of enhancing cytokine response, but human trials have not yet been published. 78 human papillomaviruses (hpvs) are now known to be the cause of cervical cancer and are also responsible for genital warts. hpvs are nonenveloped, double-stranded dna viruses that invade mucosal and epithelial tissues during sexual contact with an infected partner. it is estimated that more than 50% of the sexually active american population has been or will be infected with hpv at one point in their lives. 79 when hyperproliferation of infected cells occurs, this can lead to genital warts or cancer of the cervix, vagina, vulva, and penis, among others. there are more than 100 different types of hpv and approximately 40 of them infect genital mucosa. fifteen carcinogenic types of hpv have been identified, but 2 of them are associated with 70% of cervical cancers. 80 two vaccines have recently been introduced that prevent infection with these most common high-risk types of hpv, 16 and 18. 81 one of these vaccines can also induce protection against the most prevalent hpv types that have a low risk of malignancy, but instead cause genital warts: hpv-6 and hpv-11. hpv has the ability to persist in stratified epithelia for decades because of mechanisms that avoid immune eradication. ifn plays a large role in this cycle. 82 ifns are normally secreted by keratinocytes, but hpv reduces their expression. introduction of low-level ifn can actually increase early gene transcription and hpv replication, which may explain why use of the agent therapeutically has had mixed results. 83 overall outcomes have been positive more often for cases of genital warts than reduction of hpv lesions associated with cancers. a study comparing the in vitro activity of ifn-a2b and ifn-an3 on oncogenic hpv-16, hpv-18, and hpv-31b demonstrated that increasing concentrations did not always correlate with a stepwise inhibition of hpv replication. 84 meanwhile, a meta-analysis recently analyzed locally used and systemic ifn for genital warts. 85 seven randomized studies of ifn intralesional injection or topical gel met criteria for inclusion, and overall there was a benefit in complete response rates over placebo (44.4% vs 16.15%, relative risk 2.68, 95% confidence interval 1.79-4.02). however, there was no difference in outcomes for trials comparing systemic ifns with placebo. in comparison, clearance of genital and perianal warts occurs in 50% of patients with the topical ifn inducer imiquimod, usually after 8 to 10 weeks of use. 3 the 5% imiquimod cream (aldara) should be applied to affected areas 3 times a week for up to 16 weeks, whereas the newer 3.75% cream (zyclara) can be applied once daily for as little as 8 weeks to treat external genital warts caused by hpv. systemic ifn therapy may be useful when hpv affects areas of the body other than the anogenital region. 86 successful treatment with systemic pegifn-a and a topical retinoid has been reported for mucosal carcinomas from epidermodysplasia verruciformis, a genetic abnormality leading to persistent and widespread hpv infection of the skin. 87 recurrent respiratory hpv infection has also been effectively treated with ifn-a (12 of 18 patients), although it had no effect on viral load or replication. 88 a 20-year follow-up of patients treated with ifn-a for recurrent respiratory papillomatosis confirmed better response rates for hpv-6 than hpv-11, which had a higher likelihood of malignant transformation. 89 for recurrent conjunctival papilloma, topical plus systemic or intralesional ifn has been effective with partial excision. 90, 91 the rapid resolution of significant hpv-associated warts on the hand, foot, and face has also occurred in an hiv-infected patient on antiretrovirals while being treated for hepatitis c with pegifn-a2b and ribavirin. 92 case reports of treatment with the topical ifn inducer, imiquimod, have shown promise for its use in focal epithelial hyperplasia (heck disease), a rare disorder caused by specific types of hpv (13, 14, 32, and 55) affecting oral mucosa primarily in children. 93 in addition, imiquimod 5% cream has been used successfully in the treatment of plantar warts, a smoother, flatter manifestation of hpv-1, hpv-2, and hpv-4 on the foot. 94 of interest, the oral h2-antagonist cimetidine, along with reducing stomach acid, also induces production of ifn-g and il-2, which eliminates viral warts in some patients. 95 in the future the improved application of more effective topical ifns may become a reality, 96 which could provide a valuable treatment for hpv infections without the systemic side effects of current injectable formulations. adjuvants (adjuvare, latin for "to help") are substances that augment the immunogenicity of an antigen when mixed with the antigen for use in a vaccine. adjuvants (1) stimulate granuloma (which is a macrophage-rich mass), (2) enhance costimulatory signals, (3) stimulate nonspecific lymphocyte production, (4) prolong the antigen concentration in a site for lymphocyte exposure, 97 and (5) induce cytokines. 2, 98 research in vaccine development has shown that one of the most promising uses of ifns is as an adjuvant with specific antigens in prophylactic vaccines. 99 toporovski and colleagues 99 provide a current review of the use of ifn-a, ifn-b, ifn-g, and ifn-l in vaccine studies that focus primarily on murine, avian, porcine, and nonhuman species. regardless of the species, the use of ifns as adjuvants seems to improve the efficacy and safety of most vaccines while providing the immunomodulatory effect of stimulating the t-helper 1 response. in humans, ifn-a, predominantly produced by plasmacytoid dendritic cells, plays a large role in the body's immune response against viruses. 100 it induces plasma cell differentiation from b cells causing an increase in the serum level of influenzaspecific immunoglobulins, and channels antigen-presenting cells (apcs) to the site of infection. 101 most research on ifn-a adjuvant activity and its subsequent use in approved vaccines seems to indicate that it is a potent adjuvant. 99 when mixed with the influenza vaccine and injected intramuscularly, it is a highly effective adjuvant. 100 oromucosal administration of recombinant ifn-a, like that of natural oromucosal ifn production, has been shown to provide immunity against viral infection and tumor cell growth. 102 nonresponders low responders to a previous vaccine showed an improved immunoglobulin response with a recombinant ifn-a and hbv vaccine. 103 although research is also focused on the other classes of ifns as adjuvants, thus far they have not yielded results as promising as that of ifn-a. the use of ifn-b has yielded mixed results; ifn-g has been used primarily in dna vaccines; and even less is known about the use of ifn-l in vaccines. 99 nevertheless, the use of ifns as adjuvants shows great promise in augmenting vaccine efficiency, and should continue to be a top priority in the development of vaccines. ifns have been tested repeatedly against infectious diseases, but injections are used mostly for the treatment of viral hepatitis c and prevention of infections in patients with chronic granulomatous disease clinically. intralesional ifn and topical inducers are effective in reducing the manifestations of genital warts, but they do not eliminate cancer-causing hpv from the body. ifn has not proved to be consistently effective for treatment of respiratory tract infections from the common cold or influenza viruses, and prophylactic use is not currently feasible. the severity and quantity of adverse effects from systemic ifn therapy make it unattractive for many uses. several infections, including herpes simplex, herpes zoster, cytomegalovirus, and even viral hepatitis b have other effective pharmacologic treatments. ifn has been successfully used as a vaccine adjuvant, and further research may allow for its additional use for this application in the future. mim's medical microbiology goodman & gilman's the pharmacological basis of therapeutics role of endogenous biological response modifiers in pathogenesis of infectious diseases interferons at age 50: past, current and future impact on biomedicine immunoglobulins, vaccines or interferon for preventing cytomegalovirus disease in solid organ transplant recipients interferon: mechanisms of action and clinical value interferon-alpha therapy for chronic active epstein-barr virus infection: potential effect on the development of t-lymphoproliferative disease fda authorizes alferon ldo clinical study for treatment and prevention of influenza a multicenter, randomized, controlled trial of three preparations of low-dose oral alpha-interferon in hivinfected patients with cd41 counts between 50 and 350 cells/mm(3). division of aids treatment research initiative (datri) 022 study group vitro activity of interferon-alpha n3 (alferon n) against hiv-1. interscience conference on antimicrobial agents and chemotherapy nj: pegasysò (peg-interferon alfa-2a) nj: peg-intronò (peginterferon alfa-2b) a controlled trial of interferon gamma to prevent infection in chronic granulomatous disease. the international chronic granulomatous disease cooperative study group long-term interferon-gamma therapy for patients with chronic granulomatous disease biological response 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polymorphism determines treatment response of hepatitis c virus genotype 2 or 3 patients who do not achieve a rapid virologic response albinterferon alfa-2b dosed every two or four weeks in interferon-naive patients with genotype 1 chronic hepatitis c safety and antiviral activity of albinterferon alfa-2b dosed every four weeks in genotype 2/3 chronic hepatitis c patients albinterferon alfa-2b was not inferior to pegylated interferon-alpha in a randomized trial of patients with chronic hepatitis c virus genotype 2 or 3 albinterferon alfa-2b was not inferior to pegylated interferon-alpha in a randomized trial of patients with chronic hepatitis c virus genotype 1 phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients with chronic genotype 1 hepatitis c virus infection design and optimisation of orally active tlr7 agonists for the treatment of hepatitis c virus infection the common cold alpha 2-interferon for the common cold intranasal interferon alpha 2 for prevention of rhinovirus infection and illness intranasal interferon-alpha 2b for seasonal prophylaxis of respiratory infection prophylactic efficacy of intranasal alpha 2-interferon against rhinovirus infections in the family setting prevention of natural colds by contact prophylaxis with intranasal alpha 2-interferon ineffectiveness of postexposure prophylaxis of rhinovirus infection with low-dose intranasal alpha 2b interferon in families intranasal interferon (rifn-alpha a, ro 22-8181) for contact prophylaxis against common cold: a randomized, doubleblind and placebo-controlled field study tolerance of one-month intranasal interferon prevention of experimental coronavirus colds with intranasal alpha-2b interferon the efficacy of intranasal interferon alpha-2a in respiratory syncytial virus infection in volunteers wellferon") prophylaxis against rhinovirus and influenza virus in volunteers intranasal interferon-alpha 2 treatment of experimental rhinoviral colds intranasal 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with topical imiquimod: report of three cases plantar warts treated with an immune response modifier: a report of two cases cimetidine treatment for viral warts enhances il-2 and ifn-gamma expression but not il-18 expression in lesional skin topical delivery of interferon alpha in human volunteers and treatment of patients with human papillomavirus infections new york: w.h. freeman and co advances in vaccine adjuvants interferons as potential adjuvants in prophylactic vaccines plasmacytoid dendritic cells induce plasma cell differentiation through type i interferon and interleukin 6 adjuvant activity of interferon alpha: mechanism(s) of action oromucosal interferon therapy: marked antiviral and antitumor activity randomized comparative trial of interferon-alpha versus placebo in hepatitis b vaccine non-responders and hyporesponders key: cord-279849-zzkliu76 authors: dapalma, t.; doonan, b.p.; trager, n.m.; kasman, l.m. title: a systematic approach to virus–virus interactions date: 2010-01-20 journal: virus res doi: 10.1016/j.virusres.2010.01.002 sha: doc_id: 279849 cord_uid: zzkliu76 a virus–virus interaction is a measurable difference in the course of infection of one virus as a result of a concurrent or prior infection by a different species or strain of virus. many such interactions have been discovered by chance, yet they have rarely been studied systematically. increasing evidence suggests that virus–virus interactions are common and may be critical to understanding viral pathogenesis in natural hosts. in this review we propose a system for classifying virus–virus interactions by organizing them into three main categories: (1) direct interactions of viral genes or gene products, (2) indirect interactions that result from alterations in the host environment, and (3) immunological interactions. we have so far identified 15 subtypes of interaction and assigned each to one of these categories. it is anticipated that this framework will provide for a more systematic approach to investigating virus–virus interactions, both at the cellular and organismal levels. by design, viral infections in the laboratory almost always occur in the absence of other viruses. while this may be a logical starting point for virus research, viral infections in nature rarely occur in isolation. polymerase chain reaction (pcr)-based techniques have revealed that persistent viral infections are present in all domains of life. archaea can be hosts to several types of phage viruses (snyder et al., 2003) . most environmental and commensal bacterial isolates are infected with one or more phage viruses (ackermann and kropinski, 2007) . yeasts, including endomyces magnusii (pospisek et al., 1994) and even laboratory strains of saccharomyces cerevisiae harbor multiple types of endogenous rna viruses (fujimura and esteban, 2007) . filamentous fungi are similarly affected (nogawa et al., 1993) . phytoplankton are commonly infected by dsdna viruses (bellec et al., 2010) , while both wild and domesticated vascular plants have been shown to carry numerous persistent viral infections, including badnaviruses (kenyon et al., 2008) . finally, genomic sequencing of animals has shown that chromosomes of both invertebrates and vertebrates are permeated with genetic sequences that resemble integrated retroviruses, in various states of viability (gifford and tristem, 2003) . approximately 8% of human genomic dna, for example, is derived from retroviral genomes (tristem, 2000) . however, persistent infections in animals are not limited to retroviruses. the picorna-like nora virus has been detected in all drosophila melanogaster strains tested, both laboratory and wild caught (habayeb et al., 2006) , and life-long herpesvirus infections are well known in humans (chen and hudnall, 2006; pancharoen et al., 2001) as well as marine bivalves, fish, reptiles, birds, and other mammals from mice to elephants (vandevanter et al., 1996) . therefore in any individual, persistent, sometimes congenital, often asymptomatic viral infections provide a background onto which all future viral infections are superimposed. given the now significant evidence for ubiquitous endogenous viral infections in all domains of living things, virus coinfections would seem to be the rule rather than the exception in nature, if not the laboratory. yet, they have rarely been systematically studied for possible effects of one virus on the other, with most documented interactions having been discovered accidently. it is certainly true that coinfection may not result in interactions for all virus species. however, because many viruses induce profound changes in their host, it seems likely that virus-virus interactions are common and may be critical to understanding viral pathogenesis and evolution. therefore, in this review we identify known and potential types of virus-virus interactions (vvis) and organize them into three categories: (1) direct interactions of viral genes or gene products, (2) indirect interactions that result from alterations in the host environment, and (3) a subset of indirect interactions called immunological interactions, unique to organisms equipped with an adaptive immune system. we have so far identified fifteen subtypes of vvi and assigned each to one of the three major categories (table 1) . this framework provides a systematic approach to investigating vvi, both at the cellular and organismal levels. as a first step, it is necessary to define vvi. we define a vvi as a measurable difference in the course of infection of one virus as a result of a concurrent or prior infection by a different species or of course the simple case of a prior viral infection conferring protective immunity against future infections with an immunologically identical virus is well known. but this interaction follows directly from the role of the adaptive immune system, and is therefore a rather uninteresting sort of interaction. a trivial case also exists with virus-induced generalized immunosuppression, as seen with human immunodeficiency virus (hiv)/aids. generalized immunosuppression, regardless of the cause, results in increased replication and pathology by some viruses that have a benign disease course in immunocompetent persons. however, this effect is not specific to viral pathogens. therefore, the discussion of vvi involving virus-induced immunosuppression will be limited here to those in which there is evidence of other viruses impacting the course of infection of the immunosuppressive virus, or evidence of direct interaction of proteins or nucleic acids of the immunosuppressive virus with proteins or nucleic acids of a coinfecting virus (lisco et al., 2009) . these uninformative examples aside, we have collected reports of vvi involving unexpected direct, environmental, and immunological interactions and used them to create what is intended to be a growing online database of these important phenomena. in the sections below, the current subtypes of vvi are each described and illustrated with a few examples. some categories have many more currently known examples than those described here, which are included in the online database. we do not mean to represent either the examples below nor the online list as a complete collection. instead, our aim is to provide the framework to launch a publicly available collection of vvi which allows continuous updates by and for the scientific community. we define a direct vvi as an occurrence in which nucleic acids or proteins of one virus physically interact with the genes or gene products of a coinfecting virus. this definition encompasses six different subtypes of interaction: helper viruses, pseudotype viruses, superinfection exclusion, genomic recombination, embedded viruses, and heterologous transactivation. direct interactions require coinfection of the same cell to take place, but the infections need not take place close in time. in several cases of documented direct interactions described below, cells latently infected with one virus may be infected by the second virus years, or even millenia later if the initial viral genome is incorporated into the germline of the host. a helper-dependent virus is any virus that is replication defective on its own, and therefore requires the gene products of another virus to produce infectious progeny. engineered recombinant helper virus/helper-dependent virus pairs have been developed as safe viral vector technologies (dorigo et al., 2004; marconi et al., 2008) , but several natural pairs of such viruses are also documented. one of the first helper-dependent viruses described was bacteriophage p4, a bacteria-infecting virus that is able to replicate its own genome, but requires the presence of a coinfecting bacteriophage, such as p2, to provide capsid components and cell lysis (shore et al., 1978; six and klug, 1973) . in plants, the carrot mottle virus of the umbravirus genus has been shown to be dependent upon viruses of the luteoviridae family for encapsidation and transmission by aphids (waterhouse and murant, 2008) . a mammalian helper-dependent virus is adeno-associated virus (aav), a replication-defective parvovirus that generally requires a host cell that is coinfected with an adenovirus or herpesvirus in order for virions to be produced and escape the host cell (buller et al., 1981; goncalves, 2005) . since the original description of aav, it has been discovered that genotoxic stress and other factors may also make a host cell permissive for aav progeny production, indicating that aav is not entirely replication defective (meyers et al., 2000; yalkinoglu et al., 1988) . a more clearly defined case is that of hepatitis d virus (hdv), a human pathogen. originally, discovered in the 1970s as a subtype of hepatitis b virus (hbv), further studies led to the understanding that hdv only occurred in hbv infected individuals. although the small viroid-like hdv is able to reproduce its own rna and ribonucleoprotein capsids, it requires the hbv membrane glycoprotein, hepatitis b surface antigen (hbsag) in order to associate with cell membranes and bud as infectious particles (rizzetto, 2009) . therefore, naturally occurring helper-dependent viruses appear to be dominated by non-enveloped viruses that can replicate their genome autonomously and only require a helper virus for packaging and/or release. it should be noted that the interaction between a helperdependent virus and its respective helper virus is not necessarily unidirectional. both aav and p4 significantly inhibit the replication of their helper viruses (barrett et al., 1973; timpe et al., 2006) . in contrast, hdv coinfection with hbv results in increased activity of both viruses, often resulting in a more severe clinical course than hbv alone (rizzetto, 2009 ). virus pseudotyping occurs when two species of virus coinfect a host cell and progeny virions are produced that contain nucleic acid of one parental virus and some structural proteins of the other parental virus. this phenomenon, also called phenotypic mixing, may involve capsid proteins, or in the case of enveloped viruses, only membrane glycoproteins (zavada, 1976) . various bacteriophage, plant viruses, and animal viruses have all been observed to produce phenotypically mixed progeny from coinfections (zavada, 1976) . some coinfections result in pseudotyped virions from both parental genomes, while other interactions result in pseudotyped virions of only one type (certo et al., 1998) . for example coinfection of human syncytiotrophoblasts with human cytomegalovirus and human t-cell leukemia-lymphoma virus (htlv-i) results in htlv-1 capsids within cmv envelopes, but not the reverse (toth et al., 1995) . pseudotyped viruses are distinct from helper-dependent virus interactions since neither virus requires components of the other to complete its replication cycle. envelope glycoproteins are the main receptor binding proteins of most enveloped viruses, and therefore pseudotyped virions often have an expanded host range, able to infect the targets of both parental viruses. two herpesviruses have been shown to produce pseudotyped human immunodeficiency virus (hiv) or htlv-1 virions when coinfected with these retroviruses (heng et al., 1994; toth et al., 1995) . in addition to a hybrid virion morphology, these pseudotyped retroviruses possess an expanded host cell range: herpes-pseudotyped hiv is capable of infecting keratinocytes (heng et al., 1994) . this feature of pseudotyped viruses has been used to advantage in the production of recombinant viral vectors, to expand the range of targets they are able to infect (funke et al., 2009; li et al., 2009; wu et al., 2009 ). a third type of direct vvi is viral superinfection exclusion, which occurs when a primary viral infection induces resistance to subsequent infections by similar viruses. superinfection exclusion is known to occur among bacteriophage, retroviruses, hepadnaviruses, arboviruses, and plant viruses (brindley et al., 2008; geib et al., 2003; mcallister and barrett, 1977; nethe et al., 2005; saumet and lecellier, 2006) . mechanisms of exclusion are diverse and have not been determined in all cases, but all mechanisms described so far depend on direct interaction of products of the primary infection with the secondary infecting virus. for example, the cytoplasmic accumulation of borna disease virus (bdv) nucleocapsid components prevents a subsequent infection of a different bdv strain or arbovirus type through interference with the polymerase of the secondary virus, inhibiting early viral multiplication steps (geib et al., 2003) . the sim protein of bacteriophage p1 appears to block injection of nucleic acid by superinfecting phage at the cell membrane (kliem and dreiseikelmann, 1989) , and the equine infectious anemia virus secretes a soluble protein that masks its cell surface receptor, blocking binding of subsequent viruses (brindley et al., 2008) . in plants, interaction of heterologous viral messenger rna molecules which contain sequence homology induces destruction of both messages by the rna silencing mechanism and inhibition of replication (saumet and lecellier, 2006) . although most examples of superinfection exclusion deal primarily with different strains of the same virus, and inhibition of superinfection is not absolute, better understanding of this vvi has the potential to impact future antiviral development (federico et al., 1996) . coinfections with two or more strains of the same virus species in the same host cell can result in progeny virions that are genetic recombinants of the parental viruses. this phenomenon is arguably the vvi with the most serious consequences for human health. influenza virus coinfections, by virtue of the virus's segmented genome, readily produce recombinant progeny (gardner and shortridge, 1979; nelson et al., 2008) . worldwide surveillance networks monitor the appearance of recombinant influenza viruses as the dramatic antigenic shift that results often produces a virus to which few people have immunity, increasing the risk of a pandemic (nuzzo and lam, 2006) . however, due to the relatively short course of influenza infections, coinfections are relatively uncommon (nelson et al., 2008) . human immunodeficiency virus (hiv) in contrast, is a life-long infection and both coinfections (secondary infection prior to seroconversion) and superinfections are well documented. hiv genomic recombination has been shown to facilitate immune escape (streeck et al., 2008) , evolution of replicationdefective hiv variants (iwabu et al., 2008) , and the spread of drug resistance (burke, 1997) , all of which complicate hiv control. life-long herpesvirus infections also result in homologous recombination between strains in vivo, with similar effects on immune control and drug resistance (chou, 1989; haberland et al., 1999; poole et al., 1999) . more recently, recombination between attenuated poliovirus vaccine strains and virulent wild enterovirus strains has led to regeneration of virulent polioviruses and cases of poliolike paralysis in regions targeted for poliovirus eradication (arita et al., 2005; rakoto-andrianarivelo et al., 2008) . although not as carefully monitored as human pathogens, recombination-driven altered virulence of viral pathogens of crop plants has also been reported (ogawa et al., 2008) . whereas genomic recombination involves nucleic acid transfer between viruses with significant sequence homology and similar if not identical genomic organization, embedded viruses are retroviruses that have integrated themselves into the genomes of unrelated, large dna viruses. presumably, integration is random but only those integrations that leave the dna virus capable of productive infection propagate and are detected. two examples, a moth retroviral element embedded into the genome of autographa californica nuclear polyhedrosis virus (friesen and nissen, 1990) , and reticuloendotheliosis virus embedded in the fowlpox genome (hertig et al., 1997) , illustrate that the effects of embedded viruses can be multifaceted. in both cases, the embedded retrovirus gains an alternative transmission and entry pathway to hosts, but in the case of the retrovirus embedded in the baculovirus, retroviral gene expression is also activated (friesen and nissen, 1990) . and for reasons not yet understood, the fowlpox strains carrying the embedded reticuloendotheliosis virus cause immunosuppression in chickens while reticuloendotheliosis virus-free strains do not (wang et al., 2006) . the final type of direct vvi for which we find documentation, involves transactivation of the genes of one virus species by gene products of a heterologous virus. many viruses encode powerful promoters and transactivating proteins in order to appropriate the cellular transcription machinery for maximum viral gene expression. direct binding and transactivation of a heterologous viral promoter is documented in the case of cytomegalovirus transactivating protein ie2-86 which binds to the −120 to 20 region of the hiv-1 long terminal repeat (yurochko et al., 1999) and in the case of human foamy virus bel1 protein which recognizes and binds to the −158 to −118 region of the hiv-1 long terminal repeat (lee et al., 1992) . epstein-barr virus (ebv) and hepatitis c virus (hcv) coinfection results in significantly higher hcv production than hcv infection alone. it is known that the ebv gene product responsible for enhanced hcv replication is the transcriptional activation protein ebna1 (ebv-encoded nuclear antigen-1) (sugawara et al., 1999) and it therefore seems likely that ebna-1 enhances hcv replication by direct transactivation, however, the targeted genes in hcv have not been identified. it is possible, however, that ebna1 activates hcv genes indirectly, as discussed in more detail below. post-transcriptional heterologous transactivation also takes place. herpes simplex virus protein us11 is an rna binding protein that controls post-transcriptional expression of herpes simplex genes. however, during coinfections it also binds and controls splicing of htlv-1 and hiv-1 transcripts normally controlled by the retroviral proteins rex and rev, respectively (diaz et al., 1996) . viral infections can cause many pathogenic changes in the host. often seen during dual infections is acceleration of disease, because of the compounded nature of the two viral cytopathic effects affecting the host in a negative manner. in this section, indirect vvi resulting from alterations in the host environment created by pre-existing or simultaneous coinfections are explored. five subtypes of indirect environmental vvi are currently recognized: indirect transactivation of genes, breakdown of host physical barriers against infection, altered receptor expression, heterologous activation of antiviral pro-drugs, and modification of the interferon-induced antiviral state. while direct binding and activation of viral transactivating proteins to heterologous viral promoters has been documented, more common are reports of viral infections inducing increased expression or activation of cellular transcription factors, which then act on promoters of coinfecting viruses. for example, hepatitis b virus xprotein transactivates promoters containing kappa-b like enhancer elements, both cellular and viral, but not by binding to the elements itself (twu et al., 1989) . human herpes virus 6 (hhv-6) infection of epstein-barr virus (ebv)-infected cells results in transactivation of the ebv zebra gene, but the transactivation appears to be mediated by cellular transcription factors on the ebv promoter (flamand and menezes, 1996) . the human endogenous retrovirus k (herv-k) ltr is transactivated by hsv-1 protein icp0 via increased binding of cellular transcription factor, ap-1 (kwun et al., 2002) . cmv ie1 protein also transactivates an ltr, the hiv ltr, but does so by inducing increased binding of nf-kappa b to the ltr sequence by an unknown mechanism (kim et al., 1996) . a vvi involving another ebv latency protein, lmp2a, illustrates the potential complexity of indirect virus-virus transactivation interactions. ebv infection, and subsequent lmp2a expression in human cells, results in the activation of a superantigen gene encoded by the long integrated and inactivated human endogenous retrovirus k18 (herv-k18) with serious clinical consequences (hsiao et al., 2006) . however, lmp2a appears to cause gene expression from the remnants of this ancient viral infection by binding to an enhancer 13 kb downstream and transactivating a cellular gene encoded on the opposite strand. transcription of the cellular gene most likely displaces repressors on the herv-k18 promoter sequences, resulting in transcription of its env superantigen gene (hsiao et al., 2009) . in summary, indirect heterologous transactivation is probably the most common vvi, with many interactions yet to be discovered. the diversity of mechanisms they represent and our present incomplete understanding of transcriptional control mechanisms increase the challenge of identifying such interactions. most have been investigated because a coinfection was observed to exacerbate a viral disease. there are potentially other cases of indirect heterologous transactivation that reduce pathology, but are more difficult to detect. viral replication and progeny production are often characterized by cytopathic effects. tissue damage that results can compromise physical barriers within the host, allowing secondary infections to gain access to otherwise protected tissues. this type of vvi has been observed in plants, specifically zucchini squash (cucurbita pepo). some cucumber mosaic virus strains infect zucchini squash plants but only cause localized infections. however, in plants coinfected with cucumber mosaic virus and zucchini yellow mosaic virus, the long distance movement of the cucumber mosaic virus is facilitated and systemic infection of both viruses is observed (choi et al., 2002) . this synergistic effect, which may also be mediated by viral movement proteins (melcher, 2000) , is readily observed by the overall deterioration of the plant as well as by molecular analysis (choi et al., 2002) . examples of this type of vvi also exist for animal viruses. humans infected with herpes simplex viruses 1 or 2 (hsv-1 or -2) have a higher susceptibility for acquisition of hiv, and a higher possibility of transmission of hiv to other persons sheffield et al., 2007) . both of these situations are associated with the ability of hsv-2 to cause open skin lesions and to recruit cd4+ t cells to the sites of these lesions (celum, 2004) . the recruitment of these cells makes more potential host cells available for acquisition of hiv in a herpesvirus lesion than are found in a traumatic lesion. in the case of someone already hiv infected, active hsv coinfection increases the probability of hiv transmission because infected cd4+ t cells are recruited to the open hsv lesion, increasing production of infectious virus at the skin surface (celum, 2004) . another example of barrier compromise allowing increased viral spread involves cytokine mediated tissue damage. atencio et al. showed that newborn balb/c and nih swiss mice coinfected with wild-type polyomavirus (a2 strain) and moloney murine leukemia retrovirus (m-mulv) exhibit growth inhibition and kidney inflammation, whereas either virus alone rarely produced these symptoms. coinfection was found to elevate cytokines il-6, ifn-␥, il-1␤ and il-10 early after infection (7 days) much more than single infections, and may be responsible for the kidney inflammation and runting (atencio et al., 1995) . the density of viral receptors on a prospective host cell is a significant factor in determining whether infection is successful (agnello et al., 1999; li et al., 1999) . human immunodeficiency virus, for example, binds to a complex of cd4 protein and either ccr5 or cxcl4 as its receptor, and it therefore almost exclusively infects human cd4+ t cells. coinfections have been shown to alter the cell types infected by hiv by altering expression of cd4 or the co-receptors ccr5 or cxcl4. human herpes virus 6 (hhv6) has multiple effects in this system. it upregulates cd4 expression on t cells that are already cd4+ increasing their susceptibility to the hiv virus, but it also induces expression of cd4 on the surface of cd8+ t cells making them susceptible to hiv infection as well (lusso et al., 1991) . in addition, hhv-6 coinfection boosts the production of the ccr5 ligand, rantes, which binds to ccr5 and inhibits the complex formation between ccr5 and cd4 needed for hiv to infect cells. exogenous rantes alone, can mimic this inhibitory effect of hhv6 on hiv infection, but it is only inhibitory to hiv strains that utilize ccr5 as a co-receptor, not cxcl4-tropic strains (grivel et al., 2001) . human herpes virus 7 (hhv7) infection also alters cell surface receptor expression in a manner protective against hiv. hhv7 is a t-lymphotrophic virus which also utilizes cd4 as a receptor, and competes directly with hiv for binding sites on host cells. in a host first infected by either hiv or hhv7, cd4 expression on t cells is down-regulated, slowing the spread of a subsequent infection by the other virus (lisco et al., 2007; lusso et al., 1994) . a fourth indirect mechanism by which vvi alter infection outcomes by affecting the host environment is the activation of pro-drugs with antiviral activity. many nucleoside analog antiviral drugs, such as acyclovir, gancyclovir, and famcyclovir, specifically target herpesvirus infected cells because they must be phosphorylated by herpesvirus-encoded kinases or phosphorylases before becoming active. once activated, the drugs can be incorporated into nascent herpesvirus genomes by viral polymerases, where they act as chain terminators, preventing replication. recently, it was shown that acyclovir can be activated by one virus and act on another . hiv, lacking a thymidine kinase is usually unaffected by acyclovir. however, in herpesvirus and hiv dual infected cells, acyclovir decreases the replication of hiv as well as the herpesvirus. acyclovir is phosphorylated by herpesvirus kinases and then moves to directly inhibit hiv reverse transcriptase, having an unintended but beneficial effect for the host . a fifth category of virus-induced change in the host environment that may affect coinfecting viruses involves the innate immune mechanism induced in vertebrates by type i interferons known as the antiviral state. the antiviral state consists of increased expression of a combination of enzymes, which if activated, shut down cellular translation (galligan et al., 2006; staeheli, 1990) . the most critical of these enzymes are pkr and 2 -5 oligoadenylate synthetase (2 -5 oas). protein kinase r (pkr) has multiple roles in a cell, but its role in the antiviral state is to phosphorylate eukaryotic translation initiation factor 2 alpha (eif2␣), inactivating it and shutting down protein synthesis (garcia et al., 2007) . the 2 -5 oas synthesizes unique oligonucleotides which activate rnasel, initiating destruction of cellular and viral rna molecules necessary for translation. activation of both enzymes is dependent on the presence of molecules associated with infection, particularly dsrna, and their activation usually results in cell death (staeheli, 1990) . animals with defective type i interferon signaling pathways, pkr, or 2 -5 oas are much more susceptible to viral infections, indicating the effectiveness of the antiviral state (levin and hahn, 1985) . however, most viruses have also evolved antagonists of pkr and or 2 -5 oas (hengel et al., 2005; langland et al., 2006; levy and garcia-sastre, 2001) . from this information it would seem logical to speculate that an antiviral state antagonist from one virus could benefit a coinfecting virus, and this has been shown to be the case in several in vitro systems. murine cytomegalovirus (mcmv) has two proteins that are known to inhibit pkr, m142 and m143. when these proteins are present and active the virus can readily replicate in the host. in the absence of these two proteins, the antiviral state is activated and mcmv replication is inhibited (budt et al., 2009 ). this antiviral state activation can be overcome by introducing the pkr inhibitors encoded by vaccinia virus (e3l) or herpes simplex virus (icp gamma 34.5) (budt et al., 2009) . a herpes simplex mutant lacking icp gamma 34.5, in turn can be rescued in cv-1 cells by coinfection with the polyomavirus sv40, due to the sv40 large t antigen's inhibitory effect on the antiviral state downstream of icp gamma 34.5 (randazzo et al., 1997) , or by inserting human cytomegalovirus genes for pkr antagonists, trs1 and irs1 (cassady, 2005; shah et al., 2007) . one possible natural example of this type of vvi involves the interaction of hepatitis b and hepatitis d viruses. the hepatitis d genomic rna molecule inhibits pkr-mediated inhibition of translation in a cell free translation system, suggesting that it may protect its helper virus, hbv, from the antiviral state (robertson et al., 1996) . although the known examples of this virus-virus interaction are so far only demonstrated in artificial systems, given the multiple in vitro examples it seems likely that this type of vvi also occurs in nature. as the third main category of vvi, we define a subset of indirect virus-virus interactions that occur only in host species with an adaptive immune system. we set these types of interactions apart, because unlike the other indirect vvi, which are dependent on an overlap of the periods of infection of two viruses, immunological interactions can occur between viral infections that are completely separated in time. this is possible because the adaptive immune system of the host organism is permanently changed by its interaction with a virus, even if that infection is completely eliminated from the host, and is changed in a manner very specific to the species and strain of the infecting virus. at present, four types of immunological vvi have been identified. these include altering the activation state of cellular components of the immune system, and induction of autoimmune responses to self-antigens that cross-react with viral antigens. in addition, the humoral immune response to viral pathogens can unexpectedly give rise to antibodydependent enhancement (ade) of subsequent viral infections. and finally, coinfections, as well as sequential infections, also indirectly interact by re-shaping the t cell memory repertoire such that the immune response induced by one infection can impact the outcome of a subsequent viral infection in an interaction termed heterologous immunity (welsh and selin, 2002) . one means by which a virus may sensitize a host for a subsequent infection is by altering the activation state of potential host cells. hiv infection, for example, is associated with human cytomegalovirus (hcmv) infection in part due to hiv's induction of elevated numbers of activated lymphocytes in certain tissues. since activated lymphocytes are permissive for hcmv infection, this hiv-induced activated cellular state results in a two-to threefold enhancement of hcmv replication in these tissues . another example is seen with lactate dehydrogenaseelevating virus (ldv). ldv stimulates polyclonal b lymphocyte activation, resulting in delayed induction of antibodies needed to control other coinfecting viruses. consequently, friend virus (fv) infection, which is normally asymptomatic in mice due to timely production of neutralizing antibodies, will upon coinfection with ldv propagate and cause symptomatic disease (marques et al., 2008) . the prevalence of viral coinfections may also impact the progression of hiv disease in this manner. specifically, it is not unusual for hiv positive patients to be coinfected with gb virus c (gbv-c) and the persistence of certain genotypes of this virus has been shown to lead to slower hiv progression. (schwarze-zander et al., 2006) . this positive effect for the host appears to be mediated by elevated expression of interferon gamma and the immune cell activation that results. higher viral titers of gb virus c are directly correlated with increased serum interferon gamma, which in turn results in increased numbers of circulating mature dendritic cells that may be controlling the hiv infection (lalle et al., 2008) . sequential viral infections have also been associated with generation of autoimmunity in the host. some viruses are able to break immunological tolerance to "self" by expressing a self-like epitope, but are unable to generate the numbers of autoreactive t cells necessary to trigger an autoimmune response. however, a second infection can, by expanding the autoreactive t cell compartment, cause autoimmunity (welsh and fujinami, 2007) . mice transgenic for a lymphocytic choriomeningitis virus (lcmv) nuclear protein remain healthy when infected with lcmv, but a subsequent infection with either poliovirus or vaccinia virus leads to the development of pancreatic inflammation and autoimmune diabetes (christen et al., 2004; evans et al., 1996) . another example of this type of vvi is associated with enteroviruses such as poliovirus. enterovirus infections have been shown to be a risk factor for several autoimmune diseases including insulin-dependent diabetes mellitus (iddm) (dahlquist et al., 1995; grist et al., 1978; hiltunen et al., 1997; hyoty et al., 1995) . multiple epidemiological studies in humans have established that children that manifest iddm have had more exposures to enteroviruses than healthy subjects (andreoletti et al., 1998; clements et al., 1995; d'alessio, 1992) . interestingly, in countries where a live attenuated polio vaccine which is known to confer cross-protection to enteroviruses (juhela et al., 1998) is used to immunize children, the incidence of iddm is lower than in countries where a killed vaccine that does not induce cross-protection is used. this suggests an association between cross-reactivity of immune responses to different viruses and the development of autoimmune diseases (juhela et al., 1999) . in order to infect animal cells, virus particles usually must bind directly to a specific cell surface molecule in a virus-specific manner (flint et al., 2004) . however, several families of virus are known to take advantage of indirectly binding to the cell surface via crosslinking with antiviral antibodies or virus activated complement components which then bind to host cells bearing fc or complement receptors (takada and kawaoka, 2003) . this process in which increased viral replication is produced by exposure to immune sera, is known as antibody-dependent enhancement (ade) of viral infection. it has been observed in vitro for flaviviruses, coronaviruses and retroviruses (cummings et al., 2005) . mechanisms underlying ade are not fully understood, but seem to include increased efficiency of virus binding to host cells, resulting in higher numbers of infected cells. the most well studied case of ade in humans is dengue hemorrhagic fever (dhf). interestingly, the severe form of dengue illness, which is often fatal, is strongly associated with preexisting heterotypic immunity kliks et al., 1988; sangkawibha et al., 1984) . the presence of non-neutralizing antibodies from a previous dengue infection augment viral growth in vitro (kliks et al., 1989) and in vivo (halstead, 1979) . also, individuals suffering from a secondary dengue virus infection have higher viremia than those with primary infections (vaughn et al., 2000) , and the presence of anti-dengue antibodies in mothers has been associated with the occurrence of dengue hemorrhagic fever in newborns (kliks et al., 1988) . the phenomenon of ade is not unique to dengue virus infections. it has been demonstrated to play a role in west nile virus infections in vitro (peiris and porterfield, 1979) and yellow fever virus in vivo (barrett and gould, 1986) . moreover, infections by two other members of the flavivirus family, the yellow fever virus and the japanese encephalitis virus, have also been shown to be enhanced by ade (gould and buckley, 1989) . this process is also thought to be responsible for the enhanced pathogenicity of viral challenges after vaccination with certain formalin inactivated viral vaccines (porter et al., 1972) , including ones for measles (iankov et al., 2006) , respiratory syncytial virus (ponnuraj et al., 2003) , and rabies (prabhakar and nathanson, 1981) . heterologous immunity gives rise to virus-virus interactions when the outcome of the adaptive immune response to a new viral infection is determined in part by immune memory acquired by the host from prior viral infections. development of a primary adaptive immune response to a pathogen results in an immunological memory, which consists of expanded numbers of long-lived, circulating t and b lymphocytes recognizing epitopes of that specific pathogen (welsh et al., 2004) . due to randomized dna rearrangement processes during the generation of the unique specificities of adaptive immune cells, even genetically identical twins have unique t cell repertoires in their naïve state and therefore show some differences in their responsiveness to the same pathogen. notably, these variations seem to have limited significance for the effectiveness of the immune response to a new infection (welsh et al., 2006) . however, if even a small subset of a t cell memory pool is cross-reactive with antigens of a later encountered pathogen, it will outcompete newly activated t cell clones and dominate that response. that the degree of heterogeneity in the immunological responses between genetically identical hosts could be dramatically influenced in different directions by the history of infections with seemingly unrelated viruses was long unappreciated (welsh and fujinami, 2007; welsh et al., 2006) . however, studies in syngeneic mice have confirmed that the unique identity of memory t cells, raised towards one virus but later activated during an infection with an unrelated heterologous virus, dramatically influence the outcome of the second infection (selin et al., 1996) . this heterologous immunity can result in both beneficial and harmful effects (chen et al., 2001) . in mice, for example, immunity to influenza virus protects the host against vaccinia virus challenge, but enhances the virulence of subsequent cytomegalovirus infections (chen et al., 2003) . while difficult to study in outbred populations, syngeneic animal models allow investigation of immunological memory after heterologous infections. when the diverse viruses lcmv, poliovirus, vaccinia virus, murine cytomegalovirus, and vesicular stomatitis virus were sequentially introduced into syngeneic hosts, the immunological memory against one infection was dramatically altered after each successive infection (selin et al., 1996) . thus, the course of each infection is influenced by the t cell memory pool, and with each infection, the t cell memory to previous encountered agents is modified (welsh and selin, 2002) . the effect of heterologous immunity is seen between many different viruses and disease outcome is dependent on both the nature and the specific order in which the sequentially encountered pathogens were encountered (chen et al., 2003) . although relatively unexplored as a field of study, vvi have already been documented to have significant and unexpected effects on viral disease severity, host range, transmissability, immunopathology, and vaccine effectiveness. increased awareness of the potential for virus-virus interactions and a framework for categorizing different types of interactions as described here, would seem to be necessary steps for achieving better understanding of infectious viral diseases in nature. it has long been noted that many viral infections result in mild disease for most infected individuals, moderate disease for some, and fatal disease for a few. the epidemiology of poliomyelitis, influenza, and the recent west nile virus outbreak in the united states are prime examples of this phenomenon. when occurring in otherwise healthy individuals, these differences in susceptibility have largely been assumed to be governed by cryptic immunological defects, either inherited or acquired (kacprzak-bergman and nowakowska, 2005; trammell and toth, 2008) . while this is likely true in some cases, it is also plausible that some variations in susceptibility are determined by virus-virus interactions. evidence for this is strong in the intensively studied case of hiv/aids (lisco et al., 2009 ). investigation of similar interactions for acute viral infections will be challenging, but may allow better identification and protection of the most vulnerable populations during disease outbreaks. it was not possible, of course, to mention every known virus-virus interaction in this article. rather, the goal was to provide a framework into which all vvi can be organized. an online database has been established (www.musc.edu/vvi/) with the aim of compiling a referenced, searchable, comprehensive list of vvi, actively updated by contributions from the scientific community via moderated forum. we anticipate that the number of vvi subtypes may expand, as during the course of this investigation hints of interactions that seem plausible but are not yet documented were found; for example, stabilization of viral rnas by heterologous viral rna binding proteins. in addition, several cases of vvi described above were discovered simultaneously with a new virus, whose existence was not previously suspected. as a wobble in a star's rotation can indicate the presence of an unseen planet, so the perturbation of a viral replication cycle may indicate the presence of an unknown infection, and a virus-virus interaction. curated list of prokaryotic viruses with fully sequenced genomes hepatitis c virus and 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cord-278839-uu2wlpmp authors: alberca, ricardo wesley; pereira, nátalli zanete; oliveira, luanda mara da silva; gozzi-silva, sarah cristina; sato, maria notomi title: pregnancy, viral infection, and covid-19 date: 2020-07-07 journal: front immunol doi: 10.3389/fimmu.2020.01672 sha: doc_id: 278839 cord_uid: uu2wlpmp pregnancy comprises a unique immunological condition, to allow fetal development and to protect the host from pathogenic infections. viral infections during pregnancy can disrupt immunological tolerance and may generate deleterious effects on the fetus. despite these possible links between pregnancy and infection-induced morbidity, it is unclear how pregnancy interferes with maternal response to some viral pathogens. in this context, the novel coronavirus (sars-cov-2) can induce the coronavirus diseases-2019 (covid-19) in pregnant women. the potential risk of vertical transmission is unclear, babies born from covid-19-positive mothers seems to have no serious clinical symptoms, the possible mechanisms are discussed, which highlights that checking the children's outcome and more research is warranted. in this review, we investigate the reports concerning viral infections and covid-19 during pregnancy, to establish a correlation and possible implications of covid-19 during pregnancy and neonatal's health. pregnancy comprises a unique immunological condition, to allow fetal development and to protect the host from pathogenic infections. viral infections during pregnancy can disrupt immunological tolerance and may generate deleterious effects on the fetus. despite these possible links between pregnancy and infection-induced morbidity, it is unclear how pregnancy interferes with maternal response to some viral pathogens. in this context, the novel coronavirus (sars-cov-2) can induce the coronavirus diseases-2019 (covid19) in pregnant women. the potential risk of vertical transmission is unclear, babies born from covid-19-positive mothers seems to have no serious clinical symptoms, the possible mechanisms are discussed, which highlights that checking the children's outcome and more research is warranted. in this review, we investigate the reports concerning viral infections and covid-19 during pregnancy, to establish a correlation and possible implications of covid-19 during pregnancy and neonatal's health. keywords: covid-19, sars-cov-2, pregnancy, neonatal, immunology pregnancy pregnancy comprises a unique immunological condition, to protect the fetus from maternal rejection, allowing adequate fetal development and protection against microorganisms (1, 2) . the maternal immune system is challenged by paternal alloantigens expressed both by the fetus and the placenta. however, through a complex range of cells and molecules, the mother does not develop a classic response to this allograft (3) . during pregnancy, fetal microquimerism occurs, where fetal cells, such as nucleated erythrocytes, trophoblastic cells, and leukocytes (3), cross the placental barrier and expose the mother to fetal alloantigens. these cells can remain in the bloodstream and maternal tissues many years after delivery (4, 5) . in comparison to the post-partum period, pregnancy increases monocytes, granulocytes, pdcs, mdcs in the blood, peaking during 2 trimesters. simultaneously, during pregnancy occurs a reduction in cd3, cd4, and cd8 t cells in comparison with post-partum. b cells are decreased during the third trimesters. nk cells cd56 dim are reduced in the second and third trimester of pregnancy in comparison with the first trimester and post-partum period. during the second and the third trimesters, nk and cd4 t cells present a reduction in the production of ifn-γ, tnf, il-6 cells, compared with post-partum (6) but the variability and contradictory reports are noted (7) . maternal monocytes do not show differences in absolute numbers, however, they show some phenotypic changes including an increase in the expression of adhesion molecules (cd11a, b; cd54), and the high-affinity igg receptor, fcγr-i (cd64) (8) . the absolute number of nk cells in maternal blood increases in the first trimester of pregnancy (9) . like lymphocytes, b cells are decreased during pregnancy and remain lower until 1 month after delivery. in vitro, b cells of pregnant women were less responsive, with suppression of lymphopoiesis and exclusion of autoreactive b cells (10). despite this, vaccine response during pregnancy remains effective (11, 12) . from the 13th week of gestation, maternal peripheral blood monocytes also undergo phenotypic and functional changes. there is an increase in the ability to produce cytokines il-1β and il-12 and a reduction in the potential for tnf-α secretion (13) . the placenta is a transient chimeric organ that develops from the uterine wall and can express different receptors and dynamically delivered microvesicles through pregnancy (14) . this organ mediates hormonal, nutritional, and oxygen support to the fetus while modulating maternal's immune response (15) . the placental maternal face is formed from decidual cells, with the presence of wide range of immune cells, including uterine natural killer (unk), dendritic cells (dcs), and regulatory t cells (tregs). the fetal face consists of the placental villus, which contains fetal blood vessels surrounded by fibroblasts and placental villous macrophages of fetal origin, hofbauer cells (16, 17) . treg cells are crucial for proper gestational development and are numerically elevated during pregnancy, in peripheral, deciduous and umbilical cord blood (18) . paternal hla-c is a crucial molecule that can elicit allogeneic immune responses by maternal cell and aid in the development of maternal-fetal tolerance (19) , also t reg may regulate cd4 + and cd8 + t lymphocyte activation through the expression of il-10 and tgfβ (20) . another striking feature of the maternal-fetal interface is the accumulation of nk cells, which comprise up to 70% of deciduous leukocytes in early pregnancy (21) . these cells are important for the regulation of cytokines production, especially il-10, and act in the production of angiogenic factors, chemokines, controlling the invasion of trophoblasts and availability of adequate maternal blood at the implantation site (17, 21, 22) . during pregnancy, hormonal variations can modulate immune responses, generating a reduction in the number of dcs and monocytes, and a decrease in the activation of macrophages, t, and b cells (23) . to better establish the tolerogenic milieu, estrogen induces efficiently foxp3 t regs cells (24) (25) (26) . changes in hormonal levels and immune system function generated by pregnancy may increase women's vulnerability to infections. pregnant women show higher mortality rates and complications associated with viral infections compared to the general population (27, 28) . for example, varicella disease in children is mild, but primary infections during pregnancy can progress to varicella pneumonia and death (29) . in 2009, during the h1n1 flu pandemic, an increased ratio of female to male cases was verified, in which pregnant women developed more complications, as severe acute respiratory syndrome, and higher mortality compared to the general population (30, 31) . similarly, in 1918 the pandemic spanish flu, among 1,350 reported cases of influenza in pregnant women, 27% died as a result of the infection (32) . in 1957, with the h5n1 pandemic, 50% of influenza deaths in women of reproductive age in minnesota occurred in pregnant women (33) . although influenza viruses are restricted to maternal lungs, inflammatory cytokines can lead to fetal complications mainly preterm birth and fetus miscarriage (34, 35) . in the ebola epidemic in 1995, 46% of infected women (out of a total of 177) were pregnant (36) . some evidence suggests that during pregnancy there is a greater risk of developing serious illnesses, spontaneous abortion, hemorrhage, and death when infected with the ebola virus (37) . additionally, infection by the lassa virus in pregnant women shows high levels of placental replication, and the risk of maternal-fetal mortality increases with the duration of pregnancy (38, 39) . viruses can gain access to the decidua and placenta by ascending from the lower reproductive tract or via hematogenous transmission, viral tropism for the decidua and placenta is then dependent on viral entry receptor expression in these tissues as well as on the maternal immune response to the virus (16) . a range of viral infections in pregnancy are associated with specific placental findings, including lymphoplasmacytic villitis with associated enlargement of villi and intravillous hemosiderin deposition in the setting of maternal cytomegalovirus infection (40) , as well as rare reports of intervillositis in the setting of zika virus (41) and dengue virus (42), among others. although there is little knowledge about placental findings associated with the common coronaviruses, ng et al. reported placental pathology in seven women with sars infection in hong kong (43) . in three placentas delivered in the acute stage of sars, demonstrated increased perivillous or subchorionic fibrin, while in two women who had recovered from third-trimester infection by the time of delivery, there were large zones of avascular villi, with one of the two additionally demonstrating a large villous infarct; both contained increased nucleated red blood cells in the fetal circulation. none of the seven placentas examined had any acute or chronic inflammatory processes (43) . the covid-19 pandemic is still in its early stages, with preliminary case series of infection in pregnant women available. a study of three placentas delivered from pregnant women with sars-cov-2 infection, infected in their third trimester with emergency cesarean section, describe various degrees of fibrin deposition. the fibrin deposition occurred inside and around the villi with local syncytial nodule increases in all three placentas, multiple villous infarcts in one placenta, and a chorangioma in another case. all samples from three placentas were negative for the nucleic acid of sars-cov-2 (44) . another study with 16 placentas from patients with sars-cov-2 were examined and the most significant finding is an increase in the rate of features of maternal vascular malperfusion (mvm), most prominently decidual arteriopathy including atherosis, fibrinoid necrosis, and mural hypertrophy of membrane arterioles (45). maternal hypertensive disorders, including gestational hypertension and preeclampsia, are the major risk factors for mvm (46) , although only 1 of the patients was hypertensive in this study. notwithstanding, sars-cov-2 is a virus that is expected to induce inflammation, it is relevant that neither acute inflammatory pathology (aip) nor chronic inflammatory pathology (cip) were increased in covid-19 patients relative to the controls. however, none of the covid-19 patients in this study were severely ill or undergoing a cytokine storm and it may be possible that cip could be induced in those cases of severe systemic inflammation (45). there few knowledges about miscarriage in women with covid-19, one case was a pregnant woman with symptomatic coronavirus disease who experienced a second-trimester miscarriage. a stillborn infant was delivered vaginally and swabs from the axillae, mouth, meconium, and fetal blood obtained within minutes of birth tested negative for sars-cov-2 and bacterial infection. the fetal autopsy showed no malformations, and fetal lung, liver, and thymus biopsies were negative for sars-cov-2. furthermore, amniotic fluid and vaginal swabs sampled during labor tested negative for sars-cov-2 and bacterial infection. placental histology demonstrated mixed inflammatory infiltrates composed of neutrophils and monocytes in the subchorial space and unspecific increased intervillous fibrin deposition (47) . during the worldwide sars-cov-1 (severe acute respiratory syndrome coronavirus-1) epidemic in 2003, a notable increase in mortality and morbidity was documented in pregnant patients (48) . agreeing with previous observations that the risk of viral pneumonia is significantly higher among pregnant women compared to the rest of the population (49) . in 2012, infection with the middle east respiratory syndrome (mers-cov) coronavirus in saudi arabia after the isolation of a male patient who died of severe pneumonia (50, 51) . data on the effects of mers-cov on pregnancy are limited, whereas there is a description of stillbirth at 5 months of gestation (52) . between 2012 and 2016, the ministry of health of saudi arabia reported the occurrence of 1,308 cases of mers-cov infection, five of which were pregnant (53) . despite the few descriptions, the immunological changes in pregnancy may alter the susceptibility to mers-cov and the severity of the clinical disease (51) . in a mice model of herpes virus infection, even in the absence of herpes virus placental passage, there was a marked increase in the levels of pro-inflammatory cytokines, including ifn-γ and tnf-α, as well as changes in fetal development (30) . this scenario may result from the placenta's pro-inflammatory response generated by the infection, or it may be due to other physiological changes in the mother or placenta related to the infectious process (54) . placental cells, predominantly trophoblasts, express tlr (toll-like receptors) and this expression varies according to the gestational age and the differentiation stage of these cells. viral infections can disturb the fine immune regulation at the maternal-fetal interface and lead to fetal damage, even without the virus reaching it directly (55) . for example, tlr-3 expressed by trophoblasts in the first trimester of pregnancy (56) , mediates rapid antiviral response (57) , and induces the production of cytokines, type i interferon (ifn) and type iii ifn (58) . tlr7 is also expressed in trophoblasts, which induces the synthesis of anti-viral cytokines and plays a role in preventing intrauterine transmission of hbv (59) . however, these inflammatory responses can be associated with complications in pregnancy, such as pre-eclampsia and/or intrauterine growth deficit (1) . in general, cytokines and ifns are important mediators in a healthy pregnancy, due to their role in the regulation of cell function, proliferation, and gene expression. however, when dysregulated, they have the potential to interrupt fetal and placental development pathways (60). the world health organization (who) estimates that about 2.5 million children died within the first month of life in 2018. every day ∼7,000 newborns die, amounting to 47% of all child mortality under the age of 5 years (61) . the majority of all neonatal's deaths are due to preterm birth, intrapartum-related complications (birth asphyxia or lack of breathing at birth), infections and birth defects. regarding the highest incidence of infection observed in early-life, it is generally attributed to an immature immune system during the transitional post-natal period (62) . innate immune cells are composed of specialized cells, such as granulocytes (e.g., neutrophil), monocytes, macrophages, dcs and innate lymphocytes. around 5 weeks gestation, neutrophils are present in human fetal liver parenchyma (63) , when compared to the adult response, neonatal neutrophils have qualitative and quantitative impairments in the response under stress conditions, including reduced chemotaxis, respiratory burst, and extracellular traps formation (64) . the cytokine profile produced by antigen-presenting cells (apcs) monocyte/macrophage and dcs in newborn differs from those produced by adults. typically, apcs from neonates produce less pro-inflammatory cytokines like il-1β, tnf-α, il-12p70, and type i ifn upon stimulation on tlrs (65) . otherwise, it produces great amounts of th17-promoting cytokines (il-6 and il-23) when compared with adult cells (66) . following, the importance of anti-inflammatory response in early life is highlighted through the great amount of il-10 produced by newborn monocyte/conventional dc (cdc) compared to adults (67) . the pattern of innate cytokine response can be attributed to two mechanisms: (i) high mononuclear cell levels of intracellular cyclic adenosine monophosphate (camp), a secondary messenger that suppresses th1 but enhances th2 and anti-inflammatory cytokine production (68) and (ii) altered dna binding capacity of transcription factors, such as irf3 to the promoter regions of cytokine genes secondary to age-specific chromatin (69) . curiously, neonates' dcs activation with clr agonist dectin or macrophage-inducible c-type lectin (mincle), simultaneously with tlr7/8 potently drives caspase-1 and nf-kb activation and th1-supporting cytokine production (including il-12p70), overcoming the age-specific epigenetic barrier in early life for irf3 function and leading to a th-1 phenotype (70, 71) . on 14 weeks of gestation, mature fetal αβ t lymphocytes can be detected. during the second and third trimesters of gestation, the repertoire of fetal t cell receptors diversifies (72) . generally, neonates have a limited th1 profile response to some vaccines and pathogens, agreeing with a lower capacity of cd4 t cells to produce ifn-γ and of apcs to produce th1-skewing cytokines (73) . although there are some situations where the responsiveness of the th1 profile is efficient, for example, neonates and infants develop adult-like th1 responses to bcg or pertussis vaccines, and a fetus can develop th1 responses in congenital cmv infection (74) (75) (76) . recent studies suggested that the early life immune system could present advantages for the elicitation of broadly neutralizing antibodies (bnabs), a response highly desired for an hiv vaccine. in fact, hiv-infected children develop bnabs responses earlier and more frequently than infected adults (77) . congenital and perinatally acquired viral infections do occur and may lead to major disabilities in infancy and childhood, the main causes can be attributed to pathogens like toxoplasma gondii, rubella virus, cytomegalovirus (cmv), herpes viruses, syphilis, and zika virus (78) . while congenital rubella virus syndrome is no longer seen in countries with compulsory immunization against this virus, an outbreak of zika virus (zikv) recently occurred in brazil resulting in the zikv syndrome, with brain lesions comparable to, but more severe than congenital cmv infection (79) . neonates display an immature immune response, the first exposition to an environmental stimulus can shape the lung's immune response (80) . furthermore, there is a predominant type 2 immune response in the lungs (81), these characteristics make infants susceptible to respiratory viral infections, a common cause of infant's death (82) . rsv is an important cause of lower respiratory tract illness in infants globally and is responsible for one-third of deaths due to lower respiratory tract infections in children <1 year of age (83) . pregnant women are considered at high risk for severe influenza disease, for this reason, influenza vaccination has been recommended for pregnant women and introduced into immunization programs (84) . influenza vaccination is safe and protective on preterm birth (ptb) and low birth weight (lbw) (85) . one of the benefits of maternal immunization has also been shown to extend to neonates through the transfer of maternal antibodies, providing passive immunization against the influenza virus (86). on the severe 2009 pandemic h1n1 influenza illness, some studies suggested an association between severe h1n1 disease, preterm birth, and fetal death; however, these limited data do not permit firm conclusions (35) . sars-cov-1 infected ∼100 pregnant women during the pandemic (87), causing a high lethality and miscarriage rate (88) , but no neonatal infection has been reported (88) . in 2017, cynthia maxwell postulated possible intensive care and procedures to properly manage maternal and neonatal sars-cov-1 infections (89) . vertical transmission of mers has not been documented. in a case report by alserehi et al., a mother was diagnosed with mers, treated and a cesarean section was performed to deliver a healthy preterm baby with 32 weeks of gestation (52) . hon et al. described 14 children with mers, that presented persistent fever and cough, after treatment no fatal case was reported. all children in this report obtained the infection via adult-to-children transmission, and no children-tochildren transmission was reported (90) . iqbal et al. reported a case of spontaneous vaginal delivery in covid-19-positive pregnant, with no signs of neonatal infection up to 7-days post-partum (91) . nevertheless, it is important to highlight contact precautions were made in this report to prevent post-partum transmission. in late 2019, a respiratory infectious disease began to be investigated in wuhan, china (92) . at first, contagion occurred through contact with some infected animals but, soon there were the first reports of human-to-human transmission (93), the virus was identified as belonging to the coronaviridae family and was designated sars-cov-2 (severe acute respiratory syndrome coronavirus-2) (94). like other members from this viral family, mers and sars-cov-1, the new coronavirus causes a respiratory disease, named covid-19 (coronavirus disease−2019) (95) . although very similar, sars-cov-1 and sars-cov-2 impacted the world differently. sars-cov-1 emerged in 2002 and killed almost 800 people in 26 countries (96) and, even without a vaccine, it was taken preventive actions as patient isolation. the new coronavirus has killed more than 480,000 people in just 6 months and has spread to 5 continent (97). sars-cov-2 shares genetic similarities between sars-cov-1 and mers, 79 and 50%, respectively (98) . sars-cov-2 is an enveloped single-stranded rna virus and has a genome of ∼30,000 nucleotides that encode structural and accessory proteins-the largest known viral rna genome (99) . sars-cov-1 and sars-cov-2 enter the host's cells via the ace2 receptor (angiotensin-converting enzyme 2) (100). in the lung, the most affected organ among those infected, the main target is the type 2 alveolar cell (101) . the ace2 receptor is also expressed in cells from kidneys, esophagus, heart (102) . moreover, a small percentage of monocytes and macrophages express the ace2 receptor (94, 99) . thus, there may be another alternative receptor or infectious pathway, such as antibody-dependent enhancement (ade). however, unlike other coronaviruses, limited to respiratory disorders, sars-cov-2 caused multiple organ failure. furthermore, this receptor is more expressed in the elderly, which associated with immunosenescence and other comorbidities common among the elderly may justify the high lethality rate in this age group (103) . the viral load peaks occur during the first week of infection and then gradually decrease over the next few days. in addition, the viral load is correlated with the patient's age. igg and igm antibodies start to increase 10 days after disease and most patients are seroconverted in the first 20 days (104) . moreover, in vitro assays, has shown that the serum from sars-cov-2-infected patients were able to neutralize the virus (101) . thereby, the humoral response can be another antiviral strategy via plasma transfer (105) . in sars-cov-1 and mers, as a viral escape mechanism, the virus can suppress ifn type i response, either by cytosolic sensors of ubiquitination, inhibiting nuclear factors translocation or decreasing stat1 phosphorylation (106) . neutrophils, c-reactive protein and several cytokines (as il-6, tnf, il-10) are increased in covid-19, and this elevation is correlated with disease severity and death (97) . in serious illness, the same protein levels were detected and inflammatory cytokines increase is correlated with t cd4 + and t cd8 + lymphocytes decrease and lower ifnγ production. b-lymphocytes do not appear to be affected by the disease, regardless of severity (92, 103, 107) . these characteristics observed in patients indicate that a covid-19 can be mediated by an intense inflammatory process that follows the disease severity. as with sars-cov-1 and mers, this increase in cytokine levels-known as a cytokine storm-can be involved with the pathogenesis of the disease (92). to defend itself against an aggressive agent (such as infection, trauma, acute inflammation, among others) the body produces an exaggerated response to localize and then eliminate the damage. this response is known as the systemic inflammatory response syndrome (sirs) or, if the source infection sepsis (108) , this process leads to the release of acute-phase proteins and endocrine, hematological and immunological changes, among them, the cytokine storm can lead to tissue damage and even death (109) . cytokine storm is produced, mainly, by highly activated macrophages and can cause lung damage and start viral sepsis (110) . this inflammation leads to other complications, such as acute respiratory distress syndrome (ards) and respiratory and cardiac failure (48, 111) . studies in mice infected with sars-cov-1, also demonstrate the cytokine storm dampening adaptive immunity (112) . other factors may also influence the susceptibility for covid-19 infected persons, and some gene polymorphisms, well-documented for other viral infections (113) . at the moment no vaccine or specific treatments are available for disease control of the sars-cov-2. in pregnancy, pneumonia infections may trigger an increased mortality risk to the mother and fetus (114) , which can also lead to complications as preterm birth and small for gestational age (115) . placental syncytiotrophoblast cells express the ace2 receptor and this receptor is highly expressed in the first months of pregnancy. associated with placental immaturity, the early ace2 expression can make the first trimester the most likely period for sars-cov-2-infection (14) . a serine protease, tmprss2, is also required for viral entry (100, 116) and there is still no consensus about placenta expression. some studies report low, but present, mrna expression in human placentas (117) , others describe that expression is not detectable (118) . the association of tmprss2 and ace2 expression, in the first months of pregnancy, would make this phase more susceptible to sars-cov-2-infection. blood tests in pregnant women revealed regular covid-19 markers, such as lymphopenia, neutrophilia, and elevated c-reactive protein level in pregnant women (119, 120) . some reports also verified an increase in alt, ast, and d-dimer (120) (121) (122) ). an important report verified that 3 mothers developed anemia and dyspnea, which could potentially be a risk factor during c-section labor (123) . chen and collaborators, verified alteration in calcium and albumin levels in the blood of pregnant women with sars-cov-2 infection (124) , which could potentially increase the severity in covid-19 (125) . furthermore, in a recent report involving maternal death in consequence to covid-19, 2 cases reported a low number of platelets, which is associated with an increase in mortality by covid-19 (126, 127) . it is still under investigation the effects of sars-cov-2infection in the maternal-fetal context ( table 1) . some reports describe that symptomatic infected-mothers did not transmit the virus during pregnancy. in a case report of seven cases, showed that three babies were tested to sars-cov-2 and only 1 was positive 36 h post-partum (138) . on the other hand, another report shows increase in inflammatory cytokines and virus-specific igm levels in newborns, from infected-mothers, 2 h after birth (120) , and in another report, newborns presented virus-specific igm and igg, but no sars-cov-2-infection ( table 1 ) (128) . this lead to the possibility of the activation of the maternal immune system by sars-cov-2 may have some implication of the offspring's health and immune system development. although the number of pregnant women with covid-19 studies is limited, there is no conclusive report of vertical transmission ( table 1 ) (129, 139) . a recent case report, was described two cases of rashes and one with facial ulcerations (123) . another important factor, besides the immune activation, the maternal usage of antiviral drugs can also permanently affect the offspring's immune response (140) , as there is no current standard protocol of treatment regarding the usage of antibiotics or antivirals ( table 1 ) (115) . only a fraction of patients infected with sars-cov-2 develops severe respiratory disorders, it is unknown whether the pregnant could be more susceptible to pulmonary diseases. covid-19 can progress to a severe lung inflammation that can progress to life-threatening illness at the severe stage (141) . this inflammatory process is associated with high plasma levels of cytokines, as cytokines storm, including il-2, il-7, il-10, g-csf, ip-10, mcp-1, mip-1a, and tnfα (92) . this might play an important role in pregnancy as il-2 has been implicated to be upregulated in pre-eclampsia (142) and miscarriage (143) and il-7/il-7r signaling pathway in fetal miscarriage (144) , due to the upregulation in the ratio of th17/treg cells (145) . another relevant aspect is the possible implication of polymorphisms in covid-19 diseases, as is well-documented for other viral infections (114) . also, cytokines polymorphisms, such as tnf-α 308g/a (rs1800629) polymorphism is associated with recurrent miscarriage (146) . in fact, tnf-α and tnf-α receptor play an important role in the development of the fetus, being present in the ovary, endometrium, placenta, and fetus, and in the amniotic fluid in different concentration (147) . this increase in tnf-α during pregnancy may implicate in different health outcomes depending on the gestational period (148) , leading to tissue necrosis in the placenta and hypoxia (149) . interestingly, an acute increase of this cytokine during pregnancy in animals may cause abortion (7) . moreover, alteration in the health status of the mother during pregnancy can have long-term effects on the offspring's health (150) . inflammatory processes during pregnancy can also impact women's health, as the increase in tnf-a during pregnancy can also lead to impaired insulin sensitivity (151) and gestational diabetes mellitus (152) . in animal models, inflammation during pregnancy has been shown to alterations in the behavior (153, 154) fetal brain development (155) (156) (157) , metabolic disturbance (158, 159) , and shape offspring's immune response to antigens and infections (160, 161) . the physiological response, as stress and the control of temperature, during the infection may present a long-term effect in pregnant women with covid-19. the increase in stressrelated hormones can also affect the offspring's immune system (162) and fever during pregnancy increase the chances of neural disorders in the children (163) . moreover, an increase in anti-inflammatory cytokine il-10 in covid-19 mothers is probably a regulatory mechanism crucial to regulate the inflammation (164) and pregnancy maintenance (165). even though no vertical transmission for covid-19 has been reported until now, several reports of early-life infections have been described with very low death rates (98, 119) . reports with recommendations to the treatment of pregnant women with covid-19 (166) and for neonates with covid-19 have been published (167, 168) . another possible route for sars-cov-2 is oral transmission by fecal samples (169) , and via breastfeeding from a sars-cov-2 infected mother. regarding breastfeeding, a small study found no evidence of covid-19 in breast milk, of six patients (139) . however, the primary concern is whether an infected mother can transmit the virus through respiratory droplets during breastfeeding. other viruses in the past have also caused concern in pregnant women. the zika virus has been linked to several cases of microcephaly in newborns during an epidemic in 2015 in brazil (170) . the infection had a high point in the first trimester of pregnancy, where there were more favorable conditions for its entry and replication in placental cells. in the case of sars-cov-2, it has not yet possible due to the time of infection occurs in the world, to observe the consequences of infection in the firsttrimester pregnancy. taking into account the early pregnancy, the placental tissue immaturity together with the up-regulation of ace2 expression in placental cells, perhaps the more susceptible period for sars-cov-2 infection is around the first trimester of pregnancy. it is important to highlight that after the 2009 influenza pandemic there have been reports of reduced cytokine response to bacterial infections. this leads to the hypothesis that covid-19 can lead to impairments of the immune response to other pathogens and vaccines in the future. future investigations are needed to identify the possible implications of sars-cov-2/covid-19 in pregnancy, the possible infection of the placenta in the first trimester of pregnancy and implications of the cytokine storm to the neonatal's health. ra and ms: write, conception, and review. np, lo, and sg-s: write and review. all authors contributed to the article and approved the submitted version. ra holds a post-doctorate fellowship from fapesp (19/02679-7) and lo also holds a post-doctorate fellowship from fapesp (19/07976-0). sg-s holds a master degree fellowship from fapesp (19/22448-0). the immune system in pregnancy: a unique complexity immune mechanisms at the maternal-fetal interface: perspectives and challenges self-recognition and the role of fetal microchimerism male fetal progenitor cells persist in 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management care emergency plan for inter-hospital transfer of newborns with sars-cov-2 infection chinese expert consensus on the perinatal and neonatal management for the prevention and control of the 2019 novel coronavirus infection detectable sars-cov-2 viral rna in feces of three children during recovery period of covid-19 pneumonia zika virus in the americas: early epidemiological and genetic findings the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 alberca, pereira, oliveira, gozzi-silva and sato. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-255479-yd5cbwnx authors: vu, david m.; jungkind, donald; angelle desiree labeaud, title: chikungunya virus date: 2017-06-30 journal: clinics in laboratory medicine doi: 10.1016/j.cll.2017.01.008 sha: doc_id: 255479 cord_uid: yd5cbwnx for chikungunya virus (chikv), the long-term sequelae from infection are yet ill-defined. the prolonged debilitating arthralgia associated with chikv infection has tremendous potential for impacting the global economy and should be considered when evaluating the human burden of disease and the allocation of resources. there is much still unknown about chikv and the illnesses that it causes. developing a better understanding of the pathogenesis of chikv infection is a priority and forms the basis for developing effective strategies at infection prevention and disease control. the envelope proteins, e2 and e1, play important roles in the binding of the virus to the host cell membrane and its subsequent cellular invasion, respectively. anti-chikv antibodies directed against the envelope protein that neutralize the virus in vitro also protect neonatal mice from lethal chikv infection in vivo, suggesting that these proteins may be important antigenic lethal targets for development of naturally acquired, or vaccine-elicited protection. [5] [6] [7] epidemiology the earliest report of chikungunya fever described an outbreak of a dengue-like illness that occurred in 1952 to 1953, on the makonde plateau in the southern province of tanganyika (present day tanzania). 8 residents of all ages experienced a febrile illness with rash and arthralgia. however, certain aspects of this outbreak distinguished it from previous reports of dengue fever outbreaks. most striking was the severity of the arthralgia that "would prevent the sufferer from changing position without help." 8 the local population began to call the disease chikungunya, which is a makonde (bantu) term that means "that which bends up," referring to the contorted positions of those who were affected by the sudden and severe onset of arthralgia. additionally, many individuals affected with the disease continued to experience intermittent joint pains that persisted for months after the acute illness. the attack rate also seemed to be unusually high, often affecting entire households. between 1952 and 1953, an estimated 60% to 80% of the population in this region developed symptoms of fever, rash, and arthralgia. 8 attempts to isolate the pathologic agent from symptomatic individuals during the outbreak also diverged from previous experience with dengue virus (denv): inoculation of infant mice with serum samples from symptomatic individuals resulted in death of the animals. in contrast, denv infection is difficult to establish in mice. 9 these data suggested that the cause of the syndrome termed chikungunya indeed was distinct from the cause of dengue fever. in africa, chikv is transmitted by arboreal aedes mosquitoes (a furcifer-taylori, a africanus, a luteocephalus, and a neoafricanus) in an enzootic cycle with nonhuman primates as the principle reservoir ( fig. 1) . [10] [11] [12] between the 1960s and 1990s, incidental human infection led to numerous, small-scale chikv outbreaks in countries throughout central and southern africa, and senegal, guinea, and nigeria in western africa (reviewed in ref. 13 ). the outbreaks occurred after periods of large rainfall and associated surges in the arboreal aedes mosquito density. in contrast, chikv outbreaks in southeast asia occurred in larger cities where aedes aegypti mosquitoes were implicated as the primary transmission vector. a aegypti mosquitoes require very small amounts of water to lay eggs, and thrive in human urban environments, particularly in areas where residents store water in open containers or cisterns. in 2004, a large-scale chikv epidemic erupted, sweeping down the coast of kenya into islands on the indian ocean (comoros, mayotte, seychelles, ré union, madagascar, sri lanka, and the maldives), india, southeast asia (malaysia, singapore, thailand), and china. 14 although chikv infection in travelers returning to europe had been reported previously, autochthonous transmission of chikv was observed for the first time in italy in 2007, 15 and in france in 2009. 16 an important factor that facilitated the rapid expansion of chikv infection was a novel single amino acid substitution of alanine for valine at position 226 (a226v) in the e1 envelope protein that enhanced the ability of the aedes albopictus mosquito to transmit chikv to humans. 17 a albopictus is an anthropophilic, peridomestic species of mosquito that has an even greater geographic range than that of its relative, a aegypti, and has been implicated as having a major role in the spread of chikv epidemics across asia and to the americas. in december 2013, the first cases of locally transmitted chikv in the americas were confirmed in st. martin, 18 followed rapidly by cases identified throughout the caribbean and latin america. by january 2015, chikv infection had been identified in 42 countries or territories in the caribbean, central america, south america, and north america (local transmission in florida) with more than a million suspected cases reported, and more than 25,000 laboratory-confirmed. 19 although epidemics of febrile arthralgia have been reported in the americas since the 1700s, these outbreaks previously had been attributed to dengue fever. for example, an epidemic "break-bone fever," which referred to modern-day dengue fever, erupted on the islands of st. thomas and santa cruz in the west indies from 1827 to 1828. stedman, who reported this "anomalous disease" called "dandy fever" by local residents, noted that the illness "attacked almost every individual in the town," had "extremely low mortality," and was associated with "pains in the joints for weeks after recovery from the acute stage," which were key differences between the 1827 and 1828 west indies epidemic and previous descriptions of a "break-bone fever" (referring to modern-day dengue fever). he concluded that "the diseases, though somewhat alike in a few symptoms, are essentially different." 20 thus, although the 2013 epidemic was the first chikv outbreak in the americas to be confirmed using modern-day virologic methods, historical reports raise questions to whether this truly was the first introduction of chikv infection to the americas. 21 chikv is known to infect a variety of cell lines in vitro, including vero cells (from green monkey kidney) 22 and bhk21 baby hamster kidney cells, 23 and various insect cell lines. 24, 25 human cellular tropism was more recently described. 26 fibroblasts in the dermis, joint capsule, and muscle seem to be the major targets of chikv infection in humans. 27 human epithelial and endothelial cells 26 and muscle progenitors (satellite cells) 28 also have been observed to be infected by chikv. lymphocytes and monocytes seem resistant, yet macrophages seem susceptible to chikv infection. 26 investigations of disease pathogenesis during human chikv infection have been limited, in part, by the lack of relevant and/or accessible models of chikv disease. development of nonhuman primate models of chikv infection has been helpful for use in evaluating potential chikv vaccines. rhesus macaques immunized with an investigational chikv virus-like particle vaccine were protected against developing viremia after intravenous challenge with 10 10 pfu of chikv, whereas control monkeys that received the mock vaccine developed high levels of viremia after challenge. 29 in a separate study, cynomolgus macaques challenged with a much lower chikv inoculum, by either intravenous or intradermal injection, developed viremia, fever, and rash. viral rna remained detectable in synovial and muscle tissue for up to 1.5 months after infection, and in lymphoid tissue for up to 3 months. 30 thus, this model may be useful for studying long-term sequelae of chikv infection, such as the prolonged arthralgia experienced by many chikv individuals. because nonhuman primate models of chikv infection are not readily accessible to many investigators, some investigators have developed mouse models to study chikv disease. viral challenge of neonatal wild-type mice results in fatal infection. however, this susceptibility to chikv infection wanes quickly and adult wild-type mice are resistant to chikv infection. type i interferon receptor knock-out mice (ifn-a/br à/-) have an impaired type i ifn pathway and, in contrast with adult wildtype mice, develop viremia after viral challenge. 27 thus, it seems that activation of the type i ifn pathway plays an important role in controlling chikv infection. mice also have been used to develop models of chikv-related arthritis. 31, 32 one group of investigators demonstrated that mice injected with chikv in the footpad developed leg swelling and weight loss, and had histologic evidence of necrotizing myositis, arthritis, tenosynovitis, and vasculitis. 32 a separate group of investigators also elicited foot swelling in mice injected with chikv in the ventral footpad. these mice developed viremia, and histologic examination revealed large mononuclear cell infiltrates in and around synovial membranes, and in muscle tissue. furthermore, treatment with ifn-a before chikv inoculation reduced viremia and prevented manifestations of arthritis, 31 again highlighting the importance of type i ifns in chikv virus control. in contrast to denv, which can cause asymptomatic infection, most individuals with chikv infection are symptomatic. 8 however, chikungunya fever shares many similarities with dengue fever. sudden onset of high fever is typically the initial symptom reported and can appear within 2 days of infection. a rash sometimes is observed and is typically maculopapular, although bullous rashes have been noted in some infants with chikv infection. 33 both viral infections also are known to cause arthralgia and arthritis. however, the polyarthralgia caused by chikv frequently is characterized as debilitating and has been reported to continue well beyond the resolution of fever. for example, a third of travelers to the caribbean who acquired chikv infection in 2014 while abroad reported persistent joint or muscle pain, or joint swelling at greater than or equal to 9 months after their acute infection. 34 the knees are the most commonly involved joints; however, other large or small joints may be affected. of note, symmetric involvement of joints is frequently reported. 35 additional symptoms reported include fatigue, nausea, vomiting, and conjunctivitis. conspicuously absent among those with chikv infection are reports of retro-orbital headache, which are characteristic of dengue fever. 8 most symptoms resolve within 7 to 10 days; however, many infected individuals have reported protracted arthralgia that has lasted weeks, months, or even years. this long-term burden of disease can be devastating to local economies and represents a significant health cost: chikv was responsible for 1386 to 1,081,962 nondiscounted years of life lost in 2005. 36 these values are now significant underestimations of the true health cost burden, given its expanding distribution to the americas since those estimates were made. neurologic complications of acute chikv disease were observed with the 2006 outbreak on la ré union island, and include encephalitis 37 and guillain-barré syndrome. 38 these also were observed during chikv outbreaks in india. [39] [40] [41] additional observations of severe manifestations of disease, including myocarditis and hepatitis, 37 have re-energized investigations of chikv disease pathogenesis. chikv infection had not been associated with increased risk of mortality before 2006. during the outbreak on la ré union island, however, at least 213 people with chikv infection died. investigators estimated that the case-fatality rate was approximately 1:1000, and observed that the fatalities occurred mainly in persons greater than or equal to 75 years of age. 42 during the 2006 outbreak of chikv infection in ahmedebad, india, 18 of the 90 confirmed cases of chikv infection were fatal. fifteen of the 18 deaths occurred in persons 60 years of age and older. 43 autopsy of fatal cases of chikv infection in colombia revealed evidence of hepatocellular coagulative necrosis, tubulointerstitial nephritis, and acute pericarditis. 44 good laboratory testing services are important in the diagnosis of suspected chikv infections. chikungunya fever can easily be confused at various stages of the disease with other arboviral infections, such as dengue and zika virus. the clinical consequences of these three viruses are different, so specific diagnosis is important. situations where laboratory test confirmation for presence or absence of infection are discussed next. testing sporadic cases of suspected arboviral infection can provide early warning that the virus is in the community. this can help public health and medical personnel prevent a possible epidemic. this is especially important if conditions conducive to an epidemic are present, such as during the rainy season when the mosquito population is high, and with open housing conditions that enhance exposure of an immunologically naive population to infection. diagnosis of a significant number of cases early on to establish cause of an epidemic and the characteristic symptoms enables reliable clinical diagnosis with decreased need for laboratory confirmation if an epidemic occurs. public health departments may occasionally perform epidemiologic testing for evidence of past chikv infection in a community so that the immunologic history and state of susceptibility in a population are determined to better approximate risk for an outbreak. focused and random spot testing is important during an epidemic to detect the entrance of a second arbovirus, such as denv, entering the population during a chikv outbreak. focused testing is important in cases with serious underlying diseases and in cases with complications or a fatal outcome. in the postoutbreak period, persons not previously tested for chikv should be tested for a recent or past infection as part of the work-up in patients presenting with new chronic arthritic problems including joint pain and swelling. during interepidemic periods, patients presenting with typical chikv symptoms should be tested for chikv and other arboviruses with similar symptoms. three tests for chikv are useful in various situations. for diagnostic confirmation of current and recent infection, a molecular test (typically polymerase chain reaction [pcr]) for the virus and an assay for the presence of specific igm antibody are required. the most frequently needed assay is the chikv igm antibody assay. molecular testing for the presence of the virus is required in the early stages of disease. virus is present in the blood at the time symptoms appear and pcr testing provides reliable detection for 5 days thereafter (6 days total). during that time only a molecular test for the virus should be ordered. as igm production rises, by days 7 to 9, viremia falls to pcr undetectable levels. during days 5 to 9 when the viral load is waning and igm has not reached its peak, it is necessary to order a molecular test for the virus and the igm antibody assay to maintain good diagnostic sensitivity. after that, only the igm test is required. after 14 to 21 days both the igm and igg test are positive, and the igm test wanes over several weeks or months, whereas igg remains for years as a good marker of past infection and immunologic protection, and also as an epidemiologic tool to determine seroprevalence in a population. using grenada as a case study to illustrate the chikv diagnostic strategy, during the peak of the 2014 grenada chikv epidemic, 112 samples from typical cases were tested by pcr and the igm assay independent of the stage of disease. although all 112 had classic symptoms, only 101 were found to be positive by at least one of these laboratory tests. in the 101 samples, igm outperformed pcr: 92% were positive by igm, 17% were positive by pcr, and 9% were positive by both. reliance on pcr testing only is unlikely to accurately characterize incidence. [45] [46] [47] commercially available kits are of variable quality. 48 pcr assays with favorable performance characteristics are documented in the literature and becoming commercially available for clinical use in other countries; however, they can only be obtained for research use in the united states. good immunoassays for chikvspecific igm in patient serum are less reliable and available. [49] [50] [51] a partial list of sources for molecular viral assays and igm assays to document chikv infection is given in table 1 . these sources can serve as starting points to explore test kit choices and capabilities in what it is hoped will be an expanding menu of kits for clinical testing. until reliable food and drug administration-approved kits for molecular detection of virus and chikv antibodies are available, only the largest commercial and government reference laboratories should consider routine diagnostic testing for chikv. the centers for disease control and prevention has facilities for testing samples to establish transmission of chikv in the united states. some city and state health departments and other government agencies also have this capability. specific details related to collection and preserving serum for transportation to and testing at regional or national facilities can be found at: http://www.cdc.gov/chikungunya/hc/diagnostic.html. if an epidemic should require greater testing capacity, the centers for disease control and prevention and similar agencies in other countries may implement emergency use authorization for diagnostic tools for chikv that could be distributed to qualified clinical laboratories that demonstrate proficiency with the assays by successfully testing verification panels for each assay. if chikv infection becomes an annual endemic threat in certain regions, there will be a need for testing capability on a more local basis. it would be ideal to have chikv detection as part of a routine test panel and supported by the clinical laboratory testing industry. encouraging research studies suggest that this will be possible, first for pcr and perhaps later for the igm assays. 46, 51, 52 treatment there is presently no licensed targeted therapy for acute chikv infection. treatment is primarily supportive care and includes the use of analgesic and anti-inflammatory medication, rehydration, and rest. however, research to identify potential new antiviral therapies, or repurposing of existing compounds for treating chikv infection is ongoing ( table 2 ; reviewed in ref. 53 ). for example, chloroquine has in vitro activity against several viruses, and has been found to inhibit chikv replication in vero cells. 54 however, it has rt, reverse transcriptase. a partial list of companies providing pcr and elisa kits for detection of chikv or human antibody to the virus. none of these are fda approved for use as diagnostic tests at this time but are considered in the research use only category. one product is ce approved. these tests may prove to be useful adjuncts to assist in the development of future fda-approved or validated assays. not been shown to have anti-chikv effects in vivo. compounds that may interfere with viral entry, including phenothiazines 55 and flavaglines, 56 are being investigated as potential therapies. ribavirin has been shown to have in vitro activity against chikv, and synergized with doxycycline to reduce viral load and inflammation in infected mice. 57 monoclonal antibodies to the chikv e1 and e2 proteins have been used to protect mice and nonhuman primates from developing chikv infection after viral challenge. 27, 29 however, it is unclear whether passive immunization with monoclonal antibodies or hyperimmune serum can ameliorate symptomatology of chikv disease after infection has already been established. the past decade has seen explosive viral epidemics, from severe acute respiratory syndrome to ebola to arboviruses including zika and chikv. for some diseases, the human toll is acutely evident in the form of mortality or acute morbidity. for chikv and others, the long-term sequelae from infection are yet ill-defined. the prolonged debilitating arthralgia associated with chikv infection has tremendous potential for impacting the global economy, and should be considered when evaluating the human burden of disease and the allocation of resources. there is much still unknown about chikv and the illnesses that it causes. developing a better understanding of the pathogenesis of chikv infection is a priority and forms the basis for developing effective strategies at infection prevention and disease control. the alphaviruses: gene expression, replication, and evolution global emergence of alphaviruses that cause arthritis in humans complete nucleotide sequence of chikungunya virus and evidence for an internal polyadenylation site a structural and functional perspective of alphavirus replication and assembly identifying the role of e2 domains on alphavirus neutralization and protective immune responses exposure of epitope residues on the outer face of the chikungunya virus envelope trimer determines antibody neutralizing efficacy isolation and characterization of broad and ultrapotent human monoclonal antibodies with therapeutic activity against chikungunya virus an epidemic of virus disease in southern province, tanganyika territory, in 1952-53. i. clinical features the newala epidemic. iii. the virus: isolation, pathogenic properties and relationship to the epidemic vectors of chikungunya virus in senegal: current data and transmission cycles further studies on the chikungunya outbreak in southern rhodesia in 1962. i. mosquitoes, wild primates and birds in relation to the epidemic further studies on the chikungunya outbreak in southern rhodesia in 1962. ii. transmission experiments with the aedes furcifer-taylori group of mosquitoes and with a member of the anopheles gambiae complex changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus tracking epidemic chikungunya virus into the indian ocean from east africa infection with chikungunya virus in italy: an outbreak in a temperate region chikungunya virus, southeastern france a single mutation in chikungunya virus affects vector specificity and epidemic potential arboviral diseases branch, national center for emerging and zoonotic infectious diseases, cdc. 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diagnostics in the asia pacific region evaluation of commercially available serologic diagnostic tests for chikungunya virus sero-prevalence and cross-reactivity of chikungunya virus specific anti-e2ep3 antibodies in arbovirus-infected patients assessment of qpcr, nested rt-pcr and elisa techniques in diagnosis of chikungunya clinical and serological insights from the asian lineage chikungunya outbreak in grenada, 2014: an observational study towards antivirals against chikungunya virus assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in vero cells inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays assessment of flavaglines as potential chikungunya virus entry inhibitors a combination of doxycycline and ribavirin alleviated chikungunya infection effectiveness of chloroquine and inflammatory cytokine response in patients with early persistent musculoskeletal pain and arthritis following chikungunya virus infection administration of e2 and ns1 sirnas inhibit chikungunya virus replication in vitro and protects mice infected with the virus in vitro inhibition of chikungunya and semliki forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination thiazolidone derivatives as inhibitors of chikungunya virus mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (t-705), a broadspectrum antiviral a neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus e2 protects from disease key: cord-258333-jmk8hdk2 authors: sivier, v; odelin, m.f; gonthier, r; pozzetto, b title: place des viroses respiratoires dans les hyperthermies de sujets âgés hospitalisés au cours d’une saison hivernale date: 2001-12-10 journal: rev med interne doi: 10.1016/s0248-8663(01)00489-1 sha: doc_id: 258333 cord_uid: jmk8hdk2 purpose. – in the geriatric units of the university hospital of saint-etienne, 129 cases of fever (38° c or more) were recorded prospectively during the 1995–1996 winter period in a population of 503 hospitalised patients (25.6%), and were investigated for the detection of a viral aetiology. methods. – in febrile patients, a standard form was used to record clinical and biological parameters, including the results of investigations for respiratory viruses from a nasal swab and dual serum specimens. results. – a clinical or radiological respiratory infection was found in 69 cases (53.5% of all cases of fever), including 14 respiratory syncytial virus (rsv) infections. in comparison to nonviral respiratory infections, the rsv infections were characterised by the prevalence of anorexia (57% vs 20%, p < 0,05) and rhinorrhea (64% vs 5%, p < 0,01). no influenza infection was recorded despite the concomitant circulation of influenza virus in the community. a nosocomial outbreak of rsv infection (nine cases, attack rate of 18.7%) was identified in a long-stay care unit. conclusion. – this study illustrates the high prevalence (10.9%) of rsv infections in elderly patients with fever during this season and the importance of hygienic measures to control the spread of nosocomial outbreaks. infections, the rsv infections were characterised by the prevalence of anorexia (57% vs 20%, p < 0,05) and rhinorrhea (64% vs 5%, p < 0,01). no le patient âgé est particulièrement exposé aux infections en général tant en milieu communautaire qu'en institution. les caractéristiques des patients séjournant en institution gériatrique sont le grand âge, la dépendance pour les activités de la vie quotidienne et l'association de plusieurs maladies sous-jacentes. cette population offre un excellent terrain pour le développement des infections [1] [2] [3] [4] [5] . les infections virales en institution ont une dissémination particulièrement facile, la transmission s'effectuant par le malade lui-même, le personnel ou encore par les autres patients ou les visiteurs [6, 7] . ce risque infectieux impose un respect absolu des règles d'hygiène collective pour prévenir une dissémination épidémique [5, [8] [9] [10] . très souvent, la présentation clinique des infections est modifiée chez le sujet âgé, les signes étant volontiers frustres ou absents [11, 12] . chez la période de surveillance s'est déroulée du 1 er novembre 1995 au 30 avril 1996 dans le département de gérontologie clinique du centre hospitalouniversitaire de saint-étienne qui compte huit services répartis dans six bâtiments, dont deux situés dans un autre établissement. l'étude a porté sur 503 sujets répartis en 176 sujets (35 %) dans le service de court séjour de 29 lits (neuf chambres seules et dix chambres à deux lits), 79 sujets (16 %) dans un service de moyen séjour à orientation rééducation de 29 lits (sept chambres seules, huit chambres à deux lits et deux chambres à trois lits) et 248 sujets (49 %) répartis dans six services de long séjour comprenant un total de 200 lits (57 chambres seules, 45 chambres à deux lits, 15 chambres à trois lits et deux chambres à quatre lits). l'âge moyen de la population était de 84,1 ± 6,8 ans, dont 73 % de femmes. chez les patients résidant dans les services de long séjour, la vaccination antigrippale a été systématique, renouvelée tous les ans en automne (vaxigrip t , mérieux msd) sauf en cas de refus de la part du patient ; aucun sujet n'a été protégé du risque pneumococcique. le critère d'inclusion des sujets dans l'étude a été la constatation d'une hyperthermie égale ou supérieure à 38°c lors d'au moins une prise, avec ou sans signe(s) respiratoire(s). la surveillance a été réalisée grâce à une fiche standardisée permettant le recueil systématique et prospectif des signes généraux (dont viroses respiratoires et hyperthermies la température mesurée deux fois par jour), des signes fonctionnels et des signes physiques, ainsi que celui des résultats des investigations paracliniques mentionnées ci-après. une radiographie pulmonaire a été réalisée en fonction des données cliniques et de la mobilité du patient. la mise en place d'un traitement antibiotique à base d'amoxicilline a été systématique dans l'attente des résultats du bilan étiologique. le bilan biologique infectieux des patients fébriles a compris de façon quasi systématique trois paires d'hémocultures, un examen cytobactériologique urinaire (ecbu), une numération-formule sanguine, une vitesse de sédimentation, une détermination de la protéine c réactive, une détection par immunofluorescence indirecte, grâce à un écouvillonnage nasal, des antigènes viraux respiratoires suivants : virus grippaux a et b, virus para-influenza, virus respiratoire syncytial, adénovirus et coronavirus, et une double sérologie par technique de fixation du complément, le jour de l'épisode fébrile et 15 jours à trois semaines plus tard, à la recherche d'une infection par les agents ci-après : virus grippaux a et b, virus para-influenza, virus respiratoire syncytial et mycoplasma pneumoniae. les critères de positivité de la sérologie ont été les suivants : variation significative du taux des anticorps (multiplication par quatre ou plus) sur les deux sérums ou taux d'anticorps élevé d'emblée (supérieur à 32) dans le premier sérum. les patients fébriles ont été classés en trois groupes : ceux présentant des stigmates biologiques d'infection virale spécifique selon les critères définis ci-dessus, quelles que soient les données cliniques ou radiologiques (groupe « virose respiratoire »), ceux présentant au moins un symptôme respiratoire auscultatoire ou radiologique sans stigmate d'infection virale biologique et sans cause évidente de maladie extrarespiratoire pouvant expliquer la symptomatologie (groupe « infection respiratoire non virale ») et ceux n'entrant dans aucune des deux catégories précédentes (groupe « autre »). les variables qualitatives ont été comparées à l'aide du test du 2 , en utilisant la correction de yates pour les petits effectifs. le test t de student a été utilisé pour comparer des variables quantitatives. le seuil de signification statistique a été fixé à 0,05. sur 503 patients hospitalisés pendant la période concernée, 129 cas d'hyperthermie ont été répertoriés, soit des taux d'incidence cumulée de 25,6 % pour l'ensemble des services se répartissant comme suit : 29,5 % en court séjour (52 cas), 13,9 % en moyen séjour (11 cas la radiographie pulmonaire pratiquée chez seulement 80 malades (62 %), en raison de l'impossibilité de réaliser des images chez des malades impotents ou grabataires, a montré chez 33 d'entre eux (41 %) des anomalies systématisées ou non, de localisation pleurale ou parenchymateuse. sur les 129 malades ayant présenté une hyperthermie, 12 décès ont été observés, soit un taux de mortalité de 9,3 % : un décès dans le groupe « virose respiratoire » par surinfection bactérienne pulmonaire, sept décès dans le groupe « infection respiratoire non virale » (un choc septique, cinq décompensations respiratoires aiguës, une décompensation cardiaque aiguë) et quatre décès dans le groupe « autre » (deux cancers en phase terminale, un accident vasculaire cérébral et une cause indéterminée). parmi les 14 cas de viroses à virus respiratoire syncytial, le diagnostic a été confirmé dans 12 cas par la sérologie, dans un cas par la détection d'antigènes viraux sur le prélèvement nasal et dans un cas par la positivité simultanée des deux approches. l'âge moyen de ces cas a été un peu plus élevé que celui des autres sujets avec hyperthermie (89,1 versus 84,2 ans, différence non significative). tous ont été vaccinés contre la grippe à l'automne. les infections à virus respiratoire syncytial sont survenues entre le 8 janvier et le 23 février 1996, exclusivement dans les services de long séjour, dont neuf cas dans le service 5 (taux d'attaque de 18,7 %) (figure 2). les patients de ce dernier service cumulaient des durées de séjour particulièrement longues, notre étude a analysé prospectivement toutes les hyperthermies survenues au cours d'une saison hivernale en focalisant l'attention sur les infections d'origine virale. elle s'intègre dans le cadre d'une surveillance effectuée dans le service de gérontologie clinique du centre hospitalo-universitaire de saint-étienne entre 1990 et 1998, en particulier sous l'angle des infections respiratoires virales [13, 14] . le taux d'incidence cumulée des hyperthermies hivernales est relativement stable dans nos sites gériatriques : 21,9 % en 1990-1991 [13] , 18,2 % en 1991-1992 [13] , 21,6 % en 1993-94 [14] et 25,6 % en 1995-1996 (cette étude). l'étude prospective de jackson et al. [15] réalisée sur une période de trois ans révèle une densité d'incidence de 7,1 infections pour 1 000 patients-jours, celle de nicolle et al. [16] réalisée sur 12 mois une incidence de 59 infections pour 100 patients-ans. notre chiffre plus faible (1,43 hyperthermies pour 1 000 patients-jours) est à relier au fait que le niveau de fièvre requis pour l'inclusion des patients est relativement élevé, ce qui a sans doute contribué à méconnaître un nombre élevé d'infections. les épisodes fébriles sont survenus chez des sujets âgés présentant des maladies multiples où prédominent des troubles psychocomportementaux et des troubles locomoteurs, une polymédication, une éventuelle malnutrition et parfois un état grabataire. une maladie infectieuse chez le sujet âgé ne se manifeste pas toujours par une sémiologie franche ; elle prend fréquemment un aspect atypique ou trompeur avec des signes proprement gériatriques : absence de fièvre, majoration de l'asthénie ou de l'anorexie, décompensation des troubles mentaux ou des maladies associées. les manifestations générales comme la fièvre ou les frissons manquent souvent [17] . dans deux séries autopsiques, une série japonaise concernant 463 sujets décédés à leur domicile ou à l'hôpital [18] et une série suisse concernant 3 000 malades vivant en institution gériatrique [19] , une maladie infectieuse présente au moment du décès du patient a été documentée dans respectivement 50,2 et 42,9 % des cas, le diagnostic ayant été rarement évoqué avant le décès du patient. dans ce contexte, le parti pris pour cette étude de surveiller exclusivement les sujets fébriles peut paraître discutable, d'autant que les infections virales sont souvent non ou peu fébriles à cet âge. néanmoins, ce choix peut se justifier sur trois arguments : la surveillance pratiquée étant avant tout destinée au dépistage des infections grippales symptomatiques dans une population largement vaccinée, la fièvre demeure un signe cardinal dans cette maladie, même chez le sujet âgé ; par ailleurs, compte tenu des difficultés à interpréter des signes fonctionnels chez des sujets présentant pour un grand nombre d'entre eux des troubles psychomoteurs graves, l'hyperthermie est un critère d'inclusion objectif, facile à utiliser ; enfin, le coût non négligeable du bilan virologique restreint l'extension de ses indications à des symptomatologies cliniques franches. aucun cas de grippe n'a été décelé parmi nos patients hyperthermiques alors qu'avait sévi une double épidémie de grippe a (h3n2) et (h1n1) dès les premiers jours d'octobre 1995, suivie de cas de grippe b de janvier à mars 1996 dans la région rhône-alpes [20] . la corrélation entre ces données épidémiologiques et l'absence de cas de grippe chez nos 129 malades avec hyperthermie (dont la couverture vaccinale était globalement de 72,3 %) montre le caractère protecteur de cette vaccination chez le sujet âgé [21] . comme d'autres auteurs [22] [23] [24] , nous rapportons une proportion importante d'infection à virus respiratoire syncytial survenus sous forme de quelques cas sporadiques et d'une épidémie nosocomiale dans l'un des services. les manifestations cliniques ont été présentes sous forme de signes généraux bruyants et de signes respiratoires, notamment un catarrhe nasal relativement évocateur [25] . néanmoins, cette étude à la suite de plusieurs autres [26] [27] [28] [29] montre qu'il est très difficile de repérer les infections à virus respiratoire syncytial sur la seule sémiologie. l'écouvillonnage nasal par immunofluorescence indirecte n'a permis de faire un dépistage immédiat d'une viroses respiratoires et hyperthermies virose à virus respiratoire syncytial que dans deux cas sur 14. dans cette population âgée, cet examen doit donc être associé systématiquement à une double sérologie virale qui reste le seul moyen de faire un diagnostic de certitude, bien que tardif, des viroses respiratoires. des complications bronchopulmonaires ou de décompensation de maladies associées sont fréquentes au décours de ces viroses respiratoires, 5 à 67 % selon falsey [6] , 42 % selonagius et al. [24] . d'après han et al. [30] , aux états-unis, le virus respiratoire syncytial serait la cause de 2 à 9 % des pneumopathies du sujet âgé, avec un coût total annuel d'hospitalisation estimé entre 150 et 680 millions de dollars. dans notre étude, nous avons constaté la survenue d'une complication dans 25 % des cas d'infection à virus respiratoire syncytial, avec un taux de mortalité de 8,3 %. l'épidémie nosocomiale de virus respiratoire syncytial est survenue dans un service de long séjour avec des locaux communs et exigus, chez des patients porteurs de multiples maladies et souvent déments. il est probable que le biais du critère d'inclusion (fièvre supérieure ou égale à 38°c pour la réalisation de prélèvements) et le caractère atypique des symptômes aient sous-estimé l'ampleur de cette épidémie. pour éviter le développement de ces infections nosocomiales virales, il est important d'effectuer une surveillance microbiologique, d'appliquer les précautions standard d'hygiène et, à la moindre alerte épidémique, de mettre en place des mesures d'isolement de type « gouttelettes » et de renforcer les consignes d'hygiène des mains auprès du personnel soignant [31] [32] [33] . en conclusion, les infections respiratoires, qu'elles soient virales, bactériennes ou non documentées, apparaissent comme la principale cause d'hyperthermie chez le sujet âgé institutionnalisé pendant la saison hivernale et sont à l'origine de nombreuses complications, voire de décès. notre étude souligne le bénéfice des explorations virologiques qui ont permis de dépister, dans un contexte d'épidémie grippale communautaire, des infections fébriles à virus respiratoire syncytial de symptomatologie peu spécifique, en dehors de la plus grande fréquence du catarrhe nasal. il convient également de souligner l'importance du respect des règles d'hygiène collective pour éviter la diffusion nosocomiale de virus dans les collectivités de sujets âgés, en attendant la disponibilité d'un vaccin anti-virus respiratoire syncytial. prevalence of infections and their risk factors in geriatric institutions : a one-day multicentre survey mortimer ea. acute respiratory illness in older community residents influenza vaccination for all elderly impact of respiratory virus infections on persons with chronic underlying conditions infections chez les sujets âgés. paris : ellipses noninfluenza respiratory virus infection in long-term care facilities viral respiratory infections in the institutionalized elderly : clinical and epidemiologic findings nosocomial viral respiratory infections hygiène et prévention des infections dans les établissements de soins pour personnes âgées les infections chez les personnes âgées en institution the atypical presentation of infection in old age fever and the elderly viroses respiratoires responsables d'hyperthermie chez les sujets âgés. étude portant sur 601 patients hospitalisés dans le service du chu de saint-étienne sur une période de 6 mois investigations de 140 épisodes d'hyperthermie chez des sujets âgés hospitalisés, au cours de deux saisons hivernales intensive surveillance for infections in a threeyear study of nursing home patients twelvemonth surveillance of infections in institutionalized elderly men approach to fever and infection in the nursing home a comparison of community-acquired lung infection and nosocomial infection causes of death in a hospitalised geriatric population : an autopsy study of 3000 patients centres nationaux de référence pour la grippe france-nord et france-sud. surveillance de la grippe et du virus respiratoire syncytial en france d'octobre 1995 à avril 1996 control of influenza outbreaks in the nursing home : guidelines for diagnosis and management outbreak of respiratory syncytial virus infection in the elderly respiratory syncytial virus infection in the ederly an epidemic of respiratory syncytial virus in elderly people : clinical and serological findings concurrent respiratory syncytial virus and influenza.ainfections in the institutionalized elderly and chronically ill gravenstein s. can respiratory syncytial virus and influenza a be distinguished clinically in institutionalized older persons ? respiratory syncytial virus or influenza ? respiratory syncytial virus and influenza a infections in the hospitalized elderly acute respiratory tract infection in daycare centers for older persons respiratory syncytial virus pneumonia among the elderly : an assessment of disease burden société française d'hygiène hospitalière. isolement septique. recommandations pour les établissements de soins. paris : ministère de l'emploi et de la solidarité surveillance for outbreaks of respiratory tract infections in nursing homes viral pneumonias. epidemic respiratory viruses viroses respiratoires et hyperthermies key: cord-253197-9hjvk7p5 authors: thomas, evan; hillman, bruce j.; stanisic, thomas title: urinary tract infection with atypical mycobacteria date: 1980-11-30 journal: the journal of urology doi: 10.1016/s0022-5347(17)55642-6 sha: doc_id: 253197 cord_uid: 9hjvk7p5 abstract we present a case of disseminated atypical mycobacterial (mycobacterium intracellularis) infection of the urinary tract. the patient had anhydrous ectodermal dysplasia and an unrelated defect in cell-mediated immunity. the infection resulted in a lengthy ureteral stricture with resultant hydronephrosis and diminished kidney function. a review of the literature revealed only 13 previous cases of atypical mycobacterial infection of the urinary tract. it is important to distinguish between simple colonization by these organisms and actual infection. differentiation between atypical mycobacterial infection and urinary tuberculosis is important in determining the therapeutic regimen. although first described in 1918 it was not until runyon's classification of atypical mycobacteria in 1959 that these organisms came under closer scrutiny. runyon categorized the many varieties of atypical mycobacteria into 4 groups, based upon color and growth characteristics. 1 of these groups i and iii (the photochromogens and the non-photochromogens, respectively) are those most pathogenic in humans. 2 the vast majority of cases represent pulmonary involvement but systemic dissemination also is known to occur. 3 • 4 nonetheless, genitourinary tract infection has been reported only rarely, with only 13 cases reported previously in the literature, some of which were described incompletely. herein we describe a child with anhydrous ectodermal dyspasia in whom a ureteral stricture developed secondary to infection by mycobacterium intracellularis (battey's bacillus). we relate the features of our case to those previously reported. a 14-year-old white boy was hospitalized with increasing pedal edema, ascites and diarrhea 5 days in duration. bloody stools developed 3 days before presentation. there were no complaints referable to the urinary tract. when the patient was 33 months old anhydrous ectodermal dysplasia was diagnosed. he had had numerous hospital admissions for soft tissue infections, including meningitis, pneumonia, otitis media and cellulitis. immunologic testing revealed recently a defect in cell-mediated immunity, unrelated to the ectodermal dysplasia. 5 three years ago disseminated m. intracellularis developed, as evidenced by positive cultures of the urine, bone marrow, liver, lymph nodes, cerebrospinal fluid, stool and blood. hepatosplenomegaly had been present continuously since then. numerous antimicrobials had been tried and found ineffective, including isoniazid, para-aminosalicylate, ethambutol, streptomycin, kanamycin, capriomycin, erthyromycin, ethionamine, rifampin, levamisole, trimethoprim-sulfamethoxazole and pyrimidine. parenteral transfer factor was begun 4 months before hospitalization with some subjectively assessed reduction in the hepatosplenomegaly. however, cultures remained positive. at hospitalization the patient was receiving oral transfer factor, clofazimine and streptomycin. the family history is significant, since his brother died when he was 2 years old of diarrhea and dehydration. at autopsy acid fast organisms were present in the gastrointestinal tract. these organisms were not characterized further. the patient was an afebrile, cachetic-appearing young boy, small for his age. the teeth were pointed and ridden with cavities, the hair was sparse and white, and the skin was dry and atrophic, with numerous scars and hypopigmented areas secondary to healed subcutaneous abscesses. the abdomen was more protuberant than usual and a fluid wave was believed to be present. hepatosplenomegaly was palpably evident. there was a moderate amount of pitting pedal edema. laboratory values showed only a mild hepatic dysfunction unchanged from previous values. electrolytes were normal except for slight hyponatremia. white cell count was 5,000 with 18 per cent bands. creatinine was 0.4 mg./ dl. and blood urea nitrogen was 11 mg./dl. urinalysis was normal. the diarrhea abated soon after hospitalization, although testing for occult blood remained intermittently positive. there was no gross melena. coronavirus and m. intracellularis were cultured from the stool. an upper gastrointestinal barium examination revealed possible nodularity and thickening of the small bowel wall, as well as slow passage of barium. a gallium scan demonstrated abnormal uptake in the left upper quadrant and mid epigastrium, probably within the small bowel. these findings were interpreted as compatible with intestinal mycobacterial involvement. during the third month of hospital stay hematuria, pyuria and an increase in serum creatinine to 0.8 to 0.9 mg./dl. occurred. urine output remained normal. an abdominal ultrasound and a computerized tomographic body scan confirmed the presence of massive hepatosplenomegaly. the computerized tomographic scan also showed an unexpectedly large left kidney with hydronephrosis. a subsequent excretory urogram verified the left hydronephrosis and a normal right kidney, ureter and bladder. retrograde pyelography revealed the source of the left pyelocaliectasis to be a 2.5 cm. long stricture of the left mid ureter (see figure) . cultures of urine taken from the left renal pelvis yielded a pseudomonas species. unfortunately, cultures for mycobacteria were not performed at this time. the patient was treated for the pseudomonas. since then urine cultures for bacterial and mycobacterial growth had been negative. serum creatinine remained at 0.8 mg./dl. and mild hematuria, pyuria and proteinuria persisted. subsequently, the patient died. postmortem pathologic examination revealed diffuse foci of atypical mycobacteria with poorly organized cellular rection. organisms were present in both kidneys and in the strictured region of the left ureter. discussion the table summarizes the findings in 14 cases, including our own, of atypical mycobacterial genitourinary involvement. as indicated in the table information concerning some of these cases is scant. in others, such as those reported by lester,6 and lattimer and boyes, 7 culture of m. kansasii in the urine was not associated with evidence of urinary tract disease. this is significant in that mycobacterial colonization and infection appear not to be synonymous. in 1970 klotz noted 306 cases of urinary colonization with atypical mycobacteria, none of which was reportedly associated with observable urinary tract disease.8 thus, we believe that adequate documentation of atypical mycobacterial infection must include either positive culture of genitourinary tissue specimens and/ or positive urine cultures in conjunction with some combination of radiologic, pathologic or laboratory evidence compatible with granulomatous genitourinary disease. in view ofklotz's study the occurrence of these spinal fluid, lymph nodes, liver, bone marrow, blood combinations of findings would seem exceedingly rare. several facets of our case are uncommon with respect to those previously reported. the patient was only 14 years old. lattimer and boyes referred to 2 children in whom urine culture yielded group i atypical mycobacteria but no supporting evidence of genitourinary disease was found. 7 in no other reported case was a patient <27 years old. 9 in discussing the reasons for the rarity of urinary tuberculosis in children, ehrlich and lattimer have cited the usual lag between initial cortical dissemination and clinical or radiologic manifestations. 10 our patient possessed a defect in cellular immunity, which may have hastened the progression of the disease. while decreased immunocompetence appears to have a critical role in our case this is not generally true of others in the literature. of the 13 cases reported only 2 had a recognized immune deficiency, which was an unexplained pancytopenia. 4 • 11 pancytopenia has been described in other instances of disseminated mycobacterial infections as well but it is not ascertained whether this represents cause or effect of this disease. 12 finally, the radiographic manifestations of our case are typical of those usually associated with genitourinary tuberculosis. although pseudomonas superinfection was present it is not reasonable to expect that this was responsible for renal nonfunction and the exceedingly long left ureteral stricture. rather, the length and irregularity of the stricture are characteristic of granulomatous infection and healing, while the renal non-function may be attributed to obstruction and/or the effects of diffuse renal parenchymal infection. 11 the postmortem examination, revealing the presence of atypical mycobacteria in the kidneys and region of the ureteral stricture, supports this contention. although unusual, genitourinary infection by atypical mycobacteria is significant and potentially life-threatening. it is important to differentiate correctly this entity not only from pyogenic infections but from tuberculosis as well, because relative resistance of these organisms to conventional antituberculosis therapy may necessitate the institution of more heroic measures. anonymous mycobacteria in pulmonary disease atypical mycobacteria: what you should know about diagnosis and treatment human infection with the "yellow" acid-fast bacillus; report of 15 additional cases pancytopenia and death. disseminated anonymous mycobacterial infection renal hematuria associated with atypical acid-fast bacillus: battey type. cure by partial nephrectomy urogenital tuberculosis in children unclassified mycobacterial diseases disseminated mycobacterium kansasii infection with pancytopenia and interstitial nephritis renal tuberculosis in children disseminated mycobacterium kansasii infection: successful treatment of a patient with pancytopenia key: cord-026595-imn2jxcu authors: qamar, mariam khan; shaikh, babar tasneem; afzal, aamir title: what do the dental students know about infection control? a cross-sectional study in a teaching hospital, rawalpindi, pakistan date: 2020-06-01 journal: biomed res int doi: 10.1155/2020/3413087 sha: doc_id: 26595 cord_uid: imn2jxcu background: dental students are exposed to various infections and infective sources during their training, and on this aspect, their level of knowledge is suboptimal and practices are risky. therefore, improving their knowledge and practices would contribute significantly to infection control. objective: to ascertain the level of understanding of senior dental students regarding the infection control in the dental practice. methods: a cross-sectional study was conducted among dental students (3rd year and 4th year) of the foundation university dental college, pakistan. the sample consisted of 100 third year dental students and 88 fourth year students. a self-administrated questionnaire was used for data collection which consisted of fourteen close-ended items. frequencies of knowledge, attitudes, and practice were calculated separately by using spss 21.0 software. results: almost half of the students would not use any antiseptic for sterilizing their hands, and only two-third would ask their patient to use an oral mouth rinse before starting the treatment. many students did not the optimal temperature of the autoclave for sterilization of the instruments. only one-third would wear the personal protective equipment during a procedure. around one-third of the study participants reported that ineffective sterilization during clinical practice can transmit infection from one patient to another. conclusion: knowledge on infection control among the dental students is though weak, practices are not as per standards but attitudes are positive and encouraging for taking steps and complying with measures on infection control. dental students are one of the dental health care professionals who are at a high risk of exposure to infections because of their direct contact with the patients, infected instruments, and hospital environment. cross infection is a major concern to all the dental health care professionals. it is defined as the transmission of infection between the staff and the patient within the hospital environment [1] . among these, the most serious oral infections are caused by bacteria that colonize in the oral cavity including mycobacterium tuberculosis, influenza virus, and streptococci. students are equally vulnerable to cross infections that are caused by the hepatitis b virus (hbv), hepatitis c virus (hcv), and other viruses [2] . moreover, they are also at a risk of percutaneous occupational injuries and eye exposure while treating the patients [3] . only through strict safety precautions and implementation of guidelines for infection control, we can prevent these mishaps from happening. the center for disease control and prevention (cdc) in the united states has updated its guidelines on infection control in dental settings. the aim of these guidelines is to ensure a safe working environment to prevent the transmission of nosocomial and occupational infections among the dental health professionals [4, 5] . dental colleges are responsible for providing proper training to their students to ensure safety of the patients, implementing infection control measures, and establishing a safe working environment [6] . several studies have been conducted to assess the practices and knowledge of dental students and have demonstrated poor compliance of the students to infection control measures. a study conducted in india to assess the infection control practices among dental students showed that only one-tenth of the respondents adhere to the infection control measures [1] . similar studies have been conducted worldwide to investigate the knowledge and practices of dental students on infection control [6] [7] [8] [9] [10] [11] [12] [13] [14] , and a general consensus is that students need awareness and must be protected in the unsafe environment. fauji foundation hospital in rawalpindi city houses a huge number of medical and dental students for educational and training purposes. this hospital is a busy tertiary health facility catering to mostly ex-army servicemen and their families. it has a fully equipped dental outdoor clinic where the dental undergraduate students come on daily basis for observations, education, hands-on training, or apprenticeship. nevertheless, there is paucity of data with regard to infection control practices among dental students within the hospital environment in pakistan. for this purpose, it is imperative to ascertain the understanding of the senior dental students who have attended the module of infection prevention and control during their study course. this study will help better understand the gaps and deficiencies in the dental college curriculum and will sensitize and educate the future dental surgeons in adopting the necessary infection prevention practices. a cross-sectional study was conducted among dental students (3 rd year and 4 th year) attending the foundation university dental college, pakistan. the duration of study was two months from 14 th july 2019 to 14 th september 2019. the sample size (n = 188) was calculated by using the who sample size calculator with the following parameters: confidence level = 95%, anticipated population proportion = 95:5% [6] , and absolute precision required = 3%. a total of 228 dental students studying in the college included 121 third year and 107 fourth year students. out of these approaches, 82.6% (n = 100) students from the third year and 82.2% (n = 88) students from the fourth year gave a complete response and therefore were included in the study. as per the exclusion criteria, the students of first and second year were excluded from the study because training in infection control is provided in the dentistry curriculum of third year and fourth year. written informed consent was taken from all the participants of the study. the institutional review board of the hospital granted the requisite permission to conduct the study. a total of 188 dental students completed the questionnaire comprising of 14 items. there were 6 questions related to "knowledge," 5 questions related to practice, and 3 questions were based on "attitude." a self-administered questionnaire developed by singh et al. [6] was distributed to the dental students. the consistency of the questionnaire was assessed using cronbach's alpha (α = 381). each student was given fifteen minutes to fill the questionnaire in silence without discussing with each other. they were not asked to put their names on the forms, hence keeping the anonymity intact. data was entered and analyzed on spss v.21. descriptive statistics was done in terms of mean and standard deviation. frequency and percentage were calculated for qualitative variables. independent sample t-test was used to compare the mean score of knowledge, attitude, and practice among both groups. p value ≤ 0.05 was taken as a level of significance. written verbal consent was obtained from all the respondents. study received approval from the ethics and research committee of the foundation university, rawalpindi, dated january 2, 2019. a total of 188 dental students responded to our questionnaire completely and hence included in the study results. hence, the study population comprised of 28 male and 168 female students. table 1 shows the analysis of cross tabs between the independent (gender and class of study) and dependent variables which yielded no significant statistical association (i.e., p > 0:05). table 2 shows that although a majority (94%) reported washing their hands before and after examination of the patient, yet only half of them (49%) would use an antiseptic solution for washing their hands. there were 64% participants who preferred patients to have an oral mouth rinse before commencement of any treatment procedure. the majority (98%) of the students considered isolation as an important measure for the infection control. with regard to vaccination, 85% got vaccinated for hepatitis b, 45% for tetanus, and only 15% of the students were vaccinated for tuberculosis, but surprisingly, 18% have not received any vaccine at all. on the subject of sterilization of instruments, 95% reported use of autoclave for the purpose and only 68% reported using the same for 15 minutes (optimal time) and only 60% thought that 120 degrees is the required temperature on which the autoclave should be used. around 80% study participants considered that hepatitis b has the highest rate of transmission via saliva. in case of direct blood contact with a hiv patient, 62% would opt for anti-hiv immunoglobulins. during the work at a dental surgery, the use of face mask and gloves as an infection control measure was practiced by 38% while 32% would wear an eye protector, and only one-third of them (29%) would wear all of them. after using a pair of gloves on the patient, 57% would dispose them off but 43% think that they can reuse them after washing. only 37% reported that ineffective sterilization during clinical practice can transmit infection from one patient to another, although a large majority (98%) believe that besides instruments, disinfecting the dental chair, clinic, and doctor's office is necessary. in table 3 , it has been observed that there is a significant positive correlation between the mean score of knowledge and attitude r = 0:781, p ≤ 0:05; however, there was no differences hands are the foremost significant reservoir for many pathogens. handwashing is therefore considered as an effective method of prevention of infection [15] . in the current study, the majority of the students described that hands should be washed and the students described plain soap and antiseptic solution before and after the examination as a preferred method. similar results were seen in a study conducted in germany where most of the trainee dentists washed their hands before and after the procedure [8] . nonetheless, there are studies that show poor compliance of the students with handwashing [9] . a study conducted in yemen reported only 43% of the students adhere to handwashing [9] . in the present study, most of the dental students were vaccinated with hepatitis b vaccine which was in concurrence with the other studies [12] [13] [14] . this rate is much higher than the study conducted in india showing that only 38% of the students were vaccinated against hepatitis b [6] . the dental students had good knowledge of autoclaving and sterilization similar to other studies [6, 9] . wearing of face mask and eye protection was observed in only one-third of the students. similar results were recorded in other studies [9] . however, a good majority of the dental students in our study thought that hepatitis b infection and not tuberculosis is transmitted through saliva, which was quite surprising. another surprising finding in our study was that two-thirds of the students considered that ineffective sterilization during clinical practice cannot transmit infection from one patient to another. this highlights the lack of awareness of the concept of cross infection. the majority of the students did believe that disinfection of dental chairs and the office of the dentist is also required. this concurs with the findings of other studies included in our literature review [2, 3] . importance of continuous-based infection control lectures and training could help in raising the level of knowledge regarding the subject [16] . we recommend that every hospital needs to set up infection control measures in accordance with the guidelines. for both the students and faculty of a hospital to improve their practice and knowledge of infection control, hospitals need to address the need for quality assurance and implementation of infection control measures. it is highly recommended that reinforcement training of the students through periodic workshops on infection precaution and control should be planned to highlight the significance of infection control [17] . another recommendation is that of vaccination, especially hepatitis b, which should be mandatory for all medical and dental students prior to their admission. the strength of this study is that it was conducted with a senior group of dental students (3 rd and 4 th year) who have studied the module on infection prevention and control. the use of a self-administered questionnaire itself is one of the limitations of this study; therefore, objectivity in responding to certain questions might have been compromised. results could be generalized with caution because similar group of students in a private hospital setting with better infection control and prevention standard operating procedures might exhibit different behaviors and practices. we concluded that there was lack of correct knowledge on infection control among the dental students, yet they showed a positive attitude towards infection control measures. however, a greater compliance and supervision would be needed. further studies in similar settings and utilizing the mix methods with qualitative enquiry would be beneficial to understand certain careless attitudes and behaviors among dental students. available with first author. the authors declare that they are no conflict of interests. compliance with infection control programs in private dental clinics in jordan attitudes and practices of infection control among senior dental students at college of dentistry, university of sharjah in the united arab emirates infection control: knowledge and compliance among saudi undergraduate dental students updated cdc recommendations for the management of hepatitis b virus-infected health-care providers and students guidelines for infection control in dental health-care settings-2003 knowledge, attitudes, and practice regarding infection control measures among dental students in central india cross-infection control in malaysian dental practice rate of compliance with hand hygiene by dental healthcare personnel (dhcp) within a dentistry healthcare first aid facility knowledge, attitudes, and practice of infection control among dental students at sana'a university knowledge, attitude and practices about hepatitis b and infection control measures among dental students in patiala infection control measures in private dental clinics in lebanon knowledge, attitude, and practice of needle stick and sharps injuries among dental professionals of bangalore, india awareness of droplet and airborne isolation precautions among dental health professionals during the outbreak of corona virus infection in riyadh city, saudi arabia knowledge, attitude and practice of infection control measures among dental practitioners in public setup of karachi, pakistan: cross-sectional survey compliance with infection control practices in a university hospital dental clinic knowledge, attitude and compliance of infection control guidelines among dental faculty members and students in ksu evaluation of final-year turkish dental students' knowledge, attitude, and self-perceived competency towards preventive dentistry appreciation is hereby extended to the students of foundation medical and dental university for participating in our study. mkq, bts, and aa were all responsible for defining the initial research question and developing the protocol and study design. mkq and aa were involved in the collection of data and took lead in writing the manuscript. bts contributed to the completion of the manuscript and has critically revised it. all authors read and approved the final manuscript. key: cord-023942-vrs3je1x authors: powers, karen s. title: acute pulmonary infections date: 2011-12-16 journal: pediatric critical care study guide doi: 10.1007/978-0-85729-923-9_25 sha: doc_id: 23942 cord_uid: vrs3je1x acute lower respiratory infection is a common cause of morbidity in infants and children, and at times, requires intensive care and mechanical ventilation. viral bronchiolitis and bacterial pneumonia account for the majority of lower respiratory tract infections that lead to respiratory insufficiency and pediatric intensive care admission. twenty-seven percent of children who require mechanical ventilation for at least 24 h in pediatric intensive care units are diagnosed with bronchiolitis and 16% have the diagnosis of pneumonia. the median length of time intubated for an acute pulmonary infection leading to respiratory failure is approximately 7 days. acute lower respiratory infection is a common cause of morbidity in infants and children, and at times, requires intensive care and mechanical ventilation. viral bronchiolitis and bacterial pneumonia account for the majority of lower respiratory tract infections that lead to respiratory insuffi ciency and pediatric intensive care admission. twenty-seven percent of children who require mechanical ventilation for at least 24 h in pediatric intensive care units are diagnosed with bronchiolitis and 16% have the diagnosis of pneumonia. the median length of time intubated for an acute pulmonary infection leading to respiratory failure is approximately 7 days. viral bronchiolitis remains the leading cause for hospital admission in infancy and the most frequent cause of acute respiratory failure in children admitted to pediatric intensive care units in north america. pneumonia in children younger than 5 years of age has an annual incidence karen s. powers of 34-40 cases per 1,000. community acquired pneumonia can also lead to severe respiratory compromise especially in children with pre-existing disease. a detailed understanding of the diverse etiologies and distinct clinical courses of acute pulmonary infections is essential for the pediatric critical care practioner. this chapter will focus on bronchiolitis and pneumonia as the two leading causes of pulmonary infections leading to picu admission. approximately one third of children develop bronchiolitis during the fi rst 2 years of life. of these, only 1 in 10 (3% of all infants in the united states) will require hospitalization. although hospitalization rates have increased over the last three decades, mortality remains low. overall mortality rate is 1-2%, but as high as 5% in high risk infants. most deaths occur in infants younger than 6 months of age with co-morbidities such as prematurity, congenital heart disease, congenital or acquired lung disease or immunodefi ciency. respiratory syncytial virus (rsv) was fi rst isolated in 1957 and still represents the major cause of bronchiolitis. other causative viruses include parainfl uenza, adenovirus, enterovirus, infl uenza and most recently human metapneumovirus and human bocavirus (hbov). in the northern hemisphere, rsv outbreaks occur from october to june. human metapneumovirus (hmpv) recently has been identifi ed as the causative agent in 3-19% of bronchiolitis cases, possibly surpassing parainfl uenza as the second most common etiology. its prevalence is slightly higher in the late winter and spring. parainfl uenza infections peak at 10 months of age, representing approximately 7-10% of cases of bronchiolitis. parainfl uenza (piv-3) is endemic throughout the year, but especially common in the late spring. males are 1.5-2 times more likely to require hospitalization for bronchiolitis and are likely to have more severe disease. an x-linked genetic trait that results in a reduced tolerance to hypoxia has been postulated and would be consistent with the observation of increased mortality in newborn males with infant respiratory distress syndrome. virtually all children by the age of two will have been infected with rsv, all children by the age of fi ve will have been infected with hmpv, and all children by the age of nine will have been infected with hbov. the remainder of the discussion on bronchiolitis will be divided into rsv and non-rsv bronchiolitis. although etiologic agents may differ, clinical courses are often similar. respiratory syncytial virus (rsv) accounts for 50-80% of bronchiolitis, infecting one-half of all infants within the fi rst year of life and hospitalizing approximately 120,000 infants yearly (about 3% of affected infants). approximately 10% of these infants require mechanical ventilation. co-infection with either hmpv or rhinovirus occurs in 10-30% of young children. two types of rsv exist -types a and b. type a is more common and is believed to cause more severe disease, although data is not conclusive. both types may exist simultaneously in the community. infants less than 1 year will typically shed the virus for about 9 days. children with immunodefi ciencies may shed the virus for months. the immune response varies with age and contributes to both termination of the disease and its pathologic features. the virus is transmitted from respiratory secretions by close contact with infected persons or by contact with contaminated objects or surfaces. there is a 45% rsv transmission rate within families and about one-half of hospital workers will acquire rsv. therefore, hand washing and the wearing of gowns and gloves is of primary importance to attenuate transmission. mortality from rsv bronchiolitis continues to decline with better intensive care and the use of preventive therapies. male infants are more likely to require hospitalization and usually manifest more severe disease. about ½ of all infants will be infected with rsv bronchiolitis in their 1st year of life; 3% will be hospitalized; 10% of hospitalized infants will require mechanical ventilation. antibody-mediated immunity rsv introduced onto the nasal or conjunctival mucosal surface causes profuse rhinorrhea within a few days. during the fi rst 2 months of life, passively acquired maternal antibodies are protective. however, as maternal antibody titers gradually decrease, infants become susceptible to severe disease. cell-bound iga may develop to help clear the virus. circulating igg directed against the glycoprotein (g) and fusion (f) proteins (operative in syncytia formation) on the viral surface will develop several days later. infants less than 3 months of age appear to induce a weaker antibody response likely due to the presence of maternal antibodies. virus-specifi c ige in the respiratory tract is associated with disease severity. often, complete and effective immune responses are not induced, thus re-infections are possible even during the same season. epithelial cells and alveolar macrophages are key activators of cellular immunity. although these cells enhance viral clearing, they also contribute to airway infl ammation through the release of cytokines and chemokines. these include interleukin (il)-1, tumor necrosis factor-alpha, il-6, il-8, macrophage-infl ammatory protein (mip)-1-alpha and rantes (regulated upon activation, normal t cell expressed and secreted). release of these cytokines and chemokines are believed to be partially responsible for airway infl ammation and hyperreactivity. the effects of these mediators persist beyond the acute infection and contribute to prolonged pulmonary dysfunction. children who require mechanical ventilation have lower peripheral t cell counts compared to hospitalized infants not requiring mechanical ventilation. these infants demonstrate low t cell proliferative responses and interferon (ifn-g ) production. il-12 is required for the initiation of cellular immunity. the length of time requiring mechanical ventilation has been found to be inversely related to il-12 production. the role of th1/th2-like cytokine profi les, expressed as ifn-g /il-4 ratios, is controversial. in some studies, these ratios decreased after polyclonal stimulation in hospitalized infants with rsv. however, more recent studies have shown normal ratios following polyclonal stimulation. neutrophils are the predominant cell found in the airways of infants with rsv bronchiolitis. elevated levels of il-8 are found in high concentrations in the nasal secretions of infected children and act as a neutrophil chemoattractant. further evidence of cellular induced injury is seen in post-mortem examination where peribronchial lymphocyte infi ltration with bronchial epithelial necrosis is typically present. infants typically present with tachypnea, rhinorrhea, cough, low-grade fever, irritability, poor feeding and vomiting. respiratory rates greater than 60 breaths per minute are often associated with room air saturations of less than 96%. infants may also have tachycardia, mild conjunctivitis, otitis media, or pharyngitis. low-grade fever usually persists for 1-3 days. in addition, infants may develop a metabolic acidosis from poor caloric and fl uid intake. apnea often is the fi rst presenting symptom of rsv bronchiolitis in small infants. the etiology of apnea remains unknown; however, is likely related to the immaturity of the respiratory control center in the brainstem. the incidence of apnea in infants with bronchiolitis is approximately 16-20%. the heterogeneous nature of rsv induced lung disease can cause atelectasis in some areas and overdistension in others. chest roentgenograms often show hyperinfl ation with fl attening of the diaphragms and patchy or peribronchial infi ltrates. atelectasis, especially of the right upper lobe, is often seen. infants may have high lung volumes with the functional residual capacity often being twice normal. the decrease in dynamic compliance and increase up regulation of the infl ammatory cascade with release of chemokines and cytokines are contributory to the airway infl ammation and hyperreactivity. c hapter 25 • ac ute pu lmonary i n fections in airway resistance leads to marked increase in work of breathing, often worse during expiration from lower airway obstruction. alterations in gas exchange and hypoxemia are secondary to a ventilation-perfusion mismatch. the anatomical differences between young infants and older children contribute to the severity of the disease in the young. due to the highly compliant cartilaginous chest wall and poor thoracic musculature, the infant's chest wall has diffi culty countering the lung's inherent tendency towards collapse. this leads to a greater propensity of small infants towards atelectasis compared with older children. the absence of effective collateral ventilation in infants also contributes to the development of atelectasis and impaired gas exchange. cellular debris in small airways and peribronchial edema increase airways resistance leading to wheezing as the predominant symptom in some infants. despite the potential for severe impairment in lung function, most hospitalized infants improve within 3-5 days. typically, by 2 weeks, they have normal respiratory rates, oxygenation, and ventilation. chest radiographs usually normalize by day 9. however, about 20% of infants will have a protracted course, with some mild respiratory symptoms persisting for months. viral respiratory infections have been linked to the development of asthma later in childhood. the tucson children's respiratory study group prospectively followed for 13 years, 880 infants who had bronchiolitis and found an increased risk for subsequent wheezing episodes. some infants are at an increased risk for severe rsv disease such as those with chronic lung disease due to prematurity (bronchopulmonary dysplasia), cystic fi brosis, congenital heart disease, and immunodefi ciencies. in children with cystic fi brosis, rsv accounted for 18% of symptomatic infections, 33% of hospitalizations for infants less than 1 year, and 43% of infants requiring mechanical ventilation. in a study of hospitalized infants with congenital heart disease infected with rsv, 33% required intensive care, 19% received mechanical ventilation, and 3.4% died. children having undergone hematopoietic stem cell transplants who develop rsv infections have an extremely high mortality of 60-80% despite mechanical ventilation and antiviral therapy. environmental factors such as crowding, passive exposure to tobacco smoke, and lack of breast-feeding are associated with the development of severe disease. compared to national averages, native american and alaskan children younger than 1 year of age have higher rates of infections. there are three subtypes of human parainfl uenza viruses. hpiv-3 is most frequently isolated from children with bronchiolitis, while piv-1 and piv-2 most commonly cause croup. similar to rsv, both cell-mediated hyper-responsiveness to viral antigen and virus-specifi c ige responses are observed in children with parainfl uenza bronchiolitis. upper airway edema with concomitant obstructive symptoms may be present. children that are infected with parainfl uenza have a signifi cant likelihood of developing asthma later in life. the human metapneumoviruses (hmpv) are a group of rna viruses of the paramyxoviridae family identifi ed in humans in 2001. hmpv appears to be the second most common cause of bronchiolitis in children throughout the world. the majority of children are born with maternal hmpv specifi c igg which wanes to around 25% by 6-12 months of age. by age fi ve, essentially 100% of children have been exposed to hmpv and will have neutralizing antibody to hmpv. there are two subgroups, a and b, with group a having more severe clinical symptoms. clinical presentation of children with this virus is similar to rsv. the pulmonary infl ammation generally peaks on day 5 which includes interstitial edema and infl ammatory cell infi ltrates of the bronchioles and alveoli. these infl ammatory changes can persist for up to 21 days. about half of infected children are 0-12 months of age, and infection is primarily in the winter months. human bocavirus (hbov) was recently discovered in 2003. with amino acid sequencing, this new member of the parvoviridae family was found to be closely related to the bovine parvovirus and the canine minute virus, hence the name bocavirus (bo for bovine and ca for canine). detection of the hbov from the respiratory tract in symptomatic children and its absence of detection in non-symptomatic controls strongly suggest the virus to have a role in respiratory infections in children. co-infection is commonly described in up to 60% of samples. it remains unclear if hbov is a primary pathogen or acts to exacerbate other viral illnesses. the pathogenesis of hbov has not been well described, but with the high occurrence of wheezing and lower respiratory tract symptoms in children infected with the virus, it is speculated that this virus may be a signifi cant contributor to asthma exacerbations. the majority of infected children have rhinorrhea, cough, and wheezing, however, diarrhea has been reported in up to 25% of these children. in children with high viral loads, hbov has been detected in the serum suggesting the potential for disease beyond the respiratory tract. both infl uenza a, including novel infl uenza strains such as h1n1, and infl uenza b can cause a clinical picture consistent with bronchiolitis in the small infant. these viruses may cause severe multisystem disease and are discussed in greater detail in the viral pneumonia section. rapid diagnostic assays are available for early detection of many viruses. the older assays are antigen-based and include indirect immunofl uorescence/direct immunofl uorescence (ifa/ dfa), enzyme immunoassay (eia), optical immunoassay (oia), and neuraminidase activity assays. although still widely used because they are inexpensive and technically simple, they have a low specifi city and sensitivity. molecular assays are becoming the new "gold standard" for respiratory virus detection -replacing tissue culture that may take days. the published sensitivities and specifi cities approach 100% when compared to tissue culture or antigen assay. these assays generally use polymerase chain reaction (pcr) amplifi cation. signifi cant advancements in these assays are being made to simplify the performance of the assay and decrease the required time. the most important cause of false negative test results remains poor specimen handling or inadequate sample collection. other than aiding with cohorting of hospitalized patients, serologic detection of respiratory viruses is rarely clinically useful. regardless of the viral etiology of bronchiolitis, supportive care remains the mainstay of treatment. supplemental humidifi ed oxygen is frequently needed. due to many infants being obligate nasal breathers, frequent nasal suctioning may be benefi cial to maintain an unobstructed upper airway. the affected infant or child is often unable to take adequate fl uids complicated by increased insensible losses from the respiratory tract; hence, intravenous fl uids may be required. infants and children with severe respiratory distress should be kept npo in the event respiratory failure ensues and endotracheal intubation is required. antibiotics are not routinely indicated in previously healthy children infected with rsv. progressive disease, leukocytosis, persistent fever, consolidation on radiograph or systemic toxicity should prompt an evaluation of bacterial co-infection and the use of empiric antibiotics. high risk patients often require close monitoring and care in an intensive care unit. these include infants less than 6 weeks of age or infants with a history of prematurity, congenital heart disease, bronchopulmonary dysplasia, immunodefi ciency or neurologic disease. infants with rsv bronchiolitis typically have a combination of hyperinfl ation, pulmonary infi ltrates, supportive therapy is the mainstay of treatment for bronchiolitis. ribavirin, bronchodilators, and corticosteroids have not shown to be of benefi t. secondary bacterial infections are rare. and atelectasis. therefore, no one mode of ventilation can be recommended for all infants. non-invasive positive pressure (niipp) modes (cpap or bipap) may be attempted in infants where their primary respiratory embarrassment is secondary to atelectasis. however, this may not be suitable if the disease process appears severe or protracted as prolonged use of nipp may make feeding diffi cult, cause breakdown of facial tissue, or be diffi cult to maintain without signifi cant sedation that further compromises ventilation. if an infant requires endotracheal intubation, the mode of mechanical ventilation should be tailored to the predominant lung pathology present (i.e. atelectasis versus hyperinfl ation). children with signifi cant air trapping may need mechanical ventilation similar to a child with asthma, providing low respiratory rates and longer inspiration and exhalation times. the more typical infant will lose functional residual capacity (frc) because of atelectasis and alveolar infi ltrates. therefore, despite having some air trapping, these infants often need peep to be adjusted to recruit alveoli and return frc to normal. in the setting of elevated pulmonary vascular resistance (pvr) which may occur in infants with congenital heart disease or bronchopulmonary dysplasia, lowering pvr by traditional methods such as maintaining oxygenation, deep sedation, muscle relaxation and even nitric oxide may be indicated. ribavirin is the only fda-approved antiviral drug for rsv. ribavirin inhibits viral replication and is active against rsv, infl uenza a and b, adenoviruses, and hepatitis viruses. for lower respiratory tract diseases, ribavirin is typically administered via aerosolization. in 1996, a meta-analysis of studies involving ribavirin was discouraging and was consistent with the common clinical experience that ribavirin did not improve clinical outcomes. therapy targeted at attenuating the virus-induced infl ammatory cascade has also been disappointing. corticosteroid administration was not associated with reduction in clinical scores, the need for hospitalization, or the length of hospitalization. routine use of any corticosteroid given via any route (intravenous, enteral or aerosolized) is not indicated, except in patients with pre-existing chronic lung disease. bronchodilators have not shown a clear benefi t in patients with acute rsv bronchiolitis. in 12 randomized control trials, involving 843 infants, evaluating the effect of salbutamol or albuterol on bronchiolitis, 9 (75%) showed no effect. the remaining three studies demonstrated only a small transient improvement in the acute clinical score. although the routine use of bronchodilator therapy cannot be recommended, it has become acceptable practice to attempt to see if individual infants are beta agonist responsive or not. if no clinical response is seen after a trial of a beta agonist, its use should be discontinued. in the 1990s, fi ve randomized trials involving 225 infants, evaluating the effect of nebulized adrenaline (epinephrine) on bronchiolitis showed clinical improvement, with reductions in oxygen requirement, respiratory rate, wheezing, and decrease in pulmonary vascular resistance. two of these studies showed lower hospital admission rates and earlier discharge. a 2004 cochrane systematic review suggested a potential benefi t with epinephrine administration. however, subsequent studies have not supported its routine use. as with albuterol, a clinical trial in selected infants seems reasonable. nebulized hypertonic saline has been used for treating hospitalized, as well as ambulatory, children with viral bronchiolitis with variable success. a recent cochrane meta-analysis of nebulized hypertonic saline has shown an improvement in clinical scores and decrease in hospital duration. several studies have evaluated the benefi t of surfactant and nitric oxide for severe respiratory distress. the results have been inconclusive and do not currently support their routine use. heliox, a mixture of oxygen (20-30%) and helium (70-80%) with lower viscosity than air has been used successfully in cases of airway obstruction, croup, airway surgery, and asthma to reduce respiratory effort during the period of airway compromise. several studies have shown improved respiratory distress scores in patients on heliox with continuous positive airway pressure obviating the need for intubation and mechanical ventilation. palivizumab is a neutralizing humanized mouse monoclonal antibody directed against the rsv-f glycoprotein. it was licensed by the food and drug administration (fda) in 1998 for premature infants and infants with bronchopulmonary dysplasia. the randomized, double blind, placebo controlled impact-rsv trial involving 1,502 high risk infants found a signifi cant reduction of 55% in hospitalizations. with the exception of very rare anaphylaxis, no signifi cant adverse effects have been observed. palivizumab has been approved for use in infants with congenital heart disease. the cardiac synagis study group included 1,287 children with congenital heart disease in a randomized, double blind, placebo controlled trial; it found a 45% relative reduction in rsv associated hospitalizations with no deaths attributable to the palivizumab. since cardiopulmonary bypass can decrease serum drug concentrations by about 58%, it is recommended that an additional dose be given following surgery, if continued protection is desired. palivizumab should be administered intramuscularly as 15 mg/kg every 30 days for a total of fi ve doses during rsv season, which is generally from november through march, to high risk infants. infants or children that develop an rsv infection should continue to receive prophylaxis following recovery because the naturally acquired antibodies are not fully protective. motavizumab, a new, enhanced potency, humanized rsv monoclonal antibody has demonstrated 50-100 times greater neutralizing activity against rsv. in completion of a phase iii trial, motavizumab was found equal to palivizumab for the prevention of rsv hospitalization and superior to palivizumab for reduction of rsv-specifi c outpatient medically attended lower respiratory tract infections (malris). pneumonia describes any infl ammatory condition of the lung in which the alveoli are compromised by aspirated foreign matter, infl ammatory fl uid, or cellular debris. infection is the primary cause of parenchymal injury to the lung. pathogens include viruses, bacteria and fungi. signs and symptoms of pneumonia are non-specifi c and may be occult in the young infant. children often have fever, chills, headache, malaise, restlessness, and irritability. gastrointestinal complaints such as abdominal pain, distention, or emesis may also be present in young children. the symptoms are often preceded by minor upper respiratory tract infections characterized by low-grade fever and rhinorrhea. with more signifi cant involvement of the lower respiratory tract, tachypnea, dyspnea, cough, nasal fl aring, grunting, or retractions may be seen. the older child may demonstrate productive sputum and complain of pleuritic chest pain. on auscultation of the chest, rales and/or decreased breath sounds might be heard over areas of consolidation or pleural effusions. however, due the short path for transmission of breath sounds and the small chest size in infants, breath sounds may not be decreased, even in the presence of effusions. children with pleural irritation might prefer to lie on the affected side with legs fl exed and may complain of radiating pain to the neck and shoulder or into the abdomen. community acquired pneumonia (cap) is a common, and at times, a serious infection in children. the incidence of cap is 35-40 cases per 1,000 children less than 5 years of age and 11-16 cases per 1,000 children 5-14 years of age. the exact prevalence of the etiologic agents causing pediatric pneumonia is diffi cult to ascertain. it is often diffi cult to differentiate viral from bacterial pneumonia based solely on clinical examination. specifi c pathogens causing cap can be determined in only approximately one-third of children using commonly available cultures, antigen detection, or serologic techniques. blood cultures yield pathogens in only about 10-15% of infants and children with bacterial cap and many children do not undergo viral testing as it is often unnecessary. with these inherent limitations, it is generally thought that viruses account for approximately 80% of cap in children under the age of 2 years and approximately 50% of cap in preschool children ages 2-5 years. palivizumab should be used as preventive therapy in infants with chronic lung disease and congenital heart disease. cardiopulmonary bypass signifi cantly lowers the serum level of palivizumab, so it should be redosed following surgery if continued protection desired. viral causes decline in the school age and adolescent child and bacterial causes such as streptococcus pneumoniae and mycoplasma become important pathogens ( fig. 25-1 ) . overall, bacteria account for 20-30% of community-acquired pneumonias. the likelihood of infection with different bacteria varies by age. in the newborn period, organisms from the maternal genital tract are likely causes and include group b streptococcus , escherichia coli , enteric gram-negative bacilli, listeria , and chlamydia . in older infants, streptococcus pneumoniae becomes a signifi cant cause and remains so until 6 years of age. group a streptococcus and staphylococcus aureus are uncommon causes. moraxella catarralis is a common cause of upper respiratory tract disease, but rarely causes pneumonia. about 20% of infants with pertussis will have bacterial co-infection. in children older than 6 years of age, streptococcus pneumoniae remains the most common cause. hemophilus infl uenzae type b (hib), and most recently streptococcus pneumoniae , have decreased signifi cantly as causes of cap due to the widespread use of effective vaccines. in the older child and young adolescent, the atypical pneumonias, mycoplasma and chlamydia , become more prevalent and viral causes less common. rare bacterial pneumonias can occur with animal contact and include: francisella tularensis (rabbits); chlamydia psittaci (parrots and birds); coxiella burnetii (sheep); and salmonella choleraesuis (pigs). children with congenital anatomical defects, immunodefi ciencies, and genetic disorders are at increased risk for bacterial, viral and fungal pneumonia. the airways are normally sterile below the sublaryngeal area to the lung parenchyma. there are several protective mechanisms that include anatomic and mechanical factors, local immune defenses, and the systemic immune response. microbes are fi ltered by nasal hairs or are expelled from the airways by the epiglottic refl ex, cough refl ex, and mucociliary apparatus. immunoglobulin a (iga) is the predominant immunoglobulin present in the upper respiratory tract. iga is able to bind two antigens simultaneously, forming large antigen-antibody complexes. in this manner, the microbes are neutralized and removed by ciliary clearance, thus preventing microbial binding to the epithelium. in the lower tract, immunoglobulin g (igg) provides humoral protection by opsonizing microbes for phagocytosis by neutrophils and macrophages, activating the complement cascade, and by neutralizing bacterial it is diffi cult to determine the etiologic agent causing pneumonia, but when microbial agents are identifi ed, bacteria are isolated in 20-30%. etiology of community acquired pneumonia based on age endotoxin. alveolar macrophages produce superoxide anions, hydrogen peroxide, and hydroxyl radicals that serve an important role in the host defense; however, uncontrolled production can lead to lung injury. in addition to oxygen radicals, a number of cytokines are produced by the alveolar macrophages. these include il-1, il-6, tnf, transforming growth factor-β (tgf-b ), chemotactic factors, platelet derived growth factor, and m-csf. these cytokines play a central role in phagocytic recruitment and activation. infection occurs when one or more of the defense mechanisms is altered or if the inoculum is too large. pathogens typically gain entry through inhalation of aerosolized material or through aspiration of resistant organisms inhabiting the upper airways. less frequently, pneumonia can occur via hematogenous spread. in children with bacterial pneumonia, a signifi cant portion will have a concurrent or preceding viral infection. viral infection may predispose to bacterial superinfection by reducing clearance mechanisms and by weakening the host immune response. pathogens entering the lower airways evoke an exudative consolidation of pulmonary tissues. initially, there is hyperemia of lung parenchyma due to vascular engorgement and capillary leak causing exudation and intra-alveolar fl uid accumulation. fibrin is then deposited and the airways are infi ltrated with neutrophils. consolidation causes a decrease in lung compliance and vital capacity and a total reduction in the surface area available for gas exchange. a physiologic shunt (v/q mismatch) occurs as there is increased blood fl ow through poorly ventilated segments of lung, resulting in hypoxia. compensatory hypoxic vasoconstriction may occur in an attempt to reduce v/q mismatch and hypoxia, especially in localized areas of consolidation. with treatment, resolution of consolidation will occur in 8-10 days. the exudate undergoes enzymatic digestion and is either reabsorbed or removed by coughing. if the bacterial infection extends into the pleural cavity, an empyema may result. streptococcus pneumoniae is a gram-positive diplococcus that is frequently found in the upper respiratory tract. there are over 80 capsular serotypes with 80% of infections caused by 14 serotypes. it is the most common bacterial cause for pneumonia occurring at a peak age of 13-18 months. typically, it causes a lobar or segmental consolidation, but it may manifest as patchy infi ltrates in infants. pleural effusions occur in up to 20% of children that require hospitalization (fig. 25-2 ) . pneumatocoele formation is rare. hemolytic uremic syndrome is associated with neuraminidase-producing strains. treatment is typically with a penicillin or cephalosporin. emerging resistance may require initial therapy with vancomycin. in hospitalized patients, parenteral therapy is generally needed for 48-72 h after fever resolves, followed by completion of 7-10 days of enteral therapy. pneumococcal conjugate vaccines (pcv) have been developed that confer immunity against 7 and 13 serotypes. the 7-valent pcv (prevnar) was licensed for use in the united states in 2000. a 13-valent pcv has been recently introduced and will replace the 7-valent pcv. the pcvs have been highly effective at reducing hospitalizations among children younger than 2 years for pneumococcal pneumonia. pcv is now recommended universally for children younger than 24 months of age and older children at high risk due to underlying diseases. high risk children include those with sickle cell disease and other types of functional asplenia, human immunodefi ciency syndrome, primary immunodefi ciency, children receiving immunosuppressive therapy, and children with chronic pulmonary or cardiac disease. a 23-valent pcv is available for pneumonia occurs when one or more of the host defense mechanisms are altered. viruses enhance the host susceptibility to bacterial pathogens by affecting clearing mechanisms and by weakening the host immune response. streptococcus pneumoniae is the most common bacterial cause for pneumonia. c hapter 25 • ac ute pu lmonary i n fections high risk children who need expanded serotype coverage. children with sickle cell disease or functional asplenia should continue to receive antibiotic prophylaxis regardless of whether or not they have received pneumococcal vaccines. approximately 50-75% of infants born to chlamydia trachomatis -infected mothers will become infected at one or more anatomical site, including conjunctiva, nasopharynx, rectum, and vagina. about 30% of infants with nasopharyngeal infections will develop pneumonia. the infants usually present at about 4-12 weeks of age with cough and congestion, but an absence of fever. the cough often interferes with the ability to feed. infants generally have tachypnea and rales on examination and chest x-ray frequently shows hyperinfl ation. a peripheral eosinophilia may be present. c . trachomatis is susceptible to macrolides, tetracyclines, quinolones, and sulfonamides. erythromycin for 2-3 weeks is the treatment of choice for neonatal pneumonia. mycoplasma pneumoniae and chlamydia pneumoniae play a greater role in causing respiratory tract disease in children then previously thought. an indolent course that develops over 5-7 days manifested by low-grade fever, scratchy sore throat, aches, and headaches characterizes both pathogens. after a few days, rales may be heard, particularly in the bases where the infi ltrates tend to occur. these organisms have been associated with the initiation, promotion, and exacerbation of asthma in children. in addition, a pertussis-like illness with acute bronchitis has been described. a recent study has shown that nearly half of the cases of community-acquired pneumonia in children aged 2-14 years were associated with m . pneumoniae or c . pneumoniae . classic atypical pneumonias caused by these organisms are usually mild and self-limited. however, a number of studies have suggested that severe pulmonary infection may occur in otherwise healthy children. pleural effusions, pneumatocoeles, lung abscesses, pneumothoraces, bronchiectasis, chronic interstitial fi brosis, and acute respiratory distress syndrome although rare complications, have all been reported. serological testing is the most common means of diagnosis, but this is often retrospective. cultures obtained from swabbing the nasopharynx may take several days to grow. pcr techniques are currently being refi ned and standardized. treatment with antibiotics reduces the rate of recurrent wheezing episodes, decreases morbidity, and shortens the duration of symptoms. the organisms are susceptible to tetracyclines, macrolides, and quinolones. the optimal doses and duration of treatment is unclear; however, some data suggest that prolonged treatment for greater than 2 weeks may be more desirable to decrease symptoms and eradicate the organism from the nasopharynx. chlamydia pneumoniae have an increased prevalence in older children. chest radiograph of 3 year old female with streptococcus pneumoniae pneumonia. note the combination of consolidation and effusion affecting the right lung. (image provided courtesy of fa maffei) staphylococcus aureus is a gram-positive organism that can be found on the skin, nasal mucosa, and other mucus membranes. about 20-30% of children are carriers. it is generally spread by direct contact or by respiratory particles. s . aureus is an unusual cause of lower airway disease in otherwise healthy children. it is more typically isolated from infants and young children with debilitating conditions. primary s . aureus pneumonia presents in the winter or early spring with a short febrile prodrome and a rapid onset of pulmonary symptoms. blood cultures are positive in 20-30% of patients. secondary staphylococcal pneumonia will have a more prolonged prodrome with no seasonal predilection, but is often seen after infl uenza infections. as this secondary pneumonia is usually a result of hematogenous spread, blood cultures are positive in about 90% of patients. unilateral lobar disease is more typical with primary disease, while diffuse bilateral infi ltrates are more frequent with secondary pneumonia. effusions can be diagnosed in about 15% of children at presentation, but ultimately will develop in about 75% of cases. pneumatocoeles occur in up to 45-65% of children. treatment is with nafcillin or oxacillin, but more organisms are becoming resistant and require therapy for serious or invasive disease with vancomycin, linezolid, daptomycin, or quinupristin-dalfopristin. methicillin resistant staphylococcus aureus (mrsa) was once considered to be restricted to hospitals and long-term care facilities. however, community acquired mrsa (ca-mrsa) is now a signifi cant cause of a variety of infections (including pneumonia) in children without prior health care facility exposure. the majority of community acquired mrsa infections involve minor skin and soft tissue infections, but invasive and sometimes fatal infections can occur in otherwise healthy individuals. ca-mrsa and healthcare-associated mrsa (ha-mrsa) can be distinguished by several important features. patients with ca-mrsa by defi nition have not had recent hospitalization (acute or chronic care), prolonged antibiotic use or chronic underlying disease. toxin production also distinguishes ca-mrsa from ha-mrsa. panton valentine leukocidin (pvl) is a toxin which is present in most ca-mrsa isolates, but rarely in ha-mrsa isolates. pvl toxin lyses white blood cells leading to leukopenia and a decreased ability to kill s . aureus . its production has been implicated as a contributor to the development of ca-mrsa necrotizing pneumonia. ca-mrsa isolates, unlike ha-mrsa, lack multi-drug resistance. ca-mrsa is generally more susceptible to clindamycin, trimethoprim-sulfamethoxazole and doxycycline than ha-mrsa, probably because ha-mrsa has developed resistance to survive in the healthcare setting. group a betahemolytic streptococcus (gabhs) is a gram-positive organism responsible for about 15% of pharyngitis and tonsillitis in children. it is rare as a primary cause of pneumonia. when it does occur, the children generally have high fever and appear toxic. the pneumonia is typically lobar. associated empyemas are common and pneumatocoeles may develop. there are several virulent toxin-producing gabhs m-serotypes that are associated with toxic shock syndrome. pre-existing varicella disease with disruption of skin and soft tissue as the port of entry is reported approximately 40-50% of the time. an associated pneumonia occurs in 10-20% of children with toxic shock syndrome. gabhs are highly susceptible to penicillins and cephalosporins. in cases of toxic shock, clindamycin is often added to inhibit the production of streptococcal pyrogenic exotoxins a (spe-a) and b (spe-b). about 30-40% of infants with perinatally acquired group b streptococcus (gbs) infections will have pneumonia. the infant usually has systemic disease and blood cultures are frequently positive. late-onset gbs is predominantly caused by the type iii serotype. in these infants, the infection is usually manifest as bacteremia without a focus or with meningitis. pneumonia is rare in late-onset disease. gbs is uniformly sensitive to penicillin. while staphylococcus aureus pneumonia is uncommon, effusions ultimately develop in about 75% of cases and pneumatocoeles occur in 45-60%. pertussis, or "whooping cough" is a highly contagious respiratory tract infection caused by the gram-negative pleomorphic bacillus bordetella pertussis and less commonly bordetella parapertussis . with the development and widespread use of a vaccine in the 1940s, a significant and sustained decrease in incidence has occurred. however, despite immunization rates greater than 80%, cyclical recurrences of the disease have occurred every 3-4 years since the 1980s. this is likely secondary to the waning of immunity in adolescents and young adults. under-immunized or unimmunized infants are the most vulnerable. nearly all deaths reported from pertussis occur in infants younger than 3 months of age. pertussis is often divided into catarrahal (fever, rhinnorhea and initiation of cough), paroxysmal (severe coughing episodes, lymphocytosis, potential for complications) and convalescent stages (slow waning of cough over weeks to months). complications include secondary bacterial or viral pneumonia, apnea, malnutrition, pulmonary hypertension and neurologic involvement including seizures and encephalopathy. infants less than 6 months of age are at highest risk for complications and mortality. characteristic paroxysms of cough with an end inspiratory whoop occur in children. infants may present with a nonspecifi c cough with associated apnea and cyanosis, without a whoop. adolescents may be asymptomatic or have only a mild prolonged cough. an increased white blood count up to 100,000 with a lymphocytosis is characteristic early in the course of the disease. the preferred test for laboratory confi rmation is the detection of b. pertussis dna by pcr assay. bacteriologic culture provides a defi nitive diagnosis. if administered during the early stages of the disease (fi rst 7-10 days of illness), erythromycin for 14 days may decrease symptoms and reduce the risk of spread. a 5 day course of azithromycin or a 7-10 day course of clarithromycin has been found to be as effective with less gastrointestinal symptoms. corticosteroids, bronchodilators, or intravenous immunoglobulins have not demonstrated effi cacy. supportive care with supplemental oxygen, mechanical ventilation, intravenous fl uids, maintenance of adequate caloric intake, and treatment of secondary bacterial infections are the mainstay of therapy. the use of extracorporeal membrane oxygenation in infants with hypoxemia, pulmonary hypertension and right heart failure refractory to conventional mechanical ventilation has resulted in poorer outcomes than expected. vaccination in infancy with booster doses in adolescence is preventative. about 80-85% of pneumonias in children are caused by viruses. there is considerable evidence that viral infections often precede bacterial pneumonias and cause weakening of the host defenses. viral pneumonias with rsv and parainfl uenza are discussed in more detail in the bronchiolitis section. infl uenza is the main viral cause of pneumonia in school-aged children requiring hospitalization. there are three serotypes, a, b, and c which are further divided into subtypes based on the hemagglutinin and neuraminidase genes. hemagglutinin 1, 2, and 3 and neuraminidase 1 and 2 typically infect humans. the gene segments for the surface glycoproteins are unstable, so mutations, called antigenic shift, occur regularly. epidemics occur annually during the winter months with a short, 1-3 day incubation period. the virus causes destruction of the ciliated respiratory epithelium within 1 day of symptoms. airway edema and infi ltration with infl ammatory cells into the airway mucosa and epithelium follows. slow repair occurs over 2-4 weeks. a severe fulminating pneumonia may result in hemorrhagic exudates that contain many polymorphonuclear and mononuclear cells. destruction of the respiratory epithelium often leads to secondary bacterial infections. during the 2003-2004 infl uenza season, 143 infl uenza-related deaths occurred in children; of these, 41% were less than 2 years of age. forty-fi ve percent of the older children (2-17 years of age) did not have an underlying medical condition. rare complications of although death from infl uenza pneumonia is uncommon, a signifi cant number of the children that died were previously healthy. infl uenza include acute myositis, rhabdomyolysis, myocarditis, pericarditis, reye syndrome, encephalitis, transverse myelitis, and guillain-barré syndrome. children may present with an abrupt clinical course manifested by high fever, myalgias, headaches, scratchy sore throats, and dry cough. peripheral white blood counts are usually less than 5,000. pulmonary infi ltrates often involve multiple lobes. bacterial co-infection, especially with mrsa, increases morbidity and mortality signifi cantly. rimantidine and amantadine can shorten the course for infl uenza type a disease by limiting viral replication, but only if given within the fi rst 48 h of the disease. prophylactic dosing is 70-90% effective and does not interfere with antibody production from the vaccine. both drugs have central nervous system and gastrointestinal side effects, including an increase in the incidence of seizures. oseltamivir and zanamivir have recently been approved for the treatment of infl uenza infections in children. they inhibit neuraminidase, an enzyme produced by infl uenza a and b. the course of disease in healthy adults can be reduced by 1-2 days, if started within 48 h of the onset of symptoms. zanamivir is a dry powder aerosol that must be delivered by a special breath-activated device. bronchospasm in patients with asthma has been reported. aspirin or aspirin-containing products should be avoided due to the risk of reye syndrome. immunoprophylaxis is the most effective strategy for the prevention of infl uenza infection. inactivated vaccines have effi cacy rates from 70% to 90%. currently, the inactivated vaccine is recommended for all children older than 6 months of age with high risk conditions including chronic pulmonary or cardiac disease, immunosuppressive disorders, sickle cell disease and other hemoglobinopathies, diseases requiring long-term aspirin therapy, chronic metabolic and renal diseases; healthy children aged 6-23 months; and household contacts over the age of 6 months of high risk persons. a live, attenuated infl uenza vaccine was licensed in 2003. it is administered by the intranasal route and is approved for healthy children aged 5-17 years. avian infl uenza viruses do not normally infect species other than birds and pigs. however, in 1997, the fi rst human death from avian infl uenza occurred in hong kong in a 3 year old with reye syndrome. subsequently, an epidemic occurred among humans in hong kong with close contact to live, infected poultry. the subtype h5n1 appears to be the most ominous due to its ability to rapidly mutate and infect new species. the overall mortality rate is greater than 70%. the avian viruses are not believed to be transmissible from person-to-person, but some recent cases are being investigated for this possibility. children uniformly present with fever and cough. symptoms range from typical infl uenza-like symptoms to conjunctivitis to respiratory disease and failure. signifi cant laboratory data include leukopenia and thrombocytopenia. all children who developed pneumonia and progressed to ards died. diagnosis remains diffi cult, as no tests are widely available. of the antiviral drugs available for infl uenza a, the most recent h5n1 strains in southeast asia are resistant to rimantadine and amantadine. therefore, treatment is mainly supportive. a prototype h5n1 vaccine was made available to manufacturers in april 2004, but production is diffi cult because the standard means of producing infl uenza vaccines from specially grown chicken eggs is not feasible. h5n1 kills the embryo before enough viruses can be harvested for vaccine production. in april, 2009, the centers for disease control confi rmed the emergence of a novel infl uenza a (h1n1) virus with genes from swine viruses of the eurasian lineage and genes from avian infl uenza viruses. by june, 2009, the fi rst infl uenza pandemic since 1968 was declared, affecting over 191 countries and territories. in comparison to illnesses with seasonal infl uenza, the majority of cases occurred in individuals younger than 65 years of age, with nearly half of the cases occurring in children under 18 years of age . the clinical symptoms can be typical for infl uenza; fever, sore throat, cough, and muscle aches with the addition of vomiting and diarrhea in children. a wide range of complications although antiviral medications may attenuate the course of infl uenza when given early, immunoprophylaxis with vaccines is the most effective strategy for the control of infl uenza infections. avian infl uenza has occurred in epidemics among persons with close contact to live, infected poultry. all children with pneumonia that progressed to ards succumbed to the disease. have been reported that include mild-to-moderate (otitis media, sinusitis, myositis, and febrile seizures) to more severe complications such as myocarditis, rhabdomyolysis or encephalitis. severe complications may frequently involve invasive bacterial co-infection (i.e. mrsa) and/or exacerbation of underlying medical conditions in particular asthma. children who present initially with uncomplicated infl uenza may have rapidly progressive hypoxemic respiratory failure and multiorgan system dysfunction that is refractory to all therapies ( fig. 25-3 ) . of reported h1n1 deaths, approximately 20% were in children. the majority of these children had comorbid asthma, neuro-developmental conditions, or obesity. an american academy of pediatrics work group identifi ed children at greatest risk for life-threatening h1n1 infl uenza disease (table 25-1 ) . the centers for disease control has recommended prompt empiric antiviral therapy for infants, children, and adolescents of any age presenting with suspected or confi rmed h1n1 infl uenza and any of the following conditions: illness requiring hospitalization ■ progressive, severe, or complicated illness, regardless of previous health ■ presence of signifi cant risk factors (see table ■ 25-1 ) the h1n1 strain has been found to be resistant to amantadine and rimantadine, but is usually sensitive to neuraminidase inhibitors, specifi cally oseltamivir or zanamir. in 2009, oseltamivir was emergently approved for treatment in children less than 12 months of age. resistance to oseltamivir has been reported and is thought due to the h275y mutation. interestingly, the mutation confers resistance to oseltamivir, but not to zanamivir. peramivir, a neuraminidase inhibitor, an unapproved (investigational) antiviral available in an intravenous formulation received an emergency use authorization permit from the fda for use in children with confi rmed severe refractory h1n1 infl uenza. its use should be restricted to children that are not responding to either oral or inhaled antiviral drugs or if the parenteral route is the only dependable method of drug delivery. a vaccine was manufactured and licensed using the same standards as seasonal infl uenza by late 2009. a single dose was found to provide adequate protection in children older than 10 years of age, younger children requiring two doses separated by at least 21 days. adenoviruses have been implicated in 4-10% of pneumonias in children. adenoviruses are classifi ed into 49 serotypes with types 3, 7, 7a, 11, and 21 being the most common etiologic agents of lower respiratory disease and causing a severe necrotizing pneumonitis. these serotypes are associated with serious pulmonary sequelae, such as bronchiectasis, bronchiolitis obliterans, unilateral hyperlucent lung, and persistently abnormal pulmonary function tests. adenovirus infections peak between 6 months and 5 years of age. mortality from severe respiratory infections can be high, because the disease often involves multiple organ systems. survivors may have permanent lung injury often in the form of bronchiolitis obliterans. in the immunocompromised host, mortality rates are as high as 50-80%. cidofovir has in vitro activity against adenovirus, but proof of effi cacy is limited. therapy is supportive. severe acute respiratory syndrome is a newly described pulmonary infection caused by a novel sars-associated coronavirus. sars-cov is highly contagious and was coined "the fi rst plague of the twenty-fi rst century". the disease rapidly spreads among household contacts and healthcare personnel. children less than 18 years of age account for only approximately 5% of those affected, with a mean age of 12 years. no deaths were reported among children in the 2003 outbreak. children and adults present with fever, malaise, cough, coryza, chills or rigor, sputum production, headache, myalgia, leukopenia, lymphopenia, thrombocytopenia, mildly elevated activated partial thromboplastin times, and elevated levels of lactate dehydrogenase. radiographs of the chest show non-specifi c infi ltrates. apart from diarrhea, patients have minimal extrapulmonary symptoms. early diagnosis by reverse transcription-polymerase chain reaction (rt-pcr) can be made with 80% sensitivity on nasopharyngeal aspirates within the fi rst 3 days of the illness. the clinical course follows a triphasic pattern. there is an incubation period of 2-10 days with a prodrome of high fever, chills, malaise, headache, and myalgias. diarrhea occurs in up to 20% of adults. after 2-7 days, the disease progresses to involve the lower airways with a dry non-productive cough and dyspnea. in 10-20% of cases, acute respiratory distress syndrome (ards) follows and often patients require mechanical ventilation. deaths occur from respiratory failure. young children run a milder and shorter biphasic clinical course. cough is found in approximately half the children, and crackles are rarely heard despite radiographic evidence of infi ltrates. a regimen of antibiotics, ribavirin, and corticosteroids was proposed based on initial anecdotal success. however, ribavirin has demonstrated minimal activity against sars-cov isolates in vitro . non-randomized studies of corticosteroids have reported favorable outcomes. a pediatric series of 44 children with confi rmed sars treated with ribavirin and corticosteroids showed no adverse effects and all survived. mortality from adenovirus infections remains high because of multiple organ system involvement. sars rarely affects children, and when it does, morbidity is less, with no reported mortalities. 1. neurological disorders, such as epilepsy, cerebral palsy, developmental delay and neuromuscular disorders 2. chronic respiratory diseases associated with impaired pulmonary function and/or diffi culty handling lung secretions, moderate and especially severe persistent asthma, technology-dependent children (e.g., those requiring oxygen, tracheostomy, or a ventilator) 3. primary immunodefi ciencies or conditions that require medications or treatments that result in secondary immunodefi ciencies 5. congenital heart disease 6. metabolic (e.g., mitochondrial) or endocrine disorders, especially if cardiopulmonary function is impaired adapted from http://www.aap.org/new/swinefl u.htm hantavirus cardiopulmonary syndrome is a viral zoonotic disease that affects healthy children and adolescents who are exposed to aerosols of rodent excreta. the deer mouse is the main rodent reservoir. most cases occur in the southwestern united states, but cases have been confi rmed in 30 states. hcps presents with a prodrome of fever, chills, myalgia, headache, and gastrointestinal symptoms. respiratory compromise requiring supplemental oxygen generally occurs within 72 h. the disease can progress to respiratory distress and ards. the majority of deaths result from hypoxemia and cardiac dysfunction with marked hypotension and ventricular arrhythmias. in adults, the case fatality rate is approximately 38%. a recent case series of 13 children aged 10-16 years, revealed that 92% of infected children developed hcps, 33% died, and 67% were critically ill and required mechanical ventilation. treatment is supportive as ribavirin has not been proven to reduce mortality. extracorporeal membrane oxygenation was used on two patients, one of which survived. laboratory evaluation reveals thrombocytopenia, leukocytosis, and circulating immunoblasts. an elevated prothrombin time of ³ 14 s is predictive of severe disease. no deaths were reported in children younger than 14 years of age. diagnosis can be made by detection of hantavirus-specifi c immunoglobulin m, hantavirus-specifi c rna by polymerase chain reaction, or hantavirus antigen by immunohistochemistry. respiratory infections in children with primary or acquired immunodefi ciencies requiring intensive care are not uncommon. these infants and children are susceptible to many organisms that are rarely pathogenic in a normal host. primary immunodefciencies include abnormalities or defi ciencies in immunoglobulins and antibodies, t and b cells, phagocytes, natural killer cells, and complement. acquired immunodefi ciencies include asplenia, human immunodefi ciency virus (hiv), corticosteroid therapy, and immunosuppresion used for marrow or solid organ transplants. immunocompromised children can present with attenuated signs and symptoms of respiratory infections. in addition to physical examination and chest roentgenograms, these children often require chest computed tomography to better delineate the extent of disease. bronchoalveolar lavage, needle aspiration, or lung biopsies might be required to make a defi nitive diagnosis. pulmonary specimens should be tested for common bacteria as well as for pneumocystis carinii, acid-fast bacilli, nocardia, legionella, crytococcus, aspergillus, candida, histoplasma, coccidioides, and blastomyces. viruses such as cytomegalovirus, varicella, herpes virus, and measles should be considered. pneumocystis carinii (now known pneumocystis jiroveci ) is an opportunistic pulmonary pathogen in infants and children with human immunodefi ciency virus (hiv) and other primary immunodefi ciencies, malnutrition, hematological malignancies, solid organ and bone marrow transplant recipients, and patients on high dose corticosteroid therapy for infl ammatory and collagen-vascular diseases. it is a unicellular organism that exists as a cyst (the diagnostic form). the organism attaches to the type i alveolar cells resulting in an alveolitis characterized by ventilation-perfusion mismatch and decreased pulmonary compliance. if untreated, pcp carries a mortality rate of 25-50%, and nearly 100% in the hiv-seropositive child. fortunately, the incidence has markedly decreased with the administration of chemoprophylactic agents to high risk patients. children typically present with fever, tachypnea, non-productive cough, and hypoxia with an absence of rales on auscultation of the chest. initially, they may have an elevated ph and low carbon dioxide levels. lactate dehydrogenase levels are generally elevated. bilateral diffuse alveolar infi ltrates are seen with initial hilar involvement subsequently spreading to the periphery (fig. 25-4 ) . diagnosis is made by demonstrating the organism with the methenamine silver nitrate stain on pulmonary tissue, respiratory secretions, or lung fl uid. bronchoalveolar lavage is the most widely used technique to obtain lung fl uid for diagnosis. treatment consists of supportive therapy hantavirus is rare in infants and school-aged children. no deaths have been reported in children less than 14 years of age. with supplemental oxygen; ultimately continuous positive airway pressure or mechanical ventilation may be necessary if respiratory failure occurs. trimethoprim-sulfamethoxazole (tmp-smx) is the recommended initial treatment. in patients that cannot tolerate tmp-smx, then pentamidine isoethionate should be used. corticosteroids in anti-infl ammatory doses as an adjunct to antimicrobial therapy have improved clinical outcomes. concurrent pulmonary infections were found in 35% of patients, most frequently bacterial or cytomegalovirus pneumonia. determination of the etiologic agent in pneumonia is diffi cult. fortunately, in most community-acquired pneumonias, identifi cation of the specifi c causative organism is not critical. however, in children with a complicated course that fails to respond to standard therapies, defi nitive diagnosis of the etiologic agent is essential. complete blood counts, infl ammatory markers, and chest radiographs do not differentiate the causative agents for pneumonia. blood cultures are rarely positive outside of the neonatal period. rapid antigen tests are available for rsv, parainfl uenza, infl uenza, and adenovirus. nasopharyngeal swabs for viral cultures generally take 7-8 days to become positive, and in one study, 86% of the patients had been discharged prior to the positive results. older children and adolescents might be able to produce sputum for gram stain and culture. an adequate specimen should contain more the 25 leukocytes and fewer than 25 squamous epithelial cells per low-power fi eld. in the intubated patient, sputum can be more easily acquired. however, interpretation of the results of gram stains and cultures is at times diffi cult in differentiating colonizing from pathologic organisms. colonization of the endotracheal tube may occur as early as 12 h, but most frequently between 60 and 96 h. the oropharynx becomes colonized within 36 h, the stomach at 36-60 h, and the lower respiratory tract between 60 and 84 h. in addition, a comparison of infectious agents isolated by both tracheal aspirates and bronchoalveolar lavage found only 36% concordance. bronchoalveolar lavage (bal) can be safely used to obtain secretions from the lower airways for gram stain and culture. it is especially useful in the diagnosis of pneumonia in the immunocompromised child. however, bal performed directly through the bronchoscope carries a risk of contamination. the smallest bronchoscope that can accommodate a protected specimen brush is 4.8 mm and requires a 6.5 mm endotracheal tube for passage. the smallest fl exible fi beroptic bronchoscope with a suction channel has an external chest radiograph of severe pneumocystis carinii pneumonia in a 13 month old male with combined immunodefi ciency. note the diffuse alveolar involvement and air bronchograms. (image provided courtesy of fa maffei) diameter of 2.8 mm and is too small to admit a double-sheathed brush. non-bronchoscopic double-lumen plugged catheters can be inserted blindly through the endotracheal tube to obtain a non-contaminated specimen. the sensitivity and specifi city of these samples are similar to those obtained by a bronchoscopic guided protected specimen. transthoracic needle aspirations are performed in some centers with good results. one study reported a diagnostic success rate in 59% of patients. the incidence of pneumothorax was approximately 20%, but none required subsequent placement of a pleural drainage catheter. a lung biopsy is rarely needed to make a defi nitive diagnosis. supportive treatment with oxygen and intravenous fl uids are often standard therapies. as both pneumonia and mechanical ventilation can cause an elevation in anti-diuretic hormone levels, careful fl uid monitoring is essential to avoid overhydration, excessive lung water and hyponatremia. initial antibiotic choices should be empiric and based upon the likely organisms for each age group, because of the diffi culty in identifying the causative agent. the child's respiratory status including respiratory rate, work of breathing, pulse oximetry, and central nervous system response should be closely monitored. non-invasive bi-level positive airway pressure (bipap) has been effective for use in children with mild to moderate respiratory insuffi ciency, defi ned as an a-a gradient >100 and <250 or pao 2 /fio 2 ratio <200 but >100 mm hg. serial evaluation of mask-face contact areas is essential to avoid skin breakdown. children with moderate or severe respiratory insuffi ciency often require intubation and mechanical ventilation. children with respiratory failure secondary to pneumonia often require increased positive end expiratory pressure (peep), increased inspiratory time, and aggressive pulmonary toilet to recruit alveoli. for patients requiring high levels of peep, adequate sedation is often required to prevent patient/ventilator asynchrony and barotrauma. spontaneous respirations should be encouraged while on mechanical ventilation. rarely, the use of neuromuscular blockade is required to allow mechanical ventilation. prone positioning may improve ventilation/perfusion (v/q) mismatching in dependent lung regions. lung protective strategies allowing permissive hypercapnea with small lung volumes to ventilate and appropriate peep to maintain alveolar recruitment is recommended for children with pneumonia. high frequency oscillatory ventilation can also be utilized to maintain mean airway pressure and alveolar recruitment. airway pressure release ventilation (aprv) provides recruitment of alveoli while allowing spontaneous respirations. in children with severe respiratory distress syndrome, treatment with bovine surfactant may improve oxygenation. extracorporeal life support continues to have a role in children with reversible severe acute hypoxemic respiratory failure refractory to mechanical ventilation. pneumonias can often be complicated by the development of pleural effusions and empyemas. these occur when the fl uid production by the interstitial lung tissue exceeds the maximum pleural lymphatic fl ow. parapneumonic effusions often occur from pneumonia as white blood cells and other debris of infection block the lymphatics resulting in elevation of protein in the pleural space, increase in colloid osmotic pressure, and consequent failure of fl uid reabsorption. on physical exam, the child will have decreased breath sounds over the effusion. in older children, auscultatory percussion changes might be appreciated. plain chest radiographs can reveal most clinically signifi cant effusions. ultrasound and chest computed tomograms are useful in determining the volume and quality of the fl uid and the presence of loculations. simple parapneumonic effusions or transudates can also be differentiated from exudates by using the criteria of light et al. (table 25 -2 ). a pleural fl uid ph less than 7.2 indicates a complicated effusion that is likely exudative and requires drainage whereas a pleural fl uid ph more than 7.3 suggests that the effusion may be managed with systemic antibiotics alone. complicated parapneumonic effusions or empyemas occur when the fl uid becomes purulent. during this stage, the effusions undergo a fi brinopurulent stage with many polymorphonuclear leukocytes, bacteria, and cellular debris entering the fl uid. fibrin is deposited over the pleural surfaces and loculations begin to form. the ph and glucose levels fall as the ldh levels rise. if untreated, they often progress to a third organizing stage in which the exudate non-invasive bipap ventilation can be effective for children with moderate respiratory insuffi ciency. develops into an inelastic, fi brotic peel that restricts the lung. simple parapneumonic effusions usually resolve with thoracentesis or tube thoracostomy and antibiotic treatment of the pneumonia. more complicated parapneumonic effusions have been successfully treated with thoracotomy tubes and fi brinolytics. however, although risks for bleeding are reportedly low, this therapy requires close monitoring of chest tube drainage and instillation of expensive medications with intermittent clamping of the chest tube. no single recommendation for the choice of fi brinolytic agent or dosage has been established. also, if tried late in the organizing phase, this is often unsuccessful due to loculations and the high viscosity of the purulent fl uid. surgical debridement either by open procedure or by video-assisted thorascopic surgery (vats) is often needed for organizing, complicated parapneumonic effusions. multiple studies have reported that early vats or thoracotomy for empyema leads to a shorter hospital stay. the treatment modality is best determined by the temporal stage and nature of the effusion. acute pulmonary infections are common diagnoses that require admission to the pediatric intensive care units. understanding the pathophysiology of lower respiratory infections enables the intensivist to tailor therapy to the individual child and pathogen. early establishment of a specifi c etiology and the selection of the correct treatment plan directly impacts clinical outcome. video-assisted thorascopic surgery (vats) for the treatment of empyemas has been associated with shorter hospital stay. which of the following therapies have been proven to be a consistent benefi t for rsv bronchiolitis? a. aminophylline b. bronchodilators c. corticosteroids d. ribavirin e. supportive care 2. palivizumab is indicated for which of the following children? a. a 5 month old, former 27 week premature infant who just underwent surgical repair of a large ventricular septal defect who received palivizumab 2 weeks ago b. a 9 month old, former 28 week premature infant with mild bronchopulmonary dysplasia who received palivizumab 2 weeks ago c. a 1 month old, former 36 week premature infant with peripheral pulmonic stenosis who has never received palivizumab d. a 2 month old full term infant with a urea cycle defect who has never received palivizumab e. an 8 month old, former 25 week premature infant with bronchopulmonary dysplasia who received his fi fth dose of palivizumab a month ago pleural fl uid may be classifi ed as exudative, if one or more of the following criteria are met: ■ pleural fl uid protein divided by serum protein >0.5 (sensitivity 98%, specifi city 83%) ■ pleural fl uid lactate dehydrogenase (ldh) divided by serum ldh > 0.6 (sensitivity 86%, specifi city 84%) ■ pleural fl uid ldh is more than two-thirds of the upper limit of normal for serum ldh (sensitivity 82%, specifi city 89%) adapted from light (2002) human bocavirus and acute wheezing in children american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis. diagnosis and management of bronchiolitis the yield of fl exible fi beroptic bronchoscopy in pediatric intensive care patients immunological mechanisms of severe respiratory syncytial virus bronchiolitis human metapneumovirus infection in young children hospitalized with acute respiratory tract disease: virologic and clinical features a multicenter, randomized, controlled trial of dexamethasone for bronchiolitis natural infection of infants with respiratory syncytial virus subgroups a and b: a study of frequency, disease severity, and viral load the use of albuterol in hospitalized infants with bronchiolitis advances in the treatment and prevention of severe viral bronchiolitis the presence and sequence of endotracheal tube colonization in patients undergoing mechanical ventilation palivizumab prophylaxis reduces hospitalization due to respiratory syncytial virus in young children with hemodynamically signifi cant congenital heart disease systemic corticosteroids in infant bronchiolitis: a meta-analysis drainage, fi brinolytics, or surgery: a comparison of treatment options in pediatric empyema does vats provide optimal treatment of empyema in children? a systematic review intravenous ribavirin treatment for severe adenovirus disease in immunocompromised children clinical picture, diagnosis, treatment, and outcome of severe acute respiratory syndrome (sars) in children noninvasive therapy with helium-oxygen for severe bronchiolitis pleural effusion pleural effusions: the diagnostic separation of transudates and exudates nebulized 3% hypertonic saline solution treatment in hospitalized infants with viral bronchiolitis development of wheezing disorders and asthma in preschool children heliox therapy in infants with acute bronchiolitis nasal continuous positive airway pressure with heliox versus air oxygen in infants with acute bronchiolitis: a crossover study diagnosis and management of pneumonia in children community-acquired pneumonia in children selected populations at increased risk from respiratory syncytial virus infection human metapneumovirus and human bocavirus in children mixed respiratory virus infections mycoplasma pneumoniae and chlamydia pneumoniae cause lower respiratory tract disease in paediatric patients children's hospital respiratory syncytial virus database: risk factors, treatment and hospital course in 3308 infants and young children infection with sin nombre hantavirus: clinical presentation and outcome in children and adolescents ribavirin for respiratory syncytial virus lower respiratory tract infection: a systematic overview risk of bacterial infection in previously healthy respiratory syncytial virus-infected young children admitted to the intensive care unit infl uenza in pediatric intensive cure unit do bronchodilators have an effect on bronchiolitis? comparison of conventional viral cultures with direct fl uorescent antibody stains for diagnosis of community-acquired respiratory virus infections in hospitalized children respiratory syncytial virus immune globulin for prophylaxis against respiratory syncytial virus disease in infants and children with congenital heart disease respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years h1n1 infl uenza the impact-rsv study group. palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants high incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (rsv) bronchiolitis bench-to-bedside review: ventilator strategies to reduce lung injury -lessons from pediatric and neonatal intensive care etiological diagnosis of childhood pneumonia by use of transthoracic needle aspiration and modern microbiological methods a multicenter, randomized, double-blind, controlled trial of nebulized epinephrine in infants with acute bronchiolitis respiratory syncytial virus and other respiratory viruses effect of exogenous surfactant (calfactant) in pediatric acute lung injury. a randomized controlled trial current concepts on pulmonary host defense mechanisms in children nebulized hypertonic saline solution for acute bronchiolitis in infants bronchiolitis: recent evidence on diagnosis and management key: cord-257662-viy65y72 authors: burrack, kristina s.; morrison, thomas e. title: the role of myeloid cell activation and arginine metabolism in the pathogenesis of virus-induced diseases date: 2014-09-08 journal: front immunol doi: 10.3389/fimmu.2014.00428 sha: doc_id: 257662 cord_uid: viy65y72 when an antiviral immune response is generated, a balance must be reached between two opposing pathways: the production of proinflammatory and cytotoxic effectors that drive a robust antiviral immune response to control the infection and regulators that function to limit or blunt an excessive immune response to minimize immune-mediated pathology and repair tissue damage. myeloid cells, including monocytes and macrophages, play an important role in this balance, particularly through the activities of the arginine-hydrolyzing enzymes nitric oxide synthase 2 (nos2; inos) and arginase 1 (arg1). nitric oxide (no) production by inos is an important proinflammatory mediator, whereas arg1-expressing macrophages contribute to the resolution of inflammation and wound repair. in the context of viral infections, expression of these enzymes can result in a variety of outcomes for the host. no has direct antiviral properties against some viruses, whereas during other virus infections no can mediate immunopathology and/or inhibit the antiviral immune response to promote chronic infection. arg1 activity not only has important wound healing functions but can also inhibit the antiviral immune response during some viral infections. thus, depending on the specific virus and the tissue(s) involved, the activity of both of these arginine-hydrolyzing enzymes can either exacerbate or limit the severity of virus-induced disease. in this review, we will discuss a variety of viral infections, including hiv, sars-cov, lcmv, hcv, rsv, and others, where myeloid cells influence the control and clearance of the virus from the host, as well as the severity and resolution of tissue damage, via the activities of inos and/or arg1. clearly, monocyte/macrophage activation and arginine metabolism will continue to be important areas of investigation in the context of viral infections. tissue -resident and monocyte-derived macrophages are innate immune cells that play a key role in normal tissue homeostasis, presentation of foreign and self antigens following infection or injury, pathogen clearance, and resolution of inflammation and wound healing. depending on the microenvironment, macrophages can be programed to adopt a variety of proinflammatory, regulatory, resolving, and immunosuppressive activation phenotypes, particularly in vivo. these activation states exist as a complex continuum of overlapping phenotypes; however, macrophage subsets with distinct functions have been defined (1) . macrophages are considered m1-polarized when stimulated by ifn-γ or toll-like receptor (tlr) ligands, such as lipopolysaccharide (lps), to express inducible nitric oxide synthase (inos; nos2) and produce nitric oxide (no). nos enzymes metabolize l-arginine to citrulline and no. no is a short-lived gaseous messenger with physiological and pathological effects. nanomolar concentrations of no, generated by endothelial nos and neuronal nos, are important for maintaining homeostasis, regulating vasodilation, and for the aggregation, recruitment, and adhesion of platelets to the vascular endothelium. inos generates micromolar levels of no that modulates various pathophysiological processes and is important for killing intracellular pathogens (2) . in contrast, m2-polarized macrophages result following stimulation of cells with a variety of stimuli, including type 2 cytokines such as interleukin (il)-4 or il-13. m2-polarized macrophages express a distinct l-arginine-metabolizing enzyme, arginase 1 (arg1), which hydrolyzes l-arginine to l-ornithine and urea. l-ornithine can be further metabolized to polyamines, which participate in a variety of fundamental cellular functions (e.g., proliferation, cell membrane transport), and l-proline, which is an essential component of collagen. in addition to playing important roles in defense against extracellular parasites and tissue repair, arg1 expression and activity in myeloid cells have emerged as a major regulator of innate and adaptive immune responses (3) . other m2-like suppressive or anti-inflammatory macrophages include myeloid-derived suppressor cells (mdscs) and tumor-associated macrophages (tams). mdscs are considered to be an immature population of myeloid cells, including both monocyte-like (gr-1/ly-6c + ) and neutrophil-like (gr-1/ly-6g + ) populations, associated with tumors or infections that suppress proinflammatory responses (4, 5) . depending on the context, mdscs have been shown to mediate their suppressive activity via no-and/or arg1-dependent mechanisms. importantly, macrophages are not permanently programed, but are considered "plastic" -that is, macrophages have been shown to change activation phenotypes depending on the local environment. although the m1/inos and m2/arg1 division is generally appropriate, arg1 can be induced in m1-like macrophages under certain conditions. thus, due to the spectrum of activation states for macrophages, a framework for macrophage-activation nomenclature was recently suggested (6) . in an attempt to avoid confusion in this review, we focused on the specific effects of the l-arginine metabolizing enzymes inos or arg1 on the pathogenesis of viral infections, noting other activation markers where appropriate. increasing evidence suggests that myeloid cell programing, inos, and arg1 contribute to the pathogenesis of numerous virus infections, suggesting that therapies that target these cells and pathways may be beneficial for the treatment of some virus diseases. in this review, we highlight recent studies of viral infections where myeloid cell polarization -resulting in expression of inos or arg1 -contribute to viral control or the development of chronic virus infection and mediate the resolution of tissue damage or cause immunopathology. no has antimicrobial activity against a number of bacteria, parasites, and fungi (7, 8) . additionally, no has been shown to have direct antiviral effects in vitro and/or in vivo against several viruses, including dna viruses such as herpes simplex virus type-1 (hsv-1), ectromelia virus (ev), and vaccinia virus (vv) (9, 10), as well as some rna viruses such as vesicular stomatitis virus (vsv) (11), japanese encephalitis virus (jev) (12) , dengue virus (denv) (13) , and coxsackievirus ( table 1 ) (14) (15) (16) (17) . there are several advantages of using no as an antiviral agent. for instance, unlike complement and antibody, no can readily pass through cellular membranes into neighboring cells as well as some viruses. additionally, no is likely to act on a variety of both viral and virally exploited cellular targets, inhibiting viral replication as well as limiting the capacity of viruses to develop resistance. lastly, the effect of no is independent of immune recognition of the infected cell, in contrast to that of antiviral lymphocytes, which could be important in virusinfected cells where expression of mhc class i molecules may be downregulated and in some virally infected tissues such as the brain where expression of mhc class i and ii molecules is limited. in initial studies in vitro, inhibition of ev, vv, and hsv-1 replication in mouse raw 264.7 macrophages and in primary mouse macrophages following ifn-γ treatment was shown to be largely dependent on no production (9, 10) . additionally, pharmacologic inhibition of nos or genetic deletion of nos2 resulted in increased viral titers and mortality following ev infection in mice (9, 18) . moreover, no affects several events in the late stages of the life cycle of vv, including viral dna replication, viral protein synthesis, and virion maturation in vitro (32) . these studies provided some of the first evidence that macrophage-produced no has direct antiviral effects. in addition to inhibiting hsv-1 replication in vitro, macrophage-derived no has been shown to have anti-hsv properties in vivo. in a mouse model of hsv-1-mediated corneal disease, inos was highly induced in the trigeminal ganglion (tg) of hsv-1-infected mice, and its expression was markedly reduced in mice depleted of macrophages (22) . depletion of macrophages prior to hsv-1 infection resulted in markedly reduced inos expression and higher viral loads in the tg of infected mice (22, 23) , suggesting that macrophages were the main source of inos expression in the affected tissues following hsv-1 infection and that no had important anti-hsv-1 properties in vivo. consistent with these data, inhibition of nos activity resulted in increased viral loads in the tg (22) . additional studies showed that f4/80 + gr-1 + inflammatory monocytes were recruited to the eye via an ifn-α-driven ccl2 gradient and restricted hsv-1 replication in that tissue via no production (24) . it was further shown that no production by f4/80 + macrophages in the brains of hsv-1-infected mice blocked viral replication in a partially tlr2-and tlr9-dependent mechanism (25) . finally, following footpad inoculation, hsv-1-infected nos2 −/− mice displayed a delayed clearance of virus from the dorsal root ganglia (drg) and exhibited an increase in the frequency of virus reactivation in drg (26) . the reactivity of no and its higher oxides and nitrosothiol products (84) makes it likely that a variety of molecular targets are involved in its antiviral action. it has been shown that no can inhibit ribonucleotide reductase (85, 86) , a rate-limiting enzyme in dna synthesis, and no can lead to the deamination of mammalian and bacterial dna (87, 88) , which may be important antiviral mechanisms. indeed, hsv-1 encodes its own ribonucleotide reductase and although it is not required for hsv-1 replication in vitro, it is necessary under conditions where the intracellular pool of deoxynucleotides is limited (89, 90) . thus, by inactivating this cellular and/or viral enzyme, no may halt virus replication by directly inhibiting viral dna synthesis. in addition to hsv-1, treatment of primary human cells with an no donor following infection with human cytomegalovirus (hcmv), a beta-herpesvirus, resulted in a significant reduction of early and late viral protein expression (28) . consistent with these in vitro data, nos2 −/− mice (129/sv/ev x c57bl/6 f1) exhibited increased viral titers and mortality following infection with murine cmv (mcmv; smith vr194 strain) (29) . nitric oxide has also been shown to have antiviral properties on a chicken herpesvirus, marek's disease virus (mdv), which can cause t cell lymphomas in chickens: addition of no-generating compounds inhibited viral replication in chicken fibroblasts (33) . additionally, the treatment of chickens with an inhibitor of inos increased the level of mdv replication in vivo (34) . further studies demonstrated that no production was limited to chickens that were genetically resistant to tumor development following mdv infection or to chickens that were vaccinated before being inoculated with mdv (35) . thus, no appeared to be produced in both types of resistance to tumor development in marek's disease, either acquired after vaccination or genetic. together, these findings suggest a role of no in the protective immune mechanisms against marek's disease, possibly through its activity on viral replication. finally, studies with hbv, a hepadnavirus associated with acute and chronic hepatitis, demonstrated that hbv replicated to higher levels in the livers of hbv-transgenic nos2 −/− mice than control transgenic mice, and transgenic nos2 −/− mice had increased frontiers in immunology | antigen presenting cell biology liver disease (36) . it was further demonstrated that no production by mononuclear cells, most likely macrophages, in the liver mediated most of the antiviral activity resulting from ifn-γ production by virus-specific t cells (36) , suggesting an antiviral role for macrophage-derived no following hbv infection in mice. in addition to dna viruses, macrophage-derived no also exerts antiviral effects against a number of rna viruses. inhibition of jev, a mosquito-transmitted flavivirus that causes encephalitis in humans, in ifn-γ-activated raw 264.7 macrophages in vitro correlated with no production, and ifn-γ-activated raw 264.7 macrophage-mediated inhibition of jev replication in murine neuroblastoma n18 cells was no-dependent (12) . moreover, inhibition of nos activity led to increased mortality in jev-infected mice (12) . in terms of its mechanism of action, no was found to inhibit jev rna synthesis, viral protein accumulation, and virus release from infected cells in vitro (12) . these data suggest that no may be directly or indirectly inhibiting viral enzymes and/or other www.frontiersin.org cellular components required for viral replication, and this may subsequently block viral protein synthesis. additionally, no may interfere with the release and/or maturation of virions. monocyte/macrophage-derived no may also block replication of denv, another mosquito-transmitted flavivirus. infection with denv resulted in increased levels of no in patients with dengue fever, the classic form of the disease (37) . additionally, inos expression was induced in cd14 + monocytes from a subset of acutely infected individuals (13) . it was further shown that ex vivo infection of human monocytes with denv-1 resulted in increased inos expression, and inhibition of inos activity led to increased denv antigen detection in these cells (13) . moreover, treatment of c6/36 mosquito cells with an no donor resulted in reduced denv-positive cells (13) . these data suggest that denv replication is susceptible to no-mediated inhibition. consistent with this, nos2 −/− mice were shown to be more susceptible to denv infection, resulting in more severe disease and increased lethality in mouse models of denv-2 and denv-3 infection (38, 39) . it was further demonstrated that, following denv infection in vivo, il-12 and il-18 induced ifn-γ production, resulting in inos expression and no production, which contributed to viral control (38, 39) . in addition to monocyte/macrophage-derived no, a recent study demonstrated that platelets isolated from patients with dengue fever had increased l-arginine transport and increased no production compared to platelets from healthy controls (40) . however, no has anti-aggregatory properties, and mendes-ribeiro et al. (40) found that dengue patients exhibited decreased collagen-induced platelet aggregation, consistent with the vascular leak and hemorrhagic manifestations of dengue hemorrhagic fever/dengue shock syndrome (dhf/dss), thus establishing an association between reduced platelet aggregation, enhancement of the l-arginine-no pathway, and dhf/dss (41) . in contrast, getts and colleagues showed that experimentally abrogating no activity during west nile virus (wnv) encephalitis, a related flavivirus, in no-competent mice at a specific, relatively late time point prolonged survival of infected mice, while pharmacological inactivation throughout disease did not (42) . combined, these data suggest that although during denv infection ifn-γ-induced no production has a role in antiviral defense, it is likely that dysregulation of the il-12/18-ifn-γ-no axis leads to immune-mediated damage in certain flavivirus infections. along these lines, it has also been shown that treatment of mice with a nos inhibitor increased mortality rates following sindbis virus (sinv) infection (43) , suggesting a protective role for no during this particular cns infection. however, sinv replication in the brain was unaffected. furthermore, treatment of neuroblastoma cells with no donors had little effect on sinv replication but increased cell viability (43) . these data suggest that no protects mice from fatal sinv-induced encephalitis by a distinct mechanism that does not directly involve the inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication. nitric oxide also plays an antiviral role during cns infection with reovirus. infection of neonatal mice with the prototypic neurotropic reovirus strain (t3a) induced inos expression in brain areas demonstrating reovirus antigen expression and associated virus-induced injury (44) . reovirus also induced inos expression following in vitro infection of primary neuronal and glial cultures. reovirus was shown to infect a subpopulation of microglial cells in vitro (44) , suggesting that direct virus interaction may induce inos in this specialized population of macrophages. treatment of neuronal cultures with an no donor inhibited viral replication whereas a nos inhibitor increased viral growth (44) , suggesting inos has the potential to exert antiviral activity in vivo. finally, coxsackievirus infection has been shown to induce expression of inos in macrophages infiltrating the hearts of infected mice (17) . treatment of wt mice with a nos inhibitor and infection of nos2 −/− mice resulted in more severe coxsackievirus-induced pancreatitis and myocarditis, elevated viral loads in tissues, and decreased survival compared to wt mice following coxsackievirus b3 (cvb3) infection (14, 15, 17) . similarly, nos2 −/− mice infected with coxsackievirus b4 exhibited decreased survival and delayed viral clearance compared to wt mice (16) . these data suggest an antiviral effect of no against coxsackievirus infection. consistent with this, it was demonstrated that no inhibits the 2a and 3c proteinases of cvb3 in vitro (91) . additionally, cvb3-infected outbred mice showed significantly reduced signs of myocarditis after treatment with no donors (91) . despite its protective capacity during some viral infections, no can also contribute to immunopathology. the pathological effects of no are likely due, at least in part, to oxidative damage caused by the interaction of no with oxygen radicals such as the superoxide anion radical o − 2 and hydrogen peroxide (h 2 o 2 ). for example, although addition of an no donor to virusinfected mdck cells reduced influenza a and b viral burden in vitro (45) , treatment of mice with inhaled no (ino) did not decrease the viral load of influenza a (mouse-adapted h1n1 strain)-infected mice; in fact, prophylactic treatment with ino resulted in enhanced weight loss and decreased survival following infection (46) , suggesting a pathogenic role for no. consistent with this, chickens, which show a high level of mortality and associated pathology following avian influenza infection, had higher levels of inos expression in the lungs compared with h5n1 influenza-infected ducks, which show relatively minor symptoms following influenza infection (47) . additionally, akaike and colleagues (48) found evidence of the production of peroxynitrite, which is generated through the reaction of no and o 2 -, in the lungs of influenza a (mouse-adapted h2n2 strain)-infected mice. moreover, inhibition of nos resulted in enhanced survival and decreased pneumonia, but not decreased viral loads, in influenzainfected mice (48, 49) , suggesting that no was contributing to pathogenesis rather than having direct antiviral effects. nos2 −/− mice also survived a lethal dose of influenza a virus (pr/8/34 strain) infection with little histopathologic evidence of pneumonitis; however, in these studies no infectious virus was detected in nos2 −/− mice at day 6 after infection (49) . the enhanced viral control in nos2 −/− mice was shown to require the activity of ifn-γ (51), with nos2 −/− mice also producing increased virusspecific igg2a antibody titers (50) . additionally, genetic deletion frontiers in immunology | antigen presenting cell biology of nos2 or pharmacologic inhibition of nos enhanced survival of mice inoculated with the highly pathogenic (non-mouse-adapted) 1918 influenza virus strain, although mice exhibited similar viral loads to control mice in lung tissue at the peak of viral replication (51) . influenza infection in vitro was shown to induce apoptosis, and a reduction in influenza-mediated apoptosis was noted in cells treated with a nos inhibitor (52) . similarly, fewer apoptotic cells were found in the lungs of influenza-infected nos2 −/− mice, suggesting that no mediates cell death following influenza infection (52) . the cellular source of inos/no following influenza infection in mice was shown to be ccr2 + inflammatory monocytes that accumulate in the lungs: ccr2 −/− mice survived a lethal challenge of influenza infection (pr/8/34 strain) and had significantly reduced accumulation of inos-expressing macrophages in the lung, with no associated increase in viral titers or dissemination (53) . it was also recently shown that a subset of monocyte-derived dendritic cells (dcs), described as tnf-α/inos-producing dcs (tipdcs), accumulate in greater numbers during the course of lethal versus sublethal influenza infections, suggesting a pathogenic role for this subpopulation of myeloid cells (54) . interestingly, though, aldridge et al. (54) found that the tipdcs also stimulated a local, protective cd8 + t cell response in the virusinfected respiratory tract, indicating both protective and pathogenic roles for these cells in influenza infection. it was further shown that partially compromising tipdc recruitment via treatment with pioglitazone, a synthetic agonist of the peroxisome proliferator-activated receptor-γ (ppar-γ), was protective against lethal influenza challenge (54). pioglitazone treatment led to a reduction in the levels of ccl2 (mcp-1) and mcp-3 in the bal fluid of influenza-infected mice (54) . pioglitazone has also been shown to reduce the production of a wide range of proinflammatory molecules, including inos (55), providing further evidence for the importance of no production by monocyte-derived cells in the pathogenesis of influenza infection. pharmacologic inhibition of nos using l-nmma also decreased pneumonitis and increased survival following intranasal infection of cba/j mice with hsv-1, despite a 17-fold increase in viral titers in the lung at day 3 after inoculation (58). in contrast, treatment of balb/c mice with a different nos inhibitor [aminoguanidine (ag), administered intranasally] resulted in enhanced pneumonitis, viral titers, and mortality following infection with a different strain of hsv-1 (59) . thus, the precise role of no in hsv-1 pneumonitis remains to be determined. no and other ros/rns were also shown to be pathogenic in the brains of mice with herpes encephalitis: inos was induced in cd11b + resident microglia following intranasal infection with hsv-1, and oxidative and nitrative damage was found in the brains of infected animals (60) . a common neurological complication of hiv infection in the developed world is sensory neuronal injury accompanied by inflammation, which is clinically manifested as disabling pain and gait instability. feline immunodeficiency virus (fiv) infection of cats, which causes similar neuroinflammation together with immunosuppression in cats, resulted in induction of inos and stat-1, which were predominantly produced by macrophages, in drg (64) . additionally, inhibition of nos resulted in reduced nitrotyrosine and prevented neuronal injury in fiv-infected drg cultures in vitro (64) . these data suggest that lentivirus infection contributes to axonal and neuronal injury through a mechanism involving m1 macrophage immune activation mediated by stat-1 and inos activation. in addition to these studies, infection of mice with the retrovirus lp-bm5, which causes profound immunodeficiency, induces cd11b + gr-1 + ly-6c + mdsc-like cells that inhibit both t-and b-cell responses in an inos/no-dependent but arginase-independent fashion (65) . this study identified an important -and only recently appreciated -role for inosexpressing myeloid cell-mediated suppression of b cell responses in retrovirus infection. the oxidative effects of no have also been shown to inhibit immune cells, particularly t cells. this phenomenon has been appreciated for a number of years in the context of tumors (92), where myeloid suppressor cells can inhibit the anti-tumor t cell response via the effects of no in addition to other mechanisms (2, 4) . in a similar manner, it has been shown that no can inhibit the antiviral immune response. mcmv clearance from balb/c mice is predominantly cd8 + t cell-mediated. a recent report showed that mcmv infection in balb/c mice induced cd11b + ly-6c hi inflammatory monocyte recruitment from the bone marrow to infected tissues that was dependent on ccr2 signaling (66) . this recruitment was shown to inhibit antigen-specific cd8 + t cell activation, expansion, and cytotoxic activity via no production, thus facilitating viral persistence (66) . in a similar fashion, no may contribute to a defective immune response following infection of mice with an attenuated neurotropic coronavirus (rj2.2 strain of mouse hepatitis virus). rj2.2infected wt mice exhibited mild acute encephalitis, followed by a non-lethal, chronic demyelinating disease (68) . in marked contrast, rj2.2 infection of mice that transgenically express ccl2 in the brain (ccl2 tg) ineffectively cleared virus and rapidly succumbed to the infection (68). ccl2 tg mice mounted a dysregulated immune response, characterized by increased accumulation of inos-expressing macrophages and microglia as well as regulatory t cells, but decreased arg1 expression (68) . these data suggest that persistent ccl2 overexpression establishes and sustains an immunological milieu that may predispose mice to a defective immune response to a typically minimally virulent virus. arginase activity is important for wound healing and tissue regeneration through the production of polyamines and proline (2) . in the context of some viral infections, arginase activity and m2 macrophage activation have been shown to be beneficial for tissue repair following virus-induced damage. for instance, resolution of severe respiratory syncytial virus (rsv)-induced bronchiolitis in mice is mediated by m2 macrophages that counteract cyclooxygenase (cox)-2-induced lung pathology (19, 20) . arg1 was induced in the lungs of rsv-infected mice, and its induction was shown to be il-4rα-dependent (19) . additionally, wt macrophages adoptively transferred into rsv-infected il-4rα −/− mice restored the www.frontiersin.org m2 phenotype in the lungs and decreased lung pathology (19) . it was further shown that the lipoxogenase pathway was important for m2 macrophage activation and lung resolution following rsv infection (20) . most recently it was demonstrated that treating mice with agents that sustain arg1 expression (e.g., il-4/anti-il-4 immune complexes) limited rsv-induced lung pathology (21) . consistent with a pathogenic role for inos/no following influenza infection (described above), it was recently shown that the presence of airway bacteria polarize alveolar macrophages into a m2 phenotype, thus limiting influenza-mediated lethal lung inflammation. wang and colleagues (27) demonstrated that priming with staphylococcus aureus, which commonly colonizes the upper respiratory mucosa, attenuated influenzamediated lung injury via tlr2 signaling that recruited peripheral ccr2 + cd11b + monocytes into the alveoli (27) . these monocytes polarized alveolar macrophages into a m2 phenotype characterized by high arg1 as well as ym1, fizz1, and il-10 expression (27) . it was further shown that s. aureus-primed m2 alveolar macrophages inhibited inflammatory cell recruitment to the lung, including neutrophils, nk cells, and cd8 t cells (27) . s. aureus-primed m2 alveolar macrophages also expressed higher levels of the inhibitory ligand pd-l1 (27) , suggesting that expression of a combination of anti-inflammatory cytokines and inhibitory ligands could be the mechanisms by which s. aureusprimed m2 alveolar macrophages limit influenza-mediated lung inflammation. as discussed above, coxsackievirus b3 (cvb3) infection causes myocarditis in human beings as well as in male balb/c mice. although female mice do not develop severe myocarditis, both male and female mice have comparable numbers of infiltrating macrophages and viral titers in the heart following cvb3 infection (30) . the macrophages infiltrating the heart in male mice were skewed toward a m1 phenotype characterized by high expression of inos (17) as well as m1-associated cytokines such as ifn-γ and il-12 (30) . additionally, inhibition of nos resulted in increased viral titers and higher mortality in cvb3-infected mice (17), consistent with an antiviral role for no during cvb3 infection (see above). however, in contrast to male mice, the heart-infiltrating macrophages in female mice were skewed toward a m2 phenotype characterized by high expression of arg1 as well as il-4 and il-10 (30). moreover, adoptive transfer of ex vivo-programed m1 macrophages significantly increased myocarditis in both male and female mice. strikingly, transfer of m2-programed macrophages into susceptible male mice alleviated myocardial inflammation by modulating the local cytokine profile from a m1 to m2 phenotype and promoting peripheral regulatory t cell (treg) differentiation (30) . using different variants of cvb3, one that caused myocarditis in c57bl/6 mice and one that did not, it was additionally shown that the myocarditic variant induced a m1 macrophage phenotype (31) . in contrast, the amyocarditic variant induced a m2 macrophage phenotype, which was also associated with the activation of nkt cells that promoted a treg response (31) . the ability of nkt cells to suppress myocarditis was shown by adoptive transfer of purified nkt cells into nkt knockout (jα18 knockout) mice infected with the myocarditic cvb3 variant, which inhibited cardiac inflammation and increased treg response (31) . cardiac virus titers were equivalent in all mouse strains indicating that nkt cells did not participate in control of virus infection (31) . thus, although no appears to have antiviral properties against cvb3, these data indicate an important role for arg1-expressing m2 macrophages in controlling cvb3-induced myocarditis. as a consequence of their co-evolution with their hosts, viruses have developed numerous strategies to evade the host immune system and ensure their own replication and survival. recent studies have identified a new evasion strategy for viruses: exploitation of the host's anti-inflammatory, wound repair response to promote chronic infection. two strains of lcmv -armstrong (arm) and clone 13 (c13)have been studied for decades as models for acute and chronic infections (93) . infection of mice with the arm strain leads to a robust cd8 + t cell response that rapidly clears the virus (94) , whereas infection with c13 results in t cells with impaired functionality, enabling the virus to persist (95) . it was recently demonstrated that c13 infection led to an enhanced and sustained expansion of cells that resembled mdscs (70) . these suppressive myeloid cells inhibited t cell proliferation ex vivo via an inos/no-dependent but arg1-independent mechanism. another study, however, found that arg1-expressing immunoregulatory antigen presenting cells induced during c13 infection suppressed t cell responses (67) . most recently, it was demonstrated that t cell responses were improved -resulting in clearance of the normally chronic c13 infection -when either myeloid cells or t cells lacked il-10 production (96) . overall, these data demonstrate the importance of inos/arg1-expressing myeloid cells in viral persistence. similar to lcmv c13 infection, it was recently demonstrated that infection of mice with the arthritogenic alphaviruses ross river virus (rrv) and chikungunya virus (chikv) resulted in the induction of arg1 in macrophages in the infected and inflamed musculoskeletal tissues (69) . it was further shown that genetic deletion of myeloid cell arg1 resulted in enhanced viral control in inflamed muscle tissue and reduced tissue pathology following rrv infection in mice (69) , suggesting an important role for arg1-expressing macrophages in the persistence of these chronic viruses. infection of mice with theiler's murine encephalomyelitis virus (tmev) results in persistent virus infection in the cns, which contributes to the development of a demyelinating disease that has similarities with multiple sclerosis. bowen and olson (97) showed that cd11b + ly-6c + cells infiltrated the cns following infection and were the dominant cell type during the innate immune response. depletion of the cd11b + ly-6c + cells via administration of an anti-gr-1 ab resulted in reduced development of demyelinating disease and enhanced virus-specific cd4 + and cd8 + t cell responses (97) . additionally, tmev-infected, anti-gr-1 ab-treated mice had decreased myelin-specific cd4 + t cell responses compared to control ab-treated mice during the demyelinating disease at a later time post-infection (97) . although the expression of arg1 was not investigated in this study, tmevinfected mice had elevated expression of il-10 in the brain and spinal cord (97), suggesting a role for this cytokine in the suppression of antiviral t cell responses, potentially through the effects of arg1. interestingly, a role for the modulation of arginine metabolism in viral control versus persistence along with associated disease has recently been demonstrated for the tumor-inducing, chickenspecific herpesvirus mdv. we mentioned above that mdv was vulnerable to the antiviral properties of no, with inos being induced in genetically resistant chickens and in vaccinated chickens (35) . in contrast, mdv induced strong macrophage arginase activity in cell extracts from adherent monocytes from genetically susceptible chickens, but not in chickens that were resistant to marek's disease, either genetically or acquired after vaccination (35) . together, these data suggest that in the case of marek's disease, the state of resistance versus sensitivity to disease was correlated with a reciprocal balance of nos versus arginase activities in macrophages. this phenomenon of arg1-mediated t cell suppression has also been recognized in human viral infections. arg1 mrna and protein levels were elevated in hcv-infected liver cell lines in vitro and in hcv-infected liver samples compared with paired hepatocellular carcinoma samples from the same patients or with uninfected liver tissues (71) . additionally, the number of mdscs in chronic hcv patients correlated with levels of plasma hcv-rna (98). cai et al. (98) also found that mdscs from patients with chronic hcv infection suppressed t cell function via an arg1-dependent mechanism. an additional study found that more pbmcs from chronic hcv patients expressed the phenotypic markers of mdscs than pbmcs from healthy controls, and these cells expressed increased levels of p47 phox , a component of the nadph oxidase complex (99) , suggesting a role for ros in mdsc-mediated suppression. consistent with this, cd33 + mononuclear cells co-cultured with hcv-infected hepatocytes or hcv core protein suppressed t cell proliferation in a ros-dependent manner (99) . overall, these data suggest that multiple mechanisms -including arginine metabolism and ros -may be at play in myeloid cell-mediated suppression of anti-hcv t cell responses. it has been suggested that prolonged immune activation during chronic virus infections, such as hcv and hiv, provides an environment that drives viral replication and disease progression (100, 101) . moreover, immune activation can drive an anti-inflammatory response to limit immunopathology, which can be characterized by the presence of m2-like macrophages. indeed, similar to hcv infection, a role for arginase and m2polarized mdsc-like cells has been identified in the suppression of antiviral t cell responses following hiv infection. individuals with detectable hiv-1 infection showed an increase in the frequency of cd163 + cd16 + cd14 + monocytes, which are thought to be precursors of m2 macrophages, when compared to seronegative or hiv-1-infected persons with undetectable viral loads, and monocyte frequency correlated positively with hiv-1 viremia and negatively with cd4 + t cell counts (in patients with counts <450 cells/µl) (72) . furthermore, qin and colleagues (73) observed elevated levels of mdscs, defined as hla-dr −/low cd11b + cd33 +/high cd14 + cd15 − cells, in the peripheral blood of hiv-1-seropositive subjects compared with healthy controls, and these mdscs suppressed t cell responses in an arg1-dependent manner. moreover, pbmcs from hivseropositive patients exhibited increased levels of arginase activity (73) . cloke and colleagues (74) found that increased arginase activity correlated with lower cd4 + t cell counts, and this association was abrogated following antiretroviral treatment (75) . additionally, exposure of pbmcs to hiv gp120 expanded t cellsuppressive mdscs in vitro (76) . these data point to a direct role for arginase-expressing mdsc-like cells in the suppression of anti-hiv t cell responses. consistent with that, individuals co-infected with hiv and leishmania parasites had increased arginase activity in pbmcs and plasma compared with leishmania-only infected individuals, even though leishmania infection alone results in increased arginase activity (77) . in addition, the parasite load in the spleen was significantly higher in co-infected patients (77) . the arginase-expressing cells were identified as low-density granulocytes (77) . these results suggest that increased arginase might contribute to the poor immune responses and disease outcome characteristic of patients with leishmania and hiv co-infection. hepatitis b virus (hbv) infection is another common chronic viral infection, with estimates as high as 350 million chronically infected humans (102) . bility and colleagues (78) recently developed a humanized mouse model with both a human immune system and human liver cells, named the a2/nsg-hu hsc/hep humanized mouse model, to study the pathogenesis of hbv infection. following hbv infection, the mice developed persistent hbv infection as well as chronic hepatitis and liver fibrosis (78) . the liver disease was associated with a high level of infiltrating human macrophages with a m2-like activation phenotype (78) . similarly, m2-like macrophage accumulation was seen in chronic hbv-infected patients, and m2-like macrophage induction in the liver was associated with accelerated liver fibrosis and necrosis in patients with acute hbv-induced liver failure (78), suggesting a role for m2 macrophages in persistent hbv infection. additionally, patients with acute hbv infection had increased serum levels of arginase, and this serum inhibited ifn-γ production by cd8 + t cells (79) . das et al. (80) also found decreased l-arginine levels in the circulation of chronic hbv patients with marked liver inflammation (>100 alt) and increased arginase activity in liver extracts taken directly ex vivo from patients with chronic hbv compared with those from patients with other types of liver pathology (80) . they further showed that cd8 + t cells from chronic hbv patients, regardless of their antigen specificity, exhibited less il-2 but not ifn-γ or tnf-α production and impaired proliferation following tcr-dependent stimulation, indicating an aberrant antiviral t cell response in chronic hbv infection (80) . in the a2/nsg-hu hsc/hep humanized mouse model, hbv-infected mice had impaired liver t cell responses, and m2 macrophages were associated with t cells in the liver (78) . expression of the tcr signaling molecule cd3ζ was reduced in both peripheral and intrahepatic cd8 + t cells from chronic hbv patients; similarly, cd28 was also downregulated on cd8 + t cells from high viral load hbv patients (80) . downregulation of the cd3ζ molecule has previously been shown to occur in the arginine-depleted tumor microenvironment. consistent with this, in vitro transfection of cd3ζ and cd28 restored il-2 production and supplementation of l-arginine partially restored cd3ζ expression and t cell proliferation (80) . these data suggest a role www.frontiersin.org for arginase activity and arginine depletion in the impairment of anti-hbv t cells functions. in the absence of inkt cells, influenza a (pr/8 strain) infection was shown to induce the expansion of cd11b + gr-1 + mdscs in the lungs of mice, which suppressed influenza-specific t cell and antibody responses through the activity of both arginase and nos, resulting in higher viral titers and increased mortality (81) . adoptive transfer of inkt cells reversed this phenotype; mice had an increased survival rate, reduced viral titers, and increased virusspecific immune responses, suggesting a novel immunomodulatory role for inkt cells during influenza virus infection (81) . moreover, these authors identified that influenza infection in humans induced the expansion of cd11b + myeloid cells with suppressive activity that could be reduced by inkt cell activation or the inhibition of arginase and nos activity. similarly, it was recently shown that highly pathogenic h5n1 and h1n1 influenza virus infection induced the accumulation of cd11b + gr-1 + cells and the expression of arg1 in the lungs (82) , further supporting a role for m2-polarized mdsc-like cells in promoting viral persistence and immunopathology. helminth infection induces the expression of type 2 cytokines and is associated with m2 macrophage activation, as determined by arg1, fizz1, and ym1 expression. indeed, osborne and colleagues (83) found that arg1, fizz1, and ym1 were highly induced in the ileum of mice infected with the helminth trichinella spiralis (ts). interestingly, they further showed that co-infection of mice with ts and murine norovirus (mnv) resulted in decreased frequencies and numbers of mnv-specific cd8 + and cd4 + t cells within the small intestine and spleen as well as decreased polyfunctionality of these t cells, compared to ts-only infected mice (83) . additionally, the defective t cell responses were associated with increased viral loads in the double-infected mice compared to the mono-infected controls (83) , suggesting that ts-elicited m2-activated macrophages inhibited the antiviral t cell response to mnv. lastly, neutralization of ym1, a chitinase-like molecule, in co-infected mice partially restored antiviral immunity and was associated with enhanced control of viral replication (83) . these data point to a new mechanism by which arg1-expressing macrophages inhibit antiviral responses. cumulatively, these data are reminiscent of macrophages found in tumors (e.g., mdscs, tams) that have been shown to suppress anti-tumor t cell responses via a variety of no-and/or arg1-dependent mechanisms (4, 5) . indeed, in a mouse model of human papillomavirus (hpv)-induced cancer, arg1-expressing cd11b + f4/80 + macrophages infiltrated the tumors and inhibited t cell responses, including virus-specific t cells, by suppressing t cell proliferation and promoting a regulatory phenotype (103) . moreover, depletion of the tumor-infiltrating macrophages resulted in reduced tumor growth and increased tumor infiltration by virus-specific cd8 + t cells (103) . thus, increasing evidence points to a direct role for arginase-expressing m2-polarized cells in the suppression of antiviral t cell responses and the persistence of a variety of important pathogenic viruses. in addition to the actions of inos and arg1, mdsc-like cells can employ other mechanisms to promote chronic viral infections, which were recently reviewed by goh and colleagues (104) . in contrast to some parasitic infections where m2 macrophages limit th2 cell-mediated immunopathology, m2-polarized macrophages have been shown to promote immunopathology in some viral infections. for example, it was recently demonstrated that sars-cov infection of mice induced suppressive alveolar macrophages that inhibited the induction of antiviral t cell responses, a phenotype that was reversed by the adoptive transfer of activated bone marrow-derived dcs into mice prior to virus infection (56) . additionally, sars-cov-infected mice lacking hematopoietic stat-1 expression were shown to have greater weight loss and lung pathology, and this was associated with the activation of m2 macrophages (57) . to further test the role of m2 macrophages in enhanced pathogenesis following sars-cov infection, the authors generated stat-1/stat-6 double knockout mice due to the established role for stat-6 in driving m2 macrophage activation in response to il-4/il-13 stimulation. stat-1/stat-6 double knockout mice, which reversed the upregulation of m2 macrophages observed in stat-1-deficient mice, had reduced lung disease and prefibrotic lesions (57) . these data support the notion that m2 macrophages contribute to sars-cov pathogenesis. in another example, mice deficient in the ifn-γr exhibit more severe disease following infection with murine gammaherpesvirus-68 (mhv-68), including interstitial and intra-alveolar fibrosis that is reminiscent of idiopathic pulmonary fibrosis (ipf) in human beings. in this model, alveolar macrophages were recruited to the lungs of mhv-68-infected ifn-γr −/− mice, were associated with areas of fibrosis, and exhibited a m2-polarized phenotype characterized by the expression of fizz1, ym1, and arg1 (61) . additionally, lung tissue from patients with ipf showed increased expression of arg1 in alveolar macrophages compared with normal lung (61) . these results suggest that virus-induced upregulation of arg1 could be mediating lung fibrogenesis. mhv-68 infection in ifn-γr −/− mice also resulted in fibrosis in lymphoid tissues such as the spleen, which is a site of latent mhv-68 infection, and the liver (62, 63) . similar to the lung, mhv-68 infection in the absence of ifn-γr signaling induced a m2 macrophage response in the spleen, characterized by high arg1 expression along with fizz1 and m2/th2 cytokines such as il-13, resulting in fibrotic disease in the spleen (105) . moreover, depletion of t cells prevented mhv-68-mediated fibrosis in ifn-γr −/− mice (62) , suggesting that m2 macrophages were further driving th2 activation to possibly create a m2/th2 cytokine-induced cycle, resulting in the exaggerated pathology. in contrast to ifn-γr −/− mice, inos was induced in the spleen of mhv-68-infected wt mice (105) , indicating an important role for ifn-γ in inducing a m1-associated immune response to control gamma-herpesvirus infection and limiting arg1-mediated immunopathology. macrophages and other myeloid cells have marked phenotypic heterogeneity, as a result of distinct cellular differentiation programs, distribution in tissues, and responsiveness to various endogenous and exogenous stimuli. indeed, macrophages have well-established roles in development, tissue homeostasis, coordinating the adaptive immune response and inflammation, as well as directing tissue resolution and repair following damage -processes that are often modulated via the actions of the arginine-hydrolyzing enzymes nos2 and arg1. we have highlighted a number of viral infections in which these enzymes have a beneficial effect: no has antiviral properties against a variety of viruses, and arginase activity can mediate tissue repair and regeneration following a viral insult (table 1) . however, no production can also result in immunopathology in some virus infections, and the suppressive functions of arg1-expressing macrophages can promote immunopathology. additionally, some viruses have exploited the immune-suppressive properties of inos-and/or arg1-expressing macrophages to evade the immune response, particularly the antiviral t cell response, resulting in chronic viral infections. clearly, inos-and/or arg1-mediated responses are important in many viral infections. thus, there is the potential to develop the means to selectively stimulate or inhibit either m1 or m2 responses to mediate viral clearance or repair tissue damage. due to the overlap in immunosuppressive mechanisms of inos-and/or arg1-expressing suppressor cells, therapeutic strategies under development to limit the immunosuppressive effects of myeloid cells in cancer may be beneficial in treating persistent/chronic virus infections. however, as described above, inos and arg1 activity can be both beneficial and detrimental during certain viral infections. therefore, further research is needed to define the molecular and tissue-specific mechanism(s) by which inos and arg1 influence the clearance of viral pathogens as well as the injury and repair of tissues. in addition, a better understanding of the pathways regulating macrophage polarization (specifically inos and/or arg1 induction and activity), 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interest. key: cord-260679-tm1s6wvj authors: lim, wei shen title: pneumonia—overview date: 2020-05-20 journal: reference module in biomedical sciences doi: 10.1016/b978-0-12-801238-3.11636-8 sha: doc_id: 260679 cord_uid: tm1s6wvj pneumonia is very common and continues to exact a high burden on health. the global burden of disease study 2015 found lower respiratory infections (lris) were the leading infectious cause of death and the fifth leading cause of death overall. pneumococcal pneumonia caused 55% of lri deaths in all ages (1.5 million deaths). novel pathogens, particularly viruses, continue to emerge as causes of pneumonia. the rise of drug-resistance among common respiratory pathogens is a further challenge. pneumonia is commonly classified according to patient location at the time of infection, leading to the categories of community-acquired, hospital-acquired and ventilator-acquired pneumonia. pneumonia may be defined as an infection of the lung characteristically involving the alveolar space. the presence of microorganisms in the alveolar space without an accompanying inflammatory response represents colonization and does not constitute pneumonia. a range of other types of infection may also affect the lung and can be classified according to their principle site of infection ( fig. 1 ). the term lower respiratory tract infection (lrti) is often considered to include both acute bronchitis and pneumonia. however, it is sometimes used to designate non-pneumonic infections of the lower respiratory tract alone. in patients with chronic lung disease (e.g., chronic obstructive pulmonary disease (copd)), an infection of the bronchi often results in an exacerbation of the underlying lung illness. in these circumstances, the illness is usually designated as an exacerbation of disease (e.g., exacerbation of copd) rather than "acute bronchitis." in general, the more distal the infection within the respiratory tract, the greater the likelihood of bacterial infection and the greater the severity of illness. exceptions to this include acute epiglottitis, diphtheria and pertussis which may present as severe bacterial infections of the upper respiratory tract without causing pneumonia. pneumonia is further classified in various different manners. these are mainly clinical classifications that broadly describe differences in the likely range of pathogens involved (table 1 ). the commonest grouping is according to patient location at the time of acquisition of infection. infections arising within a hospital setting may involve more drug-resistant pathogens compared to infections arising in the community. within the grouping of hospital-acquired pneumonia (hap), further distinction is usually made according to whether the patient was on an intensive care unit, or intubated (ventilator-acquired pneumonia (vap)) at the time of infection (torres et al., 2017; kalil et al., 2016) . a specific category of healthcare-associated pneumonia (hcap) was previously advocated as describing pneumonia developing outside a hospital setting that shared features of pathogenesis, causative pathogens and antibiotic resistance patterns with nosocomial (hospital-acquired) pneumonia. this category was never fully adopted internationally and the latest evidence does not support the continued use of this classification. host factors play an important role in the manifestation and management of pneumonia. pneumonia arising in immunocompromised hosts usually warrants distinct treatment. in general, the greater the degree of immune compromise, the wider the range of potential pathogens. the classic symptoms of infection, which are partly related to the host immune response, may also be absent, altering the clinical presentation. a third common classification is according to the causative pathogen(s). until recently, a microbiological diagnosis used to take days to confirm. however, with the advance of rapid, point-of-care diagnostics, microbiological confirmation within minutes/hours of clinical presentation is becoming more realistic. hence, this classification will hopefully become more clinically relevant in guiding patient management at the time of presentation. anatomically, pneumonias may be classified as bronchopneumonia or lobar pneumonia. bronchopneumonia occurs when infection leads to multiple discrete foci of consolidation within the lung, whereas lobar pneumonia is described when the area of consolidation is confined to the affected lobe which is diffusely involved. it was once thought that the pattern of consolidation (whether described radiologically or pathologically) was indicative of the causative pathogen (e.g., lobar pneumonia caused by streptococcus pneumoniae). however, it is now recognized that such discrimination is unreliable because of the large overlap in patterns caused by infecting pathogens. aspiration pneumonia refers to a specific situation when a patient who is manifestly at risk of aspiration develops pneumonia and anaerobic pathogens from the digestive tract are implicated, usually alongside multiple other microorganisms. an accompanying pleural reaction or lung abscess may develop. micro-aspiration events are common and the aspiration of microorganisms into the lower airways likely accounts for how pneumonia develops in the majority of cases (see "pathogenesis" section). hence, the presence of micro-aspiration alone in a patient with pneumonia does not invariably denote aspiration table 1 classification of pneumonia. description/comments pneumonia. stroke-associated pneumonia has been advocated as the desired terminology for lrti occurring within 7 days of acute stroke (smith et al., 2015) . the lung is not a sterile environment. the normal lung microbiome includes bacterial species that may be implicated in the development of pneumonia, such as streptococcus spp. and mycoplasma spp. (beck et al., 2015) . these, and other microorganisms, are normally held in check by pulmonary host defenses. disruption of these host defenses allow externally transposed pathogenic microorganisms to grow and displace the normal flora, or allow overgrowth of selected resident flora, leading to infection. there is growing interest in the role of antecedent viral respiratory tract infections as triggers for the disruption of the normal lung microbiome, providing an avenue for bacterial pathogens to take hold. the acute inflammation generated by the host immune response to infection results in an influx of inflammatory cells into the alveolar space, giving rise to the radiological pattern of consolidation (fig. 2) . in most cases, the predominant inflammatory cell involved reflects the inciting pathogen; neutrophils in bacterial infections, lymphocytes in viral infections and granulomatous inflammation in mycobacterial and fungal infections. the systemic cytokine response gives rise to many of the characteristic features of infection, such as fever, myalgia and a rise in c-reactive protein levels. the introduction of microorganisms to the lung is most commonly via micro-aspiration. haematogenous spread from other sites in the body, and direct spread from a contiguous source are less common. a range of host factors that predispose to pneumonia have been identified (wunderink and waterer, 2017; almirall et al., 2017) (table 2) . these factors mostly increase the susceptibility to pneumonia through reducing host defenses. some commonly used non-immunosuppressive drugs have been associated with pneumonia, but the mechanisms of action for all of these have not been fully described. immunocompromised patients are not only at higher risk of developing pneumonia but the range of possible pathogens is also wider. as the number of therapeutic interventions that modify the immune system (such as monoclonal antibodies and tyrosine kinase inhibitors) expands, patients with widely differing levels of immune integrity are being described. in addition, multiple immune insults may exist together. for instance, the severe immune defects caused by hematological malignancies are often compounded by their treatments which may include cytotoxic drugs and/or total bone marrow ablation followed by hematopoietic stem cell transplantation. immunologically, defects can be broadly grouped into (a) cell-mediated defects, (b) antibody deficiencies and (c) neutrophil dysfunction, most commonly neutropenia. understanding the type of immune defect facing a patient can aid as a guide to the likely range of pathogens involved, prior to confirmatory microbiological diagnosis. a definitive diagnosis of pneumonia comprises four aspects: (i) symptoms and signs of a respiratory tract infection, (ii) radiological changes, (iii) identification of a putative pathogen and (iv) a treatment response, or clinical course, consistent with pneumonia. it is not always easy to make this diagnosis. in settings where investigations are not readily available, such as in primary care, a clinical (or working) diagnosis of pneumonia may be made without recourse to radiological or microbiological tests. the accuracy of a clinical diagnosis of pneumonia made in primary care is reasonable; 49-57% of patients clinically diagnosed with cap have radiologically confirmed cap. however, of patients with acute cough in whom a radiological diagnosis of cap is made, only about 30% are clinically diagnosed as cap (van vugt et al., 2013) . even in secondary care, up to 25% of cases of pneumonia diagnosed in the emergency department are eventually discharged from hospital with an alternative diagnosis (sikka et al., 2012) . the differential diagnosis of pneumonia includes other cardiac and pulmonary conditions that present acutely with features of cough and/or dyspnoea together with radiological abnormalities. (table 3 ) in patients who are mechanically ventilated, diagnosing pneumonia amidst the wide range of differential diagnoses is challenging. patients with pneumonia usually present with a combination of (i) respiratory symptoms, specifically cough ($75%), dyspnoea ($65%), sputum production ($30%) and chest pain ($30%), and (ii) systemic symptoms including fever, rigors, myalgia and confusion. confusion is commoner in older patients and those who are severely ill. immunocompromised patients, and to a lesser extent, older patients, may not mount as rigorous an immune response and therefore may present with more subtle symptoms. about 10% of patients with cap present to hospital with extra-pulmonary features alone; these include falls, generalized weakness and acute abdominal pain. a high index of suspicion is required in these circumstances. until recently, identification of consolidation on a chest radiograph (cxr) has been regarded as the "gold standard" radiological investigation in the diagnosis of pneumonia. it is recognized that in patients with chronic lung abnormalities (e.g., pulmonary fibrosis, bronchiectasis, lung cancer) or in certain settings (e.g., intensive care unit), the sensitivity and specificity of a cxr for the identification of pneumonic changes can be limited. however, computed tomography (ct) scanning ( fig. 2 ) has raised further questions regarding the reliability of the cxr for the diagnosis of pneumonia more generally. when evaluated against ct imaging, the cxr results in both over-diagnosis and under-diagnosis of pneumonia with up to a third of patients diagnosed with pneumonia on cxr having no infiltrate on ct scanning (claessens et al., 2015) . ultrasonography is also being evaluated for the diagnosis of pneumonia with promising initial findings when compared against cxr (orso et al., 2018) . it remains necessary to determine if these imaging modalities will enable discrimination of patients suspected of having pneumonia into groups that warrant different management strategies. table 2 risk factors for pneumonia. age >65 years chronic co-morbid conditions copd, cancer, diabetes, chronic liver disease, renal impairment immunosuppressive disorders hiv, solid organ transplantation, immunosuppressive agents factors that increase the risk of aspiration placement of endotracheal tube, stroke, neurological disorder lifestyle factors smoking, high alcohol intake, malnutrition drugs proton-pump inhibitors, anti-psychotic medication, inhaled corticosteroids (in patients with copd) table 3 differential diagnosis of pneumonia. identification of a causative pathogen not only aids in the diagnosis and classification of pneumonia, it also guides antimicrobial therapy and infection control measures. a wide array of microbiological tests is available (table 3) . however, even with the use of modern molecular-based microbiological investigations (e.g., pcr, antigen detection tests) in patients with cap, a pathogen is identified in only 50-75% of cases (jain et al., 2015) . in routine care where microbiology testing is still based mainly around cultural techniques (e.g., from blood and respiratory tract samples), a pathogen may be identified in only 5-10% of cases. conversely, in patients with suspected vap, extensive colonization of airways creates difficulties in the interpretation of positive microbiology results. the use of highly sensitive pcr techniques can compound the difficulty. similar challenges confront the management of immunocompromised patients. in these instances, careful attention to the coherence of microbiology test results with the clinico-radiological pattern is necessary to distinguish colonization from infection. sometimes, a diagnosis of pneumonia can only be confirmed or refuted following review of the subsequent clinical course of illness, including the clinical response to empirically initiated antimicrobial agents. microbiological investigations are rarely performed in primary care settings due in part to a lack of access to laboratory facilities, low yield and delays in obtaining results in time to influence clinical management. advances in science and technology have come together to enable the development of rapid point-of-care (poc) tests that can provide microbiology test results within 15-60 min. the benefits of definitive identification of the causative pathogen (or in some cases, definitive exclusion of a specific pathogen, such as influenza) at the time of clinical presentation need to be weighed against the resource demands of these newer technologies and their costs. because lrtis can be caused by a range of pathogens, singlepathogen specific technologies are less helpful. multi-array pcr platforms may overcome some of these hurdles, but come with increased costs. the cost-effectiveness of poc-driven management strategies compared to more empirical approaches remains to be proven in many circumstances. an alternative approach to the management of patients with lrtis is to rapidly discriminate those may have a bacterial infection and who would therefore require antibiotic therapy, versus those with a viral infection or a non-infectious condition. several hostresponse biomarkers have been investigated in this regard (table 4) . c-reactive protein (crp) and procalcitonin (pct) are the two most extensively studied biomarkers in respiratory tract infections. levels of these biomarkers increase more extensively in bacterial compared to viral infections. procalcitonin has a more responsive kinetic profile than crp, which means that levels of procalcitonin increase and decrease more swiftly in line with bacterial load. apart from informing the decision whether or not to initiate antibiotics, the other role of crp-and procalcitonin-guided treatment strategies in patients with pneumonia may be to guide the duration of antibiotic therapy through serial assessments of biomarker levels. a meta-analysis of individual participant data from 26 rcts found that pct-directed treatment in the management of acute respiratory tract infections (of varying types and severity, including cap and hap) was associated with a reduction in antibiotic exposure (5.0 vs. 7.0 days) composed of a decrease in both (a) the proportions initiating antibiotics and (b) the duration of antibiotics (6.0 vs. 8.0 days), as well as a reduction in 30-day mortality (8.6% vs. 10.0%) (schuetz et al., 2017) . proadrenomedullin, neopterin, strem-1 and pentraxin-3 are other biomarkers that have been found in a small number of early studies to be potentially of value in lrtis (saleh et al., 2018) . measuring pentraxin-3 and strem-1 in bronchoalveolar lavage fluid table 4 biomarkers studied in respiratory tract infections. c-reactive protein acute phase protein synthesized by hepatocytes procalcitonin prohormone of calcitonin which is induced by the activation and adherence of monocytes to the endothelial layer of blood vessels proadrenomedullin precursor for adrenomedullin which is involved in immuno-modulation strem-1 soluble triggering receptor expressed on myeloid cells -1 (strem-1) levels rise following an increase of trem-1 expression on neutrophils, granulocytes, monocytes and alveolar macrophages. trem-1 expression is increased by microbial products pentraxin-3 acute inflammatory marker and a component of innate immunity neopterin produced in monocytes and macrophages. a marker of cell-mediated immunity. levels rise in viral infections copeptin stable byproduct of vasopressin biosynthesis lipocalin-2 protein involved in innate immunity. it limits bacterial growth by sequestering iron-containing siderophores syndecan-4 receptor in intracellular signaling. present in alveolar macrophages midregional proatrial natriuretic peptide (mr-proanp) a byproduct of atrial natriuretic peptide (anp) biosynthesis. anp regulates macrophage activity in innate and acquired immunity samples has been found to be more discriminatory for bacterial versus viral infections than their levels in blood. other biomarkers (e.g., copeptin, lipocalin-2, syndecan-4) display poorer performance characteristics. it may be that diagnostic performance can be further improved by combining different biomarkers (e.g., pct and mr-proanp) or matching certain biomarkers to selected target patient populations (e.g., hap vs. cap). most of the investigated biomarkers aim to differentiate between bacterial or viral infections according to the host's immunological response. the role of these biomarkers in patients with impairments of the immune system is therefore more limited, depending on the nature of the immune defect. data on the epidemiology of pneumonia are drawn from two broad sources-(a) datasets that rely on the coding of pneumonia following clinical episodes, and (b) cohort studies of patients with radiology-confirmed pneumonia. the accuracy of clinical coding of pneumonia reflects the difficulties with making a firm diagnosis. in the uk, up to 20% of coded cases of hospitalized-pneumonia may have no radiographic evidence of infection (daniel et al., 2017) . changes in the way pneumonia is coded can also affect the interpretation of data. in the us, there has been a shift in recent years from assigning a primary diagnosis code of "pneumonia" in patients hospitalized with severe cap, towards a primary diagnosis code of "sepsis" with "pneumonia" as the secondary diagnosis (lindenauer et al., 2012; ruhnke et al., 2013) . prospective studies of radiology-confirmed pneumonia provide more robust data but are often less comprehensive in scope. the global burden of disease study estimated that in 2015, the incidence of lrti in children aged <5 years old was 0.15 episodes per child-year, and in all ages it was 0.04 episodes per person-year (collaborators, 2017) . most episodes of lrti/cap are treated in community settings. in the uk, cap affects approximately 1% of the uk adult population each year, accounting for over 100,000 hospital admissions. the average length of stay is 6 days and estimated direct healthcare costs are £441 million. in the us, the annual incidence of cap requiring hospitalization has been estimated at around 46 per 10,000 adults (hayes et al., 2018) . this compares with lower estimates from other parts of the world (table 5 ). incidence rises steeply in adults aged >65 year (table 6) . changes in the prevalence of risk factors for pneumonia can be expected to influence the incidence of pneumonia. in the uk, a 34% increase in the incidence of cap requiring hospitalization was observed from 1998 to 2005 (trotter et al., 2008) . such trends of increasing incidence are thought to be explained by an aging population and a higher proportion of persons living with co-morbid illnesses, the latter encompassing an increase in persons with complex multi-morbidity. some have implicated alterations in the epidemiology of causative pathogens (quan et al., 2016) . changing patient expectations may also influence how healthcare is accessed and hospital admission policies. in contrast, in the us between 2001 and 2014, a decrease in the annual age-adjusted rate of pneumonia-associated hospitalisations was noted despite an increase in the proportion of co-existing immunocompromising conditions from 18.7% in 2001 to 29.9% in 2014 (hayes et al., 2018) . in sub-saharan africa, the incidence of cap is dominated by the effect of hiv infection. the prevalence of hiv within cohorts of patients with cap is 50-75%. in a community surveillance study in kenya, the annual incidence of pneumococcal acute respiratory infection was 50 per 10,000 persons in hiv negative individuals compared to 670 per 10,000 persons in hiv positive individuals (aston, 2017) . effective vaccination against respiratory pathogens has the potential to prevent infection and decrease the incidence of pneumonia. national immunization programmes involving pneumococcal vaccines have contributed towards a reduction in overall pneumococcal infections and attendant mortality. however, in some countries, replacement disease, involving pneumococcal serotypes not covered by existing vaccines, has since begun to offset the reductions in vaccine serotype disease. further vaccine development incorporating other serotypes, or effective against all pneumococcal serotypes/serogroups, will hopefully curtail this rise in replacement disease. estimates of the incidence of hospitalisations for cap (takahashi et al., 2013; ewig et al., 2009; trotter et al., 2008; hayes et al., 2018) . annual incidence (per 10,000 adults) globally, lrtis are the leading infectious cause of death and the fifth-leading cause of death overall. in 2015, lrtis caused 2á74 million deaths in all ages, with children <5 years of age bearing a disproportionate burden (704,000 deaths). between 2005 and 2015, the number of deaths due to lrti decreased by 36.9% in children younger than 5 years, and by 3.2% in all ages; most of these decreases occurred in countries with a low to middle socio-demographic index (sdi). in high-sdi countries, the lrti mortality rate in all ages increased by 9.6% over the same period (from 36.2 per 100,000 to 39.7 per 100,000) (collaborators, 2017) . in the us, pneumonia remains the leading infectious cause of death, and the eighth commonest cause of death overall. since 2000, mortality from pneumonia and influenza in the us has stayed below 20 deaths per 100,000 population. most deaths occur in hospitalized patients. in the period 2001-14, in-hospital deaths occurred in 7.4% of pneumonia-associated hospitalisations (hayes et al., 2018) . in europe, mortality rates for hospitalized patients are mostly around 5-20% though a wide range is reported likely reflecting differences in healthcare systems, data sources and possibly microbiological patterns. in patients admitted to icu with cap, mortality rates are in the region of 20-30%. in africa, an even wider range of mortality rates have been reported, from <1% to nearly 50%. in most high-income countries, advancing age is associated with increasing mortality rates. however, in sub-saharan africa, this trend is not always evident; 55% of lrti-related deaths in adults occur in persons under 70 years, including 22% in adults aged 15-49 years. rates of admission to icu vary globally according to availability of resources and admission policies. a higher proportion of hospitalized patients are admitted to icus in north american (>15%) compared to europe (5-10%). hospital acquired pneumonia is the second commonest nosocomial infection after urinary tract infections. estimated incidence rates are 5-20 cases per 1000 hospital admissions (torres et al., 2017) . these rates are influenced by the hospital ward setting and patient groups affected. the majority of hap (65%) occurs on non-icu wards, however, the incidence of hap is greater among patients in icu compared to patients on general wards. higher rates are also observed in at-risk patient groups such as the elderly, those who have had surgery and immunocompromised hosts. the incidence of vap is 2-6 episodes per 1000 ventilator-days according to data from the us. during the first 5 days of mechanical ventilation (mv), the estimated risk of vap is 3% per day, decreasing to 2% per day from days 5 to 10 of mv, and to 1% per day from day 10 of mv onwards. patients with brain injury and trauma are at particularly high risk of vap (50%) (kalil et al., 2016) . hospital acquired pneumonia is the leading cause of death from nosocomial infections in critically ill patients. the crude mortality from hap is high (up to 70%). however, patients who develop hap are often already severely ill and the factors that predispose to hap may also increase the risk of mortality. accordingly, mortality is higher for patients with vap than for patients with hap on general wards. in some studies of vap, 30-50% of vap-related deaths are deemed to be a direct result of infection. the vapattributable mortality has been estimated at 13% with surgical patients carrying the highest associated risks (melsen et al., 2013) . infection with pseudomonas aeruginosa or acinetobacter spp. is associated with higher mortality as well. an extensive range of pathogens can cause pneumonia. the respiratory pathogens commonly implicated in patients with cap remain important aetiological agents in all other types of pneumonia, including hap and pneumonia in the immunocompromised host (table 8 ). factors that modify (usually extend) the range of pathogens that might be implicated include alterations in immune status, exposure to specific environments/pathogens and prior exposure to antibiotics. s. pneumoniae is the predominant bacterial pathogen in pneumonia, especially cap. geographical differences are important in two broad regards: (a) the spectrum and frequency of likely pathogens, (b) the patterns of drug-resistance likely to be encountered. in countries where tuberculosis (tb) is endemic, acute tb pneumonia is a well-recognized cause of cap in both immunocompetent and immunocompromised persons. in thailand particularly, and to a less extent in some surrounding countries such as northern australia, burkholderia pseudomallei (a gram-negative bacillus that is present as free-living saprophytes in soil and surface waters in endemic areas) is a common cause of fulminant cap with high mortality. time from hospital admission to the development of hap influences the likely pathogens encountered. alterations in a patient's normal flora increase with duration of stay in hospital, potentially modified by illness, medical procedures and drug (antibiotic) exposure. as a general guide, the risk of infection from drug-resistant pathogens increases with duration of hospital stay. the main additional pathogens to consider are gram-negative enteric bacilli and methicillin-resistant s. aureus (mrsa) ( table 7) . the additional pathogens to consider in immunocompromised patients with pneumonia depends on the degree of immune dysfunction at the time of infection. in patients with hiv, the risk of infection with s. pneumoniae (and subsequent bacteraemia) is increased over 10-fold even with normal cd4 counts (>500 cells/ml). similarly, the risk of infection by mycobacterium tuberculosis is increased in early hiv infection. with cd4 counts <200 cells/ml, the risk from "opportunisitc" infections increases vastly (table 8 ) (segal et al., 2011) . infections with fungal pathogens are of particular concern in patients with severely impaired immune defenses. of note, some pathogens which commonly cause systemic infections in immunocompromised hosts rarely involve the lung (e.g., candida spp.). other pulmonary infections represent reactivation of disease as immune function declines rather than the acquisition of new disease (e.g., non-tuberculous mycobacteria, toxoplasma gondii). certain pathogens are associated with specific environmental exposures, or in the case of zoonosis, exposure to animal reservoirs (table 8) . some pathogens display seasonal patterns of higher incidence. for instance, infections with influenza, respiratory table 7 common microbial pathogens in pneumonia. mycobacterium tuberculosis non-tuberculous mycobacteria syncytial virus and s. pneumoniae are commoner in winter, whilst legionella infections are commoner in summer when the weather is hotter and more humid. over the years, the list of pathogens causing pneumonia has continued to increase through a combination of factors; advances in microbiological diagnostics, better recognition of clinical syndromes and the expansion of human populations into new territories. in particular, a number of viral pathogens have been recognized in the last two decades as implicated in the development of pneumonia. these include coronavirus, human metapneumovirus and enterovirus-d68. infection by more than one pathogen can occur in up to a third of patients with pneumonia. these are mostly viral-bacterial pathogen combinations and may reflect recognized associations between certain infections. for instance, influenza-related pneumonia is associated strongly with the bacterial pathogens s. aureus and s. pneumoniae. particular attention is necessary with immunocompromised patients when multiple pathogens may co-exist to cause disease. in 2017, the world health organization (who) published a global priority list of antibiotic-resistant bacteria to guide research, discovery and development of new antibiotics. the list includes a number of important respiratory pathogens, such as penicillin non-susceptible s. pneumoniae, ampicillin-resistant h. influenzae, methicillin and vancomycin-resistant s. aureus, carbapenemresistant a. baumannii, carbapenem-resistant p. aeruginosa, and carbapenem-and third-generation-cephalosporin resistant enterobacteriaceae (such as k. pneumoniae). whilst many of these pathogens are mainly implicated in nosocomial infections, some are important in cap as well. rates of drug-resistant s. pneumoniae (drsp) vary globally. northern european countries have tended to have lower numbers of drsp (<5% of pneumococcal isolates) while some other countries (southern europe, japan and the united states) report figures of drsp approximating 20-30% of isolates. the introduction of childhood pcv vaccination programmes in many countries has generally led to a reduction in rates of drsp. the epidemiology of drsp continues to change under vaccine pressure. in europe, during the era of pcv vaccination, the most frequent serotypes to display resistance to penicillin (from samples taken in 2010 to 2011) were serotypes 14, 19a and 15a. multi-drug resistance (defined as resistance to 3 or more classes of antimicrobial agents) was commonest in serotype 15a, a non-vaccine serotype (yahiaoui et al., 2018) . an assessment of illness severity is fundamental to the management of patients with pneumonia. severity assessment guides decisions around (a) place of treatment (whether in the community, in hospital, or in intensive care), (b) depth of investigations, (c) speed of treatment and (d) type of treatment, including the choice of antimicrobial agents and route of administration. various severity assessment tools have been developed for the management of patients presenting with cap. the two most widely validated and adopted tools are the pneumonia severity index (psi) and the curb65 score. both of these were developed to predict shortterm, 30-day mortality in patients presenting to hospital with cap. prognostic tools to predict icu admission are not as widely used partly due to differences in icu admission policies influencing the predictive accuracy of tools developed in one healthcare system and applied to a different healthcare system. severity assessment tools for hap are less well validated. this reflects the much broader diversity of factors influencing prognosis in patients with hap, including type of hospital ward, reason for hospital admission, time from hospital admission, medical interventions, exposure to nosocomial pathogens and preceding antibiotic exposure. similarly, there are no pneumonia-specific tools for assessing severity in immunocompromised patients. in these patients, the degree of immune compromise is often the dominating factor in severity assessment. patients who are severely immunocompromised may lack many of the symptoms or signs associated with severe illness. a high degree of vigilance is therefore important. early treatment with appropriate antimicrobial agents is the goal. in patients who are severely ill, earlier treatment (measured in hours) is associated with improved clinical outcomes, such as mortality. in patients presenting with signs indicative of severe sepsis, antibiotic administration within an hour is advocated. the impetus for early antimicrobial treatment limits the window in which to complete investigations to confirm the diagnosis of pneumonia and associated microbiology, prior to antimicrobial administration. in many instances, empirical broad-spectrum treatment must be started whilst awaiting results. in the case of influenza infection, time from onset of illness to antiviral treatment is the critical factor. viral load peaks rapidly within the first 2 days of illness. in line with this, evidence of clinical benefit from neuraminidase inhibitors is greatest when treatment is started within 48 h of symptom onset. concepts such as "start smart, then focus" acknowledge the inevitable uncertainty that often exists at the time of commencement of antimicrobials, and emphasis the equally important role of reviewing the diagnosis and treatment plan in the light of emerging results and response to empirical treatment. this pertains not only to antibiotics, but also to antiviral and antifungal agents. the optimal duration of antimicrobial therapy in the treatment of pneumonia has not been adequately studied. traditionally, a 7-day course of antibiotics has been standard. increasing awareness of the importance of good antimicrobial stewardship has led to efforts to refine the required duration of antimicrobial therapy. shorter 5-day courses of treatment are gaining acceptance, as are biomarker-driven antibiotic prescribing strategies (see section on "biomarkers"). some infections, such as legionella pneumonia, are still treated with longer courses of antibiotics (10-14 days) based on clinical experience rather than evidence derived from clinical trials. in the treatment of pneumonia, adjuvant therapy refers broadly to interventions that are aimed at modulating the immune response to infection. the use of systemic corticosteroids has been extensively tested in patients with severe sepsis, a large proportion of whom have pneumonia. a meta-analysis of 42 trials involving >10,000 patients found, with low certainty, a small mortality benefit in favor of low-dose corticosteroids (rochwerg et al., 2018) . fewer trials have been conducted in patients with cap and the existing debate around the value of corticosteroids for this specific indication reflects the weaker evidence base; evidence from 17 trials involving >2000 patients indicate a mortality benefit in patients with severe pneumonia, but not in those with non-severe pneumonia (stern et al., 2017) . macrolide antibiotics have immunomodulatory properties apart from their antibiotic effects. large observational cohort studies of patients with both all-cause pneumonia (i.e., involving a range of respiratory pathogens) and pneumococcal pneumonia suggest the combination of macrolide with beta-lactam antibiotics is associated with improved prognosis. in contrast, data from available randomized controlled trials are conflicting. hmg-coa reductase inhibitors (statins) have a range of immunomodulatory effects and in murine models of sepsis, pre-dosing with statins has been associated with improved outcomes. studies in adults with pneumonia support the notion that patients already taking a statin at the time of infection have a better prognosis, but the value of statins started as adjuvant therapy in patients presenting with pneumonia has not been established. immunization programmes against common respiratory pathogens are cost-effective public health interventions for the prevention of cap in many countries. in relation to s. pneumoniae, two types of pneumococcal vaccines are commercially available. the current multivalent pneumococcal polysaccharide vaccine (ppv) was first introduced in 1983 and covers 23 of over 90 pneumococcal serotypes/serogroups (1,2,3,4,5,6b,7f,8,9n,9v, 10a, aa!,12f,14,15b,17f,18c,19a,19f,20,22f,23f,33f.) . the 23-valent ppv offers good protection against invasive pneumococcal disease but relatively weak protection against pneumococcal pneumonia (falkenhorst et al., 2017) . in older persons who are most at risk of pneumonia, immunosenescence adversely influences the protective effect of these vaccines. pneumococcal conjugate vaccines (pcvs) promote a more robust immune response and are able to reduce nasopharyngeal carriage of vaccine-type s. pneumoniae strains, but cover a smaller range of pneumococcal serotypes. as children are the main carriers of s. pneumoniae, vaccination of children with pcvs has been associated not only with a reduction in the incidence of childhood pneumococcal infections, but also in the incidence of adult pneumococcal pneumonia (herd protection) (tsaban and ben-shimol, 2017) . countries vary in the target groups for pneumococcal vaccination and the vaccines offered. trivalent influenza vaccines against 3 of the 4 main circulating influenza strains (2 influenza a strains and 1 influenza b strain) have been available for many years. due to antigenic shifts within influenza viruses, the components of these vaccines are reviewed (and renewed) each year to maximize vaccine-pathogen "match." vaccines against all four of the common seasonal influenza viruses (quadrivalent vaccines covering 2 influenza a strains and 2 influenza b strains) are now available. in addition, and perhaps more importantly, is the development of newer conjugated vaccines and high-dose vaccines that promote stronger immune responses in older at-risk persons compared to previous "standard" influenza vaccines, hence offering greater protection. influenza vaccines that are not strain-specific (so-called "universal" vaccines) and therefore less subject to mismatch are also being developed as are vaccines against respiratory syncytial virus. the h. influenzae type b (hib) vaccine is available for the prevention of childhood pneumonia. however, in adults, non-typeable h. influenzae (nthi) is much more important and there are some data to suggest that childhood carriage of nthi may be increasing. an effective vaccine for nthi is not currently available. smoking cessation and a reduction in alcohol use are important modifiable lifestyle factors for the prevention of pneumonia. current tobacco smokers are over two times more likely (pooled odds ratio 2.7, from meta-analysis) to develop cap compared to adults who have never smoked, while people who consume alcohol (or in higher amounts) have a 83% increased risk of cap compared to those who consume no (or less) alcohol (simou et al., 2018) . for every 10-20 g higher alcohol intake per day, there is an 8% increase in the risk of cap. a large number of specific interventions have been advocated in the prevention of vap. these may be grouped as functional (e.g., semi-recumbent position), mechanical (e.g., silver-coated endotracheal tube) and pharmacological (e.g., selective decontamination of the digestive tract) interventions. the institute of health improvement (ihi) developed the concept of "bundles" of care in the icu to improve clinical outcomes. implementation of a vap prevention bundle may enable the ideal goal of "zero vap" to be achieved (álvarez-lerma et al., 2018a,b) . recovery from pneumonia usually takes several weeks. at 4-6 weeks following discharge from hospital for an episode of cap, one or more symptoms continue to be reported by 70% of patients, including cough, dyspnoea and fatigue in roughly equal proportions. functional impairment is reported by 18-51% of patients at 4 weeks (pick et al., 2019) . cardiac complications (including heart failure, acute coronary syndrome and arrhythmias) have been observed in 18% of patients within 30 days following occurrence of cap, with higher rates among patients who are hospitalized compared to those treated in the community (corrales-medina et al., 2011) . the risk of cardiac complications remains high for at least the first 1-2 years following hospitalization (corrales-medina et al., 2015) . higher rates of long-term mortality (17% at 1 year, 40% at 5 years) have been observed in patients following hospitalization for cap compared to patients hospitalized for other reasons. the predominant causes of long-term mortality are vascular and respiratory in nature. although studies have attempted to adjust for co-existing medical illnesses, it remains uncertain whether the association of pneumonia with long-term mortality is causal or whether pneumonic events are simply markers of poorer overall health (wagenvoort et al., 2017) . many of the current treatment strategies for pneumonia have remained broadly unchanged over the last decade. efforts at reducing the burden of disease through vaccination have been rewarded with some success but continued innovation is required to maintain gains made so far. the rise of antimicrobial resistance threatens our ability to treat infections that occur, urging a more judicious approach than the empirical antimicrobial strategies that characterize much of current clinical practice. the future expectation is a shift towards targeted treatments supported by rapid diagnostics, thus enabling the use of non-antibiotic pathogen-specific therapies (such as anti-toxins) and promising the eradiation of inappropriate antimicrobial use. immune-modulating agents may offer further benefits in relation to short-term recovery, and improved post-pneumonia care could translate into better longer-term outcomes. risk factors for community-acquired pneumonia in adults: a systematic review of observational studies prevention of ventilator-associated pneumonia: the multimodal approach of the spanish icu "pneumonia zero" program the multimodal approach for ventilator-associated pneumonia prevention"-requirements for nationwide implementation pneumonia in the developing world: characteristic features and approach to management multicenter comparison of lung and oral microbiomes of hiv-infected and hiv-uninfected individuals early chest ct-scan to assist diagnosis and guide treatment decision for suspected community-acquired pneumonia estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a 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diagnosis of pneumonia in the ed has poor accuracy despite diagnostic uncertainty alcohol and the risk of pneumonia: a systematic review and meta-analysis diagnosis of stroke-associated pneumonia: recommendations from the pneumonia in stroke consensus group corticosteroids for pneumonia the incidence and aetiology of hospitalised community-acquired pneumonia among vietnamese adults: a prospective surveillance in central vietnam international ers/esicm/escmid/alat guidelines for the management of hospital-acquired pneumonia and ventilator-associated pneumonia: guidelines for the management of hospital-acquired pneumonia (hap)/ventilator-associated pneumonia (vap) of the increasing hospital admission for pneumonia indirect (herd) protection, following pneumococcal conjugated vaccines introduction: a systematic review of the literature diagnosing pneumonia in patients with acute cough: clinical judgment compared to chest radiography long-term mortality after ipd and bacteremic versus non-bacteremic pneumococcal pneumonia advances in the causes and management of community acquired pneumonia in adults distribution of serotypes and patterns of antimicrobial resistance among commensal streptococcus pneumoniae in nine european countries key: cord-267003-k7eo2c26 authors: hendaus, mohamed a; jomha, fatima a; alhammadi, ahmed h title: virus-induced secondary bacterial infection: a concise review date: 2015-08-24 journal: ther clin risk manag doi: 10.2147/tcrm.s87789 sha: doc_id: 267003 cord_uid: k7eo2c26 respiratory diseases are a very common source of morbidity and mortality among children. health care providers often face a dilemma when encountering a febrile infant or child with respiratory tract infection. the reason expressed by many clinicians is the trouble to confirm whether the fever is caused by a virus or a bacterium. the aim of this review is to update the current evidence on the virus-induced bacterial infection. we present several clinical as well in vitro studies that support the correlation between virus and secondary bacterial infections. in addition, we discuss the pathophysiology and prevention modes of the virus–bacterium coexistence. a search of the pubmed and medline databases was carried out for published articles covering bacterial infections associated with respiratory viruses. this review should provide clinicians with a comprehensive idea of the range of bacterial and viral coinfections or secondary infections that could present with viral respiratory illness. viral respiratory tract infections (vrtis) are very common in children and their presentations vary from simple colds to life-threatening infections. [1] [2] [3] [4] [5] the detection of a respiratory virus does not necessarily infer that the child has only a viral infection, 6 since outbreaks of vrtis are being linked to increased incidence of bacterial coinfections. 7 the human body is usually capable of eliminating respiratory viral infections with no sequelae; however, in some cases, viruses bypass the immune response of the airways, causing conceivable severe respiratory diseases. 8 robust mechanical and immunosuppressive processes protect the lungs against external infections, but a single respiratory tract infection might change immunity and pathology. 9 health care providers often face a dilemma when encountering a febrile infant or child with respiratory tract infection. the reason expressed by many clinicians is the challenge to confirm whether the fever is caused by a virus or bacterium. 10 acute otitis media (aom) is a usual bacterial coinfection that occurs in 20%-60% of cases of vrtis. [11] [12] [13] [14] in addition, almost 60% of children with vrti have changes in the maxillary, ethmoidal, and frontal sinuses. 11, 12 moreover, in the year 1918, it was estimated that 40-50 million individuals died from the influenza pandemic, many of which were due to secondary bacterial pneumonia with streptococcus pneumoniae. 15 a search of the pubmed database and google was carried out, using different combinations of the following terms: virus, induced, bacteria, pathogenesis, prevention, vaccine, and children. in addition, we searched the references of the identified articles for additional articles. we then reviewed abstracts and titles and included studies that were submit your manuscript | www.dovepress.com hendaus et al relevant to the topic of interest. finally, the search was limited to studies of disease in humans that were published in english and spanish from 1918 to the end of 2014 ( figure 1 ). the epithelium ( figure 2) is usually covered by a layer of mucus that functions as a boundary. 16 mucins, which are charged glycoproteins, are the main components of mucus. 17, 18 muc5ac and muc5b are the most common mucins in the human sputum, and they assist the innate immune system through their anti-inflammatory and antiviral properties. 19, 20 in addition, they facilitate trapping and clearance of viruses; however, overproduction of those mucins might have a paradoxical effect. 18, 19 the airway epithelium not only functions as a physical barrier but also recognizes microorganisms through pattern recognition receptors such as toll-like receptors (tlrs), 18 nucleotide-binding oligomerization domain (nod)-like receptors (nlrs), and retinoic acid-inducible gene (rig)like helicases. 21, 22 tlrs are single, noncatalytic, membrane-spanning receptor proteins used by the innate immune system. 23 respiratory viruses collaborate with tlr lanes, leading to extended bacterial load in the lungs. 21, 24 in comparison, nlrs and rig-like helicases activate innate immune responses through cytosolic sensing of viral and bacterial components. 22, 25 nod1 and nod2, which are family members of nlrs, are induced by molecules synthesized during the production and/or degradation of bacterial peptidoglycan. [26] [27] [28] [29] in addition, many epithelial cells express the classical antiviral interferons (infs), especially ifn-α and ifn-β. 30, 31 moreover, the respiratory virus-infected epithelia facilitates the attraction of inflammatory cells, including natural killer cells, neutrophils, macrophages, and eosinophils from the bloodstream into the infected site. 32 finally, the airway epithelium consists of many molecules including intercellular adhesion molecule 1 (icam-1), carcinoembryonic antigen-related cellular adhesion 1 (ceacam-1), and platelet-activating factor receptor (paf-r). 33 viruses have an effect in modulating these receptors, leading to an increase risk of bacterial adherence; for example, rhinovirus upregulates the expression of paf-r, leading to the binding of s. pneumoniae to bronchial epithelial cells. 34 different mechanisms might contribute to the debilitation in host defense of the respiratory tract against bacteria following viral infection. some of the mechanisms have been extrapolated from studies conducted in animal models of sequential infections by respiratory viruses and several bacterial pathogens. mammalian cells are prone to bacterial attachment during a viral illness. 8, 35 viruses can debilitate the mucociliary clearance structure, leading to the increased attachment of bacteria to mucins and colonization; moreover, the condensed mucus will impede the penetration of antibacterial material and immune cells. 36 viruses like the respiratory syncytial virus (rsv) can damage ciliated cells, resulting in ciliostasis and, therefore, deterioration of mucociliary clearance. 37 the same concept applies to an influenza virus infection, leading to decreased tracheal mucociliary velocity and clearance of s. pneumoniae. 35, 38 moreover, virus-induced cell death debilitates the mechanical elimination of the attached pathogens and displays novice receptors for bacterial adherence. 39 studies have shown that the rsv virus induces the adherence of s. pneumoniae, pseudomonas aeruginosa, and haemophilus influenza to airway epithelial cells. [40] [41] [42] [43] in addition, adenovirus and rhinovirus play the same role in the adherence of s. pneumoniae to the airway epithelial cells; 8, 44 however, the measles virus decreases the risk of adherence of streptococcal bacteria, implying that every virus has a specific mode of changing the host cell membrane. 44 moreover, bacterial adhesion might also be a result of the upregulation of surface receptors including paf-r, which is involved in pneumococcal invasion. 45, 46 in patients with cystic fibrosis, bacterial adherence forms a biofilm, creating permanent airway colonization with p. aeruginosa. 47 viruses such as rsv, rhinovirus, and influenza virus also lead to pneumococcal biofilm formation on the airway lining. 48 furthermore, rsv increases the risk of adherence of staphylococcus aureus and bordetella pertussis to hep-2 (human epidermoid cancer) epithelial cells. 49, 50 virus effect on the immune system post-viral sustained desensitization of lung sentinel cells to tlr signals may be one possible contributor to the common secondary bacterial pneumonia associated with viral infection. for instance, tlr4 and tlr5 pathways are altered after influenza virus infection, resulting in decreased neutrophil attraction, thereby leading to increased attachment of s. pneumonia and p. aeruginosa to the airway epithelial cells. 25 the interrelation between host cells and microorganisms during an infection induces immune responses that include the generation of proinflammatory molecules. despite their crucial role as a bactericidal, proinflammatory cytokines such as tnf-α produced in response to infection could be detrimental to the host cells. 51 during a viral infection, tlr and rig-i-like receptor activation induces production of type i ifns, which can augment the inflammatory response to tlr ligands including lipopolysaccharide (lps). 52, 53 in addition, certain bacteria such as s. aureus integrate into the a549 respiratory epithelial cells (adeno-carcinomic α β hendaus et al human-alveolar basal-epithelial cells) during a respiratory viral infection by increasing the expression of icam-1. 54 rsv differs from influenza virus in that the former upregulates cellular receptors including ceacam-1 and icam-1, which eventually leads to bacterial infection. 45 finally, interaction between type i ifns and nod1/nod2 signaling leads to bacterial recognition, but indicts harmful effects in the virally infected host. 55 another study showed that the rates of bacteremia and om were 18% and 44%, respectively, in children with viral-induced bronchiolitis. 11 the highest incidence of aom is usually 2-5 days after an upper respiratory infection. 64, 65 isolation of viruses alone from sinus aspirates or in concomitance with bacteria proposes the role of viruses in the induction of bacterial sinusitis, 62 with rhinovirus and parainfluenza viruses being the culprits. 66 the rate of bacteremia in children with acute bronchiolitis ranges from 0.2% to 1.4%. [67] [68] [69] [70] [71] [72] [73] [74] [75] in addition, the rate of bacterial urinary tract infection (uti) in children with bronchiolitis can be as high as 11.4%. 67 in a recent study, hendaus et al 76 the human myxovirus resistance protein 1 (mxa) is an important intermediary of the ifn-induced antiviral response against a variety of viruses. mxa expression is firmly modified by type i and type iii ifns, which also requires signal transducer and activator of transcription 1 signaling. additionally, mxa has many characteristics similar to the superfamily of large guanosine triphosphatases. 78 mxa analysis could be beneficial to differentiate between bacterial and viral infections. engelmann et al 79 conducted a prospective, multicenter cohort study in different pediatric emergency departments in france on the role of mxa in the diagnosis of viral infections. mxa blood values were calculated in infants and children with verified bacterial or viral infections, uninfected controls, and infections of unknown origin. a receiver operating characteristic analysis was used to verify the diagnostic performance of mxa. the study, which included 553 children, showed that mxa was significantly higher in children with viral versus bacterial infections and uninfected controls (p0.0001). additionally, mxa levels were significantly higher in children with clinically diagnosed viral infections than in those with clinically diagnosed bacterial infections (p0.001). 79 other authors have also reported the usefulness of blood mxa testing in patients with viral infections. 80, 81 the use mxa in diagnosing viral infection is very promising, especially in patients who are at risk of infectious complications. two separate studies have shown that blood mxa is beneficial in differentiating between viral illness and acute graft-versushost disease after allogenic stem cell transplantation. 82, 83 it has been recommended that treatment or prevention of a viral disease may be a superior method for diminishing 62 it has also been published that live attenuated influenza vaccine is effective in reducing the incidence of all-cause aom [86] [87] [88] and pneumonia 89 compared to placebo in children. in addition, the intranasal influenza vaccine can reduce om by 44%. 90 moreover, studies have shown that a combined influenza/pneumococcal vaccine is efficient in the prevention of om in children and pneumonia. 91, 92 however, the credit of protection was awarded to the influenza vaccine since studies have shown that pneumococcal vaccine has no benefit in the reduction of aom. 93, 94 in addition, the pneumococcal polysaccharide vaccine showed no efficacy in the prevention of pneumonia in adults. 95 treatment of viral infection is anticipated to prevent bacterial superinfections. currently, the only respiratory virus that is pharmacologically treatable is the influenza viruses (type a and b). 62 neuraminidase inhibitors can potentially diminish the morbidity related to influenza. 96 oseltamivir can reduce the incidence of aom in preschool children, 97 and the reduction rate can be up to 44%. 98 a meta-analysis review showed that oral oseltamivir reduces the rate of hospitalization by 25% and morbidity by 75%. 99 in addition, its use can reduce the use of antibiotics by up to 50%, 100, 101 the same concept of protection applies to vaccines that prevent against rsv infections. 62 the vaccine available for rsv is palivizumab (medimmune, gaithersburg, md, usa), a humanized monoclonal antibody that perceives the fusion protein of rsv. the other monoclonal antibody that is under clinical trials is motavizumab (medimmune), which has a higher affinity for rsv fusion protein than palivizumab and can prevent against medically attended lower respiratory tract infection. 102 the rate of concurrent serious bacterial infections with viral illness is appreciable. similar emphasis must be given to the prevention and treatment of viral illnesses, especially in young 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polysaccharide vaccine in older adults neuraminidase inhibitors: zanamivir and oseltamivir neuraminidase inhibitors for preventing and treating influenza in children oral oseltamivir treatment of influenza in children impact of neuraminidase inhibitor treatment on outcomes of public health importance during the 2009-2010 influenza a(h1n1) pandemic: a systematic review and meta-analysis in hospitalized patients impact of oseltamivir treatment on influenza-related lower respiratory tract complications and hospitalizations efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial: us oral neuraminidase study group motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial publish your work in this journal submit your manuscript here: http://www.dovepress.com/therapeutics-and-clinical-risk-management-journal therapeutics and clinical risk management is an international, peerreviewed journal of clinical therapeutics and risk management, focusing on concise rapid reporting of clinical studies in all therapeutic areas, outcomes, safety, and programs for the effective, safe, and sustained use of medicines. this journal is indexed on pubmed central, cas, embase, scopus and the elsevier bibliographic databases. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord-174036-b3frnfr7 authors: thomas, loring j.; huang, peng; yin, fan; luo, xiaoshuang iris; almquist, zack w.; hipp, john r.; butts, carter t. title: spatial heterogeneity can lead to substantial local variations in covid-19 timing and severity date: 2020-05-20 journal: nan doi: nan sha: doc_id: 174036 cord_uid: b3frnfr7 standard epidemiological models for covid-19 employ variants of compartment (sir) models at local scales, implicitly assuming spatially uniform local mixing. here, we examine the effect of employing more geographically detailed diffusion models based on known spatial features of interpersonal networks, most particularly the presence of a long-tailed but monotone decline in the probability of interaction with distance, on disease diffusion. based on simulations of unrestricted covid-19 diffusion in 19 u.s cities, we conclude that heterogeneity in population distribution can have large impacts on local pandemic timing and severity, even when aggregate behavior at larger scales mirrors a classic sir-like pattern. impacts observed include severe local outbreaks with long lag time relative to the aggregate infection curve, and the presence of numerous areas whose disease trajectories correlate poorly with those of neighboring areas. a simple catchment model for hospital demand illustrates potential implications for health care utilization, with substantial disparities in the timing and extremity of impacts even without distancing interventions. likewise, analysis of social exposure to others who are morbid or deceased shows considerable variation in how the epidemic can appear to individuals on the ground, potentially affecting risk assessment and compliance with mitigation measures. these results demonstrate the potential for spatial network structure to generate highly non-uniform diffusion behavior even at the scale of cities, and suggest the importance of incorporating such structure when designing models to inform healthcare planning, predict community outcomes, or identify potential disparities. since its emergence at the end of 2019, the sars-cov-2 virus has spread rapidly to all portions of globe, infecting nearly five million people as of late may 2020 (1) . the disease caused by this virus, denoted covid-19, generally manifests as a respiratory illness that is spread primarily via airborne droplets. while most cases of covid-19 are non-fatal, a significant fraction of those infected require extensive supportive care, and the mortality rate is substantially higher than more common infectious diseases such as seasonal influenza (2) . even for survivors, infection can lead to long-term damage to the lungs and other organs, leading to long convalescence times and enhance risks of secondary complications (3, 4) . by early march of 2020, covid-19 outbreaks had appeared on almost every continent, including significant clusters within many cities (5) . in the absence of an effective vaccine, public health measures to counteract the pandemic in developed nations have focused on social distancing measures that seek to slow diffusion sufficiently to avoid catastrophic failure of the healthcare delivery system. both the planning and public acceptance of such measures have been highly dependent upon the use of epidemiological models to probe the potential impact of distancing interventions, and to anticipate when such measures may be loosened with an acceptable level of public risk. as such, the assumptions and behavior of covid-19 diffusion models is of significant concern. currently dominant approaches to covid-19 modeling (6) (7) (8) are based on compartment models (often called sir models, after the conventional division of the population into susceptible, infected, and recovered groups in the most basic implementations) that implicitly treat individuals within a population as geographically well-mixed. while some such models include differential contact by demographic groups (e.g., age), and may treat states, counties, or occasionally cities as distinct units, those models presently in wide use do not incorporate spatial heterogeneity at local scales (e.g., within cities). past work, however, has shown evidence of substantial heterogeneity in social relationships at regional, urban, and sub-urban scales (9, 10) , with these variations in social network structure impacting outcomes as diverse as regional identification (11) , disease spread (12) , and crime rates (13) , as well as in both human and non-human networks (14) . if individuals are not socially "well-mixed" at local scales, then it is plausible that diffusion of sars-cov-2 via interpersonal contacts will likewise depart from the uniform mixing characteristic of the sir models. indeed, at least one computational study (15) using a fairly "generic" (non-covid) diffusion process on realistic urban networks has showed considerable nonuniformity in diffusion times, suggesting that such effects could hypothetically be present. however, it could also be hypothesized that such effects would be small perturbations to the broader infection curve captured by conventional compartment models, with little practical importance. the question of whether these effects are likely to be present for covid-19, and if so their strength and size, has to date remained open. in this paper, we examine the potential impact of local spatial heterogeneity on covid-19, modeling the diffusion of sars-cov-2 in populations whose contacts are based on spatially plausible network structures. we focus here on the urban context, examining nineteen different cities in the united states. we simulate the population of each city in detail (i.e., at the individual level), simulating hypothetical outbreaks on the contact network in each city in the absence of measures such as social distancing. despite allowing the population to be well-mixed in all other respects (i.e., not imposing mixing constraints based on demographic or other characteristics), we find that spatial heterogeneity alone is sufficient to induce substantial departures from spatially homogeneous sir behavior. among the phenomena observed are "long lag" outbreaks that appear in previously unharmed communities after the aggregate infection wave has largely subsided; frequently low correlations between infection timing in spatially adjacent communities; and distinct sub-patterns of outbreaks found in some urban areas that are uncorrelated with the broader infection pattern. gaps between infection peaks at the intra-urban level can be be large, e.g. on the order of weeks or months in extreme cases, even for communities that are within kilometers of each other. such heterogeneity is potentially consequential for the management of healthcare delivery services: as we show using a simple "catchment" model of hospital demand, local variations in infection timing can easily overload some hospitals while leaving others relatively empty (absent active reallocation of patients). likewise, we show that individuals' social exposures to others who are morbid or deceased vary greatly over the course of the pandemic, potentially leading to differences in risk assessment and bereavement burden for persons residing in different locations. differences in outbreak timing and severity may exacerbate health disparities (since e.g., surge capacity varies by community) and may even affect perception of and support for prophylactic behaviors among the population at large, with those in so-far untouched communities falsely assuming that the pandemic threat is either past or was exaggerated to begin with, or attributing natural variation in disease timing to the impact of health interventions. we note at the outset that the models used here are intended to probe the hypothetical impact of spatial heterogeneity on covid-19 diffusion within particular scenarios, rather than to produce high-accuracy predictions or forecasts. for the latter applications, it is desirable to incorporate many additional features that are here simplified to facilitate insight into the phenomenon of central interest. in particular, we do not incorporate either demographic effects or social distancing, allowing us consider a setting that is as well-mixed as possible (and hence as close as possible to an idealized sir model) with the exception of spatial heterogeneity. as we show, even this basic scenario is sufficient to produce large deviations from the sir model. despite the simplicity of our models, we do note that the approach employed here could be integrated with other factors and calibrated to produce models intended for forecasting or similar applications. covid-19 is typically transmitted via direct contact with infected individuals, with the greatest risk occurring when an uninfected person is within approximately six feet of an infected person for an extended period of time. such interactions can be modeled as events within a social network, where individuals are tied to those whom they have a high hazard of intensive interaction. in prior work, this approach has been successfully employed for modeling infectious disesaes ranging from hiv (16) and influenza (17) to zika (18) transmission. to model networks of potential contacts at scale, we employ spatial network models (19) , which are both computationally tractable and able to capture the effects of geography and population heterogeneity on network structure (20) . such models have been successfully used to capture social phenomena ranging from neighborhood-level variation crime rates (13) and regional identification (11) to the flow of information among homeless persons (21) . the spatial network models used here allow for complex social dependence through a kernel function, referred to as the social interaction function or sif. the sif formally defines the relationship between two individuals based on spatial proximity. for example it has been shown that many social interaction patterns obey the zipf law (22) , where individuals are more likely to interact with others close by rather than far away (a pattern that holds even for online interactions (9) ). here, we use this approach to model a network that represents combination of frequent interactions due to ongoing social ties, and contacts resulting from frequent incidental encounters (e.g., interactions with neighbors and community members). we follow the protocol of (13, 20) to simulate social network data that combines the actual distribution of residents in a city with a pre-specified sif. we employ the model and data from (13) to produce large-scale social networks for 19 cities and counties in the united states -providing a representation of major urban areas in the united states (see supplement). given these simulated networks, then implement an sir-like framework to examine covid-19 diffusion. at each moment in time, each individual can be in a susceptible, infected but not infectious, infectious, deceased, or recovered state. the disease diffuses through the contact network, with currently infectious individuals infecting susceptible neighbors as a continous time poisson process with a rate estimated from mortality data (see supplement); recovered or deceased individuals are not considered infectious for modeling purposes. upon infection, an individual's transitions between subsequent states (and into mortality or recovery) are governed by waiting time distributions based on epidemiological data as described in the supplemantary materials. to begin each simulated trajectory, we randomly infect 25 individuals, with all others being considered susceptible. simulation proceeds until no infectious individuals remain. from the simulated trajectory data, we produce several metrics to assess spatial heterogeneity in disease outcomes. first, we present infection curves for illustrative cities, showing the detailed progress of the infection and its difference from what an sir model would posit. we also present chloropleth maps showing spatial variation in peak infection times, as well as the correlations between the infection trajectory within local areal units and the aggregate infection trajectory for the city as a whole. while an sir model would predict an absence of systematic variation in the infection curves or the peak infection day for different areal units in the same city, geographically realistic models show considerable disparities in infection progress from one neighborhood to another. to quantify the degree of heterogeneity more broadly, we examine spatial variation in outcomes for each of our city networks. we show that large variations in peak infection days within across tracts are typical (often spanning weeks or even months), and that overall correlations of within-tract infection trajectories with the aggregate urban trajectory are generally modest (a substantial departure from what would be expected from an sir model). in addition to these relatively abstract metrics, we also examine a simple measure of the potential load on the healthcare system in each city. given the locations of each hospital in each city, we attribute infections to each hospital using a voronoi tessellation (i.e., under the simple model that individuals are most likely to be taken to the nearest hospital if they become seriously ill). examination of the potential hospital demand over time shows substantial differences in load, with some hospitals severely impacted while others have few cases. finally, we consider the social exposure of individuals to covid-19, by computing the fraction of individuals with a personal contact who is respectively morbid or deceased. our model shows considerable differences in these metrics over time, revealing that the pandemic can appear very different to those "on the ground" -evaluating its progress by its impact on their own personal contacts -than what would be suggested by aggregate statistics. networks are generated using population distributions from (13), based on block-level census data. hospital information are obtained from the homeland infrastructure foundation-level data (hifld) database (23) . hifld is a an initiative that collects geospatial information on critical infrastructure across multiple levels of government. we employ the national-level hospital facility database, which contains locations of hospitals for the 50 us states, washington d.c., us territories of puerto rico, guam, american samoa, northern mariana islands, palau, and virgin islands; underlying data are collated from various state departments or federal sources (e.g., oak ridge national laboratory). we employ all hospitals within our 19 target cities, excluding facilities closed since 2019. latitude/longitude coordinates and capacity information were employed to create a spatial database that includes information on the number of beds in each hospital. the dates of the first confirmed case and all the death cases for king county, where seattle is located, were obtained from the new york times, based on reports from state and local health agencies (24) . the death rate was calculated based on population size of each county from the 2018 american community survey, and employed to calibrate the infection rate (the only free parameter in the models used here); details are provided in the supplemental materials. we ran 10 replicates of the covid-19 diffusion process in each of our 19 cities, seeding with 25 randomly selected infections in each replicate and following the course of the diffusion until no infectious individuals remained. simulations were performed using a combination of custom scripts for the r statistical computing system (25) and the statnet library (26) (27) (28) . analyses were performed using r. when taken over even moderately sized regions, aggregate infection curves can appear relatively smooth. although this suggests homogeneous mixing (as assumed e.g. by standard sir models), appearances can be deceiving. fig. 1 shows typical realizations of infection curves for two cities (seattle, wa and washington, dc), showing both the aggregate trajectory (red) and trajectories within individual census tracts (black). while the infection curves in both cases are relatively smooth, and suggestive of a fairly simple process involving a sharp early onset followed by an initially sharp but mildly slowing decline in infections, within-tract trajectories tell a different story. instead of one common curve, we see that tracts vary wildly in onset time and curve width, with some tracts showing peaks weeks or months after the initial aggregate spike has passed. the cases of fig. 1 are emblematic of a more systematic phenomenon: the progress of the infection within any given areal unit often has relatively little relationship to its progress in the city as a whole. a common pattern by taking the variance on the first principal component of the standardized infection curves. as before, where different parts of the city experience similar patterns of growth and decline in infections, we expect the dimension of greatest shared variance to account for the overwhelming majority of variation in infection rates. contrary to these expectations, however, fig. 2 shows that there is little coherence in tract-level infection patterns. mean correlations of local infection curves across tracts typically range from approximately 0 and 0.5, with a mean of approximately 0.2, indicating very little correspondence between infection timing in one track and that of another. the principal component analysis tells a similar story: overall, we see that first component accounts for relatively little of the total variance in trajectories, with on average only around 35% of variation in infection curves lying on the first principal component (and no observed case of the first component accounting for more than 60% of the variance). this confirms that local infection curves are consistently distinct, with behavior that is only weakly related to infections in the city as a whole. this is a substantially different scenario than what is commonly assumed in traditional sir models. these differences in local infection curves are a consequence of the unevenness of the "social fabric" that spans the city: while the disease can spread rapidly within regions of high local connectivity, it can easily become stalled upon reaching the boundaries of these regions. further transmission requires that a successful infection event occur via a bridging tie, an event with a potentially long waiting time. such delays create potential opportunities for public health interventions (trace/isolate/treat strategies), but they can also create a false sense of security for those on the opposite side of bridge (who may incorrectly assume that their area was passed over by the infection). indeed, examining the time to peak infection across the cities of seattle and washington, d.c. (fig. 3) shows that while peak times are visibly autocorrelated, tracts with different peak times frequently border each other. residents on opposite sides of the divide may be exposed to very different local infection curves, making risk assessment difficult. the cases of seattle and washington, dc are not anomalous. looking across multiple trajectories over our entire sample, fig. 4 shows consistently high variation in per-tract peak infection times for nearly all study communities. (this variation is also seen within individual trajectories, as shown in supplemental figure s2.) although peak times in some cities are concentrated within an interval of several days to a week, it is more common for peak times to vary by several months. such gaps are far from what would be expected under uniform local mixing. variation in the timing of covid-19 impacts across the urban landscape has potential ramifications for healthcare delivery, creating unequally distributed loads that overburden some providers while leaving others with excess resources. to obtain a sense of how spatial heterogeneity in the infection curve could potentially impact hospitals, we employ a simple "catchment" model in which seriously ill patients are taken to the nearest hospital, subsequently recovering and/or dying as assumed throughout our modeling framework. based on prior estimates, we assume that 20% of all infections are severe enough to require hospitalization (29) . while hospitals draw from (and hence average across) areas that are larger than tracts, the heterogeneity shown in fig. 1 suggests the potential for substantial differences in hospital load over time. indeed, our models suggest that such differences will occur. fig. 5 shows the number of patients arriving at each hospital in seattle and washington, dc (respectively) during a typical simulation trajectory. while some hospitals do have demand curves that mirror the city's overall infection curve, others show very different patterns of demand. in particular, some hospitals experience relatively little demand in the early months of the pandemic, only to be hit hard when infections in the city as a whole are winding down. just as hospital load varies, hospital capacities vary as well. as a simple measure of strain on hospital resources, we consider the difference between the number of covid-19 hospitalizations and the total capacity of the hospital (in beds), truncating at zero when demand outstrips supply. (for ease of interpretation as a measure of strain, we take the difference such that higher values indicate fewer available beds.) using data on hospital locations and capacities, we show in fig. 6 strain on all hospitals in seattle and washington, d.c. (respectively) during a typical infection trajectory. while some hospitals are hardest hit early on (as would be expected from the aggregate infection curve), others do not peak for several months. likewise, hospitals proximate to areas of the city with very different infection trajectories experience natural "curve flattening," with a more distributed load, while those that happen to draw from positively correlated areas experience very sharp increases and declines in demand. these conditions in some cases combine to keep hospitals well under capacity for the duration of the pandemic, while others are overloaded for long stretches of time. these marked differences in strain for hospitals within the same city highlight the potentially complex consequences of heterogeneous diffusion for healthcare providers. looking across cities, we see the same high-variability patterns as observed in seattle and washington. in particular, we note that local variation in disease timing leads to a heavy-tailed distribution for the duration at which hospitals will be at capacity. fig. 7 shows the marginal distribution of hospital overload periods (defined as total number of days at capacity during the pandemic), over the entire sample. while the most common outcome is for hospitals to be stressed for a brief period (not always to the breaking point), a significant fraction of hospitals end up being overloaded for months -or even, in a small fraction of cases, nearly the whole duration of the pandemic. while most hospitals will have only brief periods of overload, some will be at or over capacity for the entire pandemic, potentially several years. it should be reiterated that the hospital load model used here is extremely simplified, and that we are employing a no-mitigation scenario. however, these results quite graphically demonstrate that the importance of curve-flattening interventions does not abate once geographical factors are taken into account. on the other hand, these results suggest that differences in hospital load may be substantially more profound than would be anticipated from uniform mixing models, creating logistical challenges and possibly exacerbating existing differences in resource levels across hospitals. at the same time, such heterogeneity implies that resource sharing and patient transfer arrangements could prove more effective as load-management strategies than would be suggested by spatially homogeneous models, as hospitals are predicted to vary considerably in the timing of patient demand. in addition to healthcare strain, the subjective experience of the pandemic will potentially differ for individuals residing in different locations. in particular, social exposures to outcomes such as morbidity or mortality may shape individuals' understandings of the risks posed by covid-19, and their willingness to undertake protective actions to combat infection. such exposures may furthermore act as stressors, with potential implications for physical and/or mental health. as a simple measure of social exposure, we consider the question of whether a focal individual (ego) either has experienced a negative outcome themselves, or has at least one personal contact (alter) who has experienced the outcome in question. (given the highly salient nature of covid-19 morbidity and mortality, we focus on the transition to first exposure rather than e.g. the total number of such exposures, as the first exposure is likely to have the greatest impact on ego's assessment of the potential severity of the disease.) to examine how social exposure varies by location, we compute the fraction of individuals in each tract who are in socially exposed to (respectively) morbidity or mortality. fig. 8 shows these proportions for baltimore, md, over the course of the pandemic. as with other outcomes examined here, we see considerable variation in timing, with many tracts seeing a rapid increase in exposure to infections, while others go for weeks or months with relatively few persons having a personal contact with the disease. another notable axis of variation is sharpness. in many tracts, the fraction of individuals with at least one morbid contact transitions from near zero to near one within a matter of days, creating an extremely sharp social transition between the "pre-exposure world" (in which almost no one present knows someone with the illness) to a "post-exposure world" in which almost everyone knows someone with the illness). by contrast, other tracts show a much more gradual increase (sometimes punctuated by jumps), as more and more individuals come to know someone with the disease. in a few tracts that are never hit hard by the pandemic, few people ever have an infected alter; residents of these areas obviously have a very different experience than those of high-prevalence tracts. these distinctions are even more stark for mortality, which takes longer to manifest and which does so much more unevenly. tracts vary greatly in the fraction of individuals who ultimately lose a personal contact to the disease, and in the repidity with which that fraction is reached. in many cases, it may take a year or more for this quantity to be realized; until that point, many residents may be skeptical to the notion that the pandemic poses a great risk to them personally. by way of assessing the milieu within each tract, it is useful to consider the "cross-over" point at which at least half of the residents of a given tract have been socially exposed to either covid-19 morbidity or mortality. fig. 9 maps these values for baltimore, md. it is immediately apparent that social exposures are more strongly spatially autocorrelated than other outcomes considered here, due to the presence of long-range ties within individuals' personal networks. even so, however, we see strong spatial differentiation, with residents in the urban core being exposed to both morbidity and mortality much more quickly than those on the periphery. this suggests that the social experience of the pandemic will be quite different from those in city centers than in more outlying areas, with the latter taking far longer to be exposed to serious consequences of covid-19. this may manifest in differences in willingness to adopt protective actions, with those in the urban core being more highly motivated to take action (and perhaps resistant to rhetoric downplaying the severity of the disease) than those on the outskirts of the city. we see a large degree of spatial heterogeneity, as some tracts are more insulated from others in terms of social exposure. however, by the end of the pandemic, most people across all tracts have been exposed to someone who has had the disease. (right) the fraction of persons in each tract who have an alter who died from covid-19 in their personal network. on average, only around 40% of people in any given tract know someone who died by the end of the pandemic, though this varies widely across tracts. figure 9 : (left) chloropleth showing the time for half of those in each tract to be socially exposed to covid-19 morbidity in baltimore, md. the central and southern parts of the city are exposed far sooner than the northwestern part of the city. (right) chloropleth showing the time for half of those in each tract to be socially exposed to covid-19 mortality. central baltimore is exposed to deaths in personal networks far sooner than the more outlying areas of the city. our simulation results all underscore the potential effects of local spatial heterogeneity on disease spread. the spatial heterogeneity heterogeneity driving these results occurs on a very small scale (i.e., census blocks), operating well below the level of the city a whole. as the infection spreads, relatively small differences in local network connectivity and the prevalence of bridging ties driven by uneven population distribution can lead to substantial differences in infection timing and severity, leading different areas in each city to have a vastly different experience of the pandemic. resources will be utilized differently in different areas, some areas will have the bulk of their infections far later than others, and the subjective experience of a given individual regarding the pandemic threat may differ substantially from someone in a different area. these behaviors are in striking contrast to what is assumed by models based on the assumption of spatially homogeneous mixing, which posit uniform progress of the infection within local areas. as noted at the outset, our model is based on a no-mitigation scenario, and is not intended to capture the impact of social distancing. while distancing measures by definition limit transmission rates -and will hence slow diffusion -contacts occurring through spatially correlated networks like those modeled here are still likely to show patterns of heterogeneity like those described. one notable observation from our simulations is the long outbreak delay that some census tracts experience, even in the absence of social distancing. this would suggest that relaxation of mitigation measures leading to a resumption of "normal" diffusion may initially appear to have few negative effects, only to lead to deadly outbreaks weeks or months later. public health messaging may need to stress that apparent lulls in disease progress are not necessarily indicators that the threat has subsided, and that areas "passed over" by past outbreaks could be impacted at any time. finally, we stress that conventional diffusion models using locally homogeneous mixing have been of considerable value in both pandemic planning and scenario evaluation. our findings should not be taken as an argument against the use of such models. however, the observation that incorporating geographical heterogeneity in contact rates leads to radically different local behavior would seem to suggest that there is value in including such effects in models intended to capture outcomes at the city or county level. since these are the scales on which decisions regarding infrastructure management, healthcare logistics, and other policies are often made, improved geographical realism could potentially have a substantial impact on our ability to reduce lives lost to the covid-19 pandemic. in this supplement, we go into more depth on spatial interaction functions, spatial bernoulli models, the setup and parameterizations of our simulations, and the paramter estimation problems that we used for this paper. we focus specifically on the more technical aspects of each of these components, showing how they have been formally specified and parameterized. a spatial interaction function (sif) describes the marginal probability of a tie between any two nodes, given the distance between those nodes, represented f(d i,j , î¸). in this representation, d i,j is the distance between the nodes, and î¸ are the parameters for the function. prior literature shows that spatial interaction functions tend to be of the power law or attenuated power law form (19) . thus, we can represent the sif as f(d i,j , î¸) = p b (1+î± * d i,j ) î³ . here, p b represents the base tie probability, which can be thought of as the probability of a tie at distance 0. î± is a scaling parameter that determines the speed at which the probability drops towards zero. î³ is the parameter that determines the weight of the tail. we draw on two sifs in this paper, using models for social interactions and face to face interactions employed in prior studies (13, 20) . the social interaction sif declines with a î³ of 2.788, while the face-to-face sif declines with î³ of 6.437. the paramters for the social interaction sif are p b = 0.533, î± = 0.032, î³ = 2.788, and the parameters for the face-to-face sif are p b = 0.859,î± = 0.035, î³ = 6.437 (13) . bernoulli models are a class of random graph models that leverage the concept of a bernoulli graph, or a graph in which the probability associated with each edge is a bernoulli trial. in a spatial bernoulli graph, tie probabilities are determined by a spatial interaction function, applied to the pairwise distances between individuals within some space (here, geographically determined using census data). spatial bernoulli models are highly scalable due to the conditional independence of edges, but allow for extremely complex structure due to the heterogeneity in edge probabilities induced by the sif; likewise, they naturally produce properties such as local cohesion and degree heterogeneity observed in many types of social networks (20) . formally, we can specify a spatial bernoulli model by p r(y ij = 1) = f(d i,j , î¸), where y ij is each dyad, and f(d i,j , î¸) is a spatial interaction function with inputs as distance d i,j , and parameters î¸. to simulate diffusion of covid-19, we require a contact network. here, we employ the above-described spatial bernoulli graphs, with node locations for each of our 19 study locations drawn based on blocklevel census data (including clustering within households, an important factor in disease diffusion). we follow the protocols described in (15, 20) to generate node positions, specifically using the quasirandom (halton) placement algorithm. node placement begins with the households in each census block, using census data from (13) . the quasirandom placement algorithm uses a halton sequence to place households in space within the areal unit in which they reside. if any two households are placed within a critical radius of each other, then the algorithm "stacks" the households on top of each other by introducing artificial elevation (simulating e.g. a multistory apartment building). once all households are placed, individuals within households are placed at jittered locations about the household centroid. (individuals not otherwise attached to households are treated as households of size 1.) given an assignment of individuals to spatial locations, we simulate spatial bernoulli graphs using the models specified above. we generate two networks for each city, one with the social interaction sif, and the other with the face-to-face interaction sif. to form a network of potential high-risk contacts, we then merge these networks (which share the same node set) by taking their union, leading to a network in which two individuals are tied if they either have an ongoing social relationship or would be likely to have extensive face-to-face interactions for other reasons (e.g., interacting with neighbors). this process is performed for each city in our sample. table s1 lists the cities that we use for our simulations. these data are drawn from (13). we conduct a series of simulations to examine the spread of covid-19 across city-sized networks. these simulations use a simple continuous-time network diffusion process, the general description of which are described in the main text. the input for the diffusion simulation is a network and a vector of initial disease states (susceptible, latent (infected but not yet infectious), infectious, recovered, and deceased), and the output is detailed history of the diffusion process up to the point at which a steady state is obtained (i.e., no infectious individuals remain). infection occurs via the network, with currently infectious individuals infecting susceptible alters as poisson events with a fixed rate. the transitions between latent and infectious, and infectious and either recovery or mortality are governed by gamma distributions estimated from epidemiological data. table s2 shows the estimated shape and scale parameters for the gamma distributions employed here. the parameters for waiting time to infectiousness are directly available in the appendix of (30), while those for the recovery and death are estimated by matching the mean and standard deviation of durations reported in the literature (31) . selection into death versus recovery was made via a bernoulli trial drawn at time of infection (thereby determining which waiting time distribution was used), with the estimated mortality probabiliy being 0.0215. to determine the infection rate (the only free parameter for the network models used in our simulations), we simulate the diffusion of virus in seattle and fit it to the over-time death rate of the king county, wa before the first shelter-in-place order went into effect on march 23, 2020. (we limit our data to this time period because our simulation employs a no-mitigation scenario.) a grid search strategy was employed to determine the expected days to transmission (which is the inverse of infection rate), and the number of days between the existence of the first infected cases and the first confirmed cases (aka the time lag, a nuisance parameter that is relevant only for estimation of the infection rate). the time lag is treated as an integer and the expected days to transmission as a continuous variable. for each lag/rate pair, we randomly take 5 draws from the expected infection waiting time distribution, add them to the lag time (i.e. the introduction of the true patient zero for the initial outbreak), and simulate 50 realizations of the diffusion process (redrawing the network each time). the diffusion rate parameter was selected based on minimizing the mean squared error between the simulated death rate and the observed number of deaths over the selected period. the first round of grid-search divided the expected days of search into 100 intervals, from (1,2) to (100,101), with days of lag ranging from 1 to 100 days. the second round of grid-search, based on the performance of the first round, divided the expected days of search into 240 intervals, from (41.00,41.25) to (100.75,101.00), with days of lag ranging from 1 to 60 days. the grid-search suggests that the expected days to transmission is 78.375 (78.25,78.50) days ( fig s1) ; that is, in a hypothetical scenario in which a single infective ego remained indefinitely in the infective state, and a single alter remained otherwise susceptible, the average waiting time for ego to infect alter would be approximately 80 days. while this may at first blush appear to be a long delay, it should be borne in mind that this embodies the reality that no individual is likely to infect any given alter within a short period (since, indeed, ego and alter may not happen to interact within a narrow window). with many alters, however, the chance of passing on the disease is quite high. likewise, we note that the thought experiment above should not be taken to imply that actors remain infectious for such an extended period of time; per the above-cited epidemiological data, individuals typically remain infectious for roughly 18-33 days (though variation outside this range does occur, as captured by the above gamma distributions). when both delay times are considered, the net probability of infecting any given alter prior to recovering is approximately 27%. using the above, we can calculate the corresponding basic reproductive number (r 0 ): where d is the mean degree of an individual in the network; î± is the infection rate (the inverse of expected days of transmission); ï� is the time spent in the infectious state (here in days). for each simulated seattle contact network, we calculate the degree for every individual. the time in the infectious statae was obtained by simulating gamma distributions for days of incubation, recovery, and death, and randomly permuting the distribution for 10 times for each simulated sif network. taking the mean of r 0 for each individual, the corresponding basic reproductive number in the diffusion simulation model is 1.7. to supplement the results on the variation in the peak infection time given in the main text, we ran a series of simulation replicates. figure 3 in the main text shows the data from the figure below aggregated across all replicates. in the supplemental figure s2, we break out the peak infection days in each city, by replicate. these data show that the significant variation in figure 3 is not due to the number of replicates that were run, but instead due to the intrinsic variation that is present (due to spatial heterogeneity). network epidemiology: a handbook for survey design and data collection the sage handbook of gis and society research human behavior and the principle of least effort: an introduction to human ecology r: a language and environment for statistical computing, r foundation for statistical computing the lancet infectious diseases this research was supported by nsf awards iis-1939237 and ses-1826589 to c.t.b, and by the uci seed grants program. figure s2 : boxplot showing the peak infection days across 10 replicates for each city in the sample. there is a large degree of heterogeneity within each city, showing that the day that the infection peaks for any given tract is not uniform at all. within any given city, there is a consistently high amount of variance in the peak infection day. in other words, the variance that we show here is a property of the spread of the disease, rather than the number of simulation replicates. key: cord-253761-wjm8ju3v authors: haidopoulou, katerina; goutaki, myrofora; damianidou, lambrini; eboriadou, maria; antoniadis, antonis; papa, anna title: human bocavirus infections in hospitalized greek children date: 2010-03-09 journal: arch med sci doi: 10.5114/aoms.2010.13515 sha: doc_id: 253761 cord_uid: wjm8ju3v introduction: the epidemiology of human bocavirus (hbov) infections has not been described in greece, a south-eastern european country. to define the epidemiological profile and the clinical characteristics associated with hbov infection in a population of children hospitalized with respiratory tract infection. material and methods: during a one-year period throat swab samples were collected from 370 previously healthy children, aged 14 days to 13 years, admitted to two different paediatric wards because of respiratory tract infection. samples were tested for hbov by pcr amplifying a part of the ns1 gene. results: human bocavirus was detected in 12 children (3.2%). four of the 12 cases were co-infections, 3 of them with influenza a and 1 with coronavirus oc43. cases were observed only during the cold months. the mean age of children was 1.8 years (range 2 months to 4 years). the most common symptoms were fever, cough and various degrees of respiratory distress. all children were clinically diagnosed as having lower respiratory tract infections, mainly pneumonia and acute laryngotracheobronchitis, and recovered uneventfully. conclusions: hbov infections occur in greece mostly among very young children. they accounted for 3.2% of children hospitalized with acute respiratory disease. cases were observed only in late autumn to early spring. human bocavirus (hbov) (genus bocavirus, family parvoviridae) has been recently identified in children with respiratory tract infection (rti), first in sweden [1] , and subsequently in different parts of the world [2] [3] [4] [5] [6] [7] [8] [9] [10] . however, most studies so far have only retrospectively studied virus prevalence and only a few have addressed whether hbov infection is associated with respiratory disease symptoms. the aim of the present study was to define the epidemiological profile and the clinical characteristics associated with hbov in hospitalized children with respiratory tract infection (rti) in greece. during a one-year period (october 2006 to september 2007) throat swab samples were collected from 370 previously healthy children, aged 14 days to 13 years (mean age ± sd 17 ±13 months) admitted to two dif-ferent large hospitals of thessaloniki, northern greece. samples were taken on the first day of admission and kept at -70°c until use. all children were admitted because of acute infection of the respiratory tract. demographic data and clinical diagnosis including acute infection of the upper respiratory tract, croup, bronchitis, bronchiolitis and pneumonia were obtained from a computer-generated discharge diagnosis based database. the case notes of children were reviewed using a standardised clinical data extraction form. the following data were recorded: sex, age, initial presenting symptoms, signs, discharge diagnosis and routine laboratory examinations upon admission and during hospitalization (complete blood count, c-reactive protein, chest x-ray). extracted dna was subjected to polymerase chain reaction (pcr) which targets the ns1 gene of the hbov genome [8] . all pcr products were sequenced and nucleotide sequences were compared with respective hbov sequences retrieved from genbank. in addition, all samples were tested by molecular methods for mycoplasma pneumoniae, respiratory syncytial virus, coronaviruses, influenza viruses, human metapneumovirus and adenoviruses. hbov dna was detected in samples of 12 children (3.3%), 6 of them males. sequencing and phylogenetic analysis revealed that hbov sequences of 11 cases were identical to each other and to the swedish strain st2 (nc007455), differing by 1 nucleotide from the 12 th case (gr186), which was identical to strain chsd4 (dq471814) from usa. all cases had clinical evidence of lower rti (fever, tachypnoea, hypoxia, retractions, and abnormal auscultation findings). four of the 12 cases were coinfections, 3 of them with influenza a virus and 1 with coronavirus oc43. concerning the 8 patients in whom only hbov was detected, they were 3 males and 5 girls, aged 2-33 months (mean age ± sd: 17 ±9 months), hospitalized with the clinical diagnosis of 2 each: laryngotracheobronchitis, bronchiolitis, pneumonia, and asthma exacerbation. common symptoms of viral respiratory tract infection such as fever, cough, rhinorrhoea and pharyngitis were found in the majority of our patients (87.5-100%). six patients (75%) presented with various degrees of respiratory distress. those patients had tachypnoea (a respiratory rate of 45-90 breaths/min) and low haemoglobin oxygen saturation levels (sao 2 ) in the range 90-94%. these children received oxygen supplementation until 1 day before discharge. wheezing was the most common clinical finding (5 patients) while another 1 child presented with stridor. hoarseness was noticed in 2 children. difficulty in feeding was also a common complaint, reported by 5 patients. other clinical findings included diarrhoea (2 patients, 25%), a symptom previously described in hbov infections [11, 12] , and otitis media (1 patient, 12.5%). the major clinical and laboratory findings in the 8 patients in whom hbov was the sole pathogen detected are presented in table i . chest x-rays were available for all 8 children. the most common finding, present in 6 patients, was bilateral interstitial infiltrates. consolidation was detected in the other 2 children. clinical expression was not different in children with hbov co-infection. the child with coronavirus oc43 co-infection also presented with conjunctivitis, while respiratory distress was the main concern in a child with influenza-a virus co-infection. all children recovered uneventfully. the median hospitalization time was 5 days (range 4-9 days). for 1 patient a second throat swab sample, taken 20 days after the first one, was found negative for hbov dna. prevalence of hbov in our study (3.3%) was found to be relatively low compared to published data from other parts of the world [2, 4, 6, 9, 10, [13] [14] [15] [16] . however, prevalence rates between 1.5 and 19% have been observed and it is possible that differences of study populations and sampling techniques account for the encountered discrepancies [17] . although the number of studies since the first description of hbov is increasing, many of these studies are retrospective on stored samples, or only on samples negative for other pathogens, or on samples collected during a few months and thus suffering from the bias that sampling may be guided from the occurrence of rsv or influenza epidemics. in the present prospective study samples were collected during 1 year and were tested regardless of positivity for another pathogen. in previous whole year studies it was found that the vast majority of hbov positive samples were collected in cold months [14, 18, 19] . we also found that positive cases were observed only during the cold months. the first hbov case was observed in mid december 2006, and the last at the end of april 2007. greece is a mediterranean country in the balkan peninsula, with hot, dry summers and cold, damp winters. nevertheless, the mean temperature of winter 2006-2007 in greece (and worldwide) was above average (warmest winter on record), and the precipitation drier than normal (national observatory of athens), conditions which might have affected the prevalence of hbov. previous studies have implicated hbov as a cause of rti, most commonly in children < 2 years old, a finding similar to our study, where 10/12 children were younger than 2 years, the youngest one being a 2-month boy with acute bronchiolitis [13, 14, [20] [21] [22] . however, frequency of hbov infection was found to be relatively low in children younger than 6 months, and the possibility of passive protection due to maternal antibodies has been questioned. these findings together with the high prevalence rates in young children led to the hypothesis that hbov may be an endemic virus with high attack rates in those susceptible; one would then expect that the majority of the population would be infected during childhood. in support of this hypothesis are the high seroprevalence rates against hbov reported in the adult population [23] . the mere presence of hbov in the airway may not be able to define its role as a pathogen; therefore it is anticipated that serology would be a useful diagnostic addition to the study of hbov infection. four of the 12 cases were co-infections, 3 of them with influenza a virus and 1 with coronavirus oc43. similar findings were reported in other studies, where co-infection was found in one third of patients [5, 24] , while other investigators have reported even higher rates [8, 15, 25] . it has been hypothesized that detection of hbov asymptomatic presence may be enhanced by the symptoms caused by another virus or that hbov may aggravate the clinical course of symptoms due to other viral infections, so that it is frequently detected in hospitalized children [26] . co-infection is frequently described with many respiratory viruses and the question whether this may result in more serious clinical outcomes has not been clarified [27] . the association of hbov with acute wheezing, a symptom found in 62.5% of our patients, has been well described in recently published studies [5, 14, 18, 28] . another 2 of our patients presented with laryngotracheobronchitis, a finding which is in accordance with the study of rihkanen et al. [29] . only 1 of our patients returned for follow-up and a second throat swab sample, taken 20 days after the first one, was found negative for hbov dna. our findings suggest that hbov may have similar clinical features to those of other respiratory viruses such as rsv. one may argue that the fact that hbov is prevalent in samples from patients with respiratory tract infection does not guarantee a causative role for the symptoms, especially when -as in this case -it is frequently detected in combination with other respiratory viruses of known pathogenic potential. on the other hand, hbov was rarely detected in asymptomatic children [16, 22] . in conclusion, in the present study we have provided a first insight into the epidemiology and clinical aspects of hbov infections in hospitalized children in greece. our findings suggest a possible association of hbov with lower rti, manifested mainly by fever, cough and wheezing, most often in infants, and a seasonal pattern with hbov cases occurring in cold months. cloning of a human parvovirus by molecular screening of respiratory tract samples detection of human bocavirus in canadian children in a 1-year study bocavirus infection in hospitalized children detection of human bocavirus in hospitalised children human bocavirus in french children detection of human bocavirus in ill and healthy spanish children: a 2-year study the first detection of human bocavirus 2 infections in china evidence of human coronavirus hku1 and human bocavirus in australian children human bocavirus in very young infants hospitalized with acute respiratory infection in northeast brazil the incidence of human bocavirus infection among children admitted to hospital in singapore human bocavirus: prevalence and clinical spectrum at a children's hospital human bocavirus, a respiratory and enteric virus viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus isolation of human bocavirus from swiss infants with respiratory infections frequent detection of bocavirus dna in german children with respiratory tract infections human bocavirus human bocavirus and acute wheezing in children high incidence of human bocavirus infection in children in spain human bocavirus infection in a neonatal intensive care unit human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand human bocavirus in italian patients with respiratory diseases seroepidemiology of human bocavirus in hokkaido prefecture the association of newly identified respiratory viruses with lower respiratory tract infections in korean children frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction human bocavirus: passenger or pathogen in acute respiratory tract infections? coronaviruses oc43 and 229e lower respiratory tract co-infections: a clinical report of two cases human bocavirus: a cause of severe asthma exacerbation in children respiratory viruses in laryngeal croup of young children we would like to thank sofia komi for technical assistance. key: cord-284845-on97zu6w authors: falcinelli, shane d.; chertow, daniel s.; kindrachuk, jason title: integration of global analyses of host molecular responses with clinical data to evaluate pathogenesis and advance therapies for emerging and re-emerging viral infections date: 2016-07-29 journal: acs infectious diseases doi: 10.1021/acsinfecdis.6b00104 sha: doc_id: 284845 cord_uid: on97zu6w [image: see text] outbreaks associated with emerging and re-emerging viral pathogens continue to increase in frequency and are associated with an increasing burden to global health. in light of this, there is a need to integrate basic and clinical research for investigating the connections between molecular and clinical pathogenesis and for therapeutic development strategies. here, we will discuss this approach with a focus on the emerging viral pathogens middle east respiratory syndrome coronavirus (mers-cov), ebola virus (ebov), and monkeypox virus (mpxv) from the context of clinical presentation, immunological and molecular features of the diseases, and omics-based analyses of pathogenesis. furthermore, we will highlight the role of global investigations of host kinases, the kinome, for investigating emerging and re-emerging viral pathogens from the context of characterizing cellular responses and identifying novel therapeutic targets. lastly, we will address how increased integration of clinical and basic research will assist treatment and prevention efforts for emerging pathogens. e merging and re-emerging viruses pose a significant threat to public health and global economies. moreover, outbreaks caused by emerging and re-emerging viruses continue to increase in frequency as a result of changing socio-economic, environmental, and ecological factors. 1 notably, the zoonotic viral pathogens, severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), ebola virus, chikungunya virus, and zika virus, have emerged on a global scale in recent years; although less widely publicized, other emerging viral pathogens such as monkeypox virus and andes virus have led to smaller recurrent outbreaks. a critical challenge for combating these outbreaks is often the discordant relationship between the economic status of outbreak "hotspots" and resource distribution or control capacity within these regions. in addition, the development and delivery of therapeutics for combating such outbreaks have been complicated by both the associated costs in design and development for novel antiinfective therapeutics and the requirements for regulatory approval and licensure. 2 importantly, emerging infectious diseases present the additional inherent challenge that they are only "emerging", and thus limited resources are made available for research until they present a significant risk. for many emerging viral pathogens, the requirement for highcontainment facilities has further impeded widespread research. from the perspective of drug development, the limited knowledge and understanding of molecular pathogenesis for these agents is a daunting challenge to overcome when outbreaks emerge. in the face of an increasing burden of emerging and reemerging pathogens, it is necessary to overcome the barriers imposed by a paucity of information regarding molecular pathogenesis for these agents. omics-based approaches present a mechanism to rapidly generate large amounts of data in regard to host responses and assist in target identification for drug development efforts. in addition, omics-based analyses allow for the characterization of molecular events that mitigate cellular responses to viral pathogens from a global perspective across multiple levels of cellular complexity (individual cell types < tissues < organs). high-throughput global analyses of host gene expression, including microarrays and rna-seq, provide important information regarding transcriptional responses during infection. although these validated approaches are among the most widespread of the omics-based technologies for infectious disease investigations, they do not provide a direct measure of the activation status of the cell signaling pathways that regulate underlying cellular responses. in contrast, global investigations of cellular kinase activities (the kinome) are able to provide insight into the activation status of cell signaling networks (including those that mediate pathogen recognition and innate immune activation, cell cycle activities, metabolic status, wound healing and repair, and cell death) at the level of individual kinase-mediated phosphorylation events. in addition, kinome investigations allow for potential identification of kinase drug targets. kinases are currently one of the top targets for drug design and development, and there is potential for repurposing of kinase inhibitors with existing regulatory approval. as emerging viral infections often result in severe illness including respiratory failure [severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and influenza] and multiorgan failure [ebola virus disease (evd)], understanding complex pathogenesis of these infections is required for effective vaccine and therapeutic design and for improved patient care. healthcare providers caring for patients with severe emerging viral infections are generally focused on clinical care and biosafety as compared to the complex molecular events that underlie pathogenesis. in contrast, basic researchers typically focus on discrete aspects of pathogenesis through a variety of in vitro and in vivo analyses rather than the complex interplay between these events and the clinical, physiologic, and pathologic abnormalities observed by the clinician. integrating basic and clinical research is needed to accelerate the translation of knowledge for emerging infections toward vaccine development and therapeutic discovery. specifically, detailed natural history studies merging multiple data streams including omics approaches (high-throughput gene expression and kinomics) and focused translational investigations utilizing relevant models that can be validated to human disease are needed to clarify disease pathogenesis, advance therapeutic discovery, and facilitate regulatory approval. although an integrated approach between basic and clinical research is ideal for investigating the connections between molecular and clinical pathogenesis, there has been a paucity of investigations for which this has been undertaken. here, we will discuss emerging pathogens for which there is available information regarding the clinical course of disease, host immune responses during natural infection, and molecular information regarding the global cellular responses to infection, with particular attention on host kinome investigations. in this regard we will focus on the emerging viral pathogens mers-cov, ebola virus (ebov), and monkeypox virus (mpxv) (figure 1 ). we will review the clinical presentation and immunological and molecular features of the diseases and summarize available omics data informing pathogenesis of these pathogens. lastly, we will discuss the benefit of improved integration of available clinical knowledge or data regarding the pathologic manifestations of disease with basic research investigations to advance treatment and prevention of severe emerging viral infections. global gene expression investigations have provided information regarding host response to emerging and re-emerging review pathogens at the level of individual genes or gene clusters. however, there is a paucity of information regarding the relationship of cell signaling networks, and in particular their activation status, with the biological/pathological events that occur throughout infection. it has been well established that many biological processes can be regulated independent of transcriptional or translational changes through post-translational modification (ptm) events. indeed, kinase-mediated phosphorylation of proteins, in which kinases catalyze the transfer of the γ phosphate group from atp to the hydroxyl group of a specific ser, thr, tyr residue, is figure 2 . generation of kinome peptide array targets and kinome peptide arrays: (1) species-specific proteomic or genomic information from a diverse range of species can be used to identify kinase recognition motifs that are composed of a central phosphorylation target and the surrounding amino acids (normally +4 and −4 amino acids from the central phosphorylated residue); (2) peptides that comprise the kinase recognition motifs identified in (1) are synthesized and covalently linked to a glass surface. peptide targets are spotted in replicates of three to nine spots on each array to account for intra-array variability. individual amino acids of the peptides are represented by orange, red, purple, and green spheres. (4) biological samples are processed to generate cell lysates that are activated with atp and applied to the kinome peptide array. (5) following the application of the cell lysate, activated kinases in the cell lysate will recognize their respective kinase recognition motifs and phosphorylate the central phosphorylated residue of the peptide. (6) kinome peptide arrays are subsequently stained with a phospho-specific fluorescent stain and imaged followed by comparative bioinformatics analyses. tissue and cell images were derived and/or modified from servier medical arts under a creative commons attribution 3.0 unported license. among the most thoroughly characterized ptm. virtually all cell signal transduction events are regulated by kinases independent of biological complexity of the host (i.e., prokaryotes and eukaryotes). 3 lending further credence to the biological importance of kinases, >500 kinases have been identified in the human genome, and ∼30% of the human proteome is modulated by kinase-mediated phosphorylation events. 4, 5 thus, considering the central role of kinases in a broad range of cellular processes (including growth and development, metabolism, and immune responses), it has been postulated that the activities of individual kinases may represent more reliable predictors of cellular phenotypes than transcriptional or translational changes. 6 indeed, transcriptional or translational-based omics approaches are often unable to account for regulatory events including gene silencing, mrna stability, translational efficiencies, protein turnover, enzyme/ substrate subcellular sequestration, or protein activation/ repression ptms. 7 given the central role of kinases in the regulation of biological processes, kinases are a logical drug target. as a testament to this, 33 kinase inhibitors have been granted licensure by the u.s. food and drug administration (fda) for a broad range of malignancies, and there are a continually increasing number of kinase inhibitors that are in various stages of preclinical trials. furthermore, kinases are the second most frequently targeted gene class in cancer therapy after the g protein coupled receptors. 8, 9 the recent prioritization for the repurposing of approved therapeutics for alternative malignancies by the national institutes of health center for advancing translational sciences (ncats) 10, 11 also provides considerable impetus for the investigation of licensed kinase inhibitors as infectious disease therapeutics. concerns exist regarding the therapeutic application of kinase inhibitors as novel therapeutics for infectious disease, in particular to the potential immunosuppressive effects following prolonged treatment. however, it should be appreciated that the application of kinase inhibitors in such cases would need to be targeted in terms of timing and dose, with appropriate molecular biomarkers guiding initiation and cessation. it should also be appreciated that the clinical symptoms associated with many emerging and re-emerging pathogens have been associated with dysregulated host immune responses (in particular pro-inflammatory responses). thus, the global analysis of the activation state of host kinases (the kinome) can provide critical insight into the specific activation state of individual kinases, cell signaling pathways, or larger biological networks. in addition, kinome investigations may offer important, and predictive, insight into the cellular mechanisms that regulate phenotypic changes within cells. 7 as many kinases recognize a particular phosphorylation motif composed of the central phosphoacceptor site and the amino acids +4 and −4 residues from the central phosphorylation site, 12 peptides representing this kinase target motif can be synthesized with relatively high efficiency and low expense. indeed, kinase target motif peptides have been shown to be appropriate substrates for their respective kinases with v max and k m values approaching those of the fulllength protein. 13 thus, peptide kinome arrays can be constructed in an analogous manner to traditional dna microarrays where kinase target motif peptides are spotted onto a glass slide representing hundreds to thousands of unique peptide targets for kinases ( figure 2 ). following this, samples in the form of cellular lysates from whole organs, tissues, or individual cell types can be applied to the kinome peptide arrays, allowing for the phosphorylation of specific peptide targets by kinases within the lysate (figure 3 ). the development of kinome-specific bioinformatics analysis software, including the platform for intelligent, integrated kinome analysis (piika), has provided a mechanism to identify the complex patterns of kinase-mediated phosphorylation events and quantitate the differences between compared conditions. 14 clinical findings during mers-cov infection. syndromic case-definition for mers infection requires a compatible clinical syndrome and an epidemiologic risk factor including travel to an affected region or contact with a known or suspected case. initial symptoms of mers-cov infection include fever, chills, cough, shortness of breath, myalgia, and malaise following a mean incubation period of 5 days, which can range from 2 to 14 days. 23 mildly symptomatic or possibly asymptomatic infections have been reported, and progression to severe disease is associated with pre-existing medical conditions including cardiopulmonary disease, obesity, and diabetes. most (98%) of reported mers cases are among adults, with a median age of 50 years. in severe cases respiratory failure requiring mechanical ventilation typically occurs within 7 days of symptom onset. 22 laboratory abnormalities include lymphopenia, leukopenia, thrombocytopenia, elevated serum creatinine levels consistent with acute kidney injury, and elevated liver enzymes. 23−27 high lactate levels and consumptive coagulopathy have also been reported. 24, 28 chest radiographic abnormalities are observed in most cases consistent with viral pneumonitis, secondary bacterial pneumonia, or acute respiratory distress syndrome. 22 cytokine levels in serum and bronchoalveolar lavage (bal) from two mers patients, one with fatal disease and one who survived. 30 higher levels of retinoic acid-inducible gene 1 (rig-1), melanoma differentiation-associated protein 5 (mda5), interferon regulatory factor (irf)-3 and -7, interleukin (il) 17a, and il-23 and lower levels of il-12 and ifnγ were observed in the fatal case compared with the survivor. more recently, min et al. performed a temporal analysis of cytokine, chemokine, and growth factor blood levels from 14 patients during the recent outbreak of mers in south korea. 31 the patients were subcategorized into four groups on the basis of disease severity: group i patients developed fever and recovered. group ii patients developed mild pneumonia without hypoxemia. group iii patients had prolonged and severe pneumonia. group iv patients had severe pneumonia and acute respiratory distress syndrome. group iv patients included five fatal cases of mers; all patients in groups i−iii fully recovered from illness. ifnα was elevated in all groups and largely peaked during the second week of illness. granulocyte-colony stimulating factor (g-csf) and granulocyte macrophage (gm)-csf were similarly elevated across all patient groups; however, patients with fatal disease had reduced gm-csf responses following antiviral treatment as compared to patients that recovered. patients with pneumonia had relative elevations of il-1, tumor necrosis factor (tnf)-α, il-6, and il-10 during the second and third weeks of illness. elevated il-6 and il-10 appeared to trend positively with the severity of illness. a robust induction of multiple chemokines was found in most patients. notably, eotaxin and regulated on activation, normal t expressed and secreted (rantes) was elevated in all patients. in contrast, il-8, monocyte chemotactic protein (mcp)-3 and macrophage inflammatory protein (mip)-1β were more prominent in groups ii and iii as compared to groups i and iv. furthermore, elevated interferon gamma induced protein (ip)-10 correlated with the development of pneumonia (groups i−iii). multiple growth factors, including epidermal growth factor (egf), fibroblast growth factor (fgf)-2, vascular endothelial growth factor (vegf), and tgf-α, were significantly elevated across all patients; however, egf was significantly higher in patients that recovered from disease as compared to the fatal cases. further evaluations are needed to characterize the natural history of immune response during acute mers-cov infection and recovery. transcriptome analyses of mers-cov. in an effort to better characterize mers-cov pathogenesis in the absence of available samples from human patients and, in particular, address pathologic changes associated with infection, multiple animal species have been employed in mers-cov investigations. while multiple small animals are not susceptible to mers-cov infection, 32 rhesus macaques and marmosets develop mild to severe lung pathology following experimental infection. transcriptome analysis in mers-cov-infected rhesus macaques revealed that genes related to antiviral immunity, chemotaxis, and inflammation were overexpressed in lesional versus grossly normal lung tissue at 3 days postinfection. 33 a significantly smaller number of differentially expressed genes was found on day 6 postinfection with no obvious trends following pathway enrichment analysis. significant changes in the transcriptome profiles of peripheral blood mononuclear cells (pbmcs) were observed at only day 1 postinfection. this global analysis suggests a key role for an initial rapid innate immune and inflammatory response (through pattern recognition receptors) followed by rapid resolution. 33 a study of mers-cov-infected marmosets evaluated lung lesions by rnaseq at days 3−6 postinfection. 34 pathway analyses demonstrated that chemotaxis and cell migration, cell cycle progression, and cell proliferation and fibrogenesis were highly over-represented relative to uninfected controls. to a lesser degree, pathways associated with inflammation, vascularization, endothelial activation, proliferation of smooth muscle, and tissue repair were also overrepresented in infected animals. differences were most significant on days 4 and 6 postinfection during illness progression relative to day 3. recently, menachery and colleagues examined the interaction between mers-cov emc/2012 and the host ifnstimulated gene (isg) response by transcriptomics. 35 isg responses in mers-cov infected calu3 cells, a lung adenocarcinoma cell line, had no discernible induction initially upon infection but were up-regulated by 12 h postinfection. 35 down-regulation of a subset of isgs resulted in altered histone modifications, a potential epigenetic contributor to early impairment of antiviral cellular defenses. in a separate analysis genetically distinct mers-cov strains, mers-cov sa1 and mers-cov eng1, produced distinct gene expression profiles in calu-3 cells. 36 these analyses may better inform early host-cell antiviral responses and the impact of viral evolution on these and other complex biological responses. proteomics analysis corroborated these transcriptional data with induction of isgs observed 18 h postinfection. significantly reduced levels of stat1 and pkr compared with uninfected controls were also noted. differential host transcriptome responses to mers-cov sa1 and mers-cov eng1 highlight both the propensity of emerging viral pathogens to evolve rapidly and the importance of additional host response analyses for augmenting and clarifying such complex biological responses. kinome analyses of mers-cov infection. host responses to mers-cov infection through kinome analysis were recently assessed using huh-7 cells, an immortalized human hepatocyte cell line, that are highly permissive to mers-cov infection. 37 temporal analysis of kinome responses by peptide arrays revealed selective modulation of extracellular signal-regulated kinases (erk)/mitogen-activated protein kinases (mapk) and phosphatidylinositol-3-kinases (pi3k)/akt (also known as protein kinase b)/mechanistic target of rapamycin (mtor) signaling responses. over-representation analysis (ora) revealed erk/mapk and pi3k/akt/mtor signaling responses were consistently up-regulated during infection. multiple erk/mapk family members formed central components of functional networks and signaling pathways throughout infection. similar results were observed for intermediates of the pi3k/akt/mtor signaling pathway at 1 and 24 h postinfection, suggesting that modulation of erk/ mapk and pi3k/akt/mtor signaling may be important for productive mers-cov infection. downstream analysis of the phosphorylation patterns of pathway intermediates from the erk/mapk and pi3k/akt/ mtor signaling supported observations from the kinome analysis. both investigations demonstrated that nuclear factor kappa-light-chain-enhancer of activated b cells (nfκb)regulated family members were important mediators of mers-cov infection. 38 il8-and ifn-mediated signalings were also modulated during mers-cov infection, consistent with prior analyses. 39, 40 these results were also in agreement with in vitro transcriptional analysis of mers-cov infection. 38 prophylactic or therapeutic addition of fda-licensed kinase review inhibitors targeting activated kinases in mers-cov infection impaired viral replication. these hypothesis-generating data may inform directed investigations into mers-cov pathogenesis and, importantly, demonstrate the potential to identify novel host-centric therapeutic targets. ebolaviruses. the filoviridae family of viruses consists of three genera: ebolavirus, marburgvirus, and the newly identified cuevavirus. structurally, filoviruses have a pleomorphic enveloped, filamentous virion particle that encapsulates a negative-sense single-stranded rna genome. ebolaviruses were first described in 1976 following disease outbreaks in the democratic republic of congo and sudan and are composed of five viral species, including ebola virus (ebov), sudan virus (sudv), bundibugyo virus (bdbv), taı̈forest virus (tafv), and reston virus (restv). sporadic outbreaks of ebov, sudv, bdbv, and tafv have occurred throughout central africa for more than three decades, resulting in thousands of infections. case fatality rates during these outbreaks have routinely exceeded 50%. 41 isolated outbreaks of restv have occurred outside africa in nonhuman primate facilities in the united states, italy, and the phillipines, and infection results in high morbidity and mortality in nonhuman primates; however, restv has only been associated with asymptomatic infections in humans. 42 although ebolaviruses have been historically associated with isolated outbreaks involving small cohorts of infected patients (<500), an outbreak of evd in west africa beginning in 2014 has resulted in 28,616 cases and 11,310 deaths (40% cfr) as of june 2016 (http://www.who.int/csr/ disease/ebola/en/). although virus transmission has greatly decreased in west africa, surveillance for sporadic infections continues. clinical findings in evd. ebov transmission occurs through exposure of infected body fluids or tissues to mucous membranes or nonintact skin. 43 the mean incubation period is 6−10 days, ranging from 2 to 21 days. 43 initial signs and symptoms are nonspecific including fever, myalgia, and malaise and cannot be reliably distinguished from other endemic illnesses in africa including malaria and enteric infections. 43 whereas mild illness has been described, most patients develop severe disease within days of symptom onset. massive gastrointestinal fluid losses of up to 5−10 l per day due to vomiting and watery diarrhea may result in progressive dehydration and hypovolemic shock. even in the setting of adequate fluid and electrolyte replacement, sequential multiorgan failure may occur. ebov infects multiple organs and cell types throughout the body with the notable exception of lymphocytes that are indirectly depleted early during infection. organ injury due to direct viral or indirect host-mediated responses results in severe complications including meningoencephalitis, uveitis, respiratory failure, secretory diarrhea, disordered coagulation, renal failure, hepatic necrosis, and myositis. the clinical presentation, laboratory values, viral kinetics, and clinical management of evd patients in west africa, europe, and the united states during the 2014−2015 outbreak have been recently well-characterized. 44 soluble immune mediators associated with ebov infections. there is a paucity of information regarding ebov pathogenesis in humans primarily due to the limited frequency of evd outbreaks prior to 2014 and limitations presented by sample acquisition from infected patients in the field as well as the overall size of patient cohorts. largely contradictory findings regarding the immune responses in those who survive or succumb to evd have further confounded the under-standing of ebov pathogenesis in human patients. for example, villinger et al. reported that serum cytokine concentrations (including ifnα, ifnγ, tnf-α, il-2, and il-4) were elevated in patients with fatal infections in comparison to survivors. 45 in contrast, additional studies have suggested that fatal infections were instead related to general immunosuppression including ifnγ, il-2, and il-4. 46−49 an investigation of sudv infection in humans by sanchez et al. demonstrated limited changes in the expression levels of cytokines, fas antigen, and fas ligand in pbmcs from infected patients relative to those found for uninfected patients. 50 furthermore, an investigation of 42 fatally infected evd patients by wauquier et al. has further confounded the role of host immune responses in fatal evd as hypersecretion of multiple cytokines and growth factors and decreased secretion of t lymphocyte-derived cytokines were associated with fatal disease. 51 transcriptome analyses of ebola virus infection. to date, no investigations of host gene expression in ebov-infected patients have been reported, although limited data are available from animal models of infection or from in vitro investigations. in a study of pbmcs from ebov-infected crab-eating macaques, rubins et al. found few notable changes in the early stages of infection (1−2 days); however, broad changes were observed over days 4−6 post-infection. pro-inflammatory cytokines (il-1β, il-6, il-8, and tnf-α) and chemokines (mip-1α and mcp1−4) were up-regulated at days 4−6 postinfection relative to healthy controls. 52 multiple genes related to apoptosis including bcl-2 family members, multiple caspases, fas-associated death domain protein, and tnf superfamily member 10 were also up-regulated at late time points. ifn-regulated genes were up-regulated by day 2 postinfection and remained so through study day 6. yan et al. investigated pbmc gene expression in ebovinfected rhesus macaques with or without anticoagulant administration. untreated animals displayed up-regulation of immune response genes, b cell receptor signaling intermediates, nk cell mediated cytotoxicity, leukocyte activation, and lymphocyte activation compared with anticoagulant-treated animals during the early stages of infection. the expression levels of these gene clusters fell to pre-infection levels at the late-stage of infection. in contrast, genes related to defense responses, apoptosis, wounding, inflammation, coagulation, and leukocyte activation remained elevated during early-and latestage infection. following the isolation of restv from pigs, 53 subsequent investigations have demonstrated that pigs were susceptible to both restv and ebov infection with preferential targeting of macrophages in the lungs. recently, nfon et al. demonstrated that ebov infection in pigs resulted in up-regulation of chemokine expression beginning on day 3 postinfection as compared to mock-infected pigs. 54 the most pronounced changes in gene expression were found on days 5 and 7 postinfection and included the up-regulation of a broad set of cytokines (il-5, il-6, il-8, il-10, il-22, il-26, il-27, resistin), chemokines (ccl2, ccl10, ccl19, ccl20, amcf-ii, ccl3l1, ccl4), cell adhesion protein (selectin), antimicrobial protein, palate, lung, and nasal epithelium clone proteins, and pro-apoptotic molecules (multiple caspases, caspase recruitment domain-containing protein 6 (card), apoptosisassociated tyrosine kinase (aatk), fas, fas-associated protein with death domain (fadd), tnf receptor-associated factor 3 (traf3), tnfα-induced protein 3-interacting protein 1 review (tnip1)). in addition, expression of multiple genes related to microbial sensing (pattern recognition receptors) or antiviral responses (isgs) was up-regulated in the lungs of infected animals. although the localization of the cytokine response of pigs and humans or nhps differs during the course of ebov infection (localized responses in the lungs of pigs versus a predominantly systemic response in humans and nhps), the cytokine profiles of pigs, humans, and nhps were quite similar. for example, comparison of nhp 52 and porcine responses 54 during ebov infection demonstrated multiple gene expression similarities between the two species (i.e., il-6, il-8, caspase family members). it is also likely that direct comparison of both data sets would likely yield many common gene signatures that are conserved in their identity as well as their directionality (upregulation vs down-regulation). macrophages are an early target of ebov infection and support high-level viral replication. ebov attachment and entry into human macrophages in vitro induces pro-inflammatory mediators including il-6, il-8, and tnf-α as early as 1 h postinfection. 55 noncardiogenic pulmonary edema is a recognized complication of evd, and human autopsy data support that alveolar macrophages are a target of ebov infection. ebov infection of alveolar macrophages in vitro resulted in an early, transient increase in cytokine and chemokine expression, 56 supporting that paracrine-soluble mediators of inflammation may contribute to vascular leakage in the lungs. gene expression responses of ebov-and marvinfected huh7 cells resulted in the global suppression of antiviral responses, including toll-like receptor (tlr), irf3, and protein kinase r (pkr)-mediated pathways. 57 however, signal transducers and activators of transcription (stat) phosphorylation in ebov-and marv-infected cells were differentially modulated. ebov-mediated ifn inhibition has been well characterized and is thought to be attributable to ebov proteins vp24 and vp35. 58 interestingly, restv infection, which does not induce clinical illness in humans, resulted in the activation of >20% of the ifn-stimulated genes (isgs). kinome analysis of ebola virus. hepatocytes are an early target of ebov infection, directly contributing to diffuse hepatic necrosis observed in fatal cases. ebov infection of huh7 cells has been evaluated by kinome analyses, shedding light on liver pathogenesis in evd. 59 ebov infection of huh-7 cells resulted in temporal modulation of the tgf-β signaling pathway as compared to mock-infected cells. pathway ora demonstrated that multiple tgf-β-mediated signaling pathways were up-regulated at 1 and 24 h post ebov infection. furthermore, these responses were associated with changes in the expression patterns of multiple cellular proteins associated with a mesenchyme-like transition. these included the upregulation of matrix metalloproteinase 9, n-cadherin, and fibronectin and down-regulation of e-cadhering and claudin 1. in this process cells lose polarity and cell-to-cell adhesion transforming into mesenchymal stem cells that contribute to wound healing or organ fibrosis; however, the role of these events in ebov infection remains to be elucidated. additional analysis demonstrated that inhibition of pi3k/akt, erk/ mapk, or pkc pathways with kinase inhibitors reduced ebov replication when administered prophylactically or therapeutically. supporting this observation, a subset of kinase inhibitors administered to ebov-infected mice reduced lethality. defining mechanisms by which kinase inhibitors show benefit in these models will better clarify their role as potential therapeutics. monkeypox virus. mpxv, a member of the genus orthopoxvirus, causes zoonotic infections with a case fatality rate of ∼11%. 60 mpxv, vaccinia virus (vacv), cowpox virus (cpxv), ectromelia virus, and variola virus (varv), the etiologic agent of human smallpox, comprise the orthopoxviridae family of viruses. mpxv was first isolated in 1958 from cynomolgus macaques in denmark; however, human mpxv infections were not recognized until 1970 following the isolation of the virus from a suspected case of smallpox infection in the democratic republic of congo. 61 mpxv is composed of two distinct clades that are genetically, clinically, and geographically distinct. the congo basin mpxv (central african mpxv) clade is considered to have both higher lethality and morbidity than the west african mpxv clade as demonstrated from comparative infection models in various animal species (including nonhuman primates, mice, prairie dogs, and ground squirrels) and as well natural infection in humans. 61 64 although human mpxv infections have been recorded in west africa, the majority of human mpxv infections have occurred in the congo basin region of central africa, largely in the democratic republic of congo. 60 clinical findings in mpxv infections. clinical and epidemiological information regarding human mpxv disease has been derived from enhanced surveillance campaigns in the congo basin. 61 from this work, it has been demonstrated that human mpxv infection and illness largely mirror those of discrete, ordinary smallpox. 61 the incubation period for both viruses (varv and mpxv) is 7−17 days with an initial febrile prodromal period of 1−4 days. this prodromal period is normally accompanied by fever, headache, backache, malaise, and prostration. 61 the rash period for both smallpox and mpxv (including lesion appearance and desquamation) normally occurs 14−28 days postinfection with highly similar appearance, distribution, and progression of lesions. 60, 61 as with smallpox, mpxv-associated rash progresses through macular, papular, vesicular, and pustular phases. a second febrile period occurs when the lesions become pustular and is often associated with deteriorating conditions in the patient. lymphadenopathy (maxillay, cervical, or inguinal) is often associated with mpxv infections prior to, or concomitant with, rash development but is absent in varv infections. it has been postulated that this reflects the effective generation of host immune responses during mpxv infection as compared to varv; however, this has yet to be validated. 60, 61 severe complications have been noted late in the course of mpxv infection, including pulmonary distress or bronchopneumonia, corneal scarring and permanent vision loss, and encephalitis. 60 severe dehydration due to excessive vomiting or diarrhea may also occur. long-term sequelae in survivors are most commonly associated with pitted scarring. soluble immune mediators associated with mpxv infections. although mpxv infections in humans have been recorded for over four decades, there has been little information review regarding host immune responses during the course of natural infection. as disease presentation is highly similar during mpxv and varv infections, it has been postulated that immune responses would likely be highly conserved. recently, johnston et al. provided the first empirical evidence for a relationship between cytokine responses and disease severity during mpxv infection. 65 serum cytokines were analyzed from 19 patients with confirmed mpxv infections ranging from mild to severe as assessed by the who smallpox lesion scoring system. 66, 67 serum concentrations of il-1β, il-1ra, il-2r, il-4, il-5, il-6, il-8, il-13, il-15, il-17, mcp-1, and rantes were elevated in all disease groups (mild to severe) as compared to normal serum concentrations. il-10 concentrations were also elevated in all disease groups and were proportional to disease severity. however, patients with serious mpxv disease had significantly higher concentrations of il-10 compared to all other disease groups. mpxv infection resulted in elevated mip-1α and mip-1β; mild cases had significantly elevated levels above the moderate or severe disease groups. serum concentrations of il-2r were elevated across all disease groups; however, patients with serious disease had significantly higher il-2r serum levels than those with mild to severe mpxv disease. gm-csf levels were significantly elevated only in those with serious mpxv disease as compared to normal serum ranges. on the basis of these observations, mpxv infection resulted in prominent t helper 2 (th2) and dampened th1 responses. transciptome analyses in mpxv infection. transcriptome analyses have largely been employed for the in vitro investigation of the molecular pathogenesis of mpxv infection. alkhalil et al. investigated the host transcriptome responses to mpxv infection during the first cycle of viral replication (3 and 7 h postinfection) in rhesus macaque kidney epithelial cells. 68 interestingly, mpxv infection resulted in a strong downregulation of host transcriptional responses. of the transcripts that met the authors' criteria for significance, 89% of the transcripts were found to be down-regulated at both postinfection time points. comparative functional analysis from both time points suggested that the primary biological functions associated with these down-regulated transcriptional responses were largely related to cell morphology, cell development, metabolic responses, and post-translational modifications. canonical pathway analysis demonstrated a general conservation in the identities of over-represented pathways at both time points including multiple growth factor signaling pathways, p53 signaling, and cell cycle-related pathways. more recently, bourquain et al. investigated host transcriptome responses in mpxv-infected hela cells, a cervical epithelial cell line. 69 at 6 h post-mpxv infection, only 1.1% of the transcripts analyzed were found to have >2fold changes in gene expression. in contrast to alkhalil et al., the majority of these transcripts (∼68%) were found to be upregulated as compared to mock-infected controls. functional analysis of all transcripts with >2-fold changes in gene expression demonstrated a strong over-representation of genes involved in the negative regulation of mapk signaling and the intracellular protein cascade. positive regulation of pathways related to toll-like receptor signaling, chemotaxis, and regulation of leukocyte migration was also predicted from the data. an investigation by rubins et al. compared the temporal host transcriptome response to mpxv in multiple human cells targeted by mpxv including primary macrophages, primary fibroblasts, and hela cells. 70 the trasncriptome of mpxv-infected fibroblasts was found to have the most significant changes where mpxv infection resulted in the depletion of ∼2000 genes by a factor of ≥3. interestingly, mpxv infection resulted in the broad repression of many transcripts related to innate immune responses in all cell types tested. in contrast, inactivated mpxv resulted in strong upregulation of innate immune responses in all of the cell types. it was also noted that mpxv infection resulted in strong cytopathic effects across all of the cell types in contrast to an almost universal repression of innate immune responses. kinome analyses in mpxv infection. human mpxv infections and infection models of mpxv in various animal species have demonstrated that the congo basin mpxv clade is more virulent than the west african mpxv clade. however, there has been a paucity of information regarding the underlying molecular mechanisms mitigating these virulence differences. furthermore, previous investigations focusing on gene expression or proteomic changes during mpxv infection have focused solely on congo basin mpxv. to address this, host kinome analysis was performed on congo basin and west african mpxv-infected human monocytes, a host cell targeted by orthopoxviruses. 71 as the genomes of both mpxv clades demonstrate considerable diversity in the regions coding host response modifier proteins, and in particular in genes associated with anti-apoptotic activities, it was postulated that the virulence differences of the two mpxv clades may be related to differential modulation of host cellular responses. hierarchical clustering of the kinome data sets suggested limited similarities at the level of host kinase modulation between the two mpxv clades. the congo basin mpxv kinome data set clustered most strongly with the kinome data set from cpxv-infected monocytes and moderately with the vacv-infected monocyte data set. both cpxv and vacv can cause serious disease in humans. the pathway ora of the kinome data demonstrated that congo basin mpxv infection resulted in strong down-regulation of a large proportion of host cell responses, most notably apoptosis, in comparison to west african mpxv. biological validation through fluorescenceactivated cell sorting (facs) and caspase 3 activity analyses confirmed this phenomenon. from the perspective of individual phosphorylation events, the kinome data also suggested that akt phosphorylation at ser473 was increased in congo basin mpxv-infected cells as compared to west african mpxv-infected cells. pharmacologic inhibition of akt phosphorylation at ser473 resulted in a >250-fold inhibition of congo basin mpxv virus yields, whereas those for west african mpxv were unaffected. prior investigations with cpxv and vacv demonstrated that pharmacological inhibition of akt resulted in decreased viral yields for both viruses. 72 overall, this investigation provided significant insight into the host cellular response differences between the two mpxv clades. emerging and re-emerging pathogens are a continual threat to global health. in recent years, disease outbreaks associated with sars and the 2009 influenza pandemic have also demonstrated that these pathogens can have considerable effects on local, national, and international economies. as a consequence, regional outbreaks of emerging and re-emerging pathogens can have deleterious effects on global stability. thus, it is prudent that a concerted effort is employed to assimilate data that bridge both clinical and molecular information in investigations review of these pathogens. these efforts will not only provide considerable context in regard to the molecular events that potentiate clinical manifestations of pathogenesis but also better inform the design and implementation of novel therapeutics. to this end, global analyses of host molecular responses can provide considerable insight into the complex molecular events that underlie cellular responses. indeed, transcriptome analyses have provided important information regarding host transcriptional responses during emerging and re-emerging pathogen infection. these investigations often provide critical insight into the kinetics of host immune responses during the course of infection as well as mechanistic information regarding the cellular intermediates involved in these processes. however, the role of ptms in the regulation of these events cannot be captured by traditional transcriptome technologies. in particular, the role of kinase-mediated regulation of cell signaling pathways has remained poorly understood. given the central role of kinases in the regulation of cellular processes (e.g., homeostasis, metabolism, proliferation, and stress responses), it is of inherent importance that future investigations also address the role of the kinome in the cellular response to pathogen insult. furthermore, kinomics also provides a mechanism for the identification of novel therapeutic targets based on the direct assessment of the activation state of cell signaling pathways. for example, proinflammatory responses during early stages of infection, and in particular the dysregulation of specific cytokines or cell signaling events that contribute to these, may represent potential therapeutic targets in the early stages of highconsequence viral pathogen infection. however, the selection of immunomodulatory therapeutics that target these dysregulated host responses is complicated by the regulatory events (i.e., kinase-mediated cell signaling events) that occur upstream of changes in gene expression. in addition, mrna is subject to a variety of regulatory processes (including gene silencing, mrna stability, translational efficiencies, protein turnover, enzyme/substrate subcellular sequestration, and/or protein activation/repression ptms). thus, from the standpoint of therapeutic discovery, the sole reliance on technologies for the global investigations of host responses that do not account for these regulatory processes or the role of ptms in the modulation of cellular responses could impede the identification of efficacious therapeutics. to this end, kinome analysis may also facilitate the identification of immunomodulatory therapeutics that have gained licensure through analysis of a quantifiable biological event (kinase-mediated phosphorylation) or for identifying novel host therapeutic targets for which therapeutics could be designed/developed. furthermore, kinase inhibitors may serve as primary or adjunctive therapies for emerging infectious diseases. in addition, preclinical data and the increasing number of kinase inhibitors that have gained regulatory approval for cancer and other maladies suggest this approach is feasible and efficacious. from the perspective of this review, kinome investigations have identified several therapeutic targets and licensed kinase inhibitors that have impaired viral replication in vitro and reduced the severity of disease in vivo (table 1) . for example, it has been demonstrated that the erk/mapk and pi3k/akt/mtor signaling pathways have a role in viral propagation during mers-cov infection. 37 indeed, licensed kinase inhibitors that targeted these pathways (i.e., everolimus, selumetinib, and trametinib) resulted in decreased viral replication in vitro when added prior to, or following, infection. furthermore, the pharmacologic inhibition of pi3k and pkc following ebov infection provided partial protection in a lethal model of evd in mice. 59 it should be noted that although the modulation of an individual kinase may have suppressive effects on infection (i.e., viral replication), this might not provide the level of inhibition required to completely negate viral escape. in addition, given the ability of many cell signaling pathways to signal through both canonical and noncanonical mechanisms, inhibition at a single intermediary point within a pathway may not provide the overall level of inhibition required to negate a deleterious response (i.e., viral replication, changes in cellular phenotypes, etc.). thus, although previous investigations have demonstrated that individual kinases or cell signaling pathways may represent novel targets for anti-infective therapies, it is prudent that future investigations also examine combinations of inhibitors for efficacy and anti-infective activities. furthermore, the targeting of cell signaling pathways at or near the origin point for the cell signaling cascade should also be examined as these likely represent stronger inhibitory targets given the generally reduced branching of cell signaling networks at or near the cell receptor. in addition to host-directed therapeutic targeting, kinomics also confers the ability to identify novel inhibitors of pathogens through detailed characterization of the viral life cycle. hostmediated ptms, and in particular kinase-mediated phosphorylation, have been implicated in the viral life cycle and pathogenesis for several members of the order mononegavirales, including ebov. 73−75 thus, therapeutic targeting of kinases may represent a novel therapeutic strategy that can be employed to modulate host-centric or pathogen-centric molecular events during infection. for example, in silico prediction of viral protein phosphorylation sites provides a mechanism for the construction and, ultimately, the annotation of viral protein ptms that are critical to the viral life cycle. furthermore, the use of kinome peptide arrays has extended beyond the human kinome and now extends to a variety of animal species. 76−78 it has been suggested that the interspecies phenotypic variability may reflect differences in phosphorylation sites found within the proteome. 78 thus, the development of species-specific kinome peptide arrays provides additional utility for kinome analysis as peptide arrays representing traditional laboratory animal species (mouse, guinea pig, nonhuman primate) can be employed to detail the species-specific host response. the results from such analyses, and the overlap between these and those described previously from the analysis of human infections, may inform review the selection of appropriate animal models that meet regulatory approval through the fda animal efficacy rule. 29 taken together, it is of inherent importance that future investigations of emerging and re-emerging pathogens address the complex nature of biological responses. thus, molecular investigations of pathogenesis should be guided by available knowledge regarding the clinical and pathologic manifestations of disease. indeed, technologies that provide further granularity into the precise molecular events that potentiate cellular responses during the course of infection will assist investigations of emerging and re-emerging pathogens and the identification of novel therapeutic targets. to this end, kinomics-based analyses of host responses provide a mechanism to directly address the cellular events at the level of specific cell 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cells variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on abl and src family tyrosine kinases the authors declare no competing financial interest. review key: cord-262892-n38r8n70 authors: sheikh, jamila; wynn, bridget a.; chakraborty, rana title: nutritional care of the child with human immunodeficiency virus infection in the united states: a historical and contemporary perspective date: 2015-05-08 journal: health of hiv infected people doi: 10.1016/b978-0-12-800769-3.00009-3 sha: doc_id: 262892 cord_uid: n38r8n70 in well-resourced settings, early infant diagnosis and administration of life-saving antiretrovirals (arvs) have significantly improved clinical outcomes in pediatric human immunodeficiency virus (hiv) infection. the dramatic increase in survival rates is associated with enhancements in overall quality of life, which reflect a multidisciplinary, holistic approach to care. current optimism starkly contrasts with the outlook and prognosis two decades ago, when failure to thrive and wasting syndrome from uncontrolled pediatric hiv infection resulted from poor oral intake, malabsorption, chronic diarrhea, and a persistently catabolic state. the tenets of care developed from that era still hold true in that all infants, children, and adolescents with hiv require comprehensive nutritional services in addition to effective combination antiretroviral therapy (cart). this chapter will review the principles of nutrition in the preand post-cart eras and discuss the etiologic factors associated with malnutrition, with an emphasis on interventions that have favorably impacted the growth and body composition of infants, children and adolescents with hiv. the global pandemic of human immunodeficiency virus (hiv) infection has had grave consequences in the lives of affected infants, children, and adolescents, with more than 33% of infant and child mortality attributed to hiv infection in endemic locations [1] . in settings where voluntary and public resources are insufficient to provide long-term care, millions of children initially cared for by relatives have now been orphaned. however, many guardians themselves get sick or become overwhelmed by the number of dependents for whom they have to provide care. the growing number of street children and child-headed households are often the outcomes of a chain of events that begin with the hiv infection of a mother, her partner, or both. in the united states, perinatal transmission has decreased to such a significant extent that current estimates indicate less than 200 infants born with hiv annually [2, 3] . with the implementation of recommendations for universal prenatal hiv counseling and testing, antiretroviral (arv) prophylaxis, scheduled cesarean section delivery, and avoidance of breastfeeding, the rate of transmission events has decreased to less than 1% in the united states and europe [4] [5] [6] [7] [8] . however, there remains an unacceptable annual rate of newly diagnosed hiv-1 infections among infants in the united states, with the persistence of marked racial and economic disparities [9] . most pediatric hiv infections (>90%) are caused by vertical transmission, with events more common to areas where antenatal hiv seroprevalence is high [10] ; 21 countries in sub-saharan africa and in south, east, and southeast asia account for more than 90% of the pregnant women needing arvs to prevent vertical transmission. however, global rates of new hiv infections and prevalence among young people have fallen in many countries, likely due to reductions in vertical transmission rates and improvement in access to effective cart, which has decreased secondary transmission events. a clinical overview perinatal infection occurs at a time of relative immunologic immaturity. the inability to control viremia exposes the thymus and other lymphoid tissue to hiv-1-mediated destruction at a time of active thymopoiesis and lymphopoiesis [11] . given that the virus is transmitted from the mother and that the degree of human leukocyte antigen class i sharing between mother and infant is high, the virus could evade the protective immune response of the newborn, which results in accelerated disease progression [12] . in contrast to adults, hiv-1-related symptoms, cd4+ t cell depletion, or both develop in most untreated vertically infected children within the first few years of life [13] . in addition, plasma hiv-1 ribonucleic acid (rna) levels remained elevated over the first 2 years among infants [14] and do not decrease to less than 10 5 copies/ml through at least the third year of life [15] . the prolonged elevation of plasma hiv-1 rna levels may be related to the kinetics of viral replication, the size of the pool of host cells that are permissive to viral replication, and immature virus-specific immune responses. ii . nutrition and lifestyle the infection in perinatally infected infants and children progresses more rapidly than in adults. although 4% of the world's population with hiv-1 infection comprises children, 20% of all aids deaths were previously in this group. early studies before the era of cart indicated that a subset of children (~25%) progressed very rapidly to aids within 1 year. the median time to aids for the remaining 75% was 7 years [13] . in adults, opportunistic infections (ois) are often secondary to the reactivation of pathogens acquired before hiv infection. in contrast, in infants and children with vertical infection, ois often reflect primary acquisition of host pathogens during ongoing hiv replication and advancing immunosuppression. for example, young children with active tuberculosis more often present with miliary disease. without effective cart, the most common ois in children include serious bacterial infections such as pneumonia and bacteremia. common copathogens and ois that are difficult to eradicate without successful immune reconstitution include chronic mucosal or disseminated infections with herpesviruses, namely, cytomegalovirus (cmv), herpes simplex virus (hsv), human herpes virus 8 (hhv8) and varicella zoster virus (vzv). primary disseminated and reactivated tuberculosis is a major cause of morbidity and mortality among children with hiv in communities where infection with the pathogen is endemic. disseminated disease with mycobacterium avium complex may occur in children with hiv and advanced immunologic deterioration. pneumocystis jiroveci (formerly carinii) pneumonia (pcp) is a common and serious oi associated with a high mortality rate. pneumonia most often manifests between 3 and 6 months of age in infants with vertically acquired hiv infection. candidiasis (topical, oral, esophageal, and tracheobronchial) is the most common fungal infection in these children. causes of acute and chronic central nervous system (cns) infections include those caused by cryptococcus neoformans, and toxoplasma gondii. less commonly observed ois include cryptosporidiosis and systemic fungal infections. clinical presentations include hepatosplenomegaly, failure to thrive, oral candidiasis, recurrent diarrhea, parotitis, cardiomyopathy, hepatitis, nephropathy, developmental delay, encephalopathy, lymphoid interstitial pneumonitis, recurrent bacterial infections, and specific malignancies. malignancies include non-hodgkin b-cell burkitt-type lymphomas, leiomyosarcomas, and kaposi sarcoma, which are commonly described in children with hiv who are of sub-saharan african ethnicity. in the united states, in clinical practice, the number of ois seen in children with hiv has decreased, reflecting the widespread use and administration of effective cart regimens. however, ois continue to be the presenting symptom of hiv infection in infants due to lack of antenatal testing in mothers or in adolescents and young adults who are increasingly infected through horizontal transmission. the intestine is a primary target organ for hiv. hiv infection causes a depletion of cd4+ t lymphocytes in gut-associated lymphoid tissue, including selective loss of a subset of t helper cells called th17 lymphocytes, which are important in gut mucosal containment of extracellular pathogens such as salmonella typhimurium. th17 cells are lost early in retroviral infection and are not replenished over time. this depletion impairs long-term gastrointestinal (gi) mucosal integrity and permeability, causing increased bacterial translocation and immune activation. the intestinal mucosa is also the main reservoir of hiv in the body despite effective virologic suppression with cart. among untreated children with hiv, as many as 80% will have one or more intestinal disorders at a given time, with iron malabsorption present nearly 50% of the time [16] . hiv enteropathy is secondary to direct hiv-mediated injury and indirect immune-mediated injury to the gi tract mucosa in the absence of specific opportunistic enteropathogens, perhaps reflecting selective loss of th17 lymphocytes. hiv enteropathy can occur in children and adolescents at all stages of hiv infection. clinical manifestations include chronic diarrhea, increased intestinal permeability, malabsorption, and malnutrition. histologic changes include lymphocytic infiltration of the gi tract mucosa, villous atrophy and blunting, and crypt hyperplasia [17, 18] . a direct cytopathic effect of hiv on the intestinal mucosa is supported by the observation that clinical signs and symptoms improve after initiation of effective cart in association with virologic suppression and immune reconstitution of cd4+ t cells [19] . acute, recurrent, and chronic diarrhea associated with malabsorption and growth impairment frequently occur in children with untreated hiv infection and advancing immunosuppression. commonly identified infective enteropathogens include bacteria (salmonella, shigella sp.), viruses (including rotavirus, adenovirus, cmv), parasites (entamoeba, giardia, cryptosporidia, microsporidia, isospora), and opportunistic fungi [20] . in children with hiv, frequent and persistent watery diarrhea is the most common presentation of cryptosporidial, microsporidial, and isosporidial infections, associated with abdominal cramps, fever, vomiting, anorexia, weight loss, and poor weight gain [7] . untreated chronic severe diarrhea may cause malnutrition, failure to thrive, severe dehydration, or a combination of all these problems. gi tract disease caused by cmv may include esophagitis, gastritis, pyloric obstruction, hepatitis, pancreatitis, colitis, ascending cholangitis and cholecystitis. signs and symptoms may include nausea, vomiting, dysphagia, epigastric pain, icterus, and watery diarrhea. stools may be bloody. sigmoidoscopy in cmv colitis provides nonspecific results, showing diffuse erythema, submucosal hemorrhage, and diffuse mucosal ulcerations. specific causes of diarrhea in representative adult subjects with aids are presented in table 9 .1 [21] . data reflect prospective follow-up of 1,933 participants in the swiss hiv cohort study; 560 diarrheal episodes were evaluated by standardized stool examination, with intestinal infections diagnosed in less than 50% of chronic diarrheal episodes [22] . the site and severity of infection vary according to the infecting organism. oral mucosal ulcerations secondary to infectious agents such as candida albicans, cmv, or hsv cause inflammation and pain during swallowing or after eating, which may lead to reduced oral intake. opportunistic enteropathogens such as cryptosporidium, cmv, and microsporidia [23] may affect the hepatobiliary system and pancreas in addition to the gi tract, resulting in vomiting, abdominal pain, and malabsorption. in resource-limited settings, disease with mycobacterium tuberculosis is the most common cause of death in subjects with hiv. hiv and tuberculosis (tb) accelerate disease progression and mortality and are associated with marked clinical wasting; the extent of wasting is related to the severity of tb [24] . the largest proportion of newly diagnosed children with hiv in many us centers are foreign born and at higher risk of prior and potentially ongoing exposure to tb. tb is almost always transmitted to children by an adult, most commonly a household contact, and the infection in children is primary infection rather than reactivated disease as in adults. there should be an increased index of suspicion of tb infection and disease in children with hiv, particularly in the context of clinical wasting and a low threshold for empiric antituberculosis therapy, even when diagnostic investigations fail to identify a tb-causing organism. the combination of underlying hiv infection, nutritional status (particularly protein-energy malnutrition), and host immunity are inextricably interdependent. in the united states, prior to the widespread administration of effective cart, the predominant effect of advancing immunosuppression on nutritional status in children with hiv was wasting and negative energy balance, which predicted both morbidity and mortality [25] . in the pre-cart era, ois were major precipitants of weight loss, necessitating prevention or prompt diagnosis and treatment to prevent wasting and to promote weight recovery [26] . growth in children with hiv was persistently below normal standards, with reduced height and weight velocities, compared with hiv-exposed but uninfected children. in 1994, the centers for disease control and prevention (cdc) defined wasting in children younger than age 13 years as (1) persistent weight loss of more than 10% of baseline; (2) downward crossing of at least two percentile lines on the weight-for-age chart in a child aged 1 year or older; or (3) less than the 5th percentile on the weight-for-height chart on two consecutive measurements at least 30 days apart, plus chronic diarrhea or documented fever for at least 30 days, whether intermittent or constant [27] . in addition to ois, other etiologies that contribute to abnormal growth in untreated hiv infection in children include a synergistic combination of inadequate dietary intake, gi malabsorption, increased energy utilization, and socioeconomic adversity. the prevalence of malnutrition in children with hiv varied among centers in the united states with up to 30-50% of children followed up in pediatric hiv programs having demonstrable evidence of protein-energy malnutrition, which, in turn, exacerbated the immunosuppressive effects of hiv [28] . common patterns of wasting included an early decline in weight and height in the first 3 months of life or early linear stunting with a normal weight-to-height ratio. progressive wasting with low weights and heights were also well recognized and more commonly associated with infectious enteropathogens. sequential follow-up demonstrated that growth in children with untreated hiv infection remained below growth in age-matched and gender-matched uninfected controls. malabsorption also results in macronutrient and micronutrient deficiencies. micronutrient deficiencies are widespread and compound the effects of hiv infection in children. deficiency can manifest in conditions such as fatigue, reduced learning ability due to anemia (iron deficiency), and impaired immunity [29] . such deficiencies reflect inadequate nutrient intake and the consequences of excessive losses due to ois, diarrhea, and malabsorption, as previously described. other micronutrients that can also be malabsorbed resulting in deficiency include vitamin b12, folic acid, thiamine, zinc, selenium, calcium, and magnesium, and fat-soluble vitamins a and d [21] . the evidence base for the specific effect of micronutrient supplementation in children with hiv is limited, but a recent cochrane review of 11 studies with 2,412 participants made the following key recommendations for practice. benefits of periodic vitamin a supplementation in children over 6 months of age with hiv infection in resource-limited settings were supported by data from three african trials and were consistent with evidence of benefits of supplementation in uninfected children. zinc supplements reduced diarrheal morbidity and had no adverse effects on disease ii. nutrition and lifestyle progression in a single safety trial in south african children. children with hiv should therefore receive zinc supplements in the management of diarrhea and severe acute malnutrition in the same way as uninfected children with the same conditions. the review emphasized that micronutrient deficiencies and immune dysfunction in children with hiv would only be restored with effective suppression of hiv replication [30] . cart consists of drugs that target the life cycle of hiv at specific enzymes or receptors to inhibit replication thereby preserving or restoring immune function. specific goals of administration of cart include maximally reduction of the plasma viral load below the limit of detection (<50 copies/ml), prevention of a selection of drug-resistant strains and maintenance of good immunologic status (repopulation with cd4 + -naã¯ve t cells), and prevention of clinical disease progression and ois. clinical trials of cart in infants and children with hiv have demonstrated dramatic reductions in morbidity and mortality (>80-90%) in the united states since widespread implementation from 1996 onward, so the vast majority of infants and children with hiv-1 can now be expected to survive to adulthood [31, 32] . five classes of arv drugs are commonly available for hiv therapy. two classes target the enzyme reverse transcriptase-non-nucleoside reverse transcriptase inhibitors (nrtis) and the non-nrtis. a third class-protease inhibitors (pis)-target viral protease, whereas integrase inhibitors target that corresponding enzyme. in addition, ccr5 inhibitors target the viral co-receptor ccr5 on permissive target cells. hiv-1 mutability is largely the result of errors introduced into the viral genome during replication. the hiv genome is approximately 10,000 nucleotides long, and each new virion has an average of one mutation. this results in a large pool of quasi-species of viral variants that are incapable of productive infection but some of which may provide an adaptive benefit, for example, the development of art resistance, to the virion. drug-resistance of the virus can develop during cart administration because of poor adherence, a regimen that is not potent, or a combination ii. nutrition and lifestyle of these factors resulting in incomplete virologic suppression. in addition, primary drug resistance may occur in arv-naive infants and children who can become infected with the resistant virus. aggressive, multidrug cart as early in infection as possible, with daily adherence for an indefinite period, is advocated to fully suppress viral replication and to preclude the selection or emergence of resistant viral variants. resistance testing has enhanced the ability to choose effective initial regimens as well as second-or third-line regimens. therapeutic strategies continue to focus on timely initiation of arv regimens that are capable of maximally suppressing viral replication in order to prevent disease progression, preserve or restore quantitative and qualitative immunologic function, and reduce the development of drug resistance [33] . difficulties with long-term adherence to cart-particularly in infants and children because of variable drug administration, absorption, and metabolism; pretreatment with maternal cart and vertical transmission of drug-resistant virus; acceptability and palatability of medications; and refrigeration of syrup formulations in warm climates-are all well documented. long-term follow-up of infected infants and children involves longitudinal determinations of prognostic markers, including number and percentage of cd4 t cells, and viral load [34] . such parameters provide a useful framework for the time to initiate and change therapy but involve frequent venipuncture in minors. long-term toxicities include lipodystrophy syndrome [35] and lipid abnormalities, cardiomyopathy, mitochondrial toxicity and lactic acidosis, renal tubular acidosis [36] , hypersensitivity reactions, and cns toxicity. fortunately, the availability of new drugs and drug formulations has led to the use of more potent regimens with reduced short-term toxicity, lower pill burden, and less frequent medication administration, all factors that are associated with better adherence and outcomes. enteral [37, 38] or parenteral supplementation and appetite stimulants [39] can improve the nutritional status and weight in children with untreated hiv infection but have little effect on the growth velocity of height. however, effective virologic suppression with cart was shown to improve mean weight, weight for height, and muscle mass in 67 children with hiv, in whom pi-based therapy was initiated and maintained for a median of 5 months. these effects were independent of virologic suppression and improved cd4 t-lymphocyte counts [40] . these findings were also noted in the pediatric aids clinical trial group 219 study, which ii. nutrition and lifestyle found that pi therapy improved both weight and height z-score annually, after adjusting for cd4 cell count, age, gender, and race [41] . in the era of cart, following the introduction of pi-containing regimens, hiv-associated mortality decreased by greater than 80-90%, with significant declines in opportunistic and related infections [31, 32] . these encouraging outcomes have been tempered by the side effects associated with arvs. altered body composition, lipid abnormalities, and abnormal regulation of glucose metabolism are consequences that result in an increased risk of cardiovascular disease, reflecting complications of inflammation with uncontrolled hiv infection and the specific arv drugs as outlined. in children, adolescents, and adults, a clear syndrome of abnormal fat redistribution or lipodystrophy and metabolic changes associated with administration of cart is well described. patterns of lipodystrophy vary from peripheral fat wasting, or lipoatrophy, in the face, extremities, and buttocks to central fat accumulation, or lipohypertrophy, in the abdomen, dorsocervical spine regions (buffalo hump), and breasts. both conditions may occur alone or in combination [42, 43] and can be difficult to assess in a growing child or adolescent, since changes in body fat occur normally during childhood and puberty [44] . lipodystrophy in children with hiv is clinically evaluated by examination or self-report and has been documented to be as high as 32% [45] . dual-energy x-ray absorptiometry (dexa) quantifies total, trunk, and limb fat. observational studies in children with lipodystrophy show decreased total and extremity fat and a greater trunk-to-extremity fat ratio in children with hiv compared with uninfected children [46, 47] these changes are drug specific and associated with duration of therapy, with prolonged treatment and older age more likely to result in lipodystrophy. treatment with nrtis, including stavudine (d4t), zidovudine (azt), and didanosine (ddi), is associated with a lower percentage of extremity fat and higher percentage of trunk fat and trunk-to-extremity fat ratio even after adjustment for wasting and stunting [21, 43] . these changes in body fat distribution often cannot be reversed even after switching to less lipodystrophic arv regimens. in cohorts of children receiving a pi regimen, higher rates of dyslipidemia have been documented, with higher fasting lipids, cholesterol, and triglycerides. lipodystrophy in patients results in much higher waist-tohip ratios and elevated fasting insulin levels and blood pressure, which ii. nutrition and lifestyle are all significant risk factors for cardiovascular disease [21] . for children without lipodystrophy, up to one fifth show symptoms of dyslipidemia. in summary, when selecting arv regimens, care must be taken to consider the above life-long side effects and their consequences. at a time when newer less lipodystrophic first-line regimens, including tenofovir, abacavir, ritonavir-boosted pis (atazanavir and darunavir), and the integrase inhibitors, most with the added advantage of once daily administration, are available in the united states, regimens that include zidovudine, didanosine, and stavudine should be prescribed less often to children with hiv to reduce these potential long-term toxicities. metabolic syndrome reflects a series of clinical conditions, including elevated triglyceride, low levels of high-density lipoprotein (hdl) cholesterol, hyperglycemia and insulin resistance, increased body fat distribution around the waist, and high blood pressure, all of which collectively increase the risk of cardiovascular disease. in individuals with hiv, the prevalence of metabolic syndrome is higher than in the general population and estimated to be 7-45% [21, 48] . although uncontrolled hiv in the absence of cart can cause low hdl cholesterol and high triglycerides, as discussed previously, arvs also induce body fat redistribution in conjunction with metabolic changes. earlier pis, including treatment doses of ritonavir (without boosting other pis), nelfinavir, and ritonavir-boosted lopinavir (kaletra) were documented to increase lipid plasma concentrations, including serum triglycerides, cholesterol, low-density lipoprotein (ldl) cholesterol, and apolipoprotein e and to lower hdl. virologic control with the newer pis, integrase inhibitors, tenofovir, and abacavir may be associated with increases in serum hdl, in the absence of these metabolic complications. when compared with population norms, children with hiv were noted to have lower-than-expected bone mineral density (bmd) for their age and gender that may have been associated with delays in growth, sexual maturity, duration of hiv infection, ethnicity, and disease severity [49] . a more recent large study of 236 american children and adolescents with hiv, aged 7-24 years, showed that males with hiv had significantly lower bmd at tanner stage 5 compared with uninfected males [50] . reduced bmd secondary to cart administration was first described in 2001 from dexa scans in vertically infected children, with the severity of osteopenia directly related to lipodystrophy [51] . however, a longitudinal study from 2013 in 66 dutch children showed an association between longer cart duration and increases in spinal bmd z-scores [52] . lopinavir-ritonavir ii. nutrition and lifestyle [50] , full-dose ritonavir [53] , and tenofovir [54] are associated with lower bmd in children. the principles of maintaining good bone health in youth with perinatal hiv infection is the same as those recommended for all youth in general. adolescents should therefore receive at least 1,300 mg calcium per day and at least 600 iu vitamin d per day through their diet, by supplementation, or both [55] . immune reconstitution inflammatory syndrome (iris) is a diseasespecific inflammatory response that can occur after treatment with arvs is initiated, reinitiated, or changed, resulting in effective virologic suppression and immune reconstitution of naã¯ve and memory cd4+ t cells. iris has been noted to occur in children who begin art while they have severe malnutrition, are severely immunosuppressed [56] , or both. risk factors therefore include a low cd4 nadir and high viral load levels prior to the initiation of cart. these children and adolescents often have numerous documented ois before, during, and after cart initiation [56] . further research is needed to reduce complications and to optimize clinical management when they do occur. the interaction between hiv infection and nutrition is of great importance, and these two factors are interdependent, since strategies to improve nutritional status both quantitatively and qualitatively have been demonstrated to have a beneficial effect on clinical outcome and the immunologic course of the hiv infection. through the course of their disease, infants and children with hiv have numerous nutritional needs, which reflects, as previously described, impaired absorption, decreased oral intake, and increased nutrient requirements. specific adverse outcomes secondary to specific nutritional deficiencies include the inability to achieve normal weight for height; malnutrition and wasting; growth failure and stunting; and neurocognitive, neurodevelopmental, and oral motor delay often from hiv encephalopathy. early nutrition intervention is, therefore, essential and must be addressed simultaneously with the administration of cart, antimicrobial prophylaxis, and neurodevelopmental interventions. collectively, a ii. nutrition and lifestyle multidisciplinary approach is most effective in improving health outcomes and overall quality of life. in the pre-cart era, the nutritional causes of malnutrition reflected (1) decreased oral intake caused by anorexia and by oral and esophageal lesions often from opportunistic pathogens, (2) gastroesophageal reflux and aspiration, (3) regression or nonattainment of key developmental milestones associated with oromotor dysfunction and impaired mastication, (4) malabsorption, (5) increased energy requirements and metabolism from ois with associated negative energy balance, (6) vomiting and diarrhea from gastrointestinal (opportunistic) enteropathogens, and (7) indirect immune-mediated enteropathy. at a time when effective cart was unavailable and faced with a debilitating catabolic disease and rapid disease progression in infants and children with hiv, nutritional interventions that were developed in the early 1990s by pediatric providers targeted four key areas: 1. prompt management of diarrhea. in addition to isolation of opportunistic enteropathogens and prescribing appropriate antimicrobials for infectious etiologies, management of diarrhea mandates assessment of hydration status and rehydration by the oral or intravenous route. modification of diet in the setting of underlying food intolerance such as lactose or fat malabsorption, including pancreatic enzyme supplementation; and vitamin and mineral supplementation. other recommendations included introduction of a mechanical soft diet and nutritional supplementation. 4. management of nausea and vomiting. in addition to appropriate antiemetic agents, treatment also included recommendation of small frequent meals, liquid intakes between meals, and nutritional supplementation. 5. management of anorexia included small nutrient dense foods, nutritional supplementation, and appetite stimulants such as megestrol acetate. these early nutrition needs related to the unique physiologic demands for growth and development, so even today, interventions should be individualized according to the child's specific needs and relate to disease stage, gastrointestinal function, and growth [57] . as a corollary, the energy and protein requirements for infants and children with hiv have not yet been established because individual needs vary, depending on age, growth, and the clinical and immunologic status that may increase energy and protein needs. infants and children with hiv who have slow weight gain are often prescribed high-protein, high-calorie diets. if nutritional needs are not met through a typical high-calorie, high-protein diet, then additional support may include oral nutritional supplements and overnight feeding through nasogastric or gastrostomy-tube feedings. a commercial formula with intact protein may be appropriate for children without underlying gastrointestinal pathology. infants and children with hiv who have gastrointestinal malabsorption should receive a semi-elemental formula to maximize absorption. elemental formulas are typically prescribed when semi-elemental formulas are not tolerated. infants and children with hiv who are unable to consume adequate calories orally often benefit from supplemental tube feeding. enteral tube feeding supplementation improves weight gain in children with hiv who have growth failure [37, 38] . nasogastric tube feedings should be initially attempted and include night-time feedings, which allow the child to eat normally throughout the day. complications relating to nasogastric tube feedings include sinusitis and the technical inability of the caregiver to place the tube or administer the feedings [21] . if delivery of feedings through a nasogastric tube improves growth, then placement of a more permanent device such as a gastrostomy tube should be considered. enteral supplementation with gastrostomy feeding has improved nutrition in a number of chronic childhood illnesses by providing adequate energy intake to promote weight gain when oral intake is poor. miller et al. [37] first investigated the effects of gastrostomy tube feeding on weight gain, height, body composition, immune parameters, morbidity, and mortality in 1995 on 26 children with hiv. weight z-scores before therapy were -1.6 and had decreased to -2.2 on initiation of nasogastric feedings. gastrostomy tube feedings significantly improved weight z-scores to get back to baseline approximately 5 months after initiation of feeding. significant predictors of response to gastrostomy tube feedings included higher cd4 counts at initiation and lower weight-for-height z-scores at baseline. these findings suggested that early intervention during acute weight loss offers the best chances of improving weight in children with hiv. children with the greatest improvement in weight after gastrostomy tube placement spent less time in hospital and had a greater likelihood of survival compared with children who did not gain weight [37] . this small but important study demonstrated that early nutritional intervention improved quality of life and reduced morbidity in children with hiv at a time when effective cart was unavailable. in the cart era, compliance with medical therapy is often improved with more reliable delivery of arvs through the gastrostomy tube and is associated with improved cd4 t-lymphocyte counts, virologic suppression, and improved longitudinal growth. guarino et al. tested the hypothesis that nutritional support improves intestinal and immune functions in 62 italian children with hiv; 16 received enteral nutrition through continuous feeding, and 46 received total parenteral nutrition. the authors documented a significant increase in cd4 cell count, xylose levels, and body weight in those receiving enteral nutrition, suggesting that nutritional intervention may restore intestinal absorption and increase cd4 cell numbers if initiated early in the course of pediatric hiv infection [58] . enteral feeding is preferred over parenteral nutrition to preserve the gut structure. parenteral nutrition should be used only in those children unable to tolerate or gain weight on enteral supplementation, those who have recurrent or chronic biliary tract or pancreatic disease, and those who have intractable diarrhea with weight loss [21] . megestrol acetate is an oral synthetic progesterone used since the early 1990s as an appetite stimulant. weight gain tends to be associated with increase in body fat rather than muscle. clarick et al. investigated the effects of megestrol acetate treatment on weight gain and linear growth in 19 children with hiv who had growth failure. the average duration of the study was 7 months. the study concluded that megestrol acetate was associated with weight gain but not linear growth during the treatment period. after the megestrol acetate treatment was discontinued, poor weight gain and weight loss were again noted [39] . given the dramatic reductions in morbidity and mortality and the improved longitudinal growth in children with hiv in the united states since the widespread implementation of effective cart, megestrol acetate and other therapeutic agents (including growth hormone and the anabolic steroid oxandrolone) are prescribed very rarely, if at all, to subjects with hiv. in the 1980s and early 1990s, the devastating effects of hiv infection on the health of infants, children, and adolescents became apparent and required a rapid and effective response globally. over time, in the united states, with the introduction of arvs, the clinical manifestations associated with hiv infection as well as its treatment were seen to increase, driven by the short-and long-term toxicities of these new formulations in combination. in children with hiv, these manifestations reflected metabolic changes; wasting and stunting from gastrointestinal dysfunction were most often described in the 1980s, but new clinical concerns in the early 2000s were related to altered body composition, lipid abnormalities, and abnormal regulation of glucose metabolism. these complications were often attributed to the first-generation nrtis and pis. the longterm cardiovascular risks of these arvs on subjects with hiv are still unknown. after 2007, newer pis and integrase inhibitors became more widely available and appear to have fewer metabolic adverse effects, although ongoing surveillance of these arvs and tenofovir will be important to evaluate incidences of renal tubular dysfunction and bmd. in the course of the changes in art over the previous two decades, optimal nutritional support has continued to be a cornerstone of pediatric and adolescent hiv care, applying the same principles developed from the early 1990s to effectively support infants, children, and adolescents with hiv. these principles include ongoing comprehensive nutritional assessments and follow-up. when cart providing effective viral suppression was unavailable, enteral and parenteral support was associated with improved weight and body composition and overall survival and is still a key part of care for children and adolescents who present with advanced hiv disease. in addition, periodic vitamin a supplementation in children with hiv who are older than 6 months of age is supported by clinical trials in africa. children with hiv should also receive zinc supplements in the management of diarrhea and severe acute malnutrition in the same way as uninfected children with the same conditions. investigators should continue to study the effects of oral hypoglycemic agents, lipidlowering medications, and lifestyle changes on cardiovascular risk factors in patients with lipodystrophy and hyperlipidemia at this time when obesity has become endemic in many communities in the united states. this unfortunate development on long-term health also has implications for children and adolescents with hiv across the united states. nevertheless, the overall outlook for children with hiv has improved significantly since the 1990s, as reflected in the reduced rates of morbidity and mortality and improved quality of life. perhaps a measure of the latter is the overall medication burden. figure 9. 1 is a child's medications, as shown by oleske et al. [57] . figure 9 .2 shows the pill burden for a number of adolescent patients in the united states in 2014. the last paragraph of dr. oleske's article still relevant for 2014. to quote directly, "compassionate, comprehensive, and coordinated clinical care services are required for all hiv-infected infants and children through adolescence. we must not underestimate their needs. as we improve their longevity with advances in primary hiv therapies, we must not let quality of life suffer due to a lack of nutritional intervention." global, regional, and national causes of child mortality in 2008: a systematic analysis achievements in public health reduction in perinatal transmission of hiv infection-united states recent trends in the incidence and morbidity that are associated with perinatal human immunodeficiency virus infection in the united states combination antiretroviral strategies for the treatment of pregnant hiv-1-infected women and prevention of perinatal hiv-1 transmission european collaborative study mother-to-child transmission of hiv infection in the era of highly active antiretroviral therapy 2 the high number of medications for an adolescent with hiv in 1 the slew of daily medications for a 13-year-old long-term surviving patient with perinatally acquired hiv in 1996 included: zidovudine (azt), didanosine (ddi), trimethoprim/sulfamethoxazole (tmp/smx), fluconzol, megase, prednisone, acyclovir, dapsone, biaxin, zalcitabine (ddc), albuterol, isonicotinylhydrazine (inh), rifampin, ranitidine (zantac) two-dose intrapartum/newborn nevirapine and standard antiretroviral therapy to reduce perinatal hiv transmission: a randomized trial low rates of mother-to-child transmission of hiv following effective pregnancy interventions in the united kingdom and ireland earlier initiation of art and further decline in mother-to-child hiv transmission rates racial/ethnic disparities among children with diagnoses of perinatal hiv infection -34 states towards universal access: scaling-up priority hiv/ aids interventions in the health sector hiv-1 infection in children: a clinical and immunologic overview evolution and transmission of stable ctl escape mutations in hiv infection the european collaborative study association of human immunodeficiency virus (hiv) load early in life with disease progression among hiv-infected infants the relationship between serum human immunodeficiency virus type 1 (hiv-1) rna level, cd4 lymphocyte percent, and long-term mortality risk in hiv-1-infected children management of gastrointestinal disorders in children with hiv infection hiv enteropathy: crypt stem and transit cell hyperproliferation induces villous atrophy in hiv/microsporidia-infected jejunal mucosa enteropathies in the developing world: neglected effects on global health ritonavir combination therapy restores intestinal function in children with advanced hiv disease aetiology and management of malnutrition in hiv-positive children nutritional aspects of hiv-infected children receiving highly active antiretroviral therapy enteric infections and diarrhea in human immunodeficiency virus-infected persons microsporidia infection in patients with human immunodeficiency virus and unexplained cholangitis nutritional status in malawian patients with pulmonary tuberculosis and response to chemotherapy prospective analysis of patterns of weight change in stage iv human immunodeficiency virus infection nutrition in paediatric hiv infection magnitude of body-cell-mass depletion and the timing of death from wasting in aids centers for disease control and prevention 1994 revised classification system for human immunodeficiency virus infection in children less than 13 years of age unicef tracking progress on child and maternal nutrition: a survival and development priority micronutrient supplementation for children with hiv infection incidence of opportunistic and other infections in hiv-infected children in the haart era declines in mortality rates and changes in causes of death in hiv-1-infected children during the haart era hiv-1 drug resistance in hiv-1-infected children in the united kingdom from national study of hiv in pregnancy and childhood collaborative hiv paediatric study increased lipodystrophy is associated with increased exposure to highly active antiretroviral therapy in hiv-infected children persistent non-gastrointestinal metabolic acidosis in pediatric hiv-1 infection gastrostomy tube supplementation for hiv-infected children effect of enteral tube feeding on growth of children with symptomatic human immunodefi-� ciency virus infection megestrol acetate treatment of growth failure in children infected with human immunodeficiency virus the effect of protease inhibitors on growth and body composition in hiv-infected children impact of protease inhibitor-containing combination antiretroviral therapies on height and weight growth in hiv-1-infected children prevalence, evolution and risk factors for fat atrophy and fat deposition in a cohort of hiv-infected men and women pediatric hiv/aids cohort study. body fat distribution in perinatally hiv infected and hiv-exposed but uninfected children in the era of highly active antiretroviral therapy: outcomes from the pediatric hiv/aids cohort study regional body fat distribution in relation to pubertal stage: a dual-energy x-ray absorptiometry study of new zealand girls and young women european paediatric lipodystrophy group antiretroviral therapy, fat redistribution and hyperlipidaemia in hiv-infected children in europe morphologic and metabolic abnormalities in vertically hiv-infected children and youth longitudinal changes in regional fat content in hiv-infected children and adolescents the metabolic syndrome in hiv predictors of bone mineral density in human immunodeficiency virus-1 infected children total body and spinal bone mineral density across tanner stage in perinatally hiv-infected and uninfected children and youth in pactg 1045 bone mineral loss through increased bone turnover in hiv-infected children treated with highly active antiretroviral therapy bone mineral density increases in hiv-infected children treated with longterm combination antiretroviral therapy antiviral therapy and bone mineral measurements in hiv-infected youths comparison of changes in bone density and turnover with abacavir-lamivudine versus tenofovir-emtricitabine in hiv-infected adults: 48-week results from the assert study vitamin d status in children and young adults with perinatally acquired hiv infection severe malnutrition and metabolic complications of hivinfected children in the antiretroviral era: clinical care and management in resourcelimited settings historical perspectives on the evolution in understanding the importance of nutritional care in pediatric hiv infection effects of nutritional rehabilitation on intestinal function and on cd4 cell number in children with hiv key: cord-023748-3kfy36hg authors: lye, patricia s.; densmore, emily m. title: fever date: 2017-05-12 journal: nelson pediatric symptom-based diagnosis doi: 10.1016/b978-0-323-39956-2.00039-x sha: doc_id: 23748 cord_uid: 3kfy36hg nan temperature is controlled by the thermoregulatory center, located in the preoptic area of the anterior hypothalamus. the thermoregulatory center receives input from peripheral receptors and the temperature of the blood bathing the hypothalamus and acts on autonomic, endocrine, and behavioral mechanisms to maintain the body temperature at a particular set point. the hypothalamic set point normally maintains body temperature around 37°c, but there can be significant variation among individuals. normal temperatures range from 36-37.8°c, depending on the time of day, with the peak in the afternoon (5-7 p.m.) and the trough in the early morning (2-6 a.m.). although the circadian rhythm is not well established in infancy, it becomes more reliable by the 2nd year of life. the febrile response not only produces an elevation in body temperature but also causes physiologic changes that enhance the individual's ability to eliminate infection. production of acute-phase reactants and alterations in metabolism and endocrine function are examples of these changes. acute-phase reactants-proteins that are produced in response to infection or injury-include ceruloplasmin, c-reactive protein, haptoglobin, amyloid a, complement, and fibrinogen. hormones and cytokines, some of which are endogenous pyrogens, regulate the production of acute-phase proteins. exogenous pyrogens, such as bacteria or endotoxins, generate the production of endogenous pyrogens, which play a vital role in prostaglandin-related set point elevation and regulation of acute-phase responses. fever results when the thermoregulatory set point is elevated above the normal set point; the hypothalamus then generates physiologic changes involving endocrine, metabolic, autonomic, and behavioral processes. diversion of blood from peripheral vessels to central vessels causes coolness of the extremities but helps increase core temperature. shivering increases metabolic activity and heat production. the affected patient may feel cold and seek a warmer environment or add clothing to feel warmer and prevent heat loss. once these processes have resulted in increasing the core temperature to match the elevated set point, the thermoregulatory center works to maintain the temperature as it does during normothermia. the thermoregulatory point returns to normal once the infection is resolved. the hypothalamus then produces physiologic changes to decrease the core temperature; these include sweating, dilation of cutaneous blood vessels, and the sensation of feeling hot, which may lead to behaviors such as removing clothing or seeking a cooler environment. fever has both positive and negative effects. high body temperatures may impair the reproduction and survival of some invading microorganisms by decreasing required nutrients, such as free iron, or by increasing immunologic responses such as phagocytosis. however, at extremely high temperatures, immunologic responses may be impaired. fever increases the basal metabolic rate by 10-12% for each degree celsius elevation of temperature. this increases oxygen consumption, carbon dioxide production, and fluid and caloric needs. fluid requirements increase 100 ml/m 2 /day for each 1°c rise in temperature above 37.8°c. heat illness must be distinguished from fever as a cause for elevated body temperature. in heat illness, there is an unregulated rise in body temperature, despite the fact that the hypothalamic set point is normal. it can result from excessive heat production or inadequate heat dissipation. temperatures may reach extreme heights and can result in multiorgan dysfunction and death. restoration of normal body temperature in heat illness is mandatory (table 39 .1). a child with fever of recent onset with no obvious historical or physical explanation for the fever is said to have fever without source (fws). bacterial pathogens account for a small but clinically significant number of cases. the risk of bacterial infection decreases with increasing age and is highest for infants less than 3 months of age, compared to infants and toddlers 3-36 months of age, and even lower for children over the age of 36 months. most of the patients in all age groups have a self-limited viral illness. the challenge is to identify which children have fever caused by bacterial pathogens, or other pathogens requiring treatment, in order to avoid the morbidity and mortality associated with delayed treatment, balanced against the risks of testing or treatment when neither is needed. bacterial infection must be considered in immunocompromised patients or those with central lines or shunts. studies in adults suggest that patients with high fever (>105 o f) and rigors have a higher risk of bacterial infection; exceptions to this include influenza and adenoviral infections. oral thermometry can be considered for cooperative patients who are older than 4-5 years of age. axillary temperatures are commonly done and tympanic membrane and temporal artery temperatures are newer modalities with some studies examining their reliability. axillary temperatures are less precise than rectal temperatures. there is a correlation between axillary and rectal temperature measurements; the axillary temperature is usually 0.5-0.85°c lower. tympanic membrane thermometers are often inaccurate in children. temporal artery temperature measurement correlates well with rectal temperature in some studies, but has been shown to be inferior when patients are febrile. it can be considered in settings when children are not likely to be febrile and are over 3 months of age. when detection of fever is critical for diagnosis and management, rectal temperatures should be used in the child 3 years of age and younger. many children will have a source for fever identified on their history and/or physical examination. if no focus of infection is found on the physical examination, the clinician must rely on history and observation to determine the appropriate next steps. the child may appear ill or well. ill-appearing children are typically lethargic or irritable. they may show signs of shock, including weak peripheral pulses, tachycardia, poor perfusion, respiratory distress, mottling, cyanosis, or decreased mental status (table 39 .2). after thorough clinical and laboratory evaluation, ill-appearing children should be admitted to the hospital, and will likely need empiric antibiotic treatment. infants and children with fever without source who do not appear ill create important decision processes in terms of evaluation and management. the physician's ability to make a hypothesis about the child's degree of illness, on the basis of observation, is critical in the evaluation. an objective scoring measure may be used in an effort to assess serious illness in young febrile children. the acute illness observation scale (aios) (table 39. 3), also known as the yale observation score, is a 6-item predictive model graded on a scale of 1-5. use of the aios in conjunction with the history and physical examination has a higher sensitivity for identifying serious illness than history and physical examination alone. the aios is most useful in patients younger than 24-36 months; it has not been shown to provide sufficient data to identify serious illness in 4-to 8-week-old infants, and has not been evaluated in infants less than 4 weeks old. most children who present with fever without source (fws) are subsequently determined to have a self-limited benign viral infection. in 1 study in 2-36 month old children who presented with fws, 76% had 1 or more known pathogenic viruses found; 57% had adenovirus, human herpesvirus 6 (hhv-6: roseola), enterovirus, or parechovirus detected. other identifiable viruses include respiratory syncytial virus, parainfluenza viruses, influenza viruses, varicella (chickenpox), human metapneumovirus and parvovirus (fifth disease/erythema infectiosum). measles, mumps, and rubella are uncommon in developed countries but have been reported in epidemics following imported cases or in underimmunized communities. although rapid testing for viral pathogens is often readily available, a detailed investigation to identify a viral pathogen is not necessary unless the confirmation of a viral infection will change the acute diagnostic plan; treatment with antivirals is an option (hsv, influenza) if the fever is prolonged and evolves into fuo or if there is end-organ involvement, as in hepatitis, myocarditis, encephalitis, or meningitis. most viral infections do not have simultaneous co-infection with a bacterial pathogen. exceptions include croup due to parainfluenza history a detailed history may reveal a potential source for infection. a complete history addresses several important issues: (1) onset and duration of fever; (2) degree of temperature; (3) by what method and in which anatomic site the temperature was taken; (4) medications given, including antipyretics, antibiotics, or home remedies; (5) environmental exposures; (6) associated symptoms; (7) ill contacts; (8) recent immunizations, and (9) recent travel. inquiry into the child's medical history may reveal important information such as recurrent febrile illnesses, primary or acquired immunodeficiency, or medications such as chemotherapy that alter host defenses. rectal temperature measurement is considered to be the gold standard for children 3 years of age or younger. the most widely accepted definition of fever is rectal temperature of 38°c (100.4°f) or higher. it is important to consider that infants, especially those younger than 2 months of age, may have a blunted febrile (or hypothermic) response to infection. hence, lack of fever should not be used as a criterion for ruling out infection in infants. although rectal temperature measurement is the gold standard, it should be avoided in neutropenic immunocompromised patients, in whom rectal manipulation may seed the blood with bacteria. two of 4 criteria, 1 of which must be abnormal temperature or abnormal leukocyte count: 1. core temperature >38.5°c (101.3°f) or <36°c (96.8°f) (rectal, bladder, oral, or central catheter) 2. tachycardia: mean heart rate >2 sd above normal for age in absence of external stimuli, chronic drugs or painful stimuli or unexplained persistent elevation over 0.5-4 hr or in children <1 yr old, persistent bradycardia over 0.5 hr (mean heart rate <10th percentile for age in absence of vagal stimuli, β-blocker drugs, or congenital heart disease) 3. respiratory rate >2 sd above normal for age or acute need for mechanical ventilation not related to neuromuscular disease or general anesthesia 4. leukocyte count elevated or depressed for age (not secondary to chemotherapy) or >10% immature neutrophils sepsis plus 1 of the following: 1. cardiovascular organ dysfunction, defined as: despite >40 ml/kg of isotonic intravenous fluid in 1 hr: • hypotension <5th percentile for age or systolic blood pressure <2 sd below normal for age or • need for vasoactive drug to maintain blood pressure or • 2 of the following: • unexplained metabolic acidosis: base deficit >5 meq/l • increased arterial lactate: >2 times upper limit of normal • oliguria: urine output <0.5 ml/kg/hr • prolonged capillary refill: >5 sec • core to peripheral temperature gap >3°c (5.4°f) 2. ards as defined by the presence of a pao 2 /fio 2 ratio ≤300 mm hg, bilateral infiltrates on chest radiograph, and no evidence of left heart failure or sepsis plus 2 or more organ dysfunctions (respiratory, renal, neurologic, hematologic, or hepatic) sepsis plus cardiovascular organ dysfunction as defined above mods presence of altered organ function such that homeostasis cannot be maintained without medical intervention virus, which may predispose to bacterial tracheitis and influenza, which may predispose to bacterial pneumonia. the sequence may be biphasic with viral symptoms followed by improvement, followed by worsening symptoms of the bacterial superinfection, or both phases may not be apparent as the child demonstrates no improvement or deterioration. respiratory syncytial virus (rsv) may predispose patients to otitis media. noninfectious conditions manifesting with fws are extremely rare. historical clues (recurrences, chronicity) or systemic signs usually indicate malignancy or rheumatic disorders. if the history and physical examination are not suggestive, these diagnoses need not be pursued. heat-related illness or drug ingestion may be considered if supported by the history. fever caused by immunizations may not be accompanied by other signs or symptoms, but the history should suggest immunization as the cause. utis are the most common serious bacterial infection in children less than 36 months of age who present with fws. utis are almost always occult in children younger than 24 months because the symptoms, except for fever, are nonspecific or nonexistent. uti occurs in 7% of febrile children younger than 2 years. the prevalence of uti varies by height of the fever, duration of the fever, age, gender, race, and circumcision status. children with fever greater than 39°c are at a higher risk of utis. boys with fever for more than 2 days and girls with fever for more than 1 day are more likely to have a uti. higher rates of utis are found in girls, especially those younger than 12 months of age. for febrile boys younger than 3 months of age, 20.1% of those who are uncircumcised have a uti; for circumcised boys the rate is 2.4%. uti rates are higher among white infants than among black infants and among children with abnormal genitourinary tract anatomy or neurogenic bladder. urine specimens should be obtained from the following children with fws: those with a history of uti, those with a history of urinary tract anomalies or vesicoureteral reflux, all infants younger than 2 months, girls younger than 12-24 months, uncircumcised boys younger than 12 months, and circumcised boys younger than 6 months. there is an age-associated risk of bacteremia with utis, particularly in infants. the incidence of bacteremia in patients younger than 2 months with uti is 10%. the incidence of bacteremia in patients younger than 2 months with uti ranges from 4-15% depending on the setting. opinions regarding when to obtain blood cultures in infants with uti differ, but a reasonable approach would be to obtain blood cultures in children younger than 2-6 months with suspected uti, and in older infants with uti if they are ill-appearing (urosepsis). occult bacteremia is defined by the presence of a positive blood culture for pathogenic bacteria in a febrile patient who does not appear extremely ill and who has no focus of infection, excluding otitis media. following the introduction of the 7-valent pneumococcal vaccine in 2007, invasive pneumococcal disease decreased dramatically. pneumococcal bacteremia decreased from 80% of the cases of bacteremia to 30%. most cases of bacteremia in children were not occult. bacteremic children were either ill or had a focus of infection, such as a uti. in 1 study, the rate of occult bacteremia after 2007 was 0.25%. after the 13-valent pneumococcal vaccine was introduced in 2010, the incidence of invasive pneumococcal disease in children less than 5 years old decreased again with 1 state-based population study showing incidence rates dropping from 46/100,000 to 23/100,000 with the age group most involved being children 2-23 months of age. escherichia coli is the most common cause of bacteremia in children aged less than 12 months, all due to utis. other less common causes of bacteremia in young as a marker for invasive disease caused by e. coli than by s. pneumoniae, thus its utility has declined with the reduction of the incidence of invasive pneumococcal disease. similarly, band counts are less commonly used, except in the 29-60 day old infant as part of identifying a low-risk cohort. a wbc count of 5,000-15,000 is generally considered normal for children over 1 month of age. a wbc count less than 15,000/mm 3 or even leukopenia may be found in children with n. meningitidis bacteremia. a minority of children with occult nontyphoidal salmonella bacteremia have been found to have a wbc count exceeding 15,000/mm 3 . c-reactive protein (crp) and procalcitonin combined with a urine dipstick (the lab score) can be used to screen for bacterial infection. this combination of tests has been validated for children 7 days to 36 months of age. pcr is useful in identifying the cause of fever for common viruses such as respiratory syncytial virus, influenza viruses, parainfluenza viruses, enteroviruses, parechovirus, adenoviruses, or herpes simplex virus. additional methods available or in development that may be helpful to identify serious bacterial infections and distinguish bacterial from viral infections utilize molecular microbiology methods. gene expression profiles of the patient's peripheral blood leukocytes demonstrate different biosignatures of rna production that may differentiate bacterial from viral infections. this method does not identify the children are n. meningitidis, nontyphoidal salmonella, staphylococcus aureus, and group a streptococcus. neisseria meningitidis bacteremia is frequently associated with serious sequelae. children with n. meningitidis bacteremia are much more likely to progress to meningitis than are those with s. pneumoniae bacteremia. nontyphoidal salmonella bacteremia is often accompanied or preceded by enteritis. in some instances, particularly in young infants, the diarrhea is mild or even absent. the prevalence of salmonella bacteremia among patients with salmonella enteritis has been reported to be between 2% and 45%; fever is not always present. salmonella infection seldom causes serious complications in patients with normal host defenses and resolves spontaneously. infants younger than 3 months, malnourished, and immunocompromised individuals are exceptions. evaluation is usually divided into 4 different age ranges: younger than 1 month, 1-3 months, 3-36 months, and older than 36 months. testing for each individual age group is based on risks for diseases and prevalence of pathogens. most acute diarrhea and fever is caused by viral pathogens in developed countries. obtaining a stool culture is indicated if bacterial enteritis is indicated by the presence of risk factors in the history, such as blood in the stool or certain exposures (petting zoos) (see chapter 11). febrile infants younger than 3 months have a higher incidence of serious bacterial infections than older infants. the relatively high incidence of bacterial disease probably results from a combination of factors unique to this age group: decreased opsonin activity; decreased macrophage function; decreased neutrophil function; poor immunoglobulin g antibody response to encapsulated bacteria; and susceptibility to bacterial pathogens such as group b streptococci (gbs), gram-negative enteric organisms, and listeria monocytogenes. the incidence of early-onset group b streptococcal infections has decreased with routine screening and the intrapartum treatment of gbs-positive pregnant women; the incidence of late-onset gbs (>1 week) has not decreased. e. coli is the most common organism causing bacterial infections in neonates and young infants. in very young infants, clinical evaluation alone is inadequate for excluding serious bacterial infections. management of febrile infants younger than 28 days includes a sepsis evaluation and hospitalization for parenteral antimicrobial therapy pending culture results. the reasoning for this conservative approach lies in the difficulty in evaluating the behavioral state of neonates, the rapid clinical deterioration of infants with bacterial infections, the immature neonatal immune system, and the possibility of life-threatening viral infections caused by herpes simplex viruses (hsv) or enteroviruses. sepsis evaluation should include culture of the csf, blood, and urine; a complete blood cell count with differential; examination of the csf for cells, protein, and glucose; and urinalysis. a chest radiograph should be considered if the patient has signs or symptoms of a respiratory infection. testing (blood and csf pcr for hsv) and treatment for possible hsv infection should be considered in ill-appearing infants, those with a seizure prior to presentation, and those with a vesicular rash consistent with hsv. a combination of clinical evaluation and laboratory studies can be used to define a specific population of infants aged 29-60 days who do not appear ill and are at low risk for bacterial infections. infants at low risk for bacterial infections are those who are previously healthy with no focus of bacterial infection on physical examination and who have negative laboratory screening results. a number of prospective studies have contributed to the development of specific low-risk screening criteria (table 39 .4). the age groups included vary by study, ranging from 0-90 days to 29-56 days. because there are differences in study criteria used to define infants at low risk for bacterial infections the most conservative values have been used in the guidelines presented in this chapter. negative laboratory screening results consist of a wbc count of 5000-15,000/mm 3 ; fewer than 1500 bands/mm 3 or a band-to-neutrophil ratio of less than 0.2; fewer than 10 wbcs/hpf and no organisms on urinalysis; and fewer than 8 wbcs/hpf and no organisms on csf gram stain. some experts also include a negative chest radiograph and, when diarrhea is present, a stool examination with fewer than 5 wbcs/hpf. most experts suggest that febrile infants 29-60 days old who meet the low-risk criteria and have access to close follow-up can be managed as outpatients. blood, urine, and csf cultures should be obtained before empirical antibiotic treatment so that viral and bacterial causes specific pathogen. rapid multiplex pcr combined with standard blood culture methods may identify a specific pathogen much sooner (~20 hours) than standard blood culture techniques. specific bacteria may be identified using 16s ribosomal rna bacterial gene detection. this method does not require bacterial growth. 16s rrna detection may be helpful when antibiotics were administered before the sample was obtained, and in patients with ventilator-associated pneumonia or bacteria that grow poorly or are present in effusions or tissues (heart valves). blood cultures are the gold standard for determination of bacteremia. although blood cultures do not provide immediate results, methods allow for continuous and more rapid detection of bacterial growth. blood cultures are easy to perform and provide essential information in the diagnosis and management of patients with possible bacteremia. preliminary blood culture results are typically available within 24 hours, with positive identification of most organisms within 48 hours. false-negative blood culture results may be due to prior treatment with antibiotics, missing an episode of bacteremia if it is intermittent, and inoculation of too little blood into the culture media. alternatively, too much blood inoculated into the blood culture bottle may yield a false-negative result because of ongoing killing of bacteria by neutrophils. three to 5 ml of blood should be added to each blood culture bottle. false-positive results may be due to inadequate skin preparation, leading to contamination with skin flora. a positive urine culture was once considered the gold standard; current recommendations include a urinalysis that has pyuria (defined as >5 wbcs/high-power field [hpf] on the microscopic examination or a positive leukocyte esterase on dipstick) and a positive urine culture for a uropathogen in an appropriately collected specimen. fifty to 100,000 colonies of a single organism is considered positive (see chapter 18). children should have a catheterized urine specimen obtained, unless they are toilet-trained and can supply a clean voided specimen. suprapubic aspiration is acceptable but requires technical expertise, and parents often perceive it as unsuitably invasive; it may be the only alternative for boys with severe phimosis. the use of plastic receptacles attached to the perineum should be discouraged because contamination from skin and fecal flora commonly occurs. lumbar puncture is indicated if the patient is younger than 28 days or if a diagnosis of sepsis, meningitis, or encephalitis is considered, regardless of the child's age. normal cerebrospinal fluid (csf) findings, including chemistry, cell count with differential, gram stain, pcr, and culture, help exclude the diagnosis of meningitis. less than 1% of children with normal preliminary csf results have a positive culture; in most of these, the pathogen is n. meningitidis. thus, even in the presence of normal preliminary csf results, close follow-up is essential. chest radiographs are usually normal in children who have fws. respiratory signs or symptoms, such as tachypnea, retractions, crackles, wheezing, rhonchi, nasal flaring, grunting, cough, or hypoxia, may predict chest radiograph findings consistent with pneumonia. in practice, pneumonia can often be diagnosed solely on the basis of the clinical findings of fever, tachypnea, and crackles; chest radiographs are not always necessary. however, chest radiographs may be useful in evaluating for the presence of pleural effusion or other complications of pneumonia. screening urinalysis (ua) for uti should be considered in children with a history of uti, children with a history of urinary tract anomalies or vesicoureteral reflux, girls younger than 12-24 months, especially when the temperature is greater than 39.0°c, uncircumcised boys younger than 12 months, and circumcised boys younger than 6 months. blood cultures are recommended for children with probable utis who are less than 6 months of age. a febrile child with moderate leukocyte esterase on urine dipstick testing or pyuria on an appropriately collected specimen should be treated presumptively for a uti. urine cultures should be obtained for any patient with a suspected uti. the choice of antibiotics should be guided by knowledge of the common pathogens that cause utis and by patterns of antibiotic sensitivity in the community. hospitalization should be considered for the child who is vomiting, is dehydrated, or appears ill; for those in whom compliance is likely to be poor; and for any patient with underlying renal or urologic anomalies. examination and culture of the csf are the only tests to exclude the diagnosis of meningitis and encephalitis. they should be considered in any child in whom the diagnosis of sepsis, meningitis or encephalitis is suspected on the basis of the history, observation, and physical examination findings. outpatient management of children with fws is acceptable for those with a low probability of meningitis, good follow-up, and reliable caregivers. blood cultures should be obtained for all children in whom sepsis or meningitis is suspected. empiric treatment with antibiotics should be considered in those suspected of sepsis or meningitis after appropriate cultures are obtained. in summary, management of children aged 3-36 months with fever is based on clinical experience and numerous study results: • child who appears ill on initial evaluation or on follow-up: admit to the hospital for parenteral antibiotics after appropriate laboratory evaluation. • well-appearing children with fws should be screened for utis, based on their number of risk factors. risk factors for girls are: age <12 months, white race, temperature greater than 39°c, and fever for 2 or more days. girls 2-24 months of age with 1 or more of these risk factors have a greater than 1% probability of having a uti, and should be screened for a uti. for boys, the risk factors are uncircumcised status, nonblack race, temperature greater than 39°c, and fever for over 24 hours. all uncircumcised boys less than 12 months old, even if they don't have other risk factors, should be screened for a uti. for boys who are circumcised, 2 or more of the other risk factors increases the risk to over 1% and they should be screened. • child with positive blood culture: reevaluation should occur in any child whose blood culture is presumptively positive. if the blood is found to contain n. meningitidis or haemophilus influenzae (which has been rare since the advent of h. influenzae b immunization), a csf sample and a repeat blood culture should be obtained, and the child should be admitted to the hospital for parenteral antibiotics, pending the results of the cultures. the child with occult pneumococcal bacteremia who appears well and is afebrile when returning for a follow-up may be managed as an outpatient with parenteral ceftriaxone followed by oral antibiotics according to the sensitivity of the organism. because of the concern of pneumococcal resistance to penicillin, a 2nd dose of intramuscular ceftriaxone may be given until sensitivity results are available. if the culture is positive for nontyphoidal salmonella organisms and the child is younger than 3 months, full sepsis evaluation and intravenous antibiotics are recommended. oral antibiotics and close follow-up are recommended for older children with salmonella bacteremia. • child with positive urine culture: if the child is afebrile and appears well, treatment with oral antibiotics is recommended, according to the sensitivity of the organism. may be distinguished. an alternative strategy is to manage such infants as outpatients, without empirical antibiotic therapy, after blood, csf, and urine cultures are obtained. although most of the original studies on outpatient management of febrile infants included infants aged 2-3 months, many experts agree that infants aged 2-3 months can be managed safely according to the guidelines for infants and children aged 3-36 months (table 39 .4). regardless of whether the clinician chooses to treat the patient with empiric antibiotics, all low-risk infants should be re-evaluated within 24 hours. those who appear ill or who have positive culture results should be admitted for parenteral antibiotics. if a child appears well and all culture results are negative, close follow-up should be continued and a 2nd return visit made in 24 hours. the risk of bacteremia for children with fws in this age group has decreased with the routine use of pneumococcal vaccines. the most common occult bacterial infection in this age group is uti. for children in this age group who appear ill, a full sepsis evaluation should be undertaken (table 39 .5). infants are at low risk if they appear well, have a normal physical examination, and have a caretaker reachable by telephone, and if laboratory tests are as follows: • cbc: <20,000 wbc/µl • urine: negative leukocyte esterase • csf: leukocyte count less than 10 × 10 6 /l infants are at low risk if they appear well and have a normal physical examination, and if laboratory tests are as follows: • cbc: <15,000 wbc/µl; band: total neutrophil ratio <0.2 • urine: <10 wbc/hpf; no bacteria on gram stain • csf: <8 wbc/µl; no bacteria on gram stain • chest radiograph: no infiltrate • stool: no rbc; few to no wbc infants are at low risk if they appear well and have a normal physical examination, and if laboratory tests are as follows: • cbc: 5,000-15,000 wbc/µl; peripheral absolute band count <1,500/µl • urine (enhanced urinalysis): 9 wbc/µl and no bacteria on gram stain • csf: 5 wbc/µl and negative gram stain; if bloody tap, then wbc:rbc ≤1 : 500 • chest radiograph: no infiltrate • stool: 5 wbc/hpf with diarrhea infants are at low risk if they appear well and have a normal physical examination, and if laboratory findings are as follows: • cbc: 5,000-15,000 wbc/µl; absolute band count ≤1,500/µl evaluation and management of ill-appearing children older than 36 months with fever without source are similar to those of younger children. for children in this age group who do not appear ill, no screening diagnostic tests are indicated. close attention should be paid to environmental exposures and ill contacts because of the high likelihood of increased contacts in this school-aged cohort. bacterial meningitis is usually a disease of infants and young children. the attack rate is highest between the ages of 3 and 8 months; 66% of cases occur in children younger than 5 years of age. bacterial meningitis is seen during all seasons; however, there may be a seasonal correlation between the presence of preceding respiratory pathogens in the upper respiratory tract and the subsequent development of bacterial meningitis. bacterial meningitis usually occurs sporadically. clusters of cases have been noted in day care centers, colleges, and other closed communities. bacterial meningitis occurs more frequently in children with traumatic fractures of the cribriform plate or paranasal sinuses or with a cochlear implant (pneumococci); in children who have undergone neurosurgical procedures such as ventricular shunts (s. aureus, s. epidermidis, corynebacterium species); in children with congenital or acquired immunodeficiencies (pneumococci, l. monocytogenes, meningococci); in children with anatomic or functional asplenia (pneumococci, meningococci); and in children with sickle hemoglobinopathies (pneumococci). there may be a genetic predisposition in some groups to the development of meningitis, inasmuch as there is an increased incidence of h. influenzae type b meningitis in navahos and eskimos. bacterial meningitis manifests in 2 patterns. in the 1st, the symptoms develop slowly over several days, the initial symptoms being those of a nonspecific illness. the signs and symptoms of meningitis develop subsequently. in the 2nd pattern, the disease develops suddenly and quickly, the 1st indications of illness being the signs and symptoms of meningitis and/or sepsis. the manifestations of meningitis depend on the child's age. in infants, the findings are usually nonspecific and may be subtle; they include vomiting, diarrhea, irritability, lethargy, poor appetite, respiratory distress, seizures, hypothermia, and jaundice. only 50% of affected infants have fever; some present only with fever. it is uncommon for affected young infants to have a stiff neck; only 30% have a bulging fontanel. older children present with more specific meningeal signs. they complain of a headache that is described as being severe, generalized, deep-seated, and constant. they complain about neck stiffness, caused by inflammation of the cervical dura and reflex spasm of the extensor muscles of the neck. there is pain and limitation of motion on flexion of the short duration and inconsistent development of increased intracranial pressure, papilledema is usually not seen at presentation. when it is present, venous sinus thrombosis, subdural effusion, or an intracranial abscess must be considered. seizures occur before hospital admission in up to 20% of affected patients. children with meningitis may also present with cutaneous findings. although commonly associated with meningococcal disease, purpura, petechiae, or a diffuse nonspecific maculopapular rash may be present in meningitis caused by any of the common bacterial pathogens (see chapter 40). septic arthritis may be seen simultaneously with bacterial meningitis. this has been assumed to be caused by simultaneous localizing infection after a primary bacteremia. reactive arthritis caused by immune complex deposition is also seen with bacterial meningitis. this arthritis affects 1 large joint and appears 5-7 days after treatment for meningitis has started. in general, arthritis occurring acutely with meningitis should be assumed to be infectious (see chapter 33). various eye disorders have also been described with acute bacterial meningitis, including transient cataracts, paralysis of the extraocular muscles, pupillary dysfunction, dendritic ulcers, endophthalmitis, cortical blindness, and conjunctivitis. recurrent episodes of bacterial meningitis rarely occur. potential etiologies include congenital csf fistulas (inner ear, dermal sinus, neuroenteric cysts, lumbosacral sinus tracts), traumatic or surgical csf fistula (skull fracture, postoperative nasal surgery, cochlear implant), immunodeficiency states and parameningeal infections (mastoiditis, sinusitis, craniofacial osteomyelitis). the definitive diagnosis of meningitis is based on examination of the cerebrospinal fluid (csf). the csf is usually obtained via a lumbar puncture (spinal tap). the lumbar puncture is performed by introducing a small-bore, short-beveled, spinal needle with a stylet into the subarachnoid space at the l3-l4 or l4-l5 level (figs. 39.2 to 39.4). a needle with a stylet is used to minimize the risk of introducing a nest of epidermal cells into the subarachnoid space that may later grow into a cord-compressing epidermoid tumor. approximately 3 ml of fluid is removed for analysis. there are a few contraindications for the performance of a lumbar puncture. the 1st is cardiorespiratory compromise. performance of the lumbar puncture requires that the child be held in flexion to open the intervertebral spaces. in seriously ill children or children with significant underlying cardiac or pulmonary disease, this positioning may be enough to cause respiratory compromise. the lumbar puncture may need to be postponed, be performed cautiously with continuous oxygen saturation monitoring, or performed with the patient in the sitting position. second, children with increased intracranial pressure from a focal central nervous system (cns) lesion, such as brain abscess or tumor, or from illnesses associated with cerebral edema have a high risk of cerebral herniation after a lumbar puncture. if signs or symptoms of increased intracranial pressure are present, the lumbar puncture should be postponed until the increased pressure is lowered with appropriate treatment. if a lumbar puncture is delayed, appropriate antibiotic therapy should be initiated without further delay. third, a lumbar puncture should not be done if the spinal needle must pass through an area of infection on its way to the subarachnoid space. to do so might introduce pathogens into the cns that could cause meningitis. epidural hematomas causing lower limb paralysis may be a complication of lumbar punctures in children with bleeding disorders. therefore, in children with hemophilia, disseminated intravascular of the neck, but lateral movement of the neck may be normal and pain-free. they also complain of nausea, vomiting, anorexia, and photophobia. on examination, they demonstrate irritability, mental confusion or altered consciousness, nuchal rigidity, and, occasionally, hyperesthesia and ataxia. the clinician demonstrates nuchal rigidity by feeling resistance and observing a painful response while flexing the patient's neck. the stiffness may not be recognized until the end of flexion. the neck usually can be rotated without symptoms. in the child who is crying and tensing the muscles, nuchal rigidity may be demonstrated if the examiner places 1 hand under the occiput of the supine patient and lifts the child. if the neck does not flex, it is stiff. alternatively, a sitting child may be observed following an object as it falls to the floor. the child who flexes the neck to look at the object does not have nuchal rigidity. in the presence of meningitis, flexion of the neck causes spontaneous flexion of the legs at the hips and knees, the brudzinski sign ( fig. 39.1) . the kernig sign is elicited when the patient lies supine and, with the knee flexed, the leg is flexed at the hip. the knee is then extended. a positive sign is present if this movement is limited by contraction of the hamstrings and causes pain. absence of nuchal rigidity is found in 1.5% of older children with meningitis; it may be absent in children who have overwhelming infections, are deeply comatose, or who have focal or global neurologic impairment. as many as 15% of children with bacterial meningitis initially present in a comatose or semicomatose state (see chapter 31). because a b specific agents) of pathogenic organisms. opening pressure measurements are obtained with the head of the bed flat and with the child relaxed and in the lateral decubitus position with the back no longer tightly flexed. the upper limit of normal value in children 1-18 years of age is less than 25 cm of water. opening pressure is less than 5 cm h 2 o in premature infants and less than 10 cm h 2 o in normal newborns. opening pressure measurements are elevated if the lumbar puncture is performed with the patient in the sitting position and if the patient is combative or performing the valsalva maneuver. obstructive hydrocephalus, hyperventilation, or removal of fluid can all lead to lowering of the measurement. children with bacterial meningitis usually have a mean opening pressure of 18 ± 7 cm h 2 o. normal csf is clear and colorless (table 39 .6). blood in the csf indicates a traumatic lumbar puncture or a cns hemorrhage. obtaining a rbc count on tubes 1 and 3 may differentiate the 2 conditions because the count is unchanged in cns hemorrhage but may decline in traumatic taps. centrifugation of the csf sample may also help differentiate between a traumatic tap and a cns hemorrhage. when blood has been present in the csf for several hours, the csf is xanthochromic after centrifugation. however, if the blood was recently mixed with csf, as in the case of a traumatic tap, the supernatant is clear. xanthochromic csf can also be caused by icterus or an elevated csf protein concentration. the normal values for wbcs in the csf are shown in table 39 .6. most children with bacterial meningitis have a wbc count of at least 1000/mm 3 in their csf, but, in general, more than 6/mm 3 in children after the neonatal period is considered abnormal. normal values for neonates are 0-18 (mean: 6) wbcs in the csf. an absolute neutrophil count exceeding 3/mm 3 (neutrophils may be as high as 35%) is also considered abnormal and evidence of a coagulopathy, or thrombocytopenia, lumbar puncture should be postponed until the bleeding disorder is corrected, and extra care should be taken to avoid a traumatic lumbar puncture. such children should be monitored after the procedure for the development of neurologic deficits. empirical therapy may be started while the coagulopathy is corrected. other, rarer complications of lumbar puncture include cortical blindness from compression of the posterior cerebral artery against the tentorium cerebelli, causing ischemic infarction of the occipital lobes. cervical spinal cord infarction, with respiratory arrest and flaccid tetraplegia, may occur if intracranial hypertension causes herniation of the cerebellar tonsils through the foramen magnum with resulting compression of the anterior spinal artery or its penetrating branches. post-lumbar puncture headache may occur in up to 10% of older children and adults; it is presumably caused by persistent csf leakage at the lumbar puncture site. the csf is examined for red blood cells (rbcs), white blood cells (wbcs) and differential, glucose, protein, and the presence (by culture, by gram stain or other stain, or by antigen or dna-pcr testing for on occasion, the spinal needle is advanced too far and passes through the subarachnoid space and penetrates the richly vascularized ventral epidural space. blood is thereby introduced into the subarachnoid space, and the csf appears bloody. this occurrence is often called a traumatic tap. it is then difficult to know whether the wbcs seen on examination of the csf are caused by csf pleocytosis or are peripheral blood wbcs contaminating the csf. to aid in this determination, the ratio of wbcs to rbcs in the csf is compared with the ratio of wbcs to rbcs in the patient's peripheral blood. a higher ratio in the csf indicates the presence of csf pleocytosis. when the csf ratio is at least 10 times higher than the blood ratio, bacterial meningitis is indicated, with a sensitivity of 88% and a specificity of 90%. conversely, the negative predictive value for the presence of bacterial meningitis of a less than 10-fold difference between the ratios is 99%. traumatic taps usually do not alter the csf glucose, gram stain, or culture findings, which are often abnormal with bacterial meningitis. when there is doubt about the validity of the cell count after a bloody tap, the lumbar puncture should be repeated after several hours by introducing the spinal needle 1 intervertebral space above the original tap site. in normal csf, the glucose concentration is two-thirds that of serum glucose concentration. the csf glucose concentration is low in most infected infants and younger children and in 45% of school-aged children with bacterial meningitis. in children older than 2 months of age, a csf/serum glucose ratio of less than 0.4 is 80% sensitive and 98% specific for the presence of bacterial meningitis. the presence of rbcs in a csf sample that is promptly analyzed does not affect the glucose concentration. the normal csf protein concentration is less than 45 mg/dl in children older than 2 months. the mean csf protein concentration is 90 (range, 20-170) in full-term infants and 115 (range, 65-150) in preterm infants. the csf protein concentration is elevated in more than 90% of younger children with bacterial meningitis but in only 60% of infected school-aged children. every 1000 rbcs in the csf (from a traumatic tap) increases the protein concentration by approximately 1 mg/dl. the presence of bacterial pathogens in the csf should be investigated. microscopic examination of a gram-stained sample of the fluid is performed first. the sensitivity of this test is directly related to the number of organisms in the csf and is inversely related to the age of the patient. the gram stain identification of certain organisms, such as h. influenzae, may be problematic. a decision whether to treat a child for bacterial meningitis should not be based on the gram stain alone; the definitive diagnosis is based on the csf culture. rapid diagnostic tests for bacterial antigens in csf, including countercurrent immunoelectrophoresis and latex particle agglutination, suffer from variations in sensitivity and specificity that limit their value in clinical practice. some patients will have been treated with antibiotics before the lumbar puncture is performed. when the csf from such a child is examined, organisms may not be seen on gram stain or recovered on culture. however, abnormalities of csf cell count (including elevated leukocytes), protein concentration, and glucose concentration usually continue to suggest the diagnosis of bacterial meningitis. in this setting, presumptive treatment for bacterial meningitis is initiated. if an organism is identified by culture or antigen detection, definitive antibiotic treatment is administered. if no organism is identified, the decision to continue treatment depends on the clinical suspicion of bacterial meningitis and the exclusion of other causes of aseptic meningitis (tables 39.7 and 39.8). newer laboratory techniques that utilize pcr to detect bacterial pathogens are being developed and may be useful in the diagnosis of bacterial meningitis in patients who have been treated with antibiotics before lumbar puncture. routine computed tomography (ct) of the head is not indicated in children with suspected meningitis. even though children with bacterial meningitis have increased intracranial pressure, most ct scans are normal. in addition, most lumbar punctures do not result in cerebral herniation in patients with meningitis. ct should be reserved for children who show clinical signs of herniation or cerebral edema and for those who may have an intracranial mass causing signs and symptoms similar to meningitis. usually, the peripheral blood wbc and platelet counts are elevated with bacterial meningitis. a low wbc count and thrombocytopenia may also be seen; these are associated with overwhelming infection and a poor outcome. the sensitivity (70%), specificity (54%), and negative predictive value (81%) of the differential wbc count are too low to render the differential wbc examination useful in making the diagnosis of bacterial meningitis. blood cultures may be useful in identifying the bacterial pathogen of meningitis. however, a negative blood culture may be found in up to 33% of children with meningococcal meningitis, 20% of children with pneumococcal cases, and 10% of patients with h. influenzae type b meningitis. these numbers increase with prior antibiotic therapy. in addition, there is a negative correlation between the length of illness before diagnosis and the rate of positive blood cultures. a bacterial meningitis score has been developed to attempt to distinguish between bacterial and aseptic (nonbacterial) meningitis in patients with csf pleocytosis. the risk of bacterial meningitis is low if none of the following criteria are present: history of a seizure with the illness, blood neutrophil count ≥10 × 10 9 cells/l, positive csf gram stain, csf protein ≥80 mg/dl, or csf neutrophil count ≥1 × 10 9 cells/l. this diagnostic tool is 99% sensitive and 62% specific for bacterial meningitis. it should only be applied to non-ill-appearing children older than 2 months without petechiae, purpura, or other concerning findings on examination who have not been pretreated with antibiotics. aseptic meningitis is an inflammatory process of the meninges, most often characterized by acute signs and symptoms of meningeal irritation; csf pleocytosis, usually with a predominance of mononuclear cells; a normal or, less frequently, elevated csf protein concentration; normal or, less often, low csf glucose concentration; and no organisms demonstrable by gram stain or bacterial cultures. there are many causes of aseptic meningitis (see table 39 .7). the most common cause is viral infection; up to 90% of cases are caused by enteroviruses and arbovirus. the definitive diagnosis is made by identifying the organism in the csf. however, this is not always possible, and other causes must be excluded by history, presence or absence of associated symptoms, and appropriate laboratory tests (tables 39.7 and 39.8). enteroviral meningitis occurs most often during the summer and early fall months. transmission is via the fecal-oral route, and young children exhibit increased transmission of the viruses and more severe disease in comparison with other age groups. initially, patients may have a respiratory tract infection, a nonspecific febrile illness, or vomiting and diarrhea. viral infection of the meninges occurs 7-10 days after initial exposure. the clinical course may be biphasic. virus from the oropharynx can be cultured only during the 1st 5-7 days of the illness but may be excreted in stool for 6-8 weeks. children with viral meningitis present with fever, nuchal rigidity, irritability, headache, and vomiting. less common symptoms are anorexia, drowsiness, photophobia, myalgia, and malaise. as in bacterial meningitis, affected young infants often lack meningeal signs. in addition, children may have an altered sensorium, but focal neurologic signs are rare. seizures are more common in infants. the number of wbcs in the csf varies from zero to several thousand (table 39 .6). up to 75% of initial (early in the illness) csf specimens contain a predominance of polymorphonuclear cells. mononuclear cells predominate by 2 days after the onset of symptoms. of children with enteroviral meningitis, 18% may have decreased csf glucose concentrations, whereas 12% may have elevated csf protein. treatment of enteroviral meningitis is supportive. admission to the hospital may be required while bacterial meningitis is being ruled out and for intravenous hydration. analgesics and antipyretics may also be indicated. the lumbar puncture performed to diagnose viral meningitis is often helpful in ameliorating the acute symptoms. the mechanism for this is not clear. the outcome is quite good for patients in whom common viral pathogens cause aseptic meningitis. sequelae in older children are rare. adverse outcomes are more common (but unusual) in children who have viral meningitis during the 1st year of life. speech and language development may be affected. treatment and outcome for the other types of aseptic meningitis depend on the underlying cause. tuberculous meningitis is an important treatable cause of aseptic meningitis. during the primary pulmonary tuberculous infection and enteroviruses and arboviruses cause most cases of infectious encephalitis in children. enterovirus encephalitis, uncommon without meningeal involvement, is suggested by epidemic occurrence and presence of typical prodrome or associated findings (table 39 .8); prompt diagnosis is by pcr for enterovirus in csf, blood, throat, or stool specimens. a csf or blood specimen is preferred because pcr may identify enterovirus in throat and especially stool for weeks after the primary infection has resolved. arbovirus encephalitis is suggested by mosquito or tick exposure and epidemic occurrence and is diagnosed by findings of arbovirus immunoglobulin m in csf or blood or by paired serologic findings for immunoglobulin g. infections with herpes simplex virus (hsv) occur throughout the year. in neonates, hsv encephalitis usually occurs between 7 and 21 days of age; may produce focal or generalized cns disease; and may occur with or without conjunctivitis, oral mucosal involvement, vesicles on skin, or disseminated disease (hepatitis, pneumonia, septic appearance). after the neonatal period, hsv encephalitis is usually isolated to the cns and classically produces necrotizing encephalitis with a focus in the temporal lobe. symptoms in persons with hsv encephalitis range broadly from those suggesting mild aseptic meningitis to the presence of status epilepticus and coma and then death. in addition to neutrophils and monocytes, csf examination may show increased numbers of erythrocytes and elevated protein. ct, mri, and an electroencephalogram (eeg) may suggest a temporal lobe focus. specific diagnosis is by pcr of csf for herpes simplex dna. csf culture is usually negative. in the appropriate clinical setting, presumptive therapy with intravenous acyclovir, 60 mg/kg/day given every 8 hours, is indicated while the results of pcr of csf for hsv are awaited. autoimmune encephalitis. anti-d-methyl-d-aspartate receptor (anti-nmdar) encephalitis is a novel and relatively common form of encephalitis. data from the california encephalitis project showed that anti-nmdar encephalitis was the most common identifiable cause of encephalitis in their cohort, which included patients from 6 months to 30 years. most of the cases occurred in children and adolescents. patients present with similar features as viral encephalitis, but seizures, language dysfunction, psychosis, autonomic dysfunction, movement disorders, and eeg abnormalities are more common in these patients. in adults, fuo is defined as an illness lasting more than 3 weeks, a fever higher than 38.3°c (101°f) on several occasions, and uncertainty subsequent lymphohematogenous spread to extrapulmonary sites, tubercle bacilli produce local microscopic granulomas in the cns and meninges. if this primary cns infection is not contained by host defense mechanisms (t lymphocytes, monocytes), or if host defense mechanisms fail at a later period, tuberculous meningitis may result. meningitis occurs weeks to months after the primary pulmonary process. the symptoms of tuberculous meningitis are insidious and subacute (weeks to months). stage 1 is a prodrome with nonspecific manifestations (apathy, poor school function, irritability, weight loss, fever, night sweats, nausea); stage 2 is heralded by the onset of neurologic signs (headache, cranial neuropathy, nuchal rigidity, signs of increased intracranial pressure); and stage 3 manifests with altered levels of consciousness (lethargy, stupor, coma). meningismus is not present in all patients. the diagnosis is supported by a history of contacts with adults with known active tuberculosis, a chronic cough, or human immunodeficiency virus (hiv) disease or by a history of immigration, poverty, or homelessness. in addition, the patient's chest radiograph is consistent with active or, more often, quiescent tuberculosis (parenchymal-hilar node calcifications, infiltrates, hilar adenopathy, and, in rare cases, endobronchial or cavitary lesions), and the patient's tuberculin skin test yields a positive result (see chapter 2). cranial ct or magnetic resonance imaging (mri) may show the most intense meningeal inflammation around the base of the brain or inflammatory mass lesions (tuberculomas). the csf results (table 39 .6) include profound hypoglycorrhachia, a high csf protein, lymphocyte-or monocytepredominant cells (usually 500 cells/mm 3 ), increased opening pressure, and, on occasion, tubercle organisms on acid-fast staining. pcr amplification of mycobacterium tuberculosis dna aids in making a more rapid diagnosis than does culture of csf, sputum, or gastric aspirates, which traditionally requires 2-6 weeks. the differential diagnosis depends on the stage of the illness. encephalitis is inflammation of the brain parenchyma, whereas meningoencephalitis is inflammation of the brain accompanied by inflammation of the meninges. meningoencephalitis is distinguished from aseptic meningitis by evidence of brain parenchymal involvement, including behavior or personality changes; altered level of consciousness (including agitation or coma); generalized seizures; focal neurologic signs, including focal seizures and focal motor defects (hemiparesis or ataxia); or movement disorders. bartonellosis in developed countries and brucellosis and typhoid in developing countries. often patients with an fuo have atypical manifestations of common childhood bacterial or viral diseases rather than unusual or uncommon disorders. the evaluation of a child with fuo centers on a detailed history and physical examination. taking the history should be repeated because parents often remember important details after the initial interview. the physical examination findings may also change during the course of the investigation revealing important clues (fig. 39 .5, table 39 .11). the history should include the time of day of the fever, who measured the temperature, and the instrument that was used to measure the temperature. increased temperatures after exercise and in the afternoon often represent normal variations. the appearance of the of diagnosis after a 1-week study in the hospital (table 39 .9). in pediatrics, the defined duration of fever is variable, from 8 days to 3 weeks (average, 2 weeks). this may be dependent on the age of the patient, with shorter periods of fever in young infants and more traditional adult standards in adolescent patients. fuo is defined as a temperature higher than 38°c (100.4°f) daily for at least 8-14 days and no diagnosis after an initial evaluation. the initial evaluation recommended varies but always includes a noncontributory history and physical examination, and nondiagnostic initial laboratory and radiologic tests. in accordance with this definition, the differential diagnosis for fuo in children is large ( child while febrile is also important. increased temperature without sweating might be seen in a child with ectodermal dysplasia or factitious fever. the pattern of fever should be noted (fig. 39.6 ). sustained fever, intermittent fever, and relapsing fever have been associated with different disease states. sustained or remittent fever remains elevated with little variation during the day and has been associated with enteric (typhoid) fever, tularemia, and rickettsial diseases such as typhus and rocky mountain spotted fever. intermittent fever normalizes at least once a day and is associated with tuberculosis, abscesses, lymphomas, juvenile idiopathic arthritis (jia), and some forms of malaria. children with relapsing fever have afebrile days between febrile episodes. relapsing fever has been associated with rat bite fever, borrelia species infection, malaria, brucellosis, subacute bacterial endocarditis, african trypanosomiasis, lymphomas, and lyme disease. saddle-back or double-hump fever lasts a few days, is followed by an afebrile day or 2, and then returns. it has been associated with some viruses and dengue fever. double quotidian fever (2 fever spikes each day) occurs in kala-azar, malaria, and gonococcal endocarditis. periodic fevers occur as acute febrile episodes separated by prolonged afebrile, healthy periods. diseases to consider include cyclic neutropenia, familial mediterranean fever, and the syndrome of periodic fever, aphthous stomatitis, pharyngitis, and adenitis (pfapa). periodic fever syndromes have different prevalence patterns in different ethnic groups and different inheritance patterns. a detailed family history is particularly important when these diagnoses are considered (see chapter 41). unfortunately, neither the fever pattern nor the duration is specific for a particular cause. fevers lasting for more than 1 year are not usually infectious; factitious fever, rheumatic or granulomatous disorders, familial diseases, or malignancies need to be considered in these patients. a history of rash is important for diagnosing lyme disease, jia, and acute rheumatic fever (see chapter 40). a history of pica is associated with visceral larva migrans and toxoplasmosis. exposure to domestic and wild animals should be identified to exclude zoonoses (see chapter 40). the food history should be detailed and should include water sources, use of game meats, cooking practices, and consumption of unpasteurized, raw milk, or soft cheese. travel history is critically important in the establishment of a differential diagnosis. areas visited, accommodations, activities, prophylactic treatments, animal and insect exposures, and water and food sources should be reviewed. coccidioidomycosis, histoplasmosis, malaria, lyme disease, and rocky mountain spotted fever have regional distributions. children who have traveled to or have emigrated from developing countries are at increased risk for endemic diseases and m. tuberculosis (table 39.12) . previous medical records should be reviewed. weight loss is important for diagnosing many chronic diseases such as lymphoma, tuberculosis, and inflammatory bowel disease. poor weight gain and growth, with or without gastrointestinal symptoms, may be the only historical clue to inflammatory bowel disease (see chapter 11). hiv risk factors in the parents and child should be reviewed. past and current medications should also be reviewed. the review of systems may reveal heat intolerance, palpitations, tremors, and declining quality of schoolwork in a child with hyperthyroidism. a history of severe head trauma may be associated with hypothalamic dysfunction and central fevers. whenever possible, the patient should be examined during a febrile episode. a high fever in the absence of an increased pulse may be present in a patient with factitious fever. to verify this diagnosis, the temperature of freshly voided urine may be recorded. tremor, tachycardia, palpitations, exophthalmos, lid lag, eyelid retraction, and smooth, flushed skin with diaphoresis are suggestive of hyperthyroidism. the ophthalmologic examination should include assessment of visual acuity, extraocular motion, visual field integrity, and gaze, as well as inspection of external structures and ophthalmoscopic examination (see chapter 32). conjunctivitis, iritis-uveitis-scleritis, or both may be seen in a variety of infectious conditions, including epstein-barr virus (ebv) infection, leptospirosis, rickettsial infection, and cat-scratch disease. conjunctivitis, uveitis, or both occur with kawasaki disease, systemic lupus erythematosus (sle), polyarteritis nodosa, and jia. sarcoidosis may be associated with conjunctival and uveal tract nodules. a thorough ophthalmoscopic evaluation (and, if needed, slitlamp examination) should be performed. sarcoidosis may be accompanied by vascular occlusions, hemorrhages, vascular sheathing, and preretinal inflammatory exudates. cytomegalovirus (cmv) produces chorioretinitis associated with white infiltrates near vessels and confluent depigmented areas. histoplasmosis causes small atrophic spots and, in rare cases, focal granulomas of the retina and choroid. toxoplasma gondii is a common cause of recurrent retinochoroiditis. retinal changes also occur with bacterial endocarditis. tuberculosis can cause formation of choroidal tubercles and also ulcerative palpebral conjunctival lesions. slit-lamp examination may also reveal iridocyclitis in jia, behçet syndrome, and inflammatory bowel disease. the frontal and maxillary sinuses should be palpated for tenderness. the nares should be inspected for inflamed mucosa and purulent discharge. tympanic membranes should be viewed and insufflated (see chapter 4). the mouth should be checked for lesions, inflammation, and tooth tenderness. behçet syndrome may manifest with oral aphthous lesions. inspection of teeth and gums may reveal a dental abscess. exudative and nonexudative pharyngitis is associated with ebv infection, tularemia, leptospirosis, and cmv. pfapa syndrome is characterized by periodic fever, aphthous stomatitis, pharyngitis, and cervical adenopathy. candida infection in the mouths of children older than 2 years may result from immunodeficiency such as hiv or from the use of inhaled steroids. the neck should be examined for adenopathy or thyroid enlargement (see chapter 36). the rest of the lymphatic system should be carefully examined. a single tender node may be seen with cat-scratch disease. generalized adenopathy can be seen in cmv infection, ebv infection, and systemic jia (see chapter 33). the musculoskeletal examination should include assessments of strength and of active and passive range of motion and evaluation for warmth, tenderness, or swelling of joints. irritability and pain on palpation over a bone or disuse pseudoparalysis may be the 1st clue to osteomyelitis. bone pain may also result from neoplastic infiltration of the bone marrow or sickle cell anemia. unexplained fever, arthralgias, and arthritis may be present with acute rheumatic fever, jia, lyme disease, kawasaki disease, sle, polyarteritis nodosa, and behçet syndrome (see chapter 33). myalgias occur commonly with viral diseases such as influenza, and may be present with rickettsial diseases, polyarteritis nodosa, takayasu arteritis, and dermatomyositis. careful auscultation of the heart and lungs is essential. a mitral or aortic regurgitant murmur may be the initial finding of endocarditis or of carditis in children with acute rheumatic fever. a pericardial friction rub may also suggest jia, sle, rheumatic fever, malignancy, or viral pericarditis. the abdomen must be carefully palpated for evidence of masses or hepatosplenomegaly (see chapters 14 and 17). abdominal tenderness may be present with abdominal abscesses, hepatosplenomegaly, and inflammatory bowel disease. a rectal examination should be performed, and stool should be tested for occult blood. sexually active girls should have a pelvic examination. pain on movement of the uterus during the pelvic examination may indicate pelvic inflammatory disease. specific serologic studies aid in the diagnosis of cmv, toxoplasmosis, brucellosis, tularemia, hepatitis a, b, and c, and leptospirosis. biopsies of lymph nodes, the skin, the liver, or bone marrow may be indicated. radiologic studies that may be of benefit if directed by the history, physical examination findings, and initial laboratory study results include sinus ct, abdominal imaging, or total body mri (to evaluate for occult osteomyelitis, malignancy, histiocytic disorders). the complete blood cell count with differential is neither specific nor diagnostic, except in rare circumstances, such as seeing lymphoblasts on the blood smear. approximately 30% of patients have abnormal white blood cell counts; 46% may have a left shift, lymphocytosis, atypical lymphocytes, or blasts. an elevated esr or crp indicates inflammation. the esr is usually (70-90% of the time) high in children with fuo caused by infectious pathogens, malignancies, and rheumatic diseases. of patients with an esr less than 10 mm/hr, 90% have a self-limited viral disease. urinalysis and urine culture identify occult infections, particularly in young girls. the urinalysis may also be abnormal in patients with endocarditis and rheumatic and other inflammatory disorders. unexpected consolidations, calcifications, interstitial changes, perihilar adenopathy, or cardiomegaly (heart failure, pericarditis) may be found on chest radiographs. chest films are abnormal in 10-15% of patients with fuo. ct of the chest may reveal abnormalities not detected by a chest x-ray. specialized radiologic studies performed without specific diagnostic clues from the history, physical examination findings, or initial laboratory evaluation results have a low yield. whole body mri is another technique that may be useful in children with fuo. it is helpful in identifying abnormal areas in bones, such as with occult osteomyelitis. a wide variety of infections have been identified in children with fuo including subacute bacterial endocarditis, urinary tract infections (uti), sinusitis, abscesses, osteomyelitis, and rheumatic fever. bacterial endocarditis. bacterial endocarditis is rare in children; incidence increases with advancing age and history of preexisting heart disease (see chapter 8). a new murmur or a change in the characteristic of an existing murmur may not be initially evident. vegetations also may not be visible initially by transthoracic echocardiography; a transesophageal approach is much more sensitive. serial blood cultures with anaerobic and aerobic media are necessary for definitive diagnosis. urinary tract infection. both upper and lower urinary tract infections may be asymptomatic, and leukocytes may not always be present in urine (see chapter 18). sterile pyuria may be present with tuberculosis, nongonococcal urethritis, viral cystitis, kawasaki disease, reactive arthritis, interstitial nephritis, and other rheumatic diseases. renal ultrasonography may show areas of decreased echogenicity, enlarged echogenic kidneys, and renal or perinephric abscesses. kidneys may be enlarged with acute pyelonephritis. a ct scan with contrast may show infected parenchyma as a nonenhancing lucency. nuclear medicine renal scans also identify active areas of infection and old scars. sinusitis. factors that decrease the size and patency of the ostium, or impair the mucociliary transport system predispose a child to sinusitis. ethmoid and maxillary sinuses are present at birth. the frontal sinuses usually appear near 5 or 6 years of age but may be asymmetric or absent. sphenoid sinuses may be seen radiographically by 9 years of age. prolonged nasal congestion, headache, purulent nasal discharge, sore throat, daytime cough, tender teeth, and halitosis may be present the skin must be inspected for evidence of rashes and other lesions (see chapter 40). jia may manifest with an evanescent, salmon-colored macular rash over the trunk and joints that may appear and disappear rapidly and be evident only during febrile periods. dermatomyositis is characterized by a heliotropic rash of the upper eyelids and an erythematous eruption (vasculitis) over the extensor surfaces (gottron sign). sle may manifest with a butterfly rash over the nose and malar regions, signs of photosensitivity in sun-exposed areas, or vasculitis. the rash of kawasaki disease is erythematous and may manifest in many forms; it is most commonly a diffuse maculopapular rash. in rocky mountain spotted fever, there are macular erythematous spots on the wrists, ankles, or forearms that may become maculopapular and expand centripetally to the proximal extremities and torso; palms and soles may be involved and petechiae may develop later in the course of the illness. endocarditis may be associated with splinter hemorrhages or janeway lesions (painless, small, erythematous or hemorrhagic lesions on the palms and soles). lyme disease usually manifests with erythema migrans. this rash begins at the site of the tick bite and is erythematous with a pale center. the rash radiates out from the bite in a circular manner and persists for weeks; satellite secondary lesions may also appear. tularemia, salmonellosis, listeriosis, and ebv infections may feature generalized maculopapular rashes. laboratory evaluation should proceed in a stepwise, focused manner with emphasis on identifying serious illnesses with defined interventions (see fig. 39 .5). initial studies should include a complete blood cell count with differential, erythrocyte sedimentation rate (esr) measurement, crp, blood cultures, urinalysis, urine culture, tuberculin skin tests with controls (anergy panel) or gamma interferon release assay, and chest radiograph. because ebv infection is common in childhood, viral-specific antibody titers may also be obtained at the initial evaluation (see chapter 36). further studies should be directed from days to months. regional lymphadenopathy with 1 or more nodes occurs proximal to the skin site 1-9 weeks after inoculation. the node or nodes become enlarged and tender and may have overlying erythema. the lymphadenopathy usually resolves after 2 months but may last up to 3 years. affected children may have adenopathy with fever, headache, malaise, anorexia, sore throat, and conjunctivitis (see chapter 36). q fever. q fever is caused by coxiella burnetii, formerly classified as a rickettsia. it manifests with headache, fever, chills, malaise, and, on occasion, respiratory symptoms. hepatic, cardiac, and cns involvement may occur. rash is usually not seen. domestic farm animals, cats, rodents, and marsupials may be infected. pasteurization destroys the organism in milk. diagnosis is made by serologic testing. rat bite fever. rat bite fever is a relapsing fever caused by streptobacillus moniliformis or spirillum minus. s. moniliformis is a pleomorphic gram-negative bacillus transmitted by rat bite or by contaminated food or water. in 1-10 days after exposure, patients may exhibit fever, chills, malaise, and muscle aches. a rash may form on the extremities; arthralgias and arthritis may occur. complications include abscesses, pneumonia, endocarditis, myocarditis, and meningitis. diagnosis is made by blood culture or culture of other infected fluid, such as abscess aspiration. tularemia. francisella tularensis is the causative agent of tularemia. the disease is spread by contact with wild animals, such as rabbits and squirrels, and by insects that bite these animals, such as mosquitoes, ticks, and deer flies, as well as by contaminated water. a maculopapular nodule forms at the portal of entry and later becomes ulcerated. the child may present with fever, chills, and headache. lymphadenopathy, pharyngitis, conjunctivitis, hepatosplenomegaly, and pneumonia may also occur. diagnosis is made by serologic study. brucellosis. brucellosis is caused by gram-negative coccobacilli: brucella abortus, b. melitensis, b. suis, or b. canis. the microorganisms are found in sheep, goats, cattle, swine, and dogs. infection may occur by airborne spread or by ingestion of meat or milk. the child may present with fever, chills, malaise, headache, arthralgias, or myalgias. pneumonia, cardiac involvement, and cns involvement occur in rare cases. diagnosis is made by special culture techniques and serologic study. leptospirosis. leptospirosis is caused by members of the spirochete genus leptospira. infection is spread by contact with the urine of wild or domestic animals. in 1-2 weeks after exposure, patients experience the abrupt onset of fever, chills, nausea, vomiting, headache, and occasionally conjunctival suffusion and rash. liver, renal, and cns involvement may also occur. diagnosis is made by special culture techniques and serologic testing. blastomyces dermatitidis is a saprophytic fungus with both yeast and mycelial forms; it is found in the soil all over the world but is common in the americas. it is endemic in the southeast and midwest regions of north america. infections with this fungus may be disseminated or pulmonary. the diagnosis is made by visualization of single-budding yeast in clinical material, culture on sabouraud agar, or serologic tests. histoplasma capsulatum is a yeast found in soil in the ohio river valley and other locations in the united states that causes pulmonary and disseminated disease. diagnosis is made by the demonstration of the microorganism in biopsy specimens or by complement-fixing antibody. in children with sinusitis. ct studies may be helpful. rhinoscopy may show purulent material at the ostium of an infected sinus. infectious complications of sinusitis include dural space empyema or brain abscesses. abscesses. hepatic, renal, perinephric, pelvic, and subphrenic abscesses may present with fuo. internal jugular thrombophlebitis may manifest with prolonged fever and severe neck pain. liver abscess may manifest with right upper quadrant tenderness and hepatomegaly. blood cultures and liver function study results are often normal. the diagnosis may be made with mri, ct with contrast, or ultrasonography. the diagnosis of perinephric abscesses is made with ct with contrast or ultrasonography. ct or ultrasound guidance may be used to direct percutaneous drainage of many abscesses. pelvic abscesses should be suspected in children with fuo who have abdominal, rectal, or pelvic tenderness. osteomyelitis. osteomyelitis usually follows bacteremia, but it sometimes follows penetrating injury. tenderness to palpation over the infected site is common. abnormalities in plain films appear late (2 weeks). mri is the imaging modality of choice. the blood or bone culture is often positive, and the esr is often elevated. suppurative myositis may mimic osteomyelitis and manifest as an fuo. rheumatic fever. acute rheumatic fever may cause fuo; the diagnosis is made by fulfillment of the jones criteria, updated in 2015 (see chapter 8). initially, a child may present with polyarthralgia and an increased esr. elbows, wrists, knees, and ankles are frequently involved. the later migratory nature of the true arthritis differentiates rheumatic fever from jia. bacterial syndromes that cause fuo in children include agents of the following: • lyme disease • cat-scratch disease • q fever • rat bite fever • tularemia • brucellosis • leptospirosis lyme disease. lyme disease is caused by the spirochete borrelia burgdorferi and is transmitted by the ixodes dammini and i. pacificus ticks. the usual manifestation of early lyme disease is with erythema migrans, an erythematous, annular, expanding rash with central clearing. the rash resolves 1-30 days (usually 2 weeks) after exposure. patients may exhibit fever, chills, fatigue, headaches, malaise, myalgias, arthralgias, and lymphadenopathy. early disseminated lyme disease follows 2-8 weeks after exposure; facial nerve palsy, peripheral neuropathy, cardiac conduction defects, myocarditis, and aseptic meningitis may occur. diagnosis is made clinically in early localized lyme disease because serology lacks sensitivity and specificity during early infection and because erythema migrans is so specific for lyme disease. diagnosis of early disseminated lyme disease requires a typical clinical illness, exposure to ticks known to carry b. burgdorferi and serologic evidence of infection with a 2-tier testing strategy. the initial test is an enzyme-linked immunosorbent assay (elisa) or immunofluorescent (ifa) test. if this result is equivocal or positive, a western immunoblot is performed. western blot should not be performed if the elisa is negative or has not been performed because it lacks specificity in this setting. cat-scratch disease. cat-scratch disease is a febrile illness associated with cats (usually kittens) and, more rarely, dogs. bartonella henselae, which may be transmitted by the cat flea and by cat saliva, is the etiologic agent. after a scratch or bite, a papule forms and may persist parasites fuo in children may be caused by parasitic infections, including (1) babesiosis, (2) toxoplasmosis, and (3) toxocariasis. babesiosis is caused by babesia microtia and is a parasite of rodents transmitted to humans by tick bite. infection may result in fever, chills, nausea, vomiting, night sweats, myalgias, and arthralgias. identification of the organism in a thick smear of red blood cells is diagnostic. t. gondii is a protozoan parasite. children become infected from eating contaminated, undercooked meat or from exposure to the feces of domestic cats. most infections acquired postnatally are asymptomatic but children may develop a mononucleosis-like illness (see chapter 36). toxocariasis (previously visceral larva migrans) results from ingestion of larvae of toxocara canis or from t. cati shed in dog and cat feces, respectively. infection results in fever, intense eosinophilia, hepatomegaly, and hypergammaglobulinemia. lung, heart, and cns involvement is rare. the eye may become infected. diagnosis is presumed with increased eosinophils and hypergammaglobulinemia, and elevated titers of isohemagglutinin to a and b blood group antigens. in a child who has traveled to or lives in a developing country, consideration must be given to the area, water sources, and activities. some causes of fuo to consider include malaria, hepatitis, typhoid fever, tuberculosis, and amebic liver abscess (table 39 .12). malaria is transmitted by the bite of an infected mosquito carrying 1 of the 5 species of the plasmodium genus that cause disease in humans. the patient experiences chills, rigors, high fever, diaphoresis, and headaches. the incubation period varies among species, from 1 week to several months. demonstration of the parasite on thick peripheral blood smear is diagnostic. risk for malaria can be checked for areas of the world on www.cdc.gov/malaria. hepatitis a may be contracted by ingestion of contaminated food or water. hepatitis b and c viruses are transmitted through blood products or sexual contact. diagnosis is by serologic testing. symptoms can include fever, malaise, jaundice, hepatomegaly, nausea, and anorexia. hepatitis b and c can become chronic (see chapter 15). enteric fever is caused by infection with 3 serovars of s. enterica, which includes s. typhi. after ingestion of contaminated water or food, incubation lasts from 1-6 weeks. persistent fever, headache, malaise, anorexia, and rose spots are clinical hallmarks of enteric fever. diagnosis is by blood culture. tuberculosis may manifest as fuo in children (see chapters 2 and 36). affected children may have pulmonary or extrapulmonary disease. the signs and symptoms of pulmonary disease may vary greatly from weight loss, tuberculin skin test conversion, and low-grade fever to mass effect from mediastinal lymphadenopathy and fulminant disseminated pulmonary involvement with miliary infiltrates or, in rare cases, cavitation. nonpulmonary tuberculosis more commonly manifests as fuo, inasmuch as positive chest radiograph findings and pulmonary signs may initiate an early work-up for tuberculosis. hematogenous spread may cause liver, heart, or renal involvement. ingested bacilli may result in gastrointestinal tuberculosis. the diagnosis requires demonstration of coccidioides immitis is found in soil in the southwestern united states. infections in humans are associated with a febrile pulmonary disease characterized by cough, rash, and chest pain. diagnosis is usually made serologically. cryptococcus neoformans is often found in pigeon droppings and can cause a variety of diseases. the diagnosis is made by culture or by identification of encapsulated yeast in collected specimens. psittacosis and lymphogranuloma venereum are chlamydial causes of fuo. chlamydia psittaci may be transmitted by infected birds and produces respiratory illness with fever. cardiac, liver, cns, and thyroid involvement are rare. diagnosis is made serologically. c. trachomatis is a sexually transmitted organism that causes urogenital infections, perihepatitis, invasive lymphadenopathy (lymphogranuloma venereum), neonatal conjunctivitis, and neonatal pneumonia. diagnosis is by cell culture and rapid antigen tests. rocky mountain spotted fever. rocky mountain spotted fever manifests with fever, headache, intense myalgias, and abdominal symptoms. a characteristic rash is usually present by the 6th day of illness. the rash covers the palms, wrists, soles, and ankles and progresses from macular to petechial. the disease can last up to 3 weeks. many end organs, including the heart, kidneys, and cns, can be involved. transmission of the causative agent, rickettsia rickettsii, occurs by tick bite. diagnosis is made by pcr testing of blood. ehrlichiosis and anaplasmosis. these infections are caused by ehrlichia chaffeensis, anaplasma phagocytophilia, and e. ewingii and are transmitted by the lone star tick. anaplasmosis is caused by anaplasma phagocytophilia and is transmitted by the black legged deer tick. these illnesses are usually seen in the southeastern and upper midwestern united states, respectively, and have manifestations similar to that of rocky mountain spotted fever. the patient presents with headache, myalgias, fever, chills, nausea, vomiting, weight loss, thrombocytopenia, and leukopenia. rash is inconsistent but may be seen after 1 week. pulmonary and renal complications can occur. mental status changes are less frequent. diagnosis is confirmed by pcr. cytomegalovirus infection. cmv may cause a mononucleosis-like syndrome in children. generalized or cervical adenopathy may be seen along with fatigue, malaise, fever, hepatosplenomegaly, and abdominal pain (see chapter 36). a morbilliform rash may also be present. retinitis, hepatitis, colitis, and pneumonia may occur in children with impaired immune systems. the virus is transmitted by contact with secretions. infection is diagnosed by culture (nasopharyngeal, blood, urine) or by the detection of specific immunoglobulin g and immunoglobulin m antibodies. infectious mononucleosis. infectious mononucleosis is typically caused by ebv and may manifest with fever, exudative pharyngitis, malaise, and fatigue (see chapter 36). the appearance of rash is sometimes preceded by amoxicillin therapy, but rash may occur without antibiotic administration. tender lymphadenopathy and hepatosplenomegaly may occur. the diagnosis may be made by nonspecific tests (heterophile antibody or monospot) in older patients, but these studies are unreliable for young children. specific antibody tests against viral capsid antigen, early antigen, and nuclear antigen are recommended in younger children. treatment is supportive. human immunodeficiency virus infection. infection with hiv or associated opportunistic infections or associated malignancies is another cause of fuo in children. neuroblastoma may manifest as abdominal, thoracic, or pelvic masses; spinal cord compression; bone pain; hypertension; hepatomegaly; diarrhea; and fever (see chapter 17). diagnosis is aided by radiologic studies and urinary catecholamine measurements and is confirmed by biopsy. both acute lymphocytic leukemia and acute nonlymphocytic leukemia may manifest with lethargy, pallor, bleeding, fever, bone pain, lymphadenopathy, and arthralgias. diagnosis is made by blood smear and bone marrow biopsy. pheochromocytomas are rare catecholamine-secreting tumors; 10% occur in children. these tumors manifest with paroxysmal or sustained hypertension, headache, excessive sweating, fever, hyperglycemia, and palpitations. the tumors are usually in the adrenal medulla, but 35% of those occurring in children are multiple or extraadrenal. diagnosis is made by measuring urinary or plasma metanephrine or catecholamine levels. localization of tumor is by ct, mri, or iodine 131-metaiodobenzylguanidine scanning. familial mediterranean fever is an autosomal recessive trait seen in sephardic jews and people of middle eastern descent. the fever may be accompanied by joint, abdominal, and chest pain. anhidrotic ectodermal dysplasia is an x-linked recessive disorder associated with decreased ability to sweat, dental abnormalities, and sparse hair. eyebrows and eyelashes may be absent. fever may result from the inability of the body to cool itself. diagnosis is made by skin biopsy that shows an absence of eccrine glands. drug fever is a diagnosis of exclusion. some drugs are more likely than others to cause drug fever (α-methyldopa, quinidine, penicillins). there is no characteristic fever pattern. there is a highly variable lag time between the initiation of the drug and the onset of fever, and there is an infrequent association with rash or eosinophilia. some drugs may cause fever by virtue of physiologic side effects. anticholinergic drugs may decrease sweating and diminish the body's ability to cool itself. chronic salicylate intoxication can cause increased heat production by uncoupling oxidative phosphorylation. kawasaki disease may manifest with a variety of signs, including rash; lymphadenopathy; conjunctival hyperemia; strawberry tongue; erythematous lips; swelling of hands and feet; arthralgia; arthritis; myocarditis; late desquamation of hands, feet, and perineal area; and sterile pyuria. fever may be high and spiking. diagnosis is by fulfillment of clinical criteria (see chapter 40). inflammatory bowel disease (ibd; ulcerative colitis, crohn disease) may manifest with fuo. ulcerative colitis may manifest with bloody diarrhea, fever, fecal urgency, and straining (see chapter 11). pyoderma gangrenosum, arthritis, and erythema nodosum can also be seen. crohn disease (regional enteritis) may manifest with abdominal pain, fever, anorexia, and growth failure. diarrhea may develop later. arthritis, erythema nodosum, and finger clubbing may also occur. diagnosis of ibd is by endoscopy and histology. acid-fast bacilli from sputum, gastric aspirate, or the affected organ. skin testing may yield negative results even with positive controls. intestinal infection with entamoeba histolytica may produce invasion of the mucosal lining and spread to other organs such as the liver. amebic liver abscess may manifest with fever, weight loss, right upper quadrant pain, and anorexia. the patient may have painful hepatomegaly without splenomegaly. the abscess may be localized with abdominal ultrasonography or ct. diagnosis is by serologic study. rheumatic diseases as a cause of fuo are the 2nd most common identified cause of fuo after infections. in a systematic review, the most common causes were jia and sle (see chapter 33). jia is a diagnosis that requires time to identify all of its manifestations and to exclude other entities. jia is defined by arthritis of unknown origin that begins in a child younger than 16 years and persists for a minimum of 6 weeks. jia is divided into 3 subtypes: systemic, polyarticular, and oligoarticular. the systemic form often manifests with prolonged high fever. affected children often have a daily fever and may have a fine macular rash, arthralgias, arthritis, hepatosplenomegaly, or pericardial involvement. polyarticular jia may manifest with arthritis, low-grade fever, morning stiffness, anorexia, and weight loss. polyarteritis is a necrotizing vasculitis that may manifest with myalgia, arthralgia, fever, vasculitic skin lesions, and abdominal pain. cardiac, cns, and renal involvement may also occur. the esr usually is markedly elevated. biopsy and the presence of antibodies to proteinase 3 and myeloperoxidase (antineutrophil cytoplasmic antibodies) are helpful. sle may manifest with fever, photosensitivity, mouth sores, weight loss, rash, myalgias, malaise, and hepatosplenomegaly. patients may also have serositis and renal involvement. laboratory tests that are helpful include lupus erythematosus cell preparation and those for antinuclear antibody, anti-smith antibody, anti-ribonuclear protein antibody, anti-ro (sjögren syndrome type a) antibody, and anti-la (sjögren syndrome type b) antibody. behçet syndrome is very rare in children but may manifest with fuo. patients may have aphthous stomatitis, arthritis, genital ulcers, uveitis, and erythema nodosum. hodgkin disease, lymphoma, neuroblastoma, and leukemia may all manifest as fuo. in young children, leukemia, neuroblastoma, and lymphoma should be suspected, whereas in adolescents, hodgkin disease and ewing sarcoma are more common as causes of fuo. hodgkin disease may manifest with firm, nontender adenopathy, fever, night sweats, and weight loss. diagnosis is made through biopsy. non-hodgkin lymphoma may manifest as painless adenopathy, cough or dyspnea from a mediastinal mass, abdominal mass, nerve compression, bone pain, fever, and weight loss. diagnosis is by biopsy. sweating, tachypnea, or tachycardia. if factitious fever is suspected, the temperature should be obtained while the patient is observed. the temperature of freshly voided urine can also be recorded. other patients may produce actual diseases that cause true fevers, such as by injecting infected pyogenic material subcutaneously or intravenously or by taking toxic levels of thyroid hormone. once the diagnosis is documented, psychiatric care is indicated. if no diagnosis is made, most patients are clinically well and asymptomatic on follow-up. some may be determined to be healthy from the start; most are in good health at follow-up, whereas few have symptoms at the end of evaluation. some may have relapses of fever for a few months. jia, inflammatory bowel disease, and pfapa syndrome may not be immediately diagnosed but usually manifest typical symptoms and signs within 2 years of the onset of the fuo. hyperthyroid states may manifest with fuo. children usually have multiple symptoms, such as irritability, tremor, eyelid lag, and exophthalmos. diagnosis is made from thyroid function studies. factitious fever may be a form of factitious disorder imposed on self (formerly munchausen syndrome) or medical child abuse (formerly munchausen syndrome by proxy) (see chapter 26). a variety of techniques have been used to falsely elevate a recorded temperature. a mercury thermometer may be rubbed between hands or placed near a light bulb. hot liquids may be placed in the mouth before an oral temperature is taken. hot rectal douches have also been reported to raise a rectally taken temperature. even with pathologic fevers, there is some circadian rhythm to the temperature curve; with factitious fever there is no rhythm. in addition, there is usually no vasoconstriction, many children with fever will have a source identified on their initial history and physical examination. red flags include patients with symptoms or signs of sepsis (tachycardia, hypotension) or meningitis or encephalitis (fever, headache, irritability, altered mental status and for the older child, meningismus). affected infants with meningitis are more likely than older children to have subtle and nonspecific symptoms. a child with fever of recent onset with no adequate historical or physical explanation for the fever is said to have fever without source (fws). because of the high volume of children with fws, it is important to have a reliable system for individual patient evaluation and management. although the majority of patients with fws have a self-limited viral illness, 5-10% have an invasive bacterial infection, with young infants at highest risk. because of the potential for morbidity and mortality from the organisms that cause invasive disease, identification of patients at high risk is essential. although there is no single, timely series of tests that correctly categorizes all patients, the combination of careful clinical evaluation and appropriate laboratory screening criteria can help identify a level of risk in children of different ages. the reduction of bacteremia due to vaccine-serotype pneumococcus has led to a careful reduction in diagnostic testing, especially in the 3-36 month old child with fws. red flags include a history of immunodeficiency or other chronic medical illness, no prior immunizations, toxic appearance, signs of shock, petechiae or purpura, poor responsiveness, and other signs of altered mental status. some children, who are initially thought to have fws, develop into patients with fuo. definitions of fuo in children vary. a practical definition balancing different recommendations is fuo is a temperature higher than 38°c (100.4°f) daily for at least 8-14 days and no diagnosis after an initial evaluation. work-up of the patient with fuo should proceed in a stepwise manner. it should be kept in mind that many patients with fuo have unusual, atypical, or complicated manifestations of common childhood illness, mainly infections. red flags include weight loss, night sweats, signs of organ system dysfunction or failure, or unstable vital 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critically ill: a multicenter study of molecular detection in bloodstream infections, pneumonia, and sterile site infections serious bacterial infection in recently immunized young febrile infants fever without source clinical practice guideline for the diagnosis and management of the initial urinary tract infection in febrile infants and young children 2-24 months of age procalcitonin and c-reactive protein as diagnostic markers of severe bacterial infections in febrile infants and children in the emergency department evaluation of child with fever without source: review of literature and update epidemiology of bacteremia in febrile infants in the united states detection of viruses in young children with fever without an apparent source redefining urinary tract infections by bacterial counts changing epidemiology of bacteremia in infants aged 1 week to 3 months changing epidemiology of outpatient bacteremia in 3 to 36 month old children after the introduction of the heptavalent-conjugated pneumococcal vaccine performance of low-risk criteria in the evaluation of young infants with fever: review of the literature impact of the 13-valent pneumococcal conjugate vaccine on pneumococcal meningitis in us children management and outcomes of care of fever in early infancy serious bacterial infections in febrile infants in the post-pneumococcal conjugate vaccine era systematic review of the diagnostic accuracy of c-reactive protein to detect bacterial infection in nonhospitalized infants and children with fever diagnostic accuracy of the urinalysis for urinary tract infection in infants < 3 months of age prevalence of urinary tract infection in childhood-a meta-analysis invasive pneumococcal disease after implementation of 13-valent conjugate vaccine diagnostic value of clinical features at presentation to identify serious infection in children in developed countries: a systematic review decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine comparison of the test characteristics of procalcitonin to c-reactive protein and leukocytosis for the detection of serious bacterial infections in children presenting with fever without source: a systematic review and meta-analysis central nervous system infection csf opening pressure in children with optic nerve head edema reference range for cerebrospinal fluid opening pressure in children normative cerebrospinal fluid profiles in febrile infants clinical features suggestive of meningitis in children: a systematic review of prospective data diagnosis and management of meningitis predictors of bacterial meningitis in the era after haemophilus influenzae the frequency of autoimmune n-methyl-d-aspartate receptor encephalitis surpasses that of individual viral etiologies in young individuals enrolled in the california encephalitis project lumbar puncture in pediatric bacterial meningitis: defining the time interval for recovery of cerebrospinal fluid pathogens after parenteral antibiotic pretreatment acute bacterial meningitis in infants and children natural history of neonatal herpes simplex virus infections in the acyclovir era safety and efficacy of high-dose intravenous acyclovir in the management of neonatal herpes simplex virus infections does this child have bacterial meningitis? a systematic review of clinical prediction rules for children with suspected bacterial meningitis cerebrospinal fluid opening pressure in children: experience in a controlled setting tuberculous meningitis meta-analysis of bacterial meningitis score validation studies central nervous system tuberculosis in children: a review of 30 cases cerebrospinal fluid protein concentration in pediatric patients report of the committee on infectious diseases incidence of rash after amoxicillin treatment in children with infectious mononucleosis fever of unknown origin in children: a systematic review do penicillins really increase the frequency of a rash when given during epstein-barr virus primary infection? prolonged fever of unknown origin prolonged fever of unknown origin in children hemophagocytic syndrome in children: an important diagnostic consideration in fever of unknown origin prolonged fever in children: review of 100 cases usefulness of scanning procedures for diagnosis of fever of unknown origin in children long-term follow-up of children with fever of unknown origin rocky mountain spotted fever in children key: cord-267132-nb0j6k3h authors: loveday, h.p.; wilson, j.a.; pratt, r.j.; golsorkhi, m.; tingle, a.; bak, a.; browne, j.; prieto, j.; wilcox, m. title: epic3: national evidence-based guidelines for preventing healthcare-associated infections in nhs hospitals in england date: 2013-12-10 journal: j hosp infect doi: 10.1016/s0195-6701(13)60012-2 sha: doc_id: 267132 cord_uid: nb0j6k3h national evidence-based guidelines for preventing healthcare-associated infections (hcai) in national health service (nhs) hospitals in england were originally commissioned by the department of health and developed during 1998–2000 by a nurse-led multi-professional team of researchers and specialist clinicians. following extensive consultation, they were first published in january 2001(1) and updated in 2007.(2) a cardinal feature of evidence-based guidelines is that they are subject to timely review in order that new research evidence and technological advances can be identified, appraised and, if shown to be effective for the prevention of hcai, incorporated into amended guidelines. periodically updating the evidence base and guideline recommendations is essential in order to maintain their validity and authority. the department of health commissioned a review of new evidence and we have updated the evidence base for making infection prevention and control recommendations. a critical assessment of the updated evidence indicated that the epic2 guidelines published in 2007 remain robust, relevant and appropriate, but some guideline recommendations required adjustments to enhance clarity and a number of new recommendations were required. these have been clearly identified in the text. in addition, the synopses of evidence underpinning the guideline recommendations have been updated. these guidelines (epic3) provide comprehensive recommendations for preventing hcai in hospital and other acute care settings based on the best currently available evidence. national evidence-based guidelines are broad principles of best practice that need to be integrated into local practice guidelines and audited to reduce variation in practice and maintain patient safety. clinically effective infection prevention and control practice is an essential feature of patient protection. by incorporating these guidelines into routine daily clinical practice, patient safety can be enhanced and the risk of patients acquiring an infection during episodes of health care in nhs hospitals in england can be minimised. we would like to acknowledge the assistance of the infection prevention society, british infection association and the healthcare infection society for their input into the development of these guidelines; and other associations, learned societies, professional organisations, royal colleges and patient groups who took an active role in the external review of the guidelines. we would also like to acknowledge the support received from professor brian duerden cbe in chairing the guideline development advisory group, and carole fry in the chief medical ofà cer's team at the department of health (england). the department of health (england). this guidance is based on the best critically appraised evidence currently available. the type and class of supporting evidence explicitly linked to each recommendation is described. some recommendations from the previous guide lines have been revised to improve clarity; where a new recom mendation has been made, this is indicated in the text. these recommendations are not detailed procedural protocols, and need to be incorporated into local guidelines. none are regarded as optional. standard infection control precautions need to be applied by all healthcare practitioners to the care of all patients (i.e. adults, children and neonates). the recommendations are divided into à ve distinct interventions: • hospital environmental hygiene; • hand hygiene; • use of personal protective equipment (ppe); • safe use and disposal of sharps; and • principles of asepsis. these guidelines do not address the additional infection control requirements of specialist settings, such as the operating department or outbreak situations. the hospital environment must be visibly clean; free from non-essential items and equipment, dust and dirt; and acceptable to patients, visitors and staff. sp2 levels of cleaning should be increased in cases of infection and/ or colonisation when a suspected or known pathogen can survive in the environment, and environmental contamination may contribute to the spread of infection. the use of disinfectants should be considered for cases of infection and/ or colonisation when a suspected or known pathogen can survive in the environment, and environmental contamination may contribute to the spread of infection. sp4 shared pieces of equipment used in the delivery of patient care must be cleaned and decontaminated after each use with products recommended by the manufacturer. all healthcare workers need to be educated about the importance of maintaining a clean and safe care environment for patients. every healthcare worker needs to know their specià c responsibilities for cleaning and decontaminating the clinical environment and the equipment used in patient care. hand hygiene sp6 hands must be decontaminated: • immediately before each episode of direct patient contact or care, including clean/aseptic procedures; • immediately after each episode of direct patient contact or care; • immediately after contact with body á uids, mucous membranes and non-intact skin; • immediately after other activities or contact with objects and equipment in the immediate patient environment that may result in the hands becoming contaminated; and • immediately after the removal of gloves. use an alcohol-based hand rub for decontamination of hands before and after direct patient contact and clinical care, except in the following situations when soap and water must be used: • when hands are visibly soiled or potentially contaminated with body á uids; and • when caring for patients with vomiting or diarrhoeal illness, regardless of whether or not gloves have been worn. class a healthcare workers should ensure that their hands can be decontaminated effectively by: • removing all wrist and hand jewellery; • wearing short-sleeved clothing when delivering patient care; • making sure that à ngernails are short, clean, and free from false nails and nail polish; and • covering cuts and abrasions with waterproof dressings. effective handwashing technique involves three stages: preparation, washing and rinsing, and drying. • preparation: wet hands under tepid running water before applying the recommended amount of liquid soap or an antimicrobial preparation. • washing: the handwash solution must come into contact with all of the surfaces of the hand. the hands should be rubbed together vigorously for a minimum of 10-15 s, paying particular attention to the tips of the à ngers, the thumbs and the areas between the à ngers. hands should be rinsed thoroughly. • drying: use good-quality paper towels to dry the hands thoroughly. when decontaminating hands using an alcohol-based hand rub, hands should be free of dirt and organic material, and: • hand rub solution must come into contact with all surfaces of the hand; and • hands should be rubbed together vigorously, paying particular attention to the tips of the à ngers, the thumbs and the areas between the à ngers, until the solution has evaporated and the hands are dry. clinical staff should be made aware of the potentially damaging effects of hand decontamination products, and encouraged to use an emollient hand cream regularly to maintain the integrity of the skin. consult the occupational health team or a general practitioner if a particular liquid soap, antiseptic handwash or alcohol-based hand rub causes skin irritation. alcohol-based hand rub should be made available at the point of care in all healthcare facilities. hand hygiene resources and healthcare worker adherence to hand hygiene guidelines should be audited at regular intervals, and the results should be fed back to healthcare workers to improve and sustain high levels of compliance. healthcare organisations must provide regular training in risk assessment, effective hand hygiene and glove use for all healthcare workers. local programmes of education, social marketing, and audit and feedback should be refreshed regularly and promoted by senior managers and clinicians to maintain focus, engage staff and produce sustainable levels of compliance. patients and relatives should be provided with information about the need for hand hygiene and how to keep their own hands clean. patients should be offered the opportunity to clean their hands before meals; after using the toilet, commode or bedpan/urinal; and at other times as appropriate. products available should be tailored to patient needs and may include alcohol-based hand rub, hand wipes and access to handwash basins. selection of personal protective equipment must be based on an assessment of the: • risk of transmission of microorganisms to the patient or carer; • risk of contamination of healthcare practitioners' clothing and skin by patients' blood or body á uids; and • suitability of the equipment for proposed use. healthcare workers should be educated and their competence assessed in the: • assessment of risk; • selection and use of personal protective equipment; and • use of standard precautions. supplies of personal protective equipment should be made available wherever care is delivered and risk assessment indicates a requirement. gloves must be worn for: • invasive procedures; • contact with sterile sites and nonintact skin or mucous membranes; • all activities that have been assessed as carrying a risk of exposure to blood or body á uids; and • when handling sharps or contaminated devices. gloves must be: • worn as single-use items; • put on immediately before an episode of patient contact or treatment; • removed as soon as the episode is completed; • changed between caring for different patients; and • disposed of into the appropriate waste stream in accordance with local policies for waste management. hands must be decontaminated immediately after gloves have been removed. a range of ce-marked medical and protective gloves that are acceptable to healthcare personnel and suitable for the task must be available in all clinical areas. sensitivity to natural rubber latex in patients, carers and healthcare workers must be documented, and alternatives to natural rubber latex gloves must be available. disposable plastic aprons must be worn when close contact with the patient, materials or equipment pose a risk that clothing may become contaminated with pathogenic microorganisms, blood or body á uids. full-body á uid-repellent gowns must be worn where there is a risk of extensive splashing of blood or body á uids on to the skin or clothing of healthcare workers. plastic aprons/á uid-repellent gowns should be worn as single-use items for one procedure or episode of patient care, and disposed of into the appropriate waste stream in accordance with local policies for waste management. when used, nondisposable protective clothing should be sent for laundering. sp29 fluid-repellent surgical face masks and eye protection must be worn where there is a risk of blood or body á uids splashing into the face and eyes. appropriate respiratory protective equipment should be selected according to a risk assessment that takes account of the infective microorganism, the anticipated activity and the duration of exposure. respiratory protective equipment must à t the user correctly and they must be trained in how to use and adjust it in accordance with health and safety regulations. personal protective equipment should be removed in the following sequence to minimise the risk of cross/self-contamination: • gloves; • apron; • eye protection (when worn); and • mask/respirator (when worn). hands must be decontaminated following the removal of personal protective equipment. sharps must not be passed directly from hand to hand, and handling should be kept to a minimum. sp34 needles must not be recapped, bent or disassembled after use. used sharps must be discarded at the point of use by the person generating the waste. all sharps containers must: • conform to current national and international standards; • be positioned safely, away from public areas and out of the reach of children, and at a height that enables safe disposal by all members of staff; • be secured to avoid spillage; • be temporarily closed when not in use; • not be à lled above the à ll line; and • be disposed of when the à ll line is reached. all clinical and non-clinical staff must be educated about the safe use and disposal of sharps and the action to be taken in the event of an injury. sp38 use safer sharps devices where assessment indicates that they will provide safe systems of working for healthcare workers. organisations should involve end-users in evaluating safer sharps devices to determine their effectiveness, acceptability to practitioners, impact on patient care and cost benefi t prior to widespread introduction. organisations should provide education to ensure that healthcare workers are trained and competent in performing the aseptic technique. the aseptic technique should be used for any procedure that breaches the body's natural defences, including: • insertion and maintenance of invasive devices; • infusion of sterile fl uids and medication; and • care of wounds and surgical incisions. this guidance is based on the best critically appraised evidence currently available. the type and class of supporting evidence explicitly linked to each recommendation is described. some recommendations from the previous guidelines have been revised to improve clarity; where a new recommen dation has been made, this is indicated in the text. these recommendations are not detailed procedural protocols, and need to be incorporated into local guidelines. none are regarded as optional. these guidelines apply to adults and children aged ≥1 year who require a short-term indwelling urethral catheter (≤28 days), and should be read in conjunction with the guidance on standard principles. the recommendations are divided into six distinct interventions: • assessing the need for catheterisation; • selection of catheter type and system; • catheter insertion; • catheter maintenance; • education of patients, relatives and healthcare workers; and • system interventions for reducing the risk of infection. only use a short-term indwelling urethral catheter in patients for whom it is clinically indicated, following assessment of alternative methods and discussion with the patient. class d/gpp uc2 document the clinical indication(s) for catheterisation, date of insertion, expected duration, type of catheter and drainage system, and planned date of removal. uc3 assess and record the reasons for catheterisation every day. remove the catheter when no longer clinically indicated. assess patient's needs prior to catheterisation in terms of: • latex allergy; • length of catheter (standard, female, paediatric); • type of sterile drainage bag and sampling port (urometer, 2-l bag, leg bag) or catheter valve; and • comfort and dignity. select a catheter that minimises urethral trauma, irritation and patient discomfort, and is appropriate for the anticipated duration of catheterisation. uc6 select the smallest gauge catheter that will allow urinary outfl ow and use a 10-ml retention balloon in adults (follow manufacturer's instructions for paediatric catheters). urological patients may require larger gauge sizes and balloons. uc22 ensure patients, relatives and carers are given information regarding the reason for the catheter and the plan for review and removal. if discharged with a catheter, the patient should be given written information and shown how to: • manage the catheter and drainage system; • minimise the risk of urinary tract infection; and • obtain additional supplies suitable for individual needs. uc23 use quality improvement systems to support the appropriate use and management of short-term urethral catheters and ensure their timely removal. these may include: • protocols for catheter insertion; • use of bladder ultrasound scanners to assess and manage urinary retention; • reminders to review the continuing use or prompt the removal of catheters; • audit and feedback of compliance with practice guidelines; and • continuing professional education this guidance is based on the best critically appraised evidence currently available. the type and class of supporting evidence explicitly linked to each recommendation is described. some recommendations from the previous guidelines have been revised to improve clarity; where a new recommendation has been made, this is indicated in the text. these recommendations are not detailed procedural protocols, and need to be incorporated into local guidelines. none are regarded as optional. ivad1 healthcare workers caring for patients with intravascular catheters should be trained and assessed as competent in using and consistently adhering to practices for the prevention of catheter-related bloodstream infection. ivad2 healthcare workers should be aware of the manufacturer's advice relating to individual catheters, connection and administration set dwell time, and compatibility with antiseptics and other á uids to ensure the safe use of devices. ivad3 before discharge from hospital, patients with intravascular catheters and their carers should be taught any techniques they may need to use to prevent infection and manage their device. ivad4 hands must be decontaminated, with an alcohol-based hand rub or by washing with liquid soap and water if soiled or potentially contaminated with blood or body á uids, before and after any contact with the intravascular catheter or insertion site. ivad5 use the aseptic technique for the insertion and care of an intravascular access device and when administering intravenous medication. ivad6 use a catheter with the minimum number of ports or lumens essential for management of the patient. ivad7 preferably use a designated singlelumen catheter to administer lipidcontaining parenteral nutrition or other lipid-based solutions. ivad8 use a tunnelled or implanted central venous access device with a subcutaneous port for patients in whom long-term vascular access is required. ivad9 use a peripherally inserted central catheter for patients in whom mediumterm intermittent access is required. ivad10 use an antimicrobial-impregnated central venous access device for adult patients whose central venous catheter is expected to remain in place for >5 days if catheter-related bloodstream infection rates remain above the locally agreed benchmark, despite the implementation of a comprehensive strategy to reduce catheter-related bloodstream infection. ivad11 in selecting an appropriate intravascular insertion site, assess the risks for infection against the risks of mechanical complications and patient comfort. ivad12 use the upper extremity for nontunnelled catheter placement unless medically contraindicated. ivad13 use maximal sterile barrier precautions for the insertion of central venous access devices. ivad36 when safer sharps devices are used, healthcare workers should ensure that all components of the system are compatible and secured to minimise leaks and breaks in the system. ivad37 administration sets in continuous use do not need to be replaced more frequently than every 96 h, unless device-specià c recommendations from the manufacturer indicate otherwise, they become disconnected or the intravascular access device is replaced. ivad38 administration sets for blood and blood components should be changed when the transfusion episode is complete or every 12 h (whichever is sooner). ivad39 administration sets used for lipidcontaining parenteral nutrition should be changed every 24 h. ivad40 use quality improvement interventions to support the appropriate use and management of intravascular access devices (central and peripheral venous catheters) and ensure their timely removal. these may include: • protocols for device insertion and maintenance; • reminders to review the continuing use or prompt the removal of intravascular devices; • audit and feedback of compliance with practice guidelines; and • continuing professional education. these are systematically developed broad statements (principles) of good practice. they are driven by practice need, based on evidence and subject to multi-professional debate, timely and frequent review, and modià cation. national guidelines are intended to inform the development of detailed operational protocols at local level, and can be used to ensure that these incorporate the most important principles for preventing hcai in the nhs and other acute healthcare settings. during the past two decades, hcai have become a signià cant threat to patient safety. the technological advances made in the treatment of many diseases and disorders are often undermined by the transmission of infections within healthcare settings, particularly those caused by antimicrobial-resistant strains of disease-causing microorganisms that are now endemic in many healthcare environments. the à nancial and personal costs of these infections, in terms of the economic consequences to the nhs and the physical, social and psychological costs to patients and their relatives, have increased both government and public awareness of the risks associated with healthcare interventions, especially the risk of acquiring a new infection. many, although not all, hcai can be prevented. clinical effectiveness (i.e. using prevention measures that are based on reliable evidence of efà cacy) is a core component of an effective strategy designed to protect patients from the risk of infection, and when combined with quality improvement methods can account for signià cant reductions in hcai such as meticillin-resistant staphylococcus aureus (mrsa) and clostridium difà cile. these guidelines describe clinically effective measures that are used by healthcare workers for preventing infections in hospital and other acute healthcare settings. three sets of guidelines were developed originally and have now been updated. they include: • standard infection control principles: including best practice recommendations for hospital environmental hygiene, effective hand hygiene, the appropriate use of ppe, the safe use and disposal of sharps, and the principles of asepsis; • guidelines for preventing infections associated with the use of short-term indwelling urethral catheters; and • guidelines for preventing infections associated with the use of intravascular access devices. the evidence for these guidelines was identià ed by multiple systematic reviews of peer-reviewed research. in addition, evidence from expert opinion as reá ected in systematically identià ed professional, national and international guidelines was considered following formal assessment using a validated appraisal tool. 3 all evidence was critically appraised for its methodological rigour and clinical practice applicability, and the best-available evidence iná uenced the guideline recommendations. a team of specialist infection prevention and control researchers and clinical specialists and a guideline development advisory group, comprising lay members and specialist clinical practitioners, developed the epic3 guidelines (see sections 1.1 and 1.2). these guidelines can be appropriately adapted and used by all hospital practitioners. this will inform the development of more detailed local protocols and ensure that important standard principles for infection prevention are incorporated. consequently, they are aimed at hospital managers, members of hospital infection prevention and control teams, and individual healthcare practitioners. at an individual level, they are intended to iná uence the quality and clinical effectiveness of infection prevention decision-making. the dissemination of these guidelines will also help patients and carers/relatives to understand the standard infection prevention precautions they can expect all healthcare workers to implement to protect them from hcai. each set of guidelines follows an identical format, which consists of: • a brief introduction; • the intervention heading; • a headline statement describing the key issues being addressed; • a synthesis of the related evidence; and • guideline recommendation(s) classià ed according to the strength of the underpinning evidence. a cardinal feature of evidence-based guidelines is that they are subject to timely review in order that new research evidence and technological advances can be identià ed, appraised and, if shown to be effective for the prevention of hcai, incorporated into amended guidelines. the evidence base for these guidelines will be reviewed in 2 years (2015) and the guidelines will be considered for updating approximately 4 years after publication (2017). following publication the dh will ask the advisory group on antimicrobial resistance and healthcare associated infection to advise whether the s12 h. p. loveday et al. / journal of hospital infection 86s1 (2014) s1-s70 evidence base has progressed signià cantly to alter the guideline recommendations and warrant an update. in addition to informing the development of detailed local operational protocols, these guidelines can be used as a benchmark for determining appropriate infection prevention decisions and, as part of reá ective practice, to assess clinical effectiveness. they also provide a baseline for clinical audit, evaluation and education, and facilitate on-going quality improvements. there are a number of audit tools available locally, nationally and internationally that can be used to audit compliance with guidance including high-impact intervention tools for auditing care bundles. signià cant additional costs are not anticipated in implement ing these guidelines. however, where current equipment or resources do not facilitate the implementation of the guidelines or where staff levels of adherence to current guidance are poor, there may be an associated increase in costs. given the social and economic costs of hcai, the consequences associated with not implementing these guidelines would be unacceptable to both patients and healthcare professionals. the guidelines were developed using a systematic review process (appendix a.1). in each set of guidelines, a summary of the relevant guideline development methodology is provided. electronic databases were searched for national and international guidelines and research studies published during the periods identià ed for each search question. a two-stage search process was used. for each set of epic guidelines, an electronic search was conducted for systematic reviews of randomised controlled trials (rcts) and current national and international guidelines. international and national guidelines were retrieved and subjected to critical appraisal using the agree ii instrument, 3 an evaluation method used internationally for assessing the methodological quality of clinical guidelines. following appraisal, accepted guidelines were included as part of the evidence base supporting guideline development and, where appropriate, for delineating search limits. they were also used to verify professional consensus and, in some instances, as the primary source of evidence. review questions for the systematic reviews of the literature were developed for each set of epic guideline topics following recommendations from scientià c advisors and the guideline development advisory group. searches were constructed using relevant mesh (medical subject headings) and free-text terms. the following databases were searched: • medline; • cumulated index of nursing and allied health literature; • embase; • the cochrane library; and • psycinfo (only searched for hand hygiene). search results were downloaded into a refworks™ database, and titles and abstracts were printed for review. titles and abstracts were assessed independently by two reviewers, and studies were retrieved where the title or abstract: addressed one or more of the review questions; identià ed primary research or systematically conducted secondary research; or indicated a theoretical/clinical/in-use study. where no abstract was available and the title indicated one or more of the above criteria, the study was retrieved. due to the limited resources available for this review, foreign language studies were not identià ed for retrieval. full-text studies were retrieved and read in detail by two experienced reviewers; those meeting the study inclusion criteria were independently quality assessed for inclusion in the systematic review. included studies were appraised using tools based on systems developed by the scottish intercollegiate guideline network (sign) for study quality assessment. 4 studies were appraised independently by two reviewers and data were extracted by one experienced reviewer. any disagreement between reviewers was resolved through discussion. evidence tables were constructed from the quality assessments, and the studies were summarised in adapted considered judgement forms. the evidence was classià ed using methods from sign, and adapted to include interrupted time series design and controlled before-after studies using criteria developed by the cochrane effective practice and organisation of care (epoc) group (table 1) . 4, 5 this system is similar that used in the previous epic guidelines. 2 the evidence tables and considered judgement reports were presented to the guideline development advisory group for discussion. the guidelines were drafted after extensive discussion. factors iná uencing the guideline recommendations included: • the nature of the evidence; • the applicability of the evidence to practice; • patient preference and acceptability; and • costs and knowledge of healthcare systems. the classià cation scheme adopted by sign was used to deà ne the strength of recommendation ( these guidelines have been subject to extensive external consultation with key stakeholders, including royal colleges, professional societies and organisations, patients and trade unions (appendix a.2). comments were requested on: • format; • content; • practice applicability of the guidelines; • patient preference and acceptability; and • specià c sections or recommendations. all the comments were collated and sent to the scientià c advisors and the guideline development advisory group for consideration prior to virtual meetings for discussion and agreement on any changes in the light of comments. final agreement was sought from the scientià c advisors and the guideline development advisory group following revision. high-quality meta-analyses, systematic reviews of rcts or rcts with a very low risk of bias 1+ well-conducted meta-analyses, systematic reviews or rcts with a low risk of bias 1-meta-analyses, systematic reviews or rcts with a high risk of bias* 2++ high-quality systematic reviews of case-control or cohort studies. high-quality case-control or cohort studies with a very low risk of confounding or bias and a high probability that the relationship is causal. interrupted time series with a control group: (i) there is a clearly deà ned point in time when the intervention occurred; and (ii) at least three data points before and three data points after the intervention 2+ well-conducted case-control or cohort studies with a low risk of confounding or bias and a moderate probability that the relationship is causal. controlled before-after studies with two or more intervention and control sites 2-case-control or cohort studies with a high risk of confounding or bias and a signià cant risk that the relationship is not causal. interrupted time series without a parallel control group: (i) there is a clearly deà ned point in time when the intervention occurred; and (ii) at least three data points before and three data points after the intervention. controlled before-after studies with one intervention and one control site 3 non-analytic studies (e.g. uncontrolled before-after studies, case reports, case series) 4 expert opinion. legislation *studies with an evidence level of '1-' and '2-' should not be used as a basis for making a recommendation. rct, randomised controlled trial. this guidance is based on the best critically appraised evidence currently available. the type and class of supporting evidence explicitly linked to each recommendation is described. some recommendations from the previous guidelines have been revised to improve clarity; where a new recommendation has been made, this is indicated in the text. these recommendations are not detailed procedural protocols, and need to be incorporated into local guidelines. none are regarded as optional. standard infection control precautions need to be applied by all healthcare practitioners to the care of all patients (i.e. adults, children and neonates). the recommendations are divided into à ve distinct interventions: • hospital environmental hygiene; • hand hygiene; • use of ppe; • safe use and disposal of sharps; and • principles of asepsis. these guidelines do not address the additional infection control requirements of specialist settings, such as the operating department or outbreak situations. this section discusses the evidence upon which recommendations for hospital environmental hygiene are based. the evidence identià ed in the previous systematic review was used as the basis for updating the searches, and searches were conducted for new evidence published since 2006. 2 hospital environmental hygiene encompasses a wide range of routine activities. guidelines are provided here for: • cleaning the general hospital environment; • cleaning items of shared equipment; and • education and training of staff. current legislation, regulatory frameworks and quality standards emphasise the importance of the healthcare environment and shared clinical equipment being clean and properly decontaminated to minimise the risk of transmission of hcai and to maintain public conà dence. [6] [7] [8] [9] [10] patients and their relatives expect the healthcare environment to be clean and infection hazards to be controlled adequately. 9 the term 'cleaning' is used to describe the physical removal of soil, dirt or dust from surfaces. conventionally, this is achieved in healthcare settings using cloths and mops. dust may be removed using dry dust-control mops/cloths. detergent and water is used for cleaning of soiled or contaminated surfaces, although microà bre cloths and water can also be used for surface cleaning. 9 enhanced cleaning describes the use of methods in addition to standard cleaning specià cations. these may include increased cleaning frequency for all or some surfaces, or the use of additional cleaning equipment. enhanced cleaning may be applied to all areas of the healthcare environment or in specià c circumstances, such as cleaning of rooms or bed spaces following the transfer or discharge of patients who are colonised or infected with a pathogenic microorganism. this is sometimes referred to as 'terminal cleaning'. disinfection is the use of chemical or physical methods to reduce the number of pathogenic microorganisms on surfaces. these methods need to be used in combination with cleaning as they have limited ability to penetrate organic material. the term 'decontamination' is used for the process that results in the removal of hazardous substances (e.g. microorganisms, chemicals) and therefore may apply to cleaning or disinfection. research evidence in this à eld remains largely limited to ecological studies and weak quasi-experimental and observational study designs. there is evidence from outbreak reports and observational research which demonstrates that the hospital environment becomes contaminated with microorganisms responsible for hcai. pathogens may be recovered from a variety of surfaces in clinical environments, including those near to the patient that are touched frequently by healthcare workers. [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] however, no studies have provided high-quality evidence of direct transmission of the same strain of microorganisms found in the environment to those found in colonised or infected patients. we identià ed one prospective cohort study that found a signià cant independent association between acquisition of two multi-drug-resistant pathogens and a prior room occupant with the same organism [multi-drug-resistant pseudomonas aeruginosa odds ratio (or) 2.3, 95% conà dence interval (ci) 1.2-4.3, p=0.012; multi-drug-resistant acinetobacter baumanii or 4.2, 95% ci 1.1-1.3, p=0.04] after adjustment for severity of underlying illness, comorbidities, antimicrobial exposure and some other risk factors. 21 a further study reported an association between mrsa and vancomycin-resistant enterococcus (vre), 22 but conclusions that can be drawn from the à ndings are limited by the retrospective study design and lack of adjustment for severity of underlying illness, colonisation pressure and antibiotic exposure. similarly, another retrospective cohort study found an association between acquisition of c. difà cile and prior room occupant with the same infection; however, this was based solely on clinical diagnosis rather than active surveillance. 23 many microorganisms recovered from the hospital environment do not cause hcai. cleaning will not completely eliminate microorganisms from environmental surfaces, and reductions in their numbers will be transient. 15 there is some evidence that enhanced cleaning regimens are associated with the control of outbreaks of hcai; 24 however, these study designs do not provide robust evidence of cause and effect. enhanced cleaning has been recommended, particularly 'terminal cleaning', after a bed area has been used by a patient colonised or infected with an hcai. we searched for robust evidence from studies conducted in the healthcare environment which demonstrated cleaning interventions that were associated with reductions in both environmental contamination and hcai. a randomised crossover study of daily enhanced cleaning of high-touch surfaces in an intensive care unit (icu) demonstrated a reduction in the daily number of s15 sites in a bed area contaminated with mrsa (or 0.59, 95% ci 0.4-0.86, p=0.006), and the aerobic colony count in communal areas (or 0.65, 95% ci 0.47-0.92, p=0.013). although the reduction in mrsa in the environment was associated with a large reduction in mrsa contaminating doctors' hands (or 0.26, 95% ci 0.07-0.95, p=0.025), there was no effect on the incidence of mrsa acquisition by patients (or 0.98, 95% ci 0.58-1.65, p=0.93). 25 disinfectants have been recommended for cleaning the hospital environment; 1,2 however, a systematic review failed to conà rm a link between disinfection and the prevention of hcai, although contamination of detergent and inadequate disinfection strength could have been an important confounder. 26 whilst subsequent studies may have demonstrated a link between disinfection and reduced environmental contamination, and sometimes the acquisition of hcai, the study designs are weak with no control groups or randomisation of intervention, and/or the introduction of multiple interventions at the same time. this makes it difà cult to draw deà nitive conclusions about the specià c effect of disinfection or cleaning. new technologies for cleaning and decontaminating the healthcare environment have become available over the past 10 years, including hydrogen peroxide, and others are in the early stages of development. whilst hydrogen peroxide has been used for decontamination of selected rooms in a us hospital following use by patients with a multi-drug-resistant organism or c. difà cile, this study found that it was not possible to use hydrogen peroxide routinely for this purpose. 27 the effectiveness, cost-effectiveness and practicality of this and other new technologies in terms of reducing hcai and routine use in the variety of facilities in uk hospitals has yet to be demonstrated. we identià ed three studies conducted in patient care environments that provided evidence for the effectiveness of different products, containing chemical or other disinfection agents, on environmental contamination but not reductions in hcai. a prospective randomised crossover study provided evidence for the effectiveness of daily cleaning of high-touch surfaces with microà bre/copper-impregnated cloths on the reduction of mrsa, as discussed above. 25 an rct demonstrated the efà cacy of daily high-touch surface cleaning with peracetic acid on mrsa and c. difà cile contamination of the environment, with a signià cant reduction in mrsa and c. difà cile isolated from samples taken from surfaces with gloved hands (p<0.001) and the hands of healthcare workers (3/27 in peractic acid group vs 15/38 in standard cleaning group, p=0.13). 28 a nonrandomised controlled trial (nrct) in two wards at a single hospital provided evidence that an additional cleaner was associated with a 32.5% reduction in environmental microbial contamination of hand-touch sites (95% ci 20.2-42.9, p<0.0001) and 26.6% reduction in acquisition of mrsa infection (95% ci 7.7-92.3, p=0.032), although the infection types were not specià ed. 29 hydrogen peroxide has been used as a method of decontamination of the environment in situations where wards/ beds can be closed or left unused for the required period of time [30] [31] [32] we identià ed a prospective, randomised beforeafter study that compared the efà cacy of hypochlorite and a hydrogen peroxide decontamination system for terminal cleaning of rooms used by a patient with c. difà cile infection in reducing environmental contamination with c. difà cile. although both methods reduced environmental contamination signià cantly compared with cleaning alone, hydrogen peroxide achieved a signià cantly greater reduction (91% vs 30% decrease in proportion of samples with c. difà cile, p<0.005). 33 a prospective cohort study provided evidence for the efà cacy of hydrogen peroxide when used for terminal decontamination after standard cleaning in signià cantly reducing the acquisition of multi-drug-resistant organisms in patients subsequently admitted to the rooms (adjusted incidence rate ratio 0.36, 95% ci 0.19-0.7). however, the effect was mainly driven by reduction in acquisition of vre, and the results could have been confounded by the concurrent implementation of chlorhexidine baths, incomplete surveillance data and nonrandom assignment of rooms to the intervention. 34 the efà cacy of antimicrobial surfaces in the clinical environment in reducing surface contamination and hcai is an area of emerging research. four non-randomised, experimental studies, conducted in clinical environments, demonstrated signià cant reductions in microbial burden of between 80% and 90% on high-touch surfaces coated with metallic copper and/or its alloys compared with similar noncopper surfaces. [35] [36] [37] [38] one rct conducted in three icus reported a signià cantly lower acquisition of hcai in patients allocated to rooms with six high-touch copper-coated surfaces (3.4% vs 8.1%, p=0.013). a multi-variate analysis suggested that both severity of underlying illness and room assignment were independently associated with the acquisition of hcai or colonisation. however, these à ndings may have been biased by poor discrimination of patients colonised on admission because of limited surveillance cultures, poor agreement in deà ning cases of hcai, and incomplete adjustment for confounders in the multi-variate analysis. 39 evidence of the effectiveness and cost-effectiveness of these technologies and their contribution to reductions in hcai is therefore not currently available. indicators of cleanliness based on levels of microbial or adenosine triphosphate (atp) contamination have been recommended; however, relationships between atp and aerobic colony counts are not consistent, and neither method distinguishes normal environmental á ora and pathogens responsible for hcai. 40, 41 benchmark values of between 250 and 500 relative light units have been proposed as a more objective measure of assessing the efà cacy of cleaning than visual assessment, although these are based on arbitrary standards of acceptable contamination that have not been shown to be associated with reductions in hcai. [42] [43] [44] we identià ed a number of uncontrolled before-after studies that used atp in various forms to highlight the extent of contamination of the healthcare environment. in addition, some studies described the use of atp monitoring as an intervention to improve cleaning, but the lack of a control group in the study design precluded their inclusion in this review. as cleaning will only have a transient effect on the numbers of microorganisms, regular cleaning or disinfection of hospital surfaces will not guarantee a pathogenfree environment. preventing the transfer of pathogens from the environment to patients therefore still depends on ensuring that hands are decontaminated prior to patient contact. the hospital environment must be visibly clean; free from non-essential items and equipment, dust and dirt; and acceptable to patients, visitors and staff. sp2 levels of cleaning should be increased in cases of infection and/ or colonisation when a suspected or known pathogen can survive in the environment, and environmental contamination may contribute to the spread of infection. the use of disinfectants should be considered for cases of infection and/ or colonisation when a suspected or known pathogen can survive in the environment, and environmental contamination may contribute to the spread of infection. shared clinical equipment used to deliver care in the clinical environment comes into contact with intact skin and is therefore unlikely to introduce infection directly. however, it can act as a vehicle by which microorganisms are transferred between patients, which may subsequently result in infection. equipment should therefore be cleaned and decontaminated after each use with cleaning agents compatible with the piece of equipment being cleaned. in some outbreak situations, the use of chlorine-releasing agents and detergent should be considered. [6] [7] [8] [9] sp4 shared pieces of equipment used in the delivery of patient care must be cleaned and decontaminated after each use with products recommended by the manufacturer. in a systematic review of healthcare workers' knowledge about mrsa and/or frequency of cleaning practices, three studies indicated that staff were not using appropriate cleaning practices with sufà cient frequency to ensure minimisation of mrsa contamination of personal equipment. 13 staff education was lacking on optimal cleaning practices in the clinical areas. the à nding of the review is reinforced by a later observational study, which noted that lapses in adherence to the cleaning protocol were linked with an increase in environmental contamination with isolates of a. baumannii. 15 a second systematic review of four cohort studies that compared the use of detergents and disinfectants on microbial-contaminated hospital environmental surfaces suggested that a lack of effectiveness was, in many instances, due to inadequate strengths of disinfectants, probably resulting from a lack of knowledge. 26 we identià ed no new, robust research studies of education or system interventions for this review. however, creating a culture of responsibility for maintaining a clean environment and increasing knowledge about how to decontaminate equipment and high-touch surfaces effectively requires education and training of both healthcare cleaning professionals and clinical staff. all healthcare workers need to be educated about the importance of maintaining a clean and safe care environment for patients. every healthcare worker needs to know their specià c responsibilities for cleaning and decontaminating the clinical environment and the equipment used in patient care. total number of articles located = 5944 abstract indicates that the article: relates to infections associated with hospital hygiene; is written in english; is primary research, a systematic review or a meta-analysis; and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = 164 full text conà rms that the article: relates to infections associated with hospital hygiene; is written in english; is primary research (randomised controlled trials, prospective cohort, interrupted time series, controlled before-after, quasi-experimental, experimental studies answering specià c questions), a systematic review or a meta-analysis including the above designs; and informs one or more of the review questions. total number of studies selected for appraisal during sift 2 = 26 all articles that described primary research, a systematic review or a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers using sign and epoc criteria. consensus and grading was achieved through discussion. total number of studies accepted after critical appraisal = 12 total number of studies rejected after critical appraisal = 14 this section discusses the evidence for recommendations concerning hand hygiene practice. designing and conducting robust, ethical rcts in the à eld of hand hygiene is challenging, meaning that recommendations are based on evidence from nrcts, quasi-experimental studies, observational studies and laboratory studies with volunteers. in addition, expert opinion derived from systematically retrieved and appraised professional, national and international guidelines is used. the areas discussed in this section include: • assessment of the need to decontaminate hands; • efà cacy of hand decontamination agents and preparations; • rationale for choice of hand decontamination practice; • technique for hand decontamination; • care required to protect hands from the adverse effects of hand decontamination practice; • promoting adherence to hand hygiene guidelines; and • involving patients and carers in hand hygiene. the transfer of organisms between humans can occur directly via hands, or indirectly via an environmental source (e.g. commode or wash basin). epidemiological evidence indicates that hand-mediated transmission is a major contributing factor in the acquisition and spread of infection in hospitals. 1, 2, 45 the hands are colonised by two categories of microbial á ora. the resident á ora are found on the surface, just below the uppermost layer of skin, are adapted to survive in the local conditions and are generally of low pathogenicity, although some, such as stapylococcus epidermidis, may cause infection if transferred on to a susceptible site such as an invasive device. the transient á ora are made up of microorganisms acquired by touching contaminated surfaces such as the environment, patients or other people, and are readily transferred to the next person or object touched. they may include a range of antimicrobial-resistant pathogens such as mrsa, acinetobacter or other multi-resistant gram-negative bacteria. 1 if transferred into susceptible sites such as invasive devices or wounds, these microorganisms can cause life-threatening infections. transmission to non-vulnerable sites may leave a patient colonised with pathogenic and antibiotic-resistant organisms, which may result in an hcai at some point in the future. outbreak reports and observational studies of the dynamics of bacterial hand contamination have demonstrated an association between patient care activities that involve direct patient contact and hand contamination. 2, [45] [46] [47] [48] the association between hand decontamination, using liquid soap and water and waterless alcohol-base hand rub (abhr), and reductions in infection have been conà rmed by clinically-based nonrandomised trials 49, 50 and observational studies. 51, 52 current national and international guidance has consistently identià ed that effective hand decontamination results in signià cant reductions in the carriage of potential pathogens on the hands, and therefore it is logical that the incidence of preventable hcai is decreased, leading to a reduction in patient morbidity and mortality. 2 patients are put at risk of developing an hcai when informal carers or healthcare workers caring for them have contaminated hands. decontamination refers to a process for the physical removal of dirt, blood and body á uids, and the removal or destruction of microorganisms from the hands. 45 the world health organization's (who) 'five moments for hand hygiene' 53 provides a framework for training healthcare workers, audit and feedback of hand hygiene practice, and has been adopted without modià cation in many countries and adapted in others (e.g. canada). 54 hands must be decontaminated at critical points before, during and after patient care activity to prevent crosstransmission of microorganisms. 1, 2, 45, 55 evidence considered by the national institute for health and clinical excellence (nice) 56 indicated increases in hand decontamination compliance before and after patient contact associated with implementation of the who 'five moments' and us centers for disease control and prevention 2002 guidelines, but no difference in compliance after contact with patient surroundings. the following recommendations are derived from the who framework and nice guidelines, 56 and include additional points of emphasis. hands must be decontaminated: • immediately before each episode of direct patient contact or care, including clean/aseptic procedures; • immediately after each episode of direct patient contact or care; • immediately after contact with body á uids, mucous membranes and non-intact skin; • immediately after other activities or contact with objects and equipment in the immediate patient environment that may result in the hands becoming contaminated; and • immediately after the removal of gloves. current national and international guidelines 2,45,53 consider the efà cacy of various preparations for the decontamination of hands using liquid soap and water, antiseptic handwash agents and abhr in laboratory studies and their effectiveness in clinical use. overall, there is no compelling evidence to favour the general use of antiseptic handwashing agents over liquid soap or one antiseptic agent over another. 2, 45, 53, 57 all hand hygiene products for use in clinical care must comply with current british standards. 58 many studies have been conducted during the past 15 years to compare hand hygiene preparations, including abhr and gels, antiseptic handwash and liquid soap. 45 rcts and other quasiexperimental studies have generally demonstrated alcoholbased preparations to be more effective hand hygiene agents than non-medicated soap and antiseptic handwashing agents, although a small number of studies reported no statistically signià cant difference. [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] many of these studies involved the use of abhr as part of a number of interventions, or multimodal campaigns, to improve hand hygiene practice, and had methodological á aws that weaken the causal relationship between the introduction of abhr and reductions in hcai. 77 we identià ed one multi-variate, interrupted time series which suggested that the amount of abhr used per patientday was the only factor associated with a reduction in mrsa incidence density (p=0.011) in a neonatal icu in japan. 78 incidence density fell over a 4-year period from an average of 15 per 1000 patient-days, with a peak of 20 per 1000 patientdays in august 2006, to 0 per 1000 patient-days in october 2008 and was sustained to july 2009 (average incidence density 7.5 per 1000 patient-days). the supporting evidence from laboratory studies of the efà cacy of abhr indicates that these products are highly effective at reducing hand carriage, whilst overcoming some of the recognised barriers to handwashing; most importantly, the ease of use at the point of patient care. these studies underpin a continuing trend to adopt abhr for routine use in clinical practice. however, some studies highlight the need for continued evaluation of the use of abhr within the clinical environment to ensure staff adherence to guidelines and effective hand decontamination technique. 75, 76 choosing the method of hand decontamination will depend upon the assessment of what is appropriate for the episode of care, the availability of resources at or near the point of care, what is practically possible and, to some degree, personal preferences based on the acceptability of preparations or materials. in general, effective handwashing with liquid soap and water or the effective use of abhr will remove transient microorganisms and render the hands socially clean. the effective use of abhr will also substantially reduce resident microorganisms. this level of decontamination is sufà cient for general social contact and most clinical care activities. 2, 45, 53 liquid soap preparations that contain an antiseptic affect both transient microorganisms and resident á ora, and some exert a residual effect. the use of preparations containing an antiseptic is required in situations where prolonged reduction in microbial á ora on the skin is necessary (e.g. surgery, some invasive procedures or in outbreak situations). 2, 45, 53 abhr is not effective against all microorganisms (e.g. some viruses such as norovirus and spore-forming microorganisms such as c. difà cile). it will not remove dirt and organic material, and may not be effective in some outbreak situations. 52, 79 we identià ed two laboratory studies which demonstrated that abhr was not effective in removing c. difà cile spores from hands. 80, 81 in the à rst study, a comparison of liquid soap and water, chlorhexidine gluconate (chg) soap and water, antiseptic hand wipes and abhr resulted in all the soap and water protocols yielding greater mean colony-forming unit (cfu) reductions, followed by the antiseptic hand wipes, than abhr. abhr was equivalent to no intervention (0.06 log 10 cfu/ml, 95% ci -0.34 to 0.45 log 10 cfu/ml). 80 in the second study, three abhr preparations with a minimum 60% alcohol s19 concentration were compared with antiseptic (chg) soap and water. antiseptic soap and water reduced spore counts signià cantly compared with each of the abhrs (chg vs isagel, p=0.005; chg vs endure, p=0.010; chg vs purell, p=0.005). in addition, 30% of the residual spores were readily transferred by handshake following the use of abhr. 81 recent evidence from a laboratory study that compared the efà cacy of liquid soap and water and abhr with and without chg against h1n1 iná uenza virus demonstrated that all the hand hygiene protocols were effective in reducing virus copies. 82 a further study that compared the use of liquid soap and water and 65% ethanol hand sanitisers for the removal of rhinovirus indicated that the hand sanitisers were more effective than soap and water. 83 two economic evaluations from the usa, included in recent nice primary care guidelines, suggest that non-compliance with hand hygiene guidelines results in increased infection-related costs. 56 although compliance increases procurement costs of hand hygiene products, even a small increase in compliance is likely to result in reduced infection costs. we identià ed a further economic analysis of a hand hygiene programme based on the introduction of point-of-use abhr and associated implementation materials. this demonstrated a reduction in episodes of hcai and a saving of $23.7 for every $1 spent on the programme when future costs were considered. sensitivity analyses showed that the programme remained cost saving in all alternative scenarios. 84 abhr is likely to be less costly and result in greater compliance. national and international guidelines suggest that the acceptability of agents and techniques is an essential criterion for the selection of preparations for hand hygiene. 2, 45, 53 acceptability of preparations is dependent upon the ease with which the preparation can be used in terms of time and access, together with their dermatological effects. 58 abhr is preferable for routine use due to its efà cacy, availability at the point of care and acceptability to healthcare workers. 45 however, abhr does not remove organic matter and is ineffective against some microorganisms; therefore, handwashing is required. use an alcohol-based hand rub for decontamination of hands before and after direct patient contact and clinical care, except in the following situations when soap and water must be used: • when hands are visibly soiled or potentially contaminated with body á uids; and • when caring for patients with vomiting or diarrhoeal illness, regardless of whether or not gloves have been worn. investigations of technique for hand decontamination are limited and generally laboratory-based or small-scale observational designs. hand hygiene technique involves both the preparation and the physical process of decontamination. 2, 45, 53 hands and wrists need to be fully exposed to the hand hygiene product and therefore should be free from jewellery and long-sleeved clothing. a number of small-scale observational studies have demonstrated that wearing rings and false nails is associated with increased carriage of microorganisms and, in some cases, linked to the carriage of outbreak strains. department of health guidance on uniforms and work wear and nice guidelines indicate that healthcare workers should remove rings and wrist jewellery, and wear short-sleeved clothing whilst delivering patient care. 56, 85 evidence for the duration of hand decontamination has been considered in previous systematic reviews underpinning guidelines, and suggests that different durations of handwashing and hand rubbing do not signià cantly affect the reduction of bacteria. 59, 76 the who guidelines indicate that decontamination using abhr should take 20-30 s for a seven-step process, and that handwashing should take 40-60 s for a nine-step process. 45 we identià ed one recent rct in a single hospital which demonstrated that allowing staff to decontaminate their hands 'in no particular order' took less time and was as effective as using the who seven-step technique using abhr or liquid antimicrobial soap and water (p=0.04 and p<0.001, respectively). all three of the protocols tested in this study were effective in reducing hand bacterial load (p<0.01). 86 a similar result was reported by authors of a laboratory study that tested the en1500 six-step technique against a range of other protocols. they reported that allowing volunteers to use their own 'responsible application' or a new à ve-step technique resulted in better coverage of the hands during hand decontamination. 87 a number of laboratory-based studies that investigated methods of hand drying suggested that there is no signià cant difference in the efà cacy of different methods of drying hands, but that good-quality paper towels dry hands efà ciently and remove bacteria effectively. 88, 89 current guidance on infection control in the built environment suggests that air and jet driers are not appropriate for use in clinical areas. 90 we identià ed one systematic review of studies on hand drying that failed to meet the quality criteria for inclusion. 91 due to the methodological limitations of studies, evidence recommendations are based on national and international guidelines which state that the duration of hand decontamination, the exposure of all aspects of the hands and wrists to the preparation being used, the use of vigorous rubbing to create friction, thorough rinsing in the case of handwashing, and ensuring that hands are completely dry are key factors in effective hand hygiene and the maintenance of skin integrity. healthcare workers should ensure that their hands can be decontaminated effectively by: • removing all wrist and hand jewellery; • wearing short-sleeved clothing when delivering patient care; • making sure that à ngernails are short, clean, and free from false nails and nail polish; and • covering cuts and abrasions with waterproof dressings. effective handwashing technique involves three stages: preparation, washing and rinsing, and drying. • preparation: wet hands under tepid running water before applying the recommended amount of liquid soap or an antimicrobial preparation. • washing: the handwash solution must come into contact with all of the surfaces of the hand. the hands should be rubbed together vigorously for a minimum of 10-15 s, paying particular attention to the tips of the à ngers, the thumbs and the areas between the à ngers. hands should be rinsed thoroughly. • drying: use good-quality paper towels to dry the hands thoroughly. when decontaminating hands using an alcohol-based hand rub, hands should be free of dirt and organic material and: • hand rub solution must come into contact with all surfaces of the hand; and • hands should be rubbed together vigorously, paying particular attention to the tips of the à ngers, the thumbs and the areas between the à ngers, until the solution has evaporated and the hands are dry. expert opinion suggests that skin damage is generally associated with the detergent base of the preparation and/or poor handwashing technique. 1, 2, 45, 53 in addition, the frequent use of some hand hygiene agents may cause damage to the skin and alter normal hand á ora. sore hands are associated with increased colonisation by potentially pathogenic microorganisms and increase the risk of transmission. 1, 2, 45, 53 the irritant and drying effects of liquid soap and antiseptic soap preparations have been identià ed as one of the reasons why healthcare practitioners fail to adhere to hand hygiene guidelines. 1, 2, 45, 53, 92 in addition, washing hands regularly with liquid soap and water before or after the use of abhr is associated with dermatitis and is not necessary. 45 systematic reviews conducted to underpin national guidelines 1,2,45,53,57 have identià ed a range of studies that compared the use of alcohol-based preparations with liquid soap and water using self-assessment of skin condition by nurses. these studies found that abhr was associated with less skin irritation than liquid soap and water. 60, 61, 64, 67, [93] [94] [95] in addition, a longitudinal study of the introduction and subsequent use of abhr over a 7-year period observed no reports of irritant and contact dermatitis associated with the use of abhr. 51 we identià ed a recent study which suggested that two abhr preparations containing a glycerol emollient were more acceptable to staff (p<0.001). 96 hand moisturisers/ emollients that are for shared use are more likely to become contaminated, and have been associated with an outbreak of infection in a neonatal unit. 97 current national and international guidance suggests that skin care, through the appropriate use of hand lotion or moisturisers added to hand hygiene preparations, is an important factor in maintaining skin integrity, encouraging adherence to hand decontamination practices and assuring the health and safety of healthcare practitioners. 2, 45, 53 clinical staff should be made aware of the potentially damaging effects of hand decontamination products, and encouraged to use an emollient hand cream regularly to maintain the integrity of the skin. consult the occupational health team or a general practitioner if a particular liquid soap, antiseptic handwash or alcohol-based hand rub causes skin irritation. national and international guidelines emphasise the importance of adherence to hand hygiene guidance, and provide an overview of the barriers and factors that iná uence hand hygiene compliance. 1, 2, 45, 53 the use of multi-modal approaches to improving hand hygiene practice and behaviour has been advocated for over 10 years. observational studies have consistently reported an association between multi-modal interventions involving the introduction of near-patient abhr, audit and feedback, reminders and education, and greater compliance by healthcare staff. 51, [98] [99] [100] [101] [102] [103] [104] [105] [106] [107] [108] [109] an early systematic review of 21 studies involving interventions to improve hand hygiene compliance concludes that: • single interventions have a short-term iná uence on hand hygiene; • reminders have a modest but sustained effect; • feedback increases rates of hand hygiene but must be regular; • near-patient alcohol-based preparations improve the frequency with which healthcare workers clean their hands; and • multi-faceted approaches have a more marked effect on hand hygiene and rates of hcai. 110 h. p. loveday et al. / journal of hospital infection 86s1 (2014) s1-s70 national hand hygiene campaigns have been modelled on the multi-modal approach and implemented across the world. 45, 51, 111, 112 in england and wales, the national patient safety agency's 'cleanyourhands campaign' was piloted and implemented between 2004 and 2008 with the aim of creating sustainable change in hand hygiene compliance. the campaign comprised the use of near-patient abhr, national poster materials, audit and feedback, and materials for patient engagement. recent cochrane reviews of randomised and controlled clinical trials, interrupted time series and controlled before-after studies have suggested that the majority of studies conducted in this à eld have methodological biases that exclude them from this review. 77, 113 we identià ed four systematic reviews of interventions to improve hand hygiene compliance. 77, [114] [115] [116] the most recent cochrane review identià ed 84 studies published after 2006 for potential inclusion, but only four studies (one rct, two interrupted time series and one controlled before-after study) were included following detailed quality assessment. 77 the heterogeneity of interventions and methods precluded the pooling and meta-analysis of results, and it was concluded that multi-faceted campaigns that include social marketing or staff engagement may be more effective than campaigns without these components, and that education or product substitution alone were less effective. an integrative systematic review of 35 studies that reported a wide range of interventions, including multi-modal interventions and hand hygiene product changes, only scored nine of the included studies as having limited or no fatal á aws. 114 the authors concluded that design limitations made it difà cult to generalise the study results or isolate the specià c effects of hand hygiene (or other interventions) on reductions in hcai. an earlier systematic review of 'bundled' behavioural intervention studies that reported hcai or rates of colonisation as the primary outcome identià ed 33 potential studies for inclusion; of these, only four had quality scores >80%. again, due to the heterogeneity of study interventions and outcomes, the results were narratively synthesised. 115 the authors concluded that the formation of multi-disciplinary quality improvement teams and educational interventions might be effective strategies to improve hand hygiene and reduce rates of hcai. the à nal systematic review focused specià cally on educational interventions to improve hand hygiene compliance and competence in hospital settings, and included all study designs that reported at least one outcome measure of hand hygiene competence and had a follow-up of at least 6 months. 116 thirty studies met the inclusion criteria for the review, but it was not possible to separate competence from compliance. educational interventions taught or re-taught the correct methods for hand hygiene and then assessed compliance. the authors concluded that educational interventions had a greater impact if compliance with hand hygiene was low. multiple interventions were better than single interventions in sustaining behaviour change, as were continuous, rather than one-off, interventions. however, it was not possible to determine the duration or sustainability of behaviour change in these studies. we identià ed six new studies in our systematic review: one cluster rct and process evaluation, 117,118 one step-wedge cluster rct, 119 two interrupted time series studies 84,120 and one controlled before-after study 121 that evaluated multi-modal interventions with varying components. in a cluster rct that also included a process evaluation, the authors tested a set of core elements in a 'state-of-the-art strategy' (sas) against a team-leader-directed strategy (tds) at baseline (t1), immediately following the intervention (t2) and 6 months later (t3) to ascertain the additional beneà ts of leadership and staff engagement components. 117, 118 in the intention-to-treat analysis (itt), an or of 1.64 (95% ci 1.33-2.02, p<0.001) in favour of the tds between t2 and t3 suggested that engaging ward leadership and the involvement of teams in setting norms and targets resulted in greater compliance with hand hygiene. however, there was no signià cant difference between the groups' compliance at t3 in the itt (p=0.187), with the sas also having a sustained effect. 117 the process evaluation examined the extent to which the content, dosage and coverage of the intervention had been delivered. 118 an as-treated analysis demonstrated a greater effect size for the tds at t3 with a signià cant difference in hand hygiene compliance (p<0.01). the process evaluation also suggested that feedback about individual hand hygiene performance at t2 and t3 (p<0.05 and p<0.01, respectively), challenging colleagues on undesirable hand hygiene practice (p<0.01), and support from colleagues in performing hand hygiene (p<0.01) were positively correlated with changes in nurses' hand hygiene compliance. 118 the second cluster rct used a step-wedge design to assess a behavioural feedback intervention in intensive therapy units (itus) and acute care of the elderly (ace) wards at sites participating in the 'cleanyourhands campaign'. 119 the primary and secondary outcome measures were hand hygiene compliance measured by covert direct observation for 1 h every 6 weeks, and soap and abhr procurement, respectively. sixty wards were recruited, of which 33 implemented the intervention. the itt analysis (60 wards) showed a signià cant effect of the intervention in the itus but not the ace wards, equating to a 7-9% increase in compliance, with estimated or of 1.44 (95% ci 1.018-1.76, p=<0.001) in itus and estimated or of 1.06 (95% ci 0.87-1.25, p=0.5) in ace wards. the perprotocol analysis (33 wards) showed a signià cant increase in compliance in both ace wards and itus of 10-13% and 13-18%, respectively, with estimated or of 1.67 (95% ci 1.26-2.22, p0.001) in ace wards and estimated or of 2.09 (95% ci 1.55-2.81, p0.001) in itus. the authors concluded that individual feedback and team action planning resulted in moderate but sustained improvements in hand hygiene adherence. the difà culties in implementing this intervention point to the problems that might be faced in a non-trial context. two interrupted time series studies of the 4-year national 'cleanyourhands campaign' in england and a 4-year hospitalwide programme in taiwan demonstrated increased hand hygiene compliance (measured by procurement of abhr and liquid soap) and reductions in hcai [mrsa and c. difà cile, and mrsa and extensively-drug-resistant acinetobacter (xdrab)]. 84, 120 in the national study, increased procurement of soap was independently associated with reductions in c. difà cile infection (adjusted incidence rate ratio for 1-ml increase per patient-bed-day 0.993, 95% ci 0.990-0.996, p<0.0001) and mrsa in the last four quarters of the study (adjusted incidence rate ratio for 1-ml increase per patient-bed-day 0.990, 95% ci 0.985-0.995, p<0.0001). 120 the 'cleanyourhands campaign' was not independent of other national programmes to reduce analysis also identià ed that the publication of the health act and the department of health improvement team visits were associated with reductions in mrsa and c.difà cile. in the hospital-wide study, 84 the authors demonstrated a decrease in the cumulative incidence of hcai caused by mrsa (change in level, p=0.03; change in trend, p=0.04) and xdrab (change in level, p=0.78; change in trend, p<0.001) during the intervention period. hand hygiene compliance was signià cantly correlated with increased consumption of abhr, and improved overall from 43.3% in 2004 to 95.6% in 2007 (p<0.001). hand hygiene compliance was also signià cantly correlated with professional categories of healthcare workers (p<0.001) in both general wards and icus (p<0.001). the controlled before-after study 121 of a range of patient safety interventions in england, including hand hygiene, as measured by abhr and soap consumption in non-specialist acute hospitals, reported no signià cant differences in the rate of increase in consumption of abhr (p=0.760 favouring controls and p=0.889 favouring intervention) and non-signià cant decreases in c. difà cile (p=0.652) and mrsa (p=0.693). alcohol-based hand rub should be made available at the point of care in all healthcare facilities. hand hygiene resources and healthcare worker adherence to hand hygiene guidelines should be audited at regular intervals, and the results should be fed back to healthcare workers to improve and sustain high levels of compliance. healthcare organisations must provide regular training in risk assessment, effective hand hygiene and glove use for all healthcare workers. local programmes of education, social marketing, and audit and feedback should be refreshed regularly and promoted by senior managers and clinicians to maintain focus, engage staff and produce sustainable levels of compliance. patient involvement in multi-modal strategies to improve hand hygiene among healthcare workers is established, and includes making it acceptable for patients and carers to request that healthcare workers clean their hands. 45 however, research suggests that many patients and carers do not feel empowered to challenge staff, particularly doctors. 108, 109, 122, 123 many nhs trusts have promoted hand hygiene among visitors by placing abhr at the entrances to wards and patient rooms, but there is no evidence that this reduces hcai. despite being highlighted as an important gap in research, the role of patients' hands in the cross-transmission of microorganisms has not been investigated systematically, other than in ecologic studies that describe hand or skin contamination [124] [125] [126] or observations of non-use of hand hygiene products. 127 studies of effective interventions to enable patients to clean their hands remain small scale and descriptive in nature. [128] [129] [130] [131] we identià ed three studies that described interventions to improve patient hand hygiene: one in an outbreak situation, one uncontrolled before-after study of parent education in a single paediatric icu, and one as part of a prospective observational study in a community hospital. none of these studies met the quality criteria for inclusion in this systematic review. [132] [133] [134] however, all of these studies suggested that improving patient/carer hand hygiene had some effect on crosstransmission of microorganisms and hand hygiene technique. national guidelines indicate that it is important to educate patients and carers about the importance of hand hygiene, and inform them about the availability of hand hygiene facilities and their role in maintaining standards of healthcare workers' hand hygiene. 56 patients and relatives should be provided with information about the need for hand hygiene and how to keep their own hands clean. patients should be offered the opportunity to clean their hands before meals; after using the toilet, commode or bedpan/urinal; and at other times as appropriate. products available should be tailored to patient needs and may include alcohol-based hand rub, hand wipes and access to handwash basins. total number of articles located = 8223 abstract indicates that the article: relates to infections associated with hand hygiene; is written in english; is primary research, a systematic review or a meta-analysis; and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = 255 full text conà rms that the article: relates to infections associated with hand hygiene; is written in english; is primary research (randomised controlled trials, prospective cohort, interrupted time series, controlled before-after, quasi-experimental, experimental studies answering specià c questions), a systematic review or a meta-analysis including the above designs; and informs one or more of the review questions. total number of studies selected for appraisal during sift 2 = 78 all articles that described primary research, a systematic review or a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers using sign and epoc criteria. consensus and grading was achieved through discussion. total number of studies accepted after critical appraisal = 17 total number of studies rejected after critical appraisal = 61 this section discusses the evidence and associated recommendations for the use of ppe by healthcare workers in acute care settings and includes the use of aprons, gowns, gloves, eye protection and face masks/respirators to prevent potential transmission of pathogenic microorganisms to staff, patients and the healthcare environment. the use of gloves for other purposes does not form part of these guidelines. where health and safety legislation underpins a recommendation, this is indicated by 'health & safety (h&s)' in addition to the classià cation of any clinical evidence underpinning the recommendations. the primary roles of ppe are to protect staff and reduce opportunities for cross-transmission of microorganisms in hospitals. 1, 2, 135 there is no evidence that uniforms or work clothing are associated with hcai. however, there is a public expectation that healthcare workers will wear work and protective clothing to minimise any potential risk to patients and themselves. 85, 136 the decision to use or wear ppe must be based upon an assessment of the level of risk associated with a specià c patient care activity or intervention, and take account of current health and safety legislation. [137] [138] [139] [140] there is evidence that both a lack of knowledge of guidelines and non-adherence to guideline recommendations are common, and that regular in-service education and training is required. 106, [141] [142] [143] [144] selection of personal protective equipment must be based on an assessment of the: • risk of transmission of microorganisms to the patient or carer; • risk of contamination of healthcare practitioners' clothing and skin by patients' blood or body á uids; and • suitability of the equipment for proposed use. healthcare workers should be educated and their competence assessed in the: • assessment of risk; • selection and use of personal protective equipment; and • use of standard precautions. supplies of personal protective equipment should be made available wherever care is delivered and risk assessment indicates a requirement. the use of gloves as an element of ppe and contact precautions is an everyday part of clinical practice for healthcare workers. 1, 2, 135 there are other indications unrelated to preventing the cross-transmission of infection that require gloves to be worn (e.g. the use of some chemicals or medications). the two main indications for the use of gloves in the prevention of hcai 1 are: • to protect hands from contamination with organic matter and microorganisms; and • to reduce the risk of cross-transmission of microorganisms to staff and patients. gloves should be selected on the basis of a risk assessment, and should be suitable for the proposed task and the materials being handled. [138] [139] [140] gloves are categorised as medical gloves (examination and surgical) and protective gloves. 145 examination gloves are available as sterile or non-sterile for use by healthcare workers during clinical care to prevent contamination with microorganisms, blood and body á uids. surgical gloves are available as sterile for use by healthcare workers during surgical and other invasive procedures. protective gloves are used to protect healthcare workers from chemical hazards. 145 gloves should not be worn as a substitute for hand hygiene. their prolonged and unnecessary use may cause adverse reactions and skin sensitivity, and may lead to crosscontamination of the patient environment. 1, 2 the need to wear gloves and the selection of appropriate glove materials requires careful assessment of the task to be performed and its related risks to patients and healthcare workers. 1, 2, 135, 146, 147 risk assessment should include consideration of: • who is at risk (patient or healthcare worker) and whether sterile or non-sterile gloves are required; • potential for exposure to blood, body á uids, secretions and excretions; • contact with non-intact skin or mucous membranes during care and invasive procedures; and • healthcare worker and patient sensitivity to glove materials. we identià ed four observational studies which suggested that clinical gloves are not used in line with current guidance, and that glove use impacts negatively on hand hygiene. [148] [149] [150] [151] in addition, a cluster rct of screening and enhanced contact precautions for patients colonised with mrsa or vre found no reduction in transmission, but also found that adherence to contact precautions was less than ideal. 152 gloves must be removed immediately following each care activity for which they have been worn, and hands must be decontaminated in order to prevent the cross-transmission of microorganisms to other susceptible sites in that individual or to other patients. gloves should not be washed or decontaminated with abhr as a substitute for changing gloves between care activities. 45 there is evidence that hands become contaminated when clinical gloves are worn, even when the integrity of the glove appears undamaged. 1, 2, 135 in terms of leakage, gloves made from natural rubber latex (nrl) perform better than vinyl gloves in laboratory test conditions. 1,2 standards for the manufacture of medical gloves for single use require gloves to perform to european standards. [153] [154] [155] [156] [157] however, the integrity of gloves cannot be guaranteed, and hands may become contaminated during the removal of gloves. 1, 2, 149, 156 the appropriate use of medical gloves provides barrier protection and reduces the risk of hand contamination from blood, body á uids, secretions and excretions, but does not eliminate the risk. hands cannot be considered to be clean because gloves have been worn, and should be decontaminated following the removal of gloves. used gloves must be disposed of in accordance with the requirements of current legislation and local policy for waste management. 157 gloves must be worn for: • invasive procedures; • contact with sterile sites and nonintact skin or mucous membranes; • all activities that have been assessed as carrying a risk of exposure to blood or body á uids; and • when handling sharps or contaminated devices. gloves must be: • worn as single-use items; • put on immediately before an episode of patient contact or treatment; • removed as soon as the episode is completed; • changed between caring for different patients; and • disposed of into the appropriate waste stream in accordance with local policies for waste management. hands must be decontaminated immediately after gloves have been removed. clinical gloves should be used by healthcare workers to prevent the risk of hand contamination with blood, body á uids, secretions and excretions, and to protect patients from potential cross-contamination of susceptible body sites or invasive devices. 1 having decided that gloves should be used for a healthcare activity, the healthcare worker must make a choice between the use of: • sterile or non-sterile gloves, based on contact with susceptible sites or clinical devices; and • surgical or examination gloves, based on the aspect of care or treatment to be undertaken. healthcare organisations must provide gloves that conform to european standards (en455-1, 455-2, 455-3), and which are acceptable to healthcare practitioners. [153] [154] [155] medical gloves are available in a range of materials, the most common being nrl, which remains the material of choice due to its efà cacy in protecting against bloodborne viruses and properties that enable the wearer to maintain dexterity. 1, 158 patient or healthcare practitioner sensitivity to nrl proteins must also be taken into account when deciding on glove materials. 146 synthetic gloves are generally more expensive than nrl gloves and may not be suitable for all purposes. 1 nitrile gloves have the same chemical range as nrl gloves and may also lead to sensitivity problems in healthcare workers and patients. polythene gloves are not suitable for clinical use due to their permeability and tendency to damage easily. 1 a study that compared the performance of nitrile, latex, copolymer and vinyl gloves under stressed and unstressed conditions found that nitrile gloves had the lowest failure rate, suggesting that nitrile gloves are a suitable alternative to nrl gloves, provided that there are no sensitivity issues. importantly, the study noted variation in performance of the same type of glove produced by different manufacturers. 158 the health and safety executive (hse) also provide a guide-to-glove selection for employers. 147 a range of ce-marked medical and protective gloves that are acceptable to healthcare personnel and suitable for the task must be available in all clinical areas. sensitivity to natural rubber latex in patients, carers and healthcare workers must be documented, and alternatives to natural rubber latex gloves must be available. national and international guidelines recommend that ppe should be worn by all healthcare workers when close contact with the patient, materials or equipment may lead to contamination of uniforms or other clothing with microorganisms, or when there is a risk of contamination with blood or body á uids. 2, 138, 159 disposable plastic aprons are recommended for general clinical use. 2 full-body gowns need only be used where there is the possibility of extensive splashing of blood or body á uids, and should be á uid repellent. 2, 159 we identià ed a systematic review of the evidence that microbial contaminants found on the work clothing of healthcare practitioners are a signià cant factor in cases of hcai. 136 the reviewers identià ed seven small-scale studies that described the progressive contamination of work clothing during clinical care, and a further three studies that suggested a link with microbial contamination and infection. 17, [160] [161] [162] [163] [164] [165] [166] [167] [168] [169] one of the three studies was conducted in a simulated scenario and demonstrated that it was possible to transfer s. aureus from nurses' gowns to patients' bed sheets, but this was not associated with clinical infection. 167 a further pair of linked studies, associated with an outbreak of bacillus cereus, showed an epidemiological link between contaminated clothing and hcai, but this occurred when surgical scrub suits became highly contaminated in an industrial laundry, rather than as a result of clinical care. 168, 169 a further study demonstrated high levels of contamination of gowns, gloves and stethoscopes with vre following examination of patients known to be infected. 170 a systematic review of eight studies that assessed the effects of gowning by attendants and visitors found no evidence to suggest that over-gowns are effective in reducing mortality, clinical infection or bacterial colonisation in infants admitted to newborn nurseries. 171 one quasi-experimental study investigated the use of gowns and gloves as opposed to gloves alone for prevention of acquisition of vre in a medical icu setting. 172 a further prospective observational study investigated the use of a similar intervention in a medical icu. 173 these studies suggested that the use of gloves and gowns may minimise the transmission of vre when colonisation pressure is high. disposable plastic aprons must be worn when close contact with the patient, materials or equipment pose a risk that clothing may become contaminated with pathogenic microorganisms, blood or body á uids. full-body á uid-repellent gowns must be worn where there is a risk of extensive splashing of blood or body á uids on to the skin or clothing of healthcare workers. plastic aprons/á uid-repellent gowns should worn as single-use items for one procedure or episode of patient care, and disposed of into the appropriate waste stream in accordance with local policies for waste management. when used, non-disposable protective clothing should be sent for laundering. healthcare workers (and sometimes patients) may use standard, á uid-repellent surgical face masks to prevent respiratory droplets from the mouth and nose being expelled into the environment. face masks are also used, often in conjunction with eye protection, to protect the mucous membranes of the wearer from exposure to blood and/or body á uids when splashing may occur. our previous systematic reviews failed to reveal any robust experimental studies that demonstrated that healthcare workers wearing surgical face masks protected patients from hcai during routine ward procedures, such as wound dressing or invasive medical procedures. 1, 2 face masks are also used to protect the wearer from inhaling aerosolised droplet nuclei expelled from the respiratory tract. as surgical face masks are not effective at à ltering out such particles, specialised respiratory protective equipment (respirators) may be recommended for the care of patients with certain respiratory diseases [e.g. active multiple drugresistant pulmonary tuberculosis, 174 severe acute respiratory syndrome (sars) and pandemic iná uenza]. 175 the à ltration efà ciency of these respirators will protect the wearer from inhaling small respiratory particles, but to be effective, they must à t closely to the face to minimise leakage around the mask. 1, 2, 176 the selection of the most appropriate respiratory protective equipment (rpe) should be based on a suitable risk assessment that includes the task being undertaken, the characteristics of the biological agent to which there is a risk of exposure, as well as the duration of the task and the local environment. where the activity involves procedures likely to generate aerosols of biological agents transmitted by an airborne route (e.g. intubation), rpe with an assigned protection factor (apf) of 20 (equivalent to ffp3) should be used. in other circumstances, such as where the agent is transmitted via droplet rather than aerosol or where the level of aerosol exposure is low, the risk assessment may conclude that other forms of rpe (e.g. apf10/ ffp2) or a physical barrier (e.g. surgical face mask) may be appropriate, such as when caring for patients with iná uenza. where rpe is required, it must à t the user properly and the user must be fully trained in how to wear and adjust it. 177 we identià ed four systematic reviews of the use of facial protection, all of which had been undertaken in the aftermath of the sars outbreak and in response to the h1n1 iná uenza pandemic. a range of study designs were considered in each of the reviews, including cluster rcts, rcts, cohort studies and descriptive before-after studies. overall, many studies were poorly controlled, with no accounting for confounders, such as poor compliance in the weaker studies. the authors of each of the reviews concluded that there was no strong evidence that masks/respirators alone are effective for the prevention of respiratory viral infections. masks/respirators should be used together with other protective measures to reduce transmission. [178] [179] [180] [181] our previous systematic review indicated that different protective eyewear offered protection against physical splashing of infected substances into the eyes (although not on all occasions), but that compliance was poor. 1 expert opinion recommends that face and eye protection reduce the risk of occupational exposure of healthcare workers to splashes of blood or body á uids. 1, 2, 159, 182 sp29 fluid-repellent surgical face masks and eye protection must be worn where there is a risk of blood or body á uids splashing into the face and eyes. appropriate respiratory protective equipment should be selected according to a risk assessment that takes account of the infective microorganism, the anticipated activity and the duration of exposure. respiratory protective equipment must à t the user correctly and they must be trained in how to use and adjust it in accordance with health and safety regulations. personal protective equipment should be removed in the following sequence to minimise the risk of cross/self-contamination: • gloves; • apron; • eye protection (when worn); and • mask/respirator (when worn). hands must be decontaminated following the removal of personal protective equipment. total number of articles located = ag (867), fp (8160) abstract indicates that the article: relates to infections associated with protective clothing; is written in english; is primary research, a systematic review or a meta-analysis; and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = ag (32), fp (25) full text conà rms that the article: relates to infections associated with protective clothing; is written in english; is primary research (randomised controlled trials, prospective cohort, interrupted time series, controlled before-after, quasi-experimental), a systematic review or a meta-analysis including the above designs; and informs one or more of the review questions. total number of studies selected for appraisal during sift 2 = ag (6), fp (6) all articles that described primary research, a systematic review or a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers using sign and epoc criteria. consensus and grading was achieved through discussion. total number of studies accepted after critical appraisal = ag (6), fp (4) total number of studies rejected after critical appraisal = ag (0), fp (2) this section discusses the evidence and associated recommendations for the safe use and disposal of sharps in general care settings. this includes minimising the potential infection risks associated with sharps use and disposal, and the use of needle protection devices. the use and disposal of sharps is subject to the health and safety at work act 1974 183 and several elements of health and safety legislation including: 188 where health and safety legislation underpins a recommendation, this is indicated by 'h&s' in addition to the classià cation of any clinical evidence underpinning the recommendations. the hse deà ne a sharp as a needle, blade or other medical instrument capable of cutting or piercing the skin. 184 similarly, a sharps injury is an incident that causes a needle, blade or other medical instrument to penetrate the skin (percutaneous injury). the safe handling and disposal of needles and other sharp instruments forms part of an overall strategy of clinical waste disposal to protect staff, patients and visitors from exposure to bloodborne pathogens. 159 the national audit ofà ce identià ed that needlestick and sharps injuries ranked alongside moving and handling, falls, trips and exposure to hazardous substances as the main types of accidents experienced by nhs staff. 189 a later royal college of nursing survey of 4407 nurses found that almost half (48%) had, at some point in their career, sustained a sharps injury from a device that had previously been used on a patient. a similar number (52%) reported fearing sharps injuries, and nearly half (45%) reported that they had not received training from their employer on safe needle use. 190 the 2012 'eye of the needle' report from the health protection agency conà rms that healthcare workers continue to be exposed to bloodborne virus infections, even though such exposures are largely preventable. 191 the average risk of transmission of bloodborne viruses following a single percutaneous exposure from an infected person, in the absence of appropriate post-exposure prophylaxis, has been estimated to be: 135, 191, 192 • hepatitis b virus, one in three; • hepatitis c virus, one in 30; and • human immunodeà ciency virus, one in 300. national and international guidelines are consistent in their recommendations for the safe use and disposal of sharp instruments and needles, and the management of healthcare workers who are exposed to potential infection from bloodborne viruses. 135, [193] [194] [195] as with many infection prevention and control policies, the assessment and management of the risks associated with the use of sharps is paramount, and safe systems of work and engineering controls must be in place to minimise any identià ed risks. 139 national 184 and european union 196 legislation requires the uk and all eu member states to provide protection for all healthcare workers exposed to the risk of sharps injuries. in summary, the health and safety (sharp instruments in healthcare) regulations 2013 require all employers, under existing health and safety law, to: • conduct risk assessments; • avoid unnecessary use of sharps and, where this is not possible, use safer sharps that incorporate protection mechanisms; • prevent the recapping of needles; • ensure safe disposal by placing secure sharps containers close to the point of use; • provide employees with adequate information and training on the safe use and disposal of sharps, what to do in the event of a sharps injury and the arrangements for testing, immunisation and post-exposure prophylaxis, where appropriate; • record and investigate sharps incidents; and • provide employees who have been injured with access to medical advice, and offer testing, immunisation, post-exposure prophylaxis and counselling, where appropriate. 184, 193 legislation also includes a duty for employees who receive a sharps injury whilst undertaking their work to inform their employer as soon as is practicable. 184, 193 all healthcare workers must be aware of their responsibility in avoiding sharps injuries. we identià ed a systematic review which included studies that focused on education and training interventions to minimise the incidence of occupational injuries involving sharps devices. 197 the authors identià ed à ve primary beforeafter studies that demonstrated a consistent reduction in the incidence of percutaneous injuries when other safety initiatives (e.g. training) were implemented before and during the introduction of safer sharps devices. [198] [199] [200] [201] [202] these studies used a range of interventions in one setting and are not generalisable. however, education is essential in ensuring that staff understand safe ways of working and how to use safer sharps devices. this should form a part of induction programmes for new staff and on-going in-service education. the introduction of new devices should include an appropriate training programme as part of staff introduction. sharps must not be passed directly from hand to hand, and handling should be kept to a minimum. needles must not be recapped, bent or disassembled after use. used sharps must be discarded at the point of use by the person generating the waste. all sharps containers must: • conform to current national and international standards; • be positioned safely, away from public areas and out of the reach of children, and at a height that enables safe disposal by all members of staff; • be secured to avoid spillage; • be temporarily closed when not in use; • not be à lled above the à ll line; and • be disposed of when the à ll line is reached. all clinical and non-clinical staff must be educated about the safe use and disposal of sharps and the action to be taken in the event of an injury. to improve patient and staff safety, legislation and the department of health require healthcare providers and their employees to pursue safer methods of working through risk assessment to eliminate the use of sharps and, where this is not possible, the use of safer sharps. 184, 203, 204 the incidence of sharps injuries has led to the development of safety devices in many different product groups. 205 they are designed to minimise the risk of operator injury during sharps use, as well as 'downstream' injuries that occur after disposal, often involving the housekeeping or portering staff responsible for the collection of sharps disposal units. the lack of well-designed, controlled intervention studies means that evidence to show whether or not safety devices are effective in reducing rates of infection is limited. however, a small number of studies have shown signià cant reductions in injuries associated with the use of safety devices 2 in cannulation, 206,207 phlebotomy 208-210 and injections. 211 it is logical that where needle-free or other safety devices are used, there is a resulting reduction in sharps injuries. a review of needlestick injuries in scotland suggested that 56% of injuries would 'probably' or 'deà nitely' have been prevented if a safety device had been used. 212 however, some studies have identià ed a range of barriers to the expected reduction in injuries, including staff resistance to using new devices, complexity of device operation or improper use, and poor training. 2 a comprehensive report and product review s29 conducted in the usa provides background information and guidance on the need for and use of needlestick-prevention devices, but also gives advice on establishing and evaluating a sharps injury prevention programme. 205 it reported that all devices have limitations in relation to cost, applicability and/or effectiveness. some of the devices available are more expensive than standard devices, may not be compatible with existing equipment, and may be associated with an increase in bloodstream infection rates if used incorrectly. 213 nice identià ed three rcts that compared safety cannulae with standard cannulae. the studies were all in hospital settings and of low/very-low quality. the quality of evidence for safety needle devices was low, with no rcts identià ed and the à ve before-after implementation studies being of very-low quality. the quality of evidence for training was similarly low, with the type of training varying across the à ve observational studies identià ed. 56 we identià ed a systematic review undertaken by the hse which reviewed 41 studies that provided evidence for reductions in the incidence of occupational sharps injuries associated with use of sharps safety devices, education and training, and the acceptability of sharps safety devices. 197 thirteen studies, predominantly with observational designs, demonstrated that safer sharps devices were associated with a signià cant reduction in the incidence of healthcare worker needlestick injury. 199, 201, [208] [209] [210] [214] [215] [216] [217] [218] [219] [220] [221] however, safety devices were not the total solution to reducing occupational injury. the beneà cial outcome of consulting with end-users of safer sharps devices before they are introduced was demonstrated in à ve studies identià ed in this review. 208, 211, [222] [223] [224] [225] in the usa, the occupational safety health administration and the national institute for occupational safety and health suggest that a thorough evaluation of any device is essential before purchasing decisions are made. 195, 226 similarly, the hse suggests that the end-users of any safer sharps device should be involved in the assessment of user acceptability and clinical applicability of any needle safety devices. 184 the evaluation should ensure that the safety feature works effectively and reliably, that the device is acceptable to healthcare practitioners and that it does not have an adverse effect on patient care. use safer sharps devices where assessment indicates that they will provide safe systems of working for healthcare workers. organisations should involve end-users in evaluating safer sharps devices to determine their effectiveness, acceptability to practitioners, impact on patient care and cost beneà t prior to widespread introduction. systematic review questions total number of articles located = 3989 abstract indicates that the article: relates to infections associated with sharps; is written in english, is primary research, a systematic review or a meta-analysis; and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = 18 full text conà rms that the article: relates to infections associated with sharps; is written in english; is primary research (randomised controlled trials, prospective cohort, interrupted time series, controlled before-after, quasi-experimental), a systematic review or a meta-analysis including the above designs; and informs one or more of the review questions. total number of studies selected for appraisal during sift 2 = 2 all articles that described primary research, a systematic review or a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers using sign and epoc criteria. consensus and grading was achieved through discussion. the term 'asepsis' means the absence of potentially pathogenic microorganisms. 227 asepsis applies to both medical and surgical procedures. medical asepsis aims to minimise the risk of contamination by microorganisms, and prevent their transmission by applying standard principles of infection prevention, including decontaminating hands, use of ppe, maintaining an aseptic area, and not touching susceptible sites or the surface of invasive devices. 56 surgical asepsis is a more complex process, including procedures to eliminate microorganisms from an area (thus creating an aseptic environment), and is practised in operating theatres and for invasive procedures, such as the insertion of a central venous catheter (cvc). 56 'aseptic technique' is a term applied to a set of specifi c practices and procedures used to assure asepsis and prevent the transfer of potentially pathogenic microorganisms to a susceptible site on the body (e.g. an open wound or insertion site for an invasive medical device) or to sterile equipment/devices. it involves ensuring that susceptible body sites and the sterile parts of devices in contact with a susceptible site are not contaminated during the procedure. the aseptic technique is an essential element of the prevention of hcai, particularly when the body's natural defence mechanisms are compromised. however, similar to nice, 56 we identifi ed no clinical or economic evidence that any one approach to the aseptic technique is more clinically or cost-effective than another. thus, all recommendations here are class d/gpp. no studies that met the inclusion criteria and compared education interventions for improving the aseptic technique generally were identifi ed. we identifi ed one systematic review that assessed education interventions to improve competence in the aseptic insertion and maintenance of cvcs. 228 the review included 47 studies of educational interventions that were designed to change staff behaviour related to: general asepsis, maximal sterile barrier (msb) precautions during insertion, cutaneous antisepsis, and other aspects of insertion and maintenance practice. the studies all described multi-modal education approaches alone or combined with demonstration, simulation, video and self-study. only one of these studies reported improvements in competence with performing the aseptic technique as a discrete outcome, and nine studies measured overall compliance with the total insertion bundle. variations in terminology are used in the literature to describe the aseptic technique. inconsistencies in the use of terms and application of the principles of asepsis in clinical practice have been addressed in a framework referred to as 'aseptic non-touch technique'. 229 this provides a practice structure and educational materials aimed at minimising variation and developing competence in practice. 229 however, no comparative evidence indicating the effi cacy of this approach was identifi ed. organisations should provide education to ensure that healthcare workers are trained and competent in performing the aseptic technique. the aseptic technique should be used for any procedure that breaches the body's natural defences, including the: • insertion and maintenance of invasive devices; • infusion of sterile fl uids and medication; and • care of wounds and surgical incisions. abstract indicates that the article: relates to infections associated with asepsis; is written in english; is primary research, a systematic review or a meta-analysis; and appears to inform one or more of the review questions. full text conà rms that the article: relates to infections associated with asepsis; is written in english; is primary research (randomised controlled trialsrct), prospective cohort, interrupted time series, controlled before-after, quasi-experimental), a systematic review or a meta-analysis including the above designs; and informs one or more of the review questions. total number of studies selected for appraisal during sift 2 = 1 all articles that described primary research, a systematic review or a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers using sign and epoc criteria. consensus and grading was achieved through discussion. total number of studies accepted after critical appraisal = 0 total number of studies rejected after critical appraisal = 1 this guidance is based on the best critically appraised evidence currently available. the type and class of supporting evidence explicitly linked to each recommendation is described. evidence identià ed in the healthcare infection control practices advisory committee (hicpac) systematic review was used to support the recommendations in these guidelines. 230 some recommendations from the previous guidelines have been revised to improve clarity; where a new recommendation has been made, this is indicated in the text. these recommendations are not detailed procedural protocols, and need to be incorporated into local guidelines. none are regarded as optional. these guidelines apply to adults and children aged 1 year who require a short-term indwelling urethral catheter (28 days), and should be read in conjunction with the guidance on standard principles. the recommendations are divided into six distinct interventions: • assessing the need for catheterisation; • selection of catheter type and system; • catheter insertion; • catheter maintenance; • education of patients, relatives and healthcare workers; and • system interventions for reducing the risk of infection. urinary tract infection (uti) is the most common infection acquired as a result of health care, accounting for 19% of hcai, with between 43% and 56% of utis associated with a urethral catheter. 231, 232 catheters predispose to infection because microorganisms are able to bypass natural host mechanisms, such as the urethra and micturition, and gain entry to the bladder. 233 most microorganisms causing catheterassociated uti (cauti) gain access to the urinary tract either extraluminally or intraluminally. extraluminal contamination may occur as the catheter is inserted, by contamination of the catheter from healthcare workers' hands or from the patient's own perineal á ora. extraluminal contamination is also thought to occur from microorganisms ascending from the perineum. intraluminal contamination occurs by reá ux of microorganisms from a contaminated urine drainage bag. 234 the bladder is normally sterile; in the non-catheterised patient, a uti is usually identià ed from the symptoms of dysuria and frequency of urination. patients who develop a uti with a short-term indwelling urethral catheter in place may not experience these symptoms, and diagnosis may be based on other signs, such as fever or suprapubic or loin tenderness. 235 after a few days of catheterisation, microorganisms may be isolated from urine and, in the absence of any symptoms of uti, this is called 'bacteriuria'. 236 the duration of catheterisation is the dominant risk factor for cauti, and virtually all catheterised patients develop bacteriuria within 1 month. 230 for the purpose of these guidelines, a duration of catheterisation of 28 days is considered to be a short-term catheterisation. 234 several factors contribute to the potential development of cauti, including the formation of bioà lms and encrustation of the catheter. bacteria on the catheter surface and drainage bag multiply rapidly, adhering to the surface by excreting extracellular polysaccharides and forming a layer known as a 'bioà lm'. bacteria within the bioà lm are morphologically and physiologically different from free-living planktonic bacteria in the urine, and have considerable survival advantages as they are protected from the action of antibiotic therapy. 234 whilst bioà lms commonly form on devices inserted into the body, they can cause additional problems on urethral catheters if the bacteria produce the enzyme urease, such as proteus mirabilis. 234 this enzyme causes the urine to become alkaline, inducing crystallisation of calcium and magnesium phosphate within the urine. these crystals are incorporated into the bioà lm and, over time, result in encrustation of the catheter. encrustation is generally associated with long-term catheterisation, as it has a direct relationship with the length of catheterisation. 234 urinary catheterisation is a frequent intervention during clinical care in hospital, affecting a signià cant number of patients. it has been estimated that 15-25% of hospitalised patients have a urinary catheter inserted during their stay. [237] [238] [239] [240] this number is much higher in icus. 241 the risk of infection is associated with the method and duration of catheterisation, the quality of catheter care and patient susceptibility. 242 bacteriuria develops in approximately 30% of catheterised patients after 2-10 days, and 24% (95% ci 23-29%) of these will develop symptoms of cauti. 242 approximately 3.6% (95% ci 3.4-3.8%) of those with cauti develop life-threatening secondary infections, such as bacteraemia or sepsis, where mortality rates range from 10% to 33%. 243, 244 cauti is associated with prolonged hospitalisation, re-admission and increased mortality. 245 patients at particular risk are those who are immunocompromised, the elderly and patients with diabetes. 246 physical and psychological discomfort associated with insertion, removal and the catheter in situ are common. 247 complications such as iná ammation, urethral strictures, mechanical trauma, bladder calculi and other infections of the renal system also occur. 237, [248] [249] [250] urine retention after catheter removal is also a frequent occurrence. 251 in some instances, especially in older people, cauti may contribute to falls and delirium. 252 the treatment of both cauti and other infection sequelae contribute to the emerging problem of antibiotic resistance in hospitals, and uropathogens are a major source of infections caused by antimicrobial-resistant organisms. 253 cauti also increases the cost of health care due to delayed discharge from hospital, antimicrobial treatment and staff resources. the à nancial burden of cauti on the nhs has been estimated as £99 million per year, with an estimated cost per episode of £1968. 254, 255 however, there are no robust economic assessments of the cost of cauti. there is a strong association between the duration of catheterisation and the risk of infection (i.e. the longer the catheter is in place, the higher the incidence of uti). 242, 256, 257 in acute care facilities, the risk of developing bacteriuria increases 5% for each day of catheterisation. approximately 24% of bacteriuric patients will develop cauti, and of these, up to 4% develop a severe secondary infection such as bloodstream infection. 242 current best practice emphasises the importance of documenting all procedures involving the catheter or drainage system in the patient's records, 255 and providing patients with adequate information in relation to the need for catheterisation, details of the insertion, catheter and drainage system, maintenance procedures and plan for removal of the catheter. 255, 258 there is some evidence to suggest that computer management systems improve documentation and are associated with reduced duration of catheterisation. 259 using a short-term indwelling urethral catheter only when necessary after considering alternatives and ensuring the catheter is removed as soon as possible are simple and effective methods to prevent cauti. the use of a short-term indwelling urethral catheter may be appropriate in patients with acute urinary retention or obstruction, those who require precise urine output measures to monitor an underlying condition, and patients undergoing certain surgical procedures (especially urological procedures and those of prolonged duration). a short-term indwelling urethral catheter may also be appropriate to minimise discomfort or distress (e.g. during end-of-life care or in the management of open sacral or perineal wounds when the patient is incontinent). 230 however, short-term indwelling urethral catheterisation should not be used as a method of managing urinary incontinence. while the use of a short-term indwelling urethral catheter is sometimes unavoidable, there is evidence that catheters are inserted without a clear clinical indication, clinicians are not always aware they are in situ, and they are not removed promptly when no longer required. 238, 260 interventions that prompt or facilitate the removal of unnecessary catheters may, therefore, reduce the risk of cauti. these interventions have been categorised as reminder systems which prompt clinicians that the catheter is in place and removal should be considered, or stop orders, which indicate that catheters should be removed after a set period of time or when deà ned clinical criteria have been met. 259, [261] [262] [263] a systematic review of 14 studies (one rct, one nrct, three controlled before-after studies and nine uncontrolled beforeafter studies) on reminder and stop order systems found that these interventions signià cantly decreased the rate of cauti and did not increase the need for re-catheterisation, although, as some of the studies were not controlled, they were susceptible to bias in favour of the intervention. 261 a second systematic review identià ed a number of uncontrolled before-after studies that used ultrasound bladder scanners to assess for urinary retention and support appropriate catheterisation. 264 when used in combination with guidelines, 265 insertion checklist/kit, education, audit and feedback, 266 and reminder/stop orders, 267 ultrasound bladder scanners were found to decrease the use of urethral catheters by 5-15%. 264 only use a short-term indwelling urethral catheter in patients for whom it is clinically indicated, following assessment of alternative methods and discussion with the patient. uc2 document the clinical indication(s) for catheterisation, date of insertion, expected duration, type of catheter and drainage system, and planned date of removal. assess and record the reasons for catheterisation every day. remove the catheter when no longer clinically indicated. evidence from best practice indicates that the incidence of cauti in patients catheterised for a short time (up to 1 week) is not iná uenced by any particular type of catheter material. 268, 269 however, many practitioners have strong preferences for one type of catheter over another. this preference is often based on clinical experience, patient assessment and materials that induce the least allergic response. smaller gauge catheters with a 10-ml balloon minimise urethral trauma, mucosal irritation and residual urine in the bladder; all factors that predispose to cauti. 270, 271 there is also a risk of urethral trauma associated with using a female length catheter in a male patient, and systems should be in place to ensure that this does not occur. 272 however, in adults that have recently undergone urological surgery, larger gauge catheters may be indicated to allow for the passage of blood clots. our previous evidence-based guidelines 1 identià ed three experimental studies that compared the use of latex with silicone catheters, which found no signià cant difference in the incidence of bacteriuria. 249, 273, 274 we identià ed one new systematic review which included three trials that compared different types of standard (nonantiseptic-/non-antimicrobial-impregnated) catheters. these studies did not provide sufà cient evidence to suggest that one type of catheter may be more effective than another for the prevention of bacteriuria. [274] [275] [276] [277] in our previous systematic review, 278 we found evidence related to the efà cacy of using short-term indwelling urethral catheters coated or impregnated with antiseptic or antimicrobial agents from four systematic reviews and one meta-analysis. in general, all of these à ve studies suggested that antiseptic-impregnated or antimicrobial-coated shortterm indwelling urethral catheters can signià cantly prevent or delay the onset of cauti compared with standard untreated urinary catheters. 235, [279] [280] [281] [282] the consensus in these à ve reviews of evidence, however, is that the individual studies reviewed are generally of poor quality; for instance, in one case, only eight studies out of 117 met the inclusion criteria, 280 and in another, of the six reports describing seven trials included, only one scored à ve in the quality assessment. the other à ve reports only scored one. 282 the studies included in these reviews investigated a wide range of coated or impregnated catheters, including catheters coated or impregnated with: silver alloy, 279, 280, [282] [283] [284] [285] [286] [287] [288] [289] silver oxide, 280 gendine, 290 gentamicin, 291 silverhydrogel, 292-294 minocycline, 294 rifampicin, 295 chlorhexidinesilver-sulfadiazine, 294 chlorhexideine-sulfadiazine-triclosan, 294 nitrofurazone 294 and nitrofuroxone. 296 four studies compared the use of silver-coated (silver alloy or silver oxide) catheters with silicone, hydrogel or teá on ® latex. 297-300 a systematic review and meta-analysis of these and other studies found that silver-alloy-coated (but not silver-oxide-coated) catheters were associated with a lower incidence of bacteriuria. 256, 280 despite their unit cost, these devices may provide a costeffective option if overall numbers of infections are reduced signià cantly through their use. however, the few studies that have explored the cost-beneà t/cost-effectiveness of using these devices have been inconclusive. 285, 287, 289, 291 we identià ed two new systematic reviews of the efà cacy of silver-coated or antimicrobial-impregnated catheters for the prevention of cauti. 235, 275 the à rst systematic review included 22 rcts, as well as one nrct, and concluded that silver-coated (alloy or oxide) short-term indwelling urethral catheters reduced the risk of bacteriuria but did not demonstrate an effect on cauti. 275 catheters impregnated with antimicrobial agents (minocycline, rifampicin or nitrofurazone) were found to reduce the rate of bacteriuria during the à rst week of catheterisation, but not for catheter durations exceeding 1 week. although antimicrobial-impregnated catheters reduced the risk of cauti, the number of cases was too small to demonstrate a signià cant effect. the second systematic review, which included nine rcts and three quasi-experimental studies, concluded that, compared with standard catheters, both nitrofurazone-impregnated and silver-alloy-coated catheters can prevent and delay the onset of bacteriuria during short-term use. however, there were no data on the risk of cauti. 235 we identià ed one multi-centre rct that compared silveralloy-coated and nitrofurazone-impregnated catheters with standard teá on-coated latex for short-term catheterisation. 301 although the nitrofurazone-impregnated and silver-alloycoated catheters were associated with a reduced risk of cauti compared with the teá on-coated latex, the effect was not considered to be clinically effective (adjusted or 0.81, 95% ci 0.66-1.01 and adjusted or 0.96, 95% ci 0.78-1.19, respectively). the nitrofurazone-impregnated catheter, but not the silver-alloy-coated catheter, was associated with a signià cantly lower incidence of bacteriuria (or 0.68, 95% ci 0.48-0.99, p=0.017). however, the nitrofurazone-impregnated catheter was associated with increased discomfort during the period the catheter was in place. a major limitation of this study was that the median duration of catheterisation was 2 s34 h. p. loveday et al. / journal of hospital infection 86s1 (2014) s1-s70 days (range 1-3 days) and the risk of cauti associated with this short period is correspondingly low. also, utis developing up to 6 weeks post randomisation were included in the outcome measurement, even though they may not have been directly associated with catheterisation. the economic analysis suggested that nitrofurazone-impregnated catheters, but not silver-alloy-coated catheters, may be cost-effective, but the measures of cost were associated with a large amount of uncertainty. overall, the evidence suggests that silver-coated urethral catheters reduce the risk of bacteriuria, but there is insufà cient evidence to indicate whether they reduce the risk of cauti in short-term catheterised patients. assess patient's needs prior to catheterisation in terms of: • latex allergy; • length of catheter (standard, female, paediatric); • type of sterile drainage bag and sampling port (urometer, 2-l bag, leg bag) or catheter valve; and • comfort and dignity. select a catheter that minimises urethral trauma, irritation and patient discomfort, and is appropriate for the anticipated duration of catheterisation. uc6 select the smallest gauge catheter that will allow urinary outá ow and use a 10-ml retention balloon in adults (follow manufacturer's instructions for paediatric catheters). urological patients may require larger gauge sizes and balloons. in our previous review, 2 we found evidence from one systematic review which suggested that the use of the aseptic technique has not demonstrated a reduction in the rate of cauti. 281 however, principles of good practice, clinical guidance 258, 302 and expert opinion, 269, 270, [303] [304] [305] [306] [307] together with à ndings from another systematic review, 256 agree that shortterm indwelling urethral catheters must be inserted using sterile equipment and the aseptic technique. expert opinion indicates that there is no advantage in using antiseptic preparations for cleansing the urethral meatus prior to catheter insertion. 230, 258, 269, 270 whilst there is low-quality evidence to suggest that pre-lubrication of the catheter decreases the risk of bacteriuria, it is also important to use lubricant or anaesthetic gel in order to minimise urethral trauma and discomfort. 230 there is no evidence suggesting a general beneà t of securing the catheter in terms of preventing the risk of cauti, but it is important in order to minimise patient discomfort. 230 ensuring healthcare practitioners are trained and competent in the insertion of short-term indwelling urethral catheters will minimise trauma, discomfort and the potential for cauti. 270, 302, 306, 307 neither we nor hicpac identià ed any additional evidence of acceptable quality whilst updating our systematic review. 230 catheterisation is an aseptic procedure and should only be undertaken by healthcare workers trained and competent in this procedure. uc8 clean the urethral meatus with sterile, normal saline prior to the insertion of the catheter. uc9 use lubricant from a sterile singleuse container to minimise urethral discomfort, trauma and the risk of infection. ensure the catheter is secured comfortably. maintaining a sterile, continuously closed urinary drainage system is central to the prevention of cauti. 258, 270, 302, 306, 308, 309 the risk of infection reduces from 97% with an open system to 8-15% when a sterile closed system is employed. 269, 307, 310 breaches in the closed system, such as unnecessary emptying, changing of the urinary drainage bag or taking a urine sample, will increase the risk of cauti and therefore should be avoided. 269, 302, 311 hands must be decontaminated, and clean and non-sterile gloves should be worn before manipulation of the catheter or the closed system, including drainage taps. a systematic review has suggested that sealed (e.g. taped, pre-sealed) drainage systems contribute to preventing bacteriuria. 281 however, there is limited evidence regarding how often catheter bags should be changed. one study showed that higher rates of symptomatic and asymptomatic cauti were associated with a 3-day urinary drainage bag change regimen compared with no routine change regimen. 312 best practice suggests that drainage bags should only be changed when necessary (i.e. according either to the manufacturer's recommendations or the patient's clinical need). 258, 302 reá ux of urine is associated with infection and, consequently, drainage bags should be positioned in a way that ensures the free á ow of urine and prevents back-á ow. 270, 302 it is also recommended that urinary drainage bags should be hung on an appropriate stand that prevents contact with the á oor. 269 a number of studies have investigated the addition of disinfectants and antimicrobials to drainage bags as a way of preventing cauti. 256 three acceptable studies 313-315 from our original systematic review 1 demonstrated no reduction in the incidence of bacteriuria following the addition of hydrogen peroxide or chlorhexidine to urinary drainage bags. these à ndings are supported by a further systematic review, which suggested that adding bacterial solutions to drainage bags had no effect on catheter-associated infection. 281 neither we nor hicpac identià ed any additional evidence of acceptable quality whilst updating our systematic review. 230 uc10 connect a short-term indwelling urethral catheter to a sterile closed urinary drainage system with a sampling port. uc11 do not break the connection between the catheter and the urinary drainage system unless clinically indicated. uc12 change short-term indwelling urethral catheters and/or drainage bags when clinically indicated and in line with the manufacturer's recommendations. uc13 decontaminate hands and wear a new pair of clean non-sterile gloves before manipulating each patient's catheter. decontaminate hands immediately following the removal of gloves. uc14 use the sampling port and the aseptic technique to obtain a catheter sample of urine. uc15 position the urinary drainage bag below the level of the bladder on a stand that prevents contact with the á oor. uc16 do not allow the urinary drainage bag to à ll beyond three-quarters full. uc17 use a separate, clean container for each patient and avoid contact between the urinary drainage tap and the container when emptying the drainage bag. uc18 do not add antiseptic or antimicrobial solutions to urinary drainage bags. our previous systematic reviews 1,2 found eight acceptable studies that compared meatal cleansing with a variety of antiseptic/antimicrobial agents or soap and water. no reduction in bacteriuria was demonstrated when using any of these preparations for meatal/peri-urethral hygiene compared with routine bathing or showering. 281, [316] [317] [318] [319] [320] [321] [322] expert opinion and other systematic reviews support the view that active meatal cleansing is not necessary and may increase the risk of infection. 56, 230, 256, 258, 269, 270 daily routine bathing or showering is all that is needed in order to maintain patient comfort. neither we nor hicpac identià ed any additional evidence of acceptable quality whilst updating our systematic review. 230 uc19 routine daily personal hygiene is all that is required for meatal cleansing. evidence from our previous systematic review did not demonstrate any beneà cial effect of bladder irrigation, instillation or washout with a variety of antiseptic or antimicrobial agents for the prevention of cauti. 1, 2, [323] [324] [325] [326] [327] [328] [329] [330] [331] evidence from best practice supports these à ndings of no beneà cial effect, and indicates that the introduction of such bladder maintenance solutions may have local toxic effects and contribute to the development of resistant microorganisms. however, continuous or intermittent bladder irrigation may be required for other urological or catheter management indications. 256 given the frequency of urinary catheterisation in hospital patients and the associated risk of uti, it is important that patients, their relatives and healthcare workers responsible for catheter insertion and management are educated about infection prevention. all those involved must be aware of the signs and symptoms of uti and how to access expert help when difà culties arise. healthcare professionals must be conà dent and proà cient in associated procedures. we identià ed two systematic reviews that reported evidence of the efà cacy of healthcare workers' education in reducing the risk of cauti within other system interventions. 264, 332 most of the studies included in these reviews provided lowgrade evidence from uncontrolled before-after studies where a combination of different system interventions focusing on reducing the use of urethral catheters and risk of cauti were introduced. the à rst systematic review 264 identià ed one small controlled before-after study 263 of an educational intervention with guideline change and posters that was associated with a reduction in use of urethral catheters [relative risk (rr) 0.86, 95% ci 0.68-1.10]. another systematic review included one controlled before-after study that demonstrated a signià cant (p<0.01) increase in adherence to a clinical guideline on the insertion and maintenance of urethral catheters in association with an education programme. 332, 333 a further study reported a reduction in cauti and an increase in adherence to protocols for hand hygiene and catheter care in association with an education programme. however, this study did not include a control group. 100 uc21 healthcare workers should be trained and competent in the appropriate use, selection, insertion, maintenance and removal of short-term indwelling urethral catheters. uc22 ensure patients, relatives and carers are given information regarding the reason for the catheter and the plan for review and removal. if discharged with a catheter, the patient should be given written information and shown how to: • manage the catheter and drainage system; • minimise the risk of urinary tract infection; and • obtain additional supplies suitable for individual needs. a number of studies have reported the effect of quality improvement programmes on the risk of cauti. 230 the components of these programmes include various combinations of clinical guidelines for catheter insertion and maintenance, education, audit and feedback of compliance with policy, physician/nurse reminder systems (to prompt removal if no longer necessary), automated or nurse-driven removal protocols [where the catheter is removed after a specià ed period (e.g. 48-72 h) unless countermanded by the physician] and the use of bladder scanners to assess urinary retention and support appropriate catheterisation. 230 we identià ed three systematic reviews relevant to this question. 261, 264, 332 the à rst was a review of interventions to remind physicians/nurses to remove unnecessary catheters and the outcome on cauti, short-term indwelling urethral catheter use and catheter replacement. it included 14 studies (one rct, one nrct, three controlled before-after studies and nine uncontrolled before-after studies). 261 interventions included prewritten or computer-generated stop orders, nurse-generated daily bedside reminders to remove catheters, and daily use of a checklist or protocol to review need for the catheter. some studies also implemented catheter placement restrictions and education. the meta-analysis suggested that the use of reminder or stop order systems reduced the rate of cauti by 52% (p<0.001) and the mean duration of catheterisation by 37%, with 2.61 fewer days of catheterisation in the intervention group compared with the control group, and no difference in re-catheterisation rates. the second systematic review was a review of interventions to minimise the placement of urethral catheters in acute care patients. 264 it included one rct, one nrct and six uncontrolled before-after studies. interventions included various combinations of clinician reminders, stop orders and indication checklists, use of bladder scanners and education. the authors concluded that the studies were too small and heterogeneous to draw a deà nitive conclusion about efà cacy in terms of reducing inappropriate catheter placement. the third systematic review included three controlled before-after studies and seven uncontrolled before-after studies measuring interventions that increased adherence to catheter care protocols or reduced unnecessary catheter use. 332 interventions included reminders, stop orders, use of bladder scanners, education and catheterisation protocols with audit and feedback on performance. physician/nurse reminders, particularly automatic stop orders, were found to reduce the duration of catheterisation, although there were insufà cient data to determine their effect on cauti. many studies in this area are uncontrolled before-after designs and therefore susceptible to bias in favour of the intervention. however, these interventions constitute best practice, and this evidence supports the use of systems to minimise the insertion of catheters and promote timely removal to reduce both the duration of catheterisation and the risk of cauti. s37 uc23 use quality improvement systems to support the appropriate use and management of short-term urethral catheters and ensure their timely removal. these may include: • protocols for catheter insertion; • use of bladder ultrasound scanners to assess and manage urinary retention; • reminders to review the continuing use or prompt the removal of catheters; • audit and feedback of compliance with practice guidelines; and • continuing professional education. uc24 no patient should be discharged or transferred with a short-term indwelling urethral catheter without a plan documenting the: • reason for the catheter; • clinical indications for continuing catheterisation; and • date for removal or review by an appropriate clinician overseeing their care. systematic review questions 1. what are the clinical indications for the use of short-term urinary catheters?(*b) 2. what is the risk associated with short-term catheterisation in terms of bacteriuria, cauti, other morbidities and mortality? (b) 3. what is the effectiveness (in terms of patient acceptability and reduced risk of bacteriuria, cauti, other morbidities and mortality) and the cost-effectiveness of different types of short-term indwelling urinary catheters (material, coatings and design)? 4. what is the most effective catheter insertion technique in terms of patient acceptability and minimisation of urethral trauma, bacteriuria, cauti and other morbidities? 5. what is the most effective and cost-effective means of maintaining meatal hygiene and a closed drainage system? 6. what is the effectiveness of system interventions in reducing the use and duration of short-term urinary catheterisation to minimise the risk of bacteriuria, cauti, other morbidities and mortality? 7. what is the effectiveness of system interventions in improving healthcare workers' knowledge and behaviour relating to the insertion, maintenance and timely removal of indwelling urinary catheters to minimise the risk of bacteriuria, cauti, other morbidities and mortality? total number of articles located = 7073 abstract indicates that the article: relates to infections associated with short-term indwelling urethral catheters; is written in english; is primary research, a systematic review or a meta-analysis; and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = 54 full text conà rms that the article: relates to infections associated with short-term indwelling urethral catheters; is written in english; is primary research (randomised controlled trials, prospective cohort, interrupted time series, controlled before-after, quasiexperimental), a systematic review or a meta-analysis including the above designs; and informs one or more of the review questions. total number of studies selected for appraisal during sift 2 = 16 all articles that described primary research, a systematic review or a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers using sign and epoc criteria. consensus and grading was achieved through discussion. this guidance is based on the best critically appraised evidence currently available. the type and class of supporting evidence explicitly linked to each recommendation is described. evidence identià ed in the hicpac systematic review was used to support the recommendations in these guidelines. 334 some recommendations from the previous guidelines have been revised to improve clarity; where a new recommendation has been made, this is indicated in the text. these recommendations are not detailed procedural protocols, and need to be incorporated into local guidelines. none are regarded as optional. intravascular access devices, including peripheral, central venous and arterial catheters, are commonly used in the management of patients in acute and chronic care settings. cvcs are frequently used during clinical care and include peripherally inserted, non-tunnelled and tunnelled, and totally implantable cvcs (table 3) . 334 the use of any of these catheters can result in bloodstream infection. catheter-related bloodstream infections (cr-bsi) associated with the insertion and maintenance of cvcs are potentially among the most dangerous complications associated with health care. 231, 232, 335 in the most recent national prevalence survey, the health protection agency reported that the prevalence of bsi was 0.5%, accounting for 7.3% of the hcai detected; 64% of bsi occurred in patients with a vascular access device. 231 a previous point prevalence survey reported that the prevalence of bsi was 0.85%, accounting for 7% of the hcai detected; of these, 70% were primary cr-bsi. 232 peripheral venous catheters (pvcs) cause phlebitis in some patients, with studies indicating mean rates of 7-27%, [336] [337] [338] [339] but evidence suggests that these devices are less frequently associated with cr-bsi. 334, [336] [337] [338] [339] [340] cr-bsi involves the presence of systemic infection and evidence implicating the intravascular catheter as its source (i.e. the isolation of the same microorganism from blood cultures as that shown to be signià cantly colonising the intravascular catheter). 334, 335 catheter colonisation refers to the growth of microorganisms on either the endoluminal or the external catheter surface beneath the skin in the absence of systemic infection. 334, 335 the microorganisms that colonise catheter hubs and the skin adjacent to the insertion site are the source of most cr-bsi. coagulase-negative staphylococci, particularly staphylococcus epidermidis, are the microorganisms most frequently implicated in cr-bsi. other microorganisms commonly involved include s. aureus, candida species and enterococci. [341] [342] [343] cr-bsi is generally caused either by skin microorganisms at the insertion site, which contaminate the catheter during insertion and migrate along the cutaneous catheter track after insertion, [344] [345] [346] or microorganisms from the hands of healthcare workers that contaminate and colonise the catheter hub during care interventions. 347 less commonly, infusate contamination or seeding from a different site of infection in the body via the bloodstream is identià ed as a cause of cr-bsi. 348,349 these guidelines are based upon evidence-based guidelines for preventing intravascular device (ivd)-related infections, developed at the us centers for disease control and prevention by hicpac and published in 2011. 334 the agree ii collaboration appraisal instrument was used by four appraisers to review the guidelines independently. 3 the appraisal process resulted in the decision that the guideline development processes were valid and that the guidelines were evidence based, categorised to the strength of the evidence examined, reá ective of current concepts of best practice. the guideline development advisory group considered that they were the most authoritative reference guidelines currently available. following the agree process, we systematically searched, retrieved and appraised additional evidence published since the search period identià ed in the hicpac technical report. 334 our search period for additional evidence spanned from 2009 to 2012. these guidelines apply to caring for all adults and children over the age of 1 year in nhs acute care settings with a cvc or pvc that is being used for the administration of á uids, medications, blood components and/or parenteral nutrition. they should be used in conjunction with the recommendations for standard principles for preventing hcai, previously described in these guidelines. these recommendations describe general principles of best practice that apply to all patients in hospital in whom an intravascular catheter is being used during an acute episode of treatment/care. they do not specià cally address the more detailed, technical aspects of the care of infants under 1 year of age, or those children or adults receiving haemodialysis or chemotherapy who will generally have long-term intravascular catheters managed in renal dialysis or outpatient settings. the recommendations are divided into nine distinct interventions: • education of healthcare workers and patients; • general asepsis; • selection of type of intravascular catheter; • selection of intravascular catheter insertion site; • msb precautions during insertion; • cutaneous antisepsis; • catheter and catheter site care; • replacement strategies; and • general principles for catheter management. to improve patient outcomes and reduce healthcare costs, it is essential that everyone involved in caring for patients with intravascular catheters is educated about infection prevention. healthcare workers in hospitals need to be conà dent and proà cient in infection prevention practices, and to be aware of the signs and symptoms of clinical infection. structured educational programmes that enable healthcare workers to provide, monitor and evaluate care and continually increase their competence are critical to the success of any strategy designed to reduce the risk of infection. evidence reviewed by hicpac demonstrates that the risk of infection declines following standardisation of the aseptic technique, [350] [351] [352] [353] [354] [355] [356] and increases when the maintenance of intravascular catheters is undertaken by inexperienced healthcare workers. 353, 357 h. p. loveday et al. / journal of hospital infection 86s1 (2014) s1-s70 we identià ed two recent systematic reviews that assessed the effectiveness of education interventions in reducing cr-bsi. 228, 358 the à rst concluded that current evidence comes predominantly from uncontrolled before-after studies that do not convincingly distinguish intervention effectiveness from secular trends. clinical practices are addressed by a wide variety of educational strategies that do not draw upon pedagogic, theoretical or conceptual frameworks and consequently do not provide generalisable conclusions about the most effective approaches to education to improve practice. 358 the second systematic review concluded à rst that educational interventions appear to have the most prolonged and profound effect when used in conjunction with audit and feedback, and when availability of clinical equipment is consistent with the content of the education provided. second, that educational interventions will have a greater impact if baseline compliance with best practice is low. third, that repeated educational sessions, fed into daily practice, using practical participation, appear to have a small, additional effect on practice change compared with education alone. 228 healthcare workers should be aware of the manufacturers' advice relating to the compatibility of individual devices with antiseptic solutions, dwell time and connections to ensure safe use. with intravascular catheters should be trained and assessed as competent in using and consistently adhering to practices for the prevention of catheter-related bloodstream infection. ivad2 healthcare workers should be aware of the manufacturer's advice relating to individual catheters, connection and administration set dwell time and compatibility with antiseptics and other á uids to ensure the safe use of devices. ivad3 before discharge from hospital, patients with intravascular catheters and their carers should be taught any techniques they may need to use to prevent infection and manage their device. hand decontamination and meticulous attention to the aseptic technique are essential during catheter insertion, manipulation, changing catheter site dressings and for accessing the system. hands should be decontaminated using abhr or liquid soap and water when hands are visibly soiled or potentially contaminated with organic material, such as blood and other body á uids. 45, 53 the aseptic technique should be used for the insertion and management of ivds. structured education should be provided to ensure that healthcare workers are trained and assessed as competent in performing the aseptic technique. gloves should be worn for procedures involving contact with blood or body á uids. sterile gloves must be worn for the insertion and dressing of cvcs. 334 ivad4 hands must be decontaminated, with an alcohol-based hand rub or by washing with liquid soap and water if soiled or potentially contaminated with blood or body á uids, before and after any contact with the intravascular catheter or insertion site. ivad5 use the aseptic technique for the insertion and care of an intravascular access device and when administering intravenous medication. the selection of the most appropriate intravascular catheter for each individual patient can reduce the risk of subsequent catheter-related infection. intravascular catheter material may be an important determinant in the development of catheter-related infection. polytetraá uroethylene (teá on) and polyurethane catheters have been associated with fewer infections than catheters made of polyvinyl chloride or polyethylene. [359] [360] [361] multi-lumen intravascular access devices may be used because they permit the concurrent administration of á uids and medications, parenteral nutrition and haemodynamic monitoring among critically ill patients. several rcts and other studies suggest that multi-lumen catheters are associated with a higher risk of infection than single-lumen catheters. 334, [362] [363] [364] [365] [366] [367] however, other studies examined by hicpac failed to demonstrate a difference in the rates of cr-bsi. 368, 369 multi-lumen catheter insertion sites may be particularly prone to infection because of increased trauma at the insertion site or because multiple ports increase the frequency of cvc manipulation. 364, 365 patients with multi-lumen catheters tend to be more severely ill, although the increased risk of cr-bsi appears to be independent of underlying illness. 366 a prospective epidemiological study in patients receiving parenteral nutrition concluded that either using a singlelumen catheter or a dedicated port in a multi-lumen catheter for parenteral nutrition would reduce the risk of cr-bsi. 359 neither we nor hicpac identià ed any additional evidence for this recommendation whilst updating our systematic review, and hicpac considered this to be a unresolved issue. 334 in a systematic review and quantitative meta-analysis focused on determining the risk of cr-bsi and catheter colonisation in multi-lumen catheters compared with single-lumen catheters, the reviewers reported that, although cr-bsi was more common in patients with multi-lumen catheters, when conà ned to highquality studies that control for patient differences, there is no signià cant difference in rates of cr-bsi for the two types of catheter. this analysis suggests that multi-lumen catheters are not a signià cant risk factor for increased cr-bsi or local catheter colonisation compared with single-lumen cvcs. 370 a later systematic review and quantitative meta-analysis tested whether single-vs multi-lumen cvcs had an impact on catheter colonisation and cr-bsi. the study authors concluded that there is some evidence from à ve rcts with data on 530 cvcs that for every 20 single-lumen catheters inserted, one cr-bsi (which would have occurred had multi-lumen catheters been used) would be avoided. 371 neither we nor hicpac identià ed any additional evidence of acceptable quality whilst updating our systematic reviews. 334 ivad6 use a catheter with the minimum number of ports or lumens essential for management of the patient. ivad7 preferably use a designated singlelumen catheter to administer lipidcontaining parenteral nutrition or other lipid-based solutions. surgically implanted (tunnelled) devices (e.g. hickman ® catheters) are commonly used to provide vascular access to patients requiring long-term intravenous therapy. alternatively, totally implantable intravascular access devices (e.g. port-a-cath ® ) are also tunnelled under the skin, but have a subcutaneous port or reservoir with a self-sealing septum that is accessible by needle puncture through intact skin. multiple studies comparing the incidence of infection associated with long-term tunnelled cvcs and/or totally implantable ivds with that from percutaneously (non-tunnelled) inserted catheters have been assessed by hicpac. 372 although most studies reported a lower rate of infection in patients with tunnelled cvcs, [373] [374] [375] [376] [377] [378] [379] [380] [381] some studies found no signià cant difference in the rate of infection between tunnelled and nontunnelled catheters. 382, 383 additionally, most studies concluded that totally implantable devices had the lowest reported rates of cr-bsi compared with either tunnelled or non-tunnelled cvcs. [384] [385] [386] [387] [388] [389] [390] [391] [392] [393] [394] however, although these devices are less disruptive for patients in terms of daily living, they have a number of disadvantages including the need for needle insertion resulting in increased discomfort. 395 additional evidence was obtained from studies of efà cacy of tunnelling to reduce catheter-related infections in patients with short-term cvcs. one rct demonstrated that subcutaneous tunnelling of short-term cvcs inserted into the internal jugular vein reduced the risk for cr-bsi. 396 in a later rct, the same investigators failed to show a statistically signià cant difference in the risk for cr-bsi for subcutaneously tunnelled femoral vein catheters. 397 an additional meta-analysis of rcts was focused on the efà cacy of tunnelling short-term cvcs to prevent catheterrelated infections. 398 data synthesis demonstrated that tunnelling decreased catheter colonisation by 39% and decreased cr-bsi by 44% in comparison with non-tunnelled placement. the majority of the beneà t in the decreased rate of catheter sepsis came from one trial of cvcs inserted at the internal jugular site. the reduction in risk was not signià cant when pooled with data from à ve subclavian catheter trials. tunnelling was not associated with increased risk of mechanical complications from placement or technical difà culties during placement. this meta-analysis concluded that tunnelling decreased catheterrelated infections; however, a synthesis of the evidence in this meta-analysis does not support routine subcutaneous tunnelling of short-term subclavian venous catheters, and this cannot be recommended unless efà cacy is evaluated at different placement sites and relative to other interventions. peripherally inserted central catheters (piccs) are increasingly used for medium term (6 weeks to 6 months) 395 intravascular access, particularly in adults and children requiring antimicrobial treatment, chemotherapy and parenteral nutrition. evidence examined by hicpac suggested that piccs are associated with a lower rate of infection than that associated with other non-tunnelled cvcs. 382, 398 retrospective studies in outpatient settings indicate that rates of picc-related bloodstream infection range from 0.4 to 0.8 per 1000 catheter-days. [399] [400] [401] [402] [403] [404] however, there is little recent robust evidence regarding comparison of rates of cr-bsi in piccs vs other long-term central venous access devices. a prospective study 405 that compared the use of inpatient piccs indicated a similar rate of cr-bsi to non-tunnelled catheters placed in the internal jugular or subclavian veins and a higher rate than cuffed and tunnelled (ct) catheters (picc 3.5 cr-bsi per 1000 catheter-days vs nontunnelled 2.7 cr-bsi per 1000 catheter-days vs cuffed and tunnelled 1.6 cr-bsi per 1000 catheter-days). a systematic review of 200 studies indicated that when used in inpatients, piccs pose a slightly lower risk of cr-bsi than standard noncuffed and non-medicated cvcs placed in the subclavian or internal jugular vein (2.1 cr-bsi per 1000 catheter-days vs 2.7 cr-bsi per 1000 catheter-days). 340 neither we nor hicpac identià ed any additional evidence of acceptable quality whilst updating our systematic review. 334 ivad8 use a tunnelled or implanted central venous access device with a subcutaneous port for patients in whom long-term vascular access is required. ivad9 use a peripherally inserted central catheter for patients in whom mediumterm intermittent access is required. some catheters and cuffs are marketed as anti-infective and are coated or impregnated with antimicrobial or antiseptic agents, e.g. chlorhexidine/silver sulfadiazine, minocycline/ rifampicin, platinum/silver, and ionic silver in subcutaneous collagen cuffs attached to cvc. evidence reviewed by hicpac [406] [407] [408] [409] [410] [411] [412] [413] [414] [415] [416] indicated that the use of antimicrobial or antisepticimpregnated cvc in adults whose catheter is expected to remain in place for more than à ve days could decrease the risk for cr-bsi. this may be cost-effective in high-risk patients (intensive care, burn and neutropenic patients) and in other patient populations in whom the rate of cr-bsi exceeds 3.3 per 1,000 catheter days even when there is a comprehensive strategy to reduce rates of cr-bsi. 406 a meta-analysis of 23 rcts published between 1988-1999 included data on 4,660 catheters (2,319 anti-infective and 2,341 control). 417 eleven of the trials in this meta-analysis were conducted in intensive care unit (icu) settings; four among oncology patients, two among surgical patients; two among patients receiving total parenteral nutrition (tpn) and four among other patient populations. study authors concluded that antibiotic and chlorhexidine-silver sulfadiazine coatings are anti-infective for short (approximately one week) insertion time. for longer insertion times, there was no data on antibiotic coating, and there is evidence of lack of effect for à rst generation chlorhexidine-silver sulfadiazine coating. for silver-impregnated collagen cuffs, there is evidence of lack of effect for both short-and long-term insertion. second generation chlorhexidine/silver sulfadiazine catheters with chlorhexidine coating on both the internal and external luminal surfaces are now available. the external surface of these catheters have three times the amount of chlorhexidine and extended release of the surface bound antiseptics than that in the à rst generation catheters (which are coated with chlorhexidine/silver sulfadiazine only on the external luminal surface). early studies indicated that the prolonged anti-infective activity associated with the second generation catheters improved efà cacy in preventing infections. 418 a systematic review and economic evaluation in 2006 concluded that rates of cr-bsi were statistically signià cantly reduced when an antimicrobial cvc was used. studies in this review report the best effect when catheters were treated with minocycline/rifampin, or internally and externally treated with silver or chlorhexidine/silver sulfadiazine. a trend to statistical signià cance was seen in catheters only extraluminally coated. investigation of other antibiotic treated catheters is limited to single studies with non-signià cant results. 419 we identià ed two additional systematic reviews and one rct in our updated search. a recent cochrane review of studies using impregnation, coating or bonding for reducing central venous catheter-related infections in adults included 56, predominantly unblinded studies, with low or unclear risk of bias. patients with impregnated catheters had lower rates of cr-bsi (actual risk reduction of 2% (95% ci, 3% to 1%)), and catheter colonisation (actual risk reduction 10% (95% ci, 13% to 7%)). in terms of catheter colonisation sub-group analysis showed that impregnated catheters were more beneà cial in studies conducted in intensive care units (rr 0.68 (95% ci, 0.59 to 0.78)) than in studies conducted in haemo-oncology (rr 0.75 (95% ci, 0.51 to 1.11)) or in patients requiring long-term parenteral nutrition rr 0.99 (95% ci, 0.74 to 1.34)). however, sub-group analysis did not identify the same beneà t in terms of cr-bsi. there were no statistically signià cant differences in the overall rates of bloodstream infections or mortality, although these outcomes were less often assessed than cr-bsi and catheter colonisation. 420 a collaborative network metaanalysis of cvc use in adults indicated that rifampicin-based impregnated cvc was the only type of impregnated/coated cvc that reduced catheter colonisation and cr-bsi compared with standard cvc. 421 in a single blind non-inferiority trial, authors concluded that cvc coated with 5-á uorouracil were non-inferior to chlorhexidine and silver sulfadiazine coated cvcs with respect to the incidence of catheter colonisation (2.9% vs. 5.3%, respectively). 422 chlorhexidine is a potential allergenic antiseptic that is present in many products and is widely used in health care for skin antisepsis, insertion of urinary catheters or coating cvcs. 406 in susceptible individuals, initial contact will cause a minor hypersensitivity reaction that, although not severe, should not go undocumented as subsequent exposures to chlorhexidine may lead to anaphylaxis. 423, 424 the medicines and healthcare products regulatory agency has alerted all healthcare providers in the uk to the risk of chlorhexidine allergy 425 and requires them to have systems in place that ensure: • awareness of the potential for an anaphylactic reaction to chlorhexidine; • known allergies are recorded in patient notes; • labels and instructions for use are checked to establish if products contain chlorhexidine prior to use on patients with a known allergy; • if a patient experiences an unexplained reaction, checks are carried out to identify whether chlorhexidine was used or was impregnated in a medical device that was used; and • reporting of allergic reactions to products containing chlorhexidine to the medicines and healthcare products regulatory agency. ivad10 use an antimicrobial-impregnated central venous access device for adult patients whose central venous catheter is expected to remain in place for >5 days if catheter-related bloodstream infection rates remain above the locally agreed benchmark, despite the implementation of a comprehensive strategy to reduce catheter-related bloodstream infection. the site at which a vascular access catheter is placed can iná uence the subsequent risk of cr-bsi because of variation in both the density of local skin á ora and the risk of thrombophlebitis. cvcs are generally inserted in the subclavian, jugular or femoral veins, or peripherally inserted into the superior vena cava by way of the major veins of the upper arm (i.e. the cephalic and basilar veins of the antecubital space). pvcs are normally inserted in the upper extremity, although alternatives, such as the foot and scalp, may be used in children and babies. hicpac examined a number of studies that compared insertion sites and concluded that cvcs inserted into subclavian veins had a lower risk for catheter-related infection than those inserted into either jugular or femoral veins. 345, 408, [426] [427] [428] [429] [430] [431] [432] [433] [434] guideline developers suggested that internal jugular insertion sites may pose a greater risk for infection because of their proximity to oropharyngeal secretions and because cvcs at this site are difà cult to immobilise. 334 however, mechanical complications associated with catheterisation might be less common with internal jugular than with subclavian vein insertion. femoral catheters have been demonstrated to have relatively high colonisation rates compared with subclavian and internal jugular sites when used in adults, and current guidelines suggest that the femoral site should be avoided because it is associated with both a higher risk of deep vein thrombosis and catheter-related infection than internal jugular or subclavian catheters. 428, [432] [433] [434] [435] [436] [437] one study also found that the risk of infection associated with catheters placed in the femoral vein is accentuated in obese patients. 409 thus, in adult patients, a subclavian site is preferred for preventing infection, although other factors (e.g. the potential for mechanical complications, risk for subclavian vein stenosis and catheter-operator skill) should be considered when deciding where to place the catheter. we identià ed a systematic review and meta-analysis 438 in which investigators reviewed two rcts, eight cohort studies and data from a national hcai programme. these provided evidence that the selection of device insertion site is not a signià cant factor for the prevention of cr-bsi. the metaanalysis demonstrated no difference in the risk of cr-bsi between the femoral, subclavian and internal jugular sites, s43 having removed two studies that were statistical outliers. the authors concluded that a pragmatic approach to site selection for central venous access, taking into account underlying disease (e.g. renal disease), the expertise and skill of the operator and the risks associated with placement, should be used. 438 two meta-analyses 439, 440 indicate that the use of real-time two-dimensional ultrasound for the placement of cvcs substantially reduced mechanical complications compared with the standard landmark placement technique. consequently, the use of ultrasound may indirectly reduce the risk of infection by facilitating mechanically uncomplicated subclavian placement. in the uk, nice guidelines provide recommendations for two-dimensional ultrasound placement of cvcs. 441 piccs may be used as an alternative to subclavian or jugular vein catheterisation. these are inserted into the superior vena cava via the major veins of the upper arm above the antecubital fossa. hicpac indicated that they are less expensive, associated with fewer mechanical complications (e.g. haemothorax, inà ltration and phlebitis) and easier to maintain than short peripheral venous catheters. 334 in a prospective cohort study using data from two randomised trials and a systematic review to estimate rates of picc-related bloodstream infection in hospitalised patients, the author concluded that piccs used in high-risk hospitalised patients are associated with a rate of cr-bsi similar to conventional cvcs placed in the internal jugular or subclavian veins (two to à ve per 1000 catheter-days). 441 to reduce the risk of cr-bsi and phlebitis, it is preferable to use an upper extremity site for inserting a pvc in adults and to replace a device inserted in a lower extremity to a site in the upper extremity as soon as possible. 334 in paediatric patients, the upper or lower extremity and the scalp (in young infants) can be used for siting a pvc. 405, 442 ivad11 in selecting an appropriate intravascular insertion site, assess the risks for infection against the risks of mechanical complications and patient comfort. ivad12 use the upper extremity for nontunnelled catheter placement unless medically contraindicated. the importance of strict adherence to hand decontamination and the aseptic technique as the cornerstone for preventing catheter-related infection is widely accepted. although this is considered adequate for preventing infections associated with the insertion of short peripheral venous catheters, it is recognised that central venous catheterisation carries a signià cantly greater risk of infection. studies examined by hicpac concluded that if msb precautions were used consistently during cvc insertion, catheter contamination and subsequent catheter-related infections could be reduced signià cantly. 345, 352, 443, 444 a prospective randomised trial that tested the efà cacy of msb precautions to reduce infections associated with long-term, non-tunnelled subclavian silicone catheters, compared with routine procedures, found that they decreased the risk of cr-bsi signià cantly. 443 msb precautions involve wearing sterile gloves and gown, cap and mask, and using a full-body sterile drape during insertion of the catheter. 334 it has been generally assumed that cvcs inserted in the operating theatre pose a lower risk of infection than those inserted on inpatient wards or other patient care areas. 372 however, data examined by hicpac from two prospective studies suggest that the difference in risk of infection depended largely on the magnitude of barrier protection used during catheter insertion, rather than the surrounding environment (i.e. ward vs operating theatre). 345, 443 a systematic review of the value of msb precautions to prevent cr-bsi deà ned the components as: the person inserting the catheter should wear a head cap, face mask, sterile body gown and sterile gloves, and use a full-size sterile drape. their search identià ed 95 papers discussing the prevention of cr-bsi. the majority of these were narrative reviews or consensus statements. three primary research studies, differing in design, patient population and clinical settings, that compared infection outcomes using msb precautions with less stringent barrier techniques, concluded that the use of msb precautions resulted in a reduction in catheter-related infections. the authors concluded that using msb precautions appears to decrease transmission of microorganisms, delay colonisation and reduce the rate of hcai. they also suggested that biological plausibility and the available evidence support using msb precautions during routine insertion of a cvc to minimise the risk of infection. they recommended that, given the lack of adverse patient reactions, the relatively low cost of msb precautions and the high cost of cr-bsi, it is probable that msb precautions will prove to be a cost-effective, or even a cost-saving, intervention. 445 neither we nor hicpac identià ed any additional evidence of acceptable quality whilst updating our systematic review. 334 ivad13 use maximal sterile barrier precautions for the insertion of central venous access devices. microorganisms that colonise catheter hubs and the skin surrounding the vascular catheter insertion site are the cause of most cr-bsi. 416, 446 as the risk of infection increases with the density of microorganisms around the insertion site, skin cleansing/antisepsis of the insertion site is one of the most important measures for preventing catheter-related infections. 334 since the early 1990s, research has focused on identifying the most effective antiseptic agent for skin preparation prior to the insertion of ivds in order to prevent catheter-related infections, especially cr-bsi. in the uk, clinicians principally use alcohol, or either povidone iodine (pvi) or chg, in various strengths, and the latter two as either aqueous or alcohol-based solutions. a prospective randomised trial of agents used for cutaneous antisepsis demonstrated that 2% aqueous chg was superior to either 10% pvi or 70% alcohol for the prevention of central venous and arterial catheter-related infections. 447 a further prospective, randomised trial demonstrated that a 4% alcoholbased solution of 0.25% chg and 0.025% benzalkonium chloride was more effective for the prevention of central venous or arterial catheter colonisation and infection than 10% pvi. 448 the use of 5% pvi solution in 70% ethanol has been shown to be associated with a substantial reduction in catheter-related colonisation and infection compared with 10% aqueous pvi. 449 clinicians may à nd this useful for those patients for whom alcoholic chg is contraindicated. a meta-analysis 450 of studies that compared the risk for cr-bsi following insertion-site skin care with any type of chg solution vs pvi solution indicated that the use of chg rather than pvi can reduce the risk for cr-bsi by approximately 49% (rr 0.51, 95% ci 0.27-0.97) in hospitalised patients who require short-term catheterisation (i.e. for every 1000 catheter sites disinfected with chg rather than pvi, 71 episodes of catheter colonisation and 11 episodes of cr-bsi would be prevented). in this analysis, several types of chg solution were used in the individual trials, including 0.5% or 1% chg alcohol solution and 0.5% or 2% chg aqueous solution. all of these solutions provided a concentration of chg that is higher than the minimal inhibitory concentration (mic) for most nosocomial bacteria and yeasts. subset analysis of aqueous and non-aqueous solutions showed similar effect sizes, but only the subset analysis of the à ve studies that used alcoholic chg solution produced a signià cant reduction in cr-bsi. as few studies used chg aqueous solution, the lack of a signià cant difference seen for this solution compared with pvi solution may be a result of inadequate statistical power. additionally, an economic decision analysis based on available evidence from the same authors suggested that the use of chg, rather than pvi, for skin care would result in a 1.6% decrease in the incidence of cr-bsi, a 0.23% decrease in mortality, and à nancial savings per catheter used. 451 several studies were examined that focused on the application of antimicrobial ointments to the catheter site at the time of catheter insertion, or during routine dressing changes, to reduce microbial contamination of catheter insertion sites. 448 reported efà cacy of this practice for the prevention of catheter-related infections yielded contradictory à ndings. [449] [450] [451] [452] [453] [454] [455] [456] [457] there was also concern that the use of polyantibiotic ointments that were not fungicidal could signià cantly increase the rate of colonisation of the catheter by candida species. 458, 459 nice 56 identià ed three rcts that compared the effectiveness of different antiseptic solutions for the insertion of pvcs in hospitalised patients. the evidence from these studies was considered to be of very low quality, and no conclusion could be drawn about the beneà ts of one particular antiseptic solution over another. however, while there is no evidence comparing different concentrations of chg, the reviewers indicated that the trend in the evidence suggests that chg in alcohol may be more effective than pvi in alcohol. we identià ed one recent systematic review of the clinical efà cacy and perceived role of chg in skin antisepsis that included studies about intravascular access. 460 the authors suggested a potential source of bias, as many studies have overlooked the importance of alcohol when assessing the efà cacy of chg. the authors assessed the attribution of chg in each study as correct, incorrect or intermediate. studies were scored and analysis was performed separately to assess chg efà ciency. the authors concluded that chg is more efà cient than pvi or any other technique alone, but that the presence of alcohol provides additional beneà t. the authors suggested that vascular catheters require the immediate antiseptic activity provided by alcohol prior to insertion. they also require a long-lasting antiseptic, as they stay in place for prolonged periods of time. ivad14 decontaminate the skin at the insertion site with a single-use application of 2% chlorhexidine gluconate in 70% isopropyl alcohol (or povidone iodine in alcohol for patients with sensitivity to chlorhexidine) and allow to dry prior to the insertion of a central venous access device. ivad15 decontaminate the skin at the insertion site with a single-use application of 2% chlorhexidine gluconate in 70% isopropyl alcohol (or povidone iodine in alcohol for patients with sensitivity to chlorhexidine) and allow to dry before inserting a peripheral vascular access device. ivad16 do not apply antimicrobial ointment routinely to the catheter placement site prior to insertion to prevent catheter-related bloodstream infection. the safe maintenance of an intravascular catheter and appropriate care of the insertion site are essential components of a comprehensive strategy for preventing catheter-related infections. this includes good practice in caring for the patient's catheter hub and connection port, the use of an appropriate intravascular catheter site dressing regimen, and using á ush solutions to maintain the patency of the catheter. following placement of a pvc or cvc, a dressing is used to protect the insertion site. as occlusive dressings trap moisture on the skin and provide an ideal environment for the rapid growth of local microá ora, dressings for insertion sites must be permeable to water vapour. 446 the two most common types of dressings used for insertion sites are sterile, transparent, semi-permeable polyurethane dressings coated with a layer of an acrylic adhesive ('transparent dressings') and gauze and tape dressings. transparent dressings are permeable to water vapour and oxygen, and impermeable to microorganisms. hicpac reviewed the evidence related to which type of dressing provided the greatest protection against infection, including the largest controlled trial of dressing regimens on pvcs, 361 a meta-analysis comparing the risk of cr-bsi using transparent vs gauze dressings 461 and a cochrane review. 462 all concluded that the choice of dressing can be a matter of preference, but if blood is leaking from the catheter insertion site, a gauze dressing might be preferred to absorb the á uid. we identià ed an updated cochrane review which concluded that bloodstream infection was higher in the transparent polyurethane group compared with the gauze and tape group. 463 the included trials were graded low quality due to the small sample size and risk of bias. there was additional low-quality evidence that demonstrated no difference between highly permeable polyurethane dressings and other polyurethane dressings in the prevention of cr-bsi. hicpac reviewed the evidence related to impregnated sponge dressings compared with standard dressings and found two rcts in adults which demonstrated that chlorhexidineimpregnated sponge dressings were associated with a signià cant reduction in cr-bsi. however, a meta-analysis that included eight rcts found a reduction in exit site colonisation but no signià cant reduction in cr-bsi. in paediatric patients, two small rcts found a reduction in catheter colonisation but not cr-bsi, and evidence of localised contact dermatitis when used for infants of very low birth weight. 334 we identià ed one systematic review and meta-analysis, undertaken as part of a quality improvement collaborative, that synthesised the effects of the routine use of chgimpregnated sponge dressings in reducing centrally inserted cr-bsi. 464 five studies were included in the analysis; two of the à ve studies were in patients in haemo/oncological icus, and the remaining three studies were in surgical and medical icus. four of the à ve studies were sponsored by the manufacturer of the product. the reviewers concluded that chg-impregnated sponge dressings are effective for the prevention of cr-bsi (or 0.43, 95% ci 0.29-0.64) and catheter colonisation (or 0.43, 95% ci 0.36-0.51). we identià ed an economic evaluation of the use of chg sponge dressings and the non-inferiority of dressing changes at 3 and 7 days. 465 the authors concluded that the major cost avoided by the use of chg sponge dressings and 7-day dressing changes rather than 3-day dressing changes was the increased length of stay of 11 days associated with cr-bsi. chlorhexidineimpregnated sponge dressings remained cost saving for any value where the cost per cr-bsi was >$4400 and the baseline rate of cr-bsi was >0.35%. 465 we identià ed a further rct of chg dressings compared with highly adhesive semi-permeable dressings or standard semi-permeable dressings for the prevention of cr-bsi in 1879 patients. 466 in the chg group, the major catheterrelated infection rate was 67% lower (0.7 vs 2.1 per 1000 catheter-days, hr 0.328, 95% ci 0.174-0.619, p=0.0006) and the cr-bsi rate was 60% lower (0.5 vs 1.3 per 1000 catheterdays, hr 0.402, 95% cl 0.186-0.868, p=0.02) than with nonchlorhexidine dressings. decreases were also noted in catheter colonisation and skin colonisation rates at catheter removal. highly adhesive dressings decreased the detachment rate to 64.3% vs 71.9% (p<0.0001) and the number of dressings per catheter to two (one to four) vs three (one to à ve) (p<0.0001), but increased skin colonisation (p<0.0001) and catheter colonisation (hr 1.650, 95% cl 1.21-2.26, p=0.0016) without iná uencing cr-bsi rates. hicpac identià ed three studies that investigated the efà cacy of a 2% chg-impregnated washcloth in reducing the risk of cr-bsi. 334 these studies were included in a subsequent systematic review and meta-analysis on the efà cacy of either 2% chg-impregnated cloths or 4% chg solution for daily skin cleansing in adult acute care settings, mostly icus. 467 twelve studies were included: one rct, one cluster nrct and 10 controlled interrupted time series. five studies that reported the insertion technique included the use of chg. there was a high level of clinical heterogeneity and moderate statistical heterogeneity, which remained following a subgroup analysis by type of chg formulation. the authors concluded that among icu patients, daily chg bathing with chg liquid (or 0.47, 95% ci 0.31-0.71) or cloths (or 0.41, 95% ci 0.25-0.65) reduces the risk of cr-bsi. similar beneà t is obtained regardless of whether chg cloths or liquid preparation is used (or 0.44, 95% ci 0.44-0.59). this review was not generalisable to paediatric care. whenever chg is used for insertion site dressings or skin cleansing, systems should be in place to ensure that it is not used for patients with a history of chlorhexidine sensitivity. 408 a single rct compared the efà cacy of two commercially available alcohol-based antiseptic solutions for preparation and care of cvc insertion sites, with and without octenidine dihydrochloride. 468 data were collected from 2002 to 2005 and published in 2010. the authors concluded that octenidine in alcoholic solution is a better option than alcohol alone for the prevention of cvc-associated infections, and may be as effective as chg in practice but a comparative trial is needed. ivad17 use a sterile, transparent, semipermeable polyurethane dressing to cover the intravascular insertion site. ivad18 transparent, semi-permeable polyurethane dressings should be changed every 7 days, or sooner, if they are no longer intact or if moisture collects under the dressing. ivad19 use a sterile gauze dressing if a patient has profuse perspiration or if the insertion site is bleeding or leaking, and change when inspection of the insertion site is necessary or when the dressing becomes damp, loosened or soiled. replace with a transparent semi-permeable dressing as soon as possible. ivad20 consider the use of a chlorhexidineimpregnated sponge dressing in adult patients with a central venous catheter as a strategy to reduce catheterrelated bloodstream infection. ivad21 consider the use of daily cleansing with chlorhexidine daily in adult patients with a central venous catheter as a strategy to reduce catheter-related bloodstream infection. ivad22 dressings used on tunnelled or implanted catheter insertion sites should be replaced every 7 days until the insertion site has healed unless there is an indication to change them sooner. a dressing may no longer be required once the insertion site is healed. research previously described in these guidelines has described the superior effectiveness of chg to minimise the density of microorganisms around vascular catheter insertion sites. 447, 448, 450 consequently, alcoholic chg is now widely used in the uk for disinfecting the insertion site during dressing changes. studies focused on the use of antimicrobial ointment applied under the dressing to the catheter insertion site to prevent catheter-related infection do not clearly demonstrate efà cacy. 454, 459 most modern intravascular catheters and other catheter materials are not damaged by contact with alcohol. however, alcohol, and other organic solvents and oil-based ointments and creams, may damage some types of polyurethane and silicon catheter tubing (e.g. some catheters used in haemodialysis). the manufacturer's recommendations to only use disinfectants that are compatible with specià c catheter materials must therefore be followed. ivad23 use a single-use application of 2% chlorhexidine gluconate in 70% isopropyl alcohol (or povidone iodine in alcohol for patients with sensitivity to chlorhexidine) to clean the central catheter insertion site during dressing changes, and allow to air dry. ivad24 use a single-use application of 2% chlorhexidine gluconate in 70% isopropyl alcohol (or povidone iodine in alcohol for patients with sensitivity to chlorhexidine) to clean the peripheral venous catheter insertion site during dressing changes, and allow to air dry. ivad25 do not apply antimicrobial ointment to catheter insertion sites as part of routine catheter site care. evidence indicates that the routine replacement of cvcs at scheduled time intervals does not reduce rates of cr-bsi. three randomised trials investigated strategies for replacing cvcs routinely at either 7 days 469,470 or 3 days 471 compared with changing catheters when clinically indicated. two studies were conducted in adult icus 469,471 and a third study was undertaken in a renal dialysis unit. 470 no difference in cr-bsi was observed in patients in the scheduled replacement groups compared with those replaced when clinically indicated. another suggested strategy for the prevention of cr-bsi is the routine scheduling of guidewire exchange of cvcs. a systematic review and meta-analysis 472 of 12 rcts concluded that when compared with insertion at a new site, guidewire exchange was associated with a trend towards increased rates of catheter colonisation (rr 1.26, 95% ci 0.87-1.84), regardless of suspected cr-bsi at the time of replacement. guidewire exchange was also associated with a trend towards increased rates of catheter exitsite infection (rr 1.52, 95% ci 0.34-6.73) and cr-bsi (rr 1.72, 95% ci 0.89-3.33), but also associated with fewer mechanical complications relative to insertion at a new site. 472 neither we nor hicpac identià ed any additional evidence for these recommendations whilst updating our systematic review. 334 we identià ed one rct that compared a routine 3-day re-siting of pvcs compared with a clinically indicated resiting. ivd-related complication rates were 68 per 1000 ivddays (clinically indicated) and 66 per 1000 ivd-days (routine replacement) (p=0.86, hazard ratio 1.03, 95% ci 0.74-1.43). re-siting a device on clinical indication would allow one in two patients to have a single cannula per course of intravenous treatment, as opposed to one in à ve patients managed with routine re-siting; overall complication rates appear similar. clinically indicated re-siting would achieve savings in equipment, staff time and patient discomfort. 473 a recent update of a cochrane review found no evidence to support changing catheters every 72-96 h. 474 evidence demonstrating that contamination of the catheter hub contributes to intraluminal microbial colonisation of catheters, particularly long-term catheters, was considered by hicpac. 346, [475] [476] [477] [478] [479] [480] catheter hubs are accessed more frequently when catheterisation is prolonged, and this increases the risk of cr-bsi originating from a colonised catheter hub rather than the insertion site. 346 evidence from a prospective cohort study suggested that frequent catheter hub manipulation increases the risk for microbial contamination. 481 additional studies concurred and recommended that hubs and sampling ports should be disinfected using either povidone iodine or chlorhexidine before they are accessed. 406, 482, 483 a randomised prospective clinical trial investigated the use of needleless connectors or standard caps attached to cvc luer connections. results suggested that the use of needleless connectors may reduce the microbial contamination rate of cvc luers compared with standard caps. furthermore, disinfection of needleless connectors with either chlorhexidine/alcohol or pvi signià cantly reduced external microbial contamination. both these strategies may reduce the risk of catheter-related infections acquired via the intraluminal route. 484 we found no rct evidence comparing the efà cacy of different methods for the decontamination of ports and hubs prior to access. expert opinion, based on consensus and evidence extrapolated from experimental studies of hub decontamination, 56, 485, 486 and studies of skin decontamination prior to insertion and during dressing changes, suggests that injection ports or catheter hubs should be decontaminated for a minimum of 15 s using chg in 70% alcohol before and after accessing the system. although most intravascular catheters and catheter hub materials are now chemically compatible with alcohol or iodine, some may be incompatible and therefore the manufacturer's recommendations should be followed. ivad30 a single-use application of 2% chlorhexidine gluconate in 70% isopropyl alcohol (or povidone iodine in alcohol for patients with sensitivity to chlorhexidine) should be used to decontaminate the access port or catheter hub. the hub should be cleaned for a minimum of 15 s and allowed to dry before accessing the system. the procedure of á ushing and then leaving the lumen of a cvc à lled with an antibiotic solution is termed 'antibiotic lock prophylaxis' and has been described as a measure to prevent cr-bsi in haemodialysis or a patient who has a history of multiple cr-bsi despite optimal maximal adherence to the aseptic technique. evidence reviewed by hicpac 334 demonstrated the effectiveness of this type of prophylaxis. however, the majority of the studies were conducted in haemodialysis patients and therefore may not be generalisable. we identià ed a systematic review of rcts which concluded that the scientià c evidence for the effectiveness of the routine use of antibiotic-based lock solutions is weak, 487 thus supporting the hicpac evidence. in addition, there is concern that the use of such solutions could lead to an increase in antimicrobialresistant microorganisms. 334 an additional placebo-rct of daily ethanol locks to prevent cr-bsi in patients with tunnelled catheters 488 found that the reduction in the incidence of endoluminal cr-bsi using preventive ethanol locks was non-signià cant, although the low incidence of endoluminal cr-bsi precludes deà nite conclusions, and the low incidence of cr-bsi in the placebo arm meant the study was underpowered in retrospect. signià cantly more patients treated with ethanol locks discontinued their prophylactic treatment due to non-severe, ethanol-related adverse effects. ivad31 antimicrobial lock solutions should not be used routinely to prevent catheterrelated bloodstream infections. hicpac identià ed no studies which demonstrated that oral or parenteral antibacterial or antifungal drugs reduced the incidence of cr-bsi among adults. however, among lowbirthweight infants, two studies on vancomycin prophylaxis demonstrated a reduction in cr-bsi but no reduction in mortality. as the prophylactic use of vancomycin is an independent risk factor for the acquisition of vre, it is likely that the risk of acquiring vre outweighs the beneà t of using prophylactic vancomycin. 334, 489 topical mupirocin is used to suppress s. aureus in nasal carriers. some studies have shown that mupirocin applied nasally (or locally to the insertion site) results in reduced risk of cr-bsi. 334 however, rates of mupriocin resistance of 12% have been reported in the uk, 490 and its incompatibility with polyurethane catheters means that it should not be used routinely. 334 long-term tunnelled cvcs are frequently used for patients with cancer who require intravenous treatments. a cochrane review published in 2003 concluded that prophylactic antibiotics or catheter á ushing with vancomycin and heparin may be of beneà t in reducing the risk of catheter-related infections in these high-risk cancer patients. 491 however, this practice should not be used routinely in order to minimise the development of antimicrobial resistance. 491 ivad32 do not routinely administer intranasal or systemic antimicrobials before insertion or during the use of an intravascular device to prevent catheter colonisation or bloodstream infection. the placement of any cvc or pulmonary artery catheter leads to thrombus formation shortly after insertion, providing a focus for bacterial growth. 492 catheters manufactured from silicone or polyethylene and placed in the subclavian vein are less frequently associated with thrombus formation. 493 between 35% and 65% of patients with long-term cvcs and piccs develop a thrombosis of the large vessels, and patients are treated with prophylactic heparin to prevent the formation of both deep vein thrombosis and catheter thrombus. 334, [494] [495] [496] [497] [498] [499] the use of anticoagulants heparin may be administered through several different routes. an early meta-analysis of rcts compared the effectiveness of heparin administration via an infusion, subcutaneously or intermittent á ush for the prevention of thrombus formation and cr-bsi in patients with short-term cvcs. 500 prophylactic heparin infusion was associated with a decrease in catheter thrombus formation, deep vein thrombosis, catheter colonisation and a trend towards reductions in cr-bsi, but this was not statistically h. p. loveday et al. / journal of hospital infection 86s1 (2014) s1-s70 s49 signià cant. hicpac identià ed an additional prospective randomised trial that demonstrated a signià cant decrease in the rate of cr-bsi in patients with non-tunnelled cvcs who received continuous heparin infusion. 501 heparin-bonded (hb) catheters have also been shown to reduce the risk of both thrombus formation and cr-bsi. [502] [503] [504] [505] we identià ed one systematic review of hb cvcs in children. 506 the reviewers identià ed two rcts of 287 children aged 1 day to 16 years who received either an hb catheter or a standard catheter. there was no signià cant difference in the median duration of catheter patency in the two groups: 7 days in the hb catheter group and 6 days in the standard catheter group. the authors also reported a trend towards a reduction in the risk of catheter-related thrombosis and catheter occlusion in the hb group. the risks of catheter colonisation and catheterrelated infection were signià cantly reduced in the treatment group, with a delay to infection in the hb catheter group. however, the reviewers considered the need for further studies to conà rm the efà cacy of hb catheters. the use of warfarin has also been shown to reduce the risk of catheter-related thrombosis in some patient groups but not in others, and is generally not associated with a reduction in infection-related complications. 501,507-509 systemic heparin, as either an infusion or á ush, has a number of side effects that contraindicate its routine use for maintaining the patency of cvcs and preventing thrombus formation; these include thrombocytopenia, allergic reactions and bleeding. 510 normal saline is an alternative to the use of heparin á ush. hicpac refer to three systematic reviews, and meta-analysis of rcts evaluating the effect of heparin on the duration of catheter patency and on the prevention of complications associated with the use of peripheral venous and arterial catheters concluded that heparin at doses of 10 u/ml for intermittent á ushing is no more beneà cial than á ushing with normal saline alone. [511] [512] [513] [514] however, manufacturers of implanted ports or opened-ended catheter lumens may recommend heparin á ushes for maintaining cvcs that are accessed infrequently. we identià ed one systematic review and two rcts that compared heparin with normal saline to maintain the patency of cvcs and pvcs, respectively. 515-517 a systematic review 515 of heparin á ushing and other interventions to maintain the patency of cvcs concluded that the evidence base for heparin á ushing and other interventions to prevent catheter occlusion is limited and published studies are of low quality. the reviewers concluded that there is no direct evidence of the effectiveness of heparin á ushes to prevent cr-bsi or other central line complications. in a single-centre rct 516 of newly placed multi-lumen cvcs in patients in medical icus and surgical/burn/trauma icus, normal saline and heparin á ush solutions were found to have similar rates of lumen non-patency. given potential safety concerns with the use of heparin, normal saline may be the preferred á ushing solution for short-term use for cvc maintenance. secondary outcomes for cr-bsi were non-signià cant between groups. a single-centre cluster rct 517 of 214 medical patients found that twice-daily heparin (100 u/ml) á ushes for maintenance of pvcs was more effective than normal saline solution. the number of catheter-related phlebitis/occlusions and the number of catheters per patient was reduced; however, infection outcomes were not measured. ivad33 do not use systemic anticoagulants routinely to prevent catheter-related bloodstream infection. ivad34 use sterile normal saline for injection to á ush and lock catheter lumens that are accessed frequently. needle-free infusion systems and connection devices have been widely introduced to reduce the incidence of sharps injuries and minimise the risk of transmission of bloodborne pathogens to healthcare workers. 334 there is limited evidence that needleless devices or valves reduce the risk of catheter colonisation compared with standard devices. 334 in addition, the design features of some of these devices pose a potential risk for contamination, and have been associated with reports of an increase in bloodstream infection rates. [518] [519] [520] [521] ivad35 the introduction of new intravascular devices or components should be monitored for an increase in the occurrence of device-associated infection. if an increase in infection rates is suspected, this should be reported to the medicines and healthcare products regulatory agency in the uk. ivad36 when safer sharps devices are used, healthcare workers should ensure that all components of the system are compatible and secured to minimise leaks and breaks in the system. hicpac reviewed three well-controlled studies on the optimal interval for the routine replacement of intravenous solution administration sets. 334 a cochrane review 522 of 13 rcts with 4783 patients concluded that there is no evidence that changing intravenous administration sets more often than every 96 h reduces the incidence of bloodstream infection. the reviewers were unable to conclude if changing administration sets less often than every 96 h affects the incidence of infection from the studies. there were no differences between participants with central vs peripheral catheters, nor between participants who did and did not receive parenteral nutrition, or between children and adults. administration sets that do not contain lipids, blood or blood products may be left in place for intervals of up to 96 h without increasing the incidence of infection. there is no evidence to suggest that administration sets which contain lipids should not be changed every 24 h as currently recommended. ivad37 administration sets in continuous use do not need to be replaced more frequently than every 96 h, unless device-specià c recommendations from the manufacturer indicate otherwise, they become disconnected or the intravascular access device is replaced. class a ivad38 administration sets for blood and blood components should be changed when the transfusion episode is complete or every 12 h (whichever is sooner). ivad39 administration sets used for lipidcontaining parenteral nutrition should be changed every 24 h. ensuring that patients receive care that is evidence based is an essential element of delivering high-quality health care. in 2005, the department of health issued a series of highimpact interventions that were derived from national and international evidence-based guidelines for the prevention of healthcare-associated infection and based on experience from the institute of healthcare improvement 100,000 lives campaign focused on reducing patient harm. 523 the high-impact interventions focused on increasing the reliability of care and ensuring that recommendations were implemented every time for every patient. the intervention for the prevention of infection associated with the use of ivds included six key interventions often referred to as a 'care bundle', together with audit tools to measure adherence. these six practices included: • aseptic insertion of an appropriate device; • correct siting of the device; • effective cutaneous antisepsis; and for continuing care of the device: • hand decontamination and asepsis for any contact with the device; • daily observation of the insertion site; and • clean, intact dressing. a small number of well-designed studies 353,524 have described the use of 'bundled' approaches to reducing cr-bsi, and have stimulated individual observational and quality improvement reports of the results of using key evidence-based practices for the prevention of cr-bsi. the most prominent of these was a study conducted in the icu setting of 108 hospitals in the usa, which was then adopted by other countries including the uk. 525, 526 the authors reported the success of à ve evidence-based practices combined with system and organisational support, which resulted in a 66% decrease in cr-bsi 18 months after the inception of the programme (incidence rate ratio 0.62, 95% ci 0.47-0.81 to incidence rate ratio 0.34, 95% ci 0.23-0.50) and sustained reductions thereafter. 525 the intervention comprised: hand hygiene using abhr; msb precautions for insertion; cutaneous antisepsis of the insertion site with 2% chg; avoiding the femoral site; and removing cvcs as soon as they are no longer clinically indicated. in addition, system changes that prompted the clinician to 'do the right thing' included placing all the equipment needed in a cart for ease of access; the use of a checklist; authorising staff to halt procedures if best practice was not being followed; daily rounds to ensure the timely removal of cvcs; feedback of cr-bsi cases to clinical staff; and organisational support to purchase essential equipment and solutions prior to the start of the study. audit and feedback are an essential component of any quality improvement intervention as this promotes a continuous 'hawthorne effect' and enables staff to maintain vigilance and sustain improvement. the use of dashboards and statistical process control charts alerts clinicians to variability outside control limits, and prompts scrutiny of practice and organisational systems, and remedial action to be taken. 527 we identià ed three additional studies that reported 'bundled interventions' to reduce cr-bsi. [528] [529] [530] none were included in the systematic review as they failed to meet study quality criteria. the features of any quality improvement initiative need to be tailored to the local conditions and may include some or all of the following: • hand hygiene, aseptic insertion using msb precautions (cvc), aseptic technique (pvc), cutaneous antisepsis using 2% chg in alcohol unless contraindicated, appropriate siting of the cvc or pvc, and prompt removal when no longer indicated; • audit and feedback; • education and training; and • accessibility of equipment and appropriate system changes developed with clinical staff to make best practice the norm. in one cost-effectiveness study, a markov decision model was used to evaluate the cost-effectiveness of a care bundle to prevent cr-bsi. 531 the care bundle included in the model was based on the bundle advocated by the institute for health improvement 100,000 lives campaign, 532 comprising optimal hand hygiene, chlorhexidine skin antisepsis, msb precautions for catheter insertion and insertion equipment kit, optimal insertion site and prompt catheter removal. costs included monitoring, education and clinical leadership activities. the authors estimated that the bundle would be cost-effective if the costs of implementation were less than aus$94,559 (£55,817) per icu. to support the appropriate use and management of intravascular access devices (central and peripheral venous catheters) and ensure their timely removal. these may include: • protocols for device insertion and maintenance; • reminders to review the continuing use or prompt the removal of intravascular devices; • audit and feedback of compliance with practice guidelines; and • continuing professional education. systematic review questions 1. what types of cvcs (material, coating, antibiotic impregnation, cuffed, tunnelled, midline, picc) and pvcs (material, coating, antibiotic impregnation) are most effective in reducing the risk of cr-bsi and related complications/adverse events including phlebitis, related mortality, catheter tip colonisation and premature line removal? 2. which cvc/pvc insertion site is associated with the lowest risk of cr-bsi and related complications including phlebitis, related mortality, catheter tip colonisation and premature line removal? 3. what is the evidence that additional ports or lumens increase the risk of cr-bsi and related complications/adverse events including phlebitis, mortality, catheter tip colonisation and premature line removal? 4. which infection prevention precautions used for inserting intravascular catheters are most effective in reducing the risk of cr-bsi and related complications/adverse events including phlebitis, catheter tip colonisation, premature line removal and mortality? 5. what levels of barrier precautions are most effective in reducing the risk of cr-bsi and related complications/adverse events including phlebitis, catheter tip colonisation, premature line removal and mortality? 6. what is the most effective skin antisepsis solution/antiseptic-impregnated product for decontamination of the skin prior to insertion of cvcs and pvcs to reduce the risk of cr-bsi and related complications including phlebitis, catheter tip colonisation, premature line removal and mortality? 7. what is the effectiveness of antiseptics vs antiseptic-impregnated products (sponges or cloths) for decontaminating skin at the insertion site or surrounding area whilst a cvc or pvc is in situ in reducing the risk of cr-bsi and related complications including phlebitis, catheter tip colonisation, premature line removal and mortality? 8. what is the evidence for the effectiveness of using antibiotics or antiseptics to lock, á ush or clean the catheter hub or entry ports of cvcs and pvcs in reducing the risk of cr-bsi and related complications including phlebitis, catheter tip colonisation, premature line removal and mortality? 9. what is the effectiveness of low-dose systemic anticoagulation to reduce the risk of cr-bsi and related complications including phlebitis, catheter tip colonisation, premature line removal and mortality? 10. which dressing type is the most clinically effective in reducing the risk of cr-bsi and related complications including phlebitis, catheter tip colonisation, premature line removal and mortality, and how frequently should dressings be changed? 11. what is the optimal frequency to change or re-site pvcs or midline catheters to reduce the risk of cr-bsi and related complications including phlebitis, catheter tip colonisation, premature line removal and mortality? 12. what is the evidence for the effectiveness of replacing administration sets to reduce the risk of cr-bsi and related complications including phlebitis, catheter tip colonisation, premature line removal and mortality? 13. what is the effectiveness of the prophylactic administration of systemic antimicrobials in reducing the incidence of cr-bsi and related complications including phlebitis, catheter tip colonisation, premature line removal and mortality? 14. what is the evidence that the needle-safe devices are associated with increased risk of cr-bsi and related complications including phlebitis, catheter tip colonisation, premature line removal and mortality? 15. what is the effectiveness of system interventions in reducing the risk of cr-bsi and related complications including phlebitis, catheter tip colonisation, premature line removal and mortality, and improving healthcare workers' knowledge and behaviour relating to the use of central venous access device (cvad) and peripheral vascular device (pvd)? total number of articles located = 8053 abstract indicates that the article: relates to infections associated with intravascular access devices; is written in english; is primary research, a systematic review or a meta-analysis; and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = 96 full text conà rms that the article: relates to infections associated with intravascular access devices; is written in english; is primary research (randomised controlled trials, prospective cohort, interrupted time series, controlled before-after, quasi-experimental), a systematic review or a metaanalysis including the above designs; and informs one or more of the review questions. total number of studies selected for appraisal during sift 2 = 30 all articles that described primary research, a systematic review or a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers using sign and epoc criteria. consensus and grading was achieved through discussion. total number of studies accepted after critical appraisal = 22 total number of studies rejected after critical appraisal = 8 the epic project: developing national evidence-based guidelines for preventing healthcare associated infections. phase i: guidelines for preventing 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sampling and chemotherapy administration complications and management of implanted venous access catheters a totally implanted venous access system for the delivery of chemotherapy implantable subcutaneous venous catheters fifty-à ve patient years' experience with a totally implanted system for intravenous chemotherapy infection rates of broviac-hickman catheters and implantable venous devices complications from long-term indwelling central venous catheters in hematologic patients with special reference to infection classical external indwelling central venous catheter versus totally implanted venous access systems for chemotherapy administration: a randomized trial in 100 patients with solid tumors comparison of infections in hickman and implanted port catheters in adult solid tumor patients experience with a totally implantable venous access device (port-a-cath) in patients with aids infectious morbidity associated with long-term use of venous access devices in patients with cancer 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infection rate risk of catheter-related bloodstream infection with peripherally inserted central venous catheters used in hospitalized patients prevention of central venous catheter-related bloodstream infection by use of an antiseptic-impregnated catheter. a randomized, controlled trial evaluation of chlorhexidine and silver-sulfadiazine impregnated central venous catheters for the prevention of bloodstream infection in leukaemic patients: a randomized controlled trial iná uence of triple-lumen central venous catheters coated with chlorhexidine and silver sulfadiazine on the incidence of catheter-related bacteremia a meta-analysis dealing with the effectiveness of chlorhexidine and silver-sufhadiazine impregnated central venous catheters anaphylactic shock induced by an antiseptic-coated central venous [correction of nervous] catheter cost-effectiveness of antiseptic-impregnated central venous catheters for the prevention of catheter-related bloodstream infection central venous catheters coated with minocycline and rifampin for the prevention of catheter-related colonization and bloodstream infections. a randomized, double-blind trial. the texas medical center catheter study group decreasing catheter colonization through the use of an antiseptic-impregnated catheter: a continuous quality improvement project a comparison of two antimicrobial-impregnated central venous catheters efà cacy of antiseptic-impregnated central venous catheters in preventing catheter-related bloodstream infection: a meta-analysis prevention of intravascular catheter-related infections prevention of bloodstream infections with central venous catheters treated with antiinfective agents depends on catheter type and insertion time: evidence from a meta-analysis prolonged antimicrobial activity of a catheter containing chlorhexidine-silver sulfadiazine extends protection against catheter infections in vivo the clinical effectiveness and cost-effectiveness of central venous catheters treated with anti-infective agents in preventing bloodstream infections: a systematic review and economic evaluation catheter impregnation, coating or bonding for reducing central venous catheter-related infections in adults effectiveness of different central venous catheters for catheterrelated infections: a network meta-analysis anti-infective external coating of central venous catheters: a randomized, noninferiority trial comparing 5-á uorouracil with chlorhexidine/silver sulfadiazine in preventing catheter colonization immediate hypersensitivity to chlorhexidine is increasingly recognised in the united kingdom anaphylaxis to chlorhexidine-coated central venous catheters: a case series and review of the literature medical device alert: all medical devices and medicinal products containing chlorhexidine. mda/2012/075. london: medicines and healthcare products regulatory agency femoral vs jugular venous catheterization and risk of nosocomial events in adults requiring acute renal replacement therapy: a randomized controlled trial impact of central venous catheter type and methods on catheter-related colonization and bacteraemia to reduce catheterrelated bloodstream infections: is the subclavian route better than the jugular route for central venous catheterization? complications of central venous catheters: internal jugular versus subclavian access -a systematic review comparison between the jugular and subclavian vein as insertion site for central venous catheters: microbiological aspects and risk factors for colonization and infection prospective multicenter study of vascular-catheter-related complications and risk factors for positive central-catheter cultures in intensive care unit patients a review of risk factors for catheter-related bloodstream infection caused by percutaneously inserted, noncuffed central venous catheters: implications for preventive strategies the micro-organism responsible for central venous catheter s62 s1-s70 related bloodstream infection depends on catheter site prospective study of arterial and central venous catheter colonization and of arterial-and central venous catheter-related bacteremia in intensive care units complications of femoral and subclavian venous catheterization in critically ill patients: a randomized controlled trial risk of infection due to central venous catheters: effect of site of placement and catheter type the incidence of infectious complications of central venous catheters at the subclavian, internal jugular, and femoral sites in an intensive care unit population the risk of catheter-related bloodstream infection with femoral venous catheters as compared to subclavian and internal jugular venous catheters: a systematic review of the literature and meta-analysis ultrasound guidance for placement of central venous catheters: a metaanalysis of the literature ultrasonic locating devices for central venous cannulation: meta-analysis guidance on the use of ultrasound locating devices for placing central venous catheters infection control in intravenous therapy prevention of central venous catheter-related infections by using maximal sterile barrier precautions during insertion effect of different sterile barrier precautions and central venous catheter dressing on the skin colonization around the insertion site using maximal sterile barriers to prevent central venous catheter-related infection: a systematic evidence-based review catheter-related sepsis: an overview -part 1 prospective randomised trial of povidone-iodine, alcohol, and chlorhexidine for prevention of infection associated with central venous and arterial catheters prospective, randomized trial of two antiseptic solutions for prevention of central venous or arterial catheter colonization and infection in intensive care unit patients alcoholic povidone-iodine to prevent central venous catheter colonization: a randomized unit-crossover study chlorhexidine compared with povidone-iodine solution for vascular cathetersite care: a meta-analysis vascular catheter site care: the clinical and economic beneà ts of chlorhexidine gluconate compared with povidone iodine guideline for use of topical antimicrobial agents catheter-related sepsis in long-term parenteral nutrition with broviac catheters. an evaluation of different disinfectants colonization of central venous catheters a clinical and bacteriologic study of infections associated with venous cutdowns application of antibiotic ointment to the site of venous catheterization -a controlled trial risk of infection with intravenous indwelling catheters: effect of application of antibiotic ointment the effects of antibiotic ointments and antiseptics on the skin á ora beneath subclavian catheter dressings during intravenous hyperalimentation a comparative study of polyantibiotic and iodophor ointments in prevention of vascular catheter-related infection the forgotten role of alcohol: a systematic review and meta-analysis of the clinical efà cacy and perceived role of chlorhexidine in skin antisepsis transparent polyurethane à lm as an intravenous catheter dressing. a metaanalysis of the infection risks gauze and tape and transparent polyurethane dressings for central venous catheters gauze and tape and transparent polyurethane dressings for central venous catheters using the collaborative evidence based practice model: a systematic review and uptake of chlorhexidine-impregnated sponge dressings on central venous access devices in a tertiary cancer centre economic evaluation of chlorhexidine-impregnated sponges for preventing catheterrelated infections in critically ill adults in the dressing study randomized controlled trial of chlorhexidine dressing and highly adhesive dressing for preventing catheter-related infections in critically ill adults the efà cacy of daily bathing with chlorhexidine for reducing healthcareassociated bloodstream infections: a meta-analysis skin disinfection with octenidine dihydrochloride for central venous catheter site care: a double-blind, randomized, controlled trial catheterrelated sepsis: prospective, randomized study of three methods of long-term catheter maintenance changing subclavian haemodialysis cannulas to reduce infection a controlled trial of scheduled replacement of central venous and pulmonary-artery catheters central venous catheter replacement strategies: a systematic review of the literature routine resite of peripheral intravenous devices every 3 days did not reduce complications compared with clinically indicated resite: a randomised controlled trial clinically-indicated replacement versus routine replacement of peripheral venous catheters source and route of microbial colonisation of parenteral nutrition catheters catheter sepsis due to coagulase-negative staphylococci in patients on total parenteral nutrition a prospective study of the catheter hub as the portal of entry for microorganisms causing catheter-related sepsis in neonates adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters pathogenesis of catheter sepsis: a prospective study with quantitative and semiquantitative cultures of catheter hub and segments a randomized trial on the effect of tubing changes on hub contamination and catheter sepsis during parenteral nutrition contamination of stopcocks mounted in administration sets for central venous catheters with replacement at 24 hrs versus 72 hrs: a prospective cohort study use of disinfectants to reduce microbial contamination of hubs of vascular catheters effectiveness of disinfectant techniques on intravenous tubing latex injection ports a randomized, prospective clinical trial to assess the potential infection risk associated with the posiflow ® needleless connector scrub the hub': cleaning duration and reduction in bacterial load on central venous catheters successful disinfection of needleless access ports: a matter of time and friction antibiotic-based catheter lock solutions for prevention of catheter-related bloodstream infection: a systematic review of randomised controlled trials prevention of catheter-related bacteremia with a daily ethanol lock in patients with tunnelled catheters: a randomized, placebo-controlled trial guidelines for the prevention of intravascular-catheter-related infections can high-level mupirocin resistance reporting be relied upon to ensure patients are prescribed appropriate treatment? prophylactic antibiotics for preventing early central venous catheter gram positive infections in oncology patients heparin bonding reduces thrombogenicity of pulmonaryartery catheters central venous access sites for the prevention of venous thrombosis, stenosis and infection the relationship between the thrombotic and infectious complications of central venous catheters thrombosis as a complication of pulmonary-artery catheterization via the internal jugular vein: prospective evaluation by phlebography central vein thrombosis associated with intravenous feeding -a prospective study a cross-sectional study of catheter-related thrombosis in children receiving total parenteral nutrition at home catheter-related thrombosis in critically ill children: comparison of catheters with and without heparin bonding a prospective study of femoral catheter-related thrombosis in children beneà t of heparin in central venous and pulmonary artery catheters: a meta-analysis of randomized controlled trials very low doses of warfarin can prevent thrombosis in central venous catheters. a randomized prospective trial randomized trial of prevention of catheter-related bloodstream infection by continuous infusion of low-dose unfractionated heparin in patients with hematologic and oncologic disease heparin-bonded central venous lines reduce thrombotic and infective complications in critically ill children surface heparinization of central venous catheters reduces microbial colonization in vitro and in vivo: results from a prospective, randomized trial use of heparincoated central venous lines to prevent catheter-related bloodstream infection heparin-bonded catheters for prolonging the patency of central venous catheters in children prevention of central venous catheter associated thrombosis using minidose warfarin in patients with haematological malignancies anticoagulation for thrombosis prophylaxis in cancer patients with central venous catheters anticoagulation in patients with cancer: an overview of reviews thrombosis prophylaxis in patient populations with a central venous catheter: a systematic review the heparin á ush syndrome: a cause of iatrogenic hemorrhage beneà t of heparin in peripheral venous and arterial catheters: systematic review and meta-analysis of randomised controlled trials a meta-analysis of effects of heparin á ush and saline á ush: quality and cost implications analysis of the research about heparinized versus nonheparinized intravascular lines heparin á ushing and other interventions to maintain patency of central venous catheters: a systematic review heparin or 0.9% sodium chloride to maintain central venous catheter patency: a randomized trial intermittent á ushing with heparin versus saline for maintenance of peripheral intravenous catheters in a medical department: a pragmatic clusterrandomized controlled study outbreak of bloodstream infection temporally associated with the use of an intravascular needleless valve increased rate of catheter-related bloodstream infection associated with use of a needleless mechanical valve device at a long-term acute care hospital increased catheterrelated bloodstream infection rates after the introduction of a new mechanical valve intravenous access port incidence of catheterrelated bloodstream infection among patients with a needleless, mechanical valve-based intravenous connector in an australian hematology-oncology unit optimal timing for intravenous administration set replacement saving lives: a delivery programme to reduce healthcare associated infection including mrsa. london: department of health eliminating catheter-related bloodstream infections in the intensive care unit an intervention to decrease catheter-related bloodstream infections in the icu sustaining reductions in catheter related bloodstream infections in michigan intensive care units: observational study results of a multicentre randomised controlled trial of statistical process control charts and structured diagnostic tools to reduce ward-acquired meticillin-resistant staphylococcus aureus: the chart project a multifaceted intervention for quality improvement in a network of intensive care units: a cluster randomized trial effectiveness of stepwise interventions targeted to decrease central catheterassociated bloodstream infections matching michigan': a 2-year stepped interventional programme to minimise central venous catheter-blood stream infections in intensive care units in england costeffectiveness of a central venous catheter care bundle the 100,000 lives campaign: setting a goal and deadline for improving healthcare quality an initial search was made for national and international guidelines and systematic reviews of randomised controlled trials. search questions were based on the scope of the original review and advice from the guideline development group. databases to be searched were identià ed together with search strategy [i.e. relevant medical subject headings (mesh), free-text and thesaurus terms]. abstracts of all articles retrieved from the search were reviewed against pre-determined inclusion criteria (e.g. relevant to a review question, primary research/systematic review/meta-analysis, written in english). full text of all articles that met the inclusion criteria was reviewed against pre-determined criteria to identify primary research which answers review questions. all articles that described primary research, a systematic review or a meta-analysis were critically appraised by two experienced appraisers. consensus and grading was achieved through discussion in the context of pre-determined grading criteria. the following organisations were approached for comment: a chemical compound that contains 'energy-rich bonds' and is used by cells to store and deliver energy an organism that requires free oxygen for life and growth a hand decontamination preparation based on alcohol that, for the purposes of these guidelines, encompasses solutions, gels or wipes antimicrobial a substance that kills or inhibits the growth of microorganisms the absence of pathogenic microorganisms antiseptic a substance that destroys or inhibits the growth of microorganisms and is sufà ciently non-toxic to be applied to skin or mucous membranes a framework for the aseptic technique based on the concept of deà ning key parts and key sites to be protected from contamination. a carefully controlled procedure that aims to prevent contamination by microorganisms the presence of microorganisms in the bloodstream the presence of microorganisms in the urine. if there are no symptoms of infection, this is called 'asymptomatic bacteriuria' a complex structure comprising microorganisms and extracellular polymers that forms over surfaces, such as those in contact with water or tissues continuous á ow of a solution through the bladder to remove clots or debris a viral infection transmitted by exposure to blood and sometimes other bodily á uids. bloodborne viruses include hepatitis b and c as well as human immunodeà ciency virus the presence of microbes in the blood with symptoms of infection an analytical observational study that compares people with the disease of interest with a group of similar 'control' people who do not in order to determine potential causes or risk factors a scientià c article that describes an individual case in detail a report describing a series of several similar events the presence of symptoms or signs attributable to microorganisms that have infection (cauti)invaded the urinary tract, where the patient has, or has recently had, a urinary catheter microorganisms present on a surface of a catheter that could potentially lead to infection an infection of the bloodstream where microorganisms are found in the blood infection (cr-bsi) of a patient with a central venous access device, the patient has clinical signs of infection (e.g. fever, chills and hypotension) and there is no other apparent source for the infection. for surveillance purposes, this often refers to bloodstream infections that occur in patients with a central venous access device and where other possible sources of infection have been excluded. a more rigorous deà nition is where the same microorganism is cultured from the tip of the catheter as grown from the blood; simultaneous quantitative blood cultures with at least a 5:1 ratio of microorganisms cultured from the central venous access device vs peripheral; differential time to positivity of at least 2 h for blood cultures cultured peripherally vs from central venous access device waste material that consists wholly or partly of human or animal tissue, blood or body á uids, excretions, drugs or other pharmaceutical products, swabs/ dressings, syringes, needles or other sharp instruments closed urinary drainage system a system where a urinary catheter is connected via tubing to a collecting bag.the system relies on gravity to drain the urine a prospective or retrospective follow-up study where groups to be followed-up are deà ned on the basis of presence or absence of exposure to a risk factor or intervention microorganisms that establish themselves in a particular environment, such as a body surface, without producing disease an estimate of the number of viable bacterial cells made by counting visible colonies derived from the replication of a single microbial cell transmission of a pathogenic organism from one person to another a comparison of the outcome between two or more groups of patients that are exposed to different regimens of treatment/intervention where the groups exchange treatment/intervention after a pre-arranged period a process that removes hazardous substances, including chemicals or microorganisms a cleansing agent that removes dirt from a surface by bonding with lipids and other particles a process that reduces the number of pathogenic microorganisms to a level at which they are not able to cause harm, but which does not usually destroy spores particles 1-10 õm in diameter comprising the dried residue formed by evaporation of droplets coughed or sneezed from the respiratory tract difà cult or painful urination urinary proteins, salts and crystals that adhere to the internal and external surface of a urinary catheter the use of equipment designed to prevent injury to the operator administration of nutrients into stomach or other part of the gastrointestinal tract using tubes infections caused by microorganisms acquired from another person, animal or the environment opinion derived from seminal works and appraised national and international guidelines the type of bacteria as identià ed by gram's staining method. gram-positive bacteria appear dark blue or purple under a microscope. such bacteria have a thick layer of peptidoglycan on their cell walls. gram-negative bacteria appear red under a microscope and have an outer layer of lipoprotein and a thin layer of peptidoglycan a wire used to facilitate insertion of the intravascular catheter into the body blood in the pleural cavity, usually due to injury. if the blood is not drained, it may impair the movement of the lungs or become infected the use of soap and water or an antiseptic solution to reduce the number of microorganisms on the hands a phenomenon in which the participants change their behaviour or performance in response to being studied infection acquired as a result of the delivery of health care either in an acute (hospital) or non-acute setting any person employed by a health service, social service, local authority or agency to provide care for sick, disabled or elderly people a central venous access device that is tunnelled under the skin with a subcutaneous port or reservoir with a self-sealing septum that is accessible by needle puncture through intact skin the number of new events (e.g. cases of disease) occurring in a population over a deà ned period of time a catheter inserted into the bladder via the urethra and left in place for a period of time microorganisms that have entered the body and are multiplying in the tissues, typically causing specià c symptoms an analysis in which the results of the study are based on initial treatment assignment and not on a treatment actually received a study in which measurements from the group under investigation are taken repeatedly before and after the intervention a device inserted into a vascular system in order to administer á uids, medicines and nutrients or to obtain blood samples. these include devices inserted peripherally, as well as those inserted into larger veins any device that requires insertion through skin or other normal body defences a system of attaching catheters, syringes, tubes and any other components of ivad to each other external opening of the urethra the combination of data from several studies to produce a single estimate of an effect of a particular intervention strains of s. aureus that are resistant to many of the antibiotics commonly staphylococcus aureus (mrsa) used to treat infections. epidemic strains also have a capacity to spread easily from person to person a long peripheral venous catheter inserted in the antecubital vein and advanced to a vein in the upper arm. designed for short-term (up to 4 weeks) intravenous access a membrane lining many tubular structures and cavities such as respiratory tractneedle-free devices (also needleless intravascular connector systems developed to help reduce the incidence of intravascular catheter connectors) needlestick injury while facilitating medication delivery through intravascular catheters. there are three types of needle-free connectors: blunt cannula (two-piece) systems, one-piece needle-free systems, and one-piece needlefree systems with positive pressure needle safety device (also needle any device designed to reduce the risk of injury associated with a protection/prevention device) contaminated needle. this may include needle-free devices or mechanisms on a needle, such as an automated resheathing device, that cover the needle immediately after use the puncture of skin by a contaminated needle or other sharp medical device abnormal decrease in the number of neutrophils in peripheral blood, which results in increased susceptibility to infections nitrile a synthetic rubber made from organic compounds and cyanide a retrospective or prospective study in which the investigator observes participants, with or without control groups any derivative of a living or once-living organism two or more cases of the same disease where there is evidence of an epidemiological link between them administration of nutrients by an infusion into a vein h. p. loveday et al. / journal of hospital infection 86s1 (2014) s1-s70 particulate à lter masks (or respirator masks) face masks designed to protect the wearer from inhaling airborne particles including microorganisms. they are made to deà ned performance standards that include à ltration efà ciency. to be effective, they must be à tted close to the face to minimise leakage pathogen a microorganism that causes disease an independent assessment or evaluation of the research by a professional with knowledge of the à eld an injury that results in a sharp instrument/object (e.g. needle, scalpel) puncturing the skin a vascular catheter inserted into the superior vena cava from the basilic or catheter (picc) cephalic vein specialised clothing or equipment worn to protect against substances or situations that present a hazard to health or safety post-exposure prophylaxis drug treatment regimen administered as soon as possible after an occupational exposure to reduce the risk of acquisition of a bloodborne virus a topical preparation used for antisepsis of the skin in a form of solution or ointment the number of events (e.g. cases of disease) present in a deà ned population at one point in time study in which people are entered into the research and then followed-up over a period of time with events recorded as they happen a small, á exible tube placed into a peripheral vein for the safe infusion of medications, hydration á uids, blood products and nutritional supplements quasi-experimental research designs specià cally lack the element of random assignment of participants (individuals or clinical settings/units) to the treatment or the control group. randomisation minimises the risk that patients entered into the control and treatment groups will be different an rct is a clinical trial where at least two treatment groups are compared, non-randomised controlled trial (nrct) one of them serving as the control group. allocation to the group uses a random, unbiased method. an nrct compares a control and treatment group but allocation to each group is not random. bias is more likely to occur in an nrct microorganisms that live in the deeper crevices of skin and hair follicles. these form part of the normal á ora of the body and are not readily transferred to other people or objects, or removed by the mechanical action of soap and water. they can be reduced in number with the use of antiseptic soap a mask that covers the mouth and nose to prevent droplets from the wearer being expelled into the environment. as they are also á uid repellent, they provide some protection for the wearer against exposure of mucous membranes to splashes of blood/body á uid research that summarises the evidence on a clear question according to a deà ned protocol using explicit and systematic methods to identify, select and appraise relevant studies and extract, collate and report their à ndings an infection where the pathogen is distributed throughout the body, rather than being concentrated in one area the decontamination of a room or patient area after a patient has been transferred or discharged in order to ensure that any dirt, dust or contamination by potentially pathogenic microorganisms is removed before use by another patient a reduction in the number of platelets (thrombocytes) in the blood. this may result in bleeding into the skin, spontaneous bruising or prolonged bleeding after injury a clot in a blood vessel caused by coagulation of blood phlebitis (vein iná ammation) related to a thrombus (blood clot) microorganisms acquired on the skin through contact with surfaces. the hostile environment of skin means that they can usually only survive for a short time, but they are readily transferred to other surfaces touched. can be removed by washing with soap and water, and most are destroyed by alcohol-based hand rubs the invasion of the tissues of the bladder by microorganisms causing symptoms or signs of infection such as dysuria, loin pain, suprapubic tenderness, fever, pyuria and confusion key: cord-031252-ji0ef0by authors: d'angelo, lawrence title: infectious disease problems in adolescents date: 2020-09-01 journal: j adolesc health care doi: 10.1016/s0197-0070(20)30007-3 sha: doc_id: 31252 cord_uid: ji0ef0by nan accounts for approximately 21% of the cases, and ebv infection, which accounts for 1-5% of the cases (12) . the rest of the cases are presumed to be viral, with the exception of a small percentage caused by neisseria gonorrhoeae (13) . it is difficult to determine the etiology of pharyngitis and to propose a course of therapy on the basis of clinical findings (14) . although illness is usually self-limited, its duration is influenced by the etiologic agent and the prescribed therapy. for example, the clinical course of a streptococcal infection is shortened significantly when appropriate antibiotics are prescribed early (15) . throat cultures are advisable for patients whose primary symptoms are throat pain, fever, and cervical lymphadenopathy or exudative pharyngitis. the optimal culture site is the tonsillar surface (16) . direct-swab rapid diagnostic tests for group a streptococcus, based on enzyme-linked immunosorbent assays (elisa technique), have been well evaluated for office use and allow the practitioner to initiate antibiotic therapy, when necessary, early in the cour.se of the illness (17) . therapy for pharyngitis is determined by the nature of the etiologic agent. the best example of a cause amenable to therapy is streptococcal infection, which ideally is treated with penicillin, either orally (250 mg of phenoxymethyl penicillin qid) or by a single intramuscular injection of benzathine penicillin (1.2 million units). gonococcal pharyngitis is best treated with 4.8 million units of intramuscular procaine penicillin g administered 30 minutes after a 1.0-g dose of oral probenecid. the most significant complication of pharyngitis is peritonsillar abscess (18) . primarily a disease of adolescents and young adults, peritonsillar abscess is characterized by dysarthria, trismus, and discomfort in swallowing. in addition to high doses of antibiotics (aqueous penicillin g, 2-4 million units q 4 hr), patients require incision and drainage, often followed by a tonsillectomy. otitis media is the second leading reason that patients under age 15 seek medical care, although it is far less common in older adolescents (19) . bacterial etiologic agents of otitis media include streptococcus pneumoniae, hemophilus influenzae, streptococcus pyogenes group a, branhamella catarrhalis, and staphylococcus aureus. a number of respiratory viruses have likewise been implicated as etiologic agents in this illness. as with many disorders, the median duration of symptoms depends upon the promptness of diagnosis and the method of treatment (20) . antibiotic therapy with amoxicillin (500 mg tid), trimethoprim-sulfamethoxazole (one double-strength tablet bid), or cefaclor (250 mg tid) is appropriate in the adolescent. with treatment, complications are rare. the sinuses are affected when the normal drainage of mucus and other secretions into the nasal passages is hindered. the usual pathogens include s. pneumoniae, h. influenzae, anaerobic organisms, mixed bacteria, s. aureus, s. pyogenes, b. catarrhalis, gram-negative bacteria, influenza, and parainfluenza. the incidence of these pathogens has varied from study to study. sinusitis can develop in the course of any upper respiratory infection. it is often associated with facial pain (periorbital or supraorbital) and purulent nasal discharge. headache and nasal obstruction, which alters the patient's sense of taste and smell, may also be present. only about half of patients have an elevated temperature. eyelid edema and tearing are indicative of ethmoid involvement. cough, particularly daytime cough, is an important sign (21) . the clinical diagnosis is based on the presence of sinus tenderness, purulent nasal discharge, and daytime cough of at least ten days' duration. although opacity of the sinus on transillumination is a helpful finding, transillumination is not a consistently reliable diagnostic technique (22) . radiography is the most specific test for sinusitis. mucosal thickening is usually visible, and the presence of airfluid levels in the sinus cavity confirms the diagnosis. once again, the duration of illness is determined by the speed of diagnosis and therapy. appropriate therapy includes antibiotics and local as well as systemic decongestants. with topical decongestants care must be taken to avoid the rebound phenomenon (rhinitis medicamentosa). amoxicillin (500 mg tid) is the most frequently prescribed antibiotic for initial oral therapy in outpatients. trimethoprimsulfamethoxazole and cefaclor serve as second-line agents. symptomatic relief of pain is best provided by acetaminophen. teenagers are at considerable risk of developing complications of sinusitis because their frontal sinuses are rather recently developed. complications include meningitis, brain abscess, orbital cellulitis osteomyelitis, and cavernous sinus thrombosis. for this reason, if a prompt response to outpatient oral therapy does not occur, hospitalization to enable in-travenous administration of antibiotics (i.e., chloramphenicol and oxacillin or cefuroxime) should be considered. the usual pathogens causing pneumonia include m. pneumoniae (which accounts for 50% of the cases), s. neul11oniae, and viruses such as influenza, parainfluenza, and adenovirus. pneumocusiis carinii, al-t~ough rare, must be considered in patients at high risk for human t-celllymphotropic virus (htlv)-iii infection. cough and fever are the most common clinical characteristics of pneumonia. the nature of the fever can sometimes suggest etiology. for example, fever associated with multiple chills suggests m. pneumoniae, whereas a single, shaking chill is more characteristic of s. pneumoniae. leukocytosis can also suggest etiology; it is less common with viruses and mycoplasmal infection than with bacterial pneumonia. the median duration of penumonia is 14 days, although adolescents may recover much sooner. teenagers can generally resume normal activities after being afebrile for 48 hours. diagnostic tests for pneumonia include a chest xray and sputum examination and culture. in adolescents, however, the routine sputum culture will not pr~vide a diagnosis for 70-80% of community-acquired pneumonias. similarly, a gram stain is often nondiagnostic and may be even less helpful than a routine culture (23) . even chest x-rays are less useful than clinical diagnosis based on an appropriate history and physical examination (24) . treatment of pneumonia is based on the most probable etiologic organism. most adolescents are candidates for an outpatient antibiotic regimen. erythromycin, 500 mg q 6 hr, is appropriate. patients with an underlying disease (i.e., sickle cell disease, malignancy) or concomitant bronchoconstriction, as well as those who fail to respond to an outpatient regimen, may require hospitalization. because complications from pneumonia are rare in adolescents, the presence of complications (i.e., empyema, lung abscess, or bacteremia) may be indicative of an unclerlying health problem. influenza is an acute, usually self-limiting, febrile illness that occurs in outbreaks of varying severity al-most every winter. adolescents play a major role in the spread of influenza and frequently account for the first cases (25) . for example, the first few cases of swine flu in new jersey in 1976 occurred in teenage army recruits at fort dix (26) . these cases gave rise to a national vaccination program. although two distinct strains of the influenza virus, a and b, can infect humans, they cannot be distinguished on the basis of clinical signs and symptoms. patients with either type of influenza usually have systemic symptoms, including fever, a chilly feeling or frank shaking chills, headache, myalgia, malaise, and anorexia. these symptoms progress rapidly to include dry cough, nasal discharge, and stuffiness. the median duration of illness is 10-14 days, depending on prior immunity and whether the patient receives the antiviral agent amantadine hydrochloride. diagnosis is based almost entirely on clinical findings as cultures are expensive and the results are not immediately available. preventing infection via strain-specific vaccine or prophylactic amantadine provides the most efficacious approach to influenza outbreaks (27) . adolescents with chronic cardiac or respiratory illness (including severe asthma), severe anemia, immunocompromise, or severe renal or metabolic illness should be given the vaccine. when prevention is impossible or unsuccessful, treatment with amantadine can shorten the duration of fever and systemic and respiratory symptoms by about 50% if the illness is caused by type a virus and the drug therapy is initiated within the first 48 hours of infection. although complications in adolescents are rare, influenza can cause croup, chronic obstructive pulmonary disease, bacterial pneumonia, myositis, and myocarditis. many of the 21 million deaths in the 1918-1919 outbreak were due to viral pneumonia in otherwise healthy young adults (28) . the most dramatic and dangerous complication in adolescents is reye's syndrome, which has been reported in the convalescent phase of the flu since the early 1970s (29) . there is apparently a strong epidemiologic association between salicylate intake and reye's syndrome (30) , with the majority of recently reported cases occurring in adolescents (8) . mycoplasma pneumoniae is responsible for 50% of cases of pneumonia in adolescents and young adults. this organism also causes many other clinical syndromes, including tracheobronchitis, myringitis, pharyngitis, gastroenteritis, hepatitis, meningitis, and myocarditis (31) . headache and multiple chills are the most important symptoms of pneumonia caused by mycoplasmal infection. the course of the illness is often protracted, and x-rays may not clear for two to six weeks (32) . diagnostic tests for pneumonia rarely reveal the etiology of the illness. x-rays may show a unilateral, lower-lobe, segmental bronchopneumonia. the peripheralleukocyte count is equivocal; it may be normal, depressed, or elevated. cold agglutinins are usually present, but in varying titer. complement fixation is the most specific diagnostic test, but it lacks sensitivity, particularly early in the illness. definitive diagnosis of mycoplasmal pneumonia is based on the combination of an antibody rise and a sputum culture showing m. pneumoniae. however, growth on agar may take two to three weeks, rendering results useless for initiating drug therapy. the treatment options for pneumonia caused by mycoplasmal infection are straightforward as the organism is sensitive to both tetracycline and erythromycin (500 mg of either medication qid). erythromycin is a particularly attractive therapeutic agent because it is equally effective against other common pneumonia pathogens. pleural effusions and spread of infection within the lung are the most common pulmonary complications of mycoplasmal pneumonia, but they are uncommon in the adolescent population. death from pneumonia in adolescents is extraordinarily rare. several points about anticipatory guidance must be emphasized. first, supportive therapy is the most important part of treatment for the adolescent with undifferentiated respiratory symptoms. most adolescents with respiratory infections will not require antibiotic therapy. second, adolescents with chronic health problems must be appropriately immunized. at this time, the most useful immunizing agent is for influenza. finally, because the salicylates are associated with an increased incidence of reye's syndrome (8, 29, 30) , adolescents with respiratory infections and/or fever should be treated with acetaminophen rather than aspirin. mononucleosis is an acute infection most commonly caused by the epstein-barr virus, a member of the herpesvirus group. the ebv was first isolated in tissue culture cell lines derived from burkitt's lyrn-phoma. the observation of ebv antibody seroconversion in a laboratory technician with infectious mononucleosis led to the etiologic connection between ebv and the mononucleosis syndrome (33) . the incidence and prevalence of infectious mononucleosis are greatest in closed groups of adolescents and young adults, i.e., at boarding schools, detention centers, colleges, and military reservations. the illness causes significant morbidity and even mortality in some individuals. the mononucleosis syndrome presents with a classic triad of sore throat, fever, and cervicallymphadenopathy, all of which can have severe clinical manifestations. other characteristic symptoms include malaise, headache, fatigue, and anorexia. splenomegaly occurs in approximately 50% of patients and is greatest during the second and third weeks of illness. illness usually lasts for two to three weeks, although fatigue and exercise-induced exhaustion can remain for several months. epstein-barr virus infections are a widespread phenomonon, particularly among people in lower socioeconomic groups and third-world countries (34) (35) (36) . in general, the incidence of mononucleosis is greatest in individuals who have not encountered the virus prior to adolescence, although ebv can cause illness in younger children (3) . individuals who enter a high-risk setting without previous exposure to the virus have a seroconversion rate of 12% per annum (37, 38) . by adulthood, 90-95% of all persons have antibodies to ebv (39) . initial viral replication takes place in the pharyngeal epithelial cells (40) , and the virus can be recovered in oropharyngeal secretions for prolonged periods (41) . exchange of infected saliva during intimate oral contact or from inanimate objects, such as eating and drinking utensils, is the usual mode of transmission (42, 43) . the ebv appears to be transmitted only minimally in standard living situations. like other members of the herpesvirus group, ebv can be maintained in a host in a latent form until it is activated or transformed, which often occurs under conditions of immunosuppression (44, 45) . this latency period accounts for the occurrence of illness or viral replication in renal transplant patients and may explain the recently described virologic findings in chronic mononucleosis syndrome (46, 47) . in adolescents and young adults, other infectious agents can cause illnesses that mimic ebv-induced mononucleosis. among these agents are cytomegalovirus, toxoplasma gondii, treponema pallidum (secondary stage syphilis), rubella virus, adenovirus, the hepatitis viruses, and s. pyogenes. noninfectious diseases that mimic ebv-induced mononucleosis include serum sickness, systemic lupus erythematosus, sarcoidosis, and lymphoma. mononucleosis is diagnosed by clinical manifestations and laboratory findings. diagnostic abnormalities in routine laboratory procedures include 1) relative or absolute lymphocytosis, 2) atypical lymphocytes (more than 10% of total lymphocytes), 3) relative or absolute neutropenia (48), 4) mild thrombocytopenia, 5) mild transaminitis, with or without overt hepatitis (49) , and 6) mild elevation of cryoglobulins. there are also specific tests for ebv antibody. heterophil antibodies, mostly of the igm class, develop transiently during the course of infectious mononucleosis and are seen in 90-95% of the cases associated with ebv. these antibodies, first described in 1932by paul and bunnell, are antibodies to antigens on cell surfaces of nonhuman species (50) . the monospot test, a single-dilution slide test that uses sensitized horse red blood cells, is almost as sensitive as the heterophil test (51) and is very useful in screening for these antibodies. although this test gives quick results and is generally specific for infectious mononucleosis, false-negative results can occur when heterophil antibody titers are low; false-positive results also occasionally occur. the clinical finding most perdictive of a positive heterophil test is the presence of axillary adenopathy (52) . the ebv-specific serologic tests may be diagnostic in the small percentage of patients who are heterophil negative (53) . these tests consist of measuring antibodies to the viral capsid antigen (vca), the viral early antigens (ea), and the viral nuclear antigens (ebna). the vca is the most common ebv-specific assay, and evidence of igm and igg antibodies is of value in making the serodiagnosis of primary ebv infection. the ea test measures two separate antigens and their respective antibodies-anti-d and anti-r-which are observed in 70-85% of patients in the acute phase of infectious mononucleosis (33) . the viral nuclear antigens appear late in the course of infectious mononucleosis, and positive ebna can document ebv infection when early serum samples are not available. supportive therapy with appropriate guidance is the best approach to treat infectious mononucleosis. sore throat and fever should be treated with antipyre tics, but because of the occasional presence of relative thrombocytopenia, aspirin should be avoided. steroids should be reserved for patients with severe, life-threatening complications, such as airway obstruction, neurologic complications, thrombocytopenia, hemolytic anemia, or myocarditis. antiviral agents have not yet been shown to have a therapeutic role, although intravenous acyclovir may eventually prove useful for serious infections (54, 55) . most complications of the mononucleosis syndrome resolve without significant sequelae. possible complications can best be grouped by organ system. hematologic complications, namely, autoimmu ne hemolytic anemia and severe thrombocytopenia, occur in up to 5% of patients (56,57). these disorders are usually self-limited but may respond to corticosteroids (58) . splenic rupture, a rare but serious complication, is most often associated with trauma (59), although it can occur spontaneously (60) . cardiorespiratory complications, including myocarditis, pericarditis, and pneumonia, have been reported in adolescents but occur more commonly in children (61, 62) . neurologic complications occur in less than 1% of cases of infectious mononucleosis (63) . when these complications do occur, they are frequently the primary manifestation of the illness. complications of the central nervous system include guillain-barre syndrome, bell's palsy, transverse myelitis, and meningoencephalitis myelitis. fortunately, most of these complications will remit without lasting neurologic deficit. death from mononucleosis is rare. it is most frequently associated with x-linked immunodeficiency (64) . viral-related malignancies such as african burkitt's syndrome (65, 66) and nasopharyngeal carcinoma (67) may also occur. the role of ebv as a cause of lymphomas and hodgkin's disease is under investigation (68, 69) . advising adolescents of ways to avoid contracting infectious mononucleosis is difficult. health practitioners can share certain information with patients and parents that will help them to cope with the disease in themselves and others. for example, supportive care is the hallmark of rehabilitative services. patients with infectious mononucleosis do not require isolation as the disease is poorly transmitted from person to person (70) . platelet abnormalities occur in a small percentage of patients, and therefore acetaminophen is preferred for symptomatic relief of fever and myalgia. finally, because of the risk of subclinical splenic enlargement, patients should avoid contact sports and strenuous activities for at least four weeks after signs and symptoms of infection remit. viral hepatitis is most frequently caused by one of three types of agent: hepatitis virus a, hepatitis virus b, and the so-called non-a, non-b agents, of which here appear to be at least two. individuals between the ages of 10 and 20 comprise 17.4% of hepatitis a cases, 9.3% of hepatitis bcases, and 11.1% of non-a, non-b hepatitis cases (70) . in 1983 alone, 1,360 cases of hepatitis a in teenagers were reported, 829 cases of hepatitis b, and 301 cases of non-a, non-b hepatitis (71) . all of these hepatitis forms have worldwide distribution. acquisition of the antibody to the hepatitis virus is closely linked to socioeconomic status and geographic location (72, 73) . as with mononucleo-, sis, individuals aquire antibodies for these viruses during childhood, usually via asymptomatic infecttion. ' personal contact with someone who has hepatitis a is the most important risk factor for acquisition of symptomatic illness (71) . for hepatitis b, the risk factors are personal contact with an infected individual, intravenous drug abuse, homosexual activity in males, recent dental work, and recent transfusion of blood or blood products. for non-a, non-b hepatitis, the risk factors are surgery, hospitalization, dental .work, and drug abuse. the classic mode of transmission of hepatitis a is by fecal-oral contact. attendance at day-care centers is currently the most important cause of the spread of this disease in both children and adults in north america, accounting for approximately 10% of cases (74) . young children acquire the infection (usually asymptomatically) and spread it to staff, siblings, and parents. hepatitis b and non-a, non-b hepatitis are usually spread through the blood or body secretions. hepatitis a is a more frequent cause of epidemics in adolescents than is hepatitis b (75,76). spread of hepatitis b among classroom contacts (77) and teenage wrestlers has been reported (78) . the type of hepatitis is difficult to distinguish by the clinical symptoms alone. all forms usually have a prodromal phase, characterized by fever, malaise, fatigue, headache, anorexia, nausea, vomiting, myalgias, and/or right upper quadrant tenderness. in general, the younger the patient, the less severe the manifestations of the illness (79) . nearly 80% of the patients with viral hepatitis develop jaundice (71) . in fact, because hepatitis is most specifically suggested by scleral and skin jaundice, many cases without jaundice may go undiagnosed or unreported. the clinical course of the illness varies but usually lasts for one to three weeks (80). although clinical manifestations alone will not reveal the etiology of the hepatitis virus, certain charac-,teristics are specific to each form of the illness. the incubation period for hepatitis a is 14-50 days, with a mean of 28 days. diarrhea is more frequent in type a than in any other type, occurring in approximately 20% of patients. upper respiratory symptoms, encephalitis, and aplastic anemia, although rare, occur more often with hepatitis a. hepatitis busually has an incubation period of 60-110 days (81) . more than the other types, it is associated with extrahepatic manifestations, including urticaria and arthritis, and, much less commonly, glomerulonephritis and vasculitis. in about 10% of patients with symptomatic hepatitis b, serologic tests continue to reveal markers of acute infection. most of these patients continue to have mildly elevated liver-associated enzymes, which are indicative of chronic persistent hepatitis (81) . this generally benign, nonprogressive disease is characterized by chronic portal inflammation, with little or no fibrosis. some patients develop cirrhosis. diagnosis of the non-a, non-b forms of hepatitis is essentially done by exclusion. the incubation period varies from two weeks to six months, with a mean of about eight weeks. half of the cases of non-a, non-b hepatitis are anicteric. although there are rare instances of chronicity, the course is usually benign (82) . hepatitis is usually diagnosed by clinical signs and symptoms-especially jaundice-and by laboratory evidence of elevated liver-associated enzymes. specific serologic tests for etiology of viral hepati-tis are now available, with certain limitations. the presence of serologic markers precedes the clinical illness itself. type a markers are antibodies directed at specific viral proteins. these antibodies are of igm and igg classes. the presence of the former suggests acute infection. the markers for hepatitis b are more complicated; they include the hepatitis b surface antigen (hbsag) and its antibody, anti-hbs, as well as core antigen antibody (anti-hbc), e antigen, and its antibody (anti-e). in the classic pattern both hbsag and anti-hbc are acutely positive, but this pattern may not occur in all cases. anti-hbc is almost always positive, but a single negative test for hbsag does not definitely exclude acute hepatitis b (83) . the persistence of antigen markers without concomitant antibody suggests chronic infection (84). specific serologic markers are available for non-a, non-b hepatitis. in the absence of specific treatment for acute viral hepatitis, the major emphasis is placed on support, symptomatic care, and prevention of transmission. there is no convincing evidence to justify the use of corticosteroids in acute hepatitis, regardless of its severity (85); prophylaxis for hepatitis a involves passive immunization with gamma globulin (86) for all close contacts of the infected patient. patients exposed to hepatitis b can be given a safe, effective vaccine (87) , which consists of highly purified and triple-inactivated hbsag obtained from the serum of chronic carriers (88). in some exposure situations, the hepatitis b vaccine is used in combination with a special high-titer immune globulin-hepatitis b immune globulin (hbig)-to allow for immediate as well as continuing protection (89) . the overall incidence of complications from viral hepatitis is low. complications are limited to those problems already discussed in the review of the specific clinical presentations. mortality rates vary from 0.2-2.0%. fatalities are less frequent with type a than with type bor non-a, non-b hepatitis. the overall mortality rate for viral hepatitis is about 1%. death can occur rapidly in patients with fulminant hepatitis (90) . to prevent hepatitis a: 1) limit the amount of raw shell fish eaten; 2) administer appropriate immune serum globulin (isg) when possible exposure has occurred; and 3) give prophylactic isg to patients traveling to third-world countries. to prevent hepatitis b: 1) promptly administer hbig and the hepatitis b vaccine to individuals with known exposure; 2) check the serologic status of adolescents with highrisk lifestyles (i.e., male homosexuals and intravenous drug users) and vaccinate those who are not protected; and 3) properly screen all blood products before use. at puberty, preadolescents undergo physiological changes that transform them into sexually mature individuals. the accompanying psychological and social changes frequently result in sexual activity. eighty percent of males and 70% of females have had intercourse during their teenage years (91) , and the average age of first sexual intercourse is 16. during these years of bodily change and emerging sexuality, symptoms attributed to the genital organs become a special concerrt of adolescents. some of the most common presenting complaints include urinary tract infections, vaginal discharge, urethral discharge, and genital lesions. in sexually active teen" agers, these complaints are especially important because they are frequently caused by sexually transmitted diseases (stds). an awareness of the most likely etiologies of each of these conditions will facilitate differential diagnoses arid medical management of urogenital disease. urinary tract infections (uti) are among the major reasons for physician visits by adolescent females. in women, the usual etiological organism is escherichia coli, but in adolescent and young adult females, staphylococcus saprophyticus is also common. chlamydia trachomaiis is a frequent cause of dysuria and acute urethal syndrome (92) . the risk factors for urinary tract infections include sexual intercourse, pregnancy, behavioral factors (i.e., diaphragm use (93) and voluntary urinary retention (94)), anatomical abnormalities, and underlying disease. the diagnosis of uti is. based on both clinical symptoms and laboratory criteria. the clinical symptoms of lower tract infection (cystitis and urethritis) include frequent urination, dysuria,â· burning pain during urination, suprapubic discomfort, and passage of cloudy and occasionally blood-tinged urine. when fever, flank or back pain, and rigors occur, upper tract involvement must be assumed to be present. because the symptoms of vaginitis often resemble those of uti (95) , it is important to determine quickly the site of infection. a bacteria count of over 100,000 organisms per milliliter of unspun urine is the old standard for making the diagnosis of a uti. new data suggest that this criterion is too restrictive and will not correctly identify women with overt infections (96) . if women are experiencing such symptoms, lower counts, 100-1,000 of organisms per milliliter of unspun urine, should be considered diagnostic of infection. treatment of utis depends upon the location of the infection within the urinary tract. lower-tract infections can usually be treated effectively with oral, single-dose therapy of trimethoprim-sulfarnethoxazole, two to four double-strength tablets. because single-dose therapy will not eradicate upper-tract infections (kidney), failure to respond to short-course therapy will establish a diagnosis of upper-tract in-.fection (97) . upper-tract infections require a 7-14-'day or longer course of therapy with trimethoprimsulfamethoxazole (one double-strength tablet bid for 14 days) or amoxicillin (500 mg po tid) for 14 days. if significant systemic signs and symptoms are present, the patient should receive intravenous antibiotics (gentamicin, 80 mg iv q 8 hr, or ampicillin, 1 g iv q 6 hr). prophylaxis is used to prevent recurrent symptomatic infection (98) . a lower-tract infection that has not been adequately treated can lead to pyelonephritis (99) . stones can result from recurrent infections. but sepsis is fortunately rare in adolescents. anticipatory guidance for utis includes the following: do not retain urine voluntarily, urinate after sexual intercourse, do not leave diaphragm in longer than necessary, drink plenty of fluids, and maintain good personal hygiene. vaginitis and cervicitis are also common in adolescent and young adult females. the rising incidence of vaginal discharge results from the physiological and behavioral changes that occur at puberty. hormonal changes and ovulation make the vagina and cervix more vulnerable to infection by a number of pathogenic organisms; sexual activity, which results from behavioral changes, makes the female even more susceptible to infection. a careful history of sexual activity and use of contraception must be obtained, and a complete pelvic exam should be performed. in adolescents the infections that cause vaginal discharge, in order of frequency, include the following: fungal vaginitis, usually caused by candida albicans; trichomoniasis; gardnerella vaginitis; gonococcal vaginitis, caused by neisseria gonorrhoeae; and nonspecific urethritis, caused by c. trachomatis. diagnostic features and therapeutic regimens for these infections are shown in table 1 . the risk factors for vaginitis and cervicitis include sexual activity and mutliple sexual partners, tight clothing, oral contraceptives, underlying illness (diabetes), poor hygiene, and pregnancy (100) . the chlamydia1and gonorrheal sexually transmitted infections can cause pregnancy loss (101, 102) , pelvic inflammatory disease (pid), and a variety of nonlocalized infections. the incidence of complications from vaginitis and cervicitis that are not sexually transmitted, however, is very small. vaginitis or cervicitis caused by either n. gonorrhoeae or c. irachomatis can ascend, causing endometritis, salpingitis, and peritonitis. any combination of these disease entities is commonly known as pelvic inflammatory disease (pid). the incidence of pid in adolescents is great; [20] [21] [22] [23] [24] year olds are the only age group with a higher rate than [15] [16] [17] [18] [19] year olds (103). adolescents face an increased risk of acquiring plo for several reasons. they tend to have a larger number of sexual partners (104) , and their immature anatomy is easily infected by a variety of sexually transmitted pathogens (105) . adolescents' infrequent use of barrier methods of contraception (106) contributes to the problem, as does poor follow-up of stds. diagnosis of pid is based on physical and laboratory findings. the major criteria for clinical diagnosis are a history of abdominal pain, lower abdominal tenderness (especially rebound), cervical motion tenderness, and adnexal tenderness. minor diagnostic criteria include fever, leukocytosis, elevated sedimentation rate, abnormal sonogram, culdocentesis revealing bacteria, and vaginal discharge. three or more major clinical features are necessary for diagnosis. if only three major criteria are identified, two or more minor criteria may be used as supplements (107) . adolescents with pid usually respond well to therapy. the decision to treat the individual as an inpatient or as an outpatient depends upon the severity of illness, a prior history of pid, coincidental pregnancy, the presence of an iud, inability to comply with the outpatient treatment, or a tubo-ovarian mass. some physicians feel that the consequences of inadequate treatment are so severe that all teenagers with pid should receive some of their antibiotic therapy as inpatients (103). inpatient and outpatient regimens for the treatment of pid are shown in table 2 . outpatients should be scheduled for follow-up visits on days 2, 7, and 14 to assess the progress of therapy. complica-tions of pid include tubo-ovarian abscess (108), recurrent infections (109), ectopic pregnancy (110) , and infertility (111) . most of the same infectious etiologies of vaginal discharge in adolescent females can cause urethral discharge and dysuria in adolescent males. as with the dolescent female, physicians must obtain a thorough history of sexual activity, preferences, and use of contraception in adolescent males. a complete genital exam must be performed, with particular attention to the testes and epididymis. urethritis is usually classified as gonococcal or nongonococcal urethritis (ngu). gonococcal urethritis classically presents with dysuria and a copious yellow-green discharge two to seven days after exposure. nongonococcal urethritis has a longer incubation period (one to three weeks) and, frequently, a more indolent onset with scantier discharge and less pain (112) . the symptoms of these two types of urethritis are similar regardless of microbiologic cause. the duration of symptomatic illness varies, depending upon when appropriate therapy has been administered. diagnostic features and therapeutic regimens for these infections are shown in table 3 . .complications of urethritis are rare in adolescents, although urethritis has been known to cause prostatitis (113) and epididymitis. also, in young males, chlamydial infection has been associated with reiter's syndrome in those genetically predisposed to this form of arthritis (114) . most genital lesions encountered in adolescents and young adults are due to stds. the most common lesions are vaginal warts and herpes simplex infection, usually with type ii virus. other causes include syphilis, chancroid, lymphogranuloma venereum, and granuloma inguinale. the clinical characteristics of genital ulceration varies, depending upon the etiologic organism, and diagnosis usually requires laboratory confirmation. diagnostic features and therapeutic regimens are outlined in table 4 . adolescent females account for a disproportionate number of the reported cases of toxic shock syndrome (tss), despite the fact that most teens do not use intravaginal catamenial products (115, 116) . those that do use these products, however, are at a higher risk for developing tss than women who are older and use the same feminine hygiene products, probably because of the immature anatomy of the adolescent genital tract. therefore, clinicians should provide anticipatory guidance aimed directly at preteen patients and their parents. because the adolescent's genital tract is not completely developed until ovulation occurs regularly, teenagers should be cautioned not to use intravaginal catamenial products until two years after the onset of menarche, except under special circumstances or for brief periods of time (117) . these products also should not be used for overnight protection. if intravaginal catamenial products are used, they should be changed at least every four hours regardless of the degree of menstrual flow, and patients who have experienced symptoms suggestive of tss should not use them for at least four years after the illness and only then after appropriate cultures have demonstrated that the s. aureus is no longer present. aids is relatively uncommon in adolescents. as of june 1985, a total of 59 cases had been reported for individuals between the ages of 10 and 19 (118) . risk factors include homosexual or bisexual practices, intravenous drug abuse, hemophilia, history of prior blood transfusion, and haitian heritage (119) . the clinical characteristics of aids are related to a deficiency in t-iymphocytes caused by infection with human t-celllymphotropic virus (htlv-lli). this deficiency in the immune system makes the host susceptible to a number of opportunistic infections, some of the most prominent being p. earl/ill pneumonia, disseminated infections with mycobacterium avium and iniracellularis, cryptococcal meningitis, c. albicans, cryptosporidium diarrhea, and disseminated herpes infections. malignancies such as kaposi's sarcoma and carcinoma of the rectum are seen in older persons but are rare in adolescents. the diagnosis of aids is based on appropriate laboratory findings, particularly the antibody to htlv-iii. there is no known treatment for the underlying immune defects in aids, and there have been no reports of spontaneous reversal of this condition. sexual preferences and orientation must be addressed honestly with teenage males because of the steady increase in the prevalence of aids in the adult male homosexual population. adolescent males need to be advised of the sexual practices that put them at high risk for this fatal disease, such as oralanal or genital-anal contact. males who have had homosexual experiences should be screened for hepatitis b antibody. when results are negative, patients should be offered the hepatitis bvaccine. using barrier contraception (condom) will prevent many sexually transmitted infec-tions in homosexual, as well as heterosexual, individuals. adolescents, like adults, need to be firmly warned about the dangers of sexually transmitted urogenital infections. in an attempt to limit the incidence and complications of stds, particular attention must be paid to a history of sexual activity and preferences, the menstrual cycle in the female, use of contraception in both males and females, and the coexistence of std syndromes. the importance of careful genital and pelvic examinations in the evaluation of adolescents cannot be overemphasized. finally, treatment of stds is incomplete without appropriate patient follow-up, therapy of partners, and counseling about prevention. the widespread use of vaccines to prevent measles and rubella has resulted in dramatic decreases in the incidence ofboth these illnesses in the united states (120) . the very success of the vaccination program has resulted, paradoxically, in a change in the epidemiology of these diseases. those who care for adolescents need to be aware of these illnesses, their manifestations, and approaches to their prevention. both measles and rubella were widely prevalent infections until the appearance of vaccines in 1963 and 1969, respectively. the vaccines abruptly reduced the incidence of these infections and changed their epidemiology. caused by rna viruses, measles and rubella are spread when droplet nuclei from infected patients are inhaled by susceptible hosts. once the virus is introduced into a susceptible population, the cases multiply quickly, as dramatically demonstrated in recent outbreaks of measles on a number of college campuses (121) . with minimal changes in production technique, the rubella vaccine has remained a highly efficacious, live attenuated vaccine (122) . the measles vaccine, on the other hand, has undergone a number of changes. the original measles vaccine, a killed vaccine, was available from 1963 until 1967. at that time, a live attenuated vaccine was introduced, which took the place of the killed vaccine. individuals who received the original killed vaccine appear to be still susceptible to measles infection and need to be revaccinated with the contemporary vaccine. also, the schedule for administering the vaccine has been altered with the realization that infants vaccinated before 15 months of age have a less reliable response to the vaccine. anyone given the vaccine as an infant needs revaccination to ensure protective antibody titer (123) . once exposed to the measles virus, a susceptible individual will develop illness in 10-14 days. the prodrome consists of fever, cough, coryza, and conjunctivitis. koplik spots-raised blue-gray specks on the buccal mucosa-appear 24 hr before the rash and are characteristic of measles. the rash is seen first on the neck and face; it proceeds downward to involve the whole body. the rash is maculopapular and erythematous. after three to five days, it fades slowly and eventually disappears. in individuals who have received killed measle vaccine, a syndrome characterized by a rash that begins on the extremities, high fever, and pneumonia can occur after exposure to measles (124) . this ill-ness, known as "atypical measles," is probably mediated by a hypersensitivity reaction to the virus in a partially immune host. in rare cases, atypical measles have been documented despite revaccination with live attenuated vaccine in persons previously given the killed vaccine (125). infection with the rubella virus produces a milder illness than that caused by the measles virus. the prodrome, consisting of fever and malaise, is similar to that of measles; mild coryza and conjunctivitis might also occur. the associated rash is less dramatic and, as with measles, it starts on the neck and/or face. the rash usually lasts three to five days. lymphadenopathy, a hallmark of rubella infection, occurs before the onset of the rash and frequently persists after the rash has subsided. the posterior auricular and suboccipital nodes are most likely to increase in size and are often the last to resolve. diagnosis of both of these rash illnesses rests primarily on recognition of the clinical symptoms. serologic confirmation of the presence of type-specific antibodies serves little function in terms of immediate diagnosis but is extremely important from a public health standpoint. both infections can be prevented by appropriate vaccination. vaccine efficacy ranges from 90-95% for both present vaccines (126) . prior to 1972, the recommendation of the advisory council on immunization practices of the ll.s. public health service was to vaccinate at 12 months. therefore, persons born before 1972 should be considered candidates for revaccination against measles. the measles and rubella vaccines can be administered singly, in combination with each other (mr), or with each other and mumps (mmr). systemic reactions, including fever and myalgia, are common but rarely severe. no specific treatment is indicated for clinical infections. although rubella remains a major public health problem because of the threat of congenital rubella syndrome in infants born to mothers who contract rubella in the first trimester of pregnancy, it rarely causes severe illness in adolescents. on the other hand, measles complications, including encephalitis, pneumonia, and subacute sclerosing panencephalitis, can be severe and even fatal in adolescents and young adults (127) . all adolescents born prior to 1972 should be considered candidates for revaccination with measles vaccine. those born before 1969 are candidates for rubella vaccine. health care providers should help ensure that students be excluded from schools unless they can prove that they are appropriately vaccinated or they have proof of prior infection. if measles or rubella is suspected in an adolescent, reassessment of vaccine status is necessary for all close contacts, particularly schoolmates. iii adolescents must be quarantined away from other potentially susceptible adolescents. although adolescents are a generally healthy group, infection can create significant morbidity and occasional mortality. as in all patients, early diagnosis and treatment can shorten the duration of infection 'and limit complications. physicians should provide teenage patients with guidance necessary to prevent transmission and recurrence. current estimates from national health interview survey: united states the national ambulatory medical care survey: united states primary epstein-barr virus infections in children clinical virologic and serologic evidence of epstein-barr virus 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persisting illness and fatigue in adults with evidence of epstein-barr virus infection granulocyte changes .in infectious mononucleosis the hepatitis of infectious mononucleosis: experience with 235 cases the presence of heterophil antibodies in infectious mononucleosis monospot: a differential slide test for infectious mononucleosis heterophil antibody in adults with sore throat epstein-barr virus specific diagnostic tests in infectious mononucleosis treatment of lifethreat ening epstein-barr virus infections with acyclovir threatment of infectious mononucleosis with intravenous acyclovir infectious mononucleosis plicated by hemolytic anemia due to anti-i severe thrombocytopenia in infectious mono hemolytic and other anemias in infectious mononucleosis ruptured spleen in infectious mononucleosis spontaneous rupture of spleen in infectious mononucleosis clinical, virologic, and serologic evidence of epstein-barr virus infection in association with childhood pneumonia mononucleosis and heart disease nervous system involvement in infectious mononucleosis: their heralding and/or major manifestation epstein-barr virus and the x-linked iyrnphoproliferative syndrome antibodies to epstein-barr virus in burkitt's lymphoma and control groups is burkitt's lymphoma related to perinatal infection by epstein-barr virus? antibodies to epstein-barr virus in nasopharyngeal carcinoma, other head and neck neoplasms, and control groups epstein-barr virus antibody in childhood hodgkin's disease central nervous system lymphoma related to epstein-barr virus failure to acquire epstein-barr virus infection after intimate exposure to the virus distribution of antibody to hepatitis a antigen in urban adult populations recent advances in the study of the epidemiology of hepatitis b a large outbreak of hepatitis a in a day care center hepatitis surveillance report 1985. centers for disease control an outbreak of type a hepatitis at the naval training center transmission of hepatitis b in a classroom setting an outbreak of hepatitis iun members of a high school sumo wrestling club infectious hepatitis: stud ies on the effect of gamma globulin on the incidence of inapparent infection the natural history of infectious hepatitis viral hepatitis. type b. studies on natural history and prevention re-examined non-a, non-b hepatitis: etiology and clinical course acute viral hepatitis type b hepatitis after transfusion with blood containing antibody to hepatitis b core antigen randornization of corticosteroid therapy in fulminant hepatitis prophylaxis and prevention of viral hepatitis hepatitis i3 vaccine: demonstration of efficacv in a controlled clinical trial in a high risk population in the~united states type b hepatitis after needle-stick exposure: prevention with hepatitis b immune globulin. a final report of the veteran's administration cooperative study recommendation of the immunization practices advisory committee: recommendations for protection against viral hepatitis fulminant hepatitis: mayo clinic experience with 34 cases teenage pregnancy: the problem that hasn't gone away causes of acute urethal syndrome in women association between diaphragm use and urinary tract infection behavioral factors and urinary tract infection dysuria in adolescent girls: urinary tract infection or vaginitis diagnosis of coliform infection in acutely dysuric women antibody-coated bacteria in the urine of infants and children with their first two urinary tract infections urinary prophylaxis with trimethoprim and trirnethoprim-sulfarnethoxasole. efficacy and influence on the natural history of recurrent bactiuria and cost control single-dose therapy for cystitis in women. a comparison of trimethoprimsulfamethoxazole, arnoxicillin, and cyclacillin symptomatic gonorrhea during pregnancy cervical chlamudia trachomatis and mycoplasmal. infections in pregnancy: epidemiology and outcomes acute salpingitis in the adolescent female incidence, prevalence, and trends of acute pelvic inflammatory disease and its consequences in industrialized countries gonorrhea in female adolescents. potential analogies to toxic shock syndrome barrier method contraceptives and pelvic inflammatory disease criteria for diagndsis and grading of salpingitis tube-ovarian abscess: a retrospective review laparoscopy in the diagnosis and management of patients with suspected salpingo-oohoritis ectopic pregnancy in the united states: 1970 through 1978 reproductive consequences of pelvic inflammatory disease nongonococcal urethritis: a clinical review prostatitis syndromes: pathophysiology and differential diagnosis chlamudia trachomatis infections in men with reiter's syndrome toxic shock syndrome surveillance in the united states epidemiology of toxic shock syndrome young teens and tampons: a crisis in waiting centers for disease control: unpublished data the acquired immunodeficiency syndrome: an update measles and rubella in adolescents and young adults some current issues relating to rubella vaccine need for measles revaccination in adolescents: correlation with birth date prior to 1972 atypical measles in adolescents and young adults failure of attenuated viral vaccine in prevention of atypical measles antibody response following measles-mumps-rubella vaccine under conditions of customary use measles outbreaks on university carnpuscs-s-indiana urogenital disease syndromes in adolescents. i. vaginitis, urethritis, and genital lesions urogenital disease syndromes in adolescents. 11. pelvic pain in the adolescent female std treatment guidelines key: cord-017959-g0nf1iwm authors: lipkin, w. ian; briese, thomas title: diagnosis, discovery and dissection of viral diseases date: 2014-02-27 journal: viral infections of humans doi: 10.1007/978-1-4899-7448-8_2 sha: doc_id: 17959 cord_uid: g0nf1iwm only a few years ago, viral diagnosis was largely an exercise for academic researchers and public health practitioners with focus on epidemiologic analyses and outbreak prevention, detection, and control. opportunities for therapeutic intervention were limited to only a few applications such as herpesvirus infections, influenza, and hiv/aids; hence, once a bacterial or fungal infection was excluded, clinicians were limited to providing supportive care for what was presumed to be a viral syndrome. public health organizations tracked the incidence of viral infections and the development of resistance to the few antiviral drugs in use and provided input to governments and the pharmaceutical industry regarding selection of vaccine targets. more recently, interest in viral diagnostics has burgeoned with the advent of new tools for detection and discovery, global recognition of pandemic risk, high-throughput drug screening, rational drug design, and immunotherapeutics. an additional impetus has been the implication of viruses in chronic illnesses not previously attributed to infection. the objective of this chapter is to review the factors responsible for the rise in awareness of viral infections, methods for diagnosis and monitoring viral infections, and future prospects for improvements in discovery, detection, and response to the challenges of clinical virology. in an era when travel and trade are increasingly global, patients with what were once considered exotic infectious diseases restricted to the developing world, like dengue fever, ebola, or chikungunya, now present in clinics and emergency rooms in north america and europe. nonstop fl ights of less than 24 h connect the world's major airports; hence, physicians must be prepared to expect the unexpected. in new york city, for example, more than 12 million passengers annually pass through john f. kennedy (jfk) airport from more than 100 international destinations. with this traffi c volume in one metropolitan airport alone, it is not surprising that human and stowaway passengers like mosquitoes have been implicated in the transmission of west nile virus, hiv, infl uenza virus, mycobacterium tuberculosis , sars coronavirus, and chikungunya virus. exotic agents can also transit internationally in legal and illegal (bushmeat) food products and companion animals. the annual traffi c in bushmeat through charles de gaulle airport in paris is estimated at 273 tonnes [ 1 ] . in work with nonhuman primate, rodent, and bat bushmeat seized at jfk by the wildlife conservation society, ecohealth alliance, and the centers for disease control and prevention, we have found evidence of infection with retroviruses, herpesviruses, and pathogenic bacteria [ 2 ] . illegal importation of companion animals such as birds, primates, and rodents has been linked to outbreaks of poxviruses and salmonella [ 3 , 4 ] . approximately 70 % of emerging infectious diseases are zoonoses-infections that are transmitted to humans from wildlife or domestic animals [ 5 , 6 ] . the majority of zoonotic diseases can be attributed to anthropogenic change. loss of wildlife habitat to development and consumption of bushmeat necessitated by poverty or due to cultural preference increases opportunities for cross-species jumps. global warming may also increase the geographic range of phlebotomus insects like mosquitoes and ticks that serve as reservoirs and vectors for infectious agents [ 7 ] . given that there are more than 50,000 vertebrate species, if we assume an average of 20 endemic viruses per vertebrate species, the potential reservoir of vertebrate viruses can be estimated at one million. although it is unlikely that all of them can be transmitted to humans and cause disease, it is sobering to consider the challenge of detecting and responding even to 1 % of them (10,000 novel viruses). in the aftermath of the fall of the twin towers on september 11, 2001 , in new york city, and the anthrax attacks that followed, many western governments became concerned about bioterrorism. early investments in surveillance for biological weapons gave way to surveillance for emerging infections when sober refl ection led to recognition that the latter were more likely threats to public health. however, advances in synthetic biology over the past decade have been so dramatic that clinicians and public health practitioners must again consider the possibility that high-threat known and novel pathogens may arise through deliberate genomic manipulation either in the form of bioweaponeering, inadvertent release of high-threat human pathogens, or legitimate gain-of-function research, whereby low-risk agents become high risk. the scientifi c and larger communities are currently grappling with the implications of gain-of-function research in the context of experiments designed to understand virulence and transmission of h5n1 (avian) infl uenza viruses [ 8 -13 ] . establishing a causative link between a virus and disease can be straightforward or complex. in some instances, the virus responsible for the induction of disease is present at a site of organ pathology, and there is precedent for the same or a related virus causing similar disease. a classic example is herpes encephalitis where the detection of herpesvirus sequences by polymerase chain reaction (pcr) in the cerebrospinal fl uid (csf) of a patient with encephalitis provides a clear diagnosis and suggests a specifi c therapeutic intervention [ 14 ] . pcr alone can be inadequate. in west nile encephalitis, pcr of csf is less reliable than assays of csf for igm antibodies to the virus [ 15 , 16 ] . in some instances, the footprints of an agent cannot be found in or adjacent to the affected organ but can be detected in other compartments. pcr detection of enterovirus, for example, in the feces of a patient with aseptic meningitis, provides strong evidence of enteroviral meningitis [ 17 ] . despite these examples of success, an etiological agent is not identifi ed by any test in up to 70 % of what is presumed to be viral encephalitis. similar fi gures pertain in viral pneumonias. there are several explanations for surveillance and diagnostic failure. in some instances, the problem is simply lack of access to the appropriate sample. infectious agents that do not shed into saliva, nasopharyngeal secretions, blood, urine, csf, or feces may be detected in tissue biopsies. alternatively, pathogenetic mechanisms may be indirect, or consequences of infection may be delayed obscuring the relationship between the causative agent and the disease. the most straightforward mechanisms for viral pathogenesis are cellular damage due to replication and lysis, apoptosis, autophagy, or immune responses to proteins expressed on infected cells. however, viruses can also induce systemic damage through cytokine storm resulting in shock, acute respiratory distress, and/or organ failure, cause immunosuppression resulting in opportunistic infection, or break tolerance for self, resulting in autoimmune disease. infection can be cryptic, impairing differentiated cell functions like hormone secretion, inducing neoplasia, or impairing developmental programs that may not become apparent for months or years. in summary, the challenge in viral diagnostics is to develop strategies for not only detecting footprints of the agent itself in target tissues but also enduring shadows of infection in accessible compartments. once the mainstay of viral diagnostics, culture now receives less emphasis in clinical microbiology, chiefl y because assays require days rather than hours; thus, information obtained is unlikely to directly impact patient management. culture nonetheless continues to play an important role in public health as well as basic and clinical research because it enables insights into pathogenesis and the effi cacy of drugs, antibodies, and vaccines. the presence of virus can be detected by changes in cell morphology at the level of light microscopy including lysis, rounding, and syncytia formation-fusion of cells as revealed by an increase in size and the presence of more than one nucleus-visualization of pathognomonic structures by electron microscopy; or viral proteins that bind antibodies as revealed through immunohistochemistry or immunofl uorescent microscopy. a wide range of cell lines has been established for culturing viruses. some are immortalized; others are primary cultures that can only be propagated for a few generations. although some viruses can grow in many cell types, others have fastidious requirements. some viruses have never been cultured despite implication in disease. in some instances, propagation failure may be overcome by adaptation with serial passaging in the presence of a second, permissive type of cell (cocultivation), the use of antibodies or rnai to suppress innate immune responses, or cells obtained from genetically modifi ed animals. however, serial passaging can lead to adaptation, including changes in virulence (the capacity of the virus to cause disease) or tropism (the cells and organs the virus can infect). indeed, serial passage may be utilized to develop less virulent strains that can be used as vaccines. a potential confound in characterizing samples that may contain more than one virus is that the culture environment can select for the agent that is more fi t to replicate-that may or may not be the agent of interest. in an attempt to address this potential confound as well as to propagate viruses that fail to grow in simple cultures, investigators have developed cultures that include more than one cell type. in some instances these complex cultures are designed to replicate the architecture of an organ like the respiratory tract. an alternative to culture in cells is animal inoculation. an advantage of animal inoculation is that the presence of a wide range of cell types is associated with expression of a wide range of receptors that may allow virus entry. most investigators use suckling mice because their innate immune responses are immature. others use mice genetically modifi ed to abrogate immune responses. nucleic acid tests (nats) have largely replaced culture in viral diagnostics due to advantages in cost, speed, and ease of use. common nat platforms include polymerase chain reaction (pcr), in situ hybridization, microarray, and highthroughput sequencing. these assays, designed to detect individual viruses, are the most common nats employed in clinical microbiology. they take several forms, but quantitative real-time pcr, wherein nucleic acid replication results in either cleavage or release of a fl uorescence-labeled probe oligonucleotide that binds to a sequence region between the regular forward and reverse primers, is the most popular. the continuous ("real time") reading of the reporter fl uorescence signal affords these systems with unprecedented dynamic range and low false-negative rate. the required equipment, thermal cycler with fl uorescence detector and (laptop) computer for data analysis, is cost competitive, and rugged battery-powered instruments are available for fi eld use. loop-mediated isothermal amplifi cation (lamp) tests do not require programmable thermal cyclers [ 18 -20 ] . in the laboratory, lamp products are detected in conventional dye-stained agarose gels, but in fi eld applications the estimation of product accumulation through turbidity or dye reading by the naked eye is also possible [ 21 ] . the sensitivity of all such assays is highest when primers and/or probe sequences perfectly match the selected single genetic target. fluorescence-based taqman or molecular beacon assays, for example, typically have detection limits of <10 molecules per assay. although ideal for detecting and quantitating a specifi c known agent [ 22 , 23 ] , these assays may nonetheless fail with templates of variable sequence composition, especially if this affects the region of reporter molecule binding. this can be particularly challenging in the diagnosis of rna virus infections as rna viruses are characterized by high mutation rates and include species with high genetic strain variability. in comparison, consensus pcr assays are less likely to be confounded by sequence divergence but are also less sensitive than the specifi c pcr assays. nested pcr tests that can employ consensus or specifi c primers in two sequential amplifi cation reactions with either one (hemi-nested) or two (fully nested) primers located 3′ with respect to the fi rst primer set may both accommodate sequence variation and be more sensitive than fl uorescent or beacon-based singleplex assays. however, whereas in quantitative fl uorescence-or beacon-based realtime assays reporter readings are taken indirectly without opening the reaction vessels ("closed system"), nested pcr systems bear a high risk of contamination because of the transfer of (amplifi ed) material from the fi rst to the second, nested reaction [ 24 , 25 ] , even if scrupulous experimental hygiene is observed. recently, automated (closed) systems have been developed that allow contamination-free transfer between separate reaction compartments of single-use cartridges that may present new opportunities for nested assay design. as signs and symptoms of disease are rarely pathognomonic of a single agent, particularly early in the course of an illness, many microbial candidates must be entertained simultaneously. multiplex nats provide such an opportunity. the number of candidates considered may range from 10 to 50 with multiplex pcr systems to thousands with microarray platforms to the entire tree of life with unbiased highthroughput sequencing approaches. however, genetic targets compete for assay components in multiplex assays, and thus they may be less sensitive than a singleplex assay. in compensation, multiplex assays provide the advantage of consistently interrogating each sample for a wide range of agents without the selection bias introduced by singleplex testing. this comprehensive coverage is particularly important for surveillance and applications. multiplex pcr assays are more diffi cult to establish than singleplex assays because primer sets may differ in optimal reaction conditions (e.g., annealing temperature or magnesium concentration). furthermore, complex primer mixtures are more likely to result in primer-primer interactions that reduce assay sensitivity and/or specifi city. to advance multiplex primer design, we developed greene scprimer, a software program that automates consensus primer design over a multiple sequence alignment with customizable primer length, melting temperature, and degree of degeneracy [ 26 ] . gel-based multiplex pcr assays are limited by size differentiation of the amplifi cation products in agarose gels and the concomitant requirement for short product sizes (approx. 90-250 base pairs) to ensure high sensitivity and fi delity [ 24 , 25 ] . multiplexing can be achieved in fl uorescence-or beacon-based real-time assays to the degree by which different fl uorescent reporter emission peaks can be unequivocally separated. at present up to fi ve fl uorescent reporter dyes are detected simultaneously, although multiplexing may be increased to some extent by double-labeling strategies and/or melting curve analyses. "sloppy molecular beacons" address this limitation in part by binding to related targets at different melting temperatures [ 27 ] ; however, they are not suited to detect targets that differ by more than a few nucleotides. the bio-plex (or luminex) platform employs fl ow cytometry to detect multiple pcr amplifi cation products bound to matching oligonucleotides that are attached to differently colored fl uorescent beads [ 28 , 29 ] . by combining multiplex pcr amplifi cation systems with various protocols for direct or indirect (tag-mediated) bead hybridization of the products, assay panels have been developed that permit detection of up to approx. 20 genetic targets simultaneously [ 30 -32 ] ; the most commonly used respiratory panels range from 9 to 20 plex [ 33 -37 ] . like real-time pcr, these assays rely for assay specifi city on a three-oligonucleotide interaction with the target sequence. they are thereby limited in their tolerance for mutated or variant templates when compared to mass spectroscopy (ms)-coupled platforms that require only two oligonucleotide-binding sites, such as masstag pcr or the ibis t5000 system. two platforms are established that combine pcr with ms for sensitive, simultaneous detection of large numbers of targets. the ibis t5000 system uses matrix-assisted laser desorption/ionization (maldi) ms to directly determine the molecular weights of the generated pcr products and to compare them for identifi cation with a database of known or predicted product weights [ 38 -40 ] . masstag pcr uses atmospheric pressure chemical ionization (apci) ms to detect molecular weight reporter tags attached via a photo-cleavable linkage to pcr primers [ 41 ] . whereas the ibis system or the subsequent electrospray ionization (esi)-based plex-id system requires analytical ms to determine the exact weight of the pcr products and thus depends on advanced mass spectroscopic data analysis, masstag pcr can be performed using smaller instruments and does not require sophisticated analyses because it only records the known masses of the 40-80 reporter tags used in a given multiplex test. the ibis system may be able to alert of variants of known organisms via a divergent pcr product weight, but like masstag pcr, it too requires subsequent sequencing of the product for detailed characterization. a wide variety of syndrome-specifi c masstag pcr panels have been developed and applied to the detection of viruses, bacteria, fungi, and parasites associated with acute respiratory diseases, diarrheas, tick-borne diseases, encephalitides/meningitides, and hemorrhagic fevers [ 41 -50 ] . although multiplex pcr methods are designed to detect known agents, they can nonetheless facilitate pathogen discovery. masstag pcr requires only two differently tagged primers per target that may include degenerate positions to address genetic variation of larger taxonomic groups such as a whole species or genus, and its use to investigate infl uenzalike illness in new york state revealed the presence of a novel rhinovirus clade by the employed conserved enterovirus/rhinovirus primer set [ 42 ] . this discovery enabled follow-up studies across the globe wherein this third species of rhinovirus, rhinovirus c, was implicated not only in infl uenza-like illnesses but also in asthma, pediatric pneumonia, and otitis media [ 44 , 51 -63 ] . whereas multiplex pcr systems support rapid highthroughput diagnosis with highest sensitivity for a limited number of agents, microarray-based systems provide detection of all known pathogens for which sequence information is available, but at the expense of some degree of sensitivity. modern printing technologies can generate high-quality arrays with several million features, a printing density that enables not only detection of a wide range of infectious agents but also discrimination of medically important types or subtypes. examples of the latter application include respiratory virus resequencing arrays that identify the different infl uenza virus ha and na subtypes [ 64 -68 ] . the discovery array platforms currently in use are the greenechip and the virochip [ 69 , 70 ] . the panmicrobial version of the greenechip, addressing viruses and in addition pathogenic bacteria, fungi, and parasites, led to the recognition of plasmodium falciparum infection in a case of unexplained fatal hemorrhagic fever during the 2004-2005 marburg virus outbreak in angola [ 70 ] . a variant of the greenechip facilitated recently the implication of reston ebola virus in a respiratory disease outbreak on pig farms in the philippines [ 71 ] . in 2003, the virochip supported the characterization of the sars coronavirus and was also used subsequently to diagnose parainfl uenza virus 4 and infection with a human metapneumovirus variant in cases of acute respiratory disease [ 69 , 72 , 73 ] . both platforms rely on random pcr strategies to amplify and label nucleic acids for detection. in comparison to multiplex consensus pcr methods employed with some targeted array applications or resequencing arrays, this limits sensitivity especially with complex sample types. in tissue specimens, for example, the sensitivity may not exceed 10 6 -10 7 copies per assay because host and pathogen nucleic acids compete for pcr reagents. thus, these platforms have been more successful with samples containing comparatively low levels of competing nucleic acid, such as virus culture supernatant, serum, respiratory specimens, spinal fl uid, or urine. improvements in sensitivity to a range of 10 3 -10 4 copies per assay have been achieved with methods for host dna digestion and/or the depletion of host ribosomal rna (rrna) prior to amplifi cation through subtraction or use of random primers selected for lack of complementarity to rrna [ 74 ] . in current array platforms, virus detection is achieved via fl uorescent reporter systems-either through direct incorporation of fl uorescent nucleotides into the pcr product that is bound to the array or with a "sandwich approach" whereby fl uorescent-branched chains of dna are added to the product after it is bound to the array [ 75 , 76 ] . however, new arrays are in development that will detect viral sequences through changes in electrical conductance. such platforms would enhance portability by eliminating the need for fl uorescent scanners. they may also increase sensitivity and reduce costs by eliminating the need for pcr amplifi cation. high-throughput sequencing has transformed microbiology by enabling discovery as well as diagnostics. unlike pcr or array methods where investigators must choose the pathogens to be considered or are limited by known sequence information, high-throughput sequencing has the potential to simultaneously detect not only all viruses but also bacteria, fungi, and parasites. although the technology is presently limited to specialized laboratories, sequencing is becoming increasingly accessible as instruments become smaller, methods become more user friendly, and costs decrease. over the past 10 years, the cost has decreased 10,000-fold from $5,000 per 1,000 nucleotides in 2001 to $0.5 per 1,000 nucleotides in 2012 [ 77 ] . even more impressive perhaps is the time required to generate sequence data. projects that required weeks only a decade ago are now completed in hours [ 78 ] . current sequencing platforms analyze libraries of amplifi ed nucleic acids. however, some platforms in development will have the capacity to directly sequence nucleic acid. irrespective of the platform, raw sequence reads are fi ltered for quality and redundance before assembly into contiguous sequence streams. these streams, known as contigs, as well as reads that cannot be assembled, are aligned to databases using bioinformatic algorithms that examine homology at the nucleotide and deduced amino acid levels in all six potential reading frames [ 79 ] . the alignments allow identifi cation of known and novel agents, as well as detection of genetic features that may be associated with drug or vaccine resistance, or provide insight into provenance and evolution. finding the nucleic acid footprint of a virus is frequently only the fi rst stage in implicating it in disease. there is no functional equivalent in viruses to the pathogenicity islands found in bacteria, wherein specifi c sequences acquired through horizontal gene transfer confer specifi c pathogenic properties. the best established criteria for proof of causation in infectious disease were developed in the late 1800s by koch and loeffl er [ 80 ] . known as koch's postulates they stipulate that an agent be present in every case of the disease, be specifi c for the disease, and be suffi cient to reproduce the disease after culture and inoculation into a naive host. in the 1930s, rivers suggested that the development of specifi c immunity to an agent following the appearance of disease could be used in demonstrating causation [ 81 ] . adapting the original postulates to the molecular era, fredricks and relman later established that microbial sequences may be used as surrogates for culturing the actual organism [ 82 ] . lipkin and colleagues recently established levels of confi dence in the strength of association between an agent and a disease that considers viral burden and distribution, specifi c immunity, and prevention or amelioration of disease with use of specifi c drugs or vaccines [ 12 ] . given the sensitivity of molecular methods, it is imperative that physicians and researchers consider the biological plausibility of an assay result and, where feasible, pursue confi rmation with an independent assay, particularly when engaged in pathogen discovery. nats are rapidly replacing classical culture methods in clinical microbiology laboratories. although some nats, such as microarrays and high-throughput sequencing, still require substantial investment in equipment and personnel, diagnostic platforms are becoming more accessible and less expensive through miniaturization and improvements in methods for bioinformatic analysis. systems using handheld microarrays, for example, are in development that will ultimately enable diagnosis at the bedside or in the fi eld. benchtop sequencers are also in production. it is inevitable that as sequencing costs continue to decrease, clinicians will seek information concerning not only the presence of a single candidate organism but also the predisposition of the host to disease based on genetic factors and coinfections with other microfl ora. these improvements will bring dramatic benefi ts to medicine and public health. the scale of illegal meat importation from africa to europe via paris zoonotic viruses associated with illegally imported wildlife products monkeypox zoonotic associations: insights from laboratory evaluation of animals associated with the multi-state us outbreak recent multistate outbreaks of human salmonella infections acquired from turtles: a 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spectrometry provides accurate characterization of two genetic marker types in bacillus anthracis analysis of nucleic acids by fticr ms rapid identifi cation of emerging infectious agents using pcr and electrospray ionization mass spectrometry diagnostic system for rapid and sensitive differential detection of pathogens masstag polymerasechain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused infl uenza-like illness in new york state during masstag polymerase chain reaction for differential diagnosis of viral hemorrhagic fever multiplex masstag-pcr for respiratory pathogens in pediatric nasopharyngeal washes negative by conventional diagnostic testing shows a high prevalence of viruses belonging to a newly recognized rhinovirus clade streptococcus pneumoniae coinfection is correlated with the severity of h1n1 pandemic infl uenza detection of tick-borne pathogens by masstag polymerase chain reaction. vector borne zoonotic dis assessment of 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broad-spectrum, sequence-based pathogen identifi cation in an urban population large-scale evolutionary surveillance of the 2009 h1n1 infl uenza a virus using resequencing arrays use of consensus sequences for the design of high density resequencing microarrays: the infl uenza virus paradigm ultrasensitive detection of rare mutations using next-generation targeted resequencing microarray-based detection and genotyping of viral pathogens panmicrobial oligonucleotide array for diagnosis of infectious diseases discovery of swine as a host for the reston ebolavirus microarray detection of human parainfl uenzavirus 4 infection associated with respiratory failure in an immunocompetent adult diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray digital transcriptome profi ling using selective hexamer priming for cdna synthesis fluorescent sample labeling for dna microarray analyses a sensitive sandwich dna array using fl uorescent nanoparticle probes performance comparison of benchtop high-throughput sequencing platforms genetic detection and characterization of lujo virus, a new hemorrhagic fever-associated arenavirus from southern africa a new arenavirus in a cluster of fatal transplant-associated diseases viruses and koch's postulates sequence-based identifi cation of microbial pathogens: a reconsideration of koch's postulates key: cord-022383-pz0htccp authors: kohn, dennis f.; barthold, stephen w. title: biology and diseases of rats date: 2013-11-17 journal: laboratory animal medicine doi: 10.1016/b978-0-12-263620-2.50010-0 sha: doc_id: 22383 cord_uid: pz0htccp nan the diversity of research for which the laboratory rat is used is probably greater than that associated with any other animal. the laboratory rat is a descendent of the wild rat, rattus norvégiens, which originated in asia and reached europe in the early 1700s. wild and albino mutants were first used for ex perimental purposes in europe in the mid-1800s and in the united states shortly before 1900. the wistar institute in phil adelphia was prominent in the development of the rat as a labo ratory animal, for here originated many of the rat strains now used worldwide. henry donaldson and his colleagues at the wistar institute used these early rat strains for a variety of stud ies dealing with neuroanatomy, nutrition, endocrinology, ge netics, and behavior. the history and evolution of the many rat strains used today have been recently summarized (lindsey, 1979) . the most commonly used outbred rat stocks in north amer ica are the wistar, sprague-dawley, long-evans, and holtzman. all are albino except the long-evans stock, which is usually marked with a black or gray hair coat over the shoul ders and is sometimes referred to as a "hooded rat." there are numerous inbred and mutant rat strains, although the number is less than that in the mouse. table i lists the more commonly used strains. there are a rather large number of commercial vendors of laboratory rats in the united states. most of the stocks and strains mentioned above can be obtained from more than one source. although the origin of an outbred stock, such as the sprague-dawley, may have been the same for a number of vendors, in many cases it has been 20 to 30 years since such a stock has been removed from its original breeding colony. ac cordingly, the genotype of outbred stocks and inbred strains may vary among sources and be reflected by differences in data when multiple sources of rats are used. a standardized scheme of identifying stocks and strains of rats has been devel oped and is now used by nearly all commercial vendors. more over, it is important that authors correctly identify stocks and strains that are used in their research since the success in re peating the work in another laboratory may be dependent upon the genotype (source of the rat). table ii summarizes the stan dardized nomenclature for outbred stocks as developed by the table i commonly used strains "national institutes of health (1981) . 1. letters preceding the colon designate the supplier/breeder code consist ing of a capital and two or three lowercase letters 2. capital letters following the colon are used by a breeder to identify his stock 3. letters in parentheses denote origin of stock 4. subscript symbols indicate rearing by means other than natural mother (f, fostered; fh, fostered by hand) international committee on laboratory animals (icla). table iii contains the scheme for designating inbred strains of rats (national institutes of health, 1981) . "animals for re search" (national academy of sciences, 1979), a directory of sources for laboratory animals sold in the united states and canada, lists all rodents according to standard nomenclature, and is a valuable aid in purchasing laboratory animals. commercial production of rats has markedly changed since the 1960s due to the development of hysterectomy-derived and barrier-maintained breeding colonies. prior to the application of this technology to production colonies, infectious diseases were ubiquitous in rats from most sources. today, vendors can be selected who offer pathogen-defined animals for most stocks and strains. concomitant with changes in commercial sources of rats are the major advances made in the design and construction of institutional animal resources and husbandry practices within them. optimum housing of rats today includes provisions for quarantining and isolation of animals according to vendor subpopulations that have a similar microbial flora. there are various levels of sophistication to provide barriers to the spread of infections in rat colonies. since many rat pathogens are spread by aerosol, ventilation control is very important. nonrecirculating room air or high-efficiency panic ulate air (hepa)-filtered air has become a design standard in modern animal facilities. as discussed in chapter 17, clean/contaminated corridor-designed facilities aid in contain ment against the spread of pathogens by aerosol, personnel, table iii nomenclature for inbred rats 1. the strain designation is given in capital letters followed by a slash 2. the substrain designation follows the slash and is given as numbers or as individual or company codes. numbers are used to denote substrains that were derived from a common strain but separated before the completion of inbreeding 3. subscript symbols indicate rearing by means other than natural mother and contaminated equipment. a more complete barrier system may include an entry area in which incoming supplies and equipment are sterilized and in which personnel shower and don sterile clothing and filter masks before entering animal rooms. more recently, laminar-flow (mass air displacement) rooms and mobile units have become popular because they can be incorporated in existing buildings that lack design charac teristics mentioned above. environmental control in rat rooms is important to the com fort and health of the animals, as well as to the consistency of data derived from the rats. room temperatures between 72 and 76°f are desirable, and the relative humidity should range be tween 40 and 60%. daily fluctuations in temperature and hu midity act as significant stressors. these fluctuations may be associated with the environmental control system of a building or may be induced by procedures such as cleaning floors with a water hose or high pressure sprayer. twenty-four-hour tem perature/humidity recorders are useful in detecting changes in environmental conditions. light intensity should be evenly distributed to all animals within a room. seventy-five to 125 fc have often been suggested as an optimal range for light inten sity. however, recent evidence indicates that this intensity can induce retinal degeneration in albino rats (anver and cohen, 1979) . light-timing devices are a convenient means to provide desired day/night cycles such as 12-12 or 14-10 hr. caution should be exercised in the use of insecticides and air-deodorizing chemicals, since some have been shown to in duce hepatic microsomal enzymes in rats. accordingly, their use in animal rooms is not usually recommended (baker et al., 1979a) . rats can be housed in either wire-or solid-bottom cages. wire-bottom cages are more frequently used since they are less labor-intensive. frequency of cage and litter pan changing is a function of animal density. solid-bottom cages should be sani tized two to three times per week, while wire-bottom cages should be sanitized on a weekly or biweekly schedule with litter pans changed two or three times per week. feed should be contained in hoppers. either automatic systems or bottles are satisfactory for providing water to rats. some caution is necessary when using automatic systems, since weanling and newly arrived rats may not drink initially from such devices. to avoid undesirable microbial contamination, water bottles should be sanitized before they are refilled and automatic sys tems should be drained and flushed when racks are sanitized. acidification of water to a ph of 2.5 to 2.8 or chlorination at 8 to 12 ppm will control pseudomonas aeruginosa contamina tion of water (weisbroth, 1979) . however, this treatment is not necessary for immunocompetent animals. wood shavings or chips are the most commonly used contact bedding mate rials. hardwoods are preferred to softwoods, since the latter are capable of inducing hepatic microsomal enzymes (baker et al, 1979a) . this section summarizes some of the anatomical characteris tics of the rat with emphasis on characteristics that are unique. the reader is advised to refer elsewhere in the literature for comprehensive descriptions (bivin et al., 1979; caster et al., 1956; hebel and stromberg, 1972; smith and calhoun, 1972; zeman and innés, 1963) . the rat dental formula is 2(1 1/1, c 0/0, pm 0/0, m 3/3) = 16. the incisors are well developed and grow continuously. the rat lacks tonsils and water taste receptors. the major pairs of salivary glands are the parotid, submandibular (submaxillary), and sublingual. the parotid gland is a serous gland consisting of three to four lobes and is located ventrolaterally from the caudal border of the mandible to the clavicle. the submandibular glands are mixed glands located ventrally between the caudal border of the mandibles and the thoracic inlet. the sublingual glands are mucous glands and are much smaller than the parotid and submandibular glands. they are located at the rostral pole of the submandibular glands to which they are closely associated. brown fat deposits are present in the ventral cervical region. these multilocular deposits are well demarcated and can be confused with salivary glands or lymph nodes. the stomach of the rat is divided into two parts; the forestomach (nonglandular) and the corpus (glandular). the two portions are separated by a limiting ridge. the esophagus en ters at the lesser curvature of the stomach through a fold of the limiting ridge. this fold is responsible for the inability of the rat to vomit. the forestomach, which is thinner than the cor pus, is linked with an epithelium similar to that of the esopha gus and extends from the cardia to a narrow band of cardiac glands at the junction of the glandular portion. the small intestine is composed of the duodenum (10 cm), jejunum (100 cm), and ileum (3 cm). the cecum is a thinwalled, comma-shaped pouch that has a prominent lymphoid mass in its apical portion. the colon is composed of the as cending colon, with prominent oblique mucosal ridges, trans verse and descending colons, with longitudinal mucosal folds; followed by a short rectum that is confined to the pelvic canal. the liver has four major lobes (median, right lateral, left, and caudate) and is capable of regeneration subsequent to par tial hepatectomy. the rat has no gallbladder. the bile ducts from each lobe form the common bile duct that enters the duo denum 25 mm from the pyloric sphincter. the pancreas is a lobulated, diffuse organ that extends from the duodenal loop to the gastrosplenic omentum. it can be dif ferentiated from adjacent adipose tissue by its darker color and firmer consistency. up to 40 excretory ducts fuse into 2-8 large ducts, which empty into the common bile duct. the nasal cavity is not markedly different from that of other mammals. the rat has a maxillary recess (sinus) located be tween the maxillary bone and the lateral lamina of the ethmoid bone. the recess contains the lateral iiasal gland (steno's gland) that secretes a watery product that is discharged at the rostral end of the nasal turbinate. it has been postulated that the nonviscous secretion contributes to the humidification of in spired air and acts to regulate the viscosity of the mucous layer overlying the nasal epithelium. the left lung has one large lobe, and the right lung is divided into four lobes (cranial, middle, accessory, and caudal). the pulmonary vein in the rat has cardiac striated muscle fibers within its wall that are contiguous with those in the heart. the rat does not have an adrenergic nerve supply to the bronchial musculature, and bronchoconstriction is controlled by vagai tone. unlike the guinea pig, the rat lung has a low concentra tion of histamine (bivin et al., 1979) . the heart and peripheral circulation in the rat differ little from that of other mammals. the blood supply to the heart is derived from both coronary and extracoronary arteries. the latter arise from the internal and subclavian arteries. the right kidney, which is more craniad than the left, has its cranial pole at the l, vertebra and its caudal pole at the level of l 3 . the rat kidney is unipapillate as are kidneys of other ro dents, lagomorphs, and insectivores. having only one papillus and calyx makes the rat useful for studies in which cannulization of the kidney is done. the presence of superficial nephrons in the renal cortex has made the rat widely used as a model for studying nephron transport in an in vivo micropuncture system. the male reproductive system has a number of highly devel oped accessory sex glands. these include large seminal vesi cles, a bulbourethral gland, and a prostate gland composed of the coagulation gland (dorsocranial lobe) and ventral and dorsolateral lobes. the inguinal canal remains open throughout the life of a rat and testes descend initially by 40 days of age. the female rat has a bicornate uterus that is classified as the duplex-type because the lumina of the uterine horns are com pletely separate with paired ossa uteri and cervices. the female urethra does not communicate with the vagina or vulva, but rather exits at the base of the clitoris. the brain of the rat has very large olfactory bulbs and a nonconvoluted cerebrum. the hypophysis is behind the optic chiasma and is attached to the base of the brain by a thin hol low stalk, the infundibulum. the blood supply to the brain is from the internal carotid and vertebral arteries. blood leaves the brain via a system of sinuses that are enclosed in the dura mater. the ventricular system is similar to that of other ani mals, but the rat lacks a foramen of magendie. it must be recognized that many of the normal values deter mined for a specific group of rats may be accurate for only that rat stock/strain, source, and conditions under which they are held. selected physiological, hematological, and clinical bio chemical parameters are listed in tables iv-vii. more com plete information on biological values is available (mitruka and rawnsley, 1977; ringler and dabich, 1979) . nutritionally adequate diets are readily available from com mercial sources. these standard rations are quite satisfactory for most applications. however, for some types of experimen tation there are factors, other than nutritional adequacy, which must be considered. the nutrient composition of diets and the contamination of feed components by mycotoxins, antibiotics, synthetic estrogens, heavy metals, and insecticides may have a profound impact on many studies. for instance, caloric intake and the percent of fat and protein in the diet of rats influence the incidence of neoplasia (altman and goodman, 1979) . sim ilarly, various contaminants have an adverse effect on data from toxicologie, gerontological, and reproductive studies. standard commercial diets are formulated from natural ingre dients and will vary in nutrient composition on a batch-tobatch basis due to differences in type and quality of ingredients used. commercial makers of rodent feeds take precautions to preclude the presence of contaminants in feeds, but only a few products have a defined profile of maximal levels of heavy metals, aflatoxins, chlorinated hydrocarbons, and organophosphates. for some investigative purposes, feeds formulated with re fined ingredients (purified diets) or with chemically defined compounds are useful when control of nutrient concentrations is essential (national research council, 1978) . these diets are, however, too expensive for general use. baker et al. (1979b) and bivin et al. (1979). rats are commonly fed ad libitum, and food intake will vary according to requirements for growth, gestation, and lactation. the nutritive requirements for the rat are listed in table viii. the duration of storage and the temperature at which feeds are stored prior to use effect the nutritive quality of diets. com mercial diets are formulated to have a shelf life of up to 6 months. however, storage in a hot or damp environment will reduce this shelf-life. to help assure that only fresh diets are used, products should be used which have milling dates identi fied on their containers (see chapter 17). sexual maturity occurs between 6 and 8 weeks for both sexes, although the onset of first estrus in females occurs at about 5 weeks. the vagina opens between 34 and 109 days, and the testes descend between 15 and 51 days, although they remain fully retractable in adults. rats ovulate spontaneously, but ovulation can also be induced by forced coitus during nonestrous intervals. vaginal stimulation during mating is impor tant in rat reproductive physiology. the more often a male inserts his penis into the vagina prior to ejaculation, the greater the probability of a resulting pregnancy. however, natural or artificial stimulation of the vagina within 15 min of a first mat ing will abrogate pregnancy from the first mating by inhibition of sperm transport. a 12-hr estrous period recurs every 4 to 5 days and after parturition, without seasonal variation. estrus can be suppressed when females are housed in groups and syn chronized in the presence of a male or its excreta (whitten effect), but this effect is not as pronounced as in the mouse. female fertility wanes at 600 to 650 days, but estrous cycles may continue through 32 months. male fertility is lost between 16 and 20 months. fertility of both sexes is generally regarded as maximal between 100 and 300 days of age (adler and zoluth, 1970; baker, 1979; farris, 1963; lane-petter, 1972; leathern, 1979) . males will mount estrous females numerous times with one or two rapid ejaculations in the course of 15 to 20 minutes. ejaculated semen coagulates, forming a copulatory plug that remains in the distal vagina for a few hours, after which time it dissolves or is extruded. copulation is usually nocturnal. du ration of gestation varies with strain, age, litter size, and other variables, and ranges from 19 to 23 days, with an average of 21 or 22 days. primiparous females tend to have a slightly longer gestation than multiparous females (farris, 1963) . estrus can be detected in a number of ways. females in es trus are hyperactive and brace themselves when touched. their ears quiver when they are stroked on the head or back, and stimulation of the pelvic region induces lordosis (farris, 1963) . the vulva becomes swollen, and the vagina becomes dry in contrast to the moist pink wall during metestrus or diestrus. as proestrus occurs (approximately 12 hr), smears of va ginal cells contain nucleated epithelium, leukocytes, and occa sional cornified cells. estrus (approximately 12 hr) begins with about 75% nucleated and 25% cornified cells, with cornified cells predominating as estrus continues. metestrus follows (ap proximately 21 hr) with large numbers of leukocytes and corn ified cells, which form abundant caseous vaginal detritus. metestrus is characterized by the presence of large flat nucle ated (pavement) cells. diestrus persists for approximately 57 hr (baker, 1979; farris, 1963) . breeding dates can be established by examination of vaginal swabs for spermatozoa or examining the distal vagina or cage pan for copulatory plugs. timed pregnancies are best achieved by placing the female in the male's cage in the afternoon and examining her for a plug or spermatozoa the following morning. abdominal enlargement becomes evident at about 2 weeks. pseudopregnancy is rare (lane-petter, 1972) . rats reproduce successfully under a variety of conditions, but husbandry practices can significantly influence fecundity. rats can be bred as monogamous pairs, taking advantage of postpartem estrus for maximal breeding efficiency. polyg amous breeding is more economical, since only one male can be kept with 6 to 9 females. females are often removed to a separate cage prior to whelping, since they may not tolerate other females in the cage while nursing. they will tolerate their mates, however. females with litters do best on clean dust-free wood shavings in solid-bottom cages. due to heat regulation, pups neither thrive in overly spacious cages with wide flutuations in ambient temperature, nor in overly crowded cages where they cannot dissipate heat. the recom mended cage floor area for a female and her litter is 150 in. 2 . ambient room temperature and humidity should be within the acceptable range with minimal fluctuation. high ambient tem perature can cause male infertility (baker, 1979; baker et al., 1979a; lane-petter, 1972) . the rat estrous cycle is particularly sensitive to variations in light. daily lighting at an average of 100 fc with a spectrum approximating natural light for 12 to 16 hr is best for breeding. constant light for as few as 3 days may induce persistent es trus, hyperestrogenism, polycystic ovaries and endometrial hy pertrophy or metaplasia (baker et ai, 1979a; gralla, 1981) . nutrition may also affect reproductive performance. re quirements for certain components are increased during preg nancy and growth, but overfeeding is deleterious. caloric re striction may actually improve fertility and possibly reproduc tive life of the female (leathern, 1979) . excess dietary protein can adversely affect female sexual development. vitamin defi ciencies can cause infertility, particularly those vitamins (a, e, riboflavin, and thiamin) that are most labile to autoclaving or deterioration (baker, 1979) . it is not necessary to add nesting material to bedding for successful breeding, but rats will utilize it if offered. shredded paper or cotton nesting material will be readily accepted and used by prepartem and nursing dams. parturition is heralded by pronounced postural stretching and rear leg extension. a vagi nal discharge may be noted li-4 hr prepartum. parturition is usually complete in 1 or 2 hr, but can range from a few minutes to several hours depending on litter size. dystocia is exceed ingly rare. litters average between 6 and 12 pups, with highest fecundity through the sixth litter. inbred rats tend to produce smaller litters. although infrequent, cannibalism is most apt to occur with nervous or primiparous females subjected to stress (farris, 1963; lane-petter, 1972; leathern, 1979) . the neonate weighs about 5i gm, depending on litter size, sex, strain, and physical condition of the dam. pups are born hairless, blind, with closed ears, undeveloped limbs, and short they are fully haired between 7 and 10 days (baker, 1979; farris, 1963; lane-petter, 1972) . maternal antibody is trans ferred in utero, via the yolk sac and by intestinal absorption of colostrum by the neonate for up to 18 days after birth (chev ille, 1976) . optimal weaning age is 20-21 days, although pups can be weaned as early as 17 days. differentiation of sex in adult rats is relatively easy after the testes descend. the adult testes can be readily retracted through large inguinal canals. male neonates have a larger genital papillus and the anogenital space is greater in males than females. from national research council (1978) . h adequate to support growth, gestation, and lactation; based on 90% dry matter. ( linoleic acid, 0.6%, is required. ^one-third to one-half can be supplied by l-cystine. ^one-third to one-half can be supplied by l-tyrosine. ^mixture of glycine, l-alanine, and l-serine. ^vitamin a, 1 iu = 0.300 ìg retinol, 0.344 ìg retinyl acetate, 0.550 ìg retinyl palmitate. vitamin d, 1 iu = 0.025 ì£ ergocalciferol. vitamin e, 1 iu = 1 mg dl-a-tocopheryl acetate. artificial insemination can be achieved in rats, but the major obstacle is the coagulative properties of their semen. sperm can be obtained by maceration of the epididymis and vasa or by electroejaculation, although the latter method is unreliable and the semen often rapidly coagulates. coagulation can be eliminated by prior surgical extirpation of the seminal vesicles and coagulating glands without significant effect on fertility. semen can be diluted with a number of media but frozen stor age of rodent semen has met with little success. insemination can be achieved surgically by direct injection of seminal fluid into the uterus and by nonsurgical means. successful concep tion seems to require not only insemination during estrus but also induction of pseudopregnancy by mating with a vasectomized male or mechanical stimulation within a few hours (before or after) insemination. egg harvest for transfer can be accomplished by excision of the preovulatory ovaries and teas ing from gravid follicles or recovery from the oviduct or uterus by flushing with transfer medium. superovulation by injection of gonadotropisms may enhance yield, but is usually not nec essary. eggs are generally injected directly into the uterus but the recipient uterus must be at the same stage of the uterine cycle (bennet and vickery, 1970) . synchronization of estrus can be achieved by vaginal inser tion of polyurethane sponges containing 0.75 mg medroxyprogesterone for 7 days. females are then put in a cage previously occupied by male rats, sponges are removed, and the rats are injected with 3 iu of pregnant mare's serum. within 34 hr, 93% will be in estrus. this can also be attained by administer ing 40 mg medroxyprogesterone in 200 ml ethanol/liter drink ing water, prepared fresh daily for 6 days, then intramuscular injection of 1 iu of pregnant mare's serum (bennet and vick ery, 1970 ). the rat has been utilized extensively in a variety of research fields, including behavioral science. rats are docile, adapt to new surroundings, tend to explore, and are easily trained to a variety of sensory cues by positive or negative reinforcement. rats sleep during daylight hours and activity, including feed ing, is greater during the night and early morning. laboratory rats are easily handled, but strain differences exist. sprague-dawley and lew rats tend to be less fractious than long evans or f344 rats. docility is improved with routine and proper handling. rats become nervous and refractory to han dling when they hear others squeal. nutritional deficiency, particularly hypovitaminosis a, and mishandling can make rats vicious. rats seek entry into small openings, a trait that is utilized for coaxing them into restrait apparatus. like other rodents, rats are coprophagic, which must be taken into con sideration when administering drugs, measuring fecal output, or performing nutritional studies. unlike mice, rats are less apt to fight, and males can be housed together. in addition, rats are not gregarious like mice, and seem to tolerate single caging well. experimental studies indicate significant changes in plasma corticosteroid levels, depending on cage cohort size. levels tend to be least in rats housed singly, to increase in groups up to 5, to decrease in larger groups up to 10-12, then rise again in groups up to 30 (lane-petter, 1972). infectious agents constitute a significant environmental vari able that impacts on research data derived from laboratory rats. as is the case with other species, infectious agents induce a wide range of diseases in the rat that vary from inapparent to overt clinical disease. most investigations use large numbers of rats in which a specific group or colony consists of several to hundreds of rats. accordingly, emphasis on disease is one of prevention and placed at the colony level rather than on a sin gle or a few animals. curative use of antibiotics, which is important in the treatment of bacterial diseases of nonrodent species, is rarely useful in the laboratory rat. administration of drugs to obtain therapeutic blood levels is difficult to achieve in a colony; also some animals may improve clinically but re main colonized by the pathogen and serve as carriers, reinfecting other animals. rats seldom show clinical signs of disease upon arrival to the laboratory from commercial sources. however, these rats may harbor pathogens that are of low to moderate virulence and that are capable of severely compromising the health of animals when the rats are exposed to various types of experimental stress. moreover, some of these pathogens may never cause clinical disease, yet induce microscopic lesions or biochemical aberrations that can have profound effects on research data. for these reasons, investigators and clinicians should be aware of the pathogen status of the animals used in studies, both ini tially and throughout the course of the studies. this section on infectious diseases contains those agents that are of principal importance to the investigative use of the rat. a. streptococcosis. the causative organism, streptococcus pneumoniae, is a gram-positive coccus that is rather ubiq uitous among humans and animals. streptococcus pneumoniae is frequently recovered from respiratory tract lesions in guinea pigs, nonhuman primates, and some domestic animals. in hu mans, it is often present in the nasopharynx in the absence of clinical symptoms of infection. upper respiratory tract infec tion of conventionally raised rats has been reported to be com mon. however, it is seldom present in barrier-maintained, commercial rat sources. as in pneumococcal disease in hu mans, a number of serological types have been associated with respiratory disease in rats. streptococcus pneumoniae infection in rats often remains lo calized in the nasopharynx without the development of overt disease. a shift in the host-parasite balance due to stress or concurrent infection with another pathogen may result in bronchopneumonia and bacteremia. the most common signs of respiratory disease are serous to mucopurulent nasal discharge and "red tears" due to porphyrin pigments secreted from the harderian glands, dyspnea, rales, and depressed activity. ani mals will often die within a few days after the onset of pneu monic signs. the severity and prevalence of clinical disease within an infected colony are associated with environmental conditions that induce stress (e.g., experimental manipulation, overcrowding, fluctuations in ambient temperature and humid ity, and copathogens). although all age groups are susceptible to infection and clinical disease, young animals are more apt to be clinically affected. transmission between rats is by aerosol droplet. although both humans and rats can carry the same serotypes of s. pneumoniae, the authors are unaware of evi dence indicating zoonotic or human-to-animal transmission. the most characteristic gross lesions are pulmonary consol idation and fibrinopurulent pleuritis and pericarditis ( fig. 1 ). an extensive fibrinopurulent peritonitis, orchitis, or meningitis may occur as well. if a bacteremia occurs early, the disease may be acute with few gross lesions. streptococcus pneu moniae induces an outpouring of exudate rich in fibrin, neutrophilic leukocytes, and erythrocytes into the alveoli. bron chioles are filled with neutrophilic leukocytes. embolie lesions may occur in multiple tissues which include the spleen, liver, kidneys, joints, and brain. streptococcosis is diagnosed by clinical signs, characteristic lesions, and isolation of s. pneumoniae from lesions. the per icarditis, pleuritis, and pleural effusion noted above differenti ate pneumococcal disease from pneumonia due to mycoplasma, although the two pathogens often are superimposed. this organism produces an á-hemolysis on blood agar plates similar to that of the streptococcus viridans group. streptococ cus pneumoniae isolates are most commonly differentiated from nonpathogenic s. viridans by the sensitivity of the former organism to optochin (hydrocuprein hydrochloride). optochin-impregnated discs are placed on a blood agar plate which has been inoculated with a pure culture of the clinical isolate. if the isolate is s. pneumoniae, a distinct zone of growth inhibition will be present around the disc. although typing of s. pneumoniae isolates is seldom done today, one can type an isolate by reacting known specific s. pneumoniae antisera with s. pneumoniae isolates. this serological test is the neufeld-quellung reaction and is based on the capsular swelling that is induced by specific antiserum. there is no effective means to control s. pneumoniae infec tion once it is enzootic in a colony. benzathine penicillin (30,000 units/200 gm body weight) may be helpful in reducing the severity of the disease and as an aid in limiting infections to a subclinical mode in some animals. however, antibiotics will not eliminate the organism from rat colonies. hysterectomy rederivation of breeding stock from infected colonies is an ef fective method of initiating new stock free from pneumococcal infection (weisbroth, 1979) . b. pseudotuberculosis (corynebacteriosis). the causative agent of pseudotuberculosis is the gram-positive bacillus, corynebacterium kutscheri. on occasion, other corynebacterium species can cause similar syndromes in rats. typically, the or ganism causes inapparent infections in rats, with exacerbation of respiratory disease under conditions of stress. when clinically ill, the most commonly seen signs include serous oculonasal discharge, dyspnea, anorexia, and loss of weight or retarded growth. animals with severe pulmonary signs usually succumb within several weeks, while rats with less severe signs often survive much longer. most rats will have inapparent infections in which c. kutscheri cannot be isolated from internal organs. little is known concerning how c. kutscheri is carried or transmitted within a colony. it has been suggested that the organism is transmitted via aerosol droplet or direct contact. once rats are infected, a hematogenous spread may be involved, since lung lesions are initially interstitial and not bronchial. gross lesions are characterized by a variable number of grayish-yellow foci surrounded by red zones, particularly in the lung (fig. 2) . in longer-standing cases, individual foci co alesce into raised lesions 1 cm or larger in diameter. occasion ally, fibrous adhesions occur between the lungs and thoracic walls. similar lesions may be seen in other organs, including the liver, brain, and kidneys. the hepatic lesions resemble tu bercles and have caseous centers and fibrous capsules. prepucial adenitis, arthritis, and otitis media may also be caused by c. kutscheri. the lesions in various target organs appear to be due to septic emboli. pulmonary lesions initially consist of a polymorphonuclear cell and macrophage infiltrate of the bronchioles and interstitial tissue with a round cell infiltrate occurring later. bronchi become impacted with polymorphonuclear cells and necrotic leukocytes. giemsa or gram staining of infected tissues will reveal the rod-shaped c. kutscheri organisms. diagnosis of c. kutscheri infection is made on clinical signs, gross and microscopic lesions, and isolation of the bacterium from infected tissues. although the respiratory signs are simi lar to those present with mycoplasmosis, the rapidity with which c. kutscheri clinically affected rats succumb helps dif ferentiate it from mycoplasma pulmonis-'mduced disease. un like streptococcosis, fibrinopurulent pericarditis, peritonitis, and pleural effusion are not seen. whereas peribronchial lymphoid hyperplasia is a dominant lesion in mycoplasmosis, it is unremarkable in c. kutscheri infections. corynebacterium kutscheri is easily recovered from lesions and upper respiratory tract exudates by culturing on blood agar plates incubated aerobically at 37°c. epizootics of pseudotuberculosis may occur in conven tionally raised breeding colonies, but rarely occur in barrierraised colonies. epizootics often can be retrospectively associ ated with an environmental stress (e.g., fluctuation in ambient temperature or ventilation). culling of ill animals will not eliminate c. kutscheri from animals remaining in a colony. isolation of the organism from animals with subclinical infec tions is not usually successful. for this reason, cortisone ad ministration has been advocated as a means for surveillance of infection in colonies prior to necropsy and culturing for c. kutscheri. in the past, most serological methods have been un satisfactory in detecting antibody in animals with inapparent infections (weisbroth, 1979) . recently, however, enzymelinked immunoabsorbant assay (elisa) has been shown to be capable of detecting antibody in animals without clinical signs of infection (ackerman et al., 1984) . hysterectomy derivation is an effective means to establish a c. kutscheri-free colony. antibiotic therapy will not eliminate c. kutscheri from a colo ny, but a 7-day regimen of penicillin has been reported to be effective in curtailing an epidemic of c. kutscheri-'mductd pneumonia (fox et al., 1979) . since c. kutscheri infection is, in most cases, inapparent and manifests itself whenever the host is sufficiently stressed, it can be a significant problem in experimentally stressed rats. c. tyzzer's disease. tyzzer's disease is caused by the gram-negative, spore-forming rod, bacillus piliformis. this organism, which is not a true bacillus, is an intracellular pathogen that has not been cultivated on artificial media, and is, as yet, taxonomically undefined. in the laboratory, b. piliformis is propagated in the yolk sac of embryonated chick eggs. this disease occurs in other rodent species and appears to be widely distributed in many nonrodent species, but there ap pears to be a degree of species specificity among b. piliformis strains. it occurs occasionally in conventionally raised rat colo nies. the vegetative form of b. piliformis is unstable in the environment. however, spores of the organism are relatively stable and are believed to be the source of transmission among animals. clinical signs associated with tyzzer's disease are not partic ularly distinctive and, accordingly, only suggestive in making a diagnosis. typically, affected rats are apt to be adolescents with signs such as lethargy, weight loss, and distended abdo mens. diarrhea is not a common sign in rats with b. piliformis infection. animals displaying clinical signs generally die with in several weeks. clinically inapparent infections occur and are most probably responsible for transmission of the organism within a colony. clinically evident tyzzer's disease is usually associated with experimentation that compromises the immunocompetence of rats. the most remarkable gross lesions involve the liver, ileum, and myocardium. hepatic lesions consist of numerous small, pale foci on the surface and within the parenchyma. the intes tinal lesion has been termed "megaloileitis" due to a segmen tai dilatation and inflammation of the ileum (fig. 3) (jonas et ai, 1970) . heal distension is not always present. in some rats, circumscribed gray foci also occur in the myocardium. the pathogenesis of the disease is believed to involve a pri mary intestinal infection with spread to the liver via the portal circulation. bacillus piliformis invades enterocytes, resulting in villus shortening, inflammation, necrosis, and hemorrhage. intracellular organisms are demonstratable in epithelium of crypts and villi. the necrotic foci in the liver are most often present near vessels. surrounding these foci are varying num bers of leukocytes, macrophages, and fibroblasts. intracytoplasmic bacteria may be seen in hepatocytes at the periph ery of the lesions, but may be present in very small numbers and thus be hard to find. organisms are also found in myocar dium around foci of necrosis (weisbroth, 1979) . a presumptive diagnosis can be made by the gross lesions, but a definitive diagnosis is dependent upon observation of the organism within hepatocytes, intestinal epithelium, or myocar dium. impression smears of liver taken at necropsy and stained with gram, giemsa, or méthylène blue stains may be useful for a rapid diagnosis. however, formalin-fixed specimens stained by giemsa or warthin-starry methods are usually per formed to confirm a diagnosis. the ileal distension seen in rat tyzzer's disease must be differentrated from other causes of adynamic ileus, particularly chloral hydrate-induced lesions. prevention of tyzzer's disease in a colony is dependent upon a barrier that excludes entry of the agent by contaminated cages, equipment, and infected animals. routine cage sanita tion probably is ineffective in killing the spores of b. piliformis, but exposure of spores to 80°c for 30 min has been shown to inactivate them. sodium hypochlorite (0.3%) is an effective disinfectant (ganaway, 1980) . although antibiotics have been shown to be effective under experimental conditions in mice, there is no evidence to indicate that antibiotic therapy can be of value under natural conditions within a colony of rats (weisbroth, 1979) . d. pasteur elio sis. pasteur ella pneumotropica frequently infects conventionally raised rats and has been recovered occa sionally in rats from barrier-and axenic-maintained colonies. it is a pathogen of very low virulence, and most infections remain clinically inapparent. only a relatively few reports doc ument p. pneumotropica as a primary pathogen in cases of penumonia, otitis media, and conjunctivitis. as a copathogen with either m'ycoplasma pulmonis or sendai virus, it has a con tributory role in the resultant respiratory lesions and otitis. its localization is not limited to the respiratory tract, since it is frequently isolated from the oral cavity, intestinal tract, and uterus. it also has been associated with mastitis and furunculosis in rats. it has been suggested that p. pneumotropica is essentially an enterotropic rather than a pneumotropic orga nism. the intestinal tract is probably the primary site for local ization of the organism in subclinical infections. horizontal transmission is by the oral-fecal route and direct contact. since p. pneumotropica is frequently carried in the uterus, vertical transmission can occur, and, accordingly, this can compromise the microbial status of axenic and gnotobiotic colonies. distinctive clinical signs and lesions do not occur with p. pneumotropica-induoed disease. accordingly, a diagnosis must be based upon its isolation as the sole pathogen or, as in many cases, as a copathogen within lesions. blood agar medi um is satisfactory for primary isolation from nonenteric sites. however, for recovery from the intestinal tract, enrichment in a medium such as gn broth is recommended before isolation is attempted on blood agar plates (weisbroth, 1979) . hysterectomy derivation and barrier maintenance are the only means to control infection. however, particular attention must be made to ensure that hysterectomy-derived young came from dams that had culturally negative uteri. antibiotic thera py is not effective in eliminating the organism from a colony. e. salmonellosis. salmonella species that infect rats in clude salmonella enteritidis, s. typhimurium, s. dublin, and s. meleagridis. salmonellosis, which was once a major cause of disease in laboratory rat and mouse colonies, is rarely reported in either species today. however, it still exists in wild popula tions of rodents and, therefore, remains a potential threat to laboratory rodents. infection in an immunologically naive colony typically re sults in an epizootic of clinically affected rats and a varying proportion of animals with inapparent infection. these latter animals act as subclinical carriers to render the infection as enzootic in a colony. acute outbreaks will occur intermittently whenever immunological and other host defense mechanisms are altered. signs associated with salmonellosis in the rat are anorexia, depressed activity, starry hair coats, and soft to formless feces. affected animals die in 1 to 2 weeks. lesions that occur in salmonellosis differ depending on the stage of the disease. salmonellae penetrate the intestinal mucosa at the level of the ileum and cecum. the earliest le sions occur in this locale and consist of a mild dilatation, thick ened intestinal walls, and a granular mucosal surface. involve ment of the reticuloendothelial system is reflected by enlarged peyer's patches, mesenteric lymph nodes, and spleen. in some infected animals, a bacteremic state occurs that results in the demise of the host before the development of further lesions. however, in animals not succumbing to septicemia, ulcération of the ileal, colonie, and cecal mucosa occurs. histologically, the villus epithelium of the ileum is markedly degenerated, and the lamina propria is infiltrated with neutrophils and mac rophages. concomitant with intestinal lesions is the develop ment of focal necrosis and granulomas in the spleen and liver due to hematogenous spread of the organism (buchbinder et al, 1935; maenzae/fl/., 1970) . in rats who are intermittent or chronic shedders of salmonel la, the most remarkable lesions are lymphadenitis of the mes enteric lymph nodes and ulcération of the cecal mucosa. rats from which salmonella is chronically shed have more ad vanced lesions than do intermittent shedders of the organism. a diagnosis of salmonellosis relies upon identification of an isolate as a salmonella sp. recovery of salmonella from the intestines, spleen, and liver is readily accomplished in rats clinically affected during an epizootic. however, this is not true for asymptomatic carriers, since some will shed the orga nism intermittently in the feces, and recovery from tissues is difficult. recovery in carrier animals is best accomplished by initial incubation of fecal pellets in an enrichment broth, such as selenite f plus cystine broth, followed by streaking onto brilliant green agar (weisbroth, 1979) . from this medium, possible salmonella colonies are inoculated into triple-sugariron slants. final identification is then made by biochemical tests and serotyping. prevention of this disease is based upon the exclusion of wild rodents from laboratory animal facilities and the use of only feed and bedding that has been properly processed and pack aged to ensure against salmonella contamination. /. pseudomoniasis. pseudomonas aeruginosa, a ubiq uitous gram-negative bacterium found in soil and water, colo nizes plants, insects, animals, and humans. it often colonizes the oropharynx and can be isolated from the intestinal tract of rodents. infection with this organism in immunocompetent rats is nearly always inapparent. however, when rats are immunosuppressed, p. aeruginosa invades the upper respiratory mucosa and cervical lymph nodes, becomes bacteremic and induces an acute, lethal disease. in some cases, rats develop facial edema, conjunctivitis, and nasal discharge. in genet ically thymic-deficient rats (nude), retro-orbital abscesses may occur prior to bacteremia. transmission in laboratory rodents occurs primarily by direct contact and contaminated water bottles and automatic watering systems. phenolics are usually effective disinfectants, but quaternary ammonium compounds may actually support its growth. diagnosis of pseudomoniasis is based upon a history of immunosuppression associated with an epizootic of acute disease and isolation of p. aeruginosa from the blood and organs of affected rats. facial edema in affected rats must be differenti ated from viral sialodacryoadenitis. pseudomonas aeruginosa grows well on blood agar and most other standard laboratory media. most strains are ß-hemolytic and produce a bluish-green pigment, pyocyanin, as well as fluorescein. the use of specialized media (pseudomonas p agar) enhances pigment production. the organism derives energy from carbohydrates via oxidation rather than fermentative me tabolism. identification of isolates as p. aeruginosa is easily made by the above characteristics and appropriate biochemical reactions (weisbroth, 1979) . in most research applications, p. aeruginosa-free rats are not necessary for the conduct of the work. it is a major problem, however, in rats used for burn research and in studies in which drugs or radiation induce immunosuppression. infection can be relatively well controlled in a colony by hyperchlorinating drinking water at 12 ppm or by acidification of water to a ph of 2.5-2.8. in a closed colony, it is also advisable to remove rats that remain culturally positive after water treatment has been instituted. in studies requiring pseudomonas-free rats, isolators are useful in which a gnotobiotic environment can be achieved. alternatively, laminar flow units may suffice if supplies and equipment are sterilized and personnel wear sterile garments. g. streptobacillosis. streptobacillus moniliformis is a commensal bacterium often present in the nasopharynx of con ventionally raised rats. although it may be involved occasion ally as a secondary invader within inflammatory lesions of the rat, the chief importance of s. moniliformis is that it is the principal agent causing rat-bite fever in humans (anderson et ai, 1983) . the other bacterium associated with this clinical syndrome is spirillum minus. clinical signs in humans usually occur within 10 days of a rat bite and consist of headache, weakness, fever, a generalized rash, and arthritis. often clini cal signs subside in several days but then recur at irregular intervals for weeks or months (see chapter 22). a. murine respiratory mycoplasmosis. murine respirato ry mycoplasmosis (mrm) is the term now accepted for a dis ease which, for many years, had an undefined etiology and a number of synonyms [i.e., infectious catarrh, enzootic bronchiectasis, chronic respiratory disease (crd), and chronic murine pneumonia]. since 1969, the causal relationship of mycoplasma pulmonis with this disease has become well estab lished (kohn and kirk, 1969; lindsey et al., 1971; whittlestone et al., 1972) . of all the pathogens occurring in laboratory rats, m. pulmonis has had the greatest negative im pact on studies. this has been primarily due to the chronicity of the disease, which often manifests itself only after months of infection. long-term studies in areas of toxicology, carcinogenesis, nutrition, and gerontology, in particular, have been affected. prior to the use of gnotobiotic techniques and barrier maintenance in rat production colonies, m. pulmonis was enzootic in nearly all commercial and institutional colo nies. today, vendors can be selected who offer mycoplasmafree rats. my coplasma pulmonis is highly contagious and in duces a disease that frequently results in debilitation or demise of the host after a long period of time. the clinical signs associated with mrm range from negligi ble upper respiratory tract signs to systemic signs associated with pneumonia. the earliest and most common signs include snuffling and serous or mucopurulent oculonasal discharge. extension of m. pulmonis infection from the nasopharynx via the eustachian tubes to the middle ears is common. however, torticollis and circling due to involvement of the inner ear are infrequently observed, even though one or both middle ear bullae may be impacted with exudate. the onset of upper res piratory signs is variable, but often occurs within several weeks postinfection. signs of penumonia include dyspnea, rales, and systemic effects such as weight loss, starry hair coat, and hunched posture. characteristically, signs of pneumonia occur 3-6 months postinfection, but this is quite variable and is a function of environmental influences, such as intracage ammonia levels and the immune competence of the host. in a small percentage of cases, the disease will be nearly subclinical even in the presence of extensive pulmonary lesions. mycoplasma pulmonis is transmitted both horizontally and vertically from dams to their litters. in most instances, trans mission from the female occurs postpartum by direct contact, but if the genital tract of the dam is infected, antenatal infec tion can occur. horizontal transmission between postweanling rats of any age readily occurs, and there appears to be no sig nificant age-related resistance to either infection or disease. although little is known about differences in resistance among rat stocks and strains, the lew rat has been shown to be more susceptible to mrm than the f344 rat. there is little evidence available to indicate that transmission occurs through fomites such as caging equipment and garments worn by personnel. since aerosol droplet and direct contact appear to be the prima ry modes by which m. pulmonis infections are spread, the rapidity with which the organism is transmitted is dependent upon environmental factors, such as ventilation rates, degree of recirculation of air, and animal density within rooms. the basis for the pathogenicity of m. pulmonis is not well understood. mycoplasma pulmonis adsorbs to the cell mem brane of the ciliated, columnar or cuboidal epithelia in the res piratory tract (fig. 4) . it has been suggested that adsorption is a means by which mycoplasmas damage host cells by uptake of essential cellular metabolites; release of cytotoxic products, such as h 2 0 2 ; or cross reaction of antibody with cell mem brane components that are antigenically similar to or altered by mycoplasmas. infection severely distorts or ablates ciliary structures (fig. 4) , interfering with mucociliary clearance mechanisms. the gross lesions in the upper respiratory tract include mucopurulent exudate in the nasal cavity, sinuses, and middle ear bullae. later, the exudate becomes caseous within the bul lae. lesions in the lower respiratory tract reflect those of a bronchopneumonia. the earliest lesion is a mucopurulent exu date within the trachea, bronchi, and bronchioles. this pre cedes grossly evident lesions of the lung parenchyma that ini tially consist of atelectasis due to bronchial occlusion. later, bronchiectatic lesions appear as numerous cream-colored nod ular abscesses on the surface of the lung. these lesions may be restricted to only a portion of a lobe or may involve nearly all of the parenchyma (fig. 5) . microscopically, the inflammatory response is characterized by a lymphocyte and plasma cell infiltrate in the submucosa and neutrophilic leukocyte response within the lumina of the epithelium. nasal cavity, eustachian tubes, middle ears, and tracheobronchial tree. a consistent and prominent lesion in the lung is the peribronchial lymphoid hyperplasia that often be comes quite massive. within the lumina of the bronchi and bronchioles, mucin and neutrophil exudation increases during the course of the disease to the point of bronchiectasis. con comitant with the impaction of bronchi is a change in the epithelia from a ciliated, columnar type to a squamoid type. this change in epithelial architecture is likely associated with cytotoxic enzymes from autolyzed neutrophils, although a di rect cytotoxic effect from mycoplasmas could be involved. a tentative diagnosis of mrm can usually be made by obser vance of the clinical signs and gross lesions described above. clinical signs alone are not particularly helpful, since nasal exudates are present in bacterial infections such as s. pneumoniae. in addition, the reddish porphyrin deposition seen in the nares and periorbitally in sialodacryoadenitis virus infec tion and water deprivation may be confused with exudation. the gross lesions of otitis media and bronchiectasis are rather distinct. however, c. kutschen lung lesions may grossly mim ic those of mrm. histopathology and serological evidence will differentiate mrm from sendai virus infection, although the two infections are often superimposed. recently a filamen tous bacterium has been associated with bronchiectasis in wild and laboratory rats (mackenzie et al., 1981) . however, the causal relationship of this organism with lesions is undefined since the rats were also infected with m. pulmonis. this fila mentous bacterium has not been successfully grown on artifi cial media, and its presence is best verified by either histology, using the warthin-starry stain, or electron microscopy (fig. 6) . although a definitive diagnosis of mrm is made by isola tion of m. pulmonis from involved tissues, it is evident that the existence of other agents must be evaluated to determine if copathogens are contributory to lesions. prevention of mrm in either breeding or experimental colo-nies is dependent upon barrier systems that preclude the entry of m. pulmonis into the facility. hysterectomy derivation is the only means of establishing an m. pulmonis-frtt breeding colo ny from a previously infected stock. due to the frequent local ization of this microorganism in the uterus, it is necessary to ensure that neonates taken by hysterectomy have not been in fected in utero. rats used in research animal facilities are ob tained from various commercial and institutional sources. ac cordingly, it is essential that the mycoplasma status of these sources is known and that the rats are housed by vendor or in groups with a similar microbial status. for assessment of whether a group of rats is m. pulmonisfree, the best sites for isolation in animals without gross lesions are the nasal cavity, middle ear, trachea, and uterus-oviduct. mycoplasma pulmonis is not particularly fastidious and grows well in several types of mycoplasma media (cassell et al., 1979; lentsch et al., 1979) . most formulations have a ph indi cator that is useful since m. pulmonis ferments glucose. in broth media, moderate to heavy growth is reflected by ph and color of the broth. in broth cultures in which the titer is low, a perceptable ph change may not occur. tissue and washing samples should be placed in broth rather than agar media, since recovery of the organism is more likely in those samples con taining few mycoplasmas. samples from broth cultures are transferred to agar media when a ph change is readily evident or at 7-10 days if no ph change occurs. mycoplasma colonies are evident in 3-4 days by observation with 40 x stereoscopic microscopy. although culturing and histopathology have been the usual means to survey rat colonies, elisa testing has recently been shown to be a very sensitive serological assay and one that can be performed quickly in most clinical laboratories (cassell et al., 1981a) . in vitro sensitivity tests show m. pulmonis to be susceptible to tetracycline and tylosin. tetracyline, given at 5 mg/ml drinking water, may be useful in some situations (lindsey et al., 1971) . however, treatment with antibiotics seldom influences the disease course of mrm in a colony situation. b. murine genital mycoplasmosis. mycoplasma pulmonis recently has become recognized as an important pathogen in the female genital tract of rats, and thus is being treated here as a distinct disease rather than as a sequella to mrm. infection of the genital tract is usually inapparent. however, reduced fertility and fetal deaths can occur. infection of the oviduct and uterus occurs frequently in rats who have respiratory my coplasmosis. it is unknown whether localization in the genital tract occurs due to a hematogenous spread or to an ascending infection of the genital tract. it has been shown that subsequent to intravenous inoculation, m. pulmonis almost invariably lo calizes in the female oviduct-uterus. gross lesions, when present, consist of a purulent oophoritis, salpingitis (fig. 7) , and pyometra. the lew strain is particu-ä^sfcvvv ( ä ß f/g. 6. electron micrograph of filamentous bacterium (large arrow) and m. pulmonis (small arrow) attached to epithelium of respiratory mucosa. the morphology of size of the filamentous bacterium are similar to that of the cilia. (courtesy of dr. w. f. mackenzie.) larly prone to develop gross lesions. mycoplasmapulmonis ad sorbs to the epithelial cells in the genital tract in a manner similar to that seen in the respiratory tract. salpingitis occurs most frequently and is characterized by exudation of neutrophils into the lumen, hyperplasia of oviductal epithelium, and a lymphoid response in the submucosa. the lesions in the ovarian bursa include edema and inflammation. uterine le sions can vary from a mild inflammatory change to pyometra (casselle/fl/., 1981b). genital mycoplasmosis in the male rat has not been well doc umented. however, it is known that experimental inoculation can include an inflammatory response in the ductus efferens and epididymis. moreover, it is known that m. pulmonis is capable of adherence to spermatozoa in an in vitro system. since pasteur ella pneumotropica can also induce similar le sions in the female rat, a diagnosis of mycoplasmosis is depen dent upon isolation of m. pulmonis from the lesions. methods for culturing and identification are similar to those used for respiratory mycoplasmosis. because the rat is widely used in various types of reproduc tive biology research, m. pulmonis colonization, even without gross lesions, would probably impact on the validity of data. the grossly evident caseous lesions in the ovary and oviduct can be mistaken for neoplasia if microscopy is not done. c. mycoplasmal arthritis. the etiological agent of this disease is mycoplasma arthritidis. this mycoplasma species colonizes the pharynx, middle ears, and lungs of rats, although few studies have been done to document the relative frequency of this mycoplasma in rat sources. within the respiratory tract, m. arthritidis colonization is thought to induce negligible le sions, and it has been shown to coexist with m. pulmonis. although it is often considered to be the principal agent in volved in arthritis in rats, the disease has been rarely reported. nearly all reports of its involvement in clinically apparent ar thritis have been made prior to 1960. it has been suggested that poor cage sanitation and abrasions of the extremities are in volved in entry of the organism to the joints by hematogenous spread or extension from surrounding tissues (ward and cole, 1970) . since the organism appears to be of low virulence, the immunocompetence of the host may be a major factor in the outcome of infection. arthritic animals limp and move with difficulty due to pain associated with the polyarthritis. any of the joints in the limbs and vertebrae can be affected, but the tibiotarsal and radiocarpal joints are most often involved. affected joints are hyperemic and swollen. incised joints reveal a purulent exudate in both articular and periarticular tissues. microscopically, there is exudation of neutrophils into the synovial spaces, and a lym phocyte and plasma cell infiltration in the synovial mem branes. destruction of the articular cartilage occurs subsequent to the inflammatory response. since polyarthritis can occur subsequent to septicemias asso ciated with other bacteria, particularly c. kutschen, a diag nosis of m. arthritidis-'mduced arthritis is contingent upon the demonstration of m. arthritidis by isolation or immunofluorescence techniques. this mycoplasma species grows well in me dia used to isolate m. pulmonis if arginine is added to the for mulation (cassell et al., 1979) . tetracyclines have been used to prevent the onset of arthritis when the organism has been inoculated intravenously, but there are no reports of its efficacy in spontaneous cases. mycoplasma arthritidis, like m. pulmonis, may contaminate transmissible tumors and caution should be exercised to ensure transplanted tissues are not contaminated. hemobartonellosis. the causative agent of this rickettsial disease is hemobartonella muris. this organism is an extra cellular parasite of erythrocytes and induces inapparent infec tions that may persist for long periods. the ability of the host to restrict the infection to a subclinical mode rests with the integrity of the reticuloendothelial system. evidence of infec tion is usually limited to splenomegaly and laboratory findings of mild parasitemia and reticulocytosis. transmission of h. muris involves the blood-sucking louse, polyplax spinulosa. transmission can occur during a blood meal or when rats crush infected lice and are inoculated via pruritis-induced abrasions. the organism can also be transmit ted inadvertantly with transplantable tumors and other biolog ical products. diagnosis of hemobartonellosis is dependent upon identifica tion of the organism in the peripheral blood of infected ani mals. the usual method of detection is by splenectomizing rats suspected of harboring the organism. in these rats, severe para sitemia and hemolytic anemia occur within 2 weeks after sur gery. hemobartonella muris can be visualized on the surface of erythrocytes in romanowsky-stained blood smears as coccoid bodies arranged singly, in clusters, or chains (cassell et al., 1979) . the rarity of reported cases would indicate h. mûris is no longer a significant problem in barrier-maintained colonies. however, conventionally maintained colonies may be exposed to infected wild rats and p. spinulosa and, accordingly, the disease still is of importance in the laboratory rat. the disease has had a negative impact on investigations of various types, but principally with those in which the host's immune compe tence has been impaired. a. parvoviral syndromes. parvoviruses that can infect rats include rat virus (rv), toolan h-l (h-l) virus, and minute virus of mice (mvm). parvoviruses are small nonenveloped viruses that resist extremes in temperature, ph, and drying. rat virus, or kilham rat virus (krv), has several antigenically related strains (rv, h-3, x-14, l5, hb, sprv, her, hhp, kirk), all of which have been isolated as inadvertant contami nants of rat tissue or rat-passaged biological material. toolan h-l related serotypes (h-l and h-t) are antigenically distinct from rv serotypes. both rv and h-l are experimentally pathogenic, producing similar lesions, but only rv has been associated with natural disease. neonatal rats can be experi mentally infected with mvm, but the virus does not seem to cause natural infection. minute virus of mice antibody reac tivity can be present in rat serum, but this is probably non specific, since it can be found in germfree rat serum and is reduced or eliminated by receptor destroying enzyme. rat virus infection is usually subclinical or latent, but a num ber of clinical syndromes have been associated with it. infec tion of pregnant females can cause fetal résorption and birth of small litters. pups are runted, atactic, or jaundiced. neonates develop similar signs following postpartem exposure. rats in troduced to an infected colony can develop ruffled fur, de hydration, and sudden high mortality. a similar syndrome oc curs in latently infected adults subjected to immunosuppressive regimens. the rat is the only natural host for rv and h-1, although experimental infection can be established in a number of other species. seroconversion to both rv and h-l virus is common, with a high prevalence of infection within an enzootically in fected colony. horizontal transmission is achieved by the oral and probably respiratory routes, with virus excretion primarily in the feces. some strains of rv can be excreted in the milk or in utero. clinical signs are manifest transiently upon introduc tion of rv into a previously uninfected population, but, there after, the virus spreads rapidly to produce subclinical or inapparent enzootic infection. rat virus can persist as a true latent infection in the presence of high circulating antibody, but dis ease can be activated by immunosuppression. it must, therefore, be assumed that seropositive rats are persistently infected and can serve as a source of infection to other rats. pups infected in utero or as neonates develop intranuclear inclusions and necrosis in the outer germinal cell layer of the cerebellum. the recovered animal has severe depletion of the internal granular layer and disorganized purkinje cells. intra nuclear inclusions are also in hepatocytes, kupffer cells, endothelial cells, and biliary epithelial cells, resulting in necrotizing hepatitis and the sequellae thereof (bile retention, jaundice, peleosis, bile ductal hyperplasia, parenchymal collapse, nodu lar hyperplasia). in adults, infection is usually inapparent, but when acute disease is precipitated, rv injures vascular walls and hematopoietic elements, causing coagulative disorders, thrombosis, hemorrhage, and infarction within the central ner vous system (hemorrhagic encephalomyelopathy). hemorrhagic and necrotic lesions have also been noted in the per itoneum, testis, and epididymis. rat virus has broad tissue tropism and lesions or clinical signs may potentially be varied, depending on virus and host factors (coleman et al., 1982; jacoby et al, 1979) . infertility and unthrifty pups caused by rv must be differenti ated from environmental and husbandry factors or infectious agents such as mycoplasma or sendai virus. adult disease must be differentiated from toxicity, nutritional deficiency, and trau ma. diagnosis is made by the typical lesions, if present, virus isolation, and serology. seroconversion to each virus (rv or h-l) can be detected by serum neutralization, hemagglutination inhibition, complement fixation, and immunofluorescence. hemagglutination inhibition is currently the most commonly used means of antibody determination (jacoby et ai, 1979) . since rv infection is usually silent and persistent and can be transmitted either vertically or horizontally, effective control is best achieved by destroying the entire population, decon taminating, and repopulating with clean stock. virus-free rats can be obtained from selected commercial vendors or by caesarean rederivation. rederived progeny must be tested for vertically transmitted strains of virus. colonies can be kept virus-free by limiting entry to seronegative, virus-free rats (as well as transplantable rat neoplasms or tissues), periodic serological testing, and adequate physical containment. although parvovirus infection of rats is usually inapparent, there can be adverse effects on the research usefulness of in fected rats. immunosuppression may exacerbate illness and mortality in latent carriers. the viruses often contaminate transplantable tumors and cell lines, can modify immune re sponsiveness or cause teratological effects. a decision to work with infected animals should be made carefully. b. other dna virus infections. rats are susceptible to rat cytomegalovirus, which has a predilection for the salivary and lacrimai glands. infection is widespread among wild, but not laboratory rats (jacoby et al., 1979) . rats also seroconvert to mouse adenovirus, but it is not known if infection is due to a mouse or rat strain of virus. adenovirus-like inclusions have been reported in the intestine of rats treated with cancer chemotherapeutic agents (ward and young, 1976) . c. siaiodacryoadenitis virus and related coronaviral infections. two strains of coronavirus have been identified as pathogens of laboratory rats: siaiodacryoadenitis virus (sdav) and rat coronavirus (rcv). furthermore, rats are experimen tally susceptible to the coronavirus of mice, mouse hepatitis virus (mhv). coronaviruses are large, pleomorphic enveloped rna viruses with surface peplomers or spikes that confer a corona-like appearance to the virion. viruses of this group have complex antigenic interrelationships and cross-react ex tensively. common antigens are shared by sdav, rcv, and mhv, particularly by complement fixation, but antibody reac tivity is highest with homologous virus. siaiodacryoadenitis virus and rcv represent different strains of the same virus, but whether different strains of the same virus or separate viruses, they are both important natural pathogens in rats. the signifi cance of mhv for rats is not known, but the virus can replicate in the respiratory tract of intranasally inoculated rats (taguchi et al., 1979) . natural antibodies to mhv can occur in rats, but this is probably due to the closely related antigenicity of mhv to sdav and rcv rather than natural mhv infection of rats (barthold, 1984) . clinical signs of sdav infection vary widely in severity, but include blepharospasm, sneezing, porphyrin-pigmented nasal and ocular discharge, and cervical edema (fig. 8) . some rats develop keratoconjunctivitis and other ocular lesions. signs persist approximately one week, but ocular sequellae can be permanent. acutely infected rats become anorectic, and estrus can cease temporarily. infection is subclinical in weanling or older rats, but intranasally inoculated neonates die and suck lings develop lower respiratory disease. siaiodacryoadenitis virus is highly contagious and spreads rapidly among susceptible rats by contact, aerosol, or fomite. susceptible rats of any age can be infected. when enzootic within a colony, clinical disease occurs only in sucklings, since adults are immune. infection is acute, lasting only about 1 week, at which time rats seroconvert with no carrier state. maintenance of sdav in a colony requires continuous intro duction of susceptible stock as weanlings or newly introduced rats. the epizootiology of rcv is presumed to be similar to sdav. within 2 days of intranasal inoculation, sdav causes rhi nitis followed by necrosis of the ductular and acinar epithelium of salivary and lacrimai glands, accompanied by intense in flammation and edema. tracheitis and peribronchial lymphoid hyperplasia can also be found. salivary glands appear swollen, pale, with interlobular and periglandular edema. harderian glands are flecked with yellow-gray foci. one, some, or all of the salivary or lacrimai glands can be affected, with the excep tion of the sublingual glands, which are spared. cervical lymph nodes become enlarged. glandular repair ensues within 1 week, with squamous metaplasia of ductular epithelium and hyperplasia of acinar epithelium. the repair phase subsides within 30 days with minimal residual lesions. interstitial pneu monia can occur in suckling, but not adult rats. conjunctivitis, keratitis, corneal ulcers, synechia, hypopyon, and hyphema can arise due to lacrimai dysfunction. eye lesions usually re solve, but can proceed to chronic keratitis, megaloglobus (fig. 9) , and retinal degeneration. rat coronavirus infection causes rhinotracheitis and focal interstitial pneumonia. salivary but not lacrimai gland infection is rare, but when present, resem bles wild sdav lesions. infection with rcv also lasts approx imately 1 week (barthold, 1984; jacoby et al., 1979) . nasal and ocular signs must be differentiated from those caused by mycoplasma, sendai virus, pathogenic bacteria, ex cess ammonia, or hypovitaminosis a. cervical swelling may fig. 8 . epiphora and swelling of the ventral neck in a rat naturally infected with sdav. (from barthold, 1984 ; courtesy of hemisphere publishing corp.) fig. 9 . megaloglobus and hyphema in a young rat naturally infected with sdav. (from barthoid, 1984; courtesy of hemisphere publishing corp.) also occur in immunosuppressed rats infected with p. aeruginosa. microscopic sdav lesions are characteristic. mild lower respiratory tract lesions associated with rcv must be differentiated from those of sendai virus or pneumonea virus of mice (pvm). seroconversion or rising complement fixing antibody titers following acute disease is confirmatory. how ever, antibody may be low or undetectable with this method. serum neutralization is another test that can be used, but the most sensitive antibody tests are immunofluorescence or elisa. either mouse or rat coronaviruses are used as antigen in these latter tests (smith, 1983) . rats can be kept free of sdav and rcv if they are isolated and if newly introduced rats are immune or unexposed. intro duction of a single subclinically infected rat can precipitate epizootic disease among naive rats. if an outbreak occurs, the infection will run its course and die out within 3-4 weeks if new rats are not introduced into the room and if breeding is temporarily ceased. routine disinfection of rooms and equip ment is sufficient to destroy environmental sources of virus. sialodacryoadenitis virus lesions can be confused with or contribute to changes induced by test compounds or nutritional deficiencies, particularly vitamin a. sialodoacyoadenitis virus disease can predispose to anesthetic death due to airway hypersecretion. eye lesions resulting from sdav infection can in terfere with eye research. both sdav and rcv can potentiate other respiratory infections. d. sendai viral infection. sendai virus commonly infects laboratory rats, but its clinical significance is less than in mice. sendai virus is a parainfluenza 1 virus of the paramyxovirus family. paramyxoviruses are pleomorphic, enveloped, labile rna viruses. sendai virus infection in rats is usually subclinical, but can be manifested as ruffled fur, dyspnea, or anorexia. a decrease in average litter size and runted pups is common during outbreaks in breeding colonies. sendai virus is highly contagious and disseminates rapidly. outbreaks subside following development of an immune popu lation, with the potential of recurrence several months later as the susceptible population enlarges. sendai virus induces an acute respiratory infection with no natural carrier state. excre tion and transmission of virus occurs via the respiratory tract (jacoby <?r ö/., 1979) . sendai viral lesions in the rat are similar to those in genet ically resistant strains of mice. following an initial necrotizing rhinitis, tracheobronchitis ensues within 1 week of infection. lungs become consolidated and plum-colored in a patchy, pri marily anteroventral, distribution. necrotizing bronchiolitis, focal nonsuppurative interstitial pneumonia, and perivascular and peribronchial lymphoplasmacytic infiltrates are the hall marks of the lower respiratory component. in the rat, acute lower respiratory lesions last only a few days and coincide with clinical disease. repair of bronchial mucosa is effected through hyperplasia and squamous metaplasia. residual le sions can last for months in the mouse, but probably not in rats. during the acute stage of sendai virus infection, mucosal necrosis and inflammation can extend up the eustachian tubes to the middle ear. bacterial otitis media is often preceded by sendai viral infection in rodents (brownstein et al., 1981; d. g. brownstein, unpublished observations, 1982) . mild nonsuppurative pulmonary lesions can be seen with sendai, pvm, rcv and to a lesser extent, sdav. sendai virus can be superimposed upon and contribute to respiratory mycoplasmosis. squamous metaplasia of respiratory mucosa can mimic hypovitaminosis a and also occurs in my coplasmosis. virus isolation and serocon version or rising titers to sendai virus confirm a diagnosis based upon characteristic lesions and clinical signs. sendai virus antibody can be accu rately detected by complement fixation and hemagglutination inhibition, but newly developed immunofluorescence and elisa are replacing these methods (smith, 1984) . rats can be kept free of sendai virus in a similar manner as with sdav and rcv. recovered sendai virus antibody-posi tive stock can be utilized as breeders to reestablish a virus-free population. pups born of these parents will be virus-free and antibody negative, following decay of their maternal antibody. mice and hamsters can serve as sources of infection with paramyxoviruses. sendai virus infection, although usually subclinical, can cause significant pulmonary changes and act as a copathogen with mycoplasma or bacteria. squamous metaplasia and hy perplasia induced by these agents influence respiratory carcinogenesis and interpretation. sendai virus can cause immunosuppression for prolonged periods in rats (garlinghouse and vanhoosier, 1978) . e. pneumonia virus of mice infection. pneumonia virus of mice is a pneumovirus of the paramyxovirus family and thus shares many features in common with sendai virus. it has not been associated with clinical disease in the rat or mouse under natural conditions. several rodent species, including hamsters, guinea pigs, and gerbils, seroconvert to pvm under natural conditions. the virus seems to spread rapidly among suscepti ble rodents, based on serocon version. it is presumed to be transmitted in a manner analogous to sendai virus. the pathogenesis of pvm has been investigated in mice, but has not been thoroughly examined in rats, even though it pro duces more significant pulmonary lesions in rats than mice un der natural conditions. in the mouse, pvm replicates in the upper respiratory mucosa without obvious cytolysis following low-dose exposure (d. g. brownstein, unpublished observa tions, 1982) . viral antigen and cytolytic lesions are seen in the bronchial and bronchiolar epithelium as well as alveolar walls following apparently higher doses of virus in a pattern reminis cent of sendai virus. the ensuring lesion, like that of sendai virus, is necrotizing bronchiolitis and interstitial pneumonia (carthew and sparrow, 1980) . the active infection lasts about 1 week, after which time lymphocytic infiltration of peribronchial and perivascular tissues and focal interstitial pneumonia can persist for several weeks. rat lungs develop prominent nonsup purative perivascular and mild interstitial inflammation in re sponse to both experimental and natural pvm infections (d. g. brownstein, unpublished observations, 1982; vogtsberger et al., 1982) . infection is usually diagnosed retrospectively in rats, where pulmonary lesions are observed following seroconversion to pvm in the absence of other respiratory pathogens. pulmonary lesions mimic those of sendai virus or possibly coronaviruses. antibody to pvm has usually been detected by hemagglutination inhibition or serum neutralization, but these tests are being replaced by immunofluorescence and elisa. hemagglutination inhibition is least expensive and highly accurate, however (smith, 1983) . pulmonary lesions induced by pvm can inter fere with the interpretation of experimental studies, or possibly pulmonary function, although this has not been established. hemorrhagic fever with renal syndrome, korean hemorrhagic fever, or muroid virus nephropathy is a human disease com plex caused by one or more serologically related bunyaviruses, including hantaan virus. this syndrome is mentioned here be cause of its emerging eminence as a major zoonotic disease in which several species of the suprafamily muroidae, including laboratory rats, can serve as carriers. the virus is transmitted to man by excretions and aerosols from the lungs, saliva, and urine of healthy rodent carriers. pathology, if any, in rats has not been described. the disease in humans includes proteinuria, azotemia, and, less often, petechiae, hemoconcentration, hypotension, and renal failure. human mortality can oc cur, but recovery with immunity to reinfection is usual. hemorrhagic fever with renal syndrome viruses cause human disease throughout much of eurasia, but on the basis of serological evidence, hfrs viruses exist world-wide in feral rodents and humans, including the united states. transmis sion of hfrs to laboratory personnel by silently infected al bino laboratory rats (rattus norvegicus) has been documented in japan and belgium. virus-infected lung and kidney cell lines provide excellent sources of antigen for sensitive immu nofluorescence tests, but serological monitoring of laboratory rats for hfrs virus antibody is not currently a common prac tice (gajdusek et al, 1982; anonymous, 1982) . g. other virus infections. rats harbor endogenous retroviruses or genomic retrovirus sequences that (in contrast to the mouse) are of minimal significance in terms of natural disease. a number of endogenous rat sequences have been artificially / / / recombined with mouse leukemia virus sequences or naturally recombined with other rat retrovirus sequences to form defec tive rat sarcoma viruses, for example kirsten and harvey rat sarcoma viruses, which are experimentally oncogenic to rats, mice, and hamsters (shih and scolnick, 1980) . natural seroconversion to reovirus-3 and mouse encephalomyelitis virus have been noted with no apparent disease. an enterovirus with neurotropic properties has been isolated from naturally infected, asymptomatic rats. although rats can be experimen tally infected with ectromelia and lymphocytic choriomeningitis virus, these agents are not of practical significance as natu ral infections of rats. testing for antibody to these agents is therefore a vacuous exercise. a number of other viruses or viruslike agents have been isolated or incriminated as causes of disease in rats, including rat submaxillary gland virus, novy virus, enzootic bronchiectasis agent, gray lung virus, and wild rat pneumonia agent. these agents, if extant at all, are of du bious importance in contemporary laboratory rat populations (jacoby et ai, 1979) . recently, clinically silent infection with an unidentified pox virus has been found in soviet rats. micro scopic necrotizing and inflammatory changes were restricted to the nose (kraft et ai, 1982) . the agent is believed to be cow pox virus. laboratory rats are host to far fewer parasites than wild rats, since husbandry practices interrupt complex life cycles and caesarean rederivation has simply eliminated many agents al together. control of these agents in infected colonies is also best achieved by these means. this section will mention only agents that have been reported in laboratory rats, although most are unlikely or rare. table ix outlines selected treatment regimens for control of common metazoan parasites, but the reader must beware that these drugs may render the treated rat useless for experimental work. decisions to treat rather than eliminate infected rodents must be made with care, in concert with the needs of the scientific investigator. a. protozoal infections. several flagellates can parasitize, rats but few are truly pathogenic. hemoflagellates (tripanosoma cruzi, t. lewisi) occur in wild rats, but these agents require anthropod vectors. nevertheless, t. lewisi can be en countered if a colony is infested with rat fleas. trypansoma lewisi is only mildly pathogenic even in heavy infections, and infection is short term. rats harboring this organism become ill when stressed or immunosuppressed. enteric flagellates (tritrichomonas sp., tetratrichomonas sp., pentatrichomas sp., trichomitis sp., hexamastix sp., enteromonas sp., retortamonas sp., chilomastix sp., monocercomonoides sp., and octomitus sp.) are very common but nonpathogenic in labora tory rats. giardia muris and spironucleus (hexamita) muris application 3-10 gm/liter drinking water for 7 days, stop 7 days, re peat 7 days 0.5 gm/liter drinking water for 3 days 3 mg/liter drinking water or 1.2 mg/kg feed for 4 weeks 0.5 mg/gm feed for 1 day 100 mg/g sc, single dose 100 mg/kg po, single dose 0.5-1.0% dust with or without pyrethrin 24 hr/week on cage top for several weeks 2-6 gm of pellets in bedding for 5 days, change bedding and cage, repeat at 12 days, change and repeat at 1 month 0.25% dip 3-5% dust, up to 0.5% spray or up to 2% dip, repeat at 12 days up to 0.5% dip 1% spray "information kindly supplied by dr. j. w. streett, yale university. ; 'a number of these compounds have adverse effects on the experimental usefulness of treated rats and therefore should be used with discretion and full knowledge of the investigator. are also common and considered potentially pathogenic, caus ing unthriftiness, diarrhea, and intestinal lesions in severely affected animals. however, these are generalizations made from other species, since natural clinical disease has not been reported among rats. sporozoan parasites, common in wild rats, are encountered only on occasion in laboratory rats. hepatozoon muris infec tion is usually subclinical, but severely affected rats can have leucocytosis, splenomegaly, anemia, hepatic degeneration, emaciation, or death. infection requires ingestion of the vec tor, laelaps echidninus. toxoplasmosis (toxoplasma gondii) is subclinical in rats, requiring the ingestion of cat feces or contaminated tissues containing asexual stages, but can also be transmitted transplacentally. intracellular t. gondii organisms occur in several tissues, particularly lung and reticuloendothelial organs, and cysts are found in the central nervous system. sarcocystis muris, once common but now rare in labo ratory rats, has a life cycle similar to ã. gondii. cysts are found in the muscle. frenkelia sp. has been described in a colony of laboratory rats, causing large thin-walled cysts and inflamma tion in the central nervous system, but no clinical signs. encephalitozoon cuniculi is apparently now uncommon in labora tory rats and seldom induces signs or lesions. its prevalence and significance are far greater in the laboratory rabbit. single or clusters of small spores form within cysts in a number of tissues. nonsuppurative inflammation can be found in the brain and kidneys. unlike t. gondii, e. cuniculi organisms are gram-positive. encephalitozoon cuniculi can be transmitted via contaminated transplantable tumors, tissue, or tissue fluids from infected rats. intestinal sporozoans include eimeria nieschulzi and e. miyairii, which infect the small intestine, and e. separata, which infect the large intestine of wild rats. they are exceedingly rare or nonexistent in laboratory rats. pneumocystis carinii, once considered to be a yeast but now considered a sporozoan, is an ubiquitous organism that infects the lung of a variety of species, including laboratory rats. prev alence of infection is high within infected colonies, but preva lence among various geographic areas in unknown. it seems to be very common among both conventional as well as barrierreared rats. normally p. carinii is not pathogenic, but infected rats can develop disease after several weeks of repeated steroid injections and starvation, particularly in younger rats. remark ably, rats from many commercial sources can be so treated and shown to be infected, even if free of other pathogens. under these circumstances, the lungs are expanded and rubbery. al veolar walls are thickened and hypercellular, and alveoli are distended with proteinaceous, eosinophilic foamy material containing macrophages and organisms in different stages of development. tissues can be stained with special stains, such as methanamine silver or periodic acid-schiff, to visualize the organisms, which are not visible with hematoxylin and eosin. other protozoa, including entamoeba muris and balantidium coli, are found in the large bowel of rats, but are not pathogenic. the reader is referred to more exhaustive coverage of protozoa for detailed means of diagnosis (flynn, 1973; hsu, 1979) . b. nematodiasis. syphacia muris is a common oxyurid (pinworm) of the cecum and colon. embryonated eggs are passed in the feces or deposited on the anus. rats are infected by ingestion of eggs or by larval migration into the colon from the anus. syphacia muris closely resembles s. obvelata, the mouse pinworm. both species of syphacia can patently infect rats or mice, but preference seems to be shown for the homolo gous host. another common oxyurid of rats and mice is aspicularis tetraptera, which is also found in the cecum and colon. aspicularis eggs are nonembryonated, and are not de posited near the anus. depending on parasite species, the pré paient period is 18-23 days. diagnosis is achieved by exam ination of large bowel contents (particularly the cecum and proximal colon) for nematodes, feces for ova, and, in the case of syphacia, cellophane tape impressions of the anus for ova. speciation is easily achieved by the differential morphology of ova. aspicularis ova are symmetrically ellipsoidal, while syphacia ova are flattened on one side. since eggs are ex tremely resistant to environmental factors and disinfectants, and autoinfection can occur with s. mûris, infection is difficult to eradicate. anthelminthic treatment ameliorates infection (table ix) , but all rats must be treated simultaneously and the room must be thoroughly cleaned. despite these actions, rein fection frequently occurs. caesarean rederivation and subse quent strict sanitation are the most effective means of control. pinworms are generally considered nonpathogenic, but heavy parasite loads can be deleterious to the host. concern has been raised regarding the effects of these agents on research but no effects have been documented in the rat. trichosomoides crassicauda is common in some rat colonies but is not generally encountered in commercially raised ani mals. it inhabits the transitional epithelium and lumen of the urinary tract. the small male resides within the reproductive tract of the female, whose anterior end embeds within the mucosa. embryonated ova with bipolar opercula are shed in the urine. following ingestion, eggs hatch in the stomach, lar vae penetrate the gastic wall, then migrate via the lungs, kidney, and ureter to the urinary bladder. migrating larvae can incite eosinophilia and formation of granulomata, particularly in the lung. drug treatment (table ix) is effective, as is cesarean rederivation. parasites do not incite an inflammatory response in the bladder, but can serve as a nidus for calculi or enhance natural and experimental bladder carcinogenesis. other nematodes are common in wild but rare in laboratory rats. trichinella spiralis adults occur in the intestine, and lar vae migrate extensively, encysting in muscle. transmission is effected by ingestion of contaminated meat. capii i aria hepatica eggs are ingested, hatch, and migrate through the cecum to the liver through the portal veins. adults mature and lay characteristic bipolar operculate ova in the liver, where they lie dormant until ingested by a carnivore. gongylonema neoplastium burrows in the squamous epithelium of the tongue and anterior stomach, where it incites a proliferative response or ulcération. the life cycle involves an intermediate insect host. angiostrongylus cantonesis, which requires the ingestion of a mollusk intermediate host, infects the brain and lung. the intestinal nematodes heterakis spumosa, nippostrongylus brasiliensis, strongyloides ratti, and trichuris muris lack in termediate hosts, but rarely occur naturally in laboratory rats. none is known to be very pathogenic. further details on nematode diagnosis and ova morphology are available (flynn, 1973; hsu, 1979; oldstone, 1967) . taenia taeniaformis, the cat tapeworm. following ingestion of ova, oncospheres migrate to the liver and form strobilocerci 113 (cysticercus fasiolaris). host connective tissue capsules can give rise to sarcomas. this parasite has been used as a model of parasite-induced oncogenesis in the rat. cysticercus fasciolaris is on occasion encountered in laboratory rodents with contaminated food or bedding. hymenolepis nana and diminuta infect the small intestine of several species, including the rat. hymenolepis nana, the dwarf tapeworm, has a 1-mm wide segmented body ranging from 7 to 100 mm in length. the life cycle may be either direct or indirect. following ingestion of ova, oncospheres penetrate the intestinal mucosa, form cysticercoid larva, then emerge into the lumen and mature into adults that shed eggs in the feces. nonimmune hosts can be autoinfected; eggs are pro duced, hatched, and complete their life cycle within the intes tine of a single host. the indirect cycle involves intermediate hosts, such as grain beetles or fleas, in which cysticercoid lar vae form and await ingestion by the definitive host. hymenolepis diminuta is 3-4 mm wide and 20-60 mm long. the eggs are passed in the feces but must be ingested by an intermediate host (flour beetle, flea, moth), which in turn must be ingested by the definitive host to complete the life cycle. enteritis can occur in heavy infestations, which are most likely to be encountered with h. nana. both can infect primates, in cluding humans. hymenolepis nana, which does not need an intermediate host and can cause autoinfection, is a particular public health hazard. anthelmintic treatment is effective (table ix) . diagnosis of hymenolepis is made by finding ova containing hexacanth embryos in the feces or adults in the lumen of the bowel. differential features are detailed else where (hsu, 1979) . d. insect infestation. two anopluran (sucking) lice infest wild rats, polyplax spinulosa (the spined rat louse) and hoplopleura pacifica (the tropical rat louse). the latter has not been reported among laboratory rats. polyplax spinulosa is now rare in most laboratory rat populations. it completes its entire life cycle within the fur of the host and is transmitted by direct contact. infested rats are unthrifty, irritable, pruritic, restless, and anemic. polyplax spinulosa can transmit a num ber of infectious agents, which include hemobartonella muris and t. lewisi. diagnosis is made by identifying organisms in the fur. rat fleas (xenopsylla sp., leptopsylla sp., and nosopsyllus sp.) are rare in laboratory rats, since their life cycles are usu ally interrupted by proper sanitation. fleas can transmit other agents and can serve as the intermediate host of h. nana and diminuta (flynn, 1973; hsu, 1979; oldstone, 1967) . see sec tion iii,a,5,e for methods of control. e. arachnid infestation. mesostigmate mites can be en countered on occasion in laboratory rats. they are rapid blood suckers and have a nonselective host range. they are on the host only during feeding, spending much of their life cycle secreted in the environment. diagnosis must be attained by finding engorged mites on bedding, cages, and crevices. other hosts, including humans, fall prey to their painful and irritating bites. the most important mesostigmate is ornithonyssus bacon (tropical rat mite). heavily infested colonies contain de bilitated, anemic rats with reduced reproductive efficiency and occasional deaths. liponyssoides sanguineus has not been re ported in laboratory rats, but may be unrecognized because of its similarity to ornithonyssus sp. laelaps echidninus (spiny rat mite) does not adapt well to the laboratory rat environment unless husbandry is poor. it is a vector of hepatozoon. prostigmate mites have a more selective host range and spend their life cycle in the fur (or follicles) of the host. radfor dia ensifera, the rat fur mite, can be common in some rat colonies. this mite produces few ill effects, but heavy infesta tions can induce self-inflicted trauma (flynn, 1973; hsu, 1979; oldstone, 1967) . demodex sp. is also encountered, but its prevalence is unknown, since it lives deep within hair follicles and sebaceous glands where it produces minimal lesions (walbtxgetal., 1981) . astigmate mites include the mange mites, which have a se lective host range and complete their life cycle on the host. notoedres muris, the ear mange mite, infests the skin of the ear and to a lesser extent, the nonglabrous regions of the body. this mite burrows in the cornified epithelium, and elicit a pruritic dermatitis (flynn, 1973; hsu, 1979) . it is seldom seen in contemporary rat colonies. control of ectoparasites is gained by preventing the introduc tion of infected animals (including wild rodents), sanitation, treatment of rats, and, in the case of mesostigmates and fleas, treatment of the environment (table ix) . treatment seldom completely eliminates infestation and can have an adverse ef fect on the usefulness of the treated rats for research. repopulation or caesarean rederivation and subsequent preven tion by proper management is the best approach. deep mycoses are generally not considered to be significant natural diseases in laboratory rats. pulmonary aspergillosis is occasionally observed in rats and mice, particularly when immunocompromised. aspergillus sp. can be readily isolated or observed in affected lung tissue with selected histochemical techniques. pulmonary lesions consist of miliary granulomata containing langhans giant cells in areas with characteristic uniformly branched, septate hyphae. phycomycotic encepha litis has been reported as a natural disease in 2-to 4-week-old rats. phycomycotic fungi have thick, irregularly branched, nonseptate hyphae in tissue. dermatomycosis is probably more likely to be encountered than deep mycosis, but is also rare in laboratory rats. ring worm in rats is restricted largely to infection by trichophyton mentagrophytes. infected rats can be lesionless carriers or dis play patchy alopecia and erythema with scale, papule, or pus tule formation. fungal elements are visible within the stratum corneum and hair follicles. ecothrix spore formation in hair shafts can be present. the disease can be diagnosed by exam ination of skin scrapings treated with 10% potassium hydrox ide, histological sections of skin prepared with fungal stains, or isolation (weisbroth, 1979) . a. genetic anomalies. genetic anomalies in coat color and character, organ development, immune responsiveness, obesity, hypertension, metabolism, and others have been iden tified and in many cases developed as specific characteristics of various stocks and strains. the reader is referred tö several more reviews for further information (altman and katz, 1979; hansen et al., 1981; robinson, 1965 robinson, , 1979 . some strains nat urally develop disease syndromes, such as brattleboro rats with diabetes insipidus, while other strains have less obvious characteristics but react uniquely to different research vari ables. the researcher must be aware of these variations and judiciously select the appropriate stock or strain to accomplish the goals of the experiment. each stock, strain, substrain, and even group of rats that has drifted from its parental stock can develop its own unique dis eases, manifest its own incidence and rate of development of various spontaneous lesions, and react in different ways to in fectious and research variables. expression of genetic traits is further influenced by extraneous factors such as husbandry and diet. b. nutritional deficiencies. more is known about the nu tritional requirements and deficiencies of rats than perhaps any other species. natural deficiencies of single dietary compo nents are rare and seldom recognized. this is because rats ef fectively store fat-soluble vitamins (and b 12 ), manufacture vi tamin c, and fulfill much of their requirement for b vitamins by coprophagy. furthermore, some deficiencies are influenced or compensated for by other dietary elements. the most com mon signs of nutritional deficiency are vague, such as poor hair coat, reduced weight gain or growth, diminished fertility, and susceptibility to infectious disease. commercially avail able rodent diets effectively supply balanced diets to rodents, but diets should not be utilized beyond their expiration date and ideally should be kept in cold storage. several components deteriorate with prolonged storage, heating, or sterilization, in cluding lysine, vitamins a and e, and, to a lesser extent, riboflavin and thiamin. nutritional requirements vary with the genetic strain, stage of life, and microbiological association. axenic or specific pathogen-free (spf) animals can lack gut microflora necessary for supplying certain vitamins, particu larly vitamin k. they must receive supplemented diets both because they need more and because autoclaving or pasteuriza tion reduce the levels of certain essential nutrients. antibiotic treatment, which affects intestinal microflora, can reduce the bacterial source of vitamins by coprophagy. nutritional ade quacy goes far beyond the needs of the rat, since the composi tion of nutritionally adequate diets is known to profoundly in fluence the biological responsiveness to research manipula tion, infectious disease, and expression of spontaneous lesions (national research council, 1978; rogers, 1979) . the signs of nutritional deficiency are often vague and must be differentiated from other syndromes. hemorrhage due to hypovitaminosis k can be confused with rv disease (which in itself can be precipitated by nutritional deficiency). infertility or poor production can be caused by rv, sdav, sendai virus, m. pulmonis, or nuturitional deficiency. squamous metaplasia in the salivary and lacrimai glands of rats recovering from sdav or in the respiratory tract of rats infected with sendai virus or m. pulmonis must be differentiated from hypo vitaminosis a, which in turn predisposes to respiratory infec tions. environmental factors, such as excess heat or low hu midity, contribute to infertility or skin disease which mimic hypovitaminosis e or other deficiencies. ordinarily mild infec tious diseases can become severe, such as pseudotuberculosis, due to nutritional deficiency. the diagnosis of many rat dis eases therefore requires review of nutritional practices. practices other than nutrition can also influence or cause dis ease in rats. sanitation is of obvious import. cage design, pop ulation density, and temperature influences have been men tioned in sections ii,d and e. outbreaks of opportunistic in fectious disease, such as pseudotuberculosis, can be precipi tated by sudden fluctuations in temperature and humidity. pseudomonas aeruginosa can be introduced and spread within a colony through inappropriate sanitation, particularly of water bottles. refilling water bottles at a faucet in the animal room is not advised for this reason. bottles and sipper tubes should be sanitized and water should be acidified or chlorinated. exces sive ammonia from dirty bedding, overcrowded cages, or poor ventilation predisposes to respiratory infections, particularly mycoplasmosis. a number of management-related syndromes can occur that are not related to sanitation or infectious disease. rats adapt well to automatic watering systems, but animals unfamiliar with these devices can become dehydrated. similarly, mal function of these systems and blockage of water bottle sippe tubes with metal filings or other detritus can have a similar effect. rats maintained for long periods on wire-bottom cages develop foot sores, which can result in severe lesions, discom fort, hemorrhage, and anemia. aging rats must be examined periodically for overgrowth of their incisors, a very common problem in rats held long term. teeth must be carefully clip ped, avoiding splitting or shattering. dusty bedding and food can result in foreign body pneumonia. plant, mineral, and even bone fragments can be found in sections of lung obtained from rats under these circumstances. softwood shavings used as bedding can cause intestinal impaction, particularly in young rats. high ambient light, or even light levels within the recom mended range, can cause retinal degeneration and cataracts in albino rats. rats maintained near ceiling light fixtures develop retinal degeneration, while those near the floor are spared. low relative humidity (<40%) in concert with high tempera ture and other factors can cause one or more annular constric tions of the tail skin (ringtail) or toes. the segments distal to the constrictions swell or undergo dry gangrene (fig. 10) . this is most apt to occur in suckling or preweanling rats and in rats kept in wire-bottom cages. increasing humidity, reducing air turnover rate, placing rats in solid-bottom cages, and providing nesting material for nursing dams and their litters are all ame liorative for this condition. axenic rats normally have enlarged ceca, which become ap proximately five times normal size. there is net efflux of fluid into the lumen in response to increased osmotic pressure of luminal content, reduced availability of ions needed for mucosal solute-coupled water résorption and vasoactive com pounds which exert their effect on smooth muscle tone. cecal enlargement can interfere with reproduction. dietary manip ulation can reduce the enlargement substantially (foster, 1980) . the effects on smooth muscle tone become pronounced in aging axenic rats, which can develop progressive cecal en largement due to atony. volvulus and torsion can result. the reader must be cognizant of the much larger list of environmental variables that significantly alter physiological re sponsiveness of test animals (baker et al., 1979a; gralla, 1981) . unlike the mouse, traumatic skin disease in the rat is more likely to be due to self-inflicted trauma than fight wounds. rats infested with ectoparasites or dermatophytes can develop excoriative dermatitis due to self-inflicted trauma. roughly sym metrical ulcerative dermatitis over the dorsal and lateral neck and shoulders has been reported (fig. 11) . the etiology re mains to be determined, but coagulase-positive staphylococcus aureus is consistently isolated from the skin lesions, and colonies of gram-positive cocci are microscopically visible on the surface of the lesion. attempts to induce the disease in other rats with this agent have yielded inconsistent results (fox et al, 1977) . the rat is often considered "resistant" to infection and ac cordingly submitted to a variety of experimental procedures without regard to aseptic techniques. peritonitis and wound in fections are often found under such conditions. inappropriate handling of large rats by the tips of their tails will cause the skin to tear and slip off when they struggle. adynamic ileus occurs in rats given intraperitoneal injections of anesthetic preparations containing chloral hydrate or related compounds (fig. 12) . for variable periods up to 5 weeks after treatment, rats become lethargic, anorectic, and constipated with distended abdomens that may lead to death. gross nec ropsy findings reveal segmental atony and distension of the bowel, usually jejunum, ileum, and cecum. focal serosal in flammatory changes and fibrosis are seen microscopically (fleischman et al., 1977) . this syndrome mimics megaloileitis (tyzzer's disease), but can be differentiated on the basis of history, lack of characteristic bacteria in tissues, and lack of liver and heart lesions. in.addition, it should not be confused with the bowel dilatation observed in axenic rats. space exists in this chapter to only summarize the more fre quently reported tumors in the rat, and the topic will not in clude experimentally induced tumors. the reader is encouraged to refer to the articles and monographs referenced in the text for more complete information. there have been numerous studies done to assess the inci dence of neoplasia in various stocks and strains of rats (burek, 1978; coleman et al., 1977; international agency for research on cancer, 1973 mackenzie and garner, 1973; ringler and dabich, 1979; sher, 1982) . some of these have led to the development of animal models and provided baseline data for experimental carcinogenesis work for which the rat is regularly used. there is a wide range of values reported in regard to the prevalence and type of tumors found in a particular rat stock or strain. these differences are a function of genetic and environ mental influences and differences in sample selection. nutri tion is well known as a factor that influences the development of neoplastic disease. for instance, it has been shown that a high fat diet enhances tumorigenesis, that the protein-calorie ratio influences the occurrence of some tumors, and that re stricted food intake of post weanlings for 7 or more weeks de creases tumor risk (ross and bras, 1971) . most of the surveys on tumor occurrence in rats either state nothing in regard to the presence of enzootic infectious disease or acknowledge that infectious diseases were present in the population. in many instances, infectious diseases can signifi cantly influence data on tumor risk because of their effect on longevity, preneoplastic changes, and masking of small tumors. with the exception of mammary fibroadenomas in many stocks and testicular tumors in f344 rats, the prevalence of tumors is quite low in rats under 18 months of age. according ly, the age at which rats are surveyed is very important in de fining the incidence of neoplasia in a particular stock or strain. the f344 rat is used in the bioassay program of the national cancer institute and is widely used in toxicology studies. it has been selected for these types of studies because of its small size, longevity, and low incidence of most tumors. however, this strain has a moderate to high incidence for some tumors, such as those of the testis, mammary gland, uterus, and hematopoietic and endocrine systems (sher, 1982) . a sprague-dawley-derived stock that is commonly used in carcinogenesis studies is the crl:cd(sd)br rat. this stock has a high inci dence of mammary tumors and pituitary adenomas. papillomas and squamous cell carcinomas occur infre quently, but have been reported in many stocks of rats. squam ous cell carcinomas are usually located on the face and head, and tend to be highly invasive but not metastatic. most of these arise as sebaceosquamous tumors from the glands of zymbal in the ear. similar tumors occur on the prepuce and clitoris. in females from most stocks and strains, the mammary gland is the most frequent site of neoplasia, with incidences of 30-60% being common. these tumors occur in males but much less frequently. benign fibroadenomas are the most common type, but adenocarcinomas may also occur. fibroadenomas have ductal epithelium and periductular connective tissue compo nents and tend to be less vascular than carcinomas. adenocar cinomas can metastasize to regional lymph nodes and the.lung. tumors of the mammary gland, which may arise at any site from the neck to the inguinal region, often attain a very large size (fig. 13) . the prevalence of tumors in the alimentary tract is extremely low, with most cases involving the colon. although hepatic tumors are readily induced by chemical carcinogens, very few spontaneously occurring malignant neoplasms have been re ported. more commonly occurring are neoplastic nodular le sions. the term ò 'neoplastic nodule" is used to describe cir cumscribed nodules of proliferating hepatocytes that compress the parenchyma (altman and goodman, 1979) . there is some 117 fig. 13 . mammary fibroadenoma in an aged rat. controversy as to whether these nodules are in all cases neo plastic. however, there is evidence that some develop into carcinomas. leukemia is rare in many stocks and strains of rats. howev er, mononuclear cell leukemia is very frequently seen in f344 and wf rats. in one survey of male f344 rats, an incidence of 16% proved leukemia second in occurrence only to the testic ular interstitial cell tumors. the leukocyte count in 9 leukemic rats ranged from 68,400 to 323,000 cells/ìà with an average of 143,190 (coleman et al., 1977) . other studies indicate a simi lar incidence for the wf rat. in the bn/bi rat, myelomonocytic leukemia has been reported to be the most common lymphoreticular tumor, with female and male incidences of 5 and 11%, respectively (burek, 1978) . both types of leukemia oc cur in rats over 18 months of age. neoplasms of the pituitary occur frequently with a higher proportion in females. surveys have shown an incidence of 15-30% in f344 rats, 18% in om (osborne-mendel) rats, and 3-13% in sprague-dawley rats. the most common pituitary tumor is the chromophobe adenoma. these are benign tumors that frequently become quite large, and, due to compression of the brain by the tumor, hydrocephalus and neurological signs may occur. they appear dark due to hemorrhagic areas arising from a vascular sinusoid component of the tumor. clinical manifestations may include neurological signs or cachexia, but these are often absent. pancreatic islet cell tumors are common in some stocks and strains. surveys have reported a 6% incidence in f344 rats, 4.4% in holtzman rats, and a 1-2.9% in sprague-dawley stocks. tumors of the thyroid are usually parafollicular cell ade nomas. these benign tumors occur in many stocks with an incidence varying between 1 and 7%. follicular cell car cinomas, which occur infrequently, can metastasize to the lung. adenomas of the adrenal cortex are very common in several strains. in at least three strains, buf/n, m520/n, and om/n, over 40% of the females and 20% of the males have cortical tumors. pheochromocytomas are the most common medullary tumors. they are particularly common in the buf/n, f344/n, m520/m, and wn/n strains, and, unlike cortical tu mors, they appear more often in males. tumors of the brain have been found to occur rarely in those studies that included examination of the cns. less is known about the prevalence of tumors in the cns than in other sys tems because many studies have not included brain and spinal cord examination. the astrocytoma is the most commonly re ported tumor of the cns. less often reported tumors include oligodendroglioma, meningioma, ependymoma, and granular cell tumors. primary tumors of the lung are rare in the rat. the most fre quently reported types are bronchogenic carcinomas, car cinomas, sarcomas, and adenomas. tumors of the bladder are rare in most stocks and strains of rats. however, the bn/bi strain has an unusually high inci dence of carcinomas of the bladder and ureter. males of this strain have been reported to have a 35% incidence of bladder carcinoma, while 22% of females have carcinoma of the ureter (burek, 1978) . nephroblastomas, renal tubular adenomas, and adenocarcinomas have been reported in the rat kidney. tumors of the prostate are common in the axc rat. in one report, adenocarcinoma of the ventral prostate was histologically detected in 70% of 30-to 46-month-old axc rats. prostatic tumors are reportedly uncommon in most stocks and strains of rats. reportedly, however, some of these surveys did not include adequate sectioning to locate small adenomas and carcinomas. interstitial cell tumors are the most common testicular tumor type. nearly all aged f344 rats and the majority of aci/n rats develop these tumors, but the tumor is rare in most other strains. tumors of the vagina and cervix are rare, except in aged bn/bi rats. tumors of the uterus and ovary are common in several strains of rats. one om strain was reported to have a 33% incidence of granulosa cell tumors, and a high incidence of endometrial tumors has been reported in the f344 and m520 strains. congenital disorders in the rat are infrequently reported. this is probably a reflection of two factors; the first being that evidence indicates the incidence is extremely low in outbred stocks, and the second being that commercial/institutional sup pliers select against breeders who have produced abnormal young. the genitourinary system and the eye have been most associated with congenital disorders. one substrain of the axc rat has a greater than 25% inci dence of unilateral renal agenesis and hydronephrosis (fujikura, 1970) . a high incidence of unilateral and bilateral hy dronephrosis has been found in bn/bi rats (cohen et ai, 1970) . pseudohermaphroditism has been reported in rats that are phenotypically female but who have an xy karyotype. testicles are present either in the abdomen or inguinal canal. this condition is due to a sex-linked recessive gene that is expressed by an insensitivity of tissues to androgens (bardin et al., 1970) . a number of ocular disorders have been reported in the rat. retinal dystrophy is inherited as a single autosomal recessive gene (rdy). homozygotes have a progressive loss of the retinal photoreceptor cells and an overproduction of rhodopsin early in the disease process (lavail et al., 1972) . a retinal degener ation reported in the wag/rij rat appears to be an expression of an autosomal dominant gene. end-stage lesions include disap pearance of photoreceptor cells, migration of the pigment epi thelium, and disorganization of remaining retinal layers. this retinal disease appears to mimic the pathogenesis of retinitis pigmentosa in humans (lai et al., 1980) . other reported devel opmental disorders of the eye include colobomas and cataracts. reports of congenital heart disease are rare. among the few reports is one involving an inbred strain of long-evans rats (fox, 1969) . in this strain, approximately 25% of the neonates have interventricular septal defects. this malformation is be lieved to be of polygenic origin. unlike the mouse, there are few reported mutants that have disorders of the central nervous system. recently, a partially inbred strain has been developed in which one subline has nearly a 50% incidence of hydrocephalus (kohn et al., 1981) . the mode of inheritance appears to be polygenic. the hydrocephalus is classified as communicating and the pathogenesis may be due to poorly developed veins in the periosteal and durai layers, and to underdeveloped pia-arachnoid cells. neoplastic and nonneoplastic lesions vary in type, onset, and prevalence due to the hereditary and environmental factors as sociated with a specific colony of laboratory rat. the rat is widely used in gerontological research. those who use the rat must be clearly aware of the factors that can influence lesions and death in aged rats (anver and cohen, 1979; burek, 1978) . neoplastic disease, a cause of death in many aged rats, has been discussed previously, and this section will summarize the more commonly seen nonneoplastic lesions in aging rats. a. chronic progressive nephropathy (cpn). chronic pro gressive nephropathy is among the most commonly encoun tered lesions in the rat, particularly in the aged animal where it is a major life-limiting disease. because of its complex nature, cpn has a large number of synonyms, often with inaccurate descriptive titles. sequential development of lesions and fac tors that modify the course of cpn have been thoroughly stud ied, but the actual pathogenesis remains undetermined. chron ic progressive nephropathy occurs in most rats, but its rate of development is significantly influenced by numerous factors, including sex, genetics, age, diet, association with microflora, hormone treatment (particularly testosterone), and others. male rats have an earlier onset and more rapid progression of cpn than females, and albino strains and stocks seem to be more predisposed. diet is an extremely important predisposing factor, particularly the quality and quantity of dietary protein. significant differences in severity of cpn can be observed among rats of the same sex, strain, age, and source when fed different diets for their lifetime. axenic rats do not seem to develop significant cpn. rats with cpn develop progressively severe proteinuria, first with the selective excretion of only small molecules such as albumin. in late cpn there is nonselective excretion, and the electrophoretic pattern of urinary protein resembles that of serum. proteinuria related to cpn should not be confused with the normal urinary excretion of a-globulin in male rats, proba bly of tubular origin. the histopathology of cpn is charac terized as progressive basement membrane thickening of the glomerulus, bowman's capsule, and proximal tubule. base ment membranes split and wrinkle as tubules become atrophie and collapse. glomeruli enlarge, with mesangial deposition of protein and lipid, adhesion to bowman's capsule, and segmen tai sclerosis. tubular epithelium may contain granules of resorbed protein, which later is mixed with lipofuscin and hemosiderin. as glomerular protein leakage becomes more severe, 119 tubules dilate and accumulate hyaline eosinophilic castes of protein within their lumina, resembling colloid. tubular epi thelium may undergo atrophy with collapse or dilatation. in terstitial fibrosis and leukocytic infiltration is frequently ob served. grossly, the kidneys become enlarged, pale with irregular surfaces and pigmented (fig. 14) . cystic tubules can be observed. hypoproteinemia, parathyroid hyperplasia with serum chemical changes indicative of hyperparathyroidism, and nephrotic syndrome are observed in rats with severe cpn (barthold, 1979) . b. nephrocalcinosis. this disease, which occurs more fre quently in females, has been reported in numerous strains of rats. in most cases, affected rats have been fed purified diets (e.g., . renal lesions usually involve the corticomedullary junction and consist of mineral deposition in the lumina of the proximal tubules (nguyen and woodward, 1980) . e. polyarteritis nodosa. this disease is of unknown etiol ogy and affects muscular arteries, most commonly the mesenteric, pancreatic, and spermatic. the lesions may be either acute or chronic. in the acute stage, there is intimai and medial fibrinoid necrosis, focal thrombosis, disruption of the elastic lamellae, and an infiltration of polymorphonuclear leukocytes and mononuclear cells. in the chronic stage, the arteries are nodular, tortuous, and thick-walled. affected vessels fre quently have aneurysms and thrombi. both acute and chronic phases may be observed in the same artery in an animal. d. myocardial degeneration. degeneration is a commonly observed lesion that occurs in most stocks and strains, with an onset at 12-18 months of age. it occurs more frequently in males. the lesions are usually microscopic but, in advanced stages, can appear grossly as grayish foci. the papillary mus cles and their attachment sites in the wall of the left ventricle are the most frequent sites of the lesion (anver and cohen, 1979) . e. radiculoneuropathy. spinal nerve root degeneration has been reported in a number of rat stocks and strains. lesion distribution may vary among strains; however, the cauda equi na and ventral spinal nerve roots are commonly involved. pos terior paresis and paralysis have been associated with these lesions, but some suggest that these signs are associated with a separate degenerative process of the skeletal muscle (anver and cohen, 1979) . an enzyme linked immunosorbent assay for detection of antibodies to corynehacterium kutschen in experimentally infected rats copulatory behavior can inhibit preg nancy in female rats neoplastic diseases inbred and genetically defined strains of laboratory animals. part 1. mouse and rat rat-bite fever in animal research laboratory personnel muroid virus nephropathies lesions associated with aging reproduction and breeding housing to contro! research variables selected norma tive data. appendix i pseudohermaphroditic rat: end organ insensitivity to testoster one chronic progressive nephropathy in aging rats research complications and state of knowledge of rodent coronaviruses reproduction and breeding techniques for laboratory animals morphophysiology sendai virus infection in genetically resistant and susceptible mice observa tions on enzootic paratyphoid infection in a rat colony age-associated pathology a comparison in germ-free mice of the pathogenesis of sendai virus and pneumonia virus infection my coplasmal and rickettsial diseases detection of natural mycoplasma pulmonis infection in rats and mice by an enzyme-linked immunosorbent assay (elisa) adherence and colonization of mycoplasma pulmonis to genital epi thelium and spermatozoa in rats tissue weights of the rat. i. normal values determined by dissection and chemical methods immunopathology. in "cell pathology heritable hydronephrosis in a mutant strain of brown norway rats pathological changes during aging in barrier-reared fischer 344 male rats naturally occurring lethal parvovirus infection of juvenile and young-adult rats breeding of the rat adynamic ileus in the rat induced by chloral hydrate parasites of laboratory animals gnotobiology ulcera tive dermatitis in the rat corynebacterium kutschen pneumonia in rats on long term tobacco inhalation studies evidence suggesting a multigenic origin of membranous septal defect in rats kidney malformations in fetuses of axc line 9935 rats bibli ography of hemorrhagic fever with renal syndrome (muroid virus nephropathies) effect of heat and selected chemical disinfectants upon infectivity of spores of bacillus piliformis (tyzzers disease) studies on adjuvantinduced arthritis, tumor transplantability and serologie response to bovine serum albumin in sendai virus-infected rats scientific considerations in monitoring and evaluating toxicological research nih rodents 1980 catalogue anatomy of the laboratory rat parasitic diseases viral diseases. /// "the laboratory rat tyzzer's disease in the rat pathogenicity of mycoplasma pulmonis in laboratory rats a new model of congenital hydrocephalus in the rat morphological evi dence for natural poxvirus infection in rats animal model: hereditary retinal degeneration in wag/rij rats the ufaw handbook on the care and management of laboratory animals photoreceptor pig ment epithelial cell relationships in rats with inherited retinal degenera tion comparison of techniques for primary isolation of respiratory mycoplasma pulmonis from rats historical foundations murine chronic respiratory disease. significance as a research complication and experimental production with mycoplasma pulmonis comparison of neoplasms in six sources of rats a filamentous bac terium associated with respiratory disease in wild rats salmonella enterocolitis in the rat clinical biochemical and hematological reference values in normal experimental animals intranephronic calculosis in rats helminths, ectoparasites and protozoa in rats and mice hematology and clinical biochemistry genetics of the norway rat nutrition lasting influence of early caloric re striction on prevalence of neoplasms in the rat tumors in control hamsters, rats, and mice: literature tabulation molecular biology of mammalian sarcoma viruses state of the art detection methods for rodent viruses the microscopic anatomy of the white rat asymptomatic infection of mouse hepatitis in the rat histological and serological response of b 6 c 3 f, mice and f344 rats to experimental pneumonia virus of mice infection demodicidosis in laboratory rats (rattus norvegicus) latent adenoviral infection of rats: intranuclear inclusions induced by treatment with a cancer chemotherapeutic agent the role of mycoplasmas and l forms of bacteria in disease bacterial and mycotic diseases respiratory disease in a colony of rats. ii. isolation of mycoplasma pulmonis from the natural disease, and the experimental disease induced with a cloned culture of this organism craigie's neuroanatomy of the rat key: cord-146391-jlu7nv6r authors: ohsawa, yukio title: covid-19 should be suppressed by mixed constraints -from simulations on constrained scale-free networks date: 2020-04-20 journal: nan doi: nan sha: doc_id: 146391 cord_uid: jlu7nv6r the spreading of virus infection is here simulated over artificial human networks. here, the real-space urban life of people is modeled as a scale-free network with constraints. a scale-free network has been adopted for modeling on-line communities so far but is employed here for the aim to represent peoples' social behaviors where the generated communities are restricted reflecting the spatiotemporal constraints in the real life. as a result, three findings and a policy proposal have been obtained. first, the height of the peaks in the time sequence of the number of infection cases tends to get reduced corresponding to the upper bound of the size of groups where all members meet. second, if we adopt the constraint on m0, the number of all other people one meets separately each at a time, to the range between 2 and 8, its effect on the suppression of infections may be weak as far as we allow group meetings of size w of 8 or larger. third, such a moderate constraint may temporarily seem to work for the reduction of infections in the early stage but it may turn out to be just a delay of peaks. based on these results, a policy is proposed here: for quickly suppressing the number of infections, restrict w to less than 4 if the constraint to make m0 at most 1 is too strict. if w is set to less than 4, setting m0 to 4 or less works for quick reduction of infections according to the result. network manipulation has been also explored, one of which is the control of the spreading dynamics by interacting with features of a network such as infection rate [4] , and another is network optimization by removing/rewiring links that is an np-hard problem [5] . the solutions for these problems include centralized control e.g. to suppress infection rates and a distributed approach where individuals discern the critical in-network contacts. the guidelines for attention and actions obtained from such researches should be considered for designing politics for controlling epidemic spreading. in the literature of general theories applicable to various influence propagation phenomena on a social network, a lot of findings have been obtained. for example, it has been found that the cascades in skewed human networks are triggered by large degree nodes, whereas small degree nodes may trigger cascade other networks [6] . we can find models for other social phenomena than virus spreading. in [7] , the diffusion of innovation has been simulated in artificial social networks and found the delayed propagation to lowdegree nodes from high-degree nodes, which is of greater impact if informative and nominal contacts from neighboring nodes are mixed. although this study is not applied for virus infection events, we find the authors' interest in the mutual influence between highdegree nodes and low-degree nodes in which the role of the latter is coming to be focused. this interest differs from the interest in tail-fat networks or from the detection of lowfrequency influential nodes. further, an encouraging fact is that the authors of [2] , [7] , and others having been working on network dynamics are contributing to providing points to consider in designing policies for controlling epidemic spreading [8] . the question we address in this paper is simple. is the spreading suppressed as expected if people are forced to reduce the contacts with others in the network? if so, what should the governmental policy be like? in this paper, we show a quick report based on a simple simulation of infection spreading in a social network of people in an urban environment. in this environment, people get interested in attending a place where a lot of others are attracted. this interest may not be directed to any particular people in the place, but to something that may be an object shown in the place or the atmospheric mood. in this place, a new member may come close to other humans, touch objects implemented in the place, and breathe the air in the place. these behaviors of people are similar to the social dynamics known to form a scale-free network [9] where new nodes get connected to highdegree nodes. however, there are real-space constraints on the dynamics that are 1: the restriction of space (the upper bound on the number of people to meet at a time), and 2: the restriction of time (the number of other people to meet if one may meet just one other at a time). in this paper, we aim to find some clues to discuss the question above by simple simulations using a constrained scale-free network reflecting the two kinds of restrictions. the established model of the process to generate a scale-free network (sfn) proposed by barabasi and albert [9] is described as follows. the process is here described to fit the revision in the next section. if degree.randomrank(j) < m0 8: add edge(i, j) to e 9: degree(i) ← degree(i)+1; 9: degree(j) ← degree(j)+1 10: end if 11: end for 12: end for v and e mean the sets of nodes and edges in the graph respectively, of which the combination is defined to be the graph g. the graph g starts from a clique of m0 nodes and incremented by adding one node node_i at each time. then m0 other nodes are chosen as new link destinations from node_i according to the probabilities given in proportion to the node degrees. here m0 is the initial degree of each node (the number of edges connected to a new node), and randomrank(j) means the rank (0, 1, 2, …) of node_j in choosing on the probability given in proportion to its degree denoted degree(j). that is, the values of degree(j)*random(0,1) for j's get compared for ranking of which the top m0 are chosen. in the real human society, m0 can be regarded as the number of touchable people i.e., all other people one can meet separately if preferable, not necessarily in a group meeting. it is well known that the distribution of the degree of nodes follows the power-law, that means a few nodes in v occupy a large portion of the edges in e. networks such as www and online communities [10] , cellular networks in biology [11] , patients of sexually transmitted diseases [12] , etc. have been known to for sfn following the power-law degree distribution. we basically regard the generation process above of sfn as a model capturing real social behaviors of people approximately, comparing with other existing models. for example, all the nodes are connected to the same number of edges in a regular graph, which is inconsistent with the real inequality of peoples' social activities in the real world. the small-world networks on the ws (watts-strgats) model start from a regular graph and move a certain number of randomly selected edges connected from each node, that we humans do not do in the daily activities [13] . in the more recently proposed mediation-driven attachment model [14, 15] , each new node first picks an existing node at random and connects not directly with this but with a certain number of its neighbors also picked at random. this is an extension of sfn supposed to result in having each new node connected to "rich" people linked to a large number of "poor" people of low degrees by choosing link destination nodes by the probability estimated to be inverse of the harmonic mean (ihm). although this may make the network fit to the real society, we do not choose as the model for this paper because the attendants of places do not have a bias to lowdegree nodes in the daily life human behaviors. thus, considering the suitability of each model to the intuitive understanding of the social life of people, we take sfn as the backbone and revise it with introducing constraints corresponding to the spatiotemporal restrictions in the real life of people as in the next section. reference [12] above encourages this choice in the sense a virus infection network has been shown to follow the power-law distribution. the sfn may be, as discussed above, regarded as a natural model for capturing human social behaviors as far as there is no restriction on the reach of social behaviors of each human. on the other hand, the physical constraints in the real living environment of humans restrict the width of the room where people may gather, and the time is also restricted so that a person cannot meet as many people one likes to meet. thus, the scalefree network cannot be used as it is to model real-space social behaviors of people. in this sense, let us revise the process above as follows. an important conceptualization here is that not only humans but also rooms/spaces of a place or things are dealt with as nodes here assuming these are working as entities attracting people, and these entities are dealt with to meet the requirements of humans who have strong influences on others. furthermore, the virus is wrapped in saliva and cast into the air in meeting places. we reflect the spatiotemporal restrictions as constraints in *1, *2, and *3 below. in the lines 3, 11, and 12 below (*1 and *2), at each time a new node is added, the degree of the link destination node (the partner of the new connection) from the new node is constrained by the given upper bound of w. if the node comes to be more popular than having w links, the node is removed as in line 12. this means the room or space of capacity of w or larger is forbidden as in the protection of covid-19 infection spreading. however, to add allowed groups of up to the capacity of w attendants by a certain frequency considering the load of the group-meeting organizer, we add the lines 16 and 17 where some nodes get larger preference than others when a new node explores link destinations (*3).  is a constant set to 5. the use of w in this line is not necessary (any sufficiently large value satisfies the requirement here) because the degree of the destination node (j here) is finally bounded by w after all due to *2. for each node in the network generated above, the three-step dynamics is considered here for spreading the infection. the first is to catch a virus from neighboring nodes (i.e., humans or things in the place), the second is to be infected, and the third is to be an infector to other nodes. these steps are supposed here to occur as follows, week by week: to use the edges to contact neighbors: the edges in e added by randomly chosen 3% of the steps marked with an asterisk (*2, *3: each step with an asterisk is regarded as a human's or a human group's social behavior at a time.) in the procedure of 2.2 above is activated, corresponding to peoples' activities in the real life. for the event of *1 and *3, the nodes joining the event form a clique and each node catches the virus from the partner if the partner has, following the rule to catch the virus below. for event *2, on the other hand, a new node contacts each of m0 link destinations one by one so that the pair-wise casting and catching of virus may occur between linked two nodes at each time. all node_j's adjacent to node_i. in other words, a node catches virus if any neighbor i.e., any adjacent node, has become an infector following the rule below. to be infected: node_i is infected if catch(i) is larger than a random value between 0 and 1 by uniform probability. this means one gets infected by 50% in the case of a sufficient catch of viruses. the strength of infection is here is given as a real value infected(i) rather than a discrete judgment considering, the uncertainty of the pcr test. the infection of a node is assumed to fade by a constant r (set to 0.7 corresponding to the recovery in 4 weeks) each week. we assume the recovery occurs by virus's inactivation or the acquisition of immunity to be an infector: if one is infected, one is expected to infect others by the probability of 0.2 here, according to the announcement of who on april 10, 2020. anyone linked to 100 other nodes is expected to infect others to generate 1.2 other infected nodes in 4 weeks (100 * 0.03 * 0.5 *0.2 *4). this is less than the reported number of infection reproductions by the government [16] , but increases with the coefficient corresponding to the number of infected people to meet in one's circumstance. the value infector(i) is here is also given as a real value and fades by r per week. following the procedure above, we made the experimental simulation for 20 trials of 100 weeks/trial, setting the starting infection from a given set of nodes as the 0-th week and the number of nodes (n) in g to 1000 and 10000. let us first show the overall tendencies obtained, then go into the time series of daily new cases for exemplified settings of w and m0. we did these experiments fixing two starting infected nodes, one in a cluster (meaning to have degree w) and another out of any cluster. as in table 1 table 1 show. on the other hand, in the white cells where w < 8 or m0 < 4 (especially for less than 2), the number of new infection cases are substantially smaller than in the shadowed part. in table 2 , the average timing of infection for all the cases is shown. these values mean an expectation about when the infection tends to occur, setting the first infections in this network as the 0-th week. in the shadowed range of this table, the third tendency is observed: the average infection week tends to be the later for the smaller m0. in other words, the number of infection cases for a small m0 is small for the same period as when the upward trend is apparent for larger m0 and then increases after the period. this section aims to show some pieces of evidence to support the tendencies above. condition for 20 trials of simulation. note each curve thus shown here shows not a history in one trial but the mixture of the sequences of the averaged trials among which the periods of steep increase differ. it is thus meaningless to measure the declination of the curves at each week or to fit the curve to exponential or power-law as in [2] or [3] . the purpose here is to see the overall tendencies such as an early or a slow increase in the larger time scale such as months and the changes in the number of infection cases in a similar time scale. the first finding above that w depends positively on the number of infection cases suggests the largest allowed number of participants in a meeting should be restricted for suppressing the number of infected cases. this suggests the policy to forbid face-to-face group meetings (i.e., w ≥ 10) is expected to work for suppressing infections spreading. the second finding is the tendency that the value of m0 causing the worst effect, i.e., the largest number of infection cases, tends to take a smaller value than the largest value taken in the experiment if w is large. the author's hypothesis for explaining this surprising result is that the people one meets tend not to have acquired immunity yet if the number of people to meet is restricted to a narrow community. this lack in immunity is supposed to occur in the sparsely connected parts of the entire graph g because people on the nodes in such parts are of lower degree and fewer opportunities to touch others. if there are large meeting groups in such a case, the hypothesis goes that the infections in these groups attack the nonimmune part of the network. however, if m0 is constrained further to 1, according to table 1 , the risk is reduced because the casting and catching of viruses occur in lower probability. thus, we should allow people to meet only one restricted member i.e., set m0 to 1, as far as we allow group meetings. the above two findings can be reviewed referring to figure 4 where the results of other that is, in the boundary between zones b and c for w = 8, td comes to be larger in the range of moderately restricted m0 (e.g., 2 ≤ 0 ≤ 4) than for m0 of a larger value. this tendency is found in the similar range of w and m0 to the second finding in section 3 where the number of infection cases was simulated, so we can assume that a policy of restriction on w and m0 that reduces the number of infections can also quicken the reduction. the zone d in the dotted circle is not meaningful for the discussion here because this zone shows the saturation of new cases reaching a large portion of the entire population. the third finding in section 3 was about the timing of infections. in figure 1 (n = 1000) and figure 2 (n = 10000) , we obtained the up-ward trend in the first five to ten weeks that is stronger for the larger m0 (note: the vertical axes has different scales) which seems to disappear for the same period if m0 is controlled to a smaller value. a moderate constraint where m0 ranges in 2 ≤ 0 ≤ 8 may temporarily seem to work for the reduction of infections in the early stage within 30 weeks from the beginning. however, this does not mean the uptrend really disappears but may be just delayed to a later period. as in figure 1 and 2, the increase in the number of new infection cases for smaller m0 continues for all the 30 weeks, which is a period that covers the entire hills (i.e., both the uptrend and the downtrend) of new infection cases for the larger m0. in figure 3 , showing 100 weeks in the conditions of smaller m0, we find the slow increase for the first 30 weeks in each condition shown in figures 1 and 2 was really a part of a long-lasting increase. in figure 3 , where n (w, m0) is 10000 (50, 2), the increase is found to last for 80 weeks i.e., 20 months. the peaks of new infection cases tend to shift to the later weeks for smaller m0 in the range of large w. this trend is consistent with the results in fig 4, where we find the delayed reduction for 2 ≤ 0 ≤ 8 in the range of large w (w ≥ 8). from the discussion above, in summary, the following policies are recommendable (1) for suppressing the number of infections, restrict w to less than 10, or m0 to 1. (2) for suppressing the infections quickly, restrict w to less than 4, or m0 to 1 (zone a in figure 4 ). coupled with (1), this is our recommendation for suppressing infections both in quantity and quickness. if the latter meaning to meet only one other person, is a too hard constraint, w should be restricted to less than 4. if w is restricted so, setting m0 to 4 or less can further quicken the reduction (zone a in figure 4 ). (3) a moderate restriction on the widths of contacts (w or m0) may seem to appear as a reduction of infections for a period, but we should take care of the real peak of infections that may come later. the infection spreading has been simulated over human networks where the constraints are given by the maximum size of a group in which people meet at once (w) and the maximum number of all other people one can meet separately if preferable (m0). the realspace urban life of people is modeled as a scale-free network with constraints on the constants on the two width factors w and m0 above. as a result, three findings have been obtained as in the discussion, that can be put within this one paragraph. that is, for quickly suppressing the number of infections, we recommend a policy to restrict w to less than 4 and m0 to 4 or less, if it is too hard to set m0 to 1 meaning each person can meet only one other person for all the period of a virus infection crisis. otherwise, i.e. if the politics uses a weaker restriction on the widths of contacts, we should take care that the infection may not be suppressed significantly or the real peak of infections may come later. the results in this paper might be associated with the assertion that there is a risk of infection spreading in a society where people in each household decide to maintain an inperson social connection with one person from households [17] . however, the suggested policy is not so strict in that we can release the constraint on one-to-one meetings to allow m0 = 4 if people stop all face-to-face group meetings of four or more participants. polynomial growth in branching processes with diverging reproductive number fractal kinetics of covid-19 pandemic mathematical theories on corona pandemic (covid-19) distributed topology manipulation to control epidemic spreading over networks optimal control for heterogeneous node-based information epidemics over social networks in ieee a simple model of global cascades on random networks informative and normative effects using a selective advertisement estimating effectiveness of lockdown for 2019 novel coronavirus diseases (covid-19), -a working paper wp2020404 emergence of scaling in random networks evolution of networks: from biological networks to the internet and www scale-free networks in cell biology scale-free networks and sexually transmitted diseases: a description of observed patterns of sexual contacts in britain and zimbabwe collective dynamics of 'small-world' networks growing scale-free networks by a mediation-driven attachment rule degree distribution, rank-size distribution, and leadership persistence in mediation-driven attachment networks can't i please just visit one friend? this study is in progress, partially supported by grant jsps kakenhi 19h05577. key: cord-258145-usr7b6dk authors: abdulah, deldar morad; hassan, a. b. title: relation of dietary factors with infection and mortality rates of covid-19 across the world date: 2020-07-04 journal: j nutr health aging doi: 10.1007/s12603-020-1434-0 sha: doc_id: 258145 cord_uid: usr7b6dk objective: poor dietary habits are considered to be the second-leading risk factors for mortality and disability-adjusted life-years (dalys) in the world. dietary patterns are different based on cultural, environmental, technological, and economic factors. nutritional deficiencies of energy, protein, and specific micronutrients have been shown to contribute to depressed immune function and increased susceptibility to infections. we aimed to explore the relation of dietary factors with global infection and mortality rates of covid-19 in this study. design: in the current ecological study, the countries that had national dietary data from the global dietary databases of the united nations and coronavirus disease statistics from the world health organization (who) were included. the countries that had coronavirus disease statistics from the who were consecutively checked for the recent data of the dietary factors. setting: world. participants: 158 countries across the world. measurements: infection and mortality rates of covid-19; dietary factors. results: the median crude infection and mortality rates by covid-19 were 87.78 (iqr: 468.03) and 0.0015 (iqr: 0.0059), respectively. the two highest percentage of the crude infection rate were between 0 and 500 (75.9%) and 500–1000 (8.9%) per one million persons. the regression analysis showed that the crude infection rate has been increased by raising consuming fruits (beta: 0.237; p=0.006) and calcium (beta: 0.286; p=0.007) and was decreased with rising consuming beans and legumes (beta: −0.145; p=0.038). the analysis showed that the crude mortality rate was increased by raising consuming sugar-sweetened beverages (beta: 0.340; p<0.001). whereas, the crude mortality rate by covid-19 has been decreased by increasing fruits consuming (beta: −0.226; p=0.047) and beans and legumes (beta: −0.176; p=0.046). conclusion: the present study showed the higher intake of fruits and sugar-sweetened beverages had a positive effect on infection and mortally rates by covid-19, respectively. in contrast, the higher intake of beans and legumes had a negative effect on both increasing infection and mortality rates. poor dietary habits are considered to be the second-leading risk factors for mortality and disability-adjusted life-years (dalys) in the world. the poor dietary habits are responsible for 10.3 million deaths and 229.1 million dalys in 2016 (1) . for example, the following dietary habits are among the leading risk factors for early death and disability in european countries. the habits are low intakes of whole grains, fruit and vegetables, and nuts and seeds, and high intakes of alcohol and sodium. the western dietary habits are consuming diet processed, high in red and processed meat, diets with high in sugar-sweetened beverages, and low in milk. these kinds of dietary habits are regarded to be a rising health concern. dietary patterns are different based on cultural, environmental, technological, and economic factors. however, the dietary patterns are becoming similar due to increasing living standards and growing globalization of the food sector (2, 3) . mertens et al. (4) explored the dietary intakes in four different european counters using individual-level dietary intake in adults in nationally-representative surveys of denmark, france, czech republic, and italy. they reported a higher intake of fruits and vegetables and lower intakes of sweetened beverages and alcohol in italy. while individuals in denmark and the czech republic had a higher intake of vegetables. a comparison of population subgroups within countries shows that there is a difference in the dietary preferences, beliefs, and practices for particular consumer groups. for example, highly-educated persons and women have a higher intake of fish, nuts, and seeds along with lower intake of red and processed meats (5) . the individual-level reported dietary data of the countries could be used as a useful tool to make a connection between health and environment with foods as their common denominator (1) . a recent review study reported that the detailed assessment of patients for the dietary and nutritional risks along with medical, lifestyle, and environmental factors with suitable risk management strategies make the sensible way to deal with the covid-19 (6) . the diet and nutrition have a variance impact on the immune system competence. in addition, they determine the risk and severity of the infections. the relation between diet, nutrition, infection, and immunity is bidirectional (7) . the macro-, micronutrients, and phytonutrients in diet, such as fruits and colorful vegetables improve healthy immune responses. the microand phytonutrients provide the antioxidants and the antiinflammatory nutrients, like beta-carotene, vitamin c, vitamin e, and polyphenolic compounds resulting in modulating the immune functions (8, 9) . nutritional deficiencies of energy, protein, and specific micronutrients have been shown to contribute to depressed immune function and increased susceptibility to infections. the sufficient intake of iron, zinc, and vitamins a, e, b6, and b12 is vital for the overall maintenance of immune function (10) . the new epidemics of coronavirus disease 2019 (covid-19) has become a pandemic to the world currently. we make a hypothesis that geographical variation in dietary factors could have a role in infection and mortality rates of covid-19 in the world. therefore, we aimed to explore the relation of dietary factors with global infection and mortality rates of covis-19 in this study. in the current ecological study, the countries that had national dietary data from the global dietary databases of the united nations (11) and coronavirus disease statistics from the world health organization (who) were included (12) . the countries that had coronavirus disease statistics from the who were consecutively checked for the recent data of the dietary factors. the countries/states met eligibility criteria for this investigation if they had the statistics from the who coronavirus disease (covid-19) situation dashboard from the website of the world health organization by 24 april 2020 (12) . the following countries were excluded from the analysis due to not having the statistics of the covid-19; comoros, north korea, kiribati, lesotho, malawi, marshall islands, micronesia, nauru, palau, samoa, sao tome, and principe, solomon islands, south sudan, tajikistan, tonga, turkmenistan, tuvalu, vanuatu, and yemen. the following countries were excluded from the study due to not having data on the national dietary factors on the website of the global dietary database (11) the populations of the countries were extracted from the united nations statistics division (13) . the estimated populations of the year 2018 were considered for the countries. some of the countries had not the population for the year 2018. therefore, the authors checked for the years 2015, 2016, and 2017. accordingly, the population of 2017 was used for the following country; algeria. the population of 2015 was considered for the following countries: lybia; sierra leone. the population of 2016 was extracted for the following countries: mali; mauritania; papua new guinea; sudan and 2017 for the following countries; bhutan; bosni; burkina faso; fiji; guyana; niger; nigeria; pakistan; uae. the populations of the following countries were not available for the 2015-2019 period. therefore, the population of the following countries was not included in this study based on the eligibility criteria. these countries were the central african republic; djibouti; djibouti; dominica; gabon; kosovo; lebanon; liberia; moldova; russia; saint kitts and nevis; syria; somalia, the democratic republic of the cong. finally, 158 countries/states were included in this study. the general characteristics of the countries were presented in median (interquartile range [iqr], mean (std. deviation), and number (percentage). the confirmed and dead cases were presented in median and interquartile range due to the nonnormal distribution of the data. the normality of the outcomes was examined in drawing a histogram and box plot. the number of confirmed cases was divided by the total population of a country multiplied by 1000,000 to obtain the infection rate of covid-19 per one million persons. the number of dead cases was divided by the total number of confirmed cases and divided by total population multiplied by 1000,000 to obtain the mortality rate/1000,000 persons.. the infection and mortality rates were determined in a median and interquartile range following dealing with the potential outliers. the upper limit values were considered for the extremely higher limit values in the infection and mortality rates. the crude infection rate was categorized into the following groups; 0-500; 500-1000; 1000-1500; 1500-2000; 2000-2500; and > 2500 per one million person. the infection and mortality rates were transformed through the ln technique to obtain a normally distributed histogram. no ethical aspect was applicable to this study. the median crude infection rate by covid-19 was 87.78 (iqr: 468.03) ranged between 0.00 and 24034.51 per 1000,000 persons. the median mortality rate by covid-19 was 0.0015 (iqr: 0.0059) ranged between 0.00 and 1.3728 per 1000,000 persons. the two highest percentage of the crude infection rate were between 0 and 500 (75.9%) and 500-1000 (8.9%) per one million persons ( table 1) . the study showed the crude infection rate was raised with increasing consuming fruits (r=0.416; p<0.001), unprocessed red meats (r=0.457; p<0.001), fruit juices (r=0.390; p<0.001), total protein (r=0.275; p<0.001), calcium (r=0.550; p<0.001), potassium (r=0.371, p<0.001), and total milk (r=0.450; p<0.001). regarding crude mortality rate per 1000,000 persons; the study showed that crude mortality rate was raised with increasing consuming unprocessed red meats (r=0.234; p=0.007), sugar sweetened beverages (r=0.224; p=0.010), fruit juices (r=0.272; p=0.002), calcium (r=0.288; p=0.001), and total milk (r=0.230; p=0.008). however, the mortality rate was decreased following increasing consuming non-starchy vegetables (r=-0.196; p=0.023), see table 2 . the regression analysis showed that the crude infection rate has been increased by raising consuming fruits (beta: 0.237; p=0.006) and calcium (beta: 0.286; p=0.007). however, the infection rate was decreased with rising consuming beans and legumes (beta: -0.145; p=0.038), table 3 . the effect of dietary factors on the crude mortality rate by covid-19 was examined in the regression analysis. the analysis showed that the crude mortality rate was increased by raising consuming sugar-sweetened beverages (beta: 0.340; p<0.001). whereas, the crude mortality rate by covid-19 has been decreased by increasing fruits consuming (beta: -0.226; p=0.047) and beans and legumes (beta: -0.176; p=0.046), as presented in table 4 . the comparison of dietary factors in countries with different infection rates was examined in table 5 and fig 2. the study showed that the countries with higher infection rates between 1500 and above had a higher intake of fruits (p=0.002), fruit juices (p<0.001), calcium (p<0.001), potassium (p<0.001), and total milk (p<0.001). however, these countries had a lower intake of unprocessed red meats (p<0.001) and total protein (p=0.013). the aim of the food-based dietary guidelines is to maintain the general health of the population and prevent non-communicable diseases (14) . most of the food-based dietary guidelines recommend intake of whole grains, fruit and vegetables, low-fat dairy and fish, and low intake of red and processed meat, sugar-sweetened food products, alcohol, and salt (15) . the present study showed that the crude infection rate by covid-19 has been increased by raising consuming fruits, calcium and decreased with increasing consuming beans and legumes. regarding the mortality rate, the analysis showed that the crude mortality rate was increased by raising consuming sugar-sweetened beverages and decreased by increasing fruits consuming and beans and legumes. the anti-inflammatory strategies inside foods, nutrients, or (16, 17) since the coronavirus has serious inflammatory consequences for acute pneumonia in persons (18) . the human coronavirus infections cause mild to severe diseases, systemic inflammation, high fever, cough, and acute respiratory tract infection and dysfunction in internal organs leading to death. this virus is classified as a ribonucleic acid (rna) virus. the virus has a genome that often escapes the innate immune system, particularly if it is malfunctioning (19) . entering coronavirus into the organism activates innate immunity, which intervenes in the first instance to engulf the invader. the severity of the diseases locates within the ability of innate immune cells to stem viral infection (20) . the virus has less ability to replicate itself and induce the pathological state in the case of the stronger innate immune system. when the immune system is suppressed by the virus, the body activates the adaptive immunity. the coronavirus enables to produce viral enzymes and proteases. these enzymes and proteases can damage the immunity and inhibit the signaling pathways of type i interferon (ifn) along with the nuclear factor-κb, facilitating innate immune evasion (21) . apart from the age-related micronutrient inadequacy, the nutritional status of a person has a role in the developing risk of sars-cov-ii infection, the clinical course, and the disease outcomes. hence, the maintenance of host macro-and micronutrient status is considered to be a crucial preventive measure for covid-19 (6) . the coronavirus infection is primarily attacked by immune cells, however, the virus has developed viral proteins overtime that counteracts with the innate immune system (22) . some of the viral proteins antagonize interferon (inf) and stimulate inflammatory proteins, such as il-1 family member cytokines (23) . the inflammatory state and pathogenesis of the disease are escalated after abnormal production of cytokines as shown in sars (24) . our hypothesis is that the higher intake of fruits makes the persons at further risk of infection by the covid-19. despite fruits and vegetables have anti-inflammatory and antioxidant factors and have an important role in enhancing the immune system responses (25) . but higher intake of these micronutrients makes a barrier in improving the human immune system or response to the pathogens due to the role of the fruits with a high glycemic index. our study showed that beans and legumes have a positive role in reducing the infection rate by the covid-19. the human body requires the substates in the plant proteins to improve or respond to the vial pathogens because of the human body unable to produce these substrates (26) . therefore, the body needs these substates to protect the organs against the coronavirus. we assume that the immune body system unable to recognize the virus at the early times. comparison of dietary factors in countries with different infection rates therefore, the available proteins are essential for the body to make a response to the pathogen. the beans and legumes have been effective to reduce the rate of mortality by the covid-19 as well. the role of age in the suppression of the immune system must not be overlooked. the population of the countries with a higher infection rate is older compared to the counters with a low infection rate (27) . for example, france and italy compared to iraq and saudi arabia. the available evidence indicates that adults aged 60 years and older and patients with preexisting medical conditions are more likely to have sever-even deadly-coronavirus infection that other population groups (28) . therefore, we can make the further hypothesis that the aged population of the countries with high infection rates has been the main factor in the low immune system. the impacts of aging on the immune system can reflect at multiple levels. the levels are decreased production of b and t cells in bone marrow and thymus and diminished functions of mature lymphocytes in secondary lymphoid tissues. so, the elderly persons do not respond to immune challenges as robustly as the young individual (29) . the higher intake of fruits and vegetables may not be beneficial to enhance the immune system in aged populations. diet alone may be insufficient and tailored micronutrient supplementation based on specific age-related needs necessary (7) . many micronutrients are required for immune-competence, especially vitamin a, c, d, e, bs, iron, selenium, and zinc. moreover, the dietary pattern is essential to maintain the nutritional status of an individual. however, the diet alone could not be adequate in certain metabolic and lifestyle conditions, such as elderlies, co-existing medical conditions, cigarette smoking, or occupational exposure to environmental toxins (7) . the fruits have several vitamins and minerals. the fruits at a ground level may not be quite suitable to make the final judgment. the older persons over the age of 60-65 experience some immune dysregulation with less ability to respond to immune challenges and response to pathogens, antigens, and mitogens decreases (30) . the decrease in the number of circulating lymphocytes and loss of immune cells are characteristics of the immune system in older people (31) . moreover, the older peoples have reduced the production of t cells in the involved thymus and consequently diminished function of mature lymphocytes in secondary lymphoid tissues (29) . the lifetime of exposure to antigens and to several sources of oxidative stress cause dysregulation in the immune system that makes them at further risk of infections than other age groups (31) . the role of fruits in enhancing immunity, such as micronutrients is in exhibiting pleiotropic roles in supporting immune function. the vitamins and minerals support to develop and maintain the physical barriers, produce and activate antimicrobial proteins (32) . some other mechanisms of micronutrients are supporting the growth, differentiation, and motility/chemotaxis of innate cells; phagocytic and killing activities of neutrophils and macrophages, and promotion of and recovery from inflammation (e.g. cytokine production and antioxidant activity (32) . the potential mechanisms of the fruits may back to the antiviral immune induction, the modulation of immunoregulatory defense, induction of autophagy and apoptosis, genetic or epigenetic regulation (33) . stimulation of defensins and cathelicidins may reduce the replication of the virus and raise the levels of anti-inflammatory cytokines, and reducing levels of pro-inflammatory cytokines (34) . here our hypothesis is that a higher intake of fruits suppresses the role of stimulation of defensins and cathelicidins. the common denominator that reflects the role of nutrition and dietary recommendations against viral infections; including covid-19 is the relation between diet and immunity (35) . this is why we made our hypothesis based on the immunological effects of a higher intake of fruits in patients with covid-19 by taking into account the patients' ages. the evidence highlights that diet has an important effect on the immune system and disease vulnerability of peoples. the role of nutrients or nutrient combinations back to their effects on the immune system through the cell activation, modification in the production of signaling molecules, and gene expression (36) . the relation of fruits and beans and legumes on crude mortality rate is weak (p=0.047 and p=0.046, respectively) in contrast with the strong relation of sugar-sweetened beverages (ssbs) (p<0.001). the possible role of sugar-sweetened beverages on infection rate may back to its role in weight gain and the risk of obesity. a review study of observational and clinical trials showed that a higher intake of ssbs raised the risk of weight gain and obesity (37) . the evidence has been confirmed elsewhere (38, 39) . accordingly, maccioni et al. (40) recruited 1455 individuals aged 18-70 in a cross-sectional study on airway infection in germany. the study reported that obese persons have a consistently higher frequency of upper and lower respiratory tract infections (rtis). the evidence has been reported elsewhere (41, 42) . obesity is responsible for the dysregulation of the immune system through mediation in different immune, metabolic, and thrombogenic responses (43) . the higher intake of ssbs has been reported in high-income countries (44) . the effect of higher calcium intake on raising infection rates could be due to the effect of calcium on the risk of some other chronic diseases rather than its direct effect. a meta-analysis showed the increased incidence of myocardial infarction in persons who consume higher levels of calcium with a pooled relative risk of 1.27, 95% confidence interval 1.01 to 1.59, p=0.038 (45) . in addition, calcium has been reported as a trigger for ischemic cell death (46) . the daily recommended allowance/intake of the dietary factors are different across the countries. it is required to mention that food intake varies markedly based on the sociodemographic factors; like age gender, and educational level. we did not make stratification the results of the study based on the socio-demographic aspects since the who has not published the covid-19 confirmed cases according to age, gender, and educational level. besides, the cross-country caparison of individual-level dietary data is challenged by the dietary surveys performed with various survey characteristics and data collection methods with a possible influence in the comparison of the results. however, we used the fao dietary data that represent the nationally representative sample of all age-sex, and educational level categories. the present study showed the higher intake of fruits and sugar-sweetened beverages had a positive effect on infection and mortally rates by covid-19, respectively. in contrast, the higher intake of beans and legumes had a negative effect on both increasing infection and mortality rates. the possible reason for the role of fruits and sugar-sweetened beverages on infection and mortally rates back to the indirect effect of weight gain and obesity and the role of age. the authors do not declare any conflicts of ineptest. global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2016: a systematic analysis for the global burden of disease study importance of government policies and other influences in transforming global diets global panel on agriculture and food systems for nutrition: food systems and diets: facing the challenges of the 21st century geographic and socioeconomic diversity of food and nutrient intakes: a comparison of four european countries meatless days" or "less but better"? exploring strategies to adapt western meat consumption to health and sustainability challenges 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between vitamin d and viral infections vitamin d supplementation could prevent and treat influenza, coronavirus, and pneumonia infections nutrition amid the covid-19 pandemic: a multi-level framework for action diet, exercise and gut mucosal immunity sugar-sweetened and artificially-sweetened beverages in relation to obesity risk intake of sugar-sweetened beverages and weight gain: a systematic review effects of soft drink consumption on nutrition and health: a systematic review and meta-analysis obesity and risk of respiratory tract infections: results of an infection-diary based cohort study obesity and infection. the lancet infectious diseases the burden of obesity on infectious disease obesity, respiratory disease and pulmonary infections group cde. global, regional, and national consumption of sugar-sweetened beverages, fruit juices, and milk: a systematic assessment of beverage intake in 187 countries effect of calcium supplements on risk of myocardial infarction and cardiovascular events: meta-analysis calcium in ischemic cell death key: cord-261756-4lybl57r authors: dubert, marie; visseaux, benoit; birgy, andré; mordant, pierre; metivier, anne-cécile; dauriat, gaelle; fidouh, nadhira; yazdanpanah, yazdan; grall, nathalie; castier, yves; mal, hervé; thabut, gabriel; lescure, françois-xavier title: late viral or bacterial respiratory infections in lung transplanted patients: impact on respiratory function date: 2020-02-24 journal: bmc infect dis doi: 10.1186/s12879-020-4877-3 sha: doc_id: 261756 cord_uid: 4lybl57r background: respiratory infections are a major threat for lung recipients. we aimed to compare with a monocentric study the impact of late viral and bacterial respiratory infections on the graft function. methods: patients, who survived 6 months or more following lung transplantation that took place between 2009 and 2014, were classified into three groups: a viral infection group (vig) (without any respiratory bacteria), a bacterial infection group (big) (with or without any respiratory viruses), and a control group (cg) (no documented infection). chronic lung allograft dysfunction (clad) and acute rejection were analysed 6 months after the inclusion in the study. results: among 99 included lung recipients, 57 (58%) had at least one positive virological respiratory sample during the study period. patients were classified as follows: 38 in the vig, 25 in the big (among which 19 co-infections with a virus) and 36 in the cg. the big presented a higher initial deterioration in lung function (p = 0.05) than the vig. but 6 months after the infection, only the vig presented a median decrease of forced expiratory volume in 1 s; − 35 ml (iqr; − 340; + 80) in the vig, + 140 ml (+ 60;+ 330) in the big and + 10 (− 84;+ 160) in the cg, p < 0.01. acute rejection was more frequent in the vig (n = 12 (32%)), than the big (n = 6 (24%)) and cg (n = 3 (8%)), p < 0.05, despite presenting no more clad (p = 0.21). conclusions: despite a less severe initial presentation, single viral respiratory infections seem to lead to a greater deterioration in lung function, and to more acute rejection, than bacterial infections. thanks to a better selection of recipients and donors, to an improvement in surgical / anaesthetic procedures and to better management of immunosuppressive therapies, early post-operative survival of lung transplant recipients (ltrs) has improved since the advent of lung transplantation. whereas most early deaths are related to primary graft dysfunction or acute rejection (ar), the long-term prognosis is threatened by chronic lung allograft dysfunction (clad), usually in the form of a bronchiolitis obliterans syndrome (bos), that affects 45 to 75% of lung recipients within 5 years after transplantation [1] [2] [3] [4] . clad represents the leading cause of death 1 year after lung transplantation [3, 5, 6] . infectious respiratory complications are also a major cause of morbidity and mortality for ltrs and are responsible for a third of deaths occurring in the first year post transplant, and half of all deaths during longterm follow-up [7, 8] . the severity of these infections results from several factors including induced immunosuppression, direct exposure of the graft to microorganisms, and finally less effective mucociliary function and lymphatic drainage and cough reflex following denervation of the graft [9] [10] [11] . moreover, stenosis and ischemic processes occurring at the surgical anastomosis decrease the clearance of secretions, and promote their colonization and invasion by microorganisms [12] . bacterial infections are the leading cause of respiratory infections in ltrs [7, 11, 13] . their association with the occurrence of clad is well established [2, 14] . many authors admit that viral respiratory tract infections (vrti) may be associated with clad [3, [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] , but this remains controversial, depending on the definition of respiratory infection, the virus panel studied, the time limit between vrti and spirometric analysis, and the consideration of intercurrent events possibly influencing the respiratory function [25] . similarly, the association between vrti and ar continues to be debated in the literature. while some studies have identified an association between these two events [17, 26, 27] some others, including a recent meta-analysis, have not found any link between them [21, 25, 28] . however, to our knowledge, the impact of viralbacterial co-infections on graft survival has not been specifically studied, and was not compared to single vrti or patients without any respiratory infections. the development of rapid antigenic tests and molecular biology techniques has facilitated the detection and diagnosis of several viruses. the new multiplex pcr methods (polymerase chain reaction) are fast, sensitive andable to detect an enlarged number of viruses not easily detected before (e.g. metapneumovirus, coronavirus nl63 and hku1, bocavirus, rhinovirus c) [29, 30] . thus, we hypothesis that the impact of the viral infection is at least as severe as bacterial respiratory infections on lung graft function among ltx. the objectives of our study were to assess, in a cohort study, the occurrence of late viral and bacterial respiratory infections in ltrs and to compare their respective impact on graft function with those without any respiratory infections. we retrospectively screened all individuals who underwent lung transplantation between september 2009 and september 2014 at the bichat-claude bernard teaching hospital, paris, france. in this cohort we evaluated the occurrence of late viral and bacterial respiratory infections and graft function overtime. exclusion criteria were: (1) death during the first 6 months after transplantation, (2) no available pulmonary function assessment at the time of documented infection or 6 months after, and (3) herpetic or cytomegalovirus pneumonia. in order to include patients at a steady state, we studied only the late respiratory infections, i.e. to censure the first 6 months after the lung transplantation, a period during which postoperative complications are frequent and usually intertwined. these patients were regularly followed up. at each routine follow-up visits with patients, symptoms were recorded. spirometric measurements, blood tests, radiological explorations, bronchoscopic procedures with bronchoalveolar fluid and transbronchial biopsies were systematically performed and tested for both bacterial and viral pathogens according to local guidelines. all respiratory samples, bronchial aspirates, bronchoalveolar fluids and nasopharyngeal samples were reviewed. several [31] [32] [33] , as confirmed by our internal method validations and the similar viral diversity observed over time [34] . their reliability was also assessed throughout the study period by regular qcmd controls (glasgow, uk). a sample was defined as bacteriologically positive if the bacteria were present at 10 7 cfu/ml or more for sputum, 10 6 cfu/ml or more for bronchial aspirates and 10 4 cfu/ml or more for bronchoalveolar fluid. according to virological and bacteriological results from respiratory samples, patients were divided into three groups, as follows: -patients presenting a viral respiratory infection: viral infection group (vig), i.e. -patients with at least one positive virological respiratory sampling, symptomatic or not. nasopharyngeal swabs that tested positive only for rhinovirus were not considered to be positive for a significant virus, and were ignored. -patients without any respiratory infection: control group (cg), i.e. patients with no virological or bacteriological positive respiratory samples during the study, or those with nasopharyngeal swabs positive for rhinovirus only; or with a positive bacteriological respiratory sample not followed by antibiotic treatment (i.e. considered as a simple bacterial carriage). the date of inclusion was defined as the date of the respiratory sample of interest for the two infected groups (vig and big), and the date of the first outpatient consultation for the cg. the primary endpoint was the occurrence of bos 6 months after inclusion. secondary endpoints were the occurrence of ar and the quantitative change of forced expiratory volume in 1 s (fev-1) at 6 months after inclusion, as well as death during the whole study period. the following definitions were used to assess the outcomes: biopsy that demonstrated at least a grade a1 of acute rejection as defined by the ishlt for cellular rejection. in patients in whom a biopsy could not be performed, acute rejection was defined by deterioration in lung function with no other identifiable aetiology and that positively responded to a high-dose corticosteroid therapy. 4. fev-1 delta was defined as 6-month-fev-1last fev-1 before inclusion. quantitative variables were presented using the median (quartile 25-75) and were compared by t-test or variance analysis. qualitative variables were compared using a chi2 test or a fisher-exact test. outcomes were compared using a logistic regression multivariable model, with adjustment for time since transplantation, age and sex. a sensitivity analysis was carried out excluding patients who had had a bos before inclusion. all of the statistical analysis was done using r software, version (3.1.1). this study was approved by the cepro (comité d'evaluation des protocoles de recherche observationnelle) ethical committee, number cepro 2016-027. between september 2009 and september 2014, 154 patients received lung transplants at bichat hospital. fiftyfive patients were excluded from the analysis: 47 (31%) because of death within 6 months following transplantation, six (4%) due to a lack of spirometry assessment at inclusion or 6 months afterwards, and two because of pathological, proven herpetic pneumonia. among the 99 remaining ltrs, 57 (57%) had at least one positive virological respiratory sample. thus, patients were divided into three groups according to the criteria described above: 38 (38%) in the vig, 25 (25%) in the big (6 patients mono-infected by bacteria and 19 co-infected patients) and 36 (36%) in the cg. the corresponding flow chart is presented in fig. 1 . nineteen 4 % of the respiratory samples were broncho-alveolar lavages or bronchial aspirations. general characteristics, type of immunosuppression and prior viral infections were similar across the three groups with the exception of renal dysfunction (egfr < 60 ml/min/1.73m 3 ), more prevalent in the vig (table 1) . previous ar requiring treatment in the 3 months prior to inclusion occurred for 9 (24%) patients in the vig, 3 (12%) in the big, and none of the cg (p < 0.01). among 13 patients presenting a bos at inclusion, 9 were classified in vig, compared to 3 in the big, and 1 in the cg (p = 0.02). clinical presentation (table 2) patients were included at a median time of 303 (iqr, 213-560) days after transplantation. both infected groups displayed similar respiratory symptoms. likewise, the biological presentations of both infected groups were close, and inflammatory surrogates (leucocytes, creactive protein, platelets) did not differ statistically. big patients were more prone to be hospitalized (64 vs. 34%, p = 0.04), and to present a more severe decrease of fev-1 at inclusion (− 260 ml (− 410-0) vs. -50 ml (− 170-60)), compared to the vig. among patients examined with a thoracic tomodensitometry, seven patients (11%) presented a new infiltrate. picornavirus was the most frequently detected virus in the vig (n = 21), followed by parainfluenzae virus (n = 13), and syncytial respiratory virus (n = 10) ( table 3) . pseudomonas aeruginosa was the main detected bacteria; found in 11 (52%) patients in the big. in the cg, the neglected bacteria were pseudomonas aeruginosa (n = 4), corynebacteriae striatum (n = 2) and others (n = 2). three patients had more than one detected virus and eight patients had more than one detected bacteria. the overall rate of worsening 6-month-bos was 13% with no significant differences between the three groups (table 4 ). sixteen patients (16%) died during the study period (6 bos, 2 strokes, 2 neoplasia, 2 severe infections, 4 from unknown cause), with no significant differences between the three groups ( in the two infected groups, these outcomes were not different between the symptomatic patients (n = 41) and the asymptomatic patients (n = 15). thirteen patients had a prior bos at inclusion: 1 (2.8%) in the cg, 9 (23.7%) in the vig and 3 (12.0%) in the big. there was a trend toward more viral respiratory investigation among patients with a bos at inclusion (median of 14.0 (9.0-21.0) samples/patients) than for patients without bos at inclusion (8.0 (4.0-15.0), p = 0.06). however, the sensitivity analysis performed on the remaining 86 patients without bos at inclusion found similar results in terms of significance and association effect with the development of a new bos or with ar (table s1 ). in the same way, infected patients were significantly more investigated than those in the cg: this study shows that, after the first 6 months following transplantation, more than half of ltrs were affected by vrti. clinical presentations for late viral and/or bacterial infections at baseline were very similar, albeit with additional signs of severity for the bacterial infections. single late vrti strongly impacted the patients' prognosis by leading to an increased risk of ar, a trend to an increased risk of bos (without significant association), and a more severe secondary decline in respiratory function compared to the late bacterial respiratory infection. the consequences of these different infections were similar whether or not the infection was symptomatic at the time of viral or bacterial detection. in this study, 57 patients (57%) exhibited at least one positive viral respiratory sampling during follow-up. this rate varies among studies as do screening techniques and reasons for withdrawal. while studies using cell cultures in their screening method report virus detection rates in respiratory samples at around 8% [2, 15] , the use of molecular biology tests significantly increases this prevalence from 17 to 52% [17, 28, 35] . we decided to exclude infections with cmv, especially because cmv diseases respond to different triggers than those infections, were prevented by different protocol evolutions during the study period, and can induce the death or graft rejection in both of our patient groups. with regard to microbiological aetiology, we confirmed that the most frequent viruses detected with the pcr test were those corresponding to the picornavirus group, followed by parainfluenzae viruses and coronaviruses, as already described [5, 6, 18, 23, 24, 27] . it is worthy of note that, in the big, picornavirus were the most frequently detected viruses in co-infections (52%). picornavirus are often considered as a contaminant with a controversial clinical impact. indeed, a recent prospective study demonstrated that rhinoviruses were frequent in ltrs, even in those patients who were asymptomatic [35] . influenza were rarely identified among lung graft patients, especially when compared to the non-lung graft patients in our hospital using the same mpcr assays (5 vs 27%) [34] . this is explained by the high vaccination rates and specific prevention measures compliance among lung graft patients and their relatives. among bacterial infections, pseudomonas aeruginosa and corynebacteriae striatum were more often detected. both bacteria are known to be responsible for serious infections in ltrs. as already shown, the symptomatic feature of the initial infection did not impact on graft survival [15, 17, 25] , suggesting that, for these patients, the symptomatic nature of the infection should not be taken into account. concerning ar, the impact of late respiratory viral and/ or bacterial infections on the graft function was significantly different with three times more ar within 6 months for both the vig and big compared to the cg. while some studies supported this association [16, 36] , other studies, including a meta-analysis, did not find any significant link [17, 25, 28, 30] . this difference could be explained by the variety of criteria used to define ar. we chose to identify ar when the histological pattern showed a stage of at least a1. indeed, previous studies demonstrated that minimal rejection (≥a1) was associated with an increased risk for bos development and progression that was comparable to a2 rejection [37] . on top on that, we noticed a significantly longer delay in ar in the vig than in the big, suggesting that the impact of viral infection on lung graft function must be screen even after several weeks. especially in asymptomatic ltrs and the lack of specific management, the morbidity of viral infection could be attributed to a trivialization of viral colonization, leading to a neglected and chronic cause of inflammation and, thus, to potential rejection. therefore, it seems important to assess the impact of respiratory viral infections on the graft function: to emphasize the prevention of viral infections for immunocompromised with more frequent sampling of patients including wide respiratory virus detection by molecular techniques and to strengthen spirometric controls after viral infections. the all-cause mortality was evaluated to 31% at 6 months in this study. this rate in consistent with other studies [8, 15] . it is explained by the high rate of comorbidity among our patients (more than half with arterial hypertension and diabetes mellitus), the deep immunosuppression required and the numerous complications of lung grafting. to our knowledge, this study is the first to allow a direct comparison of the impact of late viral and bacterial respiratory infections in ltrs. we were able to analyze all the spirometry data both at inclusion and 6 months after, and to present results of an extensive panel of viral pcr tests. this study has several limitations. firstly, the small number of patients could restrict the power of the conclusions, especially for patients classified only on nasopharyngeal swabs (n = 4). aors present wide ranges and must be interpreted accordingly. however, this study remains one of the largest available cohort on the topic to date. despite this small sample size we were able to illustrate that late vrti strongly impacted the patients' prognosis by leading to an increased risk of ar. in addition, although not significant there was a trend to higher risk of occurrence of other outcomes such as bos and death with vrti. we believe that these findings are important and should lead to the design of larger studies. because of the small number of patients among the group with bacterial infections and the group with bacterial and viral infections, we decided to merge these two groups and this could be also debated. however, this was done first because of the similar presentation of patients in these groups and second on the basis of the hypothesis that: (i) bacterial infections were responsible of the major acute part of the lung graft malfunction, and (ii) these groups were both subject to an intervention with the use of antibiotics. secondly, the retrospective design leads to several biases: an indication bias leading to higher infection detection in patients presenting ar 3 months prior to inclusion or a bos prior to infection because of more frequent followup visits and a trend to more respiratory sampling streptocoque, n (%) 1 0 (0) 1 (4) other bacteria, n (%) 2 2 (5) 0 (0) abbreviations: big bacterial infections group; vig viral infections group a among 25 patients with bacterial infections, 7 had no bacteria identification, but samples were examined after antibiotic therapy (n = 3) and/or presented a localized chest x-ray condensation (n = 3) and/or patients had purulent sputum (n = 2) for these patients. although treatment for ar 3 months prior to inclusion could be considered as a biais, this rate was not significantly different between the infected groups. because of the small number of patients, we could not perform a sensitivity analysis among these patients who had ar 3 months prior to inclusion. nevertheless, we performed a sensitivity analysis to address the hypothesis of indication bias. we repeated the multivariable model without considering patients with a bos at inclusion and found similar results, suggesting that this bias, if it existed, did not alter our conclusions. we also assume a survival bias that may explain why despite higher lung function deterioration at 6 months after inclusion in the viral infections group, the rate of bos was not significantly different. the relatively short 6 months follow-up period used in our study may not be sufficient to identify a potential difference in the progression of bos between groups. indeed, the incidence of bos development 6 months after the first infection episode was at 13% in our study, while previous studies described an incidence rate of 50% over a five-year period [2] . to conclude, viral respiratory infections and virusbacteria co-infections were frequent in lung graft recipients and led to similar clinical and biological presentations. bacterial infections strongly diminished initial lung function and led to more hospitalizations whereas single viral respiratory infections seem to lead to a greater deterioration in lung function, and to more acute rejection, than bacterial infections. supplementary information accompanies this paper at https://doi.org/10. 1186/s12879-020-4877-3. additional file 1: table s1 . multivariate analysis of association with development of bos. without patients with bos at inclusion service de maladies infectieuses et tropicales, 46 rue henri huchard, f-75018 greffe cardio-pulmonaire et 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respiratory virus infection and chronic lung allograft dysfunction viral respiratory tract infection during the first postoperative year is a risk factor for chronic rejection after lung transplantation respiratory viruses in lung transplant recipients: a critical review and pooled analysis of clinical studies the epidemiology of parainfluenza virus infection in lung transplant recipients human metapneumovirus infection in lung transplant recipients: clinical presentation and epidemiology upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbeadbased assay incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital comparison of anyplex ii rv16 with the xtag respiratory viral panel and seeplex rv15 for detection of respiratory viruses comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder 19 assay and respifinder smart 22 assay prevalence of respiratory viruses among adults, by season, age, respiratory tract region and type of medical unit role of rhinovirus load in the upper respiratory tract and severity of symptoms in lung transplant recipients respiratory metapneumoviral infection without co-infection in association with acute and chronic lung allograft dysfunction association of minimal rejection in lung transplant recipients with obliterative bronchiolitis springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions md: designed the research, monitored the database, analyzed and interpreted the patient data, contributed to the writting of the manuscript. bv: performed the virological study, interpreted the patient data and contributed to the writting of the manuscript. ab: monitored the database, analyzed and verified the patient data, contributed to the writting of the manuscript. pm: took care of the lung transplants reciepients, contributed to the writting of the manuscript. acm: took care of the lung transplants reciepients, contributed to the writting of the manuscript. gd: took care of the lung transplants reciepients, contributed to the writting of the manuscript. nf: performed the virological study, interpreted the patient data and contributed to the writting of the manuscript. yy: designed the research, monitored the database, interpreted the patient data, wrotte the manuscript. ng: performed the bacteriological study, interpreted the patient data and contributed to the writting of the manuscript. yc: took care of the lung transplants reciepients, contributed to the writting of the manuscript. hv: took care of the lung transplants reciepients, contributed to the writting of the manuscript. gt: took care of the lung transplants reciepients, contributed to the writting of the manuscript. fxl: designed the research, analyzed and interpreted the patient data, contributed to the writting of the manuscript. the author(s) read and approved the final manuscript. no funding was obtained for this study. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. data used in this study was anonymised before its use. the authors declare that they have no competing interests. key: cord-282204-j1slaefb authors: silva, josé v.j.; ludwig-begall, louisa f.; oliveira-filho, edmilson f. de; oliveira, renato a.s.; durães-carvalho, ricardo; lopes, thaísa r.r.; silva, daisy e.a.; gil, laura h.v.g. title: a scoping review of chikungunya virus infection: epidemiology, clinical characteristics, viral co-circulation complications, and control date: 2018-12-31 journal: acta tropica doi: 10.1016/j.actatropica.2018.09.003 sha: doc_id: 282204 cord_uid: j1slaefb abstract chikungunya fever is a mosquito-borne viral illness characterized by a sudden onset of fever associated with joint pains. it was first described in the 1950s during a chikungunya virus (chikv) outbreak in southern tanzania and has since (re-) emerged and spread to several other geographical areas, reaching large populations and causing massive epidemics. in recent years, chikv has gained considerable attention due to its quick spread to the caribbean and then in the americas, with many cases reported between 2014 and 2017. chikv has further garnered attention due to the clinical diagnostic difficulties when zika (zikv) and dengue (denv) viruses are simultaneously present. in this review, topical chikv-related issues, such as epidemiology and transmission, are examined. the different manifestations of infection (acute, chronic and atypical) are described and a particular focus is placed upon the diagnostic handling in the case of zikv and denv co-circulating. natural and synthetic compounds under evaluation for treatment of chikungunya disease, including drugs already licensed for other purposes, are also discussed. finally, previous and current vaccine strategies, as well as the control of the chikv transmission through an integrated vector management, are reviewed in some detail. chikungunya virus (chikv) is the etiological agent of chikungunya fever (chikf), an arthropod-borne disease transmitted mainly by aedes genus species (weaver, 2006) . first described in 1952 in present-day tanzania, the early chikf cases were treated as dengue virus (denv) infection (lumsden, 1955) . following isolation of chikv from infected patients' sera, as well as ae. (stegomyia) aegypti (linnaeus, 1762) and culex spp. mosquitoes in 1953, the virus was placed in the arbovirus group a (ross, 1956; calisher and karabatsos, 1988) . currently, chikv is grouped within the alphavirus genus, togaviridae family, and identified as member of the semliki forest virus (sfv) antigenic complex (van duijl-richter et al., 2015; ictv, 2017) . shortly after identification in east africa (1952), chikv was described in both central and southern regions of africa (uganda and subboth urban and sylvatic chikv transmission cycles have been described (caglioti et al., 2013) . the sylvatic cycle (especially in africa) may involve the participation of some aedes species, such as aedes furcifer (edwards, 1913) , aedes taylori (edwards, 1936) , aedes luteocephalus (newstead et al., 1907) , aedes vittatus (bigot, 1861) and aedes fulgens (edwards, 1917) , and different non-human primate (nhp) species, possible reservoirs or amplifiers hosts for chikv [e.g. african green monkeys (chlorocebus sabaeus) (linnaeus, 1766), patas monkeys (erythrocebus patas) (schreber, 1775), guinea baboons (papio papio) (desmarest, 1820), guenons (cercopithecus aethiops) (linnaeus, 1758), bushbabies (galago senegalensis) (geoffroy, 1796), mandrills (mandrillus sphinx) (linnaeus, 1758), red-tail monkeys (cercopithecus ascanius schmidti) (matschie, 1892) and chacma baboons (papio ursinus) (kerr, 1792)] (mcintosh, 1970; mccrae et al., 1971; diallo et al., 1999; chevillon et al., 2008; pruetz et al., 2010 caglioti et al., 2013 kading et al., 2013; althouse et al., 2018) . a vector role has also been suggested for culex and anopheles mosquitoes, which have been found infected in senegal from 1972 to 1996 (diallo et al., 1999) . in the urban cycle, ae. aegypti and ae. albopictus are known as the main vectors (weaver, 2006) . currently, they probably remain as the main vectors for chikv transmission in the americas, africa, europe, asia and oceania (horwood et al., 2013; vega-rúa et al., 2014 zeller et al., 2016 ngoagouni et al., 2017 . significantly, ae. aegypti can also be the main vector of other viruses, such as zikv and denv, and co-transmission events may be observed (carrillo-hernández et al., 2018) . however, it has been demonstrated that simultaneous infections by denv/chikv/zikv or denv/chikv in ae. aegypti does not compromise the vector competence (le coupanec et al., 2017; rückert et al., 2017) . it is known that horizontal transmission of chikv in aedes mosquitoes can occur and act positively in the maintenance of infection cycles ( fig. 1a ) (mavale et al., 2010) . vertical transmission of chikv into aedes has also been observed under natural and experimental conditions and has been pinpointed as a possible reason for viral persistence under harsh environmental conditions (fig. 1b) (agarwal et al., 2014; chompoosri et al., 2016; jain et al., 2016) . once inside the arthropod vector, chikv must replicate and reach the mosquitoes' salivary glands within roughly seven to ten days for transmission to a susceptible human (lim et al., 2018) (fig. 1c) . in human hosts, the intrinsic incubation time can vary from one to twelve days and infected individuals may present viremia of up to ten days (kam et al., 2009; simon et al., 2011; azevedo et al., 2015) . maternal-fetal transmission has also been reported in humans (fig. 1e ). neonatal encephalitis, as consequence of vertical transmission, was observed, for instance, during the brazilian epidemic in 2016 (bandeira et al., 2016a; lyra et al., 2016) . however, no breast milk transmission has been evidenced (patterson et al., 2016) . despite the fact that chikv rna has been detected in semen even after 30 days post symptom onset, indicating possible transmission via sexual intercourse, horizontal transmission between humans has not yet been reported ( fig. 1d ) (bandeira et al., 2016b; patterson et al., 2016) . approximately 50-97% of individuals infected with chikv develop clinical disease with fever and arthralgia (staples et al., 2009; ayu et al., 2010; nakkhara et al., 2013) . chikv infection has been associated with sudden onset of febrile illness (> 38.9°c) (92% of patients), arthralgia (87% of patients), back pain (67% of patients), headache (62% of patients) and fatigue (who, 2008; thiberville et al., 2013) . the most common symptom in chikf is polyarthralgia, typically of bilateral polyarticular nature, affecting mainly peripheral joints (ankles, wrists and phalanges) and some large joints (knees and elbows) (who, 2008; morrison, 2014) . cutaneous manifestations are reported in circa 50% of acute cases. the lesions are characterized by macular or transitory maculopapular eruption, which can be edematous or itchy, often occurring in the body extremities, palms, soles of the feet, torso and face (simon et al., 2011; thiberville et al., 2013) . gastrointestinal symptoms, such as diarrhea, vomiting, nausea and abdominal pain, occur in 15-47% of cases during the acute phase (thiberville et al., 2013) . other possible symptoms include erythema, asthenia, conjunctival effusion, persistent conjunctivitis and cervical lymphadenopathy (staples et al., 2009; staples and fischer, 2014; madariaga et al., 2016) . different studies have demonstrated that chikv infection can reach high viral loads, ranging from 10 5 to 10 9 copies of viral rna/ml, which seem to be correlated with the presence and severity of clinical signs and symptoms (chow et al., 2011; appassakij et al., 2013) . polyarthralgia and/or polyarthritis are hallmark symptoms of chronic chikungunya, mostly affecting small joints, such as phalanges and wrists, as well as large joints (e.g., ankles, knees and shoulders). the condition is usually severe and leads to mobility limitations of afflicted patients (hoarau et al., 2010) . polyarthralgia has been described to persist for varying periods of time, lasting from weeks to several months and, in some cases, up to five years, depending on the populations evaluated (borgherini et al., 2008; sissoko et al., 2009; manimunda et al., 2010; simon et al., 2011) . the persistence of polyarthralgia in some alphaviruses, such as sfv and sinv, seems to be associated with persistence of viral antigens and immune responses (inflammation) in the joints (atkinson et al., 1986; perri et al., 2000; hoarau et al., 2010; labadie et al., 2010; simon et al., 2011; poo et al., 2014; silva and dermody, 2017) . about the viral antigens, there is still no consensus whether they have replication competence (perhaps with mutations to promote their persistence) or are only the result of a delayed clearance of non-replicating viral antigen (atkinson et al., 1986; perri et al., 2000; poo et al., 2014; weaver and lecuit, 2015) . precisely on chikv, a 2010 study described the presence of macrophages with chikv genetic material and viral proteins in the synovial tissue of an 18-month-long chronically infected patient (hoarau et al., 2010) . experimental studies have shown chikv persistence in lymphoid organs, liver, joints, muscles and macrophages from nhps (labadie et al., 2010) . in addition, the presence of infiltrating cells, mainly macrophages, monocytes and lymphocytes, and specific proinflammatory mediators, such as il-6, il-8, and mcp-1, within the synovial fluid probably also contribute to the chronicity of the inflammation in chikungunya disease (silva and dermody, 2017) . moreover, severe cases of chikungunya may be related to age and diverse underlying medical conditions, such as hypertension, respiratory conditions and diabetes mellitus (borgherini et al., 2007 (borgherini et al., , 2008 sankari et al., 2008; economopoulou et al., 2009; sissoko et al., 2009; tandale et al., 2009) . the pathogenesis of rheumatoid arthritis in chikf is still under debate. while certain studies have suggested that viral infection may trigger initiation of this chronic inflammatory disorder, other studies did not find inflammatory markers in infected individuals with chronic symptoms (staples et al., 2009; schilte et al., 2013) . chikv infections can also lead to atypical clinical manifestations. guillain-barré syndrome (gbs), for instance, has been associated with chikv infection (lebrun et al., 2009; oehler et al., 2015) . gbs comprises an acute inflammatory demyelinating polyneuropathy of global incidence, in which about two-thirds of cases occur after bacterial (e.g. campylobacter jejuni) (heikema et al., 2015) or viral infection (oehler et al., 2015) , such as by dengue(simon et al., 2016) , west nile(leis and stokic, 2012) , influenza(choi and yeon, 2016) , cytomegalo(steger et al., 2012) , human immunodeficiency(girgin et al., 2014) , epstein-barr (phillips, 1973; kim et al., 2016) and zika viruses (rozé et al., 2017) . during the more recent chikv outbreaks, total or partial alopecia on the head or body, predominately in female patients, and ophthalmological alterations, such as uveitis and retinitis, were described during the chronic phase of infection (martínez-pulgarín et al., 2016; cunha and trinta, 2017) . in newborns, congenital infections may be accompanied by varying clinical signs, such as fever, lack of appetite, apnea, skin manifestations, distal and cerebral edema, encephalitis and hemorrhage (gopakumar and ramachandran, 2012; bandeira et al., 2016a; lyra et al., 2016) . heart-and gastrointestinal disorders and cutaneous lesions are reported to manifest up to two days after the onset of fever in chikv-infected newborns and children (ernould et al., 2008) . bullous lesions associated to chikv infection have also been reported in four-month-old babies, who had 20% of their body surface affected on the second day after the onset of fever (robin et al., 2010) . deaths from chikv infection were previously considered a rare event. this perception, however, has changed since the latest epidemics, which presented a considerably increased mortality rate, probably due to neurological affections, mainly in neonates, immunocompromised and elderly (rampal et al., 2007; kee et al., 2010 chusri et al., 2011 bandeira et al., 2016a) . it is challenging to differentiate clinical signs and symptoms of chikv infection from other pathologies, especially when zikv and denv are co-circulating in the same geographical region (hua and combe, 2017) . individuals infected by these arboviruses can present a wide range of similar clinical manifestations, such as rash, myalgia, exanthema, arthralgia, joint pain, headache, lymph node hypertrophy, neurological impairment and fever (brito and cordeiro, 2016) . in addition, it is difficult to determine the frequency and intensity of the symptoms and correctly assess pain (mild, moderate and intense) of afflicted patients (table 1 ). in this context, variations in the clinical presentation of cases can give hints as to the viral etiology; for instance, the salient and prolonged polyarthralgia, often accompanied by rash, is typically more indicative of chikungunya, while hemorrhagic manifestations and myalgia are more commonly observed in denv infections (lee et al., 2012) despite the patients co-infected with chikv/denv, chikv/zikv and chikv/denv/zikv often do not show exacerbation of clinical signs, the co-infection presents as additional obstacle during differential diagnosis (furuya-kanamori et al., 2016; villamil-gómez et al., 2016; carrillo-hernández et al., 2018) . tables 1 and 2 summarize clinical and laboratory features that should be evaluated for efficient differential diagnosis of chikv, denv and zikv infections. since the variety and intensity of symptoms associated to chikv, denv and zikv infections are so similar and make clinical diagnosis difficult in areas of co-circulation, laboratory analysis is necessary to confirm the respective viral etiology. hematology findings associated to chikv infection are commonly either unspecific, however, lymphopenia and hypocalcemia were the most frequent observation, and severe thrombocytopenia is rare (borgherini et al., 2007; brito and cordeiro, 2016; paho, 2017) . moreover, the c-reactive protein is generally elevated in the acute phase illness (table 2) (venugopalan et al., 2014; brito and cordeiro, 2016; paho, 2017) . elevation of hepatic enzymes, as well as elevated creatinine and creatine phosphokinase levels, have also been reported (danis-lozano et al., 2017) . the different laboratory patterns observed during chikv, denv and zikv infections, added to clinical findings, may be incorporated to support a correct diagnosis (tables 1 and 2) . laboratory tests for specific diagnosis of chikv infection are based on virus isolation, viral rna detection and serology (johnson et al., 2016) . despite not usually employed in routine diagnosis, viral isolation can be performed from sera collected up to seven days after onset of illness and inoculated into mosquito-or mammalian cell lines, in which cytopathic effects can appear between one to three days after inoculation. confirmation of results is possible via immunofluorescence or rt-pcr assays (panning et al., 2008; staples et al., 2009 dash et al., 2011 . recently, an immunochromatographic assay used anti-chikv e1 monoclonal antibodies to detect different chikv genotypes in samples from acutely infected patients. this highly specific and sensitive assay may also be an alternative method for chikv infection diagnosis (okabayashi et al., 2015) . molecular methods of chikv diagnosis, such as rt-pcr, rt-lamp, qrt-pcr, have gained increasing importance. they are more sensitive and faster than viral isolation, and permit rna detection from all chikv lineages with high specificity. usually, serum samples collected up to seven days of symptom-onset are suitable for chikv detection by molecular diagnostic platforms (edwards et al., 2007; litzba et al., 2008; sharma et al., 2010) . in addition, novel multiplex assays are capable of differentiating chikv from other infectious agents with a similar clinical spectrum. among them, rt-lamp assay has been shown to be capable of differentiating between zikv, chikv and denv infections (yaren et al., 2017) . a rt-qpcr capable of successfully differentiating between zikv-chikv-denv and chikv-denv-leptospira infections was also recently described (pabbaraju et al., 2016; giry et al., 2017) . in later phases of infection, chikv detection is usually based on serological methods, such as elisa and plaque reduction neutralization testing (prnt). elisa techniques are useful to distinguish between acute or convalescent infections via detection of anti-chikv igm or igg antibodies. igm can be detected from two/four days up to three months after the onset of illness, while igg can be detected for several years (grivard et al., 2007; pialoux et al., 2007; reddy et al., 2012) . moreover, elisa for chikv diagnosis are highly specific and have a high accuracy (johnson et al., 2016) . igm antibody-capture elisa (mac-elisa), via which igm antibodies can be detected in serum samples collected from four days after onset of symptoms, are the most table 1 signals and symptoms that may contribute in the differential diagnosis between dengue, chikungunya and zika illnesses. commonly used tests for laboratory-based diagnoses (reddy et al., 2012) . prnt, used as a parameter to measure circulating neutralizing antibodies, is useful to establish immunoprotection levels based on the determination of serum antibody titers required to neutralize a known amount of infectious virus particles (azami et al., 2016) . alternative techniques for anti-chikv antibody detection include immunofluorescence and hemagglutination inhibition (staples et al., 2009 ). in general, any suitable etiological diagnosis should be reached through combined epidemiological, clinical and laboratory approaches performed by experienced health professionals. an algorithm-guided infographic with specific laboratory key findings to be used for differential diagnosis of chikv, denv and/or zikv infections is summarized in fig. 2 . chikungunya dengue zika fever > 38°c (2-3d a ) > 38°c (4-7d) ≤38°c (1-2d) rash ++ (d2-d5 b ) + (d4 c ) +++ (d1-d2 there is no licensed specific antiviral available for the control of chikv replication, thus therapeutic strategies must be supportive and symptomatic, including fluid intake (jain et al., 2008; who, 2008; kaur and chu, 2013) . in this context, non-steroidal anti-inflammatory drugs (nsaids), such as paracetamol, are indicated to reduce the fever and relieve arthralgic pain (who, 2008) . however, nsaids that interfere (at secondary level) with platelet aggregation or with other mechanisms of blood clotting (e.g. aspirin) should be avoided (goupil and mores, 2016) . the co-administration of nsaids with low-dose systemic corticosteroids has been recommended to reduce pain and improve quality of life in treating acute chikungunya cases with arthralgia (padmakumar et al., 2009) . past concerns that corticosteroid treatment may exacerbate alphaviral arthritides appear unjustified, since serodiagnosis has demonstrated antiviral immunity (mylonas et al., 2004) . a succinct overview of current strategies for inhibition of chikv infection has recently been published (subudhi et al., 2018) . briefly, among the anti-chikv drugs under evaluation, there are preparations that target the viral adsorption and fusion, translation of viral protein and genome replication (mainly in viral non-structural protein 2, nsp2), maturation of viral glycoproteins and immunological molecules (brighton, 1984; briolant et al., 2004 once the molecular test is negative, serological test must be performed. *perform serological tests for samples collected ≥ 4 days after the onset of clinical signs and symptoms; **prnt is required due to the cross-reaction between denv and zikv; ***test also for other flavivirus (e.g. west nile and saint louis encephalitis viruses). reference: cdc (2016) and paho (2017). drugs and compounds under evaluation for treatment of chikv infection. licensed for malaria treatment antiviral activity has been already demonstrated against hiv a , sars-cov b and sfv. a pilot study shown that chloroquine could be employed in the treatment of arthralgia in chronic chikungunya. however, its use in the acute phase is still debated and some studies have also shown an increase of viral replication of sfv and ecmv after treatment with chloroquine. (brighton, 1984; de lamballerie et al., 2008; maheshwari, srikanta, bhartiyaet, 1991; khan et al., 2010) arbidol inhibition of the fusion between virus particle and plasma membrane, and between virus particle and the membrane of endosome ( silymarin reduction of both chikv replication efficiency and down-regulating of viral proteins involved in replication. silymarin interferes with post-entry stages of chikv infection, reducing, in a dose dependent manner, the nsp1, nsp3 and e2 proteins production. (lani et al., 2015) ribavirin probable inhibition of the viral mrna polymerase by binding to the nucleotide binding site of the enzyme. antiviral licensed for treatment of rsv f and hcv g . patients in the drug group reported improvement in the joint pains and the soft tissue swelling also reduced. together with ifn-alpha2b was able to inhibit chikv and sfv replication in vero cells (ravichandran, manian; palumbo, 2011; turner et al., 2014; briolant et al., 2004) decanoyl-rvkr-chloromethyl ketone (dec-rvkr-cmk) inhibition of the maturation of e2 glycoprotein by inhibition of furin. wichit et al., 2017) . examples of drugs and compounds under evaluation are available on table 3 . in the absence of therapeutic strategies and licensed vaccines, efficient vector control plays a crucial role in chikv prevention (huang et al., 2017) . unfortunately, uncontrolled urbanization, lack of proper basic sanitation and increasing resistance to various classes of insecticides challenge the true impact of vector control measures for the reduction of arbovirus incidence (resnik, 2014; liang et al., 2015 who, 2016 . to overcome these obstacles, integrated anti-virus control is required and should include: a) epidemiological surveillance; b) environmental management focusing on educative actions to eliminate potential mosquito breeding sites and reduce standing water sites; c) chemical control using repellents (mainly for travelers and pregnant women) and insecticides, respecting the vectors resistance; and d) biological control against eggs, larvae and mosquitoes (fig. 3) (hemingway and ranson, 2000; dumont and chiroleu, 2010; benelli, 2015; islam et al., 2017; benelli, 2018) . in this last context, larvivorous fish belonging to the genus gambusia (e.g. gambusia affinis) (baird and girard, 1853) and poecilia (e.g. poecilia reticulata) (peter, 1859) have been suggested in several countries and regions for mosquito control, mainly for ae. aegypti (hoy, 1985; das and prasad, 1991; cavalcanti et al., 2007; walton, 2007; chandra et al., 2008; seng et al., 2008; dumont and chiroleu, 2010; kweka et al., 2011; kamareddine, 2012; shulse et al., 2013; pereira and oliveira, 2014; chobu et al., 2015; . more natural mosquito predators, including copepods [e.g. mesocyclops thermocyclopoides (harada, 1931) and mesocyclops longisetus (thiébaud, 1912) ] and other invertebrate aquatic organism, have also been implemented successfully to reduce ae. aegypti and other culicidae populations (rawlins et al., 1997; manrique-saide et al., 1998; vu et al., 1998; schaper, 1999; mahesh kumar et al., 2012; benelli, 2015; pavela, 2015) . plant-borne molecules have shown activity against aedes, anopheles and culex larval instars . more recent approaches, based on "green"-synthesized nanoparticles, have been suggested for use against denv and ae. aegypti vector (madhiyazhagan et al., 2015; sujitha et al., 2015; . microbiological interventions, such as the use of bacillus thuringiensis var. israelensis (bti) (berliner, 1911) and entomopathogenic fungi [e.g. metarhizium anisopliae (metschnikoff, 1879) sorokin, 1883 and beauveria bassiana (balsamo) vuillemin, 1912)], have shown effects on malaria mosquitoes, as well as on ae. aegypti and ae. albopictus (novak et al., 1985; blanford et al., 2005; armengol et al., 2006; knols et al., 2010; lam et al., 2010; ritchie et al., 2010; paula et al., 2011a, b) . recently, bti was used to prevent zikv transmission in the continental united states (stoddard, 2018) . in another promising approach to arbovirus control, including also zikv, the release of wolbachia-infected male mosquitoes, sterile mosquitoes or insects carrying a dominant lethal gene can be used to reduce ae. aegypti and ae. albopictus populations (fig. 3) (ferreira et al., 2008; alphey et al., 2010; walker et al., 2011; boyer, 2012; alphey et al., 2013; massonnet-bruneel et al., 2013; lee et al., 2013; zhang et al., 2015a, b; dickens et al., 2016) . despite the theoretical basis for their effectiveness, additional studies are needed to verify the true risks and benefits of programs based on altered mosquitoes. this concern appears to be greater in relation to transgenic mosquitoes, especially on their efficacy, sustainability and impact on the environment and target and non-target species (wilke et al., 2018) . although many of these strategies have been initially directed against denv, their application to transmission control of chikv should be considered, mainly within an integrated approach, which rationally combines different measures. control programs for other insect-borne diseases, such as malaria (benelli and beier, 2017) , can also serve as model for chikv prevention. in a synergistic way, mass immunization against chikv would be an important tool for viral control and prophylaxis. several distinct vaccine approaches are currently under development, however, no vaccine has been licensed. anti-chikv candidates that have been already tested in humans and/or animals include inactivated-, attenuated-, virus like particle-(vlp), dna-and chimeric vaccines (eckels et al., 1970; levitt et al., 1986; muthumani et al., 2008; wang et al., 2008; tiwari et al., 2009; sharma et al., 2012 akahata et al., 2010 plante et al., 2011; wang et al., 2011; gorchakov et al., 2012; brandler et al., 2013; chang et al., 2014; garcía-arriaza et al., 2014; tretyakova et al., 2014; van den doel et al., 2014; erasmus et al., 2017) . in the past, viral inactivation strategies were the first to be tested in the course of anti-chikv vaccine development. chikv replicated in cell culture and subsequently inactivated by formalin, ether or 1,5 iodonapthyl azide (ina) was able to stimulate the production of neutralizing antibodies (eckels et al., 1970; tiwari et al., 2009; sharma et al., 2012) . particularly, the use of ina resulted in reduced binding capacity of anti-e2 neutralizing antibodies (sharma et al., 2012) , while formalin inactivation stimulated the cellular immune response with the production of anti-and pro-inflammatory cytokines (tiwari et al., 2009) . with the same aim of outlining the virulence of chikv infection, brandler et al. (2013) reported a recombinant live-attenuated measles vaccine expressing a vlp composed of the capsid (c) and envelope (e) proteins from the la réunion 06-46 chikv strain (ecsa lineage). in mice susceptible to measles virus (mv), immunization with mv-chikv induced high titers of chikv antibodies, specific cellular immune responses and protected all animals from lethal chikv challenge (brandler et al., 2013) . in phase i clinical trial (european clinical trials database, 2013-001084-fig. 3 . management for an integrated aedes vector control. *the use of insecticides and repellents should be carried out taking into account the mosquitoes' resistance profiles; ** it is important to consider and evaluate the influence of these interventions on ecosystem balance. 23), a second vaccination resulted in 100% seroconversion for all participants. the immunogenicity of the mv-chikv was not affected by preexisting anti-mv immunity and no vaccination-related serious adverse effects were recorded (ramsauer et al., 2015) . also using the vaccine platform vlp-based, akahata et al. (2010) proposed the use of a vaccinal vlp expressing chikv structural proteins. the vlp-based vaccine, vrc-chkvlp059-00-vp, was obtained by transfection of a plasmid expressing c and e proteins of the 37997 chikv strain (wa lineage). the vaccine stimulated the production of neutralizing antibodies against the e protein of different chikv strains and was able to protect challenged monkeys. further tests in humans showed vaccine efficacy and immunogenicity, without reports of arthralgia as side effect (chang et al., 2014) . the vrc-chkvlp059-00-vp is one of the most developed strategies and is currently undergoing phase ii clinical trials (national clinical trials, 02562482) . another approach to chikv vaccine development is the use of dna vaccines. in this context, a plasmid expressing consensus sequences of c, e1 and e2 chikv proteins elicited a robust cellular immune response and the production of high antibody titers capable of recognizing wild virus antigens (muthumani et al., 2008) . in another study, mice immunized with a dna vaccine containing the complete chikv genome of the 181/25 strain developed neutralizing antibodies and were protected from neurovirulent chikv (tretyakova et al., 2014) . these strategies involving inactivated viruses, vlp and dna vaccines often stimulate the humoral immune response, one of the main mechanisms for control and prevention of chikv infection (lum et al., 2013) . empirically-or reverse-engineered attenuated live vaccines, however, have been shown to be capable of inducing both cellular and humoral immune responses and have also been suggested to prevent chikv infection (levitt et al., 1986; wang et al., 2008 wang et al., , 2011 plante et al., 2011; gorchakov et al., 2012; garcía-arriaza et al., 2014) . an important example of attenuated chikv vaccine is the tsi-gsd-218, a vaccine based on an empirically attenuated chikv 15561 strain (asian lineage) isolated from patient sera from the 1962 chikv outbreak in thailand (levitt et al., 1986) . by the end of its phase ii clinical trial evaluation, 98% of the vaccinated individuals presented neutralizing antibodies, 85% remained seroconverted after one year and 8.47% presented temporary arthralgia (edelman et al., 2000) . in addition to classical attenuation via serial viral passage in cells, reverse genetics strategies have been employed as platforms for construction of recombinant attenuated viruses or vaccine chimeras (wang et al., 2008; plante et al., 2011; wang et al., 2011; garcía-arriaza et al., 2014; van den doel et al., 2014; erasmus et al., 2017 ). an anti-chikv strategy involves the development of an attenuated chimeric vaccine using the internal ribosome entry site (ires) of the encephalomyocarditis virus (ecmv). in this approach, the chikv subgenomic promoter from the lr2006-opy1 strain (ecsa lineage) is knocked via insertion of 13 synonymous mutations and the ecmv ires sequence is added as new promoter for subgenomic rna transcription (plante et al., 2011) . briefly, the addition of the ires sequence to the attenuated viral genome prevents viral propagation in mosquito cells, thus restricting the target population. this vaccine strategy has been shown to lead to the stimulation of tcd4 and tcd8 responses in mice (plante et al., 2011) . in nhp, chikv/ires vaccine was also showed to be safe and immunogenically effective (roy et al., 2014) . finally, the preclinical safety of the chikv/ires vaccine was ensured in interferonα/β receptor-incompetent mice (a129 mice) (plante et al., 2015) . other viral chimeras have been proposed in the context of anti-chikv vaccine strategies. notably, attenuated strains of the eastern equine encephalitis virus (veev) or eastern equine encephalitis virus (eeev) were used as backbones to express chikv structural proteins, creating immunogenic chimeras able to stimulate production of neutralizing antibodies and protect mice against disease and viremia after chikv challenge (wang et al., 2008) . another chimeric construction, the modified vaccinia ankara expressing e2 glycoprotein or e3-e2-6h-e1 proteins was shown to stimulate production of neutralizing antibodies in ifn-i (ifn α/β) and ii (ifn-γ) receptor knockout mice (ag129 mice), protecting against lethal infection (van den doel et al., 2014) . a replication-incompetent adenovirus vector also has been used to express the orf coding chikv structural polyproteins. a single dose of the chimera induced high antibodies titers capable of neutralizing asian and iol chikv lineages, protecting mice against viremia and arthralgia . most recently, a promising vaccine based on a chimera of eilat virus (eilv) and chikv has been reported (erasmus et al., 2017) . the eliv/ chikv possesses non-structural proteins of eilv and structural and accessory proteins of 99659 chikv strain (asian lineage). since the eilv is an alphavirus specific for insects, the eilv/chikv was unable to replicate in vertebrate hosts, thereby providing a high degree of safety. in nhp, eliv/chikv stimulated immune response and guaranteed protection against viremia (erasmus et al., 2017) . in the last decade, there has been an increase in the dissemination and co-circulation of several arboviruses. currently, areas that were endemic just to denv, for instance, are with autochthonous cases of chikungunya and zika diseases. these arboviruses have similar clinical spectrum and require an efficient laboratory diagnosis, especially for a rigorous epidemiological surveillance. in addition, chikv infections represent a serious public health problem. the high morbidity of chikungunya often results in absenteeism of afflicted individuals, incurring both psychosocial and economic impacts. in this context, the development of a specific anti-chikv drug is certainly an important demand. on the other hand, the ideal control strategy for chikv should combine an integrated vector management with mass immunization. although the different vector biocontrol strategies are promising, mainly within an integrated use, it is necessary to think about their sustainable use, assessing their real impacts on ecosystem equilibrium. the absence of chikv serotypes is considered a facilitative aspect in anti-chikv vaccine development, since a formulation developed from one strain will likely result in immunity against all chikv (smalley et al., 2016) . despite recent advances in vaccine strategies, the major challenge regarding chikv vaccine development remains the establishment of an equilibrium between immunogenicity and safety, notably a reduction of side effects, such as secondary arthralgia following immunization with attenuated virus. finally, recognizing the competence of vector and arboviruses control measures, we believe that the prevention of chikv infections should be planned within a global and multifactorial approach. this interdisciplinary strategy, currently framed within the one health concept, should thus integrate all aspects of health care for humans, animals and the environment (benelli and duggan, 2018) . the authors have no conflicts of interest to disclose. acknowledgments e.f. oliveira-filho and r. durães-carvalho are supported by fundação de amparo à ciência e tecnologia do estado de pernambuco (facepe) and mct/cnpq dcr grants. j.v.j. silva júnior is supported by coordenação de aperfeiçoamento de pessoal de nível superior (capes). t.r.r. lopes is supported by conselho nacional de desenvolvimento científico e tecnológico (cnpq). evidence of experimental vertical transmission of emerging novel ecsa genotype of chikungunya virus in aedes aegypti a virus-like particle vaccine for epidemic chikungunya virus protects nonhuman primates against infection sterile-insect methods for control of mosquito-borne diseases: an analysis genetic control of aedes mosquitoes role of monkeys in the sylvatic cycle of chikungunya virus in senegal viremic profiles in asymptomatic and symptomatic chikungunya fever: a blood transfusion threat 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in aedes mosquito populations-interim guidance for entomologists imipramine inhibits chikungunya virus replication in human skin fibroblasts through interference with transgenic mosquitoes -fact or fiction? identification of prohibitin as a chikungunya virus receptor protein assessment of flavaglines as potential chikungunya virus entry inhibitors point of sampling detection of zika virus within a multiplexed kit capable of detecting dengue and chikungunya chikungunya: its history in africa and asia and its spread to new regions in 2013-2014 combining the sterile insect technique with wolbachia-based approaches: ii-a safer approach to aedes albopictus population suppression programmes, designed to minimize the consequences of inadvertent female release combining the sterile insect technique with the incompatible insect technique: i-impact of wolbachia infection on the fitness of triple-and double-infected strains of aedes albopictus key: cord-285628-36gyix12 authors: stull, jason w.; weese, j. scott title: hospital-associated infections in small animal practice date: 2015-03-31 journal: veterinary clinics of north america: small animal practice doi: 10.1016/j.cvsm.2014.11.009 sha: doc_id: 285628 cord_uid: 36gyix12 hospital-associated infections (hais) occur in veterinary hospitals of all types and sizes, and their frequency is likely to increase. urinary tract infections, pneumonia, bloodstream infections, surgical site infections, and infectious diarrhea are the hais most frequently identified in veterinary medicine. a hospital infection control program, consisting of an infectious disease control officer, written protocols, and staff training, is critical to reducing hais and promoting patient, staff, and client health. infection control protocols (plans) should include discussion of hand hygiene and use of personal protective equipment, cleaning and disinfection, patient management, with-in hospital surveillance, and antimicrobial stewardship. compared with people. however, this may be countered with greater patient hygiene challenges, greater difficulty with patient compliance (eg, licking wounds), and a lesser "culture" of infection control. although current data are limited for the veterinary field, similar (or even higher) hai rates have been reported compared with human studies, such as hais in 16% of intensive care unit patients in one study. 3 during a 5-year period, 82% of veterinary teaching hospitals in north america and europe reported at least one hai outbreak and 45% reported multiple outbreaks. many of these outbreaks required restricted patient admissions (58%) or closure of the hospital or section (32%). 4 therefore, although hais are poorly quantified in veterinary medicine, they are undeniably a concern. there are many potential adverse events from hais in veterinary patients. animals suffering from hais may have an increased hospital stay (with accompanying increased cost to the client or clinic). these patients may also suffer permanent health consequences, or hais may result in death of the pet. multidrug-resistant organisms (mdros) are often involved in hais, complicating treatment and resulting in poor patient outcomes and extensive outbreaks. furthermore, some veterinary hospital-associated (ha) pathogens (eg, methicillin-resistant staphylococcus aureus [mrsa] , salmonella) can be transmitted to staff or pet owners, resulting in human illness. additionally, as veterinary medicine advances, there may be parallel increases in hai risk through the use of more invasive procedures, more use of invasive devices (eg, urinary catheters, intravenous catheters), more immunosuppressant therapies, and a greater intensity of critical care management. patients that might not have survived their underlying disease in the past may now be alive, but highly susceptible to infection. perhaps most important to this topic is the assumption in human medicine that 10% to 70% of all hais are preventable through the use of practical infection-control measures. 5 large economic benefits are estimated to occur with the implementation of infection-control interventions ($6-$32 billion cost savings in the united states alone). 2 the proportion of hais that are preventable in veterinary medicine is unknown, but is likely to be similar, and even a 10% reduction in infections could constitute a major impact on patient health, owner cost, and owner and clinician satisfaction. the routine use of simple infection prevention practices can likely dramatically reduce hais. infection control is the term best suited to the goal in small animal veterinary medicine of preventing (or, more practically speaking, limiting) the introduction and/or spread of pathogens with a group of patients and caregivers. central to this goal is the establishment and refinement of an infection-control program at each animal hospital. every hospital's infection-control program will be different, reflecting the unique pathogen risks, facility and personnel characteristics, animal populations served, and level of risk tolerance of the practice. however, at a minimum, each practice's program should include the following: an infectious disease control officer (otherwise known as an infection-control practitioner); a written infection-control protocol (plan); regular training of staff about hospital infection-control protocols (and documentation of this training and assessment of comprehension); monitoring of both disease rates and infection-control protocol compliance. together, the components of the program should address the hai risks for patients and staff and recommended or required protocols to reduce these risks. the end stull & weese result will be a safer working environment for staff, optimal care for all patients, and protection of public health. although good infection-control practices are not the only feature defining excellence in patient care, it is impossible to achieve excellent patient care without them. the standard of what is "acceptable" from an infectioncontrol standpoint is changing in veterinary medicine, and it is clear that the "bar" is being raised in terms of the expected standard of care. in human medicine, urinary tract infections (utis), pneumonia, surgical site infections (ssis), and bloodstream infections (bsis) account for approximately 80% of all hais. 6 in veterinary medicine, these sites along with gastrointestinal disease (infectious diarrhea) are likely to be the most common hais, although other conditions, such as upper respiratory tract infection, dermatophyte infection, iatrogenic blood-borne pathogen infection, and infections of a wide range of invasive devices can also occur. each of these main areas is discussed later, highlighting (where available) incidence, risk factors for disease, and commonly identified pathogens. because ssis are being exclusively covered in a separate article in this issue, they are not discussed here. catheter-associated utis are one of the more common hais in small animal veterinary medicine, although veterinary data are often limited by the failure to differentiate bacteriuria (a potentially benign condition) from uti (disease). studies have reported catheter-associated bacteriuria occurring in 10% to 32% of hospitalized dogs, with a subset of these exhibiting clinical signs or other evidence of infection. [7] [8] [9] [10] the interference of normal defense mechanisms by urinary catheters, such as mucosalsecreted adhesion inhibitors, along with patient comorbid factors and some catheter handling factors, facilitate bacterial colonization of the catheter and ascension of the organism or organisms into the bladder. these pathogens may be endogenous to the patient, arising from the rectum or perineum, or directly from the hospital environment or people through contamination of the drainage system or bag. if the collection system has been contaminated, bacteria can ascend into the bladder through the catheter if there is retrograde flow of urine. retrograde urine flow can occur if the collection system is elevated above the level of the patient; if collection lines are flushed, or if there is obstruction to flow in the collection system. in addition, biofilms (a complex structure of microorganisms and extracellular matrix) can be produced by bacteria on surfaces of urinary catheters. biofilm formation can be associated with poor antimicrobial penetration, antimicrobial resistance, and treatment failure. 11 in human medicine, several factors, such as recumbent position, mechanical ventilation, and use of endotracheal or nasogastric tubes, are likely to increase the risk for ha pneumonia. 12, 13 this topic has been minimally investigated in the veterinary field, in large part because of the limited use of mechanical ventilation. in one study, escherichia coli and acinetobacter spp were commonly identified in feline ha ventilatorassociated pneumonia cases. 14 although not included in the human surveillance definition, aspiration pneumonia is not uncommon in small animal medicine and can occur in patients hospitalized for a wide range of disorders as well as otherwise healthy patients undergoing sedation or anesthesia. in addition to those factors listed for human ha pneumonia, factors that increase aspiration pneumonia, such as hospital-associated infections laryngeal or esophageal disorders and decreased mentation or recumbency, likely increase hai risk. [15] [16] [17] if these patients have been hospitalized for multiple days before aspiration, it is more likely that the oropharynx is colonized with organisms from the hospital environment or hands of staff, and the pneumonia may be more likely to involve mdros, particularly if patients have been treated with antimicrobials. in the human literature, most ha bsis are associated with intravascular devices. duration of catheterization has been recognized as the most important risk factor for the development of catheter-related (cr) bsis (most developing 4-5 days after placement). 18 despite this increased risk, studies have not documented a benefit with prophylactic catheter changes (eg, every 3 days). in human medicine, the current recommendation is for catheters to be removed as soon as medically indicated, but for routine changes to be avoided. a similar approach is appropriate in veterinary medicine. veterinary studies have revealed that jugular and intravenous catheters are frequently contaminated with enteric or environmental pathogens. 9, 19 several factors have been positively associated with intravenous catheter contamination/colonization in dogs and cats, including receipt of dextrose infusion, longer duration of catheter placement, and patient immunosuppression (presence of immunosuppressive diseases or receipt of immunosuppressive drugs). 20 commonly isolated organisms include staphylococci, e coli, enterobacter spp, proteus spp, and klebsiella spp. [19] [20] [21] contamination may occur from the hands of people placing or handling the catheter, the patient's own flora, or the hospital environment. however, there is little evidence indicating that contaminated but not infected catheters (ie, catheters from which bacteria can be isolated but where the catheter insertion site and vein are clinically normal) pose a risk for subsequent bsi. as a result, routine culture of catheters at time of removal or culture of catheter insertion sites is not recommended because skin bacteria are expected to be present. veterinary outbreaks involving cr bsis have been associated with inadequate skin preparation or contaminated materials used in skin preparation, 21, 22 something that is of most concern when antiseptic solutions or wipes are prepared by refilling bottles or containers, which can become contaminated with biocide-resistant bacteria over time. ha gastrointestinal infections are usually recognized when there is a noted increase (outbreak) of infectious diarrhea in hospital patients. although identification of diarrhea is simple, determination of the cause is often difficult, even for known pathogens. in small animal veterinary facilities, salmonellosis is the most frequently reported gastrointestinal hai 4,23 ; however, it is unclear whether that is because it poses the greatest risk or (more likely) it is more readily identified and reported compared with other potential causes. in nonhospitalized small animal populations, several risk factors for salmonella colonization or infection have been identified, including animal species (eg, reptiles, amphibians, young poultry, exotics), consuming a raw animal-based diet or treats (eg, raw meat/eggs, rawhides), exposure to livestock, and recently receiving a probiotic. 24, 25 these factors may substantially increase the risk of shedding salmonella, 14% to 69% shedding in dogs with one or more of these risk factors as compared with less than 5% typically noted in dogs without these risk factors. 25, 26 however, the true scope of this issue is unclear because most outbreaks go unnoticed or testing is not performed, but, conversely, clusters of diarrhea seem to be uncommon in most facilities. pathogens involved in small animal hais often have one or more of the following characteristics: opportunistic pathogen in companion animals and/or humans, environmentally stable, or multidrug-resistant. many pathogens involved in hais are opportunistic pathogens that can be found in healthy animals, highlighting the inability to prevent entrance of all potential pathogens into a veterinary facility. the frequency of each pathogen varies for each veterinary practice (in part influenced by antimicrobial use/pressure, geography, animal species, vaccine coverage of animals in "catchment" area, level of care provided). in addition, environmentally stable pathogens (eg, parvovirus, clostridial spores, dermatophytes) have a demonstrated clear "advantage," increasing the chance of transmission. given the close interaction between veterinary staff and patients as well as the often poor hand hygiene practices documented in veterinary practices, human commensals with zoonotic potential are represented by hais in veterinary medicine. finally, increased resistance to antimicrobials is a common feature of most nosocomial bacteria. several pathogens are a concern from a small animal infection-control standpoint (box 1). although a wide range of pathogens may be involved in hais, currently there is a strong focus on the emerging epidemic of multidrug-resistant bacteria because of dramatic increases in infections, limited antimicrobial options, and potential public health consequences. these mdros are not inherently more virulent than antimicrobial susceptible organisms, but treatment options are limited, something that ultimately can worsen the prognosis. the us centers for disease control and prevention has recently assessed domestic antibiotic resistance threats for people based on box 1 pathogens of concern in a small animal clinic hospital-associated infections clinical and economic impact, incidence, transmissibility, availability of effective antimicrobials, and barriers to prevention. several pathogens of importance relative to veterinary hais were included as "serious antibiotic resistance threats," namely, acinetobacter spp, extended spectrum b-lactamase-producing enterobacteriaceae (esbls), pseudomonas aeruginosa, salmonella spp, and mrsa. 27 as animals and people may share common infection sources or transmit these pathogens to each other, this concern is equally important in the veterinary field, and all of the abovenamed pathogens can be found in veterinary patients. given these relatively novel threats and the often limited knowledge by veterinary personnel on this group of pathogens, the attention here is focused on mdros as hais. in human medicine, hais are often captured through voluntary or mandatory hospital reporting. as such, the occurrence (and trends) of hais are fairly well-established. in the united states, recent data indicate bacteria are responsible for 90% of hais, with commonly identified groups including staphylococcus aureus, enterococcus spp, e coli, coagulase-negative staphylococci (cons), klebsiella spp, p aeruginosa, enterobacter spp, and acinetobacter baumannii. 28 despite the importance of this field, current knowledge of many aspects of the epidemiology of important mdros and pathogens responsible for hais in veterinary medicine is unclear (eg, prevalence, risk factors, and transmission dynamics). unfortunately, companion animal veterinary medicine has been slow to implement surveillance systems; however, this is changing. currently, most data come from limited retrospective studies of clinical isolates, likely resulting in geographic and culturebased bias, potentially misrepresenting the frequency of these pathogens and potentially overestimating the prevalence of antimicrobial resistance if culture submissions are biased toward infections that failed to respond to empirical therapy. regardless, based on the reported veterinary ha outbreaks or supposition from the human literature, several important mdros responsible for hais are identifiable: s aureus, staphylococcus pseudintermedius, enterococci, salmonella spp, acinetobacter spp, e coli, and other enterobacteriaceae, and pseudomonas spp. the specific resistance profiles and treatment options for common multidrug-resistant (mdr) pathogens have recently been summarized. 29 the reader is directed to the article elsewhere in this issue of veterinary clinics of north america: small animal practice by guardabassi and prescott entitled, "antimicrobial stewardship in small animal veterinary practice: from theory to practice," which expands on the topic of mdros in hais and antimicrobial stewardship. staphylococcus s pseudintermedius and to a lesser extent s aureus are common causes of veterinary hais. 30 both are frequently carried on the skin and mucosal surfaces of dogs and people (respectively), creating the potential for both endogenous infection (infection caused by bacteria the animal was harboring at the time of hospital admission) and acquisition of the pathogen during hospitalization directly or indirectly from other patients, the environment, or human caregivers. the emergence of methicillin resistance in these species (methicillin-resistant s pseudintermedius [mrsp] and mrsa) has had important implications for hai prevention and control. methicillin resistance is mediated by the meca gene, which results in resistance to b-lactam antimicrobials (penicillins, cephalosporins, and carbapenems). in addition, resistance to other classes of antimicrobials is frequently observed: lincosamides (clindamycin), fluoroquinolones, macrolides (erythromycin), tetracyclines, trimethoprim-sulfonamides. 29, 31, 32 mrsa is an important pathogen in human hais, being a common cause of ssis and various other types of infections. 33 to a lesser extent, mrsa has also been noted in veterinary hais. 34 risk factors for veterinary mrsa hais have not been well studied, but prior antimicrobial use, prior hospitalization, ownership by veterinary or human health care workers/students, and longer hospitalization (>3 days) have been associated with mrsa colonization or infection in dogs. [35] [36] [37] [38] furthermore, the use of fluoroquinolones and cephalosporins has been linked to the emergence of mrsa in people and may play a role in veterinary species. 39 it is important to note that an abnormally high proportion of veterinarians are colonized with mrsa as compared with the general public. 40 as such, they may serve as a source for hais in their patients if infection-control practices (notably hand hygiene) are substandard. this also likely indicates deficiencies in standard infection control and hygiene practices that allow for transmission of mrsa between veterinary personnel and animals. mrsp has rapidly spread in canine populations, often with high levels of antimicrobial resistance, 41 something that is of tremendous concern because s pseudintermedius is the leading opportunistic pathogen in dogs (and, to a lesser degree, cats). it is the most common cause of ssis in some regions, 42 and treatment may be complicated because of the high level of resistance. in one study, more than 90% of mrsp isolates were also resistant to 4 additional antimicrobial classes. 43 recent prior hospitalization and b-lactam antimicrobial administration have been associated with mrsp infections, 44 suggesting hospital-associated transmission may be a factor in mrsp disease. the topic of cons deserves mention. veterinary diagnostic laboratories often consider these species as a group and speciation is rarely performed. cons are frequently identified as commensals in small animal species, with high methicillin resistance in healthy animals. with the exception of highlycompromised individuals, it has been generally assumed that cons, even those that are multidrug resistant, are of limited clinical concern. that assumption has been challenged to some degree and some cons species may be more clinically relevant than others; however, this group remains a less common cause for concern compared with s pseudintermedius and s aureus. however, their commonness as skin or mucous membrane commensals can complicate interpretation of culture results because differentiating infection from contamination may be challenging. e coli is a frequent component of the commensal gastrointestinal microbiota and is an important pathogen, particularly in utis. mdre coli is frequently shed in the feces of both community and hospitalized small animals. [45] [46] [47] multiple factors have been associated with dogs shedding or acquiring mdr e coli during hospitalization, including duration of hospitalization (>3 days) and treatment with antimicrobials shortly before or while hospitalized (cephalosporins, metronidazole). 38, 48 antimicrobial resistance is an important problem with e coli and other enterobacteriaceae (eg, enterobacter). although b-lactamase-producing isolates have been common for some time, there has been a recent emergence of esbls producers, which provide resistance to a broad range of b-lactam antimicrobials, including thirdgeneration cephalosporins. in addition, esbls are conferred resistance to other antimicrobial classes through genetic linkage with resistance mechanisms. 49 extended spectrum b-lactamase-producing e coli has been identified as the source of veterinary hais, occurring as ssis and catheter-associated utis, with observed hospital contamination. 50, 51 other genera in the enterobacteriaceae family (ie, klebsiella, enterobacter) are considered to be important in human hais; however, less is known of their involvement in veterinary infections. one of the most important drug classes for treatment of esbl-producing bacteria is carbapenems (eg, meropenem). unfortunately, carbapenemase-producing enterobacteriaceae (or carbapenem-resistant enterobacteriaceae; cre) (including e coli) have emerged as a significant problem in human health care. additional resistance mechanisms are often present, rendering isolates virtually pan-resistant, and ability for cres to spread rapidly in health care settings with extension into the community. 52 high mortality (>40%) has been documented for invasive human cre infections. 53 carbapenemase-producing e coli have recently been identified in small animals, with suggested nosocomial transmission. 54 nosocomial transmission currently seems to be a rare, albeit concerning, occurrence, and one that is likely to increase as cres increase in prevalence in the human population, with subsequent exposure of pets. enterococci are often found in the gastrointestinal tract of animals and humans. two species, enterococcus faecium and enterococcus faecalis, are most often involved in disease, including hais, although enterococci tend to be of limited virulence and typically cause infections in compromised hosts. enterococci are inherently resistant to several antimicrobial classes, including cephalosporins, some penicillins, fluoroquinolones, clindamycin, and trimethoprim. 55 they may also acquire resistance to various other antimicrobial classes and, although they are typically of limited virulence, they may be difficult to eliminate in cases when disease develops. vancomycin-resistant enterococci (vre) are an increasing concern in human medicine, with vancomycin resistance noted in up to 83% of e faecium involved in hais. 28 to date, vre appears to be rare in companion animals. 56 however, other mdr enterococci are regularly recognized in small animals and have been identified in hais. 30, 57, 58 enterococci are often identified as utis (including catheter-associated); however, infections at other anatomic sites occur (eg, ssis, bsis, pneumonia). the high degree of antimicrobial resistance, ability to propagate for extended periods in small animal hosts as a commensal, and environmental persistence make enterococci particularly challenging when involved in hais. it is important to note that isolation of enterococcus species (regardless of antimicrobial resistance) does not always indicate treatment is indicated. without clinical signs in an otherwise immune-competent animal, it may be warranted to withhold treatment and monitor the patient. when isolated in a patient with clinical signs (notably infections of the urinary tract, wound, or body cavity), treatment should often be directed at the organism or organisms also isolated that are thought to be primarily responsible for clinical disease. 29 often, that involves ignoring the enterococcus and targeting therapy toward another, more convincing, pathogen, such as e coli. salmonella is most frequently a concern in equine facilities, but has been identified as a source of sporadic illness and hospital-associated outbreaks in small animal hospitals. 23, 59 an important concern with salmonella hais is the occurrence of zoonotic transmission with accompanying human infections. 23, 59 because most infections in dogs and cats are subclinical, there is a high risk for inadvertent hospital-wide environmental contamination and nosocomial transmission. reported factors leading to an increased risk of salmonella shedding in small animals include consumption of raw meat diets, exposure to livestock, and receiving a probiotic in the previous 30 days. 25, 60 as with e coli, esbl-producing strains are a concern for antimicrobial resistance and have been identified in small animals. 61 given its environmental stability, potential shedding by healthy animals, and significant zoonotic health hazard to clinic staff and clients, salmonella needs to be considered an important companion animal nosocomial pathogen. acinetobacter is well-recognized as an important ha pathogen in human medicine, in part because of recently recognized high levels of antimicrobial resistance in a baumannii. more than 60% of a baumannii human isolates involved in hais were mdr in one study. 28 given its role as an opportunistic pathogen in small animals, ability to persist in the environment for extended periods, and documented outbreaks in veterinary facilities, it is also a concern for veterinary medicine. 30, 62, 63 documented hais involving a baumannii include intravenous and urinary catheters, surgical drain infections, ssis, pneumonia, and bsis. 62 multidrug resistance is frequently encountered with pseudomonas spp. this along with their noted persistence in the hospital environment makes pseudomonas spp a concern for hais. in humans, most infections are ha and occur in immunocompromised hosts. 64 in companion animal species, pseudomonas spp infections often involve the skin, urinary system, and ears, 65-67 along with ssis and invasive device infections. 68, 69 biofilm formation by pseudomonas spp can further complicate treatment. identification of within hospital clusters of pseudomonas infections should prompt investigation of potentially contaminated environmental, equipment (eg, endoscope), or consumable (eg, catheter preparation supplies) sources. the admission of sick animals occurs daily in most if not all small animal veterinary facilities. furthermore, every animal admitted to the veterinary clinic, healthy or not, can reasonably be assumed to be shedding multiple microorganisms that could cause infection in humans or animals, given the opportunity. as such, there is always a risk for the introduction and spread of hais and for exposure to zoonotic pathogens. the level of risk will be determined, in part, by the population of animals served (eg, young, elderly, immunocompromised), pathogens circulating in the community animals, proportion of patients for which protective or increased-risk practices are taken by their owners (eg, vaccination, husbandry practices to reduce pathogen acquisition), intensity of care typically provided for patients, and clinic infection-control practices and adherence to these practices by staff and clients. veterinary clinic staff will not be able to alter many of these risks; however, infection-control practices is an area that with some planning and dedicated time, can be relatively easy to address. although complete prevention of hais is the goal, given the nature of patient care, bacterial adaptation, and complexity of many pathogens (subclinical shedding, insensitive diagnostic tests), it is inevitable they will continue to occur. methods to reduce the risk of hais are paramount. in general, methods to reduce hais can be divided into the following main categories: hand hygiene and use of personal protective equipment (ppe; ie, clothing and/or gloves to reduce contamination of staff, patients, and the environment); cleaning and disinfection (environmental surfaces and patient equipment); patient management (eg, cohorting patients based on risk, isolating high-risk patients, discontinuing the use of higher risk devices when indicated); surveillance (identification of infected or colonized patients, hais, and source/ risk factors); antimicrobial stewardship (prudent antimicrobial use); education and training (clients, staff). these methods will not only reduce overt problems such as hospital-associated outbreaks but also reduce the likelihood of patient colonization with a ha pathogen, which can become part of the patient's resident microbiota, potentially increasing disease risk at a later date and posing a risk to other animals and humans. each of these areas should be addressed in a hospital's infection-control manual. several "model" plans are widely available to use as a starting point for developing an individualized hospital plan; infection-control officers are encouraged to review these resources. 70, 71 individual articles in this issue of veterinary clinics of north america: small animal practice are devoted to each of these areas, so they are only briefly discussed here. unfortunately, studies on the area indicate only a minority of small animal veterinary hospitals have written infection-control plans (0%-31%). 72, 73 given the relative ease of putting together an infection-control plan and potential health, legal, and financial benefits of doing so, every clinic should invest the time and effort to make this a priority. hand hygiene (washing hands with soap and water or using an alcohol-based hand rub) and use of ppe, such as nonsterile gloves and gowns, are simple techniques that can reduce the risk of hais. effective use of hand hygiene and appropriate ppe use reduces the risk of contamination of personal clothing, reduces exposure of skin and mucous membranes of veterinary staff to pathogens, and reduces transmission of pathogens between patients by veterinary personnel. unfortunately, several studies indicate that veterinarians and staff do a poor job at performing hand hygiene between patients (w20%) or using ppe when indicated (6%-37% depending on the situation). 72, 74 cleaning and disinfection recent evidence suggests environmental contamination in human hospitals increases the risk for hais, 75 whereas interventions that reduce environmental contamination have assisted with cessation of ha outbreaks or reduction of hais. 76, 77 the same connection is assumed to occur in veterinary medicine. effective cleaning and disinfection of hospital equipment and environmental surfaces play an important role in reducing hais. in order for a disinfectant to work properly, the surface or item must first be clean (free of visible organic material) and the product must be applied at the manufacturer's suggested dilution and contact time (amount of time the disinfectant is in contact with the item before being removed). disinfectants should be selected based on several criteria, including the product's spectrum of activity, susceptibility to inactivation by organic matter, and potential pathogens in the environment. given the close contact between veterinary patients and their hospital housing, environmental contamination is inevitable. furthermore, staff caring for these patients is at increased risk for spreading the pathogen through contact with the patient or its stull & weese environment. to protect other patients and clinic staff, special attention to patient housing is important in managing infectious patients. isolation procedures, use of dedicated medical equipment, and patient cohorting are important stopgaps in the transmission of hais for animals suspected to be infectious. in addition, specific patient care procedures may be helpful in reducing hais associated with catheters, aspiration pneumonia, bsis, infectious diarrhea, and ssis. resident small animals are sometimes kept at veterinary facilities as blood donors, companionship for staff, or other reasons. because these animals may harbor mdr pathogens and be sources or propagation of hospital contamination or outbreaks, special attention should be devoted to hospital policies for these animals regarding staff-animal contact and restricted movement (not permitting direct contact with patients or patient areas, including areas for exercise and elimination). 78, 79 surveillance the early identification of hais is critical for effective infection control. identification of "abnormal" (increases in disease incidence or patterns) depends on a reasonable understanding of "normal." understanding of endemic rates can be useful to allow for comparison with other facilities, to establish benchmarks for ongoing surveillance, to serve as a baseline for interventions, to allow for more accurate counseling of clients about risks (eg, ssi rates), and to provide a greater overall awareness of the importance of hais and corresponding control measures. it is not unusual for hai outbreaks to "smolder" below the radar of veterinary staff for extended periods because of the lack of centralized data reporting or communication, resulting in substantial environmental contamination, patient morbidity (and potentially mortality), and even increased zoonotic disease risk for staff and clients. key elements of early hai identification include (1) a surveillance program tailored to the risks and needs of the veterinary practice and (2) routine use of diagnostic culture and susceptibility data to establish practice-specific baseline levels of pathogen prevalence and antimicrobial resistance and detect changes from this baseline. careful selection and appropriate use of antimicrobials are important steps in combating patient mdro development and subsequent contamination and transmission in the hospital environment. antimicrobials should be avoided when a bacterial infection has not been confirmed. antimicrobials used in the initial treatment of an infection should be selected based on the effectiveness against the most likely organisms causing the infection (something that can be facilitated by having good passive surveillance data) as well as patient (eg, renal function, comorbidities) or drug (eg, penetration, route of administration, frequency of administration) factors. whenever possible, a culture should be submitted to determine the true susceptibility pattern of the bacteria involved. local therapy can be an important option that is often overlooked. during their careers, approximately two-thirds of veterinarians report a major animalrelated injury resulting in lost work or hospitalization. 71, 80 animal bite injuries and infections are a large contributor to this hazard, but zoonotic infections (eg, mrsa, dermatophytosis [ringworm], salmonellosis) are also frequently reported. 23, 81 educating staff and clients on zoonotic disease risks and enforcing in-hospital infection-control protocols to reduce these risks will be beneficial to the health of people and patients. all veterinary personnel and visitors should be familiar with the hospital's infectioncontrol plan and policies. the infection-control officer is integral to the successful development, maintenance, and enforcement of an infection-control plan. in the human health care field, infection-control practitioners are formally trained and certified, with the infectioncontrol program typically overseen by a physician with specialized training in infectious diseases, infection control, and/or microbiology. in veterinary medicine, this type of approach is only practiced in large facilities (mainly teaching hospitals), yet the basic concepts remain the same for veterinary facilities of any type and size. a functional infection-control program can be directed by a single infectioncontrol practitioner in a veterinary hospital, with minimal time requirements. this individual can be a technician or veterinarian who has an interest in infection control. the skills required (eg, general understanding of infection-control concepts) can be obtained on the job and need not be a prerequisite for the position, and the limited time requirement under normal circumstances means that a new position does not need to be added. rather, direction of the infection control can usually be undertaken by an existing staff member. of greatest importance for the individual filling this position is an interest in the topic, motivation to make improvements in the clinic's infection-control policies, and the support of clinic leaders (eg, practice owners, veterinarians). without full support by clinic leaders (eg, time to perform the required duties, financial investments, serving as a role model by following clinic infection-control policies), the infection-control officer, and resulting program, is unlikely to be successful. hais have been reported in veterinary medicine and their frequency is likely to increase with the increase in intensive care practices in many veterinary hospitals. prolonged hospitalization and the use of invasive devices and procedures increase the risk of hais. all staff members should be educated on the risks and signs associated with hais so that cases can be detected early and managed appropriately. ultimately, a multifaceted approach is necessary to address hais in small animal veterinary medicine, including prudent antimicrobial use, strengthening surveillance of hais in companion animals, improving infection-control practices (eg, hand hygiene, ppe, cleaning and disinfection, patient management), instilling an infection-control culture among veterinary staff, and improving health care and public education of antimicrobials. a hospital infection-control program, consisting of an infectious disease control officer, a written protocol, and staff training, is a key component to unifying these elements and successful reduction of hais in small animal veterinary practice. estimating health care-associated infections and deaths in u.s. hospitals the direct medical costs of healthcare-associated infections in u.s. hospitals and the benefits of prevention using syndromic surveillance to estimate baseline rates for healthcare-associated infections in critical care units of small animal referral hospitals van metre dc. characteristics of biosecurity and infection control programs at veterinary teaching hospitals the preventable proportion of nosocomial infections: an overview of published reports cdc/nhsn surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting evaluation of catheter-associated urinary tract infections and multi-drug-resistant escherichia coli isolates from the urine of dogs with indwelling urinary catheters incidence of catheter-associated urinary tract infection among dogs in a small animal intensive care unit nosocomial infection surveillance in a small animal intensive care unit urinary tract infection resulting from catheterization in healthy adult dogs biofilms and catheter-associated urinary tract infections circulating immune parameters predicting the progression from hospital-acquired pneumonia to septic shock in surgical patients supine body position as a risk factor for nosocomial pneumonia in mechanically ventilated patients: a randomised trial indications for and outcome of positivepressure ventilation in cats: 53 cases outcome of and postoperative complications in dogs undergoing surgical treatment of laryngeal paralysis: 140 cases postoperative pulmonary complications in dogs undergoing laparotomy: anesthetic and perioperative factors incidence of and risk factors for postoperative pneumonia in dogs anesthetized for diagnosis or treatment of intervertebral disk disease risk of local and systemic infection with polyethylene intravenous catheters. a prospective study of 213 catheterizations surveillance of infections associated with intravenous catheters in dogs and cats in an intensive care unit bacterial and fungal colonisation of peripheral intravenous catheters in dogs and cats inadequate skin preparation as a cause of intravenous catheterrelated infection in the dog a prospective study of intravenous catheter contamination salmonella typhimurium outbreak associated with veterinary clinic evaluation of pet-related management factors and the risk of salmonella spp. carriage in pet dogs from volunteer households in ontario perceptions, practices, and consequences associated with foodborne pathogens and the feeding of raw meat to dogs salmonella shedding in racing sled dogs antibiotic resistance threats in the united states antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the national healthcare safety network at the centers for disease control and prevention antibiotic treatment of resistant infections in small animals transmission of opportunistic pathogens in a veterinary teaching hospital inducible clindamycin-resistance in methicillin-resistant staphylococcus aureus and methicillin-resistant staphylococcus pseudintermedius isolates from dogs and cats methicillin-resistant staphylococcus pseudintermedius in a veterinary teaching hospital severe surgical site infection in community hospitals: epidemiology, key procedures, and the changing prevalence of methicillin-resistant staphylococcus aureus suspected transmission of methicillinresistant staphylococcus aureus between domestic pets and humans in veterinary clinics and in the household epidemiological profiling of methicillin-resistant staphylococcus aureus-positive dogs arriving at a veterinary teaching hospital methicillin-resistant and -susceptible staphylococcus aureus infections in dogs risk factors for methicillinresistant staphylococcus aureus (mrsa) infection in dogs and cats: a casecontrol study acquisition and persistence of antimicrobial-resistant bacteria isolated from dogs and cats admitted to a veterinary teaching hospital the effect of antibiotics on methicillin-resistant staphylococcus aureus methicillin-resistant staphylococcus aureus colonization in veterinary personnel clonal spread of methicillin-resistant staphylococcus pseudintermedius in europe and north america: an international multicentre study economic impact of tibial plateau leveling osteotomy surgical site infection in dogs evaluation of susceptibility test breakpoints used to predict meca-mediated resistance in staphylococcus pseudintermedius isolated from dogs factors associated with methicillin-resistant versus methicillin-susceptible staphylococcus pseudintermedius infection in dogs prevalence of antimicrobial-resistant escherichia coli in dogs in a cross-sectional, community-based study occurrence of antimicrobial resistant bacteria in healthy dogs and cats presented to private veterinary hospitals in southern ontario: a preliminary study risk factors for multidrug-resistant escherichia coli rectal colonization of dogs on admission to a veterinary hospital risk factors for dogs becoming rectal carriers of multidrug-resistant escherichia coli during hospitalization infections with extended-spectrum beta-lactamase-producing enterobacteriaceae: changing epidemiology and drug treatment choices characterization of multidrug-resistant escherichia coli isolates associated with nosocomial infections in dogs emergence and spread of two distinct clonal groups of multidrug-resistant escherichia coli in a veterinary teaching hospital in australia vital signs: carbapenemresistant enterobacteriaceae outcomes of carbapenem-resistant klebsiella pneumoniae infection and the impact of antimicrobial and adjunctive therapies emergence of oxa-48 carbapenemase-producing escherichia coli and klebsiella pneumoniae in dogs vancomycin-resistant enterococci: consequences for therapy and infection control monitoring of antimicrobial resistance in healthy dogs: first report of canine ampicillin-resistant enterococcus faecium clonal complex 17 susceptibility to vancomycin and other antibiotics of 165 enterococcus strains isolated from dogs in italy dogs leaving the icu carry a very large multi-drug resistant enterococcal population with capacity for biofilm formation and horizontal gene transfer multidrug-resistant salmonella typhimurium in four animal facilities comparison of antimicrobial resistance patterns of salmonella spp. and escherichia coli recovered from pet dogs from volunteer households in ontario (2005-06) prevalence, distribution and characterisation of ceftiofur resistance in salmonella enterica isolated from animals in the usa from 1999 to the role of acinetobacter baumannii as a nosocomial pathogen for dogs and cats in an intensive care unit multidrug-resistant acinetobacter baumannii in veterinary clinics pseudomonas aeruginosa carriage, colonization, and infection in icu patients characterization of antimicrobial resistance of pseudomonas aeruginosa isolated from canine infections evidence-based veterinary dermatology: a systematic review of interventions for treatment of pseudomonas otitis in dogs comparison of three techniques for the diagnosis of urinary tract infections in dogs with urolithiasis cardiovascular device infections in dogs: report of 8 cases and review of the literature an infected hip prosthesis in a dog diagnosed with a 99mtc-ciprofloxacin (infecton) scan infection prevention and control best practices for small animal veterinary clinics compendium of veterinary standard precautions for zoonotic disease prevention in veterinary personnel: national association of state public health veterinarians veterinary infection control committee infection control practices and zoonotic disease risks among veterinarians in the united states evaluation of specific infection control practices used by companion animal veterinarians in community veterinary practices in southern ontario evaluation of an educational campaign to increase hand hygiene at a small animal veterinary teaching hospital does improving surface cleaning and disinfection reduce health care-associated infections? a targeted strategy to wipe out clostridium difficile role of environmental cleaning in controlling an outbreak of acinetobacter baumannii on a neurosurgical intensive care unit outbreak of clostridium difficile-associated disease in a small animal veterinary teaching hospital resident cats in small animal veterinary hospitals carry multi-drug resistant enterococci and are likely involved in crosscontamination of the hospital environment a review of published reports regarding zoonotic pathogen infection in veterinarians a survey of veterinarian involvement in zoonotic disease prevention practices key: cord-275795-ee7qyw5h authors: monette, anne; mouland, andrew j. title: t lymphocytes as measurable targets of protection and vaccination against viral disorders date: 2018-10-24 journal: int rev cell mol biol doi: 10.1016/bs.ircmb.2018.07.006 sha: doc_id: 275795 cord_uid: ee7qyw5h continuous epidemiological surveillance of existing and emerging viruses and their associated disorders is gaining importance in light of their abilities to cause unpredictable outbreaks as a result of increased travel and vaccination choices by steadily growing and aging populations. close surveillance of outbreaks and herd immunity are also at the forefront, even in industrialized countries, where previously eradicated viruses are now at risk of re-emergence due to instances of strain recombination, contractions in viral vector geographies, and from their potential use as agents of bioterrorism. there is a great need for the rational design of current and future vaccines targeting viruses, with a strong focus on vaccine targeting of adaptive immune effector memory t cells as the gold standard of immunity conferring long-lived protection against a wide variety of pathogens and malignancies. here, we review viruses that have historically caused large outbreaks and severe lethal disorders, including respiratory, gastric, skin, hepatic, neurologic, and hemorrhagic fevers. to observe trends in vaccinology against these viral disorders, we describe viral genetic, replication, transmission, and tropism, host-immune evasion strategies, and the epidemiology and health risks of their associated syndromes. we focus on immunity generated against both natural infection and vaccination, where a steady shift in conferred vaccination immunogenicity is observed from quantifying activated and proliferating, long-lived effector memory t cell subsets, as the prominent biomarkers of long-term immunity against viruses and their associated disorders causing high morbidity and mortality rates. the world was forever changed by the introduction of vaccine against smallpox in the late 1700s, at the time protecting its first 100,000 individuals. this was the first demonstration that a vaccine could successfully eradicate viruses causing disorders and diseases that even when not lethal, still had the potential to cripple both surviving populations and their surrounding geographical economies. since that time, the occurrence of epidemics and outbreaks are now at lower risk, following the introduction of massive vaccination programs able to induce immune system targeting of viruses causing severe disorders affecting distinct geographical locations, and with many epidemiological reports demonstrating long-term efficacy of viral control of non-naïve populations. outbreaks of existing and emerging viral diseases or disorders vary widely in duration and frequency across geographical populations. some can be predicted annually, while others may see decades between outbreaks, therefore driving the continuous epidemiological surveillance of associated infectious disorders, the development and implementation of targeting vaccines, and development of immune-monitoring strategies measuring vaccine efficiency in target populations. despite vaccination-mediated protection of numerous nations documenting complete eradication of the causative agents of viral disorders in endemic populations, there is threat of re-emergence of pandemic proportions of these agents. threats to virus re-emergence are caused by contemporary choices made to not vaccinate children, by strain recombination, by viral spread to naïve populations by world-travelers, migrants, or climate change, causing redistribution of viral vectors, by potential use of these agents in acts of bioterrorism or war upheaval, or by depletion of vaccination stocks required for protection against pandemics. other factors include the increasing and aging global population and increased number of immunocompromised individualsd factors strongly supporting the maintenance of herd immunity against existing viruses, despite lower incidence of outbreaks in industrialized countries. in the event of a re-emergence of previously eradicated viruses or the acquisition of increased pathogenicity by existing viral strains, there is an urgent need for vaccine development strategies that can rapidly and effectively arrest global spread. important advances are continuously being made in vaccine development strategies toward the control of viruses and associated disorders. vaccine design has been modified from the use of attenuated viruses to use of more precise viral protein subunits specifically targeted by t cells. historically, vaccination immunogenicity was documented by measures of serum immunoglobulin (ig) classes and antigen-specific antibodies produced by humoral immunity. more recently, quantification of cellular components of innate immunity at the interface between innate and adaptive immunity are made, in addition to more precise measurements of adaptive immunity. long-term protection achieved by adaptive immunity can be quantified by measuring levels of circulating cytokines, along with specific phenotypic profiles of effector memory, antigen-specific t cells. though both humoral and cellular arms of immunity are integrally linked during the initial induction of immunity against pathogens, these can become disconnected with developing pathology due to their individual needs for survival factors, unequal declines in immune function, and differential cellular lifespans. this loss of correlation between memory t cells and neutralizing antibody responses varies according to different viruses, suggesting that independent time course measures of these separate immune responses are required over time for adequate recording of biomarkers of natural infection and vaccine efficacy or suggesting that t cell status may be most crucial measure of conferred long-term immunity. from the standpoint of fundamental or clinical research, it has become established that the targeted induction of specific pathogen-and tumorclearing effector memory t cell subsets is our endgame armor toward long-term human survival against infectious diseases and cancers. this chapter provides an overview of viruses that have historically caused severe lethal disorders, including those of the respiratory, gastric, skin, hepatic, neurologic, and hemorrhagic types. the features of viruses and associated disorders that we herein describe include viral genetics and replication cycles, transmission modalities, cell and organ tropism, host-immune evasion strategies, associated viral disorders and diseases, and epidemiology. we also report on well-accepted and other important documented instances of viral control by t cells, currently available and successful vaccines, and recorded measures of vaccination immunogenicity. we focus on quantification of vaccine-induced effector memory t cellemediated immunity, representing the gold standard of successful vaccination. just as it is for advances in vaccinology, investigations into the biology of t cells are currently at the forefront of many research fields examining various disorders, diseases, and malignancies not formerly considered to be controlled by immunity. although many viral infections are limited to the upper respiratory tract, it is lower respiratory tract infections (lrti) that most predominantly cause enormous disease burden in children and immunocompromised adults suffering from human immunodeficiency virus (hiv) infection or in patients having received stem cell or solid organ transplants for which immunosuppressive therapies were administered (henrickson et al., 2004; pavia, 2011; kim et al., 2007) . acute lower respiratory illnesses (alris) are a major cause of morbidity and mortality, accounting for approximately 1.6 million deaths, globally, per year (black et al., 2010) . frequently overlapping lrti syndromes include bronchiolitis, asthma exacerbation, wheezing, croup, and pneumonia. although certain specific syndromes can be more precisely associated with infection by specific viruses, syndromes overlaps can complicate diagnosis of these numerous viruses, and quite often, difficulties in differentiating between viral and bacterial pneumonias symptoms can also result in antibiotics being mistakenly prescribed during viral disorders. several viruses are normally, however, considered to be primarily responsible for lrtis, beginning with upper respiratory tract infections, most commonly caused by respiratory viruses that are typically spread from person-to-person by contact with infected respiratory droplets, and including respiratory syncytial virus (rsv), epidemic influenza a and b, h5n1 and h7n9 avian influenza a viruses (iavs), parainfluenza viruses 1 through 4, adenovirus, human metapneumovirus (hmpv), severe acute respiratory syndrome coronavirus, human coronaviruses nl63 and hku1, rhinoviruses, and bocaviruses (pavia, 2011; mahony, 2008; nichols et al., 2008) (table 1) . currently, vaccines for human influenza viruses, human parainfluenza viruses (hpivs), and adenoviruses causing upper and lower respiratory infections are used to control these infections and the resulting propagation of their morbid symptom derivations. human influenza viruses make up three of the five genera of the family orthomyxoviridae and are classified as a, b, and c types, based on their highly conserved matrix protein 1 (m1), membrane matrix protein (m2), and nucleoprotein (np). type a influenza viruses can be further sub-subtyped by the antigenicity of their hemagglutinin (ha) and neuraminidase (na) surface glycoproteins (gps). antigenic drift, caused by point mutations in ha and na and recombination of the ha genes, results in the generation of new strains that can escape pre-existing immunity, causing both the prediction of circulating strains difficult and antigenic mismatch by existing vaccines. approximately 18 ha and 9 na subtypes of influenza a are documented in aquatic birds, representing their natural hosts (i.e., vectors). influenza a h1 and h3 subtypes cocirculate seasonally, and influenza b viruses can only infect humans, via two distinct, seasonally cocirculating, lineages. type c influenza viruses are more rarely documented to infect humans and pigs (berlanda scorza et al., 2016) . influenza viruses cause acute upper and lower respiratory infections, and due to their rapid and unpredictable genetic drift, represent the most likely of pathogens to cause a human pandemics. annually, human influenza viruses have the potential to cause up to 5 million cases of severe illness, with an associated 500,000 deaths worldwide (who_influenza_(seasonal), 2018), causing great economic burden. four influenza pandemics have occurred over the past century, as a consequence of the h1n1 (1918), h2n2 (1957), h3n2 (1968), and h1n1 (1977) variants (palese, 2004) . since the most recent outbreak in 2009, an estimated 200,000 people globally have succumbed to the h1n1 variant of swine origin (dawood et al., 2012) . epithelial cells that are infected with influenza virus produce inflammatory cytokines acting as chemoattractants for homing macrophages and dendritic cells (dc). dcs take up influenza viral particles to trigger their maturation and pursuant migration to the lymph, where they initiate antigen-specific t cell maturation. these influenza-specific effector t cells then enter the respiratory tract to counteract viral titres through cytokine expression and the direct lysis of infected cells, with activated cd8 þ effector cytotoxic t cells (ctls) representing the main constituents of this response by their release of perforins and granzymes, and the engagement of tumor necrosis factor (tnf) receptors (spitaels et al., 2016) . influenza-specific cd4 þ t helper cells can act directly and indirectly in viral clearance, primarily by producing cytokines that induce the functions of b cells and cd8 þ t cells and which have also been reported to directly eliminate infected cells themselves (topham and doherty, 1998; hua et al., 2013) . while preexisting cd8 þ t cell immunity has not yet been demonstrated to prevent infection from occurring, it is hypothesized to be the result of the loss of granzyme expression by memory cd8 þ t cells and populations of iavspecific cd8 þ t cells are still importantly correlated with the control of spread and recovery in healthy populations (grant et al., 2016) . the most currently administered influenza vaccines are inactivated (iv) trivalent (tiv) or quadrivalent formulations containing equal amounts of ha of two influenza a strains (h1n1 and h3n2) and one of two influenza b strains (yamagata and victoria lineage). these are derived from viruses typically grown in fertilized chicken eggs, are mainly focused on eliciting a strainmatched humoral immune responsedrequiring yearly updatesdand are unable to provide protection to all vaccinated individuals. the requirement of memory t cell immunity for long-term protection against influenza virus promotes the development of vaccines that elicit both humoral and cellular immunity: a strategy expected to overcome the inadequacies of current vaccines against influenza and other viruses (spitaels et al., 2016) . there is broad interest in the development of a universal influenza vaccine, considered to be the "holy grail" of influenza vaccine research. this approach is being developed to use virus-infected cell-killing antibodies that produce an antiviral environment; these termed antibody-dependent cellular cytotoxicity (adcc)-mediating antibodies, which are predicted to link innate and adaptive immune responses, and is becoming possible due to new technologies for rapid isolation and characterization of monoclonal antibodies targeting conserved regions of influenza virus, reviewed in jegaskanda et al. (2017) . this approach has been postulated to work, in part, from reports of iavspecific cd8 þ t cells, promoting viral clearance in the absence of neutralizing antibodies, and can also mediate cross-reactive immunity against distinct iavs to drive a rapid recovery from severe influenza disorders (grant et al., 2016) . the induction of infection-permissive immunity is both protective and allows virus-induced cross-reactive immune responses. vaccines targeting the conserved ectodomain of m2 deliver this kind of non-neutralizing immunity since these antibodies rely on fc receptors and innate immune components (el bakkouri et al., 2011) . antagonizing antibodies inhibiting na activity represents another promising strategy, not by blocking viral entry and eliciting sterilizing immunity, but by contributing to immunity against a virus possessing a similar na type (wan et al., 2013) . there is also progress being made in the development of recombinant t celleinducing vaccines, with the most advanced version of this strategy demonstrated by modified vaccinia ankara (mva) viruses expressing influenza virus np and m1 antigens (berthoud et al., 2011) , with vaccinated individuals demonstrating increases in interferon-gamma (ifn-g) expressing cd8 þ t cells and increased protection against influenza infection (antrobus et al., 2012; powell et al., 2013) . co-administration of this mva-based vaccine with tiv formulations results in increased influenza strainespecific antibody responses and the generation of memory t cells that recognize a range of influenza a subtypes (antrobus et al., 2014) . additional research demonstrates production of antigen-specific t cell responses using alternate prime/boost regiments of combinations of vaccination regimens employing recombinant replication-deficient adenovirus or mva, expressing iav np and matrix protein 1 (lambe et al., 2013) . quality and clonal t cell receptor (tcr) characteristics of influenza-specific cd8 þ t cells, in addition to in silico predicted and peptide-based approaches for pools of minimal iav epitopes, are investigated for their induction of cellular immunity and recognition by cd8 þ t cells (reviewed in grant et al., 2016) . hpiv are enveloped negative-sense rna genome viruses of 150e250 nm, belonging to the large and rapidly growing paramyxoviridae family, causing significant human and veterinary disorders (henrickson, 2003) . hpiv is divided into serotypes 1 to 4, with hpiv-1 representing the most common etiologic agent of associated disease, but with hpiv-1, -2, and -3 representing common causative agents of respiratory illness in pediatric, geriatric, and immunocompromised populations (schmidt et al., 2011) . hpivs are a common cause of acute respiratory illness throughout all stages of human life (schomacker et al., 2012) , causing acute respiratory infections in children (cooney et al., 1975) . second, only to human respiratory syncytial virus, hpivs are the major contributors to hospitalization due to alris and global pneumonia mortalities in young children, and up to 80% of children are seropositive for hpivs by the age of 5 years (murphy, 1988; weinberg et al., 2009) . hpiv infections can induce potent humoral and cellular immune responses, including innate immune responses, local and systemic igg and iga responses, and adaptive cd8 þ and cd4 þ t cell responses (gitlin et al., 2010; hou et al., 1992) . though cellular responses can restrict hpiv replication dynamics and clear primary infections, neutralizing antibodies against virus envelope hemagglutininneuraminidase (hn) and fusion (f) gps are required for early infection (suzuki et al., 2001; zhang et al., 2005) and confer long-term protection against hpiv-related disorders (murphy, 1988; spriggs et al., 1987; schmidt et al., 2011) . currently, there is no vaccine to protect against human hpiv infection. progress in the development of hpiv vaccines using reverse genetics for serotypes à1 to à3 has generated several live-attenuated, intranasal hpiv vaccines evaluation in adults and in children, two of which, hpiv-3 are well tolerated in hpiv3-seronegative pediatric populations (schmidt et al., 2011) . ongoing pediatric trials testing live-attenuated hpiv vaccines for hpiv-1 and hpiv-2 predict these will replicate in the upper respiratory tract of infants to induce the full spectrum of humoral and cellular immune responses (karron et al., 2015) . heterologous (i.e., jennerian) vaccine design strategies using the sendai virus (sev) to control infections by hpivs and rsv are discussed in the following section. human respiratory syncytial virus (rsv) types a and b are found within the genus orthopneumovirus, family pneumoviridae, of the order mononegavirales. rsv is an enveloped, spherical virus of w150 nm in diameter, and reaching up to several micrometers in length (gower et al., 2005) . the negative-sense rna genome encodes for outer structural, np, polymerase, ns, transmembrane, and regulatory proteins (griffiths et al., 2017) . rsv is a major cause of alris, resulting in numerous pediatric hospitalizations globally, where by 3 years of age, most children have been exposed and are at risk of developing life-threatening bronchiolitis and pneumonia (glezen et al., 1986; hall et al., 2009; henrickson, 1994) . up to 120,000 infants are hospitalized due to rsv infection in the united states (marks, 1992) , and 34 million episodes of rsv-associated alri in children globally represent at least 3 million cases resulting in hospitalization, and approximately 199,000 associated deaths per year (nair et al., 2010) . a balance of adaptive immune ctls and neutralizing antibodies of the humoral immune response mediate protection and clearance of rsv infection (griffiths et al., 2017) . though neutrophils are the highest proportion of leukocytes found in the airways of those infected with rsv (everard et al., 1994) , and despite observations that natural killer (nk) cells are first to attain infected airways (hussell and openshaw, 1998) , it is cd4 þ helper and cd8 þ ctls that correlate with the early clearance of rsv-infected cells (anderson et al., 1990) . in infants and immunocompromised populations, fatalities resulting from rsv infection are associated with deficiencies in cd4 þ and cytotoxic cd8 þ t cells (hall et al., 1986; welliver et al., 2008) . later in infection, increases in neutralizing antibodies prevent reinfection by opsonizing viral epitopes required for rsv entry and infection, and rsv clearance can be associated with rsv-neutralizing nasal immunoglobulin a (iga) (mcintosh et al., 1978) . more recently, enhanced rsv clearance with reduced disease severity has been associated with vaccine-elicited memory cd8 þ t cells (lee et al., 2012) . while memory cd8 þ t cells can mediate protection against rsv infection, in the absence of antibodies and memory cd4 þ t cells, these cause mortality via systemic proinflammatory cytokine storms and local ifn-g production (stoley et al., 2016; schmidt et al., 2018) . other recent studies however indicate that the cd8 þ t cell response may not be the major determinant of severity of rsv-related pathology (collins and melero, 2011) . there are currently several recombinant rsv subunit vaccines in clinical trials, including the novavax rsv f vaccine representing the most promising candidate for licensing. this rsv f subunit targeting vaccine has been demonstrated to elicit the expression of circulating neutralizing antibodies against rsv (glenn et al., 2016) . toward the development of future adaptive immunity-inducing rsv vaccines, it has been demonstrated that the transfer of airway-resident t cells protect against rsv, where it has been suggested that, to lessen the burden of t cellemediated damage to airways, the induction of lung tissue t cells should be the focus of vaccine development (kinnear et al., 2018) . recently, the sev has been used as a component of a jennerian vaccine model for hpiv-1 and as a backbone for other viruses causing serious lower respiratory infections (lris), including other hpivs, rsv, and hmpv. sev-based vaccines have proven to be effective toward inducing b cell and t cell immune responses, and in the protection from hpiv-1, -2, and -3, and rsv, where they can also be used in combination with other vaccines to primeeboosts or to target one or more than one paramyxovirus pathogen (russell and hurwitz, 2016) . sev is attractive for use in human vaccination because it is a murine pathogen unable to infect humans (bousse et al., 2006) and therefore does not require attenuation as it can never revert to a human pathogenic phenotype (schickli et al., 2012) . another important feature supporting the use of sev as a pan-virus vaccination agent stems from its ability to grow transiently in mammalian cells, accommodating the endogenous expression of antigens with posttranslational modifications matching those of the target antigens and neutralizing epitopes (henrickson et al., 1991) , with endogenous expression of antigens ensuring robust activation of cd8 þ t cells able to destroy antigen-producing cells and terminate virus amplification (york and rock, 1996; russell and hurwitz, 2016) . in murine studies, sev could elicit rapid and durable respiratory mucosa and systemic hpiv-specific b cell and t cell responses (sealy et al., 2010; rudraraju et al., 2011) . clinical testing of human populations infected with rsv and hpivs is underway (adderson et al., 2015) . for a full review of current development of antiviral compounds and vaccine candidates tested against rsv, see costello et al., 2012. 2.4 adenovirus classification, epidemiology, immunology, and vaccinology human adenoviruses (hadvs) are classified in the mastadenovirus genus, containing seven known hadv species, from hadv-a to hadv-g, and with at least 57 unique known human serotypes (buckwalter et al., 2012) . adenoviruses are nonenveloped double-stranded dna viruses ranging from 65 to 80 nm in diameter and are composed of a protein capsid, a np core, and internal proteins. dna homology between hadv subgroups ranges from 48% to 99% (walls et al., 2003) . hadv infection rarely causes serious or fatal illness in immunocompetent individuals but may cause severe disease in immunocompromised, pediatric, and geriatric populations (lynch et al., 2011) . clinical disease symptoms associated with hadvs are dependent on hadv genotypes, with at least 69 recognized, and assigned to subgroups a through g. clinical symptoms include fever, rhinorrhea, pharyngitis, conjunctivitis, gastroenteritis, bronchitis, pneumonia, acute hemorrhagic cystitis, and meningoencephalitis (lynch et al., 2011) . recombination between hadvs are largely responsible for outbreaks of acute febrile respiratory disease in immunocompetent military recruits, where serotypes 4 and type 7 have been documented to account for approximately 60% of these respiratory illnesses (hilleman et al., 1957; dudding et al., 1973) , and are associated to other frequently occurring disorders including upper and lower respiratory illnesses, gastroenteritis, hepatitis, keratoconjunctivitis, meningoencephalitis, cystitis, and myocarditis in these immunocompetent populations, reviewed in lion (2014) . adenoviruses are endemic in pediatric populations (echavarria, 2008) . the incidence of adenovirus infection peaks in infants and children, where, globally, 5%e7% of respiratory tract infections in pediatric patients are ascribed to hadv (ghebremedhin, 2014) . recently, re-emergence of type 7d hadv has caused fatal outbreaks due to severe pneumonia syndromes in children from high-density populations . immune responses to adenovirus infection are dependent on primary sites of inoculation, methods of transmission, viral serotypes, and secretory ig antibody status of the infected host; igas are present in respiratory tract early following infection, and igg2 is present in serum and nasal secretions at later time points, reviewed in walls et al. (2003) . histopathological changes resulting from infection can be divided into two phases: the first phase of immune histopathology predominantly involves nonspecific, cytokine-mediated inflammatory recruitment of monocytes and macrophages, while the second phase involves t cell infiltration (prince et al., 1993) . t cellemediated immunity is believed to be required for hadv recovery from acute infections, and individuals lacking adaptive immunity are found to be at elevated risk of infection, with cd8 þ t cells as primary mediators of response to respiratory viruses, with relatively little contribution by cd4 þ cells (woodland et al., 2001) . however, following adenovirus exposure, cd4 þ t cells have been shown to be responsible for increasing proliferation status of peripheral blood mononuclear cells (pbmc), and cd4 þ t cells represent the major ctl subsets produced and recognize conserved antigens across adenovirus serotypes (flomenberg et al., 1995; regn et al., 2001) . adenoviruses have, however, evolved several hostevasion strategies, including inhibition of apoptosis, responses to ifn-g and tnf-a, and major histocompatibility complex (mhc) class i expression (mahr and gooding, 1999; wold et al., 1994 wold et al., , 1999 . the live, oral adenovirus vaccine was licensed in the 1970s for active immunization toward the prevention of febrile acute respiratory disease in military populations, where it initially reduced adenovirus-associated respiratory illnesses by over five-fold (dudding et al., 1973) . vaccine stock depletion and associated epidemics led to the manufacture of another vaccine in 2011, again denting adenovirus-associated disease burden by approximately 100-fold among recruits within the first 2 years of its introduction (radin et al., 2014) . these oral lyophilized vaccines replicate asymptomatically in the gut, inducing humoral and cell-mediated immunity, to confer longlasting protection from infection (berg et al., 2014) . due to their abilities to induce potent transgene product-specific t-and b-cell responses, adenovirus vectors are explored for use as vaccine carriers against a variety of many other pathogens (chen et al., 2010; harro et al., 2009; hill et al., 2010; radosevic et al., 2007) . alris by respiratory viruses are a major cause of morbidity and mortality, accounting for over 1.5 million deaths globally each year, and are predominantly resulting from human transmission of virus containing respiratory droplets. licensed vaccines are useful against several viruses causing these severe, often lethal, associated disorders. influenza has no borders and causes great economic burden, making it a prominent international concern. its rapid and unpredictable genetic drift causes human pandemics, where it has an annual potential of causing 5 million infections and 500,000 deaths worldwide. influenza vaccinology requiring constant yearly updates has stimulated interest in the development of universal t cell vaccines that can elicit both humoral and cellular immunity, whereby influenza-specific memory cd8 þ t cell responses against a range of influenza subtypes could be induced to clear infection in absence of neutralizing antibodies. rsv causes 34 million alris in children annually, resulting in 3 million hospitalizations and almost 200,000 deaths per year. rsv is cleared by balance of adaptive immune ctls and humoral neutralizing antibody responses, correlating most highly with cd4 þ helper and cd8 þ ctls during natural infections, and with memory cd8 þ and cd4 þ t cells following vaccination, with research endeavours targeting their strengthening by specific induction of lung tissue rsv targeting t cells. second, only to rsv, hpivs cause many ari-and lri-associated mortalities in children. hpivs can induce potent humoral, innate, and adaptive cd8 þ and cd4 þ t cell responses able to restrict their replication and where neutralizing antibodies can confer long-term protection against their associated disorders. hadvs infect both immunocompetent and immunocompromised humans and have been shown to cause up to 60% of respiratory disorders in hospitalized military personnel. despite their having evolved convoluted host-evasion strategies, adaptive t cell immunity against hadvs starts early in diseases phases and is key to recovery from acute natural infection, with its greatest contributions by cytotoxic cd8 þ t cells that require stimulating by cd4 þ t cells for their expansion. vaccines against hadvs induce both humoral and adaptive immunity, including potent transgene virus-specific t-and b-cell responses conferring long-term protection. success from hadv vaccinology has influenced explorations of adenovirus vectors as target carriers for vaccination against numerous other pathogens. diarrheal disorders remain a leading cause of morbidity and mortality worldwide, with these listed in the top five causes of death worldwide, and which are associated with global estimates at 4e6 million deaths per year; reviewed in clark and mckendrick (2004) . the majority of gastric infections are viral in origin, and viral gastroenteritis is one of the most common illnesses in all age groups and an important cause of morbidity in industrialized countries (chang et al., 2003) . the human risk of viral gastroenteritis in the united states alone is at least one per individual per year, with 450,000 adults and 160,000 children hospitalizations recorded and an associated 4000 mortalities per year (mead et al., 1999; mounts et al., 1999) . several viruses are responsible for viral gastroenteritis, where their transmission typically occurs from person-to-person by the oral-fecal route. viruses commonly causing gastroenteritis include rotavirus (rv; causing the most serious gastric disorders), norovirus, astrovirus, adenovirus, and coronavirus-like agents (table 1) . rvs are classified as a genus within the family reoviridae. these are nonenveloped viruses measuring 70 nm in diameter and have inner and outer capsids surrounding their cores containing double-stranded rna viral genomes encoding viral capsid (vp-1 to vp-6, and vp-7; vp4 outer capsid protein mediates virus attachment to cells) (bishop et al., 1973) and nonstructural (nsp-1 to nsp-6) proteins, reviewed in desselberger (2014) . rvs classify into seven serotypes (aeg), based on antigenic properties of the inner capsid vp6 protein, where subtypes aec represent human pathogens and are further subclassified into serotypes within these groups on the basis of differing outer capsid composition (anderson and weber, 2004; wilhelmi et al., 2003) . diarrhea is a major cause of death among children globally (liu et al., 2012) , and rv is the leading cause of severe diarrhea, globally causing an estimated 453,000 deaths in developing countries and 2.3 million pediatric patient hospitalizations parashar et al., 2003) . rv also represents a significant cause of disease in industrialized countries, with greater numbers of hospital admissions reported relative to developing countries (chang et al., 2003) . though group a rv causes the majority of endemic infections and can also lead to significant outbreaks in infant and geriatric populations (villena et al., 2003; marshall et al., 2003) , group b rvs are less common but can also lead to outbreaks and epidemics (sanekata et al., 2003; ahmed et al., 2004) , whereas group c rv is less often observed causing sporadic diseases. of the existing 10 g and eight p rv group a serotypes, g1 to 4, p (kostouros et al., 2003; clark and mckendrick, 2004) . studies of t cell responses to rv infection in humans have reported that most healthy adults and children have circulating rv-specific t cells, with approximately 50% of rv-cd4 þ t cells expressing the intestinal homing receptor a4b7, and with circulating rv-cd4 þ and rv-cd8 þ t cells secreting ifn-g or interleukin (il)-2 (makela et al., 2004; offit et al., 1992; yasukawa et al., 1990; rott et al., 1997; parra et al., 2014) . frequencies of circulating ifn-g þ rv t cells are comparable to those specific for other mucosal respiratory viruses (mesa et al., 2007) , but these often possess profiles of terminally differentiated effector cells that are usually associated to those unable to provide long-term immunity (parra et al., 2014) . (yen et al., 2014) . as in the case of natural neonatal rv infection, fair protection rates are achieved via humoral immunity using these vaccines. though these are unable to protect against rv reinfection, they do offer protection against severe associated clinical symptoms causing patient hospitalization. these vaccines offer both homotypic and heterotypic immunity, and protection often correlates with increases in rv typee specific igg or iga antibodies, reviewed in desselberger and huppertz (2011) . although rv vaccineeinduced humoral immunity substantially decreases disease burden, these vaccination strategies are less effective and difficult to implement in low-income countries requiring them most (patel et al., 2012) . as with natural rv infection, vaccines provide nonsterilizing immunity to children (angel et al., 2007) , where lack of establishment of long-term immunity against rv causes half of children's guardians to be at risk of becoming infected and presenting with severe associated disorders (rodriguez et al., 1987) . this further demonstrates that rv-specific t (rv-t) cells are crucial for the development of overall, long-term, protective immunity against rv (franco et al., 2006; offit et al., 1993) . indeed, in models of rv infection, vaccine-induced protective immune responses are dependent on antiviral cytokine production and by direct killing of rv-infected cells by t cell and b cell adaptive immune subsets (jiang et al., 2008; wen et al., 2016) . in addition, with observations that gut cd4 þ t cells may become tolerogenic or anergic in response to rv infection, stimulating t cells with rv antigen in the presence of il-2, il-12, or r59949, a pharmacological diacylglycerol kinase alpha inhibitor, causes increased pbmc frequencies of rv antigen-specific t effector cells, including rv-cd4 þ tnf-a þ , rv-cd4 þ ifn-g þ , and rv-cd8 þ ifn-g þ cells (parra et al., 2014) . diarrheal disorders cause an annual 4e6 million deaths worldwide. rv is the leading cause of severe diarrhea outbreaks in infant and geriatric populations, with global annual estimates of 453,000 deaths in developing countries and 2.3 million pediatric hospitalizations. most immunocompetent individuals have circulating rv-specific ctls at comparable frequencies to those elicited by other respiratory viruses, but which have terminally differentiated effector profiles rendering them incapable of conferring long-term protection against the reoccurrence of associated disorders. rv vaccineemediated protection from severe disorders is from humoral nonsterilizing immunity unable to protect against reinfection, yet vaccination programs are challenging to implement in countries requiring them the most. rv-specific t cells are key to long-term protection, and vaccineinduced protection is dependent on cytokine production and direct killing of infected cells by t cells. countermeasures against crucial helper cd4 þ t celledeveloping anergic states may assist the development of vaccines conferring long-term protection. an exanthem is a widespread eruptive skin rash that may be associated with fever or other systemic symptoms. more than 50 infectious agents causing exanthems have been identified (cherry, 1983) , where more than 70% of recorded cases of combined fever and widespread rash in pediatric populations were caused by viral infections, relative to the 20% resulting from bacterial infections (goodyear, laidler, price, kenny and harper, 1991) . correct diagnosis of these skin manifestations, resulting from direct inoculation of the infectious agent onto the cutaneous surface, or by dissemination from a distant site, is a main research theme on viral exanthems. this is because, while infections by many viral (i.e., paraviral) exanthems are benign and resolve spontaneously, others may rapidly lead to fatal conditions, reviewed in drago et al. (2017) . thus, special attention in diagnosing even vaccine-preventable viral exanthems must be applied to avoid the arising of serious complications in nonimmune pregnant women and their fetuses from the more harmful classes of viruses causing exanthems (white et al., 2012) . common exanthematous infections are typically caused by transmission of viruses from person-to-person (with exception of alphaviruses having a mosquito vector), and where a multitude of viruses are their causative agents, including rubeola virus, rubella virus, human parvovirus b19, human herpesvirus (hhv) type 6, varicella-zoster virus (vzv), variola, alphaviruses, and molluscum contagiosum virus (table 1) . numerous other exanthematous disorder causing viruses are not covered in this section, including ebola and zika, but which are becoming classified as emerging viral exanthems due to the increasing numbers of at-risk populations and the critical need to classify these diseases to minimize outbreaks and risk to pregnant women and fetuses (keighley et al., 2015) . rubeola, or measles virus (mev), belonging to the morbillivirus genus of the paramyxoviridae family, is a negative-sense rna virus having a nonsegmented genome and a lipid envelope, and measuring up to 250 nm in diameter, reviewed in griffin et al. (2012) . the 16 kb genome encodes eight proteins: the viral envelope is composed of hemagglutinin (h) and fusion (f) gps projecting from the matrix (m) protein lining its interior. the helical nucleocapsid is composed of the rna and nucleocapsid (n) protein packed within the envelope as a coil with the phosphoprotein (p) and large polymerase (l) proteins attached. the two ns proteins, c and v, regulate cellular response to infection and modulate ifn signaling (bellini et al., 1985) . humans are the only natural host of highly contagious mev virus spread by the respiratory route. despite the availability of a safe and efficacious vaccine, measles remains one of the most important viruses causing child morbidity and mortality worldwide (moss and griffin, 2006; wolfson et al., 2009) . infection by mev is associated with up to 10% of mortality rates in african children (grais et al., 2007; nandy et al., 2006) , and with 25% in unvaccinated refugee camp and virus-naive population mortalities (moss, 2007; shanks et al., 2011) . female mortality is a dominant feature disorders resulting from infection (garenne, 1994) , and many acute mortalities from secondary infections resulting from immune suppression induced by mev are also observed (beckford et al., 1985) . mev has a persistent and long latency infection period, often resulting in the development of subacute sclerosing panencephalitis (sspe) in males, causing fatal neurologic disease presenting itself many years following the original infection (bellini et al., 2005) . adaptive cellular immune responses are generally regarded as most important for clearance of mev. children with low plasma ig may recover from mev infection, while those with defects in cellular immunity develop progressive infections (albertyn et al., 2011; mcquaid et al., 1998) . mev-specific antibody and t cell responses coincide with the onset of the rash, whereby rash biopsies of mev-replicating, infected epithelial cells, have high levels of cd4 þ and cd8 þ t cell infiltrates (polack et al., 1999) . cd8 þ t cell subsets appear to be particularly important for control and clearance of infectious mev, where expanded circulating virus-specific ctls are found in the blood of patients suffering rash, and increases in cd8 þ t cells are also found in mev-induced pneumonias (jaye et al., 1998; mongkolsapaya et al., 1999; myou et al., 1993) . in addition, depending on the target tissue and cell type analyzed with regards to mev infection, though differentially rated, both cytotoxicity and ifn production have been implicated as key effector mechanisms for mev clearance (patterson et al., 2002; stubblefield park et al., 2011; finke et al., 1995) , with specific combinations of cd4 þ t cells, cd8 þ t cells, and b cells recorded as required for the control of primary mev infection (tishon et al., 2006) . protection against measles is based on mev-specific humoral, antibodybased, immunity. diagnostically, the current gold standard of protection is via quantification of neutralizing antibodies against the viral hemagglutinin (h) and fusion (f) surface gps (bouche et al., 2002; haralambieva et al., 2011; plotkin, 2010) . mev, however, triggers an aggressive immune response, involving both the humoral and cellular arms of the immune system (moss and griffin, 2012; de vries et al., 2012; buchanan and bonthius, 2012) . once measles has been cleared, it is memory t cells that can provide lifelong immunity against reinfection by mev (bester, 2016) . importantly, during mev infection, immune reactions to other pathogens are suppressed from weeks to years, leading to risk and susceptibility to secondary infections, and which is believed to be a driver of complications and mortality long after measles had been cleared. conversely, this measlesinduced immune "amnesia," sometimes disabling immune memory for up to 3 years, has been suggested to work toward herd protection against other infections and is supported by the association of measles vaccination with lowered mortality rates from other childhood infections (mina et al., 2015) . occasional spontaneous tumor regressions have also been observed to occur during natural measles infection, suggesting that mev infection may be adopted in the generation of safe and effective oncolytic viruses (russell and peng, 2009 ). rubella virus belongs to the togaviridae family and is the sole member of the rubivirus genus. rubella contains a single-stranded, positive-sense rna genome (frey, 1994) , and its viral particles measure between 50 and 85 nm in diameter (oshiro et al., 1969) and have a pleomorphic nucleocapsid surrounded by a host-derived lipid membrane (battisti et al., 2012) . the e1 and e2 rubella protein spikes are anchored to the external layer of the membrane, with membrane-bound e2 proteins bridging rows of e1 proteins, considered as the main immunodominant antigens responsible for controlling receptor-mediated endocytosis (petruzziello et al., 1996; katow and sugiura, 1985) . antibody levels against the neutralizing domain of e1 correlate with protection against rubella virus (mitchell et al., 1996; cordoba et al., 2000; wilson et al., 2006) . rubella virus is spread from person-to-person via the respiratory route and is the causative agent of rubella disease, commonly known as german measles (lambert et al., 2015) . although rarer in the united states, rubella infection remains a major health concern in developing countries (tosh et al., 2009) . although acquired rubella infection is not severe in adults, transplacental transfer of the virus to the developing fetus during maternal viremia can cause devastating consequences of congenital rubella syndrome (crs) (watson et al., 1998) , where more than 100,000 infants worldwide are born with crs each year . common crs symptoms include spontaneous abortion, premature delivery, fetal death, ocular abnormalities, neurological problems, abnormal cardiac development, and deafness (white et al., 2012) . congenital malformations due to crs may be present at birth, while other conditions such as diabetes mellitus, deafness, intellectual disability, and/or subacute encephalitis may develop months to years later (watson et al., 1998; white et al., 2012) . from mass immunization programs, the number of rubella cases has progressively declined and was no longer endemic in the united states as of 2004 but remains endemic in other countries, with a dramatic increase in reported cases the last decade (reef et al., 2011) . recently, africa and asian have seen 20-fold increases in rubella cases, representing a significant proportion of the over 121,000 global cases reported, but where neither of these regions has immunization policies in place to control rubella outbreaks (white et al., 2012) . a recent, 2013 rubella epidemic in japan reporting over 11,000 cases, with at least 13 crs cases (minakami et al., 2014) , has also served to demonstrate that partial vaccination strategies can lead to major outbreaks. in this case, vaccination was only provided to young women, while outbreaks affected the adult male populationsda phenomenon which has also been observed in other countries applying such vaccination strategies (paradowska-stankiewicz et al., 2013; janta et al., 2012) . once measles is cleared, memory t cells can provide lifelong immunity to mev (bester, 2016) , and distinct patterns of cellular immunity to rubella virus are observed and related to the time elapsed following vaccination (lambert et al., 2015) . predominant biomarkers of early cellular measles immunity are characterized by an immunosuppressive phenotype, with increases in il-10 and tnf-a and decreases in ifn-g and proliferative properties of circulating peripheral lymphocytes (pukhalsky et al., 2003) . late immunity is shifted to predominantly proinflammatory cytokine profiles via increased concentrations of il-6, granulocyte-macrophage colony-stimulating factor, and tnf-a, in combination with decreases in il-10 (dhiman et al., 2010) . human leukocyte antigens (hlas), known to play critical roles in immune response to viruses, contribute to the heterogeneity of the immune response to rubella virus as a result of their polymorphic nature, whereby hla class i and ii polymorphisms restrict the available repertoire of rubella antigens presented to t cells and therefore influence the subsequent immune response (mitchell et al., 1996; ou et al., 1994 ou et al., , 1998 . current efforts are placed on deciphering the immunogenetics of antirubella humoral and cell-mediated immune responses, with a focus on better understanding hla polymorphisms toward the development of vaccine candidates that utilize constructs comprised of hla-specific epitopes that can induce immunity across heterogenetic populations, reviewed in lambert et al. (2015) . both natural infection and vaccines induce humoral and cellular immune responses conferring protection against rubella (tosh et al., 2009) . while humoral responses have been conventionally used to measure and record protective immunity in human populations, cellular immune responses are intrinsic to humoral immunity (bautista-lopez et al., 2000; horstmann et al., 1985; ovsyannikova et al., 2004; nepom et al., 1997; vesikari et al., 1975; akaboshi et al., 2001; farzaneh et al., 2003) . since its induction into healthcare systems, immunization with live attenuated rubella virus vaccine has been demonstrated to be safe and effective at preventing infection, crs, and to interrupt endemic rubella transmission (lambert et al., 2015) . the live attenuated rubella vaccine strain ra27/3 has a proven track record for safety and immunogenicity efficacy (hilleman et al., 1968; plotkin, 1979) , where single doses have been demonstrated to potently induce humoral immunity and lifelong protection against infection, and where the vaccine has also been demonstrated to boost previously immunized persons (diaz-ortega et al., 2014) . from their safety and efficacy, use of recombinant rubella vectors has also been tested toward enhancing immune responses against siv and hiv epitopes, where increases in memory b cell repertoires have been observed upon re-exposure to rubella vectors (virnik et al., 2013) . durable hiv-specific cellular immunity has been observed from rubella vector boosting, with cytotoxic antigenspecific responses by central and effector memory cd4 þ and cd8 þ t cell subsets (rosati et al., 2015) . vzv, also known as hhv-3, is a virus of the varicellovirus genus from the herpesviridae family. humans are its only vector (hambleton and , where it specifically infects t cells, epithelial cells, and ganglia (gershon et al., 2015) . vzv viruses have diameters measuring up to 200 nm and are encoded by a linear double-stranded dna genome consisting of approximately 125 kb and encoding at least 70 unique genes, with all but the exception of 6, having homologs in herpes simplex virus (cohen, 2010) . vzv virions are composed of the viral dna, the capsid, the tegument surrounding the capsid, and the envelope surrounding the tegument and which incorporates the major viral gps (arvin, 1996) . during lytic infection phases, vzv produces at least 12 gps expressed on both virions and human cell surfaces. during this process, and which is common to other herpesviruses, gene expression is believed to proceed in an orderly cascade of immediate early genes, early genes, and late genes. during latent vzv infection, gene expression is restricted until reactivation for additional rounds of lytic infection (gershon and gershon, 2010) . vzv has extraordinarily high transmission rates and is highly communicable via the airborne transmission route, with concentrated virus coming from vesicles shedding from skin lesions, leading to cell-free contagious airborne viruses, and as evidenced by the fact that infected children without skin lesions are not contagious (tsolia et al., 1990; chen et al., 2004) . primary vzv infection causes varicella, also commonly known as chickenpox. as cellular immunity to vzv wanes in the elderly and immunocompromised populations, latent vzv becomes reactivated and causes zoster (i.e., shingles, herpes zoster), which is usually associated with chronic pain but also numerous other serious neurological and ocular disorders, as well as multiple visceral and gastrointestinal disorders, including ulcers, hepatitis, and pancreatitis (gershon et al., 2015; gilden et al., 2010) . available antiviral drugs and vaccines against varicella and zoster are safe and effective for treatment and prevention strategies (gershon and gershon, 2010) . varicella is globally endemic and is transmitted year-round, with frequent epidemics occurring every 2 to 3 years. outbreaks most commonly occur in nurseries and schools, in hospitals and other medical institutions, and in refugee camps and military and correctional facilities (izurieta et al., 1997; levy et al., 2003; longfield et al., 1990) . although it can often be a self-limiting disease, varicella can also result in death, where in developed countries, an estimated 5 of 1000 patients are hospitalized with serious complications, with up to three deaths per 100,000 patients (galil et al., 2002; rawson et al., 2001) . complications from varicella requiring hospitalization include bacterial superinfections of the skin, blood, bones, and lungs, as well as encephalitis and hemorrhagic manifestations in pediatric and immunocompromised populations (gershon et al., 2015) . importantly, acquiring vzv during early pregnancy often results in severe congenital defects in 1% of newborns (enders, 1984) . vzv is a great example of success through herd vaccination programs for children, dramatically influencing its epidemiology, and causing 95% declines of hospitalization cases in the united states (gershon et al., 2015) . following its transmission to the respiratory mucosa, vzv proliferates in the oral pharynx, where it infects human tonsillar activated memory cd4 þ t cells and induces their tissue-homing properties (sen et al., 2014) . vzv can be propagated to t cellerich regional lymph nodes for rapid proliferation and is then disseminated by the circulation to infect dermis, epidermis, and other organs (ku et al., 2002 (ku et al., , 2004 . lymphopenia is typically observed in patients during viral incubation, followed by an increase in leukocyte counts, correlating with the onset of rash until the resolution of viremia. during infection, vzv can be recovered from pbmcs in children exhibiting rash (ozaki et al., 1984; koropchak et al., 1992; sawyer et al., 1992) , is extensively observed in thymic lymphocytes (levin, 2014) , and observed in all t cell subsets examined (moffat et al., 1995) . though innate skin immunity can cause delays in multiplication of skin-bound vzv while the adaptive immune system mounts an attack, however, aggressive vzv replication in the skin results in characteristic varicella rash (ku et al., 2004) . high vzv titre-skin vesicles from rash provide cell-free virus for person-to-person transmission (chen et al., 2004) . vzv also latently infects neurons of cranial nerve ganglia, dorsal root ganglia, and enteric and autonomic ganglia (gershon and gershon, 2010) . vzv reactivation causes ganglia to become necrotic and hemorrhagic (head et al., 1997) , with vzv proteins found in neurons and non-neuronal cells, and where this vzv-induced ganglionitis is often also marked by the upregulation of mhc class i and ii proteins associated infiltration of cd4 þ and cd8 þ t cells (schmidbauer et al., 1992; steain et al., 2014; gowrishankar et al., 2010) . before vzv vaccines became available, approximately 30% of infected adults later developed shingles (yawn et al., 2007) . the single dose, lyophilized, live, attenuated vzv vaccine (i.e., zoster vaccine live (zvl), zostavax, merck) is indicated for prevention of latent vzv reactivation leading to shingles in individuals older than 50 years. zvl is licensed in over 55 countries, with 34 million distributed doses globally (willis et al., 2017) , and which has associated efficacy rates of over 50% in all ages tested (oxman et al., 2005; schmader et al., 2012) ; consistent with original clinical trial datasets (tseng et al., 2011; langan et al., 2013; marin et al., 2015) . however, increases in vzv susceptibility have arisen due to increasing aging populations, and in immune-suppressed organ transplant recipients, chemotherapy patients, hiv-infected individuals, and those suffering from chronic illnesses (forbes et al., 2014) . in these patients, earlier exposure to exogenous vzv protects against shingles by boosting cellular immunity (arvin et al., 1983; thomas et al., 2002) . the memory immune response following naturally acquired primary vzv infection is characterized by vzv igg and iga antibodies, as well as vzv-specific cd4 þ and cd8 þ t cells, where vzv-specific igg antibodies bind many vzv proteins and mediate virus neutralization and antibodydependent cytotoxicity, reviewed in arvin (2008). the frequency of vzv-specific memory proliferating t cells is estimated to be approximately one in 40,000 pbmc (hayward et al., 1986) . vsv-specific memory cytotoxic mhc class i-or class ii-restricted t cells producing ifn-g and tnf-a can recognize the vzv ge, gb, gc, gh, gi, ie62, and ie63 proteins and can be found to persist for over 20 years after varicella exposure (jenkins et al., 1998; huang et al., 1992; asanuma et al., 2000; diaz et al., 1989; hayward et al., 1986; sharp et al., 1992; sadzot-delvaux et al., 1997) . the ability of the live attenuated varicella vaccine to elicit vzv-specific igg and t cell immunity in naive hosts was established during its prelicensing clinical evaluations (gershon et al., 1992) , and where, as expected from its design, the magnitude of these vzv-specific immune responses correlated with infectious virus content and with antigen content of individual vaccine formulations (bergen et al., 1990; watson et al., 1993) . importantly, it was later discovered that providing two doses to children resulted in higher igg antibody titres and increased t cell proliferation and where experimental evidence suggesting that memory responses were sustained more effectively from such regimens (watson, 2008) . these observations led to the more recent recommendation of implementing of a two-dose regimen of varicella vaccine for all vaccine recipients (arvin, 2008) . studies of how regimens affect long-term protection by the adaptive t cell immune response to vaccination, as exemplified by vzv vaccination studies, have the potential to modify dosages and timelines to maximize overall and persisting beneficial long-term effects from vaccination against many other viruses. historically, smallpox was a severe human disease caused by the variola virus (varv), which was both highly lethal and highly contagious prior to its eradication from human populations in 1980 (moore et al., 2006) . varv belongs to the genus orthopoxvirus of the family poxviridae, which also includes zoonotic species: vaccinia virus (vacv), monkeypox virus, cowpox virus, and camelpox virus (shchelkunov, 2013) . orthopoxviruses are enveloped, brick-shaped viruses measuring 350 by 270 nm, and containing a double-stranded dna genomes encoding 150e200 genes, and measuring approximately 200 kb (garon et al., 1978) . unlike other dna viruses, these replicate as 'virus factories' in the cytoplasm of infected cells (pauli et al., 2010) . varv encodes approximately 200 proteins, where over 80 of these are found at terminal regions of the genome and are associated with host immune evasion. the origin of smallpox is unknown, but varv is considered to be one of the most deadly diseases of human history, decimating populations to such an extent that it significantly altered the course of human civilizations. smallpox is believed to have first appeared in 10,000 bc in africa, with the oldest credible confirmation found in sanskrit writings from 1500 bc and where smallpox lesions are believed to be observed on the mummified egyptian ruler ramses v (1100 bc) (ristanovic et al., 2016) . prior to its eradication in 1980, varv circulated in the human population for many centuries and repeatedly caused large-scale epidemics. in the 18th century for instance, smallpox caused the death of more than 400,000 europeans per year (babkin and babkina, 2015; smith and mcfadden, 2002) . despite varv eradication from the human population more than two decades ago, fears about its potential re-emergence or the threat of its use as a potential bioterrorism agent have not subsided. this has led to numerous debates concerning the destruction of existing viral stocks, currently maintained in the united states and russia. destruction of these stocks has been postponed for the benefit of further research elucidating varv mechanisms of pathogenesis toward the design of therapeutics as well as on efficacious vaccine strategies that may be required for potential future outbreak (smith and mcfadden, 2002; stone, 2002) . immune-evasion mechanisms by varv are the least understood among the orthopoxviruses due to difficulties of finding an appropriate host animal model (turner and moyer, 2002) , along with limited availability of authentic variola proteins since its eradication (massung et al., 1993) . thus only two variola proteins, namely smallpox inhibitor of complement enzymes (spice) and vaccinia virus complement control protein (vcp), have been characterized and are similar in structure (dunlop et al., 2003) . these viral antigens regulate the human complement system (yadav et al., 2008) , are important for stimulating innate immunity, and also have important features for adaptive immunity, shown to bolster antiviral t cell responses including ifn and cytokine expression (noris and remuzzi, 2013; moss and shisler, 2001) . vacv has been used more extensively for human immunization than any other vaccine and what was employed to provide cross-protection against varv toward smallpox eradication (jacobs et al., 2009 ). the first generation vacv/varv vaccines produced in the 1970s and 1980s (dryvax, apsv, lancyevaxina, l-ivp) contained live vacv , and induced robust humoral immunity is characterized by high antibody titers, neutralizing and opsonizing viral particles, fixing complement, hemagglutination, and antibody-dependent cell cytotoxicity (amanna et al., 2006; panchanathan et al., 2008) . these vaccines have since been observed to generate adaptive immune responses over many concentrations (frey et al., 2002; rock et al., 2006) , including the secretion of effector cytokines (e.g., ifn-g) and the lysing of infected cells (amanna et al., 2006; hammarlund et al., 2003) . most second-generation vaccines created for biodefense contain replication competent viruses (artenstein and grabenstein, 2008) and have comparable efficacies to dryvax. thirdgeneration vaccine formulations using attenuated vacv strains (lc16m8, mva, nyvac, dvvl) have increased safety profiles (artenstein, 2008; kennedy et al., 2009) . proof that adaptive cellular immunity is essential in preventing the spread of varv following immunization and in its generating overall protective immunity against smallpox comes from observations that individuals having t celledeficiency disorders suffered serious and sometimes fatal infections after vaccination, but that agammaglobulinemic children were not at risk of these adverse complications (rock et al., 2006) . varv vaccine induces strong cd4 þ and cd8 þ t cell responses, peaking after immunization and then contracting to provide stable memory t cell populations that remain detectable for decades (amanna et al., 2006; hammarlund et al., 2003) and with memory cd4 þ t cells persisting the longest (amara et al., 2004) . defects in cellular immunity lead to uncontrolled vaccinia infection (lane et al., 1970) , where cd4 þ and cd8 þ t cells are able to prevent mortality of b celledeficient animals infected with vacv (belyakov et al., 2003) and where cd4 þ t cells have the most protective overall effects (xu et al., 2004) , and are essential for optimal ctl function and memory formation (sun and bevan, 2003; kennedy et al., 2009) . vacv-specific cd4 þ and cd8 þ t cells recognize a diverse array of viral proteins, and cd8 þ t cell epitopes are predominantly found in early, non-structural genes and transcription factors (terajima et al., 2008) . cd4 þ t cell epitopes are from late viral products including membrane, structural proteins, and replicative enzymes (jing et al., 2008) , and linkage of b cell and cd4 þ t cell epitopes to varv proteins suggests t helper celleb cell interactions are those required for generation of robust vacv-specific antibody responses (sette et al., 2008) . in humans, varvspecific cd4 þ and cd8 þ t cells have been observed to persist for over 75 years following immunization (rock et al., 2006) . exanthem disorders by viruses represent more than 70% of cases of combined fever and widespread rash in pediatric populations, and their correct diagnosis is especially critical for the distinguishing of benign versus lethal viral strain variations that can cause lifelong morbidities in children born from infected mothers. mev is transmitted via human respiratory routes, and despite vaccine availability, still causes 10% of african children mortalities, and severe risk of sspe-derived fatalities years later in survivors. historically, in common with many other viruses, the gold standard diagnostic of protection is made by quantification of humoral neutralizing antibodies. adaptive cellular immune responses are, however, those most critical for mev clearance, where mev-specific t cell responses coincide with rashes densely infiltrated by cd4 þ and cd8 þ t cells. combinations of cd4 þ t cells, cd8 þ t cells, and b cells control primary infection, where cd8 þ t cells dominate for control and clearance, and memory t cells are able to provide lifelong protection. mev infection induces general longterm immunosuppression leading to vulnerability to other pathogens causing secondary infections, but this immunosuppression is believed, by some, to be simultaneously conferring herd protection and have been observed to induce spontaneous tumor regression. rubella virus infection has progressively declined from immunization programs but continues to be endemic in many countries, as a result of complete absence of or problematic or partial vaccination programs, still causing severe crs cases in 100,000 infants worldwide, per year. while humoral responses are conventionally used to measure protective immunity, it is adaptive immunity that confers protection. both natural infection and vaccination induce humoral and cellular immune responses, where memory t cells can provide lifelong immunity, with presence of cytolytic t cell biomarkers from vaccine-induced immunogenicity. vaccines in development can comprise hla-specific epitopes inducing immunity across heterogenetic populations. since rubella vaccines can boost the previously immunized, their vectors are being investigated for use toward immunization programs for unrelated viruses. vzv has extraordinarily high human transmission rates. primary vzv infection causes varicella, and before vaccination programs were initiated, would re-emerge from declines in adaptive immunity to cause zoster in 30% of in immunocompromised populations to cause the hospitalization of 1 of every 200 and the death of three per 100,000 patients. vzv represents a poster child of herd vaccination programs that led to 95% declines in hospitalization events. vzv infection rates are again on the rise in immunocompromised and immune-suppressed populations. it infects human tonsillar activated memory cd4 þ t cells that home to the lymph to then infect cd4 þ and cd8 þ t cells, followed by a lymphopenia resolved at rash onset. innate immunity controls vzv spread until adaptive immunity develops to fully counter the infection. vsv-specific memory ctls persist 20 years after varicella exposure, and observations that increased t cell proliferation with better-sustained memory responses result from multiple booster doses of vaccine have caused modifications in vaccination programs. smallpox by varv was one of the most deadly diseases in human history, causing more than 400,000 european casualties annually prior to its vaccine-mediated eradication. viral stocks are maintained from the necessity of developing new vaccines to counter potential future re-emergence of varv from natural-or bioterrorism-derived sources. characterized variola proteins amplify and strengthen t cell responses. first-generation vacv vaccine induced robust humoral immunity and adcc, in addition to generating adaptive immune responses marked by cytokines and cell lysis. varv vaccine induces strong initial effector cd4 þ and cd8 þ t cell responses having b cell linkage, then contacting to generate stable memory populations of varv-specific cd4 þ and cd8 þ t cells that can persist for over 75 years. accordingly, second-and third-generation vaccines created for biodefense are designed to stimulate adaptive cellular immunity. globally, liver cancer is the fifth most common of cancers, with an average of 374,000 cases per year, representing 7.2% of all cancers, and with mortality rates reflecting geographic incidence rates. almost 85% of liver cancers occur in developing countries, with over 20 of 100,000 individuals affected by these diseases. hepatocellular carcinoma (hcc) is the most common form of liver cancer, and approximately 80% of cases are associated with chronic infection by hepatitis b virus (hbv) or hepatitis c virus (hcv) (el-serag, 2012) . hepatitis viruses are so named because they display hepatotropism by preferentially infecting hepatocytes to cause liver inflammation, also known as viral hepatitis. infection by hbv and hcv promotes liver cirrhosis in most affected, leading to the development of hcc in up to 30% of patients (fattovich et al., 2004) . approximately 5% of the global population (350e400 million people) are chronically infected with hbv and strong correlations between hbv prevalence and hcc incidence and mortality. chronic hbv infection accounts for approximately 50% of hcc cases in adults and for all hcc cases in children (el-serag, 2012) . hepatitis transmission is from person-to-person contact with infected blood or body secretions or by the fecal-oral route and involving at least five specific viruses, namely hepatitis a, b, c, d, and e viruses (table 1) . infectious viral hepatitis is an important challenge to health worldwide: hepatitis a virus (hav) and hepatitis e virus (hev) are acute and endemic in many low-income countries, usually causing self-limiting hepatitis, whereas hbc and hcv also cause acute illness but usually lead to chronic and progressive liver fibrosis, cirrhosis, and an increased risk of hcc (stanaway et al., 2016) . hbv is controlled in adults but is chronically persistent from neonatal infection (shin et al., 2016) . hepatitis viruses differ in their virology. hbv is an enveloped dna virus that belongs to the hepadnaviridae family. it contains a 3200 bp, partially double-stranded relaxed-circular dna genome that is reverse transcribed via a pregenomic rna intermediate and encodes four overlapping open reading frames, which are translated to produce viral core protein, surface proteins, reverse transcriptase, and hbx (nguyen et al., 2008) . transmission of hbv results from exposure to infectious blood or body fluids containing blood, and hbv can integrate into the human genome, contributing to its genomic instability and ultimately to hcc (zhao et al., 2016) . hcv is also transmitted by infected blood; but unlike hbv, hcv does not integrate into the host genome . hcv is also a positivestranded rna virus but is classified in the hepacivirus genus within the flaviviridae family. its genome is 9.6 kb in length, includes an internal ribosome entry site, and encodes structural and ns proteins. the structural proteins form the viral particle and include the core protein and the envelope gps e1 and e2. the ns proteins include the p7 ion channel, the ns2-3 protease, the ns3 serine protease and rna helicase, the ns4a polypeptide, the ns4b and ns5a proteins, and the ns5b rnadependent rna polymerase (moradpour et al., 2007) . hepatitis d virus (hdv) is also transmitted by contact with infected blood or other body fluids. hdv is an enveloped, negative sense, singlestranded, closed circular rna virus, and requires hbv coinfection for its propagation, where infection with both viruses commonly results in severe liver pathologies. hdv genomic rna of hdv is composed of approximately 1700 bp, packaged with approximately 200 molecules of hepatitis delta antigen to form viral particles. hdv envelope surrounding its genome and hdag protein is composed of the three hbv small, medium, and large hbv hbsag envelope proteins. hdv also does not encode its own replicase or polymerase, and rather utilizes host cellular machineries for its replication (abbas and afzal, 2013) . hav and hev are positive-stranded nonenveloped rna viruses transmitted via the fecal-oral route, and unlike chronically persisting hbv and hcv, are typically cleared after acute infection of immunocompetent individuals (park and rehermann, 2014). hav is a hepatotropic virus belonging to the hepatovirus genus within the picornaviridae family. its genome consists of approximately 7500 bp and encompasses a single open reading frame coding for a single polyprotein, which is post-translationally processed into structural and ns proteins. the structural proteins of hav are divided into the polypeptides vp1, vp2, vp3, and vp4, forming the icosahedral capsid of the virus. ns proteins 2b, 2c, 3a, 3b, 3c, and 3d are involved in rna replication and viral polyprotein processing (martin and lemon, 2006) . hev of the family hepeviridae and genus orthohepevirus has a 7.2 kb genome having three opening reading frames encoding for the viral replicase, the capsid protein, and a small phosphoprotein required for the secretion of viral particles (debing et al., 2016) . hav and hev are waterborne viruses that usually cause acute hepatitis without progressing to chronic liver disease (joon et al., 2015) , where annually, over 100 million cases of hav and 28 million cases of hev infections have been recorded globally (makiala-mandanda et al., 2017) . hev outbreaks are reported in africa nearly every year, with some involving over 10,000 cases (kim et al., 2014) . hav is highly endemic in africa, infecting most children, conferring long-term immunity to reduce serious epidemics (jacobsen, 2014) . hbv, hcv, and hdv can be sexually, parenterally, or vertically transmitted and usually evolve into chronic hepatitis, liver cirrhosis, and hcc causing high morbidity and mortality rates, where globally, over 350 million people are chronically infected with hbv, 150 million with hcv, and 15 million with hdv (kramvis and kew, 2007; hughes et al., 2011; thursz and fontanet, 2014) . superinfection of hbv patients with hdv frequently accelerates the progression of hbv disease to liver cirrhosis, considerably increasing the burden of chronic liver disease (hughes et al., 2011) . hav, hbv and hcv are responsible for the majority of viral hepatitis cases, and there are similarities and differences in immune responses to infections by these three viruses, possibly explaining the distinct disease courses and outcomes of each hepatitis virus infection (shin et al., 2016) . type i and iii ifns, major components of the antiviral innate immune system, induce the expression of ifn-stimulated genes (isgs), observed to be much more highly induced by hcv than hav, and not at all by hbv, indicating that this virus is not recognized by the innate immune system (su et al., 2002; lanford et al., 2011; wieland et al., 2004) . another component of the innate immune system, nk cells, are also believed to be responsible for protection against hcv, where increased nk cells in protected individuals coincide with increased ifn-g and cytotoxicity (shin et al., 2016) . though virus-specific antibodies are produced by all viral hepatitis infections, these have differing roles according to the hepatitis virus infection. hav-specific antibodies with virus-neutralizing activity are induced by natural infection and vaccine immunization and confer lifelong protective immunity (walker et al., 2015; martin and lemon, 2006) . hbv surface antigen hbsag-specific antibodies are induced by infection and immunization with the recombinant protein and have virus-neutralizing activity conferring protective immunity (guidotti and chisari, 2006) . hcv-specific antibodies produced after infection do not offer long-term protection as these do not persist, are subject to loss of neutralizing activity from virus mutation, and are ineffective for cellto-cell hcv transmission (takaki et al., 2000; dowd et al., 2009; timpe et al., 2008) . t cells play critical roles during acute hcv and hbv infections, where robust and multiple epitope-specific cd8 þ t cell responses are assisted by cd4 þ t cells for spontaneous resolution of infection (shin et al., 2016) . this is supported by observations that the depletion of cd4 þ or cd8 þ t cells in chimpanzees delays rapid clearance and recovery from infection by these viruses (grakoui et al., 2003; thimme et al., 2003) . when hcv and hbv infections become chronically persistent, virus-specific t cells become exhausted and functionally impaired. in acute hcv infection, virus-specific t cells are only detected in the blood and liver after 8 weeks postinfection, and their appearance coincides with large declines of virus titres shin et al., 2006 shin et al., , 2011 . hbv virusespecific t cell responses are also important for spontaneous resolution of hbv infection, where their responses are observed to be vigorous, broad, and polyclonal in patients resolving primary infections and where their absences are associated with prolonged infection and delayed viral clearance (chisari et al., 2010; thimme et al., 2003) . cd8 þ t cells also play important roles in hav infection, where these have been observed to target multiple epitopes of hav, despite more recent results suggesting that hav is controlled by virus-specific cd4 þ t cells and not cd8 þ t cells (walker et al., 2015; shin et al., 2016) . finally, hepatitis virus infection results in liver injury, not directly caused by these viruses but rather by immunemediated mechanisms (guidotti and chisari, 2006) . liver injury biomarkers correlate with acute hav, hab, and hac infection (guidotti and chisari, 2006; park and rehermann, 2014; walker et al., 2015) and may result from cytotoxic activity of cd8 þ t cells, believed to induce apoptosis of hepatocytes in close proximity to their targeted cells (guidotti and chisari, 2006) , by il-22 producing th17-differentiated t cells, and by recruitment of nonspecific mononuclear cells by hbvspecific cytokine secreting cd8 þ t cells (iannacone et al., 2007; shin et al., 2016) . effective vaccines controlling hav and hbv have been available for over 2 decades, and an hev vaccine has also been licensed for use in china since 2011 (zhu et al., 2010; stanaway et al., 2016) . neonatal hbv vaccination has proven to be highly effective in inducing protective antibodies and preventing perinatal and horizontal transmission of hbv (lee et al., 1991) . however, observations that hbsag-specific ifn-g-or il-5-secreting pbmcs are absent in many adolescents suggest that booster vaccines should be administered to provide continued hbv immunization (lu et al., 2008) . hav vaccination provides long-term immunity in the general population and in immunocompromised patients infected with hiv (crum-cianflone et al., 2011). there is no existing vaccine for hcv, despite ongoing efforts toward their design and testing for their ability to generate prolonged cellular and humoral immune responses, reviewed in naderi et al. (2014) . in the absence of a vaccine, progress in hcv treatment includes oral treatments achieving cure in most patients, including those previously considered as difficult to treat cases (poordad et al., 2013; lawitz et al., 2013) . liver cancer is the fifth most common cancer, representing 7.2% of all cancers, with 374,000 annual cases from which 20 in 100,000 mortalities occur. hcc is the most common liver cancer, with 80% of cases resulting from chronic infection by hbv or hcv, with a significant 350 and 150 million chronically infected, respectively. in contrast, hav and hev cause acute hepatitis but do not progress to chronic liver diseases, and hdv infection depends on pre-existing hbv infection. hcv induces the expression of type i and iii isgs and nk cells, not at all present from hbv infection unrecognized by innate immunity. virus-specific antibodies are produced by all viral hepatitis infections but have differing roles across infections. robust and multiple epitope-specific cd8 þ t cell responses and dominant but depend on assistance from cd4 þ t cells for resolution of acute hav, hbv, and hcv infections. in chronic infections, cytolytic t cells either cause extensive liver injury to hepatocytes and/or become tolerant and functionally impaired. effective vaccines controlling hav and hbv provide protective antibodies. hav vaccination provides longterm immunity to the immunocompromised, but booster vaccination programs are required for persistence of hbv immunization. no vaccine is licensed for highly variable and rapidly mutating hcv, despite numerous ongoing efforts to generate those which will provide robust cellular and humoral immune responses. historically, the central nervous system (cns) has been considered to be an immunologically privileged site within the body (bailey et al., 2006; galea et al., 2007; engelhardt, 2008; prendergast and anderton, 2009 ). by definition, immunologically privileged sites, also including the brain, cornea, testis, and pregnant uterus, have a reduced or delayed ability to reject foreign tissue grafts compared with conventional sites within the body, such as skin (streilein, 2003; bailey et al., 2006; carson et al., 2006; mrass and weninger, 2006; kaplan and niederkorn, 2007) . though the cns is protected by a highly complex barrier system, a wide variety of viruses still manage to gain access to it and induce diseases. due to their sizes and tissue penetration strategies, the number of cns viral infections outweigh bacterial, fungal, and protozoa cns infections combined (romero and newland, 2003) . following cns infection, inflammatory events can arise in distinct anatomical regions such as the meninges (meningitis), brain (encephalitis), and spinal cord (myelitis) or can also simultaneously arise in multiple regions (meningoencephalitis, encephalomyelitis). for many neurotropic viruses, viral cytopathology plays a major role in cns dysfunction, reviewed in swanson and mcgavern (2015) . virus can breach the protective barriers of the cns in many ways, with the main route mechanism being via the blood, where inhaled or ingested viruses can move past the mucosa to establish infection in secondary lymphoid tissues and later be shed into circulating blood to cause broad systemic infections (swanson and mcgavern, 2015) . the cns parenchyma is protected from a plethora of agents carried in the circulation via an elaborate network called the blood-brain barrier (bbb) and the blood-cerebrospinal fluid barrier (ransohoff et al., 2003) . viruses have evolved and adapted to overcome these barriers (mcgavern and kang, 2011) , where some viruses can infect vascular endothelial cells, permitting direct passage across the bbb into the cns (verma et al., 2009; moses et al., 1993; coyne et al., 2007) . in addition, parts of the cns that are not completely protected by the bbb permit more rapid entry of several viruses (van den pol et al., 1999; wolinsky et al., 1974) . infected hematopoietic cells in circulating blood can also serve as "trojan horses" that can transport undetected virus into the cns (clay et al., 2007; tabor-godwin et al., 2010) . other mechanisms can include systemic viral infections leading to massive systemic inflammation and an ensuing bbb breakdown which opens the floodgates to cns infection by a variety of otherwise restricted infectious agents (arsenio-nunes et al., 1975; eugenin et al., 2006) . there are more than 30 recognized distinct virus strains that cause human neurological disease, and the majority of documented cases are caused by viruses that are transmitted to humans by blood-eating arthropod vectors, also known as arboviruses, that are mainly transmitted by mosquitoes and ticks and include polioviruses (pvs), alphaviruses, mosquito-borne flaviviruses, tick-borne orthobunyaviruses, mosquito-borne mammarenaviruses, and rabies virus (rabv; table 1 ). pv, the causative agent of poliomyelitis, more commonly referred to as polio, is a human enterovirus and member of the family of picornaviridae. typically spherical, nonenveloped picornaviruses range in diameter between 27 and 30 nm and have a positive-strand rna of 7000e9000 nucleotides, translated into a polyprotein (i.e., vp4-vp2-vp3-vp1-2a-2b poliovirus 2c-3a-3b-3c-3d), which yields 11 proteins upon its cleavage by viral proteases. picornavirus replication occurs in the cytoplasm of infected cells in association with intracellular membranes, where virions are released by cell lysis, ultimately killing cells and causing extensive damage to tissues. the host immune response against picornaviruses includes cytokine release, antibody production, and ctl activation, reviewed in dotzauer and kraemer (2012) . the viral genome of pv is a single-stranded rna of approximately 7500 nucleotides, enclosed in a nonenveloped capsid comprising 60 copies of four different polypeptides arranged with icosahedral symmetry (racaniello, 2006) . all three pv serotypes cause paralytic disease, and cd155 is the cellular receptor for all three serotypes, whereby pv interaction with cd155, expressed by many different cell types, leads to a conformational change of the virus particle and the following release of the rna genome into the cellular cytoplasm (mendelsohn et al., 1989; hogle, 2002) . once in the cytoplasm, the viral rna genome is translated, and the production of new infectious virions begins. pv infection results from ingested virus that replicates in the oropharyngeal and intestinal mucosa (sabin and ward, 1941) . from the primary sites of multiplication in the mucosa, pv virus drains into cervical and mesenteric lymph nodes and then into the blood, causing transient viremia symptoms. the person-to-person transmission of pv virus is through the fecal-oral route. once pv is shed in the feces, the majority of the natural human infection ends at this stage with a modest symptoms including sore throat, fever, and malaise (dotzauer and kraemer, 2012) . however, in 1%e2% of pv-infected individuals, the virus gains entry to the cns through neurons at neuromuscular junctions (nmjs) and replicates in motor neurons within the spinal cord, brain stem, or motor cortex and leads to the pv-characteristic flaccid muscle paralysis disorder poliomyelitis (racaniello, 2006; koyuncu et al., 2013) . approximately 10% of these paralytic cases result in death (roush et al., 2007) . following both pv infection and vaccination, neutralizing antibodies are generated to clear the virus, and these can be detected for many years, providing lifelong protection (libbey and fujinami, 2014) . vaccination with the injected inactivated pv vaccine prevents viral spread to the cns, whereas vaccination with the live-attenuated oral pv vaccine protects against infection of the intestinal tract and also prevents person-to-person spread of the virus (nathanson, 2008; griffin, 2010) . pv remains an important cause of neurologic disease as the three live-attenuated vaccine strains are at risk of recombining their genomes to revert to virulent form (griffin, 2010) . pv is endemic in afghanistan, pakistan, india, and nigeria, where political reasons, in part, are the most significant modality toward achieving pv eradication via vaccination. pv can usually be cleared by the adaptive immune response. under conditions of antibody deficiencies in humans, however, continuous fecal shedding of pv contributes to the establishment of persistent infection cycles (martin, 2006; nathanson, 2008; libbey and fujinami, 2014) . though less is known about the roles of adaptive t cell responses in controlling pv infections relative to that of neutralizing antibody responses, it is known that pv-specific cd4 þ t cells are induced in vaccinated individuals, where key epitopes have also been identified (graham et al., 1993; simons et al., 1993) . the induction of pv-specific cd4 þ t cells has been suggested to be the result of stimulating by pv-infected dcs and macrophages (wahid et al., 2005; dotzauer and kraemer, 2012) , where it has been demonstrated that hla class ii presentation remains intact in infected, antigen-presenting cells (apcs), and that cytolytic cd4 þ t cells produce ifn-g to lyse pv-infected cells for virus clearance. pv-specific cytotoxic, ifn-g-secreting cytotoxic cd8 þ t cell responses induced by infected macrophages have also been documented (wahid et al., 2005) , suggesting that both cd4 þ and cd8 þ cytolytic t cells partake in the adaptive immune reaction against pv (dotzauer and kraemer, 2012) . approximately 73 viruses are included in the flavivirus genus of the flaviviridae family of viruses, with 40 of its species associated with dengue, yellow fever (yf), japanese encephalitis (je), tick-borne encephalitis, and west nile encephalitis as the most important arboviruses causing extensive global morbidity and mortality (diamond, 2003) . flaviviruses are enveloped viruses with single-stranded rna genomes that are translated in the cytoplasm to generate a single polyprotein that is then cleaved into structural and ns proteins by virus and host proteases. the various encoded viral proteins assemble to generate the capsid, the envelope for receptor binding, membrane fusion and viral assembly, and the transmembrane proteins (prm) that assist in protein folding and function. the entry of flaviviruses into their target cells is mediated by the interaction of the e gp with host cell surface receptors (perera-lecoin et al., 2013) . flaviviruses are believed to evade the immune system to enter the brain and spinal cord via circulating blood (johnson and mims, 1968) , where these may cross the bbb by passive transport across the endothelium, by active replication in endothelial cells, or by a "trojan horse" mechanism, where virus hides in inflammatory cells during their transit into the brain (solomon and vaughn, 2002) . ifn-dependent and complement system innate immune responses, along with humoral neutralizing antibodies, protect against virus dissemination and spread, reviewed in diamond (2003) . adaptive cellular immunity is also important toward the destruction of infected cells, whereby virus-specific ctls become activated, proliferate, and release inflammatory cytokines following exposure to flavivirus-infected cells (kesson et al., 1987; kurane et al., 1989a; liu et al., 1989; murali-krishna et al., 1996) . there is also evidence that flavivirus replication is enhanced by myeloid cells, as observed for dengue, yf, west nile, tick-borne, and je viruses (diamond, 2003) . the je flavivirus is the leading cause of encephalitis and is amplified by waterfowl and only transmitted to humans by mosquito vectors, with no possibility of human-to-human transmission (erlanger et al., 2009) . pediatric and geriatric populations are at higher risk of infection by je (burchard et al., 2009) . while 99% of je infections remain asymptomatic, je can be devastating in symptomatic patients, causing mortality rates of 30% in these patient populations (batchelor and petersen, 2015) . after approximately 14 days of je incubation, these patients suffer from high fever, chills, headache, myalgia, and confusion, where pediatric patients also have symptoms of gastrointestinal pain, vomiting, and seizures. post-je infection, handicaps from persistent neurological deficits can last a lifetime in up to 50% of these survivors. as there is no treatment against je, the only method of prevention is avoidance of mosquitos and vaccination (batchelor and petersen, 2015) . the four available je vaccines are registered worldwide and used in national immunization programs for different age groups, including inactivated vero cell culture vaccine (je-vc) (ixiaro), inactivated mouse braine derived vaccine (je-mb), a cell cultureederived (primary hamster kidney) live-attenuated vaccine based on the sa 14-14-2 strain manufactured in china, and a live-attenuated chimeric vaccine based on the genes of yf 17d backbone combined with vero cellepropagated sa 14-14-2 strain (imojev) (chen et al., 2015) . t cell responses to je vaccination have been reported, where for sa14-14-2, t cell responses were detected in the majority following vaccination, and these cross-reacted with other flaviviruses (turtle et al., 2017) . je-specific t cell responses are observed in pbmcs isolated from je-infected patients and vaccinated individuals. cd4 þ and cd8 þ t cells directed against structural viral proteins were identified in vaccinated individuals, in contrast to specific cd4 þ and cd8 þ responses against ns or c proteins in infected patients (nathanson and cole, 1971) . these findings indicate that ns proteins, and especially ns3 have important roles in the initiation of t cell responses, as the main target of je-specific t cellemediated immune responses. in addition, cytolytic cd4 þ t cells clones that cross-reactive with other flaviviruses have been generated from individuals immunized with inactivated je vaccine (aihara et al., 1998) . finally, cd4 þ and cd8 þ and th1 t cells are believed to be primary determinants of protection from je infection (kumar et al., 2004a (kumar et al., , 2004b ). viruses from the alphavirus genus are members of the togaviridae family of viruses, a group of enveloped positive-sense rna viruses. these are mosquito-borne viruses causing two major types of human disease. the old world alphavirusesdsindbis, chikungunya, and ross river virusdcause arthritis and arthralgia, while the new world alphavirusesdeastern (eeev), western (weev), and venezuelan equine encephalitis virus (veev)dcause encephalitis (trobaugh and klimstra, 2017) . alphaviruses are small, icosahedral-shaped, enveloped viruses and are approximately 70 nm diameter in size (mancini et al., 2000; morgan et al., 1961; fuller, 1987) . alphavirus virions acquire host cell lipid membranes during viral assembly (fuller, 1987; acheson and tamm, 1967; vogel et al., 1986) , with 80 e1 and e2 viral gps spike protrusions, arranged in an icosahedral pattern embedded within their membranes and interacting with nucleocapsid (fuller, 1987; vogel et al., 1986; owen and kuhn, 1997) . alphavirus single-stranded, positive-sense, rna genomes are 12 kb long and consist of two large open reading frames encoding the ns and structural polyproteins that are subsequently cleaved by both viral and host proteases to create four ns proteins (nsp1 to 4) and five structural proteins (c, e3, e2, 6k, e1) (strauss et al., 1984; hardy and strauss, 1989) ; reviewed in leung et al. (2011) . of major concern are the new world eeev, weev, and veev alphaviruses, which are naturally transmitted by mosquitos, but where veev is also highly infectious via the aerosol route (zacks and paessler, 2010) . precise mechanisms of entry of alphaviruses into the cns remains elusive, however, once in, alphaviruses infect humans and equines neurons, causing neurologic symptoms from mild febrile illness to severe encephalitis resulting in death (ramakrishna et al., 2002; zacks and paessler, 2010) . development of severe encephalitis is believed to result from neuronal cell death from accelerated viral spread and host neuroinflammatory viral responses (paessler et al., 2006 (paessler et al., , 2007 . antibodies are protective against lethal meningoencephalitis when the virus is transmitted by insects, and virus-specific cd4 þ t cells are found to be important for protection from lethal meningoencephalitis from aerosol transmission routes (paessler et al., 2007; yun et al., 2009) ; reviewed in libbey and fujinami (2014) . veev remains an emerging disease threat by natural transmission as well as via its usage as a biological weapon. of the new world alphaviruses, veev is the most important human and equine pathogen, it having caused outbreaks of febrile and neurological disease primarily in latin america during the past century. past outbreaks have lasted several years and have involved up to 100,000 equine and human cases over large geographical regions, with the largest outbreaks on record were from the 1960s, where central colombia saw over 200,000 human cases and an estimated 100,000 equine deaths. more recent outbreaks in mexico and south america are behind the classification of veev as a re-emerging disease (weaver et al., 2004) . because veev can also be developed as a biological weapon amenable to use in warfare or terrorism, current global emphases on biological defenses have renewed interest in its virology (hawley and eitzen, 2001; weaver et al., 2004) . veev infection in humans typically causes nonlethal, incapacitating symptoms including fever, headache, malaise, myalgia, sore throat, and vomiting. up to 4% of rarer cases of cns involvement usually follow acute febrile phases, with associated severities of neurological disease ranging from somnolence and mild confusion, to seizures, ataxia, paralysis, and coma, with mortality rates ranging as high as 35% in infected children and 10% in infected adults (bowen et al., 1976) . veev has also been reported to cause long-term neurological deficits, abortions, and teratogenic effects (de la monte et al., 1985; rivas et al., 1997; weaver et al., 1996) . like veev, though the majority of human infections with eeev are asymptomatic, cns involvement results in severe neurological signs, lesions, and sequelae, with an estimated associated human mortality rate of 75%, and with its neurological manifestations including facial edema, paresis, paralysis, respiratory impairment, altered mental state, and seizures in children, many of these symptoms persisting long-term in surviving patients. in fatal cases of eeev, gross lesions in the brain include edema, meningeal congestion, hemorrhage, and malacia (deresiewicz et al., 1997) . as with veev and eeev, natural human cases of weev typically show an early, flu-like illness with associated fever, malaise, and headache. similar to eeev, weev results in cns involvement in a significant proportion of cases, including symptoms of somnolence, seizures, coma, and motor neuron dysfunction. ninety percent of infants infected with weev have severe cns symptoms (calisher, 1994) . human mortality rates from weev infection range from 3% to 15%, and neurological sequelae may become permanent features in survivors (steele and twenhafel, 2010) . alphavirus expression vectors based on sindbis, semliki forest, and veev have been demonstrated to induce strong cd8 þ t cell responses against their antigens (rayner et al., 2002; lundstrom, 2002 lundstrom, , 2003 riezebos-brilman et al., 2006; schlesinger and dubensky, 1999; polo et al., 2000) . both innate and adaptive immune responses can control viruses targeting cns neurons (griffin, 2003) . viral disruption of the type i ifn signaling pathways interferes with survival from veev, as well as of those infected with sindbis and west nile viruses (ryman et al., 2000; samuel and diamond, 2005; white et al., 2001) . virus-specific antibody responses are critical in limiting viral spread and facilitating clearance of infectious virus from neurons within the brain levine et al., 1991) . both alpha beta (ab) and gamma delta (gd) t cell responses have been demonstrated as being important for the control of veev (paessler et al., 2006) . t cell responses reduce mortality rates by direct killing of infected cells, producing antiviral cytokines and increasing production of virusspecific antibodies (bilzer and stitz, 1994; patterson et al., 2002; shrestha et al., 2006; sitati and diamond, 2006) . veev replicon particles delivered as an adjuvant have been demonstrated to induce activation of cd8 þ t cell responses (thompson et al., 2008) . more recently, t cells have been demonstrated to facilitate recovery from veev-induced encephalomyelitis in absence of antibodies, responsible for dramatic reduction in viral titres in cns, where cd4 þ t cells were the best t cell producers of ifn-g response and were more efficient at controlling veev in cns lesions than cd8 þ t cells, facilitating recovery from severe viral encephalomyelitis (brooke et al., 2010) . commercial equine vaccines marketed in the united states are generated with inactivated tc-83, which produces viremia, fever, and leukopenia in horses but generates robust neutralizing antibodies and veev protection from rechallenge (walton et al., 1972) . u.s. army special immunization programs provide inactivated c-84 to individuals failing to seroconvert in response to tc-83 boosters (pittman et al., 1996) ; however, neither of these vaccines can be shown to completely protect nonhuman primates against aerosol exposure (pratt et al., 1998) . a more stably attenuated veev vaccine candidate called v3526 has been produced, where preclinical testing has demonstrated it to be safe and immunogenic and possibly superior to tc-83 (pratt et al., 2003; hart et al., 2000; ludwig et al., 2001) . adaptive immune pbmc-derived biomarker signatures have been identified and able to efficiently stratify tc-83 vaccinated from naïve or nonresponding individuals (erwincohen et al., 2012) . rabv is the type species of the genus lyssavirus, within the rhabdoviridae family. rhabdoviruses are negative-sense, single-stranded rna viruses having a distinctive bullet-shaped structure. up to 10 viruses of lyssaviruses have the potential to cause rabies in humans. these have a 12,000 nucleotide genome encoding five proteins: nucleoprotein (n), phosphoprotein (p), matrix (m), glycoprotein (g), and rna-dependent-rna polymerase (l) (marston et al., 2007) . rabv causes acute encephalitis in mammals, causing fatality rates of almost 100%. rabv commonly infects many animals, including bats, skunks, foxes, and dogs and can also infect insects and plants. rabv in animal saliva spreads between hosts via bites or scratches. infected animals can survive for years, secreting infectious particles in their saliva, but untreated infection in humans generally results in rapidly fatal acute myeloencephalitis (koyuncu et al., 2013) . rabid dogs are the most important reservoirs for rabv, where dog bites account for more than 99% of human infections. rabv, like all members of lyssaviruses, is neurotropic and infects peripheral nerves close to the primary site of the bite. rabv then rapidly moves by retrograde axonal transport to the dorsal root ganglia where virus replication begins . rabv particles enter axons of motor neurons at the nmj via their binding to nicotinic acetylcholine receptors (e.g., nachr) and neural cell adhesion molecules (ugolini, 2011) . transneuronal rabv spread occurs between synaptically connected neurons, whereby viruses move from postsynaptic to presynaptic neurons. in humans, a relatively long asymptomatic incubation period after initial rabv infection can occur, sometimes lasting up to 1 year, and providing some time for cns infection intervention. however, death almost always ensues after rabv infection reaches the cns, with marked behavioral and neurological symptoms (koyuncu et al., 2013) . once rabv has entered the cns, it rapidly moves to the brain and is associated with an explosive increase in virus replication. initial symptoms include pain or paraesthesia close to the bite site and are often associated with fever, fatigue, and weakness in associated limbs. nonspecific neurological symptoms including headache and anxiety occur days prior to acute encephalitis (morrison and wenzel, 1985) . currently, there are no available therapies against disease symptoms once they develop, and death ensues within a number of days following cns-associated symptoms (jackson et al., 2003) and reviewed in johnson et al. (2010) . rabv replication begins following cns penetration, thereby limiting earlier possible detection of low-level primary antigens in the peripheral circulation. this delays antigen presentation, where antigens later but rapidly drain from the cns to local lymphoid tissues (knopf et al., 1998) . once b cells are stimulated, the next delaying obstacle is re-entry into the cns, but experimental models have demonstrated t and b cell infiltration of dorsal root ganglia, spinal cord, and brain (johnson et al., 2008) , with t cells as the major immune subsets, but where most of these cns-infiltrated t cells have fas-mediated apoptotic phenotypes (baloul and lafon, 2003) . further intrinsic complexities in immune responses are present in the cns, including tight mhc expression regulation (irwin et al., 1999) , and the expression of immunosuppressive factors by neuronal cells. additionally, the bbb remains intact during rabv infection (roy et al., 2007) . numerous studies have suggested that the virus suppresses the adaptive immune response, believed to be in part due to a deficit of adaptive immune effector cell accumulation within the cns due to a virally induced reduction in bbb permeability (libbey and fujinami, 2014; roy et al., 2007) . two rabv vaccines are licensed for human application, the human diploid cell vaccine manufactured by aventis pasteur and the purified chick embryo cell vaccine manufactured by chiron . pre-exposure vaccination given to healthcare personnel, laboratory workers, and travelers to endemic areas causes detectable igm and igg antibodies within a week following exposure, and long-term studies have provided evidence that igg antibodies provide the most effective protection against rabv due to its ability to penetrate tissues, in contrast to igm which cannot penetrate tissues (turner, 1978) . a multifaceted approach for human rabies eradication involving government support, disease awareness, and vaccination of at-risk humans and dogs will be required to achieve the goals of the world health organization in eradication of rabies by 2030 (fooks et al., 2017) . the cns is immunologically privileged and protected by a highly complex barrier system. viruses that have evolved to overcome these barriers can cause cns infections greatly outnumbering those from all bacterial, fungal, and protozoa infections combined. ingested pv multiplies in the oropharyngeal and intestinal mucosa and drains to cervical and mesenteric lymph nodes and then into the blood ahead of penetrating the cns to cause polio, with 10% of cases resulting in death. both neutralizing antibodies and the adaptive immune system can clear pv infection and may provide lifelong protection. vaccination combinations can induce pv-specific cytolytic cd4 þ and cd8 þ t cells for virus clearance, but their coadministration can pose the risk for reversion to virulence by recombination. the je flavivirus is amplified by waterfowl and transmitted to humans by mosquitoes, and while 99% of its infections are asymptomatic, mortality rates in 30% of infected individuals cause associated disorders that leave its survivors a lifetime of associated morbidities. as there is no existing je treatment, prevention involves either avoidance of mosquitoes or vaccination. flaviviruses evade the immune system to cross the bbb by an inflammatory celle mediated "trojan horse" mechanism. je dissemination is limited by innate immune responses, neutralizing antibodies produced by humoral immunity, and by virus-specific ctls. je vaccines are licensed worldwide, and the majority of vaccinated individuals have circulating je-specific cd4 þ and cd8 þ t cells that can cross-react with other flaviviruses. alphaviruses are transmitted by mosquito bites to infect neurons, causing mild to severe encephalitis resulting in death, with past outbreaks numbering in the hundreds of thousands. veev infection causes up to 35% mortality in children, 75% of which involve cns penetration, causing severe long-term neurological disorders. veev is not only a naturally emerging disease threat but is also a highly developed biological weapon amenable to warfare or terrorism due to its aerosol transmission route and associated lethal meningoencephalitis. ifn signaling pathways and ab and gd t cell response from innate and adaptive immunity can control veev targeting of cns, where virus-specific antibody responses are critical in limiting viral spread. in the absence of antibodies, veev replicon particles can induce t cell responses able to induce recovery from veev-induced encephalomyelitis, where cytotoxic cd4 þ t cells control veev in cns lesions. veev vaccines induce robust neutralizing antibodies for protection against rechallenge. tc-83 vaccine responders have circulating pbmc biomarkers, and military programs give boosters of c-84 to those failing to seroconvert. in contrast to these other viruses, rabv replication only begins after cns penetration, as facilitated by depth of bite by its canine vector, thereby limiting possible detection of primary viral antigen in the periphery and resulting in delayed and minimal innate and humoral responses. once rabv-related acute encephalitis symptoms begin, fatality is sure to follow due to absence of cns infiltration by adaptive immune effector cells as a result of virus-induced decreases in bbb permeability. other countermeasures against protection are tight mhc expression regulation and apoptotic phenotypes of bbb-infiltrated t cells. rabv vaccines cause increases in ig, but little is known concerning vaccine-associated adaptive immune responses. viral hemorrhagic fever (vhf) classification originates from the study of hantaviral hemorrhagic fever (hf) and was later extended to include crimeanecongo hf and omsk hf. vhf can results from infection by 23 enveloped rna viruses from four families: flaviviridae, filoviridae, arenaviridae, and bunyaviridae. vhf designation is given to severe febrile illnesses with abnormal vascular regulation and vascular damage (peters and zaki, 2002) . vascular dysregulation occurs early in the course of disease, visible as skin flushing, hypotension, and conjunctival vasodilation, whereby vascular damage with capillary leakage occurs as disease progresses, causing edema and serous effusions of pleural and peritoneal cavities. the terminal phase of vhf, or shock, arises from increased disease severity from combinations of vascular dysregulation and damage from capillary leakage (paessler and walker, 2013) . detailed mechanisms of hemorrhage and plasma leakage during vhf include endothelial injury, activation of the mononuclear phagocytic system, cytokine storm, platelet aggregation and consumption, activation of the coagulation cascade, and insufficiency of coagulation factors from severe hepatic damage (schnittler and feldmann, 2003; chen and cosgriff, 2000) . these mechanisms vary among diseases, cell and organ tropism of causative viruses, and host responses (paessler and walker, 2013) . flaviviruses, filoviruses, arenaviruses, and bunyaviruses are the main causes of hf (table 1) . these viruses continue to propagate as part of the life cycles of primates, bats, rodents, farm animals, mosquitoes, and ticks. infection by these viruses can cause mild vascular instability to fatal shock, with hemorrhage ranging from unnoticeable to life-threatening. pathogenic mechanisms of hfv are diverse and include hepatic necrosis leading to deregulation of coagulation factors, cytokine storm, increased permeability, and complement activation. overall disease severity by these viruses is varied, whereby ebola and marburg hf can cause high fatality rates, whereas yf and dengue infections can be asymptomatic. severe vhf is commonly correlated with ineffective immunity and high viral loads, and severe plasma leakage can occur from viral clearance and fever breaks in dengue hf (dhf). approximately 73 viruses are included in the flavivirus genus of the flaviviridae family of viruses, with 40 of these species associated with dengue, yf, je, tick-borne encephalitis, and west nile encephalitis as the most important arboviruses causing extensive global morbidity and mortality (diamond, 2003) . flaviviruses are enveloped viruses with single-stranded rna genomes that are translated in the cytoplasm to generate a single polyprotein that is then cleaved into structural and ns proteins by virus and host proteases. the various encoded viral proteins include capsid, envelope for receptor binding, membrane fusion and viral assembly, and transmembrane proteins (prm) that assist in protein folding and function. although the precise mechanism is unclear, flaviviruses are believed to evade the immune system to enter the brain and spinal cord via circulating blood (johnson and mims, 1968) , where these may cross the bbb by passive transport across the endothelium, by active replication in endothelial cells, or by a "trojan horse" mechanism utilizing inflammatory cells (solomon and vaughn, 2002) . ifn-dependent and complement system innate immune responses and humoral immunity, producing neutralizing antibodies limit dissemination of infection and protect against viral spread, reviewed in diamond (2003) . cellular immunity is also important toward eradication of infected cells, whereby infection induces the recognition of flavivirusinfected cells by virus-specific ctls, which then become activated, proliferate, and release inflammatory cytokines (kesson et al., 1987; kurane et al., 1989a; liu et al., 1989; murali-krishna et al., 1996) . however, there is also evidence that flavivirus replication is enhanced by myeloid cells and has been observed for dengue, yf, west nile, tick-borne, and je viruses (diamond, 2003) . yf virus (yfv) is important both historically and currently. it was once one of the most globally feared diseases terrorizing africa, europe, and the americas. hundreds of thousands were killed in the americas over a 250-year spandcrippling economies (watson and klimstra, 2017) . yfv is a member of the genus flavivirus of the flaviviridae family and contains a single-stranded rna genome of approximately 11 kb. yfv virions are icosahedral and are composed of nucleocapsid, composed of capsid (c) protein subunits and a surrounding lipid bilayer derived from host membranes. the viral envelope is studded with dimers of envelope (e) and membrane (m) proteins, for a total diameter of approximately 45 nm. as the major component of the virion surface, the e protein is responsible for cell-surface receptor binding, virion assembly, fusion, and immunogenicity. viral proteins are encoded in a single open reading frame and produced as a polyprotein later processed by proteolytic cleavage into structural (c, m, and e) and ns proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5) , reviewed in gardner and ryman (2010) . during most yfv infections, the virus is transmitted by the bite of an infected aedes aegypti mosquito found in urban areas. infected patients often develop severe acute illness hemorrhagic yf disease, with associated symptoms of fever, nausea, vomiting, epigastric pain, hepatitis, jaundice, renal failure, hemorrhage, and shock, with 20%e60% of cases resulting in death (watson and klimstra, 2017) . yf is the prototypical vhf, sharing many pathophysiological features with other viral disorders only associated via similarities in syndromes, but with the exception that yf causes the most severe symptoms of hepatic dysfunction (monath and vasconcelos, 2015) . yfv remains endemic in south american and african countries, with monkeys as its reservoir, causing regular outbreaks of jungle yf, and resulting in as many as 200,000 infections per year causing 30,000 deaths. millions are at risk for infection in africa, where vaccination prevalence is low. the 2016 outbreak in angola serves as an example of yfv traveler-associated spreading to neighboring countries, where it reached as far as china, then naïve for virus (watson and klimstra, 2017) , and representing a prime population for a major outbreak of epic proportions (wasserman et al., 2016) . geographical shifting of mosquito populations to north america is also creating new risk for yfv, dengue, and zika infection of naïve populations (monaghan et al., 2016) . despite the availability of vaccination against yfv since the 1940s, large epidemics have still arisen, with dramatic surges of yfv in africa in the 1960s and the late 1980s, with each reporting over 100,000 cases. recent outbreaks have also affected brazil, paraguay and argentina, uganda, and sudan and ethiopia. immunity is the critical for reducing and eliminating viral infections, but other contributing factors to virus amplification are multifactorial and elusive, including the emergence of new viral strains and prolonged periods of hot and humid weather promoting insect propagation, reviewed in monath and vasconcelos (2015) . fifty-seven million people were vaccinated against yf across africa between 2007 and 2010. five hundred million doses of the live-attenuated yf 17d vaccine, representing the most effective vaccine ever created, have been distributed over the last 50 years (monath and vasconcelos, 2015) . both humoral and cellular immunity elicited by 17d are observed and well characterized, where neutralizing antibodies provide protection, but 17d also provides a robust, long-lived, and polyfunctional adaptive t cell immune response (watson and klimstra, 2017) . neutralizing antibodies remain the accepted correlate of protection against yfv, with 90% or greater of 17d immunized individuals developing neutralizing antibodies (gotuzzo et al., 2013) . 17d also elicits a complex modulation of innate immune cytokines, with elevated levels of plasma ifn-g 15 days postvaccination (neves et al., 2009) . restimulation of innate immune cell cultures of nk cells, neutrophils, and monocytes from 17d vaccinated humans with yf antigen results in the increased production of ifn-g, il-1beta, il-10, il-12, tnf-a, and il-10 (neves et al., 2009; gardner and ryman, 2010; luiza-silva et al., 2011; silva et al., 2011) . since its development, humoral immunity, as a gold standard of general vaccine development, was the most studied aspect of human immunity to 17d. however, recent studies of adaptive t cellemediated immunity to 17d have demonstrated that both cd4 þ and cd8 þ t cells strongly respond to 17d, with activated cd8 þ t cells detected as 3 days after vaccination , and cd4 þ t cells detected several days later kohler et al., 2012; blom et al., 2013) . increased cd8 þ t cell proliferation correlates directly with the levels of virus genomes in plasma, which peaks once virus is eliminated . cd8 þ t cell clones responding to 17d differentiate into central memory and effector memory subpopulations (dewitt et al., 2015) and are still detectable 25 years following vaccination (wieten et al., 2016) . 17d-specific cd8 þ t cells respond to epitopes contained from every protein product generated by the 17d polyprotein, and upon peptide restimulation, these 17d-specific cd8 þ t cells have activated cytotoxic profiles including increased expression of ifn-g, tnf-a, and mip1-b and il-2 granzyme b and cd107a (blom et al., 2013; akondy et al., 2009) but are not exhausted and retain long-lived memory and polyfunctional phenotypes for at least 2 years following 17d rechallenge (akondy et al., 2009) . dengue virus (denv), also a member of the single-stranded positivesense rna viruses from the flaviviridae family, causes visceral and cns disease in humans and is closely related to yfv, where denv fever has often been mistaken for yfv infection. far more serious is dhf, where additional symptoms develop, including hemorrhage and shock, and have mortality rates exceeding 30% if left untreated (rogers et al., 2006) . denv is a spherical, 50-nm virion, comprising of three structural proteins: capsid (c), premembrane and membrane (prm and m), and envelope (e). the e protein directs several critical steps of the viral replication cycle, including engagement with cellular attachment and entry factors, membrane fusion, and virion assembly. denv binds to target cells via glycosaminoglycans, c-type lectins such as dc-sign, the mannose receptor cd206, and immunomodulatory proteins (tim and tam receptors; diamond and pierson, 2015) . thus targets for denv infection include monocytes, macrophages, dcs, mast cells, and possibly hepatocytes and endothelial cells. following its entry into the cellular cytoplasm, the viral genomic 10.7 kb rna is translated into a single polyprotein, later cleaved into three structural and seven ns proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5 ) by viral ns3 and host cell proteases. twenty-five percent of denv infections cause both mild symptoms including dengue fever (df) to more severe and lethal dhf, causing shock via hemorrhagic and capillary leak syndrome. df can be characterized by abrupt onset febrile illness causing headache, severe muscle and joint pain, and rash, whereas dhf is characterized by rapid onset capillary leakage accompanied by significant thrombocytopenia and liver injury (halstead, 2007) . as with yf, denv origins are believed to be that of a sylvatic virus, with a natural life cycle involving multiple mosquito and vertebrate species from asia and africa (diallo et al., 2003) . denv adaptation to human demography is via mosquito vector aedes aegypti, breeding in urban areas (trpis and hausermann, 1986) . cases of denv infection have increased since the 1960s, with an estimated 50 million cases of df, and 500,000 cases of dhf occurring globally every year. there are no cures for denvassociated disorders, and vaccine development has been complicated by antibody-dependent enhancement of future heterotypic infection induced by vaccination (vaughn, 2000; halstead and deen, 2002) . thus avoidance and control of aedes aegypti is the best approach for limiting denv infection (rogers et al., 2006) . adaptive immune cd8 þ t cells vigorously and frequently recognize denv ns3, ns4b, and ns5 proteins, whereas the capsid, envelope, and ns3 proteins are the dominant targets for cd4 þ t cells (simmons et al., 2005; duangchinda et al., 2010; weiskopf et al., 2011 weiskopf et al., , 2013 rivino et al., 2013) . both cd4 þ and cd8 þ t cells are believed to contribute to protection against denv, as denv-specific cd4 þ t and cd8 þ t cells proliferate, produce ifn-g, and lyse target cells, from primary denv infection (kurane et al., 1989b; mathew et al., 1996; gagnon et al., 1999; livingston et al., 1995) . higher frequencies of denv-specific ifn-gproducing t cells are present in children with asymptomatic denv infection (hatch et al., 2011) . both cd4 þ and cd8 þ t cells contribute to protection against denv challenge (yauch et al., 2009 (yauch et al., , 2010 zompi et al., 2012; zellweger et al., 2013) , and hla alleles associated with increased risk of denv severity correlated with weak cd8 þ t cell responses and vice versa, implying a protective role for cd8 þ t cells against severe denv disease in humans (weiskopf et al., 2013) . in 2015, the first dengue vaccine (dengvaxia) was licensed in asian and south american countries for protection against all four denv serotypes, and while it demonstrated an good safety and efficacy in clinical trials, it has recently been withdrawn in the philippines due to its causing elevated disease severity if administered following infection (wichmann et al., 2017) . it has been suggested that failure of this and other live-attenuated tetravalent dengueeyf chimeric virus vaccines (guy et al., 2011) is the result of their lacking the ns proteins ns3, ns4b, and ns5, otherwise dominantly targeted by cd8 þ t cells (simmons et al., 2005; duangchinda et al., 2010; weiskopf et al., 2011 weiskopf et al., , 2013 rivino et al., 2013) , making it critical to accurately assess not only antibody responses but rather t cell responses in the context of denv vaccine development (weiskopf and sette, 2014) . lassa virus (lasv), causing lassa fever (lf), is an enveloped virus with two single-stranded rna segments and is another virus causing hf. lasv is an old world member of the arenavirida family of viruses. the single-stranded arenavirus genome consists of a small (s) and a large (l) rna segment, measuring 3.4 and 7 kb, respectively. the large segment encodes a small zinc-binding (z) protein which regulates transcription, replication, and viral budding, along with the rna polymerase (l). the small segment encodes the np and the two envelope glycoproteins (gp1 and gp2) responsible for cell entry, reviewed in russier et al. (2012) . lasv disorder is endemic in africa and its neighboring countries (safronetz et al., 2010; gunther et al., 2000) , and though infection rates are difficult to quantify due to limited survey infrastructure, classification of its clinical symptoms is common to other diseases. lasv is predicted to be responsible for approximately 300,000 infections and up to 6000 resultant deaths each year (ogbu et al., 2007; mccormick et al., 1987) . transmission to humans is via the rodent host mastomys natalensis (mccormick et al., 1987) . apcs, dcs, and macrophages are believed to be the first cells targeted by lasv infection (baize et al., 2004; mahanty et al., 2003) , which can rapidly speed up dissemination of lasv to multisystem organs due to their widespread physiological distributions in mucosal tissues and skin. due to their ease in motility across various organs and tissues, apcs are believed to the responsible for the spread of lasv for the establishment of systemic infection (hensley et al., 2011) . apc infection results in substantial virus release in the secondary lymphoid organs, the liver, hepatocytes, fibroblasts, and endothelial cells that are subsequently infected. lymphopenia of cd4 þ and cd8 þ t cells, nk cells, and b cells is observed early during disease onset, reviewed in russier et al. (2012) . lasv infection severities range from asymptomatic infection to fatal hf (fisher-hoch et al., 1995) and commonly resulting from other viral infections, nonspecific symptoms beginning several days after infection include fever, headache, arthralgia, myalgia, and severe asthenia. these early symptoms are typically followed by more severe symptoms of pharyngitis, conjunctivitis, cough, abdominal pain, diarrhea, and vomiting. in severely affected patients, cervical and facial edema, hemorrhages, renal and liver failures, and encephalopathy occur, and death follows systemic shock (edington and white, 1972) . survivors of lasv-related disorders have persisting lifelong morbidities and disabling conditions including deafness (cummins et al., 1990) . no vaccine has been licensed against lasv, and ribavirin is the only existing treatment, but is only effective if administered very early after infection and is not available for broad distribution in countries where lasv is endemic (mccormick et al., 1986) . t cells play a crucial role in the outcome of severe lasv infection, which has been associated with defective t cell responses since the very cells responsible for stimulating t cell antigen responses are those infected by the virus. however, t cell responses have been demonstrated to play critical roles in the control of lasv, where strong memory cd4 þ t cell responses directed against lasv np and gp proteins are observed in lasvseropositive healthy individuals from endemic regions (ter meulen et al., 2004) . high serum concentrations of il-8 and cxcl10 chemokines that attract and activate t cells are associated with nonfatal lasv infections (christensen et al., 2006; dufour et al., 2002) and vice versa in fatality cases (mahanty et al., 2001) . the control of acute lf has been correlated with increases in circulating activated cd4 þ and cd8 þ t cells in response to lasv infection or antigen (baize et al., 2009) . in vaccine studies, protection against a lethal lasv rechallenge is associated with the induction of t cell immunity (fisher-hoch et al., 2000; geisbert et al., 2005) . however, in comparison to other viruses, lasv-infected dcs are unable to mount effector t cell responses (pannetier et al., 2011) . human and nonhuman primate studies have demonstrated that lasv np and gp proteins are the main viral antigens recognized by activated t cells (meulen et al., 2004; ter meulen et al., 2000; fisher-hoch et al., 2000; fisher-hoch and mccormick, 2001, 2004; geisbert et al., 2005) , suggesting that vaccines using these proteins to induce long-term memory t cell expansion will best control the spread of lf. ebola virus (ebov) causes a rapidly fatal hf for which there is currently no treatment (muyembe-tamfum et al., 2012; team et al., 2014) . ebov is a member of the filoviridae family, which are filamentous, negative-stranded rna viruses that cause severe human disease. filoviruses viruses are variable, with long filaments measuring 80 nm in diameter and which can reach lengths of up to 1000 nm, with many turns and branches and which have tendency to curve to resemble the number 6. viruses are composed of nucleocapsid, matrix, and envelope proteins, whereby seven genes encode np, the viral proteins vp24-vp30-vp35-vp40, l (polymerase), and the gp (hoenen et al., 2006) , expressed as gp1 and gp2, and regulating virus production and release (mohan et al., 2015) . nps embed the genome in complex with vp30 and vp35 for rna synthesis. vp40 and vp24 proteins are localized in virus matrix space (watanabe et al., 2007; hoenen et al., 2010) . ebov is transmitted to humans via mucosal surfaces, skin injury, and vertical transmission (feldmann and geisbert, 2011) , and with the exception of t cells, can infect almost all human cells using various different attachment mechanisms, reviewed in falasca et al. (2015) . both innate and adaptive immune responses are involved in ebov pathogenesis, where innate immune deregulation involves inhibition of type-i ifn response and perturbation of cytokine signaling, along with impairment of dc and nk cells, and adaptive immune deregulation involves both humoral and cell-mediated immunity (falasca et al., 2015) . because high levels of ebov replication are associated to multiple cell types, its associated systemic dissemination results in a highly complex pathogenesis model, including detrimental immune suppression and hyperactivation, and leading to disordered coagulation and tissue damage, that, in the absence of treatment, results in rapid multiple organ failure and death within days of symptomatic infection (baseler et al., 2017) . for 50 years, ebov and related filoviruses have been repeatedly re-emerging to cause large epidemics of highly fatal hf. ongoing ebov outbreaks in africa has brought this virus to the forefront of research, with over 20,000 reported cases of infection and an associated 8000 deaths (mcelroy et al., 2015) . natural serologic response to ebov infection involves virus-specific igm and igg antibody responses sometimes detected early, but usually later, once symptoms begin rowe et al., 1999) . ebovinfected dcs are impaired in cytokine production required for t cell activation (mahanty et al., 2003) , whereas infected macrophages are unable to mature (bosio et al., 2003) . ebov is classified as an immunosuppressive virus since numerous of its proteins interfere with immune responses by inducing t cell apoptosis, lymphopenia, and absence of antibody responses in fatal cases (basler and amarasinghe, 2009 ). classification of ebov-targeting mechanisms has been compromised by lack of infrastructure for adequate biosafety containment level facilities required to analyze this deadly virus. however, observations of the adaptive t cell immune response have shown that ebov correlates with fatal outcomes by causing aberrant cytokine responses (baize et al., 1999 (baize et al., , 2002 wauquier et al., 2010; villinger et al., 1999; ansari, 2014) , decreased cd4 þ and cd8 þ t cells, and increased apoptotic t cell phenotypes (baize et al., 1999; wauquier et al., 2010; geisbert et al., 2000; bradfute et al., 2010; gupta et al., 2007) . in recent work, ebov induced increased cd4 þ and cd8 þ t cell activation against the viral np, with cd8 þ t cells demonstrating the largest increases in expression of activation and proliferation biomarkers, with sustained activation following ebov clearance and following patient discharge, suggesting continued antigen stimulation after resolution of the disease (mcelroy et al., 2015) . recently, an rvsv-zebov recombinant, replicationecompetent vesicular stomatitis virusebased vaccine expressing a surface gp of zaire ebolavirus, demonstrate a 100% efficacy in preventing ebov disease in contacts and contacts of contacts of recently confirmed cases in guinea, west africa (henao-restrepo, 2017 #550). this vaccine produced rapid innate immune responses after a single dose, suggested to lead to longerterm full protection by providing an essential period of restricted virus replication during the development of specific adaptive responses (marzi, 2015 #551) . crimean-congo hemorrhagic fever virus (cchfv) is a tick-borne virus causing hf resulting in human fatalities. cchfv is a member of the nairoviridae family of viruses from the genus of orthonairovirus and the order of the bunyaviridae viruses. it has a single-stranded, negative-sense rna genome possessing three segments: the large (l), medium (m), and small (s) segments (casals, 1969; clerx et al., 1981) . the l segment encodes the viral rna-dependent rna polymerase responsible for mrna synthesis and rna replication (honig et al., 2004) . the m-segment encodes numerous ns and two structural gps (gn and gc) responsible for cell tropism and attachment and are targets for neutralizing antibodies. the s-segment encodes the viral np binding the rna segments toward formation of ribonucleoprotein complexes (altamura et al., 2007; sanchez et al., 2006) . though hf by cchfv infection in humans is not among the most common viral disorders reported, it remains important because it is fatal in up to 30% of cases (bente et al., 2013; goedhals et al., 2017) . transmission of cchfv to humans occurs through contact with infected animal blood, or ticks, belonging to the genus hyalomma, as its primary vectors and providing transit from one infected human to another (mousavi-jazi et al., 2012) . cchfv human infection involves sudden onset of acute symptoms, including high fever, headache, myalgia, and petechial rash, followed by hemorrhage progressing to multiorgan failure, with leukopenia, thrombocytopenia, and elevated liver enzymes as hallmarks of the overall disorder (begum et al., 1970; sanchez et al., 2006) . outbreak-associated fatality rates are varied but can reach 70% (mousavi-jazi et al., 2012) . there is currently no licensed vaccine, and use of ribavirin as treatment has been investigated but remains controversial (begum et al., 1970; bente et al., 2010) . distribution of cchfv infection and associated disease follows geographical spread of the principal vectors (bente et al., 2013; whitehouse, 2004) . clinical cchfv disorders are described in africa, asia, the middle and east eastern europe, and have recently emerged in other countries including turkey, india, spain, and greece, and with almost 10,000 cases reported in turkey between 2002 and 2015 (maltezou et al., 2010; papa et al., 2008; leblebicioglu et al., 2016) . typically, transient igm and igg antibody responses develop within days following primary cchfv infection and can persist long-term (shepherd et al., 1989; burt et al., 2013) , but where lack thereof usually results in fatality (shepherd et al., 1989) . igm and igg antibodies have however not been correlated with clearance, viral load, or outcomes (duh et al., 2007) , implying that innate and t cell immunity must be critical for viral clearance. neutralizing antibodies also do not cause protection, and nonneutralizing antibodies may assist in antibody-dependent cell-mediated cytotoxicity (bertolotti-ciarlet et al., 2005) . thus, as immune correlates of protection for cchfv are not well documented, vaccine design has aimed at targeting the cchfv np or gps. only an inactivated vaccine is available, and even though the attaining of immunogenicity has required its administration in multiple doses, it was demonstrated to reduce infections and induce both neutralizing antibody responses and t cell responses to np peptides (mousavi-jazi et al., 2012) . another promising approach is the use of modified vacv ankara recombinant vaccine expressing the viral gps, which induce cellular and humoral responses and which are observed to provide protection from lethal disease in mice (buttigieg et al., 2014; dowall et al., 2016) . although t cell responses are known to play a role in protection from and clearance of viral infections, it was only recently that specific cd8 þ t cell epitopes against gn and gc were shown to stimulate ifn-g production, whereby responses were detectable several years after the acute cchfv infection, even in the absence of continued antigenic stimulation, and where ifn-g-producing cd8 þ t cells were confirmed as responsible for providing long-term protection (goedhals et al., 2017) . vhfs causes high fatality rates and are commonly correlated with ineffective immunity and high viral loads. yfv is important for both historical and current reasons. historically, it crippled economies and families by killing hundreds of thousands over 250 years. yfv is transmitted by mosquitos in urban areas, causing as many as 200,000 infections and 30,000 deaths annually. like other mosquito vector viruses, yfv is an existing and re-emerging threat due to increased geological spread by both world travelers and shifting mosquito breeding geographies. flaviviruses evade immunity to enter the brain and spinal cord via circulating blood, crossing the bbb via "trojan horse" mechanism. dissemination of infection and protection against yfv is elicited by ifn-signaling, complement system, and other innate, humoral, and adaptive immune responses. neutralizing antibodies are still the gold standard correlates of protection, but evidence that a robust, long-lived, and polyfunctional adaptive cd4 þ and cd8 þ t cell immune response is what is provided, early after vaccination by the 500 million doses of effective yf 17d vaccine distributed globally. importantly, these are nonexhausted, polyfunctional central memory and effector memory t cell subsets that are detected for over 25 years following vaccination. far more serious is dhf by infection by denv, contributing to either mild or severe hf, with mortality rates exceeding 30% in the untreated. as with yfv, denv uses mosquitos adapted to urban areas as their vectors. fifty million cases of df and 500,000 cases of dhf occur globally each year, with no available cures for its associated disorders and where vaccine development has been complicated by antibody-dependent vaccineinduced enhancement of future heterotypic infections. cytotoxic cd8 þ and cd4 þ t cells, however, vigorously and frequently recognize denv proteins and are believed to contribute protection against primary infection. the denv vaccine dengvaxia has been recently withdrawn, with its failure posited to result from its lack of denv proteins specifically targeted by cd8 þ t cells, and demonstrates that is essential to accurately assess t cell responses in the context of denv development. lasv also causes mild and severe vhf and may cause 300,000 infections and up to 6000 deaths each year. lasv is transmitted to humans by a rodent vector and first targets apc, dc, and macrophages, contributing to rapid multisystem and organ lasv dissemination due to their widespread physiological distributions. lasv also causes lymphopenia of cd4 þ and cd8 þ t cells, nk cells, and b cells, and survivors of lasv-related disorders can be expected to maintain lifelong morbidities. there is no licensed lasv vaccine as of yet, complicated by the fact that the very cells responsible for stimulating t cell antigen responses are those infected by the virus. however, control of acute lf is correlated with increased circulating strong memory cd4 þ and cd8 þ t cells in response to lasv infection or rechallenge and lasv antigen. ebov also causes mass hf. it can infect almost all human cells with exception of lymphocytes and has no known treatment despite documented involvement of innate and adaptive immune responses. classification of ebov targeting mechanisms are compromised by lack of infrastructure for adequate biosafety containment level facilities. ebov causes dcs to be impaired in cytokine production for t cells activation and inhibits macrophages maturation, thus classifying it as an immunosuppressive virus, with fatalities correlating with its induction of lymphopenia the absence of antibody responses. ebov, however, causes increased cd4 þ and cd8 þ t cell activation, with recorded persistence of activated cd8 þ t cells in survivors. the knowledge that cd4 þ and cd8 þ t cell responses are against the ebov np will be useful for the design of efficient targeting vaccines. cchfv also causes hf yielding fatalities in up to 30% of cases. there is no current vaccine, and cchfv has recently reemerged in naïve countries in response to geographical relocation of its primary tick vector. infection by cchfv causes transient ig antibody responses, inversely correlating with sure fatality, but not correlating with clearance, viral load, or outcomes. no protection is granted by neutralizing antibodies either, implying that innate and t cell arms of immunity are critical for viral clearance. with little to no immune correlates of protection for cchfv, vaccine design has aimed at targeting the cchfv np or gps, where immunogenicity requires the administration of multiple doses to induce both neutralizing antibody responses and t cell responses. only recently, specific cd8 þ t cell epitopes against these cchfv proteins have been confirmed to stimulate cytotoxic cd8 þ t cells providing protection in survivors. despite many documented instances of virus eradication from populations by earlier vaccination strategies, there is a continuous imminent risk of outbreaks of pandemic proportions by existing and emerging viruses, as a result of anti-vaccination campaigns, unforeseen viral strain recombination, exposure of naïve populations to viruses by infected world travellers, a rapidly growing and aging and immunocompromised world population, acts of bioterrorism, and use of viruses as biological weapons in war. past strategies in the development of certain vaccines have often been lucky in their efficacies due to their eliciting both humoral and long-lasting adaptive t cell immunity, whereas others have failed and have only induced shorter lived humoral responses that do not always confer long-lasting protection. the precise and likely overlapping mechanisms dictating lifelong immunity conferred by successful vaccines against smallpox, measles, mumps, rubella, polio, and yf are not completely understood. what has become clear however is that innate immunity usually primes adaptive immunity to confer this long-term protection. it is not a lack of existing immune-monitoring methodologies but rather perhaps a lack of their correct implementation in the continued analysis of vaccinated individuals that are hampering the gaining of this important knowledge. therefore it seems there is an urgent need for the standardization of vaccine methodologies adequately measuring effector memory t cell responses and host-immune evasion mechanisms in appropriate animal models and humans and through the use of multiparametric 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presentation by the class i major histocompatibility complex fatal community-acquired pneumonia in children caused by re-emergent human adenovirus 7d associated with higher severity of illness and fatality rate cd4 þ t cells provide protection against acute lethal encephalitis caused by venezuelan equine encephalitis virus role of humoral versus cellular responses induced by a protective dengue vaccine candidate infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium genomic and oncogenic preference of hbv integration in hepatocellular carcinoma efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale protection from secondary dengue virus infection in a mouse model reveals the role of serotype cross-reactive b and t cells key: cord-028564-sltofaox authors: gutiérrez-spillari, lucia; palma m., geovani; aceituno-melgar, jorge title: obesity, cardiovascular disease, and influenza: how are they connected? date: 2020-07-06 journal: curr trop med rep doi: 10.1007/s40475-020-00207-0 sha: doc_id: 28564 cord_uid: sltofaox purpose of review: to better understand the impact of obesity and cardiovascular diseases on influenza a infection. recent findings: this infection could have detrimental outcomes in obese patients with cardiovascular diseases, such as an increased risk, length of hospitalization, disease severity, morbidity, and mortality. nevertheless, there also might be some cardioprotective benefits associated with influenza vaccination, such as a reduced mortality, hospitalization, and acute coronary syndromes, in patients with coronary heart disease and/or heart failure. summary: obesity negatively impacts immune function and host defense. recent studies report obesity to be an independent risk factor for increased morbidity and mortality following infection. obese patients might need special considerations in the treatment; however, there is not enough evidence to fully comprehend the mechanisms behind the reduced immunocompetence when influenza a infection occurs. future studies should focus on special consideration treatments when the patients have not been vaccinated and have cardiovascular diseases. this review focuses on how obesity and cardiovascular disease impact influenza response. retrospective studies demonstrate that during the 2009 h1n1 pandemic, obesity was identified as a risk factor for hospitalization, mechanical ventilation, and mortality upon infection. these data must be highlighted since it is projected that nearly 50% of the worldwide population is going to be obese by 2050. several case studies have identified possible effects of obesity on viral replication in the deep lung, progression to viral pneumonia, and prolonged viral shedding [1] . therefore, management of the influenza infection in this at-risk population has to have special consideration given that they may not respond optimally to vaccination [2] . excessive fat accumulation that results in obesity impairs health for adults [3] . its low-grade chronic inflammatoryinduced state negatively impacts immune function and host defense [2] , as shown during the 2009 influenza a virus h1n1 pandemic, where obesity resulted to be an independent risk factor for severe disease, hospitalization, mechanical ventilation, and mortality upon infection [1] . it is well known that influenza a virus infection is characterized by fever, myalgia, rhinorrhea, sore throat, and sneezing. these symptoms peak 3-5 days post-infection, with viral shedding peaking at days 2-3. usually, it is limited to the upper respiratory tract; however, in severe cases, the lower respiratory tract, including the lungs, can be affected and often requires hospitalization. this progression is more common in obese patients, leading to diminish infection resolution when compared with non-obese patients [1] . obesity also plays a role in the outcome of critical complications from influenza this article is part of the topical collection on metabolism in tropical medicine a/pdmh1n1 infection and is associated with longer mechanical ventilation for severe acute respiratory distress syndrome and shock [4] . a higher body mass index (bmi) and metabolic syndrome in patients with influenza have shown an increased risk and length of hospitalization [4] [5] [6] , increased disease severity, morbidity, and mortality during lower respiratory tract infections. this might be explained in part by increased lung permeability during infection, found in mice studies. obese mice have an increased protein leakage from the lung into the bronchoalveolar lavage fluid when compared to lean mice. additionally, lung edema and oxidative stress are also increased, which emphasizes the multiple etiologies of increased lung pathology in the obese host and the impairment in wound repair [1, 4] . the obesogenic state can also affect the influenza a virus evolution. it is well known that obese individuals are malnourished besides their excess in fat; they might also present nutrient deficiencies, such as vitamins [7] , minerals, and trace elements [8] . there are a variety of mechanisms by which nutritional imbalances could alter within-host viral evolution [8] . studies have shown that such imbalances prolong infections, delay clearance, and increase shedding (42% longer than non-obese) [9] , all of which potentially increase viral transmission [1] . in addition to the decreased immunocompetence mechanisms, other potential factors might contribute to its increased susceptibility to infection in the hospital setting. some examples, which are underlying diseases that affect mobility, can also increase risk for skin problems, prolonged visits at hospitals and nosocomial infections, alteration in the pharmacokinetics of some drugs, and an increased susceptibility to postsurgery infections [2] . thus, it is a complex problem that needs further evidence to develop better treatments for this increasing population. obesity causes a chronic state of inflammation in a generalized and constant way with negative effects on immunity. obese people have delayed immune responses to influenza virus infection and experience slower recovery from the disease. in addition, the efficacy of the treatment and the vaccine is reduced in this population causing an alteration of the viral life cycle and, coupled with an already weakened and delayed immune response, leading to a more serious condition. poor initial and adaptive responses to infection and vaccination create an impaired ability to respond appropriately to infection. the efficacy of the vaccine may decrease in obese humans; however, more studies are needed to better understand how the obese state affects infection control [1] . previous studies suggest that the severity of influenza virus infection is multifactorial and may be related to lung spread and repair, the formation of extracellular concentrates of neutrophils at the lung level; however, this mechanism in individuals is unknown [10] . the efficacy of the vaccine in human groups has shown that initial seroconversion rates are high in the obese population, but that over time there is a greater decrease in efficacy than that observed in non-obese populations [11] . influenza vaccine as a method of prevention is formulated each year, typically containing both influenza a and b. a study conducted in 2013-2015 aimed at evaluating whether obesity was associated with an increased risk of influenza for influenza and influenza-like illness among vaccinated obese and nonobese adults, finding that, among the obese, 9.8% had confirmed influenza or influenza-like illness compared with 5.1% of healthy weight participants. compared with the vaccinated healthy weight, obese participants had twice the risk of developing influenza or influenza-like illness (relative risk = 2.01, 95% ci 1.12, 3.60, p = 0.020); therefore, in this risk group, in the same way, vaccination is very important [12] . although it appears that in high-risk groups, such as the obese and overweight population, vaccination may not provide optimal protection, and because of the growing trend of obesity worldwide, the efficacy of the vaccine should be improved [10] . among cardiovascular disease patients, there is compelling evidence that shows lower risk of major adverse cardiovascular events, reduced hospitalization, and mortality [13] [14] [15] , being the greatest treatment effect was seen among the highestrisk patients with more active coronary disease [15] . the recent recommendation advocates the priority of vaccination against influenza in obese patients; a vaccination program should be fully evaluated in obese adults. high-dose vaccines designed to vaccinate people over 65 can also be used in the obese population [9, 10] . in the twentieth century during influenza epidemics, there has been an excess of mortality from cardiovascular disease [16] . a recent study that included 364 hospitalizations for acute myocardial infarction demonstrated an increased risk of acute myocardial infarction within one week after influenza virus infection to a risk that was six times higher than the risk during the year before or after the onset of infection [17] . cardiovascular complications associated with influenza infection include myocarditis, pericardial effusion, myopericarditis, right and left ventricle dysfunction, myocardial infarction, heart failure, stroke, and circulatory failure due to septic shock [13, [18] [19] [20] . the risk of myocardial infarction after mild respiratory infection returns to baseline within approximately 5 weeks, but in the case of pneumonia complicated by sepsis the risk persists up to 10 years after the infection [16] [21] . infectious agents (including influenza virus) have been implicated in the etiology of atherosclerosis [22] . there have been described several mechanisms by which influenza increases the risk of cardiovascular events; they may be related to pro-inflammatory mediators, sympathetic stimulation, and the activation of the coagulation cascade [19] . according to the fourth universal definition of myocardial infarction, there are 5 types of myocardial infarction based on clinical, electrocardiographic, and laboratory evaluation [23] . influenza infection can trigger type 1 and type 2 myocardial infarctions [16] . type 1 myocardial infarction is defined as myocardial ischemia caused by atherothrombotic coronary artery disease, and it is usually precipitated by atherosclerotic plaque disruption that can be rupture or erosion [23] . it is important to remember that atherosclerotic plaques also contain inflammatory cells, and pro-inflammatory cytokines, such as interleukins 1, 6, and 8, and tumor necrosis factor α are generated as a response of infection. these inflammatory cytokines can activate inflammatory cells in atherosclerotic plaques [16] [24] . acute influenza infection is associated to a procoagulant state that increases the risk of coronary thrombosis at sites of plaque disruption [16] [25, 26] . infection with influenza virus is associated with expression of genes that have been linked to platelet activation: h1n1 exposure increases platelet gene expression signature, which is associated with myocardial infarction [25] . type 2 myocardial infarction might be considered with a rise and/or fall of cardiac troponin values and evidence of an imbalance between myocardial oxygen supply and demand unrelated to coronary thrombosis. [23] . influenza infection produces a systemic inflammatory response with a resulting increase in heart rate and shortens the filling time during diastole compromising in that way, the coronary blood supply. if septic shock occurs, it may have a substantial adverse effect on coronary perfusion. in older patients with chronic coronary plaques, systemic inflammation causes cardiac metabolic mismatch increasing the risk of myocardial infarction [16, 18] . influenza infection is also associated with increased mortality in patients with heart failure [27] and is vulnerable to influenza-associated complications [28] [29] . this type of patients has limited cardiac and respiratory reserves and may not tolerate the metabolic demand and hypoxemia, exacerbating underlying cardiac disease probably due to an increased sympathetic nervous system activity, hypoxemia, and renal dysfunction that can lead to volume overload [28] . in a healthy heart, severe acute influenza infection produces pro-inflammatory cytokine level elevations that can cause acute myocarditis [28] [18] , characterized by a broad spectrum of symptoms that go from asymptomatic courses to signs of myocardial infarction to devastating illness with cardiogenic shock [30] [31] . myocarditis often results from common viral infections and post-viral immune-mediated responses [30] . in acute myocarditis, there is a high incidence of wall motion abnormalities. during influenza epidemics, 15% of the patients admitted to a military hospital with influenza infection had wall motion abnormalities on echocardiogram [18] . this becomes important, due to the fact that the myocardium inflammatory disease is regarded as a precursor of dilated cardiomyopathy [30] [32] . the electrocardiographic findings in patients with myocarditis range from nonspecific t-wave and st segment changes to st segment elevation resembling an acute myocardial infarction; supraventricular and ventricular arrhythmias can also be present. electrocardiographic findings that are related to poor clinical outcome include a qtc prolongation at 440 ms, an abnormal qrs axis, and ventricular ectopic beats [30] . a recent study developed to evaluate the incidence and hemodynamic consequences of right ventricular and left ventricular dysfunction in patients with h1n1 infection demonstrated that on admission 72% had abnormal ventricular function (46% had isolated left ventricular abnormalities and 39% had isolated right ventricular dysfunction) and 14% had biventricular dysfunction. on the follow-up, right ventricular function tended to worsen during hospitalization, but left ventricular function tended to normalize. however, patients with ventricular dysfunction needed more aggressive therapy and of rescue ventilatory strategies, such as inhaled nitric oxide, prone positioning, and extracorporeal membrane oxygenation [18] . during influenza epidemics, hospitalizations for cerebrovascular diseases increase [33] . an increase in incidence of ischemic stroke within 2 weeks after influenza infection has been suggested [34, 35] . protein c pathway and endogenous fibrinolysis are mechanisms associated with cerebrovascular ischemia and influenza infection [19] . influenza infection develops a prothrombotic state by increasing tissue factor expression and decreases fibrinolytic capacity by increased plasminogen-activator inhibitor-1 (pai-1) expression. this results in an unbalance between coagulation and anticoagulant pathways [36] . as in myocardial infarction, the relationship between systemic inflammation and stroke pathophysiology has shown that stroke often occurs in a pre-existing state of inflammation due to atherosclerosis, obesity, or infection [37] . the sixth joint task force of the european society of cardiology and other societies on cardiovascular disease prevention in clinical practice recommend that annual influenza vaccination can be considered in patients with established cardiovascular disease (class iib, level c) [38] , based on the fact that the risk of a cardiovascular event (myocardial infarction or stroke) is more than four times higher after a respiratory tract infection, with the highest risk in the first 3 days after infection [39] . there have been developed several studies that demonstrate that influenza vaccination reduces mortality, hospitalization, and acute coronary syndromes in patients with coronary heart disease and/or heart failure [13] . the mechanisms by which acute inflammation affect the risk of vascular events include the following: endothelial dysfunction, procoagulant state, and inflammatory changes in atherosclerotic plaques [39, 40] . it is also known that persistent systemic inflammatory activity is a risk of factor for cardiovascular disease, and higher interleukin-6 blood levels increase cardiovascular mortality at one year after pneumonia infection [41] . this systemic inflammatory response can be reduced by vaccination [13] . when estimating the cost and benefit of interventions to prevent pneumonia, the association of pneumonia with cardiovascular disease risk should also be considered [40] . patients with chronic heart failure are vulnerable to influenza-related complications (including secondary infections, such as pneumonia and acute heart failure exacerbations). recently, the paradigm-hf trial assessed associations between receiving influenza vaccine and cardiovascular death or heart failure hospitalizations, all-cause hospitalizations, and cardiopulmonary or influenza-related hospitalizations, concluding that vaccination was associated with reduced risk for death [42] . there have been described two possible mechanisms by which influenza vaccination may reduce cardiovascular events: unspecific and specific effects [13] . the unspecific mechanism is based on the fact that influenza infection causes a systemic inflammatory response, endothelial dysfunction, and a procoagulant state. these factors have negative effects on patients with previous cardiovascular diseases, such as ischemic heart disease and heart failure, causing acute heart failure, pulmonary edema, or destabilization of chronic ischemic heart disease, leading to myocardial infarction or sudden cardiac death [13] [15] . influenza vaccination reduces the risk fig. 1 possible cardioprotective mechanisms of influenza vaccination of infection and inflammation by decreasing the secretion of pro-inflammatory mediators, such as cytokines (that cause reduced myocardial contractility) and metalloproteinases (that cause adverse cardiac remodeling and plaque rupture); influenza vaccination also causes inhibition of platelet activation and cloth formation [13] [43] . the specific mechanism takes into account the immunological properties of the vaccine. the protective effect of the influenza vaccine has been demonstrated in multiple studies. to explain the pleiotropic effect of the influenza vaccine, the "antigen mimicry" between atherothrombotic plaque and influenza virus has been proposed [22] . it has also been proposed that there is an autoimmune "cross-reaction" between influenza and atherosclerosis [13] [44] . figure 1 summarizes the possible cardioprotective mechanism of influenza vaccination [13] . it is well studied that obese patients can develop cardiovascular diseases; however, it is less known that the lowinflammatory chronic state might affect host defense and immune cell dysfunction and infections, such as influenza a, could have detrimental outcomes in such patients, such as an increased risk, length of hospitalization, disease severity, morbidity, and mortality. cardiovascular diseases, such as ischemic heart disease and heart failure combined with influenza a infection, can trigger acute heart failure exacerbations that increase the overall mortality in a hospitalized setting. cardiovascular complications associated with influenza infection include myocarditis, pericardial effusion, myopericarditis, right and left ventricle dysfunction, myocardial infarction, heart failure, stroke, and circulatory failure due to septic shock. there have been described several mechanisms by which influenza increases the risk of cardiovascular events; they might be related to pro-inflammatory mediators, sympathetic stimulation, and activation of the coagulation cascade. while influenza vaccination is associated with a significant reduction in all-cause mortality risk in patients with heart failure, this cardioprotective mechanism may not function as intended in the obese population since they do not always respond optimally to vaccination. therefore, in an effort to prevent these complications and in the absence of special consideration treatments for this population, we strongly suggest a weight-loss approach. future studies should focus on developing targeted treatments that can combat the reduced immunocompetence that excess adiposity causes to the patient. particular interest, published recently, have been highlighted as: • of importance impact of obesity on influenza a virus pathogenesis, immune response, and evolution the impact of obesity on the immune response to infection immunity to influenza: impact of obesity high body mass index as a risk factor for hospitalization due to influenza: a case-control study epidemiology of severe influenza outcomes among adult patients with obesity in detroit association between vitamin deficiency and metabolic disorders related to obesity rna virus evolution, population dynamics, and nutritional status obesity increases the duration of influenza a virus shedding in adults obesity outweighs protection conferred by adjuvanted influenza vaccination obesity is associated with impaired immune response to influenza vaccination in humans increased risk of influenza among vaccinated adults who are obese cardioprotective effect of influenza and pneumococcal vaccination in patients with cardiovascular diseases influenza vaccines for preventing cardiovascular disease association between influenza vaccination and cardiovascular outcomes in high-risk patients acute infection and myocardial infarction acute myocardial infarction after laboratoryconfirmed influenza infection myocardial dysfunction during h1n1 influenza infection beneficial effects of vaccination on cardiovascular events: myocardial infarction, stroke, heart failure. cardiol cardiovascular manifestations associated with influenza virus infection severe infections and subsequent delayed cardiovascular disease influenza and cardiovascular disease: a new opportunity for prevention and the need for further studies fourth universal definition of myocardial infarction diffuse and active inflammation occurs in both vulnerable and stable plaques of the entire coronary tree: a histopathologic study of patients dying of acute myocardial infarction gene expression profiles link respiratory viral infection, platelet response to aspirin, and acute myocardial infarction. schulz c, editor sepsis, thrombosis and organ dysfunction relation of concomitant heart failure to outcomes in patients hospitalized with influenza association of influenza-like illness activity with hospitalizations for heart failure: the atherosclerosis risk in communities study decreased immune responses to influenza vaccination in patients with heart failure update on myocarditis current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases management of myocarditis-related cardiomyopathy in adults influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly temporal relationship between influenza infections and subsequent first-ever stroke incidence influenza-like illness as a trigger for ischemic stroke influenza and stroke risk: a key target not to be missed? influenza virus infection aggravates stroke outcome european guidelines on cardiovascular disease prevention in clinical practice. the sixth joint task force of the european society of cardiology and other societies on cardiovascular disease prevention in clinical practice (constituted by representatives of 10 societies and by invited experts. developed with the special contribution of the european association for cardiovascular prevention & rehabilitationg ital cardiol (rome) risk of myocardial infarction and stroke after acute infection or vaccination association between hospitalization for pneumonia and subsequent risk of cardiovascular disease inflammatory markers at hospital discharge predict subsequent mortality after pneumonia and sepsis influenza vaccination in patients with chronic heart failure: the paradigm-hf trial from vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: part ii influenza vaccine as prevention for cardiovascular diseases: possible molecular mechanism publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-019964-9leljj8j authors: nan title: recent research in infectious disease date: 2005-01-22 journal: j infect doi: 10.1016/j.jinf.2004.11.005 sha: doc_id: 19964 cord_uid: 9leljj8j nan value was 83.8%, and negative predictive value was 98.6%. the sensitivity of the assay was significantly higher than that of antepartum culture (54%). furthermore, many clinicians use a risk-based approach to gbs colonization by treating high-risk women with empiric antibiotics without waiting for culture results. the assay was also significantly more sensitive than the risk-factor approach (94% versus 42%) with regard to predicting intrapartum status. the quick and accurate results obtained with the idi-strep b assay during labor suggest that its use would lead to reduced rates of neonatal gbs colonization and the more judicious use of antibiotics. 2004;39:1129-35. how positive blood culture results are reported has an impact on outcomes of bsi st. louis (md consult)-many patients with bloodstream infection (bsi) do not receive adequate therapy, even after the final microbiologic report is available. the method of reporting microbiologic information to the physician has a significant impact on whether and how this information is used, according to the october 15 clinical infectious diseases. dr. emilio bouza and colleagues of hospital general universitario 'gregorio marañón' in madrid evaluated data from 197 bsis. microbiologic results were reported in 3 different ways. in 109 cases (group a), the physician was informed by telephone of the results and given a written report after the definitive results were known. in 99 cases (group b), physicians also received a written report at the bedside, along with written therapeutic recommendations. in 89 cases (group c), physicians also received clinical advice orally. during empiric treatment, 27.7% of patients received inappropriate treatment, and 13.7% received no treatment. these patients had significantly longer hospital stays (mean, 27.2 versus 19.4 days) and a higher risk of having clostridium difficile-associated diarrhoea (8.3% versus 1.9%), infection-related death (23.3% versus 13.6%), and all-cause mortality (30.8% versus 19.4%) than patients whose empiric therapy was appropriate. after the reports were received, therapy was changed for 52.3% of patients in group b and 53.1% in group c but for none of the patients in group a. consequently, the proportion of days during which patients received adequate treatment was significantly smaller in group a than in groups b and c (66.3% versus 92.1% and 91.2%, respectively). group a also received significantly fewer defined daily doses of appropriate antibiotics (16.4 versus 22.2 and 20.7 days) . the duration of hospitalization, mortality rates after reports were received, and costs also tended to be lower in groups b and c. these results suggest that written and/or oralalert reports providing clinical advice in addition to standard reports will improve the management of patients with bsi. dr. lynda anne szczech and colleagues of the women's interagency hiv study recruited 2,038 hiv-positive from october 1994 through november 1995. differences in the development of a new acquired immunodeficiency virus-defining illness (adi) or death were compared according to renal status and haart status. at baseline, 287 patients (14.1%) had proteinuria. these patients had significantly lower cd4c lymphocyte counts and higher viral loads than patients without proteinuria, both at baseline and before starting haart. before haart was widespread, proteinuria was associated with a significantly increased risk of developing a new adi (hazard ratio [hr], 1.37) and death (hr, 1.35). an elevated creatinine level was also a significant independent risk factor for mortality (hr, 1.72 per decrease in the inverse creatinine level). however, even after haart was initiated, proteinuria was a significant independent predictor of mortality (hr, 2.07). an elevated creatinine level was also a significant predictor of new-onset adi and death (hrs, 1.54 and 1.96 per decrease in the inverse creatinine level, respectively. whether the kidney modulates hiv disease or whether these findings merely reflect the impact of greater comorbidity is not known. what is clear, however, is that hiv-positive women with these findings need improved monitoring and more aggressive treatment. genetic and transmission analysis of helicobacter pylori strains within a family raymond j, thiberge j-m, chevalier c, kalach n, bergeret m, labigne a, et al. to look for evidence of intrafamilial infection, we isolated 107 helicobacter pylori clones from biopsied specimens taken from both parents and four children. we compared the sequences of two housekeeping genes (hspa and glmm) from these clones with those of 131 unrelated strains from patients living in different geographic regions. strain relationships within the family were determined by analyzing allelic variation at both loci and building phylogenetic trees and by using multilocus sequence typing. both hspa-and glmm-based phylogenetic trees showed east asian and african branches. all samples from family members showed natural mixed infection. identical alleles found in some strains isolated from the children and parents, but not in the strains isolated from unrelated patients, demonstrated that strains have circulated within the family. several mechanisms, such as point mutations, intragenic recombination, and introduction of foreign (african) alleles, were shown to enhance strain diversity within the family. increasing reports of the appearance of novel nonmultiresistant methicillin-resistant staphylococcus aureus mrsa (mrsa) strains in the community and of the spread of hospital mrsa strains into the community are cause for public health concern. we conducted two national surveys of unique isolates of s. aureus from clinical specimens collected from nonhospitalized patients commencing in 2000 and 2002, respectively. a total of 11.7% of 2,498 isolates from 2000 and 15.4% of 2,486 isolates from 2002 were mrsa. approximately 54% of the mrsa isolates were nonmultiresistant (resistant to less than three of nine antibiotics) in both surveys. the majority of multiresistant mrsa isolates in both surveys belonged to two strains (strains aus-2 and aus-3), as determined by pulsed-field gel electrophoresis (pfge) and resistogram typing. the 3 aus-2 isolates and 10 of the 11 aus-3 isolates selected for multilocus sequence typing (mlst) and staphylococcal chromosomal cassette mec (sccmec) analysis were st239-mrsa-iii (where st is the sequence type) and thus belonged to the same clone as the eastern australian mrsa strain of the 1980s, which spread internationally. four predominant clones of novel nonmultiresistant mrsa were identified by pfge, mlst, and sccmec analysis: st22-mrsa-iv (strain emrsa-15), st1-mrsa-iv (strain wa-1), st30-mrsa-iv (strain swp), and st93-mrsa-iv (strain queensland). the last three clones are associated with community acquisition. a total of 14 sts were identified in the surveys, including six unique clones of novel nonmultiresistant mrsa, namely, sts 73, 93, 129, 75, and 80slv and a new st. sccmec types iv and v were present in diverse genetic backgrounds. these findings provide support for the acquisition of sccmec by multiple lineages of s. aureus. they also confirm that both hospital and community strains of mrsa are now common in nonhospitalized patients throughout australia. use of multiple nucleic acid amplification tests to define the infected-patient 'gold standard' in clinical trials of new diagnostic tests for chlamydia trachomatis infections martin dh, nsuami m, schachter j, hook ew 3rd, ferrero d, quinn tc, gaydos c nucleic acid amplification tests (naats) can be used to define the infected-patient 'gold standard' for the purpose of designing studies of the performance of chlamydia trachomatis diagnostic tests. it is unclear how many test results run by different naats and what combinations of specimens comprise the best infected-patient gold standard. we approached this question with data from a large study of the performance of a new naat. data were available from three endocervical swabs and a urine specimen collected from each of 1,412 women and tested by three different naats. results from all three assays were used equally in a rotating fashion to define the infected-patient gold standard. multiple different infectedpatient gold standards for estimating swab and urine specimen sensitivity and specificity for one naat method were created by varying the number and combinations of swab and urine comparator results with two different naats, the effect of changing the infected-patient gold standard definition was determined by constructing receiver-operator-like curves with calculated sensitivities and specificities for each test. the one-positive-of-two-results or two-positive-oftwo-results (same or two different assays) infected-patient gold standard definitions produced low sensitivity and low specificity estimates, respectively. if four comparator naat results were used, the any-three-positive-offour-results definition or the at-least-one-specimen-positive-by-each-of-two-comparator-assays definition appeared to provide better combinations of sensitivity and specificity estimates. the any-two-positive-out-of-three-results definition resulted in estimates that were as good as produced with the former two definitions. this analytic approach provides a means of clearly visualizing the effects of changing naat-based infected-patient gold standards and should be helpful in designing future studies of new c. trachomatis diagnostic tests. prospective study of human metapneumovirus infection in children less than 3 years of age konig b, konig w, arnold r, werchau h, ihorst g, forster j most lower respiratory tract infections (lrtis) in children under the age of 3 years are due to respiratory syncytial virus (rsv). epidemiological, host, and viral factors eventually account for the severity of lrtis, but they do not completely explain it. human metapneumovirus (hmpv) was recently identified in children with lrtis. in a population-based prospective multicenter study (the pri.de study, conducted in germany over 2 years), we tested 3,369 nasopharyngeal secretions from children younger than 3 years of age with lrtis for rsv a and b, influenza viruses (ivs) a and b, and parainfluenza viruses (pivs) 1 to 3. of the children requiring intensive care (nz85), 18% had hmpv infections, and 60% of these children were infected with hmpv in combination with rsv. we did not detect hmpv in a randomly selected subset of rsv-positive nasopharyngeal secretions (nz120) from children not requiring intensive care support. hmpv was detected in !1% of virus-negative samples from patients without intensive care support (nz620). our data support the hypothesis that coinfections with rsv and hmpv are more severe than infections with either rsv or hmpv alone, at least in children younger than 3 years of age. candida parapsilosis is an important cause of bloodstream infections in the health care setting. we investigated a large c. parapsilosis outbreak occurring in a community hospital and conducted a case-control study to determine the risk factors for infection. we identified 22 cases of bloodstream infection with c. parapsilosis: 15 confirmed and 7 possible. the factors associated with an increased risk of infection included hospitalization in the intensive care unit (adjusted odds ratio, 16.4; 95% confidence interval, 1.8 to 148.1) and receipt of total parenteral nutrition (adjusted odds ratio, 9.2; 95% confidence interval, 0.9 to 98.1). samples for surveillance cultures were obtained from health care worker hands, central venous catheter insertion sites, and medical devices. twenty-six percent of the health care workers surveyed demonstrated hand colonization with c. parapsilosis, and one hand isolate was highly related to all case-patient isolates by tests with the dna probe cp3-13. outbreak strain isolates also demonstrated reduced susceptibilities to fluconazole and voriconazole. this largest known reported outbreak of c. parapsilosis bloodstream infections in adults resulted from an interplay of host, environment, and pathogen factors. recommendations for control measures focused on improving hand hygiene compliance. heikkinen t, silvennoinen h, peltola v, ziegler t, vainionpaa r, vuorinen t, kainulainen l, puhakka t, jartti t, toikka p, lehtinen p, routi t, juven t background: influenza vaccination of healthy children is encouraged because children are frequently hospitalized for influenza-attributable illnesses. however, most children with influenza are treated as outpatients, and scarce data are available on the burden of influenza in these children. methods: we performed a prospective study of respiratory infections in preenrolled cohorts of children %13 years old during 2 consecutive respiratory seasons (2231 child-seasons of follow-up). at any sign of respiratory infection, we examined the children and obtained a nasal swab for the detection of influenza. the parents filled out daily symptom diaries. of all the enrollees, 94% remained active participants in the study. results: the average annual rate of influenza was highest (179 cases/1000 children) among children !3 years old. acute otitis media developed as a complication of influenza in 39.7% of children !3 years old. for every 100 influenza-infected children !3 years old, there were 195 days of parental work loss (mean duration, 3.2 days). influenza causes a substantial burden of illness on outpatient children and their families. vaccination of children !3 years old might be beneficial for reducing the direct and indirect costs of influenza in children. moscicki ab, ellenberg jh, crowley-nowick p, darragh tm, xu j, fahrat s background: the risk of developing the human papillomavirus (hpv)-associated precancer high-grade squamous intraepithelial lesion (hsil) in human immunodeficiency virus (hiv)-infected adolescents is unknown. we examined the risk of developing hsil among adolescents with and without hiv infection. methods: hiv-infected (nz172) and-uninfected (nz84) girls aged 13-18 years who were participating in a multicenter study of primarily horizontally acquired hiv infections in adolescents (reaching for excellence in adolescent health care) and who did not have hsil on cytologic examination at study entry or at the first follow-up visit were followed at 6-month intervals. hiv-uninfected girls were recruited for comparison in a 2:1 ratio (hiv infected: hiv uninfected). the primary outcome was cytologic diagnosis of hsil confirmed by expert review. results: incidence of hsil by the end of follow-up was higher for hiv-infected girls than for hiv-uninfected girls (21.5% vs. 4.8%, respectively). in multivariate analysis, use of hormonal (either estrogen/progesterone oral combination or medroxyprogesterone acetate intramuscular) contraceptives, high cervical mucous concentrations of interleukin (il)-12, a positive hpv test, and persistent low-grade squamous intraepithelial lesion (lsil) were significantly associated with the development of hsil. conclusions: the incidence of hsil was alarmingly high in hivinfected adolescent girls. however, when other predictors were considered in multivariate analysis, hiv status was not retained in the model. the heightened risk for hsil associated with persistent lsil underscores the need to closely monitor hiv-infected adolescents with lsil. the risk for hsil associated with high concentrations of il-12 may be suggestive of a local immune dysregulation. the role of hormonal contraception as a risk factor deserves further investigation. little is known about the epidemiologic profile of trichomoniasis in men and its relationship to human immunodeficiency virus (hiv) infection. among men presenting for care for symptomatic sexually transmitted infections (stis) in malawi, trichomoniasis is not considered for first-line treatment. methods: we conducted a cross-sectional survey of 1187 men attending either a dermatology or sti outpatient clinic in the capital of malawi. men were interviewed, and the etiologies of the stis were determined. results: at the sti clinic (nz756 men), we identified 150 men (20%) with trichomonas vaginalis infection, 358 men (47%) with hiv infection, and 335 men (44%) with neisseria gonorrhoeae infection. at the dermatology clinic (nz431 men), we identified 54 (13%), 118 (27%), and 2 (0.5%) men, respectively. at both clinics, a lower education level and reporting never having used a condom were predictive of t. vaginalis infection. only at the dermatology clinic was older age associated with infection, and only at the sti clinic were marital, genital ulcer disease, and hiv-infection status associated with t. vaginalis infection. at the sti clinic, urethral symptoms attributable to trichomoniasis were more severe among hiv-positive men than among hiv-negative men. conclusions: given its high prevalence and the increased risk for hiv transmission, t. vaginalis infection should be reconsidered for inclusion in the malawi stitreatment regimen for men. 2004; 190(8):1448-55. pmid: 15378437 [pubmed-in process] role of human neutrophil peptide-1 as a possible adjunct to antituberculosis chemotherapy kalita a, verma i, khuller gk we report the role of human neutrophil peptide (hnp)-1 as an adjunct to antituberculosis (anti-tb) drugs. the combination of hnp-1, isoniazid, and rifampicin was evaluated against mycobacterium tuberculosis h(37)rv in vitro, ex vivo, and in vivo, and synergism was observed on the basis of reductions in minimum inhibitory concentrations (mics) of these agents. in vitro results revealed o1log unit reductions even when hnp-1 and anti-tb drugs were used at 1/16 mics. this combination was also found to be bactericidal against intracellular mycobacteria even at 1/8 mics of hnp-1 and drugs. hnp-1 used in conjunction with anti-tb drugs resulted in significant clearance of bacterial load from lungs, liver, and spleen of infected, compared with control animals. the effective therapeutic dosage of drugs could be reduced to half by supplementing hnp-1 in the therapeutic schedule. these results clearly suggest that hnp-1 can be used as adjunct chemotherapy with conventional drugs against tb. in this phase iii trial, dr. jo-anne h. van burik and the national institute of allergy and infectious diseases mycoses study group studied 882 children and adults undergoing hsct. about half were randomized to receive micafungin (50 mg; 1 mg/kg for patients !50 kg), whereas the others received fluconazole (400 mg; 8 mg/kg for patients !50 kg). drugs were administered once a day via infusion during the neutropenic phase of hsct (i.e., before engraftment), and they were continued until neutropenia resolved or day 42 after hsct, whichever came earlier, or until fungal infection developed. time to treatment success was also significantly shorter with micafungin. no excess toxicity occurred with micafungin as compared with fluconazole, and fewer patients receiving micafungin dropped out of the study due to drug-related adverse events (4.2% versus 7.2%, pz0.058). these results suggest that micafungin is an effective alternative to fluconazole for antifungal prophylaxis in neutropenic patients during hsct. the authors obtained baseline blood samples from 174 girls 12 to 15 years old (76% african-american; 24% white). seropositivity for hsv-1, hsv-2, and cmv was also assessed 3 years later. at baseline, 41% of subjects (71 girls) had a history of sexual activity; this proportion increased to 73% (127 girls) by the 3-year follow-up. overall, seropositivity for cmv increased from 71% to 81%, seropositivity for hsv-1 increased from 44% to 49%, and seropositivity for hsv-2 doubled, from 7% to 14%. among sexually experienced girls, hsv-2 seropositivity increased from 15% to 19%. these increases correspond with attack rates of 13.8 cases of cmv per 100 person-years and 3.2 cases of hsv-1 per 100 person-years in the entire cohort. among sexually experienced girls, hsv-2 attack rates were 4.4 cases per 100 person-years of sexual activity. at follow-up, generalized estimating equation modeling identified 4 factors significantly associated with hiv-2 seropositivity: it also saves money, according to a new study. dr. kent k. hu and colleagues of veterans affairs puget sound health care system in seattle, wash, and other institutions discuss their findings in the november 15 clinical infectious diseases. the authors developed a decision analysis model based on hospitalized patients most likely to require a cvc for 2 to 10 days (i.e., those in intensive care units, with immunosuppression, or receiving total parenteral nutrition). cvcs were inserted according to either msb techniques (person inserting the catheter must wear a head cap, face mask, sterile body gown, and sterile gloves and use a full-size sterile drape around the site) or in accordance with lessstringent measures (only sterile gloves and a small regional sterile drape). in the base-case analysis, the use of msbs actually lowered costs by $252 as compared with less-stringent infection control measures ($369 versus $621 per catheter inserted). the incidences of catheter-related bsi (2.8% versus 5.3%), catheter colonization (2.9% versus 5.5%), and death (0.4% versus 0.8%) were also lower with msbs. during sensitivity analyses-even over a wide range of clinical and economic assumptions-the use of msbs resulted in improved patient safety. these data suggest that the improved management of infection with the use of msbs also pays off by lowering medical costs. thus, even though msbs are sometime cumbersome and time consuming, the authors suggest that they should be routinely used during cvc insertion. 2004;39:1441-5. hypogonadism is a risk factor for gynecomastia in hiv-positive men st. louis (md consult)-some studies suggest antiretroviral therapy is a risk factor for gynecomastia in men with human immunodeficiency virus (hiv) infection. however, no association has ever been found between the duration of exposure to any antiretroviral drug and the development of gynecomastia. instead, it appears hypogonadism is a predisposing factor to gynecomastia in hiv-infected men, according to the november 15 clinical infectious diseases. dr. alejandra biglia and colleagues of the university of barcelona in spain and other institutions studied 2,275 men with hiv infection. clinical examination with confirmatory sonography showed 40 men, or 1.8%, had gynecomastia. this is similar to the prevalence of gynecomastia in the general population. these 40 cases were matched with 44 men with hiv but not gynecomastia, and clinical, demographic, and laboratory features were extensively compared between the 2 groups. the mean free testosterone index was significantly lower in the cases than in the controls (42.6% vs. 58.0%). hypogonadism was the strongest independent predictor of gynecomastia (adjusted odds ratio [or] 7.6). other factors increasing the risk of gynecomastia included hepatitis c virus infection (adjusted or 6.1) and lipoatrophy (adjusted or 5.6). furthermore, gynecomastia resolved in many patients with persistent gynecomastia after transdermal or intramuscular testosterone administration, and in fact improved without any intervention in a substantial proportion of men. the authors also compared antiretroviral drug use between the cases and controls. significantly more cases were taking stavudine (73% vs. 41%) or efavirenz (33% vs. 14%), but significantly more controls were taking zidovudine (34% vs. 10%). however, the duration of exposure to each drug did not differ significantly between cases and controls, which further argues against antiretroviral therapy as the cause of gynecomastia in hivpositive men. the authors conclude gynecomastia in hivinfected men appears to be related more to hypogonadism than to adverse effects of antiretroviral therapy. if gynecomastia proves to be related to hypogonadism, then hormone treatments may be able to reverse this lipodystrophy, and further study is warranted. clin infect dis 2004;39:1514-9. taira av, neukermans cp, sanders gd human papillomavirus (hpv) has been implicated as the primary etiologic agent of cervical cancer. potential vaccines against high-risk hpv types are in clinical trials. we evaluated vaccination programs with a vaccine against hpv-16 and hpv-18. we developed disease transmission models that estimated hpv prevalence and infection rates for the population overall, by age group, by level of sexual activity within each age group, and by sex. data were based on clinical trials and published and unpublished sources. an hpv-16/18 vaccine for 12year-old girls would reduce cohort cervical cancer cases by 61.8%, with a cost-effectiveness ratio of $14,583 per quality-adjusted life year (qaly). including male participants in a vaccine rollout would further reduce cervical cancer cases by 2.2% at an incremental cost-effectiveness ratio of $442,039/qaly compared to female-only vaccination. vaccination against hpv-16 and hpv-18 can be cost-effective, although including male participants in a vaccination program is generally not costeffective, compared to female-only vaccination. yokoe ds, noskin ga, cunningham sm, zuccotti g, plaskett t, fraser vj, et al. we evaluated antimicrobial exposure, discharge diagnoses, or both to identify surgical site infections (ssi). this retrospective cohort study in 13 hospitals involved weighted, random samples of records from 8,739 coronary artery bypass graft (cabg) procedures, 7,399 cesarean deliveries, and 6,175 breast procedures. we compared routine surveillance to detection through inpatient antimicrobial exposure (s9 days for cabg, s2 days for cesareans, and s6 days for breast procedures), discharge diagnoses, or both. together, all methods identified ssi after 7.4% of cabg, 5.0% of cesareans, and 2.0% of breast procedures. antimicrobial exposure had the highest sensitivity, 88% to 91%, compared with routine surveillance, 38% to 64%. diagnosis codes improved sensitivity of detection of antimicrobial exposure after cesareans. record review confirmed ssi after 31% to 38% of procedures that met antimicrobial surveillance criteria. sufficient antimicrobial exposure days, together with diagnosis codes for cesareans, identified more postoperative ssi than routine surveillance methods. this screening method was efficient, readily standardized, and suitable for most hospitals. miner al, sands ke, yokoe ds, freedman j, thompson k, livingston jm, et al. we investigated using administrative claims data to identify surgical site infections (ssi) after breast surgery and cesarean section. postoperative diagnosis codes, procedure codes, and pharmacy information were automatically scanned and used to identify claims suggestive of ssi ('indicators') among 426 (22%) of 1,943 breast procedures and 474 (10%) of 4,859 cesarean sections. for 104 breast procedures with indicators explained in available medical records, ssi were confirmed for 37%, and some infection criteria were present for another 27%. among 204 cesarean sections, ssi were confirmed for 40%, and some criteria were met for 27%. the extrapolated infection rates of 2.8% for breast procedures and 3.1% for cesarean section were similar to those reported by the national nosocomial infection surveillance program but differ in representing predominantly outpatient infections. claims data may complement other data sources for identification of surgical site infections following breast surgery and cesarean section. several studies have shown that nasopharyngeal sampling is more sensitive than oropharyngeal sampling for the detection of pneumococcal carriage in children. the data for adults are limited and conflicting. this study was part of a larger study of pneumococcal carriage on the navajo and white mountain apache reservation following a clinical trial of a seven-valent pneumococcal conjugate vaccine. persons aged 18 years and older living in households with children enrolled in the vaccine trial were eligible. we collected both nasopharyngeal and oropharyngeal specimens by passing a flexible calcium alginate wire swab either nasally to the posterior nasopharynx or orally to the posterior oropharynx. swabs were placed in skim milktryptone-glucose-glycerin medium and frozen at k708c. pneumococcal isolation was performed by standard techniques. analyses were based on specimens collected from 1,994 adults living in 1,054 households. nasopharyngeal specimens (11.1%; 95% confidence interval [ci], 9.8 and 12.6%) were significantly more likely to grow pneumococci than were oropharyngeal specimens (5.8%; 95% ci, 4.8 to 6.9%) (p!0.0001). few persons had pneumococcal growth from both specimens (1.7%). therefore, both tests together were more likely to identify pneumococcal carriage (15.2%; 95% ci, 13.7 to 16.9%) than either test alone. although we found that nasopharyngeal sampling was more sensitive than oropharyngeal sampling, nasopharyngeal sampling alone would have underestimated the prevalence of pneumococcal carriage in this adult population. sampling both sites may give more accurate results than sampling either site alone in studies of pneumococcal carriage in adults. microbiol 2004; 42(11):4974-6. pmid: 15528682 [pubmed-in process] effects of intrapartum penicillin prophylaxis on intestinal bacterial colonization in infants jaureguy f, carton m, panel p, foucaud p, butel mj, doucet-populaire f early-onset group b streptococcal (gbs) infections remain a leading cause of morbidity and mortality in infants. to prevent the vertical transmission of gbs and neonatal gbs infection, guidelines recommend intrapartum penicillin or amoxicillin prophylaxis. this intrapartum antibiotic prophylaxis (iap) is suspected to favor colonization by antibiotic-resistant bacteria. however, the effects of this prophylaxis on the patterns of acquisition of gastrointestinal bacterial flora in infants have never been studied. we collected stool samples from 3-day-old infants born to mothers who received intrapartum amoxicillin (antibiotic-exposed group; nz25) and to untreated mothers (non-antibiotic-exposed group; nz25). the groups were matched for factors known to affect intestinal microbial colonization: gestational age, type of delivery, and type of feeding. qualitative and quantitative differential analyses of the bacterial flora in stool samples were performed. similar numbers of infants in the nonantibiotic-exposed and antibiotic-exposed groups were colonized by aerobic bacteria and amoxicillinresistant enterobacteria (75 and 77%, respectively) (pz0.79). in contrast, significantly fewer infants in the antibiotic-exposed group than in the nonantibiotic-exposed group were colonized by anaerobic bacteria, especially clostridium (12 and 40%, respectively) (p!0.05). regarding intestinal bacterial colonization, the differences between antibiotic-exposed and non-antibiotic-exposed infants were remarkably few. the only statistically significant effect was the reduced initial bacterial colonization by clostridium in the antibioticexposed group. in our study, the use of iap did not favor colonization by beta-lactam-resistant bacteria. however, further evaluations are required to highlight the potential risks of the widespread use of antibiotics to prevent early-onset gbs infection. microbiol 2004; 42(11):5184-8. pmid: 15528713 [pubmed-in process] usefulness of routine epicardial pacing wire culture for early prediction of poststernotomy mediastinitis mekontso-dessap a, honore s, kirsch m, houel r, loisance d, brun-buisson c poststernotomy mediastinitis (psm) is one of the most serious complications of cardiac surgery, and its associated morbidity and mortality demand early recognition for emergency therapy. in this study, we investigated the usefulness of epicardial pacing wire (epw) cultures for the prediction of psm. among 2,200 patients who underwent a cardiac surgical procedure at our hospital between 1 january 1999 and 31 december 2001, 82 (3.7%) had psm; staphylococcus aureus was the organism (45.1%) most frequently isolated at the time of surgical debridement. epws from 1,607 (73.0%) patients, 73 (4.5%) of whom developed psm, were cultured. epw cultures from 466 (29.0%) were positive, most often (74.9%) for coagulase-negative staphylococci. epw cultures were truly positive in 26 cases, truly negative in 1,106 cases, falsely positive in 428 cases, and falsely negative in 47 cases (with sterile cultures in 35 cases and a culture positive for an organism different from that isolated at the time of debridement in 12 cases). epw culture had a positive predictive value of only 5.7% and a high negative predictive value (95.9%) for the diagnosis of psm, with an accuracy of 70.4%. however, the likelihood ratio of positive (1.27) and negative (0.89) tests indicated only small changes in pretest-to-posttest probability. therefore, a strategy of routine culture of epws to predict psm seems questionable. dual-probe assay for rapid detection of drug-resistant mycobacterium tuberculosis by real-time pcr wada t, maeda s, tamaru a, imai s, hase a, kobayashi k mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in mycobacterium tuberculosis. for rapid detection of drug-resistant m. tuberculosis in clinical specimens, a simple and applicable method is needed. eight taqman minor groove binder (mgb) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). the target of six mgb probes was the rpob gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpob gene, and one probe was designed as a tuberculosis (tb) control outside the rpob gene hot-spot. we also designed probes to examine codon 315 of katg and codon 306 of embb for mutations associated with resistance to isoniazid and ethambutol, respectively. our system was m. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. detection limits in direct and preamplified analyses were 250 and 10 fg of genomic dna, respectively. the system could detect mutations of the rpob, katg, and embb genes in dnas extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary tb. this system was much faster (3 h from dna preparation) than conventional drug susceptibility testing (3 weeks). results from the dual-mgbprobe assay were consistent with dna sequencing. because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drugresistant m. tuberculosis in clinical laboratories. to define methicillin-resistant staphylococcus aureus (mrsa) reservoirs in the community and their population dynamics, we studied the molecular epidemiology of a random sample (nz490) from a collection of 2154 inpatient and outpatient mrsa isolates during a 7-year period in san francisco. we noted a progressive replacement of type ii staphylococcal chromosomal cassette (scc)mec-bearing isolates with type iv sccmec-bearing isolates, which coincided with o4-fold increase in methicillin resistance between 1998 and 2002. type iv sccmec-bearing isolates involved in the increase in methicillin resistance belonged to 4 molecular genotypes. these 4 genotypes were associated predominantly with community-onset disease, rather than hospital-or long-term-care facilityonset disease (76.9% vs. 19.4% vs. 3.7%; pz0.0005), suggesting that they are not feral descendants of hospital isolates. the longitudinal results linked the dramatic increase in mrsa infections to an expanding community reservoir of mrsa genotypes with intrinsic community survival advantage. the outbreak of monkeypox in the midwestern united states during june 2003 marks the first documented human infection in the western hemisphere. consistent with those in outbreaks in africa, most cases in this outbreak were associated with febrile rash illness. we describe a cluster of monkeypox in a family with a spectrum of clinical illness, including encephalitis, and outline the laboratory confirmation of monkeypox. methods: standardized patient information was collected by questionnaire and medical chart review; all cases described were laboratory confirmed. laboratory methods included nucleic acid detection, viral culture, serologic testing, histopathologic evaluation, and immunohistochemical testing. results: of 3 family members with monkeypox, 2 had rash illness only, and 1 required hospitalization for severe encephalitis. the family member with the mildest clinical course had previously received smallpox vaccination. diagnostic testing by both polymerase chain reaction and culture revealed infectious monkeypox virus in skin lesions of all 3 patients; 2 patients had orthopoxvirus detected by immunohistochemistry in skin lesions. the patient with encephalitis had orthopoxvirus-reactive immunoglobulin m (igm) in cerebrospinal fluid. all patients had detectable igm responses to orthopoxvirus antigens. conclusions: these 3 patients illustrate a spectrum of clinical illness with monkeypox despite a common source of exposure; manifestation and severity of illness may be affected by age and prior smallpox vaccination. we report that monkeypox, in addition to causing febrile rash illness, causes severe neurologic infection, and we discuss the use of novel laboratory tests for its diagnosis. the proportion of infected cells to total cells was measured in serial breast milk samples collected from 291 hiv-1-infected women in nairobi, kenya, by use of real-time dna polymerase chain reaction amplification of bmcs. the number of infected bmcs per million cells was associated with levels of cell-free viral rna in breast milk previous studies demonstrated that the concentration of bmcs varies throughout lactation, and we used these data to transform infected bmcs per million cells to infected bmcs per milliliter. the estimated concentration of infected bmcs per milliliter was higher in colostrum or early milk than in mature milk (p!0.001) factors influencing increases in cd4 cell counts of hiv-positive persons receiving long-term highly active antiretroviral therapy smith cj, sabin ca, youle ms, kinloch-de loes s, lampe fc, madge s, cropley i, johnson ma, phillips an background: highly active antiretroviral therapy (haart) results in an improvement in immunologic function. we sought to investigate the factors associated with increases in cd4 cell count among human immunodeficiency virus (hiv)-positive antiretroviral-naive patients starting haart.methods: five hundred ninety-six subjects were followed for a median of 2.5 years (interquartile range, 1.0-4.0 years). factors associated with changes in cd4 cell counts in the first 3 months of haart and from 3 months onwards were analyzed.results: after 6, 12, and 24 months of haart, the median increases in cd4 cell counts were 114, 181, and 248 cells/mm(3), respectively; 84%, 84%, and 80% of subjects had a virus load of !400 copies/ml during the same periods. white ethnicity, higher pre-haart virus load, and lower pre-haart cd4 and cd8 cell counts were associated with greater increases in cd4 cell counts during the first 3 months of haart. from 3 months onward, a greater cumulative proportion of time spent with virus load !400 copies/ml was associated with a more favorable change in cd4 cell count (an average increase of 5.2 cells/mm(3)/year [95% confidence interval [ci], 3.8-6.7 cells/mm (3)/year] for each extra 10% cumulative time spent with a virus load !400 copies/ml) (p!0.0001). for every 100 cells/mm(3) higher in baseline cd4 cell count, the increase was 6 cells/mm(3)/year less (95% ci, 2-11 cells/mm(3)/year) (pz0.02). sex, risk group, age, and haart regimen were not associated with increases in cd4 cell counts.conclusions: these findings emphasize the importance of maintaining virological suppression and suggest other factors that influence long-term cd4 cell response. 2004; 190(10):1860-8. pmid: 15499544 [pubmed-in process] association of levels of hiv-1-infected breast milk cells and risk of mother-tochild transmission rousseau cm, nduati rw, richardson ba, john-stewart gc, mbori-ngacha da, kreiss jk, overbaugh j understanding how the level of human immunodeficiency virus type 1 (hiv-1)-infected breast milk cells (bmcs) affects hiv transmission via breastfeeding can shed light on the mechanism of key: cord-293151-g3758oes authors: nemzek, jean a.; lester, patrick a.; wolfe, a. marissa; dysko, robert c.; myers, daniel d. title: biology and diseases of dogs date: 2015-07-10 journal: laboratory animal medicine doi: 10.1016/b978-0-12-409527-4.00012-2 sha: doc_id: 293151 cord_uid: g3758oes historically, the dog played an important role as a laboratory animal in biomedical research. although numbers are declining, the use of dogs continues to be common in pharmacokinetics and cardiovascular studies. the normal biology of the dog as both a laboratory and a companion animal has been well studied and reference values are presented here as a clinical and experimental resource. this provides the necessary background to discuss the spontaneous diseases, including infectious and neoplastic conditions, prevalent in purpose bred as well as random source dogs used in biomedical research. in addition, diseases and conditions that arise secondary to the housing and experimental manipulation of dogs is discussed with emphasis on treatment and prevention. laboratory animal medicine mellitus. a comprehensive but concise review of the use of the dog as a research subject is available in gay (1984) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger dog breeds for use in surgical research studies. some specific breeds with congenital or spontaneous disorders have also been maintained by research institutions (see examples below). random-source dogs used in research are most frequently mongrels or larger dog breeds (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for "beagle" for the years 2012-2013, a significant portion of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. other common areas of research using beagles were dental and periodontal disease and surgery, orthopedic surgery, skeletal physiology, and imaging studies. other research areas that utilized beagles included canine infectious disease, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established and the organs of larger dog breeds are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies had been maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of the blood, most notably the neutrophil population. these dogs can be used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., 1995) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by the absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., 1994) . other genetic disorders studied in dog colonies include hereditary canine spinal muscle atrophy (cork, 1991) and narcoplepsy in doberman pinschers (ripley et al., 2001) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease) and the development of spontaneous diabetes mellitus and hypothyroidism has been studied in several breeds of dogs for comparisons with the human conditions. although historically the dog has been a common laboratory animal, their use in research has waned over the past 30 years. according to the u.s. department of agriculture (usda), animal and plant health inspection service (1998, 2011) , the number of dogs used in research has declined from 211,104 in 1979 to 75,429 in 1997 (prior to the previous edition of this text) and 64,930 in 2010. this decrease was caused by a variety of factors, including (but not limited to) decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), increased cost, and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purposebred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against rabies virus, canine distemper virus, parvovirus, adenovirus type 2, parainfluenza virus, leptospira serovars canicola, icterohaemorrhagiae, grippotyphosa, and pomona, and bordetella bronchiseptica (jasmin, personal communication). purpose-bred dogs are also usually treated prophylactically for intestinal helminths and ectoparasites, and possibly given a heartworm preventative. random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting and racing) or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. random-source dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations and tissue/organ harvest. options for procurement of dogs for biomedical research typically include purchase from a usdadesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title 9, chapter 1 (1-1-92 edition), subchapter a, animal welfare, 1.1 definitions, and 2.1 requirements and application (office of the federal register, 2002) . briefly, class a licensees are breeders who raise all animals on their premises from a closed colony. class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and record-keeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. in december 2013, the national institutes of health (nih) issued notice not-od-14-034 entitled notice regarding nih plan to transition from use of usda class b dogs to other legal sources (national institutes of health, 2013) . this nih policy begins in the fiscal year 2015 and prohibits the procurement of dogs from class b dealers using nih grant funds. from that point forward, dogs on nihfunded studies will have to be obtained from class a vendors, privately owned colonies (such as institutional breeding colonies), or client-owned animals (e.g., animals participating in veterinary clinical trials). the best resource for identification of possible vendors are online 'buyer's guide' sites or 'buyer's guide' issues of trade periodicals. online sites include the buyer's guide of the american association of laboratory animal science (http://laboratoryanimalbuyersguide.com), and the trade journals lab animal (http://guide.labanimal. com) and animal lab news (http://www.alnmag.com/ content/buyers-guide). a 'buyer's guide' typically lists sources for both purpose-bred and random-source dogs, and denotes such features as pathogen-free status, health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within issues of the journals. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal welfare act (7 cfr 2.17, 2.51, and 371.2[g] ) are described in 9 cfr chapter 1 (1-1-92 edition), subchapter a, animal welfare (office of the federal register, 2002) . regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats of part 3 (standards) of subchapter a. particular attention should be paid to section 3.6c (primary enclosures-additional requirements for dogs) because the space required for housing dogs is calculated using body length rather than weight (a parameter used for other species and also for dogs in the national research council (nrc) guidelines). section 3.8 (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute for laboratory animal research (ilar) has written the guide for the care and use of laboratory animals (national research council, 2011) . the 'guide' is the primary document used by institutional animal research units to develop their programs and by animal care evaluation groups, such as the association for assessment and accreditation of laboratory animal care international (aaalac international), to facilitate site visits and inspections. the primary difference between the 7th and 8th editions of the 'guide' (national research council, 1996 regarding the care of dogs is the notation that "enclosures that allow greater freedom of movement and unrestricted height (i.e., pens, runs, or kennels) are preferable." the ilar committee on dogs authored dogs: laboratory animal management (national research council, 1994) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. the information presented in the tables represents a range of normal values that can vary depending on the analytical method, as well as the age, breed, and sex of the animal. cohen, covance laboratories, inc., cumberland, va (2013) . physiological data for a mixed population of dogs of both sexes. fig. 12 .1 demonstrates the normal weights and corresponding ages for both male and female beagle and hound dogs. tables 12.2 and 12.3 feature hematology data from beagles of both sexes from two commercial facilities. tables 12.4 and 12.5 list serum chemical data for beagles of both sexes from two commercial facilities. representative blood gas, coagulation data, and normal urinalysis parameters can be found in tables 12.6-12.8, respectively. finally, the reviews in arterial and venous blood gas anaylses (rieser, 2013) and the manual of canine and feline cardiology (tilley et al., 2007) are excellent resources. good nutrition and a balanced diet are essential to the health, performance, and well-being of the animal. the nrc of the united states national academy of sciences is the leading provider of nutrient recommendations for dogs and provides average requirements needed to maintain growth and prevent deficiencies (subcommittee on dog and cat nutrition, 2006) . the nrc publications form the basis for the association of american feed control officials (aafco) nutrient profiles, which are updated periodically (baldwin et al., 2010) . the aafco is an advisory body comprising state representatives from across the united states. it provides a mechanism for developing and implementing uniform and equitable laws, regulations, standards, and enforcement policies, and establishes nutrient profiles for cat and dog foods (dzanis, 1994; thatcher et al., 2010) . additional resources should be consulted for details on the nutritional requirements for dogs of all ages (dzanis, 1994; subcommittee on dog and cat nutrition, 2006; baldwin et al., 2010; thatcher et al., 2010; hand et al., 2010) . recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment. the mer for most breeds may be calculated using the following equation: mer (metabolizable kcal/day) = bw × 0.75 × 550 kj de, where bw = body weight (kg), kj = kilojoules, de = digestable energy (kienzle and rainbird, 1991) . in-depth overviews of diets used in biomedical research are available in diet-specific literature. open-formula diets have defined concentrations of all ingredients and the information is publicly available. this allows researchers to control for this important environmental variable and enables retrospective analysis of possible diet composition effects on research results (barnard et al., 2009) . open-formula diets occasionally may require changes in formulation to maintain nutrient composition or meet changing nutrient requirements. these changes in quantitative ingredient formulation are made public when open-formula diets are modified. in contrast, closed-formula diets are commercially available, balanced diets that meet and label the minimum requirements for protein and fat and the maximum values for ash and fiber; however, the exact composition of ingredients may vary from batch to batch. ingredient composition varies as the manufacturer applies a leastcost strategy, referring to formulating diets to maximize profit by using the least-expensive ingredients. although the ingredients are listed, the quantitative ingredient formulation is not publicly available and can vary without public disclosure, due to proprietary nature of commercial diets produced and marketed under vendor trade names. closed-formula diets have also been referred to as as 'fixed formula' or 'constant nutrition' (labdiets, pmi nutrition international, st. louis, missouri) by manufacturers (barnard et al., 2009) . in fixed-formula diets, the quantitative ingredient formulation does not change; however, this information is proprietary and therefore laboratory animal medicine not disclosed publically (barnard et al., 2009) . semipurified and purified diets provide the strictest control of ingredients and are formulated from purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. although purified and semipurified diets do differ in the types of ingredients used, the terms are generally used to mean the same thing. purified-ingredient diets are generally 'open' formulas, meaning that they are published and available to the scientific community. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are safe for consumption up to 6 months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf-life, but the best strategy is to feed only fresh diets and use each lot based on the date of manufacture. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. from dr. asheley wathen, covance laboratories, inc., madison, wi, and dr. kimberley cohen, covance laboratories, inc., cumberland, va (2013) . overall health, body condition, nutrition, and age greatly influence reproductive efficiency (gavrilovic et al., 2008; johnson, 2008) . therefore, only normal, healthy animals in excellent body condition should be used in breeding programs. beagles between 2 and 3.5 years of age have the best conception rates and litter size with the lowest neonatal mortality. after 5 years of age, conception rates and litter size decline and neonatal mortality increases (johnson, 2008) . the vagina is a long, musculomembranous canal that extends from the uterus to the vulva. during physical examination, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva, avoiding the deep ventral clitoral fossa. examination should proceed at an angle of approximately 60° until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. female dogs are monoestrous, typically nonseasonal, spontaneous ovulators that have a spontaneous luteal phase approximately 5 days longer than the 65 ± 1 days of pregnancy followed by obligate anestrus. puberty (beginning of the first estrus) occurs between 6 and 14 months in most breeds. the time of onset positively correlates with the body size (concannon, 2011) . the canine cycle is divided into four phases: proestrus, estrus, diestrus, and anestrus. the duration of laboratory animal medicine proestrus is 5-20 days with an average of 9 days and reflects the follicular phase rise in estrogen. during this stage, the vulva is enlarged and turgid, and a serosanguinous vaginal discharge is present (concannon, 2011) . estrus may be from 5 to 15 days in duration but generally lasts 9 days. the endocrine feature of estrus is the first abrupt increase in progesterone (>5 ng/ml), which occurs concomitantly with the luteinizing hormone (lh) surge 95% of the time, followed by ovulation within 24-72 h. the vulva is softer and smaller than in proestrus. the vaginal discharge persists and may remain serosanquinous or become straw colored. diestrus begins approximately 9 days after the onset of standing heat. the end of this stage is 60 days later, which would be coincident with whelping if the bitch had become pregnant. defined behaviorally as starting when estrous behavior ceases (concannon, 2011) , diestrus represents the peak of serum progesterone. anestrous may last from 80 to 240 days and is the stage of reproductive quiescence. it is characterized by an absence of ovarian activity and serum progesterone levels of less than 1 ng/ml. the onset of puberty in the male ranges from 5 to 12 months of age and is affected by breed, season, nutrition, and disease status. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial or leydig's cells. at this time, the testicular growth is rapid, the seminiferous tubules begin to differentiate, and sertoli cells form the blood-testis barrier. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including, inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, whereas inhibin and estrogen provide negative feedback to the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in 45 days, with subsequent maturation of sperm occurring in the epididymis for approximately 15 days. thus, the entire process from the initiation of spermatogonial mitosis to the delivery of mature sperm to the ejaculate is 60 days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality semen production. supression of sexual behavior and problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities. animals with poor hind limb conformation or trauma to the back may be unable to properly mount the female. there is a positive correlation between scrotal circumference and the number of sperm produced. finally, the quality of sperm is assessed by motility, morphology, volume, and concentration. an ejaculate (5 ml) that contains approximately 500 million progressively motile sperm without significant morphological abnormalities is a good indicator of normal male fertility. complete anatomy of the bitch and dog can be found in miller's anatomy of the dog (evans and de lahunta, 2012) . cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. cornification occurs approximately 2 days prior to the estrogen peak and 4 days prior to standing heat. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear/ cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear 'anuclear' and are classified as 'cornified' or 'anuclear squames.' the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified 6 days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear/cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, observation of behavioral estrus is the best criterion to use in breeding management. during proestrus, the male is attracted to the bitch and will investigate her hindquarters, but she laboratory animal medicine will not accept breeding. estrus is characterized by proactive receptivity to mounting by males and increased male-seeking behavior (concannon, 2011) . during this stage, the bitch will exhibit 'flagging,' or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate and litter size, it is recommended to breed the bitch on days 1, 3, and 5 of the standing heat. due to the long life span of canine sperm, fertilization occurs in the oviduct up to 8 days after coitus. the ovulated oocyte is a primary oocyte that must undergo two meiotic divisions before fertilization can occur. this overall maturation process takes approximately 2 days. after maturation, the oocyte remains viable for 4-5 days. optimal conception rates tend to occur when the bitch is bred from 4 days before to 3 days after ovulation; best litter size is achieved when the bitch is bred 2 days after ovulation. implantation is evident by areas of local endometrial edema 17-18 days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary, indicating the placental villi are arranged in a belt, and deciduate, reflecting that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is 59-63 days. luteal progesterone is responsible for maintaining pregnancy and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than 1 ng/ml in late proestrus to a peak of 30-60 ng/ml during gestation, and then declines to 4-5 ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility (johnson, 2008; verstegen-onclin and verstegen, 2008) . pregnancy detection can be performed by several methods. abdominal palpation of the uterus may be most informative at approximately 28 days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings and are approximately 2 inches in length at 28-30 days. by day 35, the uterus begins to enlarge diffusely and the vesicles become difficult to identify by palpation. radiology can be used to confirm pregnancy and facilitate determination of gestational age, beginning 45 days after the lh surge (lopate, 2008) . bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. ultrasonography can be used to confirm pregnancy beginning on days 18-22, at which point the gestational sacs will be approximately 1 cm in diameter, and until parturition (shille and gontarek, 1985; lopate, 2008) . ultrasonography can assess fetal viability by visualizing fetal heartbeats and fetal movement beginning on gestational days 23-25 and 35, respectively (lopate, 2008) . it can also predict gestational age using the inner diameter of the chorionic cavity in early pregnancy and the biparietal diameter in late pregnancy luvoni and beccaglia, 2006) . however, ultrasonography for determination of gestational age is most accurate at day 30 of pregnancy when using correction factors for small (< 9 kg) and large (> 40 kg) body weight dogs (kutzler et al., 2003) . thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged because they may adhere to the umbilical cord and predispose to ascending infections. heat lamps may be placed 24 h prior to parturition and remain until all neonates demonstrate vigorous suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. monitoring of parturition is important, but human intervention should be minimal in order to prevent stressinduced cannibalism. an abrupt drop in body temperature to less than 100°f indicates impending parturition within 18-24 h. the process of parturition has been divided into three stages. stage 1 of labor lasts 6-12 h and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include panting and increased pulse rate (johnson, 2008) . fetal expulsion occurs during stage 2, which lasts approximately 3-6 h. as the fetus engages the cervix, there is release of oxytocin, referred to as the ferguson reflex, which strengthens the uterine contractions and may elicit abdominal contractions as well. the bitch is able to inhibit this stage of labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between deliveries of each pup is irregular, but the average is less than 1 h between pups. veterinary assistance laboratory animal medicine is necessary if the bitch remains in stage 2 for more than 5 h without delivering the first pup, or for more than 2 h before delivering subsequent pups. during stage 3 of labor, the placentas are expelled either immediately or within 15 min of delivery of each pup. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. the peripartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. oxytocin should not be used in the event of systemic illness or abnormalities precluding vaginal delivery. indications for its use include lack of delivery 24 h after onset of stage 1 labor, greater than 1 h of unproductive stage 2 labor, inadequate contractions, or abnormal vaginal discharge. in these cases, radiographs are recommended to assess fetal size in relation to the birth canal and any possible obstructions, followed by 0.25-2.00 iu of oxytocin intramuscularly or subcutaneously. the oxytocin can be repeated 30-60 min after the first dose for a total of two doses (plunkett, 1993) . in some cases, treatment with 0.5-1.5 ml/kg of 10% calcium gluconate, delivered slowly iv while monitoring closely for bradycardia, and 25% dextrose iv may be indicated. uterine involution occurs during anestrus within 4-5 weeks of parturition. during this time, a greenish to red-brown vaginal discharge, or lochia, is considered normal. the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the 6th postpartum week, with complete repair by 3 months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies, the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately 12 days, and ears are patent at approximately 12-20 days. solid food can be introduced between 4.5 and 6 weeks of age, and puppies can be weaned at 6-8 weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as a vaginal-vestibular stricture, narrow vagina, vaginal septum, and vaginal hyperplasia, or if there is a behavioral incompatibility between the male and female dogs (kutzler, 2005) . semen is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the scent of an estrous bitch and manually stimulated. the first two fractions are collected followed by a sufficient amount of the third fraction (predominantly of prostatic fluid) to bring the total semen volume to 4-6 ml. the semen can be introduced into the cranial vagina or directly into the uterus either through trans-cervical catheterization with a norwegian ai catheter or utilizing fiberoptic endoscopy. use of the norwegian ai catheter for intrauterine insemination of frozen-thawed, fresh, and chilled-extended semen results in significantly higher whelping rates than intravaginal insemination (linde-forsberg et al., 1999; thomassen and farstad, 2009) . for trans-cervical insemination, the bitch is either standing on all four legs or standing with hindquarters raised. the ai catheter and guiding tube are inserted into the vestibulum as far as the pseudocervix. firm abdominal palpation is then used to locate and fix the cervix in the other hand, at which point the catheter is further inserted along the dorsal vaginal fold until the cervical opening is located and semen is deposited into the uterus lumen (thomassen and farstad, 2009) . surgical and laparoscopic ai has been used successfully for intrauterine and intratubal insemination; however, these techniques are invasive and require anesthesia. therefore, the nonsurgical techniques mentioned above are recommended, as these approaches are less invasive and can be completed without anesthesia in nonsedated or sedated dogs depending on the experience of the personnel and personality of the dogs. ai with freshly collected sperm can be done on days 1, 3, and 5 of standing heat or on days of maximal vaginal cornification. the viability of frozen-thawed sperm is significantly reduced compared to fresh or chilled sperm that may live up to 5 or 6 days in the reproductive tract of the bitch; frozen-thawed sperm live only a few hours. therefore, the ova must be mature and insemination with frozen-thawed semen must be done 2-3 days after ovulation in the bitch as determined by serum progesterone concentrations (thomassen et al., 2006) . false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. a comprehensive review of canine pseudocyesis exploring its cause, clinical features, and treatments is covered by c. gobello (gobello et al., 2001) . reproductive performance in the bitch is optimal prior to 4 years of age. cycling does not completely cease; however, after 5-8 years of age, bitches demonstrate significant decreases in conception rate and the number of live pups whelped. by 8-9 years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia, are extremely common. dogs prefer living in a social environment. dogs have well developed olfactory glands, vision, and auditory and tactile senses that allow them to gain environmental cues and information from other dogs and humans (field and jackson, 2006; joint working group on refinement, 2004) . much of their instinctive behavior is dependent on learning to interact with other members of their species. beagles have been a popular animal model because of their docile nature. they are easily handled and, for the most part, respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiograms (ecgs), oral gavage, and venipuncture. although sexually mature by 6-9 months of age, dogs are not socially mature until 18-36 months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from 3 to 8 weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks 5 and 12, puppies are most capable of learning how to interact with people. by 10-12 weeks of age, dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the canine behavior section of the manual of clinical behavioral medicine for dogs and cats (overall, 2013) . by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of its disorders. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting, especially infectious diseases associated with the use of random-source dogs and conditions seen frequently in the beagle. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks. etiology canine infectious respiratory disease (cird) is a highly contagious illness and several organisms have been incriminated including bordetella bronchiseptica; streptococcus equi subsp. zooepidemicus; canine parainfluenza virus (cpiv); canine influenza virus (civ); canine respiratory coronavirus; canine adenovirus type 2 (cav-2); canine herpesvirus; canine reovirus types 1, 2, and 3; and mycoplasma and ureaplasma. naturally occuring infection can result in coinfection by two or more organisms (garnett et al., 1982; ford, 2012) . clinical signs cird can be subdivided into mild or severe forms. the mild form is more common and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough may be elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise, dogs are typically asymptomatic. mild tracheobronchitis usually lasts 7-14 days, even when untreated. the severe form results from poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and can be the determinant of severity (sherding, 1994) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. epizootiology and transmission the natural reservoir for b. bronchiseptica is the respiratory tract (bemis, 1992) , and it is very easily spread by aerosol and direct contact. transmission is heightened by confined housing of multiple animals. bordetella bronchiseptica is highly infectious with an incubation period of 3-10 days. pathogenesis the most common clinical isolates are cpiv and b. bronchiseptica (mochizuki et al., 2008) . however, b. bronchiseptica is often recovered from clinically healthy animals (chalker et al., 2003) . during clinical infection, b. bronchiseptica attaches to the cilia of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv laboratory animal medicine or cav-2 alone are usually subclinical but can cause necrotizing tracheobronchiolitis (dungworth, 1985) . diagnosis and differential diagnosis diagnosis is often based on clinical signs and known history; however, cough elicited by tracheal palpation may be inconsistent and should not be used for definitive diagnosis. presumptive diagnosis can be made by isolation of b. bronchiseptica or mycoplasma by nasal swabs. viral isolation or paired serology is often impractical and expensive. if cough persists for more than 14 days, other disease conditions should be considered. differential diagnoses include civ, canine distemper virus, pneumonia, heartworm disease, tracheal collapse, mycotic infections, and diseases resulting in tracheal compression (johnson, 2000) . prevention prevention is best achieved by avoiding exposure to infected animals. dogs should be vaccinated prior to or upon admission to the animal facility. intranasal vaccines protect against infection and disease and can be given to dogs as young as 3 weeks of age (greene and levy, 2012) . combination vaccines for b. bronchiseptica, cav-2, and cpiv are preferred. vaccinations should be boostered every 6 months when multiple animals are housed in a confined area. control staff must practice proper hygiene to prevent transmission by fomites. sanitation, proper ventilation, and proper humidity are critical for control. symptomatic animals should be isolated and kennels should be disinfected with agents such as bleach, chlorhexidine, or quaternary ammonium chloride. treatment bordetella bronchiseptica is sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur and should be continued for 14 days. for severe or unresponsive infection, treatment should be based on bacterial culture/sensitivity patterns. nebulized gentamicin or kanamycin may be helpful in severe cases. antitussives should be avoided if the cough is productive; however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction. research complications due to the altered respiratory tract physiology, infected animals should not be used for pulmonary studies. etiology β-hemolytic lancefield's group c streptococcus (s. equi ssp. zooepidemicus) is a gram-positive, non-spore-forming coccus that causes pneumonia and sepsis in dogs. clinical signs clinical signs vary based on the organ system affected. pneumonic disease is typically associated with sudden onset of clinical signs including coughing, weakness, fever, dyspnea, and hematemesis. the rapid progression of disease is similar to that seen in humans with toxic shock syndrome (tss) caused by streptococcus pyogenes. peracute death has been reported in research and shelter dogs (bergdall et al., 1996; pesavento et al., 2008) . epizootiology and transmission streptococcus equi ssp. zooepidemicus is not considered a commensal of healthy dogs as most of the β-hemolytic commensal organisms belong to group g, specifically streptococcus canis. asymptomatic carriers are suspected to be the route by which infection enters populations. streptococcus equi ssp. zooepidemicus is considered an opportunistic pathogen and stressful factors such as transport can predispose to disease (priestnall et al., 2010) . pathologic findings in peracute cases, hemorrhage from the mouth and nose and within the pleural cavity can be the most striking lesion. ecchymotic and petechial hemorrhages can be noted on several organs ( fig. 12 .2). 'bull's-eye' lesions may be observed on the pleural surface of affected lung lobes. histologic lesions can include fibrino-suppurative, necrotizing, and hemorrhagic pneumonia. gram-positive cocci can be found in intracellular clusters throughout the lung ( fig. 12. 3), tonsils, and spleen of affected animals (bergdall et al., 1996; priestnall and erles, 2011) . pathogenesis predisposing factors such as transport stress and viral coinfection have been shown to contribute to the virulence of s. zooepidemicus (priestnall and erles, 2011) . due to the similarities with the clinical signs seen in human cases of tss, superantigens are thought to contribute to the virulence of s. zooepidemicus in cases of acute hemorrhagic pneumonia. these superantigens work by bypassing the conventional mechanisms of antigen presentation and binding to major histocompatibilitity complex class ii receptors. as a result, there is a hyperactive proinflammatory response and an 'avalanche' of cytokines including interleukin 1β (il-1β), interleukin 6 (il-6), and tumor necrosis factor alpha (tnf-α). three novel superantigen-encoding genes have been identified from a case of acute fatal hemorrhagic pneumonia, szef, szen, and szep. however, it is currently unclear what effect these superantigens have in vivo priestnall et al., 2010) . while superantigens have been detected in some isolates, there is not enough data to determine if superantigens play a role in the pathogenesis (byun et al., 2009; kim et al., 2007) . diagnosis and differential diagnosis definitive diagnosis is based on bacterial culture of nasal swabs or transtracheal lavage. polymerase chain reaction (pcr) can be done on post-mortem lung tissue. bacterial pneumonias or bacteremias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and b. bronchiseptica. nonbacterial causes of respiratory disease include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control there is no vaccine for prevention of s. zooepidemicus. the organism has been isolated from the environment during active outbreaks (pesavento et al., 2008) , so dogs diagnosed with s. zooepidemicus should be quarantined and any potential fomites (e.g. food bowls, enrichment) should be properly disinfected. treatment antibiotic therapy should be based on culture and sensitivity. resistance to doxycycline and tetracycline has been demonstrated (garnett et al., 1982; pesavento et al., 2008) . research complications dogs with severe hemorrhagic pneumonia or systemic disease are not appropriate for research study. the association between epizootics of this disease and transportation supports operational policies that require adequate acclimation periods for animals upon arrival. etiology serovars canicola, bratislava, and grippotyphosa result in renal or hepatic disease, whereas serovars icterohaemorrhagiae and pomona predominantly result in hepatic disease . clinical signs canine leptospirosis can present as subclinical, acute, or chronic disease. clinical signs in acute infection can be nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, vomiting, muscle tenderness, and pyrexia. clinical signs can be related to renal failure including polyuria and polydipsia, with or without azotemia, oliguria, or anuria. leptospirosis can also lead to hepatic failure with signs such as icterus or bleeding abnormalities. peracute leptospirosis is characterized by shock, vascular collapse, and rapid death. uveitis, abortions, stillbirths, and pulmonary hemorrhage have also been associated with leptospirosis (klopfleisch et al., 2010; van de maele et al., 2008) . bivalent vaccines against the most common canine serovars, icterohaemorrhagiae and canicola, have resulted in the increased prevalence of other serovars including grippotyphosa, pomona, bratislava, and autumnalis. increased movement of wild animal reservoirs (rats, raccoons, skunks, opossums) into urban/suburban areas have also contributed to the greater prevalence of previously uncommon serovars (sykes et al., 2011) . transmission occurs primarily through environmental contact, although direct transmisson between hosts may also occur. leptospires passing from urine into water is the most common route of contamination (goldstein, 2010) . leptospirosis is a zoonotic disease. pathologic findings the kidneys consistently have gross and microscopic lesions. in the acute phase, the kidneys are swollen with subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and necrotic foci (searcy, 1995) . in chronic stages of leptospirosis, the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis the severity and course of leptospirosis depend on the causative serovar as well as the age and immune status of the dog. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly, reaching the renal tubular epithelium several days postinfection. acute or progressive renal failure leading to oliguria or anuria may occur. nephritis may or may not be accompanied by hepatitis, uveitis, pulmonary hemorrhage, and meningitis. disseminated intravascular coagulation is often a secondary complication. diagnosis and differential diagnosis paired serology for the microscopic agglutination test is the most reliable means of definitive diagnosis, and successive serum sampling should be done 7-14 days after the first sample. pcr can be used to identify active infection early in the disease when serologic testing is negative or in previously vaccinated animals (sykes et al., 2011) . differential diagnoses include other causes of acute renal failure and hepatitis. prevention and control according to the american animal hospital association's 2011 vaccination guidelines, vaccination for leptospirosis is recommended based on geographic location and exposure risk (welborn et al., 2011) . both quadrivalent and bivalent inactivated bacterins are available. quadrivalent bacterins protect against canicola, icterohaemorrhagiae, grippotyphosa, and pomona serovars, whereas bivalent bacterins cover only canicola and icterohaemorrhagiae. immunization does not prevent the development of the carrier state or protect against other serovars. control requires preventing contact with wildlife reservoirs as well as identification of carrier animals. treatment doxycycline is the drug of choice as it can eliminate renal colonization. if vomiting or allergic reactions prohibit treatment with doxycycline, ampicillin or other penicillins should be utilized. aggressive fluid therapy and supportive care may also be needed. research complications due to the zoonotic potential, dogs with clinical leptospirosis should not be used in research studies. etiology campylobacter spp. are thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rods. many species of campylobacter have been isolated from normal and diarrheic animals; however, the most common pathogenic species include campylobacter jejuni ssp. jejuni and c. coli (marks et al., 2011) . clinical signs most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than 6 months of age (greene, 2000; burnens et al., 1992) . in cases of clinical illness, mild and intermittent mucoid or watery diarrhea, with or without frank blood, is most commonly noted. signs typically last 5-21 days but can persist for several months. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea (marks et al., 2011) . bacteremia and cholecystitis secondary to c. jejuni have also been documented in dogs (fox, 2012) . epizootiology and transmission the role of campylobacter spp. as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, 1994) . stress or immunosuppression may make animals more susceptible. transmission is via the fecal-oral route, mostly through contaminated food or water. campylobacter jejuni can be zoonotic with immunocompromised individuals at greatest risk. pathologic findings lesions depend on the mechanism of the enteropathy (van kruiningen, 1995) . enterotoxin production results in dilated, fluidfilled bowel loops, with little or no histopathologic alteration. cytotoxin-mediated disease results in a friable, hemorrhagic mucosal surface. histologically, the mucosa is ulcerated with lymphoplasmacytic infiltration. translocation can result in edema and congestion of the lamina propria with focal accumulation of granulocytes. epithelial hyperplasia and decreased goblet cell numbers are also noted. campylobacter jejuni may be visualized between enterocytes with warthin-starry silver-stained sections. pathogenesis clinical disease may be produced by several different mechanisms as campylobacter spp. have a variety of virulence factors including enterotoxins, cytotoxins, and adherence or invasion properties. campylobacter jejuni can cause an erosive enterocolitis by invasion of epithelium and production of the cytolethal distending toxin (cdt) (fox, 2012; van kruiningen, 1995) . in addition, c. jejuni can produce illness via translocation to regional lymph nodes causing a mesenteric lymphadenitis. diagnosis and differential diagnosis fresh feces (per rectum) can be used for presumptive diagnosis by demonstration of highly motile, curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and johnson, 1994) . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of 37°c. a pcr multiplex assay for differentiation of c. jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus ssp. fetus has been developed (wang et al., 2002) . any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis. prevention and control proper environmental sanitation, waste disposal, and food storage can prevent campylobacteriosis. in enzootic situations, group housing should be avoided. outbreaks are controlled by isolation and treatment of affected individuals. treatment antibacterial treatment should be considered in severely ill dogs. erythromycin, neomycin, enrofloxacin, clindamycin, and doxycycline are all effective. resistance to quinolones and ciprofloxacin has been documented (acke et al., 2009) . treatment should be a minimum of 10-14 days with bacterial cultures repeating 1 and 4 weeks after treatment. research complications dogs with clinical campylobacteriosis have temporary derangements to digestive and absorptive functions. etiology helicobacters are gram-negative, microaerophilic, spiral bacteria that infect the gastrointestinal tract. helicobacter spp. can be separated into gastric and enterohepatic groups. the gastric helicobacters commonly identified in dogs are referred to as non(haesebrouck et al., 2011; joosten et al., 2013) . the most common enterohepatic species found in dogs include h. bilis, h. canis, and h. cinaedi (castiglioni et al., 2012; dewhirst et al., 2005; fox, 2012 (haesebrouck et al., 2009; fox, 2012) . clinical signs most infections are subclinical in the dog. gastric infections may present with vomiting, diarrhea, and fever, accompanied by anorexia, pica, or polyphagia. enterohepatic helicobacters have been linked with inflammatory bowel disease in experimental animal models. heavy infections in dogs have been associated with inflammatory lesions of the large intestine (castiglioni et al., 2012; nguyen et al., 2013) . epizootiology and transmission the epizootiology and transmission of helicobacter spp. in the dog remain unknown. both oral-oral and fecal-oral routes for transmission have been suggested in humans, but transmission via canine saliva is a less reliable source of infection (craven et al., 2011) . enterohepatic infections of pet dogs are as high as 52% (castiglioni et al., 2012) . prevalence of gastric helicobacter infections in colony or shelter dogs can be as high as 82-100% (fox, 1995; hermanns et al., 1995) . pathologic findings gastritis is usually mild and characterized by reduced mucus content of the surface epithelium with vacuolation, swelling, karyolysis, and karyorrhexis of parietal cells. multifocal infiltrates of plasma cells and neutrophils occur around blood vessels and between gastric pits (hermanns et al., 1995) . intestinal lesions include mild to moderate lymphoplasmacytic infiltration as well as crypt dilation and crypt hyperplasia (castiglioni et al., 2012) . pathogenesis gastric helicobacters are urease positive, which assists with survival in the acidic environment of the stomach (kusters et al., 2006; uberti et al., 2013) . enterohepatic helicobacters are urease negative and typically reside in the lower intestine. the mechanism by which enterohepatic helicobacters colonize the liver is thought to be through portal circulation after uptake by enterocytes or through retrograde movement from the intestine into the bile duct (fox, 2012) . diagnosis and differential diagnosis organisms may be demonstrated with histopathology on endoscopic or surgical biopsy tissue samples. warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. culture may be difficult depending on the helicobacter spp. for species that produce urease, a positive urease test on a gastric biopsy specimen may give a presumptive diagnosis. the urea breath test has been successfully used to diagnose helicobacter spp. in laboratory beagles with a sensitivity and specificity of 89% (kubota et al., 2013) . western blot has been used to detect serum antibodies to enterohepatic species and pcr can be used to detect helicobacter spp. in fecal samples (oyama et al., 2012; wadström et al., 2009) . any causes of acute or chronic vomiting and diarrhea in the dog are differential diagnoses. prevention and control until more is known about the epizootiology and transmission of helicobacter spp., specific recommendations cannot be made for prevention and control. treatment for gastric species, combination therapy of amoxicillin (10 mg/kg q12 h), metronidazole (30 mg/kg q24 h), and sucralfate (0.25-0.5 mg/kg q8 h) has proven to be most effective (hall and simpson, 2000) . replacing sucralfate with famotidine, omeprazole, or bismuth subsalicylate may also be effective (marks, 1997; jenkins and bassett, 1997; denovo and magne, 1995) . recurrence rates within 60 days of treatment can be as high as 80% (anacleto et al., 2011) . treatment of enterohepatic helicobacters may depend on species susceptibility. aminoglycosides have been successful in treating h. cinaedi, but resistance to fluoroquinolones has been documented (tomida et al., 2013) . combination therapy of amoxicillin, clarithromycin, metronidazole, and omeprazole in medicated chow has been successful in eliminating various enterohepatic helicobacters from mice (del carmen martino-cardona et al., 2010) . long-term antibiotic treatment at a minimum of 21 days is suggested for enterohepatic and gastric helicobacters. research complication dogs used in gastrointestinal physiology or oral pharmacology studies should be free from helicobacteriosis. etiology parvoviral enteritis in dogs is caused by canine parvovirus strain 2 (cpv-2) of the family parvoviridae, genus protoparvovirus, species carnivore protoparvovirus 1. currently, there are three antigenic variants, 2a, 2b, and 2c. parvoviruses are nonenveloped, single-stranded dna viruses. clinical signs while parvoviral infection can affect the gastrointestinal tract, bone marrow, myocardium, and nervous tissues, the most common manifestation of disease is acute enteritis. clinical signs usually appear 5 days after fecal-oral inoculation and include anorexia, fever, depression, vomiting, and profuse intractable diarrhea which may become hemorrhagic. excessive fluid and protein losses through the gastrointestinal tract result in rapid and severe dehydration. dogs can develop severe leukopenia with a total leukocyte count of 1000 cells/μl or less. repeated hemograms may provide prognostic value, as rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. epizootiology and transmission parvovirus can infect dogs of any age, but puppies between 6 and 20 weeks of age are particularly susceptible. puppies less than 6 weeks of age are protected by passive maternal antibody. strain cpv-2c has been associated with severe disease in adult vaccinated dogs (calderon et al., 2009) . pathogenesis canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes acute enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus, leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis parvovirus can be detected with a commercially available fecal enzyme-linked immunosorbent assay (elisa). due to intermittent and brief shedding of the virus, fecal elisas can have false-negative results. pcr can be used to confirm an elisa result and to differentiate the viral strain. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. prevention and control parvoviral-positive animals should be quarantined for at least 10 days as the infectious virus is shed for several days after onset of clinical signs. although pcr has been used to detect viral dna in feces for up to 6 weeks (decaro et al., 2005) , it is currently unknown if the material being shed at this time is still infectious. disinfection of exposed areas with dilute bleach (1:30) or a commercial disinfectant is essential for elimination of the virus. six-week-old puppies should be vaccinated every 2-4 weeks with a modified live vaccine until at least 16 weeks of age. treatment treatment is largely supportive and aimed at restoring fluid and electrolyte balance. antimicrobial therapy is recommended due to intestinal compromise and risk of sepsis. early nutritional support continued throughout the disease has been shown to decrease recovery times (mohr et al., 2003) . research complications infection with parvovirus precludes the use of a particular dog in an experimental protocol. due to the significant discomfort of the animal, as well as the intensive therapy required, humane euthanasia is usually chosen in a research setting. etiology rabies virus is a lyssavirus belonging to the family rhabdoviridae. clinical signs clinical progression of neurologic disease occurs in three stages. the first, prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, furious, stage, animals are easily excited or hyperreactive to external stimuli and will readily bite at inanimate objects. the third, paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death due to respiratory failure usually occurs after onset of the third stage. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact with infected saliva, usually via bite wounds. pathogenesis the incubation period for rabies is 3-8 weeks to the onset of clinical signs but can range from 1 week to 1 year. bites to the head and neck result in shorter incubation periods due to the close proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to neurons within the brain, resulting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis definitive diagnosis is based on immunofluorescence of the virus in negri bodies of hippocampal cells. submission of the whole, unfixed brain, including the cerebellum and proximal brain stem, should be done within 48 h of collection. the tissue should be kept refrigerated as freezing can cause delays in testing. differential diagnoses laboratory animal medicine include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. prevention and treatment puppies should be vaccinated by 16 weeks of age, again at 1 year, and then annually or triennially, depending on state and local laws. research complications immuno-prophylaxis is recommended for animal care and research personnel who may have work-related risks of exposure. due to risk of human exposure, animals with suspected infection should be humanely euthanized and brain tissue should be submitted for confirmation. giardiasis giardia lamblia, also known as g. duodenalis and g. intestinalis, is a binucleate flagellate protozoan that usually causes subclinical infestation of the small intestine. clinical disease is usually seen in young dogs and the characteristic sign is voluminous, light-colored, foul-smelling, soft to watery diarrhea, which is the result of malabsorption and hypersecretion. giardia has a direct life cycle with infection resulting after consuming cyst-contaminated food or water. the change in ph between the stomach and duodenum activates excystation and trophozoites then attach to the enterocytes. for diagnosis, direct fecal smears are considered best for observing trophozoites and zinc sulfate centrifugation is preferred for detection of cysts. a commercial elisa kit is licensed for use in dogs, but the positive predictive value is poor and zinc sulfate centrifugation techniques should be used in conjunction with elisa (rishniw et al., 2010) . pcr assays are also available for diagnosing giardiasis. differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. metronidazole at 25-30 mg/kg po q12 h for 5-10 days is effective at treating giardiasis as well as other enteric protozoans, which may be potential differential diagnoses or coinfections. albendazole, fenbendazole, pyrantel, and praziquantel are also effective. coccidiosis intestinal coccidia associated with enteropathy in dogs include isospora canis, i. ohioensis, i. neorivolta, i. burrowsi, and hammondia heydorni (dubey and greene, 2012) . coccidian oocysts can be found in feces of clinically healthy dogs, as well as animals with diarrhea. clinically affected animals are young or immunosuppressed and develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. coccidia oocysts are typically spread by fecal-oral transmission, but dogs can ingest monozoic cysts in intermediate host tissues. the coccidian life cycle is both sexual and asexual, and results in the release of unsporulated eggs, which sporulate under appropriate environmental conditions. other causes for diarrhea should be excluded before a coccidial etiology is implicated. treatment may not be necessary, as infections are typically self-limiting and clinically insignificant. treatment may help to limit the number of oocysts shed in a kennel-housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine (50-60 mg/kg po q24 h for 10-20 days) or trimethoprim sulfa (30 mg/kg po q8 h for 10 days). ascarids roundworms of dogs are most often toxocara canis; however, toxascaris leonina can also affect dogs. clinical illness is usually only seen in young animals with large worm burdens. diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization can be seen. puppies may have a classical 'potbellied' appearance. heavy infestations can cause intussusception and/or intestinal obstruction. puppies that experience lung migrations of larval worms can develop fatal pneumonia. toxascaris canis can infect dogs by transplacental migration, transmammary migration, or ingestion of infective eggs. the infective stage of t. canis is the third-stage larva (l3). in transplacental infections, puppies may be born with l3 larvae in their lungs (sherding, 1989) . for diagnosis, large (70-85 μm in diameter) and relatively round ascarid eggs can be seen by standard fecal flotation methods. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention (hall and simpson, 2000) . most anthelmintics are effective for treatment. puppies should be treated early and often (every other week until 16 weeks of age) because of the possibility of prenatal or neonatal infection. pregnant bitches can be treated with extended fenbendazole therapy (50 mg/kg po once a day from day 40 of gestation through day 14 of lactation). hookworms the most common and most pathogenic hookworm of dogs is ancylostoma caninum. ancylostoma braziliense can also be found in dogs, but only a. caninum infestation typically results in clinical illness. puppies with hookworm infections can present as anemic with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. these signs are a direct result of the worms' consumption of blood and body fluids. infective larvae (l3) are ingested from the environment and develop directly in the intestinal tract. infestation can also be transmammary, from ingestion of a paratenic host, and, less often, by transplacental migration. on histological sections, embedded worms with mouthparts may be identified. diagnosis is made by identification of eggs or larvae by either fecal flotation or direct smear. a differential diagnosis of parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in young dogs with anemia. pyrantel pamoate is the anthelmintic of choice because it is safest in young ill animals. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention and control (hall and simpson, 2000) . due to transplacental or milkborne infection, puppies should be treated q2 weeks from 2 to 16 weeks of age. whipworms trichuris vulpis, the canine whipworm, can cause acute or chronic large intestinal diarrhea. the adult worm resides in the cecum or ascending colon. most infections are subclinical, but in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss may also be seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. trichuris vulpis has a direct life cycle with eggs passed in the feces. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to diarrhea. factors that influence development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. whipworm infestation is diagnosed by the presence of barrel-shaped, thickwalled eggs with bipolar plugs on fecal flotation. adult worms intermittently release eggs; therefore, negative results do not exclude infection. differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for 3 months (jergens and willard, 2000) . several species of cestodes parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. most cestode infestations are subclinical, but severe infestations with dipylidium can cause diarrhea, weight loss, and poor growth. the cestode requires an intermediate host, which for d. caninum are fleas and lice. ingestion of these arthropods results in transmission of the tapeworm. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. the most significant means to limit cestode infestation is to control flea and/or louse exposure. praziquantel at 5-12.5 mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against d. caninum (hall and simpson, 2000) . demodicosis canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles and is passed from dams to nursing pups. localized demodicosis is typically asymptomatic, but disease can present with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and face, and around the ears (demanuelle, 2000a). generalized demodicosis can develop in juvenile or adult populations and is indicative of an underlying immunosuppressive disorder. demodex has a characteristic 'cigar shape' and can be identified from deep skin scrapings mounted on mineral oil (campbell, 2000; noli, 2000) . differential diagnoses include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma, which is also a common secondary complication of generalized demodicosis. ivermectin at 200-600 μg/kg and oral milbemycin at 1-2 mg/kg/day are effective treatments. treatment duration can be extensive and must be accompanied by repeated skin scrapings. sarcoptic mange canine sarcoptic mange is caused by sarcoptes scabiei var. canis, which is zoonotic. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas of the ear pinnae, elbows, ventral thorax, and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. adult mites, mite eggs, or mite feces may be observed on superficial skin scrapings, but diagnosis may be difficult because multiple skin scrapings may yield negative results. even if scrapings are negative, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige either in the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, 2000) . histologic examination is nondiagnostic; however, suggestive lesions include small foci of edema, exocytosis, degeneration, and necrosis . an important differential diagnosis is flea allergy dermatitis (fad). unless antiparasitic therapy would interfere with research objectives, all dogs with sarcoptic mange should be treated. in addition, their kennel mates should also be treated due to the contagious nature of the disease and its zoonotic potential. the usual means of treatment is either ivermectin at 200-400 μg/kg q14 days or milbemycin at 2 mg/kg q7 days for three oral doses . ticks ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, amblyomma, and ixodes. the primary significance of tick infestation is vector-borne infectious diseases, including rocky mountain spotted fever (rickettsia rickettsii), lyme disease (borrelia burgdorferi sensu stricto), thrombocytic anaplasmosis (anaplasma platys), and canine monocytic ehrlichiosis (ehrlichia canis). ticks alone cause minimal signs unless the dog develops a hypersensitivity reaction leading to a more granulomatous response at the bite location (merchant and taboada, 1991) . some species (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that causes an ascending flaccid paralysis (malik and farrow, 1991) . uncomplicated tick bites and tick-bite paralysis are diagnosed by identification of the tick and clinical signs of paralysis. dogs with tick-bite paralysis usually show improvement within 24 h of tick removal, with complete recovery within 72 h (malik and farrow, 1991) . formamidines (amitraz), pyrethroids, and phenylpyrazoles (fipronil) are available as spot-ons, collars, sprays, and foggers to treat tick infestations in both the animal and the environment (halos et al., 2014; beugnet and franc, 2012) . differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, 1991) . fleas the most common flea to infest dogs is ctenocephalides felis felis, the cat flea (sousa, 2010) . flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop severe fad, which features papules, crusting, and excoriations over the lumbosacral region, flanks, thighs and abdomen. these animals may require oral corticosteroids to relieve clinical signs (muller et al., 1983) . secondary bacterial and fungal infections can also develop. fleas can also transmit other parasitic diseases, such as dipylidium tapeworms. flea infestations and fad are definitively diagnosed by observing the fleas on the host's skin; however, the presence of flea excrement can support a presumptive diagnosis (demanuelle, 2000b) . treatment of flea infestations should use an integrated pest management (ipm) approach that targets adult fleas, immature stages, and environmental contamination in order to limit the risk of chemoresistance. combining ovicidal treatments, such as lufenuron and selamectin, with adulticidal treatments, such as fipronil, spinosad, selamectin, and imidacloprid, is recommended (halos et al., 2014; beugnet and franc, 2012; dryden et al., 2012) . certain chemicals (i.e., imidacloprid and selamectin) have both adulticidal and larvacidal abilities, but the principles of ipm preclude the use of one product solely for both adulticidal and larvacidal properties (schwassman and logas, 2009 ). differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs. canine dermatophytoses are commonly caused by microsporum spp., trichophyton spp., and epidermophyton spp. (moriello and deboer, 2012) . uncomplicated infections are characterized by circular areas of alopecia and crusting with or without follicular papules, usually around the face, neck, and forelimbs. dermatophytes infect the hair shaft and follicle, as well as the surrounding skin. infected hairs become brittle and broken shafts remain infective in the environment for months. dermatophytoses are zoonotic and easily transmitted to other animals through the environment or by direct contact. definitive diagnosis is made using dermatophyte test medium for culture. hair and crust material from infected sites can be plucked and placed on culture; however, the 'toothbrush' method is more effective for sampling multiple sites. the brush is used to comb hairs and scales from several infected sites and then pressed into the culture media. media plates should be visually inspected daily for 14 days. positive cultures will become red at the same time as growth of a fluffy white colony. microscopic examination of hairs and scales to visualize fungal elements can be done using skin scrapings in 20% koh or mineral oil; however, this method is not very sensitive. topical and systemic therapy should be initiated together after all suspected areas are clipped to reduce spreading of contaminated fragile hairs. wholebody topical therapies with antifungal shampoos, rinses, and creams are recommended rather than spot treatment. systemic therapy can be achieved with griseofulvin, ketoconazole, itraconazole, or fluconazole. due to the highly infective nature of this disease, animals should be isolated and the environment thoroughly disinfected. chlorhexidine and virkon ® s are ineffective at clearing environmental spores, but lime sulfur (1:33), enilconazole (0.2%), and bleach (1:10) are effective across many strains of microsporum canis (moriello and deboer, 2002) . although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, 1994) , deficiency in thyroid hormone can significantly laboratory animal medicine affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology primary hypothyroidism affects the thyroid gland directly, whereas secondary hypothyroidism has indirect effects through dysfunction of the pituitary gland (seguin and brownlee 2012) . both of these causes result in a gradual loss of functional thyroid tissue (avgeris et al., 1990; kemppainen and clark, 1994) . the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, 1962; beierwaltes and nishiyama, 1968; manning 1979) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, 1989; kemppainen and clark, 1994) . clinical signs because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including nonpruritic, bilaterally symmetrical alopecia, hyperpigmentation, seborrhea, and pyoderma (avgeris et al., 1990; peterson and ferguson, 1989; panciera, 1994) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, 1989; panciera, 1994) . normocytic, normochromic, nonregenerative anemia may be seen in approximately one-half of the cases (avgeris et al., 1990) . increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, 1989; panciera 1994) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, 1989) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, 1989) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bichsel et al., 1988; panciera, 1994) , and a true causal relationship with hypothyroidism has not been completely defined (panciera, 1994) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, 1989) . hypothyroidism can also cause abnormalities of the cardiovascular system including bradycardia, hypocontractility, increased vascular volume, and atherosclerosis (seguin and brownlee 2012) . abnormalities that may be detected by ecg include a decrease in p-and r-wave amplitude (peterson and ferguson, 1989 ) and inverted t waves (panciera, 1994) . these ecg abnormalities are caused by lowered activity of atpases and calcium channel function. an association between hypothyroidism and von willebrand disease has been suggested. however, the relationship is probably one of shared breed predilection and not a true correlation. contradictory studies have shown either deficient (avgeris et al., 1990) or normal (panciera and johnson 1994, 1996; avgeris et al., 1990) von willebrand factor antigen and bleeding times in hypothyroid dogs. most importantly, hypothyroidism does not appear to cause overt, clinical von willebrand disease. however, it may exacerbate existing subclinical von willebrand disease (seguin and brownlee, 2012) . epizootiology the prevalence of hypothyroidism in the general canine population is reportedly less than 1% (panciera, 1994) . the disorder occurs most often in middle-aged, larger breed dogs (avgeris et al., 1990) , and reports suggest a higher incidence of hypothyroidism in spayed, female dogs (panciera, 1994; peterson and ferguson, 1989) . doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism compared with other breeds (panciera, 1994; peterson and ferguson, 1989; scarlett, 1994) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, 1979; tucker, 1962; beierwaltes and nishiyama, 1968) . diagnosis and differential diagnosis because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, 1989; ferguson, 1994) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t 4 (thyroxine) and free t 4 (peterson and ferguson, 1989; ferguson, 1994) . t 4 serves primarily as a precursor for t 3 and is heavily protein bound. free t 4 represents the laboratory animal medicine unbound fraction that is available to the tissues (peterson and ferguson, 1989) . the measurement of total t 4 carries a sensitivity of around 95% and can be used as a good screening tool. with the measurement of both serum total t 4 and free t 4 , hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial may be in order (peterson and ferguson, 1989) . however, nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, 1989; ferguson, 1994) . therefore, low values do not always indicate that hypothyroidism is present and animals should not be treated solely on the basis of serum hormone levels if clinical signs are not present. if the clinical signs are equivocal or only total t 4 or free t 4 is decreased, further diagnostic testing is warranted (peterson and ferguson, 1989) . although t 3 is the most biologically active form of thyroid hormone, the measurement of serum t 3 levels is an unreliable indicator of hypothyroidism (peterson and ferguson, 1989; ferguson, 1994) . serum t 3 can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t 3 may be preferentially released and conversion of t 4 to t 3 may be enhanced by the failing thyroid (peterson and ferguson, 1989; ferguson, 1994) , particularly early in the disease. in one study, t 3 was within normal limits in 15% of the hypothyroid dogs (panciera, 1994) . autoantibodies can be responsible for false elevations in the concentrations of t 3 and t 4 found in these respective assays. it has been recommended that free t 4 , measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t 3 and t 4 . autoantibodies have been found in less than 1% of the samples submitted to one laboratory (kemppainen and behrend, 2000) . other means of diagnosing hypothyroidism have been described. in humans, endogenous thyroid-stimulating hormone (tsh) levels provide reliable information on thyroid status, and an assay is available for dogs. however, endogenous tsh levels can be normal in some dogs with hypothyroidism and high tsh levels have been noted in normal dogs and sick animals that are actually euthyroid. it is therefore recommended that tsh levels be considered along with other information (clinical signs, t 4 ) prior to diagnosis and treatment (kemppainen and behrend, 2000) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, 1989; ferguson, 1994) . another drawback of tsh testing is that the test must be postponed for 4 weeks if thyroid supplementation has been given (peterson and ferguson, 1989) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is 0.045 u of tsh per pound of body weight (up to a maximum of 5 u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and 6 h after. a normal response to the administration of tsh should create an increase of t 4 levels at least 2 μg/dl above the baseline levels or an absolute level that exceeds 3 μg/dl (peterson and ferguson, 1989; wheeler et al., 1985) . treatment the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is 0.01-0.02 mg/kg once a day (avgeris et al. 1990 ). if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in 6-8 weeks, and blood samples should be drawn 4-8 h after the morning pill. a clinical response is usually seen in 6-8 weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, 1994) . ecg abnormalities also return to normal (peterson and ferguson, 1989) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, 1994) . weight gain and eventual obesity are frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must address obesity and its potential effects on animal welfare and research results. etiology obesity is defined as a body weight 20-25% over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy, the result of overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, 1992) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household because access to food is more restricted, and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and limitation to exercise reduces energy expenditure. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy laboratory animal medicine expenditure are followed (butterwick and hawthorne, 1998) . as in humans, genetics plays an important role in the development of obesity in dogs, and certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, 1986) . several metabolic or hormonal changes are also associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese compared with intact females (macewen, 1992) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, 1986 ). in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, 1992) . differential diagnosis the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict the total body fat (wilkinson and mcewan, 1991) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see above), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to 60% of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to 50% produces no adverse health effects. however, t 3 levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of 1-2% of body weight per week (laflamme et al., 1997) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to have rebound weight gain after restrictions are relaxed. there has been a great deal of attention in humans as to the correct diet to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, 1992) . there has been much concern about the addition of fiber to the diet as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorie-restricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick et al., 1994; butterwick and markwell, 1997) . research complications it is important to control weight gain in research animals because of the association of obesity with metabolism. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, 1992) , this relationship is not consistently apparent (edney and smith, 1986) . joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, 1992; kealy et al., 1997) . in addition, diabetes mellitus has been linked to obesity and obesity-induced hyperinsulinism in several experimental models (macewen, 1992) . a recent study demonstrated metabolic disease, typified by hyperinsulinemia and hypoadiponectinemia, in approximately 20% of obese dogs (tvarijonaviciute et al., 2012) . research that requires anesthesia may be complicated by a greater risk of cardiovascular diseases (edney and smith, 1986) including hypertension and compromise to the respiratory tract. etiology in the laboratory setting, the majority of traumatic wounds will be small in size and quickly observed. occasionally, dogs may sustain minor trauma during transport or have a small, previously undetected, chronic wound upon arrival at the facility. when dogs are group housed, they may sustain bite wounds during early socialization periods. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or when the basic principles of wound management are not followed. clinical signs the signs and appearance of a traumatic wound will vary with the cause and the duration of time since wounding. abrasions, sustained by shear forces, are partial thickness skin wounds characterized laboratory animal medicine by minimal bleeding or tissue disruption. puncture wounds have a small surface opening but penetrate into deep tissues with the potential for contamination. lacerations are wounds caused by sharp separation of skin that may extend to deeper tissues. acute wounds are characterized by bleeding tissue, sharp edges and no obvious devitalization. they have variable degrees of contamination. chronic wounds generally do not exhibit active bleeding and will have curled or rounded edges. these wounds often have necrotic tissue and are considered contaminated. treatment to aid decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a 'golden period.' it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the competence of the host's defense systems (swaim, 1980; waldron and trevor, 1993) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as clean, clean-contaminated, contaminated, or dirty (see table 12 .9). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds require more aggressive therapy. postsurgical infections or complications of initial therapy would be considered dirty wounds. when in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of its classification. when first recognized, the wound should be covered with a sterile dressing until definitive treatment can be rendered. bleeding should be controlled with direct pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, 1980) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, 1980) . anesthesia or analgesia may be necessary and the choice of agent will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed. then a water-soluble lubricant gel may be applied directly to the wound to prevent it from further contamination during the hair removal process. a wide margin of hair should be clipped and a surgical scrub performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., 1990a, b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. several irrigation solutions have been recommended (lozier et al., 1992; waldron and trevor, 1993; sanchez et al., 1988) , but type may not be as important as the volume and pressure of delivery. it has been suggested that 8 psi is required to obtain adequate tissue irrigation, and this may be achieved by using a 35-ml syringe with an 18-or 19-gauge needle (waldron and trevor, 1993) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, 1993) . if one is unsure about tissue viability in areas that are devoid of waldron and trevor (1993) . extra skin, the tissue may be left (swaim, 1980; waldron and trevor, 1993) , and nonviable areas will demarcate within 2-3 days (waldron and trevor, 1993) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, 1980) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be placed. subcutaneous closure should be performed with absorbable suture such as polydioxanone, polyglactin 910, or polyglycolic acid. it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon (3-0 or 4-0). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. the wound may be covered by a nonadherent dressing such as vaseline-impregnated gauze (swaim, 1980) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within 3-5 days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after 5 days, this is considered secondary closure (waldron and trevor, 1993) . second-intention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, 1980) . it is important to note that second-intention healing will take longer than with surgical repair, and, in the case of large wounds, it will be more expensive because of the cost of bandaging materials. several factors must be weighed when considering the use of antibiotics in traumatic wound care, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. topical application of bacitracin, neomycin sulfate, and polymixin b combinations may be used in wounds with minor contamination. in skin wounds with more extensive contamination, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. when systemic antibiotics are necessary, cephalosporins, amoxicillinclavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, 1993) . prevention in facilities with good husbandry practices and a diligent staff, potentially injurious equipment or surfaces are identified quickly. appropriate attention to surgical technique and to initial wound care will generally reduce the occurrence of postprocedure wound infection. etiology pressure sores (decubital ulcers) can be a problem in long-term studies and housing situations that require chronic skin contact with hard surfaces. decubital ulcers often develop over a bony prominence such as the elbow, tuber ischii, tarsus, or carpus. the compression of soft tissues between hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, 1990) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores, including poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting coaptation devices (swaim and angarano, 1990) . clinical signs initially, the skin will appear red and irritated. over time, constant trauma can result in full-thickness skin defects and can progress to necrosis of underlying tissues. the severity of the sores may be graded from i to iv according to the depth of the wound and the tissues involved, from superficial skin irritation to involvement of underlying bone (waldron and trevor, 1993) . epizootiology the problem usually occurs in large dog breeds, but any type of dog can be affected. prevention and control minimizing or eliminating predisposing factors is important to both the prevention and treatment of this condition. if a dog will experience long periods of recumbency, adequate bedding or padding must be provided. recumbent animals should be moved frequently, ideally every 2 h, to prevent continuous compression on a specific laboratory animal medicine area (waldron and trevor, 1993) . skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain body weight will minimize compressive forces experienced over areas susceptible to ulceration (swaim and angarano, 1990) . treatment the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive diagnostics and therapy must be performed. the affected area should be radiographed to assess bone involvement and the wound should be cultured. all of the damaged tissue should be debrided and basic wound management guidelines should be followed (see above). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, 1990) . with extensive lesions, reconstruction with skin flaps may be necessary (waldron and trevor 1993) . bandaging should be performed on all full-thickness wounds; however, it is important to remember that illfitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound, in order to displace pressure over a larger area and onto healthier tissue. the doughnut is then incorporated into a padded bandage. if a cast has been applied to the area for treatment or research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, 1990) . bandages should be removed at least once or twice a day to allow wound care. etiology an acral lick granuloma is a skin lesion caused by self-trauma. in a few cases, the self-trauma is due to initial irritation caused by an identifiable neurologic or orthopedic condition (tarvin and prata, 1980) . allergy may also be a source of irritation that leads to self-trauma. however, the majority of cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, 1990) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., 1988) . the laboratory environment could promote the abnormal behavior and lead to acral lick granuloma. epizootiology the lesions associated with acral lick granuloma are seen most often in large dog breeds, particularly dobermans. however, any type of dog can be affected (walton, 1986) . clinical signs early lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, 1990) . the predilection for the limbs may be due to accessibility or possibly a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated and the wound develops a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of the aforementioned problems can be ruled out by the history of the animal. however, a complete history may be unavailable in the laboratory setting. fungal cultures and allergy testing may aid in diagnosis. biopsy of the affected area would rule out neoplasia. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, 1986) . prevention and control behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. environmental enrichment including exercise, co-housing and various toys is already a basic requirement and may be increased to combat self injurious behaviors. treatment several treatments have been reported for acral lick granuloma and the selection of a treatment should be based on the underlying cause. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, 1990; walton, 1986) . opioid antagonists have been applied as treatments for acral lick granulomas and self-injurious behaviors with the theory that this will block the effects of endogenous opioids. naltrexone and nalmefene have been used successfully to reduce excessive licking behaviors and resolve associated lesions. however, lesions did recur after the drugs were discontinued (dodman et al., 1988; white, 1990) . the topical administration of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide has also been shown to be effective (walton, 1986) . in addition, psychoactive drugs have been suggested to relief of laboratory animal medicine boredom or anxiety. these have included phenobarbital, megestrol acetate, and progestins. however, side effects have been reported (swaim and angarano, 1990) . other behavior-modifying medications such as clomipramine may be effective in the treatment of compulsive anxiety disorder. the potential for effects that could interfere with experimental results must be determined prior to initiation of treatment. it is important to note that none of the above-mentioned treatments have been successful in all cases. the overall prognosis for acral lick granuloma should be considered guarded since the lesions often recur when treatment is discontinued. etiology hygromas are fluid-filled sacs that develop as a result of repeated trauma or pressure over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., 1974) . epizootiology elbow hygromas are most frequently reported in large and giant breeds of dogs, less than 2 years of age (johnston, 1975; white, 2003; cannap et al., 2012) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment of research dogs, especially cage bottoms and cement runs, may predispose them to hygromas. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs the clinical presentation will depend upon the chronicity of the problem. a dog with an elbow hygroma usually presents with a painless, fluctuant swelling over the point of the elbow without signs of lameness. the condition may be unilateral or bilateral. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma becomes secondarily infected, the animal may exhibit pain and fever (johnston, 1975; white, 2003) . pathology the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is a yellow or red serous transudate. this fluid is less viscous than joint fluid and elbow hygromas do not communicate with the joint (johnston, 1975) . treatment the treatment of elbow hygromas should be conservative whenever possible. conservative management of the elbow hygroma is aimed at relieving the source of pressure at the point of the elbow. in early and mild cases, simply providing padding to cover hard surfaces will result in resolution of the hygroma. a soft padded bandage or doughnut bandage around the affected site may also be of benefit. neoprene/polyester sleeves that cover the elbows and fit over the shoulders are also available as an option for either prevention or treatment of hygromas (cannap et al., 2012) . more aggressive therapies, including needle drainage and injection of corticosteroids into the hygroma, have been described but are not recommended due to the risk of infection (johnston, 1975) . surgical options should be reserved for complicated or refractory cases. even simple excision can be associated with complications such as wound dehiscence and ulceration (johnston, 1975) due to the location of the bony prominence at the surgical site. this issue may be avoided by using a skin advancement flap (white, 2003) that allows intact, healthy skin to cover the boney prominence. a muscle advancement flap has also been described (green et al., 2008) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology in the research environment, corneal ulcers are most often associated with direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these would be rare in the laboratory setting. clinical signs the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may appear normal however, in cases of deeper ulceration, the cornea may appear roughened or have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. diagnosis a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers is made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies, abnormal eyelids, or abberant cilia. treatment the treatment of corneal ulcers will depend on the depth and size of the affected area, as well as the underlying cause. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given three times a day for 2-3 days usually provides adequate treatment. simple corneal ulcers are restained with fluorescein after 3 days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. deep ulcers may require further debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. ulcers caused by entropion, ectropion, or dystichiasis will not resolve until the condition is repaired, and descriptions for this can be found elsewhere. prevention the proper application of lubricant eye ointment at the time of anesthesia will prevent drying due to exposure and may also protect the eye from scrub solutions applied near the eye. early treatment of superficial ulcers should prevent self-trauma and progression of the wound. etiology research protocols often require the placement of chronic implants. implants such as cardiac or other biomedical devices may be the primary focus of the research study. implants may also be used as chronic monitoring devices, for delivery of compounds, or to collect serial samples. infection may occur at the time of implant. alternatively, the implant may serve as a nidus after hematogenous spread from other sources. one of the most common sources of infection is from colonization of the device from an external component, which is a frequent complication with indwelling catheters. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. one study (hysell and abrams, 1967 ) examined the lesions found at necropsy in animals with chronic indwelling catheters, which included traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. these lesions were primarily associated with catheter-induced trauma or secondary to embolization of fibrin. in a veterinary clinical setting, infections in peripheral catheters were more likely when the catheters were used for blood collection immediately after placement and when a 't' connector rather than a 'y' connector was used. . intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., 1993) . the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., 1993; kwei et al., 1995) . clinical signs dogs with implant infections may not exhibit signs initially . localized swelling around the implant may occur. in the case of indwelling catheters, signs may include redness and swelling of the skin around the external port or discharge from the skin wound. vascular access ports may develop fluctuant subcutaneous abscesses. in more severe cases, systemic signs may be noted (bach et al., 1998; hysell and abrams, 1967) . the systemic signs of infection are covered elsewhere in this chapter. treatment the treatment of catheter infections almost invariably requires removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, 1984; darif and rush, 1983 ). superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes, and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. localized abscesses or sinus tracts may be managed by establishing drainage and copious flushing. aerobic and anaerobic cultures of blood and locally infected sites should be performed prior to initial treatment (ringler and peter, 1984) . systemic antibiotic therapy should be initiated for a 10-day period. the choice of drug will ultimately be based on previous experience and culture results. if retention of a catheter is important, the catheter lumen may be safely disinfected with chlorine dioxide solution (dennis et al., 1989) . the solution is removed after 15 min and replaced with heparinized saline. all of the extension lines and fluids used with an infected catheter should be discarded. the blood cultures should be repeated 3 days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. prevention it is highly desirable to prevent complications that may result in loss of an implanted device. catheters and other implants should be made of nonthrombogenic material and be as simple as possible. a catheter with extra ports or multiple lumens requires additional management and supplies more routes for infection. the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. intravenous catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. ideally, catheters are secured to reduce movement and irritation of the skin, which may predispose to infection around external ports. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that long extension tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, 1984) . for intestinal access ports, catheter security may be improved with a synthetic cuff added to the end of the catheter allowing better attachment to the intestine (meunier et al., 1993) . after any catheter placement, animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide. in addition, a solution of the antibiotic ceftazidime used on alternate days with the heparin locking solution has been shown to effectively reduce infections in indwelling vascular catheters (bach et al., 1998) . throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. periodically, the placement of an indwelling catheter may be verified by radiography. when placed and managed correctly, catheters and ports of any kind may remain in place for months without complications. etiology sepsis is defined as the systemic response to infection caused by bacteria (gram negative and/or gram positive), fungi, or viruses. in laboratory animals, sepsis is most often seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course, dogs may present with signs of a hyperdynamic sepsis, including increased heart rate, increased respiratory rate, red mucous membranes, and a normal-to-increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals may show classic signs of septic shock including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, 1993) . pathogenesis the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell (wbc) count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of band neutrophils than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., 1997) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several areas should be evaluated for infection, including the urinary, reproductive, respiratory, alimentary, and cardiovascular systems, as well as the abdominal cavity (kirby, 1995) . treatment the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is infected, it should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or thirdgeneration cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. this category of sepsis treatment is the focus of much research. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no 'magic bullet' for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology in research animals, aspiration may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. pathogenesis aspirated compounds can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response, probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after 24-48 h. clinical signs the severity and clinical manifestation of aspiration lung injury are dependent upon the ph, osmolality, and volume of the aspirate. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on witness of aspiration, history consistent with aspiration, and/or the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. treatment the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. if the aspiration is witnessed, the mouth and, idealy, the upper airway should be cleared of residual material. when small amounts of a relatively innocuous substance (e.g., barium) have been aspirated, treatment may not be necessary. when severe inflammation is present, systemic as well as localized therapy may be necessary. oxygen therapy may be instituted; however, the concentration and time frame are controversial, because lung injury may be exacerbated by long-term administration of oxygen at high concentrations (nader-djahal et al., 1997) . fluid therapy may also be necessary in severe cases; however, cardiovascular support should be performed judiciously as fluid overload could lead to an increase in pulmonary edema. the use of colloids is also controversial because of the increase in vascular permeability that occurs in the lungs. several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. corticosteroids are contraindicated (raghavendran et al., 2011) . in humans, antibiotics are reserved for cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be immediately treated with antibiotics when the aspirated material is not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, 2000) . the presence of pneumonia should be verified by tracheal wash and cultures. prevention aspiration of drugs and other compounds may be avoided through careful administration of oral medications by experienced individuals. likewise, gavage or orogastric administration of liquids should be performed by experienced individuals, and the procedure should be aborted if coughing or other respiratory signs occur. the aspiration of stomach contents can largely be avoided by appropriate fasting prior to anesthesia for at least 12 h for food and 2 h for water. if appropriate fasting times are not observed, anesthesia should be postponed whenever possible, particularly if intended procedures require manipulation of the viscera or head-down positioning of the dog. if anesthesia cannot be avoided, it should be rapidly induced and the dog should be intubated. during recovery from anesthesia, the endotracheal tube should be removed with the cuff partially inflated and with the dog in a head-up position (haskins, 1993) . based on the source of energy, burn wounds may be categorized into four groups: thermal, chemical, radiation, and electrical. in laboratory animals, accidental burns are usually the result of thermal injury (heating pads, water bottles), chemicals (strong alkalis, acids, disinfectants, drugs), or experimental irradiation protocols. etiology inappropriate use of external heating devices is the most common cause of burns in laboratory animal medicine. the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs the clinical signs vary with the depth, location, and surface area of burn injury. classification systems for thermal burns are generally based on the depth of the injury, varying from superficial involvement of only epidermis to complete destruction of skin and subcutaneous tissues (bohling, 2012) . superficial burns appear erythematous and inflamed. in some cases, matting of the overlaying hair with exudate may be the first sign of a previously undetected skin lesion. progressive hair and skin loss may be evident over the first few days after injury (johnston, 1993) . although blistering is a characteristic of partial thickness burns in humans, this is rarely seen in dogs (bohling, 2012) . uncomplicated, superficial burn wounds heal by reepithelialization within 3-5 days. deeper burn wounds are characterized by a central area of nonviable tissue surrounded by edematous, inflamed tissues. a thick eschar, composed of the coagulated proteins and desiccated tissue fluid, develops over deep burn wounds. these wounds heal by granulation under the eschar, which will eventually slough. the amount of pain associated with burns depends upon several factors including the depth and area of the wound, procedural manipulations, and movement at the affected site (bohling, 2012) . pain associated with superficial burn wounds usually subsides in 2-3 days. theoretically, deep burns destroy nerve endings and result in less pain than superficial burns. however, inflammatory pain may still be present due to the tissue reaction around the necrotic site. in addition, sharp procedural pain and breakthrough pain have been described in humans during the healing phases of burn injuries and should be considered as potential complications in dogs as well (bohling, 2012) . severe and widespread accidental burn injury can result in clinical signs associated with multiple organs including the pulmonary, gastrointestinal, hematopoietic, and immune systems. in addition, extensive burn injury can predispose to infection and even sepsis. this type of injury with the associated complications would be extremely rare in the laboratory setting. treatment appropriate and timely treatment of a burn wound will reduce the extent of tissue damage and associated pain. thermal injuries should be immediately exposed to cool water (15°c) to reduce edema and pain. exposure to very cold water and ice does not improve outcomes (bohling, 2012) . topical wound dressings are recommended in the early stages of treatment for both partial-and full-thickness burns that are of small size. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, a thin film of a water-soluble, broad-spectrum antibiotic ointment should be applied to the wound surface. silver sulfadiazine has a broad spectrum, penetrates eschar, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it has been associated with pain upon application (demling and lalonde, 1989) . once a topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds (demling and lalonde, 1989; bohling, 2012) . after the initial treatment, burn wounds should be gently cleansed two to three times a day, followed by reapplication of the topical antibiotic and rebandaging (demling and lalonde, 1989) . systemic antibiotics are indicated in cases where local or systemic infection is present and their ultimate selection should be based on culture results. burn wounds can be extremely painful, and analgesia should be instituted immediately and adjusted accordingly throughout the treatment period. surgical intervention may be necessary in some cases. with small or moderately sized wounds, the eschar over the burn wound may actually impede wound contraction and reepithelialization. in such cases, once the eschar has become fully defined, a complete resection may improve wound healing. with large and severe burn wounds, repeated debridement by surgery or other means might be necessary. in the laboratory setting, a decision to pursue extensive surgical intervention would be dependent upon full consideration of the effects on animal welfare and research results. prevention thermal burns can be prevented in the research setting. electric heating pads and heat lamps should be avoided if possible. only heated water blankets or circulating warm air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore, these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. basic fire prevention precautions should be taken particularly around oxygen sources and flammable agents etiology chemical injury may be due to skin contact with concentrated solutions such as disinfectants or inadvertent exposure to laboratory chemicals. in addition, perivascular injection of certain drugs (pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin) have been associated with extensive tissue damage (swaim and angarano, 1990; waldron and trevor, 1993) . the mechanism of action will vary depending upon the ph, osmolality, and chemical composition of the agent and may include oxidation, reduction, disruption of lipid membranes, or other reactions (bohling, 2012; swaim, 1990; waldron and trevor, 1993) . clinical signs surface contact with chemicals may result in mild irritation and redness of superficial layers of the skin. however, many agents may cause progressive injury until the chemical reaction has been neutralized. this may result in tissue necrosis and secondary infection. the immediate signs of perivascular injection are withdrawal of the limb or other signs of discomfort and swelling at the injection site. the area may appear red, swollen, and painful as inflammation progresses. there may eventually be necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a 1-to 4-month period. this is because the drug is released over time from the dying cells (swaim and angarano, 1990) . treatment in cases of skin contact with chemical agents, the affected area should be thoroughly and repeatedly lavaged with warm water to dilute or remove the substance. the material safety data sheet for the substance should be consulted for any possible neutralization protocol. additional treatment will depend upon the severity of the tissue damage and will follow the same guidelines as for the thermal injury described earlier. for the treatment of perivascular injections, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions. the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, 1989) . the local infiltration of hyaluronidase accompanied by warm compresses has been suggested for perivascular vinblastine (waldron and trevor, 1993) and for doxirubicin. the use of dmso or another free radical scavenger, dexrazoxane, infused at the site has also been suggested for doxorubicin toxicities. despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, 1990) . in all cases, the condition can be painful and analgesia should be addressed. prevention prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. the material safety data sheets should be available for all compounds and storage recommendations followed closely. for intravenous administration of toxic compounds, insertion of an indwelling catheter is extremely important. prior to the injection, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. etiology radiation burns are generally a complication of therapeutic administration and are a result of free oxygen radical formation (waldron, 1993) . the severity of radiation burns and their treatment will depend upon the dose, frequency, total surface area, and location of the radiation. damage to epithelial layers of the skin can lead to desquamation. direct injury to fibroblasts results in decreased collagen production and poor wound healing. in addition, there may be fibrosis of blood vessels (pavletic, 2010) and subsequent hypoxia causing necrosis of deeper tissues. clinical signs the tissues most often affected are the skin and mucus membranes. with superficial injury, affected skin may exhibit hair loss and erythema, and produce a clear exudate. the intensity of the inflammation may increase for 1-2 weeks after the completion of radiation treatment. deeper and more serious injury manifests with subcutaneous fibrosis and can lead to disfigurement . the skin and underlying deep structures including the bone may become necrotic over several weeks (pavletic, 2010) . these deeper injuries are prone to infection due to their lack of blood supply. systemic signs such as vomiting are rare in dogs unless there has been direct radiation treatment to organs . treatment with superficial skin burns, the wound should be kept clean and should be covered if possible. in cases of oral mucous membrane damage, there may be special feeding requirements. when wounds are ulcerated, avascular tissues should be excised. treatments with silver sulfadiazine, mafenide acetate, or other topical agents are recommended to control infection. in addition, infection is avoided by closure of the wound as soon as possible. the goal of surgery is to cover the wound with healthy tissue to promote vascularization of the area. in some cases, this may require muscle and/ or skin grafts. prevention radiation burns can be limited by selection of appropriate, fractionated therapy and application of shielding to reduce exposure. prompt treatment of the injuries can reduce the occurrence of infection. since radiation is associated with poor wound healing, complications may arise when additional procedures are required. it is recommended to wait at least 1 week (pavletic, 2010) or even longer (laing, 1990) prior to administering radiation to a surgical site. after radiation, routine surgeries should be avoided for 1-2 months (pavletic, 2010 ). the prevalence of cancer in the general canine population has increased over the years (dorn, 1976) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, 1982) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. (1994) found death rates similar to the death rate of the at-large dog population (bronson, 1982) . approximately 22% of the male beagles died of cancer. the majority of the tumors were lymphomas (32%) and sarcomas (29%), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer (26% of the population studied), three-quarters had mammary cancer (40%), lymphomas (18%), or sarcomas (15%). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, 1975; benjamin et al., 1996) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. a complete review of clinical oncology in the dog is beyond the scope of this chapter but can be found elsewhere (withrow et al., 2013) . fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients with local anesthesia. an instrument such as a tru-cut ® needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a 1-mm × 1-1.5-cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies aid in histopathological examination and are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least 1 cm around the tumor and 3 cm if mast cell tumors are suspected (morrison et al., 1993) . incisional biopsies are performed when large softtissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. canine lymphoma represents 5-7% of canine tumors and a majority (85%) of canine hematopoetic disease (ettinger, 2003; vail and young, 2013) . whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide 2,4-dichlorophenoxyacetic acid as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, 1991) . in addition, tobacco smoke, environmental chemicals, and waste emissions are considered possible risk factors (marconato et al., 2009; gavazza et al., 2001) clinical signs multicentric high-grade lymphoma (mhgl) accounts for the majority of reported cases of canine lymphoma. depending upon grade, immunophenotype, and location involved, dogs with mhgl usually present with painless, enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, fever, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. less commonly, dogs develop alimentary, mediastinal, cutaneous, and extranodal lymphomas. alimentary lymphoma is associated with vomiting and diarrhea, in addition to clinical signs associated with mhgl. dogs with mediastinal lymphoma often present with respiratory signs (dyspnea and exercise intolerance) secondary to pleural effusion or cranial vena caval syndrome. hypercalcemia is most frequently associated with this form of lymphoma and may result in polyuria, polydypsia, and weakness. cutaneous lymphoma is an uncommon epitheliotrophic form of lymphoma. it is often referred to as mycosis fungoides and is typically of a cd8+ t-cell immunophenotype. it varies in presentation from solitary to generalized and may mimic any of a number of other inflammatory skin disorders including oral mucosal lesions. the lesions may occur as erythema, plaques, erosions, scales, nodules, crusts, hypopigmentation, and alopecia (fontaine et al., 2009) . approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement (e.g., nervous system: seizures, paresis, paralysis). epizootiology the incidence of lymphoma is highest in dogs 5-11 years old, accounting for 80% of cases. although the neoplasm generally affects dogs older than 1 year, cases in puppies as young as 4 months have been reported (dorn et al., 1967) . no gender predilection has been reported. diagnosis and pathologic findings a fine-needle aspirate is initially performed on accessible lymph nodes. thoracic radiographs and abdominal ultrasound ± fine needle aspiration of the liver or spleen can be used if mediastinal or abdominal involvement is suspected. additional staging can be determined through complete blood counts, serum biochemistry, flow cytometry for immunotyping, bone marrow aspiration, or surgical lymphadenectomy and histology. enlarged neoplastic lymph nodes vary in diameter from 1 to 9 cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. flow cytometry and lymphoblastic markers (cd34) can aid in diagnosis and subtyping of tumors. in addition, positron emission tomography is being explored for detection of extranodal and metastatic lymphoma (leblanc et al., 2009; marconato, 2011; elstrom et al., 2003) . classification of lymphoma types is based upon cytological, morphological, and immunological characteristics using the kiel classification criteria (vail and young, 2013) . histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. lymphoma subtypes can be further characterized based upon genetic, molecular, and immunological criteria (ponce et al., 2004) . pathogenesis all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is 4-6 weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, 1991) . median survival time with aggressive therapy is generally less than 12 months. hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. differential diagnosis differential diagnoses for multicentric lymphoma include systemic mycosis; salmon-poisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections for histopathologic examination may be needed. treatment therapy for lymphoma primarily consists of one or a combination of several chemotherapeutic agents. in addition, radiation therapy and bone marrow transplantation have been utilized. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. although treatment may induce clinical remission and prolong short-term survival, most treatment is palliative and aimed at improving quality of life. a thorough discussion of therapeutic options for the treatment of lymphomas in the dog can be found elsewhere (chun, 2009; marconato, 2011) . future directions include development of molecular and cellular targeted therapies to enhance traditional chemotherapy treatment, prolong remission, and treat immunologic subtypes of lymphoma (e.g., t-cell lymphoma). research complications given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with significant clinical illness. etiology mast cells are derived from cd34+ bone marrow progenitor cells. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog and may account for up to 21% of canine skin laboratory animal medicine tumors (bostock, 1986; welle et al., 2008) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin, lung, liver, and gastrointestinal tract. current research has linked mast cell tumor development to multifactorial causes including breed predisposition and a genetic component, chronic inflammation, and mutations in the surface growth factor, c-kit (ma et al., 1999; reguera et al., 2000; webster et al., 2006) . clinical signs well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, 1-to 10-cm nodules in the dermis and subcutaneous tissue. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema to surrounding tissues. mast cell tumors can be found on any portion of the dog's skin but frequently affect the trunk and hind limb extremeties along with perineal and preputial areas. the tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. abdominal organs are rarely involved but may be associated with anorexia, vomiting, melena, abdominal pain, and gastrointestinal ulceration. mast cell tumors have also been reported in extracutaneous areas such as the salivary glands, larynx, nasopharynx (london and thamm, 2013) and conjunctiva (fife et al., 2011) . mast cell tumors within the perineal, preputial, or inguinal areas are associated with a greater predilection for recurrence or metastasis (misdorp, 2004) . epizootiology these tumors tend to affect middleaged dogs but have been observed in dogs ranging from 4 months to 18 years (pulley and stannard, 1990) . pathologic findings because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. (1986) has become widely accepted. in this system, grade i has the best prognosis and are well differentiated, with round to ovoid, uniform cells with distinct cell borders. the nuclei are round and regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have the worst prognosis. the cells contain large, irregular nuclei with multiple prominent nucleoli and few cytoplasmic granules. mitotic figures are much more frequent. cells are pleomorphic with indistinct borders. in addition to associated skin lesions (e.g., ulceration, collagenolysis, necrosis, and infection), mast cell tumors have been associated with gastric ulcers in the fundus, pylorus, and/or proximal duodenum, most likely secondary to tumor production of histamine. histamine stimulates the h 2 receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (>75%) of dogs with mast cell tumors (howard et al., 1969) . pathogenesis although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver, and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis using fineneedle aspiration, mast cell tumors can be distinguished cytologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue to metachromatically stain the cytoplasmic granules red or purple. mast cell granules can also be stained with wright's, giemsa, and romanowsky stains. in addition, mast cells may contain tryptase, chymase, or both (fernandez et al., 2005) . histological evaluation is generally required for grading. examination of regional lymph nodes may be warranted if metastatic or systemic disease is suspected. in addition, radiographs and ultrasound with guided aspirates of the liver, spleen, or sublumbar lymph nodes can be used to determine metastatic disease. treatment depending upon the grade, initial treatment for mast cell tumors is generally wide surgical excision (3-cm margins), which may be followed by radiation, chemotherapy, or glucocorticoid therapy. aspiration or surgical removal of regional lymph nodes is recommended if lymphatic tumor drainage is suspected. if the tumor is not completely resectable or is grade ii or iii (moderately to undifferentiated), then debulking surgery and adjunct therapy may be used. treatment algorithms are outlined elsewhere (withrow et al., 2013) research complications because of the potential for systemic release of substances such as histamine, vasoactive substances, heparin, eosiniphilic chemotactic factor, and proteolytic enzymes, along with the possibility of delayed wound healing and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed monthly. grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every 3-6 months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the canine transmissible venereal tumor (ctvt) is transmitted horizontally to the genitals by coitus (nielsen and kennedy, 1990) . ctvt is a 'parasitic-like' tumor that appears to have originated from dogs or wolves thousands of years ago and despite immense mutation, ctvt adapted, survived, and spread across multiple continents making it the oldest known continuously passaged somatic cell line (rebbeck et al., 2009; murchison et al., 2014; murgia et al., 2006) . it has been described as a round cell tumor of histiocytic origin. although this tumor has been reported in most parts of the world, it is most prevalent in tropical or temperate climates (macewen, 1991) . clinical signs the tumors are usually cauliflowerlike masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to 10 cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis. less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission ctvts are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for exfoliation and transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, 1990) . extragenital lesions may be the result of oral contact with previously traumatized areas. pathologic findings histologically, cells are arranged in compact masses or sheets. the cells are round, ovoid, or polyhedral, and have large, round nuclei with coarse chromatin. the cytoplasm is eosinophilic with small vacuoles arranged in a 'string of pearls' pattern. pathogenesis tumor growth occurs within 2-6 months after mating or implantation, and then growth generally slows. metastasis is rare (<5-17% of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. spontaneous regression may occur within 6-9 months of tumor development. diagnosis and differential diagnosis transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. however, cytological examination of impression smears, swabs, and fine-needle aspirates generally provide a definitive diagnosis. although not usually required, histopathology of a biopsy from the mass can aid in diagnosis. prevention thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control removing affected individuals from a breeding program should stop further spread through the colony. treatment surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine (0.5-0.7 mg/m 2 ) iv once weekly for four to six treatments will induce remission and cure in greater than 90% of the cases (macewen, 1991) . research complications experimental implantation of ctvts has been shown to elicit formation of tumor-specific igg (cohen, 1972) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs single nodules are found in approximately 75% of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger, invasive, and coalescent with adjacent tissues. inflammatory mammary carcinomas may mimic mastitis or severe dermatitis and must be ruled out to prevent misdiagnosis. epizootiology mammary tumors are uncommon in dogs under 5 years of age with the incidence rising sharply after that. the median age at diagnosis is 10-11 years. a longitudinal study of a large beagle colony showed that significant risk for development of mammary tumors begins at approximately 8 years of age (taylor et al., 1976) . mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings the t (tumor size), n (lymph node involvement), and m (metastasis) system is commonly used to stage mammary tumors. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, 1977) . histopathologic grades are scored based upon tubule formation, nuclear pleomorphism, and mitosis (elston and ellis, 2002) . extensive discussions of classification, staging, and histopathologic correlations can be found elsewhere (moulton, 1990; sorenmo et al., 2011) . pathogenesis mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in 60-70% of tumors. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs and extraskeleton. diagnosis and differential diagnosis both benign and malignant mammary tumors must be distinguished from mammary hyperplasia, mastitis, and severe dermatitis. cytological evaluation from fine-needle aspirates correlates well with histological examination of benign and malignant tumors (simon et al., 2009) . radiographs and possibly ultrasound should be performed to rule out metastatic disease prior to surgery. prevention the lifetime risk of developing mammary tumors can effectively be reduced to 0.5% by spaying bitches prior to the first estrus (schneider et al., 1969) . this is commonly done in the general pet population at 6 months of age. the protective effects of early spay rapidly decrease after several estrus cycles. dogs spayed prior to the first estrus had a risk of 0.8%, whereas dogs spayed after the first and second estrus had risks of 8% and 26%, respectively. treatment surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. single mammary tumors should be surgically removed with 2-cm lateral margins or margins wide enough for complete resection. deep margins may include removing sections of abdominal fascia or musculature en bloc with mammary tumor. multiple mammary tumors should be removed via regional or unilateral chain mastectomies. bilateral, staged mastectomies are reserved for more aggressive tumors. there is insufficient evidence at this time to recommend routine complete unilateral or bilateral chain mastectomies. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. sorenmo et al. (2013) provide a thorough review of canine mammary gland neoplasia. research complications treatment of early-stage or low-grade mammary tumors may be rewarding, allowing dogs to continue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. beagles are subject to many of the inherited and/ or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, 2000) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm 1 gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). other defects observed include cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than 1.0% incidence. etiology benign prostatic hyperplasia (bph) is an age-related condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs. epizootiology and transmission bph typically affects older dogs (>4 years), although glandular hyperplasia begins as early as 3 years of age. approximately 95% of inact male dogs will develop bph by 9 years of age (smith, 2008) . pathologic findings in the early stages of bph, there is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular with a honeycomb appearance (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to 5α-dihydrotestosterone (kustritz and klausner, 2000) mediating bph. diagnosis and differential diagnosis bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis that may be characterized by hemorrhage. a prostatic biopsy can be performed to confirm diagnosis. differential diagnoses include squamous metaplasia of the prostate, para-prostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve with castration. prevention castration is the primary means for prevention of benign prostatic hyperplasia. treatment the first and foremost treatment for bph is castration. in pure cases of bph, castration results in involution of the prostate gland detectable by rectal palpation within 7-10 days. for most dogs in research studies, this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases where semen collection is necessary from a valuable breeding male (e.g., genetic diseases). if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. finasteride, a synthetic 5α-reductase inhibitor, has been used in dogs to limit the metabolism of testosterone to 5α-dihydrotestosterone. treatment at daily doses of 0.1-0.5 mg/kg orally for 16 weeks was shown to reduce prostatic diameter and volume without affecting testicular spermatogenesis (sirinarumitr et al., 2001) . upon discontinuation of finasteride, the prostate generally returns to its pretreatment size within several months (smith, 2008) . gonadotropin-releasing hormone analogs such as desorelin inhibit production of testosterone and estrogen via negative feedback on the hypothalamus-pituitary axis. this is available in a sustained release subcutaneous implant, which has demonstrated efficacy in reducing prostatic size in dogs (junaidi et al., 2009) . however, medical therapy has not shown to be as advantageous as castration. research complications bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. research model older dogs with benign protastic hypertrophy are used in research to evaluate the use of ultrasonic histotripsy as a precise nonsurgical urethralsparing alternative to prostate surgery (lake et al., 2008; hall et al., 2009; schade et al., 2012) . etiology juvenile polyarteritis syndrome (jps), also known as steroid-responsive meningitis-arteritis, is a painful disorder seen in young beagles (occasionally reported in other breeds) caused by a systemic necrotizing vasculitis. the cause of the vasculitis has not been established but appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to have a hunched posture and/ or an extended head and neck. most dogs seem to be in pain when touched, especially in the neck region. neurological examination may reveal proprioceptive deficits, paresis, or paralysis. the syndrome typically has a course of remissions and relapses characterized by 3-7 days of illness and 2-4 weeks of remission (scott-moncrieff et al., 1992) . there may be a component of this condition that is subclinical, given that vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission jps typically affects young beagles (6-40 months), with no sex predilection. jps has been reported in other breeds including sibling welsh springer spaniels (caswell and nykamp, 2003) . pathologic findings on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., 1995) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to mediumsized arteries are seen. these lesions are most noticeable where gross lesions are observed, but they may be seen in other visceral locations. arterial fibrinoid necrosis leading to thyroid gland hemorrhage and inflammation was also reported (peace et al., 2001) . the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries. the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., 1995) . fibrinous thrombosis of the affected arteries is also seen. a subclinical vasculitis has also been diagnosed in beagles post mortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclic nature and respond to treatment with corticosteroids, and the affected dogs have elevated α 2 -globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resulted in one in seven affected pups (scott-moncrieff et al., 1992) . diagnosis and differential diagnosis differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). prevention and control no prevention and control measures are known at this time. treatment clinical signs can be abated by administration of corticosteroids. prednisone administered orally at 1.1 mg/kg, q12 h, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of 0.25-0.5 mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. research complications because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes. the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym 'sterile pyogranuloma complex'). clinical signs dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, 1993) . a retrospective study of beagles housed in a research industry setting, linked development of interdigital cysts to body codition score, age, location of cyst, and type of caging, and may result from chronic interdigital dermatitis (kovacs et al., 2005) . pathologic findings histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at 1 mg/kg q12 h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require surgical removal. excision includes the removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, 1993) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weight-bearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, 1993) . research complications research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. etiology 'cherry eye' is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating membrane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit. excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. pathologic findings typically, the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, 1989) . prevention hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, 1989 ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland or its removal might compromise ophthalmologic studies. antimicrobial resistance profiles and mechanisms of resistance in campylobacter jejuni isolates from pets life span and cancer mortality in the beagle dog and human studies of distribution and recurrence of helicobacter spp. gastric mucosa of dogs after triple therapy plasma von willebrand factor concentration and thyroid function in dogs catheter-related infections in long-term catheterized dogs. observations on pathogenesis, diagnostic methods, and antibiotic lock technique aaha nutritional assessment guidelines for dogs and cats open-and closed-formula laboratory animal diets and their importance to research comparison of the accuracy of two ultrasonographic measurements in predicting the parturition date in the bitch dog thyroiditis: occurrence and similarity to hashimoto's struma surgical management of specific skin disorders bordetella and mycoplasma respiratory infections in dogs and cats associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles diagnostic exercise: peracute death in a research dog insecticide and acaricide molecules and/ or combinations to prevent pet infestation by ectoparasites neurologic manifestations associated with hypothyroidism in four dogs veterinary surgery: small animal neoplasms of the skin and subcutaneous tissues in dogs and cats neoplasia of the skin and mammary glands of dogs and cats pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis variation in age at death of dogs of different sexes and breeds comparison of campylobacter carriage rates in diarrheic and healthy pet animals advances in dietary management of obesity in dogs and cats effect of amount and type of dietary fiber on food intake in energy-restricted dogs effect of level and source of dietary fiber on food intake in the dog an outbreak of fatal hemorrhagic pneumonia caused by streptococcus equi subsp. zooepidemicus in shelter dogs molecular characterization of canine parvovirus strains in argentina: detection of the pathogenic variant cpv2c in vaccinated dogs external parasites: identification and control orthopedic coaptation devices and small-animal prosthetics enterohepatic helicobacter spp. in colonic biopsies of dogs: molecular, histopathological and immunohistochemical investigations intradural vasculitis and hemorrhage in full sibling welsh springer spaniels respiratory disease in kennelled dogs: serological responses to bordetella bronchiseptica lipopolysaccharide laboratory animal medicine do not correlate with bacterial isolation or clinical respiratory symptoms lymphoma: which chemotherapy protocol and why? detection of humoral antibody to the transmissible venereal tumor of the dog laboratory animal management: dogs reproductive cycles of the domestic bitch hereditary canine spinal muscular atrophy: an animal model of motor 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characteristics of canine and feline leukemia and lymphoma evaluation of the ovicidal cctivity of lufenuron and spinosad on fleas' eggs from treated dogs enteric coccidiosis the respiratory system the association of american feed control officials dog and cat food nutrient profiles: substantiation of nutritional adequacy of complete and balanced pet foods in the united states study of obesity in dogs visiting veterinary practices in the united kingdom pathological prognostic factors in breast cancer. i. the value of histological grade in breast cancer: experience from a large study with long-term follow-up utility of fdg-pet scanning in lymphoma by who classification principles of treatment for canine lymphoma miller's anatomy of the dog update on diagnosis of canine hypothyroidism immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors the laboratory canine canine conjunctival mast cell tumors: a retrospective study canine cutaneous 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chronic canine model flea control failure? myths and realities small animal clinical nutrition operating room emergencies shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs pulmonary parenchymal disease canine thyroid neoplasms: epidemiologic features magrane's canine ophthalmology helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats current veterinary therapy x: small animal practice congenital defects of the dog gastric and duodenal ulcers in dogs with mastocytoma complications in the use of indwelling vascular catheters in laboratory animals helicobacter infection diseases of the large intestine pregnancy management in the bitch tracheal collapse. diagnosis and medical and surgical treatment hygroma of the elbow in dogs thermal injuries joint working group on refinement factors contributing to the contamination of peripheral intravenous catheters in dogs and cats diversity in bacterium-host interactions within the species helicobacter heilmannii sensu stricto morphological study of the effects of the gnrh superagonist deslorelin on the canine testis and prostate gland five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism maintenance energy requirement of dogs: what is the correct value for the calculation of metabolic body weight in dogs? outbreak and control of haemorrhagic pneumonia due to streptococcus equi subspecies zooepidemicus in dogs kirk's current veterinary therapy xii: small animal practice an emerging pulmonary haemorrhagic syndrome in dogs: similar to the human leptospiral pulmonary haemorrhagic syndrome? tarsal joint contracture in dogs with golden retriever muscular dystrophy an epidemiological study of interdigital cysts in a research beagle colony value of the (13)c-urea breath test for detection of gastric helicobacter spp. infection in dogs undergoing endoscopic examination pathogenesis of helicobacter pylori infection prostatic diseases semen collection in the dog accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs problems in wound healing associated with chemotherapy and radiation therapy histotripsy: minimally invasive technology for prostatic tissue ablation in an in vivo canine model 18fdg-pet imaging in canine lymphoma and cutaneous mast cell tumor comparison of fertility data from vaginal vs intrauterine insemination of frozenthawed dog semen: a retrospective study withrow & macewen's small animal clinical oncology estimation of gestational age and assessment of canine fetal maturation using radiology and ultrasonography: a review effects of four preparations of .05% chlorhexidine diacetate on wound healing in dogs the prediction of parturition date in canine pregnancy clustering of activating mutations in c-kit's juxtamembrane coding region in canine mast cell neoplasms transmissible venereal tumors kirk's current veterinary therapy 11: small animal practice canine lymphoma and lymphoid leukemias the staging and treatment of multicentric highgrade lymphoma in dogs: a review of recent developments and future prospects association between waste management and cancer in companion animals tick paralysis in north america and australia thyroid gland and arterial lesions of beagles with familial hypothyroidism and hyperlipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think enteropathogenic bacteria in dogs and cats: diagnosis, epidemiology, treatment, and control dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs mast cells and canine mast cell tumours: a review etiologic study of upper respiratory infections of household dogs effect of early enteral nutrition on intestinal permeability, intestinal protein loss, and outcome in dogs with severe parvoviral enteritis determination of strain variability of microsporum canis to disinfectants cutaneous fungal infections diagnosis of neoplasia tumors of the mammary gland transmissible dog cancer genome reveals the origin and history of an ancient cell lineage clonal origin and evolution of a transmissible cancer notice regarding nih plan to transition from use of usda class b dogs to other legal sources (not-od-14-034) guide for the care and use of laboratory animals surgical closure of elbow hygroma in the dog colitis and colon cancer in waspdeficient mice require helicobacter species tumors of the genital system practical laboratory methods for the diagnosis of dermatologic diseases animals and animal products, subchapter a, parts 1, 2, and 3 comparison of three skin preparation techniques in the dog comparison of three skin preparation techniques in the dog. part 2: clinical trial in 100 dogs manual of clinical behavioral medicine for dogs and cats. mosby-year book identification of and screening for human helicobacter cinaedi infections and carriers via nested pcr identification of three novel superantigen-encoding genes in streptococcus equi subsp. zooepidemicus, szef, szen, and szep hypothyroidism in dogs: 66 cases (1987-1992) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and buccal bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in 83 dogs atlas of small animal wound management and reconstructive surgery what's your diagnosis? fever and leukocytosis in a young beagle. canine juvenile polyarteritis syndrome (beagle pain syndrome) a clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by streptococcus equi subsp. zooepidemicus thyroid diseases urogenital emergencies emergency procedures for the small animal veterinarian prognostic significance of morphological subtypes in canine malignant lymphomas during chemotherapy streptococcus zooepidemicus: an emerging canine pathogen characterization of pneumonia due to streptococcus equi subsp. zooepidemicus in dogs tumors of the skin and soft tissue aspiration-induced lung injury origins and evolution of a transmissible cancer canine mast cell tumors express stem cell factor receptor arterial and venous blood gas analyses dogs and cats as laboratory animals hypocretin levels in sporadic and familial cases of canine narcolepsy comparison of 4 giardia diagnostic tests in diagnosis of naturally acquired canine chronic subclinical giardiasis effects of chlorhexidine diacetate and povidone-iodine on wound healing in dogs genetic evidence for an east asian origin of domestic dogs urethral-sparing histotripsy of the prostate in a canine model factors influencing canine mammary cancer development and postsurgical survival how to treat common parasites safely muller and kirk's small animal dermatology systemic necrotizing vasculitis in nine young beagles thomson's special veterinary pathology, second ed. mosby-year book thyroid and parathyroid glands diseases of the small bowel canine infectious tracheobronchitis (kennel cough complex) diseases of the intestines the use of ultrasonography for pregnancy diagnosis in the bitch cytologic examination of fine-needle aspirates from mammary gland tumors in the dog: diagnostic accuracy with comparison to histopathology and association with postoperative outcome effects of finasteride on size of the prostate gland and semen quality in dogs with benign prostatic hypertrophy canine prostatic disease: a review of anatomy, pathology, diagnosis, and treatment pathologic features of naturally occurring juvenile polyarteritis in beagle dogs development, anatomy, histology, lymphatic drainage, clinical features, and cell differentiation markers of canine mammary gland neoplasms withrow & macewen's small animal clinical oncology nutrient requirements of dogs and cats (nutrient requirements of domestic animals) trauma to the skin and subcutaneous tissues of dogs and cats chronic problem wounds of dog limbs acvim small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment, and prevention lumbosacral stenosis in dogs mammary neoplasia in a closed beagle colony complete mitochondrial genomes of ancient canids suggest a european origin of deomestic dogs artificial insemination in canids: a useful tool in breeding and conservation artificial insemination with frozen semen in dogs: a retrospective study of 10 years using a non-surgical approach manual of canine and feline cardiology comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of helicobacter cinaedi thyroiditis in a group of laboratory dogs: a study of 167 beagles obesity-related metabolic dysfunction in dogs: a comparison with human metabolic syndrome pro-inflammatory properties and neutrophil activation by helicobacter pylori urease of agriculture, animal and plant health inspection service annual report animal usage by fiscal year. available from: <www leptospirosis in dogs: a review with emphasis on clinical aspects thomson's special veterinary pathology, second ed. mosby-year book endocrinology of pregnancy in the dog: a review immunoblot analysis as an alternative method to diagnose enterohepatic helicobacter infections management of superficial skin wounds psychodermatosis colony multiplex pcr assay for identification and differentiation of campylobacter jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus subsp. fetus the role of c-kit in tumorigenesis: evaluation in canine cutaneous mast cell tumors aaha canine vaccination guidelines canine mast cell tumours: a review of the pathogenesis, clinical features, pathology and treatment serum concentrations of thyroxine and 3,5,3′-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs surgical treatment of specific skin disorders naltrexone for treatment of acral lick dermatitits in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs mammal species of the world: a taxonomic and geographic reference withrow & macewen's small animal clinical oncology the authors thank dr. bambi jasmin of marshall farms for contributions on preventive health practices for class a dogs. key: cord-022472-q2qtl26d authors: fishman, jay a.; ramos, emilio title: infection in renal transplant recipients date: 2009-05-15 journal: chronic kidney disease, dialysis, & transplantation doi: 10.1016/b978-1-4160-0158-4.50041-0 sha: doc_id: 22472 cord_uid: q2qtl26d nan successful management of infections in the immunocompromised renal transplant recipient is complicated by a variety of factors. 1 these include increased susceptibility to a broad spectrum of infectious pathogens and the difficulty in making a diagnosis of infection in the face of diminished signs and symptoms of infection, an array of noninfectious etiologies of fever (e.g., graft rejection, drug toxicity), and the possibility that multiple processes are present simultaneously. further, because immunocompromised patients tolerate invasive and established infection poorly with high morbidity and mortality, the urgency for an early and specific diagnosis to guide antimicrobial therapy is increased. given the primacy of t-lymphocyte dysfunction in transplantation, viral infections in particular are increased and contribute to graft dysfunction, systemic illness, graft rejection, and enhancing the risk for other opportunistic infections (e.g., pneumocystis and aspergillus species) and for virally-mediated cancers. the risk of infection in the renal transplant recipient is determined by the interaction of two factors: 1. the epidemiologic exposures of the patient, including those unrecognized by the patient or distant in time ( the prevention and treatment of infection is central to the optimal management of transplant recipients, given the adverse impact of infections on quality of life. consideration of the epidemiology of infection allows the clinician to establish a differential diagnosis for a given "infectious" presentation and to design the optimal preventive strategy for each patient. donor and recipient screening are critical components to the post-transplant health maintenance of the patient (table 37-3) . of these, consideration should be given to empiric therapy for purified protein derivative (ppd) positive patients, for strongyloides stercoralis in patients from endemic regions, and for patients known to have received organs from donors with acute bacterial and fungal infections. specific antiviral strategies stratified according to individual risk should be considered for all kidney recipients. exposures of importance can be divided into four overlapping categories: donor-or recipient-derived infections, and community-or nosocomial-acquired exposures. infections that are derived from the donor tissues and activated in the recipient are among the most important exposures in transplantation. some of these are latent while others are the result of bad timing-active infection transmitted at the time of transplantation. all of the known types of infections have been recognized in transplant recipients. the activation of these infections may reflect the intensity of immune suppression or result from the allogeneic response (graft rejection), which activates latent viral pathogens. three types of infection merit special attention. first, in donors who are bacteremic or fungemic at the time of donation, these infections-staphylococci, pneumococcus, candida species, salmonella, e. coli-tend to "stick" to anastamotic sites (vascular, urinary) and may produce leaks or mycotic aneurysms. second, viral infections, including cytomegalovirus (cmv) and epstein-barr virus (ebv), are associated with particular syndromes and morbidity in the immunocompromised population (discussed later in text). the greatest risk of such infections is in recipients who are seronegative (immunologically naïve) and receive infected grafts from seropositive donors (latent viral infection). third, late, latent infections, including tuberculosis, may activate many years after the initial exposure. disseminated mycobacterial infection is often difficult to treat once established due largely to interactions between the antimicrobial agents used to treat infection (e.g., rifampin, streptomycin, isoniazid) and the agents used in immune suppressive therapy. given the risk of transmission of infection from the organ donor to the recipient, certain infections should be considered relative contraindications to organ donation. given that renal transplantation is, in general, elective surgery, it is reasonable to avoid donation from individuals with unexplained fever, rash, or infectious syndromes. some of the common criteria for exclusion of organ donors are listed in table 37 -4. infections in this category are generally latent infections activated in the setting of immune suppression. it is necessary to obtain a careful history of travel and exposures to guide preventive strategies and empiric therapies. notable among these infections are tuberculosis, strongyloidiasis, viral infections (herpes simplex and varicella zoster or shingles), histoplasmosis, coccidioidomycosis, hepatitis b or c, and human immunodeficiency virus (hiv). vaccination status should be evaluated (tetanus, hepatitis b, childhood vaccines, influenza, pneumococcal vaccine). dietary habits should also be considered, including the use of well water (cryptosporidia), uncooked meats (salmonella, listeria), and unpasteurized dairy products (listeria). common exposures in the community are often related to contaminated food and water ingestion, exposure to infected children or coworkers, or exposures due to hobbies (gardening), travel, or work. respiratory virus infection due to influenza, respiratory syncytial virus, and adenoviruses and more atypical pathogens (herpes simplex virus, herpes zoster virus) carries the risk for viral pneumonia but increased risk for bacterial superinfection. community (social or transfusion-associated) exposure to cmv and ebv may produce severe primary infection in the nonimmune host. recent and remote exposures to endemic, geographically restricted systemic mycoses (blastomyces dermatitidis, coccidioides immitis, and histoplasma capsulatum) and mycobacterium tuberculosis can result in localized pulmonary, systemic, or metastatic infection. asymptomatic strongyloides stercoralis infection may activate more than 30 years after initial exposure due to the effects of immunosuppressive therapy. such reactivation can result in either a diarrheal illness and parasite migration with hyperinfestation syndrome (characterized by hemorrhagic enterocolitis, hemorrhagic pneumonia, or both) or disseminated infection with accompanying (usually) gramnegative bacteremia or meningitis. gastroenteritis due to salmonella species, campylobacter jejuni, and a variety of enteric viruses can result in persistent infection, more severe and prolonged diarrheal disease as well as an increased risk of bloodstream invasion and metastatic infection. nosocomial infections are of increasing importance because organisms with significant antimicrobial resistance predominate in many centers. these include vancomycin, linezolid and quinupristin/dalfopristin-resistant enterococci, methicillinresistant staphylococci, and fluconazole-resistant candida species. a single case of nosocomial aspergillus infection in a compromised host should be seen as an indication of the failure of infection control practices. antimicrobial abuse has resulted in increased rates of c. difficile colitis. outbreaks of infections due to legionella species have been associated with hospital plumbing and contaminated water supplies or ventilation systems. each nosocomial infection should be investigated to ascertain the source and prevent subsequent infections. nosocomial spread of p. jiroveci between immunocompromised patients has also been suggested by a variety of case series. respiratory viral infections may be acquired from medical staff and should be considered among the causes of fever and respiratory decompensation among hospitalized or institutionalized, immunocompromised individuals. the net state of immunosuppression is a measure of all of the factors contributing to the patient's risk for infection (table 37 -2). among these are: 1. the specific immunosuppressive therapy, including dose, duration, and sequence of agents. 2. technical problems from the transplant procedure, resulting in leaks (blood, lymph, urine) and fluid collections, devitalized tissue, poor wound healing, and surgical drainage catheters for prolonged periods. 3. prolonged airway intubation 4. prolonged use of broad-spectrum antibiotics 5. renal and/or hepatic dysfunction 6. prolonged use of vascular access or dialysis catheters presence of infection with one of the immunomodulating viruses, including cmv, ebv, hepatitis b (hbv) or c (hcv), or hiv. specific immunosuppressive agents are associated with increased risk for certain infections (table 37-5) . combinations of these agents may enhance this risk or cause toxicity (e.g., nephrotoxicity) and may further enhance risk. as immunosuppressive regimens have become more standardized, the specific infections that occur most often will vary in a predictable pattern depending on the time elapsed infection i in r renal t transplant r recipients 683 (figure 37-1 ). this is a reflection of the changing risk factors (surgery/hospitalization, immune suppression, acute and chronic rejection, emergence of latent infections, and exposures to novel community infections. 1 the pattern of infections will be changed with alterations in the immunosuppressive regimen (pulse dose steroids or intensification for graft rejection), intercurrent viral infection, neutropenia (drug toxicity), graft dysfunction, or significant epidemiologic exposures (travel or food). the time line reflects three overlapping periods of risk for infection: (1) the perioperative period to approximately 4 weeks after transplantation; (2) the period 1 to 6 months after transplantation (depending on the rapidity of taper of immune suppression and the type and dosing of antilymphocyte "induction" that may persist); and (3) the period beyond the first year after transplantation. these periods reflect the changing major risk factors associated with infection: (1) surgery and technical complications; (2) intensive immune suppression with viral activation; and (3) community-acquired exposures with the return of normal activities. the time line may be used in a variety of ways: (1) to establish a differential diagnosis for the transplant patient suspected of having infection; (2) as a clue to the presence of an excessive environmental hazard for the individual, either within the hospital or in the community; and (3) as a guide to the design of preventive antimicrobial strategies. infections occurring outside the usual period or of unusual severity suggest either excessive epidemiologic hazard or excessive immunosuppression. the prevention of infection must be linked to the risk for infection at various times after transplantation. routine preventive strategies from the massachusetts general hospital are outlined in table 37 -6. it should be noted that such strategies serve only to delay the onset of infection in the face of epidemiologic pressure. the use of antibiotic prophylaxis, vaccines, and behavioral modifications (e.g., routine hand washing or advice against digging in gardens without masks) may only result in a "shift to the transplantation 684 (1) a combination of atovaquone 1500 mg po with meals once daily plus levofloxacin (or equivalent fluoroquinolone without anti-anaerobic spectrum) 250 mg once daily; (2) pentamidine (300 mg iv or inhaled q 3-4 weeks); and (3) dapsone (100 mg po qd to biweekly) +/− pyrimethamine. each of these agents has toxicities that must be considered, including hemolysis in g6pd-deficient hosts with dapsone. none of these alternative programs offer the same broad protection of tmp-smx. continued right" of the infection time line, unless the intensity of immune suppression is reduced or immunity develops. during the first month after transplantation, three types of infection occur. the first type of infection is that present in the recipient prior to transplantation, was inadequately treated, and now has emerged in the setting of surgery, anesthesia, and immunosuppression. pre-transplantation pneumonia and vascular access infections are common examples of this type of infection. colonization of the recipient with resistant organisms is also common (e.g., mrsa). the first rule of successful transplant infectious disease is the eradication of all infection possible prior to transplantation. the second type of early infection was present in the donor before transplantation. this is often a nosocomial-acquired organism (resistant gram-negative bacilli and s. aureus or candida species) due to (1) systemic infection in the donor (e.g., line infection) or (2) contamination during the organ procurement process. the end result is a high risk of infection of vascular suture lines with resultant mycotic aneurysm. uncommonly, infections have been transmitted from donor to recipient, including tuberculosis or fungal (e.g., histoplasmosis) infection that may emerge earlier in the time line than would be predicted (i.e., in the first month). the third type and the most common source of infections in this period are related to the complex surgical procedure of transplantation. these include surgical wound infections, pneumonia (aspiration), bacteremia due to vascular access or surgical drainage catheters, urinary tract infections, or infections of fluid collections-leaks of vascular or urinary transplantation 686 table 3 37-6 renal transplantation antimicrobial protocols at the massachusetts general hospital, boston, massachusetts-cont'd prophylaxis is achieved with 50% of the therapeutic dose of ganciclovir or valganciclovir (corrected for renal function). in some patients, intravenous immune globulin (ivig or hyperimmune globulin) is used as an adjunctive therapy for prophylaxis. certain subgroups merit routine prophylaxis. these include: • solid organ transplant recipients who are naïve (seronegative) and receive an organ from a seropositive donor (d+/r−) • solid organ transplant recipients who are seropositive (r+) and receive antilymphocyte antibodies or other intensive immune suppression (e.g., for graft rejection) symptoms, fever/neutropenia mo (or valacyclovir 500 bid or acyclovir 400 tid) use of cmv-negative or leukocyte-filtered blood status unknown with als intravenous ganciclovir 5mg/kg iv for first dose and qd (corrected for renal function) until sero-status determined. neutropenia: the dose of antiviral and antibacterial therapies are not, in general, reduced for neutropenia. consider other options first! + als: antilymphocyte antibodies include any of the lytic, lymphocyte-depleting antisera *note: not fda approved at these doses prevention of mucocutaneous infection can be accomplished with oral clotrimazole (may increase cya levels) or nystatin 2 to 3 times per day at times of steroid therapy or in the face of antibacterial therapy. fluconazole, at a dose of 200-400 mg/day for 10-14 days is utilized in the treatment of prophylaxis failures. routine prophylaxis with fluconazole is used for pancreas transplants. anastamoses or of lymphoceles. these are nosocomial infections and, as such, are due to the same bacteria and candida infections observed in nonimmunosuppressed patients undergoing comparable surgery. however, given the immune suppression, the signs of infection may be subtle and the severity or duration may be greater. the technical skill of the surgeons and meticulous postoperative care (i.e., wound care, endotracheal tubes, vascular access devices, and drainage catheters) are the determinants of risk for these infections. also among the common infections is c. difficile colitis. limited perioperative antibiotic prophylaxis (i.e., from a single dose to 24 hours of an antibiotic such as cefazolin) is usually adequate with additional coverage only for known risk factors (e.g., prior colonization with mrsa). for pancreas transplantation, perioperative prophylaxis against yeasts with fluconazole is used in addition, bearing in mind the interactions between azole antifungal agents and calcineurin inhibitors and sirolimus (levels may be increased significantly). notable by their absence in the 1st month after transplantation are opportunistic infections, even though the daily doses of immunosuppressive drugs are at their highest during this time. the implications of this observation are important: the net state of immunosuppression is not great enough to support the occurrence of opportunistic infections unless an exposure has been excessive; this observation suggests that it is not the daily dose of immunosuppressive drugs that is of importance but rather the sustained administration of these drugs, the "area under the curve," in determining the net state of immunosuppression. thus, the occurrence of a single case of opportunistic infection in this period should trigger an epidemiologic investigation for an environmental hazard. infection in the transplant recipient 1 to 6 months after transplantation has one of three causes: 1. lingering infection from the peri-surgical period, including relapsed c. difficile colitis, inadequately treated pneumonia, or infection related to a technical problem (e.g., urine leak, lymphocele, hematoma). fluid collections require drainage. 2. viral infections, including cmv, hsv, shingles (vzv), human herpesvirus 6 or 7, ebv, relapsed hepatitis (hbv, hcv), and hiv. this group of viruses is unique: lifelong infection; tissue-associated (often transmitted with the allograft from seropositive donors); immunomodulating-systemically immune suppressive and, potentially, predisposing to graft rejection. it is also notable that the herpesviruses are prominent due to the attenuated ability of t cells to control these infections. among the other viral pathogens of this period must be included bk polyomavirus in association with allograft dysfunction and community-acquired respiratory viruses (adenovirus, influenza, parainfluenza, respiratory syncytial virus, metapneumovirus). the suppression of antibody production (e.g., using tacrolimus and mycophenylate mofetil or with lymphopenia) may predispose to other infections. 3. opportunistic infection due to p. jiroveci, listeria monocytogenes, t. gondii, nocardia species, aspergillus species, and other agents. in this period, the stage is also set for the emergence of a subgroup of patients, the "chronic ne'er-do-wells"-individuals who require higher than average immune suppression to maintain graft function or who have prolonged untreated viral infections and other opportunistic infections, predicting long-term susceptibility to many other infections (third phase, discussed later). such individuals may merit prolonged (lifelong) prophylaxis (antibacterial and/or antiviral) to prevent life-threatening infection. the specific opportunistic infections that occur, reflect the specific immunosuppressive regimen used and the presence or absence of immunomodulating viral infection. viral pathogens (and rejection) are responsible for the majority of febrile episodes that occur in this period. during this period, anti-cmv strategies and trimethoprim-sulfamethoxazole prophylaxis are effective in decreasing the risk of infection. trimethoprim-sulfamethoxazole prophylaxis eliminates p. jiroveci pneumonia (pcp) and reduces the incidence of urinary tract infection and urosepsis, l. monocytogenes meningitis, nocardia species infection, and toxoplasma gondii. transplant recipients who are more than 6 months past the procedure can be divided into three groups in terms of infection risk. the first group consists of the majority of transplant recipients (70%-80%) who had a technically good procedure with satisfactory allograft function, reduced and maintenance immunosuppression, and absence of chronic viral infection. these patients resemble the general community in terms of infection risk, with community-acquired respiratory viruses constituting their major risk. occasionally, such patients will develop primary cmv infection (socially acquired) or infections related to underlying diseases (e.g., skin infections in diabetes). the second group (~10% of patients) suffers chronic viral infection, which, in the absence of effective therapy, will lead inexorably to one of three results: • end organ damage (e.g., bk polyomavirus nephropathy, cryoglobulinemia, or cirrhosis from hcv-hbv being relatively well managed at present) • malignancy (post-transplantation lymphoproliferative disease [ptld] due to ebv, skin, or anogenital cancer due to papilloma viruses) • acquired immunodeficiency syndrome (hiv/aids) the third group of patients (~10% of all recipients) has less than satisfactory allograft function and requires excessive amounts of immunosuppressive therapy for recurrent graft rejection. this may be associated with chronic viral infection. this is the subgroup of transplant recipients, often termed the "chronic ne'er-do-wells," who are at highest risk for opportunistic infection with such pathogens as p. jiroveci, l. monocytogenes, n. asteroides, and cryptococcus neoformans. it is our practice to give these patients lifetime maintenance trimethoprim-sulfamethoxazole prophylaxis and to consider the use of fluconazole prophylaxis. also, this group is susceptible to organisms more often associated with immune dysfunction of aids (bartonella, rhodococcus, cryptosporidium, and microsporidium species) and invasive fungal pathogens (aspergillus, zygomycetes, and the dematiaceae, or pigmented, molds). minimal signs or symptoms merit careful evaluation in this group of "high-risk" individuals. guidelines for pre-transplant screening have been the subject of several recent publications including a consensus conference of the immunocompromised host society (ichs), the american society for transplantation (ast) clinical practice guidelines on the evaluation of renal transplant candidates, and the astp clinical practice guidelines on the evaluation of living renal transplant donors. [2] [3] [4] [5] [6] [7] [8] [9] the transplant donor the critical feature of screening for deceased donors is time limitation. a useful organ must be procured and implanted before some microbiologic assessments have been completed. thus, major infections must be excluded and appropriate cultures and stored samples obtained for future reference. as a result, bacteremia or fungemia may not be detected until after the transplant has occurred. such infections have not generally resulted in transmission of infection as long as the infection has been adequately treated, both in terms of use of antimicrobial agents to which the organism is susceptible and time. in recipients of tissues from 95 bacteremic donors, a mean of 3.8 days of effective therapy post-transplantation appeared adequate to prevent transmission; longer courses of therapy in the recipient are preferred, targeting known potential pathogens from the donor. 10 bacterial meningitis must also be treated with antibiotics that penetrate the csf before procurement. similarly, due to the limited time for testing, certain acute infections (cmv, ebv, hiv, hbv, or hcv) may be undetected in the period prior to antibody formation, and viral dna detection is preferred. as a result, the donor's clinical, social, and medical histories are essential to reducing the risk of such infections. however, in the presence of known infection, such infections must be treated prior to procurement, if possible. major exclusion criteria are outlined in table 37 -4. the differences in screening of the living donor and the cadaver donor are largely based on the different time frames during which this screening takes place. the living donor procedure should be considered elective-and, thus, evaluation completed and infections treated prior to such procedures. an interim history must be taken at the time of surgery to assess the presence of new infections since the initial donor evaluation. intercurrent infections (flu-like illness, headache, confusion, myalgia, cough) might be the harbinger of important infection (west nile virus, sars, rabies, trypanosoma cruzi). live donors undergo a battery of serologic tests (table 37-3) as well as ppd skin test and, if indicated, chest radiograph. the testing must be individualized based on unique risk factors (e.g., travel). of particular importance to the renal transplant recipient is the exclusion of urinary tract infection. whether focal infections in the donor outside the procured organs merit therapy remains unresolved. mycobacterium tuberculosis. this bacterium from the donor represented approximately 4% of reported post-transplant tb cases in a review of 511 patients by singh and colleagues. 11 active disease should be excluded in ppd positive donors, including chest radiograph, sputum cultures, and chest ct, if the chest radiograph is abnormal. urine afb cultures may be useful in the ppd-positive kidney donor. isoniazid prophylaxis of the recipient should be considered for untreated, ppdpositive donors. 12 factors mitigating towards prophylaxis include donor from endemic region, use of high-dose steroid regimen, or high-risk social environment. chagas' disease (t. cruzi). this parasitic disease has been transmitted by transplantation in endemic areas and recently in the united states. schistosomiasis and infection by strongyloides stercoralis are generally recipient-derived problems. epstein-barr virus. the risk for post-transplant lymphoproliferative disease (ptld) is greatest in the ebv seronegative recipient of an ebv seropositive allograft (i.e., d+/r−). this is most common in pediatric transplant recipients and in adults coinfected with cmv or on higher levels of immune suppression. monitoring should be considered for at-risk individuals using a quantitative, molecular assay (e.g., pcr) for ebv. 13, 14 ebv is also a cofactor for other lymphoid malignancies. varicella screening should be used to identify seronegative individuals (no history of chicken pox or shingles) for vaccination prior to transplantation. hsv screening is performed by most centers despite the use of antiviral prophylaxis during the post-transplant period. vzv serologic status is particularly important in children who may be exposed at school (for antiviral or varicella immune globulin prophylaxis) and in adults with atypical presentations of infection (pneumonia or gi disease). other herpesviruses may reactivate with hhv-6 and hhv-7 serving as cofactors for cmv and fungal infections and in endemic regions, kaposi's sarcoma-associated herpesvirus (hhv-8/kshv) causing malignancies. hepatitis b virus (hbv). hbsag and hbv core antibody (hbcab) are used for screening purposes with hbsab positivity indicating either vaccination or prior infection. hbcab-igm positivity suggests active hbv infection, whereas igg positivity suggests a more remote or persistent infection. the hbsag negative, hbcab-igg positive donor may have viral dna in the liver but may be appropriate as a donor for hbvinfected renal recipients. quantitative assays for hbv should be obtained to guide further therapy. the presence of hbsag negative, hbcab-igg positive assays may be a false-positive or reflect true, latent hbv infection. hepatitis c virus (hcv) infection will generally progress more rapidly with immune suppression and with cmv coinfection. hcv seropositive renal transplant candidates are more likely to develop cirrhosis and complications of liver failure. there is no good therapy for hcv infection; management is by quantitative molecular viral assays. hiv-infected donors have not been utilized. the progression of disease is rapid and outweighs the benefits of transplantation. donors may be excluded based on historic evidence of "high-risk" behavior for hiv infection. western blot testing and molecular assays (pcr) should be obtained prior to the use of tissues from any hivseropositive donor. human t-lymphotropic virus i (htlv-i) is endemic in the caribbean and parts of asia (japan) and can progress to htlv-i-associated myelopathy/tropical spastic paraparesis (ham/tsp) or to adult t cell leukemia/lymphoma (atl). htlv-ii is similar to htlv-i serologically but is less clearly associated with disease. use of organs from such donors is generally avoided. 15, 16 west nile virus (wnv) is a flavivirus associated with viral syndromes and meningoencephalitis and may be transmitted by blood transfusion and organ transplantation. 17, 18 routine screening of donors is not advocated other than in areas with endemic infection of the blood supply. donors with unexplained changes in mental status or recent viral illness with neurologic signs should be avoided. sars (severe acute respiratory syndrome) is a recently described coronavirus, thought to be associated with exposure to civets or other animals common to the diet of certain regions of china. tissue persistence is prolonged and infection of transplant recipients appears to be severe and often symptomatic. organ procurement should exclude patients with recent acute illnesses meeting sars criteria. the pre-transplant period is useful for a thorough travel, animal, and environmental and exposure history; updating immunizations; and counseling of the recipient regarding travel, food, and other infection risks. ongoing infection must be eradicated prior to transplantation. two forms of infection pose a special risk: 1. bloodstream infection: this is related to vascular access, including that for dialysis and pneumonia, which puts the patient at high risk for subsequent lung infection with nosocomial organisms. infected ascites or peritoneal dialysis fluid must also be cleared prior to surgery. urinary tract infection (uti) must be eliminated prior to transplantation with antibiotics with or without nephrectomy. similarly, skin disease that threatens the integrity of this primary defense against infection should be corrected before transplantation, even if doing so requires the initiation of immunosuppression prior to transplantation (e.g., the initiation of immune suppression to treat psoriasis or eczema). finally, the history of more than one episode of diverticulitis should initiate an evaluation to determine whether sigmoid colectomy should be carried out prior to transplantation. 2. tuberculosis: both the incidence of active disease and the occurrence of disseminated infection due to m. tuberculosis are far higher in the transplant recipient than in the general population. active tuberculous disease must be eradicated prior to transplantation. the major antituberculous drugs are potentially hepatotoxic, and significant drug interactions are common between the anti-tb agents and the agents of immune suppression. in patients with active infection, from endemic regions or with high risk exposures, tb therapy should be initiated in all ppd positive individuals prior to transplantation. some judgment may be used as to the optimal timing of treatment in individuals without evidence of active or pleuropulmonary disease. greater risk may include: • previously active tuberculosis or significant signs of old tuberculosis on chest radiograph • recent tuberculin reaction conversion • known exposure to active disease • protein-calorie malnutrition, cirrhosis, or other immune deficiency • living in a shelter or other group housing aids for those benefiting from haart, aids has been converted from a progressively fatal disease to a chronic infection controlled by complex regimens of antiviral agents. haart has been associated with reduced viral loads, improved cd4 lymphocyte counts, and reduced susceptibility to opportunistic infections. in the pre-haart era, organ transplantation was generally associated with a rapid progression of aids. as a result, hiv-infected individuals have been excluded at most transplantation centers. however, prolonged disease-free survival with haart has lead to a reconsideration of this policy. renal transplantation in hiv has been associated with good outcomes in individuals with controlled hiv infection and in the absence of hcv co-infection. 19 management requires some sophistication regarding both the immune suppressive agents and the various haart regimens. the spectrum of infection in the immunocompromised host is quite broad. given the toxicity of antimicrobial agents and the need for rapid interruption of infection, early, specific diagnosis is essential in this population. advances in diagnostic modalities (ct or mri scanning, molecular microbiologic techniques) may greatly assist in this process. however, the need for invasive diagnostic tools cannot be overemphasized. given the diminished immune responses of the host and the frequency of multiple simultaneous processes, invasive diagnosis is often the only method for optimal care. the initial therapy will, by necessity, be broad with a rapid narrowing of the antimicrobial spectrum as data become available. the first choice of therapy is to reduce the intensity of immune suppression. the risk of such an approach is that of graft rejection. the selection of the specific reduction may depend upon the organisms isolated. similarly, reversal of some immune deficits (neutropenia, hypogammaglobulinemia) may be possible with adjunctive therapies (colony stimulating factors or igg). co-infection with virus (cmv) is common and merits additional therapy. cmv is the single most important pathogen in transplant recipients, having a variety of direct and indirect effects. 1, 27 the direct effects include: • fever and neutropenia syndrome with features of infectious mononucleosis, including hepatitis, nephritis, leukopenia, and/or thrombocytopenia • pneumonia • gastrointestinal invasion with colitis, esophagitis, gastritis, ulcers, bleeding, or perforation • hepatitis, pancreatitis, chorioretinitis with the exception of chorioretinitis, the direct clinical manifestations of cmv infection usually occur 1 to 4 months after transplantation; chorioretinitis usually does not begin until later in the transplant course. although cmv is the most common cause of clinical infectious disease syndromes, its "indirect effects" are often more important. cmv infection produces a profound suppression of a variety of host defenses, predisposing to secondary invasion by such pathogens as p. jiroveci, candida and aspergillus species, and some bacterial infections. cmv also contributes to the risk for graft rejection, ptld, hhv6, and hhv7 infections. the mechanisms for this effect are complex, including altered t-cell subsets and mhc synthesis, and the elaboration of an array of pro-inflammatory cytokines, chemokines, and growth factors. transmission of cmv in the transplant recipient occurs in one of three patterns: primary infection, reactivation infection, and superinfection. 1 virus may be reactivated in the setting of an allograft from a seropositive donor transplanted into a seropositive recipient (d+r+). control of cmv infection is via mhc-restricted, virusspecific, cytotoxic t lymphocyte response (cd8+ cells) controlled by cd4+ lymphocytes. seroconversion is a marker for the development of host immunity. thus, the major effector for activation of virus is the nature of the immunosuppressive therapy being administered. the lytic antilymphocyte antibodies, both polyclonal and monoclonal, are direct activators of viral infection (mimicking the alloimmune response) and also provoke the elaboration of tnf and the other pro-inflammatory cytokines that enhance viral replication. cyclosporine, tacrolimus, sirolimus, and prednisone (other than pulse doses) have limited ability to reactivate latent cmv while azathioprine, mycophenolate, and cyclophosphamide are moderately potent in terms of promoting viral reactivation. these agents perpetuate infection once established. allograft rejection is a major stimulus for cmv activation and vice versa. thus, the cmv infection has been linked to a diminished outcome of renal and other allografts. as a result, reinke and colleagues 27 showed that 17 of 21 patients for whom biopsy revealed evidence of "late acute rejection" demonstrated a response to antiviral therapy. further, lowance and colleagues 28 demonstrated that the prevention of cmv infection also resulted in a lower incidence of graft rejection. clinical management of cmv, both prevention and treatment, is of great importance for the transplant recipient. it is based on a clear understanding of the causes of cmv activation and the variety of diagnostic techniques available. cmv cultures are generally too slow and insensitive for clinical utility. further, a positive cmv culture (or shell vial culture) derived from respiratory secretions or urine is of little diagnostic value-many patients secrete cmv in the absence of invasive disease. serologic tests are useful prior to transplantation to predict risk but are of little value after transplantation in defining clinical disease (this statement includes measurements of anti-cmv immunoglobulin m [igm] levels). should a patient seroconvert to cmv, this is evidence that the patient has been exposed to cmv and has developed some degree of immunity. however, seroconversion in transplantation is generally delayed and, thus, not useful for clinical diagnosis. the demonstration of cmv inclusions in tissues in the setting of a compatible clinical presentation is the "gold standard" for diagnosis. quantitation of the intensity of cmv infection has been linked to the risk for infection in transplant recipients. [29] [30] [31] [32] [33] two types of quantitative assays have been developed: the molecular assays and the antigen detection assays. the antigenemia assay is a semiquantitative fluorescent assay in which circulating neutrophils are stained for cmv early antigen (pp65), which is taken up nonspecifically as a measure of the total viral burden in the body. the molecular assays (direct dna pcr, hybrid capture, amplification assays) are highly specific and sensitive for the detection of viremia. most commonly used assays include plasma-based pcr testing and the whole-blood hybrid capture assay, noting that whole blood and plasma-based assays cannot be directly compared. the highest viral loads are often associated with tissue-invasive disease with the lowest in asymptomatic cmv infection. viral loads in the cmv syndrome are variable. either assay can be used in management. the advent of quantitative assays for the diagnosis and management of cmv infection has allowed noninvasive diagnosis in many patients with two important exceptions: 1. neurologic disease, including chorioretinitis 2. gastrointestinal disease, including invasive colitis and gastritis. in these syndromes, the cmv assays are often negative and invasive (biopsy) diagnosis may be needed. the central role of assays is illustrated by the approach to prevention and treatment of cmv (table 37-6). the schedule for screening is linked to the risk for infection. thus, in the high risk patient (d+/r− or r+ with antilymphocyte globulin) after the completion of prophylaxis, monthly screening is performed to assure the absence of infection for 3 to 6 months. in the patient being treated for cmv infection, the assays provide an end point (zero positivity) for therapy and the initiation of prophylaxis. prevention of cmv infection must be individualized for immunosuppressive regimens and the patient. two strategies are commonly used for cmv prevention: (1) universal prophylaxis and (2) preemptive therapy. universal prophylaxis involves giving antiviral therapy to all "at-risk" patients beginning at or immediately post-transplant for a defined time period. in preemptive therapy, quantitative assays are used to monitor patients at predefined intervals to detect early disease. positive assays result in therapy. preemptive therapy incurs extra costs for monitoring and coordination of outpatient care while reducing the cost of drugs and the inherent toxicities. prophylaxis has the possible advantage of preventing not only cmv infection during the period of greatest risk, but also diminishing infections due to hhv6, hhv7, and ebv. further, the indirect effects of cmv (i.e., graft rejection, opportunistic infection) may also be reduced by routine prophylaxis. in practice neither strategy is perfect. both breakthrough disease and ganciclovir resistance have been observed in both approaches. given the risk for invasive infection, patients at risk for primary infection (cmv d+/r−) are generally given prophylaxis for 3 to 6 months after transplantation. we utilize 6 months of prophylaxis in patients receiving lytic antilymphocyte antibodies. other groups are candidates for preemptive therapy if an appropriate monitoring system is in place and patient compliance is good. the standard of care for treating cmv disease is 2 to 3 weeks of intravenous ganciclovir (5 mg/kg twice daily, with dosage adjustments for renal dysfunction). in patients slow to respond to therapy and who are seronegative, the addition of 3 months of cmv hyperimmune globulin in seronegative individuals (150 mg/kg/dose iv) may be useful. relapse does occur, primarily in those not treated beyond the achievement of a negative quantitative assay. therefore, we treat intravenously until viremia has been cleared and following it with prophylaxis with 2 to 4 months of oral ganciclovir (1 g two or three times daily) or valganciclovir (based on creatinine clearance). this approach has resulted in rare symptomatic relapses and appears to prevent the emergence of antiviral resistance. a number of issues remain. first, the role of oral valganciclovir in treatment has not been well studied. this agent provides good bioavailability but is not approved for this indication. further, some relapses occur in gi disease because the assays used to follow disease are not reliable in this setting. thus, repeat endoscopy should be considered to assure the clearance of infection. the optimum dosing of valganciclovir for prophylaxis in renal transplant recipients is also unclear. many centers use 450 mg/day po (given reduced creatinine clearance) although the fda approved dosing 900 mg/day. it is worth measuring the creatinine clearance to ensure appropriate dosing. alternative therapies are available in intravenous form only. these include foscarnet and cidofovir. foscarnet has been used extensively for therapy of cmv in aids patients. it is active against most ganciclovir-resistant strains of cmv, although we prefer combination therapy (ganciclovir and foscarnet) for such individuals, given the toxicities of each agent and the antiviral synergy demonstrated. cidofovir has been used in renal transplant recipients, often with nephrotoxicity. both foscarnet and cidofovir may exhibit synergistic nephrotoxicity with calcineurin inhibitors. a newer class of agents (leflunamide) has been approved for immune suppression and treatment of rheumatologic diseases but also appears to have useful activity against cmv (and possibly bk polyomavirus). ebv is a ubiquitous herpesvirus (the majority of adults are infected) that has b-lymphocytes as a primary target for infection. in immunosuppressed transplant recipients, primary ebv infection (and relapses in the absence of antiviral immunity) causes a mononucleosis-type syndrome, generally presenting as a lymphocytosis (b-cells) with or without lymphadenopathy or pharyngitis. meningitis, hepatitis, and pancreatitis may also be observed. remitting-relapsing ebv infection is common in children and may reflect the interplay between evolving antiviral immunity and immune suppression. this syndrome should suggest relative over-immune suppression. ebv also plays a central role in the pathogenesis of posttransplant lymphoproliferative disorder or ptld. [34] [35] [36] [37] the most clearly defined risk factor for ptld is primary ebv infection that increases the risk for ptld by 10-to 76-fold. ptld may occur, however, in the absence of ebv infection or in seropositive patients. post-transplant non-hodgkin's lymphoma (nhl) is a common complication of solid organ transplantation. lymphomas comprise up to 15% of tumors among adult transplant recipients (51% in children) with mortality of 40% to 60%. many deaths are associated with allograft failure after withdrawal of immune suppression during treatment of malignancy. compared with the general population, ptld has increased extranodal involvement, poor response to conventional therapies, and poor outcomes. the spectrum of disease ranges from benign polyclonal, b-cell infectious mononucleosis-like disease to malignant, monoclonal lymphoma. 38 the majority is of b-cell origin, although t-cell, nk-cell and null cell tumors are described. it should be noted that ebv-negative ptld has been described and that t-cell ptld has been demonstrated in allografts, confused with graft rejection or other viral infection. ptld late (more than 1-2 years) after transplantation is more often ebv-negative in adults. the clinical presentations of ebv-associated ptld vary: 1. unexplained fever (fever of unknown origin) 2. a mononucleosis-type syndrome, with fever, malaise, with or without pharyngitis or tonsillitis (often diagnosed incidentally in tonsillectomy specimens); often no lymphadenopathy is observed. 3. gastrointestinal bleeding, obstruction, perforation 4. abdominal mass lesions 5. infiltrative disease of the allograft 6. hepatocellular or pancreatic dysfunction 7. central nervous system disease diagnosis serologic testing is not useful for the diagnosis of acute ebv infection or ptld in transplantation. thus, quantitative ebv viral load testing is required for the diagnosis and management of ptld. [39] [40] [41] [42] serial assays are more useful in an individual patient than specific viral load measurements. these assays are not standardized and cannot be directly compared between centers. there are some data to suggest that assays using unfractionated whole blood are preferable to plasma samples for ebv viral load surveillance. clinical management depends on the stage of disease. in the polyclonal form, particularly in children, reestablishment of immune function may suffice to cause ptld to regress. at this stage, it is possible that antiviral therapy might have some utility given the viremia and role of ebv as an immune suppressive agent. with the progression of disease to extra-nodal and monoclonal malignant forms, reduction in immune suppression may be useful, but alternate therapies are often required. in renal transplantation, the failure to regress with significant reductions in immune suppression may suggest the need to sacrifice the allograft for patient survival. combinations of anti-b-cell therapy (anti-cd20 rituximab), chemotherapy (chop), and/or adoptive immunotherapy with stimulated t cells have been utilized. [43] [44] [45] [46] polyomaviruses polyomaviruses have been identified in transplant recipients in association with nephropathy and ureteral obstruction (bk virus) and in association with demyelinating disease of the brain (jc virus) similar to that in aids. polyomaviruses are small nonenveloped viruses with covalently closed, circular, double-stranded dna genomes. adult levels of seroprevalence are 65% to 90%. bk virus appears to achieve latency in renal tubular epithelial cells. jc virus has also been isolated from renal tissues but appears to have preferred tropism for neural tissues. reactivation occurs with immune deficiency and suppression and tissue injury (e.g., ischemia-reperfusion). bk virus is associated with a range of clinical syndromes in immunocompromised hosts: viruria and viremia, ureteral ulceration and stenosis, and hemorrhagic cystitis. [47] [48] [49] [50] [51] [52] [53] [54] active infection of renal allografts has been associated with progressive loss of graft function (bk nephropathy) in some individuals. this may be referred to as polyomavirus-associated nephropathy or pvan. bk nephropathy is rarely recognized in recipients of nonrenal organs. the clinical presentation of disease is usually as sterile pyuria, reflecting shedding of infected tubular and ureteric epithelial cells. these cells contain sheets of virus and are detected by urine cytology as "decoy cells." in most cases, such cells are not detected and the patient presents with diminished renal allograft function or with ureteric stenosis and obstruction. in such patients, the etiologies of decreased renal function must be carefully evaluated (e.g., mechanical obstruction, drug toxicity, pyelonephritis, rejection, thrombosis, recurrent disease), and choices must be made between increasing immune suppression to treat suspected graft rejection and reducing immune suppression to allow the immune system to control infection. patients with bk nephropathy treated with increased immune suppression have a high incidence of graft loss. reduced immune suppression may stabilize renal allograft function but risks graft rejection. polyoma-associated nephropathy manifested by characteristic histologic features and renal dysfunction is found in about 1% to 8% of renal transplant patients. risk factors for nephropathy are poorly defined. nickeleit and colleagues 51, 52 found that cellular rejection occurred more commonly in patients with bk nephropathy than in controls. other studies have implicated high dose immunosuppression (particularly tacrolimus and mycophenolate mofetil), pulse dose steroids, severe ischemia-reperfusion injury, exposure to antilymphocyte antibody therapy, increased number of hla mismatches between donor and recipient, cadaver renal transplants, and presence and degree of viremia in the pathogenesis of disease. the role of specific immunosuppressive agents has not been confirmed. the use of urine cytology to detect the presence of infected decoy cells in the urine has approximately 100% sensitivity for bk virus infection but a low (29%) predictive value. 53, 54 it is, therefore, a useful screening tool but cannot establish a firm diagnosis. the use of molecular techniques to screen blood or urine has also been advocated but is more useful in management of established cases (viral clearance with therapy) than in specific diagnosis. [55] [56] [57] [58] [59] [60] hirsch and colleagues 53 showed that patients with bk nephropathy have a plasma viral load statistically significantly higher (>7700 bk virus copies per ml of plasma, p<.001, 50% positive predictive value, 100% negative predictive value) when compared to patients without such disease. 53 given the presence of viremia in renal allograft recipients, it is critical to reduce immune suppression when possible. however, the possible coexistence of rejection with bk infection makes renal biopsy essential for the management of such patients. renal biopsies will demonstrate cytopathic changes in renal epithelial cells without cellular infiltration with the gradual evolution of cellular infiltration consistent with the diagnosis of interstitial nephritis. fibrosis is often prominent occasionally with calcification. immunostaining for cross reacting sv40 virus demonstrates patchy staining of viral particles within tubular cells. there is no accepted treatment for pvan other than a marked reduction in the intensity of immune suppression. it is possible to monitor the response to such maneuvers using urine cytology (decoy cells) and viral load measures in blood and/or urine. the greatest incidence of bk nephropathy is at centers with the most intensive immune suppressive regimens. thus, it is unclear whether reduction of calcineurin inhibitors or antimetabolites should be considered first. given the toxicity of calcineurin inhibitors for tubular cells and the role of injury in the activation of bk virus, as well as the need for anti-bk t-cell activity, we have generally reduced these agents first. other centers have selected reduction of the antimetabolite first. regardless of the approach, renal function, drug levels, and viral loads must be monitored carefully. some centers advocate the use of cidofovir for bk nephropathy in low doses (0.25-1 mg/kg every 2 weeks). [61] [62] [63] [64] significant renal toxicity may be observed with this agent, especially in combination with the calcineurin inhibitors. retransplantation has been achieved in such patients with failed allografts, possibly as a reflection of immunity developing subsequent to reduction in immune suppression. 65 infection of the central nervous system by jc polyomavirus has been observed uncommonly in renal allograft recipients as progressive multifocal encephalopathy. this infection generally presents with focal neurologic deficits or seizures and may progress to death following extensive demyelination. pml may be confused with calcineurin neurotoxicity; both may respond to a reduction in drug levels. it is thought that these are distinct entities, but further studies are underway. in addition to the endemic mycoses, transplant recipients are at risk for opportunistic infection with a variety of fungal agents, the most important of which are candida species, aspergillus species, and c. neoformans. the most common fungal pathogen in these patients is candida, with c. albicans and c. tropicalis accounting for 90% of the infections and c. glabrata for most of the rest. mucocutaneous candidal infection (e.g., oral thrush, esophageal infection, cutaneous infection at intertriginous sites, candidal vaginitis) occurs particularly when candidal overgrowth is promoted by the presence of high levels of glucose and glycogen in tissues and fluids (e.g., with poorly controlled diabetes, high-dose steroid therapy) and by broad-spectrum antibacterial therapy). these infections are usually treatable through correction of the underlying meta-bolic abnormality and topical therapy with clotrimazole or nystatin. more difficult to manage is candidal infection occurring in association with the presence of foreign bodies that violate the mucocutaneous surfaces of the body (e.g., vascular access catheters, surgical drains, and bladder catheters). optimal management of these infections requires removal of the foreign body and systemic antifungal therapy with either fluconazole or amphotericin. a special problem in renal transplant recipients is candiduria, even if the patient is asymptomatic. particularly in individuals with poor bladder function, obstructing fungal balls can develop at the ureteropelvic junction, resulting in obstructive uropathy, ascending pyelonephritis, and the possibility of systemic dissemination. a single positive culture result for candida species from a blood specimen necessitates systemic antifungal therapy, because this finding carries a risk of visceral invasion of more than 50% in this population. fluconazole (400-600 mg/day, with adjustment for renal dysfunction), because of its better safety profile, is usually used as initial therapy, unless the patient is critically ill or a fluconazole-resistant species (e.g., c. glabrata or c. krusei) is present. in these instances, therapy is with caspofungin or amphotericin b, usually in a lipid preparation. flucytosine may be useful as an adjunctive therapy in resistant infections but must be guided by drug levels and attention to hematopoietic toxicity. invasive aspergillosis is a medical emergency in the transplant recipient, with the portal of entry being the lungs and sinuses in more than 90% of patients and the skin in most of those remaining. two species, a. fumigatus and a. flavum, account for most of these infections, although amphotericin-resistant isolates (a. terreus) are occasionally recognized. the pathologic hallmark of invasive aspergillosis is blood vessel invasion, which accounts for the three clinical characteristics of this infection: tissue infarction, hemorrhage, systemic dissemination with metastatic invasion. early in the course of transplantation, central nervous system involment with fungal infection is most often due to aspergillus species; more than 1 year after transplantation, other fungi (zygomycetes, dematiaceous fungi) are increasingly prominent. the drug of choice for this infection is probably voriconazole, noting the intense interactions between this agent and the calcineurin inhibitors and sirolimus. liposomal amphotericin is a reasonable alternative, and combination therapies are under study. of note, surgical debridement is often essential for the successful clearance of such invasive infections. central nervous system (cns) infection in the transplant recipient is an important differential for the clinician. the spectrum of causative organisms is broad and must be considered in terms of the timeline for infection in this population. many infections are metastatic to the cns, often from the lungs. thus, a "metastatic workup" is a component of evaluation of cns lesions, including those due to aspergillus, cryptococcus, nocardia, or strongyloides stercoralis. viral infections include cytomegalovirus (nodular angiitis), herpes simplex meningoencephalitis, jc virus (pml), and varicella zoster virus. common bacterial infections include listeria monocytogenes, mycobacteria, nocardia, and occasionally salmonella species. brain abscess and epidural abscess may be observed with methicillin-resistant staphylococcus, penicillin resistant pneumococcus and quinolone-resistant streptococci problematic. metastatic fungi include aspergillus and cryptococcus but also spread from sinuses (mucoraceae), skin (dematiaceae), and bloodstream (histoplasma and pseudoallescheria/scedosporium, fusarium species). parasites include toxoplasma gondii and strongyloides. given the spectrum of etiologies, precise diagnosis is essential. in particular, empiric therapy must "cover" listeria (ampicillin), cryptococcus (fluconazole or amphotericin), and herpes simplex virus (acyclovir) while awaiting data from lumbar puncture, blood cultures, and radiographic studies. included in the differential diagnosis are noninfectious etiologies, including calcineurin inhibitor toxicity and lymphoma, as well as metastatic cancer. biopsy is often needed for a firm diagnosis. cryptococcal infection is rarely seen in the transplant recipient until more than 6 months after transplantation. in the relatively intact transplant recipient, the most common presentation of cryptococcal infection is that of an asymptomatic pulmonary nodule, often with active organisms present. in the "chronic ne'er-do-well" patient, pneumonia and meningitis are common with skin involvement at sites of tissue injury (catheters) also being observed. cryptococcosis should be suspected in transplant recipients present with unexplained headaches (especially when accompanied by fevers), decreased state of consciousness, failure to thrive, or unexplained focal skin disease (which requires biopsy for culture and pathologic evaluation) more than 6 months after transplantation. diagnosis is often achieved by serum cryptococcal antigen detection, but all such patients should have lumbar puncture for cell counts and cryptococcal antigen studies. initial treatment is probably best with amphotericin and 5-flucytosine followed by high dose fluconazole until the cryptococcal antigen is cleared from blood and cerebrospinal fluid. scarring and hydrocephalus may be observed. the spectrum of potential pathogens of the lungs in transplantation is too broad for this discussion. however, some general concepts are worth mentioning. as for all infections in transplantation, invasive diagnostic techniques are often necessary in these hosts. the depressed inflammatory response of the immunocompromised transplant patient may greatly modify or delay the appearance of a pulmonary lesion on radiograph. focal or multifocal consolidation of acute onset will quite likely be caused by bacterial infection. similar multifocal lesions with subacute to chronic progression are more likely secondary to fungi, tuberculosis, or nocardial infections. large nodules are usually a sign of fungal or nocardial infection, particularly if they are subacute to chronic in onset. subacute disease with diffuse abnormalities, either of the peri-bronchovascular type or miliary micronodules, are usually caused by viruses (especially cmv) or pneumocystis jiroveci. 66, 67 additional clues can be found by examining pulmonary lesions for cavitation; cavitation suggests such necrotizing infections as those caused by fungi (aspergillus or mucoraceae), nocardia, staphylococcus, certain gram-negative bacilli, most commonly with klebsiella pneumoniae and pseudomonas aeruginosa. [68] [69] [70] ct of the chest is useful when the chest radiograph is negative or when the radiographic findings are subtle or nonspecific. ct is also essential to the definition of the extent of the disease process, the possibility of multiple simultaneous processes (superinfection), and to the selection of the optimal invasive technique to achieve microbiologic diagnosis. the risk of infection with pneumocystis is greatest in the first 6 months after transplantation and during periods of increased immune suppression. 1, 66, 67 the natural reservoir of infection remains unknown. aerosol transmission of infection has been demonstrated by a number of investigators in animal models, and clusters of infections have developed in clinical settings, including between hiv-infected persons and renal transplant recipients. activation of latent infection remains a significant factor in the incidence of disease in immunocompromised hosts. in the solid organ transplant recipient, chronic immune suppression that includes corticosteroids is most often associated with pneumocystosis. bolus corticosteroids, cyclosporine, or co-infection with cmv may also contribute to the risk for pneumocystis pneumonia. in patients not receiving trimethoprim-sulfamethoxazole (or alternative drugs) as prophylaxis, most transplant centers report an incidence of pneumocystis jiroveci pneumonia of approximately 10% in the first 6 months post-transplant. there is a continued risk of infection in three overlapping groups of transplant recipients: (1) those who require higher than normal levels of immune suppression for prolonged periods of time due to poor allograft function or chronic rejection; (2) those with chronic cytomegalovirus infection; and (3) those undergoing treatments that increase the level of immune deficiency, such as cancer chemotherapy or neutropenia due to drug toxicity. the expected mortality due to pneumocystis pneumonia is increased in patients on cyclosporine when compared to other immunocompromised hosts. the hallmark of infection due to p. jiroveci is the presence of marked hypoxemia, dyspnea, and cough with a paucity of physical or radiologic findings. in the transplant recipient, pneumocystis pneumonia is generally acute to subacute in development. atypical pneumocystis infection (radiographically or clinically) may be seen in patients who have coexisting pulmonary infections or who develop disease while receiving prophylaxis with second choice agents (e.g., pentamidine or atovaquone). patients outside the usual period of greatest risk for pcp may present with indolent disease confused with heart failure. in such patients, diagnosis often has to be made by invasive procedures. the role of sirolimus therapy in the clinical presentation is unknown. a number of patients have been identified with interstitial pneumonitis while receiving sirolimus; it is not known whether this syndrome is directly attributable to sirolimus or reflects concomitant infection. the characteristic hypoxemia of pneumocystis pneumonia produces a broad alveolar-arterial po 2 gradient. the level of serum lactic dehydrogenase (ldh) is elevated in most patients with pneumocystis pneumonia (>300 international units [iu]/ml). however, many other diffuse pulmonary processes also raise serum ldh levels. like many of the "atypical" pneumonias (pulmonary infection without sputum production), no diagnostic pattern exists for pneumocystis pneumonia on routine chest radiograph. the chest radiograph may be entirely normal or develop the classical pattern of perihilar and interstitial "ground glass" infiltrates. microabscesses, nodules, small effusions, lymphadenopathy, asymmetry, and linear bands are common. chest computerized tomography (ct-scans) will be more sensitive to the diffuse interstitial and nodular pattern than routine radiographs. the clinical and radiologic manifestations of p. jiroveci pneumonia are virtually identical to those of cmv. indeed, the clinical challenge is to determine whether both pathogens are present. significant extrapulmonary disease is uncommon in the transplant recipient. identification of p. jiroveci as a specific etiologic agent of pneumonia in an immunocompromised patient should lead to successful treatment. a distinction should be made between the diagnosis of pneumocystis infection in aids and in non-aids patients. the burden of organisms in infected aids patients is generally greater than that of other immunocompromised hosts and noninvasive diagnosis (sputum induction) more often achieved. in general, noninvasive testing should be attempted to make the initial diagnosis, but invasive techniques should be used when clinically feasible. the diagnosis of p. jiroveci infection has been improved by the use of induced sputum samples and of immunofluorescent monoclonal antibodies to detect the organism in clinical specimens. these antibodies bind both cysts and trophozoites. the cyst wall can be displayed by a variety of staining techniques; of these, the gomori's methenamine-silver nitrate method (which stains organisms brown or black) is most reliable, even though it is susceptible to artifacts. sporozoites and trophozoites are stained by polychrome stains, particularly the giemsa stain. early therapy, preferably with trimethoprim-sulfamethoxazole (tmp-smz) is preferred; few renal transplant patients will tolerate full-dose tmp-smz for prolonged periods of time. this reflects both the elevation of creatinine due to trimethoprim (competing for secretion in the kidney) and the toxicity of sulfa agents for the renal allograft. hydration and the gradual initiation of therapy may help. alternate therapies are less desirable but have been used with success, including: intravenous pentamidine, atovaquone, clindamycin with primaquine or pyrimethamine, and trimetrexate. although a reduction in the intensity of immune suppression is generally considered a part of anti-infective therapy in transplantation, the use of short courses of adjunctive steroids with a gradual taper is sometimes used in transplant recipients (as in aids patients) with severe respiratory distress associated with pcp. the importance of preventing pneumocystis infection cannot be overemphasized. low dose trimethoprim-sulfamethoxazole is well tolerated and should be used in the absence of concrete data demonstrating true allergy. alternative prophylactic strategies including dapsone, atovaquone, inhaled or intravenous pentamidine, are less effective than trimethoprim-sulfamethoxazole but useful in the patient with significant allergy to sulfa drugs. tmp-smx is the most effective agent for prevention of infection due to p. jiroveci. the advantages of tmp-smx include increased efficacy, lower cost, the availability of oral preparations, and possible protection against other organisms, including toxoplasma gondii, isospora belli, cyclospora cayetanensis, nocardia asteroides, and common urinary, respiratory, and gastrointestinal bacterial pathogens. it should be noted that alternative agents lack this spectrum of activity. due to concerns about the efficacy of vaccines following transplantation, patients should complete vaccinations at least 4 weeks beforehand to allow time for an optimal immune response and resolution of subclinical infection from live vaccines. vaccinations should include pneumococcal vaccine (if not vaccinated in last 3-5 years), documentation of tetanus and mmr (measles, mumps, rubella) and polio status, as well as vaccines for hepatitis b and varicella zoster (if no history of chickenpox or shingles) (see also . after transplant, influenza vaccination should be performed yearly or as per local guidelines. recommended schedules and doses for routine vaccinations can be obtained from the united states centers for disease control and prevention (cdc) at www.immunize.org or the cdc immunization information hotline, (800) 232-2522. infection in organ-transplant recipients 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activities of various compounds against murine and primate polyomaviruses clinical pharmacokinetics of cidofovir in human immunodeficiency virus-infected patients quantitative viral load monitoring and cidofovir therapy for the management of bk virusassociated nephropathy in children and adults successful retransplantation following renal allograft loss to polyoma virus interstitial nephritis prevention of infection caused by pneumocystis carinii in transplant recipients prevention of infection due to pneumocystis carinii nocardiosis in transplant recipients recurrent nocardiosis in a renal transplant recipient central venous catheter-associated nocardia bacteremia: an unusual manifestation of nocardiosis key: cord-265751-q1ecpfyg authors: shahani, lokesh; ariza-heredia, ella j.; chemaly, roy f. title: antiviral therapy for respiratory viral infections in immunocompromised patients date: 2017-01-16 journal: expert rev anti infect ther doi: 10.1080/14787210.2017.1279970 sha: doc_id: 265751 cord_uid: q1ecpfyg introduction: respiratory viruses (influenza, parainfluenza, respiratory syncytial virus, coronavirus, human metapneumovirus, and rhinovirus) represent the most common causes of respiratory viral infections in immunocompromised patients. also, these infections may be more severe in immunocompromised patients than in the general population. early diagnosis and treatment of viral infections continue to be of paramount importance in immunocompromised patients; because once viral replication and invasive infections are evident, prognosis can be grave. areas covered: the purpose of this review is to provide an overview of the main antiviral agents used for the treatment of respiratory viral infections in immunocompromised patients and review of the new agents in the pipeline. expert commentary: over the past decade, important diagnostic advances, specifically, the use of rapid molecular testing has helped close the gap between clinical scenarios and pathogen identification and enhanced early diagnosis of viral infections and understanding of the role of prolonged shedding and viral loads. advancements in novel antiviral therapeutics with high resistance thresholds and effective immunization for preventable infections in immunocompromised patients are needed. the spectrum of immunocompromised hosts has expanded over the past decade owing to prolonged survival of patients with various malignancies and advances in both solid-organ and hematopoietic stem cell transplantation. novel immunosuppressive therapies create diverse immune deficits that generate a substrate for opportunistic infections [1] . these patients are defined by higher susceptibility to infections by organisms with lower native virulence than in immunologically normal hosts. influenza, parainfluenza, respiratory syncytial virus, coronavirus, human metapneumovirus, and rhinovirus represent the most common cause of respiratory viral infections in immunocompromised patients [1] . most of these infections are seasonal, and the viruses cause a wide range of upper respiratory tract infections (urtis) and lower respiratory tract infections (lrtis). however, adverse outcomes are far more likely in immunocompromised patients than in nonimmunocompromised individuals and include progression to pneumonia, respiratory failure, and increased mortality rates (1) (2) (3) (4) . in fact, the lrti rates and mortality rates for hematopoietic stem cell transplant (hsct) recipients and patients with hematological malignancies reportedly range from 10% to 50% [2] [3] [4] [5] . longterm complications associated with respiratory viral infections, such as airflow obstruction and bronchiolitis obliterans, have developed in hsct and lung transplant recipients [6, 7] . figure 1 highlights the high rates of progression to lrti and death among immunocompromised patients with common respiratory viral infections. the management of viral infections is challenging because viruses are intracellular parasites that use many of the host's own pathways to replicate and propagate. therefore, antiviral agents need to target specific viral components to avoid potential damage to host cells. figure 2 highlights the life cycle of viral replication and site of action of various antiviral agents. advances in the treatment of respiratory infections have been made over the past decades. table 1 highlights the current available agents for treatment of respiratory viral infections and table 2 lists the agents currently in the pipeline for these different viruses. the purpose of this review is to provide an overview of the main antiviral agents that are used in the management of respiratory infections in immunocompromised patients focusing on its clinical relevance and our experience, as well as to provide an update on the current investigational agents in the pipeline. the influenza virus is among the most common human respiratory viruses and belongs to the orthomyxoviridae family. four types of influenza viruses are a, b, c, and d. human influenza a and b viruses cause seasonal epidemics of disease almost every winter in the united states. influenza type c infections cause a mild respiratory illness and are not thought to cause epidemics. influenza d viruses primarily affect cattle and are not known to infect humans [17] . influenza a viruses are grouped into subtypes based on antigenic characteristics of 2 proteins on their surfaces, hemagglutinin (ha) and neuraminidase (na) with 18 different ha subtypes and 11 na subtypes. influenza a viruses can be further broken down into different subtypes. the most common subtypes of influenza a virus affecting humans are h1n1 and h3n2. influenza b viruses are not grouped into subtypes but can be further broken down into lineages. currently, circulating influenza b viruses belong to 1 of 2 lineages: b/yamagata and b/victoria [18] . the seasonal prevalence of influenza infections in immunocompromised patients, including solid-organ transplant and hsct recipients, closely parallels the community-wide prevalence, with peaks from december to february, with influenza b activity sometimes seen in april and may [19] . however, the illness has the potential to be more severe in this population than in healthy host [20] . without treatment reported, mortality rate range from 25% to 40% in immunocompromised patients, and is related to complications including pneumonia, and bacterial and fungal superinfections [21] . in a retrospective study, we identified profound lymphocytopenia (absolute lymphocyte count <200 cells/ ml), age greater than 65 years, and neutropenia (absolute neutrophil count <500 cells/ml) as potential risk factors associated with progression from urti to lrti [22] . early antiviral therapy within the first 48 h after presentation has been associated with improved prognosis in several studies [23] [24] [25] . the two main groups of antivirals used to treat influenza are m2 inhibitors (amantadine and rimantadine), which only act against influenza a, and na inhibitors active against influenza a and b: oseltamivir, zanamivir, and peramivir. m2 inhibitors inhibit the ion channel of the m2 protein in the influenza a virus, leading to defects in uncoating and assembly of the virus ( figure 2 ). the influenza virus enters its host cell via receptor-mediated endocytosis; thereafter, it is localized on endocytotic vacuoles. the m2 proton channel transports the ions needed for acidification of the influenza virus inside the vacuoles. this acidification is required for dissociation of the m1 protein from the ribonucleoprotein complexes and the onset of viral replication [26, 27] . the recommended dose of amantadine is 200 mg given once daily or 100 mg given twice daily (duration of therapy is generally 5 days) [8] . the most common side effects of these agents are gastrointestinal (nausea and vomiting) and effects on the central nervous system, including anxiety, insomnia, impaired thinking, confusion, lightheadedness, and hallucinations [8] . resistance of influenza a infection to m2 inhibitors results from mutations of the pore-lining residues in the ion channel, keeping adamantine and rimantadine from entering the channel [28] . according to data from the world health organization collaborating center for surveillance, epidemiology, and control of influenza at the us center for disease control and prevention (cdc), the rates of m2 inhibitors resistance have increased from 0.4% in 1994-1995 season to 12.3% in 2003-2004 [29] . however, during the 2005-2006 season, rates as high as 92% were reported for the influenza a (h3n2) virus [30] . recent cdc data demonstrate high prevalence of m2 inhibitors resistance in all influenza a (h3n2) and influenza a (h1n1) pdm09 virus isolates tested [31] . currently, the current advisory committee on immunization practices (acip) guidelines for treatment of influenza infections, do not recommend the routine use of amantadine and rimantadine in the usa for therapy or chemoprophylaxis for currently circulating influenza a virus strains [8] . nais block the active site of neuraminidase, resulting in uncleaved sialic acid residues on the host cell surface and viral envelopes ( figure 2 ). uncleaved sialic acid bound to viral ha causes viral aggregation on the host cell surface, which reduces the amount of virus released [8] . nais are virustatic, not virucidal, and early administration of them is a key factor in the development of resistance to the virus and their effectiveness. treatment with these inhibitors should neither be delayed while awaiting the results of diagnostic testing nor withheld from infected patients with indications for therapy who present more than 48 h after the onset of symptoms, particularly patients needing hospitalization [8] . in particular, the nais zanamivir and oseltamivir are first-line agents for treatment of and prophylaxis for influenza. oseltamivir is an oral nai usually prescribed as 75 mg orally twice daily (renally adjusted). the recommended duration of antiviral therapy is 5 days [8] . however, a longer duration (10 days) may be considered for severely ill patients or influenza a(h1n1) virus strains h275y substitution leads to resistance [10, 11] zanamavir nai intravenous zanamivir available through compassionate use program single dose of 600 mg administered intravenously [8] influenza a (h1n1) with both an h275y and an e119d or e119g na substitution lead to resistance to zanamivir [12, 13] . peramivir nai longer duration of 5 days in high-risk patients [9] . [8] . this antiviral therapy is most likely to provide the most benefits when initiated within the first 48 h after an infection occurs, so treatment should be initiated as soon as possible [8, 32] . some experts recommend higher dose of orally administered oseltamivir (e.g. 150 mg twice daily in adults with normal renal function) for the treatment of influenza infection in immunocompromised patients and those who are hospitalized and severely ill [8] . however, no clear evidence indicates that doubling the dose of oseltamivir is a more effective treatment than administering the normally prescribed dose in hospitalized patient, with or without severe illness [33, 34] . in a randomized trial of hospitalized patients with severe influenza, mortality rates were similar for patients who received oseltamivir at the double and standard doses [33] . however, 4 patients on the standard dose arm who were infected with influenza a (h1n1) virus without the h275y substitution at baseline acquired this substitution while on treatment. although no inferences can be made so far due to the small number of patients, using higher dose to prevent resistance and clinical failure in severely ill patients or immunocompromised patients still need to be determined in future studies [33] . in addition, a prospective study of adults hospitalized with influenza a and b infections treated with a single or double dose of oseltamivir twice daily demonstrated no differences between the groups in viral clearance, fever duration, oxygen supplementation, or hospitalization length [34] . patients receiving antiviral medications whose infections do not respond to treatment may have infections with antiviral-resistant influenza viruses. authors reported oseltamivir resistance, sometimes occurring within 1 week after treatment initiation, in immunocompromised patients with influenza a (h1n1) viral infections in the 2009 pandemic (pdm09) [35] . genotypic and phenotypic antiviral susceptibility testing are currently available to check the presence of mutations conferring resistance [36] . the more common emergence of resistance to oseltamivir in immunocompromised patients probably partly owes to prolonged viral shedding despite the use of antiviral therapy [37] . use of infection control measures is vital to reduce the risk of oseltamivir-resistant virus transmission in immunocompromised patients [32] . the pooled incidence rate of oseltamivir resistance for seasonal influenza a(h1n1) infections was estimated to be about 2.6% by a systematic review in 2009 [38] . however, in europe during the 2007-2008-winter season, rates of influenza a(h1n1) resistance were higher (up to 68%) [39] . authors reported that a specific substitution of the seasonal influenza a (h1n1) virus strains h275y (histidine-to-tyrosine substitution in neuraminidase), caused resistance in most of these cases [10, 11] . most circulating influenza a (h3n2) and influenza a (h1n1) pdm09 are still susceptible to oseltamivir and zanamivir. of the influenza a (h1n1) pdm09 infections tested during the 2013-2014 influenza season, 98.2% were susceptible to oseltamivir, and 100% were susceptible to zanamivir [40] . usually mild and limited to the first 2 days of treatment, nausea and vomiting are the most common reported toxic effects of oseltamivir, occurring in about 15% of recipients [41, 42] . zanamivir is administered via an inhaler in a dose of 2 inhalations (each inhalation of 5-mg) twice a day [27] . the chemoprophylaxis dosage of zanamivir is 10 mg (2 inhalations) administered once a day [8] . in randomized trials, this treatment shortened the duration of influenza symptoms by 1-3 days [43] [44] [45] [46] . use of intravenous (iv) zanamivir was evaluated in a recent phase iii clinical trial where the efficacy and safety of 300 mg or 600 mg of intravenous zanamivir twice daily were compared to 75 mg of oral oseltamivir twice daily for the treatment of hospitalized patients with influenza infections. the preliminary analysis showed no statistical difference in the time to clinical response (primary outcome variable) between iv zanamivir at 600 mg and oseltamivir, or between iv zanamivir at 600 mg or 300 mg [47] . it is currently available for compassionate use from its manufacturer via a us fda emergent investigational new drug application, and in a compassionate-use program in europe [48, 49] . zanamivir is currently the therapy of choice for oseltamivir-resistant influenza infections. however, the literature contains few cases of influenza virus with zanamivir resistance. infections with the h1n1 influenza strain possessing both an h275y na substitution (oseltamivir resistance) and an e119d (with aspartic acid replacing glutamic acid at position 119) or e119g (with glycine replacing glutamic acid at position 119) na substitution are resistant to zanamivir [12, 13] . the main adverse reactions to zanamivir are related to bronchospasm. use of inhalation powder to treat influenza infection is not recommended for patients with underlying airway issues (i.e. chronic obstructive pulmonary disease, asthma [8] . also, as it contains a lactose carrier, it can clog ventilator tubing nebulizers and mechanical ventilators [50] . peramivir is active against influenza a and b and was approved by the fda in 2014 for treating uncomplicated influenza infections in adults [51] . it is the first nai approved for iv use and is administered as a single iv dose of 600 mg because of its strong and prolonged affinity for the na in influenza virus. peramivir use should be considered for patients who are unable to tolerate oral or enteric drugs [51, 52] . use of a single dose of 600 mg of peramivir administered intravenously, alleviated influenza symptoms an average of 21 h sooner and fever approximately 12 h sooner than in patients given a placebo in a published report [52] . a study of patients at high risk for complications (including patients with diabetes, with chronic respiratory disease, or receiving immunosuppressive therapy), given peramivir for up to 5 days demonstrated shorter durations of illness than in patients given a single dose, and hence a longer duration of treatment for immunocompromised patients is suggested [9] . also, an open-label, randomized study of high-risk patients during the 2009 influenza a (h1n1) pdm09 demonstrated that use of peramivir (300 mg twice daily or 600 mg once daily) for 5-10 days reduced viral shedding and produced clinical improvement [53] . authors have reported cross-resistance of oseltamivir and peramivir in immunocompromised patients infected with influenza a (h1n1) virus containing the h275y variant [54] [55] [56] . therefore, patients infected with influenza a virus with a suspected or documented h275y substitution should not receive peramivir [14] . diarrhea is the most common reported adverse effect of peramivir [52] . more serious reactions associated with the central nervous system have included delirium and abnormal behavior leading to injury in patients with influenza who received oseltamivir or peramivir. primarily reported among children, these neurological events often began abruptly and resolved rapidly [57, 58] . the contribution of treatment with neuraminidase inhibitors to these events has yet to be established [59] , as some of these adverse events may have been related to the influenza infection rather than its treatment. authors have frequently reported neuropsychiatric symptoms in children with influenza infections; these symptoms were not always associated with the treatment with neuraminidase inhibitors [60-64]. das181 (ansun biopharma, san diego, ca, usa) is a recombinant fusion protein with a sialidase derived from actinomycoses viscosus that cleaves sialic acid receptors in host cells ( figure 2 ) [65] . this protein binds to cells and efficiently removes cell-surface sialic acid residues from respiratory epithelium, inhibiting viral infection. considering that das181 targets the host cells rather than the virus, it is less likely than virus-targeted drugs to induce treatment resistance. das 181 is administered via inhalation and has exhibited preclinical activity against numerous strains of influenza (a and b) and parainfluenza viruses (pivs) [65, 66] . in a phase ii double-blind, placebo-controlled clinical trial assessing influenza viral load and patient safety in otherwise healthy influenza-infected participants, an inhaled das181 dosage of 20 mg per day reduced viral loads and viral shedding in the multiple-dose group more than in patients taking a placebo as measured using quantitative polymerase chain reaction (p < 0.05); however, there was no difference in alleviation of flu-like symptoms between the placebo and the treatment arms. overall, das181 was well tolerated for up to 7 days when administered via daily inhalation for 5-7 days except for thrombocytopenia and liver test abnormalities in some instances. favipiravir (t705; toyama chemical, tokyo, japan) is an investigational antiviral drug that functions as a nucleotide analog and inhibitor of the viral rna polymerase of influenza. favipiravir is active against a broad range of influenza a, b, and c viruses, including highly pathogenic avian a (h5n1) and novel avian a (h7n9) viruses [67] , as well as influenza viruses resistant to treatment with nais or m2 inhibitors [68] . studies of preclinical cellular and mice models have demonstrated synergy of favipiravir with oseltamivir [69, 70] . this drug is currently being tested in phase iii clinical trials in the usa, europe, and latin america [69, 70] . laninamivir (cs-8958; biota pharmaceuticals, alpharetta, ga, usa) is a long-acting nai administered via a dry-powder inhaler. a phase iii randomized controlled trial demonstrated the superiority of a single inhalation dose of laninamivir octanoate to a 5-day course of oral oseltamivir in adults with seasonal influenza [71] . the drug is potentially effective against oseltamivir-resistant viruses and is currently available in japan. laninamivir has been demonstrated to be effective in reducing transmission of influenza infection from patients to household contacts. in a randomized trial, household contact of patient with influenza infection were randomly assigned to receive a single dose of laninamivir, 2 doses of laninamivir given daily for 2 days, or a placebo. family members in the laninamivir groups were less likely to develop clinical influenza as compared to the placebo group [72] . daiichi sankyo company, ltd. in japan plans to study the drug for the prevention of influenza, in single inhalation dose, in both adult and children [73] . jnj-63623872 (vx-787; janssen pharma, titusville, usa) is a nonnucleoside inhibitor targeting pb2, an influenza rna polymerase protein, inhibiting production of viral mrna, and preventing cell death [74] . it demonstrated activity against all influenza a strains tested in vitro. human studies have demonstrated significant decrease in virus shedding, when administered at a loading dose of 900 or 1200 mg on the first day followed by 600 mg once daily for 4 days [75] . a phase iib trial evaluating the dosing and frequency of the drug in healthy patients with uncomplicated influenza infection is currently under way [76] . nitazoxanide (nt-300; romark laboratories, florida, usa), an antiparasitic agent, appears to inhibit the maturation of influenza virus ha [77] . in a phase iib/iii trial, the treatment with nitazoxanide 600 mg twice daily for 5 days was associated with reduction in symptoms duration and viral titers among patients with acute uncomplicated influenza infection [15] . nitazoxanide has also shown synergistic effects in vitro with nais [78] and a current phase iii trial to investigate the efficacy of this synergism has been completed and results are awaited [79] . medi8852 (astrazeneca, gaithersburg, maryland, usa) is a monoclonal antibody targeting the highly conserved epitope in the ha stalk of influenza a virus [80] . it is currently being evaluated in a phase ib/iia clinical trial for safety and efficacy of a single intravenous dose in combination with oseltamivir, and as a monotherapy in adult patients with confirmed acute, uncomplicated influenza a infections [81] . vis410 (visterra, inc., cambridge, ma, usa) is a neutralizing human igg1 anti-ha antibody, which binds to a conserved region of the ha stalk of the influenza virus [82] . in mice, it resulted in 100% protection from influenza infection when administered prophylactically [83] . coinfections with bacterial pathogens and influenza infection may lead to significant morbidity and mortality. bacterial coinfection is associated with an increase in disease severity, hospital admission and even mortality, with streptococcus pneumoniae and staphylococcus aureus been the most common pathogens in such setting followed by, haemophilus influenzae, and group a streptococci [84] . a recent meta-analysis by klein et al. (2016) noted that older age, a higher apache ii (acute physiology and chronic health evaluation ii) score, diabetes mellitus, and sepsis were risk factors predisposing to coinfections [84] . the american college on immunization practices (acip) recommends simultaneous antiviral and antibiotic treatment for severely ill patients with influenza infections [8] . consistent with the acip guidelines, the infectious disease society of america (idsa) guidelines recommend appropriate use of diagnostic tests as guidance for targeted antibacterial therapy for hospitalized patients. recommended antibacterial therapy includes cefotaxime, ceftriaxone, and respiratory fluoroquinolones. treatment with vancomycin, linezolid, or other agents directed against methicillin-resistant staphylococcus aureus (mrsa) is recommended for patients with confirmed or a compatible clinical presentation of mrsa infection (i.e. shock and necrotizing pneumonia) [85] . respiratory syncytial virus (rsv), an enveloped, singlestranded rna virus of the family paramyxoviridae, frequently causes seasonal upper respiratory viral infections in infants and young children. symptomatic rsv reinfections in immunocompetent adults often consist of urtis lasting 2-5 days. in immunocompromised patients such as hsct and solid-organ transplant recipients, rsv infections may progress to severe and life-threatening lrtis [86] . investigators at the university of texas md anderson cancer center developed an immunodeficiency scoring index for rsv that accounts for major risk factors that identify hsct recipients who are at high risk for progression of rsv infection to an lrti and rsv-associated mortality [87] . age, neutropenia, lymphocytopenia, graft-versus-host disease, use of myeloablative conditioning regimens, use of corticosteroids, a recent hsct, and pre-engraftment are the main risk factors that are weighed in this index to categorize patients into prognostic risk groups [67] : low (0-2), moderate (3) (4) (5) (6) , and high (7) (8) (9) (10) (11) (12) risk. the authors reported a statistically significant trend of higher incidence of lrti-and rsv infection-associated mortality as the risk increased from low to moderate to high (p < 0.001). patients in the high-risk group demonstrated greatest benefit of ribavirin-based therapy at the urti stage and were at the highest risk for progression to lrti and death in the absence of antiviral therapy. we suggest using the immunodeficiency scoring index for rsv to identify high-risk patients who would benefit from treatment with aerosolized ribavirin. as seen in hsct recipients, researchers noted an association between a low lymphocyte count (mean, 580 cells/mm 3 ) and rsv infection progression to an lrti in solid-organ transplant recipients with lung transplant recipients having the highest risk of adverse outcomes [88] . ribavirin is a nucleoside analog that resembles guanosine. as a monophosphate, ribavirin inhibits the dehydrogenase enzyme, which is essential for the synthesis of guanosine triphosphate, and reduces the cellular deposits of guanidine necessary for viral growth. it inhibits the initiation and elongation of rna fragments resulting in inhibition of viral protein synthesis (figure 2 ) [89] . aerosolized ribavirin is the only fda-approved treatment of severe rsv-lrtis in hospitalized infants and young children with underlying compromising conditions (prematurity, cardiopulmonary disease, or immunosuppression) [90] . rsv infections markedly increase morbidity and mortality rates in hsct recipients. ribavirin-based antiviral therapy is recommended by european guidelines for leukemia patients and hsct recipients at high risk of complications [16, 91] . in a systematic review of the literature by shah et al. [92] and based mainly on retrospective studies, any form of ribavirinbased therapy (alone or in combination with immunomodulators) prevented urtis from progressing to lrtis (from 45% to 16%) and improved mortality rates (from 70% to 35%) when compared to no therapy in adult hsct recipients [92] . whether the benefits of aerosolized ribavirin versus the oral form justify its use in immunocompromised patients remain subject of controversy, especially given the recent drastic increase in the cost of the aerosolized form [93] . researchers have systematically reviewed the use of oral ribavirin to treat various respiratory viral infections, including rsv infections [94] . the authors concluded that mortality rates were highly variable and often dependent on the underlying severity of illness rather than the effects of oral ribavirin; however, there were not randomized or control studies available for evaluation [94] . in 2004, khanna et al. [95] reported that oral ribavirin had a good safety profile in 34 rsv-infected patients with upper or lower respiratory tract infection but could not draw a strong conclusion regarding its efficacy. the doses recommended in the european conference on infections in leukaemia (ecil-4) guidelines included a loading dose of 600 mg followed by 200 mg every 8 h the first day, 400 mg every 8 h the second day, and then escalation daily to a maximum of 30 mg/kg/day [16] . the iv formulation of ribavirin has been beneficial in some cases of rsv infection, but further trials are needed [96, 97] . various other therapies such as, intravenous immunoglobulin (ivig), rsv hyperimmunoglobulin, and palivizumab (a monoclonal rsv igg), have been used for treatment and prevention of rsv infections in immunocompromised patients with mixed results. early studies demonstrated that ribavirin in combination with rsv ivig (respigam; medimmune, gaithersburg, md, usa), an hyperimmune globulin preparation with high concentrations of rsv-neutralizing antibodies, offered a mortality advantage over ribavirin alone in rsv-infected pediatric hsct recipients with lrtis [98] . however, production of rsv ivig has since then been discontinued because of the introduction of alternatives such as palivizumab, an engineered anti-rsv monoclonal antibody. palivizumab is currently approved for prophylaxis for rsv infection in a select group of high-risk infants with bronchopulmonary dysplasia, infants with a history of premature birth (â�¤35-week gestational age), and children younger than 24 months with hemodynamically significant congenital heart disease during the rsv infection season [99] . the american academy of pediatrics recommends a palivizumab dose of 15-mg/kg body weight administered monthly throughout the rsv infection season (first dose administered prior to commencement of the season and a maximum of 5 doses per season) [99] . kassis et al. demonstrated the utility of palivizumab for prophylaxis in a hsct unit following an rsv infection outbreak. palivizumab was useful in preventing rsv infection in 16 rsv-negative patients considered to be at high risk for complications from rsv infection when combined with strict infection-control measures [100] . in contrast, palivizumab failed to demonstrate any impact on progression to lrti or mortality in a case series of 40 allogeneic hsct recipients infected with rsv [101] . given the questionable efficacy and high cost of palivizumab, mainly for adult patients, routine use of it is not encouraged in the adult immunocompromised population [102] . in adult hsct recipients with rsv pneumonia, uncontrolled studies suggested that use of combination therapy with ribavirin and ivig improved survival [103, 104] . additional studies of rsv-infected lung transplant recipients demonstrated that combined treatment with ribavirin (nebulized or iv) with ivig and/or corticosteroids reduced mortality rates, length of mechanical ventilation, and incidence of bronchiolitis obliterans [105] . although combined use of ribavirin and ivig has not been supported by a randomized trial, this expensive treatment is reserved for select patients with rsv-related lrtis and severe immune deficiency [103, 104] . aln-rsv01 (alnylam pharmaceuticals, cambridge, ma, usa) is small-interfering rna (sirna) that inhibits rsv replication by interrupting synthesis of the viral nucleocapsid protein, and treatment with this compound has demonstrated promising results in phase ii clinical trials [106] . rna interference is a natural process and sirnas induce sequence-specific degradation of mrna and thus reduce expression of the corresponding protein [106] . in a randomized, double-blind, placebocontrolled trial, researchers administered prophylactic aln-rsv01 as a nasal spray before experimental inoculation in healthy adults wild-type for rsv and observed a 38% reduction in the number of infections [106] . in a phase iia randomized, double-blind, placebo-controlled trial of adult lung transplant recipients with confirmed rsv urtis, use of aerosolized aln-rsv01 (0.6 mg/kg) daily for 3 days significantly reduced mean cumulative daily symptom scores (p = 0.035) and the incidence of progressive bronchiolitis obliterans syndrome by day 90 more so than in patients given a placebo (6% vs. 50 %; p = 0.027) [107] . also, a recent phase iib trial with lung transplant recipients demonstrated a trend of decreasing new or progressive bronchiolitis obliterans (bos) incidence (14% vs. 30%; p = 0.058) at 180 days. the treatment effect was enhanced with initiation of aln-rsv01 use fewer than 5 days after symptom onset [108] . whether further development of this compound would be pursued remains unknown at the present time. mdt-637 (microdose therapeutx, monmouth junction, nj, usa and gilead sciences, foster city, ca, usa) and the gs-5806 (gilead sciences, foster city, ca, usa) are both antiviral fusion inhibitors. oral gs-5806 has shown safety and tolerability in healthy adults [109] . currently, two phase iib trials are underway to evaluate the antiviral effects, pharmacokinetics, safety, and tolerability of gs-5806 in hsct recipients with either rsv uri or lrti [110, 111] . mdt-637 is delivered as a dry-inhalation powder and has been evaluated in a phase ii trial to assess safety and tolerability in healthy adults [112] . al-8176 (alios, south san francisco, ca, usa) is a nucleoside inhibitor of the l-protein [113] and has demonstrated efficacy in human challenge studies [114, 115] . l-protein is an rna-dependent rna polymerase of rsv, and its inhibition impact future viral replication [113] . in a randomized, double-blind, placebo-controlled phase ii challenge study conducted in healthy adult volunteers who were infected intranasally with rsv, al-8176 was well tolerated and demonstrated significant reduction in rsv viral loads (p < 0.0002) and improvement in symptom scores (p < 0.02) when compared to placebo [114, 115] . polyclonal high-titers rsv immunoglobulin (ri-001; adma biologics, inc., hackensack, nj, usa) is being tested in patients who are immunocompromised to prevent progression of urtis to lrtis. preliminary results are pending [116] . motavizumab is a newly developed monoclonal antibody targeting a highly conserved antigenic site on the fusion glycoprotein of rsv. it had antiviral effects in hospitalized children but was not superior to palivizumab in seasonal rsv prophylaxis in preterm infants with chronic lung disease of prematurity at-risk for rsv related lrti, hospitalization or death [117] . in 2010, fda antiviral drugs advisory committee declined the request for licensure of motavizumab. the concerns raised included the lack of additional benefits of motavizumab over palivizumab and the additional risk of cutaneous hypersensitivity reactions [118] . parainfluenza virus (piv) is a single-stranded, enveloped rna paramyxovirus comprising 4 antigens that share serotypes, although most clinical piv infections are caused by types 1, 2, and 3. in the general population, most clinical piv infections are caused by piv-3 followed by piv-1 and piv-2 [119] . although piv infections often occur year round, peak seasonal activity reportedly occurs from late september to december for piv-1 and during the spring and summer months for piv-3 [119] . piv most commonly affects the upper respiratory tract after an incubation period of 1-4 days and is commonly associated with urtis in children. in immunocompromised patients, authors described progression to lrti in about 37% of hsct recipients and piv-infected patients with hematological malignancies [120] . the risk factors for progression from piv-urti to piv-lrti include lymphocytopenia, neutropenia at the onset of infection, use of corticosteroids during piv-urti, and respiratory coinfections [120] . risk factors for piv-related mortality include lymphocytopenia, young age (<2 years), refractory or relapsed underlying hematological malignancies, an acute physiology and chronic health evaluation ii score greater than 15, respiratory coinfections, and steroid use at infection onset [120] . no antiviral agents are licensed to treat piv, so its management is limited to supportive care. in some instances, physicians have used oral or aerosolized ribavirin with or without ivigs for the treatment of piv lrti in immunocompromised patients with various outcomes [121] . new antiviral agents and vaccines in the pipeline may change the paradigm of piv infection management, particularly in immunocompromised patients. although, as described above, clinical providers have used oral and aerosolized ribavirin to treat piv [122] , the available data on their use for this infection remain controversial. two recent systematic reviews on hsct recipients and hematological malignancy patients demonstrated that ribavirin was not significantly more effective at preventing the progression of urti to lrti or piv-associated mortality than was supportive care alone [120, 123] . also, in lung transplant recipients with piv infections, use of oral ribavirin for 14 days at 15-20 mg/kg/day in 2 divided doses (dose length) was associated with some benefits, including a lower rate of bronchiolitis obliterans syndrome within 6 months after development of the infection than that in a non-ribavirin group (5% vs. 24%; p = 0.02) [124] . given, the lack of clear evidence of a positive outcome in piv-infected patients as well as the absence of control studies, justified recommendation for the use of ribavirin for the treatment of piv in immunocompromised patients cannot be made. as described above, das181 is a novel sialidase fusion protein with activity against piv in vivo and in vitro because it effectively cleaves sialic acid from respiratory epithelial cells, preventing piv entry into the cells (figure 2 ) [125] . das 181 have been administered on a compassionate-use basis for severe piv infections in immunocompromised patients, with apparent clinical benefits and antiviral effects [126] . in a case series, 4 pediatric hsct recipients with piv detected in respiratory specimens (2 from the upper respiratory tract and 2 from the lower respiratory tract) received inhaled das181 for 5-10 days. oxygen requirements and respiratory rates improved in all 4 patients, and their viral loads decreased within 1 week after therapy initiation [127] . in a similar case series, 16 hsct recipients received das181 daily to treat piv infections (14 lrtis and 2 urtis). of the 16 patient, 9 had complete clinical response, and 4 patients had a partial response to das181 therapy. of 7 patients with virological and spirometric data, 5 had reduction in piv viral load in nasopharyngeal secretions and 4 had improved forced expiratory volumes by the end of treatment [128] . in an ongoing phase ii double-blind, placebocontrolled trial, investigators are examining the effects of das181 in immunocompromised patients with piv-related pneumonia [129] . a recent report described the use of das181 in 13 hsct recipients: 56% of them had responses to therapy, and 24% had partial responses. they also had greater than a 1-log reduction in piv viral load [130] . bcx2798 and bcx2855 (biocryst pharmaceuticals, inc., birmingham, al, usa) are new antiviral hemagglutinin neuraminidase inhibitors and have been evaluated in mouse models of infection with a virus similar to piv, recombinant sendai virus [131] . bcx2798 and bcx2855 have demonstrated antiviral activity against piv-3 by markedly reducing pulmonary viral titers and mortality rates in rats when given intranasal within 24 h after development of infection [132] . human studies of these two inhibitors have yet to be undertaken. human rhinoviruses (hrvs) are positive-sense, single-stranded rna viruses with icosahedral symmetry. they are characterized into three genetically distinct groups designated a, b, and c within the genus enterovirus and family picornaviridae. the viral capsid that encases the rna genome is made up of four proteins: vp1, vp2, vp3, and vp4. the remaining nonstructural proteins are involved in viral genome replication and assembly [133] . hrv infections are responsible for more than one half of cold-like illnesses and cost billions of dollars annually in medical visits and missed days of work in the usa [134] . peak incidence occurs in the early fall, with a smaller peak in the spring [135] . both peak incidences are associated with urti, otitis media, and sinusitis [133] . a recent study of patients going to the emergency room with influenza-like illnesses who also had hematological malignancies demonstrated that 40% of the patients (110/272) presented with hrv infections. researchers found that the severity of hrv infection in these patients was similar to that of h1n1 influenza in the 2009 pandemic. nearly 40% of patients with hrv-associated respiratory symptoms were admitted to the hospital, 29% had lrtis, and 11% needed intensive care unit admission [136] . other studies, including those with hsct recipients, have replicated these results [137, 138] . markers for increased immunosuppression and illness severity in patients with hrv infections, including neutropenia (absolute neutrophil count â�¤500 cells/âµl), hypoalbuminemia (serum albumin level â�¤3.2 mg/dl), and infections with a respiratory co-pathogen(s) were associated with progression to hrv-related pneumonia [138] . in contrast, parody et al. [139] described a much lower rate of progression to lrti (13%) in a similar patient population. use of a different case definition for hrv infection may explain the disparity in the prevalence of lrtis in these two reports. chronic hrv infection has occurred in lung transplant patients [140] . furthermore, in a study of 36 adult lung transplant recipients, 13% of all bronchoalveolar fluid specimens obtained from 15 (42%) symptomatic patients over a 2-year period were positive for hrv [141] . currently, treatment of hrv infection consists of supportive care. antiviral medications for hrv are under investigation. the viral capsid was the initial viral protein targeted in the development of drugs to inhibit viral replication. these drugs work by binding to the hydrophobic pocket of the viral capsid, resulting in a conformational change, increasing the stability of the virion and interfering with its ability to interact with the cellular receptor [142] . vapendavir (aviragen therapeutics, alpharetta, ga, usa) is an oral agent that binds to the hrv vp1 capsid protein and prevents the release of viral rna into the target cells. vapendavir exhibits antiviral activity against hrv-a and hrv-b serotypes; however, activity against hrv-c is not yet known. a phase ii randomized, double-blind, placebo-controlled study of asthmatic adults with hrv urtis showed lower severity scores for cold symptoms, greater mean reductions in asthma scores, and higher evening peak expiratory flow in those given vapendavir than placebo [143] . pleconaril (viropharma, exton, pa, usa) was the first developed capsid-binding anti-hrv agent. two phase iii multicenter studies in the usa and canada randomized 2,096 healthy subjects with self-diagnosed colds into groups receiving pleconaril at 400 mg orally twice daily or placebo for 5 days. in the primary-efficacy population, which consisted of 1,363 subjects with hrv rna detected in nasal secretions, pleconariltreated subjects experienced a 1-day reduction in the mean duration of illness compared to the placebo group (7.3 days versus 6.3 days; p = 0.001) [144] . in another study, researchers found an association between hrv susceptibility to pleconaril and clinical outcomes [145] . the fda declined licensing of pleconaril owing to concerns of development of resistant virus strains. additionally, interactions among cytochrome p-450 3a, hormonal contraception, and antiretroviral therapy for human immunodeficiency viral infection may reduce the effectiveness of pleconaril [146] . rupintrivir (agouron pharmaceuticals, inc., san diego, ca, usa) is an in vitro 3c protease inhibitor that acts against many hrvs and enteroviruses. rupintrivir reduced viral loads and respiratory symptoms in healthy volunteers with experimentally induced rhinovirus colds and was well tolerated by the participants [147] . however, in trials of patients with natural infections, rupintrivir failed to reduce viral loads or symptom severity [148] . inhaled interferon-beta (sng001, synairgen plc, southampton, england) was tested in a phase ii, placebo controlled randomized trial of adult asthmatics receiving inhaled corticosteroids and with a history of deterioration with colds, and was associated with significant improvement in asthma symptoms, 65% fewer moderate exacerbations, improved morning peak expiratory flow rates, and reduced use of relief bronchodilators [149] . human metapneumovirus (hmpv) is an enveloped, negativesense, single-stranded rna virus. it is the first human member of the metapneumovirus genus in the pneumovirinae subfamily within the paramyxoviridae family. first identified in the netherlands in 2001, serological studies of antibodies against hmpv indicated that the virus has circulated in humans for at least 50 years [150] . phylogenetic analysis has identified two genotypes of hmpv: hmpv a and hmpv b [151] . hmpv uses a fusion mechanism to penetrate target cells. the fusion process consists of insertion of the hydrophobic fusion peptide into the target cell membrane and refolding of the f protein. this step requires the interaction of two specific domains: heptad repeats a and b [152] . investigators have studied this process for development of it as a potential antiviral treatment. hmpv causes respiratory infections and has a seasonal distribution comparable with those of influenza and rsv infections [153] . although immunocompromised patients acquire hmpv infections at the same frequency as immunocompetent individuals, they are at higher risk for severe infections. this higher risk likely can be attributed to poor viral clearance [153, 154] . a recent systematic review in hsct recipients and hematologic malignancy patients estimated the incidence of progression of hmpv-urti to lrti at 34% and an associated mortality rate of 6% [154] . factors associated with this progression in hsct recipients include early onset of infection after transplantation, steroid use, and a low lymphocyte count [155] . to date, treatment of hmpv infections has been mainly supportive. researchers have investigated several treatment regimens. standard immunoglobulin preparations have inhibited replication of hmpv in vitro [156] , and approaches such as use of selective immunoglobulins and fusion inhibitors have demonstrated antiviral activity in vitro and in animal studies. administration of oral or aerosolized ribavirin with or without polyclonal ivigs has been advocated for the treatment of severe hmpv infections and is currently used in some centers for highrisk patients [156] [157] [158] [159] [160] , although most data are still anecdotal. fusion inhibitors target the initial steps of viral fusion and penetration into the human cell. fusion inhibitors with sequence similarity with the hra and hrb domains of the viral fusion protein have demonstrated important role in viral inhibition. balb/c mice inoculated with lethal intranasal hmpv challenge were completely protected from clinical symptoms and mortality if they simultaneously received the hra2 peptide [152] . hr-1 peptides also have demonstrated effectiveness as viral inhibitors [161] . researchers developed mab 338 (medimmune, gaithersburg, md, usa) to target hmpv fusion proteins. it appeared to effectively neutralize hmpv in golden syrian hamster models and reduce the pulmonary viral titers, thereby limiting severe acute manifestations and bronchial hyper-reactivity [162] . a human monoclonal antibody fragment (human fab ds7) with biological activity against the fusion protein of hmpv demonstrated prophylactic and therapeutic potential against severe hmpv infections when tested in cotton rats [163] . human coronavirus (hcov) is a single-stranded, enveloped rna virus belonging to the family coronaviridae. in temperate climates, hcov infection is transmitted primarily during the winter and is a well-recognized cause of urtis during the respiratory viral season [164] . usually mild in immunocompetent hosts, hcov infection in immunocompromised populations may progress to lrti [16] . emerging hcovs, such as severe acute respiratory syndrome-associated hcov in 2002-2003 and the more recently identified middle east respiratory syndrome in 2012-2013, have prompted a further impetus to develop therapeutics against this infection because current antiviral agents are lacking and treatment of it remains palliative. discovery and in vitro evaluation of hcov therapy is ongoing, including investigation of entry inhibitors, human monoclonal antibodies, and proteosome inhibitors [165] [166] [167] [168] . respiratory viral infections continue to be major clinical problems in immunocompromised patients. high clinical suspicion and the use of rapid diagnostic tests remain crucial, as early treatment is associated with improved outcomes and reduced transmission. several advances in the prevention and treatments of influenza infection have occurred in recent decades. inadequate efficacy of the influenza vaccine as well as the emergence of antiviral resistance, which appears to occur more commonly in immunocompromised patients than in healthy host, underline the difficulties in management of respiratory infections in immunocompromised individuals. rsv and piv infections continue to be associated with high morbidity and mortality, and further advancements in prevention of and therapy for respiratory viral infections are needed. the impact of rhinovirus, coronavirus, and metapneumovirus infection in patients with compromised immune systems is becoming evident as new, widely available molecular testing improves the recognition of these viral infections. over the past decade, important diagnostic advances, specifically, the use of rapid molecular testing has helped close the gap between clinical scenarios and pathogen identification and enhanced early diagnosis of viral infections and understanding of the role of prolonged shedding and viral loads. respiratory viral infections can be complicated for both clinicians and immunocompromised patients. future studies that identify and validate scoring systems to ascertaining patients at highest risk for complications of respiratory viral infections including lrti, are of utmost importance. also, identification of long-term complications after respiratory viral infections in immunocompromised patients and devising interventions for prevention will be of the utmost value. last, advancement in novel antiviral therapeutics with high-resistance thresholds and effective immunization for preventable infections in immunocompromised patients are needed. to curtail the impact of respiratory viruses on our immunocompromised patients, we should focus on prevention of exposure and progression to worse outcomes. multiple interventional modalities should be studied from stimulation of the innate immune system, response to immunizations, to new antiviral therapies, to avert infection and progression to lower tract respiratory infections. one of the main challenges for immunocompromised patients is the ability to clear infections with subsequent complications associated with worsening infections, prolonged shedding, risk of resistance and coinfections. treatment targeting not only viral replication, but also the immune response to these infections may offer better outcomes. last, understanding the role of the microbiome and virome, and its implications on transmission as well as development of infection will be key for development of new strategies. â�¢ respiratory viruses are the most frequent cause of respiratory infections in immunocompromised patients, and are associated with higher rate of progression to pneumonia, respiratory failure and death. â�¢ high prevalence of m2 inhibitors resistance detected in influenza a (h3n2) and 2009 h1n1 virus strains preclude their use for prophylaxis or empiric treatment of seasonal influenza â�¢ neuraminidase inhibitors are the first line agents for treatment of influenza and treatment is most likely to provide the most benefit when initiated within the first 48 h of illness â�¢ zanamivir is currently the therapy of choice for the treatment of oseltamivir-resistant influenza infection â�¢ an immunodeficiency scoring index for rsv, that accounts for the number of risk factors, can be used to identify hsct recipients who are at high risk for progression to rsv lrti and rsv associated mortality â�¢ ribavirin-based therapy (alone or in combination with immunomodulators) can be effective in preventing progression from urti to lrti and may improve mortality in highly immunosuppressed adult hsct recipients â�¢ the safety and efficacy of das181 in immunocompromised patients with piv pneumonia, is currently being studied in an ongoing phase 2 double-blind, placebo-controlled trial. â�¢ vapendavir binds to the hrv capsid protein, preventing the release of viral rna into the target cells and has demonstrated favorable results in asthmatic adults with hrv urtis. â�¢ antiviral agents for hmpv and hcov are still under study in vitro or in animal models this paper was not funded. ej ariza-heredia has received research grants from oxford immunotec. rf chemaly has received research grants from gilead, gsk, and ansun pharmaceuticals and honoraria from adma biologics and gilead. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. papers of special note have been highlighted as either of interest (â�¢) or of considerable interest (â�¢â�¢) to readers respiratory infections in immunocompromised patients â�¢ the paper describes the immune defect in immunocompromised patients and their increased susceptibility to respiratory infections viral infections in immunocompromised patients the clinical features and outcome of 2009 h1n1 influenza infection in allo-sct patients: a 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the heptad repeat 1 region isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo prophylactic and therapeutic benefits of a monoclonal antibody against the fusion protein of human metapneumovirus in a mouse model update on rhinovirus and coronavirus infections human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing severe acute respiratory syndrome coronavirus replication is severely impaired by mg132 due to proteasome-independent inhibition of m-calpain virucidal activity of a scorpion venom peptide variant mucroporin-m1 against measles, sars-cov and influenza h5n1 viruses key: cord-021552-6jbm869r authors: hurst, christon j.; adcock, noreen j. title: relationship between humans and their viruses date: 2007-05-09 journal: viral ecology doi: 10.1016/b978-012362675-2/50015-x sha: doc_id: 21552 cord_uid: 6jbm869r nan many of the viruses that can infect humans should not be considered as viruses of humans, but rather as zoonotic. zoonotic viruses are those viruses of animals that can cross boundaries such that they occasionally infect humans. some examples of diseases induced in humans by zoonotic viruses are dengue and ebola fever, the equine encephalitids (i.e., eastern, st. louis, venezuelan, and western), hantavirus pneumonia, lassa fever, marburg fever, rabies, and yellow fever. additionally, it should be noted that the zoonotic category includes most, if not all, of the human illnesses induced either by arboviruses (viruses whose transmission is vectored by arthropods) or by the hemorrhagic fever viruses. with respect to the zoonotic viruses humans are, at best, alternate hosts. humans do in fact usually represent dead-end hosts for these zoonotic viruses, meaning that subsequent transmission of those viruses either to new humans or back to the natural host is not sustained. there is a subgroup of zoonotic viruses that, although principally remaining viruses of animals, seem to have adapted themselves to use humans as natural hosts. this adaptation is indicated by the fact that these viruses have demonstrated an ability to sustain a chain of transmission among humans. examples of zoonotic viruses that have shown this ability to adapt themselves to become viruses of humans are those members of the family flaviviridae (genus flavivirus) that induce the human diseases known as dengue and yellow fever. all of the above-mentioned zoonotic viruses contrast with the viral agents that clearly are known by their nature to be viruses of humans. examples of viruses of humans are those that induce the diseases known as acquired immunodeficiency syndrome, fever blisters, measles, mumps, polio, rubella, smallpox, t-cell leukemia, t-cell lymphoma, and type a influenza. the aim of this chapter is to address those viruses that are considered to be viruses of humans. those viruses of terrestrial mammals that are considered to be zoonotic are addressed by calisher and fenner in chapter 13. every virus species needs to have a successful overall approach for sustaining its existence. that overall approach must enable the virus to attain its two principal goals, namely, that the virus be able to reproduce itself within a host and that the virus then be transmitted onward to a new host. those mechanisms that any given virus species employs for achieving its sustainment, of course, have been developed through a process that involved an initiation of events by random chance followed by evolutionary selection. the most successful overall approaches may be those that subsequently evolve into the types of relationships between a virus and its host species that will allow the virus to persist without eliminating the host population. this latter point is very important, because the virus may in turn become extinct if it kills off the host population. it is for this reason that excessive virulence will be detrimental to the virus, and an interesting side point may be that, if an individual host cannot successfully surmount the infection, then the death of that individual host may be seen as an altruistic defense mechanism for the host population as a whole. achieving self-reproduction is the first principal goal of the virus. the processes involved can be divided into three aspects. the first aspect is the virus's overall approach with regard to the course of the infection it establishes within the host. this involves the time course of the infection and the extent to which infectious progeny viral particles are produced during the course of the infection. the second aspect is the replication strategy employed by the virus. this involves the issues of where the virus begins its march through the host body and the physical trajectory followed until the virus exits in search of a new host. the third aspect involves which approaches, if any, the virus uses to avoid the host defensive mechanisms. as mentioned in chapter 1, the goal of establishing an effective course of infection is an aspect of viral reproduction that can be attained in many ways. we can summarize the strategies that viruses use as following four basic patterns. three out of the four patterns of the course of viral infection are considered to be productive. the infection will be acquired in the form of infectious viral particles, and subsequently produced progeny viral particles then serve to infect future hosts. productive, in this context, means that the number of progeny viral particles produced during the course of an infection is sufficient for the particles to transmit the infection to a new host with some reasonable probability. the productive approach involves an evolutionary decision as to whether there will be either a very short initial but highly productive course of infection (short term-initial), a course of infection that is prolonged but only intermittently productive (recurrent), or a productive though very slow course of infection whose severity progressively increases with time to reach a dramatic end-stage (increasing to end-stage). these three patterns can be described as follows. a. short term-initial. viral production only occurs during a short time course near the time of initiation of the infection, which then abruptly ends. the human host may or may not survive beyond the course of this short infection. host survival depends on the type of virus involved, the extent to which the involved virus and humans have had time to coevolve as species, and whether or not the ancestral humans of that particular subgroup of the human host population previously have had contact with the causative virus. coevolution usually will tend to make the outcome of this pattern of viral infection sufficiently mild as to be associated with a fairly low incidence of mortality in an otherwise healthy population of human hosts. some examples of this pattern would be the infections caused by the human caliciviruses (family caliciviridae), human influenza viruses (family orthomyxoviridae), human polioviruses and human rhinoviruses (family picornaviridae), and the human rotaviruses (family reoviridae). b. recurrent. viral production, often very pronounced initially, is recurrent when the virus persists in a latent state within the host body and viral particle production periodically recurs but is not life threatening. some examples of this pattern would be the infections caused by the human herpesviruses (family herpesviridae) and the human papillomaviruses (family papovaviridae). c. increasing to end-stage. viral infection normally is associated with a slow, almost innocuous, start, followed by a gradual progression, associated with an increasing level of viral production and eventual host death. death of the host may relate to destruction of host immunological defense systems, which then results in death by secondary infections. this pattern of infection may take from l0 to 40 years to kill a human host. the infections caused by the human immunodeficiency viruses and the human t-lymphotropic viruses (all belonging to the family retroviridae) represent examples of this pattern. the fourth basic pattern of viral infection is considered to be nonproductive. a nonproductive infection is one in which the production of infectious virus particles is so limited that the virus must transmit itself through other means, usually by transferring a copy of only the nucleic acid genome of the virus. in these instances, the viral infection is normally acquired by direct transfer of the viral genetic material from the human parents to their developing fetuses, such transfer occurring via the egg and sperm cells. there may be no apparent health effects associated with such an infection. an example of this pattern is the infections caused by the endogenous retroviruses (family retroviridae), whose genomes are incorporated into the chromosomal material of every cell in the human body (villareal, 1997) . the nonproductive pattern of infection seems to suggest the highest degree of coevolution between a virus and its host, since a nonproductive virus has no means of transmitting itself to a new host without some very active, albeit perhaps unwitting, participation on the part of the present host. this section addresses the questions of where and how the virus begins its march through the host body, and how the virus then continues the course of this attack, leading ultimately to the concept of viral reproduction strategies at the host population level. discussion of the strategy of viral replication within the body of a host organism begins at the most basic level, which is attachment of the virus to a particular molecule present on the surface of host cells. such a molecule is said to be the "virus's receptor," and will be some cellular protein or lipid component naturally produced by those cells. the virus's choice of receptor is a product of viral evolution. after binding to its receptor, the virus gains entrance to the interior of the cell and viral replication begins. viruses whose genomes are composed of dna generally replicate mainly within the nucleus. in contrast, those viruses with rna genomes generally focus their center of replication in the cytoplasm. during the course of replication, the virus must decide which cellular systems and machinery it will employ. some large viruses carry the genomic coding capacity for many of their own enzymes, while others may rely almost completely on the enzymatic machinery possessed by the host cell. many viruses, including those belonging to the genus enterovirus of the family picornaviridae, are said to be highly cytopathogenic, meaning that they usually quickly kill the host cell as a result of infection. other viruses, such as those that occupy the genus rubivirus of the family togaviridae, may cause prolonged and severe crippling of the cell rather than killing it outright. a further discussion of these issues can be found in chapter 3. viruses vary greatly with respect to the tissues they tend to target for infection. on a larger scale, this then leads to identification of those organs that the viruses are affecting. this selective targeting is referred to as a "tropism." viral tropisms can be divided into those considered primary and those considered secondary. primary tropisms will be associated with production of those viral particles that subsequently contribute to transmission of the viral infection to a new host. as such, the primary tropisms tend to be related to those sites (termed "portals") through which the virus either enters or exits the host body. secondary tropisms may represent accidents. some of these accidents may come about as a result of the molecule that a virus uses as its receptor, existing on the cells of tissues that are unrelated to those that the virus must employ in order to achieve transmission. nevertheless, secondary tropisms may contribute greatly to the types and severity of the illnesses associated with infection of humans by any particular virus. when considered at the host population level, the strategy of viral replication includes the ease with which or likelihood that a virus is transmitted to new hosts plus the severity of infection and accompanying likelihood of death (including the age-related likelihood of death) for any given host individual. through the course of evolution, many viruses have developed mechanisms for either countering or evading the human immune and non-immune defenses as a means for enhancing the probability of viral success. the human immune system includes both humoral (antibody-mediated) and cellular components. the cellular components can include granulomatous reactions, which play a role in defense against protozoans, though their possible role in antiviral defenses has been incompletely explored. those mechanisms that viruses use to either avoid or minimize attack by the host immune system can be divided into the four following groups. the use of these types of mechanisms seems particularly critical in association with those viral infections that persist within a human host for very long periods of time, often up to decades. a. antigenic mimicry. the produced antigens are similar to those of the host, as with prions. b. rapid viral mutation. this mechanism includes both antigenic drifting and shifting. some viral types demonstrate rapid viral mutation during the course of an infection, as occurs with the human immunodeficiency viruses of the family retroviridae. other virus types, such as the influenza viruses of the family orthomyxoviridae, demonstrate rapid viral mutation between reinfections of the same host. c. low antigenicity. some viruses inherently seem to provoke little, if any, immune response. this often occurs because the virus persists in a latent state within host cells, during which time either little or no viral antigenic material is produced. examples include the endogenous retroviruses of the family retroviridae and the human herpesviruses of the family herpesviridae. d. infect the immune cells! the most direct attack may be the most effective. exceptionally notorious examples of this approach are the genus rubivirus of the family togaviridae and the genus lentivirus of retroviridae. aside from the above groupings, some viruses such as the norwalk virus of the family caliciviridae seem to be antigenic but provoke an immune response that is minimally effective. the body has non-immune defense mechanisms that help to protect against viral infections. these mechanisms are associated with the portals through which viruses can enter the body of a host. examples of non-immune defenses include the enzymes secreted as a part of pancreatic fluid, saliva, and tears. various glands associated with mucosal tissues secrete antimicrobial compounds into the mucus produced by those tissues. some mucosal tissues also possess cilia whose movement helps to expel both the mucus and any foreign materials, including pathogens, that become entrapped within the mucus. an additional example of a non-immune defense is the stomach acid produced to aid digestion of organic compounds. many gastroenteritis viruses, such as the rotaviruses of the family reoviridae and the astroviruses of astroviridae, have evolved such an effective resistance to attack by proteolytic enzymes that those viruses virtually need partial proteolysis to facilitate their infectivity. the influenza viruses of the family orthomyxoviridae are known for their ability to paralyze the activity of the mucosal cilia located within the respiratory tract. one of the defining characteristics for the enteroviruses of the family picornaviridae is their resistance to acidic exposure. the task of achieving viral transmission between hosting individuals involves two aspects. the first is the type of infectious material in which a virus will leave its present host, while the second involves the route by which the virus can encounter its proximate host. the types of bodily materials within which viruses can be released include substances that exit during the course of normal body functions. among these substances are feces and a variety of liquids, including menstrual blood, respiratory secretions of the upper and lower tracts, saliva, semen, tears, urine, and vaginal fluid. sweat is another fluid that is naturally released from the body; however, it is not known to contain viruses. viruses can also be found in blood released from wounds in the skin; from blood acquired by blood-consuming parasitic insects, among which are the fleas and several groups of flies, ticks, and mosquitoes; and blood leaked from swollen or ruptured capillaries into mucosal tissue and skin pores. those natural routes by which viruses are transferred to and between humans are the same routes associated with all surface-dwelling terrestrial vertebrates. these routes are tightly associated with the portals of entry and exit that any particular virus family uses as it tries to survive and find its way from one host to the next. viral transmission routes can be subdivided into two broad groups. the first is transmission by direct contact (also known as direct transfer) between two members of those species that host the virus. this includes both the possibility of transmission between two members of the principal host species and the possibility of transmission between a member of that principal host species and some alternate host species, and the latter may represent a vectoring species. the second group is transmission by indirect contact (also known as indirect transfer). these routes have been described in detail by hurst and murphy (1996) and are represented in figures 9 and 12 of chapter 1 herein. as explained in chapter 1, there are some routes of viral transmission that are considered unnatural vehicular routes. such routes represent the use of unnatural vehicles as a means to evade the host defenses associated with natural portals of entry. these unnatural routes involve invasive medical devices (such as syringes, endoscopes, and other surgical instruments) and transplanted tissues, including transfused blood and blood products. the remainder of this section describes the natural routes of viral transmission between hosts. the direct contact approach offers one major advantage and one major drawback to the virus. the advantage is that those viruses transmitted by direct contact need not be stable when exposed to ambient environments. the drawback is that the number of new hosts to which they have potential access may be smaller than for viruses transmitted by indirect contact. viruses that are endogenous by their nature will survive for as long as the host survives. although endogenous viruses can only be transmitted to host progeny, these viruses neither have to adapt themselves to nor coevolve with any other hosting species. an example of this type of endogenous agent would be the endogenous retroviruses of the family retroviridae. viruses that are venereal in nature, that is, transmitted in semen and vaginal secretions during sexual activity, have a somewhat greater potential for contacting new hosts. represented among the venereal viruses of humans are some species of the genera simplexvirus (family herpesviridae) and papillomavirus (family papovaviridae). once these venereal viruses infect a host, they remain associated with the host for the rest of the host's life in the form of a permanent infection. thus, although the frequency with which endogenous and venereal viruses can find a new host is restricted, the viruses compensate for this to some degree by remaining with the host for a very long time. the next step on the scale of host access is represented by those viruses transmitted via direct contact with insect vectors. these viruses have greatly increased access to new hosts and tend not to remain with their present host for the remainder of the host's life. those viruses transmitted by biting insects are commonly referred to as "arboviruses," which is an abbreviation of "arthropod-borne viruses." included among those arboviruses that infect humans are members of the genera alphavirus (family togaviridae); bunyavirus, nairovirus, and phlebovirus (all of the family bunyaviridae); and flavivirus (family flaviviridae). viruses transmitted by way of saliva may be perceived as bridging the categories of direct and indirect contact. if any particular type of virus that is secreted into saliva has either no stability when exposed to ambient environments in oral secretions or only limited stability under those conditions, then the virus will have to be transmitted by saliva that is transferred during oral contact between hosts. conversely, if the particular virus type has good stability when exposed to ambient environments in oral secretions, then that virus can be transmitted on shared food or in association with fomites. some of the viruses transmitted in saliva do remain associated with the host as a permanent infection, and these often are the viruses that possess limited stability in ambient environments, such as members of the family herpesviridae. many of the viruses that are secreted into saliva and can be transferred to a new host in association with fomites do not remain associated with the host as a permanent infection, such as members of the family picornaviridae. in general, those viruses transmitted by indirect contact between hosting individuals tend to produce only transient infections of their individual hosts rather than remain associated with the individual host as a permanent infection. these several latter points suggest that there may be some evolutionary relationship between ease of viral transmission to a new potential host individual or the frequency of opportunities for viral transmission, and the length of time that a virus must be capable of remaining with its present host to have a reasonable chance of achieving eventual transmission. the indirect contact approach also offers one major advantage and one major drawback to a virus. viruses transmitted by indirect contact have the advantage of potential access to a far greater number of hosting individuals than is the case for viruses transmitted by direct contact. the drawback is that the viruses must have evolved stability when they are exposed to the ambient environment. the vehicles that viruses utilize to achieve transmission between hosting individuals by indirect contact are subdivided into the following four categories: food, water, air (in actuality, this refers to aerosols), and fomites. transmission by any of these four categories of vehicles will usually be associated with some specific physical activity on the part of the present host, and will always be associated with some physical activity on the part of the proximate host. of course, foods are items intentionally ingested for their caloric or nutritional value. food contamination can occur by way of the food being a virally infected animal consumed by the proximate host. in such cases, there is no specific physical activity on the part of the present host (the one being eaten) that can be identified as having caused the proximate host to be ingesting contaminated food (indeed, perhaps it is a lack of physical activity on the part of the present host that is to blame). otherwise, viral contamination of foods can result from fecal material being transferred via contact with unwashed hands or if contaminated aerosols fall into the food. a particularly notable example of a virus of humans that is transmitted via foods is the hepatitis a virus of the genus hepatovirus (family picornaviridae). water usually serves as a vehicle after it has been contaminated with fecal material. acquisition of a viral infection from water usually results from the proximate host ingesting contaminated water. physical contact of the skin or mucosa of the proximate host with contaminated water, as may occur during recreational activities or body washing, can result in acquisition of infection. notable examples of viruses transmitted by these waterborne routes are those belonging to the viral families astroviridae and caliciviridae, and the genera enterovirus and hepatovirus of the family picornaviridae. viral contamination of air can occur by two principal mechanisms. the first, and most significant, involves release of aerosols that contain droplets of respiratory secretions (i.e., nasal, oral, or pulmonary mucus). this type of transmission route is referred to as being the route of droplet aerosols. notable examples of viruses transmitted in this manner are those belonging to the viral families coronaviridae, orthomyxoviridae, and paramyxoviridae, and members of the genus viral contamination of fomites (defined as solid environmental surfaces that can serve in transmission of infections) can occur in many ways. the variety of things that represent fomites include items used to conserve warmth (e.g., blankets, clothing), items used while eating (e.g., cups, dinner plates, and utensils), tables on which diapers are changed, doorknobs, medical devices, toilet seats, and toys. the ways by which these environmental items become contaminated include projection of droplet aerosols onto environmental objects during sneezing or coughing, aerosols falling onto objects, and unintended surface contamination (including children's clothing, blankets and toys) by blood, feces, fluid from skin lesions (rashes), nasal secretions, saliva, or urine. the task of achieving viral transmission via this route is accomplished when these objects are subsequently handled or used by a potential proximate host. among the viral genera whose members can be transmitted via fomites are orthopoxvirus (family poxviridae) and rhinovirus (family picornaviridae). twenty of the viral families contain members capable of infecting humans. together, they cause a broad range of illnesses in humans. the terminology used in describing these illnesses is given in table i . the rest of this section summarizes the ecology of these viral families. figure 1 schematizes the manner in which the different aspects of the ecology of viral infection fit together. the literature sources used for to compile this summary include hurst and murphy (1996) , ictv (1995), evans and kaslow (1997) , and white and fenner (1994) . dae) in association with a natural host. transmission of this virus between hosts occurs when an infected animal bites an uninfected animal, with the virus being transferred by saliva into the bite wound. subsequent movement of viral infection into the nervous system and salivary glands of the newly bitten host animal is considered to represent primary tropisms, as infection at these sites is directly related to movement of virus into the body of the current host and subsequent transfer of virus to the next host. infection of the adrenal cortex is considered a secondary tropism, since those viruses produced in the adrenal cortex will not be transferred to a subsequent host animal. however, infection of the adrenal cortex may play a role in the viral ecology by augmenting the aggressiveness of this newly infected host, thereby increasing the likelihood that this animal will then bite other potential host animals. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. adenopathy (the origin of the family name), conjunctivitis, coryza, encephalitis, gastroenteritis, keratoconjunctivitis, pharyngitis, pharyngoconjunctival fever, and pneumonia. infection course ~ productive, both short term-initial and recurrent. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the cervix, conjunctiva, pharynx, small intestine, and urethra; the secondary tissue and organ tropisms are toward the brain, kidney, lungs, and lymph nodes; at the host population level, these viruses generally are endemic and initially acquired at a very early age, with the infections very often asymptomatic in young children. evasion of host defenses ~ uncertain. and indirect (vehicle-borne) contact via fecally contaminated water, food, fomites, and fomites contaminated by respiratory secretions. genus affecting humans: arenavirus. familial nature with respect to members affecting humans" zoonotic. natural hosts: rodents, including commensal voles and mice, as well as commercial colonies of hamsters and nude mice. arthralgia, carditis, encephalomyelitis, encephalopathy, facial edema, fetal loss, focal necrosis of liver, gastritis, hemorrhagic fever, hepatitis, inhibition of platelet function (causes the fatal bleeding associated with this virus family), malaise, meningitis, myalgia, nerve deafness, and pneumonia. infection course ~ productive, short term-initial. at the individual host level, the primary tissue and organ tropisms presumably are toward the liver and lungs; the secondary tissue and organ tropisms are toward the brain, fetus, heart, joints, and nerves; at the host population level, these viruses can be extremely devastating to individual hosts but are poorly transferred between humans, which usually represent dead-end hosts. evasion of host defenses ~ uncertain. predominant routes of transmission between hosts: indirect (vehicle-borne) contact via inhalation of particulate aerosols bearing dried rodent urine or acquisition of infectious materials through skin abrasion (a form of surface contact). genus affecting humans: astrovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. types of illnesses induced in humans: enteritis, gastroenteritis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the small intestine; the secondary tissue and organ tropisms presently are unknown; at the host population level, these viruses are endemic, principally causing a mild enteritis seen in young adults. avoids host non-immune defenses by resistance to proteolytic attack (their infectivity is actually increased by proteolytic attack). via fecally contaminated water, food, and fomites. genera affecting humans: bunyavirus, hantavirus, nairovirus, phlebovirus. familial nature with respect to members affecting humans: zoonotic. natural hosts: largely rodents, but also hares and rabbits, and some ungulates. types of illnesses induced in humans: arthralgia, encephalitis, hematemesis, hematuria, hemolytic uremia, hemorrhagic fever, hemorrhagic pneumonia, hemorrhagic (petechial) skin rash, hepatitis, melena, myalgia, pneumonia, renal dysfunction, retinitis, retroocular pain. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the kidneys, liver, and lungs; the secondary tissue and organ tropisms are toward the brain and eyes; at the host population level, these viruses are not well sustained within human populations, and humans usually represent dead-end hosts. evasion of host defenses ~ uncertain, but may include avoiding host immune defenses by infecting immune cells. direct host-to-vector contact by gnats, midges, mosquitoes, sandflies, and ticks for the genera bunyavirus, nairovirus, and phlebovirus, and indirect (vehicle-borne) contact via particulate aerosols containing dried rodent urine, or contact with rodent excreta or contaminated fomites for the genus hantavirus. genus affecting humans: calicivirus. familial nature with respect to members affecting humans: viruses of humans and zoonotic. natural or alternate hosts: fish, terrestrial as well as marine mammals (especially swine). gastroenteritis, hepatitis (nonprogressive, but extraordinarily high fatality rate-10 to >25% --for women if contracted during the third trimester of pregnancy), myalgia. infection course ~ productive, short term-initial. ~ral replication ~ at the individual host level, primary tissue and organ tropisms are toward the small intestine; secondary tissue and organ tropisms are toward the liver; at the host population level, these tend to be epidemic within human populations; for the hepatitis e virus it seems that acquisition occurs from swine, with the result being epidemics (often very widespread) of human disease; some acquisition from animals may come from eating infected animals; subsequent transmission of all caliciviruses within human populations is by fecally contaminated waste and thus can be very widespread. evasion of host defenses ~ avoids host non-immune defenses by resistance to proteolytic attack. indirect (vehicle-borne) contact via fecally contaminated water, food, and fomites. genera affecting humans: coronavirus, torovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: some terrestrial ungulates and carnivores. coryza, gastroenteritis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, the primary tissue and organ tropisms are toward the intestines, lungs (possibly), nasopharynx, and sinuses; at the host population level, these viruses are very widespread and essentially nonfatal. predominant routes of transmission between hosts: indirect (vehicle-borne) contact via fecally contaminated water, food, and fomites. familial nature with respect to members affecting humans: generally zoonotic. natural hosts: unknown, but may include bats and rodents, with primates serving as intermediary hosts leading to human exposure. conjunctivitis, hemorrhagic fever (frequently fatal, death possibly resulting from extreme inflammatory response), hepatic necrosis, myalgia, pharyngitis. infection course ~ productive, short term-initial. viral replication w at the individual host level, primary tissue and organ tropisms are toward the immune cells and liver (possibly); secondary tissue and organ tropisms are toward the adrenal glands, kidneys, liver, and spleen; at the host population level, these viruses are transferred between humans but seem unable to be sustained in human populations; humans usually represent deadend hosts. predominant routes of transmission between hosts: direct contact via host-tohost transfer of contaminated bodily fluids. genera affecting humans: flavivirus, hepatitis c-like viruses. familial nature with respect to members affecting humans: viruses of humans and zoonotic. natural or alternate hosts: members of the genus flavivirus cross-infect a variety of birds and terrestrial mammals via mosquitoes or ticks (depending on the viral species) and most clearly are zoonotic, although those that cause yellow fever and the four that cause dengue may have become viruses of humans without time to coevolve; one species of the genus hepatitis c-like viruses affects humans and seems naturally limited to humans. arthritis with rash, encephalitis, hemorrhagic fever, hepatitis (chronic, which may lead to hepatocellular carcinoma). infection course ~ short term-initial for the genus flavivirus, increasing to end-stage for the hepatitis c virus. viral replication m at the individual host level, primary tissue and organ tropisms are toward the immune cells (principally monocytes and macrophages) and liver; secondary tissue and organ tropisms are toward the brain and liver; at the host population level, most of these viruses are zoonotic, with humans representing dead-end hosts; however, some can be sustained within human populations and occasionally have high lethality rates. evasion of host defenses ~ avoids host immune defenses by infecting immune cells. for flaviviruses, direct host-tovector contact; for hepatitis c virus, presumably direct contact via host-to-host transfer of contaminated bodily fluids. genera affecting humans: orthohepadnavirus. the hepatitis d virus (hdv) is a member of the floating genus deltavirus; it is a defective satellite virus which can coinfect humans, but only in association with the hepatitis b virus (hbv) because hdv encapsidates itself with proteins encoded by the genome of the coinfecting hbv. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: one species of viral family hepadnaviridae (hepatitis b virus) is known to infect humans, and it seems naturally limited to humans. type of illness induced in humans: hepatitis, which may become chronic in adults. infection course ~ productive, short term-initial, and increasing to end-stage. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the liver; secondary tissue and organ tropisms are toward the bile duct epithelium, circulating immune cells, and pancreatic acinar cells; at the host population level, when acquired by adults and older children, these viruses generally cause an acute but short-term illness that sometimes can be fulminant; when acquired by neonates or younger children, initially tends to be subclinical but becomes chronic, and the tendency to be chronic can be racially associated (chinese, possibly also black african). evasion of host defenses ~ avoids host immune defenses by infecting immune cells. direct contact via host-to-host transfer of contaminated bodily fluids and perinatally from contaminated maternal blood. genera affecting humans: cytomegalovirus, lymphocryptovirus, roseolovirus, simplexvirus, varicellovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans, but may pass to primates. carcinoma, carditis, chronic gastrointestinal infection, encephalitis, hepatomegaly, keratoconjunctivitis, lymphoma, myelitis, neuralgia, papular rash of skin and mucosa, paralysis, retinitis, splenomegaly. infection course ~ productive, recurrent. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the genital and oral mucosa, pharynx, and salivary glands; secondary tissue and organ tropisms are toward the eyes, kidneys, liver, lymph nodes, nervous system including brain, and spleen; at the host population level, these viruses are ubiquitous, tend to be acquired in childhood or early adulthood, and seldom directly result in host death. evasion of host defenses ~ avoids host immune defenses by infecting immune cells. direct contact via host-to-host transfer of fluid from viral-induced lesions of skin or mucosa and by saliva contaminated by chronically infected salivary glands; plus transmission to offspring either transplacentally, intrapartum (during the birth process), or via breast milk. genera affecting humans: influenzavirus a, influenzavirus b, and influenzavirus c. familial nature with respect to members affecting humans: generally viruses of humans. alternate hosts: birds (possibly), swine. types of illnesses induced in humans: coryza, malaise, myalgia, nasopharyngitis, pneumonia, retroocular pain, tracheobronchitis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the ciliated columnar epithelium of the respiratory tract (the exact tissue tropism is directly related to the virus hemagglutinin [ha] serotype); at the host population level, these viruses constantly undergo antigenic drift and antigenic shift and cause wide-scale seasonal epidemics in humans, although infection-related fatality is usually limited to humans aged 65 or older (most notably, age 75 or older). evasion of host defenses ~ avoids host immune defenses by antigenic mimicry and by rapid viral mutation. indirect (vehicle-borne) contact via droplet aerosols (from sneezing and coughing) and aerosol-contaminated fomites. genera affecting humans: papillomavirus, polyomavirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. types of illnesses induced in humans: benign tumors of skin and mucosa that may progress to malignancy, progressive demyelinating encephalopathy. infection course ~ productive, recurrent. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the mucosa and skin (genus papillomavirus) and toward the upper respiratory tract (genus polyomavirus); secondary tissue and organ tropisms are toward the brain and kidneys (genus polyomavirus); at the host population level, these viruses are ubiquitous and almost never directly responsible for host death. evasion of host defenses ~ avoids host immune defenses by antigenic mimicry. host-to-host or indirect (vehicle-borne) contact by way of fomites (genus papillomavirus); indirect (vehicle-borne) contact via aerosols (genus polyomavirus). genera affecting humans: morbillivirus, paramyxovirus, pneumovirus, rubulavirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. bronchiolitis, conjunctivitis, coryza, encephalitis, glandular enlargement (especially salivary glands), immunosuppression (morbillivirus causes an immunosuppression that is temporary, but which is arguably the most severe induced by a virus of humans, and can result in death by other coinfecting pathogens, such as enteric protozoans, that normally would not cause fatality), macular rash, nerve deafness, orchitis, pneumonitis. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the epidermis and mucosa (including conjunctival, oral and respiratory); secondary tissue and organ tropisms are toward the brain, breasts, circulating immune cells, and testicles; at the host population level, these viruses tend to be acquired at a young age and are almost never directly responsible for host death, although severe sequelae can result if acquired beyond early childhood. evasion of host defenses avoids host immune defenses by infecting immune cells. indirect (vehicle-borne) contact via aerosols. genus affecting humans: parvovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. anemia, arthralgia, erythema, myalgia. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the throat; secondary tissue and organ tropisms are toward the circulatory system, erythrocyte precursor cells in bone marrow, possibly reticulocytes in blood, and skin; at the host population level, these viruses usually cause a disease of childhood; parvoviral disease is either mild or self-limiting in otherwise healthy children or adults. evasion of host defenses ~ uncertain. uncertain, but potentially direct host-to-host contact, including transplacental, and indirect (vehicle-borne) contact via aerosols and fecally contaminated water, food, and fomites. genera affecting humans: enterovirus, hepatovirus, rhinovirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans, but may pass to primates and canines. diabetes, encephalitis, macular and maculopapular rashes of skin and mucosa, meningitis, myocarditis, otitis media, paralysis of skeletal muscles (occasionally including the diaphragm), pericarditis, retroocular pain, sinusitis (genus enterovirus); hepatitis (nonprogressive) (genus hepatovirus); coryza (genus rhinovirus). infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the nasopharynx and small intestine; secondary tissue and organ tropisms are very genus and species specific and toward the beta cells of the pancreas, conjunctiva, liver, meninges, muscles (including the heart), neurons (including those of the central nervous system), and skin; at the host population level, infections caused by members of the genus enterovirus usually are nonfatal, and both enterovirus and hepatovirus tend to result in asymptomatic infections if acquired in infancy, though the likelihood of severe symptomatology increases with age at acquisition; infections caused by members of the genus rhinovirus generally are symptomatic but essentially nonfatal regardless of host age. evasion of host defenses m members of genus enterovirus avoid host non-immune defenses by resistance to low ph (resistant to stomach acid) and to moderate alkalinity. indirect (vehicle-borne) contact via aerosols and fecally contaminated water, food, and fomites. genera affecting humans: molluscipoxvirus, orthopoxvirus, parapoxvirus. familial nature with respect to members affecting humans: viruses of humans and zoonotic. alternate hosts: one species affecting humans (smallpox) seems naturally limited to humans; monkeypox is a very notable but rare zoonotic exception and is presumably acquired from monkeys; several other species may cycle with domesticated bovines and ovines appearing as lesions on teats and udder. types of illnesses induced in humans: necrotic lesions of abdominal organs and skin, nodules and tumors in skin, papular rash. infection course ~ productive, short term-initial. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the skin; secondary tissue and organ tropisms are toward the internal organs and lymph nodes; at the host population level, these viruses have very low transmissibility but have prolonged survivability on fomites due to extreme resistance to desiccation. evasion of host defenses ~ avoids host immune defenses by antigenic mimicry. direct contact via host-to-host contact with skin lesions and indirect (vehicle-borne) contact via lesion-contaminated fomites (very notably blankets and other bedding items). genera affecting humans: coltivirus, orthoreovirus, rotavirus. familial nature with respect to members affecting humans: viruses of humans and zoonotic. natural or alternate hosts: those species of the genus coltivirus infecting humans seem zoonotic with terrestrial mammals (notably rodents and squirrels) serving as their natural hosts; species of the genus orthoreovirus cross-infect nearly all known terrestrial mammals (especially rodents); those species of the genus rotavirus affecting humans seem naturally limited to humans. hemorrhagic fever, meningoencephalitis (genus coltivirus); upper respiratory symptoms (possibly associated with genus orthoreovirus); gastroenteritis (genus rotavirus). infection course m productive, short term-initial. viral replication-at the individual host level, primary tissue and organ tropisms are highly genus specific, and are toward the immune cells (genus coltavirus), possibly upper respiratory area (genus orthoreovirus), and the small intestine (genus rotavirus); secondary tissue and organ tropisms are toward the brain and meninges; at the host population level, these viruses have high transmissibility, especially among newborns, for whom they usually produce asymptomatic infections; in older children and adults, these viruses likewise have a tendency to produce asymptomatic infections; although rarely fatal in wellnourished children, members of the genus rotavirus are estimated to cause a million deaths every year in undernourished children. evasion of host defenses ~ avoids host immune defenses by infecting immune cells (genus coltivirus), avoids host non-immune defenses by resistance to heat, low ph, and proteolytic attack (infectivity actually increased by proteolytic attack) (members of the genera orthoreovirus and rotavirus). indirect (vehicle-borne) contact via fecally contaminated water, food, and fomites with the orthoreovirus possibly also being spread by aerosols. genera affecting humans: blv-htlv retroviruses, lentivirus, spumavirus. familial nature with respect to members affecting humans: viruses of humans. alternate hosts: species affecting humans seem naturally limited to humans. carcinoma, encephalitis, leukemia (adult t-cell), lymphoma (adult t-cell), progressive chronic immunosuppression and immunodepletion (including acquired immunodeficiency syndrome), progressive myelopathy, sarcoma. infection course m productive, short term-initial, often followed by increasing to end-stage; also may seem nonproductive in the case of some endogenous retroviruses. viral replication ~ at the individual host level, primary tissue and organ tropisms are toward the immune cells (largely t-cell populations); secondary tissue and organ tropisms are toward the brain and intestines; at the host population level, those viruses considered transmissible (i.e., excluding endogenous retroviruses) have a very low transmissibility rate, produce infections whose incubation times are very long (10--40 years), and may pass through breast milk; the endogenous retroviruses are permanently integrated into the human genome and are passed genetically to all offspring. evasion of host defenses ~ avoids host immune defenses by rapid viral mutation and by infecting immune cells. direct contact via host-to-host transfer of contaminated bodily fluids. genera affecting humans: lyssavirus, vesiculovirus. familial nature with respect to members affecting humans: zoonotic. natural hosts: foxes, skunks, and vampire bats (genus lyssavirus), cattle and horses (genus vesiculovirus). neuronal infections leading to encephalitis which appears invariably fatal (genus lyssavirus); myalgia (genus vesiculovirus). infection course m productive, short term-initial. viral replication m at the individual host level, primary tissue and organ tropisms are toward the neurons, including those in the spinal cord and limbic system of the brain, and the salivary glands (genus lyssavirus); and toward either the muscles or nerves (genus vesiculovirus); secondary tissue and organ tropisms are toward the adrenal cortex and pancreas (genus lyssavirus); at the host population level, these viruses are essentially nontransmissible. evasion of host defenses ~ avoids host immune defenses by limited antigenic exposure within the host because the virus largely remains within neuronal cells until near end-stage (genus lyssavirus). direct contact via host-to-host contact associated with deposition of contaminated saliva into a bite wound and possibly associated with contamination of skin or mucosal wounds by other types of bodily fluids; in the case of the genus vesiculovirus, vesicular fluids. genera affecting humans: alphavirus, rubivirus. familial nature with respect to members affecting humans: viruses of human and zoonotic. natural or alternate hosts: species of the genus alphavirus cross-infect a wide variety of terrestrial vertebrates, mostly via mosquitoes and ticks; one species of the genus rubivirus affects humans and it seems restricted to humans. arthralgia, arthritis, diabetes, encephalitis, fetal developmental abnormalities (cardiological, diabetic, and neurological including auditory, encephalitic, and visual k caused by rubivirus if contracted during the first trimester of pregnancy), macular rash of skin, myalgia, myositis. infection course m productive, short term-initial. viral replication m at the individual host level, primary tissue and organ tropisms are toward the immune cells (specifically monocytes and macrophages in bone marrow, liver, lymph nodes, and spleen) and oropharynx; secondary tissue and organ tropisms are toward the beta cells of the pancreas, muscles, neurons of the central nervous system including the brain, skin, and synovial cells of joints; at the host population level, most members of the genus alphavirus seem poorly transmitted between humans, and humans probably represent a dead-end host; infection by the genus rubivirus is seldom fatal but highly transmissible via aerosols and usually causes a trivial exanthema of childhood or mild symptoms in adults, although infection during the first trimester of pregnancy can result in extremely severe developmental abnormalities. evasion of host defenses avoids host immune defenses by infecting immune cells. to-vector (genus alphavirus), host-to-host (genus rubivirus), or indirect (vehicleborne) contact via aerosols (genus rubivirus). there are many types of viruses that afflict humans. we have managed to coevolve with some of these to lessen our misery. the struggle will continue as new viruses appear and as the existing ones reshuffle their genes or change their antigenicity by mutation. in the end, the contest is a struggle of biology versus biology, and the basic biology of the viruses is the same as ours. viral infections of humans: epidemiology and control modeling disease transmission and its prevention by disinfection virus taxonomy: sixth report of the international committee on taxonomy of viruses on viruses, sex and motherhood key: cord-252691-757mh2mh authors: pratt, r. j.; pellowe, c. m.; wilson, j. a.; loveday, h. p.; harper, p. j.; jones, s.r.l.j.; mcdougall, c.; wilcox, m. h. title: epic2: national evidence-based guidelines for preventing healthcare-associated infections in nhs hospitals in england date: 2007-02-28 journal: journal of hospital infection doi: 10.1016/s0195-6701(07)60002-4 sha: doc_id: 252691 cord_uid: 757mh2mh executive summary national evidence-based guidelines for preventing healthcare-associated infections (hcai) in national health service (nhs) hospitals in england were commissioned by the department of health (dh) and developed during 1998-2000 by a nurse-led multi-professional team of researchers and specialist clinicians. following extensive consultation, they were published in january 2001.1 these guidelines describe the precautions healthcare workers should take in three areas: standard principles for preventing hcai, which include hospital environmental hygiene, hand hygiene, the use of personal protective equipment, and the safe use and disposal of sharps; preventing infections associated with the use of short-term indwelling urethral catheters; and preventing infections associated with central venous catheters. the evidence for these guidelines was identified by multiple systematic reviews of experimental and non-experimental research and expert opinion as reflected in systematically identified professional, national and international guidelines, which were formally assessed by a validated appraisal process. in 2003, we developed complementary national guidelines for preventing hcai in primary and community care on behalf of the national collaborating centre for nursing and supportive care (national institute for healthand clinical excellence).2 a cardinal feature of evidence-based guidelines is that they are subject to timely review in order that new research evidence and technological advances can be identified, appraised and, if shown to be effective in preventing hcai, incorporated into amended guidelines. periodically updating the evidence base and guideline recommendations is essential in order to maintain their validity and authority. consequently, the dh commissioned a review of new evidence published following the last systematic reviews. we have now updated the evidence base for making infection prevention and control recommendations. a critical assessment of the updated evidence indicated that the original epic guidelines published in 2001 remain robust, relevant and appropriate but that adjustments need to be made to some guideline recommendations following a synopsis of the evidence underpinning the guidelines. these updated national guidelines (epic2) provide comprehensive recommendations for preventing hcai in hospitals and other acute care settings based on the best currently available evidence. because this is not always the best possible evidence, we have included a suggested agenda for further research in each section of the guidelines. national evidence-based guidelines are broad principles of best practice which need to be integrated into local practice guidelines. to monitor implementation, we have suggested key audit criteria for each section of recommendations. clinically effective infection prevention and control practice is an essential feature of protecting patients. by incorporating these guidelines into routine daily clinical practice, patient safety can be enhanced and the risk of patients acquiring an infection during episodes of healthcare in nhs hospitals in england can be minimised. we would like to acknowledge the assistance we received from the liverpool reviews and implementation group (university of liverpool) who shared with us data from their health technology assessment focused on the clinical and cost effectiveness of central venous catheters treated with antimicrobial agents in preventing bloodstream infections. we are also indebted to the infection control nurses association and the hospital infection society for their input into the s2 r.j. pratt et al. made to some guideline recommendations following a synopsis of the evidence underpinning the guidelines. these updated national guidelines (epic2) provide comprehensive recommendations for preventing hcai in hospitals and other acute care settings based on the best currently available evidence. because this is not always the best possible evidence, we have included a suggested agenda for further research in each section of the guidelines. national evidence-based guidelines are broad principles of best practice which need to be integrated into local practice guidelines. to monitor implementation, we have suggested key audit criteria for each section of recommendations. clinically effective infection prevention and control practice is an essential feature of protecting patients. by incorporating these guidelines into routine daily clinical practice, patient safety can be enhanced and the risk of patients acquiring an infection during episodes of healthcare in nhs hospitals in england can be minimised. standard principles for preventing healthcareassociated infections in hospital and other acute care settings this guidance is based on the best critically appraised evidence currently available. the type and class of supporting evidence explicitly linked to each recommendation is described. all recommendations are endorsed equally and none is regarded as optional. these recommendations are not detailed procedural protocols and need to be incorporated into local guidelines. this guidance on infection control precautions should be applied by all healthcare practitioners to the care of every patient. job descriptions should reflect this and annual appraisal evidence should be available to support continuing engagement of each member of staff. the recommendations are divided into four distinct interventions: 1. hospital environmental hygiene; 2. hand hygiene; 3. the use of personal protective equipment; and 4. the safe use and disposal of sharps. these guidelines do not address the additional infection control requirements of specialist settings, such as the operating department. hospital environmental hygiene sp1 the hospital environment must be visibly class c clean, free from dust and soilage and acceptable to patients, their visitors and staff. guidelines for preventing infections associated with the use of short-term indwelling urethral catheters this guidance is based on the best critically appraised evidence currently available. the type and class of supporting evidence explicitly linked to each recommendation is described. all recommendations are endorsed equally and none is regarded as optional. these recommendations are not detailed procedural protocols and need to be incorporated into local guidelines. these guidelines apply to adults and children aged 1 year and older and should be read in conjunction with the guidance on standard principles. the recommendations are divided into five distinct interventions: this guidance is based on the best critically appraised evidence currently available. the type and class of supporting evidence explicitly linked to each recommendation is described. all recommendations are endorsed equally and none is regarded as optional. these recommendations are not detailed procedural protocols and need to be incorporated into local guidelines. these guidelines apply to adults and children aged one year and older and should be read in conjunction with the guidance on standard principles. the recommendations are divided into 9 distinct interventions: 1. education of healthcare workers and patients; 2. general asepsis; 3. selection of catheter type; 4. selection of catheter insertion site; 5. maximal sterile barrier precautions during catheter insertion; 6. cutaneous antisepsis; 7. catheter and catheter site care; 8. catheter replacement strategies; and 9. general principles for catheter management. 2 an evidence review in 2004 indicated the necessity to amend and update some of the original epic guideline recommendations to ensure that they continue to reflect new and emerging evidence, remain relevant to infection control and prevention practice and enjoy the confidence of practitioners and patients. 3, 4 additional updating systematic reviews were conducted in 2005 and the original epic guidelines have now been revised. they are referred to in this publication as the epic2 infection prevention guidelines, which now replace the original 2001 guidelines. what are national evidence-based guidelines? these are systematically developed broad statements (principles) of good practice. they are driven by practice need, based on evidence and subject to multi-professional debate, timely and frequent review, and modification. national guidelines are intended to inform the development of detailed operational protocols at local level and can be used to ensure that these incorporate the most important principles for preventing hcai in nhs hospitals and other acute care health services. why do we need national guidelines for preventing healthcare-associated infections? during the past two decades, hcai have become a significant threat to patient safety. the technological advances made in the treatment of many diseases and disorders are often undermined by the transmission of infections within healthcare settings, particularly those caused by antimicrobial-resistant strains of disease-causing microorganisms that are now endemic in many healthcare environments. the financial and personal cost of these infections, in terms of the economic consequences to the nhs and the physical, social and psychological costs to patients and their relatives, have increased both government and public awareness of the risks associated with healthcare interventions, especially that of acquiring a new infection. although not all hcai can be prevented, many can. clinical effectiveness, i.e., using prevention measures that are based on reliable evidence of efficacy, is a core component of an effective strategy designed to protect patients from the risk of infection. what is the purpose of the guidelines? these guidelines describe clinically effective measures that are used by healthcare workers for preventing infections in hospital and other acute care health services. what is the scope of the guidelines? three sets of guidelines were originally developed and have now been updated. they include: • standard infection control principles include best practice recommendations for hospital environmental hygiene, effective hand hygiene, the appropriate use of personal protective equipment, and the safe use and disposal of sharps; • guidelines for preventing infections associated with the use of short-term indwelling urethral catheters; and • guidelines for preventing infections associated with the use of central venous access devices. what is the evidence for these guidelines? the evidence for these guidelines was identified by multiple systematic reviews of experimental and non-experimental research. in addition, evidence from expert opinion as reflected in systematically identified professional, national and international guidelines was considered following formal assessment using a validated appraisal process. 5, 6 all evidence was critically appraised for its methodological rigour and clinical practice applicability and the best available evidence influenced the guideline recommendations. who developed these guidelines? the epic2 guidelines were developed by a nurseled team of researchers, senior infection control nurses and a director of microbiology and infection prevention and control in a large nhs teaching hospital trust (see 1.1). who are these guidelines for? these guidelines can be appropriately adapted and used by all hospital practitioners. they will inform the development of more detailed local protocols and ensure that important standard principles for infection prevention are incorporated. consequently, they are aimed at hospital managers, members of hospital infection control teams, and individual health care practitioners. at an individual level, they are intended to influence the quality and clinical effectiveness of infection prevention decision-making. the dissemination of these guidelines also help patients understand the standard infection prevention precautions recommended to protect them from hcai. how are these guidelines structured? each set of guidelines follows an identical format, which consists of: • a resume of the systematic review process; • the intervention heading; • a headline statement describing the key issues being addressed; • a synthesis of the related evidence; • an economic opinion, where appropriate; • guideline recommendation(s) classified according to the strength of the underpinning evidence. finally, at the end of each section there is a description of areas for further research and suggested audit criteria. all evidence is referenced in section 5. how frequently are the guidelines reviewed and updated? a cardinal feature of evidence-based guidelines is that they are subject to timely review in order that new research evidence and technological advances can be identified, appraised and, if shown to be effective in preventing hcai, incorporated into amended guidelines. the evidence base for these guidelines will be reviewed in two years (2009) and the guidelines will be updated approximately four years after publication (2011). how can these guidelines be used to improve your clinical effectiveness? in addition to informing the development of detailed local operational protocols, these guidelines can be used as a benchmark for determining appropriate infection prevention decisions and, as part of reflective practice, to assess clinical effectiveness. they also provide a baseline for clinical audit, evaluation and education, and facilitate ongoing quality improvements. how much will it cost to implement these guidelines? significant additional costs are not anticipated in implementing these guidelines. however, where current equipment or resources do not facilitate the implementation of the guidelines, or where staff levels of adherence to current guidance are poor, there may be an associated increase in costs. given the social and economic costs of hcai, the consequences associated with not implementing these guidelines would be unacceptable to both patients and health care professionals. these guidelines have been subject to extensive external consultation with key stakeholders, including royal colleges, professional societies and organisations, including patients, and trades unions (appendix a.1). the guidelines were developed using a systematic review process (appendix a.2). in each set of guidelines a resume of the relevant guideline development methodology is provided. electronic databases were searched for national and international guidelines and research studies published during the period 01 january 1999 to 31 august 2005. a two-stage search process was used. for each set of epic guidelines, an electronic search was conducted for systematic reviews of randomised controlled trials and current national and international guidelines. the following data bases were searched: • cochrane library; • national guideline clearinghouse; • national electronic library of health; • national institute for health and clinical excellence. guidelines were retrieved and subjected to critical appraisal using the agree instrument, 6 an evaluation method used in europe for assessing the methodological quality of clinical practice guidelines. following appraisal, accepted guidelines were included as part of the evidence base supporting guideline development. they were also used to verify professional consensus and in some instances, as the primary source of evidence. stage 2: systematic search for additional evidence review questions for the systematic reviews of the literature were then developed for each set of epic guidelines following recommendations from expert advisors. searches were constructed using relevant mesh (medical subject headings) and free-text terms. on completion of the main search, an economic filter was applied. the following databases were searched: • cumulated index of nursing and allied health literature; • embase; • the cochrane library. abstract review -identifying studies for appraisal search results were downloaded into a reference manager™ database and titles and abstracts printed for preliminary review. reviewers identified and retrieved all studies where the title or abstract: addressed one or more of the review questions; identified primary research or systematically conducted secondary research; indicated a theoretical/clinical/ in use study. no research designs were specifically excluded but wherever possible, in use rather than in vitro studies were retrieved. where no abstract was available and the title indicated one or more of the above criteria, the study was retrieved. due to the limited resources available for this review, foreign language studies were not reviewed. all full-text studies retrieved were independently assessed by two experienced reviewers who identified those studies meeting the above inclusion criteria for critical appraisal. included studies were appraised using an adapted data extraction process based on systems developed by the scottish intercollegiate guideline network for study quality assessment. 7 due to the limited resources available for this review, studies were not double-blind appraised. however, all studies were appraised and data extracted by one experienced reviewer and then checked by a second experienced reviewer. any disagreement between reviewers was resolved through discussion. evidence tables were constructed from the quality assessments and the studies summarised in the evidence reports. the evidence was classified using methods adopted by the national institute for health and clinical excellence (nice) from the scottish intercollegiate guideline network (sign) ( table 1) . 8, 9 this system differs from that used in the previous epic and nice infection prevention guidelines. 1, 2 the evidence tables and reports were presented to the advisors for discussion. at this stage, expert advice derived from seminal works and appraised national and international guidelines were considered. following extensive discussion the guidelines were drafted. factors influencing the guideline recommendations included: • the nature of the evidence; • the applicability of the evidence to practice; • costs and knowledge of healthcare systems. the classification scheme adapted by nice from sign was used to define the strength of recommendation (table 2 ). 8, 9 the complete series of evidence tables are posted on the epic website at: [http://www.epic. tvu.ac.uk]. type of evidence 1 ++ high-quality meta-analyses, systematic reviews of randomised controlled trials (rct), or rct with a very low risk of bias 1 + well-conducted meta-analyses, systematic reviews of rct, or rct with a low risk of bias 1 -meta-analyses, systematic reviews of rct, or rct with a high risk of bias* 2 ++ high-quality systematic reviews of case-control or cohort studies high-quality case-control or cohort studies with a very low risk of confounding, bias or chance and a high probability that the relationship is causal 2 + well-conducted case-control or cohort studies with a low risk of confounding, bias or chance and a moderate probability that the relationship is causal 2 -case-control or cohort studies with a high risk of confounding bias, or chance and a significant risk that the relationship is not causal* 3 non-analytic studies (for example, case reports, case series) 4 expert opinion, formal consensus *studies with a level of evidence ' -' should not be used as a basis for making a recommendation these guidelines do not address the additional infection control requirements of specialist settings, such as the operating department or for outbreak situations. we have previously described the systematic review process in section 1.10. for detailed descriptions of previous systematic reviews which have contributed to the evidence base underpinning these guidelines, readers should consult the original guidelines, 1 the guidelines for the prevention of health-care associated infections in primary and community care 2 and our interim report in 2004 on changes in the evidence base. 3 search questions were developed from advice received from our specialist advisors and the results of the searches are found in section 2.10. the process outlined in section 2.10 refers only to the most recent systematic review of the literature undertaken in 2005. following our reviews, guidelines were drafted which described 38 recommendations within the below intervention categories: 1. hospital environmental hygiene; 2. hand hygiene; 3. personal protective equipment; and 4. safe use and disposal of sharps. good hospital hygiene is an integral and important component of a strategy for preventing healthcare-associated infections in hospitals this section discusses the evidence upon which recommendations for hospital environmental hygiene are based. hospital environmental hygiene encompasses a wide range of routine activities including: cleaning and decontamination; laundry and housekeeping; safe collection and disposal of general and clinical waste; and kitchen and food hygiene. guidelines are provided here for: • cleaning the general hospital environment; • cleaning items of shared equipment; and • education and training of staff. our initial systematic review concluded that there was little research evidence of an acceptable quality upon which to base guidance related to the maintenance of hospital environmental hygiene. 1 however, there was a body of clinical evidence, derived from case reports and outbreak investigations, which suggested an association between poor environmental hygiene and the transmission of microorganisms causing healthcare-associated infections in hospital. 10, 11 attention had been drawn to perceived falling standards in the cleanliness of hospitals since the introduction of compulsory comprehensive tendering and the internal market. this concern was addressed by the infection control nurses association and the association of domestic managers, resulting in the adoption and publication by the department of health of quality standards for hospital cleanliness 12, 13 and more recently the nhs healthcare cleaning manual. 14 in addition, existing regulations, 15-17 specialist advice, 18, 19 and clinical governance guidance, 20 all provide a framework within which hospital environmental hygiene can be improved and monitored. the nhs code of practice on the prevention and control of healthcare associated infection came into effect in october 2006. 21 the purpose of this code of practice is to help nhs bodies plan and implement strategies for the prevention and control of hcai. it sets out criteria by which managers of nhs organisations and other healthcare providers should ensure that patients are cared for in a clean environment, where the risk of hcai is kept as low as possible. failure to comply with the code may result in an improvement notice being issued or other measures. there is new evidence highlighting that the hospital environment can become contaminated with microorganisms responsible for hcai. [22] [23] [24] [25] [26] [27] transmission of microorganisms from the environment to patients may occur through direct contact with contaminated equipment, or indirectly as a result of touching by hands. meticillin epic2: guidelines for preventing healthcare-associated infections in nhs hospitals s13 30, 31 and sink taps, 22, 26, 30 and sites where dust is allowed to accumulate. 24, 32 however, whilst the presence of the same strain of microorganism in the environment as those infecting/colonising patients demonstrates that the environment becomes contaminated with microorganisms from patients, it does not provide confirmation that the environment is responsible for contamination of patients. evidence suggesting that contamination of the environment is responsible for the transmission of hcai is therefore not conclusive. nevertheless, the evidence that pathogens responsible for hcai can be widely found in the hospital environment and hence readily acquired on hands by touching surfaces, does demonstrate the importance of decontaminating hands before every patient contact. many microorganisms recovered from the hospital environment do not cause hcai. cleaning will not completely eliminate microorganisms from environmental surfaces and reductions in their numbers will be transient. 24 there is some evidence that improved cleaning regimens are associated with the control of outbreaks of hcai. in one study, the control of an outbreak of an epidemic strain of mrsa was linked with increased cleaning hours and an emphasis on the removal of dust. 32 however, often a range of interventions are introduced in order to control an outbreak and it is difficult to clearly distinguish the effect of a single component such as cleaning. some evidence suggests that routine cleaning methods may not be sufficient to eliminate surface contamination with mrsa. 26, 32 disinfectants have been recommended for cleaning of the hospital environment but a systematic review failed to confirm a link between disinfection and the prevention of hcai, though contamination of detergent and inadequate disinfection strength could have been an important confounder. 33 the use of hypochlorite for cleaning has been associated with a reduction in incidence of clostridium difficile infection in one study but this was in the absence of a detectable change in environmental contamination when either detergent or hypochlorite was used. 25 in laboratory tests a combination of cleaning with detergent followed by hypochlorite was required to consistently eliminate norovirus from surfaces and prevent cross contamination. 23 dusting and cleaning using detergent was reported to have no effect on the number of mrsa isolated from the hospital environment, but the organism was virtually eliminated by exposure to hydrogen peroxide vapour. 26 indicators of cleanliness based on levels of microbial or adenosine triphosphate (atp) contamination have been proposed but are based on arbitrary standards of acceptable contamination and do not distinguish between normal environmental flora and pathogens responsible for hcai. 22, 34 the relationship between these proposed standards and the risk of acquiring infection through contact with the environment have not been established. since cleaning will only have a transient effect on the numbers of microorganisms, regular cleaning of hospital surfaces will not guarantee complete elimination. hand decontamination before every patient contact is therefore required to ensure that pathogens acquired by touch are not transferred to patients. the hospital environment must be visibly class c clean, free from dust and soilage and acceptable to patients, their visitors and staff. shared equipment must be decontaminated after use there is some evidence demonstrating that shared clinical equipment becomes contaminated with pathogens. one study found that more than 50% of commodes tested were contaminated with clostridium difficile. 25 a systematic review identified a number of studies demonstrating that pathogens can be recovered from a range of noninvasive clinical equipment, including stethoscopes, lifting equipment, and ultrasound probes. none of these studies demonstrated a link between the contamination and infection in a patient. 22 shared clinical equipment used to deliver care in the clinical environment comes into contact with intact skin and is therefore unlikely to introduce infection. however it can act as a vehicle by which microorganisms are transferred between patients, which may result in infection. 35 this equipment should therefore be appropriately decontaminated after each use with detergent and water. in some outbreak situations hypochlorite and detergent should be considered. sp4 shared equipment used in the clinical class d environment must be decontaminated appropriately after each use. hospital hygiene is everybody's business three studies in a systematic review of healthcare workers' knowledge about mrsa and/or frequency of cleaning practices indicated that staff were not utilising appropriate cleaning practices with sufficient frequency to ensure minimisation of mrsa contamination of personal equipment. 22 staff education was lacking on optimal cleaning practices in the clinical areas. knowledge deficits may hinder the application of cleaning practices and monitoring and evaluation was indicated. this is further reinforced by an observational study which noted that lapses in adhering to the cleaning protocol were linked with an increase in environmental contamination with isolates of acinetobacter baumannii. 24 a second systematic review of four cohort studies comparing the use of detergents and disinfectants on microbial contaminated hospital environmental surfaces suggested that a lack of effectiveness was, in many instances due inadequate strengths of disinfectants, probably resulting from a lack of knowledge. 33 the following section provides the evidence for recommendations concerning hand hygiene practice. the difficulty in designing and conducting robust, ethical, randomised controlled trials in the field of hand hygiene means that recommendations in these areas are based on evidence from non-randomised controlled trials (nrct), quasiexperimental studies and expert opinion derived from systematically retrieved and appraised professional, national and international guidelines. the areas discussed include: • assessment of the need to decontaminate hands; • the efficacy of hand decontamination agents and preparations; • the rationale for choice of hand decontamination practice; • technique for hand decontamination; • care required to protect hands from the adverse effects of hand decontamination practice; • promoting adherence to hand hygiene guidelines. why is hand decontamination crucial to the prevention of healthcare-associated infection? cross-transmission, the transfer of microorganisms between humans, which occurs directly via hands, or indirectly via an environmental source, such as a commode or wash-bowl, occurs all the time in hospitals. it is the antecedent factor to crossinfection that can result in severe clinical outcomes. overviews of epidemiological evidence conclude that hand-mediated cross-transmission is a major contributing factor in the current infection threats to hospital in-patients. 1 crosstransmission via hands has been identified as contributing to hospitals outbreaks involving both meticillin-sensitive and meticillin-resistant staphylococcus aureus (mrsa/mssa), multiresistant gram-negative microorganisms, such as acinetobacter spp and vancomycin resistant enterococci (vre). 1 hand-mediated cross-transmission from resident flora (microorganisms that are present on the hands most of the time) and transient flora (microorganisms that are acquired during healthcare activity and without hand hygiene can be deposited directly on to vulnerable patients) presents a direct clinical threat to patients. when these microorganisms are cross transmitted onto susceptible sites, such as surgical wounds, endo-tracheal tubes during pulmonary ventilation, intravascular cannulation sites, enteral feeding systems or urinary catheter drainage systems, etc., serious lifethreatening infections can arise. even the crosstransmission to non-vulnerable sites can still leave a patient colonised with more pathogenic and resistant hospital microorganisms which may, if opportunity arises, result in a healthcare associated infection at sometime in the future. epic2: guidelines for preventing healthcare-associated infections in nhs hospitals s15 current evidence-based guidelines conclude that in both outbreak and non-outbreak situations contaminated hands are responsible for crosstransmission of microorganisms and that effective and effective hand decontamination can significantly reduce both cross-transmission and crossinfection rates for the majority of hcai in all healthcare settings. 1 a recent case control study, conducted during an outbreak of klebsiella pneumoniae in a neonatal intensive care unit, demonstrated an association between being cared for by a nurse with positive hand cultures for the outbreak strain and infants developing infection or colonisation. 37 descriptive studies of the dynamics of bacterial hand contamination demonstrate an association between patient care activities that involve direct patient contact and hand contamination. 38, 39 in an observational study of hand contamination during routine patient care in a large teaching hospital, high levels of hand contamination were associated with direct patient contact, respiratory care and handling body fluids. 38 a further descriptive study of healthcare workers' hand contamination during routine neonatal care demonstrated that hands become increasingly contaminated and that gloves do not fully protect healthcare workers' hands from becoming contaminated. 39 the association between hand decontamination and reductions in infection have been confirmed by two additional clinically-based trials 40, 41 and two descriptive studies. 42, 43 a nrct introducing the use of alcohol-based hand gel to a long term elderly care facility, demonstrated a reduction of 30% in hcai over a period of 34 months when compared with the control unit. 40 a further nrct, demonstrated a 45% reduction in respiratory illness in the post-intervention period following the introduction of a hand washing programme. 41 one descriptive study conducted over a four year period during which alcohol-based handrub was introduced for routine hand hygiene demonstrated a reduction in hcai from 16.9% to 9.9%. 42 a second study that compared rates of hcai caused by mrsa, vancomycin-resistant enterococci (vre) and clostridium difficile (c. difficile) in the three years prior to the introduction of alcohol-based handrub showed reductions of 21% in mrsa and 41% decrease in vre. rates of c. difficile remained unchanged throughout the intervention period. 43 current national and international guidance consistently identify that effective hand decontamination results in significant reductions in the carriage of potential pathogens on the hands and logically decreases the incidence of preventable hcai leading to a reduction in patient morbidity and mortality. 1, 44 when must you decontaminate your hands in relation to patient care? decontamination refers to a process for the physical removal of blood, body fluids, and the removal or destruction of microorganisms from the hands, 44 current national and international guidance suggests that in deciding when it is necessary to decontaminate hands prior to patient contact, four key factors need to be considered: 1, 44 • the level of the anticipated contact with patients or objects; • the extent of the contamination that may occur with that contact; • the patient care activities being performed; • the susceptibility of the patient. patients are put at risk of developing a hcai when informal carers or healthcare workers caring for them have contaminated hands. hands must be decontaminated before every episode of care that involves direct contact with patients' skin, their food, invasive devices or dressings. current expert opinion recommends that hands need to be decontaminated after completing an episode of patient care and following the removal of gloves to minimise cross contamination of the environment. 1, 44 hands must be decontaminated class c immediately before each and every episode of direct patient contact/care and after any activity or contact that potentially results in hands becoming contaminated. is any one hand cleaning preparation better than another? current national and international guidelines 1,44 consider the effectiveness of various preparations for the decontamination of hands using liquid soap and water, antiseptic handwash agents, and alcohol-based handrubs. overall there is no compelling evidence to favour the general use of antiseptic handwashing agents over soap, or one antiseptic agent over another. 1, 44 systematic reviews conducted to underpin guidelines for community and primary care and update the 2001 epic guidance 2,3 identified nineteen studies comparing hand hygiene preparations including alcohol-based handrubs and gels, antiseptic hand washes and liquid soap. five randomised controlled trials (rct) were conducted in clinical settings and compared the use of alcohol-based preparations with other agents. [45] [46] [47] [48] [49] four rcts demonstrated alcohol-based preparations to be a more effective hand hygiene agent than non-medicated soap and antiseptic handwashing agents, [45] [46] [47] [48] while a fifth study found no statistical difference between the use of alcoholbased preparations and antiseptic soap. 49 a clinical crossover trial conducted over 11 months within a neonatal intensive care unit demonstrated no statistical difference between infection rates during the hand washing and handrub phases of the trial. 50 three clinically based, quasi-experimental studies [51] [52] [53] and nine controlled laboratory experiments 54-62 also demonstrated an association between reductions in microbiological flora and the use of alcohol-based preparations. these studies underpin a growing trend to adopt the use of alcohol-based handrubs and gels in clinical practice. however, two of the above laboratory studies highlight the need for continued evaluation of the use of alcohol-based handrubs within the clinical environment to ensure staff adherence to guidelines and effective hand decontamination. 61, 62 the first study, using european union (eu) reference standards raises the possibility that alcohol-based gels may not be as effective as handrubs for short durations of use. 61 the second laboratory study, comparing 14 different hand hygiene agents used for a 'clinically realistic' 10 second hand hygiene episode, suggests that some alcohol-based handrubs may lose efficacy after 10 consecutive uses. 62 one clinically-based quasiexperimental study compared the use of 4% chlorhexidine gluconate and 1% triclosan antiseptic handwash preparations in reducing mrsa hand carriage in a specialist surgical ward. 63 both preparations effectively reduced total hand bacterial counts but 1% triclosan was more effective at eliminating mrsa. choice of decontamination: is it always necessary to wash hands to achieve decontamination? choosing the method of decontaminating hands will depend upon the assessment of what is appropriate for the episode of care, the available resources, what is practically possible and, to some degree, personal preferences based on the acceptability of preparations or materials. in general, effective handwashing with a liquid soap will remove transient microorganisms and render the hands socially clean. this level of decontamination is sufficient for general social contact and most clinical care activities. 1, 3, 44 the use of a liquid soap preparation that contains an antiseptic will reduce both transient micro-organisms and resident flora. 1, 44 the effective use of alcohol-based handrubs will also successfully remove transient microorganisms and substantially reduce resident microorganisms. however, alcohol is not effective against some microorganisms such as c. difficile, will not remove dirt and organic material and may not be effective in some outbreak situations. 43, 63 when deciding which hand decontamination preparation to use, the practitioner must consider the need to remove transient and/or resident hand flora. preparations containing certain antiseptics that exert a residual effect on skin flora can be useful in situations where prolonged reduction in microbial flora on the skin is required e.g. surgery and some invasive procedures. they are not normally necessary for everyday clinical practice but may be used in outbreak situations. national and international guidelines suggest that the acceptability of agents and techniques is an essential criterion for the selection of preparations for hand hygiene. 1,44 acceptability of preparations is dependent upon the ease with which the preparation can be used in terms of time and access together with their dermatological effects. due to their efficacy and ease of use, alcohol-based handrubs are recommended for routine use and offer a practical and acceptable alternative to handwashing when hands are not grossly soiled. 44 there are no robust economic evaluations of the comparative costs associated with different hand hygiene agents and rates of hcai. in an unpublished study of the potential cost savings associated with a national hand hygiene campaign the cost of a single hcai is estimated at over £3,000. the authors hypothesise that even a small reduction in infections through the use of alcoholbased handrubs, would result in a cost saving. 64 hands 37, 65 the first study proposes that there is an association between effective hand decontamination and the wearing of rings by healthcare staff for clinical care. 65 it suggests that the removal of rings should result in decreased frequency of hand carriage of pathogens before and after the performance of hand hygiene. in a case control study, conducted during an outbreak of klebsiella pneumoniae in a neonatal intensive care unit, investigators suggest an association between being cared for by a nurse who wore false nails and had positive hand cultures for the outbreak strain, and infants developing infection or colonisation. 37 systematic reviews conducted to underpin guidelines for community and primary care and update the 2001 epic guidance 2,3 identified one rct comparing different durations of handwashing and handrubbing on bacterial reduction that found no significant differences between the two study groups. 45 in addition a laboratory study conducted following a period of clinical observation of hand hygiene technique identified that practitioners on average applied a product for 11.6 seconds and concluded that some alcohol-based handrubs become less effective following 10 consecutive hand hygiene episodes. the authors suggest that periodic decontamination of hands, using liquid soap and water, is advisable throughout a shift. 62 two small-scale laboratory studies investigating methods of hand drying were identified. one found no statistically significant differences between the four methods studied 66 and the other suggests that warm air drying, when the hands are not rubbed simultaneously, may be more effective at reducing the numbers of bacteria on the hands following hand washing than the use of paper towels. 67 due to the methodological limitations of the above studies, recommendations continue to be based on existing expert opinion that the duration of hand decontamination, the exposure of all aspects of the hands and wrists to the preparation being used, the use of vigorous rubbing to create friction, thorough rinsing in the case of handwashing, and ensuring that hands are completely dry are key factors in effective hand hygiene and the maintenance of skin integrity. 1 does hand decontamination damage skin? expert opinion concludes that skin damage is generally associated with the detergent base of the preparation and/or poor handwashing technique. 1 however, the frequent use of hand hygiene agents may cause damage to the skin and alter normal hand flora. excoriated hands are associated with increased colonisation of potentially pathogenic microorganisms and increase the risk of infection. 1, 44 in addition, the irritant and drying effects of hand preparations have been identified as one of the reasons why healthcare practitioners fail to adhere to hand hygiene guidelines. 1, 44 systematic reviews conducted to underpin guidelines for community and primary care and update the 2001 epic guidance 2,3 identified ten studies of which four were rct conducted in clinical settings. 46, 47, 50, 68 they compared the use of alcohol-based preparations with liquid soap and water using self-assessment of skin condition by nurses. in these studies a greater level of irritation was associated with the use of soap. three further studies, one clinically-based quasi-experimental study, one descriptive clinical study and one nonclinical experimental study concluded that s18 r.j. pratt et al. alcohol-based handrubs caused less skin irritation. 53, 69, 70 in addition, one longitudinal study of the introduction and subsequent use of alcoholbased handrub over a seven year period observed no reports of irritant and contact dermatitis associated with the use of alcohol-based handrubs. 42 a laboratory study demonstrated a strong relationship between the frequency of handwashing with a chlorhexidine preparation and dermatitis. 71 current national and international guidance suggests that skin care, through the appropriate use of hand lotion or moisturizers added to hand hygiene preparations, is an important factor in maintaining skin integrity, encouraging adherence to hand decontamination practices and assuring the health and safety of healthcare practitioners. 1 how can adherence to hand hygiene guidance be promoted? national and international guidelines emphasise the importance of adherence with hand hygiene guidance and provide an overview of the barriers and factors that impact on hand hygiene compliance. 1, 44 in a systematic review of 21 studies of interventions to improve hand hygiene compliance reviewers concluded that: • single interventions have a short-term influence on hand hygiene; • reminders have a modest but sustained effect; • feedback increases rates of hand hygiene but must be regular; • near patient alcohol-based preparations improve the frequency with which healthcare workers clean their hands; • multi-faceted approaches have a more marked effect on hand hygiene and rates of hcai. 72 recent observational studies of multimodal interventions involving the introduction of alcohol-based handrubs support findings that the use of near patient alcohol-based handrub is consistently associated with greater compliance by healthcare staff. 42, [73] [74] [75] [76] [77] however, observational studies identify that staff fail to assess risk appropriately and therefore make inappropriate choices in relation to hand hygiene and glove use. [78] [79] [80] [81] [82] one study suggests that the use of motivational strategies, for example feedback may be beneficial. 81 there is some evidence from small-scale observational studies that providing patient information and actively involving patients in hand hygiene improvement programmes has a positive effect on hand hygiene compliance. 73 this section discusses the evidence and associated recommendations for the use of personal protective equipment (ppe) by healthcare workers in general care settings and includes the use of aprons, gowns, gloves, eye protection and face masks. where appropriate, in addition to the classification of the evidence underpinning the recommendations, there is an indication of a health and safety (h&s) requirement. infection control dress code -protect your patients and yourself! expert opinion suggests that the primary uses of ppe are to protect staff and reduce opportunities for transmission of microorganisms in hospitals. 1, 18, 85 a trend to eliminate the inappropriate wearing of aprons, gowns and masks in general care settings has evolved over the past twenty years due to the absence of evidence that they are effective in preventing hcai. 1, 85 the decision to use or wear personal protective equipment must be based upon an assessment of epic2: guidelines for preventing healthcare-associated infections in nhs hospitals s19 the level of risk associated with a specific patient care activity or intervention and take account of current health and safety legislation. 18 since the mid-1980s the use of gloves as an element of ppe has become an every-day part of clinical practice for healthcare workers. 1 expert opinion agrees that there are two main indications for the use of gloves in preventing hcai: 1,85 1. to protect hands from contamination with organic matter and microorganisms; and 2. to reduce the risks of transmission of microorganisms to both patients and staff. to glove or not to glove? gloves should not be worn unnecessarily as their prolonged and indiscriminate use may cause adverse reactions and skin sensitivity. 1, 85 as with all items of ppe the need for gloves and the selection of appropriate materials must be subject to careful assessment of the task to be carried out and its related risks to patients and health care workers. 1, 85 risk assessment should include consideration of: • who is at risk (whether it is the patient or the healthcare worker) and whether sterile or nonsterile gloves are required; • the potential for exposure to blood, body fluids, secretions and excretions; • contact with non-intact skin or mucous membranes during general care and invasive procedures. gloves must be discarded after each care activity for which they were worn in order to prevent the transmission of microorganisms to other sites in that individual or to other patients. washing gloves rather than changing them is not safe. 1 gloves leak! our previous systematic review provided evidence that gloves used for clinical practice may leak when apparently undamaged. 1, 85 in terms of leakage, gloves made from natural rubber latex (nrl) performed better than vinyl gloves in laboratory test conditions. revised standards (bsi 2000) relating to the manufacture of medical gloves for single use have been devised and implemented. [92] [93] [94] these standards require gloves regardless of material to perform to the same standard. expert opinion supports the view that the integrity of gloves cannot be taken for granted and additionally, hands may become contaminated during the removal of gloves. 1, 85 an additional study provided evidence that vancomycin resistant enterococcus remained on the hands of healthcare workers after the removal of gloves. 95 therefore, the use of gloves as a method of barrier protection reduces the risk of contamination but does not eliminate it and hands are not necessarily clean because gloves have been worn. expert opinion is quite clear about when gloves must be used by healthcare workers in general clinical practice. 1, 85 having decided that gloves should be used for a healthcare activity, the healthcare worker must make a choice between the use of: • sterile or non-sterile gloves, based on contact with susceptible sites or clinical devices; • surgical or examination gloves, based on the aspect of care or treatment to be undertaken. nhs trusts need to provide gloves that conform to european standard, and which are acceptable to health care practitioners. 1, 85 gloves are available in a variety of materials, the most common being natural rubber latex (nrl) and synthetic materials. nrl remains the material of choice due to its efficacy in protecting against bloodborne viruses and properties that enable the wearer to maintain dexterity. 1, 85 the problem of patient or health care practitioner sensitivity to nrl proteins must be considered when deciding on glove materials. synthetic materials are generally more expensive than nrl and due to certain properties may not be suitable for all purposes. 1 nitrile gloves have the same chemical range as nrl and may also lead to sensitivity problems. vinyl gloves made to european standards provide the same level of protection as nrl. 1 polythene gloves are not suitable for clinical use due to their permeability and tendency to damage easily. 1 a study comparing the performance of nitrile, latex, copolymer and vinyl gloves under stressed and unstressed conditions found that nitrile gloves had the lowest failure rate, adding further evidence that nitrile gloves are a suitable alternative to latex, providing there are no sensitivity issues. importantly, the study noted variation in performance of the same type of glove produced by different manufacturers and propose a test and rating system to assist healthcare workers. we identified four small scale observational studies that investigated the potential for uniforms to become contaminated during clinical care. however none of these studies established an association between contaminated uniforms and hcai. [97] [98] [99] a further study demonstrated high levels of contamination of gowns, gloves and stethoscopes with vancomycin-resistant enterococci (vre) following examination of patients known to be infected. 100 a systematic review of eight studies reporting outcomes of 3,811 babies to assess the effects of wearing and gowning by attendants and visitors in newborn nurseries found no evidence to suggest that over gowns are effective in reducing mortality, clinical infection or bacterial colonisation in infants admitted to newborn nurseries. 101 one quasi-experimental study investigated the use of gowns and gloves as opposed to gloves only in preventing the acquisition of vre in a medical intensive care unit setting. 102 a further prospective observational study investigated the use of a similar intervention in a medical intensive care unit. 103 these studies suggest that the use of gloves and gowns may minimise the transmission of vre when colonisation pressure is high. national and international guidelines recommend that protective clothing should be worn by all healthcare workers when close contact with the patient, materials or equipment may lead to contamination of uniforms or other clothing with microorganisms or, when there is a risk of contamination with blood, body fluids, secretions, or excretions (with the exception of perspiration). 1, 85, 104 disposable plastic aprons are recommended for general clinical use. 1, 85, 104 however, unused aprons need to be stored in an appropriate area away from potential contamination. 97 full body gowns need only be used where there is the possibility of extensive splashing of blood, body fluids, secretions or excretions and should be fluid repellent. 1, 85, 104 waste. non-disposable protective clothing should be sent for laundering. when is a facemask, respiratory protection and eye protection necessary? healthcare workers (and sometimes patients) may use standard surgical facemasks to prevent respiratory droplets from the mouth and nose being expelled into the environment. facemasks are also used, often in conjunction with eye protection, to protect the mucous membranes of the wearer from exposure to blood and/or body fluids when splashing may occur. our previous systematic review failed to reveal any robust experimental studies that demonstrated that healthcare workers wearing surgical facemasks protected patients from hcai during routine ward procedures, such as wound dressing or invasive medical procedures. 1 facemasks are also used to protect the wearer from inhaling minute airborne respiratory particles. as surgical facemasks are not effective in filtering out such small respiratory particles, specialised respiratory protective equipment is recommended for the care of patients with certain respiratory diseases, e.g. active multiple drugresistant pulmonary tuberculosis, 105 severe acute respiratory syndrome (sars), pandemic influenza. the filtration efficiency of these masks (sometimes called 'respirators') will protect the wearer from inhaling small respiratory particles but to be effective, they must fit closely to the face to minimise leakage around the mask. 1, 106, 107 although the advice to use particulate filter masks is based on expert opinion, there is evidence from one study that staff exposed to patients with sars acquired the infection when they did not use particulate filter masks. 108 another study demonstrated a lack of knowledge about guidance on using particulate respirator masks among staff caring for patients with sars and suggests that focused training on the use of personal protective equipment and the transmission risk of sars is required. 109 our previous systematic review indicated that different protective eyewear offered protection against physical splashing of infected substances into the eyes (although not on all occasions) but that compliance was poor. 1 expert opinion recommends that face and eye protection reduce the risk of occupational exposure of healthcare workers to splashes of blood, body fluids, secretion or excretions. 1 this section discusses the evidence and associated recommendations for the safe use and disposal of sharps in general care settings and include minimising the risks associated with sharps use and disposal, and the use of needle protection devices. where appropriate, in addition to the classification of evidence underpinning the recommendations, there is an indication of a health and safety (h&s) legislation requirement. the safe handling and disposal of needles and other sharp instruments forms part of an overall strategy of clinical waste disposal to protect staff, patients and visitors from exposure to bloodborne pathogens. 110 113 the report draws attention to the need for nhs trusts to provide local protocols and information on the risk of bloodborne viruses in the work place and to ensure that healthcare workers are adequately trained on how to prevent injuries. the average risk of transmission of bloodborne viruses following a single percutaneous exposure from an infected person, in the absence of appropriate post exposure prophylaxis has been estimated to be: 113, 114 • hepatitis b virus (hbv) 33 .3% (1 in 3) national and international guidelines, are consistent in their recommendations for the safe use and disposal of sharp instruments and needles. 18, [115] [116] [117] as with many infection prevention and control policies, the assessment and management of the risks associated with the use of sharps is paramount and safe systems of work and engineering controls must be in place to minimise any identified risks, e.g., positioning the sharps bin as close as possible to the site of the intended clinical procedure. 88 any healthcare worker experiencing an occupational exposure to blood or body fluids needs to be assessed for the potential risk of infection by a specialist practitioner, e.g., physician, occupational health nurse and offered testing, immunisation and postexposure prophylaxis if appropriate. 118 avoiding sharps injuries is everybody's responsibility all healthcare workers must be aware of their responsibility in avoiding needlestick injuries. this should be a part of induction programmes for new staff and on-going in-service education. a national blended e-learning programme on preventing hcai is available for all healthcare workers. 36 the incidence of sharps injuries has led to the development of needlestick-prevention devices in many different product groups. 121 they are designed to minimise the risk of operator injury during needle use as well as so-called "downstream" injuries that occur after disposal, often involving the housekeeping or portering staff responsible for the collection of sharps disposal units. our previous systematic reviews 1,2 failed to identify any convincing evidence that needlestickprevention devices were responsible for any significant impact on injury rates. this was primarily due to the lack of well-designed, controlled intervention studies. more recent studies have shown significant reductions in injuries associated with the use of safety devices in cannulation, 122,123 phlebotomy 124-126 and injections. 127 it would seem to be logical that where needlefree or other protective devices are used, there should be a resulting reduction in sharps injuries. a review of needlestick injuries in scotland suggested that 56% of injuries would 'probably' or 'definitely' have been prevented if a safety device had been used. 128 however, some studies identify a range of barriers to the expected reduction in injuries, including staff resistance to using new devices, complexity of device operation or improper use, and poor training. 1 a comprehensive report and product review conducted in the united states of america (usa) provides background information and guidance on the need for and use of needlestick-prevention devices but also gives advice on establishing and evaluating a epic2: guidelines for preventing healthcare-associated infections in nhs hospitals s23 sharps injury prevention program. 121 the report identifies that all devices have limitations in relation to cost, applicability and/or effectiveness. some of the devices available are more expensive than standard devices, may not be compatible with existing equipment, and may be associated with an increase in bloodstream infection rates. 129 in the usa, the occupational safety health administration (osha) and the national institute for occupational safety and health (niosh) suggest that a thorough evaluation of any device is essential before purchasing decisions are made. 117, 130 similarly in the united kingdom, the national health service purchasing and supply agency identifies that meaningful evaluations are paramount in assessing user acceptability and clinical applicability of any needle safety devices. 131 the evaluation should ensure that the safety feature works effectively and reliably, that the device is acceptable to health care practitioners and that it does not adversely affect patient care. hazard analysis critical control points (haccp) has been used for many years in the food industry to identify and control hazards in food production. it is a systems approach involving a seven stage process starting with the development of a flowchart describing the process, identifying areas (critical control points) where a hazard may occur and then establishing monitoring and control procedures. clinical governance introduced audit and quality improvement into the nhs. winning ways recommended the use of haccp in preventing hcais and the introduction of haccp is particularly suitable for hospital environmental hygiene. 132 within the catering industry there are several good examples of cleaning and disinfection haccp flowcharts, which could be adapted for acute care settings. however all processes need to be defined locally in order to address the particular hazards within the organisation and the people responsible for monitoring them. 133 in adapting these guidelines into local protocols, one should also consider the use of haccp. total number of articles located = 7830 abstract indicates that the article: relates to infections associated with hospital hygiene, is written in english, is primary research or a systematic review or a meta-analysis, and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = 34 full text confirms that the article relates to infections associated with hospital hygiene is written in english, is primary research or a systematic review or a meta-analysis, and informs one or more of the review questions. total number of articles selected for appraisal during sift 2 = 5 all articles which described primary research, a systematic review or, a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers. consensus and grading was achieved through discussion. total number of articles accepted after critical appraisal = 5 total number of articles rejected after critical appraisal = 0 evidence tables for accepted and rejected studies were generated and used to create evidence summary reports. the summary reports were, in turn, used as the basis for guideline writing. total number of articles located = 32,088 abstract indicates that the article: relates to infections associated with hand hygiene, is written in english, is primary research or a systematic review or a meta-analysis, and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = 194 full text confirms that the article relates to infections associated with hand hygiene is written in english, is primary research or a systematic review or a meta-analysis, and informs one or more of the review questions. total number of articles selected for appraisal during sift 2 = 31 all articles which described primary research, a systematic review or, a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers. consensus and grading was achieved through discussion. evidence tables for accepted and rejected studies were generated and used to create evidence summary reports. the summary reports were, in turn, used as the basis for guideline writing. sharps -systematic review process abstract indicates that the article: relates to infections associated with protective clothing, is written in english, is primary research or a systematic review or a meta-analysis, and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = 112 full text confirms that the article relates to infections associated with protective clothing is written in english, is primary research or a systematic review or a meta-analysis, and informs one or more of the review questions. total number of articles selected for appraisal during sift 2 = 14 all articles which described primary research, a systematic review or, a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers. consensus and grading was achieved through discussion. total number of articles accepted after critical appraisal = 10 total number of articles rejected after critical appraisal = 4 evidence tables for accepted and rejected studies were generated and used to create evidence summary reports. the summary reports were, in turn, used as the basis for guideline writing. 1. what is the evidence that recommended modes of use and disposal of sharps reduce the incidence of sharps injury in health care workers? 2. what is the evidence that education and training interventions improve health care workers adherence to recommended modes of practice? 3. what is the evidence that the use of needle-free devices reduce occupational exposure to bloodborne pathogens? 4. is there any cost effectiveness evidence relating to the above? 5. what are the training and education implications for staff and patients? abstract indicates that the article: relates to infections associated with sharps, is written in english, is primary research or a systematic review or a meta-analysis, and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = 49 full text confirms that the article relates to infections associated with sharps is written in english, is primary research or a systematic review or a meta-analysis, and informs one or more of the review questions. all articles which described primary research, a systematic review or, a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers. consensus and grading was achieved through discussion. total number of articles accepted after critical appraisal = 12 total number of articles rejected after critical appraisal = 7 evidence tables for accepted and rejected studies were generated and used to create evidence summary reports. the summary reports were, in turn, used as the basis for guideline writing. this guidance is based on the best critically appraised evidence currently available. the type and class of supporting evidence explicitly linked to each recommendation is described. all recommendations are endorsed equally and none is regarded as optional. these recommendations are not detailed procedural protocols and need to be incorporated into local guidelines. these guidelines apply to adults and children aged one year and older and should be read in conjunction with the guidance on standard principles. the recommendations are divided into five distinct interventions: 1. assessing the need for catheterisation; 2. selection of catheter type and system; 3. catheter insertion; 4. catheter maintenance; and 5. education of patients, relatives and healthcare workers. background and context of the guidelines catheter associated urinary tract infection (cauti) is the most common nosocomial infection in hospitals. most bacteria causing infection associated with catheterisation gain access to the urinary tract either extraluminally or intraluminally. extraluminal contamination may occur as the catheter is inserted, by contamination of the catheter from the health care worker's hands or from the patient's own colonic or perineal flora. extraluminal contamination is also thought to occur by microorganisms ascending from the perineum. intraluminal contamination occurs by reflux of bacteria from a contaminated urine drainage bag. bacteria quickly develop into colonies known as biofilms which adhere to the catheter surface and drainage bag. a biofilm forms when bacteria attach to a surface and subsequently encase themselves in an exopolymeric material. such bacteria are morphologically and physiologically different from free-living planktonic bacteria. bacteria in biofilms have considerable survival advantages over free-living microorganisms, being extremely resistant to antibiotic therapy. these biofilms cause further problems if the bacteria produce the enzyme urease, such as proteus mirabilis. the urine then becomes alkaline, causing the crystallisation of calcium and magnesium phosphate within the urine, which then is incorporated into the biofilm resulting in encrustation of the catheter over a period of time. encrustation is generally associated with longterm catheterisation, since it has a direct relationship with the length of catheterisation. we have previously described the systematic review process in section 1.10. for detailed descriptions of previous systematic reviews which have contributed to the evidence base underpinning these guidelines readers should consult the original guidelines, 1 the guidelines for the prevention of healthcare associated infections in primary and community care 2 and our interim report. 3 search questions were developed from advice received from our specialist advisors and the results of the searches are found in section 3.10. the process outlined in section 3.10 refers only to the most recent systematic review of the literature undertaken in 2005. catheterising patients places them in significant danger of acquiring a urinary tract infection. the longer a catheter is in place, the greater the danger there is consistent evidence that a significant number of healthcare-associated infections in hospital are related to urinary catheterisation. 115, [134] [135] [136] the risk of infection is associated with the method and duration of catheterisation, the quality of catheter care and host susceptibility. urinary catheterisation is a frequent intervention during clinical care in hospital affecting a significant number of patients at any one time. the highest incidence of infection is associated with indwelling urethral catheterisation. 137 the per day risk of the development of bacteriuria appears comparable throughout catheterisation (3-6 percent) but the cumulative risk increases with duration of catheterisation. [137] [138] [139] consequently, around 50 percent of hospitalised patients catheterised longer than 7-10 days contract bacteriuria. 137 although frequently asymptomatic, 20-30 percent of patients with catheter-associated bacteriuria will develop symptoms of cauti. 137 many of these infections are serious and lead to significant morbidity and mortality. of patients with a cauti, 1-4 percent develops bacteraemia and, of these, 13-30 die. 137, 140 duration of catheterisation is strongly associated with risk of infection, i.e., the longer the catheter is in place, the higher the incidence of urinary tract infection. 137, 140 advice from best practice emphasises the importance of documenting all procedures involving the catheter or drainage system in the patient's records and providing patients with adequate information in relation to the need for catheterisation and details of the insertion, maintenance and removal of their catheter. 115, 141 there is some evidence to suggest that computer management systems improve documentation and in so doing reduce the length of time catheters are in situ. 142 only is one catheter better than another? current evidence-based guidelines 1 identified three experimental studies that compared the use of latex with silicone catheters. [143] [144] [145] no significant difference in the incidence of bacteriuria was found. four studies compared the use of silver coated (silver alloy or silver oxide) catheters with silicone, hydrogel or teflon latex. [146] [147] [148] [149] a systematic review and meta-analysis of these and other studies found that silver alloy (but not silver oxide) catheters were associated with a lower incidence of bacteriuria. 140, 150 new evidence related to the efficacy of using urinary catheters coated or impregnated with antiseptic or antimicrobial agents has emerged since our original review in 2000. two subsequent reviews, 2,3 together with the current update review undertaken by us, have identified four systematic reviews and one meta-analysis that have examined this issue. [150] [151] [152] [153] [154] in general, all of these five studies suggest antiseptic impregnated or antimicrobial-coated urinary catheters can significantly prevent or delay the onset of cauti when compared to standard untreated urinary catheters. the consensus in these five reviews of evidence however, is that the individual studies reviewed are generally of poor quality; for instance in one case only 8 studies out of 117 met the inclusion criteria and in another, of the six reports describing 7 trials included, only one scored 5 in the quality assessment the other five scored only 1. 150, 154 studies investigating a wide range of coated or impregnated catheters are explored in the new evidence including: catheters coated or impregnated with: silver alloy 150,151,154-161 ; silver oxide 150 ; gendine 162 ; gentamicin 163 and silver-hydrogel [164] [165] [166] ; minocycline 167 ; rifampicin 167 ; chlorhexidine-silver sulfadiazine 166 ; chlorhexidine-sulfadiazine-triclosan 166 ; nitrofurazone 166 ; and nitrofuroxone. 168 new evidence suggests that catheters coated with silver alloy are clinically effective in reducing the incidence of cauti, but many studies are of poor methodological quality. consequently there remains inconclusive evidence to recommend their use in preference to other types of catheter at this time. despite their unit cost, there is a suggestion that these devices might be a cost-effective option if overall numbers of infections are significantly reduced through their use. the few studies that have explored the cost benefit/ effectiveness of using these devices have, however, also been inconclusive. 157, 159, 161, 165 evidence from best practice indicates that the incidence of cauti in patients catheterised for a short time (up to one week) is not influenced by any particular type of catheter material. 136, 169 however, many practitioners have strong preferences for one type of catheter over another. this preference is often based on clinical experience, patient assessment, and which materials induce the least allergic response. smaller gauge catheters with a 10 ml balloon minimise urethral trauma, mucosal irritation and residual urine in the bladder, all factors that predispose to cauti. 135, 170 however, in adults that have recently undergone urological surgery, larger gauge catheters may be indicated to allow for the passage of blood clots. choice of catheter material will depend class d on clinical experience, patient assessment and anticipated duration of catheterisation. select the smallest gauge catheter that class d will allow free urinary outflow. a catheter with a 10 ml balloon should be used in adults. urological patients may require larger gauge sizes and balloons. epic2: guidelines for preventing healthcare-associated infections in nhs hospitals s29 catheterisation is a skilled aseptic procedure despite evidence from one systematic review 153 which suggests that the use of aseptic technique has not demonstrated a reduction in the rate of cauti, principles of good practice, clinical guidance 115, 134 and expert opinion [135] [136] [137] [171] [172] [173] [174] , together with findings from another systematic review 140 agree that urinary catheters must be inserted using sterile equipment and an aseptic technique. expert opinion indicates that there is no advantage in using antiseptic preparations for cleansing the urethral meatus prior to catheter insertion. 153, 173 urethral trauma and discomfort will be minimised by using an appropriate sterile, single-use lubricant or anaesthetic gel. ensuring healthcare practitioners are trained and competent in the insertion of urinary catheters will minimise trauma, discomfort and the potential for cauti. 115, 135, 173, 174 uc6 catheterisation is an aseptic procedure. ensure that health care workers are trained and competent to carry out urethral catheterisation. clean the urethral meatus with sterile normal class d saline prior to the insertion of the catheter. use an appropriate lubricant from a sterile class d single use container to minimise urethral trauma and infection. leave the closed system alone! maintaining a sterile, continuously closed urinary drainage system is central to the prevention of cauti. 115, 134, 135, 173, 175, 176 the risk of infection reduces from 97 percent with an open system to 8-15 percent when a sterile closed system is employed. 136, 174, 177 breaches in the closed system such as unnecessary emptying of the urinary drainage bag or taking a urine sample, will increase the risk of catheter-related infection and should be avoided. 115, 136, 178 hands must be decontaminated and clean, non-sterile gloves worn before manipulation. a systematic review suggests that sealed (e.g., taped, pre-sealed) drainage systems contribute to preventing bacteriuria. 153 there is limited evidence as to how often catheter bags should be changed. one study showed higher rates of symptomatic and asymptomatic cauti were associated with a three day urinary drainage bag change regimen when compared to no routine change regimen. 179 best practice suggests changing only when necessary, i.e., according to either the manufacturers' recommendations or the patient's clinical need. 115, 134 reflux of urine is associated with infection and consequently, drainage bags should be positioned in a way that prevents back-flow of urine. 115, 135 it is also recommended that urinary drainage bags should be hung on an appropriate stand that prevents contact with the floor. 136 a number of studies have investigated the addition of disinfectants and antimicrobials to drainage bags as a way of preventing cauti. 140 three acceptable studies from our original systematic review demonstrated no reduction in the incidence of bacteriuria following the addition of hydrogen peroxide or chlorhexidine to urinary drainage bags. 1, [180] [181] [182] a systematic review supports these findings in that it suggests that adding bacterial solutions to drainage bags has no effect on catheter associated infection. 153 connect indwelling urethral catheters to class a a sterile closed urinary drainage system. meatal cleansing with antiseptic solutions is unnecessary our original systematic review considered six acceptable studies that compared meatal cleansing with a variety of antiseptic/antimicrobial agents or soap and water. 1 no reduction was demonstrated in bacteriuria when using any of these preparations for meatal care compared with routine bathing or showering. [183] [184] [185] [186] [187] [188] our subsequent reviews 2,3 revealed two studies 153,189 that support these findings in that the outcomes indicate that the use of antiseptics provides no benefit in respect of meatal/peri-urethral hygiene. expert opinion [134] [135] [136] and another systematic review 140 support the view that vigorous meatal cleansing is not necessary and may increase the risk of infection and that daily routine bathing or showering is all that is needed to maintain meatal hygiene. class a that is needed to maintain meatal hygiene. none of our systematic review evidence demonstrates any beneficial effect of bladder irrigation, instillation or washout with a variety of antiseptic or antimicrobial agents in preventing cauti. 1, 140, [190] [191] [192] [193] [194] [195] [196] [197] [198] [199] three studies, however, suggest that an acid washout solution (suby g) is effective in reducing catheter encrustation. 196, 198, 200 evidence from best practice supports the findings in respect of bladder irrigation, instillation and washout and indicates that the introduction of such agents may have local toxic effects and contribute to the development of resistant microorganisms. however, continuous or intermittent bladder irrigation may be indicated during urological surgery or to manage catheter obstruction. 115, [134] [135] [136] 140 uc18 bladder irrigation, instillation or washouts class a should not be used to prevent catheterassociated infection. given the frequency of urinary catheterisation in hospital patients and the associated risk of urinary tract infection, it is important that patients, their relatives and healthcare workers responsible for catheter insertion and management are educated about infection prevention. all those involved must be aware of the signs and symptoms of urinary tract infection and how to access expert help when difficulties arise. healthcare professionals must be confident and proficient in procedures associated with preventing cauti. in developing the recommendations we identified several areas that were inadequately addressed in the literature. we recommend further research in the following areas. epidemiological studies of the prevalence and incidence of bacteriuria/urinary tract infection during short-term catheterisation in different populations and different care settings. these should at least encompass the predominant populations, i.e. older people and those undergoing surgery. there needs to be clear definition of the 'cases' and the populations from which they are drawn. randomised controlled trials of the efficacy of antiseptic/antimicrobial coated/impregnated urethral catheters for short-term use. these need to be high quality studies, using the hospital's actual catheter-associated uti prevalence rather than national data, and appropriate follow-up. randomised controlled trials of strategies to establish how often catheters and catheter bags need to be changed. this guidance is based on the best critically appraised evidence currently available. the type and class of supporting evidence explicitly linked to each recommendation is described. all recommendations are endorsed equally and none is regarded as optional. these recommendations are not detailed procedural protocols and need to be incorporated into local guidelines. background and context to the guidelines bloodstream infections associated with the insertion and maintenance of central venous access devices (cvad) are among the most dangerous complications of healthcare that can occur, worsening the severity of the patient's underlying ill health, prolonging the period of hospitalisation and increasing the cost of care. [201] [202] [203] [204] approximately 3 in every 1000 patients admitted to hospital in the uk acquires a bloodstream infection, and nearly one third of these infections are related to central venous access devices. 205 catheter related blood stream infection (cr-bsi) involves the presence of systemic infection and evidence implicating the cvad as its source, i.e., the isolation of the same microorganism from blood cultures as that shown to be significantly colonising the cvad of a patient with clinical features of bacteraemia. catheter colonization refers to a significant growth of microorganisms on either the endoluminal or the external catheter surface beneath the skin in the absence of systemic infection. [206] [207] [208] the microorganisms that colonise catheter hubs and the skin adjacent to the insertion site are the source of most cr-bsi. coagulase-negative staphylococci, particularly staphylococcus epidermidis, are the most frequently implicated microorganisms associated with cr-bsi. other microorganisms commonly involved include staphylococcus aureus, candida species and enterococci. 208 cr-bsi is generally caused either by skin microorganisms at the insertion site that contaminate the catheter during insertion and migrate along the cutaneous catheter track, or microorganisms from the hands of healthcare workers that contaminate and colonise the catheter hub during care interventions. 206 infusate contamination or haematogenous seeding from site of infection elsewhere in the body is more rarely implicated as a cause of cr-bsi. abstract indicates that the article: relates to infections associated with short-term indwelling urinary catheters, is written in english, is primary research or a systematic review or a meta-analysis, and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = 46 full text confirms that the article relates to infections associated with short-term indwelling urinary catheters, is written in english, is primary research or a systematic review or a meta-analysis, and informs one or more of the review questions. total number of articles selected for appraisal during sift 2 = 12 all articles which described primary research, a systematic review or, a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers. consensus and grading was achieved through discussion. evidence tables for accepted and rejected studies were generated and used to create evidence summary reports. the summary reports were, in turn, used as the basis for guideline writing. what is the evidence for these guidelines? evidence upon which practice can be based is derived from a range of sources and through varying processes. these guidelines are primarily based upon an expert review of evidence-based guidelines for preventing intravascular devicerelated infections developed at the centers for disease control and prevention (cdc) in the united states of america by the healthcare infection control practices advisory committee (hicpac) 208 which were updated in 2002. 209 using a validated guideline appraisal instrument developed by the agree collaboration, 6 three experienced appraisers independently reviewed the updated guidelines, taking into consideration supplementary information provided by hicpac at our request. we concluded that the development processes were valid and that the guidelines were evidence-based, categorised to the strength of the evidence examined, reflective of current concepts of best practice, and acknowledged as the most authoritative reference guidelines currently available. they were subsequently used by us as the principal source of evidence for updating the first version of the epic guidelines. 1 in preparing the epic2 guidance, we conducted a final updating systematic review which is described in section 4.14. this search was confined to elements of infection prevention where expert members of the guideline advisory group indicated new developments or changes in technology had occurred, or where pertinent new experimental trials or systematic reviews had been published. following our reviews, guidelines were drafted which described 47 recommendations within the 9 intervention categories listed below: 1. education of healthcare workers and patients; 2. general asepsis; 3. selection of catheter type; 4. selection of catheter insertion site; 5. maximal sterile barrier precautions during catheter insertion; 6. cutaneous antisepsis; 7. catheter and catheter site care; 8. catheter replacement strategies; and 9. general principles for catheter management. these guidelines apply to caring for all adults and children over the age of 1 year in nhs acute care settings with a cvad which is being used for the administration of fluids, medications, blood components and/or total parenteral nutrition (tpn). they should be used in conjunction with the recommendations on standard principles for preventing hcai previously described in these guidelines. although these recommendations describe general principles of best practice that apply to all patients in hospital in which a cvad is being used, they do not specifically address the more technical aspects of the care of infants under the age of 1 year or those children or adults receiving haemodialysis, who will generally have their cvad managed in dialysis centres. because these recommendations describe broad general statements of best practice, they need to be adapted and incorporated into local practice guidelines. to improve patient outcomes and reduce healthcare costs, it is essential that everyone involved in caring for patients with cvad is educated about infection prevention. healthcare workers in hospitals need to be confident and proficient in infection prevention practices and to be aware of the signs and symptoms of clinical infection. wellorganised educational programmes that enable healthcare worker to provide, monitor, and evaluate care and to continually increase their competence are critical to the success of any strategy designed to reduce the risk of infection. evidence reviewed by hicpac consistently demonstrated that the risk of infection declines s34 r.j. pratt et al. following the standardisation of aseptic care and increases when the maintenance of intravascular catheters is undertaken by inexperienced healthcare workers. 209 additional evidence demonstrates that relatively simple education programmes focused on training healthcare workers to adhere to local evidence-based cvad protocols may decrease the risk to patients of cr-bsi. 210 good standards of hand hygiene and antiseptic technique can reduce the risk of infection because the potential consequences of catheterrelated infections (cr-infections) are so serious, enhanced efforts are needed to reduce the risk of infection to the absolute minimum. for this reason, hand antisepsis and proper aseptic nontouch technique (antt) are required for changing catheter dressings and for accessing the system. 44, 209 hand antisepsis can be achieved by washing hands with an antimicrobial liquid soap and water or by using an alcohol-based handrub. 44 when hands are visibly dirty or contaminated with organic material, such as blood and other body fluids or excretions, they must first be washed with liquid soap and water if alcohol-based handrubs are going to be used to achieve hand antisepsis. appropriate antt does not necessarily require sterile gloves; a new pair of disposable non-sterile gloves can be used in conjunction with a nontouch technique, for example, in changing catheter site dressings. 209 the standard principles for preventing hcai previously described in these guidelines gives additional advice on hand decontamination, the use of gloves and other protective equipment. selecting the right catheter for the right patient can minimise the risk of infection different types of cvad are available, i.e.: • made of different materials; • have one or more lumens; • coated or impregnated with antimicrobial or antiseptic agents or heparin-bonded; • cuffed and designed to be tunnelled; • having totally implantable ports. the selection of the most appropriate cvad for each individual patient can reduce the risk of subsequent cr-related infection (cr-infection). although catheter material may be an important determinant of cr-infection, evidence available to hicpac when developing their guidelines was inconclusive and they were unable to draw any specific conclusions about the contribution of catheter material to cr-infections. 209, 215 teflon ® and polyurethane catheters have been associated with fewer infections than catheters made of polyvinyl chloride or polyethylene. there is no additional evidence that demonstrates conclusively that cr-infection rates vary with different materials. 206 in england, short-term cvad are almost always made of polyurethane and longterm tunnelled catheters are usually made of silicone. number of catheter lumens clinicians often prefer multi-lumen cvad because they permit the concurrent administration of various fluids and medications, hyperalimentation, and haemodynamic monitoring among critically ill patients. hicpac examined several randomised controlled trials and other studies which suggested that multi-lumen catheters were associated with a higher risk of infection than were single lumen catheters. 208, [216] [217] [218] [219] [220] however, other studies examined by hicpac failed to demonstrate a difference in the rates of cr-bsi. 221, 222 hicpac noted that multi-lumen catheter insertion sites may be particularly prone to infection because of increased trauma at the insertion site or because multiple ports increase the frequency of cvad manipulation. 218,219 hicpac also noted that although patients with multi-lumen catheters tend to be more ill than those without such catheters, the infection risk observed with these catheters may have been independent of the patient's underlying disease severity. 220 two additional studies were identified from our systematic reviews. a systematic review and quantitative meta-analysis focused on determining the risk of cr-bsi and catheter colonisation in multilumen catheters compared with single-lumen catheters. 223 reviewers reported that although cr-bsi was more common in patients with multilumen when compared with single-lumen catheters, when confined to high quality studies that control for patient differences, there is no significant difference in rates of cr-bsi. this analysis suggests that multilumen catheters are not a significant risk factor for increased cr-bsi or local catheter colonisation compared with single-lumen cvad. another systematic review and quantitative meta-analysis tested whether single versus multilumen cvad had an impact on catheter colonisation and cr-bsi. 224 study authors concluded that there is some evidence from 5 randomised controlled trials (rcts) with data on 530 cvad, that for every 20 single-lumen catheters inserted, one cr-bsi will be avoided which would have occurred had multi-lumen catheters been used. as authors were only able to analyze a limited number of trials, further large rcts of adequate power and rigour are needed to confirm these findings. in the meantime, it may be reasonable for patients who need a cvad to choose a singlelumen catheter whenever there is no indication for a multi-lumen catheter. tunnelled and totally implantable ports surgically implanted (tunnelled) cvad, e.g., hickman ® catheters, are commonly used to provide vascular access (and stable anchorage) to patients requiring long-term intravenous therapy. alternatively, totally implantable intravascular devices, e.g., port-a-cath, ® are also tunnelled under the skin but have a subcutaneous port or reservoir with a self-sealing septum that is accessible by needle puncture through intact skin. in developing their 1996 guidelines, hicpac examined multiple studies that compared the incidence of infection associated with long-term tunnelled cvad and/or totally implantable intravascular devices with that from percutaneously (non-tunnelled) inserted cvad. 208 although in general most studies reported a lower rate of infection in patients with tunnelled cvad, [225] [226] [227] [228] [229] [230] [231] [232] [233] some studies (including one randomised controlled trial) found no significant difference in the rate of infection between tunnelled and non-tunnelled catheters. 234, 235 however, most studies examined by hicpac concluded that totally implantable devices had the lowest reported rates of cr-bsi compared to either tunnelled or non-tunnelled cvad. [236] [237] [238] [239] [240] [241] [242] [243] [244] [245] [246] additional evidence was obtained from studies of efficacy of tunnelling to reduce cr-infections in patients with short-term cvad. one randomised controlled trial demonstrated that subcutaneous tunnelling of short-term cvad inserted into the internal jugular vein reduced the risk for cr-bsi. 247 in a later randomised controlled trial, the same investigators failed to show a statistically significant difference in the risk for cr-bsi for subcutaneously tunnelled femoral vein catheters. 248 an additional meta-analysis of randomised controlled trials focused on the efficacy of tunnelling short-term cvad to prevent crinfections. 249 data synthesis demonstrated that tunnelling decreased catheter colonisation by 39% and decreased cr-bsi by 44% in comparison with non-tunnelled placement. the majority of the benefit in the decreased rate of catheter-sepsis came from one trial of cvad inserted at the internal jugular site. the reduction in risk was not significant when pooled with data from five subclavian catheter trials. tunnelling was not associated with increased risk of mechanical complications from placement or technical difficulties during placement; these outcomes were not rigorously evaluated. this meta-analysis concluded that tunnelling decreased crinfections. however, a synthesis of the evidence in this meta-analysis does not support routine subcutaneous tunnelling of short-term subclavian venous catheters and this cannot be recommended unless efficacy is evaluated at different placement sites and relative to other interventions. neither we nor hicpac identified any additional evidence in updating our systematic reviews. some catheters and cuffs are marketed as antiinfective and are coated or impregnated with antimicrobial or antiseptic agents, e.g., chlorhexidine/ silver sulfadiazine, minocycline/rifampin, platinum/ silver, and ionic silver in subcutaneous collagen cuffs attached to cvad. evidence reviewed by hicpac indicated that the use of antimicrobial or antiseptic-impregnated cvad in adults whose catheter is expected to remain in place for more than 5 days can decrease the risk for cr-bsi. [250] [251] [252] [253] [254] [255] [256] [257] [258] [259] [260] this may be cost-effective in high risk patients (intensive care, burn and neutropenic patients) and in other patient populations in which the rate of cr-bsi exceeds 3.3 per 1,000 catheter days despite implementing a comprehensive strategy to reduce rates of cr-bsi. 250 a more recent meta-analysis analysed 23 rcts published between 1988-1999 and which included data on 4,660 catheters (2,319 anti-infective and 2,341 control). 261 eleven of the trials in this metaanalysis were conducted in intensive care unit settings; 4 among oncologic patients, 2 among surgical patients; 2 among patients receiving tpn; 4 among other patient populations. study authors concluded that antibiotic and chlorhexidine-silver sulfadiazine coatings are anti-infective for short (approximately 1 week) insertion time. for longer insertion times, there are no data on antibiotic coating, and there is evidence of lack of effect for first generation chlorhexidine-silver sulfadiazine coating. for silver-impregnated collagen cuffs, there is evidence of lack of effect for both shortand long-term insertion. second generation chlorhexidine/silver sulfadiazine catheters with chlorhexidine coating both the internal and external luminal surfaces are now available. the external surface of these catheters has three times the amount of chlorhexidine and extended release of the surface bound antiseptics than that in the first generation catheters (which are coated with chlorhexidine/silver sulfadiazine only on the external luminal surface). early studies indicated that the prolonged anti-infective activity associated with the second generation catheters improved efficacy in preventing infections. 262 the most recent appraisal of all of the evidence for the clinical and cost-effectiveness of cvad treated with antimicrobial agents in preventing cr-bsi is a systematic review and economic evaluation recently conducted by the liverpool reviews and implementation group (lrig). 263 study authors conclude that rates of cr-bsi are statistically significantly reduced when an antimicrobial cvad was used. studies report the best effect when catheters were treated with minocycline/rifampin, or internally and externally treated with silver or chlorhexidine/silver sulfadiazine. a trend to statistical significance was seen in catheters only extraluminally coated. investigation of other antibiotic treated catheters is limited to single studies with non-significant results. hicpac guidelines recommend the use of an antimicrobial or antiseptic-impregnated cvad in adults whose catheter is expected to remain in place for more than 5 days if, after implementing a comprehensive strategy to reduce rates of cr-bsi, the cr-bsi rate remains above the goal set by the individual institution based on benchmark rates and local factors. selecting the best insertion site for the patient can minimise the risk of infection several factors need to be assessed when determining the site of cvad placement, including: • patient-specific factors (e.g., pre-existing cvad, anatomic deformity, bleeding diathesis, some types of positive pressure ventilation); epic2: guidelines for preventing healthcare-associated infections in nhs hospitals s37 • relative risk of mechanical complications (e.g., bleeding, pneumothorax, thrombosis); • the risk of infection. hicpac concluded that the site at which a cvad is placed can influence the subsequent risk of crinfection because of variation in both the density of local skin flora and risk of thrombophlebitis. cvad are generally inserted in the subclavian, jugular or femoral veins, or peripherally inserted into the superior vena cava by way of the major veins of the upper arm, i.e., the cephalic and basilar veins of the antecubital space. multiple studies examined by hicpac concluded that cvad inserted into subclavian veins had a lower risk for cr-infection than those inserted in either jugular or femoral veins, but none of these was a randomised controlled trial. hicpac stated that internal jugular insertion sites may pose a greater risk for infection because of their proximity to oropharyngeal secretions and because cvad at this site are difficult to immobilise. they noted, however, that mechanical complications associated with catheterisation might be less common with internal jugular than with subclavian vein insertion. hicpac noted that no rct satisfactorily has compared cr-infection rates for catheters placed in jugular, subclavian, and femoral sites. however, both previous and new evidence examined by hicpac demonstrated that catheters inserted into an internal jugular vein have been associated with higher risks for cr-infection than those inserted into a subclavian or femoral vein. 252, 264, 265 femoral catheters have been demonstrated to have relatively high colonization rates when used in adults and should be avoided because they are presumed to be associated with a higher risk of deep vein thrombosis (dvt) and cr-infection than are internal jugular or subclavian catheters. [266] [267] [268] [269] [270] [271] thus, in adult patients, a subclavian site is preferred for infection control purposes, although other factors, e.g., the potential for mechanical complications, risk for subclavian vein stenosis, and catheter-operator skill, should be considered when deciding where to place the catheter. hicpac cited a meta-analysis of 8 studies and guidelines from the national institute for health and clinical excellence (nice) indicate that the use of bedside ultrasound for the placement of cvad substantially reduced mechanical complications compared with the standard landmark placement technique. 272, 273 consequently, the use of ultrasound may indirectly reduce the risk of infection by facilitating mechanically uncomplicated subclavian placement. peripherally inserted cvad (picc) may be used as an alternative to subclavian or jugular vein catheterisation. these are inserted into the superior vena cava by way of the major veins of the upper arm. hicpac stated that they are less expensive, associated with fewer mechanical complications, e.g., thrombosis, haemothorax, infiltration and phlebitis, and easier to maintain than short peripheral venous catheters, i.e., a reduced need for frequent site rotation. additionally, previous evidence examined by hicpac suggested that picc are associated with a lower rate of infection than that associated with other non-tunnelled cvad, perhaps because the skin at the antecubital fossa is less moist and oily and colonised by fewer microorganisms than the chest and neck. 234, 274, 275 hicpac also noted that an antecubital placement removes the catheter away from endotracheal and nasal secretions. finally, they noted that further studies were needed to adequately determine how long picc could be safely left in place and to determine whether routine replacement influenced the risk of associated infection. we examined a prospective cohort study using data from two randomized trials and a systematic review published in 2005. 276 in the review the authors reported a rate of picc-bsi of 2.1 per 1,000 picc-days. this was comparable to the rates reported in their prospective cohort study (2.1 to 3.5 per 1,000 catheter-days) and similar to that reported with prospectively studied, short-term non-cuffed cvad placed percutaneously in the internal jugular, subclavian or femoral veins in inpatients (approximately 2.3 per 1,000 days). investigators concluded that picc used in high-risk hospitalised patients are associated with a rate of cr-bsi similar to conventional cvad placed in the internal jugular or subclavian veins (2 to 5 per 1,000 catheter-days). this rate is much higher than with picc used exclusively in the outpatient setting (approximately 0.4 per 1,000 catheterdays). they question whether the growing trend in many hospital haematology and oncology services to switch from the use of cuffed and tunnelled cvad to picc is justified, particularly since picc are more vulnerable to thrombosis and dislodgment, and are less useful for drawing blood specimens. moreover, picc are not advisable in patients with renal failure and impending need for dialysis, in whom preservation of upper-extremity veins is needed for fistula or graft implantation. furthermore: '…the assumption that picc are safer than conventional cvad with regard to the risk of infection is in question and should be assessed by a larger, adequately powered randomized trial that assesses peripheral vein thrombo-phlebitis, picc-related thrombosis, and premature dislodgment, as well as cr-bsi.' cvad using maximal sterile barrier precautions during cvad placement will significantly reduce the risk of infection the primacy of strict adherence to hand decontamination and aseptic technique as the cornerstone for preventing cr-infection is widely accepted. although this is considered adequate for preventing infections associated with the insertion of short peripheral venous catheters, it is recognised that central venous catheterisation carries a significantly greater risk of infection. however, the level of barrier precautions needed to prevent infection during cvad insertion was controversial at the time of the development of the hicpac guidelines. 208 studies examined by hicpac concluded that if maximal sterile barrier precautions (msb) were used during cvad insertion, catheter contamination and subsequent cr-infections could be significantly minimised. 264, [277] [278] [279] one of these studies was a prospective randomised trial that tested the efficacy of maximal sterile barriers to reduce infections associated with long-term nontunnelled subclavian silicone catheters. 279 when msb were compared with routine procedures, they significantly decreased the risk of cr-bsi. 279 msb involve wearing sterile gloves and gown, a cap, mask and using a large sterile drape during insertion of the catheter as opposed to routine infection prevention procedures that involve wearing only sterile gloves and the use of a small drape. however, there is no specific evidence that wearing a facemask or cap during catheter insertion is important in preventing cr-bsi. it has been generally assumed that cvad inserted in the operating theatre posed a lower risk of infection than did those inserted on inpatient wards or other patient care areas. 208 data examined by hicpac from two prospective studies suggests that the difference in risk of infection depended largely on the magnitude of barrier protection used during catheter insertion, rather than the surrounding environment, i.e., ward versus operating room. 264, 279 previous expert reviewers who have examined the above evidence agree that maximal sterile barrier precautions are essential during cvad placement to reduce the risk of infection. 115, 207, [280] [281] [282] systematic review evidence a systematic review published in 2004 aimed to determine the value of msb to prevent cvadrelated infection. 283 msb were defined as: person inserting the cvad wear a head cap, facemask, sterile body gown, and sterile gloves and uses a full-size sterile drape. their search identified 95 articles discussing the prevention of cvad-related infections. the majority of these articles were review articles or consensus statements. only three primary research studies comparing infection outcomes using msb with less stringent barrier techniques were identified and included in the review. authors identified no additional unpublished or ongoing primary studies. all three studies included in the review concluded that the use of msb resulted in a reduction in catheterrelated infections. the studies differed notably in their patient populations, research designs, and healthcare settings. study authors concluded that using msb has been found to decrease transmission of microorganisms, to delay colonization, and to reduce the rate of hospital-acquired infections. they suggest that biological plausibility and the available evidence support using msb during routine insertion of a cvad to minimise the risk of infection. they recommend that given the lack of adverse patient reactions, the relatively low cost of msb, and the high cost of cr-bsi, it is probable that msb will prove to be a cost-effective or even a cost-saving intervention. appropriate preparation of the insertion site will reduce the risk of catheter-related infection microorganisms that colonise catheter hubs and the skin surrounding the cvad insertion site are the cause of most cr-bsis. 206, 260, 284 the risk of infection increases with the density of microorganisms around the insertion site. skin cleansing/ antisepsis of the insertion site is therefore one of the most important measures for preventing crinfection. 208 an important prospective randomised trial of agents used for cutaneous antisepsis demonstrated that 2% aqueous chlorhexidine was superior to either 10% povidone-iodine or 70% alcohol for preventing central venous and arterial cr-infections. 285 an additional study has since confirmed the superior efficacy of 2% aqueous chlorhexidine compared to povidone iodine in substantially reducing central venous catheter colonisation. 286 direct comparisons of aqueous versus alcoholic solutions of chlorhexidine have not been undertaken in relation to cutaneous antisepsis for preventing cr-infections. however, an alcoholic solution of chlorhexidine combines the benefits of rapid action and excellent residual activity. 287 the application of organic solvents, such as acetone or ether, to 'defat' (remove skin lipids) the skin before catheter insertion and during routine dressing changes had been a standard component of many hyperalimentation protocols. however, there was no evidence available to hicpac to show that the use of these agents provided any protection against cr-infection and their use could greatly increase local inflammation and patient discomfort. 208 several studies were examined that focused on the application of antimicrobial ointments to the catheter site at the time of catheter insertion, or during routine dressing changes, to reduce microbial contamination of catheter insertion sites. 284 reported efficacy in preventing crinfections by this practice yielded contradictory findings. [288] [289] [290] [291] [292] [293] there was also concern that the use of polyantibiotic ointments that were not fungicidal could significantly increase the rate of colonisation of the catheter by candida species. 292, 294 systematic review evidence a meta-analysis published in 2004 assessed studies that compared the risk for cr-bsi following insertion-site skin care with either any type of chlorhexidine gluconate (chg) solution versus povidone iodine (pi) solution. 295 this analysis indicated that the use of chg rather than pi can reduce the risk for cr-bsi by approximately 49% (risk ratio, 0.51 [ci, 0.27 to 0.97]) in hospitalised patients who require short-term catheterisation, i.e., for every 1000 catheter sites disinfected with chg rather than pi, 71 episodes of catheter colonization and 11 episodes of cr-bsi would be prevented. in this analysis, several types of chg solutions were used in the individual trials, including 0.5 percent or 1 percent chg alcohol solution and 0.5 percent or 2 percent chg aqueous solution. all of these solutions provided a concentration of chg that is higher than the minimal inhibitory concentration (mic) for most nosocomial bacteria and yeasts. subset analysis of aqueous and non-aqueous solutions showed similar effect sizes, but only the subset analysis of the five studies that used alcoholic chg solution produced a statistically significant reduction in cr-bsi. because few studies used chg aqueous solution, the lack of a significant difference seen for this solution compared with pi solution may be a result of inadequate statistical power. a prospective randomised trial in germany and published in 2004 investigated the optimal disinfection regimen at the time of catheter insertion to avoid catheter colonisation, comparing skin disinfection performed with either povidoneiodine 10% (pvp-iodine), chlorhexidine 0.5% propanol 70%, or chlorhexidine 0.5% propanol 70% followed by pvp-iodine 10%. 296 investigators found that significantly fewer catheter tips were colonized following skin disinfection of the insertion site with propanol/chlorhexidine followed by pvp-iodine (p = 0.006). study authors concluded that skin disinfection with sequential application of propanol/chlorhexidine followed by pvp-iodine was superior in the prevention of microbial cvad colonisation compared to either of the regimens alone. a randomised prospective multiple unit crossover trial conducted in france and published in 2004 compared the effectiveness in preventing central venous catheter colonization and infection of two protocols for pre-insertion cutaneous antisepsis using aqueous 10% povidone-iodine (pvp-i) or a solution of 5% pvp-i in 70% ethanol. 297 investigators found that the incidence of catheter colonization was significantly lower in the alcoholic pvp-i solution protocol than in the aqueous pvp-i solution protocol (relative risk, 0.38: 95% confidence interval, 0.22-0.65, p < 0.001), and so was the incidence of cr-infection (relative risk, 0.34: 95% confidence interval, 0.13-0.91, p < 0.04). study authors concluded that the use of alcoholic pvp-i rather than aqueous pvp-i can significantly reduce the incidence of catheter-tip colonization and nosocomial catheter-related infection in intensive care units.this study was designed to demonstrate the superiority of alcoholic pvp-i over aqueous pvp-i in preventing cvad colonization. however, the weight of evidence in the majority of studies appraised in our review favours alcoholic chlorhexidine for preinsertion cutaneous antisepsis. cvad infections can be minimised by good catheter and insertion site care the safe maintenance of a cvad and relevant care of the insertion site are essential components of a comprehensive strategy for preventing crinfections. this includes good practice in caring for the patient's catheter hub and connection port, the use of an appropriate cvad site dressing regimen, and using flush solutions to maintain the patency of the cvad. choose the right dressing for insertion sites to minimise infection following cvad placement, a dressing is used to protect the insertion site. because occlusive dressings trap moisture on the skin, and provide an ideal environment for the rapid growth of local microflora, dressings for insertion sites must be permeable to water vapour. 206 the two most common types of dressings used for insertion sites are sterile, transparent, semi-permeable polyurethane dressings coated with a layer of an acrylic adhesive ('transparent dressings'), and gauze and tape dressings. transparent dressings, e.g., opsite ® iv3000, tegaderm iv ® , are permeable to water vapour and oxygen, and impermeable to microorganisms. hicpac reviewed the evidence related to which type of dressing provided the greatest protection against infection and found little difference. 209 they concluded that the choice of dressing can be a matter of preference. if blood is oozing from the catheter insertion site, a gauze dressing might be preferred. gauze dressings are not waterproof and require frequent changing in order to inspect the catheter site. they are rarely useful in patients with longterm cvad. sterile transparent, semi-permeable polyurethane dressings have become a popular means of dressing catheter insertion sites. they reliably secure the cvad, permit continuous visual inspection of the catheter site, allow patients to bathe and shower without saturating the dressing, and require less frequent change than that required for standard gauze and tape dressings, thus saving personnel time. a cochrane review of gauze and tape versus transparent polyurethane dressings for cvad concluded that there was no evidence demonstrating any difference in the incidence of cr-related infections between any of the dressing types compared in this review. 298 each of these comparisons was based on no more than 2 studies and all of these studies reported data from a small patient sample. therefore it is probable that the findings of no difference between dressing types is due to the lack of adequate data. they further concluded that because there is a high level of uncertainty regarding the risk of infection associated with the cvad dressings included in this review, at this stage it appears that the choice of dressing for cvad can be based on patient preference. cvad use an appropriate antiseptic agent for disinfecting the catheter insertion site during dressing changes hicpac described compelling evidence that aqueous chlorhexidine 2% was superior to either 10% povidone iodine or 70% alcohol in lowering cr-bsi rates when used for skin antisepsis prior to cvad insertion. 209, 285 they made no recommendation for the use of any disinfectant agent for cleaning the insertion site during dressing changes. studies focused on the use of antimicrobial ointment applied under the dressing to the catheter insertion site to prevent cvad-related infection do not clearly demonstrate efficacy. 289, 294 systematic review evidence a recent meta-analysis assessed studies that compared the risk for cr-bsi following insertionsite skin care with either any type of chlorhexidine gluconate (chg) solution versus povidone iodine (pi) solution. 295 this analysis indicated that the use of chg rather than pi can reduce the risk for cr-bsi by approximately 49% (risk ratio, 0.51 [ci, 0.27 to 0.97]) in hospitalised patients who require short-term catheterisation, i.e., for every 1000 catheter sites disinfected with chg rather than pi, 71 episodes of catheter colonization and 11 episodes of cr-bsi would be prevented. in this analysis, several types of chg solutions were used in the individual trials, including 0.5 percent or 1 percent chg alcohol solution and 0.5 percent or 2 percent chg aqueous solution. all of these solutions provided a concentration of chg that is higher than the minimal inhibitory concentration (mic) for most nosocomial bacteria and yeasts. subset analysis of aqueous and non-aqueous solutions showed similar effect sizes, but only the subset analysis of the five studies that used alcoholic chg solution produced a statistically significant reduction in cr-bsi. because few studies used chg aqueous solution, the lack of a significant difference seen for this solution compared with pi solution may be a result of inadequate statistical power. most modern cvad and other catheter materials are generally alcohol-resistant, i.e., they are not damaged by contact with alcohol. however, alcohol and other organic solvents and oil-based ointments and creams may damage some types of polyurethane and silicon cvad tubing, e.g., some catheters used in haemodialysis. the manufacturer's recommendations for only using disinfectants that are compatible with specific catheter materials must be followed. cvad when and how catheters are replaced can influence the risk of infection a catheter replacement strategy is composed of two elements; the frequency and the method of catheter replacement. frequency hicpac noted that with short peripheral venous catheters, the risk of phlebitis and catheter colonisation, both associated with cr-infection, could be reduced by catheter replacement and site rotation every 48-72 hours. 208 however, decisions regarding the frequency of cvad replacement were more complicated. they considered evidence that showed duration of catheterisation to be a risk factor for infection and which advocated routine replacement of cvad at specified intervals as a measure to reduce infection. 222, 265, 299, 300 other studies, however, suggested that the daily risk of infection remains constant and showed that routine replacement of cvad, without a clinical indication, does not reduce the rate of catheter colonisation or the rate of cr-bsi. 301, 302 conclusions from a systematic review agree that exchanging catheters by any method every three days was not beneficial in reducing infections, compared with catheter replacement on an as-needed basis. 303 two methods are used for replacing cvad; placing a new catheter over a guide wire at the existing site, or percutaneously inserting a new catheter at another site. guide wire insertion has been the accepted technique for replacing a malfunctioning catheter (or exchanging a pulmonary artery catheter for a cvad when invasive monitoring was no longer needed) as they are associated with less discomfort and a significantly lower rate of mechanical complications than those percutaneously inserted at a new site. studies of the risks for infection associated with guide wire insertions examined by hicpac yielded conflicting results. one prospective study showed a significantly higher rate of cr-bsi associated with catheters replaced over a guide wire compared with catheters inserted percutaneously. 301 however, three prospective studies (two randomised) showed no significant difference in infection rates between catheters inserted percutaneously and those inserted over a guide wire. 302, 304, 305 since these studies suggest that the insertion of the new catheter at a new site does not alter the rate of infectious complications per day but does increase the incidence of mechanical complications, guide wire exchange is recommended. most studies examined by hicpac concluded that, in cases where the catheter being removed is known to be infected, guidewire exchange is contraindicated. 302, [304] [305] [306] [307] a systematic review concluded that, compared with new site replacement, guidewire exchange was associated with a trend toward a higher rate of subsequent catheter colonisation, regardless of whether patients had a suspected infection at the time of replacement. guidewire exchange was also associated with trends toward a higher rate of catheter exit-site infection and cr-bsi. however, guidewire exchange was associated with fewer mechanical complications relative to new-site replacement. 303 methods are available and techniques have been described which allow a diagnosis of cr-bsi to be made without the need for catheter removal. 308 such approaches could be used prior to the replacement of a new catheter over a guide wire in order to reduce the subsequent risk of crinfection. 308 aseptic technique is important when accessing the system hicpac considered evidence demonstrating that contamination of the catheter hub is an important contributor to intraluminal microbial colonisation of catheters, particularly long-term catheters. [310] [311] [312] [313] [314] [315] [316] in a relatively recent overview, additional evidence from a prospective cohort study suggested that frequent catheter hub manipulation increases the risk for microbial contamination. 260, 317 during prolonged catherisation, catheter hubs are accessed more frequently, increasing the likeli-epic2: guidelines for preventing healthcare-associated infections in nhs hospitals hood of a cr-bsi emanating from a colonised catheter hub rather than the insertion site. 316 consequently, the reviewer commented that hubs and sampling ports should be disinfected before they are accessed and noted that both povidoneiodine and chlorhexidine are effective. 250, 318, 319 systematic review evidence in a recent randomized prospective clinical trial conducted in england, the microbial contamination rate of luers of cvad with either posiflow ® needleless connectors or standard caps attached was investigated. 320 the efficacy of: chlorhexidine gluconate 0.5% w/v in industrial methylated spirit (ims) bp 70% w/w spray (hydrex ds ® ); sterile isopropyl alcohol (ipa) 70% w/w spray (spiriclens ® ); and 10% (w/v) aqueous povidone-iodine (betadine ® ) was assessed for the disinfection of intravenous connections. patients were designated to receive chlorhexidine/alcohol, isopropyl alcohol or povidone-iodine for pre-cvad insertion skin preparation and disinfection of the connections. after 72 h in situ the microbial contamination rate of 580 luers, 306 with standard caps and 274 with needleless connectors attached, was determined. the microbial contamination rate of the external compression seals of 274 needleless connectors was also assessed to compare the efficacy of the three disinfectants. the internal surfaces of 55 out of 306 (18%) luers with standard caps were contaminated with microorganisms, whilst only 18 out of 274 (6.6%) luers with needleless connectors were contaminated (p < 0.0001). of those needleless connectors disinfected with isopropyl alcohol, 69.2% were externally contaminated with microorganisms compared with 30.8% disinfected with chlorhexidine/alcohol (p < 0.0001) and 41.6% with povidone-iodine (p < 0.0001). these results suggest that the use of needleless connectors may reduce the microbial contamination rate of cvad luers compared with the standard cap. furthermore, disinfection of needleless connectors with either chlorhexidine/alcohol or povidone-iodine significantly reduced external microbial contamination. both these strategies may reduce the risk of catheter-related infections acquired via the intraluminal route. although now generally alcohol-resistant, some cvad and catheter hub materials may be chemically incompatible with alcohol or iodine and the manufacturer's recommendations must be complied with. although in-line filters reduce the incidence of infusion-related phlebitis, hicpac could find no reliable evidence to support their efficacy in preventing infections associated with intravascular catheters and infusion systems. infusate-related bsi is rare and hicpac concluded that filtration of medications or infusates in the pharmacy is a more practical and less costly way to remove the majority of particulates. furthermore, in-line filters might become blocked, especially with certain solutions, e.g., dextran, lipids, mannitol, thereby increasing the number of line manipulations and decreasing the availability of administered drugs. 209 in our systematic review we found no additional good quality evidence to support their use for preventing infusate-related cr-bsi. however, there may be a role for the use of in-line filtration of parenteral nutrition solutions for reasons other than the prevention of infection but these are beyond the scope of these guidelines. cvad antibiotic lock prophylaxis, i.e., flushing and then filling the lumen of the cvad with an antibiotic solution and leaving it to dwell in the lumen of the catheter, is sometimes used in special circumstances to prevent cr-bsi, e.g., in treating a patient with a long-term cuffed or tunnelled catheter or port who has a history of multiple cr-bsi despite optimal maximal adherence to aseptic technique. evidence reviewed by hicpac demonstrated the effectiveness of this type of prophylaxis in neutropenic patients with long-term cvad. 209 however, they found no evidence that routinely using this procedure in all patients with cvad reduced the risk of cr-bsi and may lead to an increase in antimicrobial resistant microorganisms. cvad 321 patients with cancer often need to be given drugs and other treatments intravenously, so are frequently fitted with long-term tunnelled cvad. infections sometimes occur. clinical trial evidence shows it may be useful to give prophylactic antibiotics prior to inserting a tunnelled cvad or to flush the catheter with combined vancomycin and heparin, but microbial resistance may occur unless this practice is limited to highrisk patients. cvad maintaining cvad patency and preventing catheter thrombosis may help prevent infections indwelling central venous and pulmonary artery catheters are thrombogenic. thrombus forms on these catheters in the first few hours following placement and may serve as a nidus for microbial colonization of intravascular catheters. 322, 323 thrombosis of large vessels occurs after long-term catheterisation in 35 to 65% of patients. [324] [325] [326] [327] [328] prophylactic heparin and warfarin have been widely used to prevent catheter thrombus formation and catheter related complications, such as deep venous thrombosis (dvt). 209, 329 two types of heparin can be used: unfractionated (standard) heparin and low molecular weight heparins. although more expensive, low molecular weight heparins have a longer duration of action than unfractionated heparin and are generally administered by subcutaneous injection once daily. the standard prophylactic regimen of low molecular weight heparins are at least as effective and as safe as unfractionated heparin in preventing venous thrombo-embolism and does not require laboratory monitoring. 330 a meta-analysis of randomised controlled trials evaluating the benefit of infused prophylactic heparin through the catheter, given subcutaneously or bonded to the catheter in patients with cvad found that prophylactic heparin: • was associated with a strong trend for reducing catheter thrombus (rr, 0. the authors of this meta-analysis concluded that heparin administration effectively reduces thrombus formation and may reduce catheterrelated infections in patients who have central venous and pulmonary artery catheters in place. they suggest that various doses of subcutaneous and intravenous unfractionated and low molecular weight heparins and new methods of heparin bonding need further comparison to determine the most cost-effective strategy for reducing catheter-related thrombus and thrombosis. there are many different preparations and routes of administration of heparin, and as yet there is no definite evidence that heparin reduces the incidence of cr-bsi, but this may reflect the heterogeneity of heparin and its administration. warfarin has also been evaluated as a means for reducing catheter-related thrombosis. a controlled trial of 82 patients with solid tumours were randomised to receive or not to receive low-dose warfarin (1 mg a day) beginning 3 days prior to catheter insertion and continuing for 90 days. warfarin was shown to be effective in reducing catheter-related thrombosis. 331 in this study, warfarin was discontinued in 10% of patients due to prolongation of the prothrombin time. heparin versus normal saline intermittent flushes although many clinicians use low dose intermittent heparin flushes to fill the lumens of cvad locked between use in an attempt to prevent thrombus formation and to prolong the duration of catheter patency, the efficacy of this practice is unproven. despite its beneficial antithrombotic effects, decreasing unnecessary exposure to heparin is important to minimise adverse effects associated with heparin use, e.g., autoimmunemediated heparin-induced thrombocytopenia, allergic reactions and the potential for bleeding complications following multiple, unmonitored heparin flushes. 332 the risks of these adverse effects can be avoided by using 0.9 percent sodium chloride injection instead of heparin flushes. a systematic review and meta-analysis of randomised controlled trials evaluating the effect of heparin on duration of catheter patency and on prevention of complications associated with the use of peripheral venous and arterial catheters concluded that heparin at doses of 10 u/ml for intermittent flushing is no more beneficial than flushing with normal saline alone. 333 this finding was in agreement with two other metaanalyses. 334, 335 manufacturers of implanted ports or opened-ended catheter lumens may recommend heparin flushes for maintaining catheter patency and many clinicians feel that heparin flushes are appropriate for flushing cvad that are infrequently accessed. hicpac reviewed all of the evidence for intermittent heparin flushes and systemic heparin and warfarin prophylaxis and concluded that no data demonstrated that their use reduces the incidence of cr-bsi and did not recommend them for infection prevention purposes. 209, [322] [323] [324] [325] [326] [327] [328] [329] [331] [332] [333] [334] [335] although their use for preventing cr-bsi remains controversial, patients who have cvad may also have risk factors for dvt and systemic anticoagulants may be prescribed for dvt prophylaxis. in addition, heparin flush solutions may be useful in helping to maintain patency in catheter lumens that are infrequently accessed and may also be recommended by manufacturers of implantable ports and for cvad used for blood processing, e.g., haemodialysis or apheresis. we did not identify and further new evidence when updating our systematic review. needle-free devices require vigilance needle-free infusion systems have been widely introduced into clinical practice to reduce the incidence of sharp injuries and the potential for the transmission of bloodborne pathogens to healthcare worker. hicpac examined evidence that these devices may increase the risk for cr-bsi and concluded that when they are used according to the manufacturers' recommendations, they do not substantially affect the incidence of cr-bsi. 209 some of the devices available are more expensive than standard devices, may not be compatible with existing equipment, and may be associated with an increase in bloodstream infection rates. 129 class d/gpp the optimal interval for the routine replacement of intravenous (iv) solution administration sets has been examined in three well-controlled studies reviewed by hicpac. data from each of these studies reveal that replacing administration sets no more frequently than 72 hours after initiation of use is safe and cost-effective. when a fluid that enhances microbial growth is infused, e.g., lipid emulsions, blood products, more frequent changes of administration sets are indicated as these products have been identified as independent risk factors for cr-bsi. 209 cvad this is a well researched area and few realistic research needs were identified in developing these guidelines. the following investigations, along with a health economic assessment, may inform future clinical practice. the effectiveness of subcutaneous low molecular weight heparins or low dose warfarin to prevent catheter thrombus, colonisation and cr-bsi. the infection risks associated with the use of peripherally inserted central catheters (picc). the impact of nurse consultants (intravenous therapy) and/or intravenous therapy teams on hospital cr-bsi rates. the efficacy and cost-effectiveness of antimicrobial impregnated cvad to provide sustained protection against crbsi in hospital patients with long term catherisation. the efficacy and cost-effectiveness of antimicrobial impregnated catheter site dressings in preventing catheter colonisation and cr-bsi. ensure that all healthcare workers are trained all healthcare worker involved in the care of people with cvad to implement these guidelines and assessed receive training and updates in the management of cvad. as competent. standard 100% support healthcare workers to consistently adhere to guideline recommendations. data collection: review of staff education records/direct observation/self-audit assess the need for continuing venous access evidence of regular and frequent assessment of the need for on a regular basis and remove a cvad as soon cvad and catheter discontinuation rates when the catheter is as clinically possible in order to reduce the no longer essential for medical management. risk for infection. standard 100% total number of articles located = 5273 abstract indicates that the article: relates to infections associated with central venous access devices, is written in english, is primary research or a systematic review or a meta-analysis, and appears to inform one or more of the review questions. total number of articles retrieved from sift 1 = 169 full text confirms that the article relates to infections associated with central venous access devices, is written in english, is primary research or a systematic review or a meta-analysis, and informs one or more of the review questions. total number of articles selected for appraisal during sift = 25 all articles which described primary research, a systematic review or, a meta-analysis and met the sift 2 criteria were independently critically appraised by two appraisers. consensus and grading was achieved through discussion. total number of articles accepted after critical appraisal = 20 total number of articles rejected after critical appraisal = 5 evidence tables for accepted and rejected studies were generated and used to create evidence summary reports. the summary reports were, in turn, used as the basis for guideline writing. the epic project: developing national evidence-based guidelines for preventing healthcare associated infections. phase 1: guidelines for preventing hospital-acquired infections infection control: prevention of healthcare-associated infection in primary and community care updating the evidence-base for national evidence-based guidelines for preventing healthcare-associated infections in nhs hospitals in england: a report with recommendations updating the evidence-base for national evidence-based guidelines for preventing healthcare-associated infections in appraisal of guidelines for research and evaluation (agree) instrument. st georges hospital medical school a guideline developers' handbook (sign 50) scottish intercollegiate guideline network guideline development methods: information for national collaborating centres and guideline developers. national institute for health and clinical excellence a new system for grading recommendations in evidence based guidelines mopping up hospital infection cdc guideline for handwashing and hospital environmental control working party for standards for environmental cleanliness in hospitals. standards for environmental cleanliness in hospitals. infection control nurses association and the association of domestic management standards for environmental cleanliness in hospitals. london: the stationery office the nhs healthcare cleaning manual. london: department of health safe disposal of clinical waste. london: hse hospital laundry arrangements for used and infected linen leeds: nhse guidance for clinical health care workers: protection against infection with bloodborne viruses. london: department of health decontamination of equipment, linen or other surfaces contaminated with hepatitis b and/or human immunodeficiency viruses. hc(91) 33 london: department of health; 1991. 20. national health service executive. a first class service: quality in the new nhs. leeds: department of health department of health. the health act 2006: code of practice for the prevention and control of health care associated infections. london: department of health reservoirs of mrsa in the acute hospital setting: a systematic review effects of cleaning and disinfection in reducing the spread of norovirus contamination via environmental surfaces role of environmental cleaning in controlling an outbreak of acinetobacter baumannii on a neurosurgical unit comparison of the effect of detergent versus hypochlorite on environmental contamination and incidence of clostridium difficile infection tackling contamination of the hospital environment by methicillin-resistant staphylococcus aureus (mrsa): a comparison between conventional terminal cleaning and hydrogen peroxide vapour decontamination environmental contamination due to methicillin-resistant staphylococcus aureus: possible infection control implications contamination of room door handles by methicillin -sensitive/methicillin-resistant staphylococcus aureus bacterial contamination of computer keyboards in a teaching hospital an evaluation of hospital cleaning regimes and standards chlorhexidine resistance in antibiotic resistant bacteria isolated from the surfaces of dispensers of soap containing chlorhexidine evidence that hospital hygiene is important in the control of methicillin-resistant staphylococcus aureus does disinfection of environmental surfaces influence nosocomial infection rates? a systematic review use of audit tools to evaluate the efficacy of cleaning systems in hospitals microbiological advisory committee to the department of health. sterilisation, disinfection and cleaning of medical equipment. london: department of health outbreak of extended beta lactamase-producing klebsiella pneumoniae in a neonatal intensive care unit linked to artificial nails bacterial contamination of the hands of hospital staff during routine patient care dynamics of bacterial hand contamination during routine neonatal care the impact of alcohol hand sanitizer use on infection rates in an extended care facility handwashing and respiratory illness among young adults in military training effectiveness of a hospital wide programme to improve compliance with hand hygiene reduction in nosocomial transmission of drug resistant bacteria after introduction of an alcohol-based hand rub guidelines for hand hygiene in health-care settings. recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force hand contamination before and after different hand hygiene techniques: a randomised clinical trial skin tolerance and effectiveness of two hand decontamination procedures in everyday hospital use assessment of two hand hygiene regimens for intensive care unit personnel efficacy of handrubbing with alcohol based solution versus standard handwashing with antiseptic soap: randomised clinical trial handwashing with soap or alcoholic solutions? a randomized clinical trial of its effectiveness effect of antiseptic handwashing vs alcohol sanitizer on health care-associated infections in neonatal intensive care units clinical assay of n-duopropenide alcohol solution on hand application in newborn and pediatric intensive care units: control of an outbreak of multiresistant klebsiella pneumoniae in a newborn intensive care unit with this measure a new alcohol solution (n-duopropenide) for hygienic (or routine) hand disinfection is more useful than classic handwashing: in vitro and in vivo studies in burn and other intensive care units assessment of alternative hand hygiene regimens to improve skin health among neonatal intensive care unit nurses limited efficacy of alcohol-based hand gels effectiveness of a nonrinse alcohol-free antiseptic hand wash effectiveness of hand-cleansing agents for removing methicillin-resistant staphylococcus aureus from contaminated hands a close look at alcohol gel as an antimicrobial sanitizing agent effectiveness of hand-cleansing agents for removing acinetobacter baumannii strain from contaminated hands limited effectiveness of chlorhexidine based hand disinfectants against methicillin-resistant staphylococcus aureus (mrsa) testing a new alcohol-free hand santitizer to combat infection comparison of waterless hand antisepsis agents at short application times: raising the flag of concern comparative efficacy of hand hygiene agents in the reduction of bacteria and viruses comparison of the antibacterial efficacy of 4% chlorhexidine gluconate and 1% triclosan handwash products in an acute clinical ward impact of ring wearing on hand contamination and comparison of hand hygiene agents in a hospital effects of 4 hand-drying methods for removing bacteria from washed hands: a randomised trial efficiency of hand drying for removing bacteria from washed hands: comparison of paper towel drying with warm air drying skin irritation and dryness associated with two hand-hygiene regimens: soap-and-water hand washing versus hand antisepsis with an alcoholic hand gel hand dermatitis in intensive care units dermal tolerance of sterillium, a propanol-based hand rub hand antiseptics: rubs versus scrubs, alcohol solutions versus alcoholic gels the effectiveness of interventions aimed at increasing handwashing in healthcare workersa systematic review use of alcohol hand sanitizer as an infection control strategy in an acute care facility reduction in nosocomial infection with improved hand hygiene in intensive care units of a tertiary care hospital in argentina effect of education and performance feedback on handwashing: the benefit of administrative support in argentinean hospitals handwashing programme for the prevention of nosocomial infections in a neonatal intensive care unit performance feedback of hand hygiene, using alcohol hand gel as the skin decontaminant, reduces the number of inpatients newly affected by mrsa and antibiotic costs differences in hand hygiene behaviour related to the contamination risk of healthcare activities in different groups of healthcare workers factors associated with hand hygiene practices in two neonatal intensive care units use of an alcohol-based hand rub and quality improvement interventions to improve hand hygiene in a russian epic2: guidelines for preventing healthcare-associated infections in nhs hospitals s51 neonatal intensive care unit compliance with hand hygiene and glove use in a university-affiliated hospital rates of hand disinfection associated with glove use, patient isolation, and changes between exposure to various body sites evaluation of a patient education model for increasing hand hygiene compliance in an inpatient rehabilitation unit evaluation of a patient-empowering hand hygiene programme in the uk protective clothing; principles and guidance. london: infection control nurses association management of health and safety at work regulations. london: hse books personal protective equipment at work regulations: guidance on regulations. london: hse books control of substances hazardous to health regulations knowledge of standard and isolation precautions in a large teaching hospital healthcare workers' knowledge of inoculation injuries and glove use critical incidents of nonadherence with standard precautions guidelines among community hospital-based health care workers medical gloves for single use part 1: specification for freedom from holes medical gloves for single use part 2: specification for physical properties medical gloves for single use part 3: requirements and testing for biological evaluation effectiveness of gloves in the prevention of hand carriage of vancomycinresistant enterococcus species by health care workers after patient care to determine the effects of gloves stress, type of material (vinyl, nitrile, copolymer, latex) and manufacturer on the barrier effectiveness of medical examination gloves bacterial contamination of nurses' uniforms: a study bacterial contamination of uniforms bacterial contamination of scrub jackets during dental hygiene procedures contamination of gowns, gloves, and stethoscopes with vancomycin-resistant enterococci gowning by attendants and visitors in newborn nurseries for prevention of neonatal morbidity and mortality (review). the cochrane database of to gown or not to gown: the effect on acquisition of vancomycin-resistant enterococci a prospective study to determine whether cover gowns in addition to gloves decrease nosocomial transmission of vancomycin-resistant enterococci in an intensive care unit national collaborating centre for chronic conditions for the national institute for health and clinical excellence. tuberculosis: clinical diagnosis and management of tuberculosis, and measures for its prevention and control the prevention and control of tuberculosis in the united kingdom: uk guidance on the prevention and control of transmission of 1. hiv-related tuberculosis 2. drug-resistant, including multiple drug-resistant, tuberculosis. london: department of health control of substances hazardous to health regulations 1999. approved codes of practice. hse books effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) appropriate use of personal protective equipment among healthcare workers in public sector hospitals and primary healthcare polyclinics during the sars outbreak in singapore safe disposal of clinical waste. sheffield: health and safety executive; 1999. 111. national audit office. a safer place to work: improving the management of health and safety risks to staff in nhs trusts. london: the stationery office monitoring sharps injuries: epinet™ surveillance results eye of the needle: united kingdom surveillance of significant occupational exposure to bloodborne viruses in healthcare workers center for disease control and prevention. workbook for designing, implementing and evaluating a sharps injury prevention program preventing hospital-acquired infection: clinical guidelines. london: public health laboratory service universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis b virus and other blood borne pathogens in health care settings enforcement procedures for the occupational exposure to bloodborne pathogens. directive number cpl2-2.44d london: department of health department of health. an organisation with a memory. london: stationery office the management of health safety and welfare: issues for nhs staff. london: stationery office sharps safety and needlestick prevention efficacy of catheter needles with safeguard mechanisms effect of implementing safety-engineered devices on percutaneous injury epidemiology a comprehensive approach to percutaneous injury prevention during phlebotomy: results of a multicenter study impact of safety devices for preventing percutaneous injuries related to phlebotomy procedures in health care workers evaluation of a safety resheathable winged steel needle for prevention of percutaneous injuries associated with intravascular-access procedures among healthcare workers a comparative user evaluation of three needle-protective devices potential for reported needlestick injury prevention among healthcare workers through safety devices usage and improvement of guideline adherence: expert panel assessment evaluation of safety devices for preventing percutaneous injuries among health care workers during phlebotomy procedures -minneapolis-st paul department of health. winning ways: working together to reduce healthcare associated infection in england post-operative endophthalmitis: the application of hazard analysis critical control points (haccp) to an infection control problem guideline for prevention of catheter-associated urinary tract infections prevention of catheter-associated urinary tract infections urinary tract infections: detection, prevention, and management 5th ed. baltimore md: williams and wilkins hospital infection 4th ed. philadelphia: lippincott-raven meatal colonisation and catheter associated bacteriuria an evaluation of daily bacteriologic monitoring to identify preventable episodes of catheter-associated urinary tract infections preventing catheter-related bacteriuria: should we? can we? how? archive of the royal marsden manual of clinical nursing procedures 6th ed computer-based order entry decreases duration of indwelling urinary catheterisation in hospitalised patients effect of a siliconized latex urinary catheter on bacterial adherence and human neutrophil activity incidence and importance of bacteriuria in postoperative short-term urinary catheterisation comparison of urethral reaction to full silicone, hydrogen-coated and siliconised latex catheters a large randomised clinical trial of a silver-impregnated urinary catheter: lack of efficacy and staphylococcal superinfection prevention of catheter-associated urinary tract infection with a silver oxide-coated urinary catheter: clinical and microbiologic correlates refinements in the coating of urethral catheters reduces the incidence of catheter-associated bacteriuria. an experimental and clinical study silver alloy coated catheters reduce catheter-associated bacteriuria the efficacy of sliver alloy-coated urinary catheters in preventing urinary tract infection: a meta-analysis types of urethral catheters for management of short-term voiding problems in hospitalised adults. (cochrane review). the cochrane library systematic review: antimicrobial urinary catheters to prevent catheter-associated urinary tract infection in hospitalized patients management of short term indwelling urethral catheters to prevent urinary tract infections. a systematic review. australia: the joanna briggs institute for evidence based nursing and midwifery is there evidence for recommending silver-coated urinary catheters in guidelines clinical application of the bardex ic foley catheter a randomized crossover study of silver-coated urinary catheters in hospitalized patients the potential clinical and economic benefits of silver alloy urinary catheters in preventing urinary tract infection a comparison of the effect of early insertion of standard latex and silver impregnated latex foley catheters on urinary tract infections in burn patients using silver to reduce catheterassociated urinary tract infections the impact of using silver alloy urinary catheters in reducing the incidence of urinary tract infections in the critical care setting effect of silver-coated urinary catheters: efficacy, cost-effectiveness, and antimicrobial resistance a rapid method of impregnating endotracheal tubes and urinary catheters with gendine: a novel antiseptic agent gentamicin-releasing urethral catheter for short-term catheterization randomized multi-centre trial of the effects of a catheter coated with hydrogel and silver salts on the incidence of hospitalacquired urinary tract infections use of silver-hydrogel urinary catheters on the incidence of catheter-associated urinary tract infections in hospitalized patients evaluation of the antimicrobial efficacy of urinary catheters impregnated with antiseptics in an in vitro urinary tract model efficacy of antimicrobial-impregnated bladder catheters in reducing catheter-associated bacteriuria: a prospective, randomized, multicenter clinical trial assessment of nosocomial urinary tract infections in orthopaedic patients: a prospective and comparative study using two different catheters continence clinic. catheters: design, selection and management study of patients with indwelling catheters entry of bacteria into the urinary tract of patients with inlying catheters technical advances in the prevention of urinary tract infection prevention of catheter-induced urinary tract infections by sterile closed drainage factors predisposing to bacteriuria during indwelling urethral catheterisation bacteriuria during indwelling catheter drainage: ii. effect of a closed sterile draining system does the addition of disinfectant to urine drainage bags prevent infection in catheterised patients? prevention of urinary infections in gynaecology reduction of mortality associated with nosocomial urinary tract infection incidence of urinary tract infections in patients with shortterm indwelling urethral catheters: a comparison between a 3-day urinary drainage bag change and no change regimens decreased incidence of bacteriuria associated with periodic instillations of hydrogen peroxide into the urethral catheter drainage bag evaluation of h2o2 prophylaxis of bacteriuria inpatients with long-term indwelling foley catheters: a randomised controlled study catheter-associated bacteriuria. failure to reduce attack rates using periodic instillations of a disinfectant into urinary drainage systems prevention of bacteriuria in female patients with indwelling catheters prevention of catheterassociated urinary tract infections. efficacy of daily meatal care regimens evaluation of daily meatal care with poly-antibiotic ointment in the prevention of urinary catheter-associated bacteriuria prevention of catheter-associated bacteriuria: clinical trial of methods to block three known pathways of infection daily meatal care for the prevention of catheterassociated bacteriuria: results using frequent applications of poly-antibiotic cream randomized trial of meatal care with silver sulfadiazine cream for the prevention of catheter-associated bacteriuria water or antiseptic for periurethral cleaning before urinary catheterization: a randomized controlled trial does instillation of chlorhexidine into the bladder of catheterized geriatric patients help reduce bacteriuria bladder irrigation with chlorhexidine for the prevention of urinary infection after transurethral operations: a prospective controlled study controlled trial of intravesical noxythiolin in the prevention of infection following outflow tract surgery a randomised study on the effect of bladder irrigation with povidone-iodine before removal of an indwelling catheter antibiotic irrigation and catheter-associated urinary-tract infections once-daily irrigation of long-term urethral catheters with normal saline. lack of benefit assessment of the use of bladder washouts/instillations in patients with long-term indwelling catheters neomycin-polymyxin prophylaxis of urinary-tract infection associated with indwelling catheters the use of bladder wash-outs to reduce urinary catheter encrustation assessment of the use of bladder washouts/instillations in patients with long-term indwelling catheters the dissolution of urinary catheter encrustation the second national prevalence survey of infection in hospital -overview of results nosocomial bloodstream infection in critically ill patients: excess length of stay, extra costs, and attributable mortality the rate and cost of hospital-acquired infections occurring in patients admitted to selected specialities of a district general hospital in england and the national burden imposed the impact of hospital-acquired bloodstream infections device-related sources of bacteraemia in english hospitals -opportunities for the prevention of hospital-acquired bacteraemia nosocomial infections related to use of intravascular devices inserted for short-term vascular access guideline for prevention of intravasculardevice-related infections guidelines for the prevention of intravascular-catheter-related infections long-term reduction of vascular access-associated bloodstream infection the effect of an education programme on the incidence of central venous catheterassociated bloodstream infection in a medical icu the effect of a nursing staff education program on compliance with central line care policy in the cardiac intensive care unit impact of an educational program and policy changes on decreasing catheter-associated bloodstream infections in a medical intensive care unit in brazil effect of an infection control program using education and performance feedback on rates of intravascular deviceassociated bloodstream infections in intensive care units in argentina influence of surface morphology on in vitro bacterial adherence to central venous catheters sepsis from triple-vs single-lumen catheters during total parenteral nutrition in surgical or critically ill patients prospective evaluation of single and triple lumen catheters in total parenteral nutrition central catheter infections; single-versus triplelumen catheters. influence of guide wires on infection rates when used for replacement of catheters infection rate for single-lumen vs triple-lumen subclavian catheters use of triple-lumen subclavian catheters for administration of total parenteral nutrition single-versus triple-lumen central catheterrelated sepsis: a prospective randomised study in a critically ill population triplevs single-lumen central venous catheters. a prospective study in a critically ill population rates of infection for single-lumen versus multilumen central venous catheters: a meta-analysis colonization and bloodstream infection with single-versus multi-lumen central venous catheters: a quantitative systematic review a prospective study of prolonged central venous access in leukaemia broviac catheter-related bacteraemia in oncology patients hickman catheter infections in patients with malignancies problems associated with indwelling central venous catheters. archives of diseases in complications of hickman-broviac catheters management of hickman catheter sepsis catheter-related septicemia in patients receiving home parenteral nutrition single-vs double-lumen central venous catheters in pediatric oncology patients bactermia related to indwelling central venous catheters: prevention, diagnosis, and treatment low infection rate and long durability of nontunneled silastic catheters. a safe cost-effective alternative for long-term venous access lack of clinical benefit from subcutaneous tunnel insertion of central venous catheters in immunocompromised patients a totallyimplanted injection port system for blood sampling and chemotherapy administration complications and management of implanted venous access catheters a totally implanted venous access system for the delivery of chemotherapy implantable subcutaneous venous catheters. archives of diseases in fifty-five patient years' experience with a totally implanted system for intravenous chemotherapy infection rates of broviac-hickman catheters and implantable venous devices complications from long-term indwelling central venous catheters in hematologic malignancy patients with special reference to infection classical external indwelling central venous catheter versus total implanted venous access systems for chemotherapy administration. a randomised trial in 100 patients with solid tumors comparison of infections in hickman and implanted port catheters in adult solid tumor patients experience with a totally implantable venous access device (port-a-cath) in patients with aids infectious morbidity associated with long-term use of venous access devices in patients with cancer effects of subcutaneous tunneling on internal jugular catheter-related sepsis in critically ill patient: a prospective randomised multicenter study use of tunneled femoral catheters to prevent catheter-related infection: a randomised controlled trial tunneling short-term central venous catheters to prevent catheter-related infection: a meta-analysis of randomized controlled trials prevention of central venous catheter-related bloodstream infection by use of an antiseptic-impregnated catheter. a randomized controlled trial evaluation of chlorhexidine and silversulfadiazine impregnated central venous catheters for the prevention of bloodstream infection in leukaemic patients: a randomized controlled trial influence of triple-lumen central venous catheters coated with chlorhexidine and sliver sulfadiazine on the incidence of catheter-related bacteremia a meta-analysis dealing with the effectiveness of chlorhexidine and silver-sulfadiazine impregnated central venous catheters anaphylactic shock induced by an antiseptic-coated central venous catheter cost-effectiveness of antiseptic-impregnated central venous catheters for the prevention of catheter-related bloodstream infection central venous catheters coated with minocycline and rifampin for the prevention of catheter-related colonization and bloodstream infections. a randomized, double-blind trial decreasing catheter colonization through the use of an antiseptic-impregnated catheter: a continuous quality improvement project a comparison of two antimicrobialimpregnated central venous catheters efficacy of antiseptic-impregnated central venous catheters in preventing catheter-related bloodstream infection: a meta-analysis prevention of intravascular catheter-related infections prevention of bloodstream infections with central venous catheters treated with anti-infective agents depends on catheter type and insertion time: evidence from a meta-analysis prolonged antimicrobial activity of a catheter containing chlorhexidine/silver sulfadiazine extends protection against catheter infections in vivo the clinical and cost effectiveness of central venous catheters treated with anti-microbial agents in preventing bloodstream infections: a systematic review and economic evaluation the pathogenesis and epidemiology of catheter-related infection with pulmonary artery swan-ganz catheters: a prospective study utilizing molecular subtyping prospective multicenter study of vascular-catheter-related complications and risk factors for positive centralcatheter cultures in intensive care unit patients risk of infection due to central venous catheters: effect of site of placement and catheter type femoral deep vein thrombosis associated with central venous catheterization: results from a prospective, randomized trial complications of femoral and subclavian venous catheterization in critically ill patients: a randomized controlled trial deep venous thrombosis caused by femoral venous catheters in critically ill adult patients incidence of deep venous thrombosis associated with femoral venous catheterization a prospective evaluation of the use of femoral venous catheters in critically ill adults ultrasound guidance for placement of central venous catheters: a meta-analysis of the literature guidance on the use of ultrasound locating devices for placing central venous catheters peripheral access options skin microbiology: coming of age risk of catheter-related bloodstream infection with peripherally inserted central venous catheters used in hospitalized patients aseptic technique is very important: maximal barrier precautions during insertion reduce the risk of central venous catheter-related bacteremia improved sterile technique diminishes the incidence of positive line cultures in cardiovascular patients prevention of central venous catheter-related infections by using maximal sterile barrier precautions during insertion infections due to infusion therapy catheter-related bloodstream infections: evaluation of cdc guidelines catheter-related sepsis: an overview -part 2 using maximal sterile barriers to prevent central venous catheter-related infection: a systematic evidence-based review prospective, randomized trial of two antiseptic solutions for prevention of central venous or arterial catheter colonization and infection in intensive care unit patients prospective randomized trial of povidone-iodine, alcohol, and chlorhexidine for prevention of infection associated with central venous and arterial catheters prospective, randomized trial of two antiseptic solutions for prevention of central venous or arterial catheter colonization and infection in intensive care unit patients guideline for use of topical antimicrobial agents catheter-related sepsis in long-term parenteral nutrition with broviac catheters. an evaluation of different disinfectants colonization of central venous catheters a clinical and bacteriologic study of infections associated with venous cutdowns application of antibiotic ointment to the site of venous catheterization: a controlled trial risk of infection with indwelling intravenous catheters: effect of application of antibiotic ointment the effects of antibiotic ointments and antiseptics on the skin flora beneath subclavian catheter dressings during intravenous hyperalimentation a comparative study of polyantibiotic and iodophor ointment in prevention of vascular catheterrelated infection chlorhexidine compared with povidone-iodine solution for vascular catheter-site care: a meta-analysis combined skin disinfection with chlorhexidine/ propanol and aqueous povidone-iodine reduces bacterial colonisation of central venous catheters (for) members of the nacre study group. alcoholic povidone-iodine to prevent central venous catheter colonization: a randomized unit-crossover study gauze and tape and transparent polyurethane dressings for central venous catheters (review) comparison of the sterility of long-term central venous catheterisation using singlelumen, triple-lumen, and pulmonary artery catheters colonization and bacteremia related to duration of triple-lumen intravascular catheter placement a controlled trial of scheduled replacement of central venous and pulmonary artery catheters safety of central venous catheter change over a guide wire for suspected catheter-related sepsis: a prospective randomised trial central venous catheter replacement strategies: a systematic review of the literature prospective study of catheter replacement and other risk factors for infection of hyperalimentation catheters catheter infection. a comparison of two catheter maintenance techniques catheter-related sepsis in patients on intravenous nutrition: a prospective study of quantitative catheter cultures and guide wire changes for suspected sepsis mechanical complications from insertion of subclavian venous feeding catheters: comparison of de novo percutaneous venipuncture to change of catheter over guide wire accuracy and cost-effectiveness of new tests for diagnosis of catheter-related bloodstream infections diagnosis of vascular catheter-related bloodstream infection: a meta-analysis source and route of microbial colonization of parenteral nutrition catheters catheter sepsis due to coagulase-negative staphylococci in patients on total parenteral nutrition a prospective study of the catheter hub as the portal of entry for microorganisms causing catheter-related sepsis in neonates adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters pathogenesis of catheter sepsis: a prospective study with quantitative and semiquantitative cultures of catheter hub and segments a randomized trial on the effect of tubing changes on hub contamination and catheter sepsis during parenteral nutrition ultrastructural analysis of indwelling vascular catheters: a quantitative relationship between luminal colonization and duration of placement contamination of stopcocks mounted in administration sets for central venous catheters with replacement at 24 hrs versus 72 hrs: a prospective cohort study use of disinfectants to reduce microbial contamination of hubs of vascular catheters effectiveness of disinfectant techniques on intravenous tubing latex injection ports a randomized, prospective clinical trial to assess the potential infection risk associated with the posiflow ® needleless connector prophylactic antibiotics for preventing early central venous catheter gram positive infections in oncology patients heparin bonding reduces thrombogenicity of pulmonary-artery catheters the relationship between the thrombotic and infectious complications of central venous catheters thrombosis as a complication of pulmonary-artery catheterisation within the internal jugular vein central venous thrombosis associated with intravenous feeding: a prospective study a crosssectional study of catheter-related thrombosis in children receiving total parenteral nutrition at home catheter-related thrombosis in critically ill children: comparison of catheters with and without heparin bonding a prospective study of femoral catheter-related thrombosis in children benefit of heparin in central venous and pulmonary artery catheters: a meta-analysis of randomized controlled trials british national formulary no. 51. london: british medical association and the royal pharmaceutical society very low doses of warfarin can prevent thrombosis in central venous catheters. a randomized prospective trial case report: the heparin flush syndrome: a cause of iatrogenic hemorrhage benefit of heparin in peripheral venous and arterial catheters: systematic review and meta-analysis of randomised controlled trials a meta-analysis of effects of heparin flush and saline flush: quality and cost implications analysis of the research about heparinized versus nonheparinized intravascular lines key: cord-288113-ex4yi28u authors: epalza, cristina; hallin, marie; busson, laurent; debulpaep, sara; de backer, paulette; vandenberg, olivier; levy, jack title: role of viral molecular panels in diagnosing the etiology of fever in infants younger than 3 months date: 2019-11-09 journal: clin pediatr (phila) doi: 10.1177/0009922819884582 sha: doc_id: 288113 cord_uid: ex4yi28u as infants with proven viral infection present lower risk of bacterial infection, we evaluated how molecular methods detecting viruses on respiratory secretions could contribute to etiological diagnostic of these febrile episodes. from november 2010 to may 2011, we enrolled all febrile infants <90 days presenting to emergency room. standard workup included viral rapid antigenic test and viral culture on nasopharyngeal aspirate. samples negative by rapid testing were tested by molecular methods. from 208 febrile episodes (198 infants) with standard techniques, rate of documented microbiological etiology was 13% at emergency department, 47% during hospitalization, and 64% with viral cultures. molecular methods increased microbiologically documented etiology rate by 12%, to 76%. contribution of molecular methods was the highest in infants without clinical source of infection, increasing documentation by 18%, from 50% to 68%. making viral molecular results rapidly available could help identifying a higher proportion of infants at low risk of serious bacterial infection. the management of febrile infants younger than 3 months in the emergency room (er) is challenging as they have a higher risk of serious bacterial infection (sbi) than older children and because clinical evaluation has a low sensitivity and specificity in identifying those infants with sbi. [1] [2] [3] [4] [5] [6] therefore, additional examinations are usually performed to diagnose sbi, or to identify those infants at higher and lower risk of sbi. accordingly, a large proportion of these infants considered at higher risk of sbi are admitted to the hospital and treated empirically with intravenous antibiotics while awaiting microbiological confirmation, whereas those at lower risk of sbi can be managed as outpatients, provided adequate surveillance can be ascertained. [1] [2] [3] [4] [5] [6] traditional viral and bacterial diagnostic techniques allow to find the etiology of febrile episodes in about half of the cases only. 7, 8 with the development of molecular techniques, the ability to diagnose viral infections has improved substantially in recent years. multiple real-time polymerase chain reactions (pcrs) have been developed, increasing the detection rate for cultivable viruses or allowing the detection of non-or difficult-tocultivate viruses. 9 more recently, techniques such as multiplex pcrs or microarrays allow the codetection of large panels of viruses in a single assay. 10 furthermore, these new tools can now generate accurate results within a few hours. 11 in several clinical situations, rapid report of microbial pathogens identification from clinical specimens has been shown to significantly improve the management and the outcome of infected patients, enabling rapid adjustment of antibiotic treatments, shortened hospital stay, and lower hospitalization costs. 12, 13 regarding the management of febrile infants younger than 3 months, several authors have shown that infants presenting a proven viral infection have a significantly lower risk of sbi. [14] [15] [16] [17] [18] an early diagnosis of a viral infection could help consider a larger proportion of febrile infants at lower risk for sbi and lead to a more conservative approach regarding additional invasive procedures, antibiotic treatments, or hospital admission. while the molecular techniques allowing this early diagnosis are more expensive than conventional methods, their real clinical benefits remain poorly studied. the aims of this prospective study were to evaluate the analytical performances of a multiplex diagnostic tool detecting the most frequent respiratory viruses as compared with our set of homemade real-time pcrs and the potential contribution of these molecular methods to the etiologic diagnosis of febrile episodes in infants younger than 3 months of life. the study was conducted at saint-pierre hospital, a university-affiliated hospital located in downtown brussels. about 24 000 patients per year attend its pediatric er. all infants ≤90 days of age admitted to the pediatric er from november 15, 2010, to may 5, 2011, reporting or presenting a rectal temperature ≥38.0°c, were eligible for this study. according to published guidelines, 1-6 our standard procedure for these patients includes a thorough clinical interview, a complete physical examination, and the following laboratory tests: complete blood count, blood bacterial culture, viral rapid antigen testing and viral culture of nasopharyngeal aspirates (npa), urinalysis, and bacterial culture. cerebrospinal fluid is obtained for cytology, bacterial culture, and enterovirus (ev) detection by pcr in all infants <28 days or in those 28 to 90 days with toxic aspect or the following laboratory findings: white blood cell ≥15 000/mm 3 or ≤5000/mm 3 or c-reactive protein ≥20 mg/l or white cell count >35/ µl in urine sediment collected by catheterization. additional tests such as chest x-ray, stool culture, and stool viral antigen tests are performed whenever clinically indicated. for all patients, an admission is proposed; intravenous empirical antibiotic is started in nearly all infants <28 days and in those older than 28 days with clinical or laboratory alterations. samples received in the laboratory during business hours were processed on receipt. sample arriving outside business hours were stored at 4°c and processed on the next working day. standard viral diagnostic procedures consisted in viral culture on confluent vero, mrc5, and llc-mk 2 cell lines (vircell, santa-fé, spain), and a combination of 3 of the following rapid detection tests according to the season: lateral flow chromatography for influenza a and b (binaxnow influenza a/b, alere inc, waltham, ma), respiratory syncytial virus (rsv; binaxnow rsv, alere inc) and adenovirus (adv; adeno respi-strip, coris bioconcept, gembloux, belgium), and direct fluorescent immunoassays for human metapneumovirus (hmpv) and parainfluenza virus (piv; argene, biomérieux, marcy l'etoile, france). if rapid tests were negative, 2 sets of molecular assays were performed: (1) the clart pneumovir dna array (genomica, coslada, spain) detecting influenza a, b, and c; piv 1, 2, 3, and 4; rsv a and b; hmpv a and b; adv; ev (solely echoviruses); rhinoviruses; coronavirus 229e; and bocaviruses and (2) a homemade real-time pcrs detecting influenza a and b; piv 1, 2, 3, and 4; rsv a and b; hmpv a and b; adv; ev; rhinoviruses; coronavirus 229e, nl63, and oc43; and bocaviruses. extraction of nucleic acids was arried out with the magna pure lc extraction system (roche diagnostics) using the total nucleic acid large volume isolation kit (input volume 800 µl, output volume 200 µl). the pneumovir assay was performed according to the manufacturer's instructions; detection and interpretation of the results were conducted by a carreader (genomica). all these molecular techniques were performed once a week, except for the pcr targeting ev, which was performed twice a week. for npa with negative rapid tests, as the molecular tests used were presumably more sensitive than the reference standard (viral culture), we constructed, as recommended, 19 a "composite" reference standard in order to avoid bias in establishing the specificity of the evaluated tests. this "composite" reference standard was constructed as follows: (1) samples were considered as positive for a viral pathogen if they tested positive by at least 2 of the 3 assays used (viral culture, pneumovir, homemade pcrs) and (2) they were considered negative if they tested negative by at least 2 of the 3 assays. the samples not fitting one of these categories were classified as undetermined. as a consequence, the analytical performances of our pcr detecting coronavirus nl63 and oc43 could not be evaluated, as it was the only method used here that was able to detect these viruses. for each patient, the following demographic, clinical, biological, and microbiological data were recorded: age, gender, duration of gestation, immunization status, duration and maximal documented temperature at home and in the er, symptoms, laboratory results, treatment administered and duration, destination after discharge from er, complications, and length of stay if hospitalized. all patients' files were reviewed by an infectious diseases senior pediatrician, and infants were classified into 4 groups according to clinical symptoms at presentation: (1) respiratory infection, (2) suspected infection. viral or bacterial suspected infection was defined as clinical signs and nonspecific laboratory tests matching either with viral or bacterial infections but with no microbiological documentation. no etiology. no etiology was defined as the febrile episodes not matching with any of the definitions described above. if a febrile episode matched with 2 diagnostic definitions, the most severe one was considered as the responsible of the fever. data were recorded on excel files (microsoft office, windows) and analyzed using descriptive statistics. verbal informed consent was obtained from all parents or legal guardians at inclusion. the study was approved by the local ethics committee. during the study period, 198 infants were enrolled on admittance to the er for a total of 208 febrile episodes: 10 infants presented 2 episodes (with a median of 27.5 days between episodes [6-41 days]). median age was 52 days (6-85 days), 56.3% were males, and 6.7% were prematurely born babies. median measured for fever in the er was 37.8°c and 38.5°c at home, for a median duration of 12 hours (0-240 hours) before arrival. for the 42.9% of infants older than 60 days, the immunization program was not yet started (belgian immunization program is free and starts at 8 weeks of age). blood culture, urine culture, and npa were performed in 96.2%, 94.2%, and 97.6% of patients, respectively. lumbar puncture was performed in 32.2% of the infants (92.6% of infants <28 days and 56% of older infants with toxic aspect or laboratory alterations), stool viral antigenic tests in 25%, stool culture in 22.6%, and skin pus culture in 0.5% (1 patient). in 200 of the 208 episodes, an npa was available for complete viral evaluation (3 patients were not sampled, 2 samples were not analyzed with rapid test and viral culture, and 3 others were not analyzed with molecular techniques due to technical issues). seventy-five out of 200 npa (37.5%) had a positive rapid antigen detection test ( figure 1 ); 62 positive results (82.7%) were confirmed by culture. among the 13 culture-negative rapid test-positive samples, 10 were hmpv, whose culture growth is known to be difficult. the remaining 125 cases were explored by molecular methods but, as previously said, 2 were not analyzed with pneumovir assay. from the 123 samples evaluable for analytical performances, the composite reference standard was negative for 65 samples, positive for 56 samples, and undetermined for 2 samples (culture was negative and molecular tests were discordant). of note, among the 65 "negative" samples, 10 coronaviruses oc43 and 2 coronaviruses nl63 were detected by pcr. as shown in table 1 , culture was, as expected, the technique with the lowest sensitivity (71%), missing mainly hmpvs, rhinoviruses, and mixed infections; its specificity was 100%. the in-house real-time pcrs showed good specificity (91%) and positive predictive value (88%), but its sensitivity did not reach 80%. the pneumovir assay, on the other hand, achieved the best sensitivity (96%) and negative predictive value (95%) but lacked specificity (58%), mainly due to 23 results considered as false positives for rhinoviruses according to the composite standard. eighty-four percent of episodes (n = 175) led to hospitalization and 87 episodes (42%) to intravenous empirical antibiotic treatment. using our standard protocol, the rate of documented microbiological etiology was 13% at er discharge, 47% at the end of hospitalization, and 64% when viral cultures results became available. molecular methods increased the documented etiology rate by 12%, to a total of 76% of all episodes (figure 2 ). the highest rate of documented episodes was achieved for infants with respiratory symptoms at presentation (92.6%), followed by those with gastrointestinal symptoms (76.5%). the contribution of molecular methods to establish the etiology of the febrile episode was the highest for infants with fever without clinical focus, increasing the rate of microbiological documentation by 18%, to a total of 68% ( figure 2 ). among the 76% of episodes with documented etiology, sbi was diagnosed in 15 patients (7%): 11 urinary tract infections (utis), 3 bacterial enteritis, and 1 finger cellulitis ( table 2 ). viruses were detected in the npa of 5 of these 15 infants (33%): 1 rsv, 1 rhinovirus, 1 piv, 1 coronavirus nl63, and 1 cytomegalovirus. the remaining 69% were "documented viral infections." twentyone of the 143 episodes (15%) with a proven viral etiology were suspected to have a bacterial coinfection on clinical basis or because they had laboratory signs of inflammation. these 21 episodes occurred all in infants with respiratory symptoms at presentation. in 105 episodes (50.5%), infants were considered as low risk for bacterial infection after workup in the er ( figure 3) ; 82 (78%) of these infants were hospitalized, 59 of them (77%) only for clinical observation, while 23 required supportive treatment. one single episode classified as "low risk" at er discharge ended to be a "bacterial proven infection," namely, a campylobacter jejuni enteritis. among the 59 infants with febrile episodes considered at low risk of sbi and hospitalized for observation, 42 (71%) ended to have a documented viral infection. this is the first prospective study reporting the contribution of a large panel of techniques, including molecular methods for multiple respiratory viruses, to establish the etiology of febrile episodes in infants younger than 3 months. we evaluated the possible contribution on clinical decisions in parallel with the chronology of the availability of the laboratory results that led to episode documentation. our study presents the limitation of having been conducted mainly during the autumn-winter season in a single center. this can explain the important rate of infants presenting respiratory symptoms. nevertheless, the rate of sbi is similar to those reported in the literature, 2,3,5 as is the predominance of uti among our sbi, demonstrating that our cohort can be considered as representative of the etiologic case mix usually observed among febrile episodes in infants younger than 3 months. identifying infants at risk of sbi among those presenting a febrile episode remains an important challenge, and several scales based on clinical, biological, and microbiological data have been recommended. 1-6 in the past years, important advances have been made in terms of laboratory techniques that either help better identify high-risk infants or confirm sbi more rapidly, such as procalcitonin level [20] [21] [22] [23] [24] or rapid bacterial identification from blood culture by maldi-tof (matrixassisted laser desorption/ionization-time-of-flight mass spectrometry). 25 other authors have focused on detecting infants at low risk of sbi using clinical scores. 26 documenting a viral infection has also been shown to contribute to identify febrile infants at low risk of sbi. [9] [10] [11] [12] [13] however, as having a proven viral infection does not fully rule out the risk of sbi (especially uti), 15-17 viral tests cannot replace blood or urine analysis but must be seen as additional tools in the management of these patients. current routine viral tests are rapid and relatively sensitive in pediatric population but are available for a limited number of viruses only. as a consequence, viral infection is not always documented on time to affect the clinical care. in our study, the infants considered at "low risk of sbi" when discharged from the er that finally turned out to have a proven viral infection represented about 20% of the total cohort (n = 42). these numbers are similar to those published by huppler et al, who identified in a large meta-analysis of 21 studies a similar rate of infants (30%) that were observed without receiving empiric antibiotic therapy or sent back home, after being identified at "low-risk." 27 this population, namely, "low risk" infants with a presumed viral infection, is probably the population that could benefit the most from adding molecular tools to their management, sparing them the bundle of downsides associated with hospitalization and empiric antibiotic therapy, including costs, adverse effects, development of resistant organisms, nosocomial infections, and psychosocial stress on family dynamics. the present evaluation demonstrates that molecular techniques greatly improve the detection rate of viral infections, especially in the challenging group of febrile infants without clinical source, among which the increase in microbiological documentation was nearly 20%. the clart pneumovir was the most powerful tool tested, multiplexing 11 viral targets with a sensitivity rate of 96% and a negative predictive value of 95%. however, it also presented 2 potential drawbacks: its lack of specificity (false-positive results for rhinoviruses) and the fact it does not target all circulating coronaviruses. unfortunately, as the molecular methods used here are expensive and need trained staff, they are not yet routinely used in the management of febrile infants younger than 3 months. 28 furthermore, to be useful in clinical practice, the results of these techniques should be available during the timespan in which patient's management, treatment, and follow-up are decided. new "sample-in, answer-out" point-of-care platforms that enable fully automated detection of comprehensive panels of respiratory pathogens in about 1 hour are now available and should soon enable this expected improvement of patient's management. 29 in conclusion, our study demonstrates that the use of molecular techniques increases to 76% the proportion of documented etiology in febrile episodes in infants younger than 3 months. we have also identified the population in which these techniques have the highest contribution. making these tests available 24 hours/24 and 7 days/7 could help lightening the management of these patients. our study provides adequate information to design a prospective study that could fully assess the contribution of new, rapid, point-of-care, multiplex molecular tools to the management of febrile infants younger than 3 months. the authors gratefully thank the fondation vésale for supporting the molecular analyses used in this study. the authors also thank jerôme de marchin for his contribution to the design of data collection instruments and beatriz epalza for her help in analyzing the data. ce: conceptualized and designed the study, coordinated patient's recruitment, designed the data collection instruments, coordinated and supervised data collection, drafted the initial manuscript, and approved the final manuscript as submitted. mh: supervised the molecular techniques, carried out the laboratory data analysis, critically reviewed and revised the manuscript, and approved the final manuscript as submitted. lb: supervised the laboratory techniques, reviewed the manuscript, and approved the final manuscript as submitted. sd: helped in patient's recruitment, reviewed the manuscript, and approved the final manuscript as submitted. pdb: helped in patient's recruitment, reviewed the manuscript, and approved the final manuscript as submitted. ov: reviewed the manuscript and approved the final manuscript as submitted. jl: conceptualized and designed the study, critically reviewed and revised the manuscript, and approved the final manuscript as submitted. all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: the fondation vésale supported the molecular analyses used in this study. cristina epalza https://orcid.org/0000-0002-6929-4903 supplemental material for this article is available online. febrile infant working group of the pediatric emergency care applied research network. accuracy of complete blood cell counts to identify febrile infants 60 days or younger with invasive bacterial infections predictive model for serious bacterial infections among infants younger than 3 months of age febrile infants at low risk for serious bacterial infectionan appraisal of the rochester criteria and implications for management. febrile infant collaborative study group c-reactive protein as a marker of serious bacterial infections in hospitalized febrile infants markers that predict serious bacterial infection in infants under 3 months of age presenting with fever of unknown origin risk stratification and management of the febrile young child outpatient management without antibiotics of fever in selected infants management and outcomes of care of fever in early infancy detection of 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evaluation of diagnostic tests for infectious diseases: general principles procalcitonin in young febrile infants for the detection of serious bacterial infections diagnostic value of procalcitonin in well-appearing young febrile infants validation of a laboratory risk index score for the identification of severe bacterial infection in children with fever without source european group for validation of the step-by-step approach. validation of the "step-by-step" approach in the management of young febrile infants use of procalcitonin assays to predict serious bacterial infection in young febrile infants impact of rapid microbial identification directly from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on patient management febrile infant working group of the pediatric emergency care applied research network (pecarn). a clinical prediction rule to identify febrile infants 60 days and younger at low risk for serious bacterial infections performance of lowrisk criteria in the evaluation of young infants with fever: review of the literature detection of viruses in young children with fever without an apparent source rapid point of care diagnostic tests for viral and bacterial respiratory tract infections-needs, advances, and future prospects key: cord-259823-ia1g5dt4 authors: gowin, ewelina; bartkowska-śniatkowska, alicja; jończyk-potoczna, katarzyna; wysocka-leszczyńska, joanna; bobkowski, waldemar; fichna, piotr; sobkowiak, paulina; mazur-melewska, katarzyna; bręborowicz, anna; wysocki, jacek; januszkiewicz-lewandowska, danuta title: assessment of the usefulness of multiplex real-time pcr tests in the diagnostic and therapeutic process of pneumonia in hospitalized children: a single-center experience date: 2017-01-15 journal: biomed res int doi: 10.1155/2017/8037963 sha: doc_id: 259823 cord_uid: ia1g5dt4 the aim of the study was assessment of the usefulness of multiplex real-time pcr tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. methods. the study group included 97 children hospitalized due to pneumonia at the karol jonscher teaching hospital in poznań, in whom multiplex real-time pcr tests (ftd respiratory pathogens 33; fast-track diagnostics) were used. results. positive test results of the test were achieved in 74 patients (76.3%). the average age in the group was 56 months. viruses were detected in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). the presence of comorbidities was established in 90 children (92.78%). on the basis of the obtained results, 5 groups of patients were established: viral etiology of infection, 34 patients; bacterial etiology, 7 patients; mixed etiology, 23 patients; pneumocystis, 9 patients; and no etiology diagnosed, 24 patients. conclusions. our analysis demonstrated that the participation of viruses in causing severe lung infections is significant in children with comorbidities. multiplex real-time pcr tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations. acute respiratory tract infections are the most common infectious diseases among children. the incidence of community-acquired pneumonia in europe is 33/100,000 in the age group < 5 years [1] . its clinical manifestation includes symptoms ranging from mild rhinitis to severe pneumonia leading to respiratory failure. the risk group of severe disease course includes children < 5 years of age, especially boys, individuals in immunosuppression, and patients suffering from chronic diseases [2] . establishing the etiological factor is a difficult task. there are no clinical, radiological, or biochemical markers that would enable the differentiation between bacterial and viral infections [3] . this frequently results in the overuse of antibiotics in the therapy of acute respiratory infections. british, american, and polish guidelines state that, in children hospitalized due to pneumonia, microbiological examinations should include blood cultures, the detection of the presence of viruses with the use of pcr (polymerase chain reaction) or immunofluorescence in material collected from the nasopharynx (smear or upper respiratory aspirate), the assessment of antibodies against mycoplasma and chlamydophila in classes igm and igg, and the comparison of antibody levels in the acute phase of the disease and during convalescence [4] [5] [6] . in children it is difficult to collect reliable material for microbiological analysis in a low or noninvasive manner and retain its representativeness of the lower respiratory tract flora. nasal smear cultures are not useful in establishing the etiology of pneumonia. the pathogens grown in a such manner include both physiological flora as well as flora that could potentially cause pneumonia. on the other hand, microbiological cultures need to be secured before antibiotic therapy is commenced, which is impossible in many cases. the solution to this problem is the detection of microbial genetic material in, for example, the material acquired from nasal smear cultures. it is, however, important to remember that pcr results may be positive in the case of persistent infections; in some pathogens prolonged shedding is observed even up to 7 months since the beginning of an infection [7] . another problem is that traditional bacterial cultures are insufficient to establish the etiology, mainly due to the significant participation of viruses in the etiopathogenesis of these infections. this especially concerns children in their first year of life, in whom viral infections may be responsible for even 67% of pneumonia cases [8] . the currently used molecular examinations enable quick identification of numerous pathogens. detecting the influenza virus with pcr is a widely accepted and utilized method of confirming infection [9] . this is also true for the respiratory syncytial virus (rsv). the main drawback of tests detecting just a single pathogen is the necessity of requesting every examination separately, collecting samples for analysis multiple times, and making decisions concerning the selection of pathogens for analysis. test panels used for establishing the presence of the most important bacterial and viral pathogens enable simultaneous detection of the most significant etiological factors. a separate problem is that, apart from the accepted viral etiology, pneumonia may also be caused by new viruses, such as the human metapneumovirus (hmpv), human coronavirus, or bocavirus [10] . molecular techniques are more sensitive and capable of diagnosing more viruses [11] . prompt and accurate diagnosis is important for infection control and surveillance, patient cohorting, treatment choices, and avoiding antibiotics. previous experience with the use of the multiplex realtime pcr tests in the population of children is limited [11] [12] [13] [14] . aim of the study is the assessment of the usefulness of multiplex real-time pcr tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. the study group was composed of children hospitalized due to pneumonia in the period between 01.2014 and 02.2015 at the karol jonscher teaching hospital in poznan, in whom multiplex real-time pcr tests (ftd respiratory pathogens 33; fast-track diagnostics) were used in the diagnostic process. ftd respiratory pathogens 33 is an in vitro test with eight multiplex real-time pcr reactions for the qualitative detection of the following viruses, bacteria, and fungi causing respiratory infections: influenza a, b, and c; parainfluenza viruses 1 the analysis included the following factors: age, sex, comorbidities, immunosuppression, low body mass, airways obstruction, and respiratory failure requiring admission to icu (intensive care unit). respiratory samples (throat or nasal swabs) were collected from all patients. the next step was extraction of pathogens' genetic material either dna or rna, followed by amplification of specific regions by real-time polymerase chain reaction. the presence of specific pathogen sequence in the reaction is reported as a cycle threshold value. apart from the multiplex real-time pcr method, also traditional microbiological culture tests were performed in the studied children: blood cultures, nasopharynx smear cultures, and respiratory aspirate cultures. positive microbiological cultures were defined a ≥105 cfu/ml. all the described tests were done as diagnostic tests during hospitalization. informed consent was obtained from parents/legal guardians on admission to hospital. analysis. data were presented as percentages or medians and range means and standard deviations. interval data were analyzed by mann-whitney test since data did not follow normal distribution (kolmogorov-smirnov test). nominal data were analyzed by chi-square test of independence or in case of zero observed frequencies an exact fisher-freeman-halton test was used. data were analyzed with the use of statistical packages statistica 10 (statsoft, inc.) and statxact 8.0 (cytel); all tests were considered significant at < 0.05. the study group included 97 patients, 52 boys and 45 girls; the average age in the group was 56 months. the presence of comorbidities was established in 90 children (92.78%), including respiratory system diseases in 25 cases, cardiac diseases in 21 cases, neurologic diseases in 13 cases, and neoplastic diseases in 9 cases. immunosuppression was identified in 11 patients (11.34%). the details are provided in table 1 . positive test results were achieved in 74 patients (76.3%). the presence of a viral factor was established in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). the presence of the genetic material of a single pathogen was found in 34 children (45% of all positive results). the details of the analysis are presented in table 2 . on the basis of the obtained results, 5 groups of patients were established: when comparing these groups, no differences were found in terms of age, day of material collection in relation to hospitalization time, or the necessity of icu stay. radiological image analysis was not sufficient to unambiguously diagnose the etiology of pneumonia or respiratory infection. the presence of airflow obstruction was significantly higher in patients with a viral etiology ( = 0.0011). on admission 54% of children with negative real-time pcr results had elevated procalcitonin levels. this proportion is significantly higher when compared to patient with established etiology ( = 0.0186). such difference was not observed while comparing proportion of patients with markedly elevated crp levels (>50 mg/dl). the prevalence of increased procalcitonin levels (>2 ng/ml) was higher in patients without established etiology. the difference is of statistical significance. such differences were not observed when comparing number of patients with increased crp levels. airflow obstruction was found in 44 patients (45%); 75% of them had viral etiology of pneumonia. the difference was of statistical significance ( = 0.0011). the most frequently identified pathogen was rsv in 23 children (23.7%), and rhinovirus (rh) was found in 19 patients (19.6%), adenovirus (adv) in 11 (11.3%), cytomegalovirus (cmv) in 10 (10.3%), and pneumocystis jiroveci (pnp) in 9 (9.3%). purely bacterial etiology was established in 8 patients (8.2%). the details are presented in figure 1 . relations were found between age and the presence of individual microorganisms: infections of rsv etiology were more frequent in younger children (median age 10.5 months versus 42 months; = 0.0133); airflow obstruction symptoms were also more frequent in this group ( = 0.0008). in children under the age of one, the dominant pathogen was rsv, which was found in 32% of patients in this age group. 56% of all positive results for rsv occurred in children in their first year of life. blood cultures were performed in 75 children, in 62 cases they were sterile (82,67%), bacterial flora growth occurred in 13 cases, and in 3 (4%) cases it was pathological (s. aureus, serratia sp., and e. coli). respiratory aspirate cultures were performed in 64 children, they were sterile in 15, physiological respiratory tract flora growth was achieved in 28 cases, and in 23 it was pathological; in 8 of these cases the results were confirmed with multiplex real-time pcr tests. nasal smear cultures were performed in 58 children, they were sterile in 15, and the presence of pathological flora was found in 17 cases (29.31%); in 5 of these cases the results were confirmed with multiplex real-time pcr tests. detailed results of microbiological analyses of patients with at least one positive microbiology results are presented in table 3 . in the group of children with negative results of multiplex pcr tests, blood cultures were positive in 3 cases (e. coli, serratia, and s. aureus), tracheal aspirate cultures were positive in 7 (in 4 of them pathological flora was grown), and nasal smear cultures indicated the presence of pathogenic flora in 5 cases (see details in table 3 ). test results enabled the implementation of targeted therapy in 13 patients; details are presented in table 4 . the acquired results confirm the usefulness of multiplex real-time pcr tests in establishing the etiology of severe pneumonia in hospitalized children. probable etiology of pneumonia was diagnosed in 76% of children with the use of this method. after the exclusion of children with neoplastic diseases from the group, this percentage increased to 79.54%. similar percentages of positive results are often encountered in the literature [8, 12, 14] . bierbaum et al. achieved positive results of multiplex real-time pcr tests detecting only viral factors in 76% of cases in a group of children below the age of six with symptoms of respiratory tract infection and the dominant pathogen was rsv [12] . in a study conducted by mengelle et al. on a group of 914 children with symptoms of respiratory infection, 90% of the collected samples were positive, with rh, rsv, and if (influenza virus) as the most frequent pathogens [15] . in our analysis, we found a more significant participation of rsv; however, only patients with pneumonia underwent the analysis. the general lower participation of the viral factor in comparison to the [16] . in an italian study within a group aged 5-14 years, etiology was established in 77% of cases with the use of molecular analysis. the presence of viruses was revealed in 65% of samples and bacteria in 40% of samples, while mixed etiology was present in 28% of children [17] . mengelle et al. demonstrated that infections caused by more than one virus are common and occurred in 30% of the children included in his study. the sensitivity of virus detection of the multiplex realtime pcr method is high and reaches almost 90% [11] . when genetic material of several pathogens is revealed with the use of the pcr method, it may result from a simultaneous infection caused by two pathogens or from an infection in a patient who is already a carrier of another pathogen. it may also result from establishing the presence of viral genetic material after a previous infection, through the so-called virus shedding. it is estimated that the etiology of pneumonia is mixed in about 30% of children [18] . some works point out that there is a relation between the type of viral factor and the risk of coinfection. martic et al. found coinfections with different pathogens in 50% of infections caused by adenovirus [19] . the high percentage of coinfections in our study may be explained by the specificity of the study group. the majority of the children were burdened with the presence of chronic diseases, and some of these patients were in immunosuppression. the presence of bacterial coinfection is frequent in viral etiology of pneumonia; however, bacteria are not always responsible for active infections. in children with pneumonia, establishing the presence of viruses in the upper respiratory tract is considered as a probable etiological factor. positive results may also be achieved in individuals in temporary asymptomatic carrier state during convalescence after a previous infection [13] . it is, therefore, necessary to remain very cautious when interpreting examination results. the clinical significance of human rv rna detection in respiratory samples remains unclear [20] . the pcr method does not enable unambiguous differentiation between infection and carriage, even with the use of the quantitative method. it needs to be stated, however, that comparative studies of carriage significantly more often indicated the presence of s. pneumoniae in children with pneumonia compared to the healthy population [21] . the available literature contains reports stating that higher numbers of viruses were usually associated with milder disease course [20] . the exception was the coinfections with rsv and hmpv [22] . infections of this type are characterized by intensified obstruction symptoms [22] . in our group, the children with infections caused by mixed pathogens did not differ from the children with infections caused by single pathogens, in terms of both the severity of disease course and intensity of inflammatory reaction. the high percentage of positive results of examinations detecting viral genetic material points to the significance of viruses in the occurrence of pneumonia in children. the literature indicates that viruses may be responsible for the majority of pneumonia cases in children [8, 16] . in our group, in the case of hospitalized children, some patients received outpatient antibiotic therapy, which was then continued during inpatient care in all cases. therefore, all samples were collected during antibiotic therapy, on the fifth day on average. hence, it is not possible to completely exclude the presence of bacterial factors, which were not identified in the upper respiratory tract later on due to the ongoing antibiotic therapy. this may explain the relatively low percentage of bacterial genetic material detected in the group we analyzed. the current guidelines recommend the use of antibiotic therapy in children hospitalized due to pneumonia, but the lack of clinical improvement despite the application of broadspectrum antibiotic therapy may suggest a viral cause of infection [4] [5] [6] . the studied group was unique due to the high participation of patients with comorbidities, some of them in immunosuppression. such patients are at a higher risk of severe course of infection and prolonged hospitalization. the diagnosis of a viral etiology of infection enables moderate use of antibiotics and allows for more emphasis on symptomatic treatment, which is the most beneficial for such patients. because viruses are easily transmitted from patient to patient, advanced infection control methods are critical in controlling the spread of viruses [20] . rsv turned out to be the most frequently detected pathogen, especially in the group of children under the age of one. moreover, it was frequently the only identified pathogen. in children from risk groups, rsv causes infections with severe course, and it is characterized by high infectiousness. diagnosing rsv as the etiological factor enables the isolation of or cohorting the patients in order to reduce the spread of the infection and the implementation of the recommended management for severe obstruction [4, 5] . research is currently being conducted on the implementation of antiviral drugs which are more effective than the previously used ribavirine [23] [24] [25] . in the case of viral infections such as influenza, the establishment of the etiological factor enables the implementation of specific treatment. in the studied group, a single case of influenza was found, but the year in which the study was conducted was not an epidemic year. molecular tests for detecting the influenza virus only are widely applied. they are very useful during epidemic seasons, but it is important to remember that the disease may occur also outside such periods. in certain situations, it may be difficult to select a diagnostic test only on the basis of symptoms. moreover, in the case of influenza, the time of treatment implementation is crucial. therefore, including influenza in the standard diagnostic set prevents the omission of this important infection factor. the conducted analysis showed limited usefulness of traditional microbiological examinations in the diagnostic process of pneumonia. taking blood cultures is also considered standard in severe pneumonia, but its usefulness is not significant. in a group of patients with radiologic confirmed pneumonia, esposito revealed the presence of s. pneumoniae in 14.3% of cases; 91.8% of the diagnoses were based on positive results of real-time pcr tests conducted on material collected from the respiratory tract, while positive results of blood cultures were present only in 8.14% of cases [22] . nasopharyngeal swabs cultures often show flora growths that may constitute colonization. in our analysis, traditional microbiological examinations revealed the presence of potentially pathogenic bacteria in material collected from 31 children; in 11 of these cases there was agreement with the results of multiplex real-time pcr tests. in 8 patients it was not possible to assess the agreement of research methods due to the fact that in the ftd respiratory pathogens 33 test microorganisms such as pseudomonas aeruginosa, escherichia coli, or serratia sp. are not detected. the majority of previous works assessed the results of multiplex real-time pcr tests conducted in admission rooms on all patients reporting with respiratory symptoms [15] [16] [17] . because of the costs associated with these tests, it is difficult to use them in such a manner in daily practice. the mentioned analyses seem to be more epidemiological in nature. in our work we made an attempt to assess the usefulness of tests which were conducted during hospitalization in children with severe pneumonia not responding to standard therapy. in this group of patients, the benefits of the applied treatment were satisfactory in relation to the diagnostic cost incurred. the confirmation of viral etiology was very important, especially in the group of children at a risk of severe course of infection. in such a unique group of patients, constituted by children burdened with comorbidities, under immunosuppression, and after marrow stem cell transplants, the implementation of targeted treatment should be taken into consideration with the use of modern antiviral drugs, as well as such steps as isolation and the reduction of immunosuppression. in the guidelines of the neutropenia management it is strongly advised to isolate patients with documented respiratory viruses until symptoms resolve [20] . there are some limitations to our study. we have collected a heterogenic group of patients (different indication for testing, different comorbidities, and chronic diseases). the number of patients and the number of specimens were too small to perform elaborate statistical analysis. sample collection for microbiological cultures was incomplete. the exact timing of sample collection relative to antibiotic administration was not accurately documented. similar problems were described in other clinical studies in this subject topic. our analysis demonstrated that coinfection with viruses is common in severe lung infections in children with comorbidities. multiplex real-time pcr tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations widely used in the applicable diagnostic process to date. informed consent was obtained from parents/legal guardians on admission to hospital. this article does not contain any studies with human participants performed by any of the authors. this study is done based on analysis of patients' medical records; all tests were done as diagnostic tests during hospitalization. estimates of world-wide distribution of child deaths from acute respiratory infections acute respiratory infections in children procalcitonin, creactive protein and leukocyte count in children with lower respiratory tract infection british thoracic society guidelines for the management of community acquired pneumonia in children: update the management of community-acquired pneumonia in infants and children older than 3 months of age: clinical practice guidelines by the pediatric infectious diseases society and the infectious diseases society of america rekomendacje postępowania w pozaszpitalnych zakażeniach układu oddechowego viral pneumonia impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses clinical and socioeconomic impact of different types and subtypes of seasonal influenza viruses in children during influenza seasons development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay multiplex pcr and emerging technologies for the detection of respiratory pathogens performance of a novel microarray multiplex pcr for the detection of 23 respiratory pathogens (symp-ari study) clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial epidemiological investigation of nine respiratory pathogens in hospitalized children in germany using multiplex reversetranscriptase polymerase chain reaction the use of a multiplex real-time pcr assay for diagnosing acute respiratory viral infections in children attending an emergency unit etiology of community-acquired pneumonia in hospitalized children based on who clinical guidelines etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections epidemiology and virology of acute respiratory infections during the first year of life: a birth cohort study in vietnam multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children viral infections in immunocompromised patients frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction antibiotic therapy for pediatric community-acquired pneumonia: do we know when, what and for how long to treat? new options in the treatment of respiratory syncytial virus disease successful treatment of parainfluenza virus respiratory tract infection with das181 in 4 immunocompromised children chemotherapy of respiratory syncytial virus infections: the final breakthrough all authors declare that they have no conflict of interests. key: cord-257114-pxmflm2c authors: burguete, sergio r.; maselli, diego j.; fernandez, juan f.; levine, stephanie m. title: lung transplant infection date: 2012-12-26 journal: respirology doi: 10.1111/j.1440-1843.2012.02196.x sha: doc_id: 257114 cord_uid: pxmflm2c lung transplantation has become an accepted therapeutic procedure for the treatment of end‐stage pulmonary parenchymal and vascular disease. despite improved survival rates over the decades, lung transplant recipients have lower survival rates than other solid organ transplant recipients. the morbidity and mortality following lung transplantation is largely due to infection‐ and rejection‐related complications. this article will review the common infections that develop in the lung transplant recipient, including the general risk factors for infection in this population, and the most frequent bacterial, viral, fungal and other less frequent opportunistic infections. the epidemiology, diagnosis, prophylaxis, treatment and outcomes for the different microbial pathogens will be reviewed. the effects of infection on lung transplant rejection will also be discussed. significant progress has been made since the first human lung transplant (lt) in 1963, and although survival after transplantation was initially plagued by issues of rejection, the advent of immunosuppression ushered in a new era in transplantation science and made long-term survival a possibility. with this success came the dilemma of post-transplant infectious complications, which, to this day, remain a significant contributor to overall morbidity and mortality in the lung transplant recipient (ltr). of all solid organ transplants, lungs are the most prone to infection, and this is likely due to several factors unique to the lung allograft. apart from constant exposure to the outside environment, the lungs are exposed to the colonized native airway and have been stripped of their usual mechanisms of defence including the cough reflex, bronchial circulation and lymphatic drainage. these factors, coupled with the induction of an immunosuppressed state collaborate to produce an environment that is ripe for the development of infection. apart from direct injury, infection leads to several complications that may then have an effect on overall survival including the development of both acute and chronic rejection with eventual graft failure. the immune modulating effects of some pathogens, such as cytomegalovirus (cmv), can also augment the risk of developing other infections further leading to increased morbidity. 1 a thorough and comprehensive screening and management approach must be undertaken to optimize the survival of these patients and minimize the risk of infectious complications. we present a review of the major infectious complications following lt as well as recent recommendations for the evaluation and management of these entities. the respiratory tract is the most common area of infection after lt, and bacterial pneumonia is the most common infectious complication. cmv is the second most common complication, and its occurrence is much higher than in other solid organ recipients. 2 it appears that the critical period for infections after lt is within the first 90 days. in a recent epidemiological study in which 51 ltr were followed for a mean of 38.2 months, 75% of infectious episodes occurred within the first year after transplantation, and nearly half (42%) occurred within the first 3 months. 3 bacterial disease accounted for the largest proportion of infections (48%) followed by viral, fungal and mycobacterial disease (35%, 13% and 4%, respectively). in the early post-lt period (days to 1 month), nosocomial organisms account for the majority of infections. following this period and for the next several months, at a time when immunosuppression is at the highest level, opportunistic organisms such as cmv and fungi account for the majority of infections. in the late post-transplant period, community-acquired bacterial and viral infections develop, although infection with health care-associated organisms remains common (fig. 1) . it is within the first year that infection makes the biggest impact on mortality. according to the registry of the international society for heart and lung transplantation, infection is listed as the leading cause of mortality, accounting for 31% of deaths within the first year after transplant. 5 thereafter, infection is a close second to bronchiolitis obliterans syndrome (bos) as a cause of death. recently, it has been increasingly recognized that infection may both predispose the airways to the development of bos and increase the mortality of those with bos, thus still contributing significantly to this mortality. 6 the lungs are unique organs in that they are constantly exposed to antigens from both the environment (inhaled antigens) and the bloodstream (blood-borne antigens). the upper airways and pulmonary tissue have defence mechanisms composed of physical barriers and cellular components. physical barriers include hairs in the nasal cavity, mucus secretions, cilia and turbulent airflow generated by the nasal cavity that prevent pathogens from reaching the lower airways. despite these barriers, pathogens may still reach and infect the pulmonary tissue. there are several risk factors that make ltr more vulnerable to infection (table 1) . immediately postsurgery, ltr may have disruption of normal physical barriers and are at risk of aspiration and infection (e.g. use of nasogastric and endotracheal tubes). 7, 8 there are also other important changes that happen postsurgery. first, during the surgical procedure of lt, there is a complete disruption of the bronchial circulation, and this may cause a loss of epithelium integrity, ciliary function and mucus production. 9 these effects are transient because of the development of collateral circulation but remain at risk of infection during the initial stages. [9] [10] [11] second, denervation of the allograft may suppress the cough reflex and promote bronchial hyperresponsiveness. 2 third, the lymphatic drainage of the allograft is also severed promoting stasis and oedema in the bronchial tissues impairing normal healing. 2 fourth, stenosis or necrosis may occur at the site of the bronchial anastomosis, which may in turn facilitate colonization and invasion by opportunistic pathogens and decrease the clearance of secretions beyond the anastomosis. 12 at the cellular level, the ltr is vulnerable to infection due to the immunosuppression regimen used to prevent rejection affecting multiple inflammatory cellular lines and cytokines. the regimen consists of induction agents (medications used immediately post-transplant) and maintenance agents for prolonged use. because immunosuppression is needed indefinitely, ltr has a life-long increased risk for opportunistic pathogens to proliferate and cause significant complications. the maintenance immunosuppression regimen consists typically of a calcineurin inhibitor, an antimetabolite and corticosteroids. 13, 14 the calcineurin inhibitors used in lt are cyclosporine a and tacrolimus. cyclosporine a binds to cyclophylin preventing the activation of the nuclear factor of activated t-lymphocytes (t cells) by calcineurin. tacrolimus binds to fk-binding protein 12 inhibiting calcineurin and preventing the activation of the nuclear factor of activated t cells. 13, 15 by reducing the activation of nuclear factor of activated t cells, both drugs reduce the production of interleukin-2 limiting the clonal expansion of activated t cells (fig. 2) . 16 azathioprine and mycophenolate mofetil (mmf) are the commonly used antimetobolites after lt. azathioprine, a derivative of 6-mercaptopurine, inhibits both ribonucleic acid and deoxyribonucleic acid production, reducing the proliferation of both t cells and b-lymphocytes. mmf is a prodrug of mycophenolic acid, an inhibitor of the inosine monophosphate dehydrogenase (fig. 2 ). this enzyme is responsible for the synthesis of guanine nucleotides, which both t cells and b-lymphocytes are critically dependent of. 17 other maintenance agents that have been used less frequently to maintain immunosuppression include sirolimus and everolimus. sirolimus binds to the fk-binding protein 12 and through the mammalian target of rapamycin pathway prevents the synthesis of deoxyribonucleic acid and proteins by t cells (fig. 2) . 18 through an independent mechanism, sirolimus also affects b-lymphocytes and decreases cytokine and antigen production. 19 everolimus reduces the mammalian target of rapamycin kinase activity, inhibiting the downstream pathways of proliferation and activation of t cells. 20 finally, through the alteration of gene transcription factors, corticosteroids can exert a wide variety of immunosuppressive effects: interruption of antigen presentation, changes in the production of cytokines and alteration in the proliferative responses of various cell lines. 21 the use of induction agents after lt varies among centres. these agents include okt3, antithymocyte globulin (atg), alemtuzumab and basiliximab. okt3 is a murine monoclonal antibody that inactivates the t cell receptor-cd3 complex preventing the activation of circulating t cells with a partial sparing of t regulatory cells. atg is a polyclonal antibody directed against lymphocytes. it depletes circulating lymphocytes through complement-mediated lysis and destruction by the reticuloendothelial system after opsonization. 13 basiliximab is a chimeric monoclonal antibody that targets the a subunit of the interleukin-2 receptor inhibiting the differentiation and proliferation of t cells. 22, 23 alemtuzumab is a murine monoclonal antibody that targets cd52. this receptor is present in macrophages, monocytes, b-lymphocytes and t cells among other inflammatory cells. the binding of cd52 causes complementmediated cytolysis and activation of pathways leading to apoptosis. 13 the use of okt3 is now significantly limited due to an increase risk of infection. [24] [25] [26] [27] for this reason, most centres have elected to use atg, basiliximab or alemtuzumab, in combination with corticosteroids for induction of immunosuppression after lt. 28 evaluation of large series of solid organ recipients has shown that this combination prevents graft rejection and improves survival. 29 atg does not increase the rate of infections in transplant recipients and has been associated with a survival benefit. 30, 31 basiliximab compared with atg does not increase the risk of infection and was safer than okt3 in heart and ltr. 22, 23, 26, 32 alemtuzumab was recently shown to improve survival compared with atg. 33 despite these positive outcomes, the immunosuppression is more profound during induction, and patients should be monitored closely for infection during this period. despite the removal of both lungs during bilateral procedures, residual colonization and/or infection can remain in the thoracic cavity, the bloodstream, the upper airways or the sinuses. those patients with cystic fibrosis (cf) present the highest risk for recipient-harboured infection due to the frequent colonization and infection with multiresistant microorganisms including bacteria (gram-negative rods and gram-positive cocci) and fungi. resistant gramnegative organisms pose perhaps the greatest risk, and some studies suggest an association between pretransplant colonizing organisms from patients with suppurative lung disease and pneumonias following lt. 34 the majority of recent data suggests that patients colonized with multi-drug-resistant pseudomonas appear to have acceptable outcomes, including survival following lt, and should not be excluded on that criterion alone. 35, 36 in contrast, a former subspecies of pseudomonas, now subspeciated as burkholderia cenocepacia due to its unique resistance patterns, can pose significant problems in transplant recipients. there have now been at least nine distinct genotypic variants (genomovars) identified in the burkholderia cenocepacia complex. 37 colonization with burkholderia cenocepacia complex (genomovar 3) can result in significant morbidity and mortality post-transplant and should be considered a strong relative contraindication to lt, 38, 39 although isolated reports of successful outcomes have been reported. 40 in one study of 75 patients, 38 there was a significant difference in 1-year-survival between those patients not infected (92%) and those colonized with a non-burkholderia cenocepacia strain (89%) compared with those colonized with burkholderia cenocepacia (29%). similar results of variable survival rates based on burkholderia cenocepacia species have been found in other studies. 37, 39 because of these overwhelming data, the majority of transplant centres will not transplant colonized or infected patients with this organism. when evaluating the potential lt donor, routine screening is done to prevent transmission of donorharboured infection to the recipient. 41 donor screening includes routine serology for viral infection including cmv, epstein-barr virus, varicella-zoster, hepatitis b and c, and human immunodeficiency virus, among others. in addition, the potential donor lungs are evaluated radiographically and bronchoscopically. despite these measures, infection may still occur. to potentially pre-empt the development of donortransmitted infection at the time of the transplant procedure, a culture swab or wash, or a portion of the donor bronchus is sent for culture. in contrast with some older studies, 42, 43 more recent data suggest that recovery of an organism from the donor lung, a schematic overview of the mechanisms of action of medications used for immunosuppression. il-2 is required for the activation of the mtor pathway and progression of the t cell cycle. both csa and tacrolimus reduce the activation of nfat, which in turn results in a decreased production of il-2. basiliximab is a monoclonal antibody that inhibits the il-2 receptor. sirolimus and everolimus inhibit the mtor pathway through inhibition of specific enzymes. alemtuzumab targets protein cd52 causing t cell dysfunction. both mmf and aza disrupt key elements of the deoxyribonucleic acid synthesis affecting the progression of the t cell cycle. aza, azathioprine; csa, cyclosporine a; fkbp12, fk-binding protein 12; impdh,inosine-50-monophosphate dehydrogenase; il-2, interleukin-2; il-2r, il-2 receptor; mmf, mycophenolate mofetil; mtor, mammalian target of rapamycin; nfat, nuclear factor of activated t-lymphocytes. including a positive gram stain, or subsequent growth in culture does not always translate into infection and/or poor outcomes in the recipient. 34, 44, 45 in one study of 80 ltr, the investigators noted that organisms were grown from 57% or 89% of donors for a total number of isolates of 149. 44 of these, most isolates were staphylococci or streptococci. post-transplant pneumonias were found in 41% of recipients in this study; however, pseudomonas, and not gram-positive organisms, was the most prevalent causative organism. the results of this study and others 45 suggest that the presence of organisms in the donor does not necessarily predict post-transplant pneumonia, and perhaps this donor criterion should be re-evaluated. despite these suggestions and because empirical bacterial prophylaxis was used in the majority of these studies, the general practice is to routinely initiate prophylactic, broad-spectrum antibiotics (regimens are discussed later) and then narrow the antibiotic therapy based on donor isolates. 41 any patient with suppurative lung disease, such as cf or bronchiectasis, being considered for lt will receive a bilateral procedure with attempts at avoiding infection from a remaining native lung. however, in those diagnoses where a single lt may be performed, such as chronic obstructive pulmonary disease or interstitial lung disease, the native lung may harbour infectious organisms that can infect the new graft, particularly when the patient is subjected to immunosuppression. alternatively, the native lung can develop severe infection leading to sepsis and further compromise. although attempts at avoiding this risk are undertaken by routine pretransplant screening, examples of infection that can be harboured in the native lung include bacteria, fungi (perhaps contained in a mycetoma) or non-tuberculous mycobacteria (ntm). 46 as part of the initial pretransplant evaluation, all potential transplant recipients should undergo careful screening for infection. although there may be some variation between transplant centres, routine screening includes serological measurement for cmv, epstein-barr virus, varicella-zoster, hepatitis b and c, and human immunodeficiency virus, and screening for latent tuberculous infection. the results obtained from this screening are used to assess the patient's overall candidacy for lt (e.g. human immunodeficiency virus is generally an exclusion) and also to stratify the patient for screening and prophylaxis in the post-lt period (e.g. cmv and epstein-barr virus). recommendations for recipient and donor presolid organ transplant screening are published from the american society of transplantation. 41 pneumonias comprise the most common cause of infection following lt, and bacterial pathogens remain the most common cause of all pneumonias. 34, 47 in a multicentre, prospective study from spain, with a median follow-up of 180 days, 85 episodes of pneumonia were documented in 236 ltr for an incidence of 72 episodes/100 lt years. 47 of these, bacteria were the most common pathogen accounting for 82.7% of the pneumonias. bacterial pneumonia is most common in the early post-transplant period (1-30 days) usually due to infection with health care-associated and nosocomial organisms (fig. 1 ). in the spanish study, 40 of 85 of pneumonias (44%) occurred in the first 30 days following transplant. nearly 3/4 of all bacterial pneumonias (72%) were due to gram-negative organisms-most commonly pseudomonas (incidence 118.6 episodes per 1000 ltr/year). staphylococcus aureus and acinetobacter infections were the second most common bacterial isolates (each with an incidence of 67.8 episodes/1000 ltr/year). the median time to development of gram-negative pneumonia was 31 days with a range of 3-394 days. grampositive cocci-related pneumonias also occurred in the early post-transplant period at a median of 35.5 days (range 2-486 days) post-transplant. other bacterial isolates from this and other studies span the spectrum of health care-acquired infectious organisms. similarly, p. aeruginosa was found to be the most common isolate accounting for 33.3%, staphylococcus aureus comprised 26.8%, and aspergillus 16%. 34 pneumonia is also seen in the late post-transplant period. throughout the lifespan of the ltr, ongoing contact with hospital settings, both outpatient and inpatient, and frequent antibiotic exposure commonly result in infections with health careassociated, often resistant, pathogens. communityacquired pneumonias can also develop in the late post-transplant period. 48 in a single-centre study, 14 out of 220 ltr (6.4%) developed invasive pneumococcal infection (pneumonia and/or sepsis) at a median of 1.3 years after transplantation (incidence rate: 22.7 cases per 1000 person-years). routine vaccination for pneumococcus with the pneumococcal polysaccharide vaccine is recommended both before and every 5 years following lt. 41 in general, the approach to suspected pneumonia at any time period post-transplant includes sputum, blood cultures and often bronchoscopy with bron-choalveolar lavage (bal), sterile brush and sometimes biopsy. the role of new biomarkers such as procalcitonin for diagnosis or follow-up has not been well established in the ltr. due to the high incidence of early post-transplant pneumonia, whether derived from the recipient, donor or nosocomially acquired, broad-spectrum postoperative prophylaxis is routinely used. prophylaxis in the post-transplant period varies by centre but typically includes a third generation cephalosporin and vancomycin and is then tailored to the results of donor and recipient cultures, or as clinically indicated for 7-10 days. prophylactic antibiotic treatment should be extended to 14 days for known pretransplant recipient colonization. for specific prophylactic regimens for viral and fungal pathogens, see later. treatment of bacterial pneumonia includes standard regimens as outlined by the american thoracic society and infectious disease society of america treatment for health care-acquired pneumonia. 49 in the setting of known prior colonization or infection, initial antibiotic selection may be based on prior culture and sensitivity results. typical antibiotics used should include coverage for gram-negative (including pseudomonas) and gram-positive (including staphylococcus aureus) pathogens. in general, 8-14 days of therapy is recommended. in the case of resistant organisms, inhaled aminoglycosides may also be added to the treatment regimen. pneumonia has significant impact on overall posttransplant survival and the eventual complication of chronic rejection. in the spanish study, attributable 1-year survival was reduced in those patients developing pneumonia of any aetiology (29.5% mortality) versus those without pneumonia (14% mortality), although bacterial pneumonia alone was not separated out in this analysis. these authors also found that the probability of survival during the first year of follow-up was significantly higher in the multivariate analysis in lt recipients who did not have a pneumonia episode compared with those that had at least one episode of pneumonia. 47 in the bonde et al. study, pneumonia was found to be an independent predictor of overall mortality. 44 viral infection after lt is common and classified into disease caused by cmv or caused by other community-acquired respiratory viruses (carv). a recent study showed that a viral pathogen was responsible for 25 of 71 infectious episodes in a cohort of ltr, with cmv accounting for 68% of those cases. additionally, the majority of cmv episodes occurred within the first 3 months following lt, while the majority of the later infections were due to influenza and occurred after 1 year (fig. 1) . 3 among the opportunistic infections following lt, cmv is the most prevalent and most important despite significant advances in both diagnosis and management. as well as contributing directly to both morbidity and mortality, mounting evidence suggests a relationship between cmv pneumonitis and chronic rejection in the form of bos and decreased survival despite treatment. 50 cmv seropositivity can range from 30% to 97% in the general population, and after infection, the patient will harbour the virus for life. of all solid organ transplants, ltr has the highest risk of developing cmv disease. 51 the incidence of cmv infection has been reported to range from 30% to 86% in post-ltr, with a mortality of 2-12%. 52 this increased incidence is thought to be due partly to the high viral load of cmv transmitted in the lymphatics of the lung compared with other solid organs, as well as the high level of immunosuppression required for lung allograft. the most important risk factor for the development of cmv infection is the donor-positive/recipientnegative serostatus of a transplant patient, as these patients will lack immunity to cmv. the lowest risk occurs in donor-negative/recipient-negative patients. 51 other important risk factors include type and intensity of both induction and maintenance immunosuppression, concurrent infections, rejection and host factors such as age or comorbities. 51, 52 there is almost a symbiotic relationship between rejection and cmv infection. both of these individual processes induce a cytokine cascade that in essence promotes the development of the other. tumour necrosis factor-alpha, a key signal in the reactivation of cmv from latency, is released during allograft rejection, thereby facilitating the onset of viral replication and subsequent infection. conversely, infection of the vascular endothelium and smooth muscle by cmv leads to an upregulation of adhesion molecules promoting an increase in the quantity of inflammatory cells in the graft and subsequent development of rejection. additionally, molecular mimicry and the production of anti-endothelial antibodies with cmv may also play a role in the development of rejection. 52 cmv serology of both donor and recipient must be checked prior to transplant. 53 there is an important distinction between cmv infection and disease. infection is defined as 'cmv replication regardless of symptoms', while disease is defined as 'evidence of cmv infection with attributable symptoms', such as 'a viral syndrome with fever and/or malaise, leukopenia, thrombocytopenia or as tissue invasive disease'. 51, 54 recent technologies have effected a shift in the diagnosis of cmv infection and disease. the previous method of diagnosis, pp65 antigen detection, has been replaced by quantitative nucleic acid-based amplification testing via polymerase chain reaction (pcr) for the recognition of viraemia by most centres, with 85% of institutions using this method for monitoring and diagnosis. 55 there are no universally accepted viral load cut-offs for positive and negative results, and that reported values may be dissimilar between different laboratories. despite this, current guidelines on the management of cmv in solid organ transplant patients do not clearly favour one test over the other and cite both as acceptable options for diagnosis. additionally, viral culture of blood or urine has a limited role for diagnosis and is not routinely recommended. 53 most recently, tests for cell-mediated immunity against cmv have shown promise for predicting risk of developing disease. lisboa and colleagues demonstrated that cell-mediated immunity to cmv, as shown by a cd8+ t cell response assay, was associated with decreased risk of developing disease in patients with detectable low-level viraemia. twenty four of 26 patients (92.3%) with a positive interferon-gamma release assay were able to clear their viraemia without disease compared with 5 of 11 (45.5%) in patients with a negative cell-mediated immunity at onset (p = 0.004). 56 in a similar study, the same group was able to show that a negative assay was associated with a higher chance of developing late-onset cmv after prophylaxis. in their study, cmv disease occurred in 2/38 (5.3%) patients with a detectable interferongamma response versus 16/70 (22.9%) patients with a negative response (p = 0.038). 57 there are two accepted approaches to the prevention of disease from cmv, universal prophylaxis and preemptive therapy, and although there are no randomized trials comparing one strategy versus the other in ltr, most centres favour the former or may sometimes employ both. 55 the first, universal prophylaxis, involves administration of antivirals to all transplant patients deemed to be at high risk by serostatus. the second, pre-emptive therapy, is comprised of monitoring at-risk patients for viral replication and administering antivirals at a predetermined level of replication in the hopes of treating patients prior to the onset of disease. a cochrane review comparing prophylaxis in different groups of solid organ transplant patients with antivirals versus placebo or no treatment showed a significant reduction in disease (relative risk 0.42), infection (relative risk 0.61), mortality from cmv disease (relative risk 0.26) and allcause mortality (relative risk 0.63). interestingly, the review also found a decrease in the risk of developing herpes-simplex virus, varicella-zoster virus and bacterial infections. 58 prophylaxis may not only be beneficial in decreasing direct morbidity and mortality from cmv disease but may also have secondary effects by decreasing the morbidity and mortality of both acute and chronic rejection. the cochrane review mentioned earlier failed to show a difference in acute rejection episodes, but other small studies have shown statistically significant differences in ltr specifically and it is generally believed that prevention of cmv decreases the risk for acute rejection. [58] [59] [60] the data for bos are more encouraging. a recent study by chmiel and colleagues was able to show a 23% absolute risk reduction of developing bos in a group of ltr on cmv prophylaxis as compared with a historical cohort that was not prophylaxed and a 35% absolute risk reduction compared with data in the literature (p = 0.002). 1 most centres provide prophylaxis for a period of 3-6 months after transplantation; however, the optimal duration of prophylaxis has not been well established and is currently under debate. 55 the guidelines recommend a minimum of 6 months for ltr. 53 recent data suggest that this window of prophylaxis should possibly be extended, especially for donor-positive/ recipient-negative patients. palmer and colleagues report the first randomized, placebo-controlled trial showing a decrease in the risk of cmv disease with extended prophylaxis. in this study, 136 ltr who completed 3 months of valganciclovir prophylaxis were randomized to an additional 9 months of valganciclovir versus placebo. the risk of cmv disease was reduced (32% vs 4%; p < 0.001) in the extendedcourse group versus the short-course group. there were also statistically significant reductions in cmv infection (64% vs 10%; p < 0.001) and disease severity as measured by viral load with extended treatment. acute rejection episodes, opportunistic infections, adverse events and cmv ul97 ganciclovir-resistance mutations were similar between both groups. 61 the international consensus guidelines list valganciclovir and ganciclovir (oral or intravenous (iv)) as the antivirals of choice for the prevention of cmv disease and state that cmv immunoglobulin may also be used in combination with these two, but there are limited data to support its use. 53 although foscarnet was commonly used in the past for cmv disease, the significant risk of nephrotoxicity with concomitant calcineurin-inhibitor use has made it fall out of favour for the relatively safer agents ganciclovir and valganciclovir. 55 and, although the recommendation for treatment of severe disease is still iv ganciclovir, the results of the valcyte in cmv disease treatment of solid organ recipients trial have made valganciclovir a viable choice in the treatment of less severe cmv. 53 the in cmv disease treatment of solid organ recipients trial randomized 321 solid organ transplant recipients with non-life-threatening cmv disease to either oral valganciclovir or iv ganciclovir. valganciclovir demonstrated non-inferiority in regard to clinical resolution of disease as well as eradication of viraemia in both the intent-to-treat and the per-protocol arms of the study. 62 the current guidelines recommend oral valganciclovir at twice-daily dosing or iv ganciclovir for the treatment of nonsevere cmv disease. as there are no efficacy data for valganciclovir in severe or life-threatening disease, iv ganciclovir is still the 'gold standard' for those patients. in both groups, serial monitoring of viraemia should occur optimally at 1-week intervals, and treatment should be continued for a minimum of 2 weeks and until viral eradication has been documented with two consecutive tests. the use of secondary prophylaxis is generally recommended for 1-3 months after treatment of disease. 53 infection with a carv is common after lt, and with the development of new diagnostic techniques, the incidence quoted in older literature is likely underestimated. a study of ltr undergoing serial surveillance and diagnostic bal over a 3-year period showed that a respiratory virus was isolated in 51.6% of patients on at least one bal sample. rhinovirus was the most common pathogen isolated, followed by parainfluenza, coronavirus, influenza, metapneumovirus and respiratory syncytial virus (rsv). 63, 64 carv is being increasingly recognized as contributors to significant morbidity in immunocompromised hosts and can cause severe and life-threatening pneumonitis. additionally, there appears to be evidence that infection with these organisms can also lead to a decrease in graft survival. a retrospective cohort study of 259 ltr followed over 5 years showed a significantly increased risk of developing bos or death from bos in the group that was diagnosed with a carv infection. 65 given the paucity of effective antiviral treatment for most of these viruses, early diagnosis is essential for both treatment and to minimize spread among other immunocompromised patients. with the exception of influenza and rsv, for which treatments exist, supportive care and a reduction in immunosuppression remain the cornerstones of care for the treatment of carv. a complete listing of all the viruses that commonly affect ltr would be beyond the scope of this article so we will focus on those that have the most clinical bearing, namely influenza, rsv, human metapneumovirus and parainfluenza. as it typically does not cause respiratory tract disease, we will not discuss epstein-barr virus, except to mention its known association with post-transplant lymphoproliferative disorder after lt. infection of normal hosts with influenza most commonly causes a self-limited disease with upper respiratory symptoms, myalgias and fever; however, infection in ltr appears to be associated with increased risk of lower respiratory tract involvement by either a primary viral or a concomitant bacterial superinfection. this was illustrated in a small series of ltr admitted for influenza where all appeared to have pulmonary parenchymal involvement on imaging and by bal as well as in another series by vilchez and colleagues, where 7 of 15 patients with influenza were found to have pulmonary infiltrates, 5 of which were attributed to a primary viral pneumonia after bal. 66, 67 novel h1n1 influenza appears to have similar clinical features, although there appears to be an increased rate of gastrointestinal symptoms such as nausea and diarrhoea; which may be prominent. 68 due to the increased severity of disease, all ltr and their household contacts should receive annual influenza vaccination for prevention of disease. 69 diagnosis is essential, and efforts should be made to establish the type, as specific therapy will depend on resistance patterns. 69 diagnosis of seasonal influenza is made by rapid antigen detection of nasopharyngeal swabs, but this method appears to be unsatisfactory for detection of novel h1n1 and molecular real-time pcr methods are currently approved for use when swine flu is suspected. 70 in addition to supportive care and isolation, treatment involves the use of the antiviral agents amantadine and rimantidine for susceptible influenza a strains, and zanamavir and oseltamivir for both influenza a and b strains. due to the variation in circulating strains from year to year, it is important to stay abreast of the current recommendations from the centers for disease control and prevention 71 for appropriate treatment. 72 in addition, given the prolonged viral shedding, the typical treatment course of 5 days may be insufficient in ltr, and prolonged therapy may be required. some experts advocate treating influenza even if symptom onset is greater than 48 h and treating until viral replication ceases. 73 treatment of novel h1n1 is limited by the resistance of the strain to the m2 inhibitors: amandatine and rimantidine. as such, current guidelines recommend treatment with oseltamivir or perhaps even zanamavir if resistance is suspected to this agent. iv or higher dose therapy is recommended for critically ill patients, and immunosuppression should be decreased. 63, 64 rsv by the age of 2, virtually, all children have been infected with rsv, although reinfection can occur throughout life, and early acquisition after transplant or with augmented immunosuppression is a risk factor for severe disease. 72 as with influenza, infection can vary from a self-limited upper respiratory illness to severe pneumonia and occurs through inhalation of infectious droplets and contact with fomites, making isolation precautions paramount for prevention. there are currently no available vaccines for rsv and no recommended therapies for prevention. due to a lack of data for effective antiviral treatment, the only universally accepted recommendations for therapy are supportive care and a reduction of immunosuppression. 72 ribavirin, which has shown in vitro activity against rsv, is approved for treatment of lower tract disease by showing benefit in stem cell recipients. 74, 75 there are otherwise no controlled studies showing efficacy with the use of inhaled ribavirin in transplant patients. despite this, inhaled ribavirin remains the most commonly used treatment for rsv with one report showing a multidrug regimen of ribavirin, steroids, rsv-iv immunoglobulin and palivizumab to be safe, effective and associated with stability of lung function. 76 two small case series have shown promise for parenteral and oral ribavirin in ltr. 77, 78 an optimal treatment strategy for disease due to rsv is yet to be determined, and further studies are needed to better delineate effective agents that can safely be used in the lt setting. like rsv, human metapneumovirus and parainfluenza are members of the paramyxovirus family and present similarly to rsv. although typically they are milder than rsv, they have been shown to cause severe disease and have also been associated with both acute rejection and bos. 67, 79, 80 real-time pcr is the diagnostic modality of choice, and a diagnosis should be pursued, as clinical features alone are not specific enough to distinguish between the carv. supportive care remains the mainstay of treatment although inhaled ribavirin appears to be increasingly used for the treatment of these pathogens in patients with lower respiratory tract involvement despite a lack of controlled trials. furthermore, some experts also consider the use of iv immunoglobulin with significant disease for both parainfluenza and human metapneumovirus. 72, 80 fungal infections are a common complication after lt with an estimated incidence of 15-35% and an overall mortality of 80%. 81 complications at the site of the anastomosis (i.e. stenosis or necrosis) create the ideal environment for these infections to thrive. other risk factors include the immunomodulatory effect of coexistent infections (i.e. viral) and neutropenia. [82] [83] [84] as previously mentioned, transmission of infection from donor to host after lt can occur, or the native lung may serve as a reservoir of fungal organisms during single lt. 85 this is particularly important in chronic obstructive pulmonary disease patients in whom the lung surfaces are irregular and may have colonized bullae. 84 pretransplant fungal colonization is common, especially in patients with cf and chronic obstructive pulmonary disease, and it has been associated with post-transplant fungal infection and bos, 86 although not all colonized patients develop active/invasive infection. 83 the most common fungal pathogens in ltr are candida and aspergillus species, while zygomycetes, scedosporium, fusarium, cryptococcus species, histoplasmosis and coccidiomycosis occur less commonly. in general, these infections are more prevalent during the first few months after transplantation and, in some cases such as with cryptococcus species, histoplasmosis or coccidiomyocosis, can present as a reac-tivation of a latent infection. fungal infections can manifest as invasive disease with a reported 1-year cumulative incidence of 8.6% in ltr. 87 similarly, disseminated disease, post-transplant empyema, and airway and anastomotic infection have been reported. aspergillus species are the most common cause of invasive fungal infection after lt with an incidence of 32%. 84 more than half the cases occur within the first six months following lt, 84 (fig. 1 ) and more often involve ltr than other solid organ recipients. 88 several species have been described as pathogenic: aspergillus terreus, aspergillus flavus, aspergillus fumigatus and aspergillus niger. among these species, aspergillus fumigatus remains the most common cause of invasive disease. 89 the majority of aspergillus isolates in sputum or bal represent colonization (23%), and only a fraction of these will develop invasive disease (<10%), which carries a high mortality. 69, 90, 91 in ltr, the risk of invasive pulmonary aspergillosis rises with airway colonization by aspergillus species. 84, 89, 92 colonization is found in up to 50% of patients with cf. despite higher colonization compared with other populations, these patients have lower risk of invasive aspergillosis, but a higher risk for aspergillus tracheobronchitis. 93 in addition to colonization, airway ischaemia and bos have also been implicated as risk factors for invasive aspergillosis. 84, 89, 92 disseminated disease has been reported with an incidence of 22%, occurring as reactivation from an occult focus and/or as a new post-transplant infection. 84 other less common manifestations, such as mediastinal masses, skin, softtissue, sinus, orbit, central nervous system, sternal wound and chest wall infections, have also been described. 89, 91 diagnosis there are limited data on the role of minimally invasive tests such galactomannan, pcr and 1,3-b-dglucan assay for the diagnosis of invasive aspergillosis in ltr. 94,95 1,3-b-d-glucan, a cell component of all fungi, has been used in the diagnosis of multiple invasive fungal infections, but unfortunately, the role in ltr has limitations. 96 diagnosis of invasive aspergillosis may require aggressive procedures (i.e. biopsy) to verify tissue involvement; however, this is not always possible, and often, the diagnosis is reached on evaluation of computed tomography chest findings and fungal staining/culture from bronchoscopy (i.e. bal). the radiological findings of invasive aspergillosis include consolidations, nodules, cavitary lesions and mass-like opacities, often with a 'halo sign'. 84 in cases where the diagnosis is not possible with a less invasive approach, a biopsy with fungal stain/culture and histopathology may be required. once the diagnosis of invasive pulmonary aspergillosis is made, computed tomography or magnetic resonance of the central nervous system is suggested to rule out disseminated disease. over the years, the use of antifungal prophylaxis has decreased the overall risk of aspergillosis. despite this, the risk of late infection after discontinuation of prophylaxis or even while using it is still present. 97 the treatment of pretransplant colonization has not been shown to reduce the incidence of post-transplant aspergillosis, but invasive disease in the pretransplant setting should be treated. 90 recent data has shown the superiority of voriconazole compared with amphotericin b deoxycholate in patients with invasive pulmonary aspergillosis, but solid organ transplant patients were poorly represented in the study. 98 a major concern with the use of voriconazole in ltr is the interaction with most of the immunosuppressants used in this population. tacrolimus, sirolimus and cyclosporine can potentially increase the serum concentrations of voriconazole. for this reason, close monitoring of drug levels is needed. other options for the treatment of invasive aspergillosis are posaconazole and itraconazole, but their roles as first-line agents are not well established. the echinocandins (caspofungin, micafungin and anidulafungin) have shown some in vitro activity against aspergillus species, but their utility as firstline antifungals for this infection has not been studied either. the evidence for combined therapy with two or more agents as initial therapy is limited and not recommended. despite several alternatives, voriconazole remains the standard therapy for invasive aspergillosis along with reduction of immunosuppression. 99 voriconazole levels should be monitored carefully, especially in cf patients where serum concentrations can be variable. 99, 100 in general, target trough levels should range between 1 and 5 mg/ml. duration is typically recommended for a minimum of 12 months and depends on clinical and radiographical improvement. finally, surgical resection might be indicated when there is progression of disease despite optimal antifungal therapy, life-threatening haemoptysis, sinus infection or lesions in the proximity of great vessels, pericardium or in the brain. 82 severe candidal infections can appear within weeks to months after transplant, especially in the presence of heavier donor or recipient colonization. 91 typically candida infections occur within the first 30 days after lt and appear to be the second most common cause of invasive fungal infection in ltr. 69 candidaemia usually occurs during the first 4 weeks and is often related to the intensive care unit stay and the surgical procedure; however, parenchymal lung infection is rare. 101 mortality for invasive candidal infections, excluding anastomotic infections, has been estimated at more than 50%. 102 cultures are essential for the diagnosis of candidal infection in ltr. identification of species and susceptibilities need to be obtained as intrinsic resistance and dose-dependent susceptibility has been reported in different candida species. 103 other methods such as b-d-glucan have not reached significant accuracy for clinical use, 104 while others such as pcr are still experimental. candida species are commonly found in the oropharynx and can potentially colonize the airway. their presence in respiratory secretions may make it difficult to differentiate between invasive infection and colonization. invasive lung infection with candida is very infrequent even in the lt recipient colonized with candida. 97 clinical suspicion, culture results and direct bronchoscopic findings should guide any decision for treatment of candidal infections. echinocandins and liposomal amphotericin b are the first-line agents for empirical therapy of suspected candidal infection. 69 this is especially true in ltr who are at risk of developing severe candidal disease. fluconazole has been put forward as an empirical agent as well but is frequently reserved for patients with mild-to-moderate disease, nonneutropenic and at low risk for candida glabrata and candida krusei, for which it has less activity. empirical therapy should then be adjusted based on susceptibilities. for candida albicans infections, fluconazole and echinocandins have been effective, but in widespread disease, amphotericin b might be considered. finally, the duration of therapy varies among patients and with the degree and severity of infection. in candidaemia, treatment can extend up to 2 weeks but may be even longer in cases of more invasive disease. 69 histoplasmosis, coccidioidomycosis and rarely, blastomycosis are endemic mycoses that can potentially cause infection in transplant recipients. when present in this population, pulmonary and disseminated disease can occur with a high mortality. 105 these are especially important in endemic areas of the united states such as the midwest for histoplasmosis and the southwest for coccidiomycosis. 106 histoplasmosis can present in the early or late posttransplant period as a consequence of reactivation of a latent infection, new exposure or donor-derived infection. 106 the diagnosis can be delayed, but in ltr, urinary antigen appears to be a better diagnostic tool than the fungal antibody serologies. 106 the presence of fever without a clear source should raise clinical suspicion for disseminated histoplasmosis in any transplant patient, especially when pancytopenia and absence of pulmonary manifestations are present. in patients whose explanted lung is found to have histoplasmosis, antifungal prophylaxis after transplant seems effective at preventing reactivation of this infection. 106 there is no clear consensus about the duration of prophylaxis, and 18 months has been reported to be effective. 106 coccidioidomycosis is typically acquired when patients are exposed to the desert soil of the southwestern united states and northern mexico. the most common mechanism of infection in lt recipients is reactivation, but donor-derived transmission has also been reported. 107 patients in whom there is evidence of prior coccidioidomycosis, either radiographically or serologically, may require lifelong antifungal prophylaxis after transplant. 91 cryptococcus infections can present in solid organ transplant recipients as a pulmonary or extrapulmonary process. 108 the incidence of cryptococcus infection in ltr has been estimated around 2% and has been commonly associated with exposure to pigeons and other birds. 90 interestingly, ltr may be less likely to have a positive cryptococcal antigen test in the setting of isolated pulmonary cryptococcosis. 38, 108 an immunosuppressive regimen containing a calcineurin inhibitor has been associated with decreased mortality possibly due to synergistic effects between calcineurin inhibitors and antifungal agents use to treat cryptococcus. 109 however, a recent study has reported the occurrence of an immune reconstitution syndrome-like illness in some transplant patients after the initiation of antifungal therapy for cryptococcal infection. 110 zygomycotic infections appear to be escalating in frequency in immunosuppressed patients, and this trend has been partially attributed to the increasing use of voriconazole for therapy and prophylaxis. 111 this infection is characterized by vascular invasion of affected tissues with subsequent infarction and necrosis. in ltr, it can manifest as bronchial anastomotic or parenchymal infection with a mortality of 87% in the latter. 112, 113 its management includes the combination of surgical debridement and antifungal agents. in the united states, 80% of transplant centres use antifungal prophylaxis, 114 and approximately 81% perform pretransplant surveillance for fungal colonization. 115 despite this, there is still no general consensus regarding the most appropriate prophylactic strategy in the peritransplant window. although there are no randomized trials evaluating their efficacy, several antifungal agents have been used for prophylaxis in ltr. for universal prophylaxis, voriconazole, itraconazole and amphotericin b are commonly used, while targeted prophylaxis with fluconazole (candida), voriconazole and itraconazole (aspergillus) are used based on the results of surveillance bronchoscopy. 114 in general, the choice for antifungal prophylaxis depends, in part, on the presence of specific risk factors such as colonization with aspergillus, presence of airway stents or ischaemia, single lung transplantation, cmv infection, hypogammaglobulinaemia or treatment of acute rejection. 69 despite a lack of controlled trials, several studies suggest potential prevention of invasive aspergillosis with the use of either compound of amphotericin b. 116, 117 inhaled amphotericin b has lower systemic toxicity, better delivery to the site of fungal exposure and a lower likelihood of resistance when compared with systemic antifungal therapy. 116, 118, 119 the data regarding voriconazole for prophylaxis in ltr is promising, especially given the excellent bioavailability, broad antifungal coverage and good drug levels achieved in lung tissue. 120, 121 unfortunately, the numerous drug interactions with some of the immunosuppressants, and its potential adverse effects may preclude its use as a first-line prophylactic agent. itraconazole has clinical effectiveness similar to the combination of voriconazole and inhaled amphotericin b and may have lower hepatotoxicity when compared with voriconazole. 114 duration of antifungal prophylaxis varies from centre to centre. the use of voriconazole or itraconazole for 3-6 months with or without amphotericin b has been shown to decrease the incidence of aspergillus infection after transplantation. 88 the use of inhaled amphotericin b is typically for 2 weeks or is discontinued at the moment of discharge. in cases where pretransplant fungal colonization is present, patients may be treated for several weeks before lt and continued for up to 3 months after transplantation. because ltr is at high risk for fungal infections, antifungal prophylaxis should be started in most patients after lt with careful consideration of sideeffects and interactions to improve outcomes and be guided by cultures from donor, graft and recipient. mycobacterial infection after lt is rare. previously, most of these infections were secondary to mycobacterium tuberculosis. 122 more recently, data have shown an increase in the incidence of ntm, particularly mycobacterium abscessus, ranging between 3% and 9%. 123, 124 chalermskulrat et al., reported higher isolation of ntm in end-stage cf patients undergoing pre-lt evaluation (19.7%) than in post-lt cf patients (13.7%). 124 colonization, especially when m. abscessus was isolated, was associated with an increased risk for invasive mycobacterial infection in cf patients. 124 over the last 10 years, multiple cases of m. abcessus in lt recipients have been reported with pleuropulmonary and disseminated disease. [125] [126] [127] in addition, there is an increase in both mortality and disseminated disease associated with m. abcessus in solid organ transplant recipients. 128 on the other hand, m. avium complex and other ntm infections are less common, and their impact on morbidity and mortality is less severe compared with m. abcessus. 129 if during the pretransplant evaluation, the clinical presentation and radiographical findings are suggestive of ntm infection, diagnostic testing and therapy should be considered before transplantation. in the cf population, the presence of ntm should not preclude lt, but careful monitoring for recurrence after transplant should be performed. 124 the diagnostic criteria of the american thoracic society and infectious disease society of america apply to pre-and post-ltr (symptoms, radiological findings and microbiology). 130 similarly, the antimicrobial therapy recommended in the ntm guidelines is applicable to ltr. 130 therapy for mycobacterial infection in the immunosuppressed patient can be problematic particularly due to drug interactions and increased toxicity. nevertheless, these infections can be controlled, and some patients achieve an appropriate response and cure. anastomotic tracheobronchitis is a unique form of pulmonary infection 131 that usually develops in the first 6 weeks to 3 months following lt. during the transplant procedure, the bronchial circulation is not reanastomosed, and thus, the bronchial anastomosis must receive collateral blood flow from the pulmonary circulation, is subject to ischaemia and may be susceptible to infection. this diagnosis is easily confirmed with bronchoscopic examination revealing purulence, ulcerations, pseudomembranes, necrotic material, dehiscence and sometimes narrowing at the site of the anastomoses, and histological and culture results. the organisms most commonly causing tracheobronchitis in this setting are bacteria-(pseudomonas, staphylococcus) and fungi aspergillus (an incidence of 32% and 20%, respectively) and candida. 84, 132, 133 treatment includes appropriate antibacterial and/or antifungal antimicrobials. the treatment of airway anastomotic infections with fungi is with a combination of both systemic and sometimes inhaled antifungal agents. 134, 135 for aspergillosis, the combination of voriconazole and nebulized amphotericin b along with reduction of immunosuppression has been advocated. 99, 134 duration of therapy for tracheobronchitis is usually determined by resolution under bronchoscopic surveillance. late sequelae may include stenosis and or stricture requiring intervention with balloon dilation or occasionally endobronchial stent placement. a study demonstrated a decrease in 5-year survival in single ltr who developed bronchial anastomosis fungal infections. 132 other types of bacterial infection described in ltr include those of the pleural space, blood stream and wounds, with organisms often isolated in the nosocomial setting, and clostridium difficile. pneumocystis jiroveci pneumonia (pjp) occurs exclusively in immunosuppressed states. the risk of infection is higher during the first 6 months after lt due to the degree of immunosuppression during this period. 136 cmv infection is also an independent risk factor for pjp. 137 despite this, pjp remains a rare complication after lt. 138 the low rate of infection is due to the use of prophylaxis with trimethoprimsulfamethoxazole as a first-line agent, and dapsone, pentamidine and atovaquone as alternatives. 139, 140 trimethoprim-sulfamethoxazole has been shown to have better tolerance, potentially treat a wider range of infections, and has fewer side-effects. 139 there is controversy regarding the duration of prophylaxis after transplant. a study revealed that the rate of pjp did not decline after 1 year of transplantation, suggesting that prophylaxis should be continued beyond this period. 141 ltr should receive at least 6 months of prophylaxis post-transplant, and if tolerated, adequately, it should be continued indefinitely. in those patients in whom prophylaxis has been discontinued, it should be resumed if the patient develops acute or chronic rejection requiring augmented immunosuppression. the standard therapy for pjp is trimethoprim-sulfamethoxazole in combination with corticosteroids. as previously noted, mmf is used frequently as part of the immunosuppression regimen after lt. interestingly, this medication has shown antimicrobial properties against several pathogens including pneumocysitis spp. 142, 143 in three comparative studies, none of a total of 1152 transplant patients who received mmf developed pjp compared with an infection rate of 1.8% in a similar group that did not receive mmf. [144] [145] [146] the mechanism for these effects remains unknown, but it is likely that mmf may benefit ltr by two different mechanisms. in lt, nocardia remains an important pathogen with a frequency of 0.6-2.1% and a directly attributable mortality of up to 30%. 147 it is important to note that some of these patients (60-100%) were on treatment with prophylactic trimethoprim-sulfamethoxazole, a medication to which nocardia is classically susceptible to, underscoring the resistance of some strains to prophylaxis therapy. 147 the treatment for nocardia is trimethoprim-sulfamethoxazole, but resistance has been documented and other alternatives have been used successfully: imipenem, amikacin, third generation cephalosporins, minocycline, moxifloxacin, linezolid and dapsone. 148 despite the relatively low frequency of nocardia in lt, because of the high risk of mortality and the ability to mimic other infections, clinicians must have awareness of this pathogen to improve an early diagnosis to initiate appropriate therapy. chronic rejection following lt is manifested pathologically by bronchiolitis obliterans and clinically by worsening obstructive dysfunction on pulmonary function, the bos. bos is the rate-limiting factor in long-term survival following lt, and up to 50% of ltr will develop bos. 5, 149 the aetiology remains unclear, although acute rejection is one of the identified risk factors. emerging evidence continues to point towards infectious aetiologies as important factors in the pathogenesis of bos. several different viral, bacterial and fungal pathogens have been implicated in this process. 150, 151 these findings are critical regarding the understanding the mechanisms of rejection and possible therapies to prevent it. cmv was the first pathogen linked to the development of bos. cmv pneumonitis is associated not only with bos but also with decreased survival despite treatment. 50 furthermore, there has been an absolute risk reduction in the development of bos with the use of cmv prophylaxis, supporting the evidence that this virus may play an important role in the pathogenesis of rejection. 1 carv infections, including rsv, human metapneumovirus and parainfluenza virus, were also identified as a significant risk factor for developing bos. 65, 67, 79, 80 bacterial colonization and infection may be a contributing risk factor to the development of bos. [152] [153] [154] [155] because macrolides are felt to slow the progression of bos, it has been postulated that this response is due to the potential treatment of a chronic infection with mycoplasma pneumoniae or chlamydia pneumoniae, 154, 156 although macrolide immunomodulation also plays an important role. it has been shown that a positive serology and pcr testing for chlamydia pneumoniae on bal samples increases the rate of bos and early mortality. 157, 158 supporting this theory further, a study recently demonstrated that macrolides can prevent the development of bos. 153 fungal pathogens have been also associated with the development of bos. 159 fungal pneumonitis and aspergillus colonization have been identified as independent risk factors for bos and mortality related to rejection. 151, 159, 160 moreover, the combination of lateonset aspergillosis and chronic allograft dysfunction was a risk factor for poorer survival. 132 despite several advances in surgical technique, immunosuppression and prophylaxis, infection continues to remain an important cause of death and disease in the ltr. although there are non-modifiable factors that are innate to the patient or to the nature of the procedure, there are several modifiable factors that can be recognized and changed so as to optimize the patient's chances for survival and further extend life. prompt recognition and treatment of these factors is paramount for appropriate management. prophylaxis strategies continue to evolve and show promise for several of the infectious agents. avoidance of these infectious complications may not only lead to a decrease in the direct consequences of infection but also to a reduction in the subsequent causes of ultimate graft failure including both acute and chronic rejection. antimicrobial resistance is a growing problem, and although newer antimicrobials will likely be of benefit, especially against viral and fungal pathogens, prevention of these diseases remains the best 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lung transplant recipients pulmonary nocardiosis: risk factors, clinical features, diagnosis and prognosis report of the ishlt working group on primary lung graft dysfunction part ii: definition. a consensus statement of the international society for heart and lung transplantation bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria effect of etiology and timing of respiratory tract infections on development of bronchiolitis obliterans syndrome pseudomonas aeruginosa colonization of the allograft after lung transplantation and the risk of bronchiolitis obliterans syndrome a randomised controlled trial of azithromycin to prevent chronic rejection after lung transplantation long-term azithromycin therapy for bronchiolitis obliterans syndrome: divide and conquer? impact of graft colonization with gram-negative bacteria after lung transplantation on the development of bronchiolitis obliterans syndrome in recipients with cystic fibrosis azithromycin therapy for patients with bronchiolitis obliterans syndrome after lung transplantation chlamydia pneumoniae infection after lung transplantation chlamydia pneumoniae serology in donors and recipients and the risk of bronchiolitis obliterans syndrome after lung transplantation aspergillus colonization of the lung allograft is a risk factor for bronchiolitis obliterans syndrome infections in lung allograft recipients: ganciclovir era key: cord-103436-y1br5hy8 authors: bourgoin, p.; soliveres, t.; barbaresi, a.; loundou, a.; arnoux, i.; bernot, d.; morange, p.-e.; michelet, p.; malergue, f.; markarian, t. title: cd64 and cd169 could help differentiate bacterial from viral infections in emergency department date: 2020-11-03 journal: nan doi: 10.1101/2020.10.28.20221259 sha: doc_id: 103436 cord_uid: y1br5hy8 background: the identification of a bacterial, viral or even non-infectious cause is essential in the management of febrile syndrome in the emergency department (ed) setting, especially in epidemic contexts such as flu or covid-19. objectives: the aim of this study was to assess discriminative performances of two biomarkers, cd64 on neutrophils (ncd64) and cd169 on monocytes (mcd169), using a new flow cytometry procedure, in patients presenting with fever to the ed. human leucocyte antigen-dr on monocytes (mhla-dr), hla-abc ratio (rhla-abc), and cd64 on monocytes (mcd64) were also assessed. methods: 85 adult patients presenting with potential infection were included during the 2019 flu season in the ed of la timone hospital. they were divided into four diagnostic outcomes according to their clinical records: no-infection, bacterial infection, viral infection and co-infection. results: mcd169 was elevated in patients suffering from flu a virus or respiratory syncytial virus, while ncd64 was mainly found elevated in subjects with streptococcus pneumoniae. in total, 38 (45%) patients were diagnosed with bacterial infections, 11 (13%) with viral infections and 29 (34%) with co-infections. ncd64 and mcd169 showed 90% and 80% sensitivity, and 78% and 91% specificity, respectively, for identifying patients with bacterial or viral infections. other biomarkers had lower discriminative performances. conclusions: ncd64 and mcd169 have potential for accurately distinguishing bacterial and acute viral infections. combined in an easy and rapid flow cytometry procedure, they constitute a potential improvement for infection management in the ed setting, and could even help for the triage of patients during emerging epidemics. agency for research and technology). this study was partially supported by beckman coulter through donations of the research reagents used in the study and participation of the two employees mentioned above. this private company had no role in the study design, or collection and interpretation of the clinical data. beckman coulter and the beckman coulter product and service marks mentioned herein are trademarks or registered trademarks of beckman coulter, inc. in the united states of america and other countries. all other trademarks are the property of their respective owners. request should be addressed to fabrice malergue (email: fmalergue@beckman.com; address: beckman coulter -immunotech, 130 avenue de lattre de tassigny, 13009 marseille, france; phone : 0033 638 290 051). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020. 10.28.20221259 doi: medrxiv preprint in the management of febrile syndrome, diagnostic guidance toward an infectious etiology is essential (1) . the characterization of a viral, bacterial or other infectious or non-infectious cause allows early appropriate patient management. nevertheless, the data in the literature report the complexity of triage and diagnostic guidance (2) . the integration of the clinical examination is essential, but unfortunately is often not enough (3) ; diagnostic elements are therefore needed as quickly as possible in the emergency department (ed), especially in epidemic contexts such as flu or covid-19 (4) (5) (6) . biological markers such as procalcitonin (pct) and c-reactive protein (crp) are commonly used in clinical practice. these markers have bacterial specificity but share a wide range of values with viral infections and do not make it possible to exclude or to confirm definitively the diagnosis (7-9). their triage capacity in ed is thus regularly challenged (10) . measuring multiple specific biomarkers simultaneously, with a simple technique and rapid time-to-results, would be better compatible with the needs of triage in emergency medicine (11) . we developed a rapid flow cytometry assay, able to measure leucocytes biomarkers expressions within 10 minutes (12) , and demonstrated promising results for the triage of patients with fever at the emergency department (13, 14) , with cd64 on neutrophils (ncd64), increased in case of bacterial infections (15) , and cd169 on monocytes (mcd169), increased in case of viral infections (16) . in these previous studies, one limitation was the low number of infected patients, especially those with viral diseases. in this new study, we thus included more patients, and focused on those with infectious symptoms during the flu season. the main goal was to confirm the relevance of cd64 and cd169 for discriminating between bacterial and viral infections in such an epidemic context. the study population included patients older than 18 years, if they presented to the adult ed of la timone university hospital in marseille, france, with infection signs, during 2019 flu season. the inclusion criteria were the presence of fever greater than 38°c or hypothermia less than 36.5°c, and any potential respiratory (cough, sputum, and dyspnea), urinary (potential urinary infection), abdominal (pain syndrome, diarrhea), cutaneous (erysipelas), or neurological (meningitis) infectious clinical signs. the exclusion criteria were: incomplete clinical files, traumatized patients or patients presenting with a known inflammatory or autoimmune disease, neoplasia, chronic infectious disease (viral, fungal, or bacterial), or antibiotic, antiviral, or immunosuppressive treatment prior to admission, and patients with extensive burns or recent surgery (less than 1 month). their routine care was not modified, and confidentiality was preserved at all levels. all enrolled patients provided informed consent and no objection authorization, so that their data could be retrieved from their clinical records by a team of emergency department specialists, and could be used in the study. this observational and noninterventional prospective study was approved by the la timone hospital ethical committee and the committee for protection of persons (cpp approval no. 181160; id-rcb approval no. 2018 a02706-49). procedures followed were in accordance with the helsinki declaration. electronic medical records were retrieved for each patient by a team of ed specialists: all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint -epidemiological data: sex, age, clinical history (evolutionary cancer, liver disease, congestive heart failure, cerebrovascular disease), medical institutionalization, altered mental status; -physiological data: cardiac parameters (systolic, diastolic and average blood pressures, pulse and respiratory rates), body temperature, fever duration, vital signs and symptoms (respiratory, abdominal, neurological, urinary, cutaneous or others), and eventual oxygen-, antibiotic-or antiviral-therapy; -clinical data: time from onset, symptoms, x-ray examination results (performed and atypical chest x-ray or ultrasound or ct scan), final diagnosis established by the ed practitioner, outcome of the ed visit (released home, conventional or critical care hospitalization), and eventually duration of the hospitalization; -and biological data: white blood cell (wbc) and polymorphonuclear neutrophil (pmn) counts, crp and pct levels, biochemical measurements (urea, sodium, glucose, hematocrit, hemoglobin), and name of the identified pathogens if isolated. wbc and pmn counts were assessed using a sysmex xn system (sysmex inc., kobe, japan). pct was measured using a dosage advia centaur brahms procalcitonin system (siemens, munich, germany) and crp using gen all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint based on all clinical and biological data, an adjudication committee classified patients in 4 groups. group iii: subjects diagnosed as having viral infections. this group contained subjects that presented with typical clinical symptoms of infections, but negative bacteriological results and/or low pct levels. in some cases, viral agents were found by antigen-based tests or serological assays. group iv: subjects diagnosed as having both viral and bacterial infections. the committee was not aware of the flow cytometry results. leftover ethylenediaminetetraacetic acid (edta)-treated blood samples were pseudonymized, and processed by flow cytometry according to a newly described one-step procedure (12) . briefly, a multicolor panel was constituted and dried as a "glassified" layer at the bottom of a 5-ml testing tube using the dura innovations drying process (beckman coulter inc.), with antibodies at their optimized amounts for a single test: anti-cd169-phycoerythrin (pe) (clone 7-239), anti-cd64-pacificblue (pbe) (clone 22), anti-hla-dr-allophycocyanin (apc) (clone immu357) and anti-all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. for each blood sample tested, 500 μl of versalyse lysing solution (beckman coulter inc.) and 5 μl of edta-treated blood were transferred to one dried tube. after incubation for 15 minutes, samples were analyzed on a three-laser, 10-color navios flow cytometer (beckman coulter inc.). flow-set beads (beckman coulter) were used before each analytical run in order to control the variability in device performance, however no harmonization between the measured values over the study period was necessary. analysis was performed using kaluza analysis software (version 2.1; beckman coulter inc.). leucocytes were gated using side scatter (ssc) and cd64 expressions, as lymphocytes (low ssc, cd64-), monocytes (intermediate ssc, cd64+) and neutrophils (high ssc), prior to the analysis of ncd64, mcd169, mhla-dr, rhla-abc and mcd64. results were expressed as mean of fluorescence intensities (mfi). statistical analysis was performed using ibm spss statistics version 20 (ibm spss inc., chicago, il, usa). quantitative data were expressed as mean ±standard deviation. qualitative variables were expressed as frequency with percent. comparisons of quantitative variables among the different groups were performed using student's t-test or mann-whitney u test. comparisons of percentage were performed using khi-2 or fisher's exact tests if conditions were missing. comparisons of more than two groups were performed by freeman-halton extension of fisher's exact test for qualitative variables and by analysis of variance or kruskall-wallis tests for quantitative variables. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. the ability of biomarker levels to discriminate between bacterial and viral infections was investigated by means of receiver operating characteristic (roc) curve analysis. roc curves were used to define the best threshold of biomarker indexes. analyses were based on area under the curve (auc), sensitivity (true positives / positives [tp / p]), specificity (true negatives / negatives [tn/n]), positive likelihood ratio (sensitivity / [100 -specificity]) and negative likelihood ratio ([100 -sensitivity] / specificity]). all values were expressed as ranges (between 0 and 100), with 95% confidence intervals. for all tests, two-sided p values less than 0.05 were considered statistically significant. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint 3. results. during the study period, 104 patients admitted to the ed of la timone hospital were included. all blood samples were processed by flow cytometry, but only 85 out of the 104 subjects satisfied the inclusion criteria. nineteen patients for whom clinical files were incomplete, and in particular for whom there was doubt about the presence of underlying or asymptomatic infections that were not sufficiently documented, were removed from the study, because it could have biased their final diagnosis and classification. the adjudication committee classified the 85 remaining patients: 7 (8%) were defined as not-infected, 38 (45%) as bacterially infected, 11 (13%) as virally infected and 29 (34%) as presenting with both a bacterial and a viral infection (co-infection). an overview of the study workflow is shown in figure 1 . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. the final cohort consisted of 85 patients, including 30 (35%) women and 55 (65%) men, with a mean age of 56 (± 25) years. their epidemiological, clinical, and biological data are presented in table 1 . bacterial infections were considered as patients presenting with bacterial infections only plus bacterial co-infections, whereas viral infections were considered as patients presenting with viral infections only plus viral co-infections. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. a wide range of infectious symptoms was observed among groups (respiratory, abdominal, neurological, urinary, cutaneous, and other). the most common clinical presentation associated with bacterial (n=42; 63%) and viral (n=34; 85%) infections was respiratory. as expected, based on symptoms, patients diagnosed as having bacterial infections were treated more frequently with antibiotics (n=59; 92%; p: 0.01), and x-ray examinations were performed more frequently (n=67; 100%; p: 0.02). after their ed medical consultation, 32 patients (38%) could have been released home, but 53 patients (62%) were admitted to hospital specialized departments or the critical care department for longer observation. however, all patients that were kept at the hospital for observation remained for a non-significantly different duration (p: 0.86). overall, 94 common pathogen species were detected ( table 2 ). the most frequent pathogens were streptococcus pneumoniae (38%) and flu a virus (26%). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. figure 2 . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint roc analysis of the five biomarkers was made for evaluating their performance to identify the bacterial or viral etiology of an infection (figure 3) . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint using a cutoff point of greater than or equal to 1.24 for patients with bacterial infections, the results indicated that the ncd64 mfi showed the best bacterial discriminative performance, with a sensitivity of 90% (80-96%), specificity of 78% (52-94%), positive likelihood ratio of 4.03 roc analysis of the three other biomarkers was more equivocal. to detect bacterial infection, using a cut-off-point greater than or equal to 8.72, mcd64 mfi exhibited good sensitivity of 90% (80-96%) but lower specificity of 33% (13-59%). conversely, using a cut-off point of less than 63.0, mhla-dr mfi exhibited good specificity of 94% (73-100%) but low sensitivity of 42% (30-55%). to detect viral infections, using a cut-off-point greater than or equal to 60.46, mhla-dr mfi exhibited good sensitivity of 90% (76-97%) and low specificity of 49% (34-64%), whereas, using a cut-off-point greater than or equal to 3.92, rhla-abc exhibited low sensitivity of 68% (51-81%) but good specificity of 89% (76-96%). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint this study aimed to investigate the value of ncd64 and mcd169 biomarkers in a population of patients with febrile symptoms in a clinical context of the ed, using a new procedure of flow cytometry. assessment of their levels, in non-infected, bacterially-infected, virally-infected or coinfected subjects, demonstrated, within this study, their relevance for identifying etiology of infections. in this new study, a total of 85 subjects were included, and divided into four diagnostic outcomes: not-, bacterially-, virally-or co-infected subjects. among these groups, each outcome was always represented by more than 10 subjects. moreover, almost all cases were confirmed by a bacterial or viral isolate in biological samples (22 different pathogens found among 94 isolates). as patients were included during flu season, the most frequent pathogens were respiratory: streptococcus pneumoniae, flu a virus and human respiratory syncytial virus. these pathogens are commonly expected during this period, as has been reported (5). cd64 on neutrophils and cd169 on monocytes were the two main biomarkers assessed in this study by flow cytometry for discriminating between bacterial versus viral infections. expressions of these two biomarkers have been shown to be directly induced, within hours, by interferons produced by the body in response to pathogen detection (14) . ability of ncd64 to discriminate between bacterial and non-bacterial infections has been largely demonstrated for years (17) , whereas mcd169 increase after infection by viruses has only been described recently. mcd169 seems to be a general biomarker of acute viral infections since it has been found in patients with hiv (16, 18, 19) , ebv (20) , rsv (21) , cmv (22) , dengue (23, 24) , zika (25), noroviruses (26) , lassa and marburg (27) . here, high levels of mcd169 have been observed for the first time for flu a virus. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020. 10.28.20221259 doi: medrxiv preprint high levels of sensitivity and specificity were found in the study for both biomarkers. interestingly, ncd64 showed a better sensitivity of 90% than specificity of 78%, whereas mcd169 showed a better specificity of 91% than sensitivity of 80%. these results further demonstrate their valuable use for infection etiology guidance in ed settings for patient triage. indeed, a biomarker used for bacterial infection identification in ed needs to be as sensitive as possible to detect the majority of cases, demonstrating at least 90% sensitivity, as any missing case could delay patient from receiving appropriate antibiotic therapy, and thus increase their risk of developing sepsis and progression to death. conversely, a viral marker in ed has to be very specific to ensure the etiology, with at least 90% specificity, as it allows the practitioner to discharge the patient, as well as avoiding the empirical use of antimicrobial drugs in cases where they are not required (28) . the global health issue of overuse of antibiotics has been illustrated in this study: the epidemiological data showed that an antibiotic therapy was initiated in 67 out of 85 patients. examining the whole cohort, 36 out of 38 subjects with bacterial infections, and 23 out of 29 coinfected with bacteria and viruses, appropriately received antibiotics. conversely, 4 out of the 11 subjects virally infected only, and 4 out 7 not-infected subjects, received antibiotics, cases where antibiotics are ineffective, or even worse, might be dangerous (29) . as a consequence of their usefulness for determining etiology, both biomarkers could be incorporated as part of the overall clinical management of patients with fever, and used for evaluation of antibiotic therapy initiation, duration and end (30) . three other secondary biomarkers were evaluated in the study: hla-dr on monocytes, hla-abc ratio of monocytes/neutrophils and cd64 on monocytes. the first two biomarkers have been identified among 13 other leucocyte biomarkers as being the most promising for determining infectious etiology (13) , and the third has been considered in some studies for comparison to all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint ncd64. firstly, hla-dr is widely known to be decreased in sepsis subjects (31) , but also, as it is the major histocompatibility class ii, it has also been seen to increase in cases of viral infection (13) . secondly, rhla-abc, although less frequently evaluated, normally increases in cases of viral infection (32) . finally, mcd64 is known to evolve similarly as ncd64, by increasing in cases of bacterial infection, although demonstrating lower performance (33) . these three biomarkers were evaluated in this study to clarify and confirm their promising use for infectious etiology determination. unfortunately, they did not show expected performances: either their sensitivity or specificity was not sufficient enough to ensure effective identification of bacterial or viral infections. finally, one major issue remaining is how to measure the biomarkers in ed clinical practice. the ed environment is complex and dynamic, and thus requires technologies tailored specifically for prevention, diagnosis, and outcome of infection, to enhance patient safety in emergency care. a new solution was demonstrated by assessing the levels of expression of the markers using an innovative, 15-minute, one-step method of flow cytometry (12) . this procedure may meet the minimum characteristics required for a bacterial versus viral bedside test, as targeted by dittrich team (11): 1) the use of a capillary blood drop could be potentially used as only 5 µl are necessary, although edta blood samples were used here; 2) reagent storage and laboratory procedures are carried out at room temperature; and 3) all steps for testing the samples are combined into one step, with no specific material or training needed. in summary, this assay yields results in less than 15 minutes from initial blood collection, with discriminative power greater than other currently existing tests. the procedure used here however, for the moment has limitations regarding the instrument: on one hand, mfi measurements would necessitate standardization all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint efforts to avoid variations due to instrument setup, and on the other hand, flow cytometer sample preparation and analysis would need automation. the study has other limitations. first, patients were enrolled from the ed during approximately 5 months, and only within one ed from one hospital. even if a good representation of the most common infections was achieved, it is not sufficient to provide a complete validation of biomarkers. in future studies, it might be preferred to extend the study to other ed from other hospitals. secondly, biomarkers seemed to be useful for infected patients, but their kinetics are not well known. it is important to understand their delay of onset after infection and their sequential evolution in blood circulation. future research should thus focus on measuring these biomarkers on sequential samples from same subjects, and evaluating their prognostic value, both for bacterial versus viral infection diagnosis and for therapy duration. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint in summary, cd64 and cd169 were confirmed to be biomarkers of interest to predict bacterial versus viral infection causes of fever, whereas hla-dr and hla-abc demonstrated lower performances in these settings. flow cytometry is currently the universally applied method for identifying cell surface markers, but in this context, its availability remains limited in emergency settings. as part of a global effort to reduce inappropriate antibiotic use, this study makes available the measurement of infection-related biomarkers using a new flow cytometry procedure, promisingly applicable at the point-of-care. this association of infection related biomarkers and flow cytometry is promising to facilitate an easy, rapid and robust discrimination of bacterial versus viral infections and thus the successful care of patients with potential infection presenting to the emergency department. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted november 3, 2020. ; https://doi.org/10.1101/2020.10.28.20221259 doi: medrxiv preprint accuracy of the cerebrospinal fluid results to differentiate bacterial from non bacterial meningitis, in case of negative gram-stained smear for the triage study group. predictors for delayed emergency department care in medical patients with acute infections -an international prospective observational study serum procalcitonin measurement as diagnostic and prognostic marker in febrile adult patients presenting to the emergency department acute respiratory failure in the elderly: etiology, emergency diagnosis and prognosis can procalcitonin help identify associated bacterial infection in patients with severe influenza pneumonia? a multicentre study challenges in laboratory diagnosis of the novel coronavirus sars-cov-2 accuracy of procalcitonin for sepsis diagnosis in critically ill patients: systematic review and meta-analysis procalcitonin as a diagnostic test for sepsis in critically ill adults and after surgery or trauma: a systematic review and meta-analysis diagnostic accuracy of c-reactive protein and procalcitonin in suspected community-acquired pneumonia adults visiting emergency department and having a systematic thoracic ct scan target product profile for a diagnostic assay to differentiate between bacterial and non-bacterial infections and reduce antimicrobial overuse in resource-limited settings: an expert consensus yansouni c, editor a novel one-step extracellular staining for flow cytometry: proof-of-concept on sepsis-related biomarkers flow cytometry evaluation of infection-related biomarkers in febrile subjects in the emergency department role of the interferons in cd64 and cd169 expressions in whole blood: relevance in the balance between viral-or bacterialoriented immune responses neutrophil cd64, c-reactive protein, and procalcitonin in the identification of sepsis in the icu -post-test probabilities sialoadhesin (cd169) expression in cd14+ cells is upregulated early after hiv-1 infection and increases during disease progression sommer p, editor neutrophil cd64 index as a superior biomarker for early diagnosis of infection in febrile patients in the hematology department no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity sialoadhesin expressed on ifn-induced monocytes binds hiv-1 and enhances infectivity epstein-barr virus lytic infection promotes activation of toll-like receptor 8 innate immune response in systemic sclerosis monocytes siglec-1 inhibits rsv-induced interferon gamma production by adult t cells in contrast to newborn t cells signatures of t and b cell development, functional responses and pd upregulation after hcmv latent infections and reactivations in nod.rag.gamma mice humanized with cord blood cd34+ cells monocyte-plasmablast crosstalk during dengue dengue virus infection induces expansion of a cd14+cd16+ monocyte population that stimulates plasmablast differentiation zika virus infection of rhesus macaques leads to viral persistence in multiple tissues capturing the systemic immune signature of a norovirus infection: an n-of-1 case study within a clinical trial lassa and marburg viruses elicit distinct host transcriptional responses early after infection infection prevention in the emergency department the antibiotic resistance crisis examining the utility of the cd64 index compared with other conventional indices for early diagnosis of neonatal infection how clinical flow cytometry rebooted sepsis immunology a rapid flow cytometric method for distinguishing between febrile bacterial and viral infections neutrophil and monocyte cd64 and cd163 expression in critically ill neonates and children with sepsis: comparison of fluorescence intensities and calculated indexes the authors would like to thank the technician team from the la timone university hospital hematology laboratory for their assistance in blood testing, the anrt for funding the phd grant, and beckman coulter for giving reagents used in the study. all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. key: cord-261867-6n0g3bz5 authors: evermann, james f.; ledbetter, eric c.; maes, roger k. title: canine reproductive, respiratory, and ocular diseases due to canine herpesvirus date: 2011-10-28 journal: vet clin north am small anim pract doi: 10.1016/j.cvsm.2011.08.007 sha: doc_id: 261867 cord_uid: 6n0g3bz5 this review documents how clinical inquiry expands as our knowledge base about canine herpesvirus (chv) increases. we must understand the various forms of chv infection that may occur in the dog population. this has prompted the veterinary community to develop more sensitive diagnostic assays. chv is more common than we considered a decade ago. up to 70% of some high-risk dog populations have been infected with and are latent carriers of chv. recognition of the various forms of chv-induced disease, availability of diagnostic assays with increased sensitivity, and the formation of reliable biosecurity measures will allow for better control steps. immunosuppressive regimens of therapy for various dermatologic conditions, as well as dogs being treated for various cancers. this review will provide a brief overview of the reproductive aspects of chv disease and will then bring together the current literature, documenting the involvement of chv in adult dog respiratory and ocular diseases. consistent with the other alpha herpesviruses, chv has a predilection for pregnant dogs and neonatal puppies. [23] [24] [25] [26] early reports focused on the effects of chv on various reproductive parameters in the dog, in part due to the severity of the clinical symptoms and the profound pathologic effects. in the review by anvik, 12 acute neonatal viremia and systemic infection of naïve pregnant females were regarded as 2 of the most important disease outcomes of chv infection. the emphasis at that time was on the recognition of clinical symptoms for a rapid diagnosis. since there are no commercial vaccines currently available for the prevention of chv-induced disease, it has become paramount to understand the clinical features of chv infections (see table 1 ) and to incorporate this knowledge with sound management practices to minimize the effects on reproductive efficiency and puppy survival. 27 as was mentioned previously, this has been the primary focus of the earlier literature on chv infections. infection may occur during pregnancy or may be acquired by puppies during the first few weeks of life. the key feature during both of these phases is that the pregnant female and puppies are immunologically naïve to chv and therefore highly susceptible to disease. puppies may acquire the infection in utero, from passage through the birth canal, from contact with oronasal secretions of the dam, or contact shedders. humans may serve as fomites of the virus if attending to an adult carrier-shedder dog, and then proceeding to a nursery setting without proper disinfection. naïve neonatal puppies, younger than 1 week, are at highest risk of fatal systemic disease, while naïve dogs older than 3 weeks are relatively resistant to disease but can still become infected. [27] [28] [29] virus infection in naïve older dogs is generally acquired via aerosol, so that replication occurs in the nasopharynx tonsils and retropharyngeal and bronchial lymph nodes. 2,30 -32 this respiratory site will become an important aspect of the ecology of the virus when both respiratory and ocular clinical outcomes are covered in subsequent sections. although neonatal infections are regarded as the most common, in utero infection with chv may occur. infertility and abortion of stillborn or of weak pups has been reported. while the mortality rate usually approaches 100% for the fetal puppy, there may be no further clinical manifestations reported in the dam. 1, 5, 26 passive immunity acquired from the dam appears to be of primary biological importance in the survival of infected pups. 12, 27, [33] [34] [35] puppies that are nursing from chv-seronegative dams usually develop the fatal multisystemic disease, while puppies that suckle from chv-seropositive dams remain asymptomatic but still become infected. the chv is usually recovered from the oropharyngeal region in these disease-resistant pups. it is generally accepted that maternal antibody and/or immune lymphocytes acquired through the milk explain why naturally infected dams that have a diseased litter will usually give birth to normal litters on subsequent pregnancies. since chv is one of the few canine viral infections that can proceed to fatal disease and there is no commercial vaccine routinely available, it has become necessary for infection management to prevent reproductive disease. the literature has focused on 3 aspects of the virus and its relationship with host immunity and its carrier-spread dynamics within a population of susceptible dogs. the risk factors associated with chv infection and reproductive disease has been intensively studied over the past 5 years. 36, 37 the studies have used various diagnostic assays including serology, virus isolation, and polymerase chain reaction (pcr). these studies have provided valuable information on controlling chv-associated reproductive diseases (ie, infertility, abortion, stillbirths, and neonatal mortality). table 2 lists the 12 risk factors that were studied and whether there was an association with reproductive disease. of the 12 factors, 8 were identified as having a positive correlation with disease: breeding kennel, age, mating experience, cycle (stage), concurrent kennel cough, kennel size, breeding management, and hygiene. the underlying risks in the aforementioned factors are chv infection and an immune susceptible dog. this has led to strategies to naturally immunize (via contact with adult dogs) susceptible female dogs prebreeding, to screen female dogs for chv infection (by serology and/or pcr) prior to breeding, and to use a defined quarantine period for pregnant dogs with an unknown chv infection status. an age-risk, immunologically naïve-risk strategy has been used by clinicians and clients to focus on the most susceptible time periods for disease. this time encompasses the pregnant female during the last 3 weeks prior to whelping, and her puppies up to 3 weeks post whelping. 12, 27, 33 this understanding has constituted the rationale for the "6-week danger period." 12, 27 the primary contributing risk factors that allow for chv infection and disease are kennel size, hygiene, and kennel cough. all 3 of these are important in the spread and retention of chv in high-risk dog populations. while the controversy over chv being a significant contributor to the kennel cough syndrome has been an ongoing debate (see subsequent section on respiratory-ocular infections), it should be noted that chv was initially reported as a respiratory pathogen as early as it was a reproductive pathogen. 2 the data from ronsse and coworkers 22 support the contention that chv is primarily maintained and spread among dogs in a multidog environment as a respiratory infection. the disease outcomes of chv infections are age dependent. in naive puppies that are less than 1 month of age, natural and experimental infection with chv may be highly fatal. natural exposure of pups occurs by ingestion or inhalation of virus containing material. the primary replication sites are nasal mucosa, pharynx, and tonsil. systemic spread of the virus is enhanced by a cell-associated viremia. 29 -32 the pathology induced by chv in the lungs of newborn pups is depicted in figs. 1 and 2. experimental infection of older dogs (3 months or older) with chv has resulted in a mild rhinitis and pharyngitis. symptoms of tracheobronchitis were produced following experimental inoculation with chv isolated from naturally infected dogs. 38, 39 experimental infection of 5-to 12-week-old pups induced mild rhinitis and pharyngitis and virus replication was demonstrated in the upper respiratory tract. although chv has been isolated from dogs with upper respiratory disease, reproduction of "kennel cough" has only been rarely reported. thompson and coworkers 32 reported that aerosol exposure of 12-week-old dogs caused a necrotizing rhinitis, broncheointerstitial pneumonia, and multifocal alveolar necrosis. more severe disease can occur when chv infects dogs that are immunosuppressed. 9 a case of generalized chv infection in a 9-year-old dog with a normal immune system was documented recently (gadsden bj, langohr im, maes r. fatal herpesviral infection in an adult dog. submitted for publication, 2011). the most severe lesions were seen in the liver. the histologic lesions observed in the lung of this dog are presented in fig. 3 . infection rates, based on serologic studies, are high enough to explain entry of chv into multidog environments, either as an active infection or as the result of reactivation of latent virus in environments associated with natural, or pharmacologically induced immunosuppression. in belgium the seroprevalence in adult dogs was found to be 45.8%. 22 rijsewijk and colleagues 21 reported a seroprevalence of 39% in the netherlands. reading and field, 20 using an antibody detection elisa, found a seroprevalence of 88% in the united kingdom. in japan, the seroprevalence was recently reported to be 21.7%. 6 since chv is regarded as a weak immunogen, these antibody-based surveys are probably an underrepresentation of the true infection rate in the dog populations. 33 canine infectious respiratory disease (cird) is most commonly seen in rescue centers, boarding kennels, and veterinary hospitals. most of the affected dogs have a dry cough of limited duration. in complicated cases, bronchopneumonia is seen and can be fatal. multiple infectious agents can play a role in the induction of cird. canine parainfluenza virus and bordetella bronchiseptica are frequently involved. canine distemper and canine adenovirus type 2 (cav-2) have been associated with cird but are not routinely detected due in part to effective vaccines, and the population immunity is fairly high. canine influenza, canine respiratory coronavirus, and, most recently, canine pneumovirus, are emerging components of cird, which have added to the complexity of this disease syndrome. 18, 19, 40 although chv infections have been documented in multidog environments, its etiologic role in cird is still being assessed. during a 2-year longitudinal study of viruses associated with cird at a rescue center in the united kingdom, chv was found in 12.8% of the tracheal samples examined and in 9.6% of the lung samples. infections with chv were seen 3 to 4 weeks after entry and were associated with more severe respiratory signs. 18 the delay in detection of the virus by pcr was corroborated by the serologic data, which also indicated that chv infections occurred at a later time point. a possible explanation offered for its detection in more severe cases was the possibility that latent chv could have been reactivated as a result of the stress induced by a primary cird episode that was triggered by other viral or bacterial agents. the virus source was not determined. the authors speculated that genetically different chv strains would have been detected if the source of virus was the result of reactivation of latent virus from different dogs. it has been reported, however, that chv strains show very low sequence variability. 41 erles and brownlie 19 monitored dogs in 2 training centers in the united kingdom for 1 year. all dogs were vaccinated against cav-2, cpv-2, and leptospira interrogans. tonsillar swabs and serum samples were collected at entry and every 3 months thereafter. blood samples were collected at entry and every 4 weeks thereafter. most cird cases were observed in autumn and winter. most dogs were healthy at arrival and were in the kennel for at least 2 weeks before developing clinical signs. seroconversion to chv was detected throughout the year. the most logical explanation for the seroconversion pattern would be continuous introduction in the kennel by acutely infected dogs or reactivation of latent virus in the resident population. the authors concluded that while chv contributed to the cird, it was not an obligate pathogen in that environment, since some asymptomatic dogs also seroconverted. kawakami and colleagues 6 described an outbreak of infectious tracheobronchitis in japan accompanied by death in adult dogs. the only pathogen identified during the outbreak was chv. molecular testing led to the conclusion that a single strain was involved, with virulence characteristics that were only slightly higher than those of previously tested chv strains. as was the case in the study reported by erles and colleagues, 18 it was not clear whether the virus was introduced into the center in the form of acute infections or was the result of reactivation of latent infections in the resident population. regardless, the authors emphasized that there was sufficient amounts of immunosuppression in shelter populations to allow for chv to be a significant primary pathogen in that environment. ocular manifestations of chv infection may develop during both primary and recurrent infection and are dependent upon host age and immune status. in fetal and neonatal dogs with primary chv infection, severe intraocular lesions are frequently present concurrent with systemic viral disease. subclinical or mild recurrent ocular surface disease is typically observed in immunocompetent mature dogs. in immunosuppressed mature dogs, ocular lesions associated with chv infected are often more severe, persist for a longer duration, and may be refractory to treatment. primary chv infection occurring after in utero or early neonatal chv transmission (ie, first 2 to 3 weeks of life) is associated with a cell-associated viremia. hematogeneous dissemination of virus results in chv infection of intraocular tissues with severe clinical ocular manifestations. ocular disease is typically bilateral and becomes evident within a short period after the development of systemic disease in many, but not all, dogs. 1,3 panuveitis, retinitis, and optic neuritis with extensive monocular and neutrophilic infiltrates, edema, hemorrhage, and necrosis are observed histopathologically within the iris, ciliary body, choroid, retina, and optic nerve. 42 intranuclear viral inclusions are frequently detected during the acute inflammatory phase in uveal and retinal tissues. as the palpebral fissures do not open until 10 to 14 days postpartum in dogs, ocular changes may not be externally visible in young animals. in dogs with open eyelids, most clinically detectable ocular lesions are sequelae to panuveitis and include keratitis, corneal edema, aqueous flare, anterior synechiae, cataracts, and chorioretinitis ( fig. 4) . 42 reduced vision or blindness may result from various combinations of the ocular lesions. following the acute inflammatory stage of infection, developmentally mature tissues (eg, cornea, uvea) undergo varying degrees of necrosis, fibrosis, gliosis, and atrophy. 42 the canine retina is incompletely developed at birth and responds by a combination of necrosis, disorganization, and reorganization. retinal dysplasia, characterized by formation of retinal folds with rosette-like structures, and retinal degeneration are the final result. in dogs surviving neonatal chv infection, blindness, cataracts, optic nerve atrophy, retinal degeneration, and retinal dysplasia are frequent residual sequelae. 43 in contrast to fetal and neonatal dogs, ocular lesions associated with chv infection in mature dogs are typically restricted to the ocular surface with a variety of corneal, conjunctival, and eyelid lesions. 44 in immunocompetent dogs these lesions are frequently mild and self-limiting; however, they are a source of discomfort and their recurrent nature may be frustrating to clients. nonspecific clinical signs associated with chv ocular infection in mature dogs include blepharospasm, photophobia, and ocular discharge. blepharospasm and ocular pain are often disproportionally severe compared to that expected from the extent of ocular lesions. ocular discharge is initially restricted to epiphora, but becomes mucoid, mucopurulent, or serosanguineous with progression of infection. 7, 44 primary and recurrent ocular chv infection may be subclinical or associated with various combinations of blepharitis, conjunctivitis, keratitis, and corneal ulceration. 7,44 -46 in all published descriptions of naturally-acquired primary ocular chv infection, clinical lesions were bilateral; however, the severity and specific manifestations of chv infection were not always symmetrical between eyes of individual dogs. in most cases, primary ocular chv infection resolves spontaneously and without permanent ocular lesions; however, recovered dogs are at risk for developing recrudescent ocular disease associated with reactivation of latent chv. recrudescent chv ocular disease may present with either unilateral or bilateral lesions. recurrent chv ocular infection may occur in dogs with no identifiable risk factors; however, an immunocompromise state is present in most dogs. 7,44 naturally acquired recurrent chv ocular infection is reported in dogs with a variety of immunomodulating systemic conditions and receiving a variety of immunosuppressive therapeutics. systemic conditions included diabetes mellitus, immune-mediated thrombocytopenia, and lymphoma. immunosuppressive therapeutics included topical ocular corticosteroids, topical ocular cyclosporine, systemic corticosteroids, and a variety of antineoplastic chemotherapeutics (eg, cyclophosphamide, doxorubicin, vincristine). in many reported dogs, potentially immunosuppressive conditions were concurrently present with the administration of multiple topical and systemic immunosuppressive medications. blepharitis is occasionally present with ocular chv and may appear as focal or generalized eyelid erythema, edema, exudates, and crusting. regions of alopecia may be present. the blepharitis may represent self-trauma resulting from discomfort associated with conjunctival or corneal disease, or active viral infection of eyelid cutaneous epithelium as described for other dermal regions in dogs with chv infection. 46 conjunctivitis is the most frequently reported ocular lesion associated with both primary and recurrent chv infection 44, 47 and can be presented with conjunctival hyperemia, chemosis, and ocular discharge. ulceration of the conjunctival epithelium may occur and appears as flat, irregular, pale or pink regions on the conjunctival surface surrounded by regions of hyperemia. conjunctival ulcerations are readily detected with application of sodium fluorescein, rose bengal, or lissamine green stains. although the clinical features of chv conjunctivitis are often indistinguishable from other etiologies, conjunctival petechiae are frequently reported in dogs with chv infection (fig. 5) . 9, 44, 47 although not specific to chv infection, this clinical finding is uncommon with most other etiologies of conjunctivitis and should be considered suggestive of chv. ulcerative keratitis and nonulcerative keratitis are frequent lesions associated with primary and recurrent ocular chv infection. 7, 8, 47 a variety of clinical manifestations are observed in the cornea associated with chv infection and these likely represent a continuum along the progression of active corneal epithelial infection. punctate keratitis is the earliest detectable chv corneal ulceration and appears clinically as a fine stippling of epithelial loss. this subtle lesion is often clinically overlooked when examination is performed without the aid of magnification, but application of corneal vital stains (particularly rose bengal or lissamine green) facilitate detection. 47 as punctate ulcerations progress, they form the classic alphaherpesvirus corneal lesion of dendritic corneal ulcers. dendritic corneal ulcerations are strongly suggestive of chv infection in the dog. these linear, branching ulcers stain brightly with sodium fluorescein, rose bengal, and lissamine green (fig. 6) . 7, 47 prominent terminal end bulbs are a consistent feature of chv dendritic ulcers in the dog and can be used to differentiate chv corneal lesions from other potential causes of linear corneal ulcers that might appear clinically similar (eg, external trauma, cilia abnormalities, entropion). terminal end bulbs are club-shaped, rounded ends to the chv dendritic ulcer branches, and are not seen with other causes of linear corneal ulcers. coalescence of dendritic ulcerations may result in the formation of geographic corneal ulcers. 47 these appear as larger, irregular-shaped areas of corneal epithelial loss. in dogs with chv ulcerative keratitis, corneal ulcers are commonly located in discrete groups or linear arrangements on the corneal surface. unless complicated by secondary bacterial infection, chv corneal ulcers remain superficial and corneal stromal loss is not appreciable. nonulcerative keratitis is a less frequent lesion reported with chv ocular infection. 47 clinically, nonulcerative keratitis appears as a circumferential ring of cornea stromal neovascularization with epithelial and subepithelial leukocyte infiltrates in the peripheral cornea. nonulcerative keratitis may represent a resolution stage of active corneal epithelial disease. the largest published case series of primary chv ocular disease described an outbreak of chv infection a closed colony of young adult laboratory beagles. 47 in this group of 27 dogs, conjunctivitis was detected in 100% of dogs, ulcerative keratitis in 26% of dogs, and nonulcerative keratitis in 19% of dogs. corneal ulcerations were further subclassified by clinical appearance as punctate (7% of dogs), dendritic (19% of dogs), and geographic (4% of dogs). this report confirmed chv-associated ocular disease in group housed susceptible dogs, and provides an overview of the spectrum and relative frequency of ocular lesions associated with primary ocular chv infection in dogs. under experimental conditions, acquisition of primary chv infection by ocular surface inoculation consistently produces self-limiting conjunctivitis in immunocompetent mature dogs. 29, 46 this route of infection likely occurs frequently under natural conditions and has direct clinical relevance. 47 viral inoculation by other anatomic routes, such as the genital tract, is associated with inconsistent development of ocular disease. 48 clinical signs were manifested in both eyes, even when viral inoculation was unilateral, but the magnitude of conjunctivitis may not be symmetric between eyes. the clinical severity of ocular lesions peak approximately 7 to 10 days after infection and lesions slowly resolve over the following 2 weeks. histopathologic findings in dogs with acute experimental chv conjunctivitis include conjunctival epithelial necrosis, subepithelial lymphocyte and macrophage infiltration, and edema of the substantia propria. 28, 29 experimental induction of recurrent ocular chv infection was demonstrated by administering immunosuppressive dosages of systemic corticosteroids to latently infected dogs recovered from primary chv ocular infection. 8 recrudescent chv ocular disease was detected in 83% of immunosuppressed dogs in one study. 8 bilateral conjunctivitis or linear corneal ulcers developed as early as 3 days after initiating corticosteroid administration. the mean duration of detectable ocular disease was 8.6 days and was shorter than the experimental primary ocular chv infection in the dogs. cellular lesions observed by in vivo confocal microscopy in the dogs included conjunctival leukocyte infiltrates, corneal leukocyte infiltrates, abnormal corneal epithelial cell morphologies, and corneal langerhans cell infiltrates. subsequent research determined topical ocular corticosteroid administration does not result in recurrent chv ocular disease in latently infected dogs under experimental conditions. 15 in this study, topical ophthalmic prednisolone acetate (1.0% suspension) was administered 4 times daily for 28 days to both eyes of dogs with experimentally induced latent chv infection. viral shedding and recurrent chv ocular disease were not detected; however, crystalline corneal opacities developed in some dogs. these bilateral corneal lesions appeared clinically as subepithelial and anterior stromal punctate, white, refractile opacities within the central cornea. it was unclear if the crystalline corneal opacities were a nonspecific result of corticosteroid administration or influenced by prior chv corneal disease. in immunocompromised dogs, such as lymphoma who are receiving chemotherapy or dogs with autoimmune systemic disorders receiving long-term immunosuppressive therapy, relatively severe ocular lesions may develop during recurrent chv infection. 7, 9 these lesions include severe ulcerative conjunctivitis and extensive corneal ulceration that is refractory to treatment. development of viremia, systemic chv dissemination, and visceral hemorrhagic necrosis, similar to what is typically observed in fetal and neonatal dogs, has been reported in a mature dog with ocular chv infection while receiving chemotherapy for lymphoma. 9 in the reported dog, it was speculated that viremia and systemic chv disease developed secondary to localized ocular chv reactivation with an insufficient immune response to contain virus to the anatomic site of recurrent disease. recent evidence suggests chv ocular diseases in mature dogs are clinically underappreciated. a survey of dogs with idiopathic conjunctivitis determined chv was the most common viral etiology of conjunctivitis in mature, vaccinated dogs and was detected in ocular samples from approximately 17% of study dogs. 44 conjunctivitis is among the most common ocular diseases in dogs presented to veterinarians and, if these results are extrapolated to the general canine population, it implies chv ocular diseases occur commonly. 49 to determine the sites of latency of chv, miyoshi and colleagues 16 experimentally inoculated adult seronegative dogs via the intranasal (n ϭ 2), intranasal and intravenous (n ϭ 3), or intravaginal (n ϭ 3) routes with a strain of chv. although clinical signs were not observed, infectious virus was isolated from swabs until 4 to 6 days postinoculation. tissues were collected 2 to 4 months postinoculation and examined for the presence of latent viral dna. it was determined that the trigeminal ganglion (tg) was an important latency site for chv, regardless of the inoculation route. latency was detected also in lumbosacral ganglia of 2 of 3 dogs inoculated intravaginally, 1 of 2 dogs inoculated intranasally, and 1 of 3 dogs inoculated both intranasally and intravenously. abortion and stillbirths could also be associated with reactivation of latent chv, but the mechanism by which this takes place has not been investigated. retropharyngeal lymph nodes were another important latency site, since latency was detected in this tissue in 7 of 8 dogs. conversely, all attempts to demonstrate latency in peripheral blood lymphoid cells were negative. in humans, herpesviruses have been detected in the inner ear and are considered to play a role in vestibular dysfunction. parzefall and colleagues 50 reported on the prevalence of canine herpesvirus dna in the vestibular ganglia (vg) and vestibular labyrinth (vl) of 52 dogs that were included in their study. chv dna was detected in the vl of 17% of the dogs and in the vg of 19% of the dogs. although no attempt was made to differentiate between acute and latent infection, it is very likely that the pcr was detecting latent virus. interestingly, infection of the vg or vl was not always associated with infection of tg. since the vg, in contrast to the trigeminal and geniculate ganglia, do not have direct connection with sensory nerve endings on body surfaces, it remains most probable that there was primary infection of the tg or geniculate ganglia, with subsequent spread to the vg. burr and colleagues 13 examined tissues from 12 adult dogs that had been euthanized for various reasons. from each dog 12 tissues that have been associated with latency in other herpesvirus infections were examined. viral dna was detected in the organs of 9 of the 12 dogs. the tissues most commonly found to be positive were lumbosacral ganglia, tonsil, parotid salivary gland, and liver. based on the data, lumbosacral ganglia are an important site of latency and potential source of reactivated virus for venereal infections and infection of pups as they pass through the birth canal. finding of latent virus in tonsils and salivary glands points to the role of oronasal spread in the transmission of chv. it was noted that viral dna was detected in the trigeminal ganglia extracts of only 2 of the dogs. none of the 12 blood samples tested were found to be positive, indicting a lack of detectable viremia. the authors commented that chv is either totally absent from peripheral blood or that the level of infection is limited to 1 genomic copy per 2000 mononuclear cells. they also pointed out that basing the incidence of chv infection on serology only may lead to an underestimation of the true infection rate. the difficulty in detecting circulating chv in a kennel situation is highlighted in a study by ronsse and colleagues. 11 dogs in a breeding facility were followed for the duration of 1 reproductive cycle. a number of dogs seroconverted (negative to positive) to chv during this period. conversely, antibody-positive dogs became seronegative. the serologic data clearly indicate that chv was circulating in this kennel in the form of acute and/or reactivated form, primary infections. however, despite the fact that samples were taken at regular intervals, the results of pcr testing with a previously validated assay were uniformly negative both on all nasal and vaginal swabs and buffy coat samples. a possible explanation is that the shedding interval after reactivation is very short. even during acute infection, shedding of chv is limited to 2 to 6 days. latent chv has been reactivated by treatment with corticosteroids. okuda and colleagues 51 treated dams with a history of chv infection with 600 mg of prednisolone for 5 consecutive days. reactivation of latent chv infection was confirmed in 4 of 5 dams. infectious chv was recovered from nasal, oral, vaginal, and ocular secretions on the 5th to 21st days after initiation of treatment and also from nasal mucosa and tonsil tissues. these results indicate that latent chv infections develop frequently and that the latent virus may be reactivated, without clinical signs, in dogs with a history of chv infection. ledbetter and colleagues 8 investigated whether systemic administration of an immunosuppressive regimen of corticosteroids (3 mg/kg/day for 7 consecutive days) to experimental adult dogs would lead to reactivation and recrudescence. group1 dogs were latently infected and received corticosteroid treatment. group 2 dogs were latently infected and received a placebo. group 3 dogs were control dogs and received corticosteroid treatment. bilateral ocular disease, consisting of conjunctivitis and keratitis, was seen in 83% of the group 1 dogs between days 3 and 18 of the experiment. ocular shedding was detected in 50% of the group 1 dogs, and a 4-fold rise in antibody titer was detected in all dogs in group 1. none of the dogs in the control groups showed ocular disease, shed virus, or seroconverted. corticosteroidinduced reactivation is likely the result of enhanced expression of both viral and cellular genes. corticosteroid also lead to host immune response suppression, as discussed by the authors, the immunosuppression could be involved directly in the reactivation event, or indirectly in facilitating the spread of reactivated virus to peripheral tissues, leading to renewed replication at peripheral mucosal sites and potential transmission to susceptible animals that are in contact with the animal in which reactivation takes place. ledbetter and colleagues 15 also administered topical ocular prednisolone acetate or a placebo to mature dogs experimentally inoculated with chv via the ocular route and previously tested for reactivatable latency by systemic administration of an immunosuppressive dose of corticosteroids. the dogs were treated 4 times daily for a total of 28 days. the results of this study showed that topical ocular prednisolone at the concentration and treatment regimen used did not result in detectable reactivation of chv latency, based on a combination of recrudescent clinical signs, confocal microscopy findings, ocular infectious virus shedding, real-time pcr findings, and serologic response. a potential explanation for the data is that the concentration of topically administered corticosteroid that is absorbed systemically is insufficient to induce reactivation. malone and colleagues described a disseminated chv infection, which led to euthanasia, in an adult dog. 9 the dog had undergone chemotherapy for the treatment of generalized lymphoma. it was not clear whether generalized infection in this case was the result of enhanced susceptibility to chv as a result of immunosuppression or whether it was due to reactivation of a preexisting latent chv infection in this dog. amplification of target sequences by pcr method is currently the most common and most sensitive molecular diagnostic approach to the detection of chv in natural or experimentally infected animals. the pcr assays described initially were gel based, implying that the amplified products are visualized by uv illumination of ethidium bromide-stained agarose gels. miyoshi and colleagues 16 combined a nested pcr with southern blotting and showed that the detection limit of this combination was equivalent to 1 tcid 50 . schultze and baumgärtner 17 described nested gel-based pcr and in situ hybridization assays to diagnose acute chv infection in formalin-fixed paraffin-embedded tissues of 1-to 3-week-old puppies that were naturally infected. the specificity of the pcr products was confirmed by restriction endonuclease digestion. viral dna was detected in a variety of cell types, such as bronchiolar and alveolar epithelial cells, hepatocytes, renal tubular epithelial cells, neurons, fibrocytes, cardiac myocytes, and endothelial cells. this is in accordance with the previously described "pantropism" of chv. when paraffin-embedded tissues are used for pcr, it has to be kept in mind that the quality of the dna can be affected by several factors, such as the length of time between tissue removal and fixation, the presence of nucleases in the tissue, and the length of storage of the paraffin blocks. burr and colleagues 13 developed a gel-based pcr for chv and used it in conjunction with southern blotting to confirm the authenticity of the amplicons. they also assessed the pcr compatibility of each sample for chv pcr by first verifying that primers specific for a portion of the canine pancreatic lipase gene-amplified their target in each of the tissue extracts. the assay was capable of detecting approximately 14 genomic copies spiked into 1 g of placental dna and approximately 3500 copies when spiked into 0.2 ml of blood. erles and colleagues 18 described a gel-based pcr targeting a 494 -base pair region of a gene homologous to hsv-1 ul 37. reubel and colleagues 52 described a nested pcr that had a sensitivity that was 100 times higher than virus isolation. ronsse and colleagues 22 described the use of 2 pcr assays for chv. one of these assays had a sensitivity of 0.01 ccid 50 . the most sensitive and specific method currently available to detect chv dna is probe-based real-time pcr. a fluorogenic real-time pcr assay was described by reubel and colleagues 52 and reported to have a detection limit of 10 copies of viral dna. the first probe-based multiplex real-time pcr for chv was reported by ledbetter and colleagues. 8 very recently, decaro and colleagues 14 reported the development and complete validation of a probe-based real-time quantitative pcr for the detection and quantitation of chv dna in clinical samples. the assay was found to be very sensitive, since it could detect as few as 10 copies of the target per sample. in comparison with the gel-based pcr assay described by schulze and baumgärtner, 17 which was used in parallel, this assay has a 10-fold lower detection limit. specificity for chv was very high, as determined by lack of amplification of other canine viruses. the dynamic range was validated by successful amplification of a number of chv-positive samples from different geographic locations. reproducibility of the assay was determined by determining both intra-assay and interassay variability between the results obtained with samples containing variable amounts of target dna. both intra-assay and interassay variability, expressed as a coefficient of variation, were fairly low, were dependent on the target concentration, and were found to increase with decreasing target copy numbers. a potential pitfall of pcr assays is that the sample contains substances that are inhibiting the reaction, thus potentially leading to false-negative results. to control for this possibility, an internal control construct was spiked into each sample at known quantity and co-amplified. this way, any inhibition would be readily detectable from a decrease in the expected signal resulting from the amplification of this internal control. a relatively simple way to avoid inhibition was to prepare a 10-fold dilution of the sample. since it allows absolute quantitation, the assay was used to determine viral loads in tissues of pups that had died from acute infection and a vaginal swab collected from the dam. the viral load in the vaginal swab was 1.57 ϫ 10 3 copies/10 l. the highest viral load in tissues was 5.76 ϫ 10 9 copies/10 l, present in kidney homogenates. the authors concluded that, since it quantitates copy numbers over a wide range, this assay will be very useful not only for diagnostic purposed, but also for future pathogenesis studies and for the testing of the effect of antivirals on the replication of chv. the phrase "carrier animal" has been used extensively to describe an animal that harbors an infectious agent beyond the usual time allowed for the incubation phase of the infection and the acute and convalescent phases of clinical disease. 53 when it comes to the herpesviruses this is problematic since there are at least 2 phases that exceed those previously mentioned and are characterized by latency and exacerbation of clinical symptoms from latency. according to povey, a carrier animal may or may not shed virus in excretions or secretions, and shedding may occur continuously or intermittently. 53 as was noted in the preceding section, latency in its strict definition is the lack of viral transcription and translation, so no mature virus is being produced. a latently infected dog with chv would be defined as a carrier dog that is not shedding virus and would not be contagious to in-contact, susceptible dogs. exacerbation of the latent state to a replicative state would result in virus replication and shedding. the dog may have mild to severe clinical symptoms during this exacerbation phase. primary, systemic neonatal chv infection is associated with extensive viral shedding from numerous anatomic sites. high chv viral titers are detected in respiratory secretions, ocular discharge, saliva, and urine and on many mucosal surfaces (eg, genital, nasal, ocular, oral, pharyngeal, rectal, tracheal 4, 28 ). viral shedding may persist for up to 3 weeks in dogs that survive neonatal infection. viral shedding from infected neonates may serve to spread chv, either through direct contact or fomites, to littermates and other dogs. primary and recurrent chv infection in mature dogs is associated with mucosal viral shedding that it detectable by pcr assay or virus isolation. the duration and anatomic site of shedding vary markedly between dogs and infection episodes in individual animals. canine herpesvirus-1 shedding often occurs from multiple mucosal surfaces simultaneously and may be detected at sites anatomically distant to regions of overt clinical disease. reports of experimentally induced primary and recurrent chv infection suggest viral shedding during primary infection is prolonged and associated with higher viral titers than recurrent infection. 8, 45, 51, 54 there is an individual dog susceptibility to chv reactivation and shedding. latent chv infection can be reactivated, with induction of viral shedding, by short durations of corticosteroid administration in some dogs; however, other dogs are resistant to corticosteroidinduced viral reactivation. 8, 51, 54 when naturally infected mature bitches that previously aborted chv-infected pups where experimentally immunosuppressed by a 5-day course of systemic corticosteroid administration, chv was shed from the nasal, oral, ocular, and vaginal mucosa. 51 viral shedding could not be induced in all dogs. viral shedding was detected by virus isolation as early as 5 days, and as late as 20 days, after initiating corticosteroid administration. the duration of detected chv shedding ranged from 1 to 7 days in individual dogs. in a similar study 54 using 3-month-and 2-year-old dogs experimentally infected with chv by nasal and intravenous routes, chv reactivation and mucosal viral shedding were repeatedly induced by systemic corticosteroid administration. primary oronasal infection was associated with nasal chv shedding of approximately 2 weeks' duration. following recovery from primary infection, systemic corticosteroid administration induced viral shedding from the nasal, oropharyngeal, and genital mucosa. the onset of detectable shedding was between 5 and 9 days after initiating corticosteroid treatment and persisted for up to 32 days with marked variation between individual dogs. a second round of corticosteroid administration was administered 3 months later and again resulted in viral shedding in some, but not all, dogs. the duration of viral shedding was shorter in all dogs during the second experimental reactivation and was associated with a tendency for lower viral titers. in studies examining ocular chv infection, a similar pattern of viral shedding is reported. experimental primary ocular chv infection in mature dogs produced by direct ocular surface inoculation resulted in conjunctival viral shedding that persisted for 10 days after inoculation. 46 virus was detected in conjunctival samples by virus isolation and chv pcr, and viral titers peaked 5 days postinoculation. chv was inoculated into a single eye, but viral shedding was detected bilaterally in some dogs. following recovery from primary ocular infection, viral shedding was not detected over the subsequent 8 months. experimental recurrent ocular chv infection induced by systemic corticosteroid administration to dogs recovered from primary ocular infection again resulted in viral shedding. 8 ocular chv shedding was detected by pcr assay in 50% of dogs between 10 and 13 days after administering the first dose of corticosteroid. in comparison to primary ocular chv infection, ocular viral shedding associated with recurrent infection was briefer and viral titers in samples were lower. experimental primary chv genital mucositis in mature dogs, produced by intravaginal and intrapreputial chv inoculation, resulted in genital viral shedding that was detected by virus isolation for up to 20 days. 46 several dogs also developed nasal, pharyngeal, and conjunctival viral shedding during this period. canine herpesvirus tracheobronchitis induced by intranasal viral inoculation was associated with viral shedding for up to 18 days. 2 in the dogs with chv upper respiratory tract infection, viral shedding from the nasal mucosa was detected by virus isolation in all dogs and a some had concurrent tracheal and rectal viral shedding. the clinical ecology and epidemiology of chv can be summarized in table 3 . it basically starts with a series of questions that inquire into the status of the virus, the host, and the environment with which both are localized. 55 the critical question is whether chv infection and disease are of economic concern? as was mentioned earlier, the reproductive diseases associated with chv were the initial driving force behind the recognition of the economic and emotional effects upon dog owners. while the costs of chv-associated reproductive diseases have not been reported, it would be conceivable that a dam that loses an entire litter to chv would result in a loss of $10,000, since multiple puppies are involved. in cases of respiratory disease and ocular disease, the costs of treatment and long-term care of recurrent infections may exceed $1000 per case. the second question pertains to the zoonotic or public health risks associated with chv infections. the virus is species specific and there is no evidence to support its involvement in human disease. 26 the third question is the key to the persistence of chv in the canine population-where is the virus when not causing disease? this has been a key factor in understanding the virus and controlling it. the virus maintains itself in subclinical carrier dogs by way of latency. it may be exacerbated throughout life by stress, which results in mild to severe clinical symptoms that most commonly affect the respiratory and ocular systems. concurrent with these clinical episodes there is shedding from excretions and secretions to susceptible dogs. the 2 most susceptible age groups are pregnant chv naïve dogs and puppies of these dams (in utero, postnatal). the fourth question revolves around the epidemiology of chv once its infection occurs in the susceptible dog. the course of the infection to disease is variable and has been reviewed earlier under the contemporary clinical observations. one important aspect to reiterate here is the importance of immunity in controlling the infection-disease process in pregnant dams and their offspring during the postnatal period. early postnatal infection (3 weeks or less) results in high morbidity accompanied by high mortality. 34 later infection (3 weeks or later) results in low morbidity and very low mortality. however, it is usually the later postnatal infection that establishes the lifelong carrier state via latency. the fifth question is a natural extension of the sequence of clinical inquiry and addresses the control of chv, so that infection is minimized during diseasesusceptible periods and maximized during disease-resistant periods. as noted previously, shedding states are important in maintaining the virus infection on the population to attain a certain degree of population immunity. knowing when dogs are potentially contagious, and maintaining the 6-week barrier to infection, allows for maximum protection during this susceptible period. since there is no reliable vaccine available, kennel hygiene and biosecurity are essential. 34 therapy for neonatal chv infection is largely supportive and carries a poor prognosis for survival once clinical disease is manifested. 23 in instances where dogs survive neonatal chv infection, cardiac, neurologic, and ocular lesions may be permanent. elevating the environmental temperature of dogs in a litter after chv infection is diagnosed may provide some protection to uninfected pups. viral replication is reduced at elevated body temperatures and there are lower morbidity and mortality rates in dogs that are subsequently infected; however, this is ineffective for individual dogs if implemented after viral infection. 23 intraperitoneal injection of immune sera obtained from chv-seropositive dogs is described as a method to reduce mortality in an exposed litter, but it must be administered prior to infection to be most effective. 26 lactoferrin possesses in vitro antiviral activity against chv and inhibits cellular infection. 56 administration of lactoferrin to dogs at risk for infection could theoretically provide protection; however, this is not demonstrated in vivo. isolated reports of apparently successful therapy of neonatal chv infection with the antivirals vidarabine and acyclovir are described. acyclovir was administered orally as a 10-mg total dose per dog at 6-hour intervals until 3.5 weeks of age. 26 the pharmacokinetics and tissue distribution of intravenous, subcutaneous, and oral acyclovir were investigated in dogs. 57, 58 additionally, a sustained release buccal tablet form of acyclovir was evaluated in the dog. 59 acyclovir is bioavailable when administered orally to dogs and is widely distributed within tissues; however, target plasma concentrations and effective dosages for chv infection are currently unknown. [57] [58] [59] acyclovir toxicosis resultant from accidental ingestion is reported in dogs with dosages as low as 40 mg/kg and the routine clinical use of this, and other systemic antiviral medications, in dogs for chv infection requires further investigation of safety and efficacy. 60 the canine pharmacokinetics of newer-generation antiherpesviral drugs, including famciclovir, are reported. similar to acyclovir, safe and effective doses for dogs with chv infection are undetermined. 61 treatment of respiratory and genital chv infection is primarily symptomatic. unless complicated by secondary bacterial infection, these conditions are typically selflimiting and specific antiviral therapy is not reported. in contrast to respiratory and genital infection, there are detailed reports of the successful clinical management of ocular chv infection. in addition to nonspecific treatments to prevent secondary bacterial infection (topical ocular antimicrobials) and improve comfort (topical ocular atropine), antiviral therapy with 0.1% idoxuridine or 1% trifluridine ophthalmic solution was used. idoxuridine and trifluridine are nucleoside analogues, possess good anti-herpesvirus activity, and are well tolerated by dogs when applied topically as ocular formulations. trifluridine is available under the trade name viroptic, and idoxuridine can be acquired from compounding pharmacies. both antivirals are administered 6 to 8 times daily for the first 48 hours and then 4 times daily until resolution of clinical signs of active infection. cidofovir 0.5% ophthalmic solution is an alternative ophthalmic antiviral for chv ocular disease that is effective with twice daily administration (e.c. ledbetter, unpublished data, 2011) . this review has documented well that our level of clinical inquiry expands as our knowledge base about chv increases. while earlier studies focused on the reproductive effects of chv in susceptible pregnant dogs and neonatal puppies, it has become apparent that in order to control chv-related diseases that we must understand the various forms of chv infection that may occur in the dog population (fig. 7) . this has prompted the veterinary community to develop more sensitive diagnostic assays, such as pcr, in order to answer the questions, where is the virus when not causing disease, and what is its relationship with respiratory and ocular diseases in adolescent and adult dogs (1 year or older)? molecular and serologic studies have clearly demonstrated that we are dealing with an infection that is more common than we considered a decade ago. reports have indicated that up to 70% of some high-risk dog populations have been infected with and are latent carriers of chv. this is important for veterinarians to know as we confer with clients on the best management steps we can take to protect our at-risk populations. while pregnant chv-naïve dams and neonatal puppies born from a chv-naïve dam are considered at high risk for disease, we must also take into consideration dogs in kennels and rescue centers. it is these dogs that are at risk for either exposure-infection or stress-induced exacerbation of latent chv, which had been acquired at an earlier age. the manifestations of chv in adolescent and mature dogs may range from subclinical to severe respiratory and/or ocular diseases. the reports by malone and colleagues, 9 gadsden and colleagues (submitted, 2011), and ledbetter and colleagues 7,47 all indicate that chv can cause disease in older dogs and that it is not just a "puppy disease." recognition of the various forms of chv-induced disease, availability of diagnostic assays with increased sensitivity, and the formation of reliable biosecurity measures will allow for better control steps to be taken in dogs at-risk for infection and disease. clinical and pathologic features of a fatal viral disease of newborn pups experimental production of canine tracheobronchitis (kennel cough) with canine herpesvirus isolated from naturally 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specific and sensitive detection of canid herpesvirus-1 the effect of topical ocular corticosteroid administration in dogs with experimentally induced latent canine herpesvirus-1 infection detection of canine herpesvirus dna in the ganglionic neurons and the lymph node lymphocytes of latently infected dogs nested polymerase chain reaction and in situ hybridization for diagnosis of canine herpesvirus infection in puppies longitudinal study of viruses associated with canine infectious respiratory disease investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kenneled dog populations a serological study of canine herpesvirus-1 infection in the english dog population prevalence of antibodies against canine herpesvirus-1 in dogs in the netherlands in 1997-1998 seroprevalence of canine herpesvirus-1 in the belgian dog population in 2000 herpesvirus canis: aspects of pathogenesis and immune response bsava manual of canine and feline reproduction and neonatology. gloucester (uk): british small animal veterinary association small animal pediatrics. the first 12 months of life infectious diseases of the dog and cat the role of neonatal canine herpesvirus infection in mixed infections in older dogs pathogenesis of canine herpesvirus in specific-pathogen-free dogs; 5-to 12-week-old pups the susceptibility of six-week of puppies to canine herpes virus canine respiratory viruses canine infectious tracheobronchitis canine herpesvirus respiratory infection canine herpesvirus infection: update on risk factors and control measures small animal pediatrics. the first 12 months of life protection of puppies against canine herpesvirus by vaccination of the dams reproductive effects of canine herpesvirus canine adenoviruses and herpesvirus herpesviruses of carnivores canine herpesvirus genomic analysis of a pneumovirus isolated from dogs with acute respiratory disease nucleotide sequence of glycoprotein genes b,c,d,g,h and i, the thymidine kinase and protein kinases and gene homologue ul24 of an australian isolate of canine herpesvirus canine herpes-induced retinal dysplasia and associated ocular anomalies lesions in puppies surviving infection with canine herpesvirus virologic survey of dogs with naturally acquired idiopathic conjunctivitis an atypical presentation for the first isolation of canid herpesvirus-1 in argentina experimental primary ocular canine herpesvirus-1 infection in adult dogs outbreak of ocular disease associated with naturally-acquired canine herpesvirus-1 infection in a closed domestic dog colony genital disease in dogs caused by canine herpesvirus health status and population characteristics of dogs and cats examined at private veterinary practices in the united states naturally-occurring canine herpesvirus-1 infection of the vestibular labyrinth and ganglion of dogs virus reactivation in bitches with a medical history of herpesvirus infection experimental infection of european red foxes (vulpes vulpes) with canine herpesvirus persistent viral infection. the carrier state repeated canine herpesvirus (chv) reactivation in dogs by an immunosuppressive drug diagnostic medicine: the challenge of differentiating infection from disease and making sense for the veterinary clinician antiviral activity of lactoferrin against canine herpesvirus pharmacokinetics and bioavailability of acyclovir in the dog the disposition of acyclovir in different species a sustained release dosage form of acyclovir for buccal application: an experimental study in dogs accidental ingestion of acyclovir in dogs: 105 reports metabolic and pharmacokinetic studies following oral administration of famciclovir to the rat and dog the authors would like to acknowledge those clinicians and veterinary researchers who provided insights and recommendations for our understanding of chv pathogenesis and the management of chv; these include dr l. carmichael, dr m. appel, dr j. gorham, dr r. ott, dr a. hashimoto, dr a. sears, and dr m. spector. the technical support of a. mckeirnan and l. tanaka is greatly appreciated. the assistance of t. pfaff in preparing the word document was essential. this manuscript is dedicated to all the men and women who serve as dog handlers in roles of community protection, rescue operations, guide dogs, and national defense. key: cord-201898-d1vbnjff authors: jha, vishwajeet title: forecasting the transmission of covid-19 in india using a data driven seird model date: 2020-06-08 journal: nan doi: nan sha: doc_id: 201898 cord_uid: d1vbnjff the infections and fatalities due to sars-cov-2 virus for cases specific to india have been studied using a deterministic susceptible-exposed-infected-recovered-dead (seird) compartmental model. one of the most significant epidemiological parameter, namely the effective reproduction number of the infection is extracted from the daily growth rate data of reported infections and it is included in the model with a time variation. we evaluate the effect of control interventions implemented till now and estimate the case numbers for infections and deaths averted by these restrictive measures. we further provide a forecast on the extent of the future covid-19 transmission in india and predict the probable numbers of infections and fatalities under various potential scenarios. the infections and fatalities due to sars-cov-2 virus for cases specific to india have been studied using a deterministic susceptible-exposed-infected-recovered-dead (seird) compartmental model. one of the most significant epidemiological parameter, namely the effective reproduction number of the infection is extracted from the daily growth rate data of reported infections and it is included in the model with a time variation. we evaluate the effect of control interventions implemented till now and estimate the case numbers for infections and deaths averted by these restrictive measures. we further provide a forecast on the extent of the future covid-19 transmission in india and predict the probable numbers of infections and fatalities under various potential scenarios. almost every continent of the planet is grappling with a large number of infections arising due to virus called coronavirus 2, sars-cov-2 [1] . these infections that may result in a mild to severe symptomatic disease called coronavirus disease 2019 or covid-19 were first detected in wuhan, a city in central china [2, 3] . later the infections spread across the globe and it has forced nations to undertake drastic measures to minimize the loss of precious human lives [4, 5] . for a populous country like india, which has a dense and large population (≈ 1.4 billion), the cause of concern is quite high. therefore, it is of special importance to study the spread of covid-19 in india, and make reliable predictions which can help in mitigation of its ensuing effects. these timely critical information may be crucial for devising strategies for containment of infections and estimating the requirements of medical facilities. in india an early complete nation-wide lock-down was imposed from 25 th march when the number of cumulative sars-cov-2 infections were around 650. these strict measures prevented any large scale disaster and slowed down the rate of infections in the initial stages and helped in geographical containment of the epidemic. however, recent days have seen no real decrease perhaps due to gradual weakening of restrictive measures owing to pragmatic social and economic reasons. from june 1 st india continues to have a complete lock-down only in the defined containment zones where the infection rates are high. these steps of gradual easing of lock-down have been necessitated as the balance between life and livelihoods are intertwined, which calls for invoking more intelligent strategies because a complete extended lockdown cannot be sustained for very long time without other competing collateral losses to the most vulnerable sections of society. alternative steps based on isolation of infected patients through the lock-down in the containment zones and more widespread testing and contacttracing are being followed for controlling the rate of infection. this represents the transition from suppression to mitigation strategy for the resolution of any potential outbreak but efficacy of these steps remains to be seen as the execution of these policies on ground level are challenging. the transmission dynamics of viral epidemics in any population is an interplay of various factors related to viral, immunologic, environmental and sociological conditions. a number of mathematical and physical models have been proposed in general to understand the evolution of epidemics, aiming to make reliable predictions so that to help governments to formulate proper policies and response plans for effective control of the disease [6] [7] [8] . simple deterministic mathematical models based on the formulation of differential equations have been extensively used to provide information on the transmission mechanism of various viral epidemics. the sir model is a one of the simplest epidemiological models that is based on dividing the population among three compartments, the susceptible, the infected and the recovered (or deceased) populations and determining their time evolution [9] . the seir [10] model is a simple extension of the sir model, where an additional compartment of exposed population with a latency period is introduced which is more appropriate for covid-19 like epidemic which has an inherent latency and asymptomatic transmission [11] . extended models have been employed that use several separate compartments for various sub-populations such as, asymptomatic, quarantined, hospitalized or components based on the variations for example, according to age, gender etc. [12, 13] . however, this entails incorporation of many unknown parameters and uncertain initial conditions about which the information is either not available or there are large associated uncertainties. in the present article, we employ a dynamic seird model with the inclusion of population of deaths as a separate compartment in the seir model. several works have been already performed in the indian context to explain the covid-19 dynamics in the initial phase of its transmission [14] [15] [16] [17] [18] . we incorporate the crucial parameter of contact period with a time variation connecting its value at the beginning of the epidemic to the current reduced value. the reduction in the values of contact rate has been achieved due to many isolation measures, primarily the imposed nation-wide lock-down. the time variation in the contact rate β(t) is determined through arxiv:2006.04464v1 [q-bio.pe] 8 jun 2020 the effective reproduction number r(t) that is in turn related to the doubling time of the rate of infection growth [19] [20] [21] . we integrate this parameter in the seird model calculations and estimate the role of interventions in preventing the number of probable infections and death till now. further, we consider different potential scenarios for the rate of growth of infections for making projections of sars-cov-2 transmission. we make a forecast for the probable numbers of infections and fatalities in the coming times. the projections provide information for the extent of suppression and containment strategies that need to be employed to mitigate the impact of covid-19 in coming times. it is to be mentioned that results obtained in this work are to be used for the research purposes only. the data for the present studies are collected from the repository hosted at website https://www.worldometers.info/coronavirus [22] for cases specific to india. the epidemiology of the covid-19 outbreak using a deterministic seird model is studied with five compartments governed by a set of ordinary differential equations where, s(t) is the susceptible population, e(t) is the exposed population, i(t) is the infectious population, r(t) is the recovered population and d(t) is the number of deaths at any instant t and n = s + e + i + r + d is the total population. we have not included separate compartments for the number of asymptomatic, quarantined, hospitalized populations or the variations according to age or gender, as these lead to increase in number of unknown parameters and therefore lead to large uncertainties in the predictions. in any case, these numbers can be estimated in an average way with their relations to populations that have been considered. in addition, assumption about the no re-infection of the recovered population is made as there is no evidence to the contrary. the parameters of the above set of equations are the latent period of being exposed a = 1/φ that is related to the incubation period of the virus, the contact period b of infection = 1/β, the period of being free from being infected g = 1/γ commonly known as the recovery time , the parameter corresponding to death d = 1/δ of the infected population. these parameters determine the transitions that occur across the compartments as the time evolves. here, the parameters a, g and d are specific to characteristics of sars-cov-2 and only weakly correlated to the health responses of the country and therefore expected to have similar values across countries. the parameter b represents the strength (speed) of the virus transmission which is intimately related to the prevailing conditions of containment measures undertaken by specific countries. apart from these parameters, the fraction of the susceptible population at the beginning to the total population α = s(0)/n is a very important parameter. taking total population of the region as s(0) may lead to gross overestimation of case numbers, because the part of population may be inherently immune or less affected by the virus or live in isolated conditions. furthermore, the extent of initial exposed latent population defined by = e(0)/i(0), parameter, may also be an important parameter that indicates the presence of a number of undetected or asymptomatic exposed individuals at the beginning. one of the most significant parameters that describes the pandemic is the basic reproduction number of infection r 0 , which is defined as the number of individuals that are infected from the uninfected, susceptible population by one infected individual under normal conditions [6, 19] . there are challenges in determining r 0 in terms of the parameters of deterministic model as one requires estimates of included parameters that are uncertain [23] . during the spread of the epidemic one can define an effective reproduction number r i (t), which is a time dependent quantity that changes because of control measures and depletion of susceptible population. it provides the dynamic information on the strength of the epidemic transmission as the time evolves. in general, the infection continues to expand if r i (t) has values greater than 1, while the epidemic stops eventually if r i (t) is persistently less than 1. the estimation of the effective reproduction number is complicated and many models have been proposed for its determination [24, 25] from the data. here we use a simple method based on fitting the incidence data growth rate by a distribution with gaussian shape to determine the behaviour of r i (t). it must be mentioned however, that reported data has an inherent delay as compared to the instantaneous population numbers that are required for the estimation of its actual value. in the sir -type models or their simple extensions, such as one described above r i (t) can be expressed as in the initial stages of the infection, s(t) ∼ n and r i = β γ , since (γ δ). the r i (t) value can be estimated using the initial doubling time t d of the number of infections [20] the t d value can be determined by fitting the reported growth in the cases of infection, which shows an exponential growth at the beginning of the epidemic, where, the daily growth rate r(t) is determined from the data of reported cases of infections. at smaller values of the values of r(t) are extracted from the reported data of daily growth rate of infections starting from 14 th march to 28 th may (day 76) with a 9-day moving average. it is fitted with a function in the following form where a, b, σ and t 0 are fit parameters. these parameters are determined from the best fit approach through the local minimization of the sum of squares of the error. the resulting fit to the daily growth rate is shown in fig. 1a along with the band with standard error on fit parameters. in addition, the projections for next days after 28 th may are also shown for various probable scenarios by the straight lines that are used for the extrapolations of infection growth rate. it is seen from the figure that india had a peak daily growth rate of ∼ 20 % at the beginning of the epidemic which reduced to ∼ 5 % after one month of imposition of lock-down. it is to be noted that the nationwide lock-down imposed on 25 th march has been continually relaxed in phased manner and exists now only in the containment zones from 1 st june. however, after the decrease in growth rate in infections in the initial phase following the lock-down, the cases of infection have continued to grow at somewhat constant rate for a while. the extrapolations for next 30 days that define various probable scenarios are approximated as a linear reduction or increase from the present value of infection growth rate. the quantity r(t) determined from the data is also used to study the evolution of r i (t) in time as shown in fig. 1b . it must be noted that r i (t) also depends on the period of infection for which, we present the result for values g = 12.7 days and g = 20 days. the r i (t) values have been extracted from the r(t) of the reported cases and also obtained through fitted value of r(t). these values are seen to decrease from a peak value of ∼ 4 to a value of ∼ 1.6 for g = 12.7 days, which is still substantially higher than the value 1 that is required for the spontaneous disappearance of the infection. the t d (t) value that is directly extracted from the data and also from the fit shows a constant value of ∼ 16 days. the value of r i (t) and t d values are also shown for one probable scenario where the rate reduces by one-half of the present value in a linear manner. this shows a moderate reduction in the value of r i (t). in addition, the rate decrease leads to a significant increase in t d values. the seird model calculations using eqn. 1 have been performed to make comparisons with the data aggregated for india using the reported cases of infected, recovered and dead populations up to 28 th may and to make forecasts about the future scenarios. the contact rate parameter β(t) is taken to be time dependent with the parameters β 0 and β 0c fixed in accordance of equation 6. the parameter a is taken as 5.1 days, which is the mean incubation period and bit larger than the latency period. the value of parameter δ = 0.025 is taken, which determines the death population and very weakly affects populations in other compartments. the parameter γ(t) is taken in the following form the time variation in this parameter with γ 0 = 0.079 corresponding to period of 12.7 days and κ=0.01 takes into account the larger value of g ≈ 26 days that is needed to explain the behaviour of data in the initial stages. as the time elapses, a reduction in the recovery period is seen and γ approaches γ 0 value. the model was applied from the day of the epidemic when cumulative number of infections were ∼ 100 as on 14 th march. the fraction of the population at day 1 in compartments are set as follows : i = 88/1.4e7, r = 10/1.4e7 and d = 2/1.4e7 as provided by the reported data. other initial conditions, defined by α and are the unknowns in the model. we take α=0.1 which is similar to value of α=0.08 extracted for european countries in ref. [26] . the parameter = 3.2a, is important for the initial description of data but it does not affect long time dynamics of the epidemic as predicted by the model. the results of calculations with these parameters that use the time varying β(t) parameter as determined above provide a good description of the evolution in the case numbers of reported infected, recovered and death population as shown in fig. 2a . in addition, the calculations have also been performed for constant β = 0.167 value, which is obtained from the best fit to the exponential distribution according to eqn. 4. while the model results as shown in fig. 2b provide a good description in initial days, it grossly over-predicts the case numbers as compared to the reported cases. it is quite evident from the figure that the time dependence of β(t) is necessary to understand the dynamics of infection spread for cases in india. the period of infection related to the recovery time of the infected individuals is also taken with a time variation. this parameter is primarily the characteristic of the epidemic and it is only mildly dependent on the responses of the health-care systems. in absence of any effective therapy or cure that may shorten the length of the infection it is relatively well known and it is taken as ∼ 12.7 days. however, larger value is required to explain the reported data both with constant β-value [17] or even when time variation in β-value is taken into account as 5 it has been found in the present study. the recovery rates are continually improving, a feature also reflected in the reported recovery data. therefore, a time dependence in the parameter γ(t) is introduced to account for this observation. we show the calculations in fig. 3 to study the role of interventions we perform calculations with different values for the contact rate β. the interventions have led to a decrease in the daily growth rate of infections which is intimately related to the β value. we use the constant values of β = 0.252 and 0.125, which correspond to the peak rate of growth and half its value. the peak β-rate is expected to have prevailed in the early stages of infection spread in the absence of any interventions such as, the lock-down or the conditions of no enhanced public awareness. in addition, we also give results obtained from the β = 0.167, β = 0.01 and time varying β-value. the infected, recovered and death populations for these β-values are shown in fig. 4a, fig. 4b and fig. 4c , respectively. from the comparison it is evident that the lock-down and other interventions have prevented any large spread of infections and kept the death numbers particularly low. these interventions could have prevented around 4 million peak infections and 200,000 deaths at the 100 day mark. the lower growth rate also means that number of active infections are low at any instant which helps to optimize the response of health care systems. the rate of infections in india have remained approximately constant after the initial reduction for last several days. after an extended lock-down slowly the restrictions have been loosened up. we extrapolate values of β(t) to predict the outcomes of various probable scenarios. the β(t) values corresponding to growth rate value r pr as on 28 th may are varied so as to attain a given value at the end of next 30 days assuming a time variation with constant slope. these scenarios are named as the best case, the optimistic case, the most likely, current, problematic and alarming scenarios respectively. the time evolution of the epidemic is studied with these time variations for the future. the resulting predictions for the populations of infected, recovered and dead are shown in fig. 5a, fig. 5b and fig. 5c respectively. the growth rate same as r pr may lead to a high number of total infections (∼ 8 million) with fatalities in excess of 300,000. in the scenario that we term as the most likely scenario, we can have a total of 3.5 million infected cases with almost 140,000 fatalities over the course of pandemic. this will correspond to a in this case, the death figures can be kept substantially low in the range of 25,000-50,000. in contrast, if the rate of growth were to increase from the present values due to pre-mature lifting of the lock-down in the affected zones and other lapses, the death numbers can be 500,000 with a rather alarming number of infected individuals in short time. higher rates of growth also mean the large number of active infected cases appearing early and that may stretch the health care systems to the brink. we have made detailed comparison of model predictions with the real data using the important parameter of contact rate and infection rate derived from the data itself from the first principles. it must be noted that there is an inherent delay in the reported rate and instantaneous rate of the infection. in addition, the effect of any restrictive measures undertaken appears with a delay in the reported rate, which is estimated by the fit parameter t 0 ∼ 15 days in the present case. the model calculations are able to describe very well the case number of infected and recovered populations of reported data till now. the imposed restrictions have led to a reduction in the r i (t) and an increase in the t d values as the time elapses. the quantitative measure of the intensity of the imposed lockdown that reduced the growth rate r(t) to almost half its value is given by the fit parameter σ = 14 days. it is seen from the calculations that a large number of infections and fatalities have been averted due to imposition of the lock-down. some part of this reduction may be ascribed to the enhanced public awareness, and growing disease monitoring and testing capabilities with the passage of time. however, effect of complete lock-down in reducing the infection rate has been quite significant. after the initial period of 40 days following the complete lock-down, there has not been much gain in the reduction of infection spread rate in last 30 days. it is probable that the gradual weakening of the lock-down due to socio-economic reasons might have offset the gains due to restrictive measures. nevertheless, the continued restrictions have prevented any rise in the rate of growth of infections, which in absence of any such measures is expected to rise again. even the growth rate of ∼ 4 − 5% attained so far implies an exponential growth and it is seen that the epidemic in india is still in early stages. current estimates of future trends in new infections in the weeks after 28 th may suggest that more severe outbreak may occur in coming times leading to high number of infections. with the estimates from the most likely scenario, over 450,000 would be clinically diagnosed at the maximum resulting in ∼ 140,000 total fatalities. impact of the severity of the disease outbreak is quantified through the case fatality ratio (cfr). it is defined as the ratio of fatality rate d(t) to the cumulative number of infections c(t). the cfr values have varied from ∼ 3.3 -2.8 as on april 15 to the present day which is less than the global average of ∼ 6.2. however, some doubts remain about the estimations of cfr because it is possible that both the number of fatalities and infections may be underestimated. it is more likely that c(t) may be underestimated more due to the presence of large number of asymptomatic or non-critical infected cases which leads to the overestimation of cfr, assuming reported d(t) cases to be true. cfr remains low as long as the health facilities are able to cope with the rate of patients requiring critical care. in the scenarios if the number of active infected cases is large as predicted by multiple scenarios described above, requirements of hospitalizations and critical care resources may increase sharply. in such a situation, the health care system is going to be severely challenged in providing the critical care facilities for prevention of fatalities. the cfr in these conditions may rise to higher values. therefore, imposing stricter measures inside the containment zones and more extensive testing and contact-tracing seems to be only viable logical preventive option that can lead to a manageable reduction in infected cases and casualties in absence of any therapies or large-scale immunity. there are limitations of the simplistic model employed here and therefore the exact quantitative numbers presented in the work are only indicative. in the present model, the asymptomatic populations are taken only in an indirect way at the start of the epidemic through the introduction of the parameter . inclusion of this population as a separate compartment however would lead to introduction of extra set of unknown parameters. further, we have not considered the regional and age specific heterogeneities in the model. while we have made a reasonable assumption for the parameter α =0.1, implying a 10% of the total population as the susceptible population, the overall numbers presented in this work may differ if it has a significantly different value. this number is going to be affected as the country has seen large scale migration from the infected areas to the other areas in recent times which may increase the pool of susceptible population. further, we have made forecasts in this work based on probable daily growth rates. the determination of contact rate parameter through the measured rate in the simple way is uncertain due to stochastic fluctuations in the early stages and inaccuracies and time delay of the reported data. further, there are challenges on designing the control mechanisms based on the basis of the numbers of daily growth as discussed in ref. [27] . however, this work shows the operational use of the r i (t) calculated from the instantaneous infection rate to provide a reasonable description of the transmission dynamics. in this article, we have presented results of seird model calculations to study the role of interventions and make future projections in the covid-19 spread in india. to make reliable forecast we have determined the time dependent reproduction number r i (t) and contact rate parameter β(t) from the data for the daily rate of increase of infections. it is shown that timely imposition of lock-down and other public health interventions have led to a substantial reduction in the effective reproduction number which decreased to a present value of ≈1.6 from the peak value of ≈3.2 corresponding to an increase in the doubling time of the infections. calculations performed using the time dependent contact rate parameter β(t) in the seird model provide a good description of the case numbers of infections, recovered and deaths. we further make the projections for different probable scenarios. in the most likely scenario the model predicts a peak of active infections around the month of september with significant number of fatalities over the course of the epidemic around end of november. the results show the impending critical challenges for health care systems due to prospective high number of people with infections. the salient feature of the simple model employed in this work is the use of minimal uncertain parameters and therefore in our opinion it makes reliable predictions of the infections and fatalities. the projections of peak infections suggest big challenges for the available critical care health facilities in the management of pandemic. new innovative solutions have to be continuously found and intelligent measures have to be effectively implemented if the covid-19 infections have to be contained with a moderate number and the ensuing fatalities have to be minimized. the most important extension of this study will be to incorporate the regional variability and apply this model by considering the state wise infection data and make predictions accordingly. situation report 133 infectious diseases of humans cal epidemiology of infectious diseases: model building, analysis and interpretation mathematical models in population biology and epidemiology 2nd edn seasonality and perioddoubling bifurcation in an epidemic model swarnajit chatterjee, mintu karmakar and raja paul medarxiv we thank v. v. parkar, d. k. mishra and g. chaudhuri for useful discussions and their interest in the work. key: cord-254766-585iu5ey authors: tauro, sharyn; su, yung-chang; thomas, sandra; schwarze, jürgen; matthaei, klaus i.; townsend, dijana; simson, ljubov; tripp, ralph a.; mahalingam, suresh title: molecular and cellular mechanisms in the viral exacerbation of asthma date: 2008-08-13 journal: microbes infect doi: 10.1016/j.micinf.2008.07.037 sha: doc_id: 254766 cord_uid: 585iu5ey the aetiology of asthma associated with viral infection is complex. the dynamics that contribute to disease pathogenesis are multifactorial and involve overlapping molecular and cellular mechanisms, particularly the immune response to respiratory virus infection or allergen sensitization. this review summarizes the evidence associated with factors that may contribute to the development or exacerbation of asthma including age, host factors, genetic polymorphisms, altered immune responses, and aspects of viral antigen expression. this review also provides an important perspective of key events linked to the development of asthmatic disease and related pulmonary inflammation from human and animal studies, and discusses their relationship as targets for disease intervention strategies. asthma is a chronic, reversible inflammatory disorder of the airways characterized by laboured breathing due to variable and reversible airway flow limitation. asthmatics have sensitive airways that react to stimuli with inflammation, swelling of the airway lining, muscle tightening and mucus production; all of which make breathing more difficult. the symptoms can be one or more of the following: wheezing, coughing, tightness in the chest and shortness of breath. the prevalence or disease burden of asthma varies from country to country. it is estimated that there are approximately 300 million affected individuals worldwide [1] . annual worldwide deaths from asthma have been estimated at 250,000. the highest prevalence of asthma is in the uk, australia, new zealand and ireland [1] . in australia, 12% of children and young adults aged 0e24 years have asthma [2] and expenditure on asthma in 2000 was estimated at aus$693 million [3] . in the usa, 20 million people are affected by asthma and the total direct and indirect costs of asthma exceeded us$11.3 billion in 1998 [4] . the underlying causes of asthma are not well understood but many factors are recognized as contributing to the disease, including an atopic (allergic) disposition (often a family history of allergies and asthma), certain genetic mutations, exertion, irritants (including smoking, indoor and outdoor air pollutants), both inhaled and food allergens (e.g. house dust mites, mould spores, pollens, peanuts, egg), and viral infections [3] . in people with underlying asthma, respiratory viruses frequently trigger exacerbation of the disease, with 80e90% of exacerbations in children associated with respiratory infections [5] and up to two thirds of cases in adults [5] . respiratory viruses are also thought to have a role in people becoming sensitive to allergens and developing asthma [9, 10] . asthma is a multifactorial disease, with the genetics of the host interacting with a variety of environmental triggers. viral infections are one of the environmental triggers, having been implicated both in the induction of the disease and, more frequently, in its exacerbation. a number of viruses are associated with the exacerbation of asthma in children and adults. the most common viruses detected in connection with the symptoms of asthma are rhinoviruses (rv); however, human respiratory syncytial virus (rsv), human metapneumovirus (hmpv), parainfluenza and influenza viruses, adenoviruses and coronaviruses are also thought to contribute to the exacerbation of asthma [5, 6] . the characteristics of the main viruses are listed in table 1 . for some of these viruses, there is evidence from both epidemiological and experimental studies to confirm the role of a viral infection in exacerbating asthma, while for other viruses the evidence is less clear. the immune responses triggered by viral infections and allergens are well established (fig. 1 ). this review focuses on the association between viruses and the exacerbation of asthma and outlines current understanding of the molecular and cellular mechanisms involved. the inflammatory cascade associated with asthma involves mast cells, dc, t cells, eosinophils, macrophages, fibroblasts and neutrophils (table 2) . when the inflammation intensifies, the airways become very sensitive to provoking stimuli and airway hyper-responsiveness (ahr) develops. the mast cells degranulate and they, and other cells, release chemical mediators into the lower respiratory tract that prolong the response and cause contraction of the bronchial smooth muscles (table 2) . these actions, and airway swelling, mucus secretion and inflammation, contribute to the bronchoconstriction and airway obstruction seen in asthma attacks. a number of factors are known to affect the likelihood of asthma being exacerbated in response to a viral infection (fig. 2 ). these factors include the age at which the first viral infection occurs, the gender, genetic makeup of the host, the ability of the virus to persistently infect or to become latent in the host and viral antigens. viral infections that occur early in life appear to have an important role in shaping immunological development. if initial infection occurs early in life, the t helper 1 (th1) response may be weak, allowing the development of stronger th2 responses to challenges later in life. mice initially infected with rsv as neonates were more likely to have a th2 type response upon secondary infection, whereas those first infected when older were more likely to have a th1 type response [7] . one interpretation of these findings is that rsv infection in neonates suppresses the development of the th1 immune response and a th2 response occurs. in contrast, if the initial infection is delayed for several weeks, a th1 immune response is mounted which lacks the pathogenic th2 component associated with asthma. there is some evidence that infection at one stage in life may lead to a reduction in atopy while, at another stage, it toll like receptor tnf-a tumour necrosis factor-alpha may potentiate the emergence of allergies [8] and that early severe rsv infections in infancy leads to allergic asthma later in life [9, 10] . however, this issue is controversial. boys are more likely to have severe asthma than girls; however at puberty (about age 13e14) the ratios reverse and many more adult women are admitted to hospital for asthma than adult men [11] . the reasons for this switch in susceptibility are not known but this observation suggests a potential role for sex hormones in the development of asthma. a predisposition towards developing asthma runs in families, indicating a genetic component to the disease that has been confirmed by studies in twins [12] . specific genes have been associated with asthma in nearly 200 studies, with 64 different genes associated with asthma or atopy identified in one study [13] . some of these genes have repeatedly been found to be associated with asthma, including the th2 cytokines il-4 and il-13, human leukocyte antigen drb1 (hla-drb1), tnf-a, lymphotoxin-alpha (la-a), high-affinity ige receptor (fc3r1a) and the il-4 receptor (il-4r). the gene b2 adrenergic receptor (adrb2) has been associated with asthma severity [13] . other genes associated with atopy and asthma include, for atopy, pattern recognition receptor cd14 (receptor for endotoxins such as lipopolysaccharide (lps)) and granulocytemacrophage colony stimulating factor (gmcsf); for asthma, clara cell secretory protein (cc16), cd14, acyloxyacyl hydroxylase (aoah), tnf-a, leukotriene c4 (ltc 4 ) synthase, il-10 and il-8; and, for asthma severity, adrb2 [14, 15] . host genetics is also thought to affect the severity of viral infections and the type of immune response to the infection. for example, specific alleles of pulmonary surfactant genes have been associated with more severe rsv infections in infants [16] . surfactant proteins line the alveolar surface of the lungs and are essential for normal respiratory function. other genetic variations have been shown to affect the expression of cytokines in response to viral infections. a variation causing an increase in il-8 transcription was found to lead to enhanced susceptibility to rsv induced bronchiolitis [17] and a variant affecting transcription of il-4 was found to be associated with severe rsv disease and elevated levels of ige [18] . similarly, variations in genes for il-10 have been identified, with those people heterozygous for the gene found to be less likely to develop rsv bronchiolitis [19] , and two distinct mutations in the gene for toll like receptor 4 (tlr-4) have been found to be associated with severe rsv bronchiolitis [20] . an association between differences in il-13 genes and severe rsv infection has also been found [21] . environmental factors, such as viral infections, may impact on genetically predisposed individuals to affect the development of asthma and the viral exacerbation of asthma. a number of respiratory viruses have been found to persist in the host, despite the host mounting an immune response. for example, hmpv can persist in the lungs of mice for up to 60 days, despite the presence of neutralizing antibody [22] . in mice, rsv was found to persist in the lungs for more than 100 days, despite normal cytotoxic t-cell responses and normal rsv specific antibodies [23] and viral proteins have been found in guinea pigs 6 weeks after rsv infection, despite the presence of neutralizing antibodies [24] . the evidence about respiratory viral persistence in humans is less clear, with several recent studies finding directly opposing results. a study by wilkinson et al. found that rsv persisted in patients with chronic obstructive pulmonary disease (copd) [25] , while falsey et al. found no rsv persistence associated with copd [26] . studies on viral persistence with the other main virus associated with the exacerbation of asthma, rv, have also produced opposing results. in one study, 95% of adults with an exacerbation of asthma were found to be clear of rv 6 weeks after the exacerbation [27] , whereas a study in children found that more than 40% still had detectable rv after 6 weeks and this viral persistence was associated with more severe acute exacerbations of asthma [28] . a possible mechanism for immune evasion is persistence at a site that avoids immune system surveillance. with respiratory viruses, such a site may be the pulmonary neurons and rsv infection of nerve cells has recently been demonstrated [29] . in addition, a role for cytokine in viral persistence was demonstrated in mice by alvarez and tripp. they showed that the persistent presence of hmpv is associated with increased il-10 expression and weak ctl activity [30] . it is currently not clear whether respiratory viruses persist in the host, despite a functional immune response. further characterization is required to understand the possible relationships between respiratory viral latency, the host's immune responses and the exacerbation of asthma. it is well established that rsv g protein can influence the immune response in murine models. the g protein seems to elicit pre-dominantly a th2 driven response characterized by lung eosinophilia and local production of il-4 and il-5 [31] . prior sensitization with a recombinant vaccinia virus expressing rsv g protein leads to a th2 driven augmented disease, contrasting with the usual th1 response seen in primary viral infections [31] . studies have also shown that the non-structural proteins of rsv and influenza virus are capable of modulating the immune response through their ability to inhibit ifn responses in the infected host [31] . released mediators derived from neural signalling pathways contribute to the bronchoconstriction associated with asthma. studies in humans and animals have shown that an intact nervous system contributes to virus-induced ahr. in a guinea pig model, cutting the vagus nerve reduced airway responsiveness to histamines in animals previously infected with parainfluenza virus [32] . in humans, virus induced ahr was also vagally mediated [33] . in guinea pigs, rats and cats, the efferent, parasympathetic bronchoconstrictor limb of the ). the production of il-12 and the binding of antigen-mhc molecules commit the differentiation of na€ ıve t cells to the th1 cell subset that secretes th1 cytokines including ifn-g. the cytokine contributes to the activation of macrophage (make more il-12), b cells (make igg2a) and cytotoxic t cell (kill infected cells). an ifn-g dominated microenvironment inhibits the development of a th2 cell subset. together, these responses result in the resolution of the infection in the airways. (b) in the early phase of allergen exposure, cross linking of antigen-specific ige on the surface of mast cells results in the activation and release of mediators that cause bronchoconstriction and inflammation. activated mast cells also produce il-4 that commits na€ ıve t cells to the th2 subset as well as b cell isotype class switching to ige production. in addition, antigenic peptide is presented to na€ ıve t cells by apcs in the context of mhc class ii and co-stimulatory signals. in an il-4 dominated microenvironment, this triggers the differentiation of na€ ıve t cells to th2 cell subset that generates th2 cytokines (il-4, il-5, il-10 and il-13). these cytokines are responsible for orchestrating the late phase of the allergic response. il-4 and il-13 contribute to mast cell activation and the synthesis of ige. il-5 is implicated in eosinophilia and is known to stimulate these cells, resulting in degranulation and release of toxic basic proteins (e.g. ecp, mbp). il-10 inhibits apcs, therefore preventing il-12 from initiating a th1 immune response. cough and bronchoconstriction reflexes was enhanced during acute viral illness [32, 34] . acetylcholine is released from the nerve fibres that innervate smooth muscle in the airways and binds to m 3 muscarinic receptors on the smooth muscle and causes bronchoconstriction. this is normally limited because acetylcholine also binds to m 2 muscarinic receptors on neurons that inhibit further release of acetylcholine [35] . dysfunction of these m 2 receptors is thought to contribute to ahr during viral infections [34, 36] . a number of factors are thought to cause this receptor dysfunction including viral neuraminidase [37] , endogenous tachykinins [35] , induced inflammatory cell products, such as eosinophil cationic proteins (ecp) [37] , eosinophil major basic protein (mbp) [38] , ifns from macrophages or macrophage-stimulated t cells or nitric oxide (no) released from virus-activated macrophages [39] . the effects on the m 2 receptor function are transient and normal functions are restored several weeks after resolution of the acute infection [34] . stimulation of the nerve fibres during viral infection augments the release of neuropeptides, including somatostatin, calcitonin gene-related peptide (cgrp) and the tachykinins substance p and neurokinin a. a number of these neuropeptides are associated with symptoms of the exacerbation of asthma. for example, cgrp can cause eosinophilia in rat lungs [40] , substance p and neurokinin a are associated with an increase in eosinophil numbers in the airways [41] , and substance p activates eosinophils and mast cells and enhances the degranulation of eosinophils and the release of histamines from mast cells [42] . cgrp also inhibits the production of th1 cytokines, therefore unbalancing the th1/th2 equation in favour of a th2 response [43] . a role for the neurotrophin nerve growth factor (ngf) in viral induced asthma is suggested by results finding that ngf is increased in rsv infected rats, compared to control animals [44] . ngf up-regulates the expression of the high affinity receptor for substance p (the nk1 receptor) and substance p contributes to the neurogenic inflammation seen in rsvinfected airways [45] . ngf expression in the lungs normally declines as animals age but rsv infection interferes with this decline [44] . ngf may lead to short-and long-term changes in the distribution and development of nerves and changes in the reactivity of sensory nerves in the respiratory tract [44] and may also contribute to ahr, inflammation and the activation of recruited eosinophils and mast cells [46] . in understanding the mechanisms underlying viral exacerbation of asthma, it is helpful to observe models of primary virus infection. respiratory viruses including parainfluenza, influenza and rsv have been shown to induce airway inflammation and lung function changes in rodent models. in rsv infection in mice, airway inflammation and the development of ahr are dependent on the presence of the th2 cytokines il-5 [47] and il-13 [48] and not on ifn-g, the most abundant cytokine produced during rsv infection. in addition, inflammation and lung function changes in this model depend on cd8 þ t cells [49] . taken together this suggests that type 2 cytokine producing cd8 þ t cells may drive virus induced reactive airway disease. further, respiratory viral infections lead to a marked expansion of mature dc in the lung [50] . these mature lung dc have a strong capacity to activate both na€ ıve and memory t cells and to induce their proliferation. in a model of influenza infection, maturation of lung dc resulted in strong immunogenicity of an otherwise non-immunogenic antigen [50] . increases in numbers of lung dc, which arise from local precursors in the lung, outlast the resolution of disease in rsv infection [51] . importantly, pulmonary dc can stimulate t-cell responses in the lung in situ without migration to the regional lymph nodes, favouring th2 and tc2 responses [52] . these observations suggest that respiratory viral infections lead to a prolonged period of increased antigen presentation in the airways resulting in de novo and memory t-cell responses not only to the virus but also to unrelated antigens including allergens. in addition to studies of primary infections, models studying the interactions between respiratory viral infections and allergen sensitization are essential in understanding the mechanisms of virus induced asthma exacerbations. many small animal models were designed to reveal the pathogenic mechanisms behind the enhancement of allergic sensitization by respiratory virus infections; the increased airway inflammation and responsiveness resulting from allergic airway sensitization following respiratory viral infection and, in those with established allergic airway sensitization, the increased airway inflammation and responsiveness due to respiratory viral infections. these studies show that the immune responses to allergen sensitization and respiratory viral infections interact to cause persistent inflammation and ahr, symptomatic of the asthmatic response (fig. 2) [53] . many studies have been done in animals sensitized to different allergens and then infected with respiratory viruses, a model that mimics viral exacerbation of asthma in sensitized individuals. in one study, guinea pigs were sensitized with ovalbumin (ova) and challenged with rsv and those that underwent both treatments were found to have more severe symptoms of ahr and inflammation (fig. 3) [53] . similar results were also found in mice sensitized to ova and then infected with rsv [54] , mice sensitized to ova and challenged with murine cytomegalovirus [55] , and mice sensitized to the house dust mite dermatophagoides farinae then infected with rsv [56] . experiments have been performed to determine the contribution of released inflammatory mediators on ahr following viral infection. il-4 is important in the exacerbation of asthma but the response is not solely dependent on il-4 and il-5 has been observed to increase in virally infected sensitized mice and has been directly associated with the viral exacerbation of asthma [57] . il-10 is thought to be necessary for the development of allergic ahr [58] , however, its role in viral exacerbation of ahr is less certain. the role of il-10 is complex, inhibiting il-5 production, eosinophilic inflammation and chemotaxis but potentially inducing a decrease in th1 cytokine production [59] . mice deficient in il-10 that were both sensitized (to ova) and infected with rsv did develop ahr including eosinophilic inflammation, th2 cytokine production and goblet cell hyperplasia. inhibition of il-13 has been shown to significantly reduce ahr in sensitized and infected mice, to the level of that seen in mice only infected or only sensitized [60] . these factors all appear to contribute in varying extents to the viral exacerbation of asthma. the cysteinyl leukotrienes (cyslts) such as ltc 4 , ltd 4 and lte 4 are products of activated eosinophils, basophils, mast cells and macrophages. these potent inflammatory mediators have a diverse range of biological activities, including the ability to exacerbate asthma by inducing the contraction of bronchial smooth muscle [61] . ltc 4 has been found at elevated levels in the nasopharyngeal secretions of children during the acute phase of rsv infection, with higher levels in patients with lower respiratory tract involvement than in those with upper respiratory illness alone [62] . lte 4 has been found to be elevated in urine samples collected from patients with asthma exacerbations [63] . cyslt levels have also been found to be increased in the lower airways during rsv bronchiolitis, although their concentrations are lower than those in acute asthma [64] . in mice, rsv infection has been found to produce a marked increase in cyslts in the balf and lung tissue, resulting in the recruitment of neutrophils and lymphocytes into the airways and ahr [65] . cyslts have been found to have multiple effects in animals, including the induction of th2 responses in the lungs through effects on dcs and cytokine generation, the recruitment and activation of cells such as eosinophils and mast cells, and inflammation [63] . interestingly, an lt synthesis blocker prevents the development of rsv induced lung function changes in mice [66] and montelukast, a cyslt antagonist, seems to reduce post-bronchiolitis wheeze in small children [67] . bronchial reactivity to stimulation by histamine has been found to be increased in asthmatic patients following infection with influenza a virus [68] . human rv infection in adults with mild asthma often has little effect on the lower respiratory tract or lung function [69] ; however, when infection in asthmatics is followed by allergen challenge, ahr and eosinophilia in balf have been observed for up to a month following challenge [70] . deliberate infection with rv has found that asthmatic symptoms tended to peak later during infection and were more pronounced and persistent during the later stages of infection [71] . although nasal and upper respiratory symptoms were similar between asthmatics with high ige levels, asthmatics with low ige levels and control subjects before infection, asthmatics with higher ige levels had more symptoms of lower respiratory inflammation before and during infection. asthmatics with high ige levels also had lower lung function results before infection, while the results for the asthmatics with low ige levels were similar to the controls. however, the lung function tests results did not vary significantly during the course of rv infection [71] . a similar study of people with allergic rhinitis (hay fever) found that rv infection increased ahr and the inflammatory reaction to allergen challenge. before rv infection, most subjects developed only short-term responses to the allergen; however, during rv infection nearly all subjects developed late asthmatic reactions [70] . these late asthmatic reactions were independent of changes in airway reactivity or the size of the immediate response to allergen challenge, and the propensity to develop them persisted for up to 4 weeks after virus infection [70] . a similar study found that the allergic response changed from nearly all subjects having only an immediate response before infection to a majority of subjects having both an immediate and late asthmatic reaction during or following rv infection [72] . changes associated with rv infection and subsequent allergen challenge include an immediate increase in the release of histamine and the recruitment of eosinophils into the airways within a 48 h period [73] . a study of peripheral blood mononuclear cells from patients with atopic asthma and normal controls found that exposure to rv induced the production of ifn-g, il-12, il-10 and il-13 in both groups, with significantly lower levels of ifn-g and il-12 and higher levels of il-10 in the cells from asthmatics than the cells from normal subjects. il-4 was induced only in the asthmatic group and the ifn-g/il-4 ratio was more than three times lower in this group. this suggests that the normal type 1 immune response to rv infection is defective in atopic asthmatic individuals, with infection inducing a th2-immune response similar to their response to allergens [74] . vaccines and therapeutics have the potential for eliminating or reducing the synergy between viral infections and asthma, as well as decreasing unrecognized but associated disease burden and health care costs. vaccination is the mainstay of prophylaxis for respiratory viruses; however, for some viruses such as rsv and rv, developing safe and effective vaccines has been difficult. despite the need for anti-viral drugs as an adjunct to vaccines, few are available that are effective. therefore, novel and new approaches are highly desirable. recently, mahalingam and tindle have demonstrated the efficacy of an hmpv ctl epitope vaccine that triggers a strong th1 immune response which was associated with accelerated viral clearance in a murine model [75] . in addition, rna interference (rnai), a naturally occurring process for controlling gene expression that occurs through a process mediated by short interfering rna (sirna) molecules, has been shown to have the remarkable ability to silence rsv replication both in vitro and in vivo (tripp et al., unpublished data). these approaches can be used to target host cell genes to silence aspects of the inflammatory pathway that include th1 or th2 cytokines, chemokines or proteins that regulate their expression. thus, disease intervention strategies that target the virus and/or host response are rational approaches for breaking the link between the synergy of virus infection and asthma. wright and nhmrc peter doherty fellowships respectively. s.m. acknowledges support from the nhmrc for research on respiratory viral infections (project grant #399701). k.m. acknowledges support from nhmrc programme grant #224207. r.a.t. acknowledges the georgia research alliance for their continued support. j.s. acknowledges support from the wellcome trust (senior fellowship grant #067454). we thank mr timothy lewis for excellent editorial assistance. the international study of asthma and allergies in childhood (isaac) steering committee, worldwide variation in prevalence of symptoms of asthma, allergic rhino conjunctivitis and atopic eczema national health survey: summary of results australian centre for asthma monitoring, in: asthma series 2, woolcock institute of medical research/australian institute of health and welfare data fact sheet: asthma statistics, national institutes of health: national heart, lung, and blood institute community study of role of viral infections in exacerbations of asthma in 9e11 year old children respiratory viral infections as promoters of allergic 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development of allergic asthma? il-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection il-13-induced airway hyperreactivity during respiratory syncytial virus infection is stat6 dependent cd8 t cells are essential in the development of respiratory syncytial virus-induced lung eosinophilia and airway hyperresponsiveness influenza virusinduced dendritic cell maturation is associated with the induction of strong t cell immunity to a coadministered, normally nonimmunogenic protein local cd11cþ mhc ii precursors give rise to pulmonary myeloid dendritic cells and are depleted in the process after rsv infection resident lung antigen-presenting cells have the capacity to promote th2 t cell differentiation in situ allergic sensitization increases airway reactivity in guinea pigs with respiratory syncytial virus bronchiolitis prior airway exposure to allergen increases virus-induced airway hyperresponsiveness murine cytomegalovirus infection alters th1/th2 cytokine expression, decreases airway eosinophilia and enhances mucus production in allergic airway disease recurrent respiratory syncytial virus infections in allergen sensitized mice lead to persistent airway inflammation and hyperresponsiveness timing of infection and prior immunization with respiratory syncytial virus in rsv-enhanced allergic inflammation il-10 is necessary for the expression of airway hyperresponsiveness but not pulmonary inflammation after allergic sensitization the failure of il-10 deficient mice to develop airway hyperresponsiveness is overcome by respiratory syncytial virus infection in allergen sensitized/challenged mice respiratory syncytial virus induced airway hyperresponsiveness is independent of il-13 compared with that induced by allergen cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses the release of leukotrienes in the respiratory tract during infection with respiratory syncytial virus: role in obstructive airway disease recovery of leukotriene e4 from the urine of patients with airway obstruction increased levels of bal cysteinyl leukotrienes in acute rsv bronchiolitis role of cysteinyl leukotrienes in airway inflammation and responsiveness following rsv infection in balb/c mice zileuton reduces respiratory illness and lung inflammation, during respiratory syncytial virus infection, in mice study group on montelukast and respiratory syncytial virus. a randomized trial of montelukast in respiratory syncytial virus postbronchiolitis bronchial reactivity following uncomplicated influenza a infection in healthy subjects and in asthmatic patients rhinovirus-16 colds in healthy and in asthmatic subjects. similar changes in upper and lower airways rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions experimental rhinovirus challenges in adults with mild asthma: response to infection in relation to ige experimental rhinovirus 16 infection potentiates histamine release after antigen bronchoprovocation in allergic subjects a common cold virus, rhinovirus 16, potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects a defective type 1 response to rhinovirus in atopic asthma cytotoxic t-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice key: cord-262104-oig3qrr7 authors: brüssow, harald title: covid‐19: test, trace and isolate‐new epidemiological data date: 2020-06-08 journal: environ microbiol doi: 10.1111/1462-2920.15118 sha: doc_id: 262104 cord_uid: oig3qrr7 in the absence of an efficient drug treatment or a vaccine, the control of the covid‐19 pandemic relies on classic infection control measures. since these means are socially disruptive and come with substantial economic loss for societies, a better knowledge of the epidemiology of the new coronavirus epidemic is crucial to achieve control at a sustainable cost, and within tolerable restrictions of civil rights. this article is protected by copyright. all rights reserved. comprising 10'800 participants (gudbjartsson et al., 2020) . very similar information was reported in data describing household transmission in wuhan, where children showed a 4% infection rate compared with 17% in adults. . likewise, in a study from hunan children had a threefold lower infection risk than adults . in contrast, the infection risk in children from shenzhen, china was similar to that in adults (bi et al., 2020) . however, all studies concur that disease in children is generally mild, if not asymptomatic. asymptomatic cases raise problems for contact tracing and containment, but it is currently not clear to what extent infected children transmit the disease. three asymptomatic sars-cov-2-positive adolescents showed transmission to family members (liao et al., 2020) while data for transmission from children are still absent. one special problem should still be mentioned in this context: in italy, pediatric hospitalization decreased substantially during the covid-19 epidemic. when the general population avoids hospitals for fear of infection, it can have a negative health impact. an increased number of deaths occurred in italy due to delayed arrival of children in hospitals, while no child died from covid-19 in italy (lazzerini et al., 2020) . the fear of hospitals has also been responsible for a deficit in the treatment against stroke in adults in the us (kansagra et al., 2020) . the statistics for the covid-19 epidemic have shown that the older population has suffered the greatest loss of life, and that nursing homes have been hotspots for transmission. two detailed studies from the us document these given facts. at the end of february 2020, a cluster of 167 epidemiologically linked covid-19 cases were reported in several long-term care facilities in washington state. of those, 144 cases were residents (median age 83 y), 55% of them were hospitalized, and 33% died. in comparison only 16 cases had been visitors, 50% of whom were hospitalized, but none died. further 50 cases were among the health care personnel, of whom only 6% were hospitalized, but again none died. factors favoring the outbreak were: health care personnel who showed up to work with symptoms; some of the personnel worked at more than one facility; and some residents were transferred between facilities. in the early stages of the epidemic, contributing factors to this outbreak were: an unawareness of the risk; a lack of diagnostic tests; and inadequate personal protection equipment (mcmichael et al., 2020) . currently, 1.3 million us americans reside in nursing homes. one in ten of >1'300 accredited nursing facilities reported covid-19 cases. an epidemiological survey in such a facility demonstrated the extent of the problem: the first infection in this nursing home was introduced by a symptomatic nurse, and then a week later the first resident tested positive. a further 5 days later, half of the residents from this unit tested positive for viral rna. during the following 2 weeks, 30 to70% of the residents in other units of this home became infected, along with 19% of the staff. notably, more than half of the residents were asymptomatic when they tested positive for viral rna. four days later, 89% of them had developed symptoms. the rest remained asymptomatic. mortality was high at 26%. importantly, asymptomatic, pre-symptomatic, and symptomatic residents did not differ with respect to viral load and infectious virus release. all viruses showed identical genome sequences guangdong cases. in guangdong province of china, all return visitors from wuhan/hubei province, their contacts, and all of the local hospitalized patients were tested for viral rna. 1.6 million tests were used to identify 1'400 sars-cov-2-positive cases; 1000 patients had had exposure to infected people from hubei. half of the local transmissions occurred within households. by mid-february, the local spread was controlled, but in march new cases were imported from abroad. sputum samples showed the highest viral titers, followed by oropharynx, stool, and finally nasopharynx samples. critical and severe cases showed higher viral titers than moderate and mild cases, but the differences were small. viruses from 53 patients were sequenced and compared with 177 sars-cov-2 sequences deposited in the database. single nucleotide polymorphism (snps) were detected at 97 nucleotide positions scattered through the viral genome; 77 variant sites were only seen in a single virus isolate. on a phylogenetic tree, the guangdong sequences were interspersed between the viral sequences from wuhan and those isolated abroad, documenting a recent, single source outbreak of a virus showing a low mutation rate (lu et al., 2020a) . usa west-to-east spread. the first case of covid-19 in the us was reported on jan 19 at the northwest coast of washington state and was imported from china. from march 1 to 19 the number of cases in the us increased from 70 to 13'000. epidemiologists investigated the first nine covid-19 cases on the east coast (connecticut) that were observed in mid-march with genome sequencing. only one sequence exactly matched the viral sequences from china, but the patient had not traveled to china. the viral sequences from seven further patients, clustered with a large us clade known from washington state, documented a rapid west-to-east national spread of the novel coronavirus in the us. international air travel restrictions had no, or low, impact on the epidemic spread in the us. the viral genome was rooted in a single ancestor coronavirus in wuhan by fewer than 10 mutations, and it had accumulated about 2 nucleotide changes per month during its spread across the us, which is a low mutation rate for a 30'000 nucleotide long viral rna genome. with the portable oxford nanopore technologies minion platform, viral genomes were sequenced within 14 hours after having received the sample, theoretically allowing near real-time molecular epidemiology of the epidemic spread (fauver et al., 2020) . china. in wuhan, 105 index cases of patients suffering from moderate covid-19 symptoms (fever, cough, fatigue) were investigated for secondary transmission to 392 household contacts. the average household size was 4 persons. the index case persons remained at home for a documented number of days before seeking medical advice. in total, 64 contacts (16%) were infected, 9 without symptoms, and the rest experienced moderate disease. the time lapse between primary and secondary infections was 6 days. the transmission probability was age-dependent: it was 4% in children and 17% in adults. the highest transmission rate was seen in 50 to 60 year-olds, but not in >60 year-old household members. spouses experienced a 28% infection transmission rate. when the index case was quarantined directly after symptom onset, transmission rate was 0% . in shenzhen, 391 cases were recorded, 77% of which were detected through the surveillance of symptoms. most cases were mild (26%) or moderate (65%). only 9% of infections were associated with severe disease, which correlated with male sex and older age; 3 patients died. the researchers identified 1'281 close contacts for the index cases; 8% of these had become infected. an increase to 16% in the rate of infection was seen in those who lived or traveled with index case persons. interestingly, infection risk was comparable for all age groups ranging from 0 to 70 y old. however, half of the children showed no fever, and severe infections were rare in people under the age of 40. notably, 80% of secondary infections were traced to only 9% of the index case persons (bi et al., 2020) suggesting an important role of "super-spreaders" in infection transmission. it would be helpful for public health if characteristic traits of super-spreaders were known. adding viral-specific igg antibody tests to the toolbox of covid-19 have allowed the connection between three previously separated infections clusters in singapore. an infected traveler from wuhan attended a church meeting, thereby infecting a secondary person (case x), who transmitted the virus to another subject during a family gathering, who then transmitted the virus to a large number of people in a second church. case x tested negative for viral rna and represented the missing link between the events. case x showed a strong serological response to sars-cov-2, then connecting the links in the chain (yong et al., 2020) . iceland. from among 9000 high risk icelanders (persons who were symptomatic, or had contact with infected persons, or who traveled to a high risk country) 13 % tested positive for sars-cov-2 rna. children under 10 years from that high risk group showed a 7% rna detection rate compared with 14% in subjects older than 10 y. before mid-march, travel exposure to austria and italy was a common denominator in the positive subjects. after mid-march, travel to uk was the biggest risk factor. in mid-march, a representative sample of 11'000 subjects from the general population of iceland (360'000 inhabitants) was tested, and 0.8% tested positive for sars-cov-2 rna. in april, another sample of 2000 subjects from the general population showed a similar rate of viral excreters. in the general population, males were more frequently virus-positive than females. the highest prevalence was seen in 40 y-old subjects, while no children under 10 y were infected. five hundred viral genomes, isolated from infected icelanders, were sequenced. the genomes were clustered into 42 distinct clades. during the early epidemic phase in iceland, nearly all sars-cov-2 isolates belonged to the a2 clade which was also frequently found in central european populations. viral genome sequencing identified networks of up to 14 linked infections. transmission occurred in the early phase via international travel, but later via infection from family members (gudbjartsson et al., 2020) . airborne: cough, sneeze and speech. australian engineers evaluated the literature about the reach of pathogen transmission by coughing and sneezing. the 1 to 2 meter rule has been set since the 1940s by photography, physical calculations, and through simulations. distinct models (turbulent jets vs. puffs) have been used, but not all used appropriate parameters for humidity and temperature. it remains unclear which conditions apply to the human respiratory excretions when handling infected patients in a clinic (bahl et al., 2020) . newer high speed pictures of a coughing volunteer show a turbulent jet plume that extends over 0.5 meter (tang and settles, 2020) . high speed pictures of a sneezing volunteer revealed exhaled air, muco-salivary filaments and drops. the turbulent puff cloud disintegrated into droplets that settled within 1 to 2 m distance. some droplets evaporate and become suspended in the puff and travel a room within a few minutes to land 6 to 8 m away from the sneezing person (bourouiba, 2020) speech generated droplets in front of the mouth increased with the loudness of the voice. holding a cloth in front of the mouth suppressed the droplet detection (anfinrud et al., 2020) . a who communication led to a controversy saying that there is not enough evidence that sars-cov-2 transmission is airborne. who defines airborne transmission as being via aerosols as opposed to transmission by droplets. since most covid-19 transmission seems to occur through close contact, droplets have been considered to be the more likely vehicles. whatever the exact transmission route by air, avoiding crowds, standing next to a person for too long, and increasing the rate of ventilation in closed rooms without air recirculation are reasonable precautions (lewis, 2020) . physicians recommended face mask use, even when scientific evidence for its effectiveness is lacking, in comments to leading medical journals. recent reviews have concluded that: no randomized trials with masks have been conducted; that the benefit of masks over no masks (but not of respirator masks over paper masks) was shown in an influenza epidemic in australia; that some benefit of masks was seen when worn by symptomatic, but not by asymptomatic cases during an influenza epidemic; that no data exist which directly support the use of mask wearing by the public; that no significant effect was seen for household use of masks against influenza transmission. therefore, who initially recommended masks only for symptomatic cases. cdc first advised against mask use by the public but have now changed their policy by recommending even self-made cloth masks for wide use. harm (e.g. increase of co 2 level under the mask) is low if not used by small children or elderly people with disabilities (cheng et al., greenhalgh et al., 2020) . as assessed by viral rna detection, air and surfaces in a wuhan hospital were widely contaminated during the height of the epidemic in china. contamination levels were greater on the intensive care unit ward than on the general ward. the transmission of sars-cov-2 might reach up to 4 meters distance. however, no medical staff in that particular hospital was infected. the authors admit two limitations of the study which prevent firm conclusions from being made. first, viral rna detection does not mean infectious virus detection, and second, the minimum infectious dose of sars-cov-2 for humans is unknown . when reviewing the official recommendations, the consensus seems to be to use a respirator for high risk interventions which create aerosols in the icu, and to use surgical masks on the general ward with low risk activities (bahl et al., 2020) . singapore hospital. the environment of three covid-19 patients from a ventilated hospital infection ward in singapore was tested for viral rna presence by rt-pcr. after routine cleaning of high touch areas and of the floor, no viral rna was found in the air, on hospital room surfaces or on the personal protection equipment of the treating physician. before routine cleaning, however, 16 of 20 room sites (table, chair, floor, window, toilet) and the shoe protection of a physician tested positive. room air samples and hospital corridor floors were, however, negative for viral rna. for infection control, regular room cleaning and handwashing were judged to be essential (ong et al., 2020) . primit study. it is more problematic to establish a control when a family member with mild infection remains at home. there is only one behavioral intervention study that has proved to reduce respiratory viral transmission within households, the primit germ defense study. the key interventions are web-based instructions about handwashing given to 10'000 intervention subjects in uk, but not to 10'000 controls. infection transmission was reduced by 20% and infection severity was also reduced, albeit modestly (little et al., 2015) . the rationale behind the idea was to reduce the viral load by which contacts are particularly exposed, such as through hand-to-eye contact, since the conjunctiva supports sars-cov-2 replication (hui et al., 2020; little et al., 2020) . in italy, the epidemic started in lombardy and veneto. lombardy strengthened their hospital capacity and increased icu beds, while veneto opted for strict containment and mass testing in 4% of the population. lombardy experienced 47'000 more cases than veneto and also had a much higher crude case fatality rate (18% vs 6%). the higher death rate is probably explained by the delayed public health response (odone et al., 2020) as also seen in the us (anonymus, 2020) . the swedish government had recommended a number of trust based measures (social distancing from old people, handwashing, home office, travel reduction), but refrained from closing borders, schools, restaurants and bars, partly because the swedish law does not allow lockdowns. the case reduction of seasonal influenza and norwalk diarrhea provided documented effectivity of the measures taken (paterlini, 2020) . however, compared with neighboring finland and norway, sweden experienced a tenfold higher number of deaths for a 1.5 fold larger population, but the absolute numbers are still small (3700 vs. 300 for sweden and finland, respectively, may 18). in the us, the individual states and cdc have many legal options for quarantine (for the segregation of exposed people) and isolation (separation of infected people from the general population) in such cases as with sars. the establishment of broad sanitary cordons in which entire geographical areas are quarantined (as has happened in wuhan) will raise constitutional questions in the us. the us recommendations say that patients who show mild symptoms should stay home and notify their employer electronically. low-wage workers cannot afford to stay off work, but the us senate is in the process of establishing bills for paid sick leave and unemployment insurance (parmet and sinha, 2020) . kong. the control measures that stopped the epidemic locally have included: intense infection surveillance of incoming travelers; isolation of covid-19 cases in hospitals; contact tracing and quarantine in holiday camps; and school closure but no lock-down, thus preventing the crisis from having a negative economic impact. a total of 715 cases were confirmed, half imported, and the rest locally transmitted with a reproduction number that quickly decreased to values around 1; and 13% of virus-positive subjects were asymptomatic. the control measures also stopped an ongoing seasonal influenza epidemic. surveys showed that the population agreed to participate in the measures. they kept social distancing and made behavioral changes (99% wearing masks outside house, 93% increased hand hygiene, 83% staying at home as much as possible). the study could not differentiate the impact of each individual measure. unfortunately the full effect of school closure is still unknown because the susceptibility of children for covid-19 and their capacity to transmit the infection has not yet been established . china has contained the covid-19 epidemic through a combination of different measures including drastic ones. an international team of epidemiologists developed a computer model that described the dynamics of the epidemic and tested the impact of the different containment measures by using computer simulations. without any intervention, a 100-fold higher number of cases would have occurred in china, resulting in over 10 million cases. without travel restrictions, the epidemic would have expanded more widely over the western provinces. early detection and isolation of patients reduced the number of cases by 5fold, while social distancing and contact reduction led to a 2.6-fold reduction. however, without contact reduction, the epidemic would have, over time, increased exponentially across the regions. initiating the intervention one week earlier would have decreased the number of cases by 66% or, if done one week later the number of cases might have increased by 3-fold. a delay of 2 or 3 weeks would have increased cases by 7-and 18-fold, respectively. lifting travel restrictions will result in a new rise in case numbers, but even moderate levels of social distancing could keep this increase in check. partial maintenance of npi may prevent, or at least delay, the arrival of second wave infections (lai et al., 2020) . contact surveys were conducted in wuhan and shanghai during the height of the covid-19 epidemic in china. before the epidemic, people reported between 15-18 contacts (two-way conversations, physical contact) per day. this number was reduced to 2 during the containment period. contact reduction was most significant for school-age children who, before the intervention, reported the greatest numbers of contacts out of all of the age groups, followed by adults at the workplace. during containment, contacts were mainly within families (80-95%). the survey was consistent with data from inner city mobility. all contacts of patients in hunan province were placed under medical observation and tested for excretion of viral rna. from these contact data it was deduced that children (<14 y) had an infection rate that was only a third as high as adults (15-65y), while older individuals had a 50% higher infection rate than young adults. based on these data and on a mathematical infection model, the authors concluded that social distancing alone is sufficient to control covid-19 spread. proactive school closure alone cannot interrupt transmission but can reduce the peak incidence of the disease by half, and it can delay the epidemic ). these are model simulations based on assumptions on infection transmission by children for which only few data are currently available. the reopening of schools in several countries will hopefully settle some questions with observational data. psychologists argue that contact-seeking is a basic human response to danger. this inclination takes over when an invisible infection threat is perceived. this instinct is only opposed by disgust when infected persons show appalling clinical signs which is not the case for sars-cov-2 infected subjects in pre-symptomatic or asymptomatic state. it will be increasingly difficult for health authorities to impose social distancing, as proven by the street demonstrations against containment measures in the us and in european countries by differently motivated opposition groups. the authors argue that the increased use of the internet as a substitute for contact can become an important public health tool to achieve physical distancing without social distancing (dezecache et al., 2020) . by may 18, the johns hopkins university registered 4.7 mio cases worldwide. the lion's share is from the us with 1.4 mio cases, compared with 84'000 cases reported by china. one should interpret these data with caution. the definition for a confirmed case of covid-19 was changed five times in china, which accounts for the increase in knowledge about the epidemic. scientists from the who collaboration center in hong kong calculated that when the fifth version is applied, the total number of cases in china would increase from 55'000 to 230'000, but the transmission patterns in mainland china would not change . the case number also depends on the intensity of viral testing and the capacity of the public health system to report the number of cases. with nearly 90 '000 deaths, the us number greatly surpasses the 4'600 deaths reported in china. it is still difficult to assess the morbidity and mortality impact of the covid-19 pandemic on the population. even mortality rate is not a clear figure since it is reported differently in different countries. while death is a clear diagnosis, the cause of death isn't. it might not be evident whether somebody died with or from covid-19, particularly in nursing homes. some countries attribute death to covid-19 if the virus was present at death. others attribute each death in nursing homes to covid-19 during the height of the epidemic -as was done in belgium, which explains its high mortality data. many deaths occurred while people also had underlying health problems (comorbidities), therefore they were already at increased risk of death. an international consortium of demographers called for the publication of excess mortality data. by comparing mortality statistics for a given epidemic period with a corresponding time period during previous years without that epidemic, the absolute impact of an infection can be assessed. such data are still largely lacking in the literature. excess mortality rates should best be calculated for both sexes and for each 5-y age range separately (leon et al., 2020) . first data have just now been reported: in march/april the care sector of england and wales alone has seen 20'000 excess deaths over the figures of previous years. covid-19 deaths at care homes were three times as high as covid-19 deaths in hospitals (burki, 2020) . excess mortality calculations have been done globally for seasonal influenza, arriving at 300'000 to 600'000 influenza-associated deaths occurring annually (iuliano et al., 2018) . while the majority of influenza mortality applies to elderly people, the death rate is also substantial for children at an estimated 34'000 deaths in 2018 . in comparison, the global death toll of covid-19 is now 315'000 (status may 18). a direct comparison of these two figures is difficult for two reasons: death levels are affected by vaccination campaigns against seasonal influenza and by strict containment measures for covid-19. it seems plausible that without any containment measures covid-19 mortality would surpass greatly the number of deaths from seasonal influenza. the presumption that the covid-19 mortality is comparable to that of seasonal influenza deaths is fundamentally flawed because it compares numbers which are obtained by different methods. the death rate for covid-19, which has just crossed the 100'000 figure in the us, is an actual count of dead patients. in contrast, the 23'000 to 61'000 annual deaths from seasonal influenza quoted after influenza epidemics in the us are estimates by the cdc of influenza deaths based on calculations from models. the death counts actually reported to us health authorities ranged from 3'400 to 15'000 deaths per year during an influenza epidemic in the us. expressed as deaths per peak week, influenza claimed a maximum 1'616 deaths per week, while covid-19 took about 15'000 lives per week at its peak in the us (faust and del rio, 2020) . wuhan. an international consortium of epidemiologists has estimated that 66'000 people were infected in wuhan and that 2800 have died; 70% were infected through household contact, 25% through public contact, and 5% in hospitals. from a peak number of 2000 new infections, wuhan currently has 10 or fewer new infections per day. safe strategies are now needed for the exit from lockdown measures. when lifting the lockdown to a 100% pre-quarantine social contact level, a computer model showed that a 95% face mask wearing would be needed to ensure a complete elimination of infections. in contrast, with only 50 % face mask use and a lifting date of strict measures before april 25 to pre-quarantine level, the conditions in this model would lead to a major second wave of infection. maintaining a contact rate below pre-quarantine level combined with a high percentage of face mask wearing is essential while now the restrictions have been lifted in wuhan-at least until a vaccine becomes available. however, face mask provision for such large populations represents logistical challenges and must not cause a shortage of protective gear for health personnel (zhang et al., 2020a) . in mainland china, 13'400 confirmed cases and 120 deaths were reported by march 18. in beijing and shenzhen, most cases have been imported from wuhan and the reproduction number r remained below 1.0. in shanghai and wenzhou, local cases dominated but r rose to greater than 1 for only one january week. case fatality was 1% compared with 6% in hubei. relaxing the restrictions could lead to a second wave of exponential infection from imported cases in a nonimmune, susceptible population. maximizing economic productivity under the < 1 constraint can, according to this study, only be possible with a real time prevalence determination of new infections through extensive testing . other epidemiologists working on outbreak data from mainland china observed a sub-exponential increase of cases from the beginning, instead of an expected exponential growth for an unconstrained epidemic. model calculations showed that the containment measures (the quarantine of exposed, and the isolation of infected persons) which depleted the number of susceptible individuals for the virus, reproduced the actually observed case development. similar strategies are recommended in the event of a future outbreak (maier and brockmann, 2020) . harvard model. epidemiologists from harvard university derived projections from model calculations about the future dynamics of the covid-19 epidemic. when anticipating short term immunity (as observed for seasonal common cold coronaviruses), they predict annual winter epidemics for sars-cov-2. with intermediate levels of immunity persistence, epidemics would become biannual. long-term immunity (as in the case of sars-cov) would lead to the extinction of the virus, even in the absence of social distancing. they also calculated that it needed 20 weeks of social distancing to reduce the peak number of infected persons. if the reproduction of the virus is reduced by more than 60% through lockdowns, the infection peak is predicted to shift to the next winter season with high numbers because no herd immunity has been achieved (kissler et al., 2020) . a central concept of epidemiology is herd immunity; the percentage of persons with protective immunity needed in a population to stop the propagation of an infectious agent. when this threshold is crossed, the remaining susceptible persons are protected from infection. the threshold level depends on the "force" of the infectious agent which is expressed by the basic reproduction number r 0 ,which is defined as the number of secondary infections caused by an index case. in infection modeling, herd immunity threshold and r 0 are linked by a simple mathematical function. sars-cov-2 has a higher r 0 "infectious force" than influenza virus, but a much lower one than "flying infections" such as chickenpox or measles. it is anticipated that a population needs a herd immunity of 50 to 80 % protected people to stop the covid-19 epidemic. the initial strategy of the uk government was to let the epidemic roll over the country to achieve this herd immunity, in contrast to containment policies which prevent exposure, but which also prevent immunity development in the population. this strategy has theoretical advantages (fewer economic losses when a lockdown is avoided, and a protected population in the event that no vaccine becomes available). however, it comes at a cost. if you allow, let's say, 60% of the population to get infected, you can calculate the cost of this strategy with the help of the infection fatality rate (ifr). in contrast to the case fatality rate (cfr) which expresses the number of deaths per clinically ill patients (which varies from 1.4% to 15% for covid-19), ifr is the number of deaths per infected individual. the number is, of course, lower than crf. while we know, approximatively, the number of covid-19 deaths and covid-19 cases, we do not definitively know the number of infected persons, since this would require large and systematic seroprevalence studies, but which are lacking. current estimates suggest 0.6% as a realistic approximation for ifr. with that figure, one can calculate that achieving herd immunity through natural infection with sars-cov-2 would cost the lives of 150'000 uk citizen or more than 1'000'000 us citizens. this death toll was considered as too high by the uk government, which then changed strategy by declaring a late containment strategy (randolphe and barreiro, 2020) . delays in imposing containment measures were predicted to lead to 10-fold or higher number of cases and fatalities. this prediction might explain why the us and uk have such high case and death statistics in international comparison. mobile phone technology. epidemiologists from the uk and us have developed a real-time datacapture platform applicable for mobile phone use for the self-guided collection of population-level data (cope consortium). the app queries location, age, health risk factors and asks daily for new symptoms and diagnostic test results. a test run with 1.6 million users in uk showed that the most common symptoms were fatigue and cough. anosmia (loss of smell) appeared as a strong predictor of covid-19, while fever was not a diagnostic criterion unless combined with other symptoms. the reported symptoms predicted that there would be changes in the number of cases as indeed reported from health authorities 5 to 7 days later. the tool will be important for a controlled safe exit from confinement measures. also, long-term effects of the disease, and the impact of the covid-19 epidemic on social relations, mental health, and financial outcome can be evaluated with this tool. machine learning could also reveal new disease manifestations of the epidemic (drew et al., 2020) . anosmia is an interesting symptom. a preliminary evaluation of a covid-19 symptom tracker smartphone app from uk users showed that the loss of smell was reported by 59 % of people with respiratory infection who tested positive for sars-cov-2, compared with 18% of respiratory patients who tested negative. clinical criteria which allow a diagnosis of covid-19 without a viral rna test would be welcome for mass screening and telemedicine in an epidemic situation. french physicians have also reported that many covid-19 patients reported loss of smell and loss of taste, without nasal congestion. when these criteria were combined in a retrospective questionnaire this combination of signs had a sensitivity of 42% and a specificity of 95% for detecting covid-19 patients (bénézit et al., 2020) . these observations are not surprising since the highest expression level of the sars-cov-2 receptor ace-2 was shown in the respiratory tract, more specifically in the nasal epithelia. us physicians even suspected that sars-cov-2 might, in addition to the respiratory and alimentary tract, also infect cranial nerves (i.e. being neurotropic) which potentially explains the observation of neurological signs in 9 % of covid-19 patients (chu et al., 2020) . seroprevalence studies. antibody tests are an important tool in a staggered release of population groups out of lockdowns because such tests identify people who have been exposed to the infection and who are potentially immune to infection. so far, only preliminary data became available from 3'300 volunteers from santa barbara county in california. one out of 66 (1.5%) showed antibodies to sars-cov-2. this number is 50-fold higher than the number of the official case count was for this area in early april. in a preliminary survey in geneva, less than 5 % of a population sample showed viral-specific antibodies in preliminary surveys. in a town with 12'000 inhabitants in germany, 500 people were antibody tested following carnival parties and an infection rate of 15% was determined. these datasets cannot be extrapolated to the population at large. in addition, the antibody tests were only validated with a small set of positive and negative test samples, raising concerns about the reliability of the results (mallapaty, 2020; sood et al., 2020) . the next challenge will be the acquisition of reliable antibody data for representative samples from entire populations. the covid-19 epidemic continues to challenge our societies by its toll in deaths, by the disruption of social life; by its disastrous impact on the world economy; by increasing the debt of many nations; by endangering the survival of many industries; and by reversing the worldwide trend for poverty relief. for microbiologists, the covid-19 crisis has also revealed shortcomings in the public health sector, particularly in that of countries which were exemplary in this field during past decades. in the us, the covid-19 epidemic has taken more lives in one month than over 8 years during the vietnam war. with more than 1.7 million cases, and more than 100'000 deaths, the "america first" slogan has become sadly ironic in the context of covid-19. an article in the leading us medical journal, the new england journal of medicine, attributes this calamity to insufficient diagnostic testing caused by the delivery of faulty tests by cdc; non-approval through the fda of working tests by who resulting in a delayed start of viral detection activities; and then followed then by a shortage of test reagents. public health workers were therefore blind to the unfolding of the us epidemic and unable to design efficient containment measures, short of a lockdown. epidemiologists were left without population data for modelling the epidemic in the us at a moment when the country started reopening economic and public activity. in comparison with other countries the us has tragically "failed the test". in the words of this article "the us once a leader, seem oddly lost" (schneider, 2020) . an editorial in the leading british medical research journal, the lancet, comes to the same conclusion: the cdc, once a pillar and international reference for combating diseases worldwide, instrumental in eradicating smallpox and coping with aids or ebola, has lost its technical competence and public trust due to contradictory scientific messages and the undermining of trust in scientific evidence by the current us administration. according to the lancet editors, the "us administration is obsessed with magic bullets-vaccines, new drugs-while only basic public health principles, like test, trace, and isolate, will see the emergency brought to an end" (the lancet, 2020). the situation is not better in the uk, once also renowned for its excellent public health research, particularly in the field of respiratory infections (remember the common cold research unit). at the end of may, the uk directly follows the us in the international mortality ranking list with more than 38'000 covid-19 deaths. the late onset of large scale testing, the lack of personal protective equipment, and a delayed introduction of containment measures have certainly contributed to this high death toll. a correspondent to the lancet deplores that the situation in europe was no better with respect to the lack of a coordinated response to the pandemic. the european center for disease prevention and control (ecdc), which was established in 2004 to create a complement to the us cdc, failed to become a hub in europe of knowledge for covid-19 and a coordination center for europe-wide epidemic counter-strategies. ecdc is underfunded (cdc in 2020: 8 billion $, ecd 60 million $) and understaffed (cdc: 10'800, ecdc: 270 employees). an emergency structure for a pandemic was not set up, and ecdc played essentially no role in pandemic crisis management, which was done according to eu laws by national organizations without any european coordination (jordana and triviño-salazar, 2020) . even the city-state of singapore, where the early handling of the covid-19 was lauded as exemplary public health action, had "blind spots" on their screen in overlooking the miserable living conditions of migrant workers that became hotspots of covid-19 transmission. of special global health concern are refugee camps from bangladesh to europe. a refugee camp on lesvos/ greece has just one water tap per 1300 residents, making efficient handwashing an impossible mission (newland, 2020) . governments plan to spend billions on rescuing what they consider to be essential national industries. it will be important that they also find the money needed for covid-19 containment among migrant workers, refugees and populations at risk in developing countries . the beneficial epidemic effect of the lockdowns, obtained at enormous economic costs, would be canceled out if a second wave epidemic should start from these settings with relatively unrestrained viral transmission. at present it is not clear which institution, if not the united nations' suborganizations, will be able to implement such measures. when the leading nation of the western hemisphere leaves now the who, this is a disastrous signal for global public health at this crucial moment of the covid-19 pandemic. 2020) visualizing speech-generated oral fluid droplets with laser light scattering 100'000 and counting. the economist presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility airborne or droplet precautions for health workers treating covid-19? utility of hyposmia and hypogeusia for the diagnosis of covid-19 epidemiology and transmission of covid-19 in 391 cases and 1286 of their close contacts in shenzhen, china: a retrospective cohort study investigation of a covid-19 outbreak in germany resulting from a single travel-associated primary case: a case series a sneeze england and wales see 20 000 excess deaths in care homes wearing face masks in the community during the covid-19 pandemic: altruism and solidarity comparative tropism, replication kinetics, and cell damage profiling of sars-cov-2 and sars-cov with implications for clinical manifestations, transmissibility, and laboratory studies of covid-19: an observational study impact assessment of non-pharmaceutical interventions against coronavirus disease 2019 and influenza in hong kong: an observational study 2020) pandemics and the great evolutionary mismatch rapid implementation of mobile technology for real-time epidemiology of covid-19 assessment of deaths from covid-19 and from seasonal influenza coast-to-coast spread of sars-cov-2 during the early epidemic in the united states asymptomatic transmission, the achilles' heel of current strategies to control covid-19 face masks for the public during the covid-19 crisis spread of sars-cov-2 in the icelandic population aerosol and surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards temporal dynamics in viral shedding and transmissibility of covid-19 estimates of global seasonal influenza-associated respiratory mortality: a modelling study where are the ecdc and the eu-wide responses in the covid-19 pandemic? collateral effect of covid-19 on stroke evaluation in the united states tropism, replication competence, and innate immune responses of the coronavirus sars-cov-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures projecting the transmission dynamics of sars-cov-2 through the postpandemic period effect of nonpharmaceutical interventions to contain covid-19 in china delayed access or provision of care in italy resulting from fear of covid-19 covid-19: a need for real-time monitoring of weekly excess deaths first-wave covid-19 transmissibility and severity in china outside hubei after control measures, and second-wave scenario planning: a modelling impact assessment is the coronavirus airborne? experts can't agree the characteristics of household transmission of covid-19 epidemiological and clinical characteristics of covid-19 in adolescents and young adults an internet-delivered handwashing intervention to modify influenza-like illness and respiratory infection transmission (primit): a primary care randomised trial reducing risks from coronavirus transmission in the home-the role of viral load genomic epidemiology of sars-cov-2 in guangdong province sars-cov-2 infection in children effective containment explains subexponential growth in recent confirmed covid-19 cases in china antibody tests suggest that coronavirus infections vastly exceed official counts epidemiology of covid-19 in a long-term care facility lost in transition covid-19 deaths in lombardy, italy: data in context air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2(sars-cov-2) from a symptomatic patient children with covid-19 in pediatric emergency departments in italy covid-19 -the law and limits of quarantine closing borders is ridiculous': the epidemiologist behind sweden's controversial coronavirus strategy herd immunity: understanding covid-19 failing the test -the tragic data gap undermining the u.s. pandemic response seroprevalence of sars-cov-2-specific antibodies among adults coughing and aerosols reviving the us cdc covid-19: pcr screening of asymptomatic healthcare workers at london hospital effect of changing case definitions for covid-19 on the epidemic curve and transmission parameters in mainland china: a modelling study global burden of respiratory infections associated with seasonal influenza in children under 5 years in 2018: a systematic review and modelling study virological assessment of hospitalized patients with covid-2019 connecting clusters of covid-19: an epidemiological and serological investigation detection of covid-19 in children in early changes in contact patterns shape the dynamics of the covid-19 outbreak in china what is required to prevent a second major outbreak of sars-cov-2 upon lifting the quarantine of wuhan city, china. preprint to the lancet acknowledgements i thank jacqueline steinhauser and sophie zuber for critical reading of the manuscript. the author consults nestlé, his former employer, on the scientific aspects of the covid-19 epidemic, but he does not consider this as a conflict of interest. key: cord-257255-n5o368ih authors: barker, j.; stevens, d.; bloomfield, s.f. title: spread and prevention of some common viral infections in community facilities and domestic homes date: 2001-12-21 journal: j appl microbiol doi: 10.1046/j.1365-2672.2001.01364.x sha: doc_id: 257255 cord_uid: n5o368ih nan nearly one thousand different types of viruses are known to infect humans and it is estimated that they account for approximately 60% of all human infections (horsfall 1965) . viruses are spread easily through closed environments such as the home, schools, workplaces, transport systems, etc. although many of the respiratory and gastrointestinal infections caused by viruses can be asymptomatic or relatively mild and self-limiting (coughs and colds, etc.), they still represent a signi®cant economic burden. increasing numbers of people who have reduced immunity to infection, for whom the consequences of infection can be much more serious, are now cared for at home. 1 at risk groups include not only the immunocompromised but also the elderly, neonates, pregnant women, hospital patients discharged into the community, individuals using immunosuppressive drugs and also those using invasive systems (indwelling catheters) or inhalation systems or devices. otherwise healthy family members with asthma or allergies also have increased susceptibility to infection. in the uk it is estimated that one in six people in the community belong to an`at risk' group (bloom®eld 2001) . world health organisation estimates suggest that, by 2025, there will be more than 800 million people over 65 years old in the world, two-thirds of them in developing countries (anon. 1998) . viruses are probably the most common cause of infectious disease acquired within indoor environments. close personal contact within the home and community settings, such as daycare centres and schools, makes them ideal places for the spread of viral infections. infected individuals can shed up to 10 12 virus particles per ml of faeces with the possibility of transfer of the virus by contaminated hands to surfaces in the bathroom or toilet. viruses that cause tonsillitis, colds, croup, bronchiolitis, in¯uenza, pneumonia and other respiratory tract infections can be spread in aerosolized droplets. aerosols produced by coughing, sneezing and talking can be inhaled directly by a susceptible host or may settle onto surfaces. touching hands or fomites, such as eating utensils, towels or doorknobs, inadvertently contaminated with fresh secretions or vomit, etc. from an infected person and then transferring the virus from the hands to the eyes, nose or mouth, are further routes of spread. infants are especially vulnerable to such infections because they frequently place objects, such as toys, into their mouths. transfer of viruses to food during handling and preparation via hands and food contact surfaces is an important route of spread of viral gastroenteritis. amongst health care professionals there is growing awareness that improved standards of hand, surface and air hygiene in community settings could do much to prevent the spread of viral infections within these environments. the purpose of this paper is to review the evidence base for this assumption. since viral infections are not easily treated, prevention of infection is still the main route of control. assessment of the impact of hygiene is made dif®cult by the general lack of quantitative epidemiological data and, even where evidence for cross-contamination as a causative factor in an outbreak exists, it is always circumstantial. a further problem in assessing whether contamination found on hands or other surfaces might represent a hazard is that the infectious dose can vary signi®cantly according to the pathogenicity of the organism and the immune status of the host. thus the case for practising good hygiene in these settings rests largely on evidence showing that cross-contamination can occur in these environments coupled with laboratory data demonstrating the ef®cacy of hygiene procedures in minimizing microbiological contamination. in developed countries it is estimated that 30±40% of infectious gastroenteritis cases are attributable to viruses (thompson 1994) . surveillance data from the uk show that reported outbreaks of viral intestinal infection have increased rapidly over the last 10 years; epidemiological data for 1995 and 1996 (evans et al. 1998) show that rotavirus, astrovirus, norwalk-like viruses (nlvs; also known as small round structured viruses) and other caliciviruses were responsible for 48% of all reported outbreaks of infectious intestinal disease (iid). other data indicate that nlvs and rotavirus are the commonest pathogens causing outbreaks of gastroenteritis in homes for the elderly (djuretic et al. 1996; ryan et al. 1997; dedman et al. 1998) . over the period 1993±96 a uk study involving some 460 000 participants was carried out to evaluate rates of iid in the community and presenting to general practice which has given valuable insights into the epidemiology of viral infections in the community (wheeler et al. 1999) . the study indicated that as many as one in ®ve people in the general uk population develop iid each year with an estimated 9á4 million cases occurring annually. it has long been recognized that, since cases and outbreaks related to viral agents are often unreported, the impact of viral intestinal infections may be much greater than national surveillance suggests. the ®ndings of the community study con®rmed the validity of this assumption. wheeler and co-workers estimated that for every one case of rotavirus and nlv reported to national surveillance a further 35 cases of rotavirus and 1562 cases of nlv occur in the community. uk surveillance covering 1995±96 showed nlv as a signi®cant cause of epidemic gastroenteritis in community residential and nursing homes, accounting for 43% of all reported general gastroenteritis outbreaks (evans et al. 1998) . the rate of reported nlv infection reaches a peak in children under 5 years and again in the elderly. foodborne outbreaks can arise from contaminated raw food such as shell®sh and also through secondary contamination from food handlers carrying the virus. foods implicated in outbreaks are mainly those eaten raw, or those not cooked after handling, e.g. salads, cold meats and fruit. worldwide, rotavirus is probably the most important viral pathogen causing diarrhoeal disease in infants, infecting virtually all children aged 3±5 years (parashar et al. 1998a ). however, a recent study by pang et al. (2000) of children between 2 months and 2 years of age with acute gastroenteritis, has shown that human caliciviruses are found as commonly as rotaviruses. in developing countries rotavirus accounts for approximately 6% of all diarrhoeal episodes and 20% of all diarrhoea-associated deaths of children under 5 years of age, resulting in an estimated 800 000 childhood deaths each year. it is second only to upper respiratory infections in infants under 2 years old as a major cause of death in the developing world (glass et al. 1997) . each year in the us, 2á7 million children under 5 years old are affected by rotavirus diarrhoea, resulting in 500 000 visits to the doctor and 50 000 hospitalizations (parashar et al. 1998b) . a large proportion of hospital admissions due to gastroenteritis in children under 5 years old were caused by rotavirus in both the uk and hong kong chan et al. 1998) . in older children 51% of hospital admissions for acute diarrhoea were associated with rotavirus (lewis et al. 1979) . a study by isaacs et al. (1986) showed that 20% of children under 14 years old who visited their doctor with diarrhoea had a rotavirus infection. indications are that hospital admissions only represent a small percentage of rotavirus infections; the majority will be treated by general practitioners. rotavirus infections are highly seasonal, peaking in the winter months (brandt et al. 1982; ryan et al. 1996; dedman et al. 1998) . it has been suggested that low humidity and people spending more time indoors contribute to the spread of rotavirus infections (anon. 1995) . such conditions may make it possible for the virus to be spread by the airborne route through environmental contamination (brandt et al. 1982) . a study in the us revealed that rotaviruses infected one or more members in 51% of families, including 28% of children and 13% of adults (rodriguez et al. 1987) . within infected families rotavirus infection was found in 57% of children and 25% of adults. some adults acquired rotavirus infections a few days after their children's illnesses, suggesting that the children rather than the parents brought infection into the home. rodriguez et al. (1979) also found rotavirus infection in 55% of adult family contacts of children hospitalized with gastroenteritis. in a community study in new zealand, in families with an index case of rotavirus infection, children were more frequently infected than adults. once a family member became infected there was a high probability of crossinfection (grimwood et al. 1983 ). among children with diarrhoea attending daycare centres, lew et al. (1991) detected astroviruses and adenoviruses. astrovirus was signi®cantly more common in children with diarrhoea than those without diarrhoea. enteric adenoviruses were detected in an equal percentage in children with and without diarrhoea. children can excrete astrovirus before the onset of diarrhoea and up to 20 d after the diarrhoea has stopped (mitchell et al. 1993) . although astrovirus primarily infects the young, the elderly can also be affected, with reported outbreaks in care homes and hospital wards for the elderly (gray et al. 1987; lewis et al. 1989) . enteric adenoviruses (generally serotypes 40 and 41) are also associated with outbreaks of gastroenteritis in schools, paediatric hospital wards and nursing homes (lebaron et al. 1990 ). they may be second to rotavirus as a cause of gastroenteritis in young children (blacklow and greenberg 1991) . globally, hepatitis a virus (hav) is the most common cause of hepatitis in man (melnick 1995) . contaminated water or food, particularly ®lter-feeding shell®sh, frequently transmit hepatitis a but other foods are occasionally implicated (raw milk, dairy products and cold meats). the virus is excreted in high numbers in faeces and is spread from person to person primarily by the faecal±oral route. when personal hygiene is not observed, food handlers may unintentionally transfer the virus to food during the incubation period of the disease (sundkvist et al. 2000) . outbreaks of viral hepatitis occur in institutions such as daycare centres, hospitals, nurseries and schools (bern et al. 1992; dickinson 1992) . these outbreaks may lead to secondary cases in the general community (hadler et al. 1980) . infections caused by in¯uenza viruses, rhinoviruses, coronaviruses and respiratory syncytial viruses (rsvs) are a major health burden. estimates suggest that adults suffer two to ®ve colds per year and infants and preschool children have about four to eight colds per year (sperber 1994) . although such infections are often regarded as trivial, taking into account lost days from work and school, hospital admissions and mortality rates in infants and the elderly, the health and economic costs are considerable. although the common cold can be caused by a number of viruses, rhinoviruses and coronaviruses predominate. rhinoviruses are responsible for outbreaks of the common cold in the general community such as schools, daycare centres and hospitals (denny et al. 1986; krilov et al. 1986; kellner et al. 1988) . rhinoviruses and coronaviruses have been found to cause a greater disease burden in elderly people living at home, compared with in¯uenza virus or rsv (nicholson et al. 1997) . in¯uenza affects all age groups, but it is the elderly and persons with underlying health problems who are at particular risk from complications of in¯uenza and are more likely to require hospitalization. respiratory syncytial virus infections occur all over the world and outbreaks are common in the cold season in temperate climates and in the rainy season in tropical climates. respiratory syncytial virus is a major cause of respiratory illness in young children, affecting about 90% of children by the age of 2 years (crowcroft et al. 1999; simoes 1999) . school-aged children often carry rsv to their homes and spread infection to younger siblings. attack rates within families are high, with about 40% of family members, including adults, becoming infected. in most family outbreaks although more than 95% of infections are symptomatic they are not usually severe (berglund 1967; hall et al. 1976a) . infants admitted to hospital with rsv bronchiolitis or pneumonia tend to shed the virus abundantly and for prolonged periods allowing ample opportunity for spread (hall et al. 1976b) . in adults rsv infection generally results in a`common cold' type illness although it can sometimes produce a`¯u-like' syndrome indistinguishable from in¯uenza. antibodies resulting from an early childhood rsv infection do not prevent further rsv infections later in life. respiratory syncytial virus is known to cause a high incidence of pneumonia and death in the elderly. in england and wales it is estimated that rsv causes 60±80% more deaths than in¯uenza, causing about 23 000 deaths each winter (nicholson 1996) . parain¯uenza viruses (pivs) are a further major group of respiratory pathogens. they cause severe colds, croup, bronchitis and pneumonia in children and adults and in infants the virus can cause life-threatening disease (hall 1987) . infection is probably spread by aerosols in addition to direct contact with contaminated surfaces (hall et al. 1980) . brady et al. (1990) noted that the persistence of piv on hospital surfaces contaminated with patients' secretions was a potential source of transmission. cross infection from an infected person to a new host depends on a number of factors, including the number of virus particles shed by the infected person, their stability in the environment, in aerosols or on surfaces and the potential for spread within a closed environment (valenti 1998) . viruses that increase¯uid secretions or irritate the respiratory epithelium induce coughing and sneezing, which in turn increases the shedding and transmission of the virus. although diarrhoea eliminates organisms from the gut, it increases the potential for contamination of the environment and spread of the virus infection. the more particles shed the greater their survival and the greater the chance of reaching a new host. equally important, the likelihood of cross infection depends on the number of particles that reach the new host, the immune status of that host and the route by which they become infected. virus particles can be shed in large numbers in various body¯uids from an infected person or a carrier, including blood, faeces, saliva, urine and nasal secretions. the nonenveloped viruses have greater resistance to drying and thus spread more easily than enveloped viruses, which are less stable in the environment. laboratory studies show that rotavirus, adenovirus, poliovirus, herpes simplex virus and hav can survive for signi®cant periods on dry surfaces (nerurkar et al. 1983 2 ; abad et al. 1994) . although few studies have been carried out in domestic homes, studies in children's daycare centres (lew et al. 1991; keswick et al. 1983b; butz et al. 1993 ) and in hospitals (samadi et al. 1983; akhter et al. 1995) show that viruses can survive on surfaces and that virus transfer and survival on hands play a part in the transmission of infections. bellamy et al. (1998) investigated the domestic environment for the presence of viruses and body¯uids that may contain viruses. haemoglobin was found on 2% of surfaces (taps, washbasins, toilet bowls and seats), indicating the presence of blood and possible contamination with bloodborne viruses. amylase (an indicator of saliva, sweat and urine) was found on 29% of surfaces, which were frequently handled or in contact with urine. this highlights that surfaces may remain soiled for some time and may not be thoroughly cleaned. bellamy et al. (1998) also detected enteroviral rna in three of 448 environmental samples (tap handle, telephone handpiece and toilet bowl). a previous study has shown that enteric viruses can survive on environmental surfaces for up to 60 d (abad et al. 1994) . virus transmission in childcare facilities was studied using modi®ed cauli¯ower virus dna as an environmental marker (jiang et al. 1998) . the viral dna, introduced through treated toy balls, spread within a few hours of handling. although the marker treated objects were removed after 1 d, the viral dna continued circulating in the facilities for up to 2 weeks. hand contact with contaminated surfaces played an important part in transmission. the markers were also detected in the children's homes, on the hands of family members and environmental surfaces, including toys. transmission of viruses in a household setting has been recently studied, using bacteriophage /x174 as a model virus with resistance properties similar to polio-or parvoviruses (rheinbaben et al. 2000) . contaminated door handles and skin surfaces were found to be ef®cient vectors of contamination. at least 14 persons could be contaminated one after another by touching a contaminated door handle. successive transmission from one person to another could be followed up to the sixth contact person. transfer from contaminated door handles to other surfaces was also con®rmed under everyday life conditions in a¯at shared by four students. studies focusing on home and community settings are providing a better understanding of how infectious disease is spread in these environments. such studies suggest that the airborne route is by no means the sole route of transmission of respiratory infections caused by rhinovirus and rsv and that iid, particularly that of viral origin, can arise from various sources of which food is only one. evans et al. (1998) reported that, whereas 174 of 233 outbreaks of infection attributed to salmonella were`mainly foodborne' and 15 regarded as`mainly person to person', for 680 reported outbreaks of nlv infection 607 were attributed to personto-person transfer and only 21 were reported as foodborne. although epidemiological and surveillance studies provide vital information on modes of infection transmission during outbreaks, they give only a limited picture of how sporadic person-to-person transmission actually occurs within community and home environments. although such data are limited, it is generally acknowledged that person-to-person transmission is associated not only with poor hand hygiene but also airborne or surface-to-surface transmission. what cannot be deduced from current data is the relative importance of these different modes of person-to-person transmission and how it may differ for different viral agents and different communities (home, daycare, etc.). in particular, it is dif®cult to assess the importance of environmental contamination as a source of secondary cases, but recent data show that this can be a signi®cant factor, particularly in the transmission of nlvs (anon. 1993) . although direct evidence is lacking, outbreaks in hotels and cruise ships in which recurrent waves of infection occurred in successive cohorts of guests strongly suggests transmission of nlv via environmental sites and surfaces (ho et al. 1989; gellert et al. 1994; mcevoy et al. 1996; cheesbrough et al. 2000) . these and other studies which can be used to assess the routes of transmission of viral infections in community and home settings are discussed in the following sections. 4.1.1 rotavirus. rotavirus is shed in large numbers from an infected person, with faeces often containing > 10 12 particles per gram. children and adults can be asymptomatic excreters of rotavirus (ansari et al. 1991a ) and rotavirus excretion can persist for up to 34 d after diarrhoea has stopped in symptomatic patients (pickering et al. 1988 ). more recently, a hospital study showed that 30% of the immunocompetent children excreted rotavirus particles for more than 21 d and as long as 57 d after the onset of diarrhoea (richardson et al. 1998) . rotavirus can survive on human hands and transfer of infectious virus to animate and non-porous inanimate surfaces has been demonstrated (sattar et al. 1986; ansari et al. 1988 ). ward et al. (1991) examined the transfer of rotavirus from contaminated surfaces to the mouth and from surfaces to hands to the mouth. all of the volunteers who licked rotavirus-contaminated plates became infected whereas, of those individuals touching the virus-contaminated plates with their ®ngers and then their mouths, only about half became infected. a number of studies in child daycare centres have shown that rotavirus can be widely disseminated when outbreaks occur. in one such centre faecal contamination of hands and the environment was demonstrated during an outbreak of rotavirus diarrhoea (keswick et al. 1983a) . other studies in daycare centres have shown that 16±30% of surfaces sampled can be contaminated with rotavirus. in particular, hand contact surfaces (e.g. refrigerator handle, toilet handles, telephone receivers and toys) and moist surfaces such as sinks, water fountains and water-play tables were contaminated with the virus (keswick et al. 1983a; wilde et al. 1992; butz et al. 1993) . soule et al. (1999) found that there was an increase in the number of environmental surfaces contaminated with rotavirus in a hospital paediatric unit when there was an increase in the number of children suffering from rotavirus gastroenteritis. of the surfaces in direct contact with children (thermometers, play mats and toys) rotavirus was detected in 63% of samples compared with 36% for surfaces without direct contact (telephones, door handles and washbasins). these ®ndings were similar to those of akhter et al. (1995) who showed that widespread rotavirus contamination of a paediatric ward and playroom correlated with the presence of patients infected with rotavirus. in a treatment centre in bangladesh, handwashings from 78% of the attendants of patients with diarrhoea (children under 5 years) were positive for rotavirus antigens (samadi et al. 1983) . rotavirus was also found in handwashings of 19% of attendants of patients with non-rotavirus diarrhoea, indicating that they may have come into contact with other attendants and patients in adjacent beds. this highlights the potential for contaminated hands to spread the infection. projectile vomiting associated with nlvs is probably a major source of cross-infection because it is estimated that 3´10 7 particles are distributed as an aerosol into the environment during a vomiting attack (caul 1994) . a recent report (anon. 1993) showed how aerosols produced by vomiting can be inhaled or can contaminate hands or work surfaces, with the potential for subsequent transfer to foods or direct hand-to-mouth transfer. the importance of airborne transmission was demonstrated in a recent outbreak in a restaurant where no food source was detected but an analysis of the attack rate showed an inverse correlation with the distance from a person who had vomited (marks et al. 2000) . the potential for secondary transmission via environmental surfaces in semiclosed communities was demonstrated following a wedding reception, where an outbreak of nlv gastroenteritis affected 50% of guests (patterson et al. 1997) . the previous day, a kitchen assistant had vomited in a sink that was subsequently used for preparing vegetables eaten by the wedding guests. further evidence for transmission of nlv via aerosols and environmental surfaces comes from reports of recurrent waves of nlv gastroenteritis occuring in successive cohorts of guests on a cruise ship over a 2-year period (ho et al. 1989) . it was found that the risk of gastroenteritis amongst passengers who shared toilet facilities was twice that of those who had a private bathroom. nosocomial spread is also a major concern. norwalk-like virus gastroenteritis in an elderly care unit in a hospital spread rapidly within and between wards, affecting both patients and staff. analysis of risk exposure showed areas where patients had vomited to be the most signi®cant factor for the spread of nlvs to staff (chadwick and mccann 1994) . a study by cheesbrough et al. (1997) showed that carpets can also harbour nlvs and serve as reservoirs of infection. two carpet ®tters became ill after removing a carpet from a hospital ward 13 d after the last case in a nlv outbreak. routine vacuuming every day since the outbreak had not removed the virus. during an outbreak of vomiting and diarrhoea due to nlv in a long-stay ward for the mentally ill, 36 environmental samples were collected on the affected ward of which 11 (30%) were positive by reverse transcriptase-polymerase chain reaction. positive swabs were from lockers, curtains and commodes and were con®ned to the immediate environment of the affected patients (green et al. 1998) . most recently the potential for environmental spread of nlvs was demonstrated in a prolonged hotel outbreak in successive cohorts of guests (cheesbrough et al. 2000) . environmental sampling demonstrated widespread dissemination of the virus on hand contact and other surfaces. from the patterns of infection it was concluded that, although infectious aerosols were probably the main route of dissemination of infection within a particular cohort of guests, contact with contaminated fomites was the most likely factor responsible for maintaining the outbreak by forming the link between successive cohorts. as with other enteric viruses hav is shed from an infected person in large numbers and is able to survive on environmental surfaces (mbithi et al. 1991) and be readily transferred to hands (mbithi et al. 1992) . fomites are potential risk factors in the spread of the virus, especially in hospital wards, daycare centres or restaurants (cliver 1983) . a recent outbreak of hav was associated with a public house whose barman had chronic diarrhoea and had served drinks while incubating hepatitis a himself (sundkvist et al. 2000) . fomite transmission by contamination of glasses was the likely route of spread. assessments of community outbreaks of hav have shown that persons involved in nappy-changing in daycare centres often handle food and that this is a potential risk for transmission (hadler and mcfarland 1986) . hepatitis a virus may be acquired from children who are excreting hav, the majority of whom are asymptomatic (fox et al. 1974) . a signi®cant percentage (23±52%) of susceptible household contacts of index cases with acute hav infection are at risk of acquiring acute hav infection from the index case (minuk et al. 1994) . a higher rate of hav infection amongst children (71±80%) compared with the parents (29%) suggests that play activity among children is a signi®cant factor for hav transmission in households. it is generally accepted that respiratory viruses, such as those which cause the common cold and¯u, are spread from person to person by aerosol transmission due to sneezing and coughing. nevertheless, there is growing evidence that a signi®cant proportion of¯u and particularly cold viruses are spread via hands and surfaces such as handkerchiefs and tissues, tap and door handles, telephones or other surfaces touched by an infected person (eccles 2000; goldman 2000 3 ). cross infection can occur either by handshaking or by touching the contaminated surface. rubbing either the nasal mucosa or the eyes with virus-contaminated hands can cause infection. sattar et al. (1993) have shown that rhinoviruses can survive on environmental surfaces for several hours. infectious viruses have been recovered from naturally contaminated objects in the surroundings of persons with rhinovirus colds (reed 1975) . clean hands can readily pick up the virus by touching or handling such objects (ansari et al. 1991b) . as much as 70% of infectious rhinovirus on contaminated hands has been shown to transfer to a recipient's ®ngers after contact of only 10 s (gwaltney et al. 1978) . after handling contaminated coffee cup handles and other objects, more than 50% of subjects developed an infection (gwaltney and hendley 1982) . hendley et al. (1973) and reed (1975) have demonstrated that rhinoviruses can survive for several hours on the hands and selfinoculation by rubbing of the nasal mucosa or conjunctivae via virus-contaminated ®ngers can lead to infection. in¯uenza virus can be shed before the onset of symptoms and for up to 7 d after onset and individuals with in¯uenza can be infectious before they develop symptoms and for up to a week afterwards. both in¯uenza a and b virus have been shown to survive on hard surfaces such as stainless steel and plastic for 24±48 h and on absorbent surfaces such as cloth, paper and tissues for up to 12 h (bean et al. 1982) . it was shown that in¯uenza a virus could be transferred from contaminated surfaces to hands for up to 24 h after the surface was inoculated. epidemiological evidence supports the laboratory data because an in¯uenza outbreak in a nursing home suggested that the virus was spread by staff, through hands contaminated directly with body¯uids, or by touching contaminated fomites (morens and rash 1995) . there is similar evidence for the environmental survival and spread of piv and rsv. ansari et al. (1991b) demonstrated the transfer of piv from stainless steel surfaces to clean ®ngers, which suggests that fomites have a role as a reservoir for the spread of the virus. further, piv could be recovered from non-absorbent surfaces for as long as 10 h when the surface remained moist. however, brady et al. (1990) found that, when material containing piv was spread and allowed to dry, virus was only recoverable for up to 2 h. these workers also showed that piv persisted on the skin for at least 1 h after contamination, which reinforces the need to perform vigorous handwashing before and after contact with patients and their environment. likewise, hall et al. (1980) showed that rsv was recovered from hands touching surfaces contaminated with fresh secretions from rsv-infected infants. evidence showing that direct and indirect contact is a key factor in transmission of rsv infection is further reviewed by goldmann 2000 4 ). humans are the only known reservoir of herpes simplex virus 1 (hsv). the virus is most commonly spread by oral secretions and can be shed by persons with or without symptoms. herpes simplex virus can be recovered from the skin for up to 2 h after inoculation of the hands with the virus (bardell 1989) . the virus was more readily transmitted from moist drops than from drops which had been allowed to dry, although touching dried virus-containing droplets on the skin with a moistened ®nger resulted in transmission of the virus. infectious hsv has also been recovered from environmental surfaces such as doorknobs and toilet seats, although it is not clear what role fomites play in the spread of herpes viruses (larson and bryson 1982; bardell 1990; bardell 1993) . a recent study demonstrated the rapid and broad contamination of the environment with varicella-zoster virus (vzv) when a family member acquired the disease (asano et al. 1999) . eight days after onset of the index case vzv dna was detected in both samples from the patient and on the surfaces of an air-conditioning ®lter, a table, television channel push-buttons and a door handle. the virus was also detected on the hands of the parents and children. two siblings developed the disease 18 d after onset of the index case. the problem in assessing whether contamination in the environment might be a hazard is that the infectious dose can vary signi®cantly according to the immune status of the individual. it is clear that increasingly the variability in immune status of individuals is becoming a signi®cant factor in community and domestic settings as well as in the hospital environment. although some viruses survive relatively poorly in the environment, the low infectious dose of many viral pathogens, even for individuals regarded as healthy', suggests that, where body¯uids naturally contaminate objects with a high viral load, the virus can persist in suf®cient numbers to act as sources of infection for several hours, weeks or even months (sattar and springthorpe 1999) . the infective dose for nlvs may be as low as 10±100 particles, indicating that both aerosol and surface contamination could be a route of transfer of infection (caul 1994) . likewise, the infective dose for rotavirus may be as few as 10 particles and person-to-person transmission probably perpetuates endemic disease (ward et al. 1986) . a minimal infective dose of less than 10 plaque-forming units has been demonstrated for poliovirus (minor et al. 1981) . respiratory viruses also have low infective doses. for rhinoviruses, the infective dose via the nasal route may be less than 1 tcid 50 (couch 1990) , i.e. the tissue culture infective dose infecting 50% of the cells. conversely, pivs have an infective dose via the intranasal route of 80 tcid 50 (smith et al. 1966) . although studies about the survival characteristics of viruses represent an important component in understanding the infection potential and the preventive role of hygiene, much of our knowledge comes from reports of infection outbreaks where hygiene procedures have been defective or from case control studies. fifteen such reports have been examined in which viral contamination was directly implicated or for which viral agents were likely to have been the cause of the infections. the effects of the hygiene intervention procedures are summarized in table 1 and relate to daycare and other community centres where the concentration of people and activity provides the most cost-effective setting for evaluation of the impact of hygiene procedures. although opportunities for cross-contamination and cross-infection may occur less frequently in the home it could be argued that, since the ratio of homes to daycare centres is very large, the impact of these environments on the overall infection rates across a community may not be so dissimilar, even though daycare centres bring more people together. none of the investigations cited relate speci®cally to the home but fornasini et al. (1992) and osterholm et al. (1992) report studies of disease transmission from daycare centres to the home where it is transferred among family members. in eight of the 15 studies only the impact of handwashing was evaluated. in a 36-week handwashing education programme in child daycare centres, black et al. (1981) showed that the incidence of diarrhoea in children was rarely washed hands associated with higher incidence of infection st. sauver et al. (1998) signi®cantly reduced compared with two control centres. kilgore et al. (1996) studied the prevalence of neonatal rotavirus infection in bangladesh. they found an increased risk for neonatal rotavirus infection among infants whose mothers reported no handwashing during care of the neonate. in a study carried out during the cold and¯u season at two daycare centres, fewer colds were reported in the test group of 3±5-year-olds using proper and frequent handwashing techniques than in the control group. in the test centre the proportion of colds remained fairly constant at 18á9% whilst in the control group the proportion of colds increased from 12á7% to 27á8% (niffenegger 1997) . carter et al. (1980) demonstrated that families who used an iodine-based 5 hand disinfectant, known to kill rhinoviruses, had lower rates of infection than families using an inactive handwash. to decrease respiratory infections in senior daycare centres, staff were educated on viral transmission and the value of handwashing (falsey et al. 1999 ). in the intervention year, the infection rate among those attending the centres was signi®cantly lower than in the previous 3 years, with an almost 50% decrease in the infection rate. roberts et al. (2000a) carried out a randomized controlled trial of the effect of infection control measures on the frequency of upper respiratory infection in childcare. the intervention measures were training of childcare staff about transmission of infection, handwashing and aseptic nose-wiping technique. when compliance with infection control practice was high, the incidence of colds was reduced by 17%. a similar study by these workers also examined the effects of infection control measures on the frequency of diarrhoeal episodes in childcare using a randomized controlled trial (roberts et al. 2000b) . they found that, for those centres in which children's compliance with handwashing was high, diarrhoeal episodes were reduced by 66%. in the us, an outbreak of aseptic meningitis due to echovirus 30 was reported amongst parents with children attending a childcare centre. it was found that more frequent handwashing among the teachers compared with the parents of young children was associated with signi®cantly lower rates of infection (helfand et al. 1994) . in six of the 15 studies handwashing combined with environmental decontamination or other control measures was considered, whilst one study which related to nlv infection highlighted only the importance of environmental disinfection. in a preschool daycare centre, respiratory and gastrointestinal infections decreased following implementation of measures which included reinforcing existing handwashing procedures and education of staff and families on issues of infection control including environmental surface cleaning and disinfection and disinfection of toys (krilov et al. 1996) . uhari and mottonen (1999) transmission of infections in child daycare centres. it was evident that most of the infections that did occur were viral. the programme included increased handwashing, cleaning of the daycare centres and regular washing of toys. both the children and staff had signi®cantly fewer infections that those in control centres. st. sauver et al. (1998) studied hygienic practices and the prevalence of respiratory illness in children attending daycare homes. never or rarely washing hands by both children and carers was associated with a higher frequency of respiratory illness in both family and group daycare homes. using shared cloth towels rather than individual paper towels and washing of sleeping mats less than once a week were also associated with a higher frequency of upper respiratory infection. isaacs et al. (1991) reported a sevenfold reduction in the incidence of rsv in a hospital when patients and staff were educated about the importance of handwashing and infected babies were segregated. before intervention 4á2% of children under 2 years old developed nosocomial rsv, whilst after intervention only 0á6% developed infection. following implementation of a hygiene intervention programme that included handwashing education, use of gloves, disposable nappy pads and an alcohol-based hand rinse the incidence of enteric illness was lowered in intervention child daycare homes as compared with control homes (butz et al. 1990) . as stated previously, contamination of the environment may be considerable during an outbreak of nlv. following outbreaks of viral gastroenteritis on consecutive cruises, a ship was cleared and disinfected at the end of the fourth cruise in order to interrupt transmission of nlv (mcevoy et al. 1996) . fewer than 10 cases presented in each of the ®fth and sixth cruises compared with 195 cases during the fourth cruise. control measures included cleaning and disinfection of cabins, crew and staff quarters and communal bathrooms and steam cleaning of soft furnishings. hygiene measures were also introduced into the kitchen. the contamination of soft furnishing in areas where individuals have vomited presents a dif®cult cleaning problem and steam cleaning has been recommended by mcevoy et al. (1996) . during a hospital gastroenteritis outbreak caused by nlv, the attack rate among patients decreased in several wards following the implementation of environmental hygiene procedures (chadwick and mccann 1994) . infection control measures implemented included cleaning and chemical disinfection (ward¯oors, toilet areas, toilet seats, taps and spillages of vomit and faeces) to reduce environmental contamination. hypochlorite solution (1%) was used for disinfection of places contaminated with vomit or faeces and 0á1% hypochlorite for general disinfection of ward oors and toilet areas. since the studies highlighted in table 1 involved the implementation of infection control programmes involving several steps, it is dif®cult to attribute the effectiveness of the hygiene procedures to one speci®c aspect because they were inherently multifaceted. indeed, the results do not prove that the various interventions had a direct effect in decreasing infection rates. it could be argued that the reduced rates were due to variations in individual susceptibility to infection or to a lower incidence of the pathogen amongst the case control groups. nevertheless, overall, there is convincing circumstantial evidence to suggest that improved standards of hygiene can have a signi®cant impact in reducing the rates of respiratory, intestinal and other viral infections in childcare facilities, domestic homes, hospitals and adult care centres and the circulation of infections between these communities. the data reviewed show how improved standards of education and integrated hygiene measures, including hand and environmental hygiene, could have a signi®cant impact in reducing infectious diseases within community and home environments. in recent years the concept of risk assessment or hazard analysis critical control point (haccp) has successfully controlled microbial risks in food and other manufacturing environments. traditionally, the public has tended to regard good hygiene as creating an environment free of germs. to devise a hygiene policy that has real public health bene®ts, it is now accepted that a risk-based approach should also be adopted (bloom®eld and scott 1997; jones 1998; scott 1999) . a risk assessment approach to hygiene starts from the premise that homes and other settings always contain potentially harmful microbes (people, pets, food, etc.) and that good hygiene is not about eradication but about targeting measures in the places and at the times that matter, in order to limit risks of exposure. for both the hands and for environmental surfaces hygiene can be achieved by physical removal of organisms from the surface. alternatively, organisms can be inactivated in situ by a disinfection process or a combination of both physical removal and disinfection. in many situations such as the hands, and cooking and eating utensils, appropriate risk reduction can be achieved using detergent and hot water. however, since, in this situation, hygiene is achieved by removal of the microbes from the surface, if it is to be effective it must be applied in conjunction with a thorough rinsing process with clean water and must take account of the strength of attachment of the microbes to the surface (eginton et al. 1995) . stevens and holah (1993) showed that wiping of contaminated abraded surfaces using a sponge followed by a 10-s rinse produced a 3-log reduction in bacterial contamination of stainless steel surfaces but only a 1±1á5-log reduction on enamelled steel, mineral resin and polycarbonate surfaces. scanning electron microscope studies showed that bacteria were typically retained in surface imperfections, such that surfaces which sustained the most extensive damage retained higher numbers of bacteria. studies by schurmann and eggers (1985) showed that enteric viruses may be more strongly bound to the skin surface and that the inclusion of an abrasive substance, such as sand or aluminium hydroxide, in the handwash preparation is advisable to achieve effective virus removal. recent studies of the transmission of viruses in a household setting using bacteriophage /x174 as a model showed that virus spread was not prevented by the usual standards of hand hygiene as practised in the household (rheinbaben et al. 2000) . the virus was reisolated after 24 h from the hands of all persons tested even after normal use and cleaning of the hands. biocides that have activity against both enveloped and non-enveloped viruses include chlorine-and iodine±releasing agents, peracids and ozone (rotter 1997; sattar and springthorpe 1999) . biocide effectiveness depends on the nature of the virus, the surface carrier, the presence of interfering substances such as organic soil and hard water salts and the contact time. although these compounds can be used for disinfection of environmental surfaces they are generally too toxic and irritant for use on the skin. in achieving decontamination of hands, although handrub and handwash products currently available may have good activity against bacterial pathogens, activity against viral contamination is variable and depends on the type of virus. rotter (1997) suggested that, although alcoholic handrubs are effective against enveloped viruses such as in¯uenza, piv, herpes and rsv, activity against non-enveloped viruses such as rotaviruses, rhinovirus, poliovirus, adenovirus, nlv and hepatitis virus is limited unless extended contact times (up to 10 min) are used. similarly, agents such as triclosan and chorhexidine have some activity against enveloped virus but are not considered effective against nonenveloped viruses. it is well established that viruses are shed in large numbers and can survive for long periods on surfaces or fomites commonly found in many environments and this emphasizes the possible role of surfaces in the transmission of viruses. faeces can contain up to 10 12 virus particles per gram and vomit up to 10 7 per millilitre so the potential for hand and environmental contamination is considerable. viral shedding may begin before the onset of symptoms and may continue for several days or even weeks after the symptoms have ceased. virus transfer from surfaces to hands, ®ngers and food has been demonstrated. other studies have shown a high rate of spread once a viral infection is introduced into a family home or institution. improved handwashing and surface hygiene procedures have been shown to interrupt the transmission of viral infections via hands, surfaces or fomites. although the importance of hygiene and most particularly handwashing cannot be over-emphasized as a means of reducing infections it can be dif®cult to enforce even in healthcare facilities where staff should be aware of the infection risks. studies have shown that handwashing compliance amongst healthcare workers is variable (daniels and rees 1999; mcguckin et al. 1999 7 ; nishimura et al. 1999; pittet et al. 1999) . in a department of surgery, clinicians washed their hands between examinations in only 41% of cases (daniels and rees 1999) . in continuous videocamera surveillance of an intensive care unit personnel complied with handwashing in 71% of entries, whereas visitors of patients complied in 94% of entries (nishimura et al. 1999) . a recent survey of beliefs and attitudes towards hygiene in domestic homes showed that over 74% recognized handwashing as a key preventative measure in ensuring food safety and 66% believed that surface cleaning was also important. however, the respondents admitted that they would not carry out these procedures as frequently as they thought they should (mathias 1999) . the importance of hands in the transmission of virus infections is well recognized and many of the studies cited in this review relate speci®cally to handwashing interventions. increasingly however, there is evidence that cross contamination via surfaces is a signi®cant contributory factor. most particularly hand contact with contaminated 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using bacteriophage /x174 as a model virus extended excretion of rotavirus after severe diarrhoea in young children effect of infection control measures on the frequency of upper respiratory infection in child care: a randomised effect of infection control measures on the frequency of diarrhoeal episodes in child are: a randomised longitudinal study of rotavirus infection and gastroenteritis in families served by a paediatric medical practice: clinical and epidemiological observations common exposure outbreak of gastroenteritis due to type 2 rotavirus with high secondary attack rates within families handwashing and hand disinfection hospital admissions attributable to rotavirus infection in england and wales outbreaks of infectious intestinal disease in residential institutions in england and wales detection of rotavirus in handwashings of attendants of children with diarrhoea chemical disinfection to interrupt the transfer of rhinovirus type 14 from environmental surfaces to hands institutional outbreaks of rotavirus diarrhoea: potential role of fomites and environmental surfaces as vehicles for virus transmission viricidal activity of biocides an experimental study on the epidemiology of enteroviruses ð water and soap washing of poliovirus 1 contaminated hands, its effectiveness and kinetics hygiene issues in the home respiratory syncytial virus infection protective effect of antibody to parain¯uenza type 1 virus monitoring rotavirus environmental contamination in a paediatric unit using polymerase chain reaction the common cold hygienic practices and acute respiratory illness in family and group day care homes the effect of wiping and spraywash temperature on bacterial retention on abraded domestic sink surfaces outbreak of hepatitis a spread by contaminated drinking glasses in a public house infectious diarrhoea in children: controlling transmission in the child care setting an open randomized controlled trail of infection prevention in child day-care centres selected viruses of nosocomial importance prevention of surfaceto-human transmission of rotavirus by treatment with disinfectant spray human rotavirus studies in volunteers: determination of infectious dose and serological response to infection study of infectious intestinal disease in england: rates in the community, presenting to general practice and reported to national surveillance detection of rotaviruses in the day care environment by reverse transcriptase polymerase chain reaction key: cord-242132-fhepdgz9 authors: burlak, gennadiy title: is it possible to suspend the spread of an epidemic infection? the dynamic monte carlo approach date: 2020-05-28 journal: nan doi: nan sha: doc_id: 242132 cord_uid: fhepdgz9 we study a dynamics of the epidemiological infection spreading at different values of the risk factor $beta$ (a control parameter) with the using of dynamic monte carlo approach (dmc). in our toy model, the infection transmits due to contacts of randomly moving individuals. we show that the behavior of recovereds critically depends on the $beta$ value. for sub-critical values $beta<beta_{c}sim 0.6$, the number of infected cases asymptotically converges to zero, such that for a moderate risk factor the infection may disappear with time. our simulations shown that over time, the properties of such a system asymptotically become close to the critical transition in 2d percolation system. we also analyzed an extended system, which includes two additional parameters: the limits of taking on/off quarantine state. it is found that the early quarantine off does result in the irregular (with positive lyapunov exponent) oscillatory dynamics of infection. if the lower limit of the quarantine off is small enough, the recovery dynamics acquirers a characteristic nonmonotonic shape with several damped peaks. the dynamics of infection spreading in case of the individuals with immunity is studied too. we study a dynamics of the epidemiological infection spreading at different values of the risk factor β (a control parameter) with the using of dynamic monte carlo approach (dmc). in our toy model, the infection transmits due to contacts of randomly moving individuals. we show that the behavior of recovereds critically depends on the β value. for sub-critical values β < β c ∼ 0.6, the number of infected cases asymptotically converges to zero, such that for a moderate risk factor the infection may disappear with time. our simulations shown that over time, the properties of such a system asymptotically become close to the critical transition in 2d percolation system. we also analyzed an extended system, which includes two additional parameters: the limits of taking on/off quarantine state. it is found that the early quarantine off does result in the irregular (with positive lyapunov exponent) oscillatory dynamics of infection. if the lower limit of the quarantine off is small enough, the recovery dynamics acquirers a characteristic nonmonotonic shape with several damped peaks. the dynamics of infection spreading in case of the individuals with immunity is studied too. the dangerous trends of the coronavirus spreading throughout the world gives rise to numerous investigations in wide scientific spectrum. we study the spread of epidemiological infection at different values of the risk factors beta with the use of the dynamic monte carlo (dmc) method. in such a model, it is accepted that the infection is transmitted through the contacts of randomly moving individuals. we show that the quantity of recovered critically dependent on the value β. it is remarkably that for sub-critical values β < β c ∼ 0.6 the number of infected cases asymptotically converges to zero, so that for a moderate risk factor, the infection can quickly disappear. our calculations showed that such a dynamic property of the system (asymptotically) is associated with the critical behavior in 2d percolation medium. we also analyze an extended system, which is currently widely used to prevent the spread of the virus and includes quarantine on/off settings. it was revealed that early exit from the quarantine state leads to irregular oscillatory dynamics of infection. however, when the lower limit of quarantine off is small enough, the dynamics of infection acquires a characteristic non-monotonic shape with several damped peaks. comparison of quarantine and immunity factors shows that in the case of immunity, the complete recovery occurs faster than in a quarantine mode. the dangerous dynamics of the coronavirus spread throughout the world gives rise to numerous studies in a wide scientific spectrum. improving known epidemic models and developing new models are complicated tasks because the lack of verified statistics on the infection spread and disease dynamics. unstable predictability of infection, ambiguity with drugs 1 , uncertainty regarding the immunity of disease 2 , and other factors (such as a viral mutation) make it difficult to predict the dynamics of pandemic. this may relay to some mathematical models, which depend on a significant number of free (statistically-driven) parameters. the known models of the sir family give solutions in a form of smooth functions 3 (solutions of differential equations) that only indirectly include important random factors. naturally, that in such a situation, most statistically reliable forecasts are obtained by methods based on the direct application of the central limit theorem with a predicted error of 1/ √ n , see 4 , 5 , 6 and references therein. in this paper, we propose the use of the dynamic monte carlo (dmc) method that self-consistently includes various dynamic random factors. such a technique was previously used to study the processes associated with aggregation, viscous flow properties, the formation of biological structures, and allows to scale the associated geometric and dynamic quantities that characterize these phenomena 7 , 8 , 9 . in our study, as a control (free) parameter, we use the generalized risk factor β, which includes some of the factors mentioned above in an integral form. in our 2d toy model, the transmission of infection occurs due to contacts of randomly moving individuals, that determines the complex dynamics of the infection spread and various critical aspects of such a dynamics. the paper is organized as follows. in section 2, we formulate our approach and examine the behavior of infected individuals (order parameter a(t, β)), which, as it turns out latter, critically depends on the value β. it also is discussed the similarity of the asymptotic behavior of the infection dynamics with the critical phase transition in a two-dimensional (2d) percolation system. in the next section, we analyze the dynamic properties of the extended system, where we deal with two additional parameters which allow to on/off the quarantine state. the next section, contains the study of dynamics of the infection spreading and the formation of immunity for infected individuals. the last section summarizes our conclusions. first we explain which the properties of dynamic monte carlo (dmc) approach we deal with. in order to study the infection dynamic in epidemiological system (that is far from equilibrium) the dmc method is used, that allows investigation both temporal and spatial properties by the numerical simulations. as a toy model, we choose a 2d l × l (where l is size) bounded system that contains a disordered population of n individuals. following the classifications commonly known from sir model 3 in our dmc model we divide the host population into a set of distinct categories, according to its epidemiological status, that are susceptible (s), currently infectious (i ), and recovered (r). the total size of the host population is then n = s+i +r and all the individuals are born in the susceptible category 3 . following the actual situations we assume that initially the maternally derived immunity is clear. (the effect of immunity is studied in the following sections). upon contact with infectious individuals, the susceptibles may get infected and move into the infectious category. to apply the dmc approach it is constructed the person class (individual, alias object) that encapsulates properties of a randomly placed and moving individual and contains the following significant attributes where x, y are the components of position, v x , v y are the components of velocity, the parameters i, m describe the states: infected/uninfected and immunized/nonimmunized, respectively. the list of persons that represents a total host population is used in our dmc simulations. one of the underlying reasons why epidemiological systems exhibit variation is due to a different way that the individuals in a population have contact with each other. in our dmc simulation we assume that the spreading (transmission) of the infection occurs because of random contacts for moving individuals. to do that in dmc simulations we use the following strategy. (i) any contact can occur only between two nearest individuals. (ii) at any contact, the state of an infected transmits to the other contact person. but the infected one can still be recovered with probability 1 − β (recall that β ∈ [0, 1] is a risk factor). this means that if β 1, the probability to a recovery is small. to take an advantage of the visualizations at applying the dmc technique, we allow each object to have a visual representation, which is a yellow circle (nonimmunized individual), a green circle (immunized but not infected individual) and a red circle (infected individual), see fig.1 . we used the interaction radius r as the unit scale to measure distances (used r = 6, see fig.1 ) between the individuals, while the time unit ∆t = 1 was the time interval between two updatings of the directions and positions. in our simulations we used the simplest initial conditions: at time t = 0 the positions and velocities for all the n individuals are randomly distributed. we use the velocity scale such that random v x , v y ∈ [2, 10] for which the individuals always interact with their actual neighbors and move fast enough to change the configuration after a few updates. according to our simulations, the variation of actual interval of values of v x , v y does not affect the results. we also investigated the cases when the basic parameter of the model, the density ρ = n/l 2 is slightly varied. when the simulation time runs a lot contacts occur between nearest randomly moving persons that leads to fast and uncontrollable transfer of infection between many individuals, see fig. 1 . it is of great interest to investigate the temporal infection dynamics at various risk factors β. such a dynamics of the infections spreading (coefficient a(t) = i(t)/n ) as function of time t is displayed in fig.2 . since a(t) is a random-valued function we will fit (see 10 ) a(t) by a suitable fitting function that is chosen as where a 0,1,2 are the fitting coefficients. we found that a 1 is very small ∼ 10 −5 and a 2 2 for all the cases, but the amplitude a 0 changes considerably at the β variation. in fig.2 the blue lines show the numerical simulations (dmc) data, while the red lines display the fitting function f (t). fig.2 (a) shows the case β = 0.99, (b) β = 0.90, (c) β = 0.80, (d) β = 0.60. we indicate a remarkable observation that for β < 0.60 the system asymptotically converges to a trivial solution with a a 0 = 0 already for t 6. such observation leads to an interesting assumption that the studied dynamics of the infection spread can (asymptotically) be associated with a critical transition in the two-dimensional (2d) percolation system, that occurs when the occupation probability of defects is p c = 0.594 12 , 11 , 13 , 14 , 15 see fig.3 . such an assumption is studied in the following section. fig. 3 displays a comparison of above mentioned dependencies. in fig. 3 the red line shows the dependence a 0 (β) (see fig.2 ) associated with the infecting parameter a(β) = i/n , and the blue line shows the percolating order parameters p (p) as function of the occupation defect probability p. we observe that both dependencies are in excellent agreement and at β c p c 0.6 the phase transition to infected/percolating state occurs. from fig. 3 we can assume that the parameter a(β) can be mentioned further as an order parameter (similarly p (p)). this results that the formalism of the percolation critical percolating phase transition 12 , 11 , 13 can be applied to investigation the asymptotic of infection spreading at 11 . red line shows the dependence of (slightly tailored) amplitude parameter the fitting function a0 corresponding to a = i/n at fixed time t = 8 as function of the risk factor β. blue line shows the dependence of the order parameters p (p) for 2d percolating material as function of the occupation probability p. we observe that both curves are very close and the phase transitions to infected/percolating state occurs similarly at βc pc 0.6 for both cases. various β. on the other hand, good agreement between the results of dmc modeling and the critical transition in 2d percolation system shows the general applicability of dmc approach to analyze the dynamics of infection spread in the epidemiological system. fig. 1 is an extremely dangerous and highly undesirable scenario for the development of epidemiological situation. this section discusses the extension of the model, which in principle allows suspending this trend. one of the simple solutions proposed recently is introducing the quarantine by localizing of infected individuals in order to significantly reduce the number of contacts that leads to the transmission of infection. this can be modeled by setting v x = v y = 0 for infected individuals and ignoring all the contacts with them in our approach. we call such a regime of simulation as a quarantine mode. in order to do this we introduce two new parameters into the model, a max (the infection level when the quarantine is automatically turned on), and a min (the infection level when the quarantine is turned off). fig.4 we observe that the system transmits to unexpected dynamic state: the generation of irregular oscillations of a with large amplitudes between a max and a min . we have calculated (by the method 16 ) that the lyapunov exponent for such irregular oscillations is about 0.3. this means that if quarantine is turned off too early, the growth of infections suppressed, but the system goes into dynamic mode with irregular oscillations. in this case, a significant number of infected and recovered individuals can be re-infected, therefore, a full recovery does not occur. fig.5 shows the infections dynamics a(t) in the quarantine mode but for large the risk factors β: (a) β = 0.88, (b) β = 0.90, (c) β = 0.92, (d) β = 0.88. we observe that for large β the evolution of the infections has monotonic shape (with small random variations) but without the oscillations as in fig.4 . however the dynamics a(t) in (b) for β = 0.90 is already suppressed and strongly differs with respect to a case without the quarantine shown in fig.2 (b) . although actually there are no reliable statistics for the congenital or acquired immunity for persons (for animals see ref. 17 ) , in this section we analyze this aspect we observe that for large β the evolution of the infections has monotonic shape (with small random variations) but without the large oscillations shown in fig.4 . one can see that the dynamics a(t) in (b) for β = 0.90 is already suppressed and considerably differs with respect to situation without the quarantine shown in fig.2 in framework of our model. following the ref. 3 , we assume that in the host population there is no innate immunity to the virus. but we suppose that the persons (at least a large majority) will acquire this immunity, as is usually the case. to this end, in our model we use the parameter m , see eq. (1). following 3 , we assume that this parameter acquires a non-zero value (the presence of immunity) only after first infection and recovery. re-infection no longer occurs even at contacts with infected persons. fig. 6 shows the dynamics of recovery at presence of immunity in the quarantine mode for fixed parameters β = 0.94 and a max = 0.24 and different a min = 0.17, 0.1, 0.05, 0.02, 0.01. one can see that now the oscillations shown in fig.6 acquire shape of strongly damped picks that results the number of infected (order parameter a) to rapidly decrease. this allows to predicts that after the first high pick of infection (that has nearly fixed amplitude for all the cases) may occur a second pick but with lesser amplitude and then the complete recover may become. now we compare the effects of quarantine and immunity factors for recovery. fig.7 shows the dynamics of infections (order parameter a) for different values of the risk factor β = 0.99, 0.94, 0.9, 0.8 at situation without the quarantine when only the personal immunity m > 0 presents (see eq. (1)). this simulation shows that in such case the complete recover can occur even for a lesser time comparing to fig.6 . we studied the dynamics of the infection spread at various values of the risk factors β (control parameter) using the dynamic monte carlo method (dmc). in our model, it is accepted that the infection is transmitted through the contacts of randomly moving individuals. we show that the behavior of recovered individuals critically dependents on the value β. for sub-critical values β < β c ∼ 0.6, the number of infected cases (the order parameters a(t)) asymptotically converges to zero, so that at moderate risk factor, the infection can quickly disappear. however such a nontrivial behavior has to be confirmed by direct calculation. fig. 8 shows the dynamics of infections fraction a(t, β) with time for different risk factors β near the critical transition β ∼ β c = 0.6 for n = 1000 and rather large the initial number of infections i 0 = 100. we observe that really for β 0.58 the number of infections rapidly reach zero. we also analyzed the extended system, which currently is widely used to prevent the spread of the virus. in our approach such a system includes two additional parameters on/off the quarantine state. it was revealed that early exit from the quarantine leads to irregular oscillating dynamics (with positive lyapunov exponent) of the infection. however when the lower limit of the quarantine off is sufficiently small, the infection dynamics acquires a characteristic nonmonotonic shape with several damped peaks. the dynamics of the infection spread in case of individuals with immunity was studied too. our comparison of the quarantine and the immunity factors on a recovery shows that in case of stable immunity a complete recovery occurs faster than in a quarantine mode. this work was supported in part by conacyt (méxico) under the grant no. a1-s-9201. a strategic approach to covid-19 vaccine r&d, science coronavirus research updates: potent human antibodies could inspire a vaccine mathematical modeling of infectious diseases dynamics estimating the infection horizon of covid-19 in eight countries with a data-driven approach estimation of covid-19 dynamics "on a back-of-envelope": does the simplest sir model provide quantitative parameters and predictions? mathematical modeling of covid-19 transmission dynamics with a case study of wuhan novel type of phase transition in a system of self-driven particles from phase to microphase separation in flocking models: the essential role of nonequilibrium fluctuations flocking with discrete symmetry: the two-dimensional active ising model numerical recipes in c++ percolation percolation, statistical topography,and transport in random media introduction topercolation theory sec optical percolation in ceramics assisted by porousclusters mirrorless lasing from light emitters in percolating clusters determining lyapunov exponents from a time series sars-cov-2 infection protects against rechallenge in rhesus macaques key: cord-254580-nhpjvgt4 authors: ricardo, jose w.; lipner, shari r. title: considerations for safety in the use of systemic medications for psoriasis and atopic dermatitis during the covid‐19 pandemic date: 2020-05-27 journal: dermatol ther doi: 10.1111/dth.13687 sha: doc_id: 254580 cord_uid: nhpjvgt4 coronavirus disease 2019 (covid‐19), is responsible for at least 2,546,527 cases and 175,812 deaths as of april 21, 2020. psoriasis and atopic dermatitis are common, chronic, inflammatory skin conditions, with immune dysregulation as a shared mechanism; therefore, mainstays of treatment include systemic immunomodulating therapies. it is unknown whether these therapies are associated with increased to covid‐19 susceptibility or worse outcomes in infected patients. in this review, we discuss overall infection risks of non‐biologic and biologic systemic medications for psoriasis and atopic dermatitis, and provide therapeutic recommendations. in summary, in patients with active infection, systemic conventional medications, the jak inhibitor tofacitinib, and biologics for psoriasis should be temporarily held until there is more data; in uninfected patients switching to safer alternatives should be considered. interleukin (il)‐17, il‐12/23 and il‐23 inhibitors are associated with low infection risk, with il‐17 and il‐23 favored over il‐12/23 inhibitors. pivotal trials and postmarketing data also suggest that il‐17 and il‐23 blockers are safer than tnf‐blockers. apremilast, acitretin and dupilumab, have favorable safety data, and may be safely initiated and continued in uninfected patients. without definitive covid‐19 data, these recommendations may be useful in guiding treatment of psoriasis and atopic dermatitis patients during the covid‐19 pandemic. this article is protected by copyright. all rights reserved. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a novel human coronavirus, with 2,546,527 confirmed cases of coronavirus disease 2019 (covid-19) and 175,812 deaths worldwide (april 21, 2020). 1 it was declared a pandemic by the world health organization. an overall case fatality rate of 3.61% has been reported, 2 however, inaccuracies may exist because those who are asymptomatic or suffer from mild disease may never receive confirmation. psoriasis and atopic dermatitis (ad) are common, chronic, inflammatory skin diseases, affecting 2-3% of the general population and 7% of adults in the united states (us), respectively. 3, 4 disease mechanisms are multifactorial, with immune dysregulation important for both conditions, and mainstays of treatment immune-modulation. 5 systemic therapy is preferred for psoriasis treatment in patients with body surface area>10%, involvement of sensitive areas or topical therapy failure. 6 systemic treatment is recommended for ad patients with severe disease or recalcitrant to topical therapy. 7 immunocompromised patients are highly vulnerable to infections, which is particularly concerning in the context of the covid-19 pandemic. in this review, we summarize the current literature regarding overall infection risks with systemic immunomodulating agents for psoriasis and ad, and provide evidence-based treatment recommendations during the covid-19 pandemic. systemic corticosteroids are immunosuppressive medications used to treat ad flares, but very rarely psoriasis. they have been shown to increase infection risk. in a systematic review of 101 studies on ad children (n=6,817) treated with systemic corticosteroids ≥15 days, infection rate was 8.7%, with 21 associated deaths. 8 in a meta-analysis of corticosteroid use in patients with influenza pneumonia (10 studies, n=6548), compared with placebo, corticosteroids were associated with higher mortality, longer intensive care unit length of stay and a higher rate of secondary infection. 9 therefore, oral corticosteroids should be avoided, weighing the risks of disease flare vs. sars-cov-2 infection, to prevent covid-19 susceptibility. before discontinuation, dose tapering may be considered to avoid a negative effect on respiratory symptoms. methotrexate and cyclosporine are amongst the most frequently used systemic medications for psoriasis and ad, with both associated with increased infection rates. there was a 58% higher overall infection risk with cyclosporine vs. methotrexate in the biobadaderm registry (spanish registry of adverse events for biological therapy in dermatological disease) including 2153 psoriasis patients. 10 in a head-to-head comparison of methotrexate (n=50) versus cyclosporine (n=47) in moderate to severe ad adults, infections rates were 32% and 24%, respectively. 11 while methotrexate and cyclosporine are associated with decreased infection rates and favored over treatment with systemic corticosteroids, 12 their impact on susceptibility to/severity of covid-19 is unknown and, if essential, precautions should be taken to avoid infection. of interest, cyclosporine has anti-coronavirus activity in vitro, but the effect in humans is unknown. 13 the systemic retinoid, acitretin, is anti-inflammatory and inhibits cell differentiation; it is food and drug administration (fda)-approved for psoriasis. 14 it does not suppress the immune system to the extent of the other conventional treatments for psoriasis. in an observational cohort study, there was no increased rate of overall serious infections among acitretin-treated psoriasis patients vs. methotrexate; acitretin increased risk of cellulitis compared to methotrexate (propensity score-adjusted hazard ratio [hr], 1.76; 95% ci, 1.11-2.80), possibly due to skin fragility and staphylococcus aureus colonization. 15 therefore, acitretin has not shown increased viral/respiratory infection risk and can be safely used during the pandemic. retinoids have been shown to inhibit human herpesvirus eight replication, their effect on sars-cov-2 remains to be established. 16 this article is protected by copyright. all rights reserved. azathioprine is used off-label in the us for ad treatment in patients recalcitrant or who have contraindications to cyclosporine and methotrexate. in 12 ad children treated with azathioprine, there were no associated infections. 17 in a double-blind, placebo-controlled, crossover study of 37 ad adults treated with azathioprine, there were five cases of upper respiratory infections (uris) (14%), two cases folliculitis (5%), and one report each impetigo (3%) and sore throat (3%). 18 in a retrospective analysis of 232,611 systemically-treated adults with ad (six months), there were increased risks of serious and opportunistic infections with azathioprine (rr=1.89) and prednisone (rr=1.78) compared with methotrexate, with a reduced risk with cyclosporine (rr=0.87). 12 therefore, azathioprine may increase susceptibility to infections, and if essential, exposure to covid-19 should be minimized. apremilast, an orally administered phosphodiesterase four inhibitor is fda-approved for moderate to severe plaque psoriasis and has been used off-label for ad. [19] [20] [21] although it does modulate immunologic cascades, this pathway does not seem to particularly increase susceptibility to infection. in a pooled safety analysis of two randomized, controlled trials (rcts) involving psoriasis patients treated with apremilast (n=1184), uris and nasopharyngitis occurred in 19.2% and 16.6% of patients, respectively; serious infections (urinary tract infection n=2; appendicitis n=3; pneumonia=2) occurred in 1.4%. 22 furthermore, in an observational cohort study including systemically treated psoriasis patients, overall serious infections were decreased with apremilast vs. methotrexate (hr, 0.50; 95% ci, 0.26-0.94). thus, apremilast seems to be a safe alternative for uninfected psoriasis patients during the pandemic, but specific covid-19 data is needed. data regarding infection risks of nonbiological therapies for psoriasis and ad is summarized in table 1. biologic medications are widely used for psoriasis and ad patients, with limited data regarding infection risk. since biologics inhibit immune-mediated pathways involving specific cytokines, there is at least theoretical risk of increased susceptibility to and severity of infection. a common reason for discontinuation of biologics is infection. 23 among the targeted cytokines for these biologics, tumor necrosis factor-alpha (tnf-α) plays a crucial role in the immune response against intracellular pathogens and formation of granulomas, 24 and interleukin (il) -12 and il-23 are involved in cell-mediated immunity by inducing interferon-γ. 25 il-23 also induces t-helper 17 cell differentiation and il-17secretion, fundamental in providing immunity against bacteria, viruses, fungi and parasites. 26, 27 il-4 and il-13 play key roles in the immune response against helminth infections. 28 five classes of biologic therapies are used for psoriasis or ad: tnf-α inhibitors (table 2) , il-17 inhibitors, an il-12/23 inhibitor, il-23 inhibitors, an il-4/13 inhibitor, and a janus kinase (jak) inhibitor (table 3) . this article is protected by copyright. all rights reserved. anti-tnf-α therapies inhibit a crucial immunological pathway, therefore an immunosuppressive effect and increased infection risk are expected. there is an fdarequired black box warning of infection susceptibility. 29 however, assessing infection risk is challenging because rcts are often not adequately powered to detect rare events and ineligibility criteria may exclude up to 30% of real-world patients. 30 certolizumab increased risks of all infections, uris and nasopharyngitis by 5%, 2% and 2%, respectively. 29 additionally, anti-tnf-α therapy is associated with latent tuberculosis reactivation, even with chemoprophylaxis; infliximab is associated with increased risk of herpes zoster. 34, 35 therefore, based on available data, anti-tnf-α biologics should be held similarly, an 11% increased risk in overall infections with secukinumab was reported based on pivotal trials, with most attributable to yeast infections; uris were increased slightly for secukinumab, but not for ixekizumab or brodalumab. 29 since il-17 plays an important role in immunological response against candida infections, there is a theoretical increased risk of yeast infections with anti-il17 therapies. 40 in a pooled analysis from 10 phase two and three clinical studies on 3,430 psoriasis patients treated with secukinumab 300 mg (n=1,1410), 150 mg (n=1,395) and etanercept (n=323), candida infections were reported in 2.9%, 1.5% and 1.2% of subjects, respectively. 41 all infections were mild, resolved spontaneously or responded to standard treatment, without causing treatment discontinuation. 41 overall, increased infection risk has been shown with il-17 inhibitors, but yeast infections may constitute a large proportion of that increase; uris are particularly uncommon. therefore, il-17 inhibitors may be safely prescribed and continued, unless the patient is symptomatic or positive for sars-cov-2. ustekinumab inhibits il-12 and il-23, with il-12 playing an important role in protection against viral infections. 29, 42, 43 however, no increased susceptibility to infection with ustekinumab has been reported. in a pooled analysis of four phase two/three studies of 3, 117 this article is protected by copyright. all rights reserved. contrary to il-12/23 inhibitors, anti-il-23 therapies do not target il-12, and il-12 plays a key role fighting viral infections. 29, 45 reduced risks of salmonella, candida and mycobacterium infections were seen in il-23p19-targeted vs. il-12/23p40-targeted animal models. 46-49 nonetheless, rcts on il-23 inhibitors have shown conflicting results regarding infection risks. in a phase iii, double-blinded, placebo-controlled study on 837 psoriasis patients randomized to treatment with guselkumab, adalimumab or placebo, overall, candida, and serious infections, occurred at comparable rates across treatment groups. 50 in 798 risankizumab-treated psoriasis patients, there was increased overall infection risk in two this article is protected by copyright. all rights reserved. phase three studies. 51 the most common infections were uris, urinary tract infections and influenza. 51 two cases of latent tuberculosis were reported in the risankizumab group; both patients tested negative at baseline. 51 data assessing infection risk with tildrakizumab is sparse. lebwohl et al reported an increase in nasopharyngitis (4%) with tildrakizumab compared with placebo. 29 risk, however, is low and comparable to placebo. therefore, based on available data, il-23 inhibitors may be continued/initiated, unless the patient is symptomatic or positive for sars-cov-2. tofacitinib is a small molecule inhibitor of tyrosine kinases of the janus family, preferentially jak1 and jak3, downregulating cytokines crucial for lymphocyte development; therefore, there is potential for increased risks of intracellular bacterial and viral infections. 52 it has been hypothesized, nonetheless, that fluctuations in plasma levels of jak inhibitors throughout the day may preserve immunogenicity against infectious pathogens. 53 tofacitinib carries an fda-required black box warning for serious infections. in one placebo-controlled phase three trial of 422 patients with psoriatic arthritis, randomized to treatment with 5-mg or 10-mg tofacitinib, adalimumab, or placebo, nasopharyngitis (in 7%, 12% and 10%, respectively) and uris (in 9%, 11%, and 8%, respectively) were the most common adverse events. 54 there were three cases of serious infections (influenza, appendicitis and pneumonia) and four cases of herpes zoster in the tofacitinib-treated group. 54 similarly, in two randomized, placebo-controlled studies of 745 and 741 psoriasis patients treated with tofacitinib 5-mg and 10-mg, respectively, nasopharyngitis and uris were the most common infections, and 5 serious infections (pneumonia, herpes zoster and erysipelas in the 5-mg group; and appendicitis, pneumonia and pyelonephritis in the 10-mg group) were reported in tofacitinib-treated patients. 55 furthermore, herpes zoster was reported in 12 tofacitinib-treated patients versus none in the placebo groups. 55 thus, tofacitinib has an association with increased infection risk in psoriasis/psoriatic arthritis patients. tofacitinibtreated patients may be more susceptible to covid-19, strict protective measures are recommended to minimize viral exposure. dupilumab targets il-4 and il-13, elements of the type two immune response. 56 as type one and type two immune responses cross-regulate each other, suppression of type one immunity can potentially facilitate uncontrolled or persistent viral and bacterial infections. 57 nonetheless, dupilumab has been associated with a reduced infection rate in ad patients. a pooled analysis of seven rcts on dupilumab-treated ad adults showed a decreased risk of serious infections, skin infections and herpes infections (eczema herpeticum or herpes zoster) in the dupilumab groups compared with placebo. furthermore, by also treating asthma, dupilumab may theoretically decrease risk for covid-19 infected patients for severe respiratory disease. 56 therefore, current evidence suggests continuing and initiating dupilumab treatment in ad patients during the covid-19 pandemic. it is difficult to make definitive conclusions about susceptibility to sars-cov-2 infection in psoriasis or ad patients on systemic treatments, solely based on general infection risk data. furthermore, the majority of studies included patients with mean age of approximately 40 years; therefore, these recommendations may not be applicable to older individuals, who on average have higher covid-19 associated mortality. there is also a potential role for some of these medications as treatments of covid-19 but this remains largely unknown. in we suggest the following algorithms for treatment of psoriasis and ad during the covid-19 pandemic (figures 1, 2) . this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. coronavirus covid-19 global cases by the cross-country comparison of case fatality rates of covid sleep disturbances in adults with eczema are associated with impaired overall health: a us population-based study national health and nutrition examination surveys atopic dermatitis and psoriasis: two different immune diseases or one spectrum? recategorization of psoriasis severity: delphi consensus from the international psoriasis council systemic treatment of adult atopic dermatitis: a review systematic review of the toxicity of long-course oral corticosteroids in children the effect of corticosteroids on mortality of patients with influenza pneumonia: a systematic review and meta-analysis infections in moderate to severe psoriasis patients treated with biological drugs compared to classic systemic drugs: findings from the biobadaderm registry methotrexate versus cyclosporine in adults with moderate-to-severe atopic dermatitis: a phase iii randomized noninferiority trial comparative safety of systemic immuno-modulatory medications in adults with atopic dermatitis suppression of coronavirus replication by cyclophilin inhibitors a review of its pharmacology and therapeutic use risk of serious infection in patients receiving systemic medications for the treatment of psoriasis retinoic acid analogues inhibit human herpesvirus 8 replication adalimumab therapy for moderate to severe psoriasis: a randomized, controlled phase iii trial efficacy and safety of guselkumab, an anti-interleukin-23 monoclonal antibody, compared with adalimumab for the treatment of patients with moderate to severe psoriasis with randomized withdrawal and retreatment: results from the phase iii, double-blind, placebo-and active comparator-controlled voyage 2 trial etanercept in the treatment of psoriatic arthritis and psoriasis: a randomised trial secukinumab in plaque psoriasis--results of two phase 3 trials infliximab monotherapy provides rapid and sustained benefit for plaque-type psoriasis infliximab induction and maintenance therapy for moderate-to-severe psoriasis: a phase iii, multicentre, double-blind trial a randomized comparison of continuous vs year in the treatment of moderate-to-severe plaque psoriasis certolizumab pegol for the treatment of patients with moderate-to-severe chronic plaque psoriasis: pooled analysis of week 16 data from three randomized controlled trials guselkumab versus secukinumab for the treatment of moderate-to-severe psoriasis (eclipse): results from a phase 3, randomised controlled trial longterm safety profile of ixekizumab in patients with moderate-to-severe plaque psoriasis: an integrated analysis from 11 clinical trials ixekizumab treatment for up to 5 years in adult patients with moderate-to-severe psoriasis: results from greater than 17,000 patient-years of exposure a prospective phase iii, randomized, double-blind, placebo-controlled study of brodalumab in patients with moderate-to-severe plaque psoriasis key: cord-259422-5ex12eun authors: graat, judith m; schouten, evert g; heijnen, marie-louise a; kok, frans j; pallast, esther g.m; de greeff, sabine c; dorigo-zetsma, j.wendelien title: a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection date: 2003-12-11 journal: j clin epidemiol doi: 10.1016/s0895-4356(03)00171-9 sha: doc_id: 259422 cord_uid: 5ex12eun background and objective: community-based elderly studies concerning microbiology of acute respiratory infections are scarce. data on subclinical infections are even totally absent, although asymptomatic persons might act as a source of respiratory infections. methods: in a 1-year community-based study, we prospectively investigated the possible virologic cause of acute respiratory infections in 107 symptomatic case episodes and 91 symptom-free control periods. participants, persons ⩾60 years, reported daily the presence of respiratory symptoms in a diary. virologic assessment was performed by polymerase chain reaction (pcr) and serology. results: in 58% of the case episodes a pathogen was demonstrated, the most common being rhinoviruses (32%), coronaviruses (17%), and influenzaviruses (7%). the odds ratio for demonstrating a virus in cases with symptoms vs. controls without symptoms was 30.0 (95% confidence interval 10.2–87.6). in 4% of the symptom-free control periods a virus was detected. conclusion: this study supports the importance of rhinovirus infections in community-dwelling elderly persons, whereas asymptomatic elderly persons can also harbor pathogens as detected by pcr, and thus might be a source of infection for their environment. elderly people have an increased susceptibility for respiratory infections and related complications [1] . on average, community-dwelling elderly people suffer from 1.2-1.6 acute respiratory infections per year [2, 3] . medical consultation and hospitalization because of such an infection has been reported in 40 and 0.8% of community-dwelling elderly people, respectively, during the winters of 1992-1993 and 1993-1994 in england [2] . viruses play a crucial role in acute upper respiratory tract infections, the most common being rhinoviruses, coronaviruses, influenzaviruses, and respiratory syncytial viruses [4] . however, laboratory diagnosis of acute respiratory infections in symptomatic elderly people so far focussed on institutionalized elderly persons [5] [6] [7] , on patients reporting for medical consultation [8, 9] , and to a far less degree on community-dwelling elderly persons [2] . besides, no data are available on the presence of respiratory pathogens in asymptomatic elderly persons. asymptomatic people with a subclinical infection might, however, transmit the pathogen to other persons and act as an unrecognized source of respiratory infections. therefore, in this prospective, community-based study, we investigated the presence of known respiratory viruses in elderly persons both with and without symptoms of an acute upper respiratory tract infection. second, we compared the clinical characteristics of the persons suffering from an acute respiratory infection, during episodes with positive and negative virologic laboratory diagnosis. persons with and without symptoms of an acute respiratory infection, hereafter referred to as cases and controls, were recruited from october 1, 1998, until october 1, 1999 , from an intervention trial investigating the effect of micronutrient supplementation on acute respiratory infections in community-dwelling elderly persons (у60 years) [3] . during the 1-year study period, a diary was used daily by all participants for reporting symptoms that indicated an acute respiratory infection. participants were requested to report the onset of symptoms of a possible infection to the study nurse. a subject was identified as case if (1) he/she had respiratory symptoms with a sudden onset; (2) rhinorrhoea/ sneezing, sore throat/hoarseness, or dry cough were present for at least 2 days; and (3) the symptoms had a pattern that differed from any usual symptoms [10, 11] . apart from a check by telephone, the study nurse evaluated the symptoms of cases during home visits. from those cases that reported their symptoms within 3 days to the study nurse every other case, with a maximum of five cases per week, was selected for virologic assessment. cases who reported their symptoms after 3 days to the study nurse were excluded for virologic assessment to overcome false negative test results. each case episode, i.e., the period during which a case had respiratory symptoms, had to have been preceded by a 7-day symptom-free period. during the 1-year study period, 624 incident case episodes were reported by 346 cases. in total 107 (17%) case episodes-reported by 97 cases-were selected for virologic assessment. for each case episode an asymptomatic control was selected as follows. participant numbers, including all cases and controls, ranged from 1-652. if the participant number of the case was 325 or lower, a closest eligible control was selected on participant number by counting back on these numbers. if the participant number of the case was 326 or higher, a closest eligible control was selected by counting forward on these numbers. controls were subjects without symptoms of a respiratory infection within a time window of 8 weeks before and 8 weeks after the symptomatic period of the index case. the study nurse checked the absence of symptoms at the time of recruitment of the control and the diary was checked for absence of symptoms in the previous eight weeks. in total, 99 controls were selected. cases and controls were matched on age (ϯ5 years) and sex, and they were not living in the same house or apartment, did not have chronic obstructive pulmonary disease, asthma, or cancer, and did not use severe immunosuppressive medication. afterwards, we excluded 8 (8%) of the 99 originally enrolled controls because they developed symptoms after a median duration of 21 days (range 9-54). for six excluded controls serologic testing was negative, while for two it was missing. with pcr two times a rhinovirus and two times a coronavirus oc43 was detected in the eight excluded controls. results presented are therefore based on the 107 case episodes and 91 control periods. this study was approved by the medical ethics committee of the wageningen university, the netherlands, and written informed consent was obtained from all participants prior to the study. all participants filled out a questionnaire concerning relevant subject characteristics at baseline. a diary was used daily for self-report of symptoms that indicated an acute respiratory infection. apart from the symptoms that had to be present because of our case definition (rhinorrhoea/ sneezing, sore throat/hoarseness, dry cough), also accompanying symptoms were recorded in the diary: (1) symptoms of a lower respiratory tract infection (sputum production, wheezing, pain on respiration), (2) systemic symptoms (fever-self-assessed by a supplied thermometer-malaise, headache, rigors, muscular pain, perspiration), (3) other symptoms (tearful eyes, pain in facial sinuses or ear), (4) restriction of activity (staying in bed, not being able to do daily activities, staying at home), (5) episode-related medication, including antibiotic use, (6) medical consultation, and (7) hospitalization [2] . if the study nurse judged during a home visit the case's symptoms as an acute respiratory infection, in both the case and the matched control an acute phase serum sample and one swab from the nose and one from the throat were taken within 3 days and a convalescent serum sample was taken within 2-4 weeks after onset of the first symptoms of the case. samples in cases and controls were taken on the same day to exclude seasonal differences. pcr or serology was used to diagnose infection with the eight most common respiratory viruses and mycoplasma pneumoniae (m. pneumoniae). pcr was performed for those viruses for which either no or only aspecific serology was available and for which validated pcr tests were available in our lab. infections with rhinovirus, enterovirus, coronavirus oc43 and 229e, and respiratory syncytial virus were diagnosed by pcr. serology was performed for those viruses for which either no pcr was available, or the nucleic acid extraction method had to be changed for dna isolation (in the case of m. pneumoniae). infections with influenzavirus a and b, parainfluenzavirus 1, 2, and 3, adenovirus and m. pneumoniae were diagnosed by serology. swabs from the nose and from the throat, hereafter referred to as "nose/throat samples," were placed together in 4-ml hanks' balanced salt solution containing gelatin, lactalbumin, yeast, and antibiotics. upon receipt of the nose/ throat samples at the laboratory, the swabs were twirled in the transport medium and removed. an aliquot of 200 µl of the sample was used for nucleic acid extraction by using the high pure rna isolation kit (boehringer, mannheim, germany). five microliters of the eluted rna preparation was used in a 25 µl single-tube rt-pcr followed by a nested-pcr using primer pairs as described previously for rhino-/enterovirus [12] . another 5 µl of extracted rna was used in a single 25 µl single-tube rt-pcr followed by a nested-pcr using primer pairs as described previously for respiratory syncytial virus (rsv) and coronavirus oc43 and 229e [13, 14] in a multiplex format. in the rna isolation procedure and pcr-method for rsv detection, sensitivity for rsv a was about one virus particle and for rsv b about 70 virus particles. the virus particle count was determined by quantitative em (advanced biotechnologies incorporated, columbia, md). positive controls from culture were used in each pcr test for the respective viruses. to prevent carryover contamination within the laboratory, preparation of the patient samples and pcr mixtures was performed in safety hoods in separate dedicated positive pressure laboratories. to check for carryover contamination of samples and for amplicon contamination during the procedure, negative controls, consisting of transport medium, were included after every fifth patient sample. subjects with a positive pcr result were considered to be infected by a known virus, which was interpreted as a laboratory-confirmed infection. paired sera from all cases and controls were analyzed for igg antibodies against influenzavirus a and b, adenovirus, and m. pneumoniae. for para-influenzavirus 1, 2, and 3, iga antibodies, combining the three antigens in one assay, were detected. analyses were performed using commercially available elisa (serion immunodiagnostica gmbh, würzburg, germany), and quantitative results, expressed in units/milliliter, were calculated using a lot-specific standard curve and calculation table as supplied in the test kit. results were interpreted as negative, indeterminate, or positive according to the manufacturer instructions. in the case of indeterminate results for the parainfluenza iga assay on paired sera, detection of total antibodies against separate parainfluenza 1, 2, and 3 antigens was repeated in a complement fixation assay (cfa), using commercially available parainfluenza 1, 2, and 3 antigens (virion, ruschlikon, switzerland). in elisas, a change from negative to positive result and in the cfa a fourfold rise in antibody titer between the paired sera was interpreted to be a laboratory-confirmed respiratory infection. data analysis concerning virologic (including m. pneumoniae) assessment was performed with the 107 case episodes and the 91 control periods. differences in the distributions for continuous data, i.e., age, self-perceived health, and illness duration were compared with independent sample student's t-test. illness duration was not normally distributed, and was log transformed to obtain normality. a chi-square test or a fisher's exact test was used to test the correlation between discrete variables, i.e., sex, influenza vaccination, smoking habits, allergy, sharing an apartment, presence of micro-organisms, symptoms of a lower respiratory tract infection, systemic and other symptoms, restriction of activity, fever, medical consultation, hospitalization, episode-related medication, and episode-related antibiotic use. a fisher's exact test was used to calculate the odds ratio for demonstrating a virus in cases with symptoms of acute respiratory infection vs. controls without symptoms of such an infection. alpha was taken as 0.05 in all analyses. the matching procedure on sex and age resulted in wellbalanced groups of cases and controls with respect to these and other relevant variables (table 1) . micronutrient supplementation related to the intervention trial was also similar between cases and controls [3] . the 97 symptomatic cases had 107 case episodes of respiratory infection, during which virologic (including m. pneumoniae) tests were performed. in 62 (58%) of these case episodes at least one micro-organism was demonstrated, whereas in two of these 62 two different micro-organisms were demonstrated. in 45 (42%) case episodes none of the applied tests was positive. of 10 cases, two case episodes were included. for seven out of the mentioned 10 cases, test results were different, i.e., different pathogens, or negative in one and positive virology in the other episode. in two cases, both episodes had negative virology. only in one case, in both episodes rhinovirus was detected. the most common viruses demonstrated were rhinoviruses (32%) and coronaviruses (17%) followed by influenzaviruses (7%), enteroviruses (2%), parainfluenzaviruses (2%) and m. pneumoniae (1%). respiratory syncytial virus and adenovirus were not detected. three of the seven cases diagnosed with an influenzavirus infection had been vaccinated against influenza. none of the titer rises on which the influenzavirus infection was diagnosed, was related to vaccination, as 2 to 4 months passed between vaccination and the diagnosis of an influenzavirus infection. presence of rhinovirus infections was almost five times higher compared to influenzavirus infections in this community-dwelling elderly population (table 2 ). in 4 out of 91 control periods (4%) a virus was demonstrated, i.e., two times a rhinovirus and two times a coronavirus. two out of these four controls with positive virology never showed symptoms of a respiratory infection during the 1-year study period. the two remaining controls with positive virology did not have any symptoms at least 3.5 and 4 months before and 8 and 4 months after sample collection, respectively. overall, the odds ratio for demonstrating a virus (or m. pneumoniae) in cases with symptoms vs. controls without symptoms of acute respiratory infection was 30.0 (95% confidence interval 10.2-87.6). despite small numbers (n ϭ 5) significantly more influenzavirus a infections were identified during symptomatic periods in winter (october-march) compared to summer (p ϭ .02). enteroviruses, parainfluenzaviruses and m. pneumoniae were only detected in summer (april-september). clinical characteristics of the persons suffering from an acute respiratory infection, during episodes with positive and negative virologic laboratory diagnosis, are described in table 3 . influenzavirus infection was associated with significantly longer illness duration and more systemic symptoms than the other infections with positive and negative virology. restriction of activity, presence of fever, medical consultation, and antibiotic use were also more frequently reported during influenzavirus infections, although not significantly different from the other infections with positive and negative virology. this study shows that subclinical respiratory infections occur in a minor part (4%) of asymptomatic elderly persons. besides, we showed the importance of rhinovirus infections in community-dwelling elderly people because of its high frequency. to our knowledge, this is the first study to investigate several common respiratory pathogens in community-dwelling elderly persons both with and without symptoms of an acute respiratory infection. so far, only two studies reported on microbiologic evidence of respiratory infection in community-dwelling healthy subjects with and without symptoms of such an infection. one study focussed on detection of rhinoviruses and enteroviruses by pcr in children and adults [15] . in 12 and 4% of the asymptomatic children and adults, respectively, virologic assessment was positive. although johnston et al. [15] tested only for rhinoviruses and enteroviruses, the frequency of subclinical respiratory infections in those healthy adults is similar to what we observed in our older population. preliminary results of a dutch study being performed in persons consulting their general practitioner for signs and symptoms of an acute respiratory infection, showed a positive virologic assessment in 19% of the controls [16] . this percentage is higher than observed in our study. however, that study population existed of participants from all age categories, including babies and children. as showed by johnston et al. [15] the percentage of asymptomatic persons with positive virologic assessment is clearly higher in children, which might explain the discrepancy. common viral pathogens demonstrated during symptomatic periods in children and adults [4, 17] , in institutionalized elderly patients [7, 11] , in patients with medical consultation [8] , and in community-dwelling elderly persons [2] are rhinoviruses, coronaviruses, influenzavirus a and b, and rsv, which is in line with our results. the frequency of the most common viruses varies between the different subpopulations. corresponding to one previously performed communitybased elderly study, we also showed that rhinovirus infections are highly prevalent, and can cause a great overall disease burden in this population [2] . corresponding to the results of nicholson et al. in community-dwelling elderly persons [2] , but in contrast to studies in more frail elderly persons as those living institutionalized and to studies with a general practitioner-based setting [7, 11, 18] , we also observed that influenzavirus infections and rsv infections seem to occur less frequent in free-living elderly people. a severe morbidity is caused by viruses such as influenzavirus and rsv [19] , which corresponds to our results on influenzavirus infections. this might explain the higher frequency of rsv and influenzavirus infections demonstrated in studies with general practitioner-based or institutionalized setting [19] . in total, 4 out of 7 patients with influenzavirus infection were not vaccinated against influenza. this might indicate the need for preventive vaccination in elderly persons. in agreement with other studies in institutionalized [7, 11] and in community-dwelling elderly subjects [2] , we found that infections with parainfluenzavirus, enterovirus, adenovirus, and m. pneumoniae are of minor importance in causing acute respiratory infections in elderly persons. we obtained a microbiologic diagnosis in 58% of the case episodes. the diagnostic deficit of 42% is relatively low, as in most studies a micro-organism was demonstrated in at maximum 50% of the case episodes [2, 7, 8] . other, partly new or unknown viruses, bacteria, and atypical microorganisms other than m. pneumoniae may be responsible for some of the clinical and possible additional subclinical infections with negative microbiology. bacterial, atypical and viral micro-organisms in adult patients consulting for respiratory infection have been shown in 12, 20, and 50% of the patients, respectively [20] . also, chlamydia species are reported to cause acute respiratory infections in communitydwelling elderly persons [2] , although the proportion of bacterial infections is reported to be rare in adult patients with common cold [4] . besides, chlamydia infections occurred in 1% only of the community-dwelling elderly people, and were mainly analyzed in patients with copd and asthma, while we excluded those patients [2, 21] . however, we cannot exclude that part of the diagnostic deficit in our study might be explained by such bacterial and atypical micro-organisms. little is known about the time period after infection during which pcr-based tests are positive [12, 14, 22] . this issue is especially crucial in interpreting pcr positive results in nose/throat samples obtained from subjects both with and without symptoms of a respiratory infection. andeweg et al. [12] demonstrated that rhinoviruses were no longer detected by pcr in patients who had recovered from disease. in our study, all cases and controls were followed day to day by using a self-reporting diary system. it was therefore possible to include only controls not having any symptoms 2 months before and 2 months after sampling. thus, it is very unlikely that the controls, in which a virus was detected, were in the postinfectious or incubation period of a symptomatic infection. moreover, in nose/throat samples of four of the eight excluded controls a respiratory virus was detected, and those controls apparently were in the incubation period. detection of rhinovirus, enterovirus, rsv, and coronavirus infections by the pcr method has been used before and is widely accepted [12] [13] [14] . although pcr-based tests are highly sensitive and specific, false positives due to contamination of negative samples with pcr product in the laboratory might have occurred [23] . however, given the strict conditions under which pcr was performed [24] , this is very unlikely. negative controls included after each fifth test sample were pcr-negative in all samples, indicating that contamination was effectively prevented. underreporting could have occurred if cases were admitted to the hospital when having an acute respiratory infection. because none of the cases reported having been admitted to a hospital because of acute respiratory infection or its complications, underreporting because of hospitalization is no issue in this study. the subjects who participated in this study were recruited from an intervention trial studying the effect of micronutrient supplementation on acute respiratory infections. random selection of participants of this double-blind intervention trial resulted in a similar distribution of supplementation between cases and controls. there was no significant correlation between positive microbiologic testing and (type of) supplementation [3] . therefore, confounding by the supplementation is likely to be negligible in this study. in conclusion, rhinovirus infections cause substantial morbidity among community-dwelling elderly persons because of its high prevalence in this population. also, although definitely more respiratory micro-organisms were demonstrated among persons with symptoms of an acute respiratory infection, asymptomatic elderly persons can also harbor respiratory pathogens, and thus might be a source of infection for their environment. the aging immune system: primer and prospectus acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden effect of daily vitamin e and multivitamin-mineral supplementation on acute respiratory tract infections in elderly persons-a randomized controlled trial viruses and bacteria in the etiology of the common cold meeting the nutritional needs of the elderly in the institutional setting non-influenza respiratory viruses may overlap and obscure influenza activity acute respiratory tract infection in daycare centers for older persons prospective study of aetiology and outcome of adult lower-respiratorytract infections in the community evaluation of clinical case definitions of influenza: detailed investigation of patients during the 1995-1996 epidemic in france the tecumseh study of respiratory illness. i. plan of study and observations on syndromes of acute respiratory disease acute upper respiratory tract viral illness and influenza immunization in homes for the elderly improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-pcr assay detection of respiratory syncytial virus in acute bronchiolitis in infants evaluation of nested polymerase chain methods for the detection of human coronaviruses 229e and oc43 use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms ari-el studie: acute respiratoire infecties in de eerste lijn acute respiratory illness in the community. frequency of illness and the agents involved a brief clinical instrument to classify frailty in elderly people respiratory syncytial virus or influenza? aetiology of respiratory tract infections: clinical assessment versus serological tests assessment of polymerase chain reaction and serology for detection of chlamydia pneumoniae in patients with acute respiratory tract infection low prevalence of chlamydia pneumoniae and mycoplasma pneumoniae among patients with symptoms of respiratory tract infections in dutch general practices polymerase chain reaction demonstration by a nested pcr for mycoplasma pneumoniae that m-pneumoniae load in the throat is higher in patients hospitalised for m-pneumoniae infection than in non-hospitalised subjects this work was funded by grant 28-2639 from the netherlands organization for health research and development (zon-mw), the hague. we are grateful to francis van rooij, division of human nutrition and epidemiology, wageningen university, the netherlands for her assistance in data and specimen collection. we want to thank berry wilbrink, henk boswijk, hans van der nat, and henk hooft, diagnostic laboratory for infectious diseases and perinatal screening, national institute of public health and the environment, the netherlands, for their technical help. key: cord-017862-9fkjjmvf authors: smith, roger p. title: respiratory disorders date: 2007 journal: primary care in obstetrics and gynecology doi: 10.1007/978-0-387-32328-2_19 sha: doc_id: 17862 cord_uid: 9fkjjmvf like it or not, patients with respiratory complaints are a part of our practice. the common cold is often referred to as the most frequent illness occurring in humans: over 40% of americans suffer from a “cold” each year, accounting for more lost productivity than any other illness. pharyngitis affects almost 30 million patients annually, with over 10% of all school-aged children seeking medical care each year. seventeen million patients a year are diagnosed with asthma, with more females than males among adult-onset patients. whether it is the reason for our patient’s visit or an incidental complaint, we are involved with the diagnosis and management of these problems. like it or not, patients with respiratory complaints are a part of our practice. the common cold is often referred to as the most frequent illness occurring in humans: over 40% of americans suffer from a "cold" each year, accounting for more lost productivity than any other illness. pharyngitis affects almost 30 million patients annually, with over 10% of all school-aged children seeking medical care each year. seventeen million patients a year are diagnosed with asthma, with more females than males among adult-onset patients. whether it is the reason for our patient's visit or an incidental complaint, we are involved with the diagnosis and management of these problems. sinusitis affects roughly 35 million people in the united states annually. the accumulation of purulent material in infl amed paranasal sinuses results in a feeling of fullness and pressure in and over the involved sinuses. these symptoms are worse with posture changes, bending, or with air travel-anything that alters pressure inside the sinuses. nasal congestion and a purulent, blood-tinged discharge, general malaise, low-grade fever, and sore throat are common. these patients will have tenderness over the involved sinuses and edematous or thickened mucosa may be noted on physical examination. these fi ndings help to differentiate sinusitis from the more common cold. the most common cause of sinusitis is bacterial infection with haemophilus infl uenzae, pneumococci, or streptococci. viral and fungal infections may occur, but are less common. upper respiratory infections, allergy, or air travel often precede the development of sinusitis, and secondary infection from a tooth abscess or from swimming in contaminated water may also create a sinus infection. affecting roughly 41% of people per year, the symptoms of the common cold are familiar. the sneezing, runny nose, and malaise usually lasts 6-10 days, but may range from 2 to 26 days. coryza (rhinorrhea and sneezing) is present in 50-66% of patients, and almost 50% of patients experience pharyngitis with a cold. hoarseness and cough develop in 25-50% of patients. between 25% and 45% of patients experience headache, muscular aches, lethargy, and malaise, though only 15-30% actually develop fever or chills. surprisingly, up to 25% of patients infected with the usual cold viruses will not develop symptoms. the patient's symptoms generally begin with the loss of a sense of well-being, scratchy eyes, and discomfort in the nose or back of the throat ( fig. 19.1 ). this is soon followed by sneezing and nasal obstruction with a clear, watery discharge. systemic symptoms, which reach their peak in the fi rst 2-3 days, resolve fi rst, followed by a change in nasal discharge to a cloudy or yellow, thick character. a sore throat, cough, or hoarseness may persist up to 10 days. transmission of the causative virus is usually by personal contact; infected droplets of respiratory discharge are spread by coughing and sneezing, and by transfer from the hands to the eyes, nose, or face. experimental evidence suggests that small doses of virus (1-30 particles) are suffi cient to produce infection. healthy people with normal immune systems are highly susceptible to cold virus infection once the virus enters the nose. in volunteer studies, approximately 95% of normal adults became infected when virus was dropped into the nose. cold viruses are carried to the back of the throat by ciliary action where they are deposited in the area of the adenoids, where the viruses attach. from the time a cold virus enters the nose, it takes 8-12 hours for the viral reproductive cycle to be completed and for new cold virus to be released in nasal secretions. cold symptoms can begin shortly after virus is fi rst produced in the nose (10-12 hours). the time from the beginning of the infection to the peak of symptoms is typically 36-72 hours. there are over 100 serotypes of rhinoviruses that may cause the common cold. these account for about 15-40% of infections. additional viral agents include coronaviruses (10-20% of cases), infl uenza types a, b, and c (1-5%), parainfl uenza (1-5%), respiratory syncytial virus (1-5%), adenoviruses (1-5%), and others. no specifi c agent is known in 30-50% of cases, though it is presumed to be viral. any exposure to an infected person places you at risk for infection. consequently, anything that brings larger numbers of people together, such as daycare, schools, or the work place, increases the chances of infection. the secondary attach rates in families is approximately 25%. cold weather, fatigue, and loss of sleep do not appear to alter the risk of infection, though colds are most prevalent in the winter months. careful hand washing may reduce the risk, especially among those chronically exposed (e.g., health care workers). data regarding the protective effects of large doses of vitamin c are inconclusive. only 12-25% of all "sore throats" seen by physicians have a true pharyngitis-most are simple viral upper respiratory infections such as the common cold. of greatest priority is the identifi cation and treatment of those with group a streptococcal infections so as to reduce the potential for rheumatic sequelae. despite roughly 30 million cases annually, the incidence of rheumatic fever has declined to approximately 64 cases per 100,000. streptococcal pharyngitis has its greatest incidence between the ages of 5 and 18 years, but is still common in patients seen in a gynecology offi ce setting. (see fig. 19 .2 for a decision tree for sore throats.) a sore throat, tonsillar enlargement (often with exudates), soft palate petechiae, and cervical adenopathy characterize true pharyngitis. hoarseness and lower respiratory symptoms should be absent. streptococcal pharyngitis usually runs a 5-7 day course, with a peak fever at 2-3 days. this time course and the presence of a moderate to high fever help to differentiate true pharyngitis from the common cold. spontaneous resolution of symptoms generally occurs, but rheumatic complications are still possible. viral agents, including rhinovirus, adenovirus, and parainfl uenza viruses, cause most pharyngitis. neisseria gonorrhoeae, corynebacterium diphtheriae, and h. infl uenzae may also cause bacterial pharyngitis. the same factors that increase the risk of the common cold (close quarters, unhygienic practices) also increase the risk of pharyngitis. viral infections with infl uenza a or b, parainfl uenza, or adenovirus, or bacterial infections by β-hemolytic streptococcus or streptococcus pneumoniae are the most common etiology for laryngitis. excessive or improper voice use (strain) or aspiration may also result in loss of voice. an upper respiratory tract infection, bronchitis, or pneumonia often precedes laryngitis. environmental causes such as smoking or being in an environment with second-hand tobacco smoke can also cause laryngitis. in the industrial or school environment, exposure to irritating chemicals can also lead to similar symptoms. the peak incidence of laryngitis parallels epidemics of the individual viruses (winter). this subacute viral illness is noteworthy for its barking cough, biphasic stridor, and risk of airway obstruction. while more common in children (the most common cause of stridor in children), the potential for serious complications (e.g., acute obstruction) makes it an illness that should be familiar to gynecologists. patients with laryngotracheobronchitis often have had an upper respiratory prodromal infection in the last 1-7 days. fatigue, malaise, low-grade to moderate fever, and a normal voice characterize the patient's symptoms. the uncomplicated disease usually wanes in 3-5 days but may persist for as many as 10 days. most cases of laryngotracheobronchitis are caused by viral infection (parainfl uenza, infl uenza a, and others). recurrent upper respiratory infections increase the risk of developing laryngotracheobronchitis. as the infection extends to the proximal trachea, diffuse infl ammation with exudate and edema of the subglottic area causes narrowing of the airway. the cricoid ring of the trachea (in the immediate subglottic area) is the narrowest portion of the airway. a small amount of edema in this region can cause signifi cant airway obstruction. (the resistance to fl ow through a tube is inversely proportional to the fourth power of the radius.) air fl owing through this narrowed subglottic area causes the characteristic stridor. with over 10 million new cases diagnosed each year, asthma affects up to one in fi ve children in the united states-more than 4.8 million children under the age of 18. direct health care costs for asthma (adults and children) in the united states total more than $9.8 billion annually; indirect costs (lost productivity) add another $2.8 billion for a total of $12.6 billion. inpatient hospital services represented the largest single direct medical expenditure, over $4.2 billion. the prevalence of asthma increased 75% from 1980 to 1994, and currently affects more than 17 million americans. researchers have yet to pinpoint the cause for the increase in asthma. allergic rhinitis is considered a risk factor in developing asthma, as up to 78% of people with asthma also have allergic rhinitis. while it is tempting to think of asthma as a childhood condition that is not seen in a gynecologic practice, half of all asthma cases occur in patients over the age of 10, and more women than men make up this adult onset group. asthma is a chronic infl ammatory disorder involving constriction of the muscles lining the bronchial airways. physical symptoms of asthma include coughing, wheezing, tightness of the chest, and shortness of breath. narrowing of both the large and small airways results in the wheezing, cough, and dyspnea typical of this condition. hyperresonance and decreased breath sounds are found on clinical examination. allergy, exposure to smoke or other pollutants, viral infections, exercise, or even aspirin intake may induce these episodic attacks. while there is a familial association of reactive airway disease, no known genetic pattern exists. other triggers that may play a signifi cant role in provoking asthma attacks are shown in table 19 .1. asthmatic patients who become pregnant can expect their condition to remain the same or improve (75% of cases). in about 25% of cases, asthma worsens during pregnancy. more common in adults than its viral cousin, bacterial pneumonia has an annual incidence of approximately 20 per 1000 population. of these, approximately 60% are community acquired and 40% are acquired in a hospital or nursing home setting. alcoholics, the debilitated, postoperative patients, patients with respiratory diseases or viral infections, and those who have weakened immune systems are at greater risk. the cardinal signs of bacterial pneumonia are a cough, fever and chills, chest pain, and a thick dark or bloody (rusty) sputum. malaise, myalgia, and abdominal, shoulder, or pleuritic pain may also be present. rales and rhonchi, decreased breath sounds, and vocal fremitus may be found on examination. the tissue of part of a lobe of the lung, an entire lobe, or even most of the lung becomes completely fi lled with liquid (consolidation). the infection may quickly spread through the bloodstream, resulting in septicemia or bacterial seeding to other sites. hematogenous spread or direct inhalation of the organism (s. pneumoniae, h. infl uenzae, staphylococcus aureus, legionella pneumophila, and allergens such as pollens, molds, animal dander, dust mites, and cockroaches irritants such as strong odors and sprays, chemicals, air pollutants, tobacco smoke, and cold air viral or sinus infections including colds, pneumonia, and sinusitis exercise, especially in cold, dry air gastroesophageal refl ux disease (gerd), a condition in which stomach acid fl ows back up the esophagus medication and foods emotional anxiety others) is the most common source of infection for most patients. s. pneumoniae is the most frequent cause of bacterial pneumonia and is the one form of pneumonia for which a vaccine is available. anything that diminishes the host's defenses increases the risk of becoming ill. alcoholism and smoking, immunosuppression and aids, chronic disease, malnutrition, and advanced age are all associated with an increased risk. groups for whom vaccination should be recommended are shown in table 19 .2. viral pneumonia has signs and symptoms similar to those of bacterial pneumonia, with fever, chills, and a productive cough the predominant symptoms. as with bacterial infections, a preceding upper respiratory tract infection is common. rales, rhonchi, and altered breath sounds are typical fi ndings on examination. while 90% of childhood pneumonia is viral, only between 1% and 5% of adult pneumonias are caused by viral infections. infl uenza a and b, as well as parainfl uenza (1, 2, 3, and 4), and respiratory syncytial virus may also be common agents. varicella, herpes simplex, and rubeola are atypical causative organisms but account for signifi cant morbidity when they are the causative agents. infection with the infl uenza virus may be severe and occasionally fatal. the virus invades the lungs and multiplies, but there are almost no physical signs of lung tissue becoming fi lled with fl uid. fatalities are most common among those who have preexisting heart or lung disease or are pregnant. because of its somewhat different symptoms and physical signs, and because the course of the illness differed from classic pneumococcal pneumonia, mycoplasma pneumonia was once believed to be caused by one or more undiscovered viruses and was called "primary atypical pneumonia." mycoplasmas generally cause a mild and widespread pneumonia that can affect all age groups, occurring most frequently in older children and young adults. the death rate is low, even in untreated cases. little separates mycoplasma pneumonia from viral pneumonia except for the presence of cold agglutinins. a prodromal period of mild sore throat, those with chronic illnesses such as lung disease, heart disease, kidney disorders, sickle cell anemia, or diabetes those recovering from severe illness those in nursing homes or other chronic care facilities age 65 or older low-grade fever, and malaise generally precedes the development of paroxysmal cough and blood-streaked sputum. confi ned living spaces (such as military bases, college campuses, and hospitals) increase the risk of mycoplasma epidemics. immunocompromised patients are also at higher risk for infection. pneumocystis carinii pneumonia is caused by an organism believed to be a fungus and is frequently the fi rst sign of illness in many persons with aids. this often insidious infection is seen almost exclusively in immunocompromised individuals. weakness, fatigue, malaise, fever and chills, and mild dyspnea on exertion are typical. a mild nonproductive cough or a cough productive of only scant amounts of clear sputum is common. the clinical signs and symptoms present most often establish the diagnosis of sinusitis. sinus x-rays will show cloudiness and air-fl uid levels with thickened mucosa in the affected sinuses, but are not required for diagnosis in most cases. computed tomography and magnetic resonance imaging are not indicated in these patients. transillumination of the affected sinuses will reveal opacity. a mildly elevated white blood cell count may be found and appropriate cultures (especially in chronic sufferers) may be of help, and can help to separate cases of viral and allergic rhinitis from those of bacterial infection. the possibility of a foreign body must always be considered. the diagnosis of the common cold is made on clinical grounds with testing rarely indicated and useful only when other conditions are suspected. viral culture or isolation is not practical and should not be undertaken. infl uenza, rubeola and rubella, mycoplasma pneumonia, group a β-hemolytic streptococcal infections, and allergic rhinitis may all be confused with the common cold and should be considered when appropriate. in addition to the patient's symptoms, a throat culture (on blood agar) or a rapid screening test for streptococcus is indicated because history and physical examination are only 50% accurate in establishing the diagnosis. because of a sensitivity of 80% and specifi city of 95% for most rapid screening tests, a followup culture is indicated even if the rapid test is used for the initial screening. a fever of greater than 39.2°c (102.5°f), white blood count of greater than 12,000, or a scarlet fever rash (punctate erythematous macules with reddened fl exor creases and circumoral pallor) is suggestive of a streptococcal infection and requires more aggressive evaluation and treatment. a gray pseudomem-brane suggests the presence of diphtheria and vesicles should suggest herpes stomatitis as alternate diagnoses. the diagnosis of laryngitis is made primarily on the patient's symptoms. the hallmarks of laryngitis are hoarseness, abnormal sounding voice, or loss of voice. feelings of throat tickling or rawness, coupled with a frequent urge to clear the throat, are also common. like the cough of the common cold, laryngitis may continue after the acute infection is over. this can be recognized by noting that the fever and ill feeling have resolved, but the hoarseness continues for several days to a week or longer. direct or indirect laryngoscopy is diagnostic but generally beyond the interest of most gynecologists. laryngotracheobronchitis must be differentiated from epiglottitis, foreign body aspiration, diphtheria, and simple upper respiratory infections. x-rays of the neck (pa and lateral) will show a characteristic narrowing in the subglottic region with a normal epiglottis (an inverted v-shaped "steeple" or "pencil" sign). direct laryngoscopy is often required to establish the fi nal diagnosis. early in the course of the disease leukopenia may be present with leukocytosis occurring in later, more severe cases. because hypoxia may be insidious and occurs in up to 80% of children with laryngotracheobronchitis, pulse oximetry should be considered. even in adults, the condition of the patient can change rapidly, necessitating early consultation and aggressive management. allergists follow the national guidelines for the diagnosis and management of asthma (national asthma education and prevention program, national institutes of health, 1997) to diagnosis and establish treatment plans for patients with asthma and other allergic diseases. the diagnosis of asthma is made mainly on the basis of the recurrent clinical pattern. allergy testing, spirometry, and chest x-ray may support or clarify the diagnosis. unusual conditions such as recurrent pulmonary emboli and cystic fi brosis, or more common processes such as congestive heart failure and tuberculosis, must all be considered. special attention should be paid to the nose and sinuses for evidence of chronic infection. a spirometer may be used to objectively measure the amount of air inhaled and exhaled and to determine the level of airway obstruction, though the simple bedside test of asking the patient to blow out a match held at arm's length can provide a quick assessment of forced expiratory volume (fev 1 ). frequently, the criteria for diagnosis have been fever, cough, and development of purulent sputum, in combination with radiologic evidence of a new or progressive pulmonary infi ltrate, a suggestive gram stain, and cultures of sputum, tracheal aspirate, pleural fl uid, or blood. unfortunately, symptoms, elevated white blood count with left shift, hemoconcentration, hyponatremia, and transaminase elevations are all nonspecifi c signs of bacterial pneumonia. the onset of bacterial pneumonia can vary from gradual to sudden. in the most severe cases, the patient may experience shaking chills, chattering teeth, severe chest pain, and a cough that produces rust-colored or greenish mucus. the patient's temperature may rise as high as 105°f. the patient sweats profusely, and breathing and pulse rate increase rapidly. lips and nail beds may demonstrate hypoxia and confusion or delirium may be present. the chest x-ray will show air bronchograms and consolidation, with pleural effusion common. blood cultures will be positive in 20-30% of patients with community-acquired infections. bronchoscopic cultures with greater than 10,000 organisms are diagnostic, but beyond the capability of most gynecologists. induced sputum for culture and gram stain may be helpful, but are less reliable. the initial symptoms of viral pneumonia are the same as infl uenza symptoms: fever, a dry cough, headache, muscle pain, and weakness. within 12-36 hours, there is increasing breathlessness; the cough becomes worse and produces a small amount of mucus. there is a high fever and there may be hypoxia and cyanosis. in extreme cases, the patient has a desperate need for air and extreme breathlessness. exclusion of the more common bacterial pneumonia and the addition of viral culture or fl uorescent antigen studies establish the diagnosis of viral pneumonia. the most prominent symptom of mycoplasma pneumonia is a cough that tends to come in violent attacks, but produces only sparse whitish mucus. chills and fever are early symptoms, and some patients experience nausea or vomiting. patients may experience profound weakness that lasts for a long time. the presence of cold agglutinins in a titer of 1 : 64 or greater, or with a fourfold rise in titers, is found in 50% of infections. cultures for mycoplasma take 7-10 days, so are of little use in making the acute diagnosis. suspicion greatly aids the diagnosis. chest x-ray shows bilateral interstitial or perihilar infi ltrates in 75% of cases, though a normal chest x-ray may be present. serum lactate dehydrogenase (ldh) is often elevated (average 340 iu) and cd4 cell counts are generally depressed (<200) in hiv-infected patients. outpatient care for patients with sinusitis is appropriate except when there is involvement of the frontal or sphenoid sinuses. for simple cases involving the other sinuses, steam inhalation will provide some comfort and promote drainage. irrigation with saline may be recommended, but is seldom required. amoxicillin (500 mg three times a day) or trimethoprim-sulfamethoxazole twice a day for 14-21 days will generally provide good coverage for the most common causative organisms. recent data suggest that treatment courses of as little as 3 days may be suffi cient in uncomplicated cases. with proper antibiotic treatment, over 90% of cases of acute bacterial sinusitis are cured. if response is not forthcoming, a switch to an antibiotic with activity against βlactamase-producing bacteria is prudent. cases of acute bacterial sinusitis that do not clear after a few months of appropriate medical treatment may require sinus surgery. analgesics, vasoconstrictors to relieve fullness, and antihistamines may all be used as needed. patients should be advised to avoid alcohol and caffeine because both may result in swelling of the sinus membranes. though rare, sinusitis may lead to meningitis, extradural, subdural, or brain abscesses, osteomyelitis, or septic cavernous sinus thrombosis. patients with chronic or recurrent sinusitis may require surgical drainage. common sense and supportive therapies are all that are required for most patients suffering from the common cold. rest, fl uids (including fruit juices), smoking cessation, and humidifi cation may all be of some help. the best strategy for treating a cold is to start treatment as soon as there is the recognition that a cold is beginning and to continue treatment on a regular basis until it appears that the cold is over (3-7 days). analgesics, oral decongestants (pseudoephedrine, phenylephrine, and phenylpropanolamine), antitussives (dextromethorphan, codeine) combined with mucolytics (guaifenesin), antihistamines, and topical decongestants (oxymetazoline) may all provide some relief. early studies of the fi rst generation antihistamines for the treatment of colds gave negative results because of inadequate precision in symptom recording. subsequent studies have demonstrated that fi rst generation antihistamines are quite effective in reducing the sneezing and runny nose of colds. the use of topical decongestants should be limited to 3-4 days to avoid rebound hyperemia and congestion. decongestants taken by mouth have less powerful and immediate activity but cause fewer problems with the cycles of recurrent nasal obstruction than topical preparations. there is some evidence that supplemental vitamin c may reduce the duration of disability. the use of zinc gluconate lozenges has been shown to reduce the duration of symptoms but they must be used frequently and are often associated with nausea. the use of zinc (gluconate) lozenges has been shown to reduce the duration of symptoms by roughly one-half if used early in the course of the infection. in clinical trials, doses of 13-15 mg every 2 hours while symptoms persist have been used. studies using intranasal ipratropium bromide (atrovent) three times daily have demonstrated a reduction in rhinorrhea and sneezing. efforts to either treat or prevent the common cold with echinacea preparations have had variable success when subjected to randomized trials. the use of antibiotics has only a limited role in the treatment of the common cold and should be discouraged. when a common cold has lasted for 7-10 days and is no better or worse, acute bacterial sinusitis may have developed and additional medical care may be required. for this reason, antibiotics may be indicated when symptoms continue unabated for more than 10 days. after this time, the probability of a secondary bacterial infection increases to roughly 80%. erythromycin, amoxicillin, and sulfi soxazoletrimethoprim are reasonable empiric choices at this point. symptomatic treatments for pharyngitis include salt-water gargles, acetaminophen, dyclonine lozenges, and use of a cool mist humidifi er. smoking cessation and voice rest are always indicated. when a streptococcal infection is suspected (i.e., when a high fever is present), treatment with penicillin, penicillin vk (250 mg three times daily), erythromycin ethylsuccinate (300-400 mg three times daily), or cephalexin (250 mg three times daily) should be started and continued for a full 10 days. azithromycin is also effective, with its higher cost partially offset by the shorter (5 day) course of therapy. patients are considered noninfectious after 24 hours of antibiotic coverage. complications from pharyngitis are rare and are generally restricted to the bacterial forms. the greatest concern is the development of rheumatic fever and its sequellae. poststreptococcal glomerulonephritis, peritonsillar abscess, otitis media, and systemic infections are also possible complications. usually, laryngitis is self-limiting. however, children's croup or acute epiglottitis can present like laryngitis. the primary treatment of laryngitis is voice rest, humidity (steam or cool mist), increased fl uids, antipyretics, and analgesics (table 19 .3). smoking should be stopped. penicillin g (250 mg every 6 hours for 10-12 days) is indicated when streptococcal or pneumococcal infections are suspected. indications for further investigation or that a change in therapy is needed are shown in table 19 .4. in mild cases, outpatient treatment with humidifi cation, fl uids, rest, and analgesics may suffi ce. dexamethasone has been shown to reduce symptoms in patients with moderate-to-severe croup and is frequently used in children. because of the possibility of acute airway obstruction, more severe cases require hospitalization and intensive monitoring by those familiar with the disease. although there is no cure for asthma, there are effective treatment methods. successful management of asthma consists of four components: (1) patient education, (2) reduction of environmental triggers (allergens), (3) measurement and monitoring of pulmonary function, and (4) pharmacologic intervention. medication therapies are designed to minimize the airway infl ammation component of asthma as well as to treat airway narrowing. environmental control measures are implemented to avoid or eliminate factors that induce or trigger asthma fl are-ups. immunotherapy may also be considered if allergies are known to be an asthma trigger, a condition that is most common in children. the treatment of asthma is based on fi ve major classes of drugs: antiinfl ammatory agents (cromoglycate and nedocromil), steroids (beclomethasone, prednisone), β-agonists (albuterol, terbutaline), methylxanthines (theophylline), and anticholinergics (atropine, ipratropium bromide). a sixth class has gained favor recently: antileukotrienes or leukotriene modifi ers. suck on cough drops, a throat lozenge, or hard candy stop smoking avoid places where cigarettes are smoked use a humidifi er (cool mist ultrasonic humidifi ers are preferred-these are more expensive than the usual vaporizer, but are safer and more effective); you can also try standing in a hot shower use aspirin, ibuprofen, or acetaminophen for temperature, muscle discomfort, and pain; do not give aspirin to anyone less than 19 years of age-it can trigger an attack of reyes syndrome gargle with warm salt solution (1/2 tsp salt in 1 cup of water) speak softly, but do not whisper; use a notebook and pencil to communicate drink warm liquids like hot tang or honey and lemon diffi culty breathing fever over 101°f diffi culty swallowing deep cough or, in a young child, a cough like the bark of a seal (suggestive of laryngotracheobronchitis) brown, green, or yellow sputum hoarseness that persists for 1 month or more inability to carry on normal activities symptoms of gasping or drooling leukotrienes are responsible for the contraction of the airway smooth muscle, increasing leakage of fl uid from cells in the lung, and further promoting infl ammation by attracting other infl ammatory cells into the airways. recently, new antileukotriene medications have been introduced to fi ght the infl ammatory response typical of allergic disease. these drugs are generally limited to the treatment of chronic asthma, though recent data have demonstrated that antileukotriene therapy can be benefi cial for many patients with less chronic asthma. it is likely that these newer medications will eventually have an increased role in asthma care as more studies are conducted. comanagement with both the patient and an asthma specialist is essential when the disease is advanced. mild cases of asthma (brief wheezing one to two times per week) are often managed by intermittent use of β-agonists or theophylline. bronchodilators are generally used as "rescue medications" to relieve coughing, wheezing, shortness of breath, and diffi culty in breathing. those patients with weekly symptoms that interfere with sleep, exercise, or work require a regular maintenance schedule with cromolyn qid or nedocromil bid, with inhaled steroids as an adjunct. theophylline is added when symptoms worsen. salmeterol is a long-acting bronchodilator that, along with an antiinfl ammatory medication, is used for maintenance in the long-term control of asthma symptoms. both patients and physicians must be aware of several potential pitfalls in management: not recommended are mist therapy, fl uid loading, breathing exercises, or intermittent positive-pressure breathing (ippb) therapy. erythromycin and ciprofl oxacin can slow theophylline clearance resulting in increased levels of as much as 15-20% and possible toxicity. in addition to common sense support measures, antimicrobial therapy for the most likely organisms is required. for community acquired infections, empiric treatment with erythromycin (500 iv every 6 hours) provides good coverage, including coverage for mycoplasma and legionella. for patients with nosocomial infections, third-generation cephalosporins (cefotaxime, ceftizoxime) plus vancomycin are recommended. in otherwise healthy adults, improvement should occur in 1-3 days. despite clinical improvement, it may take quite some time for the chest x-ray to clear, necessitating repeated studies for up to 6 weeks. mortality rates for bacterial pneumonia still runs about 5%. this rate rises when the pneumonia is associated with debilitating processes such as alcoholism and aids. for these high-risk patients, consideration should be given to prophylaxis with polyvalent pneumococcal and infl uenza vaccinations (table 19 .2). general supportive measures such as analgesics, antitussives, and antipyretics are appropriate. amantadine (100 mg q 12 h) is effective for treating infections with infl uenza a, but not against infl uenza b infections. symptoms should resolve in several days to a week. it should be noted that amantadine should be used with caution in patients with liver disease or epilepsy, and in those with a history of psychotic illness. rest, fl uids, and analgesics are appropriate initial therapy. antibiotic treatment with either tetracycline or erythromycin (500 mg every 6 hours for 7 days) provides good coverage. azithromycin, given as 500 mg the fi rst day, then 250 mg for days 2 through 5, may also be effective. antibiotics such as penicillin are not effective against mycoplasma pneumoniae. symptoms often take more than 2 weeks to resolve. a false-positive vdrl may be found in patients with mycoplasma pneumonia. trimethoprim/sulfamethoxazole (10-20 mg/kg/day of trimethoprim component divided every 6 hours for 21 days) with adjunctive corticosteroid therapy (prednisone) is standard, though a high percentage of aids patients will develop intolerance to trimethoprim/sulfamethoxazole. trimethoprim/sulfamethoxazole has been used in pregnancy for both treatment and prophylaxis. pneumocystis carinii pneumonia can be successfully treated in many cases. it may recur a few months later, but treatment can help to prevent or delay its recurrence. mortality for fi rst episode infections is now about 10-15%, down from as high as 30-40% previously. approximately 10% of patients develop respiratory failure and more than 85% of these succumb. a 23-year-old g2p2002 patient calls your offi ce complaining of a "sinus infection" and requesting that a prescription for antibiotics be called to a local pharmacy. she reports congestion, a nasal discharge that has become thick and purulent and she has had a low-grade fever. her symptoms began about 5 days ago. how should you proceed at this point? this is a typical presentation for the common cold-the symptoms, their progression and the request for antibiotic therapy are all typical. unless there is a history of previous sinus infections, or a reason to suspect that this patient is at high risk for an atypical infection or complication (e.g., immunocompromise), an offi ce visit, additional testing, or anything other than symptomatic interventions are not warranted. arroll b, kenealy t. antibiotics for the common cold. cochrane database syst rev 2000;(2):cd000247. 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current trials effi cacy and safety of a fi xed combination phytomedicine in the treatment of the common cold (acute viral respiratory tract infection): results of a randomised, double blind, placebo controlled, multicentre study the diagnosis and treatment of cough antivirals for the common cold gastroesophageal refl ux disease-state of the art pharmacologic management of common lower respiratory tract disorders in women the effi cacy of echinacea compound herbal tea preparation on the severity and duration of upper respiratory and fl u symptoms: a randomized, double-blind placebo-controlled study h1-antihistamines in asthma zinc for the common cold beat the winter bugs. how to hold your own against colds and fl u echinacea root extracts for the prevention of upper respiratory tract infections: a doubleblind, placebo-controlled randomized trial use of cough and cold preparations during breastfeeding epidemiology of viral respiratory infections zinc gluconate lozenges for treating the common cold. a randomized, double-blind, placebocontrolled study infl uenza-associated morbidity and mortality in young and middle-aged women highlights from the annual scientifi c assembly: patient-centered approaches to asthma management: strategies for treatment and management of asthma zinc lozenges for treatment of common colds drugs in pregnancy. respiratory disease recommendations of prophylaxis against pcp for adults and adolescents with human immunodefi ciency virus continuous vs intermittent beta-agonists in the treatment of acute adult asthma: a systematic review with meta-analysis heated, humidifi ed air for the common cold antibiotics for acute bronchitis nasal decongestants for the common cold the role of respiratory viruses in acute and chronic asthma the common cold at the turn of the millennium acute upper airway infections key: cord-296256-ipe92w4y authors: evelyn, obando; jaime, fernández-sarmiento; david, montoya; lorena, acevedo; jenifer, arroyave; oscar, gamboa title: prevalence, clinical outcomes and rainfall association of acute respiratory infection by human metapneumovirus in children in bogotá, colombia date: 2019-10-10 journal: bmc pediatr doi: 10.1186/s12887-019-1734-x sha: doc_id: 296256 cord_uid: ipe92w4y background: acute respiratory infections (aris) are one of the main causes of morbidity and mortality in children. viruses are the main etiological agents, and their behavior tends to be seasonal and vary by geographical location. human metapneumovirus (hmpv) has recently been described as a cause of severe acute respiratory infection and its prevalence and clinical behavior in children at moderate altitudes is unknown. methods: a cross-sectional study was carried out on patients seen at a university hospital in bogotá, colombia between october 2015 and december 2017 in a city at a moderate altitude above sea level. children with acute respiratory infections who had had a multiplex rt-pcr assay were selected. the prevalence of hmpv infection, its clinical outcomes and its relationship to rainfall were evaluated. results: out of a total of 14,760 discharged patients, multiplex rt-pcr was performed on 502 and a virus was detected in 420 children with acute respiratory infection (ari). the study group had a median age of 21 months (iqr 7–60), with similar proportion of males and females (56.4 and 43.6% respectively) and 5.2% (ci 95 3.3–7.8%) prevalence of hmpv infection. the group with hmpv infection showed a greater frequency of viral coinfection (22.7% vs 14% p = 0.03) compared with ari caused by other viruses. the rate of bacterial coinfection (p = 0.31), presence of comorbidities (p = 0.75), length of hospital stay (p = 0.42), need for mechanical ventilation (p = 0.75) and mortality (p = 0.22) were similar for hmpv and other viral infections. a moderate correlation was established between hmpv infection and rainfall peaks (spearman’s rho 0.44 p = 0.02). conclusions: human metapneumovirus was the fifth most frequently isolated virus in children with ari, had similar clinical behavior and severity to other viruses but a higher rate of viral coinfection. its peaks seem to correlate to rainy seasons. acute respiratory infections (aris) are one of the main contributors to the burden of disease in pediatrics. it is estimated that children under 5 years of age have an average of three to six episodes of ari per year, with ari being the second cause of death in this age group according to the world health organization (who) [1] . viruses are the most commonly isolated microorganisms in acute respiratory infection, both in adults and in children. every year in the united states, between 20,000 to 58,000 children under 5 years of age are hospitalized due to respiratory syncytial virus (rsv) and influenza virus infections [2] . discovered in 2001 by dr. van den hoogen, human metapneumovirus (hmpv), belonging to the paramyxoviridae family, has circulated for more than five decades [3] , but its importance as an etiologic agent of upper and lower ari, with the potential for developing severe disease is emergent. hmpv infection is responsible for approximately 4-16% of ari hospitalizations in pediatrics [4] , of those affected 15-25% needs transfer to intensive care and 8-17% requires mechanical ventilation [5] . during 2007-2013 in the united states, carly et al. estimated that each pediatric patient hospitalized for acute respiratory infection due to hmpv may spend an average of 6000 dollars per hospitalization, which is much greater than the cost of other viruses and other chronic conditions [2] . it has been suggested that the prevalence of hmpv has seasonal and geographical variations [6] . colombia, located near the equator, is a tropical country with five natural regions; each region maintains an average temperature throughout the year so the rainfall is the most important climatic variable that affects viral circulation. bogotá is located in the andean region, is one of the coldest cities in the country, located at an altitude of 2630 m above sea level, acute respiratory infections have been thought to have a different behavior at higher altitudes in terms of higher frequency and severity. our objective is to estimate the prevalence and describe the clinical behavior of ari caused by hmpv in pediatric patients hospitalized in a fourth level hospital in bogotá and evaluate its association with the rainfall variations. a cross-sectional study was performed on children under 18 years of age hospitalized at the fundación cardioinfantil-ic, a tertiary university hospital located in bogotá, colombia. a total of 14,760 patients were discharged between october 2015 and december 2017, out of whom 502 children had a multiplex rt-pcr (filmar-ray® biomériux). this city is located 2630 m above sea level with the characteristic weather and rains patterns of tropical countries and the andes mountains. it is characterized by an annual mean temperature of 14°c with variations between 6 and 19°c, and peaks of rain from march to may each year, as well as from october to november. this study was approved by the institutional ethics committee (protocol: pm-1090-2018). the data was taken from the institutional electronic charts of children who were hospitalized for acute respiratory infections and who received multiplex rt-pcr (filmarray® biomériux), the analyte used in this technique for hmpv detection was type 16, a1 ia10-2003 zeptometrix 0810161cf. patients who had had this test without experiencing respiratory symptoms and those who had been out of the city in the 2 weeks prior to hospitalization were excluded. the respiratory multiplex rt-pcr assay (filmarray® biomériux) was ordered for patients with upper and lower respiratory symptoms at the attending physician's discretion, and samples were taken from nasopharyngeal aspirates by the respiratory therapy specialist and processed within 30 min of collection, which is an institutional standard for sample handling. the rt-pcr detects 17 viruses and 3 bacteria and is currently considered to be the gold standard for detecting these microorganisms. clinical behavior was assessed in terms of the main complications previously described for hmpv, such as length of hospital stay, need for transfer to intensive care, need for ventilatory support and duration of mechanical ventilation. acute respiratory infections were classified as rhinopharyngitis, laryngitis, croup, bronchiolitis, tracheitis, pneumonia or acute respiratory distress syndrome (ards), according to the criteria of the attending physician. severe ari was defined as the need for oxygen with a fraction of inspired oxygen (fio 2 ) greater than 40%; the need for non-invasive mechanical ventilation, high flow nasal cannula ventilation or invasive mechanical ventilation; and/or hemodynamic instability requiring vasoactive support. patients in whom hmpv was identified 48 h after admission, and who did not have respiratory symptoms on admission, were considered to have a nosocomial infection. viral coinfection was established if two or more respiratory viruses were isolated on the rt-pcr assay, and bacterial coinfection was determined if blood cultures and/or orotracheal secretion cultures were positive and /or procalcitonin was > 0.5 μg/l. xadditionally, the ideam (institute of hydrology, meteorology and environmental studies) database was used. this is the governmental institution responsible for the analysis and detection of climate and meteorological changes in colombia. monthly rainfall patterns for the described study period were analyzed looking for an association with a higher or lower frequency of acute viral respiratory infections. statistical analyses were performed using stata 14.0 (statacorp lp, college station, tx), using descriptive statistics for the demographic variables and central tendency and dispersion measure for continuous variables, according to the distribution of the variable defined by the shapiro wilk normality test. absolute and relative frequencies were described for qualitative variables. the prevalence was estimated for the described observation period. for factors related to severity, a bivariate analysis was performed using student's t-test for independent samples, when the variable was quantitative and parametric. otherwise, a wilcoxon rank-sum test was used. for categorical variables, tests of independence were performed using chi-square. if this was not possible, due to the number of observations, fischer's exact test was performed. for seasonality analysis, the monthly number of infections due to human metapneumovirus was calculated, as well as the number of respiratory syncytial virus and rhinovirus/enterovirus infections, using a spearman's correlation analysis for non-parametric variables. out of a total of 14,560 pediatric patients seen during the research period, 502 multiplex rt-pcrs were performed on hospitalized patients. altogether, 82 were excluded because the test had been performed on a patient without a diagnosis of acute respiratory infection or the patient had traveled outside of bogotá during the 2 weeks prior to being hospitalized. a total of 420 patients were included; the median age in the analyzed group was 43 months (iqr 9-101) in the hmpv positive group and 21 (iqr 6-57) in hmpv negative group (p = 0.08). the proportion of affected girls and boys was similar between groups (p = 0.51) ( table 1) . in 291 (69.3%) cases, at least one microorganism was detected, the most frequently isolated etiological agents being rhinovirus/enterovirus (30% (ci 95 25-34%)), rsv (19% (ci 95 15-23%)), parainfluenza 3 (7.4%), and adenovirus (5.7%). a 5.2% (ci 95 3.3-7.8%) hmpv infection prevalence was found (22 patients), making it the fifth most frequently isolated virus ( table 2) . within this group, in 17 out of 22 cases, hmpv was the only virus detected. viral coinfection was documented in 22.7% of cases. the most frequent viral association was with the rhinovirus/enterovirus complex in 60% of children, followed by influenza a/h1-2009 and parainfluenza 3. in this regard, the frequency of hmpv viral coinfection is greater (22.7 vs 14% p = 0.03) than the viral coinfection of the other viruses detected. bacterial coinfection was documented in 27.3% of patients with hmpv and in 38% of children with other infections. of the patients studied, 26.6% (n = 112) were previously healthy and had no chronic medical conditions. the following comorbidities were found in the entire study group: congenital heart disease (16.2%), prematurity (13.1%), liver disease (12.6%), bronchopulmonary dysplasia (12.4%), cancer (11.9%), transplant (11.2%), kidney disease (9.5%), and malnutrition (35.9%). no statistically significant differences were found between those with or without metapneumovirus infection and the presence of any of the described comorbidities ( table 1) . the most frequent diagnoses for both groups were pneumonia (50%), bronchiolitis (18.3%), and rhinopharyngitis (14.1%). altogether, 77.3% of metapneumovirus infections were community-acquired, as well as 69.4% of other respiratory infections ( table 1) . the median hospital stay for the group with hmpv infection was 10.5 days (iqr 6-26) and for those without hmpv infection was 12 days (iqr 6-27) (p = 0.42). of the patients with hmpv infection, 54.5% required admission to the intensive care unit, and in the group without this infection, the rate was 48.5% (p = 0.58). the need for mechanical ventilation showed a similar behavior between both groups. therefore, 66.7% (n = 8) of patients with hmpv infection required ventilatory support (58.3% invasive and 8.3% non-invasive) with a median duration of mechanical ventilation of 7 days (iqr [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . similarly, 48.2% (93) of children without hmpv infection required mechanical ventilation (20.9% invasive and 2.5% non-invasive) with no significant difference in terms of need (p = 0.88) and duration of mechanical ventilation (6 days, iqr 3-14) between the groups ( table 3 ). the global mortality was 7.1% of the study sample (hmpv 13.6% vs non-hmpv 6.8%, p = 0.2). out of the 22 patients with hmpv, 54% were found to have severe respiratory infection (table 4 ). this group with severe hmpv, 58.3% were under 2 years old, while only 20% of those without severe hmpv infection belonged to this age group (p = 0.27). viral coinfection was more frequent in the group with severe hmpv infection (n = 12) (33% vs 10%, p = 0.32), compared to those who did not have it. likewise, the bacterial coinfection rate was considerably greater in patients with severe infection (50%) compared to those who did not have severe infection (10%) (p = 0.16). the rainfall in the city of bogotá was analyzed using the average rainfall reported by the 14 ideam (institute of hydrology, meteorology and environmental studies) monitoring stations (fig. 1) . typical tropical zone behavior was observed with a greater frequency of viral infections during the rainy season. the spearman rho correlation analysis showed a moderate correlation (r = 0.44, p < 0.02) between the presence of hmpv infection and the highest rainfall peaks, just as occurs with rsv (r = 0.32, p < 0.02) (fig. 2) . however, this correlation was not seen with the rhinovirus/enterovirus complex, which was the most frequently detected co-infecting agent for hmpv (r = − 0.13). in this study, we investigated the prevalence of hmpv in pediatric patients hospitalized in bogota, colombia. we found a 5.2% prevalence of hmpv infection (ci 95% 3-3 -7.8%). it was the fifth virus detected in hospitalized patients. in latin america, the prevalence in hospitalized patients is highly variable, being reported as high as 20% in the southern brazil [7] , 11% in argentina [8] and 9.2% in uruguay [9] . this behavior is most likely explained by the variable climate changes between the latin american countries and the techniques used for viral detection [10] . hmpv has often been described as a coinfectant with other viruses. in children, viral coinfection is highly variable for hmpv [11] . in spain, there was an estimated rate of 38% [12] , in jordan it was 52.5% [13] , and in south china it was 18.4% [14] . the viral coinfection rate has not been described for hmpv in children in colombia, which in our study was 14%. however, we found a greater frequency of viral coinfection in the hmpv group compared with coinfection of the other respiratory viruses, and this difference was statistically significant (22.7% vs 14%, p = 0.03). the most frequently described viral association in respiratory infection has been between rsv and hmpv. bear in mind that they belong to the same viral family and have a high genetic similarity, usually presented during the same epidemiological periods. in a recent article, schuster et al. found that rsv was the main coinfecting agent, with a global rate of coinfection of 26.4% [13] . in our study, rhinovirus/enterovirus was the main agent presenting simultaneously with hmpv in 60% of cases. contrary to the findings of this author, we did not find rsv coinfection in these children. with respect to bacterial coinfection, garcía-garcía et al. reported that 25.6% of the patients in their study received concomitant antibiotic treatment due to suspected bacterial infection associated with hmpv infection [12] , but the frequency of presentation is not documented. in our study, we found that 27.3% of patients positive for hmpv had bacterial coinfection documented by pcr, cultures or positive procalcitonin techniques. regarding the clinical behavior of hmpv infection, it has been reported that 2.2-7% of affected patients require admission to intensive care [12, 15] . in our study, [16] , they found that the duration of ventilatory support in patients with hmpv was 7 days and that 25% of their children required invasive ventilatory support. we found that children who required a picu stay had pneumonia more often than children with hmpv who did not need intensive care (58.3% vs 30% respectively), which could partially explain these findings. inpatient outbreaks of rsv in children have been described on hospital floors and in intensive care units [17] , but inpatient hmpv outbreaks are rarely reported [18] . in this study, one out of five children acquired the hmpv infection during their hospital stay. nosocomial hmpv infections with molecular confirmation have been described in cancer patients, and outbreaks have also been described in geriatric institutions and on psychiatric wards [19, 20] . contact seems to be required for viral transmission; the analysis of this situation forces us to look for strategies to decrease inpatient viral transmission by improving our isolation conditions and patient management [21] . more studies are needed to determine the risk factors for acquiring nosocomial hmpv infection. considering that bogotá is located in a zone without seasons, it does not have the drastic temperature changes like other countries. the most important climatic variable that can affect viral circulation is rainfall. it has been postulated that viral transmission increases in colder seasons. also, that the antiviral immune response in the nasal epithelium is attenuated under these circumstances [21, 22] . previous studies have shown that hmpv may have alternating epidemiological profiles in europe. it has been found that the percentage of annual hmpv infection may vary year to year, and may oscillate between 2.3 and 19.9% in the same region [12, 23] . the annual prevalence difference in our study was 4.4% in 2016 and 5.6% in 2017. a greater frequency was noted in april-may 2016, april-june and september-october 2017, with a low positive correlation between hmpv infection and the highest rainfall peaks (r = 0.44, p < 0.02). the differences in this circulation pattern, besides being related to changes in rainfall, may be associated with the circulation of different hmpv serotypes [24] . four different hmpv lineages are known, designated as a1, a2, b1 and b2. these may coexist in the same period or one may be predominant [25] . the respiratory rt-pcr used in this study does not allow for serotype differentiation, and therefore no conclusion can be drawn regarding their differential behavior. our study has several limitations. the findings are from a single tertiary care center, and although multiplex rt-pcr was used, it was not performed on all hospitalized patients with acute respiratory infections due limitations of health system. likewise, this test does not identify serotypes (a1, a2, b1, b2) which could partially explain some different seasonal behaviors of the virus. similarly, the study design allowed us to observe the behavior of the virus over a specific time period; this behavior may change according to serotypes and environmental variations, among others. additionally, our data only represents association because we cannot infer that hmpv was the only causal factor of the observed disease, especially in cases with viral coinfection. human metapneumovirus was the fifth most commonly detected acute respiratory infection virus in this study. its clinical behavior at moderate altitudes in terms of severity is similar to that of other respiratory viruses. these children frequently need to be transferred to intensive care and have viral coinfection. we observed that this infection presents more frequently during the rainy season. united nations children's fund (unicef) incidence, morbidity, and costs of human metapneumovirus infection in hospitalized children a newly discovered human metapneumovirus isolated from young children with respiratory tract disease human metapneumovirus: review of an important respiratory pathogen human metapneumovirus infections are associated with severe morbidity in hospitalized children of all ages population-based incidence of human metapneumovirus infection among hospitalized children human metapneumovirus in southern brazil evidence of human metapneumovirus in children in argentina investigación de metapneumovirus humano en pacientes hospitalizados: estudio multicéntrico relationship between meteorological conditions and respiratory syncytial virus in a tropical country rapid detection of respiratory organisms with the filmarray respiratory panel in a large children's hospital in china human metapnuemovirus infections in hospitalized children and comparison with other respiratory viruses. 2005-2014 prospective study human metapneumovirus infection in jordanian children. epidemiology and risk factors for severe disease epidemiological and clinical features of human metapneumovirus in hospitalised paediatric patients with acute respiratory illness: a cross-sectional study in southern china burden of human metapneumovirus infection in young children infección por metapneumovirus humano en niños hospitalizados por una enfermedad respiratoria aguda grave: descripción clínico-epidemiológica nosocomial respiratory syncytial virus infections in children's wards molecular epidemiological investigation of a nosocomial outbreak of human metapneumovirus infection in a pediatric hemato-oncology patient population serious outbreak of human metapneumovirus in patients with hematologic malignancies viral respiratory infections diagnosed by multiplex polymerase chain reaction in pediatric patients analysis of the thermal and ph stability of human respiratory syncytial virus temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells biennial spring activity of human metapneumovirus in austria severe acute respiratory syndrome in children the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank the medical, nursing and respiratory therapy team of the pediatric icu for their kind cooperation, as well as the clinical laboratory of the fundación cardioinfantil-ic. authors' contributions drs. fj, oe, md and go contributed to designing and performing the study. drs. oe, al, md and aj participated in data collection. drs. fj, oe and go supervised study development and data collection. all the authors contributed to drafting the manuscript and reviewing the final article. all the authors approved the final manuscript and agreed with all aspects of the study. none of the investigators declare conflicts of interest. all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. our funding for research are researchers' resources. we did not have financing from any other company.availability of data and materials datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. the ethics committee of the fundación cardioinfantil-instituto de cardiología approved this study with committee's reference number ceic 3635-2017 and the parents of the children approved to participate in this study. not applicable. the authors declare that they have no competing interests.author details 1 department of pediatrics and intensive care, universidad de la sabana, fundación cardioinfantil-instituto de cardiología, bogotá, colombia. key: cord-277313-5f5lrn3c authors: hayakawa, satoshi; komine‐aizawa, shihoko; mor, gil g. title: covid‐19 pandemic and pregnancy date: 2020-08-10 journal: j obstet gynaecol res doi: 10.1111/jog.14384 sha: doc_id: 277313 cord_uid: 5f5lrn3c at the end of 2019, a new coronavirus disease, covid‐19, emerged and quickly spread around the world. severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2), the causative virus of this disease, belongs to the β‐coronavirus family, together with sars and middle east respiratory syndrome, and has similar biological characteristics to these viruses. for obstetricians, the susceptibility and prognoses of pregnant women and the effects of the infection on the fetus have been the focus of attention; however, at present, the seriousness of the disease in pregnant women is not apparent, and covid‐19 does not increase the rate of miscarriage, stillbirth, preterm labor or teratogenicity. even so, carriers might transmit sars‐cov‐2 to pregnant women. thus, we must keep in mind that all medical personnel must understand and maintain standard precautions in their clinical and laboratory practices. disaster strikes when we least expect it. (1929, terada torahiko) it is easy to underestimate things or to be overly afraid of them, but it is difficult to justly evaluate them. (1935, terada torahiko) introduction: an emerging infection on december 30, 2019, the chinese health agency was made aware of "unexplained pneumonia" in wuhan, hubei province, and identified its pathogen as a new coronavirus (2019-ncov). the genome of this virus was quickly sequenced, and genetic similarity to severe acute respiratory syndrome (sars)-cov was noted. 1, 2 later, the who decided to name the emerging viral disease covid-19, and the name of the virus was designated severe acute respiratory syndrome coronavirus 2 (sars-cov-2) by the international virus naming committee due to its high homology with sars-cov. it spread from china to the whole world, and the who declared a pandemic on march 11. on april 8, the government of japan declared a state of emergency for seven prefectures (tokyo, saitama, chiba, kanagawa, osaka, hyogo and fukuoka), and the state of emergency was expanded throughout japan on april 16. in a very short period of time, covid-19 had spread throughout the world, and as of may 16, a total of 4 660 611 patients and 309 710 deaths have been confirmed in 215 countries, areas or territories. 3 the number of new cases has already declined in china, where the epidemic first began. in regions of the middle east and europe, including iran, italy, spain and france, where it had subsequently spread, the number of cases is also on the decline. although the u.s. and uk are past their peaks on the infection curve, a large number of cases are still being reported. 4 furthermore, although the disease spread to japan early in the pandemic, the number of patients and deaths is still low. 5 thus, it is unique and puzzling that no spike in the infection rate took place in japan, even though the country did not implement strict urban blockades. to date, no one can adequately explain why the infection and mortality rates are low in japan. what is sars-cov-2? coronaviruses (covs) possess an enveloped, single, positive-stranded rna genome, which encodes four membrane proteins, namely, spike (s), envelope (e), membrane (m) and nucleocapsid (n) proteins 3-5. s proteins are essential for viral entry into host cells and determine viral pathogenicity. 6 in addition to the four types of coronaviruses that cause the common cold, two other types of coronaviruses were reported in the 21st century: sars-cov, the pathogen underlying the sars epidemic in 2003, and middle east respiratory syndrome (mers)-cov, the pathogen underlying the mers epidemic in 2012. sars-cov-2 has higher genetic homology with sars-cov and mers-cov than with the other four coronaviruses and has conserved sequences responsible for immunomodulation. to date, numerous coronaviruses have been identified in various wild and domestic animals. however, humans are not susceptible to these coronaviruses. from genetic analysis, sars-cov-2 developed zoonotic infectivity in animals such as bats and curlews toward human-to-human infection in a short period of time. this fact completely matches the theoretical prediction of most emerging human infections derived from zoonotic ones. 7 early reports from china have shown two major sars-cov-2 genome types (l and s). 8 although the s type has been suggested to be the ancestral type, the most prevalent type in wuhan was the l type. the evolutionarily older but less aggressive s types have relatively weak selection pressures, so their numbers have been decreased. 9 on the other hand, the s type spread to other cities in china as well as japan during the urban blockade of wuhan. however, after global spread, both the s and l types were detected in japan. phylogenetic analyses are being performed to obtain real-time information on molecular epidemiology. on apr 18, a molecular epidemiological report was published from iceland, where viral subtypes could be determined in every patient. based on this article, each country has a slightly different type of virus that is prevalent. east asia has mainly the a and b types, the west coast of the united states has b1a types, europe has mostly a2a types, and iceland seems to have additional types, such as a2a3a, which branched off from europe (all of these types appear to have originated in china). these data suggest that certain viral variants tend to favor certain ethnicities (i.e., viruses with certain variants are more prevalent in certain ethnicities). 10 sars-cov-2 enters susceptible host cells via the angiotensin converting enzyme (ace)2 protein as its receptor. ace2 is abundantly expressed in the type ii alveolar epithelium of the lower respiratory tract but is also expressed in the upper respiratory tract, pharynx and gastrointestinal tract, especially the small intestine. 11 viruses reach mucous membranes through droplets or via fingers that touch the surfaces of contaminated objects and infect susceptible cells. an ace2-independent pathway of infection via dipipetidyl peptidase 4(dpp4) and or glucoseregulated protein 78(gpr78) also may exists in various cells including lymphocytes, which induces transient immunodeficiency; however, the details of this process are unknown. 12 viremia is not frequently observed, but we cannot rule out blood-borne dissemination of sars-cov-2 into various organs. in contrast to children and young individuals who are less susceptible to covid-19, older adults and those with complications such as diabetes mellitus and hypertension have the highest mortality rates. 13 although the majority of infected individuals are asymptomatic or have only mild symptoms, they might spread virus via cough, saliva and stool. after periods of incubation of up to 14 days, symptoms including fever, cough and malaise might appear. the asymptomatic carrier state is important because they also spread virus into the air and surroundings. in severe covid-19 cases, cytokine storms are thought to be associated with systemic tissue destruction and subsequent poor prognoses. 14 however, whether cytokine storms are more prevalent in the older adults than young people is unknown, in contrast to the novel influenza a of 2009, which evoked cytokine storms in younger people. 15 fortunately, there are no reports of pregnant women being prone to cytokine storms caused by covid-19. an autopsy report of a 85-year-old chinese male patient showed diffuse alveolar injury and pulmonary edema with infiltration of multiple lymphocyte types into the interstitial area. infected cells showed remarkable degeneration associated with nuclear enlargement, amphibole granules, multinucleation or syncytium formation. the increase in peripheral ccr4 + ccr6 + th17 cells and perforin-and granzyme-containing cd8 + t cells suggests that over activation of cytotoxic t cells might be involved in tissue destruction. one of the most characteristic features is rapid exacerbation in some cases. possible causes of rapid progression from relatively mild cold-like symptoms to acute respiratory failure are presumed to be microthrombi in the lungs. 17 morphologically, sars-cov-2 has been reported to proliferate in the vascular endothelium, 18 which can be a rationale for the effectiveness of anticoagulation therapy. two major concerns of pregnant individuals with covid-19 are their prognoses and the possibility of vertical transmission and subsequent miscarriage, malformations, fetal growth restriction and/or stillbirth. the worldwide common misunderstanding is that "pregnancy is an immunosuppressed condition, and pregnant subjects become susceptible to infectious disorders". this is not true because pregnant subjects reject allograft transplants and produce an effective immune response against infectious pathogens. as reported by our group, 19 and others, in contrast to organ transplantation, which requires constant immunosuppression, a successful pregnancy requires a robust, dynamic and responsive immune system. in general, pregnant mothers become tolerant to fetoplacental semi-allografts but maintain an adequate immune response to pathogens. enhanced induction of interferon (ifn)-β is crucial to protecting the fetus against viral infections at the maternal-fetal interface; thus, placental viral infections might threaten fetal survival even if they do not enter fetal tissues. one immunological concern is the suppressive activity of sars-cov-2 on ifn responses for its escape from the immune system. 20 however, fortunately, clinical data suggest no deleterious outcomes of pregnant women who are infected with covid-19 during pregnancy compared with those infected with sars-cov or mers. 21 the first case report from wuhan described nine cases of pregnant women diagnosed with covid-19 in late pregnancy. 21 their clinical courses and disease severities were not different from those of nonpregnant women, and no cases of intrauterine infection of their fetuses were observed. in another report from wuhan, of 13 pregnant women who developed covid-19 during pregnancy, one woman delivered a dead fetus at 34 weeks of gestation, but the cause of fetal death was speculated to be severe maternal pneumonia and multiple organ failure rather than viral infection of the fetus. 22 another report showed that 3 of 33 pregnant women who developed covid-19 during pregnancy in wuhan showed evidence of intrauterine infection by cord blood pcr test. 23 two of three suspected intrauterine infections were asymptomatic, but one neonate who was delivered at 31 weeks of gestation by emergency cesarean section due to maternal pneumonia had complications including severe pneumonia and sepsis, though the neonate was ultimately rescued. 23 another report from wuhan showed a relatively high incidence of intrauterine infection of up to 33% (two of six cases of late cesarean section) by cord blood igm test. 24 on the other hand, no cases of intrauterine infection were observed from 106 deliveries at 25 hospitals in various cities in china from january to march, although 8% of 116 cases had serious pneumonia. 25 in every case, the mothers and neonates survived without serious sequelae. only one woman had early spontaneous miscarriage, but no causal relationship was identified. 25 this discrepancy might reflect the difficulty of serum igg and igm testing because most studies employed less sensitive immunoblot detection systems. a systematic review by zaigham 26 evaluated the risk of serious maternal outcomes in 2.4% (3 of 108 cases) and 1.8% of neonates (one neonatal death and one intrauterine fetal death). although the total death rate in italy is higher than that in china, the prognoses of pregnant women in italy are almost comparable to those in china. 27 ferrazzi et al. reviewed 42 pregnant women who were diagnosed with covid-19 among 7000 deliveries from february to april in lombardy. there were two preterm births. seven of the 42 pregnant patients became seriously ill and were admitted to the icu, and all recovered quickly. no fetal or neonatal death was observed. however, a case report from texas presented severe neonatal pneumonia caused by intrauterine covid-19 infection, 28 and there have been other tragedies. for example, a case of intrauterine fetal death at 31 weeks of gestation and maternal death due to severe pneumonia was reported in iran. 29 yan et al. reviewed 116 cases and concluded that covid-19 did not increase the risk of spontaneous preterm birth before 37 weeks of gestation. 25 among 43 covid-19 pcr-positive women in new york, 86% had mild disease, 9.3% had severe disease, and 4.7% had severe disease. 30 another study reported that cases of covid-19 pneumonia during pregnancy were milder and that patients had a better recovery. 31 however, a very recent multicenter study from iran reported the deaths of 7 of 9 pregnant women who contracted covid-19 in the late second and third trimesters. 32 the death rates can be influenced by local factors, including medical accessibility, economic and political conditions and social hygiene. we need to collect and analyze possible risk factors for maternal and neonatal outcomes to reduce the mortality rates of both mothers and infants. the experience in the us has been more complex; the initial cases followed similar prognosis as those reported in wuhan; unfortunately the us has been experienced a significant higher number of cases, including pregnant women. the most recent report from 12 us institutions evaluated 64 pregnant women hospitalized with covid-19, 69% had severe and 31% critical disease. some of these patients had preexisting comorbidities including 25% with pulmonary condition and cardiac disease. gestational age at symptom onset was at a mean of 29 weeks and hospital admission at 30 weeks. eighty eight percent of pregnant women with critical covid-19 who delivered during their disease course were delivered preterm, 94% of them via cesarean. in all 75% of critically ill woman delivered preterm. there were no reports of still births or neonatal deaths or cases of vertical transmission. 33 placental infection with covid-19 may be the critical factor that may lead to pregnancy complications. although few, new reports have shown the presence of sars-cov-2 within the placenta correlating with pregnancy complications such as miscarriage and pre-eclampsia. 34 hosier et al reported a case of second trimester pregnancy with symptomatic covid-19 complicated by pre-eclampsia and placental abruption. when they analyze the placenta they found the presence of sars-cov-2 localized predominantly to syncytiotrophoblast cells at the maternal-fetal interface of the placenta. 35 while early studies showed no evidence of vertical transmission of sars-cov-2 from mother-to-child in late pregnancy, 21 recent reports have shown possible in utero transmission. 24, 36 possible cases of postpartum infections emphasize the importance of adequate physical separation between mothers and neonates. 37 the presence of cord blood igm is direct evidence of vertical transmission. one study reported a case in which anti-sars-cov-2 igm antibody was present at 2 h after birth. 36 the importance of the proper definition of vertical transmission is critical for understanding the risks associated with fetal development and maternal mortality. although a few studies have shown the presence of the virus at the placenta, these findings cannot be considered as vertical transmission. we define vertical transmission when the virus is able to reach fetal organs and they can be detected in fetal organs. presence of the virus at the placenta cannot be considered as vertical transmission. the function of the placenta as an immunological barrier includes the fact that can be infected but prevents the crossing of the virus from the maternal side into the fetal side. 38 currently, there are four methods used to diagnose covid-19: (i) detection of viral rna, (ii) detection of specific antibodies in the blood, and (iii) detection of ground-glass opacities in the lungs by ct scanning, (iv) detection of viral antigens. to detect viral nucleic acids, real-time pcr or loop-mediated isothermal amplification (lamp) are the most common and sensitive methods for clinical specimens, but both methods are time-consuming, require extensive effort and have a high false-negative rate. blood antibodies are not currently recommended due to reliability issues, such as the fact that it takes more than 1 week for antibody conversion even after infection; additionally, igm antibodies are not detected in the early stages. 39 the sensitivity and specificity of ct are good, but ct is not recommended for pregnant women due to radiation exposure; furthermore, ct cannot distinguish covid-19 from other viral pneumonias. very recently, detection of viral antigens by the immunoblot with saliva have been reported as a novel method. however, its sensitivity is still low compared to pcr and needs to be improved to make it as a routine examination. 40 at this time, there is no specific medicine or effective vaccine for covid-19. mechanisms of sars-cov-2 replication and the sites of expected antiviral medicines are shown in figure 1 . the current therapeutics that are expected to be effective and undergoing table 1 . a cocktail of protease inhibitors (kaletra [lopinavir/ritonavir]) was tested in the first clinical trials in china. lopinavir and ritonavir were originally used against hiv-1 and are effective. based on their efficacy against mers in vitro and in animal models, they have been expected to be effective against covid-19; however, they were not effective against covid-19 in 199 patients with severe disease in china. a randomized trial showed no significant difference in either clinical improvement or 28-day mortality. 41 favipiravir, a selective inhibitor of viral rna polymerase, was developed to treat novel influenza. based on its broad spectrum antiviral efficacy against various rna viruses, including ebola, clinical trials are ongoing. a nonrandomized controlled trial from china reported shorter virus disappearance times. 42 many case reports are available on the website of the japan society for infective diseases. 43 however, favipiravir is contraindicated in pregnant patients as well as men and women who wish to conceive due to its strong teratogenicity. remdesivir is an rna-dependent rna polymerase inhibitor that is active against a wide range of rna viruses. it was originally developed for the treatment of ebola virus infection and shows good activity against covid-19 in vitro. foreign and domestic clinical reports show its possible clinical importance, but an rct of remdesivir was discontinued in china. 44 although remdesivir may be effective against covid-19, serious side effects, including severe liver dysfunction, diarrhea, skin rash and renal dysfunction, are frequent. hydroxychloroquine, a derivative of the antimalaria drug chloroquine, is widely used in european and american countries. clinical studies of hydroxychloroquine as a single agent or as an adjunctive therapy are underway. in an early nonrandomized controlled trial in france, hydroxychloroquine in combination with azithromycin significantly reduced the viral road, 45 but other studies failed to show this effect. 46 ciclesonide is an inhaled steroid, originally indicated for the treatment of bronchial asthma. in addition to its anti-inflammatory effects, its specific antiviral activity has been reported. 47 in addition to many case reports, clinical trials are ongoing in japan. tocilizumab, a humanized anti-human il-6 receptor monoclonal antibody, has been used for rheumatoid arthritis and other collagen diseases. its effectiveness against covid-19 has been reported in china. a single-arm study showed of a higher survival rate for patients with severe pneumonia. 48 one important feature of covid-19 is its effect on the blood coagulation system. the royal college of obstetricians and gynecologists (rcog) advised clinicians that infection with covid-19 may increase the risk of venous thromboembolism and that this risk may be compounded by reduced mobility due to self-separation. thus, rcog recommended that pregnant individuals hospitalized with covid-19 should receive prophylactic low-molecular-weight heparin to reduce the risk of pulmonary embolism. 49 based on initial reports from china, every term or premature case with covid-19 with acute distress and/or severe maternal conditions was delivered by cesarean section. however, in an italian study, among 42 deliveries, 24 (57%) women delivered vaginally, with three cases undergoing induction of labor for obstetric reasons, while elective cesarean section was performed in 18 (43%) cases: in 8 cases, the indication was unrelated to covid-19 infection, but in 10 cases, the indications were worsening dyspnea or other covid-19-related symptoms. 50 a systematic review showed that overall, 90.1% of deliveries were cesarean sections. it is reasonable to choose cesarean section to reduce the opportunity for viral transmission from pregnant subjects with covid-19 to other patients and medical staff, but vaginal delivery must be chosen if the woman is multiparous and the uterine cervix has been fully open to complete the delivery sooner. guidelines from various countries are controversial in terms of the choice of delivery mode. this is because it depends on the availability of medical assets as well as human resources. both obstetricians and general practitioners as well as health care professionals are requested to advise pregnant women on how to avoid sars-cov-2 infection and that if they do become infected, these women need not be afraid because we have a general understanding of the emerging infection and disease. first, pneumonia might be more severe in pregnant women, regardless of covid-19, because the enlarged uterus lifts the diaphragm and compresses the lungs, inhibiting ventilation and making the lungs more prone to congestion. thus, pregnant women need to avoid any chance of infection as much as possible. to prevent sars-cov-2 infection, they are recommended to not leave their house unnecessarily, avoid crowds and wash their hands frequently. they can consult with their employer regarding the type of work they perform and their work environment. although it is desirable to wear a mask when going out to prevent droplet infection, the who does not regard wearing a mask as being effective against preventing droplet infection in healthy people but a mask is effective only in preventing people with symptoms from spreading droplets around them. as the virus is excreted in the feces, women must be sure to wash their hands with soap after toilet use and before eating. frequent hand washing and disinfection with alcohol after touching a touch panel, such as an atm, in a public place or after touching a train strap or handrail is recommended. if patients suspect that they have covid-19 and wish to be examined, they should first consult the consultation center for returnees and contact persons to prevent the spread of infection. they are not recommended to visit medical institutions except for emergencies because infection among patients in some institutions could trigger local outbreaks. the best way to protect them is to ensure satisfactory communication with their primary care physicians by telephone or email for online treatment, prescriptions and appropriate advice. we have published guidelines for pregnant women on the website of the japanese society of infectious diseases in obstetrics and gynecology. 51 although there is no need to panic because the medical system in the u.s. is capable of dealing with the disease and its treatment capacity, they recommend that citizens be careful to avoid contracting the disease. 52 the unique governmental policy in japan has been effective in preventing explosive infection. as of may 17, there was no urban lockdown, only relatively moderate furlough control, cluster control and pcr testing that is limited to those who need it; however, the numbers of new patients and covid-19 deaths remain among the lowest in the world. some argue that pcr-based testing should be performed on the entire population, even if they are asymptomatic, but the financial cost, low positivity rate and limited medical resources and manpower make this approach impractical. the epidemic in guangdong, far from wuhan, occurred when the virus was imported from outside the city and was largely controlled by traffic blocks. overall, out of approximately 1.6 million people tested by pcr, only 1300 were infected, or less than one in 1000 people in guangdong. thus, contact restrictions rather than increasing pcr-based testing is more effective in preventing the spread of infection. 53 human history has experienced numerous epidemics, including the plague of the 13-14 th century; the columbian exchange of measles, smallpox and syphilis in the 16th century; and cholera in the late 19th century. 54 the covid-19 pandemic is the first pandemic in 100 years, since the spanish influenza pandemic of 1918. however, in the last 100 years, we have developed new weapons against infection based on microbiology, immunology and molecular biology. with these weapons, we can fight new enemies in the clinical arenas of obstetrics and infectious disease control with a speed and precision that are incomparable to those of the early 20th century. we believe that victory can be achieved by using human wisdom and abandoning conspiracy theories, such as biological weapons and the economic, ideological and religious positions of each country, to fight the true enemy. evolution of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) as coronavirus disease 2019 (covid-19) pandemic: a global health emergency pattern of early human-to-human transmission of wuhan covid-19 coronavirus pandemic about coronavirus disease 2019 (covid-19). available from url focus on receptors for coronaviruses with special reference to angiotensin-converting enzyme 2 as a potential drug target-a perspective origins of major human infectious diseases phylogenetic network analysis of sars-cov-2 genomes on the origin and continuing evolution of sars-cov-2 spread of sars-cov-2 in the icelandic population sars-cov-2 productively infects human gut enterocytes targeting sars-cov-2 receptors as a means for reducing infectivity and improving antiviral and immune response: an algorithm-based method for overcoming resistance to antiviral agents risk factors for mortality in 244 older adults with covid-19 in wuhan, china: a retrospective study the pathogenesis and treatment of the 'cytokine storm' in covid-19 back to the future: lessons learned from the 1918 influenza pandemic gross examination report of a covid-19 death autopsy coagulopathy in covid-19 endothelial cell infection and endotheliitis in covid-19 the unique immunological and microbial aspects of pregnancy type 1 interferons as a potential treatment against covid-19 clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records clinical manifestations and outcome of sars-cov-2 infection during pregnancy neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan, china antibodies in infants born to mothers with covid-19 pneumonia coronavirus disease 2019 (covid-19) in pregnant women: a report based on 116 cases maternal and perinatal outcomes with covid-19: a systematic review of 108 pregnancies executive management summary and short report of outcome severe covid-19 during pregnancy and possible vertical transmission mortality of a pregnant patient diagnosed with covid-19: a case report with clinical, radiological, and histopathological findings covid-19 infection among asymptomatic and symptomatic pregnant women: two weeks of confirmed presentations to an affiliated pair of new york city hospitals pregnancy and perinatal outcomes of women with coronavirus disease (covid-19) pneumonia: a preliminary analysis maternal death due to covid-19 disease clinical course of severe and critical covid-19 in hospitalized pregnancies: a us cohort study second-trimester miscarriage in a pregnant woman with sars-cov-2 infection sars-cov-2 infection of the placenta possible vertical transmission of sars-cov-2 from an infected mother to her newborn what are the risks of covid-19 infection in pregnant women? viral infections during pregnancy first antibody surveys draw fire for quality, bias current laboratory diagnostics of coronavirus disease 2019 (covid-19) a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 experimental treatment with favipiravir for covid-19: an open-label control study. engineering (beijing) the japanese association for infectious diseases rapid review for the anti-coronavirus effect of remdesivir hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial observational study of hydroxychloroquine in hospitalized patients with covid-19 the inhaled corticosteroid ciclesonide blocks coronavirus rna replication by targeting viral nsp15 effective treatment of severe covid-19 patients with tocilizumab coronavirus (covid-19) infection and pregnancy version 10 vaginal delivery in sars-cov-2 infected pregnant women in northern italy: a retrospective analysis notice to pregnant women and those wishing to become pregnant. japanese society of infectious diseases in obstetrics and gynecology forecasting the impact of coronavirus disease during delivery hospitalization: an aid for resources utilization genomic epidemiology of sars-cov-2 in guangdong province the authors declare that they have no conflicts of interest. key: cord-256147-lfwytlj3 authors: gabriella, di mauro; cristina, scavone; concetta, rafaniello; francesco, rossi; annalisa, capuano title: sars-cov-2 infection: response of human immune system and possible implications for the rapid test and treatment date: 2020-04-16 journal: int immunopharmacol doi: 10.1016/j.intimp.2020.106519 sha: doc_id: 256147 cord_uid: lfwytlj3 the new coronavirus outbreak is an ongoing pandemic that is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the new coronavirus sars-cov-2 belongs to the subfamily of β−coronaviruses and shares 79.5% of the genetic sequence of sars-cov, the causative agent of the epidemic that started in 2002 and ended in 2004. considering the clinical impact of the new outbreak, it is highly important to study the potential responses of the human immune system during the sars-cov-2 infection as well as the role of virus-specific t cells and by b-lymphocytes. moreover, specific data on the production of igg and igm is crucial to allow the rapid identification of the infection. in this paper we also described the importance of sensitive and specific rapid test for sars-cov-2. indeed, this test represents an important immunological tool aimed at identifying the precise phase of the infection in order to undertake a more appropriate pharmacological treatment. lastly, we provided an overview of pharmacological treatments aimed to reduce inflammatory processes underlying the infection and the need for the discovery of a new vaccine against sars-cov-2. the sars-cov-2 virus belongs to the family of coronaviruses, positive-stranded rna viruses that are characterized by a spherical shape, which provides them the typical "crown" appearance. these viruses were first identified in the mid-1960s and classified into four distinct subfamilies: α−/β−/γ−/δ-coronavirus. alpha and beta-coronaviruses mainly infect mammals, while gamma and delta-coronaviruses are more inclined to infect birds [1] . some of them can induce a mild infection in the upper and lower respiratory tract, while others can cause serious symptoms that can lead to respiratory failure. to date, seven types of coronavirus able to infect humans have been identified: the most common are hcov-oc43 and hcov-hku1 (β−coronavirus) and hcov-229e and hcov-nl63 (α−coronavirus). these viruses can cause common colds but also severe lower respiratory tract infections. apart from these, three other beta coronaviruses, called sars-cov, sars-cov-2 infection can occur with fever, fatigue and dry cough and, in severe cases, with pneumonia, acute respiratory syndrome, and kidney failure. in some cases sars-cov-2 infection can be fatal. considering immunopathological aspects, about 80% of patients with sars-cov-2 infection experience mild or null symptoms. however, in severe cases patients may experience lymphopenia and interstitial pneumonia with high levels of pro-inflammatory cytokines including il-2, il-6, il-7, il-10, g-csf, ip-10, mcp-1, mip-1α and tnfα. as a result, the massive release of cytokines generates the so-called "cytokine storm" which, in turn, can induce acute respiratory distress syndrome (ards), respiratory failure, organ failure and potentially the patient's death. this mechanism is the basis of the rationale for the administration of tocilizumab, a monoclonal antibody that inhibits ligand binding to the human interleukin-6 receptor (il-6r), which was recently approved in china to reduce lung complications in patients with sars-cov-2 infection [2] . apart from tocilizumab, which counteracts inflammatory phenomena deriving mainly from activities of il-6, other drugs, mainly represented by antivirals (the combined treatment lopinavir/ritonavir, remdesivir, favipiravir, umifenovir), are currently under evaluation for the treatment of sars-cov-2 [3] . for instance, recent data shared by the italian ministry of health revealed that, among those medications, the combined treatment lopinavir/ritonavir is currently and in a decrease in the total number of lymphocytes [5] . as for all viral infections, in the adaptive immune response, virus-specific t cells, for cell-mediated immunity, and by b-lymphocytes, for humoral immunity, play a key role. indeed, the activation of th1 / th17 by helper t lymphocytes can contribute to the exacerbation of the inflammatory response, while b lymphocytes provide for the production of specific antibodies for sars-cov-2 aimed at neutralizing the virus. it is widely recognized that prior to the production of high affinity immunoglobulins g (igg) for long-term immunity and immunological memory, m immunoglobulins (igm) provide the first line of defense during viral infections. accordingly, the detection of igm in the serum reveals a recent exposure to the virus, while the detection of igg suggests that the exposure occurred several days before. however, specific data on the response of human immune system during the sars-cov-2 infection are still lacking and most of these are based on the knowledge acquired in the past years during sars-cov and mers-cov infections [6] . it was reported that after sars-cov infection, igm could be detected in patients' blood after 3-6 days, while igg could be detected after 8 days [7] . similarly, for mers-cov infection, seroconversion was observed at the second or third week of disease onset [8] . for both types of coronavirus infection, delayed and weak antibody response was associated with severe outcomes. thevarajan et al. defined a potential mechanism implemented by the immune system in the course of sars-cov-2 infection [9] . the study was carried out using blood samples from a 47-year-old patient who returned from wuhan with symptoms that included lethargy, sore throat, dry cough and fever. blood samples were taken in 4 different stages of the disease, before and after recovery. the results revealed that igm and igg progressively increase from day 7 to day 20. specifically, the researchers showed that 7-9 days after the onset of similarly, zhou pet al. found that a patient developed a virus-specific igm peak 9 days after the disease onset and that the transition to igg occurred within the second week [10] . in order to apply a rapid test able to detect the presence of specific igm and igg for sars-cov-2, it is important to consider that the igm values tend to disappear within 2 weeks since the beginning of the infection. therefore, considering that symptoms of the infection can occur within 14 days, in most cases it is difficult to accurately determine when a patient contracted the virus. consequently, if immunoglobulin values are not high enough at the time of the test, false negatives could be recorded [10] . sars-cov-2 infection can also be transmitted among asymptomatic patients, who can have a high viral load without showing any symptoms. this is why it is quite difficult to manage the spread of the virus. in order to solve this problem, the use of rapid tests for the combined detection of igg and igm would be desirable. these rapid tests are able to simultaneously detect the presence of igm and igg in the serum within 15 minutes and can predict which stage of patient's infection. the sensitivity and specificity of these tests were evaluated on 397 blood samples from patients who tested positive for the nasopharyngeal swab for sars-cov-2 infection and on 128 patients who tested negative and asymptomatic but potentially at risk of developing the infection based on epidemiological criteria [7] . the results of the study showed that out of 397 blood samples from patients with a sars-cov-2 infection, 352 tested positive. on the other hand, 12 of the 128 blood samples with sars-cov-2 nasopharyngeal swab negative tested positive as well. therefore, the test showed a sensitivity of 88.66% and 90.63% in the first and second group of patients, respectively. furthermore, 64.48% of positive patients (256/397) had igm and igg antibodies simultaneously. therefore, the rapid test for the combined detection of igg and igm specific for sars-cov-2 proves to be sensitive and specific. however, they are not 100% certain, so the risk is register false positives or false negatives cases. at the moment this test represents an important immunological test aimed at identifying the precise phase of the infection in order to undertake a more appropriate pharmacological treatment. therefore, it is desirable that these rapid tests become more sensitive and specific, in order to quickly identify patients with sars-cov-2 and prevent the rapid transmission of the virus. lastly, the development of a vaccine against sars-cov-2 is urgently needed. according to shang w et al., the combination of subunit vaccines with adjuvants may represent a good strategy to speed up the clinical development. authors also reported that researchers would bring a new sars-cov-2-based vaccine in approximately 16-20 weeks [11] . while waiting for a specific vaccine, a similar approach to that used for immunotherapies in the treatment of cancer and serious viral respiratory infections could represent an option for the prevention and treatment of sars-cov-2. for instance, a solution could be represented by the passive immunization, which is the administration of serum containing specific antibodies taken from patients from sars-cov-2 [12] . unlike active immunization, this approach does not require the activation of the recipient's immune responses and generates an immediate immune response. however, this would be an emergency solution and not a substitute for any vaccination therapy. there are no conflicts to declare. sars and other coronaviruses as causes of pneumonia the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status discovering drugs to treat coronavirus disease 2019 (covid-19) dysregulation of immune response in patients with covid-19 in wuhan, china. clin infect dis immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis t-cell immunity of sars-cov: implications for vaccine development against mers-cov breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 a pneumonia outbreak associated with a new coronavirus of probable bat origin the outbreak of sars-cov-2 pneumonia calls for viral vaccines convalescent plasma as a potential therapy for covid-19 key: cord-024795-xa7ke70d authors: kaviani, aaron; ince, dilek; axelrod, david a. title: management of antimicrobial agents in abdominal organ transplant patients in intensive care unit date: 2020-01-24 journal: curr transplant rep doi: 10.1007/s40472-020-00268-0 sha: doc_id: 24795 cord_uid: xa7ke70d purpose of review: early diagnosis of infections and immediate initiation of appropriate antimicrobials are crucial in the management of patients before and after organ transplantation. we reviewed the most recent literature and guidelines in this field and organized the current recommendations for healthcare professionals caring for critically ill organ transplant recipients. recent findings: the incidence of multidrug-resistant organisms is increasing. multidrug-resistant gram-negative bacteria comprise about 14% of organisms. vancomycin-resistant enterococci bloodstream infections are also on the rise, as 20.5% of nosocomial enterococci are now vancomycin-resistant, changing empiric antibiotic selection. fluconazole-resistant candida species comprise up to 46% of cases of candidemia in hospitalized patients. consequently, new guidelines recommend primary use of echinocandins in patients with candidemia who have moderate-to-severe disease. finally, the incidence of emergence of ganciclovir-resistant cytomegalovirus infection in patients is 5–12%, requiring early recognition and change to alternative regimens in the case of poor response to therapy. summary: bloodstream infections are a major cause of mortality and morbidity in solid organ transplantation. mortality as high as 24% and 50% have been reported with sepsis and septic shock respectively. as such, bloodstream infections should be diagnosed rapidly and intravenous antibiotics should be started immediately. appropriate resuscitation should be initiated and the number and/or dose of immunosuppressive drugs should be reduced. proper source control must also be achieved with radiologic drainage or surgical intervention as appropriate. initial antibiotic treatment of these patients should cover both gram-positive organisms, especially in the presence of intravascular catheters, and gram-negative bacteria. echinocandins like caspofungin should also be considered especially in critically ill patients, particularly if a patient has been on total parenteral nutrition or broad-spectrum antibiotics. organ transplantation is one of the major advances of modern medicine. the dream of replacing a nonfunctioning organ, like a kidney or a liver, with a functioning organ became a reality in 1954, when dr. joseph murray transplanted a kidney from one identical twin to the other [1, 2]. over the past few years, there has been a significant improvement in survival and reduction in rejection after solid organ transplantation (sot) as a result of more effective immunosuppression therapy. however, infections and particularly bloodstream infections (bsis) still remain as a common complication and a major cause of death after transplant [3] [4] [5] . transplant recipients have an increased risk of developing infections due to the lifelong treatment with immunosuppressive drugs [6] . another risk factor for infection is the presence of unrecognized infection in the donor or the recipient. rejection, which is often treated with intensified immunosuppression, also increases the incidence of infections, particularly viral and nosocomially acquired pathogens [3] . in addition, cytomegalovirus (cmv) infection may increase the risk of other infections, including bacterial and fungal infections [3, 7, 8] . bacterial infections are very common in patients with endorgan disease prior to and after sot, and represent one of the most important causes of progression of liver failure, development of complications, and death in patients with end-stage liver disease (esld) [9] . nosocomial bacterial infections are the most common cause of infection in this group of patients in the first month after transplantation [3, 5, 10] . furthermore, liver, pancreas, and intestinal recipients are especially at risk for fatal fungal infections, primarily caused by candida species which may occur together with bacterial infections. the risk of fungal infection is also higher in patients with kidney failure and history recent hospitalization. candidemia is associated with diabetes, antibiotic use, total parenteral nutrition (tpn), surgical drains, and vascular access catheters. delayed diagnosis because of low index of suspicion is common [11] [12] [13] [14] . the complexity of treating sot with life-threatening infections has been increasing dramatically given the increasing prevalence of multidrug-resistant organisms. multidrugresistant gram-negative bacteria comprise about 14% of organisms isolated in bsi after sot. recent studies show up to 20.5% of nosocomial enterococcal infections are vancomycinresistant. fluconazole-resistant candida species comprise up to 46% of cases of candidemia [12••, 15-17] . early diagnosis of infections and timely initiation of adequate antimicrobials are crucial in the management of patients before and after transplantation [9] . patients also require assessment and adjustment of their immunosuppression and subsequent comprehensive evaluation for the source of infection. the goal of this review is to provide concrete actionable guidelines for clinicians caring for abdominal sot patients with serious infections. infections are a major barrier prior to organ transplantation and all potential candidates should be evaluated for active infection [18] . although we usually have the luxury of treating infectious diseases in most transplant candidates well before transplantation, progressive organ failure in patients with elsd may mandate aggressive treatment of their infectious disease and urgent liver transplantation. patients with esld are prone to infections due to abnormalities of their immune system and bacterial translocation from bowel. bacterial infections may be an eliciting factor for episodes of hepatic encephalopathy, gastrointestinal bleeding, kidney failure, hyponatremia, and development of acute-on-chronic liver failure [9] . infections are usually caused by gram-negative bacteria from intestinal origin, but gram-positive bacteria are also common, particularly in hospitalized patients [9] . fungal infections, mainly caused by candida species, can also occur and are associated with high mortality rates [19] . spontaneous fungal peritonitis (sfp) is associated with higher mortality compared with spontaneous bacterial peritonitis (sbp) [20] . candida albicans is the most frequent fungal agent followed by cryptococcus neoformans and aspergillus species. clinicians should also consider the presence of polymicrobial fungal infections. in fact, while polymicrobial bacterial infections affect 5.2-17.4% of cases, polymicrobial fungal infections occurred in 73.3-100% of patients with systemic fungal infections in a small case series [20] . due to high mortality and morbidity rate, sbp treatment should be initiated even in absence of a positive culture especially in patients with a higher model for end-stage liver disease (meld) score. patients with a positive fungal culture of the ascitic fluid regardless of polymorphonuclear leukocyte (pmn) count should also be treated. antifungal drugs may also be started empirically for patients who are not responding to antibiotics [11] . cefotaxime and ceftriaxone constitute the first-line treatment for community-acquired sbp and/or bacteremia, while broad-spectrum beta-lactams or carbapenems with or without vancomycin are considered the first-line treatment for nosocomial sbp and/or bacteremia [9] . local resistance patterns should help guide empiric antibiotic therapy. echinocandins like caspofungin should be considered empirical or preemptive therapy for patients with suspected sfp [20] . in patients with esld and sbp who are treated with antibiotics, intravenous administration of 20% albumin reduces the incidence of renal failure and decreased mortality rates from 29 to 10% [9] . recommendations regarding empirical antibiotic treatment in patient with esld before liver transplantation have been summarized in table 1 [9] . a personalized approach for first-line empiric antibiotic therapy needs to be chosen according to local epidemiology. infections after organ transplantation are commonly classified in 3 time periods: less than 1 month, first year, and after the first year. the main sources of infection in the first month after sot include donor-derived infections, preexisting recipient infections, surgical complications, and nosocomial infections including clostridium difficile infection (cdi). early removal of lines and drains, drainage of fluid collections, careful wound care, and judicious and limited use of antimicrobial agents help to reduce infections in this period of time [14, 24] . the sources of infection in this period of time include surgical complications like anastomotic leaks, infected hematomas, cholangitis, and empyema as well as remaining nosocomial infections from previous month including cdi and residual pneumonia. furthermore, opportunistic infections including viral infections, pneumocystis jiroveci, listeria monocytogenes, toxoplasma gondii, nocardia species, aspergillus species, and endemic fungi usually happen in this period of time. prophylactic measures are needed in this period of time to prevent opportunistic infections [14, 24] . the risk of infection is less in this period of time in recipients with good graft function due to lower intensity of immunosuppressive agents. sources of infection during this period include community-acquired viruses, foodborne gastroenteritis, molds from work or gardening, primary socially acquired cmv infection, relapsing viral infection, and infections related to underlying conditions like skin infections in diabetics. patients with marginal allograft function who need higher levels of maintenance immunosuppression need more attention because of increased risk of recurrent infections including pneumonia, urinary tract infections (utis), cholangitis, and abscesses [14, 24] . infections are common after all types of sot. however, some infections are more frequent in certain sot recipients. in the following section, we address this issue in detail. the most common infections in kidney transplant recipients include uti (47%), viral infections (17%), pneumonia (8%), and wound infection (7%). utis are most commonly caused by enterococcus species and escherichia coli are the most common agents [25] . kidney allograft pyelonephritis may be associated with bacteremia and sepsis and can be complicated by impaired graft function and even death [26] . bk virus can cause graft failure in kidney transplant recipients. however, with universal screening and treatment, reported graft loss due to bkv is only 0 to 5%. careful immunosuppression adjustment is needed in these patients. antiviral agents have a limited role. intravenous immunoglobulin (ivig) is occasionally used if there is persistent bk viremia after reduction of immunosuppression [27] . the most common sites of infection in pancreas recipients include urinary tract and wound infections. intestinal leaks in patients with enteric drainage happen in 5 to 8% of cases, resulting in abdominal abscesses requiring surgical or radiologic intervention. most infections in pancreas transplant recipients are bacterial [28] . these patients are also at a higher risk for getting fungal infections and cmv given the greater intensity of immunosuppression and long-standing diabetes [12••, 28] . in case of enteric drained graft leak, an abdominal computerized tomography (ct) scan with oral contrast should be done. treatment comprises laparotomy and anastomotic revision which may include conversion of the enteric anastomosis from a side-to-side duodenojejunostomy to a roux-yduodenojejunostomy or a transplant pancreatectomy. for an early (≤ 2 weeks post-transplant) bladder-drained graft leak, low-pressure cystography or an abdominal ct with retrograde bladder contrast should be done. foley catheter should be placed and percutaneous drainage of intraabdominal fluid collections should be performed. peritonitis needs relaparotomy and direct leak repair or transplant pancreatectomy. late leaks usually need conversion from bladder to enteric drainage [29] . major infections develop in 53-56% of liver transplant recipients [30, 31] . bacterial infections are most frequent (70%), followed by viral (20%) and fungal infections (8%) [32•] . the most common bacterial infections include pneumonia, surgical site infection, abdominal cavity collections, abscesses, bsis, and uti [32•]. many of these infections are associated with technical problems including bile duct stricture, biliary leaks, and hepatic artery thrombosis [15] . laici and colleagues report pneumonia was the most frequent primary or associated infection and represented 40% of all infections after liver transplant. in 22% of cases, pneumonia led to the development of septic shock, which led to the death in more than 50% of affected recipients [32•] . enterobacteriaceae are the major pathogens in lt recipients. gram-positive bacteria are also a common cause infection [15] . lt recipients are also at a higher risk for getting fungal infections [12••] . bloodstream infections are the primary cause of mortality and morbidity in sot. mortality as high as 24% and 50% have been reported with sepsis and septic shock respectively. nosocomial bsis are associated with a higher rate of septic shock and mortality. gram-positive bacteria are the most common cause of bsis and tend to be associated with intravascular catheters. however, in kidney transplant recipients, gramnegative bacteria are more common in bsis, mostly associated with utis [12••, 16, 17, 33-37] . intravenous antibiotics should be started immediately. appropriate resuscitation with goal-directed therapy including monitoring of markers of adequacy (lactate, central venous saturation, and end-organ perfusion) should be achieved. furthermore, in severely ill patients, clinicians should rapidly reduce the intensity of immunosuppression. this includes stopping antiproliferative agents (e.g., mycophenolate mofetil) and mtors (e.g., rapamycin) and lowering calcineurin inhibitor levels. corticosteroids should be maintained to prevent adrenal insufficiency. the role of granulocyte colonystimulating factor (g-csf) drugs is controversial, and g-csf has been used by some centers for leukocyte counts of less than 3000 cells/mm [3, 38] . proper source control, if possible, should also be rapidly achieved with radiological drainage or surgical intervention. initial antibiotic treatment of sot patients with bsi should include coverage for probable source(s). especially in the presence of intravascular catheters, gram-positive pathogen coverage with vancomycin should be added to gramnegative pathogen coverage [12••, 39-41] . linezolid or daptomycin instead of vancomycin should be considered if there is a recent history of vancomycin-resistant enterococcus (vre) colonization or infection. prior microbiologic history and local antibiotic resistance patterns in addition to possible source of infection should guide antibiotic therapy [12••] . echinocandins like caspofungin should also be considered in patients with septic shock without a focus especially in patients with risk factors for invasive candidiasis such as older age, central venous catheters, candida colonization, total parenteral nutrition (tpn), prolonged neutropenia, prolonged intensive care unit stay, diabetes, renal replacement therapy, and/or broad-spectrum antibiotic therapy [42, 43] . likewise, diagnosed candidemia requires treatment in all patients with either fluconazole or an echinocandin. one of the main differences between the newer vs. older guidelines is the primary use of echinocandins in patients with candidemia who have moderate-to-severe disease. however, empiric antifungal coverage is not needed if a patient does not meet above criteria [12••, 39, 44, 45] . recommendations regarding initial antibiotic treatment of bloodstream infections and sepsis after sot have been summarized in table 2 [39, 42] . initial management of different fungal infections after sot has been summarized in table 3 [39, 46, 47] . bacterial pneumonia is the most common cause of pulmonary infection in sot candidates. timing after transplantation, age, transplanted organ, degree of immunosuppression, site of acquisition of pneumonia, and exposures all play into the differential diagnosis in solid organ transplant patients [48••] . community-acquired pneumonia and hospital-acquired pneumonia account for 40.7% and 59.3% of pneumonias in sot, respectively. hospital-or ventilator-associated pneumonia are typically diagnosed in the early post-transplant period with pseudomonas aeruginosa, gram-negative enteric bacteria, acinetobacter species, stenotrophomonas species, and staphylococcus aureus, including mrsa as the most common pathogens in the peri-operative period. opportunistic infections are most commonly seen in 1-6 months after transplant, and community-acquired pathogens, such as streptococcus pneumoniae, haemophilus influenzae, mycoplasma species, legionella species, and chlamydia species, are more frequent after 6 months, though opportunistic infections can still be seen, especially if immunosuppression is increased for treatment of rejection. empiric treatment for pneumonia needs to be based on the above risk factors and timing after transplantation as well as the pace of illness onset. in nosocomial or ventilatorassociated pneumonia, nosocomial flora, and antibiogram and in lung transplant patients or in patients with prior pulmonary infections, organisms colonizing the airways and their susceptibilities should also be taken into consideration. for hospital-associated pneumonia and ventilator-associated pneumonia, the id society of america and american society of transplantation guidelines on pneumonia should be followed in conjunction with local antibiogram [48••, 49] . broad-spectrum antibiotics with an activity against p. aeruginosa and other gram-negative bacilli should be started empirically. for patients with septic shock and need for new ventilatory support, two agents with activity against p. aeruginosa can be considered. in patients with a high risk of mortality, prior intravenous antibiotic use within 90 days, hospitalization in a unit with > 20% mrsa prevalence, an anti-mrsa agent should also be started empirically. empiric treatment should be modified based on previous microbiologic history and local antibiogram as well as patient's clinical response. an active agent against intracellular organisms and during the influenza season, anti-influenza agents are also recommended for empiric treatment until these infections are ruled out. respiratory viral infections, including cmv, are important causes of pneumonia among transplant recipients. viral pneumonia predisposes the patient to superinfection with bacterial and fungal pneumonia. treatment includes supportive care and reduction in immunosuppression (see cmv treatment below) [12••] . sot recipients have a significant risk of invasive fungal infections (ifis). small bowel transplant recipients followed by heart-lung, liver, and pancreas transplant recipients have the highest rate of ifis respectively [39, 46] . although candida species remain the most common cause of ifis in sot, molds comprise approximately 40% of ifis in this group of patients. aspergillus species cause the majority of mold infections. mucormycosis is the next most common mold infection. cryptococcosis and endemic fungi are other etiologies for ifis [39, 47] . echinocandins like caspofungin are recommended for treatment of candida sp. however, the infectious diseases society of america (idsa) guidelines recommend voriconazole as the treatment of choice for invasive aspergillosis with isavuconazole and amphotericin b listed as alternative agents [50] . high-dose lipid formulation amphotericin b is recommended for mucormycosis, in combination with aggressive surgical debridement of infected tissues [39] . recommendations regarding initial antibiotic treatment of respiratory infections after sot have been summarized in table 4 [39, 42] . initial management of different fungal infections after sot has been summarized in table 3 [39, 46, 47] . central nervous system infections can be caused by donorderived infections, community-acquired organisms, and reactivation and/or acquisition of opportunistic pathogens. cryptococcus, nocardia, aspergillus, zygomycetes, strongyloides, and toxoplasma can affect both the lungs and the brain [12] . clinical presentation depends on the organism, but may be subtle due to lack of a robust immune system. history; physical examination including detailed neurologic examination; laboratory evaluation including viral polymerase chain assays or multiplex panels; serum cryptococcal antigen; blood, urine, and sputum cultures; chest radiograph; magnetic resonance imaging of the brain with contrast; lumbar puncture; electroencephalography for seizures; and possibly neurosurgical intervention including biopsy and debridement are key elements in the evaluation of these patients [51] . recommendations regarding initial antibiotic treatment of central nervous system infections after sot have been summarized in table 5 [42] . a recently published cochrane review did not show sufficient evidence in favor or against primary and secondary preventions of seizures with anticonvulsive agents in viral encephalitis; however, some experts continue to recommend antiepileptic medications in all patients with seizures and encephalitis [52] . utis are the most common infection after organ transplantation and account for 45 to 72% of all infections and 30% of all hospitalizations due to sepsis in kidney transplant recipients. complicated uti including pyelonephritis can result in significantly impaired transplanted kidney function and even death [12••, 26] . while gram-negative bacilli are the most common cause of utis, enterococcus sp. and s. aureus can also lead to utis in patients with indwelling urinary catheters [12••] . broad-spectrum antibiotics such as piperacillin/ tazobactam or carbapenems might be warranted, with deescalation based on culture results [42, 53] . patient's history of prior urinary tract infections and the organisms identified also need to be considered. in severe infections such as septic shock, immunosuppression should be reduced and upper tract imaging should be obtained. cystoscopic, percutaneous, or surgical procedures to relieve obstruction or to drain abscesses might be needed [53] . candida is the most common cause of fungal uti in kidney transplant recipients. asymptomatic candiduria occurs in 11% of kidney transplant and does not require treatment. in these situations, urinary catheters should be removed or exchanged and second urine sample should be collected. persistent candiduria in patients who does not have urinary catheter needs upper urinary tract imaging to evaluate for anatomic abnormalities and fungus balls. asymptomatic candiduria should not be treated. fluconazole is the treatment of choice for utis with susceptible candida sp. echinocandins are considered to be drugs of choice in unstable patients with systemic candidiasis; however, they do not reach acceptable levels for treatment of utis and relapses may occur . recommendations regarding initial antibiotic treatment of urinary infections after sot have been summarized in table 6 [42] . bile leak, biloma, and biliary stricture can occur after liver transplantation especially after living donor liver transplants and can be complicated by peritonitis, abscess, and cholangitis. hepatic artery thrombosis can lead to biliary complications as well as hepatic necrosis, abscesses, and sepsis. cholangitis is a common complication after liver broad-spectrum gram-negative coverage with also anaerobic activity should be started in patients with sepsis. specific regimen needs to be tailored to the local antibiogram and follow institutional empiric treatment guidelines, which take into consideration institutional rates of extended-spectrum beta-lactamase-or carbapenemase-producing organisms as well as multidrug-resistant p. aeruginosa. if a patient has peritonitis or intraabdominal abscess, gram-positive coverage (vancomycin or daptomycin in patients with history of vre) and antifungals should be added [42, 54] . if there are any concerns for biliary stricture(s) or bile leak based on initial imaging, endoscopic retrograde cholangiopancreatography (ercp) should be performed and stent(s) placement might be needed. percutaneous transhepatic cholangiogram (ptc) is reserved for ercp failure or conditions that preclude it like roux-en-y hepaticojejunostomy or gastric outlet obstruction . recommendations regarding initial antibiotic treatment of hepatobiliary infections after sot have been summarized in table 6 [42] . diarrhea occurs in 22 to 52% of sot patients and can be due to infectious causes including bacterial, viral, or parasitic infections as well as medications including mycophenolate mofetil side effect. stool culture, ova and parasite testing, and multiplex pcr testing for a wide variety of pathogens, if available, are among the initial tests recommended. imaging studies and endoscopy (esophagogastroduodenoscopy and/or colonoscopy) may be warranted [12••] . clostridium difficile infection is the most common hospital-acquired infectious diarrhea, and fulminant colitis can happen in up to 13% of recipients with cdi [12••] . oral cmv colitis can occur after sot and can potentially cause end-organ disease. treatment includes oral valganciclovir or intravenous ganciclovir [12••] . while the incidence of ganciclovirresistant cytomegalovirus in patients on prophylactic valganciclovir is as low as 0-3%, the incidence of emergence of ganciclovir-resistant cytomegalovirus infection in patients receiving ganciclovir treatment is about 5-12% [58] . for patients whose viral loads fail to decline while on therapy, ganciclovir resistance test is recommended. for most patients with confirmed ganciclovir resistance, foscarnet is suggested [59] . cidofovir is the other option when there is a concern for resistance [12••] . letermovir has also shown promising results in these patients, though it has not been approved yet for this indication [60, 61] . cmv immunoglobulin is associated with limited efficacy [12••] . recommendations regarding initial antibiotic treatment of enterocolitis after sot have been summarized in table 6 [42] . surgical site infections (ssis) are a common and major complication in sot and are reported to occur in 5-40% of these patients [62] . risk factors for ssi following various organ transplant surgeries and the most common pathogens have been summarized by abbo et al. [63] . infected wounds should be opened and washed out. imaging for evaluation of possible deeper abscess should be performed and aspirates from these collections should be sent for cultures are recommended [42] . source control is needed for treatment of deep ssis and organ space infections. for ssis, empiric treatment should include gram-positive organisms as well as expected flora at site of transplanted organ, with broad-spectrum agents reserved for patients with risk for multidrug-resistant organisms [63] . recommendations regarding initial antibiotic treatment of wound infection after sot have been summarized in table 7 . transplant recipients have an increased risk of developing infections due to the lifelong treatment with immunosuppressive drugs. there should be a high index of suspicion in these patients and infections should be diagnosed rapidly. early imaging is required. a multidisciplinary approach to the management of these patients which should include transplant surgery, infectious disease, and possibly interventional radiology as well as gastroenterology is needed. appropriate resuscitation should be initiated and intravenous antibiotics should be started immediately. the number and/or dose of immunosuppressive drugs should be reduced. proper source control must also be achieved with radiologic drainage or surgical intervention. conflict of interest the authors declare that they have no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. papers of particular interest, published recently, have been highlighted as: management of infectious complications in solid-organ transplant recipients transplantation 50 years later-progress, challenges, and promises causes of death in autopsied liver transplantation patients highlights in solid transplant infectious diseases 2015-2017 risk factors for cytomegalovirus and severe bacterial infections following liver transplantation: a prospective multivariate time-dependent analysis antiviral medications to prevent cytomegalovirus disease and early death in recipients of solid-organ transplants: a systematic review of randomised controlled trials bacterial infections in cirrhosis: a position statement based on the easl special conference infections after liver transplantation. an analysis of 101 consecutive cases fungal infections in patients with cirrhosis this article is another up-to-date comprehensive review of infections that can be observed in intensive care units in solid organ trans invasive fungal infections among organ transplant recipients: results of the transplant-associated infection surveillance network (transnet) infection in organ transplantation bacterial infection after liver transplantation bloodstream infections among transplant recipients: results of a nationwide surveillance in spain risk factors and outcomes of bacteremia caused by drug-resistant eskape pathogens in solidorgan transplant recipients infections in solid-organ transplant recipients management of bacterial and fungal infections in end stage liver disease and liver transplantation: current options and future directions spontaneous fungal peritonitis: epidemiology, current evidence and future prospective spontaneous bacterial peritonitis -therapeutic challenges in the era of increasing drug resistance of bacteria bacterial infections change natural history of cirrhosis irrespective of liver disease severity bacterial infections in cirrhosis non-immunological complications following kidney transplantation infectious complications after kidney transplantation: current epidemiology and associated risk factors kdigo clinical practice guideline for the care of kidney transplant recipients management of bk polyomavirus infection in kidney and kidney-pancreas transplant recipients: a review article complications after pancreas transplantation incidence, distribution, and outcome of episodes of infection in 100 orthotopic liver transplantations bacterial and fungal infections after liver transplantation: an analysis of 284 patients ):e12834 this study reports a single-center experience of early (within 1 month) infections after liver transplantation and evaluates donor and recipient risk factors for early infection infective endocarditis in solid organ transplant recipients severe endocarditis in transplant recipients-an epidemiologic study epidemiology and risk factors for nosocomial bloodstream infections in solid organ transplants over a 10-year period bloodstream infection after kidney transplantation: epidemiology, microbiology, associated risk factors, and outcome bacteremia and septic shock after solid-organ transplantation granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and other immunomodulatory therapies for the treatment of infectious diseases in solid organ transplant recipients invasive fungal infections in transplant recipients. therapeutic advances in infectious disease sepsis and solid organ transplantation sepsis in solid-organ transplant patients ast handbook of transplant infections candida infections in solid organ transplantation: guidelines from the american society of transplantation infectious diseases community of practice empirical fluconazole versus placebo for intensive care unit patients: a randomized trial empirical micafungin treatment and survival without invasive fungal infection in adults with icu-acquired sepsis, candida colonization, and multiple organ failure: the empiricus randomized clinical trial invasive fungal infections in solid organ transplant recipients fungal infections in renal transplant patients 2019:e13545. this is the american society of transplantation id community's updated guideline on pneumonia in solid organ transplant patients, reviewing potential pathogens, differential diagnosis, diagnostic strategies management of adults with hospital-acquired and ventilator-associated pneumonia: 2016 clinical practice guidelines by the infectious diseases society of america and the invasive aspergillosis in solid-organ transplant recipients: guidelines from the american society of transplantation infectious diseases community of practice central nervous system syndromes in solid organ transplant recipients herpes simplex virus-1 encephalitis in adults: pathophysiology, diagnosis, and management urinary tract infections in solid organ transplant recipients: guidelines from the american society of transplantation infectious diseases community of practice american society of transplantation infectious diseases community of p. intra-abdominal infections in solid organ transplant recipients: guidelines from the american society of transplantation infectious diseases community of practice clinical practice guidelines for clostridium difficile infection in adults and children: 2017 update by the infectious diseases society of america (idsa) and society for healthcare epidemiology of america (shea) management of clostridioides (formerly clostridium) difficile infection (cdi) in solid organ transplant recipients: guidelines from the american society of transplantation community of practice surgical management of clostridium difficile colitis ganciclovir-resistant cytomegalovirus infection in abdominal solid organ transplant recipients: case series and review of the literature clinical manifestations, diagnosis, and treatment of cytomegalovirus infection in lung transplant recipients first report of successful treatment of multidrug-resistant cytomegalovirus disease with the novel anti-cmv compound aic246 cytomegalovirus in solid organ transplant recipients-guidelines of the american society of transplantation infectious diseases community of practice surgical site infections in solid organ transplantation surgical site infections: guidelines from the american society of transplantation infectious diseases community of practice publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-256827-tht5h1tu authors: jain, neemisha; lodha, r.; kabra, s. k. title: upper respiratory tract infections date: 2001 journal: indian j pediatr doi: 10.1007/bf02722930 sha: doc_id: 256827 cord_uid: tht5h1tu acute respiratory infections accounts for 20–40% of outpatient and 12–35% of inpatient attendance in a general hospital. upper respiratory tract infections including nasopharyngitis, pharyngitis, tonsillitis and otitis media constitute 87.5% of the total episodes of respiratory infections. the vast majority of acute upper respiratory tract infections are caused by viruses. common cold is caused by viruses in most circumstances and does not require antimicrobial agent unless it is complicated by acute otitis media with effusion, tonsillitis, sinusitis, and lower respiratory tract infection. sinusitis is commonly associated with common cold. most instances of rhinosinusitis are viral and therefore, resolve spontaneously without antimicrobial therapy. the most common bacterial agents causing sinusitis ares. pneumoniae, h. influenzae, m. catarrhalis,s. aureus ands. pyogenes. amoxycillin is antibacterial of choice. the alternative drugs are cefaclor or cephalexin. the latter becomes first line if sinusitis is recurrent or chronic. acute pharyngitis is commonly caused by viruses and does not need antibiotics. about 15% of the episodes may be due to group a beta hemolytic streptococcus (gabs). early initiation of antibiotics in pharyngitis due to gabs can prevent complications such as acute rheumatic fever. the drug of choice is penicillin for 10–14 days. the alternative medications include oral cephalosporins (cefaclor, cephalexin), amoxicillin or macrolides. acute respiratory infections are a major cause of morbidity and mortality in children and of particular significance in developing countries like india. outpatient attendance attributed to acute respiratory infections is as high as 20-40% of all outpatients and 12-35% of in patients. 1 in a study in rural zone of delhi with an estimated population of 3700, the prevalence of acute respiratory infection was 12.1% for the under-fives as a whole. 2 the overall incidence of acute respiratory infection in the under-fives was observed to be 2.5 episodes per child per year, of which, 2.2 episodes per year per child were upper respiratory tract infections. upper respiratory tract infections including nasopharyngitis, pharyngitis, tonsillitis and otitis media constituted 87.5% of the total episodes of infection. lower respiratory tract infections including pneumonia and bronchiolitis constituted the remaining 12.5%. 2 in a survey of acute respiratory infection in a rural area of south india, the prevalence of acute respiratory infection was 7.6 % in a total of 10,951 children below 5 years. majority of acute respiratory infection was mild (defined by cough + respiratory rate <50) only 1.7% episodes were severe in nature (cough + respiratory rate >50 + chest indrawing/inability to drink). 3 thus, acute respiratory infection constitutes a significant problem in childhood and the majority of these infections are upper respiratory infection. bacterial this bring forth the fact that, majority of respiratory infections on children are due to a viral etiology which do not need antibiotics for management and judicious use of antibiotics is most imperative in upper respiratory tract infections. the principles of judicious use of antibiotics in upper respiratory tract infections outlined below are based on recent reviews. most children have 3-8 episodes of common cold per year. 9-1~ common cold is a viral illness, the most common cause being rhinovirus; others being parainfluenza, respiratory syncytial, corona virus, adenovirus, echovirus, coxsackievirus and parainfluenza virus, n rhinovirus accounts for upto 60% of infections. 12 the etiological agents vary with host age, and time of the year. symptoms include nasal discharge, nasal obstruction, and throat irritation. associated with this there usually is lowgrade fever, malaise, sneezing and nasal secretions can become purulent. symptoms are non-specific and not characteristic for any agent. the usual duration of symptoms is about 7 days. common cold in children resolves on its own and no specific therapy is indicated in majority of cases. use of antibiotics does not change either the course or the outcome. 6 on the contrary antibiotic use is potentially harmful, because it increases the risk of colonization with resistant organisms. this may result in subsequent bacterial infection being unresponsive to standard antibiotics. 13 common cold almost always has an excellent outcome with complete recovery, but at times complications can occur. these include acute otitis media with effusion, tonsillitis, sinusitis, and lower respiratory tract infection. '~ unless specific diagnostic criteria for the diagnosis of these complications are met, antibiotics should not be used for the signs and symptoms of cold alone? studies have shown that use of antibiotics for the prevention of bacterial complication of common cold has no use. a meta-analysis of five randomized clinical trials of efficacy of antimicrobial treatment of cold to prevent lower respiratory infection found that it had no protective effect. it also did not shorten the duration of the upper respiratory illness. 14 recognizing the signs and symptoms of the usual course of common cold will help to prevent the overprescription of antibiotics. unfamiliarity with them can lead to the mistaken diagnosis of secondary bacterial infection and the inappropriate use of antibiotics. mucopurulent nasal dicharge, which is thick, opaque at times greenish or yellowish frequently accompanies common cold. n antimicrobial treatment is not indicated for such a discharge unless it persists for more than 10-14 days. 6 in a study on 142 children with mucopurulent nasopharyngitis, a comparison of cephalexin treatment with placebo was done. five to six days after initiation of treatment, cephalexin and placebo groups did not differ in the presence of nasal discharge, incidence of complications, or parental opinion of benefit of medicationy cough occurs in 60-80% of common cold and does not suggest a bacterial etiology. tm duration of symptoms is usually 2-7 days. patients usually improve within 10 days, but at times cough and nasal discharge can persist for 2 weeks or morey to summarize 1. antibiotics should not be given for common cold. 2. mucopurulent nasal discharge is feature of common cold and is alone not an indication of antibiotics unless it persists for more than 10-14 days. sinusitis is defined as inflammation of the mucosal lining of one or more of the paranasal sinusesy it can occur from infection or non-infectious (allergic) causes. in children sinusitis almost always occurs as a complication 1136 of common cold. n even in uncomplicated viral upper respiratory infection, as occurs in common cold, there is inflammation and congestion of both the nasal and the sinus mucosa. these infections should, therefore, be considered as rhinosinusitis. 19 this inflammation undergoes a spontaneous resolution in vast majority of cases. about 0.5 to10 percent of upper respiratory infections are complicated by development of acute sinusitis. tm since only a small percentage of children with symptoms of rhinosinusitis have a bacterial etiology, the use of appropriate diagnostic criteria is essential, as this will avoid the overuse of antibiotics. this is all the more important because viral rhinosinusitis is 20 -200 times more common than bacterial sinusitis. 7 sinusitis is classified as per the duration of symptoms into acute (upto 3 weeks), subacute (3-10 weeks), and chronic (>10 weeks). sinusitis is almost always precipitated when a viral upper respiratory infection produces mucosal inflammation leading to obstruction of the sinus ostia. this in turn leads to fluid trapping in the sinus cavities and the normal upper respiratory bacterial flora proliferates. the most common organisms causing sinusitis are s. pneumoniae, h. influenzae, m. catarrhalis, s. aureus and s. pyogenesj ~ but it must be emphasized that majority of rhinosinusitis is viral and therefore, approximately 60% resolve spontaneously without antimicrobial therapy. 2~ the major problem in the diagnosis of sinusitis in children is to distinguish simple upper respiratory tract viral infections and allergic inflammation from bacterial infection of the sinuses. the signs and symptoms in older children can be classical i.e. sinus tenderness, tooth pain, headache, and high fever. however, in young children these classical sign and symptoms are almost never present. persistence of signs and symptoms of upper respiratory infection i.e. rhinorrhea or cough for more than 10-14 days is a pointer towards sinusitis. the rhinorrhea mostly is purulent but can be serous or watery. thus, the character of the discharge is not helpful in distinguishing infected sinus fluid, from uninfected fluid. 21 other indicators of sinusitis are the presence of severe signs and symptoms during an episode of upper respiratory infection. these include high fever > 39 ~ c, persistent fever, periorbital swelling and facial pain. diagnosis of sinusitis is clinical and rarely are investigations warranted. radiographic evaluations are indicated when episodes of sinusitis are recurrent, when complications are suspected or when the diagnosis is not clear. 7 the plain radiograph shows air fluid level, opacification, or mucosal thickeningy ct scan or mri can be done for a more detailed examination and show the same findings. these features can also be present in viral rhinosinusitis and therefore, x-ray should only be done when indicated. in a study evaluating ct findings in 31 adults during first 48-96 hours of uncomplicated viral respiratory illnesses, 90% had abnormality of the upper respiratory tract infection maxillary sinus cavity. after two week these findings resolved in 79% even though none received any antibiotics? 9 when the child is younger than six months of age, opacification of the ethmoid air cells is normal. when the child is under the age of one year, opacification or thickening of the mucosa of the maxillary sinusitis may be normal. 11 this should be kept in mind when evaluating xray findings. the primary treatment of sinusitis is antibiotics once the diagnostic criteria have been met. amoxycillin (40 mg/kg/day) is successfij1 for the initial treatment of acute uncomplicated sinusitis in most children. 2~ about 35% of nontypable h. influenzae and 85% of m. catarrhalis strains produce beta-lactamases and are resistant to amoxicillin. 21 therefore, if there is no response within 48 hours a beta-lactamase-stable antibiotic like amoxycillinclavulanate or cephalexin or cetactor should be considered. these drugs should be considered first line if sinusitis is recurrent, fails to improve, or is a severe infection with high fever and facial swelling. the duration of therapy should be minimum of 10 days. 22 to summarize, 1. diagnosis of sinusitis should be made when symptoms of nonspecific upper respiratory infection are present for more than 10-14.days or there are severe upper respiratory signs and symptoms in the form of facial pain, facial swelling and high fever. 2. since radiological evidence of sinusitis may be seen even in common cold, radiographs should be interpreted with caution and done when indicated. 3. antimicrobial for sinusitis should be narrow spectrum against the likely pathogen. pharyngitis is an inflammatory illness of the mucous membranes and underlying structures of the throat. the clinical diagnostic category includes tonsillitis, tonsillopharyngitis and nasopharyngitis. pharyngitis is subdivided into two categories: illness with nasal symptomatology (nasopharyngitis) and illness without nasal involvement (pharyngitis or tonsillopharyngitis). nasopharyngitis nearly always is a viral etiology, whereas pharyngitis without nasal signs has diverse etiologic possibilities including bacteria, viruses, fungi and other infectious agents. adenoviruses are the most common cause of nasopharyngitis, other viruses being influenza, parainfluenza and enteroviruses. the bacterial agents causing pharyngitis include streptococcus pyogenes, h. influenzae, c. diphtheriae, and n. meningitides. 23 group a streptococci accounts for approximately 15% of bacterial pharyngitis and about 1-2 patients out of 10 with sore throat. infection in most of the others is attributable to viruses, u early diagnosis and treatment of group a streptococcal pharyngitis is important, as antibiotics initiated with nine days of onset are effective in preventing acute rheumatic fevery but since most sore throats are viral in etiology antibiotic have to be given only when diagnosis of group a streptococcal pharyngitis is certain. this will prevent the unnecessary overuse of antibiotics. symptoms of sore throat accompany pharyngitis but its presence alone should not be used for diagnosis as these complain can be present even in common cold. the objective evidence of inflammation in the form of erythema, exudate or ulceration in the pharynx is necessary for the diagnosis. but, these signs can also be present in some viral infections like adenovirus or enterovirus. the classical features of group a streptococcal pharyngitis include acute onset of pharyngeal pain, dysphagia and fever. rhinorrhea, cough, hoarseness, conjunctivitis and diarrhea suggest a viral etiology. presence of patchy exudate on the posterior pharynx, palatal petechie and enlarged and tender anterior cervical lymph nodes favour group a streptococci pharyngitis. but in an individual child the clinical distinction between streptococcal pharyngitis and viral pharyngitis is unreliable. therefore, in all cases of acute pharyngitis, streptococcal disease must be considered. diagnosis of group a streptococcal pharyngitis should be based on throat swab culture, which is the standard for diagnosis. antigen-detection tests alone can miss some cases; therefore the recommendation is to do a culture if antigen-detection test is negative in a child with suspected streptococcal pharyngitis. 26 furthermore, if antibiotics have been started in a child with suspected streptococcal pharyngitis and subsequently, the cultures are negative the treating physician should inform the parents to stop the antibiotic therapy. some studies have shown that early initiation of antibiotic on clinical suspicion can result in decreased desirable antibody response, thus allowing reinfection with type specific organisms. 27 bacteria other than group a streptococci are rare causes of pharyngitis. moreover, sequelae such as acute rheumatic fever do not occur. also, as already stated most pharyngitis in children is viral in etiology. therefore, antibiotics should not be given to a child with pharyngitis in the absence of diagnosed group a streptococcal pharyngitis or other bacterial infection. the recommendation is to use 10-day course of penicillin for the treatment of group a streptococcal pharyngitis. this is because it has a narrow spectrum of activity, low cost and high efficacy. group a streptococcal resistance to macrolide antibiotics has been observed, whereas, resistance to beta-lactam antibiotics has not been detected to date. resistance to macrolides like azithromycin or clarithromycin would be similar to that for erythromycin. a meta-analysis of 19 studies favoured cephalosporins over penicillin in terms of higher eradication of gabs from oropharynx. 28,29 these agents have a wide spectrum of activity against bacteria and thus encourage the emergence of resistance over a broad range of bacterial pathogens. alternative treatments must be used in patients with penicillin allergy, compliance issues or penicillin treatment failure. patients who do not respond to initial treatment should be given an antimicrobial that is not inactivated by penicillinaseproducing organisms (e.g., amoxycillin-clavulanate potassium, cefaclor, cephalexin or a macrolides). to summarize 1. group a streptococcal pharyngitis should be diagnosed using laboratory tests with clinical and epidemiological findings. 2. antibiotics should be given to a child with pharyngitis only when group a streptococcal or other bacterial infection has been identified. 3. penicillin is the drug of choice for group a streptococcal pharyngitis. acute respiratory infection magnitude of acute respiratory infection in under five acute respiratory infection in children-a survey in the rural community an indian hospital study of viral causes of acute respiratory infections in children resistant pneumococci: protecting patients through judicious antibiotic use the common coldprinciples of judicious use of antimicrobial agents principles of judicious use of antimicrobial agents for pediatric upper respiratory infections acute sinusitisprinciples of judicious use of antimicrobial agents pharyngitis-principles of judicious use of antimicrobial agents frequency and severity of infections in daycare the epidemiology, pathogenesis, and treatment of the common cold infections of the upper respiratory tract respiratory viral infection in childhood. a survey in general practice, rohanpton. 1967-1972 potential intervention for preventing pneumonia among young children : lack of effect of antibiotic treatment for upper resiratory infections bacteriology and treatment of purulent nasopharyngitis: a double blind, placebo controlled evaluation present concepts of the common cold the coughing child. etiology and treatment of a common symptom textbook of pediatric infectious diseases computed tomographic study of the common cold childhood sinusitis; pathophysiology, diagnosis and treatment resistance among problem respiratory pathogens in pediatrics sinusitis in children pharyngitis (pharyngitis, tonsillitis, tonsillopharyngirls, and nasopharyngitis) diagnosis and treatment of group a streptococcal pharyngitis rheumatic heart disease-a challenge red book report of the committee on infectious diseases adverse and beneficial effects of immediate treatment of group a-betahemolytic streptococcal pharyngitis with penicillin comparison of cephalosporin and penicillins in the treatment of group a beta-hemolytic streptococcal pharyngitis: a metaanylysis supporting the concept of microbial copathogenicity judicious use of antibiotics for common pediatric respiratory infections key: cord-265964-cnp5bwet authors: tumala, regie b.; almazan, joseph; alabdulaziz, hawa; felemban, ebaa marwan; alsolami, fatmah; alquwez, nahed; alshammari, farhan; tork, hanan m.m.; cruz, jonas preposi title: assessment of nursing students perceptions of their training hospital's infection prevention climate: a multi-university study in saudi arabia date: 2019-10-31 journal: nurse education today doi: 10.1016/j.nedt.2019.07.003 sha: doc_id: 265964 cord_uid: cnp5bwet abstract background the risk of acquiring and spreading infection must be minimized in nursing students because they are exposed to healthcare-associated infections during clinical training. to achieve this goal, students should be knowledgeable and competent in infection control practice before proceeding to their training hospitals. objectives this study assessed the nursing students' perception of the infection prevention climate in training hospitals in saudi arabia. it also examined the predictors of the students' perceptions. design a quantitative, cross-sectional design was used. methods this investigation was part of a large study conducted in six saudi universities. a total of 829 saudi nursing students were included in this study. data were collected using the leading culture of quality in infection prevention scale and analyzed using descriptive and inferential statistics. ethical approval was obtained from the king saud university, and permission was given by the administration of each participating university. results the overall perception of nursing students indicated a modest infection prevention climate. prioritization of quality and improvement orientation was rated as the highest dimensions, whereas psychological safety and supportive environment were the lowest. the nursing students in university f had the poorest perceptions among the six universities. the predictors of nursing student perception of their training hospitals' infection prevention climates were the university where they studied, their age, and participation in infection prevention seminars. conclusions this article describes nursing students' perception of the infection prevention climate of their training hospitals in saudi arabia. results may provide a unique theoretical underpinning on the perception and factors that effect an infection prevention climate. thereby, previous knowledge and literature may be expanded. results can be used as a guide in establishing clinical policies in efforts toward improving the infection prevention climate. practice among health care professionals (ivan et al., 2017) . as in any health profession, nurse education should promote well-equipped and competent health professional graduates in rendering quality and safety patient care (d'alessandro et al., 2014) . to diminish infection risk, nursing students should be knowledgeable and competent in infection control practice before proceeding to their training hospitals. contemporary research has investigated and recognized the critical role played by infection prevention and its proper application in a training hospital (colet et al., 2015; cruz, 2018) . substantive research findings have shown that infection prevention effects positive and negative clinical experiences and student learning in clinical settings (kim and oh, 2015) . nursing students are capable of applying their undergraduate knowledge and skills into practice to become competent health care professionals. however, despite nursing students' understanding of hais and their clinical exposure toward disease prevention and patient safety (mitchell et al., 2014) , a considerable body of evidence indicates that nursing students are constantly challenged to implement standard precautions because of their views in the infection prevention climate of their training hospitals (cruz, 2018) . despite the rich content pertaining to predictors of standard precautions compliance, systematic evidence linking nursing students' infection prevention climate perception in their respective training hospitals is lacking. with the rapid growth of resurgent infections, especially in saudi arabia , specific standards significant to infection control should be met by nursing students. to achieve this, universities and training hospitals should ascertain that their infection prevention and control education is appropriately focused. hais have become an international problem even with the advances in the healthcare system (who, 2011) . hais are among the most common diseases in hospitals with high morbidity and mortality rates (ali et al., 2018) . khan et al. (2017) have argued that hais are associated with unforeseen infection advances throughout the duration of healthcare treatment; furthermore, hais cause notable patient disease, deaths, and prolonged hospitalization, which creates additional financial burden on patients. according to the who (2011), 7 out of 100 hospitalized patients developed hais worldwide. in the us, approximately 1.7 million infections are detected annually in hospitals, with 99,000 associated mortality (kcdcp, 2011) . in europe, approximately 7.1% patients develop hais, and 37,000 people die as a result (kcdcp, 2011) . in one quantitative study in saudi arabia, 851 hais were reported among 5523 hospitalized patients; hospital stays amounted to 53,025 days, averaging 9 days of hospitalization (al-tawfiq et al., 2013) . in november 2018, the country dealt with the middle east respiratory syndrome coronavirus outbreak, which is a highly communicable and causes severe disease and result in high mortality (who, 2018) . these infectious occurrences continue to escalate at an alarming rate and pose a risk among health care professionals and patients. thus, prevention of infectious diseases should become a major health care professionals and patient-safety initiative. for the past two decades, studies have reported that the application of infection control strategies, such as standard precautions in different healthcare settings, varies in different hospitals (brosio et al., 2017; kim and oh, 2015; pogorzelska-maziarz et al., 2016) . these variations could be attributed to different infection prevention climate of healthcare settings. cruz (2018) suggested that infection prevention climate is commonly understood by health care professionals pertaining to infection prevention in clinical practice. this finding is worth noting because in a descriptive study by castro-sánchez and holmes (2015) , the infection prevention climate differences were due to standard precautions protocol, technical procedures, human resources, infection surveillance, and standard precautions compliance assessment. the variation could result in different geographical areas, healthcare facilities, and individual providers (colet et al., 2018) . with the increasing prevalence of hais across the globe (who, 2011), infection prevention climate variation most likely affects patient-care quality. nevertheless, describing this variation could support the implementation of interventions in decreasing hais, where this is most needed. similar to nurses, nursing students are also exposed to healthcare facilities through their clinical training (cruz, 2018) . colet et al. (2015) indicated that nursing students are involved during inpatient care in their clinical training, and they are not exempted in hai threats (colet et al., 2015) . thus, future nurses should be prepared, and they must have a good understanding of operating and maintaining effective infection control programs in healthcare settings. although some studies highlight the significance of standard precautions training and education in various nursing schools, controversy surrounds the nursing students' perception of infection prevention climate in training hospitals (cruz, 2018) . notably, although some findings highlighted the significance of sustaining high-quality infection prevention climate (cruz and bashtawi, 2016) , they have different views in terms of the infection prevention climate influence in training hospitals (cruz, 2018; d'alessandro et al., 2014) . although some studies showed that infection prevention was cautiously practiced by health care professionals in saudi arabia (colet et al., 2018) , training hospitals' infection prevention climate among nursing students is not well described. therefore, instituting a baseline understanding of nursing students' infection prevention climate perception in their respective training hospitals is important. this study assessed the nursing students' perception of the infection prevention climate in training hospitals in saudi arabia. it also examined the predictors of the students' perceptions. this study was part of a large quantitative, cross-sectional study investigating the saudi nursing students' standard precautions compliance, patient safety competence, and perceptions of their training hospitals' infection prevention climate. this article reports on the students' perceptions of their training hospitals' infection prevention climate. separate reports were published on students' standard precautions compliance (alshammari et al., 2018) and nursing students' patient safety competence (alquwez et al., 2019) . the settings and the samples were fully described in alshammari et al. (2018) and alquwez et al. (2019) . the study was conducted in six state universities in saudi arabia. one university (a) is in the north region of the kingdom, while two (b and c) and three (d, e, and f) universities are situated in the center and west of the country, respectively. the a convenience sample of 829 nursing students studying in 6 saudi universities was surveyed in the study. the students were included in the study if they were saudi nationals registered full-time in the 3rd and 4th years of the bsn of the six universities and if they had or having their clinical duties in an identified hospital for each university. nursing interns were also included. the response rate in the study was 77.3% (n = 1191). the largest sample was from university a (n = 254), followed by university f (n = 154), b (n = 144), c (n = 120), and d (n = 94). the lowest was from university e (n = 63). the majority of the respondents was females (69.5%, n = 576) who did not attend any infection prevention and control seminars in the past six months (67.9%, n = 563). more students were in the third year (n = 302) than 282 and 245 students in the 4th and internship years, respectively (alquwez et al., 2019; alshammari et al., 2018) . the leading culture of quality in infection prevention (lcq-ip) was utilized to gather information on the students' views of their training hospitals' infection prevention climate (pogorzelska-maziarz et al., 2016) . the tool was designed to measure a hospital's culture for quality associated with infection prevention. it has 19 items and is responded using a 5-point likert scale (1 = strongly disagree to 5 = strongly agree). the lcq-ip has four dimensions, which are central to infection prevention framework. these four dimensions are "psychological safety (7 items), quality prioritization (5 items), supportive work environment (4 items), and improvement orientation (3 items)". scores are obtained by computing the dimension means and overall scale mean. item 16 is negatively worded; hence, its score is reversed before further analysis. the four factors have cronbach's alpha from 0.724 to 0.883, whereas the entire scale has a cronbach alpha of 0.926 (pogorzelska-maziarz et al., 2016) . the arabic version of the tool was used in the present study (cruz, 2018) . cruz (2018) reported the psychometric properties of the arabic version of the tool among saudi nursing students. the exploratory factor analysis of the arabic version of the tool supported the four dimensions of the scale, which supported its construct validity. the arabic version also exhibited good internal consistency reliability, with cronbach's alpha of 0.89. for the demographic variables of the respondents, age, gender, year of study, and attendance to infection prevention and control seminars in the past 6 months (yes/no) were collected. the main study protocol was reviewed by the irb of the college of medicine of king saud university (project no.: e-17-2559). the study was also permitted by the administration of each participating university. information about the study, including its importance, participation benefits, participation risk, and voluntary participation were provided before the students were asked to sign an informed consent form. the respondents were also given time to ask questions about the study. third and fourth-year students were handed with the questionnaire in their classrooms, 15-20 min after their lectures. their lecturers were asked to leave the classroom to avoid potential undue influence bias. for the nursing interns, the questionnaires were distributed during their breaks in the hospital. the researchers approached them and explained the same information to them. the interns who agreed to participate were asked to sign an inform consent and were given the questionnaire. the same time was given to them to answer the questionnaire. means and standard deviations were computed for the lcq-ip individual items, dimensions, and overall score. t-tests, pearson correlations, and one-way anova with tukey hsd test as post hoc were performed to test the association between the nursing students' characteristics and their perceived infection prevention climate of training hospitals. a standard multiple linear regression was conducted to identify significant demographic predictors of the nursing students' perceptions. p < .05 was considered significant. the 95% confidence intervals were also calculated. all analyses were carried out using the spss version 22.0. the overall lcq-ip mean was 3.32 (sd = 0.62), indicating a modest table 1 item means, subscales means, and overall culture of infection prevention (n = 829). mean sd psychological safety 3.24 0.78 1. the climate in the organization promotes the free exchange of ideas. 2.91 1.25 2. staff will freely speak up if they see something that may improve patient care or affect patient safety. 3.26 1.15 3. i feel free to express my opinion without worrying about the outcome. 3.05 1.14 4. in general, people in our organization treat each other with respect. 3.43 1.10 5. people in this organization are comfortable checking with each other if they have questions about the right way to do something. 3.41 1.07 6. the people in this organization value others' unique skills and talents. 3.31 1.07 7. members of this organization are able to bring up problems and tough issues. 3.30 1.091 prioritization of quality 3.42 0.82 8. the health care-associated infection prevention goals and strategic plan of our organization are clear and well communicated. 3.40 1.13 9. results of our infection prevention efforts are measured and communicated regularly to staff. 3.34 1.20 10. there is a good information flow among departments to provide high-quality patient safety and care. 3.38 1.10 11. people here, feel a sense of urgency about preventing health care-associated infections. 3.57 1.16 12. employees are encouraged to become involved in infection prevention. 3.41 1.10 supportive work environment 3.24 0.58 13. senior leadership here has created an environment that enables changes to be made. table 1 ). table 2 the demographic characteristics that predict the respondents' perceptions of their training hospitals' infection prevention climate were identified. a standard multiple regression analysis was conducted whose results are indicated in table 3 . the model was significant (f[10, 818] = 11.39, p < .001), explaining approximately 11.1% variance in the students' perceptions (r 2 = 0.122; adjusted r 2 = 0.111). university, age, and attendance to infection prevention seminars in the past six months were significant demographic predictors of students' infection prevention climate perceptions. respondents from university f had a lower overall mean score in the lcq-ip by 0.41 (p < .001, 95% ci = 0.27, 0.55), 0.44 (p < .001, 95% ci = 0.30, 0.59), 0.38 (p < .001, 95% ci = 0.23, 0.52), 0.30 (p < .001, 95% ci = 0.15, 0.45), and 0.22 (p = .018, 95% ci = 0.04, 0.40) than those from university a, b, c, d, and e, respectively. a one-year increase in the students' age decreased the overall mean by 0.03 (p < .001, 95% ci = −0.05, −0.01). respondents who attended infection prevention seminars in the past six months had higher perception score by 0.23 (p < .001, 95% ci = 0.14, 0.33) than students who did not attend. this study assessed the nursing students' perception of training hospital infection prevention climate in saudi arabia. it also examined the respondents' perception predictors of infection prevention climate. five major points were highlighted in this study. first, the findings highlighted the students' infection prevention climate. the results revealed that nursing students have attained a relatively modest level of perspective on training hospitals' infection prevention climate (m = 3.32, sd = 0.62). this result was in accordance with a study conducted in china , ethiopia (wami et al., 2016) , and india (sodhi et al., 2013) . however, this result was lower than that conducted among nurses in saudi arabia (m = 3.86, sd = 0.51) (colet et al., 2018) . this finding may be because training hospitals are a complex learning environment for nursing students, and each hospital may have different infection prevention and control policies. thus, students might be unaware of the infection prevention and control protocol. baraz et al. (2015) indicated that training hospitals are unpredictable, stressful, and constantly changing. thus, such conditions may add confusion, and nursing students may be unable to handle the concepts of infection prevention and control at the required and defined time. students most likely viewed that infection control is beyond their responsibilities. they might have thought that infection control is a responsibility of the staff nurses. however, this assumption requires further investigation. the infection prevention climate dimensions "prioritization of quality" and "improvement orientation" received the highest dimensions. this statement implied a clear understanding of infection prevention climate in the organization. this result is consistent with the previous study of colet et al. (2017) , wherein nursing students have a great understanding and adherence regarding training hospitals' policies in providing quality patient safety and care. mosadeghrad (2014) suggested that training hospitals improve clinical skills and positively impact the overall quality of care among health care professionals. nursing students, as a training hospital beneficiary, have increased learning opportunities, and are also capable of identifying the influencing factors of their training hospitals' infection prevention climate. the "psychological safety" and "supportive environment" dimensions were the lowest. this result is worth noting because psychological safety and a supportive environment are intertwined with hospital organizational characteristics. this finding also suggests that efforts to improve equipment management, training and supervision, and interdisciplinary communications are imperative. in a descriptive study by livshiz-riven et al. (2014) , poor psychological safety means greater medical errors in the treatment of patients. cruz and bashtawi (2016) found that inadequate supportive environment on infection control and environment-related problems are among the crucial issues that need urgent attention. hence, improving the training hospitals' infection prevention climate is suggested, especially in promoting a supportive work environment and psychological safety, which were ranked as the lowest among the four infection prevention climate dimensions. second, the respondents' university has a significant association and influence on the nursing students' perception toward training hospital's infection prevention climate. the present study suggests that each university and its affiliated training hospital may have different infection prevention and control curricular content. different curricula mean different teaching approaches and different clinical experience, which may effect students' perceptions (bowser et al., 2017) . furthermore, the bsn programs of the 6 universities have varying amount of time for clinical experience of the students. this might have also effected the different perceptions among the students from different universities. baraz et al. (2015) found that clinical learning in a training hospital takes place in a complex social context of the clinical environment. given the complexities, it may be implied that the hospital wherein each nursing student was trained may have different infection prevention and control protocol and policies. hence, the respondents may have different degrees of awareness and practices of infection prevention and control. cruz (2018) stated that the quality of clinical training given on nursing students is the most important factor that influences their infection and prevention and control learning. however, this finding should be interpreted with caution because the factors that influence students' infection prevention and control learning were not discussed. establishing the competence and confidence of students is an essential factor of infection prevention and control success, and clinical educators should facilitate the process. third, nursing students age is significantly related to their infection prevention climate perception. the older the respondents are, the better their infection prevention climate perception. a previous study found that as an individual grows older, the more he or she acquires knowledge and motor learning (sharma et al., 2016) . the results are also in accordance with the empirical study conducted in china (cheung et al., 2015) . adults might better understand the significant health risk and are more satisfied with their clinical experience than the younger ones (rolison et al., 2014) . age likely imparts experience, and that they can perform accurately. the older the individual, the greater learning opportunities they have, which are may be appropriate to infection prevention and control study concepts. as such, they have an increased confidence level in terms of infection prevention and control practice. the effects of age toward infection prevention climate prections were not validated in the study. a deep understanding of the relationship between the students' age and infection prevention climate may improve their adherence to appropriate infection prevention and control practice. fourth, males have a better perception of infection prevention climate than females. this result is consistent with those who found that males exhibited better compliance with infection prevention and control than females (cruz, 2018; cruz and bashtawi, 2016) . however, this result negates that of another study, which reported that female nursing students have a more favourable infection prevention climate perception than males (colet et al., 2015) . extrapolated data from study of wilhemsson et al. (2011) showed that females demonstrated greater confidence in their abilities than males. females are used to work in groups, whereas males often work alone. working in groups could help identify an individual's strengths and weaknesses, exhibiting great productivity. thus, they are confident in their potential partners' skills. the research gap regarding gender complexity warrants further exploration. another highlight of the study is that infection prevention and control seminars/training was associated to and influenced nursing students' perceptions of infection prevention climate in training hospitals. respondents who participated in seminars on infection prevention in the last six months had better perceptions of infection prevention climate in their training hospitals than those who did not. this finding supports the work of other researchers that reported that the more nurses attended a workshop, the higher their motivation to practice infection control (cruz, 2018; cruz and bashtawi, 2016) . a study conducted in one tertiary care hospital in saudi arabia found that consistent training and workshop contributed to hai reduction (al kuwaiti, 2017) . a systematic review of hai prevention among 92 studies from 1996 to 2012 found that some of the components in successful infection prevention and control implementation are education, training, and positive organizational culture (zingg et al., 2015) . all studies in the review showed improvement in central-line-associated bloodstream infections after the education/training sessions. training/workshop may improve an individual's knowledge, skills, and may impart a good understanding of the nurses' responsibilities. hence, this study underscores the importance of integrating seminars/training on infection prevention and control for nursing students. limitations must be considered when the findings are evaluated. the study used a cross-sectional design, which could not distinguish other issues that might affect nursing students' perception toward infection prevention climate. longitudinal studies may provide definite information about the causal inference. moreover, the study did not explore the frequency of attendance of nursing students on infection prevention and control seminars in the last 6 months and the inclusion of curricular content on infection prevention and control. future studies should explore these variables. nevertheless, the researchers strongly believe that the above limitations have not undermined the study purpose. one of the strengths of the study is its large sample size and inclusion of six universities, which could help in generalizing the findings. the tools used in this study exhibited good psychometric properties and high response rate. the present findings contributed to the limited literature on infection prevention climate of training hospitals as perceived by nursing students. this study examined the nursing students' perception on infection prevention climate of their training hospitals in saudi arabia. the students have attained a relatively modest level of perspective on training hospitals' infection prevention climate. further, university, age, and participation to infection prevention in the past six months predicted nursing students' perception of infection prevention climate. gender was significantly related to infection prevention climate perception. finally, the results provided a unique theoretical underpinning that expanded on previous knowledge and literature on factors that affect infection prevention climate. nursing students are expected to be highly involved in the real world of the clinical practice setting. they are not exempted in the hai threats. this investigation critically examines the view of nursing students toward infection prevention climate in their training hospital. the findings can be used as a guide in establishing clinical policies in efforts toward training hospitals' infection prevention climate improvement. the finding can help nursing students to become competent and confident future healthcare professionals. overall, the nursing students' infection prevention climate perspective needs further improvement, especially in terms of psychological safety and supportive work environment dimensions. hence, organizing training and supervision and using supportive working condition strategies is necessary to make nursing students feel safe, creative, and engaged toward infection prevention and control implementation. creating and defining nursing student's engagement rules should be done so that they can be comfortable, engage deeply, and communicate clearly to other health care professionals. in this regard, nursing students may feel included, important, and part of the healthcare team. a supportive working environment and high engagement can increase the students' motivation to tackle issues pertaining to infection prevention climate, development opportunities, and good performance. facilitating meaningful connections between nursing students from various universities and their perceived infection prevention climate may improve through a comprehensive and unified course syllabus and supporting program that can empower students' learning. given the positive relationship between participation in infection prevention seminar and infection prevention climate perceptions, increasing the number of training facilities that can provide a variety of training, workshop, and seminar programs to nursing students related to infection prevention is important. this paper received funding from the deanship of scientific research through the research center of the college of nursing in king saud university, riyadh, kingdom of saudi arabia. institutional review board of the college of medicine of king saud university (project no.: e-17-2559) . none declared. impact of a multicomponent hand hygiene intervention strategy in reducing infection rates at a university 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quantitative and qualitative study of healthcare workers view in jimma zone hospitals patient safety curriculum guide: multi-professional edition. world health organization, geneva. world health organization (who) systematic review and evidence-based guidance on organization of hospital infection control programmes (sight) study group. hospital organization, management, and structure for prevention of health-care-associated infection: a systematic review and expert consensus key: cord-278935-3lgud7l8 authors: chen, zheng‐rong; mize, maximillion; wang, yu‐qing; yan, yong‐dong; zhu, can‐hong; wang, yunji; ji, wei title: clinical and epidemiological profiles of lower respiratory tract infection in hospitalized children due to human bocavirus in a subtropical area of china date: 2014-04-30 journal: j med virol doi: 10.1002/jmv.23952 sha: doc_id: 278935 cord_uid: 3lgud7l8 lower respiratory tract infection is a major cause of morbidity and mortality in children. human bocavirus (hbov) is confirmed to have an association with pediatric lower respiratory tract infection. seasonal and meteorological factors may play a key role in the epidemiology of hbov. the purpose of this study was to ascertain the frequency, season, and clinical characteristics of hospitalized children with hbov infection. in addition, an evaluation of the effects of meteorological factors on the incidence of hbov in a subtropical area in china will be conducted. children were <14 years in age and hospitalized for lower respiratory tract infection between january 1, 2009 and december 31, 2012 in the respiratory disease department at the children's hospital affiliated to soochow university. multi‐pathogens were detected in nasopharyngeal aspirate samples. the association between hbov activity and regional meteorological conditions was analyzed. the average incidence of hbov infection was 6.6% (502/7,626). of the 502 hbov positive children, the median age was 13 months (range 1–156 months). the hbov infection rate was highest among the 7–12 months groups (12.9%, 163/1,267). seasonal distribution of hbov was noted during june to november, especially during the summer season (june to august). hbov activity was associated with temperature and humidity although the lag effect between temperature and hbov activity observed. hbov is one of the most common viral pathogens in children with lower respiratory tract infection. hbov infection occurs throughout the year with a peak during the summer. temperature and humidity may affect the incidence of hbov. j. med. virol. 86:2154–2162, 2014. © 2014 wiley periodicals, inc. acute respiratory tract infection is a major cause of morbidity and mortality, especially for infants and young children under 5 years old [united nations children's fund, 2011] . the incidence of communityacquired childhood pneumonia in low-and middleincome countries in the year 2010, using world health organization's definition, was about 0.2 (interquartile range [iqr] 0.1-0.5) episodes per childyear, with 11.5% (iqr 8.0-33.0%) of cases progressing to severe episodes [rudan et al., 2013] . a variety of viruses and atypical pathogens, including influenza viruses (iv), respiratory syncytial virus (rsv), parainfluenza virus (piv), adenovirus (adv), human metapneumovirus (hmpv), and mycoplasma pneumoniae have all been associated with acute respiratory tract infection in children. human bocavirus (hbov), belonging to the family parvoviridae, subfamily parvovirinae, and genus bocavirus, was first identified from pooled nasopharyngeal aspirate specimens by large-scale molecular virus screening [allander et al., 2005] . evidence supporting hbov role as an etiologic agent in upper and lower respiratory tract infection in children under 5 years has begun to emerge [manning et al., 2006; weissbrich et al., 2006; allander et al., 2007; canducci et al., 2008; huang et al., 2008; deng et al., 2012] . global hbov activity varies substantially from year-to-year among different populations and regions. furthermore, epidemiological data shows that hbov is present year-round with different incidence rates from 2.2% to 19% in children with lower respiratory tract infection [manning et al., 2006; weissbrich et al., 2006; allander et al., 2007; canducci et al., 2008] . recent studies conducted in several countries have reported that the incidence of hbov fluctuates based on seasonal attributes with infection being higher during winter months [allander et al., 2005; manning et al., 2006; weissbrich et al., 2006] . however, in japan the infectious rate of hbov increases during the spring and summer seasons leading to the belief that regional, in addition to seasonal, factors play a role in the rate of disease transmission [moriyama et al., 2010] . while this suggests that seasonal changes may influence the development of hbov infection [naghipour et al., 2007] , meteorological factors (temperature, humidity, and precipitation) may play the greatest role in the epidemics of hbov based on research conducted in brazil [do amaral de leon et al., 2013] . although few studies have investigated the epidemiological files of hbov infection in children with lower respiratory tract infection in subtropical area in china, a possible relationship between meteorological factors and incidence of hbov is currently unknown. the purpose of this study was to ascertain the frequency, seasonal, and clinical characteristics in hospitalized children with lower respiratory tract infection and evaluate the effects of meteorological factors on the incidence of hbov in a subtropical region of china. in this retrospective study, subjects were <14 years in age and hospitalized for lower respiratory tract infection between january 1, 2009 and december 31, 2012 in the respiratory department at soochow university affiliated children's hospital. the demographic and clinical characteristics of all subjects were collected for analysis. this tertiary teaching hospital in suzhou is the only hospital that provides special care to pediatric patients and the participation rate was $60%. the city of suzhou is located in east of china and has a subtropical climate. informed consent was obtained from parents or legal guardians. this study was conducted with the approval of the institutional human ethical committee of soochow university. patients were considered eligible for enrollment if they had a clinical and radiological diagnosis of community-acquired lower respiratory tract infection including acute bronchiolitis, bronchitis, and pneumonia. chest radiography was performed using standard equipment and radiographic techniques, and reviewed by the radiologists in digital format. before hospital discharge, an attending physician recorded information such as age, gender, clinical diagnosis, underlying chronic diseases, clinical manifestation, and peripheral blood routine test. children with a history of chronic lung disease, underlying immunodeficiency, or preexisting cardiac, renal, neurologic, or hepatic dysfunction, or bronchopulmonary malformation were excluded from the study. lower respiratory tract infection was defined as the presence of wheezing, tachypnea, chest retractions, abnormal auscultatory findings (wheezing and crackles), the presence of fever and radiologic evidence indicative of a lower respiratory tract infection. pneumonia was defined as the presence of focal infiltration (bronchopneumonia) or consolidation (lobar pneumonia) in the lung by chest radiography. very severe pneumonia was defined as the presence of (i) central cyanosis (ii) inability to breastfeed or drink without vomiting (iii) convulsions, lethargy, or unconsciousness (iv) severe respiratory distress as defined by the world health organization. nasopharyngeal aspirate samples were obtained from all patients within 24 hr of admission. this involved passing a suction catheter through the nose with the intent of passing it into the lower part of the pharynx. the depth of penetration for the nasopharyngeal aspirate catheter was set at 5-10 cm. a total 2 ml nasopharyngeal aspirate sample was obtained and centrifuged at 500âg for 10 min and resuspended in 2 ml saline and divided into two aliquots for pathogen detection using direct immunofluorescence assay (dfa) and pcrs as described previously [chen et al., 2013b] . one of the equally divided samples of nasopharyngeal aspirate was centrifuged at 12,000âg for 5 min, followed by extraction of dna and rna from a 400-ml sample using dna-ez reagents (sangon biotech, shanghai, china) or trizol reagent (life technologies, carlsbad, ca) in accordance with the manufacturer's instructions. a final 200 ml of dna or rna was eluted and dna sample was divided into two aliquots for hbov and mycoplasma pneumoniae gene amplification via pcr. rna sample was used for hmpv gene detection. real-time pcr primers were designed using sequence information from the np1 gene sequence available from the genbank database. primers and probe were synthesized using the following sequences: hbov-f:5 0 -tga-cattcaactaccaacaacctg-3 0 ;hbov-r:5 0 -cagat-ccttttcctcctccaatac-3 0 ;hbov-probe:agcac-cacaaaacacctcagggg-tamra (sangon biotech). pcr was performed in a volume of 25 ml using iq5 tm bio-rad icycler (bio-rad, carlsbad, ca). the 25 ml amplification reaction contained 3 ml of sample dna, 0.25 ml of taqman (promega, madison, wv), depc treated water 14.75 ml, buffer solution 2.5 ml, 25 mm mgso 4 2 ml, dntp 1 ml, forward, reverse primers and probe 0.5 ml, respectively. amplification was performed with the following settings: 94˚c for 30 sec, 56˚c for 30 sec, 72˚c for 30 sec for 40 cycles. positive and no-template controls were included in each run. positive samples containing the target genes were used as positive controls for all four hbov subtypes and were constructed by shanghai sangon biotech co., ltd. the concentration of each detected sample was then calculated automatically according to standard curve. mycoplasma pneumoniae samples were also analyzed for seven common viruses, including rsv, iv-a and iv-b, piv-1, 2, 3, and adv using dfa with virus-specific fluorescencelabeled monoclonal antibodies (diagnostic hybrids, athens, oh) and ultraviolet light microscopy. hmpv and mycoplasma pneumoniae were detected by reverse transcription pcr and real-time pcr, respectively. briefly, for hmpv detection, primers were designed to specifically amplify the n gene (213 bps). the forward and reverse primers were 5 0 -aaccgtgtactaagt-gatgcactc-3 0 and 5 0 -cattgtttgaccggcccca-taa-3 0 , respectively. reverse transcription reactions were performed with m-mlv reverse transcriptase (promega) and random hexamers for cdna synthesis according to the manufacturer's specifications. pcr was performed in a volume of 25 ml and the pcr conditions were as follows: denaturation at 95˚c for 5 min, then 45 cycles of denaturation at 94˚c for 30 s, annealing at 55˚c for 30 s, and extension at 68˚c for 30 s, followed by a final extension at 68˚c for 7 min as described previously . for mycoplasma pneumoniae detection, another fluorescent real-time pcr was performed to identify the p1 adhesion protein gene of mycoplasma pneumoniae as described previously [chen et al., 2013a,b] . briefly, a 21 ml pcr master mixture (daan gene, guangzhou, china) containing the primers and probes was combined with 3 ml of the sample dna and 1 ml of the gotaq 1 dna polymerase (promega) for the pcr reactions. real-time pcr was performed using the iq5 tm bio-icycler (bio-rad), and the cycling conditions were as follows: 2 min at 37˚c; 10 min at 94˚c, and 40 cycles of 10 s at 94˚c, 30 s at 55˚c, and 40 s at 72˚c. the quantitation curves were plotted using several concentrations of standard control samples, which were purchased from daan gene co. positive samples were defined with a concentration of dna >2.5 â 10 3 copies/ml in case of mycoplasma pneumoniae colonization. meteorological data for suzhou, including daily mean temperature (˚c), mean relative humidity (%), total monthly rainfall (mm), sum of sunshine (h), and mean wind velocity (m/s), were obtained from suzhou weather bureau at longitude 120˚6 0 east and latitude 31˚3 0 north, which is located 8 km away from the hospital. meteorological data were obtained hourly, and average daily values were calculated. monthly means were calculated using the daily means for temperature, relative humidity, and wind velocity. total rainfall and hours of sunshine were calculated as a total measurement for the month. values were expressed as percentages for discrete variables, as mean and standard deviation for continuous variables. the continuous variables were compared using the student t-test or mann-whitney u test if the data were abnormal in distribution. categorical data were analyzed using the mentel-haenszel, chi-squared (x 2 ), or fisher's exact tests. correlations of incidence of hbov with meteorological factors were evaluated using pearson's or spearman rank correlation. because of colinearity between meteorological factors, associations and lag effects between meteorological factors and hbov incidence were also analyzed using linear regression. for the final goal of predicting the incidence of hbov on the basis of meteorological data and season, a time series analysis utilizing a seasonal model was established for the time period between january 2009 and december 2011 (estimation period). it was evaluated by comparing the predicted versus the observed incidence of hbov during the period between january 2012 and december 2012 (evaluation period). the r 2 autoregression coefficient was calculated to judge the fitness of the model. from january 2009 to december 2012, a total of 8,288 children with acute respiratory tract infection were admitted to our hospital. nasopharyngeal aspirate samples were not taken from 510 hospitalized children due to a refusal to participate from their parent or guardian. one hundred fifty-two hospitalized children were excluded on account of congenital heart disease, pulmonary tuberculosis, down's syndrome or bronchopulmonary malformation. finally, a total of 7,626 nasopharyngeal aspirate samples were collected, 3,491(45.8%) of which were positive for at least one virus or mycoplasma pneumoniae. the most commonly identified pathogen was rsv (15.7%, 1, 197/7, 626) , followed by mycoplasma pneumoniae (14.3%, 1,094/7,626), hbov (6.6%, 502/ 7,626), piv-3 (6.1%, 464/7,626), hmpv (4.2%, 318/ 7,626), iv-a (2.8%, 166/7,626), adv (1.3%, 97/7,626), iv-b (0.8, 62/7,626), piv-2 (0.1%, 8/7,626), and piv-1 (0.03%, 2/7,626). a total of 388 nasopharyngeal aspirate samples (5.1%, 388/7,626) were detected as containing at least two viruses or co-infection with mycoplasma pneumoniae and hbov. the latter combination was one of the most common co-infections accounting for 37.6% (146/388) of total co-infected samples as well as 29.1% (146/502) of total hbov positive samples. a total of 356 samples (4.7%, 356/7,626) were detected single hbov infection and rsv (27.4%, 40/146) and mycoplasma pneumoniae (25.3%, 37/146) were most common co-infection pathogens with hbov (table i) . of the 502 hbov positive children, the median age was 13 months (range 1-156 months). hbov infection incidence of different age groups are as follows: 1-6 months (3.5%, 71/2,005), 7-12 months (12.9%, 163/ 1,267), 13-36 months (8.1%, 170/2,109), 37-60 months (5.6%, 65/1,155), and >60 months (3.0%, 33/ 1,090), respectively. the infectious rate of hbov was highest among the 7-12 months old group compared with the other four groups (all p < 0.05) and lowest among the 1-6 and >60 months old group when compared to the other three groups (all p < 0.05) as shown in figure 1 . the ratio of male to female children with hbov infection was 1.67:1 and no gender difference was observed in the incidence of hbov infection compared with total children with lower respiratory tract infection (male to female, hbov infected children 1.7 vs. total children 1.7, p > 0.05). the demographics and clinical information of hospitalized children with single hbov infection or coinfection are summarized in table ii . no significant differences were observed in the demographic characteristics, clinical presentations, and laboratory tests between single hbov infection and co-infection groups except for diagnosis of lobar pneumonia. children with co-infection presented with a higher rate of lobar pneumonia compared to single hbov infection group (x 2 ¼ 5.2, p ¼ 0.02). to determine the seasonal distribution of hbov infection, we assessed incidence over a 4-year period from january 2009 to december 2012. the incidence of infection for 2012 (4.2%, 93/2,240) was significantly lower than that of 2009 (8.1%, 144/1,779), 2010 (7.6%, 122/1,599), and 2011 (7.1, 143/2,008) (all p < 0.05). hbov could be detected every year-round except for december 2010. a seasonal distribution of hbov was noted during june to november, especially during the months of summer (june to august) which accounted for 37.8% (190/502) of the total hbov cases. however, variations in this trend were observed from 1 year to another. interestingly, in 2009 two peaks of hbov activity occurred, one in june (11.8%) and the other in november (15.7%). this was also seen in 2010, however, the peaks occurred during april (11.8%) and july (14.0%) as shown in figure 2 . the suzhou area has a typical subtropical monsoon climate. from 2009 to 2012, the monthly mean temperature was 17.1 ae 9.0 (mean ae standard deviation)˚c, relative humidity was 68.4 ae 6.3%, total rainfall was 83.6 ae 69.7 mm, sum of sunshine was 148.4 ae 47.3 h, and wind velocity was 1.8 ae 0.4 m/s. the monthly mean data for these meteorological variables over the course of this study are shown in figure 2 . the associations of hbov activity with meteorological factors were performed using pearson's or spearman correlations. as shown in table iii , hbov activity was associated with mean temperature and relative humidity. because of colinearity between meteorological factors (data not shown), associations between meteorological factors and hbov incidence were also analyzed using linear regression based on a stepwise procedure. in model 0, which had no lag time for analyzing hbov activity and meteorological factors, hbov activity is positively associated with mean temperature and relative humidity. when the lag time was taken into consideration, the m1 model was a better fit, with a higher r 2 value of 0.370 when compared to the m0 and m2 models that had r 2 values of 0.308 and 0.230, respectively. these indicated hbov activity could be better explained according the meteorological factors. specifically, this study shows that hbov activity is associated with mean temperature and a lag time effect (supplementary table si) . time series analysis was also performed to predict the hbov activity in hospitalized children with lower respiratory tract infection. the r 2 and p were 0.8 and 0.006, respectively, which showed a good fitness for this simple seasonal model. taken together, hbov activity in hospitalized children with lower respiratory tract infection could be predicted based on periodicity and season (fig. 3) . the findings of this study confirmed the hypothesis that hbov is a common pathogen in children with lower respiratory tract infection. in addition, coinfection frequently occurs with rsv and mycoplasma pneumoniae. children with co-infection were prone to lobar pneumonia compared to children infected with hbov only. to our knowledge, this is the first time that an association between hbov activity and meteorological factors were taken into consideration by a 4-year respiratory virus surveillance in a subtropical region of china. the present study shows that mean temperature was the main meteorological factor associated with hbov activity and this influence also had a month lag time effect. based on the data from this study, hbov was the third most common pathogen after rsv and mycoplasma pneumoniae with an incidence of 6.6% in all hospitalized children with lower respiratory tract infection in suzhou area. this is consistent with recent reports in shanghai area (7.0%, 39/554) [zhao et al., 2013] and lanzhou area (7.1%, 29/406) [zheng et al., 2010] . although this data does differ when looking at the gansu area (2.2%) [huang et al., 2013] and beijing area (33.8%) in china . both serology and pcr methods could be applied to detect the hbov infection in children. certainly, pcr methods have a higher sensitivity when compared to the igm detection method using elisa (100% vs. 81.1%) [zaghloul, 2011] . most studies have shown that the incidence of infection with hbov is highest among young infants less than 3 years of age [nascimento-carvalho et al., 2012; zhao et al., 2013; abdel-moneim et al., 2013] . in the present study, the median age was 13 months, and the age group of 7-12 months and 13-36 months accounted for 66.3% of the total cases. furthermore, a higher incidence rate (12.9% and 8.1%, respectively) compared to that of the other age groups was observed. this parallels the findings from studies conducted in tokyo, japan and salvador, brazil [moriyama et al., 2010; nascimento-carvalho et al., 2012] . several previous studies have reported that rsv, hmpv, rhinovirus, and human coronavirus are principal causes of co-infection with hbov. presumably, this is because the seasonal distribution of hbov and these other viruses appear to overlap [allander et al., 2007; koseki et al., 2012; xu et al., 2012] . indeed, the reported prevalence of co-infections with other respiratory viruses varies from 18.3% to 71.1% [weissbrich et al., 2006; garcía-garcía et al., 2007; brieu et al., 2008; kim et al., 2011] . co-infection was also common in this study and accounted for 29.1% (146/502) of the total hbov positive samples. interestingly, mycoplasma pneumoniae was a major pathogen that co-infected with hbov after rsv and accounted for 25.3% (37/146) of total hbov co-infection cases. the major presenting clinical picture of lower respiratory tract infection due to hbov was similar to other common respiratory virus infections like rsv and hmpv including cough, fever, wheezing and tachypnea and no characteristic symptom was found. the present study indicated pneumonia and bronchiolitis were the most frequent diagnoses associated with hbov infected children, which is consistent with previous study [allander et al., 2007; garcía-garcía et al., 2007; moriyama et al., 2010] . there was no difference in clinical manifestation, laboratory test and proportion of severe pneumonia between single hbov infection and co-infection groups. coinfections were similar to simple infections in children with hbov except that hypoxia was slightly more frequent (p ¼ 0.038) in children with co-infection [garcía-garcía et al., 2007] . on the contrary, another study suggested that the association between hbov and other respiratory viruses may be of clinical importance, and that infection with hbov alone appears to produce no major symptoms in infants seen in emergency departments [esposito et al., 2008] . it might have contributed to a lower severity of this present study because of the exclusion of children with underlying diseases. however, a recent study showed that children with single hbov infection had a higher viral load compared to children with co-infection and there was a direct correlation of high viral load (>10 6 copies/ml) with increasing disease severity in children co-infected with hbov but not in children with single hbov infection [zhao et al., 2013] . meanwhile, high viral load (>10 4 copies/ ml) of hbov correlates with the duration of wheezing in children with severe lower respiratory tract infection [deng et al., 2012] . nevertheless, a study reported that there was no apparent association between the viral load of hbov and co-infection or disease severity [zheng et al., 2010] . taken together, the role of co-infection with hbov is still not clear and further studies need to be taken to explore these clinical observations. interestingly, children with coinfection had a higher rate of lobar pneumonia compared to children with single hbov infection. it is hypothesized that co-infection with mycoplasma pneumoniae maybe play a role in such phenomenon due to it being the most common cause of lobar pneumonia in children according our 8 year surveillance (data not shown). it has been described in earlier studies that hbov infection seems to have a seasonal distribution. hbov positive patients were most prevalent during january to may and the peak occurred during april to may in japan [ma et al., 2006] while the seasonal distribution is november to june with peaks occurring during march to april in france [jacques et al., 2008] . in hong kong, the peak seasons were in fall and winter [chieochansin et al., 2008] . in the guangzhou area of china with similar subtropical climate, hbov activity was year-round and the peak season was june [xu et al., 2012] , similar to what is seen in the present study. in india, however, which has a tropical climate, hbov appeared to have no seasonal distribution [bharaj et al., 2010] . these studies suggest that different areas with different climates have their own seasonal distribution of hbov incidence. however, how a seasonal epidemic of hbov starts is currently unknown. factors such as climate may impact the survival and spread of infectious disease indicating that the environment may contribute to an epidemic and the seasonal outbreaks of respiratory viruses [du prel et al., 2009 ]. in the present study, hbov activity was positively associated with mean temperature and relative humidity, which was consistent with another recent report [do amaral de leon et al., 2013] . the temperature and relative humidity during the summer was significantly higher than the other three seasons (shown in fig. 2 ). to our interest, a lag time effect existed between hbov activity and mean temperature especially for 1 month lag and no lag effect was found between hbov activity and relative humidity. taking into account that hbov causing lower respiratory tract infection possesses an incubation period, several days of medical consultation, and an average of 5-7 days medical treatment before admission to our hospital (data not show), we presume that the lag time between climate parameters and hbov, as confirmed by real-time pcr, is 2-3 weeks. this is consistent with the results of 1 month lag effect analyzed using linear regression in present study. this explains why the m1 model including mean temperature has a higher r2 value than the m0 or m2 models. taken together, this study suggests that high temperature plays a more important role than high relative humidity in hbov seasonal activity. the data on predicting hbov activity could help in geographical areas where detection of hbov infection may be difficult. some limitations of this study should be noted. first of all, it is difficult to confirm hbov infection depending solely upon pcr because of certain bacterial colonization in healthy children although positive samples were defined with a concentration of dna >2.5 â 10 3 copies/ml. secondly, the data analysis alone may not serve as a conclusive interpretation, since any associations with meteorological factors may be an indication of other social or environmental factors that also vary with the seasons. what's more, our study was based on a single center for data, which might have potential biases. despite these limitations, this study indicates that hbov is a common cause of lower respiratory tract infection in children less than 3 years. understanding the impact of meteorological factors, especially temperature, on hbov activity can be useful and important in predicting seasonal outbreaks of lower 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bocavirus (hbov) in children with respiratory tract infection by enzyme linked immunosorbent assay (elisa) and qualitative polymerase chain reaction (pcr) viral etiology and clinical profiles of children with severe acute respiratory infections in china high human bocavirus viral load is associated with disease severity in children under five years of age human bocavirus infection in young children with acute respiratory tract infection in lanzhou we thank the members of the research team, laboratory staff and all cooperating institutes for their dedication to the project. most of all, we are indebted to all participating children and their families. additional supporting information may be found in the online version of this article at the publisher's web-site. key: cord-226245-p0cyzjwf authors: schneble, marc; nicola, giacomo de; kauermann, goran; berger, ursula title: nowcasting fatal covid-19 infections on a regional level in germany date: 2020-05-15 journal: nan doi: nan sha: doc_id: 226245 cord_uid: p0cyzjwf we analyse the temporal and regional structure in mortality rates related to covid-19 infections. we relate the fatality date of each deceased patient to the corresponding day of registration of the infection, leading to a nowcasting model which allows us to estimate the number of present-day infections that will, at a later date, prove to be fatal. the numbers are broken down to the district level in germany. given that death counts generally provide more reliable information on the spread of the disease compared to infection counts, which inevitably depend on testing strategy and capacity, the proposed model and the presented results allow to obtain reliable insight into the current state of the pandemic in germany. in march 2020, covid-19 became a global pandemic. from wuhan, china, the virus spread across the whole world, and with its diffusion, more and more data became available to scientists for analytical purposes. in daily reports, the who provides the number of registered infections as well as the daily death toll globally (https://www.who.int/). it is inevitable for the number of registered infections to depend on the testing strategy in each country (see e.g. cohen and kupferschmidt, 2020) . this has a direct influence on the number of undetected infections (see e.g. li et al., 2020) , and first empirical analyses aim to quantify how detected and undetected infections are related (see e.g. niehus et al., 2020) . though similar issues with respect to data quality hold for the reported number of fatalities (see e.g. baud et al., 2020) , the number of deaths can overall be considered a more reliable source of information than the number of registered infections. the results of the "heinsberg study" in germany point in the same direction (streeck et al., 2020) . a thorough analysis of death counts can in turn generate insights on changes in infections as proposed in flaxman et al. (2020) (see also ferguson et al., 2020) . in this paper we pursue the idea of directly modelling registered death counts instead of registered infections. we analyse data from germany and break down the analyses to a regional level. such regional view is apparently immensely important, considering the local nature of some of the outbreaks for example in italy (see e.g. , france (see e.g. massonnaud et al., 2020) or spain. the analysis of fatalities has, however, an inevitable time delay, and requires to take the course of the disease into account. a first approach on modelling and analysing the time from illness and onset of symptoms to reporting and further to death is given in jung et al. (2020) (see also linton et al., 2020) . understanding the delay between onset and registration of an infection and, for severe cases, the time between registered infection and death can be of vital importance. knowledge on those time spans allows us to obtain estimates for the number of infections that are expected to be fatal based on the number of infections registered on the present day. the statistical technique to obtain such estimates is called nowcasting (see e.g. höhle and an der heiden, 2014) and traces back to lawless (1994) . nowcasting in covid-19 data analyses is not novel and is for instance used in günther et al. (2020) for nowcasting daily infection counts, that is to adjust daily reported new infections to include infections which occurred the same day but were not yet reported. we extend this approach to model the delay between the registration date of an infection and its fatal outcome. we therefore analyse the number of fatal cases of covid-19 infections in germany using district-level data. the data are provided by the robert-koch-institute (www.rki.de) and give the cumulative number of deaths in different gender and age groups for each of the 412 administrative districts in germany together with the date of registration of the infection. the data are available in dynamic form through daily downloads of the updated cumulated numbers of deaths. we employ flexible statistical models with smooth components (see e.g. wood, 2017) assuming a district specific poisson process. the spatial structure in the death rate is incorporated in two ways. first, we assume a spatial correlation of the number of deaths by including a long-range smooth spatial death intensity. this allows to show that regions of germany are affected to different extents. on top of this long-range effect we include two types of unstructured region specific effects. an overall region specific effect reflects the situation of a district as a whole, while a short-term effect mirrors region specific variation of fatalities over time and captures local outbreaks as happened in e.g. heinsberg (north-rhine-westphalia) or tirschenreuth (bavaria). in addition we include dynamic effects to capture the global changes in the number of fatal infections for germany over calendar time. this enables us to investigate the impact of certain interventions, such as social distancing, school closure, complete lockdowns and lockdown releases, on the dynamic of the infection and hence on the number of deaths. modelling infectious diseases is a well developed field in statistics and we refer to held et al. (2017) for a general overview of the different models. we also refer to the powerful r package surveillance . since our focus is on analysing the district specific dynamics of fatal infections we here make use of poisson-based models implemented in the mgcv package in r, which allows to decompose the spatial component in more depth. the paper is organized as follows. in section 2 we describe the data. section 3 highlights the results of our analysis. the remaining sections provide the technical material, starting with section 4 where we motivate the statistical model, which is extended by our nowcasting model in section 5. extended results as well as model validation are given in section 6, while section 7 concludes the paper. we make use of the covid-19 dataset provided by the robert-koch-institute for the 412 districts in germany (which also include the twelve districts of berlin separately). the data are updated on a daily basis and can be downloaded from the robert-koch-institute's website. we have daily downloads of the data for the time interval from march 27, 2020 until today. the subsequent analysis was conducted on may 14, 2020, and was performed considering only deadly infections with registration dates from march 26, 2020 until may 13, 2020 (the day before the day of analysis). the data contain the newly notified laboratory-confirmed covid-19 infections and the cumulated number of deaths related to covid-19 for each district of germany, classified by gender and age group. each data entry has a time stamp which corresponds to the registration date of a confirmed covid-19 infection. this means that the time stamp for a fatal outcome always refers to the registration date and not to the death date. due to daily downloads of the data we can derive the time point of death (or to be more specific, the time point when the death of a case is included in the database). we obtain the latter by observing a status change from infected to deceased when comparing the data from two consecutive days. the robert-koch-institute collects the data from the district-based health authorities (gesundheitsämter). due to different population sizes in the districts and certainly also because of different local situations, some health authorities report the daily numbers to the robert-koch-institute with a delay. this happens in particular over the weekend, a fact that we need to take into account in our model. we refrain from providing general descriptive statistics of the data here, since these numbers can easily be found on the rki webpage, which also gives a link to a dashboard to visualize the data (see also https://corona.stat.uni-muenchen.de/maps/) before we discuss our modelling approach in detail, we want to describe our major findings. first, table 1 shows that age and gender both play a major role when estimating the daily death toll. as is generally known, elderly people exhibit a much higher death rate which is for the age group 80+ around 100 times higher than for people in the age group 35-59. a remarkable difference is also observed between genders, where the expected death rate of females is around 40% (≈ 1 − exp(−0.503)) lower than the death rate for males. furthermore, we see that significantly less deaths are attributed to infections registered on sundays compared to weekdays, due to the existing reporting delay during weekends. our model includes a global smooth time trend representing changes in the death rate since march 26th. this is visualized in figure 1 . the plotted death rate is scaled to give the expected number of deaths per 100.000 people in an average district for the reference group, i.e. males in the age group 35 -59. overall, we see a peak in the death rate on april 3rd and a downwards slope till end of april. however, our nowcast reveals that the rate remains constant since beginning of may. note that this recent development cannot be seen by simply displaying the raw death counts of these days. the nowcasting step inevitably carries statistical uncertainty, which is taken into account in figure 1 by including best and worst case scenarios. the latter are based on bootstrapped confidence intervals, where details are provided in section 6.3 later in the paper. our aim is to investigate spatial variation and regional dynamics. to do so, we combine a global geographic trend for germany with unstructured region-specific effects, where the latter uncover local behaviour. in figure 2 we combine these different components and map the fitted nowcasted death counts related to covid-19 for the different districts of germany, cumulating over the last seven days before the day of analysis (here may 14, 2020). while in most districts of germany the death rate is relatively low, some hotspots can be identified. among those, traunstein and rosenheim (in the south-east part of bavaria) are the most evident, but greiz and sonneberg (east and south part of thuringia) stand out as well, to mention a few. a deeper investigation of the spatial structure is provided in section 6, where we show the global geographic trend and provide maps that allow to detect new hotspot areas, after correcting for the overall spatial distribution of the infection. on the day of analysis, we do not observe the total counts of deaths for recently registered infections, since not all patients with an ongoing fatal infections have died yet. we therefore nowcast those numbers, i.e. we predict the prospective deaths which can be attributed to all registration dates up to today. this is done on a national level, and the resulting nowcast of fatal infections for germany is shown in figure 3 . for example, on may 14, 2020 there are 25 deaths reported where the infection was registered on may 5th (red line on may 5th). we expect this number to increase to about 50 when all deaths due to covid-19 for this registration date will have been reported (blue line on may 5th). naturally, the closer a date is to the present, the larger the uncertainty in the nowcast. this is shown by the shaded bands. details on how the statistical uncertainty has been quantified are provided in section 5 below. the fit of this model has been incorporated into the district model discussed before, but the nowcast results are interesting in their own right. the curve confirms that the number of fatal infections is decreasing since the beginning of april. note that the curve also mirrors the "weekend effect" in registration, as less infections are reported on sundays. further analyses and a detailed description of the model are given in the following sections. let y t,r,g denote the number of daily deaths due to covid-19 in district/region r and age and gender group g with time point (date of registration) t = 0, . . . , t . here t = t corresponds to the day of analysis, which is may 14, 2020 and t = 0 corresponds to march 26, 2020. note that time point t refers to the time point of registration, i.e. the date at which the infection was confirmed. even though the time point of infection obviously precedes that of death, registration can also occur after death, e.g. when a post mortem test is conducted, or when test results arrive after the patient has passed away. we set the day of death to be equal to the day of registered infections in this case. the majority of fatalities with registered infection at time point t have not yet been observed at time t, as these deaths will occur later. we therefore need a model for nowcasting, which is discussed in the next section. for now we assume all y t,r,g to be known. we model y t,r,g as (quasi-)poisson distributed according to where we specify λ t,r,g through λ t,r,g = exp{(β 0 + age g β age + gender g β gender + weekday t β weekday the linear predictor is composed as follows: • β 0 is the intercept. • β age and β gender are the age and gender related regression coefficients. • β weekday are the weekday-related regression coefficients. • m 1 (t) is an overall smooth time trend, with no prior structure imposed on it. • m 2 (s r ) is a smooth spatial effect, where s r is the geographical centroid of district/region r. • u r0 and u r1 are district/region-specific random effects which are i.i.d. and follow a normal prior probability model. while u r0 specifies an overall level of in the death rate for district r over the entire observation time, u r1 reveals region specific dynamics by allowing the regional effects to differ for the last 14 days. • pop r,g is the gender and age group-specific population size in district/region r and serves as an offset in our model. we here emphasize that we fit two spatial effects of different types: we model a smooth spatial effect, i.e. m 2 (s r ), which takes the correlation between the death rates of neighbouring districts/regions into account and gives a global overview of the spatial distribution of fatal infections. in addition to that we also have unstructured district/region-specific effects u r = (u r0 , u r1 ) , which capture local behaviour related to single districts only. the district specific effects u r are considered as random with a prior structure for r = 1, . . . , 412. the prior variance matrix σ u is estimated from the data. the predicted values u r (i.e. the posterior mode) exhibit districts that show unexpectedly high or low death tolls when adjusted for the global spatial structure and for age-and gender-specific population size. model (1) belongs to the model class of generalized additive mixed model, see e.g. wood (2017) . the smooth functions are estimated by penalized splines, where the quadratic penalty can be comprehended as a normal prior (see e.g. wand, 2003) . the same type of prior structure holds for the region-specific random effects u r . in other words, smooth estimation and random effect estimation can be accommodated in one fitting routine, which is implemented in the r package mgcv. this package has been used to fit the model, so that no extra software implementation was necessary. this demonstrates the practicability of the method. the above model cannot be fitted directly to the available data, since we need to take the course of the disease into account. for a given registration date t, the number of deaths of patients registered as positive on that day, y t,r,g , may not yet be known, since not all patients with a fatal outcome of the disease have died yet. this requires the implementation of nowcasting. we do this on a national level, and cumulate the numbers over district/region r and gender and age groups g. this allows to drop the corresponding subscripts in the following and we simply notate the cumulated number of deaths with registered infections at day t with y t . let n t,d denote the number of deaths reported on day t + d for infections registered on day t. assuming that the true date of death is at t+d, or at least close to it, we ignore any time delays between time of death and its notification to the health authorities. we call d the duration between the registration date as a covid-19 patient and the reported day of death, where d = 1, . . . , d max . here, d max is a fixed reasonable maximum duration, which we set to 30 days (see e.g. wilson et al., 2020) . the minimum delay is one day. in nowcasting we are interested in the cumulated number of deaths for infections registered on day t, which we define as the total number of deaths with a registered infection at t is apparently unknown at time point t and becomes available only after d max days. in other words, only after d max days we know exactly how many deaths occurred due to an infection which was registered on day t. we define the partial cumulated sum of deaths as on day t = t , when the nowcasting is performed, we are faced with the following data constellation, where na stands for not (yet) available: we may consider the time span between registered infection and (reported) death as a discrete duration time taking values d = 1, . . . , d max . let d be the random duration time, which by construction is a multinomial random variable. in principle, for each death we can consider the pairs (d i , t i ) as i.i.d. and we aim to find a suitable regression model for d i given t i , including potential additional covariates x t,d . we make use of the sequential multinomial model (see agresti, 2010) and define π(d; t, x t,d ) = p (d = d|d ≤ d; t, x t,d ) let f t (d) denote the corresponding cumulated distribution function of d which relates to probabilities π() through f t (d) = p t (d ≤ d) = p(d ≤ d|d ≤ d + 1) · p (d ≤ d + 1) = (1 − π(d + 1; ·)) · (1 − π(d + 2; ·)) · . . . · (1 − π(d max ; ·)) for d = 1, . . . , d max − 1 and f t (d max ) = 1. the available data on cumulated death counts allow us to estimate the conditional probabilities π(d; ) for d = 2, . . . , d max . in fact, the sequential multinomial model allows to look at binary data such that where • s 1 (t) is an overall smooth time trend over calendar days, • s 2 (d) is a smooth duration effects, capturing the course of the disease, • x t,d are covariates which may be time and duration specific. assuming that d, the duration between a registered fatal infection and its reported death, is independent of the number of fatal covid-19 infections, we obtain the relationship note further that if we model y t with a quasi-poisson model as presented in the previous chapter, we have no available observation y t for time points t > t − d max . instead, we have observed c t,t −t , which relates to the mean of y t through (7). including therefore log f t (t − t) as additional offset in model (2), allows to fit the model as before, but with nowcasted deaths included. that means, instead of λ t,r,g as in (2), the expected death rates are now parametrized by λ t,r,g = λ t,r,g exp(log f t (t −t)), where the latter multiplicative term is included as additional offset in the model. we fit the nowcasting model (5) with parametrization (6). we include a weekday effect for the registration date of the infection with reference category "monday". the estimates of the fixed linear effects are shown in table 2 . the fitted smooth effects are shown in figure 4 , where the top panel shows the effect over calendar time, which is very weak and confirms that the course of the disease hardly varies over time. this shows that the german health care system remained stable over the considered period, and hence survival did not depend on the date on which the infection was notified. the bottom panel of figure 4 shows the course of the disease as a smooth effect over the time between registration of the infection and death. we see that the probabilities π(d; ·) decrease in d, where this effect is the strongest in the first days after registration. thus, most of the covid-19 patients with fatal infections are expected to die not long after their registration date. the effect of d becomes easier to interpret by visualizing the resulting distribution function f t (d). this is shown in figure 5 for two dates t, i.e.. april 14th and may 13th. the plot also shows how the course of the disease hardly varies over calendar time: in fact, the small differences between the two distribution functions is dominated by the weekday effect, since the red curve is related to a tuesday while the blue one is from a wednesday. in figure 3 above we have shown the nowcasting results along with uncertainty intervals shaded in grey. these were constructed using a bootstrap approach as follows. given the fitted model, we simulate n = 10 000 times from the asymptotic joint normal distribution (7), where c t −t is the observed partial cumulated sum of deaths at time point t − t. the pointwise lower and upper bounds of the 95% prediction intervals for the nowcast for y t are then given by the 2.5 and the 97.5 quantiles of the set { y (i) t , i = 1, . . . , n}, respectively. in section 3 we presented the fitted death rate, which is the convolution of a smooth spatial effect as well as region specific effects. it is of general interest to disentangle these two spatial components. this is provided by the model. we visualize the fitted global geographic trend figure 7: long term region specific level (left hand side) and short term dynamics (right hand side) of the covid-19 infections m 2 (·) for germany in figure 6 . the plot confirms that up to may 2020 the northern parts of the country are less affected by the disease in comparison to the southern states. the two plots in figure 7 map the region specific effects, i.e. the predicted long term level of a district u r0 (left hand side) and the predicted short term dynamics u r1 (right hand side). both plots uncover quite some region-specific variability. in particular, the short term dynamics captured in the right hand side plot (u r1 ) pinpoint districts with unexpectedly high nowcasted death rates in the last two weeks, after correcting for the global geographic trend and the long term effect of the district. some of the noticeable districts have already been highlighted in section 3 above, but we can detect further districts, which are less pronounced in figure 1 . for instance, steinfurt (in the north-west of north rhine-westphalia), olpe (southern north rhine-westphalia) or gotha (center of thringen) presently show a high rate of fatal infections. a large number of the registered deaths related to covid-19 stem from people in the age group 80+. locally increased numbers are often caused by an outbreak in a retirement home. such outbreaks apparently have a different effect on the spread of the disease, and the risk of an epidemic infection caused by outbreaks in this age group is limited. thus, the death rate of people in the age group 80+ could vary differently across districts when compared to regional peaks in the death rate of the rest of the population. in order to respect this, we decompose the district-specific effects u r in (2) into u 80− r = (u 80− r0 , u 80− r1 ) for the age group 80-and u 80+ r = (u 80+ r0 , u 80+ r1 ) for the age group 80+, where the age group 80-consists of the aggregated age groups 15-34, 35-59 and 60-79. we put the same prior assumption on the random effects as we did in (3), but now the variance matrix that needs to be estimated from the data has dimension 4 by 4. the fitted age group-specific random effects are shown in figure 8 , where the u 80− r are shown in the top panel and the u 80+ r in the bottom panel. most evidently, the variation of the random effects is much higher in the age group 80+ when compared to the younger age groups, as more districts occur which are coloured dark blue or dark red, respectively. when comparing the district-specific short term dynamics of the last 14 days (u r1 ) in figure 8 to those in figure 7 , we recognize that in most of the districts which recently experienced very high death intensities (with respect to the whole period of analysis), these stem from the age group 80+. as mentioned before, this can often be explained by outbreaks in retirement homes. when fitting the mortality model (1) we included the fitted nowcast model as offset parameter. this apparently neglects the estimation variability in the nowcasting model, which we explored via bootstrap as explained in section 5.3 and visualized in figure 3 . in order to also incorporate this uncertainty in the fit of the mortality model, we refitted the model using (a) the upper end and (b) the lower end of the prediction intervals shown in figure 3 . it appears that there is little (and hardly any visible) effect on the spatial components, which is therefore not shown here. but the time trend shown in figure 1 does change, which is visualized by including the two fitted functions corresponding to the 2.5% and 97.5% quantile of the offset function. we can see that the estimated uncertainty of the nowcast model mostly affects the last ten days, with a strong potential increase in the death rate mirroring a possible worst case scenario. in figure 9 we show a normal qq-plot of the pearson residuals in the nowcasting model. apart from some observations in the lower tail, the pearson residuals are distributed very closely to a standard normal distribution when considering the estimate φ = 1.766 of the dispersion parameter in the quasi-poisson model (7). overall, the model seems to fit to the available data quite well. the paper presents a model to monitor the dynamic behaviour of covid-19 infections based on death counts. it is important to highlight that the proposed model makes no use of new infection numbers, but only of observed deaths related to covid-19. this in turn means that the results are less dependent on testing strategies. the nowcasting approach enables us to estimate the number of deaths following a registered infection today, even if the fatal outcome has not occurred yet. moreover, the district level modelling uncovers hotspots, which are salient exclusively through increased death rates. a differential analysis of the number of current fatal infections on a regional level allows to draw conclusions on the current dynamics of the disease assuming a constant case fatality rate, i.e. a stable proportion of death compared to the true number of infections when adjusting for age and gender. a natural next step would now be to consider the nowcasted deaths in relation to the number of newly registered infections, which is, in contrast, highly dependent on both testing strategy and capacity. we consider this as future research, and the proposed model allows us to explore data in this direction. this might ultimately help us in shedding light on the relationship between registered and undetected infections as well as on the effectiveness of different testing strategies. there are several limitations to this study which we want to address as well. first and utmost, even though death counts are, with respect to cases counts, less dependent on testing strategies, they are not completely independent from them. this applies in particular to the handling of post-mortem tests. we therefore do not claim that our analysis of death counts is completely unaffected by testing strategies. secondly, a fundamental assumption in the model is the independence between the course of the disease and the number of infections. overall, if the local health systems have sufficient capacity and triage can be avoided, this assumption seems plausible, but it is difficult or even impossible to prove the assumption formally. finally, the nowcasting itself is not carried out on a regional level, though the model focuses on regional aspects of the pandemic. while it would be desirable to fit the nowcast model regionally, the limited amount of data simply prevents us from extending the model in this direction. analysis of ordinal categorical data real estimates of mortality following covid-19 infection countries test tactics in 'war' against covid-19 estimating the number of infections and the impact of non-pharmaceutical interventions on covid-19 in 11 european countries critical care utilization for the covid-19 outbreak in lombardy, italy: early experience and forecast during an emergency response baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 nowcasting the covid-19 pandemic in bavaria probabilistic forecasting in infectious disease epidemiology: the 13th armitage lecture bayesian nowcasting during the stec o104:h4 outbreak in germany real-time estimation of the risk of death from novel coronavirus (covid-19) infection: inference using exported cases adjustment for reporting delays and the prediction of occurred but not reported events substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data covid-19: forecasting short term hospital needs in france. medrxiv spatio-temporal analysis of epidemic phenomena using the r package surveillance quantifying bias of covid-19 prevalence and severity estimates in wuhan, china that depend on reported cases in international travelers infection fatality rate of sars-cov-2 infection in a german community with a super-spreading event smoothing and mixed models case-fatality risk estimates for covid-19 calculated by using a lag time for fatality generalized additive models: an introduction with r we want to thank maximilian weigert and andreas bender for introducing us to the art of producing geographic maps with r. moreover, we would like to thank all members of the corona data analysis group (codag) at lmu munich for fruitful discussions. key: cord-270892-ycc3csyh authors: rollinger, judith m.; schmidtke, michaela title: the human rhinovirus: human‐pathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery date: 2010-12-13 journal: med res rev doi: 10.1002/med.20176 sha: doc_id: 270892 cord_uid: ycc3csyh as the major etiological agent of the common cold, human rhinoviruses (hrv) cause millions of lost working and school days annually. moreover, clinical studies proved an association between harmless upper respiratory tract infections and more severe diseases e.g. sinusitis, asthma, and chronic obstructive pulmonary disease. both the medicinal and socio‐economic impact of hrv infections and the lack of antiviral drugs substantiate the need for intensive antiviral research. a common structural feature of the approximately 100 hrv serotypes is the icosahedrally shaped capsid formed by 60 identical copies of viral capsid proteins vp1‐4. the capsid protects the single‐stranded, positive sense rna genome of about 7,400 bases in length. both structural as well as nonstructural proteins produced during the viral life cycle have been identified as potential targets for blocking viral replication at the step of attachment, entry, uncoating, rna and protein synthesis by synthetic or natural compounds. moreover, interferon and phytoceuticals were shown to protect host cells. most of the known inhibitors of hrv replication were discovered as a result of empirical or semi‐empirical screening in cell culture. structure–activity relationship studies are used for hit optimization and lead structure discovery. the increasing structural insight and molecular understanding of viral proteins on the one hand and the advent of innovative computer‐assisted technologies on the other hand have facilitated a rationalized access for the discovery of small chemical entities with antirhinoviral (anti‐hrv) activity. this review will (i) summarize existing structural knowledge about hrv, (ii) focus on mechanisms of anti‐hrv agents from synthetic and natural origin, and (iii) demonstrate strategies for efficient lead structure discovery. © 2009 wiley periodicals, inc. med res rev, 31, no. 1, 42–92, 2010 human rhinoviruses (hrv) are the major cause of upper respiratory tract symptoms, the socalled common colds in humans. their name reflects the primary site of infection. because hrv are nonenveloped, icosahedral viruses of small size with a diameter of about 30 nm ( pico 5 small in latin) that consist of an rna genome, they were assigned to the family picornaviridae. currently, this virus family of the order picornavirales comprises the eight genera enterovirus, hepatovirus, cardiovirus, kobuvirus, teschovirus, erbovirus, aphthovirus, and parechovirus with 22 species and a multitude of serotypes. 1 because of high similarity in genome sequence and genome organization (fig. 1) , the former genera rhinovirus and enterovirus have been combined recently, keeping the existing name enterovirus (www.picornastudygroup. com/taxa/species/species.htm). an overview on the current taxonomy of picornaviruses pathogenic for humans as well as on newly proposed species of hrv is given in table i . at present the genus enterovirus includes four approved human enterovirus (hev) species (hev-a, -b, -c, and -d) and two approved hrv species (hrv-a and -b) (www. picornastudygroup.com/taxa/species/-species.htm). since 2007, the global distribution of highly divergent hrv strains was reported. [2] [3] [4] [5] based on the results of sequence, genomic, and phylogenetic analyses, it was proposed that these strains represent a new hrv species, hrv-c. [2] [3] [4] 6 in 2009, a further proposal concerning a new potential hrv-d species was published after sequencing and analysis of all known hrv genomes. 7 the approved and newly proposed species of the genus enterovirus share z70% homology (average amino acid identity) in the precapsid protein p1 as well as in 2c and 3cd. 3, [7] [8] [9] [10] different antigenic properties provide the basis for a further division of species into serotypes (table i) . about 100 rhinovirus serotypes are currently known. according to the currently approved taxonomy, most of them (75 and hrv hanks) belong to hrv-a and 25 of them to hrv-b. 11, 12 the genome of all known hrv-a and -b serotypes as well as of several field isolates of hrv-a, -b, and -c has been sequenced completely. 3, 7, [13] [14] [15] [16] [17] [18] [19] [20] viruses classified as hrv-c could not be grown in cell culture until now. phylogenetic analyses have been performed with partial sequences, 12, 21 as well as with the whole genome. 3, 7 the most recent and comprehensive analysis of all known hrv genomes revealed that (i) hrv-a and hrv-c share a common ancestor, which is a sister group to hrv-b, (ii) hrv-c represents a third species, and (iii) a basal divergence within hrv-a of three distinct strains that led to the proposal of a fourth species hrv-d. hrv-a and -b most often induce a mild, usually self-limited upper respiratory illness in humans characterized by nasal stuffiness and discharge, sneezing, sore throat, and cough. the conventional term is common cold. the common cold is a heterogeneous group of diseases caused by numerous viruses that belong to several different families e.g. rhinoviruses, coronaviruses, enteroviruses, and adenoviruses. 22 but, hrv represent the most common etiological agent worldwide. a large number of distinct strains circulate each year. 23 moreover, in a family or even in a single specimen, multiple hrv serotypes were detected simultaneously. [24] [25] [26] by using rt-pcr and culture, it was shown that hrv induce 22-50% of upper respiratory tract infections in adults as well as children. [27] [28] [29] [30] [31] higher incidence has been described from september to november, [32] [33] [34] and from april to may. 33, 34 in some years and perhaps some geographical areas, spring was a more important time for rhinovirus transmission. 33, 34 although overall rates of respiratory illness are lower in summer, rhinoviruses are the most frequently isolated at this time of year. 34 the incidence is inversely proportional to age. 25, 35 by age 2 years, 91% of the children have antibodies against rhinoviruses. 36 in addition to common cold, hrv are also involved in acute otitis media in children. 36, 37 moreover, data supporting a causative association with more severe lower respiratory tract infections of infants, elderly persons, and immunocompromised patients have been accumulated. [38] [39] [40] [41] [42] [43] [44] [45] studies of childhood and adult asthma have shown that hrv infections can also trigger exacerbations in patients with asthma, [46] [47] [48] [49] chronic obstructive pulmonary disease, [50] [51] [52] [53] [54] [55] and cystic fibrosis. [56] [57] [58] the recently discovered novel rhinovirus genotype hrv-c was associated with community outbreaks of influenza-like, acute upper respiratory infections and severe low respiratory tract infections of infants e.g. febrile wheeze, bronchiolytis, and asthma exacerbations, which peaked in fall and winter. [2] [3] [4] [5] [6] 26, 59, 60 in addition, the presence of hrv-c in the middle ear in patients with acute otitis media was demonstrated. 37 hrv spread occurs by means of virus-contaminated respiratory secretions that contain a high virus concentration. [61] [62] [63] besides direct hand-to-hand transmission, small-and largeparticle aerosol transmission of rhinoviruses has been shown. 61, 64, 65 children are important ''vectors'' for hrv transmission to family members. 25 moreover, studies with natural hrv-infected adults provided evidence that daily activities of infected people can lead to contamination of environmental surfaces with hrv e.g. light switches, telephone dial buttons and handsets, and virus transfer to fingers of healthy individuals for infection. 63, 66 because viral contamination of the hands plays an important role in transmission of hrv from person-to-person, interruption of this step of virus transmission presents a potential target for intervention. this was experimentally proved by treatment of hands by iodine 67, 68 or salicylic and pyroglutamic acid. 69 observations from experimentally induced infections in normal adult volunteers helped to understand the pathogenesis of hrv infections. [70] [71] [72] [73] [74] [75] the 50% human infectious dose of rhinovirus is low and the infection rate between 70 and 80%. after the deposition of hrv on nasal or conjunctival mucosa, viruses are transported to the posterior nasopharynx by mucociliary action of epithelial cells. specific receptors on epithelial cells in the adenoid area are used for binding and entry. already 8-10 hr after intranasal inoculation, infectious virus can be detected. 76 virus shedding peaks on the second day after infection and decreases rapidly thereafter. 75 but, small amounts of viruses were discovered in nasal secretions for up to 3 weeks after infection. virus and/or viral rna were demonstrated in the upper 70 as well as lower respiratory tract. 45, 72, 77, 78 using in situ hybridization, arruda et al. (1995) detected viral rna in a low number of ciliated cells in nasal biopsies. in the nasopharynx, a small portion of virus-positive ciliated as well as nonciliated cells was positive for viral rna. in 2000 papadopoulos et al. provided evidence that hrv may also lytically infect human bronchial epithelial cells in cell culture as well as in experimentally infected volunteers and induce the production of interleukin-6, -8, and -16. in agreement with these results, hrv rna was detected in 24-45% of children and 10-18% of adults with pneumonia. [79] [80] [81] [82] taken together, the results of natural cold studies as well as of experimental infection in human volunteers clearly demonstrate that hrv are able to replicate in the upper as well as in the lower airways. hrv infection triggers vasodilation and increased vascular permeability in the nasal mucosa, leading to nasal obstruction and rhinorrhoea. the mechanism is still incompletely understood because no histopathological changes were observed in nasal biopsy specimens from infected persons. 75 this led to the suggestion that clinical symptoms are primarily caused by the inflammatory response of the host to the virus infection and not by the cytopathic effect (cpe) of hrv. results of immunological investigations suggest a modest correlation between the concentrations of il-6 and il-8 in nasal secretions and the severity of symptoms in upper and lower hrv-induced respiratory tract disease. 78, 83 on day 2-4 after virus challenge, il-6 and il-8 concentrations were significantly greater in nasal secretions from experimentally infected symptomatic subjects than in those from infected asymptomatic or sham-challenged subjects. il-8 has been proposed as a mediator of neutrophile infiltrations that are observed during symptomatic infections. in experimental rhinovirus infection the onset of symptoms e.g. nasal stuffiness and discharge, sneezing, and cough was observed 10-12 hr after intranasal inoculation of the virus. 76 in contrast to rhinovirus infections in adults, fever is found in 15% of children with upper respiratory tract infections. 84 other symptoms in children and adults may be hoarseness, headache, malaise, and lethargy. sometimes viral infection is accompanied by bacterial complication, leading for instance to acute otitis media in about 20% of infected children, sinusitis, and pneumonia. [79] [80] [81] 85, 86 experimental infection was also used to study the causation between rhinovirus infection and asthma as well as copd exacerbations. 78, [87] [88] [89] [90] [91] [92] [93] it was shown that hrv infection enhances airway reactivity and predisposes allergic patients to develop late asthmatic reactions. 88, 91 rhinoviral colds were associated with an increase in histamine responsiveness that was accompanied by a bronchial mucosal lymphocytic and eosinophilic infiltrate. 89 in a recent study, an increased hrv-induced clinical illness severity in asthmatic compared with normal subjects was demonstrated. 93 strong relationships were shown between virus load, lower airway virus-induced inflammation, and asthma exacerbation severity. the results of this study also indicated that augmented th2 or impaired th1 or il-10 immunity are likely important mechanisms. mallia et al. provided evidence that low dose experimental rhinovirus infection in patients with copd induces symptoms and lung function changes typical of an acute exacerbation of copd. 92 viral replication and increased pro-inflammatory cytokine response were associated with symptomatic colds, increases in lower respiratory tract symptoms and reductions in forced expiratory volume in 1s or peak expiratory flow rate. the epidemiological data and pathology of hrv infections explain their high medical and socio-economic impact. millions of children and adults are taken ill with common cold every year, need medical consultations, are unable to attend school and go to work. 94, 95 direct costs include hospitalization, medical fees, and symptomatic treatment. moreover, exacerbations are the major cause of asthma and copd morbidity, mortality, and health care costs associated with these diseases. 92, 93 to date, specific drugs that prevent or reduce rhinovirus infection are not available. common cold can be treated only symptomatically with analgesics, decongestants, antihistamines, or antitussives and antibiotics are often wrongly prescribed. 96, 97 because of the large number of circulating hrv serotypes, treatment with specific antiviral drugs is considered to be more striking than vaccination. therefore, the search for new highly active synthetic and/or natural anti-hrv compounds is absolutely essential and represents an important area of antiviral research. such an anti-hrv drug would have to be (i) with broad spectrum activity because of the high number of hrv serotypes, (ii) administered very early in infection to demonstrate a good antiviral effect because of the fast infection kinetics, (iii) very safe because of the broad application by millions of people, and (iv) directed against a highly conserved target with low risk of resistance development. due to the very high error rates and the lack of proofreading ability in rna polymerases of picornaviruses, 98 naturally drug-resistant variants may exist in virus populations or resistant viruses can emerge under treatment. as with hiv, another highly variable rna virus, the risk of resistance development and/or selection of resistant virus variants could be minimized by applying combination of drugs directed against different targets. because clinical symptoms are suggested to be primarily caused by the inflammatory response of the host to the virus infection mediated by specific cytokines, a further advantage of drug combinations could be an additional immune-suppressive activity. the knowledge of structural components, nonstructural proteins that are necessary for viral multiplication, and stages of the viral life cycle is an essential precondition for the development of measures to prevent and treat hrv infection. the structure of hrv particles is well known. infectious virions consist of an icosahedral protein shell (capsid) that surrounds and protects the genome, a single positive-stranded rna molecule of approximately 7,400 nucleotides. the organization of the enterovirus genome is shown in figure 1 . the viral genomic rna is infectious and encodes a single, long, open reading frame flanked by untranslated regions (utr) at the 5 0 and 3 0 end. a small viral protein (vpg) is covalently linked to the 5 0 end. the 3 0 end is polyadenylated like cellular messenger rnas. structural components within these utrs e.g. the cloverleaf and the internal ribosome entry site (ires) of the 5 0 utr play an important role in rna replication as well as protein synthesis. 98 the nucleotide sequence of some regions within these structures is highly conserved among enteroviruses. their blockade could significantly inhibit viral replication. the molecular structure of hrv-1a, hrv-2, hrv-3, hrv-14, and hrv-16 was determined by x-ray crystallography. [99] [100] [101] [102] [103] [104] [105] the results show that the viral capsid is composed of 60 protomers of each of the three outer structural proteins vp1, vp2, and vp3 and of vp4 in the interior. a star-shaped plateau at the fivefold axis of symmetry, surrounded by a deep depression (canyon) and another smaller depression at the threefold axis were detected. moreover, a hydrophobic pocket was found beneath the canyon floor. with exception of hrv-14 and hrv-3, this pocket is occupied by a fatty acid, the so-called pocket factor. these host cell molecules have been suggested to play an important role in the viral life cycle by providing transient stability to the capsid during its movement from one host cell to another. 102 the outer surface of virions contains neutralization antigenic as well as host cell binding sites. the latter allow the virus to attach to molecules of the host cell membrane (adsorption), the receptors, and to start their life cycle. [106] [107] [108] based on their receptor use, two groups of hrv can be distinguished. the majority of hrv serotypes, the major group uses intercellular adhesion molecule-1 (icam-1) as their receptor. 109 the 12 viruses belonging to the minor group attach to low density lipoprotein (ldl) receptor, very-ldl (vldl) receptor, and ldlr-related protein on the cells whereat multiple receptors are involved. [110] [111] [112] hrv of the major group apply the canyon as attachment site for binding to icam-1. 113 in contrast, ldl receptors of minor group viruses bind near the tip of the five-fold vertex. 114 hrv-87 has been shown to utilize a sialylated glycoprotein as a cellular receptor. 115 furthermore, a hrv-89 variant as well as wild-type hrv-54 can use heparan sulphate proteoglycans for cell attachment in addition to icam-1. 116, 117 the interaction of rhinoviruses with their receptors leads to virus concentration on the cell surface. it induces the release of the pocket factor and conformational changes in the capsid and mediates viral entry via endocytosis. [118] [119] [120] [121] whereas icam-1 binding directly causes uncoating, 122, 123 release of the rna genome from the capsid of ldl-bound minor group rhinoviruses is triggered by acidification of the endosomal, ph-dependent pathway. [124] [125] [126] this detailed knowledge of the capsid structure and function as well as of the virus-receptor interaction offers a good possibility to develop antiviral drugs that interfere with the first steps of the viral life cycle, adsorption as well as uncoating. after uncoating, rhinovirus proteins are synthesized by the translation of a single, open reading frame using cellular ribosomes. the resulting polyprotein of approximately 250 kd is cleaved by viral proteases 2a pro and 3c pro into 11 final products (4 structural and 7 nonstrutural proteins) immediately after translation. 127, 128 at first, both proteinases release themselves from the polyprotein by selfcleavage. the primary cleavage of the viral polyprotein between p1 and p2 is mediated by 2a pro . thereafter, 3cd pro is released from the p3 precursor by autocatalytic cleavage. next, 3c pro and its precursor 3cd pro process proteins of the p1 (capsid proteins), p2, and p3 (nonstructural proteins) region. interestingly, 2a pro cleaves also the eif4gi/ii component of the translation initiation factor eif4f necessary for host cell protein synthesis, [129] [130] [131] and 3c pro and/or 3cd pro the rna polymerase transcription factors tfiid, tfiic, sl-1, and ubf. 98 therefore, effective inhibition of 2a pro and 3c pro would not only inhibit virus replication but could also prevent the shutoff of cellular protein and rna synthesis. moreover, the active site of proteinases is highly conserved among enterovirus serotypes. 132 this high conservation in conjunction with their important role in virus multiplication predestines these enzymes as targets for antiviral therapy. the viral rna polymerase 3d (3d pol ) represents another very important nonstructural protein of hrv. it forms a complex with both cellular and viral proteins, the rna replication complex. 98 this enzyme synthesizes viral minus-strand rna and uses it as template strand for the synthesis of genomic viral rna. vpg (3b) is the primer for negative-as well positive-strand rna synthesis. negative-strand, but not positive-strand rna synthesis, is stimulated by 2a pro . further viral accessory proteins include 2b, 2c, 2bc, and 3ab. besides 3d pol these proteins can play an important role in inhibition of viral rna synthesis by antiviral compounds. in summary, the knowledge of the structure of the viral capsid, proteases, and polymerase and their important function in the viral life cycle predestine these proteins as potential anti-hrv targets. hrv grow in several human and some primate cells expressing the minor group ldl receptors and/or the major group receptor icam-1. human cells susceptible to hrv infections include embryonic kidney, amnion, diploid fibroblasts from embryonic lung, tonsil, liver, intestine, and skin, adult fibroblast lines from aorta and gingival and the kb, hep-2, and hela continuous cell lines. 133 but, the susceptibility of hela cells and human fibroblasts to virus infection may vary. 32 hrv multiplication also occurs in primary human airway fibroblasts 134 and differentiated bronchial epithelium. 77, 78, 135 the proportion of infectible epithelial cells was shown to be between 3 and 10%. 77, 135 but, enhanced levels of viral production were detected in poorly differentiated in comparison to differentiated epithelial cells. 136 the degree of viral infection correlated with il-6 and il-8 induction in these cells virus growth causes a typical cpe characterized by ballooning, refractiveness, granularity, and shrinkage of infected cells. the hrv-induced cpe, infectious virus titers, viral protein expression, and rna synthesis can be chosen as parameters to evaluate the anti-hrv activity of compounds in cell-culture based assays. there are several methods for antiviral screening against hrv. the plaque reduction assay has been traditionally performed and accepted as the ''gold standard'' in antiviral testing. [137] [138] [139] however, this test is laborious, time consuming, and the evaluation is subjective. therefore, it is not suited for the routine antiviral testing. it was more and more replaced by methods based on quantification of protection from virus-induced cpe after drug treatment. so, the cpe in sample-treated and untreated cells has been compared by light microscopy. 137, 140, 141 but this evaluation is also subjective. another more objective approach is the spectrophotometric quantification of cpe results in neutral red or crystal violet uptake assays, 12,141-144 and the tetrazolium dye reduction method. 132, 145 it allows an excellent and rapid antiviral screening of large numbers of compounds using small amounts of extracts, natural, or synthetic compounds. active samples can be scheduled for additional testing using other assays e.g. virus yield or plaque reduction assays, and for studies on the mechanism of action. the activity of potential antiviral drugs has to be approved in vivo. because of the high degree of species-specific variations in icam-1 preventing infection by major group hrv, practical animal models have been absent for a long time. chimpanzees were infected with several hrv serotypes but without developing clinical signs. 133 hrv do not induce infection in rabbits, guinea pigs, and weanling mice injected with hrv by different routes. one minor group hrv, serotype 2, was adapted to grow in mouse fibroblasts and used in a mouse model of rhinovirus infection in which growth could be demonstrated. 146 based on the fact that the ldl receptor family is highly conserved between human and mouse, newcomb et al. examined whether hrv-1b, another minor group virus, may infect mouse airways in 6-to 8-week-old female c57bl/6 mice. 147 the authors demonstrated that this hrv serotype replicates and induces airway inflammation in vivo. these results strongly correspond to those of bartlett et al. who established three novel mouse models of rhinovirus infection in balb7c mice. 148 in the first model, 6-week-old balb/c mice were infected with hrv-1b. in the second model, transgenic balb/c mice, expressing a mouse-human icam-1 chimera, were inoculated with the major group hrv-16. rhinovirus-induced exacerbation of allergic airway inflammation is mimicked in the third model. due to the lack of a small-animal model for hrv infection until 2008, the experimental human challenge model has to be used to approve effects of potential antiviral drugs under controlled conditions in preclinical studies. volunteers were experimentally inoculated with various serotypes e.g. hrv-4, hrv-9, hrv23, hrv-29, and hrv-39 to examine the efficacy of potential antiviral drugs under standardized conditions. [149] [150] [151] [152] [153] [154] examples and results of these studies with capsid-binders, protease, and rna synthesis inhibitors, as well as interferons are described in the following sections. antiviral agents that inhibit virus attachment, capsid uncoating, protein and rna synthesis of picornaviruses are the best studied, 95, [155] [156] [157] and will be in the focus of this section. inhibition of virus attachment and/or uncoating interrupts the viral life cycle at its beginning and prevents hrv infection. options to prevent these early steps of the viral life cycle include (i) virus neutralization by hrv-specific antibodies, (ii) receptor blockade by antibodies directed against the cellular receptors icam-1 or ldl, (iii) by soluble receptor molecules, or (iv) by compounds interacting with the viral capsid. because of the high number of serotypes circulating often in parallel, application of hrv-specific antibodies is thought to be no promising approach for prevention or therapy of rhinovirus infection. in contrast, antibodies directed against the cellular receptor or soluble receptor molecules of major or minor group hrv could inhibit 90 and 10% of hrv serotypes, respectively. therefore, the strategy to prevent virus-receptor interaction by receptor antibodies or soluble receptor molecules has been extensively evaluated in vitro as well as in vivo. the antiviral activity of icam-and ldl-specific antibodies was confirmed in cell culture. 158, 159 furthermore, the prophylactic effectiveness and safety of intranasally administered rhinovirus murine icam-1 antibody was assessed in two double-blind, placebo-controlled, randomized studies of volunteers experimentally inoculated with hrv-39. 160 in the result, no toxicity related to antibody application was recognized. the higher dosage of 1 mg/subject of rhinovirus murine receptor antibody did not reduce overall infection or illness rates, but was associated with a 1-2 day delay in the onset of virus shedding and cold symptoms. viral titers and nasal symptoms were significantly reduced on the second day after challenge. in summary, the monoclonal antibody to the cellular icam-1 was demonstrated to be not effective enough. a new strategy was the creation of multivalent fab fusion proteins against icam-1. a new molecule, named cfy196 demonstrated a better avidity and in vitro potency against hrv over conventional mabs. 161 cfy196 is under development as nasal spray with the name of coldsol. antagonism of virus-receptor interaction was considered as another promising way to prevent hrv attachment to host cells. soluble forms of fully or truncated icam-1, [162] [163] [164] and ldl or vldl-receptor concatemers [165] [166] [167] exhibited antiviral activity against major and minor group hrv, respectively, in cell culture. soluble forms of icam-1 compete with receptor binding sites on the virus capsid, hinder an early infection event such as entry or uncoating, or directly inactivate hrv due to the formation of empty capsids. 163, [168] [169] [170] a soluble ldl receptor fragment neutralized viral infectivity by aggregation. 171 concatemers of the third ligand binding module of the vldl-receptor did not lead to viral aggregation but blocked the receptor binding sites and possibly inhibited viral uncoating by cross-linking the viral capsid subunits via multi-module binding. 166 the antiviral activity of a truncated, soluble form of icam-1 was proved in hrv-16 infected chimpanzee. 172 in randomized, double-blind, placebo-controlled trials, the safety and efficacy of intranasal administration of tremacamra, a soluble icam-1 in experimental hrv-39-induced colds in humans, was shown. 173 no further development was reported for these agents. 95 a further option to prevent virus attachment was described for low-molecular-weight compounds, the so-called capsid-binding agents, which enter the small hydrophobic pocket within viral capsid protein 1 beneath the icam-binding canyon of hrv. 174, 175 zhang et al. showed that drug may integrate into mature viruses by diffusion as well as into progeny viruses during assembly. 176 when hrv-14 and hrv-16 were grown in the presence of pleconaril, a higher occupancy occurred than when the drug was introduced into the alreadyassembled viruses. in doing so, capsid-binders induce conformational changes of the canyon of hrv-3 and hrv-14, hinder virus-receptor interactions, and prevent attachment to host cells. 105, 175, [177] [178] [179] in addition, uncoating of both hrv serotypes was shown to be inhibited as a result of a potential loss of flexibility of the viral capsid after drug binding. in contrast to hrv-3 and hrv-14, capsid-binding compounds did not prevent attachment of hrv-1a. results from x-ray studies showed that drug binding into the hydrophobic pocket of hrv-1a replaces the pocket factor but induces only very small conformational changes. 180 therefore, kim et al. suggested that the observed conformational changes are too small to affect receptor binding. but, capsid-binding compounds prevented attachment of hrv-16 possessing a pocket factor like hrv-1a without distinct deformation of the pocket. 102, 176, 181 further results from comparative antiviral studies with different capsid-binding compounds and hrv, representative for the major and minor group, did not reveal a correlation between inhibition of adsorption and receptor grouping or antiviral grouping. 182 the reasons for the difference in the mode of action of capsid-binding compounds related to attachment inhibition are not fully understood until now. taken together, inhibition of rna uncoating was found for all investigated serotypes after drug binding independent of receptor grouping whereas prevention of virus attachment was found to be an additional mode of action for individual viruses and/or drugs. till now, various potent compounds belonging to diverse chemical classes have been described as uncoating inhibitors. just to give an impression of diversity, the structures of disoxaril and pleconaril, 12,183-185 pirodavir and the oxime ether, 14, 141, 142, 186, 187 the isoxazole derivate compound, 19, 143, 188 the imidazole derivative sch 38057, [189] [190] [191] the chalcone ro 09-0881, 192, 193 4 0 ,6 dichloroflavan and isoflavan, 137, 194 the pyridine derivative mdl 20, 957, 195, 196 and the phenoxybenzene mdl-860 197, 198 that exhibit a potent anti-hrv activity (table ii) are shown as examples in figure 2 . they inhibit most of hrv serotypes and a couple of them also affect enteroviruses, however, with varying susceptibility. based on variability of susceptibility to capsid-binders of different length, hrv serotypes were classified into two different groups, a and b. 140 several of the given examples of compounds were also clinically tested. studying the development of clinically effective capsid-binders, the long road to the discovery of a clinically effective anti-hrv drug becomes apparent. one well-described example represents the discovery and optimization of capsid-binders from sterling winthrop pharmaceutical group, the so-called win compounds. 174 first inhibitors originated from juvenile hormone mimetics that demonstrated some activity against hrv-1a. determination of the x-ray structure of hrv-14 helped to understand the compounds' binding sites at the virus capsid. 103 results from subsequent x-ray studies of hrv-win compound complexes revealed the location and nature of binding sites and provided information concerning interactions within these sites. 175, 179, 180 this knowledge was used for optimization and design of new compounds. 199 optimized win compounds, for example disoxaril and pleconaril ( fig. 2) , consist of a methylisoxazol ring, a substituted phenoxy group, and a five-membered heteroatom ring and inhibit a broad spectrum of rhinoviruses and enteroviruses (table ii) . 12, [183] [184] [185] in 1985, the first broad-spectrum win compound disoxaril (win 51711) was tested in clinical trials. 139 the development of crystallurea in human volunteers treated with high doses as well as its low bioavailability (15%) prohibited subsequent development. thereafter, results from sar and qsar analysis were used to further enhance the potency and spectrum of activity. in 1992, another compound, win 54954, was clinically tested. it was not effective in humans infected with hrv-23 and hrv-29. 153 moreover, it was rapidly metabolized and induced a reversible hepatitis. consequently, the further clinical development was stopped. the better understanding of pharmacokinetic properties of capsid binders and synthetic chemistry efforts led to the discovery of pleconaril, an orally bioavailable, welltolerated capsid-binder that inhibits most rhinovirus as well as various enterovirus serotypes. 12, [183] [184] [185] in 2000, schiff et al. published the efficacy of pleconaril in an experimentally induced coxsackievirus a infection in humans. 200 in phase ii placebo-controlled, natural cold trials, the drug produced a moderate reduction of 1-1.5 day in the medium time to elevation of illness compared with placebo. 201 these results were confirmed in two subsequent pivotal studies. 202 besides the moderate clinical efficacy, these studies revealed that 13% of baseline isolates were not susceptible to pleconaril and 11% developed reduced susceptibility (defined as 10-fold increase in baseline value). in a subsequent study the relationship of pleconaril susceptibility and clinical outcomes in the treatment of common cold caused by rhinoviruses was demonstrated. 203 based on drug interaction, marginal treatment effect, and possibility of transmission of resistant viruses, the fda did not approve the applied oral administration of pleconaril for the treatment of common cold. the molecular mechanism of drug interaction of orally given pleconaril was shown to be based on hepatic cytochrome p450 3a activation. 204 to reduce adverse effects, shering-plough under license of viropharma completed a phase ii clinical trial with an intranasal formulation of pleconaril for the potential treatment of common cold in high-risk populations in 2007. the results were not published until now. pyridazine analogues developed by janssen research foundation represent another example for the long road to discovery of a clinically effective capsid-binder. 186 in 1992, the broad-spectrum activity of pirodavir ( fig. 2 ; table ii ) against rhinoviruses was published. 187 in the same year the results of a randomized, double-blind, placebo-controlled trail to assess the therapeutic efficacy of intranasal pirodavir in natural common colds were described. 205 possibly as a result of poor water solubility and rapid hydrolysis of pirodavir, no clinical benefit was found. the problem of ester hydrolysis was resolved by the development of oxime ether analogues of pirodavir by biota. an example is shown in figure 2 . like pirodavir these new analogues are potent inhibitors of rhinoviruses. 142 an advantage over pirodavir is their improved bioavailability. bta-798, an antiviral analogue with long half-life and good oral bioavailability, was scheduled to a phase ii clinical trial in 2008. the results have not yet been published. in summary, despite extensive research leading to the discovery of potent anti-hrv capsid-binders, no agent has been approved for prevention and/or therapy of rhinovirus-induced diseases so far. nearly, the same conclusion has to be drawn for protease inhibitors. because of their pivotal role for viral polyprotein processing and the high conservation of critical amino acids, 132 2a pro as well as 3c pro represent potential anti-hrv targets. results from cell culture-based assays provided evidence that inhibition of hrv replication is in principle possible. for example, processing of the hrv-2 polyprotein was prevented by pyrrolidine dithiocarbamate treatment in virus-infected hela cells. 206 in contrast to other enteroviruses, [207] [208] [209] [210] pretreatment of cell monolayers with different nitric oxide donors leading to s-nitrosylation of 2a pro and 3c pro had neither an effect on virus replication nor on hrv-induced il-8 elaboration. 211 the proteolytic activity of 2a pro of hrv-14 was specifically inhibited by two elastase-specific inhibitors, 212 and an antiviral peptide representing a derivative of the caspase inhibitor zvad.fmk. 213, 214 homophthalimides, e.g. ly353349 ( fig. 3 ; table ii) , were described as inhibitors of 2a pro as well as 3c pro . 215 in contrast to protease 3c, no structure-activity relationship studies have been reported for hrv 2a protease. moreover, protease 2a accomplishes only one cleavage in hrv polyprotein, while protease 3c performs all other cleavages. after elucidation of the crystal structure of 3c pro , 216 computer modeling of structural features of protease inhibitors became possible. 217 furthermore, structure-based design was used to develop mechanism-based inhibitors of the 3c protease with potent antiviral activity against multiple hrv serotypes. 218, 219 highly active compounds incorporate various michael acceptor moieties, irreversibly bind to 3c pro , and exhibit anti-hrv-14 activity in hela cells. 220, 221 structure-activity studies were performed to optimize protease inhibitors. 220, [222] [223] [224] [225] [226] these efforts resulted in the identification of a highly active anti-hrv compound, ag7088 (rupintrivir; fig. 3 ; table ii ) that entered clinical trials. in cell culture, ag7088 inhibited a broad spectrum of laboratory hrv as well as clinical isolates. 132, 183, 227 in a single-cycle, time-of-addition assay it demonstrated antiviral activity when added up to 6 hr after infection. 227 inhibition of hrv replication strongly correlated with reduction in the level of il-6 and il-8 release into cell supernatant, leading to the suggestion that this agent may not only block virus replication but also diminish symptoms. 228 the pharmacokinetics and safety of rupintrivir were proved in two double-blind, randomized, placebo-controlled studies. 229 intranasal rupintrivir, administered as single doses of 4 and 8 mg or every 3 hr, six times per day, for 7 days, was safe and well tolerated. three double blind, placebo-controlled clinical trials were conducted to assess rupintrivir nasal spray (2% solution) for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers. 230 rupintrivir prophylaxis reduced the proportion of subjects with positive viral culture by 26% and viral titers but did not decrease the frequency of colds. drug treatment led to the reduction of the mean total daily symptom score by 33%. subjects receiving rupintrivir also demonstrated significantly lower viral titers and rna levels than placebo-treated subjects on days 2, 3, and 5 and on days 2 and 3, respectively. there was no influence on the proportion of subjects with positive viral culture and the frequency of colds. clinical development was terminated because rupintrivir did not act in a subsequent natural infection study in patients. 231 in parallel research efforts, an orally bioavailable inhibitor of table ii) , was discovered. 225, 231 like rupintrivir, this compound is an irreversible inhibitor incorporating a michael acceptor moiety that forms a covalent bond with the 3c protease active site cysteine. it demonstrated an antiviral activity against all hrv and related picornaviruses tested. 231 in a phase 1 clinical study, compound 1 was shown to be safe and well tolerated. according to a publication of patick, no further clinical development was planned for this compound. 95 the blocking of viral rna synthesis during replication represents another site for chemotherapeutic interdiction. it was shown that, rhinoviral rna can be targeted in a sequence-specific manner by deoxyribozymes, 232 morpholino oligomers, 233 and small interfering ribonucleic acids. 234 the efficacy of the latter two approaches was confirmed in cell culture. in addition, 2-furylmercury chloride ( fig. 4 ; table ii ), 235 table iii) , 236 and pyrrolidine dithiocarbamate (fig. 4 ) interfered with rhinoviral rna synthesis and inhibited hrv replication in cell culture-based assays. 206, 237 the nucleoside analog ribavirin ( fig. 4 ; table ii ) that inhibits a broad spectrum of rna as well as dna viruses acts also against hrv-2 in hela cells. 238, 239 the cellular inosine monophosphate dehydrogenase that controls de novo synthesis of purine nucleosides represents the principal target in the mode of action of ribavirin. 240 moreover, when ribavirin is incorporated into picornavirus rna, it pairs equally well with either uracil or cytosine inducing mutations that can be lethal to rna viruses. 241 further identified mechanisms of action for ribavirin include inhibition of genomic rna capping, enhancement of host t-cellmediated immunity against viral infections through helping to switch the host t-cell phenotype from type 2 to type 1. 242 another compound with potent anti-hrv activity in vitro is enviroxime ( fig. 4 ; table ii ), a benzimidazole derivative. 243, 244 it inhibits viral plus strand rna synthesis. 245 in particular the 3a protein, which is involved in the initiation of plus strand rna synthesis, was implicated as likely target of drug activity. 246, 247 however, results from another study suggest that enviroxime targets a complex of proteins and/or cellular factors and that the exact mechanism remains to be studied. 248 although there was a statistically significant reduction in clinical score in a prophylactic study with hrv-9-infected volunteers, 152 enviroxime failed in experimentally induced hrv-4 and hrv-39 infection, 149, 151 and in clinical studies, 248-250 because of poor bioavailability and side effects. in an attempt to overcome the marked hydrophobicity, water insolubility, and toxicity, wyde et al. incorporated enviroxime into liposomes and then tested the anti-hrv activity and toxicity of the liposome-incorporated enviroxime in cell culture. 251 the liposome preparation of enviroxime inhibited hrv-1a and hrv13 as effective as the parent compound and was 10-to z50-fold less toxic. in contrast to free enviroxime, the liposome preparation was readily and successfully delivered by small-particle aerosol to the upper and lower respiratory tract of mice. in another attempt to overcome the disadvantages of enviroxime, several benzimidazole as well as nonbenzimidazole analogs were synthesized and studied. [252] [253] [254] [255] even though some compounds were better bioavailable and could be administered orally, 254 none of these compounds was tested in clinical studies. besides virus-specific targets, cellular inhibitors like interferons may represent a therapeutical approach. among other activities, interferons exhibit antiviral activity. the advantages of interferon application include the broad spectrum of activity and low risk of resistance development. human leukocyte and fibroblast as well as recombinant human a interferons prevent the hrv-induced cpe in cell culture whereas a variation in sensitivity was observed. [256] [257] [258] intranasally applied recombinant interferon a and interferon b have been shown to be effective in humans when provided prophylactically both in experimental and natural rhinovirus colds. [259] [260] [261] [262] [263] [264] significant reductions in illness frequency, mean symptom score, nasal secretion weights, and frequency of virus isolation were observed. in contrast, recombinant interferon g did not prevent hrv infection or illness and may enhance the symptoms. 265 little to no therapeutic effect was found in patients with common cold after interferon treatment. 150, 266 moreover, blood-tinged mucus and nasal bleeding were described as side effects. 263, 267 combining interferons with dichloroflavan, enviroxime, chalcone ro-09-0410 produced synergistic increases in antiviral activity in vitro against hrv-2 and hrv-9. 268 an attempt to demonstrate synergy between the anti-hrv effect of recombinant human rhuifn a and enviroxime in hrv-9 and hrv-14-infected volunteers failed. 269 according to the authors, the main reason for this failure may be the rapid removal of enviroxime from the nose when given intranasally. a. impact of natural products nature provides an astonishing pool of secondary metabolites biosynthesized from living organisms such as plants, fungi, protozoan, insects, and other animal sources. in contrast to synthetic compounds, natural products are characterized by an overwhelming chemical diversity. previously, the chemical diversity space between these two groups was evaluated with respect to drug substances by feher and schmidt. 270 it is shown that combinatorial compounds densely populate a small area, whereas natural products cover a wider range quite similar to the chemical space occupied by drug substances. the authors accordingly suggest that combinatorial libraries that mimic the distribution properties of natural products might be more biologically relevant. one may assume that secondary metabolites evolved as reaction to their target receptors related to defence, protection, attraction, and signalling. these adaptation processes have enriched not only the metabolites' structural diversity but have also optimized drug-like metabolic traits likely to have favorable pharmacokinetic properties. 271, 272 it is this evolutionary concept that gives the pool of natural products the greatest source of scaffold diversity with molecules of biological relevance. newman and cragg analyzed the number of drugs approved between 1981 and 2006 and circumstantiated that especially the anti-infective area is strongly dependent on natural products and structures derived from natural scaffolds. 273 the anti-infectives including the antiviral vaccines are with 22.8% or 230 launched drugs by far the major category with only about 30% being synthetic in origin. from 1981 to 2006, 78 vaccines and antiviral drugs have been approved. excluding the high number of vaccines (25) and biologicals (12) , most of the 41 small antiviral molecules are based on nucleoside structures or on peptidomimetics; only 16.7% are classified as totally synthetic drugs. however, till now, neither a synthetic nor a naturally derived anti-hrv drug substance has been approved for the treatment or prevention of hrv infections. intensive research and development efforts in the field of natural products revealed several inhibitors of viral attachment and entry, and inhibitors of viral protease from natural sources. the efficacy of natural products is not only reflected by statistics of launched drugs but also by empirical knowledge gained over centuries by successful application of naturalbased ethnomedicinal products such as plants, culinary herbs, and spices. phytochemical and pharmacological work performed with ethnomedicinal anti-hrvs mainly from plants revealed a high number of active metabolites from different chemical classes, e.g. coumarins, flavonoids, alkaloids, quinones, terpenoids, polyphenols, and polysaccharides. natural products include complex extracts and their chemical entities, which are biosynthesized by nature. for an unambiguous presentation of anti-hrv natural products it is of prior importance to first distinguish between a single chemical entity from nature, i.e. an isolated, purified natural compound, on one hand, and a natural preparation comprising hundreds to thousands of constituents, mainly secondary metabolites, on the other hand. if natural preparations are derived from plants, they might also be labelled as botanicals, phytoceuticals, or phytotherapeutic agents. these multicomponent preparations might show a varying profile of their constituents depending on the used species, origin, collection time, plant parts, extraction procedures, preparation methods, and manufacturing processes, just to mention a few important elements. these parameters affect the final product in terms of the qualitative and quantitative composition of chemical constituents, which may have an impact in biological activity. accordingly, studies performed with phytochemically not specified extracts or nonstandardized preparations often suffer from irreproducible and incomparable results a wide variety of natural preparations showed to be acting therapeutically in hrv and other viral infections with often complementary and overlapping antiviral mechanisms of action. [274] [275] [276] most of these remedies are described in ethnopharmacological sources or handed down for generations. they usually consist of simply prepared natural items whose chemical composition is complex. many of the contained secondary metabolites, possibly active principles, have never been examined chemically or biochemically using modern medical knowledge. they are however components of plant medicines, which have stood the test of time and as such may offer clues of great interest to medicinal chemists. a clear advantage of the application of these products is their absent or relatively low toxicity due to a usually long-term empirical trial. although the knowledge of the immuno-pathogenesis of rv-induced diseases remains limited, the host defense function of the airway epithelium plays an important role in the innate-immune response to hrv-infection. 277 host cells respond by the production of mediators with antiviral activity such as type i interferons and nitric oxide, and produce cytokines and chemokines that influence the subsequent induced innate-and specific-immune response. these processes are beneficial in facilitating clearance of virus from the respiratory tract, but also cause immuno-pathology. following hrv-infection, disease severity is dependent on direct, harmful effects of the virus as well as tissue damage as a result of the host antiviral immune response. accordingly, a number of agents with phenomenological effects against common cold have shown to exert their activity more in the field of regenerating tissue damage than on a direct anti-hrv effect. several herbal remedies consisting of a multitude of secondary metabolites from different chemical classes may attribute in a beneficial way for the treatment of common cold by reducing symptom severity and duration due to their immune-modulating, anti-oxidative, and anti-inflammatory properties. beside these commonly observed bioactivities of natural products, multicomponent mixtures like botanicals often show overlapping symptomatic effects as well as synergistic and/or additive properties. thus, it is a challenging endeavor to track down an observed phenomenological effect of a complex mixture on a molecular level. the following section explores the significance and current knowledge of selected botanicals for the prevention and therapy of common cold. questions about (1) clinical evidence of efficacy, (2) the constituents or at least the chemical classes that are involved in the observed anti-hrv effect, and (3) the involved pharmacological targets were covered as far as possible. echinacea preparations include expressed juice from aerial parts as well as extracts of roots or aerial parts, or both, from one or more species of the genus echinacea (e. angustifolia, e. purpurea, and e. pallida). they are the most recognized botanicals for prevention and treatment of common cold and flu, and account for the second top-selling herbal products in the us-market. 278 accordingly, echinacea has come under much scientific scrutiny. the high number of studies dealing with the effectiveness of echinacea for preventing and treating the common cold from clinical trials was recently reviewed by woelkart et al. 279 the authors summarized the findings of the meta-analyses regarding the 16 randomized controlled trials evaluated in the cochrane database, 280 the 14 randomized clinical trials analyzed by shah et al., 281 and the experimental hrv-infection studies pooled by schoop et al. 282 to sum up, the clinical data on echinacea so far are not fully consistent, mainly based on problems inherent in assessing the efficacy of echinacea preparations, such as lack of comparability of available preparations, study design, and outcome. nevertheless, the meta-analyses showed some evidence that preparations based on the aerial parts of echinacea purpurea might be effective for the early treatment of colds in adults. 280 echinacea showed to decrease the odds of developing the common cold by 58% and the duration of a cold by 1.4 days. 281 similarly, the evaluation of three induced rhinovirus prevention studies revealed the odds of experiencing a clinical cold were 55% higher with placebo than with echinacea. 282 stepping into a molecular level, several constituents found in echinacea species could potentially affect the symptoms of common cold. chemically identified substances include polysaccharides and glycoproteins, caffeic acid derivatives (especially cichoric acid and echinacoside), and lipophilic polyacetylenes and alkamides. pharmacological studies have shown that cichoric acid, alkamides, glycoproteins, and polysaccharides possess immunomodulatory activity. additionally, alkamides have been reported to exert not only antiinflammatory effects but also cannabinomimetic properties, which are suggested as molecular mode of action of echinacea alkamides as immunomodulatory agents. 283 raduner et al. showed that some echinacea alkamides exert cannabinoid type 2 receptor-dependent and independent immunomodulatory effects on cytokine expression. 284 different echinacea constituents were evaluated for their anti-oxidative effects measuring the inhibition of in vitro cu(ii)-catalyzed oxidation of human low-density lipoprotein. thereby, the major caffeic acid derivatives, cichoric acid and echinacoside, showed the highest anti-oxidative effects, which was even higher when combined with a natural mixture of alkamides. 285 sharma et al. used cytokine antibody arrays to investigate the changes in the pro-inflammatory cytokines and chemokines released from human bronchial epithelial cells exposed to hrv 14. 286 application of two chemically characterized echinacea extracts showed a reversion of the stimulated release of numerous pro-inflammatory cytokine-related molecules, e.g. for the cytokine il-6, and the chemokines il-8 and eotaxin. in a similar study, an echinacea extract rich in polysaccharides and another rich in alkamides and caffeic acid derivatives were as well able to neutralize the effects of hrv-infected epithelial cells. 287 using gene expression analysis both studies revealed the anti-hrv benefit of echinacea preparations being involved in multiple immune response signaling pathways. taken together, the numerous pharmacological findings from literature, the potential of echinacea preparations, and their constituents to combat or prevent common cold can be deduced to immune modulating, anti-inflammatory, and anti-oxidative properties that may also act in some combination of these event, rather than acting directly on hrv. garlic cloves have been used traditionally to treat a number of infectious diseases. however, only few confirmatory studies have been published regarding the traditional antiviral uses. the clinical effectiveness of garlic on the prevention of common cold was investigated by josling in 2005, who published a double-blind, placebo controlled study assessing 146 patients more than a 12-week treatment period with an allicin-containing garlic supplement. 288 common cold infections and symptoms were recorded in a daily diary. patients in the treatment group had significantly fewer colds than patients in the placebo group (24 vs. 65, po0.001) who also had a longer duration of symptoms (5.01 vs. 1.52 days, po0.001). as soon as the garlic is chewed, cut, or pressed, its main ingredient, the sulphur containing alliin, is broken down by the enzyme alliinase to the thiosulfinate allicin. by steam distillation allicin is transformed to diallyl disulfide and diallyl trisulfide that are responsible for the distinctive smell of garlic. further, allicin transformation compounds, such as e-and z-ajoene, are not found in fresh garlic, but in lipophilic extracts. by investigation of different garlic extracts and isolates against a number of different human pathological viruses, weber et al. could show that allicin was the most active virucidal component from fresh garlic and fresh extracts. 289 results of the direct pre-hrv-2-infection incubation assay let suggest allicin to bind to the viral protein capsid, leading to a subsequent inhibition of viral adsorption and penetration. although the garlic thiosulfinates are endowed with significant cytotoxicity, the antiviral effects were obtained in nontoxic concentrations. 289 beside the direct anti-hrv effect of fresh garlic extract and allicin, a number of human immune functions were found to be enhanced in vitro by aqueous garlic extract, its polar, and thiosulfinate fractions. 290 in north america, panax quinquefolium, the ginseng species indigenous to both canada and the united states, has been a popular herbal remedy to combat stress, and to modulate both natural and acquired immune responses. american ginseng root extracts, rich in poly-furanosyl-pyranosyl-saccharides, have been found efficacious in the prevention of upper respiratory infections in immunocompetent healthy adults. 291, 292 in a randomized, double-blind, placebo controlled trial, 200 mg of a proprietary american ginseng root extract was given to 43 community-dwelling elderly adults (age 465 year) twice a day more than a ''cold and flu'' season period of 4 months. one month into the study, all participants received an influenza vaccination. during the first two months, no significant differences in duration and incidence were observed when compared to placebo. however, during the last two months significantly fewer subjects of the ginseng group reported acute respiratory syndromes than the placebo group (32 vs. 62%). additionally, the duration of respiratory symptoms was reduced by 55% in the ginseng group. 291 in a similarly arranged trial, 323 healthy adults (ages 18-65 years) with a history of at least two colds the previous year commenced a 4 month study at the beginning of a cold and flu season. 292 they received two 200 mg capsules daily of standardized american ginseng root extract or a placebo. outcomes measured were number of colds including symptom severity and total number of symptomatic days. a therapeutic effect was reported regarding symptom severity and fewer symptom days that were 31 and 34.5% lower in the ginseng group than in the placebo group. a phase ii randomized, controlled trial of 2 dosing schedules of american ginseng root extracts, rich in poly-furanosyl-pyranosyl-saccharides, evaluated the safety, tolerability, and efficacy in a pediatric population already suffering from an upper respiratory tract infection. the results showed no serious adverse events and a good tolerability of both ginseng doses; however, frequency and severity of symptoms were not significantly different among each of the three treatment groups, i.e. standard dose, low dose, and placebo. 293 the most prominent constituents of the genus panax are the triterpene saponins ginsenosides. they are known to have numerous pharmacological activities such as anti-cancer, anti-diabetes, antiviral, and anti-atherosclerosis effects. some compounds of this chemical class showed to be responsible for the immunostimulant activity of ginseng. 294 on the other hand, the efficacy of a polysaccharide-rich extract of american ginseng was compared with an extract rich in ginsenosides on systemic and gut-associated immune function. the authors of this study investigated the lymphocytes isolated from spleen, mesenteric lymph nodes and peyer's patches, and immune cell proportions and cytokine production from sprague-dawley rats. they could show that the polysaccharide-rich ginseng extract modifies the rats' systemic immune responses and affects the gut-associated immunity in a manner distinct from that of the ginsenoside-containing extract of american ginseng. 295 a direct antiviral activity of ginseng constituents could be attested for the polysaccharides on rotavirus infection in ma104 cells. the triterpene saponins, however, did not exhibit any rotavirus infection-inhibitory activity in this study. 296 a moderate in vitro virucidal effect (id 50 62 mm) of the ginseng saponin chikusetsusaponin iii against herpes simplex virus type i was detected by fukushima et al. this compound exhibited an intracellular inhibitory activity, but could only marginally affect the viral proteins postinfection. 297 the ancient chinese formula bu-zhong-yi-qi-tang (japanese name hochu-ekki-to) is a traditional herbal medicine in china and japan that is composed of ten species of medicinal plants, namely astragali radix, atractylodis lanceae rhizoma, ginseng radix, angelicae radix, bupleuri radix, zizyphi fructus, aurantii nobilis pericarpium, glycyrrhizae radix, cimicifugae rhizoma, and zingiberis rhizoma. this formula is reported to have various immunomodulatory, [298] [299] [300] and anti-inflammatory activities. 301 yamaya et al. recently investigated the effects of hochu-ekki-to in cultures of human airway epithelial cells infected with hrv-14. 302 the output of virus, associated levels of viral rna, and the production of icam-1, cytokines and acidic endosomes in cells were measured. in airway epithelial cells hochu-ekki-to was able to decrease virus output and susceptibility to hrv infection by decreasing icam-1 and by blocking the entry of viral rna into the cytoplasm from the endosomes. glycyrrhizin, a major component of one herbal ingredient of hochu-ekki-to, i.e. glycyrrhiza glabra, was able to reduce supernatant virus titers dose-dependently, with a maximum effect between 0.12 and 0.6 mm. however, no clinical trials with representative numbers of subjects are published so far. the human rhinovirus k 63 5. umckaloabo (pelargonium sidoides) p. sidoides and p. reniforme form the origin of the popular drug umckaloabo. this herbal remedy from south africa has found entrance in western medicine mainly as aqueous ethanolic root extract from p. sidoides for the treatment of infections of the respiratory tract. the efficacy of umckaloabo compared with placebo has been evaluated in 103 adults suffering from common cold by lizogub et al. 303 the applied herbal preparation was well tolerated by the patients. the study demonstrated only a weak efficacy of umckaloabo compared to placebo after 5 days. after 10 days, however, the p. sidoides extract significantly reduced the severity of symptoms and shortened the duration of the common cold compared with placebo. just recently, timmer et al. selected randomized controlled trials examining the efficacy of p. sidoides preparations for the treatment of various acute respiratory infections and analyzed their efficacy and safety. 304 the authors concluded that umckaloabo may be effective in alleviating symptoms of acute rhino-sinusitis and the common cold in adults. it may be effective in relieving symptoms in acute bronchitis in adults and children, and sinusitis in adults. reliable data on the treatment for other acute respiratory infections however were not obtained. identification of the metabolites from umckaloabo revealed a high number of different chemical classes, such as phenolic and cinnamic acids, tannins, flavonoids, and coumarins. antibacterial activities of umckaloabo against different pathogens have been reported. phenols, coumarins, and tannins have been identified to contribute with moderate antibacterial activities, however, cannot explain the effect of the whole extract (reviewed by kolodziej 305, 306 ). additionally, p. sidoides extracts have been reported to significantly activate the nonspecific immune system by induction of tnf and no-release, and ifn-like activities. 305 these effects are assumed to contribute to the controversially discussed potential of p. sidoides extract for the treatment of upper respiratory tract infections. only one study reports a direct antiviral effect, i.e. a clear dose-dependent anti-herpes simplex virus acitivity for the aqueous root extract of p. sidoides. 307 further pharmacological studies are needed to elucidate potential direct anti-hrv properties of umckaloaba and its constituents. carrageenan, a mixture of different polysaccharides, which is mainly extracted from red seaweeds, has been extensively used in food, cosmetic and pharmaceutical industry as a thickener and gelling agent. it has previously shown an antiviral efficacy against several viruses. 308, 309 in a recently published study, lambda-, kappa, and iota-carrageenan were investigated for their anti-hrv inhibiting potential. at a concentration of 200 mg/ml iotacarragenan, a sulphated polysaccharide, was able to fully inhibit virus-induced cell death in hrv-2 infected hela cells. 310 based on their studies, grassauer et al. concluded that iotacarrageenan is effective against different hrv-serotypes on primary human epithelial cells. it is hypothesized by the authors that iota-carrageenan might create a hostile environment for hrv and thereby block viral entry and replication. because of its safe application and proved in vitro efficacy, iota-carrageenan deserves consideration as a candidate for clinical trials for prevention and therapy of hrv-induced common cold. the level of knowledge on the impact of the six botanicals on hrv-infection discussed above is different and heterogeneous. the best studied herbal remedy associated with common cold, i.e. echinacea, showcases the innate problem connected with multi-component mixtures: starting from the late nineties till june 2009 some 100 original articles have been published to this topic and tried to elucidate questions concerning efficacy, molecular mechanism, and bioactive ingredients of echinacea. although some evidence is provided for the effects of extracts, chemical classes as well as well-defined constituents on specific targets and pathways, the findings cannot be deduced to a common denominator. further, results from clinical trials often suffer from lack of comparability, because of using different study designs, outcome measures, and overall the application of different preparations. 279 a proper quality analysis and characterization of the preparation under investigation is mandatory and should follow the recommendations and guidelines for reporting clinical trials for herbal medicine. 311 as underlined before (sections 5.1. and 5.2.), the chemical complexity of a natural preparation might be beneficial in terms of synergistic, additive, and overlapping effects caused by the multitude of evolutionary trimmed metabolites, which may attribute with modulating multi-target effects. on the other side, exactly this fact is hardly compatible with the proper assignment of an activity to a defined chemical entity according to western medical practise. in contrast to a single compound (synthetic or naturally based), the chemistry of a botanical is not only complex but also varying. the analytical profile and in term the pharmacological profile of the investigated samples can differ substantially. accordingly, the quantitative and qualitative comparison of different studies resulting from botanicals is by far more complex than those performed with pure single compounds, and may also explain why so little emphasis from pharmaceutical industry has been put into the further development of even promising natural preparations. in general, the search for potent, selective, nontoxic compounds that might be developed further to a drug substance is a multidisciplinary, time-and cost-consuming process. therefore, strategies for a target-oriented discovery of lead-structures either from nature or synthesis are in high demand. some of them will be discussed in the following section, providing examples from anti-hrv research. as already mentioned, nature provides an extremely rich pool of bioactive natural products. it is however a challenging endeavor to find exactly those compounds that show an activity on the focused target. in natural product research hints from folk medicine are a valuable starting point to dig for lead structures of certain interest. the majority of active principles from higher plants has been discovered as a result of ethnopharmacologically directed pharmacognostic research. 312, 313 oral or written indications for a beneficial application of a natural material first need a critical evaluation as to the selection of the correct material, its medicinal preparation, the kind of application, and overall the pharmacological profile. a rational criticism of often anecdotal efficacy from traditional medicine is a mandatory attitude to avoid overinterpretation of handed-down information. 314 in case of pretended anti-hrv remedies, the reported biological efficacy obviously suffers from being restricted to respiratory diseases, which might be caused by a panel of bacterial or viral infections. thus, an approved remedy may affect the microbes, or show an immune modulating or even antiinflammatory effect, or a combination of these. the holistic access is an innate character of ethnopharmacology and needs to be tracked down to the specifically involved target/s. in a recently published study focusing on the discovery of hrv-capsid binders from nature using a pharmacophore-based virtual screening of an ethnopharmacologically biased 3d-multiconformational database, some 30 secondary metabolites were predicted to act as capsid-binding inhibitors of hrv. 144 for an in depth phytochemical and pharmacological investigation, it was mandatory to focus on one promising natural material. thus, we consulted the ethnobotanical source materia medica, which was written by pedanius dioscorides in the 1 st century ad. the treatise consisting of five books comprises some 1,000 or so drugs derived from minerals, animals, and the majority of them from plants (800). it represents a great repository of botanical, medical, and pharmacological lore. scrutinizing the natural materials underlying the obtained virtual hits we came across asafetida, which is a gum resin gained from the roots of a variety of foul-smelling ferula species from the apiaceae family. based on the descriptions given in the materia medica there is strong evidence that the juice (i.e. resin) of the popular ancient silphion originating from media and syria corresponds to asafetida (book iii, cap. 80). 315 yielded by incision of the root and stalk and frequently mixed with sagapenon, i.e. the resin of f. persica willd (book iii, cap. 81), it is reported to be effective in the context of upper respiratory diseases, e.g. ''for chronic harshness of the throat,'' ''it clears the voice,'' ''shrinks the uvulas,'' ''suitable for a cough,'' ''for pleurisy,'' ''for chest pain;'' sagapenon is described as follows: ''it clears thick matters from the lungs,'' ''given to those who are chilled.'' 315 these descriptions finally helped to prioritize those virtual hits, which have been reported to be constituents of asafetida. 316 the pharmacological investigation of asafetida and its constituents farnesiferol b and c ( fig. 5 ; table iii ) revealed a distinct anti-hrv-2 effect in the low micromolar range using a cpe inhibitory assay. 143 the results of this study provided a rationale for the ancient usage of asafetida for upper respiratory tract infections. on the other hand, the traditionally manifested evidence for asafetida for the treatment of common cold symptoms substantially helped in the selection of this plant material in the search for anti-hrv capsid binders. 144 typically, an ethnopharmacologically based discovery of an active (anti-hrv) extract is followed by a bioassay guided fractionation, and the isolation of those constituents that are responsible for the extracts' bioactivity. the concept of a bioassay-guided approach is the fractionation accompanied by simultaneous detection of the activity during the separation steps, which results in a continuous enrichment, and finally in the isolation of the active ingredient/s. in this way a large number of anti-hrv agents from natural sources have already been discovered. semple et al. investigated the active principle of the asteraceae plant pterocaulon sphacelatum, which has been used in traditional medicine of aboriginal people of australia as a favored treatment for respiratory infections, especially colds. by means of an antiviral activity-guided fractionation measuring the poliovirus-induced cpe assay, the authors identified the flavonoid 3,7,3 0 -trimethoxy-5,6,4 0 -trihydroxyflavone, i.e. chrysosplenol c ( fig. 5 ; table iii ) as potent and specific inhibitor of the picornaviral replication. 6,7,8-trimethoxycoumarin ( fig. 5 ; table iii) was also isolated as a major constituent from the ethanolic extract of p. sphacelatum, but showed no activity against poliovirus. 317 interestingly, this coumarin exhibited a significant effect in the hrv-2-induced cpe assay in a recently performed virtual parallel screening study performed in our laboratory. 318 in contrast to the isolated coumarin, chrysosplenol c was already known as a member of the 4 0 -hydroxy-3-methoxyflavones, which represent potent and specific inhibitors of picornaviral, especially rhinoviral replication. [319] [320] [321] in 1993, vanden berghe, haemers, and vlietinck provided a profound survey of antiviral agents from higher plants, and demonstrated the impact of ethnobotanical knowledge in their search for antiviral compounds from african medicinal plants. the selection of investigated plant species was mainly based upon their use in the treatment of viral diseases by african traditional healers. 320 the antiviral activity of 100 different plant species belonging to 33 families was investigated; thereof 21 species exhibited prominent antiviral properties against one or more of the tested viruses. the most pronounced activity against picornaviruses was recorded within the genus euphorbia. all compounds detected as antiviral constituents from the respective extracts were identified as 3-methoxyflavone derivatives, especially 3-methylethers of quercetin and kaempferol ( fig. 5 ; table iii ). they showed no significant cytotoxicity and were highly active in tissue culture against all human picornaviruses. in tissue culture, cells infected with different picornaviruses (among them also hrv) no cpe was observed, when cells had been treated with 2 mg/ml of these 3-methoxyflavones. 320 the selection of natural materials based on ethnopharmacology is a profound rationale for lead structure discovery and highly superior to random selection. this however rarely applies to marine organisms, fungi, and microbes. although these organisms are esteemed as highly valuable source for bioactive metabolites, 322 hardly any records from folk medicine are given for them. a medium-sized activity screening using a robust cell-based or in vitro-assay with reasonable effort as to time and costs is a strategy to get a first insight into the antiviral activities of extracts, fractions, and compounds from synthesis or nature. for the discovery of novel naturally based hrv 3c-protease inhibitors, singh et al. used a small peptide containing q-g scissile bond as substrate for the in vitro screening of extracts. the authors isolated the novel benzoisochromanquinone (1)-thysanone ( fig. 5 ; table iii ) from an extract of the fungus thysanophora penicilloides with potent hrv 3c-protease inhibitory activity. 323 a continued screening revealed a pronounced hrv 3c-protease inhibitory activity for the extract of the chinese herb polygonum cuspidatum. by bioassay-guided fractionation 2-methoxystypandrone ( fig. 5 ; table iii ), a naphthoquinone, with an ic 50 value of 4.6 mm, was isolated from the plant material. the total syntheses of this natural compound and further analogues allowed for a structure-activity relationship, and particularly the comparison between activities of ortho-vs. para-quinones. to measure the selectivity of the compound series against cystein proteases other than hrv 3c-protease, the compounds were evaluated against papain. the simple 9,10-phenanthraquinone ( fig. 5 ; table iii ) was the most active compound of the series and showed an ic 50 value of 1.4 mm with a distinctly higher degree of selectivity than 2-methoxystypandrone. 324 a cpe reduction assay was applied for the identification of raoulic acid, a bicyclic c25 terpene acid ( fig. 5 ; table iii ) isolated from raoulia australis (asteraceae). raoulic acid exerted an antiviral activity against coxsackie virus b3, b4, enterovirus 71, and hrv-2 and -3 with ic 50 values in the submicromolar range. no activity was recorded against influenza a and b viruses. 325 in the course of the systematic screening of microbial and natural products for anti-hrv activity, ishitsuka et al. identified 4 0 ,5-dihydroxy-3,3 0 ,7-trimethoxyflavone ( fig. 5 ; table iii) from the leaves of the chinese medicinal plant agastache rugosa (lamiaceae) as natural compound with high activity against all picornaviruses except mengovirus. the authors synthesized the orally active 4 0 ,5-diacetyloxy-3,3 0 ,7-trimethoxyflavone and performed investigations in tissue cultures and in mice to determine the compound's mode of action. this was assumed to be the process of viral replication, thus located between viral uncoating and initiation of viral rna synthesis. 326 the activity of flavan ( fig. 5 ; table iii ) was discovered serendipitously during a screening program by using a plaque inhibition assay. 194 based on these results, bauer et al. synthesized 4 0 ,6-dichloroflavan (bw863c, fig. 2 ; table ii) , which revealed an activity against a number of hrv serotypes in the range between 0.007 and 10 mm. as soon as an active lead compound is identified it is of utmost importance to scrutinize the derivatives' activity to (i) obtain insight into the chemical requirements mandatory for the focused biological activity, (ii) improve the compound's pharmacological profile in terms of potency, selectivity, cytotoxicity, etc., and to (iii) improve its bioavailability. the nonphenolic aporphine alkaloid glaucine is a prominent constituent of the aerial parts of gaucium flavum (papaveraveae). in a recently published study, spasova et al. investigated the antiviral potential of glaucine ( fig. 5 ; table iii) , glaucine derivatives, and its semi-synthesized 3-aminoethylglaucine cinnamoyl-and hydroxycinnamoyl amides. beside the anti-oxidative potential of the newly synthesized compounds, they all exerted an antiviral activity against the replication of hrv-14. the best anti-hrv activity was observed for oxoglaucine ( fig. 5 ; table iii; ic 50 table iii; ic 50 15 mm, si 10.3 and ic 50 13 mm, si 11, respectively). 327 the early findings of the anti-hrv active naturally derived 3-methoxyflavone inspired chemists and pharmacologists in the synthesis and the pharmacological evaluation of derivatives thereof decorated with various substitution patterns. several reports published during the last two decades reviewed the findings of antiviral flavonoid research. 320, 328, 329 by investigating the antiviral activity of a wide variety of naturally occurring flavonoids, tsuchiya et al. found chrysosplenol b and c ( fig. 5 ; table iii ) contained in chrysosplenium plants, and axillarin ( fig. 5 ; table iii ) as potent anti-hrv agents. based on their findings, the flavone skeleton decorated with a methoxy group in position 3 and a 5-hydroxyl group revealed as mandatory for an anti-hrv activity. 319 a series of antipicornaviral 4 0 -hydroxy-3-methoxyflavone derivatives was synthesized by de meyer et al. in order to establish a structure-activity relationship. thereby, different substitution patterns of the a-ring system with methyl, hydroxy, methoxy, halo, nitro, and amino was performed. their activity against polio and hrv was compared with those of naturally occurring flavonoids. further, the importance of the 4 0 hydroxyl-group and of the 3-methoxyl-group was confirmed by investigation of different derivatives lacking these features. 321 the results showed that 4 0 -hydroxy-3-methoxyflavones with a monosubstituted a-ring are less active than the corresponding compounds having a polysubstituted a-ring. within the tested series of compounds, 4 0 ,7-dihydroxy-3-methoxy-5,6-dimethylflavone emerged not only as noncytotoxic but also as most potent substance in both antiviral test systems. the lowest concentrations for this compound that protect 50% of the cells from cpe of 12 hrv serotypes were in the range from 0.016 to 0.5 mg/ml. in contrast to quercetin, this flavone was also reported to have no mutagenic properties (measured up to 2.5 mg/plate) in a short-term microbial assay. 321 the mechanism studies performed with 3-methoxyflavones have shown an interference with an early stage in the viral rna synthesis; no induction of resistance was observed. 320 in contrast to this mode of action, the anti-hrv chalcones and flavans are reported to interact directly with specific sites on the viral capsid proteins, thereby preventing uncoating and the consequent liberation of viral rna. 193, 330, 331 due to its anti-hrv potency, low toxicity, and promising bioavailability, dichloroflavan was evaluated for its protective efficacy against experimental hrv-infection in two clinical, double-blind, placebo-controlled trials. however, the drug candidate failed either when administered orally or intranasally. 332, 333 unfortunately, till now no clinical trials evaluating the efficacy of 3-methoxyflavones in common cold have been performed. the common idea of all computational approaches is to extract knowledge from a more or less large set of data in order to make predictions of new events. 334 within the lead discovery process, computational approaches, such as virtual screening, docking, quantitative structure-activity relationship, have largely enhanced the impact of computational chemistry and nowadays chemoinformatics plays a predominant role in drug research. 335 the key goal of the use of such methods is to reduce the overall cost associated to the discovery and development of a new drug by identifying the most promising candidates to focus the experimental efforts on. a number of books and reviews on the impact of computational chemistry for lead structure determination highlight these efforts. [336] [337] [338] [339] in general, in silico methods can be divided into (i) ligand-based approaches, which rely on known active compounds. based on their physicochemical properties crucial for biological affinity, activities are predicted by extrapolation on not-yet tested substances, e.g. machine learning techniques and classical quantitative structure-activity relationship (qsar). ligand-based approaches are invaluable tools in cases where no structural information about the pharmacological targets is available. (ii) on the other hand, structurebased approaches use experimentally determined 3d structures of the targets, such as molecular docking or structure-based pharmacophore modeling for virtual screening. these methods allow for gaining insight into protein-ligand interactions at an experimentally determined (static) level (however not considering flexibility). a unique platform containing 3d coordinates of experimentally solved protein structures (by x-ray crystallography or nmr) is the protein data bank (pdb) currently comprising more than 50,000 structures of biomolecules and protein-ligand complexes. 340 additionally, there are several pdb-related web services and tools, which enable to use the pdb-portal in a rich diversity of information services for students and scientists. 341 particularly in the early stage of drug development, such as lead discovery and lead optimization, computational approaches allow for a target-oriented and rationalized proceeding, and thus may substantially help to maximize the success rate. 338 a recently published review on the impact of computer-assisted approaches in antiviral research thoroughly describes underlying in silico techniques, and highlights the benefits of computational approaches for the discovery of antiviral lead structures. 342 in anti-hrv research the capsid protein and the protease 3c revealed to be promising targets (as described before). inhibitors are assumed to have a major impact for the treatment of hrv-infections. additionally, these targets are structurally elucidated, and some potent ligands are known as well. these facts enable the performance of sensible computer-assisted approaches, both ligand-and structure-based. some studies using an in silico approach for the discovery of potential anti-hrv agents focusing on the mentioned targets will be reported in the following paragraphs. for compounds acting as potential hrv-capsid binders, some classical qsar and 3d qsar studies have been performed. while in classical qsar, the relationship between 2d calculated properties derived from chemical structures and measured biological activities are explored statistically, 3d qsar techniques are aimed at deriving a correlation and in turn activity prediction based on spatial arrangements of chemical properties and atoms. applying the 3d qsar technique comfa (comparative molecular field analysis) statistical models are derived, which are visualized in color-coded contours around the molecule. therein spots indicate where electrostatic properties and spatial arrangements are favorable for biological activity. 343 in the studies of diana et al., artico et al., and verma et al., qsar techniques helped on one hand to analyze and rationalize the structural features of active compounds essential for the interaction to the hrv canyon's binding pocket, and on the other hand to search for new classes of capsid binders, thus to narrow the synthetic challenges for specific anti-hrv agents, respectively. [344] [345] [346] [347] [348] in all these investigations hydrophobicity was found to be one of the most important determinants of substance activity. qsar combined with simplex representation of molecular structure was applied by kuz'min et al. based on the selectivity index (cc 50 /ic 50 ) and the hrv-2 inhibitory concentration of a set of [(biphenyloxy)propyl]isoxazole derivatives. 188 on the basis of qsar analysis and computational design, three new isoxazoles with high activity prediction were selected and synthesized. they all revealed a strong coincidence between experimental and predicted anti-hrv activity and selectivity index. terminal benzene substituents with negative electrostatic potential and a molecule length of approximately 5.5-5.6 å have been suggested as mandatory features within this chemical class for a hrv-2 inhibitory activity. hrv-serotypes show a high level of conservation at the protease 3c binding site; sequence alignment and secondary structure predictions suggested an overall architecture and mechanism of hrv-3c proteases that correlates with cellular cystein-and serine proteases, such as chymotrypsin and trypsin. 349, 350 the identity among 3c proteases from different families is however modest and provides space for the development of specific inhibitors for hrv-3c protease. 350 in a recently published review on selective inhibitors of picornavirus replication, de palma et al. summarized all currently known chemical structures acting as peptidic or nonpeptidic inhibitors of this viral target. 156 in 2000, reich et al., used the hrv 3c protease co-crystal structure information to rationalize the target-oriented synthesis of hrv 3c protease inhibitors from the class of substituted benzamides. activity data and subsequent crystallographic studies pointed out important requirements for the inhibition of the 3c protease. 351 similarly, maugeri et al. used a structure-based approach, and performed docking studies based on the 3c protease crystal structure with a virtual library consisting of benzamide derivatives. quantum-mechanic calculation proposed substituents with most promising biological activities. this workflow guided the design and synthesis of substances virtually assumed to act as substrate analogues. synthesis of some of these compounds and biological testing confirmed the underlying hypothesis. 219 quantitative molecular modeling studies were performed to better define and predict interactions between bicyclic 2-pyridone derivatives that showed to be irreversible inhibitors of the 3c protease. in these studies molecular mechanics simulations to evaluate chemical rate of covalent bond formation and free energy calculation combined with crystallographic studies were applied to explain the differences in activity of some irreversible peptidomimetic inhibitors. these data were used as a basis for further optimization of these compounds. 224, 352 in a recent study performed by kuo et al. some 6,800 compounds were subjected to a high troughput screening in the search for novel inhibitors for both 3c and 3cl proteases from picornavirus and coronavirus, respectively. 217 five nonpeptides were identified with ic 50 values r10 mm against severe acute respiratory syndrome-coronavirus 3cl-protease; one molecule was found to additionally inhibit the 3c proteases of coxsackievirus, enterovirus, and rhinovirus. this compound (id 43146) contains a dihydropyrazole ring decorated with two phenyl groups and a lengthy n-butyl-benzimidazolylamino-toluene. it was used as starting point for the selection of further four analogs showing ic 50 values in the range of 0.5-10 mm against the tested viral proteases. by means of docking-based computer modeling, the authors tried to rationalize the binding discrepancies responsible for individual and common protease inhibitors, thus to provide a rational base for the development of nonpeptide multiple-function inhibitors against coronaviruses and piciornaviruses. in anti-hrv research, first application scenarios have been conducted using pharmacophore models. according to the official iupac definition by wermuth et al., 353 a pharmacophore describes the 3d arrangement of steric and electronic features necessary to trigger or block a biological response. pharmacophores can be represented by three-dimensional chemical features, which include hydrogen bond donors and acceptors, aromatic rings, hydrophobic groups, as well as positive and negative ionisable moieties. additionally, the shape of ligands can be represented by shape features, which essentially describe the van der waals radii of the ligand atoms. the pharmacophore concept has proven to be successful, not only in rationalizing structure-activity relationships but also by its large impact in developing appropriate 3d-tools for efficient virtual screening. 336, 354 steindl et al. elaborated ligand-and structure-based pharmacophore models implementing the essential feature of the covalent binding to the cysteine 147 in the active site of the hrv-2 3c protease. thus, the in silico approach focused on defining a new pharmacophore feature representing a target structure for nucleophilic addition in the ligands, which is a crucial step for protease inactivation. the generated hypotheses retrieved known 3c protease inhibitors in the virtual screening cycle, and proposed potential (unconfirmed) ligands of the 3d protease binding site from available databases. 355 the viral capsid of several hrvs has been elucidated by crystallization and resolution of the 3d-structure. the hrv coat protein complexed with its highly active inhibitor win 61209 was used as starting point for the generation of structure-based pharmacophore models by . the models were used for virtual screening of a large commercially available 3d database. for final selection of virtual hits worth to be subjected to biological testing, docking studies and principal component analysis were performed. six candidates were tested for their ability to inhibit hrv serotype 39 by multiple-cycle cpe inhibition assay. although all of them showed a certain antiviral potential, one longitudinal piperazine derivative inhibited the virus at a concentration below 10 mm. some of the test candidates showed difficulties in the interpretation of experimental results due to their relatively high cytotoxicity and bad solubility. this circumstance asks for more cautious estimation of molecular properties for compound selection as stated by the authors. 356 in a subsequently performed study, the best validated pharmacophore model was used for the identification of naturally derived hrv coat protein inhibitors. 144 for virtual screening experiments the in-house generated 3d-database dios was used (as described before). based on the virtually predicted ligands and considering knowledge from traditional use, sesquiterpene umbelliferons from the gum resin of ferula sp., i.e. asafetida, were finally selected as most promising candidates. for biological evaluation, the antiviral activities of asafetida and its isolated constituents were assessed by an exploratory determination of the inhibition of the cpe induced by hrv serotypes 1a, 2, 14, and 16. the results revealed a dose-dependent and selective anti-hrv activity against serotype 2 for asafetida and its virtually predicted constituents, farnesiferols b and c ( fig. 5 ; table iii ; ic 50 : 2.6 and 2.5 mm, respectively). to scrutinize the selectivity of these two compounds against hrv-2 in comparison to the other tested serotypes, the amino acid sequences of hrv-2 and hrv-16 vp1 were aligned. since all amino acid residues involved in ligand binding showed 100% match in both serotypes, the experimentally determined selectivity profile could not be explained by different binding pockets. the serotype alignment does however not reflect potential protein flexibility during binding, which might differ between the hrv serotypes. additionally, off-target effects could be a reason for the observed selectivity. in a recently performed virtual parallel screening approach, we tried to identify potential targets of human pathological relevance for 16 constituents isolated and identified from the medicinal plant ruta graveolens. 318 using the screening platform pipline pilot 357 included in discovery studio, 358 low-energy conformers of the 16 identified molecules from r. graveolens were subjected to parallel screening. for this purpose, the inte:ligand pharmacophore model collection was used. 359 it currently comprises 2.208 models covering 280 unique pharmacological targets. based on the predicted ligand-target interactions, the authors focused on three biological targets, namely acetylcholinesterase, the hrv coat protein, and the cannabinoid receptor type 2. virtual hits and nonhits were assayed on their respective targets for a critical evaluation of the performed target-fishing approach. beside other predicted bioactivities, determination of their cpes on hrv-2 revealed the virtual hit arborinine ( fig. 5 ; table iii; ic 50 : 3.2 mm) and the nonpredicted hit 6,7,8-trimethoxycoumarin ( fig. 5 ; table iii ; ic 50 : 12.9 mm) as the most active anti-hrv constituents. it could be shown that the applied in silico strategy has the capacity of catalyzing drug discovery profoundly for all those diseases where molecular targets or molecular ligands are well defined to create reliable pharmacophore models. hrv, a prominent member of the picornaviridae family, infects humans more frequently than any other virus. infections with hrv mainly lead to upper respiratory diseases, such as the common cold, but may also cause more severe lower respiratory tract disorders. although the symptomology and severity of the common cold is relatively mild and the course of disease self-limiting, the socio-economic impact is tremendous in terms of recouping lost productivity due to sick leave. in the last decades, a number of anti-hrv agents from different origin-synthetics, natural compounds, biologicals, botanicals, and nutritionals-have been discovered. different concepts have been used as strategy for their discovery either starting from a phenomenological effect, e.g. empirical knowledge from folk medicine, or from a targetbased molecular level, e.g. icams, capsid binders, and hrv protease inhibitors. some of the outcomes revealed potent and promising activities that partly have been evaluated for the management of hrv-induced common colds in clinical trials mainly with sobering benefit. since the hrv infection is not life-threatening in most cases, a potential therapy has to be safe and effective with an almost unrecognizable level of side effects. these preconditions render the search for anti-hrv-agents into a high challenging endeavor and explains why no antiviral agent is approved for the prevention or treatment of hrv-infection until today, despite the significant efforts. moreover, the high variability of rhinoviruses suggests the need of more than one active principle covering different modes of action for an effective treatment. therefore, there is a high need for an ongoing search for new synthetic as well as natural compounds on a molecular level. the increasing knowledge about the hrv life cycle, gene, and protein sequence combined with the improved technologies in the field of experimental and computational methods have continuously enabled further insights into the spectacular world of hrv. only with this fundamental research, virtual screening approaches, such as qsar, pharmacophore-and docking-based screening cycles, or computational compound design, are applicable on a rational base. with the gained expertise from different disciplines and an adequate infrastructure for further research, it is encouraging to hope that discovery and clinical development efforts will continue in the search for agents that may treat or prevent the annoying common cold. picornavirales, a 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against enteroviral cardiomyopathy an antiviral mechanism of nitric oxide: inhibition of a viral protease nitric oxide inhibition of coxsackievirus replication in vitro nitric oxide donors inhibit the coxsackievirus b3 proteinases 2a and 3c in vitro, virus production in cells, and signs of myocarditis in virus-infected mice effect of nitric oxide on rhinovirus replication and virus-induced interleukin-8 elaboration inhibition of proteolytic activity of poliovirus and rhinovirus 2a proteinases by elastase-specific inhibitors an antiviral peptide inhibitor that is active against picornavirus 2a proteinases but not cellular caspases antiviral activity of caspase inhibitors: effect on picornaviral 2a proteinase dual inhibition of human rhinovirus 2a and 3c proteases by homophthalimides structure of human rhinovirus 3c protease reveals a trypsin-like polypeptide fold, rna-binding site, and means for cleaving precursor polyprotein individual and common inhibitors of coronavirus and picornavirus main proteases structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3c protease with potent antiviral activity against multiple rhinovirus serotypes new anti-viral drugs for the treatment of the common cold structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 1. michael acceptor structure-activity studies structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 2. peptide structure-activity studies structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 6. structure-activity studies of orally bioavailable, 2-pyridone-containing peptidomimetics structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 3. structure-activity studies of ketomethylene-containing peptidomimetics structurebased design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 8. pharmacological optimization of orally bioavailable 2-pyridone-containing peptidomimetics structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 4. incorporation of p1 lactam moieties as l-glutamine replacements in vitro antiviral activity of ag7088, a potent inhibitor of human rhinovirus 3c protease inhibition of human rhinovirus-induced cytokine production by ag7088, a human rhinovirus 3c protease inhibitor pharmacokinetics and safety of an antirhinoviral agent, ruprintrivir, in healthy volunteers phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers in vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3c protease gaining target access for deoxyribozymes a morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus small interfering rna molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism mode of action of 2-furylmercury chloride, an antirhinovirus compound methoxyflavones as potent inhibitors of viral-induced block of cell synthesis antiviral effects of pyrrolidine dithiocarbamate on human rhinoviruses broad-spectrum antiviral and cytocidal activity of cyclopentenylcytosine, a carbocyclic nucleoside targeted at ctp synthetase in vitro antiviral activity of ribavirin against picornaviruses another ten stories in antiviral drug discovery (part c): ''old'' and ''new'' antivirals, strategies, and perspectives the broadspectrum antiviral ribonucleoside ribavirin is an rna virus mutagen ribavirin: recent insights into antiviral mechanisms of action inhibition of rhinovirus replication in in organ culture by a potential antiviral drug synthesis of syn and anti isomers of 6-[[(hydroxyimino)phenyl]methyl]-1-[(1-methylethyl)sulfonyl]-1h-benzimidaz ol-2-amine. inhibitors of rhinovirus multiplication comparative studies on the modes of action of the antirhinovirus agents ro 09-0410, ro 09-0179, rmi-15,731, 4 0 ,6-dichloroflavan, and enviroxime the antiviral compound enviroxime targets the 3a coding region of rhinovirus and poliovirus sequence determinants of 3a-mediated resistance to enviroxime in rhinoviruses and enteroviruses evidence that enviroxime targets multiple components of the rhinovirus 14 replication complex controlled trial of enviroxime against natural rhinovirus infections in a community the activity of enviroxime against rhinovirus infection in man activity against rhinoviruses, toxicity, and delivery in aerosol of enviroxime in liposomes 2-amino-3-substituted-6-[(e)-1-phenyl-2-(n-methylcarbamoyl)vinyl]imid azo[1,2-a]pyridines as a novel class of inhibitors of human rhinovirus: stereospecific synthesis and antiviral activity short synthesis and anti-rhinoviral activity of imidazo[1,2-a]pyridines: the effect of acyl groups at 3-position synthesis, antiviral activity, and biological properties of vinylacetylene analogs of enviroxime synthesis and antiviral activity of c2 analogs of enviroxime: an exploration of the role of critical functionality sensitivity of rhinoviruses to human leukocyte and fibroblast interferons cell and virus sensitivity studies with recombinant human alpha interferons comparative susceptibility of respiratory viruses to recombinant interferons-alpha 2b and -beta intranasal interferon-alpha 2 for prevention of natural rhinovirus colds intranasal interferon alpha 2 for prevention of rhinovirus infection and illness interferon-beta ser as prophylaxis against experimental rhinovirus infection in volunteers an effective dosage regimen for prophylaxis against rhinovirus infection by intranasal administration of huifn-alpha 2 efficacy and tolerance of intranasally applied recombinant leukocyte a interferon in normal volunteers intranasally applied recombinant leukocyte a interferon in normal volunteers. ii. determination of minimal effective and tolerable dose recombinant human interferon-gamma as prophylaxis against rhinovirus colds in volunteers ineffectiveness of postexposure prophylaxis of rhinovirus infection with low-dose intranasal alpha 2b interferon in families a randomized controlled trial of glucocorticoid prophylaxis against experimental rhinovirus infection synergism between anti-rhinovirus antivirals: various human interferons and a number of synthetic compounds failure to demonstrate synergy between interferon-alpha and a synthetic antiviral, enviroxime, in rhinovirus infections in volunteers property distributions: differences between drugs, natural products, and molecules from combinatorial chemistry a new rapid and effective chemistry space filter in recognizing a druglike database rational selection of structurally diverse natural product scaffolds with favorable adme properties for drug discovery natural products as sources of new drugs over the last 25 years ethnomedicinal antivirals: scope and opportunity plant substances as antiviral agents: an update plant substances as antiviral agents host defense function of the airway epithelium in health and disease: clinical background herb sales down 6 percent in mainstream market echinacea for preventing and treating the common cold echinacea for preventing and treating the common cold. the cochrane database of systematic reviews evaluation of echinacea for the prevention and treatment of the common cold: a meta-analysis echinacea in the prevention of induced rhinovirus colds: a meta-analysis the role of alkamides as an active principle of echinacea alkylamides from echinacea are a new class of cannabinomimetics: cannabinoid type 2 receptordependent and -independent immunomodulatory effects synergistic anti-oxidative effects of alkamides, caffeic acid derivatives, and polysaccharide fractions from echinacea purpurea on in vitro oxidation of human low-density lipoproteins echinacea extracts modulate the pattern of chemokine and cytokine secretion in rhinovirus-infected and uninfected epithelial cells modulation of immune response gene expression by echinacea extracts: results of a gene array analysis preventing the common cold with a garlic supplement: a double-blind, placebocontrolled survey in vitro virucidal effects of allium sativum (garlic) extract and compounds enhancement of in vitro human immune function by allium sativum l. (garlic) fractions efficacy of cold-fx in the prevention of respiratory symptoms in community-dwelling adults: a randomized, doubleblinded, placebo controlled trial efficacy of an extract of north american ginseng containing poly-furanosyl-pyranosyl-saccharides for preventing upper respiratory tract infections: a randomized controlled trial safety and tolerability of north american ginseng extract in the treatment of pediatric upper respiratory tract infection: a phase ii randomized, controlled trial of 2 dosing schedules inhibitory effect of ginsenoside rg3 and its derivative ginsenoside rg3-2h on no production and lymphocyte proliferation effect of cvt-e002 (cold-fx) versus a ginsenoside extract on systemic and gut-associated immune function inhibitory effect of ginseng polysaccharides on rotavirus infection antiviral activity of dammarane saponins against herpes simplex virus i effect of a traditional japanese herbal medicine, hochu-ekki-to (bu-zhong-yi-qi tang), on immunity in elderly persons effects of five kampohozais on the mitogenic activity of lipopolysaccharide, concanavalin a, phorbol myristate acetate and phytohemagglutinin in vivo immunological restoration and anti-tumor effect by japanese herbal medicine in aged mice antiinflammatory effects of bu-zhong-yi-qi-tang in patients with perennial allergic rhinitis hochu-ekki-to inhibits rhinovirus infection in human tracheal epithelial cells efficacy of a pelargonium sidoides preparation in patients with the common cold: a randomized, double blind, placebo-controlled clinical trial kern winfried v. pelargonium sidoides extract for acute respiratory tract infections traditionally used pelargonium species: chemistry and biological activity of umckaloabo extracts and their constituents aqueous ethanolic extract of the roots of pelargonium sidoides-new scientific evidence for an old anti-infective phytopharmaceutical efficacy of an aqueous pelargonium sidoides extract against herpesvirus protective effect of a natural carrageenan on genital herpes simplex virus infection in mice polysaccharides as antiviral agents: antiviral activity of carrageenan iota-carrageenan is a potent inhibitor of rhinovirus infection recommendations for reporting randomized controlled trials of herbal interventions: explanation and elaboration ethnobotany and the search for new drugs: ciba foundation symposium 185 ethnobotany and natural products: the search for new molecules, new treatments of old diseases or a better understanding of indigenous cultures? how scientific is the science in ethnopharmacology? historical perspectives and epistemological problems pedanius dioscorides of anazarbus-de materia medica sesquiterpene coumarins from ferula assafoetida l antiviral flavonoid from pterocaulon sphacelatum, an australian aboriginal medicine in silico target fishing for rationalized ligand discovery exemplified on constituents of ruta graveolens l antiviral activity of natural occurring flavonoids in vitro antiviral agents from higher plants and example of structure-activity relationship of 3-methoxyflavones 0 -hydroxy-3-methoxyflavones with potent antipicornavirus activity biodiversity: a continuing source of novel drug leads structure and stereochemistry of thysanone: a novel human rhinovirus 3c-protease inhibitor from thysanophora penicilloides discovery, total synthesis, hrv 3c-protease inhibitory activity, and structure-activity relationships of 2-methoxystypandrone and its analogues antiviral activity of raoulic acid from raoulia australis against picornaviruses antipicornavirus flavone ro 09-0179 cinnamoyl-and hydroxycinnamoyl amides of glaucine and their antioxidative and antiviral activities antiviral activity of flavones and flavans present status and prospects of flavonoids as antiviral agents inhibition of an early stage of rhinovirus replication by dichloroflavan (bw683c) effect of dichloroflavan (bw683c) on the stability and uncoating of rhinovirus type 1b failure of intranasally administered 4 0 , 6-dichloroflavan to protect against rhinovirus infection in man failure of oral 4 0 ,6-dichloroflavan to protect against rhinovirus infection in man neural networks as data mining tools in drug design chemoinformatics and drug discovery methods and principles in medicinal chemistry the impact of informatics and computational chemistry on synthesis and screening enhancing drug discovery through in silico screening: strategies to increase true positives retrieval rates neural networks for chemists the protein data bank the protein data bank (pdb), its related services and software tools as key components for in silico guided drug discovery development of antiviral agents using molecular modeling and virtual screening techniques comparative molecular field analysis (comfa). 1. effect of shape on binding of steroids to carrier proteins investigation on qsar and binding mode of a new class of human rhinovirus-14 inhibitors by comfa and docking experiments synthesis and structure-activity studies of some disubstituted phenylisoxazoles against human picornavirus comfa analysis of the interactions of antipicornavirus compounds in the binding pocket of human rhinovirus-14 dihydro-2-oxazolyl)phenoxy]alkyl]-3-methylisoxazoles: inhibitors of picornavirus uncoating understanding human rhinovirus infections in terms of qsar site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3c cysteine protease and cellular trypsin-like serine proteases the picornaviral 3c proteinases: cysteine nucleophiles in serine proteinase folds substituted benzamide inhibitors of human rhinovirus 3c protease: structure-based design, synthesis, and biological evaluation structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 6. structure-activity studies of orally bioavailable, 2-pyridone-containing peptidomimetics glossary of terms used in medicinal chemistry (iupac recommendations 1997) methods and principles in medicinal chemistry human rhinovirus 3c protease: generation of pharmacophore models for peptidic and nonpeptidic inhibitors and their application in virtual screening docking versus pharmacophore model generation: a comparison of highthroughput virtual screening strategies for the search of human rhinovirus coat protein inhibitors ca: accelrys. 358. discovery studio rollinger extended her studies to the fields of phytochemistry, ethnopharmacology, molecular modelling and its application in natural product science completing the habilitation thesis entitled ''the search for bioactive natural products rollinger is a project leader and an associate in various national projects, as well as executive guest editor for current pharmaceutical design. she has been awarded the phoenix science award (2005) and received several additional awards until 1994 she was a postdoctoral research fellow at the institute of immunology, university marburg, where she studied picornavirusimmune cell interactions and the virus-induced cytokine induction. thereafter, she was a postdoctoral fellow at the institute of virology and antiviral therapy key: cord-017364-d9zmdm23 authors: crowe, james e.; williams, john v. title: paramyxoviruses: respiratory syncytial virus and human metapneumovirus date: 2014-02-27 journal: viral infections of humans doi: 10.1007/978-1-4899-7448-8_26 sha: doc_id: 17364 cord_uid: d9zmdm23 human respiratory syncytial virus (rsv) and human metapneumovirus (mpv) are members of the family paramyxoviridae of the mononegavirales order, comprising the nonsegmented negative-strand rna viruses. paramyxoviridae has two subfamilies: paramyxovirinae, which includes the parainfluenza viruses 1–4 and measles and mumps viruses, and pneumovirinae, which includes rsv and mpv. pneumovirinae has two genera: pneumovirus, which includes human rsv, bovine respiratory syncytial virus, and pneumonia virus of mice, and metapneumovirus, which includes human mpv and avian metapneumovirus, sometimes called avian pneumovirus. human respiratory syncytial virus human respiratory syncytial virus (rsv) and human metapneumovirus (mpv) are members of the family paramyxoviridae of the mononegavirales order, comprising the nonsegmented negative-strand rna viruses. paramyxoviridae has two subfamilies: paramyxovirinae , which includes the parainfl uenza viruses 1-4 and measles and mumps viruses, and pneumovirinae , which includes rsv and mpv. pneumovirinae has two genera: pneumovirus , which includes human rsv, bovine respiratory syncytial virus, and pneumonia virus of mice, and metapneumovirus , which includes human mpv and avian metapneumovirus, sometimes called avian pneumovirus. rsv was isolated fi rst in 1956 from an ill chimpanzee in a laboratory setting that had an illness similar to the common cold. a virus causing a similar cytopathic effect in cultured cells was recovered from infants with respiratory illness shortly after, and studies of human antibodies in the serum of infants and children indicated that infection was common early in life [ 1 , 2 ] . now it is known that rsv is the most common viral agent of serious pediatric respiratory tract disease worldwide. in most areas of the world, rsv is the most common cause of pneumonia and bronchiolitis in infants less than 1 year of age. rsv causes severe disease in young infants, but disease is not restricted to the early life period. the virus can cause severe lower respiratory tract illness in large numbers of elderly subjects and also in subjects who are severely immunocompromised such as hematopoietic stem cell transplant recipients [ 3 -7 ] . mortality in infants and children caused by rsv is uncommon in developed countries with modern critical care units. estimates of the mortality rate are about 0.3 % of hospitalized children or less. mortality has been dropping over the last several decades, and by the late 1990s the estimated number of deaths in the usa was several hundred children a year or less [ 8 , 9 ] . large epidemiologic studies report that the us mortality in children may be only about 100 cases a year. interestingly, while many providers think of rsv infection as principally a pediatric illness, there are over 17,000 deaths per year in the elderly, making them the highest risk population for death [ 9 ] . in less developed countries, however, infant deaths due to rsv infection may be more common. infants with underlying illness are at highest risk among young children for morbidity and mortality from rsv infection. infants with chronic lung disease requiring supplemental oxygen following treatment for prematurity, due to bronchopulmonary dysplasia, are perhaps at the highest risk for prolonged, severe, or fatal illness due to rsv [ 10 ] . infants with severe congenital pulmonary or cardiac disease have been reported to be at risk of death in 3-4 % of cases when hospitalized, although this rate is likely decreasing in the usa [ 11 ] . both children and adults with primary immunodefi ciency or medically induced immunosuppression are at high risk of mortality due to rsv infection. the most severely immunocompromised, and thus those at highest risk of mortality, are hematopoietic stem cell transplant patients of any age [ 12 ] . in some settings, the mortality rate of rsv infection in hematopoietic stem cell transplant patients with severe immunosuppression verges on 100 % [ 12 ] . hospitalization rates of infants for rsv disease vary with the setting, probably due to variations in exposure, genetics, socioeconomic, and other risk factors and due to the local practice style of medical providers. many developed countries report hospitalization rates of about 1 in 100-200 infected infants during the fi rst year of life [ 13 , 14 ] . studies of rsv disease in developed countries suggest that of those infants hospitalized, about 9 % require mechanical ventilation [ 15 , 16 ] . there are certain populations at extraordinarily high risk of hospitalization with rsv, for example, alaskan native infants younger than 1 year have been reported to have a hospitalization rate of 53-249 per 1,000 infants [ 16 ] . low socioeconomic status is a risk factor for higher rate of hospitalization in most areas. rsv also is one of the most common viral causes of serious lower respiratory tract illness in the elderly, especially in institutionalized subjects [ 17 ] . exacerbations of chronic obstructive pulmonary disease (copd) are frequently associated with rsv infection [ 18 , 19 ] . the elderly do not exhibit a remarkably diminished level of antibodies to rsv [ 20 ] . decreased levels of t cell memory in the elderly and specifi cally in patients with (copd) may contribute to the increased susceptibility to rsv infection in these populations [ 21 ] . many think of infl uenza virus as the principal viral respiratory pathogen in this population, but in a hospitalized cohort, infl uenza a virus and rsv infection resulted in similar mortality, lengths of stay, and rates of use of intensive care [ 17 ] . rsv infection accounted for over 10 % of hospitalizations for pneumonia. seroepidemiology studies suggest that virtually all children are infected in the fi rst 2 years of life, and early infection is especially common in infants attending group day care. serological methods are helpful in epidemiology and vaccine studies, but serologies are not often used for diagnosis in clinical settings. because of the transfer of maternal rsvspecifi c antibodies across the placenta, and the high prevalence of early infection, it is unusual to fi nd infants who are rsv seronegative. in older children and adults, a fourfold rise in serum antibodies is often used as evidence of rsv infection, but asymptomatic infections in which viral shedding is low in titer often are not accompanied by serum antibody rises. serological tests in infants are even less sensitive, because young infants may not exhibit a large or durable rise in antibodies. neutralizing antibody tests are considered the best functional marker of infection, but sensitivity is much higher in antigen binding assays using individually purifi ed rsv f or g proteins [ 22 ] . isolation of the virus in cell culture provides a defi nitive test for diagnosis of active infection. various methods of obtaining respiratory virus secretions for testing have been compared. most studies suggested that aspiration or gentle fl ushing of nasal secretions using a solution like saline is best, though some types of nasal swab have given reasonable results. the virus is more thermolabile than most, and thus samples should be transported on wet ice to the diagnostic laboratory and processed rapidly. prolonged times of transport to remote reference laboratories reduce the effectiveness of isolation. after inoculation onto susceptible cell culture substrates, highly trained staff can recognize cytopathology in the cell monolayers by visual inspection and conventional bright-fi eld microscopy after about 3-7 days of incubation. detection may be more effi cient when using shell vial cultures and immunofl uorescence [ 23 ] . various cell lines have been used for rsv detection, such as hep-2 epithelial cells, mrc-5 fi broblasts, and rhesus monkey kidney cells, but the r-mix commercial mix of human and mink lung cells may perform better for detection of rsv [ 24 ] . culture is expensive and requires highly trained staff and therefore is not usually available at the point of care, which is often an outpatient clinic or emergency department. therefore, rapid diagnostic methods were developed for the detection of viral proteins or rna in respiratory secretions. rsv antigen tests mostly rely on direct immunofl uorescent assays (dfa) on exfoliated cells in secretions or enzyme immunoassay (eia). nucleic acid detection assays based on rt-pcr are now widely available for rsv, often in a multiplexed panel for detection of multiple respiratory virus pathogens. these tests are typically more sensitive than any of the virus isolation or protein-based detection assays discussed above. enhanced sensitivity is especially helpful when testing adults, who often shed virus in very low titers. positive rt-pcr tests need to be interpreted in the context of the clinical scenario, since the tests can remain positive for prolonged periods of time after infection, well beyond the period during which infectious virus can be isolated. since children may experience symptomatic respiratory infections every few weeks during the winter, caution must be used in interpretation of positive pcr tests, especially when multiple viruses are detected simultaneously in a sample. some instances of multiple pcr test positivity likely represent residual rna from a previous virus infection and a second rna type representing live virus from the active current infection. rsv typically is propagated in monolayer cell cultures of continuous cell lines of human epithelial cell origin, such as hep-2 cells. monkey kidney cells of various types are also used commonly for propagation in the laboratory. in fact, the virus replicates to some extent in most cell lines of mammalian origin. in non-polarized epithelial cells, the virus often causes a typical cytopathic effect in which multinucleated giant cells form due to cell-cell fusion, termed cell syncytia. this in vitro effect is the origin of the virus name, although it is not clear that rsv causes syncytia in vivo. in polarized epithelial cells in culture, the virus assembles and buds from the apical surface of cells, mimicking to some extent the budding of virus into the lumen of the airway. the virus has a negative-sense single-stranded rna genome with 10 genes encoding 11 proteins. figure 26 .1 compares the genomes of rsv and mpv, which are similar in many respects. the replication proteins are common to both of the viruses, as are the matrix (m) protein and the surface fusion (f) and glycosylated attachment (g) glycoproteins. the gene order differs slightly, and rsv possesses two nonstructural (ns) genes ns1 and ns2 that are not present in mpv. the functions of these genes are not fully defi ned, but involve interactions with the host response machinery, especially interferons. the presence of these host response-modifying genes may explain in part why rsv appears to cause severe disease more commonly than mpv. many of the gene sequences exhibit some clear global sequence relatedness; however, the extent of the relatedness of many of the sequences of corresponding proteins is relatively low. based on sequence homology, it is not expected that there is a signifi cant amount of immunologic cross-reactivity in responses to the two viruses. the rsv virion buds from airway epithelial cells, incorporating host cell membrane as the lipid bilayer that forms the envelope of the particle. since the virus is enveloped, chemicals that disrupt lipid bilayers (detergents) inactivate the virus, leading to the strong recommendations for healthcare provider hand washing following patient contact. the genome is a single strand of rna, which forms a helical complex with the nucleoprotein (n). the fi nal nucleocapsid structure likely is formed by the complete set of replication proteins, which also include the phosphoprotein (p) and the large rna-dependent rna polymerase (l). it is suspected that the m protein helps the particle to form by bridging the nucleocapsid and the lipid envelope with its incorporated surface proteins. the surface of the particle incorporates three integral membrane surface glycoproteins, the highly glycosylated rsv g protein suspected to be the attachment protein, the fusion protein f, and the small hydrophobic protein (sh). rsv f (a type i integral membrane protein) and rsv g (a type ii integral membrane protein) form oligomers on the surface of the particles, which appear like small spikes with globular heads when seen in electron microscopic (em) images by negative stain. the morphology of particles in em images or in fl uorescent microscopy images labeled by conjugated antibodies suggests that the virion particles are fi lamentous. however, spherical particles, fi laments, and more pleomorphic forms have been observed; therefore, it is uncertain what the morphology of infectious particles in vivo during natural infection is. the f protein is critical for entry into cells, by breaching the barrier of the cell lipid bilayer. it is thought that binding of virion particles to susceptible cells at physiologic ph triggers a conformational change of the f protein from a metastable pre-fusion state [ 25 ] to an altered post-fusion conformation [ 26 , 27 ] in which the hydrophobic fusion peptide in the protein is exposed and extended to insert into the host cell membrane. this membrane insertion event accomplishes a fusion of the viral and host membranes, allowing delivery of the nucleocapsid to the cytoplasm where viral replication occurs. this event is termed "fusion from without" when the particle enters a cell. an alternative fusion event ("fusion from within") occurs when newly expressed f protein on the surface of infected cells causes fusion of an infected cell to an adjacent cell in culture causing "syncytia." it is not certain whether this latter form of fusion (cell-cell fusion) occurs during natural infection and contributes to pathogenesis or if the formation of syncytia is a tissue culture phenomenon only. although there are many animal forms of rsv, there is no known animal reservoir of human rsv; close contact with infected humans is the only source of rsv infection. early prospective studies showed that approximately half of infants in the usa are infected during their fi rst year of life; most were infected after two winter epidemics [ 28 ] . about a quarter of infants exhibit signs and symptoms of lower respiratory tract disease (wheezing and/or pneumonia) during a primary rsv infection [ 14 , 28 -31 ] . rsv is the most common virus associated with hospitalization for respiratory illness and in fact is one of the most common of all causes of infant hospitalization. for example, rsv caused 13 hospitalizations per 1,000 us children younger than 1 year in one large study [ 32 ] . during winter rsv epidemics, over 75 % of the children hospitalized for acute lower respiratory tract infection are infected with rsv [ 33 , 34 ] . the rate of very severe disease in hospitalized infants is high, with about 2-5 % of hospitalized infants requiring mechanical ventilation for respiratory failure [ 35 ] . although primary infection of infants is probably most efficient, rsv can infect subjects of any age [ 28 , 36 , 37 ] . some adult infections are asymptomatic, and most are limited symptoms related to infection of the upper respiratory tract, such as the common cold [ 28 , 36 , 38 ] . since otherwise healthy adults all possess measurable rsv serum antibodies and rsv-specifi c t cells, it is clear that prior exposure and induction of immune responses may not prevent infection. however, disease severity is usually reduced after one or several infections early in life, thus immune effectors such as serum neutralizing antibodies do prevent severe disease during reinfections. unlike infl uenza virus, rsv does not exhibit a progressive antigenic drift. although rsv antigenic diversity is observed in fi eld strains, diversity of the antigenic proteins is not required for rsv to cause reinfection [ 39 -41 ] . most experts believe that serum neutralizing antibodies protect against severe lower respiratory tract disease but do not result in sterilizing immunity against infection at the respiratory mucosa. thus, healthy adults show signs and symptoms of the common cold in about half of cases of natural or experimental infections, even though they have experienced numerous previous rsv infections [ 39 ] . there is a single serotype of rsv, but two antigenic subgroups of rsv, with about 25 % antigenic relatedness, have been defi ned using immune sera; the subgroups are designated a and b. antigenic dimorphism for rsv had been noted in an early study [ 42 ] and subsequently was delineated using mabs, which identifi ed extensive differences in the g protein and less extensive differences in f and other proteins [ 43 ] . the two subgroups exhibit a three-to fourfold reciprocal difference in neutralization by polyclonal convalescent serum. analysis of glycoprotein-specifi c responses in experimental rodent models or human infants by enzymelinked immunosorbent assay (elisa) with purifi ed f and g glycoproteins showed that the f proteins are 50 % related antigenically and the g proteins are 1-7 % related [ 44 ] . consistent with this level of antigenic relatedness, f protein expressed by a vector immunization was equally protective in small animals against challenge with the homologous or heterologous subgroup virus, whereas the g protein was 13-fold less effective against the heterologous subgroup virus [ 45 ] . thus, the f protein is responsible for most of the observed cross-subgroup neutralization and protection. in some communities a pattern of infection with viruses of alternating subgroups has been described, but this is not a universal phenomenon. rsv subgroup b virus is more diffi cult to isolate and propagate in culture, so subgroup b viruses are less commonly associated with severe disease in some studies. however, clearly viruses of both subgroups can cause severe disease leading to hospitalization [ 46 -50 ] . infants exhibit the highest risk of severe lower respiratory tract disease. this elevated risk is explained by a myriad of physiologic, immunologic, and other factors. first, the highest point of resistance in the airways is the bronchioles, and the resistance of airways is inversely proportional to the airway radius to the fourth power (resistance ~ 1/radius 4 , from poiseuille's law). the bronchioles of infants are narrow, and infl ammation and secretion in the bronchioles leads to turbulent airfl ow, and further reduction of the airway lumen size. these physical factors lead to the clinical signs of wheezing (a sign of outfl ow obstruction) and air retention. increased respiratory rate can compensate to some extent for respiratory compromise, but prolonged tachypnea can lead to fatigue and eventual respiratory failure. also, during primary infection infants do not possess active immunity to infection, which allow this effi cient virus to replicate in the airway to titers as high as 10 7 infectious particles per ml of secretion. mothers pass rsv antibodies to infants across the placenta during the third trimester, but premature infants do not obtain maternal antibodies prior to 32 weeks' gestation. also, the airways of premature infants are smaller than those of term infants. another factor leading to respiratory diffi culty is dehydration. obstruction of the nasal passages with thick secretions can impede the feeding of infants, who are obligate nose breathers. rsv also is an important cause of serious infection in elderly adults; in fact rsv appears to cause substantially more fatalities in elderly adults than in children [ 17 , 51 ] . as above, a large study of the us population showed that rsv was associated with approximately 17,000 all-cause deaths per year among persons of all ages in the usa [ 9 ] , with most of those deaths in the elderly. virtually any serious medical or genetic condition is associated with some increased risk of severe diseases [ 32 ] . certain particular categories of subjects are at highest risk for severe rsv disease, including infants with congenital heart or chronic lung disease, immunodefi cient subjects of any age and the elderly [ 9 , 10 , 17 , 33 , 52 -57 ] . these latter subjects are thought to have reduced competency of rsv-specifi c t cells. prematurity increases risk to a small extent, but more importantly chronic lung diseases are important factors [ 10 , 33 , 56 , 58 -62 ] . infants who are born prematurely and then suffer persistent chronic lung disease, especially those needing oxygen supplementation, are at very high risk of hospitalization with rsv. children with cystic fi brosis are at high risk of severe disease [ 63 , 64 ] . in children younger than 2 years with cystic fi brosis, the consequence of rsv infection may be prolonged respiratory morbidity [ 65 ] . children with congenital heart disease also are at increased risk [ 33 , 56 , 58 , 62 , 66 , 67 ] . rsv is a common cause of severe respiratory disease in immunocompromised patients, including lower respiratory disease [ 68 ] . mortality rates in some populations of immunocompromised patients verges on 50 % [ 7 , 69 ] . infants with congenital severe combined immunodefi ciency are at special risk [ 57 , 70 ] , but any acquired immunosuppressive condition such as cancer or transplantation puts patients at risk, especially when t cell function is compromised [ 68 , 71 , 72 ] . rsv infection can cause severe lung disease in recipients of lung transplants, sometimes with a long-term outcome of obliterative bronchiolitis [ 73 -75 ] . children with hiv infection shed rsv for extended periods, but disease is not especially severe in hiv-infected children prior to onset of aids [ 76 -78 ] . interestingly, although most immunocompromised subjects appear to be at risk because of t cell problems, infants with phagocytic cell defects including those with interferon-γ receptor defi ciency or chronic granulomatous disease also are at risk of severe rsv disease. transmission of rsv in the hospital setting can lead to serious disease, especially in critical care units with neonates or other high-risk infants [ 79 -86 ] . nosocomial outbreaks in inpatient transplantation facilities are sometimes severe and unit outbreaks can be diffi cult to terminate because transplant patients can shed rsv for many months [ 57 , 58 , 69 , 84 , 87 , 88 ] . theoretically, transmission in inpatient healthcare settings should be preventable through strict compliance with infection control practices, especially hand washing and contact precautions, which are universally recommended for rsv patients. a high level of compliance with precautions is diffi cult to achieve in busy care settings but is needed to prevent transmission by healthcare providers. the use of the prophylactic monoclonal antibody palivizumab has been studied to interrupt an outbreak in a neonatal intensive care unit setting [ 89 ] , but currently palivizumab use is not recommended for this purpose. bacterial otitis media is a common complication of rsv upper respiratory tract infection. in fact, rsv infection is probably the most common precipitating factor associated with otitis media. rsv antigens and nucleic acids have been reported in middle ear fl uids [ 90 , 91 ] . the disease is predominantly due, however, to eustachian tube dysfunction, resulting in bacterial stasis in the middle ear and subsequent otitis media. rsv regularly occurs in annual epidemics. the us national respiratory and enteric virus surveillance system (nrevss) considers that the rsv season starts when the fi rst of two consecutive weeks during which the mean percentage of specimens testing positive for rsv antigen is ≥10 %. the rsv season is considered to have ended in a community when the mean percentage of positive specimens is ≤10 % in reference laboratories for two consecutive weeks. rsv infection occurs in infants and adults worldwide, in yearly epidemics. the virus has been isolated in every area of the world in which surveillance studies have been conducted. the principal season varies depending on the climate and region, but infection is ubiquitous. virtually all children in the world are infected within the fi rst few years of life. epidemics occur in the winter and early spring in the usa. onset of the annual epidemic varies by region of the country and by year but typically begins in the usa in october or november and lasts through late spring. within a region, the timing of rsv season changes slightly each year. florida often has the earliest onset of rsv epidemic and the longest lasting season in the usa. near the equator, infections may be more common during rainy season. during community outbreaks of rsv, the venues with the highest level of young infants and children exhibit the highest rates of transmission of virus, especially large families and day-care settings with large numbers of children per room. hospital infection control practices must be used to prevent rsv spread, using measures including careful hand hygiene, contact precautions for patient isolation, gowns and gloves [ 92 , 93 ] , and, when direct coughing occurs, facemasks and goggles [ 94 , 95 ] . rsv rapid diagnostic testing can be used in hospital infection control practice to identify rsv-infected patients during the admission process [ 96 , 97 ] . rsv is shed for prolonged periods. it has been reported that 92-100 % of hospitalized children are still shedding infectious virus after 7 days [ 98 , 99 ] . exposure to tobacco smoke and poor nutrition increase the incidence of disease [ 100 -102 ] . low socioeconomic status increases the risk of severe disease for uncertain reasons. lower income populations exhibit a fi ve-to tenfold increased risk of hospitalization in many studies [ 14 , 32 , 103 ]. breast-feeding may confer some protective benefi t against rsv disease, but the extent of the benefi t of breast-feeding has been controversial. the results of epidemiologic studies on benefi t have been confl icting, although recent studies suggest that breast-feeding may have a strong protective effect only in girls [ 104 ] . the sex of the infant modulates the severity of rsv infection for additional reasons that are not fully understood. even though the same high proportion of both male and female infants become infected, males have a higher incidence of rsv lower respiratory tract disease than girls [ 30 , 32 , 105 -108 ] . ethnic and genetic factors appear to play a role. alaska and other native american children are at increased risk of severe respiratory disease during rsv infection [ 109 ] . direct contact with secretions from an infected human, usually by fomites (contaminated objects, including hands), allows transmission of virus. in some cases, infected subjects may inoculate contacts at short distance by coughing via large-particle droplets. however, the virus is not spread efficiently by small-particle aerosols [ 110 , 111 ] . the nasal and conjunctival mucus membranes are probably the most common portals of entry [ 112 ] . rsv is one of the most infectious viruses spread by contact, and transmission is very effi cient even among subjects who possess partial immunity due to prior infection. spread among family members and day-care contacts is especially common. the infectious doses for humans are probably only a few infectious particles per ml of respiratory secretions, but infants routinely shed at least a millionfold higher concentration of virus during the peak of illness. the incubation period is not known defi nitely but is about 3-6 days and likely varies according to the intensity of exposure and the amount of virus in the inoculum. the period of viral shedding is many days; infants can shed infectious virus for weeks [ 98 , 99 ] . rsv can survive on hard surfaces for greater than 24 h. infection occurs by inoculation of the nasal or conjunctival mucosa, often by self-inoculation with infected secretions from a close contact. adult volunteers were infected experimentally if virus was inoculated onto the conjunctival sac or into the nose, but not following introduction into the mouth [ 112 ] . the incubation period from time of inoculation to onset of illness for rsv is likely about 4-5 days [ 113 ] . virus replication initiates in the nasopharynx and rapidly can reach concentrations of over a million particles per ml in the upper airway in children. adults, who are partially immune, typically shed much lower amounts of virus. virus spreads quickly to the lower respiratory tract, often causing symptoms within days of onset of upper respiratory symptoms. virus may spread from cell to cell, but also it is likely that small aspirations of upper respiratory secretions with virus inoculate the lower tract. higher titers of virus in respiratory secretions usually are associated with increased severity of disease, in prospective studies of natural infection [ 114 ] or of clinical vaccine trials [ 115 ] . rsv infection is an acute infection and virus shedding usually resolves within days to weeks. rt-pcr tests for virus nucleic acid may remain positive for prolonged periods. some animal studies suggest that a negligible amount of infectious virus may persist in airways far after apparent resolution of shedding, as evidenced by the recovery of low amounts of infectious virus during immunosuppression several months after infection [ 116 ] . the virus infects respiratory epithelial cells in the lung and airway. it is not clear whether rsv also replicates productively in macrophages and dendritic cells in the airway or not. rsv protein antigens have been detected in circulating mononuclear cells [ 117 ] , and viral genomic rna and mrna have been detected by rt-pcr in blood cells [ 118 ] , but this probably represents cells from the airway recirculating, as infectious virus in the blood (viremia) is not detected. rsv replicates in the cells at the luminal surface of the respiratory epithelium and virus both enters and is shed from the apical surface of infected epithelial cells [ 119 , 120 ] . studies of polarized, differentiated respiratory epithelial cells in vitro show that rsv infection preferentially infects ciliated cells at the luminal face [ 121 ] , but it is not clear if infection is restricted to such cells in humans. histopathology studies of infected humans are very limited, but show rsv antigens in the superfi cial cells of the airway in a patchy distribution, with antigen-positive cells and debris in the airway lumen. pathology caused by rsv infection during infant bronchiolitis includes necrosis and proliferation of the bronchiolar epithelium and destruction of ciliated epithelial cells [ 120 , 122 ] . there is an infl ux of a large variety of immune cell types including neutrophils, lymphocytes, and macrophages. the respiratory tissues become edematous. mucous secretion, sloughing of dead cell debris, and the infl ux of apparent infl ammatory cells obstruct the lumen of the narrow bronchioles and alveoli. the small diameter of infant bronchioles is easily obstructed in the presence of dead cells and edema of the airway tissues [ 123 ] . virus-infected cells can be identifi ed in the epithelium of the bronchi, bronchioles, and alveoli [ 120 ] . it is surprising, given the extent of disease, that rsv antigen staining is usually patchy or focal, and even in some cases of fatal rsv bronchiolitis, antigen is present only in small amounts [ 119 , 120 ] . the number of cases that have been collected is limited, however, and there is virtually no histopathology from milder cases. rsv upper respiratory infection is complicated frequently by otitis media caused by bacteria. it is unusual to observe frank bacterial pneumonia or sepsis as a complication of rsv infection, in contrast to some other respiratory viruses like infl uenza. although some clinicians may empirically use antibiotics during infant pneumonia, there is no evidence that antibiotic therapy alters the course of rsv bronchiolitis or pneumonia. antimicrobial therapy should not be used in most cases of rsv bronchiolitis or pneumonia because of the lack of benefi t and risk of selection of antibiotic-resistant colonizing organisms. nevertheless, there is some suggestion that bacterial-viral interactions may affect the overall rate of disease in the population. it is interesting that annual rsv and infl uenza virus epidemics correlate directly with the time of peak incidence of invasive pneumococcal disease in many population studies [ 124 ] . a double-blind, randomized, placebo-controlled trial of pneumococcal conjugate vaccine showed a vaccine-attributable reduction in rates of childhood viral pneumonia requiring hospitalization, caused by any of seven respiratory viruses, including rsv [ 125 ] . the immune mechanisms responsible for resolution of infection and protection against reinfection by rsv are not fully defi ned. antibodies . most experts agree that high levels of serum neutralizing antibodies are associated with relative protection against severe lower respiratory tract disease in otherwise healthy subjects. this idea is supported strongly by the observation that prophylaxis of high-risk infants with a neutralizing monoclonal antibody prevents about half of hospitalizations in that group of patients. many population studies suggest that infants born with high levels of transplacental rsv-neutralizing maternal antibodies develop milder illness or illness at an older age than infants with low maternal antibody levels [ 15 ] . most infants and children in whom maternal antibodies have declined to a low level make their own serum and secretory antibodies to both the f and g surface glycoproteins in response to rsv infection [ 126 ] . antibody responses in neonates are particularly low in quality and magnitude due to immunologic immaturity and the suppressive effect of passively acquired maternal antibodies [ 126 , 127 ] . antibody-mediated immune suppression by passive antibodies primarily affects humoral rather than cellmediated immunity [ 128 , 129 ] . high levels of serum antibodies do not appear to provide solid immunity against disease in the upper respiratory tract. mucosal secretory iga appears to contribute to local protection against reinfection in the airway, although potent protective iga responses are likely relatively short lived. in human infants, the decrease in virus shedding in nasal secretions was associated with the appearance of rsv-specifi c iga antibodies [ 130 ] . t cells . t cells clearly play a major role in resolution of active infection. rsv-specifi c t cells with cytolytic activity, thought to be cd8+ t cells, have been detected in peripheral blood mononuclear cells from infants with rsv disease [ 131 ] . immunodefi cient children, especially those with t cell defects, often fail to clear rsv infection and can shed virus for many months [ 57 ] . adults with leukemia or hematopoietic stem cell transplant also have a very high incidence of prolonged rsv infection leading to severe disease and sometimes death. patterns of host response rsv infection usually causes upper respiratory tract symptoms during primary infection in otherwise healthy term infants; asymptomatic primary infection is not common. there is often profuse rhinorrhea, and the upper tract disease is very often complicated by otitis media. symptoms of lower respiratory tract involvement occur in about a third of primary cases [ 28 ] . the principal diagnoses are bronchiolitis (manifested by tachypnea and wheezing) and pneumonia. these entities are probably not discrete processes but more likely represent a continuum of disease involving increasing tissue distribution. the typical illness starts with nasal congestion, followed in a few days by cough. the infection is sometimes associated with fever, which is usually low grade. after several days of upper tract symptoms, infants may wheeze. many infants suffer mild wheezing that resolves, but some cases progress with tachypnea, diffuse inspiratory crackles, and expiratory wheezes. most children recover in 1-2 weeks with supportive care and observation. if expiratory obstruction becomes severe, however, hyper-expansion of the chest occurs due to air retention, and the compliant nature of the infant chest wall leads to intercostal and subcostal retractions during tachypnea. with prolonged tachypnea, fatigue may occur with poor oxygenation and co 2 retention (measured by pulse oximeter or arterial blood gas measurement), markers of respiratory failure. intubation and mechanical ventilation is used in this setting to manage the respiratory failure. infection during the fi rst day or weeks of life may just be characterized by temperature instability or fever, irritability, and lethargy even in the absence of overt respiratory signs or symptoms. in very young infants, especially those born prematurely, apneic spells may occur in response to rsv infection. apnea may be the fi rst reported evidence of infection in some cases, and apneic spells may recur during the acute infection. these events thankfully are usually self-limited and rarely cause neurologic damage. apneic events are an indication for hospitalization and careful medical supervision with respiratory monitoring. because of the association with apnea, some have considered whether rsv is associated with sudden infant death syndrome (sids). although rsv has been detected in the lung tissues of some cases of sids, there is no statistically signifi cant association between rsv and sids. the reported cases likely refl ect a temporal association caused by the high prevalence of rsv in this age group, the prolonged pattern of shedding, and the simultaneous peak incidence of both rsv infection and sids in winter months [ 132 ] . it is not clear whether infection with rsv causes prolonged abnormal pulmonary function during childhood or whether children with underlying predisposition to lower respiratory tract disease of all causes manifest their susceptibility fi rst to rsv because of the young age of rsv infection. certainly measureable pulmonary function abnormalities are common after rsv lower respiratory tract disease, and these fi ndings may persist for a decade or more [ 133 ] . recurrent wheezing is common during subsequent viral infections after severe rsv bronchiolitis or pneumonia, with an incidence of 10-50 % [ 134 ] . a large case-control study of 200 children hospitalized for bronchiolitis or pneumonia in which rsv was the most common cause found that 7 years later there was a strong predisposition in these subjects toward decreased pulmonary function, recurrent cough, wheezing, and bronchitis [ 135 ] . seminal prospective studies by martinez et al. involved measurement of pulmonary function in infants at birth and then found a strong correlation between prior lower pulmonary function and the development of wheezing during rsv infection [ 136 ] . this correlation persisted in children during the fi rst 3 years of life [ 136 ] . even some individuals who do not typically exhibit recurrent wheezing have postexercise or pharmacologically induced bronchial reactivity [ 137 ] , which may be responsive in part to bronchodilators [ 134 ] . symptomatic upper respiratory tract rsv infections manifested by common cold symptoms are common in otherwise healthy adults, especially in those with frequent exposure to small children [ 80 ] . in the elderly, particularly those with underlying medical diseases, severe pneumonia may occur, leading to hospitalization or even death. astute clinicians can often make a presumptive diagnosis of infection based on the clinical signs of wheezing or pneumonia in an infant during a local epidemic. laboratory testing of nasal or lower airway secretions by antigen test (elisa) or nucleic acid detection (by rt-pcr) provides rapid diagnosis of the presence of virus in many cases. the gold standard for diagnosis is isolation of the virus in cell culture, but this test is typically only available in referral laboratories because of the need for extensive equipment and a high level of technical expertise. control and prevention primary treatment is supportive care, which includes oral or intravenous hydration; monitoring of respiratory status, especially of oxygen saturation during tachypnea; use of supplemental oxygen; removal of secretions from the upper airway; and, in the case of respiratory failure, intubation and mechanical ventilation. advances in support care in pediatric critical care units have caused a major decrease in morbidity and mortality from rsv in the developed world. infants hospitalized for rsv disease should be monitored for apnea. investigators have studied nitric oxide [ 138 , 139 ] mixtures of helium and oxygen [ 140 , 141 ] and surfactant treatment [ 142 ] in clinical experimental studies in the support of infants with severe rsv disease. nitric oxide treatment does not appear to mediate a bronchodilator effect during rsv infection [ 139 ] . therapy of rsv disease by any antiviral agent is challenging because it is a rapid acute infection, and by the time the onset of disease is recognized, it may be too late to alter the course of disease by reducing viral load. the guanosine (ribonucleic acid) analog ribavirin is a nucleoside inhibitor that inhibits viral rna synthesis and viral mrna capping. the drug has in vitro antiviral activity against rsv, and aerosolized ribavirin therapy has been associated with a small but statistically signifi cant increase in oxygen saturation during the acute infection in several small studies. decreases in mechanical ventilation and duration of rsv-associated hospitalization have not been proven (reviewed in [ 143 ] ). ribavirin was approved in 1986 in the usa for treatment of rsv infection [ 144 ] . clinically, the drug usually is administered as a small-particle aerosol using a tent, mask, or mechanical ventilator, delivered for 6-18 h daily for a period of 3-7 days. the drug now is not recommended for routine use because follow-up studies have not shown a major benefi t. the drug may be considered for use in select patients with documented, potentially life-threatening rsv infection. over a dozen other experimental small molecule inhibitors of rsv fusion to cells have been described and tested in preclinical studies for inhibition of rsv, but none have progressed in development to date. a third approach employs short interfering rnas (sirnas), taking advantage of an ancient host cell regulatory system. single-stranded and double-stranded rna molecules that exhibit rsv-specifi c small interfering rnas have been developed for treatment, which cause rna interference activity against rsv, destroying the corresponding rsv rna. these novel compounds have shown promising results in preclinical studies [ 145 ] and have been tested in small clinical trials. human immune globulin with a high titer of rsv antibodies and the rsv monoclonal antibody palivizumab have been tested as therapy of acute rsv disease, but they were not effective for treatment of established disease. anti-infl ammatory strategies have been investigated. no benefi t of corticosteroid therapy on disease severity or length of hospital stay has been demonstrated, despite studies in over a dozen randomized clinical trials of outpatients or hospitalized infants with rsv bronchiolitis. since the drug is of no benefi t on its own [ 146 ] and may prolong virus shedding, it is not recommended. in the future, a possibility might be to combine an effective antiviral treatment with an antiinfl ammatory agent [ 147 ] . intravenous antimicrobial therapy is not appropriate in hospitalized infants with rsv bronchiolitis or pneumonia unless there is clear evidence of secondary bacterial infection. otitis media occurs very often in infants with rsv bronchiolitis; oral antimicrobial agents can be used for therapy of otitis media if necessary. it is intuitive to think of using beta-adrenergic agents, commonly used for the treatment of asthma, to treat the wheezing associated with rsv infection. these agents are not usually recommended for routine care of fi rst-time wheezing associated with rsv bronchiolitis. short-term improvements in oxygenation and clinical scores can be achieved by these therapies, but it has not been established that their use results in improvements in duration or severity of illness or disease outcomes. studies in this area have been confl icting, but systematic reviews of randomized clinical trials of nebulized beta-agonist therapy for treatment of bronchiolitis suggest that they offer little benefi t [ 148 , 149 ] . alpha-adrenergic receptor stimulation results may decrease interstitial and mucosal edema [ 150 ] , and use of nebulized epinephrine (with combined alpha-and beta-adrenergic activity) has been studied with confl icting results [ 151 , 152 ] . alphaagonist stimulation of the sympathetic nervous system is expected to reduce capillary leakage by constricting precapillary arterioles, reducing hydrostatic pressure and consequently bronchial mucosal edema [ 150 ] . racemic epinephrine treatment relieves some respiratory distress but does not affect length of stay [ 153 ] . the usefulness of such agents in the management of rsv bronchiolitis is not clear. the most effective mode of prevention is to avoid contact with infected subjects. in the hospital setting, careful adherence to infection control practices is important for the protection of high-risk patients from rsv infection. careful hand washing may reduce transmission in family and daycare settings. pharmacologic intervention is indicated to prevent hospitalization for the highest risk infants, however. passive rsv immunoprophylaxis with antibodies has proven a costly but relatively effective intervention. parenteral infusion of rsv-neutralizing antibodies into experimental animals was shown early on to confer substantial resistance in the respiratory tract to a subsequent rsv virus challenge [ 154 ] . signifi cant reductions in rsv-associated hospitalizations and disease severity in high-risk human infants were fi rst accomplished with prophylactic administration of human immunoglobulin with high rsv-neutralizing activity given by the intravenous route (rsv-ivig; fda licensed in 1996) [ 155 , 156 ] . monthly intravenous infusions during the rsv season reduced the frequency of pediatric hospitalization and duration of stay by approximately 55 % and decreased the number of days spent in intensive care by 97 %. the use of rsv-ivig was superseded by the use of a monoclonal antibody (mab) that was developed subsequently that could be given by intramuscular route, and production of the former has been discontinued. several mabs were developed for immunoprophylaxis against rsv. the most successful of these was based on murine mab 1129 [ 157 ] , which is specifi c to the f protein and effi ciently neutralizes viruses of both rsv subgroups a and b. this mab was humanized by recombinant methods by transferring its variable regions onto a human igg1 backbone, resulting in a recombinant antibody now named palivizumab [ 158 ] . this mab is 50-100-fold more effective for in vitro neutralization on a per weight basis than was rsv-ivig, and thus the total amount of immunoglobulin administered could be reduced to an amount that could be given im. palivizumab (trade name synagis) was licensed in 1998 for rsv prophylaxis of high-risk infants, following studies demonstrating its safety and effi cacy [ 159 -162 ] . palivizumab is administered monthly through the rsv season and has been widely used in high-risk patients with prematurity, chronic lung disease, and hemodynamically signifi cant heart disease [ 163 ] . more potent derivatives of this recombinant antibody have now been developed [ 164 ] ; however, the lead candidate from these affi nity maturation efforts exhibited increased side effects in a large effi cacy study. prevention of severe disease probably will best be accomplished by development and use of an effective vaccine. vaccine development for rsv has proven exceptionally diffi cult, however. first, young infants are a diffi cult population to immunize. obstacles to immunization at this early age include immunologic immaturity and immunosuppression by maternal antibodies, as already noted [ 165 ] . also, severe adverse events occurred in early rsv vaccine trials. a formalin-inactivated rsv vaccine candidate (fi-rsv) was developed and evaluated in infants and children in the 1960s [ 166 , 167 ] . this vaccine suspension was made by mixing concentrated, inactivated virus with alum adjuvant and was delivered by the intramuscular (im) route. this inoculation did not protect against infection or disease, but rather during subsequent natural infection vaccinees experienced more frequent and severe disease. most fi-rsv vaccinees (80 %) required hospitalization during subsequent natural infection, compared to 5 % in the control group. autopsies of two fatalities showed evidence of rsv replication and pulmonary infl ammation [ 167 ] . this event put a chilling effect on rsv vaccine development efforts. therefore, rsv protein vaccines have been problematic for use in infants given their possible potential for disease enhancement, together with the poor immunogenicity in this population. however, an rsv protein vaccine might be useful in boosting immunity in rsv-experienced older children and adults who are at increased risk of severe rsv disease due to underlying disease or advanced age. protein vaccines for rsv have been evaluated clinically for use in such rsvexperienced individuals, in whom they appear to be safe. one experimental subunit vaccine consisted of purifi ed f protein (pfp) isolated from rsv-infected cell culture. this purifi ed protein vaccine candidate was evaluated in adults, in older children with and without underlying medical diseases, and in the elderly [ 168 ] . the pfp vaccine candidate was well tolerated and moderately immunogenic in these settings. a large multicenter study in children 1-12 years of age with cystic fi brosis did not provide evidence of signifi cant protection against rsv infection [ 169 ] . pfp also has been evaluated for maternal immunization in the third trimester of pregnancy. in the single study to date, the increase in antibody titers was only minimal [ 170 ] . maternal immunization studies are being pursued currently with newer non-replicating vaccine candidates such as emulsion vaccines [ 171 ] and nanoparticle protein preparations [ 172 , 173 ] . live-attenuated vaccines represent an attractive strategy for preventing rsv, since live infection induces a balanced immune response that is not associated with enhanced disease on subsequent natural infection. many live-attenuated rsv vaccine candidates have been developed over several decades. it has proven diffi cult to identify a candidate that is satisfactorily attenuated while remaining satisfactorily immunogenic in the youngest infants. clinical trials of a safe, live-attenuated rsv vaccine for intranasal administration have shown restriction of viral replication in infants following administration of a second dose and have been encouraging [ 174 ] , and additional attenuated vaccine candidates are being developed [ 175 ] . despite over 50 years of research on rsv, many challenges and questions remain. there are many unanswered fundamental questions about the biology of the organism and the pathogenesis of disease. why does reinfection occur throughout life? what are the defi nitive mechanisms of immunity in humans? is there a genetic basis for susceptibility to severe disease? does severe rsv disease cause asthma? what drives the seasonality of rsv? the mortality in infants has been greatly reduced in the usa through advances in critical care, but little rsv-specifi c intervention is available. currently, there is little to offer for therapy except for supportive care. prophylaxis of high-risk infant with a mab prevents some hospitalizations but is expensive and is not always effective. there are no licensed vaccines. given the disaster of early fi-rsv trials, it is not clear that a non-replicating vaccine can be proven safe enough in preclinical models to absolutely assure that enhanced disease will not occur. on the other hand, the explosion of new technologies for generation of recombinant rsv strains, the determination of pre-and post-fusion antigen structures, and new tools for the detailed study of the molecular and genetic basis of human immune responses suggest that much progress will be made in the rsv research fi eld in the coming years. mpv was discovered by investigators in the netherlands who cultivated specimens from children with respiratory infection on a variety of cell types [ 176 ] . a hitherto unknown virus produced cytopathic effects (cpe) in tertiary monkey kidney cells, but could not be identifi ed by antibody staining or rt-pcr for common viruses. electron microscopy of infected cells revealed pleomorphic enveloped particles with surface projections suggesting protein spikes, while biochemical experiments showed that the virus contained a lipid envelope and did not hemagglutinate avian or mammalian red blood cells. elegant randomly primed rt-pcr experiments yielded multiple fragments of genome, which sequence analysis identifi ed as related to avian metapneumovirus (ampv). ampv (formerly turkey rhinotracheitis virus), identifi ed in 1979, is an important global pathogen of poultry including turkeys and chickens [ 177 ] . there are four serotypes of ampv (a-d), and mpv is most closely related genetically to ampv-c [ 178 ] . phylogenetic analysis shows that mpv likely diverged from ampv-c ~200 years ago and thus mpv is of zoonotic origin [ 179 , 180 ] . however, mpv exhibits extremely restricted or no replication during experimental infection of chickens or turkeys and thus is a true human pathogen [ 176 ] . human poultry workers exhibit serological evidence of asymptomatic infection with ampv, providing evidence for the feasibility of an original trans-species transmission event [ 181 ] . while recently identifi ed, mpv is not truly a new virus. studies using archived sera collected in the 1950s revealed a 100 % seroprevalence in humans greater than 5 years old [ 176 ] . nasal washes collected prospectively during the 1970s from children with ari had detectable mpv rna upon retrospective testing by rt-pcr 25 years later [ 182 ] . the specialized cell culture requirements, slow growth, and limited cpe of mpv likely prevented the earlier discovery of this common respiratory pathogen. limited mortality data are available and consist of sporadic case reports, case series, or the identifi cation of fatal cases of mpv infection in research studies. mpv is not a reportable infection and there is no specifi c icd-9 diagnostic code. thus, an accurate estimate of the mortality associated with mpv is not feasible. however, lower respiratory infections are a leading cause of death in children worldwide, primarily in developing nations. mpv is a common cause of severe lower respiratory infection and thus likely accounts for a substantial number of deaths globally. a substantial body of literature has accumulated in the last decade describing the epidemiology, disease burden, and clinical features of mpv. many groups have used standard techniques to provide some estimate of the burden of mpv in diverse populations. most reports have been crosssectional studies of selected populations, usually based on convenience samples of patients with acute respiratory illness. these studies are thus limited by potential selection bias, narrow time periods, and incomplete demographic or clinical data and often lack controls. nonetheless, the broad application of these studies to sizable global populations has illuminated the frequency of mpv infection. many of these studies have focused on special populations, such as patients with asthma, immune compromise, or chronic obstructive pulmonary disease (copd), and thus offer valuable information about mpv among these persons. a number of prospective, well-designed studies in adults and children have been published and offer the best estimates of the population-based incidence of mpv infection. some of these used preexisting prospective data and samples collected prior to the discovery of mpv for retrospective analysis. classical methods including active day care and clinic surveillance, as well as newer approaches such as home surveillance with parent-collected swabs, have been used. these studies have been built upon the foundations of seminal longitudinal studies conducted to investigate other viruses including infl uenza, parainfl uenza viruses, and rsv. taken together, these reports provide a broad survey of mpv epidemiology across diverse geographic environments, socioeconomic populations, and high-risk groups. seroprevalence studies using diverse methods have been performed in different populations. most have used enzymelinked immunosorbent assay (elisa) techniques against whole virus or purifi ed proteins to detect igm or igg. a few have used immunofl uorescent detection of mpv-specifi c antibodies or measured serum virus-neutralizing antibodies. these data have mainly been useful in determining the ubiquity of infection with mpv. some studies have measured acute and convalescent sera to diagnose mpv infection, while others have attempted to establish a serum neutralizing titer that correlates with susceptibility to infection. inherent limitations of serological surveys include the potential for cross-reactive antibodies and the lack of standardized reagents for mpv. most studies have used rt-pcr to detect mpv due to the diffi culty in cultivating the virus. the original isolation of mpv in tertiary monkey kidney cells was possible because the investigator had access to a monkey kidney cell source that was free of the endogenous simian foamy virus (a.d.m.e. osterhaus, personal communication). primary monkey kidney cells commercially available in the usa all contain sfv, and even the addition of anti-sfv antisera cannot prevent the growth of this endogenous virus prior to the slow emergence of mpv. further, the fusion protein of mpv requires cleavage by exogenous trypsin for robust in vitro growth. trypsin is added by most clinical virology laboratories only to cultures of madin-darby canine kidney (mdck) cells for the isolation of infl uenza virus; however, mdck cells are very poorly permissive for mpv even in the presence of trypsin. finally, primary isolation of mpv often requires one or more passages prior to visible cpe and few laboratories routinely follow this procedure. unlike rsv, mpv is not particularly labile to freeze-thaw cycles [ 183 ] and thus can be retrospectively isolated from pcr-positive specimens. fluorescent antibody staining of patient specimens or shell vial cultures can facilitate more rapid identification [ 184 -187 ] . thus, molecular diagnostic techniques have been used for virtually all studies of mpv epidemiology. a number of sensitive and specifi c real-time rt-pcr assays have been described [ 188 -195 ] . many early assays were based on limited sequence data and subsequently were found to be suboptimal for detecting multiple diverse strains [ 195 ] . both individual and multiplexed rt-pcr assays offer more sensitive detection of mpv than culture; however, multiplex assays sometimes balance decreased sensitivity for a single agent with the convenience of detecting multiple viruses simultaneously [ 196 ] . another limitation of molecular detection for all viruses is the ability to detect low levels of viral nucleic acid in the absence of infectious virus. it has become common to detect more than one virus in a single specimen, and the interpretation of these data is far from clear. community respiratory viral infections are frequent in childhood, and the likelihood of detecting viral genome prior to the onset of illness or for prolonged periods after illness resolution complicates the assignment of causation to one of several co-detected viruses. mpv is an enveloped pleomorphic virus ranging in size from 150 to 600 nm, containing a single-stranded negative-sense rna genome [ 178 ] . complete genomic sequences of numerous mpv strains have been published [ 178 , 180 , 197 ] . the genome comprises eight separate open reading frames encoding nine distinct proteins (fig. 26.1 ) . mpv genes are analogous to those of rsv (though ns1 and ns2 are absent in mpv), but the organization of genes differs. ampv and mpv have been taxonomically classifi ed in the separate metapneumovirus genus based on the gene order. phylogenetic analysis of mpv genes consistently identifi es four genetic clades, two major groups designated a and b, each with two minor groups designated a1, a2, b1, and b2 [ 180 , 198 -203 ] . one group has suggested further sublineages based on partial f sequence diversity [ 204 ] , but there is no evidence that this further genetic distinction is of any antigenic or immunologic importance. the two major surface proteins are the fusion (f) and attachment (g), with a third integral membrane short hydrophobic (sh) protein. f is the target of neutralizing antibodies in animal models, f-only vaccines induce protection in animals, and f-specifi c monoclonal antibodies provide passive protection [ 205 -213 ] . in contrast, g-specifi c antibodies do not neutralize virus and g-only vaccines induce neither neutralizing antibodies nor protection [ 206 , 209 , 214 ] . thus, it appears that mpv is unique among human paramyxoviruses in that the attachment protein does not contribute to protective antibodies. further, the g protein exhibits a high degree of genetic variability between subgroups, with as low as 29 % amino acid identity between the major a and b subgroups and a minimum 60 % identity within subgroups [ 202 , 215 -218 ] . the selective pressure for this diversity is unclear. in contrast to g, the f protein is conserved, with a minimum 94 % amino acid identity between a and b subgroups and a minimum 98 % identity within subgroups [ 179 , 202 , 203 ] . presumably there are functional constraints on the diversity of f, since the mutation rate of mpv is high, similar to other rna viruses. the major question regarding the diversity between major or minor subgroups is whether it contributes to antigenic variation or escape in human populations. cross-neutralization against heterologous virus from the a and b lineages was tested using experimental infection of ferrets [ 202 ] . this study found relative neutralization of homologous to heterologous virus ranging from 12 to 96-fold difference, thus providing some evidence for antigenic serotypes. however, subsequent experiments using hamsters, african green monkeys, chimpanzees, and rhesus macaques found that the a and b groups were 64-99 % related antigenically [ 219 ] . infected animals developed neutralizing antibodies that were highly effective against heterologous virus, and previously infected primates were protected against challenge with heterologous virus. cynomolgus macaques infected with a or b subgroup viruses or with candidate vaccines exhibited only a 6-16-fold difference in neutralizing titer against homologous and heterologous viruses [ 220 , 221 ] . taken together, these data show that while mpv f exhibits some antigenic diversity, the virus does not have truly distinct serotypes. the potential implications for human epidemiology are discussed further below. descriptive epidemiology numerous studies document the fact that mpv infection is ubiquitous and that reinfection is common. serosurveys testing large sample collections in canada, china, croatia, germany, israel, japan, the netherlands, taiwan, thailand, the usa, and uruguay show that 95-100 % of children have antibodies against mpv by the age of 5 years [ 222 -231 ] . in many of these studies, 50-75 % of children are seropositive by age 2 years, suggesting that most acquire primary mpv infection early. most identify a decrease in serum mpv antibody titer from birth to 6-12 months, presumably due to the expected decline of maternally derived antibodies. studies in japan and india that compared mpv and rsv titers in the same cohort found that after the expected nadir during early infancy, rsv titers began increasing at an earlier age than mpv [ 232 , 233 ] . this fi nding is interesting in light of epidemiologic data suggesting that primary mpv infection peaks between 6 and 12 months of life compared with the peak of rsv at 2-3 months (discussed below). longitudinal studies in adults and children have documented reinfection by a fourfold rise in serum antibody titer [ 222 , 230 , 231 , 234 -237 ] . the serological data show that mpv infection is nearly ubiquitous during the fi rst years of life. further, reinfection occurs throughout life. in children, primary mpv infection is associated commonly with lower respiratory illness, while reinfection is associated with upper tract disease [ 182 , 238 ] . most epidemiologic studies of mpv in children show that the virus is the second leading cause of lower respiratory infection after rsv. the prevalence of mpv in studies of children with lri is 5-25 %. a 25-year prospective study of otherwise healthy children <5 years old detected mpv in 12 % of children with lri; several children experienced recurrent infection [ 182 ] . a 2-years multicenter study of inpatient and outpatient japanese children with ari identifi ed mpv in 57/637 (8.9 %) [ 239 ] . a 5-years observational study of otherwise healthy korean children <5 years old found mpv in 24/515 (4.7 %), similar to the rates for infl uenza and parainfl uenza virus type 3 (piv-3) [ 240 ] . a very large observational study in queensland, australia, tested specimens obtained from patients of all ages with lri from 2001 to 2004; mpv was detected in 707/10,025 (7.1 %). ninety-two percent of patients with mpv were <5 years old, and mpv was the second most common virus after rsv in these children [ 241 ] . a south african group tested specimens from children hospitalized with ari who were subjects in a prospective pneumococcal vaccine trial; mpv was present in 126/1,409 (8.9 %) and was the most common virus after rsv. the estimated incidence of mpv-associated hospitalization in hiv-negative children was 29/1,000; a number of children had repeat infections [ 242 ] . a prospective study of hospitalized children in hong kong over a 13-month period found mpv in 32/587 (5.5 %); the estimated incidence of mpv-associated hospitalization was 4.4 per 1,000 children <6 years old [ 243 ] . a chinese group conducted a prospective 2-years study of children hospitalized with ari and identifi ed mpv in 227/878 (25.9 %), with most <6 years old [ 244 ] . a large, population-based, prospective surveillance study conducted in three us cities over 6 years found that the incidence of hospitalization for mpv in children <5 years old was 1 per 1,000, lower than the rate of rsv-associated hospitalization in the same cohort (3/1,000) but similar to the rates for infl uenza (0.9/1,000) and piv-3 (0.5/1,000) [ 245 ] . respiratory disease is among the leading causes for hospitalization of adults in the usa, and "infl uenza and pneumonia" ranks among the top 10 causes of deaths annually. although the data are limited, mpv appears to be associated with a substantial burden of ari in adults, primarily those with comorbidities. a prospective study in rochester, ny, enrolled four cohorts during four winters: healthy adults >65, high-risk adults >65 with comorbidities, healthy adults 19-40 years old, and adults hospitalized for acute cardiopulmonary illness [ 246 , 247 ] . overall, mpv infection was detected in 8.5 % of ari in the cohort. the rate of mpv infection was highest in young adults at 13 %, though many of these were asymptomatic and detected only serologically. of note, this group had a mean age of 33, was predominantly female, and had daily exposure to children. of the hospitalized patients, the incidence of mpv annually ranged from 4.4 to 13.2 %. more than 85 % of the hospitalized mpvinfected subjects had underlying conditions, chiefl y cardiopulmonary disease or diabetes mellitus. there were six deaths in this study, all with comorbidities; one had concomitant bacteremia with streptococcus pneumoniae . interestingly, the incidence of mpv infection was similar to the annual average infection rate for rsv (5.5 %) and infl uenza a (2.4 %) in these cohorts during the same study period. a prospective, population-based study in nashville, tn, recruited adults hospitalized for ari at several county hospitals over three winters [ 248 ] . of 508 subjects, 23 (4.5 %) had mpv, 33 (6.5 %) infl uenza, and 31 (6.1 %) rsv. notably, mpv-infected subjects were signifi cantly older than infl uenza-infected subjects (mean 76 vs. 60 years) and had higher rates of chronic cardiopulmonary disease (78 % vs. 52 %). the overall population-based rates of hospitalization for the three viruses were similar, at 1/1,000 for mpv, 1.5/1,000 rsv, and 1.2/1,000 for infl uenza. however, for subjects ≥65 years, hospitalization rates were much higher for mpv and rsv at 2.2/1,000 for mpv and 2.5/1,000 for rsv compared to 1.2/1,000 for infl uenza, likely refl ecting the use of infl uenza vaccine for older adults. a prospective study of community-acquired pneumonia in canada found mpv in 4 % of hospitalized cases during an 18-month period, all with underlying conditions [ 249 ] . a dutch group detected mpv in 2 % of bronchoalveolar lavage specimens from intensive care unit patients. all were >50 years old with comorbid conditions and 83 % died [ 250 ] . similarly, a retrospective study in north ireland found mpv in only 0.8 % of residual respiratory specimens from adults, but 33 % of these died [ 251 ] . together, these data show that mpv is an important cause of acute respiratory disease in adults, primarily older adults or those with underlying comorbid conditions. mpv is associated with acute asthma exacerbations [ 252 -254 ] . mpv was detected in 10 of 132 hospitalized finnish children with acute wheezing [ 255 ] . similarly, mpv was isolated from 7 % of adults hospitalized for acute asthma exacerbation, only one of whom tested positive 3 months later [ 256 ] . premature infants who developed mpv bronchiolitis within the fi rst year of life had decreased lung function at 1 year of age [ 257 ] . a prospective, case-control study of children with mpv bronchiolitis during infancy compared to infants with acute gastroenteritis found that mpv infection early in life was signifi cantly associated with a later diagnosis of asthma and recurrent wheezing at 5 years of age [ 258 ] . mpv causes severe disease in children with comorbid conditions such as cardiac and pulmonary disease or prematurity [ 259 -263 ] . a prospective 1-year study of hospitalized children found that 34 % of patients with mpv had a history of prematurity, chronic lung disease, complex congenital heart disease, or immunodefi ciency [ 264 ] . vicente et al. detected mpv by rt-pcr in 6 % of adults >64 years old with acute exacerbations of copd; none had other pathogens identifi ed by culture or pcr [ 265 ] . a canadian study found that 4 % of hospitalized adults with communityacquired pneumonia or copd exacerbations tested positive for mpv, all with comorbid conditions; one also had infl uenza a and s. pneumoniae [ 266 ] . rsv was present in 9 % and infl uenza a in 6 % of the cohort. mpv was detected in 12 % of adults hospitalized for copd exacerbation during one winter in connecticut, none with other viruses codetected; rsv was present in 8 % and infl uenza a in 4 % of the entire cohort [ 267 ] . severe and fatal mpv disease can occur among immunocompromised individuals, including solid organ and stem cell transplant recipients, hiv-infected persons, and chemotherapy patients [ 268 -275 ] . mpv is associated with morbidity and mortality in adults with hematologic malignancies and stem cell transplant; [ 270 , 276 ] mpv was detected in bronchoalveolar lavage specimens from 5/163 (3 %) episodes of acute respiratory infection in stem cell transplant recipients, and four died [ 270 ] . hiv-positive south african children with mpv were signifi cantly more likely to receive a diagnosis of pneumonia and experience longer hospitalization, lower mean oxygen saturation, and bacteremia; further, hiv-positive children were fi vefold more likely to be infected with mpv than hiv-negative children [ 242 ] . mpv has been implicated in several hospital and institutional outbreaks leading to mortality [ 277 , 278 ] . kim and colleagues [ 279 ] report the transmission of mpv to pediatric hematology-oncology patients during a nosocomial outbreak. the incubation period was between 7 and 9 days. standard, but not droplet, precautions were used. in laboratory studies, infectious virus persists on metal and nonporous surfaces for up to 8 h [ 183 ] . due to the signifi cant morbidity and mortality of mpv in high-risk children, isolation precautions are important. mpv is associated with both upper and lower respiratory tract disease [ 182 , 280 -282 ] . rhinorrhea and cough are the most frequent symptoms, while hoarseness, laryngitis, sore throat, and croup are less common [ 238 -241 , 243 , 282 ] . a large prospective study of children with uri detected mpv in 5 % of patients, similar to rsv, infl uenza, and piv but less frequent than adenovirus and rhinovirus. in these children with uri associated with mpv, fever was present in 54 %, coryza in 82 %, cough in 66 %, pharyngitis in 44 %, hoarseness in 8 %, and conjunctivitis in 3 % [ 280 ] . mpv is associated with acute otitis media and has been detected in nasal secretions and middle ear fl uid [ 182 , 280 , 283 -285 ] . signs and symptoms of lri with mpv include cough, wheezing, and rhonchi. a large chinese study of children with acute respiratory infections found that wheezing was more common in children with mpv than with rsv [ 282 ] . in that study, children with mpv were diagnosed with pneumonia signifi cantly more than children with rsv, 47 % versus 31 % ( p = 0.002). conversely, a larger percentage of children with rsv were diagnosed with bronchiolitis compared to mpv, 62 % versus 42 %, respectively ( p < 0.001). other studies also note the trend toward a higher percentage of children with mpv and pneumonia compared to rsv [ 182 , 238 , 245 ] although this is not always statistically signifi cant [ 243 ] . mpv has been associated rarely with neurologic complications, including febrile seizures and altered mental status, but there is no conclusive evidence for direct neural infection. one case report describes a patient who died and had mpv isolated by rt-pcr from brain and lung tissues [ 286 ] . mpv was detected by rt-pcr in nasal specimens from 4 of 1,570 persons with encephalitis of unknown etiology [ 287 ] . several other reports describe the detection of mpv in a respiratory specimen from patients with encephalitis [ 286 , 288 -290 ] . only one case reports detection of mpv in cerebrospinal fl uid [ 291 ] . the incidence of viral infection varies between countries and years, but mpv circulates in every year [ 176 , 182 , 239 -241 , 243 , 282 , 292 -296 ] . epidemiologic studies have verifi ed the presence of mpv worldwide. mpv is present during all months in temperate regions, although predominant in late winter-early spring, often following the peak of rsv (fig. 26.2 ) [ 176 , 182 , 191 , 239 , 241 , 246 , 264 , 280 , 282 , 294 , 297 ] . in subtropical climates such as hong kong, a springsummer season similar to rsv occurs [ 243 ] . biannual peaks of seasonality have been described in some european studies [ 298 , 299 ] . annual rates of mpv-associated ari are lower than rsv and comparable to parainfl uenza virus types 1-3 combined and infl uenza [ 240 , 241 , 243 , 282 , 294 , 300 ] although mpv does on occasion surpass rsv in incidence [ 293 ] . multiple outbreaks have been reported in nursing homes and other long-term care facilities (ltcf). mpv was the only pathogen identifi ed in an outbreak of ari that occurred over a 6-weeks period at a quebec ltcf, with 6 pcr-confi rmed and 96 epidemiologically linked probable cases. there were three deaths among the confi rmed and nine deaths among the probable cases [ 278 ] . a california study described an outbreak of ari in 26/148 (18 %) residents and, importantly, 13 staff of a ltcf; mpv was confi rmed in 5 residents, 2 of whom were hospitalized, and no other viruses were detected in any case [ 301 ] . attendance in out-of-home day care, breast-feeding, and passive smoke exposure are not signifi cantly associated with mpv infection [ 245 ] . mpv infection is not associated with lower socioeconomic status. viral coinfection has been suggested with mpv; dual infection with rsv and mpv increased the risk of picu admission compared to rsv alone in one small study [ 302 ] . however, this fi nding was refuted by subsequent larger reports [ 303 -305 ] . other studies demonstrate no signifi cant difference in children with coinfections, including adenovirus, bocavirus, coronavirus, infl uenza virus, parainfl uenza viruses, or rsv [ 240 , 241 , 282 , 293 , 306 , 307 ] . mpv has four distinct genetic lineages or subgroups: a1, a2, b2, and b2 [ 202 , 203 , 218 ] . the predominant subtype varies by year and location [ 241 , 280 , 293 , 297 , 308 ] . in italy, over a 3-years period, all four subtypes were identifi ed each season but the predominant subtype changed. in 2001-2002, a1 accounted for 59 % of all strains, the following year b1 and b2 were present equally, and in 2003-2004, 72 % of strains were a2 [ 308 ] . similar variation was observed over 20 years in a us study, with multiple subgroups present in most seasons (fig. 26. 3 ) [ 280 ] . it is unclear whether viruses from different subgroups differ in virulence. a study in spain reported that children with group a infection more frequently had pneumonia and higher disease severity [ 309 ] , while in canada, group b was associated with more severe disease in hospitalized patients [ 261 ] . patients with group b strains in a french study were more likely to have abnormal chest radiographs but did not have signifi cant differences in oxygen saturation, hospitalizations, or clinical severity scores [ 310 ] . other studies have found no major distinctions in disease severity [ 239 , 241 , 2 0 0 1 2 0 0 0 1 9 9 9 1 9 9 8 1 9 9 7 1 9 9 6 1 9 9 5 1 9 9 4 1 9 9 3 1 9 9 2 1 9 9 1 1 9 9 0 1 9 8 9 1 9 8 8 1 9 8 7 1 9 8 6 1 9 8 5 1 9 8 4 1 9 8 3 1 9 8 2 311 ], laboratory abnormalities [ 228 ] , or symptoms [ 240 ] between subgroups. group a viruses replicate more efficiently in animal models, suggesting some meaningful biological differences between groups [ 219 , 220 ]. mpv is an enveloped virus and thus inactivated by soap, disinfectants, or alcohols. spread is thought to occur by direct or close contact with contaminated secretions. infectious virus can persist at room temperature, especially nonporous surfaces, for up to 8 h [ 183 ] . contact precautions are recommended as for rsv with meticulous hand hygiene. cohorting of patients and caregivers should be considered during outbreaks in care facilities. human data are limited; studies in rodents and nonhuman primates reveal mild erosive and infl ammatory changes in the mucosa and submucosa of the airways, with viral replication observed in ciliated epithelial cells in the respiratory tract [ 312 -314 ] . infl ammatory infi ltrates with a lymphocytic and monocytic predominance are present in perivascular and peribronchial areas. sumino and colleagues [ 315 ] reviewed the lung pathology of fi ve adults with mpv infection. histopathology in three patients demonstrated acute, organizing lung injury with diffuse alveolar membrane formation and the presence of smudge cells. the fourth patient had no evidence of lower respiratory tract infection, and the fi fth patient had nonspecifi c acute and chronic infl ammation. a similar study in children revealed chronic infl ammatory changes of the airways with intra-alveolar macrophages [ 316 ] . a major limitation of reports of human pathology is that patients had been mechanically ventilated prior to death, making it diffi cult to distinguish virus-induced pathology from barotrauma and nonspecifi c infl ammation. mpv lacks genes present in other paramyxoviruses that inhibit interferon responses; nevertheless, mpv is capable of blocking type i interferon responses by an unknown mechanism [ 317 , 318 ] . mpv and other respiratory viruses induce pulmonary cd8+ t cells that fail to secrete ifnγ or exhibit cytotoxic degranulation in response to viral peptides; these impaired cd8 t cells resemble exhausted cd8 t cells induced by chronic infections such as hiv and hepatitis c virus [ 319 ] . humans develop neutralizing antibodies to mpv, and passive antibodies alone can protect in animal models. however, immunity wanes over time and likely provides limited cross-protection between subgroups, since reinfections occur in children and adults [ 246 , 248 ] with genetically different strains [ 182 , 239 , 240 , 268 , 320 ] as well as strains from the same subgroup [ 280 ] . early protection against reinfection following primary infection was confi rmed in macaques; however, when challenged 12 weeks later, virus replication was detectable despite the presence of serum antibodies [ 220 ] . eleven months later, antibody levels had waned still further, and all macaques challenged with heterologous virus and two of three animals reinfected with homologous virus had no evidence of protection [ 220 ] . a prospective study in humans noted that baseline mpv antibodies were lower in patients who subsequently became infected versus those who did not become infected [ 321 ] . thus, cross-protection and duration of antibody responses are important issues for vaccine development. patterns of host response mpv causes both upper and lower respiratory tract signs and symptoms clinically indistinguishable from disease associated with rsv and other respiratory viruses [ 182 , 239 , 300 , 322 , 323 ] . fever is present in most cases, especially children [ 239 -241 , 243 , 264 , 280 , 293 ] . transient maculopapular rash has been described in a minority of patients [ 241 , 243 , 293 ] , and vomiting or diarrhea are described with low frequency [ 182 , 239 , 243 ] . laboratory abnormalities are uncommon, although one study identifi ed 27 % of patients with elevated alt and ast values [ 293 ] . white blood cell count and c-reactive protein were not signifi cantly different between rsv and mpv [ 238 , 282 ] . mpv is rarely detected in asymptomatic persons [ 182 , 191 , 324 -326 ] , though in otherwise healthy young adults, mpv infection can be subclinical [ 246 ] . the duration of shedding in healthy individuals is approximately 7-14 days [ 239 , 327 ] . detection in cell culture requires prolonged incubation and is both insensitive and often impractical. shell vial culture offers increased sensitivity over traditional culture [ 184 , 328 ] . ifa has demonstrated a sensitivity of 73 % and specifi city of 97 % with rt-pcr as the gold standard [ 329 ] . dfa has shown similar results [ 330 ] . commercial antibody kits for immunofl uorescent detection of mpv are available. rt-pcr is most commonly used for detection of the virus in epidemiologic studies and is becoming more common in clinical laboratories [ 176 , 320 , 331 , 332 ] . real-time rt-pcr targeting the conserved n gene has high sensitivity for detection of all four subgroups [ 189 , 195 ] . control and prevention the primary therapy for mpv infections is supportive care, including oral or intravenous hydration, monitoring of respiratory status and oxygen saturation, supplemental oxygen, and mechanical ventilation for frank respiratory failure. there are no licensed antivirals for mpv. reports of pharmacologic treatment of mpv are limited to severely ill or immunocompromised patients. ribavirin, an antiviral agent used in severe rsv infection, reduced infl ammation and viral replication in mice with mpv infection [ 333 ] . commercial intravenous immunoglobulin (ivig), ribavirin, and nmso3 (a sulfated sialyl lipid) effectively inhibited mpv in vitro [ 334 , 335 ] . ribavirin, with and without ivig, has been used in immunocompromised adults [ 336 ] . bonney and colleagues [ 337 ] reported successful treatment of an immunocompromised child with mpv using iv ribavirin and ivig. subsequently, oral ribavirin and inhaled ribavirin with ivig have been used [ 275 , 338 ] . however, no randomized, controlled trials have been conducted, and these data should be regarded as purely anecdotal. human and murine monoclonal antibodies exhibit therapeutic effi cacy in rodent models and thus offer potential for immunoprophylaxis [ 210 , 211 , 213 ]. a number of vaccine approaches for mpv have been investigated. the f protein is conserved between subgroups, immunogenic, and the only target of neutralizing antibodies; in contrast to rsv, the mpv g protein does not induce neutralizing antibodies and is not a protective antigen [ 206 , 209 , 214 ] . a recombinant parainfl uenza virus encoding the mpv f protein demonstrated protection against mpv [ 339 ] , and soluble f protein vaccines reduced viral titers in cotton rats and hamsters [ 207 , 208 ] . reverse genetics technology has been developed for mpv and has been used to produce recombinant strains for vaccine development [ 197 , 340 ] . viruses lacking the g, m2-1, m2-2, or sh proteins or with point mutations are attenuated and immunogenic in rodent and primate models [ 197 , 221 , 341 -344 ]. the major unresolved problems in mpv epidemiology and research involve understanding mechanisms of disease and developing therapeutic or preventive strategies. abundant evidence shows that mpv is a signifi cant 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cord-034133-tx0hciiv authors: engda, tigist title: the contribution of medical educational system of the college of medicine, and health sciences of the university of gondar in ethiopia on the knowledge, attitudes, and practices of graduate students of health sciences in relation to the prevention and control of nosocomial infections during the academic year of 2018 date: 2020-10-22 journal: bmc med educ doi: 10.1186/s12909-020-02271-6 sha: doc_id: 34133 cord_uid: tx0hciiv background: nosocomial infection, also called a hospital-acquired infection, is an infection acquired during admitting patients in health care facilities. nosocomial infection can be prevented and controlled by giving training to those responsible. this study aimed to assess the contribution of the medical education system on the knowledge, attitudes, and practices of the graduate students of health sciences about the prevention and control of nosocomial infection in the college of medicine and health sciences at the university of gondar in the academic year of 2018. method: an institution-based cross-sectional study was conducted among all graduate health science students posted in the different departments at the university of gondar in the college of medicine and health sciences from february to june 2018. a total of 422 study participants were included. data were analyzed using spss version 20. results: out of a total of 422 respondents, only 40% have taken training for infection prevention; out of which 39% had taken the training for a year ago. moreover, only 35.5% have good knowledge of nosocomial infections as a result of the training; and only 32.5% have good understanding of the practical training given on prevention and control. only 36% have good attitude towards its prevention and control. conclusion: the result shows that only a few of the respondents have taken the infection prevention training. yet, a smaller proportion of them had good knowledge, attitude, and practice on nosocomial infections. hence, the medical education system should give more attention to the training of the nosocomial infection control by developing different strategies to prepare the students on these issues before they start their clinical attachment. nosocomial infection is a localized or systemic infection that is acquired in a health care facility that may manifest 48 h after the patient's admission to or discharged from the health care facility [1] . it can be caused by bacteria, viruses, parasites, and fungi that may be present in the air, surfaces, or equipment surrounding the health institutions [2] . it can affect patients of all age groups; however, neonates, immunocompromised adults, and the elders are the most vulnerable ones [3, 4] . nosocomial infections have been recognized as a problem affecting the quality of health care services and are the principal source of adverse healthcare effects. increased hospital stays, increased costs of healthcare, economic hardship to patients, and their families, and even deaths are some of the negative effects [5] [6] [7] . the amount of nosocomial infections of low-and middle-income countries is higher because of the limited knowledge and utilization of post exposure prophylaxis (pep), limited knowledge of professional risks, low adherence to universal precautions (up), and inaccessibility of personal protective equipment (ppe) [2, 3, 5, 8] . findings of several epidemiological studies show that hcws, mainly physicians, dentists, laboratories, and nurses are involved in the transmission of nosocomial infections [8] [9] [10] [11] . it has also been reported that its transmission increases during the performances of medical procedures whenever hcws fail to follow aseptic precautions [8, 10] . the world health organization (who), in conjunction with the cdc, gives high attention to the prevention of nosocomial infections as it has developed a practical manual for the prevention of nosocomial infections globally (who, 2002) . some recommended strategies included in the manual were the use of hand decontamination, personal hygiene, utilization of personal protectives, and proper methods of handling soiled clothing when healthcare workers perform patient care activities. it also recommends methods of preventing environmental contamination including the cleaning of the hospital environment using hot superheated water, sterilizing patient equipment, and preventing the transmission of pathogens like hiv, hepatitis b and c viruses, and tb to the staff [1] . efficient pre-service and in-service training given by incorporating in the medical education system supported with good monitoring and evaluation methods of hcw practices play a pivotal role in the sustainability of the knowledge, attitude, and practice of universal precautions and infection control [12, 13] . this scheme was supported by many studies conducted worldwide. for example, in india, an educational module had effectively elevated the knowledge, attitude, and practice score of hcws from 14% before the intervention to 94% thereafter [14] . in korea, it was also investigated that a group of nurses and medical students who had received education on hais showed high knowledge (p = 0.036) and performance (p < 0.01) levels [15] . similarly, a study at seton hall university in new jersey, usa, indicated that the total score for the knowledge category was 93% [16] . likewise, a study in egypt reported that physicians had the best level of knowledge, but the least in practicing general safety measures than others in the preintervention phase. however, they increased their practice score from 54.3 to 86.1% after receiving continuing education [14] . a study in pakistan reported that the knowledge score was 3.8 with a median of 4. the dispenser had the highest knowledge score while the housekeepers had the lowest. knowledge about the mode of transmission of bloodborne pathogen and the work experience alone significantly predicted the use of universal precaution methods in multiple linear regression models [17] . this principle is also supported by studies conducted in ethiopia. in 2015, a cross-sectional study was conducted in addis ababa on hcws who received training on transmission, vaccination, and diagnosis of hbv to assess their knowledge of risk factors for hbv. the result showed that more than 80% of the respondents had the knowledge of the modes of transmission and prevention of hbv; 83.3% had a positive attitude towards following infection control guidelines [18] . similarly, a study conducted in ethiopia at dire dawa university on the medical and health sciences students reported that almost all of the respondents had good knowledge of the transmission, treatment, and prevention of hbv. also, 94.2% of them had good attitudes towards the importance of standard precautions, but 58.4% had poor practices in applying the recommended standard precautions [19] . in another study, a hospitalbased cross-sectional investigation was conducted among 150 health workers who were taking training about infection and prevention of hospital-acquired infections at debre markos hospital in ethiopia. the results showed that 84.7% of them were found to be knowledgeable; however, only 57.3% of the respondents demonstrated good practice in infection prevention. moreover, respondents with older age, longer work experience, and higher educational status excelled in both knowledge and practice of infection prevention. inservice training, availability of infection prevention supplies, and adherence to infection prevention guidelines were also associated with the practice of infection prevention [19] . on the other hand, a study conducted at the university of gondar hospital about hand hygiene compliance on 405 study participants showed that only 8.97% had the knowledge about it and 5.62% had received training about hand hygiene compliance, respectively [20] . hand hygiene is an important means for the control and prevention of nosocomial infection. therefore, the current study intended to determine the impact of the medical education system on the knowledge, attitude, and practice of graduate health sciences students about the prevention and control of nosocomial infections at the university of gondar. an institution-based cross-sectional study was conducted with graduate students from the college of medicine and health sciences at the university of gondar. data collection was made between february and june of 2018. students who attended the regular academic program at the university of gondar, college of medicine and health sciences were the source population. however, only graduate classes of health science students were taken as the study population. before data collection, eleven departments were selected for sampling: health informatics, medical laboratory sciences, health officer, physiotherapy, environmental and occupational health and safety, psychiatry, optometry, midwifery, nursing, pharmacy, and anesthesia. there were 465 graduate students of health sciences in the academic year of 2018. the sample size was determined using a single random sampling method. since no similar study was found in the area, 95% were taken as a confidence interval. then, the calculated sample size was 384 and by adding a 10% non-response rate, the final calculated sample size became 422 (fig. 1) . the questionnaire was constructed from emergent themes reviewed in the literature and items were derived from the established guidelines set by a task force committee on infection control practices advisory committee [5, 21] . the questionnaire includes 40 questions subdivided into four categories: sociodemographic, knowledge, attitude, and practice towards nosocomial infections. knowledge was assessed using 11 questions containing three alternative choices each. the answers from the given alternatives were symbolized as '1' for poor, '2' for fair, and '3' for good. a higher score in the questions concerning nosocomial infections is considered to be good knowledge of nosocomial infections. attitude was measured using 8 questions in which answers for each question were assigned as 1 for 'disagree', 2 for 'neutral', and 3 for 'agree.' higher score achieved was considered as a positive attitude toward standard precaution. moreover, their practice towards the prevention and control of nosocomial infections was assessed using 14 questions with three alternative answers which were assigned as '1' for poor, '2' for fair, and '3' for good (table 1, table 2, and table 3 ). bloom's cut-off point was used to determine the level of knowledge, attitude, and practice because the conceptual framework of the present study was based on the taxonomy of educational objectives developed by bloom (1956) . according to bloom's taxonomy (1956), human behaviors are derived from the integration of the cognitive, affective, and psychomotor domains. knowledge, attitudes, and practices could be representatives of the cognitive, affective, and psychomotor domains, respectively. knowledge refers to the factual, conceptual, procedural, and met cognitive thought [22] . attitude is an internal or covert feeling and emotion; or selective nature of intended behavior which represents the affective domain. practice represents the psychomotor domain which refers to the physical movement, coordination and use of motor or neuromuscular activities [22] . accordingly, participants' overall knowledge and practice are considered as good if the score is 80% and above; moderate if the score is between 60 and 79%; and poor if the score is less than 60%. similarly, attitude towards nosocomial infection was assessed using 8 questions. responses to questions related to attitude were graded on a 3-point likert scale with an agreement scale ranging from '1' for disagree to '3' for agree [7] . the overall level of attitude was categorized using bloom's cut-off point: positive if the score was 80% and above; neutral if the score was 60-79%; and negative if the score was less than 60%. a simple random sampling method and lottery technique were used to select the respondents and a quantitative method of data collection was employed through a self-administered questionnaire. the quantitative method involves assessment of the impact of medical education on the knowledge, attitude, and practices of 422 graduate students on the prevention and control of nosocomial infection. the data collection instrument format was developed in english by different individuals for its accuracy and desired results. the data collectors used a self-administered questionnaire for graduate students of the health sciences, class of 2018. after receiving a complete response of the questionnaires, data were analyzed using descriptive statistics by ibm spss version 20.0. demographic characteristics are presented in tabular form using descriptive statistics and reported as mean, median, standard deviation, frequency, and percentage as presented in tables. the study was conducted after a written ethical clearance is obtained from the ethical research committee of the school of biomedical laboratory sciences and college of medicine and health sciences. moreover, the consent forms of the participants were completed voluntarily by the study participants themselves. pretest to evaluate the understandability and applicability of the instruments used, pretest data were collected and checked from 10 medical laboratory graduate students using a self-administered questionnaire. self-administered questionnaires were collected from 422 respondents. out of these respondents, 32% were female while only 2% of them were above 25 years of age. the proportions of respondents were: (6.9%) health informatics, (6.9%) medical laboratory sciences, (15.6%) health officers, (4.5%) physiotherapy, (4.3%) environmental and occupational health and safety, (6.6%) psychiatry, (5.5%) optometry, (14.7%) midwifery, (17.5%) nursing, (12.3%) pharmacy, and (5.2%) anesthesia graduated students. only 40% of the respondents had been trained in infection, prevention out of which 39% took the training at least a year ago ( table 4) . even though 40% of the respondents stated that they had taken training on nosocomial infections, only 35.5% had good knowledge of nosocomial infections (fig. 2) . from the 55 questions administered, the score of knowledge of the respondents ranged from 11 to 55, with a mean score of 40.64 at std. of + 12.73 (table 5) . as reflected in table 5 , out of the total respondents, 59.5% had good knowledge of the modes of transmission and risk factors for nosocomial infections; 57.3% of the respondents also stated that they were fully aware of hand-washing guidelines; 47.2% knew where and how the contents in biohazard bags or containers are being disposed. it is also shown that table 1 questioner for the assessment of knowledge of graduate health sciences students towards nosocomial infection instruction: to complete this section, please make a tick "✓" on the number corresponds to how you agree with the given alternatives 1 = poor, 2 = fair and 3 = good table 2 questioner for the assessment of practice of graduate health sciences students towards nosocomial infection instruction: to complete this section, please make a tick "✓" on the number corresponds to how you agree with the given statement 1 = poor, 2 = fair, 3 = good 65.2% of them knew that nosocomial infections could be transmitted via fomites, and 59.6% of the respondents understood that healthcare facilities harbor a variety of microorganisms that could be transmitted by healthcare workers. then, 62.1% of respondents were fully aware of safety precautions for the disposal of used medical equipment, and 56.2% of them believed that neutropenic patients like those with diseases of the respiratory system should be kept in private rooms. furthermore, 57.8% of the graduates were knowledgeable in the use of alcohol-based formulations, and 63.5% of them stated that some microorganisms were not totally removed by alcoholbased solutions. the overall practice scores showed that 32.5% have good practice in the prevention and control of nosocomial infection. the score of the practice of the respondents ranged from 14 to 70, with a mean of 45.61 atstd+ 15.35 (table 6) . respondents reflection to correctly following guidelines for the use of alcohol-based solutions before and after patient care activities were 42.2%; before opening vascular access equipment were 39.3%; between each patient contact were 48.6%; before and after direct contact with patients' intact skin were 41.2%; moving from a contaminated body site to a clean table 3 questioner for the assessment of attitude of graduate health sciences students towards nosocomial infection instruction: to complete this section, please make a tick "✓" on the number corresponds to how you agree with the given statement 1 = disagree, 2 = neutral, 3 = agree body site were 46.7%; before and after drawing or manipulating patient's body fluid samples were 52.1%; before inserting indwelling urinary catheters were 50.0%; and after touching inanimate objects and equipment in the patients' room were 45.7%. of all, 31.8% of the respondents used their computer keyboards with their glove during busy workload. finally, 43.1% of the respondents removed their rings, watches, or bracelets during hand hygiene (table 6) . the attitudes of students towards the prevention and control of nosocomial infection were 36% (fig. 3) . of the total respondents, 61.9% believed that nosocomial infections are posing serious negative outcomes but 44.8% responded the opposite while a colleague is noncompliant with the recommended guidelines for patient safety. moreover, 51.9% of the respondents regularly (table 7) . nosocomial infection is one of the most important challenges in health institutions. therefore, this study assessed the knowledge, attitude, practice, and associated factors of infection prevention among health science graduate students. the overall score of knowledge (35.5%) was lower than the study conducted in the usa (93%) and nepal (97%) [16, 23] . similarly, 59.6% had good knowledge of their etiology, modes of transmission, and risk factors of nosocomial infections which were also lower than the study conducted in new jersey, usa (95.7%), and nepal (82%). moreover, only 59.6% of the participants knew fomites as transmission factors, which is still lower than the study conducted in the usa (98.9%) [16, 23] . this might be due to a difference in study participants. in the usa, the study participants were registered nurses who were working in health care institutions and they might develop knowledge from their experiences and/or in-service training. however, in this study, the participants were graduate students of which 60% never took training on the prevention and control of nosocomial infections. this shows that not all health science students in this college are taking training before their clinical attachments. in the findings of this study, only 31.5% had good practice in the prevention and control of nosocomial infections of which only 50.4% followed the guidelines for the use of alcohol-containing hand sanitizer which is lower than the study conducted in the usa (78%), but higher than a study conducted in china (11%) [16, 17] . this may be due to the difference in study participants, the accessibility of alcohol-containing hand sanitizer, and the large difference in course curriculum where infection prevention might not be incorporated in all of the target population. possibly for similar reasons, the attitude of study participants towards the prevention and control of nosocomial infection (36%) was lower than a study carried out in the usa (79.66%) and nepal (66.0%) [16, 23] . a 52.2% positive attitude towards following the recommended guidelines for reducing the transmission of nosocomial infections was lower than a study conducted among health care workers at addis ababa in ethiopia (83.3%) [18] . this might be due to a difference in study participants in addis ababa, where they were registered health care workers who were working in the health institution and they might develop knowledge, attitude, and practice either through their experience or in-service training. however, in this study, the participants were graduate students who reported that 60% of them never took training on the prevention and control of nosocomial infections before their clinical attachment. generally, more than half of the respondents had poor knowledge, attitude, and practice on nosocomial infection and the application of infection and prevention procedures. the medical education system is the most important and effective tool to bring a better outcome for controlling and preventing nosocomial infections. incorporating the necessary knowledge into the regular course curricula, organizing training modules to medical students before starting clinical attachment, providing different guidelines and standard operating procedures are also helpful in understanding the nature of infections and how, when, and where to prevent and control nosocomial infection. therefore, this study showed that a smaller number of respondents had taken infection prevention training on their regular medical system. consequently, smaller proportions of them had good knowledge, attitude, and practice on the nature of the infection, prevention, and control strategies for nosocomial infections. therefore, to improve the level of knowledge, attitude, and practice of students towards nosocomial infections, strengthening the medical education system through relevant seminars including short and long-term training is essential. at the same time, the availability of infection prevention guidelines, standard operating procedures, and personal protective equipment like alcohol-based solutions in health institutions are important. departments, schools, and college administrative officers should work together to facilitate infection prevention training programs for all health science students before starting their clinical attachments. the ministry of health and ministry of education should work to enforce the universities to incorporate infection prevention knowledge into the course curricula for all health science students. all health care institutions must be prepared to give vaccination of common hospital-acquired diseases by making available infection prevention materials and standard operational author's contributions te: conception of a research idea, study design, data analysis, interpretation, and manuscript write up. the author(s) read and approved the final manuscript. no one was responsible for the funding of this research. all data generated or analyzed during this study are included in this article. ethics approval and consent to participate ethical clearance was obtained from the research and ethical review committee of the school of biomedical and laboratory sciences, college of medicine and health sciences, university of gondar. moreover, written consent was taken from each participant after they understood the purpose of the study. all the subjects' data were kept in full confidentiality and were not being disclosed to an unauthorized person. not applicable. extended hospital stay days, mortality and increased cost of healthcare) nurse or physician) is non-compliant with the recommended guidelines for patient safety in my opinion, healthcare workers should be sanctioned for non-compliance with protocols for reducing transmission of nosocomial infections (for example, yearly assessment, and denied promotion) in my opinion, healthcare workers should be rewarded (for example, given plaques, certificate) for compliance with protocols aimed at reducing transmission of nosocomial the number of graduate health science students; % indicates percentage engda prevention of hospital acquired infections: a practical guide is us health really the best in the world? decreasing urinary tract infections through staff development, outcomes, and nursing process statistical abstract of the united states guidelines for hand hygiene in health care settings an outbreak of hepatitis c virus infections among patients at a hematology/oncology clinic new jersey: pearson education cluster of cases of severe acute respiratory syndrome among toronto healthcare workers after implementation of infection control precautions: a case series use of influenza a (h1n1) monovalent vaccine: recommendations of the advisory committee on immunization practices (acip) the global burden of disease attributable to contaminated injections given in health care settings guidelines for preventing the transmission of mycobacterium tuberculosis in health-care settings training self-assessment and task-selection skills: a cognitive approach to improving self-regulated learning student self-assessment: the key to stronger student motivation and higher achievement. educ horizons impact of education on knowledge, attitudes and practices among various categories of health care workers on nosocomial infections knowledge and performance of the universal precautions by nursing and medical students in korea exploring knowledge, attitudes and practices of registered nurses regarding the spread of nosocomial infections. seton hall university dissertations and theses (etds): paper 1865 poor knowledge predictor of nonadherence to universal precautions for blood borne pathogens at first level care facilities in pakistan assessment of knowledge, attitudes and practices toward prevention of hepatitis b virus infection among students of medicine and health sciences in northwest ethiopia knowledge, practice and associated factors of infection prevention among healthcare workers in debre markos referral hospital, northwest ethiopia. bmc hand hygiene compliance and associated factors among health care providers in gondar university hospital attitude and practice of nursing students regarding hand hygiene in the western region of nepal taxonomy education attitude and practice of nursing students on hospital acquired infections in western region of nepal the author declares that there is no competing interest.received: 6 june 2019 accepted: 1 october 2020 key: cord-025495-udz9i0fw authors: nowak, jan k.; walkowiak, jarosław title: lithium and coronaviral infections. a scoping review. date: 2020-04-03 journal: f1000res doi: 10.12688/f1000research.22299.2 sha: doc_id: 25495 cord_uid: udz9i0fw the current rapid spread of the novel coronavirus (sars-cov-2) causing coronavirus disease 2019 (covid-19) calls for a rapid response from the research community. lithium is widely used to treat bipolar disorder, but has been shown to exhibit antiviral activity. this brief review took a systematic approach to identify six in vitro studies reporting on the influence of lithium on coronaviral infections. we propose mechanistic investigation of the influence of lithium – alone and with chloroquine – on the sars-cov-2 infection. the current rapid spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) causing coronavirus disease 2019 (covid-19), calls for a rapid response from the research community. lithium is known to exhibit antiviral activity, but the knowledge of its potential as a possible therapy for coronoviral infections has not been summarized yet. the aim of this brief report is to draw attention to lithium as potential covid-19 treatment and prophylaxis. on february 1 st 2020 the following pubmed search was conducted with no language or time restrictions: (lithium and (coronavirus or *coronavirus or sarbecovirus or sars or "severe acute respiratory syndrome" or mers or "middle east respiratory syndrome" or nobecovirus or merbecovirus or hibecovirus or embecovirus or andecovirus or buldecovirus or herdecovirus or moordecovirus or cegacovirus or igacovirus or "microhyla lentovirus" or milecovirus or alphaletovirus or tegacovirus or setracovirus or rhinacovirus or pedacovirus or "porcine epidemic diarrhea" or nyctacovirus or "nectalus velutinus" or myotacovirus or "myotis ricketti" or minunacovirus or minacovirus or luchacovirus or duvinacovirus or decacovirus or "rhinolophus ferrumequinum" or "transmissible gastroenteritis virus" or "feline infectious peritonitis virus" or "canine coronavirus" or "murine hepatitis virus")). the search yielded 45 articles, of which all the abstracts were charted and reviewed by two researchers. six studies reporting on the influence of lithium on coronaviral infections were identified ( figure 1 ). in vero cells, lithium chloride (investigated at 1-15 mm) was shown to be dose-dependently effective in suppressing infection with the porcine epidemic diarrhea virus (pedv), a member of the coronaviridae family 1 . not only pedv entry and replication were inhibited in the presence of licl, but apoptosis as well. yet, licl at 1 mm (safe in patients) was not effective. at 5 mm licl reduced viral rna levels by 30% (p < 0.001). in marc-145 cells, licl reduced the production of rna and proteins specific to the porcine reproductive and respiratory syndrome virus. the relative viral mrna level decreased by more than 30% (p < 0.001) at the concentration of 10 mm and by 50% at 20 mm (p < 0.001). the authors, however, cautioned that the effect might have been dependent on licl presence during the early stages of viral replication (first 9 hours) and the increase of tumor necrosis factor-α, which was greater following licl alone than induced by the virus 2 . in vitro studies of another porcine coronavirus causing transmissible gastroenteritis indicated that licl (5-25 mm) acts on both early and late stages of infection and inhibits apoptosis 3 . both virus titer reduction and cell survival at 70-90% were achieved with licl at 25 mm (10-50% at 5 mm). the same research group from harbin in china reported earlier that licl (investigated at 5-50 mm) reduced the cytopathic effect of the avian infectious bronchitis virus (also a coronavirus) in primary chicken embryo kidney cells 4 . the results suggest that the dose of 5 mm was beneficial (20% inhibition) when applied one hour after infection, but not 8 hours post infection. in vero cells, african green monkey kidney-derived epithelial cells, and immortalized chicken embryo fibroblasts licl suppressed the avian coronavirus infectious bronchitis. relative virus titers in both cell lines were reduced by at least 45% at 5 mm and 70-90% at 10 mm. viral mrna concentration decreased 20 times in both cell types cultured with 5 mm licl. overall, the antiviral activity of lithium was ascribed to a cellular effect 5 . one study was identified outside the main search reports on the activity of high licl concentrations (10-60 mm) against porcine deltacoronavirus: at 10 mm 50% relative mrna reduction was found with no accompanying effect on the viral titer 6 . the available evidence comes only from studies of cell cultures and indicates that lithium effectively inhibits coronaviral infections when administered at concentrations that are toxic to humans. the major putative molecular mechanisms of antiviral activity and reduced apoptosis is the inhibition of glycogen synthase kinase 3-beta (gsk-3β) 7, 8 . however, lithium also inhibits gsk-3α, inositol monophosphatases, and may indirectly act via the electrolyte balance. pedv requires the pi3k/akt/gsk-3α/β pathway, which can be targeted at gsk-3β by lithium 9 . curiously, gsk-3β is required for template switching, a process seemingly indispensable for the production of coronaviral genomic rna. the inhibition of gsk-3β prevents longer viral subgenomic mrnas and the genomic rna from being synthesized 10 . their production would require gsk-3β-dependent phosphorylation of the viral nucleocapsid and subsequent recruitment of helicase ddx1. chloroquine (hydroxychloroquine) -which is thought to be effective in covid-19 11 -was shown to inhibit gsk-3β and potentiate gsk-3β inhibition caused by lithium. this indicates that mechanistic studies could investigate not only 0.5-1.2 mm lithium, but lithium with chloroquine as well. this also brings zinc to the spotlight since zinc inhibits gsk-3β at micromolar concentrations 12 . there is some evidence that lithium may affect the course of viral diseases in humans. in a retrospective cohort study of patients with affective disorders a decrease in the rate of recurrent labial herpes was found in the lithium group (n = 177, p < 0.001) but not in the alternative treatment group (n = 59, p = 0.53) 13 . in research previously conducted by prof. j. rybakowski at our hospital, lithium prevented labial herpes recurrence in thirteen out of 28 eligible psychiatric patients. lithium also seemed to bring improvement in a proof-of-concept randomized doubleblind placebo-controlled trial involving eleven healthy adults with recurrent hsv infections 14 and in a randomized study of ten women with genital herpes conducted by the same research group. other evidence for antiviral activity licl was shown to dose-dependently inhibit reovirus (10-60 mm) 15 and food-and-mouth disease virus (10-40 mm) 16 . at 5 mm concentration licl reduced the replication of avian leukosis virus subgroup j in chicken embryo fibroblast cells 17 . yet, lithium at 50 µm concentration (12-20 times smaller than usually maintained in bipolar disorder) significantly reduced hepatitis c virus copy number (p = 0.0002) in supernatant from huh7.5 cell culture 18 . the latter study gives hope that lithium may indeed be efficient at clinically relevant levels. lithium carbonate is an orphan drug widely used in the treatment of bipolar disorder. its safety, when used correctly, is excellent 19 . the main concern in the setting of an infectious disease unit would be the potential for interactions with other medication, possibly leading to the elevation of lithium levels and acute toxicity, mostly renal. this may be prevented by monitoring serum lithium concentrations. to our best knowledge, no interactions between lithium carbonate and ribavirin, lopinavir or ritonavir exist. a randomized study in tenofovir-treated patients with hiv revealed that 24-week addition of lithium at target serum concentrations of 0.6-1.0 mmol/l was not associated with nephrotoxicity 20 . lithium concentration may be, on the other hand, increased by loop or thiazide diuretics, angiotensin-converting enzyme inhibitors, and non-steroid anti-inflammatory drugs. it is also is not clear if the use of lithium would be safe in acute disease accompanied by dehydration and unstable electrolyte levels. cardiotoxicity of lithium may occur not only with concentrations larger than 1.5 mmol/l, but also when levels of the ion rapidly change 21 . although qtc prolongation is absent in most patients receiving lithium, qt dispersion ratio may increase; longer qt was also described in some cases. concurrent use of lithium with chloroquine would need to be especially cautious in patients with qt prolongation. in the light of the reviewed data lithium appears as a possible candidate for therapy of covid-19. we propose mechanistic investigation of the influence of lithium (0.5-1 mm) -alone and with chloroquine or other drugs -on the sars-cov-2 infection. underlying data all data underlying the results are available as part of the article and no additional source data are required. the rapid spread of covid-2019 around the world has caused more than 5 million infections and over 300,000 deaths, which undoubtedly has a huge impact on the global economy and people's health. it is necessary to explore the potential treatment of the disease. the two authors described the effects of different concentrations of lithium on coronavirus infections and discussed potential mechanisms based on existing literature. although the current reports are mostly cell studies, it seems that lithium plays a role in inhibiting coronavirus infection. in the revised version of the paper, the authors state that lithium appears as a possible candidate for therapy of covid-19. and they also made attempt to investigate the influence of lithium (0.5-1 mm) -alone and with chloroquine or other drugs -on the sars-cov-2 infection. besides, they emphasized the need to monitor blood lithium concentrations, which made their conclusions more rigorous. in summary, the prediction of the potential role of lithium in the treatment or prevention of covid-2019 in this paper is reasonable to some extent. however, so far, there have been no in vivo studies and other relevant conclusive evidence to confirm this hypothesis. it may still be necessary to carry out research on related aspects in the future. although lithium may not soon be used in the epidemic of covid-19, it may have a certain role in the next similar outbreak. overall a well-revised manuscript with interesting results. i recommend indexing. no competing interests were disclosed. we confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. the authors provided a systematic approach to identify six studies reporting on the influence of in vitro lithium on coronaviral infections. the discussion on the potential role of lithium is hypothetical and the proposed key target does not seem relevant in the context of covid-19. some comments: ref 2: at 5 mm licl reduced viral rna levels by 30% (p < 0.001), this level is commonly lethal to humans (and when not fatal, may be associated with respiratory failure, a hallmark in covid-19. in the same study, therapeutic levels did not work the same way, thus it seems that its antiviral effects are related to lithium's overall toxicity; also considering the higher levels (10 and 20mm) showed a linear decrease in viral mrna. similar effects may be applied to references 3 and 4, which also describe supra-therapeutic levels of lithium as potential anti-viral, which is hard to translate into potential therapeutics. in line with the presented findings, lithium showed to ameliorate hiv-gp120-mediated neurotoxicity. gsk3-b is a widespread target for lithium effects and based its wide distribution in several organs and having several other subunits targeted as well as gsk-3b inhibitors failed in early studies with most of the related agents and did not mimic any clinical effects of lithium. thus, the enthusiasm of its potential utility as a treatment target in drug development (e.g. mood disorders) has been significantly diminished in the last few years. also, the risk or severity of qtc prolongation can be increased when chloroquine is combined with lithium, and gsk-3 is not a target directly associated with covid-19 in any published study so far. effects of lithium on hiv seem much more beneficial than associated with worse outcomes and this potential effect seems to be relevant in the context. description of safety and limitations are generic in terms of lithium effects, except for the qt prolongation in combination with chloroquine, do not highlight the specific aspects related to the author's hypothesis, such as cell proliferation. evidence for the antiviral effects of lithium in prospective clinical studies remains to be presented. also, the suggestion of a similar role for lithium and zinc in this context of covid-19 should be further explained in terms of molecular and ionic convergent effects. the possibility that lithium may be useful for corona virus infections remains hypothetical. this is now acknowledged and the article presents the limitations of this approach. a possible supporting study would be to compare disease severity in lithium treated vs non-lithium treated subjects in patients receiving these drugs in the context of the covid 19 epidemic. since several psychoactive drugs (e.g. antipsychotic) also affect putative molecular targets of lithium (e.g.gsk3alpha and beta) , , groups treated with these drugs would be interesting to compare to lithium. however, several other factors affecting people with psychiatric illness therapies may also affect the impact of covid19 or other corona viruses on these populations. this is obviously beyond the scope of this review/hypothesis article. as a technical issue lithium not only targets gsk3b but also gsk3-alpha and inositol monophosphatases. so the emphasis on gsk3-beta may be a bit premature. a more important issue is that none of the studies shown an effect of lithium at a 1-1.5mm concentrations. effects are reported at li+ concentrations that are 5mm or higher. these concentrations are not toxic for cells in culture. however, in humans, serum lithium concentration above 1.5-2.0mm (or meq, which stands for the mm concentration of the lithium ion) are considered toxic (haussmann , 2015 ) . et al. the prescription of lithium in the context of the current epidemic thus appears not to be supportable by the findings. the cure may kill the patients. more detailed studies using lithium in animal models at tolerable concentrations would thus be needed. unfortunately these limitations are not addressed in the manuscript. we are grateful for the comments that you have provided. they helped to improve our manuscript. please find our responses below. sincerely yours, jan nowak and jarosław walkowiak we thank the reviewer for this comment. this is now referred to early in the discussion: " this important comment has triggered a number of major changes in the manuscript. in order not to mislead the readers we propose a new title, which is more neutral: "lithium and coronaviral infections. a scoping review." the conclusion of the abstract was changed: "we propose mechanistic investigation of the influence of lithium -alone and with chloroquine -on the sars-cov-2 infection." the conclusion at the end of the discuss was altered: "in the light of the reviewed data lithium appears as a possible candidate for therapy of covid-2019. we propose mechanistic investigation of the influence of lithium (0. throughout the results section we now report and comment on licl concentrations, which are clearly above the levels, which are safe for humans. examples: " licl." "one study was identified outside the main search reports on the activity of high licl concentrations (10-60 mm) against porcine deltacoronavirus: at 10 mm 50% relative mrna reduction was found with no accompanying effect on the viral titer ." crucially, the discussion now opens with: "the available evidence comes only from studies of cell cultures and indicates that lithium effectively inhibits coronaviral infections when administered at concentrations that are toxic to humans." the prescription of lithium in the context of the current epidemic thus appears not to be supportable by the findings. the cure may kill the patients. as cited above, the discussion now starts by stating: "the available evidence comes only from studies of cell cultures and indicates that lithium effectively inhibits coronaviral infections when administered at concentrations that are toxic to humans." however, hypothesizing that lithium could be useful in treating viral infections is now supported by some other evidence: "there is some evidence that lithium may affect the course of viral diseases in humans. in a retrospective cohort study of patients with affective disorders a decrease in the rate of recurrent labial herpes was found in the lithium group (n = 177, p < 0.001) but not in the alternative treatment group (n = 59, p = 0.53) . in research previously conducted by prof. j. rybakowski at our hospital, lithium prevented labial herpes recurrence in thirteen out of 28 eligible psychiatric patients. lithium also seemed to bring improvement in a proof-of-concept randomized double-blind placebo-controlled trial involving eleven healthy adults with recurrent hsv infections and in a randomized study of ten women with genital herpes conducted by the same research group from philadelphia." therefore it seems that in some instances lithium exhibits antiviral activity at concentrations, which are safe and maintained long-term (for years) in patients with affective disorders. additionally, the discussion of lithium safety is now broader. although qtc prolongation is absent in most patients receiving lithium, qt dispersion ratio may increase; longer qt was also described in some cases. concurrent use of lithium with chloroquine would need to be especially cautious in patients with qt prolongation." more detailed studies using lithium in animal models at tolerable concentrations would thus be needed. as we mentioned, this has been adapted to be the conclusion of our study. "in the light of the reviewed data lithium appears as a possible candidate for therapy of 6 13 14 21 "in the light of the reviewed data lithium appears as a possible candidate for therapy of covid-2019. we propose mechanistic investigation of the influence of lithium (0.5-1 mm)alone and with chloroquine or other drugs -on the sars-cov-2 infection." unfortunately these limitations are not addressed in the manuscript. we thank for the critique, which has helped to transform our manuscript. no competing interests were disclosed. in terms of discussion, the authors reviewed some existing literature and suggested a potential mechanism of reduced apoptosis by lithium, the glycogen synthase kinase 3-beta (gsk-3β) inhibitor. the possibility that targeting at gsk-3β by lithium may potentially affect the coronavirus is an interesting topic. however, direct evidence is lacking regarding 2019-ncov or related coronaviruses including in vitro severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronaviruses. moreover, relevant literature is still needed although the authors state that there is no interaction between lithium carbonate and ribavirin, lopinavir, or ritonavir exist. another aspect worth noting is that the authors indicate that monitoring serum lithium concentration can be helpful in preventing side effects of lithium, however it should be emphasized that the relationship between the effective dose and toxic dose in vivo of lithium is still unclear, with some studies reporting a dose-dependent manner of the inhibitory effect of lithium . thus, it warrants more data, both and , to clarify this issue. collectively, this study proposes a potential role of lithium in treating or preventing 2019-ncov or ncp with some possible mechanisms. however, by far, solid evidence is lacking to validate this hypothesis. the time of developing lithium orotate for clinical use, even in emergency, is not yet. are all the source data underlying the results available to ensure full reproducibility? yes no competing interests were disclosed. we confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. we would like to thank you for all the comments. they helped to improve the manuscript. please find our responses below. sincerely yours, the wide spread of infection of 2019-ncov has arouse an international concern since its original outbreak in wuhan, china. scientists and health workers around the world are currently working together to wipe out the virus and the novel coronavirus pneumonia (ncp), which has killed more than a thousand lives, by far, worldwide. with the current epidemic being so severe, it is necessary and urgent to make potentially reasonable recommendations for the treatment or prevention for 2019-ncov or ncp. the two authors clearly proposed that lithium might be a potential treatment or prophylaxis for 2019-ncov or ncp based on a summary of existing literature that reported the in vitro effects of lithium on coronaviral infections and discussed potential mechanisms, which sound reasonable to some extent, but still not rigorous. we appreciate the critique, which has helped to improve our manuscript. specifically, there are few related studies available and only in vitro data have been reported. the authors may need more related studies and solid evidence to support their hypothesis to make it more scientific and rigorous. more details are provided in the results. new paragraphs also discuss the antiviral activity in humans and cell cultures challenged with other viruses. as reported, lithium can be toxic due to its side effects, mainly thyroid, renal, and cognitive disturbances. readers may wish to see more clinical information of lithium in treating viral infection cases, if not available, or in treating other diseases. lithium cardiotoxicity is now discussed in more detail. "lithium concentration may be, on the other hand, increased by loop or thiazide diuretics, angiotensin-converting enzyme inhibitors, and non-steroid anti-inflammatory drugs. it is also is not clear if the use of lithium would be safe in acute disease accompanied by dehydration and unstable electrolyte levels. cardiotoxicity of lithium may occur not only with concentrations larger than 1.5 mmol/l, but also when levels of the ion rapidly change . although qtc prolongation is absent in most patients receiving lithium, qt dispersion ratio may increase; longer qt was also described in some cases. concurrent use of lithium with chloroquine would need to be especially cautious in patients with qt prolongation." a paragraph on lithium and herpes infections in patients with affective disorders was added. the results of the cited studies are the best evidence for the antiviral activity of lithium that comes from studies conducted in patients. "there is some evidence that lithium may affect the course of viral diseases in humans. in a retrospective cohort study of patients with affective disorders a decrease in the rate of recurrent labial herpes was found in the lithium group (n = 177, p < 0.001) but not in the alternative treatment group (n = 59, p = 0.53) . in research previously conducted by prof. j. rybakowski at our hospital, lithium prevented labial herpes recurrence in thirteen out of 28 eligible psychiatric patients. lithium also seemed to bring improvement in a 21 13 eligible psychiatric patients. lithium also seemed to bring improvement in a proof-of-concept randomized double-blind placebo-controlled trial involving eleven healthy adults with recurrent hsv infections and in a randomized study of ten women with genital herpes conducted by the same research group from philadelphia." more information on the activity of lithium in other viral infections was also provided. "licl was shown to dose-dependently inhibit reovirus (10-60 mm) and food-and-mouth disease virus (10-40 mm) . at 5 mm concentration licl reduced the replication of avian leukosis virus subgroup j in chicken embryo fibroblast cells . yet, lithium at 50µm concentration (12) (13) (14) (15) (16) (17) (18) (19) (20) times smaller than usually maintained in bipolar disorder) significantly reduced hepatitis c virus copy number (p = 0.0002) in supernatant from huh7.5 cell culture . the latter study gives hope that lithium may indeed be efficient at clinically relevant levels." in terms of discussion, the authors reviewed some existing literature and suggested a potential mechanism of reduced apoptosis by lithium, the glycogen synthase kinase 3-beta (gsk-3β) inhibitor. the possibility that targeting at gsk-3β by lithium may potentially affect the coronavirus is an interesting topic. however, direct in vitro evidence is lacking regarding 2019-ncov or related coronaviruses including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronaviruses. certainly, the evidence is lacking. the discussion is speculative. we have added the following: "the major putative molecular mechanisms of antiviral activity and reduced apoptosis is the inhibition of glycogen synthase kinase 3-beta (gsk-3β) . however, lithium also inhibits gsk-3α, inositol monophosphatases, and may indirectly act via the electrolyte balance." and also: "chloroquine (hydroxychloroquine) -which is thought to be effective in covid-2019was shown to inhibit gsk-3β and potentiate gsk-3β inhibition caused by lithium. this indicates that mechanistic studies could investigate not only 0.5-1.2 mm lithium, but lithium with chloroquine as well. this also brings zinc to the spotlight since zinc inhibits gsk-3β at micromolar concentrations ." moreover, relevant literature is still needed although the authors state that there is no interaction between lithium carbonate and ribavirin, lopinavir, or ritonavir exist. information on tenofovir is now provided: "a randomized study in tenofovir-treated patients with hiv revealed that 24-week addition of lithium at target serum concentrations of 0.6-1.0 mmol/l was not associated with nephrotoxicity ." another aspect worth noting is that the authors indicate that monitoring serum lithium concentration can be helpful in preventing side effects of lithium, however it should be emphasized that the in vivo relationship between the effective dose and toxic dose of lithium is still unclear, with some studies reporting a dose-dependent manner of the inhibitory effect of lithium in vitro. thus, it warrants more data, both in vitro and in vivo, to clarify this issue. throughout the results section, information on the concentrations of lithium were given and commented on. moreover, the discussion opens with: "the available evidence comes only from studies of cell cultures and indicates that lithium "the available evidence comes only from studies of cell cultures and indicates that lithium effectively inhibits coronaviral infections when administered at concentrations that are toxic to humans." collectively, this study proposes a potential role of lithium in treating or preventing 2019-ncov or ncp with some possible mechanisms. however, by far, solid evidence is lacking to validate this hypothesis. the time of developing lithium orotate for clinical use, even in emergency, is not yet. the reference to lithium orotate was removed. the conclusion was changed: "in the light of the reviewed data lithium appears as a possible candidate for therapy of covid-2019. we propose mechanistic investigation of the influence of lithium (0.5-1 mm)alone and with chloroquine or other drugs -on the sars-cov-2 infection." we would like the reviewers for their input, which has guided us in improving the text. please note that following the remarks of prof. beaulieu and that change of the nomenclature (2019-ncov no longer used) we have proposed a new title, which seems more neutral and therefore more representative of the softened conclusions: "lithium and coronaviral infections. a scoping review." the references were updated the benefits of publishing with f1000research: your article is published within days, with no editorial bias you can publish traditional articles, null/negative results, case reports, data notes and more the peer review process is transparent and collaborative your article is indexed in pubmed after passing peer review dedicated customer support at every stage for pre-submission enquiries, contact research@f1000.com antiviral effect of lithium chloride on porcine epidemic diarrhea virus in vitro lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture antiviral effect of lithium chloride and diammonium glycyrrhizinate on porcine deltacoronavirus in vitro glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3beta and caspase-3 involvement of glycogen synthase kinase-3beta in palmitate-induced human umbilical vein endothelial cell apoptosis suppression of virulent porcine epidemic diarrhea virus suppression of virulent porcine epidemic diarrhea virus proliferation by the pi3k/akt/gsk-3α/β pathway nucleocapsid phosphorylation and rna helicase ddx1 recruitment enables coronavirus transition from discontinuous to continuous transcription multicenter collaboration group of department of science and technology of guangdong province and health commission of guangdong province for chloroquine in the treatment of novel coronavirus pneumonia gsk-3 inhibitors: preclinical and clinical focus on cns a possible antiviral action of lithium carbonate in herpes simplex virus infections suppression of herpes simplex virus infections with oral lithium carbonate--a possible antiviral activity novel antiviral effect of lithium chloride on mammalian orthoreoviruses in vitro lithium chloride inhibits early stages of foot-and-mouth disease virus (fmdv) replication in vitro antiviral effect of lithium chloride on replication of avian leukosis virus subgroup j in cell culture glycogen synthase kinase 3β inhibitors prevent hepatitis c virus release/assembly through perturbation of lipid metabolism challenging the negative perception of lithium and optimizing its long-term administration inhibitory effects of lithium chloride on replication of type ii porcine reproductive and respiratory syndrome virus action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus comparative analysis of the effect of glycyrrhizin diammonium and lithium chloride on infectious bronchitis virus infection lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture antiviral effect of lithium chloride and diammonium glycyrrhizinate on porcine deltacoronavirus in vitro glycyrrhizinate on porcine deltacoronavirus in vitro glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3beta and caspase-3 involvement of glycogen synthase kinase-3beta in palmitate-induced human umbilical vein endothelial cell apoptosis suppression of virulent porcine epidemic diarrhea virus proliferation by the pi3k/akt/gsk-3α/β pathway nucleocapsid phosphorylation and rna helicase ddx1 recruitment enables coronavirus transition from discontinuous to continuous transcription multicenter collaboration group of department of science and technology of guangdong province and health commission of guangdong province for chloroquine in the treatment of novel coronavirus pneumonia gsk-3 inhibitors: preclinical and clinical focus on cns a possible antiviral action of lithium carbonate in herpes simplex virus infections suppression of herpes simplex virus infections with oral lithium carbonate--a possible antiviral activity novel antiviral effect of lithium chloride on mammalian orthoreoviruses in vitro lithium chloride inhibits early stages of foot-and-mouth disease virus (fmdv) replication in vitro antiviral effect of lithium chloride on replication of avian leukosis virus subgroup j in cell culture glycogen synthase kinase 3β inhibitors prevent hepatitis c virus release/assembly through perturbation of lipid metabolism challenging the negative perception of lithium and optimizing its long-term administration renal safety of lithium in hiv-infected patients established on tenofovir disoproxil fumarate containing antiretroviral therapy: analysis from a randomized placebo-controlled trial lithium-induced electrocardiographic changes: a complete review prisma scoping review checklist for nowak and walkowiak 2020 no competing interests were disclosed the wide spread of infection of 2019-ncov has arouse an international concern since its original outbreak in wuhan, china. scientists and health workers around the world are currently working together to wipe out the virus and the novel coronavirus pneumonia (ncp), which has killed more than a thousand lives, by far, worldwide.with the current epidemic being so severe, it is necessary and urgent to make potentially reasonable recommendations for the treatment or prevention for 2019-ncov or ncp. the two authors clearly proposed that lithium might be a potential treatment or prophylaxis for 2019-ncov or ncp based on a summary of existing literature that reported the effects of lithium on coronaviral infections and in vitro discussed potential mechanisms, which sound reasonable to some extent, but still not rigorous.specifically, there are few related studies available and only data have been reported. the authors in vitro may need more related studies and solid evidence to support their hypothesis to make it more scientific and rigorous. as reported, lithium can be toxic due to its side effects, mainly thyroid, renal, and cognitive disturbances. readers may wish to see more clinical information of lithium in treating viral infection cases, if not available, or in treating other diseases.in terms of discussion, the authors reviewed some existing literature and suggested a potential thank you for all the comments. the potential for interaction between lithium and chloroquine is discussed in the revised version of the article, which was submitted more than a week ago. as a lithium patient taking 800mg li carbonate i can vouch for its antiviral properties, i have not had a single cold or illness since commencing the drug for major depressive disorder in dec 2019. i have passed this article on to relevant authorities in the hope someone takes note. i am happy to volunteer for research trials. key: cord-017575-msc99cit authors: andersen, bjørg marit title: dangerous microbes date: 2018-09-25 journal: prevention and control of infections in hospitals doi: 10.1007/978-3-319-99921-0_80 sha: doc_id: 17575 cord_uid: msc99cit the most dangerous microbes for humans are those that are easily transmitted, virulent and invasive to central organs like the blood and lung, robust survivors in the environment, have a low infection dose and are without any specific treatment or vaccine. most of them are zoonoses transmitted from animals and often with insects as vectors. the most dangerous microbes cause a very high mortality, are identified as high-risk agents or “biohazard-level 4” agents and are treated at the highest level of infection protection with strict isolation measures. dangerous microbes occur as a problem mostly in countries with low hygiene standards/high population density and in tropical-subtropical areas. infection control must always be based on hygienic measures and strict infection protection. this chapter is a short information about the most virulent and pathogenic agents, geographic area and severity of disease. example, avian influenza viruses (h5n1 and others), htlv and other retroviruses, hpv, sindbis virus, parvovirus, bocavirus, coronavirus, or bacteria like legionella, borrelia, group a streptococci and meningococci [1] [2] [3] [4] [5] . virus not vaccine preventable or without specific treatment option, low infection dose and high capacity to survive in the environment will be a major problem if associated with incurable disease, disability or death. virulent and dangerous microbes occur as a problem mostly in countries with low hygiene standards/high population density and in tropical-subtropical areas. infection control must be based on hygienic measures and proper infection protection [2] [3] [4] [5] [6] [7] . the most dangerous microbes for humans are those that are easily transmitted, virulent and invasive to the central organs like the blood and lung, robust survivors in the environment, have a low infection dose and are without any specific treatment or vaccine [2] [3] [4] [5] . most of them are zoonosis transmitted from animals and often with insects as vectors [8] [9] [10] [11] [12] . dangerous microbes have also been used as biological weapons like anthrax, plague, botulism, coxiella, brucella, viral haemorrhagic fever (vhf) viruses, poxviruses and a number of other unusual agents [4, [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . in the united states, there was an anthrax bioterrorism attack in 2001 [18, 19] . if the infectious agent and transmission ways are unknown and the situation is uncontrollable, it is important to follow pre-planned practical measures like strict isolation in negative air pressure isolation, registering of all contacts and the use of personal protective equipment (ppe) for air-and contact-borne infection [4, 8-13, 17, 20, 24, 26-29] . [1, 4, 5, 8, 11, 12] . vhf virus and a number of other special viruses are considered to cause the world's most dangerous infections with very high mortality, lack of therapeutic possibilities and often absence of effective vaccines. such viruses are identified as highrisk agents or "biohazard-level 4" agents and are treated at the highest level of infection protection with strict isolation measures [4, 11, 12, [26] [27] [28] . • virus families, with members especially dangerous to humans, are arenaviridae, filoviridae, flaviviridae, bunyaviridae, togaviridae and paramyxoviridae (nipah encephalitis virus, etc.) and several others. they are mostly vector borne by insects and animals (zoonosis), dependent on a tropical-subtropical climate and temperature. therefore, the colder zones in europe, america, asia and australia are nearly free from more such serious infectious agents (see the table below) [1, 2, 4, 11, 12] . • some very virulent viruses may be spread on all continents: tbe, rsse, cchf, rabies virus (rhabdoviridae), avian influenza virus (ah5n1) and other highpathogenic influenza viruses (orthomyxoviridae). sars and mers (coronaviridae) may be spread globally as nosocomial infections. mers is a newly discovered coronavirus-zoonosis (dromedaries, bats, etc.) and acts as sars, also with a tendency for nosocomial spread in hospitals, still going on in the middle east. [30] these and other viruses are adapted to the climate and spread by direct transmission person-to-person, by increased mobility in the population and/or spread via new vectors, like animals, birds and insects [1, 4] . • a few of these dangerous viruses are vaccine preventable, and specific antiviral therapy is lacking in most cases. • zika virus-newly discovered flavivirus in south america, with mild to serious symptoms and teratogenic effect [11] . bacteria resistant to antibacterial drugs have developed rapidly among staphylococci (mrsa), enterococci (vre), tuberculosis bacilli (multidrug-resistant mycobacteria) and gram-negative rods (esbl) in the last 30-50 years. from 2009 to 2010 onwards, super-resistant gram-negative rods (cre, cpe) have increased rapidly with transmission of genes with total drug resistance (ndm-1) [1] [2] [3] [4] [5] . bacteria sensitive to common antibacterial agents will probably not be a major problem in the future, unless changed antibacterial sensitivity. • anthrax is still endemic or hyperendemic on all continents in the world, mainly as a zoonosis among unvaccinated cattle [1-6, 13, 33] . it has been known for more than 4000 years and was earlier called "black bane". natural anthrax in human is associated with contact with sick animals and animal products. the bacterium bacillus anthracis is producing lethal toxins and spores that can survive in the environments for 100 years or more. minimum infectious dose is 1-3 spores, and toxin-producing anthrax may cause skin, lung or intestinal anthrax, dependent on the transmission via contact, air or food. the non-skin anthrax has a very high lethality. today, about 2000 people are infected each year, from eating or handling infected meat. during peace time, there are few incidents of threats using bioterror weapons like anthrax. however, autumn 2001 anthrax was spread in the united states by "powder letters" [18, 19] . • plague, caused by yersinia pestis (enterobacteriaceae), is a zoonotic disease, vector-transmitted from infected rodents to humans via infected insects (fleas and lice) [1] [2] [3] [4] [5] 8] . it is one of the great historical diseases. in the last 1500 years, plague has killed more than 200 million people during large epidemics (black death), especially from the year 1347. still, plague causes bubonic-glandular pest, lung pest and/or septicaemia in 2000-3000 cases each year, and more than 10 outbreaks (africa, asia, australia, madagascar) have occurred since the year 2000 [8, 34] . • cholera caused by vibrio cholera is one of the great quarantine diseases, called "mother of all epidemics", still very active in asia and africa. cholera is associated with contaminated water, war and nature catastrophes, like in haiti 2010, with 670,000 cholera cases (profuse diarrhoea) [1] [2] [3] [4] [5] 35 ]. • brucella species (brucellaceae) are one of the most important zoonotic diseases both for animals and humans all over the world. they easily spread via contact and air from animals and food, survive in the environment for months especially during colder seasons, are very easily transmitted and may cause nosocomial infections, chronic febris undulans, septicaemia and lung diseases. vaccination of animals may eradicate the zoonotic disease [15] . brucella are particularly related to laboratory outbreaks but are easily transferable outside the laboratory and are considered highly infectious [36] . • francisella tularensis (zoonosis) is defined as a category a bioterrorism agent, highly infectious and increasing in the society, has low infection dose (one to ten bacteria) and can be inhaled or infected via food and water [1] [2] [3] [4] [5] 8] . occasionally, such patients are detected in hospitals, even in the operating department [37] . tularemia is a zoonotic disease or colonization among wild animals as rodents and hares and may survive at colder seasons for long periods in water, amoebas, birds and insects like ticks. the infection (skin, lung, intestine) is transmitted via contact with infected animals, inhalation of airborne bacteria, contaminated water and ticks. • botulism is caused by a bacterial-produced toxin (clostridium botulinum) that causes paresis and is common in soil as spores [1] [2] [3] [4] [5] . the disease can be associated with toxin formation in contaminated and poorly canned foods, shrimp fish, bacon, etc. under anaerobic conditions and randomly affects both healthy people and vulnerable groups, such as infants who have had honey infected with the bacterium, which has happened repeatedly [38] . the toxin is the most dangerous we know and is on the list of bioterrorism. • family rickettsiaceae is associated with many different types of small, intracellular microbes that are zoonotic and survive for a long time in the nature and in infected animals, birds, insects, water and amoeba [2, 3, 10] . coxiella burnetii is a global zoonosis among animals like sheep and goats and may be airborne for several kilometres, causing frequent outbreaks of q-fever in europe in more than 4000 cases in the netherlands during 2007-2010 [10] . rickettsia species are more than 25 types, and many of them, especially the typhus group, are causing severe diseases. humans are mostly infected by insects, including ticks from infected wild animals and rodents usually found in tropical or subtropical areas, but climatic changes, lack of good hygiene, war and natural catastrophes may spread these infections. during the period 1881-1920, 30 million people got epidemic typhus, and three million died in east europe and russia [2, 3, 10] . • borrelia (spirochetaceae) has an increasing number of species that may infect human cases all over the world. the disease may cause chronic infections in the skin, muscles, heart, nerve tissue and brain. borreliosis is a typical emerging zoonosis, spread via different ticks from animals and birds over large geographic areas and with large climatic variations [9] . • syphilis (spirochetaceae) and treponema pallidum are old, historical diseases, transmitted via sex, from mother to child during pregnancy and via blood products. now re-emerging and increasing worldwide [9] . • other spirochetes or the like may cause zoonosis, like borrelia recurrentis and leptospirosis [9] . • burkholderia pseudomallei is causing melioidosis, pneumonia, septicaemia, disseminated infection and shock. incubation period may be up to 20-40 years. it is a long survivor in the environment (2-10 years), robust at tropical-subtropical temperatures >25 °c and is very dangerous. endemic in southeast asia and may be spread by increasing globalization [1] [2] [3] [4] [5] . • fungi and mould-different types are especially related to floods, water damage, etc. and to contamination of medical products and equipment [39, 40] . especially, the newly defined candida auris may be very virulent. • prion disease-new shy-drager syndrome is rediscovered in 2015; multiplesystem atrophy (msa) [5, 41] . • ricin is a plant-derived toxin that is still used for bioterrorism in letters to, among others, president obama [42] . bennet's principles and practice of infectious diseases pathogenic bacteria-diagnostic, treatment and infection control. oslo: gyldendal akademisk bacteria and disease. oslo: gyldendal academisk serious, unusual dangerous infections. in: handbook in hygiene and infection control for hospitals. oslo: ullevål university hospital handbook in hygiene and infection control for hospitals. part 1. microbiology and infection control on the protection of workers from the risks related to exposure to biological agents at work ministry of labour and administration. regulations on protection against exposure to biological factors (bacteria, viruses, fungi and more) in the workplace zoonosis-transmitted from animals, insects and environment. in: handbook in hygiene and infection control for hospitals. part 1. norway: fagbokforlaget borreliosis and other spirochetes-zoonosis. in: handbook in hygiene and infection control for hospitals. part 1. norway: fagbokforlaget coxiella, rickettsia and other small bacteria -often intracellular -zoonosis. in: handbook in hygiene and infection control for hospitals. part 1. norway: fagbokforlaget other serious viral infections -zoonosis. in: handbook in hygiene and infection control for hospitals. part 1. norway: fagbokforlaget lassa, and other haemorrhagic viruses. in: handbook in hygiene and infection control for hospitals. part 1. norway: fagbokforlaget anthrax and emergency routines at ullevål university hospital haemorrhagic fever viruses as biological weapons: medical and public health management bioterrorism-related inhalation anthrax: the first 10 cases reported in the united states anthrax as a biological weapon, 2002: updated recommendations for management nosocomial spread of bacillus anthracis bacillus anthracis aerosolization associated with a contaminated mail sorting machine secondary aerosolization of viable bacillus anthracis spores in a contaminated us senate office anthrax inhalation and lethal human infection bioterrorism: crime and opportunity bioterrorism preparedness and response in european public health institutes the use of smallpox virus as a biological weapon: the vaccination situation in france infectious disease disaster: bioterrorism, emerging infections, and pandemics bioterrorism readiness plan: a template for healthcare facilities international infection control guidelines for ebola. hospital healthcare europe. facilities management guidance on personal protective equipment to be used by healthcare workers during management of patients with ebola virus disease in us hospitals, including procedures for putting on (donning) and removal multi-resistant infections in repatriated patients after natural disaster: lessons learned from the 2004 tsunami for hospital infection control acute neurological disease of unknown etiology in children -colorado hiv-cuba: new aggressive variant bacteria and disease. epidemiology, infections and infection protection. oslo: gyldendal akademisk promed mail in: handbook in hygiene and infection control for hospitals. part 1. norway: fagbokforlaget hospital-associated transmission of brucella melitensis outside the laboratory potential risk of aerosol-borne francisella tularensis transmission in the operating room an infant with acute paresis mould-preventing strategies and possible health effects in the aftermath of hurricanes and major floods fungi-human pathogenic. in: handbook in hygiene and infection control for hospitals. part 1. norway: fagbokforlaget prion disease updated: novel prion disease -shy-drager syndrome key: cord-257729-s0vo7dlk authors: bauer, melissa; bernstein, kyra; dinges, emily; delgado, carlos; el-sharawi, nadir; sultan, pervez; mhyre, jill m.; landau, ruth title: obstetric anesthesia during the coronavirus disease 2019 pandemic date: 2020-04-20 journal: anesth analg doi: 10.1213/ane.0000000000004856 sha: doc_id: 257729 cord_uid: s0vo7dlk with increasing numbers of coronavirus disease 2019 (covid19) cases due to efficient human-to-human transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in the united states, preparation for the unpredictable setting of labor and delivery is paramount. the priorities are 2-fold in the management of obstetric patients with covid-19 infection or persons under investigation (pui): (1) caring for the range of asymptomatic to critically ill pregnant and postpartum women; (2) protecting health care workers and beyond from exposure during the delivery hospitalization (health care providers, personnel, family members). the goal of this review is to provide evidence-based recommendations or, when evidence is limited, expert opinion for anesthesiologists caring for pregnant women during the covid19 pandemic with a focus on preparedness and best clinical obstetric anesthesia practice. t he management of obstetric patients infected with coronavirus disease 2019 (covid19) due to human-to-human transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) requires quite unique considerations-from caring for critically ill pregnant and postpartum women to protecting health care workers from exposure during the delivery hospitalization (health care providers, personnel, family members, and beyond). the goal of this review is to provide evidence-based recommendations or, when evidence is limited, expert opinion for anesthesiologists caring for pregnant women during the covid 19 pandemic with a focus on preparedness and best clinical obstetric anesthesia practice. in principle, the clinical characteristics reported in pregnant women with confirmed covid-19 infection in china have been consistent with those reported among nonpregnant adults, with better maternal and neonatal outcomes with covid-19 infection compared with the 2002-2003 severe acute respiratory syndrome (sars) outbreak from sars cov 1 infection. [1] [2] [3] the signs and symptoms of covid-19 infection in a large data set in nonpregnant patients from china were fever (99%), fatigue (70%), cough (59%), shortness of breath (31%), myalgias (35%), headache (6.5%), sore throat (17%), diarrhea (10%), nausea (10%), and vomiting (4%). 4 an additional manifestation noted among patients with covid-19 infection is the sudden loss (or reduction) of the sense of smell and taste, which is currently recommended by the american academy of otolaryngology-head with increasing numbers of coronavirus disease 2019 (covid 19) cases due to efficient human-to-human transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in the united states, preparation for the unpredictable setting of labor and delivery is paramount. the priorities are 2-fold in the management of obstetric patients with covid-19 infection or persons under investigation (pui): (1) caring for the range of asymptomatic to critically ill pregnant and postpartum women; (2) protecting health care workers and beyond from exposure during the delivery hospitalization (health care providers, personnel, family members). the goal of this review is to provide evidence-based recommendations or, when evidence is limited, expert opinion for anesthesiologists caring for pregnant women during the covid 19 pandemic with a focus on preparedness and best clinical obstetric anesthesia practice. and neck surgery as part of screening for covid-19 infection. 5 in pregnancy, presentation of covid-19 infection appears similar, but many of these nonspecific symptoms may be attributed to symptoms of pregnancy and labor. 2 for example, signs of latent labor may include myalgias and diarrhea; preeclampsia can present with headache; shortness of breath is perceived during pregnancy and labor; and chorioamnionitis may cause tachycardia and fever, thus, leading clinicians to overlook covid-19 infection as a possible diagnosis. in addition, women infected with covid-19 may be asymptomatic until their admission in labor and beyond, 6 which in itself poses a significant risk of exposure for their family members (including the newborn) and all providers involved in their clinical care. screening criteria for covid-19 infection usually include the following: (1) fever, (2) cough or shortness of breath, (3) diarrhea, and (4) any possible exposure to covid-19. however, because women with covid-19 infection may be asymptomatic at the time of admission and because some may present with overlapping pregnancy symptoms, universal screening may miss pregnant women infected with sars-cov-2 in communities with a high prevalence or high projected infection rate (eg, new york, new orleans, detroit, chicago, miami). 7, 8 universal testing with real-time reverse transcriptase-polymerase chain reaction (rt-pcr) tests for sars-cov-2 viral ribonucleic acid (rna) may improve case detection in high prevalence communities. however, current assays may return false-negative results if the viral load is low or if specimen collection is incomplete. the goals of covid-19 testing specific to pregnant patients admitted to labor and delivery units are 2fold: (1) to prevent vertical transmission and ensure separation of the neonate after birth and (2) to protect health care workers by ensuring use of appropriate personal protective equipment (ppe). besides the unclear sensitivity of rt-pcr testing, the time for nucleic acid detection varies between 6 and 8 hours or longer depending on availability. therefore, management of women on labor and delivery units located in a community with a high prevalence of covid-19 infection should err on the side of caution. for purposes of clinical management and ppe use, women may therefore be categorized as follows (1) covid-19 negative, (2) asymptomatic, (3) symptomatic (persons under investigation [pui]), and (4) personally positive for covid-19 testing. this information should be made available to all health care providers and updated at all times as it may change during the course of labor (from asymptomatic to symptomatic or, if tested, once the result becomes available). women who are covid-19 positive (or high-risk pui) should ideally be placed in an isolation room. airborne infection isolation rooms (single-patient negative-pressure rooms with a minimum of 6 air changes per hour), if available, should be used if performance of aerosolizing procedures is anticipated. in general, isolation rooms suitable for droplet and contact precautions are recommended. 9 strategies for exposure mitigation and cohorting, as well as considerations for transportation of patients who are pui or covid-19 should follow the same recommendations as for general patient cases. 10 a multidisciplinary team of anesthesiologists, obstetricians, labor and delivery nurses, neonatologists, critical care experts, infectious disease and infection control experts, employee health services, environmental health services, and telemedicine services should create and implement protocols to support the management of patients with covid-19 infection in the setting of a labor and delivery unit. a sideby-side comparison of recommendations from many professional societies for labor and delivery units is presented in table 1 . for institutions with multiple labor and delivery sites, consideration should be given to designating 1 institution to care for patients with covid-19 infection. this proved useful in managing patients during the sars epidemic and for cases in the recent covid-19 outbreak in wuhan, china. [11] [12] [13] resource allocation within the labor and delivery unit as well as other units (including intensive care unit) should be proactively addressed. it is imperative to establish a back-up team to care for patients without covid-19 infection due to the time-intensive tasks of donning/doffing ppe, transporting the patient, providing anesthetic care, and performing surgery in patients with active covid-19 infection. from a logistical standpoint, a designated operating room within the labor and delivery unit should be prepared. dedicated trays (or carts) containing the most commonly used supplies and drugs for both neuraxial labor analgesia and cesarean delivery should be available to minimize traffic and contamination of anesthesia workstations and other anesthesia equipment. a pregnant woman who is pui or covid-19 positive should be evaluated (limiting unnecessary encounters) including vital signs, physical examination, and review of laboratory tests (complete blood count, comprehensive metabolic panel, and arterial blood gas, if needed) to assess appropriate level of care and monitoring plan for potential deterioration. early multidisciplinary collaboration should be arranged to determine level of care, fetal monitoring, and delivery plan. discussion of the risks and benefits for administering steroids for fetal lung maturity, magnesium for neuroprotection, and indomethacin for tocolysis should be addressed, since there is concern those drugs may worsen covid-19 infection (table 2) . 9 avoiding urgent cesarean delivery is essential to reduce the risk for general anesthesia and provider exposure during uncontrolled transfers to the operating room. therefore, ongoing assessment of both maternal and fetal statuses are key to balance risks of prolonged labor versus cesarean delivery. it is unclear whether uterine decompression improves maternal respiratory status and how the potential benefit balances against the known operative risks in the setting of covid-19. on the other hand, prolonged maternal hypoxemia may ultimately cause fetal acidemia, leading to a more urgent cesarean delivery. 9 routine monitoring should include frequent vital signs (tailored to the current clinical status and adjusted as necessary) with the addition of continuous pulse oximetry and strict input and output measurements to assure fluid restriction. pulse oximetry goal should be an oxygen saturation ≥95%. early warning criteria systems specific for obstetric patients may aid in early detection and prompt escalation of care. 16 women requiring supplemental oxygen, who develop increasing oxygen requirements or worsening hypoxia (pulse oximetry [spo 2 ] < 95%), should have prompt arterial blood gas analysis with frequent clinical reassessment to guide the requirement for escalation of care and mechanical ventilation. highflow nasal oxygen or noninvasive ventilation may be considered as temporizing measures but are generally discouraged due to the potential for greater aerosolization. in addition, increasing oxygen requirements serve as a marker of disease progression, with increasing risk of atelectasis and pulmonary consolidation. it is recommended to perform early endotracheal intubation in a controlled manner minimizing exposure to health care workers and equipment with airborne precautions in an urgent/emergent situation. 17 one of the frequent complications of patients with covid-19 is acute respiratory distress syndrome the routine use of oxygen for fetal indications should be suspended overall, the use of oxygen for fetal indications is controversial the use of high-flow nasal cannula or facemask oxygen may be an aerosolizing procedure nitrous oxide 9 discuss the relative risks and benefits of nitrous oxide for labor analgesia and consider suspending its use (ards). ventilator management strategies for ards involve lung-protective strategies such as low tidal volumes (6 ml/kg using predicted body weight), plateau pressure <30 cm h 2 o, and the combined use of reduced fio 2 with increases in positive end-expiratory pressure (peep) to maintain a pao 2 of 65-90 mm hg. 18 useful ventilator titration techniques using the ardsnet ventilator protocol can be found in http:// www.ardsnet.org/files/ventilator_protocol_2008-07. pdf.17 pregnant patients have a physiological decrease in paco 2 , and it is recommended to maintain a paco 2 of 28-32 mm hg with ventilation to augment off-loading of oxygen to the fetus. however, the priority is maintaining oxygenation with low tidal volumes and peep, and this strategy may not allow for maintaining the physiologic paco 2 in pregnancy. multidisciplinary discussion should determine the fetal monitoring and delivery plan during mechanical ventilation. neuraxial labor analgesia remains a mainstay of obstetric care even with concurrent covid-19 infection. in fact, early epidural placement is desirable to avoid exacerbation of respiratory symptoms with labor pain and to reduce the likelihood of general anesthesia if intrapartum cesarean delivery becomes needed. the benefits of neuraxial analgesia in the setting of covid-19 pneumonia are 2-fold: (1) for the patient, it will help avoid any exacerbation of respiratory status with intubation and mechanical ventilation and (2) for health care providers, it reduces the risks associated with aerosol exposure and transmission of covid-19 infection during intubation and extubation, if general anesthesia is provided. the risk of covid-19 exposure for the anesthesiologist during neuraxial labor analgesia placement is presumably low, since this is not an aerosol-generating procedure. all health care workers in the room should wear contact (impervious gown and gloves) and droplet (surgical mask and eye protection) precautions. the patient should wear a surgical mask at all times to limit droplet spread, and the number of personnel present during placement of neuraxial labor analgesia should be minimized but with assistance readily available. several strategies may minimize contamination of equipment and covid-19 exposure in anesthesiologists, while also minimizing the consumption of ppe (box 1 and figure) . a parturient who is symptomatic pui, or covid-19 positive, should have a complete blood count before neuraxial analgesia placement. early studies from china suggested that thrombocytopenia may be associated with covid-19 infection; in a cohort of 1099 patients, 36.2% had thrombocytopenia (<150,000 × 10 6 /l). 19 a meta-analysis of 1779 patients with covid-19 infection observed that platelet counts are lower in patients with more severe disease. 20 though less common, a platelet count <100,000 × 10 6 /l can occur; 3 studies of 243 patients reported a total of 6.6% patients with that level of thrombocytopenia. [21] [22] [23] we suggest a platelet count on admission without the need to check serial counts before needle placement unless there is a major change in clinical symptoms. it is generally safe to perform neuraxial procedures at platelet counts of 70,000 × 10 6 /l or above, 24 and, given the rare risk of spinal/epidural hematoma and the much higher risk of respiratory compromise with general anesthesia, neuraxial procedures at even lower platelet counts should be considered. while theoretically possible, the risk of epidural or subarachnoid space seeding with viremic blood, causing encephalitis or meningitis, is exceedingly rare. at the time of this writing, there are 77 pregnant women reported in the literature who received uneventful neuraxial procedures for cesarean or vaginal delivery (4 combined spinal-epidural, 27 epidural, 46 spinal procedures; table 3 ). 6 tray-the required equipment (epidural kit) and drugs should be prepared and brought into the room in a bag before the procedure. 5. have the most experienced anesthesiologist perform the procedure to ensure adequate placement and reduce the risk of accidental dural puncture that may require an epidural blood patch. 6. increase the dosing of neuraxial medications for labor analgesia (eg, increasing the bupivacaine concentration from 0.0625% to 0.1%) or changing the setting of the programmed epidural intermittent bolus (eg, increasing the volume from 5 to 8 ml, or decreasing the interval from every 45 to 30 minutes) or adding neuraxial adjuvants (eg, epidural clonidine) to minimize intrapartum breakthrough pain requiring epidural top-up. 7. round on parturients with video or phone calls into the patient's room for hourly assessments of general status and effects of neuraxial analgesia. spinal-epidural, epidural procedures all are acceptable, and no technique confers more risk that the other based on the literature available. none of the patients experienced neurologic sequelae. current recommendations on the use of nitrous oxide (entonox) for labor analgesia suggest "there is insufficient information about the cleaning, filtering, and potential aerosolization of nitrous oxide in the setting of covid-19." individual labor and delivery units should consider suspending use. 9 additionally, the practice of high-flow oxygen for fetal distress does not improve fetal outcomes and should be suspended due to the risk of aerosolization. 9 anesthesia for cesarean delivery in reports from china, most women with a diagnosis of covid-19 infection underwent a cesarean delivery. 25 in the absence of universal testing and rapid availability of results, covid-19 status may not necessarily be known at the time of cesarean delivery. the baseline failure rate for conversion of labor epidural analgesia to cesarean delivery anesthesia is 5%. 30 urgent intrapartum cesarean delivery represents an important risk factor for failed conversion from intrapartum neuraxial labor analgesia to cesarean delivery anesthesia-therefore, ongoing communication with the obstetricians is crucial to allow safe transfer to the operating room, and adequate time for initiation of surgical block to avoid general anesthesia. 30 to minimize the risk of exposure during urgent endotracheal intubation, airborne protection (n95 respirator mask) is recommended for all providers in the operating room unless the patient is known to be covid-19 negative. a publication from wuhan, china, describing outcomes in 17 covid-19-positive women undergoing cesarean delivery, concluded that "excessive hypotension" occurred in 12 of 14 cases with epidural anesthesia in comparison with the 3 women who had received general anesthesia; however, information about the blood pressure trends and description of the use of vasopressors is not reported. 26 a larger case series of 49 patients receiving spinal anesthesia (45 for cesarean delivery and 4 for orthopedic procedures) was well tolerated with stable blood pressure. 29 in our early experience, maternal hypotension during cesarean delivery with epidural or spinal anesthesia has not been noted, most likely because prevention of hypotension with phenylephrine is part of our routine clinical practice. along with antihypotensive medication, antiemetic medication should also be administered. however, we recommend using an alternative to dexamethasone in a pui or patient with known covid-19 infection given the risk of worsening clinical severity. 14 specific considerations for medication use in pui or covid-19-positive patients during labor, delivery, and the postpartum period are described in table 2 . current understanding is that there is little evidence for vertical transmission in women who develop covid-19 pneumonia in late pregnancy. 2, [31] [32] [33] [34] [35] however, cases of possible in utero infection seem to be emerging including a recent report of a neonate born to a covid-19 infected mother. this suggests in utero infection during the 23 days between maternal infection and delivery 23 days later and supported by elevated immunoglobulin m (igm) antibodies, which are not transferred to the fetus via the placenta. [36] [37] [38] [39] serological testing of virus-specific igg and igm antibodies may alternatively be used if rt-pcr testing is not available or if rt-pcr seems to be yielding a false-negative result. 40 postpartum considerations for parturients with covid-19 infection include adequate management of usual postpartum issues (postpartum hemorrhage, pain, hemodynamic status) as well as judicious fluid management, surveillance for respiratory decompensation, and early involvement of subspecialty care as needed. appropriate isolation of mother and child on the postpartum unit is also recommended. 41 in the setting of postpartum hemorrhage due to uterine atony, carboprost tromethamine (hemabate) followed by endotracheal intubation was reported to have precipitated immediate and prolonged bronchospasm in a patient who was subsequently found to be covid-19 positive. 6, 42 oxytocin and methylergonovine as a second-line choice may be preferred, due to the potential for bronchospasm with carboprost tromethamine (hemabate), and aerosolization of viral particles during bronchospasm management. it has been posited that nonsteroidal anti-inflammatory drugs (nsaids) for management of symptoms suggestive of covid-19 infection may worsen the clinical course of covid-19 patients; however, this remains controversial and robust data are lacking. at this point, for women who are asymptomatic or mildly symptomatic with pain not well controlled with acetaminophen, nsaids can continue to be used, as the alternative of opioids likely poses more clinical risks. 9 there are no reported cases of accidental dural puncture resulting in postdural puncture headache (pdph) in a patient with a covid-19 infection, and consequently, no available guidance. similar to usual care, conservative measures should be initially provided. usual contraindications to the performance of an epidural blood patch (eg, fever, thrombocytopenia, or other coagulation issues) should apply in a covid-19 patient. mitigating the risk of a serious neurologic complications with untreated pdph 43 versus that of viral seeding in the epidural space with an epidural blood patch will require a case-by-case approach. postponing the epidural blood patch is recommended in women who are actively ill. individual assessment of the benefits and risks should be assessed and shared decision-making should be engaged with the patient before proceeding. because a nasal sphenopalatine ganglion (spg) block is likely an aerosol-generating procedure due to the injection/insertion directly into the nasal cavity, it should be avoided to minimize the risk of transmission to health care workers. key points emerging in the past weeks from the literature and our experience in labor and delivery units in the united states are that pregnant women may be asymptomatic on admission in labor, and that symptoms of covid-19 infection may initially be missed or obscured if chorioamnionitis is suspected during labor. although most women with covid-19 infection will not present with pneumonia and respiratory decompensation during labor, escalation of care and advanced critical care resources may become necessary in the postpartum period. in fact, most of the considerations surrounding management of the parturient with suspicion of or known covid-19 infection include not only best strategies to ensure safe care for the parturient but also those to prevent health care worker exposure to sars-cov-2 and contracting covid-19. anesthesiologists are deemed at significant risk of viral exposure during endotracheal intubations of covid-19-infected patients, and all strategies should be applied to avoid general anesthesia in women who are either untested or known to be covid-19 positive. early neuraxial labor analgesia is strongly recommended to ensure availability of neuraxial anesthesia in the event of an intrapartum cesarean delivery, and spinal anesthesia should be provided if needed. if deemed necessary and unavoidable, provision of general anesthesia should follow general recommendations for intubation and extubation in the setting of covid-19-infected patients. the changes in workflow that result from the need to ensure adequate ppe (contact/droplet protection for nonaerosolizing procedures such as [eg, epidural placement] or airborne protection for cesarean deliveries due to the possible conversion to general anesthesia) are considerable and require thorough planning and preparedness. close communication around covid-19 status of all patients admitted to the labor and delivery unit is essential, and anticipation of emergencies is of the essence. overall, providing the best clinical care for pregnant and postpartum women with covid-19 infection also must take into account strategies to prevent health care worker exposure to sars-cov-2 and contracting covid-19. e what are the risks of covid-19 infection in pregnant women? clinical characteristics and intrauterine vertical transmission 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service sermer m; maternal fetal medicine committee; infectious disease committee. management guidelines for obstetric patients and neonates born to mothers with suspected or probable severe acute respiratory syndrome (sars) society for obstetric and anesthesia and perinatology. interim considerations for obstetric anesthesia care related to covid19 are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? the maternal early warning criteria: a proposal from the national partnership for maternal safety for the zhongnan hospital of wuhan university novel coronavirus management and research team, evidence-based medicine chapter of china international exchange and promotive association for medical and health care (cpam). a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) ards in pregnancy clinical characteristics of coronavirus disease 2019 in china thrombocytopenia is associated with severe coronavirus disease 2019 (covid-19) infections: a meta-analysis clinical features of patients infected with 2019 novel coronavirus in wuhan clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study multicenter perioperative outcomes group investigators. risk of epidural hematoma after neuraxial techniques in thrombocytopenic parturients: a report from the multicenter perioperative outcomes group neuraxial procedures in covid-19 positive parturients: a review of current reports safety and efficacy of different anesthetic regimens for parturients with covid-19 undergoing cesarean delivery: a case series of 17 patients anesthetic management for emergent cesarean delivery in a parturient with recent diagnosis of coronavirus disease 2019 (covid-19): a case report emergency caesarean delivery in a patient with confirmed coronavirus disease 2019 under spinal anaesthesia spinal anaesthesia for patients with coronavirus disease 2019 and possible transmission rates in anaesthetists: retrospective, single-centre, observational cohort study risk factors for failed conversion of labor epidural analgesia to cesarean delivery anesthesia: a systematic review and meta-analysis of observational trials perinatal transmission of covid-19 associated sars-cov-2: should we worry? clin infect dis lack of vertical transmission of severe acute respiratory syndrome coronavirus 2, china. emerg infect dis an analysis of 38 pregnant women with covid-19, their newborn infants, and maternal-fetal transmission of sars-cov-2: maternal coronavirus infections and pregnancy outcomes clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia lack of maternal-fetal sars-cov-2 transmission possible vertical transmission of sars-cov-2 from an infected mother to her newborn antibodies in infants born to bothers with covid-19 pneumonia can sars-cov-2 infection be acquired in utero?: more definitive evidence is needed a case report of neonatal 2019 coronavirus disease in china eleven faces of coronavirus disease 2019 coronavirus disease 2019 (covid-19) and pregnancy: what obstetricians need to know lessons learned from first covid-19 cases in the united states major neurologic complications associated with postdural puncture headache in obstetrics: a retrospective cohort study nih nhlbi ards clinical network mechanical ventilation protocol summary key: cord-024093-5dplc9xr authors: sizun, j; soupre, d; legrand, mc; giroux, jd; rubio, s; cauvin, jm; chastel, c; mix, d; de parscau, l title: neonatal nosocomial respiratory infection with coronavirus: a prospective study in a neonatal intensive care unit date: 2008-01-21 journal: acta paediatr doi: 10.1111/j.1651-2227.1995.tb13710.x sha: doc_id: 24093 cord_uid: 5dplc9xr the aim of this prospective study was to evaluate the incidence of viral respiratory infection in hospitalized premature newborn infants and to assess the role of coronaviruses. all hospitalized premature infants with a gestational age less than or equal to 32 weeks were included. tracheal or nasopharyngal specimens were studied by immunofluorescence for coronaviruses, respiratory syncytial virus, adenoviruses, influenza and parainfluenza viruses. forty premature infants were included; 13 samples were positive in 10 newborns (coronaviruses n = 10; influenza 1 n= 2; adenovirus n= 1). none was positive at admission. all premature infants infected with coronaviruses had symptoms of bradycardia, apnea, hypoxemia, fever or abdominal distension. chest x‐ray revealed diffuse infiltrates in two cases. however, no significant difference was observed between infected and non‐infected premature infants for gestational age, birth weight, duration of ventilation, age at discharge, incidence of apnea or bradycardia. nosocomial respiratory tract infection with coronaviruses appears to be frequent. the clinical consequences should be evaluated in a larger population. viral pneumonia which develops in neonatal intensive care units is potentially life-threatening. indeed, young age (less than 1 month) and prematurity are two major risk factors (1). nosocomial respiratory infections can occur sporadically or as an epidemic. the precise incidence has not been established. the variability or even absence of clinical symptomatology, especially in very immature newborn infants ( 2 ) , makes all retrospective evaluations difficult. numerous viral agents have been implicated. the most frequently reported viruses are respiratory syncytial virus (rsv) (1, 3) , adenoviruses (4), influenza viruses a and b, and parainfluenza viruses i, i1 and 111 (5). the coronavirus (cv) has also been implicated. we have previously reported four cases of upper respiratory tract infection in premature newborns presenting with attacks of apnea of sudden onset (6). the pathogenic role of cvs during the neonatal period is, however, poorly established. the aim of this prospective study undertaken from november 1, 1991 to march 1, 1993, was to evaluate, in the pediatric intensive care unit, the incidence of upper respiratory tract viral infection in hospitalized premature newborn infants, to assess the role of cv and to analyze the associated symptomatology. the study was approved by the hospital center ethics committee. all newborn infants less than 3 days of age, with a gestational age less than or equal to 32 weeks and hospitalized in a resuscitation unit throughout the duration of the study, were entered into the trial. infants who died before day 7 of life were excluded a posteriori. samples were obtained on admission and then weekly. the methodology was as follows: administration of 0.5 ml of physiological saline via the intranasal route in non-intubated newborns or via the tracheal route in intubated newborns, followed by aspiration using a disposable vygon-type catheter. samples were transported at room temperature to the microbiology department and stored at +4"c until analysis. samples were treated with a mucolytic solution (mucomyst, eurobio, france). cells were then washed twice in phosphate buffered saline (pbs). after the last months = influenza virus. according to the manufacturer, monoclonal antibody against cv is a 110/120 kda peplomer group specific and recognizes bovine, porcine and human cv strains. clinical data and data obtained from the infant's family were recorded at the time of entry into the study and weekly by the same observer. gestational age was established from obstetric data. apnea was defined as absence of respiratory movements over a 20-s period and bradycardia as heart rate less than 90 beats/min, detected by a hewlett-packard cardiorespiratory monitor. days on which more than six episodes of bradycardia of less than 60 beats/min, or more than eight episodes greater than 60 beats/min, or more than five apneic events occurred, were noted. forty infants were included in the study. initial samples were negative in all infants. thirteen subsequent samples were found to be positive in 10 newborns: 10 for cv, 2 for myxovirus influenzae type 1 and 1 for adenovirus. two children were cv positive on two successive samples. another was positive for cv and then positive for myxovirus 4 weeks later. no sample was doubly positive. the seasonal distribution is shown in fig. 1 . previous perinatal histories are summarized in table 1 . the group infected with cv was comparable to the non-infected group in terms of gestational age, birth weight, sex ratio and past history of hyaline membrane disease. all infants infected with cv were symptomatic at the time of infection ( table 2) . symptomatology was predominantly bradycardia and apnea. digestive signs were present in five newborns but virology was not performed on the stools. mean age at the time of infection was 17 f 7 days. chest x-ray revealed bilateral interstitial infiltrate in two infants, 3 and 6 days after the date of the first positive cv sample. no statistically significant differences with respect to frequency of bradycardia or apnea were observed between the cv-infected and non-infected infants ( table 3) . laboratory signs of an inflammatory syndrome were not observed in any cv-infected newborns (normal c-reactive protein concentration). blood cultures were negative. the development of a neonatal viral infection did not significantly alter the therapeutic management of these infants, since the duration of intubation, administration of atropine, need to interrupt feeding and conceptional mean age at the time of discharge were comparable in both groups. two newborn infants in the non-infected group died , the first on day 13 with multiple organ failure and the second on day 30 due to cardiorespiratory arrest during surgical closure of a persistent ductus arteriosus; no infant in the infected group died. the incidence of viral infection in this study was high, affecting 25% of the newborn infants under investigation. no comparable evaluation exists in the literature. the negative initial sampling performed before the third day of life tends to support the diagnosis of nosocomial rather than materno-fetal contamination. rsv, the primary cause of bronchiolitis in infancy, has also been implicated as the cause of epidemics of chest infections in neonatal intensive care units (2, 3 ) and can worsen pre-existing respiratory disease. in this study, no sample was positive for rsv. on the other hand, we have seen a high rate of occurrence of infection with cv. cvs are rna viruses with a spherical shape covered with projections similar to a "crown". two main strains of human cv have been identified: 229 e and oc (7) . these viruses are commonly implicated in benign upper respiratory tract infections in adults (7), reproduced experimentally in healthy volunteers (8) . they are also implicated in acute decompensation in chronic respiratory insufficiency (9) . epidemiological data in young infants are rare and vary depending on the study. in infants, cvs can be the cause of respiratory infections such as pneumonia or bronchiolitis, without clinical or radiological specificity compared with other respiratory viruses. in a serological study carried out in infants aged less than 18 months and hospitalized for respiratory tract infection, this cause was identified in 8.2% of cases (10). on the similarly, in a prospective study by ray et al., based on serological analysis and cell culture from acute lower respiratory tract infections in children less than 3 years of age, human cv viruses, as initial infecting agents, were not found to be responsible (12) . the difficulty in culturing cvs and the absence of serological specificity probably explain these findings (1 3) . the pathogenic role of cvs in the newborn is poorly understood. an association with enterocolitis in fullterm newborn infants has been reported (14) . no data are available in the literature regarding the possibility of nosocomial respiratory tract infections in the newborn. we reported four apneic episodes associated with bradycardia of sudden onset in the absence of an explicable etiology and with positive nasotracheal samples for cv on i f (6). cv has also been isolated using i f in infants with chronic respiratory insufficiency requiring prolonged mechanical ventilation (1 5). all the infected neonates in our prospective study had symptoms at the time of infection, which supports the recent onset of an acute process. however, we were unable to prove any significant difference between infected or non-infected infants with respect to symptomatology and therapeutic measures necessary. several hypotheses can be proposed: these symptoms (apnea, bradycardia and digestive disorders) are not very specific in the premature infant and can be found in many pathological conditions ; moreover, duration of infection is short compared with duration of hospitalization. the diagnosis of a cv infection is based mainly on i f (1 5). the special culture conditions required make this procedure expensive and difficut to achieve. serological methods are not very reliable (14) . the polymerase chain reaction has been used in the diagnosis of murine cv by homberger et al. (16) . this technique appears to be faster and as sensitive as the usual techniques of inoculation of suckling mice or seroconversion after mouse inoculation, but it is not yet applicable to human cv, even though the genetic m sequence of oc43 is the counterpart of that of the murin virus. this study suggests a significant occurrence of upper respiratory tract viral infection in hospitalized premature newborns, mainly with cvs. the pathogenic role of cvs, as suggested by simultaneous viral infection and development of symptoms, requires a larger series of patients to be confirmed. viral pneumonia in the first month of life infections a virus respiratoire syncitial du nouveau-nt neonatal respiratory syncitial virus infection epidemiology of neonatal enterovirus infection nasal colonization with coronavirus and apnea of the premature newborn coronaviruses of man effects of a "new" human respiratory virus in volunteers coronavirus infections of man associated with diseases other than the common cold coronavirus infection in acute lower respiratory tract disease of infants diagnosis of human coronavirus infection by immunofluorescence : method and application to respiratory disease in hospitalized children acute lower respiratory illnesses during the first three years of life: potential roles for various etiologic agents viral infections of humans-epidemiology and control association of coronavirus infection with neonatal necrotizing enterocolitis rble pathogene des coronavirus en rtanimation ptdiatrique: analyse rttrospective de 19 prtlkvements positifs en immunofluorescence indirecte detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction bone mineralization after treatment of growth hormone deficiency in survivors of childhood malignancy 10) in "the discovery of phenylketonuria" by i. farlling is given incorrectly in the above supplement a recessive syndrome in man we apologize for this inconvenience key: cord-031876-v44o5shw authors: mounier, roman; kapandji, natacha; gricourt, guillaume; lobo, david; rodriguez, christophe; pons, stéphanie; djediat, chakib; woerther, paul-louis; mellano, vincent; aït-mamar, bouziane; demontant, vanessa; nebbad, biba; senova, yann; arnaud, melissa; cook, fabrice; dhonneur, gilles; lebeaux, david title: assessment of bacterial colonization of intracranial pressure transducers: a prospective study date: 2020-09-15 journal: neurocrit care doi: 10.1007/s12028-020-01096-x sha: doc_id: 31876 cord_uid: v44o5shw objectives: cerebral infections related to the presence of an intraparenchymal intracranial pressure transducer (icpt) are rare. we assessed the incidence of icpt-related infections and colonization using culture, molecular biology, and electron microscopy. methods: all consecutive patients in a neurosurgical intensive care unit who had an icpt inserted between march 2017 and february 2018 were prospectively included. presence of colonization on the icpts was assessed after removal using culture, scanning electron microscopy (sem), and next-generation sequencing (ngs). results: fifty-three icpts (53 patients), indwelling for a median of 4 (range 3–7) days, were studied. median patient follow-up was 3 months. sem, microbial culture, and ngs were performed for 91%, 79%, and 72% of icpts, respectively; 28 icpts (53%) were assessed using all three techniques. no patient developed icpt-related infection. microbial cultures were positive for two of the icpts (5%); colonization was identified on all icpts using ngs and sem. mature biofilm was observed on 35/48 (73%) of icpts. a median of 10 (8–12) operational taxonomic units were identified for each icpt, most being of environmental origin. there was no association between biofilm maturity and antimicrobial treatment or duration of icpt insertion. antimicrobial treatment was associated with decreased alpha and beta-diversity (p = 0.01). conclusions: we observed no icpt-related cerebral infections although colonization was identified on all icpts using ngs and sem. mature biofilm was the main bacterial lifestyle on the icpts. electronic supplementary material: the online version of this article (10.1007/s12028-020-01096-x) contains supplementary material, which is available to authorized users. the use of continuous intracranial pressure (icp) monitoring has been associated with improved outcomes in traumatic brain injury [1] . intraventricular pressure measurement using a fluid-filled catheter is considered the gold standard for icp monitoring [2] , but despite the lack of specific guidelines, intraparenchymal fiberoptic devices are now widely used because of the low frequency of complications [3] [4] [5] . icp transducers (icpts) cross the various brain-protecting barriers (the scalp and the skull, the meninges, and the blood brain barrier) and may therefore theoretically expose the brain to bacterial contamination and subsequent infection. for example, use of external ventricular drains (evd) is associated with infections in 10% of cases [6] and it has been previously demonstrated that bacteria move along evds, from the skin to the brain, mainly as biofilms [6, 7] . surprisingly, however, intraparenchymal fiber-optic icpts are rarely responsible for cerebral infections [5] . in the largest published series, none of 1000 consecutive patients experienced clinically significant intraparenchymal icpt-related infection [8] . some authors have reported infectious complications limited to superficial skin (0-2.4%) or bone, but no parenchymal infections or meningitis [3, 4, 8, 9] . among 101 patients with an icpt in situ, martínez-mañas et al. [4] reported meningitis in 2.9%, but the infections were considered to be related to an evd and not the icpt. recent studies reported that 8.5-17% of icpt tips were culture positive, mainly with gram-positive bacteria [4, [8] [9] [10] , but there were no associated cases of parenchymal infection. however, none of these large studies performed electron microscopy or next-generation sequencing (ngs) to address the infectious risk. given the numerous similarities between icpts and evds, in terms of positioning, the differences in terms of frequency of microbial colonization and related infections remain unexplained. the objective of this study was therefore to assess the bacterial burden on icpts [with biofilm formation studied using scanning electron microscopy (sem) and ngs] and to determine how it may correlate with patient characteristics, icpt colonization, and related infections. this prospective study was conducted between march 2017 and february 2018 in the neurosurgical intensive care unit (icu) of a tertiary teaching hospital. all consecutive patients with an icpt (sophysa, orsay, france) inserted were included, unless the icpt had been inserted for management of a central nervous system infection or prior to icu admission. icpts not removed in aseptic conditions were not included. the institutional ethical committee approved the study (claude galien). informed consent was collected from each patient or his/ her next of kin. culture-based colonization was defined by a positive bacterial culture (see below) of the icpt tip without clinical infection. any degree of positive culture was considered sufficient to define colonization because no cutoff threshold has been proposed in the literature [4, [8] [9] [10] . infectious meningitis was defined by the presence of a cerebrospinal fluid (csf) sample with altered laboratory variables (pleocytosis, elevated protein level, and decreased glucose level) and a positive microbiological culture [11, 12] . icpt-related infection was defined by the presence of meningitis or brain abscess with a csf or abscess culture positive for the same microbial strain (same species and same antibiotic susceptibility) as that identified in the culture of the icpt tip. patients who are admitted to our center with traumatic brain injury (tbi) all receive initial resuscitation followed by a whole-body computed tomography (ct) scan. patients with severe tbi (glasgow coma scale score ≤ 8) undergo icp monitoring per current guidelines [1] or, in case of emergency, extracranial surgery. during the study period, only icpts (sophysa, orsay, france) were used for icp monitoring. the icpt was inserted (on the healthy side, preferably on the right) under general anesthesia in the operating room (or) or at the bedside in the icu according to national guidelines. the patient's hair was clipped, and the skin scrubbed gently using a 10% povidone-iodine solution diluted 50:50 with water. the area was then dried with a sterile towel and washed a second time with the 10% povidone-iodine solution. after inserting the icpt and the bolt, a new round of alcohol-based hand rubbing and sterile glove exchange, a sterile gauze was wrapped at the base of the bolt (see supplemental fig. s1 ), and another one was used to wind the icpt cable. a tightly occlusive sterile dressing was used to maintain a closed system. icpt insertion sites were cleaned (povidoneiodine) at the bedside, and the dressing changed every 48 h, unless soiled. no antibiotic prophylaxis was administered for icpt placement, but in some patients prophylaxis was indicated for surgery. when icp monitoring was no longer clinically indicated, the icpt was removed under aseptic conditions, and three 0.5-cm portions were cut from the icpt tip (fig. 1 ). the first section was sent to the clinical microbiology laboratory for culture; the second was immediately fixed in a mixture of 4% paraformaldehyde-2.5% glutaraldehyde-0.1% picric acid in 0.1 m phosphate buffer (ph 7.0) for 1 h at room temperature and then kept at 4 °c until sem examination was performed; the third was stored at − 80 °c for later analysis using ngs. ct scans were performed routinely at admission and whenever clinically indicated. ct scans were also performed after the removal of the icpt, at icu discharge, at discharge from the neurosurgery unit, and at 3-and 6-month follow-up appointments to rule out subsequent infection. after receipt at the clinical microbiology laboratory, the first icpt tip section was vortexed for 10 s in 1 ml of sterile saline (biomérieux, marcy l'etoile, france). one hundred microliters of the obtained dilution was plated on blood and on polivitex agar plates using the automatic wasp system (wasp, beckman coulter, villepinte, france) and incubated at 35 °c for 48 h in an aerobic atmosphere and in 5% co 2 for 5 days in a chocolate broth, respectively. when cultures were positive, identification was performed using the andromas maldi-tof system (andromas, beckman coulter, villepinte, france). antibiotic susceptibility testing was performed using the walkaway microdilution automatic system (beckman coulter, villepinte, france) for species belonging to the staphylococcus genus. the agar diffusion method was used for all other species, and results were interpreted according to ca-sfm-eucast 2017 recommendations (www.sfm-micro biolo gie.org). samples were washed for 30 min in 1 ml of sörensen's phosphate buffer, then rinsed with distilled water, dehydrated with increasing ethanol concentrations, and dried with liquid co 2 (without critical-point drying). after one night at room temperature, samples were coated with gold in a jeol fjc-1200 sputter coater and examined under a hitachi su3500 sem. an examination was also performed using a new icpt tip as a control (fig. 2a) . sem pictures were categorized by two physicians experienced in sem into: • isolated bacteria (fig. 2b) : bacteria attached to the icpt without matrix; • adhesion phase (fig. 2c, d) : bacteria attached to the icpt in one or more layers, with little extracellular matrix; • mature biofilm ( fig. 2e) : bacteria encapsulated in a thick extracellular matrix. • when several phenotypes were observed on a single sem picture, for example isolated bacteria and mature biofilm (fig. 2f ) , the category representing the most advanced bacterial lifestyle was selected. uncertainty regarding classification of the images was resolved by discussion with a third expert. microbial genomic material from the third section of each catheter, from a new icpt (control), and from a negative control of milliq water was extracted according to a pre-extraction protocol based on bead-beating disruption of the biofilm and bacteria in a lysis buffer. the pre-extract material was then extracted using the dsp dna midi kit with qiasymphony instrument (qiagen, hilden, germany) according to the manufacturer's instructions. finally, the extracts were library-prepared using the "16s metagenomic sequencing library preparation" protocol (https ://suppo rt.illum ina.com/docum ents/docum entat ion/chemi stry_docum entat ion/16s/16smetag enomi c-libra ry-prep-guide -15044 223-b.pdf ) (illumina, san diego, california, usa). the universal primers used to amplify the bacterial 16s rrna gene (loop v3-v4) were 341f and 785r (see supplemental data and table s1 ). microbiome composition was analyzed with respect to alpha-and beta-diversity and differential abundance of operational taxonomic units (otus) across samples. alpha-diversity was assessed using quantitative and qualitative approaches by chao1 (richness) and simpson (evenness) indexes, respectively, as previously described [13] . we also used the pieloue index to measure evenness. relative frequency was defined at the phylum or genus/otu levels as the frequency of each clone relative to the number of total clones. this relative frequency was studied for each icpt but also for the total clone library from all removed icpts. beta-diversity was studied using jaccard dissimilarity and weighted unifrac distance [14] . samples of used reagents, materials, blanks (water used as the template in the procedure), and a control processing. after removal, the intracerebral portion of the icpt was immediately cut into three sections of 0.5 cm (labeled 1-3). section 1 was used for bacterial culture; section 2 was assessed by scanning electron microscopy; and section 3 was assessed using next-generation sequencing icpt were taken as procedure controls and also submitted to 16s rrna sequencing to determine potential sources of bacterial contamination. qualitative data are expressed as incidence and percentage, and quantitative data as median and interquartile range. categorical data were compared using a two-way fisher exact test or a chi-square test with a pearson correction-based on the number of groups. quantitative data were compared using a mann-whitney-wilcoxon u test or kruskall-wallis test as a function of the number of groups. a permanova test was used to compare beta-diversity between groups. a p value ≤ 0.05 was considered significant. statistical analyses for diversity were performed using jmp ® 14.1.0 and qiime ® (v2018.4.0) software. the differential otu abundance was assessed using phyloseq (v1.26.1) and deseq2 (v1.22.2) packages in r software (v3.5.0) to fit generalized linear models of abundance based on the negative binomial distribution. candidate otus with an adjusted p value < 0.01 were validated as being differentially abundant between groups if the otu was not driven by a single sample or an alone sample with a raw read count > 300 000. during the study period, 69 icpts were inserted in 67 adult patients (2 patients had 2 consecutive icpts). of these 69 icpts, 14 were excluded because they were not removed under aseptic conditions, and 2 were not sent to the laboratory when removed in the or. fifty-three icpts in 53 patients were therefore included in the study ( table 1 ). the median distance from the skin insertion site to the parenchymal end of the icpt was 4 (3.4-4.5) cm. forty of the patients were men (75%) and 36 (68%) were admitted for polytrauma. the median glasgow coma scale score was 7 (4) (5) (6) (7) (8) . the icpts were mainly inserted in the icu, and patients were monitored for 4 (3-7) days. the median icu length of stay was 14 (7-40) days and in-icu mortality was 43% (23/53). median follow-up was 3 months. we observed no bleeding, even minor, around the probes. during icp monitoring, 12 patients (23%) received antimicrobial treatment for extraneurological infection (table 1) ; amoxicillin-clavulanic acid and cefotaxime were the most commonly used empirical antimicrobial therapies, in 33% (4/12) and 58% (7/12) of cases, respectively. cefepime was used in one case. after microbial identification, amoxicillin-clavulanic acid and cefotaxime were used in 58% (7/12) and 42% (5/12) of cases, respectively. the median time to start of antimicrobial treatment was 4.5 (3.3-6.5) days. blood cultures were performed in 78% of our patients (52/67). no patient developed icpt-related infection or cutaneous infection at the site of bolt insertion. among the 16 patients who had an emergency neurosurgical intervention, two developed a surgical site infection: one a bone flap infection and the other a superficial surgical site infection. icpt-related infection was defined as a meningitis or a brain abscess. antimicrobial treatment means treatment during icp monitoring. breaking of the bbb means skull depression fracture, skull base fracture, or brain surgery data are shown as the median (25th-75th percentile) or number (%), unless otherwise indicated bbb blood brain barrier, evd external ventricular drain, gcs glasgow coma scale, icp intracranial pressure, icpt intracranial pressure transducer, icu intensive care unit, ngs next-generation sequencing, saps simplified acute physiology score, sem scanning electron microscopy, tbi traumatic brain injury microbial culture, sem, and ngs were performed for 42 (79%), 48 (91%), and 38 (72%) of icpts, respectively, and 28 icpts (53%) were assessed using all three techniques. among the 42 icpts cultured, only 2 (5%) showed colonization, both with s. epidermidis and one with bacillus spp. these 2 icpts were not studied using ngs, but mature biofilm was observed on both of them with sem (see supplemental fig. s2 ). these 2 icpts were from patients who did not receive antimicrobial treatment. all sem examinations showed the presence of bacteria (48/48, 100%). isolated bacteria were observed on 33 of the 48 (70%) icpts, adhesion phase bacteria on 37/48 (78%), and mature biofilm on 35/48 (73%). a single (2%) icpt showed only isolated bacteria. thirteen (25%) icpts showed isolated and adhesion phase bacteria without mature biofilm (see supplemental fig. s3 ). there was no significant association between the presence of mature biofilm and receipt of intravenous antimicrobial treatment or duration of icp monitoring ( table 2) . the median number of raw reads per sample was 37,933 (25, 695 (fig. 3) . across the 38 icpts, 91 otus were identified, but only 11 of them had a relative frequency greater than 1% (fig. 4) . many of these species can be considered to be of environmental and skin origin, including delftia spp. and herbaspirillum spp. stenotrophomonas spp. and serratia spp. were identified on 38 (100%) and 22 (58%) of icpts, respectively. commensal bacteria such as corynebacterium spp. and staphylococcus spp. were each identified on 3 (8%) icpts. data presented are limited to the 48 icpts assessed by sem breaking of the bbb means skull depression fracture, skull base fracture, or brain surgery data are shown as the median (25th-75th percentile) or number (%), unless otherwise indicated bbb blood brain barrier, evd external ventricular drain, icp intracranial pressure, icu intensive care unit, tbi traumatic brain injury the number of otus representing more than 90% of the sequences found by sample was 6 (5. . no clinical or therapeutic factor or the presence of mature biofilm on sem was associated with a decrease in the number of otus representing more than 90% of the sequences or with an alteration in the richness (chao1) of α-diversity (table 3) . icpt placement in the or, antimicrobial treatment during monitoring, and surgical antimicrobial prophylaxis were associated with reduced evenness [p = 0.01, p = 0.01, and p = 0.006, respectively (pieloue)]. icpt placement in the or and antimicrobial prophylaxis decreased the beta-diversity (weighted unifrac: p = 0.02 and p = 0.03, respectively) ( table 4) . antimicrobial treatment was associated with a decrease in serratia and curvibacter genera. some genera were differentially abundant depending on where the icpt had been inserted: microbacterium and curvibacter were more abundant in icpts inserted in the icu, whereas staphylococcus was more abundant in icpts fig. 4 relative frequencies of each genus/otu in the entire cohort of removed icpts. genus/otus are ranked according to the abundance of their clone library relative to the number of total clones. subsets were created according to callahan et al. [33] , and the category "others" represents genus clusters with a relative abundance ≤ 1% inserted in the operating room. the information provided by the sequencing of 16s rdna was not discriminative enough to differentiate all staphylococcal species. in this prospective study including 53 icpts, sem and ngs always identified the presence of bacteria, whereas bacterial culture was only positive for 5% of transducer antimicrobial prophylaxis means that icpt was placed under surgical antimicrobial prophylaxis. breaking of the bbb means skull depression fracture, skull base fracture, or brain surgery bbb blood brain barrier, evd external ventricular drain, icpt intraparenchymal intracranial pressure transducer, icu intensive care unit, otu operational taxonomic units, tbi traumatic brain injury data are shown as the median (25th-75th percentile) or number (%), unless otherwise indicated tips, suggesting that icpt colonization may be largely underestimated using standard techniques. in large clinical studies, intraparenchymal icp monitors have occasionally been associated with clinically relevant infections [8] [9] [10] 15] . however, analysis of published data may be hindered by the concomitant insertion of other intracranial devices, such as evds, which are more frequently associated with infectious complications [4, [16] [17] [18] [19] [20] [21] . when restricted to studies that included only intraparenchymal transducers, reported infections were rare and mainly superficial [3, 9] . indeed, the largest series, including more than 1000 patients, reported no infections [4, 8] . several reasons may explain this low risk of infection. first, because known risk factors for ventriculostomyrelated infection are csf leak, elevated icp, and duration of icp monitoring [4, 6, 17, 22, 23] , one can hypothesize that the shorter duration of icpt placement (< 10 days), as compared to evd (≥ 10 days), may play a key role in this observation [3, 6, 9, 10, 12, 24, 25] . second, the bolt, present for icpts but not evds, may also act as a physical barrier, limiting csf leaks or delaying bacterial progression from the skin to the icpt tip. third, icpts have a thinner diameter (1 mm) than evds (3 mm). when focusing on the frequency of tip colonization, one can also observe that icpts are less frequently colonized (8.5 to 17%) than evds (10-36%) [4, 6, 8-10, 26, 27] . using sem, one study reported the presence of biofilm on 75% of the analyzed evds, whereas colonization and ventriculostomy-related infection diagnosed by culture occurred in only 38% and 19% of cases, respectively [7] . using both sem and ngs, we identified bacteria on all studied icpts, although only 5% of icpts were culture-positive. the main bacterial lifestyle was mature biofilm, seen on 73% of icpts; ngs confirmed this high frequency of bacterial colonization. however, we did not observe any cerebral infections, despite a follow-up period of several months. the absence of icpt-related infection, despite almost universal icpt colonization (as shown with sem or ngs), may suggest a role of bacterial virulence traits [28] . indeed, most of the culture-positive icpts reported in the literature were colonized by s. epidermidis [4, 9, 10] . however, more virulent bacteria, such as escherichia coli, enterobacter spp., klebsiella spp., serratia spp. and pseudomonas spp., have also been recovered from icpt cultures, without clinically relevant infection [4, 8, 9] . in our study, ngs identified mostly environmental or skin bacteria [29] , suggesting possible icpt colonization during insertion or handling. finally, a seductive hypothesis to explain the low risk of infection despite almost universal bacterial colonization is that the mature biofilm we observed may act as a protective microbiota on the surface of the icpt [6, 30, 31] . studying another type of indwelling device (totally implanted venous access port, tivap) using ngs, stressmann et al. [30] identified two distinct patterns of biofilm composition, depending on the status of the patient at tivap removal (clinical suspicion of infection ["symptomatic"] or not ["asymptomatic"]). among samples from asymptomatic patients, they identified herbaspirillum, paracoccus and stenotrophomonas maltophilia more abundantly than in symptomatic patients. interestingly, we also identified stenotrophomonas and herbaspirillum on all icpts. furthermore, stressmann et al. reported that the mean species richness (represented by the number of otus/sample) in the overall sample set was significantly higher (p = 0.0002) in asymptomatic (5.4 otus/ sample) than in symptomatic (2.5 otus/sample) samples. strikingly, we noted that the main factor associated with a decrease in bacterial diversity was antimicrobial treatment. our study has some limitations. first, positive results achieved using ngs may have been the result of contamination during removal or contamination of commonly used dna extraction kits or pcr reagents [32] . to reduce this risk, icpts were excluded if there was any suspicion of contamination during removal. the sem findings also suggest that the bacteria were actually on the surface of the catheter (and not the result of contamination) as the process from bacterial adhesion to biofilm formation takes time. as we performed fast-acting fixation immediately after icpt removal, the observation of isolated bacteria using sem could correspond to contamination during icpt removal. however, "isolated bacteria" alone were observed for just a single (2%) icpt. second, the small number of icpts included reduced our statistical power to identify factors associated with the formation of mature biofilm. third, meningitis may have been under-recognized. head trauma patients with elevated icp are usually sedated with temperature control, which may interfere with clinical examination. in addition, because of the increased intracranial pressure, lumbar puncture may not be feasible. however, csf was sampled daily for microbiological analysis in patients with an evd in situ and when clinically indicated by lumbar puncture, when possible. fourth, the study was performed in a single center, thereby limiting its generalizability, especially as the method for icp probe insertion may differ from center to center. fifth, we limited our analysis to the bacterial kingdom, without taking into account the possible role of fungi or viruses. sixth, in the absence of infection and because of universal colonization, no multivariate analysis could be performed. finally, a quantitative analysis (such as quantitative pcr) may have improved our understanding of colonization dynamics and its microbial composition. in conclusion, we observed no icpt-related cerebral infections, although colonization was always observed using ngs and sem, mostly with bacteria of environmental origin. mature biofilm was the main lifestyle for bacteria on icpts. data from ngs are available 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a critical review ventriculostomyrelated infections: a critical review of the literature clinical, biological, and microbiological pattern associated with ventriculostomyrelated infection: a retrospective longitudinal study biological diversity quantitative and qualitative beta diversity measures lead to different insights into factors that structure microbial communities monitoring of intracranial pressure with intraparenchymal fiberoptic transducer. technical aspects and clinical reliability risk factors and complications of intracranial pressure monitoring with a fiberoptic device infection related to intracranial pressure monitors in adults: analysis of risk factors and antibiotic prophylaxis duration of intracranial pressure monitoring does not predict daily risk of infectious complications impact of intracranial pressure monitor prophylaxis on central nervous system infections and bacterial multi-drug resistance complications of intracranial pressure monitoring in trauma patients influence of broad-spectrum antibiotic prophylaxis on intracranial pressure monitor infections and subsequent infectious complications in head-injured patients ventriculostomy-related infections. a prospective epidemiologic study bacterial ventriculitis and duration of ventriculostomy catheter insertion a cost effectiveness based safety and efficacy study of resterilized intra-parenchymal catheter based intracranial pressure monitoring in developing world natural history of ventriculostomy-related infection under appropriate treatment and risk factors of poor outcome: a retrospective study relationship between bacterial colonization of external cerebrospinal fluid drains and secondary meningitis: a retrospective analysis of an 8-year period intracranial pressure monitors. epidemiologic study of risk factors and infections treatment of infections associated with surgical implants a diversity profile of the human skin microbiota comparative analysis of bacterial community composition and structure in clinically symptomatic and asymptomatic central venous catheters the skin microbiome inherent bacterial dna contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples bioconductor workflow for microbiome data analysis: from raw reads to community analyses we thank karen pickett for editorial assistance. source of support support was provided solely from institutional and/or departmental sources. all authors declare that they have no conflict of interest. the institutional ethical committee approved the study (claude galien). informed consent was collected from each patient or his/her next of kin. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-027860-s97hdhh6 authors: zeimet, anthony; mcbride, david r.; basilan, richard; roland, william e.; mccrary, david; hoonmo, koo title: infectious diseases date: 2020-06-22 journal: textbook of family medicine doi: 10.1016/b978-1-4377-1160-8.10016-8 sha: doc_id: 27860 cord_uid: s97hdhh6 nan infections of the upper respiratory tract accounted for more than 36 million ambulatory medical visits in 2005, according to the national ambulatory medical care survey (cherry et al., 2007) . although a large percentage of these infections are viral in origin, antibiotics are still prescribed for more than 50% of patients with acute respiratory tract infection (arti). acute bronchitis, in the arti category, is defined as a respiratory infection in which cough is the predominant symptom and there is no evidence of pneumonia. antibiotics are often prescribed despite limited evidence that they shorten the duration of acute bronchitis. with increasing incidence of antibiotic resistance, bronchitis allows physicians to practice "prescriptive restraint" and to provide supportive therapy. consider using the phrase "chest cold" to help patients understand the viral and benign nature of this infection. chronic bronchitis is one of the manifestations of chronic obstructive pulmonary disease (copd) and is defined clinically as cough and sputum production on most days for 3 months annually for 2 years. chronic bronchitis is thought to be primarily inflammatory in origin, although infection may be associated with acute exacerbations; with increased sputum production and worsening dyspnea, antibiotics have proved effective in acute episodes. however, systemic corticosteroids are the mainstay of copd exacerbation management. the patient with acute bronchitis presents with cough, often productive. patients may report clear or colored mucus in association with the presumed diagnosis of acute bronchitis. despite what many patients believe, the color of sputum, even purulent sputum, is not predictive of bacterial infection. the cough of bronchitis can last up to 4 weeks, sometimes even longer. typically, acute bronchitis is associated with other manifestations of infection, such as malaise and fever. respiratory viruses are thought to cause the majority of cases of acute bronchitis. influenza a and b, parainfluenza, respiratory syncytial virus (rsv), coronavirus, adenovirus, and rhinovirus are common pathogens in the viral category. clues to a specific virus may be found in the patient history; for example, rsv might be considered when there is household exposure to infected children. influenza typically presents with sudden onset of symptoms, including fever, myalgias, cough, and sore throat. neuraminidase inhibitors are modestly effective in shortening the duration of influenza in ambulatory and healthy patients (by about 1 day), if initiated in the first 48 hours of illness. the resistance patterns of influenza a and b have shifted in the last several years and may vary based on yearly viral strains. influenza b has remained, as of 2010, sensitive to zanamivir (relenza) and oseltamivir (tamiflu). currently circulating strains of influenza a, both h1n1 and h3n2, and influenza b have generally remained sensitive to both oseltamivir and zanamivir (fiore et al., 2011) . family physicians are advised to consider restraint in the prescribing of these agents, since resistance is of great concern. yearly influenza immunization and cough etiquette and hygiene are likely the most useful techniques for influenza management. studies have identified other pathogens, such as mycoplasma pneumoniae and chlamydophila pneumoniae, in a small minority of cases of clinical acute upper respiratory illness with cough as the predominant symptom. no significant benefit has been found in treating these infections with antibiotics. an exception in the treatment of acute bronchitis-like illness with antibiotics is when confirmed or probable bordetella pertussis is present. early treatment with a macrolide antibiotic and patient isolation will likely decrease coughing paroxysms and limit spread of disease (braman, 2006) . although common upper respiratory bacterial pathogens, such as moraxella (branhamella) catarrhalis, streptococcus pneumoniae, and haemophilus influenzae, may be isolated from patients with acute bronchitis, their relevance is questionable because these bacteria can be present in the respiratory tract of healthy individuals. obtaining sputum for culture when bronchitis is the diagnosis generally is not useful. antibiotics may offer a modest benefit in the treatment of acute bronchitis, with many studies showing no statistical significance in the outcome of treated versus not-treated groups. measures of function, such as duration of illness, loss of work, and limitation of activity, have not shown clinically significant improvement in those with acute bronchitis taking antibiotics. coupled with cost and the potential for side effects, the use of antibiotics for acute bronchitis is not recommended. if a provider decides to use an antibiotic in a specific patient situation, narrow-spectrum respiratory agents are preferred, such as a first-generation macrolide or doxycycline. treating the symptom of cough in acute bronchitis is an important concern for patients. in adults with acute bronchitis with signs of airway obstruction, evidenced by wheezing on examination or decreased peak expiratory flow rate, beta-2 agonists may be helpful in alleviating cough. these agents are not helpful for children with acute cough or adults with cough and no evidence of airway obstruction. side effects of tremor and an anxious feeling must be weighed against this benefit. patients often are primarily interested in alleviating symptoms caused by respiratory illness. unfortunately, there is mixed evidence for the use of over-the-counter (otc) and prescription cough medications. dextromethorphan and codeine may be somewhat effective, although they have not been evaluated in randomized, double-blinded, placebo-controlled trials for acute bronchitis. combination first-generation antihistamine-decongestant products may be effective for the cough associated with colds. naproxen showed efficacy against cough in one upper respiratory model study (sperber et al., 1992) . guaifenesin acts as an expectorant and may have some effect on cough by its mucus-thinning properties. community-acquired pneumonia (cap) is defined as an acute infection of the pulmonary parenchyma and, along with influenza, is the seventh leading cause of death in the united states. fever, cough, sputum production, pleuritic chest pain, and dyspnea are common symptoms of cap. nausea, vomiting, and diarrhea also may occur, and in elderly patients, cap may present with mental status changes. although its absence usually makes pneumonia less likely, fever can be absent in the elderly patient. other physical examination findings include an elevated respiratory rate, conversational dyspnea, tachycardia, and rales. egophony and dullness to percussion may be noted with focal consolidation. typical laboratory findings include leukocytosis. the diagnosis of pneumonia is based on the presence of symptoms and the presence of an infiltrate on chest radiograph. if infiltrate is not present, consider obtaining a chest tomography scan (which has higher sensitivity) to rule in or rule out cap. if negative, other diagnoses should be considered. the most common microbiologic agent of pneumonia is often not isolated (table 16 -1). furthermore, studies have shown that bacteriologic causes of pneumonia cannot be determined by radiographic appearance (i.e., "typical" vs. "atypical"). in the proper clinical setting, certain clinical microbes should be considered because they can affect treatment considerations and epidemiologic studies. these include legionella spp., influenza a and b, and communityacquired methicillin-resistant staphylococcus aureus (mrsa). certain diagnostic tests are performed based on clinical setting. blood cultures are not routinely done in the outpatient setting but should always be done if the patient is being admitted to the hospital, ideally before antibiotics are given. the use of gram stain and sputum culture remains controversial but can provide more evidence of a bacterial cause (e.g., many pmns). if sputum cultures are being obtained, it is recommended that the physician have the patient expectorate directly into a specimen cup and have it sent immediately for processing. this can increase the yield of isolating streptococcus pneumoniae among antibiotics for the treatment of bronchitis is not recommended because of the cost, potential for side effects, and lack of clinical benefit (braman, 2006; smith et al., 2009 ) (sor: a). in the treatment of bordetella pertussis, early administration of a macrolide antibiotic and patient isolation will likely decrease coughing paroxysms and limit spread of disease (braman, 2006) (sor: a). in adults with acute bronchitis with signs of airway obstruction, as evidenced by wheezing on examination or decreased peak expiratory flow rate, beta-2 agonists may be helpful in alleviating cough (braman, 2006) (sor: b). for acute exacerbation of copd associated with purulent sputum and increased shortness of breath, treatment with antibiotics decreases mortality by 77% and treatment failure by 53% (ram et al., 2009 ) (sor: a). 210 other respiratory pathogens. other tests include urine antigen tests for s. pneumoniae, legionella pneumophila serogroup 1, and nasal swab for influenza a and b. in young children, rsv, adenovirus, and parainfluenza in addition to influenza are common causes. nasal swab for rsv and influenza can be rapidly done, but the other causes can be determined with viral cultures, serology, enzyme-linked immunosorbent assay (elisa), and polymerase chain reaction (pcr), although results usually are received after resolution of the acute symptoms. perhaps the most important decision for clinicians is to determine the location of treatment. the american thoracic society (ats) and the infectious diseases society of america (idsa) recommend use of the pneumonia severity index (psi), which uses 20 variables to risk-stratify the patient into five mortality classes, or the curb-65, which measures five clinical variables in this decision making. the curb-65 may be the easiest and most convenient to use at the site of decision making. a score of 0 or 1 indicates treatment as an outpatient; a score of 2 requires hospital admission to the general medical ward; and a score of 3 or more indicates admission to an intensive care unit (icu) (box 16-1). treatment of cap should be targeted toward the most likely etiology (table 16 -2). outpatient therapy for patients who have no comorbidities and have not received antibiotics within the last 3 months includes doxycycline or a macrolide antibiotic. use of a fluoroquinolone antibiotic (levofloxacin or moxifloxacin) should be reserved for patients with more complicated pneumonia and those requiring hospitalization. patients who have comorbid conditions or recent antibiotic exposure, or who will be hospitalized, should receive a respiratory fluoroquinolone or combination therapy with a betalactam drug plus a macrolide, for 48 to 72 hours after fever abates (usually 5-7 days' total therapy). if an organism is isolated, therapy may be narrowed to cover the causative agent. the clinician should consider longer therapy and appropriate antibiotics to cover for infection by less common organisms such as staphylococcus aureus or pseudomonas aeruginosa. if the patient has no more than one abnormal value (temperature <37.8° c, heart rate <100, respiratory rate <24, sbp >90, o 2 saturation >90%, po 2 >60 on room air) and the patient is able to maintain oral intake and has a normal mental status, the clinician can safely switch to oral therapy and discharge the patient from the hospital. unless the etiology of the pneumonia is known, the physician should switch to oral antibiotics in the same class as the intravenous antibiotics used. the u.s. preventive services task force (uspstf) along with idsa and ats recommend annual influenza vaccinations to those over 50 years of age, those who are (or who reside with those who are) at high risk for influenza complications, and all health care workers. furthermore, the pneumococcal vaccine should be given to all those over age 65. smoking cessation is also important and should be discussed at each clinic visit. • concerns about development of resistant seasonal and h1n1 swine-derived influenza virus should be considered in the decision to administer antiviral medications to healthy patients with these infections. • the abrupt onset of fever with chills, headache, malaise, myalgias, arthralgias, and rigors during "flu season" is sufficient to diagnose influenza. • prevention of influenza is generally with vaccination. influenza deserves special mention because it is an important cause of pneumonitis and can precede a bacterial pneumonia. influenza viruses are medium-sized enveloped ribonucleic acid (rna) viruses that consist of a lipid bilayer with matrix proteins with spiked surface projections of glycoproteins (hemagglutinins, neuraminidase) on the outer surface ( figure 16-1) . both influenza a and influenza b have eight segmented pieces of single-stranded rna. the only difference between influenza a and b is that b does not have an m2 ion channel. hemagglutinins, three types of which typically infect humans (h1, h2, h3), bind to respiratory epithelial cells and allow fusion with the host cell. neuraminidase, consisting of two types (n1, n2), allows release of virus from the infected cells. a unique aspect of influenza is that antigenic variation occurs annually. antigenic shift is caused by a genetic reassortment between animal and human influenza strains, producing a novel virus that generally causes the worldwide pandemics. influenza viruses circulate mostly among humans, birds, and swine. sometimes; a human strain and an animal strain can intermingle and create a new, unique virus. this is what happened during spring 2009, heralding the most recent pandemic and creating "novel h1n1 influenza" (swine influenza). genotype analysis • score 0 or 1: outpatient treatment • score 2 : inpatient treatment on a general medical floor • score >3: inpatient treatment in an intensive care unit bun, blood urea nitrogen. locally adapted guidelines should be implemented to improve the processing of care variables and relevant clinical outcomes in pneumonia (mandell et al., 2007) (sor: b) . objective criteria or scores should always be supplemented with physician determination of subjective factors, including the patient's ability to take oral medication safely and reliably and the availability of outpatient support resources (sor: b). for patients with curb-65 score of 2 or higher, more intensive treatment (i.e., hospitalization or, where appropriate and available, intensive in-home health care services) is usually warranted (sor: c). of this strain determined that components came from an influenza virus circulating among swine herds in north america that combined with a virus circulating among ill swine in eurasia, creating a new influenza strain capable of causing disease in humans. because this virus had not previously infected humans, it had the potential to cause widespread morbidity and mortality worldwide. during pandemics, the u.s. centers for disease control and prevention (cdc) estimates an additional 10,000 to 40,000 deaths caused by influenza. although higher than in nonpandemic years, mortality was significantly less than initially predicted in 2009. no recent antibiotic therapy a respiratory fluoroquinolone alone or an advanced macrolide plus a β-lactam † † an advanced macrolide plus a β-lactam, or a respiratory fluoroquinolone alone (regimen selected will depend on nature of recent antibiotic therapy) intensive care unit (icu) a β-lactam † † plus either an advanced macrolide or a respiratory fluoroquinolone pseudomonas infection is not an issue but patient has a β-lactam allergy a respiratory fluoroquinolone, with or without clindamycin pseudomonas infection is an issue ‡ ‡ (cystic fibrosis, impaired host defenses) either (1) copd, chronic obstructive pulmonary disease; mrsa, methicillin-resistant staphylococcus aureus. * azithromycin or clarithromycin. † that is, the patient was given a course of antibiotic(s) for treatment of any infection within the past 3 months, excluding the current episode of infection. such treatment is a risk factor for drug-resistant streptococcus pneumoniae and possibly for infection with gram-negative bacilli. depending on the class of antibiotics recently given, one or another of the suggested options may be selected. recent use of a fluoroquinolone should dictate selection of a nonfluoroquinolone regimen, and vice versa. ‡moxifloxacin, levofloxacin, or gemifloxacin. § dosage: 1 g orally (po) three times daily (tid). ¶ dosage: 2 g po twice daily (bid). ** high-dose amoxicillin (1 g tid), high-dose amoxicillin-clavulanate (2 g bid), cefpodoxime, cefprozil, or cefuroxime. † † cefotaxime, ceftriaxone, ampicillin-sulbactam, or ertapenem. ‡ ‡the antipseudomonal agents chosen reflect this concern. risk factors for pseudomonas infection include severe structural lung disease (e.g., bronchiectasis) and recent antibiotic therapy, health care-associated exposures or stay in hospital (especially in the icu). for patients with cap in the icu, coverage for s. pneumoniae and legionella species must always be considered. piperacillin-tazobactam, imipenem, meropenem, and cefepime are excellent β-lactams and are adequate for most s. pneumoniae and h. influenzae infections. they may be preferred when there is concern for relatively unusual cap pathogens, such as p. aeruginosa, klebsiella spp., and other gram-negative bacteria. § § piperacillin, piperacillin-tazobactam, imipenem, meropenem, or cefepime. ## data suggest that older adults receiving aminoglycosides have worse outcomes. ¶ ¶ dosage for hospitalized patients, 750 mg/day. the abrupt onset of fever, along with chills, headache, malaise, myalgias, arthralgias, and rigors during "flu season," is sufficient to diagnose influenza. as the fever resolves, a dry cough and nasal discharge predominate. a rapid nasal swab or viral cultures can be used to confirm the diagnosis of influenza but is rarely needed. in fact, the sensitivity of these rapid tests can range from 50% to 70%, so a negative test does not rule out influenza. the primary care physician needs to determine if the patient has influenza or the common cold, because symptoms of both illnesses generally overlap (table 16-3) . treatment of influenza is generally not necessary because it is usually a self-limiting condition. treatment should be reserved for those with comorbidities who present within 48 hours of symptom onset. neuraminidase inhibitors (zanamivir and oseltamivir) prevent the release of virus from the respiratory epithelium and are approved for both influenza a and influenza b. the m2 inhibitors (amantadine and rimantadine) are approved by the u.s. food and drug administration (fda) for the treatment of influenza a because these drugs block the m2 ion protein channel, preventing fusion of the virus to host cell membrane (influenza b has no m2 ion channel). the use of m2 inhibitors is limited because of increasing resistance among influenza a viruses, as well as causing central nervous system (cns) problems that are usually exacerbated in elderly persons, who are more likely to seek treatment for influenza (table 16 -4). the major complication of influenza is a secondary bacterial pneumonia or exacerbation of underlying copd. initial improvement in clinical symptoms followed by deterioration usually suggests a secondary bacterial pneumonia, which can usually be confirmed with a chest radiograph showing an infiltrate. other, less common complications of influenza include myositis, myocarditis, pericarditis, transverse myelitis, encephalitis, and guillain-barré syndrome. prevention of influenza is generally with vaccination. box 16-2 outlines patients at risk for influenza complications who should be vaccinated yearly. although anyone wanting an influenza vaccine should be vaccinated, during periods of vaccine shortage, high-risk groups have priority. a well-matched vaccine can prevent influenza among 70% to 90% of adults and decrease work absenteeism. conversely, a poorly matched vaccine only prevents influenza in 50% of healthy adults. proper hand hygiene and covering one's cough are two additional important components in preventing the spread of influenza virus. • population-based vaccination programs have been highly effective in decreasing the incidence of many viral infections. • acyclovir can be used in adults and children with varicella to decrease symptoms if given in the first 48 hours after rash onset, but its benefit must be weighed against its cost and the possibility of development of viral resistance. • antiviral medications should be considered to decrease the incidence of postherpetic neuralgia, particularly in older patients. early treatment (within 48 hours of onset of symptoms) with oseltamivir or zanamivir is recommended for influenza a (jefferson et al., 2006) (sor: a). use of oseltamivir and zanamivir is not recommended for patients with uncomplicated influenza with symptoms for more than 48 hours (kaiser and hayden, 1999) (sor: a). oseltamivir and zanamivir may be used to reduce viral shedding in hospitalized patients or to treat influenza pneumonia (sor: c). (from treanor jj: influenza viruses, including avian influenza and swine influenza. in mandell gl, bennett je, dolin rd (eds) . mandell, douglas, and bennett's principles and practices of infectious diseases, 7th ed. philadelphia, churchill livingstone, 2010, p 2266.) • measles has had a resurgence in recent years and should be suspected when a patient presents with cough, coryza, conjunctivitis, and head-to-toe rash. • epstein-barr virus and cytomegalovirus infections are generally not clinically distinguishable, and their treatment is primarily supportive. vaccinations have dramatically decreased the incidence of a number of historically common viral infections; smallpox has been eradicated through widespread vaccination. however, recent outbreaks of measles and mumps on college campuses underscore the need to remain vigilant in administering vaccines at the population level, even though no vaccine is available for many common viruses. varicella is one of the classic viral exanthems of childhood. before routine vaccination, having chickenpox was one of childhood's "rites of passage." the virus, a herpesvirus (human herpesvirus 3), is effectively transmitted, causing outbreaks in schools and households. patients with primary varicella present with fever, headache, and sore throat. generally within 1 to 2 days of onset of symptoms, a papulovesicular rash erupts diffusely. the classic description of the chickenpox lesion is "a dewdrop on a rose petal," suggesting a central vesicle on an erythematous base. lesions continue to appear for 5 to 7 days. all lesions going from papule to vesicle to crusted lesion takes about 2 weeks. patients are considered to be infectious, primarily through respiratory secretions, during the 2 days before symptoms appear and until all lesions are crusted. treatment of varicella is generally supportive. control of spread may be a concern in group-living environments such as schools or residence halls. isolation of the infected patient away from those susceptible to varicella infection is standard practice. acyclovir can be started within the first 24 hours after rash eruption to achieve an attenuation of the infectious course. in children, this means a decrease in the duration of fever by about 1 day and a decrease in the number of lesions (swingler, 2010) . in adults, acyclovir decreases rash duration and the number of lesions, although the results are less significant than for children. adult dosing of acyclovir for varicella is 800 mg five times daily. the marginal benefit must be weighed against the possible development of resistance at a population level and the cost of the medication. complications of varicella can include secondary infection of skin lesions, pneumonitis, encephalitis, and dehydration from vomiting and diarrhea. varicella is prevented primarily through administration of vaccine. the vaccine is highly effective in children, with recommended dosing at 12 to 15 months with a second dose at 4 to 6 years. varicella is now included in a measles-mumpsrubella (mmr) vaccine, which can be given between 12 months and 12 years of age. the varicella vaccine is a live, attenuated virus and should not be given to certain immunocompromised patients. the vaccine can also be administered to exposed immunocompetent contacts, although the benefit is clearer for children than adults. severely immunocompromised patients exposed to varicella (particularly those with advanced hiv disease) may be given high-dose acyclovir to prevent development of disease. herpes zoster is a reactivation of the neurotropic varicella virus, typically in a dermatomal distribution. this is more common in elderly or immunocompromised patients but can occur in healthy people as well. patients with zoster may note generalized malaise, hyperesthesia, numbness, tingling, and pain in the skin before development of a rash. the appearance of the rash is the same as for chickenpox, although most often isolated to a unilateral dermatome. the diagnosis of herpes zoster is clinical based on the history and the classic appearance of the rash. in immunocompromised patients, however, the rash may not be dermatomally isolated. when the diagnosis is unclear, viral culture can be obtained from the base of a lesion. antiviral medications are likely to decrease the incidence of postherpetic neuralgia and are recommended, particularly in elderly patients (wareham, 2010) . valacyclovir (1 g three times daily) or famciclovir (500 mg every 8 hours) for 7 days is likely more effective than acyclovir in achieving this result. either drug should be started as soon after the diagnosis as possible, preferably within 48 to 72 hours of rash onset. when patients have established postherpetic neuralgia, gabapentin and tricyclic antidepressants are helpful in alleviating the pain. the rash of zoster is infectious to the touch. patients should be advised to keep the rash covered until all the lesions have crusted. zoster of the trigeminal nerve can extend to the eye and warrants immediate ophthalmologic intervention. a vaccine to prevent herpes zoster in adults was released in 2006. the zoster vaccine differs from the varicella vaccine in that the amount of attenuated virus is 14 times higher in the zoster vaccine. the vaccine decreases the incidence of zoster by 50%. it is recommended for administration by the american academy of family physicians (aafp) to adults over age 60, regardless of prior varicella or zoster history. although generally well tolerated, the vaccine is somewhat costly. in 2008, more measles cases were reported than in any other year since 1997 (cdc, 2010) . measles is the "first disease" of childhood from the history of medicine. in adults, measles infection may be acquired in the face of waning immunity from remote immunization. a booster dose of mmr vaccine is recommended before college entry. clinically, measles presents with cough, coryza (nasal irritation and congestion), and conjunctivitis. fever is common several days before the onset of the rash. the rash of measles typically spreads from head to toe and has an erythematous, papular appearance with a "sandpaper" feeling. koplik's spots are erythematous papules with a bluish center on the oral mucosa and appear early in measles. measles is highly contagious through droplets. lymphopenia and neutropenia are common laboratory findings with measles infection. complications of measles include primary infections such as pneumonia, gastroenteritis, encephalitis, and the rare subacute sclerosing panencephalitis. secondary infections such as otitis media, pneumonia, and adenitis may also occur. treatment is supportive, and the implications of measles infection are primarily in the public health realm. patients with measles should be isolated for at least 4 days after the appearance of the rash. it is important to recognize that patients are contagious for 2 days before the development of symptoms. careful verification of immunization status for close contacts is essential. clinical infectious mononucleosis is a common infection in adolescents and early adults. the clinical syndrome is most often caused by epstein-barr virus (ebv), although cytomegalovirus (cmv) may also be the source in this clinical syndrome, which includes fever, exudative tonsillitis, adenopathy (often including posterior cervical or occipital nodes), and fatigue. ebv is transmitted in oral secretions and may be transmitted sexually as well. b cells are infected with ebv either directly or after contact with epithelial cells, resulting in diffuse lymphoid enlargement. the diagnosis of infectious mononucleosis is made by recognizing the clinical symptoms of fever, pharyngitis, and adenopathy along with the laboratory findings of greater than 50% lymphocytes with 10% or more atypical lymphocytes (hoagland, 1952) . also, a positive serologic test for heterophile antibody assists the family physician in the diagnosis. to differentiate ebv from cmv mononucleosis, serology (igg and igm) may be obtained. results of these tests are generally not available in time to have a significant benefit clinically. splenic enlargement as part of this lymphoid hypertrophy can lead to splenic rupture (0.1% risk) (dommerby et al., 1986) . athletes with infectious mononucleosis must be managed carefully to avoid their participation in sports that could result in abdominal trauma. other risks associated with infectious mononucleosis include upper airway obstruction, asymptomatic transaminase elevation, thrombocytopenia, and rash after the administration of ampicillin or amoxicillin. routinely obtaining transaminase levels in patients without clinical hepatitis is of little value and can increase the overall cost of management. treatment of infectious mononucleosis is largely supportive. patients should be instructed to treat fever with antipyretics, rest, and expect symptom duration of 2 to 4 weeks, although symptoms can last for several months. the use of steroids, such as prednisone, has shown limited benefit. data suggest an initial benefit 12 hours after steroid administration, although this is lost within several days (candy and hotopf, 2006) . combination of steroid and an antiviral (valacyclovir) may have some positive effect on fatigue. • the most common presentation of tuberculosis is pulmonary disease. • tuberculosis is diagnosed by acid-fast bacilli smears and cultures. • standard first-line agents to treat tb are isoniazid, rifampin, pyrazinamide, and ethambutol. • high-risk patients with a positive purified protein derivative skin test or quantiferon-tb gold test should be treated for latent tb infection. • the current recommendation for first-line treatment for latent tb is 9 months of oral isoniazid. tuberculosis skin testing should be interpreted without regard to bacille calmette-guérin (bcg) history, because bcg is administered in areas where tb is endemic and bcg does not provide complete protection from tb infection. tuberculosis (tb) is a disease that has plagued humans since antiquity, with evidence of spinal tb in neolithic and early egyptian remains. at present, tb affects approximately one third of the world's population. tb is the world's second most common cause of death from infectious disease after human immunodeficiency virus or acquired immunodeficiency syndrome (hiv/aids). tuberculosis is caused by mycobacterium tuberculosis, an acid-fast bacillus. tb is acquired by inhalation of respiratory droplets. these respiratory droplets are spread by coughing. brief contact carries little risk for acquiring tb, and infection generally does not occur in open air; open-air sanatoriums were the cornerstone of tb treatment before antimicrobial therapy. in the united states, tb incidence rates have been on the decline since 1992, coinciding with the control of hivinduced aids by antiretroviral therapy. however, tb remains prevalent in certain high-risk groups (i.e. immigrants, iv drug use, homeless persons). most cases of tb are in people age 15 to 49 years. tb in elderly persons is generally caused by a reactivation of latent infection acquired in the remote past, whereas tb in young children indicates ongoing active transmission in the community. infection in children is more likely to progress to active tb and disseminated disease. persons with hiv infection have a disproportionately higher risk for acquiring tb than the general population. tuberculosis is most frequently manifested clinically as pulmonary disease, but it can involve any organ. extrapulmonary tb accounts for about 20% of disease in hiv-seronegative persons but is more common in hiv-seropositive persons. pulmonary tb typically manifest with fever, night sweats, chronic cough, sputum production, hemoptysis, anorexia, and weight loss. chest radiographs in patients with pulmonary tb typically reveal upper-lobe cavitary lesions and can reveal infiltrates or nodular lesions, as well as lymphadenopathy ( figure 16 -2). tb in the setting of advanced hiv co-infection does not generally manifest in the typical manner (table 16-5) . acyclovir started within the first 24 hours after varicella rash eruption can attenuate the infectious course, decreasing duration of fever by 1 day and reducing the number of lesions (sor: a). administration of varicella vaccine to a susceptible child within 3 days of exposure will likely modify or prevent disease (macartney and mcintyre, 2008) the diagnosis of pulmonary tb is made by the demonstration of acid-fast bacilli (afb) in sputum and the growth of m. tuberculosis in culture. these patients typically have an abnormal chest radiograph, as previously described. m. tuberculosis is a slow-growing bacterium, and cultures can take up to 6 weeks to grow. a pcr assay developed for m. tuberculosis can be run on afb smear-positive sputum to hasten the diagnosis of pulmonary tb. a positive pcr on afb-positive sputum is diagnostic of pulmonary tb, but a negative test does not rule out the diagnosis. patients with afb positive smears from sputum samples should be started on anti-tb therapy while awaiting results of pcr and cultures. the treatment of tb always uses multiple agents with anti-tb activity. single agents should never be used. the standard first-line agents are isoniazid (inh), rifampin (rif), pyrazinamide (pza), and ethambutol (emb) (figure 16 -3 and table 16-6). if administered, inh should be given with pyridoxine (vitamin b 6 ; 25-50 mg orally daily) to prevent neuropathy. treatment of active pulmonary tb is generally for 6 months regardless of hiv status, but treatment may need to be extended in certain situations. directly observed therapy (dot) is the preferred mechanism of administration to ensure compliance. many local county and state health departments have systems for dot. treatment of hiv-seropositive patients with tb who are receiving an antiretroviral (arv) regimen that contains a protease inhibitor is complicated by the latter's interaction with rifamycins (particularly rifampin). management of such patients should be coordinated with an infectious diseases specialist, who also should manage drug-resistant tb treatment. in the united states, latent tuberculosis infection (ltbi) is the most prevalent form of tuberculosis. ltbi is the term given to patients with a positive purified protein derivative (ppd) skin test without evidence of active tb. ppd has been used for more than 100 years and relies on delayed-type hypersensitivity (dth) to m. tuberculosis cellular proteins. early late figure 16 -3 treatment algorithm for tuberculosis. patients in whom tb is proved or strongly suspected should have treatment initiated with isoniazid, rifampin, pyrazinamide, and ethambutol for the initial 2 months. a repeat smear and culture should be performed when 2 months of treatment has been completed. if cavities were seen on the initial chest radiograph or the acid-fast smear is positive at completion of 2 months of treatment, the continuation phase of treatment should consist of isoniazid and rifampin daily or twice weekly for 4 months to complete a total of 6 months of treatment. if cavitation was present on the initial chest radiograph and the culture at completion of 2 months' therapy is positive, the continuation phase should be lengthened to 7 months (total of 9 months of treatment). if the patient has hiv infection and the cd4+ cell count is less than 100/mm 3 , the continuation phase should consist of daily or three-times-weekly isoniazid and rifampin. in hiv-uninfected patients having no cavitation on chest radiograph and negative acid-fast smears at completion of 2 months of treatment, the continuation phase may consist of either once-weekly isoniazid and rifapentine, or daily or twice-weekly isoniazid and rifampin, to complete a total of 6 months (bottom). patients receiving isoniazid and rifapentine, and whose 2-month cultures are positive, should have treatment extended by an additional 3 months (total of 9 months). *emb may be discontinued when results of drug susceptibility testing indicate no drug resistance. †pza may be discontinued after it has been taken for 2 months (56 doses). ‡rpt should not be used in hiv-infected patients with tb or in patients with extrapulmonary tb. therapy should be extended to 9 months if 2-month culture is positive. afb, acid-fast bacilli; cxr, chest radiograph (x-ray); emb, ethambutol; inh, isoniazid; pza, pyrazinamide; rif, rifampin; rpt, rifapentine. because ppd relies on dth, any factor that reduces the dth affects the host response to ppd. the most common clinical example is use of corticosteroids, which blunt the dth response and can complicate ppd interpretation. therefore, ppd testing should not be performed while a patient is taking corticosteroids. also, tb testing should be targeted to those with higher risk of infection and should not routinely be done in those with low risk (ats/cdc, 2000) . the ppd can also give false-positive results in patients with previous bacille calmette-guérin (bcg) vaccination or with infection by other mycobacterial infections. in the united states, this may cause difficulties in testing immigrants from countries who routinely use bcg vaccination programs. however, previous bcg vaccination should not change the interpretation of the ppd or willingness to treat such individuals accordingly. ‡when dot is used, drugs may be given 5 days per week and the necessary number of doses adjusted accordingly. although there are no studies that compare five with seven daily doses, extensive experience indicates this would be an effective practice. § patients with cavitation on initial chest radiograph and positive cultures on completion of 2 months of therapy should receive a 7-month (31 weeks, either 217 doses [daily] or 62 doses [twice weekly]) continuation phase. ¶ five-days-a-week administration is always given by dot. rating for 5 day per week regimens is aiii. ¶ ¶ not recommended for hiv-infected patients with cd4+ cell counts <100 cells/μl. ** options 1c and 2b should be used only in hiv-negative patients who have negative sputum smears at completion of 2 months of therapy and who do not have cavitation on initial chest radiograph. for patients started on this regimen and found to have a positive culture from the 2-month specimen, treatment should be extended an extra 3 months. the dth response can wane over time. to overcome this problem, nonreacting patients may undergo repeat ppd 1 week after their initial ppd. the diagnosis of ltbi is made by interpretation of a ppd and by ascertaining the patient's risk factors for progression to active tb if left untreated . interpretation of the ppd should be based on the area of induration and not the area of surrounding erythema. persons whose ppds have converted from negative to positive within 2 years are presumed to have been infected recently. the decision to use ppd means treating the patient for ltbi if the ppd test is positive. patients at increased risk for progression to active tb include those who have been recently infected (recent ppd converters); patients who are hiv seropositive; patients who have silicosis, diabetes, or chronic renal failure (including those receiving hemodialysis); solid-organ transplant recipients; patients with gastrectomy or jejunoileal bypass or head and neck cancer; injection drug users; patients with chest radiograph evidence of prior tb; and patients who weigh at least 5% less than ideal body weight. patients taking chronic corticosteroid therapy and those who are to receive tumor necrosis factor alpha (tnf-α) blockers (e.g., infliximab) are also at risk. patients taking corticosteroids also have higher risk of progression to active tb with larger doses and longer courses of corticosteroids. standard therapy for ltbi is inh, 300 mg orally daily for 9 months, regardless of hiv status. again, inh should always be administered with pyridoxine to prevent neuropathy. to overcome the false-positive results and confusion of ppd testing in certain populations, newer interferon-gamma (ifn-γ) release assays such as the quantiferon-tb gold (qft-g) test have been developed to detect latent m. tuberculosis. qft-g quantifies the release of ifn-γ from lymphocytes of the host's blood in response to three m. tuberculosis target antigens that are absent from bcg and most other nontuberculous mycobacterium spp. the advantages of using qft-g include one-time blood testing without the need for followup visit, no triggering of amnestic responses, and possibly more specific response to m. tuberculosis. however, qtf-g use in immunocompromised or anergic patients is limited, with indeterminate results. some studies also show discordant results in individuals tested with both ppd and qtf-g. in general, qtf-g may be used in all circumstances in which the ppd is used. however, whether the qtf-g is truly more specific or sensitive than the ppd in latent or active tb is yet to be determined. • the u.s. preventive services task force recommends "highintensity" behavioral counseling to at-risk adults and adolescents to prevent sexually transmitted infections. • be specific in addressing patients' sexual practices so as to provide appropriate prevention advice. hiv-positive persons recent contacts of tuberculosis patients fibrotic changes on chest radiography consistent with prior tuberculosis patients with organ transplants and other immunosuppressed patients (receiving equivalent of ≥15 mg/day of prednisone for at least 1 month) development in the primary prevention of stis is immunization against human papillomavirus (hpv). the vaccine can prevent infection with certain strains of hpv that cause cervical cancer and genital warts. trials are ongoing to determine the effectiveness of daily arv therapy in preventing transmission of hiv. vaccination investigation is ongoing for herpes simplex, chlamydia trachomatis, and hiv. this breadth of research effort holds promise for the future in the prevention of stis. the uspstf recommends "high-intensity" behavioral counseling to at-risk adults and adolescents to prevent stis. highintensity counseling involves multiple sessions and often is delivered to groups of patients. unfortunately, this type of intervention has limitations in its practicality for population-based delivery. no risk of harm was discovered in the delivery of counseling for sti prevention. vaccination is the most important form of primary prevention of common infectious diseases. two vaccines are currently on the market for hpv prevention-one that protects against four viral subtypes (6, 11, 16, 18) and is licensed for use in males and females 9 to 26 years of age, and the other against two subtypes (16,18), licensed for females 10 to 25 years of age. hepatitis b is a sexually transmitted infection, and immunization is recommended for adolescents who have not been previously inoculated. this is a requirement in many states for school entry. hepatitis a can be transmitted by oro-anal sexual contact, and vaccination should be offered to patients who are contemplating engaging in this sexual practice. recommendations surrounding the use of barrier methods for sti prevention should be tailored to the sex practices of the client. for example, a percentage of women use anal sex as a method of birth control but may not consider the need for condom use with this practice. the question, "do you regularly use condoms?" has little relevance to infection control for many sexual practices. evidence supports the advice to use barrier methods of latex or other approved material in a manner that prevents the exchange of blood and body fluids in decreasing stis. condoms confer a 30% risk reduction for herpes simplex and up to an 80% risk reduction for hiv, when used correctly (weller and davis-beaty, 2002; martin et al., 2009) . the secondary prevention of stis is achieved through direct and nonjudgmental patient assessment and screening and avoiding assumptions about patient sexual practices. screening is a tool to prevent the inadvertent spread of infection as well as the sequelae of undetected disease. infectious genital ulcers are associated with herpes simplex virus (hsv), syphilis, chancroid, lymphogranuloma venereum, and granuloma inguinale. hsv is by far the most common, affecting 50 million people in the united states. hsv-1 and hsv-2 are chronic, neurotropic viral infections that enter through epithelium and come to rest in the dorsal root ganglia. therefore, infection leads to lifetime presence of the virus, but the clinical manifestation of this condition is variable. a small percentage of those with serologic evidence of hsv-2 (10%-25%) have had symptoms of clinical herpes infection. in addition, patients with hsv infection can shed the virus in the absence of symptoms, creating a prime opportunity for spread. herpes simplex outbreak may be followed by a prodrome of malaise, fever, and regional lymphadenopathy before the appearance of grouped vesicles on an erythematous base. the vesicles are typically quickly broken and become ulcerated in appearance, with each vesicle usually less than several millimeters in size. true first-time infections tend to present more severely than secondary presentations of previously infected individuals, with a prodrome present in 80% of cases. the lesions can be in any location around the genitals or rectum, on the proximal thighs and buttocks, inside the vagina, and in and around the mouth. the lesions are most who to screen? often painful, particularly when on mucosal surfaces, or itchy. in women, herpes simplex can present with cervicitislike symptoms with bleeding and discharge and cervical ulcerations on examination, or simply mucopurulent cervicitis. herpetic lesions around the urethra tend to be extremely painful and can make urination difficult. rectal hsv can be confused with irritation, perianal fissure, and even candidiasis because of its often beefy-red appearance and itching. vesicles typically appear 6 days after infection and can last up to 2 weeks in an initial infection. subsequent outbreaks tend to have a shorter duration and to be less uncomfortable for patients. confirmation of infection is helpful, but the diagnosis can be made primarily on the clinical appearance of the exanthema. vigorous sample collection from an ulcer (which the patient may not appreciate) to be sent for pcr identification and typing is the most readily available method of laboratory diagnosis. serum antibody testing is not useful in the initial hsv diagnosis because antibody levels will not be appreciable early in infection. the appearance of convalescent immunoglobulin g (igg) and igm levels several weeks after a suspected outbreak might help to support the diagnosis of hsv infection. the value of screening for hsv immunity is debatable and should generally not be recommended for asymptomatic individuals. in addition, the uspstf recommends against screening asymptomatic pregnant women for hsv to prevent transmission to the newborn. given that many patients with hsv infection never manifest symptoms, the value of knowing that one is hsv seropositive is questionable. in addition, hsv-1 and hsv-2, although classically oral and genital, respectively, can "mix and match" based on sexual practices. it is often confusing for asymptomatic individuals to know that they have hsv antibody (do i have cold sores? do i have genital herpes? how should this change the way i live my life?). in monogamous couples with one partner known to be hsv positive and the other with unknown status, testing of the latter may indicate suppressive therapy in the seropositive partner if the other is found to be negative. regular barrier method use decreases transmission of herpes in both men and women, with patients using condoms 100% of the time having a 30% reduction in hsv acquisition from those who never use condoms (martin et al., 2009) . serodiscordant couples may also decrease transmission through antiviral suppressive therapy to the hsv-positive partner (table 16-8) . syphilis is a spirochetal infection that has resurged since 2001, the nadir year since 1996. syphilis infection rates are highest in men who have sex with men. syphilis is much less common than the other stis, with an infection rate of 5.6 per 100,000 population in the united states (vs. 496 per 100,000 for chlamydia). syphilis presents in several stages. the primary phase of syphilis is a painless ulcer called a chancre (figure 16-4) . the chancre may be visible on the genitals, although it can also be inside the vagina, mouth, or rectum, making it difficult to find. this lesion will appear within 3 weeks of transmission and will last for several weeks untreated. the secondary phase of infection is disseminated and involves a diffuse macular rash, typically with palm and sole lesions, generalized lymphadenopathy, fever, and condyloma latum (smooth, moist lesions on genitals without cauliflower appearance of condyloma acuminatum). tertiary syphilis is often asymptomatic but affects the heart, eyes, and auditory system and can be associated with gumma formation. gummas are soft, granulomatous growths in organs that can cause mechanical obstruction and weakening of blood vessel walls. latent infection often involves the cns. diagnosis of primary syphilis is challenging. the test of choice is darkfield microscopy, which is not readily available. direct fluorescent (monoclonal) antibody (dfa) testing may be available. antibody tests for syphilis, such as the rapid plasma reagin (rpr) and the less frequently used venereal disease research laboratories (vdrl), are often not positive early in infection and thus cannot be used to rule out primary syphilis based on a single reading. treponemal antigen testing (eia) may be available in some laboratories. the fluorescent treponemal antibody absorption (fta-abs) test may also be negative in the early infection. direct pcr for primary syphilis lesions has been tested but is not yet fda approved. a physician may choose to treat presumptively if a painless chancre and risk factors are present and may then do a convalescent rpr test in 1 to 2 weeks to confirm the infection by the appearance of a positive reaction. one would expect a fourfold change in titer of either test to indicate the presence of disease. primary and secondary syphilis are treated with a single injection of penicillin g, 2.5 million units. other regimens do not have proven effectiveness but can be used in the penicillin-allergic patient, including doxycycline, 100 mg twice daily for 14 days; ceftriaxone, 500 mg to 1 g intramuscularly (im) daily for 8 to 10 days; or azithromycin, 2 g as a single oral dose, although resistance to azithromycin has been observed. patients treated for primary syphilis should have periodic clinical follow-up and serologic testing to determine a fourfold decrease in rpr reactivity within 6 months. latent syphilis can be either early, meaning infection within the last year, or late, meaning infection beyond a year. early latent syphilis is treated with a single injection of penicillin g, 2.4 million units. syphilis of late latency or unknown duration is treated with three injections of penicillin g, 2.4 million units, in 3 consecutive weeks. for penicillin-allergic patients, doxycycline, 100 mg twice daily for 28 days, is required. those with latent syphilis should have ophthalmic examination as well as evaluation for vascular gumma formation. suspected neurologic involvement of latent syphilis must be evaluated with cerebrospinal fluid (csf) examination and treatment with aqueous penicillin g, 3-4 million units intravenously (iv) every 4 hours for 10 to 14 days. partners of patients with newly diagnosed syphilis are at risk for infection. partners within 90 days of a diagnosis of primary syphilis should be tested, but treated presumptively even if serologic testing is negative. for partners prior to 90 days before diagnosis, serology is generally reliable in detecting presence of infection and may guide treatment. patients with secondary syphilis should inform partners within 6 months before diagnosis, or 12 months for those diagnosed with tertiary syphilis (table 16 -9). chancroid may occur in regional outbreaks and presents with a painful genital ulcer and suppurative regional adenopathy. herpes and syphilis should both be ruled out in the patient suspected of having chancroid infection. chancroid is caused by haemophilus ducreyi and there is currently no fda approved test to directly detect this organism. treatment with azithromycin (1 g as single dose), ceftriaxone (250 mg im as a single dose), ciprofloxacin (500 mg twice daily for 3 days), or erythromycin (500 mg three times daily for 7 days) are all alternatives (table 16 -10). it may be necessary to perform incision and drainage on fluctuant inguinal nodes. patients should be reexamined in 1 to 2 weeks to ensure healing of the primary ulcer(s) and resolution of the adenopathy. partners who had contact with the infected patient starting 10 days before development of the patient's symptoms should be treated, regardless of the presence of symptoms. less common ulcerating stis include lymphogranuloma venereum (lgv) and granuloma inguinale ( figure 16 -5). lgv causes regional adenopathy and often an ulcer at the point of entry. rectal lgv may cause a proctocolitis with anal pain, discharge, bleeding, and diarrhea. lgv is caused by chlamydia trachomatis serotypes and can be detected by testing swabbed material from open lesions or aspirates from lymph nodes with culture, dfa, or nucleic acid detection. treatment is noted above (table 16-10) . granuloma inguinale, caused by klebsiella granulomatis, is rare in the united states and causes progressive ulcerative disease of the genitals. a second sti category includes those causing the clinical presentation of vaginal discharge, pelvic pain, dyspareunia, and dysuria in women and penile discharge and dysuria in men, as well as possible rectal pain and discharge in men and women. of this group, chlamydia trachomatis is the most common, causing 1.2 million infections in the united states in 2008 (cdc, 2009 ). in fact, chlamydia is the most frequently reported reportable infection. the majority of women with chlamydia infection are without symptoms. many men are asymptomatic as well. regular screening for chlamydia, as recommended by the uspstf, can significantly reduce the incidence of pelvic inflammatory disease (pid), one of the most serious sequelae of untreated infection. in women with untreated chlamydia infection, in addition to pid, tubo-ovarian abscess, tubal scarring and ectopic pregnancy, and infertility can all result. as previously mentioned, regular screening is currently recommended for all sexually active women under age 24, all pregnant women under 24, and at-risk pregnant and nonpregnant women over 24. chlamydia testing can be performed on several liquid-based papanicolaou (pap) tests. endocervical swabs for nucleic acid amplification are acceptable when a conventional pap smear is being used. given the recent liberalization of recommendations about pap testing for women under 21 years of age, urine nucleic acid amplification is a readily available alternative for chlamydia testing. this can easily be done at a contraceptive counseling clinic. urine testing is also an acceptable method of testing for men, in addition to a urethral swab. rectal chlamydia infection can occur in individuals who practice receptive anal intercourse. an fda-approved method of testing should be used for screening and diagnosis of this infection. asymptomatic chlamydia infection is treated with either a single dose of azithromycin, 1 g orally, the drug of choice, or doxycycline, 100 mg twice daily, for 7 days (table 16-11) . patient-delivered partner therapy (pdpt), the practice of dispensing treatment to diagnosed patients to treat their partner(s), has proved effective in reducing reinfection rates and further spread of infection. ept is legally allowable in 21 states and potentially allowable in another 21. chlamydia infection may present symptomatically in men or women with symptoms of dysuria and with discharge and with pelvic pain and dyspareunia in women. the discharge of c. trachomatis, versus that of neisseria gonorrhoeae, is said to be more mucoid than purulent, although this characteristic is not specific enough to provide diagnostic accuracy. symptomatic chlamydia, without evidence of pid, is treated the same as asymptomatic infection. many practitioners will treat presumptively for chlamydia and gonorrhea in patients who present with the symptoms previously mentioned while they wait for confirmatory testing. neisseria gonorrhoeae infection may be asymptomatic in both men and women. the current uspstf recommendation is for screening women at risk. men with penile gonorrhea typically present with purulent penile discharge and dysuria with n. gonorrhoeae infection. mucopurulent discharge, dysuria, pelvic pain, and dyspareunia are typical symptoms in women. in patients who engage in anal intercourse, anal discharge, rectal pain, and bleeding can be presenting symptoms. gonococcal pharyngitis is within the differential of exudative pharyngitis in sexually active patients. when symptomatic, throat pain, tonsillar exudates, and anterior cervical adenopathy may be present. testing for gonorrhea can be done using liquid-based pap technologies, cervical or urethral swabs, or urine for nucleic acid amplification. in men with visible discharge, a gram stain with white blood cells (wbcs) and gram-positive intracellular diplococci has a high degree of sensitivity. culture testing may be preferred for suspected pharyngeal and rectal specimens pending fda approval of other methods. again, physicians may opt to treat patients with mucopurulent cervicitis or urethritis presumptively for gonorrhea and chlamydia while waiting for confirmatory testing. fluoroquinolone therapy is no longer recommended because of widespread resistance (table 16-11) . because reinfection with gonorrhea is common for several months after treatment, it may be advisable to retest patients with confirmed gonorrhea in the 3 months after treatment. similarly, stis may be an indicator of risk behavior, and a complete risk history and testing for other stis is advisable if not completed at the initial visit. in male patients with symptomatic urethritis, a causative agent may not be identified, a situation often referred to as nongonococcal urethritis (ngu). technically, chlamydia is included in this category. organisms such as ureaplasma urealyticum and mycoplasma genitalium may be the cause and may be difficult to detect. treatment for these infections is the same as for symptomatic chlamydia, with azithromycin or doxycycline (table 16 -11). it is recommended that partners of patients with ngu should be evaluated and treated. in some cases, testing of partners may detect a specific organism as the cause of infection (e.g., chlamydia). trichomonas vaginalis causes vaginitis in women, who may have a stereotypic frothy, green, and foul-smelling discharge. many women are asymptomatic with trichomoniasis. in addition to causing asymptomatic infection in men, t. vaginalis may cause urethritis. this organism may be suspected in men when patients have repeated treatment failures and no other explanation for symptoms. microscopic examination of vaginal discharge is 60% to 70% sensitive in women. a first voided urine specimen or urethral swab for microscopic exam may be helpful in identifying the protozoa. culture for trichomonas, which requires a special medium, may be necessary to identify this infection accurately in men. trichomonas is effectively treated with a single 2-g dose of metronidazole (table 16-11) . for non-sti causes of vaginal discharge, see the online discussion at www.expertconsult.com. pelvic inflammatory disease can be caused by a number of organisms, including chlamydia, and presents with pelvic pain and discharge. findings that contribute to the diagnosis of pid include fever greater than 101° f, cervical or vaginal mucopurulent discharge, abundant wbcs on saline preparation of vaginal discharge, elevated erythrocyte sedimentation rate (esr), elevated c-reactive protein (crp), and evidence of n. gonorrhoeae or c. trachomatis infection. hospitalization with parenteral antibiotics may be necessary in pregnant patients, patients in whom surgical emergency cannot be ruled out, those who do not respond to oral treatment, those who cannot tolerate oral treatment, and patients who have severe illness or tubo-ovarian abscess. when treating pid parenterally, improvement of symptoms for 24 hours may prompt a change to oral therapy (table 16-12) . conversely, if oral therapy is not producing significant improvement within 2 to 3 days, admission for parenteral therapy may be necessary. patient awareness of human papillomavirus infection has greatly increased in recent years, in large part related to the patient-directed advertising of the hpv vaccine. hpv is likely the most common sti. thirty types of hpv can infect the genital area, some causing genital warts, some causing malignancies of the genital organs, and most being asymptomatic. the gross categories most often used are "high risk" (most often types 16 and 18) and "low risk" (types 6 and 11) hpv infection, the former more often associated with genital cancer. prevention of hpv infection and cervical cancer was revolutionized with the release of the hpv vaccine, which is effective in reducing the incidence of hpv-associated disease. currently, two vaccines are licensed in the united states. gardasil (merck), released in 2006, includes protection against viral types 6, 11, 16, and 18. it is approved for the prevention of vulvar and vaginal cancer and for the prevention of cervical cancer, cervical dysplasia, and genital warts in females age 9 to 26. the vaccine was recently approved for males of the same age range for the prevention of genital warts. more recently, cervarix (glaxosmithkline) was approved for the prevention of cervical cancer and cervical dysplasia from hpv types 16 and 18 in women age 10 to 25. ideally, the vaccine should be administered before initiation of sexual activity to prevent initial acquisition of these hpv types. patients who are already sexually active may also receive the vaccine. the transmission of hpv to men decreases with consistent condom use, from 53.9% in men who never use condoms to 37.9% in men who "always" use them. unfortunately, hpv can infect skin that is not covered by the use of traditional barrier methods (nielson et al., 2010) . male circumcision may decrease the transmission of hpv. patients have many questions about hpv, in particular about screening for asymptomatic infection. hpv infection occurs with high frequency in the sexually active population; up to 50% or more of sexually active individuals have hpv at some point in their life. in addition, hpv is effectively transmitted, even if contact does not involve genital-togenital touching (i.e., manual stimulation can transmit the virus). again, most hpv infections are without symptoms and resolve spontaneously through eradication by the intact immune system. for all these reasons, screening for the mere presence of hpv infection has minimal utility. there is no treatment for asymptomatic hpv infection. the most common presentation of hpv infection is in the context of an abnormal pap smear. hpv is directly linked to cervical dysplasia. for women over age 21 and under 35, hpv testing with high-risk viral detection is common. the presence of high-risk hpv informs further management of the pap result. it is currently recommended that women over 35 be automatically tested for high-risk hpv infection at the pap smear. patients may present with visible warts, or these may be detected at routine or sti screening. genital warts are often cosmetically unacceptable to patients, even though they are infrequently functionally problematic. in some circumstances, wart burden can be high enough to cause physical discomfort or relative obstruction of the vagina or rectum. vulvovaginal candidiasis and bacterial vaginosis are generally not thought to be sexually transmitted, although they are often in the differential diagnosis of sexually transmitted infection (sti). both these infections likely are related to changes in the vaginal ph and the normal flora distribution. it is not always clear which of these factors is primary and which is secondary, because at diagnosis, both ph and normal vaginal flora will often be abnormal. vulvovaginal candidiasis is a common infection causing typically white, curdlike discharge, itching, and sometimes dysuria. the causative organism is usually candida albicans but can be other candida spp. antibiotics can alter normal vaginal flora, so the recent use of antibiotics may predispose women to candidiasis. physical examination may reveal erythematous external genitalia as well as external and internal white, clumping discharge. usually, no distinctive odor is associated with vaginal yeast. wet preparation of vaginal specimen or treatment with potassium hydroxide (koh) may reveal branching pseudohyphae and yeast. when ph is performed, it should be directly on the vaginal discharge and not on the saline-diluted specimen because the saline will alter the ph of the specimen. typically, the ph of yeast discharge is less than 4.5 (normal vaginal ph,3.8-4.5). bacterial vaginosis (bv) is the most common cause of infectious vaginal discharge (spence and melville, 2007) . many different organisms are associated with the diagnosis of bv, including gardnerella vaginalis and mycoplasma hominis. women with bv may report discharge, vaginal irritation, vaginal odor, and at times, dysuria. findings of bv are often detected during a normal screening pap smear or pelvic examination. physical findings may reveal signs of vaginal irritation. the discharge is usually thin and gray. an amine (fishy) odor may be produced with the application of koh. the finding of clue cells, or epithelial cells with adherent bacteria, under saline preparation microscopy and a decrease in normal lactobacilli are common findings. the amsel criteria are useful in bv diagnosis; other scoring systems (e.g., nugent criteria) have been used but require gram staining. the specific amsel criteria are (1) milky, homogeneous, adherent discharge; (2) discharge ph greater than 4.5; (3) positive whiff test (fishy smell with addition of koh); and (4) at least 20% clue cells on microscopic examination. if three of the four criteria are present, the likelihood of bv is 90%. in routine vaginal examination and bimanual examination for patients with vaginal discharge, signs and symptoms of vaginitis are poor predictors of the microbiologic cause of infection (schaaf et al., 1990) . the clinical examination and office testing, in fact, are fair predictors of the true cause of infection (lowe et al., 2009 ). many patients with vaginal discharge will use over-the-counter preparations before consulting a physician, which can delay correct diagnosis of the etiology of symptoms. patient-collected, low vaginal swabs may be as useful as provider-collected specimen in making a diagnosis for the patient with vaginal discharge. the purpose of bimanual examination is to evaluate for signs of pelvic inflammatory disease and is not necessary in the low-risk patient with vaginal discharge. treatment of asymptomatic bv or vaginal yeast is not necessary in the nonpregnant patient or usually is not needed to test or treat partners of patients with isolated yeast or bv. when infection is recurrent, particularly when a woman's male partner is uncircumcised, treatment of the male partner for carriage of either infection may be warranted. options for treatment of recurrent infections are presented in etable 16-1. the treatment of warts is destructive and may serve to stimulate an immune response to the hpv-infected cells, which are typically "above" the surveillance mechanisms of the immune system in the epidermis. office methods of treatment include cryotherapy and trichloroacetic acid or podophyllin resin application. patients may apply podofilox 0.5% solution or gel or imiquimod 5% cream (table 16-13) . for more extensive cases of warts or intra-anal or intravaginal infections that are difficult to treat using the previous methods, surgical techniques may be necessary to achieve resolution. untreated, warts may resolve spontaneously, remain the same, or worsen. patients with pediculosis pubis, or pubic lice, most often present with pruritus or with visible nits. pubic lice are visible on inspection of the pubic area, as are nits, which are adherent to the hair shaft. partners of patients with pubic lice should also be treated to prevent reinfection. linens and clothing should be laundered or dry-cleaned or kept in a closed plastic container or bag for 72 hours. scabies diagnosis can be challenging. again, patients present with itching that can be anywhere on the body, although often in the genital area or on the buttocks when infection is sexual in origin. the pruritus associated with sarcoptes scabiei is a result of sensitization to the mite droppings underneath the skin as the mite burrows. the classic "burrow" or linear papular eruption is not always present. scraping of lesions with microscopic examination may be performed to identify the mite. as with pediculosis, close contacts should be treated. linens and clothing should be laundered or dry-cleaned or isolated in plastic containers for 72 hours. the pruritus-associated with scabies can take several weeks to resolve after treatment. patients living in group settings (dormitories or apartments) may reinfect one another as a result of inadequate primary treatment of all contacts ( cryotherapy trichloroacetic acid (tca): small amount applied until wart whitens podophyllin resin, 10% to 25% all these may be repeated every 1 to 2 weeks until warts are resolved. podofilox 0.5% solution or gel applied twice daily for 3 days, followed by 4 days of no therapy. imiquimod 5% cream applied once daily at bedtime three times a week for up to 16 weeks; washed off 6 to 10 hours after application. urinary tract infection (uti) is defined as significant bacteriuria in the presence of symptoms. uti accounts for a significant number of emergency department visits; an estimated 20% of women experience a uti in their lifetime. the urinary tract is normally sterile. uncomplicated uti involves the urinary bladder in a host without underlying renal or neurologic disease. the bladder mucosa is invaded, most often by enteric coliform bacteria (e.g., e. coli) that ascend into the bladder via the urethra. sexual intercourse can promote this migration, and cystitis is common in otherwise healthy young women. frequent and complete voiding has been associated with a reduction in the incidence of uti. complicated uti occurs in the setting of underlying structural, medical, or neurologic disease. signs and symptoms of a uti include dysuria, frequency, urgency, nocturia, enuresis, incontinence, urethral pain, suprapubic pain, low back pain, and hematuria. fever is unusual. up to 30% of patients with symptoms of cystitis have a smoldering pyelonephritis, especially when symptoms have been present for more than 1 week. a patient with pyelonephritis usually appears ill, with fever, sweating, and prostration, along with costovertebral angle (flank) tenderness in most cases. the differential diagnosis of uncomplicated uti includes use of diuretics or caffeine, interstitial cystitis, vaginitis, pregnancy, pelvic mass, pid, and benign prostatic hypertrophy (bph). if a uti is suspected, the initial test of choice is urinalysis, although with classic signs and symptoms of infection in women, this test is not always necessary. pyuria, as indicated by a positive result on the leukocyte esterase dip test, is found in the majority of patients with uti. the presence of urinary nitrites is fairly specific for uti. the combination of positive leukocyte esterase and nitrites improves sensitivity. on urine microscopy, levels of pyuria as low as two to five leukocytes per high-power field (2-5 wbcs/hpf) in a centrifuged specimen are significant in the female patient with appropriate symptoms, as is the presence of bacteriuria. urine culture and sensitivity are not needed in simple utis. cultures should be done in patients with recurrent utis, patients with pyelonephritis, and pregnant patients. antibiotic therapy can be given in a 3-day regimen for young, sexually active women. a 7-to 10-day course of antibiotics should be used in pregnant patients and patients with complicated utis. all the drugs listed in table 16 -15 can be used in a 3-day or 7-to 10-day course. clinical practice guidelines that include telephone assessment and treatment have shown a decrease in unnecessary laboratory utilization while maintaining quality of care (saint et al., 1999) . trimethoprim-sulfamethoxazole (tmp-smx) has been a mainstay of uti therapy, but in some localities, resistance of e. coli to tmp-smx is 20% (mehnert-kay, 2005) . if a urine culture is done and the organism is resistant to the drug prescribed, a change in antibiotics is indicated only if the patient is still symptomatic. for symptomatic treatment, a bladder anesthetic can be used, such as phenazopyridine (pyridium), 200 mg three times daily for 2 days. patients should be warned that this produces an orange tinge in tears and urine. patients should also be instructed to increase fluid intake. pyelonephritis is suggested by a failure of a short course of antibiotics. signs and symptoms of pyelonephritis include shaking chills and fever higher than 38.5° c (101.3° f), flank pain, malaise, urinary frequency and burning, and costover-tebral angle tenderness. the infection can produce septic shock. a patient who is unable to tolerate oral intake should be hospitalized and given empiric iv antibiotics aimed at broad-spectrum gram-negative coverage, such as third-generation cephalosporins, fluoroquinolones, or aminoglycosides, while awaiting results of blood and urine cultures. a 14-day course of antibiotic therapy (iv or po) is recommended. although the most common bacterial infection during pregnancy, the incidence of uti in pregnancy is similar to that reported in sexually active nonpregnant women of childbearing age. up to 40% of pregnant women with tmp-smx, 160/800 mg q12h trimethoprim, 100 mg q12h fluoroquinolones ‡ ciprofloxacin, 100-250 mg q12h ciprofloxacin xr, 500 mg qd gatifloxacin, 200 mg qd levofloxacin, 250 mg qd nitrofurantoin monohydrate/macrocrystals, 100 mg q12h nitrofurantoin macrocrystals, 50-100 mg qid amoxicillin, 250 mg q8h or 500 mg q12h cephalexin, 250 mg q6h, or other cephalosporin consider 7-day regimen. amoxicillin, 250 mg q8h or 500 mg q12h nitrofurantoin monohydrate/macrocrystals, 100 mg q12h nitrofurantoin macrocrystals, 50-100 mg qid cephalexin, 250 mg q6h, or other cephalosporin tmp-smx, 160/800 mg q12h male gender, diabetes, symptoms for 7 days, recent antimicrobial use, age > 65 tmp-smx, § 160/800 mg q12h fluoroquinolones, as per 3-day regimens cephalexin, 250 mg q6h, or other cephalosporin consider 7-day regimen. from hooton tm, stamm we. diagnosis and treatment of uncomplicated urinary tract infection. infect dis north am 1997;11:551. tmp-smx, trimethoprim-sulfamethoxazole; qd, every day; q12h, every 12 hours; q6h, every 6 hours; q8h, every 8 hours; qid; four times daily. * treatments listed to be prescribed before etiologic agent is known (gram stain may help); therapy can be modified when cause is identified. † characteristic pathogens are escherichia coli (85%-90%) and staphylococcus saprophyticus (5%-15%); other organisms account for less than 5% of cases and include proteus mirabilis, klebsiella pneumoniae, and enterococcus spp. ‡fluoroquinolones should not be used in pregnancy. § although classified as pregnancy category c, tmp-smx is widely used; however, avoid its use in the first and second trimesters. untreated bacteriuria in the first trimester develop acute pyelonephritis later in pregnancy. premature births and perinatal mortality are increased in pregnancies complicated by uti. therefore, in pregnant women, asymptomatic bacteriuria should be actively sought and aggressively treated with at least one urinalysis, preferably toward the end of the first trimester. nitrofurantoin, ampicillin, and the cephalosporins have been used most extensively in pregnancy and are the regimens of choice for treating asymptomatic or minimally symptomatic uti. tmp-smx should be avoided in the first trimester because of possible teratogenic effects and should be avoided near term because of a possible role in the development of kernicterus. fluoroquinolones are avoided because of possible adverse effects on fetal cartilage development. for pregnant women with overt pyelonephritis, admission to the hospital for parenteral therapy should be the standard of care; beta-lactam agents with or without aminoglycosides are the cornerstone of therapy. prevention of uti, including pyelonephritis, can be accomplished during pregnancy with nitrofurantoin or cephalexin taken prophylactically after coitus or at bedtime without relation to coitus. such prophylaxis should be considered for patients who have had acute pyelonephritis during pregnancy, patients with bacteriuria during pregnancy who have had a recurrence after a course of treatment, and patients who had recurrent uti before pregnancy that required prophylaxis. catheter-associated utis are associated with increased mortality and costs. risk factors for catheter-associated utis include the duration of catheterization, lack of systemic antibiotic therapy, female gender, age older than 50 years, and azotemia. to help prevent infection, urinary catheters should be avoided when possible and used only as long as needed. the catheter should be inserted with strict aseptic technique by trained persons, and a closed system should be used at all times. treatment of catheter-associated uti depends on the clinical circumstances. symptomatic patients (e.g., those with fever, chills, dyspnea, and hypotension) require immediate antibiotic therapy along with removal and replacement of the urinary catheter if it has been in place for a week or longer. in an asymptomatic patient, therapy should be postponed until the catheter can be removed. patients with long-term indwelling catheters seldom become symptomatic unless the catheter is obstructed or is eroding through the bladder mucosa. in patients who do become symptomatic, appropriate antibiotics should be administered and the catheter changed. therapy for asymptomatic catheterized patients leads to the selection of increasingly antibiotic-resistant bacteria. recurrence of uncomplicated cystitis in reproductive-age women is common, and some form of preventive strategy is indicated if three or more symptomatic episodes occur in 1 year. however, risk factors specific to women with recurrent cystitis have received little study (sen, 2008) . several antimicrobial strategies are available, but before initiating therapy, the patient should try such simple interventions as voiding immediately after sexual intercourse and using a contraceptive method other than a diaphragm and spermicide. ingestion of cranberry juice has been shown to be effective in decreasing bacteriuria with pyuria, but not bacteriuria alone or symptomatic uti, in an elderly population. cranberry juice may be effective for preventing uti in young, otherwise healthy women. if simple nondrug measures are ineffective, continuous or postcoital-if the infections are temporally related to intercourse-low-dose antimicrobial prophylaxis with tmp-smx, a fluoroquinolone, or nitrofurantoin should be considered. typically, a prophylactic regimen is initially prescribed for 6 months and then discontinued. if the infections recur, the prophylactic program can be instituted for a longer period. an alternative approach to antimicrobial prophylaxis for women with less frequent recurrences (<4 a year) is to supply tmp-smx or a fluoroquinolone and allow the patient to self-medicate with short-course therapy at the first symptoms of infection. a minority of patients have relapsing uti, as evidenced by finding the same bacterial strain within 2 weeks after completion of antimicrobial therapy. two factors can contribute to the pathogenesis of relapsing infection in women: (1) deep tissue infection of the kidney that is suppressed but not eradicated by a 14-day course of antibiotics and (2) structural abnormality of the urinary tract, particularly calculi. patients with true relapsing utis should undergo renal ultrasound, intravenous pyelogram (ivp), or voiding cystourethrogram, and longer-term therapy should be considered. urinary tract infection is one of the most common infections of childhood. factors predisposing to uti include taking broad-spectrum antibiotics (e.g., amoxicillin, cephalexin), which are likely to alter gastrointestinal and periurethral flora; incomplete bladder emptying or infrequent voiding; voiding dysfunction; and constipation. uti in young children serves as a marker for abnormalities of the urinary tract. imaging of the urinary tract is recommended in every febrile infant or young child with a first uti to identify children with abnormalities that predispose to renal damage. imaging should consist of urinary tract ultrasonography to detect dilation of the renal parenchyma. voiding cystourethrography is often ordered but does not appear to improve clinical outcomes in uncomplicated utis (alper and curry, 2005) . a common complication of uti in men is prostatitis. bacterial prostatitis is usually caused by the same gram-negative bacilli that cause uti in female patients; 80% or more of such infections are caused by escherichia coli. the pathogenesis of this condition is poorly understood. antibacterial substances in prostatic secretions probably protect against such infections. a national institutes of health (nih) expert consensus panel has recommended classifying prostatitis into three syndromes: acute bacterial prostatitis, chronic bacterial prostatitis, and chronic pelvic pain syndrome (cpps). acute bacterial prostatitis is a febrile illness characterized by chills, dysuria, urinary frequency and urgency, and pain in the perineum, back, or pelvis. the bladder outlet can be obstructed. on physical examination, the prostate is found to be enlarged, tender, and indurated. pyuria is present, and urine cultures generally grow e. coli or another typical uropathogen. chronic bacterial prostatitis is a clinically more occult disease and may be manifested only as recurrent bacteriuria or variable low-grade fever with back or pelvic discomfort. urinary symptoms usually relate to the reintroduction of infection into the bladder, with both pyuria and bacteriuria. a chronic prostatic focus is the most common cause of recurrent uti in men. cpps is the diagnosis for the large group of men who present with minimal signs on physical examination but have a variety of irritative or obstructive voiding symptoms; perineal, pelvic, or back pain; and sexual dysfunction. these men can be divided into those with and those without inflammation (defined as >10 wbcs/hpf in expressed prostatic secretions). the etiology and appropriate management in these patients, regardless of inflammatory status, is unknown. • laboratory findings in acute tick-borne infection often include a normal or low wbc count, thrombocytopenia, hyponatremia, and elevated liver enzymes. • doxycycline is the drug of choice for patients with rmsf. • appropriate antibiotic treatment should be initiated immediately with strong suspicion of ehrlichiosis. • if left untreated, lyme disease can progress to cognitive disorders, sleep disturbance, fatigue, and personality changes. in the united states, more vector-borne diseases are transmitted by ticks than by any other agent. tick-borne diseases can result from infection with pathogens that include bacteria, rickettsiae, viruses, and protozoa. most tick-borne diseases are transmitted during the spring and summer months when ticks are active. a knowledge of which species of tick is endemic in an area can help narrow the diagnosis (table 16-16) . rocky mountain spotted fever (rmsf) is the most severe and most often reported rickettsial illness in the united states. it is caused by rickettsia rickettsii, a species of bacteria that is spread to humans by ixodid (hard) ticks (figure 16-6) . initial signs and symptoms include sudden onset of fever, headache, and muscle pain, followed by development of rash. the disease can be difficult to diagnose in the early stage. rmsf is most common among males and children. risk factors are frequent exposure to dogs and living near wooded areas or areas with high grass. the presentation of rsmf is nonspecific, following an incubation of about 5 to 10 days after a tick bite. initial symptoms can include fever, nausea, vomiting, severe headache, muscle pain, and lack of appetite. later signs and symptoms include rash, abdominal pain, joint pain, and diarrhea. the rash first appears 2 to 5 days after the onset of fever. most often it begins as small, flat, pink, nonitchy spots on the wrists, forearms, and ankles. the characteristic red spotted rash of rmsf is usually not seen until the sixth day or later after onset of symptoms. as many as 10% to 15% of patients never develop a rash (figure 16-7) . no widely available laboratory assay provides rapid confirmation of early rmsf, although commercial pcr testing is available. therefore, treatment decisions should be based on epidemiologic and clinical clues. treatment should never be delayed while waiting for confirmation by laboratory results. routine clinical laboratory findings suggestive of rmsf include normal wbc count, thrombocytopenia, hyponatremia, and elevated liver enzyme levels. serologic assays are the most often used methods for confirming cases of rmsf. doxycycline is the drug of choice for patients with rmsf. therapy is continued for at least 3 days after fever subsides and until there is unequivocal evidence of clinical improvement, generally for a minimum total course of 5 to 10 days. tetracyclines are usually not the preferred drug for use in pregnant women. whereas chloramphenicol is typically the preferred treatment for rmsf during pregnancy, care must be used when administering chloramphenicol late during the third trimester of pregnancy because of risks associated with gray baby syndrome. three species of ehrlichia in the united states are known to cause disease in humans. ehrlichia chaffeensis, the cause of human monocytic ehrlichiosis, occurs primarily in southeastern and south-central regions and is primarily transmitted by the lone star tick, amblyomma americanum ( figure 16-8) . human granulocytic ehrlichiosis is caused by anaplasma phagocytophila or anaplasma equi and is transmitted by ixodes ticks. ehrlichia ewingii is the most recently recognized human pathogen, with cases reported in immunocompromised patients in missouri, oklahoma, and tennessee. after an incubation period of about 5 to 10 days following the tick bite, initial symptoms generally include fever, pregnant women should be screened for asymptomatic bacteriuria in the first trimester of pregnancy (wadland and plante, 1989) (sor: a). pregnant women who have asymptomatic bacteriuria should be treated with antimicrobial therapy for 3 to 7 days (nicolle et al., 2005) (sor: b) . pyuria accompanying asymptomatic bacteriuria should not be treated with antimicrobial therapy (nicolle, 2003) (sor: c1). a 3-day course of tmp-smx (bactrim, septra) is recommended as empiric therapy of uncomplicated utis in women, in regions where the rate of resistant e. coli is less than 20% (warren et al., 1999) (sor: c). fluoroquinolones are not recommended as first-line treatment of uncomplicated utis, to preserve their effectiveness for complicated utis (warren et al., 1999) (sor: c). a randomized, placebo-controlled trial of 150 women over 12 months found that cranberry juice and cranberry extract tablets significantly decreased the number of patients having at least one symptomatic uti per year (stothers, 2002) appropriate antibiotic treatment should be initiated immediately when there is a strong suspicion of ehrlichiosis on the basis of clinical and epidemiologic findings. the treatment recommendations are the same as for rocky mountain spotted fever. rifampin has been used successfully in a limited number of pregnant women with documented ehrlichiosis. babesiosis is caused by hemoprotozoan parasites of the genus babesia. the white-footed deer mouse is the main reservoir in the united states, and the vector is ixodes ticks. most infections are probably asymptomatic. manifestations of disease include fever, chills, sweating, myalgias, fatigue, hepatosplenomegaly, and hemolytic anemia. symptoms typically occur after an incubation period of 1 to 4 weeks and can last several weeks. the disease is more severe in immunosuppressed, splenectomized, or elderly patients. diagnosis can be made by microscopic examination of thick and thin blood smears stained with giemsa, looking for the parasite in red blood cells (rbcs). options for treatment include clindamycin plus quinine or atovaquone plus azithromycin. lyme disease is caused by the spirochetal bacterium borrelia burgdorferi. ixodes ticks are responsible for transmitting lyme disease bacteria to humans. in the united states, lyme disease is mostly localized to states in the northeastern, mid-atlantic, and upper north-central regions, as well as northwestern california. lyme disease most often manifests with a characteristic bull's-eye rash (erythema migrans) accompanied by nonspecific symptoms such as fever, malaise, fatigue, headache, muscle aches, and joint aches (figure 16-9) . lyme disease spirochetes disseminate from the site of the tick bite, causing multiple (secondary) erythema migrans lesions. other manifestations of dissemination include lymphocytic meningitis, cranial neuropathy (especially facial nerve palsy), radiculoneuritis, migratory joint and muscle pains, myocarditis, and transient atrioventricular blocks of varying degree. if left untreated, the disease can progress to intermittent swelling and pain of one or a few joints (usually large weight-bearing joints such as the knee), cognitive disorders, sleep disturbance, fatigue, and personality changes. the diagnosis is based primarily on clinical findings, and it is often appropriate to treat patients with early disease solely on the basis of objective signs and a known exposure. serologic testing may provide valuable supportive diagnostic information in patients with endemic exposure and objective clinical findings that suggest later-stage disseminated lyme disease. treatment for 3 to 4 weeks with doxycycline or amoxicillin is generally effective in early disease. cefuroxime axetil or erythromycin can be used for persons allergic to penicillin or who cannot take tetracyclines. later disease, particularly with objective neurologic manifestations, can require treatment with intravenous ceftriaxone or penicillin for 4 weeks or more, depending on disease severity. tularemia is caused by francisella tularensis, one of the most infectious pathogenic bacteria known. most cases in the united states occur in south-central and western states. humans can become infected through diverse environmental exposures, including bites by infected arthropods; handling infectious animal tissues or fluids; direct contact with or ingestion of contaminated food, water, or soil; and inhalation of infective aerosols. inhaled f. tularensis causes pleuropneumonitis. some exposures contaminate the eye, resulting in ocular tularemia; penetrate broken skin, resulting in ulceroglandular or glandular disease; or cause oropharyngeal disease with cervical lymphadenitis. untreated, bacilli inoculated into skin or mucous membranes multiply, spread to regional lymph nodes, multiply further, and then can disseminate to organs throughout the body. the onset of tularemia is usually abrupt, with fever, headache, chills and rigors, generalized body aches, coryza, and sore throat. a dry or slightly productive cough and substernal pain or tightness often occur with or without objective signs of pneumonia. nausea, vomiting, and diarrhea can occur. sweats, fever, chills, progressive weakness, malaise, anorexia, and weight loss characterize continuing illness. rapid diagnostic testing for tularemia is not widely available. respiratory secretions and blood for culture should be collected in suspected patients and the laboratory alerted to the need for special diagnostic and safety procedures. streptomycin (1 g im bid for 10 days) is the drug of choice, and gentamicin is an acceptable alternative. tetracyclines and chloramphenicol can also be used. colorado tick fever is an acute viral infection transmitted by the bite of the dermacentor andersoni tick (figure 16-10) . the disease is limited to the western united states and is most prevalent from march to september. symptoms start about 3 to 6 days after the tick bite. fever continues for 3 days, stops, and then recurs 1 to 3 days later for another few days. other symptoms include excessive sweating, muscle aches, joint stiffness, headache, photophobia, nausea, vomiting, weakness, and an occasional faint rash. routine blood tests might show a low wbc count, mildly elevated liver function, and mildly elevated creatine phosphokinase (cpk). diagnosis is confirmed by testing blood for complement fixation immunofluorescent antibody staining to colorado tick virus. treatment is removal of the tick and treatment of symptoms. physicians should advise patients who walk or hike in tickinfested areas to tuck long pants into socks to protect the legs and wear shoes and long-sleeved shirts. ticks show up on white or light colors better than dark colors, making them easier to remove from clothing. if attached, ticks should be removed immediately by using a tweezers, pulling carefully and steadily. insect repellents such as deet, alone or in combination with permethrin, may be helpful. • most cases of cellulitis are caused by staphylococci or streptococci, but other causes should be considered by clinical situation. • physicians must rule out more ominous causes of skin inflammation, such as necrotizing fasciitis and pyomyositis, when considering cellulitis. • edema-associated cellulitis is best treated by mobilizing edema fluid. cellulitis is an acute, spreading inflammation of the derma and subcutaneous issue. patients complain of tenderness, warmth, swelling, and spreading erythema. in contrast to erysipelas, cellulitis usually lacks sharp demarcation at the border. factors that predispose to cellulitis include trauma, an underlying skin lesion (furuncle, ulcer), or a complication arising from a wound, ulcer, or dermatosis. occasionally, cellulitis results from a blood-borne infection that metastasizes to the skin. pain and erythema usually develop within several days and are often associated with malaise, fever, and chills. the area involved is often extensive, red, hot, and swollen. patchy involvement with skip lesions can be seen. regional lymphadenopathy is common, and bacteremia can occur. several clinical entities resemble cellulitis, including pyoderma gangrenosum, gout, and insect bites. necrotizing fasciitis and gas gangrene are surgical emergencies. given that the predominant organism involved in most cases of cellulitis is a grampositive coccus, clinical history and morphology on physical examination usually suffice in the diagnosis and treatment of cellulitis. a history of freshwater exposure may implicate aeromonas hydrophila as the causative organism; saltwater appropriate antibiotic therapy should be initiated immediately when there is suspicion of rocky mountain spotted fever, ehrlichiosis, or relapsing fever rather than waiting for laboratory confirmation (bratton and corey, 2005; spach et al., 1993) (sor: c). treatment with doxycycline (vibramycin) or tetracycline is recommended for rmsf, lyme disease, ehrlichiosis, and relapsing fever (bratton and corey, 2005; spach et al., 1993) (sor: c). recommended actions to prevent tick-borne disease include avoidance of tick-infested areas; wearing long pants and tucking the pant legs into socks; applying diethyltoluamide (deet) insect repellents; using bed nets when camping; and carefully inspecting oneself frequently while in an at-risk area (bratton and corey, 2005; spach et al., 1993) (sor: c). antibiotic prophylaxis is not routinely recommended for a tick bite to prevent lyme disease, unless the risk of infection is high (wormser et al., 2006) (sor: b). recommended treatment for suspected tularemia is streptomycin or gentamicin given empirically before evidence of laboratory confirmation (bratton and corey, 2005; spach et al., 1993) (sor: c). exposure suggests vibrio spp. cellulitis in a patient with liver disease and shellfish ingestion moves vibrio vulnificans to the top of the differential. patients with soft tissue infection should have blood drawn for laboratory testing if signs and symptoms of systemic toxicity are present (e.g., fever or hypothermia, tachycardia, hypotension). laboratory testing should include blood culture and drug susceptibility tests; wbc count with differential; and measurement of creatinine, bicarbonate, cpk, and crp levels. hospitalization should be considered for patients with hypotension or an elevated creatinine level, low serum bicarbonate level, elevated cpk level (i.e., 2-3 times upper limit of normal), marked left shift, or crp level greater than 13 mg/l (123.8 nmol/l). gram stain with culture and culture of needle aspiration or punch biopsy specimens should be performed to determine a definitive etiology, and a surgical consult should be considered for inspection, exploration, and drainage. findings that may signal potentially severe, deep, soft tissue infection and that may require emergent surgical evaluation include cutaneous hemorrhage, gas in the tissue, pain disproportionate to physical findings, rapid progression, skin anesthesia, skin sloughing, and violaceous bullae. radiologic studies may be helpful if abscess or osteomyelitis is a possibility. ultrasonography is helpful in detecting a subcutaneous collection of fluid. magnetic resonance imaging (mri) is also useful in differentiating cellulitis from necrotizing fasciitis. the diagnosis of necrotizing cellulitis is by direct surgical examination or by frozen pathology sections. empiric antibiotics for immunocompetent patients with cellulitis should be targeted toward gram-positive cocci (table 16-17) . broader coverage should be initiated for diabetic patients to include gram-positive aerobes, gram-negative aerobes, and anaerobes. patients who present with severe infection or whose infection is progressing despite empiric antibiotic therapy should be treated more aggressively; the treatment strategy should be based on results of appropriate gram stain, culture, and drug susceptibility analysis. in the case of staphylococcus aureus, the physician should assume that the organism is resistant, and agents effective against mrsa, such as vancomycin, linezolid (zyvox), or daptomycin (cubicin), should be used. the antibiotic may be switched from an intravenous drug to an oral drug when fever has subsided and the skin lesion begins to resolve, usually in 3 to 5 days. the total duration of therapy should be 7 to 14 days. longer duration may be required if the response is slow or is associated with abscess, tissue necrosis, or underlying skin processes (infected ulcers or wounds). treatment of cellulitis should include elevation and immobilization to decrease swelling. patients with interdigital dermatophytic infections should be treated with a concomitant topical antifungal applied once or twice daily. topical antifungals can also help reduce the risk of recurrence of the cellulitis. support stockings, good skin hygiene, and prompt treatment of tinea pedis helps with prevention of cellulitis in patients with peripheral edema, who are predisposed to recurrence. in patients who continue to have frequent episodes of cellulitis or erysipelas, prophylactic treatment with penicillin v, 250 mg or 500 mg orally twice daily, or erythromycin, 250 mg once or twice daily (for penicillin-allergic patients), may be indicated. • the majority of furuncles and carbuncles are caused by staphylococcus spp., increasingly, community-acquired methicillin-resistant s. aureus. • drainage of pus is of primary importance in treating skin and soft tissue infections. • culture of sstis is important in guiding antibiotic treatment when initial measures of drainage are not effective. • for recurrent boils, consider referral to infectious disease specialist, possibly to eradicate carriage state. furuncles, or boils, are infections of the skin and soft tissue usually associated with a hair follicle. carbuncles are an extension of this skin and soft tissue infection continuum and involve more of the surrounding and subcutaneous tissue. the broad category skin and soft tissue infections (sstis) is used to describe this continuum that includes furuncles and carbuncles. sstis are common in both healthy and immunocompromised patients and likely initiate with some breach of the skin integrity, such as irritation of hair follicles from friction or microscopic trauma to the skin. up to 74% of furuncles and carbuncles are caused by community-acquired methicillin-resistant staphylococcus aureus (ca-mrsa) (cdc, 2010). other potential causative organisms include nonresistant staphylococcus spp. and streptococcus spp. it has become increasingly important to obtain culture of a lesion to direct antibiotic coverage given the increase in ca-mrsa. there is no reliable historical or examination element that will distinguish a ca-mrsa from a methicillin-sensitive staphylococcal skin lesion. stereotypically, patients report ca-mrsa lesions starting like a spider bite. furuncles and carbuncles can occur anywhere on the body, although the axillae, groin, and buttocks are particularly common sites. in addition, practices that cause skin trauma (e.g., shaving, waxing) are often noted in patients with these sstis. fever and malaise are uncommon with milder lesions but become more frequent with the increasing scope of localized infection. of primary importance in the management of carbuncles and furuncles is facilitation of drainage of any purulent material. with smaller lesions, this may be accomplished by heat application by the patient at home. as lesions increase in size and fluctuance, surgical drainage is essential to facilitate resolution of an ssti. it is important to consider culture penicillin, given parenterally or orally depending on clinical severity, is the treatment of choice for erysipelas (sor: a). for cellulitis, a penicillinase-resistant semisynthetic penicillin (amoxicillin/clavulanate) or a first-generation cephalosporin should be selected, unless streptococci or staphylococci resistant to these agents are common in the community (sor: a). for suspected mrsa skin infections, oral treatment options include trimethoprim-sulfamethoxazole, clindamycin, and doxycycline of purulent material when performing incision and drainage in the event that the patient fails to improve and antibiotic coverage becomes necessary. cure rates of lesions with drainage alone exceed 90%. careful follow-up after drainage is essential to ensure clinical improvement; daily dressing changes in the office after surgical drainage is effective. the addition of postdrainage antibiotics has not shown much added benefit. to prevent the spread of infection to others who come into contact with the patient recovering from an ssti, an occlusive dressing to prevent leakage of lesion fluid and careful hygiene are indicated. there is no evidence that extensive cleaning of common spaces (e.g., locker rooms) prevents the spread of ssti-causing bacteria more than routine cleaning measures. towels and soiled clothing should be laundered in hot water, and any common equipment should be cleaned per manufacturer recommendations. when lesions do not respond to heat, or when lesions are larger yet not amenable to drainage, antibiotics may be used. reasonable first-line antibiotic coverage for nonfluctuant lesions may include dicloxacillin, first-or secondgeneration cephalosporins, macrolides, or clindamycin. in patients with suspected ca-mrsa, better choices include tmp-smx, tetracycline, or clindamycin. it is important to note that up to 50% of ca-mrsa species will be resistant to clindamycin, particularly if the patient has been treated with other antibiotics in the previous weeks to months . oral administration of these antibiotics is acceptable in the nontoxic patient. patient signs and symptoms that would warrant hospital admission include fever or hypothermia, tachycardia, or hypotension as signs of sepsis and lesions greater than 5 cm in size (table 16-18) . for patients with recurrent sstis, evaluation for the presence of nasal carriage with a nasal culture is indicated. the value of eradication of bacterial carriage is unclear. referral for infectious disease specialist evaluation may be indicated to guide decision making in the patient with recurrent furuncles and carbuncles. • the existence, severity, and extent of infection, as well as vascular status, neuropathy, and glycemic control, should be assessed in patients with a diabetic foot infection. • visible bone and palpable bone on probing suggest underlying osteomyelitis in patients with a diabetic foot infection. • before an infected wound of a diabetic foot infection is cultured, any overlying necrotic debris should be removed to eliminate surface contamination and to provide more accurate results. patients with diabetes are prone to skin ulcers caused by neuropathy, vascular insufficiency, and diminished neutrophil function. minor wounds can be secondarily infected, leading to ulcer formation. these ulcers often have extensive undermining with necrotic tissues and are often close to the anus, thus promoting an environment suitable for multiple species of microorganisms, including anaerobes. diabetic foot infections range in severity from superficial paronychia to deep infection involving bone. non-limb-threatening infections involve superficial ulcers with minimal cellulitis (<2 cm from portal of entry), no signs of systemic toxicity, and no significant ischemia in the limb. cure rates of fluctuant skin lesions with drainage alone is over 90%. postdrainage antibiotics do not significantly improve outcomes rajendran et al., 2007) (sor: a). trimethoprim-sulfamethoxazole (tmp-smx), clindamycin, and tetracycline are first-choice antibiotics when ca-mrsa is suspected. up to 50% of ca-mrsa species will be resistant to clindamycin, particularly in the patient previously treated with other antibiotics (sor: c). subcutaneous tissues, and prominent ischemia. infection in patients who have recently received antibiotics or who have deep, limb-threatening infection or chronic wounds are usually caused by a mixture of aerobic gram-positive, aerobic gram-negative (e.g., escherichia coli, proteus spp., klebsiella spp.), and anaerobic organisms (e.g., bacteroides, clostridium, peptococcus, and peptostreptococcus spp.) . surgery is necessary to unroof encrusted areas, and the wounds need to be examined and probed to determine the extent of the infection and check for bone involvement (dinh et al., 2008) . debridement or drainage should be promptly performed. deep wound cultures should be obtained if possible. if deep culture is not feasible, gram stain and culture from the curettage of the base of the ulcer or from purulent exudates may be needed to guide antibiotic therapy (figure 16-11) . plain radiography of the foot is indicated for detection of osteomyelitis, foreign bodies, and soft tissue gas. when plain radiography is negative but osteomyelitis is clinically suspected, radionuclide scan or mri should be performed. mri provides more accurate information regarding the extent of the infectious process. the presence of peripheral artery disease and neuropathy should be assessed. the antibiotic regimen should be based on meaningful bacteriologic data. however, the initial regimen for a previously untreated patient with non-limb-threatening infection should focus on s. aureus and streptococci. mild infections may be treated with dicloxacillin or cephalexin for 2 weeks. amoxicillin/clavulanate may be used if polymicrobial infection is suspected. if msra is suspected, oral treatment options include tmp-smx or doxycycline. for limb-threatening infections, broad-spectrum antibiotics are recommended for coverage of group b streptococci, other streptococci, enterobacteriaceae, anaerobic gram-positive cocci, and bacteroides spp. treatment regimens include ampicillin-sulbactam or ertapenem (invanz), clindamycin plus a third-generation cephalosporin, and clindamycin plus ciprofloxacin. intravenous vancomycin should be added if mrsa infection is suspected. ciprofloxacin as a single agent is not recommended. in addition to antibiotic treatment, good glycemic control should be obtained and open wounds gently packed with sterile gauze moistened with ¼-strength povidone-iodine (betadine) solution. edema should be reduced by bed rest, elevation, and diuretic therapy as indicated. for prevention of diabetic foot ulcers, all patients with diabetes should have an annual foot examination that includes assessment for anatomic deformities, skin breaks, nail disorders, loss of protection sensation, diminished arterial supply, and inappropriate footwear. • the use of prophylactic antibiotics may be necessary in the initial management of bite wounds, particularly if the bite is on the hand or face or from a cat. • first-generation cephalosporins (e.g., cephalexin) are not effective as monotherapy for bite wounds because of resistance issues. • avoid primary wound closure in the management of bite wounds. it is estimated that bites account for 800,000 medical visits annually in the united states, making up 1% of emergency department visits. bite wounds consist of lacerations, evulsions, punctures, and scratches. the microbiology of bite wounds is generally polymicrobial, with an array of potential bacteria from the environment, the victim's skin flora, and the biter's oral flora. dog bites account for approximately 80% of all animal bites requiring medical attention, in which 85% are provoked attacks. most dog bites occur on the distal extremities, but children tend to sustain facial bites. patients who present for medical attention are often concerned about the care of disfiguring wounds or the need for appropriate vaccination (i.e., tetanus, rabies). however, up to 30% of medically treated wounds may become infected. these wounds are often contaminated with multiple strains of aerobic and anaerobic bacteria. local signs of infection with erythema, edema, pain, and purulent drainage are common with animal bite wounds. although the most frequently isolated pathogen related to dog and cat bite wounds is pasteurella multocida, the array of potential organisms is much greater. anaerobes such as bacteroides tectum, prevotella spp., fusobacteria, and peptostreptococci can be isolated from animal bite wounds 75% of the time, mostly from wounds with abscess formation. capnocytophaga canimorsus has also been associated with fatal infection from fulminant sepsis in asplenic patients. wounds inflicted by cats are often scratches or tiny punctures located on the extremity and are likely to become infected and lead to abscess formation. in the united states, venomous snakes bite approximately 8000 people yearly. envenomation in such snakebites account for the majority of morbidity and mortality associated with such bites. however, infection of soft tissue structures may also occur as a result of oral flora from the snake, which tends to be fecal in nature because live prey usually defecate in the snake's mouth with their ingestion. human bites are not uncommon, especially in children. human bites have a higher complication and infection rate than do animal bites. human bite wounds most often affect the hand and fingers and in some cases may present as "love routine wound swabs and cultures of material from sinus tracts are unreliable and strongly discouraged in the management of diabetic foot infection (pellizzer et al., 2001; senneville et al., 2006) nips" to the breast and genital areas. self-inflicted bites often include wounds of the lip and tissues surrounding the nail, such as paronychia. also included in this are clenched-fist injuries or "fight bites," which result in small lacerations to the knuckles when striking a person in the mouth. normal human oral flora, rather than skin flora, is the source of most bacteria isolated from human bite wound cultures (viridans streptococci, eikenella corrodens). management of bite wounds is the same as for any other wound: good wound care in the form of adequate irrigation and debridement of nonviable tissue as needed (table 1619) . bite wounds in general do not require primary closure, but after adequate irrigation and debridement, wounds may be approximated and closed by delayed primary or secondary intention. an exception to this rule may include bite wounds to the face. general wound management measures such as tetanus toxoid administration should also be employed. bite wounds involving the hands should be evaluated by a hand surgeon, given the risk of adjacent tendon sheath, bone, or joint involvement and the dire consequences if such structures are involved. the transmission of rabies through the bites of domestic pets in the united states and developed countries is rare. in fact, the dog strain of rabies is considered eliminated in the u.s. dog population, and cat bites are often managed through observation of the animal, without the immediate need for rabies postexposure treatment (pet). however, wild mammal exposure, especially bat, skunk, or raccoon, often warrants pet, which involves thorough cleaning of the bite wound, ideally with povidone-iodine solution, along with rabies immune globulin given at the wound site and rabies vaccine given on days 0, 3, 7, and 14. bite wounds should be considered contaminated wounds from presentation, given the oral microbial flora of humans and animals, and most patients should probably receive antibiotics early. empiric antibiotics are used to eradicate oral flora inoculated from the mouth of the biter, whether human or animal, into the wound. all moderate to severe animal bite wounds, or wounds that have an associated crush injury or that are close to a bone or joint, should be considered contaminated with potential pathogens, and these patients should receive 3 to 5 days of "prophylactic" antimicrobial therapy. gram stains with culture of bite wounds are specific but not sensitive indicators of bacterial growth. nonetheless, gram stain can be used to help guide initial empiric antibiotic therapy. amoxicillin-clavulanic acid (amoxicillin-clavulanate; augmentin) or penicillin plus a penicillinase-resistant penicillin are normally first-line agents for empiric therapy directed at bite wounds. first-generation cephalosporins (e.g., cephalexin) are not effective as monotherapy because of resistance of some anaerobic bacteria and e. corrodens. a 5-to 10-day course of antibiotics is usually adequate for infections limited to the soft tissue, and a minimum of 3 weeks of therapy is required for infections involving joints or bones. close follow-up is required in all bites to ensure adequate healing. of special consideration in human bite wounds is the potential for spread of viral pathogens, most notably hepatitis b virus (hbv) and hiv, if the source person is positive. hbv exposure in this setting should be handled in the same manner as other exposures, with administration of hbig and hbv vaccination. with regard to hiv, cdc guidelines for managing nonoccupational hiv exposure recommend handling each case individually in consultation with an infectious diseases specialist. • the diagnosis of osteomyelitis is based on radiographic findings (plain radiograph or mri) showing bony destruction along with histologic analysis and culture results. • chronic osteomyelitis is not an emergency, and antibiotics can be safely withheld until an etiologic diagnosis is established. • diabetic foot infections require a careful evaluation to assess perfusion and vascular supply, and corrective measures should be undertaken to reestablish adequate perfusion if necessary. • in diabetic foot ulcers, if one can probe to bone, the patient most likely has osteomyelitis. • orthopedic hardware infections are best managed in conjunction with an infectious diseases specialist and orthopedic surgeon. osteomyelitis is defined as progressive, inflammation leading to destruction of the bone, usually secondary to an infectious agent. bacteria can enter bone through hematogenous seeding or a contiguous focus after trauma, implantation of a foreign device, or a local soft tissue infection. acute osteomyelitis is defined as infection that evolves over a few weeks. chronic osteomyelitis implies persistent infection of several weeks to months. hematogenous osteomyelitis occurs primarily in children within the metaphyses of long bones (tibia and femur) and vertebrae in adults. in addition to local signs of inflammation and infection, patients generally have various systemic signs, including fever, irritability, and lethargy. physical findings include tenderness over involved area and decreased range of motion in adjacent joints. chronic osteomyelitis usually occurs in adults, caused by an open injury to bone and surrounding soft tissue. erythema, drainage around area, and bone pain are usually present on physical examination. systemic symptoms occur less frequently. the diagnosis of osteomyelitis is based on the clinical picture and supporting laboratory and radiologic findings. leukocytosis and elevations in crp and esr may use of antibiotic prophylactic after bites of the hand reduces the incidence of infection (medeiros and saconato, 2005) (sor: b) . antibiotic prophylaxis after bites by humans reduces incidence of infection (sor: c). animal bite: ascertain the type of animal, whether the bite was provoked or unprovoked, and the situation/environment in which the bite occurred. if the species can be rabid, locate the animal for 10 days' observation or sacrifice. patient: obtain information on antimicrobial allergies, current medications, splenectomy, mastectomy, liver disease, and immunosuppression. record a diagram of the wound with the location, type, and depth of injury; range of motion; possibility of joint penetration; presence of edema or crush injury; nerve and tendon function; signs of infection; and odor of exudate. infected wounds should be cultured and a gram stain performed. anaerobic cultures should be obtained in the presence of abscesses, sepsis, serious cellulitis, devitalized tissue, or foul odor of the exudate. small tears and infected punctures should be cultured with a minitipped (nasopharyngeal) swab. copious amounts of normal saline should be used for irrigation. puncture wounds should be irrigated with a "high-pressure jet" from a 20-ml syringe and an 18-gauge needle or catheter tip. devitalized or necrotic tissue should be cautiously debrided. debris and foreign bodies should be removed. radiographs should be obtained if fracture or bone penetration is possible to provide a baseline for future osteomyelitis. wound closure may be necessary for selected, fresh, uninfected wounds, especially facial wounds, but primary wound closure is not usually indicated. wound edges should be approximated with adhesive strips in selected cases. prophylaxis: consider prophylaxis (1) for moderate to severe injury less than 8 hours old, especially if edema or crush injury is present; (2) if bone or joint penetration is possible; (3) for hand wounds; (4) for immunocompromised patients (including those with mastectomy, liver disease, or steroid therapy); (5) if the wound is adjacent to prosthetic joint; and (6) if the wound is in the genital area. coverage should include pasteurella multocida, staphylococcus aureus, and anaerobes. treatment: cover p. multocida, s. aureus, and anaerobes. use oral medication if the patient is seen early after a bite and only mild to moderate signs of infection are present. the following can be considered for cat or dog bites in adults: • first choice: amoxicillin/clavulanic acid, 875/125 mg bid or 500/125 mg tid with food. • penicillin allergy: no alternative treatment for animal bites has been established for penicillin-allergic patients. the following regimens can be considered for adults: 1. clindamycin (300 mg po qid) plus either levofloxacin (500 mg po daily) or trimethoprim-sulfamethoxazole (2 double-strength tablets po bid). 2. doxycycline, 100 mg po bid. 3. moxifloxacin, 400 mg po daily. 4. in the highly penicillin-allergic pregnant patient, macrolides have been used, but the wounds must be watched carefully. on emergency department discharge, a single starting dose of parenteral antibiotic, such as ertapenem (1 g), may be useful in selected cases. if hospitalization or closely monitored outpatient follow-up is required, intravenous agents should be used. current choices include ampicillin/sulbactam and cefoxitin. the rising incidence of community-acquired s. aureus isolates that are methicillin resistant and therefore resistant to the drugs recommended here emphasizes the importance of susceptibility-testing any s. aureus isolates. indications include fever, sepsis, spread of cellulitis, significant edema or crush injury, loss of function, a compromised host, and patient noncompliance. give tetanus booster (td; tetanus and diphtheria toxoids for adults) if original three-dose series has been given but none in the past 5 years. adults who have not received acellular pertussis vaccine (tdap), should be given this instead of td. give a primary series and tetanus immune globulin if the patient was never immunized. rabies vaccine (on days 0, 3, 7, 14, and 28) with hyperimmune globulin may be required, depending on the type of animal, ability to observe the animal, and locality. elevation may be required if any edema is present. lack of elevation is a common cause of therapeutic failure. be seen but can also be normal. blood cultures may be positive in up to half of children with acute osteomyelitis. if plain radiographs show bone destruction and inflammation; the diagnosis of osteomyelitis is confirmed. typical findings on plain-radiographs will include osteolysis, periosteal reaction, and sequestra (segments of necrotic bone separated from living bone by granulation tissue). findings seen on plain radiographs usually denote a process that has been ongoing for at least 2 weeks. bone scintigraphy (bone scan) is often performed on patients with suspected osteomyelitis; however, sensitivity is quite low, and a negative result can offer false reassurance to the physician, so its routine use is not recommended. if the plain-radiographs are negative but the suspicion for osteomyelitis is still high, an mri scan should be considered. once the diagnosis of osteomyelitis has been made, the next step is to obtain an etiologic diagnosis. histopathologic and microbiologic examination of bone is the "gold standard." cultures of sinus tracts are not reliable for identifying the causative organism. common causative bacteriologic organisms in neonates include staphylococcus aureus, group b streptococci, and escherichia coli. later in life, s. aureus is most common, and in elderly persons, gram-negative organisms such as pseudomonas aeruginosa and serratia spp. have increased incidence. empiric antibiotics are rarely required for chronic disease but are often necessary for acute osteomyelitis. ideally, surgical debridement of all necrotic tissue and inflammatory debris (pus) should be undertaken and multiple surgical cultures with bone histology samples obtained. antimicrobial therapy will be dictated by test results. generally, treatment is for 4 to 6 weeks. with the exception of the fluoroquinolone class of antibiotics, which achieve high serum levels with oral administration, bone antibiotic levels cannot exceed the minimum inhibitory concentration (mic) for the infecting organism; therefore, antibiotics must be given intravenously. this underscores the importance in obtaining a bacterial diagnosis so that the appropriate antibiotic can be used for the duration of treatment. acute osteomyelitis is usually readily curable; however, chronic osteomyelitis is generally more refractory to therapy and requires repeat debridement and antibiotic courses. patients with uncontrolled diabetes are at increased risk for development of osteomyelitis, especially in the presence of neuropathy or venous or arterial insufficiency. s. aureus and beta-hemolytic streptococci are the predominant organisms, although other gram-positive or gram-negative aerobic or anaerobic bacteria may also be seen. plain radiographs should be the initial test to evaluate for the presence of osteomyelitis, followed by mri if negative. if there is a draining sinus, the "probe to bone" test should be performed with a sterile probe; if bone is palpated, the diagnosis of osteomyelitis is highly likely. further evaluation of the diabetic patient should be to assess for vascular insufficiency with the use of ankle-brachial indices and transcutaneous oximetry. if significant compromise is found, arteriography followed by revascularization should be undertaken. surgical debridement is again the cornerstone of treatment, along with antibiotics directed toward the causative microorganism. infections secondary to orthopedic hardware devices have become common problems with the increasing incidence of hip, knee, and shoulder replacement surgeries. also, patients with traumatic injury resulting in a fracture often have hardware implanted to stabilize the bone. these patients present in one of the three following ways: 1. early: symptoms develop less than 3 months after surgery and have an acute presentation with pain, erythema, and warmth, usually caused by s. aureus and gram-negative bacilli. 2. delayed: symptoms develop 3 to 24 months after surgery, generally with subtle signs of infection, including implant loosening and persistent pain, and usually caused by less virulent organisms such as coagulasenegative staphylococci and propionibacterium acnes. 3. late: symptoms develop 24 months after surgery and are usually caused by hematogenous seeding from skin, dental, respiratory, and urinary infections. treatment requires debridement of the surrounding tissue and hardware removal, although this cannot always be done in patients with bone instability. it is recommended that treatment follow-up should occur at 24 hours and perhaps 48 hours for outpatients. reporting the incident to a local health department may be required. from goldstein ejc. bites. in mandell gl, bennett je, dolin rd (eds). mandell, douglas, and bennett's principles and practice of infectious diseases, 7th ed. philadelphia, churchill livingstone--elsevier, 2010. po, orally; bid, twice daily; tid, three times daily; qid, four times daily. of these infections be done in conjunction with an infectious diseases specialist working with the orthopedic surgeon. septic arthritis is defined as infection within the joint space of two bones. the major causative organisms include s. aureus and in the sexually promiscuous individual, neisseria gonorrhoeae. intravenous drug users are likely to develop septic arthritis within unusual joints (e.g., sternoclavicular, sacroiliac). rheumatoid arthritis, presence of joint prostheses, and steroid use are predisposing factors for development of septic arthritis. diagnosis is usually based on clinical presentation of a warm, swollen joint with limitation in range of motion. a joint aspiration should be completed and the synovial fluid sent for gram stain with culture, wbc count with differential, and crystal analysis to rule out gout and pseudogout. blood cultures should also be drawn before initiation of antibiotics. gonococcal arthritis usually presents as an acute arthritis involving one or more joints in a sexually active individual. two thirds of patients have dermatitis with one or multiple, usually asymptomatic, lesions that progress from macular to papular and finally vesicular or pustular. joint fluid, urethral, and rectal cultures should also be obtained. treatment is generally with a third-generation cephalosporin intravenously until improvement, followed by oral therapy to complete a 1-week course of therapy. treatment of nongonococcal arthritis requires proper draining of the infected joint. this is often done surgically, although repeat needle drainage may also be successful if the joint is easily accessible. treatment generally depends on the gram stain and includes a third-generation cephalosporin, with the addition of vancomycin if gram-positive cocci in clusters are seen. duration of therapy is 3 to 4 weeks. • a comprehensive history and physical examination with laboratory and radiologic evaluation are important in the workup for fever of unknown origin (fuo). • if routine information is unrevealing, more specific testing for fuo is undertaken based on the patient's age, travel history, and disease process to develop a differential diagnosis. • the serum ferritin level (often elevated with malignancy) and naproxen test (reduces fever with malignancy) may be helpful in determining an underlying malignant process. • initiation of empiric antibiotics should be done only in specific fuo situations to prevent skewing culture results, thus maximizing isolation of the causative organism. patients who have a persistent fever despite workup are generally classified as having a "fever of unknown origin" (fuo). in 1961, petersdorf and beeson described 100 patients with persistent fever, otherwise known as fever of unknown origin. they introduced the standard, classic definition of fuo: fever higher than 38.3° c (101° f) on several occasions, persisting without diagnosis for at least 3 weeks, with 1 week of investigational study in the hospital setting. with advancing technology, this definition has been revised to allow for more than two outpatient visits, or 3 days if investigation is in the hospital setting. most patients with fuo have chronic or subacute symptoms and can be safely evaluated in the outpatient setting, with a median time to diagnosis of 40 days. the differential diagnosis of fuo is quite broad and extensive. determining an etiologic diagnosis of an fuo depends on generating a differential diagnosis compatible with the patient's history and physical examination. the principal disease categories for fuo include infection (30% overall), neoplasms (18%), collagen vascular diseases (12%), and miscellaneous (14%) (box 16-5). because of this broad differential, a newer classification system divides fuo into four groups: classic, nosocomial, neutropenic, and hiv associated, which helps narrow the differential diagnosis. furthermore, classic fuo can be broken down into three subgroups: infants and children, elderly, and travelers. despite an extensive workup, the etiologic diagnosis usually remains elusive in 7% to 30% of patients (box 16-4) . the diagnostic workup of fuo should begin with a thorough history and physical examination, including documentation of the fever. the patient may provide a diary noting the date and time of fever. routine noninvasive investigations are recommended in all patients before diagnosing fuo (box 16-6). acute febrile illness is never called an fuo. the patient's medication profile is reviewed because numerous drugs can be the cause. if unrevealing, a workup is initiated based on the differential diagnosis for the patient's age, travel history, geographic location, and disease process. dukes criteria for infective endocarditis have 99% specificity in patients with fuo. when the initial investigations are not helpful in identifying a cause, imaging should be considered, such as computed tomography (ct) scans of the chest, abdomen, and pelvis; ct may reveal an abscess or suggest an underlying malignancy. an elevated serum ferritin level can suggest a neoplasm or myeloproliferative disorder and, if normal, greatly decreases the chance that the patient has an underlying malignancy. lower-extremity doppler ultrasound should be considered in the sedentary or obese patient to rule out deep venous thrombosis. a temporal artery biopsy should be considered in the elderly patient to rule out temporal arteritis. liver biopsy has a high diagnostic yield with minimal toxicity, whereas bone marrow cultures usually have a low yield and should be considered only in special situations. empiric therapy with antibiotics is rarely appropriate for the patient with fuo. a diagnosis is essential to guide treatment of osteomyelitis requires surgical debridement followed by a 4-to 6-week course of intravenous antibiotic therapy (sor: c). septic arthritis is usually caused by a gonococcus in a sexually active adult, and use of a third-generation cephalosporin is the mainstay of therapy (sor: a). nongonococcal arthritis should be treated with surgical debridement or repeated needle aspirations, with a third-generation cephalosporin and vancomycin if gram-positive cocci are seen (goldenberg, 1998) (sor: b). treatment, and use of antibiotics may delay determining a causative infectious agent. the naproxen test (naprosyn; 375 mg po every 12 hours for 3 days) is helpful in determining if the fever is secondary to infection or malignancy. a dramatic decrease in the patient's temperature during the test generally indicates a malignant focus, whereas minimal or no response indicates an infectious etiology. the prognosis of fuo depends on the etiologic category. undiagnosed fuo has a very favorable outcome. patients in whom diagnostic investigations fail to identify an etiology should be followed clinically with serial history reviews and physical examinations until the fever resolves or new diagnostic clues are found. connective tissue diseases are more prominent. infections: malaria, hepatitis, pneumonia/bronchitis, uti/pyelonephritis, dysentery, dengue fever, enteric fever, tb, rickettsial infection, acute human immunodeficiency virus (hiv) infection, amebic liver abscess. postoperative urinary and respiratory tract instrumentation; use of intravascular devices; drug therapy; immobilization. septic thrombophlebitis, pulmonary embolus, clostridium difficile colitis, drug fever. fungal: 40% susceptible to empiric antifungals, 5% will be resistant to empiric therapy. bacterial: 10% not responding to empiric antimicrobial therapy and usually with cryptic focus. unusual pathogens: 5% will be toxoplasmosis (toxoplasma gondii) reactivation, atypical mycobacterial, tb, fastidious pathogens (legionella, mycoplasma, chlamydophila). viral: 5% of causes (hsv, cmv, ebv, hhv-6, vzv, rsv, influenza, parainfluenza). other: 10% will be transplant related (e.g., gvhd) following stem cell transplant, 25% will be undefined. infections: mycobacterium avium complex (mac), pneumocystis carinii pneumonia (pcp), cytomegalovirus (cmv), histoplasmosis, viral (hcv, hbv, adenovirus, hsv esophagitis, vzv encephalitis), tb, other fungi, cerebral toxoplasmosis, disseminated cryptosporidiosis. neoplasms: lymphoma, kaposi's sarcoma. other: drug fever, castleman's disease. hsv, herpes simplex virus; ebv, epstein-barr virus; hhv, human herpesvirus; vzv, varicella-zoster virus; rsv, respiratory syncytial virus; gvhd, graft-versus-host disease; hcv, hepatitis c virus; hbv, hepatitis b virus. abscesses: hepatic, subhepatic, gallbladder, subphrenic, splenic, periappendiceal, perinephric, pelvic, and other sites. granulomatous: extrapulmonary and miliary tuberculosis, atypical mycobacterial infection, fungal infection. intravascular: catheter-related endocarditis, meningococcemia, gonococcemia, listeria, brucella, rat-bite fever, relapsing fever. viral, rickettsial, and chlamydial: infectious mononucleosis, cytomegalovirus, human immunodeficiency virus, hepatitis, q fever, psittacosis. parasitic: extraintestinal amebiasis, malaria, toxoplasmosis. collagen vascular diseases: rheumatic fever, systemic lupus erythematosus, rheumatoid arthritis (particularly still's disease), vasculitis (all types). granulomatous: sarcoidosis, granulomatous hepatitis, crohn's disease. tissue injury: pulmonary emboli, sickle cell disease, hemolytic anemia. familial mediterranean fever fabry's disease cyclic neutropenia intra-abdominal infections may either be uncomplicated (limited to the gut lumen, such as gastroenteritis or colitis) or complicated (extending through to the peritoneum) . the clinical presentation of complicated intra-abdominal infections can range from mild symptoms such as nausea, mild abdominal pain, and cramping to lifethreatening septic shock. clinical findings result from local or diffuse inflammation with or without abscess formation. fever and abdominal pain are typically present, with additional symptoms depending on the organ involved. elderly and immunocompromised patients present with atypical, usually milder symptoms. imaging studies form an important adjunct to diagnosis. management involves empiric antibiotic coverage for bowel flora-mainly streptococci, enterococci, enteric gram-negative rods, and anaerobes-as well as controlling the source of infection, usually through surgery. • spontaneous bacterial peritonitis usually occurs in the setting of ascites and chronic liver disease. • spontaneous bacterial peritonitis is a diagnosis of exclusion. • ascitic fluid culture yield improves with inoculation into blood culture bottles at bedside. spontaneous bacterial peritonitis (sbp) is a form of infectious peritonitis without a surgically correctable cause and is therefore a diagnosis of exclusion. the route of infection in sbp is usually not apparent and is often presumed to be hematogenous, lymphogenous, by transmural migration through an intact gut wall from the intestinal lumen, or in women, from the vagina via the fallopian tubes (levison and bush, 2010) . sbp occurs in the setting of ascites in most cases, and it is particularly common in patients with cirrhosis. in pediatric populations, those with postnecrotic cirrhosis or nephrotic syndrome are more often affected. in adults, almost 70% of patients who develop sbp have child-pugh class c liver disease, and 10% to 30% of hospitalized patients with cirrhosis and ascites have sbp (mowat and stanley, 2001) . sbp is almost always caused by a single organism, typically enteric gram-negative rods, most often e. coli, followed by klebsiella pneumoniae. gram-positive cocci account for about 25% of episodes of sbp, and streptococci are isolated most often. sbp caused by anaerobes is rare. growth of more than one organism should raise the suspicion of secondary peritonitis. signs and symptoms of sbp are subtle and require a high index of suspicion. fever greater than 100° f (38° c) is the most common presenting sign, occurring in 50% to 80% of cases. abdominal pain, nausea, vomiting, and diarrhea are usually present. peritoneal signs (abdominal tenderness or rebound tenderness) are common but may be absent in patients with ascites. in adults, mental status changes may also occur. sbp is often confused with acute appendicitis in children. in adults, sbp should be suspected in any patient with previously stable chronic liver disease who undergoes acute decompensation in clinical status. spontaneous bacterial peritonitis is diagnosed by analysis of ascitic fluid obtained by abdominal paracentesis. infection has been typically defined as an ascitic fluid wbc count higher than 250 cells/mm 3 , which is considered diagnostic even when the culture of the ascitic fluid is negative. in cases where bloody fluid is obtained ("traumatic paracentesis"), the wbc count should be corrected by 1 wbc per 250 rbcs/mm 3 . the use of bedside dipstick for leukocyte esterase has a high false-negative rate and is not recommended (nguyen-khac et al., 2008) . ascitic fluid culture yield can be increased by inoculating blood culture bottles with 10 ml of ascitic fluid at the bedside. blood cultures should also be obtained as part of the workup. after the diagnosis of peritonitis is established, secondary peritonitis should be ruled out. ct of the abdomen with oral and intravenous contrast can help direct the surgeon to a particular source of infection, as opposed to doing a full exploratory laparotomy. a high ascitic fluid total protein (>1 g/dl) or amylase level is suggestive of secondary peritonitis. the treatment of choice is generally a third-generation cephalosporin such as cefotaxime (2 g iv every 8-12 hours) or ceftriaxone (2 g iv once daily). patients who have an ascitic fluid wbc count higher than 250 cells/mm 3 should be given empiric intravenous antibiotics without delay. oral amoxicillin-clavulanic acid can be used for mild, uncomplicated cases (navasa et al., 1996) . duration of treatment varies diagnosis of fuo may be assisted by the dukes criteria for endocarditis, ct scan of the abdomen, nuclear scanning with a technetiumbased isotope, and liver biopsy (mourad et al., 2003) (sor: b) . routine bone marrow cultures are not recommended in the fuo workup (mourad et al., 2003) (sor: b) . empiric antibiotics should be initiated only in specific situations, to avoid skewing culture results and thus maximizing potential isolation of the causative organism (mourad et al., 2003) (sor: b). from 5 to 14 days depending on clinical response. patients usually respond to appropriate antibiotic therapy within 48 to 72 hours; otherwise, a repeat paracentesis should be performed. if the ascitic fluid wbc count does not decrease by more than 25%, alternative diagnoses should be considered. prophylaxis with a fluoroquinolone or trimethoprim-sulfamethoxazole should be considered, particularly in high-risk patients (garcia-tsao and lim, 2009 • bacterial meningitis is life threatening and requires urgent medical attention and treatment. • viral encephalitis should be treated with acyclovir until herpes simplex virus is ruled out. • most brain abscesses are caused by streptococci and staphylococcus aureus. • the cns infections most likely to be encountered in clinical practice include meningitis, encephalitis, and abscess. • all cns infections can be difficult to diagnose, and a high index of suspicion by the health care provider is sometimes indicated to ensure patient survival. • mri is the most sensitive neuroimaging test for encephalitis. • acyclovir should be started immediately and continued until hsv pcr testing is obtained. meningitis can be acute, subacute, or chronic. in otherwise healthy children, the three most common organisms causing acute bacterial meningitis are streptococcus pneumoniae, neisseria meningitidis, and haemophilus influenzae type b (hib). isolation of an organism other than these three organisms from the csf of a child older than 2 months always requires an explanation or evaluation for unusual host susceptibility. children with cochlear implants, asplenia, hiv infection, or csf leak from basilar skull or cribriform fracture are at greater risk for pneumococcal meningitis. deficiencies in terminal components of complement lead to greater risk for meningococcal infection (saez-llorens and mccracken, 2008) . in adults, the common etiologic agents of acute meningitis include s. pneumoniae, n. meningitidis, and listeria monocytogenes. patients with acute meningitis most often present with fever, headache, meningismus, and altered mental status. infants can present with nonspecific symptoms such as inconsolable crying, irritability, nausea, vomiting, and diarrhea. lethargy, anorexia, and grunting respirations indicate a critically ill infant. older children may complain of headache, vomiting, back pain, myalgia, and photophobia; may be confused or disoriented; and may verbalize specifically that the neck is stiff or sore. seizures are noted in up to 20% to 30% of children before hospital admission or early in the course of the illness. in contrast, patients with subacute or chronic meningitis may have the same symptoms with a much more gradual onset, lower fever, and associated lethargy and disability. mycobacterium tuberculosis, treponema pallidum (syphilis), borrelia burgdorferi (lyme disease), and fungi (e.g., cryptococcus neoformans, coccidioides spp.) are the most common agents (tunkel et al., 2010) . physical examination should look for papilledema, middle ear and sinus infections, petechiae (common with n. meningitidis), nuchal rigidity, and in infants, a bulging fontanel. blood cultures should be taken. a lumbar puncture (lp) for csf analysis should be done as soon as possible. a brain ct scan before lp is not necessary if the patient has no evidence of immunocompromise, cns disease, new seizure, papilledema, altered consciousness, or focal neurologic deficit, and if a subarachnoid hemorrhage is not suspected. if neuroimaging is necessary, blood cultures should be taken and antibiotics given before the study; a delay in administration of antibiotics leads to a worse outcome. csf should be sent for cell count, wbc differential, glucose, protein, and gram stain with culture. acid-fast bacilli stain and cryptococcal antigen may be obtained when indicated. empiric antibiotics for the initial treatment of bacterial meningitis are listed in table 16 -20, but these should be tailored to the isolated organisms whenever possible. adjunctive dexamethasone is recommended for children and infants with hib meningitis, but not if they have already received antibiotics. in adults, adjunctive dexamethasone is recommended for pneumococcal meningitis (tunkel et al., 2004) . close contacts of patients with n. meningitidis should receive rifampin, 20 mg/kg (not to exceed 600 mg) twice daily for 2 days, or ciprofloxacin, 500 mg as a single dose, or ceftriaxone, 250 mg im as a single dose. unimmunized persons exposed to h. influenzae meningitis should receive rifampin (turkel et al., 2010) . pregnant women should not receive rifampin or doxycycline. a repeat lp should be done if no clinical response is seen after 48 hours of appropriate antibiotic therapy, particularly for patients with resistant pneumococcal disease and those who received dexamethasone. neonates with gram-negative bacilli and patients with ventriculoperitoneal (vp) shunts require documentation of csf sterility. the duration of antimicrobial therapy is 7 days for patients with n. meningitidis or hib, 10 to 14 days for pneumococcal meningitis, and 14 to 21 days for streptococcus agalactiae. spontaneous bacterial peritonitis is treated with third-generation cephalosporins (cefotaxime or ceftriaxone), with ampicillin-sulbactam, fluoroquinolones, or carbapenems as alternative agents (solomkin et al., 2010) (sor: b) . patients with diffuse peritonitis should undergo an emergency surgical procedure as soon as possible, even if ongoing measures to restore physiologic stability need to be continued during the procedure (sor: b). viral meningitis viral meningitis manifests similar to bacterial meningitis, although its course is rarely aggressive. the diagnostic process and examination are similar to those for bacterial meningitis. viral meningitis is usually caused by enteroviruses, hsv, mumps virus, and hiv. along with the signs of meningitis, signs that suggest a viral etiology include genital lesions (hsv-2), diarrhea, or a maculopapular rash (enteroviruses). diagnosis is made by the history, examination, and csf results. early in the course, the csf might show predominantly neutrophils that can resemble bacterial meningitis. treatment is symptomatic. suppressive therapy should be offered to patients with recurrent hsv meningitis. although encephalitis can also be caused by bacteria and fungi, the great majority of cases are caused by viruses. herpes simplex accounts for 10% of cases. patients present with fever, acute decreased level of consciousness, and occasionally, seizures and language, memory, or behavior disturbances. mri is the most sensitive neuroimaging test for encephalitis and might show temporal lobe inflammation in early hsv encephalitis. csf studies and electroencephalography (eeg) are also recommended for all patients with encephalitis. herpes simplex pcr should be done, and acyclovir should be given immediately until hsv encephalitis is ruled out. during late summer and early fall, doxycycline should be considered to cover for tick-borne illnesses, and testing should include the mosquito-borne encephalitides such as west nile, st. louis, eastern equine, and western equine. treatment depends on the suspected etiologic agent but is generally supportive (tunkel et al., 2008) . a brain abscess is a focal, intracerebral infection that develops into a collection of pus surrounded by a well-vascularized capsule. although fungi and protozoa (particularly toxoplasma) can also cause brain abscesses, bacterial causes are much more common. streptococci are found in 70% of bacterial abscesses and are usually from oropharyngeal infection or infective endocarditis, whereas staphylococcus aureus accounts for 10% to 20% of isolates and is more often found after trauma. community-associated mrsa strains have been increasing. enteric gram-negative bacilli (e.g., e. coli; proteus, klebsiella, and pseudomonas spp.) are isolated in 23% to 33% of patients, often in patients with ear infection, septicemia, or immunocompromise and those who have had neurosurgical procedures. most clinical symptoms are caused by the size and location of the abscess rather than the systemic signs of an infection. headache is the most common complaint and may be accompanied by fever, mental status changes, evidence of increased intracranial pressure (nausea, vomiting, papilledema), or focal neurologic deficits. diagnosis is usually made by ct scan with iv contrast showing the characteristic hypodense center with a peripheral uniform ring enhancement, with or without a surrounding area of brain edema. mri is becoming the preferred imaging modality because of increased sensitivity, particularly for detecting satellite lesions. additional testing depends on risk factors and the likely underlying source of infection and may include blood cultures, chest imaging, testing for hiv and antibodies to toxoplasma, and transesophageal echogram. empiric therapy typically involves vancomycin, ceftriaxone, and metronidazole. optimal management also includes surgical drainage for most abscesses, both to find an etiologic microorganism and to improve chances of cure (turkel, 2010) . • most acute diarrheal illness is viral and can be managed symptomatically and with appropriate attention to hydration. • travelers' diarrhea is usually caused by diarrheogenic escherichia coli. • the infection in travelers' diarrhea is usually self-limited. • antibiotics may shorten the duration of diarrhea by 1 to 3 days. • the most common cause of antibiotic-associated diarrhea is clostridium difficile. • treatment of antibiotic-associated diarrhea involves discontinuing the offending agent, if possible. adjunctive dexamethasone is recommended for children and infants with h. influenzae type b meningitis, but not if they have already received antibiotics (tunkel et al., 2004) (sor: a). in adults, adjunctive dexamethasone is recommended for pneumococcal meningitis (tunkel et al., 2004) (sor: b). diarrhea is a common presenting complaint in the primary care physician's office. not all causes of diarrhea are infectious, and not all infectious causes of diarrhea require specific antibiotic therapy. diarrhea remains a major cause of morbidity and mortality, particularly for children in the developing world. diarrhea is an alteration of normal bowel function, characterized by an increase in the water content, volume, or frequency of stools. acute diarrhea is typically defined as present less than 14 days, and diarrhea is considered chronic when symptoms persist longer than 30 days (figure 16-12 ). infectious diarrhea seen in the primary care physician's office is most frequently caused by viruses. a number of viral agents can cause diarrheal illness (box 16-7). rotaviruses are the principal enteric pathogens in children less than 5 years of age and the most important cause of hospitalization and infant mortality related to diarrheal illnesses. noroviruses evaluate severity and duration obtain history and physical examination [1] [2] [3] [4] [5] treat dehydration report suspected outbreaks 6 check all that apply: 7 are the most common cause of food-borne disease worldwide. viral gastroenteritis is usually an acute self-limited illness, referred to as the "stomach flu." enteric viruses are easily spread by fecal-oral transmission, through contamination of food and water, fomites, and person-to-person spread. secondary attack rates can be high. nausea and vomiting are the most prominent symptoms of viral gastroenteritis. diarrhea, fever, headache, and constitutional symptoms may also be experienced. these viral infections can occur at any time during the year, but tend to occur more often in the winter. there is no specific therapy. treatment is supportive, with particular emphasis on adequate replacement of fluids and electrolytes. if rehydration can be accomplished enterally, it is preferred. both the pentavalent bovine-human reassortment (rv5) and the oral, live-attenuated monovalent (rv1) rotavirus vaccines are effective for prevention of severe gastroenteritis. the rv5 vaccine series is recommended for children at ages 2, 4, and 6 months, whereas the rv1 vaccine should be administered to children 2 and 4 months of age. approximately 40% of travelers to developing regions of the world will develop diarrhea. bacteria are responsible for approximately 80% of diarrhea acquired by travelers. other important causes include viruses and parasites. the onset of the majority of cases of travelers' diarrhea is usually within 5 to 15 days after arrival. the presentation is typically a noninflammatory, nonbloody diarrhea associated with abdominal discomfort, fever, nausea, or vomiting. the duration is usually 1 to 5 days. enterotoxigenic e. coli is responsible for approximately 30% of travelers' diarrhea. enteroaggregative e. coli is the second most common bacterial agent and causes 20% of cases. salmonella, shigella, and campylobacter spp. are less often detected but are important causes of dysentery, particularly in asia and africa. dysentery is severe inflammatory diarrhea manifested by fever and bloody stools. most cases of travelers' diarrhea are self-limited, but chronic postinfectious irritable bowel syndrome may occur in up to 10% of those who experience diarrhea. prevention of travelers' diarrhea is an important component of pretravel counseling for high-risk countries. food should be boiled, cooked, or peeled and water boiled to avoid consumption of fecal contamination. if a person develops travelers' diarrhea, a short course of antibiotics with rifaximin, ciprofloxacin, or azithromycin can shorten the duration of illness by 1 to 3 days. antibiotic therapy is recommended for persons with bloody diarrhea or fever. rifaximin, a nonabsorbed antibiotic, is not effective against invasive pathogens and should not be administered for dysentery. ciprofloxacin or azithromycin should be used for dysenteric symptoms based on local antimicrobial susceptibilities. antibiotics are frequently prescribed in the primary care physician's office for a variety of infections. unfortunately, antibiotics can alter the normal host microflora that can be protective against other infections. antibiotic effects on the normal gastrointestinal tract microbiome can lead to antibiotic-associated diarrhea, which causes significant morbidity and mortality. administration of antibiotics usually precedes symptoms of antibiotic-associated diarrhea by about 1 week but can be as distant as 2 or 3 months. strong associations with clindamycin (cleocin), cephalosporins, penicillins, and fluoroquinolones have been demonstrated, but any antibiotic can lead to antibiotic-associated diarrhea. the most important cause of antibiotic-associated diarrhea is clostridium difficile, an anaerobic, gram-positive, spore-forming rod. c. difficile is implicated as the cause in up to 25% of antibiotic-associated diarrhea cases, in 50% to 75% of antibiotic-associated colitis cases, and in more than 90% of antibiotic-associated pseudomembranous colitis cases. risk factors for c. difficile diarrhea include antibiotics, health care exposure (recent stay in hospitals or long-term care facilities), older age (>60), and comorbid conditions. the clinical presentation of c. difficile colitis is usually diarrhea, abdominal pain or cramping, and fever in a patient who recently received antibiotics. leukocytosis is common and may be profound; levels can be consistent with leukemoid reaction. a rare but potentially fatal complication is toxic megacolon. toxic megacolon manifests as acute colonic dilation to a diameter greater than 6 cm, associated with systemic toxicity and the absence of mechanical obstruction. with its high associated mortality, any patient who develops toxic megacolon requires immediate surgical evaluation for possible colectomy. diagnosis of c. difficile diarrhea is achieved by demonstration of c. difficile toxin a or b in the stool by enzyme immunoassay (eia) or cell culture cytotoxicity assay in a symptomatic patient with a previous history of antibiotic use. asymptomatic patients should not be tested. with the improved sensitivities of these diagnostic assays, one stool sample is usually sufficient to test for c. difficile, unless symptoms recur. test of cure after therapy with repeat stool for c. difficile toxin is not recommended because stools may remain positive for c. difficile toxin despite clinical resolution. endoscopy can demonstrate pseudomembranes in the colon. pseudomembranes are diagnostic of c. difficile infection, but are often not present. endoscopy may only reveal the presence of nonspecific colitis. clostridium difficile colitis is treated by discontinuing the offending agent(s) if possible and initiating antibiotic therapy (box 16-8). antimotility agents should be avoided. oral metronidazole (flagyl), 500 mg three times daily for 10 to 14 days, is recommended for mild-moderate c. difficile diarrhea. severe diarrhea should be treated with oral vancomycin. oral vancomycin is currently not recommended for all patients with c. difficile diarrhea because of concerns for the promotion of vancomycin-resistant enterococci (vre) and its expense. about 10% to 20% of patients experience relapse in travelers' diarrhea, in which enterotoxigenic e. coli or other bacterial pathogens are likely causes, prompt treatment with a fluoroquinolone, azithromycin, or rifaximin or, in children, azithromycin 10 mg/kg/day once daily can reduce the duration of an illness from 3 to 5 days to 1 to 2 days (dupont, 2010) (sor: a). after therapy. for relapse, a repeat course of the original c. difficile treatment should be administered. patients who have mild to moderate cases without volume depletion or systemic toxicity can be treated as outpatients. discussions of the following infections can be found online at www.expertconsult.com: • infectious viral hepatitis • endocarditis treat mild-moderate c. difficile diarrhea with metronidazole (zar et al., 2007) evidence-based reviews of the diagnosis and treatment of many common clinical problems. www.mdcalc.com/curb-65-severity-score-community-acquired-pneumonia curb-65 score calculator to determine the severity of communityacquired pneumonia and need for hospitalization. the complete reference list is available online at www.expertconsult.com. anthony zeimet hepatitis is defined as inflammation of the liver that is commonly induced by viruses that include the hepatitis viruses a through e, which will be the focus of this discussion. other viruses that can induce hepatitis include epstein-barr virus (ebv), cytomegalovirus (cmv), herpes simplex virus (hsv), varicella zoster virus (vzv), adenovirus, and coxsackievirus. various medications and alcohol abuse are two important nonviral causes. most infectious causes of hepatitis are self-limiting; however, hepatitis b and c viruses can cause a chronic infection that may lead to cirrhosis and eventual liver failure, as well as hepatocellular carcinoma. hepatitis a virus (hav) and hepatitis e virus (hev) are spread by the fecal-oral route and only cause an acute infection. hepatitis b, c, and d viruses (hbv, hcv, hdv) are spread through the blood and have an acute form of disease that sometimes can become chronic. the clinical presentation of hepatitis is clinically indistinguishable. asymptomatic infections are more common than symptomatic infection. symptoms generally include right upper quadrant (ruq) abdominal pain, anorexia, nausea, vomiting, diarrhea, dark-colored urine, pale stools, and generalized malaise; patients may notice a yellow hue to their skin or eyes. pruritus is common, caused by deposition of bilirubin in the skin. the physical examination generally reveals jaundice and sclera icterus in addition to ruq pain. hepatomegaly is seen in 85% and splenomegaly in 15% of patients with hepatitis. liver function tests reveal elevated levels of aspartate transaminase (ast), alanine transaminase (alt), and bilirubin, and to a lesser extent, alkaline phosphatase (alp). hepatitis a virus is the most common cause of viral hepatitis worldwide. poor hygiene practices in both the industrial and the developing world account for its prevalence. in the united states, hav is common among lower socioeconomic groups, daycare attendants and workers, men who have sex with men (msm), and illicit drug users. hepatitis a is often acquired by travelers to endemic areas. the incubation period is 15 to 45 days (mean, 30 days). hav is highly contagious, and peak fecal shedding generally occurs at the onset of illness in most infected patients. viremia averages 30 to 90 days. hav infection manifests as an acute, self-limited illness, with the prodromal symptoms lasting about a week before the onset of jaundice. jaundice generally resolves after 2 weeks, and most patients recover. fulminant hepatic failure is possible but extremely rare. diagnosis of acute hav infection is made by demonstration of anti-hav immunoglobulin m (igm) in the patient's serum. this may be negative if the patient presents early, and repeat testing may be necessary if hav is strongly suspected. anti-hav igg in the serum indicates remote infection or immunization (efig 16-1). treatment is primarily supportive, except in patients with fulminant liver failure, who may require a liver transplant. vaccination should be administered to all patients who are seronegative and to persons at increased risk for acquiring hav, including those about to travel to endemic areas, patients with chronic liver disease or receiving clotting factor concentrates, msm, hiv-positive patients, and illicit drug users. certain areas of the united states now require mandatory vaccination of children as well as those who work in the restaurant industry. the vaccine is safe and highly efficacious and is given as a two-dose series at 0 and at 6 to 12 months. passive immunization with immune globulin is recommended for those exposed to the virus by a known contact, including household and sexual contacts, and those who are traveling to an endemic area for less than 4 weeks but never vaccinated. any person who receives immune globulin should also start the vaccination series. hepatitis b virus infection can be acute or chronic. about 40,000 people die from acute hbv infection annually, and 500,000 die of cirrhosis and hepatocellular carcinoma caused by chronic infection. about 400 million people worldwide are living with chronic hbv infection. in the united states, an estimated 1.25 million residents have chronic hbv infection, with 4000 to 5500 deaths each year. significant burdens of disease are seen in asia, pacific islands, sub-saharan africa, amazon basin, and eastern europe. most adults with acute hbv will clear the virus, with less than 5% progressing to chronic infection. chronic infection will develop in almost all children infected perinatally and in 50% of those who become infected at 1 to 5 years of age. hbv is transmitted through exchange of body fluids, sexually and perinatally. in the united states, most hbv cases are acquired during adolescence and early adulthood with onset of sexual activity, experimentation with drug use, and sometimes occupational exposure. fever, polyarthralgia, rash, and a serum sickness-like illness are features of hbv infection in addition to jaundice and may be seen in association with polyarteritis nodosa. clinicians have the most difficulty in interpreting the various serologic tests for diagnosis of hepatitis b (etable 16-3) . the mean incubation period is 60 to 70 days, with a range of 30 of 180 days after infection. diagnosis of acute infection can be detected by obtaining hbv surface antigen (hbsag), which can appear as early as 1 week after exposure but generally by day 50. in a patient strongly suspected to have hbv infection, the clinician can consider checking the hbv dna viral load; which can be detected as early as 1 week after exposure. eventually the patient will develop an anti-hbv surface antibody, which indicates recovery from the illness. the other viral serologies for hbv are rarely obtained in acute illness. in chronic hbv infection, there are three major phases of infection: 1. immune tolerant. active viral replication in the liver with high levels of hbv dna levels but essentially normal or minimal elevation of ast and alt. most patients eventually progress to the next stage. 2. immune active. more robust liver inflammation with alt elevation, and liver biopsy shows inflammation with or without fibrosis. hbv early antigen (hbeag) is detected along with hbsag. 3. inactive carrier state. as patients enter this phase, they clear the hbeag and develop anti-hbe antibody and have undetectable or low levels of hbv dna, with normalization of alt and liver inflammation. if patients become hbsag negative, they then develop anti-hbs and have resolved their infection; otherwise, they are considered a chronic carrier. treatment of acute hbv is primarily supportive. in the last decade, however, there have been significant advances in the treatment of chronic hbv infection. the use of interferon has long been the mainstay of treatment and has a defined, limited course but is generally poorly tolerated. with the advent of the hiv/aids epidemic and research into treatment of hiv disease, antiviral medications are now starting to replace interferon as the preferred treatment option for hbv patients. nucleoside/nucleotide analogs such as lamivudine, adefovir, entecavir, tenofovir, and telbivudine are generally given for long-term, indefinite therapy to prevent progression of liver disease and development of hepatocellular carcinoma. any patient with chronic hbv infection should be referred to an infectious diseases specialist or a hepatologist to determine the appropriate treatment course. universal vaccination of newborns and infants is routine in the united states since 1991, and the incidence of hbv infection has declined. during primary care visits, the vaccination status of any adult or adolescent born before 1991 should be reviewed and the vaccine offered. the vaccine requires three doses given at 0, 1, and 6 months. an unvaccinated person or neonate who is exposed to the body fluids of a hbv-infected individual should start the vaccination series in addition to receiving the hepatitis b immune globulin (hbig). hepatitis c virus infection is the most common cause of chronic viral hepatitis in the united states. hcv does have an acute form of infection but is usually subclinical and rarely diagnosed. the cdc estimates that there are more than 2.7 million people with hcv infection. hcv is generally transmitted parenterally, as in injection drug users who share needles. before 1992, those who received a blood transfusion may have contracted hcv. sexual transmission acute hbv has been reported in monogamous couples, with one partner who has hcv infection and the other without infection who eventually acquires the virus. this occurs in 3% to 5% of couples and represents a rare mode of transmission. because the most common mode of acquisition is sharing needles, any patient who is hcv positive should be screened for hiv because these two infections often occur together (ebox 16-1). the diagnosis of acute hcv infection can be made by obtaining a hcv rna viral load; although this is rarely done because the initial infection is subclinical. chronic disease is generally discovered by a positive anti-hcv antibody along with an elevated hcv rna viral load. hcv genotype should also be obtained in any positive individual, because this has important prognostic factors with regard to therapy, with genotype 1a and 1b the predominant type in the united states and unfortunately having a poor response to therapy. as with hbv, chronic hcv infection can lead to cirrhosis and the development of hepatocellular carcinoma. treatment consists of 24 to 48 weeks of interferon and ribavirin therapy. any patient being considered for therapy should be referred to an infectious diseases specialist or hepatologist. a liver biopsy is often needed to determine appropriate treatment candidates. also known as the hepatitis delta antigen virus, hdv is a defective virus that requires the presence of hbv to be infectious. hdv should be suspected in any patient with chronic hbv who develops acute hepatitis. hepatitis d is endemic in the mediterranean, balkans, africa, middle east, and amazon basin. diagnosis is made through an anti-hdv antibody in the presence of someone with positive hbsag or anti-hb core antibody igm or igg. treatment is supportive. any person vaccinated against hbv cannot become infected with hdv. similar to hav infection, hev is spread by the fecal-oral route. hev only has an acute form and does not progress to chronic infection. most reported epidemics have been related to consumption of contaminated drinking water. hev is endemic to southeast and central asia, north africa, middle east, mexico, brazil, venezuela, and cuba. hepatitis e can be considered a cause of infectious hepatitis in the united states in the traveler returning from an endemic area. the incubation period is 40 days. infection is of major concern during pregnancy, which can cause death in late pregnancy. diagnosis is made by demonstration of anti-hev antibody in serum. treatment is supportive. • endocarditis prophylaxis is now recommended solely for patients at high risk of a complicated course with a more narrow range of cardiac conditions. • routine prophylaxis for gi and gu procedures is no longer recommended. • the duke criteria represent a reliable scoring system for diagnosing endocarditis. • echocardiography is indicated to confirm suspected endocarditis. bacterial endocarditis is one of the most feared infections; although uncommon, it carries high morbidity and mortality. increase in antibiotic resistance among bacteria causing this infection has created challenges for effective treatment. the fundamental view of the american heart association (aha) in preventing infective endocarditis has shifted in recent years. views on pathophysiology have not changed substantially, but it is now recognized ebox 16-1 persons for whom hepatitis c virus (hcv) screening is recommended persons who have injected illicit drugs in the recent and remote past, including those who injected only once and do not consider themselves to be drug users. persons with conditions associated with a high prevalence of hcv infection, including: persons with human immunodeficiency virus (hiv) infection persons with hemophilia who received clotting factor concentrates before 1987 persons who have ever received hemodialysis persons with unexplained abnormal transaminase (aminotransferase) levels prior recipients of transfusions or organ transplants before july 1992, including: persons who were notified that they had received blood from a donor who later tested positive for hcv infection persons who received a transfusion of blood or blood products persons who received an organ transplant children born to hcv-infected mothers health care, emergency medical, and public safety workers after a needle stick injury or mucosal exposure to hcv-positive blood current sexual partners of hcv-infected persons * modified from centers for disease control and prevention. recommendations for prevention and control of hepatitis c virus (hcv) infection and hcv-related chronic disease. mmwr 1998;47(rr):1-39. *although the prevalence of infection is low, a negative test in the partner provides reassurance, making testing of sexual partners of benefit in clinical practice. • universal vaccination of infants with hepatitis b vaccine reduces the risk of acute hepatitis, chronic carrier state, and complications of chronic infection and may be more effective than selective vaccination of high-risk individuals (lee et al., 2006) (sor: a). • as part of a comprehensive health evaluation, all persons should be screened for behaviors that place them at high risk for hepatitis c infection (ghany et al., 2009 ) (sor: b). • liver biopsy may be considered in patients with chronic hcv infection to determine fibrosis stage for prognostic purposes or to make a treatment decision (ghany et al., 2009 ) (sor: b). that cumulative daily episodes of bacteremia likely carry more risk than the transient bacteremia caused by dental procedures. infective endocarditis likely begins with turbulent flow and damaged endothelium around heart valves, which allow platelet aggregation and thrombus formation, causing a "nonbacterial thrombotic endocarditis" (wilson et al., 2007) . the presence of bacteremia then allows this vegetation to become seeded with infection. bacterial "adhesins" are present to a greater degree in some species and allow for more effective attachment to the injured area of endothelium. with high concentrations of bacteria in the mouth, vagina, gi tract, and perhaps gu system, antibiotic prophylaxis was initiated when these anatomic locations were manipulated. recommendations for infective endocarditis prevention changed in 2007-2008, with aha recognizing more likely benefit from providing adequate population-based dental care and good oral hygiene, and thus less significant ongoing bacteremia at home in brushing, flossing, and "toothpicking," than in providing antibiotic prophylaxis to patients undergoing a dental procedure. no prospective rct has shown that dental prophylaxis prevents infective endocarditis. with recognition of the risk associated with administration of antibiotics (gi upset, diarrhea, rash, anaphylaxis) and the risk of contributing to increasing antibiotic resistance, versus the likely negligible benefit, aha has substantially changed its advice on this long-held practice. a preexisting cardiac condition produces a predisposition to the development of infective endocarditis (ebox 16-2). for example, those who have valve replacement for infection of an infected native valve carry a lifetime risk of 630 per 100,000 patient-years. the risk in the general population without known heart disease is 5 per 100,000 patient-years. more concerning, however, is the risk to a given patient of poor outcome if the patient develops endocarditis, which drives current aha recommendations. those with an infected mechanical valve have a mortality rate of about 20%, versus 5% or less for patients with an infected native valve (wilson et al., 2007) . a summary of current recommendations for endocarditis prophylaxis is provided in etable 16-4. of note, gi and gu procedures have been removed from those for which antibiotics are recommended, unless those systems are actively infected at the time of the procedure. the same is true for skin and soft tissue procedures, in that only infected tissue would warrant antibiotics to prevent infective endocarditis. it is still recommended to provide prophylaxis for respiratory tract procedures, if the respiratory wall will be invaded through biopsy or the procedure. in addition, respiratory procedures to treat infections (e.g., empyema) should be combined with antibiotic administration (nishimura et al., 2008) . antibiotic regimens for prophylaxis for dental procedures are still based primarily on synthetic penicillins as their cornerstone. this is with recognition that streptococcus viridans is both a mouth floral inhabitant and a common agent causing infective endocarditis. with other procedures, antibiotics should be targeted to bacterial pathogens causing any active infection in the system being manipulated. ebox 16-2 cardiac conditions associated with the highest risk of adverse outcome from endocarditis for which prophylaxis with dental procedures is reasonable prosthetic cardiac valve or prosthetic material used for cardiac valve repair previous ie congenital heart disease (chd) * unrepaired cyanotic chd, including palliative shunts and conduits completely repaired congenital heart defect with prosthetic material or device, whether placed by surgery or by catheter intervention, during the first 6 months of the procedure † repaired congenital heart defect with residual defects at the site or adjacent to the site of a prosthetic patch or prosthetic device (which inhibit endothelialization) cardiac transplantation recipients who develop cardiac valvulopathy *except for the conditions listed above, antibiotic prophylaxis is no longer recommended for any other form of chd. †prophylaxis is reasonable because endothelialization of prosthetic material occurs within 6 months after the procedure oral antibiotic william osler discussed "malignant endocarditis" in 1885 and its great diagnostic challenge. in 2009 the modified duke criteria remains a reliable tool for assessing patients with endocarditis. endocarditis is suspected in febrile patients without an obvious source, in those with recent bacteremia (including iv drug use), in those with underlying cardiac predisposition, and perhaps in patients with the clinical finding of a new cardiac murmur. in establishing a diagnosis of infective endocarditis, a patient is considered to have definite disease if two major or one major and three minor or five minor criteria are present. possible disease is defined as one major and one minor or three minor criteria (ebox 16-3). pathologic specimens showing changes consistent with endocarditis would make a definitive diagnosis. echocardiography is indicated in making the diagnosis of infective endocarditis. transthoracic echocardiography (tte) is helpful if vegetations are seen, although size of the patient and other disease (e.g., copd) may limit the ability of tte to view the cardiac valves adequately. if tte is negative and suspicion remains, transesophageal echocardiography (tee) is indicated. tte may be more widely available, depending on regional and institutional variation, and should be used rather than delaying this diagnostic test. bacteria present within valvular vegetations are often less metabolically active, which partly explains the requirement for longer courses of antibiotics for this type of infection. clearly, therapy for endocarditis should be targeted at the organism identified on blood culture, if any. the counting of antibiotic days should begin when the blood culture becomes negative and not at the start of the particular agent. recommendations for antibiotic use in infectious endocarditis are highly variable and based on the presence or absence of synthetic valvular material and the infectious agent (etable 16-5). generally speaking, a minimum of 4 weeks of iv antibiotics is indicated. in cases of resistant organisms, up to 8 weeks may be required. in either case, synergistic use of agents such as gentamicin may be indicated for the first several weeks of treatment, which then can be discontinued. the ability of a given patient to complete this course at home versus in a health care facility is dependent on the dosing frequency of the antibiotic, availability of inhome nursing services, and the type of intravenous access through which the antibiotic will be delivered. at the completion of endocarditis therapy, echocardiography should be repeated to re-assess the function of the valve(s) in question. valvular dysfunction at the completion of therapy is a good indication that the patient will need valve replacement in the future. there are circumstances, like the development of congestive heart failure in the face of endocarditis, in which primary surgery is indicated. typical microorganisms consistent with ie from 2 separate blood cultures: viridans streptococci, streptococcus bovis, hacek group, staphylococcus aureus; or community-acquired enterococci in the absence of a primary focus; or microorganisms consistent with ie from persistently positive blood cultures, defined as follows: at least 2 positive cultures of blood samples drawn >12 hours apart; or all of 3 or a majority of ≥4 separate cultures of blood (with first and last sample drawn at least 1 hour apart). single positive blood culture for coxiella burnetii or anti-phase 1 igg antibody titer >1:800 echocardiogram positive for ie (tee recommended for patients with prosthetic valves, rated at least "possible ie" by clinical criteria, or complicated ie [paravalvular abscess]; tte as first test in other patients) defined as follows: oscillating intracardiac mass on valve or supporting structures, in the path of regurgitant jets, or on implanted material in the absence of an alternative anatomic explanation; or abscess; or new partial dehiscence of prosthetic valve; new valvular regurgitation (worsening or changing or preexisting murmur not sufficient) predisposition, predisposing heart condition, or idu fever, temperature >38° c vascular phenomena, major arterial emboli, septic pulmonary infarcts, mycotic aneurysm, intracranial hemorrhage, conjunctival hemorrhages, and janeway's lesions immunologic phenomena: glomerulonephritis, osler's nodes, roth's spots, and rheumatoid factor microbiologic evidence: positive blood culture but does not meet a major criterion as noted above * or serological evidence of active infection with organism consistent with ie echocardiographic minor criteria eliminated echocardiography should be performed in all patients with suspected infective endocarditis (baddour et al., 2005) there is no evidence that antibiotic prophylaxis is effective or ineffective for preventing infectious endocarditis after dental procedures in patients at risk (chung, 2009 ) (sor: c). regimen dosage * and route duration (wk) chronic cough due to acute bronchitis: accp evidencebased clinical practice guidelines interim recommendations for the use of influenza antiviral medications in the setting of oseltamivir resistance among circulating influenza a (h1n1) viruses, 2008-09 influenza season national ambulatory medical care survey: 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disease society of america acute meningitis guidelines on acute infectious diarrhea in adults. the practice parameters committee of the american college of gastroenterology a comparison of vancomycin and metronidazole for the treatment of clostridium difficileassociated diarrhea, stratified by disease severity acute cholecystitis nett's principles and practices of infectious diseases diagnosis and management of complicated intra-abdominal infection in adults and children: guidelines by the surgical infection society and the infectious diseases society of america infectious viral hepatitis national institutes of health consensus development conference statement: management of hepatitis b acute viral hepatitis hepatitis b virus infection chronic viral hepatitis chronic hepatitis diagnosis, management and treatment of hepatitis c: an update acute viral hepatitis hepatitis c virus infection effect of hepatitis b immunisation in newborn infants of mothers positive for hepatitis b surface antigen: systematic review and meta-analysis chronic hepatitis b: update infective endocarditis: diagnosis, antimicrobial therapy, and complications prescription of antibiotics for prophylaxis to prevent bacterial endocarditis experience with a oncedaily aminoglycoside program administered to 2184 adult patients acc/aha 2008 guideline update on valvular heart disease: focused update on infective endocarditis prevention of infective endocarditis: guidelines from the american health association. circulation key: cord-258336-zs04l3s0 authors: leotte, jaqueline; trombetta, hygor; faggion, heloisa z.; almeida, bernardo m.; nogueira, meri b.; vidal, luine r.; raboni, sonia m. title: impact and seasonality of human rhinovirus infection in hospitalized patients for two consecutive years date: 2017-06-30 journal: jornal de pediatria doi: 10.1016/j.jped.2016.07.004 sha: doc_id: 258336 cord_uid: zs04l3s0 abstract objectives to report epidemiological features, clinical characteristics, and outcomes of human rhinovirus (hrv) infections in comparison with other community acquired respiratory virus (crv) infections in patients hospitalized for two consecutive years. methods this was a cross-sectional study. clinical, epidemiological, and laboratory data of patients hospitalized with acute respiratory syndrome in a tertiary care hospital from 2012 to 2013 were reviewed. results hrv was the most common crv observed (36%, 162/444) and was present in the majority of viral co-detections (69%, 88/128), mainly in association with human enterovirus (45%). most hrv-infected patients were younger than 2 years (57%). overall, patients infected with hrv had a lower frequency of severe acute respiratory infection than those infected with other crvs (60% and 84%, respectively, p =0.006), but had more comorbidities (40% and 27%, respectively; p =0.043). however, in the adjusted analysis this association was not significant. the mortality rate within the hrv group was 3%. detection of hrv was more prevalent during autumn and winter, with a moderately negative correlation between viral infection frequency and temperature (r =−0.636, p <0.001) but no correlation with rainfall (r =−0.036, p =0.866). conclusion hrv is usually detected in hospitalized children with respiratory infections and is often present in viral co-detections. comorbidities are closely associated with hrv infections. these infections show seasonal variation, with predominance during colder seasons. human rhinoviruses (hrvs) belong to picornaviridae family, genus enterovirus, and are divided in three species (hrv-a, hrv-b, and hrv-c) with about 100 serotypes within these species. 1, 2 the development of highly-sensitive molecular techniques for characterization of the hrv genome has recently allowed recognition of the hrv-c species. 3 there is already evidence that this new species may be more virulent and more strongly associated with lower respiratory tract infections than hrv-a and hrv-b. 4 hrv is the most common cause of upper respiratory tract infections, being responsible for at least 50% of cases of the common cold. this leads to considerable economic burden in terms of medical visits and both school and work absenteeism. 2, 4 hrvs have also been linked to lower airway effects that result in significant morbidity and mortality, 1 such as exacerbations of chronic pulmonary disease, severe bronchiolitis in infants and children, as well as fatal pneumonia in elderly and immunocompromised adults. 4, 5 in general, hrv infections occur during spring and autumn, 6 and manifest differently depending on whether the lower or upper respiratory tract is infected. infections of the upper respiratory tract ordinarily include symptoms of the common cold, but can present as acute otitis media or rhinosinusitis. on the other hand, infections of the lower respiratory tract can cause severe symptoms and result in bronchiolitis and pneumonia. 4 in brazil, since the influenza a pandemic of 2009, referral hospitals have been conducting active surveillance to detect respiratory viruses. such surveillance includes notification and laboratory investigation of cases meeting the diagnostic criteria of severe acute respiratory infection (sari). this viral respiratory infection monitoring has resulted in important information about the circulation of other community-acquired respiratory viruses (crv). the present study reports the epidemiological features, clinical characteristics, and outcomes of hrv infections in comparison with other crv infections in patients hospitalized in a referral hospital in southern brazil, for two consecutive years. patients hospitalized at an academic tertiary care center in southern brazil from whom respiratory samples were collected and sent for investigation, or who were diagnosed with sari in 2012 or 2013, were included in the study. respiratory samples (nasal swab or nasopharyngeal aspirate, or bronchoalveolar lavage) were collected in different periods of hospitalization according to medical recommendation. individuals with more than one sample collected during the same symptomatic period were considered as a single case and only the first result was evaluated, so that the number of respiratory viral detections was not overestimated. the medical records and influenza notification forms of patients with detectable respiratory virus were reviewed, focusing on epidemiology, clinical manifestation, outcome, laboratory findings, and diagnosis of sari. sari was defined as a flu-like syndrome with signs of severity (dyspnea or oxygen saturation below 95%). during the study, a total of 1002 cases were identified in both databases. of these, five cases were excluded because the researchers could not access the medical records and a further 242 were excluded as no samples had been sent for virus detection. thus, 755 patients with respiratory samples investigated for respiratory virus detection were included. the institutional ethics review board approved the study (no. #18714013.4.0000.0096). rvs were detected using a multiplex reverse transcription polymerase chain reaction (rt-pcr) technique. the viral genome was extracted using a high pure viral rna kit (roche inc., mannheim, germany) in accordance with the manufacturer's instructions. first strand cdna synthesis was achieved using random primers and an improm-ii reverse transcription system (promega inc., wi, usa). the resulting cdna was then subject to pcr by using a seeplex ® rv15 ace detection kit (seegene inc., korea), in accordance with the manufacturer's protocol. this multiplex pcr technology enables a simultaneous detection of 15 respiratory viruses: human adenovirus (adv), human metapneumovirus (mpv), parainfluenza virus type 1, 2, 3, and 4 (piv-1, piv-2, piv-3, and piv-4), influenza a (flua), influenza b (flub), respiratory syncytial virus type a and b (rsv-a, rsv-b), human rhinovirus types a/b/c (hrv), human enterovirus (ev), human bocavirus (bov), and human coronavirus (cov) types 229e/nl63 (alpha coronaviruses) and oc43/hku1 (beta coronaviruses). data were compiled using jmp version 5.2.1 (sas institute inc., cary, nc, usa) and analyzed using graphpad prism version 5.03 (graphpad software inc., ca, usa). parametric and non-parametric tests were used as appropriate. the nonparametric spearman correlation coefficient was used to analyze meteorological data. variables with an associated pvalue < 0.05 in the univariate analysis and those considered as confounding factors (age and length of hospitalization) were subjected to multivariate logistic regression to identify independent predictors for severe disease. all p-values were two-tailed and a value of <0.05 was considered significant. curitiba is located in southern brazil and has a temperate climate. data on monthly measures of temperature and rainfall were supplied by the meteorological system of paraná (sistema meteorológico do paraná [simepar]). were compared with those with other crv infections, groups 1 and 2, respectively) ( table 1) . for this analysis, only cases with single infections were included, excluding nosocomial infections, since given that patients with nosocomial infections were already in the hospital, it would be expected that they would have some underlying illness, which could overestimate the severity of infections. most hrv infected patients were younger than 2 years (57%), 53% were younger than 1 year, and 37% were younger than 6 months of age. the groups showed no statistically significant difference in median age. gender was unequally distributed between the groups. the main clinical manifestations observed in both groups included fever, cough, and dyspnea. however, the hrv positive group had a significantly lower proportion of cases with fever (62%, p = 0.001), and more comorbidities than the other group (40% vs. 27%, p = 0.043). although immunosuppression, chronic lung disease, and heart disease were the major medical conditions present in both groups, chronic lung disease was more frequent in the hrv group (17%), but without significant difference. concerning the diagnosis of sari, there were fewer patients diagnosed with this syndrome in the hrv group (60%, p = 0.006) than in the other crv group. in the multivariate analysis of the relationship between clinical characteristics significantly different between the hrv-and other crv-groups, only the higher prevalence of previous comorbidities in the hrv group was significant. patients with no comorbidities were 1.6 (95% ci: 1.04---2.34) times more likely to be infected crvs other than hrv, in comparison to patients with comorbidities. despite the fact that most of chest x-rays of hrv infected cases were missed (53%), radiologic alterations were described in 83% of the performed exams. most of the findings observed were interstitial infiltrate (33%) and pulmonary consolidation (28%), with no significant difference between both groups. severe disease was defined as that requiring mechanical ventilatory support, or intensive care unit (icu) admission, or death during hospitalization; no significant difference was found between the groups. two hrv infected patients died during hospitalization, both from respiratory infection complications. hrv had the highest rates of coinfection (69%) of the 128 samples with more than one crv detected. the viruses most co-detected with hrv were ev (40/88; 45%) and rsv (29/88; 33%), as shown in fig. 1 . in 52% of cases, the co-detection involved hrv plus another virus, and in 16% of cases, hrv was associated with two or more viruses. in 65 cases, hrv was the only virus detected. a comparison of the clinical characteristics of monoinfected hrv patients with co-infected hrv patients was performed, and no relation between viral co-detection and disease severity (p = 0.717) was found. the clinical features significantly associated with hrv co-detection were as follows: age 6---48 months (or = 3.2; 95% ci: 1.3---8.1), length of hospitalization more than 30 days (or = 9.7; 95% ci: 2.5---36.8), and no sari diagnosis (or = 4.0; 95% ci: 1.7---9.3). as co-infection with hrv alone was not related with severe disease, a second analysis was performed comparing disease severity between hrv co-infected patients and patients infected with other crvs. neither the presence nor absence of hrv co-detection was associated with disease severity (p = 0.196). cases of hrv infection occurred throughout the study period (fig. 2) . the months of may to august demonstrated the highest number of hrv infections during 2012, peaking in august (17 cases; 10%). in 2013, the seasonality of hrv infection was from march to may, peaking in may (19 cases; 12%). comparing the monthly distribution of hrv cases with average temperature ( • c) and rainfall (mm), a negative correlation between the number of hrv cases and the average temperature (rs = −0.636, p < 0.001) was demonstrated, but there was no significant correlation with rainfall (rs = −0.036, p = 0.866). since the implementation of systematic investigation into respiratory viruses in hospitalized patients with sari, hrv has been found with high frequency, either alone or codetected with other respiratory viruses. since some of these infections may have a poor prognosis, including death, it is critical to know and better characterize this infection to be prepared for its early diagnosis, appropriate clinical management, and prevention of nosocomial spread. the high frequency of hrv found in clinical samples from patients with sari was similar to that reported by kim et al. 7 in a tertiary hospital in korea. he et al. 8 analyzed a hospitalized pediatric population and found a prevalence of 48%, while walker and ison 9 found a prevalence of 14% in a hospitalized adult population. this difference between age groups was observed in this study, emphasizing the greater vulnerability of children to this infection, probably as consequence of a more intense inflammatory process triggered by primary infection or by favorable anatomical conditions of the respiratory tract in younger children. rsv was the first pathogen identified to be associated with the severe respiratory disease in children. however, since the development of molecular techniques to detect hrv, this virus has been the focus of the most recent studies in this field. nowadays, hrv infections have been linked with exacerbations of chronic lung diseases and fatal pneumonia in patients at the extremes of age or with pre-existing comorbidities. 4, 5 the importance of one study that evaluated the characteristics of hrv infections in a tertiary hospital is based on these new concerns. marcone et al. 10 reported a higher number of hrv infections in hospitalized children in argentina when compared with pediatric outpatients with acute respiratory infection, demonstrating a scenario that contributed to the increased concern about hrv infection in the hospital setting. previous studies have reported that the hrv infection rate in hospitals varies from 21% to 41%. 8, 11, 12 however, this variation is probably accounted for by the differences in age and clinical characteristics of patients analyzed in each study. although this study included all age groups, a predominance of pediatric patients infected with hrv was observed, mostly younger than 1 year old. in addition, the hrv-positive group had a greater proportion of patients with pre-existing comorbidities than the group with other identified crvs. 6,7,11---14 among the comorbidities observed in this study, chronic lung diseases were frequent in the hrv group, a correlation that has already been demonstrated in another similar study, 14 which corroborates the close association of hrv infection and exacerbations of chronic lung diseases, such as chronic obstructive pulmonary disease (copd), asthma, and cystic fibrosis. 4 of 444 samples positive for crvs, 128 samples (29%) had at least two co-detected viruses, in keeping with the expected 10---31% encountered in hospitalized individuals. 7, 8, 15 the viruses most frequently co-detected with hrv were ev (45%), rsv (33%), and adv (18%); consistent with previously reported patterns. 14, 15 however, the high frequency of ev and hrv co-detection may be as a result of using the 5 nrt region of the viral genome to identify both pathogens, which may have lowered the specificity of the rt-pcr test. 16 a more appropriate means to discern between these species would be to carry out the rt-pcr followed by nucleotide sequencing of amplicons. 17 the high level of dual infection involving hrv and rsv is often explained both by the coexistence of these viruses throughout the year and their similar seasonal variation, 15 but this hypothesis is not unanimous. 12 in contrast to findings reported by goka et al., 15 this study did not show an association between disease severity and viral co-detections. furthermore, hrv was not found to have a protective influence in cases where it was involved in codetection, as there was no significant difference in disease severity when comparing the groups with or without hrv co-detection. likewise, in contradiction to the present findings, asner et al. 18 observed an increased disease severity in children infected with hrv alone. among the probable factors associated with these contrasting findings, the following may be cited: (i) analysis in different groups of patients (outpatient and hospitalized), (ii) assessing a small number of patients, and (iii) adoption of different severity criteria. fever was less frequent in patients infected with hrv compared to other crvs. 19, 20 chest x-ray findings were normal in 17%, showed interstitial infiltrate in 33%, and demonstrated pulmonary consolidation in 28% of cases, with no significant difference between the hrv and other crv groups. these patterns are similar to those reported by fica et al. 11 in hospitalized adults in chile. there were significantly fewer cases of sari diagnosed in the patients infected with hrv than other crvs. overall, 14% (110/770) of patients with sari were infected with hrv. this concurs with a previous study. 20 reports about the seasonality of hrv infection, including a study from argentina, 10 which is in close geographical proximity of southern brazil, show that it circulates mainly in autumn and spring. 7, 8, 19 although hrv occurred in almost all months of the study period, different peaks were observed in 2012 and 2013, and neither peak included spring. in 2013, the highest prevalence of hrv infection was in autumn, followed by winter, and vice versa in 2012. the analysis of meteorological data found a negative correlation between the number of hrv cases and the average temperature, but no significant correlation with rainfall. however, in order to more accurately establish the seasonality of hrv infections, the analysis should include additional years. this research had some limitations: (i) it was a retrospective study and some medical records were incomplete; (ii) hrv species were not identified, which would have yielded important data since the genotypes reportedly have different virulence; (iii) this analysis was carried out only with hospitalized patients, which may have overestimated the impact of hrv infection; and (iv) it was not possible to evaluate the frequency of nosocomial hrv infection, data critical to guide preventive measures in the most affected settings. however, this is the first report about hrv infections in the region and a critical analysis of the data is important to obtain greater awareness of the dynamics of dispersion and impact of these respiratory viruses in the community. in conclusion, hrv has a high prevalence in the hospitalized children and was present in cases of severe disease, including death. however, a dependent relationship between the presence of hrv in viral co-detections and severity of disease was not observed in this study. conflicts in the literature demand a closer analysis of cases of co-detection involving hrv with review of the data to determine the impact of this, since the lack of standardization between studies probably contributes to the divergent data. hrv infection is closely associated with comorbidities, mainly chronic lung diseases, and is an important factor in exacerbations of these underlying lung diseases. the colder seasons were the period with higher frequency of hrv infections in southern brazil, and therefore must be a period to warn clinicians about young age children affected by respiratory infections, especially those with comorbidities such as chronic lung disease. patients with this profile should have respiratory samples collected to identify possible viral infection, and if hrv is detected, the medical staff should be ready for adequate management, taking into consideration the possible poor outcomes. smr is sponsored by a cnpq fellowship. the authors declare no conflicts of 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authors: miller, tracie l.; cushman, laura l. title: gastrointestinal complications of secondary immunodeficiency syndromes date: 2010-12-27 journal: pediatric gastrointestinal and liver disease doi: 10.1016/b978-1-4377-0774-8.10042-9 sha: doc_id: 22521 cord_uid: r72jtoso nan secondary immunodeficiency syndromes constitute a spectrum of disorders. infections of the gastrointestinal tract pose the greatest risk for children with secondary immunodeficiencies. cellular changes in the gastrointestinal tract (the largest immune organ in the body) that lead to diarrhea and malabsorption, peptic disease, dysmotility, and liver disease are among some of the other disorders of the gastrointestinal tract faced by these children. worldwide, human immunodeficiency virus (hiv-1) infection and malnutrition are by far the most common secondary immunodeficiency states. however, in the united states and other developed countries, severe malnutrition and new cases of perinatal hiv-1 disease are rare because of relatively high standards of living and effective highly active antiretroviral therapies (haart) given to pregnant hiv-infected women that prevent transmission of hiv to the infants. 1 between 2004 and 2005, there were 67 reported cases of perinatally acquired hiv and 4883 new diagnoses of unspecified origin in adolescents 13 to 24 years of age. 2 hiv-infected children and adolescents are now surviving because of effective antiretroviral strategies, yet there is increased horizontal acquisition of hiv in adolescents owing to risky social behaviors. furthermore, children with chronic illness are among the highest population at risk for malnutrition and its sequelae. 3 thus these two disorders serve as models for complications of other secondary immunodeficiency states. the first cases of the acquired immunodeficiency syndrome (aids) were described in the early 1980s. later, in 1984, 4 hiv-1 was determined to be the causative agent, and hiv-1 infection was recognized as a spectrum of disease, ranging from asymptomatic infection to full-blown aids. the aids epidemic claimed an estimated 2 million lives in 2007, and an estimated 2.7 million people acquired hiv-1 in 2007. an estimated 33 million people globally are living with the virus. 5 with the successful preventive strategies of elective cesarean section delivery and chemoprophylaxis of pregnant hiv-1-infected women, the transmission rates plummeted from 15 to 30% to less than 1 to 2% of all hiv-1-infected women delivering infants. 6 the advent of haart in 1996 changed the natural history of hiv-1 in children in many countries. 7 however, the successes of prevention and prophylaxis have not been realized as much in developing countries, where hiv infection continues to increase. for this reason, there are disparate accounts of opportunistic infections and other diseases in regions with high haart accessibility and those with limited haart accessibility. 8 hiv-1 is an rna virus that belongs to the lentivirus family. it has a particular tropism for the cd4 surface antigen of cells, and the binding of hiv-1 to the cd4 receptor initiates the viral cycle. the virus may subsequently replicate within the host cell or, alternatively, the proviral dna within the host cells may remain latent until cellular activation occurs. human t lymphocytes and monocytes-macrophages are the primary cells that are infected with hiv-1, although other cell lines may be infected as well. the net effect is suppression of the immune system and a progressive decline in cd4+ t lymphocytes, which leaves patients susceptible to opportunistic and recurrent bacterial infections. the gastrointestinal tract is the main source of hiv-1 infection when parenteral transmission is excluded. in vertical transmission, hiv-1 is found in the gastrointestinal tract after the fetus swallows infected amniotic fluid, blood, cervical secretions, or breast milk. the virus, inoculated in the gastrointestinal tract, infects the fetus as it enters into the gut-associated lymphoid tissue (galt) through the tonsil or upper intestinal tract. examination of both acute simian immunodeficiency virus (siv) and hiv infection have documented reduced cd4 cell levels in galt prior to a detectable reduction in t cells of the peripheral blood, highlighting the gastrointestinal tract's role and susceptibility. [9] [10] [11] [12] the rates of acquisition of hiv-1 through the gastrointestinal tract are likely related to the quantity of virus in the person transmitting it [13] [14] [15] and the immunologic function and maturity of the patient being infected. mucosal infections with opportunistic infections may increase hiv-1 transmission. mycobacterial infections up-regulate cc chemokine receptor 5 (ccr5) expression in monocytes, which facilitates the entry of ccr5-tropic hiv-1. other factors, such as tumor necrosis factor-α (tnf-α), which is induced by nuclear factor (nf)-кb (which itself is pathogen induced), are potent inducers of hiv-1. 16, 17 cellular routes that potentially can transmit hiv-1 across the gastrointestinal tract include m cells, dendritic cells, and epithelial cells. m cells are specialized epithelial cells that overlie the peyer's patches and transport large macromolecules tracie l. miller • laura l. cushman and microorganisms from the apical surface to the basolateral surface. human transport of hiv-1 by m cells in vivo has not been reported. dendritic cells bind hiv-1 through a dendritic cell-specific adhesion molecule. in vitro studies support the role of dendritic cells in transmitting hiv-1 [18] [19] [20] [21] ; however, the role of the dendritic cell in in vivo transmission of hiv-1 has yet to be determined. epithelial cells express ccr5 and can selectively transfer ccr5-tropic hiv-1. the epithelial cell can transport hiv-1 in vitro from the apical to the basolateral surface. 22, 23 the r5-tropic viruses are transferred in vitro through epithelial cell lines. 24 once transmitted, the lamina propria lymphocytes express ccr5 and cxc4 chemokine receptor 4 (cxcr4), which support hiv-1 replication. 25, 26 early after infection, there is a greater proportion of infected lymphocytes in the lamina propria than in peripheral blood. 27, 28 for the patients actively receiving haart, poles et al. 29 described "cryptic replication" occurring in galt reservoirs in which viral replication is actively taking place at slower rates but hiv-1 rna levels remain undetected in peripheral blood. further, galt contained more than twice as many lymphoid cells (160, 000) than peripheral blood mononuclear cells (70,000) possessing hiv-1 dna with viral replication capacity. there was no significant reduction in these values when the analysis was repeated after 12 months. lymphocytes are able to disseminate the virus to distant sites, with depletion of cd4 cells in the lamina propria 27, 30 and then in the blood. even with aggressive suppression of hiv-1 during the primary stages by highly active antiretroviral agents, cd4 cell depletion is observed in the effector subcompartment gut mucosa when cd4 levels in the peripheral blood have stabilized. 10 as mucosal and peripheral t cells are depleted, monocytes and macrophages become important reservoirs for the virus. the intestinal macrophages do not promote inflammation and do not carry the receptor for ccr5 or cxcr4; however, the blood monocytes are different in their profile and are infected by hiv-1. they are found infected in the blood and thereafter take up residence in the gut. 31 they are stimulated by opportunistic agents and proinflammatory cytokines. 32 recent in vitro studies have implicated the integrin receptor α4β7 on which the hiv-1 envelope binds and transmits signals mediated by an epitope in the v2loop of gp120. this in turn activates lfa-1 and is pivotal in virological synapse formation, allowing rapid cellular dissemination of hiv-1. 33 villous atrophy and gastrointestinal tract dysfunction are coincident with high levels of hiv-1 viral load in the gut. 34 altered epithelial permeability may permit microbial translocation and generalized immune activation leading to localized cytokine production and further replication of hiv. 35 a dysfunctional gastrointestinal tract can produce clinical symptoms that contribute to both morbidity and mortality in children with hiv-1 infection. these symptoms include weight loss, vomiting, diarrhea, and malabsorption (table 42 -1). the advent of antiretroviral treatment induces debilitating effects on mechanisms within the gut that promote chronic hiv infection. mainly, high levels of lipopolysaccharides (lps) are associated with marked systemic immune activation sustaining this infection; antiretroviral therapy decreases levels of lps, promotes cd4+ t cell reconstitution, and may subsequently decrease the systemic immune activation. 36 additional studies examining this relationship stand to offer greater insight into hiv pathogenesis. as mentioned, there are distinct changes in the cellular milieu of the gastrointestinal tract in hiv-1-infected patients. previous studies have shown that activated mucosal t cells play a role in the pathogenesis of enteropathy in the human small intestine 37 and can affect the morphology of the villi and crypts in a manner similar to that seen in patients with hiv-1 infection. the magnitude of viral burden in the gastrointestinal tract is associated with villous blunting and other abnormal morphology. 34 a number of studies in the 1980s associated a distinct enteropathy with hiv-1. 38 diarrhea, weight loss, an abnormally low d-xylose absorption, and steatorrhea, without evidence of intestinal infection, were common findings. jejunal biopsies showed partial villous atrophy with crypt hyperplasia and increased numbers of intraepithelial lymphocytes. this was the first histologic description of a specific pathologic process that occurred in the lamina propria of the small intestine in some patients with hiv-1. others 39 found low-grade small bowel atrophy and maturational defects of enterocytes, supporting an hiv-1 enteropathy characterized by mucosal atrophy with hyporegeneration. however, some investigators have challenged this concept, suggesting that the findings could be attributed to an undiagnosed enteric infection. recently, genotype profiling for genes responsible for endothelial barrier maintenance and metabolic functioning has shown a decreased expression in the presence of increased viral replication in the galt and reduced cd4+ t cell levels. 40 these findings are significant, because they offer an additional modality for evaluating microenvironmental alterations within the gastrointestinal tract of the patient. additional studies will help to determine the efficacy of gene expression profiling in hiv-infected individuals. miller et al. 41 published histologic findings in 43 children with hiv-1 infection. the majority of patients had normal villous architecture, and many of the children with villous blunting had an associated intercurrent enteric infection. distinct features of hyperplasia of the lamina propria and increased intraepithelial lymphocytes were not apparent. bjarnason et al. 42 studied intestinal inflammation and ileal structure and function in patients with a wide spectrum of hiv-1 disease states. hiv-1-infected patients who were minimally symptomatic had normal intestinal absorption and permeability, yet had greater gastrointestinal dysfunction as they progressed to aids. malabsorption of bile acids and vitamin b 12 did not correlate with morphometric analysis of ileal biopsies and was unremarkable in these patients. thus, there was significant mucosal dysfunction with only minor ileal morphologic changes. malabsorption of bile acids may play a pathologic role in patients with aids diarrhea. the absorptive defect of aids enteropathy using a d-xylose kinetic model of proximal absorption was studied 43 and correlated with the results of a schilling test for cobalamin absorption, which measures distal intestinal function. there were minimal histologic abnormalities in both the proximal and distal biopsy sites in patients with diarrhea and no enteric infection. d-xylose absorption was low, and the absorptive defect was more severe and greater than would be expected from the histologic abnormalities found. thus, these findings support the theory that there is little association between histologic characteristics of the small bowel and its absorptive function in patients with hiv-1 infection. most studies do not support a direct role for gastrointestinal malabsorption on growth failure or weight loss. ullrich et al. 39 described gastrointestinal malabsorption in hiv-1-infected patients who had low levels of lactase enzyme in the brush border, crypt death, decreased villous surface area, and decreased mitotic figures per crypt when compared with control patients. in addition, keating et al. 44 described absorptive capacity and intestinal permeability in hiv-1-infected patients. malabsorption was prevalent in all groups of patients with aids, but was not as common in the asymptomatic hiv-1-infected patients. malabsorption correlated with the degree of immune suppression and with body mass index. there were mild decreases in the ratio of jejunal villous height to crypt depth, yet not as severe as the subtotal villous atrophy found in celiac disease. lim et al. 45 found disaccharidase activity decreased proportionately with greater hiv-1 disease severity, although there was no association between disaccharidase levels and weight loss. in addition, mosavi et al. 46 found no correlation between diarrhea and weight loss in hiv-1-positive patients. taylor et al. 47 found mild histologic changes accompanied by severe disaccharidase abnormalities; however, symptoms were severe enough to withdraw lactose in only 25% of the patients. collectively these studies suggest that gastrointestinal malabsorption may be present, but is not always associated with weight loss and diarrhea. formal studies of intestinal absorption in children with hiv-1 are more limited. malabsorption occurs frequently in hiv-1-infected children and may progress with the disease. in one study, 40% of children had nonphysiologic lactose malabsorption and 61% had generalized carbohydrate malabsorption that was not associated with gastrointestinal symptoms or nutritional status. 48 these findings have been confirmed by others. 49 another study in children revealed an association between diarrhea and nutrition. 50 abnormal d-xylose absorption has also been associated with enteric infections in children. 48 fat and protein loss or malabsorption have also been described. sentongo et al. 51 evaluated fat malabsorption and pancreatic exocrine insufficiency using fecal elastase-1 enzyme assay in 44 hiv-1-infected children. hormone-stimulated pancreatic function testing and 72-hour stool and dietary fat sample collection were performed in children with abnormal fecal elastase levels. the prevalence of steatorrhea was 39% and that of pancreatic insufficiency was 0% (95% confidence interval 0 to 9%). there were no associations between steatorrhea and pancreatic insufficiency, growth, hiv-1 rna viral load, cd4 status, or type of antiretroviral therapy. other studies support the absence of association. 52 thus, the clinical significance of steatorrhea in pediatric hiv-1, similar to absorption of other nutrients, is unclear. the etiology of malabsorption in hiv-1 infection is probably multifactorial. the cellular milieu of the lamina propria is altered significantly with hiv-1 infection. 34, 53 the depletion of the cd4 t lymphocytes in the intestinal tract may cause change in the cytokine environment and alter intestinal function. viral load in the intestinal tract may be considerably higher than that measured peripherally, and this can also affect mucosal gastrointestinal structure and function. recently, the hiv-1 tat protein was found to decrease glucose absorption through decreasing the activity of the sodium d-glucose symporter. 54 studies suggesting these hypotheses include that of kotler et al., 55 which looked at intestinal mucosal inflammation in 74 hiv-1-infected individuals. these authors found abnormal histopathology in 69% of the patients, and this finding was associated with altered bowel habits. high tissue p24 antigen levels were observed, and these correlated with more advanced hiv-1 disease. tissue p24 detection was associated with both abnormal bowel habits and mucosal histology. the tissue content of cytokines, including tnf, α-interferon, and interleukin-1β, was higher in hiv-1-infected individuals than in controls, and these increases were independent of intestinal infection. thus, hiv-1 reactivation in the intestinal mucosa could be associated with an inflammatory bowel-like syndrome in the absence of other enteric pathogens. small bowel bacterial overgrowth can be another source of gastrointestinal dysfunction leading to malabsorption. bacterial overgrowth may be due to aids gastropathy, 56, 57 in which the stomach produces only small amounts of hydrogen chloride, allowing bacterial pathogens to escape the acid barrier of the stomach and colonize the duodenum. additionally, iatrogenic hypochlorhydria may be due to the use of acid-blocking agents as treatment for peptic disease. interestingly, some authors have found no relationship between gastric ph and small bowel bacterial colonization and diarrhea in hiv-1-infected patients. 58 enteric pathogens 59 have been associated with enteric dysfunction, as discussed later. with the advent of haart, gastrointestinal symptoms, especially those associated with opportunistic infections, are less common. 60 as viral burden decreases, immunosuppression has less effect on gastrointestinal function. compared to untreated patients, haart-treated patients had greater integrity of intestinal mucosal barrier and decreased villous atrophy. 61 ritonavir, a protease inhibitor, in combination therapy resulted in restoration of gastrointestinal function in 10 children with carbohydrate malabsorption, steatorrhea, protein loss, and iron deficiency. 62 however, one study in adults found similar rates of fat malabsorption in patients taking haart and in those not taking haart. 63 the gastrointestinal tract is a major target for opportunistic infections in hiv-1-infected children. the spectrum of these infections is dependent on hiv-1 disease progression. in developed countries, with improved hiv-1 viral suppression associated with haart, opportunistic infections of the gut and elsewhere are less common. 64 however, immunocompromised children are still at risk for infections with cytomegalovirus (cmv), herpes simplex virus (hsv), cryptosporidium, and microsporidia. previous dogma that much of the diarrhea found in children with hiv-1 infection is not associated with enteric pathogens has been challenged. unusual viral and parasitic infections can be diagnosed as a result of better diagnostic techniques. however, the cause of diarrhea in a significant number of patients with hiv-1 remains undiagnosed. 65 occurrence of opportunistic disease and infection of the gastrointestinal tract in immunocompromised patients relies heavily on the accessibility of haart. as a result, there is great disparity of documented incidence of gastrointestinal infections dependent on access to haart in particular regions. for this reason, we have divided this section into two subsections: gastrointestinal infections in regions with low haart accessibility or in patients with cd4 t-lymphocyte counts less than 200 cells/mm 3 , and gastrointestinal infections in regions with high haart accessibility and successful viral suppression. this does not imply that any of the infections discussed here occur in isolation contingent on haart accessibility, because all hiv-1 patients regardless of haart may encounter these complications. in the post-haart era, it is helpful for a physician to know which backdrop lends itself to specific vulnerabilities. the detection of viral gastrointestinal infections in hiv-1infected children can sometimes be difficult owing to the limitations of diagnostic techniques. the most common gastrointestinal viral pathogen in hiv-1-infected children is cmv. other pathogens, such as hsv, adenovirus, epstein-barr virus, and a variety of other unusual viruses, can also contribute to intestinal dysfunction and diarrhea. hsv infection in an immunocompromised child usually represents reactivation of a latent virus that had been acquired earlier in life. gastrointestinal infection with hsv most commonly involves the esophagus and causes multiple small, discrete ulcers. hsv can also involve other areas of the intestinal tract, including the colon and small bowel. the diagnosis of hsv relies on recognizing the multinucleated intranuclear inclusion bodies (cowdry type a) with a ground-glass appearance and molding of the nuclei. the squamous epithelium is usually infected, although there may also be involvement of intestinal glandular epithelium in the mesenchymal cells. hsv monoclonal antibody staining is confirmatory for the diagnosis. in extensive involvement, there may be transmural necrosis and development of tracheoesophageal fistulas. treatment of hsv and other common gastrointestinal pathogens and their primary sites of involvement are outlined in table 42-2. other herpes viruses have also been detected in the gut of hiv-1-infected individuals. a case report of one 34-year-old hiv-1-infected man with intestinal pseudo-obstruction and disseminated cutaneous herpes zoster revealed positive immunohistochemistry against herpes zoster in a resected portion of the terminal ileum. this area had focal ulceration. the virus was localized to the muscularis propria and myenteric plexi throughout the entire length of the specimen. the authors postulated that the location of the virus in the gut may have been the etiologic factor for the pseudo-obstruction. 66 cytomegalovirus cmv in the immunocompromised child, like hsv, represents reactivation of a latent virus that was acquired in earlier life. cmv is one of the more common viral pathogens of hiv-1infected children. the reported incidence of gastrointestinal involvement in the pre-haart era varied from 4.4% to 52% of patients studied. the incidence rates may have varied based on the techniques of diagnosis. 57 cmv infection is rare in patients with cd4 t-lymphocyte counts greater than 50 cells/mm 3 . 67 cmv may involve any part of the gastrointestinal tract, with an increased incidence in the esophagus or colon. cmv infection usually results in one or two discrete single and large ulcers of the esophagus and colon. lesions may lead to severe gastrointestinal bleeding and hemodynamic instability. cmv inclusion bodies can be discovered incidentally in an asymptomatic patient, and this does not necessarily reflect disease. in patients with upper intestinal cmv disease, there can be dysphagia and upper abdominal symptoms, whereas diarrhea is more common with colitis. the diarrhea can be watery or bloody. children may be systemically ill. 68 the colitis from cmv infection is patchy and can be associated with severe necrotizing colitis and hemorrhage. 69 cmv usually affects the cecum and the right colon. diagnosis is confirmed by endoscopy and biopsy. the histologic appearance of cmv-infected cells is unique (figure 42 -1). these cells are enlarged and contain intranuclear and cytoplasmic inclusion bodies. the nuclear inclusion bodies are acidophilic and are often surrounded by a halo. cytoplasm inclusion bodies are multiple, granular, and often basophilic. cells that are dying may appear smaller and smudged, with poorly defined inclusion bodies. staining for cmv antigen shows that many of the infected cells are endothelial cells with others being perivascular mesenchymal cells. cmv can cause vasculitis because of its target cell population. thus, the spread of cmv occurs with circulating infected endothelial cells. treatment options are outlined in table 42 -2. once haart is established, with decreased viral burden (both hiv-1 and cmv) and improved cd4 counts, cmv treatment may be discontinued without concern for reactivation. 70 infections with other unusual viral pathogens have been described. these include the human papilloma virus and epstein-barr virus, which have been identified in esophageal ulcers of patients with hiv-1. adenovirus of the stomach and colon have also been reported and are often difficult to identify. 71 in the pre-haart era, patients who excreted adenovirus from their gastrointestinal tract had a shorter survival. 72 there are unusual enteric viruses that have been associated with diarrhea in hiv-1-infected children. 73 these viruses, among others, include astrovirus and picobirnavirus. 74 cegielski et al. 75 studied 59 children with hiv-1 infection in tanzania. they looked for enteric viruses identified by electron microscopy of fecal specimens. small round structured viruses (srsvs) were found more frequently in hiv-1-infected children than in uninfected children with chronic diarrhea. rotavirus and coronaviruslike particles were not associated with hiv-1 infection. these authors considered that these srsvs may be associated with hiv-1 infection and could lead to chronic diarrhea in tanzanian children. bacterial infections that involve the gastrointestinal tract of children with hiv-1 infection may be divided into three groups: bacterial overgrowth of normal gut flora; pathogens that can affect immunocompromised children as well as immunocompetent children (salmonella, shigella, campylobacter, clostridium difficile, and aeromonas); and bacterial infections that are more common in immunocompromised children (mycobacterium avium-intracellulare complex; mac). few studies have evaluated bacterial overgrowth in hiv-1-infected children, although gastric hypoacidity has been associated with opportunistic enteric infections and bacterial overgrowth in adult patients with hiv-1. 76 other studies have not found this association. small bowel bacterial overgrowth was not a common finding in a group of 32 hiv-1-infected patients, regardless of the presence of diarrhea, and it was not associated with hypochlorhydria. 58 lactose hydrogen breath testing has shown high baseline readings in children that may indirectly suggest bacterial overgrowth of the small intestinal tract. 48 detection of bacterial overgrowth in the small bowel is usually performed by quantitative duodenal aspirate for bacterial culture, with therapy directed at treating the organisms, which are often anaerobic. common bacterial pathogens include salmonella, shigella, campylobacter, clostridium difficile, and aeromonas. infection with these organisms occurs more frequently in immunocompromised patients. combined morbidity and mortality rates associated with hiv-1 and these bacterial pathogens in developing countries approach 50% in some studies. 77 hiv-1-infected patients with campylobacter infection have higher rates of bacteremia than the general population. deaths from sepsis due to this organism have been reported in severely immunodeficient patients with aids, despite haart. 78 other entities, such as bacterial enteritis, have been described in adults with hiv-1. a study by orenstein and kotler 79 evaluated ileal and colonic biopsies in patients with aids and diarrhea and found bacteria similar to adherent e. coli along the intestinal epithelial border. similar findings were documented by kotler et al., 80 who showed adherent bacteria in 17% of all adult patients with aids. the infection was localized primarily to the cecum and right colon, and three distinct histopathologic patterns of adherence were observed: attachment on effacing lesions, bacteria intercalated between microvilli, and aggregates of bacteria more loosely attached to the damaged epithelium. the bacterial cultures of frozen rectal biopsies yielded e. coli in 12 of the 18 patients. these findings suggest that chronic infection with adherent bacteria can also produce the syndrome of aids-associated diarrhea. in a "look back" evaluation, orenstein and dieterich 71 found that enteropathogenic bacterial infections were overlooked on initial examination and concluded that, for accurate diagnoses, specimens should be evaluated by laboratories with expertise in hiv. intestinal infections with mycobacteria, including mycobacterium tuberculosis, mac, and other atypical mycobacteria, were the most frequently encountered bacterial infections in hiv-1-infected patients in the pre-haart era 81 and became more prevalent in the pre-haart era as patients were living longer with cd4 counts below 200 cells/mm 3 . 82, 83 in the haart era, disseminated mac in colonized patients can be successfully prevented; however, the effects of haart on restoration of cd4 counts do not prevent mac colonization. 84 infection with mac usually occurs in the very late stages of aids in children, when cd4 counts are lower than 200 cells/mm 3 . the most common clinical manifestations of gastrointestinal infections with mac include fever, weight loss, malabsorption, and diarrhea. intestinal obstruction, resulting from lymph node involvement and intussusception; terminal ileitis, which resembles crohn's disease; and refractory gastric ulcers are often found. severe gastrointestinal hemorrhage has also been described. 85 endoscopically, fine white nodules may be seen in the duodenum, or the duodenal mucosa may look velvety and grayish in appearance. segments of the gastrointestinal tract can become infected with mac. histologically, there is a diffuse histiocytic infiltrate in the lamina propria with blunting of the small intestinal villi. these histiocytic infiltrates can be recognized on hematoxylin and eosin staining and on acid-fast stains and are pathognomonic for infection ( figure 42-2) . with the advent of haart, immune reconstitution disease has been described. 86, 87 this is likely an immune reaction in which previously quiescent organisms become active because of the improved immune function associated with haart. this can occur in as many as 25% of patients who respond to haart. 88 lymphadenitis is the most common condition, although abscesses can appear anywhere. severe abdominal complaints may result. appropriate therapies are outlined in table 42 -2, yet this organism is often frustrating to treat. azithromycin 600 mg, when given in combination with ethambutol, is an effective agent for the treatment of disseminated m. avium disease in patients infected with hiv-1. 89 caution must be exercised in administering these multidrug regimens for mac in patients receiving concurrent haart. rifamycins induce cytochrome p450 enzymes and accelerate the metabolism of clarithromycin and hiv-1 protease inhibitors. conversely, clarithromycin inhibits these enzymes, resulting in increased rifabutin toxicity. the net result is treatment regimens that can be extremely difficult to tolerate and manage, especially for sicker patients. clarithromycin and azithromycin must be administered in combination with other agents, such as ethambutol, to prevent the emergence of macrolide resistance. 90 in the early 1980s, cryptosporidiosis was regarded as an aidsdefining disease and an opportunistic intestinal pathogen. it became an important cause of chronic diarrhea, leading to high morbidity and mortality rates in immunocompromised patients. to date, no effective chemotherapy is available. with the introduction of protease inhibitors in haart regimens, the incidence of cryptosporidiosis in patients with aids has declined substantially in developed countries. 91 however, in developing nations, gastrointestinal infection with c. parvum is prevalent and carries high morbidity and mortality rates. 92, 93 although cryptosporidium was initially described in animals, it was first noted to cause an enterocolitis in both immunocompromised and immunocompetent humans in 1976. 94, 95 an intact t-cell response is the primary mechanism that confers protection against this organism; thus, patients with abnormal t-cell function or number are at risk. the spectrum and severity of disease in immunocompromised individuals with cryptosporidiosis correlates with most severe disease found in individuals with defects in the t-cell response. 91 the overall frequency of infection seems to be related to the severity of immunodeficiency and not the specific disorder. 96 cryptosporidium usually affects the gastrointestinal tract, although it has been found in other organs including the biliary tract, 97 pancreas, 98 and respiratory tract. 99 in immunocompetent individuals, the diarrhea is self-limiting, whereas in immunocompromised patients, it may be protracted and associated with significant malabsorption and nutritional compromise. the small intestine is the primary target, although it can occur in any part of the intestinal tract. esophageal cryptosporidiosis has also been described in one child 100 and in adults. clayton et al. 101 described two patterns of enteric cryptosporidiosis. one was accompanied by severe clinical disease with significant malabsorption, with the majority of the organisms found in the proximal small bowel, whereas less severe clinical disease was seen in patients with colonic disease or with infection noted only in the stool. patients with proximal small bowel infection with cryptosporidium showed crypt hyperplasia, villous atrophy, lamina propria inflammatory infiltrates, abnormal d-xylose absorption, greater weight loss, and shorter survival, with greater need for intravenous hydration and hyperalimentation than patients with colonic disease. in other studies, absorption of nutrients showed an inverse correlation with active infection, 102 as shown by altered vitamin b 12 and d-xylose absorption and lactulose and mannitol urinary excretion ratios. intestinal function improved in patients whose oocyte counts were reduced by treatment with paromomycin. symptomatic cryptosporidiosis has been documented in as many as 6.4% of immunocompetent children and 22% of immunodeficient children, whereas in an asymptomatic population, cryptosporidium was found in 4.4% of immunocompetent and 4.8% of immunodeficient children. 103 spiramycin at 100 mg per kg daily for 14 days caused a significant reduction in the shedding of infectious oocysts, and no gastrointestinal symptoms developed in children treated for asymptomatic infection, whereas children who were not treated developed gastrointestinal symptoms. 103 the diagnosis of cryptosporidiosis is made by identifying the organisms in a duodenal aspirate, stool, or tissue sample (biopsies). on hematoxylin and eosin-stained sections, these organisms can be found as rows or clusters of basophilic spherical structures 2 to 4 μm in diameter, attached to the microvillous border of the epithelial cells (figure 42-3) . the tips in the lateral aspect of the villi show the greatest number of organisms in the small intestine. in the colonic epithelium, the crypt and surface epithelial involvement appears equal. cryptosporidium also stain positively with giemsa and negatively with mucous stains. the acid-fast stain on a stool sample is one of the most widely used methods of determining whether a patient has cryptosporidiosis. more recent sensitive and specific methods for diagnosing cryptosporidiosis include fluorescein-labeled igg monoclonal antibodies. 104, 105 treatment of cryptosporidiosis in children with hiv-1 infection is often difficult. the disease can be chronic and protracted with diffuse watery diarrhea and dehydration. several different agents are used to eradicate the organism, with varying success rates. the most effective treatment is to improve immunologic function and nutritional status. with the advent of haart, many children's immune function has been restored with a lower incidence and prevalence of cryptosporidium infection. 106 the introduction of haart in a patient with severe debilitating cryptosporidium infection not only resulted in an increased cd4 count in the peripheral blood and clearance of the organism, but also produced a marked increase in cd4 count in the rectal mucosa on biopsy, suggesting this may have been the main mechanism of clearing the parasite. 107 octreotide therapy of acute and chronic diarrhea, with coincident improvement in nutritional status, eradicated cryptosporidium in one patient. 105, 108 other investigators have used bovine hyperimmune colostrum with benefit. 109, 110 the macrolides, such as azithromycin, have shown some promise in the treatment of cryptosporidium infection. 111, 112 the effect of protease inhibitors as therapy against cryptosporidium has been tested in a cell culture system. 113 nelfinavir moderately inhibited the host cell invasion over a period of 2 hours. indinavir, nelfinavir, and ritonavir inhibited parasite development significantly. the inhibitory effect was increased when the aminoglycoside paromomycin was combined with the protease inhibitors indinavir, ritonavir and, to a lesser extent, saquinavir, compared with the protease inhibitor alone. thus, protease inhibitor therapy may directly (rather than indirectly, through its effects on the immune system) inhibit growth of cryptosporidium. amadi et al. 92 found that a 3-day course of nitazoxanide improved diarrhea, helped eradicate the parasite, and improved mortality in hiv-1-seronegative, but not hiv-1seropositive, children in zambia. treatment with nitazoxanide on immunocompetent patients demonstrated parasitic load reduction, but its effects on immunocompromised patients are not yet palpable. 114 microsporidia are obligate intracellular protozoal parasites that infect a variety of cell types in many different species of animals. these organisms were first described in 1857, when recognized as a cause of disease in nonhuman hosts. 115 the first description of microsporidia (enterocytozoon bieneusi) as a human pathogen was in 1985, and microsporidia have since been described as more common human pathogens. 116 infection with microsporidia typically occurs in patients with severely depressed cd4 t-lymphocyte counts. one of the largest case studies of intestinal microsporidiosis in patients with hiv-1 infection was described by orenstein et al. 117 in 67 adult patients with aids and aids-related complex and chronic nonpathogenic diarrhea. e. bieneusi was diagnosed by electron microscopy in 20 of the patients. jejunal biopsies were more positive than duodenal biopsies. the parasites and spores were clearly visible by light microscopy in 17 of the 21 biopsies. infection was confined to enterocytes located at the tip of the intestinal villus, and the histologic findings included villous atrophy, cell degeneration, necrosis, and sloughing. other investigators [118] [119] [120] found microsporidia in as many as 50% of hiv-1-infected patients with chronic and unexplained diarrhea evaluated in the pre-haart era. e. bieneusi has been documented in 15 to 25% of children with 121 or without 122 diarrhea in developing countries, making it fairly ubiquitous in these regions of the world. other species of microsporidia, including encephalitozoon (septata) intestinalis, can cause significant enteric disease with diarrhea, wasting and malabsorption. encephalitozoon intestinalis differs from enterocytozoon bieneusi in its tendency to disseminate, and it can infect enterocytes as well as macrophages, fibroblasts, and endothelial cells. microsporidia are found with increasing frequency in hiv-1negative patients. 123 infection has been documented in almost every tissue and organ in the body, and in epithelial, mesenchymal, and neural cells. microsporidia can cause inflammation and cell death and a variety of symptoms including shortness of breath, sinusitis, and diarrhea with wasting. if left untreated, microsporidiosis can be a significant cause of mortality. treatments for microsporidia include albendazole, which can relieve clinical symptoms and eliminate microsporidial spores in the feces, especially of the less common pathogen, e. intestinalis. e. bieneusi is more challenging to treat, although therapy with fumagillin or its analogue, tnp-470 (antiangiogenesis agents), has shown promising results. [124] [125] [126] other studies show atovaquone as an effective treatment as well. 127 indirect treatment by improving the immune system with haart has also effectively cleared these organisms. 106, 128, 129 isospora belli is recognized as an opportunistic small bowel pathogen in patients with hiv-1 infection. this organism is most common in tropical and subtropical climates. isosporiasis can be diagnosed by identification of the oocyte in the stool or by biopsy. the diagnosis is critical because, in contrast to cryptosporidiosis or microsporidiosis, the therapy is very effective. i. belli is found within the enterocyte and within the cytoplasm. the organism stains poorly, although the central nucleus, large nucleolus, and perinuclear halo give it a characteristic appearance. the infection produces mucosal atrophy and tissue eosinophilia. a 10-day course of trimethoprim-sulfamethoxazole is effective therapy, and recurrent disease can be prevented by ongoing prophylaxis with this combination drug. 130 ciprofloxacin, although not as effective, is an acceptable alternative for those with sulfa allergies. 130 other therapies for isospora include pyrimethamine, also indicated for patients with sulfa allergies. 131 blastocystis hominis is usually considered a nonpathologic parasite, but it has been described in patients with chronic diarrhea and hiv-1 infection. 132 this organism is more pathogenic in immunocompromised patients and can cause mild, prolonged, or recurrent diarrhea. effective therapy includes diiodohydroxyquinoline 650 mg orally three times daily for 21 days. other protozoan infections that can be found in hiv-1-infected patients are entamoeba histolytica, entamoeba coli, entamoeba hartmanni, endolimax nana, and giardia lamblia in 4% of cases. candidiasis of the gastrointestinal tract is the most common fungal infection in hiv-1-infected children. the esophagus is the primary target of candida, and this infection occurred in the majority of patients during the course of their illness in the pre-haart era. it was also the second most frequent aidsdefining disease, second in prevalence only to pneumocystis jirovecii. patients with candida esophagitis complain of odynophagia or dysphagia and may often have vomiting and recurrent abdominal pain. children often have oral thrush, coincident with more disseminated and invasive candida esophagitis, although the absence of oral thrush does not preclude the diagnosis of candida esophagitis. 41 in one study, oral candidiasis preceded the diagnosis of candida esophagitis in 94% of children. 133 other risk factors include low cd4 count and prior antibiotic use. 133 histopathologically, yeast forms within an intact mucosa confirm invasive disease. this is in contrast to colonization, where the yeast is found overlying intact mucosal surfaces or necrotic tissue. these organisms are best seen with grocott's methenamine silver method or periodic acid-schiff stain. upper gastrointestinal studies are suggestive of candida esophagitis with diffuse mucosal irregularities (figure 42-4) . upper gastrointestinal endoscopy with biopsy and appropriate staining is the most sensitive test for determining invasive candidiasis of the esophagus. candidiasis can also occur in the stomach, as well as the small bowel if the acid barrier has been suppressed either through an intrinsic decrease in gastric acid production or iatrogenically with the use of potent acid blockers. numerous effective therapies have been described to treat candida of the upper gastrointestinal tract, including fluconazole, ketoconazole, and itraconazole. 134, 135 ketoconazole has more hepatic side effects than fluconazole. itraconazole is usually well tolerated and is effective. in severe and invasive disease, either topical or intravenous amphotericin can be used. agents such as oral miconazole and nystatin are not indicated for invasive candida. disseminated histoplasmosis develops in 5% of adult patients with aids in the midwestern region of the united states, and elsewhere. the clinical signs and symptoms related to this infection may be indolent, but left untreated can carry significant morbidity and mortality. 136 the likelihood of disease is higher in patients with cd4 counts under 200 cells/mm 3 . 137 there is enterocolitis associated with infection, and at colonoscopy, plaques, ulcers, pseudopolyps, and skip areas are frequently seen. cryptococcal gastrointestinal disease has been identified in patients with disseminated cryptococcus infection. the esophagus and colon are involved most frequently. p. jirovecii infection of the gastrointestinal tract has also been described. 59 gastrointestinal pneumocystosis develops after hematogenous or lymphatic dissemination from the lungs, or reactivation of latent gastrointestinal infection. the administration of aerosolized pentamidine has increased the risk of developing extrapulmonary spread of p. jirovecii pneumonia. p. jirovecii pneumonia infection can occur throughout the gastrointestinal tract. in the lamina propria there are foamy exudates with p. jirovecii organisms found within them. although more rare, infection of the colon can also cause diarrhea. the effect of haart on rates of infection of the gastrointestinal tract are twofold. first, on a macro level, the advent of haart has led to a dramatic decrease in perinatal transmission of hiv, and therefore the rates of newly infected children have plummeted, 138 with reports of vertical transmission falling between 1 and 2%. 139 second, haart has been successful in immune reconstitution, and therefore in regions with high haart accessibility, there has been a marked decrease in the number of hiv patients presenting with opportunistic infections. these infections have not been eradicated, but the majority of patients adhering to haart are able to achieve cd4+ cell reconstitution and therefore stave these off. patients who sustain chronically low cd4+ t lymphocyte levels in spite of haart accessibility and usage continue to be at risk for the opportunistic infections 140 described in the previous section. when comparing incidence rates of opportunistic infection in the pre-(before january 1, 1997) and post-haart era, there was an overall decrease. 8, 141 specifically: incidence rates of cmv decreased from 1.4 to 0.1; esophageal candidiasis from 0.9 to 0.4; herpes simplex virus from 0.2 to 0; and chronic intestinal and cryptosporidiosis from 1.3 to 0 per 100 persons per year. 141 additionally, nachman et al. 142 reported successful withdrawal of opportunistic infection prophylaxis for a period of 132 weeks in pediatric hiv-positive patients more than 2 years old who had achieved cd4 reconstitution without significant incidence when compared to demographically matched hiv-negative patients. in light of this progress, we have integrated additional immunocompromised patient populations into our discussion of gastrointestinal vulnerabilities. colitis from c. difficile is also more common in the immunosuppressed population owing to chronic antibiotic use and impaired immune system. 143 pulvirenti et al. 144 studied 161 hiv-1-infected patients with c. difficile and found that they had longer hospital stays and more admissions than patients without c. difficile infection, as well as other opportunistic infections such as herpes virus. they found c. difficile-associated diarrhea in 32% of all study patients with diarrhea. however, infection with c. difficile appeared to have little impact on morbidity or mortality. in a 1998, new york state screening study 145 of hospitalized hiv-1-infected patients in the haart era, 2.8% were admitted with a diarrheal diagnosis, with 51.3% of these having a c. difficile infection. thus, even with haart, diarrhea is prevalent and is often associated with identifiable pathogens. c. difficile infection has been reported to be one of the most common bacterial diarrheal pathogens among hiv-infected patients although its rates have decreased with haart. 146 because of the serious complications that are associated with active bacterial enteric infections in immunodeficient children, treatment options are outlined in table 42 -2. h. pylori prevalence is not significantly different between hiv-1-infected patients and hiv-1-negative patients. 147, 148 some investigators have found the seroprevalence of h. pylori to be lower in hiv-1-infected patients, 149 especially as cd4 counts decline with advancing disease. 147 the protection from h. pylori may be a result of frequent antibiotic use or correlated with a more advanced, dysfunctional immune state that results in a decreased inflammatory response to the organism. 150 remission of a high-grade gastric mucosa-associated lymphoid tissue (malt) lymphoma followed h. pylori eradication and haart in a patient with aids. 151 however, a recent study of hivinfected adults showed that 32% of patients with peptic symptoms had h. pylori on biopsy, with those having cd4 counts above 200 cells/mm 3 at a higher risk. 152 treatment of h. pylori in hiv-1-infected children is similar to that in noninfected children, with special attention to drug interactions. in up to 15 to 25% of hiv-1-infected children, the etiology of the diarrhea is unclear. autonomic dysfunction is another potential mechanism of noninfectious diarrhea not previously described. clinically, children with autonomic neuropathy have sweating, urinary retention, and abnormal cardiovascular hemodynamics. it is possible that this autonomic denervation contributes to diarrhea in patients with hiv-1 infection, as suggested by griffin et al. 153 when neuron-specific polyclonal antibodies were applied to jejunal biopsies, there was a significant reduction in axonal density in both villi and pericryptal lamina propria in patients with hiv-1 infection compared with controls, with the greatest reduction in patients with diarrhea. octreotide therapy has shown promising results in some patients. 154 finally, drug side effects should be considered, with many of the antiretroviral therapies causing chronic diarrhea and other gastrointestinal toxicities (table 42-3) . motility problems of the esophagus and stomach have been reported [155] [156] [157] and can be a source of upper gastrointestinal complaints including vomiting, dysphagia, nausea, and dyspepsia. the motility abnormalities may be primary, or they may be secondary to infectious or inflammatory disease of the respective organ. hypertension of the lower esophageal sphincter with incomplete relaxation, esophageal hypocontraction, and nonspecific motility disorders have been described in patients with normal intact esophageal mucosa. 156 gastric emptying, especially in patients with infections or advanced disease, may be delayed, as documented by gastric scintigraphy. 155 however, delayed gastric emptying does not always correlate with upper gastrointestinal symptoms or small bowel motility studies. in adults with hiv-1 infection and minimally advanced disease, gastric emptying of solids was delayed and emptying of liquids accelerated compared with that in controls. no abnormal esophageal motility patterns were found. all patients had a normal endoscopy prior to the motility studies. 157 thus, in the absence of infectious and inflammatory disease in patients with appropriate symptoms, motility studies or empiric trials of prokinetic agents should be considered, with careful consideration of drug interactions. esophageal ulceration can be a result of an intercurrent opportunistic infection. idiopathic oral and esophageal ulcers have been described in both children and adults with hiv-1. 158 these ulcers are characteristically large and may be single or multiple (figure 42-5) . the ulcers are located in the mid to distal esophagus. controversy exists regarding the pathogenesis of these ulcers; some investigators have identified hiv-1 at the ulcer base, 159 whereas others have not. 160 treatment options for these ulcers are limited, but include steroid therapy, with encouraging results, 159 and thalidomide. 161, 162 however, chronic low-dose thalidomide does not prevent recurrence of the oral or esophageal aphthous ulcers. 163 in addition to the potentially teratogenic effects, a significant portion of children receiving thalidomide develop a rash, which precludes use of the drug. significant caution should be exercised when using thalidomide. overall, haart has had a positive impact on esophageal disease occurrence and relapse. 164 the diagnostic approach to the child with hiv-1 or other immunodeficiencies and gastrointestinal symptoms is outlined in table 42 table 42 -2). every effort should be made to correlate timing of the initiation of a drug with onset of symptoms. the clinician should keep in mind that children with active enteric infections may also have secondary problems with malabsorption. if the clinical history and physical examination are suspicious for malabsorption without enteric infection, the next step should include an evaluation of specific nutrient absorption. carbohydrate malabsorption can be detected through lactose breath hydrogen testing, which measures hydrogen production as a response to an oral lactose load. a raised baseline breath hydrogen or early peak of hydrogen production suggests bacterial overgrowth, and appropriate treatment can be initiated. lactose malabsorption results in a level of hydrogen production more than 10 to 15 parts per million over baseline, 60 minutes after ingestion. dietary changes can then be made. d-xylose absorption testing also helps to determine the absorptive capacity of the gastrointestinal tract. d-xylose is an absorbable sugar that does not require active transport for uptake by enterocytes. thus, the d-xylose serum level, after administration of a test dose, reflects the absorptive ability of the gastrointestinal tract and the integrity of the mucosal surface. in younger children, the administered dose is 0.5 g per kg bodyweight, given orally after an overnight fast. in older children and adolescents, the maximum dose is 25 g. a serum level is obtained 1 hour after ingestion. urine samples may be obtained for 5 hours after ingestion as well. plasma citrulline levels correlate with enterocyte mass and function and may be used to indicate gastrointestinal function. 165 fat malabsorption is determined by a 72-hour fecal fat collection. a high-fat diet is administered several days before the collection is initiated and throughout the collection period. an alternative method is to keep a dietary fat intake record during the period of fecal fat collection. the stool is analyzed for total fat content, and the fecal fat is compared with the amount ingested; a coefficient of fat absorption is then calculated. ten percent or more of ingested fat in the stool is considered abnormal. alternatively, a sudan stain may be performed on a random stool sample. this may be helpful as a quick test for fat malabsorption, although it is not so reliable. quantification of fecal elastase may help to determine whether the fat malabsorption is pancreatic in origin. lastly, raised fecal α 1 -antitrypsin levels suggest protein loss from the gut. if noninvasive studies, such as those described above, are not helpful in documenting and determining the etiology of the malabsorption, diarrhea, or vomiting, endoscopy (either upper or lower) with biopsy and appropriate culture of fluid may be useful. miller et al. 41 confirmed histologic abnormalities in 72% of children undergoing upper endoscopy. in 70% of patients in this series, the clinical management of the child was changed because of the endoscopic evaluation. a high diagnostic yield has been supported by other investigators. 166 specific gastrointestinal symptoms are not predictive of abnormal findings at endoscopy; advanced hiv-1 disease stage and an increased number of symptoms seem to be more predictive. 41 histologic studies of the small bowel may aid in determining the degree of the villous blunting, and electron microscopy and special staining for opportunistic pathogens can be performed. quantitative bacterial cultures and parasite evaluation of the duodenal fluid should be obtained when an endoscopy is performed. characteristically, the detection of more than 10 5 organisms per milliliter of duodenal fluid confirms bacterial overgrowth. it is important to obtain both anaerobic and quantitative cultures. however, other studies have shown that endoscopy does not improve the diagnostic yield compared with stool examination in patients with intestinal infection. the only exception is the diagnosis of cmv. 167 an additional study found that flexible sigmoidoscopy was as useful as a full colonoscopy for diagnosing infection. 168 special histologic stains for fungal, mycobacterial, or viral infections did not increase the diagnostic yield over routine hematoxylin and eosin staining. 169 treatment for intestinal infections has been outlined in table 42 -2 and previous sections. therapy for gastrointestinal malabsorption should be directed toward the underlying diagnosis. if clinically symptomatic lactose malabsorption is found, a lactose-free diet should be initiated. compliance may be difficult, because many foods contain lactose. children can limit the effects of dietary lactose by taking exogenous lactase or using lactase-treated milk. there should be careful consideration of calcium and vitamin d intake, because children with hiv-1 infection are susceptible to low bone mineral density. [170] [171] [172] if there is malabsorption of protein and fat, a protein hydrolysate diet should be tried. many of these supplements are poorly tolerated because they are unpalatable. in some circumstances, specialized supplements may need to be administered through a supplemental feeding tube. 173, 174 peptic and motility disorders can be treated as in other, non-hiv-1-infected children, paying careful attention to potential drug interactions with antiretroviral regimens. a variety of other disorders (table 42 -5) can cause secondary immunodeficiencies with effects on the gastrointestinal tract. overall, these disorders are more prevalent than either primary or hiv-1-associated immunodeficiencies. premature infants, children with cancer and associated exposure to immunosuppressant and cytotoxic medications (including children with graft-versus-host disease), and children with protein-losing enteropathy with associated loss of immunoglobulins from the gastrointestinal tract can all be immunodeficient because of the underlying disorder. in general, children with these immunodeficiencies are at risk for many of the same complications that are experienced by children with hiv-1 infection. gastrointestinal tract infections are among the most common problems facing children with other secondary immunodeficiencies. malnutrition is the most common cause of immunodeficiency worldwide. nutritional status and immunity have long been linked in many disease states. before hiv-1 was described, p. carinii (now jirovecii) pneumonia and kaposi's sarcoma, known opportunistic diseases, were first described in otherwise healthy, but malnourished, children and adults in developing nations. 175, 176 this association led investigators to conclude that nutrition alone can affect the immunologic response of an individual. in malnourished children there is a profound involution of lymphoid tissues, including thymic atrophy and diminished paracortical regions of lymph nodes. 177 in young infants and children, protein-calorie malnutrition increases the risk of death by severalfold by increasing the susceptibility to infection. 178 in many countries, the mortality rate increases from 0.5% in children whose weight-for-height percentage of standard is greater than 80%, to 18% in children whose weight-for-height percentage of standard is less than 60%. 179 in other diseases such as cystic fibrosis and cancer, nutritional status has been linked closely to survival and morbidity. malnourished children with leukemia and lymphoma have a higher risk of p. jirovecii pneumonia than children who have normal nutrition. 175 biochemically, protein-calorie malnutrition leads to changes in several aspects of the immune system. cell-mediated immunity, microbial function of phagocytes, complement systems, secretory antibodies, and antibody affinity are consistently impaired in patients with significant malnutrition. additionally, deficiencies of micronutrients, especially zinc and iron, as well as many others, may also have deleterious effects on the immune system. other aspects of immunity that are altered by proteincalorie malnutrition include impaired chemotaxis of neutrophils, decreased lysozyme levels in serum and secretions, and interferon production in antibody response to t-cell-dependent antigens. a child with protein-calorie malnutrition may also have impaired mucosal immunity with lowered concentrations of secretory iga in saliva, nasopharynx, tears, and the gastrointestinal tract compared with well-nourished control children. similar to children and adults with hiv-1 infection, patients with malnutrition have depressed t-cell function not only in the peripheral circulation but also in the intestinal tract. subsequently, plasma cell function and macrophage activity may be impaired, leading to more frequent intestinal infections in children with severe protein-calorie malnutrition. not only does nutrition improve the immunologic functioning of the intestinal tract, but nutrients themselves are trophic and essential for the maintenance of the absorptive capacity of the intestines. in some studies, weight loss greater than 30%, due to other disorders, is associated with a reduction in pancreatic enzyme secretion of over 80%, villous atrophy, and impaired carbohydrate and fat absorption. 180 these disorders are promptly reversed with appropriate nutritional rehabilitation. with villous blunting, antigen uptake can increase, leaving the child at higher risk of enteric infection. the pathogenesis of villous blunting is unclear but may be due to crypt hyperplasia as the primary event with premature sloughing at the villus tip 181 versus loss of enterocytes at the villus tip with resultant proliferation at the crypts. 182 malnutrition and its associated immunodeficiency are of global concern, and researchers have experimented with both dietary regimens and micronutrient supplementation to improve, and perhaps ultimately restore, adequate immunological function. 183, 184 because of the low cost of many micronutrients when compared to pharmacological agents, success in such experimentation could have profound implications for those suffering from malnutrition, as well as other immunocompromised patients. zinc is accepted as a promoter of immune function and consequently has been evaluated in hiv-1 immunocompromised patients. in a south african study that treated patients with 10 mg of zinc (elemental) daily for 6 months and compared them to a control group receiving a placebo, there was a significant difference in patient presentation of diarrhea favoring zinc supplementation. 185 further demonstrating its potential alleviatory effects, canani et al. 186 observed that the transactivating peptide's (tat) secretory mechanism was inhibited by zinc and subsequently prevented diarrhea in pediatric hiv-1 patients. both of these positive outcomes, although documented in hiv-1 patients, present zinc as an additional treatment option for symptoms of malnutrition-related immunodeficiency. some studies have administered multivitamins to hiv-1 patients including adults and children and evaluated effects of specific micronutrients. adequate levels of vitamin a, when bolstered by supplementation in hiv-1 positive and hiv-1 negative children older than 6 months, correlated with reduced mortality and morbidity. 187 although not yet well-documented in children, multivitamin intake of hiv-1 positive adults demonstrated retarded progression of the virus. 187 improved hematologic status, mainly decreased rates of anemia, was observed in women and their children in tanzania who were treated with iron supplements during and after pregnancy. this was marked by an average hemoglobin count that was 0.33 g/dl greater than that of the patient group who did not receive multivitamin treatment. 188 these findings underscore the need for additional randomized control trials in pediatric populations to further understand the role of micronutrient supplementation as a complimentary treatment component. immunosuppressant medications are the mainstay of therapy for many diseases in children with autoimmune disorders, inflammatory bowel disease, chronic pulmonary disease, cancer, and organ transplantation. the best known immunosuppressants include corticosteroids, azathioprine, cyclosporin, tacrolimus, and anti-thymocyte globulin. unfortunately, the effects of these medications are not targeted toward specific organs, but rather indiscriminately suppress immune function throughout the child. thus, several immunologic functions including a decrease in monocyte adherence, neutrophil chemotaxis, and overall suppression of the inflammatory response are present. children are at risk of enteric infections, similar to those described in children with hiv-1 infection. pediatric patients undergoing transplant procedures, specifically solid organ transplant, are at increased risk of acquiring gastrointestinal infections when compared to their healthy counterparts. acutely, these complications present with vomiting, diarrhea, and cramping. the most frequently diagnosed posttransplant infection is cmv, [189] [190] [191] but its onset has been reduced significantly with the administration of prophylactic drugs such as ganciclovir. [189] [190] [191] [192] another aspect of the gastrointestinal tract that renders importance in secondary immunodeficiency is the liver. although data regarding liver complications in pediatric hiv-1 patients are lacking, there are considerable reports on hiv-1 infected adults. hepatitis coinfection, biliary tract disease, and drugderived illness are some of the major complications we discuss in this section. liver complications in secondary immunodeficiency, in their own right, deserve extensive coverage beyond the scope of this chapter, and so this section offers only a snapshot of this complex topic. both hepatitis and hiv infections share similar transmission pathways, and for this reason, it is not surprising that rates of viral coinfections are considerable. hepatitis a is often thought of as the less serious of the hepatitis viruses when treated promptly, and coinfection with hiv does not seem to predispose an individual to adverse outcomes. in 2006, coinfection rates of hepatitis b were reported to be between 5 and 10% in the global hiv population. prolonged hepatitis b infection is one of the greatest concerns of for coinfected patients. 193 hepatitis b virus e antigen (hbeag) is the protein associated with active hepatitis b in patients and is used by clinicians to determine efficacy of medications. for monoinfected hepatitis b patients, proper treatment can result in significant reduction of hbeag in 90 to 95% of patients; however, this success is not mirrored in hiv-coinfected populations. 194 there is also evidence for greater reactivation and replication in hepatitis b-hiv coinfected patients. 193, [195] [196] [197] explanation for this focuses on hiv patients' increased proinflammatory cytokine production and their inability to eliminate hepatocytes infected with hepatitis b. 196, 198 balancing dual treatment for both hepatitis b and hiv viruses presents a unique challenge for clinicians. acquired mutations of hepatitis b render additional treatment considerations, deepening the challenge. resistant hepatitis b strains have been identified, many of them falling under the tyrosine-methionine-aspartate-aspartate category, ymdd. iacomi et al. 199 determined that 28 of 29 hepatitis b-hiv coinfected patients exhibited this specified resistance. lamivudine, once commonly prescribed, has become less effective because of developed resistance. of the various treatment options, only tenofovir is used in hiv treatment and is effective in both the wild-type hepatitis b virus and the resistant ymdd strain, and it is often concomitantly administered with emtricitabine. 200, 201 mothers coinfected with hepatitis c and hiv are more likely to transmit hiv to their children, indicating greater risk of perinatal transmission in coinfected patients. 202, 203 liver disease in coinfected patients may be accelerated when compared to their monoinfected counterparts. pathways that have been proposed to accelerate fibrosis in coinfected patients include direct viral effects, dysregulation of the immune system toward a profibrotic state, and other metabolic pathways that lead to liver toxicity and processes such as steatosis and insulin resistance. 204 giovaninni et al. 203 studied 49 hiv-infected children, 11 of whom were coinfected with hepatitis c. six of the coinfected patients had abnormal alanine aminotransferase (alt) levels. three of the six children had aids, and three had aids-related complex. five children with normal aminotransferase had no detectable viral progression. following acute hepatitis c infection, hepatitis c-hiv coinfected patients are over 20% more likely than monoinfected patients to develop chronic hepatitis c infection. [205] [206] [207] interferon with ribavirin therapy is widespread in chronic hepatitis-c monoinfected patients, and its efficacy in hepatitis c-hiv coinfected patients is yet to be fully realized. 208, 209 when compared to conventional interferon therapy (ifnα-2a), 180 μg/week pegylated interferon (pegifnα-2a) plus 800 mg/day ribavirin demonstrated enhanced histological effects that were also associated with improved virological response in hepatitis c-hiv coinfected patients. 210 end-stage liver failure, cirrhosis, and hepatocellular carcinoma have been observed with greater frequency in hepatitis c-hiv coinfected patients. 205 these findings reflect adult populations and may or may not be indicative of outcomes in pediatric patients. children with acute biliary tract disease may present with rightsided abdominal pain, vomiting, and jaundice. also, elevated serum bile levels may induce pruritus. despite a disproportionate increase in alkaline phosphatase levels, alt levels may or may not be elevated. an ultrasound of the biliary system may be needed when serum sampling is equivocal. biliary tract disease is often obstructive; thus, use of endoscopic retrograde cholangiopancreatography (ercp) will help to identify the obstruction, perhaps provide bile sampling, and even mitigate the obstruction via sphincterotomy. bile sampling may indicate the presence of cmv, cryptosporidium, mycobacterium, or microsporidia, which were all discussed in previous sections. in one study employing sonography, 26 of 41 hiv-infected children displayed hepatobiliary abnormalities, yet there was no evidence of infection. 211 aids cholangiopathy is defined by intraand extrahepatic sclerosing cholangitis and is commonly linked to cmv and cryptosporidium infections. incidence in pediatric hiv populations remains to be thoroughly evaluated. the advent of haart, as discussed previously, has had profound effects on reducing many infections within the gastrointestinal tract. in the liver, however, there has been a negative association between receipt of antiretroviral therapy and development of liver complications, defined as immune restoration hepatitis. 193 serum alt elevation is associated with liver tissue damage and therefore is used to detect liver disease. hepatitis-hiv patients on haart present with elevated alt levels in the blood principally derived from haart-induced hepatotoxicity, resistance to antiretrovirals, and haart noncompliancy. 212 specifically, mitochondrial damage has been implicated as a contributor to liver death, which stems from coinfection and drug treatments. 213, 214 current research aims to determine effective methodology for detecting hepatic mitochondrial toxicity as a means to identify patients at risk of developing liver complications due to treatment. 213 gastrointestinal disorders in children with secondary immunodeficiencies cause considerable comorbidity. infection of the gastrointestinal tract is one the most common complications associated with secondary immunodeficiencies. however, malabsorption, peptic disease, and liver and biliary tract disease are also prevalent. these gastrointestinal complications contribute negatively to the overall clinical outcomes of children who have other chronic medical conditions, and they can often become life-threatening. thus, clinicians should be vigilant and aggressive in the evaluation and treatment of gastrointestinal tract dysfunction in children with secondary immunodeficiencies. incidence of opportunistic and other infections in hiv-infected children in the haart era microbial translocation is a cause of systemic immune activation in chronic hiv infection ritonavir combination therapy restores intestinal function in children with advanced hiv disease plasma citrulline is a biomarker of enterocyte mass and an indicator of parenteral nutrition in hiv-infected patients interactions of nutrition and infection coinfection with hiv-1 and hcv -a one-two punch see 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immunodeficiency virus infection and gastrointestinal disease effect of hiv co-infection on mutation patterns of hbv in patients with lamivudine-resistant chronic hepatitis hepatitis b in hiv patients: what is the current treatment and what are the challenges? diagnosis and management of hepatitis b virus and hiv coinfection hepatitis c in patients with human immunodeficiency virus infection: diagnosis, natural history, meta-analysis of sexual and vertical transmission, and therapeutic issues maternal-infant transmission of hepatitis c virus and hiv infections: a possible interaction coinfection with hiv-1 and hcv -a one-two punch hiv and hepatitis c coinfection protection against persistence of hepatitis c the natural history of hepatitis c virus infection: host, viral, and environmental factors ribavirin in the treatment of chronic hepatitis c peginterferon-α-2a (40kd) plus ribavirin: a review of its use in hepatitis c virus and hiv co-infection histological response to pegifnα-2a (40kd) plus ribavirin in hiv-hepatitis c virus co-infection hepatobiliary abnormalities on sonography in children with hiv infection hiv and chronic hepatitis: co-infection with hiv and hepatitis b virus infection disease-and treatment-related predictors of hepatic mitochondrial dysfunction in chronic hiv infection assessed by non-invasive 13 c-methionine breath test diagnostic alteration of cytochrome oxidase subunit i labeling is associated with severe mitochondropathy in nrti-related hepatotoxicity in hiv patients key: cord-286368-kdwh4hgf authors: hui, david s.c.; lee, nelson; chan, paul k.s. title: a clinical approach to the threat of emerging influenza viruses in the asia‐pacific region date: 2017-07-05 journal: respirology doi: 10.1111/resp.13114 sha: doc_id: 286368 cord_uid: kdwh4hgf seasonal influenza epidemics and periodic pandemics are important causes of morbidity and mortality. patients with chronic co‐morbid illness, those at the extremes of age and pregnant women are at higher risks of complications requiring hospitalization, whereas young adults and obese individuals were also at increased risk during the a(h1n1) pandemic in 2009. avian influenza a(h5n1) and a(h7n9) viruses have continued to circulate widely in some poultry populations and infect humans sporadically since 1997 and 2013, respectively. the recent upsurge in human cases of a(h7n9) infections in mainland china is of great concern. sporadic human cases of avian a(h5n6), a(h10n8) and a(h6n1) have also emerged in recent years while there are also widespread poultry outbreaks due to a(h5n8) in many countries. observational studies have shown that treatment with a neuraminidase inhibitor (nai) for adults hospitalized with severe influenza is associated with lower mortality and better clinical outcomes, especially when administered early in the course of illness. whether higher than standard doses of nai would provide greater antiviral effects in such patients will require further investigation. high‐dose systemic corticosteroids were associated with worse outcomes in patients with severe influenza. there is an urgent need for developing more effective antiviral therapies for treatment of influenza infections. severe acute respiratory infections such as avian influenza a(h5n1) 1 and a(h7n9) viruses 2 with pandemic potential have continued to circulate widely in some poultry populations and infect humans sporadically. infection caused by these viruses may result in severe disease and high case fatality rates because most humans have no background immunity to these viruses. sporadic human cases due to avian a(h5n6), 3 a(h10n8) 4 and a(h6n1) 5 have also emerged in recent years. the global circulation of oseltamivir-resistant seasonal influenza, the emergence of a(h1n1)pdm09 virus in 2009 followed by its continual circulation, 6 the rising number of a(h7n9) infections in humans 2 and the ongoing spread of a(h5n8) in recent months in the poultry populations in many countries in asia, africa, europe and middle east with pandemic potential 7 all point to an urgent need for developing more effective antiviral therapies to reduce morbidity and mortality. this article reviews the epidemiology, clinical, diagnostic and treatment aspects of these important and emerging influenza viruses that are posing threats to human health in the asia-pacific region. seasonal influenza is a major health problem. for instance, in hong kong, influenza a and b accounted for 39.4% and 10.2%, respectively, of hospital admissions confirmed to have viral infections. 8 the average annual incidence of hospitalization with laboratoryconfirmed influenza a infection was 107.7/10 000 and 18.3/10 000 among children aged <5 years and elderly ≥65 years, respectively. furthermore, influenza a was the most common (45.5%) virus detected from patients who died of respiratory viral infections. although influenza b is usually associated with a less degree of disease burden, its impact on human health is substantial. our previous study conducted in hong kong revealed that 24% of laboratory-confirmed influenza-associated hospital admissions were due to influenza b, and was much higher (41.9%) among older children and adolescents (aged 5-14 years). 9 this observation is similar to data in europe and north america. in the temperate region, influenza exhibits an annual seasonal peak in winter. however, the seasonality in tropical and subtropical regions is more variable. for instance, in hong kong, a subtropical city, most of the time, influenza exhibits two peaks during january-march and june-august, and occasionally, the two peaks merge without an obvious gap. 8, 10 such bimodal and variable seasonality of influenza is also observed in countries within the subtropical and tropical regions, and poses a challenge to implement annual influenza vaccination campaign as well as a question on whether the northern or southern hemisphere version of influenza vaccine would be more appropriate. a schematic diagram of the structure of influenza virion is shown in figure 1 . influenza represents one of the few examples of viruses with segmented genome. this, together with the widespread infection with different subtypes in various animal species, allows robust evolution through the process of 'reassortment'. the prerequisite for reassortment is a simultaneous infection with more than one strain of viruses in a host referred to as the 'mixing vessel' that allows replication and swabbing of gene segments between different strains of viruses. water birds are well-known mixing vessels, but the reassortants are more likely to have a tropism restricted to avian species. these new reassortants may pose problems in the land birds and poultry, and subsequently be of human concern. swine is another well-known mixing vessel. the unique property of swine is their susceptibility to both avian and human influenza viruses. thus, reassortants from swine are more likely to gain tropism for infection in humans. field observations made as early as 1990s have pointed out that influenza outbreaks that began with either pigs or people were then rapidly transferred to each other. 11 swine are likely to play the role of a mixing vessel in creating pandemic strains. although the 1918 pandemic strain was most probably an entirely avian-like virus, 12 the 1957 and 1968 pandemic strains were reassortants of human-and avian-origin viruses. 13 while the most recent pandemic, a(h1n1)pdm09, was not as severe as those that occurred before, there is no guarantee that the next pandemic will be as 'mild'. at present, a few subtypes (h5, h7 and h9) are of particular concern. cross-species infections, especially the recent strong wave of human a(h7n9) infection in mainland china underscores that the next pandemic could be imminent. 14, 15 countries should have pandemic preparedness in place, including antiviral stockpiling and pandemic vaccine preparations. these would be better achieved under joint multigovernment industrial partnership. human cases of the highly pathogenic avian influenza a(h5n1) were first detected in hong kong in 1997. 1 the number of affected countries increased between 2003 and 2008, with expansion from east and southeast asia, then to west asia and africa, with a sharp rise in egypt since november 2014. 16 as of 16 march 2017, there have been 453 deaths out of 858 human cases in 16 countries since 2003. 17 a canadian tourist died of a(h5n1)-related meningoencephalitis in alberta in january 2014 after visiting beijing in december 2013 without any obvious exposure to infected avian sources or environmental contamination. 18 a history of exposure to dead or sick poultry/wild birds occurs in over 60% of cases of human a(h5n1) infection. the incubation period for a(h5n1) infection ranges from 2 to 8 days but may be as long as 17 days. the clinical spectrum may range from asymptomatic infection, mild influenza-like illness to severe pneumonia, with multiorgan failure. 19 some of the a(h5n1) human cases have been linked to consumption of dishes made of raw, contaminated poultry blood. however, slaughter, defeathering, handling carcasses of infected poultry and preparing poultry for consumption, especially in household settings, appear to be important risk factors. 19, 20 among the fatal cases, the median duration from symptom onset to death was 9-10 days (range: 6-30 days). 20 viral rna was present in the blood of fatal a(h5n1) cases and this was associated with higher pharyngeal viral loads. nasopharyngeal swab samples, bronchoalveolar lavage and cerebrospinal fluid samples were positive in the canadian case for influenza a(h5n1) virus by various molecular testing methods, including sequencing. 18 in addition, lymphopenia and high chemokine/cytokine levels have been observed in severe a(h5n1) infection and these correlated with pharyngeal loads. 21 a new reassortant genotype of a(h5n1) containing the haemagglutinin (ha) and neuraminidase (na) genes from clade 1.1.2 and the internal genes from clade 2.3.2.1 was detected in 2013 and associated with the highest number of cases (n = 26) and deaths (n = 14) in cambodia. 22 complete viral genome analysis of the fatal case in canada has shown an ha gene of clade 2.3.2.1c and was a reassortant with an a(h9n2) subtype lineage polymerase basic 2 gene without mutations conferring resistance to adamantanes or na inhibitors (nai). 18 human-human transmission remains rare as a meta-analysis has shown that only 1-2% of more than 12 500 study participants from 20 studies exposed to patients with a(h5n1) infection had serological evidence for prior a(h5n1) infection. 23 delay in the delivery of appropriate treatment to patients with influenza a(h5n1) infection in indonesia was mainly related to delay in diagnosis rather than late presentation to healthcare settings. 24 age, country, per capita government health expenditure and delay from symptom onset to hospitalization were the risk factors for mortality related to a(h5n1) infection, emphasizing the importance of early diagnosis, treatment and supportive care. 25 a systematic review has shown that females were at higher risk of death (or: 1.75, 95% ci: 1.27-2.44) following a(h5n1) infection, whereas young age in particular <5 years was protective (or: 0.44, 95% ci: 0.25-0.79). 26 human infections with a novel avian influenza a (h7n9) virus were first reported in china in march 2013 in patients hospitalized with severe pneumonia. 27 there have been five seasonal epidemics in china since the virus was first discovered in 2013. the estimated risk of death (hospitalization fatality risk, hfr) among hospitalized cases of a(h7n9) infection in the second epidemic was 48% (95% credibility interval: 42-54%), which was slightly higher than the corresponding risk of 36% in the first wave. in the second epidemic, the hfr was estimated at 36% (95% ci: 28-45%) in patients aged <60 years but at 59% (95% ci: 51-67%) among those aged ≥60 years. 28 an upsurge in human a(h7n9) infections has been observed since november 2016 in mainland china. during this fifth epidemic wave, the number of human cases is higher than during the previous two waves during 2014-2015 and 2015-2016. the majority of recently reported human cases are associated with exposure to infected live poultry or contaminated environments, including wet markets where live poultry are sold. in addition, the human cases are more geographically widespread and cases are also reported from rural areas, unlike in previous epidemics (fig. 2) . 29, 30 the phylogenetic analysis on ha genes of isolates collected from humans suggested that the virus is evolving and with the more recent isolates clustered to a separate branch (supplementary figure s1 ). 31, 32 as of 5 april 2017, 1364 laboratory-confirmed cases of human infection with avian influenza a(h7n9) virus have been reported to world health organization (who). 33 human cases of a(h7n9) infection exported from the mainland of china have been confirmed in hong kong (n = 21), taiwan (n = 4), macau (n = 2), canada (n = 2) and malaysia (n = 1) in visitors who developed illness after returning from the mainland of china to their home cities. 29, 30, 33 the incubation period of human infection with the a(h7n9) virus ranges from 1 to 10 days, with an average of 5 days. the median time from poultry exposure to disease onset was 6 days, whereas the median time from illness onset to hospitalization, acute respiratory distress syndrome (ards) development, antiviral therapy and death were 4, 7, 6 and 21 days, respectively. 34 preexisting co-morbid conditions occurred in >60% of these cases. the prominent clinical features on admission were those of a severe influenza syndrome with fever, cough, fatigue and dyspnoea, whereas the most striking laboratory findings were marked lymphopenia and thrombocytopenia. elevated cytokine levels have been observed in patients and the excessive cytokine responses may contribute to the clinical severity of a(h7n9) infection. 35, 36 limited human to human transmission with at least two epidemiologically linked cases due to close household contact with the index patients has been reported in a number of family clusters in both a(h5n1) 20, 37, 38 and a(h7n9) infections. 34, 39, 40 nosocomial transmission of a(h7n9) infection between two patients sharing a hospital room, 41 and co-transmission of avian influenza a(h7n9) and a(h1n1)pdm09 viruses between two patients with haematological disorders sharing the same room with bed distance <1 m have been reported. 42 comparisons of the clinical features between human cases of a(h5n1) and a(h7n9) are shown in table 1 . 34, 43 closure of live poultry markets (lpm) in the mainland of china has tremendously reduced the risk of a(h7n9) infection temporarily but closure is difficult to sustain due to cultural preference for live poultry. an ecologic modelling study estimated that closure of lpm reduced the mean daily number of a(h7n9) virus infections in the four most affected cities by 97-99%. 44 a retrospective serological study of blood specimens taken during january-may and october-november in 2012 from 1544 subjects who worked in lpm, farms, slaughter houses or kept backyard poultry in eastern china before the epidemic occurred in 2013 revealed no evidence of a(h7n9) infection. 45 other novel avian influenza subtypes the first human case of avian a(h6n1) infection was reported in a 20-year-old female with pneumonia in taiwan in may 2013, with subsequent full recovery following treatment with oseltamivir. 5 the first human case of avian a(h5n6) was confirmed in a 49-year-old male in sichuan province, onset of illness to death (median, days) 21 11 43 case fatality rate in hospital 34% 53% china, in may 2014 with a fatal outcome 3 and a second case was confirmed in a 58-year-old male with history of exposure to live poultry in guangdong province in december 2014. 46 the virus was a reassortant that contained seven genes from a(h5n1) and the na gene from an a(h6n6) virus circulating in ducks. 47 (fig. 3 ). 29 the latest case was reported on 1 december 2016. 48 in addition, china has confirmed three human infections, two fatal, with avian a(h10n8) viruses that contain the internal genes from a(h9n2), as does a(h7n9). 4, 49 similar to the a(h7n9) virus, the a(h6n1) and a(h10n8) viruses have low pathogenicity in poultry and are therefore more difficult to detect in birds in contrast to the highly pathogenic a(h5n1) virus. highly pathogenic avian influenza a(h5n8) viruses belonging to clade 2.3.4.4 of the a/goose/guangdong/ 1/1996 lineage were detected in 2014 in wild birds and poultry in china, japan, south korea, netherlands, germany, italy, russia, the uk and northern ireland. the virus was detected sporadically in canada and the usa in wild birds and poultry until mid-2015. the a(h5n8) viruses were also detected in taiwan, china and in hungary and sweden in 2015. since june 2016, many more countries in both europe and asia have detected infections in wild birds and/or domestic poultry with a(h5n8). many of these recent detections were associated with mortality in wild birds. 7,50 although human infection with a(h5n8) has not been detected, the ongoing spread of this virus to different countries in recent months is worrisome and may increase the risk of human infection. visitors who have returned from areas affected by avian influenza and developed respiratory symptoms and fever within 10 days after their return should be enquired about their relevant history (travel history, occupation, contact history and clustering) to facilitate early diagnosis and treatment. it is important to obtain any history of contact with poultry (live or sick/dead poultry as a(h7n9) virus may cause no or mild symptoms in poultry in contrast to a(h5n1)), exposure to wet markets or known avian influenza cases, and laboratory exposure including staff handling with spillage. patients with influenza-like illness who present with dyspnoea, tachypnoea, evidence of hypoxaemia and pulmonary infiltrates on chest radiograph should be hospitalized. 51 early case identification and isolation precautions would facilitate clinical management which may reduce the risk of severe infection and related complications for seasonal epidemic, zoonotic and pandemic influenza, in addition to reducing the risk of influenza transmission and mitigating the impact of outbreaks on the healthcare system. common findings in severe influenza infection include low or normal white cell counts, lymphopenia and thrombocytopenia, and increased serum transaminases, lactate dehydrogenase (ldh), creatine kinase and urea/creatinine. 6, 20, 27, 51 in the influenza clinical information network (flucin) study, early elevation of c-reactive protein ≥100 mg/l was an independent predictor of severe outcome. in a study of 1770 patients hospitalized with community-acquired pneumonia (cap), procalcitonin concentration had a strong association with need for invasive mechanical ventilation (imv) or inotrope support (in 6.5% of those studied). 52 higher levels of certain biomarkers on presentation linked to inflammation, coagulation or immune function (interleukin 6 (il-6), cluster of differentiation 163 (cd163), interferon gamma-induced protein 10 (il-10), lipopolysaccharide binding protein (lbp), il-2, monocyte chemoattractant protein-1 (mcp-1) and interferon gamma-induced protein 10 (ip-10)) have been associated with disease progression in both outpatients and those hospitalized with influenza. 53 laboratory diagnosis plays an important role in the management of influenza. the narrow window of an effective antiviral intervention for both seasonal and avian influenza demands a rapid turnaround time. 6, 54 nowadays, most laboratories employ either real time reverse transcription polymerase chain reaction (rrt-pcr)-based methods or antigen detection-based assays. while the rrt-pcr approach is most sensitive and can be applied to a wide variety of specimens including the less invasive throat swab samples, it requires a certain degree of laboratory setup and technical skills, which are not readily available in many healthcare settings. the antigen-based rapid influenza diagnostic test (ridt), although less sensitive, is still the test of choice particularly as a point-of-care test. our experience on a few ridt suggested that the sensitivity could be lower for patients who presented more than 2 days after the onset of illness, for young children compared to elderly, and for cases with pneumonia compared to those without. 55 it is therefore crucial to evaluate the performance with respect to the setting that ridt will be applied. the specimen of choice is critical. while in most situations, nasopharyngeal aspirate/swab is the preferred choice of specimen for seasonal influenza, falsenegative results may occur in patients with severe pneumonia where the viral load remains high in the lower respiratory tract (lrt) but has become undetectable in the upper respiratory tract (urt). 56 similarly, lrt specimens such tracheal aspirates or sputum are the specimen of choice for diagnosis of avian influenza infection in humans as the viral load in the urt is typically low. the development and availability of more affordable and reliable molecular diagnostic tests including multiplex pcr would facilitate future clinical management of patients with severe cap due to viral or bacterial aetiology. 57 the advantages and limitations of different diagnostic tests for influenza are summarized in table 2 . 55, 57 the m2 inhibitors (amantadine and rimantadine) and the nai (oseltamivir, peramivir, zanamivir and laninamivir) are the main antiviral agents approved for the prevention of and treatment for influenza. in general, antiviral treatment should be commenced as soon as possible for any patient hospitalized with confirmed/ suspected influenza with severe, complicated or progressive illness, and also for outpatients at higher risk for influenza complications. 58 the m2 inhibitors (adamantanes) are not effective against influenza b viruses and recently circulating influenza a(h3n2) and influenza a(h1n1)pdm09 viruses, which are resistant due to an s31n mutation in the m2 ion channel. however, as some avian influenza a(h5n1) strains are still susceptible, 59 combination of an adamantane with an nai may enhance antiviral activity for susceptible isolates. 60 oseltamivir is effective in reducing mortality (or: 0.17; p = 0.04) in influenza a(h5n1) infection when given before the onset of respiratory failure, 61 and may provide some survival benefit (49% reduction in mortality) when treatment is started within 6-8 days after symptom onset. 62 several observational studies have shown that treatment with oseltamivir for adults hospitalized with severe influenza is associated with lower mortality and better clinical outcomes, especially when antiviral treatment has been initiated within 2 days of illness onset but even as late as 4-5 days after symptom onset. 63, 64 a systematic review has shown that in prophylactic studies, oseltamivir could reduce the proportion of symptomatic influenza whereas in treatment studies it modestly reduced the time to first alleviation of symptoms by 16.8 h, but it caused nausea, vomiting, headaches and renal/psychiatric side effects. 65 a meta-analysis of randomized controlled trials (rct) has shown that oseltamivir in adults with influenza shortens time to clinical symptom alleviation by 21%, reduces risks of lrt complications and hospitalization, but increases the occurrence of nausea and vomiting. 66 oseltamivir resistance is infrequent in a(h5n1), and <3% in circulating a(h1n1)pdm09, a(h3n2) and b viruses but it is important to monitor for antiviral resistance when managing patients with severe influenza. 67 all h7n9 viruses are amantadine resistant due to the s31n substitution in the m2 ion channel protein. in patients hospitalized with severe a(h7n9) infection, reduction of viral load following oseltamivir treatment was associated with improved outcome, whereas the emergence of virus resistant to nai harbouring an r292k mutation was associated with poor outcomes and lack of response to oseltamivir and peramivir, and reduced susceptibility to zanamivir and laninamivir (50-and 25-fold rises in inhibitory concentration by 50% (ic 50 ), respectively). two patients with severe a(h7n9) infection and r292k mutation requiring extracorporeal membrane oxygenation had received systemic corticosteroid treatment which might have contributed to treatment failure and a fatal outcome. 68 the recommended treatment duration of oseltamivir is generally 5 days, but longer treatment (10 days, then guided by clinical and virological testing as recommended by the who) is advisable for critically ill patients with respiratory failure with persistent viral replication in the lrt. 51, 58, 69 whether higher than standard dose of nai would provide greater antiviral effects in such patients requires further investigation. one rct of hospitalized patients (76% being children) revealed no clinical or virological advantages when comparing double dose of oseltamivir against standard dose. 70 no additional benefit of higher dose oseltamivir treatment was observed in adults hospitalized with influenza a, although a faster virological response was noted in influenza b. 71 however, an rct of 18 critically ill patients with a(h1n1)pdm09 in canada found that therapy with a triple dose of oseltamivir was associated with higher proportions of viral clearance at 5 days than standard therapy (78% vs 11%; p = 0.015). 72 intravenous (i.v.) peramivir treatment was associated with viral rna decline as well as culture and rna negativity, which occurred at similar rates to those on oral oseltamivir by day 5 among adult patients hospitalized for influenza-associated lrt complications. 73 the various nai dosages for adults with adjustment for renal failure are shown in table 3 . 58 zanamivir and laninamivir have quite similar profiles of drug susceptibility. one example is that h275y mutation, which confers high level resistance to oseltamivir carboxylate and reduced susceptibility to peramivir in n1-containing viruses, does not reduce susceptibility to zanamivir and laninamivir most ridt are chromatographic immunoassays (some are fluorescence-based immunoassays); applicable to np swabs, nasal and/or throat swabs and np aspirates/washes (training and protection equipment are required). performance is best if applied within 48-72 h from onset before a significant drop in viral load (up to 4-5 days in selected populations). lower sensitivity for a(h1n1) pdm09 virus has been reported. viral cell culture detects viable viruses, including those contained in the live-attenuated influenza vaccines (laiv). isolates can be subjected to phenotypic resistance assays (e.g. neuraminidase enzyme inhibition assay). viral load, specimen quality, transport, storage and processing techniques may affect test performance. pcr assays can either provide universal detection of influenza a virus by targeting the matrix (m) gene or subtype-specific virus detection (e.g. h1n1pdm09, h3n2, h5n1 and h7n9) by targeting the haemagglutinin (ha) gene. viruses that cannot be subtyped may indicate a novel strain. newer molecular-based point-of-care tests may improve accessibility and reduce processing time and technical demands; some may allow detection of multiple viruses. cost-effectiveness of pcr is variable, depending on the circumstances. some multiplex pcr platforms may provide detection of >14 respiratory viruses (e.g. rsv, human metapneumovirus, parainfluenza virus, rhinovirus and coronavirus) and atypical pathogens (e.g. mycoplasma pneumoniae and chlamydophila pneumoniae). dfa, direct fluorescent antibody test; ifa, immunofluorescence assay; np, nasopharyngeal; ridt, rapid influenza diagnostic test; rsv, respiratory syncytial virus. significantly. 74 intravenous zanamivir was used widely on a compassionate ground for late treatment of critically ill adults with influenza a(h1n1)pdm09 and those with suspected or proven oseltamivir resistance. 75 there were no drug-related trends in safety parameters and in a subset of 93 patients with positive pcr tests at baseline for influenza, showed a median decrease in nasopharyngeal viral rna load of 1.42 log 10 copies/ml after 2 days of treatment. 76 intravenous zanamivir was used with a favourable outcome in a patient with severe a(h7n9) infection complicated by pneumonia which did not respond to oseltamivir initially. 77 favipiravir, which is an inhibitor of a viral rna-dependent rna polymerase, has strong antiviral activity against nai-resistant and sensitive influenza viruses. 67, 78 systemic corticosteroids and other immunomodulating agents for severe influenza systemic corticosteroid has been used frequently for the treatment of influenza-related ards. however, a meta-analysis of data predominantly related to treatment of severe influenza a(h1n1)pdm09 has shown that systemic corticosteroid was associated with an increase in mortality (or: 3.06, 95% ci: 1.58-5.92). pooled subgroup analysis of adjusted estimates of mortality from four studies found or of 2.82 and 95% ci of 1. 61-4.92. 79 in comparison to controls, high-dose corticosteroids (>150 mg/day methylprednisolone eqv) was associated with increased risks in 30-day mortality (38.5% vs 7.7%, p = 0.021) and 60-day mortality (50% vs 15.4%, p = 0.022) and longer viral shedding (15 days vs 13 days, p = 0.039) in patients with influenza a (h7n9) viral pneumonia while there was no difference between low dose (25-150 mg/day methylprednisolone) and controls. 80 in a study of 2649 adults hospitalized with influenza in hong kong, singapore and beijing during 2008-2011, 23.1% had received systemic corticosteroids. bacterial superinfections increased risk of death (adjusted hazard ratio (hr): 2.2, 95% ci: 1.5-3.1). systemic corticosteroids increased risks of superinfections (from 2.7% to 9.7%) and deaths when controlled for indications (adjusted hr: 1.7, 95% ci: 1.1-2.6). 81 among adults with severe sepsis not in septic shock, use of low-dose hydrocortisone versus placebo did not reduce the risk of septic shock within 14 days. the findings do not support the use of hydrocortisone in these patients without septic shock. 82 the role of low-dose systemic corticosteroid needs further investigation by a properly planned rct. 83 in a study of critically ill patients infected with influenza a (h1n1)pdm 09 requiring imv, addition of a mammalian target of rapamycin inhibitor, sirolimus 2 mg/day for 14 days to oseltamivir and prednisolone (n = 19), was associated with a higher frequency of liberation from imv (84.2% vs 47.4%, p = 0.04), a shorter duration of imv (13.8 days vs 33 days, p = 0.03) and a higher chance of achieving lrt viral rna negativity by day 7 (75% vs 33%, p < 0.05) than without addition of sirolimus (n = 19). 84 the role of sirolimus plus oseltamivir versus oseltamivir alone in a larger sample size should be examined without systemic corticosteroid. a recent study of adults hospitalized for a(h3n2) has shown that a triple combination treatment of 2 days of clarithromycin 500 mg, naproxen 200 mg and oseltamivir 75 mg twice daily (bd), followed by 3 days of oseltamivir reduced both 30-and 90-day mortalities and length of hospital stay versus oseltamivir 75 mg bd without placebos for 5 days as control. 85 confirmatory studies of the role of this triple combination would be of interest. exploratory post hoc meta-analysis of studies of severe acute respiratory syndrome (sars) and severe influenza showed a significant reduction in the pooled odds of mortality following convalescent plasma versus placebo or no treatment (or: 0.25; 95% ci: 0.14-0.45). 86 thus, convalescent plasma and hyperimmune globulin may 88 nosocomial opportunistic airborne transmission of a(h3n2) has been implicated in a medical ward with imbalanced airflow and use of non-invasive ventilation (niv) for an index patient with acute exacerbation of chronic obstructive pulmonary disease (copd). 89 standard, contact and droplet precautions are recommended for routine management of patients hospitalized for influenza. 90 the main infection prevention and control measures for managing influenza are droplet precaution (by wearing a surgical mask within 1 m of the patient) and contact precaution (by wearing gown and gloves on entering the room and removing them on leaving). droplet precautions should be added to the standard precautions when providing care to all patients with symptoms of acute respiratory infection. contact precautions and eye protection should be added when caring for probable or confirmed cases of avian influenza infection (table 4 ). 90 a study that measured the amount of influenza a(h1n1)pdm09 rna in aerosols in the vicinity of h1n1-positive patients undergoing aerosol-generating procedures (agp) has shown that bronchoscopy and respiratory/airway suctioning were the most definite procedures to produce aerosols above background baseline values. 92 thus, it is important for healthcare workers to take airborne precautions and perform agp preferably in an isolation room with negative pressure. in order to reduce room contamination in the hospital setting, major health organizations have recommended the application of a minimum room ventilation rate of 6 air changes per hour (ach) in existing facility whereas a higher ventilation of 12 ach is recommended for new or renovated construction, especially when caring for patients receiving mechanical ventilation and during agp. 91, 93 as single circuit niv may lead to room contamination by exhaled aerosols, 94, 95 it has been recommended to apply niv via a helmet and double circuit tubes for patients with mild to moderate respiratory failure during the influenza a(h1n1) pandemic in 2009. 96 however, it is important to ensure a good seal at the neckhelmet interface to prevent nosocomial transmission through environmental contamination by exhaled aerosols. 97 post-exposure prophylaxis with a daily dose of nai (e.g. oseltamivir 75 mg daily for adults) for 10 days is recommended for unprotected close contacts of patients with seasonal influenza who are at risk of complicated influenza. 98 who does not recommend routine post-exposure antiviral chemoprophylaxis for a(h7n9) virus. however, for some asymptomatic persons in which a substantial unprotected or prolonged exposure to an ill patient with a(h7n9) infection has occurred, initiation of empiric post-exposure antiviral treatment (e.g. oseltamivir 75 mg orally bd for 5 days), on the presumption that influenza virus infection has occurred, may be considered. this is likely to be limited to healthcare or other settings involving substantial exposure of those at higher risk for complications from table 4 infection prevention and control measures when assessing patients with complicated influenza 51,90,91 1. clinicians should pay attention to cases of community-acquired pneumonia and patients' travel history for early case detection 2. clinicians should remain vigilant against avian influenza and elicit any relevant clinical and epidemiological information from patients (fever, travel history, occupation, contact history and clustering) 3. standard, contact and droplet precautions are recommended for routine management of patients hospitalized for influenza. droplet precaution (by wearing a surgical mask within 1 m of the patient) and contact precaution (by wearing gown and gloves on entering the room and removing them on leaving) when providing care to all patients with symptoms of acute respiratory infection. contact precautions and eye protection should be added when caring for probable or confirmed cases of avian influenza infection 4. risk assessment should be conducted before performing agp. for cases with severe influenza, it is advisable to perform agp in an airborne isolation room. when an airborne isolation room is not available, the following minimum hourly averaged ventilation rates should be provided for natural ventilation: a. 160 l/s/patient (hourly average ventilation rate) for airborne precaution rooms (with a minimum of 80 l/s/patient) (note that this only applies to new healthcare facilities and major renovations); b. 60 l/s/patient for general wards and outpatient departments; and c. 2.5 l/s/m 3 for corridors and other transient spaces without a fixed number of patients 5. in view of the increasing influenza activity during winter season and the emerging threat of avian influenza, all healthcare workers and visitors are recommended to wear surgical masks when entering patient care areas and strengthen hand hygiene agp, aerosol-generating procedure. influenza virus infection, including patients with severe immunosuppression, neonates and infants, pregnant and early post-partum women, elderly adults, persons with co-morbidities and other highly vulnerable patients; or, unprotected healthcare workers, especially those involved in agp. 99 annual seasonal influenza vaccinations are recommended for the high-risk groups, including residents of institutions for elderly and disabled, any age with chronic illness, age > 65 years; 6 months to 6 years, poultry workers in countries affected by avian influenza, healthcare workers especially those frequently in contact with high-risk persons and household contacts of high-risk persons. while vaccination rates among healthcare workers are highly variable in different countries, 100 there is no conclusive evidence that seasonal influenza vaccination of healthcare workers may protect their patients in terms of reduction in risks of mortality and influenza-like illness. 101 a survey in hong kong assessing the acceptability of an additional ad hoc influenza vaccination among the healthcare professionals following seasons with significant antigenic drift has shown that the strongest factors associated with accepting an additional influenza vaccine included immunization with influenza vaccines in the past 3 years, higher perceived risk of contracting influenza and higher perceived severity of the disease impact. 102 the current influenza vaccines induce a strainspecific immunity that is not ideal for this rapidly mutating virus. a universal influenza vaccine capable of inducing a broad cross-protection across strains is needed. to this end, the m2e antigen, a linear peptide that is conserved across influenza a strains, is being evaluated a vaccine target. 103 the other approach is to prepare virus-like-particles based on ha, na and m1 proteins. 104, 105 virus-like particles with a cocktail of seasonal and potential pandemic strains have been prepared and demonstrated promising results. 106 as a(h7n9) is now endemic in over half of the provinces in mainland china and will continue to cause recurrent zoonotic disease in the winter months, public health measures to control the source such as some rest days every month for the poultry workers to thoroughly clean the lpm environment, and banning the stock of live poultry in markets overnight are important. separation of live ducks and geese from landbased (i.e. non-aquatic) poultry in lpm systems can reduce the risk of emergence of zoonotic and epizootic viruses at source. in the long run, it is advisable to adopt a complete transition from sale of live poultry in wholesale and retail lpm to centralized slaughter and sale of chilled or frozen poultry. 107 seasonal influenza epidemics and periodic pandemics are important causes of morbidity and mortality. apart from influenza a(h5n1) virus and sporadic human cases of a(h5n6), a(h10n8) and a(h6n1) infections, infection due to a(h7n9) virus, especially the recent 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cord-248301-hddxaatp authors: howard, daniel title: genetic programming visitation scheduling solution can deliver a less austere covid-19 pandemic population lockdown date: 2020-06-17 journal: nan doi: nan sha: doc_id: 248301 cord_uid: hddxaatp a computational methodology is introduced to minimize infection opportunities for people suffering some degree of lockdown in response to a pandemic, as is the 2020 covid-19 pandemic. persons use their mobile phone or computational device to request trips to places of their need or interest indicating a rough time of day: `morning', `afternoon', `night' or `any time' when they would like to undertake these outings as well as the desired place to visit. an artificial intelligence methodology which is a variant of genetic programming studies all requests and responds with specific time allocations for such visits that minimize the overall risks of infection, hospitalization and death of people. a number of alternatives for this computation are presented and results of numerical experiments involving over 230 people of various ages and background health levels in over 1700 visits that take place over three consecutive days. a novel partial infection model is introduced to discuss these proof of concept solutions which are compared to round robin uninformed time scheduling for visits to places. the computations indicate vast improvements with far fewer dead and hospitalized. these auger well for a more realistic study using accurate infection models with the view to test deployment in the real world. the input that drives the infection model is the degree of infection by taxonomic class, such as the information that may arise from population testing for covid-19 or, alternatively, any contamination model. the taxonomy class assumed in the computations is the likely level of infection by age group. the quaranta giorni or forty day isolation by the venetians, as the name implies, was a measure applied to incoming ships [12] which evolved into containment practices to handle recurrent epidemics 1 . at the time of writing, owing to ubiquitous world travel, covid-19 'quarantines' or lockdowns keep millions of people around the world confined mostly to the home for months before an 'easing of measures' gradually re-opens society. the 2020 lockdowns in response to the covid-19 pandemic enforce in different ways. argentine and spanish lockdowns are strictly policed with citizens required to make written application for outings. in contrast, in north western europe and the united states, lockdows are not as strict, with denmark and the united kingdom entrusting their citizens not to infringe lockdown rules, and liberal sweden choosing not to lock down in an official sense but instead practicing a small number of restrictive measures. the manner of lockdown and how to exit a lockdown are problems that are short of informed solutions. it is useful to discuss the generic lockdown problem as requiring an innovation capable of overcoming the following trade-off: generally, the longer and more extensive is a lockdown the more effective becomes society's ability to prepare hospital facilities to control the spread of the disease but the higher are important negative factors: loss of personal freedoms; damage to the economy; poorer personal psychology; social unrest; abusive relations; undetected crime; higher incidence of other disease because people are too scared to visit the emergency room or doctor and negative effects on care of both vulnerable and elderly. the idea is to enable lockdown whilst minimizing many of its negative consequences, e.g., to personal psychology, economics and health. indeed, it would be nice if life could continue as normal while in lockdown, a seemingly contradictory statement. inspiration comes from a crude approach taken by governments to ensure that those who venture outdoors are fewer in number. panama [9] used the last number of an identity document to assign two hour time slots to venture away from the home for essentials and, as spain eased measures, it allowed certain age groups to go out at different times of the day. the open literature also explores changing a general lockdown to a number of partial ones [11] . the solution presented here is for citizens in lockdown to enter into smart-phone, handset, tablet or computer, a schedule of the places that they wish to visit on that day, or future dates, together with a rough idea of the part of the day they would prefer for such outings to take place. a method of optimization, in this proof of concept this is a genetic programming [7] method, takes these requests and simulates the outings by means of an infection model, to discover a nearly optimal allocation of precise time slots for visits that reduce the likely hospitalization and death numbers. it then communicates the time allocations to citizens on their devices such that they will carry out the journeys more safely than at any times of their choice. the solution is proactive. specifically, it is useful to discuss the problem within the implementation of this proof of concept. a data file captures all requests as shown on the left side of figure 1 . the visit requests on each of three consecutve days can be many, and each is denoted by a three symbol key. requests are separated by the colon symbol. the first letter concerns a broad time of day requested for the visit limited to: m wishes for the visit to take place during 'morning' hours; p wishes for the visit to take place during 'afternoon' post meridiem hours; n wishes for the visit to take place during 'night' hours; a anytime: does not mind at what hour on this day. and the second letter is the desired place of visit, limited here to six types of establishment: f may represent a 'supermarket' selling food; c may represent a sports 'club'; p could represent a 'park'; d may stand for 'doctor' a doctor's surgery centre; r may stand for 'restaurant'; s could represent a 'social' establishment; the third symbol can be 1 or 2, because there are only two of each type of establishment, or a total of 12 establishments available for visits. for example, one such request is from person id 20 who wants to go to doctor's surgery 2 at any time of day. the day is divided into eight two hour slots. after analysis of all request, by working with an infection model, the computer generated optimization minimizes the total risk of covid-19 infection, hospitalization and death. it allocates the time slots to the requests and the solution is communicated back to citizens. in this case person id 20 is allocated a time slot within the constraint that they imposed as shown on the right side of figure 1 . there are other data fields such as age and health in the left side of the figure that are discussed further in this text. the proof of concept simulations cover three consecutive days denoted as: monday, tuesday and wednesday, involving 1704 visits as requested by 282 people. a day consists of eight two-hour visitation slot periods available to schedule the visits. details of the data for the purpose of the approximate reproduction of results is as in table 1 . the data is an entire fabrication but common sense governed its choices: older people carry out relatively fewer outings than the young, and are in a poorer state of health. the degree of health of a person is a number that ranges from 1 to 10. in any future real-world application of this research, the general health of a person, which is a measure of their immune system response to the pandemic and assumed to abate the probability of hospitalization or death could be gathered from patient records. as seen in later sections, this level of health combines with infection and plays a pivotal role in determining which solutions are better than others. the idea is to reduce hospitalizations and fatalities. the solution must simulate the infection process cumulatively and longitudinally in time as people go from place to place to optimize the allocation of time slots. this requires as component a model of covid-19 infection. the proof of concept develops a model based on person to person transmission based on simple probabilities of meetings between people in a confined location and is necessarily a very basic model but one that illustrates the potential of the solution. two models are presented: a partial infection probability developed for this work and a simple standard probability model. both are simple and would need to be improved by epidemiologists for their real world application. the idea of a 'partially infected' individual is developed here because it will only be possible in some average sense to know who might be infected 2 . the idea is to assign a 'partial infection level' to all members of a taxonomy class of interest, and then simulate how they will infect others and further infect each other. 2 if we knew precisely who was infected they could be isolated and the solution becomes unnecessary age 20 30 40 50 60 70 80 total number of people 35 65 49 43 27 43 20 if an infected individual i is in close proximity to a susceptible individual s then the possibility exists of transmission of the disease from i to s. without loss of generality the assumption is made that every such encounter will result in infection transmission. an infection probability based on a count of such encounters between s and one or more i can be expressed as p n where n is the number of infected who may come into contact with susceptible in a fixed interval of time that denotes the duration of a visit, e.g., an hour or two. more sophisticated relations should consider this time dimension and may model it with poisson distributions but such complications are ignored for the purpose of this presentation. if the location of the encounters is for example, a store of a certain physical size, and s is the number of sublocations of that are available to visitors that together comprise the walkable area of that store, they are sub-units of area small enough such that people may position themselves and come into close proximity of each other, then a simple count of probability for such encounters between s and one or more i leads to: p n = 1 − ((s − 1)/s) n . a property of this relation is the convergence to one as n grows large: n → ∞ then p ∞ → 1.0 meaning that a non-infected person will surely come into contact with one or more infected at that store. moreover, also p 2 < 2p 1 such that if doubling i then the probability of infection for s is increased but never doubled. in driving a contact based infection model, simulations must assume a certain number of persons are infected a priori at the start of the simulation. this presents a challenge because it requires assumptions of who is infected and why, and also a huge number of stochastic computation and an averaging of results. as each person is different and not a clear member of a taxonomy class 3 , for example, people may be of the same age but of different health level granting them less resistance to the infection, the computations would necessitate carefully balancing assumptions about who should be assumed to be infected to drive the computations. the partial infection model that is presented, while not being itself a perfect solution, does not have this 3 the idea will not be unfamiliar to mathematical biologists and epidemiologists [6] onerous requirement and simplifies the computational effort requirement for this proof of concept study because it can take an idea about how infected are members of the population, assigning a probability of infection to all members of that taxonomy grouping in order to drive the infection simulation. the concept of a partially infected individual is a modelling tool. each person p j is represented by a vector of size two, p j = (s j , i j ) with s j + i j = 1. for example a person with id label j = 1 that is forty percent infected is represented by s 1 = 0.6 and i 1 = 0.4 and another with id label j = 102 who is one percent infected by s 102 = 0.99 and i 102 = 0.01. consider the meeting at the store of n p persons of which n have some degree of partial infection. the resulting infection pressure, pi n , that is the resultant partial infection probability of the encounter that is brought about by the infection contributions of the n partially or fully infected persons, can be obtained for two cases: s max is the maximum s j out of all n persons. with the exception of the owner of s max each i k of the other partially or fully infected persons is multiplied by s max and by a multiplicative numerical constants g j soon to be discussed. these product terms are summed to obtain the probability of infection for the encounter. when n p = n, the number of such product terms is therefore n − 1. however, when n p > n, s max is considered to be from one of the fully susceptible persons in n p and thus s max = 1. now the number of such product terms in the sum is n. a formula to compute pi n given n p = n with every participant infected or partially infected, is developed as and for the case n p > n with s max = 1 as the constants g j emanate from simple overlap counts in probability trees as in appendix b.2 and this author's earlier technical communication [4] . deliberately the products are arranged or ordered so that the largest g j corresponds to the largest i j . this represents the worst infection case which subsumes all others. once the probability of infection pi n is calculated, the partial infection of all participants in n p is updated for encounter v in readiness for the next visit v + 1 as follows: as an example, assume s = 4 and n p = n the infection probability indeed follows a similar simple 'probability count' philosophy as in the previous section but as obtained with a monte carlo process that generates the probabilities. each evaluation involves q trials for one fully susceptible s and twenty fully infected i persons and it counts the times when the susceptible is co-located with any infected. for example, it does this on 40 separate occasions, i.e., once these forty trials are computed for the one susceptible and the twenty infected with results as in figure 2 . the procedure checks to see whether a susceptible and one or more infected shared an encounter from those forty, (q = 40 sampling). it is however fashioned in a more elaborate way to try to account for differing times spent in different sub-areas. the random sampling is made to fit into 7200 seconds of time (two hours) but the random number indicating the location is chosen to weigh certain locations more. it crudely captures that some areas of the store or establishment are more popular than others. for example, browsing a magazine rather than simply walking through some area of the store. random numbers are clustered to represent three types of areas: one where the person spends 2 seconds or another where the person spends 30 seconds, and yet another where the person spends 102 seconds in the selected area. the procedure is outlined in appendix a. the constants in the figure are calculated prior to the run and give the probability of susceptible getting infected depending on how many people are infected in the store. all visits are of the same duration so no attempt is made to model probability of infection through time with a poisson process. this model of infection transmission is once again a very simple one. when n > 20 the probability of infection is assumed to be equal to one. note that a lower or higher rate of infection can be obtained by changing the sampling from 40 to a smaller or larger number. at any meeting place and time, three types of person can participate in the encounter: i or infected; s or susceptible and r or immune (recovered). no action is taken if fewer than two people participate n p < 2. no action is taken if all are immune. no action is taken if all are infected. no action is taken if all are susceptible. no action is taken if there are no infected. no action is taken if there are no susceptible. otherwise the aforementioned infection probability is selected for the number of infected and that real number is multiplied by the number of susceptible and then truncated to obtain the integer number that will become infected. in no particular order that many susceptible are labelled as infected. the method adopted to optimize the allocation of requested visits is a genetic programming (gp) scheme developed by this author to discover a set of precise numerical constants that serve as coefficients of a polynomial representing the direct solution of the one dimensional homogeneous convection diffusion equation [5] . this and other puiblications [3] [1] demonstrate that standard genetic programming trees are capable of computing very precise real numbers when and if needed. when gp trees are evaluated they deliver a vector of real numbers of self-determined variable-length. table 2 lists the functions and terminals that comprise the gp tree. they manipulate two pointers p r and p z to the output variable length vector of numbers. they also make use of two working memories, two real numbers, m 1 and m 2 . terminals are small and large numbers that become arithmetically manipulated. certain functions write to the variable length result vector, shrink it or expand it as the gp tree evaluates. the result of evaluating a gp tree is a variable length vector of real numbers. these numbers can be of any size and can be positive or negative. a subroutine then operates on these numbers to bound them as positive real numbers in size between 0.0 to 1.0 [5] . for example, if a number in the vector is -21.27625 it now becomes 0.27625. also if a number is 29.00000 it now becomes 0.0001. how is this vector of real numbers used? consider 1704 visitation requests of table 1 . the real numbers vector is probably much smaller. this is consulted from left to right as when a child reads words letter by letter. consider the outing request by a person is ad2, e.g. person 20 monday in figure 1 wishing to go to doctor's surgery 2 at any time of the day. the day is divided, into eight two hour slots of time. as each number ranges from 0.0 to 1.0 consider that this might be 0.2763. this number would indicate prescription of the third slot of time since 2/8 < 0.2763 < 3/8. the allocation for that visit complete, the next real number in the variable length vector gets consulted to deal with the next visit request. as the variable length vector of numbers is usually shorter than the total number of visits requested, when the last element of the vector is reached it cycles back to the first number in the vector and continues until allocations for all 1704 visitations are dealt with, allocations of time as in figure 1 = m 2 setmem2(2) = r and m 2 = (m 2 + l)/2 table 2 : constituent functions or building blocks of gp trees in [5] are of four varieties: number; arithmetic; record; and memory with number of arguments as shown in brackets: if (2), one is l or'left' input and the other r or'right' input (corresponding to left and right branches of the subtrees below the node) and (1) is a tree terminal or leaf. the resultant variable length vector of constants r has elements r j that must never exceed j = p max . two pointers are manipulated p r and p z current position and vector length respectively. working memory locations are two: m 1 and m 2 . in the above 'increment' means to increase by one, and 'decrease' to decrease by one. the symbol '=' denotes what the function returns to its parent node in the tree. small to moderate sized vectors. practical use of the method may require a small number of additional strategies. on some problems for a small number of initial generations the darwinian fitness is set as the vector size. it stimulates production of large output vectors. when a desired variable length vector output size is reached by all members of the population the fitness is reset. from then onwards the fitness is the problem's darwinian fitness, typically a measure of solution error. the procedure is often not necessary but can become useful when required solution complexity or dimension is very high. once seeded in this way the solution vector will grow or shrink as crossover and mutation operations on the gp tree create new and improved gp trees. as the method makes use of gp trees and standard gp, then all that we know about standard genetic programming including modularization options such as adfs [8] , and subtree encapsulation [10] are applicable. as will be revealed in the numerical experiments, the approach works, discovers good solutions, and appears to be quick on a portable computer, the compiled c++ visual studio 2019 executable delivering solutions in seconds for the proof of concept experiments. regardless of the infection model used the procedure is similar. there will exists a taxonomy of persons by some parameter(s), for example, age group. information about the likely degree of infection for different taxonomy classes is input to the computations. as time progresses and visits take place, people get infected. at the end of the three days, account is taken of who is infected and their prior health level. this calculates how many people in the proof of concept study will become infected a few weeks hence and how many may die. there are many assumptions but if correctly taken they drive genetic programming to discover allocations that reduce hospitalizations and fatalities. central to darwinian fitness are such calculations of who and how many will perish or fall very ill and use the intensive care unit (icu). in this implementation taxonomy is assumed to depend on age group. covid-19 testing is unlikely to become universal and frequent for all citizens but limited testing and other measurements are sufficient to serve as indications of the degree of infection likelihood for sectors, partitions, of the population. the proof of concept uses age as taxonomy. figure 3 reveals how this works. if testing reveals that more people of age groups a or b have covid-19 than of age groups c or d, then percentages are entered at the start of numerical experiments. for example, 20=0.03; 30=0.01 means that the 20-year-olds have a suspected level of covid-19 infection of three percent, while the 30-year-olds are suspected to have one percent. each simulation proceeds from monday to wednesday and for each day from morning to night (8 time slots). at each time slot all twelve establishments are considered and calculations of partial infection update the partial infection level of all. at the end of the day, a calculation is made to determine who should go into self-isolation and participate no longer in the simulation. the rules to identify those persons are presented in the left side of table 3 . at the very end of the simulation another procedure calculates the total number of hospitalized in the intensive care unit (icu), denoted by the symbol n h . these are those in hospital who eventually recover but who never the less put pressure on the health service. the procedure also calculates those who unfortunately pass away, denoted by the symbol n d . the rules to determine these two numbers are presented in the right side of table 3 . note the assumption is made here that both the state of health and the age of the subject correlate similarly but are assumed to be separate causal factors. the gp darwinian fitness function f f is the measure of solution goodness. it combines n h and n d weighted by some desirability constant w c . f f is by choice a negative quantity because the evolution is set to maximize or make bigger this quantity, with a perfect score being zero implying n h = n d = 0: all of the numerical results in this proof of concept use w c = 0.65, reflecting desire to reduce fatalities. where w 3 > w 2 > w 1 > 0 reflects that the mortality of the young from covi-d19 is low and that of the elderly is very high. however, it suffices to show by the small worked out example of figure 4 (and as seen in the figures) that lower average partial infection does not necessarily reflect in smaller values of n h and n d . in this type of model persons can only become fully infected, i. hence, the rules to determine n h and n d are different and only apply to those infected. additionally, the decision to self-isolate depends on the number of days that the person is infected and their health level. consider that some of the people in the input data file are already fully infected. table 4 shows the decision schema for self-isolating persons as well as for deciding on n h and n d after the simulation is complete. note the assumption is again made here that both the state of health and the age of the subject correlate similarly but are assumed to be separate causal factors. the fitness measure for these computations is also equation 4. the numerical experiments compare the solution to three round robin uninformed allocations. the first of these comp1 sends all those: (a) requesting 'morning' visits or 'any time' visits to the first morning time slot, the 8:00 there is also a third uninformed allocation by round robin for comparison, comp3. it is a round robin of three. however, as the morning has only two time slots this sends two thirds of the requests for case (a) to the first slot and the other half to the second slot. for cases (b) and (c) it uses all three time slots distributing the visitation requests evenly. comparison could be made to other allocations but these are broadly representative of what would transpire in the real world without the benefit of the solution and in conditions of partial lockdown or no lockdown. it turns out, as expected, that comp1 results in the highest number of hospitalizations n h and deaths n d for the model problem and comp3 in the lowest as revealed in comparative experiments presented in this section. each experiment follows common gp practice executing a large number of parallel independent runs (pirs). each pir differs in its initial random seed (uses pc clock timer) seeding the population randomly and differently for each pir. pirs can have different population sizes of gp trees. all runs use 80 percent crossover and 20 percent mutation to generate new gp trees. this is a steady-state gp with tournament selection of four individuals to select a strong mate for crossover, whereby two gp trees swap branches, or the winner of the tournament simply mutates, with a kill tournament of two to choose the weaker gp tree to replace. the maximum possible tree size for gp is set at 2000 and if this is exceeded then the shorter side of the crossover swap is taken. the maximum variable length vector size is set at 10,000 but never remotely approached: excellent solutions have vector sizes of between 10 and 100. it is an 'elitist' gp for it does not destroy the fittest solution, i.e., solutions do not have a lifespan inside a pir. all pirs implement standard 'vanilla' gp with no parsimony pressure or any other non-standard approach. this proof of concept study does not employ explicit reuse: adf [8] or subtree encapsulation [10] . when a better solution emerges during the execution of a pir, this is separately stored. a pir typically only for persons who become infected self-isolation conditions outcome rules age days infected health age health actions outcomes 20 produces up to around ten of which two are highly fit and interesting. as subsequent pirs produce more solutions, all become ranked by fitness, a global list of solutions, from which the user can select one to inspect. for each solution, full details of the participants to all visitations, infection levels, numbers self-isolating, in icu and sadly passed away can be inspected, as well as the variable length vector of real numbers that is the solution and visitation schedule and identity, age and level of infection of all participants recovered, in icu or deceased. for the partial infection experiments it also computes partial infections by groups: young, middle aged and elderly every four hours. the comprehensive result panes in figures in this section merit closer inspection. as pirs build, a pareto front picture can emerge of non-dominated solutions involving the two criteria n d and n h . table 5 shows the vast superiority of the solution to the uninformed round robin allocation schemes. the superiority is marked and so there is no need to check this against other possible random allocation schemes. it makes common sense that the genetic programming solution is always superior. note that with s = 6 the problem becomes easier than with s = 4 because the chances for meetings between people are lower. this can be seen to be true for both round robin allocations and genetic programming solutions. it also appears to be that the superiority of genetic programming solutions over round robin allocations is correlated with higher s. this is probably because the genetic programming has more degrees of freedom to discover a better solution. therefore, even if the intuitive idea is that with a smaller density of people there should table 5 : summary of results attained by the numerical experiments that made use of the partial infection model. with s = 4 the solution is three times better than a round robin allocation and with s = 6 it is ten times better. be less need for the solution, this is not always the case. note that in the cases where s = 6, genetic programming managed to discover an allocation where n d = 0, with no deaths. perhaps such a counter-intuitive conclusion can be discerned that the solution is needed more so when the problem seems simpler. close inspection of the figures referred to in table 5 reveals that the system of pirs delivers a number of pareto non-dominated solutions to choose from. also, there are solutions that achieve an identical result in terms of n d and n h but which are quite different. then one is also able to inspect the age and health of the fatalities, again this may be a tertiary factor that could come into play when selecting among the many equivalent solutions. finally, it can also be discerned that the average partial infection level and its final levels as shown in the figures do not always correlate with lower error and higher darwinian fitness. a different set of computations to the real world problem is included here for completion. it pertains to knowing who is infected a priori and trying to optimize what could have happened had their visits been scheduled differently. it could either be useful to a retrospective study or, alternatively, such computations can potentially address the same real world problem but only by the carrying out of a plethora of experiments with different seeds of infected individuals, assumed infected according to some taxonomy knowledge of level of infection in the population, and then averaging the results in some way to prescribe safer allocations of visits, as alternative to the experiments (presented in the previous section) with the partial infection model. a set of 15 persons, as shown in figure 6 , out of the 282 in the proof of concept data, as described in section 3, come already fully infected a priori to drive the computations 4 . this is about 5.3 % of total people, and they undertake 105 visits or 6.2 % of the 1704 total visits that appear in the proof of concept dataset. individuals with good health levels are chosen as already infected. for completion, the figure also shows six people who have immunity and therefore cannot become infected in the computations. they are included in computations but there is no need for them. it is envisaged that the optimization problem by gp is challenging. hence, the infection model is implemented at different values of q (see section 4.2 and appendix a) to allow the solution room to make gains but also to understand the dynamics under various levels of contagion (perhaps representing adherence to social distancing and use of masks). there are eight types of combinations of the events that can befall a person: 1. person comes to the simulation already infected and does not use the intensive care unit (icu); 2. person comes to the simulation already infected and does make use of the icu (adds to n h ); 3. person comes to the simulation already infected passes through the icu or not and dies (adds to n d ); 4. person comes to the simulation immune (has had the disease before and has recovered) and stays that way; 5. person comes to the simulation susceptible and does not get infected; 6 . person comes to the simulation susceptible, gets infected but does not use the intensive care unit (icu); 7. person comes to the simulation susceptible, gets infected and does make use of the icu (adds to n h ); 8. person comes to the simulation susceptible, gets infected, goes or not to icu and dies (adds to n d ); figure 7 illustrates how to interpret that part of the result figures pointed to in table 6 . as there are generally no cases of infected ending in icu or dying only six result summaries show as in the figure. the results of all numerical experiments that use the full infection model are as in table 6 . the difficulty with this infection model is that it multiplies the probability of infection by the number of susceptible and then it simply truncates the result to the lower integer. moreover, it simply goes down the list of susceptible infecting the first bunch it sees up to that integer number. this gives the solution opportunities to play games. for example if q = 30 then p(9) = 0.947 and if there is only one susceptible and nine infected then the susceptible will not become infected. notwithstanding the weaknesses of such computations and their erroneous assumptions this can be said about the table 6 figures. as q is increased then the gp search becomes much harder. best solutions come from bigger gp populations run for longer (longer numbers of generations). also the advantage of the solution over the round robin assignments is less valuable than at lower q values. this is to be expected because at high q the disease is far more contagious and there is not much room for the optimization. note that with q = 30 all round robin solutions have the same n h and n d possibly indicating little room for schedule optimization by the solution. table 6 : summary of results attained by the numerical experiments that made use of the full infection model. with q = 4 ( see appendix a and the text), the solution is between two and three times better than simple round robin allocations. large improvements in both lower mortality and lower use of the icu are available with the solution for the proof of concept data described in section 3 in computations with both infection models. the improvements as compared to round robin allocations in tables: table 5 and table 6 clearly show it. the parameters s and q respectively in the two numerical experiments have a similar but inverse influence as the former gives the number of possible sub-locations that can be occupied while the latter is the number of checks performed inside the monte carlo calculation that detects co-presence. however, even at low s and at high q, the worst possibilities, the solution keeps outperforming round robin by a significant margin. at high s and low q the solution really shines and outperforms round robin by a very considerable margin. it augers well because with good cleaning at locations, washing of hands and even moderate social distancing, the rate of infection is expected to be low (corresponding to higher s and lower q in a sense). of course, a more serious real-world model needs to be considered in further research. such a model would need to consider: 1. is it correct that a difference in infection rates exists between taxa? if so, what is this taxonomy and how to construct it? is it possible to discover this taxonomy through covid-19 testing and other data collection? 2. is it possible to develop a reasonably accurate infection model for certain stores and places that people wish to, or need to, frequent? 3. the infection model must also include a dynamic related to object contamination and transmission through contact of surfaces and objects; 4. travel by public transport to such locations also needs to be accounted for by the model; 8. is the round robin a fair reflection of how people wish to go out in the unrestricted normal case? are there invariant principles gathered from mobile phone roaming data that could inform how and when people go out? 9. what is the effect of non-compliance on the solution? could the solution account for it and still be gainful? 10 . the solutions arrange into pareto non-dominated sets involving n d and n h : what is the difference between these and also between equivalent solutions, same score of f f ? is there a difference between such solutions of further interest? 11. the solution is designed to work even with very little idea or precision about the rates of infection and contamination as it aims for an improvement rather than precise values. how valid are these assumptions? 12. the solution out pefoms round robin but how does it fare in terms of infection rate against strict lockdowns, or exiting lockdown with phased lockdown strategies suggested in [11] ? 13. can economic, psychological and other benefits of the solution be quantified to understand the cost benefit analysis of adopting it versus strict lockdown policies? 14. will people be willing to undergo numerous lockdowns waiting for the availability of a vaccine if the solution is adopted? genetic programming is known to scale well with problem size. however, even if millions of people were considered there would be a degree of clustering that could be treated differently by different discovered solutions. the partial infection model is developed here for the first time. if there is something similar in the literature then this author does not know of it. it must be tested and developed better. it is possible that the pessimistic approach of multiplying the terms of worst case is not entirely reasonable. lastly, how well infections can be avoided will depend on the number of visitations, the number of establishments visited, the number of people and the frequency of visits. in this research genetic programming was handed a tough challenge as the number of visitations was in the order of 2000 and the times slots very few per day. also the number of establishments was only a few. in general, it is said that washing one's hands is far more effective than social distancing. it is probably true that contamination is more important than person to person transmission. contamination can be easily incorporated into the model. although the model is incipient, it is considered something few have considered if any. most research is involved in exploiting data sources to predict infection levels or explain the disease. this contribution is different in nature because its deployment could generate data and would also need only tendency of disease data. this contribution is markedly different from contact tracing approaches that are reactive. the work described here and its implementation would be proactive but it could also inform and be informed by contact tracing. sophisticated relations can be determined in function of the number of encounters between a susceptible and one or more infected that also incorporate time of exposure modelled as a poisson process [2] 6 . an important characteristic of such relations is that as the number of infected individuals n grows then p n increases but not linearly, so that for example one can expect p 2 < 2p 1 . this proof of concept assumes a trivially simple infection function based on counting co-locations between s and i. these increase with the number of infected but the ratio of these to the total possible encounters never exceeds one, that is, as n → ∞ then p ∞ → 1.0. the essence of this behaviour can be crudely emulated with monte carlo or, as discussed here, by counting on simple probability trees of figure 8 . we ignore dependency on time of exposure to assume all visits to places are of similar duration. the genetic programming approach that uses such infection functions, however, is general and able to incorporate any candidate infection function. from this figure, first, consider the case of two individuals only: p1 and p2. individual p1 is susceptible while p2 is infected. the number of encounters between these two, the red boxes at the p2 level in the figure, or where both individuals share a same location, is four and the total number of possible outcomes is sixteen. hence, the opportunity for an encounter taking place and therefore for infection, assuming each individual should have an equal presence for each of the four locations and spend the same amount of time at any visited, is p 1 = 0.25. next consider the case where three individuals participate with p1 susceptible and p2 and p3 both infected. now we recognize opportunities for all three individuals to exist at the same location, and also opportunities for p1 to share a location with either infected p2 or p3. if we count such opportunities we arrive at p 2 = 28/64 = 0.4375 and we verify that p 2 < 2p 1 . for the particular scenario of figure 8 the coarse emulation leads to a simple relation to obtain the probability of encounter for any n that is: p n = 1 − (3/4) n . considering rather large n it tends to one: p 20 = 1 − (3/4) 20 = 0.9968. we now introduce perhaps what may be an unusual idea. the real world objective of this initiative is to leverage off covid-19 testing. imagine that upon testing for covid-19 the incidence is found to be higher in age groups 20 and 50. it is unlikely that covid-19 testing will be undertaken by all people all of the time. moreover, tests are still unreliable. hence, we need to work with partial knowledge. an option to explore might be to work directly with the probable levels of infection that are informed by the testing for different groups of people as organized in some taxonomy: for example, consider the probability of infection to be 5 percent and 8 percent respectively and zero in other age groups? 7 . it motivates use of a 'partial infection' but why? if we knew who was or was not infected we would let them out or keep them in isolation. however, as we cannot test every single person, it is useful to assign to them uncertainty, a probability level 8 . in such a case, we must consider that any of the people in figure 8 namely p1, p2 or p3 may be partially infected (and partially susceptible). figure 8 shows the encounters between persons, i.e., two people sharing one location (1, 2, 3, 4) at the same moment in time, thus coming into contact with each other. we are especially interested in two encounters: p1-p2 and p1-p3 as we can assume that one person p1 is susceptible while the other two are infected. note that under permutations assigning i and s there is no need to count encounters p2-p3 as these would represent two infected, and neither of interest are encounters between two susceptible. the thirty two encounters that determined the probability of infection result in the union that gives twenty eight encounters in the figure note that in spite of p3 already carrying a small probability of infection, its new infection level 0.4247 < 0.4375. this is because others were not one hundred percent infected and the result was close to but less than 0.4375. here is a third example, consider the interactions between three people with partial infection: notice that p1 and p3 have an infection level 0.2568 > 0.2500 this is because the people were already one percent infected. an example involving four people is illustrated with the help of figure 9 , consider the interactions between three individuals with partial infection: note from previous examples that if we simply identify the participant with the highest component of s then that will be the one that indicates the highest contribution. in this case that is p1 and so siii should be highest, and it is and is 0.5708. this number is very close to p 3 = 1 − (3/4) 3 = 0.5781. it is a higher value because all participants contribute a significant level of infection. now we compute the new partial infections for our four participants: these factors can be seen to be in descending order. if n p people participate in an encounter but the number of partially infected people n is less than n p then we assume one fully susceptible individual with i = 0 and s = 1 will meet with the n partially infected individuals and thus s = 1 is used in the formula. the calculation follows this ten step procedure (pseudo-code): identify s max and that participant with the highest value of s v prepare the products (s max i v j g j ) and sum them to get the infection level 10. use it to update infection levels of all n p participants: i v+1 = ps v + i v predicting the structure of covert networks using genetic programming, cognitive work analysis and social network analysis genetic programming of the stochastic interpolation framework: convection-diffusion equation genetic programming visitation scheduling in lockdown with partial infection model that leverages information from covid-19 testing genetic programming solution of the convection-diffusion equation differential susceptibilty epidemic models genetic programming: on the programming of computers by means of natural selection genetic programming ii: automatic discovery of reusable programs .'s organización internacional para las migraciones panamá. cuarentena total en suelo panameño evolving modules in genetic programming by subtree encapsulation a phased lift of control: a practical strategy to achieve herd immunity against covid-19 at the country level. biorxiv the origin of quarantine a pseudo-code for the monte carlo routinethe following pseudo-code with q = 40 produces the numbers of figure 2 : do m, 20 times (iterate over 20 infected) ninfection[m] =0 (counts the encounters for this many infected) end iteration index m do 100,000 times (use a big number for good accuracy) do k, q times (typicaly q is between 10 key: cord-280184-91d8i6ix authors: querido, micaela machado; aguiar, lívia; neves, paula; pereira, cristiana costa; teixeira, joão paulo title: self-disinfecting surfaces and infection control date: 2019-06-01 journal: colloids surf b biointerfaces doi: 10.1016/j.colsurfb.2019.02.009 sha: doc_id: 280184 cord_uid: 91d8i6ix according to world health organization, every year in the european union, 4 million patients acquire a healthcare associated infection. even though some microorganisms represent no threat to healthy people, hospitals harbor different levels of immunocompetent individuals, namely patients receiving immunosuppressors, with previous infections, or those with extremes of age (young children and elderly), requiring the implementation of effective control measures. public spaces have also been found an important source of infectious disease outbreaks due to poor or none infection control measures applied. in both places, surfaces play a major role on microorganisms’ propagation, yet they are very often neglected, with very few guidelines about efficient cleaning measures and microbiological assessment available. to overcome surface contamination problems, new strategies are being designed to limit the microorganisms’ ability to survive over surfaces and materials. surface modification and/or functionalization to prevent contamination is a hot-topic of research and several different approaches have been developed lately. surfaces with anti-adhesive properties, with incorporated antimicrobial substances or modified with biological active metals are some of the strategies recently proposed. this review intends to summarize the problems associated with contaminated surfaces and their importance on infection spreading, and to present some of the strategies developed to prevent this public health problem, namely some already being commercialized. microorganisms' spreading, and consequent infection propagation is a serious concern worldwide, yet there are still very few guidelines or legislation for infection propagation control in public spaces. the exception is made for healthcare facilities with the existence of few guidelines and orientations for infection prevention and control, nevertheless hospital acquired infection (hai) remains a tremendous problem. one of the main causes for the high number of hai reported by the world health organization (who) is related to the ability of the microorganisms to survive for long periods in hostile environments such as dry surfaces [1, 2] . rooms occupied with infected patients often have their surfaces contaminated with pathogens leading to the contamination of hands and gloves of medical staff, and consequent transfer of these microorganisms to patients or onto other surfaces. this cross-contamination mechanism has already been recognized and lead to the implementation of hygiene procedures for hands when contacting with patients. still, it is more likely for a patient to get an infection when staying in a room previously occupied by an infected patient. thus, infected patients are the primary source of surface and material contamination. visitors and/or asymptomatic carriers also contribute to the spread of microorganisms, especially in situations where there is an apparent sense of safety [3] . as said before, there are guidelines for surface cleaning and disinfection in hospitals, however, those recommendations are not sufficient to solve such a problem and the incorrect following of the disinfection instructions can even cause greater contamination problems [4] . this review considered english-language articles retrieved from pubmed database literature searches, bibliographies from published articles, and infection-control books and chapters, in a total of 205 references published between 2000 and 2018, considering the following criteria: the most recent studies performed on microbiological analysis on different surfaces reporting samplings performed on food contact surfaces, public spaces and hospital surfaces, where microorganisms occur naturally. surfaces studies, the most recent and relevant works developed on surface modification or functionalization were selected and two main types of surfaces were considered for this review -anti-adhesive surfaces and antimicrobial surfaces, along with their subtypes. finally, products already commercially available with enough reliable manufacturers' information about the product and its adequacy to the topics discussed in this review were also included. contamination spreading by contact can occur either by direct contact with an infected patient, or indirectly through a contaminated object or surface [5] . surfaces represent an important way of transmission of diseases since they can act as a reservoir of microorganisms that may spread to whoever contacts with the surface. especially in crowded spots as public transports or public spaces and places susceptible of the presence of microorganisms like healthcare facilities, surfaces may represent a serious source of infection spreading [6] . table 1 describes the microorganisms detected in hospital, food handling, and other environmental surfaces. it is estimated that each year, in the united states alone, 9.4 million episodes of foodborne illness occur resulting nearly 56,000 hospitalizations and over 1300 deaths. the microorganisms most commonly associated with this phenomenon are norovirus, salmonella spp., clostridium perfringens and campylobacter [7] . food contamination can result from several factors and occur at different points of the preparation process. from farming to the moment of consuming, food goes through several steps where contamination by microorganisms can occur. temperature and storage conditions are important factors to prevent food spoilage however, there are other factors that can cause microbiological contamination [8] . food-contact surfaces' contamination can cause food contamination with pathogens during processing or packaging. bacteria are naturally present in plants and animals and it is easy for them to attach to a food contact surface during handling. due to adhesion mechanisms bacteria can remain on the surfaces and contaminate other foods [9] . chopping boards, knifes and preparation tables are just some of the food preparation instruments found contaminated with bacteria [10] . saad et al. [11] evaluated hygiene conditions on food preparation facilities by analysing food-contact surfaces and the results were quite table 1 examples of microorganisms isolated from different surfaces and some of their associated pathologies. adverse, suggesting fecal contamination. coliform bacteria were found in dining tables (58%), food trays (33%), cooking pots (33%) and kitchen faucets (8%). in addition, e. coli was identified in some cooking pots. in this study, surfaces with a count of total mesophilic aerobes of > 10 cfu/cm 2 or > 1 cfu/cm 2 for e. coli and coliforms were considered to fail hygiene criteria, according to the commission of the european communities guidelines [12] for hygiene conditions in fresh meat processing facilities. although most countries have guidelines about hygiene measures and good practice on food processing facilities not always the cleaning procedures are sufficient to prevent microorganisms propagation [4] . most of the times the cleaning cloths used in cleaning can become contaminated if soaked with disinfectants in the wrong dilution (common fault in food handling facilities), contributing to microorganisms' spread to previously uncontaminated surfaces [13] . in the food industry, the occurrence of biofilm formation is also a common event due to inefficient cleaning and disinfection. the remaining microorganisms can survive on the surfaces, especially if food residues remain. this will promote the development and multiplication of bacteria with consequent biofilm formation that is extremely hard to eliminate, becoming even more difficult to disinfect the surface [14] . schools and day-care centers are hot spots of infection spreading. lack of hygiene habits, common among very young children, is one of the main causes of contamination between kids, leading to the contamination of other children, staff, parents or people in the community, either by direct contact or by contaminating surfaces. [15] . ibfelt et al. [16] evaluated the presence of bacteria and viruses in day-care centers in denmark and different surfaces from toilets, kitchens and playrooms were analyzed. the results proved contamination by several bacteria and virus. despite the main bacteria present were non-pathogenic, several surfaces revealed the presence of coliform and nasopharyngeal bacteria. respiratory viruses were mainly present on toys but also on pillows and playroom tables. gastrointestinal viruses although less prevalent were found in some surfaces, mainly on playroom pillows and toilet surfaces. a study by jerković-mujkić et al. [17] analyzed potential contamination in 60 public telephones and the results showed high presence of microorganisms on the surfaces. staphylococcus epidermidis (73.3%) and bacillus subtilis (40%) were the most common bacteria identified, however more species were isolated. mukherjee et al. [18] evaluated the presence of microorganisms on different surfaces of a fitness center. the samples were collected from skin-contact surfaces as floor mats and exercise instruments, regularly shared by many people. in total, 63 species were identified with the presence of some pathogenic or potential pathogenic bacteria like salmonella, staphylococcus or klebsiella. staphylococci were present in the majority of the surfaces being one the most prevalent species, namely s. aureus, s. epidermidis and staphyloccocus saprophyticus. kandel et al. [19] developed a study with stupefying results. they compared microbiological contamination of toilet surfaces with buttons on hospital elevators, and the buttons showed higher colonization (61%) than the toilets (43%). the most common bacteria on both surfaces were staphylococcus and streptococcus, however other bacterial species were founded, as wells as fungi. otter and french [6] studied the presence of microorganisms in hand-touch surface in public transport system. the samples were collected from several surfaces in trains, stations and buses of london. the presence of bacteria was confirmed, and most sites presented a median bacterial concentration of 12 cfu cm −2 . even though there are no guidelines for acceptable values for bacteria's presence on surfaces of public spaces this value is higher than the recommended for foodprocessing surfaces [12] and hospital hand-contact surfaces [20, 21] . it has been recognized that hospitals' contaminated surfaces and medical equipment contribute to the contamination of patients and medical staff [3, 5] . several factors contribute to this occurrence but the mains reason is associated with the high prevalence of microorganisms in healthcare facilities. pathogens with the ability to cause high rates of infection come from different sources such as patients' endogenous flora (40-60%), hands of medical staff and assistants (20-40%), antibiotic-driven changes in patients' flora (20-25%) and other, including contamination of the environment (20%) [22] . carling et al. [23, 24] have shown that in some cases less than 50% of hospital surfaces can be considered clean after terminal disinfection procedures. however, for specific surfaces the rate can be even more alarming with less than 30% of bedpan cleaners, bathroom hand-holds, light switches, and doorknobs being cleaned [25] . contact between hands and gloves of medical staff with contaminated surfaces leads to pathogens' spreading to other surfaces and people [5] . particularly if they are immunocompromised patients, the risk of developing an infection is very high [26] . stiefel et al. [27] found out that the level of contamination of gloves on medical staff after contact with patients' skin sites was actually quite similar (40% vs 45%) to the level of contamination after contacting with surfaces on methicillin-resistant staphylocococcus aureus (mrsa) carriers' rooms, suggesting that environmental surfaces may pose serious risks. an identical study from guerrero et al. [28] found similar results for c. difficile contamination, proving that the risk of hand contamination after contact with patients' skin was the same than when contacting with rooms' highly-touched surfaces (50% vs 50%). this issue is also related to the compliance of hospital workers on performing disinfection protocols. a recent study from stahmeyer et al. [29] showed that only 42% of healthcare workers washed their hands before contact with a patient, and 50% washed their hands after contact with the patient. also, this study found out that 94.3% of healthcare workers' hand hygiene procedure lasted less than 15 s (average 7.6 s), when the recommendations is that it should last around 30 s. another study evaluating disinfection compliance by healthcare workers showed that only 26% of the enquired radiologists affirmed to disinfect their workstation daily, 24% admitted never to have disinfected their workstation, and 100% said to have never received any recommendation on how to perform the disinfection [30] . several studies have proved that surfaces in rooms of patients infected with important pathogens are frequently contaminated, and that a person admitted to a room previously occupied by an infected patient has an increased likelihood of developing colonization or infection with that pathogen [25, 31] . a study performed in portugal by geadas farias et al. [32] revealed hospital surfaces' contamination with different microorganisms, mostly pseudomonas and staphylococcus. several surfaces were contaminated but some of those with higher microbiological presence were surfaces associated with water distribution system as sinks, taps, showers and drains. viana et al. [33] performed an analysis on hospital mattresses, and resistant bacteria were found on about 50% of the evaluated mattresses. a. baumannii was the main species found, being present in 73.1% of the mattresses, but also other pathogens were collected as k. pneumoniae and p. aeruginosa. this study also identified the microorganisms found on mattresses and showed that in 54% of the cases the microorganisms were not related to the current patient admitted in the room but to the previous one, concluding that even after cleaning microorganisms still remain on the mattresses being a possible source of contamination to other patients. despite most studies evaluating the effectiveness of room disinfection have focused on some specific surfaces considered dirty (sink, toilets, etc.), all spots must be considered since contamination can be present on unsuspected surfaces [34] . actually, white et al. [35] found out in an hospital that microbiologically the sinks and floors were cleaner than hand touch sites as chairs, beds and cardiac monitors buttons. shek et al. [36] developed a study where hospital curtains of burn and plastic surgery units were microbiologically tested. the results proved a considerable presence of bacteria on the curtains, even multi resistant bacteria as mrsa. levin et al. [37] found that in situations of poor infection control practices it is possible for patients to become a source for unresolved contamination of portable radiographic equipment with pathogenic bacteria, namely resistant bacteria. lestari et al. [38] performed a similar study but evaluating ecg lead wires, and among the 451 lead wires analyzed only 2 were non-contaminated with some type of microorganism. coagulase negative staphylococci and aerobic spore forming bacteria, two important types of pathogens, were present in 96% and 71% of the wires respectively. the ineffective cleaning associated with the ability of microorganisms to survive on surfaces under hostile conditions, for long periods of time, is a serious cause of pathogens spreading to people and to other surfaces [31] . the presence of biofilms, the substances used in the cleaning process, and the method and frequency of cleaning, all interfere in the grade to which microorganisms resist to the cleaning and disinfection procedures. badly executed cleaning procedures may bring bigger problems than not cleaning at all. the transmission of microorganisms from a contaminated surface to a clean one can occur if the cleaning method is not correct. also, most detergents do not have antimicrobial activity they only act in the microorganisms' removal, leading to the contamination of the cleaning cloths with viable microorganisms, and its consequent spread to non-contaminated surfaces [39] . the frequency of cleaning is also an important factor, since surfaces such as toilets, sinks, and other known as dirty surfaces tend be cleaned frequently [40] . however, other surfaces thought safe but still highly touched are often neglected, being poorly cleaned. for example, according to the practical guidelines for infection control in health care facilities [41] , mattresses without plastic covers must be steam cleaned if contaminated with body fluids. however, if body fluid contamination does not occur the mattresses are not so often cleaned, allowing microorganisms to accumulate with time and use, as proved by viana et al. [33] . also geadas farias et al. [32] have found that surfaces usually not considered dirty but highly touched as light switches or nursing trays were contaminated with several microorganisms. a very recent study by johani et al. [42] revealed the presence of biofilms in many surfaces of intensive care units despite regular disinfection procedures. several highly touched surfaces were analyzed and in 70%, the presence of biofilm was proved by scanning electron microscopy. in many situations the lack of adequate training of the environmental cleaning services or of the medical staff ultimately promotes the use of inappropriate detergents or germicides promoting the survival of microorganisms on the surfaces, even after cleaning [43] . among the most used disinfectants are substances based on hypochlorite, alcohols, aldehydes, phenols and quaternary ammonium compounds [44] . however, not all of these substances are efficient against some specific types of microorganisms, namely spore forming pathogens like c. difficile [45] . some disinfectants may cause resistance on the microorganisms if not used within specific parameters [46] . there are several guidelines [41, 47, 48] for infection control that suggest cleaning and disinfection methodologies to be applied in healthcare facilities. these guidelines not only suggest how to disinfect medical equipment but also how to clean environmental surfaces as floors, furniture or toilet seats. however, different guidelines present different opinions on the frequency of cleaning or advisable disinfectants/detergents for each situation since there is no standardization for the methods and cleaning agents to be used. in addition, there is a lack of guidelines suggesting effective standardized methods to assess environmental cleaning. visual evaluation is still the most used method to determine a surface cleanness, however even if a surface is apparently clean it can contain a high microbial load [49] . according to the commission of the european communities [12] guidelines for hygiene conditions in fresh meat processing facilities, food-contact surfaces with > 10 cfu/cm 2 for total viable count and > 1 cfu/cm 2 for enterobacteriaceae are considered unacceptable. there are no established limit values for microorganisms on surfaces for public spaces, and there is poor control on cleaning performance. as depicted in section 2.2 surfaces in these places are often contaminated showing values higher than those allowed on healthcare facilities (2.5-5 cfus/cm 2 ). more regulation should be made to assess the cleaning and disinfection efficacy and compliance on such places, namely on spaces with more susceptible people as schools, day-care centers or elderly homes. more guidelines about how to clean and how often would be a good option, as well as a higher microbiological assessment on surfaces, that is rarely or never done in public spaces. for healthcare facilities, there is some controversy about the acceptable number of microorganisms on surfaces since there are no established thresholds, only tentative suggestions from researchers. for some authors, clean hand-contact surfaces in hospitals must have less than 5 cfus/cm 2 , with an increased risk of infection for values above that [20] . other researchers have proposed the maximum value of 2.5 cfus/cm 2 as the limit level of contamination in clean hospital surfaces [21, 50] . for some specific microorganisms, considered indicator organisms, the limit value must be even lower. the presence of such microorganisms (for example s. aureus, c. difficile, vancomycin-resistant enterococci or acinetobacter spp.) is considered indicative of the need for cleaning even at levels of 1 cfus/cm 2 [20] . some reference limit-values either from governmental entities or from research studies are listed in table 2 . the limit of < 1 cfus/cm 2 for indicator microorganisms makes sense since it has been shown that an infectious dose of 1 cfus/cm 2 was sufficient to cause c. difficile infection on mice [45] . also, it was observed that very low doses of norovirus have the capacity to cause infection [51] . these results show that microbiological contamination on surfaces poses a real risk of infection, since the environmental infectious dose can be very low for some microorganisms. there is still the need for reaching a consensus and creating general guidelines for healthcare facilities. establishing clear limit values and implementing frequent microbiological assessment protocols are some of the steps that should be in the horizon. to avoid contamination and consequent infection propagation on surfaces, several alternative strategies have been developed recently. application of aerosols or uv light on contaminated surfaces are good examples of no-touch strategies, and they seem to be a good option to be applied in hospital environment [52] . however, these approaches still present some limitations. uv light, in hospital facilities, can only be applied in empty rooms, therefore it cannot be used for a daily cleaning routine. also, it only disinfects areas directly reached by the uv light, so many objects and surfaces remain contaminated after this disinfection approach. aerosols are efficient but take a long time to decontaminate the surfaces compared to conventional cleaning or uv light. besides that, these strategies are also exclusively used in empty rooms. this factor increases the necessary time to do the final disinfection of the room and perform the beds turnover in hospitals [53] . cold plasma technology is another disinfection strategy that has recently been applied on surfaces. plasma is the fourth state of mater and it can be generated from gases that became ionized with lots of ions, electrons and free radicals that will provide good electric conductivity. plasma is known to have high levels of energy and it can change molecules composition making them highly ionized and with many free radicals moving randomly in all directions. for example, when a molecule of hydrogen peroxide is exposed to this technology the double bond on the molecule is broken, creating reactive oxygen species like hydroxyl radicals that in need to seek equilibrium will bind to surrounding microorganisms destroying them by oxidation [54, 55] this technology has already been tested in food industry to decontaminate vegetables [56] and meat [57] with good results obtained and also to decontaminate surfaces as packaging materials. puligundla et al. [58] have tested the elimination of different bacteria such as e. coli, salmonella typhimurium and s. aureus, from several materials often used on food packages such as glass, polypropylene, polyethylene, nylon and paper foil. the results were quite promising with several log reductions after 5 to 10 minutes of treatment. even though the results of this emerging technology are quite encouraging as a possible strategy to disinfect surfaces, more studies are needed to assure its safety and profitability to disinfect larger surfaces. surface modification/functionalization is another common procedure to obtain anti-adhesive or antimicrobial properties on different materials. this approach has several advantages over conventional disinfection techniques. for example, antimicrobial surfaces are in constant process of activity oppositely to no-touch technologies or conventional cleaning. this way the antimicrobial charge on the surfaces is reduced immediately after contact preventing its propagation and consequent contamination of surrounding surfaces or people [59, 60] . other advantage is the possibility of these surfaces to be present in populated environments since they cause no harm to people with no need to remove people from the rooms to perform the disinfection. in addition, modified surfaces are not restricted to external surfaces. medical devices such as urinary catheters, central venous catheters, prosthesis or contact lenses are some of the surfaces that have been functionalized to obtain better compatibility properties and lower infection risk when inserted on the human body [61] [62] [63] [64] . self-disinfecting surfaces are an emerging topic with more and more products coming up. some of the most recent strategies developed to obtain surfaces with anti-adhesive and antimicrobial properties are discussed below. several natural surfaces have suffered diverse evolutionary processes that turned them resistant to microorganisms' colonization. natural and bio-mimicked surfaces of insects' wings, sharks' skin, and lotus' leaves exhibit antibiofouling properties by preventing different cells, particles or microorganisms from attaching to their surface [65] . considering this, some of these natural anti-adhesive or self-cleaning surfaces have been investigated for their potential application on micro/nanostructured surfaces development. however it is also possible to give anti-adhesive properties to a surface by modifying the material's chemistry, namely using self-assembled monolayers or polymer brushes immobilized on the surface (fig. 1 ) ( [66] ; [67, 68] ). there are several ways to produce materials with anti-fouling properties acting at the level of surface modification with chemical structures. functional groups exhibited by the material's surface interact with those in the microorganism' cells determining the kinetics of microbial adhesion. for instance, surfaces highly negatively charged, i.e. polyanionic surfaces have the ability to repulse the bacteria with polyanionic glycocalice (most of gram-positive bacteria) through electrostatic interactions. however, gram-negative bacteria have policationic glycocalices, so this surface modification mechanism is only effective against some bacterial species and sometimes only against specific strain types [69] . zwitterionic materials, such as carboxybetaine, sulfobetaine, and phosphobetaine also have anti-fouling properties. zwitterionic polymers have an equal number of anionic and cationic groups present in their repeating unit. this fact gives these polymers ultra-hydrophilicity and consequently great hydration ability [70, 71] . studies concerning surface modification with zwitterionic polymers have proved to be a promising strategy to reduce microorganisms' adhesion [72, 73] . very recently, a study using a tyramine-conjugated sulfobetaine polymer, grafted on polyurethane showed a great reduction in s. aureus adhesion to the surface [74] . coating with polymeric brushes can also prevent microorganisms' adhesion. apart from avoiding direct contact between microorganisms and the surfaces, the polymers used for brushes are usually hydrophilic, so water will be attracted into the brush forming a repellent layer in aqueous environment. proteins and microorganisms encountering the brush surface will be repelled by steric hindrance due to the water bound in the brush and the elasticity of the polymer chains [75] . polyethylene oxide (peo) and polyethylene glycol (peg) are two examples of polymers used to produce hydrophilic brush coatings on biomaterials. in a recent study by. hadjesfandiari et al. [76] it was proved that peo brushes covalently immobilized reduced 98% the adhesion of staphylococci and e. coli to a surface. apart from the chemistry, also topography of a surface can be structured to increase anti-adhesive properties. as an attempt to find efficient alternatives over more classic antimicrobials, micro/nanostructured materials present themselves as possible solutions. recently, have been done an attempt to elucidate if alterations on micro or nanotopography of a surface could influence colonization and consequent contamination. an example of structure modification is the application of superficial nanostructures (nanoparticles, nanofibers or nanotubes) reducing the area available for microorganisms to attach [77] [78] [79] . as said previously, mimicking the topography of anti-adhesive surfaces innately present in nature is an innovative way to obtain new self-disinfecting surfaces. many plants have special surface properties namely wettability. for instance, lotus leaves have micropapillae structures covered by nanostructures with fine branch-like shape. these structures along with epicuticular needle-shaped wax tubes that cover them give the lotus leaf a superhydrophobic surface and consequently antifouling properties. water droplets roll down the lotus leaf due to its great hydrophobicity dragging out any possible particles present, and maintaining its surface clean [80] . other example of nature that inspired anti-adhesive surfaces' coating is sharkskin. sharks have a special scale micropattern, which consist of a rectangular base embedded in the skin with tiny spines on the surface. the ribbed texture of these scales is responsible for the selfcleaning, anti-biofouling, hydrophobic and drag reducing properties of shark skin [81, 82] . such properties have made of sharkskin one of the most mimicked surfaces, not only in research environment but in industrial area too. sharklet af (sharklet technologies, alachua, fl, usa) is a company that uses shark skin topography in different surfaces in order to avoid bacterial adhesion and biofilm formation [83, 84] . a study comparing sharklet micropattern surfaces with regular surfaces in a clinical simulated scenario proved that shark skin microtopography reduced the number of attached bacteria by about 5 fold [85] . there are several materials with intrinsic antimicrobial properties, as silver, zinc, cooper, or chitosan. nevertheless, materials can also be modified to acquire bactericidal activity [86] . chemical or physical changes in the surface can produce antimicrobial effect. chemical substances such as antibiotics or biocides can be incorporated in the materials to give it antimicrobial properties. however, a balance between these properties and biocompatibility must be observed since the materials will be in direct contact with users' organism raising toxicity concerns. photo-activated surfaces are also an example of self-disinfecting materials recently developed. fig. 2 presents some examples of different types of antimicrobial surfaces. some materials have intrinsic antimicrobial properties. they do not need antimicrobial loading to exert its activity since the material by itself has the natural ability to eliminate microorganisms. among the most known natural materials with antimicrobial properties are metals as silver, copper, or zinc and polymers like chitosan [68] . for many metals with antimicrobial properties, it is the ionic form that presents higher bactericidal activity and not the elemental metal. silver and copper are good examples of that. silver has been used in medical field for many centuries due to its antimicrobial properties. its bactericidal mechanism is based on the binding of silver atoms with thiol (sh) and disulfide (s-s) groups present in the proteins of bacterial cell membranes, leading to its disruption and eventual cell death [87] . silver has been used for many applications on medical field especially when added in the form of silver sulfadiazine to creams or wound dressings [88] . several wound dressing containing silver are available on market, however, not always they comply the expectations. cavanagh et al. [89] analyzed 6 different silver dressings on market and only two of them showed bactericidal activity against s. aureus. apart from that, silver dressing may be related with increased serum silver levels with an associated decrease on white blood cells [90] . this leads us to conclude that the delivery system of the antimicrobial is very important. a delivery mechanism that acts by contact without leaching silver ions to the surrounding areas or tissues would be good strategy to achieve antimicrobial action without compromise the safety of the product. more recently, silver nanoparticles have been greatly studied and used to produce potential antimicrobial materials such as catheters and surgery sutures [91, 92] . however, for biomaterials implanted inside the organism there are some possible risks associated with releasing of silver ions causing local toxicity and possible accumulation in organs [93] . silver may also have different ranges of efficacy according to the class of bacteria. gram-positive bacteria's cell walls do not have an outer membrane as gram-negative bacteria do, but they have several layers of peptidoglycans, making their cell wall thicker [94] ; this, along with their negative charge results in the trapping of silver ions (positively charged) preventing their entrance into the bacteria [95] . agc glass (ags glass uk ltd., rugby, great britain) developed an antibacterial glass by incorporating silver ions inside the glass. the company claims that this glass eliminates 99% of bacteria and prevents fungi proliferation which they demonstrated in the three bacterial and two fungi strains evaluated [96] . surfacine development company (tyngsborough, ma, usa) is another company that developed a silver coating (surfacine ® ) that can be applied in several materials, from medical devices to food preparations and packaging industry or water distribution systems. this coating showed good antimicrobial activity against several bacteria, fungi and yeast [97] . other company, purethread technologies inc. (cary, nc, usa) has developed purethread ® , a system that embeds silver salts into textile fibers, obtaining antimicrobial textiles. according to the company this system does not weaken or disappears during washing and kills 99.9% of microorganisms after four hours of contact with the fabric [98]. a scientific paper about a study involving hospital curtains incorporating the antimicrobial textile developed by this company proved that those curtains take more time to be contaminated for the first time when compared to regular curtains [99] . similarly, to silver, copper has been used for many centuries as an antimicrobial agent especially for water treatment and transportation. copper was also very used in the nautical field, to prevent adhesion and growth of organisms in hulls of ships [100] . the bactericide mechanism of copper is related to the release of copper ions that cause damage in the bacterial envelop and consequent leakage of the cell content and influx of copper ions into the bacteria. this will generate toxic radicals causing oxidative damage to cellular components and dna degradation [101] . more recently copper came up as an alternative for preventing hospital acquired infections through its application in hospital surfaces and medical equipment [102] . a study by souli et al. [103] performed on a hospital intensive care unit, compared two rooms, one with copper coated equipment (beds, side table, intravenous pole stands, etc.) and the other with regular equipment. the copper coated equipment room presented a reduced percentage of colonized surfaces (55.6%) compared to the regular compartment (72.5%). this study showed both reductions in colonization by gram-negative and gram-positive bacteria and in total bioburden (2.9 vs 7.6 cfu/100 cm 2 ). sifri et al. [60] also developed a study where a section of an acute care unit was equipped with copper impregnated materials, hard surfaces and textiles-patients' gowns, sinks, bed rails, tray tables, sheets and blankets. the results were quite promising with copper equipped section presenting patients' infection reductions of 83% when caused by c. difficile and 68% when caused by multi drug resistant organisms, comparing to a non-modified section in the same hospital unit. two companies that collaborate with each other, cupron medical textiles (cupron inc., richmond, va, usa) and cupron enhanced eos surfaces (eos surfaces, norfolk, va, usa) provided the copper impregnated materials used for this study. the product developed is a hard-antimicrobial surface impregnated with copper that continuously kills 99.9% of harmful bacteria in two hours, according to its specifications. according to the company this surface is effective against s. aureus, enterobacter aerogenes, mrsa, e. coli and p. aeruginosa [104] . there are several substances with antimicrobial activity that can be added to a surface to obtain an antimicrobial surface. antibiotics were one of the first substances to be applied to surfaces such as prosthesis or textiles for wound dressing production, to obtain antimicrobial action. despite the promising results, it is well known that the major disadvantage of antibiotics use is the microorganisms' ability to develop resistance. this drawback has led to an increasing search for alternatives to be used as antimicrobials. quaternary ammonium compounds, chlorhexidine, benzalkonium chloride or nitric oxide are some examples of substances that were already tested as an alternative [105] [106] [107] . quaternary ammonium compounds are disinfectants used in hospitals and healthcare facilities for several clinical purposes as disinfection of surfaces and medical material. however, more recently the quaternary ammonium compounds have been tested as antimicrobial loading agents to different surfaces and materials, namely polymers [108, 109] . antimicrobial peptides (amps) are a class of peptides, components of innate immune system, with a broad activity against bacteria, virus, fungus and more, providing a non-specific defense against a broad spectrum of invaders [110] . recently, numerous studies [111, 112] refer its use as antimicrobial loading agents for their excellent characteristics, presenting several advantages over classic antibiotics, namely fast and broad spectrum of action with low susceptibility to induce bacterial resistance [113] . in contrast with the mechanisms of action of antibiotics, which are based in slow processes of enzymatic inhibition and target specific cellular activities as dna or protein, the amps are effective against different species of microorganisms and also reduce the risk of resistance development [113] [114] [115] . for all these reasons, amps are being applied in the production of antimicrobial surfaces, either by simple adsorption on the surface or covalent immobilization [116] . nevertheless, studies suggest that covalent immobilization offers many advantages toward physical adsorption, including higher local and long-term stability, and lowering toxicity [116, 117] . all those antimicrobial substances are loaded to the surface either by immobilization or by incorporation on the bulk material; recent studies on the application of each type of loading strategy are summarized next. in materials' incorporation process, the antimicrobial substances are added to the ingredients during the phase of production, to obtain a homogeneous mixture of the bulk material with the antimicrobial. this system allows antimicrobial activity throughout the bulk material, even in deeper layers and not only on the surface. this allows the material to retain its antimicrobial activity even when worn out [118, 119] . in a very recent study, ferreira et al. [120] used levofloxacin to load a bone cement, achieving good results against s. aureus not only on its planktonic form but also on biofilm. that was quite positive since this bacterial strain is greatly associated with biofilm formation on bone implants. ciprofloxacin incorporation on textile fibers by electrospinning was tested by li et al. [79] this strategy aimed to produce a bandage to prevent wound infection and the results in vivo (rats animal model) were quite positive for e. coli and s. aureus. in han et al. [121] study they incorporated quaternary ammonium methacrylates in dental adhesives to prevent caries by avoiding the accumulation of bacteria and biofilm formation on teeth, testing the presence of three staphyloccocal species (staphylococcus mutans, staphylococcus gordonii, and staphylococcus sanguinis) and the results showed a decreased biofilm formation for the adhesives containing the quaternary ammonium methacrylates. nanoparticles containing quaternary ammonium polyethylenimine were tested in a study about endodontic sealers by barros et al. [118] . the results were quite positive for enterococcus faecalis, with the endodontic sealers showing good antimicrobial activity. the amp, ll-37, was incorporated by cassin et al. [122] in a membrane of collagen and hyaluronic acid for the production of antiinfective films to cover injured tissues. antimicrobial coatings can be obtained either by adsorption of the antimicrobial substance, by covalently binding it to the material or by immobilizing it using self-assembled monolayers. this strategy can be an advantage for some substances that are too toxic to be used as bulk material but that immobilized in small amounts as surface coatings can exert their antimicrobial activity without the toxicity drawback [116, 123] . the coatings can also be divided according to their mechanism of action. they can deliver the antimicrobial substance by leaching, releasing it to the surrounding area or direct contact with the surface where the coating is immobilized. some coatings release substances with antibacterial properties that will interact with microorganisms acting away from the bulk. this leaching mechanism has the advantage of a larger perimeter of action around the surface where it is immobilized. it is important, though, that this release into the surrounding area is controlled to avoid toxic effects by excess of substance [124] . coatings can also attach certain antimicrobial molecules to the materials' surface. several coatings can be classified as "contact biocides" since they use non-leachable substances that kill by contact with the bacteria and with no need to be released from the surface. this strategy is very interesting for both the self-sterilizing effect and the long-lasting activity, acting just through a direct contact with bacteria without consuming itself releasing from the surface without need [117, 125] . alt et al. [126] realized a study where coating of rifampicin and fosfomycin was applied to prosthesis showing good efficacy against s. aureus, even the methicillin resistant strain, on a rabbit animal model. neut et al. [127] performed a similar study using a gentamicin-releasing coating that was able to prevent staphylococci growth on a prosthetic hip surface for at least 60 hours, showing promising results. in a work by iyamba et al. [106] , quaternary ammonium compounds (hexadecyltrimethyl ammonium bromide and hexadecylbetainate chloride) were immobilized on medical catheters reducing s. aureus adhesion to its surface. zanini et al. [128] also developed a work in which quaternary ammonium silanes were used to coat polyurethane catheters. the results were promising with this coating proving its antimicrobial activity against e. coli bacteria. in a recent study, casciaro et al. [111] successfully covalently immobilized frog skin derived amp on contact lenses. this strategy not only reduced the adhesion of p. aeruginosa, a species highly associated with contact lenses associated keratitis, but it also proved to be nontoxic to mammalian cells and it did not compromise the lenses physical properties. amps have also been immobilized on nanoparticles as loading strategy. ma et al. [112] immobilized the amp hhc-36 on titanium oxide (tio 2 ) nanotubes by physical adsorption with the nanotubes acting as nanocarriers for peptides delivery. they obtained an antimicrobial and anti-fouling surface showing a reduction in s. aureus adhesion and great antimicrobial activity (> 99% activity). braun et al. [129] immobilized the human peptide ll-37 on porous silica nanoparticles, achieving an antimicrobial delivery system. all the studies presented above prove the good potential of modified surfaces to be applied on infection control in different scenarios; still some aspects must be taken into account and more studies are still needed. most studies evaluating the antimicrobial/anti-adhesive activity potential of engineered surfaces do not take into account realistic physiological environments. this can affect the results, since the host's conditioning film may affect the surface in many different ways, for example, covering the surface creating a deterrent layer preventing the antimicrobial substance to leach or to contact directly with the microorganisms, reducing its antimicrobial efficacy [130] . in addition, the composition of the surface may determine which components of the conditioning film will adhere and that on its turn will influence other cells adhesion, as bacteria. felgueiras et al. [123] showed that different concentrations of octadecyl acrylate (c18) immobilized on polyurethane surfaces will affect the deposition of albumin and fibrinogen when the surface is in contact with human plasma, that on its turn will affect microorganisms' adhesion to the surface since albumin avoids bacteria adhesion and fibrinogen promotes it. besides, coatings with antimicrobial peptides can be degraded by some components of conditioning films such as proteases [131] . despite the pointed weaknesses there are already on the market some successful cases of such as biocote limited (coventry, united kingdom) that developed an antimicrobial additive technology, biocote ® , that can be incorporated on textiles, polymers, ceramics and more. these additives are based on different antimicrobial substances such as silver, zinc or phenolic compounds, chosen according to their application and support material [132] . sanitized ag (burgdorf, switzerland) is another company that developed an antimicrobial additive that can be incorporated in polymers and textiles for different areas of application such as healthcare, public transportation and food industry. this additive technology, sanitized ® , uses different active ingredients as silver, zinc pirithione, silane quat or isothiazolinone that can be added on liquid form, powder or paste according to specific production requirements of the final product [133] . photo-activated materials have been used in different technological fields namely in antimicrobial surfaces development. the most studied material used in photo-activated surfaces production is tio 2 . tio 2 has high photocatalytic properties and is highly used in cosmetics and skin care products since it is not absorbed by human skin [134] becoming a very interesting material for development of antimicrobial surfaces. in fact, its antimicrobial efficacy has already been proved against bacteria, fungi, protozoa and virus. the main advantage of this strategy is that tio 2 is not degraded so its activity can be maintained for long periods. tio 2 surfaces are only activated when irradiated by specific photon energy. when this irradiation occurs, reactions of photo-oxidation involving o 2 and h 2 o take place on the surface with consequent formation of oxygen free radicals. these free radicals will attack the cell wall and cytoplasmic membrane of the microorganism by peroxidizing its lipids, leading to leakage of cellular components and lately cell lysis [135, 136] . recently, adán et al. [137] published a study where a water microfiltration system using photocatalytic membranes produced with tio 2 and porous steel showed good inactivation for the retained bacteria. in another study by joost et al. [138] thin films with tio 2 nanoparticles were illuminated with uv light and after 20 min of exposure no bacteria (e. coli) had survived. the analysis of bacterial cell membrane showed modifications in the chemical structure of unsaturated fatty acids and decomposition of saturated fatty acids faster than normal, confirming the peroxidation of membrane lipids. in the market, there are already some products for microorganism elimination based on tio 2 photocatalytic action. purehealth™ (orion, florence, italy) is a system developed by orion that coats walls and floors and is activated by special lamps of solar spectrum. this system promises to eliminate virus and bacteria [139] . this review points out the great contribution of surfaces for infection spreading, not only in healthcare facilities but also in other public spaces and food processing facilities. there is an urgent need to pay more attention to surface contamination in different spaces since there is still a lack of guidelines for microbiologic assessment and established safe thresholds for health care facilities. in addition, it is important to establish infection control procedures for public spaces, namely schools since they often present high microbiological charges on surfaces, posing a real risk for children whose immunological system is still developing. both on public spaces and on healthcare facilities, it should be determined how to assess microbiologic presence on surfaces as well as how frequently this assessment should be made, and which methods should be used to obtain a realistic analysis rather than performing visual assessments. the implementation of standard acceptable limits of microorganisms on surfaces is also a key point for a better control of infection spreading. standardized definition of which microorganisms can be considered indicator microorganisms should also be achieved. the existing guidelines for cleaning and disinfection methodologies in healthcare facilities should come to a consensus about the disinfectants to be used in each setting, the frequency of cleaning and the methods to apply. the implementation of self-disinfecting surfaces is a good strategy for infection control, and this review presents some successful research already developed in this area. these surfaces show clear advantages over the regular surfaces with traditional cleaning: the state of continuous disinfection and the antimicrobial activity that permanently eliminates the microorganisms. there are still some issues to improve, like the long-term efficacy of the antimicrobial/anti-adhesive action, or the high cost of implementation of these surfaces in large areas. biomaterials, modified surfaces, or new scientific products in general, have to face some big challenges before they reach the market. problems such as costs of manufacturability, scalability on production, intellectual property issues or regulatory aspects are just some of the main obstacles those products will have to overcome in order to be commercialized [140] . some products even with great results on laboratory will fail when brought to larger scale due to their high cost of production, making them commercially unattractive. but, looking at the numbers nosocomial bacteremia alone can cost a hospital over 1 million euros per year [141] , and antibiotic resistance-associated infections may cost more than 900 million euros per year in european union. in fact the societal cost of infections due to the selected antibioticresistant bacteria were estimated at about eur 1.5 billion each year, between hospital related costs and losses in productivity due to patients' absence from work [142] . therefore, maybe it is worthy to invest on new technologies, betting on infection prevention and control not only to avoid higher costs but also to avoid the harm and loss of human lives. self-disinfecting surfaces are a step forward to the future of infection control policies. more studies involving self-disinfecting surfaces should be performed in order to realize its full potential to improve infection control and safety strategies. the reports on the application of these materials in healthcare facilities show promising results and there are already few companies supplying products with self-disinfecting properties showing that investment on prevention may be the best way to reduce the tremendous problem of infection spreading. the authors declare no conflict of interest. microbiology of healthcare-associated infections and the definition accuracy to predict infection by potentially drug resistant pathogens: a systematic review the role of the surface environment in healthcareassociated infections the role played by contaminated surfaces in the transmission of nosocomial pathogens guideline for disinfection and sterilization in health care facilities: what clinicians need to know transfer of multidrug-resistant bacteria to healthcare workers' gloves and gowns after patient 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physical mechanisms of biofouling prevention micropatterned endotracheal tubes reduce secretion-related lumen occlusion surface micropattern resists bacterial contamination transferred by healthcare practitioners silver coordination polymers for prevention of implant infection: thiol interaction, impact on respiratory chain enzymes, and hydroxyl radical induction development of silver sulfadiazine loaded bacterial cellulose/sodium alginate composite films with enhanced antibacterial property evaluating antimicrobial efficacy of new commercially available silver dressings silver absorption in patients with stevens-johnson syndrome and toxic epidermal necrolysis treated with silver-impregnated dressings. a case series biobased silver nanocolloid coating on silk fibers for prevention of post-surgical wound infections antimicrobial activity and cytocompatibility of silver nanoparticles coated catheters via a biomimetic surface functionalization strategy oral toxicity of silver ions, silver nanoparticles and colloidal silver -a review the bacterial cell envelope antibacterial effect of silverzeolite on oral bacteria under anaerobic conditions novel hospital curtains with antimicrobial properties: a randomized, controlled trial antimicrobial applications of copper killing of bacteria by copper, cadmium, and silver surfaces reveals relevant physicochemical parameters role of copper in reducing hospital environment contamination reduction of environmental contamination with multidrug-resistant bacteria by copper-alloy coating of surfaces in a highly endemic setting component release and mechanical properties of endodontic sealers following incorporation of antimicrobial agents adherence of staphylococcus aureus to catheter tubing inhibition by quaternary ammonium compounds inhibition of staphylococcus aureus adhesion to the surface of a reticular heavyweight polypropylene mesh soaked in a combination of chlorhexidine and allicin: an in vitro study bactericidal specificity and resistance profile of poly(quaternary ammonium) polymers and protein-poly(quaternary ammonium) conjugates antibiofilm properties of model composites containing quaternary ammonium methacrylates after surface texture modification will new generations of modified antimicrobial peptides improve their potential as pharmaceuticals? esculentin-1a derived peptides kill pseudomonas aeruginosa biofilm on soft contact lenses and retain antibacterial activity upon immobilization to the lens surface local delivery of antimicrobial peptides using self-organized tio 2 nanotube arrays for peri-implant infections dairy-derived antimicrobial peptides: action mechanisms, pharmaceutical uses and production proposals antimicrobial peptide structure and mechanism of action: a focus on the role of membrane structure antimicrobial properties of membraneactive dodecapeptides derived from msi-78 covalent immobilization of antimicrobial peptides (amps) onto biomaterial surfaces dhvar5 antimicrobial peptide 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release high-density antimicrobial peptide coating with broad activity and low cytotoxicity against human cells rifampicin-fosfomycin coating for cementless endoprostheses: antimicrobial effects against methicillin-sensitive staphylococcus aureus (mssa) and methicillin-resistant staphylococcus aureus (mrsa) a gentamicin-releasing coating for cementless hip prostheses-longitudinal evaluation of efficacy using in vitro bio-optical imaging and its wide-spectrum antibacterial efficacy development of antibacterial quaternary ammonium silane coatings on polyurethane catheters membrane interactions of mesoporous silica nanoparticles as carriers of antimicrobial peptides the effects of blood conditioning films on the antimicrobial and retention properties of zirconium-nitride silver surfaces cecropin a-melittin mutant with improved proteolytic stability and enhanced antimicrobial activity against bacteria and fungi associated with gastroenteritis in vitro lack of significant dermal penetration of titanium dioxide from sunscreen formulations containing nano-and submicron-size tio 2 particles photocatalytic disinfection using titanium dioxide: spectrum and mechanism of antimicrobial activity understanding the antimicrobial mechanism of tio 2 -based nanocomposite films in a pathogenic bacterium bacterial inactivation and degradation of organic molecules by titanium dioxide supported on porous stainless steel photocatalytic membranes photocatalytic antibacterial activity of nano-tio 2 (anatase)-based thin films: effects on escherichia coli cells and fatty acids translational biomaterials -the journey from the bench to the market -think "product analysis according to microorganism and antimicrobial sensitivity in a university hospital in barcelona the bacterial challenge: time to react contamination of medical charts: an important source of potential infection in hospitals hospital-wide comparison of health care-associated infection among 8 intensive care units: a retrospective analysis for 2010-2015 cleaning and disinfection of biofilms composed of listeria monocytogenes and background microbiota from meat processing surfaces microbial evaluation of foodservice surfaces in texas child-care centers clinical infection in burn patients and the consequences hospital-acquired pneumonia and ventilator-associated pneumonia in adults at siriraj hospital: etiology, clinical outcomes, and impact of antimicrobial resistance relationship between environmental fungal contamination and the incidence of invasive aspergillosis in haematology patients fungal contamination of hospital healthcare workers' overalls high burden of aspergillus fumigatus infection among chronic respiratory diseases rapid fulminant case of aspergillus prosthetic valve endocarditis advances in the diagnosis and treatment of fungal infections of the cns primary cutaneous aspergillosis due to aspergillus tamarii in an immunocompetent host prevalence of pathogens and indicator organisms in home kitchens and correlation with unsafe food handling practices and conditions transferred from naturally contaminated chicken legs to cooked chicken slices via a cutting board the microbiological quality of washing-up water and the environment in domestic and commercial kitchens transfer of campylobacter and salmonella from poultry meat onto poultry preparation surfaces etiology of diarrhea among children under the age five in china: results from a five-year surveillance importation, mitigation, and genomic epidemiology of candida auris at a large teaching hospital candida infections of the genitourinary tract supragingival and subgingival microbiota from patients with poor oral hygiene submitted to radiotherapy for head and neck cancer treatment a novel quantitative sampling technique for detection and monitoring of clostridium difficile contamination in the clinical environment identification of clostridium difficile reservoirs in the patient environment and efficacy of aerial hydrogen peroxide decontamination environmental contamination in households of patients with recurrent clostridium difficile infection sanitary status and incidence of methicillin-resistant staphylococcus aureus and clostridium difficile within canadian hotel rooms clostridium difficile infection: a worldwide disease isolation and identification of human coronavirus 229e from frequently touched environmental surfaces of a university classroom that is cleaned daily detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units severe acute respiratory syndrome coronavirus on hospital surfaces epidemic and emerging coronaviruses (severe acute respiratory syndrome and middle east respiratory syndrome) performance of food safety management systems in poultry meat preparation processing plants in relation to campylobacter spp. contamination the evolving nature of infective endocarditis in spain: a population-based study enterococcal meningitis: a clinical study of 39 cases and review of the literature prospective study on central venous line associated bloodstream infections detection of ctx-m-15-producing escherichia coli isolates of lineages st131-b2 and st167-a in environmental samples of a tunisian hospital urinary tract infection syndromes. occurrence, recurrence, bacteriology, risk factors, and disease burden van der poll, expression and function of granzymes a and b in escherichia coli peritonitis and sepsis community-acquired escherichia coli enteritis in korean children: the clinical application of a stool polymerase chain reaction assay the occurrence of influenza a virus on household and day care center fomites the environmental deposition of influenza virus from patients infected with influenza a(h1n1)pdm09: implications for infection prevention and control influenza virus contamination of common household surfaces during the 2009 influenza a (h1n1) pandemic in bangkok, thailand: implications for contact transmission within-host evolution of human influenza virus contamination of the hospital environment with gastroenteric viruses: comparison of two pediatric wards over a winter season environmental monitoring for gastroenteric viruses in a pediatric primary immunode ciency unit widespread environmental contamination with norwalk-like viruses (nlv) detected in a prolonged hotel outbreak of gastroenteritis evaluation of a new environmental sampling protocol for detection of human norovirus on inanimate surfaces detection and molecular characterization of enteric viruses in children with acute gastroenteritis in northern italy prevalence, antimicrobial susceptibility, and clonal diversity of pseudomonas aeruginosa in chronic wounds microbiology and antimicrobial susceptibility of otitis externa: a changing pattern of antimicrobial resistance community-associated methicillin-resistant staphylococcus aureus infection in portugal clinical, microbiologic, and epidemiologic characteristics of pseudomonas aeruginosa infections in a university hospital dissemination of human adenoviruses and rotavirus species a on fomites of hospital pediatric units group a rotavirus detection on environmental surfaces in a hospital intensive care unit survival of salmonella in bathrooms and toilets in domestic homes following salmonellosis diversity of bacterial communities on four frequently used surfaces in a large brazilian teaching hospital enteric fever caused by salmonella enterica serovars with reduced susceptibility of fluoroquinolones at a community based teaching hospital of nepal evaluation of food handler practices and microbiological status of ready-to-eat foods in longterm care facilities in the andalusia region of spain the role of primary care prescribers in the diagnosis and management of community-associated methicillin-resistant staphylococcus aureus skin and soft tissue infections community-acquired pneumonia with methicillin-resistant staphylococcus aureus in a patient admitted to the intensive care unit: a therapeutic challenge catheter-related infections in patients with haematological malignancies: novel preventive and therapeutic strategies the authors acknowledge the funding provided by fundação para a ciência e a tecnologia (fct -portugal) through the scholarship sfrh/ bd/130203/2017 (micaela machado querido) and the project "b-safecoat -desenvolvimento de novas tintas com propriedades autodesinfetantes" (poci-01-0247-feder-017875). key: cord-273536-h7mzqef2 authors: surpure, j. s. title: pediatric emergencies: newsletter 9 date: 1989 journal: indian j pediatr doi: 10.1007/bf02726635 sha: doc_id: 273536 cord_uid: h7mzqef2 nan somnia, fatigue, difficulty concentrating, nervousness, and depression. one hundred mg twice daily in adults reduced influenza attack rates by 70-90%. equally effective was a dosage of 100 mg once daily in teenagers. the dosage in children is 5-8 rag/ kg/day divided every 12 hours (maximum 200 rag/day). protection from symptomatic infection is increased by 10-20% with combined vaccine and chemoprophylaxis compared to either modality used alone. for treatment of documented influenza infection, medication should be started within 48 hours of onset at dosages identical to the prophylaxis dosages and continued for 7 days. rimantadine is an analogue of amantadine and will probably soon be licensed for general clinical use. indications are essentially the same as those for amantadine. adverse reactions associated with amantadine are uncommon with rimantadine. ribavirin is a broad spectrum virustatic agent with activity against rsv, influenza a and b, parainfluenza, and adenovirus. it is presently approved by the food and drug administration in its aerosolized form for the therapy of rsv infections in hospitalized children who do not require assisted ventilation. although direct fluorescence and enzyme-linked immunosorbent (elisa) assays for rsv infection are commercially available, these are difficult to establish in the usual community hospital. aerosolized ribavirin has also been 303 shown to offer therapeutic benefits in the treatment of uncomplicated influenza a and b infection. studies comparing oral amantadine or rimantadine to aerosolized ribavirin for the treatment of influenza a pneumonia have not been done. toxicity of aerosolized ribavirin in infants appears minimal. a number of published reports have indicated that interferon given as a nasal spray reduced the incidence of subsequent rhinovirus upper respiratory infections. general application of interferon therapy has become more available because advances in genetic engineering allow production of large amounts of purified material at relatively low cost. interferon may have antiviral activity against a broad spectrum of pathogens, including rhinovirus, influenza a and b, parainfluenza, adenovirus, and coronavirus. resistant viral strains following therapy have not been observed. systemic toxicity does not occur except with extremely large doses. between 12 and 15% of volunteers experienced nasal dryness, bleeding ulcerations, and erosion. interferon has not been effective for, the treatment of established rhinovirus infection. furthermore, prophylactic efficacy data are available only for rhinovirus and not for other virus infections. vitamin c (ascorbic acid) does not seem to be effective for prevention and treatment for the common cold. clinical trials have also failed to demonstrate any efficacy of zinc for treatment of rhinovirus infections. one intriguing approach to therapy utilizes murine monoclonal antibodies by intranasal administration to block cellular receptor sites for rhinoviruses. transmission of cold viruses commonly occurs from colonized hands and fomites. such spread can be interrupted by requiring infected individuals to use handkerchiefs impregnated with citric acid, malic acid or other virustatie agents. however, investigations have not been conducted to establish that treated products do indeed control transmission of cold viruses. acyclovir is probably effective for treating pulmonary infections caused by herpes simplex type i or ii and varicella zoster. ganciclovir, an investigation',d compound, offers promise for the prevention and treatment of cytomegalovirus disease. finally, combination chemotherapy plus immunotherapy and other forms; of combined antiviral agents warrant further consideration in the prophylaxis and treatment of viral respiratory infections. considerable attention has been focused recently on the alteration of antimicrobial regimens in an effort to improve outcome from bacterial meningitis in infants and children. conventional therapy with ampicillin and either an aminoglycoside or chloramphenicol continues to be recommended by the american academy of pediatrics. however, the new cephalosporins have been used recently for initial empiric treatment of mening gitis because of their increased activity against the common meningeal pathogens or because some of these compounds can be adminiin several recent controlled, prospective studies, some newer cephalosporins did not sterilize csf cultures more rapidly nor did they improve case4atality rates compared with conventional antibiotic regimens in neonates and older children (providing that the pathogens were susceptible to the antibiotic used). however, a potential advantage of the new cephalosporins is the avoidance of the need to monitor serum concentrations of aminoglycosides and chloramphenicol, especially in patients who have underlying abnormalities in renal or hepatic function, respectively. after comments from several experts, the authors conclude that there is no longer a regimen of choice for initiating treatment of bacterial meningitis. in fact, each of the experts has more than one preferred regimen tailored to specific clinical situations. there was partial consensus on the following points : (1) the reluctance to accept 7-day therapeutic regimens (too short), (2) cefuroxime may not be an ideal choice, (3) third generation cephalosporins seem to be preferred as initial therapy for meningitis in older infants and younger children. the new cephalosporins, therefore, are attractive for reasons of simplicity and safety, but clearly there are cases in which the older regimens are appropriate. injuries of the elbow in children are difficult to diagnose accurately, because the multiple ossification centers are primarily cartilaginous and not visible on routine xrays. arthrography has been suggested as a means of discerning the injury pattern before complete ossification of the elbow. is arthrography helpful in diagnosis of these injuries? yates and sullivan (j pediatr orthop, 1987; 7 : 54) 3 studied 36"children under 8 years of age with elbow injuries to evaluate the diagnostic accuracy and clinical efficacy of elbow arthrograms. the authors used double-contrast arthrography, with equal volumes of contrast material and air (usually 89 ml of each). all arthrograms were obtained within 24 hours of injury with the exception of one child referred for evaluation 10 days following trauma. all arthrographie diagnoses were confirmed by subsequent clinical and radiographic courses. many elbow injuries are misdia~nosed by routine x-ray methods and are subsequently managed inappropriately. the most common arthrographic diagnosis in this study was supracondylar fracture, followed by lateral condylar fracture, distal humeral epiphyseal fracture/separation, olecranon fracture, medial epicondylar avulsion with elbow dislocation, contusion, nursemaid's elbow, and septic elbow. the additional information gained by arthrographic evaluation not only better defined the anatomic lesion, but also favorably influenced subsequent treatment in 7 of the 36 children. the authors conclude that arthrography can be helpful in the skeletally immature patient whose diagnosis is in doubt and is particularly useful in condylar fractures and periarticular fractures in which anatomic alignment cannot be ascertained from plain x-ray. however, arthrography should be needed infrequently overall as long as proper radiographs (including comparison views) are obtained. fever is the most common presenting symptom in most pediatric clinics. many health care providers believe that a fever of benign (usually viral) etiology responds better to antipyretics than a fever caused by a more serious (bacterial) infection. is this belief fact or fiction? weisse et al (pediatric infect dis j 1987; 6 : 1091) 1 studied the effect of acetaminophen on fever in bacterial vs viral infections in 100 children (ages 9 days to 17 years) with rectal or oral temperature of 1020 f (38.90 c) or greater. study patients were given 15 mg/kg (maximum 650 mg) acctaminophen, and their temperatures were rechecked 1 hour later. previous studies have indicated that the risk of bactercmia increases with certain threshold elevations of white blood cell count, erythrocyte sedimentation rate, and temperature. results have been ambiguous concerning the use of subjective analysis of a patient's "toxicity" to predict bacteremia. these authors agree with previous studies which showed that the risk of bacteremia increases with increased white blood cell count. furthermore, they found that the fever response to acetaminophen was a poor discriminator between bacterial and viral infections. they suggest that additional study including a more prolonged period of patient temperature measurements might be helpful to more fully understand this clinical question. asthma is the most common lung discase in children beyond infancy. a number of clinical scoring systems have been devised that are intended to help assess the severity of the asthma episode. one such scoring system is the clinical asthma score (cas) developed by wood and co-workers in 1972. the use of cas has not been validated by adequate clinical trials. does the cas have prognostic value? baker (ajdc, 1988 ; 142 : 183) 2 studied 210 asthma patients to evaluate the usefulness of the cas in determining outcome in childhood asthmatics. all patients received standard treatment, consisting of beta-adrenergic agents and theophylline compounds. the cas is the summation of values assigned to 5 different clinical characteristics, which include cyanosis, inspiratory breath sounds, accessory muscle use, expiratory wheezing, and cerebral function. the decision to admit or discharge a patient was based on the treating physician's clinical assessment. follow-up information was gathered by telephone on each child 10 days after emergency department disposition. the author found that the pretreatment cas was not useful for the early identification of children requiring prolonged inpatient asthma management. although the cas might be reflective of the severity of illness at the time of assessment, it is of little use in predicting the direction of illness as reflected either by the length of hospitalization or the continuation of clinically significant symptoms at home. these data indicate that the cas alone is not a reliable indicator of severity of acute asthma of childhood as judged by subsequent disability. fever response to acetaminophen in viral vs bacterial infections pitfalls in the use of clinical asthma scoringajdc arthrographic diagnosis of elbow injuries in chijdren antiviral agents for respiratory infections. pediatrb,fect consensus report : antimicrobial therapy for bacterial meningitis in infants and children pediatr inject key: cord-265005-e6rpryrh authors: tomasello, elena; pollet, emeline; vu manh, thien-phong; uzé, gilles; dalod, marc title: harnessing mechanistic knowledge on beneficial versus deleterious ifn-i effects to design innovative immunotherapies targeting cytokine activity to specific cell types date: 2014-10-30 journal: front immunol doi: 10.3389/fimmu.2014.00526 sha: doc_id: 265005 cord_uid: e6rpryrh type i interferons (ifn-i) were identified over 50 years ago as cytokines critical for host defense against viral infections. ifn-i promote anti-viral defense through two main mechanisms. first, ifn-i directly reinforce or induce de novo in potentially all cells the expression of effector molecules of intrinsic anti-viral immunity. second, ifn-i orchestrate innate and adaptive anti-viral immunity. however, ifn-i responses can be deleterious for the host in a number of circumstances, including secondary bacterial or fungal infections, several autoimmune diseases, and, paradoxically, certain chronic viral infections. we will review the proposed nature of protective versus deleterious ifn-i responses in selected diseases. emphasis will be put on the potentially deleterious functions of ifn-i in human immunodeficiency virus type 1 (hiv-1) infection, and on the respective roles of ifn-i and ifn-iii in promoting resolution of hepatitis c virus (hcv) infection. we will then discuss how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the subtypes and dose of ifn-i produced, (ii) the cell types affected by ifn-i, and (iii) the source and timing of ifn-i production. finally, we will speculate how integration of this knowledge combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments in patients. specifically, we will discuss how induction or blockade of specific ifn-i responses in targeted cell types could promote the beneficial functions of ifn-i and/or dampen their deleterious effects, in a manner adapted to each disease. type i interferons (ifn-i) were the first cytokines discovered, over 50 years ago, based on their potent anti-viral effects (1, 2) . ifn-i play a crucial, non-redundant role in vertebrate anti-viral defenses (3) (4) (5) . ifn-i also mediate protective effects in other physiopathological contexts, including cancer (6) and multiple sclerosis (ms) (7) . on the contrary, ifn-i responses can be deleterious in a number of circumstances, including bacterial or fungal infections (8-10), many autoimmune diseases (11) , and, paradoxically, certain chronic viral infections (12) (13) (14) . it is only recently that an integrated picture has emerged of the cellular and molecular mechanisms regulating the production of ifn-i and underlying their functions. much knowledge was gained recently on another class of potent innate anti-viral interferons, the lambda, or type iii ifns (ifn-iii). we will review knowledge on ifn-i/iii (ifns) and discuss how it could be harnessed to develop innovative therapeutic strategies aimed at surgically tuning ifn activity toward protective responses in a manner adapted to each disease. we will focus on ifn-α/β/λ because they are the best characterized ifns and already used therapeutically. recent reviews are covering information on other ifn-i subsets including ifn-ε, which is produced at mucosal sites and promotes local anti-viral defenses (15, 16) . dendritic cells (dcs) are rare heterogeneous mononuclear phagocytes functionally characterized by their unique efficacy for antigen-specific activation of naïve t lymphocytes. dcs are sentinel cells of the immune system, able to sense and integrate a variety of danger input signals for delivery of output signals instructing the activation and functional polarization of effector immune cells. in mammals, five major dc subsets exist, which differ in their expression of innate immune recognition receptors (i 2 r 2 s) and in their functional specialization: monocyte-derived dcs (modcs), langerhans cells, cd11b + dcs, xcr1 + dcs, and plasmacytoid dcs (pdcs) (17) . a recurrent theme of this review will be the intricate relationships between ifns and dcs, since these cells can be a major source and/or target of these cytokines under various conditions. www.frontiersin.org the first section will synthesize current knowledge on ifn production and protective anti-viral functions. the i 2 r 2 s and downstream signaling pathways responsible for ifn-i production during viral infection will be listed. the roles of different cell types for this function will be discussed. the two main mechanisms through which ifn-i promote anti-viral defense will be reviewed, succinctly for direct anti-viral effects and in greater details for immunoregulatory functions. the second section will focus on the detrimental functions of ifn-i. selected diseases will be discussed to illustrate how different, and sometimes opposite, processes underlie deleterious ifn-i responses depending on the physiopathological contexts. ifn-i induction of unbridled inflammatory responses causing lethal tissue damage will be discussed as a major pathological mechanism during bacterial encounters secondary to influenza infection or in some autoimmune diseases. inappropriate functional polarization of immune responses by ifn-i will be discussed as one potential cause for enhanced susceptibility to bacterial or fungal infections. the complex and disputed role of ifn-i in chronic viral infections will be reviewed, with emphasis on the physiopathology of the infections by human immunodeficiency virus type 1 (hiv-1) and human hepatitis c virus (hcv), with an outlook for the development of novel immunotherapeutic strategies to combine with anti-viral drugs. the third section will recapitulate how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the source and timing of ifn-i production, (ii) the cell types affected by ifn-i, and (iii) the signaling pathways activated by ifn-i. in the last section, we will speculate how integration of all the knowledge discussed before combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments, based on induction or blockade of specific ifn-i responses in targeted cell types. this"activity-by-targeting"concept is based on the design of novel "immuno-ifns" consisting in covalent association between a mutated ifn-i with decreased affinity for its receptor and an antibody with high avidity for a molecule specifically expressed on target cell types (18) . this design ensures lack of activity of the immuno-ifns on all cell types but those targeted, contrary to previous strategies using ifns with close to maximal potency that were still able to mediate strong off-target effects despite their coupling to cell-type specific antibodies and/or their local delivery. type i interferons expression is not detectable under steady state conditions in vivo using classical methods such as gene expression analysis by rt-pcr or protein titration by elisa or bioassays. however, mice deficient for the expression of the alpha chain of the ifn-i receptor (ifnar1) harbor alteration in the ontogeny or functions of various cell types (19) (20) (21) (22) (23) (24) (25) (26) . hence, extremely small or localized but functionally relevant quantities of ifn-i must be produced under steady state conditions (27) . indeed, the existence of steady state responses to ifn-i in various organs in vivo was demonstrated by using reporter mice expressing the firefly luciferase under the control of the promoter of ifnb1 (28) or of mx2 (29) , a canonical ifn-i-stimulated gene (isg). steady state ifn-i responses are promoted by gut commensals (30) . early and transiently after many viral infections, large amounts of ifns can be detected, in blood and spleen in the case of systemic infections or locally in the case of confined infections. ifn induction during viral infections results from the detection of specific danger signals by specialized i 2 r 2 s. this includes the detection of pathogen-associated molecular patterns as well as the sensing of stress signals or damage-associated molecular patterns (31, 32) . based on the nature and intracellular location of the danger signals that induce the production of the cytokines, the cellular sources of ifns during viral infection can be classified in two main groups. infected cells often contribute to ifn production as a response to their sensing of endogenous viral replication, or consecutive to the metabolic stress induced during massive translation of viral structural proteins, or as a result of plasma membrane perturbations upon viral entry. specific subsets of uninfected cells can also significantly contribute to ifn production upon engulfment of material containing viral-derived nucleotide sequences and sensing of these molecules in endosomes by specific i 2 r 2 s. all sensing pathways leading to ifn induction converge on the activation of interferon response factors 3 or 7 (irf3/7), which are the master transcription factors inducing ifn genes. most cell types constitutively express irf3 but not irf7 or only at low levels. irf7 expression requires ifn-i stimulation. ifn-β can directly be induced by irf3. all but one of the ifn-α subtypes require irf7 for their induction. hence, ifn-β secretion promotes its own production and that of ifn-α in an autocrine manner (33, 34) . this positive feedback loop strongly amplifies ifn production during viral infections, promoting fast and widespread induction of cell-intrinsic anti-viral defenses in uninfected cells to prevent virus dissemination. other feedback loops tightly regulate ifn-i production positively or negatively. this section reviews different mechanisms controlling ifn production and how they could play different roles in host/virus interactions. different innate immune recognition receptors are involved in sensing various types of viral nucleic acids in distinct categories of cells during viral infections, which may promote different types of anti-viral defenses. for each selected sensor shown, the types of viral nucleic acids recognized and the downstream signaling cascade induced are represented in a simplified, schematic manner. the potential specific role of each cell type in anti-viral defenses is also indicated at the bottom of each panel. (a) potentially all types of infected cells can detect endogenous viral replication through cytosolic sensors triggering their local production of ifn-β/λ to control viral replication in an autocrine and paracrine fashion in infected tissues. (b) uninfected xcr1 + dcs selectively produce high levels of ifn-λ and ifn-β upon engulfment of materials containing dsrna and the consecutive triggering of tlr3 in endosomes. the receptor of ifn-λ is mostly expressed by epithelial cells. hence, xcr1 + dcs might be involved in inducing local ifn responses in virally infected epithelial tissues. since xcr1 + dcs are especially efficient at producing ifn-iii upon hcv stimulation, they might contribute to local or systemic ifn production during infection with this virus, to promote ifn-λ-mediated protection of hepatocytes. uninfected xcr1 + dcs and other uninfected cells may produce some ifn-β upon engulfment of materials containing ssrna and the consecutive triggering of tlr8 in endosomes. the contribution of this pathway to anti-viral defense is not well understood yet, in part because mouse tlr8 is deficient for this function. (c) uninfected pdcs selectively produce high levels of all subsets of ifns upon engulfment of materials containing ssrna or cpg dna and the consecutive triggering of tlr7/9 in endosomes. however, pdcs seem to be activated for this function only in lymphoid tissues. hence, pdc might contribute to systemic ifn production during blood-borne viral infections or as a failsafe mechanism activated upon abnormal widespread dissemination of a viral infection once it has escaped local confinement at its portal of entry. cm, cell membrane; nm, nuclear membrane. particular nucleotide sequences or tertiary structures, their signaling pathways and their physiological significance have been recently reviewed (31, 32) . cytosolic rna sensors encompass dexd/h helicases among which the retinoic-acid-inducible gene (rig)-i-like receptors (rlrs) have been the most studied, namely rig-i and melanoma differentiation associated gene 5 (mda5). rig-i recognizes rna with a 5 -ppp or 5 -pp (38) (uncapped) moiety, or double-stranded rna (dsrna), both structures being present in viral, but not in cytosolic eukaryotic, rna molecules. mda5 might specifically recognize long dsrna fragments. both rig-i and mda5 contain a dexd/h box-containing rna helicase domain, and 2 caspase recruitment domains (card1/2), which bind to mitochondrial anti-viral signaling protein (mavs). rna/rlr/sting molecular complexes initiate a signaling cascade leading to irf3/7-dependent induction of ifns (figure 1 ). other dexd/h helicases can promote ifn-i production in dcs, www.frontiersin.org although their physiological roles for in vivo immune defenses against viral infections remain to be established (32) . cytosolic dna sensors able to induce ifn-i (mostly ifn-β) and ifn-iii encompass molecules belonging to different protein families, including dexd/h helicases, the inflammasome component ifn-γ-inducible protein 16 (ifi16) , the z-dna binding protein 1 (zbp1), and the cyclic gmp-amp (cgamp) synthase (cgas) (31, 32) . most of the cytosolic dna sensors activate sting and lead to irf3/7-and nfκb-dependent induction of ifn-β and ifn-iii. many cell types express zbp1 and are able to produce ifn-i upon triggering of this molecule, including macrophages, dcs, and fibroblasts following an hsv-1 infection (39, 40) . upon dna binding, cgas catalyzes the production of cgamp. cgas is critical for the detection of lentiviruses including hiv-1/2 (41, 42) and can contribute to sensing of, and protection against, other rna viruses, including in vivo in mice (43) . cgamp also acts as a secreted second messenger signal alerting uninfected cells to directly induce their expression of intrinsic immune anti-viral defenses. the cgas/sting/irf3 signaling cascade and the irf1 transcription factor are "master" inducers of cell-intrinsic immunity able to control the replication of most dna and some rna viruses at least in part independently of ifns (43) . infected cells become a factory for production of viral particles. hijacking of the translation apparatus of the host cell for massive production of viral structural proteins leads to an overload of the capacity of the er for correct folding of newly synthesized proteins. er overload induces a homeostatic response of the cell, the unfolded protein response (upr). upr aims at restoring normal er functions by inhibiting translation. upr activation in infected cells contributes to prevent viral replication, including through inhibition of the production of viral proteins, promotion of ifn-i production, and induction of cell suicide (44) . toll-like receptors (tlrs) are among the first and best characterized i 2 r 2 s. tlrs are transmembrane proteins with a leucine-rich repeat extracellular domain involved in ligand recognition and an intracellular toll/interleukin-1 receptor domain essential for signaling (45) . among the nine tlrs conserved between mouse and human, tlr3, tlr7, tlr8, and tlr9 are located in endosomes where they can detect the abnormal presence of nucleic acids such as occurs upon endocytosis of virions or of virally infected cell material. tlr3 recognizes dsrna, tlr7/8 ssrna, and tlr9 dna sequences containing unmethylated cytidinephosphate-guanosine (cpg) motifs. tlr fine specificity and signaling pathways have been reviewed recently (32) and are summarized in figure 1 . we will discuss the expression patterns and functions of endosomal tlrs with regards to ifn production in uninfected specialized immune cell types, pdcs and xcr1 + dcs. plasmacytoid dcs uniquely produce very large amounts of ifns in response to in vitro stimulation with many viruses, without being infected (46) . ifn-i mrnas represent up to 40% of all mrnas in pdcs at the peak of their activation (47) . in vitro, upon exposure to influenza virus, herpes virus type 1, cytomegaloviruses, or vesicular stomatitis virus, individual pdcs produce 100-1000 times more ifns than total pbmcs, monocytes, modcs, cdcs, neutrophils, and fibroblasts (47) (48) (49) (50) (51) (52) . however, in vitro, high molarity infection of cdcs with certain viruses unable to inhibit ifn-i production in their target cells can also induce massive ifn-β secretion (53) . pdcs produce high levels of all subtypes of ifns, contrary to many other cell types including infected cells, which often preferentially produce ifn-β (46, 47) . in vivo, pdc depletion during systemic viral infections leads to over 95% decrease of ifn-i production, while the total number of pdcs producing ifn-i (<100,000 in one mouse) is much lower than the total number of infected cells (54) (55) (56) (57) (58) (59) . this shows that in vivo also individual activated pdcs produce much more ifn-i/iii than most other cell types, including virus-infected cells. the professional ifn-producing function of pdcs largely results from their high constitutive and selective expression of irf7, tlr7, and tlr9 (figure 1) . these molecules are pre-associated in readyto-signal complexes located in specialized endosomes specific to pdcs (60, 61) . pdcs must also be equipped for efficient sensing and up-take of virions and virus-infected cells. the corresponding cell surface i 2 r 2 s remain to be identified. selective expression of tlr3 in xcr1 + dc endows them with a unique ability to produce very high amounts of ifn-β and ifn-iii upon stimulation with dsrna or hcv irrespective of their own infection. xcr1 + dcs are very potent for antigenspecific activation of cd8 + t cells, in particular through crosspresentation of exogenous antigens that they have captured from other cells and processed for association with class i major complex histocompatibility (mhc-i) molecules (62) . in mice, xcr1 + dcs are crucial for the initiation of protective adaptive immune responses against tumors and a variety of viruses (63) . mouse and human xcr1 + dcs constitutively and selectively express high levels of tlr3 (figure 1) . they produce large amounts of ifn-iii and ifn-β upon stimulation with a synthetic mimetic of dsrna, polyinosinic:polycytidylic acid (polyi:c) (64, 65) . human xcr1 + dcs uniquely respond to stimulation with hcv by producing large amounts of ifn-iii in a tlr3-dependent manner (66, 67) , irrespective of their own infection. positive feedback loops. in addition to irf7 induction, other positive feedback mechanisms exist to amplify the production of ifns rapidly after initiation of a viral infection as illustrated by the following selected examples. ifns induce the expression of many cytosolic rna/dna sensors and of tlr7. this broadens the spectrum of host's cell types able to detect endogenous viral replication for ifn induction. induction of oasl by ifns in human cells enforces rig-i signaling, counteracting viral immune frontiers in immunology | microbial immunology evasion genes interfering with this sensing pathway (68) . the ifninducible ribonuclease l (rnasel) generates viral and cellular rna degradation products, which engage rlrs for amplification of ifn production (69, 70) . the ifn-inducible protein kinase r (pkr) stabilizes ifn-i mrna (71) . to prevent unbridled responses deleterious for the host, ifn activity must be tightly controlled including during viral infections. several negative feedback loops exist to terminate ifn production, after anti-viral defenses have been activated. the isg ubiquitin specific peptidase 18 (usp18) binds to ifnar2, preventing it from recruiting signal transducer and activator of transcription 1 (stat1). ifns induce the expression of tam receptor tyrosine kinases in dcs, monocytes, and macrophages. tam receptors associate and signal in part through ifnar1. they activate the suppressors of cytokine signaling-1/3 (socs-1/3). socs inhibit tlr and rlr signaling, thereby terminating ifn production (72) . tam receptor ligands, gas6 and pros, bind phosphatidylserine on dying cells and are produced by activated dcs and monocytes/macrophages. thus, ifn induction of tam inhibitory receptors on uninfected phagocytic immune cells could limit their propensity to produce the cytokines upon engulfment of dying virally infected cells. ifns induce tetherin on most cell types. pdcs express a receptor for tetherin, leukocyte immunoglobulin-like receptor, subfamily a (with tm domain), member 4 (lilra4). lilra4 triggering on pdcs inhibits their production of ifn-i. hence, through lilra4 engagement by tetherin, pdcs can monitor their efficacy at inducing an antiviral gene expression program in neighboring cells through ifns, and timely terminate their ifn production. how positive and negative feedback loops integrate in time and space to promote optimal kinetics and intensity of ifn production in order to efficiently control viral infection without causing severe immunopathology is not completely understood. positive feedback loops may occur very rapidly after initiation of viral infection to allow rapid secretion of high levels of the cytokines for fast and strong induction of anti-viral cell-intrinsic immunity. negative feedback loops occur likely later to terminate the response and thus avoid chronicity of cytokine production and its ensuing deleterious effects. pdcs do not constitute the major source of ifn production upon local infections by several viruses in the lung or in the female reproductive tract. pdcs are dispensable for resistance against these infections (56, 73, 74) . during pulmonary infection by newcastle disease virus (ndv), ifn-i are produced locally in the lungs mainly by infected alveolar macrophages. lung pdcs do not express the cytokines (73) . selective depletion of lung alveolar macrophages leads to systemic dissemination of ndv, and to a strong activation of pdcs for ifn-i production specifically in the spleen. even in the case of systemic viral infections such as caused by intravenous injection of ndv or intraperitoneal injection of mouse cytomegalovirus (mcmv), pdc ifn production is confined to the spleen. it is not detected in other organs even those with high viral replication (59, 73) . hence, splenic pdcs are especially prone to high level ifn production upon systemic acute viral infections. pdcs located in non-lymphoid organs, in particular mucosal barrier tissues, appear to be inhibited for ifn production. thus, ifn production by infected cells serves as first line of defense to block virus replication at its portal of entry in the body. ifn production by uninfected pdcs might constitute a failsafe mechanism mainly activated in the spleen when viral infection gets systemic (75) . under these conditions, to promote health over disease, the benefits for the host of producing high circulating levels of ifns in order to induce widespread cell-intrinsic anti-viral defenses might prevail over the deleterious effects that this could cause on certain cell types or tissues. indeed, pdcs are required for protection against hsv-2 and hsv-1 in mice only in systemic but not local infections (56) . this observation is consistent with the crucial role of pdcs for protection of mice against systemic infection by mouse hepatitis virus (mhv), a fast replicating coronavirus (55) . conflicting results have been obtained on the role of pdcs during intranasal influenza infection (74, (76) (77) (78) . a possible explanation is that pdc ifn production contributes to resistance to highly pathogenic influenza strains that might systemically spread from the lung early after infection, even if at low levels. another intriguing observation is that ifns are critical for host resistance to mcmv and that pdcs are the major source of ifns in this infection model but are dispensable for virus control (54) . studies are ongoing to understand this apparent paradox. patients bearing genetic mutations disrupting endosomal tlr signaling do not appear to suffer from life-threatening viral infections (79, 80) , contrary to patients impaired in ifnar signaling (4, 81) . a notable exception is the specific susceptibility to severe herpes virus encephalitis in individuals' deficient for tlr3 signaling (82, 83) . however, contrary to extracellular tlr, endosomal tlr have evolved under strong purifying selection in human beings (84) . hence, while pdcs and endosomal tlr might have been required for protection of our species against viral infections in the past, this appears not to be the case anymore perhaps due to improved social, hygiene, and health care in modern society (75) . attesting to the importance of ifns for anti-viral defense in vertebrates, many mammalian viruses encode immune evasion genes specifically inhibiting the production of ifns in infected cells (39, 85) . pdcs or xcr1 + dcs might be essential for ifndependent host protection against these viruses, because they are spared from the intracellular functions of viral immune evasion genes (75) . to the best of our knowledge, mcmv does not encode for immune evasion genes inhibiting ifn production. however, mcmv manipulates ifn-i responses through specific inhibition of stat1 functions in infected cells. thus, pdcs might be dispensable for resistance against systemic mcmv infection due to sufficient levels of ifn production by infected cells locally in all infected tissues. hepatocyte responses to ifn-iii appear to play a www.frontiersin.org critical role in human resistance to hcv. in infected hepatocytes, hcv induces the expression of cellular micrornas binding to ifn-iii mrna and leading to its degradation. uninfected xcr1 + dcs produce high levels of ifn-iii in vitro upon hcv stimulation (66, 67) . hence, during acute hcv infection in vivo, xcr1 + dc may be a strong and early source of ifn-iii not subjected to virus immune evasion strategies, therefore, contributing to protect naturally resistant individuals. in secondary lymphoid organs, a subset of macrophages is critical for the clearance of viruses from the lymph (86) . these macrophages are located on viral entry routes, near to subcapsular sinuses where the afferent lymph drained from non-lymphoid tissues flows. contrary to other subsets of macrophages, subcapsular sinus macrophages are highly susceptible to viral infection, because they constitutively express only low levels of effector molecules of cell-intrinsic anti-viral immunity and because their responses to ifns are inhibited by their high constitutive expression of usp18. subcapsular sinus macrophages rapidly become infected by viruses incoming from the lymph and produce large amounts of ifns. this altruistic suicide prevents virus dissemination to other adjacent cell types and promotes the induction of innate and adaptive anti-viral immunity (87) . upon instruction by ifns, cells express a wide variety of viral restriction factors, whose combined action blocks pathogen invasion by interfering with the different stages of viral life cycle (figure 2a ). this has been extensively reviewed recently (88) and will only be described succinctly here. virus fusion with host cell membrane can be blocked by cholesterol-25hydrolase (ch25h) that inhibits sterol biosynthesis. some viruses enter cells by escaping from endosomes/lysosomes, which can be blocked by interferon inducible transmembrane (ifitm) proteins. virus uncoating can be blocked by tripartite motif (trim) proteins, such as trim5α, which bind to hiv-1 capsid thus promoting its degradation, and by myxoma resistance gtpases, mx1, and mx2, which efficiently trap viral structural proteins at an early stage following virus entry into the cell. mx1 inhibits a number of viruses, including influenza virus through sequestration of its nucleocapsid. mx2 associates with host cyclophilin a and hiv-1 capsid protein. virion assembly can be blocked at transcriptional, translational, and posttranslational levels. the adenosine deaminase acting on rna 1 (adar1) and the apolipoprotein b mrna editing enzyme, catalytic polypeptide-like (apobec) deaminases induce viral rna destabilization and hypermutation (89, 90) . the sterile alpha motif and histidine-aspartic domain (hd) containing protein 1 (samhd1) blocks reverse transcription by hydrolyzing dntps (91) . adar1, apobec, and samhd1 functions have been mainly studied in infections by hiv-1 and other retroviruses. the 2 ,5 -oligoadenylate synthase (oas) proteins, the ifn-induced proteins with tetratricopeptide repeats (ifit), and pkr inhibit viral and host protein translation by using complementary mechanisms (88) . the major post-translation modification induced by ifns is the binding of the ubiquitinlike modifier isg15 to several viral and host proteins, a process called isgylation. most of the known isgylated proteins are targeted for degradation, with few exceptions that are on the contrary stabilized like irf3 (88) . finally, the egress and budding of virions of many enveloped viruses can be inhibited by tetherin or by viperin (88) . many anti-viral isgs have been functionally characterized only recently, largely thanks to large-scale screening approaches. they display a variable degree of viral specificity (43, 92) that might inversely relate to the extent of their side effects on host cells ( figure 2b ). anti-viral effectors acting on a broad spectrum of viruses often target key metabolic pathways that are also crucial for host cell functions. this is the case for the control of cholesterol metabolism by ch25h (93) or of protein translation by pkr, oas, or ifits (88) . other anti-viral restriction factors such as mx2 may specifically target one molecule of a very restricted set of viruses with no apparent side effects on host cells. some anti-viral isgs target specific functions critical for only a restricted array of viruses and might similarly exert side effects only on a subset of host cell types. for example, samhd1 inhibits retrovirus replication through dntp depletion, which might more specifically affect proliferating host cells. hence, the infected host must balance the intensity, breadth, and location of isg induction to circumvent viral replication while preventing life-threatening damages to vital cell types or tissues. one of the mechanisms contributing to this balance is translational control of the expression of isgs, especially those with pro-apoptotic or anti-proliferative functions (94) . while many anti-viral isgs are transcriptionally activated in most ifn-stimulated cells, their translation can be specifically blocked in uninfected cells by cellular microrna. this inhibition is relieved upon cell infection through negative regulation of the function of the rna-induced silencing complex. hence, ifn stimulation of uninfected cells prepares them for rapid and strong induction of cell-intrinsic anti-viral defenses upon viral infection while avoiding their unnecessary exposure to the toxic effects of certain isgs. further knowledge on the functions and the dynamic regulation of isgs is essential to develop novel therapeutic strategies against viral infections aiming at modulating ifn responses to promote their protective anti-viral cell-intrinsic functions over their deleterious toxic effects. a better understanding of the immunoregulatory effects of ifns will also help. type i interferon can modulate the functions of a broad spectrum of immune cells ( figure 3a ). we will review this knowledge, focusing on the functions of dcs, nk cells, t cells, and b cells, since they are involved in the control of most viral infections. we will discuss the hypothesis that dcs play a central role in ifn-i orchestration of innate and adaptive immunity for the induction of optimal anti-viral defenses (figure 3) . during viral infections and cancer immunosurveillance, ifn-i constitute one of the most important input signal acting on dcs to promote their delivery of appropriate output signals to t cells, b cells, and nk cells for protective immunity ( figure 3a ). dcs deliver three types of signals to activate and functionally polarize t cells. signal 1 is the triggering of the t cell receptor by viral peptide-mhc complexes. signal 2 is the triggering of activating t cell co-stimulation receptors such as cd28 or cd27 by the cd80/86 and cd70 co-stimulation molecules expressed on dcs. signal 3 corresponds to cytokines, which can promote t cell proliferation and acquisition of specific effector functions. under steady state conditions, most dcs are in an immature state characterized by low level expression of mhc-ii (signal 1) and co-stimulation molecules (signal 2) and by the lack of production of t cell-activating www.frontiersin.org cytokines (signal 3). upon activation, including early after viral infections in vivo, dcs up-regulate their expression of signal 1 and activating signal 2 and secrete t cell-activating cytokines. this process is called dc maturation. gene expression profiling of dcs stimulated by microbial stimuli identified a core set of genes upregulated in mature dcs irrespective of the stimulus they receive, irrespective of the subset they belong to, and conserved across evolution (95) . most of these genes are induced during dc maturation in part through cell-intrinsic ifn-i signaling (95) . consistently, cell-intrinsic ifnar signaling in dcs is required in many circumstances for the induction of protective immunity, including efficient cd8 t cell responses during viral infection or tumor development (96) (97) (98) , th1 responses upon polyi:c injection independently of il-12 or ifn-γ effects (99, 100) , as well as follicular helper t cell and humoral responses (101, 102) . mechanistically, ifn-i promote dc immunogenicity for efficient t cell activation through a variety of effects ( figure 3b) . it drives dc up-regulation of signal 2 in vivo during viral infections (103) and boosts their capacity to cross-present antigens for increased delivery of signal 1 to cd8 t cells (96) (97) (98) . it shapes their delivery of activating signal 3, in particular inducing il-15 and promoting or inhibiting il-12 depending on experimental conditions (58, 104) . finally, it is necessary to induce their metabolic shift from mitochondrial oxidative phosphorylation to aerobic glycolysis, which fuels the increased needs in energy and the expansion of the intracellular organelles required for the production and proper intracellular routing of the signal 1 and 2 proteins (100, 105). selective inactivation of ifnar on cdcs compromises mouse resistance to mcmv and mhv infections (103, 106) . in contrast, ifnar expression is not required on nk cells for protection against mcmv and on pdcs, t cells, and b cells for early control of mhv replication (103, 106) . although cell-intrinsic ifn-i signaling in nk cells can promote their activation (107) (figure 3a) , ifn-i-induced il-15 trans-presentation by dcs plays a more prominent role for this function in many conditions including in vivo during mcmv infection (103, 108) ( figure 3c) . cell-intrinsic ifn-i signaling in cd4 t cells (109), cd8 t cells (110, 111) , and b cells (112) can also contribute to their efficient activation and functional polarization (figure 3 ). this depends on experimental settings. cd8 t cell-intrinsic ifn-i responses are crucial for mounting efficient cytotoxic cd8 t cell responses against lcmv but are less critical against vaccinia virus and vesicular stomatitis virus (110, 113, 114) . mechanistically, cell-intrinsic ifn-i signaling in cd8 t cells can promote their survival during their antigen-induced proliferation (110) . cell-intrinsic signaling in dcs and cd8 t cells may act in a synergistic manner. indeed, conditional inactivation of ifn-i responsiveness was required to occur simultaneously in both of these two cell types to dramatically affect cd8 t cell expansion upon vaccination with a modified ankara vaccinia virus (115) . in summary, ifn-i generally play a crucial, beneficial, role in immune defenses against viral infections, both through the induction of cell-intrinsic anti-viral defenses and through the orchestration of innate and adaptive immunity. however, if these responses are not properly regulated, they can contribute to diseases as we will now discuss. a frequent side effect of ifn-i treatment against cancer or chronic viral infections is the induction of autoimmune reactions. consistently, isg expression is a hallmark of many spontaneous systemic or tissue-specific autoimmune diseases, including systemic lupus erythematosous (sle), sjogren's syndrome, psoriasis, and other skin disorders (11) . the dysregulation of ifn-i responses observed in patients with these autoimmune diseases likely results from both genetic and environmental factors. genome-wide association studies show that polymorphisms in genes involved in ifn-i responses strongly correlate with increased susceptibility to many autoimmune diseases (11) . diverse environmental factors can also contribute to the onset of autoimmune diseases. microbial infections often precede first clinical manifestations of autoimmune diseases. whether infections (116) and/or alterations in the commensal microbiota of the affected barrier tissues (117, 118) are the cause or rather the consequence of autoimmunity is still matter of debate. infection-or dysbiosis-induced tissue damages and unbridled ifn-i responses can contribute to initiate autoimmune reactions. gender is another prominent factor affecting susceptibility to autoimmune diseases. women are more prone to autoimmunity, which may result from endocrine regulation of ifn-i responses. pdc ifn-i production is enhanced in human and mouse females, due at least in part to cell-intrinsic enhancement of tlr7/9 responses by the female hormone estradiol (119). in autoimmune diseases, different mechanisms could operate to initiate the dysregulation of immune responses leading to a vicious circle of reciprocal activation between innate ifn-i responses and adaptive self-reactive lymphocyte responses (figure 4) . adaptive immune cells are educated to spare "self." this occurs through negative selection of potentially autoimmune b and t cells during their development in the bone marrow or thymus, respectively, a process called central tolerance. self-reactive b or t cells that have escaped this pruning can be either deleted or functionally inactivated once they have egressed in secondary lymphoid organs or non-lymphoid tissues, a process called peripheral tolerance. in some individuals, polymorphisms in genes involved in the promotion of central or peripheral tolerance lead to a higher number, diversity, and/or responsiveness of self-reactive lymphocytes in the periphery, in particular of b cells secreting anti-dna or anti-rnp antibodies (120, 121) . mammalian dna or rna are poor inducers of pdc ifn-i induction under normal conditions. however, pre-existing anti-dna or anti-rnp autoantibodies can break this innate tolerance of pdc. indeed, antibodies binding to self nucleic acids can protect them from degradation and compact them into nanoparticles that are very effective for the induction of ifn-i in pdc (figure 4) . dna-containing immune complexes (ics) are frequently found in the serum of sle patients (sle-ics) and can activate pdc ifn-i production (122) . in turn, pdc ifn-i activate cdcs, monocytes (123) , and b cells, leading to a vicious circle of reciprocal activation between dcs and frontiers in immunology | microbial immunology figure 4 | a simplified model of the deleterious role of ifn-i in several autoimmune diseases. when exposed to different kinds of injuries (microbial infection, commensal microbiota dysbiosis, chemical or physical insults), healthy tissues can undergo cell damage and death. these events induce the release of apoptotic bodies encompassing self rna or dna. neutrophil recruitment and activation in inflamed tissues can also constitute a potent source of self nucleic acids, through the release of neutrophil extracellular traps (net). self rna or dna can associate with cationic peptides (e.g., ll37) as shown in psoriatic patients or with inflammatory molecules (e.g., high mobility group box 1, hmgb1) to generate nanoparticles that are extremely efficient for ifn-i production by pdc and eventually other cell types. pdc can also be efficiently activated for ifn-i production by immune complexes (ics) generated by the association between self nucleic acids and auto-antibodies as frequently found in the serum of systemic lupus erythematosus patients. ifn-i promote the differentiation and/or the maturation of antigen-presenting cells, in particular different subsets of dc. activated dc can then present self-antigens for activation of auto-reactive t cd4 + cells, including follicular helper lymphocytes, which in turn activate auto-reactive b cells for auto-antibody secretion, leading to a vicious circle of reciprocal activation between innate and auto-reactive adaptive immune cells. idc, immature dc; mdc, mature dc; mo-dc, monocyte-derived dc. see main text for further details. self-reactive lymphocytes and to the exacerbation of autoimmune responses (figure 4) . certain infections or dysbiosis of the commensal microbiota of the affected barrier tissues could promote chronic production of host amphiphatic peptides able to combine with eukaryotic dna or rna, likely released from dying cells, thus forming pdc-activating nanoparticles. indeed, in psoriatic skin, both a high expression of ll37 and a massive infiltration of pdcs is observed (124) (figure 4) . hence, to treat many autoimmune diseases, novel therapeutic strategies could be designed to target dysregulated pdc ifn-i production or b cell activation by ifn-i. one of the most common complications of primary infections by many respiratory viruses, in particular influenza virus, is a lifethreatening pneumonia due to secondary pulmonary infections by bacteria, such as streptococcus pneumoniae, staphylococcus aureus, or haemophilus influenza (125, 126) . these pathologies affect especially infants, elderly, and immunocompromised patients. retrospective studies indicate that secondary bacterial pneumonia was highly recurrent in lung tissues isolated from patients who died during last century influenza pandemics, independently of antibiotic availability (127, 128) . influenza virus induces high ifn-i responses in human beings and mice. in both hosts, secondary bacterial infections are lethal only when they occur in a limited time window following primary viral infection (3-7 days), around the peak of ifn-i responses, before complete virus clearance. mouse models of viral/bacterial coinfections are being used to dissect disease mechanisms (129) . ifnar1-deficient mice appear more resistant to secondary pulmonary bacterial infections, showing that ifn-i responsiveness contributes to disease (130) . similarly, after lymphochoriomeningitis virus (lcmv) infection, wild-type but not ifnar1-deficient mice are more susceptible to lpsinduced septic shock (131) . several mechanisms may contribute to the detrimental role of ifn-i in secondary bacterial infections ( figure 5) . early during viral infection, ifn-i decrease the host ability to control bacterial replication, by dominantly polarizing immune responses toward anti-viral functions, simultaneously inhibiting the development of the types of immune responses required for protection against most bacterial infections. ifn-i can inhibit the production of chemokines required for the recruitment to the respiratory tract of antibacterial effector innate immune cells, in particular neutrophils or monocytes/macrophages (132, 133) (figure 5) . depending on the experimental models used, ifn-i can on the contrary induce a ccr2-dependant recruitment of classical monocytes (134) . in infected tissue, ifn-i might skew the functional polarization of resident or infiltrating monocytic cells toward immunosuppression, because it does limit their antibacterial functions by inhibiting their il-1 production (135) (136) (137) while it might promote their production of il-10 and nitric oxygen intermediates. the exact nature of infiltrating monocytic cells is not clear and could correspond to activated classical monocytes, modcs, monocyte-derived macrophages, or myeloidderived suppressor cells (mdscs). the boundaries between these putatively different cell types are currently ill-defined (138) . these cells could fuel local replication of monocyte/macrophage-tropic bacteria (134) , be immunosuppressive (139) or contribute to local immunopathology (140) . the role of ifn-i on monocytes/macrophages is complex and will require further investigations to determine when it is protective versus deleterious and what the underlying mechanisms are. depending on the context, ifn-i can either promote or inhibit the induction of th1 cytokines such as il-12 and ifn-γ, and myeloid cell responses to ifn-γ (10, (141) (142) (143) . ifn-i can also polarize cd4 t cell responses toward th1 at the expense of th17, while the th17-type cytokines il-17a and il-22 are required for host defense against pulmonary www.frontiersin.org bacteria by inducing the production of anti-microbial peptides and of tissue repair molecules ( figure 5 ) (141) (142) (143) . ifn-i may not only affect host resistance to bacterial infection, but also host tolerance, i.e., the ability of the host to tolerate a given burden of pathogen without undergoing excessive tissue damages (143, 144) . hence, to counter ifn-i deleterious effects during secondary bacterial infections, it will be important to better delineate the respective contribution of lung tissue tolerance modulation and of immune-mediated resistance weakening. another well documented example of deleterious effects of ifn-i due to their inappropriate functional polarization of immune responses is the enhanced susceptibility to fungal infections of patients with genetically determined hyperactive ifn-i responses, as exemplified in the hereditary disease chronic mucocutaneous candidiadis (cmc) (figure 5 ) (145) . patients with cmc have a significant deficit in th17 cd4 t cells, at least in part as a consequence of altered responsiveness to il-6 or il-21. several stat1 mutations were identified in patients with autosomal dominant cmc. gain-of-function stat1 mutations were found to hard wire cd4 t cell responses to cytokines toward stat1 signaling, compromising their stat3-dependent ability to produce il-17 upon il-6 or il-21 stimulation. this was associated to induction of a global ifn-i transcriptomic signature in blood (145) . deleterious ifn-i effects on immunity to candida might not only occur in cmc patients but also in other types of individuals upon secondary fungal infections occurring shortly after a primary viral infection, likewise to the situation discussed above for secondary bacterial infections. indeed, polyic induced ifn-i abrogate innate immunity to systemic candidiasis in mice (146) , and ifnar-deficient mice can be more resistant to candida infection under certain experimental settings (147) . however, the role of ifn-i in the modulation of the ability of immunocompetent hosts to control fungal infection is disputed (148, 149) . the inhibition of th17 responses by ifn-i could be protective in at least one important human pathology, ms (figure 5) . ms represents a striking exception to the previously discussed detrimental role of ifn-i in autoimmune diseases. indeed, a large proportion of ms patients have low serum ifn-i activity and low isg levels. these ms patients present a significant reduction of ms relapse upon ifn-β administration (150) . the underlying mechanisms are not yet completely unraveled. however, in the experimental autoimmune encephalitis mouse model of ms, th17 responses bear a major contribution to nervous system damages and are inhibited by the il-10 and il-27 induced upon ifn-i administration (151) . in summary, ifn-i responses can be deleterious in autoimmunity by promoting a vicious circle of reciprocal activation between innate immune cells and auto-reactive cd4 t or b lymphocytes. ifn-i responses can also be deleterious upon secondary bacterial or fungal infections in the lung or the kidneys occurring shortly after a primary viral infection, by compromising the recruitment of anti-microbial innate effector cells and/or by preventing the proper functional polarization of immune responses. we will now discuss how ifn-i responses can also compromise host immune defenses against certain viruses and promote chronic infections. different lcmv strains such as armstrong and clone-13 (cl13), respectively, lead to acute versus chronic infections in mice. a hallmark of chronic lcmv infection is the loss of the proliferative potential and effector functions of anti-viral cd8 t cells, a process called exhaustion. exhausted cd8 t cells are characterized by a high expression of the inhibitory receptors pd-1, ctla4, and lag-3 (152) . in vivo blockade of these inhibitory receptors can reverse t cell exhaustion and allow resolution of the chronic infection (152) . ifn-i and isgs are induced early after infection with all strains of lcmv, albeit to lower levels with those leading to chronic infection. this early ifn-i production is critical to limit viral replication (3). in models of acute infection, ifn-i responses rapidly return to normal, undetectable, levels, before viral replication is completely controlled. in contrast, isg induction is maintained in chronic infection, including the expression of pd-1 ligands on apcs and of the immunosuppressive il-10 cytokine, consistent with a prolonged expression of ifn-i albeit at low levels (13, 14) . in vivo neutralization of ifn-i by antibody administration promoted resolution of chronic lcmv cl13 infection, allowing the frontiers in immunology | microbial immunology restoration of functional anti-viral cd8 t cell responses at least in part through cd4 t cell-and ifn-γ-dependent mechanisms (13, 14) . during persistent lcmv cl13 infection, chronic low level ifn-i production polarizes cd4 t cell responses toward t follicular helper (tfh) rather than th1 functions. thus, chronic ifn-i responses promote enhanced anti-viral b cell responses but facilitate cd8 t cell exhaustion due to deficient cd4 t cell help, therefore contributing to host failure to prevent chronic infection (153) . strikingly, establishment of chronic infection by lcmv cl13 could also be prevented by early administration of two shots of a high dose of exogenous ifn-i, at days 2 and 5 post-lcmv inoculation. this treatment allowed viral clearance by rescuing anti-viral cd8 t cell from exhaustion (154) . altogether, these studies show that the timing and duration of ifn-i production during viral infections is critical in determining how this response will impact the balance between the virus and the host. an early and robust but transient production of ifn-i promotes strong induction of cell-intrinsic viral restriction mechanisms as well as adequate polarization of adaptive anti-viral immune responses, which combined effects lead to viral clearance. in contrast, if the production of ifn-i is too low and/or too late, both viral replication and low ifn-i responses become chronic, their combined action leading to induction of immunosuppressive effects and to inadequate functional polarization of cd4 t cells. this results in cd8 t cell exhaustion and maintenance of chronic infection. chronic viral replication and cd8 t cell exhaustion is also a hallmark of hiv-1 infection. we will now discuss the complex and disputed role of ifn-i in this disease. both in hiv-1 infection and in its most relevant animal model, infection of non-human primates with simian immunodeficiency virus (siv), disease progression after the acute phase of the infection is associated with high and chronic expression of isgs while ifn-i production is inconsistently detected (155) (156) (157) . in contrast, the individuals that do not progress toward disease despite persistent high viral loads show much lower immune activation, in particular low isg expression, after the acute phase of the infection (158) (159) (160) (161) . hence, chronic low levels of ifn-i are associated to disease progression independently of the level of viral replication. therefore, an outstanding question still open for a better understanding of the physiopathology of hiv-1 infection is whether chronic ifn-i responses are merely a marker of progression, or whether they are implicated in driving disease development. in addition to mechanisms similar to those uncovered in the mouse model of chronic lcmv infection, during hiv-1 infection other effects of ifn-i could promote a vicious circle of reciprocal activation between chronic viral replication and sustained, deleterious immune responses (figure 6 ). very early after hiv-1 infection, in most individuals, ifn-i production might be too weak or too late to induce a combination of cell-intrinsic defense mechanisms and of immune responses efficient enough to prevent later establishment of chronic infection. on the contrary, as demonstrated in the case of the mouse model of lcmv infection, ifn-i responses could favor cd8 t cell exhaustion, either by direct cell-intrinsic effects on cd8 t cells (figure 6 , ) or by contributing to deprive them from cd4 t cell help (figure 6, ) . several effects of ifn-i might compromise anti-hiv-1 th1 responses or more generally contribute to the global depletion of cd4 t cells. these mechanisms include functional polarization of anti-hiv-1 cd4 t cells toward tfh rather than th1 responses, cxcl10 production leading to enhance recruitment of memory cd4 t cells to the sites of viral replication where they fuel chronic viral replication with new hiv-1 target cells (figure 6, to ) , direct pro-apoptotic and anti-proliferative effects on cd4 t cells (figure 6 , ), as well as trail induction on pdcs licensing them for killing cd4 t cells irrespective of their infection (figure 6 , ) (162, 163) . altogether, these mechanisms entertain chronic viral replication and continuous depletion of cd4 t cells, leading to the dramatically enhanced susceptibility to opportunistic infections (figure 6 , ) characteristic of the acquired immunodeficiency syndrome (aids) (figure 6, ) . other lines of evidences have been reported to support a deleterious role of pdc activation during hiv-1 infection. women undergo faster hiv-1 disease progression than men with similar viral loads, which may result in part from the highest ifn-i production of women's pdcs including in response to hiv-1 stimulation (164) . pdc recruitment and activation in the vaginal mucosa of female macaques early after local siv inoculation contribute to attract and activate cd4 t cells, which can then be infected and promote virus dissemination from its portal of entry (165) . however, in vivo blockade of pdc ifn-α production by administration of tlr7/9-antagonistic oligonucleotides early after siv infection of macaques did not decrease t lymphocyte activation, which suggests that additional sources of ifn-i likely contribute to the immune dysfunction observed in siv/hiv-1 infections. targeting dysregulated ifn-i responses during hiv-1 infection might represent an interesting adjuvant therapeutic strategy to highly active antiretroviral treatments. administration of ifn-i in the non-pathogenic siv infection model of sooty mangabeys was not sufficient to switch it into a pathogenic model. no cd4 t cell depletion ensued, no hyperactivation of immune responses were observed. viral loads were even significantly decreased. however, this could be consistent with the positive impact of early and high dose ifn-i administration in chronic lcmv infection (154) . indeed, during the review process of this manuscript, it was reported that, early during primary siv infection in the pathogenic rhesus macaque model, a high dose injection of ifn-i was protective while neutralization of endogenous ifn-i was deleterious. in contrast, in the same animal model, prolonged ifn-i administration accelerated disease development in the chronic stage of the infection (166) . in mice with a humanized immune system, pdc depletion strongly decreased isg induction and enhanced viral replication both in the acute and chronic phases of hiv-1 infection. however, pdc depletion during chronic infection decreased infection-induced t cell apoptosis and increased t cell numbers in lymphoid organs (167) . these results further emphasize the dual role of ifn-i and pdcs in the physiopathology of hiv-1 infection. a strong and transient production of ifn-i early after infection benefits the host by lowering the set-point of viral replication during chronic infection. sustained production of low levels of ifn-i during chronic infection contributes to immune dysregulation and cd4 t cell depletion. further studies will be necessary to examine whether complementing standard-of-use antiretroviral drugs with pdc www.frontiersin.org depletion, ifn-i neutralization, or selective inhibition of t cell responses to ifn-i could yield additional benefits to chemotherapy in non-human primates during chronic siv infection. ifn-i administration has been used for many years to treat another human chronic viral disease, hcv infection. roughly, half of the patients do not show sustained virological responses (svr). the treatment causes severe side effects in many individuals. new chemotherapeutic drugs very potent at blocking hcv replication in vivo have recently become available. hence, whether ifn-i administration still constitutes a viable treatment against chronic hcv infection is being questioned (168, 169) . we will now discuss this issue. chronic hcv infection is the main cause of liver cirrhosis and hepatocellular carcinoma. there is currently no vaccine against hcv. the most common therapy for chronic hcv patients is the administration of recombinant pegylated ifn-α (peg-ifn-α) combined with the anti-viral drug ribavirin. however, because of ifnar pleiotropic expression, ifn-α administration induces severe side effects including flu-like syndrome, fever, fatigue, myalgia, and nervous depression ( figure 7a ) (170) . moreover, only about half of treated patients harbor svr (171) . prior-totreatment high hepatic isg expression is a negative predictor of svr upon peg-ifn-α therapy. high isg expression in untreated patients likely results from chronic but low ifn-i production triggered by persistent hcv replication. indeed, hepatocytes from non-responder patients were found to be infected at a greater frequency and to exhibit dampened antiviral and cell death responses (172) . what the cellular sources of ifn-i production are and why they persist only in non-responder patients still remain to be established. in chronic hcv infection, cytotoxic effector lymphocytes may contribute to the development of hepatocarcinoma by causing low level but sustained hepatocyte death and renewal. in contrast, local production of ifn-γ in the liver by nk and t lymphocytes could promote resistance to disease through non-cytolytic control of viral replication. as discussed previously for lcmv and hiv-1, low chronic production of endogenous ifn-i in hcv patients could compromise both innate and adaptive anti-viral immune responses. chronic exposure to ifn-i could dampen the ability of frontiers in immunology | microbial immunology utr of ifnl3 mrna to promote their degradation. the favorable ifnl3 allele associated with responsiveness to peg-ifn-α treatment may allow endogenous expression of sufficient levels of ifnl3 for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes. this process is, however, hampered by the limited expression of the receptor for this cytokine (ifnλr1) in these patients. peg-ifn-α treatment might promote resolution of the infection by inducing ifnλr1 in these patients, potentiating their response to their endogenous production of ifnl3. in the patients that do not respond to peg-ifn-α treatment, endogenous levels of ifnl3 are insufficient for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes, due to the degradation of the corresponding mrna in infected hepatocytes. in these patient's hepatocytes, however, ifnλr1 is already expressed to high levels prior to treatment due to their high endogenous ifn-i responses. administration of exogenous ifn-λ might cure these patients. see main text for further details. nk and cd8 t cells to produce ifn-γ (173, 174) and promote cd8 t cell exhaustion (175) . it could also induce an antagonist form of cxcl10, a chemokine required for recruitment to the liver of anti-viral nk and cd8 t cell effectors (176) . it may also polarize monocytes toward immunosuppressive functions (177) . therefore, better understanding ifn-i effects in hcv infection is critical to improve care of both responders and non-responder patients to peg-ifn-α. for responder patients, the issue is to modify the treatment to favor beneficial antiviral and immunoactivating effects over side effects strongly affecting patient's quality of life (figure 7a ). this might be achieved by specific delivery of ifn-i to targeted cell types as discussed later. for non-responder www.frontiersin.org patients, the issue is to understand the mechanisms underlying treatment failure to determine whether alternative therapies could be designed (figure 7a) . genome-wide association studies identified various single nucleotide polymorphisms (snp) in the gene encoding il-28b/ifn-λ3, one of the ifn-iii, as well in its 5 and 3 non-coding regions (178) (179) (180) (181) . one snp, called rs12979860, is located 3 kb upstream of the ifnl3 gene. patients harboring the cc genotype have a favorable prognosis to ifn-i treatment. patients with the tt genotype are at high risk of treatment failure (178, 179) . in europeans, the favorable cc genotype is the major, most common, ifnl3 allele. the unfavorable tt snp is the minor allele. the frequency of these alleles is reversed in africans. the favorable allele allows escape of ifnl3 mrna from degradation by cellular microrna induced upon hcv infection (181) . until recently, ifnl3 genotypes and hepatic isg expression were considered as independent predictors of response to peg-ifn-α treatment in hcv patients (171) . here, we propose a potential explanation, which integrates both factors in a relatively simple model ( figure 7b ). our main hypothesis is that efficient control of hcv infection depends on hepatocyte response to ifn-λ rather than ifn-α. this is supported by reports that ifn-λ induces a stronger and more sustained isg expression in hepatocyte cell lines in vitro (182) , and that polyi:c-induced control of hcv replication in humanized liver in chimeric mice is correlated to the induction of ifn-λ but not ifn-i in human hepatocytes (183) . responder patients harboring the favorable ifnl3 allele preventing the degradation of the corresponding rna in infected cells might express significant levels of endogenous ifn-λ3, although this is disputed. however, they express only low levels of ifn-λr1, which limits ifnλ3 efficiency ( figure 7b ) (184) . how these patients benefit from peg-ifn-α treatment could be that it induces ifn-λr1 expression on hepatocytes thus boosting endogenous ifn-λ3 effects (184) . in contrast, high isg-expressing non-responder patients harboring the unfavorable ifnl3 allele might not express enough ifn-λ3 for virus control. however, they do express ifn-λr1 as a result of their endogenous production of ifn-i. hence, peg-ifn-α might be ineffective in these patients because they already express ifn-λr1 but fail to produce endogenous ifn-λ3 due to the degradation of its mrna in infected hepatocytes (figure 7b) . these patients may be good candidates for peg-ifn-λ therapy, currently undergoing clinical development. since the expression of ifn-λr1 is mainly restricted to epithelial cells, melanocytes, and hepatocytes, some of the side effects related to ifn-i treatment might be strongly attenuated in peg-ifn-λ therapy. however, as ifn-i are key to induce anti-viral immune responses, it will be critical to determine whether, beside viral clearance, peg-ifn-λ therapy can also induce long-term immune protection against hcv. ifn-i transduce intracellular signals through a single receptor, ifnar, but via a multitude of downstream signaling pathways. the janus activated kinase (jak)/stat pathway was the first to be identified (185) . ifnar is composed of two distinct subunits, ifnar1 and ifnar2, which are constitutively associated with members of the jak family, tyrosine kinase 2 (tyk2) and jak1, respectively (186) . the binding of ifn-i to their receptor leads to the phosphorylation of jak1 and tyk2, which in turn induce the phosphorylation and activation of the stat proteins (186) . different stat complexes can form upon triggering of ifnar (figure 8) . a transcriptional complex that forms in most conditions of ifn-i stimulation and induces the expression of many molecules of cell-intrinsic anti-viral immunity is interferonstimulated gene factor 3 (isgf3), a heterotrimer composed of pstat1, pstat2, and irf9 (187) (figure 8) . following its translocation into the nucleus, isgf3 binds to isre regulatory sequences in target genes. many molecules playing a key role in the function of innate or adaptive immune leukocytes are also induced by isgf3, including cd80, cd86, or il-15 in dc, and granzyme b in nk cells. isgf3 is generally composed of stat1 phosphorylated on tyr701 and ser727 and of stat2 phosphorylated on tyr689. however, alternative isgf3 complexes have been described in various contexts which could participate to the diversity of ifn-i effects (188) . the pstat1 homodimer also plays a prominent role in cell-intrinsic ifn-i-dependent gene induction. it binds ifnγactivated sequences (gas) and controls the expression of many pro-inflammatory molecules (187) . pstat1 homodimers can form upon stimulation with either ifn-i or ifn-γ. many gasregulated genes can be induced by either cytokines. depending on cell types, jak signaling downstream of ifnar can lead to the activation of virtually all stat proteins and to their combinatorial association into a variety of complexes with different affinities for specific gas elements (189) (190) (191) (figure 8) . this diversity contributes to ifn-i induction of different transcriptional programs in distinct cell types (39) . stat complex formation depends in part on the relative abundance of stat molecules in the cell (192) . while stat1, stat2, stat3, and stat5 can be activated in most cell populations, stat4 and stat6 are mainly activated in lymphocytes (193) . for example, quiescent nk cells express more stat4 than stat1, leading to constitutive association of ifnar to stat4 in these cells. hence, quiescent nk cells mount pstat4 homodimer-dependent responses to ifn-i stimulation, including ifn-γ production and t-bet-driven proliferation (figure 8 ) (194, 195) . changes in stat levels can also occur upon the differentiation/activation of a given cell type and lead to a shift in its functional response to the cytokines (196) . upon activation, nk cells decrease their expression of stat4 and increase that of stat1, shifting their ifn-i response from stat4-dependent in a quiescent state to stat1dependent in pre-activated cells. this translates into opposite ifn-i effects on ifn-γ production and proliferation for quiescent versus pre-activated nk cells (194) . however, this outcome can be modulated by simultaneous exposure to other cytokines such as il-15 or il-12/18. a reverse stat1-to-stat4 shift occurs in dc during their maturation, shifting their functional responses from inhibition to activation of il-12 production in response to combined stimulations with ifn-i and cd40l (197) . this frontiers in immunology | microbial immunology ifn-i binding to ifnar triggers the phosphorylation of tyk2 and jak1, which in turn phosphorylate a variety of stat proteins. activated stats are able to form complexes, as homo-or hetero-dimers. the heterodimer stat1-stat2 binds to a third partner, ifn-regulatory factor 9 (irf9), in order to form the isgf3 complex. this complex translocates into the nucleus and binds to specific regulatory sequences, ifn-stimulated response elements (isre), to activate the expression of many interferon-stimulated genes (isgs). in particular, isgf3 induces most, if not all, of the isgs encoding effector molecules of cell-intrinsic anti-viral defenses such as oas or mx1. alternative jak/stat pathways include the formation of stat1 or stat4 homodimers, which may drive different functional responses to ifn-i. stat1 homodimers bind to ifnγ-activated sequences (gas) in the promoter of certain isgs, which may promote inflammatory, anti-proliferative, and pro-apoptotic responses. stat4 homodimers also bind to gas but promote ifn-γ production and pro-proliferative responses. enables mature dc to efficiently activate cd8 t cells. other yet unknown mechanisms control the formation of different stat complexes in distinct cell types. the nature and dynamics of the signaling pathways triggered by ifn-α or -β were evaluated in bulk cultures of human blood leukocytes using flow cytometry (191) or high throughput mass cytometry (190) . a diversity of phosphorylation patterns of stat1/3/5 was observed upon ifn-i stimulation. ifn-α activation induced phosphorylation of stat1, stat3, and stat5 in most cell types, peaking at 15 min (190) . ifn-β-induced stat1 phosphorylation was found to be poor in b cells as compared to monocytes and t cells (191) . however, the underlying mechanism remains to be identified since b cells did not express lower amount of ifnar2 or stat1 or enhanced levels of the inhibitory socs1 molecule. the high stat1 activation in monocytes led to their induction of ifn-i-dependent pro-apoptotic genes while this was not the case in b cells. these results strikingly differ from those obtained in the other study upon ifn-α stimulation, where stat1 phosphorylation was on the contrary lower in cd14 + monocytes and was prolonged in b cells and nk cells (190) . the differences between these two studies might have resulted from the use of different subsets and doses of ifn-i. in any case, both studies consistently reported that cd4 t cells showed the highest activation of stat5. all cd4 t cells but not all cd8 t cells activated stat5 and for a longer time (190) . ifn-β activation of stat3 was delayed in cd4 t cells and b cells as compared to cd8 t cells and monocytes (191) . different stat complexes may lead to distinct transcriptional programs linked to different biological functions (figure 8) . more systematic studies are needed to understand this complexity. besides changing stat levels between cell types or www.frontiersin.org activation states, the processes controlling differential formation of stat complexes downstream of ifnar triggering remain to be identified. in addition to jak/stat signaling, other pathways can be activated downstream of ifnar, including those involving the phosphatidylinositol 3-kinase (pi3k), mitogen-activated protein kinases (mapk), and the crk adaptor molecules (39, 198) . this leads to the activation of other transcription factors such as irf, nf-κb, or pu.1, which contribute to orchestrate cell responses to the cytokines by regulating both distinct and overlapping sets of genes as compared to stat (199, 200) . in summary, ifnar signals through a remarkable diversity of pathways, including but not limited to diverse combinations and kinetics of stat phosphorylations. this explains at least in part the diversity of ifn-i effects, including their induction of opposite responses depending on the physiopathological contexts and/or the nature of the principal responding cell types (200, 201) . ifn-iii induce the same signaling pathways as ifn-i, although they engage a different heterodimeric receptor, composed of the il-28ra and il-10rb chains and preferentially expressed on epithelial cells including hepatocytes. in mice and human beings, numerous ifn-i subtypes exist. functional and population genetic analyses showed that these ifn-i subtypes significantly differ in their functions (202) (203) (204) (205) (206) (207) . hence, one of most extraordinary feature of ifn-i biology is how ifn-i subtypes can elicit so many pleiotropic and diverse functions by interacting with the same receptor complex (208) . both ifnar1 and ifnar2 are required for the initiation of ifn-i-dependent signals, as mice deficient in either one are highly susceptible to viral infections (3, 5) . the assembling of the ifnreceptor ternary complex is a two-step process. first, a binary complex is formed by the binding of one side of the ifn molecule to ifnar2. then, a single binary complex interacts with ifnar1 via the other side of the ifn molecule. the stability of the ternary complex will be determined in part by the association and dissociation kinetics between the cytokine and the two receptor chains, as well as by ifnar expression levels since the cell surface concentrations of the receptor subunits are relatively low. hence, both the affinity of ifn-i subsets for ifnar and the amounts of ifn-i, ifnar1, and ifnar2 will regulate their biological effects ( figure 9a ) (209, 210) . cell membrane density of ifnar1 and ifnar2 is also involved in differential ifn-β-versus ifn-α-induced functional activities, such as anti-proliferative function (211) . a variety of cell-intrinsic parameters can also impact the lifetime of the ifn-receptor ternary complex, such as the rate of endocytosis/degradation/recycling of signaling complexes, and negative isg regulators such as usp18 that decrease the affinity of ifn-ifnar1 binding (203, 212) . based on a definition of a prototypic cytokine-receptor binding module and by analogy with the epo receptor system, ifn-i subtypes were originally postulated to form ternary complexes of differing architectures, resulting in distinct geometry and assembling of intracellular signaling components (213) . experimental evidence rejected this hypothesis. rather, the differential activities of ifn-i subtypes are determined by the stability of the ligand/receptor ternary complex (207, 212) . differential affinities of the ifn-i subtypes for ifnar1 and ifnar2 extracellular domains generate subtype-specific signaling cascades and biological outcomes ( figure 9a ) (210, 214) . crystal structure of ternary ifn-i/ifnar1/ifnar2 complex illuminated the biochemical complexity of ifn-i interaction with their cognate receptors (215) . the main conformational features of ifn-i/ifnar1/ifnar2 ternary complexes are conserved among the different ifn-i, but are quite different from the other cytokine receptors (214, 215) . in the formation of the binary ifn-i/ifnar2 complex, ifn-i ligand discrimination resides on differential energetics during the interaction of anchor points with ifnar2, shared by all ifn-i, as well as on key amino acid substitution among ifn-i subtypes (215) . ifnar1 then performs major conformational changes to interact with ifn-i associated in the binary complex, thus displaying an optimized functional plasticity (215) . these differences in the chemistry of ifn-i subtype interaction with ifnar2 and ifnar1 thus explain the different affinities of ifn-α versus ifn-β within ternary complex and their differential activities (210) . the functions regulated by ifn-i strongly depend on the main responding cell types ( figure 9b ). this has been studied in vitro by examining the functional consequences of the stimulation of different cell types with ifn-i, and in vivo by determining the contribution of cell-intrinsic ifn-i responses of different cell types to resistance or susceptibility to various diseases. an emerging concept is the central role of dc responses to ifn-i for induction of protective immunity against viral infections or tumors (figure 3) . the development of mutant mice allowing conditional genetic inactivation of ifnar1 in a cell-type specific manner using the cre-lox system (216) has been instrumental in accelerating our understanding of how different cell types respond to ifn-i in vivo and what their respective contribution is to protective or deleterious ifn-i responses. this has been investigated most extensively in viral infections (106, 111, 112, 115, 217) but also in cancer (97, 98) , bacterial infections (218), autoimmunity (216, 219) , sepsis (220), or inflammatory diseases (221) . efforts are being pursued to better understand which cell types respond to ifn-i in a manner promoting protective versus deleterious effects in different physiopathological settings. that knowledge will considerably help to develop novel strategies to modulate ifn-i functions for promoting health over disease. the development of mutant mice allowing conditional genetic inactivation of stat1, stat3, and stat5 (222-226) will help better understanding how different signaling pathways in different cell types determine the outcome of ifn-i response in vivo in various conditions. this knowledge might lead to the development of strategies aiming at targeting a given cell type with a specific subset of ifn-i, or in the presence of antagonists of certain signaling pathways, to surgically tune ifn-i responses in vivo toward the most desirable outcome. frontiers in immunology | microbial immunology for example, the affinity of ifn-β for ifnar1 is 100-times higher than that of ifn-α2, and ifnβ is much more potent in inhibiting cellular proliferation or (continued ) ifn-i can also determine distinct functional outcomes. for example, during viral infections, early and transient high levels of ifn-i promote protective dc and t cell responses, while delayed, chronic and low level ifn-i production compromises host immune defenses and promotes chronic viral infections. within a given cell type, the outcome of ifn-i stimulation also depends on time of exposure to these cytokines relative to other modulatory signals (timing relative to other stimuli). for example, in naïve cd8 t cells, tcr signaling prior to ifn-i stimulation leads to increased expression of stat4 and promotes ifn-γ production and proliferation, while ifn-i stimulation prior to tcr triggering leads to stat1-dependent anti-proliferative and pro-apoptotic effects. the formation of specific stat complexes is a highly dynamic process. it depends not only on the cell type but also on its specific state at the time it sees ifn-i. hence, major parameters controlling the effects of ifn-i in a given cell type also include its microenvironment ( figure 9c ) and the timing of its exposure to the cytokines both in terms of duration of the stimulation and of previous activation history ( figure 9d) . the tam receptor ligand gas6 is expressed within tumor cells in various solid cancers (227, 228) . elevated gas6 expression is of bad prognosis in different cancers (228, 229) . in a mouse model of ovarian cancer, early during tumorigenesis tumor-infiltrating dcs were found to be immunogenic and promote antitumor immunity, but they were later altered in the course of tumor development to acquire immunosuppressive properties beneficial to the tumor (230) . one may thus hypothesize that expression of tam soluble ligands in certain tumors and of tam receptors on tumor-infiltrating dcs might contribute to dampen dc response to ifn-i and therefore facilitate their polarization by the tumor microenvironment into immunosuppressive cells (figure 9c) . acute versus chronic exposure to ifn-i can lead to strikingly opposite effects on a given cell type (13, 14, 231) . in addition to duration, the time when a cell is exposed to ifn-i can also dramatically impact its functional response, depending on its previous activation history ( figure 9d) . in vitro stimulation of dcs with ifn-β can lead to opposite outcomes depending whether it occurs simultaneously to, or after, tnfα-induced maturation. ifn-β polarizes dcs toward th1 induction in the former case, and toward il-10-secreting t cells in the latter case. these opposite effects result at least in part from the differential expression of il-12/18 by dcs (232) . similarly, ifn-i effect on the functional polarization of cd4 t cells is strongly modulated by the other cytokines present in the lymphocyte microenvironment at the same time (233) . ifn-i can also mediate opposite effects on cd8 t cells depending whether it occurs before or after cognate engagement of the t cell receptor. indeed, while cd8 t cells have the potential to respond to ifn-i by inducing both stat1-and stat4-dependent genes, this depends upon their activation history. naïve cd8 t cells respond mostly to ifn-i through stat1 signaling, leading to the inhibition of their proliferation and eventually to the induction of their apoptosis. however, cognate triggering of the t cell receptor causes a decrease in stat1 and an increase in stat4 expression in cd8 t cells. this leads to a shift of their ifn-i response from stat1-to-stat4 signaling, resulting in the promotion of their proliferation and ifn-γ production. during lcmv infection, this mechanism promotes stat4-dependant expansion of anti-viral cd8 t cells, but stat1-dependant inhibition of naïve cd8 t cell proliferation (234) . since the late 70s the clinical potential of ifn-i for the treatment of patients suffering of viral infection or cancer diseases has been widely acknowledged (235) . today, this expectation is tempered because ifn-i treatment can induce severe side effects and sufficient doses cannot be administered in patients. therefore, there is a strong need to create tuned ifn molecules devoid of side effects. based on our current understanding of ifn-i responses as reviewed above, many parameters could be tuned individually or in a combined manner to modulate ifn-i activity to promote their beneficial effects over the deleterious ones in a number of diseases. these parameters include modifying the affinity of ifn-i for its receptor, playing with the local quantity/concentration of ifn-i and with the duration of its delivery, and modulating the nature of the cells that are responding to ifn-i. we will discuss here novel strategies being developed to deliver ifn-i to, or block ifn-i responsiveness of, a specific target cell type in vivo (figure 10 ). if ifn-i-induced side effects are a consequence of the pleiotropic nature of ifn-i, and if the bioactivities mediating deleterious effects have some degree of independence from those mediating beneficial effects, one could mutate the ifn-i molecules in order to skew their activity toward a desired bioactivity. indeed, introducing key mutation in ifn-α2 allowed increasing its affinity to ifnar1 by a factor of 100. accordingly, this ifn-α2 mutant is 100times more potent in inhibiting cell proliferation, but as potent as wt ifn-α2 in inducing an anti-viral state (236) (237) (238) . hence, it is possible to tune ifn activity by modifying its binding to ifnar. however, translating such an approach for the design of molecules for clinical application is severely hampered by the poor understanding we have on the ifn-i bioactivities mediating the side effects. furthermore, we are far from having established the list of bioactivities that could be differentially modulated by changing the stability of the ifn-i/ifnar complex. we know more about the frontiers in immunology | microbial immunology cell types that mediate beneficial versus deleterious ifn responses in various diseases. hence, we will now discuss strategies aimed at focusing ifn activity to specific cell types to promote health over disease. several strategies have been developed to specifically target ifns on tumor cells, tumor-infiltrated immune cells or infected tissues. these strategies include intra-lesional injection (239, 240) , adenoviral-mediated gene transfer (241) (242) (243) , engineered tumorinfiltrating monocytes (244) , and fusion of ifns with a cleavable protecting shell (245) . another strategy to increase cytokine accumulation within the tumor or infected tissue is antibody-mediated targeting of cytokine delivery, where a cytokine moiety is fused to an antibody directed against a specific cell surface marker (figure 10) . the fusion molecule retains both antigen-binding and ifn-i bioactivities, and is enriched at the targeted site upon in vivo injection (246) (247) (248) (249) . when targeted to human cd20, ifn-i inhibited the proliferation of lymphoma cells engrafted in immunodeficient mice (250) . an ifn-i targeted to a tumor antigen can also amplify the therapeutic effect of the antibody by acting on tumor-infiltrated dcs, thus increasing antigen cross-presentation and antitumor cytotoxic t cell responses (249) . on non-targeted cells, the antibody conjugation negatively impacts ifn-i potency, but only modestly (18, 248, 251) (figure 10a) . fusion molecules generally retain full ifn-i biological activity on the cells expressing the antibody target ( figure 10b) . hence, this difference only leads to a modest ratio between the ifn-i specific activity measured on target and non-target cells (figure 10b) . such a targeting efficiency is definitely too low to reduce the toxic effect of ifn-i administration, because it will not specifically focus ifn-i activities on "beneficial cells" without stimulating "deleterious cells." the engineering of immuno-ifn-i must be improved to reach the very high targeting efficacy required to significantly diminish the treatment side effects. we recently reported an innovative strategy reaching this goal (18) . it is based on the postulate that the antibody moiety of an immuno-ifn-i stabilizes the ifn-i/receptor-complex by avidity. it also takes into account the fact that the biological potency of an ifn-i is proportional to the stability of the ifn-i/receptor complex up to a certain threshold beyond which increasing the stability does not increase its potency (238, 252) . ifn-α2 and ifn-β are used in most immuno-ifn-i studies. they have evolved to retain close to maximal potency. hence, their targeting by an antibody that only provides a modest gain in terms of biological potency. however, it is expected that decreasing the affinity of the ifn-i for its receptor, by introducing a mutation, would increase the targeting effect of the antibody (figures 10 c,d) . this is indeed the case. using an ifn-i with a single point mutation that dramatically decreases its affinity for ifnar2 ( figure 10c ) allows engineering immuno-ifns that are up to 1000-fold more potent on cells expressing the antibody target ( figure 10d) . the three log targeting efficiency of these novel types of immuno-ifns is found for various activities measured in vitro or in vivo when delivered in mice. if the toxic side effect experienced by the patients treated with ifn-i is due to systemic ifn-i activity, this targeting technology may find considerable clinical applications since such engineered immuno-ifns are virtually inactive while "en route" and are activated only after binding of the fused antibody to the desired target. it remains to define the useful targets according to pathologies, for example, tumor cells themselves and professional cross-presenting xcr1 + dcs for cancer (97, 98, 249) , or hepatocytes for chronic hcv infection. to treat autoimmune diseases, novel therapeutics targeting ifn-i have been developed, including two ifn-α-neutralizing monoclonal antibodies currently in clinical trials (sifalimumab and rontalizumab) (253, 254) . however, long-term systemic neutralization of ifn-i activity may increase susceptibility to viral infection and tumor development. alternative strategies are needed to specifically inhibit ifn-i deleterious effects in these diseases without globally compromising ifn-i anti-viral and www.frontiersin.org anti-tumoral functions. the sequential nature of the assembling of the ifn-i/receptor complex opens the possibility to design ifn-i antagonists specifically targeting the cell subsets responsible for ifn-i deleterious effects. an ifn-α2 carrying a single amino acid substitution that blocks the ifn-i/ifnar1 interaction engages ifnar2 in a complex, which cannot bind ifnar1 (255) . since the binary ifn-i/ifnar2 complex is devoid of any ifn-i activity, such mutant behaves as a potent ifn-i antagonist. when linked to an antibody specific for a cell surface marker, the antagonistic activity of the mutant ifn-i should be significantly reinforced specifically on the cells expressing the target. hence, it should be possible to design and construct targeted antagonists that inhibit responsiveness to endogenous ifn-i specifically on the cell subsets on which the cytokines act to promote autoimmunity or severe side effects, leaving the other cells fully responsive. for example, in chronic hcv patients treated with peg-ifn-α, one of the most deleterious side effects is nervous depression, which might be prevented by co-administration of an ifn-i antagonist specifically targeting neurons or other cells of the central nervous system. in the last decade, several major technological breakthroughs and the generation of novel animal models have remarkably advanced our understanding of the mode of action of ifns. in vitro high throughput screening allowed systematically studying the functions of isgs by ectopic expression or knock-down. advance biophysical investigation of the interactions between ifn-i and the ifn-i receptor allowed to rigorously investigate the mechanistic basis for the differential bioactivities of ifn-i subtypes. the analyses of the responses of different cell types to ifns or to viral infection, in vitro but also in vivo in various pathologies, demonstrated that ifn-i often mediate beneficial versus deleterious roles by acting on different cell types. from integrative analysis of these data, a picture is now emerging suggesting that it will be possible to segregate protective from deleterious ifn-i effects, based (i) on their differential induction depending on ifn-i subsets or on the magnitude/timing of ifn-i production, (ii) on their conditioning in different tissues, (iii) or on their occurrence in different cell types. hence, innovative immunotherapeutic treatments are being designed to tune ifn-i activity toward desired effects in order to promote health over disease in a manner adapted to each physiopathological condition. in particular, a proof-of-concept has been made in vitro that it will be possible to target ifn-i activity on given cell types or tissues to administer to patients sufficiently high doses of the cytokine at the site of interest while limiting unwanted effects in other tissues or cell types. the next steps will be to demonstrate efficacy of this strategy in vivo in preclinical animal models. importantly, to foster the development of these innovative immunotherapies, major efforts are still warranted to continue delineating which cell types are mainly responsible for the protective versus deleterious effects of ifn-i in different diseases. combining high throughput technologies and systems biology approaches will also advance our understanding of the molecular mechanisms dynamically controlling ifn-i responses in health and diseases, which should reveal potentially novel therapeutic targets. virus interference. i. the interferon virus interference. ii. some properties of interferon functional role of type i and type ii interferons in antiviral defense inborn errors of interferon (ifn)-mediated immunity in humans: insights into the respective roles of ifn-alpha/beta, ifn-gamma, and ifn-lambda in host defense suppressor of cytokine signaling 1 regulates the immune response to infection by a unique inhibition of type i interferon activity new insights into 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hepatitis c virus infection human blood dendritic cell antigen 3 (bdca3)(+) dendritic cells are a potent producer of interferon-lambda in response to hepatitis c virus antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor activation of ifn-&#946; expression by a viral mrna through rnase l and mda5 rnase l releases a small rna from hcv rna that refolds into a potent pamp protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity tam receptors are pleiotropic inhibitors of the innate immune response alveolar macrophages are the primary interferon-alpha producer in pulmonary infection with rna viruses differential type i interferon induction by respiratory syncytial virus and influenza a virus in vivo crosstalk between components of the innate immune system: promoting anti-microbial defenses and avoiding immunopathologies plasmacytoid dendritic cells and toll-like receptor 7-dependent signalling promote efficient protection of mice against highly virulent influenza a virus plasmacytoid dendritic cells are dispensable during primary influenza virus infection clearance of influenza virus from the lung depends on migratory langerin+cd11b− but not plasmacytoid dendritic cells human tlr-7-, -8-, and -9-mediated induction of ifn-alpha/beta and -lambda is irak-4 dependent and redundant for protective immunity to viruses pyogenic bacterial infections in humans with myd88 deficiency impaired response to interferon-alpha/beta and lethal viral disease in human stat1 deficiency herpes simplex virus encephalitis in a patient with complete tlr3 deficiency: tlr3 is otherwise redundant in protective immunity herpes simplex encephalitis in children with autosomal recessive and dominant trif deficiency evolutionary dynamics of human toll-like receptors and their different contributions to host defense type 1 interferons and the virus-host relationship: a lesson in detente subcapsular sinus macrophages prevent cns invasion on peripheral infection with a neurotropic virus enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus interferon-stimulated genes: a complex web of host defenses protein kinase pkr and rna adenosine deaminase adar1: new roles for old players as modulators of the interferon response dna deamination mediates innate immunity to retroviral infection samhd1 host restriction factor: a link with innate immune sensing of retrovirus infection a diverse range of gene products are effectors of the type i interferon antiviral response the transcription factor stat-1 couples macrophage synthesis of 25-hydroxycholesterol to the interferon antiviral response reciprocal inhibition between intracellular antiviral signaling and the rnai machinery in mammalian cells plasmacytoid, conventional, and monocyte-derived dendritic cells undergo a profound and convergent genetic reprogramming during their maturation crosspriming of cd8+ t cells stimulated by virus-induced type i interferon type i interferon is selectively required by dendritic cells for immune rejection of tumors host type i ifn signals are required for antitumor cd8+ t cell responses through cd8{alpha}+ dendritic cells dendritic cells require a systemic type i interferon response to mature and induce cd4+ th1 immunity with poly ic as adjuvant direct type i ifn but not mda5/tlr3 activation of dendritic cells is required for maturation and metabolic shift to glycolysis after poly ic stimulation type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo johansson-lindbom b. type i interferon signaling in dendritic cells stimulates the development of lymph-noderesident t follicular helper cells differential responses of immune cells to type i interferon contribute to host resistance to viral infection a type i interferon autocrine-paracrine loop is involved in toll-like receptorinduced interleukin-12p70 secretion by dendritic cells tlrdriven early glycolytic reprogramming via the kinases tbk1-ikkvarepsilon supports the anabolic demands of dendritic cell activation type i ifn-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection direct action of type i ifn on nk cells is required for their activation in response to vaccinia viral infection in vivo dendritic cells prime natural killer cells by trans-presenting interleukin 15 cutting edge: the direct action of type i ifn on cd4 t cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection type i interferons act directly on cd8 t cells to allow clonal expansion and memory formation in response to viral infection direct stimulation of t cells by type i ifn enhances the cd8+ t cell response during cross-priming cutting edge: enhancement of antibody responses through direct stimulation of b and t cells by type i ifn cd8 t cells specific for lymphocytic choriomeningitis virus require type i ifn receptor for clonal expansion innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd8 t cells for clonal expansion and memory formation concomitant type i ifn receptor-triggering of t cells and of dc is required to promote maximal modified vaccinia virus ankara-induced t-cell expansion antiviral immune responses: triggers of or triggered by autoimmunity? is chronic plaque psoriasis triggered by microbiota in the skin? genetic dysbiosis: the role of microbial insults in chronic inflammatory diseases the tlr-mediated response of plasmacytoid dendritic cells is positively regulated by estradiol in vivo through cell-intrinsic estrogen receptor alpha signaling accumulation of peripheral autoreactive b cells in the absence of functional human regulatory t cells cvid-associated taci mutations affect autoreactive b cell selection and activation human lupus autoantibody-dna complexes activate dcs through cooperation of cd32 and tlr9 ifn priming is necessary but not sufficient to turn on a migratory dendritic cell program in lupus monocytes plasmacytoid dendritic cells sense self-dna coupled with antimicrobial peptide the pathology of influenza virus infections how do viral infections predispose patients to bacterial infections? predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness pathology of human influenza revisited immune dysfunction and bacterial coinfections following influenza type i interferon: friend or foe? a role for ifn-alpha beta in virus infection-induced sensitization to endotoxin type i ifns mediate development of postinfluenza bacterial pneumonia in mice synergistic stimulation of type i interferons during influenza virus coinfection promotes streptococcus pneumoniae colonization in mice intranasal poly-ic treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population innate and adaptive interferons suppress il-1alpha and il-1beta production by distinct pulmonary myeloid subsets during mycobacterium tuberculosis infection host-directed therapy of tuberculosis based on interleukin-1 and type i interferon crosstalk inflammation. 25-hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type i interferon dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny cytomegalovirus impairs antiviral cd8+ t cell immunity by recruiting inflammatory monocytes tnf/inos-producing dendritic cells are the necessary evil of lethal influenza virus infection influenza a inhibits th17-mediated host defense against bacterial pneumonia in mice influenza a virus exacerbates staphylococcus aureus pneumonia in mice by attenuating antimicrobial peptide production role of tissue protection in lethal respiratory viral-bacterial coinfection disease tolerance as a defense strategy gainof-function human stat1 mutations impair il-17 immunity and underlie chronic mucocutaneous candidiasis type i interferon inhibits interleukin-1 production and inflammasome activation type i interferons promote fatal immunopathology by regulating inflammatory monocytes and neutrophils during candida infections ifnalpha/beta signaling is required for polarization of cytokine responses toward a protective type 1 pattern during experimental cryptococcosis interferonbeta production via dectin-1-syk-irf5 signaling in dendritic cells is crucial for immunity to c. albicans aberrant type i interferon regulation in autoimmunity: opposite directions in ms and sle, shaped by evolution and body ecology janus-like effects of type i interferon in autoimmune diseases coregulation of cd8+ t cell exhaustion by multiple inhibitory receptors during chronic viral infection type i interferon suppresses de novo virus-specific cd4 th1 immunity during an established persistent viral infection timing and magnitude of type i interferon responses by distinct sensors impact cd8 t cell exhaustion and chronic viral infection distinct transcriptional profiles in ex vivo cd4+ and cd8+ t cells are established early in human immunodeficiency virus type 1 infection and are characterized by a chronic interferon response as well as extensive transcriptional changes in cd8+ t cells chronic cd4+ t-cell activation and depletion in human immunodeficiency virus type 1 infection: type i interferon-mediated disruption of t-cell dynamics host genes associated with hiv-1 replication in lymphatic tissue global genomic analysis reveals rapid control of a robust innate response in siv-infected sooty mangabeys nonpathogenic siv infection of african green monkeys induces a strong but rapidly controlled type i ifn response downregulation of robust acute type i interferon responses distinguishes nonpathogenic simian immunodeficiency virus (siv) infection of natural hosts from pathogenic siv infection of rhesus macaques comparative transcriptomics of extreme phenotypes of human hiv-1 infection and siv infection in sooty mangabey and rhesus macaque hiv turns plasmacytoid dendritic cells (pdc) into trail-expressing killer pdc and down-regulates hiv coreceptors by toll-like receptor 7-induced ifn-alpha plasmacytoid dendritic cells express trail and induce cd4+ t-cell apoptosis in hiv-1 viremic patients sex differences in the toll-like receptor-mediated response of plasmacytoid dendritic cells to hiv-1 glycerol monolaurate prevents mucosal siv transmission type i interferon responses in rhesus macaques prevent siv infection and slow disease progression plasmacytoid dendritic cells suppress hiv-1 replication but contribute to hiv-1 induced immunopathogenesis in humanized mice chronic hepatitis c: future treatment host-targeting agents in the treatment of hepatitis c: a beginning and an end? the interferons: 50 years after their discovery, there is much more to learn immune responses to hcv and other hepatitis viruses interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness altered interferon-alpha-signaling in natural killer cells from patients with chronic hepatitis c virus infection natural killer cells are polarized toward cytotoxicity in chronic hepatitis c in an interferon-alfa-dependent manner coexpression of pd-1, 2b4, cd160 and klrg1 on exhausted hcv-specific cd8+ t cells is linked to antigen recognition and t cell differentiation evidence for an antagonist form of the chemokine cxcl10 in patients chronically infected with hcv monocyte activation by interferon alpha is associated with failure to achieve a sustained virologic response after treatment for hepatitis c virus infection genetic variation in il28b and spontaneous clearance of hepatitis c virus genetic variation in il28b predicts hepatitis c treatment-induced viral clearance a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus the favorable ifnl3 genotype escapes mrna decay mediated by aurich elements and hepatitis c virus-induced micrornas distinct and overlapping genomic profiles and antiviral effects of interferon-lambda and -alpha on hcv-infected and noninfected hepatoma cells targeted induction of interferon-lambda in humanized chimeric mouse liver abrogates hepatotropic virus infection ifn-lambda receptor 1 expression is induced in chronic hepatitis c and correlates with the ifn-lambda3 genotype and with nonresponsiveness to ifn-alpha therapies how cells respond to interferons jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins regulation of type i interferon responses stat2 and irf9: beyond isgf3. jakstat (2013) single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators major differences in the responses of primary human leukocyte subsets to ifn-beta critical role for stat4 activation by type 1 interferons in the interferongamma response to viral infection immunomodulatory functions of type i interferons high basal stat4 balanced by stat1 induction to control type 1 interferon effects in natural killer cells type 1 interferon induction of natural killer cell gamma interferon production for defense during lymphocytic choriomeningitis virus infection changing partners at the dance: variations in stat concentrations for shaping cytokine function and immune responses to viral infections dendritic-cell maturation alters intracellular signaling networks, enabling differential effects of ifn-alpha/beta on antigen cross-presentation mechanisms of type-i-and type-ii-interferon-mediated signalling type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors complex modulation of cell type-specific signaling in response to type i interferons alternate interferon signaling pathways ifn-beta induces serine phosphorylation of stat-1 in ewing's sarcoma cells and mediates apoptosis via induction of irf-1 and activation of caspase-7 usp18-based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response interferon-alpha and -beta differentially regulate osteoclastogenesis: role of differential induction of chemokine cxcl11 expression differential activity of type i interferon subtypes for dendritic cell differentiation evolutionary genetic dissection of human interferons the receptor of the type i interferon family interferons as biomarkers and effectors: lessons learned from animal models protection against progressive leishmaniasis by ifn-beta multifaceted activities of type i interferon are revealed by a receptor antagonist receptor density is key to the alpha2/beta interferon differential activities structural and dynamic determinants of type i interferon receptor assembly and their functional interpretation the type i interferon receptor: structure, function, and evolution of a family business differential receptor subunit affinities of type i interferons govern differential signal activation structural linkage between ligand discrimination and receptor activation by type i interferons distinct and nonredundant in vivo functions of ifnar on myeloid cells limit autoimmunity in the central nervous system type i interferons protect t cells against nk cell attack mediated by the activating receptor ncr1 lymphadenopathy in a novel mouse model of bartonella-induced cat scratch disease results from lymphocyte immigration and proliferation and is regulated by interferon-alpha/ beta cytosolic rig-i-like helicases act as negative regulators of sterile inflammation in the cns expression of type i interferon by splenic macrophages suppresses adaptive immunity during sepsis myeloid type i interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions stat3 activation is responsible for il-6-dependent t cell proliferation through preventing apoptosis: generation and characterization of t cell-specific stat3-deficient mice generation of mice with a conditional stat1 null allele conditional stat1 ablation reveals the importance of interferon signaling for immunity to listeria monocytogenes infection loss of stat1 from mouse mammary epithelium results in an increased neu-induced tumor burden inactivation of stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation meta-analysis of microarray data identifies gas6 expression as an independent predictor of poor survival in ovarian cancer axl and growth arrest-specific gene 6 are frequently overexpressed in human gliomas and predict poor prognosis in patients with glioblastoma multiforme gas6 expression identifies high-risk adult aml patients: potential implications for therapy ovarian cancer progression is controlled by phenotypic changes in dendritic cells ifnalpha activates dormant haematopoietic stem cells in vivo timing of ifn-beta exposure during human dendritic cell maturation and naive th cell stimulation has contrasting effects on th1 subset generation: a role for ifn-beta-mediated regulation of il-12 family cytokines and il-18 in naive th cell differentiation combinatorial flexibility of cytokine function during human t helper cell differentiation regulating type 1 ifn effects in cd8 t cells during viral infections: changing stat4 and stat1 expression for function interferons at age 50: past, current and future impact on biomedicine inquiring into the differential action of interferons (ifns): an ifn-alpha2 mutant with enhanced affinity to ifnar1 is functionally similar to ifn-beta an interferon alpha2 mutant optimized by phage display for ifnar1 binding confers specifically enhanced antitumor activities the stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities intra-lesional low-dose interferon alpha2a therapy for primary cutaneous marginal zone b-cell lymphoma intratumoral injection of interferon-alpha and systemic delivery of agonist anti-cd137 monoclonal antibodies synergize for immunotherapy delivery of interferon alpha using a novel cox2-controlled adenovirus for pancreatic cancer therapy the efficacy of radiotherapy relies upon induction of type i interferondependent innate and adaptive immunity a trial of intrapleural adenoviral-mediated interferon-alpha2b gene transfer for malignant pleural mesothelioma tumortargeted interferon-alpha delivery by tie2-expressing monocytes inhibits tumor growth and metastasis targeting cytokines to inflammation sites livertargeting of interferon-alpha with tissue-specific domain antibodies antibody-based targeting of interferon-alpha to the tumor neovasculature: a critical evaluation targeting ifn-alpha to b cell lymphoma by a tumor-specific antibody elicits potent antitumor activities targeting the tumor microenvironment with interferon-beta bridges innate and adaptive immune responses targeted delivery of interferon-alpha via fusion to anti-cd20 results in potent antitumor activity against b-cell lymphoma targeted delivery of interferon-alpha to hepatitis b virus-infected cells using t-cell receptor-like antibodies variations in the unstructured c-terminal tail of interferons contribute to differential receptor binding and biological activity safety and pharmacodynamics of rontalizumab in patients with systemic lupus erythematosus: results of a phase i, placebo-controlled, double-blind, dose-escalation study safety profile and clinical activity of sifalimumab, a fully human anti-interferon alpha monoclonal antibody, in systemic lupus erythematosus: a phase i, multicentre, double-blind randomised study mutation of the ifnar-1 receptor binding site of human ifn-alpha2 generates type i ifn competitive antagonists the studies performed in the laboratories are supported by funding from inserm, cnrs, aix-marseille university, the labex mabimprove, and the european community's seventh framework programme fp7/2007-2013 (grant agreement 223608 for gilles uzé, european research council starting grant agreement number 281225 for marc dalod including salary support to emeline pollet and thien-phong vu manh). we thank past and present laboratory members for their contribution to studies on dcs or ifns. we apologize for not quoting certain studies because of space limitations. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-282668-bs634hti authors: niang, mbayame ndiaye; diop, ndeye sokhna; fall, amary; kiori, davy e.; sarr, fatoumata diene; sy, sara; goudiaby, déborah; barry, mamadou aliou; fall, malick; dia, ndongo title: respiratory viruses in patients with influenza-like illness in senegal: focus on human respiratory adenoviruses date: 2017-03-22 journal: plos one doi: 10.1371/journal.pone.0174287 sha: doc_id: 282668 cord_uid: bs634hti background: human adenoviruses (hadvs) are highly contagious pathogens that are associated with a wide spectrum of human illnesses involving the respiratory tract. in the present study, we investigate the epidemiologic and viral molecular features of hadvs circulating in senegal after 4 consecutive years of sentinel surveillance of influenza-like illness cases. methodology and results: from january 2012 to december 2015 swabs were collected from consenting ili outpatients. adenoviral detection is performed by rrt-pcr with the anyplex(™) ii rv16 detection kit (seegene) and molecular characterization was performed using a partial hexon gene sequence. 6381 samples were collected. more than half of patients (51.7%; 3297/6381) were children of ≤ 5 years. 1967 (30.8%) were positive for hadv with 1561 (79.4%) found in co-infection with at least one another respiratory virus. the most common co-detections were with influenza viruses (53.1%; 1045/1967), rhinoviruses (30%; 591/1967), enteroviruses (18.5%; 364/1967) and rsv (13.5%; 266/1967). children under 5 were the most infected group (62.2%; 1224/1967; p <0.05). we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitudes without any clear seasonality. phylogenetic analysis revealed species hadv-c in majority, species hadv-b and one hadv4 genome type. the 9 hadv-b species like strains from senegal grouped with genome types hadv-7, hadv-55 and hadv-11 as shown by a phylogenetic branch with a high bootstrap value of (88%). conclusion: in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to 5 years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c and hadv-b species. the circulation of though hadv-7 and hadv-55 genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. from january 2012 to december 2015 swabs were collected from consenting ili outpatients. adenoviral detection is performed by rrt-pcr with the anyplex™ ii rv16 detection kit (seegene) and molecular characterization was performed using a partial hexon gene sequence. 6381 samples were collected. more than half of patients (51.7%; 3297/6381) were children of 5 years. 1967 (30.8%) were positive for hadv with 1561 (79.4%) found in co-infection with at least one another respiratory virus. the most common co-detections were with influenza viruses (53.1%; 1045/1967), rhinoviruses (30%; 591/1967), enteroviruses (18.5%; 364/1967) and rsv (13.5%; 266/1967). children under 5 were the most infected group (62.2%; 1224/1967; p <0.05). we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitudes without any clear seasonality. phylogenetic analysis revealed species hadv-c in majority, species hadv-b and one hadv-4 genome type. the 9 hadv-b species like strains from senegal grouped with genome types hadv-7, hadv-55 and hadv-11 as shown by a phylogenetic branch with a high bootstrap value of (88%). in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to 5 years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c and hadv-b a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 species. the circulation of though hadv-7 and hadv-55 genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. human adenoviruses (hadvs) are highly contagious pathogens that are associated with a wide spectrum of human illnesses involving the respiratory, ocular, gastrointestinal, and genitourinary systems [1] . they belong to the family adenoviridae, genus mastadenovirus with seven species (a-g), including each various types [2] . ubiquitous in the environment, hadvs are non-enveloped, double stranded dna viruses that vary in size from 70 to 100 nm [3] . hadvs are recognized as a common cause of respiratory infection in persons of all ages. the illnesses range from influenza-like fever and discomfort to pneumonia and death [4] . indeed, hadvs infections are usely mild but some groups such as very young children, elderly, immunocompromised persons, or persons with underlying pulmonary or cardiac disease, might be at higher risk degree for severe disease [5, 6, 7, 8] . the most common hadvs species that cause respiratory tract infections in children are b (hadv-b3 and b7) and c (hadv-c1, c2, and c5). serotypes b3, b7, and b21 are the most frequent strains responsible for epidemics of acute febrile respiratory disease [9] . circulating hadvs can vary temporally and geographically with possibility of emergent genomic variants which can be associated with more severe illness [10, 11] . in the present study, we investigate the epidemiologic and viral molecular features of hadvs circulating in senegal after 4 consecutive years of sentinel surveillance of influenzalike illness cases. from january 2012 to december 2015 we collected specimens (nasal-pharyngeal and oral-pharyngeal swabs) and surveillance data for influenza and other viral respiratory pathogens from outpatients presenting with influenza-like-illness (ili) at different sentinel sites in senegal. once collected, swabs are placed in 2-ml cryovials with viral transport medium (universal transport medium; copan diagnostics inc., murrieta, ca), and transported at a controlled temperature of 2˚c-8˚c to the laboratory. an ili patient was defined as a person presenting with sudden onset of fever (>38˚c) or history of sudden onset of fever in the recent past ( 3 days) and either cough or sore throat and/or rhinorrhea in the absence of other diagnosis, according to the cdc case definition. each sample is accompanied by a case report form collecting demographic and clinical data. the questions included information on date of enrollment and symptom onset, sex, age, clinical symptoms, previous treatments, travelling history, vaccination status for influenza, and whether or not the patient was hospitalized. upon arrival at the laboratory, the specimens were processed immediately for virus diagnosis. aliquots of samples were also stored at −80˚c for additional analysis (isolation and/or molecular characterization). the data obtained daily were entered into an epi info database (centers for disease control and prevention, atlanta, ga) and analyzed using epi info. total viral nucleic acid (dna and rna) was extracted from 140 μl of each clinical specimen using the purelink™ viral rna/dna mini kit (invitrogen, carlsbad ca, usa) according to the manufacturer's recommendation. dna/rna are eluted with 60 μl nuclease-free water and stored at −80˚c until use. a two-step multiplex real-time rt-pcr was performed with a bio-rad cfx-96 thermocycler (bio-rad laboratories) and the anyplex™ ii rv16 detection kit (seegene) for a simultaneous testing of influenza viruses (flua and flub), human respiratory syncytial virus (rsva and rsvb), human adenoviruses (hadv), human metapneumovirus (hmpv), human coronavirus (229e, nl63, oc43), human parainfluenza virus (piv1, -2, -3 and -4), human rhinovirus (hrv), human enterovirus (hev) and human bocavirus (hbov), as previously described [12] . in consideration with low ct-values, 80 hadv positives samples (20 per year) were selected using a random number generator on ms excel for further molecular characterization using classical pcr and sequencing. viral dna was extracted as previously described and eluted with 50 μl water nuclease-free. dnas were stored at −20˚c until pcr reactions. for hadv molecular characterization the last 300 base pairs (bp) of the hexon gene were amplified with the following specific primers: adeno3 (5'-cctttggcgcatcccattct-3') and adeno4 (5'-tgggcacctatgacaagcgc-3') previously used by garcia et al, [13] . the phusion high-fidelity pcr master mix with hf buffer (new england biolabs, ipswich ma, usa) was used for amplifications. for each sample, pcr was carried out in a total reaction volume of 50 μl consisting of 15 μl h2o rnase free, 2.5 μl of each primer (diluted at 10μm), 25 μl of 2x phusion master mix and 5 μl of dna template. cycling conditions were as follows: denaturation step of 15 min at 95˚c, 40 pcr cycles including 30 s at 95˚c, 60 s at 55˚c, 60 s at 72˚c followed by an extension step of 10 min of 72˚c. five microliters of the pcr product was then mixed with 1 μl of 10x 5prime loading dye and loaded on to a 1% agarose gel along with an appropriated molecular weight markers (100 bp ladder, new england biolabs), and gels were stained with ethidium bromide (0.5 μg/ml) before visualization under uv. for positive samples (380 bp size band), amplicons were cut and purified using the gene-jet gel extraction kit (thermo scientific). purified products are then sent for sequencing to beckman coulter services. sequencing was performed in both directions with the same pcr primers (adeno3 and adeno4) on an abi prism bigdye terminator v3.1 ready reaction cycle sequencing kit (applied biosystems) on a 96-capillary abi prism 3730-xl (applied biosystems). data in fasta format were then sent to the laboratory for analysis. sequences successfully obtained were aligned with representative genbank sequences of previously published genotypes using the bioedit sequence alignment editor [14] . the search for sequence similarities were carried out using the basic local alignment search tool (blastn) from ncbi blast web portal. phylogenetic trees were performed in mega 6 software [15] using the neighbor-joining method, and the statistical significance of the tree topology tested by bootstrapping (1,000 replicates). the evolutionary distances were derived using the tamura-nei method. bootstrap replicates with values !70 are shown on the trees. statistical analysis. regarding hadv infection comparisons between age groups were performed using the fisher's exact test. p value < 0.05 was considered statistically significant and the 0-5 year age group was used as reference group. hadv mono-infections were also compared to hadv co-infections. the r.3.0.1 tool was used to perform the analyses. this study is a component of the 4s network syndromic surveillance [12] . the principles of the 4s network were approved by the ministry of health in its guidelines for influenza surveillance policy, finalized with the support of pasteur institute in dakar and the strengthening influenza sentinel surveillance in africa (sisa) project funded by the who. the protocol and oral consent were determined as routine surveillance activity, and therefore non-research by the senegalese national ethics committee and the steering committee for 4s network, an entity representing moh, ipd, who and clinicians in compliance with all applicable national regulations governing the protection of human subjects. data were collected in an objective of surveillance and are anonymous. the information provided to participants was an informal description of the study. respiratory specimens were collected, only after informed consent was granted, verbally, to local health care workers by the patients or parents in the case of minors. oral consent was documented in the patient form with two questions about received information and about oral consent. patients could refuse to participate, no specimen will be taken. for the surveillance activities, written consent is judged not necessary by the senegalese national ethics committee, which has also previously approved the work of the national influenza center. collections of non-sensitive data or an observation from normal care in which participants remain anonymous do not require ethics committee review. the patients included in this study were of all ages and consulted the sentinel sites due to influenza-like symptoms; the patients, or parents in the case of minors, accept the tests for respiratory viruses largely because they are free and safe. of 6381 specimens tested, 1967 (30.8%) were positive for hadv (table 2) . detection rates over the study period are almost similar in the first 3 years (2012, 2013 and 2014) while in 2015 there is a marked decrease in adenoviral infections. the mean age of infected patients was 8 years 7 months and median age was 3 years. regarding the viral detection per age group, most of hadv infected cases (62.2%; 1224/1967) were under 5 years patients, a statistically significant finding (p <0.05). however, the detection rates in the other groups including the elderly (above 50 years old) remain high. no significantly gender distribution of adenoviral infection was observed. the comparison of symptoms prevalence between ili patients with adenoviral infection and patients without adenoviral infection showed that cases of myalgia (p = 0.0014), cough (p = 0.0028), diarrhea (p < 0.001), rhinitis (p < 0.001) and headache (p = 0.01) are significantly higher in patients infected by adenoviruses ( table 3 ). the fig 1 shows the temporal distribution of hadv positivity rate per month in senegal from 2012 to 2015. we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitude. the highest peak, with 62% of detection rate, was recorded on december 2013. hadv circulation pattern shows no seasonality even if results suggest a higher activity of these viruses during cold periods. it should be pointed out that the cold periods (between december and february) experience some instability in senegal with possibilities of shifting. for phylogenetic analysis, we were able to obtain the partial hexon gene sequence from 54 hadv-positive samples: 8 were from samples in 2012, 13 from 2013, 11 from 2014, 16 from 2015 and 6 from 2016. unfortunately, some samples showed no amplification or poor-quality sequences. the low sensitivity of conventional pcr compared with real time pcr on samples with low viral load, and certainly non-specific amplifications could be the cause of these failures. the nucleotide sequence alignment clustered the majority of senegalese isolates into hadv-c species (44/54). 9 isolates grouped with hadv-b species and the remaining isolate, from 2012, seems close to the hadv-4 genome type belonging to the hadv-e species. in all cases bootstrap values are high (more than 85%). within the hadv-c species, 16 senegalese isolates are grouped with the type hadv-6 (36.4%); 2 isolates with hadv-2 type (4.5%), 4 with hadv-5 type (9%), and 22 isolates formed a subcluster with hadv-1 and 57 types (fig 2) . the 9 hadv-b like species from senegal grouped with genome types hadv-7, hadv-55 and hadv-11 as shown by a phylogenetic branch with a high bootstrap value of (88%). we also noted that this dominance of species c and b is confirmed over the years. in this four-year retrospective study, we characterized hadv isolates derived from an ili surveillance program conducted as collaboration between pasteur institute of dakar and the senegalese ministry of health between 2012 and 2015. it is the first nationally molecular epidemiology investigation of hadvs and even in west africa. our study suggests that hadv are strongly associated with ili syndrome in senegal with an overall detection rate of 30.8% among 6381 patients. this rate seems very higher in comparison with available data. indeed, much lower rates are reported in similar other studies conducted in other countries. a study conducted in kenya [16] on refugees from different countries (somalia, sudan, ethiopia and kenya) yielded a detection rate of 21.7%, in gabon douki et al [17] detected hadv in 16.3% of outpatients with ili. these detection rates are still lower in other geographical regions: south korea with 10.1% or 0.6% [18, 19] , china with 2.7% or 6.3% [20, 21] , philippines with 0.9% [22] , malaysia less than 2% [23], usa with 5.7% or 2.8% reported [24, 25] , canada in the ontario provence with 0.9% [26] , peru with 6.2% [27] , venezuela with 1.6% [28] , england with 6.6% [29] . the analysis of these data tends to confirm a higher prevalence of adenoviruses in the respiratory sphere in african populations. this trend was largely confirmed when we investigated the importance of hadv in children with acute respiratory infections. indeed we observed that proportions in cameroon (27.3%) [30] and senegal (29.2%) (fall et al, on submission) were considerably higher than those found in other geographical areas: nascimento-carvalho et al., [31] in brazil with 3%, moe et al., [32] in norway (1.7%), wansaula et al., [33] in usa (1%) or lu et al., [34] in china. however, these discrepancies in hadv detection rates can be also due to differences in technical approaches, virus burden geographical differences, the number of patients tested, the periods during which samples were collected and even the duration of the study. it should be also noted that adenoviral detection does not necessarily prove disease causation as coincidental upper airway infection, asymptomatic viral carrier state [35] , or prolonged shedding [36] in a previous infection could explain adenoviral detection. regarding the group age, as expected, results showed that most patients with hadv infection were younger than 5 years (62.2%), a statistically significant finding. these results are in concordance with those of other studies which findings concluded that most children are infected by adenovirus at an early age [25, 37, [38] [39] [40] [41] . indeed, it is well established that by 5 years of age, 70% to 80% of children demonstrate antibodies to at least one serotype [42] . additionally more than 80% of diagnosed hadv infections occur in children < 4 years old (due to lack of humoral immunity) [43] . although most cases exhibit low to mild symptoms are and indistinguishable from other viral causes, acute respiratory infections caused by hadv can be severe [44] , or even fatal [7, 45] , and are associated with the highest risk of long term respiratory sequelae [46] . consistent with the report from many other studies, results here showed that 79.4% of hadv infected participants were co-infected with one or more other respiratory tract viruses. the most frequently co-detected viruses were influenza viruses (53.1%), rhinoviruses (30%), enteroviruses (18.5%) and rsv (13.5%). however, we noted no significant differences in clinical characteristics and laboratory findings between patients with single hadv infection and those co-infected. the same observation was reported in studies conducted in diverse geographical contexts [27, 41] . a previous study conducted in chilean children stated that the clinical severity in patients with single hadv infection and those with mixed infections was the same [47] . the overall finding is that the clinical value of such co-infections is not clear and still requires independent investigations in order to assess the association between co-infection and severe illness or symptoms. regarding the four years of surveillance, hadv circulation pattern shows no clear seasonality even if results suggest a higher activity of these viruses during cold periods. this lack of seasonality of hadv infection has been largely reported elsewhere [27, 48, 49] . however, seasonal peaks for hadv infection were noted in summer in some china areas [50] or in spring in northern china [51] , mexico [52] and taiwan [53] . in our study, the last 300 bp region of the hexon gene were used for molecular studies of the different hadv isolates. phylogenetic analysis showed that among the 54 sequenced strains hadv-c species were the most common hadv detected (81.5%) in patients with ili in senegal from 2012 to 2016. despite some divergences, the strains from senegal were close to types 1, 2, 5, 6 and 57. this hadv-c species predominance was reported in malaysia [23], in italy [54] , in many latina america countries [27, 52, 55] in contrast with studies done in the united states of america [56] , united kingdom [37] , korea [57] , in argentina [58] and china [59] , where hadv-b species were the most commonly isolated hadv. hadv-b species were the second most common in senegal with 9 strains, and only one type belonging to hadv-e species was sequenced. hadv-b species from senegal clustered with genome types hadv-7, hadv-7d, hadv-55 and hadv-11 (88% bootstrap value). hadv-7d serotype, firstly identified in 1980 in beijing [59] , is of particular concern as it was often associated with illnesses presenting with more severe and higher levels of morbidity than other respiratory hadv pathogens, and also may result in higher levels of fatalities [60] [61] [62] . the hadv-55 genome type, formerly known as hadv-11a, is a genotype resulting from recombination between hadv-11 and hadv-14 [63] . the serotype has recently reemerged as a highly virulent pathogen, causing severe [64] and sometimes fatal pneumonia among immunocompetent adults, particularly in asia [65] [66] [67] . so the circulation of such hadv genome types in senegal emphasizes the need to reinforce hadv surveillance, especially in hospitalized patients, by including hadv genome detection and genotyping in the documentation of severe respiratory infections. the single hadv-e species strain was typed as hadv-4, the unique human type in this species, which is more commonly associated with high rates of febrile respiratory illness in us military recruits [68] though associated with viral conjunctivitis outbreak in australia [69] for example. we observed some limitations in our study. first, considering the vast number of hadv positive samples, only a small number of hadv were typed. so the sequencing results do not reflect the full spectrum of hadv strains that may be circulating in ili patients in senegal, and even for selected samples it may have a bias toward samples with a high viral load. another limitation concerned the molecular methods used for typing hadvs in this study, a method which targeted a short hexon hypervariable region that has been shown to correlate closely with serotype. this method does not provide genomic detail and might miss recombination events located in other regions of the genome. therefore, full-genome sequencing would be more informative on senegalese strains, especially for hadv-b7 and hadv-b55 types. the results of this study should also be interpreted with caution especially for hadv ili causality (carriage in healthy or asymptomatic individuals). in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to 5 years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c, hadv-b species. the circulation of though hadv-7d and hadv-55 genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. so in the interest of global public health we strongly suggest molecular surveillance and genotyping of newly detected hadv strains in senegal and even by whole genome sequencing for some especial strains. our study offers also an important perspective on the burden of adenovirus-associated respiratory illness in senegal. such a perspective, especially among children, should include asymptomatic controls, sari cases, information on disease outcome, atypical clinical signs, duration of symptoms, and treatment. data regarding viral load, shedding, and other possible etiologies 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adenovirus genome type 7d caused an acute respiratory disease outbreak in southern china after a twenty-one year absence fatal community-acquired pneumonia in children caused by re-emergent human adenovirus 7d associated with higher severity of illness and fatality rate. sci rep characterizing, typing, and naming human adenovirus type 55 in the era of whole genome data severe pneumonia associated with adenovirus type 55 infection outbreak of febrile respiratory illness associated with adenovirus 11a infection in a singapore military training camp epidemiology of human adenovirus and molecular characterization of human adenovirus 55 in china fatal pneumonia cases caused by human adenovirus 55 in immunocompetent adults respiratory diseases among u.s. military personnel: countering emerging threats outbreak of adenovirus type 4 conjunctivitis in south australia this study would not have been possible without the excellent support from all the health-care workers of the 4s network who contributes, every day, to the surveillance network. we convey our special thanks to kathleen victoir from the international network of pasteur institutes for her unwavering support to the 4s network. we acknowledge the senegalese ministry of health for their help in implementing the 4s network. key: cord-029032-s9geepsc authors: vargas-garcía, cesar; lis-gutiérrez, jenny paola; gaitán-angulo, mercedes; lis-gutiérrez, melissa title: parasite-guest infection modeling: social science applications date: 2020-06-22 journal: advances in swarm intelligence doi: 10.1007/978-3-030-53956-6_55 sha: doc_id: 29032 cord_uid: s9geepsc in this study we argue that parasite-host infections are a major research topic because of their implications for human health, agriculture and wildlife. the evolution of infection mechanisms is a research topic in areas such as virology and ecology. mathematical modelling has been an essential tool to obtain a better systematic and quantitative understanding of the processes of parasitic infection that are difficult to discern through strictly experimental approaches. in this article we review recent attempts using mathematical models to discriminate and quantify these infection mechanisms. we also emphasize the challenges that these models could bring to new fields of study such as social sciences and economics. considering that the evolution of parasites and pathogens is important for human health, agricultural systems and wildlife [1, 2] , there is a theory that focuses on how the mechanisms of infection can evolve. because viruses are the most abundant and simple entities on the planet, they are often used as models to study the evolution of parasitic infections. in particular, parameters such as replication, mortality rate of the infected host, infection rate (absorption rate), among others, have been suggested as possible control parameters used by parasites to optimally infect hosts [3] [4] [5] [6] . this paper reviewed the different mathematical models that describe the traditional and recently proposed infection mechanisms. in addition, we reviewed how these are used in the optimal dispersion of infections through susceptible host populations. in the first section, the classic theory of the evolution of the parasite is reviewed. this theory states that natural selection maximizes the number of secondary infections resulting from infection of a susceptible host through free channels that do not involve direct contact between infected and susceptible hosts [7] . one way of doing this is by the evolution of the infection rate, which is the probability of a parasite infecting a host after direct contact. in restricted environments, the classical theory predicts that a parasite will evolve to an infinite maximum infection rate. however, experiments using bacteria as a host and viruses as parasites show the unexpected appearance of viruses with a moderate or intermediate infection rate [8, 9] . how and under what conditions this intermediate rate evolves is still an open question. the proposed section reviews the classical and recent models that try to explain this phenomenon it has been suggested that infection channels between infected and susceptible hosts may provide an advantage, either by allowing parasites to evade the host's immune response [10] , reducing antiviral drug activity [11] , or simply having a more efficient mode of infection. in the second section, a novel model of parasite-host interactions is proposed that accounts for transmission, both through free channels (not involving contact between infected and susceptible hosts), and through infections produced by contact between hosts. the last section examines the possible social and economic science applications that could result from this modeling. first consider a basic model for parasite dynamics introduced by [12] . let h, i and p be the number of healthy and infected hosts and parasites, respectively. host -free infection a healthy host reproduces at a rate λ and dies at a d h rate. the parasite attacks hosts at a rate of rph, where r is the rate of infection. once the infected host dies (with latency period 1/d i ), a set of b-size parasites is released. alternatively, the term bd i i in (3) can be replaced by bi in situations where infected hosts release parasites throughout their life cycle rather than dying before releasing them. parasites that are free in the environment (outside the infected host) can die at a d p rate. the level of parasites in the steady state system is: equation (4) can be seen as a way of measuring the parasite's ability to infect. note, that for the parasite to develop its maximum infective capacity, the infection rate should be infinite (the maximum population of the parasite in a stable state is λ(b − 1)/d p ). alternatively, the number of secondary infections can be used to represent the performance of the parasite. remember that the infection-free steady state, given by h = λ/d h , i = 0, y p = 0, is an unstable point (meaning that infection will take place) if where r 0 is the number of secondary infections and can be interpreted as the number of newly infected hosts produced by an infection. r 0 can be used to infer the evolutionary outcome of the system (3). for example, from (5) it is derived that the parasite should evolve towards infinite infection rates to obtain the maximum fit. the experiments shown in the literature challenge the theory that parasites can evolve to an infinite infection rate, suggested by the previous model. these experiments show the unexpected appearance of parasites with moderate or low infection rates [8, 9] . intermediate infection rates can be explained by the presence of the spatial structure of the host [13] . presumably, parasites with high infection rates tend to create a shielding effect in which the local availability of healthy hosts is reduced, resulting in more interactions with the parasite-infected host, leading to a rate of new parasites equal to zero [7] . figure 1 shows the shielding effect. this shielding effect can be incorporated into the previous model, assuming the number of parasites released by the death of an infected host as a function of the infection rate [14] . where b is the maximum number of parasites released that can be obtained from the death of infected cells. d f represents the ability of the newly released parasite to escape from the harmful residues produced by the death of the infected host. a larger d f means that the parasite has a high probability of finding a healthy host to infect and reproduce. x represents the average amount of waste generated by the death of a single infected host. this modification produces a new level of parasites in a stable state with an optimal finite infection rate given by (7) in addition, there is an optimal number of secondary infections given this finite rate of reproduction [14] . the our system (1-3) was modified to include the ability of the parasite to carry out transmission by direct contact between infected and susceptible hosts. let s the number of parasites sent through the channel formed between an infected and a susceptible host. in addition to the infections produced by the traditional mechanism of infection (without direct contact) r p h, we add an additional production of infections represented by p(s)βp h. here β is the rate of interaction between infected and uninfected hosts. the p(s) function is the probability that an uninfected host will become infected by receiving s parasites through the channel formed between an infected and a susceptible host. the probability p(s) is defined as: where σ (s) is the probability that the infected and uninfected host will form a host-tohost channel. f (s) is the probability that sending pathogens through a given host-to-host channel will result in an infection, and can be any monotonously increased function in s. assuming the probability of parasites infecting a cell as a binomial distribution. if each copy of the parasite has an r probability of successful infection, then that is, f(s) is the probability that at least one of the parasites will have a successful infection given that there is an enabled host-to-host channel. there are two possible scenarios for host-to-host channel formation: channels between infected and uninfected hosts; and channels between infected hosts. the first scenario leads to an infection with probability p(s). therefore, there is a reduction in the number of s σ(s) h i parasites that cannot be used in other infections. the other scenario arises because there is no discrimination mechanism that causes the infected host to form channels with the uninfected host. channels between infected hosts produce a waste of parasites s σ(s) s σ (s) ri 2 that does not produce additional infections, because both cells are already infected. figure 2 shows the three possible routes of infection from host to host: transmission of parasites without involving direct contact between hosts; transmission of parasites through channels between infected and uninfected hosts; and transmission of parasites through channels between infected hosts. including the mechanism described above in the system (1-3) results in the increased. an application to the aids virus of this augmented system is available at work [15] . host -to -host infection (10) host -to -host infection according to fig. 2 , a parasite has the ability to infect cells through (a) a free channel without direct interaction; (b) through contact. in the former, infected cells produce chains of rna (red lines) that use information from the parasite stored in their genome (blue and red line), encapsulate them (blue and red concentric circles) and send these out of the cell. this paper reviewed current approaches in ecology through the use of mathematical tools such as ordinary differential equations (odes). extended models were presented that address issues under debate in ecology, such as optimizing parasite-host interactions and why host infection mechanisms can be beneficial to parasites. whether these models and their projections of infection spread can be applied to fields such as economics, business administration and public policy is relevant for future research. for example, recent studies have suggested studying crime in a region in a manner similar to an epidemic. one could, for example, predict how many additional crimes occur in a given season and design public policy using these models. in the medical field, one could determine which transmission model (host-to-host or non-host) is most effective in spreading and mitigating infections, or in agronomic science [16, 17] . computational modeling approaches to the dynamics of oncolytic viruses chapter 2 -parasite-host interactions fitness of rna virus decreased by muller's ratchet experimental selection reveals a trade-off between fecundity and lifespan in the coliphage qß conditions for invasion of synapse-forming hiv variants fitness benefits of low infectivity in a spatially structured population of bacteriophages are parasites "prudent" in space? local interactions select for lower pathogen infectivity viral infection: changing sides to get in cell-to-cell hiv-1 spread and its implications for immune evasion cell-to-cell spread of hiv permits ongoing replication despite antiretroviral therapy population dynamics of immune responses to persistent viruses emergence of increased frequency and severity of multiple infections by viruses due to spatial clustering of hosts optimal adsorption rate: implications of the shielding effect synaptic transmission may provide an evolutionary benefit to hiv through modulation of latency developing a thief: haustoria formation in parasitic plants prediction rules in e-learning systems using genetic programming key: cord-260472-xvvfguht authors: papadopoulos, nikolaos g.; konstantinou, george n. title: antimicrobial strategies: an option to treat allergy? date: 2007-01-31 journal: biomedicine & pharmacotherapy doi: 10.1016/j.biopha.2006.10.004 sha: doc_id: 260472 cord_uid: xvvfguht abstract respiratory infections by bacteria and viruses often trigger symptoms of asthma in both adults and children. this observation and subsequent mechanistic studies have demonstrated important interactions among allergens, microbes and the atopic host. the mechanisms responsible for microbe-induced asthma exacerbations are only incompletely understood. a focal point of current research is the inflammatory response of the host following an encounter with a pathogenic microbe, including variations in chemokine and cytokine production and resulting in changes in bronchial hyper-responsiveness and lung function. direct bronchial infection, exposure of nerves with resulting neurogenic inflammation and a deviated host immune response are among the mechanisms underlying these functional disorders. lately, suboptimal innate immune responses, expressed as defective interferon production, have gained attention as they might be amenable to intervention. this review describes the suggested mechanisms involved in the complex interactions between ‘asthmagenic’ microbes, the immune system and atopy, based on in-vitro and in-vivo experimental models and epidemiological evidence. in addition, it provides a synopsis of potential therapeutic strategies either directly against the microorganisms or in respect to the associated inflammation. the prevalence of allergies and asthma has been increasing, especially in children, for the last several decades. modern lifestyle and various environmental factors significantly influence the onset of these complex, chronic disorders. considerable research effort has focused on the potential effects of exposure to pollutants, aeroallergens and infectious agents that could adversely affect lung development or function but also precipitate asthma exacerbations [1] . it is widely recognized that respiratory viral infections are among the most important triggers of asthma exacerbations, both in children and adults [2e4] . the association between upper respiratory viral infections and asthma exacerbations in children was demonstrated almost three decades ago using virus cultures and serological techniques [5] . these findings were subsequently confirmed and expanded using more sensitive techniques for virus detection, such as reverse transcription polymerase chain reaction (rtepcr) assays in well-designed longitudinal studies [6e9]. after implementation of these techniques more than 80% of reported episodes of wheeze or drop in lung function could be attributed to respiratory viral infections [7] , rhinovirus (rv) being the most prevalent virus. similar studies in adults implicate respiratory pathogens in almost half of the exacerbations, rhinovirus being once again the prevailing virus [8, 10, 11] . abbreviations: rtepcr, reverse transcriptionepolymerase chain reaction; rv, rhinovirus; piv, parainfluenza virus; rsv, respiratory syncytial virus; mpv, human metapneumovirus; icam-1, intracellular adhesion molecule-1; ifn-b, interferon-beta; ngf, nerve growth factor; sp, substance p; nk1, neurokinin 1 receptor; mbl, mannose-binding lectin; laba, long-acting b 2 agonists. in addition, there is the evidence for an association between 'atypical' bacterial respiratory pathogens and the pathogenesis of asthma, with mycoplasma pneumoniae and chlamydophila pneumoniae most commonly implicated. however, many studies investigating such a link have been uncontrolled and have provided controversial evidence, mainly reflecting the difficulty in accurately diagnosing infection with these pathogens [12, 13] . taking into account these strong associations, it is conceivable that antimicrobial agents and/or strategies may have the potential to reduce the burden of asthma-associated morbidity. the majority of viruses implicated in the pathogenesis of asthma exacerbations are single-stranded rna viruses, including rv, influenza and parainfluenza (piv) viruses, respiratory syncytial virus (rsv) [3, 7, 14] , coronaviruses [15] and the newly described human metapneumovirus (mpv) [16] and bocavirus [17e19]. among the double-stranded dna viruses adenoviruses have also been involved [20] . in the human respiratory tract, all the above agents are able to produce a spectrum of clinical acute infection phenotypes, ranging from the common cold, croup and acute bronchiolitis, to pneumonia, although each virus has increased propensity for a particular clinical disease (e.g. parainfluenza for croup, rsv for severe bronchiolitis, influenza for pneumonia) [21, 22] . there is some evidence that adenovirus can also cause a latent infection in the human lung [23] . the spectrum of viral-triggered asthma exacerbations and reported viral prevalence may vary according to various factors. the age of the subjects is important [24] , since, overall, the frequency of viral respiratory illness is highest in children up to 4 years of age, gradually declines in teenagers, rises again in young parents exposed to children, re-declines in older adults, while the elderly are again more susceptible [25] . in addition respiratory viral infections have strong seasonal patterns although sporadic cases or nosocomial outbreaks can occur [6,26e30] . the presence or absence of a lipid-containing envelope affects viral survival in the environment [31, 32] . in temperate areas, the enveloped viruses, (e.g. influenza virus, rsv and coronavirus), are, characteristically, prevalent during middle-winter periods, whereas nonenveloped ones, such as rvs, are found most often in spring and fall. rv characteristically produces epidemics soon after children return to school. september epidemics of asthma exacerbations coincide with such increase in the rate of respiratory track infections [28, 29] . there is evidence that different infectious agents may induced asthma exacerbations of varying severity, however, stronger evidence is needed in this respect [6,33e35] . finally, the type of diagnostic test used to identify infection may considerably affect epidemiological results [7, 36] . although rsv remains the agent associated with the majority of cases of bronchiolitis requiring hospitalization, recent evidence suggests that in the community rv is the most prevalent virus at all ages [35] . furthermore, with a few exceptions, studies assessing virus-induced exacerbations of asthma have shown that rvs are the prevalent agents, attributing for 50e80% of virus-confirmed cases or around half of all exacerbations studied [7, 8] . there is also evidence that rv may be more 'asthmagenic' than other viruses (papadopoulos et al, unpublished) . rvs belong to picornaviridae family and probably represent the most abundant pathogenic microorganisms universally. these viruses have small rna genomes are nonenveloped and are capable of surviving on surfaces for several hours under ambient conditions [37] . more than 100 serotypes of rvs are identified and numbered. they are divided into major (90%) and minor (10%) groups depending on their receptor specificity. major rvs attach to the intracellular adhesion molecule-1 (icam-1) while minor group rv binds the low-density lipoprotein receptor. in vitro and in vivo, rv infects the bronchial epithelium and upregulates a range of pro-inflammatory cytokines, chemokines, adhesion molecules, mucins and growth factors, all of which are thought to contribute to lower airway inflammation and consequent effects on lung function [38, 39] . a large number of these mediators are upregulated partly or solely through the transcription factor nf-kb [40e42]. several mechanisms have been suggested as part of the complicated pathways leading from a viral infection to an acute asthma exacerbation. these include direct infection of the lower respiratory tract [43, 44] , induction of local inflammation [38, 41, 45] , increase in bronchial reactivity [43, 44, 46, 47] and induction of bronchial obstruction [48] . local inflammation produced after bronchial epithelial cell infection, neurogenic inflammation induced directly or indirectly through the epithelium, and the immune response of the host are probably the most important. there is increasing evidence that the epithelium of the lower airway does not simply act as a physical barrier. not only it has important regulatory role on the immune response inasmuch it may act as an antigen presenting structure [49] but also contributes to the inflammatory response following a viral infection through the production of cytokines and chemokines (e.g. il-6, il-8, il-11, tnf-a, rantes, gm-scf, eotaxin 1 and eotaxin 2) that attract inflammatory cells involved in asthma exacerbations [50, 51] . the epithelial cells' structure and function is altered after the infiltration with inflammatory cell and the oedema of the airway wall. it has been recently suggested that epithelial cells from asthmatic subjects may have an abnormal innate response to infection by rvs, resulting in increased virus replication and cell lysis compared with cells from healthy normal controls. cells from asthmatic individuals did not produce enough interferon-beta (ifn-b) in response to infection, leading to a reduced apoptosis rate, a consequent increase in viral replication within the cells and finally increased cytotoxicity because of the increased viral load [52] . another proposed mechanism by which acute infections might enhance airway narrowing and hyper-responsiveness is the stimulation of the airway neural network which may lead to neurogenic inflammation [39, 44, 53, 54] . virus-mediated damage to the epithelial layer can expose the dense subepithelial nerve endings, increasing stimulation of sensory nerves by inhaled particles or pro-inflammatory mediators. sensory nerves can directly release neuropeptides which may trigger reflex bronchoconstriction. among the mediators of neurogenic inflammation, nerve growth factor (ngf) may have an important role in the pathogenesis of hyper-responsiveness induced by respiratory viruses. it has been documented that ngf induces a selective up-regulation of the high-affinity neurokinin 1 receptor (nk1) for the tachykinin substance p (sp) [55] . sp is a neuropeptide released from sensory nerves with both bronchoconstrictive effects and immunomodulatory properties which regulates the functions of all white blood cells by affecting their migration and response to various mitogens and allergens [56] . one recent study [57] focused on neural development in the lungs during early life and has proposed that this process is under the control of ngf and its corresponding receptor trka. these factors control the branching of nerves into the developing lungs and are downregulated with age. ngf is strongly upregulated during rsv infections, especially in infants and such overexpression may result in prolonged viral clearance from the infected epithelial cells. finally, another interesting pathway attributes to protracted inflammation, associated with an imbalance in t h 1/t h 2 immune responses. in the lower airways of atopic asthmatic individuals a t h 2 environment predominates. although ifng, and il-12 (t h1 1 cytokines) are produced both in normal and atopic asthmatic subjects, the ratio between ifn/il-4 is considerably reduced in asthmatics compared with normal subjects [58] . furthermore, atopic individuals may have impaired antiviral responses concerning ifn-a [59, 60] or/ and ifn-b [52] or/and ifn-g [50, 61, 62] reduced secretion, something that may result in prolonged bronchial inflammation and increased asthma severity. it has been also demonstrated that this impairment is extended to cell recruitment, since in asthmatic patients there seems to be an increased number of eosinophils, compared with normal individuals which also indicates a difference in the immune response to viral infections [54, 63] . in the natural history of asthma exacerbations, interactions between viral infections and other environmental stimuli are often noted. in several occasions synergistic effects have been shown. this is important as it implies that therapeutic results may occur with treatment of only one of such factors. an association between upper respiratory tract infection and air pollution, especially no 2 , and tobacco smoke exposure, has been observed in children [64e66]. there are several potential mechanisms by which pollutants can exacerbate asthma interacting with respiratory viral infections. direct effects of the pollutant on the airways include epithelial damage and an acquired ciliary dyskinesia; release of pro-inflammatory mediators and increases in ige concentration may follow. indirectly no 2 can also impair local antiviral immunity in the airways [67e69]. recent studies have suggested that viruses and allergens may have a synergistic effect on individuals with asthma [43, 70] . this has been shown for both clinical outcomes [71] (symptoms severity) and in experimental models, where there is evidence that viral infections enhance allergen induced inflammatory responses, eosinophil recruitment, histamine release and late phase airway response [72] . although there is increasing evidence from controlled studies to support an association between atypical bacterial infection and both chronic stable asthma and acute exacerbations of asthma, it is still unclear whether such association is causal or patients with asthma are just more susceptible to colonization and/or infection with atypical bacteria. nevertheless, case reports, but also controlled trials during several decades have suggested that postinfectious wheezing may respond to antibiotic therapy, in particular to macrolides [73, 74] . problems with diagnostic techniques for acute and chronic infections with mycoplasma pneumoniae and chlamydophila pneumoniae have made difficult to conduct and interpret epidemiologic studies of the potential relation between these microorganisms and asthma. to add to this complexity, macrolides and ketolides have been shown to have anti-inflammatory properties [75, 76] , making it difficult to assess the true role of infection. in asthmatic patients, exacerbations can be associated with an increase in antibody titres to chlamydophila pneumoniae or mycoplasma pneumoniae [13, 74, 77, 78] . it has recently been proposed that c. pneumoniae might modulate epithelial cell apoptosis by upregulating both pro-apoptosis and anti-apoptosis genes. it has been suggested that c. pneumoniae-induced inhibition of apoptosis increases the longevity of the host cell, enhancing the survival of c. pneumoniae in patients with chronic asthma [79] . consequently, bronchial infection with atypical bacteria is likely to be associated with increased airway inflammation and thus possibly increasing asthma severity and airway remodelling. although, these organisms are common causes of infection, not all infected patients develop or exacerbate their asthma. this suggests that certain individuals may be genetically predisposed to the chronic effects of atypical bacteria, or be genetically susceptible to infection [52] , rendering them more likely to be persistently infected. only a few studies have investigated the possibility of such susceptibility. in one of them [80] , isotype-specific serologic tests have been performed for c. pneumoniae, and the results have been compared with variations in mannose-binding lectin (mbl), a complement component that is important in clearance of respiratory pathogens. the presence of variant alleles in mbl was associated with increased susceptibility to other types of respiratory infections, significantly increasing the risk of asthma development among children infected with c. pneumoniae. on the other hand, a recent study, detailed below, showed improved outcomes when a ketolide was used in patients with asthma exacerbations [81] . although there has been much progress in understanding the mechanisms of microbe-induced asthma exacerbations, there is a need for the development of new therapeutic agents as well as preventive strategies. both antimicrobial and immune modulators could have therapeutic benefits in this respect. rhinovirus is the key virus accounting for the majority of exacerbations both in children and adults and thus the effective treatment or prevention of that infection would be a major asset in asthma therapy. unfortunately, there is currently no available antiviral therapy of clinical value and vaccination seems to be far away because of the large number of rvs' serotypes (more than 100). although this genetic diversity has hampered vaccine development, modern vaccination strategies (such as recombinant proteins, reverse genetics, replication defective particles and other techniques) may make it feasible to induce cross-reactive neutralizing antibodies to the majority of serotypes and produce an effective vaccine [82] . in contrast to rv, rsv has gained more attention because of its association with severe bronchiolitis in infancy. ribavirin, although initially promising, did not find a place in majority of cases, although still included among possible choices for severe bronchiolitis. passive immunoprophylaxis by monthly administration of anti-f monoclonal antibody (palivizumab) reduces the risk of lower respiratory tract rsv disease and hospitalization in high-risk infants and children [83] . however, it cannot be used widely or in an outpatient basis. no vaccine against rsv is available yet, but studies of intranasal live-attenuated vaccine in children and injected subunit vaccine in elderly persons are ongoing [84] . influenza viruses a and b cause annual outbreaks of illness worldwide. a variety of antiviral agents are available for treatment of influenza. the previous generation of agents, amantadine and rimantadine, have demonstrated clinical efficacy, however, potential side effects and most importantly resistance considerably reduced its usefulness [85e87]. the more recent neuraminidase inhibitors, zanamivir and oseltamivir, are active against both a and b viruses, including the avian influenza a/h5n1 strain [87, 88] , and are promising as important tools against a pandemic. more possibilities for anti-influenza agents are being explored [89] . influenza vaccination is available in two forms: an intramuscular preparation containing formalin-inactivated virus and purified surface antigen and an intranasal spray containing live attenuated viruses [90] . the efficacy of these vaccines is approximately 70e90% in young adults, especially when the vaccine antigen and the circulating strain are closely matched. immunization in healthy working adults is associated with fewer upper respiratory illnesses and fewer visits to physicians' offices [90e93]. however, the use of influenza vaccines in reducing virus-induced exacerbations remain controversial [94, 95] . concerning the other asthmagenic viruses (coronaviruses, adenoviruses, human metapneumovirus, bocavirus), clinically available therapeutic or prophylactic agents are still awaited. as mentioned above, a causal link between deficient interferon-impaired apoptosis and increased virus replication has been demonstrated, suggesting that type i interferons might be useful in the treatment or prevention of virus-induced asthma exacerbations. type i ifns include the numerous ifn-as, ifn-b and the newly identified ifn-ls [96] . in the past, ifn-a2 was shown to be effective when given prior to experimental rv infection [97e99], or as a prophylactic therapy [100, 101] , in a context of natural rv infections, however cost and side-effects have prevented its exploitation in the common cold and/or asthma exacerbation fields. ifn-b has not been very effective in preventing experimental or natural rv colds [52,102e104] , but its effects on asthma exacerbations have not been investigated. promising data have been recently published about ifn-ls [105] . it should be noted that in addition to the antiviral approach, combinations of ifn-a with intranasal ipratropium, or oral naproxen, or chlorpheniramine, or ibuprofen have been tested with promising results, but these were also not commercialized [106, 107] . although quite active in vitro [108] , glucocorticosteroids (gcs) so far have been disappointing in their ability to control symptoms in models of experimental rv challenge of asthmatics [109, 110] and high-dose steroids remain only partially effective at controlling virus-induced exacerbations of asthma [111, 112] . a synergistic effect of gcs with long-acting b 2 agonists (laba) has been shown both in in vitro studies and clinically. labas act via a g protein coupled receptor, activate adenylate cyclase and through the second messenger camp, induce intracellular signalling events affecting a broad range of physiological processes, providing by this way an extra potentiality to enhance the anti-inflammatory properties of gcs when acting together in a combination therapy [113] . studies have confirmed clinical benefit in exacerbations, although the viral origin of these events has not been confirmed [114, 115] . evidence suggests that leukotrienes play a key role in viralinduced respiratory illness [116, 117] . the leukotriene receptor antagonist, montelukast, has proven efficacy in the control of asthma exacerbations in adults [118] , but also in preschool and school children with persistent [119, 120] and intermittent asthma [121] . in addition, montelukast significantly reduced symptoms and exacerbations from respiratory syncytial virus postbronchiolitis in infants without asthma [122] . other agents, including antihistamines [123] and antioxidants [124] can block pro-inflammatory mechanisms induced by virus infections in airway epithelial cells, although in vitro evidence has not been paralleled by convincing clinical data. although viral infections are of major interest, the potential role of antibacterial therapy should also be discussed. a number of different antibacterial agents, namely tetracyclines, macrolides, quinolones, azalides and the ketolide telithromycin have in vitro and in vivo activity against the common atypical bacteria c. pneumoniae and m. pneumoniae [125e128]. clarithromycin, roxithromycin, azithromycin [73, 74, 129, 130] and recently telithromycin [81] , have shown some clinical benefit in patients with chronic stable asthma or acute exacerbations. in the most recently published double-blind, randomized, placebo-controlled study [81] telithromycin (at a daily dose of 800 mg for 10 days) provided improvement in symptoms and lung function among adult patients with acute exacerbations of asthma. the study design did not incorporate an analysis of the mechanism by which telithromycin was associated with improvement, but the data imply a benefit not 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in the respiratory tract during infection with respiratory syncytial virus: role in obstructive airway disease montelukast, a once-daily leukotriene receptor antagonist, in the treatment of chronic asthma: a multicenter, randomized, doubleblind trial. montelukast clinical research study group montelukast once daily inhibits exercise-induced bronchoconstriction in 6-to 14-year-old children with asthma montelukast for chronic asthma in 6-to 14-year-old children: a randomized, double-blind trial. pediatric montelukast study group montelukast reduces asthma exacerbations in 2-to 5-year-old children with intermittent asthma a randomized trial of montelukast in respiratory syncytial virus postbronchiolitis effect of desloratadine and loratadine on rhinovirusinduced intercellular adhesion molecule 1 upregulation and promoter activation in respiratory epithelial cells oxidants in asthma and in chronic obstructive pulmonary disease (copd) activity of gemifloxacin and other new quinolones against chlamydia pneumoniae: a review effects of respiratory mycoplasma pneumoniae infection on allergen-induced bronchial hyperresponsiveness and lung inflammation in mice activity of telithromycin, a new ketolide antibacterial, against atypical and intracellular respiratory tract pathogens review of macrolides and ketolides: focus on respiratory tract infections macrolides for chronic asthma macrolides for chronic asthma effect of inhaled formoterol and budesonide on exacerbations of asthma. formoterol and corticosteroids establishing therapy (facet) international study group key: cord-018058-n3majqes authors: modrow, susanne; falke, dietrich; truyen, uwe; schätzl, hermann title: historical overview date: 2013-08-12 journal: molecular virology doi: 10.1007/978-3-642-20718-1_1 sha: doc_id: 18058 cord_uid: n3majqes “poisons” were originally considered as the causative agents of illnesses that we know as viral diseases today. at that time, there were no standard methods to detect pathogenic (disease-causing) organisms such as bacteria and protozoa in the supposed “poisonous materials”. only animal experiments performed by louis pasteur at the end of the nineteenth century, in which no dilution of the poisonous properties was achieved even after several passages, suggested that the disease-causing agent was able to multiply in the organism. therefore, there was talk of a reproducible “virus” (latin for “poison” or “slime”) in living organisms, and later also in cells. in st. petersburg in 1892, dimitri i. ivanovski demonstrated that tobacco mosaic disease is caused by an “ultrafilterable” agent, whose size is significantly smaller than that of bacteria: tobacco mosaic virus (bacteria filters have a pore size of approximately 0.2 μm, however, most viruses are smaller than 0.1 μm). soon afterwards, martinus willem beijerinck came to the same conclusion: he developed, for the first time, the notion of a self-replicating, “liquid” agent (contagium vivum fluidum). the discovery of foot-and-mouth disease virus by friedrich loeffler and paul frosch in greifswald in 1898 was the first evidence of an animal pathogenic virus. since when have we known of viruses? "poisons" were originally considered as the causative agents of illnesses that we know as viral diseases today. at that time, there were no standard methods to detect pathogenic (disease-causing) organisms such as bacteria and protozoa in the supposed "poisonous materials". only animal experiments performed by louis pasteur at the end of the nineteenth century, in which no dilution of the poisonous properties was achieved even after several passages, suggested that the disease-causing agent was able to multiply in the organism. therefore, there was talk of a reproducible "virus" (latin for "poison" or "slime") in living organisms, and later also in cells. in st. petersburg in 1892, dimitri i. ivanovski demonstrated that tobacco mosaic disease is caused by an "ultrafilterable" agent, whose size is significantly smaller than that of bacteria: tobacco mosaic virus (bacteria filters have a pore size of approximately 0.2 mm, however, most viruses are smaller than 0.1 mm). soon afterwards, martinus willem beijerinck came to the same conclusion: he developed, for the first time, the notion of a self-replicating, "liquid" agent (contagium vivum fluidum). the discovery of foot-and-mouth disease virus by friedrich loeffler and paul frosch in greifswald in 1898 was the first evidence of an animal pathogenic virus. however, it can be retrospectively documented that as long as 3,000 years agowithout knowledge of the nature of the pathogens -practices were implemented which today would be described as vaccinations against viral diseases. in ancient china, india and egypt, devastating smallpox epidemics must frequently have occurred; pharaoh ramses v -as his death mask shows -most likely died of an infection of smallpox virus. as observed at that time, people who had survived the disease were spared from the disease in further epidemics; therefore, they had to have developed some kind of protection caused by the first disease -they were immune. this protective status could also be induced artificially; when dried scabs of smallpox were transmitted to healthy people, they were at least partially protected against smallpox -a measure that we now denominate variolation (the medical term for smallpox is "variola"; ▶ sect. 19.6). historical descriptions indicate that smallpox was used as a biological weapon at that time. in the eighteenth century, it was discovered in england and germany that overcoming milker's nodule disease, which is triggered by a virus related to smallpox, confers protection against genuine smallpox. edward jenner must have been aware of these observations in 1796 when he transmitted swinepox and cowpox material, as a sort of vaccine, initially to his first-born son and later to james phipps, a young cowherd. both boys remained healthy following exposure to the human pathogenic smallpox virus by inoculation of smallpox pus; in fact, a protective effect was generated by this first deliberate "virological experiment". knowledge of this vaccination spread very rapidly from england to the european continent and the usa. the term "vaccination" is derived from the latin vacca, which means "cow". vaccinations were soon prescribed by law and this led to a gradual reduction of the dreaded disease. in the former german reich, a first vaccination law was enacted in 1871. however, it still took about 100 years until a human, ali maov maalin, was naturally infected with smallpox (in somalia) for the last time (in 1977), after the who had conducted a worldwide vaccination programme. today, the disease is considered eradicated. on a similar basis, i.e., without precise knowledge of the nature of the pathogen, louis pasteur developed a vaccine against rabies (▶ sect. 15.1.5) in paris in 1885. he transmitted the disease intracerebrally to rabbits in 1882, seeing the causative agent rather in unknown and invisible microbes. as he demonstrated, the pathogen lost its disease-inducing properties by continuous transmission in these animals. in this way, pasteur achieved the basis for a vaccine virus (virus fixe), which, in contrast to the wild-type pathogen (virus de rue), was characterized by a constant incubation period. rubbed and dried spinal cord of rabbits that had been inoculated with the virus fixe was no longer infectious, but caused (initially in dogs) protection against rabies. for the first time, in 1885, pasteur inoculated a 9-year-old alsatian boy named joseph meister with this material. the boy had been bitten by a rabid dog 2 days before, and finally survived, by virtue of the protective effect induced by the vaccine. they especially noticed the striking ability of these agents to lyse bacteria and, therefore, called them bacteriophages -according to the greek word phagein, which means "to eat". the exploration of the nature of bacteriophages has provided virology with important findings and impulses both in methodological and in conceptual terms. many of the steps that characterize a viral infection were first discovered in experiments with bacterial viruses: such processes include attachment and penetration, the reproduction-cycledependent regulation of gene expression that results in early and late synthesized proteins, and lysogeny, which is associated with the existence of prophages. the study of viruses and their attributes was particularly difficult because they, in contrast to bacteria, could not be propagated in artificial culture media. however, it could be ascertained that some of the pathogens isolated from diseased people were transmissible to animals, in which they were able to reproduce. for example, human herpes simplex virus was transmitted from human skin blisters to the cornea of rabbits by wilhelm gr€ uter in marburg in 1911. the extraordinary susceptibility of ferrets allowed the isolation of influenza a virus by christopher andrews, wilson smith and patrik laidlaw from pharyngonasal fluid of a sick person for the first time in 1933. animal experiments also provided many insights into the pathogenesis of viral infections from another point of view. richard e. shope discovered rabbit papillomavirus in 1935, and thus the first tumour virus, which -as was later shown -contains a dna genome. he suspected that such a virus could exist in a latent form as a provirus in the organism. in addition, the discovery that skin cancer can develop from benign skin papillomas is attributed to him. hence, malignant tumours develop in two or more steps -nowadays a universally accepted notion. shope further observed that the incidence of cancer differs in different rabbit breeds, and thus genetic traits of the host also influence the development of cancer. in the framework of animal experiments, erich traub made an important observation while studying the virus of lymphocytic choriomeningitis in princeton in 1935: when pregnant mice were infected with the virus, the virus was transferred to the embryos; mother animals became sick from meningitis, and produced protective antibodies in the further course of the disease. by contrast, the newborn mice remained healthy, but secreted large quantities of the virus for life without developing a specific immune response against the pathogen. this discovery was the first example of an immune tolerance induced by a virus, but the general significance of this phenomenon was not recognized, and the now popular term was not coined (▶ sect. 16.1.5). later, lymphocytic choriomeningitis was shown to be an immunologically related disease. the restriction of cytotoxic t lymphocytes by certain genetically determined types of mhc proteins was demonstrated for the first time in this model by rolf m. zinkernagel und peter c. doherty in 1974. the above-mentioned experiments of traub evidenced also for the first time the intrauterine transmission of a virus. this raised the question of similar ways of infection in humans. in fact, after a severe rubella epidemic in australia in 1941, sir norman gregg observed embryopathies when pregnant women were affected by the infection. as demonstrated later, these malformations were the result of intrauterine transmission of rubella virus. in 1947, coxsackievirus was discovered after transmission of virus-containing stool extracts into newborn mice (coxsackie is a small town in the us state of new york). later in new york, ludwik gross isolated murine leukaemia virus from blood cells. besides the importance for tumour virus research, these observations aroused interest in the question concerning the basis of the high susceptibility of newborn animals to viral infections, and suggested investigations on the innate resistance of an organism to infections as well as the time and the causes of its formation. laborious and time-consuming experiments were initially the only way to prove the existence of viruses: therefore, there simpler methods were sought. one way involved the observation of so-called inclusion bodies in virus-infected tissues, which were soon judged as an indication for proliferation of the pathogen; as we now know, inclusion bodies are the accumulation of viral proteins and particles in the cytoplasm or the nucleus. the first inclusion bodies were discovered by dimitri i. ivanovski; at the same time, guiseppe guarnieri discovered similar deposits in cells infected with smallpox virus, then in 1903, adelchi negri found inclusion bodies in ganglion cells of rabid animals, which were later named after him. thus, there were at least simple dye detection methods for some viral diseases. however, virus culture methods became available later. in the 1930s, it was found that embryonated chicken eggs are appropriate for the propagation of some viral species. between 1918 and 1920, a pandemic emerging viral disease, spanish flu, claimed more than 20 million lives, i.e., more than in the first world war. after cultivation of the virus responsible in embryonated chicken eggs in 1933, their haemagglutinating properties were discovered in 1941 (i.e., their ability to agglutinate red blood cells), thereby laying the basis for the development of haemagglutination tests to detect viruses. another important step in the history of modern virology was the development of the first ultracentrifuges, which became available at about the same time. they made possible the sedimentation and concentration of the minute virus particles. however, the breakthrough in the elucidation of the pathogenetic mechanism of influenza viruses was only possible by the development of molecular biological techniques that allowed the investigation of the genetic material of this pathogen, which exists in the form of singlestranded rna. its sequencing revealed the genetic reasons for the previously not understood ability of influenza viruses to change their antigenic properties at periodic intervals (▶ sect. 16.3.5). however, it was particularly the donation-funded research of poliomyelitis (▶ sect. 14.1.5) which provided crucial new insights. retrospectively, it represents the actual transition to molecular biological research of viral infections. the strong increase in the incidence of polio and the number of deaths -a result of enhanced hygiene standards and the shift of infection rates into later years of life -brought about in the usa the establishment of the national polio foundation by franklin d. roosevelt, himself a victim of this disease, in the early 1930s. with the funds raised, a major research programme was initiated whose coronation was the discovery of the cytopathic effect by john f. enders, thomas h. weller and frederik c. robbins in 1949. in 1928, hugh b. and mary c. maitland had already introduced the method of tissue culture, in which the cells of small tissue pieces were cultivated in serumcontaining liquid media and infected with viruses. successful viral replication was then demonstrated in animal experiments or by detecting the presence of inclusion bodies. when antibiotics became available in the 1940s, it was then possible to largely prevent bacterial contaminations in cultures, which led to much simpler handling in this method. polioviruses were cultivated in embryonic human cells of fixed kidney tissue fragments, and thereby cellular alterations were easily identifiable. this diagnostically valuable cytopathic effect drove the development of virology forwards. it was the basis for the plaque test that was developed by renato dulbecco and marguerite vogt in 1952, which rendered possible, for the first time, the quantitative determination of the number of infectious particles in cell culture. the capability to cultivate polioviruses under controlled conditions in vitro was the basis for the development of the two polio vaccines: the inactivated vaccine developed by jonas e. salk and the live vaccine with attenuated, i.e., weakened polioviruses, developed by albert b. sabin. both vaccines are still in use today. later, vaccines against measles, rubella and mumps were also produced following the principle applied by sabin. by using the method of cell culture, it was possible to cultivate even yellow fever virus, vaccinia virus and rabies virus in vitro. wallace p. rowe isolated adenoviruses from cultures of human tonsil tissue after a long cultivation period in 1953. a further development of viral cultivation in vitro provided the method of co-cultivation, which consists in the addition of indicator cells to the tissue cultures, which indicate viral replication by the occurrence of a cytopathic effect. in this manner, the existence of herpes simplex virus was verified in latently infected human dorsal root ganglia in 1971, whereas direct virus detection was not possible at that time. until then, it had generally been assumed that in the course of viral infections the pathogen would be eliminated completely from the body by the resulting antibodies. the occurrence of herpes blisters as recurrent disease in people with antibodies -known as herpes immunological paradox -refuted that notion (▶ sect. 19.5). ernest w. goodpasture had previously suggested that the trigeminal ganglia should contain a "latent" form of the virus. after such a virus had been detected by co-cultivation, it was recognized that there are a number of infections with latent or persistent viruses, which -independently of the illness symptoms -are excreted either intermittently (e.g., herpes simplex virus) or permanently (such as epstein-barr virus). the primary isolation of human immunodeficiency virus (hiv) 1 was also accomplished by co-cultivation of lymph node biopsy material from an aids patient with suitable t lymphocytes. concurrently with the rather practical benefits from developments in viral cultivation, interest in general biological issues became increasingly important. the crystallization of tobacco mosaic virus from liquid media by wendell stanley in california in 1935 stimulated discussions as to whether viruses are dead or living matter. the main question concerned, however, the nature and structure of the genetic material, which was found to be nucleic acid by the seminal experiments of oswald t. avery, colin mcleod and maclyn mccarty with pneumococci in 1944. in 1952, alfred d. hershey and martha chase proved that during bacteriophage t4 infections only dna, but not the protein shell of the virus, penetrates into the bacterial cell. this demonstrated that nucleic acids are the carrier of genetic information. a few years later, in 1955, gerhard schramm and heinz fraenkel-conrat showed independently in tobacco mosaic virus that rna can also be infectious. schramm and his colleagues had already described in germany in 1944 that tobacco mosaic virus is composed of rna and proteins; however, these findings attracted initially only little attention. the base ratios in dna molecules (a ¼ t and c ¼ g) that were discovered and developed by edwin chargaff enabled james d. watson and francis h. crick, in connection with the rosalind franklin's x-ray structural analysis, to develop their model of the dna double helix in 1953. in 1958, matthew meselson and franklin w. stahl demonstrated that dna is replicated semiconservatively during cell division. these fundamental insights created the way for the elucidation of basic molecular biological processes which are nowadays generally familiar. the now common molecular genetic, biochemical and immunological methods allow the detection of viruses in the organs and the study of their spread in the organism. the function and effect of viral genes can be explored in isolation and in interaction with other viral or cellular components. today, many viruses can be cultivated in large quantities in vitro in order to resolve their morphology and particle structure as well as to sequence their genetic information. in the case of non-cultivable viruses, modern molecular biological methods are available which make possible the investigation of the pathogens. virus-cell interactions can be explored, and provide important insights into the mechanisms of viral replication. on the other hand, many of the molecular processes in eukaryotic cells have been elucidated by using viruses as cell research tools. in this way, the process of rna splicing was originally described in adenoviruses, in which widely separated gene segments are assembled into single messenger rna molecules after transcription. the fact that dna is arranged with histone proteins into nucleosome structures within the nucleus was also first discovered in a virus, simian virus 40. in addition, even enhancers were originally described in viruses, i.e., the specific dna regions that increase the expression of certain genes in a localization-and orientationindependent manner. several of these viral regulatory elements have been used for alternative purposes: for example, the most frequently used promoter/ enhancer sequences to control the expression of heterologous genes in commercially available vectors are derived from cytomegalovirus. this immediate-early promoter/enhancer region actually regulates the expression of early genes of the virus (see ▶ sect. 19.5). furthermore, the transfer of nucleic acid sequences and foreign genes by viral transduction, e.g., using vector systems based on the functions of adenoviruses or retroviruses, is today an indispensable constituent of molecular and cell biology, and has essentially contributed to the development of gene therapy procedures. what is the importance of the henle-koch postulates? the study of the epidemiology and pathogenesis of infectious diseases raises the fundamental question of how can it be proved that an illness is caused by infection with a bacterium or a virus. robert koch derived four postulates from his work with anthrax bacteria between 1882 and 1890, which his teacher jacob henle had previously developed as a hypothesis from the study of so-called miasma and contagions, i.e., the animate or inanimate disease and infection agents: 1. a pathogen must be detected in all cases of a certain disease, but it must be absent in healthy organisms. 2. the pathogen must be cultivable on culture media or in suitable cell cultures in the form of pure cultures. 3. healthy animals must develop the same disease after inoculation of the pathogen. 4. the causative agent must be reisolated from the infected animals. koch noted that the postulates do not comply in all cases. he acknowledged that there are healthy and long-term carriers and that a normal flora of facultative pathogenic bacteria exists. in the realm of virology, not all pathogens comply with these postulates. positive examples are measles virus (▶ sect. 15.3.5), human poxviruses (▶ sect. 19.6.5), canine and feline parvoviruses (▶ sect. 20.1.6) and classical swine fever virus (▶ sect. 14.5.7). with regard to viral diseases, charles river proposed modifications of the postulates in 1937. the exceptions concern preferentially latent or persistent viral infections and the fact that tissue and organ damage or tumour formation cannot always be reproduced as consequences of infection. taken from epidemiology, the "evans postulates" are a worthy supplement (▶ table 11 .1). they show the aetiological importance of a pathogen for a clinical picture if, among others, the pathogen is significantly more frequent in an exposed group and the disease in this population is commoner than in a non-exposed group. similarly, an immune response should be detectable in the affected collectives. the evans postulates are valuable particularly for multifactorial infectious diseases, such as canine kennel cough and porcine circovirus infection (▶ sect. 20.2.6). the further development of virological and immunochemical detection methods in recent years has allowed the use of additional criteria for the causal relationship between a virus and a disease. these include the detection of a specific humoral or cellular immune response against the pathogen, i.e., igm or igg antibodies and specific stimulatable lymphocytes, and the detection of viral proteins, enzyme activities, dna or rna by in vitro and in situ methods. the specific detection of viral nucleic acids in tissues is especially important for the aetiological correlation between persistent viral infections and cancer. the fulfilment of koch's postulates or their modifications is still essential for the development of aetiological relationships between the pathogen and the host. what is the interrelationship between virus research, cancer research, neurobiology and immunology? as early as in 1911, it was demonstrated that rous sarcoma virus can cause cancer. in 1959, margarete vogt and renato dulbecco observed the transformation from benign to malignant cells after infection with murine polyomavirus in vitro. after animals had been inoculated, these cells generated tumours. shortly afterwards, it was also discovered that rous sarcoma virus can transform cells in vitro. thus, tumour virus research became a driving force of virology. it has enriched the realm of cancer research with decisive impulses both in conceptual and in methodological terms. experiments with the oncogenic polyomavirus also showed that its dissemination within mouse populations can be followed by serological methods. this aroused the hope that it would be possible to reduce cancer development to viral agents and to study its nature using the classical methods of epidemiology such as virus isolation and antibody detection. in particular, the study of oncogenic retroviruses in animal systems provided seminal insights into the molecular processes that lead to carcinogenesis (▶ sect. 18.1). by investigating oncogenic retroviruses in 1970, howard temin and david baltimore discovered reverse transcriptase -an enzyme that transcribes the single-stranded rna of retroviruses into double-stranded dna. after integration of the viral genetic information into the genome of the host, these viruses lose their individual existence. a few years earlier, temin had described that an inhibitor of dna synthesis prevents replication of rous sarcoma virus, which should not be the case in a typical rna virus. the integration of a viral genome, which is then present as a provirus, has been associated with tumour development. this event interrupts the continuity of the genome of the cell, as cellular and viral genes can be amplified and destroyed, or their expression can be activated by recombination with viral promoters. as mentioned above, shope had already described that carcinomas arise from papillomas by a two-stage or multistage process. the development of cervical carcinoma caused by human papillomaviruses and that of primary liver cancer caused by hepatitis c virus or hepatitis b virus are similar. even epstein-barr virus exerts its tumorigenic effect in a complex way: the viral dna is detectable in nasopharyngeal carcinoma tumour cells and in various lymphomas (african burkitt's lymphoma). the cells are infected and immortalized, but do not produce infectious virus particles. furthermore, chromosomal translocations are found in b-cell lymphomas (▶ sect. 19.5). however, malignant transformation develops in a multistep process by interaction with other factors, such as malaria, which contributes to a chronic stimulation of cells. research on the molecular processes that occur in infections with papillomaviruses, hepatitis b virus and retroviruses has led to the development of vaccines which induce protection against the respective viral infection and are capable of preventing the development of cancer as a long-term consequence. the vaccination strategy in southeast asia that was initiated and promoted by the who 25 years ago has led to a significant decrease of primary liver carcinoma, which is caused by hepatitis b virus infections (▶ sect. 19.1). in veterinary medicine, vaccines against feline leukaemia virus have proved that cats are protected against infections by this exogenous retrovirus and that tumour formation can be prevented (▶ sect. 18.1). the detailed investigation of the molecular biology and pathogenesis of human papillomavirus infections by the research group of harald zur hausen at the german cancer research center (dkfz) paved the way for the development of appropriate vaccines. these have been available for several years and protect against infections with the highly oncogenic papillomaviruses: they prevent the possible development of cervical carcinoma -one of the commonest cancers in women (▶ chap. 10, ▶ sect. 19 .3). in september 2008, harald zur hausen was awarded the nobel prize in physiology or medicine for his work concerning the role of papillomaviruses in cervical cancer. the term "slow virus infections" was initially coined by björn sigurdsson for maedi disease of sheep in iceland in 1954. maedi-visna virus causes respiratory symptoms in a slow and progressive disease after very long incubation and latency periods. maedi-visna virus thus became a model for a range of pathogens that cause diseases with a similar protracted course (▶ sect. 18.1.6). the exploration of its pathogenesis revealed that most slow virus infections are caused by pathogens which are usually associated with other diseases. slow virus infections principally affect the central nervous system and are caused, for example, by measles virus and jc polyomavirus. subacute sclerosing panencephalitis is probably caused by mutations in measles virus genes, which lead to the emergence of defective virus particles (▶ sect. 15.3.5) . in progressive multifocal leucoencephalopathy, which is triggered by jc polyomavirus, the virus seemingly enters the brain very early and persists there for a long time before the disease breaks out as a result of damage to the immune system (e.g., by infection with hiv; ▶ sect. 19.2.5). infections with hiv can also be considered as a slow virus disease. similarly, prion diseases also progress along the lines of a slow virus infection, but they are caused by non-viral pathogens and have a fundamentally different pathogenesis (▶ chap. 21). working on yellow fever virus, m. hoskins, g.m. findlay and f. maccallum discovered the phenomenon of interference in 1935: if an avirulent virus was injected into an experimental animal, the animal was protected from the consequences of infection by a virulent strain when it was applied within the next 24 h, i.e., before the onset of an immune response. in 1957, alick isaacs and jean lindenmann showed that interferon is responsible for the interference effect. it is species-specific, inducible and belongs to a group of substances that are known as cytokines today. interferons play an important role in the primary, non-specific defence against viral infections and in stimulating the immune system. the observation that antiviral interferon preparations also have tumour-inhibiting effects was surprising. generally, cytokines are synthesized when a suitable inducer binds as a ligand to its receptor on the cell membrane, thus triggering specific signal transduction processes in the cell (▶ chap. 8). attempts have been made to develop antiviral chemotherapeutic agents since about 1960. in retrospect, this search can be divided into three stages: the first successful experiments for therapy of a viral infection were performed by josef wollensak and herbert e. kaufman around 1960 in herpetic keratitis. they used substances that inhibit viral replication in vitro and were known from experimental cancer therapy. however, the selectivity of these substances, i.e., their ability to selectively influence viral and not cellular processes, was only slight because of the high cytotoxicity of the compounds. after the discovery of virus-coded enzymes such as thymidine kinases, dna polymerases and proteases, it was possible to address the development of specific inhibitors. antiviral drugs such as amantadine against influenza a virus (▶ sect. 16.3) and adenine arabinoside and acyclovir (acycloguanosine) against herpes simplex virus (▶ sect. 19.5) were found by targeted empiricism, i.e., by attempting to find a compound that selectively influences viral reproduction among many compounds with similar effects. the achievement of gertrude elion and her staff to use acyclovir as a systemically applicable and selective antiviral drug in herpes encephalitis was an important milestone in chemotherapeutic research. she was awarded the nobel prize in physiology or medicine in 1988 for her work. after the development of dna sequencing techniques, the experimental chemotherapy of viral infections entered its third stage. virus-encoded enzymes were discovered such as the retroviral protease and neuraminidase of influenza viruses, and it was possible to build structural models of enzymes by comparison with proteins of similar functions and known three-dimensional structures. this allows one to identify potential active centres and to develop compounds, also known as "designer" drugs, which accommodate within the active centres and inhibit the viral enzymes. that means deviating from purely empirical research, and is a first step towards a more rational development of antiviral compounds (▶ chap. 9). molecular virology has achieved significant successes in recent decades: many infectious diseases can be prevented through the use of modern vaccines or have been completely eradicated (▶ chap. 10). this ultimately made possible the global elimination of infectious agents such as smallpox virus. poliovirus, which causes poliomyelitis, is no longer found on some continents, and is currently confined to fewer than ten countries worldwide. in cases in which no preventive vaccination is possible today, e.g., against hiv and some herpesviruses such as cytomegalovirus and herpes simplex virus, a large number of antiviral drugs are available. although these drugs do not provide a cure, they substantially allow the control of symptoms. these successes might tempt one to assume that virus research has become unnecessary. the assessment of the epidemiological situation by the who and the many sensationalistic headlines in newspapers and the media with which we are repeatedly confronted imply the opposite. because of their frequent and high rates of mutation, viruses are subject to continuous change and development: viruses are permanently compelled to cope with the infected organism and its immune defence systems, always trying to undermine and circumvent them. in particular, viruses that persist in the organism are capable of evading the host immune defence systems by very skilful strategies. the worldwide increase in travel leads not only to contact with new human pathogens, but also to their rapid dissemination. this is demonstrated, for example, by sars virus infections (▶ sect. 14.8), the pandemic with the new influenza a virus variant (mexican flu, "swine flu") and the threatening potential with regard to humans of new highly pathogenic influenza viruses (▶ sect. 16.3). new and novel viral diseases which have their origin in the animal kingdom (zoonoses) are also expected owing to increased environmental changes and their serious consequences. outbreaks of infection with ebola virus, nipah virus and hendra virus are examples. deforestation of rainforests has led to a change in living conditions for bats, which then infect horses and pigs and, via these intermediate hosts, also humans. birds carried west nile virus from africa to north america, and avian flu virus h5n1 was transported from asia to europe by migratory birds. the aids pandemic that was induced by human immunodeficiency viruses was originally the result of a zoonotic transmission from monkeys to humans, followed by efficient further dissemination within the human population. the threat from both new and already well-known viral infections will not decrease because of reduced vaccination, particularly in industrialized countries; therefore, scientists who conduct research in the field of molecular virology will continue to have an ample sphere of activity. behbehani am (1988) the smallpox story in words and pictures. university of kansas medical center, kansas city causation and disease. the henle-koch postulates revisited helmut ruska and the visualisation of viruses mikrobenj€ ager. ullstein, frankfurt levine aj (1991) viruses. palgrave macmillan m€ uller r (1950) medizinische mikrobiologie. parasiten, bakterien, immunit€ at, 4th edn history of virology key: cord-118119-it3q17rp authors: odagaki, takashi title: self-organized wavy infection curve of covid-19 date: 2020-10-16 journal: nan doi: nan sha: doc_id: 118119 cord_uid: it3q17rp exploiting the siqr model for covid-19, i show that the wavy infection curve in japan is the result of fluctuation of policy on isolation measure imposed by the government and obeyed by citizens, assuming the infection coefficient be a 2-valued function of the number of infected individuals, i show that when the removal rate of infected individuals is between these two values, the wavy infection curve is self-organized. on the basis of the infection curve, i classify the outbreak of covid-19 in each country into five types and show that these differences can be related to the relative magnitude of the transmission coefficient and the quarantine rate of infected individuals. since november 2019, the pandemic covid-19 is still expanding in the world. the infection curves of some countries like australia, austria, japan, luxemburg and serbia [1] show clearly a wavy structure. since the period of the wave is much shorter than that of the wave in the siqr model [2, 3] , population are separated into four compartments; susceptible individuals, infected individuals at large (will be called infecteds for simplicity), quarantined patients in hospitals or at home who are no longer infectious in the community and recovered (and died) patients. the population in each compartment are denoted by s, i, q and r, respectively, and the total population is given by n(= s + i + q+ r). the basic equations for the time evolution of the populations are given by a set of ordinary differential equations. where t is the time. the term βs i n denotes the net rate at which infections spread, where β is a transmission coefficient determined by lockdown measure including social-distancing and self-isolation of people and by the trait of virus. infected individuals at large, regardless of whether they are symptomatic or asymptomatic, are quarantined at a per capita rate q and become non-infectious to the population. the quarantine rate is determined by the government policy on pcr (polymerase chain reaction) test. quarantined patients recover at a per capita rate γ ′ (where 1/γ ′ is the average time it takes for recovery) and infected individuals at large become non-infectious at a per capita rate γ (where 1/γ is the average time that an infected patient at large is capable of infecting others). it is apparent that eqs. (1) ∼ (4) guarantee the conservation of population n = s + i + q + r. at the end of september 2020, the total number of infected, quarantined and recovered people is much smaller than the entire population in any countries, and thus the pandemic can be regarded as in its early stage far from the stage of herd immunization. therefore, i can assume that i + q + r << n is satisfied and s = n − (i + q + r) ≃ n. then the basic equation governing the time evolution of the number of infecteds is written as where the net rate of change of the number of infecteds denoted as determines the short-term behavior of the number of infecteds. the number of infecteds increases when λ > 0 and decreases when λ < 0. it is straightforward to obtain the solution to eq. (5): where i(0) is the initial number of infecteds. the observed data for the outbreak of covid-19 is the daily confirmed new cases ∆q(t) ≡ qi, which is given by a convolution of the incubation period distribution function ψ(t) and the number of infecteds i(t). therefore ∆q(t) can be expressed as since the incubation period distribution is a well behaved function with a single peak [12, 13] , the convolution can be evaluated by the saddle-point method of integration and it is given by [3] where τ is a characteristic time representing the peak position of ψ(t) and ψ ′′ (t) = d 2 ψ(t) dt 2 . therefore, the short-term behavior of the number of daily confirmed new cases can be described by a function similar to eq. (7). the first wave of the outbreak of covid-19 in various countries has been analyzed on the basis of eq. (7), where ∆q(t) is approximated by a piece-wise exponential function [3, 4, 5, 6, 7, 8, 9] . as can be seen in eq. (7), the time dependence of ∆q(t) is determined by λ(t) alone and it is a difficult task to convert it to β, q and γ [10, 11] . here, i first assume that γ is a constant since no treatment could be given to infecteds and set γ = 0.04 [12, 13, 14] . next, i assume that β(t) and q(t) change in time continuously between two values represented by a hyperbolic tangent function table 1 . the quarantine rate is increased from q(0) = 0.02 to q(200)= 0.029 as explained in the text. fig. 1 . + b is set to satisfy β(t 2i ) = β 2i and β(t 2i+2 ) = β 2i+2 for t i 's listed in table 1. for t 2i ≤ t ≤ t 2i+2 with β(t 2i ) = β 2i and β(t 2i+2 ) = β 2i+2 and t 2i+1 as the transition point and dt 2i+1 as the width of the transition (i = 0, 1, 2, . . .). table 1 summarizes parameters determining the time dependence of β(t) used for fitting in fig. 1 . figure 2 shows the time dependence of β(t), q(t) and λ(t). it should be emphasized that the assignment of β and q from λ is not unique since adding any amount to β and q at a given time does not change λ. in the present study, i assumed that the time dependence of q is weak since the procedure for the pcr test did show no drastic change in the period for identifying infected individuals at large. the time dependence of the transmission coefficient β(t) must be attributed to the attitude of people to self-isolation under government policy and massive information from news media. here i consider the transmission coefficient is a function of i and di dt , and i introduce a model country in which β is given by β h when di dt > 0 and i ≤ i h β ℓ when di dt < 0 and i ℓ ≤ i < i h , where β ℓ < q + γ < β h is satisfied. figure 3 (a) shows β(i). the infection curve for β(i) given by eq. (13) is shown in fig. 3 according to the data available at coronavirus resource center, johns hopkins university [1] , the daily confirmed new cases in each country up to the end of september seems to be classified into the following five types: type i the infection curve keeps increasing, like india, iraq and argentina. type ii after the first peak with a certain length of tail, the infection curve increases again like type i. this is seen in russia and many european countries. type iii the infection curve shows oscillation like in austria, australia, luxemburg, japan and serbia. type iv infection curve is characterized by a sharp peak followed by more or less constant infection for a long time. this infection curve is seen in usa and sweden. type v after a small peak, few new cases have been observed like in china, taiwan, thailand and viet nam. as discussed in sec. 3, the relative magnitude of β and q + γ must be responsible for the structure of the infection curve. here, keeping q + γ constant, i discuss the relative magnitude of these parameters for different types of infection curve. it should be emphasized that the difference β − (q + γ) determines the infection curve. here, for the sake of simplicity, i fix q + γ and attribute all effects to change in β. it is possible to discuss in the same way by changing q with a fixed β. for type i infection curve, β > q + γ is satisfied ( fig. 4(a) ) and thus the infection curve keeps increasing. the infection curve will reach eventually its maximum and start to decline because of the non-linear term si/n in eqs. (1) and (2) . the spanish flu belongs to this type. type ii infection curve will be realized when a strong lockdown measure is introduced at the outbreak and it is lifted in fear of economic break down ( fig. 4(b) ). after a little peak and some length of tail, the infection curve will follow the same trend as type i. wavy infection curve (type iii, fig. 4 (c) ) has already been discussed in sec. 3. infection curve of type iv is characterized by a fixed point in the β -i plane which is reached after the first peak ( fig. 4(d) ), and i ℓ determines the size of the daily confirmed new cases. type v infection curve represents the most efficient measure; the transmission coefficient is brought below q + γ (or q is increased) so that β < q + γ is satisfied, and the measure is kept. the trajectory in the β -i plane has a fixed point near i = 0 as shown in fig. 4(e) . in this paper, i discussed the infection curves of covid-19 observed in many countries and showed that the infection curve can be classified into five types. in particular, a wavy infection curve can be self-organized due to change in self-isolation and/or quarantine measures making β above or below q + γ. it is shown that these different infection curves are caused by relative strength of lockdown measure and quarantine measure. it will be possible to formulate the optimum policy specific to the country for controlling the outbreak on the basis of the present theoretical framework, if the cost function and the aim of policy in each country are given [15] . the pandemic in countries whose infection curve is of type i or type ii will stamp out when sufficient number of population get immunity. according to percolation theory, the condition for herd immunity is that the fraction of immunized individuals is larger than a critical value [16] p c = 1 − 4.5 nβ , where β is the transmission coefficient and n is the average number of people with whom an infected individual meets while it is infectious. the critical value depends on β and n and it could be as large as 50 ∼ 80 %. therefore, it could take much longer time before the herd immunity is realized in any countries in the world. the conversation the physics of connectivity key: cord-265699-0socw0hp authors: ortega, miguel ángel; guzmán merino, alberto; fraile-martínez, oscar; recio-ruiz, judith; pekarek, leonel; g. guijarro, luis; garcía-honduvilla, natalio; álvarez-mon, melchor; buján, julia; garcía-gallego, sandra title: dendrimers and dendritic materials: from laboratory to medical practice in infectious diseases date: 2020-09-14 journal: pharmaceutics doi: 10.3390/pharmaceutics12090874 sha: doc_id: 265699 cord_uid: 0socw0hp infectious diseases are one of the main global public health risks, predominantly caused by viruses, bacteria, fungi, and parasites. the control of infections is founded on three main pillars: prevention, treatment, and diagnosis. however, the appearance of microbial resistance has challenged traditional strategies and demands new approaches. dendrimers are a type of polymeric nanoparticles whose nanometric size, multivalency, biocompatibility, and structural perfection offer boundless possibilities in multiple biomedical applications. this review provides the reader a general overview about the uses of dendrimers and dendritic materials in the treatment, prevention, and diagnosis of highly prevalent infectious diseases, and their advantages compared to traditional approaches. examples of dendrimers as antimicrobial agents per se, as nanocarriers of antimicrobial drugs, as well as their uses in gene transfection, in vaccines or as contrast agents in imaging assays are presented. despite the need to address some challenges in order to be used in the clinic, dendritic materials appear as an innovative tool with a brilliant future ahead in the clinical management of infectious diseases and many other health issues. infectious diseases are produced by pathogenic microorganisms, mainly bacteria, viruses, parasites, and fungi. since the dawn of civilization, these diseases have persisted as sources of human morbidity and mortality, representing the second cause of death worldwide and the main reason of b groups are dormant/protected and will react in a subsequent step after deprotection/activation. iterative growth and activation steps lead to the desired generation, while the end-groups are available for further postfunctionalization. the divergent growth is the most viable approach, as it employs an excess of inexpensive reagents, but could lead to structural defects at high generations due to incomplete substitutions. the convergent growth approach was initially developed by hawker and fréchet, to improve the weaknesses of the divergent approach. this outside-in strategy relies on the coupling of monomers to generate monodisperse dendrons, which are finally attached to a multifunctional core through their focal points. while the risk of structural defects is minimized, the synthesis of higher generation dendrons and dendrimers are challenging due to steric hindrance, leading to low yields. importantly, new strategies, namely "accelerated growth" approaches, are continuously evolving to simplify the synthetic routes while keeping their perfection. these include the orthogonal chemoselective strategy and the one-pot approaches, among others. the number of steps is thus reduced, as the chemoselective moieties avoid the need for protection/deprotection steps. the reader is referred to excellent reviews on this topic [23, 27] . pharmaceutics 2020, 12, x for peer review 4 of 28 routes are prevalent ( figure 1 ): the divergent strategy and the convergent strategy [23] . in the divergent growth approach, first developed by tomalia et al. [26] , the dendrimer synthesis proceeds inside-out from the core. the core reacts with abn monomeric units through the a functional group, while the b groups are dormant/protected and will react in a subsequent step after deprotection/activation. iterative growth and activation steps lead to the desired generation, while the end-groups are available for further postfunctionalization. the divergent growth is the most viable approach, as it employs an excess of inexpensive reagents, but could lead to structural defects at high generations due to incomplete substitutions. the convergent growth approach was initially developed by hawker and fréchet, to improve the weaknesses of the divergent approach. this outside-in strategy relies on the coupling of monomers to generate monodisperse dendrons, which are finally attached to a multifunctional core through their focal points. while the risk of structural defects is minimized, the synthesis of higher generation dendrons and dendrimers are challenging due to steric hindrance, leading to low yields. importantly, new strategies, namely "accelerated growth" approaches, are continuously evolving to simplify the synthetic routes while keeping their perfection. these include the orthogonal chemoselective strategy and the one-pot approaches, among others. the number of steps is thus reduced, as the chemoselective moieties avoid the need for protection/deprotection steps. the reader is referred to excellent reviews on this topic [23, 27] . a broad variety of dendritic scaffolds have been described in the literature, with a purpose-driven design. for details on their preparation, the reader is referred to excellent books and reviews on the literature [28] [29] [30] . in the biomedical field, the dendritic families which stand out are poly(amino amide) (pamam) [31] , poly(propylene imine) (ppi), poly(l-lysine) (pll) [32] , carbosilane [33] , poly(phosphorhydrazone) (pph) [34] , and polyester dendrimers [35] (figure 2 ). the biocompatibility, flexibility, and commercial availability are behind their prevalence in this field. pharmaceutics 2020, 12, x for peer review 5 of 28 a broad variety of dendritic scaffolds have been described in the literature, with a purpose-driven design. for details on their preparation, the reader is referred to excellent books and reviews on the literature [28] [29] [30] . in the biomedical field, the dendritic families which stand out are poly(amino amide) (pamam) [31] , poly(propylene imine) (ppi), poly(l-lysine) (pll) [32] , carbosilane [33] , poly(phosphorhydrazone) (pph) [34] , and polyester dendrimers [35] (figure 2 ). the biocompatibility, flexibility, and commercial availability are behind their prevalence in this field. pamam dendrimers are probably the most studied dendritic architectures, reaching up to the tenth generation, with different cores and terminal groups (mainly nh2 or oh), figure 2a . pamam dendrimers exhibit appealing properties for biomedical studies [31] , such as a high water solubility, a peptide-mimicking backbone, and readily modifiable amine termini. ppi dendrimers, also known as popam or dab, present multiple tertiary amines on the scaffold and primary amines as terminal groups, figure 2b . they are comparatively smaller than pamam and present a more hydrophobic scaffold, but are also commercially available, prevalent in the biomedical field and similarly cytotoxic due to the peripheral amino groups [36] . pll dendrimers, which comprise the amino acid lysine in their entire structure ( figure 2c ), stand out due to their biocompatibility, biodegradability, and the ability to maintain its activity in environments with high and low salinity [32] . pll differ from other dendrimers such as pamam and ppi in the asymmetry of their branching cell, which inevitably influences the encapsulation properties as they possess no interior void space [37] . however, they share the presence of multiple nh2 peripheral groups, which can cause certain cytotoxicity. carbosilane dendrimers comprise carbon-carbon and carbon-silicon bonds in their scaffolds, conferring flexible, non-polar, inert, and thermally stable properties, very interesting in biomedicine [33] , figure 2d . they are often decorated with polar groups in order to achieve water solubility. they are pamam dendrimers are probably the most studied dendritic architectures, reaching up to the tenth generation, with different cores and terminal groups (mainly nh 2 or oh), figure 2a . pamam dendrimers exhibit appealing properties for biomedical studies [31] , such as a high water solubility, a peptide-mimicking backbone, and readily modifiable amine termini. ppi dendrimers, also known as popam or dab, present multiple tertiary amines on the scaffold and primary amines as terminal groups, figure 2b . they are comparatively smaller than pamam and present a more hydrophobic scaffold, but are also commercially available, prevalent in the biomedical field and similarly cytotoxic due to the peripheral amino groups [36] . pll dendrimers, which comprise the amino acid lysine in their entire structure ( figure 2c ), stand out due to their biocompatibility, biodegradability, and the ability to maintain its activity in environments with high and low salinity [32] . pll differ from other dendrimers such as pamam and ppi in the asymmetry of their branching cell, which inevitably influences the encapsulation properties as they possess no interior void space [37] . however, they share the presence of multiple nh 2 peripheral groups, which can cause certain cytotoxicity. carbosilane dendrimers comprise carbon-carbon and carbon-silicon bonds in their scaffolds, conferring flexible, non-polar, inert, and thermally stable properties, very interesting in biomedicine [33] , pharmaceutics 2020, 12, 874 6 of 27 figure 2d . they are often decorated with polar groups in order to achieve water solubility. they are classified as "inorganic dendrimers" and exhibit relevant differences compared to traditional "organic dendrimers" such as pamam. furthermore, they have a great variability by altering the core and the amount and length of the branches. pph dendrimers, which can be quantitatively prepared up to generation 12 [38] , present phosphorus atoms throughout the entire dendritic scaffold and have been widely studied for biomedical applications [34] , figure 2e . like carbosilane dendrimers, pph are also "inorganic dendrimers" with a huge variability in cores, branches, and peripheral groups, and require the attachment of polar groups in the periphery to become water-soluble. polyester dendrimers attract widespread attention in the biomedical field due to their biocompatibility and biodegradability. in particular, dendrimers based on 2,2-bis(hydroxymethyl) propanoic acid (bis-mpa, figure 2f ) are commercially available. since the first reports in the early 1990s, bis-mpa dendrimers have undergone an extraordinary increase in their structural complexity and control, which capitalized on a constant evolution of the synthetic strategies, from the traditional divergent and convergent routes to accelerated approaches based on chemoselective reactions [35, 39] . since their first reports, dendrimers have been tested in a large number of in vitro and in vivo studies for multiple biomedical applications. the most explored use of dendrimers is their ability to carry drugs to the desired site of action, being an important resource in precision medicine [24] . dendrimers protect the encapsulated or bound drug and allow the delivery to the desired site of action. as they can be customized, dendrimers improve the drug pharmacokinetics and solubility, control the drug release, enable more comfortable administration routes, and reach target sites with difficult accessibility such as the ocular system [40] . another application that has raised great interest is the use of dendrimers in gene therapy. several dendrimers have been explored as non-viral vectors for dna and rna, enabling gene transfection to specific cells. this has been especially useful in in vitro cancer studies, where rna transfected to tumor cells can alter their mechanisms, making them more susceptible to treatment or hindering their uncontrolled division [41] . on the other hand, dendrimers can act as immunomodulators, by either reducing or enhancing the immune response [42] . the first approach can be very useful towards autoimmune diseases and allergies [43] , while the second has been employed, for example, in cancer immunotherapy [44] . the attachment of multiple antigen copies to the dendritic scaffold produces an increase in the immune response related to the multivalency and the decrease in the conformational freedom of the antigen. in infectious diseases, dendrimers can support the development of vaccines by acting as antigens carrier, providing stability, safety, and a sustained release. in addition, they can serve as adjuvants or can promote the uptake of the antigen by the antigen-presenting cells, thus enhancing its recognition and improving the effectiveness of the vaccine [45] . another outstanding application is the use of dendritic materials in diagnosis [46] , such as iron oxide magnetic nanoparticles decorated with dendrimers, which can be monitored through magnetic resonance, or the oxygen sensors, very useful in pathologies such as diabetes [47] . dendrimers also enable a combined therapeutic and diagnostic action in a single platform, the so-called "theranosis" [48] . the diagnostic capacity is provided by a specific molecule (e.g., a radionuclide) which, bound to the surface or encapsulated inside the nanoparticle, serves to detect its position in vivo by means of diagnostic imaging such as single photon emission computed tomography (spect). in order to fully benefit from the use of dendrimers as nanocarriers, it is essential to understand the mechanisms of interaction between the dendrimer and the different cargo ( figure 3 ) [49] [50] [51] : • encapsulation. the drug is physically trapped within the dendritic scaffold due to the spheroidal or ellipsoidal hollow cavities found between the different branches. these cavities are frequently hydrophobic, so they exhibit affinity towards drugs with poor water-solubility, and can also lead to h-bonding due to the presence of oxygen and nitrogen atoms. the main drawback of this approach is the tendency of the drug to rapidly leak in biological fluids, compared to a covalent conjugation approach [52] . • electrostatic interactions. the multivalent structure of the dendrimer enables the formation of multiple bonds in the periphery, which depend on the nature of the end groups. a common example are electrostatic interactions between the drug and a dendrimer bearing cationic (e.g., ammonium groups) or anionic (e.g., carboxylate) moieties. pamam and ppi dendrimers frequently employ this mechanism, due to the multiple ionizable amino groups in the periphery as well as in the interior of their scaffolds. the ph, the ionic strength and the presence of proteins such as albumin have a remarkable impact on dendrimer-cargo electrostatic interactions [52] . this approach is widely employed in gene therapy to generate dendrimer-nucleic acid complexes, or "dendriplexes" [53] . • covalent conjugation. drugs and other molecules can be attached to dendrimers through covalent bonds. sometimes labile or biodegradable bonds are employed, such as amide or ester bonds, to enable the release under chemical or enzymatic scission. other strategy relies on the use of spacers, such as poly(ethylene glycol) (peg), which also generates a hydrophilic surface with a hydrophobic interior, an amphiphilic unimolecular micelle to improve drug encapsulation. furthermore, the attachment of peg reduces the interaction with blood proteins and cells, prolongs the circulation in blood and increases the overall molecular weight, improving the permeability and retention of the drug [54] . other types of ligands have also been covalently bound, such as antibodies or contrast agents. this type of interaction increases the stability of the drug towards degradation, alters the release kinetics, and improves the therapeutic efficiency. pharmaceutics 2020, 12, x for peer review 7 of 28 • electrostatic interactions. the multivalent structure of the dendrimer enables the formation of multiple bonds in the periphery, which depend on the nature of the end groups. a common example are electrostatic interactions between the drug and a dendrimer bearing cationic (e.g., ammonium groups) or anionic (e.g., carboxylate) moieties. pamam and ppi dendrimers frequently employ this mechanism, due to the multiple ionizable amino groups in the periphery as well as in the interior of their scaffolds. the ph, the ionic strength and the presence of proteins such as albumin have a remarkable impact on dendrimer-cargo electrostatic interactions [52] . this approach is widely employed in gene therapy to generate dendrimer-nucleic acid complexes, or "dendriplexes" [53] . • covalent conjugation. drugs and other molecules can be attached to dendrimers through covalent bonds. sometimes labile or biodegradable bonds are employed, such as amide or ester bonds, to enable the release under chemical or enzymatic scission. other strategy relies on the use of spacers, such as poly(ethylene glycol) (peg), which also generates a hydrophilic surface with a hydrophobic interior, an amphiphilic unimolecular micelle to improve drug encapsulation. furthermore, the attachment of peg reduces the interaction with blood proteins and cells, prolongs the circulation in blood and increases the overall molecular weight, improving the permeability and retention of the drug [54] . other types of ligands have also been covalently bound, such as antibodies or contrast agents. this type of interaction increases the stability of the drug towards degradation, alters the release kinetics, and improves the therapeutic efficiency. these three strategies have also been exploited in the treatment of infectious diseases, as previously reported [55] [56] [57] [58] . the present review, however, focuses on a broader overview to cover the prevention, treatment, and diagnosis of these diseases, as detailed in section 3. the interest in the dendrimer field has continuously increased over time. as recently reported by tomalia (2020) [59] , more than 60,000 articles/patents have been published on dendritic materials, with an approximate increase of 1500 publications and 3000 patents per year since 2013. key commercial successes include the stratus cs acute care diagnostic system (siemens healthcare gmbh, erlangen, germany), for emergency diagnosis of cardiovascular infarctions; vivagel ® products (starpharma, melbourne, australia), for the prevention and treatment of sexually transmitted infections (stis); targeted dep ® and priostar ® (starpharma), for the delivery of anticancer drugs and agrochemical products, respectively; or spherical (polymer factory, stockholm, sweden), as mass spectrometry standards [59] . these three strategies have also been exploited in the treatment of infectious diseases, as previously reported [55] [56] [57] [58] . the present review, however, focuses on a broader overview to cover the prevention, treatment, and diagnosis of these diseases, as detailed in section 3. the interest in the dendrimer field has continuously increased over time. as recently reported by tomalia (2020) [59] , more than 60,000 articles/patents have been published on dendritic materials, with an approximate increase of 1500 publications and 3000 patents per year since 2013. key commercial successes include the stratus cs acute care diagnostic system (siemens healthcare gmbh, erlangen, germany), for emergency diagnosis of cardiovascular infarctions; vivagel ® products (starpharma, melbourne, australia), for the prevention and treatment of sexually transmitted infections (stis); targeted dep ® and priostar ® (starpharma), for the delivery of anticancer drugs and agrochemical products, respectively; or spherical (polymer factory, stockholm, sweden), as mass spectrometry standards [59] . from the low rate of issued patents turning into commercial products, it becomes apparent that dendritic materials must face several challenges for the bench-to-bedside translation in the biomedical field. mignani et al. (2017) summarized the requirements to become a clinical candidate [60] . to secure a successful development, the authors highlight the importance of complying with the good laboratory practice (glp) requirements to ensure the quality, reproducibility and reliability of in vitro and in vivo data. furthermore, the good manufacturing practice (gmp) is desirable, but emerges as one of the main challenges in dendrimer translation. indeed, dendrimer defects have been related to the failure of relevant preclinical trials [61] . an accurate dendrimer synthesis and a thorough purification process are deemed necessary to ensure monodispersity and batch-to-batch reproducibility. this is a highly demanding challenge especially for high-generation dendrimers, multipurpose platforms, and large-scale production. many different strategies are currently explored to overcome these challenges in dendrimer translation, including engineering through critical nanoscale design parameters (cndps) [37] ; accelerated synthetic approaches [27] ; the improvement of analytical techniques, such as mass spectrometry; the accurate and simplified design of multipurpose platforms [62] ; or the dendronization of materials to expand their uses [63] . viruses are simple acellular organisms, which have coevolved with living beings to replicate and reproduce inside their cells, after the binding to specific receptors [64] . nearly 5000 species of viruses have been reported; many of them can cause human diseases [65] . some viral infections, such as respiratory ones, represent an important economic burden and a serious public health concern [66] . antiviral drugs are the most common clinical tool to address these pathologies. to date, 86 different drugs have been approved for the treatment of 17 viral infections [67] . however, some drugs have serious side effects, including nausea, insomnia, vomiting, allergic reactions, behavior disorders, cardiovascular complications, and dependency [68] . opening new therapeutic windows, decreasing the side effects while maintaining their efficacy, is a key action. dendrimers contribute to the fight against viral infections [69] , acting as microbicides per se or as drug nanocarriers, with relevant properties such as low systemic absorption, biocompatibility, water solubility, or simple formulation [70] . the main uses of dendrimers in viral infections are herein addressed, the human immunodeficiency virus (hiv) being one of their most important targets. stis are highly prevalent worldwide, despite the efficient preventive tools (e.g., preservatives) [71] . one of the most illustrative examples is the human immunodeficiency virus (hiv). hiv is responsible for the deterioration of immune cells, especially the target cd4+ t cells [72] , thus aiding the entry of opportunistic pathogens that cause the acquired immunodeficiency syndrome (aids) [73] . hiv transmission mainly occurs through body fluids exchange, mostly by sexual contact, but blood, breastfeeding or vertical transmission have also been described. according to the who, 37.6 million people were infected by hiv in 2015 [74] . fortunately, around 23% decrease in infections was registered from 2010 to 2019, but the treatment, diagnosis, and prevention remain as a global challenge, especially for developing countries with lack of resources. to date, antiretroviral therapy has shown an excellent outcome in the clinical management of aids. however, these drugs produce important side effects, including hiv resistance to the treatment [75] . dendrimers represent an interesting alternative to minimize these side-effects and prevent the transmission of hiv and other viral or bacterial stis, figure 4 [76] . a promising approach relies on the use of dendrimers bearing anionic, sugar, or peptide moieties to prevent the entry of the virus in the target cell. these dendrimers block either the host cell or the viral receptors ( figure 4 , top), such as the glycoproteins gp120 and gp41 located at the hiv envelope, two key proteins for the interaction and fusion of hiv with cd4 t cells. the most representative example is the anionic pll dendrimer spl7013 [77] . spl7013 is a component of two approved and marketed products (starpharma): vivagel ® antiviral condom, for the treatment and prevention of hiv and hsv (herpes simplex virus); and vivagel ® bv for bacterial vaginosis (a second product under phase iii clinical trial nct01577537). furthermore, it also shows significant activity against other viruses, such as the coronavirus sars-cov-2 [78] , enabling a fast-track development of tools to fight covid-19. polyanionic carbosilane dendrimers are also promising microbicides against hiv infection, as shown in different animal models [79] . besides their own antiviral activity, muñoz-fernández et al. showed that their combination with tenofovir and maraviroc (two antiviral agents) produce almost complete inhibition of hiv infection and transmission [80] . carbosilane dendrimers are also efficient towards hiv-hsv coinfection [81] and can be employed in the development of fast diagnostic assays based on dendronized magnetic nanoparticles [82] tools. dendrimers can also contribute to the design of efficient vaccines against hiv, such as the peg-citrate g2 dendrimer bearing multiple hiv epitopes which produced a significant cellular immune response in vivo and a higher th1 response compared to th2 [83] . pharmaceutics 2020, 12, x for peer review 9 of 28 fusion of hiv with cd4 t cells. the most representative example is the anionic pll dendrimer spl7013 [77] . spl7013 is a component of two approved and marketed products (starpharma): vivagel ® antiviral condom, for the treatment and prevention of hiv and hsv (herpes simplex virus); and vivagel ® bv for bacterial vaginosis (a second product under phase iii clinical trial nct01577537). furthermore, it also shows significant activity against other viruses, such as the coronavirus sars-cov-2 [78] , enabling a fast-track development of tools to fight covid-19. polyanionic carbosilane dendrimers are also promising microbicides against hiv infection, as shown in different animal models [79] . besides their own antiviral activity, muñoz-fernández et al. showed that their combination with tenofovir and maraviroc (two antiviral agents) produce almost complete inhibition of hiv infection and transmission [80] . carbosilane dendrimers are also efficient towards hiv-hsv coinfection [81] and can be employed in the development of fast diagnostic assays based on dendronized magnetic nanoparticles [82] tools. dendrimers can also contribute to the design of efficient vaccines against hiv, such as the peg-citrate g2 dendrimer bearing multiple hiv epitopes which produced a significant cellular immune response in vivo and a higher th1 response compared to th2 [83] . besides hiv and stis, dendrimers are effective towards other virus such as the enterovirus a71 (ev71). ev71 belongs to the picornaviridae family, associated with the hands-feet-mouth disease in children, a syndrome characterized by the presence of cutaneous vesicles and ulcerations and frequently with severe neurological manifestations [84] . currently, neither vaccines nor therapies have been approved to prevent or treat ev71 infection, representing an important global problem but specially in the asian southeast [85] . a tryptophan-decorated pentaerythritol dendrimer was especially active towards ev71 in some clinical isolates in the low nm-high pm range [86] . as ev71 is mainly transmitted through fecal-oral route, these dendrimers could be used as a prophylactic method after their oral administration, thus avoiding the transfer of ev71 from the gut to the bloodstream. similar dendrimers have also shown a dual activity towards ev71 and hiv [87] . dendrimers and dendronized materials, such as fullerenes and carbon nanotubes, have also promising activity against other viruses like sars-cov-2 [78] , figure 5a ; ebola virus [88] , figure 5b ; zika and dengue viruses [89] , figure 5c ; hsv [90] ; cytomegalovirus [91] ; some flavivirus, such as the responsible of the japanese encephalitis [92] ; and different human or aviary flu viruses [93] . besides hiv and stis, dendrimers are effective towards other virus such as the enterovirus a71 (ev71). ev71 belongs to the picornaviridae family, associated with the hands-feet-mouth disease in children, a syndrome characterized by the presence of cutaneous vesicles and ulcerations and frequently with severe neurological manifestations [84] . currently, neither vaccines nor therapies have been approved to prevent or treat ev71 infection, representing an important global problem but specially in the asian southeast [85] . a tryptophan-decorated pentaerythritol dendrimer was especially active towards ev71 in some clinical isolates in the low nm-high pm range [86] . as ev71 is mainly transmitted through fecal-oral route, these dendrimers could be used as a prophylactic method after their oral administration, thus avoiding the transfer of ev71 from the gut to the bloodstream. similar dendrimers have also shown a dual activity towards ev71 and hiv [87] . dendrimers and dendronized materials, such as fullerenes and carbon nanotubes, have also promising activity against other viruses like sars-cov-2 [78] , figure 5a ; ebola virus [88] , figure 5b ; zika and dengue viruses [89] , figure 5c ; hsv [90] ; cytomegalovirus [91] ; some flavivirus, such as the responsible of the japanese encephalitis [92] ; and different human or aviary flu viruses [93] . bacteria are unicellular prokaryote organisms with great implications in human health, as they compose the core of microbiota [94] , but also in human disease. certain bacterial populations can colonize and infect different tissues, leading to the development of a wide range of pathologies [95] . antibiotics have long been one of the most effective solutions to fight against bacterial infections. however, their improper use drove a global public health problem: bacterial resistance. only in europe, a total of 672,000 multiresistant bacterial infections were estimated, being responsible for up to 33,000 deaths in 2015 [96] . dendrimers emerge as a potential solution, as they employ an unspecific mechanism that prevents the development of resistances (figure 4 bottom) . some representative examples against resistant bacteria are herein collected. biofilms are one of the most important adaptive mechanisms of bacteria, enabling them to survive in an adverse environment. it is activated under different stress conditions, like limited oxygen or iron levels or the presence of some antimicrobial agents in sublethal concentrations [97] . p. aeruginosa represents one of the most important biofilm-forming bacteria, with outstanding impact in some chronic diseases like cancer [98] or cystic fibrosis [99] . this is the main problem associated with p. aeruginosa infection, especially in non-immunocompetent patients, hindering the clinical management. p. aeruginosa represents a clear example of how dendrimers can address resistant bacteria infection, including the inhibition of biofilm formation. it has been described that a bacterial specific lectine (lecb) plays a key role in biofilm formation by promoting the adhesion to cells [100] . lectines are proteins which show a high specificity for sugars and bacteria are unicellular prokaryote organisms with great implications in human health, as they compose the core of microbiota [94] , but also in human disease. certain bacterial populations can colonize and infect different tissues, leading to the development of a wide range of pathologies [95] . antibiotics have long been one of the most effective solutions to fight against bacterial infections. however, their improper use drove a global public health problem: bacterial resistance. only in europe, a total of 672,000 multiresistant bacterial infections were estimated, being responsible for up to 33,000 deaths in 2015 [96] . dendrimers emerge as a potential solution, as they employ an unspecific mechanism that prevents the development of resistances (figure 4 bottom) . some representative examples against resistant bacteria are herein collected. biofilms are one of the most important adaptive mechanisms of bacteria, enabling them to survive in an adverse environment. it is activated under different stress conditions, like limited oxygen or iron levels or the presence of some antimicrobial agents in sublethal concentrations [97] . p. aeruginosa represents one of the most important biofilm-forming bacteria, with outstanding impact in some chronic diseases like cancer [98] or cystic fibrosis [99] . this is the main problem associated with p. aeruginosa infection, especially in non-immunocompetent patients, hindering the clinical management. p. aeruginosa represents a clear example of how dendrimers can address resistant bacteria infection, including the inhibition of biofilm formation. it has been described that a bacterial specific lectine (lecb) plays a key role in biofilm formation by promoting the adhesion to cells [100] . lectines are proteins which show a high specificity for sugars and their derivatives, being able to successfully recognize and agglutinate cells with glycosylated proteins or lipids. lecb binds to fucose, a mucin located in the epithelial mucosa, playing a key role in p. aeruginosa biofilm formation, in conjunction with leca, specific to galactose, although this binding is weaker and less important [100] . in this context, some peptidic dendrimers have been developed to directly inhibit lecb-fucose interactions at low concentrations, such as fd2 (ic 50 = 0.14 µm) depicted in figure 6a . dendrimers decorated with fucose-derived groups prevent the formation of p. aeruginosa biofilms (ic 50~1 0 µm) and even disperse formed structures, by inhibiting the agglutination of the pathogen and acting as antimicrobial nanocarriers, thus increasing the efficacy of the established treatments [100, 101] . pharmaceutics 2020, 12, x for peer review 11 of 28 less important [100] . in this context, some peptidic dendrimers have been developed to directly inhibit lecb-fucose interactions at low concentrations, such as fd2 (ic50 = 0.14 µm) depicted in figure 6a . dendrimers decorated with fucose-derived groups prevent the formation of p. aeruginosa biofilms (ic50~10 µm) and even disperse formed structures, by inhibiting the agglutination of the pathogen and acting as antimicrobial nanocarriers, thus increasing the efficacy of the established treatments [100, 101] . one of the most representative examples of resistant bacteria is gram-negative bacteria. these microorganisms present a peptidoglycan wall located between the inner and the outer membranes, which is responsible for the higher resistance of gram-negative bacteria to immune system, and in case of lysis, promotes the release of proinflammatory substances known as lipopolysaccharides (lps), exacerbating the infection [106] . this structure also confers gram-negative bacteria resistance to some external agents through multiple mechanisms, which are heterogeneous between species [107] . examples of common mechanisms of bacterial resistance are the expelling of toxic residues that eliminate antibacterial agents; a decrease in bacterial permeability, through the alteration of the membrane channels; or the production of antibiotic inactivating enzymes [108] . cationic dendrimers may be a solution to fight against resistant bacteria, as they can bind efficiently to the negatively-charged walls, destabilize it by displacing sodium and calcium ions and increase the membrane permeability [109, 110] , figure 4 bottom. however, despite their promising biocide activity, cationic dendrimers exhibit high toxicity towards mammal cells and require structural modifications to reduce their cytotoxicity, without affecting the efficacy [111] . cationic antimicrobial peptides (amp) arranged as multiple antigen peptide (map) dendritic structures can also exert a potent antibacterial activity, decreasing the minimum inhibitory concentration (mic) and minimum bactericide concentration (mbc) of the peptide alone, while dramatically increasing the peptide stability to proteolysis [112] . for example, a dendritic map structure based on the peptide qkkirvrlsa effectively inhibited diverse gram-negative bacteria (mic = 4-8 g/ml for e. coli atcc 25922, p. aeruginosa atcc 27853, and a clinical isolate of k. pneumoniae) [112] . the higher local concentration of amp allows a multivalent binding and enhances the destabilizing effect of the bacterial membrane. dendrimers may also be used in the rapid diagnosis to discern between gram-negative or gram-positive bacteria, through a ph-dependent bacteria-selective aggregation occurring within 5 min of adding the dendrimer to a bacterial suspension [113] ; and as carriers of different drugs such as vancomycin or agents like led209, both specific to gram-negative microorganisms [114, 115] . chorioamnionitis is an infection in the amniotic liquid, which may cause neurological problems in the fetus due to the production of proinflammatory cytokines, escherichia coli being one of the most important etiological agents [116] . this disease often occurs due to the ascent of microorganisms from vagina to uterus, although other pathways have also been reported like transplacental infection, retrograde seeding from the peritoneal cavity through the fallopian tubes or accidental invasive procedures [117] . the use of antibiotics such as penicillins, cephalosporins, macrolides, and corticosteroids reduce the risk of developing chorioamnionitis [118] . in this sense, dendrimers can increase their efficacy. for example, a study conducted by wang et al. (2010) in a guinean pig model demonstrated that hydroxyl-and amino-functional pamam dendrimers successfully encapsulate ampicillin [119] . both dendrimers significantly decreased the uterus cytokines, compared to the usual therapy, but the amino-dendrimer exhibited a higher toxicity. dendrimers have been studied against other bacterial infections like chlamydia trachomatis, increasing the efficacy of vaccines by conjugating a peptide mimic of a chlamydial glycolipid antigen to a g4-pamam-oh dendrimer [120] ; some opportunistic agents, such as s. aureus, using different generation gn-pamam-nh 2 dendrimers [121] ; or genital infections, through a sustained and localized delivery of amoxicillin in the cervicovaginal region by pamam-peg dendritic hydrogels [122] . overall, these studies show the potential role of dendrimers in bacterial infections, although a long road is still to cover, especially to decrease their cytotoxicity and increase their specificity. fungi are eukaryotic organisms responsible of a wide range of human infections. the prevalence of these diseases has increased in some countries, particularly in hospital areas and immunocompromised patients, candida, cryptococcus, pneumocystis, and aspergillus being the most representative families [123] . data collected by the national nosocomial infections surveillance system (nnis) from january 1990 to april 1996 showed that up to 9% of nosocomial infections in eeuu were due to fungi, mainly candida species [124] . however, current trends indicate the prevalence of aspergillus in different european states [125] . the treatment of fungal infections is performed through antifungal (antimycotic) drugs, which produce some alterations in their cellular structures, thus inhibiting their development, viability and survival in a direct or indirect way. the most representative antifungal drugs include [126] : polyenes (e.g., amphotericin b); azoles (e.g., imidazole, triazole); allylamines; lipopeptides; and miscellaneous agents, as griseofulvin, which inhibits microtubules and mitotic fuse, affecting cell division. unfortunately, the resistance developed by some fungi may hinder the clinical management of these infections [127] . in addition, the great similarity between mammal and fungal cells can lead to cytotoxicity problems, being necessary to find molecules which selectively target fungal cells in a particular tissue [126] . in this context, the use of nanoparticles like dendrimers may be an effective method to carry all these substances, maintaining their benefits and reducing their side effects. candida albicans, which is responsible of more than half of total fungal infections around the world [128] , is often treated with ketoconazole, a dual-action drug capable of inhibiting both ergosterol synthesis and the transformation of spores to micellar infectious forms [129] . however, ketoconazole is poorly water-soluble and can greatly benefit from the use of nanocarriers, which increase its bioavailability in the bloodstream. gn-pamam-nh 2 dendrimers improve the administration of ketoconazole (up to 16-fold increase of antifungal activity using g2 dendrimer, compared to the drug alone), being even more efficient when used as hydrogel formulation [129] ; as well as clotrimazole (up to 32-fold increase with g2 dendrimer) due to its hydrophobic and electrostatic interactions [130] . similarly, these dendrimers have significantly improved the antifungal activity of amphotericin b, overcoming the low water solubility and nephrotoxicity issues [131] . on the other hand, peptide dendrons have shown efficient anti-candida activity per se [102] . the representative example shown in figure 6b , which displays four tryptophan residues in the periphery and a dodecyl chain in the focal point, produced 100% growth inhibition at 16 µg/ml, as well as affected the biofilm viability and the hyphal and cell wall morphology. on the other hand, dendrimer-assisted gene therapy can prevent fungal infections. cationic pamam dendrimers, bearing -nh 2 , -nme 2 , and -nme 3 + peripheral groups, were used with a ribozyme extracted from an intronic region of c. albicans, an rna molecule capable to cut other rna chain or even itself [132] . these dendrimers inhibited the catalytic activity of candida ribozymes, with a generation-dependent activity (g4 > g3 > g2). however, the nature of the peripheral group did not produce a significantly different inhibition. consequently, the construction of the dendrimer depended on the size of the rna to inhibit and the charge ratio between dendrimer and rna [133] . the use of rna:dendrimer complexes may have multiple applications, such as inhibiting protein synthesis, splicing or even rna delivery in an era where non coding rna are beginning to be used, thus showing the potential of dendrimers in targeted therapy [134] . unfortunately, the diagnosis of fungal diseases is currently a challenge. the current golden standard for the detection of these infections relies on poorly sensitive and invasive methods such as cell culture and histopathological study of the infected tissue [135] . new diagnostic tools are demanded, such as polymerase chain reaction (pcr), immunoassays, or tests capable of detecting specific fungal antigens such as beta-glycans [136] . in this context, the use of nanostructures can play a key role in the development of new techniques that are more sensitive and effective in the early diagnosis of fungal infections. at present, very few studies report the use of dendrimers in the diagnosis of fungal diseases. takano et al. (2003) performed cdna microarray analysis using highly sensitive dendrimer-based technology in the detection of the rice pathogen magnaporthe grisea and the stage of infection in this pathogen [137] . another potential approach is the dendrichips ® technology (dendris), relying on a phosphorous dendrimers coating which dramatically increases their sensitivity. this tool is capable of discerning up to 11 respiratory bacterial pathogens from a single sample [138] , and could be refined and targeted to other types of infectious agents such as fungi. parasites are organisms characterized by the need of other living organism or "host" in order to survive. parasites comprise an important variety of species of diverse complexity, from the simplest organisms such as protozoans to the more complex ones, such as plants [139] . helminths and protozoans entail the main threat of human parasitosis; the clinical expression and its severity depend on the condition of the immune system of the host, as part of a tight interrelation [106, 140] . prevention could be the most efficient mechanism of controlling parasitic infections but, despite the considerable efforts, there are no effective vaccines against any of the main parasites. accordingly, antiparasitic drugs are the pillar in protozoan control, when the simple prevention measures fail. however, the drug resistance of protozoans is becoming an alarming public health problem [141] . dendrimers could be an effective tool in the early diagnosis or prevention of parasitosis, as well as a new treatment for some of these infections. protozoans comprise a diverse group of eukaryotic unicellular microorganisms that belong to protista kingdom. most common human infections caused by protozoans are related to plasmodium spp. and toxoplasma gondii, as well as trypanosoma and leishmania spp. protozoan parasites are responsible for a considerable mortality and morbidity all over the world, that affect more than 500 million people [142] . malaria is par excellence the main parasitic infection and it is caused by intracellular plasmodium parasites transmitted by mosquitoes of genus anopheles. approximately, 40% of world population lives in areas where malaria is transmitted, causing 300-500 million infections and 2.7 million deaths per year. in specific regions, such as sub-saharan africa, children below 5 years-old conform 90% of the total deaths from malaria [143] . an important epidemiological study of the prevalence of malaria in salomon islands revealed the high rate of asymptomatic patients, highlighting the need for a diagnostic tool with high sensibility and specificity to detect plasmodium [144] . this study relied on pcr and rapid diagnostic tests (rdt), which are more sensible than a simple inspection with a microscope, but also far more expensive. in order to find a sensible detection method with a lower cost, a dendrimer-based assay was approved in south korea [145] . the coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (flisa) could detect two specific antigens of malaria: histidine-rich protein ii (hrp2) and lactate dehydrogenase (ldh). flisa has good spectroscopic properties, such as photostability [146] , and a better performance than traditional elisa, enabling the quantification of the number of parasites in a sample even if they are present in low concentrations. accordingly, flisa method could be useful to detect asymptomatic cases at a modest price and with a high capacity. the process is depicted schematically in figure 7 . the role of dendrimers against helminthic schistosoma parasites has also been tested. the disease, known as schistosomiasis [147] , begins when the larvae form penetrates in the organism through the skin and settle in mesenteric and pelvic veins of the host, turning into the adult form. here, the female parasites lay eggs, which could be eliminated through feces or urine or lead to complications like granulomas or intestinal, hepatosplenic and urogenital damage. this highlights the need for an early diagnosis. wright et al. (2019) confirmed the promising activity of magnetic particles coated with g4-pamam-nh 2 to concentrate the schistosoma circulating anodic antigen (caa), resulting in a 200-fold improvement in caa limits of detection for lateral flow assays [148] . preventive measures like vaccines are key to stop the impact of schistosoma parasites in specific regions where they cause endemic infections. for example, infections by s. haematobium, s. japonicum, or s. mansoni affect over 200 million people worldwide [149] . lysine-decorated pamam dendrimers showed excellent behavior as vaccine vector, enhancing the immunoreactivity and efficacy of dna vaccine against s. japonicum infection [150] . along the same lines, anderson et al. (2016) developed a dendrimer-based vaccine platform which encapsulate antigen-expressing replicon mrnas and generate a protective response towards others parasites such as toxoplasma gondii, and relevant viruses like ebola and h1n1 influenza, with a single dose [151] . the vaccine nanoparticle comprised an ionizable g1-pamam dendrimer, a lipid-anchored peg and rna. these studies show the role of dendrimers in the development of new generation vaccines against different infections. specificity to detect plasmodium [144] . this study relied on pcr and rapid diagnostic tests (rdt), which are more sensible than a simple inspection with a microscope, but also far more expensive. in order to find a sensible detection method with a lower cost, a dendrimer-based assay was approved in south korea [145] . the coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (flisa) could detect two specific antigens of malaria: histidine-rich protein ii (hrp2) and lactate dehydrogenase (ldh). flisa has good spectroscopic properties, such as photostability [146] , and a better performance than traditional elisa, enabling the quantification of the number of parasites in a sample even if they are present in low concentrations. accordingly, flisa method could be useful to detect asymptomatic cases at a modest price and with a high capacity. the process is depicted schematically in figure 7 . leishmaniasis is a parasitic disease produced by leishmania protozoans that infects and multiplies in macrophage-rich organs and tissues of the reticuloendothelial system, mainly the liver and spleen. estimations indicate an increase of 1.5 to 2 million cases per year [152] . for decades, leishmaniasis has been treated with pentostam and glucantime, leading to drug resistance and serious side effects like pancreatitis. alternative broad-spectrum drugs with less toxicity, such as amphotericin b, have been used but they present disadvantages such as the high cost, low solubility, the side effects, and its less efficacy as antiparasitic agent. mannose-decorated g5-ppi dendrimers improved the activity of amphotericin b for the treatment of leishmaniasis, reducing the toxicity by increasing the targeting in macrophage-rich organs [153] . other nanocarrier, a dendritic-linear-dendritic hybrid based on peg and citric acid, figure 6c , also improved the solubility of amphotericin b (478 times) and reduced the in vitro/in vivo toxicity. it resulted as potent as free amphotericin and glucantime in reducing the parasite burden and number [103] . toxoplasmosis is a zoonosis caused by the ingestion of the parasite toxoplasma gondii. this disease has a chronic and silent course in the majority of population without immune system disorders, mainly causing symptoms such as low fever and muscular pain. this infection is usually treated with sulfadiazine [154] , which has two disadvantages: it requires a high dose of the drug, which can produce severe side effects like high fever or allergic reactions; and it cannot reach target tissues where the parasite is typically localized. cationic g4 pamam dendrimers and anionic g4.5 dendrimers efficiently solubilize sulfadiazine (up to 30 molecules per dendrimer) and improve the penetration into the parasite, thus greatly reducing the required sulfadiazine dose and localizing the drug in muscle and brain, where t. gondii is usually present [155] . the main dendrimer-drug interactions found were electrostatic, for cationic dendrimers, and hydrogen bonding, for anionic counterparts. furthermore, the anionic dendrimer showed intrinsic antiparasitic effect. other successful examples of antiparasitic dendrimers include pegylated pll dendrimers coated with chondroitin sulfate a, as targeted unimolecular micelles for the delivery of the antimalarial drug chloroquine phosphate [156] ; and pamam dendrimers decorated with ethynil estradiol against trypanosoma cruzi, the parasite responsible for chagas disease, where the g2 dendrimer is 8 times more effective than benznidazole at 24 h and 48 h (ic 50 = 1.25 µm) [157] . amoebae are eukaryotic protozoa extensively distributed in nature and human habitats, often acting as a host and reservoir of other microorganisms like giant viruses and some class of bacteria [158, 159] . amoebae infecting humans are classified as parasitic, such as entamoeba organisms, or opportunistic free-living amoebae, such as acanthamoeba spp., balamuthia mandrillaris, sappinia diploidea, and naegleria fowleri (known as the brain-eating amoebae) [160] . free-living amoebae do not require human infection for their life cycle, which presents two stages: trophozoite (active form) and cyst (inactive form). acanthamoeba and other free-living amoebae are responsible of serious diseases such as keratitis, encephalitis, and infections in immunocompromised patients in the central nervous system, skin, and lungs. fortunately, amoebae infections are relatively rare, although the high mortality associated with meningoencephalitis are of concern, mainly due to the late diagnosis and the lack of effective antimicrobial treatments [161] . low generation cationic carbosilane dendrimers bearing ammonium or biguanide moieties have shown strong amoebicidal activity against trophozoites and cysts of acanthamoeba spp., figure 6d (ic 50 = 2.4 mg/l; minimum cysticidal concentration mcc = 256 mg/l) [104, 162] . furthermore, they exhibit a synergistic effect with traditional drugs such as chlorhexidine, decreasing the required drug concentration 5-10 times using g1 dendrimers [163] . the dendrimers target the amoeba membrane and produce inhibition and cell death. in this context, dendrimers could be a promising therapeutic alternative to properly manage amoebae infections. prions (prp) are infectious pathogens that cause neurodegenerative transmissible diseases such as spongiform encephalopathies, after a long period of incubation from 2 to 10 years [164, 165] . prp sc are glycoproteins with an abnormal folding that are originated from a conformational change of normal prion proteins (prp c ), acquiring pathological properties [166] . prion diseases can occur for two reasons: the infectious prion agent can transmit the pathological folding to the prp c ; or the prnp gen mutates, leading to a genetic variation of the prion disease [167] . infectious prion diseases have gained importance since the epidemic bovine spongiform encephalopathy in united kingdom in 1986 that was transmitted to humans as a variation of the creutzfeldt-jakob prion disease [168, 169] . the spongiform encephalopathies are named after the shape the brain acquires at final stages of the disease, caused by an increase of vacuoles and the holes that appear in the tissue. it is a rare but severe pathology, which leads to neuronal loss and, eventually, dementia. nowadays, there are some pharmacological interventions that delay the progression of prion diseases; however, there is no effective treatment to stop or avoid it in a significant way [170] . in this field, nanomaterials such as dendrimers can exert a relevant activity, preventing the conversion of prp c to prp sc [171] . due to the high affinity of amine groups to prions, phosphorus dendrimers decorated with tertiary amines in their surface are promising agents in the therapy against prion diseases, figure 6e [105]. these dendrimers showed an inhibitory effect in the generation of prions (ic 50 = 10 (g3), 1.5 (g4) and 5 (g5) µg/ml) and they had anti-infectious action in vitro and in vivo for some spongiform encephalopathies, some of them causing creutzfeldt-jakob disease [105] . other dendritic families with even higher anti-prion activity include g4-pamam-nh 2 and g4-ppi-nh 2 (both ic 50 = 80 ng/ml) [172, 173] . for example, ottaviani et al. (2012) showed that ppi glycodendrimers, comprising maltose or maltotriose units, prevented the aggregation of prp and aβ(1-28) proteins due to the interference of dendrimers with the latency phase (nucleation) of the prion protein [174] . dendrimers can also be useful for the diagnosis of prion infections. usually, elisa is used to determine whether the causing agent of the disease is a prion, but this method has important limitations as it cannot distinguish between prion chains [175] . the nature of the prion agent will determine the etiology and course of the disease, changing the pathology parameters like the incubation period or the type of lesion [176] . importantly, different generation pamam and ppi dendrimers were capable of distinguishing between the different types of prions [177] . this study demonstrated that the susceptibility of a prion towards a particular dendrimer could be used to diagnose and predict the course of a disease. on the other hand, korri-youssoufi et al. developed an electrochemical biosensor comprising multiwalled carbon nanotubes modified with dendrimers and aptamers to detect prion proteins with a high sensibility (min. 0.5 pm) [178] . in conclusion, dendrimers constitute a promising research line for fighting against prion diseases as they could slow down their progression and allow a reliable diagnosis of the responsible etiological agent. dendritic nanosystems could have a principal role as excipients in the pharmaceutical industry, providing that they are compatible, safe and effective nanocarriers in several administration routes [179] . for example, ocular administration is still a challenge due to the unique physiology of the eye and the presence of numerous barriers. in this context, recent studies showed that dendrimers improved the time that a drug remains in the cornea after topical administration and they supply directed and sustained neuroprotection for retina [180] . moreover, dendrimers could act as dna vectors, providing safety and reducing cytotoxicity compared to the viral vectors and intraocular injections used at present [53] . dendritic polymers can easily improve the properties of different materials, such as cotton, to generate antimicrobial activity. for example, the addition of amine-functional dendrimers confers antibacterial activity against gram-positive bacteria (e. coli, p. aeruginosa) and gram-negative bacteria (s. aureus) and fungi (c. albicans) [181, 182] , even after several washing cycles. sepsis is a life-threatening response to an infection, which happens when the immune system overreacts and starts to damage the patient's own tissues and organs. peptide-decorated pamam dendrimers inhibited the acetylation of transforming growth factor β-induced protein, thus improving the mortality and organ damage in the septic mouse model [183] . additionally, gadolinium-containing g4 dendrimers can be used as mri diagnostic and prognostic biomarker of sepsis-induced acute renal failure [184] . these studies open up a field in the treatment and diagnosis of sepsis with a not very high cost and that could be protective against infectious pathogens in certain circumstances like during hospital stays, surgeries or risky exposures. the design of metallodendrimers is a promising field to explore, which can open new avenues in the fight against infectious diseases [185] . dendrimers are excellent platforms to control the attachment of a great variety of metal complexes with antimicrobial activity, such as silver(i), copper(ii), and zinc(ii), among others. the resultant metallodendrimers often exhibit a synergistic activity and improve the therapeutic response of the dendrimer or the metal complex alone. for example, demonstrated the impact of a single metal ion in the prevention of hiv infection [186] . a carboxylate-decorated g2 ppi dendrimer, which inhibited hiv-1 infection of hec-1a cells around 30%, reached 90% inhibition by attaching a single cu(ii) ion through its ethylenediamine core. the remarkable control on the dendritic structure enables an optimized antimicrobial activity, difficult to accomplish with other nanomaterials. the reader is referred to a recent review on this topic [185] . dendrimers are an innovative tool in the treatment, prevention and diagnosis of serious infectious pathologies, as summarized in table 1 . they emerge as a unique opportunity to overcome problems of traditional approaches, such as microbial resistance. nevertheless, the transition from the laboratory to the clinical practice still requires facing several challenges, such as the big scale production, the batch-to-batch consistency, the purity for clinical tests, or the regulatory obstacles. it should be kept in mind that most investigations have been conducted in experimental models in vitro or in animal models, so it is not possible to extrapolate these results to humans yet. however, the outstanding results obtained by vivagel ® in the prevention of viral and bacterial infections predict a brilliant future of dendritic materials in the fight against infectious diseases. 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replication of sars-cov-2 in vitro development of sulphated and naphthylsulphonated carbosilane dendrimers as topical microbicides to prevent hiv-1 sexual transmission triple combination of carbosilane dendrimers, tenofovir and maraviroc as potential microbicide to prevent hiv-1 sexual transmission carbosilane dendrons with fatty acids at the core as a new potential microbicide against hsv-2/hiv-1 co-infection dendronized magnetic nanoparticles for hiv-1 capture and rapid diagnostic conjugated anionic peg-citrate g2 dendrimer with multi-epitopic hiv-1 vaccine candidate enhance the cellular immune responses in mice immunohistology of infectious diseases antiviral drug discovery for the treatment of enterovirus 71 infections optimization of a class of tryptophan dendrimers that inhibit hiv replication leads to a selective, specific, and low-nanomolar inhibitor of clinical isolates of enterovirus a71 structure-activity relationship studies on a trp dendrimer with dual activities against hiv and enterovirus a71. modifications on the amino acid nanocarbon-based glycoconjugates as multivalent inhibitors of ebola virus infection synthesis of highly efficient multivalent disaccharide/[60]fullerene nanoballs for emergent viruses dendrimers functionalized with membrane-interacting peptides for viral inhibition peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate antiviral and neuroprotective role of octaguanidinium dendrimer-conjugated morpholino oligomers in japanese encephalitis antiviral potential of 3 -sialyllactose-and 6 -sialyllactose-conjugated dendritic polymers against human and avian influenza viruses the healthy human microbiome bacterial-host interactions: physiology and pathophysiology of respiratory infection attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the eu and the european economic area in 2015: a population-level 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enhanced antibacterial effect of vancomycin hydrochloride against gram-negative bacteria development of a guinea pig model of chorioamnionitis and fetal brain injury micronutrients and intrauterine infection, preterm birth and the fetal inflammatory response syndrome mid-trimester preterm premature rupture of membranes (pprom): etiology, diagnosis, classification, international recommendations of treatment options and outcome inhibition of bacterial growth and intramniotic infection in a guinea pig model of chorioamnionitis using pamam dendrimers dendrimer-conjugated peptide vaccine enhances clearance of chlamydia trachomatis genital infection antimicrobial efficacy and mechanism of action of poly(amidoamine) (pamam) dendrimers against opportunistic pathogens injectable pamam dendrimer-peg hydrogels for the treatment of genital infections: formulation and in vitro and in vivo evaluation general epidemiology of invasive fungal disease international surveillance of bloodstream infections 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malaria diagnostics in an elimination setting performance of coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (flisa) to detect malaria antigen development of a novel fluorophore for real-time biomonitoring system poly(amidoamine)-coated magnetic particles for enhanced detection of schistosoma circulating anodic antigen in endemic urine samples schistosomiasis: drugs used and treatment strategies pamam-lys, a novel vaccine delivery vector, enhances the protective effects of the sjc23 dna vaccine against schistosoma japonicum infection dendrimer-rna nanoparticles generate protective immunity against lethal ebola, h1n1 influenza, and toxoplasma gondii challenges with a single dose leishmaniasis impact and treatment access surface-engineered dendrimeric nanoconjugates for macrophage-targeted delivery of amphotericin b: formulation development and in vitro and in vivo evaluation antibiotics for human toxoplasmosis: a systematic review of randomized trials nanomolar cationic dendrimeric sulfadiazine as potential antitoxoplasmic agent pegylated peptide dendrimeric carriers for the delivery of antimalarial drug chloroquine phosphate pérez-campos, e. in vitro activity of steroidal dendrimers on trypanosoma cruzi epimastigote form with pamam dendrons modified by "click"chemistry genetic and physiological interactions in the amoeba-bacteria symbiosis giant virus vs amoeba: fight for supremacy human infections caused by free-living amoebae pathogenic and opportunistic free-living amoebae: acanthamoeba spp., balamuthia mandrillaris, naegleria fowleri, and sappinia diploidea in vitro evaluation of the effectiveness of new water-stable cationic carbosilane dendrimers against acanthamoeba castellanii uah-t17c3 trophozoites in vitro anti-acanthamoeba synergistic effect of chlorhexidine and cationic carbosilane dendrimers against both trophozoite and cyst forms transmissible spongiform encephalopathies and prion protein interconversions the prion concept and synthetic prions prion disease a new variant of creutzfeldt-jakob disease in the uk prion disease genetics toward therapy of human prion diseases nanomedicine for prion disease treatment: new insights into the role of dendrimers elimination of prions by branched polyamines and implications for therapeutics influence of surface functionality of poly(propylene imine) dendrimers on protease resistance and propagation of the scrapie prion protein kinetics of amyloid and prion fibril formation in the absence and presence of dense shell sugar-decorated dendrimers rapid typing of transmissible spongiform encephalopathy strains with differential elisa adaptation of the bovine spongiform encephalopathy agent to primates and comparison with creutzfeldt-jakob disease: implications for human health differentiating prion strains using dendrimers electrochemical aptasensor of human cellular prion based on multiwalled carbon nanotubes modified with dendrimers: a platform for connecting redox markers and aptamers dendrimers as pharmaceutical excipients: synthesis, properties, toxicity and biomedical applications dendrimeric systems and their applications in ocular drug delivery the antimicrobial activity of the cotton fabric grafted with an amino-terminated hyperbranched polymer preparation, characterization, and antimicrobial property of cotton cellulose fabric grafted with poly (propylene imine) dendrimer. cellulose dual peptide-dendrimer conjugate inhibits acetylation of transforming growth factor β-induced protein and improves survival in sepsis dendrimer-enhanced mri as a diagnostic and prognostic biomarker of sepsis-induced acute renal failure in aged mice metallodendrimers as a promising tool in the biomedical field: an overview hiv-1 antiviral behavior of anionic ppi metallo-dendrimers with eda core the authors declare no conflict of interest. key: cord-019009-3ngfv96u authors: gea-banacloche, juan title: risks and epidemiology of infections after hematopoietic stem cell transplantation date: 2016-02-15 journal: transplant infections doi: 10.1007/978-3-319-28797-3_6 sha: doc_id: 19009 cord_uid: 3ngfv96u infections following hct are frequently related to risk factors caused by the procedure itself. neutropenia and mucositis predispose to bacterial infections. prolonged neutropenia increases the likelihood of invasive fungal infection. gvhd and its treatment create the most important easily identifiable risk period for a variety of infectious complications, particularly mold infections. profound, prolonged t cell immunodeficiency, present after t cell-depleted or cord blood transplants, is the main risk factor for viral problems like disseminated adenovirus disease or ebv-related posttransplant lymphoproliferative disorder. understanding the epidemiology of infections after allogeneic hematopoietic stem cell transplantation (hct) is important to implement appropriate preventive strategies as well as to effectively diagnose and treat individual patients. several groups of experts and professional organizations publish guidelines that provide specifi c recommendations for prophylaxis and management of infections after hct [ 1 -8 ] , including vaccinations [ 1 , 9 , 10 ] . many of these recommendations are necessarily based on low-quality evidence and rely heavily on expert opinion. guidelines should not be followed blindly, but understood as tools that may help to provide the best possible care. risk factors for infection include individual characteristics (e.g., indication for hct, prior infections, cmv serostatus, particular genetic traits) and type of transplant (based on conditioning regimen, stem cell source, degree of hla homology, and immunosuppression). the development of graft-versus-host disease (gvhd) is frequently the decisive contributor to infectious morbidity and mortality. different indications for hct are associated with their own infectious risks. primary immunodefi ciencies (pid), hemoglobinopathies, and hematologic malignancies present different challenges. even in hematologic malignancies, the risk may vary depending on the specifi c condition: patients with chronic myelogenous leukemia (cml), acute myeloid leukemia (aml), and chronic lymphocytic leukemia (cll) present different risks based on both the biology of the disease and prior treatment. these factors should be considered when assessing individual patients. prior infections must be considered. a history of infection or colonization with a multidrug-resistant organism (mdro) like carbapenem-resistant enterobacteria (cre), extended-spectrum beta-lactamase (esbl)-producing gram-negative bacteria, vancomycin-resistant enterococcus (vre), or methicillin-resistant staphylococcus aureus (mrsa) has implications regarding optimal management of fever during neutropenia [ 6 , 11 , 12 ] , which is a common complication of hct. transplant candidates are routinely screened for serologic evidence of latent infections that may reactivate (hsv, vzv, cmv, ebv, hepatitis b and c, toxoplasmosis); some of these will be discussed later in this chapter. some transplant centers will perform screening for tuberculosis with tuberculin skin test (tst) or interferon-gamma release assay (igra), at least for patients who are considered at signifi cant risk for the disease. prior invasive fungal infections may reactivate following transplant, and secondary prophylaxis is required [ 13 -15 ] . even active fungal infection has been reported to be controllable. there are, however, cases of progression of prior aspergillosis after transplant; myeloablative conditioning, prolonged neutropenia, cytomegalovirus (cmv) disease, and graft-versus-host disease (gvhd) are risk factors [ 15 , 16 ] . as the correlates of native and adaptive immunity are better understood, genetic associations are coming to light. there is evidence that some donor haplotypes of tlr4 , the gene that encodes the toll-like receptor protein 4 (tlr4) are associated with increased risk of invasive aspergillosis after hct [ 17 ] . recipient's mutations in mbl2 , the gene that encodes mannose-binding lectin (mbl), have been associated with increased risk of infection after neutrophil recovery following myeloablative transplant [ 18 ] . other polymorphisms of mbl2 may be important for infection through a direct infl uence on the risk of developing gvhd [ 19 , 20 ] . different genotypes of activated killer immunoglobulin-like receptors (akir) in the donor have been found to protect from cmv reactivation [ 21 ] . many of these associations are preliminary and require more data to be confi rmed, but they hold the promise of a more individualized approach to infectious prophylaxis. from a practical standpoint, it is helpful to consider three distinct periods during transplant: pre-engraftment (until neutrophil recovery), early post-engraftment (from engraftment until day 100) , and late post-engraftment (after day 100) . this framework originated with myeloablative transplants, and is eminently pragmatic. the pre-engraftment phase may be accompanied by profound neutropenia and signifi cant mucositis, which results in increased risk of bacterial infections from the resident gastrointestinal fl ora, candidiasis, aspergillosis (in cases of prolonged neutropenia) and herpes simplex virus reactivation. after engraftment, with neutropenia no longer being a factor, many infections are related to the profound defect in cellular immunity caused by the conditioning regimen and the immunosuppression administered to prevent gvhd. cmv reactivation and the development of acute gvhd and its treatment play a central role during this time. the day 100 landmark derives from the standard time at which immunosuppression (e.g., cyclosporine a or tacrolimus) is frequently tapered. infections after this point would be primarily related to lack of immune reconstitution and, in the absence of gvhd, become progressively less common. not all allogeneic stem cell transplantations are the same. several characteristics of the transplant infl uence the risk of infection: the conditioning preparative regimen, the source of stem cells, the degree of hla identity between donor and recipient, and the prophylactic strategy adopted to prevent gvhd (use of t cell depletion or immunosuppressive medications). table 6 -1 summarizes the impact of these factors on infections. matching for ucb transplants focuses on three loci (hla-a, hla-b, and hla-drb1). the majority of ucb transplants are mismatched by at least one locus (often two). among transplants mismatched at two loci, mismatching at hla-c and hla-drb1 was associated with the highest risk of mortality [ 24 ] . the degree of mismatch between the donor and the recipient affects the infectious risk mainly through the likelihood of gvhd. more gvhd usually results in more infections. to prevent gvhd in a mismatched transplant, more potent immunosuppression may be required, increasing the risk of infection. it is also possible that immune reconstitution proceeds more slowly (even with the same immunosuppressive regimen) after a urd hct. these factors may result in increased risk of infections associated with t cell immunodefi ciency, like cmv, pneumocystis jirovecii pneumonia (pcp), and epstein-barr virus (ebv)-related posttransplant lymphoproliferative disorder (ptld). however, provided the number of stem cells administered is the usual (>3 × 10 6 kg −1 ), neutrophil recovery proceeds at the standard pace and there is no increased risk of neutropeniarelated infections. the problems with ucb transplants include a markedly decreased stem cell dose (often <1 × 10 5 kg −1 ) which results in prolonged neutropenia (up to 6 weeks), with the attendant risk of bacterial and fungal infections [ 27 ] . in addition, the cord blood does not have antigen-specifi c memory t cells that can expand in a thymus-independent fashion to provide protection against viruses and opportunistic pathogens. this results in high frequency of late severe infections following cord transplantation, even when the neutropenic period is shortened by coadministration of stem cells from a thirdparty donor [ 28 ]. stem cells may be given using the bone marrow, g-csfmobilized peripheral blood stem cells (pbscs), or ucb. frequently bone marrow will result in more prolonged neutropenia compared with pbsc, and increased infections during neutropenia should be expected. however, a multicenter randomized trial comparing peripheral blood stem cells with the bone marrow from unrelated donors showed no difference in the relapse or infectious mortality between both groups, but confi rmed that chronic gvhd is more common with mobilized pbsc [ 29 ] . the particular features of ucd transplants were discussed on the preceding paragraph. manipulation of the stem cells, immunosuppressive drugs, or a combination gvhd may be prevented by decreasing the amount donor t cells or by limiting t cell function with immunosuppressive agents. the stem cells, whether from the bone marrow or the periphery, may be administered unmanipulated (sometimes called "t cell replete") or enriched by cd34 selection (also called "t cell depleted"). if unmanipulated bone marrow or pbscs are used, the dose of cd3+ t cells administered with the graft varies between 24 × 10 6 kg −1 when bone marrow is used and 300 × 10 6 kg −1 when pbscs are used [ 30 ] . reductions in the amount of t cells of 2-3 log 10 are possible, and in some haploidentical transplant regimens, as few as 12.5 × 10 3 cd3+ cells are given, which still results in detectable immune reconstitution starting 2-3 months after transplant [ 31 ] . t cell depletion may minimize or altogether prevent gvhd but may result in prolonged immunodeficiency, depending on the degree of depletion. if an unmanipulated product is used, t cell depletion may be attained in vivo by using alemtuzumab or atg. these agents produce a profound depletion of t cells in vivo, and their long halflife makes them still be present and active in the recipient when the stem cell product is administered. if no in vitro or in vivo t cell depletion is used, one of a variety of immunosuppressive regimens will be given to prevent gvhd (e.g., tacrolimus + methotrexate, tacrolimus plus mycophenolate mofetil, cyclosporine a, sirolimus, posttransplant cyclophosphamide). a randomized controlled trial documented more infections in patients randomized to (moderate) t cell depletion than in the group who received pharmacologic immunosuppression [ 32 ] . t cell depletion in vivo with alemtuzumab has been associated with increased risk of infection [ 33 ] . it is possible that different pharmacological regimens may result in different infectious risks, but this has not been adequately studied. preliminary evidence suggests that a sirolimus-based regimen may result in less cmv reactivation [ 34 ] and that posttransplant cyclophosphamide result in relatively decreased risk of ptld [ 35 ]. the above categories may combine in several ways, compounding the risk of infection. these variations should be considered both when designing a regimen of anti-infective prophylaxis and when considering an individual patient who may have an infection. gvhd is the most important cause of non-relapse mortality following hct, and it is frequently complicated by infection. gvhd is categorized as acute or chronic based on its time of onset. acute gvhd develops before day 100 and is characterized by gastrointestinal disease (secretory diarrhea, nausea, vomiting), liver dysfunction, and skin rash. stages of gvhd in the skin, gut, and liver combine to give a grade (i-iv) of the severity of the disease. acute gvhd grades iii-iv is associated with signifi cant mortality. the treatment of choice is high-dose systemic corticosteroids. gvhd is associated with signifi cant immune dysregulation [ 36 , 37 ] and is frequently accompanied by cmv reactivation [ 38 ] . the combination of disruption of the gi mucosa (and sometimes skin) and high-dose corticosteroids (in addition to the immunosuppressive agents concurrently given, like tacrolimus and mmf) constitute a high-risk setting for infection. bacterial, fungal, and viral infections are common under these circumstances. chronic graft-versus-host disease (cgvhd) has been traditionally defi ned chronologically: gvhd starting after day 100. it has been classifi ed based on its relation to prior gvhd (progressive when acute gvhd continues after day 100, quiescent when there is a period of time during which the patient is free of gvhd, or de novo when chronic gvhd is the fi rst manifestation of gvhd) and its extension (limited or extensive, reformulated as clinical limited, or clinical extensive). the clinical syndrome of typical chronic gvhd is quite distinct from the acute form, and a new classifi cation focusing on the clinical characteristics of the disease as well as on the timing is being increasingly used [ 39 ] . from the standpoint of infectious diseases, the important consideration is that the presence of chronic gvhd is associated with high risk of infection [ 40 , 41 ] . multiple immune defects have been described during chronic gvhd, involving humoral and cellular immunity [ 42 , 43 ] as well as functional hyposplenism [ 44 , 45 ] . besides these abnormalities, that result in delayed immune reconstitution and poor response to immunizations, the risk is of infection is increased by the treatment of extensive cgvhd [ 41 ], which typically includes systemic corticosteroids and a variety of steroid-sparing agents. notably, cgvhd is a well-documented risk for pneumococcal infections [ 45 , 46 ] , fungal infections, and late cmv disease. however, all types of infections are more common during cgvhd, particularly during the fi rst few months [ 47 ] . when gvhd is not controlled by corticosteroids, it is called " steroid refractory ," and there is currently no universally accepted standard treatment. this situation is important from the infectious disease standpoint because patients are usually treated with a variety of highly immunosuppressive regimens (e.g., atg, cyclophosphamide, mmf, infl iximab, daclizumab, alefacept, alemtuzumab, sirolimus, visilizumab, denileukin diftitox, and others) [ 48 ] that result in a wide array of infectious complications. reactivation of cmv is very common, as are fungal infections [ 49 , 50 ] , epstein-barr virus-related ptld [ 51 ] , as well as human herpesvirus 6 (hhv-6) [ 52 ] and adenovirus [ 53 ] . there are no controlled studies to support any particular infection prevention strategy during this period of increased immunosuppression, but some authors have emphasized that early use of prophylactic antibiotics and antifungals is an essential part of a successful approach to this problem [ 54 ] . unfortunately, this is a condition for which controlled trials are unlikely to be performed, and different centers will have to decide on a particular approach of close monitoring versus prophylaxis based on local experience and published case series. in the following sections, the epidemiology of bacterial, fungal, viral, and parasitic diseases will be discussed. the implications for prophylaxis and management will be mentioned. immunizations for transplant recipients, (as well as their caregivers and immediate contacts) are discussed in chap. 48 6.6 risks and epidemiology of bacterial infections after allogeneic hct 6.6.1 early bacterial infections: pre-engraftment approximately 20% of hct recipients will experience at least one episode of bacteremia during the fi rst few weeks, and a similar proportion after engraftment [ 55 ] . these infections are usually related to either neutropenia with subsequent bacterial translocation through the gi mucosa (mucosal barrier injury laboratory-confi rmed bloodstream infection or mbi-lcbi) or the intravascular catheter (central lineassociated bloodstream infections or clabsis) [ 56 ] . the relative frequency of gram-positive and gramnegative infections during neutropenia varies in different series and with the use of prophylactic antibiotics. in some centers, the most frequent gram-positive isolates are viridans group streptococcus [ 55 ] ; this may be a function of the conditioning regimen or the patient population. enterococcus faecium , frequently vre, is another gram-positive organism that tends to cause bloodstream infection relatively early, although this seems to be rather institution dependent [ 57 ] . the gram-negative bacteria are commonly enterobacteriaceae . these infections are generally related to the disruption of the gi mucosa due to the preparative regimen. the role of reduced diversity of the microbiota with subsequent bacterial domination and ultimately bacteremia is an area of intense study [ 58 ] . the risk of bacteremia during neutropenia may be decreased by the use of prophylactic antibiotics [ 59 , 60 ] . this had been shown in multiple studies over the years, but the recommendation of using antibiotics did not become part of practice guidelines until recently. it is not clear whether this recommendation will continue amidst the increasing concern over the role of antibiotic-induced decreased microbiome diversity on the outcome of hct [ 61 ] . in this regard it is of interest that fl uoroquinolones seem to have less detrimental effects on biodiversity of the fecal fl ora than beta-lactams. levofl oxacin at a dose of 500 mg/d for patients who are going to be profoundly neutropenic for longer than 1 week is the current recommendation of the idsa [ 11 ]. following engraftment in a large study from the sloan kettering cancer center, the risk factors for post-engraftment bacteremia included acute gvhd, renal dysfunction, hepatic dysfunction, and neutropenia [ 55 ] . enterococcus (vre) and coagulase-negative staphylococcus were the most common gram-positive isolates. enterobacteriaceae and non-fermentative gram-negative bacteria (including pseudomonas , stenotrophomonas , and acinetobacter , possibly related to the indwelling catheter) were the most common gramnegative isolates. bacteremia following engraftment often happens in the setting of patients with a complicated clinical course, acute gvhd, and multiple medical problems or else is catheter related. daily bathing with chlorhexidine-impregnated washcloths decreased the risk of acquisition of mdros and development of hospital-acquired bloodstream infections in transplant recipients in a randomized trial [ 62 ] , and this practice should be considered by every transplant program. the advantages and disadvantages of active screening for colonization by resistant pathogens have not been adequately studied in hct recipients. it is likely that local epidemiology determines whether screening is an effi cacious and costeffective approach to either prevent infection or improve outcomes. a retrospective study on vre bacteremia from the sloan kettering cancer center showed that vre carriage was predictive of subsequent vre bacteremia, but failed to detect the pathogen in many patients [ 63 ] . performing surveillance cultures for resistant organisms in vulnerable patient populations is part of the cdc recommendations "management of multidrug-resistant organisms in healthcare settings, 2006" [ 64 ] , and has been vigorously advocated by some experts [ [ 66 , 67 ] . both early and late (beyond day 100) pneumococcal disease has been reported, with late infections strongly associated with active cgvhd [ 46 ] . these have been attributed to inadequate antibody production and functional hyposplenism [ 44 , 67 ] . vaccination against s. pneumoniae should be given to all hct recipients, starting 3-6 months after transplant and using the 13-valent conjugate vaccine [ 9 ] (see chap. 48 for details). four doses of the vaccine result in enhanced antibody response and tolerable side effects [ 68 ] . antibiotic prophylaxis against s. pneumoniae prophylaxis for adults with active cgvhd has been recommended [ 69 ] , although there is only weak evidence supporting its effi cacy. penicillin v-k is safe and well tolerated, but the local patterns of penicillin resistance may make other antibiotics (e.g., trimethoprim, sulfamethoxazole, azithromycin, or levofl oxacin) preferable, although their long-term safety is not well established. late bacterial infections often involve the respiratory tract. pneumonia is the most common cause of fatal late infection [ 40 , 70 ] . chronic gvhd is the risk factor most commonly identifi ed. besides s. pneumoniae , multiple other pathogens have been reported. nocardia also tends to occur late and in patients with cgvhd [ 71 , 72 ] . mycobacterial infections are uncommon and diffi cult to diagnose [ 73 ] . risk factors for the development of active tb include gvhd, corticosteroid treatment, and total body irradiation (tbi) [ 74 ] . the need for universal testing for tuberculosis is controversial, given the unknown sensitivity and specifi city of the tests in this population and the fact that tuberculosis is a relatively uncommon complication after hct (albeit still approximately three times higher than in the general population) [ invasive candidiasis follows prior colonization and favorable conditions for the yeast: disruption of the gi mucosa during chemotherapy or acute gvhd, overgrowth in the presence of broad-spectrum antibiotics, and/or presence of indwelling catheters (the catheter seems to be the main risk factor in the case of c. parapsilosis ). early studies showed that fl uconazole during the pre-engraftment period could decrease the incidence of invasive candidiasis [ 76 , 77 ] . accordingly, fl uconazole is recommended as part of the standard prophylactic regimen during the pre-engraftment period. the prevalent use of fl uconazole has resulted in substantial decrease in the incidence of infections caused by c. albicans with relative increases in the incidence of other species of candida with decreased susceptibility to this agent (e.g., c. glabrata , c. krusei ) [ 78 ] . invasive aspergillosis occurs during specifi c "at risk" periods following hct, with a fi rst peak around the time of neutropenia pre-engraftment, a second peak between days 40 and 70 (the time of acute gvhd and its treatment), and a third peak late after transplant, usually in the midst of actively treated cgvhd [ 79 ] (figure 6-1 ) . a variety of risk factors for invasive aspergillosis have been identifi ed over the years, but the most consistently found to be signifi cant in multivariate analyses are acute gvhd, chronic extensive gvhd, and cmv disease [ 80 -82 ] . systemic corticosteroids are almost always present as part of the treatment of acute and chronic gvhd. non-aspergillus mold infections (e.g., fusariosis, mucormycosis, scedosporiosis), sometimes referred to as emerging mold infections, have been reported with increasing frequency [ 83 ] . the increased use of prophylaxis with activity against aspergillus would be expected to result in a relative increase of other opportunistic mycoses like mucormycosis [ 84 ] . considering the diversity of fungal infections after transplant and the current antifungal armamentarium, it is controversial which antifungal prophylaxis is appropriate at what point during transplant. for instance, although fl uconazole is a safe and well-established intervention during the preengraftment period of myeloablative transplants [ 76 , 77 ] , it is reasonable to question how necessary it is in transplants with conditioning regimens that result in shorter neutropenia. micafungin showed to be equivalent to fl uconazole in a randomized controlled trial [ 85 ] , and the same question (what kind of transplant patient would benefi t most) applies. regarding the duration of antifungal prophylaxis, fl uconazole up to day 75 posttransplant was associated with improved survival mainly due to decreased incidence of systemic candidiasis [ 86 ] , but it is uncertain whether this strategy should be used for all patients or should be received for some selected subgroups considered at higher risk. similarly, it is reasonable to question the indication for fl uconazole during periods when the main fungal infection is aspergillosis. several randomized controlled trials have compared fl uconazole with another azole with activity against molds (itraconazole [ 87 , 88 ] , voriconazole [ 89 ] , or posaconazole [ 90 ] ) either as standard posttransplant prophylaxis or during periods of increased risk. the general conclusion of these trials is that the aspergillus-active drugs are, indeed, more effective than fl uconazole in preventing ia, but the benefi t in survival in the context of a clinical trial with careful monitoring of galactomannan antigen is hard to demonstrate [ 91 ] . the 2009 asbmt/ebmt guidelines recommend posaconazole or voriconazole as antifungal prophylaxis in the setting of gvhd and micafungin in the setting of prolonged neutropenia [ 1 ] . of note, posaconazole prophylaxis was superior to fl uconazole or itraconazole and improved survival in prolonged neutropenia in non-transplant patients [ 92 ] . now, there are even more options of mold-active prophylaxis with posaconazole delayed-release tablets, intravenous posaconazole, and the new agent isavuconazole. infections after allogeneic hct viral infections remain a challenge because newer transplant modalities result in severe prolonged t cell immunodeficiency and because the current antiviral armamentarium is very limited. multiple latent viruses may reactivate following hct [ 93 ] . the role of monitoring by pcr is well defi ned mainly for cmv. latent viral reactivation is of particular concern in recipients of cord [ 94 ] or t cell-depleted transplants. table 6 -3 presents a summary of this section. members of the herpesvirus family that have caused significant disease after transplant include hsv-1, hsv-2, vzv, ebv, cmv, and hhv-6. posttransplant complications of hhv-7 are not well defi ned, although multiple associations have been described. hhv-8 infection and disease (primary effusion lymphoma and kaposi's sarcoma) occur only infrequently after hct. hsv-1 and hsv-2 may reactivate following the preparative regimen and complicate chemotherapy-induced mucositis, so it is customary to administer prophylaxis with acyclovir or valacyclovir at least until engraftment. in patients with common recurrences, long-term suppression may be appropriate. vzv predictably reactivates following transplant (approximately 25% in the fi rst year), either as shingles, multidermatomal, disseminated, or even without a rash ("zoster sine herpete"). in patients who are at risk for vzv reactivation, the use of long-term acyclovir safely prevents the occurrence of vzv disease [ 95 , 96 ] , and currently it is recommended for at least 1 year following hct. cmv remains latent in a variety of human cells. cmvseropositive hct recipients are at risk for cmv reactivation and disease after transplant. the term "cmv infection" is used to denote the presence of cmv in the blood detected by pcr or pp65 antigenemia [ 97 ] . following reactivation, cmv may cause disease typically in the form of pneumonia and/or gastrointestinal disease (most commonly colitis). other cmv diseases like retinitis or cns involvement are rare after hct but have been described: retinitis has been associated with high cmv viral load [ 98 ] sometimes in the context of chronic gvhd and cns disease (encephalitis and ventriculitis), sometimes with resistant virus in the cns [ 99 , 100 ] . the risk for reactivation may be related to the presence of cmv-specifi c immunity in the donor. the rate of cmv infection in the donor-recipient (d/r) pairs often follows the progression d r d r d r d r -+ + + + , suggesting that cmv-specifi c memory t cells administered with the stem cells may play a role in preventing reactivation and disease. cmv infection or disease in cmv-seronegative recipients of seronegative donors (r−/d−) is rare when leucodepleted or cmv-negative blood products are used [ 101 ] . every transplant program must decide on a strategy to monitor cmv and prevent disease. depending on a variety of factors, either universal prophylaxis with ganciclovir up to day 100 or a preemptive strategy of weekly monitoring and early therapy may be used. both approaches resulted in similar overall mortality when compared in a randomized controlled trial, but universal prophylaxis was followed by more cases of late cmv disease [ 97 , 102 ] . late cmv disease has emerged as a signifi cant problem, as it occurs when patients are not being under close monitoring by the transplant center. risk factors include lymphopenia and chronic gvhd [ 103 ] . preventing late cmv disease may be accomplished by either prophylaxis with valganciclovir or the preemptive approach with weekly cmv pcr monitoring [ 104 ] . the effect of cmv serostatus of donor and recipient on overall survival is complex (for a review, see [ 105 ] and chap. 24 ). ptld is a spectrum of lymphoid proliferations that may happen after solid organ or allogeneic stem cell transplantation, usually (but not always) driven by ebv [ 106 ] . pathologically the spectrum goes from polymorphic, polyclonal tissue infi ltration of lymphocytes to monomorphic involvement with high-grade b cell lymphoma. after allogeneic hct, the proliferating cells may be from donor (most commonly) or recipient origin. this disorder is typically related to insuffi cient or abnormal t cell responses against ebv [ 107 ] , and accordingly it is more common in the setting of hla-mismatched transplants, t cell depletion, or intense immunosuppression for the treatment of gvhd [ 108 -110 ] . some cases have followed the use of alemtuzumab for in vivo t cell depletion or gvhd prophylaxis [ 110 ] , despite the fact that anti-cd52 also results in depletion of b cells and earlier had been reported to be associated with relatively less risk. interestingly, the use of posttransplant cyclophosphamide to prevent gvhd seems to be associated with lower risk of ptld [ 35 ] . monitoring of ebv viral load by quantitative pcr is now recommended in those transplants considered at high risk. preemptive management of increasing ebv viral load in patients at risk has been associated with good outcomes [ 111 ] , although it is not clear when exactly this treatment should be given. a ct/pet may be useful to localize areas amenable to biopsy (figure 6 -2 ). hhv-6 is acquired early in life, when it may cause roseola infantum and nonspecifi c febrile illnesses. it frequently reactivates following hct. using quantitative pcr, hhv-6 can often be detected in peripheral blood 2-5 weeks after transplant. most of the time the reactivation seems to be asymptomatic [ 112 ] , but a number of associations (rash, delayed engraftment, gvhd, thrombocytopenia, increased overall mortality) as well as actual clinicopathological entities (hepatitis, pneumonitis, encephalitis) have been described [ 113 -115 ] . hhv-6 is possibly the most common cause of infectious encephalitis after hct [ 116 ] . it seems to be particularly frequent after cord blood transplant. cases of encephalitis tend to be accompanied by higher viral loads of hhv-6 in plasma [ 117 ] , but the role of systematic monitoring of hhv-6 in plasma is unknown at this time, as reactivation seems much more common than disease [ 118 ] and attempts to use a preemptive strategy using foscarnet have not been successful [ 119 ] . the european conference on infections in leukemia has proposed evidence-based guidelines to address the diagnostic and therapeutic uncertainties related to this infection [ 120 ] . respiratory viruses , a heterogeneous group of virus that is responsible for most upper acute respiratory infections in normal hosts, result in signifi cant morbidity and mortality after hct, particularly during the fi rst 3 months following transplant [ 121 ] . even asymptomatic carriage of respiratory viruses at the time of transplant has been reported to result in increased risk of unfavorable outcomes [ 122 ] . besides respiratory syncytial virus (rsv) [ 123 ] , infl uenza, parainfl uenza virus (piv) [ 124 ] , rhinovirus [ 125 ] , and adenovirus, newly identifi ed viruses including metapneumovirus [ 126 ] , coronavirus [ 127 ] , and bocavirus [ 128 ] have emerged as signifi cant pathogens. these infections present signifi cant risks both acutely and in the long term. during the acute infection, hct recipients are at risk of developing viral pneumonia that sometimes progresses to respiratory insuffi ciency, mechanical ventilation and death, and also at risk of concomitant or secondary bacterial or fungal infections that are associated with increased mortality [ 124 , 129 , 130 ] . longterm, there seems to be an association between early infection (pre-day 100) with some of these viruses (most notably piv and rsv) and later development of chronic airfl ow obstruction [ 131 ] . the most signifi cant risk factor overall for progression of these infections from the upper respiratory tract to the lungs seems to be lymphopenia [ 132 ] . corticosteroid use seems to contribute to progression to pneumonia in rsv and parainfl uenza infections but not so in infl uenza [ 129 , 130 ] (see table 6 -3 ). besides its role among the community-acquired respiratory virus, adenovirus may cause disease in transplant recipients following reactivation in the gastrointestinal tract followed by dissemination and end-organ damage [ 133 ] . de novo acquisition of adenovirus may also result in disseminated disease. there are more than 60 types of human adenovirus, with dif-ferent tropisms and possibly varying susceptibilities to antiviral agents. they can cause a variety of diseases, including upper and lower respiratory tract infection, colitis, hemorrhagic cystitis (hc), nephropathy, and cns disease. systemic adenovirus disease seems to be more common in children, particularly in recipients of cord blood or t cell-depleted transplants [ 134 -136 ] . patients with gvhd on treatment with high-dose corticosteroids are also at risk (figure 6-3 ) . some studies have documented that sustained high levels of adenoviremia are associated with disease [ 137 ] . it is not known yet whether a preemptive approach with cidofovir can successfully prevent disseminated disease and death [ 133 , 138 ] . bk virus infects 90% of humans by age 12. it predictably reactivates in most patients following hct and causes hemorrhagic cystitis (hc) in a minority of them [ 139 ] . detection of high levels of bk in the peripheral blood seems to correlate with the presence of bk-induced hc [ 140 , 141 ] . in a large study from the fred hutchinson cancer research center (fhcrc), no association was found between bk virus-associated hc and lymphopenia, corticosteroid use, and gvhd-the typical risk factors for viral infections after hct [ 140 ] . in contrast, other smaller studies have found an association with gvhd. the pathogenesis of this disease remains unexplained. bk-induced nephropathy, a common problem after kidney transplant, remains infrequent after hct and does seem to be related to profound immunosuppression [ 142 ] . bk pneumonitis has also been described, but it is distinctly rare [ 143 ] . jc virus is also acquired by most people during childhood. in immunocompromised hosts, it may cause encephalitis (jc encephalitis, previously called progressive multifocal leukoencephalopathy (pml)) with multiple areas of demyelin-ation without edema detectable by mri. some studies have suggested that detectable viral load after hct may be more common than currently thought [ 144 ] . ascertaining risk factors for this disease is diffi cult because some transplant recipients may have conditions known to be associated with it and also received medications like mmf, rituximab, or brentuximab, which have been associated with pml even in the absence of allo-hct. pcp is an opportunistic infection of patients with profound cellular immunodefi ciency, and prophylaxis is recommended after hct. it is now relatively uncommon: 1.3-2.4% of patients transplanted from several series [ 145 , 146 ] most cases seem to occur relatively late, after discontinuing prophylaxis or during periods of intensive immunosuppression for the treatment of gvhd [ 147 ] . hypoxemia is characteristic at presentation. atypical radiological manifestations, including nodular infi ltrates and pleural effusions (in contrast to typical interstitial pneumonitis), are described frequently, as is the presence of co-pathogens [ 148 ] . the preferred prophylaxis is trimethoprim/sulfamethoxazole (tmp/smx) , and several dosing regimens are effective (one single-strength tablet daily, one double-strength tablet daily, or one double-strength tablet three times/week) [ 149 ] . tmp/ smx may be poorly tolerated because of hematologic toxicity, skin rash and/or gastrointestinal toxicity [ 150 ] . it is unclear which is the prophylaxis of choice if tmp/ smx cannot be used. aerosolized pentamidine is convenient, obviates the problem of compliance, and is less toxic than dapsone and better tolerated than atovaquone. however, it has been reportedly associated with more failures than dapsone [ 150 ] . dapsone seemed to be effective and well tolerated in one study [ 151 ] but not in another when it was given only three times per week [ 152 ] . dapsone should not be given to patients with g6pd defi ciency. methemoglobinemia is a well-known complication of dapsone [ 153 ] that should be considered in the presence of unexplained shortness of breath. atovaquone suspension 1500 mg/d may be used, but published experience in hsct recipients is limited [ 154 , 155 ] . atovaquone is expensive and poor tolerance has made compliance for some patients diffi cult. absorption is better in the presence of signifi cant amount of fat, and breakthroughs are well documented ( figure 6-4 ) . pcp prophylaxis is recommended at least until all immunosuppression has been stopped but it is unclear how much longer to continue it [ 156 ] . most cases of toxoplasmosis after hct represent reactivation, although rare cases of transmission with bone marrow transplant have been suspected [ 157 ] . recipients should be tested for anti-toxoplasma igg antibody, and if they are found to be positive, prophylaxis is recommended. rare cases of toxoplasmosis after hct have occurred in seronegative recipients [ 158 , 159 ] . the disease tends to occur within the fi rst 6 months after transplant, but it can happen later in the presence of persistent immunosuppression [ 160 -162 ] . the risk of toxoplasmosis varies with the type of transplant and the immunosuppression: cord blood and use of atg were found to be risk factors for disease in a prospective study [ 162 ] ; most cases in another series occurred in urd or mismatched transplants [ 107 ] . tmp/smx as given for pcp prophylaxis is considered adequate to prevent toxoplasmosis, although there have been cases on hct recipients who were receiving it [ 162 ] . the best alternative for patients who are intolerant to tmp/smx is unknown. dapsone and atovaquone showed some effi cacy in hiv-infected patients and there is increasing experience after hct [ 163 ] , although failures have been reported. other regimens include clindamycin with pyrimethamine and leucovorin, pyrimethamine with sulfadiazine, or pyrimethamine and sulfadoxine and leucovorin [ 107 ] . if a reliable quantitative pcr assay is available, frequent monitoring and preemptive treatment may be appropriate, since pcrdetected reactivation seems to precede symptoms by 4-16 days [ 162 ] . retrospective data suggest this strategy may result in improved outcome [ 164 ] . in summary, infections following hct are frequently related to risk factors caused by the procedure itself. neutropenia and mucositis 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prevention of pneumocystis carinii pneumonia in human immunodefi ciency virus-infected subjects intolerant of trimethoprim or sulfonamides regionally limited or rare infections: prevention after hematopoietic cell transplantation transmission of toxoplasmosis by bone marrow transplant associated with campath-1g disseminated toxoplasmosis in marrow recipients: a report of three cases and a review of the literature. bone marrow transplant team disseminated toxoplasmosis after allogeneic stem cell transplantation in a seronegative recipient toxoplasmosis after hematopoietic stem cell transplantation report of a 5-year survey from the infectious diseases working party of the european group for blood and marrow transplantation early detection of toxoplasma infection by molecular monitoring of toxoplasma gondii in peripheral blood samples after allogeneic stem cell transplantation atovaquone for prophylaxis of toxoplasmosis after allogeneic hematopoietic stem cell transplantation molecular diagnosis of toxoplasmosis in immunocompromised patients: a three-year multicenter retrospective study risks and epidemiology of infections after hematopoietic stem cell transplantation key: cord-257299-z9u12yqb authors: mansi, n.; de maio, v.; della volpe, a.; ripa, g.; malafronte, l.; de filippis, c. title: ear, nose and throat manifestation of viral systemic infections in pediatric patients date: 2009-12-31 journal: international journal of pediatric otorhinolaryngology doi: 10.1016/s0165-5876(09)70006-0 sha: doc_id: 257299 cord_uid: z9u12yqb abstract objective/methods an exhaustive review of literature was performed to investigate available data and evidences regarding pediatric otolaryngologic manifestations of viral systemic infections. results/conclusions modern otolaryngologists should be familiar with viral systemic infections since many have head and neck manifestations. cooperation between otolaryngologist, paediatrician and virologist can be considered and excellent tool in diagnosis and treatment of these diseases in particular when complications occur. there are multiple systematic viral infections that can manifest themselves in orl related organs. their actions can work directly or indirectly causing an alteration in the human immune system and a consequent secondary bacterial invasion. notable advances in the diagnosis and treatment of viral infections have been mitigated by the appearance of new pathological processes, for example aids, which often has its initial manifestations in orl regions. table 1 is a list of illnesses affecting different anatomical sites and the viral etiologies that commonly strike each particular location. considering the vast nature of the subject, we subdivided our treatment into three parts corresponding to the same groups of interrelated viral illnesses: -viruses that can cause deafness. -viruses that can cause inflammation in the upper respiratory tract. -viruses particularly relevant to ent (infectious mononucleosis, papillomatosis, herpes infections). ascertaining specific viral causes of most infections is neither necessary nor cost-effective, and should be reserved only for specific cases. clinical and epidemiological acumen remain the basis for a presumptive diagnosis. when a specific diagnosis is necessary, diagnostic procedures based on biochemical and molecular biological processes provide sensitive, specific and rapid results [1] . in most viral infections, immunity to re-infection generally lasts a short period of time due to the host's limited immunological response or, rather for an antigenic change in the virus. viral pathologies that can cause deafness can be congenital, appear in either the pre-natal or postnatal period and can also be acquired upon contact with the pathogen [2] [3] [4] (table 2 ). in particular, the hearing damage caused by congenital infections can be part of a severe syndrome (such as "congenital rubella syndrome") but more frequently it is the first and only manifestation of intrauterine infection. common childhood viral infections, such as measles and mumps are probably an unrecognized cause of acute or progressive damage to hearing [5] . in prenatal deafness, a pathogen introduced during pregnancy can provoke an arrest or alteration of the normal development of the ear, even causing lesions on the already-formed hearing mechanism [6] . the most serious lesions manifest themselves in the first three months of pregnancy, especially between the seventh and tenth week, when the cochlea is developing; this would be considered a case of embriopathy. fetopathy refers instead to lesions that form between the fourth month of pregnancy and birth. since the hearing organ has already formed in these cases, patients do not generally suffer serious alterations although the inner ear is certainly sensitive. the viruses that most frequently cause prenatal deafness are rubella and citomegalovirus (cmv). rubella is caused by an rna virus of the togaviradae family of the rubivirus genus. congenital rubella is typically passed on to the fetus from a primary infection in the mother. the virus invades the upper airways of the mother causing viremia and spreading into different sites including the placenta. it has been hypothesized that in the first gestational phases, the rubella virus provokes a chronic intrauterine infection. fetal infection in the first trimester, particularly in the first 8-10 weeks, has an extremely high risk of malformations such as hypoacusia, cardiac and ocular defects (gregg triad); however, if the rubellum infection is contracted in the second or third trimester, it results in hypoacusia and pigmented retinas. thus, the more precocious the maternal rubellum, the greater the risk of fetal infections and the more serious the fetal malformations (100% in the first month, 80% in the first trimester, 70% in the second trimester and 30% in the third). this reduction is likely due to either a maturation of the placenta after the first trimester which limits the transfer of the virus, or the greater resistance of the differentiated cells [5] . the deriving hypoacusia is generally sensorineural and bilateral and at birth can already be progressive or it can manifest itself later. the hearing damage seems to be caused by a "teratogenic" effect of interference with the normal development of the organ at the cochlear level [6, 7] . unlike other congenital infections, rubella is easily prevented. between 12 and 15 months of age, a livevirus rubellum vaccine is administered along with a measles and mumps vaccination, giving the patient immunity to rubellum for about 15 years (mmr); a booster vaccination is administered before elementary or middle school. women of child-bearing age who are not immune to rubella must undergo vaccination and not get pregnant in the following three months. vaccination immediately after giving birth is advisable for mothers at risk of being infected. citomegalovirus (cmv) is a dna virus that belongs to the herpesviridae family. it can go into latency and then reactivate and has been isolated in various sites including saliva, urine, breast milk, sperm, brain fluid, and amniotic fluid. congential cmv infection is thought to be derived from transplacental infection from a primary or recurring maternal infection occurring in the first half of pregnancy. prenatal cmv infection is contracted by contact with infected cervical secretions, breast milk, or blood derivatives. it is believed that maternal antibodies have a protective function and that most of these newborns are either born asymptomatic or are not infected by the virus in the case of contact. many women who are infected by cmv during pregnancy are asymptomatic, but occasionally develop an illness similar to mononucleosis. it is still unclear if more serious lesions are a consequence of a precocious maternal infection or of a later one during the course of gestation. nearly 10% of children with congenital cmv infection are symptomatic at birth. manifestations include delayed intrauterine growth, premature birth, microcephalus, jaundice, petechia, hepatosplenomegalia, periventricular calcification, corioretinitis e pneumonia. the virus causes deafness by infecting the inner ear and altering the organ of corti. moreover, it can cause malformations of the labyrinth of ethmoid and at the same time also lesions on the auditory tract due to secondary toxicity. the hypoacusia that establishes itself is sensorineural, almost always bilateral, and profound, generally regarding acute tones. symptomatic newborns have a mortality rate of up to 30% and 70-90% of those who survive have neurological deficits such as hearing loss, mental retardations and visual disturbances [8] [9] [10] . a vaccine for cmv is still under research. exposure to the disease in non-immunized pregnant women must be controlled, despite the fact that cmv is ubiquitous everywhere. since it is frequent in children who attend preschools, pregnant women must observe all the common norms of good hygiene after contact with or being exposed to the urine or expectorate of such children [11, 12] . in post-natal deafness, numerous infective illnesses can be responsible for serious damage to the viii nerve and the cochlear apparatus. a large part of hearing defects arising in childhood can be traced back to the intrauterine period. the most frequent forms are: viral meningoencephalitis (arbovirus, herpesvirus, mixovirus, poxvirus, etc.), mumps, chickenpox and measles. meningoencephalitis can be primitive or constitute the secondary complication of a viral infection. the forms of primitive meningoencephalitis can be both epidemic (arbovirus, poliovirus, echovirus, coxsackievirus illnesses), and sporadic (herpes simplex, varicella zoster, parotite) [13] . secondary encephalitis, such as complications of a viral infection, probably have an immunological, pathgenic mechanism. secondary encephalitis to rubella, mumps, measles, smallpox, cow's pox and other less defined illnesses are all examples. currently, deafness caused by meningoencephalitis is not infrequent among the post-natal causes of deafness; in some cases it can be caused by a virus that, before birth, reach the cochlea via the external hearing conduct by way of the vascular system, causing a relatively symmetrical bilateral sensorineural hypoacusia which is either mild-serious or profound. in other cases, deafness is due to a meningoencephalital localization of liquor infection in the first four weeks of life as a complication of neaonatal sepsis (25%). deafness caused by meningoencephalitis occurs more often in males and is found in 2/10,000 neonates born at full term and in 2/1,000 low-weight neonates. no vaccines exist for the described viral forms. mumps (parotitis) is instead caused by a paramixovirus, spread through drops of infected saliva or through direct contact with material contaminated by infected saliva. the virus probably penetrates the body through the mouth. it can be found in saliva 1-6 days before the appearance of the swelling of the salivary glands and lasts throughout the duration of the illness (usually 5-9 days). an infection usually results in permanent immunity, even when there is unilateral swelling of the salivary glands. although the illness can occur at any age, most cases occur in children between 5 and 10 years of age; it does not usually occur in children under 2 years old. breast-fed children less than one year old are usually immune. the incubation period is 14-24 days. deafness can be a complication in 5/10,000 cases of mumps. in 80% of cases, deafness is sudden and unilateral in the context of an acute infection in association with aseptic meningitis and often accompanied by tinnitus, ataxia, and vomiting. hearing loss is profound and permanent for high frequencies and can go unrecognized. damage is confined to the cochlear duct and consists in the degeneration of the vascular strip of the corti organ, the degeneration of the upper membrane, usually more serious on the basal curve of the cochlea. active immunization is obtained through a single-dose, live-virus inoculation between 12 and 15 months of age with a mmr vaccine. a booster dose is administered before starting elementary or middle school. measles, caused by a paramixovirus, is extremely contagious and is spread primarily through either the nasal and oral excretions of an individual in the prodrome or precocious eruptive stage of the disease or through the nuclei of drops dispersed in the air. the contagious stage of the illness extends from 2 to 4 days before the appearance of the eruptions until 2-5 days after their appearance. the virus disappears from the nose and pharynx secretions as soon as the eruption on the skin has cleared up. the incubation period is 7-14 days. measles virus causes permenant, bilateral deafness in 1/1000 cases. deafness appears suddenly at the same time as the cutaneous rash. the viral infection of the inner ear spreads through the vascular strip and destroys the structures of the cochlea as well as most of the nervous and ganglionic and fibers [14] . measles infection can be avoided by administering a reduced, live-virus vaccine to children between the ages of 12 and 15 months (mmr). the vaccine confers long-term immunity and provokes an antibody response similar to that of natural measles. in some cases, the vaccine can provoke a light or asymptomatic infection that is not contagious. sensitive subjects at risk of contracting measles can be protected if the live-vaccine is administered within 2 days of exposure. in other cases, such as pregnant women, or children under one year of age, an immunoglobuline specific measles vaccine (mig) or a 0,25 ml/kg im dose of serum immunoglobulin may be administered. chickenpox is caused by the varicella-zoster virus (herpesvirus) and represents its acute, invasive phase, while the reactivation from its latent phase causes the herpes zoster illness. it is believed that chickenpox, which is extremely contagious, is transmitted through drops of saliva which are infected and even more infective during the brief prodromic period and the first phase of eruption. incubation period is 14-16 days and transmission is considered possible 10-21 days after exposure. the deafness it causes can lead to the destruction of the nervous and sensorial cells of the neuroepithelia through a process of neurolabyrinthitis which can result in severe bilateral, sensorineural hypoacusia [15] . chickenpox can be prevented by inoculating with a reduced, live-virus vaccine in all healthy children between 12 and 18 months of age, after which children who lack immunity to chickenpox may be vaccinated at any time. subjects over 13 years old who have not been immunized must receive two doses of the vaccine, with a period of 4-8 weeks between doses. the severity of the disease can be lessened by administering an immunoglobuline anti-zoster (zig) or anti-varicella-zoster (vzig) within 96 hours after exposure. its use, however, is restricted to subjects at risk, like those affected by leukemia, immunodeficiency syndromes or other serious pathologies, and pregnant women. neonates with mothers who were infected by chickenpox five days before giving birth or two days after are also candidates for such treatment [16, 17] . viral respiratory infection is almost always a benign pathology. its beginning is connected with the socialization of the child, and as such is most frequent during preschool. it noticeably interferes with the child's wellbeing and provokes significant medical-social costs.unfavorable environmental factors (atmospheric pollution, passive smoking, etc.), precocious socialization, and predisposing immunological factors with immunological immaturity could all be predisposing factors. viral respiratory infections are characterized by a series of acute episodes that can involve the entire respiratory system or a single sector (pharyngotonsillitis, otitis, rhinosinusitis, laryngitis, bronchitis, pneumonia) [18] [19] [20] [21] . the damage that a viral infection can inflict on the mucous membranes of the upper respiratory tract are influenced by the reduction of the mucous flux and phagocytes as well as the increase in the bacteria's adhesiveness to mucus cells [22, 23] . an upper-resipiratory viral infection is characterized by multiple processes. first, the virus replicates itself in the epithelium, spreading fragments of the disintegrated cells into respiratory secretions and demonstrating the presence of the virus, viral peptides, or viral nucleic acids [24, 25] . the host then responds to the infection by producing a range of cellular products. some, such as alpha-interferon, are specifically anti-viral. others, such as interleukin, are aspecific. specific antibodies are produced in sequence, for example igm followed by igg and iga; the comparison of a high level of igm without an increase in igg is an index of recent infection (the comparison of blood examinations confirms the diagnosis but is only clinically useful for epidemiologic purposes. acute rhino, pharengeal and tonsillar inflammation, caused by viral infections [26] are some of the most common deseases found in pediatric populations. less frequently, the pharangeal-tonsillar forms are accompanied by an involvement of the oral and or respiratory tract mucosa [27] [28] [29] . such infections, in anglo-saxon countries, the term, upper respiratory tract infection (urti) or "common cold" is used to describe an inflammation of the upper airways [30] . the episodes, which often reoccur, effect mostly preschool-age children and have socioeconomic repercussions, which are not related to the gravity of the pathology, but mostly to the increase in the requests for visits to the doctor, the costs incurred for treatment and the days of school and work lost by the children and the parents who must look after them. the etiology of the acute forms in the respiratory airways is, initially, of a viral nature in most patients, with later, secondary bacterial infections on the mucous lesions caused by the viral agents [31] . the transmission of the pathogens responsible for the urti frequently occurs in public locations by direct or indirect contact with the nasal secretions or plugge drops from infected subjects. seasonability, which could increase or reduce sensitivity to such infections, constitutional factors, and an incomplete maturation of the immune system of the child are hypothesized to be pathogenic mechanisms. multiple viruses are responsible for these infections as described in table 1 . the course of acute, unspecific viruses is quite variable but almost always concludes with a recovery within 2-5 days as long as other complications do not develop. the most common complications are infections such as otitis media (mostly in younger children), rhinosinusitis, satellite lymphoadenitis, spreading of the infection into the lower respiratory tract and obstructed respiration, and bronchial spasms in subjects with bronchial hyperactivity. occasionally respiratory viruses are responsible for clinical pictures described as the common cold [32] , caused by picornavirus (rhinovirus, echovirus and coxackievirus), and influenza syndrome [8, [33] [34] [35] , caused by influenza viruses that initially strike the epithelium of the respiratory mucous membranes which other viral strains attach themselves to, aggravating and complicating the original clinical portrait [36, 37] . both herpangina and laryngotracheitis have a unique clinical picture. herpangina is an extremely contagious illness caused by a coxackievirus characterized by a presence of a vesicular exanthema at the velopharyngeal mucous level and acute or croup laryngotracheitis [38] [39] [40] [41] when viral infections are associated. the infections are caused by diverse viruses but more frequently by type 1 and type 2 parainfluenza viruses [42] [43] [44] , which cause an inflammation of the tracheal and subglottic muscous membrane with a charateristic symptomology (inspiratory stridor and barking cough) occasionally relapsing with serious, obstructing respiratory complications [45] [46] [47] . the varicella-zoster virus (vzv) belongs to the herpesvirus group. it is a dna virus that gives rise to chickenpox as a primary infection and to herpes zoster (hz) as a localized relapse due to modifications of the pathogenic power of the virus and/or alterations of cellular immunity [38, 48, 49] . hz is an acute cutaneous-nervous illness that is locally circumscribed and provoked by a resurgence of the vzv acquired during infancy and latent in one or more of the more sensitive ganglia of the dorsal roots of the spinal marrow and/or cranial nerves for a prolonged period of time (often decades); during the latency period the virus does not replicate itself or give any sign or symptom of its presence [50] [51] [52] . in particular, at the auricular level, the illness takes on the name herpes zoster oticus but it is also described in the auricular zone as herpes zoster auris or ramsay hunt illness, in honor of the author who, in 1907, described its characteristics and its correlation with the geniculate ganglion [53, 54] . the illness is caused by a reactivation of the vzv in the geniculate ganglion of the facial nerve; through the sensitive nervous fiber, the virus reaches the skin and causes a characteristic centrifugal root vesicular eruption. we can clinically define four stages according to the involvement of the vii n.c. the diffusion of the virus from cell to cell must be impeded with the use of antiviral drugs (5 mg/kg acyclovir administered intravenously per day in three daily doses for 7-10 days followed by an oral administration for another 7 days); results of treatment with more recent antiviral drugs (such as famciclovir and valaciclovir) are promising, while a significant inflammatory reaction reactive in the nerve must be treated with cortisonebased anti-inflammatory medication (1 mg/kg/daily for ten days) and sometimes with surgical decompression [13] . analgesics and local antiseptics should be added to treament with cortisones and antiviral medication. laryngeal papillomatosis (lp) [55, 56] is caused by subtypes of the human papilloma virus (hpv) which is a member of the papova family of viruses [57] . seventy subtypes have been described, but only hpv 11, hpv 6, and more rarely hpv 16 are specifically associated with laryngeal papillomas [58] [59] [60] . typical of such a pathology is the multifocal nature of the lesion (>85% of cases), localized on the vocal cords in 60% of cases but also involving the upper-glottic plain (35%), the oropharynx, the bronchial tree and, rarely, the cervical esophagus. in the united states, the annual incidence rate of laryngeal papillomatosis is 4.3/100,000 in children and 1.8/100,000 in adults, with a prevelance of 5.7/100,000 [61, 62] . this data is substantially analogous to that revealed in a 1991 danish study [63] which registered an annual incidence in children of 3.6/100,000. histologically, papilloma cosists of a cartilaginous fiber scaffolding with the presence of connective vascular tissue its surface surrounded by squamous epithelium. keratinizing aspects are not observed on its surface. the course of this pathology is characterized by frequent relapses and aggravations that require frequent and repeated laryngoscopy and bronchoscopy to ablate the rapidly forming pappillose formations and to avoid obstruction of the airways [64] . even if the illness is usually resolved by a spontaneous recovery, some cases can move toward an unfavorable prognosis at any time for no recognizable reason and involve the trachea, the bronchi and the lungs [65] . it is believed that hpv 11 has a greater propensity for a distal, pulmonary diffusion and that, moreover, therapeutic action, such as tracheotomy or repeated endolaryngeal ablation can favor the distal insemination of papillomas. the tracheal, bronchial, and pulmonary involvement occurs, according to various authors, in 2-4% of cases. malignant degeneration of the laryngeal papillomatosis, is a rare, but serious event and leads to an unfavorable prognosis. most described cases involve adults who have other risk factors, such as the use of tobacco, long-term illnesses, and previous exposure to radiation due to radiation therapy for papillomatosis. there is a greater probability of malignant transformation with hpv 16 but also hpv 6 and 11 are capable of oncologically transforming the nature of a cell culture [66] . in surgical treatment of laryngeal papillomatisis, many techniques are used: asportation with tweezers via indirect laryngoscopy, electro-cauterization, cryosurgery, direct asportation in larynfissure, endoscopic asportation with microlaryngoscopy in suspension. currently, most surgeons prefer endoscopic laser surgery for its high precision and the haemostatic control which the technique permits [10] . more recent techniques include argon laser photo sensitization and hematoporphyrin derivatives, known as photodynamic therapy [67, 68] , and the technique with scaples and rf ("coblation") which seems to avoid the modest heat damage and the edema which results from the use of the laser [69, 70] ; the same advantage is shared by ablation with microdebrider which is also more rapid than excision by laser. that is why it is coming ever closer to replacing the co 2 laser as the ablative method of choice for use on children [71] . despite the radical nature of the treatment, relapse is the norm. this makes reiterated procedures necessary with the possibility, occasionally, of having to resort to a tracheotomy. such a procedure should be avoided for as long as is possible because of the recognized possibility of colonization on the part of the papillomas in the region of the tracheostomy and tracheobronchial tree. the risk of the tracheobronchial tree being colonized is also believed to be a consequence of the repeated intubations. the need to lengthen the amount of time between surgical asporation of the papillomas and the possibility of complete medical resolution have spurred many researchers to find adjuvant or resolving treatments. recent studies have supplied a way to identify adjuvant therapies to control papillomatosis and its relapses such as interferon-alpha [56] , acyclovir [72] , indolo-3-carbinolo [73] , retinic acid [74] , metotressato, cidofovir [75] [76] [77] [78] . even if the above therapies have sometimes significantly reduced relapses of papillomas, we believe the most effective to be intralesional injection of cidofovir associated with surgical treatment [76] . however, none of them are able to eradicate the hpv genome from the mucous cells of the respiratory tract [79] . the most promising therapies for pl are based on both therapeutic and prophylactic hpv vaccines that are currently in experiemental phases [56, 80, 81] . the epstein-barr virus (ebv) or human herpesvirus 4 is a ubiquitous gammaherpesvirus that infects more than 95% of the world's population. the most common manifestation of the primary infection of this organism is infective mononucleosis (im), a sometimes acute, but often asymptomatic clinical syndrome which more often strikes children, adolescents, and young adults [82] . it is a self-limiting lymphoproliferative illness connected to a first contact with the epstein-barr virus. the virus generally comes into contact with the mucous membranes of the oropharanx where it causes a localized primary infection from which it can circulate through the bloodstream.the period of incubation is not known, and to be the source of infection, is often misunderstood, even if it is known that it is mainly spread orally. in particular, the cells which host cells are mainly the b-lymphocytes and the cells of the human nasalpharanx are where the virus replicates itself. the b-lymphocytes transformed by ebv are the target of a multiform immune response. the immune response (production of antibodies) documents a primary ebv infection. the cellular immune response, consisting in part of the induction of an activated, postive t-lymphocyte cd8, is mostly responsible for atypical lymphocytes which is the consequence of a primary ebv infection. the virus can be found in the oropharangeal secretions of 15-25% of healthy adults who test positive for ebv. the reactivation of ebv is generally asymptomatic, the opposite of that of the herpes simplex and varicella-zoster virus. ebv is relatively labile. it has not been isolated from environmental sources and it is not very contagious. in the majority of cases, it is believed that the incubation period is 30-50 days. the virus can be spread by the transfusion of blood derivatives but is more frequently passed on by oropharangeal contact (kissing) between a non-infected subject and a healthy carrier that asymptomatically secretes the virus from the oropharynx. during early childhood, infection occurs more frequently in lower socioeconomic classes and in conditions of overcrowding [83] . ebv has also been associated with african burkitt lymphoma and some b-cell neoplasias in immunodepressed patients (especially transplant recipients, hiv or ataxia-teleangectasia patients) and to nasalpharangeal carcinoma [29, 84, 85] . these associations are based on seriologic evidence of an increased ebv activity and on proof of nuclear antigens (epstein-barr nuclear antigens, ebna) and of ebv dna found in tumor biopsies. it has been postulated that ebv places a role in some b-cell lymphomas, polyclonally stimulating and transforming the b-lymphocytes, making them more susceptible to a successive chromosome transfer to an evolution toward an oligoclonal or monoclonal lymphoproliferation. the classic symptomology of mi includes fatigue, fever, inflammation, lyphoproliferation, however, patients can also present all or only some of these symptoms. ebv infection in children is usually asymptomatic or with a light symptomology. usually patients present with an illness that has lasted several days to a week, followed by fever, inflammation, and adenopathy. fatigue is usually highest in the first 2-3 weeks. fever reaches its peak in the afternoon or early evening with a temperature of around 39.5â°c, but can even reach 40.5â°.when fatigue and fever are the dominant signs (the so-called typhoid form), the beginning and the resolution can take longer. inflammation can be serious, painful and sedating and can resemble streptococcica inflammation. lymphoadenopathy can involve almost any group of lymphnodes but is usually asymmetrical; anterior and posterior cervical adenopathy is often relevant. the enlargement of only one lymphnode or a group of lymphnodes can be the only manifestation; in these cases, studies of the heterophiles can forgo lymph nodal biopsy or help the interpretation of alarming histopathological aspects. splenomegaly, present in around 50% of cases, is at its maximum during the second and third weeks, manifesting itself through pain the upper left quadrant. slight hepatomegaly can also be present as well as a pain on the hepatic percussion. less frequent signs are malcularpapular eruptions, jaundice, periorbital edema, and palatal exanthema [40] . infective mononucleosis is usually self-limiting. the duration of the illness is variable, usually about 2 weeks, but generally 20% of patients can go back to school or work after a week, and 50% after two weeks. patients can usually begin their normal activites again after this period but sometimes the complete resolution of asthenia requires several weeks. only in 1-2% of patients does asthenia last months. the decline happens in less than 1% of all cases and is generally caused by complications of the primary ebv infection (encephalitis, rupture of the spleen, airway obstruction). generally, the diagnosis is clinical but it must always be confirmed by laboratory testing, and, in particular, identification of ebv. it should be mentioned that the tendency of a late positive score and the elevated possibility of false negatives. treatment of mi is generally supportive and consists of antipyretic and/or analgesic drugs. the use of antibiotics is controversial while therapy is underway with cortisones, which is considered by some to be routine, but considered by other aa to be exclusively reserved for the most serious cases or complications. mi is generally considered to be a benign and self-limiting illness. however, in some, rare cases, complications can arise putting the life of the young patient at risk. serious hepatic complications are those which progress toward cirrhotic forms, reye's syndrome or in extreme cases, toward duncan's syndrome, a syndrome characterized by massive hepatitis linked to the x chromosome and caused by a defect in the immune response to ebv. respiratory complications are, in general, obstructive and linked to adenotonsillar hypertrophy or to serious interstitial pneumonia. hematologic complications are particularly alarming and can lead to the bursting of the spleen as well as neurological complcations where encephalitis is the leading cause of death. in this regard, particular attention should be paid to guillain-barrã©'s syndrome which is an inflammatory, demyelinating form that can complicate infective mononucleosis and cause a progressive paralysis of the respiratory muscles or rather a more or less diffused involvement of the cranial nerves. none declared. evaluation of recurrent respiratory tract infection in children genetic evaluation guidelines for the etiologic diagnosis of congenital hearing loss temporal relationship between human parechovirus 1 infection and otitis media in young children immune-mediated inner ear desease and parvovirus b19 the 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papillomatosis treatment of severe laryngeal papillomatosis with intralesional injections of cidofovir systemic cidofovir in papillomatosis adjuvant drug strategies in the treatment of recurrent respiratory papillomatosis can mumps vaccine induce remission in recurrent respiratory papilloma? world health organization initiative for vaccine research: state of the art of new vaccines -research & development. world health organization clinical manifestations and quantitative analysis of virus load in taiwanese children with epstein-barr virus-associated infectious mononucleosis the merck manual of diagnosis and therapy nasopharyngeal carcinoma in tunisian children: retrospective epidemiological, clinical and biological study about 48 cases unilateral tonsillar lymphoepithelioma with ipsilateral parapharyngeal space involvement: a case report key: cord-103893-p9ul6k5m authors: omame, a.; okuonghae, d. title: a co-infection model for oncogenic hpv and tb with optimal control and cost-effectiveness analysis date: 2020-09-18 journal: nan doi: 10.1101/2020.09.15.20195297 sha: doc_id: 103893 cord_uid: p9ul6k5m a co-infection model for oncogenic human papillomavirus (hpv) and tuberculosis (tb), with optimal control and cost-effectiveness analysis is studied and analyzed to assess the impact of controls against incident infection and against infection with hpv by tb infected individuals as well as optimal tb treatment in reducing the burden of the co-infection of the two diseases in a population. the co-infection model is shown to exhibit the dynamical property of backward bifurcation when the associated reproduction number is less than unity. furthermore, it is shown that tb and hpv re-infection parameters ($varphisst{p} neq 0$ and $sigmasst{t} neq 0$) as well as tb exogenous re-infection term ($varepsilonsst{1} neq 0$) induced the phenomenon of backward bifurcation in the oncogenic hpv-tb co-infection model. the global asymptotic stability of the disease-free equilibrium of the co-infection model is also proven not to exist, when the associated reproduction number is below unity. the necessary conditions for the existence of optimal control and the optimality system for the co-infection model is established using the pontryagin's maximum principle. uncertainty and global sensitivity analysis are also carried out to determine the top ranked parameters that drive the dynamics of the co-infection model, when the associated reproduction numbers as well as the infected populations are used as response functions. numerical simulations of the optimal control model reveal that the intervention strategy which combines and implements control against hpv infection by tb infected individuals as well as tb treatment control for dually infected individuals is the most cost-effective of all the control strategies for the control and management of the burden of oncogenic hpv and tb co-infection. 1 introduction of treatment failure in the drug-sensitive tb infectious individuals and the treatment of individuals with drug-resistant tb is the most cost-effective in curbing the co-infections of drug-resistant tb and hiv/aids. okosun and makinde [20] considered a malaria-cholera co-infection model, in the presence of prevention and treatment controls for both diseases. applying the pontryagin's maximum principle, they derived the necessary conditions for optimality and equally showed using simulations, that to successfully curb malaria spread, control strategies must include cholera intervention strategies as well. tilahun et al. [32] investigated a pneumonia-typhoid co-infection model with cost-effective optimal control analysis, incorporating preventive and treatment controls for both diseases. they showed that the strategy that combines pneumonia treatment and typhoid fever prevention is the most cost-effective in reducing the burden of the co-infections of pnemonia and typhoid fever. the researchers in [9] analyzed an optimal control model for hiv and tb co-infection, capturing resistance of hiv to anti-retroviral therapy. the authors showed that a combination of tb treatment and anti-retroviral therapy for hiv is the most effective in reducing the burden of the co-infection of the two diseases. in addition, tanvi and aggarwal [31] studied an hiv-tb co-infection, incorporating detection and treatment for both diseases. they observed that the strategy that implements detection for both diseases yielded the most economic and epidemic gains infighting the co-infections of the two diseases. to the best of the authors' knowledge, optimal control analysis has never been applied to the coinfection of human papillomavirus and tb in the literature. hence, it will be imperative to investigate the optimal control and cost-effectiveness analysis of the co-infections of oncogenic hpv and tb. in particular, this paper extends the work of omame et al. [24] by assessing the impact of hpv prevention and tb treatment controls on the control and management of the co-infections of the two diseases. the rest of the paper is organized as follows. the model is formulated in section 2, alongside the basic properties of the model. the co-infection model without controls is analyzed qualitatively in section 3. the optimal control model is considered in section 4 while section 6 gives the concluding remarks. the total sexually active population at time t, denoted by n h (t), is divided into eleven mutually-exclusive compartments: susceptible individuals (s(t)), individuals infected with hpv (i hp (t)), individuals who have recovered from or cleared hpv infection (r hp (t)), individuals with persistent hpv infection (p hp (t)), individuals with latent tb (e t (t)), individuals with active tb infection (a t (t)), individuals treated of tb (t t (t)), individuals dually infected with hpv and latent tb (i p he (t)), individuals dually infected with hpv and active tb i p ha , individuals dually infected with persistent hpv and latent tb, (p p he (t)) individuals dually infected with persistent hpv and active (p p ha (t)). thus n h = s + i hp + r hp + p hp + e t + a t + t t + i p he + i p ha + p p he + p p based on the above formulations and assumptions, the hpv-tb coinfection model is given by the following deterministic system of non-linear differential equations (flow diagram of the model is shown in figure 1 , the associated state variables and parameters are well described in tables 1 and 2) : where: λ hp = β hp [i hp + η p p hp + κ t (i p ha + η t i p he + η p p p ha + η t η p p p he )] the parameters κ t (κ t ≥ 1) is a modification parameter accounting for the increased infectiousness of dually infected individuals due to tuberculosis, η t (η t ≤ 1) is a modification parameter accounting for reduced infectiousness of dually infected individuals due to latent tb. ω p (ω p ≥ 1) is a modification parameter accounting for the increased infectiousness of dually infected individuals due to hpv while η p (η p ≤ 1) is a modification parameter accounting for the reduced infectiousness of singly infected and dually infected individuals due to persistent hpv infection. this population is further reduced by natural death (at a rate µ h natural death occurs in all epidemiological compartments at this rate). in (2) , β hp is the effective contact rate for hpv transmission of hpv infection. 2.1 basic properties of the co-infection model (1) without controls the basic qualitative properties of the oncogenic hpv-tb co-infection model (1) will now be considered. specifically, we establish the following results. for the oncogenic hpv-tb co-infection model (1) to be meaningful in the concept of epidemiological sense, it is necessary to prove that all its state variables are non-negative over time. the approach adopted by omame et al [23] will be used to establish the results below. theorem 2.1 let the initial data be 3 mathematical analysis of the model without controls the co-infection model (1) has a dfe, obtained by setting the right-hand sides of the equations in the model (1) to zero, given by ξ 0 =(s * , i * hp , p * hp , r * hp , e * t , a * t , t * t , i p* he , i p* ha , p p* he , p p* ha ) the basic reproduction number of the oncogenic hpv-tb co-infection model (1), using the approach in van den driessche and watmough [35] , is given by r 0 = max{r 0h , r 0t } where r 0h and r 0t are, respectively, the oncogenic hpv and tb associated reproduction numbers, given by figure 1 : schematic diagram of the model (1) 8 all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 18, 2020. . where, using theorem 2 of [35] , the following result is established. 3.2 global asymptotic stability(gas) of the disease-free equilibrium(dfe) ξ 0 of the co-infection model we shall apply the approach illustrated in [5] to investigate the global asymptotic stability of the disease free equilibrium of the co-infection model. in this section, we list two conditions that if met, also guarantee the global asymptotic stability of the disease-free state. first, system (1) must be written in the form: where v ∈ r m denotes (its components) the number of uninfected individuals and k ∈ r n denotes (its components) the number of infected individuals. u 0 = (v * , 0) denotes the disease-free equilibrium of this system. the conditions (w 1) and (w 2) following must be satisfied in order to guarantee local asymptotic stability: (w 1): for dv dt = p (v, 0), v * is globally asymptotically stable (gas), where b = d k q(v * , 0) is an m-matrix (the off-diagonal elements of b are nonnegative) and ω is the region where the model makes biological sense. if system (1) satisfies the above two conditions then the following theorem holds: is a globally asymptotic stable (gas) equilibrium of (1) provided that r 0 < 1 (las) and that assumptions (w 1) and (w 2) are met where v denotes the number of non-infectious compartments and k denotes the number of infectious it is clear from the above, that,q(v, k) 0. hence the dfe may not be globally asymptotically stable, suggesting the possibility of a backward bifurcation. this supports the backward bifurcation analysis in the proceeding section. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.15.20195297 doi: medrxiv preprint we shall carry out analysis in this section to know the type of bifurcation the model (1) may undergo, using the centre manifold theory as illustrated in [6] . the following result can be established. then model (1) exhibits backward bifurcation at r 0 = 1. if a < 0, then the system (1) exhibits a forward bifurcation at r 0 = 1. suppose represents any arbitrary endemic equilibrium of the model. the existence of backward bifurcation will be studied using the centre manifold theory [6] . to apply this theory, it is appropriate to do the following change of variables. further, using the vector notation (1) can be re-written in the form all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.15.20195297 doi: medrxiv preprint as follows: with: x i consider the case when r 0h = 1. assume, further, that β hp is chosen as a bifurcation parameter. solving evaluating the jacobian of the system (8) at the dfe, j(ξ 0 ), and using the approach in [6] , we have that j(ξ 0 ) has a right eigenvector (associated with the simple zero eigenvalue) given by w = [ω 1 , ω 2 , ω 3 , ω 4 , ω 5 , ω 6 , ω 7 , ω 8 , ω 9 , ω 10 , ω 11 ] t all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 18, 2020. . where, the components of the left eigenvector of where, the associated bifurcation coefficients defined by a and b, given by: 13 all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.15.20195297 doi: medrxiv preprint since the bifurcation coefficient b is positive, it follows from theorem 4.1 in [6] that the model (1), or the transformed model (8), will undergo a backward bifurcation if the backward bifurcation coefficient, a, given by (11) is positive. setting the hpv and tb re-infection paramters ϕp = 0, σt = 0 and the tb exogenous re-infection term ε t 1 = 0, the bifurcation coefficient, in this section, we shall use the pontryagin's maximum principle to determine the necessary conditions for the optimal control of the oncogenic hpv-tb co-infection model. we incorporate time dependent controls into the model (1) to determine the optimal strategy for curbing the co-infections of the two diseases. thus, we have, subject to the initial conditions the control functions, u1(t), u2(t), and u3(t) are bounded, lebesgue integrable functions. the control u1(t) represents the efforts at preventing incident hpv infections. the control u2(t) aims at preventing infection with hpv by tb infected individuals so as to reduce the co-infection cases. tb treatment control for individuals dually infected with oncogenic hpv and tb is denoted by u3(t). the controls u1 and u2 satisfies 0 ≤ u1, u2 ≤ 1 while the control u3 satisfies 0 < u3 ≤ h, where h is the tb drug efficacy used for the treatment of co-infected individuals. our optimal control problem involves a scenario where the number of hpv-infected, tb-infected, the co-infection cases and the cost of implementing preventive and treatment controls u1(t), u2(t) and u3(t) are minimized subject to the state system (12) . for this, we consider the 14 all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in t is the final time. we seek to find an optimal control, u * 1 , u * 2 , u * 3 , such that where t ] is the control set. we shall now establish the existence of such an optimal solution which minimizes the objective functional j. theorem 4.1 given the objective functional j, defined on the control set u , and subject to the state system (12) with non-negative initial conditions at t = 0, then there exists an optimal control triple u * = (u1, u2, u3) such that j(u * ) = min {j(u1, u2, u3)|u1, u2, u3 ∈ u }. we shall prove the existence of the given optimal control u * , along with the corresponding solution trajectories by following the approach used in mohammed-awel and numfor [18] . proof : the state functions are positive and the controls are lebesgue measurable, therefore we have that j(ui) ≥ 0 for all ui ∈ u . as a result, inf u i ∈u j(ui) exists and is finite. therefore, a minimizing sequnce of controls (ui) ∈ u exists such that let s n , i n hp , p n hp , r n hp , e n where, ξ * = (s * , i * hp , p * hp , r * hp , e * t , a * t , t * t , i p* he , i p* ha , p p* he , p p* ha ) since ||ui||l∞ < k for some k > 0, it follows that ui ∈ l 2 ([0, t ]), such that on a sub-sequence applying the lower semi-continuity of l 2 norm with respect to weak convergence, we have that considering the convergence of the sequences and passing to the limit in the ordinary differential equation system (12) , we have that s * , i * hp , p * hp , r * hp , e * the pontryagin's maximum principle [28] gives the necessary conditions which an optimal control pair must satisfy. this principle transforms (12) , (13) and (14) into a problem of minimizing a hamiltonian, h, pointwisely with regards to the control functions, u1, u2, u3: proof of theorem 4.2 is an optimal control and s * , i * hp , p * hp , p * hp , rhp * , e * t , a * t , t * t , i p* he , i p* ha , p p* he , p p* ha are the corresponding state solutions. applying the pontryagin's maximum principle [28] , there exist adjoint variables satisfying: with transversality conditions; we can determine the behaviour of the control by differentiating the hamiltonian, h with respect to the controls(u1, u2, u3) all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.15.20195297 doi: medrxiv preprint at t. on the interior of the control set, where 0 < uj < 1 for all (j = 1, 2, 3), we obtain therefore, we have that [14] u in this section, we shall explore uncertainty and sensitivity analyses on the parameters of the model due to lack of precision in the estimates of some of the co-infection model parameters and the uncertainty which may occur when gathering data for the simulations. we shall also carry out numerical simulations of the optimal control model (12) , in order to assess the impact of different intervention strategies on the dynamics of oncogenic hpv-tb co-infections. the oncogenic hpv-tb co-infection model (1) (1) per lhs were run. using the total number of individuals dually infected with hpv and latent tb (i p he ) as the response function, the parameters that strongly influence the dynamics of the co-infection model (1) are the demographic parameter, µh, the effective contact rate for hpv transmission, βhp, the parameter accounting for increased susceptibility to hpv by tb infected individuals, p 1 , and the recovery rate from hpv for individuals in i p he compartment. also, using the population of individuals dually infected with hpv and active tb (i p ha ) as the input, the five top ranked parameters are the effective contact rate for hpv transmissibility, βhp, the effective contact rate neccesary for tb transmission, βt, the modification parameter accounting for increased susceptibility to hpv infection by active tb infected individuals, p 2 , the recovery rate from hpv for individuals in i p ha compartment and the recovery rate from tb for individuals in i p ha class. taking the population of individuals dually infected with persistent hpv and latent tb (p p he ) as the input, the three top ranked parameters are the effective contact rate for hpv transmissibility, βhp, the fraction of individuals who recover from hpv infection and do not progress to persistent hpv infection, θ p 2 , and the recovery rate from persistent hpv infection, ρ p 3 , for individuals in p p he compartment. when the population of individuals dually infected with persistent hpv and active tb is used as the response function, the two top-ranked parameters are the effctive contact rate for tb transmission, βt and the tb treatment rate, r t 3 , for individuals dually infected with persistent hpv and active tb. considering the hpv associated basic reproduction number, r0h, as the response function, it is observed in table 3 all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.15.20195297 doi: medrxiv preprint that the top four prcc-ranked parameters are the demographic parameter, µh, effective contact rate for hpv transmission, βhp, the fraction of infected individuals who recover from hpv and do not develop persistent hpv, θ p 1 and the recovery rate from hpv φ p 1 . finally, using the tb associated reproduction number, r0t, as the response function the two key parameters that drive the dynamics of the model are the effective contact rate for tb transmission, βt and the tb treatment rate r t 1 . numerical simulations of the optimal control problem (12), adjoint equations (18) and characterizations of the control (21) are implemented by the runge kutta method using the forward backward sweep (carried out in matlab). following the choice of the weight function for hpv vaccination control used in malik et al [17] , the balancing factor χ1 = 10 3 . we also assume χ2 = 500 and χ3 = 400. the demographic data related to the shanxi province in rural china is used [8] . we assume the initial conditions to be: iii. strategy c: control against infection with hpv by tb infected individuals (u2 = 0) and tb treatment control for dually infected individuals (u3 = 0). applying this control strategy, we note that the total number of individuals dually infected with hpv and tb in latent and active stages, respectively, (figures 2 (a) and 2(b) ), and the total number of individuals dually infected with persistent hpv and tb in latent and active stages, respectively (figures 2(c) and 2(d) ) are less when this control is applied than when the control is not applied. to be specific, this control strategy averts almost 420,352 new co-infection cases, which is the greatest number of averted cases in comparison with other control strategies implemented. the control profile for this strategy given in figure 5 , reveals that control u1 is at its upper bound for the first 3.9 years before ultimately declining to zero. similarly, the control u2 is at the maximum value of 90% for the first 8 months before declining to zero at time, t=4.5 years. the simulations of the total number of dually infected individuals in the presence of tb treatment controls are depicted in figures 3(a) -3(d) . applying this control, we observe that the total number of individuals dually infected with hpv and tb in latent and active stages of infection respectively, is less than the total population when no control is applied as expected (figure 3(a) and 3(b) ). similarly, it is noticed from figures (3(c) and 3(d) , that high population level impact is observed in the total number of individuals dually infected with persistent hpv and tb in latent and active stages of infection, respectively, when this control strategy is applied. specifically, when this control strategy is implemented, about 300,549 new co-infection cases were averted. the control profile for this strategy presented in figure 7 shows that control u1 is at its upper bound for the first 4.0 years before steadily declining to zero at final time. also, the control u3 is at the maximum value of 100% for the first 1.2 years and then steadily declines to zero at time, t=4.5 years. this simulation results conforms with the epidemiological report in the introduction section that gynaecologic tb is a risk factor for oncogenic hpv infection (and subsequent cancer infection) [37] . hence, if we focus on tb treatment controls, it can significantly reduce the burden of the co-infection of oncogenic hpv and tb in a population. the simulations are also in line with the point opined by [37] , that prior tb infection was associated with persistent hpv and increased susceptibility to cervical cancer. as a result, treating tb infections in dually infected individuals will significantly curb the mixed infections of both diseases. 20 all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 18, 2020. (figures 2c and 2d) , respectively, the presence of optimal vaccination control against incident hpv infection (u 1 = 0) and control against infection with hpv by tb infected individuals (u 2 = 0). here, β hp = 2.0, β t = 8.557. all other parameters as in table 2 table 2 all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 18, 2020. figure 9 reveals that control u2 is at its upper bound for the first 4.5 years before gradually falling down to zero at final time. moreso, the control u3 is at its minimum value for the first 2.1 years and then steadily rises to its peak value of 100% at time, t=2.7 years, before gradually declining to zero at final time. it is imperative to state categorically, that the results of the simulations are based on the parameter values given in table 2 and the initial conditions and weight constants given in section 5.2 the cost-effectiveness analysis is used to evaluate the health interventions related benefits so as to justify the costs of the strategies [4] . this is obtained by comparing the differences among the health outcomes and costs of those interventions; achieved by computing the incremental cost-effectiveness ratio (icer), which is defined as the cost per health outcome. it is given by: difference in costs between strategies difference in health effects between strategies . we calculated the total number of co-infection cases averted and the total cost of the strategies applied in table 4 . the total number of co-infection cases prevented is obtained by calculating the total number of individuals when controls are administered and the total number when control is not applied. similarly, we apply the cost functions 1 2 χ1u 2 1 , 1 2 χ2u 2 2 , 1 2 χ3u 2 3 , over time, to compute the total cost for the various strategies that we implemented. we now compare the cost-effectiveness of strategy b (optimal hpv vaccination control for sexually active susceptible individuals against incident hpv infection and tb treatment control for dually infected individuals) and strategy c (control against hpv infection by tb infected figure 10 : plots of the total number of individuals dually infected with hpv and tb in latent and active stages, respectively (figures 10a and 10b ) as well as total number of individuals dually infected with persistent hpv and tb in latent and active stages (figures 10c and 10d) , respectively, in the presence of control against hpv infection by tb-infected individuals (u 2 = 0) and tb treatment control dually infected individuals (u 3 = 0). here, β hp = 2.0, β t = 8.557. all other parameters as in table 2 individuals and tb treatment controls for dually infected individuals). from icer (b) and icer(c), we observe a cost saving of 0.01298 observed for strategy c over strategy b. this implies that strategy b strongly dominated strategy c, showing that strategy b is more costly and less effective compared to strategy c. therefore, strategy b is removed from subsequent icer computations, shown in table 5 . we shall now compare strategies c and a. comparing strategy c (control against hpv infection by tb infected individuals and tb treatment controls preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 18, 2020. , showing that strategy a strongly dominated strategy c and is more expensive and less effective compared to strategy c. consequently, strategy c (the strategy that combines and implements control against hpv infection by tb infected individuals as well as tb treatment controls for dually infected individuals) has the least icer and is the most cost-effective of all the control strategies for the control and management of oncogenic hpv and tb co-infection. this clearly agrees with the results obtained from acer method in table 4 that strategy c is the most cost-effective strategy. in this work, we have developed and presented a co-infection model for oncogenic hpv and tb with cost-effectiveness optimal control analysis. the full co-infection model was shown to undergo the phenomenon of backward bifurcation when the associated reproduction number is less than unity. it was further shown that tb and hpv re-infection parameters (ϕp = 0 and σt = 0) as well as the exogenous re-infection term (ε1 = 0) induced the phenomenon of backward bifurcation in the oncogenic hpv-tb co-infection model. the global asymptotic stability of the disease-free equilibrium of the coinfection model was also proven not to exist, when the associated reproduction number was below unity. the necessary conditions for the existence of optimal control and the optimality system for the co-infection model was established using the pontryagin's maximum principle. uncertainty and global sensitivity analyses were also carried out to determine the top ranked parameters that influence the dynamics of the co-infection model, when the associated reproduction numbers as well as the infected populations were used as response functions. when the population of individuals dually infected with hpv and latent tb (i p he ) was used as the response function, the parameters that strongly influence the dynamics of the co-infection model (1) are the demographic parameter, µh, the effective contact rate for hpv transmission, βhp, the parameter accounting for increased susceptibility to hpv by tb infected individuals, p 1 , and the recovery rate from hpv for individuals in i p he compartment. in addition, using the population of individuals dually infected with hpv and active tb (i p ha ) as the input, the five top ranked parameters are the effective contact rate for hpv transmissibility, βhp, the effective contact rate neccesary for tb transmission, βt, the modification parameter accounting for increased susceptibility to hpv infection by active tb infected individuals, p 2 , the recovery rate from hpv for individuals in i p ha compartment and the 25 all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.15.20195297 doi: medrxiv preprint recovery rate from tb for individuals in i p ha class. taking the population of individuals dually infected with persistent hpv and active tb as the response function, the two top-ranked parameters are the effctive contact rate for tb transmission, βt and the tb treatment rate, r t 3 , for individuals dually infected with persistent hpv and active tb. numerical simulations of the optimal control model showed that: i. a combination of hpv prevention control and tb treatment control has a positive population level impact in reducing the burden of oncogenic hpv and tb co-infection cases in a population. ii. a strategy that implements control against hpv infection by tb infected individuals and tb treatment controls for dually infected individuals can significantly reduce the burden of oncogenic hpv and tb co-infections. iii. the strategy that combines and implements control against hpv infection by tb infected individuals as well as tb treatment control for dually infected individuals has the least icer and is the most cost-effective of all the control strategies for the control and management of the burden of oncogenic hpv and tb co-infection. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted september 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study tuberculosis and oncogenic hpv: potential co-infections in women at high-risk of cervical cancer in rural china key: cord-159554-50077dgk authors: shan, fei; gao, yaozong; wang, jun; shi, weiya; shi, nannan; han, miaofei; xue, zhong; shen, dinggang; shi, yuxin title: lung infection quantification of covid-19 in ct images with deep learning date: 2020-03-10 journal: nan doi: nan sha: doc_id: 159554 cord_uid: 50077dgk ct imaging is crucial for diagnosis, assessment and staging covid-19 infection. follow-up scans every 3-5 days are often recommended for disease progression. it has been reported that bilateral and peripheral ground glass opacification (ggo) with or without consolidation are predominant ct findings in covid-19 patients. however, due to lack of computerized quantification tools, only qualitative impression and rough description of infected areas are currently used in radiological reports. in this paper, a deep learning (dl)-based segmentation system is developed to automatically quantify infection regions of interest (rois) and their volumetric ratios w.r.t. the lung. the performance of the system was evaluated by comparing the automatically segmented infection regions with the manually-delineated ones on 300 chest ct scans of 300 covid-19 patients. for fast manual delineation of training samples and possible manual intervention of automatic results, a human-in-the-loop (hitl) strategy has been adopted to assist radiologists for infection region segmentation, which dramatically reduced the total segmentation time to 4 minutes after 3 iterations of model updating. the average dice simiarility coefficient showed 91.6% agreement between automatic and manual infaction segmentations, and the mean estimation error of percentage of infection (poi) was 0.3% for the whole lung. finally, possible applications, including but not limited to analysis of follow-up ct scans and infection distributions in the lobes and segments correlated with clinical findings, were discussed. the outbreak of 2019 novel coronavirus in wuhan, china has rapidly spread to other countries since dec 2019 [1] [2] [3] [4] [5] [6] [7] . the infectious disease caused by this virus was named as covid-19 by the world health organization (who) on feb 11, 2020 8 . to date (mar 5 th 2020), there have been 80,565 confirmed cases in china and 95,333 confirmed cases all around the world 9 . each suspected case needs to be confirmed by the real-time polymerase chain reaction (rt-pcr) assay of the sputum 10 . although it is the gold standard for diagnosis, confirming covid-19 patients using rt-pcr is time-consuming and has been reported to suffer from high false negative rates. on the other hand, because chest ct scans collected from covid-19 patients frequently show bilateral patchy shadows or ground glass opacity (ggo) in the lung 11, 12 , it has been used as an important complementary indicator in covid-19 screening due to high sensitivity. chest ct examination has also shown its effectiveness in follow-up assessment of hospitalized covid-19 patients 13 . due to fast progression of the disease, subsequent ct scans every 3-5 days are recommended to evaluate the therapeutic responses. although ct provides rich pathological information, only qualitative evaluation has been provided in the radiological reports owing to the lack of computerized tools to accurately quantify the infection regions and their longitudinal changes. thus, subtle changes across follow-up ct scans are often ignored. besides, contouring infection regions in the chest ct is necessary for quantitative assessment; however, manual contouring of lung lesions is a tedious and time-consuming work, and inconsistent delineation could also lead to subsequent assessment discrepancies. thus, a fast auto-contouring tool for covid-19 infection is urgently needed in the onsite applications for quantitative disease assessment. we developed a deep learning (dl)-based segmentation system for quantitative infection assessment. the system not only performs auto-contouring of infection regions, but also accurately estimates their shapes, volumes and percentage of infection (poi) in ct scans of covid-19 patients. in order to provide delineation for hundreds of the training covid-19 ct images, which is a tedious and time-consuming work, we proposed a human-in-the-loop (hitl) strategy to iteratively generate the training samples. this method involves radiologists to efficiently intervene dl-segmentation results and iteratively add more training samples to update the model, and thus greatly accelerates the algorithm development cycle. to the best of our knowledge, there are no literatures that have reported the utilization of hitl strategy in identifying covid-19 infection in ct scans. the protocol of this retrospective study was approved by the ethics of committees of shanghai public health clinical center. informed consent was waived because of the respecpective nature of the study, and all the private information of patients was anonymized by the investigators after data collection. totally 300 ct images from 300 covid-19 patients (from shanghai) were collected for validation. 249 ct images of 249 covid-19 patients were collected from other centers (outside shanghai) for training. the inclusion criteria are list as follows: (a) patients with a positive new coronavirus nucleic acid antibody and confirmed by the cdc; (b) patients who underwent thin-section ct; (c) age >=18; (d) presence of lung infection in ct images. patients with ct scans showing large motion artifacts or pre-existing lung cancer conditions were excluded in this study. 51 of 300 patients have been previously reported 23 . the prior article investigated the clinical, laboratory, and imaging findings of covid-19 pneumonia in humans, whereas in this manuscript we develop a deep learning system to quantify covid-19 infection in ct scans. the patient data were used for validation of system performance. all covid-19 patients underwent thin-section ct scan (scenaria 64 ct, hitachi medical, japan). the median duration from illness onset to ct scan was 4 days, ranging from 1 to 14 days. the ct protocol was as follows: 120 kv; automatic tube current (180 ma-400 ma); iterative reconstruction; 64 mm detector; rotation time, 0.35 sec; slice thickness, 5 mm; collimation, 0.625 mm; pitch, 1.5; matrix, 512×512; and breath hold at full inspiration. the reconstruction kernel used is set as "lung smooth with a thickness of 1 mm and an interval of 0.8 mm". during reading, the mediastinal window (with window width 350 hu and window level 40 hu) and the lung window (with window width 1200 hu and window level-600 hu) were used. due to the low contrast of the infection regions in ct images and large variation of both shape and position across different patients, delineating the infection regions from the chest ct scans is very challenging. we developed a dl-based network called vb-net for this purpose. it is a modified 3-d convolutional neural network that combines v-net 14 with the bottle-neck structure 15 . vb-net consists of two paths ( figure 1 ). the first is a contracting path including down-sampling and convolution operations to extract global image features. the second is an expansive path including up-sampling and convolution operations to integrate fine-grained image features. compared with v-net 14 , the speed of vb-net is much faster because the bottle-neck structure is integrated in vb-net, as detailed in figure 1 16, 17 . the bottle-neck design is a stacked 3-layer structure. the three layers use 1×1×1, 3×3×3 and 1×1×1 convolution kernels, where the first layer with 1×1×1 kernel reduces the number of channels and feeds the data for a regular 3×3×3 kernel layer processing, and then the channels of feature maps are restored by another 1×1×1 kernel layer. by reducing and combining feature map channels, not only the model size and inference time are greatly reduced, but also cross-channel features are effectively fused via convolusion, which makes vb-net more applicable to deal with large 3d volumetric data than traditional v-net. training samples with detailed delineation of each infection region are required for the proposed vb-net. however, it is a labor-intensive work for radiologists to annotate hundreds of covid-19 ct scans. we, therefore, adopted the human-in-the-loop (hitl) strategy to iteratively update the dl model. specifically, the training data were divided into several batches. first, the ct data in the smallest batch are manually contoured by radiologists. then, the segmentation network was trained by this batch as an initial model. this initial model was applied to segment infection regions in the next batch, and radiologists manually correct the segmentation results provided by the segmentation network. these corrected segmentation results were then fed as new training data, and the model can be updated with increased training dataset. in this way, we iteratively increased the training dataset and built the final vb-net. in the testing stage, the trained segmentation network segments the infection area on a new ct scan via a forward pass of neural network, and the hitl interaction also provides possible intervention and human-machine interaction for radiologists in clinical application. according to our experience, this hitl training strategy converged after 3~4 iterations. figure 2 illustrates the process of the proposed hitl training strategy. after segmentation, various metrics were computed to quantify the covid-19 infection, including volumes of infection in the whole lung, and volumes of infection in each lobe and each bronchopulmonary segment. in addition, the pois in the whole lung, each lobe and each bronchopulmonary segment were also computed, respecrively, to measure the severity of covid-19 and the distribution of infection within the lung. the hounsfield unit (hu) histogram within the infection region can also be visualized for evaluation of ggo and consolidation components inside the infection region. figure 3 shows the entire pipeline for quantitative covid-19 assessment. a chest ct scan is first fed to the dl-based segmentation system, which generates infection areas, the whole lung, lung lobes, and all the bronchopulmonary segments, respectively. then, the aforementioned quantitative metrics are calculated to quantify infection regions of the patient. the quantification provides the basis for measuring the severity of covid-19 from the ct perspective and for tracking longitudinal changes during the treatment course. statistical analysis was performed by r version 3.6.1 (r project for statistical computing, vienna, austria). because a majority of the continuous data did not follow a normal distribution, they were expressed as the median and interquartile range (iqr, 25th and 75th percentiles). the dice similarity coefficient (dsc) was used to evaluate the overlap ratio between an automatically segmented infection region ( ) and the corresponding reference region ( ) provided by radiologist(s). it is calculated as follows: where |•| is the operator to calculate the number of voxels in the given region, and ∩ is the intersection operator. the pearson correlation coefficient 18 was used to evaluate the correlation of two variables: where is the total number of observations, and , = 1, ⋯ , , are the observations of the two variables. to demonstrate the effectiveness, figure 4 shows typical cases of covid-19 infection in three different stages: early stage, progressive stage and severe stage. coronal images without and with overlaid segmentation are presented in parallel for comparison. in addition, 3d rendering of each case is also provided to give a more vivid understanding of covid-19 infection within the lung. all three cases show that the contours delineated by the deep learning system match well with the visable lesion boundaries in ct images. to quantitatively evaluate the accuracy of segmentation and measurement, infection regions on 300 ct scans of 300 covid-19 patients were manually contoured by two radiologists (w.s. and f.s., with 12 and 19 years of experience in chest radiology, respectively) to serve as the reference standard. each case was manually contoured by one radiologist and reviewed by the other. in case of disagreement, the final results were determined by consensus between the two radiologists. the automatically segmented infection regions are compared to the reference standard in terms of overlap ratio (measured by dice similarity coefficient), volume, the percentage of infection (poi) in the whole lung, poi in each lung lobe, and poi in each bronchopulmonary segment. inter-rater variability was assessed by randomly sampling 10 ct scans of covid-19 patients from the entire validation set. the two radiologists first independently contoured the infection regions in these ct scans. their manual segmentation were then compared using the same metrics as mentioned above. ). the mean poi estimation difference is 0.2% for whole lung, 0.3% for lung lobes, and 0.4% for bronchopulmonary segments. 91.4% of lung-lobe pois and 85.9% of bronchopulmonary-segment pois are consistently estimated with equal or less than 1% difference. by comparing table 1 and table 2 , it can be seen that the segmentation and measurement errors of the deep learning system are close to the inter-rater variability. this demonstrates the effectiveness of using deep learning to quantify the covid-19 infection in ct images. two metrics were used to evaluate the hitl strategy. first, the time of manual contouring was recorded to compare labeling time of a ct scan with the deep learning model. second, the segmentation accuracy of deep learning models at different stages was assessed to see whether the accuracy improves with more annotated training data. table 3 shows the labeling time and segmentation accuracy at different stages. without any assistance of deep learning, it takes 211.3±52.6 minutes to contour covid-19 infection regions on one ct scan. the contouring time drops dramatically to 31.1±8.1 minutes with the assistance of the first deep learning model trained with 36 annotated ct scans. it further drops to 12.0±2.9 minutes with 114 annotated data, and to 4.7±1.1 with 249 annotated data. meanwhile, the segmentation accuracy of deep learning models was evaluated using dice similarity coefficient on the entire 300 validation set. it improves from 85.1±11.4%, to 91.0±9.6%, and to 91.6%±10.0 with more training data added. the improved segmentation accuracy greately reduces human intervention and thus reduces significantly the time of annotation and labeling. ct imaging has become an efficient tool for screening covid-19 patients and for assessing the severity of covid-19. however, radiologists lack a computerized tool to accurately quantify the severity of covid-19, e.g., the percentage of infection in the whole lung. in the literature, deep learning has become a popular method in medical image analysis and has been used in analyzing diffuse lung diseases on ct 19, 20 . in this work, we explored deep learning to segment covid-19 infection regions within lung fields on ct images. the accurate segmentation provides quantitative information that is necessary to track disease progression and analyze longitude changes of covid-19 during the entire treatment period. we believe that this deep learning system for covid-19 quantification will open up many new research directions of interest in this community. the first potential application of this system is to quantify longitudinal changes in the followup ct scans of covid-19 patients. hospitalized patients with confirmed covid-19 typically take a ct examination every 3-5 days. as currently there is no effective medicine to target covid-19, most patients recover with different degrees of supportive medicine intervention. given lots of such patients, it is interesting to see how disease progresses under different clinical management. figure 5 gives a case with three follow-up ct scans. with infection region segmented, the changes of infection volume as well as consolidation and groundglass opacities can be easily visualized using surface rendering technique. the poi estimated by our system can be used to indicate the severity of covid-19 from the radiology perspective. it is of great interst to find out how this poi correlates with clinical pneumonia assessment. pneumonia severity index (psi) is a clinical prediction rule that is often used to calculate the probability of morbidity and mortality among patients with community acquired pneumonia 21, 22 . it is calculated based on demographics, the coexistence of comorbidity illnesses, and physical and laboratory examinations. in our study, covid-19 patients were classified into non-severe (psi level ≤2) and severe groups (psi level ≥3). the pois in the whole lung were calculated from their ct scans by the system. based on 196 patients with both psi and poi available, the pearson correlation coefficient between these two variables gives 0.5, which means moderate correlation between these two scores. this result indicates the poi estimated from ct scans is clinically relevant with the severity of pneumonia. ongoing research works are being carried on to study whether poi or its derived coefficients are helpful in predicting covid-19 disease progression. another application of our system is to explore the quantitative lesion distribution specifically related to covid-19. according to recent literature 23, 24 , covid-19 infection happens more frequently in lower lobes of the lung. however, so far no researches have reported quantitatively the severity of covid-19 infection in each lung lobe and bronchopulmonary segment. with this deep learning system, the pois of lung lobes and bronchopulmonary segments can be automatically calculated. thus, statistics of infection distribution can be summarized in a large-scale dataset, e.g., 300 ct scans in our study. figure 6 show the boxplots of these pois calculated from 300 ct scans of covid-19 patients in shanghai district. figure 6 (a) shows that the mean pois of left and right lower lobes are higher than those of other lobes, which coincides with the findings reported in 23, 24 . moreover, infection distribution can be analyzed further down to the bronchopulmonary segment level, as shown in figure 6 (b). to the best of our knowledge, this is the first work that reveals the covid-19 distribution in bronchopulmonary segments in terms of a largescale patient ct data. our results show that the following segments are often infected by covid-19 (listed with decreasing mean poi): right lower lobe -outer basal, right lower lobe -dorsal, right lower lobe -posterior basal, left lower lobe -outer basal, left lower lobe -dorsal, left lower lobe -posterior basal, and right upper lobe -back. using hitl strategy in training the segmentation network is a novel feature of our system. existing ai-based systems for automatic quantitative assessment always requires a large amount of annotation ct data, whereas collecting the annotated data is very expensive or even difficult. moreover, these ai systems are always trained as a black box to users, who however always want to know what has happened behind the model. our experimental results indicate that the hitl strategy makes the manual annotation process faster with the assistance of deep learning models. also, the hitl strategy makes the system more comprehensible. that is, with manual intervention in hitl, the radiologists are aware of how good the system performs in the training process. besides, the hitl strategy helps radiologists accustomed to the ai system because they are involved in the training process. it integrates the professional knowledge from radiologists in an interactive way. it is worth noting the limitations of our work in several aspects. first, the validation ct datasets were collected in one center, which may not be representative of all covid-19 patients in other geographic areas. the generalization of the deep learning system needs to be further validated on multi-center datasets. second, the system is developed to quantify infections only, and it may not be applicable for quantifying other pneumonia, e.g., bacterial pneumonia. finally, in our future work, we will extend the system to quantify severity of other pneumonia using transfer learning. with this automatic dl-based segmentation, many studies on quantifying imaging metrics and correlating them with syndromes, epdemicology, and treatment responses could further reveal insights about imaging markers and findings towards improved diagnosis and treatment for covid-19. tables table 1 . quantitative evaluation of the deep learning segmentation system on the validation dataset. the dice coefficients, volume estimation error, and poi estimation error in the whole lung, lung lobes and bronchopulmonary segments were calculated to assess the automatic segmentation accuracy. table 2 . inter-rater variability analysis between two radiologists on randomly sampled 10 ct cases. the dice coefficients, volume estimation difference, and poi difference in whole lung, lung lobes and bronchopulmonary segments were estimated to serve as the reference for assessing the automatic segmentation accuracy. a novel coronavirus from patients with pneumonia in china a novel coronavirus genome identified in a cluster of pneumonia cases -wuhan importation and human-to-human transmission of a novel coronavirus in vietnam first case of 2019 novel coronavirus in the united states a novel coronavirus outbreak of global health concern the response of milan's emergency medical system to the covid-19 outbreak in italy. the lancet a new coronavirus associated with human respiratory disease in china severe acute respiratory syndrome-related coronavirus-the species and its viruses, a statement of the coronavirus study group chest ct for typical 2019-ncov pneumonia: relationship to negative rt-pcr testing clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china imaging profile of the covid-19 infection: radiologic findings and literature review fully convolutional neural networks for volumetric medical image segmentation deep residual learning for image recognition image-guided procedures, robotic interventions, and modeling; 2019: international society for optics and photonics automatic mr kidney segmentation for autosomal dominant polycystic kidney disease applied multiple regression/correlation analysis for the behavioral sciences automatic lung segmentation based on texture and deep features of hrct images with interstitial lung disease lung segmentation on hrct and volumetric ct for diffuse interstitial lung disease using deep convolutional neural networks a prediction rule to identify low-risk patients with community-acquired pneumonia validity of pneumonia severity index and curb-65 severity scoring systems in community acquired pneumonia in an indian setting emerging coronavirus 2019-ncov pneumonia chest ct findings in coronavirus disease-19 (covid-19): relationship to duration of infection key: cord-018545-fk17n2bx authors: dorofaeff, tavey; mohseni-bod, hadi; cox, peter n. title: infections in the picu date: 2012 journal: textbook of clinical pediatrics doi: 10.1007/978-3-642-02202-9_268 sha: doc_id: 18545 cord_uid: fk17n2bx nan effective control of infections starts at the community level, outside the hospital. there are a number of important initiatives that, although simple and not necessarily intensive care related, have the greatest impact on the outcomes of infections. these include provision of adequate and age appropriate foods, breast feeding, drinkable water, provision of mosquito nets and shelter, avoidance of overcrowding, sanitization, and prevention of disease by vaccination. these are basic needs and requirements of mankind as a basis of good health. they are attainable in the largest cities or the most remote areas. infections are one of the commonest causes of mortality in the pediatric intensive care unit (picu), with a mortality of up to 50%, depending on the origin of the infection. infections in the intensive care unit can be divided into those that occur outside the hospital (community acquired) and those that occur within the walls of the hospital and beyond 48 h of admission (nosocomial). preventive measures give the most benefit, both outside and inside the hospital. good hand washing, good respiratory care practice, and judicious use of antibiotics are examples of effective interventions that reduce the rate of nosocomial infections. sepsis comprises up to 25% of admissions to a typical pediatric intensive care unit. shock and management of septic shock are discussed elsewhere in this text. however basic principles of management are the same and are not, and should not be, limited to the intensive care unit. treatment should commence as soon as the recognition of any septic process is underway be it in the field, the clinics, emergency departments, or on the wards. in the community, on the hospital wards, and in the picu, timely identification of illness and access to skilled healthcare personnel are crucial steps limiting the development of organ dysfunction and failure. early identification means early resuscitation and early treatment. this may be hours or in some cases days prior to the admission to the picu. this early recognition and intervention gives the patient the greatest chance of surviving a significant infection. crucial to the management of any serious infection in the intensive care unit and elsewhere is the early use of appropriate antibiotics. early identification of most bacteria is almost universally by the way of a gram stain. this can be performed in any microbiology lab, in the field, or a clinic that is suitably equipped. though references such as the ''red book'' (american academy of pediatrics) give invaluable information on the appropriate antimicrobial therapy for a given microbe or infectious syndrome, there is no substitute for a well informed, up-to-date infectious diseases physician or microbiologist. they are able to provide information on local isolates, patterns of sensitivity, and best management practices for a large variety of infections. the majority of the bacteria listed below will be referenced elsewhere; they are highlighted to reflect their frequency of identification in the picu. additionally, the immunocompromised host will be at risk from a number of opportunistic infections that will be discussed later in this chapter. these bacteria have a tendency for multiple antibiotic resistances. there are a number of other medically significant bacteria that only periodically present in the pediatric intensive care unit. these bacteria are prevalent in some regions and not elsewhere. they will not be discussed further in this chapter. fungi (of importance in the picu) classification • yeasts (e.g., candida or cryptococcus sp.) • molds -filamentous fungi (e.g., aspergillus sp. and trichophyton sp.) • dimorphic fungi -yeasts in tissue but grow in vitro as molds (e.g., histoplasmosis) candida. c. albicans has the highest incidence in the critical care environment followed by c. parapsilosis, c. tropicalis, c. glabrata, and c. krusei. localized and systemic infections in neonates and immunocompromised children. any organ can be involved: mucous membranes, larynx, esophagus, brain, eyes, lungs, heart, kidneys, liver, and spleen. aspergillus. a. fumigatus is the most common species in invasive aspergillosis, followed by a. flavus, and a. nigra. localized and systemic infection in immunocompromised children (particularly post stem cell transplant and in children with aml); in the skin, subcutaneous tissues, nasopharynx, lungs, brain, and virtually any other organ. endemic mycosis (histoplasmosis, blastomycosis). pneumonitis, hepatosplenomegaly, fever in children with t-cell dysfunction and in those with hiv infection. coccidioides immitis. pneumonia, meningitis in children with t-cell dysfunction and in those with hiv infection. cryptococcus neoformans. pneumonia, meningitis in children with t-cell dysfunction and in those with hiv infection. human herpes viruses 1. herpes simplex virus (hsv, types 1 and 2) (a) systemic infection in the neonate with shock and coagulopathy and severe liver failure (b) encephalitis, hepatitis (c) local (mouth, esophagus, larynx, lungs, heart, liver, kidneys, cns) or systemic disease in organ and stem cell transplant and immunocompromised patients 2. cytomegalovirus (cmv) (a) congenital infection in the neonate with systemic involvement (b) localized (liver, lungs, heart, kidneys, gi, cns, eyes) or systemic infection in solid organ and stem cell transplant as well as immunocompromised patients 3. epstein-barr virus (ebv) (a) infectious mononucleosis (b) burkitt's lymphoma, x-linked lymphoproliferative disorder (xl-lpd), post transplant lymphoproliferative disorder (ptld) (c) localized (liver, heart, lungs, kidneys, gi, cns) it is likely that viral infections have been underestimated both in their frequency and the degree of morbidity they cause. indisputably, in the modern day, hiv (human immunodeficiency virus) is one of the most significant viral pathogens worldwide, particularly in africa and developing nations that do not have the available resources to prevent spread among the community and more importantly maternal infection of the newborn. this leads to a range of morbidities as discussed in the immunocompromised section of this chapter. viral infections are mostly diagnosed clinically on the basis of history and physical examination as well as the regional prevalence of viral diseases. there are a number of ways to test for the presence of a particular virus from either patient blood or other body fluids. these are: tissue culture, serology and seroconversion, immunoflouresence, and pcr (polymerase chain reaction). only two of these are of any use to the intensivist: pcr and immunoflouresence. the turnaround time for viral culture and serum serology is inefficient in the critical care context. respiratory viruses (respiratory syncytial virus, influenzae, adenovirus, parainfluenzae, and human metapneumovirus) are the main contributors to viral disease in the picu, as they are in the general pediatric population. in the picu the majority of patients that develop significant degrees of illness are those who have significant comorbidities. conditions such as ex-prematurity, chronic lung disease, neuromuscular diseases, and congenital heart disease are probably the most common of these. herpes virus family, particularly herpes simplex virus (hsv), is the next important contributor to the burden of viral disease. all members of this family (hsv, ebv, cmv, and vzv) can cause serious infections in the neonate and in immunocompromised children. all services that treat the acutely unwell child (and adult) are at risk of being overwhelmed in an epidemic. national and regional planning needs to be undertaken prior to the advent of any serious infection where ever possible. (examples are sars -severe acute respiratory syndrome, or h1n1 ''swine flu.'') this is encapsulated in the worldwide pandemic planning taking lessons from the sars epidemic and the last major (in terms of mortality) influenza epidemic, the ''spanish flu'' that was prevalent from 1918 to 1920. at a hospital or an organizational level the concept of ''surge strategy'' is used. this is an organization based contingency plan to deal with large numbers of patients admitted simultaneously (i.e., mass trauma casualties or epidemics). in the case of influenza (or sars) this is relevant to the intensive care in that there is a finite capacity of any unit to provide mechanical ventilation. in addition to this, the institution is responsible for the protection of health care workers who are at high risk to contract an infectious illness and become a patient themselves. this would further increase the burden of illness and has the potential to limit available human resources. the prevalence of hiv in the general population and, in particular, in children in many developing countries poses significant stress on limited resources. hopefully, with more effective preventive programs to control vertical transmission of the infection and with availability of affordable anti-hiv medications, the quality of care for hiv-infected children will improve and the need for intensive care will diminish. regional or local experience is crucial in the management of many infections. dengue fever and viral hemorrhagic fevers, which are of more global importance, will be reviewed in more detail. dengue infections, caused by the four antigenically distinct dengue virus serotypes (den1, den2, den3, den4) of the family flaviviridae, are the most important arbovirus diseases. dengue is the most widely distributed mosquito-borne viral infection of humans, affecting an estimated 100 million people worldwide annually. dengue hemorrhagic fever usually occurs in children, with peaks in incidence at 7 months of age (with dengue-immune mothers), and at 3-5 years of age (during a second infection with a new serotype). it is spread throughout the tropical and subtropical zones between 30 n and 40 s where environmental conditions are optimal for viral transmission by aedes mosquitoes, principally aedes aegypti. the disease is endemic in se asia, the pacific, west africa, the caribbean, and the americas. global warming, by increasing the range of aedes mosquito, has the potential to lead to more widespread disease. who has classified the severity of dengue infection on the basis of a combination of clinical and laboratory findings (presence of hypotension and shock, tourniquet test, lowest platelet count, plasma leakage represented by high hematocrit level) in to: • dengue fever • dengue hemorrhagic fever • dengue shock syndrome (dss) viral hemorrhagic fever is a loosely defined category that includes infections from a host of viruses leading to similar clinical syndromes and sharing a similar severity of illness. otherwise, these viruses are different from each other with regard to their reservoir hosts, geographic distribution, and taxonomy. risk factors for exposure also vary among these infections and hence the control methods are geared to specific infections and their causative agents and intermediate hosts. in endemic areas diagnosis is by and large clinical and is confirmed by serological tests and viral pcr or culture. there are vaccines developed for some of these viruses. as a group, the treatment for these infections in the picu is mainly supportive and includes measures to: • optimize hemodynamic state and treat shock • monitor and control brain edema and intracranial hypertension • support ventilation and gas exchange with noninvasive or invasive ventilation • treat coagulopathic state if symptomatic • provide renal replacement therapy if needed; monitor and optimize glucose and electrolyte levels exhaustive discussion of this topic is beyond the current chapter, so the most important ones are briefly mentioned here. yellow fever is endemic in tropical africa between 15 n to 10 s and in parts of central and south america between 10 n and 40 s. in the life-cycle of this virus, in different parts of the world, mosquitoes (a. aegypti, haemagogus, and sabethes), monkeys, and people are involved; however, epidemic mosquito-borne human-to-human transmission can occur. after an incubation period of 3-10 days, fever, headache, malaise, nausea and vomiting, and musculoskeletal pain occur suddenly. initially, the clinical signs may include conjunctivitis, flushing of the skin, and relative bradycardia. in about 10% of cases the illness deteriorates with development of shock, systemic toxicity, gi bleeding, renal dysfunction, liver failure and jaundice, encephalopathy, and systemic bleeding. this latter picture is associated with a high mortality rate (30-50%). differential diagnosis includes other viral hemorrhagic fevers, viral hepatitis, leptospirosis, malaria, typhus, typhoid fever, brucellosis, rickettsial disease, and some intoxications. therapy is supportive and these patients may need intensive care admission for hepatic, renal and circulatory failure. who has recommended routine childhood vaccination in endemic areas (for children >4 months of age). vector control is important in highly populated areas to reduce the risk of epidemic transmission. lassa fever causes as many as 300,000 cases and 5,000 deaths each year in west africa and is a leading cause of maternal and fetal deaths. the virus is carried by mastomys huberti and mastomys erythroleucus, the rodent reservoirs whose infectious excretions are the source of human infections in west africa. in adults and children, early illness includes fever, malaise, headache, and musculoskeletal pain. these nonspecific symptoms progress over 4-5 days to include pharyngitis, cough, chest pain, diarrhea, and vomiting. in endemic areas, a purulent pharyngitis, with conjunctivitis, head and neck edema, and mucosal bleeding are highly specific signs of lassa fever. in severe cases, the illness may be complicated by hypovolemic shock, encephalopathy, respiratory distress caused by laryngeal edema, pleural effusions, or pneumonitis. liver failure, systemic and gi/gu bleeding, and myocarditis can occur. mortality is between 15% and 30%. there are anecdotal reports of the use of intravenous ribavirin in critically ill children with lassa fever but treatment is mainly supportive. lassa fever has been transmitted from person to person during hospitalization. universal exposure precautions should be observed as well as contact and droplet precautions. cchf is caused by a nairovirus (family bunyaviridae), and is transmitted by hyalomma ticks and by contact with infectious body fluids. the geographical distribution of the hyalomma ticks covers africa, the middle east and mediterranean areas, eastern russia, and west asia. the incubation period is from 2 to 9 days. illness onset is abrupt and nonspecific, with fever, chills, rigors, intense headache, and generalized muscle pain. onset of bleeding in the skin, mucous membranes, and the gi tract usually occurs after 3-6 days of illness. hepatitis, liver failure, circulatory failure, shock, and ards can ensue with mortality in up to 30% of cases. treatment is mainly supportive. the virus is sensitive in vitro to ribavirin, and this agent has been used in management of cchf with variable success (who). the value of immune plasma from recovered patients for therapeutic purposes has not been demonstrated, although it has been employed on several occasions (who). patients with suspected or confirmed cchf should be cared for by staff using added droplet and contact precautions. hfrs is caused by old world hantaviruses (family bunyaviridae). the reservoirs are small rodents, and humans are infected percutaneously or by direct exposure. clinical illness has an abrupt onset with fever, severe musculoskeletal pain, renal failure, systemic and gi bleeding, circulatory failure, and shock. this form of the disease is more common in asia and eastern europe. in hantavirus pulmonary syndrome (hps) (mainly seen in the americas), within 12-24 h of onset of symptoms, most patients develop some degree of hemodynamic instability and pulmonary edema accompanied by hypoxemia to full blown ards. petechiae of the head and neck are common but overt hemorrhagic symptoms are not. treatment is supportive and in those who survive, recovery is usually rapid. when given early in the course of illness, intravenous ribavirin has improved survival rate in hfrs but not in hps. steroids reduce the severity of the symptoms but do not increase the survival rate. malaria is singled out here because it is the most significant parasitic disease in humans with an estimated 500 million infections annually that result in 1-3 million deaths. the majority of these deaths are in children younger than 5 years of age and most are in africa. in developed countries malaria is the most common cause of febrile illness with no localizing signs in travelers returning from developing countries. the most important aspects of severe malaria are reviewed, which, for the most part, is caused by plasmodium falciparum. indicators of severe and complicated falciparum malaria and prognostic signs (world health organization 2000) cerebral malaria unrousable coma (gcs < 11/15), with peripheral p. falciparum parasitemia after exclusion of other causes of encephalopathy severe anemia hgb < 5 g/dl in the presence of parasitemia >10,000 per ml impaired consciousness of any degree, prostration, jaundice, intractable vomiting, parasitemia >2% in nonimmune individuals. levels of parasitemia should be interpreted in the light of immunity. patients with complicated malaria should be managed as severe malaria, i.e., with parenteral antimalarials even though they do not necessarily meet the criteria of severe disease. for details of management, review > chap. 101, ''malaria''. the world health organization defines cerebral malaria as unrousable coma in the presence of p. falciparum parasitemia when other causes of encephalopathy have been excluded. the precise etiology of cerebral malaria is not certain. most likely it is caused by sequestration of infected erythrocytes. this condition has a high mortality that likely results from brain micro vascular ischemia, infarction, and secondary cerebral edema. cerebral malaria is a medical emergency that requires: 1. supportive care: (a) continuous monitoring of vital signs. for a detailed discussion on sepsis, and the diagnosis and management of shock, please review the appropriate chapters (> chap. 61, ''bacterial sepsis and shock''). toxic shock and necrotizing fasciitis are two particular sepsis syndromes that require a special reference. toxic shock syndrome (tss) is caused by two bacteria: staphylococcus and streptococcus. s. aureus is a gram-positive coccus that is grouped in clusters. it is responsible for a number of infections ranging from skin sepsis, pneumonia, and joint infections to endocarditis. phage transformed staphylococcus produces a toxin that initiates a syndrome known as toxic shock syndrome (tss). this came to light in the 1980s with the ''menstrual shock'' syndrome. a non menstrual form was also identified. this was associated with staphylococcus sepsis at surgical sites, skin or joint infections, and with staphylococcal pneumonia. this syndrome is said to be ''superantigen'' mediated. the toxin proteins produced by the staphylococcus are able to ''cross-link'' the t-cell receptor without being processed by an antigen presenting cell (apc). this leads to an uncontrolled cascade of cytokines and immune system up regulation. at the level of the capillary this leads to inflammation and increasing permeability with secondary organ dysfunction (renal impairment, cardiac, pulmonary, and liver dysfunction). clinically this is manifested by skin erythema, tachycardia, hypotension, hypoxia and other critical organ dysfunction. initially this is subtle but rapidly develops into multi organ dysfunction. see the table below for the criterion upon which a diagnosis of staphylococcal toxic shock is made. treatment consists of recognition of the process, draining any collections of pus, and debridement, if that is appropriate. at the same time initiation of large volume fluid resuscitation, inotropic support and support of failing lungs with oxygen and ventilation if needed. antistaphylococcal antibiotics should be administered (this includes an antibiotic to cover for methicillin resistant staphylococcus). clindamycin being an anti-ribosomal antibiotic (50s bacterial ribosome) has a theoretical advantage in reducing the amount of toxin produced prior to antibiotic induced death of the bacterium. intravenous immunoglobulin (ivig) is a treatment for severe toxic shock that is progressing to multi-systems dysfunction. it has a proven efficacy in toxic shock in reducing the mortality of severe disease. this is thought to be via two general mechanisms. the first is by binding directly to the toxin. the second is by its immuno-mediatory properties. major criteria (all required) 1. fever !38.8 c 2. hypotension (orthostatic or shock) 3. rash (erythematous early and desquamative later) minor criteria (any three required) 1. gastrointestinal: vomiting or diarrhea 2. muscular: severe myalgia or cpk !2x upper limit of normal 3. mucous membranes: vaginal, oropharyngeal, or conjunctival hyperemia 4. renal: urea or creatinine !2x upper limit of normal, or urinalysis with >5 wbc per high-power field 5. hepatic: total bilirubin, ast or alt !2x upper limit of normal 6. blood: platelet count <100,000/ml 7. cns: disorientation or change in level of consciousness without focality, noted when fever and hypotension are absent streptococcal toxic shock is a syndrome that is analogous to staphyloccal toxic shock syndrome in that it is a superantigen mediated toxin related dysfunction of the immune system. group a beta-hemolytic streptococcus is most commonly associated with streptococcal toxic shock syndrome. clinical presentation is very similar to staphylococcal toxic shock. see table below. treatment consists of appropriate antibiotics. clindamycin is used for antimicrobial and antitoxin producing properties as previously mentioned. ivig here too has a role in reducing the mortality of severe disease. intensive care therapy consists of fluid resuscitation (large volume) and support of organ dysfunction (inotropes, ventilation, renal replacement therapy). hypotension or shock, plus any two of the following: 1. scarlet fever rash 2. abnormal liver function tests 3. renal insufficiency 4. disseminated intravascular coagulopathy (dic) 5. acute respiratory distress syndrome (ards) 6. soft tissue necrosis definite: preceding requirements + isolation of group a streptococcus from a normally sterile body site probable: preceding requirements + isolation of group a streptococcus from a non sterile body site necrotizing soft tissue infections are aggressive soft tissue infections that cause extensive necrosis, and include necrotizing cellulitis, fasciitis, and myonecrosis. the following clinical findings may be present: • erythema or discolored skin also the following systemic signs may be present: • local pain and tenderness out of proportion to physical findings • pain or tenderness that extends past the margin of apparent affected skin area necrotizing fasciitis is a surgical emergency. it is caused by a number of organisms: group a beta-hemolytic streptococci and other streptococci, staphylococcus, clostridium, pseudomonas, klebsiella, serratia, neisseria, escherichia, morganella, proteus, shigella, vibrio, salmonella, pasturella, enterobacter, corynebacterium, cryptococcus, fusobacterium, peptococcus, eikenella, bacteroides. the most common causative agent is group a streptococcus. pathologically it is characterized by micro angiopathic thrombosis and necrosis along superficial and deep fascial planes. the illness is associated with a breach of the integument. this can be by superficial infection, surgery or trauma. non steroidal antiinflammatory drugs are implicated in the pathogenesis. in children there is an association with varicella (chickenpox) infection. clinically the lesions appear either pale or have violaceous discoloration, often edematous, and there may be crepitus from gas forming bacteria. pain and tenderness in excess of that expected is a feature. the concern for the intensivist is the physiological decompensation that can lead to rapid cardiovascular collapse. broad-spectrum (and appropriate) antibiotics are indicated, and mechanical ventilation and cardiovascular support may be needed. urgent and wide surgical debridment of the affected areas is indicated. in cases of streptococcal necrotizing fasciitis there may be additional benefit from human immunoglobulin (ivig) therapy. though this has not been subjected to clinical trials, given the high mortality rate of necrotizing fasciitis and the biologically plausible consideration that ivig could neutralize the effects of streptococcal superantigens, its use can be justified. other treatments that have been used are: • vacuum-assisted wound closure (particularly in patients who have had large wound debridement) • hyperbaric oxygen (anecdotal evidence) the child, especially the infant, presenting with upper airway obstruction (uao) demands immediate attention. acute inflammation of the upper airway is of greater importance in small children because of the smaller diameter of the airway, hence the greater degree of obstruction from a similar amount of inflammation (resistance changes inversely to the fourth power of the radius of the airway). the following signs and symptoms are particularly worrisome: • inspiratory and expiratory stridor • active expiration (use of the rectus abdominis muscle when exhaling) • apnea or irregular breathing • increasing tachycardia (if no intervention is done tachycardia may be followed by decreasing heart rate which is usually a pre-arrest sign) • hypoxemia (late sign) • change in neurological status (becoming increasingly inconsolable and restless, or a child who ''stops fighting'' and becomes fatigued and hypotonic) there are many scoring systems for severity of the uao in children. the following is one suggested by downes et al. in 1980 . of note there is no mention of the neurological status in this scoring system. level of alertness and consolability of a small child are very important indicators of the severity of the uao. immediate management of acute severe stridor outside the picu, independent of underlying cause: • keep the child and the parent as calm as possible. do not separate the child from parent. • give the parent an oxygen mask to hold near the child's face. • call for help urgently from someone with expertise in airway management (usually an anesthesiologist). • give nebulized epinephrine (im epinephrine if airway obstruction is due to anaphylactic reaction). (skip nebulized epinephrine if you suspect epiglottitis.) • do not send the child to the radiology department for a lateral x-ray of the neck. • do not administer any sedative medications to the patient. • do not do attempt to draw blood for investigations. • place ecg monitoring leads and pulse oximetry probe without disturbing the child. • do not attempt to place an iv line (obviously you would place an iv/io access if the child has already had a respiratory or cardiac arrest). • the airway expert will decide to take the child to the or for intubation, or transfer to the picu. in children, there are many causes of acute uao, including infections (viral, bacterial) such as infectious mononucleosis, croup, epiglottitis, tracheitis, peritonsillar abscess, retropharyngeal abscess, diphtheria. noninfectious causes include foreign body, severe allergic reactions, acute angioneurotic edema, airway burn, trauma, and post-extubation in the picu. there are many causes of chronic/recurrent uao. in the history there may be chronic/recurrent symptoms. these patients may become symptomatic acutely (often with a viral respiratory infection) mimicking acquired acute upper airway obstruction. examples are: choanal atresia, laryngotracheomalacia, vascular ring, laryngeal web, subglottic stenosis, subglottic haemangioma, vocal cord palsy, recurrent angioneurotic edema. in this section the infectious causes of uao are addressed to. the more common infectious etiologies that may present with severe uao in children are: • croup or viral laryngotracheobronchitis • bacterial tracheitis • epiglottitis viral croup is the most common form of uao in children 6 months to 6 years of age (mostly 6 months to 2 years) and is more common in the autumn and early winter. the site of obstruction is the subglottic area. obstruction is caused by inflammation and edema. the most common viral etiology is parainfluenza, but influenza, enterovirus (coxsakie and echovirus), rsv, adenovirus, paramixovirus, rhinovirus, and hsv can cause a similar clinical picture. human metapneumovirus has been implicated in a few reports. in immunocompromised children, candida sp. can cause a similar presentation. there is a prodrome of mild fever and uri symptoms for 1-2 days before the onset of stridor. the stridor is characteristically harsh, dry, high pitched, and inspiratory. a ''barking'' or ''seal-like'' cough is prominent and usually worse at night. these children do have a voice, though hoarse, and they do not have trismus, dyphagia, or significant drooling. children with stridor at rest should be admitted for observation, while those with severe uao should be admitted to a picu. up to 15% of children with croup require hospitalization. usually no investigations are needed. administration of steroids (oral route is as good as intramuscular) in the emergency room has decreased the rate of hospitalization. hospitalized children with croup should receive a short course of oral or intravenous steroids (an example of a regimen is: dexamethasone 0.6 mg/kg iv/ po as an initial dose followed by 0.15 mg/kg q 6 h iv/po). inhaled nebulized epinephrine 1:1,000 solution (0.5 ml/kg, up to 5 mg) reduces the severity of obstruction and stridor. this can be repeated as required. the child must be observed for at least 2 h after a dose of nebulized epinephrine as the effects are transient. the decision on when to intubate a child with croup is a clinical one. if, despite maximum medical treatment there is not a clinical improvement or perhaps deterioration, a decision to intubate should be made or at least considered. a gentle and smooth intubation, using a tube one size smaller than usual for the age of the child, should be performed by a skilled and experienced practitioner. these children are at risk of accidental extubation and need proper securing of the ett, skilled nursing care, and adequate sedation only once the airway has been secured. most clinicians extubate the child 2-6 days later, when an audible air leak has developed around the ett and fever has settled. epiglottitis, or acute bacterial supraglottitis, is a bacterial infection of the laryngeal inlet, and is usually caused by h. influenzae type b (hib). with ''classical'' hib epiglottitis, the peak age of involvement is 2-3 years of age. since the introduction of the hib vaccine, the incidence of this disease has fallen dramatically, but the vaccine does not offer 100% protection. also, other organisms like s. aureus, s. pneumoniae, group a + b streptococcus, and n. meningitidis have been implicated as causative agents. the incidence of these latter organisms is higher in adolescents and older children. noninfectious causes of epiglottitis have been described in the following conditions: kawasaki's disease, stevens-johnson syndrome, airway burn, caustic ingestion, post-radiotherapy, angioneurotic edema, trauma (including trauma from intubation), leukemia, and lymphohistiocytosis. granulomatous states can cause a more chronic picture (sarcoidosis, tb, or wegener's granulomatosis). as fewer and fewer physicians have seen even one case of epiglottitis, it is important to have a high index of suspicion in any febrile child with uao. the following signs are highly suspicious of epiglottitis: • usually there are no prodromal signs and symptoms. • a few hours of high fever and tachypnea. • pain with swallowing, hence drooling is common. • reluctance to speak. • the child looks ill, with circumoral pallor, and a ''toxic'' appearance. • there is minimal or no coughing. • stridor is low-pitched and muffled, more like a snore. • child prefers to sit forward in the tripod position with mouth open and is reluctant to move his head or neck. if you have suspicion (on clinical grounds) that a child may have epiglottitis: • do not make the child lie down. • do not separate the child from parent. • do not examine the throat. • do not place an iv cannula. • do not order a lateral x-ray of the neck. • do not order any blood work. • do not transport a child with epiglottitis between hospitals unintubated. • the child should be accompanied by an expert in difficult airway management to the operating room for examination under anesthesia and securing airway if needed. the technique for induction of anesthesia is beyond the scope of this chapter. generally the inhalational method is performed in the sitting position (position of comfort for the child), and once the child loses consciousness intravenous access is secured and the rest of the monitoring is applied. laryngoscopy and intubation is only attempted after adequate depth of anesthesia has been obtained. blood cultures and a swab from the inflamed epiglottis should be sent and a 3 rd generation cephalosporin should be given once an iv is in place. when back in picu, accidental extubation can have disastrous consequences. skilled taping of the ett, nursing care, and adequate anaelgesia/sedation cannot be over emphasized. usually after 12-48 h of intravenous antibiotics the patient can be safely extubated, once the fever has subsided and presence of a leak is documented and the child is able to swallow (the child is not drooling). some practitioners prefer to reevaluate by direct laryngoscopy with the patient deeply sedated or anesthetized. if the causative organism is proved to be hib, in families with siblings under 4 years of age or families with an immunocompromised child, prophylaxis with rifampicin should be provided. bacterial tracheitis is characterized by profuse purulent secretions or sometimes by pseudomembrane formation in the tracheal lumen. the median age of the patient is 5 years. s. aureus is the most common etiology, though other gram-positive and less commonly gram-negative microorganisms might be causative. in immunocompromised children candida and aspergillus can cause tracheitis. these children usually have a high fever, they look toxic, and the stridor characteristically is high pitched and composed of both inspiratory and expiratory components. cough is usually prominent and they may have dysphonia or aphonia. drooling can be seen with bacterial tracheitis. these patients are at risk of airway obstruction. they require appropriate antimicrobial therapy, observation, and intubation by experts if warranted. community-acquired pneumonia (as opposed to nosocomial or hospital acquired pneumonia) is a common pediatric diagnosis that leads to admission to hospital for intravenous antibiotics and supportive respiratory therapy. pneumonia means inflammation of the lung parenchyma caused by infection and the diagnosis is made clinically in a febrile child with respiratory signs and symptoms who has evidence of consolidation on cxr. blood cultures frequently fail to reveal the infecting organism in pneumonia. tracheal aspirate, or more reliably, bronchoalveolar lavage (bal) and on occasions lung biopsy are required. children with immunodeficiency or malignancy undergoing therapy are a common example of where bal and/or lung biopsy may be necessary. • mycobacterium tuberculosis s. pneumoniae and s. aureus are the most important bacterial pathogens in children with pneumonia who need intensive care admission. as a general rule, truly focal disease, confined to a single lobe, is more likely to be due to bacteria. an ill child with unilateral pleural effusion most likely has s. pneumoniae or s. aureus pneumonia. viral • rsv • influenza a, b, c • parainfluenza routes to acquire infection: • inhalation of infected particles (most common) • aspiration • hematogenous invasion of the lower respiratory tract with viruses and bacteria leads to inflammatory changes characterized by migration of neutrophils into the alveoli. together with alveolar macrophages they provoke the production of inflammatory exudates and cellular debris that lead to consolidation of the lung parenchyma. the surrounding areas can be affected by atelectasis. • spo 2 <90% in high concentrations of inspired oxygen (>60% fio 2 ) • excessive work of breathing which may lead to exhaustion • shock and hemodynamic instability • change in neurological status (agitation or alteration of the level of consciousness) reasons of failure to respond to treatment on the pediatric ward or as outpatient: • development of an empyema or less commonly a lung abscess • underlying lung disease such as: bronchopulmonary dysplasia (bpd, in ex-premies), cystic fibrosis, inhaled foreign body, tracheobronchomalacia or post tracheal surgery, or infected congenital lung cyst • diagnosed or undiagnosed immunodeficiency states (primary, hiv, leukemia) • children with neuromuscular diseases, weakness, or spasticity such as muscular dystrophies, myasthenia, spinal muscular atrophy, or cerebral palsy • inappropriate antibiotics, inappropriately low dose or resistant bacteria • non bacterial pneumonia (viral pneumonia or alternative pathogen such as tuberculosis) once the culture results (bal, blood culture, sputum culture) and sensitivities are known the therapy should be tailored to the antibiotic sensitivities of the causative organism(s). • empyema. more commonly seen with s. pneumoniae and s. aureus pneumonia. generally a chest drain is needed. the use of fibrinolytics and surgery are areas for debate and local advice from thoracic or general surgeons and physicians from the respiratory and infectious diseases services should be sought. bronchiolitis is a seasonal viral infection of the lower respiratory tract that mainly affects infants. the usual cause is respiratory syncytial virus (rsv), although influenza, parainfluenza, adenovirus, and human metapneumovirus can cause a similar syndrome. in young infants, chlamydia and b. pertussis can cause respiratory illness with a more prolonged course that initially may resemble bronchiolitis. 20-25% of infants with bronchiolitis may have a secondary bacterial infection. infants with bronchiolitis have fever, cough, difficulty in feeding, and, on occasion, audible wheezing. on examination bronchiolitis is a syndrome characterized by respiratory distress, hyperinflation of the chest, and wheezes with fine inspiratory crackles heard on auscultation. apnea may occur even before onset of clinically significant respiratory distress, especially in ex-premature infections in the picu and very young infants. though not common, neonates may present with hypothermia and a sepsis like syndrome. the following groups are at increased risk of severe infection: • ex-premature infants and neonates • infants with congenital heart disease • infants with immune deficiency • infants with neuromuscular disease the virus causes direct damage to the respiratory epithelium with resultant inflammation, increased secretions, small airway obstruction. areas of hyperinflation and atelectasis exist simultaneously throughout the lung. this leads to ventilation and perfusion (v/q) mismatch and hypoxia. hyperinflation flattens the diaphragm and makes breathing less efficient. should they require respiratory support, many of these infants can be managed with noninvasive continuous positive airway pressure (cpap) at 4-8 cm h 2 o. this reduces the work of breathing and improves oxygenation. suction to maintain patency of airways is of crucial importance. if the saturations remain low and/or the infant continues to have frequent apneas despite providing noninvasive ventilatory support intubation of the trachea is indicated. • intubation is usually required for several days. • inadequate humidification and inadequate tracheal suctioning cause endotracheal tube blockage or lung atelectasis followed by increasing pressure and fio 2 requirements. • as a general rule, the best ventilatory mode is one that assists spontaneous respiratory efforts; keep the child's own respiratory and coughing efforts by providing enough comfort (sedation) and pressure support. • peep or cpap (initially at 4-6 cm h 2 o) may reduce the work of breathing. • apply enough peak inspiratory pressure (pip) to achieve visible chest excursions and if higher pressures (>30 cm h 2 o) are needed, let the paco 2 gradually rise to 75-80 mmhg with arterial ph > 7.2 (permissive hypercapnia). particular issues in infants with congenital heart disease and bronchiolitis: • infants with left to right shunts have more frequent viral and bacterial respiratory infections, and have higher morbidity and more prolonged course with bronchiolitis. • infants with palliated single ventricle physiology and those with limited cardiac output (for example severe valvar aortic stenosis) have high morbidity and mortality with bronchiolitis. • bronchiolitis and other viral respiratory infections in infants with congenital heart disease lead to operative delays and increasing complications post cardiac bypass surgery (e.g., pulmonary hypertension). in any infant with rsv bronchiolitis and congenital heart disease awaiting surgery, it is suggested to wait for 2-3 weeks before proceeding with bypass and surgery. the american academy of pediatrics has specific recommendations on prophylactic monthly injection of rsv monoclonal antibody in ''at risk'' infants during the cold season. however, the use of this approach has only been shown to aid a small number of patients. this section reviews: • myocarditis • infective endocarditis • infectious pericarditis • wound infection after cardiac surgery myocarditis is an inflammatory disease of the heart muscle characterized in its active phase by cellular infiltrates and myocardial necrosis. however myocarditis can have cellular infiltrates with little or no myonecrosis. most cases of myocarditis are thought to have a viral etiology; however, viruses are infrequently isolated. the most common viral causes include the enterovirus family particularly coxsackievirus b and adenovirus. other viral causes are influenza, cmv, hsv, parvovirus, rubella, varicella, mumps, hiv, and ebv. myocarditis has a number of other non viral etiologies, some infective and some not. they include bacteria, rickettsia, fungi, protozoa, pharmaceuticals, toxins, and connective tissue/autoimmune disorders. typically viral myocarditis begins as a systemic viral illness with flu-like symptoms. as the virus infects the myocytes the immune system is up regulated and cd4 t-helper cells and cd8 cytotoxic t cells are stimulated along with proinflammatory cytokines. persistence of the viral rna and production of no by the myocytes have been linked to myocardial tissue damage. myocarditis can present in a number of ways: • out of hospital cardiac arrest/sudden death • cardiogenic shock (may mimic sepsis) • congestive heart failure (increasing dyspnea, lethargy) • dysrhythymias -bradycardia, tachycardia whilst sepsis and hypovolemic shock are more common than cardiogenic shock from myocarditis, it should always be in the differential. sometimes acute ''decompensation'' of these children is heralded by abdominal distension and vomiting. teenagers may complain of a feeling of ''impending doom'' or severe chest discomfort. clinically, signs of tachycardia/tachypnea, gallop rhythm, hyperdynamic precordium, and displaced apex are often present. hepatomegaly and in older children elevated jugular venous pressure (jvp) are usually present. crackles on auscultation are often present in the chests of older children. chest x-ray (cxr) may show an enlarged heart, pulmonary venous congestion, alveolar edema, kerley b lines, and in some cases pleural effusions. in acute myocarditis the heart may often look normal in size on the cxr. a 12-lead ecg is useful to assess underlying rhythm, assess for ischemia and for the subtle ecg changes that are sometimes evident with myocarditis; st-t changes, reduced qrs voltage, widened qrs. echocardiography is absolutely necessary to assess structure and function of the heart and to assess for a pericardial effusion. involvement of appropriate specialists is important -cardiologists, intensivists, and cardiothoracic surgeons work cooperatively to manage and stabilize these patients. treatment of myocarditis is largely supportive. immunomodulation using steroids, intravenous immunoglobulin, and immunosuppressive agents is controversial. identifying any modifiable contributors, i.e., toxins and drugs, is of crucial importance. supportive therapy for heart failure associated with myocarditis ranges from diuretics and afterload reduction, addition of inotropic support to placing the patient on mechanical circulatory support. dopamine and dobutamine increase contractility but also heart rate and myocardial oxygen consumption. milrinone is an intravenous phosphodiesterase inhibitor that improves contractility and at the same time, reduces the afterload. enoximone is an oral phosphodiesterase inhibitor that is available in europe, but not in north america. levosimendan is a calcium sensitizer and improves contractility. it has limited availability world wide. positive intrathoracic pressure, given noninvasively via a face mask (cpap), reduces lv afterload and may improve cardiac output in the setting of lv dysfunction. failed medical therapy or deteriorating function will usually indicate the need for extracorporeal support and ultimately heart transplantation. mechanical support (ecls, ''berlin'' heart) is frequently used as a ''bridge'' to recovery or transplant. for a complete review of endocarditis review the cardiology chapter in this book. the major reasons a child with endocarditis may need admission to picu are: infections in the picu 1. congestive heart failure due to worsening valvar regurgitation 2. congestive heart failure with abrupt onset due to valve apparatus rupture/perforation, or dehiscence of a prosthetic valve 3. systemic to pulmonary artery shunt obstruction 4. arrhythmia 5. renal failure 6. embolic events to (a) brain (b) heart (c) lungs (d) bowel (e) extremities for a complete review of pericarditis and tamponade, please review the cardiology chapter in this book. acute inflammation of the pericardium in a previously healthy child has usually been assumed to be viral. in most cases a causative agent is not detected (hence the term ''idiopathic'' pericarditis). an upper respiratory infection usually precedes the onset of symptoms by 10-14 days. the reported viral pathogens include coxsackievirus, adenovirus, rsv, varicella, hepatitis b, hiv, and post influenza vaccine. primary infectious pericarditis that may need picu care is usually purulent bacterial pericarditis. these patients are generally toxic looking. the infection in the pericardium rarely occurs in the absence of infection elsewhere (hematogenous spread). in comparison with the viral (idiopathic) pericarditis, the incidence of tamponade and hemodynamic instability is much higher with purulent pericarditis. s. aureus is the most common cause of purulent pericarditis. other bacteria include h. influenzae, n. meningitides, and s. pneumoniae. in developing countries, tubeculous pericarditis is a common cause of chronic constrictive pericarditis. therapy depends on the hemodynamic status of the patient. a toxic-looking child with physiological signs and symptoms of tamponade should be transferred urgently to the catheterization laboratory or to the intensive care unit for percutaneous drainage of the pericardial collection. sometimes the pus in pericardium is so thick or organized (esp. with h. influenzae) that percutaneous drainage may not be sufficient and the child will need open surgical drainage. with tamponade physiology, administration of fluid boluses can temporarily increase the intracardiac ''filling'' and stabilize the patient until the definitive treatment (percutaneous or surgical drainage) is performed. broad-spectrum intravenous antibiotics with good antistaphylocccal coverage should be commenced promptly if purulent pericarditis is suspected. surgical wound infections after cardiac surgery can be categorized as superficial (cellulitis) or deep (mediastinitis). the patient usually presents a few days after the procedure, but may occur up to 2 months after the initial operation. the important signs are erythema and induration at the surgical incision. the child may be irritable and have a mild fever. there may be a leukocytosis with ''left shift'' and elevated inflammatory markers such as c-reactive protein (crp) or erythrocyte sedimentation rate (esr). in addition to the wound erythema with or without purulent discharge there may be signs of sternal instability with ''crepitus'' on direct pressure over the sternum. diagnosis nevertheless is a clinical one and relies on a high index of suspicion. the risk factors are: neonates, long cardiopulmonary bypass time, delayed sternal closure, and reexploration of the chest for postoperative bleeding. the most common organisms associated with sternal wound infections are s. aureus, s. epidermidis, enterococcus species, and candida species. antibiotic treatment should begin as soon as a sternal wound infection is suspected and a wound swab has been sent. blood cultures should be sent from both peripheral and central venous sites whenever possible. the initial antibiotic regimen should consist of broad-spectrum gram-positive (anti-staphylococcal) coverage, with the addition of gram-negative coverage if the patient is septic or mediastinitis is suspected. if there is not a rapid improvement or the patient deteriorates the sternal wound may need to be surgically explored and debrided. antibiotics should be given for 10 days to 2 weeks for cellulitis and for 4-6 weeks for deep wound infections. meningitis is an inflammation of the leptomeninges of the brain. for a review of ''aseptic'' meningitis which also includes viral causes of meningitis, please look at the neurology and infectious disease chapters in this textbook. suffice to mention that patients with ''aseptic'' meningitis are usually not as sick as those with purulent meningitis, and the csf abnormalities are not as prominent. patients with bacterial meningitis have a number of reasons for requiring intensive care. the most common clinical scenarios are coma and seizures. the local inflammatory response to bacteria multiplying in the csf involves polymorphonuclear leukocytes, the endothelium, complement, and cytokines. this results in an alteration in the cerebral blood flow and venous drainage, vascular inflammation, and obstruction to csf flow and reabsorption. the infection within the meninges may extend to the surrounding brain parenchyma. the commonest bacteria are s. pneumoniae, n. meningitidis, and h. infuenzae. in the neonatal period, the likely causative organisms are different: group b streptococcus, l. monocytogenes, and gram-negative bacilli are the commonest. this profile will be modified depending on the local vaccination policy, socioeconomic status of the children in the area, and local/regional epidemics of disease. none the less the intensive care management is similar: 1. broad-spectrum cns penetrating antibiotics with narrowing of spectrum of antibiotic cover once results of cultures of the blood and csf are known. antibiotics with high csf/brain tissue penetrance must always be used. in areas with high incidence of s pneumonia penicillin resistance (including the united states), empiric therapy for community-onset bacterial meningitis is both vancomycin and a 3rd generation cephalosporin. acute complications of bacterial meningitis are: • hyponatremia (serum sodium <135 micromole/l): this is usually due to the syndrome of inappropriate secretion of antidiuretic hormone (siadh). there is hyponatremia and low serum osmolarity without signs of hypovolemia. hyponatremia can cause convulsions. cerebral salt wasting is a much rarer condition that gives hyponatremia with signs of volume contraction. siadh is treated by free water restriction and cerebral salt wasting is treated with sodium (either iv or po) supplementation. in a hyponatremic child with convulsions give 3% nacl 3-5 ml/kg intravenously. it is important to note that the change of serum sodium (and hence serum osmolarity) is of more importance in some cases than the absolute serum sodium. a sudden drop in serum sodium (greater than 0.5-1.0 micromole/h) should be treated with hypertonic saline. • seizures: convulsions that occur early in the course of purulent meningitis are usually generalized and have less prognostic significance than those occurring later. etiology of convulsions in meningitis can be any of the following: brain edema, diffuse ischemia, hyponatremia, subdural collection, sinus venous thrombosis, or focal infarction. • subdural effusion: this complication is seen more commonly in neonates and infants. a good practice is to measure the head circumference daily in any infant with meningitis. the diagnosis is made or confirmed by neuroimaging. if the effusion is large, or if it is associated with focal signs, convulsions, or signs of increased intracranial pressure, a neurosurgical consultation is necessary. • obstructive hydrocephalus: obstructive hydrocephalus occurs when the pus (often with high protein content) in the ventricles blocks the outflow of csf. this situation occurs more frequently in small infants and neonates. similar to meningitis, seizures, focal or generalized signs, and coma, are common presentations. the list of differential diagnosis is long and includes: aseptic meningitis, post infectious encephalitis and noninfectious encephalopathies (metabolic, vascular, demyelinating disease, tumor). the most frequent causes of acute encephalitis include: enteroviruses, hsv, vzv, ebv, adenovirus, influenza virus, and m. pneumoniae. mumps, measles, and rubella infections are rarely seen in developed countries. in many parts of the world arboviruses are major causes of endemic encephalitides. tuberculosis is always high on the list of differential diagnosis in developing countries. diagnosis: csf can be completely normal, but usually contains >10 leukocytes/mm 3 (mainly lymphocytes), mildly increased protein level and mildly reduced to normal glucose level. in children with hsv encephalitis, the csf may contain red blood cells. csf in addition to cell count, chemistry and culture should be sent for pcr for viral agents. csf pcr can be helpful in a number of the infectious encephalopathies. for example: m. pneumoniae, mycobacterium, cmv, ebv, vzv; where the organism may not be cultured from the csf. brain imaging (ct or mri) can be helpful in the diagnosis. in hsv encephalitis there may be focal edema and enhancement seen in the temporal area. this is relatively specific for hsv infection of the brain. the electroencephalogram may show focal periodic epileptiform activity in frontal and temporal parts of the brain. this is common in hsv disease. diffuse slow waves generalized over the cerebral cortex may also be seen. this may either represent encephalitis or be secondary to sedatives and anticonvulsants used in the picu. the primary use of eeg is in the management of seizures. finally, when the etiologic diagnosis is not clear, or the patient is deteriorating despite treatment, brain biopsy may be performed. treatment is largely supportive. as for meningitis this consists of appropriate antimicrobial (antiviral) therapy. there should be no delay in starting acyclovir if hsv is suspected or considered. the dose is 30 mg/kg/day for 10 days. when m. pneumoniae is suspected antibiotics with good penetration into the brain (ciprofloxacin, or azithromycin) should be used. additional management includes airway protection for coma and seizures. medical and surgical therapies for management of intracranial hypertension, as discussed above should be adhered to. adem is an acute or sub acute inflammatory demyelinating disease of the cns (brain and spinal cord). in contrast to multiple sclerosis it is a monophasic illness. adem is considered a parainfectious disease and the precipitants include infection with upper respiratory tract viruses, influenza, group a streptococcus, ebv, vzv, measles, mumps, rubella, and mycoplasma. clinical presentation may involve fevers, seizures and a constellation of neurological phenomena. commonly these are coma, focal neurological deficits or alterations in personality and behavior. often a recent ''viral infection'' is present in the history. the lumbar puncture is done to exclude infections. csf may have normal white cell count or mild pleocytosis (mainly lymphocytes), and mild to significant protein elevation. the cultures and pcr should be negative. neuroimaging is the main diagnostic tool for adem mri is the modality of choice, as the ct is normal in 40% of cases. the typical mri findings are multiple disseminated asymmetrical hyperintense lesions on t2wi and flair in the white matter and basal ganglia. the cerebrum is more involved than the cerebellum. treatment: (1) continue antibiotics and antivirals until final csf cultures and pcr results are confirmed to be negative. (2) once infection is ruled out methylprednisolone ''pulse'' dose at 30 mg/kg/day (maximum 1 g) for 3 days followed by oral prednisolone 2 mg/kg/day for 2 weeks. this is followed by a 4 weeks weaning regimen. (3) plasma exchange or ivig for relapsed or refractory adem. gbs is an immune-mediated polyneuropathy that is usually preceded by a viral or bacterial infection of the respiratory or gi tract 1-3 weeks prior to presentation. gbs is the most common cause of acute paralysis in developed countries and is characterized by progressive, ascending, symmetric motor weakness and loss of reflexes. the sensory symptoms (extremity pain, paresthesia) and autonomic irregularities (tachycardia, bradycardia, hypertension, hypotension, arrhythmia) can be prominent. there are usually no sensory deficits in physical examination. infections known to precede the onset of paralysis are: cmv, ebv, vzv, campylobacter jejuni, mycobacterium tuberculosis, hiv, and m. pneumoniae. the pathogenesis involves an immune response against the infectious agent and has components that cross-react with those of the peripheral nervous system. diagnosis is clinical but is aided by csf and electrophysiology testing. the csf shows a high protein content and low/normal white cell count. this may be missed if the lumbar puncture is done early in the first week of the illness. electrophysiology will show decreased conduction velocity in the peripheral nerves. treatment: ivig at 2 g/kg/day for 2-5 days or plasma exchange. (two courses for mild gbs and four to five courses for severe illness.) corticosteroids have not demonstrated effectiveness in gbs and are not recommended. indications for picu admission: • for respiratory support • for plasma exchange • autonomic instability (hypo-or hypertension, arrhythmia) botulism is a toxin mediated disease caused by c. botulinum. symptoms start a few hours and up to 6 days after exposure to the toxin. the cranial nerves are involved initially with difficulty swallowing, abnormal speech (abnormal cry in infants) and eye movements (ptosis). other symptoms may include nausea, vomiting, constipation, and abdominal distension. as the illness progresses it causes paralysis of the extremities and respiratory muscles to various degrees. in infants the disease can be mild with hypotonia and constipation as the main findings. also in infants there is sometimes a history of ingestion of honey before the onset of symptoms (honey may contain the spores of c. botulinum). diagnosis is made from a combination of clinical findings and electromyography. stool for botulinum toxin or serum serology is confirmatory but takes time. treatment is with antitoxin to remove circulating toxin but this will not affect the toxin already present at the neuromuscular junction. specific botulinum immunoglobulin is not readily available world wide. the cost is prohibitive for a lot of countries and the cost-benefit analysis is only favorable for those patients requiring mechanical ventilation. penicillin and metronidazole are given to eradicate the source of toxin production. aminoglycosides and steroids should not be given as they may worsen the neurosmuscular transmission defect and increase muscular weakness. if the source of the c. botulinum is a wound (i.e., wound botulism) then it will need surgical debridement. indications for admission to picu: • respiratory support • autonomic instability children are increasingly surviving diseases that until recently were considered untreatable. there are more potent, intense chemotherapy regimens being used and increasingly there are more patients who undergo solid organ and stem cell transplants. these treatments and interventions particularly with immunosuppressants, though frequently successful, leave patients at considerable risk for severe infections. neutropenia is defined as the absolute neutrophil count (anc) [anc = pmn + band count] <1,000/mm 3 , and is generally associated with cancer and its treatment. the risk of infection is particularly high with: • rapid drop in anc • anc < 100/mm 3 (profound neutropenia) • prolonged neutropenia fever in neutropenic patients is defined as an oral/ tympanic membrane temperature >38 c in two repeated measurements over a 4 h period or one measurement above 38.5 c. the portals of entry of infectious agents are usually: the oral mucosa, the gut, the upper/lower respiratory tract, and central vascular lines. the most common organisms are gram-positive cocci (s. aureus, s. epidermidis, and strep. viridans), gram-negative bacilli (e. coli, k. pneumoniae, p. aeroginosa), and fungi (candida, aspergillus). recently the spectrum of pathogens has begun to change, with the emergence of more gram-negative and fungal infections. this is likely due to an increase in resistant pathogens in the face of the use of very broadspectrum antibiotics, intensity of therapy (high-dose chemotherapy and stem cell transplant) and prolonged neutropenia. the single most important risk factor for fungal infection is the duration of neutropenia. it is standard of practice to start antibiotics for a child with anc < 500 who is febrile. initial empiric therapy for febrile neutropenia consists of a b-lactam antibiotic and an aminoglycoside, plus a glycopeptide if a coagulase negative staph or enterococcus is suspected or isolated, if the child is in shock, has an endoprosthesis or a vascular tunnel infection. if patient has a history of arabinoside-c administration and has severe mucositis strep viridans infection is highly suspected and vancomycin should be added. if perianal infections in the picu tenderness is present add anaerobic coverage (metronidazole or clindamycin). after 4-5 days of fever and neutropenia adding an antifungal is the usual practice of most oncologists. infants with a severe combined or t-cell immune deficiency usually present early in the first few months. defects in cell-mediated immunity can result from congenital disorders such as digeorge syndrome, severe combined immunodeficiency disease (scid) and wiskott-aldrich syndrome. they can be secondary to lymphomas, immunosuppressive medications or chronic illness. acute viral infections such as measles and pertussis are also known to decrease a patient's cellmediated immunity. typical infections are pneumocystis jiroveci pneumonia (formerly carinii), cmv pneumonitis, rsv pneumonitis, disseminated enteroviral infection, and invasive fungal infection. patients are highly susceptible to infections with intracellular organisms such as salmonella, listeria, mycobacteria, herpes family viruses (cmv, ebv, and hsv), as well as fungi and protozoa. in older children and those with secondary immunodeficiencies, these infections tend to be reactivated disease. children with chronic mucocutaneous candidiasis and chronic granulomatous disease typically present early in life with recurrent candida and staphylococcal infections. neonates with adhesion molecule deficiency usually present with delayed separation of the umbilical cord stump, increased polymorphonuclear count, and increased incidence of bacterial infections. children with primary humoral immune deficiency usually present between 6 months and 5 years of age. the onset is consistent with the time when the level of placentally transferred maternal antibodies (igg) has declined. these defects as well as complement deficiency and asplenia are more commonly associated with infections by encapsulated microorganism such as h. influenza, n. meningitides, and s. pneumoniae. although patients with these conditions are mainly susceptible to bacterial infections involving the upper and lower respiratory tract, they can have protracted diarrhea with giardia or echovirus. defects in the late complement component (the ''attack'' component, c5-9) are prone to recurrent neisseria infections. children with early complement defects usually have autoimmune and rheumatologic manifestations. fulminant meningococcemia is also associated with properdin deficiency (alternative complement pathway). hsct is now an established treatment for a host of immunologic, metabolic, hematological, and neoplastic disorders. the ''stem cells'' may be obtained from the patient (autologous). alternatively from an hlacompatible related or unrelated donor (allogeneic). there is little risk of acute or chronic graft versus host disease (gvhd) with autologous hsct. currently there are three sources for stem cells: bone marrow, peripheral blood, and umbilical cord blood. generally with the umbilical cord stem cell transplant the speed of engraftment is lower, but so is the risk of gvhd. with peripheral blood stem cell transplant engraftment occurs faster but the risk of gvhd is also higher. with bone marrow stem cell transplant the speed of engraftment and the risk of gvhd is somewhere between the other two sources. with unrelated umbilical cord blood and t-cell depleted bone marrow or blood stem cell transplant, the risk of graft failure is higher. there is a higher risk of infection with occurrence of gvhd, and with graft failure. the conditioning regimens used to prepare the patients generally consists of high-dose chemotherapy with or without regional or total body irradiation. post transplant, there is a period of pancytopenia and though the neutrophil count usually normalizes after 3-4 weeks it is not unusual for these patients to need red cell or platelet transfusions for much longer. the risk of infection is influenced by rapidity of myeloid recovery and the rate of lymphoid reconstitution. the speed of restoration of adequate immune function is highly variable. the stem cell source, hla compatibility, purging or t-cell depletion of the graft prior to transplant, and severity of gvhd are important factors. in the early post-transplant period (first 100 days), transplant centers employ prophylactic measures to reduce the risk of infection. these measures vary between different centers, and include: • prophylactic antibiotics (for pcp, candida, hsv, cmv) • administration of ivig • environmental precautions (isolation and barrier nursing) in the early post-transplant period, patients are most susceptible to infections caused by both gram-negative and gram-positive organisms and by fungi. there is a higher risk of cmv pneumonitis in patients with gvhd (largely due to the need for immunosuppressive treatments). cmv negative recipients who receive transplant from a cmv positive donor are at highest risk for cmv pneumonitis. children who have undergone hsct and are admitted to picu have a particularly poor prognosis. pneumonia, mechanical ventilation, and the need for renal replacement therapy are especially poor prognostic factors. those with septic shock and line sepsis have the best prognosis. in addition to bacteria, the most commonly isolated organisms are cmv, rsv, adenovirus, candida, aspergillus, and pcp. in a recent report from great ormond street hospital in london, uk, only 56% of these patients survived to discharge. similar to children who have received hsct, children receiving solid organ transplants are prone to infections before and after the transplant. the main differences are: 1. patients after solid organ transplants are usually less immunosuppressed than hsct patients and are not at risk of immune reconstitution syndrome and its associated inflammatory and infectious complications. however they are at risk of surgery-related complications and postsurgical infection issues (wound infection, bacteremia, atelectasis/pneumonia, urinary tract infection). 2. infection of the transplanted organ due to latent or colonizing organisms present in either the donor or recipient can lead to invasive widespread disease in the immune suppressed post-transplant recipient. an example of this is the child with cystic fibrosis colonized with pseudomonas species; those colonized with b. cepacia are particularly at risk of developing resistant infection post lung or combined heart/lung transplants. 3. children receiving solid organ transplants are at risk of reactivation of latent infections, such as cmv. but, unlike hsct patients who most commonly present with cmv pneumonitis in recipients of solid organ transplants the cmv disease depends on the sites where the virus is latent and on the organ that has been transplanted. lung, heart/lung, and liver transplant patients are most vulnerable to systemic disease. cmv infection can precipitate rejection and increase vulnerability to other infections such as fungal infections. 4. ebv-associated post transplant lymphoproliferative disorder (ptld) is a potentially fatal complication of solid organ transplant. the risk of ptld is higher in children who were ebv seronegative prior to the sot. ebvrelated infection in sot patients may present in several different ways: asymptomatic or nonspecific viral syndrome, mononucleosis syndrome, and ptld. the latter can have a spectrum from fever, lymphadenopathy and diarrhea to full blown lymphoma. tissue biopsy is necessary to establish the diagnosis of ptld. the mainstay of therapy consists of decreasing immunosuppression. chemotherapy and biological treatments (such as anti-cd20 monoclonal antibodies) have been used. the most common reason children with hiv/aids are admitted to the picu is respiratory distress. septic shock and cns involvement (encephalopathy, encephalitis, and meningitis) are other common conditions leading to admission. in addition to the bacteria, viruses and mycoplasma that cause infections of the lower respiratory system in the non-hiv patient, there are a number of other opportunistic infections and inflammatory conditions to consider in the hiv-infected patient. these are commonly: • pneumocystis jiroveci • cmv pneumonitis • tuberculosis • fungal infections • lymphoid interstitial pneumonitis (lip) • immune reconstitution inflammatory syndrome (iris) most infants with respiratory failure will not have a previous diagnosis of hiv infection when they present. in non-endemic areas, especially during the colder season, such infants would be diagnosed and treated initially as cases of bronchiolitis. the possibility of an underlying immune deficiency and or aids should be considered in any infant who responds poorly to treatment or who has risk factors. these include failure to thrive, history of recurrent chest infections, hepatosplenomegaly, adenopathty, severe persistent oral thrush, or abnormal neurological signs. in sub saharan africa, tb and other bacterial pneumonias were common in both hiv-infected and uninfected children who presented with respiratory failure. however pneumocystis and cmv pneumonitis occurred almost exclusively in infants who were hiv-infected. the majority of cases of p. jiroveci pneumonia present in the first 6 months of life. usually these children are quite hypoxemic. high fever is uncommon compared with bacterial pneumonia. there is a diffuse, bilateral air space or interstitial involvement on the chest x-ray. occasionally there is a ''ground glass'' appearance. in an hiv positive patient with bilateral diffuse parenchymal or interstitial infiltrates on cxr, the development of pneumothorax is suggestive of p. jiroveci infection. diagnosis is by bronchoalveolar lavage (bal). even if a child develops pneumocystis while on prophylactic therapy, high-dose intravenous trimethoprim/sulfamethoxazole should be started. prophylaxis may have failed because of poor compliance. if one suspects drug resistance then other agents should be used (pentamidine, dapsone). methylprednisolone at 2-4 mg/kg/day divided in four doses should be administered in moderate to severe cases (practically all children with pneumocystis who are admitted to picu) for 5 days and then tapered. untreated, it is universally fatal. with proper therapy the mortality is less than 10%. the risk factors for mortality are the severity of respiratory failure and the severity of immunosuppression. lip in children with aids is associated with increased risk of lower respiratory tract infections including bronchiectatsis. this condition can produce severe ventilation/perfusion mismatch and hypoxemia but may also be asymptomatic. the cxr shows diffuse infiltrates and hilar lymphadenopathy persisting for >2 months despite antibiotic therapy. usually cxr changes are worse than clinical symptoms. lip can be related to ebv infection or to an exaggerated immunological response to inhaled or circulating antigens or both. steroids have been used in the treatment of symptomatic lip. coexistence of hiv and tb accelerates the course of both of these infections. the risk of miliary and extra pulmonary tb is higher in children with aids and the course is more likely to be severe and rapid. a child with hiv infection is five to ten times more at risk of active tb. in countries with high prevalence of tuberculosis, the who suggests bcg vaccination of all neonates at birth but not in any child/infant with clinical aids. for treatment of tb, please refer to the chapter in this book. mac can produce a systemic illness in children infected with hiv that is characterized by fever, chronic diarrhea, abdominal pain, malabsorption, lymphadenopathy, and obstructive jaundice. mac usually would not present with significant lung disease in these children. iris occurring weeks after the initiation of specific anti-hiv treatment may on occasion be severe enough to cause respiratory failure warranting admission to the icu, though it can also indicate latent or incipient mycobacterial disease. this (iris) is a diagnosis of exclusion and requires a bal and possibly a transbronchial biopsy. management is usually with corticosteroids. other than sepsis and respiratory failure, the other conditions that may bring the child with aids to the intensive care unit are (to name the more common ones): • cns infections (bacterial, mycobacterium, fungi, cryptococcus, viruses, and rarely cns toxoplasmosis), acute hiv encephalopathy • hiv-related cardiomyopathy • severe diarrhea and shock due to cryptosporidium or other microorganisms • liver failure due to infections or drugs • complications of antiviral medications such as acute pancreatitis, acute liver failure, stevens-johnson syndrome an important aspect of care for these children in the picu for the staff is risk of exposure to body fluids and of needle stick injury. universal exposure precautions should be strictly adhered to. it is imperative that all staff be aware of the guidelines and procedures after exposure to biological fluids in their institutions and seek advice from the occupational health department immediately if exposed. as increasing numbers of immunocompromised children with fungal and viral infections are admitted to the pediatric intensive care units, common antifungal and antiviral medications that are used against these infections are briefly reviewed. for a complete review of these topics, the reader is referred to chapters on individual fungal and viral infections in this book. increasingly systemic fungal infections have become more significant in morbidity and mortality of immunocompromised patients in intensive care units. factors that have been associated with this increase are: • use of more potent and broad-spectrum antibacterial agents • prolonged and severe neutropenia • prolonged and severe immune dysfunction (primary or secondary) • having central venous lines and invasive devices • total parenteral nutrition (tpn) the most common fungal pathogens causing systemic illness in critically ill children are candida and aspergillus species. in recent years there has been an increasing importance of uncommon fungal pathogens such as non-albicans candida species, fusarium species, trichosporon species, and dematiaceous fungi. in an immunocompromised patient with a positive fungal culture from a central venous line, current guidelines strongly advocate removal of the line. traditionally with invasive candidiasis, amphotericin b (amb) has been the first-line drug to use. however, intravenous flucanozole and itraconazole could be considered. in non-neutropenic patients positive for c. albicans (but not other candida species) fluconazole is as effective as amb. in empirical treatment of prolonged febrile neutropenic patients (>4-5 days) amb should be started. clinical trials have shown that liposomal preparations of amphotericin b (l-amb) have similar, but not better, efficacy compared with conventional amb preparations. some authors recommend a liposomal preparation of amphotericin as a preferred first-line treatment. unfortunately, high cost can be prohibitive in many parts of the world. in general, the l-amb agents cause less fever, rigors, nausea, and vomiting. they are also less nephrotoxic. voriconazole, a second generation triazole, can be used for empirical treatment of febrile neutropenic patients in place of l-amb. in patients with invasive aspergillosis it has been shown that initial therapy with voriconazole leads to a better response and improved survival with fewer side effects. echinocandins such as caspofungin have been used in combination with amb or voriconazole in more resistant cases of invasive aspergillosis with persisting fevers. micafungin and anidulafungin are two other agents in this family. antifiungals (old and new) have a large potential for side effects and drug-drug interactions. clinicians need to be aware of the specific profiles of the drugs they use from this antimicrobial family. below are the common agents likely to be used in the intensive care unit. hiv drugs have not been discussed. please refer to the chapter on hiv for more detailed discussion of these agents. there are two main groups of antiviral agents: various types of nosocomial infections in pediatric intensive care units, blood stream infections had the highest incidence, followed by lower respiratory infections and urinary tract infections. a basic mandate of medicine is ''primum non nocere'' -first do no harm. while it is inevitable that some patients may acquire nosocomial infections, these infections cause significant morbitiy and mortality. the overall mortality attributable to the various nosocomial infections within the picu has been estimated to be between 10% and 15% and infections acquired in the picu are associated with an increased risk of death, with a relative risk of 3.4. it is widely appreciated that these infections can be minimized by a number of simple interventions, most important of which is hand washing; ''clean hands save lives.'' in a review of the related literature between 1990 and 2002, it was shown that between 11% and 48% of nosocomial infections could have been prevented. blood stream infections are common. not surprisingly they are most common in those patients who are the most debilitated, receiving mechanical ventilation and have central venous lines in situ for longest time, urinary catheters and other artificial surfaces. the spectrum of infections also has a predictable frequency. gram +ve infections are the most common bacteremias (whether or not associated with a central line) followed by gram àve and then fungi. typically fungi are found in those patients on tpn or long term, broad-spectrum antibiotics and immuosuppressed. central venous lines (cvl) are used to provide secure intravenous access for administration of medications such as vasopressors and inotropes, to monitor pressures, blood oxygen saturations, and for intravenous nutrition. in a survey of picu's in the united states the rate of cvl infection was 7.6 per 1,000 catheter-days. in neonates the corresponding figure was 11.3 per 1,000 catheter-days. in europe the cvl infections occurred at a rate of 10.9 infections per 1,000 catheter-days. measures taken at time of insertion of the cvl significantly reduce the incidence of infection. strict aseptic technique (gowns/gloves/mask and wide sterile field), use of chlorhexidine (as opposed to povodine/iodine), and minimal trauma (use of ultrasound and experienced operators) are all very important factors at the time of insertion. chlorhexidine disks topically placed upon the skin at the insertion site and antibiotic impregnated lines are used by some units but not proven to be of value. cuffed and tunneled lines such as hickman lines, port-a-cath lines and picc (peripherally inserted central catheter) have a significantly lower rate of infections than standard central lines that are inserted in the intensive care. where long term therapy >1 week is required consideration should be undertaken to the insertion of one of these types of lines. site of insertion is important. the femoral vein is easy to cannulate with fewest insertion complications. however, it is more likely to become infected and thrombosed. it is good as a temporary line but early consideration should be given to removing and/or repositioning access. to prevent central line infection minimization of the ''opening'' of the line on a daily basis is important. asepsis on line ports prior to use (with alcohol or chlorhexidene) is critically important. ultimately to reduce infection rates lines should be kept for the briefest time possible. difficulty of insertion and type of ongoing therapy come into this cost-benefit analysis. when there is a suspicion that a central venous line has become infected then blood cultures should be drawn from the line and from a peripheral puncture. broad-spectrum antibiotics that cover the bacteria above (vancomycin and gentamicin for example) should be commenced. the line should be removed if at all possible. an attempt to sterilize the line may be made in the circumstances where the line is ''precious'' and not easily replaced. this can involve alternate infusion of antibiotics through all lumens and the use of antibiotic ''locks.'' this is defined as a respiratory infection that occurs 48 h post admission for mechanical ventilation. the respiratory infection is defined by: fever/hypothermia, crackles on physical exam, new respiratory infiltrates on cxr, deteriorating ventilatory status (tachypnea), cough, deteriorating gas exchange, elevated or depressed white cell counts. this may or may not be in the presence of bacterial isolates from a sterile respiratory sample (i.e., bal). there are age specific criteria for the diagnosis of vap. the cdc has produced a document that lists the specific criteria. this can be found at: http://www.cdc.gov/ncidod/hip/nnis/members/pneumonia/final/pneumocriteriav1.pdf. the incidence of vap in the picu is 6-11.6 per 1,000 ventilator-days. the diagnosis of vap is challenging and controversial. there are a number of simple interventions to reduce the incidence of vap. they are: 1. elevate head of the bed to 30 2. ventilator tubing: (a) dependant positioning of ventilator tubing to avoid aspiration (b) removal of excess condensate (c) limit frequency of tubing change unless required 3. suctioning: (a) limiting amount of saline lavage when suctioning (b) sterile technique (gloves and sterile catheter) + (gowns/masks and eye wear for protection of staff) 4. mouth care: frequent mouth cares with chlorhexidene-based wash 5. feeding: (a) early institution of feeding (b) avoidance of gastric over distension (c) limiting use of antacid therapy to high risk patients (i.e., burns, head injury) 6. avoid/limit antibiotic therapy to minimize chance of colonization with antibiotic resistant flora urinary tract infection is directly proportional to the length of time that a foley catheter is in place. frequently, patients are on antibiotics that will suppress urinary infections. however, virtually all intensive care patients with urinary catheters will acquire urinary sepsis if their stay is prolonged. more than 90% of hospital acquired uti's occur in catheterized children. the best intervention (as with central lines) is early removal of the catheter. when strict measurement of urinary output is not needed and the likelihood of urinary retention (due to illness or drugs) is not an issue then catheters should be removed. intermittent catheterization can be considered as an intervention to avoid a permanent foley catheter where retention is an issue in a longer term patient. if an icu patient develops fever or unexplained sepsis then it is mandatory that a urine specimen be sent for microscopy and culture. this is especially important in the patient with a catheter. if urinary sepsis is proven then consideration for catheter removal should be given. broad-spectrum antibiotics that cover the spectrum of bacteria listed above should be commenced. antibiotics should be specifically weighted to cover the gramnegative bacteria as these are most common. surgical site infections are a less frequent infection but none the less important source of infectious morbidity. if a wound is ''dirty'' or contaminated such as a traumatic soiled wound or contaminated peritoneum from perforated appendicitis then broad-spectrum antibiotics should be commenced in high dose. at the same time, appropriate surgical management should be undertaken to deal with the contaminated wound. the surgical team will usually offer guidance on this issue. if a wound is ''clean,'' for example a surgical incision, the surgical team will generally have a preference for antibiotic prophylaxis. commonly a second generation cephalosporin will be used. this should be given at time of the operation and for a defined and limited time thereafter. prolonged prophylaxis has been shown not to prevent inevitable wound infections and promotes emergence of multiple antibiotic resistances. for wounds that become infected in the intensive care unit, swabs should be taken of any discharge. surgical review should be initiated and the wound dressed (with frequent changes). appropriate antibiotics should commence. opening of the wound and drainage/debridement of infected tissue is the responsibility of the managing surgical team. all intensive care units should have the ability to isolate for airborne and body fluid infectious organisms. simple hand washing is very important (before and after examining patients or attending the bed side). where this is not infections in the picu practical an alcohol-based hand gel can be used. from simple hand washing a graduated appropriate degree of isolation and infection control processes should be undertaken, i.e., gowns, gloves, respiratory protection -masks with increasing filtering ability to full respirators. negative pressure rooms (with antechambers) are usually reserved for respiratory isolation for the protection of staff and other patients. positive pressure rooms are for protective isolation of the patient who is immunocompromised. negative pressure isolation and strict barrier isolation is reserved for highly infectious pathogens. sars and ebola virus are examples where this may be necessary. all intensive care staff should strictly adhere to hand washing practices (with a chlorhexidine based product). unfortunately this is not the case and medical staff are often the worst offenders in this regard. active and repeated awareness campaigns should be carried out to reinforce this basic but very important healthcare related activity. severe infectious processes are common reasons for admission to the pediatric intensive care unit. children in the picu are at risk for developing severe infections. increasingly children with a dysfunctional immune system survive their primary illnesses and are admitted to the picu with severe infections. secondary immune deficiency is common in the course of prolonged critical illness. rapid sampling of body fluids and commencement of broad-spectrum antibiotic cover is of the utmost importance. it is shown that even 5 min of delay in starting appropriate antibiotics has been associated with increased mortality. if there are reasons to believe there is an anatomical source of infection (collection of pus, infected central venous line, infected prosthesis etc.) often the antibiotics would not achieve their effects until the source of infection is dealt with effectively (surgical evacuation/ removal, drainage). optimizing the hemodynamic status of the patient (oxygen delivery, addressing preload, after load, and contractility) should start from the moment one considers the diagnosis of sepsis or severe life threatening infection. diagnostic and therapeutic interventions all go hand in hand and start in parallel from the initial encounter with the patient. it is vital that every unit has an updated knowledge of the prevalence and sensitivity of the micro organisms prevalent in their community and in the hospital. colleagues in clinical microbiology or infectious disease departments are invaluable members of any picu team in dealing with these issues. prophylactic measures such as effective hand washing, observing strict sterility while placing central venous lines, measures to reduce incidence of the vap, discontinuing the invasive lines and catheters when not indicated anymore, and adherence to universal exposure precautions should be implemented and monitored and audited regularly. they save more lives and money than much more expensive interventions. effective antibiotic stewardship, tailoring the antibiotic coverage when sensitivities of the causative organisms are known, and discontinuing broad-spectrum antibiotics as soon as clinically prudent will decrease the burden of antimicrobial resistance in the intensive care unit. supportive care of children with cancer: current therapy and guidelines from the children's oncology group incidence of pediatric and neonatal intensive care unit-acquired infections. national nosocomial infections surveillance system; pediatric prevention network viral sepsis in the pediatric intensive care unit bts guidelines for management of pleural infection in children infection control and the prevention of nosocomial infections in the intensive care unit ventilator-associated pneumonia in the pediatric intensive care unit: characterizing the problem and implementing a sustainable solution necrotizing fasciitis in children: diagnostic and therapeutic aspects clinical practice parameters for hemodynamic support of pediatric and neonatal septic shock: 2007 update from the american college of critical care medicine goal-directed management of pediatric shock in the emergency department cdc guidelines on the diagnosis of ventilator associated pneumonia toxic shock syndrome in children epidemiology, pathogenesis, and management manson's tropical disease british thoracic society guidelines for the management of pleural infection prognostic factors in pediatric cancer patients admitted to the pediatric intensive care unit epidemiology and outcome of necrotizing fasciitis in children: an active surveillance study of the canadian paediatric surveillance program acute bronchiolitis and croup ventilator-associated pneumonia in neonatal and pediatric intensive care unit patients risk factors for healthcare-associated infection in a pediatric intensive care unit a national point-prevalence survey of pediatric intensive care unit-acquired infections in the united states; pediatric prevention network concept of operations for triage of mechanical ventilation in an epidemic outcome of children requiring admission to an intensive care unit after bone marrow transplantation infections of the airway the global neonatal and pediatric sepsis initiative infections in the intensive care unit urgences et soins intinsifs paediatriques severe malaria: lessons learned from the management of critical illness in children sepsis and septic shock: a global overview pediatric critical care surge capacity critical care outcomes in the hematologic transplant recipient clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 update by the infectious diseases society of america management of severe dengue in children infectious diseases in the pediatric intensive care unit rogers' textbook of pediatric intensive care red book: 2009 report of the committee on infectious diseases nosocomial infections in pediatric patients: a european, multicenter prospective study; european study group hand washing in the intensive care unit: a big measure with modest effects evidence behind the who guidelines: hospital care for children: what treatments are effective for the management of shock in severe dengue? epiglottitis and croup a prospective study of ventilator-associated pneumonia in children predictors of mortality in patients undergoing autologous hematopoietic cell transplantation admitted to the intensive care unit london world health organisation (2005) pocket book of hospital care for children: guidelines for the management of common illness with limited resources. who, geneva zar hj, apolles p, argent a et al (2001) the etiology and outcome of pneumonia in human immunodeficiency virus-infected children admitted to intensive care in a developing country infections in the picu key: cord-022453-xe5v7947 authors: babiuk, l.a. title: viral gastroenteritis in ruminants date: 2013-11-17 journal: virus infections of ruminants doi: 10.1016/b978-0-444-87312-5.50076-x sha: doc_id: 22453 cord_uid: xe5v7947 nan diarrhea is one of the leading causes of morbidity and mortality in infants and young animals in both developing and developed countries. it has been estimated that over 500 χ 10 6 cases of diarrhea occur annually in humans, leading either directly or indirectly to approximately 10 χ 10 6 deaths (editorial, 1978) . accurate estimates of diarrhea cases in ruminants are not available, but they would appear to be at least as high as in humans on a percentage basis, thus indicating the economical importance of this disease syndrome. until recently, the agents responsible for most cases of nonbacterial gastroenteritis were not identified. however, since the discovery in 1969 by mebus (mebus et al., 1969 ) that a virus was present in feces of calves suffering from diarrhea, it has been proven that rotaviruses can infect calves and cause diarrhea. this discovery prompted the search for related viruses as a cause of diarrhea in other animals. as a result of these investigations, rotavirus has been found to be a major cause of nonbacterial gastroenteritis in most mammals and in fowl (flewett and woode, 1978; mcnulty, 1978; woode, 1982) , and it is now accepted that up to 60% of nonbacterial gastroenteritis cases may be caused by rotavirus. the remaining cases of diarrhea are caused by a variety of viral agents, which may include coronavirus, torovirus, calicivirus, parvovirus, enterovirus, adenovirus, astrovirus, minireovirus or rota-like viruses (table 35) . however, the role and prevalence of many of these agents in causing diarrhea has not been firmly established, neither in ruminants nor in other mammals. although as microbiologists we try to identify a specific etiological agent as a cause of diarrhea, it must be emphasized that diarrhea is often multifactorial and that interactions of various factors with infectious agents can exacerbate the disease. these factors can be broadly grouped into immunological, environmental and nutritional. in each category there are large numbers of components that can interact and alter both the degree of diarrhea and the final outcome of the disease. for example, diarrhea is often associated with inclement weather, i.e. storms, sleet, cold, etc. this is probably associated with increased stress due to temperature fluctuations which can alter the animals' defense mechanisms and increase the probability of infection due to increased animal congregation. the viruses that cause gastroenteric infections can generally be divided into two groups. for viruses of the first group, replication is restricted to the gastrointestinal tract; they induce disease as a result of direct effects only on the cells of the intestine (fig. 179) . most of these agents enter the host directly, via the oral cavity, and pass into the gastrointestinal tract. viruses of the second group enter the host via the oral cavity and replicate in the gastrointestinal tract but they do not remain localized. these viruses may spread to other target organs such as lymphoid tissue (kahn, 1978) or even the cns (nathanson, 1979) . at least eleven different viruses can cause intestinal damage and diarrhea in ruminants under appropriate conditions (table 35 ). all of these agents produce the most severe clinical signs during the first few weeks of life little and shadduck, 1982) . however, there are reports of virus shedding associated with diarrhea in older animals as well (von bonsdorff et al., 1976; mcnulty et al., 1978; jones et al., 1979) . in most cases the infection of older animals is subclinical, and it has been suggested that they serve as virus carriers and are a source of infection for young susceptible animals. in fact, recent reports suggest that removal of carrier animals from a herd reduces or eliminates the disease. the reason for increased severity of diarrhea in younger animals and a higher mortality is that the viruses generally cause greater villous atrophy in the younger animals. rotavirus-induced disease was first described by cheever and mueller (1948); later rotaviruses were established as important infectious agents in mice by kraft (1957 kraft ( ,1958 . however, rotavirus was not recognized as an important pathogen in domestic animals until mebus et al. (1969) identified it as a cause of neonatal diarrhea in calves. since then it has been demonstrated to play a major role in nonbacterial gastroenteritis in most mammalian species. in most outbreaks the disease occurs suddenly and spreads rapidly to other susceptible individuals. the reason for this rapid spread is that the concentration of virus can reach 10 11 particles per gram of feces, which is equivalent to 10 7 infectious doses (flewett and woode et al., 1976a, b) . experimental inoculation of bacteria-free filtrates containing rotavirus causes diarrhea within 12-24 h in susceptible young animals. diarrheic animals are also anorexic and can vomit. the reason for such rapid clinical signs is that in the absence of passive antibody or local acquired immunity, the virus infects the enterocytes of the villi, rapidly killing them. the replication cycle of rotavirus is approximately 12 h (carpio et al., 1981) . rotavirus infection is generally limited to the small intestine in calves, pigs and humans (middleton et al., 1974; mebus and newman, 1977; mcadaragh et al., 1980) , but antigen can be found in the colon of lambs (snodgrass et al., 1977) , pigs (theil et al., 1978) and mice (little and shadduck, 1982) . viral infection occurs in the enterocytes of the upper half of the villi of the small intestine, resulting in rapid death and sloughing of the cells (see fig. 179 ). with death, the villi become shortened and loose their adsorptive capacity (woode and crouch, 1978) . the cells at the tips of the villi are responsible for production of lactase which aids in digestion of lactose. thus the combination of reduced adsorptive capacity and reduced enzyme activity accounts for the diarrhea. since the crypt cells are not damaged, regeneration of the enterocytes and recovery of the villi is generally rapid after the infection is overcome. animals that recover from the disease return to normal body weight within 10-28 days after infection. coronaviruses can cause both respiratory and gastrointestinal infections in humans and animals (robb and bond, 1979) . transmissible gastroenteritis virus of swine was one of the first coronaviruses identified as a cause of diarrhea in animals (doyle and hutchings, 1946) . coronaviruses have also been identified as a major cause of calf diarrhea (stair et al., 1972; mebus, 1978; storz et al., 1978a) . bovine coronavirus diarrhea, like rotavirus diarrhea, occurs within 15-24 h p.i. early in infection the villous epithelial cells appear morphological normal but they contain large amounts of antigen. since diarrhea occurs before denudation and loss of enterocytes it is postulated that it is a direct result of infection of the cell and the ensuing redirection of cellular functions from absorption to virus replication. if absorption does not occur there is accumulation of digestive fluids. as the infection proceeds cells are lost from the villi and are replaced by immature squamous to cuboidal epithelial cells which lack the enzymes required for digestion of milk. they also have a reduced absorptive capacity as is the case in all other virus infection of the gastrointestinal tract. caliciviruses, astroviruses (woode and bridger, 1978) and parvoviruses (storz and bates, 1973 ) may be responsible for causing gastroenteritis and may account for a significant portion of the cases of diarrhea which are not caused by rotaviruses or coronaviruses. however, there are still 20-30% of diarrhea cases for which no etiological agent has been identified. as the search continues for etiological agents other viruses are discovered. one such new agent is the breda virus which has recently been identified. in the case of breda virus infection, the lesions at first appear similar to coronavirus infections with respect to location of lesions. however, on closer examination it becomes obvious that the disease is different, since the lesions and infected cells are visible in the lower 50% of the villi and in the crypts of the small intestine. in the colon, infected cells are present throughout the villi and in the crypts. in addition, structurally this virus is not identical to coronaviruses and is related to an equine virus described in europe (horzinek et al., 1984) . recent studies also suggest that other species may have antibody to this group of viruses, which has been tentatively designated as toroviruses (horzinek et al., 1987) . parvoviruses can infect a wide variety of animals, ranging from pets to large domestic animals (kahn, 1978; storz et al., 1978b) . in contrast to the other viruses discussed so far, this virus family can produce systemic disease as well as enteritis. since the virus generally replicates in rapidly dividing cells, the lesions are seen in the crypts of both the small and large intestines as well as in lymphoid tissue. because of the replication in lymphoid tissue this disease can be more severe, especially in small animals, than other viral infections, because of interference with immune responses and damage to the crypts. however, the role of parvoviruses in diarrhea of ruminants is probably small compared to that played by the other agents. one common feature of all of the agents described to date is that infection occurs by ingestion of the virus. since the virus does not have to spread systemically, the incubation period is extremely short, with villus shortening and reduced fluid adsorption, dehydration and death if diarrhea is severe enough. for a differential diagnosis of the actual cause of diarrhea attempts must therefore be made at demonstrating the presence of the specific agent. however, regardless of the agent treatment will be the same. although these viruses have been associated with diarrhea, there are also many instances where they are present but no disease occurs. furthermore, it is not always possible to reproduce the disease in conventional or even gnotobiotic animals to the extent that it occurs naturally. under experimental conditions most attempts to reproduce the disease are made with a single pathogen and more importantly with plaque purified isolates. replication of these pure populations may be restricted to very localized regions of the intestine; therefore, they do not cause sufficient damage throughout the intestine (longitudinally) to cause the damage required for disruption of intestinal function and diarrhea. under natural conditions there may be multiple strains of a virus (sahara et al., 1982) that infect different areas of the intestine, thus causing severe diarrhea. another explanation for poor reproducibility of diarrhea under experimental conditions could be that avirulent strains grow in culture more rapidly than the virulent strains (woode, 1982) . if these are used to challenge animals no disease occurs. another explanation could be related to immunity. if low levels of active or passive immunity are present this would keep the virus infection rate low so as to allow only few viruses to initiate new infections, as is often the case with persistent local infections. however, during stress the immune defense is reduced and virus shedding and diarrhea may occur. when the immune system returns to normal, diarrhea stops but virus may continue to be shed. this type of carrier state would insure the continued presence of the virus in the environment for infection of susceptible neonatal animals. such carrier states have been demonstrated to occur both in vivo (leece and king, 1980; benfield et al., 1982; crouch et al., 1985) and in vitro (misra and babiuk, 1980) . many of the viral agents involved in causing diarrhea are not easily cultivable in vitro by conventional methods. the reason for this may be related to the virus tropism for differentiated cells. because of the difficulty in growing enteric viruses a large variety of tests have been developed to diagnose these agents directly (yolken, 1982) . most tests are based on the observation that there are high levels of virus particles present in the feces of diarrheic animals and humans (woode et al., 1976a, b; flewett and woode, 1978; mcnulty, 1978) . it is therefore easy to observe virus in feces by em techniques. however, direct observation is less efficient than if combined with serological tests, as is the case with iem, where the virus is aggregated by specific sera and can be visualized much more easily. the availability of specific antisera and monoclonal antibodies to numerous viruses makes this a very attractive means of diagnosis. some enteric viruses may infect cells without causing a cpe or producing infectious virus. in these cases viruses may be identified by culturing in vitro and testing for the presence of viral antigen in infected cells. a number of points must be considered when choosing a test, including efficiency, speed, relative costs and the specific purpose for making a diagnosis (table 36 ). if trained personnel and proper equipment are available then iem, ria and elisa appear to be very good, with respect to specificity and speed. elisa tests are especially useful for automation and diagnosing large numbers of samples. furthermore, elisa can be read by eye if readers are not available, making this test very attractive. if serotyping is also desired then many of the tests listed can be adapted if specific antisera and preferably monoclonal antibody produced against each serotype are prepared. in most virus infections of the gastrointestinal tract, regardless of whether the virus has a predilection for the epithelial cells at the tips of the villi or the crypts, there is shortening and occasional fusion of adjacent villi resulting in a reduced absorptive surface (keenan et al., 1976; leece et al., 1976; pearson and mcnulty, 1977) . infection generally begins in the proximal part of the small intestine and spreads progressively to the jejunum and ileum and sometimes to the colon (mebus et al., 1973; snodgrass et al., 1977) . this will, however, depend on the initial infective dose, the virulence of the virus strain (woode and crouch, 1978) , and the host's immunological status. thus in the presence of passively acquired antibody, infection can occur but replication is limited to such an extent that either no or only mild diarrhea occurs. in rotavirus and coronavirus infections, which are limited to the cells at the tips of the villi, the absorptive cells are replaced with immature squamous to cuboidal epithelial cells. until these cells mature their absorptive capacity and enzymatic activity is greatly reduced. since they also appear to be relatively resistant to virus infection the disease is often self-limiting if dehydration is not so significant as to cause death (woode, 1982; garwes, 1982) . crypt cells are not damaged and the rate of recovery is therefore generally rapid. in contrast, after infection with viruses that replicate in the crypt cells there is a limited number of new cells available to migrate up the villi and recovery tends to take longer. it should be stressed that although the degree of villous damage may be influenced by the virulence of the virus and the immunological status of the animal, the rate of regeneration of enterocytes and enterocyte maturation may also vary with the age of the animal and the site of virus infection. since glucose and sodium adsorption are highest in the proximal and middle part of the jejunum (shephard et al., 1979; bachmann and hess, 1982) , damage here would cause most severe diarrhea. in viral infections, the mechanism of fluid loss is considered to be different from that in bacterial infections; however, the net losses may be the same. in viral infections, water is predominantly lost from the extracellular fluid due to impaired adsorption and osmotic loss primarily due to the presence of undigested lactose in the lumen rather than to active secretion (lewis and philips, 1972; philips and lewis, 1973; tennant et al., 1978; graham et al., 1982) . however, replacement of mature adsorptive cells with immature cells, which retain some of their secretory functions, also increases the rate of secretion (pensaert et al., 1970; butler et al., 1974; kerzner et al., 1977) . as the virus kills the adsorptive cells there is also a loss of enzymes which are responsible for digestion of disaccharides. furthermore, loss of differentiated villous cells diminishes glucose, sodium carrier and (na + -k + )-atpase activities, which result in a loss of sodium, potassium, chloride, bicarbonates and water. the loss of bicarbonate leads to the development of acidosis. however, acidosis also develops as a result of increased microbial activity in response to fermentation of undigested milk (lewis and philips, 1978) , as well as the increased lactic acid production and decreased utilization in dehydrated animals (tennant et al., 1972; lewis et al., 1975) . acidosis can create a k + -h + ion exchange across the cellular membrane and inhibit cellular functions required for maintaining normal potassium concentration with a net loss of potassium from cells. the next step that occurs is hypoglycemia due to decreased intestinal adsorption, minimal glycogen reserves in young animals, inhibited glyconogenesis and increased glycolysis (lewis and philips, 1978) . this series of complex pathophysiological changes, if not promptly corrected, results in death. effective management of diarrhea requires prompt action to prevent continued loss of fluids and electrolytes. this is most economically achieved by removal of milk from the diet. this reduces the amount of undigested lactose in the lumen and therefore reduces fluid loss and acidosis. therapy should include administration of balanced electrolyte solutions either orally or by the intravenous route. the use of intravenous fluid replacement and careful monitoring of animals could save a large percentage of severely affected animals; however, the costs are generally too high to recommend this as a standard procedure. severity of diarrhea is not only related to the virulence of the infectious agent and the age of the animal but also due to the presence of multiple infections. only a minority of cases of diarrhea in animals is caused by a single virus pathogen (house, 1978) . furthermore, it has been suggested that even if a single pathogenetic virus is involved in an infection there may be heterogeneity within the pathogen (sahara et al., 1982; spencer et al., 1983) . therefore, if two viruses co-infect an animal and have different sites of replication, the combined effect may be much more severe than if they infected the animal individually. this may help explain why it is difficult to reproduce enteric infections in conventional calves with single plaque-purified virus isolates. another important factor is the presence of viral-bacterial synergistic interactions. there is accumulating evidence that many bacterial infections can be more severe if combined with a virus infection (runnels et al., 1980; leece et al., 1982) . thus escherichia coli generally produces scours only during the first few days of life. however, if an animal is infected with, for example, rotavirus or coronavirus, ε. coli can colonize and produce a more severe disease at a later age (fig. 180 ). the exact mechanisms by which this occurs is unknown; however, viruses may alter fluid transport by virtue of infecting some cells. this alteration allows the build-up of toxin by bacteria that are normally nonpathogenic. thus the combination of toxin build-up and decreased mobility of the intestine results in diarrhea. in addition, reduced adsorption of nutrients occurs as a result of virus infection. this provides a more suitable nutritional environment for bacteria to grow, adhere and secrete more toxin. additionally, virus infection may reduce the rate of cell maturation. since it is known that the physiological state of cells may alter adherence, it can be postulated that bacteria actually colonize virus-infected intestines but not normal ones. the virus infection may alter the cells in such a way as to allow direct attachment of bacteria to the viral glycoprotein expressed on their membranes or to altered host cell glycoproteins, as has been convincingly shown for virus-bacterial interactions in the respiratory tract (sanford et al., 1978; davison and sanford, 1981) . finally, some viruses induce fc receptors on the surface of host cells. if this occurs, antibody-coated bacteria can bind via the fc receptor and anchor to the cell, allow secretion of toxin, activation of cyclic amp and increased fluid loss. although herpesviruses are the only viruses reported to induce fc receptors (westmoreland and watkins, 1974; lehner et al., 1975; costa et al., 1977) , preliminary evidence suggests that bovine coronaviruses may also induce them (l.a. babiuk, unpublished data, 1985) . chapter 54 references, p. 563 a major problem with controlling gastroenteric infections in animals is the age at which the animals get the disease. even if the adult animals are immune and transfer antibody to the young, the antibody must be present continuously in the lumen of the intestine to prevent infection, since serum antibodies are not protective wells, 1976, 1978a,b) . since many mammalian species do not continue to secrete high levels of antibody in their milk after parturition, the antibody in the intestinal lumen drops rapidly and the young becomes fully susceptible even if it has acquired high levels of serum antibody. thus, in cattle, the colostrum generally has high antibody levels to most enteric viruses, since the infection rate in adults is high. however, within 5-7 days after parturition antibody levels drop below the threshold required to neutralize virus in the lumen. this is the reason that most enteric virus infections causing neonatal diarrhea in mammals do so after 1 week of age. the observed requirement for local immunity has stimulated the interest in immunizing the newborn. there is presently an oral vaccine on the market for use in newborn calves to provide protection against rotaviruses and coronaviruses. unfortunately, if the colostral antibodies can protect against virulent virus they will also prevent the attachment of vaccine virus to enterocytes. thus the supposed early nonspecific protection, possibly by interferon and the later specific immunological protection do not occur unless the animals are immunized prior to ingestion of colostrum. this is often not possible and, therefore, this vaccine has not proven to be as successful as hoped. to overcome this problem a few attempts have been made at in utero immunization but this is impractical at present under field situations (newman et al., 1978) . however, recent advances may make this approach feasible in the near future. the most recent trend to overcome the requirement of local immunity in gastrointestinal virus infection is hyperimmunization of the dam. this results in a much higher initial level of antibody in the colostrum, which provides excellent early protection. more importantly, even though antibody levels drop they remain above a threshold level which is protective against normal virus challenge doses. the final method of providing high levels of antibody in the lumen is by feeding monoclonal antibody to the animal. this has proven to be very effective in preventing e. coli induced diarrhea in calves (sherman et al., 1982) . the combination of various antiviral monoclonal antibodies with anti-z?. coli antibody should prove effective under certain situations but is probably of limited value in field situations where animals cannot be handled routinely. furthermore, the presence of serotypes, especially in rotaviruses (woode et al., 1983) dictates that each serotype is represented either in the vaccine or in the monoclonal antibody mixture. the recent advances in recombinant dna technology have great potential for helping control gastroenteric infections in animals. they are especially relevant to producing vaccines against viruses that do not replicate well in culture. identification of the antigens involved in protection and the genes coding for them should make it feasible to produce sufficient antigen for immunization. furthermore, it should be possible to identify the sequences involved in protection and synthesize them (lerner, 1983) for immunization of dams during pregnancy so as to elevate colostral and milk antibodies. the problem of multiple serotypes combined with multiple agents that can cause diarrhea in ruminants emphasizes the need for inclusion of many agents in a vaccine before a great decrease in disease incidence will be seen. in this regard economical production of these vaccines is mandatory. a final method of reducing enteric infections is by proper management. since it is assumed that infections either occur as a result of virus shedding from small numbers of adults or from virus in the environment, animals should not be crowded into contaminated areas. movement of young into clean environments, away from other animals, will greatly reduce the rate of infection and economic loss. finally, if only a limited number of animals are carriers, it may be possible to eliminate these and thus break the infection cycle. however, this hypothesis is in need of proper testing. although there is a wide variety of viruses that can cause infections of the gastrointestinal tract of animals, most of them are localized in either the crypts or the enterocytes. infections are initiated in the proximal part of the small intestine and progress sometimes to the colon. infections result in loss of adsorptive cells, villous atrophy, fluid loss and ion imbalance. these pathophysiological events lead to anorexia, dehydration and death. since young animals do not have large reserves of fluids and glycogen, mortality can reach 50-80% in severe outbreaks. however, removal of milk and administration of oral electrolytes can significantly reduce losses. effective immunization requires that local immunity is present at an early age. oral immunization with live attenuated vaccines is difficult due to the high levels of maternal antibody in the milk during the first few days of life. to overcome this problem the present trend is to immunize the dam so as to increase the level of antibody in milk above the threshold level required to prevent infection. as more agents and serotypes involved in gastroenteritis are identified, vaccines will have to combine various pathogens and serotypes for protection. although in some cases vaccines may be produced by conventional methods, recombinant dna technology may aid in providing sufficient quantities of antigens to vaccinate against viruses that do not replicate well in culture. however, it will be more difficult to test the efficacy of these vaccines under field conditions than that of many other vaccines due to the difficulty in reproducing the disease and its variable incidence from year to year in herds under natural conditions. comparative aspects of pathogenesis and immunity in animals shedding of rotavirus in feces of sows before and after farrowing transmissible gastroenteritis: mechanisms responsible for diarrhea in acute viral enteritis in piglets bovine rotavirus cell interactions: effect of virus infection on cellular integrity and macromolecular synthesis epidemic diarrheal disease of suckling mice: iii the effect of strain, litter and season upon the incidence of the disease immunoglobulin binding to herpesvirusinduced fc receptors inhibits virus growth chronic shedding of bovine enteric coronavirus antigen-antibody complexes by clinically normal cows pathogenesis of rotavirus infection in mice pathogenesis of rotavirus enteritis in gnotobiotic pigs: a microscopic study rotavirus infection in avian species pathogenesis of coronaviral infection in calves scanning electron, light and 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virus infected cells: a cell culture model of bacterial superinfection the mucosal lesion in viral enteritis protection of calves against fatal enteropathogenic colibacillosis by orally administered k99-specific monoclonal antibody conf rotavirus infection in lambs: studies on passive protection the immunoprophylaxis of rotavirus infections in lambs passive immunity in rotavirus infections rotavirus infection in lambs: pathogenesis and pathology analysis of human rotavirus mixed electropherotypes neonatal calf diarrhea: purification and electron microscopy of a coronavirus-like agent parvovirus infections in calves coronavirus morphogenesis and ultrastructural changes in intestinal infections of calves parvovirus associated with diarrhea in calves physiological and metabolic factors in the pathogenesis of neonatal enteric infections in calves clinical management and control of neonatal enteric infections of calves pathogenesis of porcine rotavirus infection in experimentally inoculated gnotobiotic pigs rotavirus associated with acute gastroenteritis in adults the igg receptor induced by herpes simplex virus: studies using radio-iodinated igg rotaviruses in animals viral enteritis in calves isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves naturally occurring and experimentally induced rotaviral infections of domestic and laboratory animals levels of colostral antibodies against neonatal calf diarrhoea virus the isolation of reoviruslike agents (rotavirus) from acute gastroenteritis in piglets morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice and foals studies with an unclassified virus isolated from diarrheic calves antigenic relationships among some bovine rotaviruses, serum neutralization and cross protection in gnotobiotic calves enzyme immunoassays for detecting human rotavirus key: cord-267041-i94lyfsh authors: ellner, jerrold j. title: management of acute and chronic respiratory tract infections date: 1988-09-16 journal: the american journal of medicine doi: 10.1016/0002-9343(88)90456-1 sha: doc_id: 267041 cord_uid: i94lyfsh abstract pharyngitis, bronchitis, and pneumonia represent the most common respiratory tract infections. with a view to establishing effective management strategies, the origins of these illnesses and the diagnostic techniques that have been developed to discover them are reviewed. therapeutic regimens with documented efficacy are outlined with emphasis on specific rather than empiric treatment. although many respiratory tract pathogens remain exquisitely sensitive to penicillin, the emergence of resistant strains underscores the need for safe and effective alternative therapies. respiratory tract infections jerrold j. ellner, m.d. cleveland, cm pharyngitis, bronchitis, and pneumonia represent the most common respiratory tract infections. with a view to establishing effective management strategies, the origins of these illnesses and the diagnostic techniques that have been developed to discover them are reviewed. therapeutic regimens with documented efficacy are outlined with emphasis on specific rather than empiric treatment. although many respiratory tract pathogens remain exquisitely sensitive to penicillin, the emergence of resistant strains underscores the need for safe and effective alternative therapies. r espiratory tract infections include both the most common and the most life-threatening illnesses plaguing humankind. the ordinary "sore throat"pharyngitis-accounts for approximately 40 million visits to physicians per year in the united states, as well as significant time lost from work. although often considered more of a nuisance than a disease, unrecognized and/or untreated pharyngitis can lead to serious suppurative and nonsuppurative complications. acute bronchitis-the painful cough with sputum and possibly fever that develops after an upper respiratory infection-does not necessarily represent a bacterial infection. in contrast, persons with chronic bronchitis often experience episodes of acute infectious exacerbation of their condition, which may respond to antibiotic therapy. the most serious of the respiratory tract infections is acute pneumonia. it is a fairly common cause of hospitalization among adults, accounting for 10 percent of adult hospital admissions per year. acute pneumonia ranks sixth among all causes of death in the united states and should be considered a potentially fatal illness. pharyngitis can be caused by a number of etiologic agents, of which about a third are viral. in as many as 40 percent of cases, attempts to identify any specific agent-viral or bacterial-will be unsuccessful. from a therapeutic standpoint, the most important pathogen in pharyngitis is group a streptococcus, which accounts for 10 percent to 30 percent of cases. less common causes of pharyngitis, which may require specific isolation procedures or serologic assay, include neisseria gonowhoeae (isolation requires thayer-martin medium), corynebacterium hemolyticum (sore throat and skin rash), group c streptococcus, mycoplasma pneumoniae, and epstein-barr virus (infectious mononucleosis). diagnosis and treatment of streptococcal pharyngitis are clearly indicated, for a number of reasons. several prospective, placebo-controlled trials have demonstrated that treatment decreases duration of fever and other symptoms of sore throat due to streptococcal infection [l-4] . treatment of streptococcal sore throat also contributes to controlling and reducing the spread of such infections within families and classrooms. clinically, prompt and appropriate treatment prevents suppurative complications-not only can bacteria spread from the pharynx to the tonsils but also to the retro-and lateral pharyngeal spaces. even meningitis was a not-uncommon consequence of streptococcal pharyngitis before the availability of antibiotic drugs. finally, perhaps the best recognized reason to treat group a streptococcal infections is to prevent the development of acute rheumatic fever, which appears, after years of declining incidence, to be on the rise again in the united states. the standard laboratory procedure for diagnosing pharyngitis is culture of a throat swab. if the culture result is positive for group a streptococcus, the specificity of the test is high. on the other hand, the sensitivity of the test is not as high as most clinicians would like. cultures of two sequential specimens, for example, taken from the same patient will show identical results only about 90 percent of the time [5] . moreover, a study conducted by saslow and colleagues [6] showed that 24 percent of throat culture specimens failed to reveal streptococcal infection that was later identified when the patients' tonsils were removed and examined. one of the most frustrating drawbacks of the culture method of diagnosis is the 24-to 36-hour delay before results are available. delay also may compromise treatment efficacy; in one study, immediate institution of antibiotic drugs was associated with less incidence of morbidity than when treatment was delayed until culture results became available 131. for these reasons, rapid tests for identifying streptococcal infection will soon become standard. based on the use of an antibody to detect type-specific carbohydrate of group a streptococcus in throat swabs (latex fixation or an enzyme-linked immunoassay), these tests can yield a diagnosis with both high specificity and high sensitivity within seven to 70 minutes. in the absence of positive test results, the decision to treat a patient with pharyngitis is primarily based on clinical findings. centor and colleagues ['7] have performed a decision analysis based on four clinical markers of streptococcal pharyngitis: tonsillar exudates; swollen, tender anterior cervical lymph nodes; fever or history of fever; and absence of cough. if all four features are present, the chance that the infection is streptococcal ranges from 26 percent to 65 percent; if only three of the four are present, the likelihood of streptococcal infection decreases to between 11 percent and 38 percent; if none of the features is present, the infection can be assumed to be nonstreptococcal in origin. the setting, age, and contacts of the patients help determine whether the probability of streptococcal infection is at the lower or higher end of the range. an older person who has no contact with children, for instance, will have a much lower chance of contracting streptococcal infection than a preschool youngster, or a first-grade teacher. decision analysis indicates that anyone with three or four clinical features indicative of streptococcal pharyngitis should be treated, without proceeding to a throat culture. if and when rapid tests become widely available, treatment without confirmation would be reasonable only when the prevalence of group a streptococcus in the population at risk is at least 20 percent and the patient manifests all four streptococcal clinical features. in practical terms, the availability of a rapid test would virtually eliminate the question of whom to treat. regimens of choice for treatment of streptococcal pharyngitis include oral penicillin for 10 days or a single, intramuscular injection of benzathine penicillin g. the importance of taking the oral medication for the full 10 days to prevent nonsuppurative complications should be stressed to patients. erythromycin is an appropriate alternative for penicillin-allergic patients. other regimens, such as cefaclor or amoxicillin plus clavulanic acid, have been used effectively to treat streptococcal pharyngitis but offer no advantages compared with penicillin. finally, although diagnosis and treatment of pharyngitis are usually straightforward, certain signs warrant immediate investigation. respiratory difficulty, particularly stridor associated with sore throat in adults, may be indicative of epiglottitis due to haemophilus influenxae. other signs and symptoms of potentially dangerous clinical conditions include difficulty swallowing or handling secretions, severe pain without visible erythema, or a palpable mass in the pharynx. blood in the pharynx or ear suggests an impending disastrous suppurative complication such as erosion in a carotid artery. even untreated, the symptoms of streptococcal infection will disappear. therefore,, persistence of symptoms for more than one week also warrants further evaluation. in previously healthy persons, acute purulent bronchitis usually is not the result of a bacterial process. rather, a productive cough, characterized by sputum, fever, and retrosternal pain on coughing, develops at the end of a typical upper respiratory tract infection. if the person is well with minimal systemic symptomology and has no underlying conditions, antibiotic treatment is generally unnecessary. antibiotic therapy should be started, however, if cough productive of purulent sputum is protracted, and bacteria are seen on a gram's stained preparation of the sputum. the choice of drugs is dictated by the findings on smear and culture specimens. a much more frequent and important type of bronchitis is acute exacerbation of chronic bronchitis. the classic definition of chronic bronchitis is almost daily production of sputum for at least three consecutive months and for two consecutive years. the origins of chronic bronchitis are cigarette smoking, inhalation of toxic substances, and possibly infection, although the role of infection usually is not clear. acute infectious exacerbation of chronic bronchitis describes some combination of the following: a change in sputum color, consistency, or amount; increasing cough or dyspnea; chest tightness; and general fatigue without other systemic manifestations. if the patient presents with fever and chills, the diagnosis will probably be something other than simple acute infectious exacerbation. the relationship between infection and exacerbation is sometimes difficult to determine. potentially pathogenic bacteria can be isolated in most sputum specimens from persons with chronic bronchitis, even in the absence of symptoms of acute infectious exacerbation, although it is true that streptococcus pneumoniae is isolated in increased quantities when patients experience exacerbation [sl, it is not clear in the symposium on cefixime / ellner determination of the cause of pneumonia * is the current condition accurately termed "acute," or is it really chronic? is it perhaps an acute illness superimposed on several months of progressive respiratory symptoms? * is it community-or hospital-acquired? @ who is the host? young and healthy? edlerly? is there serrous underlying disease? * )ias there been an unusual exposure? * is the patient a member of an at-risk group for aids? * is there a pleural friction rub or has there been a rigor? * is there lobar consolidation or large pleural effusions! * is the pneumonia cavitating? individual case whether culture specimen results are indicative of the normal flora for the patient or are pathogenic. about 30 percent to 50 percent of patients with acute bronchitis will have nonencapsulated h. influenxae or s. pneumoniae (or both) as persistent colonizers of the bronchial tract. presumably, these are the agents responsible for exacerbation. in another 25 percent to 50 percent of patients, the bronchitis will have a viral (influenza, parainfluenza, respiratory syncytial virus, rhinovirus, coronavirus) rather than bacterial origin. staphylococcus aureus (often following influenza) or enteric gram-negative rods are found in 5 percent to 10 percent of cases, and, occasionally, m. pneumoniae is implicated based on serologies. since antibiotic treatment appears to decrease the incidence of morbidity and shorten time missed from work; it is indicated in patients with acute exacerbation of chronic bronchitis [9, 10] . several regimens have been proposed and tested based on the bacteriology of bronchitis. although none has been shown to be clearly superior, amoxicillin plus clavulanic acid, ampicillin, cefaclor, erythro-my&n, tetracycline, or trimethoprim-sulfamethoxazole appear to be effective. to initiate appropriate, life-saving therapy for acute pneumonia, it is vitally important to establish its specific origin. clues about the cause of pneumonia can be elicited by addressing a number of questions while obtaining the history and during the physical examination (table i) if the patient is young and otherwise healthy, he or she probably has infection with s. pneumoniae, m. pneumoniae, or a viral pneumonia. on the other hand, an elderly person is more likely to have influenza or s. pneumoniae, which osler dubbed "the old man's friend." elderly people, particularly those with refractory pneumonia, also may be infected with ivycobacterium tuberculosis, and in those older patients who are especially debilitated, a search for gramnegative eriteric organisms may be indicated. aspiration pneumonias and infections with possibly antibiotic-resistant s. aureus or gram-negative bacilli are a problem in hospitalized patients, particularly in those who have suffered seizures. underlying alcoholism may predispose a person to s. pneumoniae or gram-negative organisms such as klebsiella, whereas underlying diabetes increases the risk of infection with gram-negative enteric organisms or m. tubercu-symposium on cefixime i ellner losis. patients with hemoglobinopathy, specifically children with sickle-cell disease, are particularly prone to infections with s. pneumoniae, h. influenxae, and m. pneumoniae. possible causes of pneumonia in 'patients with chronic lung disease include s. pneumoniae, h. influenxae: pasteurella multocida, gram-negative enteric bacilli, and m. tuberculosis. it is important to determine where the patient lives and whether he or she has had contact with animals. exposure to farm animals such as cattle, goats, and sheep, for example, may be associated with q fever, bracellosis, or anthrax. rabbit hunters may have tularemia and,exposure to birds can lead to psittacosis or histoplasmosis. people who live in semi-arid regions of the united states southwest could have coccidioidomycosis, and those from areas around the mississippi and ohio river valleys could have histoplasmosis or blastomycosis. armed services personnel,travelers, and visitors who have been in southeast asia may be ill with tuberculosis or melioidosis. the patient with community-acquired purulent pneumonia classically presents with sudden pleuritic chest pain, productive cough with purulent sputum, high fever (up to 4o"c), and profound shaking chills (rigors). physical examination and chest radiograph reveal signs of lobar consoliddtion. the white blood cell count is elevated with an increase in circulating immature neutrophils, and lobar infiltrate is tonfirmed radiologically. acute community-acquired purulent pneumonia is most commonly caused by s. pneumonia (50 percent to 90 percent). depending on where the patient lives, 17 percent to 23 percent of these cases may be due to legionella pneumophila, 2 percent to 18 percent to h. influenxae, and 2 percent to 10 percent to s. aureus. anaerobes and non-haemophilus gram-negative enteric organisms may also account for some of these community-acquired infections. the initial diagnostic decision concerns whether the patient has acute purulent bacterial or nonbacterial pneumonia. the first step in establishing origin is obtaining a good, uncontaminated specimen for gram's stain. if the sample is heavily contaminated with oral flora, it is unlikely to be useful and may be misleading. the adequacy of the specimen can be ascertained by the absence of squamous epithelial cells (less than five per high-power field), and the presence of neutrophils (10 to 15 per high-power field), as well as alveolar macrophages and bronchial epitheiial cells. nasotracheal or transtracheal aspiration may be necessary to obtain a good specimen of loiver respiratory tract secretions. the presence of a predominant organism, particularly if found within white blood cells, suggests that it is pathogenic; aspiration pneumonia may be associated with multiple organisms. although the gram's stain may suggest that the cause of a pneumonia is not bacterial by the finding of inflammatory cells and no organisms, the clinical presentation can be even more useful for distinguishing a bacterial from a nonbacterial infection. nonbacterial pneumonia usually begins less abruptly than bacterial pneumonia, with three or four days of symptoms. constitutional symptoms rather than respiratory tract symptoms comprise the patients' chief com-symposium on cefixime/ellner plaints. the white blood cell count remains within normal limits. importantly, the appearance on radiographic examination is much worse than would be anticipated based on physical examination. this constellation of symptoms suggests m. pneumoniae, adenovirus, q fever, psittacosis, or l. pneumophila. a stain for acid-fast bacilli also is warranted because tuberculosis may present as an apparently nonbacterial pneumonia. occasionally, patients with a more chronic set of respiratory symptoms will present with a nonlobar, unusual infiltrate that does not seem to fit the picture of a viral pneumonia. in this situation, it is particularly important to determine whether there are pockets of cavitation in the lesion. although both pneumococcus and haemophilus can sometimes cause cavitation, its presence suggests tuberculosis, s. aureus, necrotizing infection due to gram-negative bacilli, or mixed anaerobic or fungal infection. clearly, the finding of cavitation shifts the differential diagnosis of pneumonia as well as the antibiotic drugs that may be required for its treatment. cavitation can also result from noninfectious sources, such as septic pulmonary emboli and carcinoma. because of the importance of recognizing necrotizing pneumonia as early as possible, investigators in cleveland have been assessing the feasibility of a specific sputum test based on searching for elastin fibers in sputum [l&12]. elastin fibers are found in the alveoli and terminal bronchioles; their presence in respiratory tract secretions, therefore, indicates a destructive process. these elastin fibers can be identified by dissolving the constituents of sputum with 10 percent potassium hydroxide. the presence of elastin fibers in sputum precedes both the development of cavities and the appearance of pulmonary infiltrates on radiographic examination of the chest. when the sputum test for elastin fibers is positive in an intubated or tracheostomized patient, its predictive value is 100 percent-the patient will progress to a full-blown nosocomial pneumonia [121. since not all nosoeomial pneumonias are necrotizing, the sensitivity of the finding of elastin fibers is only 52 percent. the search for elastin fibers appears to be a generally useful adjunct for diagnosis not only in hospitalized patients but in all persons with pneumonia, particularly when symptoms do not fit the classic patterns of either bacterial or nonbacterial syndromes. clinicians managing patients with pneumonia should strive for specific treatment based on a definite origin of the illness. nevertheless, when obtaining sputum or interpreting the gram's stain is difficult, an empiric regimen may be necessary. penicillin g is the drug of choice for s. pneumoniae, which remains exquisitely sensitive to that agent. parenteral ampicillin would be appropriate treatment for infection with h. inzfluenxae, although beta-lactamase-producing h. inzfluenxae is presenting an increasing problem in adult patients. initial treatment with a combination of trimethoprim and sulfamethoxazole or a third-generation cephalosporin, therefore, is a prudent choice for a serious infection presumed to be caused by h. in.uenxae. s. aureus should be treated with a semisynthetic penicillin such as nafcillin. anaerobes are exquisitely sensitive to penicillin, which is an appropriate starting regimen. if there is no clinical response, it may be useful to switch to a specific antianaerobic agent such as clindamycin. the finding of gram-negative enteric organisms on gram's stain requires combination therapy with an aminoglycoside plus a third-generation cephalosporin. choice of aminoglycoside should be based on the prevailing patterns of bacterial sensitivities in the hospital. although penicillin remains a mainstay of therapy for many patients with bacterial pneumonia, the emergence of resistance should provide the impetus for discovery and testing of alternative therapies. although l. pneumophila can be diagnosed by culture or serologies, confirmation usually requires days to weeks. a diagnosis based on clinical findings is necessary, therefore, and empiric treatment should be initiated. erythromycin can be prescribed for suspected l. pneumophila as well as for ill. pneumoniae. when empiric therapy for a seriously ill patient is clearly indicated, combination regimens, such as an aminoglycoside plus a third-generation cephalosporin, will provide the broad-spectrum coverage that ensures efficacy against possibly resistant gram-negative organisms. in summary, the management of any respiratory tract infection-whether relatively minor or lifethreatening-is most successful when an anti-infective agent is selected on the basis of a definite causative organism. effect of penicillin and aureomycin on the natural course of streptococcal tonsillitis and pharyngitis treatment of acute sore throat with penicillin streptococcal pharyngitis: early treatment and management by nurse practitioners effect of antibiottc therapy on the clinical course of streptococcal pharyngltis bergner-rabinowitz s: primary prevention of rheumatic fever in jerusalem children: ii. identification of beta-hemolflic streptococci beta hemolytic streptococci in tonslllar tissue throat cultures and rapid tests for diagnosis of group a streotococcal oharvneitis a&t: bronchiolitis: a study of 207 cases antibiotic therapy of acute exacerbation of chronic bronchitis the role of infection during exacerbation of chronic bronchitis sputum elastln fibers and the diagnosis of necrotizing pneumonia: a prospective trial diagnosis of nosocomial pneumonia in lntubated intensive care unit patients key: cord-264159-e9071tyv authors: lin, weikang nicholas; tay, matthew zirui; lu, ri; liu, yi; chen, chia-hung; cheow, lih feng title: the role of single-cell technology in the study and control of infectious diseases date: 2020-06-10 journal: cells doi: 10.3390/cells9061440 sha: doc_id: 264159 cord_uid: e9071tyv the advent of single-cell research in the recent decade has allowed biological studies at an unprecedented resolution and scale. in particular, single-cell analysis techniques such as next-generation sequencing (ngs) and fluorescence-activated cell sorting (facs) have helped show substantial links between cellular heterogeneity and infectious disease progression. the extensive characterization of genomic and phenotypic biomarkers, in addition to host–pathogen interactions at the single-cell level, has resulted in the discovery of previously unknown infection mechanisms as well as potential treatment options. in this article, we review the various single-cell technologies and their applications in the ongoing fight against infectious diseases, as well as discuss the potential opportunities for future development. five months since the first reported infection cluster, covid-19 has turned into a vicious worldwide pandemic that infected more than 3.6 million people and caused over 250,000 deaths [1] . the pandemic will also have large spillover effects in terms of economic damage both in the form of healthcare costs and in monetary losses from the disruption of global supply chains, with world trade expected to fall between 13% and 32% in 2020 [2] . the covid-19 pandemic serves as a grim reminder that infectious disease is, and will always be, a major threat to the continued existence of mankind. to date, there are about 1400 microorganisms known to be pathogenic to humans. these pathogens can be broadly classified as viral, bacterial, fungal, and parasitic pathogens [3] . in particular, there have been 3 infectious diseases that have been persistently difficult to eradicate, namely, human immunodeficiency virus and acquired immune deficiency syndrome (hiv/aids), tuberculosis, and malaria. aids, due to hiv, is responsible for nearly 1 million deaths per year [4] . the death toll from tuberculosis, caused by mycobacterium tuberculosis (mtb) bacteria, is the highest amongst all infectious diseases, which is a problem that is exacerbated by the rise of antimicrobial resistance variants of the disease [4] . malaria, a parasitic infection, has afflicted humans for thousands of years and continues to do so today [5] . in light of the above-mentioned examples, among other infectious diseases, further efforts have to be directed for the continued management of the global burden of these diseases. the covid-19 pandemic has highlighted many questions that are relevant in the context of infectious disease as a whole. why are certain people more susceptible to infections? why are some infected individuals asymptomatic or display only mild symptoms? why are there differences in terms of disease progression and outcomes among patients? this diverse response to infection could be explained by the interactions of inherently heterogeneous populations of pathogens, host cells, and immune cells. however, discerning this heterogeneity is difficult in conventional bulk analyses, as they fail to recognize the following: (1) the genomic variability of pathogens, (2) the coexistence and interactions of infected host cells and bystanders, and (3) the diverse functional roles of immune surveillance participants. aside from the limited resolving power in pathophysiological studies, bulk analyses often fall short in terms of the level of precision and the amount of derived information needed for early diagnostics and high-efficacy vaccine development against infectious diseases. just as how microscopy revolutionized our understanding of biology, the enhanced resolution, precision, and breadth of information offered by single-cell technologies has brought an exciting overhaul to our perception of infectious diseases in recent years. the use of single-cell genomics, transcriptomics, proteomics, and epigenetics (referred to as omics altogether in this article) has flourished in many areas of the battlefield against infectious diseases. table 1 presents commonly used single-cell technologies in infectious disease studies alongside several of other non-single-cell systems. a scoring heatmap is used to represent the complexity of information in various aspects that they can provide (e.g., genetic, epigenetic, proteomic, spatial). the heatmap also provides an overall ranking of throughput, cost, and downstream assay compatibility amongst all the listed techniques. hence, it serves as a general guide for future users to select the methods that match their desired outputs. for instance, if the primary target for the study is to collect genetic information (e.g., analysis of invading virus gene heterogeneity in single host cell), single-cell sequencing might be the best candidate. likewise, if the focus is on proteomics orchestrating the immune responses, mass cytometry can furnish the most detailed insight. for infectious disease models that involve the interplay of genomics and proteomics, both cite-seq and reap-seq can be the suitable candidates. while flow cytometry has the highest throughput and lowest cost per experiment amongst the listed single-cell methods, it yields very limited aspects of information. other single-cell assays capable of providing more complex data typically come at the expense of a decreased throughput and increased cost. it is also worth mentioning that microfluidics has made great success in boosting the throughput and cost efficiency of existing single-cell assays. one prominent example would be the transition of well plate systems into microchambers or microdroplets, which ultimately reduces the required amount of reagent required per experiment and in turn reduces costs. in this review, we identified infection pathophysiology, therapeutic discovery, and disease diagnostics as three major areas in which single-cell omics has contributed substantially in the past decade. in pathophysiological studies of infectious disease, single-cell omics offer excellent spatial-temporal resolution that help to not only reconstruct the uneven subcellular distribution of pathogen across the entire host cell population, but also reveal the sequence of immune events accompanied by the change of immune cell profiles. single-cell omics also extrapolates meaningful molecular details that describe the dynamic host-pathogen interplay and immune activation. furthermore, single-cell omics identifies the rare molecules and cell subtypes that exhibit significant functionality in the pathogen-host immune interactions. insights in fundamental pathophysiology naturally have spillover benefits for translational science, such as in vaccine development where single-cell omics has the capability to enhance the discovery of mechanistic correlates of protection through multi-parameter measurements of the immune state with respect to disease, and it enables precision quality control checkpoints to aid the evaluation of vaccine efficacy. in the field of antibody discovery, single-cell omics can simultaneously interrogate antigen specificity and recover the b cell receptor gene sequences, which in turn shortens the previously prolonged and labor-intensive research cycle in the search for effective therapeutic or diagnostic antibodies. in the application of infectious disease diagnostics, single-cell omics are on the verge of practical clinical deployment, as demonstrated by some examples of automated and miniaturized devices. the diagnostic power of single-cell omics can be further enhanced by incorporating digital assays or integrating with other label free single-cell technologies. while there are many merits of single-cell analysis, we also discuss the new sets of challenges that need to be addressed in these systems. finally, we will conclude with our insight on future prospects of single-cell research in infectious disease and highlight several emerging single-cell technologies that may further enrich our arsenal against infections. understanding the pathophysiology of infection is critical to the rational design of prophylactic and therapeutic strategies to tackle infectious diseases. the course of infection, determined by the encounter of pathogens and host cells, is often measured as population-averaged results, leaving the important cell-to-cell heterogeneity out of the picture. the heterogeneity arises from both the pathogens and the infected cells. for example, pathogen heterogeneity can be reflected in the case of viruses, as a mixture of mutated viral particles displaying different infection ability [6] , or in the cases of bacteria, as a population of cells having different resistance to the same antibiotics [7] . host cellular heterogeneity is a combined result of variances in metabolism, composition, activation status, cell cycle, or infection history [6] . recent advances in single-cell analysis provide an attractive approach to probe the cellular population diversity and characterize infection pathophysiology at single-cell resolution. in this section, we will review how the recent advancement of single-cell technologies has helped deepen the understanding of pathogen and host cell heterogeneity and how the complex immune system reacts against infectious pathogens, with a focus on the contributions of single-cell sequencing. pathogen heterogeneity can be inherent or as a result of heterogeneous host-pathogen interactions. it is a favorable feature for pathogens because varied genomic sequences or functional properties enable immune evasion, colonization in novel hosts, and drug resistance acquisition; therefore, they increase the possibility of survival. besides, stochastic fluctuation in biochemical reactions may also contribute to cell-to-cell variability. single-cell technologies provide high-resolution insights into different aspects of intracellular pathogen replication. one area of virology that has benefited from the enhanced resolution of single-cell technologies is the study of variation in infection across single cells and the reasons for such variation. in the study by heldt et al., cells were infected in a population, isolated into microwells, and incubated. the supernatant was subjected to viral plaques measurement, and viral rna was quantified from lysed single infected cells [8] . it was shown that cells infected by influenza a virus (iav) under the same conditions produced largely heterogenous progeny virus titers, ranging from 1 to 970 plaque-forming units (pfu) and intracellular viral rna (vrna) levels varied three orders of magnitude. similarly, using scrna-seq, another study determined the percentage of viral transcripts in the total mrna generated from iav-infected cells, and it revealed that while most cells contained less than 1% of viral transcripts, some cells generated more than 50%, demonstrating infection heterogeneity from the angle of viral load [9] . reasons for this variation can be further explored through the use of high-throughput imaging technology. for instance, akpninar et al. used virus expressing red fluorescence protein (rfp) to study the effect of defective interfering particles (dip) on viral infection kinetics. dip are noninfectious progeny particles lacking genes essential for replication, and they are commonly produced during infection due to the high mutation rate. when participating in infection along with viable viral particles, they compete for host cellular machinery and result in viral replication inhibition. in this study, cells in a bulk population were infected with a mixture of vesicular stomatitis virus (vsv) expressing rfp and vsv-dips, and they were either untreated or isolated by serial dilution. rfp expression was observed during incubation as a surrogate for viral replication levels. the results showed that dip inhibited viral replication 10 times more on single cells, suggesting that the inhibition of viral replication is mitigated by cell-cell interactions when infection happens in a population [10] . table 1 . overview of commonly used single-cell technologies and their respective characteristics: a higher color intensity corresponds to a higher score (e.g., higher throughput, ease of moving cells of interest onto subsequent assays, higher information content, higher cost). the genomic mutation of pathogens during infection can be also detected directly. the sequencing of transcriptome and viral genes in single infected cells showed that iav is highly prone to mutation during infection [11] . detected mutations can cause consequences include viral polymerase malfunction and failure to express the interferon (ifn) antagonist protein, which is correlated to heterogeneous immune activation among infected cells [11] . the sequencing of 881 plaques from 90 vsv-infected cells detected 36 parental single nucleotide polymorphism (snp) and 496 snp generated during infection ( figure 1a -e) [12] . although extremely low multiplicity of infection (moi) was adopted, resulting in 85% of the cells statistically infected with only one pfu, 56% contained more than one parental variant, indicating that pre-existing differences in viral genomes can be spread within the same infectious unit, in this case, the host cell population. moreover, by measuring the viral titers produced by each infected cell, a significant correlation was found between the number of mutations in the viral progeny and the log yield of the initially infected cell. (e) correlation between the abundance of each type of substitution in single-cell-derived plaques and natural isolates. all panels adapted with permission from [12] . copyright 2015, elsevier. genomic variability also widely exists among bacteria populations. fluorescence labeling enables the quantification of bacterial growth in single host cells [13] [14] [15] , and by correlating the heterogenous growth with host response, it was found that the salmonella population exhibits different induction levels of the phop/q two-component system, which modulates lipopolysaccharides (lps) on the surface of individual bacteria [14] . to understand the pathophysiology of infectious diseases, it is important to study the identities of targeted cells. mounting evidence has shown that even under identical conditions, individual host cells manifest differential susceptibility and responses to infection in a population. how does this preference arise? do they share similar features that might be reasons for their susceptibility of infection? how do the states of infected cells affect pathogen replication and infection outcome? furthermore, how are host cells' phenotypes influenced by infection individually and temporally? answers to these questions are critical for the identification of target cells and individuals of novel pathogens, as well as for the understanding of infection pathophysiology. analysis of cells exposed to pathogens at single-cell resolution requires, first and foremost, strategies to distinguish infected cells from uninfected ones. pathogen-specific proteins, such as viral glycoproteins embedded in the cell membrane, or intracellular proteins such as viral capsid or polymerases, as well as pathogen nucleic acids, including genomic dna/rna and transcripts, can serve this purpose. these microbial elements can be labeled with specific antibodies or oligonucleotide probes for detection and quantification. alternatively, pathogen nucleic acids can be directly captured in deep sequencing. by combining tools for pathogen identification with host cell phenotyping assays, infected cells can be profiled at the single-cell level. xin et al. investigated the effects of host cell heterogeneity on both acute and persistent infection by foot-and-mouth disease virus (fmdv) [16] . by sorting single infected cells with facs based on cellular parameters, and quantifying viral genome replication with rt-pcr, they showed that the host cell size and inclusion numbers affected fmdv infection. cells with larger size and more inclusions contained more viral rna copies and viral protein and yielded a higher proportion of infectious virions, which is likely due to favorable virus absorption. additionally, the viral titer was 10-to 100-fold higher in cells in g2/m than those in other cell cycles, suggesting that cells in the g2/m phase were more favorable to viral infection or for viral replication. such findings have also been reported for other viruses [9, 17, 18] , revealing a general effect of heterogeneous cell cycle status in a population on virus infection. golumbeanu et al. demonstrated host cell heterogeneity using scrna-seq: they showed that latently hiv-infected primary cd4 + t cells are transcriptionally heterogeneous and can be separated in two main cell clusters [19] . their distinct transcriptional profiles correlate with the susceptibility to act upon stimulation and reactivate hiv expression. in particular, 134 genes were identified as differentially expressed, involving processes related to the metabolism of rna and protein, electron transport, rna splicing, and translational regulation. the findings based on in vitro infected cells were further confirmed on cd4 + t cells isolated from hiv-infected individuals. similarly, enabled by scrna-seq and immunohistochemistry, several candidate zika virus (zikv) entry receptors were examined in the human developing cerebral cortex and developing retina, and axl was identified to show particularly high transcript and expression levels [20, 21] . scrna-seq can also be used to identify potential target cells of novel pathogens and facilitate the understanding of disease pathogenesis and treatment. the spike protein of the virus sars-cov-2, the pathogen responsible for the covid-19 pandemic, binds with the human angiotensin-converting enzyme 2 (ace2) [22, 23] . this binding, together with a host protease type ii transmembrane serine protease tmprss2, facilitates viral entry [22, 23] . by analyzing the existing human scrna-seq data, it was identified that lung type ii pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells co-express ace2 and tmprss2, which suggests that they might be the putative targets of sars-cov-2 [24] . in the preparation of scrna-seq library, standard poly-t oligonucleotide (oligo-dt) is commonly used to capture mrna from single cells, which can also capture polyadenylated viral transcripts from dna virus or negative-sense single stranded rna virus. a simultaneous analysis of host transcriptome profiles and viral dna/rna offers information on the presence of the studied pathogen and its activities and allows a more accurate characterization on the dynamics of host-pathogen interactions. wyler et al. profiled the transcriptome of single human primary fibroblasts before and at several time points post-infection with herpes simplex virus-1 (hsv-1), and they described a temporal order of viral gene expression at the early infection stage [25] . more importantly, by simultaneously profiling the host and viral mrna, they identified that transcription factor nrf2 is related to the resistance to hsv infection. the finding was verified with the evidence that nrf2 agonists impaired virus production. steuerman et al. performed scrna-seq of cells from mice lung tissues obtained 2 days after influenza infection [26] . facs was applied to sort immune and non-immune cells based on cd45 expression. nine cell types were clustered (figure 2a) , and viral load was determined by the proportion of reads aligned to influenza virus gene segments, with higher than 0.05% considered infected. the authors found that viral infection can be detected in all cell types, and the percentage ranges from 62% in epithelial cells to 22% in t cells. however, the high variability of viral load was only observed among epithelial cells, while the majority of infected cells of other cell types showed to have low viral load (less than 0.5%) ( figure 2b ). for positive sense rna virus whose transcripts lack polyadenylation and cannot be captured by oligo-dt, a reverse complementary dna oligo probe to the positive-strand viral rna was employed. zanini et al. described this method and correlated gene expression with virus level in the same cell to study the infection of dengue virus (denv) and zika virus (zikv). they identified several cellular functions involved in denv and zikv replication, including er translocation, n-linked glycosylation, and intracellular membrane trafficking [27] . interestingly, by contrasting the transcriptional dynamics in denv versus zikv-infected cells, differences were spotted in the specificity of these cellular factors, with a few genes playing opposite roles in the two infections. genes in favor of denv (such as rpl31, tram1, and tmed2) and against denv infection (such as id2 and ctnnb1) was also validated with gain/loss-of-function experiments. analysis methods have been advancing for the detection of genetic variant-based scrna-seq data [28] [29] [30] . they could contribute, in the study of infectious diseases, to the characterization of temporal changes in viral mutational prevalence [31] . moreover, viral mutation can be correlated with host gene expression status at the single-cell level to further investigate their potential mutual effect on one another throughout the course of infection and reveal the dynamic host responses and pathogen adaptations in the progression of infection [32] . in spite of the above-mentioned examples characterizing virus presence with scrna-seq, it is worth noticing that viral mrna or genome occurrence is not necessarily equivalent to viral progeny, due to reasons such as missing essential genes caused by mutations. experimental techniques enabling the joint analysis of host transcriptional responses and viral titers will be needed to reveal the underlying mechanisms of virus production levels and host cell heterogeneity. another challenge of analyzing viral rna data is distinguishing infected cells with intracellular viral transcription from uninfected cells acquiring exogenous viral rna. combining single-cell transcriptomics data with flow cytometry or mass cytometry by time-of-flight (cytof) to measure the intracellular viral protein may help overcome this issue. immune and non-immune single cells were isolated from the whole lung of control and influenza-treated mice for massively parallel single-cell rna sequencing. host and the viral mrna were simultaneously measured, allowing the identification of infected as opposed to bystander cells, the quantification of intracellular viral load, and the profiling of transcriptomes. nine cell types were distinguished based on their transcriptional identities (b) the single-cell heterogeneity of intracellular viral load during influenza infection. percentages of low (yellow), medium (light brown), and high (dark brown) viral-load states (y axis) within the population of infected cells are shown for each of the nine cell types (x axis; total numbers of infected cells are indicated). (c) host genetic responses across all cell types. differential expression in influenza-treated and control mice (color bar) of nuclear-encoded genes (rows) across the nine major cell types (columns). right column indicates membership in four type i interferon (ifn)-related categories. all panels adapted with permission from [26] . copyright 2018, elsevier. immune responses activated by infection, since it is the innate immune responses that are primarily initiated in infected cells, or adaptive immune responses by lymphocytes carrying specific roles, are dynamic and complex, and they often happen in specific tissue microenvironments. heterogeneity in immune responses is also a long-recognized phenomenon. for instance, the activation of antiviral responses in dendritic cells (dcs) by bacterial lps starts with a small fraction of cells initiating the reaction, followed by the response by the rest of the population via paracrine responses [33] . technologies that enable the simultaneous measurement of multiple parameters facilitate high-resolution characterization of transcripts and protein at the single-cell level and boost our understanding of how host immune responses are initiated and orchestrated against infection. although pathogens usually dominate the war with host immune responses, hence the prevalence of infectious diseases, in-depth understanding of the interplay provides valuable information for the design of strategies to fight against infectious diseases. in this section, we cover the single-cell characterization of both innate immune responses from infected cells and adaptive immune responses activated in infected units. type i interferon (ifn), a key cytokine in innate immunity, orchestrates the first line of host defense against infection. its production is initiated upon host cells sensing pathogen-specific molecules, and it turns on the antiviral state of host cells by activating the transcription of hundreds of ifn-stimulated genes (isgs), some of which are crucial for coordinating adaptive immune responses. many studies have shown a large variability of ifn expression among infected cells. in the case of influenza virus infection, this can be partially explained by the high mutation rate during replication, revealed by sequencing viral genes in single infected ifn reporter cells [11] . however, such viability was also found to exist in infected cells expressing unmutated copies of all viral genes, which might be a result of the stochastic nature of immune activation irrelevant to viral genotypes [11] . in another study, pbmcs from patients with latent tuberculosis infection (ltbi) or active tuberculosis (tb), and from healthy individuals were analyzed with scrna-seq [34] . t cells, b cells, and myeloid cells were distinguished, and 29 subsets were clustered. the novel finding in this work is the consistent depletion of one natural killer (nk) cell subset from healthy individual samples to samples from ltbi and tb, which was also validated by flow cytometry. the discovered nk cell subset could potentially serve as a biomarker for distinguishing tb from ltbi patients, which is valuable for predicting disease outcome and developing treatment strategies. by analyzing scrna-seq data of pbmcs derived from individuals before and at multiple time points after virus detection, kazer et al. investigated the dynamics of immune responses during acute hiv infection [35] . after identifying well-established cell types and subsets in pbmcs, the authors examined how each cell type varies in phenotype during the course of infection. genes involved in cell-type specific activities, including monocyte antiviral activity, dendritic cell activation, naïve cd4 + t cell differentiation, and nk trafficking manifested similar changes with plasma virus levels: peaking closer to detection and gradually descending with time. phenotypic variations in bacteria populations were shown to influence host cell responses. avraham et al. investigated macrophage responses against salmonella infection with fluorescent reporter-expressing bacteria and scrna-seq on host cells [14] . transcriptional profiling revealed the bimodal activation of type i ifn responses in infected cells, and this was correlated with the level of induction of the bacterial phop/q two-component system. macrophages that engulfed the bacterium with a high level of induction of phop/q displayed high levels of the type i ifn response, which was presumably due to the surface lps level related to phop/q induction. with a similar setup, saliba et al. studied the salmonella proliferation rate heterogeneity in infected macrophages [13] . the varied growth rate of bacteria, indicated by fluorescent expression by engineered salmonella in single host cells, influenced the polarization of macrophages. those bearing nongrowing salmonella manifested proinflammatory m1 macrophages markers, similar with bystander cells, which were exposed to pathogens but not infected. in comparison, cells containing fast-growing salmonella turned to anti-inflammatory, m2-like state, showing that bacteria can reprogram host cell activities for the benefit of their survival. the above-mentioned strategy to simultaneously profile host cell transcriptome and viral rna also plays an important role in characterizing immune responses against infection by identifying infected immune cells and analyzing the transcriptomes simultaneously. for instance, it was applied to study the heterogeneous innate immune activation during infection by west nile virus (wnv) [17] . high variability was revealed for both viral rna abundance and ifn and isgs expression. interestingly, the expression of some isgs, with tnfsf10, ifi44l, and mx1 being the most prominent examples, was found to be negatively correlated with viral rna abundance, which could be a direction for future studies on wnv-mediated immune suppression in infected cells. similarly, zanini et al. studied the molecular signatures indicating the development of severe dengue (sd) infection by analyzing single pbmcs derived from patients [36] . facs was employed to sort pbmcs into different cell types (t cells, b cells, nk cells, dcs, monocytes), and then scrna-seq was performed. the majority of viral rna-containing cells in the blood of patients who progressed to sd were naïve immunoglobulin m (igm) b cells expressing cd69 and cxcr4 receptors, as well as monocytes. transcriptomic profiling data indicated that various ifn regulated genes, especially mx2 in naive b cells and cd163 in cd14 + cd16 + monocytes, were upregulated prior to progression to sd. comparison of the single-cell transcriptomes of lung tissue from health and influenza-infected mice revealed that 101 genes, among which the majority are isgs and targets of antiviral transcription factors, were consistently upregulated among all nine identified infected cell types, including both immune and non-immune cells [26] . this finding suggested that antiviral innate responses against influenza infection generically exist ( figure 2c ). moreover, by contrasting the expression profiles among infected, bystander, and unexposed cells, it was shown that the non-specific ifn gene module is a result of extracellular exposure and responses of environmental signals. while single-cell transcriptomics analysis provides an unbiased determination on host cell states, proteomics analysis offers direct characterizations of proteins expressed upon pathogen activation. going beyond traditional flow cytometry, mass spectrometry, or cytometry by time-of-flight (cytof) offers vastly increased numbers of parameters that can be investigated simultaneously, exponentially increasing the depth of the dataset collected. for instance, to investigate the effect of a precedent dengue virus infection on the outcome of subsequent zika infections, pbmcs derived from patients with either acute dengue infection or health individuals were incubated with dengue virus or zika virus, and the treated pbmcs were assessed by multiparameter cytof [37] . cytof in this study allowed the simultaneous detection of changes in the frequency of immune cell subpopulations and quantification of functional activation markers and cytokines in distinct cell subsets. while secondary infection with dengue virus led to increases of cd4+ t cells and t cell subsets, which are involved in adaptive immunity, secondary infection with zika virus induced the upregulation of several functional markers including ifnγ and macrophage inflammatory protein-1β (mip-1β) in nk cells, dcs, and monocytes, indicating an intact innate immunity against zika virus in the cases of possible concurrent dengue infection. hamlin et al. compared two denv serotypes (denv-2 and denv-4) in their infection in human dcs using cytof, which allowed simultaneous analysis on denv replication, dc activation, cytokine production, and apoptosis [38] . the tracking of intracellular denv proteins and extracellular viral particles showed different replication kinetics yet similar peak viral titers by these two serotypes, as well as the percentage of infected dcs. moreover, denv-4 infection was found to induce a higher expression of cd80, cd40, and greater production of tumor necrosis factor-α (tnfα) and interleukin-1β (il-1β), compared to denv-2 infection. additionally, bystander cells, which were identified by the absence of intracellular viral proteins, were identified to produce less tnfα and il-1β, but show more activation of interferon-inducible protein-1 (ip-1), which is a member of isgs. besides cytof, host cell secretomes can also be measured with customized miniatured systems, and the level of multiplexing and flexibility of sample handling is often improved. for instance, lu et al. showed the co-detection of 42 secreted proteins from immune effector cells stimulated with lps [39] . in a similar setup, chen et al. performed a longitudinal tracking of secreted proteins from single macrophages in response to lps treatment [40] . these studies provide valuable insights into the dynamic and comprehensive responses to pathogen over time. notably, such methods require microfabrication tools and skills, which is not always available and thus hinder their accessibility, compared with flow cytometery and cytof. epigenetic profiling at the single-cell level is also important, especially for elucidating the influence of host immune responses in chronic infection. the assay for transposase-accessible chromatin with high throughput sequencing (atac-seq) utilizes tn5 transposase to insert sequencing adapters into regions of open chromatin, in order to study genome-wide chromatin accessibility. buggert et al. applied atac-seq and established the epigenetic signatures of hiv-specific memory c8 + t cells resident in lymphoid tissue [41] . yao et al. used chromatin immunoprecipitation followed by high-throughput sequencing (chip-seq) to examine the histone modification of progenitor-like cd8 + t cells from mice chronically infected with lymphocytic choriomeningitis virus (lcmv) [42] . they found that progenitor-like cd8 + t cells showed distinct epigenomic features compared with memory precursor cells, exhibiting more abundant active histone markers (h3k37ac modification) at genes co-expressed with tox, which encodes the thymocyte selection-associated high mobility group box protein tox. this might promote the long-term persistence of virus-specific cd8 + t cells during chronic infection. in some cases, deep sequencing can be implemented together with other single-cell technologies for a comprehensive and systematic profiling of immune responses against infection. for instance, michlmayr et al. performed 37-plex cytof on peripheral blood mononuclear cells (pmbcs), rna seq on whole blood, and serum cytokine measurement of blood samples from patients with chikungunya virus (chikv) infection [43] . moreover, samples collected at acute and convalescent phases were compared to study the disease progression. such multidimensional analysis allows the large-scale, unbiased characterization of gene expression, cytokine/chemokine secretion, and cell subpopulation changes in response to infection. one important result of this study is revealing monocyte-centric immune response against chikv, with the frequency of two subsets both related to antibody titers and antiviral cytokine secretion. in addition, significant viral protein expression was found in two b cell subpopulations. while multiple assays can be done on the same bulk sample to obtain different data parameters (e.g., transcriptomic, proteomic), such datasets are not able to correlate the data parameters at the resolution of a single cell. newer advances allow the simultaneous collection of multiple types of parameters for the same cell. for instance, cellular indexing of transcriptomes and epitopes by sequencing (cite-seq) and rna expression and protein sequencing (reap-seq) are techniques for the simultaneous collection of transcriptomic and high-dimensional information on specified proteomic targets. by using antibodies tagged with unique nucleotide sequences, the subsequent transcriptomic sequencing simultaneously sequences these tags to allow the quantification of the antibody targets. corresponding transcriptomic and proteomic data at the single-cell level allows the opportunity to study the role of post-translational gene regulation in the immune response. the increased dimensionality of the information obtained may also allow more accurate machine learning to identify signatures of healthy or dysfunctional immune responses. for instance, using cite-seq, kotliarov et al. were able to identify a common signature of activation in a plasmacytoid dendritic cell-type i interferon/b lymphocyte network that was associated both with flares of systemic lupus erythematosus (sle) and influenza vaccination response level [44] . as noted above, the ability to study biological processes at the single-cell level gives an unprecedented to attribute bulk phenotypes in immunology and host-pathogen interaction to specific cell subpopulations, including rare cell populations, in a relatively unbiased fashion. apart from basic science discovery, how do these insights affect clinical practice in infectious disease? biomarker discovery is one obvious area of impact-the molecular differences found to underpin broader disease phenotypes can be used to diagnose or even predict disease. in particular, diagnosis is a notable problem in infectious disease, where identification of the causative pathogen can take days to weeks for culture-based systems, which may delay appropriate, targeted treatment [45] . apart from biomarker discovery, single-cell technology is also revolutionizing the discovery of vaccines and therapeutics, which will be elaborated upon in the sections below. other clinical uses of single-cell technology may require an increased uptake of such technologies within the hospital setting. for instance, one potential area of impact is antimicrobial resistance. the bulk genotype or phenotype of a pathogen population may not accurately identify its ability to become resistant to antimicrobials, since antimicrobial resistance can involve the selection of a previously rare, resistant population. should single-cell technology become routinely used in hospitals, the increased resolution could enables the identification of such rare populations, which can inform the choice of antimicrobials prescribed. to generalize, this similarly applies to any disease phenotype that can be triggered by a rare host or pathogen cell population. the complexity of current single-cell technologies hinders their implementation in the clinic, and in the section titled diagnostics, we highlight various steps that have been taken toward simplifying single-cell technology platforms to allow their clinical use. the first step of the vaccine development pipeline would be to identify a promising disease antigen, which could be in the form of a recombinant protein or inactivated/attenuated virus. unlike traditional vaccinology where vaccines were generated via pathogen growth and inactivation, the reverse vaccinology approach relies on predicting antigen features that are likely to trigger protective functions and engineering the antigen accordingly [46] . to predict these antigen features, two main approaches have been used: via whole genome sequencing and more recently, identifying and mapping the structural epitopes of neutralizing antibodies using the methods discussed later in this review [47] . after identifying a vaccine candidate, the next step would be to verify its efficacy. this efficacy is quantified based on its ability to bring about a set of specific immune responses which are specifically linked with protective functions, which are known as correlates of protection (cops) [48] . it is important to identify the cops for each vaccine for multiple reasons, including the following: (1) to understand the mechanisms of vaccine protection for improvement of vaccines, (2) to understand the mechanisms of vaccine protection for improvement of vaccines, (3) to determine the consistency of the vaccines produced, (4) to evaluate the levels of protection to patients before and after treatment, and (5) for the licensure of said vaccine [49] . historically, most of the cops in commercial vaccines typically involve quantifying the titer of neutralizing antibody produced by antigen-specific memory b cells. in the past decade, better understanding of the in vivo vaccine response has led researchers to identify several relevant memory t-cell responses as cops, and these t-cell responses are usually quantified by measuring the expressed cytokines via techniques such as elispot, flow cytometry, and elisa [50] . however, it remains difficult to define vaccine cops for a number of diseases. these include those diseases that cannot yet be eliminated by vaccine or infection-elicited immune responses (e.g., hiv-1 infection, tuberculosis), since a suitable end point of protection is not attainable. they also include those diseases for which vaccines do not yet exist but vaccine cops may be expected to differ from infection-related cops, including diseases for which natural clearance occurs via the innate immune response or early adaptive immune response (e.g., . even when immune parameters that correlate with disease risk are found, the causative mechanism of immune protection, or mechanistic cop, may remain elusive if multiple immune parameters are elicited in parallel by a protective response. as seen in the excellent review by plotkin [51] , the cops may not always be as obvious or limited to humoral immunity, and since vaccines typically elicit multiple immune responses. this is especially true for the case of vaccines against complex pathogens such as hiv and malaria, where the resultant network of immune responses may not always be easily identifiable. single-cell approaches may define a greater space of immune parameters to be explored as cops. furthermore, the increased breadth of data that can be obtained from a single sample is useful in increasing the number of hypotheses that can be probed, especially in longitudinal analyses, which are most useful for mechanistic immune studies but where the sample volume is often limited. furthermore, using a systems vaccinology approach via omics technology, researchers have begun to uncover these potential cops early in the vaccine development process [52] . in one of the earliest proof-of-concepts, querec et al. successfully identified a cop for vaccine efficacy on humans vaccinated against yellow fever. a gene marker present in cd8+ t cells which could predict for protection was discovered by using a multivariate analysis of the immune response via a combination of flow cytometry and microarray techniques [53] . with the rapid developments in single-cell omics technology, a deeper understanding of vaccine response can be obtained through an even more detailed mapping of the interactions between the various immune cell populations at the single-cell level, as well as identify the causes of heterogeneous vaccine response in individual immune cells [54] . this could be seen from the recent work by waickman et al. [55] where a dengue vaccine elicited a highly polyclonal repertoire of cd8+ t cells that was identified using scrna-seq. combined with transcriptional analysis of the cd8+ t cells, the authors established a set of metabolic markers that could be potential cops for vaccine efficacy evaluation. combining the simultaneous analysis of single-cell transcriptomic and tcr sequence data, tu et al. identified preferential transcriptional phenotypes among subsets of expanded tcr clonotypes. this is a strategy that may be highly valuable in assessing the functionality of t cells and their correlation to protection in vaccine responses [56] . antibodies are widely used in therapeutics and diagnostics due to their high specificity and generally low toxicity. antibodies are capable of mediating protective functions against infectious diseases, including pathogen neutralization, antibody-mediated phagocytosis, antibody-mediated cellular cytotoxicity, and complement-dependent cytotoxicity. antibody-containing sera remains in use for diseases where there are no other therapeutic options, including for viruses such as hepatitis a or b, rabies, vaccinia, sars-cov-2 at the point of writing, and for toxins (e.g., snake venom). however, there are limitations to this approach: serum therapy from animal sources causes a risk of serum sickness due to immune reaction against animal protein, while pooled hyperimmune sera from humans is difficult to collect and standardize. instead, the appropriate b cell clone that secretes antibody with protective activity can be isolated, and its antibody sequence can be obtained and expressed in culture to obtain monoclonal antibodies as therapeutics. similarly, in diagnostics, monoclonal antibodies provide the specific recognition of pathogen antigens that allow the rapid diagnosis of infection. in order to identify the correct b cell clone from thousands or millions of b cells, its antigen specificity and/or protective activity must be interrogated. this is classically done by cell immortalization (such as by hybridoma production or epstein-barr virus infection to generate b lymphoblastoid cell lines), followed by single-cell plating and expansion to obtain sufficient antibody from a single clone, and then the well-based screening of the antibody-containing cell supernatants. however, these techniques are low in throughput and efficiency, losing more than 99% of potential cells [57, 58] for hybridomas, and 70-99% of potential cells for b lymphoblastoid cell lines [59, 60] . moreover, there remains a bottleneck in throughput at the subsequent stage of subcloning and screening the resulting clones to determine which clones are antigen-specific and functional for the desired purpose-even large experiments are limited to screening several thousand cells [61, 62] , or up to 100,000 cells for robot-assisted operations [63] , whereas a single 30 ml human blood draw contains an order of magnitude more (approximately 900,000) candidate cd27+ igd-class-switched memory b cells [64] . more recently, techniques that avoid the need for cell expansion have been developed-these speeds up the life cycle for monoclonal antibody discovery. primary b cells expressing antigen-specific b cell receptors (bcrs) are labeled using fluorescent antigens, allowing flow cytometry-based single-cell sorting to isolate these antigen-specific b cells [65] . this technique is useful especially for the interrogation of memory b cells, which express the bcr on their surface. the interrogation of plasmablasts and plasma cells, which secrete antibodies but have low or no surface expression of the bcr, require other procedures such as the formation of an ig capture matrix on the b cells [66] , or alternative methods of screening that allow the physical separation of single cells such as droplets [67] , nanowells [68] [69] [70] [71] , or microcapillaries [72] . following the isolation of the desired b cells, they are lysed and their rna is interrogated to recover the antibody heavy and light chain genes. with these techniques, both antigen interrogation and antibody gene recovery do not require large clonal cell populations, removing the need for inefficient and time-consuming cell expansion processes. for the recovery of antibody genes, rt-pcr is commonly used, but recovery rates are typically low (<70% success rate for each pair of heavy and light chains) due to the large variability across the v gene families. single-cell rna-seq (smart-seq2) is an alternative to rt-pcr, which results in improved recovery rates (>90%) [73] . bcr recovery can also be done in the same step as antigen-specific sorting via the use of dna-barcoded antigens, such that both the antigen barcodes and bcr sequence are recovered simultaneously during single-cell ngs [74] . this has been used to successfully isolate broadly neutralizing hiv-1-specific antibodies and influenza-specific antibodies simultaneously from a single sample, although the resulting antibody candidates had variable neutralization functions, which required subsequent in vitro confirmation. another method for monoclonal antibody discovery is the use of phage display libraries, where phages expressing antibody genes are selected for using an antigen-coated surface in an iterative process of biopanning [75] . this has been a fast and effective method for monoclonal antibody discovery. the main limitation of phage library display is the random, largely non-native pairing of vh and vl genes, which may cause problems in subsequent antibody expression and production, and it may also have a higher likelihood of triggering anti-idiotypic allergic responses. more recently, a single-cell emulsion technique has been used for the interrogation of antigen specificity and high-throughput sequencing, allowing the interrogation of a yeast library utilizing natively paired human antibody repertoires [76] . using it, rare broadly neutralizing antibodies against hiv-1 could be identified, albeit with the correct antigen required for identifying the desired b cell clones. antigen binding is the most common form of screening for monoclonal antibodies due to its compatibility with high-throughput methods including flow cytometry, biopanning, and nanowell-based elisa. however, antigen binding may not correlate with functional activity against the intended target. for example, this may occur if the protein antigen used does not accurately mimic the native form of the antigen; monoclonal antibodies generated against the protein antigen may not be active against the native target [77] [78] [79] [80] . another example would be if functional activity requires binding in a specific orientation, such as virus neutralization requiring the monoclonal antibody to disrupt the receptor binding site [81, 82] . assays for monoclonal antibody function include assays for virus neutralization, opsonophagocytosis, antibody-dependent cellular cytotoxicity, and receptor agonism/antagonism [83] . currently, these assays are typically done in bulk with relatively low throughput, creating a bottleneck in monoclonal antibody screening. microfluidic technologies, such as water-in-oil emulsions or nanowells, are being developed to increase the throughput of such assays. for instance, a high-throughput screen for enzyme antagonism using a droplet-based assay has been reported [84] . using water-in-oil microdroplets, el debs et al. co-encapsulated single hybridoma cells with an enzyme (ace-1) and an enzyme substrate that emits a fluorescence signal upon enzyme hydrolysis, and they were able to sort out hybridomas secreting ace-1-inhibiting antibodies through fluorescence-activated droplet sorting. another group has recently also reported assays that are capable of assaying cellular internalization, opsonization, and the functional modulation of cellular signaling pathways [67] , and several companies have also reported proprietary platforms that may be able to carry out some other functional assays [85] . however, the specificity and sensitivity of these assays have not been reported. the ability to immobilize single cells in nanowells allows repeated longitudinal profiling, which is a property that was utilized by story et al. to obtain antibody-antigen binding curves that can classify related populations of b cells [71] . the characterization of diverse bacterial populations, including microbiome studies, has traditionally been done at the bulk level. for instance, the selection of particular organisms out of a diverse population has been done by the plating and amplification of single colonies. however, this method is limited in throughput. the enhancement of throughput can be done via miniaturization-for instance, one study isolated antibiotic-resistant e. coli mutants by encapsulating and culturing single bacteria in nanoliter-scale droplets containing the antibiotic [86] . this approach can be applied to accelerate the identification of targets acted upon by antibiotics of unknown mechanisms. apart from being limited in throughput, traditional microbial selection systems also require the ability to culture the microorganism of interest in vitro. however, it is estimated that the bulk of microorganisms cannot be cultured and expanded in typical cell culture media [87] . one potential solution is to use microfluidic devices to physically separate and phenotype individual bacteria while immersing them in media derived from their natural environment. this method was adopted to identify a new antibiotic, teixobactin, from a previously unculturable β-proteobacteria belonging to a group of gram-negative organisms not previously known to produce antibiotics [88] . in the clinical setting, single-cell analysis techniques are currently rarely routinely used in infectious disease diagnostics and monitoring. it is still impractical to apply most of the other conventional single-cell analysis techniques for diagnostic applications due to the associated high costs, long workflow durations, and high degree of technical expertise required. one notable exception would be flow cytometry, where aside from its high initial equipment cost, its fast turnaround times, high sensitivity, and ease of operation make it a staple tool in clinical institutions worldwide [89] . flow cytometry is mainly used to perform the immunophenotyping of blood cells against various disease-specific biomarkers [90] [91] [92] . the most prominent example would be in the routine monitoring of human immunodeficiency virus (hiv) progression by counting the number of cd4 + t cells in a patient's blood sample [91] . the other single-cell technique that has seen some use in diagnostics against pathogens would be fluorescence in situ hybridization (fish). as a diagnostic tool, fish has numerous advantages that include low cost and complexity; its rapid turnaround time allows the diagnosis of fastidious bacteria and the ability to distinguish between mixed populations of pathogens at a single-cell resolution [93] . while fish has been successfully used for the direct identification of panels of pathogens from blood samples [94, 95] , its reliance on image analysis as the readout limits the throughput of this technique, and the results are subject to user-to-user variation and bias [96] . to resolve these issues, a variant of the technique, fish-flow, was developed. fish-flow combines fish with flow cytometry to achieve higher throughputs as well as automates the signal readout through the cytometric system [97] , and it has been used to detect hiv reservoirs in t cells [98] as well as bacteria from blood [99] . while the ability to identify biomarkers at a single-cell resolution is certainly invaluable in the fight against infectious diseases, current flow cytometer systems are typically bulky and expensive, thereby limiting their use in a laboratory setting [100] . fortunately, advancements in microfluidics and low-cost electronics have given rise to the development of portable platforms that can perform single-cell analysis in a point-of-care (poc) setting. recent examples of portable cytometric systems that are relevant to infectious disease diagnosis include a miniaturized modular coulter counter capable of label-free detection and the differentiation of particles of varying sizes [101] , a low-cost and portable image-based cytometer for the quantification of malaria-infected erythrocytes [102] (figure 3a) , and a portable miniaturized flow cytometer that is capable of multi-channel fluorescence interrogation of whole blood samples [103] . the portability of such cytometers could mean faster turnaround test timings through on-site diagnostics and disease monitoring, hence expediting clinical decisions and improving healthcare outcomes in general [104] . in addition, the portability of such microfluidic systems lends to other practical applications of flow cytometry, especially in pathogen detection in water and food sources. particularly, diarrheal diseases (a leading cause of death for children under the ages of 5) are closely linked to the consumption of contaminated water sources and could be mitigated via regular, on-demand pathogenic testing of drinking water [105] . however, adapting current single-cell technologies into a portable format holds its own set of unique challenges. most of the existing literature surrounding such technologies still report separate sample enrichment or staining steps prior to cell analysis [106] [107] [108] ; such additional preparatory steps increase assay complexity, which may not be desirable in a poc setting [109] . while a gamut of existing microfluidic technology has already been established for sample purification as well as for reagent addition and mixing, integrating the various modules into a single platform is typically not a trivial process [110] . for single-cell technology to make the successful transition from the lab to the bedside, such practicalities must be considered and successfully implemented. digital assays are a relatively recent assay format comprised of the following steps: (1) the discretization of a single initial larger sample volume into multiple smaller volumes (typically via microwell, microvalve, or droplet emulsion partitioning techniques [111] ), and (2) performing the chemical or biological assay on each individual volume to obtain a quantifiable signal [112] . due to the ability to individually assay a large number of cells at the single-cell level, the digital assay format has been widely employed in single-cell omics studies [113] . in the field of infectious disease research, while most of the applications of digital assays have been centered on answering fundamental questions relating to pathophysiology, there are other single-cell diagnostic applications that can benefit tremendously from such an assay format. an example mentioned earlier in the review would be rapid antimicrobial-susceptibility testing (ast) to address the surge of antimicrobial-resistant infections worldwide as a result of the misuse of antimicrobials. phenotypic ast, which involves the culture of the pathogen in the presence or absence of antibiotics, may help guide treatment options, but existing conventional assays have low sensitivity and require a long time of 12-48 h for cell regrowth to achieve measureable assay outcomes [114, 115] . higher-sensitivity single-cell digital assays that have been recently reported can obtain measurable signals without requiring cell regrowth and could be the answer to reducing ast turnaround times ( figure 3b ) [116] [117] [118] . another application of digital assays could be in quantifying viral reservoirs in patients at a single-cell resolution. in hiv eradication studies, latent reservoirs are reactivated using latency-reversing agents (lras) for subsequent inhibition via antiretroviral therapy [119] . the ability to isolate and individually assay the patients' blood to obtain the distribution of reactivation states in the heterogenous cell population can give clinicians an idea of antiretroviral treatment efficacy in the future [120, 121] . pump-free droplet emulsion generation system that is capable of performing antimicrobial-susceptibility testing (ast) of different species of bacteria with a turnaround time of ≈5 h. image reproduced with permission from reference [117] . copyright 2020 royal society of chemistry. (c) microfluidic impedance cytometry is able to differentiate between healthy and malaria-infected red blood cells at a single-cell resolution based on the difference in electrical impedance measured across two electrodes. image reproduced from [122] under a creative commons license. the development of label-free single-cell analysis techniques has been gaining considerable attention over the last decade with the advent of microfluidics because of their numerous advantages over their counterparts that require cell labeling. some of the advantages include: (1) lower technical complexity and turnaround times in assay workflow because the preparatory step is omitted, (2) not requiring knowledge of cell biomarkers beforehand, making them suitable for assaying novel cell populations, and (3) by avoiding the use of labels that might affect the natural state of the cells, results might be more representative of actual in vivo cellular conditions [123] . coupled with the precise fluid handling capabilities afforded by microfluidics systems, there is a burgeoning number of label-free single-cell analysis platforms that have been reported in recent years that are able to measure infection based on the inherent properties of the cells. one prime example would be the identification of cells via their electrical properties, specifically electrical impedance. this impedance is derived from the change of voltage or current signal when single cells flow across a pair of miniaturized electrodes, and it has been shown to be able to differentiate between healthy and malaria-infected erythrocytes ( figure 3c ) [122] , as well as the viability and species of parasitic protozoa [124] . another promising direction for label-free single-cell analysis is via measuring the inherent optical properties of cells. this has been shown in recent work such as the single-cell identification of parasites through their raman spectra [125] , the quantification of single-cell viral infection titer through laser force cytology [126] , and single bacteria detection via refractive index measurements [127] . lastly, the mechanical and size properties of cells have also been exploited for identifying infected single cells. for example, using inertial microfluidics, white blood cells could be hydrodynamically isolated from lysed blood containing ring-stage malaria parasites as a result of the white blood cells' larger sizes [128] . other recent works that demonstrate potential applications for single-cell label-free infectious disease analysis includes cell identification via their acoustophoretic responses [129] , as well as their deformability and hydrodynamic resistance [130] . evidently, there is a host of promising label-free single-cell analysis technology that could be translated to clinical diagnostic applications. in the near future, these technologies would be useful complement poc applications where labeling steps in the assay workflow would increase the technical complexity and hinder the transition from the lab to bedside. among the methods discussed, scrna-seq is the primary tool for single-cell studies. in the following section, we briefly cover some important points that needs to be considered when designing and conducting such experiments. for a more in-depth coverage of this subject matter, the reader is invited to read other excellent reviews from luecken [131] , see [132] , and lähnemann [133] . as covered earlier in this review, the main applications of scrna-seq in infectious disease study comprise of the following: (1) studying effect of host cell heterogeneity on infection, (2) identifying host immune responses, and (3) antibody discovery. however, the number of sequenced cells and depth of sequencing ultimately depend on the end goal of each experiment as well as the amount of financial resources at hand. the availability of a variety of commercial platforms for single-cell analysis with different throughput and sensitivity can provide users with different options to best suit the purpose of their studies [134] . for studies that involve identifying the cell types of a heterogeneous sample, a minimum of 50,000 reads per cell would be sufficient [135] , while testing on a significantly large number of cells would ensure that rare subpopulations do not get missed out. one such application in infectious disease studies would be the systemic characterization of immune cell populations in response to an infection, wherein a large number of cells has to be screened in order to encompass the extensive diversity of b and t cells [136] . on the other hand, for studies which the main goal is to obtain a high resolution readout of the transcriptome for a small number of cells, 1,000,000 reads per cell would be a reasonable estimate [33] . in typical bulk analysis, multiple biological and technical replicates can be performed in order to ensure the reproducibility of data. however, for single-cell experiments, particularly for scrna-seq, there are two main issues to contend with. firstly, measurements typically have high technical variability as replicate measurements cannot be performed on the same cell, which is lysed as part of the rna extraction process. secondly, the resulting single-cell data are typically noisy due to technical variations from the multitude of steps in scrna-seq, as well as biological variation stemming from cell heterogeneity [137] . as such, great care has to be taken at each step of the scrna-seq workflow (i.e., sample preparation, library preparation and sequencing, data analysis) to minimize such technical variability and batch effects. one of the major sources of such variability arises from the initial sample preparation process. regardless of how the cells are dissociated, purified, or enriched, cell expression is likely to change in response to the stress induced from these processes. to minimize such undesired changes which might affect downstream data analysis, the sample preparation protocol should be optimized iteratively for each cell type [138] . to reduce technical variability, one common method would be to spike = 0 in known quantities of synthetic rna into the samples as controls to normalize read counts prior to data analysis [139] . a recent advancement in such rna spike-in normalization methodology would be the bearscc (bayesian ercc assessment of robustness of single-cell clusters), which generates simulated technical replicates based on the readout signal variation from spike-in measurements [140] . an alternative to rna spike-ins would be the use of unique molecular identifiers (umis) incorporated into the primers during reverse transcription, which essentially act as unique barcodes that allows the identification and subsequent tracking of transcribed mrna. then, the resulting data can be normalized against the umi levels to account for amplification bias during the library generation step [141] . however, both rna spike-in and umi have their own set of limitations to consider; rna spike-ins are unsuitable for protocols that utilize poly-t priming and template switching, and since they are typically used in large amounts relative to the endogenous rna, they could potentially occupy a lot of reads. protocols utilizing umi need to ensure that library sequencing is sufficiently deep to cover all umi transcripts; otherwise, there will be a risk of incorrectly quantifying the initial sample rna [142] . another source of error for scrna-seq comes from batch effects, which are brought about by unavoidable variations between batches of experimental runs due to changes in environmental conditions, temperature, reagent lot, etc. in response, several computational methods have been developed to mitigate said batch effects from the scrna-seq data. for example, one of the more commonly used batch-effect correction methods, combat, utilizes an empirical bayesian framework that removes batch effects via a linear model, which factors in both the mean and variance of the scrna-seq data [143] . for a more in-depth study on the comparative performance between the various batch-effect correction methods, we urge readers to consult a recent study by tran et al. [144] . while single-cell platforms have indeed come a long way in the past two decades, the plethora of existing techniques still face a few general concerns that could present themselves as opportunities for development in the near future. one of the inherent challenges in single-cell studies stems from the simple fact that the total amount of biological material present in a single cell is pretty limited and as a result, the resulting data are typically noisy from multiple biological and technical sources. making sense of the data requires downstream data pre-processing and analysis, which are non-trivial components of the workflow that limits the accessibility of such studies to groups with the essential background. additionally, with the increasing number and complexity of parameters at which single-cell assays are being performed, the curse of dimensionality is a pertinent problem that still requires further examination [133] . another concern for single-cell platforms would be inter-experiment variability, as mentioned in the previous section. single-cell technologies innately have high measurement sensitivity and thus are more susceptible to variations in results obtained from technical replicates, and methods to bioinformatically correct for such differences are required. coupled with the fact that single-cell studies are typically expensive and therefore sample sizes are small, ensuring that results are comparable between each sample becomes an even more important issue. finally, the inability to maintain viable cells after analysis, particularly for high-throughput methods such as flow cytometry or scrna-seq, gives rise to a couple of problems. firstly, a majority of the conventional single-cell studies are limited to a single time point of study, following which the cells are discarded. secondly, the irrecoverability of the cells makes it difficult to integrate back-to-back assays, which required measuring different parameters. as such, improvements in cell handling to improve cell viability would be invaluable in obtain multi-parametric datasets required for a more holistic understanding of cellular behavior. to date, the applications of single-cell technology have revolutionized our understanding of host-pathogen interactions. while many studies have focused on immune cells from the blood, the study of immune responses in the context of solid tissues or foci of infection (in both acute and chronic disease phases) is important to understand the local context of host-pathogen interaction. for this, techniques allowing the integration of spatial information with other single-cell technologies will be useful. for instance, imaging mass cytometry has been used to obtain quantitative information on 32 proteins at a spatial resolution of 1 µm [145] . this is done by systematically ablating a formalin-fixed tissue sample spatially line by line. the increased number of markers allows the fine distinction of cell subsets and activation states, providing valuable information on cellular roles in immune effector function or immunopathogenesis. it may even be possible to simultaneously obtain information on specific dna and rna targets via in situ hybridization. similarly, several techniques have been recently developed to obtain simultaneous spatial and transcriptomic data, including multiplexed error-robust fish (merfish) [146] , laser capture microdissection sequencing (lcm-sequencing) [147] , tomo-seq [148] , slide-seq [149] , and spatial transcriptomics [150] . these techniques may similarly be helpful in infectious disease to better define the interplay of immune cells, susceptible cells, stromal cells, and pathogens. another important gap that remains to be bridged is the ability to comprehensively access the state of a single cell across time. both immune and infection processes are highly dynamic, but because most of the single-cell technologies listed above are destructive, changes over time must be assessed either by careful time-point studies, or by assuming the presence of a range of cells in a population that represent early and late stages of the process (e.g., cellular activation or infection stage). with the advent of microfluidic devices that can immobilize single cells for continued study, the same cell can be assessed at multiple points for longitudinal study. in addition to typical proteomic marker analyses and rna or dna in situ hybridization techniques with live cell imaging, it is already possible even to measure more complex phenotypes such as bioenergy metabolism [151] . since microfluidic devices are also used for 3d organoid growth to simulate in vivo conditions, it is conceivable that future developments in technology will allow similar types of information to be collected in the context of organoids. this will represent one approximation toward high-dimensional in vivo data, which remains impossible with current methods. most of the single-cell studies reviewed in this article are based on end-point assays that are destructive and can therefore only measure a single time point of these single-cell targets. however, several recent studies outside the sphere of infectious disease have highlighted time as a prominent variable that influences the level of heterogeneity in host cells, immune cells, and pathogens. to that end, microfluidic platforms that enable the automated and precise control of media and reagents are ideal for performing such dynamic studies. for example, wu et al. [152] , using a customized microwell-microvalve system, performed a continuous measurement of a disintegrin and metalloproteinases (adams) and matrix metalloproteinase (mmps) secretions by single hepg2 cells upon a phorbol 12-myristate 13-acetate (pma) challenge. using their microfluidic platform, heterogenous changes in the secretion rates of adam and mmps were observed in response to pma stimulation, which may be used to predict hepg2 cell fates. in another recent study, a microfluidic platform that combined mutation visualization (mv) and microfluidic mutation accumulation (µma) enabled real-time tracing of mutations of single bacteria [153] . evidently, the utilization of microfluidic technology could enable high temporal resolution single-cell studies suited for uncovering the pathophysiology of infectious diseases. the advantages offered by microfluidics technologies include the efficient capture and compartmentalization of single cells, the precise control of fluid exchange, and ensuring a viable microenvironment for cell survival. these characteristics enable the dynamic study of large populations of single cells in parallel, which may eventually provide us with a more comprehensive understanding of the causes and effects of single-cell and single-pathogen heterogeneity. through the various applications of single-cell technology, we have gained a more thorough understanding of infectious disease pathophysiology at an unprecedented resolution. revealing the heterogeneity within populations of pathogens has allowed a finer dissection of virulence factors, and similarly, heterogeneity within populations of infected cells has given us a deeper understanding of host immune defenses. in addition, the high-dimensional single-cell information that can be collected even from primary cells has allowed us to identify rare but important cell subtypes, and it has shed light on the complex interplay between the different cells of the immune system. with the advent of antibody and t cell-based therapeutics, and antibody-based diagnostics, the contributions of single-cell technology to the high-throughput identification of candidate b and t cell receptor sequences that are target-specific have also accelerated the development of new therapeutics and diagnostics for both newly emerging and existing diseases. the adoption of single-cell technologies is likely also to revolutionize clinical studies for both drugs and vaccines, given its immense potential for biomarker discovery. with the field of single-cell technology only just taking off in the last decade, there remain vast prospects in both the increased adoption of existing technologies and the development of new technologies. the authors declare no conflict of interest. covid-19 coronavirus pandemic trade set to plunge as covid-19 pandemic upends global economy microbiology by numbers history of the discovery of the malaria parasites and their vectors the use of single-cell rna-seq to understand 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10.1007/978-1-60327-534-7_23 sha: doc_id: 18220 cord_uid: 8m11ig06 although influenza remains indisputably the most significant viral pathogen in adults, other viruses such as respiratory syncytial virus, parainfluenza viruses, and human metapneumovirus are now recognized as significant pathogens in older populations. oseltamivir and zanamivir are antiviral agents that are effective for the treatment and prophylaxis of influenza a and b. for treatment and for optimal effect, therapy should be initiated within 48 h of symptom onset. infection with hepatitis viruses may be more severe in older adults with more fulminate disease as observed with acute hepatitis a and a more rapid progression to cirrhosis with hepatitis c. outbreaks of viral gastroenteritis are common in long-term care facilities, and infection may lead to death due to dehydration and oliguria. the incidence of herpes zoster increases with advancing age and carries with it a significant risk of post herpetic neuralgia. the use of antiviral medications and corticosteroids may reduce the incidence and severity of chronic pain. parainfluenza viruses (piv), human metapneumoviruses (hmpv), coronaviruses (co-v), and rhinoviruses may cause severe disease in adults and result in hospitalization (2) . together pneumonia and influenza comprise the fifth leading causes of death in persons aged 65 years and older. influenza viruses are enveloped ribonucleic acid (rna) viruses that are classified as a, b, or c, based on stable internal proteins (3) . the virus contains two major surface proteins: hemagglutinin (h) and neuraminidase (n), which can undergo minor antigenic changes leading to yearly epidemics or major changes resulting in influenza pandemics. currently, there are two circulating influenza a viruses, h1n1 and h3n2, in addition to influenza b, present in the united states. h1n1 viruses do not appear to cause serious problems in older persons, possibly due to previous immunity. in 1997, influenza a (h5n1), which was previously seen only in birds, crossed the species barrier and human infection occurred in southeast asia (4) . this highly pathogenic avian influenza has spread in bird populations throughout asia and into europe. to date, human infection has been rare and transmission has occurred primarily by direct contact with infected birds. unlike seasonal influenza, illness due to avian influenza is more severe in young children as compared to older adults. in a community, peak influenza activity typically lasts 6-8 weeks, with attack rates highest in preschool and school-aged children and lowest in older persons (3) . despite lower attack rates, mortality from influenza rises dramatically with age and the presence of underlying medical conditions. the presence of one high-risk medical condition (cardiovascular, pulmonary, renal, metabolic, neurologic, or malignant disease) increases the risk of death from influenza 39-fold. despite increasing vaccine coverage, current centers for disease control and prevention (cdc) data indicate increasing influenza-related morbidity and mortality over the past decade (5) . in the united states, approximately 226,000 hospitalizations and 34,000 deaths occur each year in persons age 65 years and older (6, 7) . the devastating impact of influenza is most dramatically seen in long-term care facilities (ltcfs) where explosive epidemics may occur. during outbreaks, rates of pneumonia and hospitalization are as high as 52% and 29%, respectively, with case fatality rates of 30%. the classic presentation of influenza is an abrupt onset of fever, chills, headache, and myalgias (see table 1 ). dry cough, sore throat, and ocular pain are also common (3) . fever remains a common finding in the elderly, although the height of the fever may be lower compared with young persons. although many elderly adults have classic symptoms, a substantial number may have more nonspecific presentations such as fever and confusion or worsening of chronic medical conditions. in contrast to young healthy persons, the triad of fever, cough, and acute onset of symptoms has a positive predictive value of only 30% in elderly adults. given the protean manifestations of influenza in older persons, it is always important to consider influenza in the differential when evaluating an acutely ill elderly adult during the winter. influenza lower respiratory tract involvement increases steadily with advancing age with the rates of pneumonia 4-8% in persons aged 5-50 years and rising to 73% in persons over age 70 (8) . secondary bacterial pneumonia following acute influenza also occurs more frequently in older persons. although the rates of pneumonia rise with age, hospitalization most frequently results from exacerbation of chronic medical conditions. in addition to the immediate complications of influenza, residents of nursing homes who survive influenza experience a significant functional decline in activities of daily living. although many physicians use clinical features to make a diagnosis of "the flu," laboratory confirmation is best, especially if therapeutic decisions are needed because influenza may be difficult to distinguish from other respiratory viruses. winter-spring 1-2 non-specific none rsv, respiratory syncytial virus. *100 mg daily for severe hepatic dysfunction, renal failure (crcl 10 ml/min) and for elderly nursing home patients. **dosage should be reduced in persons with creatinine clearance of < 30 ml/min ***ribavirin is not fda approved for use in adults. bid twice a day; po orally older persons typically shed the virus for 3-5 days, although shedding up to 14 days has been documented in hospitalized patients (9) . rapid antigen testing may be done directly on nasopharyngeal specimens using an enzyme immunoassay (eia) (10) . although not as sensitive as viral culture, rapid tests offers quick turnaround times and may be useful for infection control and treatment decisions. sensitivity of rapid testing in adults is estimated to be 50-60% for influenza a strains and 10-20% for influenza b (11) . when available, rt-pcr offers rapidity while retaining excellent sensitivity. as of this writing, zanamivir or rimantadine can be used for the treatment or prophylaxis of influenza a h1n1 strains (*). for influenza a h3n2 or influenza b, oseltamivir or zanamivir can be used. if the strain of influenza is unknown, currently, zanamivir by itself or a combination of oseltamivir and rimantadine is appropriate coverage (3). resistance to zanamivir remains rare. zanamivir is not recommended for patients with reactive airway disease, because it may exacerbate bronchospasm. future influenza treatment and prophylaxis recommendations for influenza will need to be guided by close monitoring of cdc reports on influenza resistance patterns. treatment should begin < 48 h after onset of symptoms (table 1 ). in practice, physicians are often faced with the question of whether to treat patients who present outside the 48-h period. at present, only one observational study addresses this question in hospitalized adults (12) . in 327 adults hospitalized with influenza, mortality was significantly lower in those who received antiviral treatment as compared to those who did not receive antiviral treatment. of the 100 patients who were treated, only 29% had symptoms < 48 h. the cornerstone of reducing the morbidity and mortality of influenza in the elderly is vaccination (see also chapter "vaccinations"). although the degree of protective efficacy for current inactivated vaccines in the elderly has recently become a subject of controversy, older adults clearly benefit from vaccination (13) . a multi-layered approach for protecting the elderly from influenza is needed and includes vaccinating elderly persons, their close contacts and care givers, and providing oseltamivir if exposure to influenza has been documented (3, 14) . when staff was highly vaccinated, several studies have demonstrated a benefit to elderly residents of ltcfs (15) . the recommendations of the advisory committee on immunization practices (acip) 2007 relating to the elderly, include vaccination of all persons ³ 50 years, vaccination of residents of nursing homes and chronic-care facilities, vaccination of healthcare personnel, and vaccination of healthy household contacts (including children) and caregivers of adults ³ 50 years (3) . * http://emergency.cdc.gov/coca/ppt/antivirals_update_010809_fiore.pps at the present time, only trivalent inactivated virus (tiv) vaccine, which contains killed h1n1, h3n2 and b strain influenza, is recommended for use in persons 50 years and older (3) . mild acute local reactions occur in approximately one-third of vaccines and systemic reactions such as fever, and myalgias are uncommon in older persons. influenza vaccine may be safely given simultaneously with pneumococcal vaccine, and the only contraindications to vaccination are anaphylactic hypersensitivity to eggs or other components of the vaccine and a history of guillian-barré syndrome. by using adjuvants and higher doses of antigens, active research continues to improve the immunogenicity and efficacy of inactivate influenza vaccine. live attenuated influenza virus vaccine is not approved for persons over age 49; however, it may be given to healthcare workers and close household contacts of older adults. antiviral prophylaxis is recommended for all residents of nursing homes and chronic care facilities and to unvaccinated healthcare providers once influenza a has been documented in the institution (13, 14) . chemoprophylaxis is given regardless of vaccination status and is continued until 1 week after the onset of the last influenza case. rsv has long been recognized as the leading cause of lower respiratory tract disease in children; however, recently, it has been recognized as a serious adult pathogen (16) . it is estimated that rsv results in approximately 178,000 hospitalizations and 14,000 deaths annually in the united states yielding healthcare costs in excess of $1 billion. a number of epidemiologic studies and mathematical models indicate that rsv is second to influenza as a cause of serious viral respiratory disease in adults (7) . rsv was initially recognized as a pathogen in older persons when several outbreaks were described in long-term care facilities (17) . attack rates are variable and may be as high as 90% during outbreaks, but more commonly they range from 1 to 7% when residents are followed prospectively. in published reports, rates of pneumonia range from 0 to 53% and death from 0 to 55%. rsv appears to cause serious disease in community-dwelling older persons as well. in a 2-year prospective study of elderly persons in the united kingdom (uk) nicholson et al., identified rsv in 3% of illnesses using serology for diagnosis (18) . with the advent of sensitive molecular testing, a more accurate assessment of the true incidence of rsv has emerged. in a 3-year study from the united kingdom by zambon et al., rsv was identified by rt-pcr in 10-18% of adults age 65 years and older who were visiting a general practitioner during the winter for a respiratory illness (19) . in comparison, during the same period, influenza a was identified in 13-42% of subjects. in a prospective study from rochester, ny, using a combination of viral culture, rt-pcr and serology for diagnosis, rsv infection was documented in 3-7% of 608 healthy elderly and 4-10% of adults with chronic cardiopulmonary conditions over four winter seasons (16) . serious disease was more common in high-risk patients: 9% visited the emergency room, 16% required hospitalization, and 4% died. finally, a large study of community-acquired pneumonia in adults found rsv to be the third most commonly identified pathogen at 4.4% compared with 6.2% due to streptococcus pneumoniae and 5.4% due to influenza (20) . manifestations of rsv infection can be difficult to distinguish from other viral respiratory infections, particularly influenza. most individuals with rsv have nasal discharge, cough, sputum production, and constitutional symptoms. although overlap exists, there are some helpful clues to differentiate rsv from influenza. high fever, sore throat, myalgias, and gastrointestinal complaints are more characteristic of influenza, whereas rhinorrhea, dyspnea, sputum production, and wheezing are more frequently associated with rsv infection . unfortunately, because of the labile nature of the virus and low titers of virus in nasal secretions in adults, diagnosis of acute rsv by standard testing is difficult. under ideal circumstances, viral culture is only 50% sensitive when compared with serology using eia. commercial rapid antigen tests also have poor sensitivity in adults (21) . rt-pcr offers the best combination of sensitivity and specificity for the diagnosis of acute rsv in adults but is not widely available to most clinicians. the treatment of rsv infection in adults is largely supportive. supplemental oxygen and bronchodilators may be useful, and antibiotics should be considered if bacterial super-infection is suspected. ribavirin is a nucleoside analogue, which has broad antiviral activity, including rsv. anecdotal experience suggests it may be beneficial in selected cases, particularly in persons with immunosupression. however, due to lack of data in the elderly, general recommendations on its use cannot be made (22) . the major problems with ribavirin are its high cost and difficulty with administration. the recommended 12-18 h/day of aerosol at 20 mg/ml concentrations may be quite difficult for the elderly adult to tolerate. higher concentrations (60 mg/ml) given three times a day may also be effective and more tolerable (23) . in healthy elderly patients and in adults with chronic pulmonary disease, low serum neutralizing antibody titers are associated with increased risk of hospitalization with rsv infection suggesting a vaccine may be beneficial. although research is ongoing, an effective rsv vaccine has yet to be developed. rsv is spread primarily by large droplet inoculation and fomites, and handwashing is the single most important measure in the control of rsv. the parainfluenza viruses (piv) are most commonly thought of as the etiologic agents of croup, bronchiolitis, and pneumonia in young children (24) . four serotypes and two subgroups of piv are recognized (1, 2, 3, 4a, and 4b); piv-3 is endemic throughout the year, whereas piv-1 and piv-2 tend to occur during the fall. although piv infections are not commonly documented in older adults, several studies of community-acquired pneumonia and chronic obstructive pulmonary disease (copd) exacerbations implicate piv as a cause in 2-17% of cases (25, 26) . the piv-1 and 3 serotypes account for the majority of isolates in older persons, with piv-2 being relatively uncommon (27) . similar to rsv, outbreaks of piv infections in nursing homes have been described (27, 28) . variable morbidity and mortality has been reported. clinical characteristics of piv infection are not distinctive and include rhinorrhea, sore throat, hoarseness, and cough with high rates of pneumonia ranging from 20 to 30%. in an institutional outbreak of piv-3, the attack rates among residents and nursing staff were 31% and 11%, respectively. antecedent parainfluenza infection in long-term care residents has been linked to outbreaks of pneumococcal pneumonia. in clinical practice, a diagnosis of piv infection is usually made by viral culture, although rt-pcr is available in some settings (24) . diagnosis can also be made serologically; however, piv-1 and 3 infections result in cross-reactive antibody responses and cannot be distinguished. no antiviral agents have been approved for the treatment of piv infection. in 2001, human metapneumovirus (hmpv) was first identified in the netherlands from archived respiratory cultures collected from infants and young children in whom other pathogens could not be isolated (29) . it is an enveloped rna virus closely related to rsv and piv. since its discovery, infection has been widely reported each winter in young infants with an illness similar to rsv and characterized by wheezing and bronchiolitis (30) . however, as with many pediatric respiratory viral pathogens, hmpv infection induces incomplete immunity and reinfections occur later in life at all ages (31, 32) . in a 2-year study, hmpv infection was identified in 4.1% of elderly and high-risk adults, using rt-pcr and serology for diagnosis (32) . impact was greatest in subjects with cardiopulmonary diseases, who were ill twice as long as healthy elderly. in a study of adult pneumonia, 4% of subjects were diagnosed by rt-pcr with hmpv (33) . seventy-five percent of those infected were 65 years of age and older. such hmpv outbreaks can also occur in ltcfs. in a recent outbreak of severe hmpv illness in an ltcf in quebec, canada, 17% of those infected had pneumonia and 50% died (31) . autopsy material from an elderly woman with an extensive right middle and lower lobe pneumonia confirmed the presence of virus in the lower airways by immunohistochemical staining. the clinical characteristics of hmpv pneumonia in older adults do not appear to be distinctive from the other wintertime respiratory viruses. cough is universal and wheezing, dyspnea, and sputum production are common symptoms (32) . in part due to its fastidious growth characteristics, hmpv remained undetected for many years. although isolation by viral culture is possible, this method of diagnosis is not practical, and rt-pcr is the diagnostic method of choice (30) . rapid tests have been developed for direct antigen detection in respiratory secretions; however, there are little data regarding sensitivity in the elderly. currently, there is no known effective antimicrobial therapy against this virus. therapy is primarily symptomatic, supportive, and managing any complications. rhinoviruses are the most commonly identified cause of the "common cold," accounting for 25-50% of upper respiratory infections (34) . rhinoviruses circulate at all times of the year, but peak activity tends to be during the spring and fall. because the virus does not replicate well at 37°c, infection of the lower airways was previously considered rare. however, recent studies utilizing experimental challenge and specimens obtained at bronchoscopy clearly demonstrate rhinovirus infection of the lower respiratory epithelium (35) . a prospective study from the united kingdom indicates that rhinoviruses are an important cause of debility and lower respiratory disease in elderly people in the community (36) . rhinoviruses accounted for 24% of respiratory illnesses in a cohort of 533 persons over a 2-year period. although death and hospitalization rates were low, the mean length of illness was 16 days, and 26% of people were unable to perform their normal household activities. the presence of chronic medical conditions and smoking increased the likelihood of lower respiratory tract complications. because of the frequency of rhinovirus infection, the overall burden of disease in the elderly may approach influenza. rhinovirus has also been identified as the cause of 3.1%-19.6% of copd exacerbations (37) . lastly, outbreaks of severe respiratory illness due to rhinovirus have been described in nursing homes with attack rates as high as 56% and a mortality as high as 21% of those affected (38, 39) . in elderly persons, nasal congestion (79-89%), cough (71-94%), constitutional symptoms (43-91%), and sore throat (21-51%) characterize illnesses. rhinoviruses are now appreciated as a common trigger for asthma exacerbations and, therefore, in persons with preexisting lung disease, the dominant symptom may be wheezing (34) . the diagnosis of rhinovirus is usually made by a viral culture of nasopharyngeal secretions. if available, the use of rt-pcr greatly increases detection rates (34) . treatment is supportive and care should be exercised when prescribing "cold" medications to elderly persons because many of these "cold" medications contain combinations of sympathomimetics and antihistamines. coronaviruses are rna viruses of which two are major serotypes: human coronavirus 229e (hco-229e) and hco-oc43, which, for decades, have been known to cause respiratory disease in humans (34) . two recently discovered coronaviruses (hco-nl63 and hco-hku1) cause lower and upper respiratory tract infections with similar frequency (40) (41) (42) . in temperate climates, peak viral activity occurs in the winter. reinfections with coronaviruses are common throughout life, and, similar to rhinoviruses, illnesses are generally mild upper respiratory infections in healthy adults; symptoms include malaise, headache, sore throat, and nasal congestion. exacerbations of chronic obstructive pulmonary disease have been linked to coronavirus infections in several studies. coronavirus infections have been evaluated in the community-dwelling elderly and have, in one prospective study from the united kingdom, accounted for 9.5% of the respiratory illnesses (18) . coronavirus's were associated with lower respiratory tract symptoms in more than 40% of cases; infections have also been documented in ltcfs and in frail elderly people attending daycare centers (43) . coronaviruses have also been implicated in severe respiratory infections requiring hospitalization in older adults (44) . rt-pcr is now available for diagnosis of coronavirus infection but frequently a specific viral diagnosis is not made (34) . no antiviral agents are available, and treatment is supportive. hepatitis a virus (hav) is an rna virus in the picornavirus family (see table 2 ). the virus is easily transmitted by the fecal-oral route. in countries where the virus is endemic and sanitation is poor, most people become infected in early childhood when the disease is mild and life-long immunity results (45) . recently, a shift in the prevalence of cases from childhood to adulthood has occurred, presumably due to improved living conditions. the incidence of hepatitis a in the united states has fallen 88% from a peak in 1996 to an all-time low of presently 1.2 cases per 100,000 persons (46) . the prevalence of anti-hav antibodies increases with advancing age (47) . in a 1994 study from colorado, the prevalence of anti-hav antibodies at ages 60, 70, and 80 was 40%, 60%, and 80%, respectively (45) . an acute hepatitis a, advancing age correlates with more severe clinical manifestations, higher bilirubin levels and increased hospitalization rates (48) . liver failure and death are also more common with increased age (49) . in the united states, the overall case fatality rate for hav infection is 0.3%; however, it rises to 1.5% in persons 60 years or older (46) . an inactivated hepatitis a vaccine has been available since 1993, and clinical studies have shown the vaccine to be safe, very well tolerated, and highly immunogenic in all age groups. immunization of toddlers in israel resulted in a > 95% reduction of hepatitis a in the general population (50) . although the benefit was least for ages ³ 65 years, a 77.3% reduction in cases was observed in this age group. immunogenicity in frail elderly persons such as residents of long-term care has not been reported. although disease may be more severe in older adults, current vaccination policies do not specifically target the elderly. however, vaccination is recommended for older travelers who plan to visit countries endemic for hav. hepatitis b virus (hbv) is a complex deoxyribonucleic acid (dna) virus transmitted by percutaneous and mucous membrane exposure to infectious body fluids. serum, saliva, and semen have been shown to contain hepatitis b surface antigen (hbsag). since 1990, the incidence of acute hepatitis b has declined in all age groups with the largest decline (98%) being in children <15 years (46) . because the primary risk group in the united states and europe is intravenous drug abusers, acute infection is not common in the elderly. transfusion-related hbv infection is now an uncommon event with risk estimated to be 1 in 205,000 transfusions (51) . when several outbreaks occurred during the 1970s-1980s, ltcf's were thought to be a risk area for hbv (47) . however, recent surveys of geriatric hospitals indicate the prevalence of hbsag is similar to the general geriatric population (<1%) (47) . acute hbv in older adults is usually mild, and many cases are subclinical or presents with manifestations of cholestasis. in addition to the typical symptoms of jaundice, anorexia, and fatigue, diarrhea is a common complaint in elderly persons. complaints reflecting immune complex disease such as myalgias and arthalgias are rare in older adults. although acute hbv is generally not a severe disease in older adults, the mortality from fulminant hbv increases with age (47) . in a multivariate analysis of prognostic factors in 115 patients with hbv, age was an independent predictor of survival (46) . mortality for persons over age 60 years was 3.1% compared with 1.2% for persons ages 40-59. when individuals are infected at older ages, chronic carriage rates also increase. compared with a 10% carriage rate in young adults, approximately 60% of older persons become chronic carriers (47) . in addition to cirrhosis from chronic active hepatitis, one of the major complications of hbv infection is hepatocellular carcinoma (hcc). the length of time infected is an important factor in the development of cancer, and, thus, elderly persons who have been infected for many years are at the greatest risk. the rate of hcc rises from 197 per 100,000 in 30-to 39-year olds to 927 per 100,000 in 60-to 69-year-old chronic hbv carriers (52) . most cases of acute hepatitis b clear spontaneously and do not require treatment. in young patients with compensated disease, alpha-interferon is primarily used in the treatment of chronic hepatitis b. those who respond favorably may see suppression of viral replication and a decrease in the risk of progression to cirrhosis or to cancer. side effects of therapy are frequent and increase with advancing age (47) . therapy should be reserved for patients in overall good health, except for their liver disease, and who have evidence of active viral replication. five nucleotide/nucleoside analogs (lamivudine, adefovir, entecavir, tenofovir, and telbuvidine) are currently approved by the food and drug administration to treat chronic hepatitis b (53) . these drugs are usually better tolerated than interferon, although they have not been studied specifically in the elderly. patients with chronic hepatitis b should be tested for immunity to hepatitis a and, if seronegative, should be vaccinated against hepatitis a to prevent acute decompensation that could occur with hepatitis a superinfection. in addition, patients should be counseled to abstain from alcohol, to maintain a healthy body weight, and to use condoms to protect their sexual partners from infection. the currently licensed hepatitis b vaccine is a genetic recombinant vaccine; it is very well tolerated and highly immunogenic with excellent protective efficacy in children and young adults. however, response rates to hbv vaccine diminish significantly with increased age. ninety percent of persons under age 40 achieve an adequate seroresponse compared with only 50% in persons over age 60. hepatitis c virus (hcv) is an rna virus in the flavivirus family. exposures to contaminated blood, either through occupation or through intravenous drug abuse, may transmit hcv. although 40-50% of community-acquired hcv cases do not report a parenteral exposure, nonparenteral transmission of hcv is not well understood. sexual transmission, if it occurs, is not efficient. the major risk factor for hcv in older persons is transfusion, and most became infected prior to routine screening of the blood supply in 1990 (47) . for southern europe and japan, the peak era of transfusion-associated hcv was between 1940 and 1960 whereas in the united states and northern europe, the peak transmission was between 1960 and 1980. the current risk of acquiring hcv from transfusion in the united states is approximately 1 in 1,935,000 (51) . in southern europe, numerous population-based studies have shown that the prevalence of hcv is 16-42% in persons over 60 years and 33-50% in those over 80 years; infection is extremely rare in subjects <40 years (54) . the seroprevalence of 1.4-2.2% in ltcf residents is approximately that of the general elderly population (55) . the clinical manifestations of acute hcv are generally mild and nonspecific. in a series of 20 older people with acute non-a non-b hepatitis, approximately 30-40% had fever, abdominal pain, and jaundice (47) . fulminant hepatitis is rare with hcv, but development of chronic liver disease is very common (56) . virtually all persons become chronically infected, and a significant number develop chronic liver disease. on average, 20 years after initial infection, chronic active hepatitis or cirrhosis develops in 29-76% of persons. age affects the rate of progression to cirrhosis in two important ways: the younger a patient is when hcv is acquired, the slower the rate of progression to cirrhosis; however, the longer one is infected, the greater the risk of development of cirrhosis and hcc (54) . hepatocellular carcinoma is clearly associated with chronic hcv infection, and the relative risk of cancer from hcv may be even greater than that from hbv. in a study of 25 older persons with hcv in the united kingdom, 36% developed hcc (56) . chronic hcv infection is currently treated with pegylated interferon alpha and oral ribavirin. goals of therapy include clearance of viremia and prevention of decompensated cirrhosis and hcc. persons with high viral load and viral genotype 1 have a low response rate to a -interferon, and, many patients who initially respond, relapse when therapy is discontinued (47) . most studies of interferon treatment of hcv have not included older participants. in one study from japan, interferon was administered to 19 patients aged 65 and older with hcv, and the response rate was 26% compared with 33% in younger persons (57) . of note, the older subjects had higher hcv-rna titers and more severe fibrosis on their liver biopsy as compared to their younger subjects. response rates in elderly persons correlated with lower hcv-rna titers. because older persons have more side effects and a lower response rate to interferon, treatment should be carefully considered on an individual basis; it should be proposed only in patients up to 75 years who have a significant risk of progression of liver disease, no serious co-morbidities, and an otherwise good life expectancy (54) . patients with chronic hepatitis c should be tested for immunity to hepatitis a and b and, if seronegative to either, they should be vaccinated to prevent acute decompensations that could occur with hepatitis a or b superinfection. they should be counseled to abstain from alcohol, to maintain a healthy body weight, and to use condoms to protect their sexual partners from infection. although deaths related to diarrhea have traditionally been thought to be a problem of young children in developing countries, in the united states from 1979 to 1987, 51% of the 28,538 diarrhea-related deaths occurred in adults over the age of 74 as compared to 11% in children < 5 year old (58) . the odds ratio of dying during a hospitalization involving gastroenteritis was 52.6 for adults over the age of 70 as compared with children less than 5 years of age. residents of nursing facilities are at particular risk for infectious diarrhea illness because of the outbreaks that can occur in closed populations. the majority of nursing facility outbreaks of gastroenteritis are probably viral and include rotavirus, enteric adenoviruses, norovirus, snow mountain agent, and small round structured viruses (srsv) (58, 59) . rotaviruses are small rna viruses in the retrovirus family and are the most important cause of gastroenteritis in infants and young children worldwide. the mode of transmission is assumed to be fecal-oral, and the virus is relatively resistant to common disinfectants and facilitating nosocomial dissemination. several outbreaks of rotavirus infection in elderly residents of ltcfs have been reported with attack rates ranging from 36 to 66% and mortality rates of 1-10% (60, 61) . a typical illness lasts 2-3 days and includes voluminous vomiting and watery diarrhea with low-grade fever. death may result from dehydration progressing to oliguria and acidosis (60, 61) . in an analysis of specimens from gastroenteritis outbreaks in aged care facilities in australia, rotavirus was detected by electron microscopy in 18 of 29 individuals in 7 out of 53 outbreaks (62) . although rotavirus infections are most common in winter, outbreaks can occur at other times of the year (63) . rotavirus serum antibody titers offer some protection against severe disease and tend to diminish with increasing age (61) . two rotavirus vaccines were released in 2006 for use in children, a pentavalent human-bovine reassortant rotavirus vaccine called rotateq ® and a live-attenuated human rotavirus vaccine called rotarix ® . no data on safety or immunogenicity in the elderly exists; however, given the mortality rates in this age group, further study would be reasonable. norovirus (formerly called norwalk-like virus) is also a frequent cause of diarrhea in adults including the elderly (see also chapter "infectious diarrhea"). in a study of 20 gastroenteritis outbreaks in maryland, 52% of stool samples were positive for norwalk-like virus (nlv) (64) . the median duration of symptoms with nlv gastroenteritis is 2-3 days (65) . varicella-zoster virus (vzv) is a dna virus and a member of the herpes virus family. it causes two distinct clinical syndromes: primary disseminated infection (chickenpox) and reactivation of latent virus in the dorsal root ganglia, leading to herpes zoster or "shingles" (66) . herpes zoster is a painful, vesicular exanthem that erupts in one to two dermatomes after a prodrome of days to weeks and may take up to a month to heal. most patients report a deep aching or burning sensation, altered sensation to touch with paresthesias, dysesthesia, or hyperesthesia. herpes zoster is a common condition with a cumulative lifetime incidence of 20-30% with most of the risk concentrated in older age. the overall incidence is 1.2-3.4 per 1,000 personyears, but rates rise sharply with increasing age to 11.8 per 1,00,0 for persons age 65 years and older (67) . chapter "herpes zoster" is devoted to the topic of herpes zoster, and the reader is referred to this section for further details. the epstein-barr virus (ebv) is a herpes virus that establishes lifelong infection. primary infection may occur in childhood when infection is asymptomatic or during adolescence when the symptoms of classic mononucleosis are most often observed (66) . although primary infection is uncommon in old age, the manifestations may be different than those in youth, which makes diagnosis challenging. seroepidemiologic studies indicate that 3-10% of older adults are at risk for primary infection (66) . diagnosis is also often delayed because symptoms may be misleading. lymphadenopathy, pharyngitis, and splenomegaly are significantly less common and jaundice is more common in older persons as compared with the young persons (67) . fever is more common and often lasts more than 2 weeks (68, 69) . the neurologic manifestations of ebv infection are protean, and acute ebv encephalitis has been described (70) . adding to the difficulty of making a correct diagnosis, development of atypical lymphocytosis may be absent or delayed in the elderly. diagnosis of primary ebv is made by the presence of heterophile antibodies or ebv-specific igm. although acyclovir has in vitro activity against ebv, no benefit has been demonstrated in the treatment of acute ebv infection. trends in hospitalizations for pneumonia among persons aged 65 years or older in the united states improved diagnosis of the etiology of community-acquired pneumonia with real-time polymerase chain reaction prevention and control of influenza. recommendations of the advisory committee on immunization practices (acip) current concepts: avian influenza a (h5n1) infection in humans impact of influenza vaccination on seasonal mortality in the us elderly population influenza-associated hospitalizations in the united states mortality associated with influenza and respiratory syncytial virus in the united states lung involvement in influenza duration of influenza a virus shedding in hospitalized patients and implications for infection control rapid tests for influenza . current opinions in pediatrics performance of six influenza rapid tests in detecting human influenza in clinical specimens antiviral therapy and outcomes of influenza requiring hospitalization in ontario, canada effectiveness of influenza vaccine in the community-dwelling elderly antivirals and the control of influenza outbreaks effects of influenza vaccination of health-care workers on mortality of elderly people in long-term care: a randomised controlled trial respiratory syncytial virus infection in elderly and high-risk adults respiratory syncytial virus infection in adults acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults lack of sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults ribavirin therapy of adult respiratory syncytial virus pneumonitis high-dose, shortduration ribavirin aerosol therapy compared with standard ribavirin therapy in children with suspected respiratory syncytial virus infection parainfluenza viruses impact of respiratory virus infections on persons with chronic underlying conditions parainfluenza virus infection among adults hospitalized for lower respiratory tract infection parainfluenza infections in the elderly 1976-82 influenza-like illness in residential care homes: a study of the incidence, aetiological agents, natural history, and health resource utilization a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus . seminar respiratory critical care medicine an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility human metapneumovirus infections in young and elderly adults human metapneumovirus pneumonia in adults: results of a prospective study rhinovirus and coronavirus infections quantitative and qualitative analysis of rhinovirus infection in bronchial tissues risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study effect of interactions between lower airway bacterial and rhinoviral infection in exacerbations of copd rhinovirus outbreak in a long term care facility for elderly persons associated with unusually high mortality two outbreaks of severe respiratory disease in nursing homes associated with rhinovirus human coronaviruses: what do they cause? a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection the novel human coronaviruses nl63 and hku1 human coronavirus oc43 causes influenza-like illness in residents and staff of aged-care facilities in melbourne spectrum of clinical illness in hospitalized patients with "common cold" virus infections history and epidemology of hepatitis a virus surveillance for acute viral hepatitis -united states viral hepatitis in older adults hepatitis a epidemic in the elderly acute hepatitis a virus infection: a review of prognostic factors from 25 years experience in a tertiary referral center incidence of hepatitis a in israel following universal immunization of toddlers current prevalence and incidence of infectious disease markers and estimated window-period risk in the american red cross blood donor population liver disease in the elderly new drugs for chronic hepatitis b: a review hepatitis c virus infection in the elderly: epidemiology, natural history and management prevalence of hepatitis b surface antigen, hepatitis c antibody, and hiv-1 antibody among residents of a long-term care facility hepatitis c virus infection in the elderly interferon therapy for patients more than 60 years of age with chronic hepatitis c approach to acute diarrhea in the elderly outbreaks of gastroenteritis in elderly nursing homes and retirement facilities associated with human calciviruses rotavirus infection in a geriatric population an epidemic of rotavirus-associated gastroenteritis in a nursing home for the elderly rotavirus detection and characterisation in outbreaks of gastroenteritis in aged-care facilities report of a rotavirus outbreak in an adult nursing home population a predominant role for norwalk-like viruses as agents of epidemic gastroenteritis in maryland nursing homes for the elderly clinical manifestation of norovirus gastroenteritis in health care settings epstein-barr virus and the elderly host clinical and laboratory evaluation of elderly patients with heterophil-antibody positive infectious monoculeosis infectious mononucleosis in middle age infectious mononucleosis in patients aged 40 to 72 years: report of 27 cases, including 3 without heterophil-antibody responses epstein-barr virus causing encephalitis in an elderly woman hepatitis c virus infection in the elderly: epidemiology, natural history and management recommendations for the management of herpes zoster antiviral therapy and outcomes of influenza requiring hospitalization in ontario, canada key: cord-281249-89eycq64 authors: falsey, ann r; becker, kenneth l; swinburne, andrew j; nylen, eric s; snider, richard h; formica, maria a; hennessey, patricia a; criddle, mary m; peterson, derick r; walsh, edward e title: utility of serum procalcitonin values in patients with acute exacerbations of chronic obstructive pulmonary disease: a cautionary note date: 2012-02-23 journal: int j chron obstruct pulmon dis doi: 10.2147/copd.s29149 sha: doc_id: 281249 cord_uid: 89eycq64 background: serum procalcitonin levels have been used as a biomarker of invasive bacterial infection and recently have been advocated to guide antibiotic therapy in patients with chronic obstructive pulmonary disease (copd). however, rigorous studies correlating procalcitonin levels with microbiologic data are lacking. acute exacerbations of copd (aecopd) have been linked to viral and bacterial infection as well as noninfectious causes. therefore, we evaluated procalcitonin as a predictor of viral versus bacterial infection in patients hospitalized with aecopd with and without evidence of pneumonia. methods: adults hospitalized during the winter with symptoms consistent with aecopd underwent extensive testing for viral, bacterial, and atypical pathogens. serum procalcitonin levels were measured on day 1 (admission), day 2, and at one month. clinical and laboratory features of subjects with viral and bacterial diagnoses were compared. results: in total, 224 subjects with copd were admitted for 240 respiratory illnesses. of these, 56 had pneumonia and 184 had aecopd alone. a microbiologic diagnosis was made in 76 (56%) of 134 illnesses with reliable bacteriology (26 viral infection, 29 bacterial infection, and 21 mixed viral bacterial infection). mean procalcitonin levels were significantly higher in patients with pneumonia compared with aecopd. however, discrimination between viral and bacterial infection using a 0.25 ng/ml threshold for bacterial infection in patients with aecopd was poor. conclusion: procalcitonin is useful in copd patients for alerting clinicians to invasive bacterial infections such as pneumonia but it does not distinguish bacterial from viral and noninfectious causes of aecopd. guidelines from the global initiative for chronic obstructive pulmonary disease (gold) recommend antibiotics for the treatment of moderate to severe acute exacerbations of chronic obstructive pulmonary disease (aecopd). 1 several meta-analyses support these recommendations to reduce mortality and treatment failures. 2 although purulent sputum and growth of a bacterial pathogen suggest infection, definitive causality with an aecopd is difficult because patients may be chronically colonized. 3, 4 in addition, recent studies using molecular diagnostics indicate that a substantial proportion of aecopd are associated with viral infection. 5, 6 accurate methods to differentiate viral and bacterial respiratory infections to allow targeted antibiotic therapy would be beneficial. measurement of serum procalcitonin has been proposed to discriminate bacterial infection from viral or noninfectious causes. 7 procalcitonin is a calcitonin precursor that is normally produced in neuroendocrine cells of the thyroid and lungs. however, in response to bacterial infections, procalcitonin is produced by cells throughout the body. 8, 9 stimuli of procalcitonin include bacterial products, including endotoxin and proinflammatory cytokines such as tumor necrosis factor alpha, whereas procalcitonin is attenuated by viralinduced interferon-γ. 8 , 10 procalcitonin testing has been used successfully as a guide to predict serious bacterial infections and medical outcomes in patients with sepsis. 11, 12 recently, procalcitonin has been assessed as a guide to antibiotic therapy in patients with respiratory illnesses, including copd. 7, [13] [14] [15] [16] [17] [18] however, microbiologic assessment to prove the presence or absence of bacterial infection was not performed in the majority of subjects in most trials, which is a concern for the food and drug administration and infectious disease society of america, as noted in a recent workshop. 19 therefore, we examined the utility of serum procalcitonin levels as predictors of viral and bacterial infection in patients hospitalized with aecopd using rigorous viral and bacteriologic studies. the study was performed at rochester general hospital, a 520-bed general medical-surgical hospital located in rochester (monroe county), ny. subjects were participating in a larger ongoing study of the relationship between procalcitonin level and definitive bacterial and viral diagnosis (to be described in a separate publication). adults $21 years of age admitted through the emergency department to rochester general hospital with an admitting diagnosis compatible with acute respiratory tract infection were recruited from november 1 to may 30 during 2008 may 30 during -2009 may 30 during and 2009 may 30 during -2010 . a subset of subjects with a past medical history of copd and smoking and symptoms consistent with aecopd according to the gold criteria (increased dyspnea, cough, and/or increased sputum volume or purulence) were identified for the present analysis. confirmation of copd was sought as available in the hospital medical records. patients were screened within 24 hours of admission and those given antibiotics prior to admission, or who had immunosuppression, cavitating lung disease, or witnessed aspiration were excluded. 20 in addition, subjects with other conditions known to increase serum procalcitonin levels (burns, trauma, pancreatitis, renal failure, thyroid tumors) were excluded from the study. subjects or legal guardians provided written informed consent. the study was approved by the university of rochester and rochester general hospital institutional review boards. at enrollment, demographic, clinical, and laboratory information was collected. chest radiographs were classified by a pulmonary specialist as infiltrate or no infiltrate. testing for bacterial pathogens included: blood cultures, sputum culture, and gram stain; nose and throat swabs for mycoplasma pneumoniae and chlamydophila pneumoniae polymerase chain reaction; and urine for streptococcus pneumoniae antigen and pneumococcal serology. sputum was induced with normal saline if subjects were unable to expectorate an adequate sputum sample spontaneously. specimens were considered adequate by standard criteria of .25 polymorphonuclear leukocytes and ,10 epithelial cells per high power field. nose and throat swabs and sputum were tested for viruses by reverse transcriptase polymerase chain reaction. sera were collected on hospital day 1 (prior to antibiotics) and 4-6 weeks later for viral serology, pneumococcal serology, and procalcitonin measurements. serum was also collected on hospital days 2 and 28 for procalcitonin measurement. standard microbiological testing including blood cultures, sputum gram stain and culture, influenza antigen testing, and viral cultures were performed by the rochester general hospital clinical laboratory. sputum samples were plated on blood, chocolate, and macconkey agar. legionella testing was performed at the discretion of the treating physician. pneumococcal surface protein a antigens covering families 1 and 2, obtained from the university of alabama bacterial respiratory pathogen reference laboratory, were used in an enzyme immunoassay. 21 a $4-fold rise in titer was considered evidence of infection. urine samples were assayed for pneumococcal antigen using binaxnow ® (binax inc, scarborough, me) urine assay. 22 procalcitonin procalcitonin was measured by resolved amplified cryptate emission technology (kryptor pct, brahms, henningsdorf, germany). functional sensitivity is 0.06 ng/ml (normal 0.033 ± 0.003 ng/ml). 12, 23, 24 immunoglobulin g titers in acute and convalescent serum for each virus was determined using established eia methods. 25 a $4-fold rise in viral specific immunoglobulin g was considered evidence of infection. submit your manuscript | www.dovepress.com real-time polymerase chain reaction assays for respiratory syncytial virus, human metapneumovirus, m. pneumoniae, and c. pneumoniae used published methods. [25] [26] [27] primers and probes for other viruses were as follows: influenza a (matrix gene), influenza b (nonstructural 1 [ns1] gene), coronaviruses (polymerase gene), and parainfluenza viruses (nucleocapsid gene). sequences can be supplied on request. bacteriology was considered unreliable if sputum was not obtained or was obtained after 6 hours of antibiotics, or was of inadequate quality. subjects were not considered to be viral infection alone or no infectious diagnosis unless "reliable" bacteriology was negative. fisher's exact and t-tests were used to compare distributions of categorical and continuous clinical variables for pneumonia versus aecopd patients. differences in procalcitonin levels were evaluated using the wilcoxon test, summarized using both means and standard deviations and medians with interquartile range, and receiver operator curves were plotted and area under the curve tabulated. multiple logistic regression was used to model maximum (procalcitonin over days 1-2) $0.25 as a function of clinical covariates. sas 9.2 was used for all analyses, with tests performed at the two-sided 0.05 level. during two winters from 2008 to 2010, we enrolled 532 subjects, of whom 224 had a history of copd and a respiratory illness with symptoms of aecopd. of these 224 subjects, 213 had a single illness, 12 had two, and one subject had three hospitalizations for a total of 240 illnesses evaluated. the average time between aecopd admissions for subjects with more than one illness evaluation was 7 ± 4 months. a diagnosis of copd was confirmed by pulmonary function testing, pulmonary physician evaluation, or radiographic changes in 90% of subjects. chest radiographs on admission revealed pneumonia in 56 cases, and 184 illnesses were considered to be aecopd alone ( figure 1 ). bacteriology was considered reliable in 134 (56%) of the 240 illnesses (104 aecopd and 30 pneumonia). notably, 47 viral infections alone (37 aecopd and 10 pneumonia) were excluded from further analyses of microbiology, primarily because sputum samples were collected more than 6 hours after antibiotics had been started. no patient was bacteremic. a variety of viral pathogens were identified and 45% were associated with bacterial infection (table 1 ). s. pneumoniae (22 cases) was the most common bacterial organism identified and was associated with viral infection in 13 (59%) and mixed bacterial infection in three (14%). of the 22 subjects with pneumococcal infection, half had more than one positive test (ie, sputum culture, pneumococcal serology, or urine antigen). of the 31 subjects considered to have bacterial infection based on sputum culture alone, all had 3-4+ growth of a potential pathogen and 87% had a consistent gram stain. procalcitonin values were measured on day 1 (admission), day 2, and at one month convalescence. mean procalcitonin table 4 , analysis c), procalcitonin values were significantly higher in the mixed viral bacterial infection group. given the lack of difference noted in analysis a, this finding might suggest that the presence of a viral infection is necessary for invasive bacterial infection. using the $0.25 ng/ml threshold, the specificity for bacterial infection among patients with a documented viral infection was 96% but sensitivity was low at 31%, with a positive predictive value of 83% and negative predictive value of 69% (figure 2) . although a significant difference in the mean and median procalcitonin values in this group could be demonstrated, discrimination between viral and bacterial coinfection by procalcitonin was poor (figure 3 ). although only one of 25 patients with virus infection alone would have been misclassified as possible bacterial infection, interestingly, the single patient with a viral infection alone and elevated procalcitonin levels had influenza complicated by pericardial tamponade, necessitating surgical intervention on the second hospital day. as demonstrated in figure 3 , most subjects in any category (viral alone, viral + bacterial, or bacterial alone) fell below the 0.25 ng/ml threshold on day 1 or 2 of hospitalization. infection is a major cause of morbidity and mortality among patients with copd. 28 in copd patients presenting to the hospital with dyspnea, our study demonstrates a wide variety of viral and bacterial pathogens, as well as a high incidence of pneumonia. recently, use of procalcitonin to guide clinical decisions has been shown to reduce antibiotic use significantly in patients with respiratory illnesses without compromising composite patient outcomes in a number of randomized interventional trials. [13] [14] [15] [16] [17] [18] 29 although procalcitonin-guided decisions have been promulgated as a method to "rule out" bacterial infection, the 0.25 ng/ml threshold for recommending antibiotic treatment is not based on microbiologic data. recovery without antibiotics or with an abbreviated course has been equated with the absence of a bacterial infection. the lack of microbiologic correlation with procalcitonin levels is likely to slow endorsement of procalcitonin-guided management in respiratory illness. 19, 30, 31 ours is the first study in the us to examine the utility of procalcitonin levels in patients with copd, and consistent with the european literature, we found that a high procalcitonin level was relatively specific for invasive bacterial disease such as pneumonia. 32, 33 in addition, elevated procalcitonin values in the aecopd group correlated with higher temperature, white blood cells, and more severe illness, suggesting the possibility of occult pneumonia. thus, high procalcitonin values may alert clinicians to the presence of bacterial pneumonia when the chest radiograph results are negative or ambiguous. however, our results also indicate that minor elevations at the low end of the procalcitonin spectrum do not correlate with bacterial infection for subjects with aecopd alone. it has been estimated that approximately 40%-50% of aecopd cases are due to bacterial infections. 34 the precise contribution of bacterial infection is difficult to define because the airways of copd patients may be chronically colonized. 3, 4 acquisition of new strains of haemophilus influenzae and moraxella catarrhalis, rather than bacterial load, appears to be the most important factor in the pathogenesis of acute exacerbations. 35 this factor has not been accounted for in aecopd antibiotic trials and might explain the modest beneficial effects of antimicrobial treatments observed. we accept that some subjects who were classified as having bacterial infection in our study were colonized rather than infected. however, only 17% of patients overall with aecopd and without pneumonia, and only 25% of those with evidence of bacterial infection had procalcitonin levels $0.25 ng/ml. while it is possible that this small subgroup represents those who will actually benefit from antibiotic treatment, such a conclusion is premature. notably, of those with procalcitonin levels ,0.25 ng/ml, 57% had three anthonisen criteria, suggesting they would benefit from antibiotic treatment based on past studies. 36 thus, only two conclusions are possible, ie, either the contribution of bacterial infection in aecopd has been markedly overestimated or procalcitonin values do not differentiate bacterial bronchitis from viral or noninfectious etiologies. our study adds to the growing body of literature which questions the utility of procalcitonin levels to discriminate viral-associated from bacterial-associated aecopd. in a study by daniels et al, procalcitonin levels were measured in outpatients enrolled in a trial of doxycycline for aecopd. 37 a significant benefit of doxycycline was noted for patients with procalcitonin levels ,0.1 ng/ml. in this study, as well as in two additional reports, no differences in procalcitonin levels were noted in patients with or without bacteria in sputum during exacerbation. 32, 37, 38 our study provides the most rigorous microbiologic analysis of moderate to severe illness requiring hospitalization to date, particularly for subjects who were deemed negative for bacterial infection. unlike prior studies, we did not consider patients to be free of bacterial infection unless adequate samples were taken in a timely fashion and without antibiotic use prior to hospitalization. our study had several limitations. the number of patients with documented bacterial infection was relatively small. however, large numbers of subjects are not needed to show that a test is insensitive, especially if missing even a few patients with potentially treatable bacterial infections is considered unacceptable. because medical information was restricted to that available in the hospital medical record, we could not classify the stage of copd using gold criteria in our subjects. lastly, we did not test for rhinovirus, a common pathogen in this population, although this omission does not invalidate the findings for those patients with a viral or bacterial infection identified. in conclusion, we found that elevated serum procalcitonin levels are associated with more severe illness in patients hospitalized with symptoms of aecopd and that high values may alert clinicians to the possibility of pneumonia. however, low procalcitonin values do not "rule out" bacterial infection in aecopd. before procalcitonin-based treatment algorithms are endorsed, additional studies in copd patients should be performed. clinical trials focusing on antibiotic global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. nhlbi/ who global initiative for chronic obstructive lung disease (gold) workshop summary antibiotics for exacerbations of chronic obstructive pulmonary disease bacterial infection in chronic obstructive pulmonary disease. a study of stable and exacerbated outpatients using the protected specimen brush pathogen-directed therapy in acute exacerbations of chronic obstructive pulmonary disease viral infections as a cause of chronic obstructive pulmonary disease (copd) exacerbation importance of viral and bacterial infections in chronic obstructive pulmonary disease exacerbations procalcitonin for triage of patients with respiratory tract symptoms: a case study in the trial design process for approval of a new diagnostic test for lower respiratory tract infections clinical review 167: procalcitonin and the calcitonin gene family of peptides in inflammation, infection, and sepsis: a journey from calcitonin back to its precursors ubiquitous expression of the calcitonin-i gene in multiple tissues in response to sepsis comparison of procalcitonin with c-reactive protein, interleukin 6 and interferon-alpha for differentiation of bacterial vs viral infections pneumonitis-associated hyperprocalcitoninemia procalcitonin in sepsis and systemic inflammation: a harmful biomarker and a therapeutic target procalcitonin guidance of antibiotic therapy in community-acquired pneumonia: a randomized trial effect of procalcitonin-guided treatment on antibiotic use and outcome in lower respiratory tract infections: cluster-randomised, single-blinded intervention trial antibiotic treatment of exacerbations of copd: a randomized, controlled trial comparing procalcitonin-guidance with standard therapy effect of procalcitoninbased guidelines vs standard guidelines on antibiotic use in lower respiratory tract infections: the prohosp randomized controlled trial antibiotic treatment interruption of suspected lower respiratory tract infections based on a single procalcitonin measurement at hospital admission -a randomized trial procalcitonin-guided antibiotic use vs a standard approach for acute respiratory tract infections in primary care procalcitonin as a biomarker in respiratory tract infection procalcitonin in peritoneal dialysis -a useful marker of inflammation? bacterial respiratory pathogen reference laboratory rapid diagnosis of pneumococcal pneumonia in adults using the binax now streptococcus pneumoniae urinary antigen test procalcitonin in young febrile infants for the detection of serious bacterial infections the future diagnostic role of procalcitonin levels: the need for improved sensitivity human metapneumovirus infections in young and elderly adults comparison of quantitative reverse transcription-pcr to viral culture for assessment of respiratory syncytial virus shedding simultaneous detection of chlamydophila pneumoniae and mycoplasma pneumoniae by use of molecular beacons in a duplex real-time pcr infection in the pathogenesis and course of chronic obstructive pulmonary disease procalcitonin algorithms for antibiotic therapy decisions: a systematic review of randomized controlled trials and recommendations for clinical algorithms 1-2. and drug administration and infectious disease society of america. fda-idsa public workshop: advancing clinical development of molecular and other diagnostic tests for respiratory tract infections value of procalcitonin, c-reactive protein, and neopterin in exacerbations of chronic obstructive pulmonary disease procalcitonin and c-reactive protein in hospitalized adult patients with community-acquired pneumonia or exacerbation of asthma or the role of bacteria in exacerbations of copd. a constructive view new strains of bacteria and exacerbations of chronic obstructive pulmonary disease antibiotic therapy in exacerbations of chronic obstructive pulmonary disease procalcitonin vs c-reactive protein as predictive markers of response to antibiotic therapy in acute exacerbations of copd upper-respiratory viral infection, biomarkers, and copd exacerbations the authors wish to thank jamie biear, karen leavenworth, and kyle quinn for their technical support, susan romansky, tashara smalls, and the staff of the rochester general hospital clinical microbiology, hematology and chemistry laboratory for assistance with specimen retrieval, and members of the respiratory therapy department for help with sputum induction. this work was supported by the national institute health at the national institute of allergy and infectious diseases (1r01ai079446-01). dr falsey has served as a consultant for sanofipasteur, gsk biologics, medimmune, astrazeneca, and novartis. dr walsh has served as a consultant for novartis, alnylam, astrazeneca, medimmune, and boehringer ingelheim. otherwise, the authors have no financial conflicts of interest which could potentially bias the results of the study. submit your manuscript here: http://www.dovepress.com/international-journal-of-copd-journalthe international journal of copd is an international, peer-reviewed journal of therapeutics and pharmacology focusing on concise rapid reporting of clinical studies and reviews in copd. special focus is given to the pathophysiological processes underlying the disease, intervention programs, patient focused education, and self management protocols.this journal is indexed on pubmed central, medline and cas. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors.international journal of copd 2012:7 key: cord-284216-4sl8xfur authors: sinha, anirban; lutter, rené; dekker, tamara; dierdorp, barbara; j. sterk, peter; frey, urs; delgado-eckert, edgar title: can measurements of inflammatory biomarkers be used to spot respiratory viral infections? date: 2020-10-17 journal: viruses doi: 10.3390/v12101175 sha: doc_id: 284216 cord_uid: 4sl8xfur accurate detection of human respiratory viral infections is highly topical. we investigated how strongly inflammatory biomarkers (feno, eosinophils, neutrophils, and cytokines in nasal lavage fluid) and lung function parameters change upon rhinovirus 16 infection, in order to explore their potential use for infection detection. to this end, within a longitudinal cohort study, healthy and mildly asthmatic volunteers were experimentally inoculated with rhinovirus 16, and time series of these parameters/biomarkers were systematically recorded and compared between the preand post-infection phases of the study, which lasted two months and one month, respectively. we found that the parameters’/biomarkers’ ability to discriminate between the infected and the uninfected state varied over the observation time period. consistently over time, the concentration of cytokines, in nasal lavage fluid, showed moderate to very good discrimination performance, thereby qualifying for disease progression monitoring, whereas lung function and feno, while quickly and non-invasively measurable using cheap portable devices (e.g., at airports), performed poorly. infections of the respiratory tract in humans are a major cause for global morbidity and mortality. about 80% of such infections are caused by viruses, the most prevalent being influenza, parainfluenza, respiratory syncytial virus (rsv), coronavirus, adenovirus, and rhinovirus [1] . early detection and accurate identification of the virus in potentially affected individuals are necessary for appropriate and timely remedies and management. however, the differential diagnosis can be difficult due to nonspecific clinical presentation and symptoms [2] , particularly in premorbid patients with, for example, bronchial asthma. historically, examination of clinical symptoms in combination with virus isolation, serological tests, imaging, and chest radiography [3] have served as mainstays of viral detection [4] . later, chest computed tomography [5] , antibody staining and detection methods complemented with immune assays paved the way for better diagnostics but still lacked the specificity and sensitivity required for accurate detection. in the last two decades, there has been a remarkable improvement in the diagnosis of respiratory pathogens with the availability of molecular assays. indeed, mainly due to the advent of polymerase chain reaction (pcr) techniques, highly sensitive and robust methods such as nucleic acid sequence-based amplification (nasba), loop-mediated isothermal amplification (lamp), and micrornas [1, [6] [7] [8] have come into the picture. multiplex assays coupled to microfluidics and high-resolution imaging constitute further developments towards more accurate diagnostics [1] . these latest diagnostic tools rely on either finding the virus or traces of it in the patient's body, or virus specific antibodies generated by the patient's immune system. a topical example is sars-cov-2. currently, on the one hand, the most accurate diagnosis method to detect sars-cov-2 infections in humans is based on detecting amplified nucleic acid sequences of the virus genome [9] . however, pcr technology may not be fully accurate [10, 11] and often requires multiple testing for reliable results [12] . on the other hand, clinically available parameters for airways assessment such as inflammatory and lung function indices have not been traditionally used for the diagnosis of viral respiratory infections. this is mainly due to the fact that pathological changes in such indices have been found to lack pathogen-specificity [13, 14] . furthermore, to the best of our knowledge, the discrimination accuracy of such parameters for the detection of any viral respiratory infection in humans, regardless of the specific virus involved, has never been systematically tested. whether a physiological parameter or biomarker qualifies for the detection of a viral respiratory infection depends on how accurately it can discriminate between infected and uninfected states. this can only be the case if the change in the parameter/biomarker elicited by the infection is stronger than its natural fluctuations during uninfected phases. moreover, ideally, this should be independent of other underlying disease processes, such as chronic ailments. therefore, we aimed at investigating how reliable inflammatory biomarkers and lung function are for the detection of respiratory viral infections in humans, by systematically comparing the values of these parameters/biomarkers during the participants' pre-and post-infection state within a longitudinal study cohort. our approach consisted of using longitudinally measured inflammatory biomarkers and lung function parameters to assess whether post-infection values exceeded their natural variability during uninfected phases. because of ethical reasons, we focused on experimental infection of humans in vivo with rhinovirus 16 (rv16), which is a not particularly virulent pathogen that causes the common cold. in a prospective, longitudinally designed study comprising 12 healthy and 12 mildly asthmatic subjects, we measured time series of a set of inflammatory/immune biomarkers and standard lung function [15] . the cohort demographics have been previously published in [15] and are also summarized in table 1 below. the serum antibody titer of rv16 required had to be less than 1:8 for all participants before enrollment in order to exclude any participants whose immune system had already been exposed to the virus. according to our previous findings, a serum antibody titer less than 1:6 is sufficient to exclude such participants [16] . however, to increase recruitment of participants and without compromising the validity of the study outcomes, a relaxed titer of 1:8 was chosen. the participants were within 18-30 years of age and without concomitant disease or pregnancy. asthmatic subjects were not on steroids, at least in the previous 6 weeks before study screening. the inclusion/exclusion criteria for participants along with the study design have been published in detail before [15] . a summary of inclusion criteria can be found in table 2 below. the parameters screened were cellular immune markers including eosinophil and neutrophil cell numbers in nasal lavage fluid. molecular immune markers, also measured in nasal lavage fluid, were the following 10 cytokines: ifn-gamma, il-1β, il-10, il-13, il-17a, il-33, il-6, il-8, ip-10, and tnf-alpha, which are biomarkers known to play a role in asthma, inflammation, and rv16 infection [17] . the inflammatory biomarker, measured in the exhaled air, was nitric oxide (feno). the lung function parameters measured were peak expiratory flow (pef), forced expiratory volume in 1st second (fev1), forced vital capacity (fvc), and the ratio fev1/fvc. these parameters/biomarkers were repeatedly captured during two months prior to and one month following an experimental rv16 infection induced by controlled and deliberate inoculation with rv16 of healthy and mildly asthmatic volunteers [15] . the sampling frequencies of the various parameters/biomarkers are detailed below in table 3 . in order to minimize the influence of diurnal variations of the cytokines measured in nasal lavage fluid, for any given participant, the sampling visits were scheduled in such a way that samples were collected at approximately the same time of the day. the study protocol was approved by the hospital medical ethical committee. all study participants provided written informed consent of participation. the cohort study has been registered in the netherlands trial register, nl5317 (ntr5426). the efficacy of the inoculation with rv16 was established using serum antibody tests (rv16 seroconversion), clinical symptoms, and rhinovirus polymerase chain reaction (pcr) conducted on nasal lavage fluid taken from every participant after the inoculation. the viral load just after a few days from rhinovirus challenge, along with the seroconversion for antibodies against rhinovirus (measured at the end of the study for each participant), are summarized in table 4 below. see also supplementary materials for symptoms data. in addition, the development of a host response was carefully assessed for each cohort participant, as described previously [15] . all these tests provided strong evidence supporting the fact that all 24 participants were effectively infected with the rv16 after inoculation. nevertheless, we excluded participants 13a and 11h from the analysis due to their negative outcomes of the viral load test during the first 10 days after inoculation and negative seroconversion tests at the end of the study (see table 4 ). thus, only those participants were included in our analysis who, either had a positive viral load in nasal lavage fluid when measured during the first 10 days after inoculation, or a positive seroconversion test at the end of the study, or both. all participants were continuously monitored throughout the study. clinical symptoms and occurrence of any concomitant infection due to any exposures to pathogens apart from the experimental challenge were carefully recorded using a detailed questionnaire (see supplement materials), which was filled out at every visit. indeed, during the pre-challenge phase, none of the participants reported symptoms of any concomitant infection that could possibly confound the results during this phase of the study. apart from that, pcr tests using primers from a panel of common respiratory viruses were performed for every individual before experimental challenge to rule out the occurrence of concomitant infections. table 4 . overview of the viral load (copies/ml) measured in nasal lavage fluid from each participant. samples of nasal lavage fluid were collected on days 1, 3, and 6 after challenge. na means no sample was collected. the rightmost column displays, for each participant, the results of the antibody test against rhinovirus (rv) (from seroconversion) performed at the last visit of the study (approximately 30 days after challenge). 0 = no response and 1= response. for every inflammatory/immune/lung functional biomarker separately, we calculated the average value over the first 10 days after inoculation and compared it to the average calculated over every possible time interval of 10 consecutive days prior to the inoculation. previously published work has suggested that all important pathophysiological and immunological changes elicited by a rhinovirus infection in humans were most likely to happen within a time interval of 10 days after exposure. this constituted the rationale behind our choice of time interval or "window" length [18] . in order to assess the time dependency of the above-mentioned comparison of averages, the 10-day windows prior to the inoculation were glided, one day at a time. the start position of the gliding window is expressed relative to the day of the viral inoculation, which is marked as day 0. chronologically speaking, the first pre-inoculation window considered in our analysis started on day -50 relative to the day of the inoculation, and the last one at position -9 (the last window contains the day of the inoculation). see figure 1 below for more details. viruses 2020, 12, x for peer review 5 of 12 for every inflammatory/immune/lung functional biomarker separately, we calculated the average value over the first 10 days after inoculation and compared it to the average calculated over every possible time interval of 10 consecutive days prior to the inoculation. previously published work has suggested that all important pathophysiological and immunological changes elicited by a rhinovirus infection in humans were most likely to happen within a time interval of 10 days after exposure. this constituted the rationale behind our choice of time interval or "window" length [18] . in order to assess the time dependency of the above-mentioned comparison of averages, the 10day windows prior to the inoculation were glided, one day at a time. the start position of the gliding window is expressed relative to the day of the viral inoculation, which is marked as day 0. chronologically speaking, the first pre-inoculation window considered in our analysis started on day -50 relative to the day of the inoculation, and the last one at position -9 (the last window contains the day of the inoculation). see figure 1 below for more details. figure 1 . graphical representation of a participant's biomarker time series xi. the chronological order of measurements of the biomarker is expressed in days from the day of inoculation, which is marked as "day 0". negative indexes represent measurements taken before the viral challenge, whereas positive indexes belong to measurements conducted after the challenge. note that for those biomarkers not measured on a daily basis, some of the values in the time series are missing. a moving figure 1 . graphical representation of a participant's biomarker time series x i . the chronological order of measurements of the biomarker is expressed in days from the day of inoculation, which is marked as "day 0". negative indexes represent measurements taken before the viral challenge, whereas positive indexes belong to measurements conducted after the challenge. note that for those biomarkers not measured on a daily basis, some of the values in the time series are missing. a moving window (depicted in green), covering ten days, glides, one day at a time, during the pre-challenge phase, starting from day −50 down to day −9. for each window, the average over the ten days the window covers is calculated. for example, the window starting on day −25 yields the average µ -25 . after the challenge, only one window is contemplated, namely the window covering the first ten days immediately after inoculation. this window yields the average µ 1 . for every participant, there is such an average µ j and µ 1 , respectively, where j = −50,9. for a given day j between -50 and -9 during the pre-challenge phase, and for a given subgroup of the cohort (healthy, asthmatics, and pooled), the empirical distribution of averages µ j were compared to the empirical distribution of averages µ 1 using a two-tailed paired mann-whitney u test. the results (p-values) of these comparisons are depicted for the biomarkers il-6 and feno in the top panels of figures 2 and 3 below. summary statistics for the pooled group can be found in tables "summary statistics of the averages of il-6 and feno concentration within each window" in supplementary material data. furthermore, roc curves were constructed assuming that the collection of averages µ j is characteristic of uninfected individuals, and the collection of averages µ 1 is characteristic of infected individuals. the areas under these roc curves (auc) are depicted for the biomarkers il-6 and feno in the bottom panels of figures 2 and 3 below. the above-mentioned comparison of within-window averages was not done individually, but rather for all cohort participants using a two-tailed paired mann-whitney u test. thus, the test aims at establishing whether the distributions of values of a given parameter/biomarker prior to and during the infection are significantly different. for a given pre-inoculation window, the p-value resulting from this test is indicative of the ability of a given inflammatory/immune or lung function biomarker to discriminate between healthy and infected states as a function of the starting position of the pre-inoculation window at hand (see top panels in figures 2 and 3) . thereby, we looked at how the range observed during the healthy state varied in time and whether it remained, over time, statistically distinct from the range observed during the infected state. furthermore, this discriminatory ability was also quantitatively assessed by constructing a receiver operating characteristic (roc) curve [19] and calculating the area under the same (see bottom panels in figures 2 and 3) . the area under a roc curve (auc) is an effective way to summarize the overall discrimination accuracy of a given parameter/biomarker at a given time point during the pre-challenge phase. on the one hand, if there is no apparent distributional difference between the values of the parameter/biomarker for a given pre-challenge window and the values for the post-challenge window, the auc will have a value of about 0.5. on the other hand, if there is a perfect separation of the values for a given pre-challenge window and the values for the post-challenge window, the auc will be equal to 1. to relate the auc to its discriminatory performance, we followed [20] , and used the following denominations of auc ranges: 0.9-1.0 excellent, 0.8-0.9 very good, 0.7-0.8 good, 0.6-0.7 sufficient, and 0.4-0.6 bad. this was how we quantitatively compared the discrimination accuracy of a given parameter/biomarker at a given time point during the pre-challenge phase to its discrimination accuracy at another time point during the pre-challenge phase (bottom panels in figures 2 and 3) . moreover, we used the auc to compare two different parameters/biomarkers in terms of their discrimination accuracy. for this comparison, however, it was also important to consider the stability over time of the discrimination accuracy of each of the two biomarkers. in order to explore whether the discrimination performance of the parameters/biomarkers at hand was independent of other underlying disease processes, such as chronic ailments, the above described analysis was conducted on the groups of asthmatics and healthy individuals separately. however, we also carried out the analysis on the data resulting from pooling these two groups. the cytokines il-6 ( for cellular immune markers measured in nasal lavage fluid, the emerging picture is more complex (see supplementary materials data). the eosinophil cell numbers were found to be a sufficient discriminator (auc values from 0.6 to 0.7), although bad during limited time intervals, whereas for neutrophil cell numbers, this appeared to be the other way around, with some episodes of good discrimination performance ( figures s10a,b and s11a,b in supplementary materials data) . surprisingly, the overall cell numbers in nasal lavage fluid oscillates between being a sufficient and a good to very good discriminator ( figure s12a ,b in supplementary materials data). feno is, in general, a bad discriminator (auc 0.5-0.6), except for non-asthmatic patients, for which it is sufficient, and good only during a limited time interval (figure 3 , see also "summary statistics of the averages of feno concentration within each window" in supplementary materials data for summary statistics of the pooled group). viruses 2020, 12, x for peer review 8 of 12 to the best of our knowledge, this is the first study in which the utility of inflammatory/immune biomarkers and lung function has been tested for detecting respiratory viral infections, while the lung function parameters are throughout bad discriminators, whether measured in the morning or in the evening (see figures s13a,b-s20a ,b in supplementary materials data for the occasional exceptions to this rule). a general pattern, observed in our results, was how the ability to discriminate between healthy and rv16-infected states varied over time. the amplitude of these temporal fluctuations was found to be relatively highest among the cellular immune markers and some of the cytokines. when comparing asthmatics and non-asthmatics in terms of the ability of the various parameters/biomarkers to discriminate between healthy and rv16-infected, it appeared that for feno and the lung function parameters, with the exception of pef and the ratio fev1/fvc, the discriminatory performance was higher in the non-asthmatics group. this difference between the two groups could be observed over time intervals of weeks (see figure 3 and figures s13a,b-s20a ,b in supplementary materials data). for the cellular immune-markers and the cytokines, however, discriminatory performance differences were less pronounced or less consistent over time (see figure 2 and figures s1a,b-s12a ,b in supplementary materials data). to the best of our knowledge, this is the first study in which the utility of inflammatory/immune biomarkers and lung function has been tested for detecting respiratory viral infections, while considering the natural temporal fluctuations of the parameters/biomarkers studied. our findings indicate that the concentrations of the various cytokines and the overall cell count in nasal lavage fluid are consistently at least sufficient discriminators of rv infection, the cytokines at times even excellent discriminators. furthermore, eosinophil and neutrophil cell numbers in nasal lavage fluid were found to reach, at times, the level of a sufficient discriminator. however, this was not consistently the case throughout the observation period. finally, feno and the lung function parameters were, in general, bad discriminators, except within the group of non-asthmatics, where sufficient discrimination power was reached during certain time intervals of the observation period. within a period of time of seven weeks prior to experimental and controlled inoculation with rv16, each cohort participant was closely monitored, and no symptoms of any respiratory disease were reported during this time interval. nevertheless, our analysis has revealed a time dependency of the discriminative power of all the parameters/biomarkers studied herein. this can be interpreted in two ways as follows: (1) during the phase prior to inoculation, the parameters/biomarkers fluctuate with an amplitude large enough to occasionally reach or nearly reach values characteristic of an infection. (2) the effect elicited by the rv16 infection on these parameters/biomarkers is not strong enough, and thus the values remain within a "normal" range that can be observed during infection-free phases. in the context of discriminating between the non-infected and the infected state, the first interpretation hints at the possibility of finding false positives, whereas the second interpretation indicates that false negatives may result. whilst feno and lung function parameters can be quickly and non-invasively measured using cheap portable devices (e.g., at airports), our results suggest, on the one hand, that these parameters do not qualify as accurate discriminators of rv infection. on the other hand, we found that the concentrations of the various cytokines in nasal lavage fluid may indeed be successfully used for the detection of rv infection. in particular, il-6 ( figure 2 ) and ifn-gamma ( figure s1a ,b in supplementary materials data) are the best discriminating cytokines, despite the natural temporal fluctuations in their concentrations. this may not be surprising, as these cytokines are known to play critical roles in local anti-viral responses [21] . furthermore, the influence of the asthma condition on ifn-gamma's discrimination performance is almost negligible. what is the clinical implication of these data? a nasal lavage is a relatively bothering procedure that needs to be carried out by trained health care workers. moreover, the laboratory assays to measure the concentration of these cytokines are expensive and require proficient staff and laboratory facilities. consequently, we conclude that measuring these cytokines as biomarkers in nasal lavage fluid will be most suitable in a hospital setting. in fact, some cytokines have been shown to be good predictors of disease progression in flu patients [22] . our data analysis strategy of averaging biomarkers over a sliding 10-days window has, for our purposes, an undesirable effect of smoothing the time series data, thereby artificially reducing the true variability of the parameters/biomarkers. in fact, moving average filters are a widely used time series smoothing technique [23] . however, on which days after rhinovirus infection a given patient will display the strongest changes in the parameters/biomarkers measured in our study was highly variable from patient to patient. previous research work has indicated that the strongest changes typically occurred within the first 10 days after infection [18] . therefore, we used the average of a given parameter/biomarker over the first ten days after inoculation as the magnitude that would be representative of the changes occurring upon infection, without having to choose a particular day after infection that may not be suitable for all individuals. consequently, and for the sake of consistency, we also used averages of the parameters/biomarkers over 10-day windows prior to inoculation. in previous work [15] , we explored the potential of the whole parameter/biomarker time series to characterize each participant's state upon inoculation. those findings suggest that a "personalized time-series analysis" would most likely have more power and lead to better discriminatory performance. however, such an approach is challenging from a practical point of view, as patients would need to be constantly monitored, such as weekly, in order to detect a potential infection that no one knows in advance when it will hit. this would constitute a significant burden for most patients or healthy individuals, which discouraged us from pursuing such an approach in the context of rhinovirus infection detection. nevertheless, it is conceivable that, in the future, there could be portable patient telemonitoring technologies that allow for passive patient monitoring, i.e., longitudinal measuring of relevant parameters/biomarkers without the patient's active participation. with such technologies, a personalized time-series analysis approach would indeed become feasible. however, we believe that the development of such technologies is still in early stages. furthermore, and more importantly, we think that the potential ethical issues arising from constantly telemonitoring patients or healthy individuals need to be traded-off against the presumptive health benefits of such technologies. the choice of the age group for our study volunteers (18-35 years) was determined by the fact that, in this study, the participants were inoculated with an active virus. indeed, ethical approval by the hospital board for the inclusion of pediatric or older patients would have been very difficult to obtain. our results may have been influenced by the presence of atopy/allergy in our cohort. however, our study was designed to mimic a real-life setting in which the majority of the asthma patients have atopy as opposed to non-atopic asthma [24] . moreover, some of the cytokines we decided to measure in this study (e.g., ifn-g) are known for their increased secretion during rhinovirus infections and less for their appearance during allergic reactions. furthermore, we selected mediators that would be released at sufficient abundancy in the nasal compartment to enable detection, even in the absence of any respiratory infection, and that have been consistently reported in the field [25, 26] . although our study has an important clinical and epidemiological message, rhinovirus infections represent a milder form of respiratory infection as compared with other more virulent pathogens. hence, the conclusions derived from this study can only serve as potential reference for other viruses infecting the human respiratory system. moreover, the sample size of our study is relatively small owing to the number of sampling visits every volunteer was subjected to. however, this shortcoming was compensated for by the unprecedented exceptionally high sampling frequency of parameters/biomarkers. the following are available online at http://www.mdpi.com/1999-4915/12/10/1175/s1. figures s1 to s20: discrimination performance (panel a: p-value plot; panel b: auc plot) of biomarker/parameter (respectively) ifn-gamma, il-10, il-13, il-8, tnf-alpha, ip-10, il-1β, il-17a, il-33, percentage of eosinophils, percentage of neutrophils, cell density, normalized morning fev1, normalized morning fev1/fvc, normalized morning fvc, morning pef (% predicted), normalized evening fev1, normalized evening fev1/fvc, normalized evening fvc, and evening pef (% predicted). table s1 : summary of windowed averages for ifn-gamma. table s2 : summary of windowed averages for il-10. table s3 : summary of windowed averages for il-13. table s4 : summary of windowed averages for il-8. table s5 : summary of windowed averages for tnf-alpha. table s6 : summary of windowed averages for ip-10. table s7 : summary of windowed averages for il-1β. table s8 : summary of windowed averages for il-17a. table s9 : summary of windowed averages for il-33. table s10 : summary of windowed averages for percentage of eosinophils. table s11 : summary of windowed averages for percentage of neutrophils. table s12 : summary of windowed averages for cell density. table s13 : summary of windowed averages for normalized morning fev1. table s14 : summary of windowed averages for normalized morning fev1/fvc. table s15 : summary of windowed averages for normalized morning fvc. table s16 : summary of windowed averages for morning pef (% predicted). table s17 : summary of windowed averages for normalized evening fev1. table s18 : summary of windowed averages for normalized evening fev1/fvc. table s19 : summary of windowed averages for normalized evening fvc. table s20 : summary of windowed averages for evening pef (% predicted). table s21 : summary of windowed averages for il-6. table s22 : summary of windowed averages for feno. table s23 : symptoms from viral challenge and antibody detection. furthermore, a copy of the study questionnaire filled out for each participant at each visit. for every parameter/biomarker, each cohort participant's pre-challenge time series of windowed averages can be used by researchers as reference values to compare to values measured on their own patients infected with similar/different viruses known to affect the respiratory system. these time series can be made available upon request. detection of respiratory viruses by molecular methods clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study chest radiological findings of influenza a h1n1 pneumonia laboratory diagnosis of viral infection molecular imaging of influenza and other emerging respiratory viral infections identification of micrornas in throat swab as the biomarkers for diagnosis of influenza reliable detection of respiratory syncytial virus infection in children for adequate hospital infection control management improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-pcr assay false negative tests for sars-cov-2 infection-challenges and implications reverse transcriptase pcr diagnostic assay for the coronavirus associated with severe acute respiratory syndrome evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus laboratory diagnosis of respiratory tract infections in children-the state of the art cytokines in the respiratory airway as biomarkers of severity and prognosis for respiratory syncytial virus infection: an update loss of adaptive capacity in asthmatic patients revealed by biomarker fluctuation dynamics after rhinovirus challenge systemic tryptophan and kynurenine catabolite levels relate to severity of rhinovirus-induced asthma exacerbation: a prospective study with a parallel-group design new possible biomarkers for diagnosis of infections and diagnostic distinction between bacterial and viral infections in children lower airway rhinovirus burden and the seasonal risk of asthma exacerbation receiver-operating characteristic (roc) plots: a fundamental evaluation tool in clinical medicine measures of diagnostic accuracy: basic definitions host immune responses to rhinovirus: mechanisms in asthma the association between serum biomarkers and disease outcome in influenza a(h1n1)pdm09 virus infection: results of two international observational cohort studies digital signal processing: a practical guide for engineers and scientists upper airway·1: allergic rhinitis and asthma: united disease through epithelial cells cxc chemokines and antimicrobial peptides in rhinovirus-induced experimental asthma exacerbations nasal cytokine responses to natural colds in asthmatic children we would like to acknowledge all the participants who volunteered to participate in our study along with all master students, research technicians, and study nurses who helped us with parts of the project. the authors do not have any commercial interests or associations to disclose that could result in a conflict of interest. key: cord-022084-hap7flng authors: arruda, eurico; cintra, otavio a.l.; hayden, frederick g. title: respiratory tract viral infections date: 2009-05-15 journal: tropical infectious diseases doi: 10.1016/b978-0-443-06668-9.50064-8 sha: doc_id: 22084 cord_uid: hap7flng nan acute respiratory infections (aris) are prevalent worldwide 1 and rival diarrhea as the leading cause of death in developing countries. 1, 2 in some impoverished urban populations in south america, ari symptoms may be present on an almost continuous basis, making it difficult to determine symptom-free days and estimate attack rates. 3, 4 children from these areas may spend 40% to 75% of their time with respiratory symptoms, 1,5 mostly caused by upper respiratory infections (uris). the most striking disparity between developing and developed countries with regard to ari epidemiology is the case-fatality rate of lower respiratory infection (lri), mainly pneumonia, bronchiolitis, and influenza, 6, 7 in children under 5 years of age, which may reach 16% in some areas. 1, 8 several community-based studies have established the importance of common respiratory viral infections in tropical countries 5 (table 59-1) . in impoverished populations, these common viral infections may occur simultaneously with measles, diarrhea, and malnutrition, resulting in complex interactions of pathologic conditions that carry the potential to become life-threatening diseases. 1, 9 unlike certain pathogens restricted to tropical areas, the respiratory viruses have worldwide distribution, efficient person-to-person transmission, and an impact on all age groups. except for a few agents (e.g., adenoviruses, severe acute respiratory syndrome [sars] coronavirus) and rare cases of extrapulmonary dissemination with other respiratory viruses, replication is generally restricted to the respiratory mucosa of humans. in most health-care facility-based studies of acute lri (alri) conducted in tropical countries (table 59 -2), respiratory syncytial virus (rsv) is the virus most frequently detected (11% to 33%), followed by parainfluenza viruses (1% to 13%), adenoviruses (2% to 34%), and influenza viruses (1% to 4%). with few exceptions, human rhinovirus (hrv) and human coronaviruses (hcov) have not been reported frequently in studies in tropical countries, 5 probably because of difficulties in their detection. although previous studies have shown that attending daycare centers can be a risk for ari, 10 providing day care for children has become an important economic issue in developing countries, where mothers must join the workforce to contribute to the family income. consistent with studies in the united states and elsewhere, a study found a high burden of ari in young, low-income children attending day care in salvador, brazil. 11 few specific interventions are available to reduce the impact of respiratory viruses, 2 and the application of these interventions may be further hampered by epidemiologic patterns in ari and socioeconomic differences in temperate, developed countries compared with equatorial regions. for example, housing conditions and crowding pose challenges for optimizing health-care strategies in the tropics. while the incidences of some respiratory viruses, particularly rsv and influenza, show seasonal trends in some tropical areas, the association of seasonal peaks of respiratory viruses in general may be less apparent where fluctuations in temperature are smaller. 12 nutritional and educational interventions, such as reinforcing breast-feeding, 13 vitamin a supplementation for measles, 14 and facilitation of access to oral rehydration therapy, 15 may have significant effect on the morbidity and mortality due to lri alone or in association with diarrhea. in this chapter we focus attention on the most common viral respiratory infections, whose main features are summarized in table 59 -3, and try to highlight features unique to the developing world. in tropical countries influenza activity may occur yearround as well as in outbreaks more typical of temperate regions. these infections cause serious disease in populations weakened by malnutrition, with limited access to medical care. 16 of note, the predisposition induced by influenza to superimposed bacterial infections, mainly streptococcus pneumoniae, may greatly affect morbidity and mortality, mainly among impoverished populations. 17 in addition, influenza viruses can reassort or sometimes cross species barriers to generate emergent strains that may cause localized outbreaks or potentially pandemics with enormous impact for health on a global scale. 18 influenza viruses are pleomorphic, enveloped, with segmented negative-strand rna genomes and belong to the family orthomyxoviridae. influenza viruses are distributed in three genera-a, b, and c-based on the antigenicity of the nucleoprotein (np) and matrix protein. influenza a virus is further classified in subtypes based on its two surface glycoproteins: hemagglutinin (ha) and neuraminidase (na). 19 among the 15 ha and 9 na subtypes recognized in nature, 6 ha (h1, h2, h3, h5, h7, and h9) and three na (n1, n2, and n7) subtypes have now been identified in human isolates of influenza a viruses. 18 however, only three subtypes of ha (h1, h2, and h3) and two of na (n1 and n2) have caused pandemics and sustained circulation in human populations in recent years. 20 the genomes of influenza viruses contain eight rna segments in influenza a and b viruses, and seven rna segments in influenza c. 19 the glycoprotein ha is responsible for the attachment of the virus to sialic acid-containing cell receptors, and it mediates fusion and penetration. proteolytic cleavage of ha by cellular serine proteases exposes hydrophobic fusion domains that mediate membrane fusion. the na cleaves terminal sialic acid from glycoconjugates present on respiratory mucins, cells, and progeny virions. this action destroys receptors recognized by ha and allows budding virus to be released from infected cells and to spread within the respiratory tract. influenza c virus contains a single surface glycoprotein that binds to receptor, promotes fusion of membranes, and also cleaves sialic acid. 19 virus binding to receptor is followed by internalization into endosomes, fusion of viral and endosomal membranes, and release of the genome to the cytoplasm, from where it is transported to the nucleus. in influenza a viruses, the m2 protein serves an ion channel function that facilitates dissociation of the rna segments from the virion interior. transcription of the negative-strand genomic rna into positive-strand messenger rna (mrna) and complementary rna (crna) is mediated by a viral rna polymerase complex in the nucleus. crna serves as a template for the synthesis of negative-strand virion rna genome segments, and mrna directs viral protein synthesis. newly assembled nucleocapsids acquire an envelope as they bud through the cell surface. only viruses with a full complement of genome segments are infectious. 19 influenza a viruses are primarily viruses of aquatic birds, particularly ducks and shore birds, which harbor all subtypes recognized to date. selected subtypes naturally infect a range of terrestrial (swine, horses, humans) and aquatic (seals) mammals; influenza b virus infects humans and uncommonly seals, dogs, cats, and swine, and influenza c virus is primarily a virus of humans. depending on the virus type and subtype, experimental infection can be induced in mice, ferrets, chickens, swine, and primates, and the viruses can be propagated in primary cultures of kidney cells, continuous cell lines (mdck, vero, per.c6, and llc-mk2), and also in embryonated hen' s eggs. 20 the biologic property of influenza viruses to bind erythrocytes is exploited for early detection of the virus in cell culture and for the development of serologic assays by hemagglutination inhibition. 20 influenza viruses are inactivated by temperatures above 50°c and by lipid solvents, acid, formaldehyde, ionizing radiation, and ultraviolet (uv) light. 20 influenza viruses occur throughout the world, causing highly contagious respiratory infections with high morbidity and excess mortality (in seasonal peaks), particularly in infants and the elderly. in developing tropical countries, influenza has been associated with an average of 5% of aris leading to physician contact. 6, 21 this apparently low proportion probably represents only the most severe cases, since 30% to 50% of children under 5 years of age in tropical africa have been found to seroconvert in one outbreak. 21 previously healthy children younger than 1 year of age are hospitalized for influenza at rates similar to those for adults at high risk for influenza, and influenza accounts for a great number of outpatient visits and courses of antibiotics in children of all ages. 22 when human influenza virus is introduced into a malnourished population with limited access to care, high morbidity and mortality rates can occur, as was observed in madagascar in 2001 where a conventional influenza a/h3n2 subtype virus was associated with case-fatality rates of approximately 3%. contrary to the remarkably sharp seasonality of influenza a outbreaks in temperate countries, seasonal patterns in tropical countries have varied between studies. in southern india 23 and thailand 24 influenza has occurred throughout the year with sporadic outbreaks, whereas there have been consistent outbreaks in june-july and november-january, coinciding with the winter seasons in the southern and northern hemispheres, but with no apparent association with meteorologic factors. 12 in the philippines influenza has been more frequent between november and january, 25 while in senegal, nigeria, and taiwan there has been clear association with increased rainfall. 12 in southeastern brazil, 26 argentina, 27 as well as in south africa, 28 seasonal outbreaks of influenza a have occurred annually from may through august (mid autumn through winter) in association with cooler temperatures but not with rainfall. influenza b outbreaks occur periodically, yet less frequently than influenza a, in both temperate and tropical regions, 12, 20 whereas influenza c is generally nonseasonal. 20 minor changes in antigenicity, called antigenic drift, are caused by accumulation of point mutations in the genes coding for influenza ha and na, generating new strains that spread in annual epidemics. influenza viruses b and c are less prone to antigenic drift. major antigenic changes in influenza a are called antigenic shift and result in the emergence of a novel ha subtype with or without new na to which humans lack significant immunity. 18 this may be caused by the acquisition of new gene segments through genetic reassortment in a host infected simultaneously by a human and an animal (typically avian) virus, or by reappearance of a subtype from reservoir. swine are susceptible to both avian and human influenza viruses and may be hosts for reassortment or serve as the mammalian species in which avian viruses can adapt. novel influenza virus subtypes generated by shift have caused catastrophic pandemics, including three in the last century. the h1n1 "spanish flu" pandemic of 1918 is estimated to have caused up to 100 million deaths worldwide, 18 while the h2n2 "asian flu" in 1957 and the "hong kong flu" in 1958 caused an estimated 1 to 3 million deaths. recent clusters of human infections due to avian influenza, particularly h5n1 subtype viruses in asia, have raised concerns about new pandemic threats. in 1997 a highly pathogenic avian influenza h5n1 virus resulting from reassortment among several avian viruses caused lethal outbreaks in domestic poultry and severe illness with 6 deaths among 18 human cases in hong kong. the outbreak was due to exposure to infected poultry in live poultry markets and was later contained by their slaughter. this virus was transmitted inefficiently from person to person. 18, 20 in february 2003 an h5n1 virus caused deaths in a family visiting fujian province in china. since late 2003 wide-scale poultry outbreaks due to h5n1 virus have occurred in at least 10 asian countries. interspecies transmission to humans occurred in at least two countries, causing over 45 cases and 32 deaths in vietnam and thailand. 29 these h5n1 viruses have continued to reassort, evolve antigenically, and extend their host range with documented infections in swine and felids. prolonged nonsymptomatic excretion in ducks and detection in migratory birds indicate that this virus has become endemic in southeast asia. 18, 30 an avian h9n2 virus with human receptor specificity has also spread throughout asia in domestic poultry and pigs and caused mild disease in humans in hong kong and china. 18 an avian h7n7 virus caused conjunctivitis in at least 82 people and one fatal pneumonia in the netherlands in 2003; the outbreak was contained by culling and quarantine of poultry. in february 2004 a highly pathogenic h7n3 virus emerged in domestic poultry in british columbia with two documented human cases of conjunctivitis and mild "flulike" illness. 18, 29 influenza virus is transmitted from person to person by large droplets and small-particle aerosols, as well as possibly by fomites with hand contamination and subsequent selfinoculation. the relative importance of these routes is uncertain for natural influenza. ingestion of infected birds has led to infection by avian viruses in cats. secondary attack rates may reach over 70% in semiclosed populations, especially among schoolchildren and patients debilitated by underlying conditions who live in relative confinement, such as nursing home residents. 20 children play a major role in influenza outbreaks with respect to propagation of the epidemic virus in families and communities. 20 classic influenza starts abruptly after an incubation period of 1 to 2 days, with fever, chills, malaise, headache, myalgia, and prostration, often accompanied by nonproductive cough, sore throat, and mild rhinorrhea. systemic complaints last 3 to 5 days, whereas sore throat, hoarseness, and cough, with substernal discomfort, may increase in severity as the systemic symptoms subside. cough and asthenia often persist for 2 weeks or longer. respiratory symptoms may be minimal or absent initially, especially in the elderly or infants. in frail elderly persons, lassitude, lethargy, confusion, lowgrade fever, and sometimes gastrointestinal complaints may be the primary findings. influenza b tends to be milder than influenza a, and influenza c typically causes colds or bronchitis. 20 influenza may also present as unexplained fever, croup (laryngotracheobronchitis), vomiting, diarrhea, and neurologic manifestations in young children. 31 up to 50% of influenza virus infections in adults are subclinical. 20 influenza causes a variety of viral respiratory complications, including otitis media, sinusitis, tracheobronchitis, pneumonia, and, in young children, bronchiolitis and croup. secondary bacterial infections, especially pneumonia caused by staphylococcus aureus, streptococcus pneumoniae, and haemophilus influenzae, are common complications and should be suspected in relapses of fever, chest pain, and cough. 20 influenza is also associated with invasive meningococcal infections. other complications include exacerbations of asthma, chronic bronchitis, and congestive heart failure. myositis, myoglobinuric renal failure, meningoencephalitis, transverse myelitis, polyneuritis, parotitis, myocarditis, arthritis, and disseminated intravascular coagulation rarely occur after influenza. reye' s syndrome occurs in fewer than 1 per 100,000 cases of influenza in patients under 18 years of age following the use of salicylates. pregnant women, human immunodeficiency virus (hiv)-infected patients, and other immunocompromised hosts are at higher risk for severe disease and complications. 20 the virus infects the respiratory mucosa, where it causes lytic infection of cells and desquamation of the respiratory epithelium, mononuclear cell infiltrates in the lamina propria, and altered mucociliary clearance. tracheobronchitis is a typical feature and often associated with prolonged abnormalities in small airway pulmonary function and airway hyperreactivity. primary influenza viral pneumonia results in diffuse alveolar damage, alveolar hemorrhage and exudate, hyaline membranes, and later reactive fibrosis. fatal cases show pathologic changes in nonrespiratory organs, such as brain congestion and swelling, myocardial inflammation, and fibrinoid changes in arterioles. 20 viral replication in the upper respiratory tract generally peaks within 1 or 2 days of symptom onset and, depending on age and prior immunologic experience, continues for about 3 to 8 days. the severity of illness broadly correlates with upper respiratory tract viral levels. constitutional symptoms with influenza are due in part to the release of proinflammatory cytokines and chemokines. levels of interferon (ifn-α and ifn-γ), tumor necrosis factor (tnf-α), interleukins and chemokines (il-1β, il-6, il-8, il-10, mcp-10, mip-1α and mip-1β) are increased in nasal secretions, and ifn, il-6, and tnf-α are increased in blood in human influenza. 20 the tissue tropism of a strain of influenza virus depends, among other factors, on a combination of susceptibility of its ha to be cleaved by, and tissue availability of proteases with specificity to cleave it, thus rendering the virus infectious. 32 extrapulmonary dissemination of virus has been uncommonly documented in humans, but systemic spread is a regular feature of highly pathogenic avian viruses in chickens and sometimes in rodents or other mammalian hosts. serum and secretory antibodies directed to ha and na appear about 10 days after infection. protection against reinfection by the homologous strain is durable following natural infection and is correlated with serum and nasal neutralizing antibody levels, principally directed against ha. vaccine-induced protection may last for up to 2 to 3 years against homotypic virus. infection also induces cell-mediated immunity, which is detectable 3 to 6 days after infection and seems to be important for recovery. 30 cytotoxic t-lymphocyte responses against internal proteins may provide some degree of heterosubtypic immunity. the diagnosis of influenza is frequently made on the basis of clinical and epidemiologic information. a higher index of suspicion and laboratory diagnostics is needed outside the season, particularly in sporadic individual cases or unexplained outbreaks of febrile respiratory illness. viral isolation from respiratory specimens can be done in several types of cells (e.g., prmk, mdck, llc-mk2) and remains the current standard. 20 the presence of virus may be detected in cell cultures by hemadsorption with guinea pig erythrocytes before or after cytopathic effect (cpe) is visible. blind hemadsorption is positive 3 days after inoculation in almost all positive samples. 20 confirmation of isolates can be done by hemagglutination inhibition or immunofluorescence with typespecific antisera. diagnosis can also be made in 1 to 2 days by immunofluorescence of monolayers of mdck cells inoculated by centrifugation (shell-vial). 33 conserved influenza antigens (m or np) directly in clinical samples can be detected by one of several techniques (e.g., immunofluorescence [if], enzyme immunoassay [eia]), and multiple point-of-care kits are commercially available with turnaround time of 15 to 30 minutes. one commercial assay is based on detection of influenzaspecific na activity. the sensitivities of these assays are higher in children (up to 90%) than in adults (generally 50% to 70%) and depend on duration of illness and sample type. 20 several formats of reverse transcription-polymerase chain reaction (rt-pcr) assays have been used for the detection of influenza a and b rnas in clinical samples, with the advantage of detecting genomes of noninfectious virus. 20 the time to perform rt-pcr is longer but the cost may be lower than for commercial rapid antigen detection kits, especially in developing countries. real-time rt-pcr has enabled the development of assays that provide rapid quantitative detection of influenza a and b with high sensitivity. 34, 35 these assays have great potential to replace other methods, because they are simultaneously rapid, highly sensitive, quantitative, and amenable to being used in multiplex format, which might include probes for several different respiratory pathogens. 35 however, the costs are still prohibitive for most laboratories in developing nations. serologic diagnosis of influenza using paired acute and convalescent serum can be done retrospectively by a variety of techniques but mainly for serologic survey purposes. 20 amantadine and rimantadine are m2 ion channel blockers that inhibit influenza a virus replication at the uncoating step. 20 in uncomplicated influenza a in adults without underlying diseases, treatment with either drug can reduce the duration of influenza illness by approximately 1 to 2 days if started early, within 48 hours from the onset of symptoms. amantadine is excreted in an unchanged state in the urine, while rimantadine is extensively metabolized after absorption and less than 10% of the dose is excreted unchanged in the urine. elderly persons need only half the dose to achieve similar plasma levels. amantadine or rimantadine may cause gastrointestinal upset and central nervous system side effects. central nervous system (cns) intolerance is more common with amantadine and, when severe, can be manifested as agitation, psychosis, seizures, and coma. mild complaints including insomnia, dizziness, anxiety, dry mouth, anorexia, and nausea are reversible upon discontinuation. amantadine and rimantadine are marketed as 100-mg tablets and 10-mg/ml syrup. the recommended dose is 100 mg twice daily for adults older than 65 years of age (100 mg/day for patients ≥65 years of age). for children under age 10 years, a rimantadine dose of 5 mg/kg/day (maximum, 150 mg/day) has been suggested. 20 dose reductions proportional to the creatinine clearance (clcr) are suggested for patients with renal insufficiency (amantadine for clcr <60 to 80 ml/min/1.73 m 2 ; rimantadine for clcr <10 to 20 ml/min/1.73 m 2 ). influenza virus resistant to amantadine-rimantadine emerges in approximately one third of treated patients; such viruses are transmissible to close contacts and cause typical influenza illness. resistance to these drugs renders them ineffective and is sometimes present naturally, including in recent human isolates of h5n1 virus. 20 the neuraminidase inhibitors zanamivir and oseltamivir inhibit both influenza a and b viruses by blocking the active site of the enzyme for cleavage of sialic acid, thus inhibiting virus release from infected cells and spread within the respiratory tract. 36 in adults and children older than 5 years, inhaled zanamivir (10 mg twice daily for 5 days) provides 1to 2.5-day reduction in illness 37 and reduces antibiotic use for lower respiratory complications by 40%. zanamivir is generally well tolerated but may uncommonly induce bronchospasm, particularly in those with influenza and pre-existing airway disease. 20 oseltamivir (75 mg orally twice daily for 5 days) reduces illness severity, time to resumption of daily activities by 1 to 3 days, and rates of complications leading to antibiotic prescription and hospitalization by about 50% in adults. in children 1 to 12 years of age, oseltamivir reduces the frequency of otitis media and, consequently, antibiotic prescriptions. side effects include mild-to-moderate nausea or emesis. dosage of neuraminidase inhibitors does not need to be adjusted for the elderly. 20 resistance emergence is uncommon with both drugs, 20 although a recent study of children treated with oseltamivir detected drug-resistant viruses in 18%, often in association with prolonged viral excretion, and showed that children can be a source of viral transmission, even after 5 days of treatment. 38 antipyretic-analgesic drugs may be used for influenzainduced fever and aches. aspirin should be avoided because of its association with reye' s syndrome. immunization with formalin-inactivated or live-attenuated multivalent influenza virus vaccines and chemoprophylaxis for influenza virus a are the methods available for preventing influenza. influenza vaccine is used prior to the influenza season and currently includes one strain of influenza b and two strains of subtypes h3n2 and h1n1 of influenza a virus, chosen by the world health organization (who) surveillance network among the viruses most likely to circulate in the next influenza season. 20, 39 the inactivated vaccine has an approximate 70% to 90% efficacy in preventing illness in healthy children and adults. 39 it also reduces influenza-related hospitalizations and mortality in elderly and high-risk patients. the centers for disease control and prevention (cdc) recommends the immunization of persons aged 50 years and older; residents of nursing homes; children and adults with chronic cardiovascular or pulmonary disease, including asthma; persons chronically ill with diabetes mellitus, renal dysfunction, or hemoglobinopathies; immunosuppressed patients including those with hiv infection; children and adolescents on chronic aspirin therapy who may develop postinfluenza reye' s syndrome; women who will be pregnant during the influenza season; children aged 6 to 23 months; those who can transmit influenza to persons at high risk, such as health-care workers and household contacts of those at high risk including children 0 to 23 months of age; crew members of cruise ships; providers of essential services; and unimmunized travelers to areas where influenza may be circulating, including the tropics, the southern hemisphere between april and september, and those traveling in large organized tourist groups. in addition, vaccine is made available to anyone interested in reducing the likelihood of becoming ill with influenza. 20, 39 the inactivated vaccine, administered as a single intramuscular (im) dose shortly before influenza season (two doses in previously unimmunized children <9 years of age), is safe during pregnancy but should be avoided in persons with history of anaphylactic reactions to eggs. 39 vaccine safety and efficacy in children has been extensively evaluated and has shown a favorable safety profile with efficacy in 1-to 15-year-old children of 77% to 91%. inactivated vaccine is not currently recommended for children younger than 6 months, but vaccination of household contacts and caregivers should reduce the risk of these high-risk children contracting influenza. healthy people aged 5 to 49 years who are not contacts of immunosuppressed patients can receive either inactivated or intranasal live-attenuated vaccines. 39 influenza inactivated vaccine has been recently introduced in many tropical areas of the world, with a composition based on influenza viruses circulating in the southern hemisphere. the vaccine is given prior to the influenza season, which for most countries in the southern hemisphere is between may and july. 40 in south america, annual vaccination of the elderly has reduced hospitalizations and mortality for respiratory diseases. 41 continuous surveillance has already shown that regional variations of circulating influenza virus strains should be taken in consideration in the formulation of influenza vaccines with compositions more appropriate for south america. 42 live-attenuated, cold-adapted vaccines administered intranasally are well tolerated, genetically stable, and rarely transmissible and have the advantage of inducing local secretory immunoglobulin a (iga) responses. because of potential interference between components, two doses may be required in young children. 20 this vaccine was licensed in the united states in 2003, where it has become an option for healthy persons aged 5 to 49 years, including those in close contact with groups at high risk and those wanting to avoid influenza. 39 this vaccine is not recommended for persons with asthma and other chronic disorders of the pulmonary or cardiovascular systems; persons with underlying medical conditions, including diabetes, renal dysfunction, and hemoglobinopathies; persons with known or suspected immunodeficiency diseases or who are receiving immunosuppressive therapies; children or adolescents receiving aspirin or other salicylates; persons with a history of guillain-barré syndrome; pregnant women; and persons with a history of hypersensitivity to eggs. 39 cold-adapted trivalent influenza vaccine is highly effective (92% in phase 3 studies) in preventing cultureconfirmed influenza in healthy children and has provided protection against drift variant strains in some studies. in young and middle-aged adults, efficacy is generally comparable to that of inactivated vaccine. 39 other investigational approaches have been explored in influenza vaccine development, including recombinant ha produced in insect cells, virosomes incorporating surface glycoproteins, m2 protein conjugated with hepatitis b virus core, and naked dna encoding influenza virus nucleoprotein or ha. 20 cell culture-based vaccines (mdck, vero) have been approved in europe and may offer an alternative to the limitations of the current egg-grown vaccines. the technique of reverse genetics has been used to rapidly produce candidate vaccines against potential pandemic threat viruses. amantadine and rimantadine are approved for use, and are 70% to 90% effective in the prophylaxis of influenza a during outbreaks. unvaccinated elderly persons, immunodeficient patients, patients in chronic care institutions experiencing outbreaks, persons who could not be vaccinated, and those who received a vaccine strain different from the outbreak strain may receive prophylaxis with amantadine or rimantadine. prophylaxis should be started as early as possible at doses equivalent to those used for therapy, and continued until 1 week after the end of the outbreak for a total of at least 2 weeks. 39 amantadine-and rimantadine-resistant mutants of influenza a virus occur in up to 30% of treated patients and may be associated with failure of drug prophylaxis. 43 both oseltamivir (75 mg twice daily) and inhaled zanamivir (10 mg/dose twice daily) are more than 80% effective in the prophylaxis of influenza during outbreaks, but only oseltamivir has been approved for this indication in the united states. 20, 39 antiviral agents, especially the neuraminidase inhibitors, could significantly help in the control of a future influenza pandemic by reducing lower respiratory complications and hospitalizations as well as potentially person-to-person transmission. however, supply limitations 44 pose a real difficulty. therefore, policies to ensure a reasonable supply of these drugs, as well as directions to optimize the use of limited supplies, are important issues to be considered. 18 respiratory syncytial virus (rsv) is the single most important viral cause of lower respiratory disease and a major cause of morbidity and mortality in children worldwide. rsv is the leading cause of hospitalization in young children in developed and developing countries. 45 in tropical areas, rsv has been the most frequently isolated virus in hospital-based ari studies of children. 5 agent rsv, the only known human pathogen of the genus pneumovirus in the family paramyxoviridae, is a pleomorphic rna virus with helical nucleocapsid and lipid-containing envelope. antigenic differences in the surface glycoprotein g permit the classification of rsv into groups a and b, each with antigenic subgroups. 46 the interaction of rsv envelope glycoprotein g with glycosaminoglycans enables adherence to the cell surface. however, g protein-independent mechanisms of attachment must exist, since mutants devoid of g protein can also enter host cells. rsv enters the cell by fusion of viral envelope with cell membranes, a process mediated by binding of the viral f protein to the cell gtpase rhoa. 46 the syncytia resulting from fusion of the infected cells to adjacent ones are the major feature of the cytopathic effect of paramyxoviruses. once in the cytoplasm, the negative-strand rna is transcribed by viral transcriptase into mrnas, which then direct viral protein synthesis. an intermediate positivestrand full-length crna serves as a template for the synthesis of progeny negative-strand rna. as they bud through the cell membrane, the virions acquire a glycoprotein-containing envelope. 46 rsv causes asymptomatic infection in a variety of experimental animals, but natural infection occurs only in humans and chimpanzees. 47 rsv grows well in several human heteroploid cell lines, such as hep-2, hela, and a549, and is sensitive to ether, chloroform, detergents, and a ph less than 5. rsv is inactivated at 55°c, survives poorly on porous surfaces, and loses infectivity significantly by slow freezing and storage at temperatures above 4°c. 46 rsv occurs worldwide and causes annual outbreaks in temperate climates in the winter and early spring, with sporadic cases throughout the year. 47 in tropical regions, where temperature fluctuations are smaller and the only significant seasonal variable is often rainfall, rsv outbreaks tend to occur in the rainy seasons. such has been the case in malaysia, hong kong, india, papua new guinea, colombia, kenya, and the gambia. 12 interestingly, in singapore rsv peak activity occurs from march to august, a period of higher temperature, higher day-to-day temperature variation, and lower relative humidity. 48 in southeast brazil, rsv occurs seasonally, within a broader range of months from february through july, after the rainy season and when temperatures tend to be cooler, with slight variations from year to year. 49 in regions where average winter temperatures are colder, such as in são paulo city and the southernmost parts of brazil, as well as in argentina, rsv peak activity tends to occur in july and august. [50] [51] [52] most children have specific serum rsv antibody by age 2 years, but reinfections occur throughout life. more than one subtype of either rsv group may cocirculate in one season, with group predominance changing from year to year, without apparent correlation with clinical or epidemiologic characteristics of the illness they cause. 49,53 rsv transmission requires close contact and occurs either by large-particle aerosols or by contamination of hands and inoculation into the eye or nose. secondary infections in family contacts of an index case are common, after an average incubation period ranging from 2 to 8 days. 45 it is estimated that 30% of all infants will have rsv infection that requires medical attention and that 2% of them will be hospitalized. 45 an estimated 10% of children will have bronchiolitis in their first year of life, with 60% to 90% of those infections caused by rsv. 54 in southeast brazil, rsv is the leading cause of lower respiratory tract infections in children younger than 1 year of age and is responsible for up to 85% of hospitalizations in this age group during peak months. 52 the spectrum of illnesses caused by rsv ranges from mild uri to severe lri, including pneumonia, bronchiolitis, tracheobronchitis, and croup. 45 in infants and young children, uri with fever and otitis media is common. during outbreaks, rsv rna has been detected in up to 75% of middle ear effusions in children with rsv infection and acute otitis media. 55 the most frequent lri caused by rsv in infants is bronchiolitis, usually preceded by 2 to 3 days of uri symptoms, and progressing to lower respiratory tract involvement characterized by tachypnea, dyspnea, cough, expiratory wheezing, air trapping, and in more severe cases, intercostal muscle retractions and cyanosis. fever is present in only 50% of infants. chest radiographs may show hyperaeration of the lungs and sometimes segmented atelectasis. 45 blood counts usually show lymphocytosis, and an increase in neutrophils with a left shift could be associated with bacterial superinfection. the most frequent bacterial superinfection in children with rsv infections is acute otitis media, which may be found in up to 60% of children with brochiolitis. 56 however, more serious bacterial infections that may require sepsis work-up is uncommon in previously healthy infants with rsv infections. 57 this may be different, however, in developing tropical areas, where rsv frequently causes infections in children previously debilitated by other diseases and malnutrition. infants with congenital heart disease, premature infants, or infants with underlying pulmonary conditions, such as cystic fibrosis and bronchopulmonary dysplasia, as well as immunocompromised hosts of any age, are at risk for severe and fatal rsv infections. hiv-infected children with rsv infections have a higher rate of pneumonia and prolonged illness and virus shedding, but the general severity of the rsv disease is not increased. 45 differential diagnosis of acute bronchiolitis includes asthma, pneumonia, congenital heart and lung diseases, and cystic fibrosis. particular clinical signs are generally not accurate predictors of specific viral causes, but in a study conducted in the philippines, wheezing was a significant predictor of viral lri, while manifestations of higher severity, such as chest indrawing and cyanosis, were more often associated with bacterial lri. 58 the most frequent rsv illness in children over 3 years of age and adults is uri with coryza and cough, sore throat, and hoarseness, often accompanied by low-grade fever. exacerbations of chronic pulmonary diseases and wheezing can also be seen in adults with rsv infection. 45 the role of rsv infections in causing wheezing and asthma exacerbations in infants is well established in studies conducted in temperate areas. 59 similar observations have been made in an emergency room study conducted in southeast brazil, which found that infection with respiratory viruses, especially rsv, and a family history of allergy were independently associated with wheezing. 60 similar findings have been observed in urban nigerian preschool children. 61 rsv has been increasingly recognized as a cause of lri in the elderly, mainly characterized by interstitial pneumonia, prolonged cough, and dyspnea in persons with chronic pulmonary conditions, and it should be considered in the differential diagnosis of flulike illnesses. 62 rsv replicates in respiratory epithelium to reach titers as high as 10 6 tcid 50 /ml in nasal secretions of infected babies, and virus shedding may be as prolonged as 3 weeks after the symptoms disappear. 46 rsv spreads from cell to cell and may involve the entire respiratory tree, reaching bronchioles in 1 to 3 days after the onset of rhinorrhea. replication in the bronchiolar epithelium causes necrosis of ciliated cells, syncytia formation, peribronchiolar inflammation with abundant lymphocytes and macrophages, and impairment of secretion clearance, resulting in small airway obstruction and the hyperinflation characteristic of bronchiolitis. pneumonia frequently coexists, evidenced by interstitial mononuclear infiltrate, eosinophilic cytoplasmic inclusions in epithelial cells, and multinucleated giant cells. the most severe rsv disease occurs in young babies, whose immature airways may be unable to compensate for the pathologic changes. 46 naturally acquired immunity to rsv is incomplete and short-lived, but the severity of illness tends to decrease with reinfections. local secretory iga correlates better with protection than does serum antibody level and age, and pre-existing virus-specific maternal antibodies influence the development of neutralizing antibodies. cell-mediated immune response is central to recovery from rsv infection, and patients with suppressed cell-mediated immune response are at risk of severe rsv pulmonary disease and fatal outcome. 45, 46 the type of immune response to the virus is probably a major factor in the development of wheezing and asthma exacerbations. a bias toward a th2 cytokine response seems to be associated with more severe disease, whereas a th1 response leads to effective viral clearance and milder illness. the virus itself generally triggers a th1 response, but a preexisting th1 deficiency may be associated with disease severity in some children. it has been suggested that rsv bronchiolitis may be a marker of predisposition to wheezing or asthma later in life. 46, 63 children vaccinated with a formalin-inactivated rsv vaccine developed in the 1960s had severe disease when exposed to natural infection, apparently as a consequence of an imbalance between protective and immunopathologic t-cell responses elicited by previous parenteral immunization with inactivated rsv. this would favor a cd4+ th2 cytokine pattern in response to subsequent rsv infections, whereas a previous natural infection would favor a cd4+ th1 pattern in response to reinfection. 64 nasopharyngeal aspirates or swabs, nasal washings, and lower respiratory samples are all appropriate specimens for rsv isolation. this is usually accomplished in cultures of hep-2 cell line, in which rsv induces syncytia in 3 to 5 days. rsv antigen detection by eia, including membrane-based eia, is sensitive and specific and requires virtually no equipment, making it ideal for field studies. rapid rsv detection by if of exfoliated respiratory cells may be even more sensitive than eia-based methods. 45, 46 the increasing use of rapid tests has facilitated the assessment of rsv in tropical areas. 1 ideally, a combination of a rapid method with viral isolation should be used for maximal rsv detection, but the cost may still be prohibitive for the meager resources available in some tropical areas. detection of rsv rna by conventional rt-pcr has shown suboptimal sensitivity, especially when compared with easy-to-perform, more sensitive rapid methods. 45 however, more recently developed assays based on real-time rt-pcr are proving to be more sensitive than conventional rt-pcr assays, with the added conveniences of being rapid, quantitative, and amenable to simultaneous detection and subtyping of rsv directly from clinical specimens. 65 rsv serology has limited value for case management but may be useful for epidemiologic surveys. 45 treatment uri caused by rsv requires no specific treatment, and antibiotics are needed only when bacterial otitis media or sinusitis are present. 45 the supportive treatment of infants with rsv bronchiolitis consists basically in preventing hypoxemia and electrolyte imbalance, in addition to aerosolized bronchodilators. the lack of obvious correlation between radiologic findings and disease severity suggests that a chest film should be recommended only for severely ill or deteriorating infants. 54 to prevent hypoxemia, requirements may vary from simple removal of respiratory secretions and proper positioning of the infant to mechanical respiratory assistance and even extracorporeal membrane oxygenation (ecmo). pulse oximetry has been advocated to assess oxygen needs, but in tropical developing areas, where oximeters may not be available, serial clinical assessment is essential to monitor disease progression. for this purpose, crackles and cyanosis seem to correlate better with hypoxemia than tachypnea and intercostal retraction. 54 correction of hypoxemia can be accomplished with 40% or lower oxygen concentrations. 45 oxygen should be humidified with saline and delivered by mask if head boxes or tents are unavailable. the role of corticosteroids remains unclear with some evidence that they are not beneficial. 54 the only antiviral drug currently approved for the treatment of infants with rsv is the synthetic nucleoside ribavirin, delivered by small-particle aerosol via a mist tent, mask, oxygen hood, or ventilator. it is recommended only for infants and young children with an underlying condition, such as congenital heart disease, cystic fibrosis, or immunosuppression. premature infants, infants younger than 6 weeks of age, and severely ill infants may also be considered for therapy. 66 aerosolized ribavirin is well tolerated, but it is expensive and its prolonged administration requires facilities that may not be available in impoverished tropical areas. passive immunotherapy with rsv immunoglobulin, in combination with aerosolized ribavirin, improved the outcome of rsv pneumonia in bone marrow transplant patients. 67 the use of rsvintravenous immunoglobulin (ivig) or humanized monoclonal antibody against rsv has shown no benefit for the treatment of rsv infections in infants. 45 no vaccine is currently available for rsv prophylaxis. the disease enhancement caused by formalin-inactivated vaccine in the 1960s plus results of more recent unsuccessful trials of live-attenuated vaccines, have significantly slowed progress toward an rsv vaccine. 2 purified fusion protein vaccine has been tested for safety and immunogenicity in seropositive children older than 18 months, and was associated with reduction of lower respiratory tract illness, but not of rsv infection rates, in children with cystic fibrosis. 45 these and other candidate subunit vaccines, as well as intranasal liveattenuated vaccines, should be tested in high-risk children with underlying bronchopulmonary diseases. passive immunization of high-risk infants with monthly infusions of rsv immunoglobulin during the rsv season reduced the incidence and severity of rsv infections in highrisk children. 68 this costly intervention is the only available means of protecting high-risk children against serious rsv lri. monthly intramuscular injections of humanized monoclonal antibody should be considered for passive immunoprophylaxis during rsv season for high-risk infants such as preterm infants less than 6 months old, children with congenital heart disease, and children less than 2 years of age with bronchopulmonary dysplasia. 45 hospitalized infants with rsv infection should be isolated or grouped to prevent cross-infection. hand washing; use of eye-nose goggles, gowns, and gloves; and decontamination of surfaces and fomites are additional nosocomial infection control measures. 45 human parainfluenza viruses (hpivs) are the single most frequent cause of croup in infants and children worldwide and are second only to rsv as cause of lri in infants. 45, 69 little is known about the epidemiology of hpivs in tropical countries, but these viruses have been detected in up to 13% of children in hospital-based ari studies in developing countries. 5,21 hpivs are distributed in two genera of the family paramyxoviridae, sharing the structural and biological characteristics already mentioned in the rsv section. hpivs are classified antigenically into types 1 to 4, and hpiv-4 has subtypes a and b. hpiv types 1 and 3 are classified in the genus respirovirus, while hpiv types 2 and 4 are in the genus rubulavirus. hpiv-1 and -3 are the types most frequently associated with lri in children, the immunocompromised, the chronically ill, and the elderly, whereas piv-4 causes mostly uri in both children and adults. 69 binding of hpiv to sialic acid in the cell membrane is mediated by the glycoprotein hn, which contains hemagglutinin and neuraminidase activities. fusion of viral and cell membranes is mediated by the viral f protein, which is cleaved by cellular proteolytic enzymes. 45 once inside the cell, the cycle is similar to other paramyxoviridae, as summarized in the rsv section. hpivs can be propagated in primary simian or human kidney cells and in several cell lines, such as hep-2, vero, mdck, llc-mk2, bhk, and hela. 69 a variety of experimental animals undergo asymptomatic infection with piv, but only higher primates develop symptoms. 45, 69 epidemiology primary hpiv infection occurs early in childhood, and by age 5 virtually all children are seropositive. 19 an estimated one third of all viral lris in children in the united states are caused by hpiv-1 and -3. 69, 70 in most temperate regions, hpiv-1 and -2 cause epidemics in the fall of alternate years, either in co-circulation or alternating with one another. the biennial pattern of hpiv-1 is found in both hemispheres. 69 hpiv-1 causes most croup epidemics, whereas hpiv-2 more frequently causes illness with milder manifestations, although it can also cause croup. 69 hpiv-3 occurs endemically throughout the year, with sporadic spring outbreaks mainly among infants, and hpiv-4 occurs sporadically throughout the year in children and adults. 45, 69, 70 in tropical areas hpivs may account for up to 15% of child hospital admissions due to lri. 21 community-based ari studies in children under age 5 years show higher hpiv activity during rainy seasons in tropical countries. 3, 24 hpivs were the most frequent viruses detected in school-aged children with bronchial asthma exacerbations in urban nigeria. 61 hpivs spread mainly within families and closed communities, such as nurseries, day-care centers, and pediatric wards, with high secondary attack rates. in a longitudinal study conducted with children less than 2 years of age with ari in a day-care center for low-income families in northeast brazil, hpivs represented 11% of the viruses detected. 11 the virus does not persist long in the environment and is transmitted mainly by large droplets and fomites. 45 viral shedding usually lasts 3 to 10 days, but shedding of hpiv for months has been reported in very young children and immunosuppressed hosts. 71 primary hpiv infection may cause rhinitis, pharyngitis, laryngotracheobronchitis (croup), bronchiolitis, or pneumonia. approximately two thirds of all piv infections in children result in febrile uri with associated otitis media in 10% to 34%. the remaining one third of piv infections are cases of croup, bronchiolitis, or pneumonia. 61, 63 hpivs, mainly of types 1 and 2, cause up to 74% of all cases of croup. 69 croup is the most striking clinical presentation of hpiv infection and is most common between the ages of 6 and 36 months. 72 croup is manifested by inspiratory stridor, barking cough, and hoarseness caused by subglottic edema, preceded by rhinorrhea, mild cough, and low-grade fever. 45, 72 most children recover in 2 to 5 days, but some may develop bronchiolitis and pneumonia and present with a bronchopneumonia-croup syndrome. 45, 69, 72 since immunity to hpivs is incomplete, infections tend to occur throughout life, but little is known about hpiv infections in adults. in general, adults have only nonspecific uri, commonly with hoarseness. 45 hpivs can cause particularly severe diseases in immunocompromised hosts, especially children with severe combined immunodeficiency and bone marrow transplant patients. mortality in bone marrow transplant patients with hpiv infection varies from 10% to 20% in most series. 45, 69 hpivs replicate in ciliated epithelial cells, causing cytolysis of the respiratory mucosa. the infection begins in the upper respiratory tract and tends to disseminate down the respiratory tree. the larynx and trachea are mostly involved in the croup syndrome, and extensive involvement of the lower respiratory tree may be present in tracheobronchitis, bronchopneumonia, and bronchiolitis. 69, 71, 72 similar to influenza, factors determining the extent of hpiv infection include the susceptibility of the viral f protein to be cleaved and tissue-specific differences in the production of proteases to cleave it. 45 host immunity is largely mediated by humoral immunity to the two surface proteins hn and f. virtually all children by the age of 3 years will have seroconverted to hpivs, generally first to hpiv-3 but later also to hpiv-1 and -2. at school age, a significant proportion of children will have seroconverted also to hpiv-4. secretory antibody targeted to the hn glycoprotein is the best marker of protection against piv, 71 but the protection conferred by antibodies is limited, and repeated infections will develop. t-cell immune response seems to be involved in the clearance of virus and additionally in the development of inflammatory infiltrate, edema, and excess mucus secretion, 69 and immunocompromised hosts may develop progressive and even lethal disease. 73 like rsv, pivs cause mononuclear interstitial infiltrate, epithelial necrosis, inflammatory exudate into the alveoli, and hyaline membrane formation in the lungs. 45 piv is present in respiratory secretions until about 8 days from the onset of symptoms and can be isolated in monkey kidney primary cells and several continuous cell lines. virus can be detected in the monolayers by hemadsorption with guinea pig erythrocytes in around 3 days after inoculation and confirmed by if. 69, 71 shell-vial assays have been developed for hpiv detection but with mixed results. 69 if of exfoliated respiratory epithelial cells has produced conflicting and sometimes disappointing results, with most studies reporting sensitivities between 50% and 75% at best. 69 detection of viral rna by rt-pcr, including commercially available multiplex assays for several respiratory viruses, has enhanced the sensitivity of detection of hpiv from clinical samples. 69, 74 real-time pcr for respiratory viruses in multiplex format is sensitive and specific for hpiv. 35 at present, only supportive and symptomatic treatment is available for piv infections. management of croup includes supplemental oxygen and racemic epinephrine nebulization in hospitalized patients. mist therapy, although traditional, has no proven value. 72 short-term, high-dose systemic corticosteroids may reduce the need for intubation, and nebulized budesonide has a rapid effect and is as safe and efficacious as nebulized epinephrine in moderately severe croup. 72 several antiviral agents have in vitro activity against hpivs, but none has reached clinical testing. 69 there have been anecdotal reports of reduced hpiv shedding in immunocompromised patients treated with ribavirin, but this finding has not resisted scrutiny. 69 future possibilities include the bcx 2798 and bcx 2855 compounds, whose design is based on the threedimensional structure of the hn protein, which inhibit the hemagglutinin and neuraminidase activities of the protein and were effective in vitro and in an animal model against hpiv-1, -2 and -3. 75 no interventions are available for the prevention of hpiv infections. early trials with inactivated hpiv vaccine in the 1960s were unsuccessful. 69 recently, a live-attenuated, coldadapted hpiv-3 vaccine was found to be immunogenic for children as young as 1 month of age and holds promise for further development. 69 the same vaccine was tested in combination with a live-attenuated rsv vaccine candidate, showing that this approach is feasible and deserves further study. 76 characterization of hpiv proteins hn and f has led to development of subunit immunogens that showed efficacy in animal models. 69 human rhinoviruses (hrvs) are the most frequent respiratory pathogens of humans. 77 they were the most frequently isolated viruses in children under 5 years of age with ari in an urban slum in northeast brazil. 3 human rhinoviruses are small, nonenveloped, positivestrand rna viruses in the family picornaviridae, with over 100 identified serotypes. 77 hrv serotypes have been classified according to receptorspecificity into three groups. the major group includes 91 serotypes whose receptor is intercellular adhesion molecule-1 (icam-1); the minor group contains 10 serotypes whose receptor is the low-density lipoprotein receptor (ldlr); and the remaining serotype, hrv-87, utilizes a sialoprotein as cell receptor. unlike other picornaviruses, hrvs are acid-labile, a property that distinguishes them from enteroviruses. 77 the hrv genome is a monocistronic single-stranded rna, packed in an icosahedral capsid composed of 12 pentamers. surrounding the fivefold vertex, each pentamer contains a 1.2-to 3.0-nm-wide canyon that contains the receptor binding site. following receptor binding, the viral positivestrand rna is released into the cytoplasm and directs the synthesis of a polyprotein, whose cleavage products include an rna polymerase. this enzyme will produce an expanding pool of positive-strand rna using as a template an intermediate negative-strand rna. the positive-strand rna can be either translated into virion proteins or packaged as a genome into newly assembled virions. the hrv replication cycle takes place in the cytoplasm, and mature virions are released when the host cell is lysed. 77 hrvs are resistant to ethanol, ether, chloroform, and nonionic detergent but are sensitive to uv light; to ph lower than 5 and higher than 9; and to halogens such as chlorine, bromine, iodine, and phenolic disinfectants. they are stable for days on environmental surfaces and for years at minus 70°c. 77 hrv infects only higher primates and causes illness only in humans. several cell lines of primate origin support hrv propagation, but certain strains of hela cells and human embryonic fibroblasts provide higher sensitivity for hrv isolation from clinical specimens. 78 the optimal growth temperature for hrv is 33°c to 35°c. 77 hrv infections occur in people from all continents, including remotely located population groups, such as bushmen from the kalahari desert, native alaskans, and an isolated amazon indian tribe. 79 hrv has been estimated to cause up to 80% of all autumn colds in temperate climates. 80 in tropical countries, very few community-based studies of viral ari have used adequate hrv detection methods, 5 and this has limited the assessment of the actual impact of hrv in those areas. however, available evidence indicates that hrv is frequently associated with ari in children in brazil. in fortaleza, a city in northeast brazil, hrv detected by isolation in cell culture represented 46% of the viruses in children under 5 years of age with ari. 3 in salvador, another city in the same region, hrv represented 52% of the viruses detected by rt-pcr in association with ari in children younger than 2 years of age attending a day-care center for the underprivileged. 11 data on the frequency of hrv among adults in tropical countries are even more scarce. in singapore, hrv was detected in 20% of the samples obtained from adults with ari symptoms attending primary care centers. 81 hrv transmission requires close exposure and occurs mainly by hand-to-hand contact, followed by self-inoculation into the eye or nose, but also by airborne spread. once hrv reaches the nasal cavity, infection occurs in virtually 100% of susceptible subjects, and approximately 75% of those infected develop illness after a 1-to 2-day incubation. 77 children play a central role in spreading the virus in the household. evidence suggests that indoor hrv transmission is favored by high relative humidity and crowding of young children, as occurs in the united states at the beginning of the school term, which may explain the autumn seasonal peak of hrv. 77 in tropical northeastern brazil, however, where relative humidity remains above 70% reaching 90% during the rainy season, longitudinal studies have found no obvious hrv seasonality. 3, 11 hrv colds are indistinguishable from colds of other viral causes and consist of nasal discharge, nasal obstruction, sneezing, sore or scratchy throat, hoarseness, cough, and headache. facial and ear pressure may be present. fever and malaise are uncommon. these symptoms last approximately 7 days but may persist for up to 2 weeks in 25% of cases. infants and toddlers may display only nasal discharge and be otherwise asymptomatic. 77 the majority of patients have obstruction and mucosal abnormalities of the sinus cavities, eustachian tubes, and middle ear, which predispose to secondary bacterial sinusitis and otitis media, each complication found in approximately 2% of all colds. 82 hrv rna may be detected by rt-pcr in maxillary sinus brushings in 40% of adults presenting with acute sinusitis, 83 and in 24% of the samples of middle ear fluid from children less than 7 years of age with diagnosis of acute otitis media. 84 hrv is frequently associated with exacerbations of chronic obstructive pulmonary disease and asthma attacks in children over 2 years of age and in adults. 59, 77, 85 hrv replication is restricted to the respiratory epithelium, taking place in scattered ciliated cells of the nose and in nonciliated cells of the nasopharynx. 86 this tropism seems to be a consequence of receptor availability. infection of a limited number of cells triggers the release of cytokines, chemokines, and inflammatory mediators, which together with stimulation of the local parasympathetic nerve endings, results in the cold symptoms. kinins, prostaglandins, and proinflammatory cytokines and chemokines may contribute to vasodilation, increased vascular permeability, influx of polymorphonuclear leukocytes, exocrine gland secretion, and nerve ending stimulation, resulting in nasal obstruction, rhinorrhea, sneezing, cough, and sore throat. 77 serotype-specific neutralizing igm, igg, and iga antibodies develop in most infected persons in 7 to 21 days and persist for years. protection from infection is partially attributed to the presence of iga antibody in nasal secretions, and recovery from illness is more dependent on cell-mediated immunity. hrv-induced blastogenesis, natural killer cell activity, mitogen-stimulated cell production of il-2 and ifn-γ have been documented during hrv infection. 77, 87 hrv induces the expression of human β-defensin 2 (hbd-2) in the respiratory epithelium, which supports a role for hbd-2 in host defense to hrv infection. 88 hrv can be detected in respiratory secretions by isolation in cultures of susceptible cell lines. 78 hrv shedding peaks around 48 hours after infection and declines rapidly, but may remain at low levels for up to 3 weeks. 77 cultures should be kept at 33°c to 35°c in a roller drum and examined for 10 to 14 days. the presence of hrv, indicated by the typical cpe, is confirmed by the acid sensitivity of the isolate. rapid immunocytochemical methods are not available because of the large number of serotypes. rt-pcr in clinical samples is more sensitive and less tedious than hrv isolation, 83 and the recently introduced real-time pcr-based assay is more sensitive than conventional rt-pcr. 89 pcr-based assays have been useful in studies to assess the impact of hrv in different settings. the homotypic nature of hrv antibodies restricts serology to experimental settings. 77 trials of antiviral agents for hrv have been conducted, but no specific treatment suitable for routine use has yet been identified, mainly because of lack of potency, untoward side effects, and drug delivery problems. 90 ruprintrivir, a selective inhibitor of hrv 3c protease, has potent, broad-spectrum anti-hrv activity in vitro. a double-blind, placebo-controlled clinical trial of intranasal ruprintrivir in experimental hrv infection reduced symptoms by 33% and also decreased viral titers and nasal discharge. 91 symptomatic relief from cold symptoms can be obtained with a broad variety of nonprescription medications. systemic sympathomimetic decongestants, such as pseudoephedrine, may reduce nasal obstruction, first-generation antihistamines may reduce sneezing and rhinorrhea, and nonsteroidal antiinflammatory drugs such as naproxen or ibuprofen may reduce headache, cough, and systemic symptoms. 77 the large number of hrv serotypes with minimal crossantigenicity has hampered the development of an hrv vaccine. it may be possible to reduce exposure to hrv by hand washing after contact with a cold sufferer or after handling objects that may have been contaminated with respiratory secretions. 77 studies in experimentally infected volunteers show that application of the virucidal agents salicylic acid or pyroglutamic acid to the hands reduced recovery of rhinovirus from the hand skin of treated persons as compared with controls. 92 this result suggests that rhinovirus transmission can be prevented by virucidal hand treatments. short-term, postexposure prophylaxis by intranasal ifn-α significantly reduced the incidence of hrv colds in household contacts of an index case. 93 however, the cost and difficulty of making the drug available to homes in a timely fashion reduce the utility of this approach for extended use by populations, especially in tropical countries. ruprintrivir has also been evaluated for prophylaxis of hrv colds starting 6 hours prior to inoculation of human volunteers. this approach reduced the proportion of subjects with positive viral cultures and viral titers but did not affect the frequency of colds. 91 respiratory infections caused by adenoviruses are among the most frequent illnesses that these viruses cause, particularly in children under age 5 years. 94 adenoviruses have been frequently isolated in ari studies in tropical countries. 5 in the south cone of south america, adenoviruses were the second most frequent virus recovered from children hospitalized for ari. 95 adenoviruses are nonenveloped, icosahedral dna viruses of the genus mastadenovirus in the family adenoviridae. 94 adenoviruses are distinguished antigenically by group-specific (a through f) and type-specific (1 through 49) antigens and by genomic subtypes identified by restriction site mapping. 93, 94 the adenovirus capsid consists of three morphologically, antigenically, and functionally distinct types of capsomers: hexons, penton bases, and fibers that project from the penton bases. the hexon and penton bases contain complementfixing, group-specific antigens common to all human adenoviruses, whereas the fibers have primarily neutralizing and hemagglutination-inhibiting, type-specific antigens. adenoviruses are commonly accompanied by small, singlestranded dna parvoviruses known as adenoassociated viruses, which do not seem to cause any specific disease. most people have antibodies to at least one of the four serotypes of adenoassociated virus by age 10 years. 96 the fiber protein binds to the host cell, through the protein coxsackie and adenovirus receptor (car) of the immunoglobulin superfamily, which serves as a high-affinity receptor for adenoviruses. the class i major histocompatibility complex (mhc) also may serve as receptor for adenovirus 5. 94 ligand-receptor interaction facilitates interaction of the penton base with cell surface integrins, which triggers entry. after endocytosis, the double-stranded linear genomic dna is transported to the nucleus, where "early" and "late" sets of viral genes are transcribed, resulting in mrnas coding for structural and nonstructural proteins. virus assembly takes place in the nucleus, and the infectious cycle is completed by the release of up to 1 million virions upon cell lysis. 94 adenoviruses replicate well in continuous cell lines of epithelial origin, such as hep-2, hela, and a549, and can be adapted to grow in human embryonic lung fibroblasts. they are stable over a wide ph range (5 to 9), resistant to isopropyl alcohol, ether, and chloroform, stable for weeks at room temperature and for years at approximately 20°c or colder, and can be lyophilized. they are inactivated by sodium hypochlorite and a temperature of 60°c for 2 minutes. 97 respiratory transmission of adenoviruses occurs at all ages but is of prime importance during epidemics among military recruits. ocular transmission has been associated with swimming pools and physician offices where sterilization or hand washing has been inadequate. asymptomatic infection and a prolonged carrier state are common. 94 low-number adenovirus serotypes (1, 2, 3, and 5) are more frequent before age 5 years and account for 5% to 20% of cases of uri and approximately 5% of cases of lri in children. 98 in adults, adenoviruses occur sporadically and cause mostly uri. infections by adenoviruses types 4 and 7 are usually epidemic, with attack rates of 6% to 16% per week in newly assembled military recruits, whose adenovirus carriage rate may be as high as 18%. 94 in this group, the adenoviral syndromes vary from mild colds to severe lri, but overall attack rates may reach 80%, with 20% to 40% of the individuals needing hospitalization. 94 in temperate climates, adenoviral infections are more frequent in late winter, spring, and early summer, whereas in northeast brazil they seem to occur year-round. 5 in salvador, also in northeast brazil, adenoviruses were detected in 11% of children younger than age 2 years with ari in a day-care center. 11 in tropical areas the incidence of adenovirus infections in military recruits is lower, and different serotypes may be involved. 98 pharyngoconjunctival fever, commonly caused by adenoviruses types 3 and 7, may be epidemic or endemic among children during the summer in temperate climates. inadequate chlorination or filtration of swimming pools and lakes has been associated with epidemics. 99 the incubation period of adenovirus infections averages 10 days. 98 adenovirus respiratory diseases may involve all parts of the respiratory tract, and up to 50% of nonepidemic infections are asymptomatic. in fact, adenoviruses were discovered because of their propensity for latency in adenoidal tissue. 94, 98 in southeast brazil, adenoviruses were detected with equal frequency in wheezing young children and asymptomatic controls. 60 most adenoviral illnesses consist of febrile colds, and in children the fever may be high and long-lasting. pharyngitis is common and may be associated with fever, pharyngeal exudate, granular appearance of the mucosa, and anterior cervical adenopathy, similarly to streptococcal pharyngitis. 94 adenoviruses can be recovered from up to 20% of cases of pharyngitis in small children. pharyngitis may be concurrent with pharyngoconjunctival fever, a syndrome caused by adenovirus types 3 and 7 and characterized by conjunctivitis, frequently unilateral, which may last for 1 to 2 weeks, preauricular adenopathy, cough, rhinitis, malaise, and fever. 99 the most frequent complication of adenoviral colds is acute otitis media, which occurs in up to 30% of cases. 100 adenovirus lris consist mainly of bronchitis and pneumonia, and may make up over 10% of childhood lris in temperate areas. 101 adenoviruses may cause permanent lung parenchymal damage, especially when concurrent with measles. 101 epidemic adenoviral infections in military recruits have a spectrum of clinical manifestation ranging from colds to severe pneumonia. typically, however, the manifestations are fever, pharyngeal symptoms, cough, chest pain, headache, and malaise. 98 overwhelming pneumonitis may be part of disseminated adenoviral infections in newborn infants and patients with immunodeficiencies, including acquired immunodeficiency syndrome (aids). however, the frequent concomitance of other respiratory pathogens in aids patients and the high prevalence of asymptomatic adenovirus infection shed doubt on the causal role of the adenovirus in these patients. 102 adenoviruses are also an important cause of epidemic keratoconjunctivitis. 94, 98 while most adenoviral aris are self-limited and uncommonly associated with death or permanent sequelae, 94 adenoviruses alone or associated with other pathogens have been recovered from 20% of fatal cases of lri in argentina. 103 adenoviral respiratory disease results from necrosis of cells of airway epithelia, and viremia may result in disseminated infection in immunocompromised persons. bronchiolitis, interstitial pneumonitis, and mononuclear cell infiltrates are part of the inflammatory process in the lungs. it remains unclear why certain strains are more virulent than others. for example, the genomic variant b7h was associated with the majority of fatal lower respiratory disease in south america. 95 in addition to lytic infection, adenoviruses may become latent in epithelial and lymphoid cells, which is probably important to maintaining the virus in populations. 94 a possible role of latent adenovirus in the pathogenesis of chronic airway inflammation has been suggested. 94, 96, 104 protection from adenovirus infection and disease is mainly due to type-specific neutralizing antibody, but reinfections, mostly asymptomatic, may occur. a long-lived t-cell immune response develops in most infected immunocompetent persons and is not only responsible for recovery but also is involved in tissue pathologic changes. 94 adenoviruses can be detected in respiratory, ocular, or ear secretions, but clinical correlation is required, because asymptomatic virus shedding is common. isolation of adenoviruses in cell culture with identification by if constitutes the standard diagnostic method, but direct detection of viral antigens or viral dna by pcr in clinical samples is an attractive rapid alternative. 94 rapid antigen detection by immunochromatography is around 95% sensitive in comparison with cell culture, and easily can be used in point-of-care diagnosis of adenovirus. however, both conventional and real-time pcr are more sensitive than cell culture is. 105 positive results by pcr should be interpreted with caution, given the propensity of adenoviruses to cause latency. adenoviruses cause a characteristic cpe in a variety of cell lines of human origin, and maintenance of cultures for 2 weeks combined with blind passage (i.e., passage of cells even without obvious cpe to see if it develops after passage) may increase adenovirus recovery. 3, 94 inoculation of cells by centrifugation followed by immunostaining may shorten the detection time. 106 several serologic tests can detect antibodies to the common hexon antigen. 94 however, their clinical utility is restricted. at present, there is no routine effective antiviral treatment for adenovirus infections. successful therapy of severe adenoviral infections in immunocompromised patients with iv ribavirin has been reported. 107 cidofovir has shown some efficacy in the rabbit ocular model of adenoviral infection. iododeoxyuridine and adenine arabinoside were unsuccessful in the treatment of adenoviral keratoconjunctivitis. 94 a live vaccine consisting of wild-type adenovirus packaged in enteric-coated capsules induces immunity by ensuring enteric infection without infection of the respiratory tree. this approach has been used successfully to vaccinate military recruits against adenoviruses types 4 and 7. 98 proper sterilization, hand washing, and chlorination can prevent adenovirus spread via tonometers, hands, and swimming pools. coronaviruses are enveloped viruses with distinct virion morphology, displaying widely spaced, long petal-shaped spikes at the surface, that confer a crownlike appearance, the origin of the name corona. the envelope contains a long helical nucleocapsid with single, positive-stranded rna, 27 to 32 kb in size, which is the largest known viral rna genome. 108 until very recently, only three human coronaviruses (hcovs) were known to exist: hcov-229e, hcov-oc43, and the cov associated with severe acute respiratory syndrome (sars-cov). recently, two groups in the netherlands almost simultaneously published studies that resulted in the identification of two new strains of hcov: hcov-nl63 109 and hcov-nl. 110 in addition, pcr primers directed to conserved replicase 1a sequences of animal covs led to the identification of yet another hcov detected in 8.8% of children from new haven, connecticut with symptoms of ari. this agent was designated hcov-nh and is likely to represent the same species of hcov-nl and -nl63. 111 on the basis of antigenic and genetic studies, the known human coronaviruses are distributed in three of the four coronavirus groups so far identified. hcov-229e, -nh, -nl, and -nl63 belong to group i, hcov-oc43 belongs to group ii, and sars-cov is the only known constituent of group iv, while group iii contains no known human viruses and consists only of the avian infectious bronchitis virus. coronavirus rna synthesis occurs in the cytoplasm via a negative-strand rna intermediate. the viral rna possesses a 5′ cap followed by a leader sequence and an untranslated region, with another 3′ terminal untranslated region followed by a poly(a) tail. the genome is polycistronic and the synthesis of subgenomic negative-sense rnas is done by discontinuous transcription to originate a nested set of subgenomic mrnas that share the 5′ leader sequence and overlap at the 3′ end. the envelope contains the structural proteins s (spike), m (membrane), e (envelope), and only in the case of some group ii coronaviruses, ha (hemagglutinin). the s glycoprotein contains neutralizing and t-cell epitopes and functions as the cell receptor ligand, thereby determining tissue tropism. the m protein is embedded in the envelope and interacts with the n (nucleocapsid) protein during maturation. in addition to the nucleocapsid and envelope proteins, a replicase is present in cells infected by all coronaviruses. new virions assemble by budding through intracellular membranes and are released through vesicles of the secretory pathway. 108 hcov-229e and -oc43 are considered to be second only to rhinoviruses as agents of common colds, causing infections with variable frequency, depending mainly on the detection method and season of the study. up to 35% of mild upper respiratory tract infections in adults have been attributed to hcov-229e. 112 while hcov-229e and -oc43 are documented causes of colds in temperate regions, their impact as causes of respiratory infections in tropical regions has not been defined. human coronaviruses were first isolated in england, almost 40 years ago, in human organ cultures of tracheal and nasal tissues. there have been relatively few field studies based on hcov isolation in cell culture, likely because these viruses are too fastidious to be propagated, but most respiratory isolates obtained so far have been antigenically similar to either hcov-229e or -oc43. 112 these agents have the same structural features as the other members of the family. the s protein of hcov-229e binds to the metalloprotease human aminopeptidase n at the cell surface, and entry is independent of enzymatic activity of the receptor. the hemagglutinin of hcov-oc43 binds to sialic acid present in glycoproteins on the cell surface and this interaction facilitates infection, but to the best of our knowledge, a specific receptor has not been identified for this agent. 108 hcovs have been found throughout the world and are considered to be the second most frequent cause of common cold, accounting for an average rate of 15% of respiratory illnesses in the general population in the united states. however, the rates may be quite variable from year to year, ranging from 1% to 35% in years of peak activity. hcov infections occur mainly in the winter and spring months, but summer activity has also been documented. during the autumn peak of rhinovirus activity, 8% of the adults with a cold negative for rhinovirus were positive for hcov by rt-pcr in charlottesville, va. 80 hcov-229e has caused well-documented winter outbreaks at 2-to 4-year intervals in temperate regions. 112, 113 similarly, winter outbreak of hcov-oc43 has also been detected in europe. 114 in contrast, little is known about the prevalence of hcov-229e and -oc43 in tropical countries. in brazil, the activity of hcov-229e as cause of respiratory infections in nonhospitalized children was first documented by serology in the early 1970s, with a seropositivity rate of 26% in adults by complement fixation assay. 115 the usual manifestations of hcov infection are typical common colds. the incubation period tends to be 1 day longer that that for rhinovirus colds, with illness duration of 6 to 7 days. low-grade fever may occur in up to 20% of the patients, and in addition to nasal symptoms, cough and sore throat occur frequently. more serious infections of the lower respiratory tract caused by hcov have also been documented, either sporadically in infants with pneumonia and immunocompromised patients, or in up to 33% of previously healthy marine corps recruits with pneumonia. 112, 113 in addition, hcov-229e and -oc43 have been recognized in association with influenza-like illnesses in frail elderly patients. eight of 100 (8%) nasopharyngeal swabs from older patients hospitalized for cardiopulmonary illnesses during the influenza seasonal outbreak in rochester, n.y., were positive for hcov (five for hcov-229e). 116 respiratory hcov infections have been associated with exacerbations of asthma, chronic bronchitis, and recurrent wheezing in children. 112, 113 hcov was detected by rt-pcr in 38 of 292 (13%) episodes of asthma in children 9 to 11 years old in england. 117 in brazil, hcov was detected in respiratory samples from 3 (2 oc43 and 1 229e) of 73 (4%) children younger than 2 years of age who came to the er with wheezing. 60 similarly to hrv, hcov infections have been frequently recognized in association with otitis media and maxillary sinusitis in children and adults. hcov was detected by rt-pcr in the middle ear effusion or nasopharyngeal aspirate from 16 of 92 (17%) children with acute otitis media in finland 84 and in nasal swabs from 3 of 20 adults with acute maxillary sinusitis. 83 there is no convenient small animal model to study the pathogenesis of hcov, and humans naturally or experimentally infected are the only source of information obtained in vivo. hcovs are transmitted by the respiratory route, and experimentally infected volunteers shed virus for approximately 5 days, beginning 48 hours after infection, which is approximately the time of onset of symptoms. 112, 113 the peak of symptoms occurs 2 to 4 days postinoculation. 112 hcov-229e is known to infect airway epithelial cells from the apical surface, where the receptor is constitutively expressed, and to exit productively infected cells through the same route. 118 ultrastructural studies of nasal epithelium of volunteers experimentally infected with hcov-229e revealed significantly greater epithelial cell damage, ciliary loss, and cytolysis in virus-inoculated subjects than in sham-inoculated ones on day 3 postinfection. 119 in the united states, seropositivity to hcov-oc43 and -229e rises during the first 5 years of life, and around 40% of adults are seropositive. symptomatic reinfections are possible, despite the presence of antibodies, suggesting rapidly waning immune response or circulation of closely related but antigenically different viruses. 113, 120 several studies indicate that respiratory hcovs are able to reach the central nervous system. 112, 113, 120 the recently reported temporal association between hcov-nh infection and kawaski disease 121 awaits confirmation. laboratory diagnosis in clinical samples by isolation is tedious, because the two best characterized strains of hcov are difficult to grow in routine cell cultures. since primers can be developed for relatively constant parts of the genome, rt-pcr-based assays for hcov-229e and -oc43 have recently become the best alternative to other methods of detection. 80 more recently, a quantitative real-time pcr-based assay for hcovs has been developed, providing a faster means for detection and determination of viral load with potential applications in clinical studies. 122 serologic diagnosis of hcov by eia is sensitive and specific and has been useful in epidemiologic surveys. 113 intranasal interferon protects against experimental infection with hcov-229e, 123 but no specific antiviral therapy is available, and treatment of hcov-induced colds remains largely symptomatic. no vaccines are currently available for hcov. the hcov that fulfills koch`s postulates as the causative agent of sars 127 shares structural features and genome organization of the family coronaviridae ( fig. 59-1) . the prompt recognition of the peculiar morphology of a coronavirus in the electron microscopic studies of vero e6 cells inoculated with oropharyngeal material from a patient was the initial finding that resulted in the identification of sars-cov. 128 the viral genome is 29,727 nucleotides in length, with more than 11 open reading frames coding for 23 putative proteins, some of which have unknown functions. sars-cov is phylogenetically different and equidistant from all previously known coronaviruses, but isolates from different origins are relatively homogeneous genetically. genome analysis reveals that sars-cov is neither a host-range mutant nor a recombinant of respiratory tract viral infections ■ 651 previously known coronaviruses but rather an independently emerged virus. sars-cov seems to have evolved from an animal sars-like virus, acquiring greater fitness in humans during the course of the outbreaks, probably through the appearance of nucleotide deletions in open reading frame 8. 129 it is also noteworthy that genetic signatures present in the genomes allow for differentiation of isolates obtained from different clusters. 129 the replicative cycle of sars-cov is thought to follow the same main steps as other coronaviruses. sars coronavirus (sars-cov) probably emerged around november 2002 in the province of guangdong, china, where there was no serologic evidence of infection caused by this virus in sera of healthy humans sampled prior to that time. 130 at the beginning of the outbreak, many affected individuals in guangdong were directly or indirectly involved with game trade, and indeed, palm civets and raccoon dogs from wildgame markets in the area were later found to harbor a cov 99% homologous to sars-cov at the nucleotide level. this suggests that animal-to-human interspecies transmission was involved in the outbreak, providing the source of an agent that later adapted to efficient human-to-human transmission. 131, 132 interestingly, shortly after the lifting of a wildlife trade ban that originally had been imposed to control the sars outbreak, new cases were again detected in guangdong, all of them caused by viruses newly introduced from animals. since the ban was reinstalled, there have been no further naturally acquired human cases of sars in guangdong. 131 remarkably, 1.8% of 938 serum samples from adults recruited in 2001 in hong kong tested positive for sars-cov antibodies, suggesting that a small proportion of healthy people from hong kong, as opposed to guangdong, china, had been exposed to sars-related viruses at least 2 years before the outbreak. 133 it is probable that sars-cov precursors previously crossed the species barrier and may even have caused subclinical human infection, but perhaps only occasionally this event generated strains adapted to successful human-to-human transmission. 131 sars-cov is mainly transmitted between humans by the deposition of infected droplets or aerosols on the respiratory epithelium. the number of confirmed secondary cases generated by one index case of sars is relatively low, ranging from 2.2 to 3.7, suggesting relatively inefficient transmission. in addition, transmission is infrequent during the first 5 days of illness, partly because of the low viral load in respiratory secretions during that phase. for reasons not completely understood, some sars patients, identified as superspreaders, disproportionately contribute to the generation of a high number of secondary cases. 131 excretion of sars-cov in sputa and stools may average 21 and 27 days, respectively, after symptom onset, but an excretion period as prolonged as 126 days has been documented in stools. 134 such prolonged shedding of virus in feces raises the possibility of oral-fecal transmission and, in fact, one outbreak of sars was attributed to a faulty sewage system. 131 case-fatality rates estimated based on cases admitted to hospital have been around 13% for patients younger than age 60 and 43% for those older than age 60 years. however, it is likely that case-fatality rates based on all infections occurring in the community would be lower. 135 transmission of sars-cov among health-care workers and between patients in the hospital setting played a pivotal role in outbreak propagation. analysis of data from initial outbreaks indicates that close contact is the most important factor leading to nosocomial transmission of this agent. despite the lack of complete studies on the sensitivity of sars-cov to different environmental conditions, there have been reports of sars-cov persisting for up to 2 days on environmental surfaces and 4 days in diarrheal stools. 136 the median incubation period of sars is 4 to 6 days. clinical symptoms and signs of sars appear 2 to 10 days after exposure, and systemic symptoms, such as fever, chills, myalgia, and malaise, usually appear first. respiratory symptoms appear 2 to 7 days later, represented most frequently by nonproductive cough, dyspnea, chest pain, headache, and sore throat. diarrhea and vomiting may occur. chest radiograms frequently reveal infiltrates consistent with viral pneumonitis, consisting mostly of consolidations and ground-glass opacifications. computed tomography (ct) scans in patients with normal or equivocal chest radiograms may show unilobar or multilobar abnormalities. fever generally subsides in 48 hours, but one or two relapses within 8 to 15 days are frequently observed. lymphopenia with reduction of both cd4+ and cd8+ cells, slight decrease in platelet counts, prolonged coagulation profile, and elevated serum enzymes (lactic dehydrogenase [ldh], creatinine kinase [ck], and c-reactive protein [crp]) are often observed. around one third of patients may have cd4+ lymphocyte counts below 200 cells/mm 3 and higher susceptibility to secondary infections. watery diarrhea with an average of six evacuations per day is common. 130, 131, 137 radiologic worsening of the pulmonary lesions seen at admission, with or without appearance of new lesions, is a frequent observation, with development of diffuse groundglass changes frequently heralding the development of acute respiratory distress syndrome (ards). hypoxemia is noted in approximately half of the patients at around 9 days after the onset of symptoms, and a high proportion of those admitted to the intensive care unit (icu), especially older males, require mechanical ventilation around day 13. development of spontaneous pneumomediastinum during follow-up is not uncommon, probably as a consequence of ruptured peripheral lung lesions into the pleural space. 130 prognosis is related to the level of viral replication in tissues, and patients with high viral loads in serum, nasopharyngeal aspirates, or feces, as well as those in whom virus can be detected from multiple sites, tend to have poor clinical outcome. 131 in addition to old age and severe underlying diseases, ck and crp levels have been identified as predictors of poor outcome. 138 the n-terminal portion of the spike glycoprotein is needed for virus attachment to the virus receptor, identified as the metallopeptidase angiotensin-converting enzyme homolog (ace-2), 139 but it is unclear whether the mechanism of entry is contingent on ph-dependent endocytosis. 131 some inconsistencies between ace-2 and sars-cov tissue distribution suggest that ace-2 may not be the only receptor, or that a coreceptor molecule may be needed for cell infection. sars-cov spike protein can also bind the dendritic cell-specific c-type lectin intercellular adhesion molecule 3-grabbing nonintegrin (dc-sign), which does not result in dendritic cell infection by the agent but allows for sars-cov to be transported to susceptible target cells elsewhere. 140 sars-cov has been detected in studies using different combinations of immunohistochemistry, in situ hybridization, and electron microscopy in pneumocytes and on the apical surface of enterocytes. marked inflammatory infiltrates and mucosal atrophy have not been observed in the intestine, and the pathogenesis of the sars-cov-related diarrhea remains largely unknown. 131 sars-cov viral load in the upper airways is low in the 4 initial days, with the peak at day 10 of illness. quantitative rt-pcr for sars-cov in nasopharyngeal aspirates from patients who tested positive at admission revealed viral loads around 10 5 copies/ml on days 5 and 15 after clinical onset, and peak 10 7 copies/ml on day 10. 130 higher viral loads can be detected in the lower respiratory tract than in the upper airways. pulmonary tissue shows diffuse alveolar damage, mixed infiltrate, lung edema, hyaline membrane, abundant macrophages in alveoli and interstitium, and syncytia formation. besides respiratory secretions and stools, sars-cov can be detected in urine in up to 30% of patients, with titers averaging 10 4.4 copies/ml, in association with abnormal urinalysis results. 141 the effect of sars-cov infection on the immune system is highlighted by pronounced t-cell lymphopenia and elevation of several inflammatory cytokines (il-1β, il-6, and il-12) and chemokines (mcp-1 and ip-10) observed in sars patients. while mcp-1 is likely to be involved in the lung monocytic/macrophagic infiltrate, its role is not firmly established, since other viral diseases that are associated with elevated mcp-1, such as influenza, do not include such prominent histologic features. in addition, since immunologic markers in the peripheral blood may not reflect what happens in the microenvironment of the lung, the pathogenic importance of these findings is not clear. co-inheritance of hla-b*0703 and -b60 is higher among sars patients than in the general population, favoring a role for the genetic background in susceptibility to sars-cov. 131 the pathogenesis of the t-cell lymphopenia remains unknown. seroconversion has been documented in 93% of the patients at around 20 days and the rise in igg titers correlates with decrease in viral load. 130 paradoxically, clinical worsening also occurs during this phase, suggesting that, rather than unchecked viral replication, immunopathologic factors may be responsible for the lung lesions. 130 while infection in experimental animals, such as cynomolgus macaques, ferrets, cats, golden syrian hamsters, mice, and african green monkeys, does not induce disease that mimics that in humans, these models are important for studies of pathogenesis and development of vaccines and therapy. 129 in addition, the development of an infectious cdna clone of sars-cov should permit reverse genetics experiments and may help elucidate determinants of viral pathogenesis. 142 low viral loads in the upper respiratory tract in the first few days of illness account for the relatively poor sensitivity (35% to 65%) of first-generation rt-pcr for diagnosis in that period. sars-cov is detectable by rt-pcr in nasopharyngeal aspirates in only one third of patients at presentation and in two thirds at day 14. rt-pcr may be positive for sars-cov in stools from as much as 97% of patients at day 14, and in urine in 42% of samples at day 15. 130 testing multiple nasopharyngeal, serum, and fecal samples increases the sensitivity of the diagnosis by rt-pcr. 143, 144 to overcome the low sensitivity of conventional rt-pcr, quantitative real-time pcr-based assays for sars-cov have been developed that improve sensitivity and turnaround time, allow for amplification and analysis to be done in a closed system, and thus reduce cross-contamination. in addition, the capability of the assay to quantitate viral load has contributed not only to understanding viral pathogenesis but also to predicting outcome, since high viral loads are associated with poor prognosis. 143 the ability to grow sars-cov in vero e6 cell cultures was critical to identifying the agent. sars-cov can be recovered by isolation from respiratory secretions, feces, and urine in the first 3 weeks of illness, but the overall sensitivity is relatively low and recovery is more likely to be successful from respiratory secretions than from stools and urine. 143 recent small outbreaks of sars-cov originating in laboratories 143 have heightened concern about laboratory safety issues regarding sars specimens. the who guidelines for biosafety in the diagnosis of sars (updates available at the who web site) recommend that propagation of sars-cov in cell culture for isolation or for preparation of viral stocks and cell slides be performed in biosafety level 3 (bsl3) laboratories, whereas handling serum and blood specimens for routine tests and serology can be performed in bsl2 laboratories. nucleic acid extraction procedures, inoculation of bacterial or mycologic cultures, and preparation of sample smears can be done in bsl2 laboratories, observing bsl3 work practices (use of safety cabinets, sealed centrifuges, protective equipment, 5% bleach spillage decontamination, and proper waste disposal). although not useful for early diagnosis, seroconversion determined by ifa or eia remains the gold standard for confirming sars diagnosis. igg seroconversion is detectable in over 90% of patients at around day 28. 130 antibody crossreaction with other human coronaviruses, however rare, remains a possibility; therefore, confirmation of positive serology by an independent neutralization assay should be performed if available. 143 the main component of treatment of sars patients is supportive therapy, chiefly the management of hypoxemia and ards. during the 2003 outbreak, treatment included a broadspectrum antiviral agent (ribavirin) and immunosuppressive doses of corticosteroids, aimed at reducing the immunopathologic damage to the lungs. the use of high-dose steroid therapy is controversial and for the most part supported by anecdotal evidence, whereas the use of ribavirin is based on the broad antiviral spectrum of the drug. however, sars-cov is only modestly susceptible to ribavirin in vitro, and therapeutic doses are difficult to achieve clinically. since it became possible to grow sars-cov in culture, many potential antiviral compounds have been evaluated in vitro, but just a few have been tested in animal models and even fewer are in clinical testing. 131 interferons (ifn-αn1/n3, leukocytic ifn-α, ifn-β) and hiv protease inhibitors were consistently active in vitro and may be considered for animal testing and clinical trials. 131 the resolution of the structure of sars-cov principal protease has prompted studies of the inhibitory capacity of known anti-hiv protease inhibitors for treatment of sars. in one open-label study, a combination of hiv protease inhibitor (lopinavir plus pharmacokinetic booster ritonavir) and ribavirin was used to treat sars patients and the outcomes were compared with historical controls treated with ribavirin alone. at day 21 after onset of symptoms, development of ards or death was significantly less frequent in the group treated with the combination (2.3%) than in historical controls (28.8%). in addition, peak viral loads in respiratory samples and stools were reduced in the group treated with the combination as compared with controls. 145 however, since there were differences in outcome predictors, such as sex, platelet counts, and ldh levels, between the two groups, these results should be interpreted with caution. a preliminary open-label study found that a restricted number of patients treated with subcutaneous interferon alfacon-1 in association with corticosteroids showed reduced oxygen-saturation impairment and faster resolution of radiographic chest findings than those treated with corticosteroids alone. 146 convalescent plasma has also been tested in the treatment of sars patients. in one preliminary uncontrolled study, convalescent plasma may have reduced the frequency of poor outcome when given before 14 days of illness. 147 it is impossible to predict whether naturally reemerging sars-cov would be likely to cause a global outbreak. nevertheless, a vaccine for this agent would be relevant for high-risk individuals, such as workers in laboratories, hospitals, and game-animal farming. therefore, considerable effort has been directed at developing such a vaccine. it has been shown that sars-cov spike protein produced in bacteria and expressed on chimeric parainfluenza virus, as well as spike protein-encoding dna, induced neutralizing antibodies and protected experimental animals from challenge with live virus. at present, no sars-cov vaccine is available for human use. therefore, in the absence of person-to-person transmission of sars-cov worldwide, prevention of future outbreaks of sars requires careful surveillance. the goal is to maximize early detection of new cases of sars to implement control measures, thereby minimizing social disruption. 131 to reach this goal, the cdc recommends testing for sars-cov in patients who require hospitalization for radiographically confirmed pneumonia or ards without identifiable etiology and who have one of the following risk factors in the 10 days before the onset of illness: (1) travel to mainland china, hong kong, or taiwan, or close contact with an ill person with a history of recent travel to one of these areas, or (2) employment in an occupation associated with a risk for sars-cov exposure (e.g., health-care worker with direct patient contact; worker in a laboratory that contains live sars-cov), or (3) belonging to a cluster of cases of atypical pneumonia without an alternative diagnosis (updates on these recommendations are made available at the cdc web site http://www.cdc.gov/ncidod/sars). during times of overt sars activity, prevention of humanto-human transmission is pivotal to curtailing outbreaks. although sars infectiousness relative to the onset and termination of clinical symptoms has not been accurately determined, it is clear that shortening the time from onset to hospital admission and isolation reduces the risk of transmission, thus contributing substantially to curtailing of outbreaks. identification of new cases through contact tracing played an important role in the control of the outbreaks registered so far. stringent isolation procedures must be adopted for confirmed and suspected cases, which require a high level of alertness among health-care workers for early identification of sars cases. the scenario may be further complicated in situations in which other diseases such as influenza and hantavirus pulmonary infections may occur simultaneously. 135 rates of transmission of sars-cov among health-care workers vary, depending on stringency of control measures adopted, presence of so-called superspreaders in the hospital, and kind of activities carried out by personnel, especially as related to proximity to the index case. assisting during intubation, suctioning, and manipulating ventilatory apparatuses seem to be high-risk activities. while studies conducted in different settings have produced conflicting results, one study in toronto found that up to 25% of the nurses who cared for sars patients in critical care units became infected. 148 the presence of severe watery diarrhea may add to the challenge for the infection control team. 130 an updated set of recommendations for health-care and laboratory personnel is available at the cdc web site (http://www.cdc.gov/ncidod/sars). a new paramyxovirus was described in the netherlands in 2001, in association with respiratory illness in children. the agent was first detected by analysis of previously unidentifiable viral isolates that induced cytopathic effect in llc-mk2 cell cultures. the isolates were recovered over a 10-year period in respiratory secretions from 28 children with ari occurring in the winter time. electron microscopy of cell culture isolates revealed paramyxovirus-like particles, and rna sequencing revealed genome sequences and organization consistent with a paramyxovirus of the subfamily pneumovirinae, most closely related to avian pneumovirus of the genus metapneumovirus. rather than an avian virus that can also infect humans, this agent is now recognized as a primarily human pathogen, and thus has been named human metapneumovirus (hmpv). 156,157 hmpv antibodies detected in sera collected in 1958 in the netherlands indicate that this agent has been in circulation for at least 4 to 5 decades. 156 agent hmpv particles are enveloped, pleomorphic, spherical, and filamentous particles, with a mean diameter of about 209 nm. 156,157 complete genome sequences of hmpv are available and, in contrast to the genomic organization of pneumoviruses, metapneumoviruses have different positioning of the genes between m and l and lack ns1 and ns2 genes. 156,158 similar to hrsv, genetic and antigenic studies indicate that hmpv isolates cluster into two main serotype named a and b, with n gene sequences 83% to 85% similar at the nucleotide level, each subgroup including two genetic lineages (a1, a2, b1, and b2). 156, 159, 160 both are globally distributed. there have been no detailed studies of the hmpv replication cycle, but it is likely to be similar to that of other human paramyxoviruses. hmpv is a frequent cause of community-acquired ari in children and adults in all continents, although with variable incidence in different settings. 157, 159, [161] [162] [163] [164] [165] [166] [167] [168] [169] in the united states, hmpv has been reported in up to 20% of lower respiratory tract illnesses whose etiology would have been unidentifiable prior to the development of assays for the detection of hmpv. 170 in canada, during the 2001-2002 winter season, hmpv was detected in 15% of patients of all age groups from four different provinces. 167 similar to hrsv, hmpv infections are more frequent in the colder months in temperate regions, and different strains of both subgroups a and b cocirculate during the same year. 157, 162 however, only limited knowledge is available about hmpv seasonality in more tropical climates. peaks of hmpv activity have been documented in the spring/summer in hong kong, 171 while in south africa hmpv has been detected in 6% to 9% of children with ari admitted to hospitals in the winter season. 161 ,165 hmpv was detected alone or simultaneously with rsv in 24% of children younger than 3 years of age admitted to health-care facilities in aracaju, northeast brazil, in the months of april and may, 2002. 164 interestingly, hmpv was not detected by the same methods in that same city, in the following year. 166 this apparent variability in hmpv incidence from year to year has also been observed in studies conducted in argentina 168 and italy, where hmpv frequencies varied from 7% to 43% in three consecutive annual respiratory virus seasons. 172 long-term prospective studies will be needed to establish whether there is a seasonal pattern in hmpv circulation in tropical regions of the world. clinically, hmpv infections resemble closely those caused by hrsv, ranging from mild upper ari to severe bronchiolitis and pneumonia. the median age of children hospitalized with hmpv infection is older than those with hrsv. hrsv in hospitalized infants and young children may require intensive care and mechanical ventilation, 156,173,174 and dual infection with hmpv and hrsv appears to increase the likelihood of severe illness. 175, 176 the most frequent symptoms in all age groups are fever, dyspnea, cough, wheezing/stridor, rhinitis, and sore throat. 162, 173 all infected children in one study had either pneumonia or bronchiolitis, frequently accompanied by otitis media. 162 hmpv may cause more serious infections in patients with comorbid or immunosuppressive conditions, as well as in the very young and the elderly. 162, 167 in one study, all individuals older than 65 with lower respiratory infection caused by hmpv had at least one underlying chronic or debilitating condition, including lymphoma, leukemia, or neurologic or cardiovascular diseases. 162 hmpv infection in adults may present as influenza-like illness, acute bronchitis, or common cold. 162 in england, in the winter of 2000-2001, hmpv was detected by rt-pcr in association with 2.2% of samples taken from patients in all age groups with influenza-like illnesses negative for hrsv and influenza viruses. 159 hmpv has been increasingly recognized as cause of acute wheezing in children. one study conducted in finland found hmpv in 8% of wheezing children, who presented significantly higher levels of il-8 in nasal secretions as compared to children with hrsv-associated wheezing. 174 a study conducted in brazil found that 47% of the children with hmpv had wheezing and 31% had chest indrawing. 164 previous history of asthma has been more frequently associated with hmpv than with hrsv infection and hmpv-infected patients are more often treated with bronchodilators and corticosteroids than hrvs-infected patients. 173 little is known about specific mechanisms of pathogenesis and host immune response in hmpv infections. hmpv is a pathogen of both the upper and lower respiratory tracts. 162 hmpv replicates efficiently in the respiratory tract of monkeys, with virus shedding peaking between days 2 and 8 following infection. 156 serologic data indicates that hmpv infects young individuals, and by the age of 5 virtually all children have become seropositive for the agent; reinfections at later ages are common. 156, 157, 159 interestingly, coinfection with hmpv has been reported to correlate with increased severity of hrsv infections. a study conducted in the united kingdom found that this coinfection caused a tenfold increase in the relative risk of admission to the icu for mechanical ventilation in children under 2 with hrsv bronchiolitis. 173 a similar finding was also reported in germany. 176 hmpv can be isolated in llc-mk2 cells from nasal aspirates or nasopharyngeal swabs. the cytopathic effect, characteristically negative on hemadsorption testing, develops usually late after inoculation (up to 23 days). 160 sensitive rt-pcr assays for this agent have been developed in many different laboratories and have rapidly become standard for hmpv diagnosis. 159,169 a real-time pcr assay for hmpv showed to be more sensitive than conventional rt-pcr, even when hybridization was used to increase sensitivity of the detection of amplicons generated by the conventional method. 169 using real-time pcr, hmpv was detected in 10% of 329 samples collected from patients with ari in australia from march to october 2001 that were negative for other pathogens. 169 other than supportive measures, oxygen therapy, bronchodilators, corticosteroids and mechanical ventilation, there is no specific antiviral treatment for this agent. 173 ribavirin is inhibitory for hmpv in vitro. 176 although a hmpv vaccine is not available at this time, the demonstration that hamsters, ferrets, and 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metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis prospective study of human metapneumovirus infection in children less than 3 years of age identification of small animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hmpv) and implications for hmpv design were protected from challenge with either a or b hmpv serotypes opens possibilities for hmpv vaccine design. 176 humanized neutralizing monoclonal antibody to f protein is active in experimentally infected animals. 177 key: cord-024134-ym7ce5ux authors: chawla, sonam; saxena, shailendra k. title: preparing for the perpetual challenges of pandemics of coronavirus infections with special focus on sars-cov-2 date: 2020-04-30 journal: coronavirus disease 2019 (covid-19) doi: 10.1007/978-981-15-4814-7_14 sha: doc_id: 24134 cord_uid: ym7ce5ux covid-19, arising from novel, zoonotic coronavirus-2, has gripped the world in a pandemic. the present chapter discusses the current internationally implemented pandemic preparedness strategies succeeding/recommended to curb the covid-19 threat to humankind. the updated phase-wise categorization of a pandemic as recommended by the who is described, and associated innovations in surveillance, response, and medical measures/advisory in practice across the globe are elaborated. from a bird’s eye view, the covid-19 pandemic management relies on revolutionizing the disease surveillance by incorporating artificial intelligence and data analytics, boosting the response strategies—extensive testing, case isolation, contact tracing, and social distancing—and promoting awareness and access to pharmaceutical and non-pharmaceutical interventions, which are discussed in the present chapter. we also preview the economic bearing of the covid-19 pandemic. the present-day pandemic spotlight on covid-19 (coronavirus disease-2019) was earlier placed on zika virus, h1n1, severe acute respiratory syndrome (sars), chikangunya, middle east respiratory syndrome (mers), and ebola. the "advancements" of the human race-increased urbanization, global travel, changes in land use, and fervent exploitation of the nature-are also the prime reasons for zoonosis and emergence of novel infectious diseases such as above (madhav et al. 2017; ahmed et al. 2019) . this rapid emergence of novel infectious diseases transmitting from surrounding animal life to humans and then from human to human, traveling quickly across the globe can trigger worldwide public health emergency situations, as prevalent today (https://www.who.int/dg/speeches/detail/who-director-general-sopening-remarks-at-the-media-briefing-on-covid-19-11march2020, http://www. emro.who.int/fr/about-who/rc61/zoonotic-diseases.html). the world health organization (who) declared covid-19 a pandemic on march 11, 2020. mesh database defines pandemics as-"epidemics of infectious disease that have spread to many countries, often more than one continent, and usually affecting a large number of people." such emergencies compromise human health, society, economics, and politics-a case in point: the covid-19 pandemic is forecasted to cost the global economy one trillion us dollars (https://www.ncbi.nlm.nih.gov/mesh/? term¼pandemics, https://news.un.org/en/story/2020/03/1059011). as against the earlier guidelines of who where it classified a pandemic into six stages, the 2009 revision in pandemic descriptors and stages stands today as follows: • predominantly animal infections, few human infections. this corresponds with the stages 1-3 of earlier classification, starting with phase 1 where the virus is in its animal host and has caused no known infection in humans, phase 2 where zoonosis has occurred and the virus has caused infection in humans, and phase 3 where sporadic cases or clusters of infectious disease occur in humans. humanto-human transmission is limited in time and space and is insufficient to cause community-level outbreaks. • sustained human-human transmission. corresponds with the stage 4 of the classical description wherein animal-human and human-human transmissions have sustained a community-level outbreak. the risk for pandemic is greatly increased. • widespread human infection or the stage 5-6 from the classical description where the same identified virus has caused a community-level outbreak in another country in another who region. • post-peak period where there exists a possibility of recurrence of infection. • post-pandemic phase when the disease activity is seasonal (https://web.archive. org/web/20110910112007/http://www.who.int/csr/disease/influenza/ gipa3aidememoire.pdf, https://www.reuters.com/article/uk-china-health-who-idukkcn20i0pd). at the time of writing this chapter, coronavirus-2 (cov-2)/covid-19, though originated in wuhan, china, the first case being reported in november 2019, had pervaded africa, americas, europe, south-east asia, eastern mediterranean, and the western pacific nations with 191,127 confirmed cases of covid-19 and claimed 7807 lives, globally (https://www.who.int/docs/default-source/coronaviruse/situa tion-reports/20200318-sitrep-58-covid-19.pdf?sfvrsn¼20876712_2). europe was declared the new epicenter of the pandemic on march 13, 2020. the number of new cases in china though declining and is believed to be in post-peak stage, the numbers are alarmingly increasing worldwide (https://www.nbcnews.com/health/ health-news/europe-now-epicenter-pandemic-who-says-n1158341). who and other leading epidemiology organizations unanimously agree on the indispensable role of pandemic preparation and planning at global and national levels to mitigate through the present public health emergency of covid-19 and any future outbreaks. pandemic preparation is not a job of single individual or organization. it requires inputs from each person susceptible to the infection agent as well as policy makers at national and international levels, frontline healthcare providers, infrastructure developers and maintenance personnel, pharmaceutical industry and researcher community, and so forth. moreover, the pandemic preparedness plan needs constant reviewing and improvisation (https://www.ecdc.europa.eu/ en/seasonal-influenza/preparedness/why-pandemic-preparedness). in line with the magnitude of the covid-19 pandemic, worldwide action plans have been activated on national and international levels. the united nations' strategic preparedness and response plan (sprs) against covid-19, in layman terms, is designed to control human-human transmission, preventing outbreaks and delaying spread; provide optimal care for all patients; and minimize the impact on healthcare systems and socioeconomic activities. under sprs each nation is assessed for risk and vulnerability, and the resource requirements to support the country to prepare for and respond to covid-19 are estimated. several nations are well placed to implement this action plan with minimal support. however, otherwise partners are to be introduced to facilitate implementation of measures where there is a gap in capacity, on either a national or a subnational level, in additional support to national governments. thus, an extensive analysis and identification of an affected nation's gaps and needs shall be the basis to develop a covid-19 country preparedness and response plan (cprp). these cprps will need constant monitoring and reviewing using indicators charted in the sprp and updated as the situation evolves (https:// www.who.int/docs/default-source/coronaviruse/covid-19-sprp-unct-guidelines.pdf). grossly, the extent of success of each pandemic action plan stands on the following pillars: • surveillance of coronavirus-2 and covid-19 infection: characterization of the virus, infection modes, diagnosing and detecting infection, contact tracing, annotation of data from confirmed cases, predicting mass infection outbreak, keeping a count, and estimation of mortality. • response management: bulk production and supply of protective/preventive pharmaceutical interventions or non-pharmaceutical interventions. • facilitating timely medical help: access to hospitals/healthcare providers, personal and public hygiene, disinfection, and quarantine services. • lesson learning from the present outbreak of covid-19 to facilitate future action plans and preparedness. hereafter we discuss the present salient strategies under the aegis of the covid-19 pandemic preparation plan, globally, which are helping the humankind mitigate through this emergency. we also discuss the impact of covid-19 on world economy and its bearing on future preparedness plans. as defined by the who, surveillance during pandemics is defined as "the ongoing collection, interpretation and dissemination of data to enable the development and implementation of evidence-based interventions during a pandemic event" (https:// www.who.int/influenza/preparedness/pandemic/who_guidance_for_surveil lance_during_an_influenza_pandemic_082017.pdf). the present-day key worldwide surveillance activities against covid-19 include: (a) detection of coronavirus-2 and verification of covid-19 (b) risk and severity assessment (c) monitoring the pandemic the rapidly expanding array of pcr/reverse transcriptase pcr-based diagnostics which are quick and efficient in identifying the virus (pang et al. 2020 ; lake 2020, https://www.finddx.org/covid-19/) are basic requirements for surveillance at every phase of the pandemic for identifying and segregating the infected from non-infected and risk assessment, as well as monitoring recurrences and seasonal disease activity. covid-19 diagnostics have been discussed previously in this publication. an emerging exciting field of disease surveillance is infectious disease modeling and incorporation of artificial intelligence. notably, these are predictive techniques applicable to each of the three facets of disease surveillance (siettos and russo 2013) . a classical epidemiological surveillance parameter is quantitation of r 0 (r nought, basic reproductive number) using mathematical models (https:// wwwnc.cdc.gov/eid/article/25/1/17-1901_article). r 0 is a crucial metric indicating that on average the number of new infection cases are generated by a confirmed infection case, i.e., the potential transmissibility of an infectious disease. r 0 of covid-19 infection is estimated as 2-3.5 in the early phase, as even the asymptomatic patients or with mild pneumonia extruded large amounts of virus . important characteristics of r 0 are: • it is a dynamic number and changes with -each stage of the disease -with interventions, e.g., vaccination, antivirals -precautionary measures such as personal/community disinfection, social distancing, and travel restrictions • with the knowledge of r 0 , one can predict: -new cases expected on a daily basis, and hence facilitate arrangement of healthcare services and interventions locally -outbreak size and the dates of peak infection of the pandemic -probable decline timeline -the extent of vaccination coverage required to prevent future outbreaks • an r 0 > 1 indicates that each infected individual is transmitting the disease to more than 1 new individual, and the infection is spreading increasingly. r 0 ¼ 1 indicates stable transmission and r 0 < 1 indicates the decline in disease transmission ( fig. 14.1 ). the aim of pandemic action plans is to monitor and depreciate the r 0. for instance, the median daily r 0 in wuhan declined from 2.35 a week before the introduction of travel restrictions (january 23, 2020) to 1.05 one week later. the study used a stochastic transmission model of cov-2 transmission with four datasets from within and outside wuhan and estimated how transmission in wuhan varied between december 2019 and february 2020 (kucharski et al. 2020) . the most prominent mathematical model of covid-19 infection is the seir (susceptible-exposed-infected-recovered) model put forth by wu and coworkers and also endorsed by the who. this model estimates the size of epidemic in wuhan between december 2019 and january 2020 and forecasts the extent of domestic and global public health risks of taking into account for social and non-pharmaceutical prevention interventions. it is a compartmental model comprising four compartments and the individuals comprising the sample population move through each compartment-"susceptible" (not immune to infection) and get infected from other infection individuals and move to the "exposed" compartment for the incubation period. hereafter the infectious individuals move to the "infected" compartment and eventually to the "recovered" compartment after the disease has run its course, and they now have some immunity (fig. 14. 2). the changes in the population in each compartment are estimated using ordinary differential equations to simulate the progression of an infectious disease. the critical parameters associated with this model are: • force of infection (λ) is the rate at which susceptible individuals are exposed. it depends on the transmission rate (β). • incubation rate (e) is the rate at which exposed people become infectious. • recovery rate (γ) is the rate at which infected individuals recover from the infection. through this model, it was predicted that the r 0 was 2.68, each confirmed case infected 2-3 other people, and the epidemic doubling time was 6.4 days. also, the size of the outbreak in wuhan was estimated to be up to 75,815 people (statistical uncertainty presented at 95% credible intervals). most striking feature of this model was that it took into account the travel data from and to wuhan over the period of study. thus, it was able to predict that multiple major chinese cities-guangzhou, beijing, shanghai, and shenzhen-had already imported the infection to trigger local epidemics. it also recommended that controlling the transmissibility by 25-50% could eventually rein the local epidemics, and a control of 63% would phase out the epidemics . the application of artificial intelligence (ai) in close conjunction with technology in pandemic surveillance has demonstrated manifold advantages in surveillance activities as exemplified by china and other severely hit nations during the present covid-19 outbreak. ai, data analytics, and technological support amalgamated to facilitate: • track and forecast community outbreaks: bluedot is a canadian ai company using natural language processing and machine learning algorithms to monitor news outlets, worldwide official healthcare reports in several different languages, and air-travel data and flag the mention of contagious or novel diseases such as coronavirus. importantly this is followed by scrutiny by epidemiologists and thus also has a component of human analytics. bluedot alerted its clients to the potential outbreak in wuhan, china, on december 30, 2019, 9 days prior to the who recognized it as an epidemic (https://bluedot.global/products/). chest computed tomography (ct) scans have been endorsed as a primary diagnostic tool for covid-19 (ai et al. 2020 ). ali-baba group's research academy has developed a deep-learning ai-enabled system that can diagnose covid-19 in 20 s with 96% accuracy and hence possibly automate the diagnosis activity in the face of overburdened healthcare systems. the ai system identifies an infectious individual based on the chest ct scans. the algorithm has been trained with data and ct scans from nearly 5000 confirmed coronavirus cases from across china. it can be used to track the efficacy of treatment during the course of infection as well as rapidly diagnose covid-19 (https://www.alizila. com/how-damo-academys-ai-system-detects-coronavirus-cases/). • implementing public hygiene guidelines: risk communication in places of potential communication is critical to alert the public and implement mass hygiene measures such as use of sanitizers and face masks when in public places. google trends was used in taiwan to monitor the public risk awareness following the first imported case of covid-19 which correlated with the increased search keywords "covid-19" and "face masks." moreover, search for "handwashing" increased coinciding with the face mask shortage. high to moderate correlations between google relative search volume and covid-19 cases were evident in several major cities of taiwan (husnayain et al. 2020) . similarly, in china an ai-based company sensetime has developed a "smart ai epidemic prevention solutions"-a quick and effective system based on facial recognition and thermal imaging to screen for individuals with fever in a crowd without physical contact and hence preventing transmission. it can also monitor if any individual is wearing a face mask or violating the quarantine rules (https://www.sensetime.com/en/news/view/id/140.html). health code, a chinese government monitoring system, for which users can sign up through alipay or wechat, assigns individuals a color code (red-14 days self-quarantine/yellow-7 days self-quarantine/green-free movement) based on their travel history, time spent in outbreak hotspots and exposure to potential carriers of the virus. the software can be used to check the color of an individual on entering their identity numbers. • characterization of the cov-2 and vaccine/therapy development: the genome detective coronavirus typing tool is a web-based, user-friendly software application that can identify the novel severe acute respiratory syndrome (sars)-related coronavirus (sars-cov-2) sequences isolated in the original outbreak in china and later around the world. the tool accepts 2000 sequences per submission, analyzing them in approximately 1 min. this tool facilitates tracking of new viral mutations as the outbreak expands globally, which may help to accelerate the development of novel diagnostics, drugs, and vaccines to stop the covid-19 disease (cleemput et al. 2020 ). google's ai platform deepmind-based protein structure prediction tool alphafold has predicted and released the 3d structures of several understudied proteins of the cov-2 as an open source. these can be useful in designing antivirals/vaccines against covid-19 in future outbreaks (jumper et al. 2020) . in summary, the new-age pandemic surveillance using ai, data analytics, mathematical modeling of infectious diseases, and risk prediction has significantly contributed to the management of the present covid-19 pandemic and is laying the foundation for future improvisation of the pandemic action plans. critical issues to be addressed while recruiting laboratory diagnostic services during the novel covid-19 pandemic are: • the authenticity of the diagnostic tool in light of the novelty of the coronavirus-2 • bulk of samples to be processed • the ease of obtaining the sample for use in the diagnostic tool the coronavirus causing covid-19 was first isolated from a clinical sample on january 7, 2020, and within weeks, several reliable and sensitive diagnostic tools were developed and deployed. by mid-january, the first rt-pcr assays for covid-19 were accessible in hubei. the viral sequences and pcr primers and probe sequences were open-sourced and uploaded to public platforms by the centre for disease control, china. by february, there were ten kits for the detection of covid-19 approved in china by the national medical products association-six were rt-pcr kits, one isothermal amplification kit, one virus sequencing product, and two colloidal gold antibody detection kits (https://www.who.int/docs/defaultsource/coronaviruse/who-china-joint-mission-on-covid-19-final-report.pdf). as of today, when the disease has assumed pandemic proportions, the volume of diagnostic tools needs to be multiplied, and hence, the united states food and drug administration (usfda) has provided regulatory relief to several testing companies like thermofisher, hologic, and labcorp under the emergency use authorizations, to facilitate ease of diagnosis. also it is keeping a tight watch on fraudulent companies claiming to sell interventions against covid-19 (https://www.fda.gov/ emergency-preparedness-and-response/mcm-issues/coronavirus-disease-2019covid-19). moreover, the world's first crispr (clustered regularly interspaced short palindromic repeats)-based diagnostic kit has been developed for covid-19 by mammoth biosciences, usa, and university of california. notably, the diagnostic kit is a simple strip-based assay and is easy to use and allows rapid detection without the need of transporting samples over long distances. the kit is still under approval evaluation by the fda (https://www.medrxiv.org/content/10.1101/2020.03.06. 20032334v1). nose and mouth swabs, the most widespread samples for cov-2 diagnostics, require trained personnel to procure samples. however, the study by to and coworkers recommends saliva as an easy-to-procure, noninvasive sample not requiring any trained personnel in covid-19 screening. they detected cov-2 in 11 out of 12 patient samples and also could trace the declining titers post-hospitalization . additionally, a long-term goal of evolving the diagnostics for this novel disease is to develop a prognostic marker of covid-19. although it is too soon to say, qu and coworkers have proposed platelet counts and platelet to lymphocyte ratios as prognostic marker to distinguish between severe and non-severe patients. severe patients had higher platelet peaking and platelet to lymphocyte ratio correlating with deranged chest ct and longer hospital stays against the lower platelet peaks and platelet to lymphocyte ratios and lesser hospitalization stay (qu et al. 2020 ). vaccines and antiviral drugs are the prophylactic and therapeutic measures against a viral disease cov-2. on march 17, 2020, pfizer inc. and biontech announced to co-develop and distribute a potential mrna-based coronavirus vaccine which is likely to enter clinical testing by the end of april 2020 (https://www.pfizer.com/ news/press-release/press-release-detail/pfizer_and_biontech_to_co_develop_poten tial_covid_19_vaccine). however, in pandemic scenarios of new infectious diseases such as covid-19, vaccine supplies will be limited or nonexistent at the early phase in lieu of the novelty of the disease and the unpredictability of the pandemic occurrence. thus, vaccines cannot be stockpiled, and production can only start once the novel virus has been recognized. with the current state-of the-art, the first doses of vaccine are not likely to become available in the early months of the pandemic. however, the pandemic action plans have accounted for forward planning to increase the likelihood that the vaccine will progressively become available as the pandemic unfolds. importantly, national or regional priorities need to be fixed in the action plan for the rational use of the building/limited supply of the novel vaccine. also, production and use of vaccines during the inter-pandemic period will influence their availability during a pandemic. thus, improving the infrastructure and logistics for vaccine production, administration, cold-chain, and professional training with the novel vaccines are important to avert/cruise through future outbreak events. the who advisory on prioritizing the population groups are as enlisted in descending order. however, the priorities need tailoring in each country/region according to local needs and epidemiological circumstances. recommended prioritizing of the groups is as follows: • healthcare workers and essential service providers • groups at high risk of death and severe complications requiring hospitalization • individuals (adults and children aged more than 6 months) in the community who have chronic cardiovascular, pulmonary, metabolic or renal disease, or are immunocompromised • persons without risk factors for complications (https://www.who.int/csr/ resources/publications/influenza/11_29_01_a.pdf) antivirals are a crucial adjunct to vaccination as a potential strategy for managing covid-19. several drugs such as chloroquine, arbidol, remdesivir, and favipiravir are currently undergoing clinical studies to establish their efficacy and safety against covid-19. it is important to establish a regular supply chain of antivirals and a high surge capacity in the face of cov-2 pandemic and future outbreaks. antivirals have a significant impact in reducing morbidity and mortality in light of unlikelihood of availability of vaccine against in early phases of pandemic. it is important to evaluate the non-interference of the antiviral interventions with the eventual vaccination, as well as the epidemiology of the group of individuals most seriously affected. it is also advisable to make available the information about the performance characteristics, side effects, and costs of antiviral therapy to public. also the commonly used neuraminidase inhibitors in influenza pandemics are ineffective in the case of cov-2 mediated infection and outbreaks (pang et al. 2020; dong et al. 2020a , https://www. who.int/csr/resources/publications/influenza/11_29_01_a.pdf). the incorporation of antiviral therapy can be categorized as a prophylactic and for treatment use. as with vaccines, prioritizing of groups for antiviral therapy is advised as follows: • essential service providers, including healthcare workers (prophylaxis or treatment). especially, healthcare providers are in a position to be in direct contact with infectious individuals and are thus entitled to priority antiviral therapy. other community services such as those responsible for vaccine manufacture and delivery and personnel responsible for enforcing law and order and public safety. • groups at high risk of death and severe complications requiring hospitalization. the goal of prophylaxis or treatment here is to rein the mortality and morbidity. thus, high-risk persons living in the community outbreaks, seriously ill hospitalized patients, patients for whom a potential cov-2 vaccination is contraindicated are prioritized. • persons without known risk factors for complications from covid-19. here the approach is generally therapeutic and aims to rein the morbidity and rationalize the use of healthcare resources such as antibiotics. though, the logistics of this strategy are extensive and expensive (requires large quantities of antivirals and access to healthcare service providers), it is most likely to limit the economic and social destabilization associated with a pandemic (monto 2006; henry 2019, https://www.who.int/csr/resources/publications/influenza/11_29_01_a.pdf). a major step in ensuring bulk supplies of antivirals and vaccines is the setting up of medical stockpiles as a part of the pandemic preparedness plans. the usa has constituted a strategic national stockpile which is the nation's largest supply of potentially life-saving pharmaceuticals and medical supplies-antibiotics, chemical antidotes, antitoxins, vaccines, life-support medication, iv administrations, airway maintenance supplies, and other emergency medical and surgical items, for use in a public health emergency severe enough to deplete the local supplies. the facility also houses a data bank of other stockpiles and supply agencies, so that any emergency requirements can be procured in the shortest possible time (https:// www.phe.gov/about/sns/pages/default.aspx). healthcare personnel, maintenance of hygiene and disinfection during pandemics • rapid facilitation of treatment/prophylaxis, hospital centers, quarantine centers • caring for the patients as well as the health service providers to ensure uninterrupted care • communicating awareness about public and personal hygiene and implementing measure to ensure personal and public hygiene in the face of a highly infectious disease causing novel virus, china has successfully executed an ambitious, swift, and aggressive disease containment effort in the history of mankind. the laudable of its responses to facilitate timely medical care for infected was construction of two dedicated hospitals-1000-bed huoshenshan facility and the 1600-bed leishenshan hospital in 2 weeks (https://www.wsj.com/arti cles/how-china-can-build-a-coronavirus-hospital-in-10-days-11580397751). moreover, it ensured coordinated medical supplies, reserve beds were used and relevant premises were repurposed medical care facilities, and prices of commodities were controlled to ensure the smooth operation of the society (https://www.who.int/docs/ default-source/coronaviruse/who-china-joint-mission-on-covid-19-final-report.pdf). ensuring the care of the medical service providers is a key response strategy to warrant efficient care for the general public. under the china's response plan, the healthcare workers were facilitated with personal protective equipment. nosocomial infections accounted were reported to be nearly 2055 from 476 hospitals across china. the majority of this nosocomial infection (88%) were reported from hubei. a deeper contact tracing indicates infection of the healthcare worker from households than the workplace and were pinpointed to the early stages of the epidemic when understanding of the covid-19 transmission and medical supplies were limited (https://www.who.int/docs/default-source/coronaviruse/who-china-joint-mis sion-on-covid-19-final-report.pdf). in fact a novel infection control system for averting nosocomial infections of covid-19 was proposed by chen and coworkers, titled "the observing system." designated personnel called "infection control observers" were appointed by the department of infection control and nursing in guangdong second provincial general hospital who underwent training to familiarize infection control requirements in the negative pressure isolation wards. the wards were under camera surveillance and the infection control observer monitors medical staff in real-time via computer monitors outside the ward. the observer ensures normal operation of the negative pressure isolation wards, supervise the implementation of disinfection, ensure a sufficient supply of protective materials, arrange specimens for inspection, and relieve anxiety of the medical personnel while treating patients . personal and public hygiene/disinfection implementation is a key step to control transmission and prevent community outbreaks. good hand hygiene and respiratory hygiene have been aggressively promoted by who worldwide in this covid-19 pandemic. the basic personal and public hygiene practices are depicted in fig. 14.3. fig. 14.3 the basic personal and public hygiene practices a key parameter to consider while disinfecting in households and public places hence avoid contact with infected surfaces is the estimation of decay rates of cov-2 in aerosols and on various materials composing the surfaces. it has been estimated that cov-2 in an aerosol (<5 μm, similar to those observed in samples obtained from the upper and lower respiratory tract in humans) was viable for up to 3 h, up to 4 h on copper surfaces, up to 24 h on cardboard, and 2-3 days on plastic and stainless steel (van doremalen et al. 2020 ). on the basis of these findings, disinfection protocols can be set up for public places/medical facilities depending on the surfaces involved, and even in households' frequently touched surfaces like door handles, slabs, and tables be disinfected. the exponential transmission of cov-2-starting with few infectious individuals with covid-19 quickly increasing manifold in a geographical location within a short time-is a recurring observation (dong et al. 2020b , https://www.who.int/ emergencies/diseases/novel-coronavirus-2019/situation-reports, https://www.ecdc. europa.eu/en/publications-data/download-todays-data-geographic-distributioncovid-19-cases-worldwide). figure 14 .4 depicts the exponential surge in new confirmed cases per day plotted against time lapse post outbreak. the highly implemented global covid-19 management strategies of social distancing, travel restrictions, implementation of personal and public hygiene (non-pharmaceutical interventions), and pursuit of pharmaceutical interventions aim to delay the peaking of the outbreak, avoid burden on the healthcare infrastructure and personnel to ensure quality care for all in need, and rein overall mortality and declined health effects. this phenomenon has been described as "flattening of the curve" and is much shared on social media platforms facilitating awareness in the public (https://www.cdc.gov/flu/pandemic-resources/ pdf/community_mitigation-sm.pdf). hereafter, we discuss the key, successful globally adopted strategies for covid-19 management. it is a non-pharmaceutical infection prevention and control intervention to avoid or control contact between infectious and uninfected individuals to rein the disease transmission in a community eventually culminating into decreased infection spread, morbidity and mortality. the individuals infected with cov-2 shed the virus from their respiratory tract during the early infection stage when there are minor clinical manifestations leading to the extensive community transmission. while practicing social distancing, each healthy individual behaves like an infected individual, self-restricting contact with others. the main advantage is gleaned when an individual in incubation period/early infectious stage of covid-19 restricts coming in contact with other healthy individuals. this strategy involves the policymakers, and strict advisories/orders are issued under the pandemic action plan to restrict public gatherings and events, shutting down of educational institutes/offices/restaurants, avoiding nonessential travel and use of public transport, maintaining a distance of 1 m at least between two individuals, restricting visit to hospitals, and avoiding online shopping. additionally, the elderly and individuals with hypertension, cardiovascular disease, diabetes, chronic respiratory diseases, and cancer are at a higher mortality risk (https://www.mohfw.gov.in/socialdistancingadvisorybymohfw. pdf, https://onlinelibrary.wiley.com/doi/pdf/10.1111/ijcp.13501; zou et al. 2020) . the who director-general at a media briefing on covid-19 on march 16, 2020, recommends testing of any suspected case, isolation to break the chain of transmission, and contact tracing for the prior 2 days the subject came in contact with and then testing them as well (https://www.who.int/dg/speeches/detail/who-director-gen eral-s-opening-remarks-at-the-media-briefing-on-covid-19%2d%2d-16-march-2020). a case in point-south korea carried out 286,716 covid-19 tests until march 17, 2020-second highest across the world trailing behind china (https:// ourworldindata.org/coronavirus-testing-source-data), combined with strict social distancing implementation and is now seeing a fall in the number of covid-19 cases (https://ourworldindata.org/grapher/daily-cases-covid-19-who?time¼1..52& country¼kor, https://www.sciencemag.org/news/2020/03/coronavirus-caseshave-dropped-sharply-south-korea-whats-secret-its-success). social distancing combined with case isolation and contact tracing has demonstrated its effectiveness in controlling the transmission from imported infection cases to the community transmission scenario (wilder-smith and freedman 2020). the relevance of case isolation and contact tracing are highlighted in a study by hu and coworkers where they have performed a clinical characterization of 24 asymptomatic patientsconfirming infection by a laboratory-confirmed positive for the covid-19 virus nucleic acid from pharyngeal swab samples. the asymptomatic cases did not present any obvious symptoms while nucleic acid screening. about 20.8% developed classical symptoms of fever, cough, and fatigue, during hospitalization; 50.0% presented ct images of ground-glass chest and 20.8% presented stripe shadowing in the lungs; 29.2% cases presented normal ct image and had no symptoms during hospitalization, comprising the younger subset (median age: 14.0 years). none developed severe covid-19 pneumonia, and no mortality was observed. however, epidemiological investigation indicated at typical asymptomatic transmission to the cohabiting family members, which even caused severe covid-19 pneumonia. this study puts a spotlight on close contact tracing and longitudinally surveillance via virus nucleic acid tests. case isolation and continuous nucleic acid tests are also recommended (hu et al. 2020) . de-isolating of suspect cases where the first confirmatory test has returned negative is also an emerging issue of concern and needs improvement in testing capacity as well as strict implementation of social distancing during pandemic scenarios (tay et al. 2020 ). approaching january 13, 2020, the daily risk of exporting at least a one covid-19 infected individual from mainland china through international travel exceeded 95%. for containment of the global spread of cov-2 and a covid-19 epidemic, china implemented border control measures-airport screening and travel restrictions, and were later also adopted by several other countries. wells and coworkers in their study in proceedings of natural academy of sciences used daily incidence data of covid-19 outbreak from china from december 8, 2019, to february 15, 2020, as well as airline network data, to predict the number of exported cases with and without measures of travel restriction and screening. the group put forth that the lockdown of wuhan and 15 more cities in hubei province on january 23-24, 2020, averted export of 779 more covid-19 cases by mid-february. however, it is to be highlighted that these travel restrictions and lockdown measures only slowed the rate of infection exportation from china to other countries. the global spread of covid-19 was based on most cases arriving during the asymptomatic incubation period. the authors recommend rapid contact tracing at the epicenter and at importation sites to limit human-to-human transmission outside of the location of first outbreak (wells et al. 2020 ). the covid-19 pandemic in today's times of excessive electronic connectivity and several social media platforms has a potential to alleviate the global mental stress levels. especially, if local myths and rumors are circulated in the already homequarantined population, restriction in fear of the disease can lead to additional mass hysteria. thus it is important to create awareness and dispel any negative myth, as put forth by the who in context of cov-2 (https://www.who.int/emergencies/ diseases/novel-coronavirus-2019/advice-for-public/myth-busters). some myths dispelled are: • covid-19 virus can be transmitted in areas with hot and humid climates or cold and snowy climates. cov-2 can be transmitted in all areas. • hot bath cannot prevent cov-2 transmission. • cov-2 is not transmitted by mosquito bites. it is a respiratory illness and respiratory hygiene is a key protective strategy. • heat generated from hand dryers is not effective against killing cov-2 on your hands. the only way to disinfect hands is washing with soap-water or alcoholbased sanitizers. • thermal scanners are effective in detecting only the elevated body temperatures. a confirmatory nucleic acid test is the most guaranteed test. • all age groups can be infected by cov-2. elderly people and people with pre-existing medical conditions-asthma, diabetes, cardiovascular disease-are more vulnerable to exhibit severe covid-19. thus, who advises people from all age groups to protect themselves by implementing good hand hygiene and respiratory hygiene. • sars-cov-2 is not a bioengineered organism arising out of manipulations from earlier sars-cov. andersen and coworkers have analyzed the genome sequence of the novel coronavirus-2 and compared it with several other zoonotic viruses. they claim that the viral-human point of contact-the high affinity receptor binding domain of cov-2 binding with the angiotensin-converting enzyme 2 (ace2) of the target host-is acquired through natural selection. also they have shed light of probable animal host being rhinolophus affinis, as another virus ratg13, sampled from a bat, is~96% identical overall to cov-2 (https:// www.nature.com/articles/s41591-020-0820-9). the covid-19 pandemic can cause short-term fiscal distress and longer-term damage to the global economic growth. if we trace the economic trajectory of a pandemic: • early phase measures to contain/limit outbreak-shutting down of workplaces and public businesses, mobilization of healthcare supplies and surveillance activities, contact tracing, social distancing by isolation of contacts/quarantines incur significant human resource and staffing costs (achonu et al. 2005 ). • as the scale of the epidemic expands, new medical infrastructure will need to be constructed to manage building number of confirmed infected individuals, the surge in demand for medical consumables can increase the health system expenditures (herstein et al. 2016 ). • the quarantine and lockdown impositions disrupt trans-national supply chains, transportation industry, agriculture, entertainment, and travel industry. hoarding and black marketing of essential and medical commodities are expected. • behavioral changes during pandemic-avoiding association with other people to avoid infection-are the major determinant for economic impact of pandemics ( fig. 14.5) and not mortality. the behavioral change fear driven which in turn is driven by awareness and ignorance (burns et al. 2008 risk_jonas.pdf). • the high-income countries in scenario of a moderate pandemic can offset fiscal distress by providing official development assistance to affected countries and budgetary support. however, during a severe pandemic, a high-income country will also confront the same fiscal stresses and may be unwilling to provide assistance. on march 19, 2020, worldwide 191,127 confirmed cases were reported, mortality was 7807 against 118,319 confirmed cases, and mortality of 280 on march 13, 2020 when the covid-19 crisis was declared a pandemic (https://www.who.int/emergen cies/diseases/novel-coronavirus-2019/situation-reports). it is increasingly becoming evident that no region of the world will remain untouched by cov-2 invasion. however, pandemic preparedness is the key to tackle the present-day covid-19 health emergency worldwide. the rapid and effective enforcement of existing international and national action plans, as well as parallel review and improvisation, is facilitating the affected countries to contain transmission and possibly delay the peak of outbreak and mortality and garner recovery. it is notable that in the present pandemic scenario, innovative ai-powered surveillance, quick and strategic response actions-the trinity of testing-isolation-contact tracing, committed social distancing measures-travel restrictions, self-isolation, implementation of personal and public hygiene, and extensive mobilization of medical care facilities are helping the world mitigate through. the most insightful trend emerging from the global clinical and epidemiological data is that the identification of asymptomatic infected individuals is a crucial step to contain community outbreaks. for preventing future outbreaks of cov-2 infection, high-volume cutting-edge investigations are warranted in understanding the covid-19 pathology, cov-2 origin, biology, structural data of potential surface antigens, and precise anti-cov-2 antiviral therapies. although the global economy is suffering at the hands of cov-2, it is important to review the current action plans and suitably improvise the future action plans to avoid potential recurrence. pandemics are unforeseen. national and international preparedness are crucial to tackle a pandemic. world over, humanity is grappling with covid-19. the pandemic preparedness charter prescribed by international and national agencies to tackle covid-19 is evolving on the go. the key facets of pandemic preparedness emerging from global success and failure scenarios are: • active surveillance employing state-of-the art technology: -development of mathematical models for simulating the infection of cov-2 in a given country. the models can help predict the basic reproductive number which in turn facilitates monitoring the pandemic on day-to-day basis. -incorporation of contactless artificial intelligence-based technologies for mass thermal screening, track and forecast community outbreaks, implement public hygiene and use of masks, and contact tracing. • expanding the diagnose capacity/testing for cov-2 (the infectious agent) to catch any asymptomatic carriers, but at the same time ensuring: -precision of the test. -test is adaptable to processing bulk samples. -easy procurement of sample from suspected infected individuals. • management of bulk antiviral interventions and vaccines: -national or regional priorities need to be fixed for rational use of antiviral/emerging vaccines -prioritizing the population has been recommended: the healthcare workers and essential service providers are top priority. -medical stockpiles should be established. • promoting facilities for healthcare/healthcare workers and facilitating public disinfection and hygiene via: -rapid expansion of hospitalization facilities for treatment and isolation/ quarantine centers. -ensuring minimal nosocomial infections to the healthcare workers. -issuing and implementation of guidelines for ensuring good hand and respiratory hygiene. (continued) -public disinfection (and personal household as well) based on the established life of cov-2 on different surfaces. • globally successful strategies are: -social distancing and lockdowns to ensure minimal human-human contact. -travel restrictions to facilitate containment. -trifecta of test-isolate-contact tracing. • the economic cost of the covid-19 pandemic management is expanding due to: -disruption of economic activities due to implementation of social distancing via lockdowns. -battling the diseased and the increasing mortality. -diversion of resources to expansion of healthcare systems. -international and national relief funds are being constituted. -fiscal reliefs and aid from developed nations to the developing or underdeveloped countries can release the surmounting economic pressure. the financial impact of controlling a respiratory virus outbreak in a teaching hospital: lessons learned from sars does urbanization make emergence of zoonosis more likely? evidence, 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implicate special control measures pii: 202002616) impact of international travel and border control measureson the global spread of the novel 2019 coronavirus outbreak isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus (2019-ncov) outbreak nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study sars-cov-2 viral load in upper respiratory specimens of infected patients key: cord-017583-72mbsib7 authors: devarajan, padma v.; dawre, shilpa m.; dutta, rinku title: infectious diseases: need for targeted drug delivery date: 2014-09-01 journal: targeted drug delivery : concepts and design doi: 10.1007/978-3-319-11355-5_3 sha: doc_id: 17583 cord_uid: 72mbsib7 infectious diseases are a leading cause of death worldwide, with the constant fear of global epidemics. it is indeed an irony that the reticuloendothelial system (res), the body’s major defence system, is the primary site for intracellular infections which are more difficult to treat. pro-inflammatory m1 macrophages play an important role in defence. however, ingenious pathogen survival mechanisms including phagolysosome destruction enable their persistence. microbial biofilms present additional challenges. low intracellular drug concentrations, drug efflux by efflux pumps and/or enzymatic degradation, emergence of multi-drug resistance (mdr), are serious limitations of conventional therapy. targeted delivery using nanocarriers, and passive and active targeting strategies could provide quantum increase in intracellular drug concentration. receptor mediated endocytosis using appropriate ligands is a viable approach. liposomes and polymeric/lipidic nanoparticles, dendrimers micelles and micro/nanoemulsions could all be relied upon. specialised targeting approaches are demonstrated for important diseases like tuberculosis, hiv and malaria. application of targeted delivery in the treatment of veterinary infections is exemplified and future possibilities indicated. the chapter thus provides an overview on important aspects of infectious diseases and the challenges therein, while stressing on the promise of targeted drug delivery in augmenting therapy of infectious diseases. of the microorganism. intracellular infections result when the organisms cleverly evade destruction following phagocytosis. the intracellular location of these microorganisms protects them from the host defence mechanisms, such as antibodies or complement, and from the action of drugs that are unable to penetrate the cell effi ciently. hence, while adequate drug concentrations are readily achieved at extracellular infection sites to enable effi cient therapy, intracellular infections are more diffi cult to treat. some common intracellular and extracellular infectious diseases and their causative organisms are listed in table 3 .1 . the res also known as the mononuclear phagocytic system (mps)/macrophage system is the primary defence mechanism of the human body and hence the site of intracellular infections. the macrophages constitute the major defence cells of the res. derived from the bone marrow the res also contributes to both non-specifi c and specifi c immunity. recognition by the res is facilitated by opsonins, with the step of opsonisation being a precursor to phagocytosis. opsonisation is the process by which bacteria are altered by opsonins so as to become more readily and effi ciently engulfed by phagocytosis. opsonisation is mediated by the complement system: c3b, c4b, and ic3b, antibodies igg and igm and mannose-binding lectin. mannose binding lectin initiates the formation of c3b. opsonisation of particles enables recognition by the fc receptors, complement receptors or specifi c receptors for phagocytosis. opsonins are generally proteins which can bind to pattern-recognition receptors (prrs) or other specifi c receptors expressed on the surface of macrophages. pentraxins [c-reactive protein and serum amyloid p] [ 1 ] , mindin, collectins [ 2 ] and fi colins [ 3 ] are such opsonins. the function of pattern-recognition receptors (prrs) is to recognise and enhance phagocytosis of pathogen-associated molecular patterns (pamps), specifi c patterns present on microbial pathogens like lipopolysaccharide (lps) in gram-negative bacteria, lipotechoic acid (lta) in gram-positive bacteria and mannans in yeast. toll-like receptors (tlrs) are prrs essential for recognition of microbial components such as tlr4 (lps) [ 4 -6 ] , tlr3 [double-stranded rna] [ 7 ] , tlr6 [mycoplasmal macrophage-activating lipopeptide-2 kda] [ 8 ] , tlr9 [cpg bacterial dna] [ 9 ] , tlr5 [bacterial fl agellin] [ 10 ] , and tlr2 [peptidoglycan] . however, the exact mechanisms of tlr recognition of microbial components remain unclear. opsonisation facilitates adherence of pathogens to macrophages, and is facilitated by integrins. adherence induces membrane protrusions, called pseudopodia, to extend around the attached material. following fusion with the macrophage, the pseudopodia forms a phagosome that encloses the pathogen within a membrane, which then enters the endocytic process. phagosomes coalesce with intracellular organelles to mature into phagolysosomes, which have an acidic environment with many digestive proteins which fi nally degrades the internalised material. phagocytised material is eliminated by exocytosis. the process of phagocytosis is mediated by several proteins such as actin, dynamin and cortactin. while actin is connected to the lipidic membrane and responsible for invagination of the membrane to form the endosome, cortactin is an actin-binding protein which stimulates its polymerisation. dynamin hydrolyses guanidine triphosphate and uses the resulting energy for the contraction of actin and formation of endosome. particulates that cannot be digested remain sequestered in residual bodies within the cell. other cells such as fi broblast, endothelial and epithelial cells also exhibit phagocytic activity and can engulf microbes like shigella, listeria and yersinia [ 11 ] . such phagocytosis is mediated by laminin and fi bronectin receptors/heparan sulfate present on the membrane surface [ 11 ] . however, the major cells responsible for phagocytosis are macrophages. macrophages (greek: makros means large and phagein means eat) are cells formed by the differentiation of monocytes in tissues. macrophages play an important role in both innate and adaptive immunity in vertebrates. these specialised phagocytic cells engulf and destroy infectious microbes, foreign particles and cancer cells [ 12 ] . the macrophages also regulate lymphocyte, granulocyte populations and important tumor growth modulators [ 13 ] . macrophages act by both oxygen-dependent killing and oxygen independent killing mechanisms. the mediators for oxygen-dependent killing are reactive oxygen intermediates (rois) (superoxide anion, hydroxyl radicals, hydrogen peroxide and hypochlorite anion), reactive nitrogen intermediates (rnis) (nitric oxide, nitrogen dioxide and nitrous acid) and monochloramine, while the mediators for oxygen independent killing are defensins, tumor necrosis factor (macrophage only), lysozyme and hydrolytic enzymes. floating macrophages predominate in the vascular system, while tissue macrophages are localised in specifi c tissues. based on the tissue of residence they have specifi c nomenclature ( fig. 3.1 ). macrophages can be classifi ed mainly into two groups: (1) pro-infl ammatory or classically activated macrophages (m1) and (2) anti-infl ammatory or alternatively activated macrophages (m2). m1 macrophages are immune effector cells that aggressively work against microbes and cause their destruction much more readily. m1 is mainly associated with gastrointestinal infections (e.g. typhoid fever and helicobacter pylori gastritis) and active tuberculosis. m1 macrophages are stimulated by interferon (ifn)-g or lipopolysaccharide (lps) to release nitric oxide (no), important for killing intracellular pathogens. activated macrophages are characterised by expression of major histocompatibility molecule like mhc class ii and cd86 and their ability to secrete proinfl ammatory cytokines such as tumor necrosis factor (tnf)-a, il-1b, il-12, il-18 and the chemokines ccl15, ccl20, cxcl8-11 and cxcl13 [ 14 ] . activated m1 macrophages facilitate killing of microorganisms by endocytosis, synthesising reactive oxygen intermediates (roi), limiting the uptake of nutrients and iron essential for the growth of bacteria and replication of viruses, or production of nitric oxide facilitated by ifn-g-inducible no synthase (inos). m2 macrophages are important for killing extracellular parasites, wound healing, tissue repair, and to turn-off immune system activation. m2 macrophages are activated by interleukin (il)-4 or il-13 (m2a) to produce il-10, transforming growth factor (tgf)-b and arginase-1 (arg1), to enable this function [ 14 ] . m2 macrophages are mostly observed in lepromatous leprosy, whipple's disease and localised infections (keratitis, chronic rhinosinusitis). a number of infectious organisms which manage to overcome the res defence develop unique adaptive mechanisms which enable them to survive within the cell for prolonged periods of time. eradication of such intracellular organisms poses immense challenges. many pathogens have an innate ability to develop adaptive mechanisms under stress conditions to fi ght for their survival. such adaptive mechanisms or protective strategies, enables them to exhibit greater defence to the host and there by prolong survival. the different adaptive mechanisms employed by pathogens are discussed below. strategies adopted by microorganisms to inhibit phagolysosome formation include interference with the transformation of primary endosomes into late endosome, fusion with lysosomes and or phagosome acidifi cation. this delays the fusion of endosomes with lysosomes [ 15 ] or blocks the same [ 16 ] . the strategies to inhibit phagolysosome formation and the pathogens which exhibit the same [ 17 ] are summarised in table 3 .2 . pathogens which exhibit this adaptation survive and multiply in vesicles formed by fusion of endosomes with cell organelles other than the lysosome, such as the rough endoplasmic reticulum, ribosome or mitochondria [ 29 ] and thus avoid phagolysosome formation. they thereby bypass destruction due to the enzymatic activity in the lysosome [ 30 ] . escape from endocytosis is a crucial step for intramacrophagic survival. pathogens from this category contain lytic enzymes which enable them to break the endosomes membrane and disrupt membrane of the vacuole [ 31 ] , and hence evade degradation in the phagolysosome, and enter the cytosol rich in nutrients [ 32 ] . specifi c enzymes are produced by the microorganisms for instance, l. monocytogenes pujol et al. [ 17 ] disturbs the formation of lipid rafts by producing beta-1,2 glucans brucella spp. brucellosis roy [ 27 ] alteration of host cell signaling by dephosphorylation of signal regulated kinase leishmania spp. leishmaniasis ghosh et al. [ 28 ] produces listeriolysin o (llo) [ 33 ] and haemolysin c [ 34 ] while phospholipases are produced by the rickettsia spp. [ 35 ] . the microbes in this category exhibit virulence factors which allow them to survive in lytic enzymes, acidic conditions and oxidants, the harsh conditions in the phagolysosome environment. intramacrophagic resistance employing multiple virulence factors enables alternative pathways for survival and multiplication [ 36 ] . pathogens are internalised into macrophages by alternate routes. they traverse inside the cell by receptor mediated pathways like clathrin [ 37 ] and lipid rafts [ 38 ] . formation of vesicles with new properties after fusion between the pathogen and membrane of the cell, like the parasitophorous vacuole formed by toxoplasma gondii [ 38 ] also provides protection. in certain infections successful fusion of microorganisms with the macrophage is followed by secretion of antiapoptotic molecules (e.g. bcl2). this results in impairment of apoptosis of the infected cells. in addition to the adaptive mechanisms certain microbes employ highly specifi c strategies for persistence inside the cell. such strategies are discussed with reference to some important diseases. the adaptive mechanisms of mycobacterium tuberculosis to survive inside the macrophages are prevention of fusion of the phagosome with lysosomes by producing tryptophan-aspartate-containing coat protein (taco). transformation of primary endosomes into phagolysosomes is prevented by a number of actions that occur simultaneously. these include reduced levels of proton atpase inside the endosomes [ 18 ] and scavenger receptors [ 59 , 60 ] have also been implicated in mycobacterial uptake. uptake of mycobacteria by the complement receptor pathway protects it from the aggressive lysosomal compartment ensuring relatively hospitable conditions. salmonella specifi cally forms a glycolipid capsule or biofi lm. biofi lm formation in salmonella is related to the multicellular and aggregative response of rdar [ 61 ] , rugose [ 62 ] , or lacy [ 63 ] . this multicellular behavior is a property of salmonellae [ 64 ] and is responsible for elaboration of thin fi mbriae like tafi , curli [ 65 ] , cellulose [ 66 ] , and other uncharacterised extracellular polysaccharides. together, these components form the extracellular matrix that confers resistance to acid and bleach and facilitates environmental persistence [ 62 , 64 , 67 -70 ] . pathogens which cause fungal infections adapt various mechanisms to increase their pathogenesis and survive inside macrophages. c. albicans contains superoxide dismutases (sod) and catalase enzymes which are able to convert o 2into molecular oxygen and hydrogen peroxide, thereby decreasing the scavenging and toxic effects of o 2and h 2 o 2 levels by certain reactions [ 71 ] . further, c. neoformans evade phagocytic uptake by phenotypic switching. this mechanism is observed in yeast cells that express glucuronoxylomannan mucoid capsule that resist phagocytic uptake and cause high lethality in mice [ 72 ] . in case of aspergillus conidia infection collectins, pentraxin proteins are essential for opsonisation, but their defi ciency is responsible for high susceptibility to infection in immunocompetent mice. furthermore, several enzymes such as elastases and proteases released by the fungus enable conidia to escape from phagocytic uptake by alveolar macrophages. in hiv-1-infected macrophages, the viral envelope protein induces macrophage colony-stimulating factor (m-csf). this pro-survival cytokine down regulates the trail (tumor necrosis factor-related apoptosis-inducing ligands) receptor and up regulates the anti-apoptotic genes bfl -1 and mcl-1 enabling hiv to survive inside the macrophages. hiv invades the macrophage through ccr5 a chemokine receptor and through binding of gp120 to cd4 [ 73 ] . macropinocytosis as a route of entry of hiv-1 into macrophages [ 74 ] also enables intracellular protection. leshmania prevent activation of macrophages by inhibiting secretion of cytokines such as the infl ammatory response il-1 and tumor necrosis factor beta (tnf-beta) or t-lymphocyte activation (il-12) and produce various immunosuppressive signaling molecules, such as arachidonic acid metabolites and the cytokines tnfbeta and il-10. l. chagasi induces tnf-beta production in the immediate environment of the infected human macrophage, and this may lead to inhibition of immune responses [ 75 ] . further, this pathogen induces alteration of host cell signaling. macrophages infected with l. donovani or l. mexicana have shown altered ca 2+ dependent responses, such as chemotaxis and production of roi [ 76 , 77 ] . based on the adaptive mechanisms microorganisms reside in different cells and at different locations in the cells. treating diseases therefore, necessitates an understanding of both the resident cells and target organelles. illustrative examples of microorganism and their cellular/organelles targets are listed out in table 3 .4 . the granulocytes are classifi ed as neutrophils, eosinophils, or basophils on the basis of cellular morphology. neutrophils play the major role in the body's defence. neutrophils are produced in the bone marrow by hematopoiesis. they are released into blood where they circulate for 7-10 h and migrate into tissues where they have a life span of a few days. during infection the bone marrow releases more than usual tularemia -cytosol al-khodor [ 91 ] number of neutrophils, which migrate to the site of the infection. they act by both oxygen-dependent and oxygen-independent pathways to kill microbes. neutrophils exhibit a larger respiratory burst than macrophages and consequently are able to generate more reactive oxygen intermediates and reactive nitrogen intermediates. in addition, neutrophils express higher levels of defensins than macrophages do. hence, neutrophils are more active than macrophages in killing ingested microorganisms. dendritic cells are antigen-presenting cells and constitute 0.5-1 % of the leukocyte population in the peripheral blood mononuclear cells. they are found mostly in nonlymphoid tissues and organs such as skin, heart, liver, lungs, and mucosal surfaces. the function of these cells is to initiate, stimulate and regulate a t cell response which includes antigen-specifi c t lymphocytes, th1/th2 modulation, regulatory t cell induction and peripheral t cell deletion. there are four types of dendritic cells, i.e. langerhans cells, myeloid dendritic cells, plasmacytoid dendritic cells and infi ltrating infl ammatory dendritic epidermal cells. cd1b, cd11a, cd11b and cd11c, the thrombospondin receptor (cd36), and the mannose receptor (cd206), present on infl ammatory dendritic epidermal cells, are known to be involved in the uptake of bacterial components. in case of mycobacterium tuberculosis infection, alveolar macrophages (dust cells), along with dendritic cells engulf bacteria and exhibit innate as well as an adaptive immune response. combined efforts by macrophages and dendritic cells establish protective immunity in 90 % of infected individuals. natural killer cells (nkc) are non-phagocytic cells present mostly in mammalian and avian species [ 92 ] . nkc express surface receptors for the fc portion of igg and their function is to mediate antibody-dependent cytotoxicity against tumor target cells [ 93 ] . it is also suggested that nkc play a role in resistance against some microbial infections. nkc also play a role in natural genetic resistance to infections caused by cytomegalovirus and herpes simplex type i [ 94 , 95 ] . however, there is also evidence against the role of nkc in resistance to some other viruses [ 96 ] . lymphocytes are cells present 99 % in the lymph and constitute 20-40 % of the body's white blood cells. there are approximately ~10 10 -10 12 lymphocytes in the human body, and this can vary with body weight and age. they circulate in the lymph and blood, and can migrate into tissue spaces and lymphoid organs, enabling integration with the immune system. the two main categories of lymphoid cells that can recognise and react against a wide range of specifi c antigens are b lymphocytes or b cells and t lymphocytes or t cells. the main function of b cells is to produce antibodies against antigens [ 97 ] . each of the approximately 1. natural t lymphocytes mature in the thymus region and survive in the periphery. the chief function of t cells is to respond to signals associated with tissue destruction and to minimise the collateral tissue damage they cause [ 98 ] . t cells express t-cell receptors (tcr) which are a composite of polypeptides including cd3 and either of one of the two membrane molecules, cd8 and cd4. tcr recognises virus infected cells and cancer cells. however, unlike b cells, tcr does not recognise free antigen, unless it is bound to mhc molecules on the membrane of antigen presenting cells. the main function of t cells is to induce death of virus infected cells by secretion of cytotoxins and cytokines which activates b cells, macrophages and cytotoxic t cells. t cells also play role in infectious diseases such as leishmaniasis [ 99 ] , infection by hepatitis c virus (hcv), etc. their ability to confi ne exuberant immune reactivity, associated with many chronic infections is benefi cial the host due to limited tissue damage [ 100 ] . infectious diseases are also located in cells other than cells of the res. such cells include hepatocytes, epithelial cells and erythrocytes. hepatocytes are located in the liver and are major site for infections such as hepatitis b/c and malaria. the hepatocytes are discussed in greater detail in chapter 6 of this book. epithelial cells bind together to form the epithelial tissue which is held together by adherens, tight junctions, gap junctions and desmosomes. the functions of epithelial cells are boundary and protection of vital organs, transportation, absorption, secretion, lubrication and movement. these epithelial cells can be readily attacked by microbes such as hiv virus, infl uenza, herpes simplex virus (hsv-2) and cause infections. furthermore, erythrocytes are infected and act as hosts for plasmodium causing malaria, one of the current fatal infections posing serious challenges. the introduction of antimicrobial agents such as penicillin resulted in a major breakthrough to decrease morbidity and mortality caused by infectious diseases. antibiotics represented one of the greatest discoveries. this euphoria was short lived due to adverse effects and the emergence of drug resistance. conventional therapy when associated with side effects or necessitates long term treatment, results in low patient compliance. further inadequate drug concentration within cells is a major barrier for effective treatment of intracellular diseases. increasing the dose, however, resulted in enhanced toxicity. mono-drug therapy evolved into multi-drug therapy, and enabled a good degree of success and continues to form standard therapy, even today. classic examples include the multi-drug combination for tuberculosis akt2, akt3, akt4 comprising 2, 3 or 4 drugs, respectively. the haart combination for aids and two drug combinations for malaria are also examples of successful therapy. nevertheless, the alarming rate at which drug resistance is occurring, and more so the emergence of multi-drug resistance are a matter of great concern. tuberculosis is one such major disease which has evolved from resistant to multi-drug resistant(mdr) to total drug resistant (tdr), the latest being extremely drug resistant tuberculosis (xxdr), wherein, resistance is seen to almost all known antitubercular drugs. the emergence of multi-drug resistance is attributed to a number of factors. pathogens resort to different mechanisms to avoid intracellular killing. some pathogens secrete exotoxins which destroy phagocytes and prevent phagocytosis. bacteria with pore forming cytolysins avoid the phagosome and also escape lysosomal destruction [ 101 -105 ] . certain bacteria interfere with the production of cytotoxic metabolites of phagocytes or contain the antioxidant proteins, thereby overcoming the effects of rnis or rois and cause obstruction in phagocytosis [ 106 , 107 ] . bacteria adhere to surfaces, aggregate and form a hydrated polymeric matrix comprising of exopolysaccharide known as biofi lms [ 108 ] . biofi lms are developed by various bacteria such as salmonella , streptococcus, vibrio cholerae, klebsiella pneumonia and haemophilus infl uenzae. further some cells in the biofi lm experience nutrient limitation and therefore survive in the starved state. such cells are slow growing cells and less susceptible to antimicrobial agents [ 109 ] . certain cells in a biofi lm adapt a different and protected phenotype. biofi lms are resistant to antibodies, phagocytes, and antibiotics . although p hagocytes reach the biofi lms, they become frustrated and release their enzymes, which cause damage to the tissue around the biofi lm. release of bacteria through the damaged biofi lm results in dissemination of the infection, leading to acute infection in the surrounding tissues [ 110 , 111 ] . effl ux pump genes and proteins are present in almost all organisms. effl ux pumps thwart the entry of an antibiotic in the bacterial cell and export an antibiotic from the cell. as effl ux pumps can be specifi c for one substrate or for drugs of dissimilar structure, they can be associated with multi-drug resistance. multi-drug-resistance effl ux pumps are a known cause for the development of bacterial resistance against antibiotics. bacterial effl ux-pump proteins related with mdr are divided into fi ve families namely the atp binding cassette (abc) superfamily, the major facilitator superfamily (mfs), the multi-drug and toxic-compound extrusion (mate) family, the small multidrug resistance (smr) family and the resistance nodulation division (rnd) family [ 112 ] . multi-drug resistance occurs, when effl ux proteins are overexpressed on the cell, and easily identify and effi ciently expel a broad range of antibiotics from the cells [ 113 ] . gram-negative bacteria express several families of transporters which cause resistance [ 114 ] . gram-positive bacteria mainly staphylococcus aureus and streptococcus pneumoniae express mdr effl ux pumps. s. aureus (responsible for skin and soft-tissue infections) overexpress mfs effl ux pump nora which enables resistance to chloramphenicol and fl uoroquinolones. the s. pneumoniae mfs effl ux pumppmra exports the fl uoroquinolones ciprofl oxacin, norfl oxacin, and also expels the dyes acrifl avine and ethidium bromide [ 115 -117 ] escherichia coli emre express a member of the small multi-drug resistance (smr) superfamily and acrab-tolc, a member of the resistance-nodulation-cell division (rnd) superfamily. vibrio parahaemolyticus overexpress norm, a member of the multi-drug and toxic compound extrusion (mate) superfamily. multi-drug-resistant tuberculosis (mdr-tb) is appearing as a ghost among the mdr bacteria because tb patients are at high risk of death due to failure of treatment. it is evident that mdr exhibits p55 effl ux pumps which play a crucial role in the pathogenicity of the microorganisms, and is responsible for the effl ux of tetracycline and aminoglycosides. this has opened a vast array for research in identifying mutants which are responsible for overexpressing these protein pumps in cases of elevated virulence [ 112 ] . chemical modifi cation of antibiotics resulting in their inactivation and hence, ineffective dug concentration can be a cause of bacterial resistance. the inactivation reactions include hydrolysis, redox, and group transfer. hydrolysis is the major cause of degradation of beta lactam antibiotics. the group transfer approach is the most varied and includes modifi cation by thiol transfer, glycosylation, acyl transfer, ribosylation, nucleotidylation and phosphorylation transfer. drugs which are degraded by group transfer are aminoglycoside, chloramphenicol, rifamycin, macrolides, etc. [ 118 ] . one important strategy to overcome the limitation of conventional drug delivery is to deliver high therapeutic payloads intracellularly. this could ensure high efficacy, coupled with low toxicity to provide major advantages. targeted nanocarriers provide high promise as potential drug delivery systems with the capacity to address this specifi c challenge. targeted nanocarriers could therefore prove to be the magic wand. passive and active targeting approaches could be relied on to achieve organ based targeting (fi rst order), specifi c cell based targeting in an organ (second order) and cell organelle based targeting (third order) [ 119 ] . a major requirement, however, besides reaching the targeting site is to ensure adequate concentration and adequate retention at the site. passive targeting can be described as deposition of drug or drug-carrier systems at a particular location due to pharmacological or physicochemical factors [ 120 ] . passive targeting can be achieved by exploiting pathophysiological and anatomical opportunities. introduction of drugs directly into various anatomical sites for example lungs and the eye by using non-invasive or invasive methods such as catheters or direct injections can enable local targeting. these site specifi c drug delivery methods limit systemic toxicity of the drug thus reducing adverse effects of drugs in the nontarget tissues [ 121 ] . exploiting altered pathological conditions in diseased tissues are strategies that can be adopted for passive targeting for example chemotactic factors released in infected or infl amed tissues increased permeability of vascular tissues, decreased ph and/or increased temperature [ 122 , 123 ] . increased vascular permeability specifi cally in cancers has enabled passive targeting of nanocarriers and is cited as the enhanced permeation and retention effect (epr) effect [ 124 ] . surface properties such as particle size, shape, hydrophobicity and surface charge have great impact on macrophage activation and phagocytosis. particle size plays essential role in distribution and elimination of nanocarriers [ 125 ] . particles size can infl uence attachment, adhesion, phagocytosis, distribution, circulation half-life and endocytic pathways [ 126 , 127 ] . the opsonisation and phagocytosis of particles is strongly affected by size of nanocarriers. although macrophages engulfed 0.2 versus 2 μm igg-coated spherical particles by different mechanisms, they followed similar kinetics [clathrin endocytosis versus fc-receptor mediated phagocytosis]. phagocytic uptake is generally observed with polymeric particles and liposomes with high particle size [>200-microns] [ 128 ] . table 3 .7 highlights the size of a number of nanocarriers evaluated for targeted delivery in infectious diseases. a broad range of non-spherical shaped particles studied including cylinders, cubes, hemispheres, ellipsoids, cones and complex shapes like fi lamentous, biconcave discoid showed varying effects on phagocytosis [ 169 ] . non-spherical shaped particles bypassed phagocytosis due to incomplete actin structure formation. particle shape affected attachment and internalisation during phagocytosis [ 170 ] . for instance oblate ellipsoids show best attachment and internalisation by phagocytosis, while prolate ellipsoids showed good attachment but poor internalisation. champion et al. reported that worm-like particles showed low phagocytosis as compared to spherical particles of the same volume [ 169 ] . asymmetric polymer lipid nanostructures (lipomer) of doxycycline hydrochloride (dh) in the range of (250-400 nm) [ 171 ] revealed enhanced splenic delivery. the irregular shape of the lipomer coupled with rigidity resulted in fi ltration and non-phagocytic accumulation to reveal splenotropy in sinusoidal spleen models, rat, rabbit and dog. a high spleen liver ratio of 6.7:0.53 was seen in the dog model (fig. 3.2 ) [ 172 ] . surface properties like hydrophobicity and surface charge also impact opsonisation, phagocytosis and biodistribution of nanoparticles [ 173 ] . hydrophobic nanocarriers are readily coated by complement proteins, albumin, and immunoglobulin and scavenged by res [ 174 ] . surface charge of particles also infl uences interaction and stability with cells [ 175 ] . reports suggest that positively charged particles showed high phagocytic uptake over negatively charged particles probably due to better interaction with the negatively charged cell membrane. cationic and neutral nanocarriers are less taken up by res as compare to negatively charge [ 176 -178 ] . however, negatively charged nanoparticles can potentially attach to cationic sites on the macrophages namely the scavenger receptors, which facilitate their uptake by res [ 179 ] . for details on infl uence of particle size, shape and charge readers are directed to the following reviews [ 126 , 180 ] . active targeting, defi ned as specifi c targeting of drugs or drug containing nanocarriers by anchoring active agents or ligands, provides selectivity, recognisability and potential to interact with specifi c cells and tissues in the body [ 181 ] . targeting by attaching ligands has been investigated as an additional strategy to enhance translocation of antimicrobials inside cells. attaching ligands facilitates greater uptake and can be mediated by various mechanisms. the membrane of macrophages expresses various receptors to facilitate the internalisation of cargoes inside the cell and their degradation. receptor mediated endocytosis (rme) permits the rapid internalisation of ligand attached particles as compared to untargeted particles [ 182 ] . the common rme mechanisms are macropinocytosis, clathrin dependent endocytosis (cde), caveolae-mediated endocytosis and clathrin independent endocytosis (cie). each approach exhibits different binding and internalisation mechanisms. further, the predominant uptake mechanism is often dictated by the nature of the ligand. receptor mediated processes are relatively slower than phagocytic processes, with the ligand playing an important role. the sizes, geometry, charge and density of the ligand signifi cantly infl uences receptor mediated endocytosis [ 183 ] . for more references readers can refer to [ 182 , 184 , 185 ] . table 3 .5 lists the endocytic pathways, endosome morphology and the proteins involved in the endocytic pathways. macrophages possess large number of surface receptors which help in the process of recognition and endocytosis of engineered particulate carriers. infection of macrophages leads to changes in the expression pattern of the concerned receptors, which can be exploited for targeted drug delivery employing nanocarriers. table 3 .6 is a summary of the important receptors on macrophages and illustrative examples of ligands for the same that could play a role in designing targeted nanocarriers for infectious disease therapy. cd14 [ 213 ] , decay accelerating factor (cd55), endo180 [ 214 ] are also receptors which could be targeted. nevertheless, ligands for the same need to be explored. all known nanocarriers can be effectively employed for targeted delivery in intracellular infections. both passive and active targeting approaches have been evaluated. the following tables 3.7 and 3.8 illustrate examples of nanocarriers, limited to major anti-infective agents for active and passive targeting, respectively. size being a major parameter infl uencing targeting to res. table 3 .7 also highlights the size of nanocarriers, which is a primary factor in passive targeting. tuberculosis is persistent and deadly infectious disease, caused mycobacterium tuberculosis which is non-specifi cally phagocytosed by alveolar macrophages. malaria is a complex disease caused by plasmodium and majorly resides in non-res cells like red blood cells (rbcs) and hepatocytes. entry of the parasite into the brain causes cerebral malaria. malaria can be targeted at the exoerythrocytic stage by targeting rbcs, or targeting the hypnozoites to tackle malarial relapse and further in case of cerebral malaria targeting the brain. increased permeability of infected rbcs is seen after 12-16 h of plasmodium invasion through formation of channels. these channels are "new permeability pathways" (npps) which allow entry of molecules such as dextran, protein a and igg2a antibody thereby differentiating the non-infected and infected rbcs. such pathways could be targeted to enable high drug loading in the erythrocytes specifi cally through design of nanocarriers of <80 nm [ 241 ] . this could be supported through design of stealth nanocarriers which could enable long circulation, using various stealth agents like poly(ethyleneglycol) (peg), pluronic, etc. [ 242 ] . chloroquine liposomes anchored with anti-erythrocyte f (ab′)2 were studied for targeting to erythrocytes [ 243 ] . hepatocytes the residence of hypnozoites expresses the asialoglycoprotein receptor (asgpr), which is overexpressed in infections. targeting this receptor using nanocarriers anchored with asgpr ligands is a strategy for hepatocyte targeting. joshi et al. prepared in situ primaquine nanocarboplex of primaquine phosphate anchored with pullulan as the asgpr ligand for specifi c targeting to hepatocytes. signifi cantly, enhanced hepatic accumulation with preferential accumulation in the hepatocytes and a high hepatocytes/nonparenchymal cells ratio of 75:25 confi rmed hepatocyte targeting [ 244 ] . transferrin (tf)-anchored solid lipid nanoparticles (slns) were intravenously administered for targeting quinine dihydrochloride to the brain, in cerebral malaria. compared to conventional slns or drug solution the tf-slns signifi cantly enhanced the brain uptake of quinine [ 234 ] . a major feature of hiv that complicates therapy is the existence of hiv in multiple reservoirs, which include various cellular and anatomical sites [ 245 ] . the typical reservoirs are the liver, spleen, lungs, git and genital tract with the brain and bone marrow representing remote sites [ 246 ] . targeted delivery for hiv therefore needs to address delivery to maximum sites simultaneously to achieve remission. one strategy that we propose is a combination of nanocarriers of size <100 nm to target remote sites and size >200 nm target major res organs (unpublished data). viral replication is inhibited by the antioxidant glutathione. erythrocytes containing glutathione (gsh) in combination with azidothymidine (azt) and didanosine (ddi) showed higher reduction in viral dna in bone marrow and brain as compared to ddi + gsh alone [ 247 ] . immunoliposomes containing sirna for targeting the lymphocyte function-associated antigen-1 (lfa-1) integrin, which is expressed on all leukocytes, was selectively taken up by t cells and macrophages, the primary site of hiv. further, in vivo administration of anti-ccr5 sirna resulted in leukocytespecifi c gene silencing that was sustained for 10 days [ 248 ] . nanogels comprising non-reverse transcriptase inhibitors (nrits) decorated with a peptide for brain specifi c apolipoprotein e (apoe) receptors, showed tenfold suppression of retroviral activity and decrease infl ammation in humanised mouse model of hiv-1 infection in the brain [ 249 ] . targeted drug delivery for the therapy of veterinary infections assumes immense importance not only for improved animal health but due to the challenges posed by zoonotic diseases. about 13 zoonotic diseases including brucellosis, tuberculosis, trypanosomiasis, cysticercosis and others are related to 2.4 billion cases of infection in humans and over two million deaths annually [ 166 , 167 ] . such infections exist both in domestic animals and wild life. the close proximity of humans especially with such domestic animals is a cause of global concern. the who policy of "cull and kill" results not only in the loss of lives but also heavy monetary losses to the farmer. targeted treatment strategies using nanodrug delivery systems could provide a revolutionary strategy to benefi t both the animals and man. the benefi ts of targeted nanomedicine strategies are slowly gaining recognition as evident from a number of reported studies. liposomes have been used by many researchers for treating various veterinary diseases such as leishmaniasis [ 250 , 251 ] , brucellosis [ 252 ] , blastomycosis [ 253 ] , babesiosis [ 254 ] , etc. patil et al. [ 171 ] developed an asymmetric lipomer. this is a combination of polymer-lipid containing doxycycline which could have application in the treatment of intracellular infections that are primarily resident in the spleen like brucellosis, ehrlichiosis, etc. a number of studies are reported on horses infected with babesiosis, streptococcus equi, t. gondii and strongylus vulgaris infections using liposomes [ 254 ] , polymeric nanospheres [ 255 ] , dendrimers [ 256 ] and micelles [ 257 ] respectively. a recent study revealed the improved therapy of theileiriosis in cattle with solid lipid nanoparticles (sln) of buparvaquone [ 258 ] . sln revealed comparable effect with the intramuscular injection at signifi cantly lower doses. nanodrug delivery systems have also been evaluated in dogs, sheep and pigs. for details on nanodrug delivery applications in targeted delivery in veterinary infections, readers are directed to the following reference [ 259 ] . targeted delivery for infectious diseases 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diamidine (imidocarb), preparation and animal studies enhanced mucosal immunoglobulin a response and solid protection against foot-and-mouth disease virus challenge induced by a novel dendrimeric peptide evaluation of immune responses in sheep induced by dna immunization with genes encoding gra1, gra4, gra6 and gra7 antigens of toxoplasma gondii effi cacy of ivermectin in injectable and oral paste formulations against eight-week-old strongylus vulgaris larvae in ponies buparvaquone loaded solid lipid nanoparticles for targeted delivery in theleriosis targeted nanomedicine strategies for livestock infections. nanotechnology for animal health and production key: cord-257467-b8o5ghvi authors: smith, barbara a. title: anesthesia as a risk for health care acquired infections date: 2010-12-31 journal: perioperative nursing clinics doi: 10.1016/j.cpen.2010.07.005 sha: doc_id: 257467 cord_uid: b8o5ghvi anesthesia is delivered in a variety of modalities including general, regional, or local. patients are most vulnerable when receiving anesthesia, as they must depend on the anesthesia team to provide this care without untoward effects. it is expected that patients will be protected from health care acquired infections (hais) by appropriate use of infection prevention measures. in addition, the anesthesia team may be at risk of hais because of their intimate contact with the patient's blood and respiratory system. adequate adherence to infection prevention methods should reduce the risk of occupation exposure and infection to the anesthesia team members. health care associated infections involving anesthesia have been transmitted from health care worker to patient, patient to patient, and patient to the anesthesia provider. this article further discusses the risks for hais apparent in intravascular cannulation, endotracheal intubation, and the development of surgical site infections, and examines occupational measures to prevent infections in the health care worker. regardless of the health care setting or the level of provider, the standard of care for infection prevention and managerial oversight of this care should remain the same. of provider, the standard of care for infection prevention and managerial oversight of this care should remain the same. the anesthesia team has direct and indirect responsibility for the prevention of infections, which may manifest most commonly as bloodstream infections, local injection site infections, abscesses, meningitis, respiratory tract infections, and surgical site infections (ssis). bacterial and viral infections may also be attributed to anesthetic care. because anesthesia care interrupts two of the body's significant defense mechanisms, there is the potential for risk of infection to the patient. first, the body's intact skin functions as a barrier to pathogenic organisms. however, intravascular cannulation for conscious sedation disrupts the skin integrity and permits a local or systemic infection to occur. these risks also apply to central venous vascular catheters inserted by the anesthesiologist, whether in the operating room or as part of a critical care team. insertion of a needle, cannula, or implantable device into the spinal column for anesthesia or analgesia disrupts the skin integrity and provides a direct portal of entry for organisms. second, surgery will often be performed with general anesthesia that involves the insertion of an endotracheal tube to maintain an open airway and permit artificial ventilation during the procedure. although endotracheal intubation during surgery is generally a controlled safe procedure, this artificial airway predisposes the body to exposure to respiratory pathogens whether from the health care provider, the environment, or equipment. the same risk arises when intubation is performed during an emergency situation such as cardiopulmonary resuscitation. measures to prevent this exposure, including the role of the environment and equipment, are discussed in this article. third, although not physically involved at the sterile operative field, the anesthesia team can have influence over the development of ssis by their collaboration with the surgical team in achieving normothermia, glycemic control, and appropriate antibiotic prophylaxis for the patient. 6 finally, the author examines occupational measures to prevent infections in the health care worker. access to the spinal column is used to provide regional anesthesia, for example with epidural anesthesia, or to deliver medication such as analgesics or steroids. other examples of procedures that enter the spinal column include diagnostic procedures such as lumbar puncture and myelography. the overall risk of infection from these procedures appears to be low. miller 7 cites a rate of less than 1 per 10,000 cases of serious infection (ie, meningitis or spinal abscess). he notes 2 factors that had a relation to infection: the duration of epidural anesthesia and the patient's medical conditions. in a review conducted through 2005, schulz-stü bner and colleagues 8 noted rates of 3.7 to 7.2 spinal abscesses per 100,000 cases and 0.2 to 83 epidural abscesses per 100,000 procedures. baer 9 lists 179 cases of postdural meningitis occurring after spinal or epidural anesthesia and other types of instrumentation. spinal or epidural anesthesia accounted for 65% of the cases ( table 1) . some investigators have argued that the true incidence is unknown because there is no uniform reporting mechanism in the united states. nonetheless, ruppen and colleagues 10 attempt to define the risk in obstetric epidural anesthesia and cite a rate of 1 deep epidural infection per 145,000 procedures; an admittedly low smith incidence. nonetheless, in an american society of anesthesiology newsletter, hughes 11 urges his obstetric colleagues that they can lower the risk to their patients. evidence of infection transmission is also documented in the descriptions of outbreaks. five women in two separate states (new york and ohio) developed bacterial meningitis after intrapartum anesthesia ( table 2) . 1 the article reveals that the causative organism was recovered from a nasal swab of one of the anesthesiologists linked to the 2 cases in ohio. in each outbreak, unmasked personnel (including the anesthesiologist delivering the spinal anesthesia in ohio) were present in the room during the procedures. eight cases of meningitis following myelography were reported by the centers for disease control and prevention (cdc) that appear related to contamination from mouth flora from the clinicians who performed the procedure. 12 as part of the investigation, the cdc eliminated equipment and fluids as a potential source for these infections, and confirmed that adequate aseptic technique had been followed. as described by baer, 9 the mechanism of infection in these cases occurs through: droplet transmission of aerosolized mouth organisms, contamination from skin bacteria or hematogenous or direct spread from an endogenous site of infection. regarding this first method of transmission, the likelihood is that some of these infections are related to the clinician who performed the procedure. this assumption is supported by arguments that several cases were related to clusters among specific operators, or were linked by dna testing of nasal swabs from the operator and positive cerebrospinal fluid cultures, as in the ohio case cited. these data suggest that the clinician can implement preventive practices related to anesthesia or analgesia given through the spinal cord to reduce the likelihood of infection. these practices include skin disinfection with an appropriate antiseptic, sterile gloves and sterile drapes, and aseptic technique. because of the growing evidence for droplet transmission of oropharyngeal flora during the procedures that puncture the spinal column, the cdc's guidelines for isolation precautions recommend the use of a surgical mask by personnel placing a catheter or injecting material into the spinal canal or subdural space. 12 a recent practice advisory prepared by the american society of anesthesiologists (asa) concurs with the implementation of aseptic technique when handling neuraxial needles and catheters, and states it should include "hand washing, wearing of sterile gloves, wearing of caps, wearing of masks covering both the mouth and nose, use of individual packets of skin preparation, and sterile draping of the patient." the same advisory does not make a specific recommendation regarding the type of skin antisepsis to use. 13 intravascular catheters, including central and peripheral venous catheters and arterial catheters, play an integral part in the delivery of anesthesia or analgesia. once again, these devices provide an opportunity for organisms to enter the normally sterile vasculature. the insertion and care of these catheters should remain the same regardless of whether they are inserted in an operating room, a critical care unit or a free-standing practice. there are 2 key mechanisms by which a catheter can lead to an infection. 14 the first occurs with colonization of the device and is referred to as catheter-associated infection. the pathogenesis of these types of infections is: skin organisms gain entry via the puncture site at time of insertion or shortly thereafter, the catheter hub can become contaminated during use or organisms spread hematogenously from another site of infection in the body. catheter-associated infections can lead to local site infections or systemic infections including bacteremia, sepsis, or endocarditis. the second mechanism occurs with contamination of the medication or substance being injected, which is referred to as infusate-associated infection. although intrinsic contamination of intravenous fluids or medications from the manufacturer is rare in the united states, improper procedures by the anesthetist or technician during medication preparation and administration can led to infusate-associated infections. in this situation the bacteria, virus, or fungus is directly infused into the patient's bloodstream. the rate of catheter-associated infections varies by the type of device used. for peripherally inserted, short-term catheters the risk is low. lee and colleagues 15 report a local site infection rate of 2.1% to 2.6% among 3165 patients with short peripheral intravenous catheters. no patients in this group developed a bloodstream infection. rates of arterial line catheter infections are similarly low, with lucet and colleagues 16 and koh and colleagues 17 reporting rates of 1.0 and 0.92 bacteremias per 1000 catheter days, respectively. these investigators report low rates of bacteremias related to central venous lines as well. the risk of central line-associated bloodstream infections in critical care patients ranges from 1.3 infections per 1000 catheter days in pediatric medical units to 5.5 in burn units. 18 table 3 lists the central line-associated bloodstream infection rates in a sample of units in which the anesthetist may have involvement. although it is possible for individual solutions to become contaminated and subsequently infused into patients, it is difficult to attribute an individual infection to a specific medication, vial, or infusion bag without direct causal evidence. hence, data related to infusate-related infections derives mainly from experiences with outbreaks. for example, blossom and colleagues 19 describe an outbreak of 162 serratia marcescens bacteremias in 9 states related to manufacturer's contamination of prefilled heparin and saline syringes. these circumstances are clearly beyond the control of the anesthesia team, although the team must respond promptly to alerts regarding potential contamination. there are, however, reports of medication contamination occurring under the control of the anesthesia personnel. in 2003, morbidity and mortality weekly report reported hepatitis b and c virus transmission occurring in 3 separate locations. 3 in each of these practices, reuse of needles and syringes and contamination of multidose medication vials led to patient-to-patient transmission of viral infections. as depicted in table 4 , more than 200 people were affected. despite this significant number of patient-to-patient transmissions, practitioners have continued to demonstrate unsafe medication practice. as recently as 2008, 6 patients were infected with hepatitis c due to unsafe injection practice used during sedation for endoscopic procedures. 2 the investigation revealed that the anesthesia provider contaminated vials of propofol by repeated aspiration into the vial with a syringe contaminated with hepatitis c from backflow of the index patient's blood. although the vial was labeled for single-patient use, the practitioner used the vial on the next patients. a graphical example is shown in fig. 1 . the cdc has published extensive guidelines for the prevention of intravascular infections. 20 key elements intended to reduce catheter-associated infections that apply to peripheral, central, or arterial catheters handled by the anesthesia team include: skin disinfection of the intravenous insertion site with an appropriate disinfection (a chlorhexidine-based preparation is preferred), aseptic technique during insertion and care and decontamination of ports and stopcocks with a disinfectant such as 70% alcohol before accessing the device. the cdc guidelines and the asa recommend additional specifications for the insertion and maintenance of central venous catheters because of their higher risk of infection. 21 many facilities have adopted a bundle approach to the insertion of central line catheters both in the operating room and elsewhere. the elements of the bundle are: 1. hand hygiene before insertion 2. full barrier precautions: sterile gown, gloves, masks, and large sterile drapes 3. skin antisepsis with chlorhexidine 4. subclavian vein as preferred anatomic site versus internal jugular or femoral 5. daily review of line necessity. from the anesthetist's perspective, the elimination of infusate-related infections demands preventing contamination of medications and infusions. most practitioners presumably would report adherence to safe medication handling. yet a survey conducted by the american association of nurse anesthetists (aana) revealed that 1% to 3% of clinicians reuse needles or syringes on multiple patients. 22 the aana has joined with the cdc, two state medical societies, the association for practitioners in infection control, and other advocacy groups in the safe injection practices and awareness campaign to further educate health care providers and the public about the importance of these practices. the campaign poster is displayed in fig. 2 . 23 because of the aforementioned outbreaks of hepatitis transmission, the cdc's 2007 guidelines for isolation precautions 12 highlights safe injection practices that are outlined in fig. 3 . the asa supports these practices 21 and makes further recommendations: cleanse rubber septum of vials and the neck of glass ampoules with a disinfectant. medications should be drawn up as close as possible to the time of use. medications in a syringe should be discarded within 24 hours unless specified by the manufacturer or pharmacy. expiration times for medications must be followed, especially the time limits for the use of lipid formulations such as propofol. according to the national health care safety network, the range of postprocedure pneumonias varies greatly by procedure. 18 for example, among those procedures reporting more than 1000 cases, patients undergoing knee prosthesis had a rate of 0.06 postoperative pneumonias per 100 procedures compared with cardiac surgery patients who had a rate of 1.19 pneumonias per 100 procedures. some additional information is gleaned from examining rates of ventilator-associated pneumonias (vap). among surgical type critical care units, the pooled mean rate per 1000 ventilator days was a low of 0.6 in pediatric cardiothoracic units to a high of 8.1 in trauma units. however, it is difficult to distinguish how much of a direct impact intubation and anesthesia had on the development of these pneumonias. one study by rello and colleagues 24 examined the development of pneumonia within the first 48 hours of intubation. eighteen of 250 intensive care unit (icu) patients developed pneumonia within the first 24 hours. there were 65 surgical patients included in this study. the 2 most important risk factors for pneumonia in patients were undergoing cardiopulmonary resuscitation and receiving conscious sedation. the investigators conclude that variables directly related to the intubation had less of an impact on the occurrence of pneumonia. nonetheless, intubation places the patient at risk of infection for several reasons. because intubation interrupts the defense mechanisms of the upper airway, it increases the risk of aspiration. aspiration of oral pharyngeal secretions is a prime cause of health care acquired pneumonia. this condition may be further aggravated by mechanical damage to the larynx or trachea from the endotracheal tube or stylet. 7 furthermore, mechanical ventilation increases the risk of infection. measures to reduce infection risk associated with intubation and mechanical ventilation deal with technique and equipment. cheung and colleagues 25 reviewed the literature to determine the impact of sterile handing of the endotracheal tube and the incidence of pneumonia. of note, the investigators found very few data on the topic yet noted that intubations performed under unsterile conditions do occur, although they do not provide a recommendation. it is prudent for intubation to be performed as aseptically as possible, with personal protective equipment worn for the safety of the health care worker. oral intubation is preferred over nasal intubation because the latter is more likely to lead to sinusitis, thereby increasing the risk of aspiration of infected secretions. 26 care should be taken to drain condensate in the ventilator tubing away from the patient. although there are other measures to reduce vap such as mouth care and semirecumbent position of the patient, these apply after the intraoperative period. equipment utilized by the anesthesia team includes endotracheal tubes, laryngoscope handles and blades, fiberoptic endoscopes, and anesthesia circuits, machines, and carts. there are also ancillary devices used by the team such as pulse oximetry, invasive temperature probes, and airways. this equipment may become contaminated from contact with the patient's skin, blood, secretions, splashes from the operative field, or contact with contaminated hands of the health care worker. the cdc, asa, and aana each have comparable standards for cleaning and disinfection of these items. these standards are based on the spaulding classification method 27 that stratifies items based on their likely contact with a sterile body site, mucus membrane, or intact skin, as noted in table 5 . neither the cdc 12 nor the asa 21 recommends the routine use of a bacterial filter for the breathing circuits or anesthesia ventilators. conversely, the aana states "protective anesthesia as a risk for hai use of bacterial filters is recommended," although they do acknowledge its use is controversial. 28 each of these organizations supports the use of a bacterial filter when caring for an infectious tuberculosis patient. another debated topic is the disinfection of laryngoscope handles. because they do not enter sterile tissue or touch mucous membranes, the spaulding classification would indicate cleaning and low-level disinfection. there is, however, the risk of contamination with body fluids. call and colleagues 29 challenged a common practice of wiping blades with low-level disinfectant between operative cases. after culturing 40 handles that had been cleaned according to the facilities' standard practice, they found 75% had positive bacterial cultures. most importantly, standard protocols should be developed that outline the correct cleaning, disinfection, or sterilization process for each item used by the anesthesia team. the manufacturer of the equipment should be consulted for their recommendations. an oversight mechanism should be included in the policy to ensure adherence with the correct practice. adequate training must be provided. overall, the documented transmission of infection from anesthesia equipment appears to be low. yet loftus and colleagues 30 raise the issue of transmission of bacteria in the anesthesia work area. these investigators cultured 2 specific areas of the anesthesia machine and the sterile stopcock just before the beginning of the case and again at the end of the case. their results showed that 32% of the stopcocks were contaminated by the end of the case. the work area showed a significant increase in bacterial contamination as well. two cases of methicillin-resistant staphylococcus aureus (mrsa) were transmitted to the work area intraoperatively. one case of vancomycin-resistant enterococcus transmission was documented between the anesthesia work are and the stopcock. the investigators also noted a trend toward increased hais among patients with contaminated stopcocks. the machine and stopcocks appear to have become contaminated by contact with providers' hands or lapses in aseptic technique. these results reinforce the need for rigorous attention to hand hygiene not only before the start of surgery but also intraoperatively. most studies indicate that adherence to hand hygiene by health care workers needs to be improved. mcguckin and colleagues 31 report only modest improvement to 51% compliance among non-icu staff after a year-long program of observation and feedback. a 2004 report by pittet and colleagues 32 found a 23% compliance rate among anesthesiologists. hand hygiene is clearly a challenge in the operating room because of the multiple functions being performed. limited access to hand hygiene products within the operating room undoubtedly contributes to poor compliance in the room. koff and colleagues 33 address this latter challenge through a study utilizing a portable device that dispenses alcohol-based hand rub. the device has the added benefit of tracking the frequency of use and providing a reminder if too long a time has elapsed between hand hygiene events. the introduction of the device was considered the study phase. during the study phase, hand hygiene events increased among attending anesthesiologists and other caregivers by 6.9 and 8.3 times per hour, respectively. the investigators also monitored the frequency of stopcock contamination and the occurrence of hais, and demonstrated decreases in both of these indicators as noted in table 6 . while the reduction in hais is promising, koff and colleagues caution that additional research is needed to confirm these results. ssis are the second most frequent hai. 34 control measures for the prevention of ssis include preoperative preparation of the patient, sterile attire and draping, surgical hand preparation, skin antisepsis, air handling, and sterile surgical instrumentation. there are additional conditions that can influence the occurrence of ssis when the anesthesiologist may be involved. one measure aimed at reducing bacteria at the surgical site is the delivery of antibiotic prophylaxis. the national surgical infection prevention project (sip) proposes a 25% reduction in national surgical complication rates by adherence to 3 indicators 35 : 1. administration of an appropriate antibiotic as described by the sip. the antibiotic is selected based on the organisms most likely to cause infection and varies by the type of surgery. 2. timely administration of the antibiotic. to reach adequate blood and tissue concentrations, the antibiotic should be administered within the 60 minutes prior to the surgical incision. (vancomycin may be given up to 120 minutes prior.) 3. discontinuation of prophylactic antibiotics with 24 hours (48 hours for cardiac surgery). a second intervention to reduce ssis is the maintenance of normothermia. hypothermia is thought to contribute to infection because of a decrease in subcutaneous tissue perfusion. 6 lastly, glycemic control has been shown to reduce the rate of infections. anesthesia providers are at risk of occupational infections from direct contact with blood and respiratory secretions. in addition, they may be exposed to microorganisms via the airborne or droplet route. diseases transmitted through the airborne route include tuberculosis, measles, and varicella. most clinicians should be immune to measles and varicella because of effective vaccines. surgery should be delayed for patients with these active infections. if the case cannot be postponed, the air handling in the operating room should ideally have negative pressure relative to the corridor. as previously mentioned, a bacterial filter should be placed on the anesthesia breathing circuit for patients with active tuberculosis. the health care provider should wear an n95 respirator approved by the national institute for occupational safety and health. when called to intubate patients on airborne isolation, again the n95 respirator is indicated. a large number of sars cases in canada were occupationally acquired. fowler and colleagues 5 determined in one small series that physicians and nurses involved in intubation had a relative risk of 3.82 and 13.29, respectively, of developing sars. this result stresses adequate respiratory protection. infections spread through the droplet route include pertussis, mumps, and influenza. as most of these cases are unlikely to undergo elective surgery or procedures while symptomatic, the exposure may occur from undiagnosed cases or from people who are shedding organisms in the few days prior to symptoms. respiratory protection is indicated for known or suspect cases. immunization is strongly encouraged. (for the 2009-2010 influenza season, the cdc recommended use of an n95 for contact with patients with influenza-like symptoms. current recommendations can be found at http://www.cdc.gov/flu/professionals.) hand hygiene is indicated. because of their contact with blood and other body fluids, anesthesia providers may be exposed to viral pathogens such as hepatitis b or c and human immunodeficiency virus (hiv). it is difficult to determine the actual number of occupationally acquired blood-borne infections in the discipline. in a 1998 study among anesthesia personnel, the estimated average 30-year risks of hiv or hepatitis c virus infection per full-time equivalent was 0.049% and 0.45%, respectively. 36 in addition, there may be exposure to bacterial pathogens such as mrsa and clostridium difficile. the aana supports the cdc's recommendations to use standard precautions in the care of all patients. 28 in summary, standard precautions entail: consider all blood and body fluid as potentially infectious. use of personal protective equipment (ppe) (gloves, gowns, protective eye wear, and masks) when anticipating contact with blood or body. the ppe worn will depend on the task being performed and the possibility that splash or aerosolization can occur. handle and dispose of all needles and syringes properly. the practitioner should be aware of his or her facility's protocol for managing occupational exposure to blood and body fluid. body fluid exposures should be evaluated promptly to determine the need for antiviral or other prophylaxis. transmission-based precautions may be added for particular diseases that are highly transmissible or of epidemiologic importance. there are 3 categories: airborne isolation, droplet precautions, and contact precautions. the documented risk of infection related to anesthesia is low, yet the potential exists for serious infectious outcomes including death. the risk of infection can be minimized by adherence to hand hygiene, aseptic technique, safe infection practices, equipment decontamination, and use of ppe by all members of the anesthesia team. bacterial meningitis after intrapartum spinal anesthesia-new york and ohio acute hepatitis c virus infections attributed to unsafe injection practices at an endoscopic clinic-nevada transmission of hepatitis b and c viruses in outpatient settings clinical anesthesia. philadelphia: lippincott, williams and wilkins treatment of severe acute respiratory syndrome during intubation and mechanical ventilation the anesthesiologist's role in the prevention of surgical sire infection nosocomial infections and infection control in regional anesthesia post dural puncture meningitis incidence of epidural hematoma, infection and neurologic injury in obstetric patients with epidural analgesia/anesthesia neuraxial blockade in obstetrics and complications related to infection: can we lower the risk? park ridge (il): american society of anesthesiology newsletter guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings practice advisory for the prevention, diagnosis, and management of infectious complications associated with neuraxial techniques: a report by the american society of anesthesiologists task force on infectious complications associated with neuraxial techniques intravascular device infection risk factors for peripheral intravenous catheter infection in hospitalized patients: a prospective study of 3165 patients infectious risk associated with arterial catheters compared to central venous catheters prospective study of peripheral arterial catheter infection and comparison with concurrently sited central venous catheters national healthcare safety network report (nhsn): data summary for multistate outbreak of serratia marcescens bloodstream infections caused by contamination of prefilled heparin and isotonic sodium chloride solution syringes guidelines for prevention of intravascular catheter-related infections american society of anesthesiologists. recommendations for infection control for the practice of anesthesiology aana condemns unsafe injection practice. press release risk factors for developing pneumonia within 48 hours of intubation endotracheal intubation: the role of sterility apic text of infection control and epidemiology guideline for disinfection and sterilization in healthcare facilities american association of nurse anesthetists. aana infection control guide for certified nurse anesthetists. park ridge (il): aana nosocomial contamination of laryngoscope handles: challenging current guidelines transmission of pathogenic bacterial organisms in the anesthesia work area hand hygiene compliance rates in the united states-a one year multicenter collaboration using product/volume usage measurement and feedback hand hygiene among physicians: performance, beliefs and perceptions reduction in intraoperative bacterial contamination of peripheral intravenous tubing through the use of a novel device apic text of infection control and epidemiology multicenter study of contaminated percutaneous injuries in anesthesia personnel key: cord-104317-t30dg6oj authors: parker, michael t. title: an ecological framework of the human virome provides classification of current knowledge and identifies areas of forthcoming discovery date: 2016-09-30 journal: yale j biol med doi: nan sha: doc_id: 104317 cord_uid: t30dg6oj recent advances in sequencing technologies have opened the door for the classification of the human virome. while taxonomic classification can be applied to the viruses identified in such studies, this gives no information as to the type of interaction the virus has with the host. as follow-up studies are performed to address these questions, the description of these virus-host interactions would be greatly enriched by applying a standard set of definitions that typify them. this paper describes a framework with which all members of the human virome can be classified based on principles of ecology. the scaffold not only enables categorization of the human virome, but can also inform research aimed at identifying novel virus-host interactions. the term "microbiome" was coined by whipps, lewis, and cooke in 1988, defined as "a characteristic microbial community occupying a reasonably well defined habitat which has distinct physico-chemical properties" [1] . many attribute the popularization of the term to nobel laureate joshua lederberg, where he (anthropocentrically) defined the microbiome as "the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease" [2] . the community of microorganisms referenced in these definitions includes viruses, bacteria, fungi, protozoa, and archaea. within this community there is an inherent plasticity arising from the interaction of organisms with one another and with their environment. such meta-interactions lead to complex repercussions for each level of life, and this review will focus on the consequences that have implications for human health. the viral component of the microbiome, termed the "virome" [3] is a poorly understood facet of the microbiome, despite having the potential to significantly impact human health. from a philosophical perspective, viruses were likely integral to the very existence of man as the primordial precursor to all current life on earth [4] . more tangibly, during the course of human evolution, the viruses in and around humans not only drove evolution via selective pressure, but also contributed novel genetic material. in fact, approximately 42 percent of the human genome is composed of viral sequence [5] . this role as a major agent driving horizontal (i.e., non-reproductive) gene transfer between biomes [6] [7] [8] [9] and the fact that the diversity, complexity, and abundance of viruses surpasses any other biological entity make it apparent that understanding viromes will impart unparalleled understanding of the organisms they inhabit [10] . however, the obvious importance of viruses in the composition of all biomes has not (yet) been met with an appropriate fervor for the characterization of the viral review recent advances in sequencing technologies have opened the door for the classification of the human virome. while taxonomic classification can be applied to the viruses identified in such studies, this gives no information as to the type of interaction the virus has with the host. as follow-up studies are performed to address these questions, the description of these virus-host interactions would be greatly enriched by applying a standard set of definitions that typify them. this paper describes a framework with which all members of the human virome can be classified based on principles of ecology. the scaffold not only enables categorization of the human virome, but can also inform research aimed at identifying novel virus-host interactions. component of microbiomes. the original explosion of microbiome information was spurred by the utilization of 16s sequencing technology, which can give vast information about microbial communities using universal primers [11] . however, this technique is specific to organisms with ribosomes, so early research focused on analysis of bacterial sequences rather than much more technically challenging viral sequences [12] . thankfully, recent advances in next generation sequencing technologies are making virome characterization more technologically and financially possible and will ensure an explosion of virome description in the near future [13, 14] . in an effort to facilitate such classification of the human virome, it is prudent to use basic ecological interactions both to organize current knowledge as well as identify areas where new information may be found. this review will build a framework of the human virome from an ecological perspective, identify currently underappreciated and possibly undiscovered roles of the virome, and apply these principles to analysis of the role of the virome in human health. such a systematic conception of the structure of these ecological interactions can also facilitate the application of similar conventions to the other components of the microbiome. while appreciation of the possible benefits of the human microbiome accumulated in the latter half of the 20th century, a concordant acknowledgement for the human virome was not so quickly realized [15] . it is likely this was because human viruses rely on invasion of host cells to replicate and this provokes an overwhelmingly negative impression. however, all interactions of viruses with their hosts are symbioses in the classical sense [16] and fall along a spectrum from exclusively antagonistic to exclusively mutualistic [17] . in general, these interactions are facultative for the human and obligate for the virus, but cases like the presence of viral sequences in human genomes exemplify where this simplistic view breaks down [5] . while the position of a particular virus along this spectrum is not fixed, it is obvious that many viruses have varied, intimate relationships with their hosts. botanists and entomologists were among the first to notice the intricacy of virus-host interactions, which challenged the purely parasitic dogma of virus-host interaction (reviewed in [17] [18] [19] [20] ). for example, parasitoid wasps harboring polydnavirus genomes utilize virally packaged host genes to prevent rejection of wasp eggs introduced into the parasitized caterpillar [21] . in a more benign example, the white clover suppresses nodulation in conditions of sufficient nitrogen in a manner dependent on viral coat proteins of a persistently infecting cryptic virus [22] . while the breadth of knowledge of interactions types is surely incomplete, their presence in other organisms al-ludes to the possibility of the existence of similar interactions between humans and viruses. recent reviews have addressed the issue of the current, primarily negative, view of the human virome [18, [23] [24] [25] [26] . virologists are beginning to realize that the viruses within us may be more important than previously appreciated. the discovery of intimate interactions of viruses with humans, like the role of endogenous retrovirus (erv †) syncytins in placentation [27] , are categorically dissimilar to the classical view of viruses only as parasites and brings to issue how scientists are approaching the study of the virome. a lack of structure in this endeavor has arguably hindered the true understanding of non-pathogenic consequences of human-virome interactions. to address this issue, an ecological framework can be constructed for more informative classification of the virome based on how viruses fill niches in humans. here, i describe a conception of the human virome with three general classifications of virus types: parasitic, commensal, and mutualistic ( figure 1 ). within these groupings are subcategories containing more specific characteristics germane to different viral groups. table 1 illustrates how representative members of the human virome spanning a vast array of families, replication schemes, genome architectures, and tropisms can be succinctly classified with this scheme. this arrangement provides functional information about the nature and plasticity of virus-host interaction, the most critical defining characteristic of a virus in the context of human health, in a way absent in the more classical types of organization mentioned above. in the rest of this review, i will characterize each grouping and provide examples from the literature supporting this classification. the application of this scaffold will not only deepen the understanding of known virus-host interactions in the ecological context of the virome, but will also identify logical next steps and gaps in current knowledge that are tantalizing areas for future exploration. the canonical classification of viruses is as parasites. in general, parasitic viruses are transmitted horizontally from host to host, and this process is necessary for the virus' survival. their obligate life cycles necessitate manipulation of cellular processes and have been classically linked to disease manifestation. while the role of viruses in human disease was not appreciated until the 19th century [28] , throughout human history, the diseases caused by particularly pathogenic viruses have been some of the most feared and deadly. currently, there are 129 known human viral pathogens [29] and at least 219 viruses that can infect humans [30] , with more being discovered every year [31, 32] . the apparent bias toward pathogenic species is undeniably because phenotype (read: pathogenesis) has traditionally been a major factor in the discovery of new viruses. while parasitic viruses are a morose facet of human history, they have contributed to man's evolution as a species. the biologist's viewpoint includes the selective pressure on man in which viruses kill susceptible hosts, leading to the emergence of fitter genotypes. some viruses play a similar role by limiting an infected host's reproductive fitness [33] . the presence of deadly viruses has also supplied pressure for behavioral and psychological adaptation via influence on practices in mate selection, food preparation, and sanitation [34] . a modern example of such behavioral adaptation is the development of vaccination, kick-started by edward jenner's famous work with cowpox [35] . however, not all parasitic viruses are deadly. in fact, excessive pathogenicity is an evolutionarily poor strategy for a virus' survival unless balanced with a number of other trade-offs such as transmission rate and recovery rate [36] . most parasitic infections occur horizontally (though there are rare exceptions [37] ) and the equilibrium between pathogenicity and other trade-offs is crucial to ensuring continuation of the viral lineage. while parasitic viruses by definition have some level of pathogenic effect on their host, it is not the intent of the virus to do so, because viruses don't have intentions. viral genomes (particularly those of rna viruses) exist as a pool of non-identical, related sequences, termed quasispecies, and are constantly adapting in response to selective pressures [38] . in some cases, the virus' environment can select for changes that create a balance of the above-mentioned trade-offs where pathogenicity may be a successful, or at least not detrimental, strategy [39] . in this light, i will define types of parasitic viruses according to both their degree of pathogenesis and the way in which they interact with their host. the key characteristic of pathogenic viruses is that they cause an acute disease in which the host either lives or dies, with the virus simply using it as a vessel to move to the next host. in these interactions, hosts typically mount strong immune responses and literally fight for their lives. this is not to say that all pathogenic infections are deadly or resolved immediately, since the degree of pathogenicity and time to resolution vary widely, as will be discussed in following sections. rather, the virus-host interactions classified here as "pathogenic" represent perturbations of homeostasis for which the only resolution is that one side must prevail. either the host completely clears its body of the virus, or it succumbs to infection. while this sounds like a poor transmission strategy, history has shown it can be quite effective, as long as the proper trade-offs are satisfied (see the parasitic virome, above). influenza virus is a prime example of the success of a pathogenic strategy. most humans can expect to be infected with at least one influenza virus per year, and in general they will observe a fairly robust set of symptoms including, but not limited to, fever, chills, headache, nasal congestion, and coughing [40] . while infection can be severe and even cause death, this is typically limited to hosts in an immunocompromised state, as selective pressure typically drives virus evolution to less virulence [41] . in general, virus is shed one to two days after infection, with symptoms usually presenting one day later, ensuring that a person shedding virus will be out and about among currently uninfected individuals [42] . severe symptoms may be due to viral replication and destruction of tissue; however, over-reaction of the immune system is often the cause [41] . this is exemplified by secondary bacterial pneumonia where blooms of pathogenic and/or commensal bacteria can result in severe disease or death [38] [39] [40] [41] . however, by the time a person has become ill, they have likely spread the virus by direct contact with an uninfected individual, contaminated fomites, or aerosolized respiratory droplets [46] . the fact that many pathogenic viruses have circulated in humans for hundreds to thousands of years, despite our best efforts to eliminate them, speaks to their success. as far back as the 16th-11th centuries b.c., there is evidence of poliomyelitis caused by poliovirus in egypt [47] . a more recent example is measles virus, which appears to have diverged from rinderpest sometime in the 11th or 12th century b.c. [48] . the long histories of human disease caused by these and other pathogenic viruses made them low hanging fruits for study in the new field of microbiology in the early 19th century [47] . this can explain much of the bias of current knowledge toward pathogenic viruses. while it can be argued that in the majority of human history these viruses were the most formative in terms of social, cultural, and behavioral characteristics of humans, it cannot be overlooked that this point may also be similarly biased. the notion that some viruses could live in relative homeostasis with the organisms they infect is very recent, and such interactions may prove to be a renaissance in the thinking of the importance of different portions of the virome. in fact, some of the most clinically important viruses of the last century were not classically pathogenic, but rather, persist within the host in relative silence for long periods of time. the characteristic strategy of persistent viruses is one of immune evasion and long term interaction with the host. typically, these viruses exhibit mild and short-lived acute phase infections followed by establishment of a long term niche within a host. in this niche, the virus may go through stages of dormancy and reactivation, or slow replication for a long period of time [50] . this ability to progress to persistence for months, years, or the entire life of the host clearly delineates these viruses from the pathogenic viruses [51] . these relationships straddle the border somewhere between symbiosis and pathogenesis, traditionally making it difficult to classify them. in this context, i will typify long-term infections with well-described detriment to the host as persistent, and other longterm interactions as either commensal or mutualistic (see respective sections, below). while the virus has a safe home during persistence, the question arises that if a virus is not replicating, or is present at very low levels, how is it transmitted to the next host? the answer lies in the spread of virus via close intrahost interactions that may occur many times during the host's life. the most conventional method by which persistent viruses are transmitted is contact between mucous membranes. this includes kissing and sexual intercourse, but can also include transmission vertically from mother to child, in rare cases [37] . the prototypical example is herpes simplex virus 1 (hsv-1) which, after an initial acute or subclinical infection, moves into a sensory neuron and enters a period of latency [52] . later in life the virus can break latency, move down the neuron to the skin, and begin to replicate, the prototypical presentation of which is a cold sore. these lesions release infectious virus that can be transmitted to a new host. after a short time, the virus returns to latency and the lesion will heal [52] . the success of this strategy of latency and mild pathogenic effect is made obvious by the fact that approximately two-thirds of the world's population is infected with hsv-1 [53] . non-conventional methods of inoculation have arisen concomitant with certain human tendencies and interactions. the most common examples include intravenous drug use, blood transfusion, and organ transplantation, all of which break typically impassable barriers [54] . for viruses like hepatitis c virus (hcv) and human immunodeficiency virus (hiv) that circulate in the blood of their host, these disruptions of natural barriers created a heyday for transmission. an important development of modern medicine was the implementation of standard blood screening for these and other persistent viruses and has led to a decrease in medically acquired disease [55, 56] . because of the longevity of asymptomatic interaction, those who are infected with persistent viruses may not know for years. this has consequences not only for transmission to others, but also for eventual progression to disease, as treatment is delayed until symptoms emerge when the damage is often irreparable. for hiv, the condition of acquired immune deficiency syndrome (aids) weakens the immune system drastically, allowing opportunistic microbes to cause fatal disease [57] . hcv is the most common cause of chronic liver disease, cirrhosis, and liver cancer in the form of hepatocellular carcinoma, all caused by years of persistent viral presence [58, 59] . the delayed onset and long-term homeostasis of persistent viruses within their hosts makes them a unique class within the human virome. understanding the biology of these viruses and the development of methods to control and eliminate them is an area of much importance in future medical science. additionally, further characterization of the human virome is likely to uncover more viruses that persistently infect humans [31] , and such discoveries could pave the way for the treatment of diseases of currently unknown etiology. as will be discussed in later sections, not all viruses are pathogenic. herein, i classify viruses that impact host physiology without directly eliciting a disease state as atypically pathogenic viruses. for example, some viruses infect humans without ever causing disease while others inhabit and change the composition of the human microbiome without ever infecting human cells. since our immune system can't tell the difference between harmful or benign viral signatures, the mere presence of these viruses harbors potential to cause disease. so far, this type of interaction has not been defined as a facet of the virome, but in this section i will review examples from the scientific literature that hint at such scenarios. endogenous retroelements (eres) represent a unique class of human-colonizing viral material that is resident in the germ line and include endogenous retroviruses (ervs), retrotransposons, and retrotranscripts [60] . while the general classification of these genetic elements will be defined in this work as mutualistic (see the mutualistic virome, below), it is evident from a number of studies that they can sometimes contribute to pathogenesis. in the autoimmune disease multiple sclerosis (ms), human endogenous retrovirus (herv) syncytin expression is upregulated in lesions and leads to an increase of cellular protein oxidation and destruction of oligodendrocytes [61] . another possible autoimmune contribution of eres is by their engagement of nucleic acid pattern recognition and downstream immune activation during both their rna and cdna synthesis [62, 63] . carcinogenesis may also occur, as exemplified by the rec protein of herv-k(hml2), which can facilitate tumorigenesis when expressed in mice [64] . a final example of deleterious consequences conferred by eres is their ability to destabilize the genome via insertion, rearrangement, and deletion [65] . continued exploration is necessary to expand upon these data to elucidate the mechanisms behind the negative impacts of eres on human health. bacteriophages (from here, referred to as phages), which are canonically classified as commensals (see the commensal virome, below), may also play an atypically negative role. while these viruses do not infect human cells, the estimated 10 15 phages present in the human gut [66] and those elsewhere on the body are not likely to be completely benign. while it is known that phages can be co-opted for benefit to the host [66] , i postulate that the presence of this amount of genomic and proteinaceous material is unlikely to be immunologically inert. an example of how a phage could affect human health is as an endogenous ligand for immune activation. it is known that humans harbor antibodies to phage proteins [67] [68] [69] [70] and that phagocytic cells of the immune system can ingest phages [71] , but whether this has any negative impact on health is unknown. toll-like receptors (tlrs) -7 and -9 play key roles in the production of autoantibodies, presumably via sensing of nucleic acid containing immune complexes (reviewed in [72] ). these autoantibodies could be derived from any number of endogenous sources, including those of phages. interestingly, identifying the particular nucleic acids responsible for this activation in vivo has been challenging, though functional work has vetted this mechanism with immune complexes containing nucleic acids [73, 74] . it is possible that b-cell receptor (bcr)-mediated endocytic or plasmacytoid dendritic cell (pdc) phagocytic internalization of phages could deliver ligands necessary for these responses. in this light, i would argue that correlating phage localization and expression with disease state is a particularly intriguing area of research moving forward. such research may also elucidate mechanisms by which specific bacteria (and their viral cargo) are linked to autoimmune, allergic, and pathogenic phenotypes. protozoan parasite-associated viruses have emerged as another source of atypically pathogenic effects in humans [75] . such interactions are reminiscent of the uses of viruses as agents for forwarding their host's survival in parasitoid wasps and plants (see an ecological framework of the human virome, above). the recognition of the leishmania-infecting lrv1 virus by human macrophages can promote inflammation, subvert the immune response, and ultimately promote persistence of the parasite [76] . trichomonas vaginalis harbors a trichomonasvirus that triggers ifn responses via tlr-3, leading to inflammatory sequelae [77] . at present, there is insufficient data as to whether these are evolutionarily adapted mechanisms meant to perturb host immunity to benefit the parasite, but the extensive literature of such interaction in insects and plants makes this a tantalizing conjecture. additional characterization should explore the nature of the interactions of protozoan viruses with humans. while all the above postulates are conjecture on the limited knowledge base regarding atypically pathogenic virus-host interactions, it must be noted that these are certainly not overlying principles. associated pathologies likely require particular genetic dispositions and/or external stressor states. it cannot be understated, however, that a better understanding of the viruses that typically inhabit humans will likely lead to a concomitant rise in the cases of viruses identified as atypically pathogenic. the term commensal was originally coined to describe an organism that benefitted from being on or in a host but did not have any direct detriment or benefit for the host [78] . as the field of microbiome research blossomed, it became clear that many of the microbes that had originally been considered commensal truly did have direct effects on host health (reviewed in [79] ). these organisms may be more accurately defined as atypically pathogenic or mutualistic (see sections above and below, respectively) if these effects are common or stable, but in general, scientists have followed the practice of innocence until proven guilty. however, it is likely that many commensals exist that either rarely or never have effects on the host. while at the local level some commensal viruses may have detrimental effects on individual cells, many eukaryotic and prokaryotic viruses are associated with healthy human tissues [80] [81] [82] [83] [84] [85] [86] [87] . the prototypical examples of commensal viruses are phages that infect bacteria [88] . no phages are known to infect human cells and therefore their presence may be primarily innocuous. the effect of phages on the composition of the microbiome, in that they may kill or inhibit growth of some bacteria, could be considered a possible detriment or benefit depending on the situation, albeit indirect. in fact, the targeted utilization of phages to eliminate unwanted bacterial pathogens (phage therapy) was introduced in the early 1900s and has many potential benefits including, but not limited to, low toxicity, treatment of antibiotic resistant infections, and rapid discovery/generation [89] . however, concern over unknown health implications and the rise of antibiotics stalled the field, though a renaissance has been building since the 1990s [90] . many different levels of this application exist, such as treatment of animals and plants with phages to ensure better yield as well as to kill human pathogens that may grow on or in these food sources [91] . phages are also known to be able to stimulate and/or modulate immune action in humans at multiple levels including antibody generation, development of adaptive immune cells, and innate pattern recognition (reviewed in [88] ), and the possible applications of phages in these contexts are intriguing. in the future, sections of this group may be able to re-classified as atypically pathogenic or mutualistic if their presence can be linked to roles in specific disease phenotypes, but for now they are most appropriately classified as commensal. viruses can also infect fungal members of the human microbiome (mycoviruses) and protozoa of the human macrobiome [77, [92] [93] [94] [95] [96] [97] [98] . while much less is known about these particular types of viruses, similar conjectures have been made as for phages in regard to their potential as both atypical pathogens and mutualists. direct effects on human health are likely rare, though they have been observed for protozoan parasite viruses (see atypically pathogenic viruses, above). the presence of these viruses may ultimately have an indirect effect on human health of unknown consequence. investigation of the breadth and impact of such viruses is an interesting and important area of future research, particularly for the benefit of the immunocompromised and those in developing countries who are most affected by diseases caused by fungi and protozoa. there is also an abundance of prototypically planttropic viruses found in the human gut [99] . it is likely these viruses have been acquired via agricultural crops in the diet and the effect of their presence is unknown, but assembly of plant viral particles in escherichia coli has been exhibited, so it is possible that these viruses may interact with gut bacteria [100] . some commensal viruses can infect human cells and use the human as a vessel for transmission. these viruses establish asymptomatic infections that do not result in a change in host health or behavior. this represents a veritable antithesis to the strategy of parasitic human viruses (see the parasitic virome, above), wherein viruses have prioritized trade-offs other than pathogenicity to ensure their transmission to a new host. the most well documented examples of asymptomatic infection of humans by commensal viruses are the rhinoviruses and other infections of the nasopharynx and upper respiratory tract [80, [101] [102] [103] [104] . many of the human papilloma viruses (hpvs) interact with human cells as an asymptomatic infection of mucous membranes or skin [105] . anelloviruses are also well described human commensals [106] [107] [108] [109] [110] . while immune detection of non-human tropic commensal viruses is avoided by physical separation, the human-tropic commensal viruses assuredly engage with host innate and adaptive immune responses, and so must avoid or subvert these mechanisms to maintain their immunological silence. description of the viral techniques enabling this evasion is an important direction for future research on these viruses, as this may point to novel avenues of therapeutic intervention for suppression or activation of immune responses. while commensal viruses do not have any direct effect on human health during homeostatic equilibrium, perturbation of homeostasis may cause them to contribute to pathogenesis (see atypically pathogenic viruses, above). contrarily, the presence of viruses from the final major group of the human virome, mutualistic viruses, exhibit positive effects on host health via an assortment of mechanisms. perhaps the most underappreciated portion of the virome are those viruses that have a positive health benefit for humans. a mutualistic interaction is one that benefits both organisms involved [111] . it is reasonable to assume that it may be beneficial for a virus to encourage and aid in its host's health to facilitate a longer lasting and amenable environment for survival. but this is a bit counterintuitive considering that above i discussed that viruses are intrinsically parasitic. this can be rationalized with the assertion that nearly all mutualistic interactions were probably at some point a form of parasitism, then passed through a commensal stage, and finally adjusted to mutualistic harmony through co-evolution. this stipulation is based on the reasoning that the amount of evolutionary change necessitated in the virus and/or the host to switch interaction types requires significant adaptation and thus is more likely to proceed stepwise [112] . a relationship must become sufficiently non-pathogenic to allow interaction without rejection as well as develop benefit for both sides, and an intermediate state seems a logical platform for this. indeed, this postulation seems bolstered by the spectrum of mutualistic relationships that exist, spanning obligate to conditional mutualistic benefits [18] . a classic non-viral example that illustrates this point is the integration of the mitochondrion into the cells of ancient prokaryotes or eukaryotes. this kind of relationship is considered symbiogenic, meaning that once separate organisms have now fused into a distinct new species [18] . either by phagocytosis or infection, an endosymbiosis was established in which the mitochondrial bacterial progenitor supplied some benefit to its host. millions of years later, mitochondria are part of all eukaryotic organisms with functions spanning energy production, innate immunity, and membrane potential, to name a few [113, 114] . it is now appreciated from data on the microbiome that it would be to the host's benefit if the power of the genetic information in and around them could be harnessed as an evolutionary (and medical) tool for self-betterment [115] [116] [117] . this could occur in a number of ways, either through encouraging colonization by beneficial microbes or even by integrating the genetic material of these microbes into the human genome. a small body of literature characterizing virus-human mutualism has given a glimpse into this facet of the virome. in the next sections, i will review two levels of symbiotic relationships and discuss important gaps in knowledge important for the understanding and possible utilization of these interactions for human health. symbiotic viruses that are transmitted vertically from parent to child in the germ line are considered primary symbionts [118] . these relationships are often ancient and obligate for both parties [119] . the most obvious example is the eres that have integrated themselves into human genomes at many points throughout millions of years of evolution. while some have been inactivated by recombination, deletions, and random mutations, many still appear to be expressed [65] . it is also obvious the control of these elements is an important challenge for the host, as many strategies have evolved to control replication and reinfection of inserted eres [120, 121] . to date, the role of most eres in the human genome is unknown, but a few human and mammalian examples indicate that their presence confers a number of benefits. some of the most important effects of ere colonization come in the form of genomic diversity and plasticity. eres can not only insert new genetic material, including protein coding genes, but also play roles in genetic mobility and control. the recombinatorial ability of eres is conferred upon insertion and has facilitated recombination and duplication of large swathes of genomic material [122, 123] . where an ere inserts itself can be an important factor in how it affects the genome, as insertion into a protein coding gene could be innocuous or catastrophic. alternatively, insertion in or near promotion and regulatory elements can result in changes in transcriptional control [124] , as can the native regulatory sequences in the eres themselves [125, 126] . of course, all of these benefits can have similarly damaging consequences, as destabilization of the genome or particular genes could have profound negative effects on the host (see atypically pathogenic viruses, above). however, natural selection dictates such effects will not successfully continue in the host lineage. eres also have important immunologic and developmental roles. building off of the transcriptional control effects mentioned in the last paragraph, eres appear to have a role in gene upregulation via pattern recognition during the initiation of adaptive immune responses [127, 128] . adaptive responses to eres can also play a role in restricting viruses by neutralizing incoming exogenous virus via antibody development against ere surface glycoprotein antigens [129] . examples from mice also indicate that the expression of the erv glycoprotein env can competitively bind surface receptors important for exogenous virus entry and that a short product of an erv gag gene can inhibit the nuclear translocation of incoming capsids. [130, 131] . developmentally, the acquisition of erv syncytins has been a critical step in the evolution of placentation in humans [27, 132, 133] . the presence of retroviral nucleic acids in human cells also suggests that ere reverse transcription may be able to activate host innate immunity [62] . whether humans can harness the beneficial properties of eres is of much scientific import. for years it has been debated whether gene therapy using retroviral vectors for insertion of genes into an individual would be of more help than harm. overall the utility of such methods have been tempered by the observed oncogenic potential of such therapies [134] . recently, studies with retroviral-modified chimeric antigen receptor t-cells (car tcells) have shown impressive application for the activation of adaptive immunity against cancers, though there have also been morbid failures in this field [135] . the application of retroviral-modified car t-cells could presumably be expanded to infectious disease as well. these progressive areas of medical science will continue to be explored and debated well into the future, and better understanding the relationship of eres with their human hosts will be indispensable for these studies. similar to the primary symbionts, secondary symbionts form mutualistic relationships with their host, but are instead acquired via non-germline vertical transmission or horizontally during the post-natal life of the host. a popular example is that of phages. beginning with the microbial colonization of a child during birth, phages hitch a ride with their host bacteria. phages can regulate bacterial species in the gut, as it is known that phages can be coopted into mucosal surfaces for this purpose [136] . free phages may also serve a similar role as sentinels maintaining a balanced microbiota. these benefits have been studied extensively as possible medical tools in phage therapy (reviewed in [137, 138] ) and there has recently been a resurgence of interest as the problem of antimicrobial resistance has become more dire [139] . these same principles can likely be extended to viruses that parasitize other microbes and macrobes as well. other examples of secondary symbiotic viruses are isolated, but numerous. in general, these viruses likely compete for receptors, cellular resources, or niches within hosts to confer protection. hepatitis g virus (hgv) is known to slow development of hiv infection to aids pathogenesis [140, 141] and human cytomegalovirus (hcmv) infection can suppress hiv infection by limiting availability of the coreceptor ccr5 on macrophages [142] . infection with hepatitis a virus (hav) can suppress infection with hcv and may actually promote clearance [143] . evidence from mice supports the assertion that viruses can confer resistance to non-viral disease as well, as infection with murine gammaherpesvirus results in protection from challenge with listeria [144] and type 1 diabetes can be prevented in mice infected with lymphotropic viruses [145] . oncolytic viruses, which specifically target, infect, and kill tumor cells are also notable in both their novelty and their clinical utility (reviewed in [146] [147] [148] ). viruses may even be able to provide developmental and immunological benefits not unlike that of the bacterial microbiota, as evidenced by the establishment of gut homeostasis in mice colonized with norovirus [149] . endogenous retroelements could also presumably be included here in the case of their population of a nongermline subset of cells within an individual and conferral of benefit to the host. one of the main functions of secondary symbionts may be to help maintain a baseline of immune activation in order to give the host a head start upon challenge with incoming pathogens. it has been estimated that healthy humans are infected with > 10 viral infections at any given time [51] . this constant barrage of immunogenic material presumably drives a smoldering activation of both innate and adaptive immunity. from this, it may be concluded that a state of continual challenge may be integral for the immune system to be fortified against incoming pathogens. while there is a plethora of research about secondary symbiotic bacteria, similar attention must to be paid to the virome. the examples outlined above point to incredible benefit derived from the interaction of humans with these mutualists, and significant effort must be made to discover and harness such potentially impactful relationships. moving forward, consideration of the effects of the human virome will be crucial in disease treatment and discovery of disease etiologies. from purely pathogenic viruses to those mutualists helping humans adapt to their world, the human virome has the potential to lend insight into the basic and applied biology of human health. in light of this, further exploration into the composition, tropism, and evolution of the human virome will undoubtedly make way for innovations in the treatment of human disease. throughout this manuscript, i have assembled the components of the human virome into broad classifications of parasitic, commensal, and mutualistic. while these definitions are logical, the intermixing of the groups and exceptions to rules drives home the point that the virome has inherent plasticity. disruption of the yin and yang of virus-host interactions can contribute to this transitory nature and may blur the lines that delineate the three main groups. this acknowledgement of convention and the departures from it gives both structure and flexibility to this conception of the human virome. the culmination of these ideas provides a template that identifies gaps in our current knowledge, postulates interactions that are currently unknown, and describes future areas of research, the results of which can then be classified in this same manner. i look on with anticipation to the continuation of the scientific soul search that is the 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and may lead to clearance of hcv herpesvirus latency confers symbiotic protection from bacterial infection prevention of type i diabetes in nonobese diabetic mice by virus infection recent progress in the battle between oncolytic viruses and tumours gene therapy progress and prospects cancer: oncolytic viruses apoptin, a tumor-selective killer an enteric virus can replace the beneficial function of commensal bacteria any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the national science foundation. key: cord-023669-3ataw6gy authors: masur, henry title: critically ill immunosuppressed host date: 2009-05-15 journal: critical care medicine doi: 10.1016/b978-032304841-5.50056-x sha: doc_id: 23669 cord_uid: 3ataw6gy nan as the population of patients with cancer, organ transplants, vasculitides, and human immunodefi ciency virus (hiv) infection has grown, intensivists are seeing more and more patients with altered immunity. these patients may come to the intensive care unit (icu) because of life-threatening opportunistic infections, or they may develop life-threatening infection while in the icu for an unrelated problem. intensivists must recognize how these patients differ from immunologically normal patients in terms of clinical presentation and management of these infections. this chapter emphasizes the important ways in which immunosuppressed patients differ from immunologically normal individuals in terms of infectious complications. clearly, however, immunosuppressed patients also develop complications from their underlying diseases and the drugs used to treat these underlying processes. these noninfectious complications are not the focus of this chapter but are reviewed in chapter 81. patients who are at increased risk for infectious complications because of a defi ciency in any of their host defense mechanisms are referred to as compromised hosts. patients in icus are almost universally compromised either by virtue of their underlying disease or by virtue of the invasive devices utilized to support and monitor them. patients are termed immunocompromised or immunosuppressed if their defect specifi cally involves immune response. often, patients who have defi cient infl ammatory response (e.g., neutropenia) are grouped into the category of immunocompromised or immunosuppressed, although technically they have a different category of defi cient host response. patients in icus are often immunosuppressed as a result of their underlying disease, therapy, or nutri-tional status. this chapter focuses specifi cally on patients who are immunocompromised or immunosuppressed. the microbial complications that any patient develops are determined by general, nonspecifi c barriers; innate immunity; acquired specifi c immunity; and environmental exposures. nonspecifi c barriers include anatomic barriers such as intact skin and mucous membranes; chemical barriers such as gastric acidity or urine ph; and fl ushing mechanisms such as urinary fl ow or mucociliary transport. organisms that breach these barriers encounter nonspecifi c and innate host factors termed the acute phase response. acute phase responses include trigger molecules and effector molecules. organisms also encounter acquired specifi c immune response systems including mononuclear phagocytes and antibodies. 1 infections that occur may result from normal fl ora that colonize mucosal or cutaneous surfaces. infections may result from abnormal fl ora that have invaded or replaced normal fl ora because of environmental exposures, disrupted barriers, or selective pressure of antimicrobial agents. table 54-1 lists organisms that cause disease when specifi c anatomic defenses are disrupted in individuals with normal microbial fl ora. infections may also result from common defects in the infl ammatory or immunologic systems; examples are detailed in table 54 -2. [1] [2] [3] [4] [5] [6] [7] [8] [9] infl ammatory and immunologic barriers can be disrupted by the primary disease process (e.g., tumor can invade the bone marrow, immunologic abnormalities associated with aplastic anemia or collagen vascular disease can destroy cells either in the bone marrow or the periphery). infl ammatory and immunologic mechanisms can also be disrupted by drugs. cytotoxic drugs, for instance, can reduce neutrophil number and function. certain monoclonal antibodies can destroy lymphocyte populations or interfere with cytokine attachment to receptor sites. some agents such as corticosteroids have multiple effects on neutrophils, lymphocytes, and soluble factors. infections may result from organisms that are usually not pathogenic, but become opportunistic because of poor host defense mechanisms. opportunistic infections are defi ned as those that occur with enhanced frequency or severity in a specifi c patient population compared with a normal patient population. pneumocystis jiroveci, for example, never causes disease in immunologically normal individuals but can cause frequent episodes of pneumonia in certain immunosuppressed patients. candida can cause mild mucosal disease in normal patients receiving antibacterial drugs but causes more frequent and more severe mucositis when patients have impaired cell-mediated immunity. recognition of which host defense mechanisms are disrupted enables the clinician to focus diagnostic, therapeutic, and prophylactic management and optimize patient outcome. for instance, if a patient presents with severe hypoxemia and diffuse pulmonary infi ltrates, a health care provider who recognizes a prior splenectomy as the major predisposition to infection would focus the diagnostic evaluation and the empiric therapy on streptococcus pneumoniae and haemophilus infl uenzae. by contrast, if the patient's major predisposition to infection were hiv infection with a cd4+ t lymphocyte count below 50 cells/µl, the health care provider would focus on pneumocystis jiroveci and s. pneumoniae; if a cytomegalovirus (cmv)-negative patient's major predisposition were a recent allogeneic stem cell transplant from a cmv-positive donor, then cmv would be a prime consideration. [2] [3] [4] [5] [6] [7] [8] [9] immune competence should ideally be measurable by objective laboratory parameters. in fact, the risk for opportunistic infection in patients with hiv infection can be assessed by clinical laboratories with a high degree of accuracy by measuring the number of circulating cd4+ t lymphocytes. 5 the susceptibility of cancer patients to opportunistic bacterial and candida infections can be assessed by measuring the number of circulating neutrophils. 7, 10, 11 the predisposition of patients with certain congenital immunodefi ciencies can be assessed by measuring serum immunoglobulin levels. 12 unfortunately, however, for a large number of immunodefi ciencies, no objective laboratory measures have been validated as predicting the risk of infection. moreover, laboratory measures must be interpreted in context. cd4+ t lymphocyte counts have great prognostic value in patients with hiv infection but not in most other patient populations; neutrophil counts are relevant in all patient populations, but low counts are associated with disrupted mucosal surfaces compared with those with intact mucosa. thus laboratory parameters must be interpreted in the context of the patient's underlying disease-risk is not always easily manageable by measuring one laboratory parameter. most importantly, most patients have multiple overlapping predispositions to infection. knowledge of the infectious complications associated with specifi c diseases, specifi c immune defects, and specifi c laboratory abnormalities is helpful for predicting and managing infectious complications. however, a specifi c diagnosis should be established in each patient: knowledge of the immune defect helps guide empiric therapy or helps determine therapy if a diagnostic procedure is not safe to perform. immunocompromised patients, by defi nition, are susceptible to a broader array of pathogens than immunocompetent patients. understanding the specifi c immune defect can be enormously helpful in understanding the likely location and source of infection. however, the immune defect must be assessed in the context of the specifi c disease: the clinical manifestations of hiv infection, for instance, are quite different from the clinical manifestations of patients with other diseases that alter cellmediated immunity such as lymphoma. the immune defect must also be interpreted with the understanding that predisposition to infection is usually multifactorial: in addition to neutropenia or lymphocyte depletion, patients often have impaired mucosal barriers, poor ciliary function, or breaches in their skin (i.e., from catheters) that can increase their risk of infection. effective management of opportunistic infections requires understanding of several basic tenets of care. 1. diseases may present with subtle symptoms and signs, and patients are predisposed to deteriorate precipitously. because immunocompromised patients may lack infl ammatory and/or immunologic mediators, the clinical manifestations of infections are often less prominent and less impressive than immunocompetent patients with similar complications. thus clinicians must recognize that even subtle changes in skin color, catheter site appearance, chest radiograph, or abdominal examination may warrant an aggressive diagnostic evaluation and early institution of broad-spectrum empiric therapy. although all icu patients demand prompt attention and vigorous diagnostic and therapeutic management, many types of immunosuppres-sion can be associated with especially precipitous clinical deterioration despite their innocuous presentation. 2. fever is not invariably present when patients are infected. although fever is not invariably present in any patient population with infection, immunosuppressed patients are notorious for developing infection in the absence of fever. thus infection must be considered as part of the differential diagnosis among patients with afebrile syndromes that might not appear to be infectious. conversely, patients with fever may not have infection: fever may be a manifestation of the underlying disease, an allergic response to a drug, or an underlying neoplastic or collagen vascular disease. 3. diagnostic evaluation needs to be prompt and defi nitive. as indicated earlier, patients with life-threatening infection may present with subtle symptoms and signs that progress rapidly: these early manifestations merit aggressive attempts to defi ne the anatomy of the lesion and the causative microbial pathogen. because the spectrum of potential pathogens includes a wide array of microorganisms (e.g., viruses, fungi, protozoa, or bacteria), clinicians must be certain that appropriate specimens are obtained and the appropriate microbiologic and histologic tests are ordered to identify common, as well as uncommon or unusual, pathogens. invasive diagnostic techniques such as bronchoalveolar lavage or tissue biopsies should be performed with less hesitancy than in immunologically normal patients. patients often have enhanced risk factors for invasive procedures, such as thrombocytopenia, coagulation factor defi ciencies, or compromised organ function. however, the benefi t of defi nitive diagnosis often outweighs these risks when the procedures are performed by experienced operators. 4. the threshold for initiating broad-spectrum empiric therapy should be low. because patients can deteriorate rapidly and because they are susceptible to such a wide array of microbial pathogens, clinicians should have little hesitation in instituting empiric antimicrobial therapy. this therapy must be directed at the full range of bacterial, fungal, viral, protozoal, and helminthic infections to which patients are predisposed. this therapy should be administered promptly, preferably within an hour of suspecting an infectious process. clinicians should initiate comprehensive regimens: antimicrobial agents can be discontinued or reduced when culture results and clinical events clarify the scenario. 5. foreign bodies and infectious foci should be addressed. patients may need careful imaging to be certain that they do not have an obstructed viscus or localized collection that should be drained. such imaging is appropriate even when signs or symptoms are unimpressive. similarly, patients often have multiple intravascular catheters that may need to be removed, as discussed in chapter 51. 6. consideration should be given to augmenting the immune or infl ammatory response. there may be opportunities to augment immunologic or infl ammatory responses by administering pharmacologic or biologic agents such as granulocyte colony-stimulating factor (g-csf) or intravenous immunoglobulin. [12] [13] [14] [15] eliminating immunosuppressive drugs or reducing the dose can also improve the patient's prognosis. 7. effi cacy and toxicity of therapy should be assessed serially. icu patients characteristically require attentive monitoring to assure the adequacy and safety of therapy. immunocompromised patients often have multiple prior and concurrent insults to their renal and hepatic function, and they often receive multiple drugs that can produce drug-drug interactions. thus monitoring the pharmacokinetics and assessing potential toxicities are especially important in these patient populations. moreover, because response to therapy may be less robust than in immunocompetent patients, antigen titers or pcr titers, as well as serial imaging studies, can be important to assure the adequacy of the management plan. therapy must often be continued longer than in immunologically normal patients. cytotoxic therapy-induced neutropenia is a major predisposition to infection. 7, 11 counts below 500 cells/mm 3 (the total of polymorphonuclear neutrophils and bands) increase susceptibility to infection in a linear fashion (i.e., the lower the neutrophil count, the greater the degree of susceptibility). the absolute neutrophil count is not the only factor that determines susceptibility, however, because some patients with cyclic neutropenias, druginduced neutropenias, or hiv-induced neutropenias, for example, are not nearly as susceptible to infection as are cancer patients receiving cytotoxic therapy. other important contributors to susceptibility, in addition to the absolute neutrophil count, are the duration of neutropenia, the functional capability of neutrophils, the integrity of physical barriers such as the skin and gastrointestinal mucosa, the patient's microbiologic environment (endogenous and exogenous fl ora), and the status of other immune mechanisms. for example, a patient with vancomycin-induced neutropenia during therapy for a staphylococcal infection may not develop any complications if the neutropenia is brief and defense mechanisms are otherwise intact. a patient with hiv-induced neutropenia may have prolonged or even lifelong neutrophil counts below 500/µl yet suffer few serious bacterial complications. 14 the presence of intact physical defense barriers is a major difference compared with cancer patients, whose skin and mucous membranes are disrupted by cytotoxic therapy in which the skin and gastrointestinal tracts are portals of entry for infections that are not controlled by diminished host immunologic or infl ammatory defenses. thus the patient with hiv infection is usually at a much lower risk for a bacterial infection than is a cancer patient, despite a comparable neutrophil count. in the 1960s and 1970s, aerobic gram-negative bacilli such as escherichia coli, klebsiella pneumoniae, and pseudomonas aeruginosa predominated as pathogens in neutropenic patients. anaerobic bacteria and aerobic gram-positive cocci were recognized less commonly. aerobic gram-negative bacillus infections were also associated with a poorer outcome than infections from gram-positive cocci. given the spectrum of pathogenic organisms that were seen in that era, combination therapy was usually advocated. 11,16-24 a number of reasons were proposed to justify combination therapy: (1) broad coverage of potential pathogens; (2) prevention of emergence of resistance; and (3) synergy. in general, these principles are reasonable concepts on which to base a preference for using combination therapeutic regimens. however, no study unequivocally demonstrated that combination therapy provided better outcomes than did monotherapy, assuming that both study arms contained drugs that had activity against the causative organism. in addition, predicting synergy proved diffi cult. 25 in the 1990s the spectrum of causative pathogens in neutropenic patients shifted from a predominance of gram-negative bacilli to a majority of gram-positive cocci including streptococci, staphylococci (including oxacillin-resistant staphylococcus aureus), and enterococci (including vancomycin-resistant enterocci). 20, 24, 26 the development of potent broad-spectrum β-lactam and quinolone drugs in the 1980s and 1990s has provided single agents that can probably provide comparable outcomes to combination therapy when used empirically or specifi cally. in the current era the choice of single or combination regimens is based predominantly on the spectrum of organisms that needs to be covered rather than attempting a strategy of trying to obtain more potency through additive or synergistic combinations. 10, 27 promptly initiating broad-spectrum antibacterial therapy for all cancer patients who are febrile and who are neutropenic (neutrophil count <500/mm 3 ) as a result of cytotoxic chemotherapy is standard practice. 7,10,27 for febrile neutropenic patients who have no apparent source of infection, there is no evidence that the initial antibacterial regimen is any more effective if a broad-spectrum antibacterial regimen consisting of two or more drugs is used instead of a single broad-spectrum antibacterial drug. for stable "low-risk" patients outside the icu, an oral regimen is now considered a reasonable approach. 7, 10, 17 such oral regimens would not be used for inpatients in most circumstances and would not be appropriate for high-risk or unstable patients. 7, 10 antifungal and antiviral drugs are generally not used empirically when neutropenic patients are initially treated unless there is a specifi c reason to have a high suspicion for a fungal or viral process. historically, an infectious cause of fever has been found in about two thirds of febrile, neutropenic cancer patients. when a specifi c causative organism is identifi ed, antimicrobial therapy is modifi ed to include an agent or agents determined to be active by in vitro susceptibility tests and that penetrate to the site of the infection. 10 combination therapy is advocated by some authorities for the specifi c (compared with empiric) therapy of either gram-positive or gram-negative bacteria, although, as noted earlier, there are little data for most pathogens that indicate that a combination regimen produces a better outcome than an appropriate single agent. therapy is generally not narrowed in terms of spectrum, however, because alteration of broad-spectrum coverage to focused therapy has been associated with more complications (e.g., "breakthrough bacteremias") unless the neutropenia resolves. whenever fever persists, therapy has generally been continued during the entire course of neutropenia because cessation of antimicrobial therapy has been associated with recurrent bacteremia resulting from the initial causative organism or a newly identifi ed pathogen. a 10-to 14-day course of antibacterial therapy is usually the minimum recommended if a causative infection is identifi ed. therapy is usually stopped promptly when the neutrophil count exceeds 1000 cells/µml if fever resolves and no source was ever identifi ed. empiric antibacterial therapy has been a successful strategy for reducing morbidity resulting from bacterial processes but has been associated with the emergence of fungal infections, as well as resistant bacterial pathogens. candida and aspergillus organisms, in particular, have become major causes of morbidity and mortality over the past 2 decades. these fungal processes can be diffi cult to diagnose because they are not always associated with detectable fungemia. the emergence of fungi as important pathogens, especially in patients with prolonged neutropenia, has led to the recommendation that empiric antifungal therapy be added to neutropenic patients who do not have an identifi ed bacterial process and who do not defervesce within 4 to 7 days of empiric antibacterial therapy. 7,10 fluconazole or an amphotericin b compound (e.g., liposomal amphotericin b) are often used, although echinocandins or certain other azoles such as voriconazole are being used by some investigators and clinicians. [28] [29] [30] [31] as patients receive chemoprophylaxis with quinolones and/or azoles during periods of intense neutropenia or immunosuppression, breakthrough pathogens are more and more likely to be resistant to the prophylactic agents. 32, 33 thus empiric regimens must be chosen with keen attention to the drugs that patients have received in the recent past, as well as pathogens they have previously been colonized or infected with. 34 patients with fever and neutropenia require aggressive diagnostic efforts to identify the cause of fever so that the appropriate antimicrobial agent is used and appropriate procedures (e.g., surgical drainage, removal of foreign body such as a catheter) can be performed. regular physi-cal examination is necessary to identify sites that merit more focused investigation: with impaired infl ammatory response, fi ndings on examination may be subtle. knowledge of the specifi c immunologic defect is important so that when cultures of blood, sputum, urine, or other appropriate body fl uids or body sites are performed, special microbiologic approaches can be used to detect viruses, fungi, helminths, protozoa, and bacteria. imaging studies are also important because intra-abdominal, intrathoracic, intracerebral, or musculoskeletal processes can be clinically subtle and may not be associated with identifi able organisms in the bloodstream. a growing array of antigen, nucleic acid, and gene detection systems including polymerase chain reaction and microarray gene assays are being investigated to facilitate diagnosis. some antigen or nucleic acid detection systems for blood or other body fl uids can be useful for detecting cryptococcus, histoplasma, hepatitis b and c, hiv, mycobacteria, pneumococci, and legionella. some of these approaches, despite their promising initial reports, are not yet clinically practical because of their level of sensitivity, specifi city, or the cost or expertise required to perform them adequately. careful attention to antimicrobial susceptibility patterns is also important. patients are exposed to repeated courses of antimicrobial agents. patients come into contact with contaminated environments in a variety of health care settings. resistance is no longer an issue exclusively for aerobic gram-negative organisms but is a concern for anaerobes, gram-positive cocci, viruses, fungi, and protozoa. clinicians must recognize that pathogens may be resistant when they are acquired by the patient, or they may become resistant during therapy if there is an inducible resistance mechanism or drug concentrations are not adequate to inhibit or kill the organism. a broad-spectrum agent used as monotherapy for febrile, neutropenic patients should have activity against aerobic gram-positive cocci and aerobic gram-negative bacilli including p. aeruginosa. 7, 10, 19, 35 potential drugs for this indication include certain cephalosporins (e.g., cefepime), carbapenems (e.g., imipenem or meropenem), and βlactam/β-lactamase combination agents (e.g., piperacillintazobactam). ceftazidime is an option chosen by some, but its poor activity against gram-positive cocci has caused some clinicians to use other agents. 18 intensivists must recognize, however, that these monotherapy regimens may not be appropriate in an icu. patients in icus, by defi nition, are either unstable hemodynamically or have a potentially life-threatening process such as diffuse pneumonia or are "fragile" because of concurrent processes. thus combination regimens are preferred by many authorities in icu settings, even though no study clearly documents superior outcomes from such combination regimens. the decade that started in 2000 is an era when microbial resistance is becoming an increasingly important problem for many types of bacteria including aerobic grampositive cocci and anaerobes, as well as aerobic gramnegative bacilli. multiple drug empiric regimens are more likely than monotherapy regimens to include an agent with activity against the offending pathogen(s). thus in a situation in an icu when failure to use an active drug is more likely to be lethal than in other settings, and when enhanced potency is a logical goal, combination therapy is prudent as an initial management strategy. thus adding vancomycin or linezolid or daptomycin for better grampositive coverage, adding a quinolone for better gramnegative bacillus coverage, and adding metronidazole to cefepime would be prudent in this patient population pending results of initial diagnostic studies. of note, however, is that although this strategy is logical, no study has shown convincingly that such an approach improves outcome. 22 a substantial number of febrile, neutropenic patients fail to improve in terms of fever or other manifestations. failure to improve may result from poor immune response, a need for drainage or necessity to remove foreign bodies, the use of drugs without activity against the causative organism, or a noninfectious process including drug allergy (i.e., fever resulting from a drug including an antimicrobial agent). the potential causative processes need to be aggressively reassessed on a regular basis by physical examination, history, cultures, and imaging techniques. most centers add antifungal therapy empirically at day 4 or day 7 of therapy if patients remain febrile. 10, 27, 29, 36, 37 fluconazole, liposomal amphotericin b, caspofungin, or voriconazole may be used: in some situations fl uconazole would be less attractive either because the patient has received fl uconazole prophylaxis or because molds are suspected. 28, 38, 39 the toxicity profi le of amphotericin b, even in its liposomal form, has led many clinicians to prefer voriconazole or one of the echinocandins (i.e., caspofungin, micafungin, or anidulafungin). 30, 40, 41 after empiric antimicrobial therapy is initiated, the optimal duration of therapy is a complex issue that depends on the type and severity of the infectious process and the duration and severity of immunosuppression, especially the neutropenia. if a causative bacterium is identifi ed, a minimum of 7 to 10 days of therapy is generally advocated, with at least 3 to 4 days being administered after neutropenia has resolved. longer courses may be required in certain settings. the duration of antifungal therapy is a complex issue and depends on the specifi c mycosis, the location and extent of disease, and the patient's immune status. 15 this is discussed in chapter 53. the use of combination therapy for fungal diseases remains controversial. 42, 43 a common problem in febrile, neutropenic patients is managing indwelling intravascular lines. [44] [45] [46] in general, these lines can be left in place initially if examination of the site reveals no indication of infection. blood cultures should be drawn through the catheter. although some experts advocate drawing a culture through each port of each catheter, obtaining this many blood cultures is often not feasible. if a patient is hemodynamically unstable and fails to respond promptly to fl uid administration, it is prudent to remove the line in case an infected catheter is the source of the sepsis. failure to remove the foreign body in this situation probably increases the likelihood of an unfavorable outcome. should blood cultures become positive and should the suspicion be high that the catheter is the source, antibacterial therapy may be successful in some settings (e.g., if the pathogen is a bacteria that is relatively sensitive to antibacterial therapy), thus avoiding the need to remove the catheter. situations suggesting that catheter removal is necessary include hemodynamic instability despite aggressive fl uid resuscitation, tunnel infection, or infections resulting from fungi or relatively antibiotic-resistant bacteria such as p. aeruginosa. a major determinant of prognosis is the immunologic status of the patient. prompt return of neutrophil number to normal improves the outcome. the use of g-csf or granulocyte-monocyte colony-stimulating factor (gm-csf), if not contraindicated by the underlying disease, can improve clinical status by hastening the return of neutrophil numbers and function. [12] [13] [14] [15] 47 granulocyte transfusions have not been proved useful in most clinical settings because of the inability to administer a large number of cells with adequate frequency. 48 the manipulation of immune response with cytokines, cytokine inhibitors, or immunoglobulins is the subject of considerable investigation: such interventions may reduce the duration of fever or the incidence of infections when used empirically, but in no setting have they been clearly shown to improve survival when administered after an infection has been documented. an algorithm for managing fever in neutropenic patients is provided in figure 54 -1. table 54 -3 suggests modifi cations of standard empiric regimens in certain common clinical scenarios. given the experience with frequent and severe infectious complications in cancer patients with neutropenia, it has been logical to attempt to prevent infection. 33 most microorganisms causing disease in this patient population arise from endogenous gastrointestinal, cutaneous, or respiratory fl ora. total protected environments probably reduce frequency of infection, but this approach is expensive and inconvenient. trying to prove a consistent benefi cial impact on survival has been diffi cult, and thus such isolation is rarely used anymore. some experts are enthusiastic about placing patients in positive pressure rooms so that pathogens do not enter via particles and droplets from outside the room. this type of isolation has not clearly improved outcome, however, and is not a standard of care. prophylactic bacterial therapy has also been controversial. 32 systemic antibacterial prophylaxis and systemic antifungal prophylaxis have been shown in some studies to reduce the number of infections, but their lack of effect on patient survival, their cost, and their impact on the emergence of resistance have made many clinicians reluctant to use them. selective gastrointestinal decontamination has not consistently improved survival and thus is not recommended by most authorities in the united states. antipneumocystis prophylaxis is, in contrast, highly effective in susceptible populations. prophylaxis for cmv is highly effective in well-defi ned, high-risk patients (e.g., some recipients of organ transplants who are either sero-positive for cmv or who are seronegative but received a graft from a seropositive donor). 2, 4, 49 strategies that reduce the period of immunologic susceptibility (e.g., reduce the duration of neutropenia), such as adding g-csf to a regimen or reducing the intensity of chemotherapeutic regimens, are promising. because so many patients are receiving highly active antiretroviral therapy (haart), opportunistic infections are not complicating the course of hiv infection to the same degree that they did in the 1980s and early 1990s. [50] [51] [52] [53] opportunistic infections continue to occur, however, in three groups of hiv-infected patients: (1) those who are unaware of their hiv status until they develop a clinical syndrome; (2) those who are unable or unwilling to receive appropriate therapy; and (3) those who fail haart and opportunistic infection prophylaxis. although haart has dramatically reduced the incidence of opportunistic infections, a surprisingly large fraction of patients either never respond virologically and immunologically or lose their response within the fi rst 12 to 24 months of therapy. these patients, most of whom have dominant viral quasispecies that are highly resistant to currently licensed antiretroviral drugs, will likely experience immunologic decline over the next few years and will again become more susceptible to opportunistic infections. severe necrotizing mucositis or gingivitis add specifi c antianaerobic agent (e.g., metronidazole, meropenem, imipenem, or piperacillin-tazobactam) plus agent with activity against streptococci; consider acyclovir. ulcerative mucositis or gingivitis add acyclovir and anaerobic coverage. add fl uconazole or caspofungin; consider adding acyclovir. pneumonitis, diffuse or interstitial add trimethoprim-sulfamethoxazole and azithromycin or levofl oxacin or moxifl oxacin (plus broad-spectrum antibiotics if the patient is granulocytopenic). perianal tenderness include anaerobic agents such as metronidazole, imipenem, meropenem, or piperacillin-tazobactam. abdominal involvement add antianaerobic agent (e.g., metronidazole, meropenem, imipenem, or piperacillin-tazobactam). patients with hiv infection develop clinical disease as a result of three basic processes: the direct effect of hiv on specifi c organs (e.g., cardiomyopathy, enteropathy, dementia); immunologically mediated processes (e.g., glomerulonephritis, thrombocytopenia); or opportunistic infections and tumors that are enabled by hiv-induced immunosuppression. hiv appears to cause direct organ damage. 50,53-57 this damage may be mediated by cytokines, lymphocytes, monocytes, or infl ammatory cells. cardiomyopathy, for example, can be a profound and lethal process that can lead to icu admission or complicate other processes. 55 when patients present with or develop pulmonary manifestations such as shortness of breath or diffuse bilateral infi ltrates on chest radiograph, cardiogenic causes must be considered. hiv also causes a diffuse pneumonitis, 56 profound encephalopathy, 54 and a diffuse enteropathy. 57 patients with compatible syndromes need a comprehensive evaluation to look for other specifi c opportunistic infections or tumors, especially those that can be specifi cally treated. in all of the hiv-caused syndromes, hiv as the etiology remains a diagnosis of exclusion. the institution of antiretroviral therapy appears to be benefi cial for patients with susceptible isolates, although data regarding such effects for these hiv-related entities are largely anecdotal. hiv-related thrombocytopenia and anemia appear to be immunologically mediated. 58, 59 both can be severe: platelet counts below 10,000/mm 3 and hemoglobins below 10 g/dl can be seen with the expected complications. these disorders are related to the development of antigenantibody complexes and may improve dramatically with the institution of antiretroviral therapy and a decline in viral load. for thrombocytopenia, intravenous immunoglobulin (or anti rhd antibody), corticosteroids, or splenectomy may also be useful. hemolytic anemia can also be severe: hemoglobin levels below 5 g/dl can be seen. the most prominent manifestations of hiv continue to be the opportunistic infections and tumors that occur as a consequence of hiv-induced immunosuppression. the cd4+ t lymphocyte cell number is a useful marker for predicting the occurrence of opportunistic infections in patients with hiv infection. 5, 9 this relationship of cd4+ t lymphocyte count to the occurrence of opportunistic infection continues to be as valid in the era of haart as it was before the licensing of the fi rst antiretroviral agent, zidovudine, in 1987. [60] [61] [62] figure 54 -2 demonstrates the typical relationship of cd4+ t lymphocyte counts to the occurrence of opportunistic infections. knowledge of this relationship permits the focusing of diagnostic, therapeutic, and prophylactic management. for instance, if a patient with hiv infection and a cd4+ t lymphocyte count of 700 cells/µl presents with diffuse pulmonary infi ltrates, the diagnostic evaluation and empiric antimicrobial regimen should focus on s. pneumoniae; h. infl uenzae; mycoplasma, legionella, and chlamydia organisms, as well as common community-acquired viruses. in contrast, if the same patient had a cd4+ t lymphocyte count of 50 cells/µl, the evaluation and empiric regimen would focus on pneumocystosis and cmv, although the previously mentioned processes that occur at high cd4+ t lymphocyte counts can also occur at lower cd4+ t lymphocyte counts. keeping in mind that cd4+ t lymphocyte counts are useful predictors of susceptibility to infection is important, but they are not perfect. occasionally, patients will develop opportunistic infections at "uncharacteristically" high cd4+ t lymphocyte counts. for instance, 5% to 10% of cases of pneumocystosis occur at cd4+ t lymphocyte counts greater than 200 cells/µl. 61 clinical parameters can provide additional clues; for example, oral candidiasis, a previous opportunistic infection, a prior episode of pneumonia, or high viral load are independent risk factors for the occurrence of pneumocystis jiroveci carinii pneumonia (pcp), and logically for other infections as well. 9 a frequent question is whether an hiv-infected patient's prior cd4+ t lymphocyte count nadir affects the likelihood of an opportunistic infection occurring if haart has stimulated a cd4+ t lymphocyte count rise. specifically, if a patient has a cd4+ t lymphocyte count of 400 cells/µl while receiving haart and that patient's cd4+ t lymphocyte count was 50 cells/µl before haart, is that patient at greater risk for developing an opportunistic infection than another patient whose current cd4+ t lymphocyte count is 400 cells/µl but whose nadir before haart was 250 cells/µl? the data suggest that these two patients have comparable risk (i.e., the current cd4+ t lymphocyte count is the most important predictor of risk and the earlier nadir has only minor infl uence on opportunistic infection susceptibility). 62 in evaluating the differential diagnosis of infectious syndromes in patients with hiv (and in every other patient population as well), geography is an important part of the history. tuberculosis is always a concern because of the extraordinary susceptibility of hiv-infected patients for developing active disease. 63 in many urban settings in the united states, each pulmonary evaluation should include smears and cultures for m. tuberculosis, both to diagnose the appropriate cause of the pulmonary dysfunction and to assist in determining what respiratory precautions are appropriate. in some areas of the country, such as the ohio river valley and indianapolis, histoplasmosis is as common as pneumocystosis in causing diffuse pulmonary infi ltrates. 64 in the southwestern united states, coccidioidomycosis must be recognized as a cause of pulmonary infi ltrates. the clinical presentations of tuberculosis, histoplasmosis, coccidioidomycosis, and other processes such as cmv can be clinically indistinguishable from pcp. thus for patients with pulmonary infi ltrates in an icu, prolonged empiric therapy is discouraged in favor of vigorous efforts to establish a specifi c diagnosis. hiv-infected patients are admitted to icus for several major syndromes: respiratory insuffi ciency, cerebral dysfunction, septic shock, hepatic or renal failure, and drug toxicities. 50 however, patients with hiv infection also come to icus for routine procedures and routine postoperative care. in those situations their management ordinarily requires no extraordinary measures, with two exceptions. first, the staff must be fully aware of how hiv is transmitted, the danger of injuries resulting from sharp objects, and the procedure for managing injuries involving sharp objects contaminated with blood or other biologic fl uids from infected or potentially infected patients. 65 second, drug interactions involving drugs used during procedures and certain antiretroviral drugs can have important clinical consequences. 66, 67 many of the protease inhibitors and the non-nucleoside reverse transcriptase inhibitors that are now the backbone of antiretroviral therapy can inhibit or enhance the metabolism of drugs that depend on the cytochrome p450 system. thus the half-lives of certain analgesics, sedatives, and hypnotics can be prolonged in hiv-infected patients who are taking ritonavir, for example. this pharmacokinetic effect is also relevant for a host of other therapeutic agents used in the icu and may affect their effi cacy or safety. clinicians need to be familiar with these interactions when selecting new therapies for procedures or for clinical entities. patients with hiv infection can develop severe pulmonary dysfunction because of common community-acquired pathogens such as s. pneumonia, legionella, mycoplasma, and chlamydia; adenovirus; infl uenza; or respiratory syncytium virus, as well as other opportunistic viruses and fungi. thus the diagnostic evaluation needs to be comprehensive, emphasizing direct smears of sputum or bronchoalveolar lavage. it is important to recognize that the clinical presentations produced by many causative agents can be similar. for instance, histoplasmosis, tuberculosis, and nonspecifi c interstitial pneumonitis can present identically to pcp. 50, 61, 63, 64, 68 thus although empiric diagnosis and empiric therapy may be reasonable as initial approaches to some patients with hiv infection and mild pneumonitis, such an approach is usually not appropriate for patients in an icu. evaluation of induced sputum is the fi rst step in the diagnostic approach to pcp. sensitivity can be 80% to 95% at many hospitals (at some institutions the yield is considerably lower). 69 specifi city should be 100% in an experienced laboratory. other pathogens, including mycobacteria, fungi, and routine bacteria, can be identifi ed in sputum as well. for intubated patients, respiratory secretions obtained by deep intratracheal suctioning are also likely to be useful, although they have not been as carefully studied as induced sputum. should the diagnosis not be established by evaluation of sputum or intratracheal secretions, bronchoscopy should be performed. bronchoalveolar lavage should diagnose almost 100% of cases of pcp, even if patients have received 7 to 10 days of empiric therapy. 70 a diagnosis of pcp is established by visualizing one or more clusters of organisms. diagnostic criteria for other opportunistic infections are reviewed in chapters 12 and 43. in patients with hiv, cmv merits special mention. culture of sputum or bronchoalveolar lavage does not provide useful information because patients with cd4+ t lymphocyte counts below 100 cells/µl will predictably have cmv present in their secretion independent of whether or not pulmonary disease is present. 71 a diagnosis of cmv pneumonia in this patient population is suggested by cytology and confi rmed by the presence of multiple inclusion bodies in lung tissue obtained by transbronchial or open lung biopsy. similarly, mycobacterium avium complex (mac) and hsv can often be found in respiratory secretions, but these organisms almost never cause pneumonia in patients with hiv infection. in other patient populations they can clearly cause pneumonia, but the dearth of cmv, mac, and hsv pneumonia in this patient population emphasizes the point that it is important to know from published literature what the clinical likelihood is for different microbial processes. fungal pneumonias other than pcp are generally diagnosed by direct microscopy or culture of respiratory secretions (sputum or lavage). candida organisms almost never cause pneumonia in patients with hiv infection. the frequency of cryptococcus, histoplasma, blastomyces, and coccidioides organisms as causes of pneumonia depends on the geographic exposure of the patient. among these mycoses, antigen detection techniques can be useful for fi nding cryptococcus and histoplasma organisms. mycobacteria frequently infect the respiratory tract of patients with hiv infection. as noted earlier, m. avium complex almost never causes pulmonary dysfunction in this patient population. when acid-fast bacilli are seen (as opposed to cultured) in respiratory secretions or tissue, m. tuberculosis is almost always the pathogen; m. kansasii and other mycobacteria less commonly cause disease. screening all patients with acid-fast bacillus smears is important for preventing transmission of tuberculosis and text continued on p. 1126 should be considered as part of a routine respiratory evaluation for patients with radiographic infi ltrates in most areas of the united states. therapy of opportunistic infections is summarized in table 54 -5. 72 while awaiting a specifi c diagnosis, it is reasonable to initiate empiric therapy in patients ill enough to merit admission to an icu. for patients with a cd4+ t lymphocyte count greater than 250 to 300 cells/µl, azithromycin and ceftriaxone or azithromycin and ampicillin-sulbactam would be reasonable choices. for patients with cd4+ t lymphocyte counts below 200 to 250 cells/ µl, levofl oxacin or moxifl oxacin plus trimethoprimsulfamethoxazole or pentamidine plus levofl oxacin or moxifl oxacin would be potential regimens. if pcp is documented, trimethoprim-sulfamethoxazole is always the drug of choice in patients who can tolerate it. table 54-5 lists alternatives for sulfa-intolerant individuals. regardless of which specifi c antipneumocystis regimen is used, corticosteroid therapy is indicated for any patient who presents with an oxygen pressure (po 2 ) below 70 mm hg or an alveolar-arterial gradient higher than 30 mm hg. [73] [74] [75] [76] patients with an initial po 2 lower than 70 mm hg are the subgroup with substantial mortality for whom corticosteroids have been shown to provide a survival benefi t. corticosteroids may provide more rapid and perhaps more complete resolution of pulmonary manifestations in patients who present with better pulmonary function, but survival in this population is so high that clinical trials have not been able to show survival benefi t. some experts are concerned that corticosteroid use will be associated with reactivation of latent infections such as cmv or tuberculosis. however, reactivation of life-threatening infections has not been associated with this corticosteroid regimen. how should a patient with aids-associated pcp be managed if there is no improvement, or if there is deterioration, after 5 to 10 days of therapy? the median time to improvement in clinical variables is 4 to 8 days; therefore, changes in therapy are probably not warranted before 5 to 10 days. at that point the accuracy of the diagnosis should be reassessed: consideration should be given to repeat bronchoscopy with transbronchial biopsy to determine if cmv, fungi, mycobacteria, or a nosocomial bacterial process is present. noninfectious processes such as congestive heart failure or tumor (e.g., kaposi's sarcoma) must also be considered. if pneumocystosis is the only causative process that can be identifi ed, corticosteroids should be added to the regimen if they have not been already. whether switching from one antipneumocystis agent to another or whether adding a second agent is helpful has not been determined by clinical trials. some human pneumocystosis isolates are resistant to sulfonamides, but such testing is available only in a few research centers. most clinicians add parenteral pentamidine to trimethoprim-sulfamethoxazole. parenteral trimetrexate or clindamycin-primaquine could be used as salvage regimens as well. patients who have not improved after 14 to 21 days of therapy with specifi c chemotherapy plus corticosteroids have an exceedingly poor prognosis. should patients with aids-related pcp be intubated and provided with mechanical ventilation? mortality for such patient populations was 70% to 80% in several series in the early 1980s. [77] [78] [79] [80] since that era, supportive care has improved, and treatment modalities for concurrent infectious and noninfectious processes have become more effective. patient selection for ventilatory support is probably also improving. patients who have multiple active opportunistic infections, substantial weight loss, and no response to 14 days of therapy have a worse prognosis than ambulating patients who develop respiratory failure the third day of therapy. thus decisions about icu support for patients with hiv infection and respiratory failure need to be individualized on the basis of a realistic assessment of prognosis, the availability of resources, and the preference of the individual patient. a frequent question for any hiv-infected patient in the icu is whether antiretroviral drugs should be continued or initiated during the critical or life-threatening illness. although there is no specifi c study of various strategies, most authorities discourage the use of antiretroviral drugs in the icu because of drug interactions and drug toxicities. in addition, the initiating haart can be associated with dramatic "immune reconstitution" syndromes that can complicate the process that brought the patient to the icu. [81] [82] [83] finally, almost all antiretroviral drugs that are commercially available are oral: in most situations it is better to discontinue all antiretroviral drugs for a few days or weeks or months rather than risk poor absorption and suboptimal serum levels. the latter would enhance the emergence of drug-resistant hiv. an important cause of admitting hiv-infected patients into the icu is either seizures or altered mental status. either can result from infectious or neoplastic processes caused by meningeal disease or parenchymal involvement. the differential diagnosis of meningeal disease includes pneumococcal and staphylococcal meningitis, cryptococcal meningitis, tuberculous meningitis, and lymphomatous meningitis, as well as involvement from other endemic mycoses and common community-acquired viral and bacterial processes. 24,84 diffuse central nervous system parenchymal disease can be caused by hiv itself, by progressive multifocal leukoencephalopathy, and occasionally by herpes viruses such as cmv or herpes simplex virus. focal mass lesions may be caused by toxoplasmosis or lymphoma. less often, tuberculosis, fungi, conventional bacterial abscesses, nocardia, and other tumors are the cause of focal lesions. these lesions can be diffi cult to distinguish clinically and radiologically. the cd4+ t lymphocyte count can help narrow the differential diagnosis, but csf or brain tissue is usually necessary for defi nitive diagnosis. the routine therapies for many of these processes are outlined in table 54 -5. toxoplasmosis deserves particular mention because of its frequency. [85] [86] [87] toxoplasmosis occurs mainly in patients with hiv infection who have cd4+ t lymphocyte counts below 100 cells/µl, have a positive igg antibody titer against toxoplasma, and who have not been receiving trimethoprim-sulfamethoxazole or dapsone prophylaxis. patients present with altered cognition, focal motor or sensory defi cits, or seizures. lesions may be unifocal or multifocal. they usually enhance with contrast, but this is not invariably true. for patients who fi t the profi le for high risk of toxoplasmosis, and with a compatible presentation, it is reasonable to establish an empiric diagnosis and institute specifi c therapy with sulfadiazine plus pyrimethamine or, for patients unable to tolerate sulfa, clindamycin plus pyrimethamine. corticosteroids may be needed for patients with considerable intracerebral edema or elevated intracranial pressure. antiseizure medication is usually instituted only after a seizure has occurred rather than prophylactically. most patients improve clinically and radiologically within 7 to 10 days. if patients fail to improve, a stereotactic needle biopsy is appropriate, especially because the prevalence of lymphoma is increasing. organisms can be diffi cult to see in brain specimens obtained by this technique. patients with hiv infection develop hypotension resulting from the same types of disorders as with non-hiv infected individuals-sepsis from a primary infection or a wound or device (especially an intravascular access device), fl uid depletion from vomiting or diarrhea, and hemorrhage from a gastrointestinal lesion are examples of common causes. the evaluation of hypotension in a patient with hiv infection must take into account factors particular to this patient population: it is susceptible to opportunistic infections; it undergoes many procedures that can be associated with infectious complications; and it receives an array of drugs, some of which have cardiovascular effects. thus evaluating hypotension in this patient population requires a comprehensive and thorough approach. a differential diagnosis of the major causes is shown in table 54 -6. adrenal function always deserves special attention because several viral processes, fungal and mycobacterial diseases, hiv, and drugs can suppress the adrenal axis and either cause hypotension or exacerbate it. patients with hiv infection typically receive several antimicrobial agents to reduce the likelihood they will acquire opportunistic infections. 9 primary prophylaxis is the term used to indicate strategies that reduce the likelihood of an initial episode of a disease process. secondary prophylaxis is the term used to indicate strategies that prevent recurrences or relapses. chronic suppressive therapy is identical to secondary prophylaxis: this refers to regimens that are continued after the initial therapeutic course to prevent relapses. all patients with hiv infection and cd4+ t lymphocyte counts below 200 cells/µl typically receive antipneumocystis prophylaxis. trimethoprim-sulfamethoxazole is the regimen of choice. patients who actually take this drug have very few breakthroughs of pcp and receive considerable protection against toxoplasmosis and certain routine bacterial infections. alternative regimens include monthly dapsone, weekly dapsone-pyrimethamine, or daily aerosol pentamidine. prophylaxis against m. avium complex is recommended for patients with cd4+ t lymphocyte counts under 100 cells/µl; clarithromycin and azithromycin are currently the drugs of choice. 9 many clinicians also use fl uconazole or acyclovir prophylaxis to reduce the frequency of fungal and viral processes, respectively, although this is not recommended because of issues of cost, pill burden, and the emergence of resistant pathogens. isoniazid prophylaxis is important for any patient with a tuberculin skin test that shows more than 5 mm of induration or a history of substantial recent exposure. 9 transmission of tuberculosis from patients to other patients, from patients to staff, or from staff to patients is an urgent concern in icus. patients with hiv infection are extraordinarily susceptible to tuberculosis. thus an infected patient poses a substantial risk, especially when hospitalized for pneumonia or when undergoing procedures at high risk for producing aerosols such as intubation, bronchoscopy, sputum induction, or aerosol pentamidine treatment. identifying potentially infected patients early and placing them in appropriate isolation until their tuberculosis status is fully examined is important. in many centers, patients with syndromes compatible with pulmonary or upper airway tuberculosis are maintained in isolation at least until three specimens of respiratory secretions have been examined for tuberculosis. hiv-infected health care practitioners need to carefully assess their risk of acquiring tuberculosis by their exposure in the icu. transmission of hiv is an issue that requires attention in the icu. 65 no evidence exists that hiv-infected health care professionals can infect patients, regardless of what procedure they perform, outside of two unusual events. hiv patients pose a risk to health care professionals, however. this risk can be substantially reduced by education, by strict monitoring for compliance with universal precautions, and by having proper equipment. almost all hiv transmission in an occupational setting occurs as a result of injuries involving sharp instruments (e.g., needles, scalpels). the risk of such injuries is about one case of hiv transmission per 250 injuries, but the likelihood of transmission in an individual accident depends on the amount of viremia at the time of the accident (late-stage patients generally have more circulating virus than do early-stage patients) and the nature of the accident. most authorities recommend immediate prophylaxis if a signifi cant injury occurs involving an hiv-infected patient. considerable debate exists over the optimal choice of drugs and the optimal duration of therapy, but it is clear that initiating therapy within a period of hours rather than days is best. many authorities now advocate a haart regimen for any situation when the patient and health care provider determine that therapy is appropriate, and continue that for 4 to 6 weeks. increasingly, icus are caring for organ transplant recipients, either in the period immediately after the procedure or during a crisis that occurs days, weeks, months, or years after engraftment. managing each type of organ transplant recipient has unique features depending on whether bone marrow, kidney, heart, lungs, liver, or other organs are transplanted. 2, 6 laboratory monitoring provides useful predictive information about the status of cellular immunity, humoral immunity, and neutrophil number and function. ultimately, however, clinical experience is necessary with each type of organ transplant and each immunosuppressive regimen to predict the most likely pathogens, when they most characteristically occur in relation to the transplant procedure, and what infl uence each immunosuppressive therapy has. an example of the temporal pattern of infectious complications after bone marrow transplantation is shown in figure 54 -3. 9 although such fi gures are useful conceptually, however, the immunosuppressive regimens are changing rapidly, and such fi gures may be misleading when applied to current transplantation protocols. organ transplant recipients share a complex interaction between immunosuppression and infection. immunosuppression is usually necessary in allogeneic transplantation to permit graft survival. the more potent the immunosuppression, the more likely infection is to occur. strategies that use antimicrobial agents (drugs, vaccines, and other biologic products) aggressively may reduce the risk of and damage from infection in a manner that allows more potent immunosuppression and better graft survival. such approaches may include prophylactic antibacterial and antiretroviral treatment, as well as prompt empiric therapy for emerging febrile episodes. patients receiving hematopoietic stem cell transplantation (hsct) or solid organ transplants are often receiving antimicrobial prophylaxis. acyclovir for hsv, valacyclovir for cmv, fl uconazole for yeast, voriconazole for yeast and molds, trimethoprim-sulfamethoxazole for pcp, and quinolones for bacteria are used in various combinations at different transplant programs. these agents dictate which organisms will break through to cause disease, and what their antibiotic susceptibility patterns will be. several pathogens deserve special mention. cmv is one of the most prominent pathogens for solid organ and bone marrow transplant recipients. [88] [89] [90] most disease is secondary (i.e., disease results from reactivation of a previously acquired, latent infection) in a seropositive organ recipient. in urban areas of the united states, 60% to 70% of the population is seropositive for cmv, and thus 60% to 70% of the transplant recipients will have latent infection that could potentially be reactivated. some cmv seronegative patients acquire primary infections from a cmvinfected organ or from cmv-infected blood or blood products. a few cmv seropositive individuals develop superimposed cmv disease from cmv acquired through a seropositive donor. laboratory monitoring of patients for evidence of cmv disease by using a dna amplifi cation assay, or surveillance of cmv antigen in buffy coat smears, is an important feature in efforts to reduce morbidity and mortality resulting from cmv. 89, [91] [92] [93] [94] [95] intensivists need to understand how to interpret these assays in terms of starting empiric, pre-emptive, or defi nitive therapy. strategies to reduce the frequency of cmv disease with acyclovir, intravenous or oral ganciclovir (or oral valganciclovir), the investigational agent proganciclovir, or immune globulin are used by many programs. cmv disease can cause substantial morbidity and mortality including fever, hypotension, pneumonitis, hepatitis, glomerulitis, enteritis, and allograft injury. the availability of ganciclovir, foscarnet, and cidofovir has enabled these conditions to be treated successfully in many instances, although all three of these drugs are associated with substantial toxicity. whether immune globulin (either immune globulin or specifi c hyperimmune globulin) adds anything to the potency of therapeutic regimens is not clear, although these products are usually administered when they are available. pcp has been reported in recipients of most types of organ transplants. most organ transplant programs use pcp prophylaxis. 6, 33, 96 trimethoprim-sulfamethoxazole is usually the prophylactic agent of choice because it is more effective than other agents, is well tolerated, and reduces the frequency of urinary tract infections and other potential complications (e.g., disease resulting from nocardia, s. pneumoniae, and haemophilus organisms). fungal infections have been common, but the causative pathogens are changing because of changes in prophylactic regimens. with the use of fl uconazole, candida albicans infections became less common. molds, especially aspergillus, became more important pathogens, as did fl uconazole-resistant candida. some programs are now using voriconazole prophylaxis. for such patients, mucormycosis and non-albicans candida are becoming more prominent causes of morbidity. thus clinicians must know what antifungal prophylaxis has been used in order to anticipate which complications will occur. mold infections can be diffi cult to diagnose: serum galactomannan assays can yield specifi c information, but the test has low sensitivity. mold infections almost never cause fungemia. thus diagnosis depends on cultures, which can be highly suggestive if obtained from sources such as bronchoalveolar lavage or biopsy. viral respiratory infections require particular mention because some are treatable and most are transmissible. community-acquired respiratory viruses such as adenoviruses, coronaviruses, or infl uenza can occur in immunocompetent or immunosuppressed patients. when respiratory infections occur in immunocompromised patients, health care professionals need to be certain that a transmissible virus is not the cause because of the potential to infect other patients, families, or hospital staff. of the respiratory infections, rsv deserves special attention in hsct patients. although rsv can, like other community-acquired viruses, cause disease in any patient population, it is especially lethal in solid organ, bone marrow, and stem cell transplants. thus rsv must be specifi cally sought in this patient population, as well as their visitors and health care providers, so that it does not spread to highly susceptible patients. similarly, when caring for immunosuppressed patients, attention to mycobacterium tuberculosis is important because this pathogen can also spread to other patients, families, and hospital staff. with more immigrants in the united states and more patients having travel exposure, m. tuberculosis needs to be considered in the differential diagnosis and specifi cally sought by gene probe, smear, or culture where appropriate. diagnosis and therapy of opportunistic infections and nosocomial infections should follow the guidelines given in chapters 43, 51, and 54. in choosing therapies, attention must be focused on the toxicities of antimicrobial agents and how they infl uence the outcome of the transplanted organ. in addition, drug interactions are important, especially with cyclosporine. drugs that alter hepatic metabolism, such as rifampin, rifabutin, and fl uconazole, can have substantial infl uence on cyclosporine levels and thus need to be used with careful pharmacologic attention. finally, clinicians must recognize that new immunosuppressive regimens and changing prophylactic regimens are changing the spectrum of infectious complications. as mentioned earlier, fungal infections are increasingly likely to be caused by species other than c. albicans: non-albicans candida, fusarium, and rhizopus are recognized with increasing frequency. similarly, prophylaxis with valganciclovir is reducing cmv disease and pushing disease that does occur later and later in relation to the transplant procedure. viruses such as hhv-6 and bk virus are causing disease. thus clinicians need to look for changing spectrum of pathogens, as well as changing manifestations if the morbidity and mortality caused by infection is to be managed optimally. ■ knowledge of a patient's specifi c defects in immunologic and infl ammatory response helps predict which opportunistic pathogens are most likely to occur. ■ icus are increasingly successful in enabling immunosuppressed patients to survive acute crises, especially if the defect in immunologic or infl ammatory function is reversible over time or by replacement therapy. ■ for neutropenic patients, gram-positive cocci are becoming more frequent than gram-negative bacilli as causes of life-threatening illness. ■ resistance to antimicrobial agents is becoming a major problem including bacteria (e.g., vancomycin-resistant enterococci and penicillin-resistant pneumococci), fungi (e.g., fl uconazole-resistant candida organisms), as well as pcp, and viruses (e.g., acyclovir-resistant herpes simplex and ganciclovir-resistant cmv). ■ in neutropenic patients, combination therapy should be considered when treating any life-threatening bacterial process. ■ a substantial fraction of hiv-infected patients with pcprelated respiratory failure can survive mechanical support and be discharged from the hospital. ■ adjunctive corticosteroid therapy is indicated for respiratory failure related to pcp. ■ tuberculosis is a concern in any immunologically abnormal individual with pulmonary disease but is a special concern in hiv-infected patients. tuberculosis in these cases often warrants respiratory isolation until appropriate specimens are evaluated for mycobacteria. ■ organ transplant recipients develop opportunistic infections at relatively predictable points depending on the type of transplantation and the specifi c immunosuppressive regimen used. innate (general or nonspecifi c) host defense mechanisms infections in solid organ transplant recipients infectious diseases associated with complement defi ciencies infection in organ-transplant recipients cd4 counts as predictors of opportunistic pneumonias in human immunodefi ciency virus (hiv) infection recent advances in the diagnosis and management of infection in the organ transplant recipient infections in recipients of hematopoietic stem cell transplantation bacteremia and fungemia in patients with neoplastic disease guidelines for the preventing opportunistic infections among hiv-infected persons-2002 recommendations of the u.s. public health service and the infectious diseases society of america guidelines for the use of antimicrobial agents in neutropenic patients with cancer fever in immunocompromised patients use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodefi ciency committee of the american academy of allergy, asthma and immunology colony-stimulating factors for chemotherapy-induced febrile neutropenia: a meta-analysis of randomized controlled trials neutropenia, neutrophil dysfunction, and bacterial infection in patients with human immunodefi ciency virus disease: the role of granulocyte colony-stimulating factor update of recommendations for the use of white blood cell growth factors: an evidence-based clinical practice guideline monotherapy with meropenem versus combination therapy with ceftazidime plus amikacin as empiric therapy for fever in granulocytopenic patients with cancer. the international antimicrobial therapy cooperative group of the european organization for research and treatment of cancer and the gruppo italiano malattie ematologiche maligne dell'adulto infection program a double-blind comparison of empirical oral and intravenous antibiotic therapy for low-risk febrile patients with neutropenia during cancer chemotherapy monotherapy for fever and neutropenia in cancer patients: a randomized comparison of ceftazidime versus imipenem treatment of febrile neutropenia with cefepime monotherapy additional anti-gram-positive antibiotic treatment for febrile neutropenic cancer patients fever in the pediatric and young adult patient with cancer. a prospective study of 1001 episodes gram-positive infections and the use of vancomycin in 550 episodes of fever and neutropenia ceftazidime monotherapy for empiric treatment of febrile neutropenic patients: a meta-analysis staphylococcus epidermidis: an increasing cause of infection in patients with granulocytopenia antibiotic synergism and response in gram-negative bacteremia in granulocytopenic cancer patients vancomycin-resistant enterococcal infections an evidence-based evaluation of important aspects of empirical antibiotic therapy in febrile neutropenic patients effi cacy of caspofungin against invasive candida or invasive aspergillus infections in neutropenic patients antifungal therapy in patients with fever and neutropeniamore rational and less empirical? caspofungin versus liposomal amphotericin b for empirical antifungal therapy in patients with persistent fever and neutropenia a randomized, double-blind comparative trial evaluating the safety of liposomal amphotericin b versus amphotericin b lipid complex in the empirical treatment of febrile neutropenia. l amph/ablc collaborative study group levofl oxacin to prevent bacterial infection in patients with cancer and neutropenia guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients. recommendations of cdc, the infectious disease society of america, and the american society of blood and marrow transplantation breakthrough zygomycosis after voriconazole treatment in recipients of hematopoietic stem-cell transplants beta-lactam antibiotic therapy in febrile granulocytopenic patients. a randomized trial comparing cefoperazone plus piperacillin, ceftazidime plus piperacillin, and imipenem alone risk assessment and riskbased therapeutic strategies in febrile neutropenia randomized, double-blind clinical trial of amphotericin b colloidal dispersion vs. amphotericin b in the empirical treatment of fever and neutropenia voriconazole versus amphotericin b for primary therapy of invasive aspergillosis review of treatment of zygomycosis with posaconazole in a patient with acute myeloid leukemia intravenous and oral itraconazole versus intravenous amphotericin b deoxycholate as empirical antifungal therapy for persistent fever in neutropenic patients with cancer who are receiving broad-spectrum antibacterial therapy. a randomized, controlled trial anidulofungin: a new echinocandin with a novel profi le combination antifungal therapy for invasive aspergillosis treatment of solid organ transplant patients with invasive fungal infections: should a combination of antifungal drugs be used? prevention of intravascular catheter-related infections guidelines for the management of intravascular catheter-related infections guidelines for the prevention of intravascular catheter-related infections eortc guidelines for the use of granulocyte-colony stimulating factor to reduce the incidence of chemotherapyinduced febrile neutropenia in adult patients with lymphomas and solid tumours return of granulocyte transfusions metaanalysis: the effi cacy of strategies to prevent organ disease by cytomegalovirus in solid organ transplant recipients 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with human immunodefi ciency virus infection relations among cd4 lymphocyte count nadir, antiretroviral therapy, and hiv-1 disease progression: results from the eurosida study tuberculosis in patients with human immunodefi ciency virus infection disseminated histoplasmosis in the acquired immune defi ciency syndrome: clinical fi ndings, diagnosis and treatment, and review of the literature public health service guidelines for the management of occupational exposures to hiv and recommendations for postexposure prophylaxis interactions among drugs for hiv and opportunistic infections drug interactions in infectious diseases tuberculosis in patients with human immunodefi ciency virus infection. how often does it mimic pneumocystis carinii pneumonia? diagnosis of pneumocystis carinii pneumonia: improved detection in sputum with use of monoclonal antibodies the diagnosis of pneumocystis carinii pneumonia in patients with the acquired immunodefi ciency syndrome using subsegmental 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surveillance should prophylaxis for pneumocystis carinii pneumonia in solid organ transplant recipients ever be discontinued? key: cord-259050-482nk9je authors: mätz‐rensing, k.; winkelmann, j.; becker, t.; burckhardt, i.; van der linden, m.; köndgen, s.; leendertz, f.; kaup, f.‐j. title: outbreak of streptococcus equi subsp. zooepidemicus infection in a group of rhesus monkeys (macaca mulatta) date: 2009-06-07 journal: j med primatol doi: 10.1111/j.1600-0684.2009.00359.x sha: doc_id: 259050 cord_uid: 482nk9je background a severe upper respiratory tract infection occurred in a breeding group of rhesus monkeys housed together in one of six indoor/outdoor corals of the german primate center. the clinical signs of the disease included severe purulent conjunctivitis, rhinitis, pharyngitis, respiratory distress and lethargy. six of 45 animals died within a few days after developing signs of infection. methods and results histopathologic and microbiologic examinations of the dead animals were consistent with a severe fibrinopurulent bronchopneumonia. microbiology revealed a lancefield group c streptococcus identified as streptococcus equi subsp. zooepidemicus as the causative agent of infection. conclusions the infection was passed on from animal to animal but did not spread to the other five breeding groups nearby. extensive diagnostic testing failed to reveal the consisting presence of copathogens in individual cases. a visitor with upper respiratory disease was suspected as source of infection. streptococcus (s.) equi subspecies zooepidemicus belongs to the b-hemolytic group c streptococci. it is able to cause disease both in animals and humans. streptococcus equi subsp. zooepidemicus primarily causes equine infections. the agent may be found in the nasopharynx, on the tonsils, in the respiratory tract and on the genital mucous membranes of healthy horses and cattle. it is an important cause of respiratory tract infections in foals and young horses and it is involved in uterine infections in mares. the agent has also been associated with a wide variety of infections including mastitis in pigs, sheep, cows, goats and several other mammalian species [8] . these hosts can be a reservoir for human infections. the clinical manifestation includes pharyngitis, septicemia, meningitis, purulent arthritis and endocarditis. the source of human infection is often traced back to contact with domestic animals, especially horses, or ingestion of unpasteurized milk or milk products. streptococci are colonizers of mucous membranes and are transmitted through droplets or direct contact. streptococcus equi subsp. zooepidemicus has sporadically been described in non-human primates. one outbreak occurred in the national zoological park, washington d.c., leading to the death of several callithrichids after contact to infected horse meat fed to armadillos kept in the same exhibition area [4, 7] . another outbreak of group c streptococcal infection with a high mortality rate occurred in a group of wanderoos (macaca silenus) of the zoological garden of rheine/germany. the source was suspected to be a human being [1] . the russian primate center in sochi background a severe upper respiratory tract infection occurred in a breeding group of rhesus monkeys housed together in one of six indoor/outdoor corals of the german primate center. the clinical signs of the disease included severe purulent conjunctivitis, rhinitis, pharyngitis, respiratory distress and lethargy. six of 45 animals died within a few days after developing signs of infection. methods and results histopathologic and microbiologic examinations of the dead animals were consistent with a severe fibrinopurulent bronchopneumonia. microbiology revealed a lancefield group c streptococcus identified as streptococcus equi subsp. zooepidemicus as the causative agent of infection. conclusions the infection was passed on from animal to animal but did not spread to the other five breeding groups nearby. extensive diagnostic testing failed to reveal the consisting presence of copathogens in individual cases. a visitor with upper respiratory disease was suspected as source of infection. reported an outbreak of septicaemia caused by s. equi subsp. zooepidemicus in representatives of five species of lower monkeys [2] . in 1994, an outbreak among the pig and monkey population was reported from the island of bali, indonesia. the infection spread from pigs to monkeys and infected animals died within a few days with signs of bronchopneumonia, pleuritis, epicarditis, endocarditis and meningitis [6] . however, most of the reported disease outbreaks were characterized by symptoms of an enteric infection. the present case report describes an outbreak of respiratory diseases among rhesus monkeys induced by s. equi subsp. zooepidemicus. the outbreak involved one of six breeding colonies housed in an indoor-outdoor facility. each unit was composed of an indoor area, a heated and roofed outdoor room and a large henced outdoor enclosure. contact to any other animal species except wild birds was not possible. the animals could move freely between the different compartments of the unit. they were fed twice a day with a primate specific diet, composed of standard commercial monkey pellets (ssniff, spezialdia¨ten gmbh, soest, germany), fresh fruits and vegetables. water was available ad libitum. the animals are kept in accordance with the guidelines of the european union for the accommodation and care of animals used for experimental and other scientific purposes (2007/526/eg). the primate husbandry is controlled by local and regional veterinary authorities in accordance with the german animal protection law. all procedures are supervised by an animal welfare officer and the ethical committee for experiments using animals in the federal state of lower saxony. a total of 12 animals died in one of these breeding colonies in a short time period. at the moment of the outbreak, the group was composed of 49 animals; most of them were adult females with their offspring and one adult male. necropsies were performed on all carcasses and samples for histology, parasitology, microbiology and molecular diagnostics were taken. for histopathology, samples were taken from all thoracic, abdominal and pelvic organs, fixed in 10% neutrally buffered formalin, embedded in paraffin and stained with hematoxylin and eosin. for parasitological diagnosis fresh samples were examined microscopically. for microbiological, investigations swabs from liver, spleen, kidney, heart and lung were streaked immediately onto columbia blood agar plates. swabs recovered from the intestine were swabbed onto columbia agar, emb, mcconkey and skirrows campylobacter agar plates and inoculated into rapaport and skirrow's enrichment broth. biochemical characterization of the isolated bacterial colonies was achieved using the crystel-system from becton dickinson. bacterial isolates were sent to the german national reference center for streptococci for further characterization. pulsed field gel electophoresis (pfge) of bacterial dna was carried out on a bio-rad chef-dr iii system using the bio-rad (bio-rad laboratories gmbh, munich, germany) gene path gel kit and the restriction enzyme smai. the outbreak started at the end of october 2007. two adult females were found seriously ill with signs of severe upper respiratory tract infection and severe purulent conjunctivitis (fig. 1 ). antibiotic therapy with baytril ò (dosage 2.5 mg/kg) and aviapen ò (dosage 40.000-80.000 ie/kg) was started but both animals died within 2 days after onset of the first symptoms (animals no. 1, 2). one month later, four more female animals developed severe upper respiratory tract infection. two died suddenly without obvious clinical findings (animals no. 3, 4), one of these was a pregnant one (animal no. 3). the other two died after 10 days on antibiotic therapy with the antibiotics mentioned above (animals no. 5, 6). necropsy findings were similar for all six animals. the main findings were severe inflammatory alterations of the upper respiratory tract and the lung ( table 1) . all six animals showed a severe subacute tonsillitis and pharyngitis and a severe subacute fibrinopurulent pleuritis and pneumonia. the inflammation spread to the heart and developed into a fibrinopurulent epi-and pericarditis with exception of animal no. 3 (figs 2 and 3 ). variably sized and poorly demarcated atelectatic areas were present in all lung lobes. histologically, there was a fibrinosuppurative pneumonia with extensive to lobular obliteration of the airways and alveolar spaces with neutrophils (fig. 5a) . large and medium sized airways were unaffected but terminal airways were intensely altered and showed signs of a purulent bronchopneumonia (fig. 5b) . the inflammatory process extended from the pleura to the diaphragm leading to severe purulent myositis (fig. 5c ). one pregnant animal additionally developed a severe purulent endometritis. the aborted fetus was nearly fully developed and showed signs of a purulent hepatitis and splenitis (animal no. 3a). splenitis and hepatitis were mild side effects in four more animals. only one animal developed a purulent encephalomeningitis (animal no. 6). clusters and chains of gram-positive cocci were present within the altered organs. they were found within the cytoplasm of macrophages and free within the extracellular spaces laying in pairs or chains (fig. 4) . septic thromboemboli were less consistently observed (fig. 5d) . septicemia was attributed as cause of death in all cases. septicemia and bacteriemia were confirmed by microbiological investigations on samples taken during necropsy. pure cultures of large colonies of b-hemo mä tz-rensing et al. lytic streptococci were grown not only from the respiratory tract, respectively tonsils and lung, but also from nearly all internal organs including the central nervous system. bacterial characterization was based on the observation of gram-positive, catalase-negative chain forming cocci which fermented lactose and trehalose. agglutination tests showed that they belong to lancefield group c. pulsed field gel electophoresis patterns were identical for all seven isolates analyzed, showing that the infections were caused by the same strain in all cases (fig. 6) . polymerase chain reaction based on amplification of common primate respiratory pathogens was performed on the lung tissue of the necropsied animals to exclude the presence of other important viral pathogens. the methods used are summarized in ko¨ndgen et al. 2008 [3] . all samples were negative for influenza a and b, parainfluenza, rhinovirus, coronavirus, adenovirus, human metapneumovirus, respiratory syncytial virus and enterovirus. the six dead animals were high ranked closely related animals. their loss induced severe ranking fights, a problem which is well known in rhesus monkey colonies. as a consequence of these heavy fights six more animals (animal no. 7-12) died due to severe injuries, stress and shock-symptomatic. streptococcus equi subsp. zooepidemicus was isolated from the tonsils of three of these animals (animal no. 7-9). a fatal outbreak of streptococcus infection occurred in one of six breeding colonies housed in an indoor/outdoor facility. the source of infection with s. equi subsp. zooepidemicus in the present outbreak remains unknown. contact to horsemeat, equines or domestic animals could reliably be excluded. instead a human to animal transmission was suspected. generally s. equi subsp. zooepidemicus can be transmitted by aerosols, via the oral route or through wound contamination. aerosol transmission is most likely in the present case. it was assumed that contact to a visitor with upper respiratory disease led to the initial infection of two elderly and closely related animals. streptococci of lancefield group c can be recovered from the pharynx of 1.5% of normal humans. infections of the upper respiratory tract in humans are rare but may occur after contact with infected horses [5] . it was assumed that the infection was subsequently transmitted from animal to animal because ill and dead animals were closely related to each other. there was a high degree of relationship and close contact among them. close contact seems to be necessary for the transmission of this agent. none of the animals in adjacent corrals situated at a distance of 2-3 m became infected. none of the staff members was ill before or during the time of the outbreak. in horses s. equi subsp. zooepidemicus is a commonly isolated mucosal commensal that opportunistically invades after virus infections, transport stress or other immunosuppressive conditions. in the described monkey group the situation seems to be different. bacteriologic investigation of tonsillar swabs indicates that there was no colonization or asymptomatic infection with s. equi subsp. zooepidemicus among the group members which could have been activated by a stressor. the only exceptions were three animals (animal no. 7-9) that died in the consequence of heavy position fights. in these animals streptococci were found in the tonsils but they developed no clinical findings. the three animals belonged to the same family and had been in close contact to the dead animals. they developed a purulent tonsillitis and it is speculative if these animals may have established a latent infection comparable to horses if they survived the attacks. the streptococcus outbreak among rhesus monkeys clearly demonstrates the high susceptibility of these animals. this is confirmed by data from the literature describing outbreaks caused by s. equi subsp. zooepidemicus in non-human primates marked by sudden, explosive appearance and high fatality rate [1, 4, 7] . this observation should lead to a critical discussion of housing primates and other animal species in close vicinity of each other as is often the case in zoological gardens. particularly equines should not be kept in the vicinity of primate facilities and there should be a strict separation between the different units regarding animal keepers and other staff members. the problem of the described outbreak for the breeding colony was not only the initial fatal infection but also the death of the first six animals. the loss of these animals caused ranking fights, injuries and death of six more animals. as a consequence the group had to be split up and newly composed. the outbreak ended after the dramatic depopulation of the group and the intense cleaning and disinfection of the facility. the disease caused heavy losses; not only the loss of the animals, but also a reduced reproductive rate and high costs for therapy. the experience from this outbreak leads to the conclusion that contact of important and valuable breeding colonies to visitors should be strictly avoided. an outbreak of streptococcus equi ssp. zooepidemicus infection of probable human origin in wanderoos (macaca silenus) -case report outbreak of infection caused by streptococcus zooepidemicus among laboratory primates pandemic human viruses cause decline of endangered great apes diseases of zoo marmosets, tamarins and goeldi's monkeys streptococcus zooepidemicus (group c) pneumonia in a human persistent occurrence of a single streptococcus equi subsp. zooepidemicus clone in the pig and monkey population in indonesia streptococcus zooepidemicus infections of possible horsemeat source in red-bellied tamarins and goeldie's monkeys identification and molecular characterization of serological group c streptococci isolated from diseased pigs and monkeys in indonesia key: cord-022305-uvor9rts authors: jacoby, robert o.; bhatt, pravin n.; jonas, albert m. title: viral diseases date: 2013-11-17 journal: the laboratory rat doi: 10.1016/b978-0-12-074901-0.50018-6 sha: doc_id: 22305 cord_uid: uvor9rts nan the number of viruses known to be naturally infectious for laboratory rats is small, and most cause inapparent infections which usually are detected by serological monitoring (table i) . significance: latent, vertically-transmissible agent isolated from submaxillary gland; no signs or lesions; induces hai antibody; unre lated antigenically to rat coronaviruses or cytomegalovirus; must dif ferentiate isolates from coronaviruses, cytomegaloviruses c. novy virus natural host: rat significance: extremely stable filterable agent recovered from rat blood. agent and its pathogenicity not well studied d. viruslike pneumotropic agents (enzootic bronchiectasis agent; gray lung virus; wild rat pneumonia agent) natural host: rat; mouse? significance: incompletely characterized agents associated with pneumonias of laboratory and/or wild rats. a hai, hemagglutination inhibition; cf, complement fixation; nt, neutrali zation; fa, fluorescent antibody. ft mousepox virus may be mildly infectious for experimentally inoculated rats, but natural infections have not been reported and evidence of natural immunoreactivity to the virus has not been detected. agents from three families of viruses are especially well dis seminated among rat colonies: rat virus (rv) and related strains (parvoviridae), sialodacryoadenitis virus (sdav) and rat coronavirus (rcv) (coronaviridae), and sendai virus (parainfluenza 1) (paramyxo viridae). rat parvo viruses nor mally induce latent infections, but, under conditions to be dis cussed, they may be lethal or cause vascular, neurological, and hepatic lesions. sialodacryoadenitis virus causes nonlethal, self-limiting disease characterized clinically by oculonasal ac cumulations of lacrimai porphyrin pigment, photophobia, and cervical swelling from enlarged salivary glands and morphologically by necrotizing inflammation in the upper res piratory tract, salivary glands, and lacrimai glands. there is recent evidence that keratoconjunctivitis, sometimes resulting in permanent eye damage, can also accompany sdav infec tion. rat coronavirus may cause sialoadenitis, but appears to be more pneumotropic than sdav. it can induce mild intersti tial pneumonia in adult rats and can cause fatal pneumonias in sucklings. sendai virus has received increased recognition as an important virus of rats, and current evidence indicates that natural infection causes pneumonia. the impact of other viruses naturally infectious for rats has not been thoroughly studied. humoral antibodies to minute virus of mice (mvm), pneumonia virus of mice (pvm), reovirus 3, mouse encephalomyelitis virus, and murine adenovirus have been detected in rats. the corresponding viruses, however, are currently considered nonpathogenic for rats. similarly, cytomegalovirus inclusions have been found in rat tissues, but are not associated with clinical disease or signif icant lesions. several other viruses [mgh virus, rat submaxil lary gland virus (rsmg), and novy virus] and viruslike agents (enzootic bronchiectasis, gray lung, wild rat pneumonia) have been reported in rats, but their significance is unclear. there fore, this chapter concentrates primarily on rat parvoviruses, rat coronaviruses, and sendai virus. the viruses are reviewed individually, but the reader is encouraged to peruse section vii for broad approaches to the detection, diagnosis, and control of virus infections in rat colonies. oncogenic viruses of rats, po tential or confirmed, are not discussed. a. general. parvoviruses are extremely small (18-26 nm) icosahedral viruses that are remarkably stable to heat, acid, lipid solvents, and prolonged exposure to room temperature. some are defective and require adenovirus "helpers" to rep licate, but rat parvoviruses are not defective. parvoviruses have been isolated from rodents, birds, dogs, cats, pigs, cattle, nonhuman primates, and humans. they appear to have a pre dilection for tissues containing rapidly dividing cells and may induce latent or subclinical infections. recent reviews offer additional background information (134, 142) . rat virus (rv) is the type species for the genus parvovirus and was the first virus isolated of the family parvoviridae. the literature on rat parvoviruses is extensive, but much of it per tains to experimentally induced infections in hamsters and sev eral other species, as well as in rats. since this report centers on natural parvovirus infections of rats, only experimentally range in size from 18 to 30 nm depending on conditions of purification and measurement and appear to have 32 capsomeres (69, 142, 158) . rat virus has a bouyant density in csci of about 1.40 gm/ml, and its molecular weight is approximately 6.6 x 10 6 daltons (138) . rat virus and h-l virus retain infectivity and hemagglutinating activity after exposure to ether, chloroform, or alcohol and are stable at ph's from 2 to 11 at 4°,25°, and 37°c (47, 78) . they also are remarkably temperature resistant. rat virus can remain infectious after exposure to 80°c for up to 2 h, for more than 6 months at -40°c, and for up to 60 days at 40°c (162) . the stability of rv and h-l virus to variations of temperature and ph may, however, depend on virus strain and supporting medium (18, 86) . these viruses are inactivated by ultraviolet light at room temperature (25, 155) . they are, however, resistant to ultrasonication, rnase, dnase, papain, trypsin, and chymotrypsin-properties which facilitate preparation of purified virus from infected cell cultures (20, 46, 97, 154, 158) . ii. hemagglutination and hemadsorption. hemagglutination is a common property of rat parvoviruses and is the simplest method to detect them. guinea pig erythrocytes are preferred to detect rv, h-l virus, and h-3 virus. the test can be run at 4°, 25°, or 37°c (78, 110) . rat parvoviruses agglutinate erythrocytes of other species to variable degrees, and it has been suggested that differential agglutination be used to identify virus strains (153, 154) . since the hemagglutinin is cell associated, cell debris should be pretreated with deoxycholate, receptor-destroying enzyme, or, preferably, alkaline buffer (48) . portella (126) showed that rv, h-l, and h-3 caused adsorption of guinea pig erythrocytes to virus-infected cells, but this technique is not generally used as a diagnostic test. iii. antigens. neutralizing (nt), hemagglutination inhibition (hal), and complement fixation (cf) tests have shown that rat parvoviruses consist of two major antigenic groups (36, 86, 110, 126, 151, 154) . one group includes rv, h-3, x-14, and her viruses, and the other includes h-l and ht viruses (table ii) . some cross-reactivity has been detected by immunofluorescence (fa) among rv, h-l virus, and mvm (36, 54) . d. in vitro cultivation. rat virus and h-l replicate well in primary monolayer cultures of rat embryo cells (78) . hamster embryo cultures support viral growth less well, and cells of mouse, chicken, calf, or human origin are unsuitable for rv, but the h viruses can grow in human, monkey, rat, and hamster cells [reviewed by siegl (142) ]. primary cultures should be prepared from rats free of rv and h-l virus infection, and all cell lines should be checked for latent contamination with parvoviruses. rat parvovirus-infected cultures usually develop cytopathic changes characterized by intranuclear inclusions and necrosis (78, 110, 156 (81) . the morphology of viral replication and inclusion body formation have been described in detail (8, 38, 99, 126) . e. host range. laboratory and wild rats appear to be the only natural hosts for rat parvo viruses. there is still debate as to whether the h viruses also may be naturally infectious for humans (154) . rat parvo viruses are, however, infectious for several species by experimental inoculation. rat virus and h-l virus can infect neonatal, suckling, and adult rats and hamsters (70, 73, 78, 111, 112, 149, 155) . experimental rv infection also has been reported in neonatal mice (40, 96) , praomys (mastomys) natalensis (129) , and newborn kittens (72) . h-l virus infec tion has been induced in newborn and adult rhesus monkeys (macaca mulatta) and in human volunteers (152) . attempts to induce rv infection in newborn rhesus monkeys were unsuc cessful (154) . /. clinical disease. kilham and margolis (73) were first to report natural rv disease. they had purchased 13-day preg nant rats from a commercial breeder, and the embryos were harvested to prepare primary tissue cultures. embryos cultured from one rat with three uterine résorption sites contained rv. rat virus also was isolated from three undersized pups in a second litter of eleven, and one other pup from the litter de veloped ataxia from cerebellar hypoplasia and was euthanatized at 45 days. a third litter consisted of two small pups. they developed severe jaundice shortly after birth and were killed at 4 days; the dam had nine intrauterine résorption sites. a fourth pregnant rat was killed at gestation day 16. one of 10 fetuses was abnormal with a mottled placenta, and rv was isolated from the fetus. the strain of rv isolated from these rats was designated sprv. it proved highly infectious for pregnant and nonpregnant rats and could be isolated from tissues, milk, and feces after experimental inoculation. furthermore, it crossed the placenta effectively and caused fetal death or teratogenesis when inoculated early in gestation, and postnatal cerebellar de struction and hepatitis when inoculated late in gestation. simultaneous intraperitoneal (ip) and intracerebral (ic) inocula tion of newborns with sprv also caused cerebellar and liver lesions and pups usually died by 8 days. one control suckling developed cerebellar disease from contact infection. nevertheless, natural rv disease among newborn and suck ling rats is sporadic (73) , despite the high prevalence of rv infection among commercial and institutional colonies. in con trast, reduced litter size, runted litters, or decreased production with increased numbers of résorption sites in uteri of breeding females should be considered potential signs of parvovirus in fection. rv infection of nonimmune pregnant dams in a pro duction colony can be expressed initially by decreased litters or litter size followed by a progressive return to normal produc tion as immunity to rv builds. parvovirus infection in adult rats is usually asymptomatic, but latent infection can be activated by immunosuppression (40) . we have, however, followed a natural enzootic of severe rv infection in a colony of sprague-dawley rats (66) . two groups of nonimmune male weanling rats, numbering 100 and 50, respectively, were introduced to a colony known to be latently infected with rv. two weeks later most of the newly introduced rats developed ruffled haircoats, dehydration, and cyanotic scrotums. twenty-five rats from the first group and seven from the second group died. rat virus was isolated from affected rats and hemorrhagic lesions compatible with rv infection were detected (see section ii, a, 1, g). the spectrum of clinical signs associated with rv infection likely depends on the strain of virus involved in a given out break. strains capable of infecting the gravid uterus of nonim mune dams may affect production. strains restricted to hori zontal transmission probably produce largely silent infections, but may occasionally provoke acute fatal disease in neonates or adult rats. the transmission of parvo viruses is discussed in more detail in section ii, a, 1, h. g. pathology. parvo viruses can be distributed widely in tissues of infected rats, but they most commonly cause lesions in tissues containing mitotically active cells, such as the de veloping liver and cerebellum. since resistance to these agents usually develops during the first week or two of life, much information about the pathogenesis of parvovirus lesions stems from study of infected pregnant dams, neonates, or young sucklings. rat parvo viruses also have been studied extensively in heterologous hosts, particularly suckling hamsters, in which they not only cause cerebellar lesions but also induce severe mongoloid developmental abnormalities and dental defor mities. the reader is referred to several reviews for details about these lesions (37, 89, 154) . the cerebellar lesions in pups, either naturally or experi mentally infected with rv, are characterized by development of intranuclear inclusions in rapidly dividing cells of the ex ternal germinal layer of cerebellum ( fig. 1 ) and then by necrosis ( fig. 2) (73) . since cells of the external granular layer migrate to form the internal granular layer, their deple tion aborts cerebellar development and results in granuloprival cerebellar hypoplasia with ataxia (fig. 3) . the liver lesions also begin with intranuclear inclusions in hepatocytes (fig. 4) , and less frequently in kupffer cells, biliary epithelium, endothelial cells (fig. 5) , and connective tissue cells (73) . ensuing cytopathic changes are characterized by nuclear pyknosis, cytoplasmic eosinophilia, ballooning de generation, dissociation of affected cells from adjacent cells and finally cell lysis. other changes may include hepatocytic bile retention and pigmented casts (ostensibly bile) in renal tubules. margolis et al. (94) reported that fresh isolates of rv and h-l virus were more virulent for experimentally infected new born rats than tissue culture passaged strains. furthermore, rats inoculated as sucklings (3 to 8 days) rather than as neonates (fig. 7) , adenomatoid distortion of lobular architecture, and formation of blood-filled spaces (peliosis hepatitis) (fig. 8) ; and (c) postnecrotic stromal collapse, fibrosis, and nodular hyperplasia (fig. 9) . peliosis hepatitis has been produced in several strains of suckling rats and is presumably a sequel of rv-induced hepatic necrosis (7). hemorrhagic encephalomyelopathy was first detected in about 2% of more than 1500 adult lewis rats given a single dose of 100-225 mg/kg of cyclophosphamide (40, 112) . le sions developed 10 to 25 days after treatment and consisted of multiple hemorrhages in medulla and spinal cord (fig. 10) . they were manifested clinically by paralysis. an agent, desig nated her virus, was isolated from affected rats, and sub sequently was shown to be a strain of rv. hemorrhagic en cephalomyelopathy was produced experimentally by intracerebral inoculation of suckling rats. it also occurred after intraperitoneal inoculation of virus into young adult rats given cyclophosphamide. typical neurological disease and hemor rhagic lesions appeared in 1 to 4 weeks, but in only 20% of inoculated rats. margolis (30) reported that large doses of her inoculated intracerebrally into newborn rats produced cerebellar destruction with hemorrhages throughout brain and spinal cord, whereas small doses produced jaundice and runting. sucklings infected after day 10 remained well. we have recently observed hemorrhagic lesions with throm bosis, and necrosis in the testicles and epididymis of weanling rats with naturally occurring rv infection (figs. 13 and 14) (see section ii, a, 1, f.). the hemorrhagic lesions appear to result from viral-induced injury to vessel walls. inclusion bodies have been demon strated in the vascular endothelium of small blood vessels and in arteriolar smooth muscle before extravasation of blood be gins (fig. 15 ) and hemorrhage is accompanied by vascular thrombosis and liquifaction necrosis (infarction) in the central nervous system (fig. 16 ) (30, 91) . baringer and nathanson (5) found that platelet-fibrin aggregates attached perferentially to rv-infected cells and suggested that endothelial injury and hemorrhage followed activation of clotting near infected cells rather than from a direct cytolytic effect of rv. there is indi rect evidence indicating rv may also interfere with blood coagulation. margolis and kilham (91) hypothesized that rv could cause coagulative disorders and promote hemorrhagic lesions through infection of hematopoietic tissues. they dem onstrated, in a retrospective histological study (92) , that rv inclusions can be found in megakaryocytes, and earlier reports by portella (126) showed that rv had an affinity for erythrocytes. corroborative coagulation studies on rv-infected rats have not been reported. since rat parvoviruses have a broad tissue tropism, clinical signs and lesions other than those described may occur. it seems likely that lesions of natural parvo virus infection will depend, as do their experimental counterparts, on the age and strain of the host and on the virulence and tropism of the strain of virus. h. epizootiology. serological surveys show clearly that natural infections with rv and h-l virus are common among laboratory and wild rats. robey et al. (130) found that about 85% of more than 350 sera collected from sprague-dawley rats from three commercial breeders and from their own colony had hai antibody to rv. kilham (71) surveyed several popu lations of wild rats near hanover, new hampshire, and de tected hai antibody to both rv and h-l virus. the proportion of rats with antibody to each virus varied independently for each population. robinson and co-workers (131) made an ex tensive seroepizootiological study of rv in a closed colony of 2000 mccollum rats with an annual production of about 3000 rats. forty percent of rats without maternal antibody seroconverted to rv by 7 weeks of age, and 67% had anti-rv anti body by 7 months (fig. 17 ). if several pups in a litter sustained infection, most cagemates developed antibody (fig. 18 ). pas sive antibody titers disappeared by 2 months. about 70% of 1000 juvenile rats were susceptible to infection at any given time, whereas 30% were actively immune (fig. 19 ). several attempts to isolate rv from immune rats were unsuccessful. seroconversion to h-1 virus was not detected, but about half of the litters had transient maternal antibody to h-1 virus. these studies demonstrated that rv is readily transmitted among rats held in confined quarters and that infections are perpetuated by sustained introduction of susceptible hosts. the epizootiological studies suggest that oral and perhaps respiratory infection are primarily responsible for natural hori zontal transmission of rv. the studies of kilham and olivier (78) and kilham and margolis (73) indicated that natural verti cal transmission can occur. experimental studies from several laboratories support these impressions. kilham day of gestation and showed that transplacental and fetal infec tion with fetal death occurred although dams remained asymptomatic. they also found that rv replication was wide spread in tissues of pregnant dams and that dams developed viremia which contributed to placental infection. these obser vations parallel those of natural rv infection where signs may be limited to fetal résorption and reduced litter size (see sec tion ii, a, 1, f). in contrast, h-1 virus infected rat fetuses only after intraperitoneal inoculation but not after oral inoculation of pregnant mothers. these workers suggested that in rats h-1 virus is more likely restricted to horizontal transmission primarily among sucklings and that adults are susceptible to clinical disease only if they are debilitated by intercurrent in fections or leukemia. lipton et al. (84) reported that viremia and virus infection of intestine, lung, liver, spleen, brain, and kidney followed intragastric inoculation of adult rats with the her strain of rv. furthermore, virus was excreted in feces but not in urine for up to 12 days, and inoculated rats were infectious for contact cohorts for 15 to 20 days. they were also able to infect rats by intranasal inoculation. novotny and hetrick (116) did find rv in urine of suckling rats inoculated parenterally as newborns with high doses (10 6 ld 50 ) of rv. dams and siblings of infected sucklings developed hai an tibodies to rv which indicated that horizontal infection had ensued. vertical transmission of rv was also confirmed, since litters born 2 to 5 days after intraperitoneal or intravenous (iv) inoculations of mothers had 100% mortality, whereas rats in oculated at least 2 weeks before conception delivered normal litters. furthermore, litters of infected dams developed disease when nursed by normal mothers, whereas normal litters nursed on infected mothers remained well. kilham and margolis (73) showed that rv may be excreted briefly in milk by rats inocu lated with rv in late pregnancy. dams infected on the first postpartum day had rv in milk by 24 h and could excrete virus in milk up to 12 days and up to 5 days after hai antibody appeared in their serum. there is additional evidence that rv can persist as a latent infection in the presence of high titers of hai antibody. robey et al. (130) isolated rv from five clinically normal laboratory rats, all of which had hai antibody to rv, and the rat with the highest antibody titer had the most widespread infection. la tent rv infection also was activated in clinically normal rats by a single nonlethal dose (150 mg/kg) of cyclophosphamide (40) . the repository of latent rv is unknown, but margolis and kilham (91) suggested that, since rv favors rapidly dividing cells, the gastrointestinal tract or hematopoietic system are likely sites. in addition, factors that contribute to rv latency and the role of latency in h-l virus infection are not under stood. nevertheless, it must be assumed that seropositive rats are latently infected and can serve as a potential source of infection to all susceptible rats in a colony. furthermore, since some strains of rat parvoviruses can be transmitted vertically, even caesarian-derived or germfree rats can not be assumed free of infection until they have been tested serologie ally. clinical signs in adult rats include neurological disturbances, sudden death, and hemorrhagic diatheses (e.g., scrotal hemor rhage and cyanosis). the incidence of clinical signs among infected adults is likely to be low, but may be exacerbated by immunological crippling. in breeding colonies, production may decrease, or neurological deficits and jaundice may occur among sucklings. morphological evidence of parvovirus infection is more dif ficult to detect since viruses are usually nonpathogenic for adults. an increase in fetal résorption sites in gravid uteri may occur. spontaneous lesions rarely develop in young animals, but histological examination for persisting inclusion bodies oc casionally may be rewarding. other lesions in suckling and adult rats have been described in section ii, a, 1, g. inapparent infection is detected most easily by serological testing. four serological tests are available (hai, cf, nt, and fa), but only the first three distinguish reliably between rv and h-l infection (36, 110, 151, 154) . hemagglutination inhibi tion tests are the simplest and least expensive to perform (131) . the use of kaolin to remove nonspecific inhibitors from test serum has been recommended (49, 110, 131) , but we and others have not found this necessary (31) . serological evidence of infection should be confirmed, whenever possible, by viral isolation (see section ii, a, 1, d). virus may be found in blood early in disease (viremia can persist for 2 to 10 days after experimental infection) (74) . fetuses, placenta, hematopoietic tissue, or gastrointestinal tract should, however, yield virus more consistently. confir mation of rv infection in our laboratory is made by inocula tion of test material into primary rat embryo cultures and into rv-free suckling rats. cultures are checked for hemagglutinin production and cpe. the identity of isolates is confirmed by hai assay with anti-rv and anti-h-1 virus immune serum. sera are collected from inoculated rats after 2 to 4 weeks and are tested for anti-rv hai antibody. j . differential diagnosis. rat virus infection may be subclinical or may cause lesions in the central nervous system, liver, vascular system, testes, and fetal death or résorption. sporadic illness affecting these organs must be carefully evaluated for infection by other viruses, chemical toxicity (e.g. dicoumarin toxicity), neoplastic diseases including leukemia or pituitary adenomas, trauma, and genetic abnormalities such as hydrocephalus. reduction in litters or litter size may be as sociated with sendai virus and possibly with environmental factors. since rv-infected colonies are common, specific an tibodies per se only indicate subclinical infection has occurred. associations between infection and clinical signs or lesions must be carefully made to confirm a cause and effect relation ship. for example, dual viral infections may occur. further, environmental or experimental factors may activate latent rv, while simultaneously causing lesions unrelated to rv infec tion. k. control. since rat parvoviruses cause latent infections and may be transmitted vertically and/or horizontally, proce dures to control infection must be planned carefully and adhered to stringently. once a colony has been infected, infec tion can be eliminated effectively only by destroying the col ony and repopulating from parvo virus-free stock. for experi mental colonies, this usually requires purchase of rats from vendors who have adequate serological monitoring programs. for production colonies, it means rederiving breeding stock by caesarian section, nursing sucklings on parvo virus-free foster dams, and testing weanlings for antibody before they are used for breeding. physical facilities for rederived stock should in clude pathogen-free barriers with personnel locks and proper air balancing to prevent reinfection. periodic serological sur veillance also is essential to detect possible reinfection as early as possible. /. interference with research. the effects of rat par voviruses on biological research are only partially understood and are potentially serious. for example, they can contaminate transplantable neoplasms (78, 150) and established cell lines (49, 163) . therefore, it is reasonable to assume, because of their predilection for rapidly dividing cells, that they could alter the growth characteristics of affected cells. furthermore, parvoviruses are pathogenic for fetal and suckling rats and hamsters, so inadvertent inoculation, especially of young ani mals, with virus-contaminated cells could cause fatal infection. immunosuppression also can activate latent rv infection in older rats (40) , so rats subjected to immunosuppression or severe stresses, such as intercurrent infections or surgical trauma, may be at risk. conversely, there is recent evidence that rv can suppress proliferative responses of infected lym phocytes to concanavalin a, phytohemagglutinin, or allogeneic lymphoid cells (25, 26) . if infected adult rats are used for experiments unaffected by latent viral infections, parvovirus infection can be tolerated, because it is unlikely that rats will develop clinical signs or lesions. since all factors relevant to latent infection have not been identified, a decision to work with infected animals should be made cautiously. fi nally, since these viruses are so hardy, instruments should be thoroughly disinfected after contacting infected animals to re duce the spread of infection. this is a widely disseminated latent virus of mice. experi mentally inoculated neonatal rats developed viremia and mild necrosis of ependyma and choroid plexus, and infected cells contained intranuclear inclusion bodies. virus also was de tected on postinoculation day 6 in brain, liver, intestine, and urine (76) . low titers (^ 1 : 40) of hai antibody to mvm have been found in wild rats (76) and in laboratory rats (119) . titers were reduced, however, by pretreating sera with receptor-destroying enzyme and attempts, to recover mvm from more than 100 seropositive rats proved unsuccessful. therefore, the significance of anti-mvm antibody in rat serum is unresolved. clinical signs or lesions due to natural mvm infection of rats have not been reported, and mvm has not been isolated from rats. cytomegalovirus is the only known herpesvirus of rats. similar viruses have been described for many species including humans, nonhuman primates, mice, guinea pigs, and rats (133) . they have physical and chemical characteristics typical of the herpetoviridae, including a penchant for latency. as with other cytomegaloviruses, rat cytomegalovirus appears to have a predilection for salivary glands and has also been found in lacrimai glands (87) . typical lesions include cytomegaly, intranuclear inclusion body formation in acinar or ductal epithelium, and mild nonsuppurative interstitial inflammation (79, 87, 128) . kuttner and wang (79) also showed that salivary glands of affected wild rats contained infectious virus by transmitting disease by intraglandular and ic inoculation of several "young" rats with emulsions of submaxillary gland. the epizootiology of rat cytomegalovirus has not been well studied, but virus and/or antibody have been detected in wild rats in several widely separated areas of the world (79, 128) . the potential the virus has for interfering with research has not been evaluated. infection is usually diagnosed by histological examination. anti-viral antibody can be detected by nt test (128) , but rapid serological tests are not available. rat cytomegalovirus can be grown in primary rat fibroblasts, rat kidney cells, and hamster kidney cells (6, 128) . we have found that some rat sera contain cf antibodies to mouse adenovirus, but clinical disease or lesions attributable to infection of rats with this virus have not been detected, and virus has not been recovered from rats. coronavirus) coronaviruses are lipid solvent-labile, pleomorphic, 60 to 220 nm particles with characteristic clublike projections (corona) uniformly arranged on their surfaces. they multiply in the cytoplasm and mature by budding through endoplasmic reticulum. coronaviridae are fairly species specific and have been identified as etiologic agents in diseases of humans, pigs, bovines, rats, mice, dogs, chickens, and turkeys. they gener ally infect the gastrointestinal tract and its associated glandular organs and/or the respiratory tract. reviews of coronavirus biology are available (17, 103) . two strains of coronavirus have been identified as important pathogens of laboratory rats; sialodacryoadenitis virus (sdav) (15) and rat coronavirus (rcv) (120) . in 1961, innes and stanton reported two outbreaks of clinical disease in weanling rats characterized by cervical edema and "red tears" (59) . they described the morphology of the dis ease in considerable detail and named it sialodacryoadenitis from the characteristic lesions: inflammation and edema of salivary and lacrimai glands. hunt (58) described a similar disease of young rats, but inflammation was restricted to the intraorbital lacrimai glands and was accompanied by keratoconjunctivitis. innes and stanton suggested an infectious agent caused the disease, and hunt detected acidophilic intra nuclear inclusion bodies in affected harderian glands and in conjunctival mucosa, but viral isolations were not attempted in either study. ashe and co-workers (3,4) isolated a transmissi ble cytopathic viral agent from the submaxillary glands of gnotobiotic rats that hemagglutinated rabbit erythrocytes. ashe's virus apparently was not associated with clinical signs or lesions in infected rats (see section iv,b). jonas et al. (67) induced sialodacryoadenitis in germfree rats by intranasal in oculation of an ultrafiltrate of diseased submaxillary salivary gland. virus particles were detected in ducts of submaxillary glands from experimentally infected rats by electron micro scopy, but attempts to isolate an agent in vitro were initially unsuccessful. however, when neonatal mice were inoculated ic with submaxillary gland homogenate they developed severe neurological deficits and died in 3 to 6 days. brain homogenates from affected mice caused sialodacryoadenitis in intranasally inoculated rats. bhatt and co-workers (15) subsequently isolated a virus from salivary glands of affected rats by inocu lation of neonatal mice and primary rat kidney (prk) monolayer cultures. the isolate had serological and physicochemical characteristics of a coronavirus. it was lethal for infant mice after ic inoculation but not for weanling mice. mouse brainpassaged virus induced sialodacryoadenitis in susceptible rats. in 1964 hartley and associates found that some rat sera con tained antibody to mouse hepatitis virus (mhv) (51) . they suggested that an agent antigenically related to mhv could elicit anti-mhv antibody in rats. parker et al. (120) offered support for hartley's theory by isolating a coronavirus antigen ically related to mhv from lungs of infected but clinically normal rats. parker's virus (rcv) was subsequently shown to be antigenically related to both sdav and mhv (15) , and the current view is that sdav and rcv may be different strains of one rat coronavirus. the biology of each virus is discussed separately in each of the following sections. a. sd a v. the diameter of sdav particles, determined by ultrafiltration, is 100 to 220 nm. jonas et al. (67) described a 60-to 70-nm particle in ductal epithelium of experimentally infected submaxillary glands by electron microscopy. prelimi nary ultrastructural studies of sdav propagated in vitro indi cate it has the typical morphology of a coronavirus (fig. 20) and is about 114 nm in diameter (83) . the differences in size reported probably reflect differences in the methods of mea surement and the source of virus as well as the pleomorphism characteristic of coronaviruses. sialodacryoadenitis virus is sensitive to lipid solvents, but it is relatively stable at acid ph (3.0). it also is stable in 3% fetal bovine serum (in phosphatebuffered saline) at 4°c for up to 7 days, at 37°c for up to 3 h, and at 56°c for less than 5 min. it can be stored at -60°c for more than 7 years but loses infectivity rapidly if stored at -20°c. the virus replicates intracytoplasmically and replica tion is not affected by 5-bromodeoxyuridine (budr). detailed morphological studies of viral maturation and release have not been reported. the virus does not hemagglutinate rabbit, guinea pig, or goose erythrocytes at 4°, 25° or 37°c (15) . sialodacryoadenitis virus is closely related antigenically to rcv, to mhv (tables iii and iv ) and a human coronavirus (oc38), but its antigenic relationship to other coronaviruses has not been tested. the virus is not antigenically related to the pox-, herpes-, areno-, paramyxo-, rhabdo-, reo-or togavirus groups (15 b. rcv. methods for and results of cultivation of rcv resemble those described for sdav (120). a. sdav. early work showed that sdav is pathogenic for in inoculated adult rats and for ic inoculated infant mice (15, 61, 67) . recent evidence indicates that sdav also is infec tious and pathogenic for weanling mice (12) . contact-exposed mice developed nt antibody to sdav, whereas in inoculated mice developed nt and cf antibody. the virus was recovered from the respiratory tract for up to 7 days postinfection, and mice developed interstitial pneumonia. anti-sdav and anti-mhv antibody also has been found among retired breeder mice from colonies thought to be free of mhv. since sdav and mhv are antigenically related, sdav infection should be considered if unexpected or unexplained seroconversions to mhv occur in mouse colonies. seroconversions to mhv from infection of mice or rats exposed to human coronaviruses (e.g., carried by animal technicians) also should be considered (hartley et al., 1964) but has not been studied. extensive host range studies of sdav have not been done, but preliminary trials with several strains of rats and mice suggest that various strains of sdav may vary in infectivity and antigenicity (14) . for example, during spontaneous out breaks, wag/rij rats developed severe clinical disease, whereas da rats developed primarily subclinical disease. fur thermore, some strains of mice developed both cf and nt antibody following experimental sdav infection, whereas others produced only nt antibody. conversely, one strain of sdav induced only nt antibody in a given mouse strain whereas a second strain of sdav induced both cf and sn antibody. these variations are important for interpretation of diagnostic and epizootiological data. b. rcv. host range studies with rcv also have been lim ited. rat coronavirus is infectious for rats and induces seroconversion to rcv, mhv, and sdav (11, 15, 120) . its pathogenicity varies with strain and age but is greatest for suckling rats. for example, mortality among intranasally in oculated fischer 344 rats less than 48 h old approached 100%, whereas comparable wistar rats had only 10 to 25% mortality. furthermore, deaths among fischer 344 sucklings occurred 6 to 12 days after infection, whereas wistar sucklings usually died after 12 days. resistance to mortality, however, among even highly susceptible sucklings, increased rapidly so that rats inoculated after 7 days of age had nonfatal respiratory disease and weanlings were asymptomatic (120) . the pathogenicity and infectivity of rcv for other species have not been re ported. a. sdav. susceptible rats can be infected at any age, but clinical disease usually occurs in one of two patterns: endemic infection of breeding colonies or explosive outbreaks among nonimmune rats exposed to virus as weanlings or adults. in the former setting, adults may have clinical signs, but more com monly they are immune. therefore, clinical disease develops among susceptible sucklings and is characterized by so-called ' 'winking and blinking ' ' associated with acute inflammation of the eye and adnexae. signs are transient (1 week or less) among individual sucklings, but affected animals will be prev alent among the suckling population as long as newly suscep tible litters are available to become infected. in the latter situa tion, either new, sdav-susceptible rats are placed in a room with infected rats or an infected rat(s) is placed in a room housing nonimmune weanlings or adults. generally, within 1 week, the susceptible population will develop typical signs of sdav infection. for individual rats they include cervical swell ing (edema) with palpable enlargement of submaxillary sali vary glands, sneezing or repeated wiping of the external nares with the forepaws, photophobia and nasal, and ocular dis charges which are often red-tinged due to a high content of porphyrin pigment. clinical signs last about 1 week. they may be mild or severe, and all signs do not occur in every infected rat. this last point is especially significant, since a single subclinically infected rat placed in a susceptible colony can initiate a full enzootic episode. keratoconjunctivitis has been associated with several natural outbreaks of sdav (80, 161) and may be the only clinically detectable evidence of sdav infection. signs and lesions commonly begin by the time of weaning, but also can occur in adults. they include photophobia, lacrimation, circumcorneal flush, diffuse corneal opacities, corneal ulcers, pannus, hypopyon, and hyphema. lesions usually resolve completely in 1 to 2 weeks, but chronic active keratitis and megaloglobus may develop in some rats. the morbidity of eye lesions during an acute outbreak of sdav infection varies from 0 to 100%, but is usually 10 to 30%. the prevalence of eye lesions seems greater among breeding colonies chronically infected with sdav (65) . the severity of lesions also may vary among strains of rats. in our experience, inbred lewis and wag/rij rats are more susceptible to sdav-associated eye disease than da rats or outbred cd rats (65). weisbroth and peress (161) found that the spontaneously hypertensive strain tacshr/n also was highly susceptible. the pathology of the eye lesions is discussed in greater detail in section iii, a, 7. b. rcv. rat coronavirus infection is subclinical in postweaned rats. nonfatal respiratory disease can occur in in tranasally inoculated sucklings, and intranasally infected sus ceptible neonates may die (11, 120) . a. sdav. the lesions of sdav infection have been de scribed in detail by several groups of workers (59, 61, 67, 80, 161) . gross lesions of sdav infection usually are restricted to mixed or serous salivary glands, lacrimai glands, cervical lymph nodes, thymus, and occasionally lung. submaxillary and parotid salivary glands and cervical lymph nodes are un-smk* fig. 21 . swollen pale submaxillary gland (arrow) in a rat inoculated intranasally with sdav 5 days previously. the cervical lymph nodes are also moderately enlarged. ilaterally or bilaterally enlarged, pale yellow to white, and edematous (fig. 21) , although they may have red spots or a red tinge if they are congested. periglandular connective tissue usually is severely edematous. the exorbital and intraorbital lacrimai glands also may be swollen, and the harderian gland may be flecked with yellow-gray foci. brown-red mottling of the harderian gland is due to its normal content of porphyrin pigment and should not be interpreted as a lesion. the thymus may be small and the lungs may be spotted with small grey foci. histological changes of sdav infection are found in the respiratory tract, salivary glands, thymus, cervical lymph nodes, eye, exorbital and intraorbital lacrimai glands, harder ian gland and other ocular adnexae. nasopharyngeal lesions include multifocal necrosis of respi ratory epithelium with inflammatory edema of the lamina pro pria (figs. 22 and 23) . nasal meatuses may contain neutrophils, mucus, and necrotic cell debris. focal necrosis of glands in the lamina propria also occurs. mild, nonsuppurative tracheitis with focal necrosis of mucosal epithelium also may occur, and hyperplastic peribronchial lymphoid nodules de velop. pneumonia has not been reported in either naturally or experimentally infected adult rats, but interstitial pneumonia may occur in naturally infected sucklings (65). salivary gland lesions (submaxillary and parotid) begin as necrosis of ductular epithelium which rapidly progresses to diffuse acinar necrosis (fig. 24 ). ultrastructural and immunofluorescence studies of experimentally infected rats have con firmed that sdav has a predilection for salivary ductal epithelium (fig. 25) (61,67) . moderate to severe interstitial inflammatory edema develops and glandular architecture is rapidly effaced (fig. 26) . inflammatory edema and occasional focal hemorrhage are prominent in periglandular connective tissue. repair begins 5 to 7 days after infection and is charac terized by prominent squamous metaplasia of ductular epithelium and by proliferation of hyperchromatic regenerating acinar cells (fig. 27) . the sublingual salivary gland which is exclusively of the mucus-secreting type, is not affected. lacrimai gland lesions develop in essentially the same pat tern as salivary gland lesions, i.e., necrosis and inflammation is followed by regeneration with squamous metaplasia of ducts or tuboalveolar units (harderian gland) and acinar regeneration (figs. 28 and 29) . squamous metaplasia in all glands subsides by 30 days postinfection, and the cytoarchitecture is restored to normal be cause basement membrane is not destroyed during necrotic and inflammatory phases of the disease. cervical lymph nodes may be hyperplastic with focal ne crosis and perinodal edema, and focal necrosis of thymus with widening of interlobular septums is common. after experimental intranasal inoculation of adult rats, viral replication and lesions occur first in the respiratory tract, then in the salivary glands and exorbital glands, and finally in the intraorbital lacrimai glands (fig. 30) (61) . it is common, how ever, in either spontaneous or experimental infection, to en counter a varied distribution of lesions. for example, a rat may have marked submaxillary sialoadenitis, whereas the parotid gland may remain normal or dacryoadenitis may occur without sialoadenitis. furthermore, lesions may be unilateral or bilat eral. the factors responsible for these variations have not been identified. also it is not clear how virus spreads from the respiratory tract to the salivary glands, since attempts to detect viremia or retrograde infection of salivary excretory ducts were not successful (61) . several groups have reported keratoconjunctivitis in rats with naturally occurring sdav infection, but eye lesions have produced experimentally (52, 80, 161) . lai et al. (80) recently described an outbreak of keratoconjunctivitis in a closed col ony of inbred lewis rats. lesions were most prominent in weanlings and included focal or diffuse interstitial keratitis, corneal ulcération, synechia, hypopyon, hyphema, and con junctivitis (figs. [31] [32] [33] [34] . lesions resolved in most rats by 6 weeks of age, but about 6% of affected rats developed chronic keratitis with megaloglobus and lenticular and retinal degener ation. similar lesions in adult rats, excluding chronic sequellae, were reported by weisbroth and peress (161) . affected rats from both outbreaks had a high incidence of dacryoadenitis and serum antibody to sdav. in the yale study (80) sdav was recovered from the respiratory tract of some rats, but virus was not detected in the eyes either by tissue culture isolation techniques or by immunofluorescence. it has been suggested that inflammation and necrosis of lacrimai glands during sdav infection results in impedence to flow of lacrimai fluids, proptosis, and keratitis sicca. ostensibly, normal conjunctival bacteria could proliferate under these conditions and increase the severity of lesions (80, 161) . included hyperemia, mononuclear cell infiltrates, focal atelectasis, and emphysema. sialodacryoadenitis was not observed. bhatt and jacoby (11) have described rhinotracheitis and focal interstitial pneumonia in adult germfree rats inoculated intranasally with rcv. virus was recovered from the respira tory tract for 7 days (fig. 35) , and viral antigen was detected in mucosal epithelium of the nasopharynx and in alveolar septae of some rats. upper respiratory lesions were nearly identical to those described for experimental sdav infection. gross pul monary lesions were limited to scattered red-brown to gray foci. histologically, peribronchial lymphoid cell hyperplasia was detected by day 5. alveolar septa contained mononuclear cells and neutrophils, and inflammatory cells occupied some adjacent alveolar spaces. pneumocytes, macrophages, and edema fluid also filled alveolar spaces in some lungs. lesions were mild and focal and subsided by day 7 (fig. 36) . salivary gland lesions were rare, but, when present, were identical to those caused by sdav. dacryoadenitis was not detected. a. sdav. sialodacryoadenitis virus infection is nonfatal, but is highly contagious and spreads easily and rapidly among susceptible rats in a colony room by contact, aerosol, or fo mite. morbidity is normally highest among late suckling or weanling rats, but susceptible rats of any age can be infected. infected rats excrete virus from the respiratory tract for about 7 days, at which time anti-sdav antibody is first detectable in serum by either nt or cf tests ( fig. 30 and table v) (61) . there is no evidence for a carrier state, so recovered rats should be considered free of virus and immune. therefore, propagation of virus in a colony depends on continuous intro duction of susceptible rats. the possibility remains, however, that as with some human coronavirus infections (109) rats im mune to a strain of sdav could be reinfected with the same strain or with an antigenically different strain. there also is no evidence that sdav can be vertically transmitted, but this point has not been adequately tested. the incidence of sdav infection cannot be determined reli ably by clinical signs, since infection may remain subclinical. therefore, serological methods are preferred. serum titers of cf and nt antibody should be determined simultaneously, since recent studies of natural and experimental sda indicate that cf antibody titers rise during outbreaks then decline rela tively rapidly, whereas nt titers increase during infection, but decline more slowly (fig. 37) (14) . therefore, cf antibody titers appear be a better marker for current or recent infection, whereas nt antibody titers are a more reliable indicator of previous infections. sialodacryoadenitis virus infection is probably widespread among rat colonies. we recently tested retired breeders from six vendors, and all had anti-sdav antibody (10) . neverthe less, confirmation that antibody detected during a given out break is due exclusively to sdav infection may be difficult to obtain, since rcv and sdav are closely related antigenically, and seroconversions for both viruses and to mhv follow infec tion by either rcv or sdav. bhatt et al. (15) have shown, however, that antibody titers to the homologous virus are usu ally higher than those to the heterologous, antigenically related viruses (tables iii and iv) fig. 37 . serum antibody titers to sdav in specific pathogen-free rats after a single intranasal inoculation of virus. rats were kept in germfree isolators for this study. arrows = titer equal to or greater than indicated value; x = average titer. b. rcv. the epizootiology of rcv has not been reported in detail aside from parker et al. (120) who demonstrated that rcv-infected rats developed anti-mhv antibody. rat coronavirus is cleared from tissues of experimentally inocu lated rats by postinoculation day 7, and there is no evidence for a carrier state. it may be assumed, for the present, that rcv is highly infectious and that it follows epizootiological patterns similar to those of sdav. infections of sdav or rcv can be diagnosed on the basis of clinical signs, lesions, and serological profiles of nt and cf antibody and confirmed by isolation of the causative virus. active sdav infection should not be diagnosed solely on the basis of serological data, since anti-sdav antibody can persist in previously infected rats for many weeks (see section iii, a, 8). the duration of anti-rcv antibody in rat serum after infec tion has not been reported. the viruses can be demonstrated in tissues by immunofluorescence for about 7 days after exposure and can be isolated in prk cultures or by ic inoculation of neonatal mice (15) . it is difficult to differentiate rcv from sdav infection virologically or serologically since the viruses are similar antigenically, physicochemically, and in their in vitro growth characteristics. as noted previously, however, if cf and nt antibody titers to sdav and rcv are compared, titers to the homologous virus will likely be slightly higher than those to the heterologous virus (table iii) (15) . comparison of experi mental infections with sdav and rcv have revealed additional differences (table vi) . first, clinical signs of rhinitis and sialodacryoadenitis are common during sdav in fection and keratitis may occur, whereas rcv infection is asymptomatic. second, rcv causes mild interstitial pneu monia in adults, whereas sdav does not. third, rcv rep licates poorly in salivary and lacrimai glands and only rarely produces lesions, whereas sdav is highly pathogenic for these tissues. nevertheless, suitable caution should be main tained in differentiating these infections since the experimental data described were obtained solely from cd rats. mitigating factors such as strain of virus, strain of rat, and age remain to be examined. clinically, cervical swelling from inflammatory edema and salivary gland enlargement is virtually pathogonomic for rat coronavirus infection and is more characteristic of sdav in fection than of rcv infection. porphyrin-tinged nasoocular exudates can accumulate during sdav infection, but they also occur in murine respiratory mycoplasmosis. ammonia fumes, especially from urine-soaked bedding, can cause acute in flammation of the eye and nose resembling sdav infection. morphologically, sialoadenitis and dacryoadenitis are characteristic of rat coronavirus infections. salivary and lacri mai gland lesions may, however, be mild and transient or may not develop, especially during rcv infection, and detectable lesions may involve only the respiratory tract. they must then be differentiated from infections caused by mycoplasma pulmonis, sendai virus, or pathogenic bacteria. lesions asso ciated with these agents are generally more severe than those caused by coronaviruses, and have been described elsewhere* (21, 82) . dual infections, for example, with sdav and sendai virus, may also complicate interpretation of lesions unless adequate serological and virological tests are performed. infection with sdav also must be differentiated from rat cytomegalovirus infection. the latter is clinically silent, le sions are usually mild and are characterized by enlarged sali vary ductal epithelial cells with intranuclear inclusions (79) . hunt (58) junctivitis can also occur in rats, but sdav must be eliminated as an underlying cause (53, 164) . rat colonies can be maintained free of coronaviruses if they are kept under rigid barrier conditions and are handled by personnel familiar with proper disinfection procedures for spe cific pathogen-free facilities. the key to effective control in infected colonies stems from recognition that infected rats shed virus for about 7 days and then are immune and that latent infections do not occur. since sdav spreads rapidly through a susceptible colony, all rats in a room should be infected and immune in 3 to 5 weeks. thus, infected holding or experimen tal colonies should be quarantined for at least 4 weeks and preferably 6 to 8 weeks after infection is detected. in produc tion colonies, breeding should cease for 6 weeks and weanlings should be removed from the room, since sucklings and wean lings of infected dams are particularly susceptible to infection. the quarantine period may be reduced by increasing contact exposure among susceptible rats. if susceptible rats are elimi nated (e.g., weanlings and rats introduced from other colonies) the infection will disappear (14) . however, once immune rats are replaced by susceptible rats, the opportunity for infection again increases. in addition, the possibility of reinfection with the same strain or with a different strain of coronavirus has not been ruled out. the immune status of the colony and of any rats to be introduced can be established by serological monitor ing. there are no published reports of effective vaccination protocols for sdav or rcv infection. rat coronaviruses are quite labile, so routine disinfection of facilities and equipment will destroy environmental sources of infection. there is no evidence that rcv or sdav are com municable to humans. it is not known whether human coronaviruses can infect rats. rat coronavirus infections may hamper, if only transiently, studies on the respiratory system or salivary glands of rats. furthermore, infected rats may be a greater risk for inhalation anesthesia, since excess mucus produced during acute coronavirus rhinitis can obstruct major airways. eye research may be hampered by sdav-associated keratoconjunctivitis, and chronically affected rats are obviously unsuitable for study. the eye lesion may be particularly troublesome for long-term studies such as those required for toxicological pro grams (80) . finally, since both sdav and rcv are primary pathogens for the respiratory tract, it has been suggested they may act as initiators or as copathogens in murine respiratory mycoplasmosis (61, 120) . sendai virus was isolated originally from mice in japan (44) , but it can infect hamsters and guinea pigs (127) . in mice, it causes silent respiratory infections, particularly in adults, but severe or even fatal bronchopneumonia and interstitial pneu monia can occur in sucklings and in genetically susceptible adults (13, 118, 121, 122) . the infectivity and pathogenicity of sendai virus for rats is not well characterized, and there are few reports of natural outbreaks. serological studies from our laboratory indicate, however, that sendai virus is widely dis seminated in rats from commercial breeders. (rats from six of nine colonies had anti-sendai virus antibody.) therefore, we feel that sendai virus infection is common among rats. sendai virus is a pleomorphic, filamentous virus with a lipid coat derived from host cell plasma membrane. therefore, it is readily inactivated by organic solvents such as ether. it can also be inactivated by ultraviolet light and is unstable at tem peratures above 37°c. sendai virus has hemolytic activity and two major surface antigens: a hemagglutinin and a neuramidase. it also has a nonhemagglutinating internal cf antigen. sendai virus is antigenically related to parainfluenza types 2 and 3, but can be differentiated from them serologie ally by cf, hai, or nt tests. the virus grows well in the amnion and allantois of embryonated eggs and in a variety of cell cultures, including human and monkey kidney and in perfused rat lung organ cultures (19) . we prefer to propogate sendai virus in bhk-21 cells to minimize the danger of contamination with latent passenger viruses which are particularly troublesome in pri mary monkey kidney cultures (13) . sendai virus induces syncytial giant cells in culture as is typical for other parainfluenza viruses. the biochemical, physical, antigenic, and cultural characteristics of sendai virus has been described at length by others (27, 33, 44, 60) . spontaneous sendai virus infection of rats appears to follow patterns described for mice (121) . in our experience and in the experience of others, infection is usually asymptomatic (21) , but it may be associated with signs of pneumonia (88). makino and colleagues (88) also observed a decrease in average litter size and retarded growth of young rats during a cyclic epizootic of sendai virus infection. jonas (65) has also noticed transient decreases in production during active sendai infection in a large breeding colony. intranasally inoculated rats developed extensive pulmonary lesions with respiratory difficulty, anorexia, and starry haircoats within 1 week after exposure. tyrrell and coid (157) found that clinical disease in experi mentally infected weanlings was self-limiting and that mortality was negligible. contact exposed rats seroconverted, but remained asymptomatic. coid and wardman (28) examined the effects of sendai virus on pregnant rats. nine of 12 rats exposed to aerosols of virus at 4 to 5 days of pregnancy resorbed all embryos, but virus was recovered from the conceptus of only 1 of 14 additional rats. each dam developed respiratory distress, inappétence, and a rough haircoat within 1 week postinfection, and virus was recovered from lungs of all rats. the authors concluded that résorption was probably related to systemic distress from res piratory disease in the dams, rather than to direct virus infec tion of embryos. the pathogenesis and lesions of natural and experimental sendai virus infection have been well described for mice (2, 132, 160) , but only limited information on rats is available. burek and co-workers (21) found that naturally infected rats seroconverted to sendai virus and developed multifocal inter stitial pneumonia. unfortunately, limited attempts to isolate sendai virus from affected rats were unsuccessful. lesions included perivascular and peribronchial lymphocytic and plasmacytic infiltrates; focal necrosis and hyperplasia of bronchiolar epithelium with infiltration by lymphocytes and neutrophils; and accumulation of mucus, inflammatory cells, and necrotic cell debris in some airways. in some rats, interstitial inflammation was accompanied by syncytial giant cell forma tion. rats younger than 8 months seemed predisposed to severe interstitial lesions compared to older rats in which peribron chial and perivascular inflammation were prominent and inter stitial lesions were less severe. the frequency and severity of interstitial and perivascular lesions subsided over a 7-month period, and antibody titers to sendai virus also decreased. peribronchial lesions persisted in many rats, however, for at least 7 months postinfection. they also reported that rats seroconverted to pneumonia virus of mice (pvm) as well as to sendai virus. since pvm can cause interstitial pneumonia in mice, they could not elimi nate the possibility that pvm contributed to the sendaiassociated lesions they described in rats. sendai virus lesions have been produced in rats experimen tally, but macroscopic descriptions were sketchy and histological findings were not reported (28, 157) . an example of an early lesion in a germfree rat inoculated intranasally with sen dai virus is shown in fig. 38 . epizootiological studies of sendai virus infection must be expanded before its natural history in rats is well defined, but based on the few reports available and our own experience, it seems likely that infection of rats will follow patterns reported previously for mice (13, 121) . sendai virus is highly infectious and can be expected to disseminate widely and rapidly through a colony. a typical outbreak was reported by makino and co-workers (88) in a breeding colony of 500 rats. infection spread rapidly as deter mined by development of hai antibody in exposed rats, but clinical signs of respiratory disease and retarded growth lasted for several weeks during each episode. furthermore, sendai virus was isolated from lungs of affected rats. outbreaks re curred at 8-to 10-month intervals among rats born after each preceding outbreak had subsided. susceptible rats remained seronegative until the colony was reinfected. hemagglutination inhibition antibody titers to sendai virus increased during each outbreak and were detectable for at least 1 year postinfection. burek et al. (21) reported an epizootic of sendai virus infection in breeding and aging colonies of wag/rij rats. rats seroconverted and had interstitial pneumonia, but they remained asymptomatic. attempts to isolate virus from six seropositive rats were unsuccessful. sendai virus infection may provoke clinical signs, such as ruffled haircoats, inappétence, respiratory distress, growth re tardation, or decreased litter size. sendai virus infection should also be considered if deaths occur during routine anesthesia or if unexplained shifts in immunological responses occur (45) . more commonly, however, infection remains subclinical, so laboratory diagnostic tests are required for diagnosis. detec tion of anti-sendai antibody in serum provides strong evidence for infection. there are hai and nt tests available, but the cf test appears to be most sensitive (118, 141) . since a single antibody survey may indicate past or current infection, diag nostic procedures should include histopathological examina tion for pneumonia characterized by epithelial necrosis in bronchi and bronchioles and, when possible, virus isolation from lung during acute stages of infection. virus can be iso lated in embryonated eggs, primary monkey kidney cells (121) , or bhk-21 cells, but in our laboratory the last is pre ferred. if cpe does not appear by 1 week, it has been recom mended that cultures be checked for sendai antigen by hemadsorption with guinea pig erythrocytes (27) . in our experience, however, bhk-21 cultures that are cpe negative are never hemadsorption positive. sendai virus infection of rats must be differentiated from respiratory infection of rats due to coronaviruses, my coplasma, and bacteria. it is helpful to remember that uncompli cated sendai virus infection involves the lungs, but not the upper respiratory tract, whereas coronavirus and mycoplasma infections regularly induce rhinitis. furthermore, in contrast to sendai virus infection, coronavirus infection is not associated with necrotizing bronchitis and bronchiolitis, and mycoplasma infection produces chronic inflammation of the lung culminat ing in bronchiectasis and bronchiolectasis (11, 61, 82) . it has been suggested that pvm may cause pneumonia in rats, but this has not been confirmed (21) . since sendai virus infection also has been associated with embryonic résorption and re tarded growth, rv infection must also be ruled out. based on studies in mice (13, 121) , it is likely that sendai virus infection in rats is self-limiting. persistence of infection in a colony probably depends on continued introduction of susceptible animals, especially sucklings and weanlings. thus, infection should be controlled by halting introduction (includ ing breeding) of susceptible rats into a colony for 4 to 8 weeks. this will allow infection to run its course in exposed rats. rats can be kept sendai virus-free in suitable barrier facilities serviced by technical personnel schooled in the con trol of infectious diseases. filter lids for animal boxes have, in our experience, neither prevented nor contained sendai virus outbreaks. furthermore, since sendai virus infection is preva lent among mice and hamsters, susceptible rats should not be housed with infected mice and hamsters if they must remain sendai virus-negative. if valuable animals from an infected colony must be placed in a sendai virus-free colony, seroposi tive animals should be selected and quarantined for 30 days in a laminar air flow unit or other appropriate isolation unit. production colonies, experimental colonies, and, if possible, animal vendors should be monitored serologically for sendai virus infection at regular intervals (e.g., semiannually). additional discussion on control of virus infections in rat col onies is found in section vii. sendai virus can cause pneumonia in rats, but infection is frequently subclinical. therefore, any experimental procedure involving the respiratory system, from anesthesia to inhalation toxicology, could be at risk during active infection. in addi tion, histological interpretation of experimentally induced pulmonary lesions may be complicated by residual lesions of sendai viral pneumonia. tissue harvests from inapparently in fected rats also present a danger of tissue culture contamination or inadvertent infection of recipient rats subsequently inocu lated with virus-infected material. there is recent evidence that selected immunological responses of rats may be suppressed during sendai virus infection (45) and that the virus can induce numerous long-lasting immunological changes in mice (69a) . sendai virus also should be considered a potential copathogen or aggrevating factor in other respiratory diseases of rats. fi nally, infected rats may disseminate infection to other suscep tible rodents. encephalomyelitis virus] these viruses are not known to be naturally pathogenic for rats. subclinical infections may occur, however, since an tibodies to them have been detected in rat sera. mammalian reoviruses consist of three serotypes: 1, 2, and 3. they cross-react by cf and immunofluorescence assays, but can be separated antigenically by nt and hai tests (135) . reovirus 3 is a natural pathogen of mice and produces a syn drome in sucklings characterized clinically by runting, oily skin, jaundice, conjunctivitis, hair loss, neurological distur bances, and sporadic deaths and pathologically by focal nee-rosis of liver, pancreas, heart, and brain with nonsuppurative encephalitis (32, (143) (144) (145) . spontaneous reo virus infection has not been described for rats, but we and others (10, 31) have detected hai antibody to reo virus 3 in rat serum. experimental infections of rats with reoviruses 1 and 3 have been reported as described in section vi. this virus is closely related to respiratory syncytial virus of humans. it produces silent infections in mice. however, virulent, mouse lung-passaged strains can cause severe intersti tial pneumonia after intranasal inoculation in mice (56, 57) . it can be cultivated in vitro in bhk-21 cells and produces cpe in about 1 week (50) . neutralizing and hai antibodies have been detected in rats (55), but clinical signs or lesions have not been reported. burek et al. (21) detected seroconversions to pvm among rats sustaining sendai virus-associated interstitial pneumonia and suggested that pvm may have contributed to the development of pneumonia. mouse encephalomyelitis virus is a picornavirus that causes persistent latent infection of mice, but which can occasionally cause a paralytic syndrome similar clinically and morphologic ally to human poliomyelitis (24, 147) . there are no published reports documenting infection of rats either by isolation of virus or by detection of serum antibody. we and others (10; j. c. parker, personal communication) have, however, detected occasional low titers of hai or nt antibody to the gd vii strain of mouse encephalomyelitis virus in rats. the biolog ical significance of these antibodies is unknown. mcconnell and co-workers (101) isolated a neurotropic agent from adult sprague-dawley rats that was biologically and physically similar to an enterovirus. a clinical disease characterized by circling, incoordination, tremors, torticollis, and high mortality was produced in suckling rats and mice by ic inoculation. virus was isolated from brain, lung, and intes tine. lesions included hydrocephalus, brain edema, neuronal destruction, and gliosis in the gray matter of spinal cord and brainstem and mild encephalitis. the agent was nonhemag-glutinating with erythrocytes from a variety of species, but it reacted at low titer (up to 1 : 16) with antibody to mouse encephalomyelitis virus (gd vii strain) by cf assay. interestingly, these workers also found cf antibody to their agent in human sera. additional characterization of the agent has, to our knowledge, not been reported. ashe and co-workers (4) isolated a cytopathic agent from submaxillary glands of 74 of 97 clinically normal conven tional, monoinfected, or germfree rats and designated it rsmg virus. it measured approximately 50 to 300 nm by filtration techniques and was not sensitive to lipid solvents (in contrast to cytomegalovirus). it was, inactivated, however, at phs below 5, after exposure to bactericidal light or by heating to 60°c for 30 min. the rsmg virus was originally isolated by inoculat ing salivary gland extracts into primary monolayer cultures of rabbit kidney and was passed repeatedly in similar cultures. cytopathic effect developed in 2 to 5 days, and cultures were rapidly destroyed. efforts to grow rsmg virus in cultured cells from monkeys, rats, hamsters, mice, and chickens were unsuccessful. virus could be adapted to hela cells and to human skin cells. clinical signs did not develop in experimen tally inoculated suckling, weanling, or adult rats, mice, or hamsters. infected salivary glands were histologically normal, but con tained a hemagglutinin which was first detected at 8 weeks, was apparently specific for rabbit erythrocytes, and was osten sibly virus associated. neutralizing antibody to rsmg virus and hai antibody to the hemagglutinin were detected in sera of affected rats, the latter antibody being detected in rats as young as 3 weeks. the hemagglutinin was not inhibited by antisera to rv, h-l virus, mvm, or reo virus 3. additional studies of rsmg virus have not been reported, but ashe's work indicates that it is a persistent latent virus that can probably be transmit ted vertically and that is not related to other sialotropic rat viruses such as rcv, sdav, or rat cytomegalovirus (3). jordan et al. (68) recovered a filterable agent from virusinfected rat blood stored in glycerin at about 9°c. remarkably, the blood had been collected by novy 35 years prior to jor dan's reisolation (117) . it was subsequently shown to cause fatal infection of ic-or ip-inoculated weanling rats and mice, but lesions were not described. further work with this agent has not been reported. pneumonia agent) nelson et al. [reviewed by nelson (114) ] found that suspen sions of pneumonic lung from mycoplasma-free rats caused chronic pneumonia in intranasally inoculated mice and rats. the putative etiological agent was filterable, but was not iso lated. nelson indicated that it may be a copathogen with m. pulmonis in chronic respiratory disease of rats, but this view has not been confirmed. andrews and glover (1) found a pneumotropic agent in mice inoculated with bovine and human material. it caused red-gray consolidation of lungs due to interstitial pneumonia and pul monary edema. vrolijk et al. (159) described similar naturally occurring lesions in laboratory and wild rats. a viruslike agent infectious for mice was isolated from affected animals, but it has not been characterized in laboratory rats. nelson (113) also recovered an agent from lungs of several wild rats which produced interstitial pneumonia in mice. he was not able to isolate the etiological agent, but suggested it was related to the agent of gray lung pneumonia (114) . the association of these agents with rat pneumonias needs further definition. none has been adequately characterized in vitro or in vivo, and serological tests to detect them have not been developed. excluding studies of natural infections, rats have been used sparingly as experimental animals for in vivo virological inves tigations compared to mice and hamsters. examples of ex perimental infections of rats with heterologous viruses are listed in table vii . three viruses have been selected for additional comment either because they induce interesting le sions in inoculated rats or because they are natural pathogens for other rodents. wild mice are the natural hosts for lcm virus, an arenavirus which may occur as a latent infection of mice and hamsters and as an acute infection of guinea pigs. the päthobiology of lcm infection has been studied extensively, since in mice it is a prototype for cell-mediated immunological injury to the ner vous system of animals infected as adults and a prototype for immune complex-mediated glomerular disease in adult mice infected as fetuses or neonates [reviewed by lehmann-grube (81a)]. lcm infection has not been detected in laboratory rats, but infection can be induced experimentally. monjan and col leagues (104, 105, 107, 108) have induced cerebellar 4 'hypo plasia" and retinopathy in ic inoculated suckling rats. cerebel lar hypoplasia was secondary to necrosis of the cortex which was most severe in rats inoculated 4 days postpartum. interest ingly, cerebellar lesions were prevented by immunosuppressing rats with anti-lymphocytic serum (106) and were elicited by adoptive immunization of infected rats with lcm virus-immune splenic cells (105) . therefore, the pathogenesis is similar to that observed for lcm disease in mice in that cell-mediated immunity to lcm virus is required to cause acute lesions. the retinal lesions were characterized by progressive destruction of all retinal layers and modest inflammation, and the presence of lcm virus in all layers of retina. the retinal lesions were also inhibited by immunosuppression (108). löhler and associates (85) showed that the we strain of lcm virus caused widespread infection of the brain after ic inoculation of adult sprague-dawley rats. lesions were characterized by lymphocytic infiltration of méninges, choroid plexus, and paraventricular areas. they closely resembled changes seen in brains of inoculated adult mice. recently, zinkernagel et al. (165) have shown that rats sensitized with live lcm virus develop potent cell-mediated immune re sponses to the virus. the infectivity and pathogenicity of reoviruses for naturally infected rats are believed to be low; however, suckling rats inoculated ic and ip with reovirus 1 at 3 days of age develop encephalitis. some rats also develop hydrocephalus secondary to viral infection of ependymal cells inoculated with reovirus 3. pregnant rats (75, 90) developed viremia in the presence of maternal serum antibodies, and transient nonfatal infection of fetuses occurred. when virus was inoculated directly into fetuses, however, there was a high rate of death and résorption (77, 93) . we are not aware of adequately documented studies showing that mousepox virus produces natural infections of rats. burnet and lush (22) found that rats inoculated intranasally with large doses of virus developed inapparent infection of olfactory mu cosa. neutralizing antibody was detected in serum of infected rats. rats inoculated intradermally developed either a minute papule at the injection site or no lesions, but hai antibodies appeared in serum (41) . intraperitoneally inoculated rats also developed antiviral antibody. the demonstration of anti-viral antibody in serum is good evidence for infection with the homologous virus or with an antigenically related virus. serological testing, in fact, often provides the first evidence of asymptomatic or latent viral in fections and, in laboratories with limited capabilities in tissue culture or pathology, may be the only method to detect infec tion. sampling protocols must reflect the population under study and the goals of the surveillance program. consideration must be given to the number, age, and sex of rats to be tested for each sampling period, to the geographical distribution of rats selected from a colony or a vendor shipment, and to the frequency of sampling. these variables are discussed further in section vii. blood can be collected at necropsy by cardiac, intraaortic, or intravenacaval puncture and should be allowed to clot at room temperature. if an animal must be saved, smaller samples can be obtained under ether anesthesia by aseptic cardiac puncture or by retroorbital bleeding with a heparinized capillary tube. use of nonheparinized tubes frequently results in premature clotting. heparinized samples (plasma) should be held in ice, separated shortly after collection, and assayed immediately or frozen as described below. clotted blood samples can be held overnight at 4°c and centrifuged and 0.5-to 1.0-ml aliquots of serum should be stored at -20°c or lower until tested. we recommend that samples not be pooled. frozen or refrigerated samples may be packed for shipment as described at the end of section vi, b. if serum samples are collected with reasonable care (aseptically) and rapid shipment is assured, they can be shipped unrefrigerated. it is best, however, to discuss shipping details with the respective testing laboratory. clinical signs, serological profiles, lesions, and immunofluorescence staining can incriminate a particular virus and thus narrow the selection of tissues for virus isolation. conversely, there is considerable overlap in the spectrum of tissues suscep tible to the common rat viruses, so it is better to "overcollect" than to "undercollect." timing is also critical for successful isolations during acute, self-limiting infection such as those caused by sdav or sendai virus, since rats may harbor virus before lesions develop or only during early or florid stages of disease. attempts to isolate infectious virus once seroconver sion has occurred may therefore be unsuccessful. on the other hand, isolation of latent viruses such as rv may be difficult without proper culturing techniques, regardless of when tissues are harvested. as a rule fetal, suckling, or weanling rats with low levels of antibody should be selected (table viii) . regardless of the tissues to be harvested, aseptic techniques should be followed. the pelt should be wet with an antiseptic solution (e.g., 70% ethyl alcohol), but care must be taken not to contaminate the tissues so that virus is not inactivated. in struments should be autoclaved or flame-sterilized (after im mersion in 95% or absolute alcohol) and allowed to cool at room temperature. small pieces of tissue 0.5-1.0 cm 2 should be placed in labeled sterile vials (2 dram screwcap glass vials are adequate) and held on ice until they are frozen. clotted or anticoagulant-treated blood also can be stored in vials. tissues should be stored at -60°c or below. if deep cold storage must be delayed, tissues may be held overnight in ice. storage of tissues at -20°c should be avoided, since virus titers may drop quickly. if tissues are triturated before freez ing, antibody-free protein (e.g., fetal bovine serum) should be added to the suspension to a final concentration of 50% to if facilities for serological testing or virus isolation are not available locally, samples can be shipped in sealed ampules encased in rigid cardboard carriers placed in a styrofoam or other suitable insulated shipping carton filled with dry ice. alternatively, serum can be shipped in containers containing ice cubes or crushed ice. containers should be clearly marked according to federal regulations,* and the receiving laboratory should be notified of the time and mode of shipping in ad vance. a healthy laboratory rat has been defined as one free of all currently detectable known viruses, with a defined microbial flora and maintained in a protective barrier. ever, investigators usually use rats with a varied history of exposure to viruses, so documentation of exposure of infection should be integrated into quality assurance programs for breed ing and experimental colonies. recognition of viral infection may be relatively simple if mortality or characteristic clinical signs occur, but detection is more difficult if infection is asymptomatic or latent. detection procedures must, therefore, be designed to encompass all pos sibilities so that preventive measures can be instituted to reduce the negative impact of infection on animal-related research. infectious disease can be detected by intramural routine sur veillance programs. "routine surveillance" is, however, a general term which, depending on the needs and judgment of the professional staff, may imply minimal effort or a thorough multidimensional program. minimal surveillance procedures may be limited to clinical spot checks of production or research colonies or newly received vendor rats with follow-up clinical checks. this may detect active infections such as sdav, but will usually miss latent infections such as those caused by rat parvo viruses. effective surveillance must, therefore, include adequate diagnostic laboratory support in virology, serology, and pathology as well as thorough clinical evaluations (63, 64) . signs of disease may be reported to the veterinary staff by veterinary assistants or animal health technicians, animal care technicians, research technicians, or the investigator. the role of investigators in the early recognition of disease can be ex tremely helpful, since they often have unique opportunities to observe effects of potential viral infection through unexplained changes of responses in experimental procedures. increased mortality, decreased survival times, increased or unexplained anesthesia-associated deaths, altered metabolic or immunological responses, or variations in tumor growth in vivo or in vitro all may be potentially associated with viral infection. the detection of subclinical or latent infections depends on well conceived, statistically valid sampling procedures, since it is impossible to test every animal. sampling protocols must consider the age, sex, and number of rats to be tested for each sampling period, the distribution of rats selected from a colony room or vendor, and the frequency of sampling. as a rule, we recommend testing equal numbers of males and females at 90 days of age and also retired breeders at 9 to 12 months of age. the number of rats tested may vary according to the goals of the program, but we suggest that a minimum of 10 rats per age group from a given room be screened twice yearly for a total of 40 rats annually. for intramural production colonies or longterm holding colonies, no more than 1 rat per cage should be included in a single sample population, but blood or tissues from all rats selected should be collected on the same day. in addition, rats should be chosen from as many different racks or shelves in a room as possible. a useful technique, especially for intramural sampling, is to place sentinel weanling rats in separate cages among other rats in a room and test them when confidence limit = 95%. 6 see text for additional explanation. c from formulas contributed by dr. c. vviiite, yale university school of medicine and described in "long-term holding of laboratory rodents." a report of the committee on long-term holding of laboratory rodents, institute of laboratory animal resources. ilar news, 19, no. 4 (1976) . they reach the appropriate age. vendor sampling is more difficult to control, but if the source of the rats (specific room or area) is requested, reliable data can be obtained. we recommend testing only 90-day-old vendor-derived rats according to the protocol outlined above, unless screening of retired breeders can be justified. the recommendations for sample size were derived from a statistical formula provided by white and extrapolated to table ix . for example, from the table one can infer that if a popula tion of rats with a 30% incidence of viral infection is properly sampled, there is a 95% probability that at least one infected rat will be detected in a sample of nine rats from that population. the sampling protocol must include a thorough serological and pathological evaluation. the reader is referred to table ix for additional information. accurate diagnosis is critical for control of infection and for evaluating the impact of infection on research, not only with respect to requirements of individual investigators but also with respect to hazards infection may present to other animals. seroconversion or increasing titers of antibody to a particular viral antigen over several weeks is commonly accepted as good presumptive evidence of active or recent viral disease. anti body alone, however, indicates only that exposure to a viral agent or to an antigenically related agent may have occurred. for example, it was known that rats developed humoral anti body to mhv (51); however, signs or lesions of mhv infec tion were never seen in rats. parker et al. (120) and bhatt et al. (15) showed subsequently that rat antibodies to mhv were elicited by rat coronaviruses (sda and rcv), viruses which are antigenically related to mhv. thus, antibody induced by one virus reacted with all three viruses. similarly, isolation of a virus per se during a suspected outbreak may be misleading, since passenger viruses, multiple viral infections, or latent viruses with no influence on the problem under investigation may occur. therefore the significance of serological and virological findings must be assessed after evaluating clinical signs, lesions, and epizootiological data. furthermore, before a final diagnosis is confirmed, the veterinarian must critically evaluate information about an outbreak and compare it to his or her knowledge about the suspected infection derived from per sonal experience or from the scientific literature. diagnosis of viral infection must be combined with a search for its source. for example, rats lose colostrum-derived antibody pro gressively and, around the time of weaning, can become sus ceptible to viruses to which they were passively immune. therefore, although infection may be present in a vendor col ony, it is possible that weanling rats can be infected by an "in-house" source after arrival when passive immunity has decayed. it may be helpful in such situations to test whether rats received from a vendor become seronegative and remain free of infection when held in isolation for several weeks.* effective control of viral infection requires that the veteri nary clinician have thorough knowledge of the epizootiology of the etiological agent. for example, the clinician must be aware of the influence viruses may have on research so that control procedures are neither overzealous nor inadequate. the clinician must also consider the implications of spread from a colony where the impact of the virus may be negligible to a colony where its impact may be major and engender significant losses in time and money. generally, infections such as those caused by sdav or sendai virus can be eliminated, as discussed in previous sections, by appropriate quarantining procedures. on the other hand, persistent latent infections, such as those due to rv and which can occasionally be transmitted vertically, may require that all stock be killed, that rooms and equipment be thoroughly de contaminated, and that colonies be restocked with virus-free rats. *the mouse antibody production (map) test has been used to detect viruses in mouse tissues and has been useful in detecting viral contamination of tumors (136) . the test is based on the principle that inoculation of test tissues into a nonimmune recipient will elicit antibody to viruses in the inoculum. this test can be adapted for rats and is particularly valuable when dealing with latent infections such as those caused by rv. spread of air-borne virus from infected to susceptible ani mals can be reduced by methods ranging from (a) improved air handling procedures, such as 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microscopic study of the rodent "picodnavirus" x-14 persistent infections caused by borna virus isolation and characterization of a neurotropic agent (mhg virus) from adult rats the dna's of three parvoviruses coronaviruses: a comparative review pathogenesis of cerebellar hypoplasia produced by lymphocytic choriomeningitis virus infection of neonatal rats. i. evolution of disease following infection at 4 days of age pathogenesis of lcm disease in the rat pathogenesis of cerebellar hypoplasia produced by lymphocytic choriomeningitis virus infection of neonatal rats: protective effect of immunosuppression with anti-lymphoid serum cerebellar hypoplasia in neonatal rats caused by lymphocytic choriomeningitis virus lym phocytic choriomeningitis virus-induced retinopathy in newborn rats the tecumseh study of respira tory illness. vi. frequency of relationship between outbreaks of coronavirus infection characteristics of certain viruses isolated from transplantable tumors lethal infection and pathological findings in axc rats inoculated with h-virus and rv viral hemorrhagic encephalopathy of rats. i. isolation, identification and properties of the her strain of rat virus observations on a pneumotropic virus obtained from wild rats respiratory infections of rats and mice with emphasis on indigenous mycoplasms untersuchungen über die experimentella bornavirus-infektion bei der ratte pathogenesis and transmis sion of kilham rat virus infection in rats the rat virus suscepti bility of inbred and outbred mouse strains to sendai virus and preva lence of infection in laboratory rodents minute virus of mice: prevalence, epidemiology and occurrence as a contaminant of transplanted tumors rat coronavirus (rcv): a prevalent, naturally occurring pneumotropic virus of rats natural history of sendai virus infection in mice enzootic sendai virus infections in mouse breeder colonies within the united states virus studies with germfree mice. i. preparation of serologie diagnostic reagents and survey of germfree and mono contaminated mice for in digenous murine viruses a virus from mammary tissue of rats treated with x-ray or methylcholanthrene (mc) experimental infection with herpes simplex virus type 2 in newborn rats: effects of treatment with iododeoxyuridine and cytosine arabinoside hemadsorption and related studies on the hamster-osteolytic viruses enzootic sendai infection in laboratory hamsters isolation and growth of rat cytomegalovirus in vitro intranuclear inclusions in rattus (mastomys) natalensis infected with rat virus studies on the natural infection of rats with the kilham rat virus seroepidemiological study of rat virus infection in a closed laboratory col ony the pathogenesis of sendai virus infection in the mouse lung the replication of herpe svini ses parvovirus reproduction reoviruses polyoma and other indigenous mouse viruses the induction of hepatitis by prior partial hepatectomy in resistant adult rats injected with h-l virus characterization of the kilham rat virus linear, single-stranded dna isolated from kilham rat virus the effect of altered immune reactivity on experimental measles en cephalitis in rats application of a microtechnique to viral serological investigations the parvoviruses studies on the pathogenesis of a hitherto undescribed virus (hepato-encephalomyelitis) producing unusual symptoms in suckling mice studies on the hepatoencephalomyelitis virus (hev) murine infection with reo virus. ii. the chronic disease following reovirus type 3 infection zoster-like lesions from herpes simplex virus in newborn rats spontaneous encephalomyelitis of mice, a new virus disease production of a "mongolian-idiot" like abnor mality in hamsters experimental production of mongoloid hamsters a virus associated with transplantable human tumors studies on h-viruses susceptibility of the rhesus monkey (macaca mulatta) to h-l virus agglutination of the h-viruses with various types of red blood cells the picodnaviruses: h, rv and aav an unidentified, filtrable agent isolated from transplanted human tumors growth and cytopathogenicity of h-viruses in human and simian cell cultures sendai virus infection of rats as a convenient model of acute respiratory infection purification and fine structure of kilham's rat virus virus pneu monia (snuffling disease) in laboratory rats and wild rats due to an agent probably related to grey lung virus of mice naturally occurring sendai virus infection of athymic nude mice ophthalmic lesions and dacryoadenitis: a naturally occurring aspect of sialodacryoadenitis of the laboratory rat purification of the kilham rat virus persistent infection of a rat nephroma cell line with kilham rat virus conjunctivitis in a colony of rats cellmediated immune response to lymphocytic choriomeningitis and vaccinia virus in rats key: cord-260605-smkr7b15 authors: vestby, lene k.; grønseth, torstein; simm, roger; nesse, live l. title: bacterial biofilm and its role in the pathogenesis of disease date: 2020-02-03 journal: antibiotics (basel) doi: 10.3390/antibiotics9020059 sha: doc_id: 260605 cord_uid: smkr7b15 recognition of the fact that bacterial biofilm may play a role in the pathogenesis of disease has led to an increased focus on identifying diseases that may be biofilm-related. biofilm infections are typically chronic in nature, as biofilm-residing bacteria can be resilient to both the immune system, antibiotics, and other treatments. this is a comprehensive review describing biofilm diseases in the auditory, the cardiovascular, the digestive, the integumentary, the reproductive, the respiratory, and the urinary system. in most cases reviewed, the biofilms were identified through various imaging technics, in addition to other study approaches. the current knowledge on how biofilm may contribute to the pathogenesis of disease indicates a number of different mechanisms. this spans from biofilm being a mere reservoir of pathogenic bacteria, to playing a more active role, e.g., by contributing to inflammation. observations also indicate that biofilm does not exclusively occur extracellularly, but may also be formed inside living cells. furthermore, the presence of biofilm may contribute to development of cancer. in conclusion, this review shows that biofilm is part of many, probably most chronic infections. this is important knowledge for development of effective treatment strategies for such infections. bacteria form biofilms as part of their survival mechanisms, and biofilms are thus ubiquitous in nature. already in 1683, antoni van leeuwenhoek observed and described biofilms by using his primitive microscope on matter from his own teeth. however, the biofilm lifestyle of microorganisms were of no interest to medical microbiologists until the early 1970s when nils høiby observed a link between the etiology of a persistent infection and aggregates of bacteria in cystic fibrosis patients [1] . since then, biofilms have been recognized to be involved in many clinical infections [2, 3] , and evidence is accumulating that biofilms contribute to the pathogenesis, especially in chronic infections [4] . bacterial biofilms are clusters of bacteria that are attached to a surface and/or to each other and embedded in a self-produced matrix. the biofilm matrix consists of substances like proteins (e.g., fibrin), polysaccharide (e.g., alginate), as well as edna. in addition to the protection offered by the matrix, bacteria in biofilms can employ several survival strategies to evade the host defense the primary infection in endocarditis is a biofilm composed of both bacterial and host components located on the cardiac valve. this biofilm causes disease in the following ways: (1) the biofilm physically disrupts valve function, causing leakage when the valve is closed and turbulence as well as diminished flow when the valve is open; (2) the biofilm provides a source for near-continuous infection of the bloodstream that are difficult to remove by antibiotic treatment; (3) pieces of biofilm can break off and be carried to a terminal point in the circulation causing the brain, kidneys, and extremities particularly vulnerable to emboli. it is generally accepted that biofilms is involved in infective endocarditis (ie) and it has been so for several years since costerton et al. recognized native valve endocarditis as a biofilm infection caused by viridans group streptococci [2] . treatment with antibiotics are often difficult even if the bacteria are sensitive to the selected antibiotic. successful treatment with antibiotics often require prolonged intravenous administration. in cases where antibiotic treatment is unsuccessful, surgical excision and replacement of the infected valve might be an option. most of what is known about the pathogenesis of endocarditis involving biofilms is learned from animal studies using rabbits [48] . the biofilm on the valve consists primarily of bacteria and biofilm matrix components, platelets and fibrin derived from the circulation. first the endothelial surface of the valve gets injured, second a formation of the sterile clot like lesion of platelets and fimbrin occurs at the site of the injury. then bacteria starts to adhere to the thrombus before microcolonies are formed and lastly a mature biofilm is formed and pieces of the mature biofilm can cause embolization [48, 49] . the most commonly isolated microorganisms from ie cases are staphylococci, streptococci, and enterococci. these species are responsible for more than 80% of ie cases. electron microscopy is used for identification of biofilm in relation to endocarditis [49] . already before costerton et al. recognized endocarditis as a biofilm infection [2] , marrie et al. published a study in 1987 where bacterial colonies embedded in a matrix material on valves of six ie cases were shown using electron microscopy [50] . to diagnose ie, the patient is evaluated for several different criteria called duke criteria. the criteria are based on clinical, echocardiographic, and microbiologic (blood culture) evaluation. however, bacteria of the biofilm rarely enter the blood stream as planktonic bacteria, and for this reason the blood culture may be negative when testing for microorganisms [51] . consequently, immunodiagnostic assays (elisa) have been developed to detect serum antibodies against biofilm matrix components. for example, an elisa has been developed to detect antibodies against staphylococcal slime polysaccharide antigens. to date, the elisa assays developed do not have the sensitivity and specificity to alone determine biofilm-associated infections [49] . in atherosclerosis, fatty deposits and calcium accumulate as plaques in the arterial wall. this leads to reduced arterial elasticity, narrowed lumen of the artery, and subsequently to cardiovascular diseases caused by reduced blood flow. in addition, sudden rupture of a plaque may be life threatening [52] . a number of studies, including two recent meta-analyses, show that periodontal disease and cardiovascular disease, including atherosclerosis, are significantly related [53] . furthermore, oral bacteria have been identified in the atherosclerotic plaques in several studies. however, it is not known whether the bacteria are involved in initiation of plaque formation or colonize the plaques after they are formed. recently, bacterial biofilms in atherosclerotic arteries have been identified by fluorescence microscopy and fluorescence in situ hybridization (fish) [54, 55] . this may indicate biofilms to be involved in the pathogenesis of atherosclerosis, and it is hypothesized that the presence of biofilm may contribute to enhanced risk of plaque rupture [55] . sialolithiasis is a condition where calcified masses (called sialoliths or salivary stones) form within a salivary gland. this can cause pain and swelling in about 0.5% of the general population. earlier studies have suggested that bacteria may be involved in sialolithiasis [56] , and new studies now link this to biofilm production. in a descriptive case-control study, sections of submandibular glands with chronic obstructive sialadenitis were compared with those of healthy controls, using confocal laser scanning microscopy [57] . morphological evidence of bacterial biofilm was observed in half of the histological sections of the chronic obstructive sialadenitis group, whereas no sign of bacterial biofilm formation was seen in the control group. interestingly, two recent studies report observations of biofilm structures in the center of the stones, thus indicating that biofilm formation may be part of the etiology of salivary stone production [58, 59] . fusconi et al. observed structures resembling bacterial cells embedded in amorphous material, when investigating the stones by scanning electron microscopy [59] . furthermore, the presence of bacterial dna was demonstrated by qpcr. in a later study, kao et al. reported light and scanning electron microscopy observations of biofilm together with host immune cells, platelets, and erythrocytes, as well as calcium nanoparticles [58] . they proposed a hypothesis where biofilm formation leads to local injury, followed by inflammation and calcium deposition. biofilm formation has also been observed on the surface of salivary stones. in a study on 54 patients with sialolithiasis, biofilm was observed on 71% of the removed stones by fluorescence microscopy, and common oral bacteria were found on half of the stones [60] . the observation that bacterial biofilms were found in 75-100% of patients with clinical post-operative infections, recurrent sialadenitis or pus drainage, indicates that the presence of bacterial biofilms may contribute to more severe cases of sialadenitis. typhoid fever is an acute food borne illness, predominantly caused by salmonella enterica serovar typhi that is often characterized by high fever, weakness, headache, abdominal pain, and constipation. untreated, serious complications may arise, including intestinal bleeding, bowel perforation, septicemia, meningitis, and death [61, 62] . there were an estimated 21.7 million cases of typhoid fever worldwide in 2000, resulting in approximately 217,000 deaths [63] . three to 5% of typhoid fever patients become chronic carriers after the acute phase of the illness [64] [65] [66] . these chronic carriers are generally asymptomatic and constitute an important reservoir of bacteria that can shed in feces and urine and thereby spread the disease. the chronic carrier state, in which s. typhi is typically detected in the gall bladder, is often associated with pre-existing hepatobiliary disease and approximately 90% of chronic carriers have gall stones [61, 64, 67, 68] . complications related to chronic carriage of s. typhi include hepatitis, cholecystitis, cholangitis, chronic diarrhea, and pancreatitis as well as hepatobiliary antibiotics 2020, 9, 59 6 of 29 carcinomas [62, 69] . antibiotic treatment generally resolves the acute infection, but it is often ineffective against the chronic colonization of the gall bladder by s. typhi [64, 70] . there are indications that chronic colonization of the gall bladder by s. typhi, involves attachment and invasion of epithelial cells and biofilm formation on gall stones [48, 71, 72] . consistent with this, s. typhi was observed to cover a large part of the surface of gall stones in patients presenting with colilithiasis and asymptomatic typhoid carriage ( figure 1 ) [73] . this is in agreement with in vitro studies that showed biofilm formation by s. typhi on human gall stones, in a medium supplemented with bile [74, 75] . recent studies indicate that both the host and the bacteria adapt to the chronic gall bladder infection. the host immune response changes from an early pro-inflammatory response to a later anti-inflammatory response [76] whereas s. typhi adapts to the gall bladder environment by increasing the biofilm forming ability and the capacity for persistence and simultaneously reducing the ability to cause acute infections [77, 78] . it has also been shown that s. paratyphi a can persist in the gallbladder [79] and accumulating evidence indicate a role for non-typhoidal salmonellae in persistent infections. bacteria, other than salmonellae, can occasionally be detected in gallbladder tissues, but gallstones from these patients are generally not covered by bacterial biofilms [73] . this is consistent with the experiments showing that many bacteria causing acute gallbladder infections do not form biofilms on gallstones in the presence of bile [73, 80] . antibiotics 2020, 9, 59 6 of 27 the chronic gall bladder infection. the host immune response changes from an early proinflammatory response to a later anti-inflammatory response [76] whereas s. typhi adapts to the gall bladder environment by increasing the biofilm forming ability and the capacity for persistence and simultaneously reducing the ability to cause acute infections [77, 78] . it has also been shown that s. paratyphi a can persist in the gallbladder [79] and accumulating evidence indicate a role for nontyphoidal salmonellae in persistent infections. bacteria, other than salmonellae, can occasionally be detected in gallbladder tissues, but gallstones from these patients are generally not covered by bacterial biofilms [73] . this is consistent with the experiments showing that many bacteria causing acute gallbladder infections do not form biofilms on gallstones in the presence of bile [73, 80] . studies performed in a murine gall stone model using s. typhimurium support a role of biofilm formation in the gall bladder in the chronic typhoid carrier state. mice with diet-induced cholelithiasis had a higher number of salmonellae associated with the gallbladder epithelium and bile compared to gallstone negative controls and this was associated with increased fecal shedding [72, 73] . in addition, biofilms covering approximately half the surface of gall stones that were isolated from mice 21 days post infection were detected by scanning electron microscopy. using the murine gallstones model gonzales et al. showed that s. typhimurium was more resistant to antibiotic treatment in mice fed a cholelithiasis-inducing diet compared to mice fed a standard diet [81] . these experiments mimicking the chronic carrier state support an important role for biofilms in recalcitrance of chronic s. typhi infections to antibiotic treatment and are consistent with clinical data [70, 82, 83] . studies performed in a murine gall stone model using s. typhimurium support a role of biofilm formation in the gall bladder in the chronic typhoid carrier state. mice with diet-induced cholelithiasis had a higher number of salmonellae associated with the gallbladder epithelium and bile compared to gallstone negative controls and this was associated with increased fecal shedding [72, 73] . in addition, biofilms covering approximately half the surface of gall stones that were isolated from mice 21 days post infection were detected by scanning electron microscopy. using the murine gallstones model gonzales et al. showed that s. typhimurium was more resistant to antibiotic treatment in mice fed a cholelithiasis-inducing diet compared to mice fed a standard diet [81] . these experiments mimicking the chronic carrier state support an important role for biofilms in recalcitrance of chronic s. typhi infections to antibiotic treatment and are consistent with clinical data [70, 82, 83] . in healthy people, a protective mucosal layer covers the colon epithelium and separates it from the luminal microbiota. breaches in this protective layer result in increased contact between microbes and the epithelial cells, which can result in biofilm formation on the epithelium. these changes constitute a pathogenic state that has been implicated in development of inflammatory bowel disease (ibd). studies also suggest an association between colonic biofilm formation, dysbiosis and colorectal carcinogenesis [84] [85] [86] . ibd (ulcerative colitis and crohn's disease) is characterized by chronic inflammation of the digestive tract. common symptoms are pain, diarrhea, weight loss, and fatigue. furthermore, patients suffering from ibd have a 10-30% cumulative risk of developing colorectal cancer (crc) within 30 years after the onset of ibd [87] . ibd has been linked with occurrence of biofilms adhering to the epithelium and dysbiosis of mucosa-associated bacteria that result in stimulation of an inflammatory response. this might be associated with failure of maintaining the integrity of the mucosal barrier resulting in a reduced ability to clear the infection [88] . using fluorescence in situ hybridization (fish) and fluorescence microscopy two log higher numbers of bacteria were detected associated with the mucosa of patients with ibd compared to patients with irritable bowel syndrome (ibs; a disease not generally associated with intestinal inflammation) and healthy controls [89] . in addition, the density of bacteria associated with the mucosa has been shown to be significantly higher in patients with intestinal inflammation [88] . bacteria adhering to colonic epithelium were detected in ibd patients only, and these biofilms were dominated by bacteriodes fragilis [89] and enterobacteriaceae [88] . in contrast, mucosa-associated biofilms with different species composition compared to the ibd associated biofilms were sporadically detected in healthy controls and ibs patients [89] . several studies have detected higher incidence of escherichia coli in patients with ibd compared to healthy individuals [90] and although biofilms were not demonstrated in vivo, the isolates were isolated from biopsies after removal of the mucosal layer, indicating adherence to the colonic epithelium and the isolates displayed biofilm forming capacity in vitro [90] . colorectal cancer (crc) is one of the most common cancer forms worldwide in both males and females [91] . it is generally considered that crc develops from normal colorectal epithelium as a result of accumulating genetic mutations and epigenetic changes as described by the adenoma-carcinoma sequence model [92] ). accumulating evidence from studies comparing the colonic microbiota compositions of crc-patients and healthy individuals indicates that crc is associated with microbial dysbiosis, which is associated with an aberrant inflammatory response ( [93, 94] ). based on the adenoma-carcinoma model and the accumulating microbiota data, tjalsma et al. suggested a bacterial driver-passenger model for crc [95] . in this model, certain bacteria that are part of the normal microbiota can facilitate dna damage in epithelial cells that may result in initiation of crc (bacterial drivers). biological changes that occur during tumorigenesis result in changes of the microenvironment that favor colonization by opportunistic bacteria (bacterial passengers). the new microenvironment provided by the tumor may result in that the bacterial drivers are eventually outcompeted by bacterial passengers [95] . recent evidence indicates an important role of biofilms in the colorectal carcinogenesis and li et al. have suggested that biofilms may be regarded as a driver in the adenoma-carcinoma sequence at an early stage of carcinogenesis [85] . it is currently not known exactly how biofilms influences carcinogenesis, if a specific species is responsible or if it is due to sequential or synergistic activities of different members of the microbial community. however, it has been suggested that accumulation of pro-inflammatory species affects the normal processes of the colorectal epithelium leading to disturbed regulation of inflammation, apoptosis, and cell proliferation. in fact, there are antibiotics 2020, 9, 59 8 of 29 a number of species that have been shown to directly affect these processes. toxigenic b. fragilis has been proposed as an initiator of colorectal cancer due to its ability to disturb the epithelial homeostasis and influence cell proliferation [96, 97] . increased levels of enteropathogenic e. coli (epec) have been detected in tumors and certain strains have been shown to produce toxins that can induce double strand breaks and cause chromosomal instability [97, 98] . simultaneous colonization of tumor prone mice by toxin-producing b. fragilis and e. coli resulted in increased dna damage, faster initiation of tumorigenesis, and greater mortality compared to mice colonized with either species alone, suggesting a potential link between a tumorigenic microbiota and early neoplasia of the colon [97] . fusobacterium nucleatum can influence both inflammatory processes and cell proliferation. it is often found enriched in tumor tissues, and high numbers of f. nucleatum correlates with poor prognosis [85, 99] , however f. nucleatum may not be involved in the early stages of carcinogenesis, but may be involved in cancer progression [100, 101] . polymicrobial biofilms adhering directly to the epithelium were detected in colorectal tumors by sem and fish [102] . biofilms were detected in 89% of right-sided tumors compared to 13% of left-sided tumors. right-sided and left-sided tumors also differ in microbiota composition, molecular characteristics, response to chemotherapy, and prognosis; however it is not clear how these aspects are connected (reviewed by kim et al. [103] ). tomkovich et al. recently reported that human colon mucosal biofilms were carcinogenic in murine crc-models, whether the biofilm associated microbiota were from crc-patients or healthy individuals [104] . yu et al. detected biofilms associated with crc, adenomas, and polyps by sem, fish, and fluorescence microscopy and found that the prevalence of f. nucleatum was significantly higher in biofilms from crc than the other sample groups [105] . although a connection between the composition of the colorectal microbiota and crc is becoming increasingly evident, more research is needed to determine the exact role of the microbial community of the biofilm and its constituting species in carcinogenesis. wounds are damaging to living tissue caused by e.g., a trauma like cuts, abrasions, burns, and surgery, or as a consequence of underlying illnesses such as diabetes. most wounds that contain microorganisms heal successfully. however, sometimes microorganisms, and particularly bacteria, multiply, healing is disrupted and wound tissues are damaged resulting in an infection [106, 107] . both chronic and acute wounds are susceptible to infection as a result of the loss of the innate barrier function of the skin and dermal appendages [108] . it is generally accepted that chronic wound infections harbor several different microorganisms and the number of species are thought to be underestimated because of the limitations in culturing techniques. to circumvent this problem, molecular methods can be used to identify viable, but non-culturable bacteria. also novel microscopy techniques, like e.g., confocal scanning electron microscopy, fluorescence microscopy, and electron microscopy, can be used both in the visualization and to identify bacteria in wounds [108, 109] . chronic wounds can be colonized with several different bacterial species whereas staphylococcus aureus is most commonly isolated [110] . aerobic bacteria, like s. aureus, s. epidermidis, and pseudomonas aeruginosa, are often found on the surface of chronic wounds while anaerobic species are predominant in deeper tissue [111] . the anaerobic bacteria that predominate chronic wounds of both humans and animals are bacteroides spp., fusobacterium spp., peptostreptococcus spp., as well as clostridium spp. [108] . as for other bacterial infections, it has historically been assumed that wound infections are caused by planktonic bacteria. more recently, researchers have suggested that chronic wound infections are due to the biofilm mode of growth of the bacteria [2, 48] . this idea is supported by recent studies that have demonstrated that chronic wound infections in fact are biofilm infections and observations indicate the presence of biofilms also in acute infections [4, 111, 112] . it is claimed that bacterial biofilms are located on the surface of wounds and they have been implicated in the failure of wound healing and contribution to chronic inflammation [108] . for instance, it has been suggested that p. aeruginosa may significantly contribute to inflammation of the wound by producing rhamnolipids [108] . biofilm has been postulated to be the reason why wounds like e.g., venous leg ulcers and pressure ulcers often develop into chronic stages [108] . the first scientific study that showed a link between biofilm and wound infections, analyzed the biofilm forming ability of a burn wound isolate of p. aeruginosa in vitro using light microscopy [113] . later, most evidence that wound infections are biofilm related has been shown by different advanced microscopy methods. the study by bjarnsholt used confocal laser scanning microscopy and a specific peptide nucleic acid-fluorescence in situ hybridization probe to visualize p. aeruginosa biofilm in non-healing chronic wounds [4] . the study by davis et al. used light microscopy, scanning electron microscopy, and epifluorescence microscopy to look for biofilm-like structures in biopsies from wounds on pigs infected with s. aureus [112] . a study by james et al. examined biopsies from both acute and chronic wounds using electron microscopy. they found that as many as 60% of chronic wounds contained biofilm, as opposed to only 6% of acute wounds [111] . lately also an international consensus for clinical indicators of wound infection and biofilm has been published [114] . a total of 14 experts agreed on a list of ten clinical indicators of possible biofilm in a wound, including failure of antibiotic treatment and delayed healing in spite of optimal wound management. biofilm infections in wounds are also frequently evaluated macroscopically because of the absence of advanced microscopy equipment and knowledge in clinics. using macroscopic examination of slough in a wound combined with microscopy of the wound slough has been suggested as a clinical marker of biofilm. other markers such as a "shiny or sheen" appearance of a wound has also been used as an indication that biofilm is present. these macroscopic observations have the limitation of being highly subjective [108] . a rapid and easy to perform method to diagnose biofilm infections in wounds is needed. bacterial vaginosis (bv) is the most common genital tract infection in women during their reproductive years, and tends to have a high rate of relapse and recurrence [115] . typical for bv are increased numbers of anaerobic bacteria like gardnerella vaginalis, atopobium vagnae, and others accompanied by decreased numbers of protective lactobacilli [116] . interestingly, since early 1980s one of the criteria used to diagnose bv in clinical practice has been the presence of so called "clue cells", i.e., epithelial cells covered with bacteria [117] . however, it was not until the studies of swidsinski et al. thirty years later that the clue cells were finally understood to be desquamated biofilm-coated epithelial cells [118] . in this study, they found that an epithelial, multispesies biofilm dominated by g. vaginalis was present in as many as 90% of the bv vaginal biopsies [119] . currently, it is generally agreed that bv involves the presence of a dense, structured, and polymicrobial biofilm, primarily constituted by g. vaginalis clusters strongly adhered to the vaginal epithelium [116] . g. vaginalis is probably the first species to adhere to the vaginal epithelium creating a scaffold for other species to adhere [120] . g. vaginalis may also be present in the vagina of "healthy" women and sexually inexperienced women and does not necessarily cause bv. studies by swidsinski et al. on g. vaginalis in urine, showed that the bacteria were attached to desquamated epithelial cells in all patients with proven bv and their partners, and dispersed when it was present in urine of healthy controls [121] . furthermore, pathogenic strains displayed higher in vitro phenotypic levels of virulence traits like cytotoxicity, adherence, and biofilm production than commensal strains [122] . this might indicate genetic differences between pathogenic and commensal g. vaginalis strains. however, recent transcriptome studies have suggested that g. vaginalis is able to exhibit different phenotypes through large changes in gene expression [123] , and studies on mixed biofilms indicate that expression of g. vaginalis virulence genes may be significantly influenced by other bacterial species in the biofilm [124] . consequently, the composition of the biofilm may also influence the pathogenesis. accordingly, verstraelen and swidsinski suggest that "environmental pressures or ecological disturbances of the vaginal niche might be a more determining factor in biofilm formation and development of bacterial vaginosis in a given woman, than gardnerella genotype alone" [120] . the uterus has traditionally been assumed to be free of bacteria, but recent studies have identified a functional microbiome of the endometrium under physiological conditions [125, 126] . lactobacillus was found to be the most abundant followed by gardnerella, prevotella, atopobium, and sneathia. in approximately 20% of the women investigated, the bacterial community varied greatly from that of the vagina, suggesting that the endometrial and vaginal microbiota are not necessarily identical [127] . in chronic endometritis (ce), the endometrial mucosa may be colonized by common bacteria like enterococcus faecalis, e. coli, g. vaginalis, klebsiella pneumoniae, proteus spp., p. aeruginosa, staphylococcus spp., and streptococcus. spp. [128, 129] . the prevalence of ce has been estimated to be 19% in the general population and 45% in infertile patients [125] . however, because ce is often asymptomatic, it is seldom suspected and diagnosed. swidsinski et al. found that a structured polymicrobial g. vaginalis biofilm present in bv often was accompanied by a similar biofilm in the endometrium or the fallopian tubes ( figure 2 ) [130] . such biofilms were not found in any of the women without bacterial vaginosis. however, the clinical significance of these endometrial biofilms is still not clear. [128, 129] . the prevalence of ce has been estimated to be 19% in the general population and 45% in infertile patients [125] . however, because ce is often asymptomatic, it is seldom suspected and diagnosed. swidsinski et al. found that a structured polymicrobial g. vaginalis biofilm present in bv often was accompanied by a similar biofilm in the endometrium or the fallopian tubes ( figure 2 ) [130] . such biofilms were not found in any of the women without bacterial vaginosis. however, the clinical significance of these endometrial biofilms is still not clear. studies performed in horses may shed more light. bacterial endometritis is an important cause of subfertility in mares, contributing to a major economic loss for the equine industry [131, 132] . ferris et al. used an in vivo model of infectious endometritis where the mares were inoculated with equine p. aeruginosa isolates isolated from clinical cases. bioluminescence imaging of the endometrium displayed focal areas with bacteria surrounded by a "biofilm-like" matrix [133] . furthermore, the biofilm matrix component pel and the biofilm-regulatory molecule cyclic di-gmp were detected in such areas. [134] . these observations support the hypothesis of biofilm formation in the uterus by clinical strains. in addition, ferris et al. made some interesting observations on the local immune responses in their model. although inflammatory cells were observed both in areas with and areas without adherent bacteria, neutrophils were decreased and gene expression of the immune-modulatory cytokine interleukin-10 was increased in the areas with biofilm. whether this modulation of the host immune response is actively caused by the biofilm bacteria, or by the immune system's reaction to the biofilm structure, is not known. mastitis is an inflammatory condition of the mammary gland primarily occurring during lactation. the who estimates a global prevalence of approximately 10% of breastfeeding women [135] . s. aureus is considered to be the main etiological agent of infectious mastitis, whereas coagulasenegative staphylococci (cns), e. coli and streptococci may also be found. the number of studies dealing with the microbiological aspects of human mastitis is low, especially compared with the vast studies performed in horses may shed more light. bacterial endometritis is an important cause of subfertility in mares, contributing to a major economic loss for the equine industry [131, 132] . ferris et al. used an in vivo model of infectious endometritis where the mares were inoculated with equine p. aeruginosa isolates isolated from clinical cases. bioluminescence imaging of the endometrium displayed focal areas with bacteria surrounded by a "biofilm-like" matrix [133] . furthermore, the biofilm matrix component pel and the biofilm-regulatory molecule cyclic di-gmp were detected in such areas. [134] . these observations support the hypothesis of biofilm formation in the uterus by clinical strains. in addition, ferris et al. made some interesting observations on the local immune responses in their model. although inflammatory cells were observed both in areas with and areas without adherent bacteria, neutrophils were decreased and gene expression of the immune-modulatory cytokine interleukin-10 was increased in the areas with biofilm. whether this modulation of the host immune response is actively caused by the biofilm bacteria, or by the immune system's reaction to the biofilm structure, is not known. mastitis is an inflammatory condition of the mammary gland primarily occurring during lactation. the who estimates a global prevalence of approximately 10% of breastfeeding women [135] . s. aureus is considered to be the main etiological agent of infectious mastitis, whereas coagulase-negative staphylococci (cns), e. coli and streptococci may also be found. the number of studies dealing with the microbiological aspects of human mastitis is low, especially compared with the vast amount of studies on mastitis in dairy animals. in cattle, mastitis is one of the most frequent and costly diseases in the dairy industry [136] . also in dairy animals, s. aureus is the main pathogen. other bacteria associated with mastitis are s. agalactiae (causing contagious mastitis), cns, e. coli, klebsiella spp., enterobacter spp., citrobacter spp., s. dysgalactiae, s. uberis, enterococci, and pseudomonas spp. chronic and recurrent infections are frequent. the mechanism of persistence of s. aureus in its host is still not fully understood. s. aureus has been shown to be able to adhere to and internalize into mammary gland epithelial cells in vitro [137] . intracellular cocci have also been demonstrated in mammary epithelial cells of infected mice, cultured mammary epithelial cells from cows, and epithelial cells isolated from mastitic milk (reviewed by [138] ). invasion may be a way to evade host defenses in vivo. biofilm formation might be another. direct evidence that biofilms are involved in the pathogenesis of mastitis is scarce. however, an in vivo study by hensen et al. with microscopic examination of s. aureus in mammary tissue did indicate the presence of biofilm [139] . both in the early and chronic stages of infection, clusters of s. aureus was observed in the lumen of alveoli or lactiferous ducts, in association with the epithelium. the clusters appeared approximately 24 h after exposure to the pathogen, and polymorphonuclear neutrophils were also often present. in vitro, both lactose and milk have been shown to increase biofilm formation by s. aureus [140, 141] . for cns, a positive correlation between biofilm formation and days in milk was observed, and cns isolated later in the lactation were better biofilm formers than those isolated earlier [142] . on the other hand, simojoki et al. found no association between cns biofilm formation in vitro and the persistence or severity of mastitis in vivo [143] . bap is a surface protein involved in biofilm formation, and studies on the presence and expression of the bap gene may contribute to elucidate a possible role of biofilm in mastitis pathogenesis. the bap gene has been identified in up to 25% s. aureus isolates and up to 95% of cns isolates from bovine mastitis [144, 145] , and up-regulation of the bap gene with the presence of low concentrations of milk or lactose in the growth medium has been shown in vitro [141, 146] . zuniga et al. studied the presence of genes encoding bap and a group of adhesins in staphylococci isolated from subclinical mastitis [147] . the median somatic cell counts, which are markers of sub-clinical mastitis, were higher in milk samples where the bacteria had the bap gen and the adhesin genes eno, fnba, fib, than in samples with staphylococci without these genes. thus, the presence of biofilm may contribute to a higher intensity of the inflammatory process. in a study on sub-clinical s. aureus mastitis, bap-positive isolates were observed to be more able to colonize and persist in the bovine mammary gland in vivo, and anti-bap antibodies in the serum confirmed that bap was produced during infection [146] interestingly, bap promoted adhesion and prevented entry of s. aureus into epithelial cells in vitro, whereas bap deficient bacteria displayed increased invasion into mammary gland epithelial cells in a lactating mice mastitis model [148] . altogether, these results may indicate that biofilm formation is correlated with persistence of s. aureus in the bovine intramammary gland. another indication of the possible contribution of biofilm to the pathogenesis of mastitis is seen in vaccination studies. when mice immunized with formalin-killed biofilm s. aureus were compared to those immunized with formalin-killed planktonic bacteria, they showed significantly lower s. aureus colonization, as well as less severe clinical symptoms and tissue damage in mammary glands, [149] . likewise, in a sheep mastitis vaccination study, crude bacterial extracts from strong biofilm formers gave the highest production of antibodies and the best protection against infection and mastitis, when compared with extracts from weak biofilm formers and controls [150] . rhinosinusitis (rs) is an inflammation of the nose and the paranasal sinuses, characterized by nasal blockage, obstruction, congestion, or nasal discharge. additional symptoms may include loss of smell and facial pain and pressure. according to the duration of the disease, it can defined as acute when lasting less than 12 weeks, or chronic when lasting more than 12 weeks [151] . viruses account for up to 80 to 90% of the acute rs, and the most commonly involved viruses are rhinovirus, respiratory syncytial virus, influenza virus, coronavirus, parainfluenza virus, adenovirus, and enterovirus. the host immune response to a viral infection consists of non-specific and specific components, which will eventually eliminate the invading agent, but also generate dead epithelial and immune cells, creating an environment opportune for secondary bacterial infections. during viral infection in chronic rs, a similar inflammatory process can occur as in acute rs [152, 153] . the presence of biofilms in crs patients, as well as in animal models, has been reported in a relatively large number of studies, mainly using scanning electron microscopy and confocal laser microscopy on biopsies (figure 3 ) [154] [155] [156] . biofilms have also been identified in healthy controls, although to a lesser extent than in crs patients [157] . the presence of biofilms in crs patients, as well as in animal models, has been reported in a relatively large number of studies, mainly using scanning electron microscopy and confocal laser microscopy on biopsies (figure 3 ) [154] [155] [156] . biofilms have also been identified in healthy controls, although to a lesser extent than in crs patients [157] . once established, biofilms may induce changes in the mucociliary blanket like destruction of the epithelial layer and absence of cilia, and a local inflammatory response [158] [159] [160] [161] . this suggests epithelial damage being a part of the pathogenesis of biofilm-associated crs. interestingly, a study of crs patients by tan et al. indicated a link between intracellular s. aureus and biofilm colonization [162] , indicating that biofilm also can facilitate cellular invasion by pathogens. the clinical relevance of biofilms in the pathogenesis of crs has been demonstrated in several studies. the presence of biofilm has been associated with worse pre-operative radiological scores and post-operative outcome, as well as higher risk of recurrence [163] [164] [165] [166] . s. aureus biofilm in particular appears to be more pathogenic than other bacterial species, and this has been suggested to be due to a severe local inflammatory response to s. aureus superantigens [167] . colonization in the form of biofilm seems to have a different function in the pathogenesis of s. pneumoniae infections. the human nasopharynx is the main reservoir for s. pneumoniae and pneumococcal colonization always precedes infection [168] . striking differences between biofilm once established, biofilms may induce changes in the mucociliary blanket like destruction of the epithelial layer and absence of cilia, and a local inflammatory response [158] [159] [160] [161] . this suggests epithelial damage being a part of the pathogenesis of biofilm-associated crs. interestingly, a study of crs patients by tan et al. indicated a link between intracellular s. aureus and biofilm colonization [162] , indicating that biofilm also can facilitate cellular invasion by pathogens. the clinical relevance of biofilms in the pathogenesis of crs has been demonstrated in several studies. the presence of biofilm has been associated with worse pre-operative radiological scores and post-operative outcome, as well as higher risk of recurrence [163] [164] [165] [166] . s. aureus biofilm in particular appears to be more pathogenic than other bacterial species, and this has been suggested to be due to a severe local inflammatory response to s. aureus superantigens [167] . colonization in the form of biofilm seems to have a different function in the pathogenesis of s. pneumoniae infections. the human nasopharynx is the main reservoir for s. pneumoniae and pneumococcal colonization always precedes infection [168] . striking differences between biofilm residing and dispersed pneumococci indicate that the biofilm phase serves as a non-pathological reservoir. in mouse models, dispersed bacteria displayed inflammatory infiltration, whereas biofilm pneumococci were quickly cleared from the blood without causing invasive disease [169, 170] . these observations correspond with gene expression studies where the dispersed cells displayed a higher expression of virulence, stress-response, and bacteriocin production/excretion genes than their biofilm residing counterparts [170] . pharyngitis, or sore throat, is a very common condition. most cases are viral, but 10-25% are caused by bacteria. the tonsils and adenoids are lymphoid structures, and recurrent bacterial infections may result in hypertrophy of the tonsillar or adenoid tissue [171] . s. aureus, haemophilus spp., and streptococcus spp. are the most common bacterial causes. biofilms have been identified in situ after adeno-and tonsillectomy in several studies, using scanning electron microscopy, confocal microscopy, and light and transmission electron microscopy [23, [172] [173] [174] [175] [176] . whether biofilm also can contribute to development of clinical symptoms is uncertain, although a couple of studies may indicate an association between the presence of biofilms and chronic inflammation. al-mazrou and al-khattaf found that biofilms were present in a significantly higher proportion of patients with chronically inflamed tonsils and adenoids than in patients with obstruction [172] . diaz et al. report that symptoms like harsh raucous sound, tonsillar, and adenoids hypertrophy, apnea, and cervical adenopathies were related to the presence of biofilm in tonsils [174] . interestingly, studies indicate that adenoid biofilms may also serve as reservoirs for infections in other parts of the respiratory system, as well as in the middle ear. children with recurrent acute om were found to have large parts of their adenoid mucosa covered with polymicrobial biofilms containing middle ear pathogens [177] . similar findings have been reported in children with crs [178] . on the other hand, in vitro experiments showed that when epithelial cells were covered by s. oralis and s. salivarius biofilms, the cells were protected from gas adherence, internalization, and cytotoxic effects [179] . this may indicate protective effects by biofilms produced by such respiratory tract streptococci. chronic laryngitis is believed mainly to be non-infectious, and this may be the reason for the sparse research activity on biofilm in relation to this disease. however, when investigating true vocal fold biopsies with confocal scanning laser microscopy and pcr, biofilm was found in 62% of the patients with chronic laryngitis, but only in 20% of the controls, thus supporting a hypothesis that chronic laryngitis also may be biofilm related [180] . pertussis, also called whooping cough, is a highly contagious disease, which for decades has been controlled by mass vaccination. unfortunately, we now observe a resurgence. pertussis is mostly caused by bordetella pertussis, but also to some extent by human associated b. parapertussis. both are human-specific pathogens, which most likely have evolved from b. bronchiseptica or a common ancestor. b. bronchiseptica, on the other hand, infects a number of mammal species, including humans, mostly leading to a chronical, subclinical infection. in addition, it causes infectious respiratory disease in dogs and atrophic rhinitis in pigs [181] . bordertella biofilms have been demonstrated in mouse models (reviewed by [182] ). in both a nose and a trachea model, distinct architectural features adherent to ciliated epithelium were observed; in the form of mats, towers or pillars for b. bronchiseptica, and clusters and macrocolonies for b. pertussis. furthermore, cattelan et al. observed an association between increased biofilm formation by b. pertussis and higher levels of bacterial colonization in the nose and trachea of mice [183] . direct observations of b. pertussis biofilm in humans have not been reported, but abundant extracellular bordetellae were observed in respiratory tissue samples from 15 infants who had died from confirmed b. pertussis pneumonia [184] . several other data support the notion of biofilm being present in whooping cough. during the biofilm lifestyle, increased production of the surface-associated b. intermediate protein a (bipa) can be observed. expression of the bipa gene has also been shown during respiratory tract infection of mice, and anti-bipa antibodies were present in whooping cough patients [185] . when mice were vaccinated with bipa, colonization of the lungs was significantly reduced, and antibodies to bipa were found to opsonize bacteria. promising results in a murine model have also been obtained with vaccines with other biofilm antigens like bamb and lptd [186] . consequently, specific biofilm proteins may be candidate antigens for improved pertussis vaccines. cf was the first infection where biofilm was recognized as part of the etiology, and is probably the most thoroughly studied biofilm infection to date [187] . cf is a genetic disease primarily affecting the respiratory and the digestive system, and is characterized by production of viscid mucus and chronic infections. lung infection is the main cause of morbidity and mortality [188] . in young patients, primarily s. aureus and h. influenzae colonize in the airways. p. aeruginosa dominates at later stages, although other bacterial species also have been seen to form biofilm in the lungs of cf patients [189] . h. influenzae in biofilm-like structures have been observed in lung lavage samples from children with cf [190] . furthermore, clinical isolates formed biofilms on the apical surface of airway epithelium in vitro, and this stimulated epithelium to increased secretion of factors that mediate inflammation. colonization with p. aeruginosa often starts with biofilm in the paranasal sinuses, which serves as reservoirs for repeated lung infections that finally become chronic. p. aeruginosa biofilm has been observed in lung tissue, lung abscess, and sputum of cf patients [191] [192] [193] [194] . microscopic analyses have shown that p. aeruginosa in sputum grows as microcolonies adherent to sputum components [195] . in response to the presence of biofilm, large numbers of polymorphonuclear leukocytes (pmns) infiltrate the area, producing a chronic inflammation with subsequent tissue damage, loss of lung function, and obstruction of the airways. the metabolic activity of bacteria and cells consume available oxygen and produce anerobic conditions [195] , which unfortunately seems to favor the biofilm mode of p. aeruginosa even more [196] . diagnostic criteria for p. aeruginosa biofilm infection in cf patients are recommended in the escmid guideline for the diagnosis and treatment of biofilm infections of 2014 [197] . one criterion is the detection of bacterial aggregates embedded in an alginate-including matrix in sputum or other samples from the lower airways. mucoid growth with hyperproduction of alginate by bacteria isolated from lung tissue and sputum is also considered diagnostic for biofilm infection (figure 4) . the presence of alginate in sputum is a good indication. serum igg antibodies to p. aeruginosa antigens, including the major biofilm matrix component alginate, is usually present. likewise, siga in saliva or in the mucosa of the paranasal sinuses indicates biofilm in this location. is the detection of bacterial aggregates embedded in an alginate-including matrix in sputum or other samples from the lower airways. mucoid growth with hyperproduction of alginate by bacteria isolated from lung tissue and sputum is also considered diagnostic for biofilm infection (figure 4) . the presence of alginate in sputum is a good indication. serum igg antibodies to p. aeruginosa antigens, including the major biofilm matrix component alginate, is usually present. likewise, siga in saliva or in the mucosa of the paranasal sinuses indicates biofilm in this location. colonies. the mucoid variant over-produces alginate, which is the matrix in the p. aeruginosa biofilm in the respiratory tract of cystic fibrosis patients. mucoid colonies are only found in patients with chronic biofilm infection and alginate from mucoid colonies is therefore a biofilm-specific antigen. høiby et al. [197] . bacterial prostatitis (bp) generally presents with urinary tract infection (uti), pain in the pelvic and genital region, and occurrence of bacteria in expressed prostatic secretions. acute bp may result colonies. the mucoid variant over-produces alginate, which is the matrix in the p. aeruginosa biofilm in the respiratory tract of cystic fibrosis patients. mucoid colonies are only found in patients with chronic biofilm infection and alginate from mucoid colonies is therefore a biofilm-specific antigen. høiby et al. [197] . bacterial prostatitis (bp) generally presents with urinary tract infection (uti), pain in the pelvic and genital region, and occurrence of bacteria in expressed prostatic secretions. acute bp may result in complications such as reduced fertility, bladder infections, prostatic abscesses, urosepsis, and death. the disease can also progress to chronic prostatitis (cp) or chronic pelvic pain syndrome (cpps) [198, 199] . if symptoms of bacterial prostatitis last for more than 3 months, it is considered to be chronic bacterial prostatitis (cbp). similar to uti, the species most commonly associated with bp are e. coli, proteus mirabilis, p. aeruginosa, klebsiella spp. and other enterobacteriaceae as well as e. faecalis [199] [200] [201] . pathogenesis of cbp has been suggested to involve biofilm-forming bacteria [202] [203] [204] [205] . this hypothesis is based on the observations that bacteria can persist in the prostate for long periods of time, a high percentage of cases are refractory to treatment with antibiotics [200, 202] , and even when the treatment is successful, as judged by negative microbiological tests, symptoms may remain [202] . a few studies support the involvement of biofilm forming bacteria in the pathogenesis of cbp. nickel et al. used an animal model of experimental bp and showed that chronic symptoms correlated with the presence of glycocalyx-enclosed bacterial microcolonies inside the ducts and acini of the prostate [206] . occurrence of microcolonies seemed to be associated with a chronic inflammation of the gland. in support of this finding; scanning electron microscopy of prostate calcifications from patients with cbp showed structures reminiscent of bacterial microcolonies on the surface of, and embedded in, the calcifications [205, 207] . these observations were associated with isolation of bacteria, from the surface of calcifications, with the potential to form biofilms in vitro [207] . in addition, prostatitis appears to be correlated with occurrence in urine of e. coli with high capacity to form biofilm in vitro [203, 204] . however, biofilm formation may not be the only explanation for persistent bacterial infections in the prostate. uropathogenic e. coli (upec) strains, which are often isolated from bp-patients, can invade prostate cells in a mouse model (nod and c57bl/6j mice) as well as in cultured rwpe-1 and pec-1 cell lines [208, 209] . in the mouse model, the upec strains were able to proliferate within the epithelial cells following invasion, and although the effect was strain dependent, the infection could induce and sustain pelvic pain in the nod mice [209] . upec strains can also invade bladder epithelial cells, proliferate and form matrix-enclosed biofilm-like structures that has been referred to as intracellular bacterial communities (ibc) [210, 211] . interestingly, the invasive capacity of different upec strains into the prostate cell line rwpe-1 correlated with both the ability to adhere to epithelial cells and the capacity to form biofilm on a plastic surface, in vitro [208] and similar results were observed with an adherent/invasive e. coli (aiec) strain, which is highly similar to upec, isolated from an ileal lesion in a crohn's disease patient [212] . it is therefore possible, but to our knowledge has not been demonstrated, that bacteria can persist in the prostate using a similar strategy. more research is needed to understand the pathogenesis of chronic bacterial prostatitis and if bacterial persistence involves biofilm formation. utis are very common infections in humans and occur when bacteria, often from the rectum and perineum, enter the urethra and colonize the urethra, bladder, ureters, and/or kidneys. symptoms depend on the anatomical location of the infection and generally include urinary frequency, urgency, dysuria, and/or pain in the lower abdominal region. systemic symptoms and sepsis may occur, especially in infections involving the kidneys. a urinary tract infection often clears up on its own within a few days or after a short course of antibiotic treatment, but the relapse rate is high. the etiology of these chronic, or recurrent, urinary tract infections is not fully understood and has been attributed to reintroduction of bacteria via the rectal-peritoneal route, or from a vaginal reservoir. however, a growing body of evidence suggests involvement of a persistent infection in the bladder due to ibc [213] [214] [215] [216] [217] [218] [219] . ibc were first described in a mouse model using an immunosuppressed mouse strain and upec [210] , the primary uti-pathogen accounting for 70-95% of cases. scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy combined with fluorescently labeled bacteria and specific staining methods for type i fimbriae and polysaccharides revealed large protrusions on the urothelium which consisted of bacterial populations surrounded by a matrix of polysaccharide and fimbrial proteins that were not localized to specific organelles [210] . these results indicate bacterial biofilm formation inside the bladder epithelial cells. ibc can facilitate bacterial immune evasion as shown by video microscopy where luminal bacteria of the bladder were consumed by neutrophils whereas bacteria that had invaded the epithelial cells were protected from neutrophil attack and could proliferate in the ibc [220] . in the mouse model, upec undergo three-stage cycles of invasion of epithelial cells, followed by proliferation as ibc and dispersion into the bladder lumen, which allows for a new round of infection of adjacent cells [220] . this mechanism could explain persistent uti with periods of quiescent and relapsing infections. there is also evidence that ibc can contribute to chronic and recurrent uti in humans [213, 217, [221] [222] [223] [224] , and ibc has been detected in the epithelium of the urinary bladder. the majority of upec isolated from urine of women with urinary tract infections were able to form ibc in a mouse model [213, 221] . in addition, ibc have been observed by electron microscopy in shed epithelial cells in urine from 18% of women presenting with acute uti (figure 5 ) [222] . in this study, ibc were not detected in the urine samples from asymptomatic women. ibc have also been detected in 36.8% of urine samples from children presenting with cystitis [223] as well as in 44% of renal transplant patients tested for uti [224] . of upec isolated from urine of women with urinary tract infections were able to form ibc in a mouse model [213, 221] . in addition, ibc have been observed by electron microscopy in shed epithelial cells in urine from 18% of women presenting with acute uti (figure 5 ) [222] . in this study, ibc were not detected in the urine samples from asymptomatic women. ibc have also been detected in 36.8% of urine samples from children presenting with cystitis [223] as well as in 44% of renal transplant patients tested for uti [224] . an increasing number of diseases have been suggested to be biofilm related. in the majority of cases, this is based on observations of biofilm-like structures in biopsies, autopsies, and exudates of patients and /or research animals ( figure 6 ). additionally or alternatively, the presence of biofilm is indicated by studies on bacterial phenotypes during infection, immune responses, and vaccination experiments. however, even when biofilm is present, there is still a question whether the biofilm is the cause of the disease or the bacteria are just taking advantage of a favorable environment for colonization caused by the disease. an increasing number of diseases have been suggested to be biofilm related. in the majority of cases, this is based on observations of biofilm-like structures in biopsies, autopsies, and exudates of patients and /or research animals ( figure 6 ). additionally or alternatively, the presence of biofilm is indicated by studies on bacterial phenotypes during infection, immune responses, and vaccination experiments. however, even when biofilm is present, there is still a question whether the biofilm is the cause of the disease or the bacteria are just taking advantage of a favorable environment for colonization caused by the disease. our review of the literature shows that biofilm may potentially contribute to the pathogenesis of a disease in several ways. biofilm formation increases the bacteria's resistance against the defense mechanisms of the body, as well as antimicrobial treatments, thereby promoting chronic infections. biofilms may also function as an environment that accumulate different bacterial species as well as bacterial numbers in certain locations. this can result in deleterious effects on host cells due to concentrated, sequential, and/or synergistic activities by the present bacteria. furthermore, the mere presence of persistent biofilms may modulate the local immune response in several ways, e.g., by stimulating a local inflammatory response that can cause or aggravate tissue damage. these biofilm-mediated mechanisms have also been suggested to be involved in initiation and/or progression of cancers, such as crc. in wounds, an additional effect of biofilm is that the physical presence may obstruct wound healing. biofilm might also facilitate cellular invasion by pathogens, as indicated by the observed link between intracellular s. aureus and biofilm colonization in the upper airways. interestingly, biofilm-like intracellular bacterial communities have been identified, e.g., within the epithelial cells of the bladder of the urinary tract, which appears to result in bacterial immune evasion and persistent or recurrent infections. rhinosinusitis and cystic fibrosis. however, colonization in the form of biofilm may also serve as a sub-clinical reservoir for pathogens preceding clinical infection with planktonic bacteria, as observed for s. pneumoniae in the nasopharynx and upec in the urinary bladder. in addition, biofilms by nonpathogenic bacteria may even offer protection against pathogen infection, as observed for biofilms by s. oralis and s. salivarius in the upper airways. based on present research, it is clear that both diagnosis and treatment of a number of chronic diseases need to take into account the importance of biofilm. diagnostic criteria for biofilm infections are needed, and have already been suggested for a few diseases like cystic fibrosis and chronic wounds. development of effective treatment against such infections is also imperative. presence of biofilm can be linked to the severity and prognosis of disease, e.g., as in chronic rhinosinusitis and cystic fibrosis. however, colonization in the form of biofilm may also serve as a sub-clinical reservoir for pathogens preceding clinical infection with planktonic bacteria, as observed for s. pneumoniae in the nasopharynx and upec in the urinary bladder. in addition, biofilms by non-pathogenic bacteria may even offer protection against pathogen infection, as observed for biofilms by s. oralis and s. salivarius in the upper airways. based on present research, it is clear that both diagnosis and treatment of a number of chronic diseases need to take into account the importance of biofilm. diagnostic criteria for biofilm infections are needed, and have already been suggested for a few diseases like cystic fibrosis and chronic wounds. development of effective treatment against such infections is also imperative. author contributions: all authors have contributed to the design of the work, performed literature studies, written parts of the manuscript, and approved the submitted version. all authors have read and agreed to the published version of the manuscript. funding: this research received no external funding. a short history of microbial biofilms and biofilm infections bacterial biofilms: a common cause of persistent infections evolving concepts in 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lungs are infected with bacterial biofilms effects of reduced mucus oxygen concentration in airway pseudomonas infections of cystic fibrosis patients pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis escmid* guideline for the diagnosis and treatment of biofilm infections clinical courses following acute bacterial prostatitis acute bacterial prostatitis: how to prevent and manage chronic infection? chronic bacterial prostatitis (nih type ii): diagnosis, therapy and influence on the fertility status chronic prostatitis: a thorough search for etiologically involved microorganisms in 1,461 patients. infect the impact of biofilm-producing bacteria on chronic bacterial prostatitis treatment: results from a longitudinal cohort study increased biofilm formation in escherichia coli isolated from acute prostatitis biofilm formation in uropathogenic escherichia coli strains: relationship with prostatitis, urovirulence factors and antimicrobial resistance prostate calcifications: a case series supporting the microbial biofilm theory pathogenesis of chronic bacterial prostatitis in an animal model biofilms in chronic bacterial prostatitis (nih-ii) and in prostatic calcifications features of uropathogenic escherichia coli able to invade a prostate cell line uropathogenic escherichia coli induces chronic pelvic pain intracellular bacterial biofilm-like pods in urinary tract infections urothelial cultures support intracellular bacterial community formation by uropathogenic escherichia coli the adherent/invasive escherichia coli strain lf82 invades and persists in human prostate cell line rwpe-1, activating a strong inflammatory response persistence of uropathogenic escherichia coli in the bladders of female patients with sterile urine after antibiotic therapies subversion of host innate immunity by uropathogenic escherichia coli urinary tract infections in women with urogynaecological symptoms intracellular bacterial communities: a potential etiology for chronic lower urinary tract symptoms detection of intracellular bacterial communities in a child with escherichia coli recurrent urinary tract infections intracellular bacterial communities of uropathogenic escherichia coli in urinary tract pathogenesis host subversion by formation of intracellular bacterial communities in the urinary tract differentiation and developmental pathways of uropathogenic escherichia coli in urinary tract pathogenesis escherichia coli from urine of female patients with urinary tract infections is competent for intracellular bacterial community formation detection of intracellular bacterial communities in human urinary tract infection intracellular bacteria in the pathogenesis of escherichia coli urinary tract infection in children urinary atp and visualization of intracellular bacteria: a superior diagnostic marker for recurrent uti in renal transplant recipients? springerplus the work was funded by norwegian veterinary institute, oslo university hospital and university of oslo. the contribution of kjell arild danielsen (department of otorhinolaryngology, akershus university hospital, norway; department of otorhinolaryngology, østfold regional hospital, østfold, norway; and university of oslo, norway) to figure 2 is highly appreciated. the authors declare no conflict of interest. key: cord-016601-gp259urb authors: bonadonna, lucia; briancesco, rossella; coccia, anna maria title: analysis of microorganisms in hospital environments and potential risks date: 2017-03-24 journal: indoor air quality in healthcare facilities doi: 10.1007/978-3-319-49160-8_5 sha: doc_id: 16601 cord_uid: gp259urb this report provides information on indoor air quality and on associated potential risks in hospitals. spread and persistence of microbial communities in hospital environments are of huge interest to public health. hospitals are characterized by high infective risk, firstly cause of the compromised immunologic conditions of the patients that make them vulnerable to bacterial, viral, parasitological and fungal opportunistic infections. evidence suggests that microbial agents spread through air, surfaces, aerosol and hands. if surfaces may act as a reservoir for some pathogens, hands are an important transmission route. airborne and aerosolized waterborne microorganisms are taken into consideration, and their presence into the hospital environments is reviewed. the term hospital environment includes hospital buildings and healthcare settings with all indoor components that differentiate them: occupying people (sick people, visitors and hospital staff), indoor air, surfaces, medical equipment, drugs, medical devices, food and wastes (bottero et al. 2015; capolongo et al. 2016) . all these components may potentially support survival and growth of biological agents. how microbial communities persist and change in indoor environments is of great concern to public health. in fact, recent studies demonstrated that when humans occupy a space, human being there alters the microbiota of that space (smith et al. 2013; capolongo et al. 2015b) . within hospitals, people can be exposed to bioaerosols, particles of biological origin suspended in the air, and the potential for contracting a microbial pathogen is high. the human exposure to pathogens may be associated with a wide range of major public health issues, such as infectious diseases, acute toxic effects and allergies. hospital environments are characterized by high infective risk, firstly cause of the compromised immunologic conditions of the patients that make them vulnerable to bacterial, viral, parasitological and fungal opportunistic infections (d'alessandro et al. 2016) . the potential transmission of biological matter during surgery operations and medical treatments of infected individuals makes hospital environments strongly designated to become easily contaminated with spread of pathogens among patients (baglioni and capolongo 2002) . furthermore, in the last decades, if the use of antibiotics has been an excellent tool into preventing nosocomial infections, the extensive employ of these drugs has inevitably conducted to the insurgence of antibiotic resistance events. hospital buildings may be considered as dynamic environments affected by several factors that actively contribute to define the infective risk for patients. aspects that have to be considered are represented by the number of occupants (in addition to patients, medical employees and visitors), their effective state of health, hygienic habits and activity occurring at any time in the hospital (capolongo et al. 2015a; astley et al. 2015) . hygienic conditions of sites and rooms, building materials and equipment, furnishings also influence the microbial community composition (signorelli et al. 2016 ). in addition, technological devices such as hydraulic, heating and air-conditioning systems may represent a potential source of bacteria, fungi (moulds), virus and other organisms if not adequately designed and submitted to a planned preventive maintenance. microclimatic conditions and accidental events can support microbial and fungal growth (water infiltration and condensation) causing harmful indoor conditions (buffoli et al. 2007 ). outdoor microbial load and seasonal climatic characteristics also affect the microbiological quality of the hospital indoor air. hospital-acquired infections are emerging as important cause of morbidity and mortality in immunocompromised patients and severe underlying illnesses. each year, 2 million patients suffer from hospital-acquired infections and nearly 100,000 of them die (klevens et al. 2007) . data from the world health organization show that on 100 hospitalized patients, 7-10 are expected to contract, at least, one healthcare-associated infection (who 2011). however, the real burden is unknown because of the difficulty to gather reliable data. in fact, the diagnosis of nosocomial infections is complex and based on multiple criteria and not on a single laboratory test. in healthcare facilities, the main sources of infection are the patients and the healthcare employees, although the environment plays also an important role. in fact, environment may act as a reservoir for potential infective microorganisms and may contribute to their dissemination. consequently, bacteria are also common on inanimate surfaces, equipment and indoor air. infected patients spread microorganisms in the hospital sites through the release of expectorate drops, fluids from infected wounds, excrements, urine, blood, other corporeal fluids, but also through clothes and blankets. in addition to pathogenic microorganisms, the patients' endogenous flora could be a consistent source of microbes. microbial spread occurs mostly via large droplets, direct contact with infectious material or through contact with inanimate objects contaminated by infectious material. the direct contact between patients is rare; hands of clinical personnel can spread infective microorganisms and represent the most frequent vehicle of nosocomial infections. thus, hand hygiene is recognized as the primary measure to reduce infections. even healthy people and staff may act as carriers when infected or colonized. pathogens such as staphylococcus aureus, staphylococcus pyogenes, neisseria meningitidis, corynebacterium diphtheriae, hepatitis b virus, cytomegalovirus can be transmitted by symptomless carriers. pathogens and opportunistic pathogens may be present in water distribution systems and in aerosol released by water-cooling systems (e.g. legionella sp., mycobacterium sp.). microbial contamination can also occur in pharmaceuticals during the distribution among patients and in improperly processed food. in addition, hospital wastes not rightly and quickly eliminated can become a harmful contamination source. microorganisms that can be spread by contact include those associated with impetigo, abscess, diarrhoeal diseases, scabies and antibiotic-resistant organisms (methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococci). vectorborne transmission is limited to areas in which insects, arthropods and parasites are widespread. water and aqueous solutions used in healthcare facilities are often associated with the hospital-acquired infections. despite water treatment and chlorination, water entering in the hospital distribution systems may contain low concentrations of various autochthonous microorganisms such as pseudomonas sp., legionella sp., nontuberculous mycobacteria, acinetobacter sp., aeromonas sp., sphingomonas sp., enterobacter sp., aspergillus sp. and amoebae, which may cause clinically important opportunistic infections. remaining embedded in a matrix of extracellular organic polymers combined with nonorganic particles, these microorganisms can induce the development of biofilms in the plumbing system of healthcare facilities, hot water tanks, air-conditioning cooling towers, sinks, shower heads and faucet aerators. in addition to own each group characteristics, the biofilm constitutes a barrier, thus preventing both the complete cleaning of the environment that the total elimination of the microorganisms, with the consequent presence of survivors that, in the same time, can develop resistance to biocides and transmit this resistance, whether genetic, even in microorganisms of other species. some biofilm-forming bacteria such as legionella, klebsiella, pantoea agglomerans, acinetobacter baumannii and enterobacter cloacae can cause hospital infections and are more resistant to disinfectants and antibiotics than their planktonic states. biofilm can act as microbial reservoir that constantly releases viable microbes into the water stream. tap water may then contaminate surfaces, medical devices and instruments as well as endoscopes, dialysis machines, nebulizers, humidifiers and ventilators (exner et al. 2005) . the routes of transmission of waterborne pathogens include direct contact, ingestion of water, indirect contact and inhalation of bioaerosols. pseudomonas aeruginosa and legionella pneumophila are the most significant waterborne pathogens in healthcare facilities. p. aeruginosa is an environmental common microorganism. it is frequently associated with nosocomial infections, particularly among mechanically ventilated or immunocompromised patients in intensive care units. the major reservoir of p. aeruginosa is considered the patients' endogenous flora, and horizontal transmissions among patients have long been considered the most frequent source of p. aeruginosa infections. other studies have shown patient-to-patient spread via hands of healthcare workers, or via fomites. however, during the last years, the application of molecular typing methods made it possible to identify tap water supplied by intensive care units as a significant source of exogenous p. aeruginosa isolates. a review of prospective studies showed that between 14.2 and 50% of infection/colonization episodes in patients were due to genotypes found in intensive care unit water (trautmann et al. 2005) . l. pneumophila has been recognized as the first emerging waterborne pathogen transmitted by inhalation. its transmission represents a considerable risk for patients with chronic lung disease and those who undergo general anaesthesia. in hospitals, the immunosuppressive status of patients and other risk factors induce not only a higher risk of infection but also a higher incidence of lethality than in other settings. from 5 to 20% of notified legionellosis are of healthcare-associated origin (exner et al. 2005) . in healthcare settings, not only humidifiers, respiratory devices and cooling towers, but also showers and taps are specific reservoirs of legionella (joly and alary 1994; who 2007; ansi/ashrae 2015) . nontuberculous mycobacteria (ntm), even called environmental mycobacteria, are also responsible for healthcare-associated infection by inhalation route and direct contact. the structure of their cellular wall particularly rich of long-chain lipids and the ability to form biofilms contribute to their resistance to chemicals and support their persistence. indeed, ntm are frequently found in water distribution systems and can be aerosolized through showers and taps. a microbiological survey carried out by the authors confirmed ntm presence in the water plumbing of a hospital after the occurrence of some cases of atypical mycobacteriosis in a hospital wards. the ntm load ranged between 2 × 10 2 and 4 × 10 4 cfu/l and human pathogenic opportunistic nmt species (m. intracellulare, m. chelonae, m. llatzerense and m. gordonae) were found in addiction to other harmless environmental species (briancesco et al. 2014) . since the risk resulting from the presence of ntm in water is not controllable by classical water disinfection procedures, filters at the point-of-use are now recommended to be the best option for minimizing the risk. moreover, water distribution systems may be potential indoor reservoirs of moulds such as aspergillus sp., zygomycetes, fusarium sp. and other fungi. showers and taps can be the sources of risk for aerosolization of fungal spores (anaissie et al. 2003) . moulds are ubiquitous in nature and grow almost anywhere indoors or outdoors. persons can be exposed to mould through skin contact, inhalation or ingestion. because of the ubiquity of mould in the environment, some level of exposure is inevitable. inhalation is usually presumed to be the most important mechanism of exposure to viable (live) or nonviable (dead) fungi, fungal fragments or components. the majority of fungal spores have aerodynamic diameters of 2-10 µm, which are in the size range that allow particles to be deposited in the upper and lower respiratory tract. inhalation exposure to a fungal spore requires that the spore be initially aerosolized at the site of growth. in general, persons with impaired host defences suffer the most severe types of fungal infections. airborne hospital microorganisms are apparently harmless to healthy people. nevertheless, they can cause adverse health effects in immunocompromised individuals. the hospital itself and its technological systems can offer detrimental sources to the indoor air quality. air-conditioning systems and aeraulic plants can become contaminated over time and trap various contaminants such as dust and biological organisms. moisture from them can condense within the ducts and support microbial growth. thus in hospitals, special air handling and ventilation are required to prevent airborne transmission (ansi/ashrae 2016). inadequate ventilation is implicated in the airborne transmission of bacteria (obbard and fang 2003) . bioaerosol spread through the air cover in a wide size range. droplets are larger than 5 μm and their source is primarily the act of coughing, sneezing or talking. in hospitals, particular medical performances such as suctioning and bronchoscopy spread particles of this size. among droplet-transmitted infections, smallpox, measles, chickenpox, tuberculosis, meningococcal disease, pneumonia caused by mycoplasma, sars and flu are the most relevant. small particles residual from evaporated droplets (5 μm or smaller in size) and dust particles containing infectious agents may remain suspended in air for a long time. in this way, microorganisms can be dispersed widely by air currents over a longer distance from the source. the airborne transmission of infections regards only microorganisms spread in large number into the air with low infective dose. key factors influencing the level of airborne microbial burden are the occupant density and dampness depending on the particular location within the hospital. in hospital indoor air quality moulds are frequently recovered, especially during the construction/repair activities. fungal spores have low settling velocities remaining in the air for a long time. the hospitalized weakened patients are more susceptible to infections from naturally occurring mesophilic fungi, and in last decades, high mortality rates have been reported in transplant patients and leukaemia patients (taccone et al. 2015) . in a survey study that followed the occurrence of numerous post-surgery infections at a transplant centre of a hospital in rome, the levels of bacteria and fungi occurring in air and surface samples from an operating block (operating rooms, intensive care units, surgery recovery rooms and annexes corridors) were assessed. low concentrations of fungi were found in air and surface samples (ranging from 0 to 70 cfu/m 3 and from 0 to 21 cfu/cm 2 , respectively). other than numerous pathogenic opportunistic species were isolated (alternaria infectoria, alternaria tenuissima, epicoccum nigrum, purpureocillum lilacinum, cryptococcus laurentii), many other environmental opportunistic fungi belonging to the genera penicillum, aspergillus, cladosporium, mucor, stemphylium, conidiobolus and trichoderma were found. bacterial densities in bioaerosol ranged from 9 to 174 cfu/m 3 with the highest values characterizing an emergency room. staphylococcus aureus and other opportunistic staphylococcus species were isolated in many areas. several bacterial opportunistic species were also recovered (leclercia adecarboxylata, enterobacter cloacae, bacillus cereus and kokuria varians). in general, a moderate microbial pollution affected the examined surfaces with the exception of a massive bacterial density (>1 × 10 3 cfu/cm 2 ) observed on a drug carriage where pseudomonas stutzeri, opportunistic pathogenic bacteria, was isolated as prevalent microbial species (bonadonna et al. 2015) . although recommendations exist, there is a regulatory lack of a referential standard for microbiological parameters of indoor air quality in healthcare facilities because of the deficiency in the relationship between microbiological survey data and their epidemiological implications. in hospital rooms, the surfaces are frequently contaminated with pathogens able to survive for a long time on room surfaces (beds, sheets, floors, walls and furniture) and medical equipment (de oliveira and damasceno 2010; capolongo et al. 2013) . biological agents may be transmitted to the patients by personnel gloves and visitor hands or through dust that, once deposited on the surfaces, may be contaminated and then resuspended by natural convection or conditioning air systems. hospitalization in a room in which the previous patient had been colonized or infected with methicillin-resistant staphylococcus aureus (mrsa), vancomycinresistant enterococci (vre), clostridium difficile, multidrug-resistant acinetobacter, multidrug-resistant pseudomonas or yeasts as candida auris can represent an additional risk factor for the next patient admitted to the room. the most relevant nosocomial pathogens persisting on dry inanimate surfaces and the duration of their persistence are reported in how long do nosocomial pathogens persist on inanimate surfaces? a systematic review by kramer et al. (2006) . gram-negative bacteria persist longer than gram-positive bacteria. moisture improves the persistence for several types of bacteria (e.g. chlamydia trachomatis, listeria monocytogenes, salmonella typhimurium, pseudomonas aeruginosa and escherichia coli) while only staphylococcus aureus persists longer at low dampness. no health-based standards or exposure limits for indoor biological agents exist. differences in season; climatic and meteorological conditions; type, construction, age and use of the building and ventilation systems; and differences in measurement protocols used in the various studies (e.g. viable versus nonviable microorganism sampling, sampler type and analysis) make it difficult to interpret sampling data relative to information from the medical literature (alfonsi et al. 2014) . these difficulties are exacerbated in hospitals where the patient health status, the activities that take place and the potential spread of pathogenic biological agents increase the level of complexity respect to other indoor environments. moreover, the global burden of healthcare-associated infection is unknown because of the difficulty of gathering reliable diagnostic data. the definition of the role that the environment has on the acquisition of hospital infections is highlighted by the need for multiple strategies to control the dissemination of pathogenic microorganisms and the adoption of prevention measures. because nearly 10 6 skin flakes containing viable microorganisms are shed daily from normal skin, it is not unexpected that patients-through gowns, bed linen, bedside furniture and other objects close to them-become contaminated with other patient flora. the clarification of the role that surfaces have in the spread of infections could provide support to increase adherence to control measures. improving and intensifying the cleaning routine may reduce the dissemination of pathogens. more attention should be given to the adequacy of the length, the frequency and specific care when cleaning surfaces, because removing dirt helps to reduce biofilms. the spread of pathogens could be prevented by using engineering and environment control strategies. thus, in addiction to cleaning and disinfection standard procedures, the maintenance of appropriate hygienic targets may be obtained by employment of durable antimicrobial materials, such as copper and copper alloys (brasses and bronzes), especially for high-touch surfaces. antimicrobial copper touch surfaces can lower the number of microbes on surfaces, reducing the risk and preventing the transfer of antibiotic resistance between bacterial species (michels et al. 2015; gião et al. 2015) . cause of microbial biocide multiresistance issue, in recent years new sanitation procedures, based on the use of probiotic products, have been studied. this technique connoted as biostabilization is based on the principle of competitive microbial exclusion and does not imply a biocidal action. surfaces sanitizing probiotic products containing vegetative and spore forms of bacillus species, in association with good hygienic practices, seem to provide 80-90% reduction of pathogenic agents and more than 60% reduction of infection events (mazzacane et al. 2014; caselli et al. 2016) . in order to avoid infections caused by airborne microorganisms, it is very important to maintain protective barriers that control the microbiological quality of the air. for aerosolized waterborne pathogens, faucets are easily accessible for preventive measures, and the installation of single-use filters on hospital water outlets appears to be an effective concept to reduce water-to-patient transmissions of nosocomial pathogens. infection control programs have been defined by who and the centres for disease control. the improvement of the surveillance systems for hospital infections and the implementation of standard procedures for reduction of microbial spread represent the main commitments (who 2011; cdc 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investigators ecology of pseudomonas aeruginosa in the intensive care unit and the evolving role of water outlets as a reservoir of the organism legionella and the prevention of legionellosis. copenhagen: who report on the burden of endemic health care-associated infection worldwide. copenhagen: who key: cord-019051-gtruu1op authors: weber, olaf title: the role of viruses in the etiology and pathogenesis of common cold date: 2009-11-10 journal: common cold doi: 10.1007/978-3-7643-9912-2_5 sha: doc_id: 19051 cord_uid: gtruu1op numerous viruses are able to cause respiratory tract infections. with the availability of new molecular techniques, the number of pathogens detected in specimens from the human respiratory tract has increased. some of these viral infections have the potential to lead to severe systemic disease. other viruses are limited to playing a role in the pathogenesis of the common cold syndrome. this chapter focuses on the viral pathogens that are linked to common cold. it is not the intention to comprehensively review all the viruses that are able to cause respiratory tract infections—this would go beyond the scope of this book. the list of viruses that are briefly reviewed here includes rhinoviruses, respiratory syncytial virus, parainfluenza virus, adenovirus, metapneumovirus and coronavirus. bocavirus is discussed as one example of a newly identified pathogen with a less established role in the etiology and pathogenesis of common cold. influenza virus does not cause what is defined as common cold. however, influenza viruses are associated with respiratory disease and the clinical picture of mild influenza and common cold frequently overlaps. therefore, influenza virus has been included in this chapter. it is important to note that a number of viruses are frequently co-detected with other viruses in humans with respiratory diseases. therefore, the viral etiology and the role of viruses in the pathogenesis of common cold is complex, and numberous questions remain to be answered. numerous viruses are able to cause respiratory tract infections. some of these may also cause severe diseases. others are limited to a role in the pathogenesis of the common cold syndrome. with the availability of new molecular techniques, the number of pathogens detected in specimens from the human respiratory tract has increased. the association of some of these agents with human respiratory disease is not always clear. it is not the intention of this chapter to comprehensively review the virology of all the viruses that cause or potentially cause respiratory tract infections. the chap-ter focuses on some pathogens that are linked to common cold. the list of viruses described includes rhinovirus, respiratory syncytial virus, parainfluenza virus, adenovirus, metapneumovirus and coronavirus. bocavirus is discussed as an example of a newly identified pathogen with a less established role in the etiology and pathogenesis of common cold. influenza virus does not cause what is defined as common cold, but is associated with respiratory disease and the clinical picture of mild influenza frequently overlaps with that of the common cold. therefore, influenza viruses are briefly described in this chapter. for details on the biology of the individual viruses and their role in pathogenesis of respiratory diseases further reading of standard literature and text books of virology is recommended. viruses with an established role in common cold are rhinoviruses, adenoviruses, parainfluenza viruses, coronaviruses and the respiratory syncytial virus, and these are reviewed in greater detail here. their structure and replication, the transmission and epidemiology and the clinical symptoms are described. in addition, some brief comments about current models of pathogenesis and animal models, respectively, complete the respective subchapters. table 1 provides an overview of the viruses that cause respiratory tract infections and that do, or may, play a role as a cause or in the pathogenesis of common cold. a number of viruses are frequently co-detected with other viruses in humans with respiratory diseases. therefore, the viral etiology and the role of viruses in the pathogenesis of common cold is complex and it is safe to say, not fully understood for each and every virus that is linked to respiratory tract infection. recent developments in the field of antivirals are described in the chapter by tom jefferson in this book. rhinoviruses cause the vast majority of the common colds in humans. although the infection usually is self limiting and the symptoms of the disease are mild in healthy adults, rhinovirus infections may cause serious illness in children or patients with pre-existing medical problems [1] . taxonomy, structure and replication rhinoviruses more than 100 serotypes, strains and isolates of rv have been isolated from humans. two human rv species have been described: human rhinovirus (hrv) a and b. eighteen serotypes and 2 subtypes (hrv 1a and 1b) belong to hrv a. five serotypes are assigned to hrv b and 82 serotypes are not yet assigned to a species including bovine rhinoviruses (brv) 1-3. rv are non-enveloped viruses with an icosahedral symmetry. the virus is small and has a diameter of approximately 30 nm. four capsid proteins, vp1-4 have been described. a protomer is composed of one copy of vp1, vp3 and vp0 (a precursor where vp4 and vp2 are covalently linked). cleavage of vp0 is the final step of the assembly process [3] . one or two copies of vp0 will remain uncleaved; no role for this uncleaved vp0 has been established yet. five protomers are arranged symmetrically about a fivefold axis, forming a pentamer that represents a corner of the icosahedron. the capsid is formed by 12 pentamers. rv have, similar to human enteroviruses, the same comparatively uneven surface with its characteristic canyon around the fivefold axis [4] . the canyon serves as an attachment site for the cell receptor [5] . in cscl, rv have a buoyant density of 1.38-1.42 g/ cm 3 . virions are unstable at a ph below 5-6, a feature, which distinguishes rv from other enteroviruses. however, as they are non-enveloped viruses, rv are stable against detergents and most organic solvents. on the other hand, alcohol and phenol are effective virucidal agents. the rv genome is organized as a single-stranded positive-sense rna of approximately 7100-7200 nucleotides in length with its 5' terminus covalently linked to a small protein, vpg. the 5'-untranslated region (utr) of approximately 0.65 kb is shorter than that of other enteroviruses, owing to a deletion of approximately 100 nucleotides between the internal ribosomal entry site (ires) and the translation start site. one open reading frame (orf) of about 2150 codons, a 3'-utr of approximately 40 nucleotides and a 3' poly(a) tail complete the structure of the genome. rv have a characteristic nucleotide composition with a preponderance of a and u, particularly in the third position of the codons. the genome is fully sequenced and has the accession number replication is initiated through attachment to the cell receptor. for most rv serotypes this is intercellular adhesion molecule-1 (icam-1) [6, 7] . it is hypothesized that the viral canyon structure releases a lipid moiety upon icam-1 binding, which, in turn, leads to a change in conformation, destabilization and the release of the viral rna into the cytoplasm [8] . rv shut off host cell protein synthesis by inactivating the cap binding complex. their ires allows them to replicate despite this inactivation. the rna serves as a messenger rna encoding a single polyprotein which is cleaved post-translationally by virus-encoded proteases. once the first round of translation and subsequent processing is complete a 3d pol rna-dependent rna polymerase produces negative-sense rna from the genomic template, which in turn serve as template for the production of positive-sense genomic rna. the synthesis of a single virus polyprotein requires post-translational processing to facilitate subsequent steps in viral replication. at least two proteolytic activities are encoded by the virus: 2a pro performs the first cleavage releasing capsid precursors and 3c pro catalyzes most other cleavage reactions. the protease has a trypsin-like structure but the active site of the enzyme is a cysteine sulfydryl [9] . rv are transmitted mainly by direct contact and less frequently through aerosols (for details see the chapter by diane pappas and owen hendley). virus can frequently be isolated from the hands of an infected individual and is transmitted to other individuals or to objects in the environment. during rv infection virus titers in nasal secretions are as high as 10 2 -10 3 tcid 50 /ml of nasal lavage fluid [10, 11] . infection of humans is very effective and less rv may be needed for infection of seronegative volunteers by nasal drops than needed for infection of a human embryonic fibroblast tissue culture [12] . in contrast, when the same virus was used for inhalation of aerosols a 20-fold disparity in infectious dose has been described [13] , suggesting that the lower respiratory tract is less susceptible for infection than the nasopharynx. after a short incubation period of 1-4 days virus is shed, peaks after another 2-3 days, and declines thereafter [13, 14] . the primary site of viral replication is ciliated epithelial cells as detected by in situ hybridization [15] . the histopathology of rv infection is not as yet very detailed. nasal mucosa biopsies reveal only few or no histopathological abnormalities despite active virus shedding. explant cultures inoculated with rhinovirus failed to develop cytopathic effects (cpe) [16] . biopsies showed marked edema of connective tissue, sparse infiltration of inflammatory cells, hyperemia and exudation of seromucous fluids [17] [18] [19] . infection of bovine tracheal organ cultures with brv leads to shedding of ciliated cells. it has been suggested that the immune response of the host contributes to the symptom complex. increased concentrations of the pro-inflammatory cytokines il-8, il-1 and il-6 [20] [21] [22] have been found in nasal secretions of subjects with symptomatic rv infection. a direct correlation between concentration in nasal fluids and symptom severity has been described for il-6 [22] . rv infection is usually accompanied by the typical common cold symptoms: nasal discharge and obstruction, sneezing, coughing, sore throat and, less frequently, fever. gastrointestinal symptoms are sometimes observed in children. the infection is usually limited to the upper respiratory tract. it is commonly believed that rv infection increases the risk for subsequent or secondary bacterial infections. in patients predisposed with existing under-lying diseases, like cystic fibrosis or chronic bronchitis, and in immunocompromised patients, elderly and infants, rv can cause serious infections of the lower respiratory tract. rv infection of the lower respiratory tract (lrti) was demonstrated by papadopoulos and co-workers [23] using in situ hybridization techniques. these authors demonstrated rv infection not only in epithelial cells but also in underlying submucosal cells. exacerbation of chronic bronchitis or asthma may be a consequence in these patients [24, 25] . indeed, approximately 80% of asthma exacerbations in children [26] and about 70% in adults [27] are associated with respiratory virus infections, and the vast majority of these are rv infections [28] . examination of the early innate immune responses to rv infection in asthmatic bronchial epithelia revealed profound impairment of virus-induced interferon (ifn)-expression leading to impaired apoptotic responses and enhanced rv replication [29] . rv infections lead to the production of type-specific iga, igg and igm antibodies. however, frequencies of response to natural infection have been reported to vary between 37% and 92% [30] . infected patients usually develop neutralizing antibodies to the infecting virus within 1-3 weeks after infection. iga is the dominant immunoglobulin in nasal secretions, has a protective role and may prevent re-infections with homotypic viruses or reduce the symptoms upon reinfection. the involvement in the viral clearance process is less clear and other mechanisms like the induction of an innate immune response are being discussed. both serum and secretory antibodies persist for several years after infection. in the absence of effective antiviral treatments, the diagnosis of rv infection for guiding an anti-rv therapy is not useful. the diagnosis of rv infection largely relies on the clinical symptoms. approximately 60-80% of the patients with a common cold syndrome of afebrile prominent nasal symptoms but minimal systemic disease that occurs between august and early november have rv infection [31] . however, the general method for identifying the etiological agent is the isolation and propagation in cell culture. in addition, polymerase chain reaction (pcr) can be used for rapid identification of rv in specimens. point-of-care diagnostics are under development. a major obstacle to understanding disease pathogenesis has been the lack of a small-animal model for rv infection. rv have shown a high degree of species specificity, limiting the use of experimental animal systems. therefore, aspects of pathogenesis have been studied in experimentally induced colds in human volunteers. infection of rabbits, guinea pigs or weanling mice by parenteral routes was not successful with certain strains of the virus [32] [33] [34] , but infection of mice was possible using a tissue-culture adapted hrv-2 [35] . chimpanzees or gibbons has been experimentally infected using specific strains of hrv [36, 37] . approximately 90% of the rv use human icam-1 as their cell receptor and do not bind mouse icam-1; the remaining 10% use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. recently, three novel mouse models of rv infection: minor-group rv infection of balb/c mice, major-group rv infection of transgenic balb/c mice expressing a mouse-human icam-1 chimera and rv-induced exacerbation of allergic airway inflammation were described by bartlett et al. [38] . these models have features similar to those observed in rv infection in humans, including augmentation of allergic airway inflammation, and may be useful in the development of future therapies for colds and asthma exacerbations. association between common cold symptoms and inflammatory mediators is an important aspect of understanding common cold and rv infection. although this association seems obvious, the exact mechanisms are less clear and one might expect a better understanding of the detailed mechanism(s) once inhibitors of viral replication are available for studies in humans. in addition, the new animal models developed by bartlett et al. [38] can be expected to support efforts to study pathogenesis of rv infection in vivo in greater detail. human respiratory syncytial virus (rsv) was first isolated from a laboratory chimpanzee with upper respiratory tract infection (urti) in 1956 [39] . rsv is today recognized as the leading viral agent in upper respiratory tract disease in infancy and childhood. the spectrum of rsv-caused diseases includes rhinitis, otitis media, pneumonia and bronchiolitis. the latter two diseases can be associated with a substantial morbidity and mortality. in addition, there is growing recognition for its importance as a causative agent for diseases in elderly and immunocompromised patients [40] . the world health organization estimates that rsv causes 64 million infections and 160 000 deaths annually [41] . a bovine rsv (brsv) has been described causing economically important respiratory diseases in cattle [42] . another animal rsv is the pneumonia virus of mice (pvm) [43] , suggesting that there was an interspecies spread in the evolution of these viruses. however, an animal reservoir for human rsv has not been described so far [44] . despite the importance of rsv as a leading cause for respiratory diseases, the pathogenesis of rsv infection is not fully understood and efficacious vaccines are not available. rsv is a member of the order mononegavirales, which includes several non-segmented negative-strand rna viruses. rsv is a member of the family of paramyxoviridae, subfamily pneumovirinae and represents the type species for the genus pneumovirus [43] . rsv virions consist of an envelope and a nucleocapsid. the viral gene expression and nucleic acid replication occur in the cytoplasm. the envelope is acquired by cell budding. virions are spherical to pleomorphic; filamentous and other forms are common. they measure 150-300 nm in diameter and up to 1000-10 000 nm in length [45] . the surface of the virion is covered by projections (spikes) formed by fusion (f) glycoproteins. the spikes are 11-20 nm long and are spaced 6-10 nm apart; they mediate attachment and penetration. the helical nucleocapsid is filamentous with a length of 600-800(1000) nm and a width of 12-15 nm [40] . the nucleocapsid does not enter the cells by surface fusion typical for paramyxoviruses but rather by membrane fusion which may involve clathrin-mediated endocytosis [46] . the unsegmented genome contains a single molecule of linear negativesense, single-stranded rna. virions occasionally contain a positive-sense single-stranded copy of the genome (partial self-annealing of extracted rna may occur). the complete genome is approximately 15 300 nucleotides long and fully sequenced. the genome has the accession number(s) [d00386] -[d00397] [43] . the rna genome has a 3'-extragenic leader region, followed by the ten viral genes and a 5' trailer region. each gene is transcribed into a separate mrna encoding for a single viral protein with the exception of the m2 mrna. this contains two overlapping orf, expressed by a ribosomal stop-restart mechanism into two proteins, m2-1 and m2-2 [47] . although gene expression is consistent with that of other members of the order of mononegavirales m2-1 and m2-2 have some regulatory features unique to rsv [44] . the five nucleocapsid-associated proteins are the n (nucleocapsid) protein, the phosphoprotein p (co-factor for rna synthesis), the l protein (large, a 2165-amino acid subunit of viral polymerase), the m2-1 (transcription processivity factor) and the m2-2 protein (which possesses regulatory functions) [44, 48] . the n protein binds the genomic and the antigenomic (positive-sense intermediate) rna and protects is against degradation. in addition, it reduces the detection and responses by the host's immune system (for instance toll-like receptors, tlrs) and intracellular rna recognition helicases, which initiate innate immune responses [44, 49, 50] . the viral envelope is formed by four rsv proteins that associate with the lipid bilayer: a matrix (m) protein that is located at the inner surface and is important for the assembly of the virion [51] , a glycosylated (g) protein, a fusion (f) protein and a small hydrophobic (sh) protein. two other rsv proteins, the ns1 and ns2 proteins are a minor part of the virion [44] . ns1 and ns2 are thought to modulate the host response to rsv infection. the g glycoprotein (~90 kda) has a peptide backbone with 24-25 side chains and is important for viral attachment to the host cell [52] . a second secretory form of the g protein exists that arises from a second initiation codon in the g orf. proteolytic trimming removes additional amino acids, and the final protein lacks the 65 n-terminal residues, including the membrane anchor [44, 52] . the ectodomain of the g protein has a mucin-like structure that differs from attachment proteins of other paramyxovirus. its function is not clear but it is thought to contribute to virus spread or to prevent trapping by mucus [44] . the f protein has two distinct functions: penetration into the host cell by membrane fusion and a syncytia-forming property. the f protein matures by activation through a furin-like intracellular protease that cleaves the precursor, f 0 into three fragments, f 1 , f 2 and p27. f 1 and f 2 are linked by a disulfide bond and represent the active form of f [53] . the hydrophobic n terminus of f 1 is conserved within the rsv and it is thought that this domain inserts into the host cell membrane when fusion occurs [44] . ns1 and ns2 are thought to modulate the host's immune response to rsv [44] . lack of m2-1 results in reduced expression of ns1 and ns2, and it is thought that this down-regulation of the host-defense antagonists may help to facilitate persistent rsv infection [44] . infection occurs through direct contact, through large respiratory droplets and, to a lesser extent, through small droplets. the site of the first replication is the nasopharynx. after an incubation period of 4-5 days the virus spreads to the lower respiratory tract [54, 55] . the clinical signs include cough, rhinitis, fever and signs of bronchiolitis like air trapping, wheezing and increased airway resistance. the most prominent clinical symptoms are cough and rhinorrhea, which occur in approximately 90% of primary rsv infections in infants and to some lesser extent in reinfected adults [56] . fever occurs in 30-40% of both infected infants and adults and otitis media is reported in approximately 20% of infected infants. ear and sinus pain is reported in 20-30% of infected adults. symptoms of lrti, including bronchiolitis, pneumonia, croup, wheeze and tracheobronchitis, are observed in 30-40% of infected infants and to some lesser extent in adults, with wheeze and tracheobronchitis being the most prominent disease symptoms. hospitalization is necessary in approximately 3% of infected children and below 0.1% of infected adults. rsv is the single most important agent in children younger than 3 years of age. importantly, children with a mild rsv disease have also been reported to have recurrent wheezing for up to 10 years after the primary acute disease [57] . extrapulmonary dissemination may occur in immunocompromised patients [58] . the virus may spread to kidneys, liver, the central nervous system and the heart. virus can be isolated from the nasopharynx of children for up to 14 days. in immunocompromised patients, virus recovery is possible for up to 1 month or even longer. in immunocompetent individuals the viral infection is usually restricted to the superficial cells of the epithelia and viral spread outside the respiratory tract is uncommon [54, 59] . an exception, however, is the middle ear: the virus frequently causes otitis media [60] . the typical pathological findings in rsv-infected tissue include epithelial necrosis and infiltrates of monocytes, t cells and neutrophils [61] . airways appear obstructed due to sloughed cells, mucus secretion, proliferation of bronchoalveolar epithelium or cellular infiltration. formation of syncytia in the bronchoalveolar epithelium is sometimes observed [61] . however, giant cell pneumonia or syncytia formation are related to severe t cell-deficient patients [44] . specific host factors that may influence the clinical signs and outcome of rsv infection including the general health status, the nutritional status [56, 62, 63] , gender, ethnic group, levels of maternal antibodies [64] , age of first rsv infection [65] and underlying cardiac or pulmonary diseases [66] . in addition, there are several environmental factors (e.g., tobacco use in household, stress, day care) that may influence the course of the disease or severity of symptoms. the role of inflammation and a bias of the host's immune response towards a humoral th2 response (the typical cytokines are il-4, il-5, il-10 and il-13) are discussed controversially in the literature. a strong inflammatory response does not seem to be determinative for the severity of clinical symptoms [67] , a finding supported by the fact that in many clinical studies patients receiving anti-inflammatory therapy did not significantly benefit from that treatment [68] . however, strong inflammatory responses also have been suggested to enhance the severity of clinical symptoms in rsv disease. the role of inflammatory chemokines in rsv pathogenesis has been supported by many preclinical and clinical studies (reviewed in [44] ). for example, genetic polymorphisms that increase il-8, a major chemoattractant for neutrophils, and ccr5 expression have been associated with increased rsv disease [69] . a link between an increased ratio of th2/th1 immune response (a bias toward the humoral vs the classical cytotoxic response) has been suggested by several authors. this discussion is based on evidence for elevated th2/th1 response ratios in clinical studies, the role of key th2 cytokines in the pathogenesis of asthma and the experience with a formalin-inactivated rsv vaccine in the 1960s [70] [71] [72] . this vaccine was poorly protective and vaccinated children and infants developed dramatically enhanced disease compared to naive patients upon natural rsv reinfection [48] . subsequent preclinical studies confirmed a bias toward a th2-specific cd4 + t cell response in animals treated with formalin-inactivated rsv vaccine vs naturally infected animals [73] . il-4 and il-13 support isotype switching to ige, which is bound to mast cells and eosinophils and, upon antigen contact, induces release of inflammatory mediators like histamine or leukotrienes by these cells. these mediators contribute to the development of the typical clinical symptoms associated with rsv disease. several viral proteins play an important role in the pathogenesis of rsv disease (reviewed in [44] ). the soluble g protein has been suggested to modulate the innate immune response by down-regulating inflammatory mediators such as il-6 or il-8 in epithelial cells as a response to rsv infection [74] . g protein also modulates inflammatory responses of monocytes by acting as a general antagonist for tlr activity [75] . the g-mediated suppression of tlr-4 signaling appears to be counteracted by the f protein, which has been described to induce signaling through this tlr [76] , although the significance of this activity is unclear. importantly, rsv infection can block the maturation of dendritic cells (dc), which serve as major antigen-presenting cells (reviewed in [44] ). this alteration of dc biology may support the shift of the th2/th1 balance towards th2, reduce antiviral interferon activity and limit the mobility of mature antigen-presenting cells, thus qualitatively altering the immune response to rsv infection (reviewed in [44] ). in summary, there are several host factors or viral factors that play roles in the pathogenesis of rsv infection. the picture, however, is highly complex and relative contributions of the various factors to rsv pathogenesis are not entirely understood. as mentioned above rsv is a leading cause of respiratory diseases in children and has increasing importance as a causative agent for respiratory diseases in elderly. in a prospective study of infants and children in the unites states, rsv was detected in 43% of pediatric hospitalizations for bronchiolitis, 25% for pneumonia, 11% for bronchitis and 10% for croup [54] . approximately 90% of infants have been infected at least once by 2 years of age [44, 77] . although rsv is represented by one serotype, a protective immunity against rsv is generally weak and reinfection occurs. virus-neutralizing antibodies, including secretory iga found in the respiratory tract, contribute to viral clearance and may play a role in protection against reinfection [54] . however, the iga response is short [44, 54] . in the lower respiratory tract, the igg response has been described as more efficient. the role of antibodies as a down-modulator of clinical symptoms has been confirmed by the clinical experience with palivizumab. in temperate regions, rsv circulates quickly during winter/early spring, but timing varies more elsewhere. effective vaccines are not available and effective therapies are not available. however, an rsv-neutralizing human-ized monoclonal antibody, palivizumab, reduces rsv-associated hospitalization if used as a passive immunoprophylaxis [78] . rsv infection is assumed to be frequently misdiagnosed, particularly in adults [56] , because the symptoms are similar to those caused by other respiratory viruses like influenza. laboratory diagnostic tests are usually performed on secretion samples obtained from the nasopharynx. novel rapid elisa-based or rt-pcr-based tests are useful particularly in a hospital setting to identify outbreaks, prevent further transmission, initiate therapies or reduce inappropriate use of antibiotics [56] . animal models are comprehensively reviewed by moore and stokes peebles [79] . rsv is species specific; however, some animal species exhibit semipermissive infection with rsv. chimpanzees were productively infected with rsv and exhibited upper respiratory tract illness, whereas adult squirrel monkeys, newborn rhesus monkeys, and infant cebus monkeys did not show symptoms but shed low levels of virus [80] . bonnet monkeys, which are more widely available than chimpanzees, can be infected with rsv [81] . non-human primate models of rsv infection, especially chimpanzee, have advantages but certainly limitations associated with the high cost and genetic variability, which limits the reproducibility of results. the cotton rat which is susceptible to both urti and lrti with rsv is seen as one of the best animal models of rsv infection and disease [79, 82] . in these animals, rsv infection led to histologically confirmed proliferative rhinitis, bronchiolitis, and the pulmonary infiltration of lymphocytes and neutrophils [82] [83] [84] . the cotton rat model was used to study the mechanism of antibody-mediated clearance of rsv [84] . the advantages of mouse models are obvious: they are inexpensive, inbred strains are available and a wealth of reagents (e.g., antibodies), arrays, probes or information (e.g., the genome sequence) is available. although there is variability between the strains regarding susceptibility and viral load, viral load does not vary much within the strains [79] . balb/c is the most widely used inbred mouse strain to study rsv infection [79] . rsv-infected balb/c mice show signs of clinical illness including weight loss, ruffled fur and ataxia peaking at day 8 after infection [85] . interestingly, the susceptibility to rsv replication in nose and lung increased with age. the predominant histological findings in rsv-infected 3-week-old balb/c mice were peribronchiolar and perivascular accumulations of mononuclear cells [86] . the role of th1 and th2 cell response to rsv infection has been the dominant focus of the balb/c mouse studies in this context [79] . rsv infection induces a th1 response dominated by high ifn-levels in the lungs of infected mice, abundant ifn--producing cells in the bronchoalveo-lar lavage fluid (balf) and rsv-specific cytotoxic t cell (ctl) response [87, 88] . stat1 (-/-) mice with a balb/c background have been described as having an excellent rsv disease phenotype (reviewed in [79] ). although this model has the limitation that it probably does not exactly mirror the complexity of a natural infection in humans, it is regarded as an attractive tool to study rsv pathogenesis and evaluating novel therapies. in addition to other mouse models and the infection in chinchillas [89] , infection of natural hosts has been studied in detail. bovine respiratory syncytial virus is a major cause of respiratory illness in calves and has been studied in this context. the clinical signs after experimental infection include cough, lung sound, dyspnea, fever, increased respiratory rate and pulmonary resistance. prominent histological findings include proliferative bronchiolitis, alveolitis, syncytia, and, to some extent, emphysema [90] . rsv infection of calves might be useful for evaluating vaccination strategies; however, it is not an animal model for the evaluation of novel therapies. the list of experimental models also includes pneumonia virus infection of mice (pvm) [91] . although the pvm model of respiratory disease is interesting because it shows the phenotype of a natural infection, pvm differs from rsv including the g and the ns1 proteins that fulfill important functions during rsv infection. human parainfluenza viruses (hpiv) are important causes of respiratory diseases in infants and children. they usually cause urti of which 30-50% may be accompanied by otitis media. hpiv may also cause lrti, about 0.3% of which require hospitalization. hpiv1-3 infections are second to rsv infections as the viral cause of serious acute respiratory infections in young children that occur primarily in the first 6 months of life [92, 93] . hpiv3 may cause severe diseases. approximately 80% of infants and children infected with hpiv3 developed febrile illness and one third of these infected individuals developed lrti, resulting in bronchitis or pneumonia [93] [94] [95] . most children have been infected with hpiv3 by the age of 2 years. croup is the main clinical manifestation of infection with parainfluenza viruses, especially hpiv1 and 2, and these infections may extend to the lower respiratory tract and result in pneumonia [94] . hpiv4 mainly causes mild urti in children and adults [93] . along with rsv, hpiv are also a leading causative agent of serious acute respiratory infections and community-acquired respiratory disease requiring hospitalization in adults. the proportions of hospitalizations associated with hpiv infection vary widely in hospital-based studies. according to the who, hpiv1 is estimated to account for 5800-28 900 annual hospitalizations in the usa, hpiv2 for 1800-15 600 hospitalizations, and hpiv3 for 8700-52 000 hospitalizations [92] . parainfluenza viruses belong to the order of mononegavirales, family paramyxoviridae, subfamily paramyxovirinae. hpiv1 and 3 belong to the genus respirovirus, and hpiv2 and 4 to the genus rubulavirus [96] . the virions are spherical enveloped particles of approximately 150-250 nm in diameter with an internal helical nucleocapsid. virions are enveloped by a lipid bilayer membrane that bears spike-like projections composed of hemagglutinin-neuraminidase (hn) and fusion (f) protein [97] . as for other paramyxoviruses, all hpivs contain a negative strand, ~15 500-nucleotide-long non-segmented rna genome [93] encoding two envelope glycoproteins, the hn, and the f protein, a matrix protein (m), a nucleocapsid protein (np) and several nonstructural proteins including a polymerase-associated protein (p/v) and the viral replicase (l) (reviewed in [93] ). the replication of piv is similar to that of other paramyxoviruses with the rna genome serving as a template for the transcription of the mrnas. binding of the hn glycoprotein to its cell receptor initiates the infection [98] . in addition to having this function, hn is thought to enhance the fusion activity of f, which, following virus attachment to the host cell, mediates the fusion of virus and subsequent penetration. f also mediates fusion of infected and uninfected cells, allowing virus to spread. f is synthesized as an inactive precursor (f 0 ) that is post-translationally cleaved by a host cell protease to yield two subunits, f 1 and f 2 that remain linked by a disulfide bond [93] . the mucous membranes of the upper respiratory tract are the common sites of infection. prominent clinical symptoms of hpiv infection can be characterized by rhinitis, pharyngitis and bronchitis. the incubation period is about 4 days [95] . coughing, hoarseness and fever that last for approximately 2-3 days are frequent. involvement of the trachea results in croup and extension to the lower respiratory tract may lead to pneumonia. severe disease characterized as bronchopneumonia or bronchiolitis has been observed with hpiv3 infections [93] [94] [95] . specific virus and host properties that determine the severity of hpivrelated disease are not yet understood. infection with piv induces an immune response to hn and f. neutralizing antibodies to piv correlate with partial resistance to infection or clinical symptoms but usually do not prevent re-infection [99] . secretory iga neutralizing antibodies are more important in adults than in children [100] . as mentioned above, the hpivs are important causes of respiratory tract diseases in infants and children. epidemics of hpiv3 usually occur in the early spring [101] . viruses do not persist for a long time in the environment [93] . the seasonal peak of hpiv1 and 2 infections is reflected in the seasonality of croup, which is the highest in autumn in the usa [102] . croup during the winter months is more likely to be caused by other viruses, such as influenza virus or rsv [93] . the diagnosis of piv infection is mainly clinical, and molecular diagnostic procedures are usually not performed. there is no specific antiviral treatment against piv available, and therapeutic intervention is mainly targeted against symptoms of croup. early treatment will reduce the severity of the symptoms, the rates at which patients return to a health care practitioner for additional medical attention, visits to the emergency department, and admission to the hospital [103] . effective vaccines are currently not available. attenuated strains have been studied and a virosomal formulation of an hpiv3 vaccine is currently under development [92] . the national institute of allergy and infectious diseases (niaid) is studying the safety and immunogenicity of a recombinant live-attenuated chimeric bovine/human parainfluenza type 3 virus, rb/ hpiv3, vaccine in a phase i study. the test vaccine is delivered as nose drops to adults 18-49 years of age, hpiv3-seropositive children 15-59 months of age, and hpiv3-seronegative infants and children 6-36 months of age [104] . hpiv vaccines are also developed by some companies [105]. adenoviruses cause infections of the respiratory and gastrointestinal tract, kidney, eye and other organs, the latter mostly as a consequence of immunosuppression [106] . they are known to frequently cause respiratory infections among people in institutional environments -outbreaks among children are reported at boarding schools and summer camps [107] . outbreaks have also been reported in military camps [108] . most infections with adenovirus result in infections of the upper respiratory tract. in addition, adenovirus infections may result in conjunctivitis, tonsillitis, ear infection or croup [106] [107] [108] [109] . adenoviruses are responsible for approximately 5% of acute respiratory infections in children under the age of 5 [107, 109] adenoviruses belong to the family adenoviridae, genus mastadenovirus. there are 6 species including human adenovirus a-f with 51 immunologically distinct human adenovirus serotypes [110] . the most common human adenoviral pathogens belong to the c adenoviruses and those mainly infect the upper respiratory tract [107] . the virions are not enveloped. they consist of a capsid and a core with proteins associated to it. the icosahedral capsid has a diameter of 70-100 nm [111, 112] . all capsids consist of 252 capsomers. the surface structure reveals a regular pattern with distinctive features. surface projections are often lost during preparation. distinct filaments protrude from the 12 vertices/ pentons [112] [113] [114] . the genome is not segmented and contains a single molecule of linear double-stranded dna with terminally redundant sequences, which have inverted terminal repetitions (itr). the complete genome of mastadenoviruses is approximately 31-36 kpb long and has a guanine + cytosine content of 48-61%. the genome has a terminal protein, which is covalently linked to the 5'-end of each dna strand [114, 115] . the viral genome encodes structural proteins and non-structural proteins. virions consist of 11 proteins located in the capsid, fibers, and core. the capsid is comprised of seven polypeptides, polypeptide ii which is the basis for the hexon (three tightly associated proteins). polypeptides vi, viii, ix are associated with the hexon, polypeptides vi and viii serve as bridge between the capsid and the core. five polypeptide iii copies are the basis for the penton. polypeptide iv forms the trimeric fiber [116] , which has a knob domain that serves as a viral receptor for the target host cell. the cell receptor is the receptor for coxsackie b virus and adenovirus (car) for adenoviruses a, c, d, e and f and cd46 for adenoviruses b with the exception of serotypes 3 and 7 [117] . the core consists of four proteins (v, vii, mu and the terminal protein, which is covalently linked to the 5´ end of the dna) and the dna (reviewed in [112] ). the replication cycle of adenoviruses is divided into two phases. the early phase includes adsorption of the virus to the host cell, penetration, transcription and translation of early genes. the early gene products mediate gene expression and dna replication, block apoptosis and promote the cell cycle progression. in addition, they possess potent immunomodulatory functions (reviewed in [112] ). the viral e1a gene product should briefly be mentioned: in the nucleus, e1a activates the expression of a number of genes by interacting with cellular transcription factors and other cellular regulatory proteins [112] . e1a has been postulated to play role in the pathogenesis of chronic obstructive pulmonary disease (copd) [118] [119] [120] . human adenovirus a-f can cause human infections ranging from respiratory disease, and conjunctivitis (b and d), to gastroenteritis (f serotypes 40 and 41) [106, 112] . the most common clinical picture after adenovirus infection of the respiratory tract is mild self-limiting upper respiratory disease with nasal congestion, coryza and cough [106, 113] . some patients develop exsudative tonsillitis that is clinically indistinguishable from streptococcus tonsillitis [120] . these infections are commonly caused by serotype 1, 2, 5 and 6 c adenoviruses and serotype 3 b adenovirus [107, 109] . respiratory symptoms may be accompanied by systemic manifestations including generalized malaise, chills, fever and headache [106] . however, adenoviruses may infect alveolar and bronchiolar epithelial cells [121] and cause pneumonia, bronchiolitis or bronchiolitis obliterans [122] [123] [124] . adenoviruses account for approximately 10% of pneumonias in children [106] . in contrast to many other respiratory viruses, both persistent and latent infections have been described, particularly in lymphocytes [106] . adenoviral dna may persist in the nuclei of infected cells or even integrate into the host dna. adenoviral e1a protein has been postulated to play a role in the pathogenesis of copd [121] . adenoviral dna was found in the lungs of copd patients and expression of e1a correlated with disease severity [125] [126] [127] . in response to inflammatory stimuli, e1a increases icam-1 and il-8 expression along with nuclear factor-b (nf-b) activation in lung epithelial cells ( [128] , reviewed in [113] ). while these factors support emphysema, e1a up-regulates transforming growth factor-1 (tgf-1) in bronchiolar epithelial cells [129] , supporting a role for e1a in airway remodeling [130] . in summary, adenoviruses are pathogens that frequently cause mild or severe acute infections of the respiratory tract. the importance of adenovirus infections, however, goes beyond acute airway disease. adenoviruses are non-enveloped pathogens and thus very stable to chemical or physical agents and adverse ph conditions. it is believed that respiratory adenoviruses are mainly spread via aerosols; however, other routes (fecal, waterborne) also frequently lead to infection. antibodies to one or more adenoviruses are found in approximately 50% of infants and nearly 100% of adults and since there are many different types of adenovirus, repeated adenoviral infections can occur [109, 131, 132] . although adenovirus infections can occur at any time of the year, respiratory tract disease caused by adenovirus is more common in late winter, spring, and early summer [106] . virological diagnosis can be performed using a variety of molecular or immunological approaches. this is, however, only important in the context of severe or epidemic diseases. development efforts for vaccines were discontinued [133] . antiviral therapy is only important in infected immunocompromised patients. in these patients, cidofovir has shown some promise [122] . human metapneumovirus (hmpv) was identified in 2001 [134] , and is today considered as a major cause of acute respiratory infections worldwide, especially in children. virtually all children have experienced an infection with hmpv by the age of 5-10 years (reviewed in [135] ). hmpv is a member of the order mononegavirales, family paramyxoviridae, subfamily pneumovirinae (as is rsv), genus metapneumovirus [136] . there are two major groups and at least four subgroups of hmpv [137] [138] [139] [140] . hmpv particles are enveloped, pleomorphic, filamenteous and spherical and have a mean diameter of approximately 210 nm [137] . the genome consists of a single-stranded negative rna of approximately 13.3 kb and contains eight genes in the order 3'n-p-m-f-m2-sh-g-l-5' coding for a nucleoprotein (n), a phosphoprotein (p), matrix protein (m), fusion protein (f), a transcription elongation factor (m2-1), a protein regulating rna synthesis (m2-2), a small hydrophobic protein (sh), attachment protein (g), a polymerase subunit (l) and probably additional proteins [141, 142] . the f protein is the major immunogenic viral protein [143] . replication is generally comparable to that of other members of mononegavirales. this virus infection occurs primarily during winter months or early spring and can manifest as both upper and lower respiratory tract disease [144] . after a severe hmpv infection, virus was detected in alveolar and airway epithelial cells [145] . it was also reported in this study that the virus caused acute organizing lung injury, tissue damage and, which is not observed in other paramyxovirus infections, induction of smudge cell formation. the clinical symptoms associated with hmpv infection are indistinguishable from those of rsv infection [146] and range from common cold to pneumonia. otitis media is observed in up to 50% of the infected individuals (reviewed in [135] ). as for other respiratory viruses, hmpv may cause exacerbation of underlying chronic diseases like asthma, congestive heart disease or chronic obstructive pulmonary disease. the importance of coinfections with rsv or influenza viruses is not clear (reviewed in [135] ). as for many other respiratory viruses, serious disease caused by hmpv is observed among immunosuppressed patients. results from a recent retrospective study suggested that hmpv infection may be an important cause of idiopathic pneumonia syndrome after stem cell transplantation [147] . hmpv is thought to be the second or third cause of severe acute respiratory tract infection in children, just ranking behind rsv and influenza virus [146, 148] . infections occur very early in life and up to 100% of children have experienced an hmpv infection by the age of 10 years. reinfections occur frequently. incidences in hospitalized children with acute respiratory tract infection range from 5% to 10%. incidence is up to 20% in patients consulting at an outpatient clinic (reviewed in [135] ). the routes of transmission are believed to be similar to those of rsv (respiratory droplets, hand-to mouth or hand-to eye contact) [149, 150] . the diagnosis of hmpv infection is mainly clinical. molecular diagnostic procedures are usually not performed routinely but are possible using standard technologies like rt-pcr or immunological techniques. there is no specific antiviral treatment available. however, ribavirin has shown some promise [151] and monoclonal antibodies are under development that could potentially be used to prevent hmpv infections [152] . several vaccines are being developed but are still in an early stage. human bocavirus (hbov), a parvovirus, was detected in children with lrti in 2005 using random amplification methods [153] . hbov infection is predominantly associated with respiratory and/or gastrointestinal symptoms in children at around 2 years of age [154] . seroprevalence reaches 95% in adults [155] . however, the role of hbov in the pathogenesis of human respiratory disorders is not yet fully understood -hbov infections are frequently accompanied by coinfections with other viral and bacterial pathogens [154] . hbov is classified into the parvoviridae family, subfamily parvovirinae, genus bocavirus. as other parvoviruses, hbov virions are icosahedral nonenveloped particles with a diameter of 21-25 nm [154] . the hbov genome (a linear single-stranded dna that encompasses approximately 5.2 kb) is organized like that of other parvoviruses: conserved genes encoding for the two non-structural proteins are located in the 5' region and genes for two structural proteins are located in the 3' region of the genome [156] . the structural proteins vp1 and vp2 are identical in sequence but differ in an n-terminal extension that is only present in vp1 (vp1 unique region, vp1u) and possesses a phospholipase a2-like activity (pla2) [157] . the functions of the two nonstructural proteins ns1 and np1 of hbov are not known; however, regulatory functions of ns1 of other parvoviruses have been described [154] . hbov has been detected in children with respiratory disease. the range of clinical manifestations is broad. diseases of the upper (rhinitis or coughing) and the lower respiratory tract (including pneumonia, bronchiolitis and wheezing) or even the gastrointestinal disease have been described (reviewed in [154] ). other symptoms include fever or rashes [158] . however, hbov infections are linked frequently with coinfections with viral and bacterial pathogens in up to approximately 69% of hbov dna-positive individuals [159] which makes it rather difficult to distinguish between symptoms that have been caused by hbov or by other pathogens. the role of vertical transmission, seen with other parvoviruses, is not known. antibodies against the viral structural protein vp1 have been detected in approximately 95% of children older than 2 years and adults [155] . in addition, igg1 subclass antibodies against hbov vp2-virus-like particles (vlp) were detected in approximately 98% of samples that were obtained from healthy adult blood donors [154] . the same authors found igm antibodies in 41.7% of sera from hbov dna-positive children but not in samples from dna-negative children. cellular immunity also plays a role in hbov infections and frequent cd4 + t helper cell reactions have been observed against hbov vlp [160] . many epidemiological aspects have been discussed above. however, it is noteworthy that the majority of the analyses have been performed in symptomatic individuals and more data from asymptomatic healthy chil-dren and adults would add to the understanding of the hbov epidemiology. diagnosis of hbov infection is mainly done by pcr amplification of viral dna or the detection of anti-hbov antibodies by elisa [154, 161] . at present, no specific treatment of hbov infections is available. coronaviruses are known to cause a variety of diseases in animals [161] . human coronaviruses are mainly associated with respiratory disorders; some may cause enteric infections [162] . the human coronaviruses hcov-229e and hcov-oc43 were identified in the 1960s [163] [164] [165] . a coronavirus causing a severe acute human respiratory syndrome (sars), the sars-cov, was first described in 2003 [166, 167] and two additional human coronaviruses, hcov-nl61 and hcov-hku1 that were both linked to respiratory disorders have been identified recently [168, 169] . because of the economic importance of coronaviruses in veterinary medicine (for instance in swine), development of vaccines is more advanced in veterinary medicine than in human medicine. however, with the appearance of the sars coronavirus, human coronaviruses gained a greater share of interest. coronaviruses belong to the order nidovirales, family coronaviridae, genus: coronavirus. in addition to the five human coronaviruses (hcov-229e, hcov-hku1, hcov-nl 63, hcov-oc43 and sars-cov), a specific human enteric coronavirus has been reported [170] . coronaviruses have been assigned to three groups based on antigenic relationships between species of different groups (reviewed in [171] ). hcov-229e and hcov-nl63 are included in group 1, hcov-hku1 and hcov-oc43 in group 2 and sars-cov represents an early split from group 2 [172] . coronaviruses are enveloped viruses, approximately 120 nm in diameter with large (20 nm) club-shaped surface projections (spike protein, s). the structure and function of the s protein have been reviewed elsewhere [173] . apart from s, coronaviruses have a smaller membrane protein, m (reviewed in [174] ). in addition, coronaviruses have a third envelope protein, the very small, non-glycosylated e protein [175] . e and m have been found to be essential for virus particle formation (reviewed in [176] ). group 2 coronaviruses also have an he (hemagglutinin esterase) protein that forms a layer of approximately 7 nm [175] . he is a neuraminic acetylesterase that hydrolyses the 9-o-acetylated sialic acid on erythrocytes, thus potentially destroying receptors [177] . another coronavirus protein, n, is closely linked to the rna genome (and forms a ribonucleoprotein, rnp). n, which may have a functional role in replication and transcription, is phosphorylated (reviewed in [178] ). coronaviruses have positive-sense single-stranded rna genomes of about 30 kb. the genome is generally organized in the following manner: 5'-utr-polymerase gene-structural protein genes-utr3' where the utr are untranslated regions each up to 500 nucleotides. the structural proteins are encoded in the following order: he (only group 2 coronaviruses) -s -e -m -n [171] . the infection is initiated by binding of the s protein to the cell receptor [179] . cd13 (human aminopeptidase n, apn), a metalloproteinase located on the surface of epithelial cells, has been identified as the cell receptor for hcov-229e [180] . a metallopeptidase, angiotensin converting enzyme 2 (ace 2) may be the cell receptor for sars-cov [181] . binding of the s protein to the cell receptor induces conformational changes in s that triggers fusogenic activity [182] . the coronavirus genome can only be released into the cytoplasm after fusion of the envelope with the cell membrane which is mediated through the s2 region of the s protein [173, 183] . replication occurs within the cytoplasm. early during infection the genomic rna is released and acts as an mrna for the translation of the first gene, the polymerase. mrnas for the other genes are generated subsequently. in general, coronaviruses have several 3' co-terminal subgenomic mrnas, socalled 'nested set'. the unique part of each mrna (which is not within the next smaller mrna) is translated during the replication cycle. at the 5' end of each gene there is a sequence that is common to all genes, the so-called 'transcription-associated sequence', which, as the name implies, is associated with the discontinuous transcription process (reviewed in [171] ). the various mechanisms that have been proposed for the production of subgenomic mrnas have been reviewed elsewhere [184] . human coronaviruses are generally thought of as common cold agents. this role was confirmed when healthy volunteers were infected with hcov-oc43 and hcov-229e and developed classical common cold symptoms [185] . whereas most infections result in mild disease or are even asymptomatic, additional factors like immunosuppression or coinfections might cause severe disease, even pneumonia [186, 187] . it is not clear whether hcov-229e and hcov-oc43 infect the lower respiratory tract in otherwise healthy people, because only urti was observed in this population. it has been suggested that the lower respiratory tract is more susceptible to hcov infection in children [187] . most studies were performed in healthy adults and there is less information about the most susceptible and vulnerable population, children and elderly. however, hcov-229e and hcov-oc43 nucleic acid were frequently detected in children with respiratory tract disease (11%) using rt-pcr, whereas in an otherwise healthy control group (asymptomatic bone marrow recipients) only one sample tested positive (0.37%, p < 0.01) [188] . this finding suggests that these coronaviruses cause upper and lower respiratory tract diseases in children that are more severe than in adults. it has been suggested that up to 30% of wheezing episodes in asthmatic children may be due to coronavirus infections [189] . hcov-nl63 was detected in young hospitalized children with severe lrti [190] . this virus has also been detected in elderly patients with fatal respiratory disease [191] . the risk of developing croup was about 6.6 times higher in children shown to be positive for hcov-nl63 than in those who tested negative [192, 193] . hcov-hku1 was first detected in elderly and children with underlying disease [194] . symptoms of hcov-hku1 include rhinorrhea, fever, coughing, and wheezing as well as bronchiolitis and pneumonia [195] . hcov-hku1 might also cause gastrointestinal disease [196] . hcov infection results in antibody titers in serum. secretory antibodies can be detected in the respiratory and enteric tracts (and in milk or colostrum) [197] . infections with hcov peak during winter season [198] . it is estimated that approximately 25% of common cold cases are caused by coronaviruses [162] . outbreaks of different human coronaviruses have found to alternate every 2-3 years [197] . although early studies demonstrated that antibodies against coronaviruses are frequently present in adults [199] , new studies suggest that there are differences with respect to the hcov species. hofmann et al. [200] demonstrated that infections with hcov-229e occur less frequently than with hcov-nl63 by measuring specific antibodies neutralizing either hcov-nl63 or hcov-229e. in addition, co-detection of coronaviruses with other viruses is common [187] . as with most coronaviruses, human coronaviruses are species specific. however, the sars coronavirus probably originated from an animal reservoir, presumably bats [201, 202] but was transmitted to humans by civet cats. the transmission of human coronaviruses from human to human occurs via secretions like aerosols and respiratory droplets (or, in case of enteric infection, feces) [197] . adults with acute symptomatic or inapparent infection transmit the virus to infants who develop clinical disease [162] . diagnosis of hcov infection is more clinical; an etiological diagnosis, however, can be performed using molecular or immunological techniques. vaccines against coronavirus diseases have been developed for domestic animals because of the economic importance [197] , but not for humans. recent antiviral strategies against coronavirus infection have been reviewed elsewhere [203] . these strategies explore small interfering rna, blocking of viral entry (e.g., using carbohydrate-binding agents) or neutralizing antibodies. in addition, viral enzymes like protease or helicase are studied as potential targets for novel antivirals (reviewed in [203] ). according to estimates from the who, the burden of influenza in the usa is currently estimated to be 25-50 million cases per year, leading to 150 000 hospitalizations and 30 000-40 000 deaths per year. if these numbers are extrapolated to the rest of the world, influenza virus would infect 5-15% of the world population, causing 3-5 million cases of severe disease and approximately 0.5 million deaths per year. although this number is high, it would only characterize inter-pandemic influenza [204] . epidemics and outbreaks of influenza follow a seasonal pattern, which differs according to the region in the world: in temperate climate zones, seasonal epidemics typically begin in the late fall with a peak in late winter. the seasonal pattern is less pronounced in tropical zones (reviewed in [205] ). the focus of this book is on 'common cold'. for details on influenza and influenza viruses, further reading of standard literature is recommended. influenza viruses are members of the orthomyxoviridae, genus influenzavirus and include influenza virus types a, b and c. the viruses are enveloped pleomorphic particles with a size ranging from 100 to > 300 nm, their genome is organized on eight (influenza virus a and b) or seven (influenza virus c) negative-sense single-stranded rna segments [206] . spikes consist of hemagglutinin (ha) and neuraminidase (na). the nomenclature for human influenza virus includes type, geographic location of the first isolation, isolate number and year of isolation. in addition, subtypes of influenza a are described by their ha and na designations. to date, 16 ha and 9 na types have been described. the viral envelope is also associated with a matrix protein (m) that, after infection, forms a tetrameric ion channel. several polymerase proteins (pb1, pb2, pa) form, together with the nucleoprotein (np) and the rna, a ribonucleoprotein (rnp) complex (reviewed in [206] ). influenza viruses are transmitted via the respiratory route and bind to a cell receptor that consists of oligosaccharides and that is present on the surface of respiratory epithelial cells. the sialic acid-2,6-galactose linkage (sa 2,6gal) that is associated with binding to human influenza virus ha is present in the human respiratory tract (reviewed in [207] ). after binding, the virus enters the host cell by endocytosis [208] . further steps include fusion of the virus to the endosome [206] and the release of the rnp into the nucleus through an ion channel that is formed by m2 [209] . the viral rna is a template for complementary rna and the mrna. the nonstructural nep/ns2 as well as m1 play a role in the nuclear export of novel rna. assembly occurs at the apical surface of the cell, budding occurs and the novel virus is released (reviewed in [206] ). influenza viruses are transmitted via the respiratory route [206] . host specificity is largely determined by the availability of host cell receptors on the surface of epithelial cells [210] . usually, influenza in humans is an urti that is characterized by cough, headache, malaise and fever [211] . however, complications are frequent, and encephalitis, reye's syndrome, myelitis [212] as well as muscular manifestations of the infection including myocarditis [213] , disseminated intravascular coagulation and toxic and septic shock [214] may occur. major airway congestion, inflammation and necrosis have been found in histopathological examinations [215] . although much progress in the understanding of the pathogenesis of human influenza has been made during the past decade, the molecular mechanisms responsible for the virulence of particular strains of influenza virus are not yet understood. influenza a virus can infect humans as well as waterfowl and chickens, swine, horses, and other species. influenza b virus, on the other hand, has a restricted host range and circulates predominantly in humans. however, influenza b virus was recently isolated from seals [216] . different types of ha mediate species-specific binding of the virus [217, 218] . it would be beyond the scope of this book on common cold to review all the recent literature that has been published regarding the epidemiology of influenza viruses. however, one important aspect should be mentioned here. influenza virus is a changing virus and the repetitive occurrence of the yearly epidemic is supported by 'antigenic drift', an accumulation of point mutations in the viral receptors (ha and na). the drift is attributed to a low fidelity of the viral rna polymerase [219] . these new variants then infect a 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replication in tissue culture sialic acid species as a determinant of the host range of influenza a viruses does this patient have influenza influenza virus and cns manifestations influenza-associated deaths among children in the united states influenza b virus infection associated with non-bacterial septic shock-like illness histopathologic and immunohistochemical features of fatal influenza virus infection in children during the 2003-2004 season influenza b virus in seals differential sensitivity of human, avian, and equine influenza a viruses to a glycoprotein inhibitor of infection: selection of receptor specific variants receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the h3 hemagglutinin based on species of origin positive darwinian evolution in human influenza a viruses the epidemiology of influenza and its control influenza and influenza vaccination waiting for a pandemic key: cord-169428-g6k0vqrm authors: schurwanz, max; hoeher, peter adam; bhattacharjee, sunasheer; damrath, martin; stratmann, lukas; dressler, falko title: infectious disease transmission via aerosol propagation from a molecular communication perspective: shannon meets coronavirus date: 2020-10-31 journal: nan doi: nan sha: doc_id: 169428 cord_uid: g6k0vqrm molecular communication is not just able to mimic biological and chemical communication mechanisms, but also provides a theoretical framework regarding viral infection processes. in this tutorial, aerosol and droplet transmission is modeled as a multiuser scenario with mobile nodes, related to broadcasting and relaying. in contrast to data communication systems, in the application of pathogen-laden aerosol transmission, mutual information between nodes should be minimized. towards this goal, several countermeasures are reasoned. the findings are supported by experimental results and by an advanced particle simulation tool. this work is inspired by the recent outbreak of the coronavirus (covid-19) pandemic, but also applicable to other air-borne infectious diseases like influenza. and innovative solutions to urgent problems. the need for novel solutions has already created rapid infection control solutions during the pandemic. these include "social distancing" rules [5] , mouth and nose protection in public spaces, and large-scale disinfection measures as suggested by the various institutes of health care and virology around the world. just as mc combines biological/chemical and communication/information theoretical approaches to provide systematic solutions, a bridge can be built between mc and infectiology to apply known techniques from information engineering to the field of health care and infection prevention. findings from health research dealing with the spread of aerosols and the viruses they may contain, can be joined with insights from mc in order to predict infection processes and to identify more sophisticated and safer protective measures. recent research in combining these areas has dealt with possible use cases for aerosol communications [6] , and with channel modeling considering infectious aerosols in a point-topoint scenario [7] , [8] . an infected person acts as a transmitter of pathogen-laden aerosols and a device or another person acts as a receiver of the infectious particles. the close relation to the domain of macroscopic mc, where a transmitter emits molecules into a fluid environment with an absorbing receiver to recover the information, was exploited. in [9] , the concept has been expanded to include several features: • the transmission of infectious aerosols is suggested to be a multiuser mc scenario. • in each mc transmitter, the modulator is modeled by respiratory-event-driven higher order molecular variableconcentration shift keying [10] . • channel modeling is aided by air-based experiments using a fluorescence dye in conjunction with optical detection, motivated by the macroscopic testbed for airbased mc published in [11] . • an advanced computer simulation tool has also been designed based on the work in [12] . parameters feeding this tool were obtained by own experiments, among other established sources. a large variety of safety measures can be rethought from a communication and information theory perspective to optimize the goal of infection prevention. building on the novel contributions revealed in [9] , this tutorial presents the following communication aspects in greater depth: • the transmission of infectious particles is modeled as a multiuser mc scenario with mobile nodes in a timevarying environment. infected users are transmitters that broadcast pathogen particles, whereas the remaining users are potential receivers. after a certain delay, some of the receivers may become active transmitters, like in relaying. • the reception of the particles is represented by effective apertures, which map specific body regions of the user responsible for the infection, similar to antennas in wireless communications. • infection is considered to be a generalized threshold detection problem: if the density of infectious particles received exceeds a user-dependent threshold, an infection is likely to occur. furthermore, the viral load may also affect the course of the disease if it is above the infection threshold. in the field of mc, the corresponding analogy is reliability information. • the viral infection rate is modeled by the concept of mutual information [13] . contrary to data transmission, the aim is to minimize the mutual information between an infected user and the healthy ones. the remainder of this tutorial paper is structured as follows: section ii describes the duality between communication and information theory on the one hand and the field of airborne aerosol infection on the other. in section iii, the concept of mutual information is introduced to the area of infectious disease transmission. prevention techniques from different areas are discussed to minimize the mutual information in order to reduce the risk of infection. section iv presents a particle simulation tool suitable for the problem under investigation, and corresponding simulation results for a multiuser scenario. section v discusses the challenges that arise from the duality concept, before finally drawing the conclusions in section vi. in the area of wireless communications, a general multiuser scenario consists of multiple participants distributed in a three dimensional (3d) environment, acting as transmitters and/or receivers. the communication links between all users are distorted channels, influenced by various environmental parameters and the emitted signal powers. in the scenario under investigation, the signal powers are modeled in terms of the parameters of the emitted aerosols and the contained viral load coming from the infected user(s). analogous to infectiology, in this multiuser scenario one assumes at least one infected person who, unlike the other participants, emits infectious aerosols and droplets. this situation can be understood as a point-to-multipoint communication scenario at the physical layer, related to broadcasting and relaying. in case of a successful transmission -a necessary condition is that the ingested amount of infectious aerosols exceeds the infection threshold -the receiver itself becomes an active transmitter after a certain incubation period. the relays act as virus spreaders or even superspreaders, i.e., they boost the viral load. table i features some duality aspects between air-based mc on the one hand and infectious particle transmission on the other. details will be presented next. the emission of aerosols and droplets is triggered by respiratory events such as breathing, speaking, singing, coughing or sneezing. these actions can be divided into three categories according to their type of occurrence, cf. fig. 1 : 1) breathing can be modeled as continuous emission, following a breathing pattern, mainly influenced by the physical stress on the human body. other factors like size of the body or age may also be considered. 2) speaking/singing can be described as a two-state markov process with the two states being either silence or singing/speaking. the model is defined by the transition probabilities between the two states and retention probabilities for staying in the current state. 3) coughing/sneezing can be emulated as a poisson random process. the density parameters are influenced by the infection state of the user, resulting in a higher probability for an infected user to cough or sneeze than for a healthy user. based on these actions, the users emit aerosols into the environment. the different respiratory actions are responsible for the main parameters of the emission of aerosols, namely the • number of emitted aerosols, • diameter distribution, • initial velocity distribution, • temperature distribution, and • viral load or viral distribution. each respiratory event causes a time-varying particle cloud, even in the absence of air movement. due to a counteraction between air drag, buoyancy, and gravity, aerosols survive for a longer period than droplets [7] , [8] . the particle clouds of different respiratory events superimpose. all parameters are of stochastic nature where the actual emitted aerosol distribution changes between users and with each emission. the infection state of the human ultimately decides if an emission contains pathogens or not, so every healthy user emits aerosols, but these have no influence on the infection status of the other users when received. as an alternative to the experiments conducted in [9] , the mentioned respiratory events can be emulated in the macroscopic-scale air-based testbed introduced in [11] . mouth and nose are replaced by transmitter-side sprayers. in order to visualize particles, a fluorescent dye solution is emitted. intensity and duty cycle are software-controlled. the angle of departure is adjustable. all mentioned actions can be emulated by respiratory-event-driven higher order molecular variableconcentration shift keying (movcsk) modulation [9] . in this context, an movcsk modulator is capable to release infected and non-infected particles. this can be further refined by distinguishing between particles of different sizes. the intensities of the respiratory events are mapped to the different concentration levels. the random occurrence of the events over time can be represented by a sequence of movcsk symbols. several parts of the human body are actively or passively involved in the absorption of aerosols, as depicted in fig. 2 . the facial region with the mouth, the nose and the eyes is the most sensitive receiving area [8] , implying that a small amount of received aerosols can lead to an infection of the user. other parts of the body like hands and feet or shoes are passively involved by receiving infected aerosols and either transferring them to more sensitive parts of the user by smear infection or contaminating other surfaces, which in turn can cause infection. the reception-sensitive body areas can be interpreted as effective apertures, similar to spatially distributed antennas, with individual antenna gains and sensitivity levels. starting with the highly sensitive body regions in the face, which can trigger an infection even with a low viral load, to the feet or shoes, which are only infectious to humans in certain cases and with a very high viral load. the sensitivity levels of the individual sensors contribute to the user-dependent infection threshold, which depends on other criteria as well including the health conditions. the amount of viral molecules that the receiver absorbs per time unit, i.e. the viral load, has an impact on the probability of infection. according to the state-of-the-art, the human-dependent infection threshold is checked by hypothesis testing [7] , [8] : the probability of infection corresponds to the situation where the viral load exceeds the threshold. however, the viral load additionally is likely to have an influence on the severity of the course of the disease. this situation is related to reliability information. in communication and information theory, receiver-side reliability information describes the quality of information transmission in terms of the error rate. from the perspective of our testbed, the water-based dye solution from the sprayer can be monitored and recorded by a digital camera at the receiver side under the influence of an ultraviolet (uv-a) light source. the intensity recorded by the camera can be interpreted as the viral load carried by the transmitted droplets over a certain distance through active transmission. the region between the sprayer and the camera also contains those droplets which settle down under the influence of gravity. the intensities from these droplets can be recorded to formulate a model for passive transmission of pathogens. the spatially-distributed time-varying aerosol clouds that are emitted into the environment from the various users, are subject to a dynamic channel with turbulences and changing parameters. various effects change the channel and cause the aerosols to propagate through the space with different velocities and ranges. the turbulences in the air are caused by the movement of the users, ventilation devices, air streams introduced through windows and doors, temperature gradients, and/or weather phenomena. turbulence can either increase the range of the aerosols by constructively influencing the propagation vector, or reduce it when the particles are forced to the ground. in the testbed environment, turbulence can be modeled by introducing an external fan into the setup to cause an artificial air movement. the turbulent channel can be described by shannon's "mother of all models" [14] . this analogy is exploited subsequently. the scenario under investigation comprises multiple users in a predefined environment, see fig. 3 . initially, at the physical layer level, the multiuser scenario can be described by a point-to-multipoint network, the nodes of which comprise of the entire set of users. the infectious user broadcasts pathogens. some of these can be absorbed by one or more healthy users. those users can be modeled as store and forward relays, which after some delay may broadcast even more pathogens. when the number of infectious users increases on average, the reproduction factor is said to be greater than one. in the case of a testbed, a point-to-multipoint scenario can be emulated by having multiple cameras placed at various locations acting as receivers. the intensities are recorded by the cameras and if the measurements breach a certain threshold level, the receivers may be termed as "infected" which may further trigger new sprayers to activate and start spraying the water-based dye solutions, mimicking a relay mechanism. the users are able to dynamically move and change their absolute and relative positions. the absolute position change results in a random emission pattern, where the infectious aerosols are emitted at multiple locations throughout the environment. depending on the channel characteristics, the aerosols are able to stay suspended in the air for some time, resulting in a risk of infection for other users that move with their apertures through the aerosol clouds or that are hit by moving clouds. infection prevention by means of "social distancing" is focused on the relative positions among the users. keeping appropriate relative distances may lead to a reduction of mutual information transfer, but ignores the possibility of the presence of suspended aerosols in the environment from previous respiratory events from other users at the same absolute position. in addition, the movement of the users create movement and turbulences of the surrounding air, which in itself results in a change of aerosol movement. in this section, the concept of mutual information is introduced to the area of infectious disease transmission. contrary to a maximization of mutual information in data communication systems, the aim of infection prevention is to minimize the mutual information between nodes. this can be accomplished by employing several methods that are discussed afterwards. infection is strongly related to the probability that a certain density of pathogen-laden particles successfully come into contact with the effective aperture area of the receiver. if the receiver-side density exceeds the user-dependent infection threshold, after a certain incubation time, the receiver will become infected with a certain probability. in terms of shannon information theory, this situation can be modeled mathematically by means of mutual information. starting off with a point-to-point channel [14] , let x denote the channel input symbol and y the corresponding observation. the mutual information is a function of the channel input distribution, p(x), and the joint probability p(x, y). in our case, the channel input distribution depends on the respiratory events and the joint distribution on the nature of the turbulent atmospheric channel. this concept can be generalized to multiuser channel models. details, however, are beyond the tutorial style of this article. the analogy between aerosol-based infection and mutual information demonstrates three important facts: the risk of infection depends on the channel input distribution, the turbulent channel, and the infection threshold. although a minimization of mutual information is counterproductive in classical communication scenarios, it is a common design objective in cryptosystems. fig. 4 summarises several techniques/approaches that reduce the risk of infection. these are described next in connection with mutual information. spatial actions: maximization of euclidean distances between the users in the multiuser scenario under investigation, i.e., "social distancing", aims to reduce the probability of direct infection caused by respiratory events. other techniques like pruning/thinning the user density in a certain space as well as route optimizations to reduce the interference of users trajectories with infectious aerosol clouds have a similar impact on the propagation conditions. despite these spatial actions, sometimes it is desirable that the users are still able to communicate with each other. hence, another physical approach is air humidification. if the relative humidity of the room air is below 40 percent, the emitted particles absorb less water, stay lighter and move around the room for a longer period. in addition, the nasal mucous membranes in our noses become drier and more permeable to viruses when the air is dry. this has an impact on the infection threshold. additional precautions like indoor fans, air ventilation, and air purification devices are also useful, as well as fresh air through open windows to prevent an accumulation of infectious aerosols in the ambient air. temporal actions: the allocation of individual time slots on user or group level leads to a more orderly procedure and this results in a lower probability of infection. this technique is known as time-division multiple access and is widely used in communication engineering. chemical actions: reducing the viral load on surfaces is a key factor in infection prevention. this can be accomplished by cleaning of shoes or disinfection of the hands, as they are part of the effective aperture of the receiver. household bleach solution containing 5.25 to 8.25 percent sodium hypochlorite could be used to disinfect surfaces while at least a 70 percent ethyl/isopropyl alcohol solution proves to be effective when it comes to proper disinfection of hands. optical actions: the viral load in airborne aerosols that were expelled into the air can be reduced by ultraviolet germicidal irradiation. this technique can be used in air purifiers to filter stale air, to clean handles, and other surfaces. it is essential to ensure that the skin and eyes are protected from radiation. furthermore, uv-c irradiation can be employed for cleaning protection masks, even online when integrated into exhalation valves embedded in medical masks. biological actions: medical methods with a direct effect on the receiver's detection mechanism provide the best protection against transmission of infection. at the same time, a biological solution is also the most difficult to apply to a wide range of users. an example of a biological action would be vaccination. at the same time, this is also the least broadly based mechanism, as vaccination usually only protects against a specific pathogen. ongoing research on biochemical nose sprays and mouthrinses is aimed at improving the infection threshold as well. note that all these techniques/approaches have an influence on the mutual information. some target the propagation conditions while others affect either the particle emission or the infection threshold, respectively. the propagation of aerosols can be simulated with high accuracy with the help of existing computational fluid dynamics (cfd) tools [15] . this approach, however, is very demanding on computational resources. if the goal is to simulate the spreading of disease in a multiuser scenario with mobility and over longer periods of simulated time, mc simulation tools optimized for the simulation of pathogen-laden particle transfer may become useful. in previous work [9] , we have extended the mc simulator presented in [12] aiming to replicate a coughing event. in the current contribution, this scenario is extended further towards a multiuser network. an emitter as presented in [9] is an object which can be freely moved or duplicated in the simulation space. at a specified time, the emitter releases 20 particles in intervals of 0.25 ms simulation time for a fixed duration of 100 ms. the angle and velocity of emission are determined randomly based on the configured random distributions. after emission, the trajectory of each particle is guided by its inertia and the forces acting on it, namely air drag, buoyancy, and gravity. when a particle eventually collides with an absorbing receiver such as the ground or a spherical shape, it will be removed from the simulation and the position and speed of collision can be logged. as an example, let us consider a scenario with three people. each of the three people is modeled as having one point emitter at mouth height (1.65 m), one absorbing spherical receiver with a radius of 5 cm immediately behind the point of emission, as well as two additional spherical receivers of the same size to model the hands at a height of 70 cm with a lateral offset of 30 cm. initially, person a represents an emitter and coughs with the parameters we determined experimentally in [9] . as one alteration to these parameters, the emission opening angle is now sampled randomly for each ejected particle based on the empirical cumulative distribution function of observed emission angles. previously, the emission opening angle was approximated with a normal distribution. at a distance of 2 m opposite of person a, person b is a passive receiver. 75 cm to the left of person b is person c, who acts as both a receiver as well as a second transmitter with a delay of 3 s. in our previous work, the trajectory of simulated particles was only determined by their initial velocity vector at the time of emission and the forces acting on them, assuming a completely stationary volume of air for computing the drag force. in order to emulate the initial ejection of air in a coughing event in a simplified fashion, we can emit the simulated particles inside a volume of constant air speed. for this demonstration, we chose a 3 m long and 1 m wide cylindrical volume aligned with the vector of emission at the mouth, and an air speed of 5 m s −1 . as shown in fig. 5 , the receiving person b standing 2 m in front of the emitter a is surrounded by the bulk of particles that have been emitted. this receiver's facial region has received 112 simulated particles and the hands have received 14 particles, out of a total of 8000 emitted particles per cough. the second receiver, offset by 75 cm to the left of the first receiving person, did not receive any particles from the cough. when this receiver c becomes the second emitter, the situation is mirrored and the first emitter a, now a receiver, is only reached by two particles at the closest hand. in future work, this simulation concept can be extended further to account for both the aspect of mobility and the remaining types of aerosol emissions outlined in section ii. mobility can be realized, for example, by letting simulated persons follow predetermined paths collected from real-world mobility traces, or paths hand-drawn using 3d animation software. alternatively, such paths may be generated randomly. when incorporating other emission types, like with the airbased testbed as also outlined in section ii, movcsk can be used as a modulation scheme to control the emission of particles. this article extends recent advancements with respect to the symbiosis of mc and infectiology [6][9] . emphasis is on a conceptual level by pointing out dualities and similarities between these fields. a key aspect is to treat the transmission of pathogen-laden particles as a dynamic multiuser communication scenario. nevertheless, especially when combining different disciplines like engineering and health care, it should be checked whether the proposed approaches also lead to comparable results and are therefore fundamentally correct. new findings from existing and refined mc techniques can be applied in infectiology and evaluated from a health care point of view. furthermore, a reverse check of established infection prevention techniques can be accomplished by means of tools established in communication and information theory. these reviews will show whether the dualities and similarities described above can lead to new prevention approaches being useful in the real world and thus reducing the incidence of infection. the fact that both research areas are mature, opens up the possibility for a reciprocal examination of the respective other side. air-based mc with fluorescent particles offers new possibilities with regard to the investigation of the propagation of aerosols. the testbeds and measurement tools can be used to verify precautions like face masks and gain further insights into infectious ranges. due to the continuing spread of sars-cov-2, it is assumed that the social objective is now to "live with the virus" and to control viral spreading or at least its impact on individuals, on the society, and on economical aspects. other diseases such as influenza show a similar behavior. warning apps, already used in many countries in the context of the sars-cov-2 virus to track contacts between infected and non-infected people, are already laying the foundations for this objective. they can perhaps be extended with an online functionality to warn users in real time of a possible infection with the virus. it is also conceivable that mc-based contributions including channel modeling can be exploited to place future virus detectors at the best possible positions. we studied infectious disease transmission via aerosol propagation as viewed from a communication and information theory perspective. towards this goal, dualities and similarities between macroscopic air-based mc and infected particle transmission are worked out. in the concept, for the first time, users are modeled as mobile nodes in a multiuser scenario. infected users emit aerosol clouds in the sense of broadcasting, whereas healthy users may become infected with a certain probability if they absorb a sufficient viral load by viral-sensitive aperture areas, like spatially distributed antennas in the area of wireless communications. in turn, after some incubation time, these users may become pathogen-laden spreaders or superspreaders as well, related to store and forward relaying. apart from being a pure binary problem of getting infected or not, the viral load frequently has an impact on the course of the disease, similar to reliability information in mc. in the sense of shannon information theory, the goal is to minimize the mutual information between infected and non-infected users, where the information corresponds to infected and non-infected particles. in this stochastic approach of infection prevention, numerous actions are discussed regarding channel input distribution, channel propagation, and channel output characteristic. for example, protective masks affect the channel input distribution. spatial and temporal actions mainly have an impact on the channel propagation. biochemical actions as well as health conditions affect the infection threshold at the channel output. additionally, challenges regarding "living with the virus" are discussed. the conceptual approach is supported by an experimental macroscopic-scale molecular communication testbed and by an advanced simulation tool. as an example, simulation results are presented for a 3d environment with three users who emit aerosols that are subject to air movement due to the ejection action. molecular communication. cambrigde nanonetworks: a new communication paradigm a comprehensive survey of recent advancements in molecular communication channel modeling for diffusive molecular communication -a tutorial review targeted social distancing designs for pandemic influenza communication through breath: aerosol transmission modeling of viral aerosol transmission and detection a molecular communication perspective on airborne pathogen transmission and reception via droplets generated by coughing and sneezing duality between coronavirus transmission and air-based macroscopic molecular communication exit chart analysis of higher order modulation schemes in molecular communications a testbed and simulation framework for air-based molecular communication testbed using fluorescein efficient simulation of macroscopic molecular communication: the pogona simulator mutual information and maximum achievable rate for mobile molecular communication systems a mathematical theory of communication numerical investigation of aerosol transport in a classroom with relevance to covid-19 since 2020, he is a research assistant at the chair of information and coding theory, kiel university. his current research interests include air-based macroscopic molecular communication and radar signal processing since 1998 he is a full professor of electrical and information engineering at where he is currently pursuing the dr.-ing. (ph.d.) degree at the faculty of engineering. since 2019, he has been a research and teaching assistant at the chair of information and coding theory, kiel university. his current research interests include physical layer issues of key: cord-018785-tcr5xlf8 authors: nambiar, puja; silibovsky, randi; belden, katherine a. title: infection in kidney transplantation date: 2018-06-27 journal: contemporary kidney transplantation doi: 10.1007/978-3-319-19617-6_22 sha: doc_id: 18785 cord_uid: tcr5xlf8 infection is an important cause of morbidity and mortality after kidney transplantation. it has been estimated that 70% of kidney transplant recipients will experience an infection episode within the first 3 years after transplantation (dharnidharka et al. 2007). after cardiovascular disease, infection is the second leading cause of death in recipients with allograft function (snyder et al. 2009). the immunosuppressive therapy required to prevent organ rejection places the kidney transplant recipient at increased risk for donor-derived, nosocomial, and community-acquired infections as well as reactivation of latent pathogens. pretransplant screening, immunizations, and optimal antibacterial and antiviral prophylaxis can help to reduce the impact of infection. awareness of the approach to infection in the transplant recipient including diagnostic and management strategies is essential to optimizing outcomes. a total of 17,600 kidney transplants were performed in the united states in 2013. as the incidence of acute rejection has declined, the probability of graft and patient survival continues to improve (usrds 2015) . infection, however, remains an important cause of morbidity and mortality after kidney transplantation. it has been estimated that 70% of kidney transplant recipients will experience an infection episode within the first 3 years after transplantation (dharnidharka et al. 2007 ). after cardiovascular disease, infection is the second leading cause of death in recipients with allograft function (snyder et al. 2009 ). the immunosuppressive therapy required to prevent organ rejection places the kidney transplant recipient at increased risk for donor-derived, nosocomial, and community-acquired infections as well as reactivation of latent pathogens. the kidney transplant recipient's net state of immune suppression and epidemiologic exposures determine the risk for infection at a given time. a traditional timeline has been used to predict patterns of infection after organ transplantation. this timeline has been altered in recent years with changes in immunosuppressive therapy and the routine use of antibacterial and antiviral prophylaxis. treatment for acute rejection and coinfection with viruses such as cytomegalovirus (cmv) and epstein-barr virus (ebv) may also alter predictable patterns of infection (fishman 2007) . the basic concepts of the traditional timeline, however, are still used to establish a differential diagnosis for infection at varied intervals posttransplantation (fig. 1) . within the first month, infections are noted to include those related to surgical complications, nosocomial exposures, and donor-derived pathogens. multidrug-resistant organisms including methicillin-resistant staphylococcus aureus (mrsa), vancomycin-resistant enterococcus (vre), and carbapenem-resistant enterobacteriaceae (cre) are important considerations, as is clostridium difficile. urinary tract infections are common within the first 6 months. opportunistic infections are more likely to occur 1-6 months after transplantation, reflecting the greater impact of immune suppression during this time. reactivation of latent pathogens such as polyoma virus bk, hepatitis c virus (hcv), and mycobacterium tuberculosis may also occur. prophylaxis for pneumocystis jiroveci, herpes viruses including cmv, and hepatitis b virus (hbv) makes these infections less common during this time period. beyond 6 months, the degree of immune suppression for most patients decreases. risk remains, however, for community-acquired infection, environmental exposures, recurrent infection, and the late presentation of viral infection, in particular cmv, once prophylaxis has been discontinued (fishman 2007; karuthu and blumberg 2012) . interventions can be undertaken to reduce the impact of infection after kidney transplantation. pretransplant screening of donors and recipients for infection that can be transmitted with organ donation or reactivated in an immune suppressed recipient is essential for optimizing transplant outcomes. guidelines for pretransplant screening are available from the american society for transplantation (fischer et al. 2013) , kidney disease: improving global outcomes (kdigo 2009) and the us public health service (seem et al. 2013) . recommended screening tests for donors and recipients are listed in table 1 . screening of living donors is performed prior to transplantation with varied timing. if there is a hiv(þ) consider if hiv is well controlled hcv: anti-hcv and hcv nat hcv(þ) hcv(à) reject, may be a consideration in the future hcv(à) hcv(þ) consider, hcv(þ) candidates should have a liver biopsy, improved outcomes if hcv is treated pretransplant hcv(þ) hcv(þ) consider (as for dà/rþ) hbv: hbsag, hbsab and hbcab (igm/igg); hbv nat (center dependent) sag(à), cab(à) sag(à), cab(þ), sab(þ/à) accept, vaccinate sab(à) candidates consider, with prophylaxis posttransplant sag(à), cab(þ) sag(à), cab (þ/à), sab (þ/à) accept if donor is cigm(à) and vaccinate sab(à) candidates, offer prophylaxis posttransplant if sab(à) or lost; reject if donor is cigm(þ) sag(þ), cab(þ) sag(à), cab (þ/à), sab (þ/à) reject cmv igg cmv(þ) or (à) cmv(þ) accept; will need posttransplant prophylaxis or preemptive therapy cmv(þ) cmv(à) accept; high risk for cmv infection, will need posttransplant prophylaxis ebv igg ebv(þ) or (à) ebv(þ) accept ebv(þ) ebv(à) accept; at risk for primary ebv and ptld, monitor posttransplant hsv 1/2 igg hsv(þ) hsv(þ) or (à) (fischer et al. 2013) . deceased donor screening, in contrast, is under time constraints and is usually performed within hours of transplantation in coordination with organ procurement organizations. infection with hiv, hbv, and hcv may not be detected in the early stages of infection. many transplant centers now perform more sensitive rapid molecular testing on potential organ donors including nucleic acid amplification (nat) testing for hiv, hbv, and hcv. a comprehensive medical and social history on potential organ donors is required in order to identify risk factors for blood borne pathogens. in efforts to expand the pool of available organs, recipients may consent to receipt of a kidney from a nat negative donor who is deemed "high risk" for blood borne infection based on identified risk factors. recipients of such organs are monitored posttransplantation with testing for hiv, hbv, and hcv between 1 and 3 months and for hbvagain at 12 months (fischer et al. 2013; seem et al. 2013; kovacs et al. 2014; len et al. 2014 ). use of hcv-and hbv-positive organs can be considered in respective positive recipients. furthermore, in 2013 the hiv organ policy equity act lifted a long-standing ban on allowing hivpositive organs to be donated to hiv-positive recipients (mgbako et al. 2013; muller et al. 2015) . donors who have active bacterial infection at the time of kidney procurement may transmit infection to the recipient. screening for bacterial infection in kidney donors includes assessing for urinary tract infection and bacteremia. urine and blood culture data are reviewed. if a kidney donor is known to have a urinary tract or systemic infection with a virulent organism such as staphylococcus aureus, pseudomonas aeruginosa, or candida species, the organ recipient is usually treated with a 10-14 day course of targeted antimicrobial therapy since these bacteria can compromise vascular and urinary anastomoses leading to mycotic aneurysms, anastomotic, and organ failure (fischer et al. 2013 ). allograft contamination can occur during organ procurement or processing. interpretation of organ preservation fluid cultures is challenging. the risk of transmission of infection to the organ recipient from contaminated preservation fluid, however, is low (fischer et al. 2013; len et al. 2014 ). candidates for kidney transplantation should have their vaccine status reviewed and updated in accordance with recommendations issued by the advisory committee on immunization practices with the centers for disease control and prevention (cdc 2012). while vaccinations in end stage renal disease patients may be less effective and durable than in healthy patients, a better response can be anticipated prior to transplantation than after (janus et al. 2008; kausz and pahari 2004) . special consideration should be given to vaccination for pneumococcus, influenza, and hbv. two pneumococcal vaccines are currently licensed for use in the united states: the 13-valent pneumococcal conjugate vaccine (pcv 13, prevnar 13) and the 23-valent-pneumococcal-polysaccharide vaccine (ppsv 23, pneumovax 23). current guidelines recommend that unvaccinated patients with chronic renal failure receive pcv 13 followed at least 8 weeks later by ppsv 23 (kobayashi et al. 2015) . a second dose of ppsv 23 is recommended 5 years after the first dose (cdc 2012). influenza vaccination should be administered annually. there are a number of influenza vaccine formulations available. live attenuated influenza vaccination (flumist) is not recommended in chronic kidney disease patients. an inactivated vaccine option should be used (cdc 2012) . a high dose inactivated influenza vaccine is now available and was shown to induce a higher antibody response than traditional vaccines in adults over the age of 65 (diaz-granados et al. 2014) . the use of this vaccine in transplant candidates and recipients is currently under investigation. transplant candidates not immune to hbv should receive high dose hbv vaccination (40 micrograms antigen per dose) due to decreased response rates with standard dosing (huprikar et al. 2015) . human cytomegalovirus-human herpes 5 (cmv), a member of the family herpesviridae, is an opportunistic pathogen occurring in 20-60% of solid organ transplant recipients (brennan 2001) . cmv is a cause of significant morbidity and mortality in this population (mwintshi and brennan 2007) . the incidence of cmv in the renal transplant population is estimated to be between 8% and 32% (patel and paya 1997) . renal transplant patients are at lower risk for primary cmv compared with other organ transplant recipients owing to a lower burden of latent virus in renal allograft tissue. the risk factors for development of cmv disease include donor seropositivity/recipient seronegativity(dþ/rà), use of induction immunosuppression (antilymphocyte antibodies), donor age >60 years, simultaneous kidney-pancreas transplantation, treatment for acute rejection, impaired transplant function, and concurrent infection from other viruses (like ebv and hhv-6 and 7) (de keyzer et al. 2011) . cmv-seronegative recipients of cmv-seropositive donors (d + /r à ) are at the highest risk, whereas d + /r + or d à /r + transplantations are considered to be moderate risk with d à /r à being lowest risk, with an incidence of cmv disease <5% (de keyzer et al. 2011) . immunosuppressive drugs also influence the incidence and severity of cmv disease. for instance, cyclosporine increases the risk of cmv disease, whereas use of sirolimus seems to have a protective effect (san juan et al. 2008) the use of antilymphocyte antibody (antithymocyte globulin or muromonab-cd3) is associated with a two to fivefold increase in the rate of cmv, but basiliximab and daclizumab do not seem to increase its incidence (de keyzer et al. 2011) . cmv infection may occur in solid organ transplantation recipients as primary infection when a cmv seronegative individual receives cells latently infected with cmv from a seropositive donor, donor-derived reinfection, or reactivation of latent recipient infection (patel and paya 1997) . the following definitions are commonly used in the transplant literature to differentiate cmv infection from cmv disease. cmv infection is evidence of cmv replication regardless of symptoms, and cmv disease is evidence of cmv infection with symptoms, such as viral syndrome, leukopenia, thrombocytopenia, or invasive tissue disease (e.g., pneumonitis, hepatitis, retinitis, gastrointestinal disease) (humar and snydman 2009) . cmv disease and even asymptomatic cmv infection have been shown to be independent risk factors for reduced graft survival and overall mortality beyond 100 days posttransplantation . infection with cmv has been implicated in acute allograft dysfunction and chronic allograft nephropathy (mclaughlin et al. 2002; tong et al. 2002) . cmv disease is also associated with posttransplant lymphoproliferative disorder (ptld), posttransplant diabetes mellitus, and transplant artery stenosis (pouria et al. 1998; hjelmesaeth et al. 2004; manez et al. 1997) . cmv infection can occur as acute infection between the first and 6 months following transplant, when immunosuppression is at its maximum or as delayed infection from reactivation of latent virus after antiviral prophylaxis has completed, later in the first year. given the significant effect of cmv on patient outcomes, prevention plays an important role. serologic screening for cmv should be performed on both donor and recipient prior to transplant to categorize high risk patients. several cmv vaccine candidates are under investigation although none are currently available. universal prophylaxis involves giving antivirals to those recipients at risk posttransplant before the onset of infection, whereas in preemptive therapy patients are monitored at regular intervals and started on antivirals when there is early evidence of replication prior to onset of clinical disease. chemoprophylaxis in high risk patients (dþ/rà) has shown to reduce the incidence of cmv disease by 60% and has decreased cmv associated mortality and opportunistic infection (hodson et al. 2005) . preemptive therapy in high risk patients based on cmv viral load monitoring has not shown reduction in acute rejection or all-cause mortality (strippoli et al. 2006) . a randomized controlled trial by kliem et al. in 2008 comparing oral ganciclovir chemoprophylaxis with viral load monitoring revealed improved graft survival in those who received ganciclovir chemoprophylaxis (kliem et al. 2008) . a recent cochrane review from 2013 concluded that the efficacy of preemptive therapy compared with prophylaxis to prevent cmv disease remains unclear due to significant heterogeneity between studies and that additional head-tohead studies are required to determine the relative benefits and harms of preemptive therapy and prophylaxis to prevent cmv disease in solid organ transplant recipients (owers et al. 2013) . standard prophylactic guidelines recommend therapy in dþ/rà, dþ/rþ, and dà/rþ using oral ganciclovir or valganciclovir for a minimum of 3 months posttransplant and 1-3 months after treatment of rejection with antilymphocyte therapy (humar and snydman 2009; kotton et al. 2013 ). valganciclovir has replaced ganciclovir because of better bioavailability, lower pill burden, and reduced availability of oral ganciclovir (paya et al. 2004 ). the optimal length of prophylaxis is unknown, but recent trials have shown that 6 months of prophylaxis is more effective in decreasing the incidence of cmv disease in dþ/ rà kidney transplant recipients (humar et al. 2010; doyle et al. 2006) . current guidelines recommend dosing valganciclovir at 900 mg daily (adjusted for renal dysfunction) if tolerated in dþ/ rà recipients. some centers have successfully treated patients with half of this dose (450 mg daily) with less drug toxicity. however, dþ/rà recipients may be at higher risk of breakthrough infection and the development of resistance with this lower dosing strategy (kotton et al. 2013) . the diagnosis of cmv disease can be made by several techniques including cmv antigenemia assay, nucleic acid testing (nat), serology, antibody testing, viral culture, and histopathology. nat is generally more sensitive than antibody testing or culture. higher values by nat are suggestive of cmv disease and weekly viremia testing can be used to monitor response to therapy. the interlaboratory variability of nat is expected to be reduced with the recent establishment of international standards, intended to be used in the standardization of nucleic acid amplification technique (nat)-based assays for hcmv (karuthu and blumberg 2012) . patients with gastrointestinal and neurologic cmv disease often fail to exhibit cmv viremia and histopathology is necessary to establish diagnosis in these instances. treatment of active cmv disease requires a combination of immunomodulation, antiviral therapy with or without adjuvant therapy and if possible, reduction of immunosuppression (kotton et al. 2013; green et al. 2004 ). the mainstay of therapy is intravenous ganciclovir. the victor trial (valcyte in cmv disease treatment of solid organ recipients) demonstrated oral valganciclovir was not inferior to intravenous ganciclovir in mild to moderate cmv disease in solid organ transplant recipients (asberg et al. 2009 ). the current guidelines recommend renally adjusted intravenous ganciclovir 5 mg/kg twice daily or oral valganciclovir, 900 mg twice daily for mild cmv disease (kotton et al. 2013) . in severe cmv disease, intravenous ganciclovir is preferred with reduction of immunosupression despite the increased risk of rejection (de keyzer et al. 2011) . the use of adjuvant therapy with cmv-specific hyperimmune globulin or standard intravenous immunoglobulin may be considered in individuals with hypogammaglobulinemia, severe systemic infection, or in failure to respond to standard therapy (humar et al. 2010) . cmv resistance to ganciclovir has been noted in renal transplant recipients due to mutations in ul 97, the gene responsible for the first phosphorylation step in ganciclovir activation and ul 54, the gene responsible for dna polymerase (limaye et al. 2000) . cmv resistance should be considered when patients have worsening disease or persistent, unchanged viremia at 2 weeks of therapy and in such cases, genotype testing for mutations of the genes encoding ul 97 and ul 54 should be performed (weikert and blumberg 2008). treatment options for drug resistant cmv include the use of high dose ganciclovir, foscarnet, and cidofovir; however, no clinical trial data are available regarding optimal therapy options for resistant cmv. the use of novel agents including leflunomide and artesunate has been attempted as salvage therapy with varying success. several new antiviral treatment options are currently under investigation including maribavir and brincidofovir (an oral prodrug of cidofovir with less nephrotoxicity) for use in the treatment of drug resistant cmv (limaye et al. 2000) . epstein barr virus -human herpesvirus 4 (ebv) is a ubiquitous gamma herpes virus that remains latent in lymphocytes following primary infection. it is responsible for posttransplant lymphoproliferative disorder (ptld) which increases morbidity and mortality in the transplant population. approximately 62-79% of ptld cases have been associated with ebv (karuthu and blumberg 2012) . ptld most commonly occurs in the first year posttransplant (cockfield et al. 1993) . the risk factors for ptld include ebv naïve recipients who receive ebv seropositive organs, active primary ebv infection, younger recipient, coinfection by cmv and other viruses, prior splenectomy, second transplant, acute or chronic graft versus host disease, immunosuppressive drug regimen (okt3 or polyclonal antilymphocyte antibody), and the type of organ transplanted. kidney transplant recipients are at lower risk compared with other types of transplants and have an incidence of approximately 1-3% (gulley and tang 2010; allen et al. 2009; taylor et al. 2005) . the majority of symptomatic ebv infections in renal transplant recipients are primary infection likely related to transmission of donor virus. ebv disease can be asymptomatic or presents with a nonspecific febrile syndrome, lymphadenopathy, hepatosplenomegaly, atypicalþ lymphocytosis, hematologic disorders including anemia, leukopenia, thrombocytopenia, and organ-specific diseases like gastroenteritis, hepatitis, or pneumonitis (allen et al. 2009 ). ptld typically follows primary infection and frequently presents as a rapidly enlarging mass in the grafted organ, lymph nodes, bone marrow, or extranodal sites (manez et al. 1997) . ptld is divided into four major histopathologic subtypes as per the world health organization (who): early lesions, polymorphic ptld, monomorphic ptld, and classical hodgkin lymphoma type ptld. definitive diagnosis of ptld requires histopathologic confirmation by tissue excision biopsy with immunologic cell typing, cytogenetics, immunoglobulin gene rearrangements, and ebvspecific staining (allen et al. 2009 ). staging is performed by histologic types (monoclonal versus polyclonal, t cell versus b cell) and location (allograft, other organs, metastasis) (weikert and blumberg 2008) . clinical management of ptld typically involves reduction of immunosuppression which can lead to remission in 23-86% of the patients (weikert and blumberg 2008). antiviral therapy with acyclovir or ganciclovir is controversial and no evidence supports its efficacy (taylor et al. 2005) . rituximab (monoclonal antibody to cd20) is commonly used for treatment of ptld in recipients who failed reduction of immunosuppression alone (allen et al. 2009 ). in isolated graft ptld, surgical resection is an option (weikert and blumberg 2008) . in patients that fail the above strategies, ifn and ivig have been used with varying success and cytotoxic chemotherapy with radiation remains salvage therapy (green et al. 2004 ). there is no standardized therapy to prevent ptld. kdigo guidelines recommend monitoring ebv viral load in high risk renal transplant patients within the first week after transplant, then at least monthly for 3-6 months and then every 3 months for the rest of the first posttransplant year. additional viral load monitoring is recommended after treatment for acute rejection in high risk groups (children, ebv dþ/rà). outcomes with ptld in renal transplant patients vary according to the site involved. patients with isolated graft involvement have a 5-year survival of 68% compared with those patients with ptld extending beyond the allograft whose survival varied between 36% and 38% (weikert and blumberg 2008). human herpesvirus 1herpes simplex virus types 1 and 2 (hsv)and human herpesvirus 3varicella zoster virus (vzv)are alpha herpes viruses. hsv 1 has a seroprevalence of 60% in the adult population, while hsv 2 has a seroprevalence of 15% and vzv rates can be as high as 90% (green et al. 2004 ). the incidence of hsv disease in renal transplant recipients is approximately 53% and vzv 4-12% (patel and paya 1997) . hsv may cause primary infection following which the virus remains latent in the sensory nerve ganglia or more commonly causes reactivation infection. hsv may be seen as early as in the first posttransplant month in the absence of prophylaxis. hsv infection usually presents with oral or genital mucocutaneous lesions, occasionally pneumonitis, tracheobronchitis, esophagitis, hepatitis, encephalitis, or disseminated infection (green et al. 2004) . vzv causes localized dermatomal or multidermatomal or disseminated zoster with or without visceral involvement (pneumonitis, hepatitis, pancreatitis, encephalitis). pretransplant screening for prior vzv infection should be performed, and naïve patients should be vaccinated with live attenuated varicella vaccine before transplant whenever possible in order to avoid primary vzv infection posttransplantation (fehr et al. 2002) . since vzv is a live vaccine, it should not be given if transplant is expected within 4-6 weeks in order to avoid active shedding of virus at the time of transplant. posttransplant prophylaxis is recommended with acyclovir, valacyclovir, or ganciclovir (in those who need cmv prophylaxis) for approximately 1-3 months posttransplant in order to avoid hsv and vzv reactivation (green et al. 2004) . diagnosis of hsv and vzv infection can be made with pcr or direct fluorescence antibody for hsv from vesicular lesions, csf, or visceral tissue samples. serologies are rarely helpful in active infection owing to high seroprevalence. kdigo guidelines recommend that renal transplant recipients who develop less severe hsv or vzv infections can be treated with an appropriate oral antiviral agent (e.g., acyclovir, valacyclovir, or famciclovir), and those with systemic infection should be treated with intravenous acyclovir and a reduction in immunosuppressive medication and subsequently switched to an appropriate oral antiviral agent (green et al. 2004 ). the use of foscarnet, cidofovir, or topical trifluridine may be considered in patients with acyclovir resistant virus with careful monitoring of renal functions (kotton and fishman 2005; tan and goh 2006) . human herpesvirus 6 and human herpesvirus 7 (hhv 6 and hhv 7) are ubiquitous with high seroprevalence in adults. these viruses are common causes of fever in children and remain latent in lymphocytes following primary infection. hhv 6 uses the cd46 molecule as its receptor but may also infect other cell types, such as monocytes, and epithelial and endothelial cells. hhv 7 uses the cd4 molecule as its receptor and is more strictly lymphotropic. infection occurs as a result of reactivation in the first 4 weeks following transplant often in recipients not on cmv prophylaxis (singh and carrigan 1996) . clinical manifestations include fever, rash, hepatitis, interstitial pneumonitis, encephalitis, leukopenia, and myelosuppression. owing to its immunomodulatory effects, it is hypothesized that hhv 7 may act as a cofactor for hhv 6 and cmv reactivation, while both hhv 6 and hhv 7 may act as cofactors in the pathogenesis of cmv disease and acute rejection (kidd et al. 2000; chapenko et al. 2000; dockell and paya 2001) . the diagnosis of hhv 6 and hhv 7 is made by tissue immunohistochemistry or nat testing of peripheral blood lymphocytes. treatment includes reduction in immunosuppression and ganciclovir, but cidofovir and foscarnet have also been utilized (green et al. 2004; kotton and fishman 2005; dockell and paya 2001) . hhv 8 is associated with primary effusion lymphoma, kaposi's sarcoma, and multicentric castleman's disease. infection can be acquired as primary through the allograft or through reactivation of latent virus (diociaiuti et al. 2000; regamy et al. 1998 ). hhv 8 causes kaposi's sarcoma, the most common presentation in renal transplant recipients, through upregulation of vascular endothelial growth factor (vegf) receptor f1 k1/kdr in endothelial cells (stallone et al. 2005) . treatment includes reduction in immunosuppression and cytotoxic chemotherapy. sirolimus, an immunosuppressive drug used in renal transplant patients is thought to inhibit not only the production of vegf but also dampens its effect on endothelial cells (stallone et al. 2005 ). bk polyomavirus (bkv) and jc polyomavirus (jcv) belong to the family polyomaviridae. bkv is responsible for causing polyomavirus associated nephropathy (pvan) in 95% of cases and jcv in less than 5% of the cases. pvan occurs in 1-10% of patients with renal transplantation and causes renal allograft loss in 10-80% of cases dadhania et al. 2008) . the risk factors for bkv associated pvan include the use of potent immunosuppressive regimens, caucasian race, older age, diabetes mellitus, cadaveric renal transplant, and combined kidney and pancreas transplant trofe et al. 2003) . bkv is known to cause interstitial nephritis, ureteral stenosis, and ureteral stricture of the allograft kidney most commonly occurring within the first 3-4 months after renal transplant patients when immunosuppression is at its highest (randhawa and brennan 2006) . jcv less commonly causes pvan and is more frequently associated with progressive multifocal leukoencephalopathy (pml), a demyelinating disorder of the white matter presenting as neurologic impairment and dementia (phillips et al. 2004) . diagnosis of bkv includes the use of viral load assays (blood, urine), detection of viral cytopathic effect (decoy cells), nat, bkv-specific antibody, or histopathology (hariharan 2006) . kdigo guidelines recommend screening all renal transplant recipients for bkv with quantitative plasma nat at least monthly for the first 3-6 months after transplantation, then every 3 months until the end of the first posttransplant year, whenever there is an unexplained rise in serum creatinine, and after treatment for acute rejection. the guidelines suggest reducing immunosuppressive medications when bkv plasma nat is persistently greater than 10,000 copies/ ml (107 copies/l) (kdigo 2009). sustained high bk viremia in spite of reduction in immunosuppression may need additional antiviral therapy, although data regarding optimal treatment options are unknown. there are limited data regarding the effectiveness of leflunomide and/or cidofovir or the use of fluoroquinolones or ivig for treatment of bkv infection (randhawa and brennan 2006; josephson et al. 2006) . to date there is no effective treatment for pml. patients with allograft loss due to pvan have undergone successful retransplantation (hariharan 2006) . patients with chronic renal failure, in particular those receiving hemodialysis, are at increased risk for contracting hepatitis b virus (hbv). the prevalence of hepatitis b surface antigen (hbsag)positive patients has declined because of hbv vaccination, strict segregation of hbsag-positive patients in dialysis units, improved screening of blood products, and the use of erythropoiesis stimulating agents (karuthu and blumberg 2012) . approximately 2-10% of patients with a history of hbv prior to transplant will reactivate posttransplant (weikert and blumberg 2008) . serial monitoring of hbv dna every 3-6 months is required after transplantation as liver enzyme levels do not reflect infection status and elevated viral loads suggest resistance to therapy (levitsky et al. 2013) . in a meta-analysis conducted by fabrizi and his colleagues, hbsag seropositivity was an independent risk factor for allograft loss and posttransplant death (fabrizi et al. 2005) . the treatment options currently approved for chronic hbv include: ifn alpha, pegylated ifn, lamivudine, entecavir, telbivudine, tenofovir, and adefovir (fabrizi et al. 2005; chan et al. 2002; chang et al. 2010) . kdigo recommends that interferon treatment generally be avoided because of the high associated incidence of rejection. tenofovir or entecavir are preferable to lamivudine, to minimize the development of drug resistance, unless medication cost requires that lamivudine be used. during therapy with antivirals, hbv dna and alt levels should be measured every 3 months to monitor efficacy and to detect drug resistance. all hbsagpositive renal transplant recipients should receive prophylaxis with tenofovir, entecavir, or lamivudine. hbsag-positive patients with cirrhosis should be screened for hepatocellular carcinoma every 12 months with liver ultrasound and alpha feto-protein. patients who are negative for hbsag and have hbsab titer <10 miu/ml should receive booster vaccination to raise the titer to >100 miu/ ml (kdigo 2009). hepatitis c virus (hcv) infection has been increasingly recognized in end stage renal disease patients (esrd). donor-derived hcv may uncommonly occur after transplantation. screening of patients with esrd and testing renal transplant patients for newly acquired hcv should include nat (levitsky et al. 2013) . hcv-positive donors can be considered for hcv-positive recipients and possibly will be considered for hcv-negative recipients in the future given improved treatment options for cure of hcv that could be administered post transplant. hcv-infected renal transplant recipients have decreased survival and increased complication rates. posttransplant complications include glomerulonephritis (gn), posttransplant diabetes mellitus, and accelerated progression to cirrhosis with fibrosing cholestatic hepatitis (morales et al. 2010) . liver biopsy within 6-12 months of transplantation and subsequent biopsies are required for evaluation of liver disease posttransplant as 20-51% of patients may have normal liver enzyme levels with abnormal histologic features (ashry ahmed gheith 2011). hcv-infected recipients should be tested for proteinuria every 3-6 months, and patients with new onset proteinuria should undergo allograft biopsy (kdigo 2009) . the effect of immunosuppression on the progression of hcv-related liver injury and the management of immunosuppression in the hcvinfected renal transplant recipient remain uncertain. thus, it is preferable to treat hcv in transplant candidates prior to transplantation given the potential for improved outcomes with successful hcv treatment and the complications associated with treatment posttransplant. patients with a sustained virologic response to pretransplant treatment have a reduced risk for hcv recurrence and decreased posttransplant gn (domınguez-gil and morales 2009). options for treatment include interferon/ peginterferon alone or in combination with ribavirin. the risk of toxicity with the addition of ribavirin has limited the use of combination therapy in chronic kidney disease (ckd) patients. the availability of direct acting hcv protease and polymerase inhibitors has sparked new enthusiasm for treating hcv-infected ckd patients and studies are ongoing evaluating the use of these agents in ckd. if treatment cannot be given prior to transplant, kdigo recommends monotherapy with standard interferon for hcv-infected renal transplant recipients in whom the benefits of antiviral treatment clearly outweigh the risks (kdigo 2009). the use of direct acting hcv antivirals posttransplantation can also be considered and will likely be preferred in the future given improved tolerance and efficacy with these agents with an understanding that drug interactions with calcineurin inhibitors may occur.a study looking at 20 hcv-positive kidney transplant recipients (60% treated pre-transplant with interferon unsuccessfully) treated with direct acting antivirals posttransplant found that 100% cleared the virus and had a sustained virologic response at 12 weeks. the most common agents used were sofosbuvir and simeprevir (sawinski et al. 2016 ). human immunodeficiency virus (hiv) belongs to the family of retroviridae. with the introduction of antiretroviral therapy (art) in the mid-1990s, the incidence of hiv related deaths has been reduced. renal diseases related to hiv infection include hiv associated nephropathy (hivan), immune complex diseases, and thrombotic microangiopathy (frassetto et al. 2009 ). a total of 10% of patients with hiv develop hivan and it remains an important complication of hiv infection, progressing rapidly to end stage renal disease (esrd) (shahinian et al. 2000) . a large prospective clinical trial examining outcomes among 150 hiv + kidney transplant recipients reported 3-year patient and graft survival rates of 88.2% and 73.7%, respectively, which were similar to survival rates among a cohort of unmatched elderly (>65 years) hivnegative (hiv à ) kidney recipients (stock et al. 2010 ). the candidates for transplantation include those with well-controlled hiv infection with undetectable viral loads, cd4 >200 cells per microliter, and absence of untreatable infections or malignancies (blumberg et al. 2009 ). the most significant complications in this patient population posttransplant include increased rejection rates (up to 25%), managing drug interactions between art and immunosuppressive therapy and complications related to cardiovascular risk factors and hepatitis coinfection (blumberg et al. 2009 ). the choice of art should be based on susceptibility results and if possible, the use of protease inhibitors should be avoided owing to significant drug interactions with this class of art. with regards to immunosuppressive therapy, the use of thymoglobulin may result in prolonged depression of cd4 counts, whereas monocloncal anti-il2 receptor antibodies, such as basiliximab/daclizumab, have been shown to increase cd4 cell counts (ciuffreda et al. 2007; carter et al. 2006) . the risks of antilymphocyte therapy should be balanced with the risks of rejection in hiv-infected recipients. of note, hiv-positive donors can now be considered in hivpositive recipients. the various respiratory viruses that cause infection affecting the renal transplant patient population include adenovirus, respiratory syncytial virus (rsv), influenza, parainfluenza, human metapneumovirus, rhinovirus, and coronavirus (green et al. 2004) . clinical manifestations include upper respiratory tract infection, bronchitis, and pneumonia. in addition to respiratory illness, adenovirus is known to cause gastroenteritis, hemorrhagic cystitis, pancreatitis, meningoencephalitis, necrotizing hepatitis, and nephritis/renal dysfunction in renal transplant recipients (pham et al. 2003; alsaad et al. 2007) . infection with these viruses may also be associated with rejection (weikert and blumberg 2008) . prevention involves hand hygiene and the use of droplet precautions for those suspected of having infection. influenza vaccination is recommended prior to transplant and yearly following transplant. treatment of respiratory viral infection involves supportive care and antiviral medications. influenza can be treated with oseltamivir or zanamavir. ribavirin is approved for the treatment of rsv. adenovirus infection is treated with reduction of immunosuppression with consideration of cidofovir (ison 2006) . emerging viral pathogens include newly recognized viruses or previously known viruses that are either increasing or threatening to increase in incidence. some of the emerging viruses causing infections in renal transplant population include human t-cell leukemia virus type 1 (htlv-1), hepatitis e virus (hev), measles virus, rabies virus, lymphocytic choriomeningitis virus (lcmv), dengue virus (denv), west nile virus, and zika virus. case reports of adult t-cell leukemia (atl) following renal transplantation in htlv-1-positive patients have been documented, though in a case series of renal transplant recipients with long-term follow-up, no cases of atl or htlv-1-associated myelopathy (ham) developed (nakamura et al. 2005; tanabe et al. 1998) . hev may induce kidney injury with significant reduction in glomerular filtration rate. glomerular injuries such as membranoproliferative glomerulonephritis have been described in kidney transplant patients with acute and chronic hev infections (kamar et al. 2012) . the incidence of measles in transplant recipients is unclear. cases of subacute measles encephalitis (sme) have developed in renal transplant recipients. the clinical course is one of deteriorating mental status and treatment refractory seizures (waggoner and deresinski 2013) . worldwide, vector-borne viral disease is increasing in incidence and can be transmitted with blood products and organ transplantation. fatal cases of dengue have been reported within the first month following renal transplant (waggoner and deresinski 2013) . west nile virus has also been reported in transplant recipients with a high incidence of neuroinvasive disease and poor outcomes. zika virus is also now a concern. cases of donor-derived rabies in the sot population have been reported. patients typically developed encephalitis between 1 and 2 months posttransplant, and all symptomatic reported patients died (srinivasan et al. 2005) . cases of lcmv causing severe disease in organ transplant patients have been documented. the 4 clusters of lcmv infection occurred in the united states and involved kidney, liver, and lung transplants; symptoms included fever, abdominal pain, nausea, diarrhea, and altered mental status (srinivasan et al. 2005; barry et al. 2008; fischer et al. 2006) . two renal transplant recipients survived lcmv infection. ribavirin has been employed in some cases, though the benefits remain unclear (waggoner and deresinski 2013) . data regarding the incidence, screening and treatment options of the above-mentioned emerging viruses are limited. given the risk of donor-derived viral transmission, organs should not be accepted from donors with unexplained febrile or neurologic illness. in unclear cases, the risk of donor-derived infection should be balanced with the benefit of the transplant. bacterial infections after renal transplantation can be due to surgical complications at the time of transplantation, nosocomial infection, immunosuppression, or community-acquired infection. donorderived bacterial infections from the transplanted kidney or blood stream can occur as well. about 47% of kidney transplant recipients develop bacterial infections (patel and paya 1997) . occurring any time posttransplantation, urinary tract infections account for the overwhelming majority of these infections and are the most common bacterial infections prolonging or leading to re-hospitalization (wyner 1994) . enterococci, staphylococci, enteric gram-negative organisms, and p. aeruginosa are the most common bacteria isolated (wyner 1994) . bacterial pneumonia, postoperative wound infections, and bacteremia or sepsis, although less common, also prolong or lead to rehospitalizations after transplantation (karuthu and blumberg 2012) . common bacterial pathogens for these infections are gram-negative organisms, including multidrug resistant bacteria; gram positive organisms, including methicillin-resistant staphylococcus aureus (mrsa), and vancomycin-resistant entercococci (vre), as well as organisms more typically seen in immunocompromised patients such as listeria. months after the operation, bacterial pathogens include streptococcus species, mycoplasma, legionella, listeria, salmonella, and nocardia. trimethoprim-sulfamethoxazole (tmp-smx) prophylaxis has been shown to reduce the incidence of some of these infections. increased antimicrobial resistance, urgency of treatment, drug interactions, and toxicities, as well as the risk for clostridium difficile colitis all contribute to the complex decision making required for antimicrobial management. risk factors for urinary tract infection after transplantation are a prolonged period of hemodialysis before transplant, prolonged bladder catheterization, female sex, deceased donor transplant, kidney-pancreas transplant with bladder drainage, uretero-vesical stents, and an increased immunosuppressed state (karuthu and blumberg 2012; lapchik et al. 1992) . prophylaxis to lower the risk of infection after transplant with trimethoprim-sulfamethoxazole is routine (karuthu and blumberg 2012) . controversy regarding the exact dosing and duration of prophylaxis exists. typically it is given at a dose of 160 mg trimethoprim and 800 mg of sulfamethoxazole daily for 6-12 months (kdigo 2009 ). trimethoprim-sulfamethoxazole reduces the risk of uti and bacteremia (karuthu and blumberg 2012; patel and paya 1997) . symptoms of uti include frequency, urgency, and dysuria as well as nausea and vague abdominal complaints. some patients are asymptomatic. escherichia coli is the most common pathogen and an increasing number of pathogens are multidrug resistant. sensitivity testing is required. treatment of asymptomatic bacteriuria in the renal transplant recipient is controversial and is not routinely recommended (coussement and abramowicz 2013) . although not well studied, since utis in renal transplant patients are complicated, 7-14 days of antibiotics is a typical duration. removal of stents and catheters as well as drainage of abscesses are frequently required to prevent relapse and for cure. surgical wound infections, occurring at a rate of 3-4%, usually present within the first 4 weeks after transplant (ramos et al. 2008) . obesity, urine leaks, re-operation through the original incision, diabetes, high creatinine levels in plasma, and prolonged bladder catheterization are risk factors for wound infections (humar et al. 2001; khoury and brennan 2005) . improved organ procurement, preservation, and surgical techniques along with preoperative antibiotics all reduce the risk of subsequent postoperative wound infection. bacterial organisms causing these types of infections may be nosocomial and multidrug-resistant making antibiotic treatment difficult due to limited options or toxicities. source control with good wound care is critical in the management of these types of infections. although pneumonia is the most common bacterial infection in all solid organ transplant recipients, its incidence is lowest in those who have received a kidney (khoury and brennan 2005) . occurring early in the posttransplant period, cmv infection and rejection treatment with antilymphocyte preparations increase the pneumonia risk. hospital-acquired pneumonia due to resistant pathogens, such as mrsa, and extended spectrum beta lactamase (esbl) or carbapenemresistant (cre) gram-negative organisms are increasing in incidence and sometimes require nephrotoxic agents for treatment. communityacquired pneumonia can occur any time after transplantation and the incidence of communityacquired pneumonia specifically due to streptococcus pneumoniae can be lowered with vaccination. bacteremia and sepsis are most commonly due to a urinary source, followed by lung, wound, and abdomen (khoury and brennan 2005) . intravenous catheters also play a role. diabetes mellitus and posttransplant dialysis increase the incidence of sepsis which decreases the survival rate in these patients (abbott et al. 2001) . prompt treatment with broad spectrum antibiotics followed by rapid de-escalation to pathogen-specific therapy based on sensitivities is required. removal of foreign bodies such as intravenous catheters and stents is also necessary for cure. nocardia is a rare infection seen in the renal transplant recipient occurring in less than 4% of patients (wilson et al. 1989 ). trimethoprim-sulfamethoxazole prophylaxis used after transplant to prevent pneumocystis jiroveci pneumonia (pcp) likely prevents nocardia infection as well. nocardia asteroides is the most common species and causes pulmonary infections, including cavitary lesions and pleural effusions (patel and paya 1997) . other common sites of infection, due to dissemination, are central nervous system (cns) and cutaneous. all patients with nocardia should be evaluated for cns disease. allograft rejection, high-dose prednisone, azathioprine, instead of cyclosporine based immunosuppression, and neutropenia are risk factors for this infection (patel and paya 1997) . diagnosis is made by the identification of branching and beading rods on gram and modified acid fast staining and cultures of infected sites. antimicrobial susceptibility testing should be performed on all isolates. high dose trimethoprim-sulfamethoxazole sometimes in combination with amikacin is the treatment of choice, but allergic reactions and other side effects sometimes limit their use. alternatives include imipenem, minocycline, and ceftriaxone, but choices should be based on susceptibilities and site of infection (spelman 2016) . nocardia infections can relapse and prolonged therapy up to a year is recommended followed by chronic suppressive therapy (spelman 2016; arduino et al. 1993 ). listeria monocytogenes is a bacterial organism that is transmitted most commonly during summer and early fall to humans via the gastrointestinal tract from contaminated dairy products, raw vegetables, and meat. although more common during the first 2 months after transplantation, infection may occur at any point, and risk is increased with rejection therapy (patel and paya 1997) . infections involving the central nervous system, such as meningitis and meningoencephalitis, are most common and present with headaches, fever, meningismus, altered mental status, and possibly focal neurologic deficits including cranial nerve palsies and seizures (patel and paya 1997) . cerebrospinal fluid examination typically reveals a pleocytosis, mostly polymorphonuclear leukocytes, decreased glucose, and elevated protein, but as the name implies, a mononuclear predominance may occur instead. gram staining has a low sensitivity and may be negative or reveal gram positive bacilli which may be confused with diphtheroids. other sites of infection include bacteremia, pneumonia, endophthalmitis, and septic arthritis. while trimethoprim-sulfamethoxazole, used for p. carinii prophylaxis, may also prevent infection with listeria, the treatment of choice is intravenous ampicillin and gentamicin for up to 8 weeks in those with cns infections to prevent relapses. gentamicin is usually continued for a shorter duration, about 2 weeks if kidney function is stable. (gelfand 2016) . trimethoprim-sulfamethoxazole is an alternative treatment for those who are allergic to penicillin. decreasing immunosuppressive agents is sometimes, but not always necessary. legionella infections in renal transplant recipients most commonly occur early in the posttransplantation period, but can be seen any time, especially during episodes of rejection. legionella pneumophila is the most common species to infect humans, and although more commonly community-acquired, nosocomial transmission occurs (patel and paya 1997) . most infections are pulmonary including pneumonia, and abscess with cavitation. symptoms are typical of lung infections but also may include headache and diarrhea. a legionella urinary antigen test and culture of lower respiratory secretions on selective media are used for diagnosis. empiric treatment for legionella is appropriate while waiting for results. quinolone antibiotics, such as levofloxacin, are preferred over macrolides in renal transplant patients because of drug interactions between macrolides and immunosuppressive medications. initially given intravenously, quinolone antibiotics can be quickly deescalated to oral treatment when the patient has defervesced. renal transplant patients, especially those who are severely ill at presentation, should receive 21 days of treatment (yu 2016) . along with pcp and listeria, as noted above, prophylaxis with trimethoprim-sulfamethoxazole may also prevent legionella infection. immunosuppression increases the risk of developing mycobacterium tuberculosis (tb) disease. although the majority of tuberculosis infections in renal transplant recipients occur in the first 18 months, tb can occur any time after transplantation (khoury and brennan 2005) . its overall incidence is lower in the united states when compared to the rest of the world, and foreign-born recipients are at greatest risk. having a high index of suspicion is important in renal transplant patients because presentation can be atypical and pretransplant screening with tuberculin skin tests or ifn-gamma release assays are unreliable in chronic kidney disease patients due to anergy. extra-pulmonary sites of infection and disseminated disease occur in about a third of cases (karuthu and blumberg 2012). laryngeal, meningeal, skeletal, cutaneous, intestinal, and renal infections are examples of extra-pulmonary disease. fevers are common, but sweats and weight loss may be absent (patel and paya 1997) . screening prior to transplant should include a history regarding prior exposures, and treatment for tb, as well as a chest x-ray and urine afb culture. prophylaxis with isoniazid or rifampin should be offered to patients prior to transplantation with a history of inadequately treated tb, an abnormal chest x-ray suggestive of prior tb exposure, a positive ppd or ifn gamma assay, contact with someone with active tb, or a kidney from a ppd-positive donor in order to minimize reactivation disease after transplantation (khoury and brennan 2005) . patients receiving treatment for latent tb may undergo renal transplantation and complete their defined course afterwards with special attention to potential drug interactions and toxicities (karuthu and blumberg 2012) . diagnosis of tb after renal transplantation often requires a biopsy of the infected site with stains for acid fast bacilli and cultures for sensitivity testing. treatment of active disease after transplantation requires multiple drugs and should follow the american thoracic society, center for disease control, and infectious disease society of america guidelines (mmwr 2003) . special attention to drug toxicities and interactions with immunosuppressive agents is required. rifampin, in particular, decreases cyclosporine levels and increases the risk for rejection. fungal infections in kidney transplant recipients occur less frequently than in other solid organ transplant recipients. most present within the first 6 months after transplantation (hagerty et al. 2003 ) and can represent primary, reactivated, or donor-derived infection. those associated with geographic and environmental exposures include histoplasmosis, coccidioidomycosis, blastomycosis, and paracoccidioidomycosis. others are considered opportunistic and include infections such as candida, aspergillus, and cryptococcus (karuthu and blumberg 2012) . broad spectrum antibiotics, corticosteroids, diabetes mellitus, rejection therapy, cmv infection, and duration of pretransplant dialysis are risk factors (khoury and brennan 2005) . esophageal candidiasis, urogenital candidiasis, and pneumonia are the three most common sites of fungal infections in these patients (abbott et al. 2001) . clinical presentation may be nonspecific and diagnosis difficult due to testing limitations. positive cultures may represent colonization rather than infection with pathogens such as candida and aspergillus. cultures, antigen assays, serum galactomannan assays, and radiography may be helpful, but are not always diagnostic. subsequently, biopsy with pathology and cultures is considered the gold standard for diagnosing fungal infections (karuthu and blumberg 2012) . drug interactions and toxicities as well as immune reconstitution, due to lowering of immunosuppressive medications, further complicate the management of fungal disease in these patients and require expert advice (karuthu and blumberg 2012) . pneumocystis jiroveci (formerly pneumocystis carinii (pcp), protozoa) is a pathogen currently considered a fungus based on nucleic acid and biochemical analysis. presenting as pneumonia with interstitial infiltrates on chest x-ray within the first year after transplantation in those not receiving prophylaxis, mortality may be high. nonproductive cough and shortness of breath with rapid progression to hypoxia is a classic presentation. diagnosis is based on silver staining of deep respiratory specimens from induced sputum, bronchoalveolar lavage, or transbronchial biopsy (martin and fishman 2013) . the treatment of choice is high dose trimethoprim-sulfamethoxazole for 21 days with corticosteroids in hypoxic patients (partial pressure of oxygen of <70 mmhg on room air) tapered over 14 days. atovaquone or clindamycin plus pyrimethamine are alternative agents (martin and fishman 2013) . trimethoprim-sulfamethoxazole prophylaxis for 6-12 months after transplantation is highly effective in preventing this infection and should be administered to all renal transplant patients if tolerated. frequently used alternatives for prophylaxis in allergic patients include dapsone (if glucose-6 phosphate dehydrogenase levels are normal) and atovaquone. infection remains an important concern in patients undergoing kidney transplantation. attention to pretransplant screening of the potential organ donor and recipient is essential to optimizing transplant outcomes. advances in the management of transplant-related infections include the increasing use of rapid molecular diagnostic testing as well as improvements in the approach to prophylaxis and treatment. ongoing challenges include the need for prolonged immunosuppression to prevent organ rejection, drug-drug interactions, and the management of resistant and emerging pathogens. continued awareness of the risks, timing, and presentation of infection posttransplant and strategies to reduce its impact will contribute further to progress in the field of kidney transplantation. hospitalizations for bacterial septicemia after renal transplantation in the united states epstein-barr virus and posttransplant lymphoproliferative disorder in solid organ transplant recipients late-onset acute haemorrhagic necrotizing granulomatous adenovirus tubulointerstitial nephritis in a renal allograft nocardiosis in renal transplant recipients undergoing immunosuppression with cyclosporine long-term outcomes of cmv disease treatment with valganciclovir versus iv ganciclovir in solid organ transplant 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recipients viral infections affecting the skin in organ transplant recipients: epidemiology and current management strategies long-term results in human t-cell leukemia virus type 1-positive renal transplant recipients post-transplant lymphoproliferative disorders (ptld) after solid organ transplantation the association of viral infection and chronic allograft nephropathy with graft dysfunction after renal transplantation polyomavirus in kidney and kidney-pancreas transplant recipients epidemiology of kidney disease in the unites states. national institutes of health, national institute of diabetes and digestive and kidney diseases in: safdar a (ed) principles and practice of transplant infectious diseases nocardial infections in renal transplant recipients the evaluation and management of urinary tract infections in recipients of solid organ transplants treatment and prevention of legionella infection key: cord-266985-9qwttt2y authors: gale, p.; hill, a.; kelly, l.; bassett, j.; mcclure, p.; le marc, y.; soumpasis, i. title: applications of omics approaches to the development of microbiological risk assessment using rna virus dose–response models as a case study date: 2014-11-04 journal: j appl microbiol doi: 10.1111/jam.12656 sha: doc_id: 266985 cord_uid: 9qwttt2y t e in the amount of ‘omics’ data available and in our ability to interpret those data. the aim of this paper was to consider how omics techniques can be used to improve and refine microbiological risk assessment, using dose–response models for rna viruses, with particular reference to norovirus through the oral route as the case study. the dose–response model for initial infection in the gastrointestinal tract is broken down into the component steps at the molecular level and the feasibility of assigning probabilities to each step assessed. the molecular mechanisms are not sufficiently well understood at present to enable quantitative estimation of probabilities on the basis of omics data. at present, the great strength of gene sequence data appears to be in giving information on the distribution and proportion of susceptible genotypes (for example due to the presence of the appropriate pathogen‐binding receptor) in the host population rather than in predicting specificities from the amino acid sequences concurrently obtained. the nature of the mutant spectrum in rna viruses greatly complicates the application of omics approaches to the development of mechanistic dose–response models and prevents prediction of risks of disease progression (given infection has occurred) at the level of the individual host. however, molecular markers in the host and virus may enable more broad predictions to be made about the consequences of exposure in a population. in an alternative approach, comparing the results of deep sequencing of rna viruses in the faeces/vomitus from donor humans with those from their infected recipients may enable direct estimates of the average probability of infection per virion to be made. classical microbiological risk assessments (mras) consist of four steps: hazard identification, hazard characterization, exposure assessment and risk characterization (cac 1999) . there is now a wealth of literature on this subject, and mras have been developed for many pathogen/commodity combinations spanning a wide range of food types from ready-to-eat produce to animal products cooked in the home (e.g. cassin et al. 1998; gale 2005; nauta et al. 2007) . despite their potential contribution to mra, the use of 'omics' data in mra has to date been limited. therefore, the aims of this paper were to elucidate how omics data and the techniques used to generate and analyse such data can be used to improve and/or complement mra, to identify where barriers may exist to the incorporation of omics data into mra, and to consider where future omics data may prove useful. the focus is on the human oral dose-response relationship (drr) not only because some of the molecular processes governing the pathogen/host interaction are established, but also because there are known data gaps in the drr journal of applied microbiology 117, 1537--1548 © 2014 the society for applied microbiology for many pathogen/commodity combinations. the drr is used to translate the exposure through the oral route into a risk of infection/disease. in classical mras, the drr is based on human studies where different doses are fed to groups of volunteers (ward et al. 1986 ) and the proportion infected (or ill) at each dose is plotted against the dose. mathematical models are then fitted to those data and used to predict the probability of infection for doses not included in the volunteer study (haas et al. 1993; teunis et al. 2008) . over the last decade, there has been a huge increase in the amount of omics data available and in our ability to interpret those data using bioinformatics techniques. for genomics, the introduction of high-throughput next-generation sequencing (ngs) technologies in 2005 greatly increased the capability to sequence whole genomes from individual living organisms rapidly and cheaply, providing information on variation across the host population (pareek et al. 2011) . ngs has also enabled the 'deep sequencing' of viral genomes and effectively gives the sequence of every virus in the exposure dose in terms of a mutant spectrum (bull et al. 2012) . proteomics allows the identification of those proteins important to the cell in any given situation (neidhardt 2011) . the relative proportions of proteins represent the protein signature which changes in response to a challenge and could be used to identify proteins expressed in a host cell in response to infection by a virus, for example. the great potential of proteomics is to be able to detect previously unknown proteins and assign their cellular location and their contacts with other proteins (marchadier et al. 2011) . another approach which is considered here is 'glycoproteomics'. many proteins, particularly those on cell or pathogen surfaces and in the host immune system, have sugar units, called glycans, added to them. indeed, these glycans serve as receptors on cell surfaces for a number of pathogens including norovirus (nov). glycoproteomics characterizes proteins containing glycans. glycosylation (the synthesis of glycans) still remains practically unexplored at the proteome scale (doerr 2012) . there are relatively few pathogens for which human volunteer dose-response data are available, and in general, such data are absent for newly emerged pathogens. while animal models have been used together with calibration from human outbreak data (teunis et al. 2004) , other techniques are needed to obtain drr parameters. furthermore, account needs to be taken of the more susceptible members of the human population. the focus of the first part of this paper is on the development of a mechanistic dose-response model for estimating the probability of initial infection through the oral route. the second part of this paper considers the exciting possibility that ngs data of virus isolated from donor and recipient human cases may facilitate a direct estimation of the probability of initial infection. the final part addresses the problems in applying omics approaches to predicting disease progression and spread to other tissues once infection has established in the gastrointestinal tract. nov is selected as an example to demonstrate the concepts because there are dose-response data from human volunteer studies (teunis et al. 2008) together with a wealth of molecular information on receptor binding for this pathogen. an omics-based approach for estimating the probability of initial infection by virus in oral exposure infection by an oral pathogen is defined as the multiplication of organisms within the host, followed by excretion (haas et al. 1999) . biologically, the infection process for a virus such as nov is comprised of a number of steps as set out in table 1 . each pathogen particle of genomic form, i, has a probability p ij of initiating infection in a host individual of genomic form, j. the probability, p ij , is expressed as:where each p xij is the probability of a virus particle i completing the step x of the four steps (table 1) in human j. calculating p ij for every j in the host population and every i in the virus population would allow definition of the drr for initial infection if the relative proportions of each genomic form in the virus and human populations were known. with ngs, it is conceivable that gene sequences for at least some js and most is could be available in the future, thus giving the relative proportions of genomic forms. the data required and feasibility of assigning a quantitative probability to p xij for steps 1-4 in table 1 are now discussed using nov as the primary case study. probability of pathogen overcoming the immediate host defences to reach its cellular receptor there are a number of immediate host defences in the mouth and gastrointestinal tract including the mucus barrier, microbial interference, decoy receptors and the innate immune system. the histoblood group antigens (hbgas) are glycans to which certain viruses, including nov and rotavirus, selectively bind. hbgas are present on both decoy receptors in the saliva and on mucin, the main protein component of mucus (boshuizen et al. 2005; huang et al. 2009; mcguckin et al. 2011 ). shanker et al. (2011 propose that local flexibility in the viral capsid protein could allow the nov to disassociate from salivary hbga due to the ph change during its passage through the gastrointestinal tract. however, the molecular mechanism is not understood, and the problem for estimation of p 1 is that the effect of ph on protein structure is going to be very difficult to predict from gene sequences. a further complication is that while mucin production is increased during rotavirus infection through a mechanism involving the innate immune system, other pathogens such as helicobacter pylori suppress synthesis of gastric mucin (mcguckin et al. 2011) . thus, information on p 1 from one pathogen cannot be applied to another. mucin glycosylation patterns change with age of the host altering the antiviral capabilities (boshuizen et al. 2005; mcguckin et al. 2011 ) but providing a molecular basis for understanding of variation in p 1 with age of the host. the gastrointestinal tract microbiota prevent enteric disease by pathogens, including rotavirus, a trait referred to as colonization resistance or microbial interference (wardwell et al. 2011) . however, the relationship between gastrointestinal viruses and commensal bacteria or how gut microbial populations affect infection by enteric viruses is not well understood (wardwell et al. 2011) . interestingly, the microbiota modulate mucin production and glycosylation (mcguckin et al. 2011 ) and therefore indirectly affect p 1 . sequencing the human microbiome will provide a 'deep' genetic perspective on individual bacteria (hmp 2012), thus shedding light on the host-pathogen-microbiome interaction, although it will be difficult to translate this into an estimate of p 1 . the innate immune system provides an immediate defence for the host against pathogens in a nonspecific and generic manner. central to the innate immune system is the recognition of specific, repeating structures (pathogen-associated molecular patterns or pamps) on the pathogen by the pathogen recognition receptors (prrs) of the host (de koning et al. 2012) . a goal towards developing a mechanistic drr would be to predict whether pamps on a given pathogen could be detected by the prrs in a given host j. this requires an understanding of the molecular structure of the prr in combination with its pamp ligand. crystal structures of prr proteins complexed with pathogen pamp ligands have been published (takeuchi and akira 2010) , and in combination with genomics, data could assist in predicting the strength of binding in different individuals. however, predicting effects of changes in gene sequence on binding may not be straightforward because although polymorphisms in host prr genes attenuate recognition of pathogen pamp ligands, the amino acids corresponding to these polymorphisms do not seem to directly contact the ligand molecules themselves in the case of salmonella flagellin at least (uenishi et al. 2012) . indeed, the differences may be subtle because it is not yet understood how the innate immune system distinguishes commensal bacteria from pathogenic bacteria, that is friend or foe (de koning et al. 2012) . furthermore, there are numerous prrs with each recognizing different parts of the virus (triantafilou et al. 2011) giving alternative pathways, which will greatly complicate the estimation of the probability of a virus escaping the innate immune system prior to binding to its cellular receptor in step 2. if the virus fails to bind to a receptor on a cell in the lumen of the gastrointestinal tract, it will pass harmlessly through. the binding of virus to its receptor(s) on the host cell surface is a highly specific molecular interaction and controls host cell susceptibility and hence defines which cell types and tissues are infected (tissue tropism). the binding of nov capsid protein to its hbga receptor table 1 breakdown of the initial infection process into four steps for building a mechanistic dose-response relationship for rna viruses through the oral route: information needs step description current information gaps and understanding requirements 1 overcoming initial host defences including mucus barrier, the microbiome, decoy receptors and the innate immune system to reach the cellular receptor in the gastrointestinal tract* mechanism of dissociation of virus from the decoy receptor and mucus, changes in glycosylation. mechanisms of microbial interference 2 binding of virion to its cellular receptor in the gastrointestinal tract predicting specificity of glycan binding by viruses. genetic markers of resistance and frequency of specific alleles in host population 3 entry of virion to the host cell better understanding on proteolytic cleavage of viral proteins by host cell proteases 4 replication of the virus in the infected cells (to give a mutant cloud) and capsid assembly proteomics may facilitate identification of host protein/virus protein interactions that control the regulatory pathways within the infected cell *microbiome includes the indigenous bacteria in the gastrointestinal tract, and decoy receptors are host molecules which mop up viruses before they reach their target cells. on the intestinal cells is well understood at the molecular level through x-ray crystallography studies (bu et al. 2008; choi et al. 2008) . the ultimate goal would be to predict pathogen binding on the basis of nucleotide sequences of the viral capsid protein and the known (or ultimately predicted) structures of the hbga glycan receptors. however, despite high conservation of the amino acid residues of the viral capsid protein that interact with hbga, sequence similarity is not a predictor of the hbga binding pattern (bull and white 2011) . indeed, closely related capsids can display distinct hbga binding patterns, whereas genetically unrelated capsids from separate genogroups can display comparable hbga binding patterns (bull and white 2011) . this will complicate the application of omics approaches to predicting pathogen specificity from viral protein sequences, and hence estimating p 2 . further complications are that structures of nov from genogroups gi and gii in complex with hbgas reveal different modes of interaction (hansman et al. 2011) , and novs recognize hbgas in a distinct strain-dependent manner (shanker et al. 2011) . the novs are constantly evolving with antigenic drift in the capsid protein giving different hbga binding specificities shanker et al. 2011) . genomics data, together with crystal structure data, could be used to identify changes in surface moieties of viral capsids although interpretation of impact on function may be more difficult. an example of this has been observed by ngs of fmdv in laboratory-infected cattle (wright et al. 2011) . the laboratory strain of virus had several positively charged amino acids in its coat protein selected for by negatively charged heparin sulphate in cell culture. in the cow, those positive charges reverted. while rotavirus targets the intestinal epithelium, it is not known exactly which cells in the intestine nov binds to (chan et al. 2011) . to develop a mechanistic drr, it is a requirement to know which cells are targeted initially by the virus as different cells express different surface proteins. it is concluded that even for the best understood systems, prediction of p 2 for individual virus i x human j interactions will be extremely difficult. using genomics data to assess the proportion of host population which is resistant to virus on the basis of receptor binding in step 2 the genetics of resistance to nov infection provides an excellent example of how genetic markers could be used for calibrating a mechanistic drr. lindesmith et al. (2003) reported that 29% of humans studied in one population were homozygous recessive for the fut2 gene and were unable to synthesize a functional enzyme to make the glycan part of the hbga receptor to which the nov binds. these populations do not express the receptor required for nov binding and are thus highly protected against nov infection, that is p 2 % 0. indeed, none of those individuals developed an infection after exposure, however large the dose of virus ingested. about 20% of europeans are in this highly protected category (kindberg and svensson 2009) . de rougemont et al. (2011) could not accurately calculate the dissociation constant for nov and its hbga receptor, although the association is likely to be strong. thus, in the case of nov, it may be possible to simplify step 2 into assuming that p 2 ? 1 for those individuals who are genetically susceptible in having a functional fut2 gene, while p 2 = 0 for those who are genetically resistant through having a nonfunctional fut2 gene. this simplifies the calibration of p 2 in the drr into determining the proportion of the population which is resistant, an application to which genomics approaches are particularly well suited. however, the possibility that the pathogen could use alternative receptors would need to be eliminated, as in the case of the bacterium h. pylori, which is known to use other carbohydrate receptors in addition to hbga (de mattos 2012) such that p 2 is the sum of probabilities of parallel routes, each with a different p value. the breakdown of volunteer dose-response data of ward et al. (1986) into a two component model has been proposed previously for rotavirus (gale 2005) . however, unlike the case for nov, there are limited molecular data on binding of rotavirus to its receptor or indeed on the genetic basis for variation between populations in susceptibility. also, the binding process for rotavirus may be more complicated than for nov with several cell surface molecules implicated (isa et al. 2008 ) and evidence that rotavirus surface protein may open up tight junctions between cells in the intestinal lumen allowing access for binding to the basal side of the cell (nava et al. 2004; boshuizen et al. 2005) in addition to the apical side of the cell used by nov. predicting such sequences of events would be difficult even if proteomics approaches identified the hubs of protein-protein interactions involved. the probability of host cell entry (p 3 ) by the virus (given it has bound to the cellular receptor in step 2) depends, for some viruses at least, on the efficiency of cleavage of a viral protein by a host cell proteolytic enzyme. this specific cleavage process typically occurs after the newly synthesized virions are released from the cells in the previous round of infection. the ability of the host cells' proteolytic enzymes to cleave the viral proteins may control entry to the cell in the next round of infection, as in the case of highly pathogenic avian influenza virus, for which the tissue tropism and pathogenicity are determined by whether the host cells' proteolytic enzymes can cleave the viral haemagglutinin protein. this explains the ability of highly pathogenic h5 and h7 avian influenza strains to replicate in many organs outside the respiratory tract, thus defining their highly pathogenic phenotype (chaipan et al. 2009 ). predicting p 3 depends in part on being able to predict whether the viral protein is not only specifically cleaved (i.e. in the right place) but also efficiently cleaved. this is more of a traditional biochemistry/bioinformatics problem dependent on understanding the structure of the virus protein and the mechanism/specificity of the host proteolytic enzymes. thus, for example, misasi et al. (2012) found that specific differences in the requirements of filoviruses for a host cell proteolytic enzyme (called cathepsin b) are correlated with sequence polymorphisms at residues 47 and 584 in the viral glycoproteins. the use of cathepsin proteases as host factors for virus entry is a general property of members of the filovirus family. those authors were able to predict that a newly identified ebola virus is cathepsin b dependent. this approach may be applicable to assessing cell entry requirements for other viruses in the future and would contribute to our understanding of p 3 in different host cell types. in this respect, the rate of cleavage in different tissues would affect the magnitude of p 3 . cell entry by sars coronavirus also requires cleavage of the virus spike protein by cellular cathepsin (bosch et al. 2008) . the mechanism of nov entry is not clear although there is some evidence that a proteolytic process could be necessary for replication (tan et al. 2006) . isolation of viruses by cell culture relies on replication of the virus within the cells, and using several types of cell culture provides a suitable environment for many viruses (leland and ginocchio 2007) . indeed, transgenic expression of the appropriate receptor (step 2) may make a previously nonsusceptible cell susceptible (e.g. as for poliovirus (pv)) (leland and ginocchio 2007) . thus, the default position for step 4 would be to assume that p 4 ? 1, and that viral rna replication, capsid synthesis and assembly can occur in the host cell given the virus nucleoprotein core has gained entry through steps 2 and 3. supporting this, replication of the viral rna is performed by rna-dependent rna polymerase (rdrp) present in the virus core, and synthesis of new virus capsid proteins takes place on the host cell ribosomes. indeed, viral proteins seize control of cellular translation factors, and host signalling pathways not only ensuring viral proteins are produced but also stifling the innate host defences that limit the capacity of infected cells to produce virus (walsh and mohr 2011) . understanding the regulation of these pathways may enable refinement of the estimate of p 4 . thus, viruses commonly use host cell survival mechanisms to their own advantage and in particular the pi3k/akt pathway, which suppresses apoptosis (programmed cell death) so giving time for completion of viral replication (eden et al. 2011) . phosphorylation of nov rdrp by the host akt pathway decreases the polymerase activity (eden et al. 2011 ) and may be an antiviral strategy, which should be considered in estimating p 4 . central to understanding virus replication in step 4 is the application of proteomics to determine protein-protein contacts in situ in the infected cell and thus how the individual virus proteins associate with host cell proteins and hence work together. it is envisaged that proteomic techniques could be used to identify hubs in virus-infected host cells in a similar way to those in the bacterium bacillus subtilis (marchadier et al. 2011) . of importance for assessing the infectivity of a virus in terms of the drr is how each viral protein interacts with the host proteins in the different hubs, many of which correspond to regulatory pathways (marchadier et al. 2011) . a proteomic analysis (using mass spectrometry) of human host proteins associated with the rdrp of h5n1 avian influenza virus identified 400 proteins, which may have a role in the induction of apoptosis and in innate antiviral signalling or are cellular rna polymerase accessory factors, that is, involved in the regulation of gene expression in the cell (bradel-tretheway et al. 2011) . such proteomics data by identifying host cell proteins bound to viral polymerases and so defining protein hubs may enhance our understanding of virus replication and assembly and would enable qualitative refinement of the estimation of the p 4 . nov is an example of an rna virus. indeed, many viruses which infect humans, including pv, hepatitis c virus (hcv) and human immunodeficiency virus (hiv), have rna genomes and differ from dna pathogens such as bacteria and protozoa in that the replication of the viral rna during infection of a cell (step 4) is prone to error (smidansky et al. 2008) , so generating a cloud of mutants (i.e. the progeny virions differ in sequence, albeit by only one or two bases in 10 000). this is called a mutant spectrum and is central to quasispecies theory (lauring and andino 2010) . the key point is that the individual viruses excreted by an infected person in faeces or in vomitus, and therefore present in an oral exposure, are slightly different in sequence, albeit related. ngs (or deep sequencing) provides sequences for all the viral variants in an exposure together with their relative frequencies (i.e. the mutant spectrum) and has been used to detect minority sequence variants for a number of human viruses (wright et al. 2011) . this is clearly an important first step for making predictions on the drr. indeed, ngs of rna viruses recovered from an infected person may give an indication of the number of virions that initiated the infection and has shown that in the case of hcv and hiv, infection arises from a single variant often representing initial infection by one or a few viral particles (wang et al. 2010; bull et al. 2012) . moreover, the infection process may represent a bottleneck such that only one or two viral variants in the exposure dose successfully establish infection . thus, in the case of nov, only minor variants at frequencies as low as 0á01% were successfully transmitted to establish a new infection (bull et al. 2012 ). this suggests the risk of infection from a nov virion, on average, is approx. 0á0001 (assuming the exposure dose was high, which is likely through faeces or vomitus, such that the complete spectrum of variants was present). if every nov had a high probability of infection, then bull et al. (2012) would have seen a range of nov sequences in the infected persons corresponding to most of the individual virions in the exposure dose. indeed, the two major variants in the donor person accounted for 99% of the variants but were not present in either of the two recipient persons. similarly, for hcv, only one or two viral variants successfully established infection (wang et al. 2010; . wang et al. (2010) write that, 'nothing in our data rules out the possibility that many additional particles were in the initial inoculum but did not replicate in the new host'. this raises the question of how many viruses were in the dose in the original exposure event but had very low, or even negligible, probabilities of infection due to the bottleneck. the number of footand-mouth disease virus (fmdv) rna molecules/ml of serum measured in infected pigs by rt-pcr is three to four orders of magnitude higher than the numbers of plaque-forming units (pfu) measured by conventional methods (rodriguez-calvo et al. 2011). thus, each pfu 'dosage' in the human rotavirus volunteer study (ward et al. 1986 ) may also comprise many individual virions, again consistent with the id 50 being high in terms of virions. thus, although haas et al. (1993) estimated the risk of infection from one pfu of rotavirus to be 27%, that pfu may comprise thousands of virions. this is not inconsistent with the results of bull et al. (2012) for nov, which suggest the id 50 is in the order of 7000 virions as only 0á01% can get through the bottleneck and initiate infection. thus, if the probability of infection by a virion is 0á0001, then according to the negative exponential dose-response model, about 7000 virions would be needed for a 50% chance of infection. ngs data on the viral mutant spectrum give complementary information on some of the steps of the mechanistic infection process in table 1 . thus, bull et al. (2012) speculate that only a few nov variants can bind to hbga in step 2 effectively filtering out the majority of nov variants in an exposure (representing a bottleneck). indeed, the minor variant transmitted differed from the dominant donor variant in an amino acid change directly adjacent to the primary hbga binding site in the capsid. for hiv, the variants responsible for establishing infection in a new host have unique phenotypic properties that contribute to the establishment of infection with specific amino acids that increase viral entry efficiency (step 3) as well as unique glycosylation patterns that aid attachment to host receptors (step 2) (bull et al. 2012 ). thus, a challenge for developing a mechanistic drr for initial infection is determining which variants are important for a given step (table 1) . central to disease progression and viral pathogenesis by rna viruses is the fidelity of replication by rdrp smidansky et al. 2008) , which controls the complexity of the mutant spectrum produced from step 4. the spreading of pv, for example, to the nervous system is secondary to the initial establishment of infection in the gastrointestinal tract. thus, pv titres peak in the small intestine and brain at day 1 and 5, respectively (vignuzzi et al. 2006) . different individual progeny viruses in the mutant spectrum have different cell and tissue tropisms reflecting their specific gene sequences compared to the original infecting virus. specifically, a pv mutant with a high-fidelity rdrp (i.e. produces a reduced range of variants) replicated in the small intestine, kidney and spleen but failed to reach the brain and spinal cord and to produce neuropathology in mice (vignuzzi et al. 2006) . however, restoration of the standard mutant spectrum complexity by subjecting the mutant pv to chemical mutagenesis led to a neuropathogenic mutant spectrum with the virus reaching the brain and spinal cord (vignuzzi et al. 2006; smidansky et al. 2008 ). thus, enhanced opportunities for co-operation between more diverse clades of mutant spectra (arising from the high mutation rates) facilitate the virus's reaching specific target organs, thereby increasing viral loads and chances of transmission. however, too much genotypic variability may retard the progress of the virus infection by producing too many defective variants (smidansky et al. 2008) . a conclusion of smidansky et al. (2008) is that rdrp fidelity is tuned by natural selection to achieve the optimal genotypic diversity in the mutant spectrum and, consequently, viral competitiveness in the dynamic and hostile environment of the host cell. furthermore, the level of diversity and type of diversity needed may change in different tissues as different host challenges are encountered. it may also change during the infection cycle and may vary depending on the route of transmission (wright et al. 2011; bull et al. 2012) . bull et al. (2012) speculate that deep sequencing may yet identify differences between nov transmitted through faeces compared to those in vomitus, for example. the huge complexity of the interactions together with the lack of understanding of how rdrp controls fidelity at the molecular level (smidansky et al. 2008 ) severely limits prediction of disease progression. a further complication is that the host response to pathogen is also dynamic with the host hbga specificity to nov changing over time together with changes in host mucin glycosylation during rotavirus infection such that the mucins become more potent in inhibiting the virus (boshuizen et al. 2005) . there is also evidence that disease progression could to a certain extent depend on chance (i.e. it is probabilistic). thus, deep sequencing has shown that fmdv may take different evolutionary trajectories even within the same cow, albeit in different isolated compartments, namely the front left foot vs the back right foot (wright et al. 2011 ). this specifically confounds prediction for the individual host. although it is not possible at present to interpret pathogenic potential from viral gene sequences, there are examples of markers in the virus sequence known to be associated with disease-related traits. thus, the amino acid threonine at residue position 33 in rdrp is associated with pandemic potential in nov gii (eden et al. 2011) , and in the case of fmdv in cattle, an amino acid change in one of the four capsid proteins is associated with persistent infection (horsington and zhang 2007) . identification of such markers may enable more broad predictions to be made about disease potential in a population. genetic markers on the host may give an indication of resistance to disease progression through being able to mount an effective immune response. for example, understanding polymorphisms in the prr genes (wang et al. 2011 ) and human leucocyte antigens (hla) genes (hoof et al. 2012 ) together with interferon coding regions (thomas et al. 2009 ) may indicate the proportion of the population able to mount more effective responses to an rna virus such as hcv. the hla proteins present viral peptides to the t cells in the acquired immune response, and it is well documented that individual persons differ in their hla proteins and that some are more protective than others against viruses including hiv (wang et al. 2012) , hcv, hepatitis b virus and herpes simplex type 1 virus. hoof et al. (2012) found that protective hla molecules show a preference to present peptide fragments from conserved hcv proteins (e.g. core and ns5b), while nonprotective hla molecules preferentially target hcv proteins that are significantly less conserved (e.g. ns5a). taken together, their analysis suggests that by targeting the most constrained, and thereby conserved, parts of the hcv genome, 'protective' hla molecules reduce the potential of hcv to escape the cytotoxic t-cell response of the infected host. thus, it may be possible to predict viral clearance from the type of viral protein, which is presented by the hla. carlsson et al. (2009) identified capsid residues in nov that are evolutionarily conserved. x-ray crystallography structures are available for human hla molecules bound to viral peptides including sars coronavirus (roder et al. 2008a) , and other studies for swine leucocyte antigen (the porcine hla equivalent) complexed with peptides from 2009 pandemic h1n1 influenza virus or ebola virus have recently been published (zhang et al. 2011 ). however, predicting whether conserved or less conserved peptide fragments are presented by the hla and the effectiveness of viral peptide presentation for the purpose of estimating disease progression will be difficult. an additional problem is that glycans play a role in antigen presentation by the hla, with glycosylation perhaps fine-tuning the binding properties (ryan and cobb 2012) . some viral proteins target the hla proteins and interfere with antigen presentation (roder et al. 2008b) . other viral countermeasures in disease progression include the ability of the nov capsid to evolve to evade the memory immune response and escape from herd immunity while retaining its ability to bind any of several hbgas . thus, many individuals are susceptible to reinfection by nov (lindesmith et al. 2010) . ngs of viral sequences may allow identification of immune escape mutations which impair the ability of both t-cell responses and neutralizing antibodies to maintain immune control as shown for hiv-1 (henn et al. 2012) . these rapid, low-frequency viral variants are not detected by conventional sequencing approaches and demonstrate the application of genomics in understanding the dynamic interplay in immune control. some viruses, for example porcine reproductive and respiratory syndrome virus (prrsv), prevent the protective immune response by suppressing of certain host genes. as an example of the use of transcriptomics, wysocki et al. (2012) compared differences in rnas from lungs of high and low prrsv burden pigs and found that in high burden pigs, expression of cellular genes associated with host protection was delayed. this review summarizes some of the potential applications of omics approaches to developing drrs for infection through the oral route by rna viruses, with a view to better defining the role of omics data in mra. while some specific issues of the drr can be addressed by omics data (e.g. estimating resistant/susceptible ratios in the host), there are clearly other areas where substantial progress in our understanding of the molecular interactions is required before omics data can be applied to mra (if they can ever be applied). although viruses have far fewer genes than bacterial pathogens, the nature of the mutant spectrum in the case of rna viruses greatly complicates the application of omics approaches to the development of mechanistic dose-response models. this is compounded by the fact that the frequencies of some important variants that initiate infection are as low as 0á01% as in the case of nov and hcv (wang et al. 2010; bull et al. 2012) , raising the question of which sequence to look at in terms of specific interactions with the host. here, the initial infection process in the gastrointestinal tract has been broken down into four mechanistic steps (table 1 ). the breadth of the fields considered in these steps demonstrates the need for a multidisciplinary approach including not only omics data but also conventional biochemistry and molecular biology inputs, which provide detail on the molecular components of each step. thus, currently protein structure determination by crystallography studies underpins our understanding of the specific molecular interactions (e.g. prr with pamp ligands in step 1 or nov capsid binding to hbga in step 2). the overall conclusion is that the molecular mechanisms of each step in table 1 are not sufficiently well understood at present to enable quantitative estimation of probabilities on the basis of omics data. however, omics approaches will make a major contribution to specific areas of the initial infection process in particular in accommodating genetic variation both in the pathogen and in the host population through extensive sequencing. the human microbiome should be included here too because the gastrointestinal microbiota play a major role in the protection against infection in step 1, although the molecular mechanisms are not established. genome sequencing for individual humans enables identification of genetic differences between susceptible and nonsusceptible persons, and it may be possible in future to tailor risk assessment to different host populations in terms of genetic susceptibility to initial infection. a promising application of such genetic markers is to define the proportion of the host population, which is more susceptible to initial infection by a specific pathogen. current examples include the fut2 gene, which affects nov binding in the gastrointestinal tract in step 2 and potentially polymorphisms in the host prr genes in step 1 (wang et al. 2011) . thus, the great strength of genome sequencing is in giving information on the distribution and proportion of susceptible genotypes in the host population rather than in predicting specificities of interactions from the amino acid sequences concurrently obtained. indeed, even for the best characterized step in the infection process, namely nov capsid/hbga binding for step 2, changes in amino acid sequence cannot be used to predict changes in binding specificity and affinity. furthermore, not only are there many gaps in our understanding of how the few viral proteins actually function and interact with host cell proteins but also there are often multiple mechanisms and alternative molecular pathways used by the virus, for example inhibition of host gene expression and translation by sars coronavirus (lokugamage et al. 2012) . the potential of proteomics approaches is in contributing to our understanding of how the few viral proteins interact with a multitude of cellular proteins within the host cell particularly in step 4. host glycans play a central role in the pathogen infection process including binding of virus to specific receptors in steps 1 and 2 and also in the immune system. the example with hbga glycans in nov binding (step 2) is simple in the sense that a single mutation in a gene (the fut2 gene coding for the enzyme that synthesizes hbga) results in the absence of a functional receptor. however, not all relationships involving glycans and omics data are as simple as this, and the link between a single mutation in a protein-coding gene and susceptibility to infection may be the exception rather than the rule. glycans have complex structures which may be changed during infection as the host tries to evade the pathogen, and also during development of the host (mcguckin et al. 2011) . prediction of glycan structures from dna sequences is not possible currently, and unlike gene expression, it is not clear how cells control glycan structures. thus, our current understanding of glycoproteomics and glycan function is a major limitation in developing a mechanistic drr for viruses. the ultimate aim in the application of omics and molecular biology approaches would be in taking the drr beyond the classic volunteer-derived model for infection in a broad group of (typically) healthy individuals into the realms of predicting disease progression and morbidity at the individual host level, based on the genetic information of the host and virus. however, although genomics approaches may enable consideration of the virus as a dynamic and evolving system, as opposed to a static single entity within the infected host, predicting the progression of an rna virus in terms of its ability to spread to new tissues (e.g. nervous system) or to evade the host acquired immune system and so persist, resulting in chronic infection, is fraught with difficulties. this is due to both the complexity of the pathways and the nature of how variants in the rna virus mutant spectrum interact. thus, the variants collectively and cooperatively contribute to the characteristics of the viral population and can express different phenotypic traits such that our ability to predict the outcome of an infection is limited (lauring and andino 2010) . however, interaction of mutant spectra could help elucidate in the future the mechanisms by which higher exposure doses increase the probability of illness, given infection has occurred. thus, for nov, teunis et al. (2008) reported that infected subjects had a dose-dependent probability of becoming ill, ranging from 0á1 (at a dose of 10 3 genomes) to 0á7 (at 10 8 genomes). genomics data should increase our repertoire of genetic markers both in the host and in the virus giving better understanding of those factors affecting disease progression. as an alternative to attempting to use omics data to develop a mechanistic drr, ngs may provide empirical data for calibrating a drr. thus, the data obtained by ngs of virus in donor and recipient hosts (e.g. that obtained for nov by bull et al. 2012) show great promise for directly estimating the probability of infection by a virion while avoiding the uncertainties associated with the role of viral proteins, glycan interactions and mutant spectra. ngs of virus in donor and recipient provides a unique insight into the infection process in terms of the frequency of the variant which initiated infection and demonstrated in the case of nov that 99á99% of virions, including the major variants, do not initiate infection (bull et al. 2012) . this suggests most of the virions in an exposure have a very low probability of infection. ngs has shown that different virions in a high-dose exposure have different sequences and some are better adapted at initiating infection than others (bull et al. 2012) . in conclusion, it is anticipated that omics approaches can make a contribution to specific areas of the initial infection process. the emphasis will be on omics approaches, together with traditional biochemistry techniques, as a tool to understand more fully the mechanisms of each step in the initial infection process, although it seems certain that it will be an extremely difficult task to interpret this information quantitatively. genomics data will continue to give information on the frequency of genes affecting susceptibility to initial infection within the host population. lack of knowledge on glycans together with difficulties in predicting the functional effects of amino acid changes in key proteins poses challenges. it is concluded that predicting disease progression (given initial infection has occurred) for rna viruses at the level of the individual host is not possible. this reflects the lack of understanding in variability of the virus, evolution of the virus during progression of the disease, cooperation between different clades of mutant spectra and the countermeasures by both the host and virus, together with chance factors. however, molecular markers in the host and virus may provide useful information on disease progression for risk assessment. the potential use of ngs to compare mutant spectra of viruses in the exposure dose presented by the infected donor and in the infected recipient as a method to give direct estimates of probability of infection should be explored further. cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class 1 fusion protein upstream of rather 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subversion of the host protein synthesis machinery hepatitis c virus transmission bottlenecks analysed by deep sequencing tlr7 and tlr8 gene variations and susceptibility to hepatitis c virus infection high-throughput, high fidelity hla genotyping with deep sequencing human rotavirus studies in volunteers: determination of infectious dose and serological response to infection current concepts of the intestinal microbiota and the pathogenesis of infection beyond the consensus: dissecting within-host viral population diversity of foot-and-mouth disease virus by using next-generation genome sequencing identifying putative candidate genes and pathways involved in immune responses to porcine reproductive and respiratory syndrome virus (prrsv) infection crystal structure of swine major histocompatibility complex class 1 sla-1*0401 and identification of 2009 pandemic swine-origin influenza a h1n1 virus cytotoxic t lymphocyte epitope peptides we thank prof. trevor drew of ahvla for his helpful comments. no conflict of interest declared. key: cord-015487-iugrhyaq authors: nicolson, garth l. title: chronic bacterial and viral infections in neurodegenerative and neurobehavioral diseases date: 2008-05-01 journal: lab med doi: 10.1309/96m3bwyp42l11bfu sha: doc_id: 15487 cord_uid: iugrhyaq often, patients with neurodegenerative or neurobehavioral diseases have chronic, neuropathic infections that could be important in disease inception, disease progression, or increasing the types or severities of signs and symptoms. although controversial, the majority of patients with various neurodegenerative or neurobehavioral conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, alzheimer’s disease, parkinson’s disease, and autistic spectrum disorders, show evidence of central nervous system or systemic bacterial and viral infections. for example, using serology or polymerase chain reaction evidence of chlamydia pneumoniae, borrelia burgdorferi, mycoplasma species, human herpesvirus-1 and -6, and other bacterial and viral infections revealed high infection rates that were not found in control subjects. although chronic infections were not found in some studies, and the specific role of chronic infections in neurological disease pathogenesis has not been determined or is inconclusive, the data suggest that chronic bacterial or viral infections could be common features of progressive neurodegenerative and neurobehavioral diseases. neurodegenerative diseases are chronic degenerative diseases of the central nervous system (cns) that cause dementia. for the most part, the causes of these brain diseases remain largely unknown. 1 they are characterized by molecular and genetic changes in nerve cells that result in nerve cell degeneration and ultimately nerve dysfunction and death, resulting in neurological signs and symptoms and dementia. 1, 2 in addition to neurodegenerative diseases, there are also neurobehavioral diseases that mainly, but not exclusively, appear in the young, such as autistic spectrum disorders (asd) that encompass autism, attention deficit disorder, asperger's syndrome, and other disorders. 3 there appear to be genetic links to neurodegenerative and neurobehavioral diseases, but the genetic changes that occur and the changes in gene expression that have been found in these diseases are complex and not directly related to simple genetic alterations. 1, 4 in addition, it is thought that nutritional deficiencies, environmental toxins, heavy metals, chronic bacterial and viral infections, autoimmune immunological responses, vascular diseases, head trauma and accumulation of fluid in the brain, changes in neurotransmitter concentrations, among others, are involved in the pathogenesis of various neurodegenerative and neurobehavioral diseases. [1] [2] [3] [5] [6] [7] [8] one of the biochemical changes found in essentially all neurological, neurodegenerative, and neurobehavioral diseases is the overexpression of oxidative free radical compounds (oxidative stress) that cause lipid, protein, and genetic structural changes. [5] [6] [7] [8] [9] oxidative stress can be caused by a variety of environmental toxic insults, and when combined with genetic factors, pathogenic processes could result. 10 an attractive hypothesis for the causation or promotion of neurological disease involves chronic bacterial or viral toxic products, which result in the presence of excess reactive oxygen species and culminate in pathologic changes. 11, 12 infectious agents may enter the cns within infected migratory macrophages, they may gain access by transcytosis across the blood-brain barrier, or enter by intraneuronal transfer from peripheral nerves. 11 cell-wall-deficient bacteria, principally species of chlamydia (chlamydophila), borrelia, brucella (among others), bacteria without cell walls, such as mycoplasma species, and various viruses are candidate infectious agents that may play important roles in neurodegenerative and neurobehavoral diseases. [12] [13] [14] since they are usually systemic, such infections can affect the immune system and other organ systems, resulting in a variety of systemic signs and symptoms. [15] [16] [17] [18] amyotrophic lateral sclerosis amyotrophic lateral sclerosis (als) is an adult-onset, idopathic, progressive neurodegenerative disease affecting both central and peripheral motor neurons. patients with als show gradual progressive weakness and paralysis of muscles due to destruction of upper motor neurons in the motor cortex and lower motor neurons in the brain stem and spinal cord, ultimately resulting in death, usually by respiratory failure. 19, 20 the overall clinical picture of als can vary, depending on the location and progression of pathological changes found in nervous tissue. 21 in als, the role of chronic infections has attracted attention with the finding of enterovirus sequences in a majority of spinal cord samples by polymerase chain reaction (pcr). 22, 23 this finding is not without controversy, since others failed to detect enterovirus sequences in spinal cord samples from patients with or without als. 24, 25 nonetheless, infectious agents that penetrate the cns may play a role in the etiology of als, although evidence for a transmission of an infectious agent or transfer of an als-like disease from man-to-man or man-toanimal has not been demonstrated. 26 the presence of systemic mycoplasmal infections in als patients has been investigated with pcr methods. 27, 28 our studies indicated that 100% of gulf war veterans diagnosed with als (n=8 from 3 different nations) had systemic mycoplasmal infections. 27 all but 1 patient had mycoplasma fermentans, and 1 veteran from australia had a systemic m. genitalium infection. in approximately 80% of nonmilitary (unrelated to military patients) als patients from the united states, canada, and great britain (n=28), blood mycoplasmal infections were also found. 27 of the mycoplasma-positive civilian patients who were further tested for m. penetrans, m. fermentans, m. hominis, and m. pneumoniae, most were positive for m. fermentans (59%), but other mycoplasma species, such as m. hominis (31%) and m. pneumoniae infections (9%), were also found. some of the civilian als patients had multiple mycoplasmal infections; however, labmedicine.com multiple mycoplasmal infections were not found in the military patients with als. 27 in another study in mexico, 10 of 20 als patients showed evidence of systemic mycoplasma species by analysis of their blood by pcr. 28 another chronic infection that is commonly found in als patients who live in certain areas is borrelia burgdorferi, the principal etiologic agent of lyme disease (ld). for example, als patients who live in new york, an ld-intense area, were examined for b. burgdorferi infections, and over one-half were found to be seropositive for lyme borrelia compared with 10% of matched controls. 29 in addition, some patients diagnosed with als were subsequently diagnosed with neuroborreliosis. 30 a survey of the literature indicates that spirochetal forms have been observed for some time in the cns tissue of als and other neurodegenerative diseases. 31 thus, a byproduct of ld may be progression to als, but this is probably only possible in some ld patients who have the genetic susceptibility genes for the neurodegenerative disease and who have other toxic exposures. 32, 33 amyotrophic lateral sclerosis patients also have other chronic infections, including human herpesvirus-6 (hhv6), chlamydia pneumoniae, and other infections. 34, 35 a suggestion that retroviruses might be involved in als and other motorneuron diseases 36 prompted mccormick and colleagues 37 to look for reverse transcriptase activity in serum and cerebrospinal fluid (csf) of als and non-als patients. they found reverse transcriptase serum activity in one-half of als cases but in only 7% of controls (p<0.008). interestingly, only 1 of 25 als csf samples contained reverse transcriptase activity. 37 the exact role that infections play in the pathogenesis or progression of als is not known. they could be cofactors in als pathogenesis, or they could simply be opportunistic infections that cause morbidity in als patients, such as the respiratory and rheumatic symptoms and other problems often found in als patients. they could also be involved in the progression of als rather than in its inception. although the exact cause of als remains unknown, there are several hypotheses on its pathogenesis: (a) accumulation of glutamate causing excitotoxicity; (b) autoimmune reactions against motor neurons; (c) deficiency of nerve growth factor; (d) dysfunction of superoxide dismutase due to mutations; and (e) chronic infection(s). [22] [23] [24] [27] [28] [29] [31] [32] [33] [34] none of these hypotheses is exclusive, and als may have a complex pathogenesis involving multiple factors. 34 future studies should determine more precisely the role of chronic bacterial and viral infections in the pathogenesis and progression of als. multiple sclerosis (ms) is the most common demyelinating disease of the cns, and it can occur in young as well as older people. in ms, inflammation and the presence of autoimmune antibodies against myelin and other nerve cell antigens are thought to cause the myelin sheath to break down, resulting in decrease or loss of electrical impulses along the nerves. 38, 39 in the progressive subset of ms, neurological damage occurs additionally by the deposition of plaques on the nerve cells to the point where nerve cell death occurs. in addition, breakdown of the blood-brain barrier in ms is associated with local inflammation caused by glial cells. 38, 39 the clinical manifestations of demyelinization, plaque damage, and blood-brain barrier disruptions are variable but usually include impaired vision, alterations in motor, sensory, and coordination systems, and cognitive dysfunction. often these are cyclic (relapsing-remitting subset) over time, but a substantial ms subset progresses without remitting. 39 there is strong evidence for a genetic component in ms. 40, 41 although it has been established that there is a genetic susceptibility component to ms, epidemiological and twin studies suggest that ms is an acquired, rather than an inherited, disease. 42 the possibility that ms is linked to chronic infections has attracted attention. 43, 44 in fact, ms patients show immunological and cytokine elevations consistent with chronic infections. [44] [45] [46] a possible infectious cause for ms has been under investigation for approximately the last decade, and patients have been examined for various viral and bacterial infections. 44, 47 one of the most common findings in ms patients is the presence of antibodies and dna of c. pneumoniae in their csf. [47] [48] [49] for example, sriram and colleagues 48 examined relapsing-remitting (n=17) and progressive (n=20) ms patients for the presence of c. pneumoniae in csf by culture, pcr, and immunoglobulin reactivity with c. pneumoniae elementary body antigens. they were able to isolate c. pneumoniae from 64% of ms patients' csf versus 11% of patients with other neurological diseases. high rates of pcr-positive (momp gene) patients (97% ms-positive versus 18% with other neurological diseases) as well as serology-positive patients (86% ms-positive, confirmed by enzyme-linked immunosorbant assays [elisa] and western blot analysis) were found in ms. 48 further examination of ms patients for oligoclonal antibodies against c. pneumoniae revealed that 14 of 17 patients were positive, whereas none of the control non-ms patients had antibodies that were absorbed by c. pneumoniae elemental body antigens. 49 other studies have also found evidence for the presence of c. pneumoniae in ms patients but at lower incidence. fainardi and colleagues 50 used elisa techniques and found that highaffinity antibodies against c. pneumoniae were present in the csf of 17% of 71 ms cases compared with 2% of 52 patients with noninflammatory neurological disorders. they found that the majority of the progressive forms of ms were positive compared with patients with remitting-relapsing ms. the presence of c. pneumoniae antibodies were also found in other inflammatory neurological disorders (n=51), and thus it was not specific to ms. 50 using immunohistochemistry, sriram and colleagues 51 performed a study of formalin-fixed cns tissue from ms and non-ms neurological disease controls and found that in a subset (7 of 20) of ms patients, chlamydial antigens were localized to ependymal surfaces and pariventricular regions. staining was not found in 17 cns tissue samples from other neurological diseases. frozen tissues were available in some of these ms cases, and pcr amplification of c. pneumoniae genes was accomplished in 5 of 8 cns tissue samples from ms patients but none in 17 frozen cns tissues from other neurological diseases. in addition, they examined csf sediment by immuno-goldlabeled staining for chlamydial antigens and found by electron microscopy that the electron-dense bodies resembling bacterial structures correlated with pcr-positive results in 10 of 11 ms cases. 51 the same group also used different nested pcr methods to examine additional c. pneumoniae gene sequences in the csf of 72 ms patients and linked these results to mri evidence of ms-associated lesions. 52 similarly, grimaldi and colleagues 53 linked the presence of c. pneumoniae infection with abnormal mri results in 23 of 107 ms patients with more progressive disease. in addition, a higher rate of c. pneumoniae transcription was found by dong-si and colleagues 54 in the csf of 84 ms patients. the above, among other data, 55-57 support the presence of c. pneumoniae in the cns of ms patients, at least in a subset of more progressed patients that are most likely the progressive forms of ms. not all studies have obtained evidence, however, for the presence of c. pneumoniae 58, 59 or other bacteria 60 in the cns of ms patients. hammerschlag and colleagues 61 used nested pcr and culture to examine 12 frozen brain samples from ms patients but could not find c. pneumoniae in any of the tissue samples. alternatively, in one study, c. pneumoniae was found at similar incidence in ms and other neurological diseases, but only ms patients had c. pneumoniae in their csf. 59 swanborg and colleagues 62 have reviewed the evidence linking c. pneumoniae infection with ms and have concluded that they are equivocal due to negative reports, and they also speculated that specific genetic changes may be necessary to fulfill the role of such infections in the etiology of ms. another possible reason for the equivocal evidence linking ms etiology with infection, such as c. pneumoniae, is that multiple coinfections could be involved. in addition to c. pneumoniae found in most studies, ms patients could also have mycoplasma species, b. burgdorferi, and other bacterial infections as well as viral infections. 63 when multiple infections are considered, it is likely that >80% of ms patients have obligate intracellular bacterial infections caused by chlamydia (chlamydophila) or other bacteria that can be intracellular, such as mycoplasma, borrelia, and other infections. these infections were found only singly and at very low incidence in age-matched subjects. 63 in spite of these findings, others did not find evidence of mycoplasma species in brain tissue (n=30), csf, or peripheral blood (n=57) of ms patients. 64 viruses have also been associated with ms. certain viruses have been found in ms patients, such as hhv6, but these viruses have also been found at lower incidence in control samples. 62 sanders and colleagues 65 used pcr to examine postmortem brain tissue (n=37) and controls (n=61) for the presence of neurotrophic viruses. they found that 57% of ms cases and 43% of non-ms neurological disease controls were positive for hhv6, whereas 37% and 28%, respectively, were positive for herpes simplex virus (hsv1 and hsv2) and 43% and 32%, respectively, were positive for varicella zoster virus; however, these differences did not achieve significance, and the authors concluded that "an etiologic association to the ms disease process [is] uncertain." they also found that 32% of the ms active plaques and 17% of the inactive plaque areas were positive for hhv6. 65 challoner and colleagues 66 used sequence difference analysis to search for pathogens in 86 ms brain specimens. using pcr, they found that >70% of the ms specimens were positive. they also used immunocytochemistry and found staining around ms plaques more frequently than around white matter; nuclear staining of oligodendrocytes was also seen in ms samples but not in controls. 66 using immunofluorescent and pcr methods, hhv6 dna has also been found in peripheral leukocytes in the systemic circulation of ms patients. 67, 68 however, using pcr methods, others did not find herpesviruses in the peripheral blood or csf of ms patients. 69, 70 although significant information (reviewed in 43, 44, 70 ) points to an infectious process in ms, this remains a controversial concept. as evidence emerges of new possible pathogens in ms, such as a new putative retrovirus, 71 these reports must be intensively examined and further studies initiated. since most studies have found that the progressive form of ms, rather than relapsingremitting forms of ms, were associated with chronic infections, infections might be more important in ms progression than in its inception. various infections may also nonspecifically stimulate the immune system. 43 as in other neurodegenerative diseases, multiple factors appear to be involved in the pathogenesis of ms. thus, like als, ms progression may turn out to be more likely linked to chronic infections, rather than its inception. alzheimer's disease (ad), the most common cause of dementia, is a collection of brain disorders usually found in aged patients. the disease is characterized by slow, progressive loss of brain function, especially notable by lapses in memory, disorientation, confusion, mood swings, changes in personality, language problems (such as difficulty in finding the right words for everyday objects), loss of behavioral inhibitions, loss of motivation, and paranoia. the prognosis and course of ad varies widely, and the duration of illness can range from a few years to over 20 years. during this time, the parts of the brain that control memory and thinking are among the first affected, followed by other brain changes that ultimately result in brain cell death. 72 alzheimer's disease is characterized by distinct neuropathological changes in the brain. among the most notable are the appearance of plaques and tangles of neurofibrils within brain nerves that affect nerve synapses and nerve-nerve cell communication. both of these structural alterations involve the deposition of altered amyloid (ab) proteins. 73, 74 although the cause of ad is not known with any certainty, the formation of the amyloid plaques and neurofiber tangles may be due to genetic defects and resulting changes in the structure of ab proteins, neurotoxicity caused by chemicals or other toxic events, inflammatory responses, oxidative stress and increases in reactive oxygen species, loss of nerve trophic factors important in nerve physiology, and loss of nerve cell transmission. [73] [74] [75] [76] [77] brain infections in ad have only recently become an important topic. [78] [79] [80] one pathogen that has attracted considerable attention is c. pneumoniae. 81, 82 as mentioned above, this intracellular bacterium has a tropism for neural tissue, 81 and it has been found at high incidence in the brains of ad patients (17 of 19 patients in brain areas of typical ad-related pathology) by pcr and immunohistochemistry methods. 82 chlamydia pneumoniae has also been found in nerve cells in close proximity to neurofibrillary tangles. 82, 83 this microorganism can invade endothelial cells and promote the transmigration of monocytes through human brain endothelial cells into the brain parenchyma. 84 although c. pneumoniae has been found in the brains of most ad patients studied, 77, 81 and this infection results in amyloid beta (ab) plaque formation in mice injected with c. pneumoniae, 85 some investigators have not found an association of c. pneumoniae infection with ad using pcr or immunohistochemistry. 86, 87 in addition to c. pneumoniae, evidence has been forthcoming that ad patients also have other bacterial infections, such as b. burgdorferi. 88 this infection has been examined in ad cases by serology, culture, western blot, and immunofluorenscence 89, 90 ; however, others could not find evidence of b. burgdorferi in ad patients. 91, 92 the presence of intracellular infections, like b. burgdorferi in ad patients, has been hypothesized to be a primary event in the formation of labmedicine.com ad amyloid plaques by forming "congophilic cores" that attract amyloid materials. 93 multiple reports show that ad nerve cells are often positive for b. burgdorferi. [88] [89] [90] 93, 94 in addition to the hypothesis that intracellular microorganisms may provide "cores" for the attraction of amyloid materials, the induction of reactive oxygen species, lipid peroxidation, and the breakdown of the lysosomal membranes releasing lysosomal hydrolases are also thought to be important in amyloid deposition. 94 although the possibility that infections may be important in ad pathogenesis is attractive, some negative reports where investigators have not confirmed the presence of infections, such as b. burgdorferi, in ad patients, indicate that this is still controversial (reviewed in 91, 94 ). herpes simplex virus infections have also been found in ad, and an interesting relationship has developed between the presence of hsv1 in ad. 95 it had been noted previously that hsv1 but not a related neurotrophic virus, varicella zoster virus, was found often in ad brains and may be linked to patients who have the ad risk factor apoe e4 allele. 96, 97 in ad, hsv1 is thought to be involved in the abnormal aggregation of beta amyloid or ab fragments within the brain by reducing the amount of full length amyloid precursor protein and increasing the amount of the ab fragment from this precursor. 98 recently, wozniak and colleagues 99 showed that hsv1 infection of cultured glial and neuronal cells results in a dramatic increase in the intracellular levels of beta amyloid forms, whereas the levels of native amyloid precursor protein decreased. this is similar to what has been found in mice infected with hsv1, indicating that hsv1 is probably involved directly in the development of senile-associated plaques. another herpesvirus, hhv6, has also been found in ad patients, but it is thought that this virus is not directly involved in ad pathogenesis, but it may exacerbate the effects of hsv1 in apoe e4 carriers. 100 in spite of the evidence that ad has been associated with, for example, c. pneumoniae, hsv1, or other infections, robinson and colleagues 101 have stressed caution in concluding that infections act as a trigger or cofactor in ad. in particular, there is a paucity of experimental evidence that pathogens can elicit the neuropathological changes and cognitive deficits that characterize ad. they also stress that there is a need for consideration of systemic infections as potential contributors to the pathogenesis of ad. parkinson's disease (pd) is characterized by akinesia, muscular rigidity, and resting tremor. also present are autonomic dysfunction, olfactory disturbances, depression, sensory and sleep disturbances, and frequently dementia. 102 parkinson's disease pathology indicates a progressive loss of the dopamine neurons of the substantia nigra together with the presence of lewy bodies and alpha-synuclein. more extensive brain degeneration also occurs, from the medulla oblongata to the cerebral cortex. 103, 104 although there is consensus among investigators that age-related inclusion bodies and protein aggregations or defects in their degradation occur in pd, their cause and role in pd pathogenesis remains a subject of intense research. 103, 104 some evidence suggests a relationship between pd and specific genetic changes, such as changes in the genes affecting mitochondria, protein degradation, organelle trafficking, and vesicular fusion, and in proteins involved in oxidative stress or antioxidant function. 102 inflammation has also been associated with pd pathology. 105 similar to other neurodegenerative diseases, the pathogenesis of pd has been proposed to be due to multiple genetic and neurotoxic hits that produce oxidative damage and cell death; however, in the case of pd the relevant targets of toxic events are neuromelanin-containing dopaminergic neurons of the substantia nigra. 104, 106 a case-control study in italy indicated that multiple environmental factors and genetic background were statistically related risk factors for pd. 107 among these, toxic exposures (over many years) and trauma early in life may be important in the pathogenesis of pd. 108 for example, early life exposure to brain injury, chemicals, or infections may initiate a cyclic inflammatory process involving oxidative damage, excitotoxicity, mitochondrial dysfunction, and altered proteolysis that later in life results in substantia nigra neuron death by apoptosis. [109] [110] [111] the possible role of chronic infections in pd pathogenesis has been proposed for a number of years. 109 one infection found in pd that has aroused considerable interest is the presence of chronic gastrointestinal helicobacter pylori, as treatment of this infection offered some relief to late stage cachexia in pd patients who were receiving l-dopa. 112 indeed, pierantozzi and colleagues 113 found that helicobacter pylori-infected pd patients had reduced l-dopa absorption and increased clinical disability, while treating this infection increased l-dopa absorption and reduced clinical disability. 114 helicobacter pylori may not be directly involved in pd, but its systemic presence could affect the progression and treatment of pd, probably by stimulating inflammation and autoimmunity. 115 inflammation and autoimmune responses have also been attributed to other chronic infections found in pd. [115] [116] [117] indeed, experimental models of pd have been developed using neurological, viral, or bacterial infections to initiate the pathogenic process. 118, 119 in humans, spirochetes have been found in lewy bodies of pd patients. 29 other infections, such as viral encephalitis, 120 aids-associated opportunistic infections of the basal ganglia, 121 coronavirus, 122 and other infections, 63, 123, 124 have been found in pd and could be important in stimulating inflammation and autoimmune responses. richy and mégraud 125 have stressed, however, that more rigorous investigations will be required to establish whether a causal link exists between infections and pd. autism spectrum disorders (asd), such as autism, attention deficit disorder, asperger's syndrome, etc, are neurobehavioral diseases of primarily the young where patients generally suffer from an inability to properly communicate, form relationships with others, and respond appropriately to their environment. such patients do not all share the same signs and symptoms but tend to share certain social, communication, motor, and sensory problems that affect their behavior in predictable ways. these mostly children often display repetitive actions and develop troublesome fixations with specific objects, and they are often painfully sensitive to certain sounds, tastes, and smells. 3, 126, 127 the causes of asd are unknown but appear to include genetic defects, heavy metal, and chemical and biological exposures, which are probably different in each patient. 3, 4, [126] [127] [128] moreover, in asd patients, there may be similarities in genetic defects and environmental exposures that are important in patient morbidity or in illness progression. chronic infections appear to be an important element in the development of asd. 14, 129 in some patients, there are a number of nonspecific chronic signs and symptoms, such as fatigue, headaches, gastrointestinal and vision problems, and occasional intermittent low-grade fevers and other signs and symptoms that are generally excluded in the diagnosis of asd. 130 although nonspecific and not always related to infection, these signs and symptoms suggest that asd patients could suffer from bacterial or viral infections. 14, 130 indeed, increased titers to various viruses as well as bacterial and fungal infections have been commonly seen in asd patients, 14, [129] [130] [131] although epidemiological evidence for an association of childhood infections in the first 2 years of life and asd is lacking. 132 in addition, environmental exposures to chemicals and heavy metals also appear to be important in the development of asd. 126, 127, 133 although controversial, the relationship between asd and heavy metals may involve the role of multiple vaccines in asd pathogenesis. 126, 127 asd patients often show their first signs and symptoms after multiple childhood immunizations. 126 rimland 126 noted that the sharp rise in autism rates occurred only after the multiple vaccine mmr came into widespread use, and now in some states in the u.s., children receive as many as 33 vaccines before they can enroll in school. such vaccines often contain mercury and other preservatives, 127 and some may also contain contaminating bacteria, as found in veterinary vaccines. 134 previously we found that 42 veterans of the gulf war with chronic fatiguing illnesses (gulf war illness) exhibited multiple nonspecific signs and symptoms similar to chronic fatigue syndrome/myalgic encephalomyopathy (cfs/ms). 135, 136 interestingly, their symptomatic children (n=35) were often diagnosed with autism or attention deficit disorder, 2 disorders that fall under asd. 137, 138 in our study, approximately 42% of gulf war illness patients had mycoplasmal infections, and almost all of these (approximately 82%) were single infections, usually m. fermentans. when the few multiple-infection cases were examined, most were found to have combinations of m. fermentans plus either m. pneumoniae, m. hominis, or m. genitalium. in contrast, in healthy control subjects (n=70), only 8.5% were positive for any mycoplasmal infection and all of these were single infections of various types. 137, 138 when we examined the immediate family members (n=107) of veterans with gulf war illness, we found that more than 53% had positive tests for mycoplasmal infections and showed symptoms of cfs. among the cfs-symptomatic family members, most (>70%) had mycoplasmal infections compared with the few nonsymptomatic family members who had similar infections. when the incidence of mycoplasmal infections was compared within families, the cfs-positive family members were more likely to have mycoplasmal infections compared with nonsymptomatic family members (p<0.001). 137 symptomatic children (mostly asd, n=35) were also infected with m. fermentans, and this was not seen in aged-matched control subjects. although some nonsymptomatic family members did have mycoplasmal infections (approximately 10%), this was not significantly different from the incidence of mycoplasmal infections in healthy control subjects (8.5%). 137, 138 examining asd patients (n=28) who were not in military families for systemic mycoplasmal infections showed that the majority (approximately 54%) were postive for mycoplasmal infections. however, in contrast to the children of gulf war illness patients who for the most part had only 1 type of mycoplasmal infection, m. fermentans, the civilian children tested positive for a variety of mycoplasma species. we also tested a few siblings without apparent signs and symptoms, and for the most part few had these infections. 138 in another study we examined the blood of asd patients (n=48) from central and southern california and found that a large subset (>58%) of patients showed evidence of mycoplasma infections compared with age-matched control subjects (odds ratio=13.8, p<0.001). 14 asd patients were also examined for c. pneumoniae (8.3% positive, odds ratio=5.6, p<0.01) and hhv6 (29.2% positive, odds ratio=4.5, p<0.01). the results indicated that a large subset of asd patients display evidence of bacterial and/or viral infections (odds ratio=16.5, p<0.001). 14 recently, asd patients have been examined for b. burgdorferi infections. various studies revealed that 22% to 30% of asd patients (n=76) have borrelia infections. 139 the incidence of borrelia infections in asd patients may be related to lyme disease (ld) distribution, with some ld-intense areas having high prevalence and other areas having low prevalence. chronic fatigue syndrome (cfs) is characterized by unexplained, persistent, long-term disabling fatigue plus additional signs and symptoms, including neurophysiological symptoms. 140 brain imaging studies showed that cfs patients are dysfunctional in their ventral anterior cingulate cortex function, and they also have other brain mri abnormalities. 141, 142 most cfs patients also have immunological abnormalities (reviewed in 143, 144 ) . a large subset of cfs patients can be characterized by the presence of chronic bacterial and viral infections, 16, 17, [144] [145] [146] [147] [148] although this has not been seen in all studies. 149 for example, using the blood of cfs patients (n=100) and pcr procedures, an overwhelming majority of patients showed evidence of multiple, systemic bacterial and viral infections (odds ratio=18.0, p<0.001). 17 chronic fatigue syndrome patients had a high prevalence (51%) of 1 of 4 mycoplasma species (odds ratio=13.8, p<0.001) and often showed evidence of co-infections with different mycoplasma species, c. pneumoniae (8%, odds ratio=8.6, p<0.01), and active hhv6 (30%, odds ratio=4.5, p<0.001). 17 in a separate study, the presence of these infections has also been related to the number and severity of signs/symptoms in 200 cfs patients. 146 a a sizable percentage of cfs/me patients are also infected with b. burgdorferi. 148, 150, 151 other infections are also found in cfs patients, such as cytomegalovirus, enteroviruses, and human herpesvirus-7 (reviewed in 43 ). lyme disease (ld) is the most common tick-borne disease in north america. it is caused by a tick bite and the entry of the spiral-shaped spriochete b. burgdorferi and other coinfections. 152 after incubation for a few days to a month, the borrelia spriochete and coinfections migrate through the subcutaneous tissues into the lymph and blood where they can travel to near and distant host sites. 153 transplacental transmission of b. burgdorferi and coinfections can occur in pregnant animals, including humans, and blood-borne transmission to humans by blood transfusion is likely but unproven. the tick-borne ld coinfections can and usually do appear clinically at the same time. 154 labmedicine.com since the signs and symptoms of ld overlap with other chronic conditions, ld patients are often diagnosed with other illnesses, such as cfs or chronic arthritis. 150, 151, 153 about onethird of ld cases start with the appearance of a round, red, bulls-eye skin rash (erythema migrans) at the site of the tick bite, usually within 3 to 30 days. 154 within days to weeks, mild flulike symptoms can occur that include shaking chills, intermittent fevers, and local lymph-node swelling. after this localized phase, which can last weeks to months, the infection(s) can spread to other sites (disseminated disease), and patients then show malaise, fatigue, fever and chills, headaches, stiff neck, facial nerve palsies (bell's palsy), and muscle and joint pain and other signs and symptoms. 154 lyme disease can eventually become persistent or chronic and involve the cns and peripheral nervous system as well as ophthalmic, cardiac, musculoskeletal, and internal organ invasion. at this late chronic stage, arthritis, neurological impairment with memory and cognitive loss, cardiac problems (mycocarditis, endocarditis causing palpitations, pain, bradycardia, etc), and severe chronic fatigue are often apparent. 154, 155 the signs and symptoms of the late chronic phase of the disease usually overlap with other chronic conditions, such as cfs, chronic arthritis, among others, as well as neurodegenerative diseases, such as ad, pd, als, etc, causing confusion in the diagnosis and treatment of the chronic phase in ld patients. 29, 30, [88] [89] [90] 141, [156] [157] [158] these late stage neuroborreliosis patients exhibit neuropathologic and neuropsychiatric disease similar to the neurodegenerative diseases discussed above. 31, 89, 128, [156] [157] [158] [159] the involvement of coinfections in ld has not been carefully investigated; however, such infections on their own have been shown to often produce comparable signs and symptoms. diagnostic laboratory testing for ld at various clinical stages is, unfortunately, not foolproof, and experts often use a checklist of signs and symptoms and potential exposures, along with multiple laboratory tests, to diagnose ld. 156 coinfections are common in ld, and we 148, 155 and others 160 have found that the most common coinfection found with b. burgdorferi are various species of mycoplasma, usually m. fermentans. in some cases, multiple mycoplasmal infections are present in ld patients. 148 other common ld coinfections include: ehrlichia species, bartonella species, and babesia species, and 10% to 40% of cases of ld show such coinfections. [148] [149] [150] ehrlichia and bartonella species are usually found along with mycoplasma species in ld. 155, bartonella species, such as b. henselae, which also causes cat-scratch disease, 164 are often found in neurological cases of lyme disease. 154, 164 in addition, protozoan coinfections have been found with b. burgdorferi, such as intracellular babesia species. 165 the combination of borrelia, mycoplasma, and babesia infections can be lethal in some patients, and approximately 7% of patients can have disseminated intravascular coagulation, acute respiratory distress syndrome, and heart failure. 165 this review suggests that various infections could be directly or indirectly involved in the pathogenesis and/or progression of neurodegenerative and neurobehavioral diseases. although the evidence is far from conclusive for their general involvement, 43 certain cases may eventually be explained by considering infections in the mix of multiple toxic events, such as head trauma, excitotoxicity, nutritional deficiencies, local inflammation, and genetic susceptibilities. these toxic events occur over time and likely participate in pathogenic processes. 2, [5] [6] [7] an argument can be made that neurodegenerative and neurobehavioral disease progression are affected by various infections. even if infections are not directly involved in the pathogenesis of neurodegenerative and neurobehavioral diseases, patients with these diseases are at risk for a variety of opportunistic infections that could result in comorbid conditions or promote disease progression. infections can complicate diagnosis and treatment, and late-stage patients with complex neurological manifestations, meningitis, encephalitis, peripheral neuropathy, psychiatric conditions, or with other signs and symptoms could have infections that are not recognized or treated by their physicians. patients with neurodegenerative and neurobehavioral diseases are particularly difficult to treat using single modality approaches. this may be due, in part, to the multifocal nature of their disease and to the fact that often treatments are given to suppress signs and symptoms, rather than treat causes of the disease or its progression. however, even if the causes of neurodegenerative and neurobehavioral diseases are known, by the time therapeutic intervention is undertaken, it may be entirely too late to use this approach. moreover, if complex, chronic infections are ignored or left untreated, recovery may be difficult to achieve. 139 central nervous system infections can stimulate glial responses, and the presence of viral and bacterial infections in nerve cells, in particular, may stimulate autoimmune responses against nerve cell antigens. in the case of ms, some 20 different bacterial and viral infections have been found, but the link between these infections and the pathogenesis of ms is still being debated. 43, 70 perhaps this is the reason that 1 or even a few types of infections cannot be causally linked to ms-there are just too many possibilities. eventually, certain infections may eventually be linked, at least in a subset of ms patients, to the pathogenesis of this neurodegenerative disease. further research may shed more light on this important subject. does the overall evidence suggest that chronic infections may be involved in the pathogenesis of neurodegenerative and neurobehavioral diseases? at the moment, the evidence is inconclusive. some individuals can harbor chronic infections without any observable signs or symptoms, although the incidence of infection in such individuals is usually very low, only a few percent (for example [14] [15] [16] [17] ). animal models have provided some additional insight, 117-119 but this area will require much more intensive investigation. some information outside the area exists on the infection of nonhuman primates with neuropathologic microorganisms, such as mycoplasma fermentans, which results in cns infection and a fatal disease with neurological signs and symptoms. 166 animal models that can be reproducibly infected with specific microorganisms to reproduce a similar disease will be an important resource. future basic and clinical research may ultimately elucidate the involvement of chronic infections in the pathogenesis and progression of neurodegenerative and neurobehavioral diseases. finally, one problem that will have to be overcome is the disparity of results from different laboratories. 43 this may be due, in part, to differences in the sources of clinical materials, qualities of reagents, and techniques used. indeed, in some procedures, such as pcr, there are various problems that must be overcome in the handling of specimens, their stability, presence of interfering substances, contamination, sensitivity and specificity of the tests, and interpretation of the results. interlaboratory variability will remain a problem unless laboratories work closely together to solve these problems. for 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which lyme disease is endemic lyme disease-a tick-borne spirochetosis? borrelia burgdorferi migrates into joint capsules and causes an upregulation of interleukin-8 in synovial membranes of dogs experimentally infected with ticks tick-borne diseases diagnosis and therapy of chronic systemic co-infections in lyme disease and other tick-borne infectious diseases recognition and management of lyme disease borrelia burgdorgferi persists in the brain in chronic lyme neurobor-reliosis and may be associated with alzheimer disease lyme-associated parkinsonism: a neuropathologic case study and review of the literature psychiatric manifestations of lyme borreliosis evidence for disseminated mycoplasma fermentans in new jersey residents with antecedent tick attachment and subsequent musculoskeletal symptoms immunoserologic evidence of coinfection to tick-borne pathogens of babesiosis, ehrlichiosis and lyme borrelosis in human sera clinical and epidemiological features of early lyme disease and human granulocytic ehrlichiosis in wisconsin concurrent infection of the central nervous system by borrelia burgdorferi and bartonella henselae cat-scratch disease encepthalopathy: a cause of status epilepticus in school-aged children when to suspect and how to monitor babesiosis fatal systemic infections of non-human primates by mycoplasma fermentans (incognitus strain) comparative study of the presence of chlamydia pneumoniae in cerebrospinal fluid of patients with clinically definite and monosymptomatic multiple sclerosis key: cord-022119-bzd9e1q6 authors: orzell, susannah; suryadevara, amar title: pharyngitis and pharyngeal space infections: fever, sore throat, difficulty swallowing date: 2018-10-15 journal: introduction to clinical infectious diseases doi: 10.1007/978-3-319-91080-2_5 sha: doc_id: 22119 cord_uid: bzd9e1q6 pharyngeal infections are very common in patients of all ages. they are typically associated with a sore and swollen throat that makes it difficult for the patient to swallow. they may also be accompanied by fevers, swollen lymph nodes, cough, voice hoarseness, and classic symptoms of an upper respiratory infection such as rhinorrhea. the majority of these infections are benign and run a self-limited course, although they often cause patients significant discomfort. a small but still significant number of cases will be due to a more serious process, such as a deep neck space infection. it is important to recognize the clinical and radiographic differences between these patients and patients with simple pharyngitis and triage them appropriately. failure to recognize and treat patients with more serious infections can have dire consequences and can lead to increased morbidity and mortality. it is also important to recognize that certain populations of patients will be more susceptible to atypical infections. specific conditions, particularly those that lead to immunocompromise, should prompt the clinician to expand their differential and consider atypical causes of pharyngeal infections. understand that most cases of pharyngitis are caused by viruses and will resolve with supportive care alone. 5 describe common symptoms associated with a case of uncomplicated pharyngitis. 5 recognize the warning signs of a more serious infection requiring prompt attention such as a deep neck space infection, epiglottitis, or ludwig's angina. 5 recognize some of the predisposing factors associated with atypical causes of pharyngeal infections. acute pharyngitis affects a significant number of people across the united states and the world each year and impacts patients from all age groups. it accounts for 1-2% of all ambulatory visits and causes missed days from work and school [1, 2] . the cause is typically infectious in etiology, with viruses being most common pathogens involved, followed by bacteria, fungi, and rarely parasites. noninfectious conditions may cause pharyngitis, and if symptoms are not self-limiting or do not respond to appropriate medical treatment, they should be considered. symptoms range from a mild sore throat lasting for several days to severe manifestations that threaten the patency of the airway. deep neck space infections can also extend directly into the mediastinum or the lungs causing life-threatening mediastinitis or pneumonia, underscoring the importance of early recognition and treatment. important cavities, spaces, and structures in the head and neck. see . figs. 5.1, 5.2, and 5.3 for pictographic demonstrations of select neck spaces. the glossopharyngeal nerve courses through it in a minority as an anatomic variant. the peritonsillar space communicates with the parapharyngeal space. parapharyngeal space -this anatomic space is traditionally described as an inverted pyramid lateral to the peritonsillar space and nasopharyngeal cavity. it is bound superiorly by the base of the skull, inferiorly by the greater cornu of the hyoid bone, anteriorly by the pterygomandibular raphe and medial pterygoid, posteriorly by the cervical vertebrae and paravertebral muscles, and laterally by the parotid gland. the parapharyngeal space is further divided into preand post-styloid compartments by a band of fascia called the aponeurosis of zuckerkandl and testut, which connects the styloid process of the temporal bone to the tensor veli palatini. the pre-styloid compartment contains fat, muscles, lymph nodes, the deep lobe of the parotid gland, the internal maxillary branch of the external carotid artery, and several branches of cranial nerves. the post-styloid compartment contains the internal carotid artery, internal jugular vein, sympathetic chain, lymph nodes, and cranial nerves ix through xii. this space communicates with the submandibular space, peritonsillar space, retropharyngeal space, and sublingual space. retropharyngeal space -as its name implies, this anatomic space lies posterior to the pharynx. it is bound anteriorly by the visceral . fig. 5 . 3 shown is a normal axial ct scan with intravenous contrast outlining the carotid sheath, retropharyngeal, sublingual, and submandibular spaces division of the middle cervical fascia, posteriorly by the alar division of the deep cervical fascia, laterally by the carotid sheaths, superiorly by the skull base, and inferiorly to the mediastinum. this space typically only contains loose areolar connective tissue and lymph nodes, but may contain the internal carotid arteries as an anatomic variant referred to as retropharyngeal carotids. infections in this space are particularly concerning due to their ability to spread inferiorly to the mediastinum or superiorly to the skull base. the retropharyngeal space communicates with the parapharyngeal space. danger space -this anatomic space lies just posterior to the retropharyngeal space, and like the retropharyngeal space, it extends from the skull base to the mediastinum. it is bound posteriorly by the prevertebral fascia and laterally by the transverse processes of the vertebrae. it is called the "danger space" because bacterial infections in this area can spread to the thorax very quickly. prevertebral space -this anatomic space lies posterior to the prevertebral fascia and danger space and extends from the skull base to the coccyx. it is bound posteriorly by the vertebral bodies and laterally by the transverse processes of the vertebrae. it contains muscles, the vertebral arteries and veins, and the phrenic nerve and roots of the brachial plexus. the anterior belly of the digastric muscle, posteriorly by the posterior belly of the digastric muscle, medially and superiorly by the mylohyoid muscle, laterally by the platysma, and inferiorly by the hyoid bone. it contains the submandibular gland, lymph nodes, facial vessels, and hypoglossal nerve. it communicates with the parapharyngeal and sublingual spaces. sublingual space -these anatomic paired spaces are each bound superiorly by the floor of the mouth, inferiorly by the mylohyoid muscle, anteriorly and laterally by the mandible, posteriorly by the hyoid bone, and medially by extrinsic muscles of the tongue. they communicate with each other beneath the lingual frenulum, and they communicate with the submandibular and parapharyngeal spaces. masticator space -this anatomic space is bound medially by the fascia medial to the pterygoid muscles, laterally by the mandibular ramus, superiorly by the base of the skull, inferiorly by the lower border of the mandible, anteriorly by the pterygomandibular raphe, and posteriorly by the parotid gland. it is subdivided into the masseteric and pterygoid spaces, which are delineated by the superficial layer of the deep cervical fascia. the masticator space contains muscles of mastication, the ramus and posterior body of the mandible, the inferior alveolar nerve, and the internal maxillary branch of the external carotid artery. this space communicates with the parapharyngeal space, submandibular space, and sublingual spaces. waldeyer's tonsillar ring -an arrangement of lymphoid tissue in the pharyngeal cavity that forms a circular loop consisting of the lingual tonsils, the adenoids, and the palatine tonsils. carotid sheath -paired anatomic structures lateral to the retropharyngeal space extending from the base of the skull to the sternum containing the carotid arteries, internal jugular veins, cranial nerves ix through xii, and the sympathetic trunk. the vast majority of patients with pharyngitis have mild to moderate illness that is either self-limiting or rapidly responsive to appropriate antibiotic treatment. the initial approach to a patient with suspected pharyngitis or parapharyngeal space infection, however, should always include a careful assessment for signs of airway compromise. findings of concern include respiratory distress, drooling, orthopnea, muffled speech ("hot potato voice"), difficulty turning the head, and elevation of the tongue and floor of the mouth. a patient in acute distress due to a retropharyngeal abscess, for example, may exhibit opisthotonic posturing with neck hyperextension. these findings should prompt immediate transfer to an emergency department for evaluation with flexible laryngoscopy, if expertise is available. in extreme cases, emergency endotracheal intubation or surgical interventions are necessary to establish and protect the airway. intravenous broadspectrum antibiotics should also be administered as soon as possible in critical patients. the laboratory evaluation typically includes a complete blood count, erythrocyte sedimentation rate, c-reactive protein, blood cultures (both aerobic and anaerobic), and pharyngeal, laryngeal, tracheal, or epiglottis cultures for those undergoing a procedure that protects their airway. imaging should be deferred for the critically ill patient until their airway has been secured. the recognition of and immediate intervention for a compromised airway are always the top priority (airway, breathing, circulation, in that order). in the stable patient, the extent of the diagnostic evaluation depends on the history and physical examination findings. the history should include a determination of the onset, progression and duration of symptoms, the quality and severity of any associated pain, and factors that aggravate or alleviate the discomfort. the presence or absence of associated symptoms, such as fever, cough, dysphagia, trismus, nasal congestion, rhinorrhea, neck pain, or dental pain, should be noted. symptoms that are more prominent on one side suggest the presence of an abscess or phlegmon. the presence or absence of underlying chronic medical conditions, particularly those that are associated with immunosuppression such as diabetes, hiv infection, collagen vascular diseases, or malignancy, should be determined. a recent history of dental work, endotracheal intubation, surgery on the upper aerodigestive tract, and/or penetrating neck trauma is also important to obtain [7 call out box 5.1]. the different infectious agents varies by age, medical comorbidities, and exposures, so a complete history is necessary to guide clinical decision-making or to raise the possibility that a more unusual pathogen may be responsible for a patient's illness. rarely, autoimmune disorders, previously unrecognized immunodeficiency, anatomic abnormalities, or malignancies can exacerbate or mimic symptoms of neck infections. patients with persistent symptoms, who do not respond to empiric therapy based on the suspected diagnosis of infection, should undergo additional diagnostic testing to evaluate for the presence of the more unusual infectious and noninfectious entities. most cases of pharyngitis and tonsillopharyngitis are caused by viruses [3] . since effective antiviral medications are not available for the majority of relevant viruses, treatment is nearly always focused on reducing pain and fever. patients may refuse to eat and drink for a sufficient period of time, making intravenous fluids necessary to reestablish and maintain hydration. . table 5 .1 lists the more common causes of viral pharyngitis. pharyngitis is usually one of several symptoms that patients complain about when infected with any of the common "cold and flu" viruses. since respiratory viral pathogens do not discriminate between mucosal targets, symptoms of conjunctivitis, rhinitis, and laryngitis with cough and/or hoarseness are typically present along with the sore throat. if only a single mucous membrane appears to be involved, a bacterial cause should be considered. any of the respiratory viral infections can be associated with fever, especially in young children, but influenza viruses and adenoviruses are notorious for causing fevers at any age. viral pharyngitis is a clinical diagnosis. respiratory virus diagnostic testing is available, however, for patients with severe infection or confusing atypical presentations. at the present, polymerase chain reaction (pcr)-based testing can be performed on nasopharyngeal or oropharyngeal samples. some platforms allow for the rapid diagnosis of more than a dozen viruses simultaneously. respiratory "cold and flu" viruses account for most cases of viral pharyngitis, but several others are also worthy of discussion. for example, exudative pharyngitis is a classic finding in patients with infectious mononucleosis, a syndrome most commonly caused by epstein-barr virus (ebv). the syndrome is diagnosed clinically in patients with exudative pharyngitis, lymphadenopathy, and splenomegaly. fevers are common and can persist for 3 weeks or longer. patients with infectious mononucleosis also complain of extreme fatigue, sometimes lasting for months. the diagnosis of ebv-associated infectious mononucleosis is confirmed serologically by testing for igm and igg antibodies against the ebv viral capsid antigen (ebv vca igm and igg). the detection of ebv vca igm is consistent with acute ebv infection. a rapid screening test for ebv infection, the serum heterophil antibody assay, is still used commonly. results should be interpreted with caution since the assay is both less sensitive and less specific than antibody testing. treatment for ebv infection is primarily supportive care. in severe cases, the combination of pronounced pharyngeal swelling and impressive cervical adenopathy may lead to concerns for impending airway compromise. under such circumstance, systemic glucocorticoids can be administered to reduce the swelling, perhaps avoiding the need for a medical or surgical procedure to maintain airway patency. the use of glucocorticoids should otherwise be avoided because of their immunosuppressive activity. the second most common cause of infectious mononucleosis is cytomegalovirus. the clinical presentation can be identical to that seen from ebv, but the ebv-specific serologic testing will not indicate an acute ebv infection, and the rapid heterophil antibody assay will be negative. a positive cmv igm antibody test confirms the diagnosis of acute cmv infection. . acute infection with human immunodeficiency virus (hiv) can also present with an infectious mononucleosislike illness called acute retroviral syndrome. the syndrome is characterized by self-limiting fevers, malaise, myalgias, pharyngitis, and cervical lymphadenopathy occurring several days to several weeks after exposure. hiv should, therefore, be suspected in all patients who present with an infectious mononucleosis syndrome. the diagnosis of hiv infection is made using combined, fourth-generation tests designed to detect both hiv-specific antibodies and hiv p24 antigen. patients who are infected recently, who have not yet seroconverted by making anti-hiv antibodies, often have detectable circulating hiv p24 antigen. a major advantage of fourth-generation testing is the ability to detect hiv p24 antigen and diagnose hiv infection as early as 1 week after exposure. the earliest detectable anti-hiv antibody is present after 2-4 weeks. herpes simplex virus (hsv) types 1 and 2 can also cause impressive pharyngitis. patients may present with fevers, malaise, headaches, cervical lymphadenopathy, and sore throat with or without the formation of vesicles visible in the oropharyngeal cavity. when vesicles are seen, they tend to rupture forming ulcerative plaques with grayish exudate. infants and young children who develop primary oral hsv infection typically present with gingivostomatitis. a very painful vesicular eruption is seen on the mucous membranes in the anterior part of the mouth including the gingiva and the tongue, with lesions extending onto the lips. in contrast, primary oral hsv infection in adolescents and adults most commonly presents as severe pharyngitis. since vesicles are not always present, it is likely that a substantial number of such cases go undiagnosed. the diagnosis of hsv pharyngitis (or gingivostomatitis) is made either by hsv-specific polymerase chain reaction (pcr)-based testing or by viral culture of material collected by swabbing the affected mucous membranes. in immunocompetent patients, oral hsv infections will eventually resolve spontaneously; however, outpatient treatment of antiviral medication acyclovir, or one of its derivatives, offers the potential to reduce symptoms, reduce virus shedding, and hasten recovery. immunosuppressed patients who develop active hsv disease benefit most by treatment with intravenous acyclovir. another group of viruses capable of causing painful vesicular lesions in the mouth are the enteroviruses. the term herpangina is used to describe these lesions when present on the roof of the mouth and in the back of the throat. despite its name and the vesicular nature of the lesions, herpangina refers to an enteroviral infection, not a herpetic infection. when herpangina is associated with a maculopapular or vesicular rash of the hands and feet, it is referred to as hand, foot, and mouth disease. among the enteroviruses that cause herpangina with or without the rash, coxsackie a16 is the most notorious because it continues to be responsible for a large number of pediatric cases of hand, foot, and mouth disease each summer during enterovirus season. the diagnoses of herpangina and hand, foot, and mouth disease are easy to make on clinical grounds alone, but if viral diagnostic testing is pursued, pcr testing is pre-ferred. enterovirus typing is only performed by reference laboratories and usually only for the purposes of outbreak investigations. oral candidiasis, commonly called "thrush, " is the most common fungal infection of the upper aerodigestive tract. thrush leads to white, cheese-like plaques on the tongue and buccal mucosa that are not easily scraped away. some bleeding may occur if scrapings are done for diagnostic purposes. the diagnosis of oral candidiasis is made clinically; however if scrapings of the affected area are cultured, the yeast candida albicans or a related candida species is recovered. two less typical presentations of oral candidiasis include the erythematous and the chronic hyperplastic types. erythematous oral candidiasis presents with a red, very sore oropharyngeal cavity, while chronic hyperplastic candidiasis presents with leukoplakia at the corners of the mouth and tongue. c. albicans is a normal flora of the human skin and mucous membranes. when there is a disturbance in immune function or an imbalance in the usual bacteria flora, as occurs with antibiotic use, c. albicans can cause infection. thrush is quite common during the first few months of life, at least in part because of the immaturity of the newborn's cellular immune function. in almost every other instance, c. albicans requires a conditional opportunity to cause an opportunistic infection. patients who are being treated with long-term antibiotics or immunosuppressive medications, including glucocorticoids, and those with primary and acquired immune deficiencies commonly develop thrush. untreated, the infection can progress and extend to posterior pharyngeal cavity structures, the esophagus, and the airway. pharyngitis secondary to candidiasis can be extremely painful causing severe dysphagia. when oral candidiasis progresses to visibly involve the structures of the posterior oropharyngeal cavity, it is important to consider that it may have also spread to the esophagus, larynx, or trachea. direct visualization using nasopharyngeal laryngoscopy may be indicated. the presence of advanced oral candidiasis in a patient without known risk factors should always prompt an evaluation for immunocompromising conditions, including hiv infection. other fungal infections of the pharynx and parapharyngeal space caused by a variety of opportunistic yeasts and molds have been described as case reports, nearly always in patients with significant immunocompromising conditions. parasitic infections of the pharynx and parapharyngeal spaces are exceedingly rare but should be considered in certain circumstances. in developed countries, infection with toxoplasma gondii has been described in organ transplantaalthough virulent pathogens such as staphylococcus aureus and s. pyogenes are also identified on a regular basis. the etiologies and sequelae of these infections have been shown which vary by patient characteristics, particularly by age and by the presence of comorbid conditions. for example, dental infections are the most common source and predisposing factor for deep neck space infections in adults, but tonsillitis and pharyngitis are the most common predisposing factor among children [10] [11] [12] . adolescents and young adults have higher rates of peritonsillar abscess compared to the younger children or older adults, while retropharyngeal abscesses are most common in preschool-aged children [13] . group a streptococcus (gas) is the most common cause of acute bacterial pharyngitis and tonsillopharyngitis. less frequent causes are listed in . table 5 .2. clinically, streptococcal pharyngitis is associated with fever, tender anterior cervical lymphadenopathy, pharyngeal erythema, and tonsillar swelling. exudate may be seen on the posterior pharyngeal wall and on the tonsils. streptococcal pharyngitis may also be associated with the presence of palatal petechiae and a skin rash. the diagnosis of gas pharyngitis is based on both the clinical presentation and the results of laboratory testing. the centor and modified centor criteria chlamydophila psittaci rare a chlamydia trachomatis can be detected regularly from pharyngeal swabs, but is only rarely associated with symptoms of pharyngitis (. table 5 .3) have been used as diagnostic adjuncts to help clinicians identify patients who are likely to have gas infection and unlikely to have a viral infection. a score of 0 or 1 on the modified centor score indicates a low probability of gas infection, and no further diagnostic testing or empiric antibiotic therapy is recommended. score of 2 or 3 indicates the possibility of strep throat, so throat cultures are recommended, with initiation of antibiotics if the cultures are positive for group a streptococcus. a score of 4 or more once indicated that empiric treatment with antibiotics should be started while waiting for culture results; however, in recent years, the us centers for disease control and prevention (cdcp) and national professional societies have since recommended against empiric antibiotic usage [7 call out box 5.2]. antibiotics should only be used when cultures are positive [14, 15] . overall, evidence has shown only a modest benefit of antibiotics in reducing the duration of a sore throat symptoms; however treatment is highly effective at preventing acute rheumatic fever and reduces the frequency of peritonsillar abscess formation [16] . antibiotic treatment does not, however, reduce the possibility of developing post-streptococcal glomerulonephritis. penicillin is the antibiotic of choice for gas pharyngitis unless the patient is allergic to it. amoxicillin is an acceptable alternative. patients who are allergic to, or cannot tolerate, î²-lactam antibiotics can be treated with clindamycin, azithromycin, or clarithromycin. unlike many other common bacterial pathogens, s. pyogenes has not developed resistance to î²-lactam antibiotics. resistance to clindamycin is rare in most communities but well described in others. occasional resistance to azithromycin and clarithromycin is also seen. clinical treatment failures with penicillin and amoxicillin do occur despite the absence of antibiotic resistance. such failures are best explained by failure of the penicillin pharmacodynamics, not by a resistance mechanism acquired by the streptococcus. some patients develop recurrent gas pharyngitis and/or tonsillitis because unlike many other common infections, natural disease does not confer protective immunity to reinfection. the american academy of otolaryngology clinical practice guidelines for streptococcal pharyngitis advocate for watchful waiting if a patient has had fewer than seven documented episodes of streptococcal pharyngitis in the last year, fewer than five episodes per year for at least 2 years, or fewer than three infections per year for at least 3 years [17, 18] . for patients who do not meet these criteria, tonsillectomy may be offered. in addition, tonsillectomy may be considered in patients with comorbidities, including obstructive sleep apnea, chronic tonsillitis unresponsive to medical therapy, cardiac valvular disease, recurrent febrile seizures, tonsiliths, history of peritonsillar abscess, or allergy/intolerance to antibiotic therapy. ideally, a tonsillectomy should be performed in the absence of an active infection to reduce the chances of a surgical complication such as postoperative bleeding. occasionally an emergency tonsillectomy, historically referred to as a quinsy tonsillectomy, needs to be performed because of airway compromise. deep neck space infections can present as discrete organized abscesses within specific neck spaces, as a soft tissue phlegmons without clearly forming collections of pus, or rarely, as a very rapidly destructive life-threatening process called necrotizing fasciitis. these conditions typically arise from the direct spread of a less serious infectious process present in an adjacent space. deep neck space infections become particularly concerning when compartments that communicate directly with the mediastinum are involved, such as the retropharyngeal, danger, and prevertebral spaces. depending on their specific location, these infections can also cause significant airway compromise over a relatively short period of time. the underlying predisposing cause for deep neck space infections varies based on age. children who develop deep neck infections usually do so after starting with pharyngitis, while the most common initial source of infection in adults comes from an odontogenic process [7 call out box 5.3]. the . pharyngitis is the most common source for developing deep neck space infections in children, whereas odontogenic infections are the most common underlying source in adults. microbiology of deep neck space abscesses is often polymicrobial with a mix of aerobic and anaerobic organisms. . box 5.1 includes some of the more common bacterial isolates from deep neck space abscess cultures [11, 19] . some bacterial species require very specific conditions in order to be successfully cultured in the laboratory and thus are not detected in standard cultures [7] . evolving changes in the antibiotic susceptibility profiles for several of these bacterial types further complicate approaches to patient management [11, 20, 21] . differentiating between an abscess and phlegmon on clinical grounds is difficult since both conditions cause sore throat, dysphagia, otalgia, voice changes, and swelling of the neck and oropharyngeal cavity. abscesses are usually discovered by computed tomography (ct) scan; however the level of suspicion for an abscess is raised if there is marked swelling of the oropharynx or neck or when an infection is not responding to broad-spectrum antibiotics. abscess formation is the result of the host reaction to an infection and serves to encapsulate the infected area. the lack of blood flow into the abscess, together with the low ph and low or absent oxygen context, can render antibiotics ineffective in treating these infections due to their inability to penetrate the abscess intact at concentrations that are bactericidal. three of the more common deep neck space abscesses include peritonsillar abscesses (ptas), parapharyngeal abscesses, and retropharyngeal abscesses (rpas). as their names imply, these are conditions where there is abscess formation within their respective deep neck spaces. because of their proximity to each other and relative ease of bacterial spread between the compartments, it is not uncommon for two or more neck spaces to be involved [13, 22] . for example, . fig. 5.4 shows the ct scan findings from a case of a parapharyngeal abscess with a contiguous rpa. the clinical presentation of ptas, parapharyngeal abscesses, and rtas has many similarities; however there are some distinctions between the three that may help differentiate them. similarities between the three include the presence of sore throat, dysphagia, voice changes, odynophagia, and, in advanced cases, difficulty breathing. the classic presentation of ptas also includes a visible, asymmetrical bulge of the tissues on the affected side with deviation of the uvula to the contralateral side, a cellulitic appearance of the anterior tonsillar pillar and lateral soft palate (. fig. 5.5) , and trismus. when considering the appearance of a pta on ct scan (. fig. 5.6 ), it is easy to imagine the abscess displacing the surrounding tissue and thus creating the bulging appearance of the anterior tonsillar pillar and deviation of the uvula away from the affected side. parapharyngeal abscesses can also cause trismus and neck swelling above the level of the hyoid bone. anterior parapharyngeal space abscesses may result in a bulging appearance of the tonsil and lateral pharyngeal wall associated with swelling in the areas of the parotid gland and angle of the mandible. in contrast, posterior parapharyngeal space abscesses result in posterior pharyngeal wall swelling. trismus is less frequent and, when present, less severe. posterior parapharyngeal space abscesses are also associated with a higher incidence of sepsis due to their proximity to the carotid sheath. patients with rpas often present in extremis, appearing toxic with severe respiratory distress, holding their neck in hyperextension and drooling. rpas also cause bulging of the posterior pharyngeal wall (. fig. 5.7) . despite the numerous clinical findings that can use to diagnose and potentially differentiate between deep neck space infections, most cases will require imaging. the imaging modality of choice is a contrast-enhanced ct scan of the neck. some patients will not be able to tolerate lying supine and may require endotracheal intubation prior to the imaging procedure. needle aspiration or incision and drainage is a common method of treating abscesses in the head and neck, although intratonsillar abscesses are usually not treated surgically unless the tonsils become obstructive. peritonsillar abscesses may be treated in a conscious patient using either needle aspiration or incision and drainage. general anesthesia is necessary for surgical drainage of other deep neck space abscesses. new evidence suggests that small ptas, particularly those that are not associated with severe symptoms, can be treated medically, avoiding an incision and drainage procedure altogether [23] . similarly, some small, clinically mild, parapharyngeal space abscesses [20, 24] and rpas [25, 26] can be treated medically with close follow-up in case a surgical procedure becomes necessary due to progression of the illness [7 call out box 5.4]. although suppurative infections of the deep neck spaces are often regarded as being more acute and in need of surgical drainage, nonsuppurative infections can be just as severe and, in some cases, more serious than deep neck space abscesses. one of these conditions is ludwig's angina. this condition refers to a rapidly spreading cellulitic infection of the submental, sublingual, and submandibular areas, typically due to an infection originating in the teeth. the swelling of the sublingual space displaces the tongue superiorly and posteriorly. the swelling impairs speech and ability to swallow. as the infection progresses, the patency of the airway becomes compromised. on physical examination, the patient has a muffled voice and appears distressed. trismus may prevent examination of the oropharynx. the structures of the superior neck are indurated with a marked elevation of the tongue and floor of mouth. ludwig's angina is a surgical emergency. the treatment priority is establishing a protected airway, typically by tracheostomy. once the airway is protected, the infection is incised and drained and the area debrided as necessary. broad-spectrum intravenous antibiotics are used. ideally the dental source of the infection is also addressed, but severe trismus typically prevents access during the incision and drainage procedure. cervical necrotizing fasciitis (cnf) is an uncommon, life-threatening infection of the head and neck associated with substantial morbidity and mortality. these infections typically arise from an existing infection of the adjacent deep neck spaces that propagates along the relatively avascular fascial planes. because of the poor vascularity of the fascia, these infections are poorly responsive to antibiotic therapy and demand surgical debridement. clinically and radiographically, cnf is difficult to differentiate from cellulitis or phlegmon. symptoms typical of other pharyngeal . fig. 5.7 shown is a sagittal ct scan showing a retropharyngeal abscess (arrow) . fig. 5.6 shown is an axial ct scan establishing the presence of a left-sided peritonsillar abscess (arrow) small, clinically mild peritonsillar abscesses, retropharyngeal abscesses, and parapharyngeal abscesses can be managed medically with close follow-up, whereas larger, more symptomatic abscesses require a surgical drainage procedure. infections, such as sore throat, dysphagia, and odynophagia, are present; however cnf may also cause bullae, skin necrosis, subcutaneous emphysema, and pain out of proportion to the findings on examination. secondary obstruction of lymphatic drainage can give the skin an "orange-peel" appearance [27, 28] . the classic ct scan finding of gas bubble formation within the soft tissues of the neck (. fig. 5.8) is found in 65% of cnf cases [29] . other ct scan findings typically include the presence of diffuse edematous changes of the surrounding soft tissue and scattered areas of hypodensity that do not enhance in the presence of contrast material. laboratory-based predictors of necrotizing fasciitis, such as the laboratory risk indicator for necrotizing fasciitis (lrinec) score [24] and the model developed by wall et al. [30] , which are validated from other anatomic areas of the body, have not proven useful in diagnosing cnf [29] . when clinical and radiographic findings suggest the possibility that a patient has cnf, surgical exploration is necessary. if the surgical team encounters necrotic tissue that is easily pulled apart, the diagnosis of cnf is confirmed, and debridement is performed. tissue is debrided until there is a bleeding, viable edge. repeat visits to the operating room for additional surgeries are expected. medical treatment involves meticulous intensive care support and the administration of long-term, broad-spectrum intravenous antibiotics. even with prompt recognition and treatment, cnf has a mortality rate approaching 40% [31] . the radiographic study of choice for the evaluation of a deep neck space infection is a contrast-enhanced ct scan. a ct scan provides excellent characterization of the soft tissues and bony structures. this is particularly important when there is a suspicion of an odontogenic source of infection (. fig. 5.9 ). abscesses appear as areas of hypodensity with rim enhancement. other imaging modalities, including plain radiography, ultrasonography, and magnetic resonance imaging (mri), may be considered. plain radiographs and ultrasonography do not provide the resolution detail that a ct scan does, but may be useful in some instances. panoramic radiographs used to evaluate dentition may also be used to identify odontogenic sources of infection. ultrasonography allows for the possibility of concurrent evaluation and treatment of an abscess with drainage under guidance, although the inability of ultrasound to visualize deeper structures of the neck often limits its use. mri is rarely used in the evaluation of acute head and neck infections, but may be considered in cases where extension to the skull base, intracranial compartment, prevertebral space, or other complications, such as thrombosis of critical vessels, are suspected. . fig. 5.8 shown is an axial ct scan of the neck revealing hypodense areas without peripheral enhancement (red arrows) and the abnormal presence of gas pockets (blue arrows) within the soft tissue. surgical debridement of this area revealed necrotic tissue, confirming a diagnosis of necrotizing fasciitis . fig. 5.9 shown is a coronal ct scan demonstrating a periapical lucency (arrow) in a patient with a submandibular abscess, indicating the dental source of the infection complications of bacterial pharyngitis and deep neck space infections are uncommon, particularly if appropriate antibiotic therapy has been instituted and immediate surgical concerns have been addressed; however direct extension and invasion into surrounding structures or spaces are always a risk. other complications include bacteremia, lemierre's syndrome, carotid artery aneurysm/rupture, and spread along adjacent neck spaces or along fascial planes. lemierre's syndrome occurs when infection, usually with the anaerobic bacterium fusobacterium necrophorum, extends from the pharynx or parapharyngeal space to the internal jugular vein causing an infectious thrombophlebitis. this can lead to bacteremia as portions of the infected clot break away causing septic emboli. the lungs are typically affected first. patients with septic metastasis of infection to the lungs may develop pulmonary nodules, cavities, and abscesses. patients may also develop septic arthritis, particularly of the knee, hip, or shoulder [32] . the diagnosis of lemierre's syndrome is confirmed by performing a contrastenhanced ct scan of the neck to demonstrate the presence of a thrombosed internal jugular vein. treatment consists of intravenous antibiotics that include reliable activity against anaerobic bacteria. some experts also recommend anticoagulation therapy. in rare instances, ligation or excision of the internal jugular vein may be necessary if the patient fails to respond to standard treatment [7 call out box 5.5]. other potential complications of pharyngeal and parapharyngeal infections involving the carotid sheath are carotid artery aneurysm or rupture. these complications are extremely rare and only described in case reports. when they occur, they are life-threatening and demand immediate attention. similar to lemierre's syndrome, carotid artery pseudoaneurysm or rupture secondary to infection occurs due to spread of the infection from the pharynx or parapharyngeal spaces into the carotid sheath. unlike lemierre's syndrome, this condition tends to affect nonvascular structures in the carotid sheath, resulting in horner syndrome and palsies of cranial nerves ix through xii. damage to the walls of the carotid artery may produce a pulsatile neck mass on palpation, and in the case of carotid artery rupture, an expanding neck hematoma may be accompanied by a very large volume of bright red blood coming from the oropharynx. treatment involves protecting the patient's airway and either surgical ligation or embolization to prevent circulatory collapse from blood loss. spread of infection to the retropharyngeal space can lead to retropharyngeal abscesses as well as spread to other important areas of the body, such as the prevertebral space, danger space, or the mediastinum. the retropharyngeal, danger, and prevertebral spaces are bound by the skull base superiorly, and the retropharyngeal and danger spaces extend to the mediastinum, whereas the prevertebral space extends to the coccyx. once infection reaches these spaces, there is relatively easy access to the mediastinum. the complication of mediastinal infection is rare, estimated to occur at a rate of 4.1/100,000 [33] , but is always life-threatening [34, 35] . complications specific to gas pharyngitis include two noninfectious conditions: acute rheumatic fever (arf) and post-streptococcal glomerulonephritis (psgn). these entities are believed to arise from autoimmune complications secondary to the host's immune response to the gas infection. the autoimmune sequelae seen with arf are believed to be due to molecular mimicry, while psgn occurs secondary to a type 3 hypersensitivity reaction. arf is rare within the developed world due to the availability of antibiotics; however it is still a significant cause of morbidity and mortality in developing countries [36, 37] . untreated or multiple episodes of arf may progress to chronic rheumatic heart disease causing lifelong complications. acutely, patients with arf may present with fever, arthritis, arthralgia, carditis, first-degree heart block, erythema marginatum, sydenham chorea, or subcutaneous nodules. inflammatory biomarkers are typically elevated. a diagnosis of arf is made by a combination of clinical and laboratory findings as outlined by the modified jones criteria [36] . psgn typically occurs several weeks after gas infection. unlike arf, psgn may occur despite effective treatment of the gas infection. symptoms of psgn include hematuria, edema, and hypertension although many cases present with asymptomatic microscopic hematuria alone. acute renal failure may need to be supported temporarily with dialysis, but psgn typically resolves with time. a small number of patients go on to develop chronic renal failure. pharyngeal infections are very common, and not often lifethreatening. they are usually caused by viruses and resolve without the need for any intervention. sepsis, muffled voice, difficulty breathing, drooling, inability to turn the head, and unilateral throat swelling on exam are not normal and demand immediate attention. additionally, findings suggestive of an abscess on imaging warrant further evaluation and possibly surgical drainage. failure to treat these patients in a timely manner may lead to further extension of the abscess, either within the same neck space or spread to a different neck or chest space, sepsis, invasion into surrounding call out box 5.5 lemierre's syndrome refers to infectious thrombo phlebitis of the internal jugular vein that occurs as a complication of pharyngitis. it is most commonly caused by the obligate anaerobe, fusobacterium necrophorum. direct extension of the infection from the infected veins to the lungs causes a serious anaerobic bacterial pneumonia. vasculature, bacteremia, lemierre's syndrome, or even death. it is also important to recognize that immunocompromised patients are at risk for different pathogens compared to immunocompetent patients. please refer to the supplementary information section for answers to these exercises. ? 1. a 21-year-old male patient presents to your office for evaluation of a sore throat. he states that his sore throat began 3 days ago and is associated with a runny nose, cough, and a hoarse voice. he is afebrile and nontoxic in appearance and does not have tender cervical lymphadenopathy, but does have tonsillar exudates. he has had two episodes of gas pharyngitis when he was a child. what is the most appropriate next step in treatment? a. manage conservatively b. begin empiric treatment with antibiotics c. perform a throat culture and begin empiric treatment with antibiotics d. perform a throat culture and use results to guide antibiotic treatment ? 2. a 2-year-old male patient presents to the ed in respiratory distress. his mother states that he has complained of throat pain for several days and that his voice has sounded muffled. he also developed fevers to 39 â°c, and his oral intake has decreased to the point where he has not eaten for the past days. on physical examination, he is tachypneic, has a toxic appearance, and appears tired. he is sitting forward with his neck in a position of hyperextension, and he is drooling. what is your next step in the management of this patient? a. obtain an emergency ct scan of the neck b. obtain blood, urine, and cerebrospinal fluid cultures; initiate antibiotic treatment c. perform an airway evaluation, and establish a secure airway if needed d. perform a bedside ultrasound to assess for a drainable abscess ? 3. a 35-year-old female presents for evaluation of a sore throat. she has had the sore throat for approximately 2 weeks, and it has been worsening during this time. additionally, she notes that her mouth hurts. she denies voice changes and difficulty breathing and states that her pain is not any worse on one side or the other. she denies any significant past medical history or use of medications, but does reveal that she is an intravenous drug user and occasionally will share needles. on physical examination, you find a diffuse white deposit over her oral mucosa that bleeds slightly when scraped. a presumptive diagnosis of oral candidiasis (thrush) is made. in addition to starting the patient on antifungal treatment, what other tests or treatments would be prudent in this patient? a. antibiotics b. fungal culture c. hiv testing d. none ? 4. a 7-year-old female presents to the emergency department for evaluation of a sore throat and difficulty eating. for the past 5 days, she has had worsening right-sided throat pain associated with fevers, chills, and a slight muffled voice. in the ed, she is febrile to 38.8 â°c, but not in any respiratory distress. she is able to swallow without significant difficulty. during your physical examination, you note some fullness and erythema of the right anterior tonsillar pillar. a ct scan shows a small collection of hypodense fluid with rim enhancement in the right peritonsillar space. acceptable options for the management of this patient include (more than one answer may apply): a. hospitalize for observation and treatment with intravenous antibiotics b. discharge home on oral antibiotics with follow-up in 24 h c. perform a needle aspiration now d. take the patient to the operating room for an incision and drainage under general anesthesia ambulatory medical care utilization estimates for burden and economic cost of group a streptococcal pharyngitis acute pharyngitis spectrum and management of deep neck space infections: an 8-year experience of 234 cases molecular methods for diagnosis of odontogenic infections antimicrobial treatment of head and neck infections the human oral microbiome pharyngitis and epiglottitis the role of fusobacterium necrophorum in pharyngotonsillitis -a review deep neck infections: a constant challenge microbiology and management of peritonsillar, retropharyngeal, and parapharyngeal abscesses head and neck space infections. otolaryngol head neck surg incidence and microbiology of peritonsillar abscess: the influence of season, age, and gender appropriate antibiotic use for acute respiratory tract infection in adults: advice for high-value care from the american college of physicians and the centers for disease control and prevention clinical practice guideline for the diagnosis and management of group a streptococcal pharyngitis: 2012 update by the infectious diseases society of america antibiotics for sore throat clinical practice guideline: tonsillectomy in children. otolaryngol head neck surg tonsillectomy versus watchful waiting for recurrent throat infection: a systematic review increased incidence of head and neck abscesses in children. otolaryngol head neck surg children with deep space neck infections: our experience with 178 children microbial flora and antibiotic resistance in peritonsillar abscesses in upstate new york parapharyngeal abscess: diagnosis and treatment comparison of medical versus surgical management of peritonsillar abscess: a retrospective observational study. laryngoscope to drain or not to drain -management of pediatric deep neck abscesses: a casecontrol study a prospective study of 113 deep neck infections managed using a clinical practice guideline pediatric deep space neck infections in necrotizing soft-tissue infection: diagnosis and management objective criteria may assist in distinguishing necrotizing fasciitis from nonnecrotizing soft tissue infection retrospective evaluation of laboratory-based diagnostic tools for cervical necrotizing fasciitis a simple model to help distinguish necrotizing fasciitis from nonnecrotizing soft tissue infection hospital epidemiology of emergent cervical necrotizing fasciitis septic arthritis of the hip by fusobacterium necrophorum after tonsillectomy: a form of lemierre syndrome? retropharyngeal and parapharyngeal abscesses among children and adolescents in the united states: epidemiology and management trends retrospective review of management and outcomes of pediatric descending mediastinitis mediastinitis in infants from deep neck space infections revision of the jones criteria for the diagnosis of acute rheumatic fever in the era of doppler echocardiography: a scientific statement from the american heart association the global burden of group a streptococcal diseases key: cord-018302-lmly43rd authors: renaud, christian; englund, janet title: respiratory syncytial virus and human metapneumovirus infection in transplant recipients date: 2016-02-15 journal: transplant infections doi: 10.1007/978-3-319-28797-3_31 sha: doc_id: 18302 cord_uid: lmly43rd respiratory viral infections due to respiratory syncytial virus (rsv) and human metapneumovirus (hmpv) cause infections in immunocompromised transplant patients ranging from mild upper respiratory infections to severe lower respiratory tract disease with respiratory failure. these viruses are more readily diagnosed due to improvements in sensitive molecular diagnostic methods. the epidemiology of rsv and hmpv is similarly becoming more readily appreciated in hematopoietic stem cell transplant (hsct) patients of all ages as well as solid organ transplant (sot) patients, with lung transplant recipients having evidence of more frequent and severe complications related to these viruses. rsv and hmpv infection typically but not always present with upper respiratory signs and symptoms that progress to lower respiratory tract disease. treatment options for rsv are limited, with aerosolized, intravenous, and oral ribavirin all studied in hsct and lung transplant patients. no antiviral therapy for the treatment of hmpv is available, although ribavirin has shown some effectiveness in vitro. new antiviral agents including rsv fusion inhibitors and nucleoside analogs are being developed, with some under clinical evaluation. respiratory viruses may cause serious morbidity and mortality in the immunocompromised host, and the transplant recipient appears particularly vulnerable. the impact of infection with respiratory viruses and the subsequent development of severe lower respiratory tract disease has been increasingly appreciated as respiratory viruses become more readily detectable. respiratory syncytial virus (rsv) and human metapneumovirus (hmpv) are among the best-documented respiratory viruses causing a wide range of respiratory disease in transplant recipients, ranging from asymptomatic shedding to fatal respiratory failure. understanding the epidemiology and clinical characteristics of rsv and hmpv permits clinicians to intervene pretransplant, provide appropriate infection control and prevention measures, potentially treat patients, and manage immunosuppressive therapy. severe respiratory disease in the seriously immunocompromised host in the mid-twentieth century was originally attributed to infection with opportunistic pathogens such as gram-negative bacteria, fungi, pneumocystis jiroveci , and mycobacteria, as well as cytomegalovirus (cmv) and adenovirus. in the later twentieth century, episodes of acute upper respiratory infection (uri) and lrti without an identifi ed etiology were often considered to be "idiopathic" pneumonia or attributed to regimen-related toxicity or acute respiratory distress syndrome (ards). in the classic 1982 study of 215 non-bacterial, non-fungal pneumonias in 525 allogeneic hematopoietic stem cell transplant (hsct) recipients, 44% of the episodes of pneumonia remained undiagnosed. the overall mortality rate associated with idiopathic pneumonia in these hsct recipients was 60%, a strikingly high fi gure [ 1 ] . the potential morbidity of rsv infections was fi rst recognized in immunocompromised children in the 1970s, more than a decade earlier than in immunocompromised adults [ 2 -6 ] . the recognition of respiratory viruses as an important clinical problem likely refl ects the increasing number of severely immunodefi cient patients, more aggressive attempts to identify the cause of respiratory illness in high-risk patients, and the increasing ability of clinical virology and pathology laboratories to identify respiratory viruses in clinical specimens. although uncommon or atypical pathogens may be responsible for respiratory disease in the transplant recipient, the same viruses that cause typically mild but acute respiratory illness in the general population are responsible for hospitalizations in persons of all ages with underlying medical conditions [ 7 ] . these same viruses are also a common cause of respiratory disease in transplant recipients [ 8 -15 ] . with the widespread availability of sensitive and reliable molecular diagnostic methods, rsv and hmpv have been detected worldwide in transplant recipients and shown to be common causes of respiratory disease in the immunocompromised host [ 16 , 17 ] . in both the general population as well as in transplant recipients, rsv and hmpv may produce a wide constellation of clinical syndromes ranging from the common cold to bronchiolitis to severe pneumonia, but in contrast to the general population, rsv and hmpv may signifi cantly impact the morbidity and mortality of the transplant recipient. rsv was fi rst identifi ed in 1956 and became appreciated as a major cause of epidemic bronchiolitis and pneumonia in young children in the 1960s [ 18 , 19 ] . hmpv was fi rst identifi ed in 2001 by molecular techniques in symptomatic children by van den hoogen et al. as a paramyxovirus causing bronchiolitis and uri in children [ 20 ] . both viruses are classifi ed within the pneumovirinae subfamily of the paramyxoviridae family of non-segmented, negative-strand, enveloped rna viruses [ 21 ] . hmpv belongs to the metapneumovirus genus whereas rsv is a member of the pneumovirus genus. both viruses are highly pleomorphic and their sizes vary from 150 to 600 nm. the rsv and hmpv genomes are approximately 13-15 kb in length and closely resemble each other, excluding a few differences in the order of the genes and the absence of the non-structural genes (ns1 and ns2) from hmpv genome. the remaining eight genes code for nine proteins present in both viruses: the nucleoprotein (n protein), the phosphoprotein (p protein), the matrix protein (m protein), the fusion glycoprotein (f protein), the putative transcription factor (m2-1 protein), the rna synthesis regulatory factor (the m2-2 protein), the small hydrophobic glycoprotein (sh protein), the attachment glycoprotein (g protein) and the viral polymerase (l protein). the rna core of the virion is associated with p, n, l, m2-1, m2-2 proteins, surrounded by m protein and covered by a lipid envelope. f is the most highly conserved of the envelope glycoproteins within each virus and between rsv and hmpv. the fusion glycoprotein is essential in promoting attachment and fusion of the virus with the cell membrane during viral entry. the fusion protein is the target of many vaccines under development as well as that of monoclonal antibodies such as palivizumab, which is used to prevent rsv disease in preterm infants. by contrast, the g gene is the most variable. whole genome analysis of both rsv and hmpv has shown the existence of two genotypes, a and b. in hmpv, those two major genetic groups are further divided into subgroups a1, a2, b1, b2 based upon the sequence variability of the g and f genes. subgroup a2 is again divided into a2a and a2b. rsv and hmpv infections produce both humoral and cellular immune responses. humoral immunity protects against reinfection while cellular immunity controls established infection and terminates viral shedding. protective immunity in immunocompetent hosts is thought to be relatively shortlived. both viruses interfere with the host's innate immune system resulting into incomplete clearance and partial immunity. prompt and accurate identifi cation of respiratory viral pathogen is critically important in the transplant recipient because it enables specifi c infection control precautions to be instituted, the initiation of specifi c antiviral therapy, impacts the use of immunosuppressive therapy, and potentially affects whether transplantation should proceed [ 22 -24 ] . furthermore, identifi cation of a respiratory viral pathogen can assist in avoiding unnecessary therapy, procedures, and surgical procedures (such as open lung biopsy), as well as assist in the identifi cation of a potential cluster or epidemic of infections within the medical unit, hospital, or community. in hospitalized adults (both immunocompromised and immunocompetent), rapid viral diagnosis has been shown to reduce mortality and decrease the length of hospital stay and total cost [ 25 , 26 ] . laboratory diagnosis of respiratory viruses including rsv and hmpv has evolved considerably; adequate specimen collection is still essential for the successful identifi cation of viruses in clinical samples. newer types of nasopharyngeal swabs have shown improved viral diagnostic sensitivity compared to previous swabs, with similar sensitivity to nasal washes when using sensitive molecular methods in patients [ 27 , 28 ] . for example, nylon fl ocked swabs and foam swabs increase cell capture within the swab and then release into the transport media, increasing viral recovery [ 29 , 30 ] . nasal wash or aspiration methods are superior for isolation of viruses by culture and increase the sensitivity of culture, antigenic assays and quantitative molecular assays [ 31 ] . nasal washes are well tolerated in cooperative adults and offer the advantage of visualizing the quality of the specimen. bronchoalveolar lavage remains the specimen of choice to diagnose lower respiratory tract infections because of the ability to simultaneously test for potential co-pathogens such as fungi, pneumocystis jiroveci , and bacteria, as well as to document viral infection in the lower airways. discordance in viral detection between upper and lower respiratory tract samples have been described with both viruses; negative upper tract and positive lower tract specimens in immunocompromised patients are possible but more discordance has been noted for hmpv compared with rsv. molecular diagnosis of rsv and hmpv is faster and more sensitive than viral culture or antigen detection, and most laboratories currently use commercial or in-house molecular assays to detect rsv and hmpv (table 31-1 ) . many genes have been targeted to detect rsv, including the n, f and l genes, with similar genes also targeted to detect hmpv. many rapid assays have been approved including some highly multiplexed respiratory panels allowing detection of rsv and hmpv as well as many other respiratory viruses and bacteria. several different primer sets may be utilized simultaneously in the reaction mix, and the virus identifi ed by the size of the amplicon or following hybridization with a virus-specifi c probe. some commercial assays are very rapid and require minimal technical expertise, with only 1-2 h of turn around time [ 32 , 33 ] . some laboratories have developed quantitative assays using hydrolysis probe technology with standard curves to help understand the signifi cance of positive results and to follow viral loads under therapeutic management [ 34 -36 ] . no quantitative commercial assays are yet available. molecular assays have also been used to detect viral rna from blood/serum as a prognostic marker [ 37 ] . unlike molecular methods, isolation of virus by culture confi rms the presence of a complete infectious unit capable of further multiplication. positive culture results may be obtained with as little as a single infectious virion, below the threshold of detection for most other detection methods, including some nucleic acid amplifi cation test (naat) methods . another advantage of viral culture is that multiple viruses may be identifi ed from a single sample and viruses can grow independently of point mutations that could potentially create false negative results by naat. the major limitations of viral isolation include the time, expense, and expertise required for virus isolation. a variety of cell lines can be used to grow rsv (hep-2, a549, rhmk) or hmpv (llc-mk2, vero), and detection by cell culture can also be accomplished using several types of cells together, such as the r-mix cells (mixture of mink lung cells and a549 cells) [ 38 ] (diagnostic hybrids, athens, oh). centrifugation combined with viral antigen detection methods permits more rapid diagnosis [ 39 ] . rsv-and hmpv-specifi c monoclonal antibodies have been used for immunofl uorescence (ifa) techniques either directly on respiratory specimens or in cell culture [ 40 , 41 ] . the sensitivity of ifa is lower than that of naat for detection of rsv and hmpv. however, results can be reasonably fast, the method is relatively inexpensive, and importantly, this method also confi rms that an appropriate specimen has been properly obtained by looking at ciliated epithelial cells. sensitivity of ifa when performed by an experienced laboratory is as high as 70-90% of the samples positive by pcr-at least in children [ 34 ] . ifa can detect viruses that would be missed by naat because of point mutations. ifa positivity also has good clinical correlation while low grade naat positivity can be detected for longer periods of time with unclear signifi cance and transmissibility. enzyme immunoassays (eias) and rapid antigenic diagnostic tests ( radts) are commercially available for rsv and to a lesser extent for hmpv. these assays lack sensitivity and/or specifi city and are not recommended in transplant populations. more than one diagnostic method should be used, since no method is perfect. molecular assays have the greatest sensitivity-although perhaps at times can perhaps be too sensitive. paradoxically, rare point mutations causing mismatches have been described causing false negative results of naat in transplant units. viral cell culture requires time, is becoming less available in laboratories, and is more expensive and less sensitive, although it can catch those strains with mismatches and provide information on viral replicative nature while receiving treatment. further characterization of rsv and hmpv strains obtained from culture or directly from the clinical specimen is frequently desirable. antigenic differences among virus strains isolated from different geographic locations or at different times may also be examined. pools of monoclonal antibodies and "rna fi ngerprinting" have been used in the analysis of rsv strains in nosocomial outbreaks [ 42 , 43 ] but direct sequencing of the f and g glycoproteins is more commonly utilized [ 44 , 45 ] . next-generation sequencing is a very promising tool to characterize rsv or hmpv strains during severe infection. this could provide information on phylogenicity to identify outbreaks and also detect mutations that could be associated with antiviral or monoclonal resistance as well as increased virulence. some human gene alleles in the human genome may increase rsv severity in infants, but little is known yet on these genomic variations in transplant recipients. next-generation sequencing has the potential to provide information on the virus and the human genomes simultaneously. rsv is well known to cause annual winter outbreaks in the community ( figure 31-1 ). surveillance studies of respiratory viruses from transplant centers have established the high frequency and the signifi cant clinical impact of respiratory viral infections in hsct recipients overall [ 8 -15 , 46 , 47 ] as well as the relative importance of rsv in terms of morbidity and mortality (table 31 -2 ). a 1988 retrospective review conducted at the children's hospital of philadelphia revealed a [ 47 ] . one of the earliest studies demonstrated fatal rsv pneumonia in four of 11 immunocompromised adult hsct and solid organ transplant recipients with rsv infection [ 5 ] . most studies conducted in the twentieth century utilized classical virological methods that were relatively insensitive for the detection of rsv and/or did not detect recently described respiratory viruses such as hmpv. thus, these studies likely underestimated the true frequency of rsv overall. nonetheless, early studies from large transplant centers reported serious sequelae in small numbers of patients who developed pneumonia where rsv was detected [ 5 , 9 , 13 ] . at the fred hutchinson cancer research center (hutch), a prospective surveillance study conducted in 1987 documented respiratory viral infections in 15 (19%) of 78 immunocompromised patients who were followed until hospital discharge [ 13 ] ; fi ve (33%) patients developed pneumonia and two (13%) patients died (one with rsv, the other with adenovirus). a subsequent prospective surveillance study described 127 viral infections and revealed an overall frequency of viral infections of approximately 4% [ 11 ] ; 49% of rsv isolates were from bal. this study demonstrated the relatively high numbers of patients with rsv lower respiratory tract disease, which was higher than rates of lrti caused by piv (22%) infl uenza (10%), or rhinovirus (3%). results of viral surveillance in transplant units vary depending on the type of surveillance protocols utilized, time of year surveillance was conducted, type of clinical samples evaluated, and laboratory tests utilized. at the huddinge university hospital, stockholm, a prospective surveillance study conducted among hsct recipients between 1989 and 1996 detected 39 (7.1%) respiratory viral infections in 545 patients, including rsv in 21% [ 14 ] . at m.d. anderson cancer center (mdacc), prospective surveillance conducted using culture techniques among hospitalized adult hsct recipients during two 6-month winter periods detected 67 (31%) respiratory viral infections in 217 hospitalized patients with acute respiratory symptom. nearly half of these were due to rsv (33 patients, 49%). the impact of these illnesses was considerable: 20 (61%) patients had rsv infection progressing to pneumonia, and 12 patients with rsv pneumonia died. during the winter period when community viruses were frequent and nosocomial transmission was high, the frequency and mortality of respiratory viral-associated pneumonias was more than four times as high as cmv-associated pneumonia. recent prospective studies conducted in transplant centers worldwide continue to demonstrate the importance of rsv and report incidence of infections and risk factors for fatal disease (table 31 -2 ). a 2005 study from barcelona, spain, described the 2-year incidence of symptomatic respiratory viral infections in over 400 patients followed for up to several years posttransplant. altogether, 29% of allogeneic hsct recipients and 14% of autologous hsct recipients had respiratory viruses detected, with 19 patients (4.6%) receiving either autologous or allogeneic transplants having symptomatic rsv disease [ 55 ] . risk factors associated with having a respiratory virus identifi ed included close household contacts with children under the age of 12 years and chronic graft versus host disease. lymphopenia was identifi ed as a major risk for uri progression. similar rates have been demonstrated in other centers in the usa [ 15 ] , south america [ 56 ] , and europe [ 57 ] . less data is available documenting the impact of rsv in sot recipients (table 31 -2 ), rsv has been shown to cause lower respiratory tract disease and has been associated with other complications, such as organ rejection and bronchiolitis obliterans. lung transplant recipients have the highest rates of rsv lower respiratory tract disease in diverse types of organ transplant. rsv infections in lung transplant recipients have also been associated with organ rejection and progressive bronchiolitis obliterans, both of which have been observed to be seasonally related [ 58 , 59 ] . adult renal transplant recipients have also been reported to develop rsvassociated lower respiratory tract disease. the mortality has been low, and most patients have recovered without specifi c antiviral therapy [ 5 , 60 ] . rsv infections in pediatric liver transplant recipients have been associated with signifi cant morbidity and some but relatively low mortality [ 51 ] . among 483 pediatric liver transplant recipients cared for at the university of pittsburgh between 1985 and 1991, 17 (3.4%) children developed rsv infections, three-quarters of which were nosocomially acquired. the majority of the children had lower respiratory tract involvement, and two (12%) children died. specifi c antiviral therapy was not administered. the risk factors for more severe disease included onset of infection early after transplant, preexisting lung pathology, augmented immunosuppression prompted by rejection, and younger age. infections occurring late after transplantation in the absence of rejection were usually not severe. an intensive prospective approach to determining the incidence and risk factors for respiratory viruses was carried out at the hutch among 122 hsct recipients, who were pro-spectively enrolled between 2000-2004 and tested weekly through 100 days posttransplant using both culture and rt-pcr detection methods [ 50 ] . the cumulative incidence estimates of hmpv and rsv at day 100 were similar, at 6.2% and 5.8%, respectively. multivariable analysis demonstrated that only recipient cmv seropositivity was associated with increased risk for acquisition of a respiratory virus (hazard ratio = 4.1, ci 1.7-10.1, p = 0.002). the frequency of rsv infections and associated morbidity and mortality differs substantially, potentially accounting for the variability reported by different institutions. these differences refl ect the intensity of viral surveillances, the time of surveillance, the viruses prevalent in the community, the degree of immunosuppression of the patients, infection control policies, the inclusion of potential as well as actual transplant recipients, surveillance in outpatients as well as inpatients, the types of laboratory assays utilized, and the case defi nition utilized (i.e., both clinical and laboratory defi nitions). hmpv has been detected worldwide with a seasonal distribution. community outbreaks occur yearly mainly in winter and spring (january to may in the northern hemisphere; june to july in the southern hemisphere) (figure 31-1 ). often hmpv outbreaks will be concomitant with or subsequent to rsv outbreaks. hmpv most commonly affects young children less than 2 years old and is second only to rsv as a cause of bronchiolitis. seroprevalence studies have shown a high percentage of children have contracted the virus by age 5-10 years. however, reinfection can occur later secondary to insuffi cient immunity or infection with different genotypes. predominant hmpv strains can vary from location to location and from year to year. vicente et al. have reported higher virulence by genotype a [ 61 ] , while papenburg et al. reported higher virulence by genotype b [ 62 ] . however, the interaction or impact of hmpv with other viruses or bacteria remains unclear, particularly in immunocompromised patients. the importance of hmpv in transplant recipients has not been as well studied as rsv (table 31 -2 ). it was fi rst reported shortly after the detection of hmpv by boivin et al. [ 17 ] . an early prospective longitudinal study from spain documented hmpv in both autologous and allogeneic hsct recipients, with the incidence and clinical impact of hmpv and rsv disease documented to be quite similar [ 55 ] . one early prospective study documented hmpv infections in 22 adults with hematologic malignancies that progressed from upper respiratory infection to pneumonia, with a case-fatality rate close to 14% [ 63 ] . lower respiratory tract disease and pneumonia due to hmpv infection in hsct recipients has been reported to have an overall incidence of 1-4% [ 48 , 55 , 64 , 65 ] . a single case series described hmpv-positive nasal aspirate samples in 86% of 21 adults following hsct, many of whom were asymptomatic [ 66 ] . this study demonstrated very high rates of genetically similar viruses and differs from most other studies due to high rates of genetically identical viruses. these authors suggested that these hmpv infections may have originated in the hospital nosocomially; nonetheless, this study is an outlier compared to other reports of hmpv in transplant centers. pneumonia rates following hmpv infection have been reported at 20-28% with mortality rates of 0-4% [ 67 -69 ] . among 163 hsct recipients who underwent bal for investigation of lower respiratory tract disease with pulmonary infi ltrates by radiographic imaging, hmpv was detected in bal samples from 5 of 163 (3%) patients; four of these fi ve died with acute respiratory failure highlighting the potential severity of hmpv pneumonia [ 64 ] . a retrospective cohort study at the hutch described a high mortality rate of 43% among patients with hmpv pneumonia, a rate similar to rsv pneumonia mortality [ 49 ] . studies from other transplant units continue to document the potential of hmpv to cause severe lower respiratory tract disease, with clinical presentations and outcomes generally similar to rsv [ 49 , 70 ] . the signifi cance of hmpv infection in sot recipients remains less well defi ned, with the exception of hmpv disease in lung transplant recipients [ 70 ] . case reports of severe disease have been described following liver and renal transplantation [ 71 , 72 ] . rates of hmpv infection in lung transplant recipients have been reported to be similar to those seen in studies of hsct recipients, varying from 4-6% [ 57 , 73 ] . in most of these patients, hmpv appears to frequently be the sole pathogen detected. detection of hmpv in lung transplant recipients may not necessarily signify disease, as was noted in a study of 93 lung transplant recipients undergoing bal mainly for surveillance purposes; four cases of hmpv was detected in asymptomatic patients [ 74 ] . hmpv infection has been found in 4-6% of lung transplant recipients, but prevalence may be higher during nosocomial outbreaks [ 39 , 75 ] . one study in the setting of a community outbreak identifi ed hmpv in bal samples from 9 of 26 (35%) patients; their clinical presentation varied from asymptomatic infection to severe disease [ 39 ] . acute allograft rejection was more frequent in the hmpvinfected group than in the non-hmpv-infected group (33% vs. 6%, respectively; p = 0.0257); and overall mortality was also higher (33% vs. 0%, respectively; p < 0.0025) [ 39 ] . another prospective study found hmpv infection as frequent as rsv after lung transplantation, and to cause as much pneumonia and acute allograft dysfunction (63% vs. 72%, respectively), but only rsv was associated with chronic allograft dysfunction at 6 months [ 76 ] . in another study, 25% of hmpv infections in lung transplant recipients were associated with acute allograft dysfunction compared with 88% for rsv [ 49 ] . a meta-analysis of hmpv respiratory infections and allograft rejection, among lung transplantation recipients indicated that detection of hmpv from airway secretions may be a signifi cant posttransplantation occurrence. a total of 2883 samples from 1007 lung transplant recipients, were analyzed for virus detection; 337 samples had viruses identifi ed and 57 (17%) were positive for hmpv. twenty of these 57 (35%) cases of hmpv had acute rejection within 3 months of viral detection. there were fi ve (9.4%) cases of chronic rejection in association with hmpv. all studies included in the meta-analysis, with the exception of one, identifi ed rejection within 3 months. another study has also described cases of chronic rejection within 6 months [ 77 ] . disease manifestations of rsv are dependent on many factors including the immunity and immune competence of the host, the time of infection related to transplant, the type of transplant, the age and underlying health of the patient, and the degree and duration of immunodefi ciency. rsv infections in hsct recipients typically follow the same clinical sequence as rsv infections in previously healthy children: signs and symptoms of a uri such as rhinorrhea, sinus congestion, sore throat, or otitis media frequently precede signs of lower respiratory tract disease including cough, wheezing, hypoxia, and pneumonia [ 5 , 6 , 9 ] (tables 31-3 and 31-4 ). the presence of wheezing with respiratory symptoms during the respiratory virus season may provide the clue that rsv may be present. progression of uri to lri has been associated with patients who are early (<1 month) posttransplant, risk factors for the progression of rsv upper respiratory disease to lower tract disease or pneumonia and factors relating to fatal disease have been evaluated. the most common risk factor described in multiple centers using different methods of case ascertainment and viral detection remains lymphopenia. in a prospective multicenter study carried out by the european group for blood and marrow transplantation, lymphopenia but not neutropenia significantly increased the risk for lower respiratory tract disease [ 80 ] . older age and donor status are also signifi cant risk factors in some studies, whereas cmv serostatus, acute graft versus host disease, time relative to engraftment, and preemptive aerosolized ribavirin at a low dose of 2 h daily were not signifi cant [ 81 ] . investigators from mdacc have demonstrated that season of year, relapse of malignancy, presence of graft versus host disease, increasing age, and lack of engraftment are inpatient risk factors for the development of rsv pneumonia [ 9 , 12 , 22 , 82 ] . recently, large retrospective studies have identifi ed graft source including cord or marrow (adjusted hazard ratio (hr), 4.1, 95% ci 1.8-9.0) and oxygen requirement (adjusted hr 3.3, 98% ci, 15-6.7) to be independently associated with death due to respiratory failure in hsct recipients [ 83 ] . smoking history, conditioning with high-dose total body irradiation, and an absolute lymphocyte count < 100/mm 3 at the time of uri onset are also signifi cantly associated with disease progression [ 84 ] . since initiation of therapy hinges on prompt diagnosis, the possibility of false negative laboratory tests must be considered in individual patients and the diagnosis should be aggressively pursued by other means, such as bal. regardless of the therapeutic intervention, high rates of mortality due to rsv pneumonia are documented in seriously immunocompromised patients when therapy has been initiated after the development of respiratory failure (figure 31-2a ) . survival rates remain low for severely immunosuppressed patients who are intubated due to progressive rsv pneumonia unrelated to super-imposed pulmonary hemorrhage, pulmonary edema, or bacterial superinfection [ 5 , 50 , 55 , 81 , 83 ]. hmpv may cause upper or lower respiratory tract infections in hsct recipients. asymptomatic shedding from upper respiratory tract has been reported, indicating that not all hmpv infections result in severe lower tract disease [ 50 , 66 , 85 ] . hsct recipients with hmpv disease in the immediately posttransplant period typically present with respiratory symptoms including nasal congestion, sore throat, cough, or fever. once lower respiratory tract disease develops, rapidly progressive pulmonary infi ltrates frequently accompanied by hypotension, septic shock, or both may be present [ 64 ] ( figure 31-2b and c ) . in a prospective viral surveillance in hsct recipients where samples for respiratory viruses were obtained weekly, regardless of the presence of respiratory symptoms, a cumulative incidence estimate of hmpv of 6.2% (95% ci, 1.3-11.2) over 1 year was determined; all cases of hmpv had clinical respiratory symptoms identifi ed ranging from mild to more severe disease with single or multiple symptoms [ 50 ] (table 31-4 ) . among 15 hsct recipients with hmpv detected by rt-pcr in bal, 10 (67%) had positive hmpv rt-pcr in nasal wash sampled within 11 days prior to or following the bal [ 49 ] . viral rna was detected in the serum of one of these severely immunocompromised hsct recipients at two time points, 4 days apart, with a viral load of ~8 log10 copies/ml in each sample. this patient died of severe respiratory disease. in assessing risk factors associated with overall mortality at day 100 posttransplant, the use of bone marrow as the stem cell source, steroid treatment and oxygen use have been associated with overall mortality [ 49 ] . radiographic fi ndings associated with hmpv infection in the hsct recipient may consist of centrilobular nodules, ground glass opacities, tree-in-bud to diffuse bilateral alveolar infi ltrates [ 86 , 87 ] (figure 31 -2b-f ). centrilobular nodules have been associated with less mechanical ventilation while ground glass opacities tended to be associated with increased rates of hypoxemia [ 49 ] . alveolar consolidation corresponds to more extensive damage on histological examination. histological evaluation has also shown hyaline membrane formation, foci of bronchiolitis obliterans and organizing pneumonia and diffuse alveolar hemorrhage. variable disease severity has been reported following hmpv infection in sot recipients. in one study of 114 lung transplant recipients, hmpv was detected in four symptomatic and one asymptomatic patients (4.3%), but viral infection was not persistent and resolved without major complications [ 88 ] . in another study, nine lung transplant recipients with hmpv were compared with 17 transplant recipients without hmpv infection; hmpv infection was associated with signs of acute graft rejection and increase overall mortality (three of nine with hmpv-infected patients died and none died in the hmpv-negative group) [ 54 ] . outbreaks of respiratory viruses occur annually in the community, with potential for patients, families, or health care workers to become infected following exposure outside the hospital. however, nosocomial transmission within the hospital setting becomes a serious concern because of the high rates of morbidity and mortality in immunocompromised patients documented in nosocomial outbreaks. hospitalbased oubreaks of rsv infection in hsct recipients can occur through introduction of circulating community strains as well as transmission of identical viral strains among patients [ 42 , 44 ] . outbreaks of rsv have been associated with high mortality rates ranging up to 45% of infected patients [ 89 , 90 ] . the transmission of identical strains of rsv within the outpatient setting into the hospital setting has also been shown [ 91 ] , demonstrating the importance of infection control measures in both the inpatient and outpatient setting. nosocomial transmission of hmpv can also occur, with outbreaks possible in both inpatient and outpatient units. in one study, 15 patients were diagnosed with hmpv within 7 weeks in a tertiary care cancer unit [ 92 ] . molecular subtyping revealed infection with genotype a2a virus, implicating nosocomial transmission. four patients (26.6%) died from hmpv-associated pneumonia and consequent multi-organ failure. current options for the antiviral therapy of rsv disease in immunocompromised hosts are limited. large, controlled, therapeutic trials for rsv pneumonia or lower tract disease in immunocompromised patients have not been conducted. aerosolized ribavirin, licensed in the usa for the therapy of rsv bronchiolitis and pneumonia in infants and young children in 1986, is the antiviral agent currently utilized for the treatment of rsv disease in immunocompromised patients. in general, ribavirin is given as an inhaled aerosolized solution via a face mask in a protective environment, such as a "scavenging tent," to protect health care workers from potential drug contamination. ribavirin was initially licensed for use when given for 12-18 h/day, but the use of intermittent aerosolized ribavirin given over 2 h, three times daily, was found to have similar effectiveness to 12-16 h/day continuous infusion ribavirin in healthy children [ 93 -95 ] and has been utilized in adults because of ease of administration and enhanced tolerability. a randomized trial in hsct patients at risk for lrti evaluated intermittent dosing of ribavirin given over 2 h three times daily versus continuous ribavirin administration using an adaptive randomized trial design in 50 hsct patients, with the authors concluding that the intermittent schedule was preferable because of ease of administration and evidence of higher effi cacy [ 96 ] . only one randomized, controlled, multicenter clinical trial of aerosolized ribavirin for the prevention of rsv disease progression to lri has been conducted in hsct recipients early posttransplant, and despite an enrollment period of several years, only 15 patients were enrolled [ 97 ] . none of the ten patients randomized to high dose, short duration aerosolized ribavirin (administered as 2 g/100 ml water given over 2 h three times daily) had disease progression compared to 2/5 control patients, a trend that was not statistically significant ( p = 0.08). viral loads appeared to be reduced during the ribavirin treatment, but did rebound after cessation of therapy. data demonstrating effectiveness of ribavirin is mainly retrospective. in an open trial in adult hsct recipients with "rsv-induced acute lung injury," monotherapy with aerosolized ribavirin was reported to be of benefi t if initiated prior to the development of radiographic infi ltrates [ 6 ] . in another open trial in adult hsct recipients with radiographically proven rsv pneumonia, combination therapy with aerosolized ribavirin and high rsv-titered ivig was reported to be of benefi t only if initiated prior to the onset of respiratory failure [ 8 , 98 ] . a retrospective mdacc study of confi rmed rsv infections in 280 allogeneic hsct recipients from 1996 to 2009 utilized multivariable logistic regression to demonstrate that lack of ribavirin aerosol therapy at the upper respiratory tract disease stage was an important risk factor associated with rsv lrti and all-cause mortality [ 99 ] . in a retrospective study of hsct recipients with confi rmed lower respiratory tract rsv infection based on analysis of bronchoalveolar lavage fl uid at the hutch, viral rna detection in the blood was detected in 30% of 92 patients at a median of 2 days following diagnosis of lower respiratory tract disease [ 37 ] . neutropenia, thrombocytopenia, and mechanical ventilation increased the risk of rsv rna detection in the plasma or serum but lymphopenia and steroid use did not. the detection of rsv rna in the serum or plasma increased the risk of overall mortality with an adjusted hazard ratio (ahr) of 2.09 ( p = 0.02). data in solid organ transplant recipients is even more limited. favorable responses have been reported in an open trial of lung transplant recipients with lower respiratory tract disease who received monotherapy with aerosolized ribavirin [ 58 ] , as well as open trials with oral ribavirin [ 100 -102 ] , although controlled studies have not been performed. oral ribavirin was found to be well-tolerated, result in less hospitalization, and be less expensive than intravenous or inhaled ribavirin in a retrospective study of 52 lung transplant recipients [ 102 ] . the treatment of rsv disease with a combination of antiviral therapy and passively administered immunoglobulin has been investigated in animal models and in children [ 103 -106 ] . therapy with ivig containing high levels of rsvspecifi c antibodies alone does not seem to be effective in placebo-controlled trials in children who were not immunocompromised [ 104 , 107 ] . in small open trials at mdacc, combination therapy with aerosolized ribavirin (18 h/day) and high rsv-titered ivig (0.5 g/kg every other day) was associated with a favorable response in adult hsct recipients and patients undergoing induction chemotherapy for leukemia who had rsv lower respiratory tract disease in whom therapy was initiated prior to respiratory failure [ 8 , 98 ] . at the dana farber cancer institute, combination therapy with aerosolized ribavirin (18 h/day) and rsv-ivig (1.5 g/kg for one dose) was similarly associated with a favorable response in 2 hsct recipients with clinically severe rsv pneumonia occurring early following transplant [ 105 ] . in subsequent years, mdacc has utilized a combined regimen with similar response, although standard ivig in frequent and large doses (500 mg/kg qod) has been substituted for high-titered ivig. other therapeutic options for the treatment of rsv include the use of oral ribavirin, which has been studied in hsct recipients and found to be safe and less expensive than intravenous or aerosolized ribavirin [ 100 , 108 , 109 ] , and is recommended as an option in addition to intravenous and inhaled ribavirin by the european conference on infections in leukemia [ 94 ] . other options include iv ribavirin (an investigational drug, icn) and topical immunoglobulins administered with aerosolized or intravenous ribavirin [ 105 , 110 , 111 ] . the relative ease of administration of iv ribavirin is attractive, but high rates of mortality (80%) and signifi cant cases of hemolytic anemia (20%) make this option currently problematic. although the european experience with combination aerosolized/intravenous ribavirin has been favorable [ 112 ] and intravenous ribavirin is relatively simple to administer, the high rates of mortality and signifi cant cases of hemolytic anemia make this approach controversial. monotherapy with iv ribavirin may be more toxic in these patients than has been previously reported in patients with hemorrhagic fevers [ 113 , 114 ] . the decision to initiate therapy with aerosolized ribavirin with or without immunotherapy for a rsv-uri must take into consideration many factors, including the patient's risk of developing serious lower respiratory tract disease (and specifi cally, the degree of anticipated lymphopenia), the potential exposure of health care workers to the medication, the psychological and physical discomfort to the patients of aerosol therapy, the adverse effects of aerosolized ribavirin such as bronchospasm, the high cost of these drugs as well as the intensive respiratory therapy needed to safely administer aerosolized ribavirin, and the need for hospitalization with more frequent or prolonged ribavirin dosing regimens. in patients who have already undergone conditioning therapy and stem cell infusion but have not yet engrafted, the initiation of antiviral therapy at the uri stage may be beneficial. early studies conducted in the 1990s were small and uncontrolled. one study conducted at fhcrc treated 25 hsct recipients with upper tract rsv disease with low dose aerosolized ribavirin administered at a high concentration (60 mg/ml) for 2 h each day (total: 2 g/day [ 8 ] ). unfortunately, 8/25 patients developed pneumonia, and seven of these died. another study evaluated combination therapy with aerosolized ribavirin and 500 mg/kg ivig every other day in 12 patients, two of whom developed pneumonia and died [ 8 , 115 ] . this "preemptive" strategy, similar to that used in the prevention of cmv disease and cmv pneumonia, is used at some transplant centers for those patients at highest risk of rsv disease progression, such as pre-engrafted patients with rsv detected in the fi rst weeks following transplantation. other options for preemptive therapy include immunotherapy with ivig or rsv-specifi c monoclonal antibodies, although little data on effi cacy of immunotherapy is available. no antivirals for the therapy of hmpv are currently available or routinely utilized. ribavirin is active in vitro and in vivo against hmpv, although there are no controlled studies or evidence from large retrospective reviews for the treatment of hmpv pneumonia in humans and no drug has yet demonstrated clinical effectiveness in humans [ 116 , 117 ] . intravenous, oral or inhaled ribavirin alone or in combination with ivig has been reported as potentially successful therapeutic options. a retrospective analysis compared the outcome between 13 immunocompromised patients with hmpv pneumonia treated with ribavirin â± ivig and ten untreated patients. ribavirin treatment was associated with more hypoxemia and similar mortality, possibly related to late initiation of therapy [ 49 ] . a seattle study describing hmpv lower respiratory tract disease in 55 immunocompromised children, including nine undergoing hsct and eight sot recipients, demonstrated that hsct recipients had more evidence of severe disease [ 91 ] . five of eight hsct recipients but no sot recipients had lower tract disease and were treated with aerosolized ribavirin; three had been diagnosed with hmpv pretransplant and during the posttransplant period received both ribavirin and ivig. two additional children received aerosolized ribavirin only. ribavirin was generally administered at a dose of 2 g given three times daily for 5-11 days. two of the three patients diagnosed with hmpv pretransplant who received ribavirin and ivig died [ 91 ] . an aggressive infection control strategy can be effective in reducing the nosocomial acquisition of rsv by transplant recipients [ 91 , 118 ] . infection control strategies play a crucial role in the prevention of respiratory viral infection [ 89 , 118 -120 ] . an effective strategy is based on understanding the potential seriousness of these infections in transplant recipients, knowledge of the viruses circulating in the community, and ongoing surveillance in high-risk patients. continuing education of patients, family members, visitors, and staff regarding the potential seriousness of these infections must be repeatedly emphasized. frequent and routine clinical screening of high-risk patients for acute upper and/or lower respiratory tract illness or fl u-like illness must be conducted, with sampling of respiratory secretions from symptomatic high-risk patients routinely performed both pretransplant and posttransplant. each health care-acquired infection should be viewed as a sentinel event warranting an investigation and reaffi rmation or modifi cation of the preventative strategy. infection control strategies should be designed to prevent spread by multiple modes of transmission [ 90 , 91 ] . multiple respiratory viruses may circulate in the community concurrently and can be spread by different means. infection control measures may need to be intensifi ed during community or hospital outbreaks, and the intensity and duration of infection control measures should be tailored to the risk of serious disease in different subsets of transplant recipients, and to what works in the "real world." guidelines for the prevention of opportunistic infections among hsct recipients have been issued by the centers for disease control and prevention (cdc), the infectious diseases society of america, and european and american societies for blood and marrow transplantation. the guidelines clearly present evidencebased recommendations rated by the strength of the recommendation and the quality of the supporting evidence, similar to guidelines previously issued for the prevention of opportunistic infections in those with human immunodefi ciency virus [ 120 ] . preventive strategies for hsct recipients, their household contacts and other close contacts, and health care workers are clearly outlined in this document. the prevention of nosocomial acquisition of respiratory viral infections in hsct recipients has been demonstrated in one prospective study comparing rates of infection in patients cared for in a "protected environment" with patients cared for on a transplant unit where infection control measures were strongly encouraged, but not rigidly enforced [ 9 ] . the effectiveness of infection control interventions has also been demonstrated by the dramatic decline in the frequency of nosocomial crv infections among hsct recipients cared for in this transplant unit after the implementation of an aggressive, multifaceted infection control strategy [ 118 ] . although this intensive multifaceted approach has been effective, modifi ed versions of this strategy have also been effective. for instance, the seattle cancer care alliance (scca ) adult inpatient transplant unit uses a similar strategy with the exception that health care workers and other visitors do not wear masks when entering the patient room [ 91 ] . however, these workers are intensively screened for signs and symptoms of respiratory illnesses prior to entering the unit, and restricted from entering the unit if they are symptomatic. similarly, hsct recipients with respiratory symptoms are not transferred to other units, but are cared for on the transplant unit using modifi ed droplet precautions with all persons entering the room wear gloves, gown, and mask, and the door to the room is kept closed. it may not be feasible to so intensively protect patients for the duration of increased susceptibility to respiratory viral diseases. protective strategies are costly and cumbersome, and pose unpleasant restrictions on the freedom and quality of life of the patient and their families. this problem is further compounded by the growing trend to discharge patients early from the hospital and to perform outpatient hsct or posttransplant care. transplant recipients residing in the community and followed frequently in the outpatient setting are another group in which infection control practices must become priority [ 91 ] . the prevention of exposure to respiratory viruses is particularly challenging among high-risk transplant recipients living in the community because respiratory infections are so prevalent and so contagious. examples of protective measures for outpatients include washing hands frequently and thoroughly, avoiding close contact with individuals suffering from respiratory illnesses, and encouraging close contacts to vigorously practice respiratory hygiene. in many cases, such as individuals living with children, such efforts may be nearly impossible. consideration of removing day care exposures for young children or decreasing exposure to transplant recipients to children (including siblings), can and should be discussed with families. the rigor and duration of prophylactic measures need to be individualized based on the immunologic status of the patient and the risk for serious disease, the needs of the patient, and quality-of-life issues. passive immunization with immunoglobulin, immunoglobulin products, and humanized monoclonal antibodies have been actively studied in the pediatric infant population. palivizumab , a humanized monoclonal antibody directed against the rsv f protein, is licensed for the prevention of rsv disease in premature infants and infants with congenital heart disease, and is administered as a monthly injection during the 4-5 months of rsv season. the cost of this therapy has led to new guidelines for use in the pediatric population [ 121 ] . similar interventions have been utilized to attempt to decrease the morbidity of serious rsv disease in hsct recipients but direct proof of effectiveness has not yet been demonstrated [ 115 ] . for example, passive immunoprophylaxis in immunocompromised patients has been evaluated in an open trial conducted [ 122 ] using high-titered human rsvig. in this adult study, signifi cant antibody titer increases to other respiratory viruses were extremely variable, although the subset of patients with the lowest titers appeared to receive the greatest increase in viral-specifi c antibody. the cost of this potential therapy remains quite high. the monoclonal rsv antibody palivizumab (synagis) has been studied in an open label study in adult hsct recipients [ 123 ] . immunoprophylaxis with monoclonal rsv antibodies would be prohibitively expensive in older children and adults, but may have the potential to protect against rsv infection, based on pediatric studies. new antibody products, including long-lasting monoclonal antibodies with enhanced activity against rsv, are under development and if available at a less expensive price, could potentially provide protection against rsv for patients in the pretransplant and immediate posttransplant phase. newer monoclonal antibodies that have the potential to neutralize against both rsv and hmpv have also been described, offering hope for newer preventive modalities that may protect against both these viruses [ 124 ] . screening of transplant recipients for respiratory viruses prior to transplant is not routinely recommended based on current us and international transplant guidelines [ 94 , 125 ] . however, the assessment of viral shedding or infection in symptomatic transplant candidates prior to transplant is recommended. delay of transplant based on detection of rsv or hmpv may be warranted depending on the evaluation of the risk and benefi ts of continuing on with transplantation. consequences of postponing transplantation should be considered, including progression of underlying malignancy, logistical issues regarding the donor availability, and accessibility of services for the patient. an early study of rsv diagnosed prior to hsct demonstrated that delaying transplant reduced the risk of pneumonia following transplant [ 23 ] . more recently, a large prospective study conducted in 458 patients at the hutch demonstrated that nearly 25% of subjects had respiratory viruses detected pretransplant [ 24 ] . overall, patients with a virus detected prior to transplant had fewer days alive and lower survival at day 100 (ahr 2.4; 95% ci 1.3-4.5) compared with patients who did not have viruses. in hsct recipients who had respiratory symptoms and a virus detected prior to transplant (mainly adults), an increased overall mortality was seen compared with patients without respiratory viruses detected. higher rates of pretransplant infection and sequelae of infection were seen in pediatric patients. detection of respiratory viruses in asymptomatic patients was not associated with increased mortality. this data strengthens current guidelines that recommend patients with respiratory symptoms prior to transplant should be tested for respiratory viruses and transplant delayed, when feasible. however, this study was performed chiefl y in adults. the higher rates of respiratory viruses documented in children pretransplant make routine screening of pediatric patients worthy of further investigation. experimental approaches to the therapy of rsv antiviral therapy include novel fusion inhibitors [ 126 ] , nucleoside agents, small rna inhibitory molecules [ 109 ] , and high-titered monoclonal antibody preparations. two promising rsv antivirals that have shown effi cacy in challenge studies in healthy adults include the alios compound al8176 (alios biopharma, south san francisco, ca), an oral anti-rsv nucleoside designed to inhibit rsv replication by acting on the viral polymerase, and the gilead sciences compound gs5806 (gilead sciences, foster city, ca), an orally bioavailable rsv fusion inhibitor [ 126 , 127 ] . clinical trials of these agents in healthy young children with rsv have been proposed [ 128 ] . an international multicenter, placebo-controlled clinical trial of gs5806 was initiated in july 2014, and remains ongoing in adult hsct recipients with documented rsv infections (clinica ltrials.gov identifi er nct02135614). other antiviral agents are under development. no rsv or hmpv vaccine is currently available. in general, active immunization of transplant recipients will be unlikely to prevent severe disease seen in the fi rst several months posttransplant. however, prevention of rsv infection in families, staff, and nosocomial spread of virus by the use of vaccines holds true promise to benefi t transplant recipients themselves. promising advances in new vaccines directed against both rsv and hmpv have been reported over the past decade, with progress evident in both rsv fusionprotein based vaccines and live attenuated vaccines. advances in the understanding of the pre-and post-fusion nature of the rsv f protein [ 129 ] has led to increased work in developing protein-based vaccines appropriate for older children, adults, and pregnant women. advances in technology and better molecular understanding of rsv and hmpv have resulted in new potential rsv candidate vaccines [ 130 ] . at least 12 rsv vaccines are in clinical studies in phase 1 or 2 clinical studies, with one rsv f vaccine under study in pregnant women. examples of live rsv vaccine candidates under study include live-attenuated vaccines relying on genetic manipulation of the rsv genome [ 131 ] , vectored virus vaccines utilizing the chimpanzee adenovirus or vaccinia virus ankara [ 132 ] , or chimeric viruses containing a backbone of attenuated parainfl uenza with the f gene of rsv added [ 133 , 134 ] . a chimeric hmpv vaccine containing a backbone of avian hmpv and f genes of the hmpv [ 135 ] . although live viral vaccines are unlikely to be given to transplant recipients pretransplant or early 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respiratory syncytial virus challenge study chemotherapy of respiratory syncytial virus infections: the fi nal breakthrough preventing severe respiratory syncytial virus disease: passive, active immunisation and new antivirals structural basis of respiratory syncytial virus neutralization by motavizumab the changing landscape of respiratory syncytial virus live-attenuated respiratory syncytial virus vaccines chimpanzee adenovirus-and mva-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults phase 1 study of the safety and immunogenicity of a live, attenuated respiratory syncytial virus and parainfl uenza virus type 3 vaccine in seronegative children attenuated human parainfl uenza virus type 1 (hpiv1) expressing the fusion glycoprotein of human respiratory syncytial virus (rsv) as a bivalent hpiv1/rsv vaccine new approaches for immunization and therapy against human metapneumovirus key: cord-027679-89yt6fzo authors: mcloud, theresa c.; boiselle, phillip m. title: pulmonary infections in the normal host date: 2020-06-22 journal: thoracic radiology doi: 10.1016/b978-0-323-02790-8.00003-2 sha: doc_id: 27679 cord_uid: 89yt6fzo nan the computed tomography (ct) features of lobar pneumonia are similar to those seen on standard radiography ( fig. 3-3 ). there is usually evidence of confluent opacification with air bronchograms. the air bronchograms are often more easily visualized with ct examination. table 3 -2 summarizes the radiographic clues to the cause of pneumonia. bronchopneumonia (i.e., lobular pneumonia) results when organisms are deposited in the epithelium of peripheral airways (i.e., distal bronchi or bronchioles), resulting in epithelial ulcerations and formation of a peribronchiolar exudate. the inflammatory process spreads through the airway to involve the peribronchiolar alveoli, which become filled with edema and pus. lobules may be affected in a patchy pattern initially, and further spread results in involvement of contiguous pulmonary lobules. eventually, a confluent bronchopneumonia may resemble lobar pneumonia. offending organisms that produce this type of pathologic response include s. aureus, gram-negative organisms, anaerobic bacteria, and l. pneumophila. the radiographic appearance of bronchopneumonia pneumonia is most frequently that of multiple, ill-defined nodular opacities that are patchy but that may eventually become confluent and produce consolidation with airspace opacification (fig. 3-4) . the opacification may be multifocal and involve several lobes, or it may be diffuse. as the disease progresses, segmental and lobar opacification develops, similar to the pattern of a lobar pneumonia. early necrosis and cavitation can occur. the nodular opacities of bronchopneumonia can be identified with facility on ct scans. the small nodules, usually less than 1 cm in diameter, represent peribronchiolar areas of consolidation or ground-glass opacity. they are called acinar or airspace nodules, but these nodules histologically are found in a peribronchiolar location. they are ill-defined and may be of homogenous soft tissue opacity and obscuring vessels, or they may be hazy and less dense so that adjacent vessels are clearly seen (i.e., ground-glass opacity). these nodules usually have a centrilobular location because of their proximity to small bronchioles. this type of pneumonia is usually produced by viral organisms, which result in edema and mononuclear cell infiltration around the bronchi and bronchiolar walls and extend into the interstitium of the alveolar walls. bronchopneumonia or an acute interstitial pneumonia may be seen with viral infections . the early radiographic appearance is that of thickening of end-on bronchi and tram lines. however, this often evolves into a reticular pattern that may be seen extending outward from the hila. hematogenous spread to the lungs from bacterial infection may occur, although this is unusual. one of the most frequent manifestations is septic infarcts. they usually originate from right-sided tricuspid endocarditis or infected thrombi within major systemic veins. this phenomenon is seen in intravenous drug abusers and patients with longstanding indwelling central catheters. septic infarcts tend to be multiple and peripheral and to abut the pleural surface. they occur more frequently in the lower lobes. these nodules or wedge-shaped opacities may show evidence of cavitation ( fig. 3-6) . ct often demonstrates a vessel connected to the area of infarction. on ct, the septic infarcts appear as wedge-shaped, peripheral opacities abutting the pleura. they may contain air bronchograms or rounded lucencies of air, sometimes referred to as pseudocavitation. true cavitation is common. occasionally, septic bacterial infection may result in diffuse massive seeding of the lungs with a miliary pattern (i.e., very small nodular pattern), although this is much more common with hematogenous dissemination of granulomatous infections. box 3-1 outlines the complications of pneumonia. necrosis of lung parenchyma with cavitation ( fig. 3-7) may occur in pneumonia, particularly that produced by virulent bacteria, including s. aureus, streptococci, gram-negative bacilli, and anaerobic bacteria. if the inflammatory process is localized, a lung abscess will form. it is usually rounded and focal, and it appears to be a mass ( fig. 3-8) . with liquefaction of the central inflammatory process, a communication may develop with the bronchus; air enters the abscess, forming a cavity, which often contains an air-fluid level. the walls of the cavity may be smooth, but more often, they are thick and irregular. multiple, small cavities or microabscesses may develop in necrotizing pneumonia ( fig. 3-9 ). they are recognized as multiple areas of lucency within a consolidated lobe or segment. a similar appearance may be produced by consolidation superimposed on areas of preexisting emphysema. if the necrosis is extensive, arteritis and vascular thrombosis may occur in an area of intense inflammation, causing ischemic necrosis and death of a portion of lung. this is a particular complication of klebsiella pneumonia and other pneumonias producing lobar enlargement. the radiographic features include multiple areas of cavitation, often with air-fluid levels. portions of dead lung may slough and form intracavitary masses. coned-down view of the right lung demonstrates a fine reticulonodular pattern, which is more prominent centrally. pneumatoceles are usually associated with pneumonia caused by virulent organisms; the classic offender is s. aureus (fig. 3-10) . they usually form subpleural collections of air, which result from alveolar rupture. radiographically, they appear as single or multiple, cystic lesions with thin and smooth walls. they may show rapid change in size and location on serial radiographs. intrathoracic lymphadenopathy that can be recognized on standard radiographs is uncommon in most bacterial and viral infections; some notable exceptions include mycobacterium tuberculosis, pasteurella tularensis, and yersinia pestis. adenopathy may be associated with fungal infections or bacterial infections that are long-standing or virulent, as in lung abscesses. ct may show slightly enlarged nodes (>1 cm) in patients with common bacterial infections that are not visible on standard radiography. pleural effusion is a common complication of pneumonia, occurring in about 40% of cases ( fig. 3-11 ). most effusions are parapneumonic, but infection of the pleural space with empyema requiring drainage is an important but uncommon complication of some pneumonias. empyemas can be recognized by the presence of gross pus within the pleural space, by a white blood cell count in the pleural fluid of greater than 15,000 cells/mm 3 , by the presence of bacteria within the pleural fluid, or by a ph less than 7.2. chapter 18 provides more detail on the pleural complications of pneumonia. parenchymal necrosis in an underlying pneumonia may produce a fistula between the bronchus and the pleural space (i.e., bronchopleural fistula), and this results in an empyema with an air-fluid level. further discussion of these entities can be found in chapter 18. rapidly progressive and fulminant bacterial or viral pneumonia may result in the acute respiratory distress syndrome (ards). in the preantibiotic era, bronchiectasis was an extremely common complication of bacterial pneumonia, but the incidence of bronchiectasis has declined with the advent of antibiotics. most pneumonias clear within 2 or 3 weeks, but in elderly patients, resolution may take 3 to 4 months. necrotizing pneumonias also tend to resolve slowly. recurrent pneumonias are frequently found in patients with predisposing factors such as chronic obstructive lung disease, bronchiectasis, alcoholism, and diabetes. although recurrent or persistent pneumonia in the same location raises the possibility of an obstructing endobronchial lesion due to lung carcinoma, cancer accounts for less than 5% of such cases. the most common gram-positive bacteria causing pneumonia include s. pneumoniae (pneumococcus), s. aureus, and streptococcus pyogenes. s. pneumoniae (box 3-2) is responsible for one third to one half of community-acquired pneumonias in adults. these infections occur more frequently in the winter and early spring. pneumococcal pneumonia occurs in healthy people, but it is much more common in alcoholic, debilitated, and other immunocompromised individuals. the radiographic features include consolidation that is usually unilateral, although it may be bilateral, and it typically affects the lower lobes (see fig. 3-1) . although it is a lobar pneumonia, it is uncommon for the lobe to be completely consolidated. cavitation is rare, and large pleural effusions are uncommon. when present, they suggest the development of empyema. sometimes, especially in children, the pneumonia may have a rounded, masslike appearance ( fig. 3-12) . this is called a round pneumonia; it results from centrifugal spread of the rapidly replicating bacteria by way of the pores of kohn and canals of lambert from a single primary focus in the lung. s. aureus (box 3-3) is a gram-positive coccus, and the spherical organisms occur in pairs and clusters. this pneumonia rarely develops in healthy adults, but it is sometimes a complication of viral infections and is much more common in infants and children. in infants, unilateral or bilat-eral consolidation involving the lower lungs is the most frequent radiographic presentation. pneumatoceles, thinwalled cysts filled with air or partially filled with fluid, may develop and occasionally rupture into the pleural space, resulting in pneumothorax. in adults, the disease is usually bilateral and is preceded by an atypical pneumonia such as influenza. cavitation is a common feature, and the cavities may be multiple, thick walled, and irregular ( fig. 3-13 ). there is a high incidence of large pleural effusions, and empyema resulting from bronchopleural fistula is a common complication. methicillin resistant staphylococcus aureus (mrsa) pneumonia usually occurs as a nosocomial infection in health care centers particularly in older, immunocompromised or intensive care unit patients. staphylococcal infection in the lungs may occur by way of the hematogenous route. this is usually the result of septic emboli, which arise in the central veins or as vegetations on cardiac valves, particularly in intravenous drug abusers and patients with indwelling intravenous catheters. the radiographic appearance is that of multiple nodular masses with or without cavitation, as previously described. streptococci (box 3-4) are gram-positive cocci that occur in pairs and chains. the pneumonia occasionally occurs in epidemic proportions. this form of pneumonia is much less common than that caused by staphylococcus or s. pneumoniae (pneumococcus). the radiographic features include lower lobe consolidation, often occurring with a segmental distribution. pleural effusions occur frequently, but localized empyema is unusual. pneumonias caused by gram-negative organisms usually are nosocomial pneumonias that affect hospitalized patients. these pneumonias tend to occur in patients maintained on artificial ventilators or in those who have intravenous catheters or a variety of other ancillary support systems. the incidence of gram-negative pneumonia acquired in the community is increasing, which may be related to the a b c klebsiella pneumonia (box 3-5) usually occurs in middle-aged or elderly patients, in those with underlying chronic lung disease, and in alcoholic individuals. radiographic features consist of an upper lobe consolidation. cavitation is common, and the lobar consolidation may lead to an expanded lobe with bulging interlobar fissures (see fig. 3 -2). if necrosis is extensive, pulmonary gangrene may develop. e. coli pneumonia (box 3-6) may be caused by direct extension from the gastrointestinal or genitourinary tract across the diaphragm or result from bacteremia. as is true of most of the gram-negative pneumonias, it is frequently characterized by the development of necrosis and multiple cavities. the lower lobes are more frequently involved. p. aeruginosa pneumonia (box 3-7) usually occurs in hospitalized patients, particularly those with debilitating disease (see fig. 3 -9). organisms that affect the lungs often result from contamination of suction and tracheostomy devices. radiographic features include a lower lobe predilection. however, the consolidation may spread rapidly to affect both lungs. pleural effusions are uncommon. multiple, irregular nodules may develop and are usually associated with bacteremia. these nodules may cavitate. h. influenzae pneumonia (box 3-8) usually develops in patients with copd. the appearance is typically that of a bronchopneumonia with homogeneous segmental opacities, usually in the lower lobes. cavitation and pleural effusions are rare. chemical pneumonitis and acute lung injury diffuse consolidation resembling pulmonary edema aspiration of particulate matter or foreign bodies may produce different clinical syndromes, depending on the size of the aspirated material and the level of airway obstruction. large food particles or foreign bodies may be aspirated into the larynx and upper trachea, resulting in the so-called café coronary syndrome, which is caused by acute upper airway obstruction. these patients exhibit respiratory distress and aphonia. results of chest radiographs are usually normal for patients who have aspirated foreign bodies. if the foreign body is opaque, it may be visible in the airways. air trapping may occur if the foreign body causes airway obstruction of one of the major bronchi. this can be demonstrated by inspiratory and expiratory radiographs, decubitus views, or chest fluoroscopy. occasionally, complete obstruction of the bronchus results in atelectasis and, if the foreign body is unrecognized, in the development of distal pneumonitis or bronchiectasis. ninety percent of aspiration pneumonias and lung abscesses are caused by anaerobic organisms. the pathogens include prevotella, bacteroides, fusobacterium, and peptostreptococcus. because of the presence of oxygen in the lung, the progression of anaerobic infection is slow, beginning in the dependent lung zones. if the patient is in a supine position when the aspiration occurs, the superior segments of the lower lobes are most commonly affected, with the right side affected more frequently than the left (fig. 3-14) . aspiration can also affect the posterior segments of both upper lobes. chronic or recurrent aspiration, particularly in patients who are in the upright position, usually results in consolidation involving the basilar segments of the lower lobes. the middle lobe and lingula are uncommon sites for aspiration pneumonia. aspiration is the most common cause of a primary lung abscess (see fig. 3-8) . a primary lung abscess refers to a focal, walled-off area of anaerobic pneumonia with central liquefaction necrosis. it is most commonly identified in the superior segments of either lower lobe. lung abscesses have a fairly thick wall and may or may not have an air-fluid level. a rounded, masslike lesion may precede the development of cavitation. occasionally, aspiration of nontoxic material that contains insufficient bacteria to produce an infection or insufficient volume to produce atelectasis may occur. the radiographic appearance usually consists of basilar patchy opacities resembling atelectasis, and these areas clear within several days. mendelson's syndrome is a specific form of aspiration that results from the aspiration of gastric acid. this event produces a chemical pneumonitis and acute lung injury. the radiographic manifestations of gastric aspiration are similar to those of noncardiogenic pulmonary edema. the distribution is usually diffuse. atypical pneumonia syndrome (box 3-10) describes pneumonias that do not respond to usual empiric antimicrobial therapy or do not have clinical features distinctive from the usual bacterial pathogens responsible for communityacquired pneumonias. originally, these atypical pneumonias were thought to be caused by viruses. however, other treatable organisms have emerged as important causes of atypical pneumonia, including m. pneumoniae, l. pneumophila, and chlamydia. these nonviral, atypical pneumonias are for the most part readily treatable with antibiotics. most patients with atypical pneumonia present with a nonspecific syndrome consisting of fever, usually without shaking chills, and nonproductive cough, headache, myalgias, and some degree of dyspnea. this contrasts with the classic presentation of bacterial pneumonia, which is characterized by abrupt onset with fever, shaking chills, and purulent sputum, often with chest pain. patients with the latter signs and symptoms usually have a bacterial pneumonia attributable to pneumococci, group a streptococci, klebsiella, s. aureus, or h. influenzae. many of the atypical pneumonias are associated with extrapulmonary manifestations. for example, diarrhea is a prominent part of legionella and mycoplasma infection. m. pneumoniae (box 3-11) accounts for approximately 20% of all cases of pneumonia. it usually occurs during the winter months in enclosed populations, such as students in college dormitories. the incubation period is 2 to 3 weeks, and the onset is often insidious, with low-grade fever and nonproductive cough. extrapulmonary manifestations may include otitis, nonexudative pharyngitis, and diarrhea. the radiographic features are usually those of a fairly diffuse, interstitial, fine reticulonodular pattern. this may evolve to patchy airspace consolidation, particularly in the lower lobes ( fig. 3-15 ). hilar adenopathy is seen in approximately 20% to 40% of patients. the radiographic appearance is very similar to that of many viral infections. the diagnosis is made by serologic evaluation. the first outbreak of legionnaires' disease was recognized in philadelphia at a legionnaires' convention (box 3-12). clinical features include acute febrile illness without pneumonia; systemic disease with primarily pulmonary manifestations; a peak incidence in patients older than 60 years; a predisposition in smokers and those with alcoholic liver disease; high fever, shaking chills, and cough with small amounts of mucoid sputum; pleuritic chest pain; watery diarrhea in about one half of patients; and headache. the organism is spread by airborne transmission, usually through moist air exhaust or cooling towers. the radiographic features of legionnaires' disease often consist of segmental opacification and consolidation, particularly of an upper lobe. rapid development of coalescence with complete consolidation of an involved lobe and rapid extension to adjacent lobes are common features ( fig. 3-16 ). parenchymal changes are extensive, but pleural effusions are uncommon. the diagnosis of legionnaires' disease is usually made by serology using indirect fluorescent antibody. direct identification of the organism may be confirmed by direct fluorescent antibody (dfa) techniques using properly collected specimens. chlamydia, a long recognized cause of pneumonia in neonates, is an increasingly frequent cause of communityacquired atypical pneumonia in adult patients (box 3-13). it is caused by the twar agent (chlamydia pneumoniae). chlamydia pneumonia may occur in compromised and noncompromised adults as an atypical pneumonia. the disease is characterized by fever and nonproductive cough. it is often preceded by pharyngitis. radiographic features may be similar to those of mycoplasma pneumonia. however, more commonly there is a localized area of consolidation in the middle or lower lobes, which may be patchy or homogeneous ( fig. 3-17 ). atypical nonviral pneumonias are rare. they include psittacosis; q fever, a rickettsial disease; and tularemia. chlamydia psittaci is the etiologic agent of psittacosis, which may be transmitted by any avian species, and it is contracted by inhalation of infected aerosol material. the clue to the diagnosis is the history, which should include information about any contact with birds. psittacosis usually mimics a standard bacterial pneumonia on chest radiography. coxiella burnetii is the etiologic agent of q fever, which is a rickettsial disease. it is most common in the western and southwestern parts of the united states, and it can be transmitted by infected dust from animals. the radiographic features vary, but the most specific pattern simulates mycoplasma or viral pneumonia and usually consists of bilateral, diffuse reticulonodular opacities. tularemia, another animal-associated, atypical pneumo nia, is transmitted by ticks in summer and rabbits in winter. there is an ulceroglandular form, which produces a skin papule that eventually ulcerates at the port of entry. regional lymph nodes may become enlarged and eventually drain and ulcerate. in the typhoidal form, no portal of entry is apparent, but patients are characteristically extremely ill with gastrointestinal symptoms. pneumonia may occur in patients with either of these presentations. the most common radiographic feature is that of a localized and homogenous opacity, but lobar consolidation has also been reported. occasionally, multiple lobes are involved. bilateral hilar adenopathy may occur. primary respiratory viruses (box 3-14) include the parainfluenza and influenza group of viruses, respiratory syncytial virus (rsv), adenovirus, and picornavirus. the incidence of these infections varies with the age of the patient. for example, in children, rsv is responsible for up to 85% of epidemic lower respiratory tract infections and up to 60% of all pneumonias; in adults, the influenza and parainfluenza groups are responsible for most of the epidemic viral pneumonias. they usually occur during late winter. adenovirus and picornavirus cause nonepidemic respiratory infections. other viruses (e.g., cytomegalovirus) produce pneumonia as part of a systemic infection. in all cases, the infection usually begins in the larger central airways. at this stage, the chest radiograph frequently appears normal. the radiologic correlates of severe inflammation and edema of the bronchial walls include coarse reticular opacities in the form of rings and parallel lines (i.e., tram tracks) due to bronchial wall thickening in the central perihilar lung zones. when the small airways are involved, bronchiolitis develops. involvement of terminal bronchioles may lead to airway obstruction. this is more likely to occur in infants and young children because the cross-sectional area of the airways is small. diffuse overinflation and air trapping can be visualized. when the infection spreads to the alveoli, the disease is usually limited to the parenchyma around the terminal airways. the radiographic features in children and adults usually consist of a diffuse reticulonodular pattern, often with focal and patchy areas of consolidation (see fig. 3 -4). multiple lobes are usually involved. ct may reveal the anatomic localization of the disease. the bronchiolitis and surrounding inflammation produces nodular opacities, which are located in the center of the lobules. branching centrilobular opacities represent impaction of small airways, and their appearance has been referred to as the treein-bud pattern ( fig. 3-18 ). other common ct findings of viral pneumonia include ground-glass attenuation with a lobular distribution and foci of segmental and subsegmental consolidation. influenza is one of the most frequently reported contagious diseases. symptoms include fever, nonproductive cough, weakness, and myalgias. most patients who develop severe pneumonia have underlying disease or superinfection with bacterial organisms. radiographic features may reflect the complicating bacterial pneumonia. however, a diffuse reticulonodular pattern may be seen in infants and children with the disease. adenovirus may occur in epidemic or pandemic proportions. when pneumonia develops, there may be destructive changes involving the peripheral airways, leading to chronic bronchitis, bronchiectasis, and bronchiolitis obliterans. symptoms tend to persist after resolution of pneumonia. radiographic features are very similar to pneumococcal pneumonia in pattern and distribution. rsv, rarely reported in adults, is the most prevalent respiratory viral pathogen in the first 6 months of life. it usually produces focal and diffuse bronchiolitis. if radiographs are abnormal, they usually show increased lung volumes and air trapping, and linear interstitial opacities occasionally may be identified. varicella-herpes zoster (i.e., chickenpox) infection may be responsible for severe pneumonia in adults. the radiographic features are fairly characteristic. they consist of nodules ranging from 4 to 6 mm in diameter, with illdefined margins diffusely distributed throughout both lungs (fig. 3-19) . radiographic resolution usually occurs over many weeks. one of the interesting sequelae of chickenpox pneumonia is the development of diffuse, discrete pulmonary calcifications that can be identified on routine radiographs obtained after the infection (fig. 3-20) . histoplasmosis should be considered in the differential diagnosis of this radiologic appearance. cytomegalovirus infection is discussed in chapter 4. the epstein-barr virus is the presumed etiologic agent for infectious mononucleosis. although upper respiratory symptoms predominate, patients may develop a nonproductive cough. the chest radiograph is usually normal, but occasionally, pronounced hilar lymph node enlargement with an ill-defined, diffuse reticular pattern in the lungs may be seen. mycobacteria are aerobic, nonmotile, non-spore-forming rods that have in common the characteristics of staining bright red with carbol fuchsin and resistance to discoloration by strong acid solutions. the organisms are therefore referred to as acid-fast bacilli (afb). there are several mycobacterial species, but the most important include mycobacterium leprae, the cause of leprosy; m. tuberculosis and mycobacterium bovis, responsible for tuberculosis; and the nontuberculous mycobacteria that are important etiologic agents in the development of pulmonary disease. in the latter part of the 19th century, tuberculosis (box 3-15) was a leading cause of death in the united states. the advent of drug therapy and improved public health measures led to a steady decline in the incidence of tuberculosis after world war ii until 1985. for the next 7 years, a slow but steady increase in the incidence of tuberculosis was observed. this rise was primarily attributed to a large number of cases associated with acquired immunodeficiency syndrome (aids). immigration into the united states of individuals from third world countries also might have contributed to the increased prevalence of tuberculosis. since 1993, the rate of tuberculosis has declined considerably. in 2006, the rate of tuberculosis in the united states was the lowest since the beginning of national record keeping in 1953. the tuberculosis rate is continuing to decline, but the rate of decline has recently slowed. respiratory or systemic symptoms patients older than 60 years diarrhea common airborne spread through moist air exhaust or cooling towers diagnosis by serology with indirect fluorescent antibody affects upper lobes rapid spread to other lobes from 1993 to 1997, there was also a decrease in the percentage of multidrug-resistant tuberculosis cases among persons with no prior history of tuberculosis, with a reduction from 2.4% to 1.1%. since 1997, the rate has remained steady at approximately 1%. in the united states, tuberculosis case rates vary considerably among different racial and ethnic populations and are lowest among whites. for example, compared with whites, the case rates are nearly 25 times higher for asians and 10 times higher for blacks and hispanics. the rate of tuberculosis among foreign-born persons in the united states is nearly 10 times higher than that of persons born in the united states. other susceptible populations include the aged and the immunocompromised, particularly patients with aids. infection with tuberculosis occurs as the result of inhalation of airborne droplets containing the tubercle bacilli. the initial infection, referred to as primary tuberculosis, is most common in the lower lobes. the bacteria are ingested by macrophages and initially spread to local lymph nodes at this stage, and they then may disseminate throughout the body. the infection is usually contained if the host is immunocompetent. however, walled-off tubercle bacilli representing a dormant focus of tuberculosis may activate under appropriate conditions. this may occur in the second type of tuberculosis, referred to as reactivation or postprimary tuberculosis. reactivation or post primary tuberculosis can occur any time after the primary infection, but the highest rate of reactivation occurs during the first and second years after the initial infection. reactivation tuberculosis usually involves the lung apex, but a dormant focus of tuberculosis may become active in other organs, such as the bones, kidney, or brain. clinically active disease may develop at the time of primary tuberculous infection (i.e., primary progressive tuberculosis) or when dissemination occurs (i.e., miliary tuberculosis). clinical reactivation disease results when there is an ineffective t-cell immune reaction. the typical pathologic feature of tuberculosis is the caseating granuloma. chlamydia pneumoniae (twar agent) nonproductive cough preceding pharyngitis localized consolidation in lower lobes patchy or homogeneous pattern patients with primary tuberculosis are usually asymptomatic but occasionally may have a symptomatic pneumonia. patients with acute or chronic reactivation tuberculosis usually present with a chronic cough, weight loss, and occasionally with hemoptysis and dyspnea. the symptoms are often insidious. ninety-five percent of patients with active tuberculosis have a positive tuberculin skin test result. the diagnosis must be made on the basis of culture of the organism, although the presence of afb on the smear from the sputum is strong presumptive evidence of tuberculosis. classification of tuberculosis into primary or reactivation phases is based on the radiographic appearance. in third world countries and in the united states during 19th and early 20th centuries, primary tuberculosis was a disease of children, and reactivation tuberculosis was typically a disease of young adults. however, a significant change in the pattern of adult tuberculosis has occurred in the past several decades. because of diminished exposure of children to tuberculosis, the disease often occurs in the primary form in adults. this has resulted in atypical radiographic manifestations of tuberculosis in adults, attributable to primary infection rather than reactivation of the disease. the radiographic features of primary tuberculosis are summarized in box 3-16. primary tuberculous pneumonia can occur in any lobe of the lung but is more common at the lung bases (fig. 3-21) . in more than one half of cases, the disease occurs in the lower lobes. any chronic consolidation, particularly in the bases of the lungs, may suggest tuberculosis. cavitation, although rare in primary tuberculosis, is more frequently reported in adults than in children with the primary form of disease. mediastinal and hilar adenopathy is another feature of primary tuberculosis (fig. 3-22) . it may occur alone or in association with consolidation in the lung. it tends to be particularly predominant in children. ct may be helpful in identifying and localizing adenopathy. on ct scans, tuberculous adenopathy has a predilection for the right paratracheal, right tracheobronchial, and subcarinal regions. occasionally, atelectasis may result from extrinsic obstruction of a bronchus by enlarged lymph nodes. on ct scans obtained with intravenously administered contrast material, these nodes often demonstrate low-attenuation necrotic centers. pleural effusion due to tuberculous pleurisy, also a feature of primary infection, develops when subpleural foci of tuberculosis rupture into the pleural space. patients present 3 to 7 months after the initial exposure. organisms are rarely found in the fluid, and the diagnosis must be confirmed with a pleural biopsy. the ghon lesion (fig. 3-23) is a manifestation of primary tuberculosis, which usually occurs in childhood and is selflimited. the host defense mechanisms handle the initial infection, and the area of consolidation in the lung slowly regresses to a well-circumscribed nodule. this nodule then shrinks and may disappear completely or remain as a solitary, calcified granuloma. the adenopathy regresses and may also exhibit calcification (i.e., rhanke complex). reactivation tuberculosis usually occurs in the apical and posterior segments of the upper lobes and in the superior segment of the lower lobes. it is characterized by chronic, patchy areas of consolidation (fig. 3-24) . cavitation is a hallmark of reactivation tuberculosis (fig. 3-25) . cavities result when areas of caseation necrosis erode into the bronchial tree, expelling liquefied debris. ct is more sensitive than plain radiography in the detection of small cavities (fig. 3-26) . they may have thick or thin walls, which can be smooth or irregular. bronchogenic spread of tuberculosis occurs when a cavity erodes into an adjacent airway and organisms spread endobronchially to other parts of the lung. the typical radiographic features (fig. 3-27) consist of ill-defined nodules that usually are 5 to 6 mm in diameter. they are numerous and often bilateral. on ct, the pattern of bronchogenic spread can easily be recognized by a tree-in-bud pattern. this consists of centrilobular, branching, linear opacities with or without the presence of centrilobular nodules within 3 to 5 mm of the pleural surface or interlobular septa. this pattern is best appreciated on high-resolution ct (hrct). it is not specific for bronchogenic spread of tuberculosis and may occur in other inflammatory diseases involving the peripheral airways. the chronic lesion of reactivation tuberculosis usually consists of fibronodular opacities in the upper lobes, often with the presence of calcification (fig. 3-28) . it is usually associated with volume loss and retraction of the hila. another feature of chronic reactivation tuberculosis is bronchiectasis. tuberculosis should be considered in the differential diagnosis of upper lobe bronchiectasis. the activity of tuberculous disease cannot be determined by radiographs; it is confirmed only by positive cultures. however, tuberculosis is considered radiographically stable if there has been no change over 6 months. unusual patterns of tuberculosis (box 3-17) may occur in the patient who has altered host resistance to the primary infection. miliary tuberculosis is a term used to describe diffuse hematogenous dissemination of tuberculosis that has progressed when the host defense system is overwhelmed by massive hematogenous dissemination of organisms. it may occur at any time after the primary infection. the radiographic appearance (fig. 3-29) is that of multiple, tiny nodules in the interstitium of the lung that are approximately 1 to 2 mm in diameter. ct may allow earlier detection than standard radiography (fig. 3-30) . miliary disease takes up to 6 weeks to become apparent on plain radiographs. pneumothorax occasionally results from tuberculosis. tuberculosis may also cause ulceration of the bronchi, and advanced endobronchial tuberculosis may produce lobar atelectasis and strongly simulate a primary carcinoma of the lung. a localized nodular focus of tuberculosis, referred to as a tuberculoma (fig. 3-31) , occurs in any portion of the lung and may result from primary or reactivation tuberculosis. it is usually solitary, spherical, and smooth. it may contain a central calcification, but tuberculomas occasionally may be multiple and simulate metastatic disease. tuberculous empyema and bronchopleural fistula may result from a tuberculous pleural effusion. such effusions can become loculated and remain dormant for years. radiographic patterns of tuberculous disease in patients with acquired immune deficiency syndrome (aids) may vary. they are described in chapter 4. nodule communicating with the right upper lobe posterior segment bronchus (single arrow), with associated centrilobular nodular opacities in the superior segment of the right lower lobe (three arrows). b, ct of another patient shows a typical tree-in-bud pattern. centrilobular nodules and branching opacities can be identified close to the pleural surface (arrows). characteristics some nontuberculous mycobacteria (box 3-18) are pathogenic in humans. the most important of these organisms are mycobacterium avium-intracellulare, often referred to as the mac complex, and mycobacterium kansasii. these organisms often exhibit common features. they are usually found in the soil and water. bronchopulmonary disease is caused by inhalation of the organisms, but no human-tohuman transmission occurs. unlike tuberculosis, nontuberculous mycobacterial infections do not manifest separate patterns of primary or reactivation disease. certain geographic areas have a preponderance of these forms of nontuberculous mycobacterial disease. for example, m. kansasii is more prevalent in the western and southern united states, and mac is found more often in the southeastern united states. the three major clinical presentations depend to some degree on the immune status of the host (chapter 4 describes mac disease in aids patients). in human immunodeficiency virus (hiv)-negative hosts, mac typically affects male patients who are heavy smokers with underlying copd. similar infections may occur in patients with silicosis or bronchiectasis. the radiographic features of m. kansasii and mac in this group of patients are indistinguishable from tuberculosis. however, mac lung disease may develop in older women who are considered immunologically competent and who do not have a background of thickening and multiple, calcified nodular and irregular opacities can be seen in the left upper lobe (arrows). volume loss is not a prominent feature in this case. although such an appearance suggests inactive disease, serial radiographs are necessary to determine stability. viable organisms may be present, and the development of clinically active disease may rarely occur. hematogenous dissemination diffuse, 1-to 2-mm nodules pneumothorax endobronchial tuberculosis lobar or segmental atelectasis tuberculoma single or multiple nodules larger than 1 cm tuberculous empyema bronchopleural fistula copd. this disease is usually noncavitary. many women with this form of nontuberculous mycobacterial infection share similar clinical characteristics and bodily features, including scoliosis and pectus excavatum. it is uncertain whether these skeletal features predispose patients to infection due to poor tracheobronchial secretion drainage and ineffective mucociliary clearance or they are associated with markers for specific genotypes that affect body morphotype and susceptibility to infection. because nontuberculous mycobacteria are common contaminants, the identification of invasive disease caused by these infections should be made only when defined clinical, radiographic, and microbiologic criteria have been met as defined by the american thoracic society (ats) and infectious disease society of america (idsa) guidelines. radiologic criteria include the presence of nodular or cavitary opacities on the chest radiograph or an hrct scan that shows multifocal bronchiectasis with multiple small nodules. establishing a diagnosis of nontuberculous mycobacteria does not necessitate the need for treatment in all cases: rather, the decision to institute multidrug therapy should be based on an assessment of the relative risks and benefits of therapy on an individual patient basis. the classic form of atypical mycobacterial infection produces features almost identical to those of reactivation tuberculosis (fig. 3-32) . involvement occurs in the apical and posterior segments of the upper lobes and superior segment of the lower lobes. cavitation is common, and multiple cavities may be observed. the disease tends to be slowly progressive. mac lung disease occurring in older women who are usually nonsmokers without evidence of copd is noncavitary and is associated with bronchiectasis. the classic radiographic features are best appreciated on ct ( fig. 3-33) . the findings are those of cylindrical bronchiectasis associated with multiple, small, focal lung nodules that are approximately 5 mm in diameter. any lobe may be involved, but disease in the lingula and middle lobe has the highest prevalence. occasionally, airspace disease may be delineated. evidence indicates that patients with these findings are truly infected and not colonized with mac and that the mac infection causes the bronchiectasis rather than colonizing preexisting disease. the wide variety of fungi that may produce lung disease can be divided into two groups. some are truly pathogenic and can produce pulmonary infection in normal hosts. they include histoplasma, coccidioides, blastomyces, and cryptococcus. a second group of fungi are secondary invaders or opportunistic organisms, which produce disease in immunosuppressed patients. this group includes aspergillus, candida, cryptococcus, and mucor. the latter group is discussed in chapter 4. histoplasma capsulatum (box 3-19) is a dimorphous fungus that gains entry to the lung by inhalation. distribution is worldwide, and in the united states, it occurs along river valleys, particularly the ohio, mississippi, and st. lawrence. the organism exists in the soil, particularly when it is contaminated by the excrement of birds (e.g., pigeons) or bats. many epidemics may occur when there is heavy exposure due to demolition or construction in areas containing these droppings, such as bat caves, chicken houses, or attics of old buildings. in endemic areas, up to 80% of the population may be infected, but most individuals are asymptomatic. inhalation of spores results in a localized infection of the lung, which then migrates to mediastinal and hilar lymph nodes and eventually migrates to the spleen and liver. the organisms usually are destroyed, and there is no residual of the initial infection, although a scar or calcification may occur. if individual foci of infection and necrosis persist, they may enlarge, resulting in a chronic cavitary lesion indistinguishable from that of tuberculosis. pathologically, well-defined granulomas may be found during the acute phase of disease in the lung, in the mediastinum, and in the various organs to which the organism disseminates. when healed, these granulomas are small and densely calcified. outbreaks of histoplasmosis are usually associated with constitutional symptoms and nonproductive cough. many cases never come to medical attention. the radiographic manifestations of histoplasmosis vary. the acute phase of the disease is characterized by single or multiple areas of consolidation, which are usually segmental or sublobar in distribution. these areas may be accompanied by ipsilateral hilar or mediastinal adenopathy, and occasionally, adenopathy alone may be the only finding. in the epidemic form of the disease, multiple, discrete nodules may be seen throughout both lungs; nodules may occur alone or be associated with hilar adenopathy (fig. 3-34) . they are usually 1 to 5 mm in diameter, discrete, and poorly marginated. with healing, the nodules may remain visible as multiple, discrete, calcified lesions less than 1 cm in diameter with or without calcified hilar lymph nodes (fig. 3-35) . a third radiographic pattern consists of a solitary granuloma or histoplasmoma, which is usually well defined and can range in size from several millimeters to 4 cm. it typically contains a central or target type of calcification. these lesions usually occur in the lower lobes, and they may have associated smaller, calcified satellite nodules. additional radiographic features may be identified in patients with histoplasma infection. they include calcifications in the spleen, which often are best detected on ct. mediastinal lymphadenopathy is common as a sole manifestation of histoplasmosis or accompanying pulmonary consolidation or nodules. nodes frequently calcify as healing occurs. calcified lymph nodes may lead to two complications: broncholiths and fibrosing mediastinitis. calcified lymph nodes may over time erode into a bronchus, producing broncholithiasis and its resulting symptom complex. patients may have unexplained chronic cough and hemoptysis. ct can best identify the intrabronchial calcification that may be associated with distal atelectasis of a segment or lobe (fig. 3-36) a rare chronic form of histoplasmosis can simulate tuberculosis. it usually consists of thin-or thick-walled cavities with patchy areas of consolidation, particularly involving the upper lobes with fibrosis and retraction. disseminated histoplasmosis, which may occur in normal individuals, is much more common in immunosuppressed patients. radiographically, the appearance is identical to that of miliary tuberculosis. coccidioides immitis infection (box 3-20) follows inhalation of infected spores in endemic areas such as desert areas of the southwestern united states and central and south america. clinical manifestations vary. most individuals are asymptomatic, or they may experience a mild flulike illness of the lower respiratory system. acute, severe disease may be associated with fever, cough, and pleuritic chest pain. with the initial inhalation of the spores, a local response or pneumonitis occurs. the immune system eventually destroys the organism, with resolution of the pneumonia. about 5% of individuals may have a chronic, often asymptomatic pulmonary lesion, such as a pulmonary nodule or cavity. similar to tuberculosis, reactivation of the initial focus can occur. dissemination of the organism to hilar and mediastinal nodes is common, and diffuse dissemination is rare but almost universally fatal. the initial pneumonic form of the disease is characterized by an area of consolidation anywhere in the lung but most commonly in the lower lobes. it is usually sublobar, segmental, or patchy. it may be bilateral. hilar and mediastinal lymph node involvement occurs in about 20% of cases, and rarely, it can be seen in the absence of the parenchymal consolidation. most of these lesions resolve spontaneously without therapy. the radiographic features of chronic coccidioidomycosis include solitary or multiple nodules. these tend to cavitate rapidly, and the cavities typically have very thin walls ( fig. 3-37) . the thin-walled cavity is the classic lesion of coccidioidomycosis, but it occurs in only 10% to 15% of cases. disseminated coccidioidomycosis is rare and is characterized radiographically by nodules ranging from 5 mm to 1 cm in diameter. a classic miliary pattern can also be observed. (box 3-21) is a dimorphic fungus that grows in a mycelial form in the soil. infection can occur by inoculation of the skin or by inhalation of organisms into the lungs. the organism is endemic in north america, occurring mostly in the same areas where histoplasmosis occurs but also in the southeastern united states. blastomycosis is an infection associated with hunters because the organisms are prevalent in wooded areas. the organism is usually inhaled from the soil, and if the initial port of entry is the lung, a focal pneumonic process will occur. the disease can be self-limited, or a disseminated form can occur. the radiographic findings are nonspecific but consist of areas of inhomogeneous consolidation in a segmental or nonsegmental distribution in any area of the lung. the next most common manifestation is that of solitary and multiple pulmonary nodules. the solitary nodules may simulate lung carcinoma. these nodules are 3 to 6 mm in diameter. a third pattern results from disseminated disease and consists of a diffuse nodular or micronodular pattern. cryptococcus neoformans (box 3-22) is an encapsulated, yeastlike fungus that exists in the soil and in the yeast form in humans. the soil may be contaminated by pigeon or chicken excreta. seventy percent of individuals who have clinical disease are immunocompromised (see chapter 4). the central nervous system is the most frequently affected site. in the normal host, the most common finding is that of single or multiple pulmonary nodules that are approximately 1 to 5 cm in diameter and that usually occur in the lower lobes (fig. 3-38) . cavitation, lymph node enlargement, and pleural effusion are uncommon. adenopathy is rarely identified. characteristically, the single or multiple nodules tend to abut the pleura. characteristics candidiasis (box 3-23) may be caused by a group of various organisms in the candida genus, of which candida albicans is the most important species. c. albicans lives in human and animal sources and may be a normal inhabitant of the spores in soil contaminated with pigeon and chicken excreta of patients with clinical disease, 70% are immunocompromised central nervous system involvement single or multiple nodules larger than 1 cm affects lower lobes oral pharynx. as a result, short of an open lung biopsy, the true invasiveness or pathogenicity of this organism when recovered from the sputum is difficult to determine. it is an unusual infection found in immunocompromised individuals. the most common sites of infection are the mucous membranes and skin. pulmonary candidiasis is unusual but may occur as a primary infection of the lungs, presumably resulting from aspiration of the organisms from the oral cavity. in most immunocompromised patients, pulmonary infection accompanies a diffuse, widespread fungemia. the radiographic findings are usually nonspecific. although most fungal diseases, particularly in immunocompromised hosts, are characterized by multiple nodules with cavitation, candida pneumonia is more likely to produce areas of consolidation that are multiple and patchy and involve both lungs. cavitation and hilar adenopathy are rare, and pleural effusion occurs in approximately 25% of cases. characteristics actinomyces (box 3-24) is a rod-shaped bacterium rather than a fungus, but it is often considered a fungus because of its clinical presentation and radiographic findings. the organism is found in the mouth, and pulmonary infection usually occurs in people with extensive dental caries and poor oral hygiene. involvement results from aspiration of these organisms. there are three forms of actinomycosis: cervicofacial, gastrointestinal, and thoracic. the hallmark of the pulmonary disease is a focal abscess with extension to the chest wall, with secondary complications such as osteomyelitis, bronchopleural fistula, and pericarditis. the organism is an anaerobe, and anaerobic cultures must be obtained to confirm the diagnosis. typical sulfur granules may be identified on pathologic specimens. the radiographic features initially consist of an area of consolidation in the lung. this area may become rounded and suggest an abscess. classic signs include extension of the disease process into the chest wall with bone destruction and osteomyelitis (fig. 3-39) . chest wall invasion is best appreciated on ct. pleural effusions are moderately common. invasion of the ribs or vertebral bodies characteristically causes bone destruction and fairly extensive reactive periostitis. notice the erosion of the cortex of the overlying rib (arrows). characteristics nocardia (box 3-25) is a gram-positive organism, and although it is classified as a bacterium, it shares many features with fungal disease. it is weakly acid fast and can be confused with mycobacteria or legionella. it is similar to actinomyces, but the disease usually occurs in immunocompromised patients rather than in normal hosts (see chapter 4). focal consolidation is the most common finding, although the disease can appear as single or multiple nodules with cavitation. unlike aspergillosis, progression of disease usually is rather slow. chest wall involvement may occur but is rare. aspergillus (table 3 -3) is a dimorphic fungus. the most common of the many species is aspergillus fumigatus. aspergillus grows widely in soil and water, in decaying vegetation, and in animal material. aspergillosis occurs in several different forms in the lung, including noninvasive (mycetoma) and semi-invasive aspergillosis, invasive aspergillosis, and allergic bronchopul-monary aspergillosis. the type of involvement depends on the immune status of the host. infection is initiated by the inhalation route, and aspergillus spores may exist in the mouth and airways of normal hosts. immunocompetent or mildly immunosuppressed patients may acquire mycetomas or semi-invasive aspergillosis, whereas those who are severely immunosuppressed develop invasive aspergillosis. allergic bronchopulmonary aspergillosis usually occurs in asthmatic patients. the most common radiographic form of aspergillosis is the mycetoma or fungus ball. the fungus ball consists of aspergillus hyphae, mucus, and cellular debris developing within a preexisting cyst, cavity, bulla, or area of bronchiectasis. it grows as a saprophytic organism and usually is noninvasive. a high prevalence of mycetoma has been found among patients with sarcoidosis or cystic fibrosis. symptoms usually include hemoptysis, which may be life threatening. the radiographic appearance of a fungus ball or mycetoma can be quite characteristic (fig. 3-40) . typically, there is a solid, round opacity within a cavity or thin-walled cyst. air may dissect into the solid mass, creating the appearance of an air crescent. in most cases, the fungus ball is mobile, and changes in position occur with changes in body posture. extensive pleural thickening at the apex of the thorax frequently accompanies the development of a mycetoma. in making the differential diagnosis, necrotizing squamous cell carcinoma and an intrapulmonary abscess should be considered. no treatment is necessary for asymptomatic individuals, but for those who develop severe hemoptysis, there gram-positive, acid-fast bacilli immunocompromised hosts single or multiple nodules with or without cavitation slow progression focal consolidation are several therapeutic options. one is an interventional radiologic technique that consists of embolization of bronchial arteries that supply the cavity. direct installation of amphotericin b in the form of a paste inserted through a percutaneous catheter into the cavity has been successful in some cases. semi-invasive aspergillosis occurs in mildly immunosuppressed patients, such as those with alcoholism, chronic debilitating illness, or advanced malignancy. the lesion usually begins as a focal consolidation in the apex of one or both lungs that progresses over a period of months to become cavitary. it may form a crescent of air (i.e., air crescent sign) similar to that seen in a mycetoma. a thick-walled cavity, which later becomes thin walled and contains a fungus ball, is then formed. the appearance may be identical to that of a mycetoma. it consists of a cavity with or without a fungus ball and air crescent, or it may be a localized area of consolidation. extensive pleural thickening can be identified. the features of invasive aspergillosis are described in chapter 4, which discusses pulmonary infections in the immunocompromised patient. characteristics allergic bronchopulmonary aspergillosis (see chapter 13) occurs almost exclusively in asthmatic individuals. aspergillus spores contained within mucous plugs in the tracheobronchial tree incite an allergic reaction. the syndrome consists of blood eosinophilia with positive precipitins and marked elevation of ige antibodies. large masses of mucus and aspergillus hyphae can become trapped in the airways, producing mucoid impaction of the bronchi. the most characteristic pattern is that of mucoid impaction of the bronchus. central branching opacities, which sometimes are referred to as a finger-in-glove or v pattern, are identified. a more extensive description is provided in chapter 13. atelectasis distal to the areas of mucoid impaction usually does not occur because of collateral air drift. air trapping may be identified, and lobar consolidation may be present. as the mucous plugs are expectorated, areas of central bronchiectasis can be identified, particularly on ct scans. patients usually respond to steroids, but in the chronic form of the disease, scarring and upper lobe bronchiectasis are prominent features. mucormycosis, almost exclusively a disease in immunocompromised patients, is discussed in chapter 4. pneumocystis jiroveci (formerly called pneumocystis carinii) is discussed in chapter 4. in the united states, parasitic infection of the lung is rare. pneumonia is caused by a hypersensitivity reaction to the organisms, or it results from systemic invasion of the lungs and pleura. toxoplasma gondii pulmonary involvement usually develops as part of a more generalized disease. the congenital variety is the most common, and it results from transmission of the organism from mother to fetus. it is associated with a consolidative and hemorrhagic pneumonia in neonates. in adults, toxoplasmosis, like pneumocystosis, occurs in patients who are immunocompromised. the radiographic appearance is that of fairly diffuse reticulonodular opacities. echinococcus granulosus (box 3-26), the cause of most cases of human hydatid disease, occurs in two forms: pastoral and sylvatic, which differ in definitive and intermediate hosts and in geographic distribution. the pastoral variety is more common and occurs in sheep, cows, or pigs as the intermediate hosts, and in dogs as the definitive host. it is particularly common in sheep-raising areas. the sylvatic variety has as the definitive host the dog, wolf, or arctic fox. approximately 65% to 70% of echinococcus cysts occur in the liver, and 15% to 30% occur in the lungs. the hydatid cyst is composed of two layers, an exocyst and an endocyst. daughter cysts may be formed within the endocyst. cysts may rupture in the lung parenchyma, with resulting intense inflammation. rupture into the bronchus may result in severe hypotensive shock. echinococcal cysts are usually well-circumscribed, spherical or oval masses that may be single or multiple (fig. 3-41) . they are usually located in the lower lobes. if communication develops between the cysts and the bronchial tree, air may enter between the pericyst and exocyst, producing the appearance of a thin crescent of air around the periphery of the cyst, sometimes called the meniscus or crescent sign. bronchial communication occurs directly into the endocyst. occasionally, an air crescent sign and air-fluid level can be identified. the membrane of the cyst, which has ruptured into the bronchial tree, may float on the fluid within the cyst, giving rise to the classic water lily sign. ct can differentiate cystic from solid lesions and may identify the pathognomonic features in ruptured or complicated hydatid cysts, such as the presence of daughter cysts and endocyst membranes. calcification of a pulmonary hydatid cyst is rare. pulmonary amebiasis is rare and is usually a sequela of hepatic or gastrointestinal involvement. amebiasis is caused by the protozoan entamoeba histolytica. this organism causes dysentery and has a worldwide distribution. pleuropulmonary complications usually occur when the liver is involved. patients present with right upper quadrant and right-sided pleuritic chest pain. the common radiographic features are right-sided pleural effusion with basal consolidation. involvement of the lung may result from rupture of an amebic abscess in the liver. occasionally, areas of consolidation in the right lower lobe may progress to abscess formation with cavitation. schistosomiasis is a common disease in many areas of the world, including central and south america, the middle east, and the far east. the intermediate host of this parasite is the snail. humans contact the parasites in water. the parasites penetrate the skin, reach the circulation, and eventually grow in the mesenteric or pelvic venous plexus, where they mature into adult worms and lay eggs. pulmonary symptoms may occur during the larval migration phase in the lungs due to a hypersensitivity reaction. a progressive diffuse endarteritis and thrombosis may result from impaction of ova in the pulmonary circulation, with the eventual development of pulmonary arterial hypertension. pathologic changes in the lungs result from deposition of eggs or ova, which are released directly into the systemic venous blood or occasionally into the portal system, where eggs can reach the lungs through anastomotic channels as the liver becomes cirrhotic. the embolized ova become impacted in pulmonary arterioles and then extruded into the surrounding tissue. this causes an obliterative arteriolitis, which can result in increased pulmonary artery pressure. the ova may mature into adult worms in the lungs and can cause lung damage. pulmonary arterial hypertension is the most common finding in patients with pulmonary schistosomiasis (fig. 3-42) . the appearance consists of dilation of the central pulmonary arteries with rapid tapering. the passage of larva through the pulmonary capillaries can cause a transitory eosinophilic pneumonia, simulating loeffler's syndrome. this is characterized by the presence of peripheral areas of consolidation. the lungs may be infected by a number of worms, causing ascariasis, strongyloidiasis, trichinosis, ancylostomiasis (i.e., hookworm disease), and filariasis (i.e., tropical eosinophilia). most of these organisms produce hypersensitivity reactions in the lungs, similar to loeffler's syndrome (see chapter 9). outbreaks of several newly recognized viral infections, including avian influenza, severe acute respiratory syndrome-associated coronavirus, and hantavirus, have been associated with high mortality rates. these infections have presented challenges to clinicians, radiologists, scientists, and public health officials. avian influenza is caused by the h5n1 subtype of the influenza a virus. human transmission occurs through close contact with infected birds, usually from ingestion of infected poultry. the first documented case occurred in 1997 in hong kong. in 2003, the virus resurfaced in vietnam. approximately 180 people throughout southeast asia have been infected, with a nearly 50% mortality rate. affected patients present with a rapidly progressive pneumonia that may lead to respiratory failure and ards. chest radiographs usually show abnormalities at the time of presentation. the most common finding is multifocal consolidation (fig 3-43) , which is bilateral in 80% of cases. consolidation may infrequently be complicated by areas of cavitation. bilateral pleural effusions occur in about one third of cases. severe acute respiratory syndrome (sars) is caused by the sars-associated coronavirus. it results in a systemic infection that is manifested clinically as a progressive pneumonia. the first reported case in humans occurred in china in 2002. in 2003, sars spread to hong kong and subsequently to canada, singapore, and vietnam. before the infection could be contained by vigorous public health measures, more than 8000 persons were infected, with a nearly 10% fatality rate. no additional human infections have been reported since 2003. after an initial incubation period of 2 to 10 days (mean, 6 days), affected patients typically present with headache, malaise, fever, and nonproductive cough. chest radiographs show abnormalities at the time of clinical presentation in about 80% of cases. the most common radiographic finding is poorly defined airspace consolidation. although about one half of cases appear to have a focal distribution at the time of presentation, progression to multifocal involvement is common. areas of consolidation have a predilection for the lower lobes and lung periphery. ct shows abnormalities at the time of clinical presentation, even when chest radiographs do not. the most common ct finding is ground-glass opacification (fig 3-44) , which is often accompanied by small foci of consolidation and interlobular and intralobular thickening. severe sars may progress to diffuse alveolar damage. overall, 20% of patients with sars require mechanical ventilation, and 10% of patients do not survive the infection. survivors often have residual abnormalities seen on ct, reflecting interstitial fibrosis and small airways disease. hantaviruses are carried by rodent vectors. human infection occurs after inhalation of aerosolized rodent feces or urine. the sin nombre hantavirus (translated as "the nameless virus") was initially discovered in the southwestern united states in 1993 as a cause of pulmonary edema and respiratory failure accompanied by hematologic abnormalities. this clinical entity is referred to as the hantavirus pulmonary syndrome (hps). hps is caused by endothelial damage to the lung. the initial interstitial edema manifests radiographically as kerley lines, bronchial wall thickening, and subpleural edema. although some patients recover fully from the initial stage of infection, many progress to diffuse alveolar edema, which is manifested by symmetric perihilar and basilar airspace consolidation (fig 3-45 ). this phase of illness requires mechanical ventilation and is associated with a high mortality rate. as the disease progresses, it may be accompanied by myocardial depression, which worsens tissue hypoxia and contributes to the high mortality rate associated with this syndrome. the centers for disease control and prevention (cdc) lists several infectious agents as a category a threats, denoting the highest potential for public health impact. these agents include inhalational anthrax (bacillus anthracis), plague (y. pestis), smallpox (variola major), botulism (clostridium, botulinum), tularemia (francisella tularensis), and hemorrhagic fever (ebola and marburg filoviruses). among these infections, anthrax has the unique distinction that imaging studies may allow prompt diagnosis and institution of life-saving therapy before organ damage is irreversible. for this reason, the discussion focuses on anthrax. anthrax has been used as a biologic weapon since world war ii. the most recent episode occurred in 2001, when highly refined anthrax spores were placed in envelopes and mailed through the united states postal system. this act of bioterrorism resulted in 22 diagnosed cases of anthrax, which were evenly split between inhalational and cutaneous forms. almost one half of those with the inhalational form died. b. anthracis is a sporulating, gram-positive bacterium that may result in cutaneous, gastrointestinal, or pulmonary infection. the latter, which is also referred to as inhalational anthrax, is the deadliest form. the spores are 2 to 6 μm, an ideal size for deposition in the distal respiratory tract after inhalation. once inhaled, the spores are ingested by macrophages. surviving spores are transported to mediastinal lymph nodes, where they germinate for 2 to 30 days (mean, 1 week). radiologic findings have not been identified before germination. after germination, the organisms synthesize a toxin, resulting in the prodromal phase of the disease. this is manifested by flulike symptoms of fever, chills, fatigue, and cough. the prodromal phase lasts about 4 days and is rapidly followed by the second phase of the illness, which is characterized by stridor, respiratory failure, and shock. in many cases, death occurs despite antibiotic therapy. imaging findings for anthrax reflect hemorrhagic lymphadenitis and mediastinitis caused by the release of anthrax toxin within the mediastinum. in the prodromal phase of the illness, the chest radiograph typically demonstrates mediastinal widening and unilateral or bilateral hilar enlargement. these findings are frequently accompanied by pleural effusions. although limited peribronchovascular airspace opacities may be present, extensive consolidation is uncommon. imaging findings of mediastinal widening and pleural effusions are helpful for differentiating inhalational anthrax from a community-acquired respiratory infection. ct may provide convincing evidence of inhalational anthrax before confirmatory laboratory tests have returned (fig 3-46 ). unenhanced ct may show high-attenuation (46 to 62 hounsfield units) mediastinal and hilar lymph nodes, which may rapidly enlarge over a period of days. these findings reflect the presence of hemorrhage and edema within lymph nodes. because of this characteristic appearance, unenhanced ct is considered the imaging modality of choice for the diagnosis of inhalational anthrax. after contrast administration, rim enhancement and central low attenuation of lymph nodes may be seen. rapidly enlarging pleural effusions are commonly identified by ct, and they may contain dependently layering, highattenuation fluid, reflecting serosanguineous exudates. peribronchovascular thickening correlates with the presence of edema, hemorrhage, and necrosis of the airways and adjacent lymphatics. the constellation of these ct findings is almost pathognomonic for inhalational anthrax, but a variety of other causes of mediastinitis may produce similar findings in the appropriate clinical setting. inhalational anthrax is treated with an antibiotic regimen that includes ciprofloxacin or doxycycline combined with two other agents, usually rifampin and clindamycin. early recognition of anthrax and prompt administration of antibiotics before the onset of fulminant illness can dramatically improve patient survival. tree-in-bud pattern: frequency and significance on thin section ct aspiration and inhalation pneumonias pulmonary coccidioidomycosis reported tuberculosis in the united states. department of health and human services, cdc pulmonary manifestations of mycobacterium intracellulare epidemiology of tuberculosis chlamydia pneumoniae pneumonia in hospitalized patients an overview of pulmonary fungal infections mycobacterium tuberculosis coccidioidal pneumonia legionella pneumonias gram-negative bacillary pneumonias tuberculous pleural effusions the chest radiograph in legionnaires' disease: further observations aspiration pneumonia, lung abscess, and empyema the roentgen manifestations of thoracic actinomycosis fraser and pare's diagnosis of diseases of the chest inhalational anthrax semi-invasive pulmonary aspergillosis: a new look at the spectrum of aspergillus infections of the lung the spectrum of pulmonary aspergillosis the acute bacterial pneumonias histoplasma capsulatum the varied roentgen manifestations of primary coccidioidomycosis an official ats/ idsa statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases pulmonary blastomycosis: radiologic manifestations mycobacterium aviumintracellulare complex: evaluation with ct mycoplasmal, viral, and rickettsial pneumonias viral pneumonia radiology of severe acute respiratory syndrome (sars): the emerging pathologic-radiologic correlates of an emerging disease radiology of bacterial weapons-old and the new? hantavirus pulmonary syndrome: radiologic findings in 16 patients thoracic cryptococcosis: immunologic competence and radiologic appearance viral pneumonia in adults: radiologic and pathologic findings clinicoradiographic correlation with the extent of legionnaire disease ct features of thoracic mycobacterial disease pulmonary septic emboli: changes with ct anaerobic pleural and pulmonary infections pneumonia caused by mycoplasma pneumoniae infection ct features of pulmonary mycobacterium avium complex infection studies in chronic allergic bronchopulmonary aspergillosis. i. clinical and physiological findings thoracic manifestations of tropical parasitic infections: a pictorial review pneumatocele formation in adult pneumonia tuberculosis: frequency of unusual radiographic findings pulmonary infections granulomatous infections of the lung tuberculosis in the normal host: radiological findings spectrum of pulmonary non-tuberculous mycobacterial infection atypical mycobacterial infection in the lung: ct appearance pulmonary aspiration complexes in adults guidelines for the management of adults with community-acquired pneumonia: diagnosis, assessment of severity, antimicrobial therapy, and prevention pneumococcal pneumonia in hospitalized patients' clinical and radiological presentations pulmonary cryptococcosis adult respiratory distress syndrome infectious pneumonias-including aspiration states the radiologic manifestations of h5n1 avian influenza hilar and mediastinal adenopathy caused by bacterial abscess of the lung viral pneumonitis thoracic histoplasmosis other mycobacterium species tuberculosis among elderly persons: an outbreak in a nursing home imaging of bacterial pulmonary infection in the immunocompetent patient severe acute respiratory syndrome: radiographic appearance and pattern of progression in 138 patients pulmonary aspiration of gastric contents update: the radiographic features of pulmonary tuberculosis pulmonary bacterial and viral infections key: cord-019089-oots4fe4 authors: laya, bernard f. title: infections date: 2013-08-31 journal: radiology illustrated: pediatric radiology doi: 10.1007/978-3-642-35573-8_13 sha: doc_id: 19089 cord_uid: oots4fe4 lower respiratory tract infection is a very common illness in children and is a significant cause of morbidity and mortality. clinical signs and symptoms are nonspecific especially in infants and younger children and some even present with nonrespiratory complaints. infectious agents causing pneumonia is not limited to viruses and bacteria, but it could also be due to mycoplasma, mycobacteria, fungi, protozoa, and parasites. coinfection with two or more microbial agents can also occur. the etiologic agent of lower respiratory infection in a child is often difficult to obtain, but the patient’s age can help narrow the possible cause. microbiological tests are important but could be difficult to obtain especially in younger children. various medical imaging modalities not only play an important role as an aid in diagnosis but can also help during and after therapy. imaging can also help evaluate complications to pneumonia and exclude other causes of respiratory distress including underlying developmental anomalies, foreign body, gastroesophageal reflux disease, and aspiration. in this chapter, the imaging modalities utilized in the detection of pulmonary infections will be discussed. the spectrum of typical imaging findings for various etiologic agents in both immunocompetent and immunocompromised children will be presented. lower respiratory tract infection is the most common cause of illness in children and is a signifi cant cause of morbidity and mortality. there are also associated complications, which should be recognized in order to make correct decisions regarding interventions or management. clinical signs and symptoms are nonspecifi c, especially in infants and younger children. some children with pneumonia even present with nonrespiratory symptoms including fever, malaise, decreased appetite, irritability, weakness, chest pain, and abdominal symptoms. physical examination is also less reliable in children than adults (donnelly 2001 ) . microbiological tests are important but could be diffi cult to obtain especially in younger children. various medical imaging modalities not only play an important role as an aid in diagnosis but can also help during and after therapy. diagnosis of pneumonia calls for a combination of clinical awareness, appropriate microbiological tests, and radiological studies. the cause of pneumonia in a child is often diffi cult to identify, but the patient's age can help narrow the possible etiologies. viral pneumonia is rare in the neonatal period because of conferred maternal antibody protection. group b streptococcus and gram-negative enteric bacteria are the most common pathogen in neonates (birth to 20 days), obtained through vertical transmission from the mother during birth. from age 3 weeks to 3 months, streptococcus pneumoniae is the most common pathogen. viruses are the most frequent cause of community-acquired pneumonia in infants older than 4 months and in preschool-aged children, with respiratory syncytial virus (rsv) being the most common. for school-aged children (6-16 years old), the incidence of bacterial infections from streptococcus increases, although viral disease remains the most common cause (condon 1991 ; ostapchuk et al. 2004 ) . bacterial pneumonia can occur at any time in preschool and school-aged children and adolescents. mycoplasma pneumoniae causes 30 % of lower respiratory tract infections in school-aged children (condon 1991 ; donnelly 2001 ) . infectious agents causing pneumonia are not limited to viruses and bacteria, but it could also be due to mycobacteria , fungi, protozoa, and parasites. co-infection with two or more microbial agents can also occur. evaluation of suspected pulmonary infection is a very common indication for an imaging study in children. the role of imaging including chest radiographs, ultrasound, computed tomography (ct), and even magnetic resonance imaging (mri) is to detect the presence or exclusion of pneumonia, determine its location, characterize and describe the extent of pneumonia, exclude other causes of respiratory symptoms, and show complications. it is also an important tool for image-guided interventions. the cornerstone of imaging in children suspected of having pulmonary infection is the chest radiograph. the radiographic appearance refl ects the pathologic process occurring in the respiratory system (bramson et al. 2005 ) . frontal and lateral views are obtained when possible because hyperinfl ation and lymphadenopathy are more accurately evaluated on a lateral radiograph especially in younger children. lateral decubitus views may be useful in distinguishing free fl owing pleural fl uid versus loculated fl uid collections. chest radiographs have inherent limitations, but despite of this, there is moderate evidence to suggest that chest radiographs are suffi ciently sensitive and highly specifi c for the diagnosis of community-acquired pneumonia (westra and choy 2009 ). 13.5.20 tuberculosis: progression of lymph node disease .................................................................. 460 13.5 the use of ultrasound as an imaging tool for pulmonary infections has been increasing especially for assessment of complications. its utility is even more important because there is no associated radiation, no sedation, no specifi c preparation, and the ultrasound machine can be transported to the patient's bedside. it can be used for planning thoracentesis, thoracotomy, and image-guided drainage procedures. lower-frequency (3.5-7 mhz) sector transducers are initially used for overview through inter-and subcostal scanning, but higher-frequency (10-12.5 mhz) linear transducers are helpful for more detail in the near fi eld. the development of helical and multidetector ct has revolutionized imaging evaluation of pulmonary infections. the use of intravenous contrast medium also helps optimize the assessment of pleura, mediastinum, and pulmonary parenchyma in cases of complicated pneumonia. high-resolution ct (hrct) shows greater accuracy in characterizing diseases into interstitial, airway, and airspace processes and gives a more accurate depiction of the extent of the disease. ct has an important role when a complication is suspected, to exclude an underlying abnormality in recurrent and persistent infections, for image-guided interventions, and for the evaluation of immunocompromised children (westra and choy 2009 ) . radiation-associated risks are important to consider, and thus, a clear indication for the procedure has to be present. low radiation dose technique with 80-120 kvp, age and thoracic thickness-adjusted low milliampere-seconds, along with radiation dose modulation should be utilized. evaluation of lung parenchyma with conventional mr imaging has limitations because of the inherent low proton density and weak mr signal as related to the low physical density of the lung. however, lung parenchymal, pleural, and lymph node infl ammatory abnormalities can be visualized and characterized by mri in children with pulmonary infections. peripheral airways disease or bronchiolitis are common terms ascribed to lower respiratory infection secondary to viruses. it commonly occurs in children less than 2 years of age, typically presenting with cough, coryza, and wheezing. rsv is the most common cause, but other viral causes include rhinovirus, parainfl uenza virus, human metapneumovirus, adenovirus, infl uenza virus, coronavirus, and human bocavirus (eslamy and newman 2011 ) . following inhalation of infected aerosols, the virus migrates to small airways and alveoli resulting to bronchoconstriction and increased mucous secretion (aherne et al. 1970 ; swischuk and hayden 1986 ) . typical chest radiographic appearances are peribronchial thickening/opacities, hyperaeration, and subsegmental atelectasis ( fig. 13.1 ). the peribronchial infl ammation and edema manifests as increased peribronchial cuffi ng or thickening of the bronchial walls, which is usually asymmetric and radiates from the hila into the lung. narrowed distal airway lumen due to bronchiolar wall edema and mucus results in hyperinfl ation with areas of segmental and subsegmental atelectasis (condon 1991 ; donnelly 2001 ) . patchy areas of airspace consolidation have also been described in viral pneumonia. ct is rarely required in the investigation of viral lower respiratory infection, but the most common ct feature is peribronchial thickening and ground-glass attenuation without consolidation (tanaka et al. 1996 ) . varicella zoster virus infection is a highly contagious but a relatively benign, self-limited disease in childhood. varicella pneumonia is regarded as a serious manifestation in adults, but immunocompromised children are also at risk. clinically, cough, fever, dyspnea, chest pain, and vesicular rash are generally accompanied by mild constitutional symptoms (kim et al. 1999 ) . radiographs of the chest initially reveal nodular infi ltrates that may progress to large segmental areas of patchy consolidation, predominantly in the bases and perihilar regions. total clearing is virtually guaranteed, although punctate calcifi cations maybe evident within 2 years after acute illness ( fig. 13 .2 ). airspace disease associated with chicken pox in children occurs most often in the immunocompromised host (blickman 1998 ) . certain groups of viruses have been recently reported to cause severe respiratory infection leading to respiratory failure and even death. the severe acute respiratory syndrome (sars) caused by coronavirus a (sars-cov) created a scare in 2003 with over 8,000 cases reported from 29 different countries. sars presents with a prodrome of fl u-like illness, followed by cough, dyspnea, and possibly acute respiratory distress. the initial radiographic manifestation is the presence of focal or diffuse interstitial opacities but rapidly progresses to bilateral areas of consolidation (thibodeau and viera 2004 ) (fig. 13 .3 ). another pandemic virus is the infl uenza virus a h5n1 (avian infl uenza virus), originating from asia and spreading over many parts of the world from 2003 to 2007. more recently, infl uenza virus of swine origin, designated as infl uenza a h1n1, was fi rst reported in mexico in 2009 and has rapidly spread globally. symptoms range from asymptomatic infection to mild upper respiratory illness, viral syndrome, diarrhea, severe pneumonia, acute respiratory distress syndrome (ards), and progression to multiorgan failure. initial chest radiographs in children with a mild and self-limited clinical course are often normal, but they may demonstrate prominent peribronchial markings with hyperinfl ation and multifocal areas of consolidation (lee et al. 2010 ) (fig. 13.4 ) . the most common complication of viral pneumonia is a secondary bacterial infection. viral infection can compromise the respiratory mucosa and render the host pulmonary respiratory system susceptible to develop superimposed bacterial pneumonia (donnelly 1999 ) . postinfectious bronchiolitis obliterans (constrictive bronchiolitis or obliterative bronchiolitis) is a clinical syndrome of chronic airfl ow obstruction associated with infl ammatory changes in the small airways as response to epithelial injury associated with infections. it is particularly associated with adenovirus , rsv, varicella , and severe mycoplasma infection. the chest x-ray fi ndings are often nonspecifi c and can appear normal, but the most common abnormality is hyperaeration (yalcin et al. 2003 ). on hrct, there is a mosaic perfusion pattern ( fig. 13 .5 ). perfusion is diminished in areas of parenchymal attenuation due to vasoconstriction secondary to hypoxia. inspiratory and expiratory phases of ventilation are important in hrct to better assess air trapping in this condition (hansell et al. 1997 ) . peribronchial thickening, atelectasis, bronchiectasis, and sometimes lung volume reduction can also be seen. swyer-james is a subtype of postinfectious bronchiolitis obliterans, which is typically unilateral. it can affect one lung segment, a lobe, or the entire lung. the characteristic chest radiographic and ct fi ndings are hyperlucency due to the pulmonary hypoperfusion, reduction of vascular and hilar markings, and volume reduction of the affected lung or lobe (daltro et al. 2011 ) (fig. 13 .6 ). bacterial pneumonia occurs with the inhalation of the infectious agent into the airspaces. it is most commonly caused by s. pneumoniae , haemophilus infl uenza type b, and staphylococcus aureus . staphylococcus commonly occurs in early infancy, haemophilus most often between 6 and 12 months, and s. pneumoniae more commonly between 1 and 3 years of age. gram-negative aerobic bacteria such as pseudomonas aeruginosa and s. aureus are a major problem in patients with cystic fi brosis. patients present with cough, chest pain, and high fever. following inhalation of the infectious agent into the airspaces, acinar exudate and edema ensues, manifesting as localized airspace consolidation with air bronchogram on chest radiographs. the typical distribution is lobar or segmental, depending on the stage of progression at the time the x-ray was obtained ( fig. 13 .7a ) (condon 1991 ; donnelly 1999 ) . round pneumonia is a spherical pneumonia, usually caused by s. pneumoniae (rose and ward 1973 ) (fig. 13.7b ). it is common in children less than 8 years old, maybe due to poor development of collateral pathways of ventilation (pores of kohn and channels of lambert). when round pneumonia is seen in children over 8 years old, other etiologies should be considered. the ct manifestations of bacterial infection are areas of consolidation with or without air bronchogram, typically with a segmental or lobar distribution and involving the lung periphery (tanaka et al. 1996 ) . parapneumonic effusions occur most commonly in bacterial pneumonia. it represents a spectrum of infl ammatory fl uid collections that ranges from transudative effusion to empyema. parapneumonic effusions complicate pneumonia in 36-56 % of cases in pediatric patients (kurt et al. 2006 ) , and empyema complicates an estimated 0.6 % of all childhood pneumonias (jaffe and balfour-lynn 2005 ) . pleural fl uid can usually be detected on a frontal chest radiograph, but layering of fl uid on the lateral decubitus view distinguishes a free fl owing fl uid from a loculated fl uid. ct scan gives a better characterization of parapneumonic effusions compared to radiographs. ct fi ndings include enhancement and thickening of the parietal and visceral pleura, thickening of the extrapleural subcostal tissues, and increased attenuation of the extrapleural subcostal fat (muller 1993 ) (fig. 13.8 ). these ct characteristics do not accurately predict empyema and should not be used to distinguish between empyema and transudative effusions. in ultrasound, pleural fl uid can be characterized as simple effusion, complicated effusion, or fi brothorax (pleural thickening or fi brosis) ( fig. 13.9 ) . a simple effusion appears as a clear anechoic or cloudy hypoechoic fl uid with or without swirling particles. a complicated effusion appears as a septated or multiloculated, hypoechoic fl uid with fi brinous septations, with no clear demarcation between the lung and pleural components, while a fi brothorax appears as a thickened, echogenic rind of pleural plaque (kim et al. 2000 ) . suppurative lung parenchyma complications represent a spectrum of abnormalities including cavitary necrosis, lung abscess, pneumatocele, bronchopleural fi stula, and pulmonary gangrene. necrotizing pneumonia or cavitary necrosis is a complication of severe lobar pneumonia, characterized by massive necrosis and liquefaction of lung tissues resulting to multiple cavities rather than a solitary one. it is most commonly caused by s. pneumoniae although aspergillus and legionella have also been implicated in the pediatric population (hodina et al. 2002 ) . evidence of cavitary necrosis complicating pneumonia is often seen on ct before or in the absence of fi ndings in chest radiography. ct fi ndings include lung consolidation with decreased parenchymal enhancement, loss of lung-pleura margin, and multiple thin-walled cavities lacking an enhancing border. the adjacent visceral pleura is particularly fragile and tends to rupture, causing bronchopleural fi stula (hoffer et al. 1999 ) (fig. 13 .10 ). cavitary necrosis indicates an intense and prolonged illness, but it usually resolves without surgical intervention (donnelly and klosterman 1997 ) . lung abscesses are thick-walled cavities containing purulent material resulting from pulmonary infection. an airfl uid level with reactive rim is a typical imaging appearance, as compared to necrotizing pneumonia where cavities occasionally have air-fl uid level but without rim of enhancement (donnelly and klosterman 1997 ) (fig. 13.11 ). differentiating the two is important because abscess not responding to therapy may require drainage, whereas necrotizing pneumonia does not require invasive treatment, and intervention may even be harmful resulting in complications such as bronchopleural fi stula (hoffer et al. 1999 ) . pneumatoceles are thinwalled cysts without septations that develop within the lung parenchyma after an acute pneumonia (fig. 13.12 ) . it may represent a later or less severe stage of resolving or healing necrosis and is most often associated with s. aureus (daltro et al. 2011 ) . bronchiectasis is the most common long-term sequelae of lung parenchymal damage from pneumonia. it is best demonstrated on high-resolution chest ct scan, and the main diagnostic features are as follows: internal diameter of the bronchus is wider than its adjacent pulmonary artery, failure of the bronchus to taper peripherally, and visualization of bronchi in the outer 1-2 cm of the lung zones (eslamy and newman 2011 ) (fig. 13.13 ). pertussis is a highly contagious respiratory bacterial infection caused by bordetella pertussis . it infects mainly infants and young children causing symptoms that include mild fever, runny nose, and cough, which develops into a paroxysmal cough followed by whooping (whooping cough). pneumonia is a common complication, and untreated patients may be contagious for 3 weeks or more following onset of the cough. the spread of pertussis can be prevented by immunization. histopathologic examination reveals an infection dominated by necrotizing bronchiolitis, intra-alveolar hemorrhage, fi brinous edema, and angiolymphatic leukocytosis (paddock et al. 2008 ) . conventional radiographs reveal streaky perihilar infi ltrates with most often unilateral hilar adenopathy, a pattern sometimes called the shaggy heart appearance (blickman 1998 ) (fig. 13.14 ) . m. pneumoniae is a common ubiquitous organism and treatable cause of community-acquired pneumonia, occurring primarily in children and young adults. it accounts for up to 30 % of all pneumonia in the general population, but the highest incidence is seen in children between 3 and 14 years of age. of those infected, 50 % get tracheobronchitis, 30 % pneumonia, 10 % pharyngitis, and 10 % otitis media. clinically, symptoms are less severe but more common than in true bacterial pneumonia (blickman 1998 ) . the radiographic fi ndings are nonspecifi c, have a broad spectrum of appearances, and may present with a pattern intermediate between the classic viral and bacterial pneumonia patterns (hsieh et al. 2007 ). some authors reported that a reticulonodular pattern or nodular opacities are typical radiographic pattern (john et al. 2001 ) , while others stress the occurrence of confl uent and patchy consolidation (reittner et al. 2000 ) (fig. 13.15 ). hrct fi ndings are thickened bronchovascular bundles, ground-glass attenuation and consolidation, centrilobular nodules, and lobular distribution (tanaka et al. 1996 ; reittner et al. 2000 ) . chlamydia trachomatis is an obligate intracellular parasite. genital chlamydial infection is recognized as the world's most common sexually transmitted disease, and the high prevalence in women of childbearing age results in exposure of neonates during childbirth. chlamydia pneumonia is a neonatal infection acquired after passage of the fetus through the cervix and vagina. the infant typically presents at 3-6 weeks of age with respiratory symptoms and occasional pulmonary hemorrhage. c. trachomatis should be suspected in infants who are afebrile or nontoxic and have a dry cough. these patients often have a peripheral eosinophilic pleocytosis, sometimes with concomitant conjunctivitis (ostapchuk et al. 2004 ). most chest radiographs show bilateral hyperaeration and diffuse infi ltrates with a variety of radiographic patterns including interstitial, reticular nodular, atelectasis, coalescence, and bronchopneumonia (radkowski et al. 1981 ) (fig. 13.16 ). tuberculosis (tb) is caused by infection with the mycobacterium tuberculosis complex. once inhaled, the infected aerosolized droplet in the alveoli cascades a series of infl ammatory reaction, and the bacilli also spread to nearby mediastinal lymph nodes. the alveolar site of infection (ghon focus), the infected lymph nodes, and the associated lymphangitis form the "primary (ranke's) complex." in most immunocompetent children, the infection goes into latency and the bacilli become dormant. these children usually have a reactive tuberculin skin test (tst) and/or a positive interforon-gamma release assay (igra) test, but without clinical evidence of tb and generally no abnormalities on chest radiograph apart from the primary complex residual. primary tuberculosis disease occurs if the host is unable to contain the infection, and disease progression occurs in the lungs, the lymph nodes, and adjacent structures in the thorax or could disseminate in any part of the body. lymphadenopathy (present in 92 %) with or without a visible ghon focus is the radiographic hallmark of tb infection and usually involves the hilar and paratracheal regions. the ghon focus may be too small to be radiographically visible but can also undergo caseation and calcify ( fig. 13.17 ). disease progression may occur at the site of ghon focus, within the regional lymph nodes, or following disease spread (fig. 13.18 ). parenchymal involvement in primary pulmonary tb most commonly appears as homogeneous consolidation, although it can appear patchy, linear, nodular, and mass-like. caseation necrosis, liquefaction, or calcifi cations can be seen within the consolidation and can progress into extensive lung damage (marais et al. 2004 ) (fig. 13.19 ). enlarged and edematous hilar, paratracheal, and subcarinal lymph nodes may cause compression of the adjacent bronchus and can lead to hyperinfl ation or atelectasis of the affected lung segment. contrast-enhanced ct shows a characteristic appearance consisting of central areas of low attenuation with peripheral rim enhancement and obliteration of perinodal fat (kim et al. 1997 ) (fig. 13.20 ) . pulmonary dissemination, usually seen in very young and immunocompromised patients, leads to the formation of pulmonary nodular interstitial granulomas, usually 1-2 mm in size, throughout the lungs. chest radiographs demonstrate the usual miliary nodular pattern but ct is more sensitive for the detection of miliary tb (kim et al. 1997 ) (fig. 13.21 ) . adult-type disease presentation is common after primary infection in children over 10 years of age or via endogenous reactivation (postprimary tb) or reinfection. chest radiograph shows ill-defi ned, fi bronodular parenchymal disease and cavitation mainly involving the apical segments of the upper lobes (perez-velez and marais 2012 ) (fig. 13.22 ). aspergillus fumigatus is a ubiquitous saprophytic mold found in many environmental sites, and infection is usually via inhalation of spores, although other routes of entry also occur. infection can manifest as colonization of airway cavities and necrotic tissue, allergic disease, and invasive disease, which is usually acute and rapidly progressive severe disease (foster and alton 2003 ) . airway colonization occurs in patients with underlying airway disease such as asthma and bronchiectasis. intertwined fungal hyphae, called as mycetoma or aspergilloma, form in the pulmonary cavity or ectatic bronchi. important underlying causes are pulmonary tb with cavitation and cystic fi brosis with bronchiectasis. rounded soft tissue mass within a cavity forming an "air-crescent" sign is a typical appearance ( fig. 13.23 ). allergic bronchopulmonary aspergillosis is characterized by mucoid impaction of the proximal bronchi presenting as fi ngerlike shadows involving the upper lobes on the chest radiograph. ct demonstrates the mucoid impaction of the central airways and the bronchiectasis of the segmental or subsegmental airways (foster and alton 2003 ) . invasive disease is an aggressive, rapidly disseminating and destructive disease and occurs when host defenses are impaired. it is characterized by the occlusion of large-or medium-sized arteries by plugs of hyphae causing pulmonary hemorrhage, arterial thrombosis, and infarction. radiographic fi ndings are nonspecifi c, with multiple nodules or areas of consolidation (fig. 13.24 ) . the typical ct fi nding is the halo sign due to ground-glass attenuation representing hemorrhage surrounding the pulmonary nodule or mass (foster and alton 2003 ; eslamy and newman 2011 ) . histoplasmosis, caused by the fungus histoplasma capsulatum , is usually an asymptomatic and self-limited disease that rarely requires therapy in children other than the very young or immunocompromised. it is found in the soil of endemic areas including central united states, central america, and northern south america but has also been reported in some parts of asia (houston 1994 ) . after inhalation, the spores germinate within the alveoli inciting an intense tissue reaction characterized by granulomas, which may calcify. it spreads to the lymphatics and into the hilar or mediastinal lymph nodes, and systemic dissemination may occur in patients with impaired t-cell immunity (mcadams et al. 1995 ) . histoplasmosis falls in one of three categories: acute, chronic pulmonary, and disseminated disease. acute pulmonary histoplasmosis is a self-limited illness. chronic pulmonary histoplasmosis occurs in patients with chronic lung disease and presents similar to tuberculosis with predilection for apical and posterior segments of the lung. disseminated histoplasmosis in children is characteristically a fulminant illness, which may or may not have pulmonary involvement. radiologic manifestations parallel the clinical syndromes. acute disease usually manifests as focal parenchymal consolidation with or without ipsilateral hilar adenopathy (fig. 13.25 ) . with healing, a nodule representing a histoplasmoma may result. chronic histoplasmosis radiographically manifests as an upper lobe fi brocavitary disease indistinguishable from postprimary tuberculosis. chest radiographs of patients with disseminated disease may show miliary or diffuse reticulonodular pattern that could progress to diffuse airspace opacifi cation (mcadams et al. 1995 ) . echinococcosis, also known as hydatid disease or hydatidosis, is a parasitic infection in humans caused by dog tapeworm, echinococcus granulosus , in its larval stage. it is endemic in many sheep and cattle-raising countries throughout the world. humans are intermediate hosts and become infected through ingestion of contaminated water or vegetables. when eggs of adult tapeworm are ingested, embryos are freed and migrate through the host's gastrointestinal mucosa and enter the portal vein and lymphatic system to various parts of the body where the embryo develops into a cyst. the wall of the cysts contains three layers: the outermost, pericyst; the middle laminated membrane layer, ectocyst; and innermost germinal layer, endocyst (czermak et al. 2001 ) . the lungs are the most common sites of infection in children but majority remain asymptomatic until the cyst enlarges to cause symptoms due to mass effect or due to cyst rupture (santivanez and garcia 2010 ) . diagnosis is obtained by imaging evaluation, supported by serology. a high proportion of lung lesions are discovered incidentally on a routine x-ray, and the most prominent radiological fi nding is a dense, round, well-demarcated opacity that can resemble a neoplasm (fig. 13.26a ) . when the growth of the cyst produces erosion in the bronchioles, air between the endocyst and pericyst can produce a "crescent or inverse crescent sign." if air continues to enter the cyst cavity, endocyst membrane can be seen fl oating in the most dependent part of the pericyst cavity producing the "waterlily sign" (fig. 13.26b ) . ct recognizes the appearance of the cystic lesion including smaller cysts, assesses signs of cyst rupture, evaluates the surrounding structures, and helps exclude alternative differential diagnoses (santivanez and garcia 2010 ) . ascariasis and hookworms remain the most common intestinal nematodes in the world (sarinas and chitkara 1997 ) . in the western hemisphere, parasitic pneumonia secondary to toxoplasma gondii is associated with compromised hosts, particularly acquired immunodefi ciency syndrome (aids) patients. strongyloides stercoralis infestation is seen in patients receiving glucocorticoids or chemotherapy and in patients with aids or other causes of t-cell dysfunction (berk and verghese 1998 ) . ascaris infestation generally occurs through hand-to-mouth ingestion of food contaminated with parasite eggs, while hookworms are transmitted through larval penetration of the skin. symptomatic pulmonary disease may present with fever, cough, chest pain, hemoptysis, dyspnea, and wheezing. these pulmonary symptoms could be due to loffl er's syndrome, effects of larval tissue migration, airway reactivity or bronchospasm, superimposed bacterial infection, and chronic eosinophilic pneumonia (sarinas and chitkara 1997 ) . ascaris and hookworm infections present with peripheral eosinophilia during larval migration phase. chest radiographs could be normal or demonstrate nonspecifi c patchy pulmonary infi ltrates (fig. 13.27 ) . ct scan could depict abnormalities better and could show ground-glass pulmonary lesions with ill-defi ned margins as well as nodules (sakai et al. 2006 ). causes of immunodefi ciency can be divided into congenital (primary) and acquired (secondary). the range of respiratory complications encountered is broad and is infl uenced by both the type and degree of immunodefi ciency. chest radiographs are insensitive and may show only subtle change. hrct detects abnormalities not visible on the plain fi lm such as bronchial wall thickening, bronchial dilatation, and air trapping. various noninfectious pulmonary processes including alveolar hemorrhage, pulmonary edema, graft versus host disease, and drug reaction are also seen in the immunocompromised hosts, which can mimic pulmonary infection on imaging. the primary or congenital immunodefi ciency disorders are inherited group of disorders resulting from innate defects of the immune system. clinical manifestations are diverse and nonspecifi c, which include recurrent infections, infection with opportunistic organisms, failure to thrive, skin rashes, recurrent skin sepsis, and unusual wound healing (jeanes and owens 2002 ) . primary immunodefi ciency can be broadly divided into t-cell (cellular) immune defi ciency versus b cell (humoral defi ciency). humoral immunodefi ciencies are the most commonly encountered type characterized by defective antibody production with increased susceptibility to pyogenic infections but able to recover from viral infections (fig. 13.28 ). examples are x-linked agammaglobulinemia, iga defi ciency, and common variable immunodefi ciency. cellular immunodefi ciencies have increased susceptibility to disseminated viral and opportunistic infections. cellular immune disorders include digeorge syndrome and severe combined immunodefi ciency (collingsworth 2005 ) . acquired immunodefi ciencies in childhood can be caused by chemotherapy, radiation therapy, immunosuppressive therapy aimed at treating childhood malignancies, transplant rejection, rheumatologic disorders, or infl ammatory or infectious diseases. it can also be due to human immunodeficiency virus (hiv) infection, malnutrition, or any state of chronic debilitation. bone marrow transplant requires complete eradication of the immune system. early infectious complications are frequently caused by bacteria and fungi, most commonly gramnegative bacteria ( pseudomonas and klebsiella ) and aspergillus (fig. 13.29 ). widespread use of long-term indwelling catheters has led to an increased incidence of both staphylococcal and streptococcal pneumonia. chest radiographs may show classic focal or lobar consolidation although atypical appearance can also be seen. children are also at increased risk of viral infections, most importantly rsv, herpes simplex , adenovirus , and varicella . immunosuppressive therapy following solid organ transplantation predisposes a patient to recurrent pulmonary infections. in these patients, viral infections can be life-threatening, but pneumocystis and fungal infections ( aspergillus and candida ) can also be seen (collingsworth 2005 ) . children represent 2 % of the reported cases of human immunodefi ciency virus (hiv) infection. most children are infected after vertical transmission from their mother, and majority develop acquired immunodefi ciency syndrome (aids) early in life. there is increased susceptibility to bacterial, viral, fungal, protozoal, and opportunistic infections. lobar or segmental consolidations are the most common patterns (marks et al. 1996 ) . mycobacterial infection can be seen in aids patients, and the radiographic appearance mimics that seen in immunocompetent children with primary tuberculosis. mycobacterium avium-intracellulare is also encountered later in the course of disease and imaging fi ndings cannot be distinguished with other forms of mycobacterial infections (collingsworth 2005 ) . pneumocystis jiroveci is the most common opportunistic pulmonary infection in children with aids, occurring in up to 50 %, and is the leading pulmonary cause of death (jeanes and owens 2002 ) . radiographic appearances are variable and include hyperinfl ation with diffuse bilateral interstitial or nodular infi ltrates from the perihilar region to the periphery, which often progresses to widespread alveolar opacities with air bronchogram (fig. 13.30 ) . cavitary nodules and cysts can be seen, with pneumothorax and/or pneumomediastinum as common complications. hrct fi ndings include patchy or diffuse ground-glass opacity, consolidation, cyst or cavities, centrilobular opacities, nodules, peribronchial cuffi ng, and interlobular septal thickening (jeanes and owens 2002 ; collingsworth 2005 ) . pathological changes in virus infection of the lower respiratory tract in children parasitic pneumonia infl ammatory lung disease interpretation of chest radiographs in infants with cough and fever thoracic disorders in the immunocompromised pneumonia in children echinococcus granulosus revisited: radiologic patterns seen in pediatric and adult patients pulmonary infections maximizing the usefulness of imaging in children with community-acquired pneumonia practical issues concerning imaging of pulmonary infection in children pneumonia in children: decreased parenchymal contrast enhancement-ct sign of intense illness and impending cavitary necrosis pneumonia in normal and immunocompromised children: an overview and update chronic lung infection in children obliterative bronchiolitis: individual ct signs of small airways disease and functional correlation imaging of cavitary necrosis in complicated pneumonia lung abscess versus necrotizing pneumonia: implications for interventional therapy histoplasmosis and pulmonary involvement in the tropics mycoplasma pneumonia: clinical and radiographic features management of empyema in children chest imaging in the immunocompromised child spectrum of clinical and radiographic fi ndings in pediatric mycoplasma pneumonia pulmonary tuberculosis in children: evaluation with ct high resolution ct fi ndings of varicella zoster pneumonia ultrasound in the diagnosis of pediatric chest diseases therapy of parapneumonic effusions in children: video-assisted thoracoscopic surgery versus conventional thoracostomy drainage swine-origin infl uenza a (h1n1) viral infection in children: initial chest radiographic fi ndings the natural history of childhood intrathoracic tuberculosis: a critical review of prechemotherapy literature thoracic diseases in children with aids thoracic mycoses from endemic fungi: radiologic-pathologic correlation imaging of the pleura community acquired pneumonia in infants and children pathology and pathogenesis of fatal bordetella pertussis infection in infants tuberculosis in children chlamydia trachomatis in infants: radiography in 125 cases mycoplasma pneumoniae pneumonia: radiographic and hrct features in 28 patients spherical pneumonias in children simulating pulmonary and mediastinal masses pulmonary lesions associated with visceral larva migrans due to ascaris suum and toxocara canis : imaging of six cases pulmonary cystic echinococcosis ascariasis and hookworm viral versus bacterial infections in children: is roentgenographic differentiation possible? high resolution fi ndings in community acquired pneumonia atypical pathogens and challenges in community acquired pneumonia what imaging should we perform for the diagnosis and management of pulmonary infections? postinfectious bronchiolitis obliterans in children: clinical and radiological profi le and prognostic factors key: cord-017531-fm8gl5b3 authors: andersen, bjørg marit title: scenarios: serious, infectious diseases date: 2018-09-25 journal: prevention and control of infections in hospitals doi: 10.1007/978-3-319-99921-0_82 sha: doc_id: 17531 cord_uid: fm8gl5b3 scenarios for serious, infectious diseases are important procedures used to understand the special microbe’s behaviour (clinical illness, spread of infection, etc.) and how to act most rational during special dangerous outbreaks. furthermore, scenarios describe how to handle patients, personnel and others possibly exposed to infections,outside and inside the hospitalto stop spread of the infection as soon as possible. today, it is not acceptable to place a patient with a known high-risk, serious infection in the same hospital room as other patients with not the same disease (who). in this chapter, some seldom but realistic scenario is described to better understand how to react and treat patients to stop spread of microbes during the primary phase of dangerous transmittable diseases. • all personnel who are the first in contact with the infected/exposed person, relatives, other contacts and environment. • it is not acceptable to place a patient (with not defined same disease) in the same room with a patient with known high-risk infectious disease (who). the hospital's management provides written plans for how to react in situations where personnel and others may be exposed to known/unknown serious communicable disease and a practical arrangement for how to handle the situation. the infection control officer at the level of where the problem occurs, and at the departments/ward where the infection may spread, is responsible for following local emergency plans. personnel who unprotected have come in a seriously contagious situation and may have been exposed to infectious agents are responsible for contacting the nearest responsible/infectious unit for advice and of following written guideline and practical advice. [4] • the patient (infected/suspected infected) is usually relatively easy to deal with since there are guidelines for preventing spread of infection and treating the patient for the current disease; see isolation routines. • contacts exposed to infection are often worse to handle since it may be larger numbers of people (travel company, etc.) and because fear of being infected itself creates uncertainty. therefore, some imaginable scenarios are made that deal with infected contacts. • transport by ambulance. all transport of infectious patients from the place of arrival to the hospital should take place in ambulances using the same infection control regime as for the individual infectious disease (contact infection, airborne infection, strict isolation); see isolation regimes; chaps. 14 proper use of protective equipment is used when handling such patients. the ebola outbreak in 3 african countries in 2014 showed that almost 30,000 were registered ill, more than 11,000 died, 800 health professionals became ill and more than half of them died. lack of use of ppe and proper infection control led to escalation of the epidemic. the staff used only 1 m distance from the patient as a zone of infection, as recommended by the who and cdc, and lacked protection for the head, hair and neck when within the 1 m zone and used only ordinary masks [7] [8] [9] [10] . this occurred despite the fact that ebola is defined as a high-risk, biosafety level 4 infection in which airborne infection could be relevant [11] [12] [13] [14] . the epidemic declined from 14 september 2014, following the introduction of more proper use of personal protection equipment and infection control routines [11, 12, 15] . contacts are differentiated after infection risk: 1. high-risk contact: physical contact with vhf, or with blood secretion or excretion. healthcare staff, ambulance staff, laboratory staff, family or others who have treated the patient before admission. 2. low-risk contact: been in the same room with the patient after the onset of the disease, but not in direct contact with the patient, equipment or others in the room. examine the contacts; measure temperature two times for 3 weeks. 3. transmission from healthy contacts is considered unlikely. however, everyone who has been in the same place at the same time with a vhf sick patient should be informed and followed up. if probable or verified vhf, inform the contacts-low chance of infection: • the contacts keep calm at home and measure the temperature daily two times for 3 weeks after the last contact with the index patient. no crowding with many people and no use of collective traffic. • if temperature 38 °c or more, or rash/flu symptoms/sickness, contact the infection medical department. • if this cannot be achieved at home, the contact may come to defined outpatient clinic for temperature measurement by appointment or is admitted to hospital. • served with food, etc. brought out from store to door, possibly, while isolated at home. mrsa (methicillin-resistant staphylococcus aureus), vancomycin-resistant mrsa, penicillin-resistant pneumococci, super-resistant gram-negative bacteria (esbl, cre,cp, ndm-1 bacteria), multidrug-resistant tubercle bacteria (tuberculosis), vancomycin-resistant enterococci (vre), etc. evaluated in collaboration with microbiological laboratory. this was especially observed during the major tsunami disaster in 2004 [16] . serious problems can occur in areas with melioidosis and other highly virulent bacteria. in hospital, not usually many at the same time, but dependent on endemic situation. the patient can be contact or air isolated, depending on the infectious agent. • registering of direct contacts-depending on the infectious agent (name, address), and include where the patient have been earlier (information). • ambulance personnel and other personnel use routines for the relevant infection type according to isolation procedures and in accordance with emergency department's report. in case of doubt, contact infection control personnel. • use respiratory protection, p3 mask, if suspecting pulmonary tuberculosis, and put a surgical mask or p2/p3 mask without a valve on the patient in case of suspected pulmonary tuberculosis or respiratory tract infection. contacts/carriers-differentiated follow-up-low chance of getting sick: example: three people in a norwegian travel company get voluminous, watery, painless diarrhoea on return from bangladesh, just before landing at oslo airport, gardermoen. due to a loss of fluid, they were transported with an ambulance equipped for "import infection" and admitted directly into contact isolation. municipal infection control doctor is notified and is responsible for reporting, measures and follow-up outside the hospital together with the municipal emergency response group. this is one of the old, major quarantine diseases. only a few get sick (top of the iceberg), i.e. 1-4 patients out of 1000 infected. there is a low mortality by proper treatment (<5%). the transmission risk is relatively low due to good hygiene and good sanitation in today's norway and other developed countries. most patients are shedding bacteria in large amounts and are also carriers without symptoms. patients are isolated with contact isolation regimens. the infection can reach unmanageable heights in disaster areas, by hunger, contaminated water supply and destroyed infrastructure. following the natural disaster in haiti, cholera was introduced with infected helper crew from asia (carriers), and an epidemic started in 2010, which in 2013 had increased to 670,000 cholera patients, 370,000 hospitalized and 8200 deaths [17] . • registering of contacts and remaining passengers in the airplane and from same travel company (name, address, telephone number). • ambulance staff and other personnel use the contact regime. if risk of spills, etc., use also surgical mask, visor and cap in addition to gloves and gown/overall. example: an elderly woman who recently attended a bus trip to moscow became sick 2 days after returning to oslo. she had sore throat, fever, cough and eventually a white, firm-sitting "plaque" in the throat. she was hospitalized after 3 days because of suspected diphtheria. contact persons and other close contacts were contacted for follow-up and treatment with erythromycin (according to resistance pattern). municipal infection control doctor is notified and is responsible for reporting, measures and follow-up outside the hospital together with the municipal emergency response group. diphtheria may still be periodic problems in eastern europe and many places in the world. vaccination status is good in children in most countries but more uncertain in elderly, especially in women. this is a contact and airborne infection, relatively highly infectious. there are probably few cases in an outbreak due to herd immunity. patients are isolated with air and contact isolation regime until free from bacteria (negative culture). a historically serious infectious disease also in norway, with high mortality rates until the middle of the last century [18] . ullevål hospital, oslo, introduced treatment with the diphtheria serum in 1914 and achieved an impressive response-from 20-35% mortality to 3-5% [19] . • registering: all exposed persons (name, address, telephone number) and followup; see below. • ambulance staff and other personnel use the contact and airborne infection regime when picking up and transporting a patient. use respiratory protection, visor and cap in addition to gloves and gown/overall, and put a surgical mask on the patient. contacts/carriers of diphtheria are differentiated-vaccination protects against disease: 1. sampling (nasopharynx samples) of all exposed persons (even if vaccinated and are not sick, you may be a carrier). 2. prophylactic/therapeutic treatment with erythromycin may be initiated rapidly. 3. if living at home, others should not be exposed to infection/carrier state. 4. short-time airborne isolation may be relevant for carrier or exposed to infection until the infection state is clarified/effect of antibacterial therapy. 5. booster vaccine against diphtheria is considered for all contacts. example: a person of a family of five who has stayed in madagascar for a month got sick on his way home to norway. he coughs, has fever and develops skin rashes that resemble big boils, especially in the groin. he was admitted directly to strict isolation in hospital and suspected of serious import infection. the municipal infection control doctor is notified and is responsible for reporting, measures and follow-up outside the hospital together with the municipal emergency response group. • quarantine disease: periodic problem in the east asia, especially india, and ongoing outbreaks in africa, namibia and madagascar. pest may be a warrelated disease (biological warfare). untreated dies more than 50% of the cases while, with streptomycin treatment, less than 5%. this is a typical airborne disease, relatively highly infectious when respiratory tract symptoms. there is usually small outbreak with few cases (1-2). air and contact infection regime until free for bacteria. in november 2014, new pest outbreaks were reported in madagascar. about 120 patients got sick, of whom 40 died of bubonic plague [20] . the infection spread rapidly between humans and "killed quickly" [20] . multidrug-resistant yersinia pestis is described in this country [20] . • registering: all infected persons (same travel company, all in the same flight home) are registered (name, address, telephone number) and followed up. • ambulance staff and other personnel use the contact and airborne isolation regime when picking up and transporting a patient. use respiratory protection (p3 mask), visor and cap in addition to gloves and gown/overall, and put a surgical mask on the patient. all close contacts/carriers are isolated to infection state is clarified-little chance of getting sick: 1. sampling from all exposed persons 2. prophylactic/therapeutic treatment, eventually vaccine 3. short-time airborne isolation of exposed cases until the infection state is clarified/effect of antibacterial therapy 82.5.6 anthrax after staying in turkey, sick on the plane home 82.5.6.1 patient: strict isolation-air pressure isolate with pressure [21, 22] example: two out of six people who have been on family visits in turkey for a week, on farms with goats and skin production, are acutely ill on the plane home with cough, shortness of breath and fever. upon arrival, the emergency outpatient clinic was contacted by the patients who were immediately transferred to intensive care unit for airway symptoms and suspected import infection. at the hospital, anthrax is suspected, and patients are strictly isolated in air isolate and treated. municipal infection control doctor is notified and is responsible for reporting, measures and follow-up outside the hospital together with the municipal emergency response group. • endemic problem in several places (asia, africa, middle east, north and south america), war-related (biological warfare). varying number of cases of anthrax at outbreaks during peacetime, depending on how many people have eaten, for example, infected food, etc. • symptoms: 95% are cutaneous, 5% are respiratory, and a few cases are gastrointestinal anthrax. the latter two are two-phasic and almost always fatal. • the bacterium bacillus anthracis is a spore-forming, resistant, gram-positive rod that survives nearly infinity in the environment if not removed. the bacteria are usually penicillin-sensitive. • infection: person-to-person infection is unlikely, but hospital infection is described. air and contact regime is conducted around such patients in hospitals until free of bacteria (ca 24 h treatment); however spores may survive for a long time. • registering: all directly exposed persons are registered (name, address, telephone number) and followed up. • ambulance personnel use contact and airborne regime for patient pickup and transport. use respiratory protection (p3 mask), visor and cap in addition to gloves and gown/overall, and put a surgical mask on the patient. contacts/carriers-low risk of getting sick: 1. sampling and antibacterial treatment are offered to all contacts that may have a common source of infection with the index patients (travel company, co-passenger). person-to-person transmission is unlikely. 2. vaccine may, in addition to antibacterial treatment, be applicable to people with a common source of infection with the index patients-when risk of large outbreaks (note that vaccination should be discussed due to some serious adverse reactions). 3. while waiting for result of the sampling, close contacts live at home with contact isolation restrictions. 4. in case of detected anthrax in contact (incubation phase for disease 3-7 days), the person is isolated and treated, and vaccination may be assessed for close contacts. this is an endemic problem among wild animals in most countries. person-toperson transmission is unlikely. it is almost never reported more than one case at a time, infected by animal bites or licking but occasionally without known exposure. close contacts/exposed persons are registered. • registering: all exposed persons are registered (name, address, telephone number) and followed up. • ambulance staff and other personnel use the contact and airborne regime when picking up and transporting a patient. use respiratory protection and ppr; put a surgical mask on the patient. contacts/exposed-low chance of getting sick: 1. close contacts/exposed persons are assessed for vaccine and rabies immunoglobulin. 2. followed up at the infection outpatient clinic. patients and contacts are treated just like at vhf. patients and contacts are treated mainly like vhf. see also sars and bird flu. new infectious diseases and agents still appear, for example, sars, avian viruses (h5n1), htlv and other retroviruses, hpv, sindbis virus, parvovirus, bocavirus, coronavirus, etc., or bacteria like legionella and borrelia, or agents with virulence changes, like group a streptococci, meningococci, etc. • biological terrorism made anthrax, plague, botulism, coxiella, brucella, vhf, poxviruses and a number of other unusual agents more appropriate as biological weapons . • bacteria sensitive to common antibacterial agents will probably not be a major problem. • viruses with no vaccine or treatment against low infection dose and high capacity to survive will be a major problem if associated with incurable disease, disability or death. • it is probable that this will be a problem first in countries with low hygiene standards/high population density. if the infectious agent is unknown, transmission ways are unknown and the situation is uncertain or uncontrollable, this practical measure may be followed: patient and contacts: strict isolation-negative air pressure isolation 1. serious illness: isolation of index case and all contacts 2. less severe disease: isolation of index case and close contacts • registering: all exposed persons are registered (name, address, telephone number) and followed up. • ambulance personnel use contact and airborne regime for patient pickup and transport. use respiratory protection (p3 mask), visor and cap in addition to gloves and gown/overall/shoe covers/dedicated shoes, and put a surgical mask on the patient. • botulism is caused by a bacteria-produced toxin (clostridium botulinum) that causes paresis and is common in soil as spores. the disease can be associated with toxin formation in contaminated and poorly canned foods, shrimp fish, bacon, etc. under anaerobic conditions and randomly affects both healthy people and vulnerable groups, such as infants who have had honey infected with the bacterium, which has happened repeatedly [42] . the toxin is the most dangerous we know and is on the list of bioterrorism. • brucella bacteria (zoonosis) are particularly related to laboratory outbreaks but are easily transferable outside the laboratory and are considered highly infectious. [43] • francisella tularensis (zoonosis) is defined as a category a bioterrorism agent, highly infectious and increasing in the society, has low infection dose (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) bacteria) and can be inhaled or infected via food and water. occasionally, such patients are detected in hospitals, even in the operating department [44] . • mers-middle east respiratory syndrome-newly discovered coronavirus zoonosis (dromedaries, bats, etc.) and acts as sars, also with a tendency for nosocomial spread in hospitals. in 2015 there were 1100 cases, of which 40% died [45] . • polio-like illnesses, especially among children, were discovered in august 2014 in different states in the united states. probably caused by enterovirus d68 [46] . • hiv-aggressive variants (crf19) have been detected in cuba in 2015 and earlier in africa, with a faster course from infection to aids development [47] . • prion disease-new shy-drager syndrome is rediscovered in 2015; multiplesystem atrophy (msa) [48] . • ricin is a plant-derived toxin that is still used for bioterrorism in letters to, among others, president obama [49]. • fungi and mould: different types that are especially related to floods, water damage, etc. and to pollution of medical products [50] [51] [52] [53] . in norway, a recent overview of candida in blood cultures showed a stable state for the past 22 years [54] . • zika virus: newly discovered flavivirus with mild to serious symptoms and teratogenic effect [5] . ministry of labour and administration. regulations on protection against exposure to biological factors (bacteria, viruses, fungi and more) in the workplace guidelines for environmental infection control in health-care facilities disinfection, sterilization, and control of hospital waste serious, common contagious scenarios. in: handbook in hygiene and infection control for hospitals. olso: ullevål university hospital other serious viral infections-zoonoses. in: handbook in hygiene and infection control for hospitals. part 1 fagbokforlaget lassa, and other haemorrhagic viruses. in: handbook in hygiene and infection control for hospitals. part 1. fagbokforlaget interim-infection prevention and control guidance for care of patients with suspected or confirmed filovirus haemorrhagic fever in health-care settings, with focus on ebola infection prevention and control recommendations for hospitalized patients with known or suspected ebola haemorrhagic fever in us hospitals department of health. management of hazard group 4 viral haemorrhagic fevers and similar human infectious diseases of high consequences ebola guideline-from the norwegian institute of public health 09/11 international infection control guidelines for ebola. hospital healthcare europe. facilities management transmission or ebola virus from pigs to nonhuman primates violent expiratory events: on coughing and sneezing guidance on personal protective equipment to be used by healthcare workers during management of patients with ebola virus disease in us hospitals, including procedures for putting on (donning) and removal multi-resistant infections in repatriated patients after natural disaster: lessons learned from the 2004 tsunami for hospital infection control in: handbook in hygiene and infection control for hospitals. part 1. fagbokforlaget tonsillitis in europe's outskirt-when the diphtheria toxin came to the county of romsdal blood is a very special juice"-introduction of serum therapy for diphtheria in norway preparedness at ullevål university hospital in connection with biological weapons anthrax and emergency routines at ullevål university hospital the threat of biological attack: why concern now? haemorrhagic fever viruses as biological weapons: medical and public health management bioterrorism-related inhalation anthrax: the first 10 cases reported in the united states anthrax as a biological weapon, 2002: updated recommendations for management anthrax of the gastrointestinal tract nosocomial spread of bacillus anthracis bacteria and disease. epidemiology, infections and infection protection. oslo: gyldendal akademisk, gyldendal norsk forlag as can anthrax infect from patient to patient? bacillus anthracis aerosolization associated with a contaminated mail sorting machine secondary aerosolization of viable bacillus anthracis spores in a contaminated us senate office risk assessments of anthrax threat letters. defense research establishment suffield. dres technical report 2001-038 anthrax inhalation and lethal human infection bioterrorism: crime and opportunity bioterrorism preparedness and response in european public health institutes deliberate releases of biological agents. initial lessons for europe from events in the united states the use of smallpox virus as a biological weapon: the vaccination situation in france are there «new » and «old » ways to track infectious diseases hazards and outbreaks ? infectious disease disaster: bioterrorism, emerging infections, and pandemics. apic text of infection control and epidemiology bioterrorism readiness plan: a template for healthcare facilities an infant with acute paresis hospital-associated transmission of brucella melitensis outside the laboratory potential risk of aerosol-borne francisella tularensis transmission in the operating room acute neurological disease of unknown etiology in children-colorado hiv-cuba: new aggressive variant prion disease updated: novel prion disease-shy-drager syndrome mould-preventing strategies and possible health effects in the aftermath of hurricanes and major floods fungi-human pathogenic. in: handbook in hygiene and infection control for hospitals. part 1. fagbokforlaget mucormycosis-usa: fatal, premature infant, probiotic supplement, recall, alert twenty-two years of candidaemia surveillance: results from a norwegian national study key: cord-021069-v9f9874x authors: morrison, lynda a.; fields, bernard n. title: viral pathogenesis and central nervous system infection date: 2004-11-23 journal: nan doi: 10.1016/1044-5765(91)90002-6 sha: doc_id: 21069 cord_uid: v9f9874x both host defense and viral genetic factors influence the development of viral infection and disease. due to the presence of the blood-brain barrier, infection of the central nervous system creates additional complexities in interactions between a virus and its host. stages in viral pathogenesis defined as (1) virus entry, (2) spread, (3) tropism, (4) virulence and injury to the host, and (5) the outcome of infection are discussed for viral infections in general and those aspects unique to infections of the central nervous system. information about neuronal physiology and function has also been revealed through studying virus infection. an increased understanding of viral pathogenetic mechanisms and host response to infection raises interesting possibilities for vaccine development and for basic studies in neurology and neurobiology. seminars in the neurosciences, vol 3, 1991 : pp 8 3 -91 viral pathogenesis and central nervous system infection lynda a . morrison and bernard n. fields both host defense and viral genetic factors influence the development of viral infection and disease. due to the presence of the blood-brain barrier, infection of the central nervous system creates additional complexities in interactions between a virus and its host. stages in viral pathogenesis defined as (1) virus entry, (2) spread, (3) tropism, (4) virulence and injury to the host, and (5) the outcome of infection are discussed for viral infections in general and those aspects unique to infections of the central nervous system . information about neuronal physiology and function has also been revealed through studying virus infection . an increased understanding of viral pathogenetic mechanisms and host response to infection raises interesting possibilities for vaccine development and for basic studies in neurology and neurobiology . key words : virus / infection / pathogenesis / central nervous system viral pathogenesis has been defined as the process by which a virus causes disease in a susceptible host .' several stages in pathogenesis can be identified that represent unifying themes in this process . all viruses must successfully penetrate the host's barriers to entry, undergo primary replication and then spread to their ultimate target tissue . any virus able to cause disseminated infection has the potential to infect the cns . viruses infecting the cns can either circumvent the blood-brain barrier by entry into peripheral nerve endings and axonal transport into the cns, or they can invade from the blood stream by infection of or transport across the vascular endothelium in the cns, in regions of greater permeability, or by passive transport in infected monocytes . but with all potentially neurotropic viruses successful invasion of the cns is rare, limited by host defenses, the mechanics of the blood-brain from the department of microbiology and molecular genetics, and the shipley institute of medicine, harvard medical school, 25 activity and are thought to gain entry through small immune host defenses . breaks in the integrity of the mucosa, or possibly by uptake at the intact mucosal surface . infection of the conjunctiva, presumably by direct inoculation from contaminated fingers, can occur with enterovirus 70, hsv and some coxsackie viruses . 3 regardless of the route of entry, viruses must evade local phagocytic cells that make an important contribution to early host defense . alveolar macrophages and histiocytes of the reticuloendothelial system efficiently phagocytose and eliminate invading viruses, but some (lymphocytic choriomeningitis virus, cytomegalovirus and the lentiviruses visna and hiv) escape destruction by productively infecting these cells . 1,2,5 elements of the host immune system, immunoglobulin a in mucous secretions and intraepithelial lymphocytes in the intestine, must also spread tissue invasion a period of replication in epithelial or subepithelial tissue at the site of inoculation typically occurs, except when virus is directly inoculated into the blood . whether a virus infection will remain localized at the site of entry or become disseminated is influenced by the temperature of the epithelial surface (e .g . rhinovirus), the propensity of a virus to bud from the apical (influenza) or basolateral (hsv) surface of epithelial cells, 3 following replication at the site of inoculation, virus enters the subepithelial lymphatic capillaries and is carried into the blood stream 3 where it must survive encounters with phagocytic cells and elements of the immune system . primary viremia permits the rapid dissemination either of free virus or of bloodcell associated virus to other susceptible tissues :' successful penetration of the vascular endothelium and replication in these organs amplifies the infection and augments viremia . viremia must be of a magnitude and duration adequate to sustain infection and, for some viruses, to permit an assault on the blood-brain barrier . replication of some arboviruses in cerebral capillary endothelium precedes viral invasion of the neural parenchyma, 6 and replication in or passive transport across the endothelium has been postulated for several other viruses, including enteroviruses, arboviruses and retroviruses .6' 7 because of its fenestrated endothelium and sparse basement membrane, the choroid plexus is also a target for replication or passive transport of mumps and arboviruses ; 1,3 through it, virus enters the cerebral spinal fluid to infect ependymal cells lining the ventricles and subseqently the underlying brain tissue . 3,5 migrating infected monocytes, lymphocytes or leukocytes may be an important vehicle for virus transport into the cns, especially in lentivirus infection (see zink and narayan, this issue,$ and refs 9,10) . neural spread from the periphery to the cns can be considered as two processes, penetration and propagation . penetration probably occurs at nerve terminals, either at the site of initial entry into and replication in the host or at a secondary site of replication in extraneural tissues to which virus is disseminated by the blood . electron microscopic observations have documented accumulation of neuroinvasive rabies virus particles in infected myocytes at neuromuscular junctions and neuromuscular 85 spindles, followed by entry into motor or sensory nerve terminals . 3 receptors used by viruses may be concentrated at nerve terminals and synapses, facilitating uptake and possibly transport of virus (see below, tropism) . once within nerves, propagation is mediated by axonal transport, replication and transsynaptic transfer of virus . hsv, rabies, poliovirus and reovirus are known to spread along nerves by the system of fast axonal transport ." transport has been demonstrated both towards and away from the cell body in somatic motor, somatic sensory and autonomic nerves, under various experimental conditions . bidirectional transport is important in the establishment of, and reactivation from, latency seen with hsv and varicella zoster . 12,13 viruses may have a predilection for a subset of nerve types, as evidenced by the more rapid uptake of reoviruses in motor than sensory fibers innervating the footpad 14 and by the restriction of rabies mutants to transport by sensory but not autonomic paths . 15 in contrast to the fast axonal transport of most viruses, the scrapie agent has been postulated to use the system of slow axonal transport . 3,6,16 disruption of the neurofilament cytoskeleton that supports slow axonal transport has been observed in scrapie-infected neurons and has been implicated in generation of the spongiform changes that characterize this degenerative neurological disease (see hope, this issue, 17 and refs 16, 18) . viral replication occurs once virus particles reach the neuronal cell body. newly synthesized virion components and virus particles then accumulate at post-synaptic sites, probably reflecting directed transport of virus components analogous to directed budding in polarized epithelial cells (see entry into the host, above ; ref 11) . both rabies and vesicular stomatitis virus accumulate at synaptic terminals and beneath the nodes of ranvier . when antiviral antibody is added to infected neuronal cultures, vesicular stomatitis virus buds preferentially from the synapses, indicating that exocytosis by neurons may be facilitated at this point .3 neurons thus transfer virus at synaptic junctions in the periphery . in the cns, the high density of neurons and glia permits many viruses to spread from cell to cell with less fidelity . the site of virus inoculation can influence the ultimate distribution of lesions within the cns, especially for viruses that spread along neural routes . neurons can transport viruses great distances intraaxonally both to and within the cns, and the final destinations of a virus will be determined in part by the particular synaptic connections made with individual neurons whose processes took up virus in the periphery . 19 hsv gaining entry through the genitourinary tract infects and becomes latent in sacral ganglia, whereas hsv entering in the oral mucosa becomes latent in trigeminal sensory neurons . though cases of strictly blood-borne or neural spread of viruses to the nervous system have been documented, a combination of the two routes may be used by certain viruses, or under certain conditions . for example, flaviviruses can spread through the blood to exposed neurons of the olfactory bulb which acetylcholine receptor, localized on muscle cells in the region of neuromuscular junctions, has been identified as a receptor for rabies virus . phosphatidyl serine may function as an alternative receptor for rhabdoviruses, 5,26 indicating that lipid molecules (and carbohydrate residues) as well as proteins can serve as neurotropic virus receptors . other molecules acting as receptors for neurotropic viruses are listed in table 2 . specific interactions with receptors may regulate binding of a particular virus at epithelial surfaces and vascular endothelium as well as nerve terminals, and may explain to some degree tissue specificity and differential susceptibility to infection . numerous viral cell-attachment proteins have also been identified ( gp350, gp220 heparin sulfate proteoglycana gb, gc, basic fibroblast growth gd/gh1 factor receptorb 87 selected with antibody against the e2 envelope protein revealed that a single amino-acid substitution in the e2 molecule determines loss of tropism for neurons without change in tropism for oligodendrocytes . 6,26 little is known about the murine coronavirus receptor protein on neurons or its similarity to the receptor on oligodendrocytes . x-ray crystallographic analysis of the poliovirus virion has provided a detailed molecular view of a ligand in virus-receptor molecular interactions . the analysis has revealed a series of peaks in the vp1 protein surrounded by a broad valley composed of vpi and vp3 that forms the receptor binding pocket (see almond, this issue 29 ; ref 13) . other members of the picornavirus family are known or postulated to have cell-attachment proteins of similar topology . transcriptional regulatory elements are an important post-receptor-binding determinant of viral tropism . 5,30 an enhancer element confers tissuespecific expression for jc papovavirus in human oligodendrocytes . similarly, promoter elements of some neurotropic murine retroviruses determine their species-specificity . on the other hand, host cells that lack enzymatic activities required for maturational cleavage of viral proteins or completion of the virus replicative cycle (as observed for poliovirus) will not be productively infected, thereby limiting tropism .5,25 a number of host factors modulate viral tropism and virulence, influencing viral pathogenetic potential and ultimately the potential for cns infection . 3,31 many neurotropic viruses more readily infect the young. this has been shown to variously result from the immaturity of the immune response, reduced capacity to produce interferon, increased susceptibility to infection of some cell types, and age-specific nature and distribution of receptor proteins2 '3 (see coyle, this issue 32) . genetic determinants of disease susceptibility have been found for infection of mice with strains of most neurotropic viruses, in at least one case of coronavirus reflecting lack of a gene encoding a virus receptor protein . determinants have been linked to histocompatibility genes that regulate immune responses only in chronic diseases of immunopathological etiology . last, sex of the host and nutritional status can influence resistance to infection . the selective vulnerability of different species or one species at different ages, coupled with viral determinants of neurotropism, may explain differences in clinical manifestations of cns disease . the efficiency with which a virus can multiply and extend infection after it has invaded the nervous system, its neurovirulence, determines in part the extent of injury suffered by the host . the effectiveness of the host response and the resulting immunopathology also play a role . although each virus has evolved its own genetic determinants of virulence, some generalizations can be drawn . outer capsid and envelope glycoproteins are frequently implicated as virulence factors because changes in their amino-acid sequence usually weaken their infectivity (attenuation), although this may be a subtle reflection of altered tropism . genetic reassortants between virulent and avirulent strains of bunyaviruses and reoviruses have also been used to identify determinants of neurovirulence by segregation of phenotype with a particular viral gene . 3 genetic recombination between strains of poliovirus and of hsv have similarly been used to identify genes critical for neurovirulence . 5 more than one region of a viral protein identified as a virulence factor may be important3 and, when neurotropism and neurovirulence are determined by the same protein, changes in that protein can have multiple and dramatic effects on the resulting infection . in studies of theiler's murine encephalomyelitis virus, a model of human demyelinating disease (see nash, this issue 33), variants selected with l .a . morrison and b .n. fields antibody to the vp1 protein possess tissue-specific alterations in extent and duration of infection and in capacity to induce demyelination . 13 but not all changes in viral genes are attenuating : mutations causing reversion to neurovirulence have been identified in poliovirus isolates from the feces of recipients of the attenuated trivalent oral vaccine . 34 mutations in genes encoding proteins other than those of the capsid or envelope can also result in attenuation, for example, sequences encoding a retroviral core protein or in the 5' noncoding region of poliovirus rna . 34 transacting transcriptional activators may play a role in determining virulence, exemplified by a mutant of hiv with a defective tatiii gene that shows reduced replication and cytopathic effect .3,6 importantly, neurovirulence may be under multigenic control and determinants of virulence may differ depending on the host species infected . 3 cns injury resulting from viral infections viral infections of the nervous system can have very severe consequences because the neurons vital to host function and survival cannot be replaced once injured or destroyed ; the effect of even minor damage may be catastrophic .2 furthermore, once infection in the cns has begun, the reduced permeability and dense structure of the blood-brain barrier which often impedes the initial spread of virus to the cns may reduce the capacity of the host to resolve infection and limit injury. 9 injury at a cellular level may be directly due to virus infection, occurring when cytopathic effect is significant enough to damage irreparably the plasma membrane (lysis) or to arrest metabolic activity by inhibition of protein, rna or dna synthesis, e .g . poliovirus, vesicular stomatitis virus and herpesvirus, respectively . 35,36 viruses such as rabies may alter nerve impulse conductance without histologically perturbing infected neurons . 19 accumulations of virus capsids or even individual viral proteins may in themselves be toxic . the sequence homology between hiv gp120 envelope protein and certain essential neurotransmitters and neuropeptides has evoked the hypothesis that competitive binding of gp120 may block normal neuronal function (see tillman and wigdahl, this issue 37). gp120 also mediates another potential form of direct injury by virus, syncytium formation . indirect injury to cells has been postulated to occur by release of toxic`factors' from neighboring cells damaged by infection . 36 systemic injury occurs by both direct and indirect mechanisms as well . the consequences of infection vary with the location and function of tissue injured by the virus : for example, motor neuron destruction in poliomyelitis results in paralysis ; demyelinating reactions to virus infection cause incoordination ; and virus infections of cells in the developing nervous system produce a variety of congenital abnormalities and neurological diseases (see coyle, this issue, 32 and refs 19, 38) . indirect mechanisms of viral injury to the host cns include the actions of interferons, 39-41 and the virus-specific cellular and humoral immune response (see sedgwick and dã¶rries, this issue, 42 and refs 2, 40, 43) . paradoxically, the elaborate system of host response to virus infection that may be protective outside the cns can be destructive when generated within this normally isolated compartment, 1,43 where cell lysis and complement activation may injure the host while helping to clear virus . virus replication in macrophages or immunocompetent t or b cells can also induce immune disregulation . 41,43 last, autoimmune reactions may be provoked by viral infections, resulting in indirect injury to the host even without continued presence of virus. 41,44 autoimmunity can be triggered in genetically susceptible individuals by non-specific inflammatory stimuli that alter immune regulation or by cross-reactivity between the viral inducing agent and normal host proteins (molecular mimicry) . 44,45 exposure of normally sequestered antigen due to virus-induced cytolysis and destruction or dysfunction of suppressor t cells have also been postulated as mechanisms in the development of autoimmune reactions . 41 autoimmune demyelination reactions, initiated in the cns against nervous system antigens, are especially prevalent . 46 several outcomes are possible in viral infections of the cns that do not acutely result in death . virus may be effectively cleared from the cns, may remain latent in the nervous system and cause recurrent disease, or may produce persistent disease . clearance is thought to be highly dependent on the immune response, though limitation of viral replication and the type of disease produced can be influenced by nonspecific factors . failure to clear varicella zoster, cytomegalovirus, adenovirus and 89 measles infections in natural cell-mediated immunodeficiency states suggest that t cell responses may be more important than antibody in eventual clearance of many infections . ) successful clearance may still leave the host impaired, depending on the nature and severity of the injury sustained .9" 10 a chronic autoimmune reaction may continue even after virus clearance . when latency is established by some viruses, the viral genome remains within the nervous system after acute infection ceases . no infectious virus is produced until reactivation occurs and the absence of viral protein products transcribed during latency may permit the infected cell to escape immune surveillance . capacity for latent infection in the nervous system is characteristic of the herpesviruses, hsv and varicella zoster (see stevens, this issue, 47 and refs 12,13) . persistent viral infections, in contrast to latency, are characterized by continued production and shedding of virions from infected cells . they are especially prevalent in cns tissues, 9 possibly because these contain non-dividing cells, are isolated behind the blood-brain barrier, have low expression of major histocompatibility complex proteins and may permit increased generation of defective viruses . 19 immune responses are usually generated and maintained but are ineffectual in clearing virus .48 as a result, chronic immune reactions may contribute much of the tissue injury .43 possible mechanisms of establishment and maintenance of persistent infection are listed in table 3 . persistent infection takes many forms . in lymphocytic choriomeningitis virus infection of newborn mice, chronic production of virus occurs without serious detriment to cells . 43 a more thorough understanding of these stages in pathogenesis of viral cns infections may engender new or more effective measures for prevention of disease . development of more efficacious vaccines is a primary objective but requirements for protection from individual viruses differ . 40,50 knowledge of pathogenetic stages of each virus may reveal points of greatest vulnerability and will help to determine requirements for stimulating immunity with a minimum of immunopathological side effects . ideally, infection by any virus with potential to invade the cns should be stopped before entering the nervous system . characterization of neurotropic virus infections also offers interesting possibilities for studying structure and function of the nervous system . basic neurological and neuroscience research has begun to benefit from using the strongly neurotropic rabies, hsv and pseudorabies viruses for tracing neural pathways . 51 more speculative is the use of attenuated neurotropic viruses for delivering foreign proteins into regions of the cns defined through studies of viral pathogenesis . foreign genes can be packaged by many viruses and may be selectively expressed, permitting intracerebral production of pharmacological agents or neurotransmitters . thus studies of viral pathogenesis of the cns may offer tools for the future as well as an understanding of disease states . viral infections of the nervous system viral pathogenesis and immunology pathogenesis of viral infections host defense mechanisms at mucosal surfaces pathogenesis of neurotropic viral infections molecular and genetic aspects of the pathogenesis of viral infections of the central nervous system vascular endothelium in immunology and infectious disease the neuropathogenesis of visna virus infection in sheep narayan 0 (1988) the neurobiology of human immunodeficiency virus infections the human immunodeficiency virus : infectivity and mechanisms of pathogenesis implications of axoplasmic transport for the spread of virus infections in the nervous system the use of molecular techniques in studying viral pathogenesis in the nervous system penetration of the nervous system of suckling mice by mammalian reoviruses pathways of the early propagation of virulent and avirulent rabies strains from the eye to the brain slow infections, in infections of the nervous system the key role of the nerve membrane protein ap in scrapie-like diseases fields viral pathogenesis and cns infection hypothesis : interference with axonal transport of neurofilament as a common pathogenetic mechanism in certain diseases of the central nervous system persistence of rna viruses in the central nervous system enteroviruses, in handbook of poliovirus type 1 enters the human host through intestinal m cells intestinal m cells : a pathway for entry of reovirus into the host isolation of cellular receptors for viruses viral receptors : expression, regulation and relationship to infectivity host and viral factors that influence viral neurotropism . i . viral cell attachment proteins and target cell receptors fibroblast growth factor receptor is a portal of cellular entry for herpes simplex virus pathogenesis of reovirus infections of the central nervous system enhancers and eukaryotic gene transcription host and viral factors that influence viral neurotropism . ii . viral genes, host genes, site of entry and route of spread of virus viral infections of the developing nervous system virological and pathological processes involved in theiler's virus infection of the central nervous system the attenuation of poliovirus neurovirulence impact of virus infection on host cell protein synthesis virus-host-cell interactions neuropathogenesis of human immunodeficiency virus infection the viral infections of the developing nervous system : an overview, in virus infections and the developing nervous system the pharmacology and toxicology of the interferons : an overview immunological aspects of the prevention of viral diseases interactions of viruses with the immune system the immune system response to viral infection of the cns viruses perturb lymphocyte functions : selected principles characterizing virusinduced immunosuppression autoimmune diseases : the failure of self tolerance molecular mimicry and autoimmune disease virus-induced autoimmune demyelinating disease of the central nervous system herpes simplex virus : neuroinvasiveness, neurovirulence and latency viral persistence pathogenetic aspects of persistent measles virus infections in brain tissue modern approaches to vaccines viruses as transneuronal tracers the authors are grateful to dr k tyler for much helpful advice and discussion, and for reviewing this manuscript . work on the manuscript was supported by public health service grants a108207-02 and program project 5 p50 ns16998 from the national institutes of health . key: cord-006586-49btg9w7 authors: golfieri, r.; giampalma, e.; morselli labate, a. m.; d'arienzo, p.; jovine, e.; grazi, g. l.; mazziotti, a.; maffei, m.; muzzi, c.; tancioni, s.; sama, c.; cavallari, a.; gavelli, g. title: pulmonary complications of liver transplantation: radiological appearance and statistical evaluation of risk factors in 300 cases date: 2000 journal: eur radiol doi: 10.1007/s003309900268 sha: doc_id: 6586 cord_uid: 49btg9w7 the aim of this study was to evaluate the incidence, radiographic appearance, time of onset, outcome and risk factors of non-infectious and infectious pulmonary complications following liver transplantation. chest x-ray features of 300 consecutive patients who had undergone 333 liver transplants over an 11-year period were analysed: the type of pulmonary complication, the infecting pathogens and the mean time of their occurrence are described. the main risk factors for lung infections were quantified through univariate and multivariate statistical analysis. non-infectious pulmonary abnormalities (atelectasis and/or pleural effusion: 86.7 %) and pulmonary oedema (44.7 %) appeared during the first postoperative week. infectious pneumonia was observed in 13.7 %, with a mortality of 36.6 %. bacterial and viral pneumonia made up the bulk of infections (63.4 and 29.3 %, respectively) followed by fungal infiltrates (24.4 %). a fairly good correlation between radiological chest x-ray pattern, time of onset and the cultured microorganisms has been observed in all cases. in multivariate analysis, persistent non-infectious abnormalities and pulmonary oedema were identified as the major independent predictors of posttransplant pneumonia, followed by prolonged assisted mechanical ventilation and traditional caval anastomosis. a “pneumonia-risk score” was calculated: low-risk score ( < 2.25) predicts 2.7 % of probability of the onset of infections compared with 28.7 % of high-risk ( > 3.30) population. the “pneumonia-risk score” identifies a specific group of patients in whom closer radiographic monitoring is recommended. in addition, a highly significant correlation (p < 0.001) was observed between pneumonia-risk score and the expected survival, thus confirming pulmonary infections as a major cause of death in olt recipients. abstract. the aim of this study was to evaluate the incidence, radiographic appearance, time of onset, outcome and risk factors of non-infectious and infectious pulmonary complications following liver transplantation. chest x-ray features of 300 consecutive patients who had undergone 333 liver transplants over an 11year period were analysed: the type of pulmonary complication, the infecting pathogens and the mean time of their occurrence are described. the main risk factors for lung infections were quantified through univariate and multivariate statistical analysis. non-infectious pulmonary abnormalities (atelectasis and/or pleural effusion: 86.7 %) and pulmonary oedema (44.7 %) appeared during the first postoperative week. infectious pneumonia was observed in 13.7 %, with a mortality of 36.6 %. bacterial and viral pneumonia made up the bulk of infections (63.4 and 29.3 %, respectively) followed by fungal infiltrates (24.4 %) . a fairly good correlation between radiological chest x-ray pattern, time of onset and the cultured microorganisms has been observed in all cases. in multivariate analysis, persistent non-infectious abnormalities and pulmonary oedema were identified as the major independent predictors of posttransplant pneumonia, followed by prolonged assisted mechanical ventilation and traditional caval anastomosis. a ªpneumonia-risk scoreº was calculated: low-risk score ( < 2.25) predicts 2.7 % of probability of the onset of infections compared with 28.7 % of high-risk ( > 3.30) population. the ªpneumonia-risk scoreº identifies a specific group of patients in whom closer radiographic monitoring is recommended. in addition, a highly significant correlation (p < 0.001) was observed between pneumonia-risk score and the score), the technical quality of the transplant surgery, the type, intensity and duration of the immunosuppressive therapy and epidemiological exposure to environmental agents. because one or more of these factors are important at different points in the posttransplant course, it should be possible to predict which complications are most likely to occur at different moments [16] : an accurate chest x-ray follow-up could therefore lead us to the type of pulmonary abnormality (infectious or non-infectious) and to the possible infecting agent, thus improving the final patient outcome. the aim of the present study was to: 1. describe the radiological pattern on chest x-ray film (crx) and prevalence of non-infectious and infectious pulmonary complications during the follow-up of olt 2. verify the type of infecting pathogens and the mean time of their occurrence 3. identify the main risk factors for lung infections, and their quantification through univariate and multivariate statistical analysis, in order to evaluate the likelihood of developing pneumonia. particular emphasis was placed on the predisposing factors for pneumonia occurring during the first posttransplant month, because this is considered to be the most critical period, when technical and clinical problems are associated with fatal infections [12, 16] . many papers have been published previously on this topic [2, 3, 4, 5, 6, 7, 8, 9, 10, 11] , but, to our knowledge, the present study represents the largest series to date of radiologically demonstrated pulmonary complications following liver transplantation involving a follow-up of over 11 years. the first 300 patients who underwent olt at the surgical department and transplant centre of the university of bologna, between september 1986 and september 1997, were included in this retrospective study. among these, 269 patients received only a single graft, 29 cases were retransplanted and 2 cases received three grafts for a total number of 33 retransplantations (14 due to vascular ischaemic events, 10 for ªprimary non-functionº and 9 for acute rejection). of the 31 patients who needed retransplantation, only 12 survived (38.7 %). the medical records of all patients were reviewed from the time immediately preceding olt until death or throughout the complete follow-up (maximum 11.8 years). the following clinical data were recorded and correlated to the onset of pulmonary infections and to the outcome: 1. the mean age of the present patient population was 45.3 years (sd 12.3 years), with 105 females and 195 males. mean follow-up after the transplant was 3.4 years (median 2.7 years; interquartile range 1.2±4.8 years). two hundred fifteen olt recipients are still living (71.7 %). in addition to the six major indications listed in table 1 , the ªotherº group included the following 28 cases: 4 wilson's disease; 2 diffuse angiomatosis; 3 giant hemangiomas; 1 hemangio-endothelioma; 1 hepatoblastoma; 1 macronodular hyperplasia due to venocclusive disease; 2 crigler-najjar disease; 2 polycystic disease; 5 amyloidosis; and 7 different metabolic liver diseases. the patients' pre-operative clinical conditions, presented in table 1 , were assessed according to the united network of organ sharing (unos) classification [21] . orthotopic liver transplantation (olt) was carried out with two standard surgical techniques: the traditional technique, as first described by starlz [22] in 189 patients (63.0 %), and the ªpiggy-backº caval anastomosis technique [23] in 111 patients (37.0 %). in this population, piggy-back caval anastomosis was performed only once in each patient. selective bowel decontamination with colomycin, nystatine or gentamicin sulphate was routinely performed in the immediate pre-transplant period and continued for the 3 weeks following olt. a standard triple immunosuppressive therapy [cyclosporine a (cya)-azatioprine-steroids] was routinely used. in 34 non-responsive cases (11.3 %), monoclonal anti-t cells antibodies okt3 was administered, combined with steroids, antihistamines and lasix iv on the first day and immunoglobulins. in 29 patients (9.7 %) as an alternative to okt3 (in steroid-refractory rejection episodes or in cases of neuro-/nephrotoxicity secondary to cya) fk 506 (tacrolimus fuji, sawa, japan) alone, together with steroids and/or azatioprine was used. in 4 cases fk 506 was associated with okt3. intensive care unit (icu) risk was considered to be duration of stay in icu and days of assisted mechanical ventilation (amv). data distribution is reported in table 1 . rejection was histologically diagnosed after liver biopsy and its severity was graded from i to iv according to the current classification system (table 1 ) [24] : it was observed in 124 patients (41.3 %) and needed retransplantation in 9 cases (7.3 %). in all patients chest roentgenograms (cxr) were performed daily, as a bedside semi-erect film, in the immediate posttransplantation period and throughout the entire hospitalisation as an erect film; in the follow up, cxr was performed according to the study protocol: an erect cxr every 2 months during the first 6 months and every 6 months during the following 2 years. chest x-rays were independently reviewed by two radiologists (r. g. and e. g.) who were unaware of the clinical pa-rameters at that time: in every cxr the following parameters were evaluated and described according to the commonly used terminology, fleishner society nomenclature [25] , and quantified as described below. atelectasis was classified as slight (involvement of less than one subsegment or discoid atelectasis), moderate (involving one or more segments or lobar hypoventilation) or severe (atelectasis of one or more lobes). pleural effusion was scored as slight (in the case of loss of the sharpness of costo-phrenic sulci and diaphragmatic profiles or subpulmonary effusion), moderate (effusion involving less than 25 % of a hemithorax) or severe (involving more than 25 %, including a massive effusion with mediastinal shift). for statistical analysis, normal pulmonary patterns were considered as score 0; slight and moderate atelectasis and/or slight and moderate grades of pleural effusion were considered as score 1; and severe atelectasis and/or severe effusion and/or when lasting > 1 week were included in score 2. pulmonary oedema was classified as score 1 (interstitial: localised, basal and/or diffuse) and score 2 (alveolar: localised or diffuse). vascular pedicle width (vpw) was classified as normal when it was approximately 6 cm. hydrostatic oedema, due to hyperhydration, was considered in all cases where a non-cardiogenic oedema with homogeneous pattern was associated with an increase in vpw diameters. adult respiratory distress syndrome (ards) was defined as the acute onset of a diffuse ªpatchyº alveolar oedema documented at cxr, with no enlargement of the vascular pedicle (no clinical evidence of cardiogenic pulmonary oedema), with reduced pulmonary compliance, needing assisted ventilation at fi o 2 0.50 and, when measurable, pulmonary artery occlusion pressure of less than 18 mmhg [5, 12, 14] . infectious pneumonia was classified according to three main radiological patterns: (a) focal pulmonary consolidation; (b) nodules or rapidly growing masses (with or without central cavitation); and (c) diffuse pulmonary infiltrates (interstitial or alveolar pattern) [13, 19] . in order to identify early lung infections, control cultures from biological fluid specimens were obtained (bacteriology of sputum, bronchoscopy aspirates, pleural fluid, bile, blood) and serological samples were controlled daily. a pathogen or a potential pathogen obtained from a normally sterile site (sputum, specimen obtained through a bronchoscopy or pleural fluid) was considered responsible for pulmonary infection. the combination of a chest x-ray positive for a new or increasing infiltrate, clinical symptoms such as fever or dyspnoea, and positive cultures were considered to be pulmonary infection [5, 12] . in order to limit our observations to ªmajorº infections only, febrile episodes with no positive cultures or isolated cultural positivities without clinical±radiological positivities were considered as ªminorº infections or ªcontaminationsº and were excluded [7, 12] . when superimposed on ards, pneumonia could not be identified by cxr: the diagnosis could only be obtained by positive cultures from biological fluids. for the evaluation of the time of occurrence, in polymicrobial pneumonia, the most aggressive pathogen of the association was considered prevalent (e. g. bacterial vs viral and fungal over bacterial). the yates corrected chi-square was applied to compare proportions between different groups of patients. the putative risk factors for development of pneumonia after olt were tested by means of univariate and stepwise multivariate survival analysis based on the cox proportional hazard regression model [26] . the exponentials of the coefficients calculated by these analyses (or) were reported to quantify the effect of each putative risk factor on the hazard function. these ors estimate the fractional increase of the risk of developing pneumonia either determined by the increase of one unit in the score of the putative risk factors or determined by the presence of dichotomous risk factors. the 95 % confidence intervals (ci) of the ors were also evaluated. the same univariate and multivariate procedures were applied to analyse the mortality rate after olt. the product-limit estimate [27] was used to plot both the time course of the appearance of pneumonia and the survival after olt. statistical analyses were performed by means of the bmdp statistical software [28] running on a personal computer. a two-tailed p-value of less than 0.05 was used to define statistical significance. a summary of non-infectious pulmonary abnormalities is presented in table 2 . in the early postoperative period, pulmonary non-inflammatory changes involved 260 patients (86.7 %). atelectasis of different degrees was a common finding (73.7 %): it was lamellar or subsegmental and it accompanied pleural effusion in most cases (128 of 221, 57.9 %), whereas only in 29 cases did it prevail against effusion, appearing as lobar or multilobar atelectasis. in the majority of patients it resolved itself spontaneously within 15 days on average: in 6 of the 20 cases of severe and prolonged right lobar atelectasis, a bacterial superinfection appeared (table 2) . pleural effusion was very frequent (68.6 %) in the first week: in the majority of cases (185 cases: 89.8 %) it was slight or moderate, situated mainly or exclusively at the base of the right lung, the site of surgical manipulation, with complete resolution within 5 days on average (range 2±7 days). only in 59 cases did it last more than 7 days and in 37 of them (62.7 %) it was treated with thoracentesis. pulmonary oedema was very common in the early postoperative period. the interstitial involvement was prevalent (72.4 %). the cause was overhydration (hydrostatic oedema) in 67 cases (69.1 %) and it regressed after 3±4 days of diuretics and fluid restriction. a cardiomegaly was always absent. the radiographic pattern of interstitial pulmonary oedema was characterised by bilateral, diffuse or central ªground glassº opacities accompanied by kerley b lines. the vpw was always increased in hydrostatic oedema. in alveolar oedema a diffuse or basilar air-space consolidation was present, differing from the patchy focal opacities of interstitial pneumonia. in 12 patients (4.0 %) a disseminate pattern of ards was observed in the final phase and al-ways superimposed on sepsis complicating a pulmonary infection: the ards mortality was 100 %. the appearance of ards was characterised by an extensive patchy diffusion of air-space opacities (more extensively than the previous pneumonia infiltrates), with air bronchogram and without an increase in vpw diameters. the frequency of ards was significantly higher (p = 0.029) in retransplanted patients (4 of 31, 12.9 %) than in single graft patients (8 of 269, 3.0 %). of 34 patients who received immunosuppressive therapy with okt3 (24 due to hyperacute rejection and 10 due to the onset of cya toxicity), 21 developed acute pulmonary hydrostatic oedema with diffuse interstitial involvement. no okt3-related alveolar oedema was observed. infectious pneumonia was observed in 41 patients (13.7 %), and was fatal in 15 (36.6 %). in 10 patients a polymicrobial infection was present and in 29 a single agent was isolated (a total of 49 pathogens were isolated on biological fluids or confirmed by serological tests). in addition, in two bacterial abscesses the specific agent could not be identified. bacterial infections were the most common (26 of 41, 63.4 %) followed by viral (12 of 41, 29.3 %) and fungal (10 of 41, 24.4 %) pneumonia: the most frequent agents were gram-negative (mainly represented by pseudomonas aeruginosa as a single or coinfecting agent) isolated in 41.5 % (17 of 41), cytomegalovirus (cmv) isolated in 26.8 % (11 of 41) and candida albicans observed in 22.0 % (9 of 41) of cases, respectively. in 36.6 % of cases, pulmonary infection was the cause of death (12 with final ards). the majority of infections (38 of 41 cases, 92.7 %) had their onset within the first 2 months (ªearly infectionsº). in 32 cases (78.0 %) the pneumonia occurred during the first month, in 6 cases (14.6 %) within the second month, and in only 3 cases did the infection have later onset. all the lethal infections developed within the first 2 months after olt, 10 of which developed during the first month. twenty-seven of 38 ªearly infectionsº had onset during the icu stay (65.9 % of the whole infections); among these, 20 patients were still subjected to amv at the time of the onset of pulmonary infections and the causal agents were: pseudomonas in 11 cases; staphylococcus aureus in 5 cases; and coinfections of candida with pseudomonas and cmv in 2 cases each. in all 7 cases of infection solely by cmv, pre-olt serology was positive (high igg rate anti-cmv): 5 of these had complete recovery. two of the 3 cases of cmv pneumonia superinfected by candida were seronegative in pre-transplant screening, and both had lethal pneumonia: this confirms a more severe outcome of seronegative pre-olt patients. there was a general correlation between the type of radiographic pattern and the microorganism producing the pneumonia, since the radiological patterns observed in the 41 lung infections were: . 4) ; in 2 cases of isolated cmv and 3 cases of fungal superinfections the radiological findings were small multiple alveolar sub-segmental consolidations, associated with little pleural effusion, resembling those of bacterial pneumonia ( fig. 4 b,c) . in all cmv infections, either isolated or coinfecting agent, a previous non-infectious pulmonary abnormality was present. the only case of herpes virus pneumonia showed a diffuse symmetrical reticular interstitial thickening, without pleural effusion at cxr (fig. 5 ). fungal infections (candida, aspergillus) had late onset ± median day 18 (range 5±98 days). the radiological pattern in 4 cases was as a single pulmonary infiltrate, and the six remnants showed multiple focal rapidly growing lesions, always associated with pleural effusion. in 2 cases a central cavitation occurred, quickly evolving towards an ards syndrome with fatal outcome (figs. 3, 4 a). in all fungal infections a previous non-infectious pulmonary abnormality had been present: 7 of slightmoderate and 3 of severe entity. mortality related to pneumocystis carinii pneumonia had onset on the twenty-eighth postoperative day: cxr showed an interstitial ªground glassº pattern, mainly in the perihilar or basal lung regions (fig. 6) , which resolved after therapy in 2 weeks. univariate analysis for infections demonstrated six risk factors associated with pneumonia, as reported in ta non-infectious pulmonary abnormalities are strong risk factors for pneumonia: the increase of one unit in the score scales of both atelectasis/effusion and pulmonary oedema elevates the pneumonia-risk by three times. atelectasis and pleural effusion were observed in 97.6 % of patients who developed pneumonia. pulmonary oedema preceded pneumonia in 68.3 % of cases. among the patients who developed infections, pulmonary oedema, especially with alveolar pattern, was observed significantly (p = 0.002) more frequently (68.3 %) when compared with uninfected cases (40.9 %). these findings indicate the pre-existing pulmonary abnormalities as a local risk factor predisposing to infection. the variables which entered the stepwise procedure are shown in table 3 ; four independent risk factors for pneumonia were identified: pulmonary non-inflammatory abnormalities and oedema, both doubling the risk of pulmonary infections, and prolonged amv; on the contrary, piggy-back anastomosis reduces the risk of pneumonia as compared with traditional caval anastomosis. on the basis of the coefficients computed by the stepwise multivariate survival analysis and the pattern of the variables that entered into the procedure, a score for the risk of developing pulmonary infections was calculated for each patient: pneumonia-risk score = 0.8049 (if no piggyback) + 0.4143´amv + 1.0735´pni + 1.0244´ede, where amv, pni and ede represent the score values of mechanically assisted ventilation, pulmonary non-infectious abnormalities and oedema, respectively. patients with different risks of developing pulmonary infections were identified according to the tertile values of the pneumonia-risk score (low risk: score less than 2.25; medium risk: score from 2.25 to 3.30; high risk: score greater than 3.30). the time-course of the appearance of pulmonary infections (fig. 8) shows that the cumulative 1-year incidence of pneumonia in high-risk patients was 28.7 %, 10.3 % in medium-risk patients, and 2.7 % in low-risk patients. in univariate analysis (table 1) , a statistically significant higher risk was demonstrated in cases of ahf patients, retransplantation, immunosuppression with okt3, prolonged stay in icu and amv and in protracted pulmonary oedema; instead, surgical piggyback caval anastomosis is a factor reducing hazard. multivariate analysis taking into account all the risk factors expressed in table 1 , also including pneumonia and age, was performed. two of the four independent risk factors are represented by prolonged amv, which triples the risk, and pulmonary oedema, which doubles the risk (table 3) , whereas the piggy-back technique and a prolonged icu stay both seem to constitute a relative ªprotectionº against fatal complications, since their or were significantly less than one (0.45 and 0.43, respectively). a highly significant (p < 0.001; or = 1.66, ci = 1.23±2.23) correlation was also observed between the survival rate and the pneumonia-risk score in the same groups of patients: kaplan-meier curves depicting survival rates in pneumonia-risk scores groups are shown in fig. 9 . the expected 5-year survival rate in low-, medium-and high-risk patients is 84.1, 74.3 and 60.3 %, respectively. unlike the experience with respiratory disorders occurring after transplantation of organs such as the kidney, bone marrow, lung and heart [2, 13, 14, 15, 16, 17, 18, 19, 20] , the majority of the pulmonary complications we identified following olt were non-infectious in origin. non-infectious pulmonary complications were re[11] , respectively, in 77 % in a preliminary report of our institution [9] and in 86.7 % of the present series. these complications are directly related to surgical manipulations for diaphragmatic dissection and to the type of technique and the surgical time spent in performing caval anastomosis: the right phrenic nerve is often injured during surgery. therefore, atelectasis due to diaphragmatic hypomobility in the early postoperative period is a common finding, involving one or more lung segments, mainly on the right side [15] . reduced compliance of the pulmonary basis secondary to the increase of intravascular volume, retained secretions or compression from perioperative pleural effusion can also be responsible for postoperative atelectasis [14] . usually the recovery is complete after intense respiratory therapy, with no need for bronchial aspiration. afessa et al. [12] reported atelectasis in 74 % of patients after olt (unilateral right in 31 % and bilateral in 44 % of cases) with spontaneous resolution in 95 % of the cases after 6 weeks. similarly, in our experience, atelectasis was observed in 73.6 %, bilateral (34.6 %) or right-sided (32.0 %), and it resolved itself spontaneously within 15 days on average. a severe and prolonged right lobar atelectasis is rare [14, 15] and a superimposed bacterial pneumonia should be suspected in these cases, as it was observed in 6 of 20 patients (30 %) of the present series. pleural effusion is an expected consequence of olt, observed in 54±100 % of patients in the first postoperative week [11, 12, 14, 15] : mainly on the right side or bilateral, it is a transudate (as demonstrated by the performed thoracentesis) also named ªhepatic hydrothoraxº [15] due to residual ascites or to surgical trauma. usually less than 20 % of the effusions need thoracentesis [9, 11, 14, 15] . in duran et al.'s series, pleural effusion was noted in 61.9 % of patients, 31.4 % of them requiring thoracic tube drainage [11] . in the present series it was observed in 68.6 % of the cases, with spontaneous resolution within a week in the majority of cases and thoracentesis was required in only 18 % of them. pulmonary oedema with hemodynamic or hydrostatic origin is a common non-infectious complication of cardiac, renal, bone marrow and liver transplant. in liver transplants pulmonary oedema is due mainly to overhydration from fluid infusion, excess or massive blood transfusion during surgery, to fluid retention related to preoperative renal dysfunction or to renal failure due to cya nephrotoxicity [12, 14] . in the majority of cases, it regresses after diuretics. in our series pulmonary oedema was observed in 44.7 %, with interstitial involvement in the majority of them: the main cause was overhydration (hydrostatic oedema) and it regressed after 3±4 days of diuretic therapy. ards has been reported in the literature in from 4.5 to 17.5 % of cases following olt [3, 5, 14] . the rate of mortality approaches 80 % and the onset is generally within the first postoperative week [15] . multiple etiological factors have been described (peri-surgical events such as infraoperative hypotension, prolonged surgical time, haemorrhage and blood transfusions). despite the complexity and dura-tion of the olt surgical procedure, which often involves extensive blood transfusions, ards has rarely been described in the early postolt period. sepsis appears to be, in our and in other authors' experiences [3] , the most common cause of ards in olt patients. the incidence of ards in our experience was 4.0 %. it was always associated with sepsis from pulmonary infections and had a fatal outcome in all cases due to toxic shock syndrome. similarly, in a recent report [14] , ards was observed in 3.5 % of cases during the postolt course, sepsis was always the causal factor and, as in non-transplant patients, there was a very high mortality rate [3, 5] . retransplantation is a significant risk factor for ards, as seen in a recent series in which ards occurred in 21 % of the patients receiving multiple grafts as compared with 2.7 % of patients receiving one graft [14] . accordingly, in our series, ards was observed in 12.9 % of second graft patients and in 3.0 % of the single graft cases (p = 0.029). pulmonary infections after olt have been observed in a range from 15 to 52 % [2, 3, 4, 5, 6, 7, 8, 11] , with mortality around 40 %. these prevalences are much higher than those observed after routine hepatic surgery, as shown in approximately 25 % of cases [29] . the 13.7 % prevalence of pneumonia (lethal in 36.6 %) in the olt population of this series was similar to some previous reports [4, 7, 10] and lower in comparison with previously reported prevalences of 43.3 % of infectious events (fatal in 28 %) [11] . a wide variability among the reported experiences about rate of infections, prevalence and type of infecting agents in postolt follow-up is observed in table 4 , depending mainly on bowel decontamination, patient selection criteria for olt, immunosuppressive therapy, percentage and type of environmental agents. during the first month after olt, the majority of bacterial pneumonia is reported by almost all series [7, 8, 12] . the most frequent pathogens in our series were gram bacteria and viruses (cmv) followed by candida in 9 cases. after olt, the majority of lung infections have onset within the first 6 months of the posttransplant course. after the first month graft recipients have passed the major global risk of lethal infections (figs. 7, 8) . in our experience, in the first month 78.0 % of the infections appeared. almost all the infections (92.7 %) and all the fatal infections appeared by the end the second month (fig. 9) . the different opportunistic pathogens tend to appear at predictable times during the posttransplant course, following the timetables suggested by rubin [16] after liver transplant and recently by leung [20] after bone marrow transplant. the knowledge of the time lines of the different pathogens is helpful in the differential diagnosis. accordingly, in our series, during the first 2 weeks, bacteria and fungi were the only agents observed; subsequently (days 15±60), cmv and fungi were the most important pathogens, followed by p. carinii. the only difference in our data is the pcp onset, which is reported to occur, in the majority of cases, 3±5 months after olt and which in the sole case of our series had onset approximately the twenty-eighth day. after the second month, there is no predominant patho-gen: bacterial pneumonia from the environment, viral infections from cmv and herpes simplex and p. carinii pneumonia are common [30] . bacterial pneumonia is the most common pulmonary infection after liver, lung and cardiac transplants [7, 14, 18] . in olt, bacterial pneumonia constituted up to 50 % of pulmonary infections and arises during assisted ventilation: pre-transplant prolonged intubation, aspiration pneumonia or postoperative atelectasis and lengthy surgical procedure seems to represent promoting factors [4, 5, 6, 7, 10, 12] . in our series, bacterial infections constituted 63.4 % of all pulmonary infections. the bacteria most frequently described are enterogenic gram-and particularly pseudomonas aeruginosa, as observed in 41.5 % of our series, as single or co-infecting microorganism. all but three bacterial infections had an onset within the first month, during assisted mechanical ventilation in 52.3 %. the radiographic findings of bacterial pneumonia are believed to be identical in immunocompetent and immunocompromised patients: in the present series they appeared as dense air-space consolidations with lobar, segmental, localised or patchy distribution, usually associated with a small amount of pleural effusion. the same characteristics were also observed in two cases of candida superinfection. in patients with sepsis and bacteraemia, fulminant disease with rapid progression to ards may occur, with a pattern changing into diffuse, irregular (patchy) air-space consolidation. cytomegalovirus pneumonia is reported to be the most frequent pulmonary infection after bone marrow transplantation [16] and it has been described in up to 35 % cardiac and kidney transplants and in more than 50 % lung and lung-heart transplant recipients. in olt recipients the major cmv-infected organs are the liver, intestine and lung. a high prevalence of cmv pneumonia is, however, reported [4, 5, 7, 17] and it is confirmed in our series, where it was second in frequency, with 26.8 % overall incidence. due to frequent superinfections (three candida superimpositions in our series), the prognosis is poor. coinfection with other opportunistic pathogens has been previously described [3] , particularly with pneumocystis carinii, as observed in one case of the present series. strong antirejection therapy, such as antilymphocytic antibodies, such as okt3, has been reported to be the highest risk factor of serious cmv infections [4, 15, 31] . our experience excludes a specific cmv-promoting effect of okt3. as predictors of cmv pneumonia have also been identified: the donor seropositivity and recipient seronegativity, advanced unos status, invasive fungal diseases, abdominal reinterventions and bacteraemia, all events reducing immunological defences [30] . in our series, reactivation or reinfection mechanisms were present in the majority of seropositive recipients: preoperatively cmv-seropositive recipients have a higher percentage of reactivation infections, yet rarely develop severe infections, whereas cmv seronegative recipients have a higher risk of developing severe infections [15, 30, 32] . furthermore, as the most significant effect of primary cmv infection, a broad-based depressant effect on the host defences is described, responsible for the onset of a great number of opportunistic infections, elevating the mortality rate [15, 16] . moreover, in the present series, the immunodepressant effect of primary cmv infection and the frequent fungal superinfection was confirmed, as 3 cases of primary cmv pneumonia were superinfected by candida and led to a fatal outcome. the chest radiographic findings of cmv have been variably described as consisting of a diffuse fine reticular or a haziness pattern of interstitial pneumonia, diffuse micronodular patterns, focal air-space consolidation resembling that of bacterial pneumonia [33] or, eventually, with normal findings [20] . the majority of our cases demonstrate diffuse parenchymal haziness and in 2 cases we found a pattern of parenchymal consolidation distributed in the middle and lower lung zones, similarly to what has been observed in previous studies performed on bone marrow transplant recipients [20] : in these cases the basilar predominance has been reported as related to hematogeneous cmvdissemination, with preferential distribution to the lower lung zones, due to their greater blood flow perfusion. candida superinfections appeared as nodular or focal airspace consolidations. herpes simplex pneumonia is rarely described in liver transplant recipients. it usually has a late onset, after the first month, and is due, like cmv, to the reactivation of a latent virus, as a result of immunosuppressive therapy. the only case of herpes pneumonia observed in our series showed the same radiological pattern of uncomplicated viral pneumonia, which consisted in all cases of diffuse interstitial infiltrates, having a grossly reticular pattern. pleural effusion was absent. fungal pneumonia in solid organ transplantations is less frequent than bacterial and viral (cmv) infections, but it is by far the most severe and it has the highest mortality rate. the two main agents are usually represented by candida or, more rarely, aspergillus. these infections are frequently observed within the first 2 months [14] . the starting point of candida infection is the gastroenteric tract, colonised by this fungal agent in 30±60 % of the normal subjects, and the manipulations at the time of the transplant can cause the diffusion of the pathogens. surgical instrumentation and central catheters are the major source of aspergillus, criptococcus and mucor, which are non-endogenous agents, but are always acquired from the environment. as significant risks of postolt fungal infections, a surgical time > 12 h, intratransplant transfusional requirements, bacterial infections within the first 2 months after olt together with prolonged systemic antibiotic therapy, reinterventions and high dose immunosuppression have been cited [4, 12, 34] . accordingly, a significant correlation has been observed in our population between okt3 immunosuppression and fungal pneumonia. the incidence of candida infections in the different olt series varies from 11 to 42 % according to the type of bowel decontamination and antifungal prophylaxis performed. the usual site of infection is abdominal. the mortality rate from fungal infection after olt ranges from 70 to 90 % and is higher in fungal superinfections of pre-existing infiltrates. accordingly, in the present series, the majority of candida pneumonia superinfected bacterial or viral infections and had a fatal outcome in 70 % of the cases [12, 34] . aspergillus infection most commonly appears as pneumonia or disseminated infection with mortality rates that approach 100 %. the one observed case of aspergillosis in our series was present as an isolated pathogen with lethal pneumonia. the chest radiographic features of fungal infections consisted of multiple nodular opacities with intense alveolar infiltrates, with tendency to central cavitation and air bronchograms or nodules with hazy margins or clusters of fluffy nodules. the nodules of fungal pneumonia tended to be multiple rather than solitary, and a prevalent involvement of the upper lobes is frequently observed. pleural effusion and adenopathy may be seen [20] . in the present study, the radiological pattern of candida infections or superinfections showed multifocal areas of air space consolidations, usually not associated with pleural effusion. in those cases which do not respond to therapy, the nodules may coalesce to larger regions of consolidation and, eventually, to the typical appearance of ards. in aspergillosis the nodules were rapid growing and showed a crescent-shaped area of hyperlucency (ªair crescent signº) representing cavitation around the pre-existing dense central nodule. pneumocystis carinii pneumonia (pcp) in organ transplants has a reported incidence of 0±14 %, depending on the specific prophylaxis adopted [2, 4, 5, 6, 7, 10, 12, 14] . usually pneumocystis pneumonia has a later onset as compared with the other agents, appearing between the third and sixth month: the mortality rate is between 25 and 50 % [4] . it has rarely been observed in olt recipients (5 % of cases) and more often is a coinfection with cmv infiltrates. in this case it is associated with a higher mortality rate [4] . the radiological pattern is similar to viral pneumonia, with smooth interstitial reticular involvement or typically with central and bilateral perihilar linear processes, which progress to a homogeneous diffuse alveolar consolidation. pneumocystis carinii pneumonia sometimes appears with atypical infiltrates (focal and non-diffuse pattern) [4, 13] . the sole cases observed in our series appeared late in the postolt course (twenty-eighth day) and showed a radiological pattern of diffuse interstitial impairment, ªground glassº appearance and mild symptoms (fever). there was a complete and rapid recovery after therapy. previously reported risk factors of pulmonary infections following olt [6, 8, 15, 16, 30] included: poor preoperative unos status; technical quality and duration of the transplant surgery; surgical complications needing major surgery (retransplantation); the type, intensity and duration of the immunosuppressive therapy; infections due to immune-modulating viruses; metabolic disturbances and epidemiological exposure to environmental agents. in a previously published study on the first 100 cases of olt [9] , prolonged icu and amv, advanced unos status, okt3 immunosuppression and pulmonary oedema were identified as highly predictive risk factors for the onset of pulmonary infections. the present study, performed on a wider patient population, demonstrated, in univariate analysis, a significantly increased association of pulmonary infection with the following five risk factors: caval traditional anastomosis, retransplantation, okt3 immunosuppression, icu stay and amv duration, pulmonary non-inflammatory abnormalities, such as effusion and atelectasis, and oedema. in our multivariate analysis, the strongest independent risk factors for pneumonia were non-infectious abnormalities (atelectasis and pleural effusion) and pulmonary oedema, followed by prolonged amv, whereas piggy-back caval anastomosis is shown to be significant as a protection factor, preventing the onset of pneumonia, as compared with traditional caval anastomosis. in our experience, which differs from previous studies [6, 8, 9, 30] , basal liver disease, unos score and graft rejection do not represent statistically significant risk factors for pneumonia. about surgically related risks, traditional caval anastomosis is demonstrated in our series to be more risky than the piggy-back technique. liver transplantation with preservation of the recipient vena cava (the piggyback technique), which avoids retrocaval dissection [23] , reduces overall time of surgery, need of blood transfusions and postoperative renal failure, with earlier extubation, shorter icu and total hospital stay. due to these advantages, the follow-up of olts performed with the piggy-back technique showed, in the present study, non-infectious pulmonary abnormalities of a less severe degree, thus resulting in a significantly lower rate of pneumonia as compared with traditional caval anastomosis and a significantly reduced overall mortality rate. in multivariate analysis the piggy-back technique represents a significant factor preventing postoperative pneumonia and mortality. in accordance with the studies of the mayo clinic and other authors [6, 7] , our results confirm a significantly higher percentage of major infections after retransplantation than in a single graft. retransplant also represents a significant risk of mortality. anti-t-cell antibody (okt3) immunosuppressive therapy has previously been described as a strong risk factor for serious infection in the weeks following treatment [31, 35] . a significantly higher incidence of severe pneumonia with fatal outcome in the majority of cases has been also confirmed in our series. a further serious complication of okt3 is the onset of acute pulmonary oedema during the first 2 days of treatment, which could predispose to superinfections. this usually appears in patients already overloaded with fluid prior to treatment. in 21 of 30 cases in our series, diffuse interstitial oedema was evident, with a slight increase in vpw: clear alveolar oedema was never observed due to diuretics routinely administered in the days before the injection of okt3. a prolonged stay in icu has been reported as the only significant risk factor for the onset of infections in liver graft recipients [6] . our statistical univariate analysis confirms this as one of the major risk factors for infection: 27 of 41 infections in our series (65.9 %) developed in icu, and in 20 cases during amv (48.8 %). multivariate analysis, instead, demonstrates that the icu stay risk in the global population is paradoxically ªa protectionº against infection and mortality, and that it is only indirectly significant because it is related to the real ªmajorº risk for pneumonia and related mortality, represented by prolonged intubation and amv. protracted amv has been previously identified as a risk factor for developing nosocomial infection [15] . the high percentage of pseudomonas pneumonia observed in the present series (41.5 %) was directly related to a longer duration of amv during the icu stay. prolonged intubation and amv were shown to be, in multivariate analysis, the greatest independent risk factors for mortality. among the variables studied, the pre-existing pulmonary abnormalities ± pleural effusion, atelectasis and pulmonary oedema ± were shown to be the greatest independent predictors of pneumonia in our olt population. persistent effusion, atelectasis and pulmonary oedema triple the risk of developing infectious complications. the vast majority of infections (37 of 41) were ipsilateral and superimposed on these previously non-infectious lesions, which constitute ªlocus minoris resistentiaeº especially in nosocomial bacterial infections (pseudomonas, staphylococcus). pulmonary oedema was demonstrated, in multivariate analysis, to be the second most important predictor for mortality. by associating the selected major risk factors for pneumonia and mortality, piggy-back anastomosis, prolonged amv and icu stay, pulmonary non-inflammatory abnormalities and pulmonary oedema, we defined a pneumonia-risk score in order to identify which patients needed closer cxr and clinical screening for pneumonia. the cumulative 1-year incidence of pneumonia was 28.7 % in the high-risk score, 10.3 % in the medium-risk score, and 2.7 % in the low-risk score. the time course of the appearance of pneumonia confirms that the majority of infections had their onset within the second month, but the time of onset is more protracted as the risk score increases. in low-risk patients, all pneumonia appeared by the twentieth day after olt, whereas the pneumonia risk period is prolonged in the high-risk group, in which a higher percentage of pneumonia can appear after the first month. clinical and radiological follow-up and intensified preventive measures should therefore be prolonged according to the risk score, in order to reduce morbidity and, consequently, mortality. previously published data [7, 12] demonstrated that infections are the leading causes of death after olt. our study confirms that in all fatal infections the lung was the primary site of infection, as shown by the close correlation between pneumoniarisk score and survival: the expected 5-year survival rate differs significantly in low, medium and high pneumonia-risk populations, being 84.1, 74.3 and 60.3 %, respectively. as previously reported [20] , fungi constituted the pathogenic group most highly associated with mortality within 30 days of diagnosis (mortality 70 %) followed by bacteria (27 %) and cmv (25 %). in conclusion, in olt recipients, as in all immunocompromised patients, prophylaxis, when possible, per-sistent infection surveillance and an aggressive diagnostic and therapeutic approach help to reduce the potentially fatal impact of pulmonary abnormalities. bacteria were the most frequent (63.4 %) pathogenic group in our series, rarely fatal, followed by cmv (29.3 %) and fungi (24.4 %), the latter being associated with higher mortality rate. during the posttransplant course these agents show a predictable time of onset ± bacterial infections prevailing during the first 2 weeks, fungal agents later (median 18 days) and viral (mainly cmv) and p. carinii pneumonia appearing last. this knowledge could be an important aid to the radiological diagnosis in indicating the nature of the infiltrate and, therefore, in predisposing therapy, thus influencing the final clinical outcome. upon radiographic cxr features of the different agents, a fairly good correlation was found between the radiological cxr pattern and the microorganism, with an air-space consolidation pattern in bacterial, a nodular or focal consolidation pattern in fungal and a reticular interstitial pattern prevalent in viral and pcp pneumonia, with the only exception being cmv which could appear as focal alveolar consolidation in a minority. our study demonstrated three main independent risk factors for developing posttransplant pneumonia: prolonged amv/icu stay, pulmonary oedema and non-infectious abnormalities such as atelectasis and pleural effusion. the knowledge of the probability of the onset of infections based on the calculated pneumonia-risk score identifies the high-risk patient population, in whom closer, even daily radiographic monitoring is justified, in the early posttransplantation period, in order to control the pneumonia-risk factors. persistent or severe non-infectious pulmonary abnormalities, such as atelectasis and pleural effusions, triple the risk of pneumonia onset and therefore daily cxr monitoring is mandatory. pulmonary oedema, more frequently due to overhydration, should be surveyed and quantified by close cxr and clinically prevented, reducing postoperative water overload. furthermore, olt recipients having a high pneumonia-risk score are particularly at risk, since our data also confirms that pulmonary infections remain the leading cause of death after liver transplantation, as demonstrated by the close correlation between the pneumonia-risk score and mortality in the present series. very few studies have been done on the extensive employment of ct in early posttransplant follow-up. a recent report on ct studies of pulmonary complications after lung transplant [17] , in which a comparison with histopathological studies has been carried out, demonstrated that ct findings were not helpful in differentiating between the different parenchymal complications. when matched with proper clinical data, close cxr monitoring could be of value in orienting the diagnosis of the different pulmonary abnormalities which complicate the postoperative course after olt. a precise knowledge of the probability of the onset of the different opportunistic agents of pneumonia which could superimpose on pulmonary non-infectious abnormalities at specific phases of the posttransplant course together with the consciousness of the differential risk of infection according to the calculated pneumonia-risk score is mandatory for whomever is involved with liver transplantation. infections complicating orthotopic liver transplantation early infections in kidney, heart, and liver transplant recipients on cyclosporine karchmer aw (1986) pulmonary complications of orthotopic liver transplantation infections after liver transplantation. an analysis of 100 consecutive cases pulmonary complication and disease severity in adult liver transplant recipients infectious complications in liver transplantation ) incidence, distribution and outcome of episodes of infection in 100 orthotopic liver transplantations early severe infections after liver transplantation pulmonary complications following orthotopic liver transplant: radiologic appearances and epidemiologic considerations in a series of 100 cases pulmonary infections in liver transplant recipients receiving tacrolimus. changing pattern of microbial etiologies pulmonary complications following orthotopic liver transplant pulmonary complications of orthotopic liver transplantation pulmonary infections in the immunocompromised host pathologies pulmonaires dans la transplantation cardiaque, høpatique et rønale chez l'adulte state of the art: pulmonary considerations of organ transplantation infection in the organ transplant recipient diagnosis of pulmonary complications associated with lung transplantation in children: value of ct vs histopathologic studies infectious complications following isolated lung transplantation pulmonary complications after bone marrow transplantation pulmonary infections after bone marrow transplantation: clinical and radiographic findings the unos optn waiting list and donor registry: 1988±1996 factors in the development of liver transplantation orthotopic liver transplantation with preservation of the inferior vena cava liver allograft rejection: an analysis of the use of biopsy in determining the outcome of rejection glossary of terms for thoracic radiology: recommendations of the nomenclature committee of the fleishner society regression models and life tables bmdp statistical software manual: to accompany the 1990 software release major liver resection: perioperative course and management the boston center for liver transplantation cmvig study group (1996) incidence and predictors of cytomegalovirus pneumonia in orthotopic liver transplant recipients a randomized trial comparing okt3 and steroids for treatment of hepatic allograft rejection cmv reinfection/reactivation after liver transplantation cytomegalovirus pneumonitis and lobar consolidation risk factors of invasive candida and non-candida fungal infections after liver transplantation pulmonary complications from monoclonal antibody (okt3) immunosuppresion in patients who have undergone an orthotopic liver transplant key: cord-017815-0t7jvvz5 authors: andersen, bjørg marit title: general information date: 2018-09-25 journal: prevention and control of infections in hospitals doi: 10.1007/978-3-319-99921-0_15 sha: doc_id: 17815 cord_uid: 0t7jvvz5 many bacteria, viruses, parasites, fungi and prions may cause serious infections and lead to the isolation of those who are infected from those who are susceptible. isolation may be done in single rooms or in special isolation units. a modern isolate for patients with infections comprises (1) a sluice with a good space for dressing and undressing of personal protective equipment (ppe) and for hand hygiene, (2) a large patient room and (3) a bathroom/disinfection room with own decontaminator or autoclave and with separate entrance from the patient’s room. isolates for airborne and droplet-transmitted infections have in addition a defined negative air pressure and hepafiltered exhaust. in all isolates, doors must be closed in such a way that contaminants do not escape the isolate. a modern isolate for patients with impaired immune defence is similar to the infection isolates, with following exceptions: usually no need for decontaminator, hepafiltered clean air into the room and with a defined positive air pressure. a positive pressure isolate should never be used for patients with infections, and a negative pressure isolate should never be used for patients with impaired immune defence, except if the patient also has an infection that needs isolation. to prevent transmission from an infectious patient to other patients, personnel, visitors and the environment and to protect patients with impaired immune defence against infection [1] [2] [3] [4] [5] [6] [7] [8] . • all patients having contagious disease that can easily be transferred directly or indirectly via contact, blood and body fluids, air/droplets or via equipment, textiles and surfaces. • all patients with significant reduced infection defence or otherwise infection vulnerable and who should be protected against infection. hospital management should ensure necessary capacity and type of isolating units: contact-and air-droplet isolates and protective isolates. updated isolation routines, adequate protective equipment-including ppe-routines for disinfection of rooms and surfaces and disinfectants and hand hygiene facilities should be available. department management should implement isolation procedures, train the use of ppe, control the use of routines and provide sufficient stock and capacity of ppe and means for disinfection and hand hygiene. the staff should follow current guidelines for treatment of patients with infections and for patients that should be extra protected against infections. the need for isolates varies with type of hospital activity, epidemic and endemic conditions and the condition of the hospital [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] . single rooms with separate bathroom and anteroom prevent the spread of infection more than single rooms and multiple bedrooms with shared toilet and bathrooms. modern isolates, well designed, ensure other patients and personnel against infection, streamline hospital activity and simplify work [21, 22, [25] [26] [27] . updated status should be available as to the type (infections or protective) and quality of isolates and single rooms-by national and county health authorities. an overview of the number and types of infection isolates in a radius of at least 10 mils around densely populated areas should be available, and collaboration should be established between the hospitals. isolates should be checked at least once a year. contact isolate should have a sluice system (priority) and an own disinfection room with decontaminator. an air-droplet isolate should, in addition, have a defined negative air pressure in the rooms and separate ventilation. protective isolates should have hepafiltered, positive air pressure. it is also important to have overview over single rooms with separate bathrooms-which can be used for contact isolation. hospital buildings or other healthcare facilities that can be quickly converted into cohort isolates for defined types of outbreaks like the pandemic influenza should be appointed to the emergency. different measures may be done relatively to preparedness whether there is a normal epidemiological situation or if it is smaller or larger outbreaks in the community that also affects the hospitals. normal epidemiological condition: in a developed country, like norway, 20-25% of hospitalized patients have one or more infections, including hospital-associated infections (hai); 20-30% receive antibacterial agents, and approximately 20% need a minimum-size single room with separate bathroom, due to infection [21, 22, [28] [29] [30] . at least 10% of the hospital infections are transmitted via air-droplets. local outbreaks, often seasonal, may double isolate needs: this happens nearly every year concerning norovirus, rotavirus, influenza, metapneumovirus and rsv (toddlers and the elderly) and is an increasing problem concerning resistant bacteria from home and abroad (cd, mrsa, vre, esbl, etc.). large outbreaks, more difficult to control, such as the norovirus, may be organized by cohort isolation of patients with the same type of infection [21, 22] . closing departments and hospitals happens occasionally during greater nosocomial outbreaks of norovirus or influenza when many are sick at the same time, both patients and personnel. the intake of new patients is often discontinued during norovirus outbreaks; the ward may be emptied and disinfected thoroughly before opening it again [21, 22] . a study of 194 nosocomial outbreaks in medical departments that ended up closing the ward showed that it took a long time before the unit was reopened, median 14 days [16] . influenza pandemics and other serious pandemics: in the case of pandemic, up to 0.3% of the population will require hospitalization, and approximately 13% need outpatient treatment [1, 31] . capacity and readiness for escalation should be calculated and included in preparedness plans for countries and counties [30] [31] [32] [33] [34] contact isolation: 25-30% of the bed capacity of surgical, paediatric, postoperative department and intensive care unit, preferably with some negative pressure and separate ventilation. in the case of special wards for patients with infections, 50-75% of the capacity should be contact isolates and the rest airborne infection isolates. at all other clinical wards, the minimum is one contact isolate per ward. airborne infection isolation units: at least 10% of the bed capacity for adults and 10-15% of children in hospitals should be isolates well equipped with negative pressure, separate ventilation and private bath/disinfection room with decontaminator. there should be minimum one isolate per 30 beds. contagious and severe infections like tuberculosis, pandemic influenza, sars, mers, ebola, lassa and other haemorrhagic viral infections should not have shared ventilation with other patient rooms, service rooms, etc. [12, 21, 22, [30] [31] [32] [33] [34] . single rooms can be used as for contact isolation if separate bathroom. all rooms with the same air pressure as the rest of the ward may result in risk of airborne transmission. single rooms may only be used an emergency solution for common infectious diseases. there are many who need single bedrooms in hospitals, not only for infectious diseases. lack of isolation beds may cause inefficient hospital activity and involves risk of spread of infections [21, 22-24, 28, 29, 35-37] . protective isolation protects the patient from infection. this patient group is highly immunosuppressed, vulnerable and/or transplanted. the isolate is on positive, hepafiltered air pressure compared to the corridor and neighbouring rooms. it has its own, separate bathroom [21, 22] . the number of isolates may be in accordance with the hospital activity concerning this type of patients. nb! a positive pressure isolate should never be used for patients with infections, and a negative pressure isolate should never be converted and used for patients with impaired immune defence. this is due to the high risk of cross infection from microbes in filters and ventilation ducts. patients: the usual need of isolates for the population is covered if-in additionuse of isolates at other hospitals in the area, flexible. most outbreaks are related to a few patients, one to five [30] . close contacts/exposed to infection/disease carriers: a short isolation period may be actual if the index case has diphtheria (adults, elderly), plague, ebola and possibly anthrax and in some cases where infection-prone person or carrier cannot take care of himself/follow preventive measures. information and training are important [19] . everyone must implement appropriate routines in connection with the isolation. do not forget the information to other departments (including requisitions)! contact isolation: suspected or detected transmittable human pathogenic microbes by contact with body fluids, skin and mucous membranes, textiles, clothing, furniture and contaminated equipment (telephone, computer, keyboard, blood pressure set, tourniquet, stethoscope, temperature measure, etc.) • use: gloves and gown. • surgical cap, surgical mask and eye protection/visor at risk of splashing of blood/ liquid and aerosols. • yellow sign on the door. blood contamination isolation: suspected or detected transmittable microbes like hepatitis viruses, hiv and other pathogens. all biological materials can be infectious, which blood and blood products are the dominant carriers of the pathogen. • use: gloves and gown/protective clothing on direct contact with blood/body fluids. • surgical cap, surgical mask and eye protection/visor at risk of splashing of blood/ liquid and aerosols. • the patient may be placed on ordinary patient rooms, if not unrestricted or uncontrolled secretion/excretion of tissue fluids. airborne isolation: suspected human pathogenic microbes transmitted through air, dust particles, aerosols, droplets and droplet nuclei. there is always simultaneous contact contamination. air and contact isolation with satisfactory negative pressure, a proper sluice system and a separate bathroom/decontamination room the isolation unit has a defined, negative air pressure (in pascal), separate ventilation with disinfection of air extraction, a properly interlock function and also direct access from the outside. the waste water is decontaminated (autoclave). the patient room has sluice systems and bathroom with throughput decontaminator/ autoclave with direct entrance from the patient room. • red sign on the door isolation may be necessary for patients with infections to prevent others from getting the same infection. this does not mean that the patient is particularly in a bad condition but that some bacteria or viruses can spread, for example, by coughing, secrets, blood, faeces or urine. in order to prevent spread of infection, the ward needs participation of relatives and other visitors. the patient must be informed of the following: 1. the patient's room cannot be left without consultation with a nurse or doctor. use the ring bell. 2. the bedpan and urine bottle must be used if the patient room does not have a separate toilet. 3. avoid touching wounds, bandages or tubes. 4. pus or blood/secret on the bandage, in bed linens, or loosened dressing; notify the nurse. 5. good hand hygiene is important. always wash your hands after toilet visits; after using the bedpan or urine bottle; after any touch of wounds, bandages or tubes; before eating; before leaving the room; and when you return to the room. 6. use clothes that can be washed at higher temperatures (>65 °c); preferably use the hospital textiles. 1. contact the nurse for the necessary precautions before entering the patient room. 2. follow routines for personal use of infection control equipment-you will be notified by a responsible nurse. 3. it may be necessary to reduce the number of visitors. nurses will report this. small children should not enter the room without special agreement with the responsible nurse. 4. disinfect your hands before entering the isolate. 5. do not sit on the bedside. use a chair by the bed. 6. do not eat or drink in the patient's room. 7. do not use the ward's kitchen or refrigerator or serve on the corridor, etc. 8. there is no access to the ward's service room (disinfection room, textile room, storeroom, etc.). 9. leave the department immediately after visit-avoid living rooms, etc. 10. thoroughly wash your hands before leaving the isolate (sink in the sluice), even if you only need a short errand outside. 11. close all doors after you in a calm and careful manner as you walk in and out of the isolate. • dedicated contacts among nurses that the patient accept/like. there should be more contacts so that someone is always present. interpreting may be used if language problems. • visit the patient unasked. it is better with frequent, short visits than one long. • good information and update with regard to the patient's illness is important [38] . • use a flexible visit time. check that visitors understand, accept and follow the protective routines and other advices from healthcare professionals. • private food to the patient is not allowed, possibly delivered directly to the department by appointment in special situations. • good aids are tv, video, radio, telephone, data and papers that are appropriate for the patient. • isolates should not have curtains, but washable posters, etc. can be used on the wall. • large windows and a good daylight are important. • the room should be clean, neat and well maintained. • isolation involves no more depression or fear than what the patient has upon admission [39] . • isolation does not involve a poorer patient care experienced by the patient, quite the contrary [40] [41] [42] [43] . • close all doors behind you in a calm and cautious manner when going in and out of the isolate in order not to create unfortunate air currents [44] . contact isolation: gloves and disposable infection gown with cuffs ( fig. 15.1) . when certain agents are suspected (norovirus, cd, mrsa, etc.), or gastroenteritis, respiratory tract symptoms etc., use in addition surgical mask, cap, and eventually visor, room-bound shoes/shoe covers. in the sluice, clean off the shoes on a "cloth" with chloramine 5% outside the door to get rid of the agents if not using shoe covers. replace at each shift. used especially during cd isolation. blood contamination isolation: gloves and disposable infection gown with cuffs by direct contact with blood/body fluid-and double gloving when sharp procedures. airborne isolation: surgical mask or respirator, cap, gloves and disposable infection gown with cuffs. strict isolation: respiratory protection, surgical cap and hood and disposable fluid-resistant gown with long arms and cuff or overall with hood, boots or shoe covers, double gloving. protective isolation: good hand hygiene, clean gown, surgical mask, surgical cap, room-bound shoes. a clean, not used ppe can be taken on in the sluice or on the corridor outside the sluice. a used, contaminated ppe is taken off in the patient sluice, nearby the door of the patient room and is placed directly in an infection waste container. if there is no sluice present, the ppe is removed inside the patient room-at the exit door. nb! never take off used ppe in the hallway or corridor-except if a separate sluice is made here. • surgical cap shall always be used when the surgical mask or respirator mask is used (by airborne infection, strict isolation and when protective isolation). the surgical cap makes the mask easier to fasten on the head and removes hair from the face and neck. covering ears and all the hair may protect against exposure to microbes when the patient coughs or sneezes. • surgical masks (gastroenteritis virus, mrsa, respiratory infections/droplets and protective isolation; see isolation procedures). the mask is attached to the back of the head-one strap-and in the neck, the other strap. make sure that the mask is properly fitted over the nose. do not use a mask that attaches to the ears. • respiratory protection mask instead of a surgical mask (severe airborne infections, tuberculosis and strict isolation-see isolation procedures) was attached to the back of the head-one strap/tie-and in the neck, the other strap/tie. check that respiratory protection is properly fitted over the nose and around the mouth; check the leak test. • gown (for contact, blood contamination, airborne infection, strict isolation), waterproof, long and with good cuffs, tightly around the neck, preferably single use gown that is placed in the infection waste bag after use. nb the gown is tied on the back-not tied on the front. • gloves with long cuffs-(for contact, contaminated blood, airborne infection, strict isolation) solid and comfortable. double gloves at high risk of transmission and blood-borne infection. • shoe cover-room-bound shoes, boots, overdrafts, etc. (norovirus, c. difficile, vomiting, diarrhoea, high pollution in the room, strict isolation). [21, 22] • shoe protection, overdrafts, etc. if used, disinfect gloves (outside) before carefully taking off shoes/cover and place them in infection waste bag or waste container for shoes that can be autoclaved/disinfected. put a doffed foot on a clean area in the sluice or directly in your own shoes. • hand hygiene-disinfect the outside of the glove. • gloves; grab the left glove at the wrist (not higher up) and gently turn inside out when doffing. -the inside of this glove is usually clean and can be used as a "cloth" placed on the right hand to grab through to remove the other glove. -hand hygiene. -grab the cuffs (clean because the gloves have covered them) and pull successively the cuffs over the wrists. -do the rest of the doffing from inside the gown; arms off and carefully roll the entire coat away from both sides to the middle and then roll downwards-as "packing in" the contaminants of the gown-learn the technique. place the used gown carefully in the waste container. • hand hygiene. • goggles: if used, take careful behind the head and bend forward, so the goggles do not come into contact with skin, hair or textiles-put in container for decontamination if using reusable goggles. • hand hygiene. • surgical mask/respirator mask; do not touch the front of the mask directly-be careful behind the head and neck. bend forward and gently tilt so that the front of the mask does not come into contact with skin, hair or textiles. loosen the band in the neck first and then on the head (the mask should not fall on the chest). carefully place it in the waste bag. • hand hygiene. • cap or hood; grab the cap carefully back on the head and gently remove it. bend forward to avoid contact with skin, hair or textiles. the cap is placed in the waste bag. • hand hygiene. nb! in the case of airborne isolation and strict isolation, it is never appropriate to remove the ppe before going out from the patient's room, in the sluice, and the door to the patient's room should be closed. this applies only to an established sluice function. if this function is missing, approved alternatives may be available, for example, a separate and enclosed corridor defined for this use. contact infection control personnel. • take care with the movements during doffing to not release contaminants from the ppe into the environment. • take care when opening and closing doors to avoid unfortunate airflows [44] . • multi-use of gowns where the same gown can be used for several persons entering the patient's room is not recommended because of problems with cross-contamination. if still in use, it must be properly hang up after use; with the outside of the gown turned out-in the patient room (at the door)-and the outside of the gown turned in-in the sluice. gowns that are hang up incorrectly or are dirty or wet should be carefully put into the bag for contaminated textiles. hand hygiene is done before taking on a new gown. the multi-use gown is changed for each shift or more often when contaminated. never enter the corridor with a gown used for protection. isolation regimens should not be a hindrance for-but included in-diagnostics and treatment. see chap. 21. handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control no. 55 of protection against infectious diseases regulations in infection control in health facilities -hospital infections, established by the health and social affairs action plan for infection control in norwegian hospitals, health directorate's guidance series 2-92. directorate of health use of isolation to prevent spread of infection in hospitals. health directorate's guidance series: directorate of health on the protection of workers from the risks related to exposure to biological agents at work cdc draft guideline for isolation precautions in hospital guideline for isolation precautions in hospitals cdc draft guideline for preventing the transmission of mycobacterium tuberculosis in health care facilities the use of adult isolation facilities in a uk infectious disease unit an audit of the use of isolation facilities in a uk national health service trust risk factors for the isolation of multi-drug-resistant acinetobacter baumannii and pseudomonas aeruginosa: a systematic review of the literature closure of medical departments during nosocomial outbreak: data from systematic analysis of the literature cdc management of multidrug-resistant organisms in healthcare settings prevention of transmission of multidrug resistant organisms guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings strategies two prevent methicillin-resistant staphylococcus aureus transmission and infection in acute care hospitals: 2014 update variability of contact precaution policies in us emergency departments in: handbook of hygiene and infection control in hospitals. oslo: ullevål university hospital in: handbook of hygiene and infection control in hospitals. part 2. practice and theory. moss: elefantus forlag a predicted outbreak in an overcrowded, administratively neglected and run-down haemodialysis unit as an offer of "new public management" in norwegian hospitals spread of methicillin-resistant staphylococcus aureus in a neonatal intensive unit associated with understaffing, overcrowding and mixing of patients the design of isolation rooms hospital and community acquired infection and the built environment-design and testing of infection control rooms evaluation of the contribution of isolation precautions in prevention and control of multi-resistant bacteria in a teaching hospital a three-year survey of nosocomial and community-acquired infections, antibiotic treatment and re-hospitalization in a norwegian health region hospital-acquired infections before and after healthcare reorganization in a tertiary university hospital in norway isolation of dangerous infections. in: handbook of hygiene and infection control in hospitals. oslo: ullevål university hospital scenario pandemic influenza and pandemic fulgeinfluensa avian influenza with pandemic potential handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control hospital infections at ullevål hospital. occurrence and additional expenses a norwegian nosocomial outbreak of methicillin-resistant staphylococcus aureus resistant to fusidic acid and susceptible two other anti-staphylococcal agents a multi-drug resistant, methicillin-susceptible strain of staphylococcus aureus from a neonatal intensive care unit in oslo psychological effects of source isolation nursing (2) patient satisfactory depression, anxiety, and moods of hospitalized patients under contact precaution care of children isolated for infection control: a prospective observational cohort study adverse outcomes associated with contactless precautions: a review of the literature contact isolation for infection control in hospitalized patients: is patients satisfaction affected? adverse effects of isolation in hospitalized patients: a systematic review door-opening motion can potentially lead to a transient breakdown in negative-pressure isolation conditions: the importance of vorticity and buoyancy airflows key: cord-023168-cd7adns8 authors: thachil, jecko; owusu-ofori, shirley; bates, imelda title: haematological diseases in the tropics date: 2013-10-21 journal: manson's tropical infectious diseases doi: 10.1016/b978-0-7020-5101-2.00066-2 sha: doc_id: 23168 cord_uid: cd7adns8 nan haematological disorders are common in low-income countries. they make a substantial contribution to morbidity and mortality of individuals in these regions and have a negative impact on the growth and development of under-resourced nations. genetic red cells abnormalities are common in lowincome countries because they provide protection against malaria and they often co-exist with other causes of anaemia such as malnutrition and chronic illnesses. there is a close association between haematological abnormalities and infections which are a major cause of illness and death in these populations. morphological abnormalities of blood can often provide clues about the underlying diagnosis and blood film examination is particularly important where diagnostic facilities are limited. abnormal blood counts can manifest as various combinations of alterations of numbers of red cells, white cells or platelets. this section will outline some of the most common causes of abnormal blood counts likely to be encountered in clinical practice in low-income countries. anaemia is one of the most common causes of morbidity in the world and its impact is reflected in several of the health-related millennium development goals. although anaemia by itself is not a diagnosis, it suggests that there is an underlying disease state which needs to be recognized and treated. it is also a useful indicator of the general health of the population. the causes of anaemia may be identified systematically by considering the life cycle of the red cells (figure 65 .1). nutrients necessary for red cell production are absorbed from the gastrointestinal tract and carried through the portal vein to the liver and ultimately reach the bone marrow where erythropoiesis occurs. this process is regulated by erythropoietin, a hormone released from the kidneys mainly in response to hypoxia. mature • africa and asia have more than 85% of the world's anaemic populations and anaemia burden is highest among children and women of reproductive age. • the accurate diagnosis of anaemia has been neglected; clinical assessment of anaemia is unreliable unless the anaemia is severe. • in low-income countries, anaemia in an individual is often due to multiple interdependent factors. removing or treating a single factor may not resolve the anaemia. • early diagnosis of sickle cell disease and rapid access to a specialist centre for emergencies such as severe pain crises, strokes and acute chest syndrome, can help to prevent permanent long-term complications. • beta-thalassaemia major is fatal in the first few years of life unless regular blood transfusions are given; unless they are accompanied by iron chelation, these transfusions will eventually cause death due to irreversible organ damage from iron overload. • malarial anaemia is a particular problem for children and pregnant women and severe anaemia can be caused by p. falciparum and p. vivax. malarial anaemia can be reduced with chemoprophylaxis and intermittent treatment, and by anti-mosquito measures such as insecticidetreated bed nets and vector control. • anaemia occurs in 70% of hiv-infected patients and is an independent risk factor for death. prompt treatment of factors associated with anaemia, such as infections and poor nutrition, and commencement of antiretroviral treatment will reduce deaths. • blood shortages are common in tropical countries. to increase the availability of blood, transfusions should be prescribed in accordance with guidelines and efforts made to encourage blood donors to donate regularly as repeat donors are the safest type of donor. reaction' , characterized by circulating myelocytes and metamyelocytes, can be mistaken for leukaemia but, unlike leukaemia, there is an orderly maturation and proliferation of neutrophils. leukaemoid reactions have also been described in patients with tuberculosis, juvenile rheumatoid arthritis and dermatitis herpetiformis. 2, 3 decreased margination of neutrophils with egress of cells into the circulation can occur with exercise, adrenaline (epinephrine) injection, emotional stress and postoperatively or in response to drugs (e.g. steroids, β-agonists). other drugs, such as lithium and tetracycline, produce neutrophilia through increased production. neutrophilia is also a feature of bone marrow proliferation which occurs in myeloproliferative neoplasms, particularly chronic myeloid leukemia and myelofibrosis. teardrop cells and nucleated red blood cells are features of myelofibrosis on the blood film; basophilia and eosinophilia are common with chronic myeloid leukaemia. molecular testing for the jak-2 mutation or bcr-abl fusion gene can also help to differentiate between myeloproliferative neoplasms. rebound neutrophilia can occur following treatment of megaloblastic anaemia or after recovery from neutropenia induced by drugs. acute haemorrhage can cause neutrophilia, especially if bleeding occurs into the peritoneal cavity, pleural space, joints or adjacent to the dura. this is possibly due to the release of adrenaline and chemokines in response to local inflammation. the presence of neutrophilia can be useful in raising suspicions about the onset of complications in infections that are not primarily associated with neutrophilia. examples include meningitis in tuberculosis, orchitis in mumps, bowel perforation in typhoid fever and superadded bacterial infection in measles. the absence of neutrophilia can be helpful in differentiating typhoid and paratyphoid fever from pyogenic infections. neutropenia is defined as an absolute neutrophil count <1.5 × 10 9 /l. it is usually classified into severe (<0.5 × 10 9 /l), moderate red cells are released into the circulation from the bone marrow and percolate through the tissues and organs. anaemia can result from defects in any of these stages. inadequate production of red cells in the bone marrow can be due to lack of nutrients (e.g. iron, b 12 , folate, vitamin a, copper or zinc), abnormal haemoglobin synthesis (i.e. haemoglobinopathies) or ineffective erythropoeisis from myelodysplasia or infections. red cells can be lost from the body (e.g. gastrointestinal bleeding) or removed prematurely if they are abnormal or the spleen is enlarged (i.e. haemolysis). kidney disease can result in decreased erythropoietin. anaemia of chronic disease (or 'anaemia of inflammation') is due to an inadequate response to erythropoieitin or to increased cytokine-induced hepcidin release in inflammatory states which interferes with iron absorption or iron utilization. diagnostic algorithms to determine the cause of anaemia are usually based on a combination of the mean cell volume of the red cells, the reticulocyte count and blood film appearance (figures 65.2, 65.3 ). this approach is based on the availability of a haematology analyser and an experienced microscopist. several conditions which cause anaemia may co-exist in the same individual (e.g. intestinal parasites, malaria and sickle cell disease) and hence a thorough investigation is crucial to identify all potential causes of anaemia. neutrophils released from the marrow after maturation can either enter the 'circulating pool' or they can remain in the 'marginal pool' where they are loosely attached to the blood vessel wall. cells in the marginal pool are not sampled when blood is taken for a full blood count. 1 neutrophilia can therefore result from increased bone marrow synthesis and also from decreased margination which increases the circulating pool. there are many causes of neutrophilia (box 65.1) but the commonest is bacterial infection in which there is increased bone marrow production of neutrophils and release of neutrophil precursors into the peripheral blood. this 'leukaemoid (0.5-1.0 × 10 9 /l) or mild (1.0-1.5 × 10 9 /l). the propensity to develop infections is related to the degree and duration of neutropenia, with higher risk associated with counts below 0.5 × 10 9 /l. africans, african americans, yemenite jews, palestinians and saudi arabians generally have slightly lower neutrophil counts compared with other races. this is thought to be due to an increase in the bone marrow storage pool as ethnic neutropenia is associated with good neutrophil responses to infections. neutropenia can be due to impaired or ineffective (intramedullary death of neutrophil precursors despite normal bone marrow production) synthesis by the bone marrow (e.g. myelodysplasia, megaloblastic anaemia, treatment with phenytoin or methotrexate); a shift from the circulating pool to marginated pool (pseudoneutropenia) and increased peripheral destruction (e.g. secondary to antibodies against the neutrophils or increased reticulo-endothelial activity in sepsis or haemophagocytic syndrome) (box 65.2). increased consumption of neutrophils can result from increased attachment of cells to endothelium or other leukocytes in inflammatory states. neutropenia is often the result of a combination of several of these mechanisms. infants of hypertensive mothers may have moderate to severe neutropenia, which can last for several days. this is probably related to bone marrow suppression. moderate to severe neutropenia can also occur in newborn infants as a result of the transfer of maternal igg anti-neutrophil antibodies in a manner similar to rhesus haemolytic disease of the newborn. 4 although neutropenia has been described with typhoid fever, minimum neutrophil count seldom falls below 0.6 × 10 9 /l and the box neutropenia may not develop until after the first week of illness. infectious hepatitis and yellow fever can both cause neutropenia. overwhelming infections can lead to a failure of bone marrow production of neutrophils, especially in undernourished individuals and alcoholics. individuals with severe neutropenia can develop lifethreatening septicaemia, often from endogenous flora (e.g. oral cavity), and stringent measures should be taken to avoid situations which may predispose these individuals to infections. they may need prophylactic antimicrobials and should have rapid access to medical care. fungal infections are less common than bacterial infections in neutropenic individuals, and viral or parasitic infections rarely occur with isolated neutropenia. granulocyte colony stimulating factor (gcsf) injections can be helpful in raising the neutrophil count in patients with complicating infections since it stimulates the release of neutrophils from the marrow, but gcsf is only useful if there is some bone marrow reserve. patients with some congenital or immune forms of neutropenia can tolerate persistently low counts without any increase in the incidence of infections. monocytosis occurs in chronic infections and inflammatory conditions. protozoan infections such as typhus, trypanosomiasis and kala-azar may be associated with monocytosis. chronic and juvenile myelomonocytic leukaemias are malignant disorders in which monocytosis may be severe; acute monocytic leukaemias may present with mild to moderate monocytosis. monocytosis, and particularly a monocyte : lymphocyte ratio greater than 0.8-1.0, may indicate active progression of tuberculosis and an unfavourable prognosis. the normal ratio of 0.3 or less is restored when the healing process is complete. a decreased absolute monocyte count occurs in bone marrow failure states such as aplastic anaemia or after chemotherapy. low monocyte counts can occur with overwhelming sepsis and with splenomegaly. monocytopenia is a characteristic feature of hairy cell leukaemia and is considered to be a diagnostic hallmark of this disease. peripheral blood contains only around 2% of the total body lymphocyte population since these represent the cells present in the blood during their transit into secondary lymphoid organs. wide variations exist in lymphocyte counts between individuals especially in childhood. lymphocyte counts exhibit a diurnal pattern; peaking at night with a nadir in the morning. lymphocytosis is characteristic of infectious mononucleosis and many atypical and large lymphocytes can be seen in the peripheral blood film. these atypical cells can also occur in cytomegalovirus infection and infectious hepatitis. absolute lymphocytosis can occur with chronic infections such as brucellosis and in the recovery stages of tuberculosis. lymphocytosis is unusual in bacterial infections except in the case of pertussis. heavy smoking is also an often overlooked cause of lymphocytosis and is probably one of the commonest reasons for a mild to moderate increase in the lymphocyte count. malignant bone marrow disorders, predominantly acute lymphoblastic and chronic lymphocytic leukaemia and non-hodgkin's lymphomas, can cause lymphocytosis. these lymphocytes may have characteristic morphological changes identifiable in the blood film (e.g. smear cells with chronic lymphocytic leukaemia) and the correct diagnosis can be confirmed by immunophenotyping for specific combinations of cell markers. lymphopenia is due to decreased production, redistribution or increased rate of death of lymphocytes. decreased production usually results from cytotoxic drugs and radiotherapy, while increased lymphocyte death can occur in infections such as influenza and hiv. occasionally, an isolated low lymphocyte count in the context of an otherwise normal full blood count can be a clue to the diagnosis of hiv. this reflects the destruction of cd4+ t cells by the virus although an expansion of cd8+ t cells may raise the total lymphocyte count to normal levels. redistribution rather than depletion of total body lymphocyte numbers occurs with steroid treatment or with endogenous secretion of corticosteroids during acute illnesses due to the retention of lymphocytes in secondary lymphoid organs. eosinophilia eosinophils are involved in innate immunity and hypersensitivity. their number in the circulation is relatively small compared to other leukocytes because they predominantly reside in tissues such as the gut, skin and lungs which are entry points for allergens and infections. the commonest causes of eosinophilia are helminthic infections, atopy and allergic diseases, and adverse drug reactions. less common causes are classified under the umbrella term of hypereosinophilic syndromes (table 65 .1). since parasitic infections are likely to be the commonest cause of eosinophilia in the tropics and in returning travellers, an extensive search for such infections should be undertaken in patients with persistent eosinophilia; initial investigations should be determined by the patient's history of geographical exposure (figure 65.4) . [5] [6] [7] the absolute number of eosinophils in the peripheral blood may not correlate with their tissue distribution or with their potential to cause tissue damage from their granule release. this is because the degree of eosinophilia depends on the extent of tissue invasion and is therefore modest with tapeworms and roundworms resident in the bowel but much higher where invasion occurs, for example with, toxocara canis or filaria. schistosomiasis almost always causes eosinophilia. strongyloides stercoralis has the capacity to remain in the host for decades after initial infection and causes varying degrees of eosinophilia, with or without other symptoms. steroid treatment, which may be necessary in cases of eosinophilic tissue damage, can exacerbate clinical problems in patients with strongyloides infection so this parasitic infestation should be excluded before starting steroids for hypereosinophilia. mild to moderate eosinophilia is common in asthma although a very high count should prompt a search for churg-strauss syndrome or allergic bronchopulmonary aspergillosis. most drugs including penicillins can cause eosinophilia but the diagnosis can only be made by noting recovery when the drug is discontinued. eosinophilia can be a feature of hodgkin's lymphoma. it signifies a more favourable prognosis and may precede the original diagnosis of lymphoma or relapses. in immunocompromised patients, such as those with hiv infection, the finding of eosinophilia may be crucial since the success of antiretroviral treatment may depend on concomitant eradication of parasites. thrombocytopenia is often discovered incidentally in patients during full blood count estimation. a platelet count above 20-30 × 10 9 /l is usually not associated with any symptoms such as bleeding. if clinically evident haemorrhage does occur at counts above this level, other conditions such as coagulation defects, vascular problems or rarely platelet dysfunction should be suspected. although the prime role of platelets is in haemostasis, several other important roles have been recognized in recent years including wound repair, tissue healing, antimicrobicidal properties, lymphangiogenesis, tumour metastasization and maintenance of blood vessel integrity. congenital platelet disorders are often part of a syndrome. patients with wiskott-aldrich syndrome have small platelets in association with eczema and recurrent infections. other congenital platelet disorders, such as myh9-related disorders, can present with deafness or cataracts while skeletal deformities and oculocutaneous albinism are common in other syndromic presentations. blood film morphology can provide important clues about the causes of thrombocytopenia (figure 65 .5). fragmented red cells (schistocytes) increase the possibility of microangiopathic haemolytic anaemia, where an altered vessel wall and fibrin formation in the blood vessels shred the erythrocytes and consume platelets. thrombotic thrombocytopenia purpura, haemolytic uremic syndrome and disseminated intravascular coagulation can all present with thrombocytopenia. dysplastic red or white cells should raise the suspicion of myelodysplasia which can be confirmed by bone marrow examination and cytogenetic analysis. it is important to exclude in vitro platelet agglutination as a cause for apparent thrombocytopenia. this can be an anticoagulant (edta)-dependent phenomenon so a repeat sample should be examined using citrate anticoagulant. rarely, platelet satellitism where the platelets clump round the neutrophils, can cause artefactual thrombocytopenia. anaemia affects nearly two billion people globally with a much higher prevalence in developing countries compared with more wealthy nations (43% vs 9%). 8 the continents of africa (highest prevalence) and asia (greatest absolute burden) account for more than 85% of the anaemic population. anaemia burden is highest among children and women of reproductive age. anaemia contributes to more than 115 000 maternal deaths and 591 000 perinatal deaths globally per year. 9 who have defined anaemia according to various haemoglobin concentrations (table 65. 2) 10 but the appropriateness of these thresholds has been questioned because there are wide variations in haemoglobin concentration among people of different races. 11 the prevalence of anaemia can be a useful indicator of public health status of a nation because: • the prevalence of anaemia is objective and quantifiable • anaemia is a major complication of several infections, including malaria, hiv, tuberculosis, and the neglected tropical diseases, which are among the commonest problems in most tropical countries • the incidence of anaemia changes in a predictable fashion with alterations in disease burden • the prevalence of anaemia can be used to assess whether an intervention has reached the poorest communities. haemoglobin concentration of <90 g/l has been recommended for disease surveillance in high-prevalence countries where changes in haemoglobin are used for monitoring the impact of interventions. 11 anaemia in tropical countries (box 65.3) is often due to infections but chronic health problems, such as diabetes and chronic respiratory disease, and cancer and related complications are increasing as causes partly due to lifestyle changes. the body to compensate for the drop in haemoglobin content. for this reason the haemoglobin level can drop to extremely low levels before symptoms develop. anaemia presents with symptoms such as exertional breathlessness, palpitations and in some cases, syncopal attacks. patients with chronic anaemia may also have a multitude of nonspecific symptoms including poor concentration, decreased work performance and easy exhaustion (table 65. 3). a thorough history and clinical examination may provide clues about the cause of anaemia but further investigations are often necessary to confirm the diagnosis and guide treatment. however, in many resource-poor settings, access to routine biochemical and haematological testing is scarce, so much reliance is placed on clinical examination. the international guidelines for the integrated management of childhood illness recommend that a diagnosis of anaemia in sick children is based on the assessment of palmar pallor. for pregnant women, symptoms of fatigue and dyspnoea, combined with signs of conjunctival and palmar pallor, and increased respiratory rate suggest anaemia. however, making a diagnosis of anaemia based on clinical assessment alone is unreliable unless the anaemia is severe. 12 no specific anatomical site is particularly accurate for the prediction of anaemia 11 though sensitivity may be increased by using multiple sites. 12 most central laboratories in low-income countries have automated haematology analysers and several manual methods exist for assessment of haemoglobin concentration, which are suitable for rural areas where there is no mains electricity (e.g. haemoglobin colour scale; hemocue technique). [13] [14] [15] haemoglobin colour scale 16 principle. the colour of a finger-prick blood sample, soaked into special chromatography paper, is compared with the clinical symptoms and signs of anaemia vary and depend on the cause and the speed of onset. a rapid drop in haemoglobin is much more likely to cause symptoms of anaemia than chronic anaemia. slowly developing anaemia allows time for in many cases, there will be more than one of these conditions coexisting in the same individual. an adequate response to the treatment of anaemia requires management of all the contributory factors. intrauterine growth. thus, low birth weight and prematurity are both associated with iron depletion in the postnatal period. several interventions have been suggested to improve infantile iron deficiency, including: [17] [18] [19] • delayed cord clamping at delivery; the short delay of 2-3 minutes allows a small but important amount of blood to continue to flow to the foetus from the placenta • improvement of infant feeding practices • prevention and treatment of infectious diseases • interventions to prevent low birth weight, such as maternal nutritional supplementation, the control of infections and chronic health problems in pregnancy. anaemia in young children can be due to increased nutrition requirements during periods of rapid growth; these requirements may be up to 10 times higher per kilogram of body weight than that of an adult male. in addition, infant and toddler diets often lack bio-available iron. a case-control study of preschool children in malawi with severe anaemia (haemoglobin concentration, <50 g/l) identified bacteraemia, malaria, hookworm, hiv infections and deficiencies of vitamins a and b 12 as the commonest causes of anaemia. lack of folate and iron were uncommon. in low-income countries multiple interdependent causes of anaemia often operate in one individual so rectifying a single factor is unlikely to make a big impact on resolving anaemia. interventions which are useful in preventing anaemia in younger children include micronutrient supplementation (food fortification), de-worming, prevention and treatment of infectious diseases, school nutrition programmes and community-based nutrition promotion. who defines anaemia of pregnancy as a haemoglobin level less than 110 g/l, or haematocrit less than 33%, at any time during pregnancy. about one-fifth of maternal mortality is attributable to anaemia in pregnancy 20 and anaemia affects nearly half of all pregnant women worldwide. maternal anaemia is associated with many factors that might also be causally associated with mortality including poverty, infections and inadequate health-seeking behaviour. globally, the most important cause of anaemia in pregnancy is iron deficiency although hookworm, malaria, hiv infection, and deficiencies in folate and other micronutrients may contribute. pregnancy-associated complications, including septicaemia, pre-eclampsia and other obstetric problems can precipitate anaemia. 21 it is important to note that a diagnosis of iron deficiency in pregnancy which relies on ferritin measurements may be misleading because of high-quality digital examples of known haemoglobin concentration. the colours are represented in 20 g/l increments from 40 g/l to 140 g/l. this method is inexpensive, does not depend on skilled scientists, is durable in dusty, hot, dry and humid conditions and is probably better than clinical diagnosis for detecting mild and moderate degrees of anaemia. the disadvantages are that it requires specific chromatography paper and good natural light and it cannot detect changes in haemoglobin less than 10 g/l. this is a small battery-or mains-operated machine, which uses a drop of blood in a plastic cuvette to produce a direct read-out of haemoglobin in a few seconds. it is simple to use, produces accurate and consistent results to one decimal place and it has an in-built quality-checking mechanism. the hemocue hb-301 has been specifically designed for tropical conditions and operates in temperatures up to 50°c, in dusty and humid conditions. however the recurrent costs associated with disposable plastic cuvettes mean there is little opportunity for cost-saving with high-volume workloads. the iron status of an infant is directly proportional to its body mass and blood volume, both of which are reflections of the major cause of anaemia in most of these cases is iron deficiency. some of the effects have been described in individuals with iron deficiency without obvious features of anaemia. there are three intervention strategies recommended by who to prevent anaemia in pregnancy: 1. weekly iron and folic acid supplementation in women of reproductive age 22 2. daily iron and folic acid supplementation during pregnancy 3. presumptive treatment of hookworm infection during pregnancy in areas where hookworm infection is known to be endemic. 23 several factors may interfere with the efficacy of these interventions. under-participation in antenatal care may be common due to factors such as geographic distance, low motivation and poor interpersonal skills of health staff, poor quality of supplies and facilities, insufficient supply of iron and folic acid pills and womens' poor understanding about the daily use of supplements, especially in the face of common side effects. 23 in sub-saharan africa, the acute shortage and high turnover of health workers, and lack of time have also been shown to contribute to ineffective antenatal measures for reducing anaemia. 24 interestingly, a study from bangladesh showed that the first 20 pills (whether taken on a daily basis or less frequently) yielded most of the benefit for raising haemoglobin levels, which suggests that currently recommended doses may be higher than necessary to achieve optimal outcomes, except when anaemia is very severe. 25 the global burden of iron deficiency has been estimated from anaemia prevalence surveys, which include many different causes of anaemia so data may be unreliable as they are often not based on proven cases of iron deficiency. who estimates that globally 41% of women and 27% of pre-school children are affected by iron-deficiency anaemia, making it number 15 of selected risk factors for preventable death and disability worldwide. 20 iron deficiency begins in childhood, worsens during adolescence in girls and is aggravated during pregnancy. poor iron stores at birth, low iron content of breast milk and low dietary iron intake throughout infancy and childhood result in high prevalence of anaemia in childhood. anaemia is exacerbated by increased requirements during adolescence and iron loss from menstruation and is often compounded by the lack of adequate nutrition. the situation is worsened by pregnancy when iron requirement is approximately two times higher than in a nonpregnant state. iron deficiency should not be considered a diagnosis but a secondary outcome due to an underlying medical condition. although it may be a physiological response to rapid growth or increased requirements during childhood and pregnancy, it still requires treatment due to potential deleterious consequences. many of the chronic effects of iron deficiency may develop before the clinical and laboratory evidence of anaemia becomes apparent. the biochemical evidence for iron deficiency occurs in several steps. 26 initially, iron stores in the bone marrow are depleted as reflected by a decreased serum ferritin. the total iron-binding capacity then starts to rise, while the serum iron saturation begins to fall before microcytosis and a drop in haemoglobin ensue. there have been attempts to identify this early iron deficiency before anaemia develops in order to improve neurological and psychomotor functions in children and work performance in adults through widespread iron supplementation. however, there are concerns that iron excess may promote infections, especially in malarious areas. a range of laboratory investigations are usually necessary if iron deficiency is suspected (table 65 .4) 27-30 because once the diagnosis is confirmed, a search for the precise cause is necessary. a systematic approach to the investigation of iron deficiency (see below) is required based on an understanding of alterations in the iron absorption and transport cycle. • deficient intake (cow's milk has poor iron content and can cause gut blood loss in some infants) • rare defects of haem biosynthesis and iron transport. iron-deficient individuals may have no symptoms. excessive fatigue and other nonspecific signs of anaemia become more pronounced as anaemia develops. consumption of unusual 'foods' such as ice and paint or 'pica' only occurs in a minority of individuals. physical examination may reveal stomatitis, glossitis, koilonychia (spoon-shaped nails) and hair loss. oesophageal webs have been described in the plummer-vinson syndrome but are rare and may respond to iron replacement. since iron is important in neuromuscular development, several features of anaemia described in table 65 .3 may be related to iron deficiency. treatment of iron deficiency is with dietary modifications and oral or parenteral iron. 31 blood transfusions should be reserved for those with severe symptoms especially if the anaemia developed rapidly. haemoglobin levels alone should not be considered as a criterion for transfusion since very low levels (e.g. 10-30 g/l) may be appropriately treated with oral iron if anaemia has developed slowly. intravenous iron should only be considered in cases of poor response or intolerance to oral iron. cereals, poultry and green leafy vegetables, contain non-haem iron, which is often poorly absorbed. if dietary history suggests a deficiency, diet with foods rich in haem iron, such as red meat or liver should be recommended if social and religious customs and financial status allow, ideally with a drink containing vitamin c to facilitate iron absorption. absorption is also facilitated by taking supplements on an empty stomach although side effects of dyspepsia may not always allow this strategy. heavy tea intake can interfere with iron absorption and should be avoided. multivitamin or dietary supplements containing calcium, zinc or copper can also interfere with iron absorption. absorption may be delayed by tetracyclines, milk and soft drinks. since acid is necessary for iron absorption, antacids may account for a poor response to oral iron. iron is usually prescribed as a daily dose of 150-200 mg of elemental iron, commonly ferrous sulphate, 1 tablet three times daily. the dose in children is 3-6 mg/kg per day split into divided doses. assuming good compliance and absorption, this should result in an increase in haemoglobin within 4 weeks. once the haemoglobin is normalized, iron should be continued for 3 months to replenish the iron stores. the major problem with oral iron is upper gastrointestinal side-effects, which can be dose-dependent. a reduction in the dose or change in the formulation to gluconate or fumarate or even liquid forms, may be successful. liquid iron preparations may stain the teeth and should therefore be taken through a straw. oral iron can also cause constipation or diarrhoea which is not dose-dependent. parenteral iron is best given intravenously because intramuscular iron is painful and has been associated with development of soft tissue sarcomas. 32 high-molecular-weight iron dextran carries a low but significant risk of anaphylaxis, but the newer formulations including low-molecular-weight iron dextran, iron sucrose, ferumoxytol and iron gluconate have minimal risks. vitamin b 12 or cobalamin deficiency is a well-recognized cause of macrocytic anaemia (box 65.5). although some microorganisms can synthesize cobalamin, humans need to obtain this essential vitamin from foods, mainly meat, poultry and dairy products. vitamin b 12 is an essential co-factor in dna synthesis, serving as a co-factor in two key biochemical processes involving methylmalonic acid and homocysteine as precursors. consequently vitamin b 12 deficiency can interfere with dna synthesis. clinical manifestations include haematological (megaloblastic anaemia and pancytopenia), and neuropsychiatric disorders (paraesthesia, peripheral neuropathy, psychosis and dementia) and an increased risk of cardiovascular disease because of hyperhomocystinaemia. [32] [33] [34] a systematic approach to the investigation of vitamin b 12 deficiency requires an understanding of the absorption cycle. 35 ingested vitamin b 12 is broken down in the acidic environment of the stomach. it binds to r-binders in gastric secretions and saliva which stabilize the vitamin b 12 . in the alkaline environment of the small intestine, vitamin b 12 is released from r-binders to bind to intrinsic factor, synthesized in the gastric parietal cells. this vitamin b 12 -intrinsic factor complex is absorbed from the terminal ileum. recently, an alternative absorption system independent of intrinsic factor and the terminal ileum has been postulated which provides a rationale for mean cell volume useful as a diagnostic clue but not confirmatory can also be low in thalassaemia, sideroblastic anaemia and rarely lead poisoning can be falsely normal in the presence of iron deficiency in older people or with coexistent megaloblastic anaemia anaemia of chronic disease can occasionally cause microcytosis serum ferritin the most useful laboratory measure of iron status low value is diagnostic in the presence of anaemia very high values (>100 µg/l) usually exclude iron deficiency' being an acute-phase protein, it increases in inflammatory conditions, and certain malignancies, making it unreliable also increased in tissue damage especially of the liver levels are falsely decreased in vitamin c deficiency and hypothyroidism erythrocyte zinc protoporphyrin an intermediate in haem biosynthesis and elevated concentrations indicate interrupted haem synthesis due to iron deficiency when zinc is incorporated in place of iron can be measured on a drop of blood with a portable haematofluorometer small sample size makes it very useful as a screening test in field surveys, particularly in children, and pregnant women where inflammatory states may not co-exist red cells should be washed before measurement (serum bilirubin and fluorescent compounds like some drugs can give falsely high values) although not often done lead poisoning can give falsely high values rarely acute myeloid leukaemia and sideroblastic anaemia give slightly high values useful in that it is not increased in thalassaemias who recommends normal level >70 µmol/mol haem iron studies serum iron concentration represents the iron entering and leaving the circulation. its range varies widely with age, circadian rhythm, infections and iron ingestion total iron binding capacity measures iron bound to transferrin. raised levels are suggestive of iron deficiency transferrin saturation is the ratio of serum iron and the tibc expressed as a percentage -it is probably more useful in detecting iron overload rather than low levels. sensitive indicator that falls within days of onset of iron-deficiency reduced levels shown to be predictor of iron deficiency especially in the setting of renal insufficiency false normal values can occur when mcv is increased or in thalassaemia serum transferrin receptor it is not increased in inflammatory conditions may be upregulated by increased erythropoiesis (haemolytic diseases) giving falsely high values -serum transferrin receptor to ferritin ratio has been suggested in these cases bone marrow examination with special iron staining (perl's) absence of stainable iron in a sample that contains particles can establish the diagnosis without other laboratory tests a simultaneous control specimen containing stainable iron should also be assessed useful in differentiating from anaemia of chronic disorders or α-thalassaemia or milder forms of thalassaemia can help in identifying the sideroblastic anaemias (ring sideroblasts with perls stain), and some forms of congenital dyserythropoietic anaemia which can also cause microcytosis. an improvement in haemoglobin and clinical symptoms with iron replacement is probably the simplest way to diagnose iron deficiency. peripheral smear may help by demonstrating pencil cells, anisopoikilocytosis and high platelet number in cases of blood loss. the treatment of vitamin b 12 deficiency can be by the oral or parenteral route. 37 increasing evidence suggests that oral supplementation may be adequate even in the presence of malabsorption or pernicious anaemia. 38, 39 the recommended initial oral replacement dosage is 1-2 mg but higher doses may be needed for malabsorption or pernicious anaemia. 40 for patients with severe anaemia and/or neurological disease, daily or alternate day intramuscular injections should be initiated for the first 2-4 weeks before reverting to the maintenance threemonthly dose. reticulocytosis is an early marker of response to treatment and is noticeable within 1-2 weeks. folic acid deficiency causes similar haematological manifestations to vitamin b 12 deficiency though neuropsychiatric manifestations are less common. the ability of nerve tissue to concentrate folate to levels five times greater than those in the plasma has been suggested as a reason for the absence of neuropathy in folate deficiency. folic acid deficiency is associated with fetal neural tube defects, and possibly with an increase in atherosclerosis and arteriovenous thrombosis, dementia and colonic cancer. dietary folic acid is present in the form of polyglutamates, which are converted to folate monoglutamates by the enzyme folate conjugase in the intestinal brush border, prior to absorption. the monoglutamates function as a carbon transporter and are essential for dna biosynthesis. folate is found in green vegetables and fruits and deficiency can result from decreased intake, impaired absorption and increased utilization, although the commonest cause is dietary insufficiency. 41 in some wealthy countries, cereals have been fortified with folic acid to successfully prevent vitamin deficiency. however folate deficiency continues to be a problem in less wealthy countries and particularly among children and pregnant women. 42, 43 exclusive feeding of goat's milk to infants can lead to folate deficiency. other causes include alcoholism, excessive cooking of vegetables, and malabsorption (e.g. abnormalities of the small bowel). increased demand for folic acid occurs in pregnancy because the growing foetus has a high avidity for folate. for this reason, folate supplementation has been widely recognized as an essential part of routine antenatal care to reduce the risks of neural tube defects. high folate utilization also occurs in haemolytic anaemias such as sickle cell disease due to high red cell turnover and exfoliative dermatitis. several drugs, including sulfasalazine, trimethoprim, methotrexate, pyrimethamine and phenytoin, can also interfere with folate metabolism. folate-deficient individuals develop a macrocytic anaemia with peripheral blood and bone marrow findings similar to that found in vitamin b 12 deficiency. 32 diagnosis of folate deficiency is confirmed by the presence of low serum folate. red cell folate levels decrease more slowly than serum levels during the 120-day turnover of the red cells. red cell folate levels may be a better indicator of tissue folate levels than serum folate, although red cell folate can be more expensive and falsely low in vitamin b 12 deficiency. 45, 44 treatment of folate deficiency is with oral folate (5 mg daily) which is sufficient even in malabsorptive states. it is crucial that any co-existing vitamin b 12 deficiency is ruled out before initiating folic acid therapy, otherwise the neurological manifestations of b 12 deficiency may deteriorate rapidly. it is also important increasingly popular oral replacement therapies. once absorbed, vitamin b 12 binds to transcobalamin ii to be transported around the body. the diagnosis of vitamin b 12 deficiency is based on the measurement of serum vitamin levels in a patient with clinical evidence of deficiency. a note of caution is that folic acid deficiency can cause falsely low serum vitamin b 12 levels. diagnostic clues for vitamin b 12 deficiency include marked macrocytosis (often >130 fl), neutrophil nuclear hypersegmentation and oval macrocytes in the peripheral blood film. blood tests may demonstrate increased lactate dehydrogenase and low haptoglobin levels due to haemolysis within the bone marrow. the cause of the macrocytosis can be confirmed by bone marrow examination which reveals a megaloblastic picture. although macrocytic anaemia is a typical feature of vitamin b 12 deficiency, it can be absent in older individuals who may only have neuropsychiatric features. measurements of methylmalonic acid and homocysteine levels, two markers which are very sensitive for detecting b 12 deficiency, have shown that vitamin b 12 deficiency can occur with normal haemoglobin levels and without macrocytosis. 36 pernicious anaemia is probably the commonest cause of vitamin b 12 deficiency. the presence of parietal cell or intrinsic factor antibodies supports a diagnosis of pernicious anaemia. [33] [34] [35] [36] schilling tests are rarely performed because of the unavailability of the radio-labelled vitamin b 12 and the difficulty in interpreting the results in the presence of renal insufficiency. • pernicious anaemia (begins after 40), increased risk of gastric carcinoma and carcinoid tumours • rare congenital disorders, e.g. imerslund-grasbeck syndrome. the neglected tropical diseases are a group of infections which are endemic in developing countries. several of these neglected tropical diseases cause anaemia and many can be managed using inexpensive interventions to treat the underlying parasitic infections. 14 the mechanisms of anaemia in these conditions are predominantly blood loss from the gastrointestinal or genitourinary tracts but also poor nutrition, bone marrow suppression, inflammation, hypersplenism and haemolysis. 58 anaemia is a common consequence of infections with soiltransmitted helminths or schistosoma with a strong correlation between haemoglobin level and worm load or faecal egg count. even mild infections can lead to anaemia. 59 polyparasitism (i.e. infection with several parasites simultaneously) can be responsible for unresponsiveness of the anaemia to eradication of one organism. 60 treatment of communities at high risk of soiltransmitted helminths improves growth and iron stores in children and reduces anaemia in pregnant women. 61 the treatment of anaemia due to neglected tropical diseases depends on eradication of the parasite with drugs such as albendazole and praziquantel though anaemia resolution may be less successful if it is due to trichuriasis. 62-64 the addition of iron to anthelmintic treatment has met with variable success rates probably because there is associated anaemia related to inflammation. however it is still generally recommended that iron supplementation should be included with anthelmintic therapy in treatment programmes for neglected tropical diseases. [65] [66] [67] introduction haemoglobin s (hbs) has a prevalence of 25-30% in many parts of africa and also some areas in the middle east ( figure 65 .6). hbs tends to be common among ethnic groups that have traditionally had high exposure to plasmodium falciparum malaria. in sub-saharan africa approximately 230 000 infants are born with sickle cell disease each year, mostly with hbss. sickle cell disease (scd) is an autosomal recessive disorder characterized by production of an abnormal haemoglobin, sickle haemoglobin. sickle haemoglobin (hbs) arises from a mutation in codon 6 of the β-globin gene resulting in replacement of the normal glutamic acid residue by a valine. 68 scd is most commonly caused by the co-inheritance of two sickle cell genes (homozygous hb ss disease) but patients who are heterozygous for hbs and for another haemoglobin mutation such as hbc (haemoglobin sc disease) or β-thalassaemia (sβ 0 and sβ + ) can also present with features of scd. 69 ss disease and sβ 0 disease are more severe than sc disease and sβ + disease (box 65.6). 70 scd can affect multiple organs and its clinical course is punctuated by episodes of acute illness on a background of progressive organ damage, especially of the central nervous system and the lungs. 70 the first description of scd was in 1910 in an anaemic grenadian dental student 71 and over the next 30 years it was that the underlying cause of folate deficiency is identified and treated. vitamin a is important in erythropoiesis, iron metabolism (enhances iron absorption and its release from stores to the bone marrow) and for decreasing the risk of infections. 45 vitamin a deficiency is a major public health problem in lowincome countries, with an estimated 200 million preschool children affected. 47 pregnant women and women of childbearing age also constitute high-risk groups for vitamin a deficiency. 46 vitamin a given to thai school children with conjunctival xerosis led to a significant increase in haemoglobin level 47 and in anaemic school children in tanzania, vitamin a supplementation produced a marked increase in haemoglobin which was enhanced by co-administration of iron. 48 vitamin a can also improve anaemia in pregnant women, depending on the local prevalence of deficiency [49] [50] [51] [52] though the response may be suboptimal in pregnant women infected with hiv. 53 copper is a trace element necessary for normal haematopoiesis and myelopoiesis. anaemia in copper deficiency is due to decreased activity of the copper-dependent enzymes, hephaestin, ceruloplasmin and cytochrome c oxidase. these are important in ferrous-ferric iron conversions and their decrease leads to abnormalities in iron absorption and its incorporation into the haemoglobin molecule. acquired copper deficiency occurs with malnutrition and gastrointestinal malabsorption syndromes. coeliac disease, cystic fibrosis and individuals who have had gastrectomy or surgery resulting in 'short bowel' are also at risk. copper deficiency has also been described in persons ingesting excessive amounts of zinc-containing supplements and those who have swallowed zinc-containing coins. 54, 55 anaemia related to copper deficiency is normocytic or macrocytic and can be associated with neutropenia; thrombocytopenia is rare. bone marrow findings are characteristic with cytoplasmic vacuolization of both erythroid and myeloid precursor cells with ringed sideroblasts and an unusual finding of iron granules in plasma cells. these findings may be misdiagnosed as myelodysplastic neoplasm. measurement of serum copper levels is helpful in confirming the diagnosis although the test is fairly insensitive. since almost complete haematological recovery can occur with copper replacement, this may be a useful diagnostic test. oral copper supplements can be started with 8 mg of elemental copper a day slowly decreasing over the next few weeks to 2 mg until a good response is noted. although low zinc levels do not cause anaemia they have been linked to growth retardation, heightened susceptibility to infection and male hypogonadism in relation to sickle cell disease. 56 zinc deficiency has been described in nearly half of children and 70% of adults with sickle cell disease possibly due to increased loss of zinc in the urine and high cell turnover with decreased dietary intake. 57 in contrast zinc excess can cause anaemia through interference with copper absorption by sequestering it in the gut lumen. for this reason, zinc compounds have been used to treat wilson's disease which is characterized by copper excess. however repeated sickling and unsickling eventually causes irreversible changes, 77 so early management to avoid repeated crises is important to prevent disease progression. polymerization, and therefore the clinical features of scd, are influenced by three main factors 78 ; hypoxia, the intracellular hbs concentration and the co-existence of other genetic haemoglobin abnormalities (e.g. α-thalassaemia or hereditary persistence of fetal haemoglobin-haemoglobin f). 79 sickled red cells lead to vaso-occlusion and haemolysis due to the entrapment of sickled erythrocytes in the microvasculature and upregulation of adhesion receptors. 76, 80, 81 white blood cells contribute to this process by providing an inflammatory discovered hypoxia led to red cell sickling 72 scd arises from the tendency of hbs to polymerize in hypoxic states. this phenomenon occurs where there is deoxygenation and is due to the binding between β1 and β2 chains of two haemoglobin molecules, a property unique to haemoglobin variants that have the glu-6-val substitution. 74 the polymerized haemoglobin fills the erythrocyte and deforms its architecture and flexibility to form a sickle shape. this alteration in the structure promotes cellular dehydration, 70, 75, 76 upon reoxygenation, the polymers dissolve thus reversing the sickling process. 90 exposure to cold, fever, menstruation, alcohol intake and dehydration can precipitate pain crises. unlike acute pain crises, chronic pain in scd usually has an identifiable basis such as femoral head necrosis, osteoarthritis or chronic skin ulcers. sickle erythrocytes have an average life span of 17 days and anaemia can be due to several causes (box 65.8). red cell haemolysis causes anaemia and gall stones and can cause fatigue out of proportion to the anaemia. 91, 92 there are suggestions that patients with low haemoglobin concentrations and high haemolytic rates are more likely to develop vascular problems compared with those with higher haemoglobin concentrations. splenic sequestration with a sudden rapid drop in haemoglobin occurs in those who have not yet developed autosplenectomy so it can occur in young children with hbss and adults with hbsc disease or sickle cell-β + -thalassaemia. treatment may require blood transfusion and in rare cases, sequestration can be fatal. splenectomy may be needed for recurrent severe sequestration. parents can be taught to feel their infant's abdomen for an enlarging spleen and report to hospital if there is a sudden increase in spleen size. red cell aplasia can develop due to secondary parvovirus infection which has a predilection for erythroid progenitors. alloimmunization is common in scd patients who have had frequent transfusions so, if possible, extended red cell phenotyping should be undertaken. hyperhaemolytic crisis is suspected when there is sudden exacerbation of anaemia with increased reticulocytosis and bilirubin level. infectious complications of scd are a major cause of morbidity and mortality, even with adequate vaccination and prophylactic antibiotic regimens. this propensity to infection is related to impaired splenic function although tissue ischaemia, especially in the lungs and renal system, can contribute. hyposplenism is demonstrable in the peripheral blood film by the presence of howell-jolly bodies. most children with scd have undergone autosplenectomy by the age of 5 years and therefore have increased risk of infection from encapsulated microorganisms. 93 typical infectious complications include pneumococcal sepsis, neisseria meningitis, osteomyelitis caused by salmonella species, urinary tract infections and pyelonephritis due to escherichia coli. anatomical abnormalities such as renal papillary necrosis can predispose to urinary complications which may require long-term antibiotics. acute chest syndrome (acs) is defined as a new pulmonary infiltrate on the chest radiograph combined with one or more environment. activation of platelets and the coagulation system also contribute to the vaso-occlusion in scd. [82] [83] [84] [85] [86] infants with scd are protected during the first few months of life by the high levels of haemoglobin f in the red cells. anaemia usually develops by 3 months. at all ages, chronic haemolysis of abnormal red cells means that scd is associated with steady state haemoglobin levels of 60-80 g/l. although any organ can be affected by scd and complications can occur at any age, certain features tend to predominate in different age groups (box 65.7). pain is the hallmark of scd 87 and four different patterns of pain have been described with scd each with different underlying mechanisms: 88 • vaso-occlusive (acute and intermittent) • pain from bone and tissue necrosis (chronic) • neuroplasticity (chronic, neuropathic) -functional brain changes • opioid-induced hyperalgesia (acute or chronic). painful crises often start in young children as dactylitis or handfoot syndrome, in which painful swelling of the hands and feet results from the inflammation of metacarpal and metatarsal periosteum. these crises are the result of vaso-occlusion of the bone marrow causing bone infarction and release of mediators that activate pain receptors. 82 the number, severity and frequency of painful episodes vary widely in individuals. half may never have any episodes whereas about 3% may need hospital admission up to 6 times a year. 89 more than three pain episodes requiring hospitalization per year is associated with increased mortality among patients over 20 years old. in under-resourced settings, hospital visits underestimate the frequency of pain box manifestations such as fever, cough, sputum production, tachypnoea, dyspnoea or new-onset hypoxia. 94 acs is the most common cause of death in scd patients and a frequent cause of hospitalization, second only to painful crisis. mortality in patients with acs in a wealthy country setting is 1% in children and 4.3% in adults. 95 the peak incidence for acs is 2-4 years of age and gradually declines to 8.8 per 100 patient-years in subjects older than 20 years. 96, 97 fever and cough are more common in children with acs and chest pain and dyspnoea are more common in adults. 96 acs is often preceded by febrile pulmonary infection in children and by vaso-occlusive pain crisis and lung infarction in adults. it is important to note that although tachypnoea, wheezing and features of chest infection may be identified, a third of the patients may have a normal physical examination. more than one-third of patients with acs are hypoxaemic (oxygen saturation <90%). 98 chest radiography is essential although infiltrates may lag behind clinical symptoms by up to 3 days. repeat chest x-rays are recommended if there is a strong clinical suspicion of acs. bilateral infiltrates or involvement of multiple lobes may predict a poorer prognosis. risk factors for acs (box 65.9) include fat embolus which can be confirmed by finding stainable fat in pulmonary macrophages. 99 chronic complications such as pulmonary hypertension occur in as many as 60% of patients and do not appear to be associated with prior episodes of acs. high serum phospholipase a2, and the surrogate marker c-reactive protein, have been noted in patients admitted with vaso-occlusive crisis 24-48 hours before the development of acs. 100, 101 stroke neurological complications occur in at least 25% of patients with scd and scd is one of the most common causes of stroke in children. 102, 103 in scd, the risk of having a first stroke is 11% by the age of 20, 15% by age 30 years and 24% by age 45 years. both thrombotic and haemorrhagic strokes occur, although the former is more common in children and those over 30 years of age, whereas the latter is more common between the ages of 20 and 30 years. 108 this age-specific pattern may be related to the higher cerebral flow rates in early childhood. although the prevalence of clinically overt stroke is of the order of 11%, clinically silent infarction, detectable by magnetic resonance scans, affect nearly double this number by the age of 20. silent infarcts are associated with cognitive impairment and the majority of these children require lifelong specialist care. 104 cerebral thrombosis, which accounts for 70-80% of all strokes in scd, results from large-vessel occlusion whereas silent infarcts are the result of microvascular occlusion or thrombosis or hypoxia secondary to large-vessel disease. in a third of scd patients, major-vessel stenosis is accompanied by collateral vessels that appear as 'puffs of smoke' (moyamoya) on angiography. risk factors for ischaemic strokes in scd include increased cerebral blood flow velocity, previous silent infarcts, nocturnal hypoxaemia, severe anaemia, acute chest syndrome and elevated systolic blood pressure. an elevated leukocyte count is a risk factor for haemorrhagic stroke. [105] [106] [107] [108] diagnosis often the family history and clinical findings clearly point towards a diagnosis of scd and during an acute crisis, abundant sickled red cells can be seen on a blood film. white cell counts are higher than normal in scd disease, particularly in patients under age 10 years. the presence of sickle haemoglobin in different sickle syndromes (e.g. hbas, hbss, hbsc) ( table 65 .5) can be confirmed by a simple sickle slide or solubility test. haemoglobin electrophoresis will distinguish between many of these variants but high-performance liquid chromatography and iso-electric focusing are preferred for a definitive diagnosis. haemoglobin mass spectrometry and dna analysis are being increasingly used. antenatal screening is available to women in some countries to help to identify couples who are at risk of having a baby with scd. community acceptance of reproductive genetic services however depends on the effectiveness of education and counselling. the use of prophylactic penicillin and the provision of comprehensive medical care during the first 5 years of life have reduced mortality related to scd from 25% to less than 3%. 109 management (box 65.10) individuals with scd are best managed by a multidisciplinary team as they may require a variety of specialist inputs including haematology, ophthalmology, nephrology, obstetrics, orthopaedics and physiotherapy. the cornerstones of scd therapy are disease modification and prompt and effective management of crises. severe pain crises generally require intravenous fluids and adequate, often opiate, analgesia (box 65.11), while disease modification is based on interventions to increase hbf levels. in steady state it is usual practice to give sickle cell patients folate supplements (1-5 mg/day) because their high rates of haemopoiesis put them at risk of deficiency. scd is associated with functional asplenia so patients should also receive prophylactic oral penicillin (250 mg twice a day) and vaccinations against encapsulated organisms. hydroxycarbamide is the main agent used to increase hbf (box 65.12) and is associated with significant reductions in acute pain crises, hospitalization rate, time to first and second pain crises, episodes of acute chest syndrome, and the need for transfusions and the number of units transfused. 110 other beneficial effects of hydroxycarbamide, which are independent of the increase in hbf, include reduced neutrophil count, increased cellular water content, decreased hbs concentration, changing expression of adhesion molecules and nitric oxide generation. 111 hydroxycarbamide may also be an alternative to frequent blood transfusions for the prevention of recurrent stroke in children as it can lower transcranial doppler velocities. 112, 113 under-use of this cheap, effective drug is related to concerns about leukaemogenicity but this has not been shown to be a problem when used for a non-malignant condition like scd. the two main approaches to transfusion 74 in scd are simple top-up transfusion and exchange transfusion. target haemoglobin level in scd therapy is 100 g/l or a haematocrit of 30%; higher target levels are associated with hyperviscosity and box 65.10 management of complications of sickle cell disease • inability to maximally concentrate urine (hyposthenuria) in response to water deprivation is an early finding • renal tubular acidosis • increased urinary tract infections • glomerular hyperfiltration, increased creatinine secretion, and a very low serum creatinine are characteristic of young patients with sickle cell anaemia, so renal dysfunction can be present even with normal serum creatinine values • microalbuminuria is common in childhood and up to 20% of adults develop nephrotic-range protein loss • gross haematuria can develop due to microthrombin in renal vessels, renal medullary carcinoma, and nocturnal enuresis • treatment is based on the early use of hydroxycarbamide and angiotensin-converting enzyme inhibitors in children with clinically significant albuminuria. • noted in up to 60% of scd cases • no relationship to acute chest syndrome (different pathophysiology) • mortality risk with even mild pulmonary hypertension is high • regular blood transfusions and long-term anticoagulation have been tried • hydroxycarbamide may decrease the risk • prostacycline analogues (epoprostenol, and iloprost), endothelin-1 receptor antagonists (bosentan), phosphodiesterase inhibitors (including sildenafil), and calcium channel blockers are being evaluated. • brief but recurrent (stuttering); may occasionally last for many hours and can lead to impotence • usually ischaemic, or low-flow, priapism • patients should be educated to seek medical attention if more than 2 hours duration • detumescence within 12 hours is necessary to retain potency • intravenous hydration and analgesia initially with consideration for α-adrenergic agonists (etilefrine or phenylephrine) • penile aspiration and irrigation with saline and α-adrenergic agents or shunting may be required in severe cases in combination with an exchange transfusion. • assess pain intensity • choose the analgesic, dosage, and route of administration • paracetamol and hydration should be considered in all patients • oral, sustained-release morphine is as good as intravenous morphine infusion in children and young adults • manage mild pain with rest, hydration, and weak opioids (such as codeine). admit patients in whom pain that does not subside promptly or require opioid treatment; fever, pallor, or signs of respiratory compromise; a low likelihood of receiving appropriate care at home • pain management should be individualized and dosing should take into account prior pain management and use of opioids • the pain pathway should be targeted at different points with different agents, avoiding toxicity with any one class • always look for a cause, e.g. infection, dehydration, etc. • education about avoiding exposure to precipitants • be empathetic, reassuring, and supportive • benzodiazepines may be helpful to reduce anxiety • re-examine the patient often to ensure adequate pain relief, to assess sedation and respiratory rate (to avoid opioid overdose). in assessing patient responses to conventional doses of analgesia, it must be remembered that those with sickle cell disease metabolize narcotics rapidly • re-search for evidence of any complications such as acute chest syndrome or anaemia • always look for a cause, e.g. infection. of multi-organ failure. 116 both simple transfusion and exchange transfusions have been used and neither appears to be superior. a short course of steroids may attenuate acs but it may also increase the risk of re-hospitalization. 117 bronchodilators may help patients with wheezing 118 but inhaled nitric oxide has not shown any clear benefits. 119 since coagulation activation is important in the pathophysiology of acute chest syndrome, treatment with low-molecular-weight heparin may reduce clinical complications. 120 transcranial doppler measurement of cerebral blood flow has been a major step forwards in identifying individuals with an increased risk of ischaemic stroke. a value more than 200 cm/ second imparts a 40% risk of stroke within the next 3 years. 121 regular blood transfusions can reduce the incidence of stroke in children. 105 due to a high recurrence of stroke (60%) on stopping transfusions, continuation of transfusions should be guided by transcranial doppler measurements. 122, 123 once a stroke has developed, the best therapeutic strategy is exchange transfusion which probably needs to be done monthly. 70, 73 neurosurgical re-vascularization should be considered for moyamoya-like syndromes when new strokes occur despite transfusion. haemoglobin sc results from the co-inheritance of hbs and hbc and has its highest prevalence in west africa. clinical features and disease management are similar to those of hbss disease but splenomegaly, splenic infarcts and splenic sequestration may occur into adulthood. proliferative retinopathy necessitates regular ophthalmic review in those aged over 10 years. compared with hbss, anaemia is less marked in hb sc (8-140 g/l) and there are fewer sickle cells and more target cells on the blood film. the diagnosis can be confirmed by haemoglobin electrophoresis, hplc or iso-electric focussing. worsening of complications. in exchange transfusion, the aim is to achieve an hbs% of <30%. complications of transfusion in scd include alloimmunization, 114 delayed haemolytic transfusion reactions and iron overload. the high rates of red cell antibody formation (30%) noted in wealthy countries are due to minor blood group incompatibilities between the recipient and the blood donor who is often of a different ethnicity. leukocyte reduction of transfused blood, routine abo, rh and kell matching for all patients and extended phenotype matching for those with alloantibodies may be useful for reducing transfusion reactions. treatment for acs is predominantly supportive and includes adequate pain relief, antibiotics (e.g. a macrolide with a cephalosporin), continuous pulse oximetry and delivery of supplemental oxygen to patients with hypoxaemia. incentive spirometry can prevent atelectasis and infiltrates 115 and blood transfusion is indicated when a patient develops respiratory distress, a clinically significant fall in the haematocrit or signs the following predict a more severe clinical course and are additional reasons to consider offering hydroxyurea: hb <70 g/l, wbc >15 × 10 9 /l, hbf <6% and renal insufficiency due to scd. • start at 10-15 mg/kg per day (to the nearest 500 mg/day) • if no or poor response, increase dose by increments of 5 mg/ kg per day every 4 weeks (max: 30 mg/kg per day). most good responses require about 1-2 g/day in adults • monitor fbc, hbf%, and reticulocytes every 1 or 2 weeks initially, then every 4 weeks when on a stable dose • monitor biochemistry profile (hydroxyurea has renal excretion and hepatic toxicity). • less pain • persistent increase in hbf (usually measured every 6-8 weeks) or mean cell volume • persistent increase in haematocrit if severely anaemic • decrease in ldh • acceptable toxicity. improvement in symptoms and blood parameters may take 3-4 months of therapy, but can be seen after approximately 6 weeks. if the reticulocyte count is less than expected for the degree of anaemia, erythropoietin deficiency should be considered. • aim in all cases to reduce hbs level to <30% • exchange transfusions may be considered in cases of stroke, acute chest syndrome not responding to top-up transfusion and major surgeries • target haemoglobin concentration of 100 g/l may be considered in cases of organ failure and surgery. individuals with sickle cell trait (hb as) have 10-fold protection against severe malaria compared to individuals with normal haemoglobin (hbaa) probably due to both innate and immune-mediated mechanisms. individuals with sickle cell trait (hbas) are generally asymptomatic and they have a normal haemoglobin and normal life expectancy. uncommonly, complications such as poor perfusion of the renal papillae and increased bacteruria may occur. 124 the blood film is generally normal and the diagnosis can be confirmed by haemoglobin electrophoresis, hplc or iso-electric focusing. the original descriptions of thalassaemia originated from areas round the mediterranean and the term derives from the greek thalassos (sea) and haima (blood). [125] [126] [127] epidemiology thalassaemia is one of the most common single gene disorders and approximately 5-7% of the global population are carriers. α + -thalassaemia occurs throughout the tropics, whereas α 0thalassaemia, which is responsible for haemoglobin bart's hydrops fetalis, is concentrated predominantly in south-east asia and to a lesser extent around the mediterranean. 128, 129 β-thalassaemia is common in the mediterranean countries, parts of africa, throughout the middle east, the indian subcontinent and south-east asia. haemoglobin e prevalence is highest in cambodia, laos and thailand and can reach 50-60% with lower prevalence rates in indonesia, malaysia, singapore and vietnam. β-thalassaemia β-thalassaemia is an inherited quantitative deficiency of β-globin chains which are required to make normal adult haemoglobin. 130 more than 200 mutations have been associated with the development of β-thalassaemia (a complete list is available at the globin gene server website, at: http://globin.cse. psu.edu) and they affect protein synthesis 130, 131 leading to reduced (designated β + ) or absent (designated β 0 ) production of the β-globin chains. the clinical severity of thalassaemia can be lessened by co-existing haemoglobin abnormalities such as the co-inheritance of α-thalassaemia and increased production of haemoglobin f. 132, 133 α-thalassaemia normal α-globin synthesis is regulated by duplicate α-globin genes on chromosome 16. the genotype is usually represented as αα/αα and α-thalassaemia usually results from deletion of one or both α-genes. occasionally point mutations in critical regions of the α-genes may cause non-deletional α-thalassaemia (α t ). 130 mutations can completely abolish expression of the αgenes (i.e. α 0 -thalassaemia) or partially down-regulate expression (α + -thalassaemia). 130 both α 0 and α + thalassaemias can occur in the heterozygous or homozygous state or as a compound α 0 /α + heterozygote form (table 65 .6). underproduction of α-globin chains due to three or four gene deletions gives rise to excess γ (fetal) or β (adult) globin chains which form tetramers, called hb bart's (fetal) or hbh (adult). 134 rare forms of α-thalassaemia occur in association with other conditions such as mental retardation and myelodysplastic/ leukaemia syndrome. 135, 136 pathophysiology β-thalassaemia (figure 65 .8) thalassaemias 131, 137, 138 cause an imbalance of αand β-globin chain synthesis. in homozygous β-thalassaemia, excess α-chains precipitate in the red cell precursors and up to 75% of cells are destroyed in the bone marrow resulting in ineffective erythropoiesis and a shortened red cell survival. the red cells released from the bone marrow contain abnormal α-chains and these inclusions promote destruction of the cells by the spleen leading to clinical symptoms and signs of haemolysis. in heterozygotes, the α-chain excess and the degree of inadequate erythropoiesis is much less than in homozygous β-thalassaemia. hbf production normally tails off within a few months of birth but in β-thalassaemia hbf production can continue into adulthood. the effect of increased hbf production is to prevent precipitation of the excess globin chains and consequent ineffective erythropoiesis. however hbf has a high oxygen affinity, which can lead to increased erythropoietin production and thus, increased bone marrow expansion. the pathophysiology of α-thalassaemia, and hence the clinical manifestations, is quite different from β-thalassaemia. the excess non-α-globin chains form soluble tetramers rather than precipitates so there is only minimal ineffective erythropoiesis. the only clinical abnormality in those with hbh may be splenomegaly secondary to increased work load from destruction of red cells containing inclusions. rarely anaemia may be severe enough to require blood transfusions. classification of α-thalassaemia divide β-thalassaemia into thalassaemia major (transfusiondependent), thalassaemia intermedia (able to maintain adequate haemoglobin without transfusions or requiring less than 8 units/year) and thalassaemia minor (asymptomatic). 139 infants with β-thalassaemia are protected from severe anaemia by the presence of haemoglobin f and are usually asymptomatic. clinical manifestations of thalassaemia major depend on whether adequate blood transfusions are available and the stringency with which iron chelation is undertaken. untreated patients with thalassaemia major will die in late infancy or early childhood from the effects of severe anaemia. those who receive sporadic transfusions may survive longer but suffer from the secondary effects of anaemia, bony deformities and growth retardation. the clinical features of β-thalassaemia major are divided into those resulting from anaemia, bony changes and iron overload. anaemia from defective erythropoeisis, decreased red cell survival and increased haemolysis in thalassaemia major leads to cardiac decompensation, failure to thrive and growth retardation in children. splenomegaly, from the increased work load of culling red cells with inclusion bodies, can cause dilutional anaemia and a further drop in haemoglobin. compensatory extra-medullary haematopoiesis can lead to hepatomegaly and occasionally vertebral compression and neurological defects. haemolysis from increased red cell destruction is associated with gall stones in up to 20% of individuals with β-thalassaemia. 140 another consequence of accelerated haemolysis is the increased incidence of thromboembolism (4% in thalassaemia major and 10% with intermedia) from the exposure of negatively charged phospholipids on the red cell membrane and the generation of red cell and platelet microparticles. 141 splenectomy with postoperative thrombocytosis is a risk factor for thrombosis especially if combined with endothelial oxidative stress from iron overload, or procoagulant co-morbid conditions such as diabetes mellitus, hormone therapy, thrombophilic mutations and atrial fibrillation. 142 folate deficiency, hyperuricaemia and occasionally gout have been observed in thalassaemia major due to the high turnover of red cells. the enhanced erythropoietic drive from anaemia in thalassaemia can lead to increased marrow expansion with in homozygous β-thalassemia, β-globin synthesis is markedly reduced or absent. the excess α-chains cannot form a tetramer but form a precipitate in the red cell precursors leading to intra-medullary destruction of these cells. this destructive process of the red cell membrane occurs from the formation of α-chain hemichromes (shown as red cell inclusions) and degradation products of the excess α-chains. the red cells which may be released from the bone marrow are destroyed by the spleen leading to clinical symptoms and signs of haemolysis. since only the β-chain is affected in these individuals, the synthesis of hbf and hba 2 continues unabated. these haemoglobins have very high oxygen affinity, which can lead to increased erythropoietin production and thus, increased bone marrow expansion splenomegaly, which may be massive, and growth retardation in children. bony changes are unusual. other complications include infections, leg ulcers, gall stones and acute haemolysis in response to drugs and infections. the severity of the clinical features is related to the molecular basis with non-deletional types of hbh disease more severely affected. haemoglobin bart's (−/−) occurs almost exclusively in asians, especially chinese, cambodian and thai populations. an infant with hb bart's hydrops fetalis syndrome has pallor and gross oedema with signs of cardiac failure, marked hepatosplenomegaly and skeletal and cardiovascular deformities. there is often gross hypertrophy of the placenta. many of the clinical manifestations of this condition can be explained by the characteristic bossing of the skull and overgrowth of maxillary region, radiologically noted as 'hair on end' or 'sun-ray' appearance. metatarsal and metacarpal bones are the first to expand so measurement of the metacarpal bones has been considered a good indicator for initiation of transfusion therapy. 143 other skeletal deformities include shortening of long bones due to early epiphyseal fusion and overgrowth of the maxilla causing dental malocclusion. the marrow expansion can also lead to pathological fractures, early bone thinning and osteoporosis 144, 145 while ineffective drainage of the sinuses and middle ear from skull bone overgrowth can cause chronic sinus and ear infections. growth retardation is primarily the result of anaemia with contributions from iron overload, hypersplenism, deficiencies of thyroid and growth hormone, hypogonadism, zinc deficiency, chronic liver disease, malnutrition and psychosocial stress. 146 patients with β-thalassaemia have increased iron absorption mediated by reduced hepcidin and those who receive regular transfusions may also develop transfusion siderosis if they are inadequately chelated. the iron is deposited in the parenchymal tissues with a variety of clinical consequences (box 65.14), [147] [148] [149] [150] [151] [152] [153] [154] a process which may be modulated by variants in the haemochromatosis (hfe) gene. 155 thalassaemia intermedia is characterized by haemoglobin concentrations of 70-100 g/l and children usually present at around 2-4 years of age with symptoms of anaemia, jaundice and hepatosplenomegaly. 156 there may also be skeletal changes such as expansion of the facial bones and obliteration of the maxillary sinuses. 157 several molecular factors including: (a) coinheritance of α-thalassaemia; (b) hereditary persistence of haemoglobin f; (c) δβ-thalassaemia and (d) the specific gγxmn1 polymorphism contribute to the 'conversion' of thalassaemia from major to intermedia type. 158 in contrast to patients with thalassaemia major, iron loading in thalassaemia intermedia occurs mainly as a result of increased intestinal iron absorption rather than transfusion therapy. ineffective erythropoiesis with resultant chronic anaemia and hypoxia can suppress hepcidin, the regulator of iron metabolism, leading to increased iron absorption. 158 the excess iron tends to accumulate in the liver rather than the heart. 159 other clinical complications in thalassaemia intermedia include gallstones, extramedullary haemopoiesis leg ulcers, thromboembolic events and pulmonary hypertension, which is the major cause of heart failure in these individuals. 160 although individuals with thalassaemia intermedia do not usually need regular blood transfusions, there is some evidence that complications, particularly later in life, may be less common in regularly transfused patients. 159 α-thalassaemias 130, 134 carriers of α-thalassaemia (traits, with loss of 1 or 2 α genes) are usually asymptomatic and may only be detected through a routine blood count which shows mild to moderate microcytic, hypochromic anaemia. antenatal counselling may be indicated if the mother has αα/− as there is a possibility that the fetus may be at risk of having haemoglobin bart's. haemoglobin h disease occurs mainly in asians and occasionally in the mediterranean population. it is the result of deletion of three α genes (α−/−) and can produce anaemia varying from 30-130 g/l. there is usually associated • hypogonadism is the most frequent complication in patients with prevalence over 50% in both males and females. it is usually hypogonadotrophic suggesting iron damage to the anterior pituitary or hypothalamus. the features range from total absence of sexual development to delayed puberty. in females with normal menstrual function, fertility is normal with the ovarian function preserved in most although secondary amenorrhoea can develop. damage of the ovaries is rare and is more likely to appear in older women (around 30) because of high vascular activity on the ovaries at this age. secondary hypogonadism is common (50%) in older men. serum ferritin >2000 ng/ml is a risk factor. • hypothyroidism is the second most common endocrine disorder (about 20%) although many of them may have the subclinical variety. most commonly hypothyroidism is of the primary type with secondary, central hypothyroidism increasingly being diagnosed in recent years. • the prevalence of diabetes mellitus is around 5% with the mean age of diagnosis being 18 years. impaired glucose tolerance occurs first with microvascular damage like retinal changes being less common than the conventional form. erythroid precursor destruction. osmotic fragility is reduced, sometimes strikingly so since in some cases the red blood cells do not haemolyse even in distilled water. 161 for this reason, if sophisticated tests are not available, osmotic fragility can be used as a screening test for thalassaemia trait. serum zinc levels may be low and this may be related to abnormal growth. 162 vitamin c levels may also be low due to its increased conversion to oxalic acid in the presence of iron overload. 163 care may be needed if folic acid is commenced on a background of bone marrow failure due to folate deficiency as it may precipitate painful erythropoietic crises. 164 management a comprehensive management plan for patients with thalassaemia may involve transfusion therapy, iron chelation, splenectomy, prevention or early treatment of complications and stem cell transplant. the mainstay of treatment for the severe forms of thalassaemia is blood transfusion with the aim of reducing anaemia and erythropoietic drive. however, in many low-income settings blood supplies are inadequate and many thalassaemic patients are chronically under-transfused (table 65 .7). 165 transfusion frequency should be guided by clinical symptoms and signs such as poor growth and facial or other bone abnormalities, and should take into account any potential disease-modifying comorbidities. 156 although the decision to transfuse should not be based purely on haemoglobin levels, a value of <70 g/l is often used as a trigger for regular transfusions. to prevent alloimmunization, extended red cell antigen typing for c, e and kell in addition to abo and rh(d) typing should be carried out prior to the first transfusion, and before each transfusion, full cross-match and screening for new antibodies should be undertaken. 166 the risk of alloimmunization appears to be greater in patients who begin transfusion therapy after the first few years of life. 128 development of alloantibodies and autoantibodies may result in increased transfusion requirements or haemolysis. use of leukodepletion techniques can result in less alloimmunization and fewer febrile transfusion reactions. since storage of red cells in anticoagulant solutions may decrease their efficacy, the use of blood that has been stored for less than 7-10 days may be beneficial for patients who require frequent transfusions. the use of 1st-degree relatives as blood donors should be discouraged, especially if the patient is a candidate for stem cell transplant. patients with thalassaemia major need lifelong regular blood transfusions, 15 ml/kg per month or 1-2 units of blood every 2-5 weeks, to maintain the pre-transfusion haemoglobin level above 90-105 g/l. the clinical benefits of this regular transfusion programme include normal growth, suppression of erythropoiesis and bone marrow expansion, reduced hepatosplenomegaly and an overall sense of wellbeing, which allows normal age-appropriate activities. a higher target pretransfusion haemoglobin level of 110-120 g/l may be necessary for patients with heart disease or other medical conditions and for those patients who do not achieve adequate suppression of bone marrow activity at the lower haemoglobin level. shorter intervals between transfusions may reduce overall blood requirements but need to be balanced against the patient's work or school schedule and other lifestyle issues. iron chelation therapy 167, 168 has improved survival rates for thalassaemic patients, and prevented hepatic fibrosis and ironinduced cardiac disease; most patients who are compliant with chelation therapy have normal growth and sexual development. iron chelators (box 65.16) are usually initiated in children over 2 years who have received 10 units of blood and/or have a steady-state serum ferritin level above 1000 ng/ml on at least two occasions. this level of iron overload typically occurs after 1-2 years of transfusions. desferrioxamine is started at 25-30 mg/kg per day in these children initially, to avoid toxicity due to over chelation. marked splenomegaly, often treated with splenectomy, was common in thalassaemia patients before the advent of regular transfusion programmes. severe haemolysis in thalassaemia is related to a hyperactive spleen, which aggravates anaemia and can increase transfusion requirements. although early the initiation of regular transfusion therapy for severe thalassaemia usually occurs in the first 2 years of life. some patients with thalassaemia intermedia who only need sporadic transfusions in the first two decades of life may later need regular transfusions because of a falling haemoglobin level or the development of serious complications. haemoglobin should be monitored to assess the rate of fall in the haemoglobin level between transfusions and this can be used to indicate the frequency of transfusions. exchange transfusions have been tried as a way of reducing iron loading and are associated with a reduction in blood requirements by about one-third. (table 65. since each unit of red cells can contain up to 200 mg of iron, cumulative iron burden is an inevitable consequence of a longterm transfusion programme. in addition there is increased iron absorption from the gut (0.3-0.6 mg/kg per day) as a response to severe anaemia and down-regulation of hepcidin. transfusion therapy can avert splenomegaly, hypersplenism still can develop, usually in children between 5 and 10 years of age. in these individuals, splenectomy can limit the complications from extramedullary hematopoiesis. splenectomy should be considered when the annual transfusion requirement reaches 200-250 ml red blood cells/kg per year and usually results in a halving of the transfusion requirements. splenectomy complications include opportunistic infections with encapsulated organisms. patients should therefore receive appropriate vaccinations preoperatively and should be advised to seek medical advice at the first sign of infection. it is advisable to delay splenectomy until patients are at least 5 years old because of the increased risk of overwhelming sepsis below this age. thalassaemia patients can develop thromboembolic complications and pulmonary hypertension after splenectomy so partial splenectomy and splenic embolization have been attempted to minimize these complications but have not been studied in large trials. iron overload can occur in any organ in thalassaemia patients but particularly affects the heart, liver, the endocrine system, the bone and occasionally the pancreas and lungs. iron overload needs to be detected early and treated to prevent long-term damage. annual assessment of the iron loading of the liver and heart can be achieved using non-invasive methods such as magnetic resonance scanning to detect early changes. children should have regular growth and endocrine assessments and appropriate investigations should be carried out if there are any signs of developmental delay or hormonal deficiencies. osteoporosis is increasingly being recognized and should be prevented by ensuring adequate dietary calcium intake and sun exposure. vitamin supplementation with folic acid, zinc, vitamin e and vitamin c may be useful although the combination of vitamin c and desferrioxamine carries a risk of cardiac toxicity. allogeneic stem cell transplant 169, 170 is currently the only means of curing thalassaemia. the outcome in carefully selected patients, measured by overall event-free survival, is around 90% with a transplant-related mortality of 3%. hepatomegaly, liver fibrosis, and inadequate iron chelation therapy predict a poor outcome. the best results from transplant have been obtained with hla-matched siblings. umbilical cord blood is a useful source of stem cells for young children. other potential treatment options for thalassaemia are outlined in box 65.16. [171] [172] [173] [174] prevention of severe thalassaemia births by prenatal diagnosis and termination of pregnancies has been successful in countries with a high prevalence of thalassaemia. 175 early identification of couples at risk and culturally sensitive genetic counselling facilitate decision-making for termination or continuation of pregnancy. the mean corpuscular haemoglobin (mch) is used to screen for the presence of thalassaemia using a cut-off of less than 27 pg. rarely, silent β-thalassaemia mutation may present with an mcv over 27 pg and should be considered in those with a positive family history. at-risk couples should be referred for detailed counselling on the options for prenatal diagnosis. these include chorionic villous sampling or amniocentesis, which are used to obtain fetal dna samples for genetic analysis. polymerase chain reactions and precise hybridization assays to detect single point mutations using very small dna samples have also been developed. a less invasive and less risky option is to isolate fetal dna circulating in the maternal blood for genetic analysis. pre-implantation genetic diagnosis is a newer technique where dna from the blastomere is used for genetic diagnosis. ultrasound can be used from the 2nd trimester for fetuses suspected of having α-thalassaemia to detect signs of hydrops fetalis and enlarged placenta (figure 65.9) . 175 • hydroxyurea -helpful in some patients with β-thalassaemia intermedia, but not as effective in thalassaemia major • histone deacetylase inhibitors -derivatives of butyric acid; intermittent pulses with hydroxycarbamide has been tried • kit ligand • decitabine • knockdown of bcl11a (regulator of γ-globin expression) • erythropoietin. • vitamins c and e • fermented papaya preparations. • successful in β-thalassaemia animal models using a retroviral vector transferring the human β-globin gene sequence and its promoter region into mice stem cells • β-globin gene transfer into progenitor hematopoietic cells of humans is also being studied • other molecular approaches being tried include using different mutations of stop codons and aberrant splicing. partner testing in all cases β-thalassaemia trait protecting red cells. red cells lack any other source of nadph and are solely dependent on the pentose phosphate pathway so g6pd deficiency leaves these cells with no defence against oxidative damage. oxidative damage results in denatured haemoglobin aggregates which form heinz bodies (denatured haemoglobin precipitates). these damaged cells bind to the membrane cytoskeleton resulting in decreased cellular deformability, and are also destroyed in the spleen, resulting in haemolysis. the level of enzyme activity is higher in young erythrocytes than in more mature cells so older cells are more susceptible to haemolysis. the global distribution of g6pd deficiency mirrors that of malaria, and where malaria has historically been prevalent, and it provides a degree of protection against malaria. 181 • class iv -normal (60-150% enzyme activity) • class v -increased activity (>150% enzyme activity). g6pd enzyme variants can be distinguished by their electrophoretic mobility. 184 g6pd b, the wild-type enzyme, and g6pd a + , a common variant in populations of african descent, demonstrate normal enzyme activity and are not associated with haemolysis. g6pd a − is the most common variant associated with mild to moderate haemolysis with approximately 10-25% hb e is caused by a substitution of glutamic acid by lysine at codon 26 of the β-globin gene. 177 this causes reduced synthesis of the β-e chain and leads to a thalassaemia phenotype. hb e β-thalassaemia affects at least a million people worldwide and is an important health problem particularly in the indian subcontinent and south-east asia. in some areas, it has replaced β-thalassaemia as the most common thalassaemia disorder. the frequency of hbe reaches 60% in many regions of thailand, laos and cambodia with estimates of at least 100 000 new cases of hbe β-thalassaemia expected in the next few decades in thailand alone. the natural history of hbe thalassaemia is highly variable; some patients are asymptomatic (e.g. heterozygotes, hbe 20-30% or homozygotes hbe, 80-90%) while others (e.g. hbe with β-thalassaemia) may be transfusiondependent. pathophysiology glucose-6-phosphate dehydrogenase (g6pd) deficiency was originally recognized through its association with haemolysis related to eating fava beans ('favism') and primaquine ingestion. 178 g6pd deficiency is the most common enzyme defect in humans and is present in about 400 million people worldwide (figure 65 .10). 179, 180 it is an x-linked, hereditary defect caused by mutations in the g6pd gene. g6pd is an enzyme that catalyses the first reaction in the pentose phosphate pathway, to produce nadph, which is an important antioxidant used to preserve the reduced form of glutathione. 178, 181 reduced glutathione acts as a scavenger for oxidative metabolites thereby <0.5% 0.5-2.9% 3-6.9% 7-9.9% 10-11.9% 15-25% of africans carrying this variant. g6pd mediterranean, present in all countries surrounding the mediterranean sea, middle east, india and indonesia, has the same electrophoretic mobility as g6pd b but the enzyme synthesis and catalytic activity are reduced. in several populations, g6pd a − and g6pd mediterranean co-exist. the clinical manifestations of g6pd deficiency can be classified into: (i) asymptomatic; (ii) acute haemolytic anaemia; (iii) favism; (iv) neonatal jaundice; and (v) chronic non-spherocytic haemolytic anaemia. acute haemolytic anaemia in g6pd deficiency can be secondary to infection (e.g. pneumonia, hepatitis a and b, and typhoid fever) or oxidant drugs, or may be precipitated by diabetic ketoacidosis, myocardial infarction and strenuous physical exercise. 185, 186 a list of the drugs which may cause haemolysis in g6pd-deficient individuals (table 65 .9) 187 can be obtained from: http://www.g6pd.org/favism/english/index.mv. a drug which is deemed to be safe for some g6pd-deficient individuals may cause haemolysis in others due to the heterogeneity of the underlying genetic variants. haemolysis typically occurs within 1-3 days after commencing the drug and can produce intense haemoglobinuria. fortunately, the disorder is self-limiting and most patients do not develop renal impairment or anaemia requiring transfusion. the spontaneous recovery reflects replacement of the older, enzyme-deficient red cells by younger reticulocytes which can withstand oxidative injury. 186 if the precipitating cause has been removed the haemoglobin begins to recover after 8-10 days. acute renal failure due to acute tubular necrosis and tubular obstruction by haemoglobin casts can develop as a complication of haemolysis in g6pd deficiency. this occurs more often in adults than children and may require haemodialysis. this occurs predominantly in boys aged 1-5 years in mediterranean countries, but it has also been observed in the middle east, asia and north africa. both intravascular and extravascular haemolysis, occasionally severe enough to cause renal impairment, can occur after eating fresh or cooked fava beans, and favism has been reported in breastfed babies of mothers who have eaten fava beans. 179 divicine and isouramil have been implicated as the toxic components of fava beans. 188 neonatal jaundice this occurs in one-third of male babies in areas where g6pd deficiency is common and is likely due to g6pd deficiency. 179 it presents 1-4 days after birth and can lead to kernicterus. 189, 190 maternal exposure to oxidant drugs, and even naphthalenecamphor mothballs, can precipitate haemolysis in affected babies. breast-feeding mothers should therefore be warned to avoid offending drugs, umbilical potions containing fava, triple dye or menthol, and should not apply henna to the skin or use clothes that have been stored in naphthalene. 190 premature infants and babies who have co-inherited the mutation for gilbert's syndrome are at particular risk. phototherapy and exchange transfusion therapy may be required to reduce the level of unconjugated bilirubin. the diagnosis may be easily missed so assessment of g6pd status should be undertaken for any jaundiced infant whose family history or ethnic or geographic origin suggest the likelihood of g6pd deficiency, and in infants who respond poorly to phototherapy. 191 this is an unusual manifestation of g6pd deficiency and usually presents in childhood. 179, 184 there may be a history of severe neonatal jaundice, episodic or worsening anaemia which requires blood transfusions, and complications from gallstones. although these individuals usually have a well-compensated anaemia, and require transfusions only for exacerbations, rarely some may become transfusion-dependent. antioxidants such as vitamin e and selenium may be of benefit in some cases. the haemolysis does not resolve following splenectomy. folic acid supplementation is necessary to support the increased compensatory erythropoiesis. the diagnosis of g6pd deficiency is usually suspected when neonatal jaundice occurs in an area where g6pd deficiency is drugs which may cause haemolysis in g6pd-deficient individuals common or when an episode of non-immune haemolytic anaemia occurs in association with an infection or drug. the appearance of the red cells on the blood film is characteristic because denatured haemoglobin concentrates in one area within the cell creating 'helmet' or 'bite' cells. 192 denatured haemoglobin precipitates in peripheral red blood cells as heinz bodies which can be detected by staining with methyl violet. definitive diagnosis of g6pd deficiency is by quantitative spectrophotometric analysis of the rate of nadph production. point of care tests for g6pd deficiency are being developed but have not yet been validated for routine use. measuring enzyme activity during an episode of acute haemolysis is not helpful since reticulocytosis, which is a feature of acute haemolysis, produces a false-negative result because of the high enzyme levels in younger erythrocytes. 179, 186 management the most effective management strategy for g6pd deficiency is to prevent haemolysis by avoiding triggering agents like infections, drugs and fava beans. for the milder variants (e.g. class iii and iv), drugs known to trigger haemolysis may be given to individuals with g6pd deficiency if the benefits outweigh the risks and the blood count is closely monitored (e.g. use of low-dose primaquine for individuals with g6pd avariant). screening programmes have been established in some mediterranean and other populations where g6pd deficiency is prevalent. 193 haematological complications of malaria (see chapter 43) the pathophysiology of anaemia in malaria is multi-factorial and influenced by the age of the individual and their antimalarial immune status. anaemia mechanisms in malaria involve: • haemolysis with increased red cell destruction of both infected and bystander erythrocytes • dyserythropoiesis • hypersplenism • haemolysis • co-existent conditions which can cause anaemia. haemolysis is more common in non-immune individuals with acute malaria, whereas dyserythropoiesis is the predominant mechanism for anaemia in recurrent falciparum malaria. 207, 194 haemolysis is the result of red cell phagocytosis by the reticuloendothelial system and is triggered by damage to the red cell membranes and exposure of abnormal surface antigens on their surface. [195] [196] [197] [198] ten uninfected red cells are removed from the circulation for each infected red cell destroyed, 199 possibly related to loss of red cell complement regulatory proteins and increased levels of circulating immune complexes. 200 this may partly explain the persistent or worsening anaemia following parasite clearance and the poor correlation between parasitaemia and the severity of anaemia noted in some studies. 207 an increased incidence of anaemia has been noted in malaria vaccine trials possibly due to enhanced clearance of uninfected red blood cells. 201 decreased erythropoeisis with abnormalities in red cell precursors and reticulocytopenia is found consistently on examination of bone marrow from malaria-infected patients. 202 the decreased erythropoiesis is due to many factors including low levels of tnf-α, high levels of interleukin-10, abnormalities of erythropoietin, a decrease in burst colony forming units, cytokine-induced suppression of red cell production and the inhibitory effect of the malarial pigment haemozoin. [203] [204] [205] [206] epidemiology malaria-related anaemia is most commonly seen in children and pregnant women. the prevalence of malarial anaemia in sub-saharan africa in children is 30-90% and in pregnant women it is 60-80%. 207 the highest prevalence is in infants and children less than 3 years of age. infants may acquire malaria through the placenta. 208, 209 individuals living in malarious areas may have multiple reasons for anaemia such as bacteraemia, hookworm infections and vitamin a deficiency 208 making it difficult to assign anaemia solely to malaria. however, animal studies and the fact that anaemia improves with anti-malarial treatment suggest a direct relationship between malaria infection and anaemia. 210, 211 for example, in tanzanian children about 60% of anaemic episodes were thought to be caused by malaria. 212 who defines severe anaemia attributable to malaria as: (i) haemoglobin concentration <50 g/l or haematocrit <15%; (ii) parasitaemia with >10 000 parasites/µl of blood and (iii) normocytic blood film (to exclude other common causes of anaemia). 213 however, aspects of this definition have been criticized because blood films are not examined routinely and parasite density varies with endemicity and age. 207 although traditionally it is p. falciparum that has been associated with the most severe malaria-related anaemia, p. vivax is also a major risk factor for severe anaemia especially in young children or those with chronic and recurrent infections. p. vivax anaemia is associated with recurrent bouts of haemolysis of predominantly uninfected erythrocytes with increased fragility. 214 symptoms of malarial anaemia can vary from negligible to profound depending on the degree of anaemia and the rapidity of onset. splenomegaly is a common feature of malarial anaemia because of the role of the spleen in the removal of both infected and uninfected red cells. 215 blackwater fever, characterized by intense intravascular haemolysis with haemoglobinuria and occasionally renal failure in a patient with malaria, may be related to underlying glucose-6-phosphate deficiency. 216, 217 factors such as poor nutrition, deficiencies of vitamins and micronutrients, bacteraemia, and hookworm or hiv infection may co-exist with malaria and contribute to anaemia 209 so nonmalarial causes of anaemia should be considered in patients whose anaemia does not respond to malaria treatment. the management of severe malarial anaemia involves supportive care and treatment of the malaria and any other underlying conditions. recovery from malaria-associated anaemia can be slow, taking 6 weeks or even longer if there are episodes of re-infection. 212 in children, blood transfusion is usually reserved for those with haemoglobin levels of less than 40 g/l (<50 g/l if there are complications such as respiratory distress 207 ). there of parasitized red cells 227 and the release of von willebrand factor multimers which cause widespread platelet aggregation leading to thrombocytopenia 228, 84, 231 platelet synthesis by the bone marrow is relatively well maintained during infection 231, 229 but antiplatelet antibodies, immune complexes and splenomegaly all contribute to thrombocytopenia. 230 thrombocytopenia occurs in 60-90% of individuals infected with malaria irrespective of the species of plasmodium. 214, 231 thrombocytopenia in febrile patients in an endemic area increases the probability of malaria by a factor of 5 232 and in individuals returning from tropical countries with a fever, thrombocytopenia is highly specific for malaria infection. 233 profound thrombocytopenia is unusual and malaria-associated thrombocytopenia is rarely associated with haemorrhagic manifestations. 234 the clinical consequences of platelet aggregation and endothelial binding are primarily microvascular ischaemia. this may manifest as renal impairment, cerebral ischaemia, and occlusion of retinal vasculature or even in some cases, skin necrosis. bleeding is unlikely, although in severe thrombocytopenia, petechiae or purpura may develop which denotes extravasation of red cells into the subcutaneous tissue. 235 continued platelet activation and consumption can exacerbate bleeding and decreased circulating platelets are associated with increased vascular leakage and the development of oedema. 236 platelet transfusions are rarely required because the platelet count generally rises rapidly on treating the underlying malaria. coagulopathy is a disturbance of the whole coagulation system involving not just coagulation factors but platelets, anticoagulant factors, fibrinolytic system and, in the case of malaria, the parasitized red cells and the vascular endothelium. parasitized red cells induce expression of tissue factor on endothelial cells and monocytes, release of microparticles, cytokine release and platelet clumping, all of which initiate blood coagulation and tilt the balance towards the pro-coagulant state ( figure 65.12) . 234, [237] [238] [239] [240] [241] [242] anticoagulant factors are severely depleted in malaria. protein c and antithrombin levels are inversely correlated with severity of falciparum malaria and return to normal with treatment of the malaria. 245 have been some concerns about a possible increased risk of infection associated with iron supplementation for children in malarious areas 218, 219 but current recommendations advocate that where iron deficiency and malaria are common, iron supplements should not be withheld and appropriate anti-malarial treatment or prevention should also be offered. 220 the best way to prevent malarial anaemia is to prevent malaria infection by avoiding mosquito bites (e.g. through the use of bed nets) or through chemoprophylaxis. malaria chemoprophylaxis during infancy can reduce both malaria and anaemia. 221 children who have been hospitalized with severe malarial anaemia may benefit from intermittent preventive malarial therapy after discharge to prevent recurrence of anaemia. 222 daily co-trimoxazole prophylaxis which is used for hiv-infected individuals has been shown to reduce malaria parasitaemia and anaemia. 223 the normal platelet life span of 7-10 days is reduced to less than 4 days in malaria infection. 224 several factors are responsible for thrombocytopenia in malaria infection, the most common being increased platelet activation and aggregation ( figure 65 .11). 225 platelet activation is by parasitized red cells which express surface tissue factor and initiate coagulation and platelet aggregation. the resultant activated endothelium binds platelets and sequesters them in vascular beds including in the cerebral vasculature. 226, 234 these platelets facilitate the adhesion and to 46% after a year. 253, 247 although transfusions may be required in severe life-threatening cases of anaemia, aggressive transfusion therapy has been associated with fatal pulmonary emboli due to accelerated haemolysis and disseminated intravascular coagulation. 254 in those who do not respond to art, erythropoietin may be considered since reduced responsiveness to this hormone and antierythropoietin antibodies have been noted in hiv patients. erythropoietin is particularly useful in individuals whose erythropoietin levels are less than 500 iu/l 255 because in addition to increasing the haemoglobin it can also improve the quality of life. 256 erythropoietin may take several weeks to achieve full effect and patients should be replete in haematinics. erythropoietin can very rarely be associated with thrombosis or pure red cell aplasia. thrombocytopenia is a common finding in hiv-infected patients and it may be the initial manifestation of hiv infection in as many as 10% of patients. data from wealthy countries demonstrate platelet counts less than 150 × 10 9 /l in 11% of patients, and less than 50 × 10 9 /l in 1.5%. overall the 1-year coagulopathy in malaria infection is unusual, occurring in less than 5% of cases. it appears to be most common in adults with cerebral malaria who may present with gastrointestinal bleeding 243 or with microvascular ischaemia in the brain, kidneys, retina and occasionally, the dermal vasculature. 244 prolongation of prothrombin time and activated partial thromboplastin time only occur in 4-8% of patients with p. falciparum infection and coagulopathy does not appear to be a feature of p. vivax infection. 245 since coagulation factors need to be depleted to less than 20% of normal to prolong the clotting times, these tests can be normal despite active coagulopathy. management of coagulopathy aims to restore the balance between pro-and anticoagulant processes. this is complex and requires input from a coagulation specialist and ideally, access to plasma, heparin and factor concentrates and a well-equipped coagulation laboratory. anaemia anaemia is very common in hiv-infected individuals occurring in up to 20% at initial presentation and about 70% at some stage during their disease. 246 thirty-seven percent of patients with clinical aids have a 1-year incidence of anaemia (haemoglobin <100 g/l) 246 and high rates of anaemia persist despite combination anti retroviral treatment (art). 247 anaemia is directly related to mortality in hiv infection and is independent of other risk factors including cd4 count. 248 there are multiple reasons for anaemia in hiv-infected patients (box 65.17), which often co-exist in individual patients. bone marrow infection by mycobacteria species, histoplasma, cryptococcus and penicillium marneffei can all decrease red cell production 249 and can be detected by bone marrow examination and cultures. parvovirus has a predilection for the erythroid progenitor cells and can cause severe anaemia in hiv-infected patients. serological tests for parvovirus are unhelpful in hivinfected patients and viral polymerase chain reaction is needed to confirm the diagnosis. 250 the likelihood of parvovirusinduced anaemia increases with the severity of anaemia and has been found in 31% of individuals with hiv and haemoglobin less than 70 g/l. 251 haemophagocytosis occurs in hiv infections and may be secondary to co-infection with mycobacteria, cytomegalovirus, epstein-barr or other herpesviruses. poor nutrition due to socioeconomic reasons, hiv-related anorexia, malabsorption from conditions affecting the gastrointestinal tract, and achlorhydria may contribute to anaemia. haemolytic anaemia occurs secondary to drugs or concomitant glucose-6 phosphate dehydrogenase deficiency and because reticulocytopenia is common in those with hiv infection, reticulocyte counts cannot be used to exclude haemolysis. although the direct coombs test may be positive in patients with hiv infection, autoimmune haemolysis is not a common cause of anaemia. a reduction in red cell precursors has also been noted in children in africa with severe anaemia. 252 treatment of hiv-related anaemia should focus on starting art and eliminating any other factors, such as infections or vitamin deficiencies, which may contribute to the anaemia. in wealthy countries art has been shown to reduce anaemia prevalence from 65% to 53% within 6 months of starting treatment, non-hodgkin's lymphoma (nhl) was noted to be associated with hiv infection early in the epidemic and is an aidsdefining illness. 270 the incidence of nhl is up to 200 times greater in hiv-infected adults than in those who are not infected, and it is responsible for nearly one-sixth of the deaths attributable to aids. since the introduction of haart, the incidence of all types of nhl has decreased by approximately 30-50% 271, 272 and the outcome of hiv-infected patients with lymphoma has improved. in the setting of clinical trials, the 60% 1-year survival rate is comparable to those without hiv infection. 273 the incidence of hodgkin's lymphoma has increased in the post-haart era, possibly due to immune reconstitution and increased cd4 cells. 274, 275 evidence of epstein-barr virus (ebv) infection can be found in virtually all cases of hodgkin's disease. 276 hiv-related lymphomas (box 65.18) 277 (see also lymphomas, below), are broadly divided into systemic lymphomas (80%) and primary central nervous system lymphomas. 278 the incidence of highly aggressive lymphomas, either burkitt's lymphoma (approx. 25%) or diffuse large b-cell lymphoma (approx. 75%), is much higher in hiv-infected patients than in those without infection. 279 although t-cell lymphomas are uncommon in hiv disease (1%), there has been an increase in recent years. the incidence of primary central nervous system lymphoma in hiv-affected individuals is 2-6% and it is 1000 times more common than in the general population. 280 the pathogenesis of nhl in hiv infection is related to the inadequate host immune responses to viruses with oncogenic potential, predominantly ebv and human herpesvirus 8 (hhv8)/kaposi's sarcoma-associated herpesvirus. this allows unregulated lymphoid growth and an accumulation of genetic abnormalities in b cells. 272 markers of b-cell activation such as serum immunoglobulins and free light chains, and cd4 cell count have been suggested as predictive markers for the development of nhl in hiv infection. 281, 282 extranodal and leptomeningeal involvement, and b-symptoms occur in the majority of hiv-infected patients with nhl and the bone marrow is commonly involved. the most common extranodal site to be involved is the incidence of moderate thrombocytopenia (<50 × 10 9 /l) is 3.7%, though this is higher in those with clinical aids (8.7%). 257, 258 thrombocytopenia is more common in those who abuse drugs, have opportunistic infections and malignant disorders of the bone marrow (e.g. lymphoma), and it may also be a side-effect of therapeutic drugs. 259 the most common cause of thrombocytopenia in hiv infection is immune thrombocytopenia which may be associated with hepatitis c co-infection, and produces decreased platelet survival, particularly at cd4 counts below 200/µl. the anti-platelet antibodies, immune complexes and cross-reacting antibodies to hiv envelope proteins and platelets, which occur in hiv-associated thrombocytopenia 259, 260 may also contribute to generation of reactive oxygen species. 261 platelet production can also be affected in hiv infection and may explain the high levels of thrombopoietin that have been documented in hivrelated thrombocytopenia. 262 some cases of hiv-related thrombocytopenia may undergo spontaneous remission so treatment of thrombocytopenia is usually only initiated if it is associated with bleeding, which is unusual. 259 the first line of treatment involves antiretroviral therapy with the aim of achieving undetectable plasma hiv viraemia. 263, 264 any drugs that may be associated with causing thrombocytopenia should be withdrawn and opportunistic infections or secondary malignancies treated. the treatment of immune thrombocytopenia is the same as in non-hiv cases and options include a short course of steroids, intravenous immunoglobulin (short-lived response), anti-d, interferon-α or splenectomy. although there are multiple causes of thrombocytopenia in hiv-positive individuals, one of the most devastating is the thrombotic microangiopathy of thrombotic thrombocytopenic purpura (ttp). this is because the combination of haemolytic anaemia and microthrombi has a very poor prognosis. symptoms are nonspecific and may include fever, headache, bleeding and changes in consciousness. if ttp is suspected, an urgent blood film should be requested and the combination of thrombocytopenia with red cell fragmentation is highly suggestive of ttp. ttp associated with hiv infection was more frequent before the introduction of art and is more common if adherence to treatment is poor or resistance to therapy has developed. 265 ttp is thought to be due to endothelial damage, but unlike the situation in non-hiv-infected individuals, low levels of adamts-13 are not a useful predictor of outcome. 266 treatment of ttp involves plasma exchange, and although refractoriness may occur, this can be corrected by art in some cases. 266 if art is administered in these cases it is important to maintain adherence throughout the period of plasma exchange. if apheresis facilities are limited, plasma infusions alone (30 ml/ kg per day) may also produce a response. 267 art should also be administered immediately after plasma exchange to minimize drug removal. patients with a viral load of less than 500 000 copies/ml generally require fewer plasma exchanges for remission than those with a higher load. 265 survival of patients with hiv-associated ttp in the pre-art era was rarely longer than 2 years, even with plasma exchange and steroid treatment, but for patients who are compliant with art the mortality is around 4%. 268 which immediately limits the amount of blood loss. exposure of the subendothelial space releases factors such as von willebrand factor multimers which bind to platelets and initiate platelet adhesion to the endothelium. the adherent platelets release their granules and attract more platelets, which in combination with fibrinogen, form an aggregate. the activated platelets also attract coagulation factors thereby promoting the clotting process. the critical parts of clot formation are the conversion of prothrombin to thrombin and the thrombinfacilitated conversion of fibrinogen to fibrin (figure 65.13) . haemostatic control mechanisms operate throughout the clotting process to prevent excessive clot formation and involve proteins c and s, and anti-thrombin and antifibrinolytic systems. any alteration in these regulatory pathways can lead to either bleeding or thrombotic complications. bleeding can result from: • inadequate vasoconstriction, due to vascular problems which can be acquired (e.g. viral haemorrhagic fevers or immune vasculitis) or congenital (e.g. collagen vascular disorders) • qualitative or quantitative abnormality of von willebrand factor causing von willebrand's disease • decreased number or function of platelets which can be either acquired (e.g. aspirin, nsaids) or congenital (e.g. platelet function defects) • qualitative (e.g. caused by inhibitors to coagulation factors, commonly factor viii) or quantitative (e.g. haemophilia) abnormality of coagulation factors • increased fibrinolysis (e.g. viral haemorrhagic fevers, snake bites). acquired bleeding disorders are commonly caused by vitamin k deficiency, disseminated intravascular coagulation (dic) or platelet disorders (box 65.19) but may sometimes be due to acquired inhibitors of coagulation factors. the initial laboratory tests in a patient with excessive bleeding should therefore include a platelet count, clotting screen (prothrombin time (pt) and activated partial thromboplastin time (aptt)), and gastrointestinal tract, often the stomach or the perianal region. 283 hepatic involvement, seen in a quarter of cases, is associated with a particularly poor prognosis. cns disease may be asymptomatic so diagnostic lumbar puncture may be required. 284 hiv-related lymphomas frequently present with poor prognostic features such as elevated serum lactate dehydrogenase levels. 285, 286 older age, lowest nadir cd4 cell counts prior to nhl diagnosis, developing nhl while on art, and cumulative hiv viraemia are also poor prognostic features. 282 a formal prognostic scoring system has been developed which takes into account the cd4 count (<100 cells/µl). 287 some types of hiv-related lymphoma are associated with characteristic clinical and laboratory features. primary effusion lymphoma is an aggressive lymphoma characterized by effusions in serosal cavities in the absence of any other tumour masses. 288, 289 it is strongly associated with hhv8 infection and the virus can be identified in the nuclei of the malignant cells. plasmablastic lymphoma mainly affects the oral cavity and the mucosa of the jaw and is typically associated with epstein-barr virus. 290 histological examination of biopsied tissue is necessary to confirm the diagnosis and type of lymphoma. diagnostic difficulties may arise because hiv-related hyperplasia in lymph node biopsies may be confused with lymphoma, the histological appearance of hiv-related lymphomas may be different from those of non-infected individuals 291 and many opportunistic pathogens may mimic the appearances of nhl, or co-exist with it, and will need to be identified or excluded before making a diagnosis of lymphoma. prior to the widespread use of art, conventional lymphoma chemotherapy resulted in considerable toxicity, increased opportunistic infections and high mortality. art has facilitated the use of conventional doses of chemotherapy in conjunction with haematopoietic growth factor support. this has markedly improved the outcome of patients with hiv-related lymphomas who now have overall response rates of 60%. 292 the concomitant use of art and chemotherapy is therefore recommended, especially in those with cd4 counts of less than 100/µl. anti-cd20 antibody is now included in treatment regimens for nhl, and studies that include patients with hivrelated lymphomas all report favourable outcomes. 293, 294 some antiretroviral agents such as zidovudine are best avoided in combination with chemotherapy, because it adds to the myelosuppression of chemotherapy. didanosine may worsen the peripheral neuropathy caused by taxanes and vinca alkaloids. hiv-infected patients undergoing chemotherapy should receive adequate anti-infective prophylaxis due to the high risk of opportunistic infections such as pneumocystis, herpes simplex and zoster and candida. consolidation chemotherapy and stem cell transplant have been used successfully in relapsed hivrelated lymphomas. haemostasis is maintained by interactions between vessel walls, platelets and a balance between pro-and anticoagulant factors. although the process of haemostasis is usually considered to occur in a stepwise fashion, in vivo the steps happen virtually simultaneously. activation of the lining of the endothelium by trauma, cancer cells or cytokines triggers vasoconstriction, the tests for each pathway is given with arrows corresponding to each box complications, malignancies and infections and is a serious condition with a high mortality. patients present with spontaneous bruising or excessive bleeding from minor wounds such as venepuncture sites, and they may also have signs of complications such as renal failure, acute respiratory distress syndrome and microangiopathic haemolytic anaemia. dic is associated with a combination of depleted clotting factors (i.e. prolonged pt and aptt), a falling platelet count, red cell fragments on the blood film, raised d-dimers or fibrin degradation products, and reduced fibrinogen levels. management of disseminated intravascular coagulation includes treating or removing the underlying cause, and correcting the haemostatic abnormalities with combinations of platelets, cryoprecipitate and fresh frozen plasma. although bleeding due to thrombocytopenia is unusual unless the platelet counts falls below 10-20 × 10 9 /l, bleeding may occur with a normal platelet count and normal clotting screening tests (i.e. pt and aptt) if platelet functions are impaired (e.g. myelodysplastic syndromes). platelet transfusions are generally not required unless there is active bleeding or prior to surgery. idiopathic thrombocytopenic purpura. idiopathic thrombocytopenic purpura is due to immune destruction of platelets. it is usually primary but can be associated with conditions such as lymphomas and infections including hiv. it may present incidentally or with petechiae, bruising or bleeding from the nose or gums, especially if the platelet count is less than 20 × fibrinogen levels, which may be helpful in cases of excessive fibrinolysis (table 65. 10). a difficult venepuncture can cause in vitro activation of the clotting system resulting in a shortened pt or aptt. similar findings may occur in chronic dic due to in vivo activation. the pt and aptt are not necessarily good predictors of the bleeding risk because some clotting disorders associated with thrombosis (e.g. anti-phospholipid antibodies) can prolong the aptt. a shortened aptt can be associated with marked elevation of factor viii levels (e.g. pregnancy) and may be a predictor of deep vein thrombosis. a prolonged thrombin time is caused by quantitative or qualitative fibrinogen deficiency, heparin and fibrin degradation products. reptilase time is helpful to distinguish between fibrinogen abnormalities (prolonged reptilase time) and heparin therapy (normal reptilase time). deficiency of vitamin k can be due to poor diet, small bowel disease or bile flow obstruction. clotting factors (ii, vii, ix and x) are dependent on vitamin k which is a fat-soluble vitamin. vitamin k deficiency therefore causes prolongation of the pt and aptt. in newborn infants, vitamin-k-dependent clotting factors can drop precipitously within a couple of days of birth. this causes haemorrhagic disease of the newborn which particularly affects infants that are premature, exclusively breast fed or have been exposed to drugs for tuberculosis, convulsions or anticoagulation in utero. these babies develop bleeding into the skin and gut, or bleeding from the umbilical stump or circumcision. vitamin k deficiency will respond to intravenous vitamin k (10 mg/day for 3 days orally or by intravenous injection) and in severe bleeding the clotting abnormality can be treated with fresh frozen plasma. haemorrhagic disease of the newborn can be prevented with 1 mg of intramuscular vitamin k given at delivery. disseminated intravascular coagulation (dic) is characterized by activation of haemostasis with widespread fibrin formation, activation of fibrinolysis and consumption of platelets and clotting factors. it may be precipitated by tissue injury, obstetric desmopressin (ddavp) is a relatively inexpensive drug that increases fviii levels and vwf activity within 30 minutes of administration. it is useful in mild haemophilia and mild von willebrand's disease. 299 the major side effects are headaches and hyponatraemia so fluid intake should be restricted to 1.5 l/ day. tranexamic acid mouthwashes may be helpful for oral mucosal bleeding. danazol can increase both factor viii and ix levels within 5-7 days 300 and has therefore been recommended for patients with recurrent haemarthrosis or with central nervous system bleeding which both carry a high risk of recurrence. most thromboembolic episodes are single events and may be associated with precipitating events or underlying risk factors. thrombophilia is the clinical state of hypercoagulability and should be suspected in patients who have a strong family history of thrombosis, or who have recurrent or unusual thromboses. increasing affluence and consequent lifestyle changes mean that the prevalence of thromboembolism is rising in some low-and middle-income countries. risk factors such as sedentary work, obesity, excessive alcohol intake, smoking and additional cardiovascular risk factors are compounded by other 10 9 /l. spontaneous recovery occurs less commonly in adults than in children. it is important to exclude other causes of thrombocytopenia such as drugs, dic or sepsis. the diagnosis can be suspected from a bone marrow examination which shows increased numbers of platelet precursors. treatment with prednisolone (0.25-0.5 mg/kg) is usually only necessary if there is bleeding or excessive bruising and the dose should be reduced slowly once the platelet count improves. second-line treatments include immunosuppressive agents and danazol. splenectomy may also be beneficial but carries an increased risk of infection. platelet transfusions or intravenous gammaglobulin can temporarily increase the platelet count in an emergency or prior to surgical procedures. inherited bleeding disorders can be classified broadly into coagulation factor deficiencies (e.g. factor viii and factor ix deficiencies), von willebrand's disease and platelet disorders. the frequency of genes for inherited bleeding disorders is the same throughout the world. haemophilia a has a prevalence of about 10/10 000, von willebrand's disease of >10/10 000 and haemophilia b of <0.1/10 000. these conditions occur more frequently among populations where consanguineous marriage is common and where prenatal diagnostic facilities are unavailable. in general, individuals with inherited coagulation factor deficiencies present with soft tissue bleeds such as haemarthroses or intramuscular bleeds. those with platelet disorders or von willebrand's disease tend to present with mucosal bleeds, however severe (type iii) von willebrand's disease can present with severe soft tissue bleeds. many of these conditions are diagnosed following excessive and uncontrolled bleeding after trauma or surgical procedures. menorrhagia and delayed severe postpartum haemorrhage may be presenting features of bleeding disorders, particularly von willebrand's disease or hypothyroidism, which can cause decreased synthesis of von willebrand factor. some inherited platelet function disorders are associated with characteristic syndromes (e.g. oculocutaneous albinism or skeletal defects) which may provide a clue to the diagnosis. early recognition of symptoms by clinicians, teachers and the public is important so that early treatment can be established. patients with inherited bleeding disorders are usually managed with blood products (box 65.20) or chemotherapy designed to reduce bleeding and associated complications. 295, 296, 298, 299 clotting factor concentrates may be imported or produced locally by fractionation of plasma and are included in the who list of essential medicines. 297, 298 one international unit (iu) of fviii clotting factor concentrate per capita is recommended as the minimum requirement for countries wishing to achieve optimal survival for their haemophilia population but only about 25% of the estimated 400 000 people in the world with haemophilia receive adequate treatment. management of patients with bleeding disorders relies on a wellequipped and quality assessed laboratory for accurate diagnosis and monitoring of treatment and access to plasma and components for replacement therapy. appropriate support services such as physiotherapy, orthopaedics and counselling should also be available. in many countries inherited bleeding disorders are associated with stigma, which is particularly directed against the mothers of affected children, 297 acute and chronic leukaemias are usually associated with a high white cell count but acute leukaemias can present with normal or even sub-normal white cell counts. morphology of peripheral blood and bone marrow specimens is crucial to confirm the diagnosis. this is particularly important in the case of acute leukaemia in children which may be mistaken for an acute viral infection. staining methods including sudan black b, myeloperoxidase and nonspecific esterase are important to distinguish between the different subtypes of acute myeloid and lymphoid leukaemias and therefore to guide treatment. acute myeloid leukaemia (aml). prevalence of this increases with age and the success rate with chemotherapy protocols is not high even in the most sophisticated centres. neutropenia and myelosuppression requiring intensive blood component support occur during chemotherapy 314 and bone marrow transplantation offers the best option for cure for patients who relapse. management of aml is therefore complex and expensive. hydroxycarbamide or subcutaneous cytarabine may be used as a palliative treatment. acute promyelocytic leukaemia (aml subtype m3). this must be distinguished from other types of acute myeloid leukaemia because it has a high cure rate with early treatment. it predominantly affects young adults and it has a high incidence in certain ethnic groups especially those of latin american descent. 315 a treatment protocol which includes all-transretinoic acid with combination chemotherapy has been developed which is feasible in low-income countries. 315, 315 another regimen based on intravenous arsenic trioxide has been developed in india, 316, 317 which has an 86% response rate with good diseasefree and overall survival. conditions that are associated with thrombosis such as hiv infection, and chronic infections including tuberculosis 301, 302 and helminth-induced eosinophilic myocarditis. 303 african americans are more likely to be diagnosed with pulmonary embolism rather than deep-vein thrombosis compared to other racial groups 304 and african patients with thrombosis tend to be younger than those reported in literature 305 with higher mortality rates (around 28%) possibly due to late presentation and poor access to health facilities. asian populations 306-308 seem to have a lower prevalence of symptomatic venous thrombosis compared to african americans. 304 very little is known about the prevalence of prothrombotic factors such as mutations of the prothrombin gene or deficiencies of antithrombin, protein c and protein s in tropical countries, although high rates of factor v leiden, a risk factor for venous thrombosis, have been described in tunisia. 309, 310 lupus anticoagulant and anti-phospholipid syndrome, which are associated with increased thrombosis risk, are increased in afro-caribbean populations, especially in the presence of hiv, and have also been described in nigerian women with pre-eclampsia. 311, 312 the management of venous thrombosis is initially with heparin and then with warfarin for 3-6 months. compliance may be difficult in low-resource settings because of the requirement for regular monitoring of warfarin. it is therefore important to try to prevent thromboses by removing any underlying risk factors and by treating individuals at risk of thrombosis with a short course of prophylactic heparin to cover procedures known to be associated with thrombosis risk. this can present as venous or arterial thromboembolism and it may be inherited (e.g. deficiencies of thrombin, protein s or protein c) or acquired (e.g. antiphospholipids). the patient's personal and family history, and the results of clinical and imaging examinations to confirm thrombosis, may suggest the diagnosis. the laboratory tests needed to determine the cause and classify the type of thrombophilia, and their interpretation, are complex, so patients with recurrent or unusual thromboses should be referred to a specialist centre. haematological malignancies are predominantly leukaemias, lymphomas and myelomas. some of the general approaches for managing these conditions in low-income countries are outlined in box 65.21 313 but definitive treatment should be undertaken by a specialist haematology unit. leukaemias can be broadly classified as acute or chronic, and lymphoid or myeloid. the presenting symptoms and signs are related to the disturbed blood cell production from the bone marrow due to the effects of the malignant cell clone (box 65.22) . acute leukaemias are characterized by rapid progression and poor prognosis if left untreated whereas chronic leukaemias generally follow a much slower course. • mobilization of the community (especially parents and families) to raise awareness among local councils and government bodies about the treatability of the cancers and benefits from curing them • find an external partner unit locally, nationally or internationally which is already well-established and willing to help but will not dictate terms • improvement of supportive care facilities, especially protection from those with infectious diseases • development of a safe and reliable blood transfusion service • provision of subsidized travel, and satellite clinics to lessen the burden • development of appropriate protocols for each disease entity which is locally practicable with minimum cost and maximum efficacy • development of medical, nursing and paramedical expertise in the diseases to be treated -initially by offering visiting fellowships and in the long term for the trained individuals to arrange regional and local teaching programmes • formation of a cooperative group bringing together all the professionals involved in the speciality within a country or region to share expertise and develop training programmes. acute lymphoblastic leukaemia (all). this is the most common type of leukaemia in children. it has a good prognosis when treated with modern chemotherapy protocols with cure rates in the best centres exceeding 80%. 318 in low-income countries, cure rates are much lower at around 35% 319 primarily because of failure to complete therapy and deaths caused by treatment. considerable improvements in all outcomes have been achieved by twinning institutions in developing countries with specialist centres elsewhere in the country or internationally. 313 measures that may improve outcomes focus on preventing abandonment of therapy (e.g. providing funding for transport, satellite clinics and support groups) and prompt treatment of infection. 320 treatment in a dedicated paediatric oncology unit using a comprehensive multidisciplinary team approach and protocol-based therapy, is also associated with improved outcomes in resource-poor settings. 321 chronic myeloid leukaemia (cml). management has been revolutionized by tyrosine kinase inhibitors (e.g. imatinib) which can produce complete remission in over 80% of cases. once the diagnosis of cml is established, hydroxycarbamide can be used to reduce the white cell count, followed by treatment with a tyrosine kinase inhibitor. manufacturers will provide the drug free of charge to patients in low-income countries with confirmed cml and generic forms of tyrosine kinase inhibitors are now becoming available. chronic lymphocytic leukaemia (cll). this occurs predominantly in older people and usually presents with lymphadenopathy and recurrent infections. treatment is with chlorambucil and prednisolone although aggressive forms require combination therapy with rituximab, fludarabine and cyclophosphamide. treatment is generally not curative but the disease may be indolent and drugs may only be required if the patient has symptoms or if there is a risk of hyperviscosity from a very high lymphocyte count. approximately 30 000 cases of non-hodgkin lymphoma (nhl) occur in the equatorial belt of africa each year (table 65 .11). 322 there are marked geographical variations in prevalence but up to 50% are thought to be related to hiv infection. 322 burkitt's lymphoma, a b-cell nhl, was originally described in children from africa and has an estimated incidence of 30-70 per million. lymphomas are broadly classified into hodgkin's lymphoma and nhl; nhl are divided into b-cell, t-cell and nk-cell, and immunodeficiency-associated types. the clinical presentation of lymphomas is characterized by enlargement of the lymphoid organs and subsequent compression of the adjacent structures, infiltration of organs by the malignant lymphoid cells and a dysfunctional immunological system which can manifest as immunosuppression or excessive but dysregulated immune activation associated with, for example, autoimmune conditions. the diagnosis and management of the various types of lymphomas are complicated and should be undertaken in a specialist box 65. 22 clinical features of leukaemias • fatigue and cardiac symptoms from anaemia • bleeding from thrombocytopenia • increased risk of infections despite a higher number but dysfunctional white cells • lymphadenopathy and hepatosplenomegaly occur with all although lymphadenopathy may be observed in the monocytic variety of aml • blindness due to hyperviscosity from hyperleukocytosis • tumour lysis syndrome due to spontaneous cell lysis presents as renal failure • pustules or pyogenic infections of the skin from minor wounds • bleeding gums are a characteristic feature of acute monocytic leukaemia • disseminated intravascular coagulation can occur with acute promyelocytic leukaemia • gout can arise from breakdown of the excess white cells and release of uric acid • oral aphthous ulceration is seen with severe neutropenia in both aml and all • granulocytic sarcoma or chloroma represent extramedullary deposits of leukaemic cells in any organ but mainly the skin. this may occur in the absence of peripheral blood involvement and is more common with chromosomal translocation (8; 21) of aml • central nervous system manifestations due to sludging of the cerebral circulation by the malignant cells or increased intracranial pressure due to ventricular blockade can occur. monocytic myeloid leukaemia can also involve the meninges • intracranial haemorrhage can occur in all with very high white cell counts (>400 × 10 9 /l) • bone pain and arthralgia can be a presenting feature of all in children in more than a quarter. these children may present with a limp or unwillingness to walk due to marrow infiltration by leukaemic cells. rarely, they may have normal blood counts delaying the diagnosis of all • anterior mediastinal mass (thymus enlargement) can also occur in children and young adults with all which may present as superior venocaval obstruction • painless enlargement of scrotum is a sign of testicular leukaemia or hydrocele from lymphatic obstruction. priapism can result from hyperleukocytosis rarely. • most often asymptomatic and usually suspected on blood counts • the chronicity of cml or cll tends to cause gradual-onset symptoms since the patients get adjusted to the slowly developing anaemia • abdominal discomfort and early satiety are a feature of cml due to excessive splenomegaly compressing the stomach and reducing the luminal volume • sternal tenderness may be noted in cml • hyperleukocytosis in cml can occur more often than with aml or all due to the gradual increase in white cells. this can cause symptoms like hyperuricaemia and gout, tinnitus, priapism or central nervous system disturbances • left shoulder tip pain can arise from splenic infarction from the massive splenomegaly in cml • cml can rarely present with features of thyrotoxicosis (heat intolerance, weight loss and excessive sweating) due to hyper-metabolism • cll is often associated with lymphadenopathy and rarely with mild to moderate splenomegaly. all, acute lymphoid leukaemia; aml, acute myeloid leukaemia; cll, chronic lymphocytic leukaemia; cml, chronic myeloid leukaemia. centre. diagnosis depends on clinical history and examination, radiological investigations to document the extent of disease, and morphology, immunohistochemistry and molecular studies on tissue samples to confirm the lymphoma subtype. guidance on the diagnosis and treatment of lymphoma in settings where resources are limited includes recommendations about panels of immunostains and chemotherapy regimens that minimize the need for supportive care. 322 tele-pathology, which involves transmitting histological images via the internet to experts overseas, may be helpful in certain circumstances though it is dependent on the quality of the histology preparations and the images of appropriate diagnostic regions in the sample. treatment regimens for lymphomas differ according to the subtype but may involve chemotherapy and radiotherapy. high remission rates can be achieved in burkitt's lymphoma with a combination of cyclophosphamide, vincristine and methotrexate and progressive disease can be managed with ifosfamide, mesna and cytosine arabinoside. 322, 323 adult t-cell leukaemia-lymphoma (atll) adult t-cell leukaemia-lymphoma (atll) is an uncommon lymphoid malignancy which occurs in patients infected with human t-lymphotropic virus type i (htlv-i). 324 htlv-1 is endemic in the caribbean, western africa, peru and southern japan. less than 5% of those infected with htlv-i develop atll and up to 30 years can elapse between the primary infection and the development of atll suggesting additional factors are needed for malignant transformation. 325 atll presents acutely in approximately 60% of cases, although chronic forms have also been described. 326 the clinical presentation is with generalized lymphadenopathy in most cases and hepatosplenomegaly in over half. 324 atll is associated with a high risk of hypercalcaemia which occurs in more than two-thirds of patients during the course of their disease and may be associated with central nervous system disturbances and renal impairment. lytic bone lesions occur as a para-neoplastic types of lymphomas identified from selected countries in sub-saharan africa phenomenon due to production of parathormone-like peptides. as with other t-cell disorders, atll can involve the skin, producing, e.g. erythrodermic plaques. the diagnosis of atll can be suspected from a high peripheral blood white blood cell count in combination with hypercalcaemia and characteristic lymphocytes with convoluted and hyperlobulated nuclei. 324 the diagnosis is confirmed by histological examination of a tissue (lymph node or bone marrow), immunophenotyping for specific cell markers and proof of htlv infection, usually by serological methods. management of atll is primarily with combination chemotherapy with intrathecal prophylaxis. 327,328 a combination of zidovudine and interferon, as agents against htlv, has also been tried with some success. 329 hypercalcaemia and opportunistic infections should be sought and treated early in these patients. the high white cell count is associated with a significant risk of tumour lysis syndrome and should be prevented by adequate hydration and the judicious use of allopurinol and other urate-reducing agents. myeloma is a monoclonal proliferation of plasma cells and it particularly affects older people. myeloma appears to be less common in asian countries than elsewhere, although during the last 25 years, an almost four-fold increase in incidence of myeloma has occurred in taiwan. 330 in the united states, the incidence of multiple myeloma in the black population is twice that of the white population. the abundant plasma cells infiltrate the bone marrow and interfere with normal haematopoiesis. this leads to anaemia, which is a presenting feature in 70% of individuals. bony infiltration by the malignant plasma cells can produce osteoporosis, lytic lesions and pathological fractures in 60% of patients with myeloma. involvement of the bones can lead to hypercalcaemia, which may be a presenting feature, and vertebral fracture leading to spinal cord compression. the malignant plasma cells produce a paraprotein which can cause renal impairment in 20-50% and hyperviscosity may ensue in 10% of patients if the paraprotein production is not controlled. patients with myeloma may need a variety of supportive interventions including management of anaemia, renal failure, hypercalcaemia, hyperviscosity, infections and bone pains. specific anti-myeloma treatment should be managed within a specialist unit and has undergone a radical change in the last decade with the use of thalidomide and its newer formulations, and the more expensive, proteasome inhibitors (e.g. bortzomib). thalidomide is relatively safe and effective although somnolence and constipation can sometimes be troublesome. there is a risk of thrombosis with thalidomide especially at the initiation of therapy, and prophylaxis with heparin, warfarin or antiplatelet agents, depending on an assessment of the risk, may be warranted. melphalan may also be useful, particularly if resources are limited and there is no specialist centre. however it is myelosuppressive, so regular monitoring of the blood count is essential. maintaining an adequate blood supply is a major challenge for low-income countries. only 39% of the global blood supply is donated in the poorest countries where 82% of the world's population lives. 331 blood transfusion is a vital component of every country's health service. it can be a life-saving intervention for illnesses such as severe acute anaemia, but mistakes in the transfusion process can be life-threatening, either immediately or years later through transmission of infectious agents. clinicians need to understand how blood is acquired and its risks and benefits, and to use it appropriately. governments and transfusion services need to put measures in place to ensure that blood is safe for transfusion and that it reaches those who need it in a timely manner. only 16% of member states meet all the world health organization's (who) recommendations for a national quality blood transfusion system. 331 at the national level the transfusion service should have a director, an advisory committee and clear transfusion policies and strategies (table 65 .12). 331 who recommend standardization of blood collection, testing and distribution. although centralization of these services may offer the best guarantee of quality, it is often not practical in countries with poorly developed communications and transport infrastructure. two systems, centralized and hospital-based, exist in lowincome countries for managing blood supply. in the centralized system, voluntary blood donors are recruited, screened and bled by regional centres and the blood collected is distributed to peripheral hospitals. hospital-based systems are the predominant source of blood across sub-saharan africa. hospital-based systems obtain blood predominantly from relatives of patients, and blood is screened and used within the local vicinity. 332 blood from the centralized system costs at least three times as much per unit as that from a hospital-based system. 333 although centralized systems can save costs through batching and bulk purchasing, the quality assurance processes and donor recruitment components are expensive and difficult to maintain without dependence on external funds. in hospital-based transfusion services, testing quality is variable and the families of patients bear the cost of finding blood donors. the vast majority of blood in low-income countries is transfused as whole blood. in high-income countries it is standard practice to optimize the use of each donation of blood by separating it into individual components but whether this approach is cost-effective in low-income countries, where indications for transfusion are different, is not known. these components, which may include plasma, platelets and cryoprecipitate, are prepared by centrifugation using a closed, sterile system and each component has different storage requirements. plasma and cryoprecipitate are kept frozen, red cells are stored at 1-5°c, and platelets at 18-22°c with constant agitation. recent evidence suggests that warm, fresh, whole blood may be better than component therapy for resuscitation of acidotic, hypothermic and coagulopathic trauma patients 334 and for patients needing massive transfusions. 335 many infections can be transmitted through blood transfusions and transfusion of infected blood causes morbidity and mortality in the recipients, and has an economic and emotional impact on their families and communities. those who become infected through blood transfusion are infectious to others and contribute to the spread of disease thereby increasing the burden on health services and reducing productive labour. strategies for recruiting blood donors have to provide blood for all who need it in a timely manner while ensuring that the blood is as safe as possible. the safest type of blood donor is one who donates regularly (i.e. repeat donors). who states that the safest source of blood is altruistic, voluntary, unpaid donors. only 32% of who member states report having at least 90% of their blood supply from voluntary donors, and low-income countries have not been able to increase the recruitment of voluntary donors for several years. 331 recent evidence from sub-saharan africa indicates that the focus on voluntary donors may be misplaced since first-time voluntary donors have a similar prevalence of transfusion-transmitted infections as family replacement donors. 336 in order to limit blood shortage and maintain constant blood supply in poorer countries, both voluntary and replacement donors should be accepted and encouraged to donate regularly. mechanisms to convert family replacement donors into repeating voluntary donors have the potential to significantly increase blood donations in africa. political will and open-mindedness about ways to improve the supply and safety of blood are essential to promote more evidence-based approaches to blood transfusion practice in poorer countries. 337 supporting strategy in wealthy countries, the majority of transfusions are carried out electively. by contrast, in poorer countries, and particularly those where the malaria transmission rate is high, most transfusions are given for life-threatening emergencies. in low-income countries, 50-80% of transfusions are administered to children, predominantly for malaria-related anaemia, and pregnant women. transfusion can significantly reduce the mortality of children with severe anaemia within the first 2 days of hospital admission 347 and successful malaria control can reduce paediatric transfusion requirements. 348 in sub-saharan africa, 26% of in-hospital maternal deaths from severe bleeding were due to lack of blood for transfusion. 349 other specialities which are significant users of blood are surgery, trauma, emergency medicine and general medicine. in low-income countries the most effective way to avoid transfusions is to reduce the prevalence of anaemia. more studies on the efficacy and cost of combinations of interventions including insecticide-treated bed nets, nutritional supplements and anthelmintic drugs to prevent anaemia are needed. when resources are very limited, governments may need to make some difficult decisions in order to achieve an equitable balance between investing in a transfusion service and public health measures to reduce anaemia. whether a patient needs a blood transfusion or not is ultimately a clinical decision. emergency transfusions can be lifesaving for patients in whom anaemia has developed too quickly to allow physiological compensation, as in severe malariarelated anaemia in children, and sudden, severe obstetric bleeding. in contrast, if the anaemia has developed slowly, for example due to hookworm infestation or nutritional deficiency, patients can generally be managed conservatively by treating the cause of the anaemia and prescribing haematinic replacements. iron supplements should be continued for at least 3 months after the haemoglobin has returned to normal, so that body stores can be replenished. clinical guidelines. it is possible to avoid unnecessary transfusions by adhering to clinical transfusion guidelines. most institutions have developed guidelines to help clinicians make rational decisions about the use of blood transfusions (box 65.23) 344, 350 and strict enforcement of transfusion protocols can significantly reduce avoidable transfusions. 351 the principles underlying most transfusion guidelines are similar and combine a clinical assessment of oxygenation, with haemoglobin measurement being used as a surrogate measure for intracellular oxygen concentration. increasingly, transfusion guidelines are making use of evidence which shows that adequate oxygen delivery to the tissues can be achieved at haemoglobin levels that are significantly lower than the normal range. 352 implementation of transfusion guidelines is particularly difficult if clinicians do not have access to reliable haemoglobin high-risk donors, such as commercial sex workers and their contacts, intravenous drug abusers, or those with an itinerant lifestyle such as traders, drivers and military personnel, should be deterred from donating. 338 even in areas where hiv infection rates in the general population are high, donor deferral can be effective in excluding hiv-infected donors. 339 the whole donation process, including tests for hiv and other infections, should be explained to the donor before blood is collected and donors should have the option of knowing the results and receiving counselling. it is imperative that complete confidentiality is maintained throughout all procedures. infections with organisms such as hiv, hepatitis viruses, cytomegalovirus, syphilis, lyme borreliosis, malaria, babesiosis, american trypanosomiasis (chagas disease) and toxoplasmosis can all be acquired through blood transfusions. some 5-10% of hiv infections worldwide are thought to have been transmitted through the transfusion of infected blood and blood products. there have also been reports of transmission of variant creutzfeldt-jakob disease through blood transfusion and there is a theoretical risk of transmission of severe acute respiratory syndrome (sars). 340, 341 who recommends that all donated blood should be screened for hiv, hepatitis b and syphilis and, where feasible and appropriate, for hepatitis c, malaria and chagas disease. malaria can be transmitted by blood transfusion and, depending on the local infection prevalence, 2-55% of blood donors in africa screen positive for malaria. 342 however, there is very little evidence to suggest that these donors transmit malaria to transfusion recipients. although who recommends screening donors in endemic areas for malaria, none of the screening methods that would be practical for transfusion services are sufficiently sensitive. furthermore, in some countries with high malaria transmission, exclusion of parasitaemic donors could result in deferral rates exceeding 50% which would have a major impact on blood supply. 343 there is no evidence to support the widespread practice of routine treatment of transfusion recipients for malaria. fresh blood is potentially infectious for syphilis, but storage at 4°c for more than 5 days can inactivate treponema pallidum. the high demand for blood in low-income countries means that blood is generally not stored for long enough to inactivate t. pallidum and syphilis seroconversion associated with transfusion has been reported from africa. 344 globally, the prevalence of hepatitis c, htlv-1 and -2 and chagas disease is variable and the decision to introduce donor screening for these infections should be based on local assessments of the risks, benefits, feasibility and costs. blood should not be separated into components if the residual risk of infection is high, as this will increase the number of potentially infected recipients. a unit of blood is usually stored until screening tests for infections have been completed. this means that potentially infected blood may be mixed up with units that have already been screened, and costly blood collection bags are wasted. screening potential donors before venesecting a unit of blood may therefore be a more cost-effective way of ensuring safe blood. 345 tests for screening blood donors need to be highly sensitive, and infected blood should be rejected. before informing the donor of the outcome, all positive results should be confirmed using a test with a high degree of specificity. where blood or that the blood may become infected with bacteria during the process. intraoperative blood salvage. this involves collecting blood lost during the operation and reinfusing it into the patient either during or after surgery. although this technique is practical and safe, and reduces the need for donor blood by 27-53%, 355 it requires specialized equipment and training, and may be more expensive than routinely donated blood. 356 other measures. normal saline or intravenous replacement fluids can be used judiciously in acute blood loss, and in certain circumstances may be as effective as whole blood, red cells or plasma. erythropoietin, which stimulates endogenous red cell production is well-established for use in chronic anaemias such as those due to renal failure, cancer and hiv infection but its delayed action makes it unsuitable for use in acute anaemias. synthetic oxygen carriers, such as perfluorocarbons, are not yet routinely available. 357 in low-income countries, the recommended haemoglobin threshold for transfusions is often well below that which would be accepted in more wealthy countries. randomized controlled studies in wealthy countries indicate that for most adults and children undergoing critical care, a haemoglobin threshold of 70 g/l for transfusion is safe 358 whereas paediatric blood transfusion protocols in sub-saharan africa often recommend transfusions for stable children only when the haemoglobin level is less than 40 g/l. 351 complications such as cardiac failure or infection may necessitate transfusion at a higher haemoglobin level. transfusion should be combined with adequate haematinic replacements and underlying conditions should be treated. 359 early evidence suggests that intermittent preventive treatment with anti-malarials may reduce the high hospital readmission rates experienced by children post-transfusion. 360 complications can occur immediately during transfusion, within a few hours of its completion, or be delayed for many years, as in the case of viral infections (box 65.24). measurements. when they doubt the haemoglobin result, clinicians rely entirely on clinical judgement to guide transfusion practice which can lead to significant numbers of inappropriate transfusions. 353 a lack of investment in the quality of a critical test, such as haemoglobin measurement, can waste significant resources downstream in the transfusion process, and unnecessarily expose recipients to the risk of transfusion-related infections. minimizing surgical blood loss. where blood is in short supply, it is particularly important to ensure that the best anaesthetic and surgical techniques are used, to minimize blood loss during surgery. drugs which improve haemostasis or reduce fibrinolysis, such as aprotinin and cyklokapron, and fibrin sealants, can be effective in reducing perioperative blood loss. these drugs can therefore reduce the need for blood transfusion but they may be too expensive for use in low-income countries. a cost-effectiveness study of surgical bleeding in four sub-saharan countries indicates that the antifibrinolytic, tranexamic acid, could save lives in countries with blood shortages, reduce healthcare costs and prevent transmission of infections. 354 preoperative autologous blood deposit. patients undergoing planned surgery who are likely to require a blood transfusion can have units of their own blood removed and stored in case they have significant intraoperative blood loss and need a transfusion. this process, known as preoperative autologous donation, can reduce the need for allogeneic transfusions by 46-74% 355 but it requires careful organization: the surgeon needs to predict how much blood will be required, the patient has to be fit enough to withstand removal of one or more units of blood over the weeks preceding the surgery and the surgery must take place within the shelf-life of the blood. as the blood has to be stored in the blood bank there is still a risk that the patient may receive blood which is not their own box 65.23 prescribing blood: a checklist for clinicians always ask yourself the following questions before prescribing blood or blood products for a patient: 1. what improvement in the patient's clinical condition am i aiming to achieve? 2. can i minimize blood loss to reduce this patient's need for transfusion? 3. are there any other treatments i should give before making the decision to transfuse, such as intravenous replacement fluids or oxygen? 4. what are the specific clinical or laboratory indications for transfusion in this patient? 5. what are the risks of transmitting hiv, hepatitis, syphilis or other infectious agents through the blood products that are available for this patient? bacterial contamination and should be investigated and managed accordingly. allergic reactions are due to infusion of plasma proteins and manifestations include erythema, rash, pruritus, bronchospasm and anaphylaxis. the transfusion should be stopped and the patient treated with antihistamines. if the reaction is mild and the symptoms and signs completely disappear, the transfusion can be restarted. if this type of mild reaction occurs repeatedly with more than one unit of blood, the red cells can be washed before transfusion. this should only be done if absolutely necessary, as it carries the risk of introducing potentially fatal bacterial infection. severe allergic reactions with evidence of systemic toxicity should be managed as acute anaphylaxis. blood should always be transfused slowly to avoid overloading the circulation, unless the patient has active and severe bleeding. fluid overload may be a particular problem when paediatric blood bags are not available, as children may be over-transfused due to miscalculation of the required volume, lack of accurate infusion devices or inadvertent administration of an adult-sized unit of blood. four units of blood contain the equivalent amount of iron stored in bone marrow (approx. 1 g). repeated transfusions for chronic haemolytic anaemia, as in thalassaemia major and sickle cell disease, lead to iron deposition in parenchymal cells. eventually failure of the heart, liver and other organs supersedes. adequate doses of iron chelators, such as injectable desferrioxamine or oral deferiprone, are able to maintain acceptable iron balance in patients with chronic anaemia who need regular transfusions. it is not usually necessary to warm blood unless large quantities are transfused rapidly. this may lower the temperature of the sino-atrial node to below 30°c at which point ventricular fibrillation can occur. if blood needs to be warmed, an electric blood warmer specifically designed for the purpose should be used. this keeps the temperature below 38°c and avoids the haemolysis associated with overheating blood. graft-versus-host disease occurs when donor lymphocytes engraft in an immune-suppressed recipient. the lymphocytes recognize the recipient's bone marrow as foreign and induce aplasia. graft-versus-host disease is almost universally fatal and can be prevented by irradiating the donor blood, which inactivates the donor lymphocytes. transfusion of blood into a recipient who possesses antibodies to the donor's red cells can cause an acute, and occasionally fatal, intravascular haemolysis. this could occur, e.g. if group a cells are transfused into a group o recipient who has naturally occurring antibodies to group a cells. the profound haemolysis induces renal vasoconstriction and acute tubular necrosis. treatment involves stopping the transfusion, cardiorespiratory support and inducing a brisk diuresis. in addition to abnormalities indicating renal failure, laboratory findings include haemoglobinuria and haemoglobinaemia. proof of the diagnosis involves rechecking the whole transfusion process including all documentation stages, regrouping the donor and the recipient, and screening for antibodies on red cells with a direct antiglobulin test. these tests are usually available in any hospital laboratory capable of providing a transfusion service. delayed haemolysis has a similar physiological basis to acute intravascular haemolysis but it tends to be less severe, it occurs 7-10 days after the transfusion and it is less likely to present as a clinical emergency. limited data from sub-saharan africa show rates of bacterial contamination in donated blood of around 9% 361,362 but the clinical consequences for transfusion recipients are unknown. bacteria can enter the blood bag during venesection or if the bag is breached, e.g. when reducing the volume for a paediatric recipient or during component preparation. gram-negative bacteria, including pseudomonas and yersinia, grow optimally at 4°c and infected blood may not necessarily appear abnormal to the naked eye. reactions following infusion of infected blood are often due to endotoxins and may occur several hours after the transfusion has finished. although these reactions are rare, they can be severe and fatal. if bacterial contamination is suspected, the transfusion should be stopped and samples from the patient and the blood bag sent to the laboratory for culture. cardiorespiratory support may be needed and broad-spectrum antibiotics should be started immediately and continued until culture results are available. non-haemolytic febrile reactions are episodes of fever and chills associated with transfusion and for which no other cause can be found. they are due to the recipient's antibodies reacting against antigens present on the donor's white cells or platelets. these reactions are most common in patients who have had transfusions in the past and have therefore 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guidelines in kilifi intermittent preventive therapy for malaria with monthly artemether-lumefantrine for the post-discharge management of severe anaemia in children aged 4-59 months in southern malawi: a multicentre, randomised, placebocontrolled trial bacterial contamination of pediatric whole blood transfusions in a kenyan hospital bacterial contamination of blood and blood components in three major blood transfusion centres in accra, ghana access the complete references online at www.expertconsult.com key: cord-257539-01s21vh0 authors: delvecchio, rodrigo; higa, luiza m.; pezzuto, paula; valadão, ana luiza; garcez, patrícia p.; monteiro, fábio l.; loiola, erick c.; dias, andré a.; silva, fábio j. m.; aliota, matthew t.; caine, elizabeth a.; osorio, jorge e.; bellio, maria; o’connor, david h.; rehen, stevens; de aguiar, renato santana; savarino, andrea; campanati, loraine; tanuri, amilcar title: chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models date: 2016-11-29 journal: viruses doi: 10.3390/v8120322 sha: doc_id: 257539 cord_uid: 01s21vh0 zika virus (zikv) infection in utero might lead to microcephaly and other congenital defects. since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. here we show that chloroquine exhibits antiviral activity against zikv in vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. we demonstrate that chloroquine reduces the number of zikv-infected cells in vitro, and inhibits virus production and cell death promoted by zikv infection without cytotoxic effects. in addition, chloroquine treatment partially reveres morphological changes induced by zikv infection in mouse neurospheres. zika virus (zikv) is an arthropod-borne virus, transmitted by aedes mosquitoes, that belongs to the flavivirus genus, which also includes other pathogens such as west nile virus (wnv), yellow fever virus (yfv), japanese encephalitis virus (jev), and dengue virus (denv). zika virus was first isolated from a sentinel monkey in the zika forest in uganda in 1947 [1] . since then, zikv has been isolated from humans and mosquitoes throughout africa and southeast asian countries. phylogenetic analysis of the nonstructural protein 5 encoding region has disclosed three zikv lineages: east african, vero cells (atcc, manassas, va, usa) are derived from the kidney of african green monkey and were grown in dmem high glucose (gibco, thermo fisher scientific, waltham, ma, usa) supplemented with 5% fetal bovine serum (fbs) (gibco). human brain microvascular endothelial cells (hbmec) were a kind gift from dr. julio scharfstein (federal university of rio de janeiro, rio de janeiro, brazil), and hbmec isolation was performed as previously described [25] . these cells were cultured in dmem high glucose supplemented with 20% fbs. the c6/36 cell line is derived from aedes albopictus. c6/36 cells (atcc, manassas, va, usa) were grown in leibovitz l-15 medium (gibco) supplemented with 2.95 g/l tryptose phosphate broth (sigma aldrich, boston, ma, usa), 2 mm glutamine (gibco), 0.075% sodium bicarbonate (gibco), 1x non-essential amino acids (gibco) and 5% fbs. neural stem cells (nscs) were derived from human induced pluripotent stem cells (ipscs). ipscs were provided by the biobank of ipscs of the brazilian ministry of health (conep b-027 # 25000.111598/2014-04). according to the supplier, fibroblasts were reprogrammed using the protocol developed by paulsen et al. [26] , and transduced with the cytotune-ips sendai kit (thermo fisher scientific, waltham, ma, usa). ipscs presented a normal karyotype and the expression of pluripotency markers. these cells were cultured with e8 culture media (gibco) on a matrigel (bd biosciences, san jose, ca, usa) coated surface. ipsc colonies were manually passaged every 5-7 days until they reached 70%-80% confluence and were maintained at 37 • c in humidified air with 5% co 2 . to produce nscs, human ipscs were exposed to the serum-free neural induction medium (gibco), containing neurobasal medium (gibco) and the pluripotent stem cell neural induction supplement (gibco), according to the manufacturer's protocol [27] . briefly, the medium was changed every other day until day 7, when initial nscs are split and grown on neural expansion medium (advanced dmem/f12 and neurobasal medium (1:1) with neural induction supplement; gibco). nscs were used after four passages in neural expansion media. mouse central nervous system (cns) cells were harvested from swiss mouse embryos at embryo day 14 (e14) and grown for 72 h as free floating neurospheres in neurobasal culture media (gibco) supplemented with 1x b27 (gibco). chloroquine diphosphate was kindly supplied by farmanguinhos (fiocruz, rio de janeiro, brazil). the lyophilized powder was diluted in double distilled water to 20 mm. the chloroquine solution was filtered through a 0.22 µm membrane and stored at −80 • c. zikv strain mr 766 (uganda/africa, accession no.: nc012532.1, kindly provided by dr. davis ferreira, federal university of rio de janeiro, rio de janeiro, brazil) was isolated from a rhesus monkey and injected intracerebrally on swiss mice for several passages [1] and zikv br (recife/brazil, zikv pe/243, accession no: kx197192.1, kindly provided by dr. marli tenório cordeiro, centro de pesquisas aggeu magalhães, recife, brazil) was isolated from a patient presenting classical symptoms of zikv infection [28] . these viruses were propagated in vero and c6/36 cells, respectively. briefly, the cells were infected with zikv at a multiplicity of infection (moi) of 0.01 and incubated at 37 • c. after 1 h, the inoculum was removed and replaced with dmem high-glucose (vero) and leibovitz l-15 (c6/36) growth media supplemented with 2% fbs. after 4 to 6 days, the conditioned medium was harvested, centrifuged at 300× g, and sterile-filtered to remove cells and cellular debris. virus stocks were stored at −80 • c. virus titers were determined by plaque assay performed on vero cells. virus stocks or samples were serially diluted and adsorbed to confluent monolayers. after 1 h, the inoculum was removed and cells were overlaid with semisolid medium constituted of alpha-mem (gibco) containing 1% carboxymethyl cellulose (sigma aldrich) and 1% fbs (gibco). cells were further incubated for 5 days when cells were fixed in 4% formaldehyde. cells were stained with 1% crystal violet in 20% ethanol for plaque visualization. titers were expressed as plaque forming units (pfu) per milliliter. vero cells, hbmec, and nsc were infected with zikv mr 766 or zikv br strain at an moi of 2. after 1 h, the inoculum was removed and medium containing chloroquine (6.25 to 50 µm) was added to the cells. after 4 to 5 days, cells were fixed with 4% paraformaldehyde (sigma aldrich) in phosphate buffered saline (pbs) for 15 min at room temperature and washed with pbs. cells were permeabilized with 0.1% triton x-100 (sigma aldrich) in pbs, washed with pbs, and blocked with pbs with 5% fbs. cells were incubated with 4g2, a pan-flavivirus antibody raised against the zikv envelope e protein produced in 4g2-4-15 hybridoma (atcc), diluted 1:5 in pbs with 5% fbs. cells were labeled with donkey anti-mouse alexa fluor 488 antibody (thermo scientific, waltham, ma, usa) diluted 1:1000 in pbs with 5% fbs, and were analyzed by flow cytometry in a bd accuri c6 (becton, dickinson and company, franklin lakes, nj, usa) for zikv infection. vero, hbmec, and nsc were seeded on black 96-well plates with clear bottoms and infected with zikv mr 766 or zikv br strain at an moi of 2 for 1 h. neurospheres were seeded on coverslips and infected with zikv mr766 with 2.5 × 10 5 pfu after infection, the viral inoculum was removed and cells were incubated with medium containing chloroquine (1.56 to 50 µm) for 4 to 5 days, depending on cell type. cells and neurospheres were fixed with 4% paraformaldehyde in pbs for 20 min at room temperature. the fixative was removed and the samples were washed three times with pbs. blocking of unspecific binding of the antibody and permeabilization were performed with pbs supplemented with 3% bovine serum albumin (bsa, sigma aldrich) and 0.1% triton x-100 for 40 min at room temperature. incubation with anti-map2 antibody was performed following the manufacturer's instructions (abcam, cambridge, cambridgeshire, uk, #32454; 1:1000) and the 4g2 antibody was diluted 1:4 in pbs with 3% bsa and incubated with cells for 1 h. after washing three times with pbs, cells were incubated with secondary antibodies coupled to alexa fluorochromes (thermo fisher scientific) for 40 min and washed five times. coverslips with neurospheres were mounted with prolong gold mounting medium (thermo fisher scientific). samples were imaged using either leica sp5 or leica spe (leica biosystems, wetzlar, hesse, de) confocal microscopes and a nikon te300 (tokyo, japan) inverted microscope coupled to a leica dfc310fx camera (leica biosystems, wetzlar, germany). vero, hbmec, and nsc were exposed to zikv mr 766 at an moi of 2. after 1 h, inoculum was removed and chloroquine-containing medium (6.25 to 50 µm) was added to the cells. five days post-infection, 20 µl of celltiter blue reagent (promega, madison, wi, usa) was added in each well, incubated for 12-16 h, and fluorescence was measured (560/590 nm), except for nscp14 cells when celltiter blue was added at three days post-infection. mean fluorescence intensity (mfi) and standard deviation were displayed. in order to calculate the half maximum effective concentration (ec50) that protects cells from death caused by zikv infection, mfi values from zikv mr766 control were subtracted from every condition and then these values were normalized over the mock control. these data were plotted and hill 4 parameter sigmoidal regression was performed on sigma plot v.12.0 (systat software inc., san jose, ca, usa). vero cells, hbmec, and nsc were incubated with medium containing chloroquine (1.56 to 200 µm) for 3 days (nsc) or 5 days (vero and hbmec). celltiter blue reagent (promega) was added in each well, incubated for 12-16 h, and fluorescence was measured (560/590 nm). in order to calculate the 50% cytotoxicity concentration (cc50), mfi values were normalized over the untreated control. these data were plotted and hill 4 parameter sigmoidal regression was performed on sigma plot v.12.0 (systat software inc.). vero cells were inoculated with zikv mr 766 at an moi of 10 for 1 h at 4 • c. cells were washed three times with cold pbs to remove unbound virus and treated with 50 µm chloroquine that was added at different time points: 0, 0.5, 3, 12, and 24 h post-infection. conditioned media were collected at 30 h post-infection to analyze the production of virus particles through viral rna content or the amount of infectious virus particles. viral rna was extracted and quantitative reverse transcription polymerase chain reaction (rt-qpcr) was performed. the titer of infectious virus particles was determined by plaque assay. vero cells were infected with zikv mr766 or zikv br strain at an moi of 2 for 1 h at 4 • c. virus input was washed three times with cold pbs and cells were treated with chloroquine (6.25 to 50 µm) for 48 h, and then the supernatant was collected and the rna was extracted and analyzed by relative quantification by rt-qpcr. the supernatant was also evaluated by plaque assay to quantitate the infectious virus particles. viral rna was extracted from 200 µl supernatant of infected cells using qiamp minielute virus spin (qiagen, hilden, düsseldorf ,de), following the manufacturer's recommendations. zikv detection was performed using one step taqman rt-qpcr (thermo fisher scientific) on a 7500 real-time pcr system (applied biosystems) with primers and the probe described elsewhere [2] . threshold cycle (ct) was determined and ∆ct (ct chloroquine treated − ct untreated) was calculated. the fold reduction of virus particles' release, including defective viral particles, were calculated by 2 ∆ct . mean and standard deviation (sd) were calculated for each assay. one way analysis of variance (anova) was conducted using the non-parametric test (kruskal-wallis) followed by dunn's multiple comparisons test. a p-value of <0.05 was considered significant. all analyses were performed on graphpad prism v.7 (graphpad software, san diego, ca, usa). the sample size is provided in the respective figure legends. we characterized the antiviral properties of chloroquine in vero cells, a model widely used to study viral infections. vero cells were infected with zikv mr766 at an moi of 2 (i.e., 2 pfu/cell) and were then treated for 5 days with chloroquine in concentrations ranging from 6.25 to 50 µm. viral infectivity was assessed using the 4g2 antibody, which detects flavivirus envelope e protein. we observed that chloroquine treatment decreased the number of zikv-infected cells in a dose-dependent manner. flow cytometry analysis showed a reduction of 35% and 65% in zikv-infected cells when cultures were treated with 25 µm and 50 µm chloroquine, respectively, compared to untreated infected cells ( figure 1a ). immunofluorescence staining corroborated these results ( figure 1b ) and additionally, chloroquine decreased the production of infectious ( figure 1c ) and total ( figure 1d ) virus particles, including defective viral particles, by zikv-infected cells. to confirm that viral inhibition is independent of chloroquine cytotoxicity, the viability of uninfected cells treated with chloroquine (1.56 to 200 µm) for 5 days was analyzed. chloroquine did not impact cell viability at concentrations of 50 µm or lower ( figure 1e ). we further analyzed whether chloroquine treatment could protect vero cells from zikv infection as assessed by cell viability. chloroquine, ranging from 12.5 to 50 µm, increased cell viability from 55% up to 100% ( figure 1f ). microcephaly cases and neurological disorders have only been associated with infection with strains of zikv from the asian lineage, detected in french polynesia and in the americas [6, 9, 29] . to determine the inhibition spectrum of chloroquine against zikv infection, vero cells were infected with the brazilian isolate of the asian lineage (zikv br). chloroquine decreases the percentage of cells infected with the brazilian isolate from 70% to 30% and 5% at 12.5 and 25 µm, respectively ( brazilian strain at an moi of 2 and exposed to chloroquine for 48 h. the supernatant was collected and viral rna was relatively quantified over the untreated infected control (b) or infectivity was analyzed by immunofluorescence with 4g2 antibody (c). the dashed line represents fold reduction on virus production of 1. data are represented as mean ± sd from two independent experiments. statistical significance was assessed by kruskal-wallis test and multiple comparisons with infected and untreated control corrected by dunn's test (* p < 0.05). inhibition of viral infection mediated by chloroquine can occur in both the early and later stages of the viral replication cycle [30, 31] . to evaluate which step of the viral cycle was susceptible to inhibition, chloroquine was added to vero cells at different time points post-infection with zikv mr766. the supernatant was collected 30 h post-infection and virus production was evaluated by relative quantification of viral rna over the untreated control by rt-qpcr. virus titers were also determined by plaque assay in vero cells. incubation of vero cells with chloroquine at 0 h postinfection had a greater impact on the production of zikv particles, decreasing viral rna 64-fold over the controls ( figure 3a ). the addition of chloroquine from 30 min to 12 h post-infection was able to reduce virus release 9-20 fold over untreated, infected-cells. however, chloroquine added at 24 h post-infection had only a minor effect on viral production ( figure 3a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure 3b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. inhibition of viral infection mediated by chloroquine can occur in both the early and later stages of the viral replication cycle [30, 31] . to evaluate which step of the viral cycle was susceptible to inhibition, chloroquine was added to vero cells at different time points post-infection with zikv mr766. the supernatant was collected 30 h post-infection and virus production was evaluated by relative quantification of viral rna over the untreated control by rt-qpcr. virus titers were also determined by plaque assay in vero cells. incubation of vero cells with chloroquine at 0 h post-infection had a greater impact on the production of zikv particles, decreasing viral rna 64-fold over the controls ( figure 3a ). the addition of chloroquine from 30 min to 12 h post-infection was able to reduce virus release 9-20 fold over untreated, infected-cells. however, chloroquine added at 24 h post-infection had only a minor effect on viral production ( figure 3a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure 3b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. reduce virus release 9-20 fold over untreated, infected-cells. however, chloroquine added at 24 h post-infection had only a minor effect on viral production ( figure 3a) . these results were confirmed by quantification of zikv infectious particles released after chloroquine treatment ( figure 3b ). these data confirm that chloroquine interferes with the early stages of the zikv replication cycle. considering that zikv infects hbmecs [32] , we investigated whether chloroquine could inhibit viral infection of these cells. chloroquine reduced 45% and 50% of the number of zikv mr766-infected hbmecs at 25 and 50 µm, respectively ( figure 4a,d) . these concentrations are non-cytotoxic ( figure 4b ), and protected approximately 80% of hbmecs from zikv infection as demonstrated by the increase in cell viability ( figure 4c ). considering that zikv infects hbmecs [32] , we investigated whether chloroquine could inhibit viral infection of these cells. chloroquine reduced 45% and 50% of the number of zikv mr766-infected hbmecs at 25 and 50 μm, respectively ( figure 4a,d) . these concentrations are non-cytotoxic ( figure 4b) , and protected approximately 80% of hbmecs from zikv infection as demonstrated by the increase in cell viability ( figure 4c ). neural stem cells are key cells in the process of corticogenesis, giving rise to the three main cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. depletion of the nsc pool is one of the main mechanisms responsible for primary microcephaly [33] . to evaluate if chloroquine could protect these cells from zikv mr766 infection, nscs were exposed to up to 50 μm chloroquine for 4 days. chloroquine treatment decreased the number of nscs infected with zikv mr766 by 57%, and, through cell viability assessment protected 70% of nscs from zikv infection, without cytotoxicity effects ( figure 5a-d) . similar results were observed when nscs were infected with the brazilian strain ( figure 5e ). neural stem cells are key cells in the process of corticogenesis, giving rise to the three main cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. depletion of the nsc pool is one of the main mechanisms responsible for primary microcephaly [33] . to evaluate if chloroquine could protect these cells from zikv mr766 infection, nscs were exposed to up to 50 µm chloroquine for 4 days. chloroquine treatment decreased the number of nscs infected with zikv mr766 by 57%, and, through cell viability assessment protected 70% of nscs from zikv infection, without cytotoxicity effects ( figure 5a-d) . similar results were observed when nscs were infected with the brazilian strain ( figure 5e ). neuroprogenitor-enriched neurospheres, when subjected to differentiation culture conditions, can generate neurons. our group recently demonstrated that zikv infection affected neurosphere size, neurite extension, and neuronal differentiation [34] . as we previously observed, neurospheres infected with zikv strain mr766 showed convoluted and misshapen neurites. neurite extension was evaluated in chloroquine treated cultures by microtubule-associated protein 2 (map2) staining and phase contrast microscopy and although many neurospheres were severely impacted by the infection, many others displayed the same general characteristics of mock-infected spheres indicating that chloroquine treatment rescued the neurite extension phenotype (figure 6a-c) . furthermore, zikv infection decreased when neurospheres were treated with 12.5 μm chloroquine, as evaluated by 4g2 staining (figure 6d-f) . data are represented as mean ± sd from two to three independent experiments. statistical analysis was performed with kruskal-wallis test and multiple comparisons with infected and untreated control corrected by dunn's test (* p < 0.05). neuroprogenitor-enriched neurospheres, when subjected to differentiation culture conditions, can generate neurons. our group recently demonstrated that zikv infection affected neurosphere size, neurite extension, and neuronal differentiation [34] . as we previously observed, neurospheres infected with zikv strain mr766 showed convoluted and misshapen neurites. neurite extension was evaluated in chloroquine treated cultures by microtubule-associated protein 2 (map2) staining and phase contrast microscopy and although many neurospheres were severely impacted by the infection, many others displayed the same general characteristics of mock-infected spheres indicating that chloroquine treatment rescued the neurite extension phenotype (figure 6a-c) . furthermore, zikv infection decreased when neurospheres were treated with 12.5 µm chloroquine, as evaluated by 4g2 staining (figure 6d-f) . chloroquine is known to be a non-specific antiviral agent, but its effect on the zika virus replication has not been evaluated yet. this is the first report of inhibitory effects of chloroquine on zikv replication, which, given the ongoing epidemics, may become interesting both for the scientific knowledge of the virus and for the clinical perspective. although zika virus was first identified in uganda in 1947, from january 2007 to april 2016, zikv transmission has been reported in 64 countries and territories [35] . the zika virus disease is in general mild, but the recent positive correlation between infection, congenital malformations, and neurological damage in adults has intensified the need for therapeutic approaches. prophylactic treatments for women intending to get pregnant in epidemic areas and travelers going to affected countries would represent relevant tools to reduce zikv transmission and avoid the spread of the disease by travelers. moreover, a drug that blocks placental transfer of the virus could decrease the figure 6 . chloroquine inhibits zikv infection in mouse neurospheres. mouse neurospheres were infected with zikv mr766 (2.5 × 10 5 pfu and were treated with chloroquine for 3 days. neurospheres were analyzed by phase contrast microscopy (a-c), and triple stained for envelope viral protein (green), microtubule-associated protein 2 (map-2, red), a neuron-specific protein, and dapi (blue) (d-f). chloroquine is known to be a non-specific antiviral agent, but its effect on the zika virus replication has not been evaluated yet. this is the first report of inhibitory effects of chloroquine on zikv replication, which, given the ongoing epidemics, may become interesting both for the scientific knowledge of the virus and for the clinical perspective. although zika virus was first identified in uganda in 1947, from january 2007 to april 2016, zikv transmission has been reported in 64 countries and territories [35] . the zika virus disease is in general mild, but the recent positive correlation between infection, congenital malformations, and neurological damage in adults has intensified the need for therapeutic approaches. prophylactic treatments for women intending to get pregnant in epidemic areas and travelers going to affected countries would represent relevant tools to reduce zikv transmission and avoid the spread of the disease by travelers. moreover, a drug that blocks placental transfer of the virus could decrease the chance of vertical transmission in viremic pregnant women as was shown for hiv-infected pregnant women treated with antiretroviral therapy [36] . here we demonstrated that chloroquine decreases the number of zikv-infected cells and protected cells from zikv infection as measured by cell viability at non-cytotoxic concentrations (figures 1, 4 and 5) . the ec50 or concentration of chloroquine that protected 50% of the cells from zikv infection assessed by cell viability, was 9.82-14.2 µm depending on the cell model and the cc50 ranged from 94.95 to 134.54 µm ( table 1) . the values of ec50 obtained for zikv mr766 are lower than those obtained for denv inhibition (~25 µm) and hiv inhibition (100 µm) [20, 22] . furthermore, we observed similar zikv inhibitory effects of chloroquine when tested on different zikv lineage infections (figures 2 and 5) , supporting the idea that chloroquine could help to manage recent infections caused by asian zikv lineage. although chloroquine has shown antiviral activity against a large spectrum of viruses in vitro, few clinical studies have been performed to evaluate chloroquine effects on patients with viral infections. two clinical trial studies of chloroquine have been conducted to assess chloroquine treatment in patients infected with denv [37, 38] . one of the trials evaluated the benefits of chloroquine treatment for 3 days in patients infected with denv and showed no reduction in the duration or intensity of denv viremia or nonstructural 1 protein (ns1) antigenemia clearance [37] . however, a trend towards a reduction in the number of dengue hemorrhagic fever cases was noticed in the chloroquine-treated group [37] . a more recent clinical trial of chloroquine administration to denv-infected patients, also for 3 days, showed that 60% of the patients in the chloroquine-treated group reported feeling less pain and showed improvement in the performance of daily chores during treatment [38] . moreover, the symptoms returned after medication withdrawal. however, chloroquine treatment did not reduce the duration and intensity of the fever or duration of the disease [38] . the antiviral effect of chloroquine may be insufficient to produce a decrease in viral load or improvement of the disease progression when chloroquine/hydroxychloroquine is used in monotherapy. however, chloroquine may produce a significant antiflaviviral effect when used in combination therapies, as recently shown in a clinical trial of hydroxychloroquine plus ribavirin and interferon alpha in individuals infected with hepatitis c virus (hcv) [39] . in regard to the potential antiviral therapeutic combinations for zika, a freshly published screening of drugs already approved for other clinical indications has resulted in the identification of more than 20 candidate drugs [40] . of note, one of these is mefloquine, a compound related to chloroquine. in terms of safety for pregnant women, however, mefloquine is included in the b category, i.e., a drug for which the animal reproduction studies have failed to demonstrate a risk to the fetus and there are no adequate and well-controlled studies in pregnant women. be that as it may, the aforementioned study corroborates our results using chloroquine, and provides new anti-zikv drugs that could be tested in combination with chloroquine. differing from mefloquine administration, the use of chloroquine during pregnancy was thoroughly evaluated and when prophylactic doses of chloroquine were administered for malaria (400 mg/week), no increment in birth defects was observed [41] . higher concentrations of chloroquine (250 mg to 500 mg/day) were administered to pregnant women with severe diseases, such as lupus or rheumatoid arthritis. in these studies, few cases of abortion and fetal toxicity were observed. however, fetal toxicity or death could not be discarded as direct consequences of the disease itself. in addition, all term deliveries resulted in healthy newborns [19, 42] . chloroquine has been successfully tested in animal models. twice daily administration of chloroquine (90 mg/kg) has been shown to increase the balb/c mice survival rate to 80%-90% after infection with ebola virus (ebov) [43] . in a c57bl/6 mice model for coronavirus infection, chloroquine (50 mg/kg) protected 100% of 5-day-old suckling mice infected with human coronavirus oc43 (hcov-oc43) when administered to pregnant mice 1 day prepartum [44] . a survival rate of 70% was observed in balb/c mice infected with avian influenza h5n1 virus treated with chloroquine at 50 mg/kg/day [45] . these results suggest that chloroquine has the potential to inhibit zikv in in vivo mouse studies. chloroquine is widely distributed to body tissues as well as its analogue hydroxychloroquine. the concentration of hydroxychloroquine in the brain is 4-30 times higher than in the plasma [46] . the concentration of chloroquine in the plasma reached 10 µm when a daily intake of 500 mg was prescribed to arthritis patients [47] . chloroquine is able to cross the placental barrier and is supposed to reach similar concentrations in both maternal and fetal plasma [48] . concentrations of chloroquine, similar to the ec50 values calculated here (table 1) , are achieved in the plasma in current chloroquine administration protocols and might reach the brain. different mechanisms for the chloroquine inhibition of viral infection have been described [49] [50] [51] . we observed a strong reduction in the release of zikv particles when the drug was added at 0 h post-infection (figure 3) , suggesting a higher impact on early stages of infection, possibly during fusion of the envelope protein to the endosome membrane. chloroquine inhibits acidification of the endosome, consequently inhibiting the low ph-induced conformational changes required for the fusion of the envelope protein of flaviviruses with the endosomal membrane [52] . chloroquine was also effective in decreasing virus release, although less pronouncedly and not statistically significant, when added after the early stages of virus infection (from 0.5 to 24 h post-infection), suggesting that later stages of the zikv replication cycle might also be affected (figure 3) . zikv was detected in the cerebrospinal fluid of zikv-infected adult patients that manifested meningoencephalitis, indicating that zikv invades the central nervous system through yet unknown mechanisms. transcytosis through the endothelial cells of the blood brain barrier is a known mechanism of viral access to the central nervous system [53, 54] . here we demonstrated, by different methodologies, that chloroquine protects hbmec, an immortalized cell line widely used as in vitro model of the blood-brain barrier [25] , from zikv infection (figure 4) . recent studies showed that neural stem cells are highly permissive for zikv infection and one of the mechanisms proposed for the cause of microcephaly would be the depletion of the stem cell pool induced by zikv [10, 11, 55] . our data showed that chloroquine inhibits the infection of human neural stem cells ( figure 5 ). using the mouse neurospheres model to study neural stem cell differentiation into neurons, another process that might be disturbed in microcephaly, we observed that chloroquine inhibited the infection of neuronal progenitors and partially protected the ability of these cells to extend neurites ( figure 6 ). the protective effect of chloroquine on stem cells and committed progenitors is potentially a groundbreaking feature of this compound, as it would be prescribed to women at childbearing age that are traveling to affected countries and women planning pregnancy in endemic areas. this would decrease the chances of infection and thus fetal damage, especially to the developing brain. our results suggest that the chloroquine concentrations inhibiting zikv replication in vitro may overlap the highest drug concentrations detected in humans [56] . we therefore suggest that the therapeutic potential of chloroquine for zika be subjected to further study. zika virus. i. isolations and serological specificity genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia molecular evolution of zika virus during its emergence in the 20th century zika virus infection in pregnant women in rio de janeiro-preliminary report notes from the field: evidence of zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses -brazil zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg? congenital zika virus infection: beyond neonatal microcephaly detection and sequencing of zika virus from amniotic fluid of fetuses with microcephaly in brazil: a case study zika virus impairs growth in human neurospheres and brain organoids 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infectivity bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells altered oxygen metabolism associated to neurogenesis of induced pluripotent stem cells derived from a schizophrenic patient efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells full genome sequence and sfrna interferon antagonist activity of zika virus from recife, brazil zika virus, french polynesia, south pacific endocytosis is a critical step in entry of subgroup b avian leukosis viruses characterization of herpes simplex virus-containing organelles by subcellular fractionation: role for organelle acidification in assembly of infectious particles type iii interferons produced by human placental trophoblasts confer protection against zika virus infection genetic causes of microcephaly and lessons for neuronal development the impact of african and brazilian zikv isolates on neuroprogenitors virus 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developments in the understanding of the pharmacokinetics and mechanism of action of chloroquine dose refinements in long-term therapy of rheumatoid arthritis with antimalarials transfer of chloroquine and desethylchloroquine across the placenta and into milk in melanesian mothers mechanism of borna disease virus entry into cells weak bases affect late stages of mayaro virus replication cycle in vertebrate cells effects of chloroquine on viral infections: an old drug against today's diseases? rodenhuis-zybert, i.; wilschut, j. flavivirus cell entry and membrane fusion human immunodeficiency virus-1 uses the mannose-6-phosphate receptor to cross the blood-brain barrier mechanism of west nile virus neuroinvasion: a critical appraisal brain-region-specific organoids using mini-bioreactors for modeling zikv exposure chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/aids acknowledgments: this work was supported by conselho nacional de desenvolvimento e pesquisa (cnpq), key: cord-025628-9611eglg authors: bonagura, vincent robert; rosenthal, david walter title: infections that cause secondary immune deficiency date: 2020-05-29 journal: stiehm's immune deficiencies doi: 10.1016/b978-0-12-816768-7.00049-1 sha: doc_id: 25628 cord_uid: 9611eglg discriminating between patients with microbial infections that cause secondary immune effects from those with the same infection who have primary immune deficiency disease can be difficult. there are many microbes that temporarily “stun” innate and/or adaptive immunity during a primary infection. the common result of temporary inhibition, or permanent depletion of host immunity during some primary infections is the development of superinfections of other microbes that cause significant morbidity, and on occasion, mortality. in addition, microbes that cause persistent infection, such as the human immune deficiency virus, deplete effective immunity over time and can also lead to secondary infections with other microbes ultimately leading to death if not appropriately treated. in some cases, such as influenza virus infection, mortality can be dramatic due in large part to acquired secondary bacterial infections. many microbes manipulate host immunity and thereby inhibit the ability of patients to combat secondary microbial infections. the overall survival of patients primarily infected with some viruses, parasites, or bacteria, is closely linked to the ability of secondary infections to take advantage of the immune modulation induced by the primary infection. herein we discuss some of the secondary immune defects caused by select viruses (measles, influenza, hiv1, htlv), parasites, (leishmania, malaria), and bacteria (bordetella pertussis). furthermore, the genetic susceptibility of a given host to become infected, or develop severe disease, also determines whether an infected individual who becomes infected with a secondary microbe survives. future studies are needed to explore not only the immunomodulation caused by select microbes, but also the repertoire of immune responses expressed by infected individuals in order to predict clinical outcomes of these infections. thus, understanding the delicate balance that exists between “immune altering” microbes that suppress immune responses during primary infections leading to severe secondary infections versus those infections found in patients with inborn errors of innate or adaptive immunity, is essential in predicting the clinical outcome and the appropriate treatment for these two different patient populations. immune deficiencies can be subdivided into two categories: primary or secondary. this chapter describes secondary defects related to infection, and other forms of secondary immunodeficiency are described elsewhere in this book. some microbes manipulate or exhaust effector immune responses, leading to secondary infections by other microbes. while the manipulation or exhaustion of immune responses is an important defense mechanism of some microbes to evade effective immune clearance, it is also likely that the genetic immune response repertoire that programs how an infected individual will respond to these microbes contributes significantly to whether the resulting immune responses will protect against, or facilitate, superinfection by other microbes. in this chapter, we discuss how some microbes manipulate or, in the case of the human immune deficiency virus (hiv), exhaust protective innate and/or adaptive immunity, ultimately leading to severe microbial superinfections by other infectious organisms that cause significant morbidity and, on occasion, mortality from secondary infections. a critical component of host defense includes the expression of immunosuppressive cytokines. il-10, and tgf-b, and the generation of regulatory t cells that can express these cytokines serve to limit the immune-mediated damage related to host defense. these same elements also cause secondary immune deficiency by suppressing or blocking effector th1-like responses. these immunoregulatory elements, generated in response to the original infection, as a consequence, can lead to secondary immune deficiency and the development of severe or fatal infection with other microbes. some of the microbes that cause temporary or permanent changes in immune responses to other organisms are shown in table 49 .1 (see below). in addition, fig. 49 .1 shows the functional diversity of cd4 þ t cells that has evolved to control the microbiome present on mucosal surfaces. in concert with the repertoire of these t cells, and the cytokines and chemokines that they express, the continuum of macrophages ( fig. 49. 2) 1 and their respective cytokine/chemokine repertoires expressed in response to microbial infection, form the critical immune response elements that are required to mount and maintain an effective immune response against these microbes. the amount of immunomodulatory cytokines, such as il-10, expressed during infection contributes to the development of secondary infections during and after recovery from the primary infection. alteration in the balance between immunity and immune suppression is in part, based on how much il-10 is expressed during a given infection. il-10 promoter polymorphisms that control high, intermediate, or low expression of il-10 2e4 during and after microbial infection may influence the development of resistance or susceptibility to secondary microbial infection. excessive il-10 levels defined by a given host's il-10 genotype could predispose an individual to develop secondary infections by blocking appropriate pro-inflammatory responses. in contrast, low il-10 levels, also defined by a given host's genotype, could protect against secondary infection, at the expense of limiting collateral tissue damage and t cell memory that higher il-10 expression would support. 5 taken together, the successful balance of cellular innate and adaptive immune responses, and the cytokine/chemokine repertoires that they express during a primary infection to an infectious microbe, can temporarily or permanently "hijack" effective immune responses that are necessary to prevent other infections by microbes it is often difficult to distinguish secondary immune deficiency in a normal individual with a significant initial primary infection, where the offending microbe temporarily subverts or paralyzes the adaptive immune response to other microbes, from a patient with primary immune deficiency disease (pidd) who presents with a serious initial infection (box 49.1). several clues help distinguish between an initial infection in these different patient populations. 6e8 among these clues that patients with pidd are more likely to have are: patients with normal immune systems who become infected with viruses, bacteria, or parasites known to temporarily subvert or paralyze adaptive immunity without the above history are more likely to have secondary immune defects caused by microbes that prevent appropriate immunity to other organisms. thus, the challenge for clinicians is to distinguish between patients with pid versus those with microbe-induced, secondary immune deficiency at the time of first presentation of a serious infection. in this review, we address the patient population with secondary, infection driven immune compromise, in contrast to patients with pidd who are the focus of the remainder of this textbook. sepsis with a wide variety of organisms causes a profound secondary immune deficiency. it is not an uncommon scenario to have to rule out a pidd in the intensive care setting where both the underlying disease and medications may alter the immunologic findings. sepsis is one of the more common causes of secondary lymphopenia (box 49.1). furthermore, there are both acute disruptions of immunologic function and longer term findings that are poorly characterized that are seen after trauma and burn injuries as well. in fact, increased mortality persists for years after an episode of sepsis. immunologic findings occurring with acute sepsis include: examples of microbes that cause temporary or long-term secondary immune deficiency 9 shows some of the microbes that are known to induce secondary immune deficiency, the cells that they affect, and the pathogens causing secondary infection. in some cases, secondary immune deficiency occurs as a result of a cytokine storm. some infections known to cause a cytokine storm are shown in box 49.2. below, we describe examples of how some of these microbes suppress immunity and thereby cause secondary immune deficiency, predisposing patients to secondary microbial infections. measles virus (mv) continues to cause child morbidity and mortality worldwide, despite the availability and use of an effective live attenuated measles vaccine. 10e14 part of the reason why control of mv continues to be elusive is that it is highly contagious for susceptible individuals and there are difficulties with vaccine delivery. mv infection begins in the respiratory tract, spreads systemically in lymphoid, epithelial and endothelial cells, and ultimately infects multiple organs, 15 causing a characteristic fever, rash, and conjunctivitis 10e14 days after respiratory infection ( fig. 49.3a and b). high fever, rhinorrhea and conjunctivitis typically precede the rash and the rash migrates from the head and neck to the hands and feet over 3e4 days. many of these manifestations are caused by the immune response made to mv, and commonly this response clears mv in infected tissues and prevents re-infection for life (fig. 49 .3c and d). however, mv infection can cause several weeks of immune suppression after resolution in select individuals. this is the primary cause of measles-associated deaths: mv-induced secondary infection. 16 although vaccination against measles is very high in the united states, 92.7% of children aged 19e35 months were vaccinated in 2017, 17 there are pockets of unvaccinated people who are susceptible to local outbreaks of measles. since "herd immunity" is primarily effective when the vaccination rate is around 96%, 18 vaccination levels below this level leave children and adults at risk for primary mv infection and secondary microbial infections. begin approximately 10 days after infection, with prodromal symptoms of fever, conjunctivitis, and oral koplik's spots, followed by a maculopapular rash for 3e5 days. (c) the rash represents adaptive immune responses with infiltration of cd4 þ and cd8 þ t cells into sites of mv replication and clearance. there is a rapid activation, expansion, and then contraction of mv-specific cd8 þ t cells. cd4 þ t cell responses appear at the same time, but activation is prolonged. mv-specific igm appears with the rash, and this is commonly used for diagnostic purposes. this is followed by the sustained mv-specific igg synthesis. immune suppression is evident during acute disease and for many weeks after recovery. (d) cytokines and chemokines produced during infection are found in plasma in elevated amounts. early, il-8 is increased. during the rash, ifn-g and il-2 are g and il-2 are produced by activated t h 1-like, cd4 þ and cd8 þ t cells. after rash resolution, t h 2-like t cells and regulatory cd4 þ t cells produce il-4, il-10, and il-13. measles was the first virus clearly identified to cause increased susceptibility to other microbial secondary infections. most often, measles-associated deaths are caused by severe, overwhelming pneumonia and diarrhea. 16 suppression of delayed hypersensitivity has been identified in tuberculin-sensitized individuals many weeks after complete resolution of mv infection (fig. 49 .3c). 19 furthermore, several weeks after successful mv recovery, increased susceptibility to other infections has been reported, and t cell function and in vitro proliferation of t cells in response to mitogens has been shown to be markedly decreased (fig. 49.4a and b) . 1, 20, 21 immunosuppression occurs during a period of intense immune activation that occurs during the onset of the mv rash and anti-mv immune responses (fig. 49.3c and d) . lymphopenia, skewing of th2-like chemokine polarized responses, and suppression of lymphocyte proliferation have also been documented (fig. 49.3d ). mv infection causes decreases in t and b cells in the blood during the mv rash period. 22e25 altered trafficking and increased apoptosis of mv-infected and uninfected lymphocytes contribute to the development of lymphopenia. 22,26e30 while lymphocyte numbers rapidly return to normal in the blood after the rash resolves, immunologic abnormalities persist. 21, 22, 31, 32 immune suppression, th2 cytokine polarization of cd4 þ t cells, and treg induction have been associated with indirect immunosuppression caused by mv infection. 33, 34 mv infection is also associated with suppression of il-12 expression, lymphocyte cd30 expression, and il-4, il-10, and il-13 expression after rash resolution. 35e37 reduction of il-12 production reduces t cell expression of type i cytokines, particularly ifn-g 10,32 (fig. 49.3d ). it is possible that mv interacts with the complement regulatory molecule cd46 in polarizing th2-like cytokine production, causing activation of signaling cascades that modify cell function, although this interaction is not firmly established. 38, 39 the mv-cd46 interaction may alter innate immunity by selectively downregulating receptor expression. 40e46 this would increase susceptibility to complement-mediated lysis of mv-infected cells, and decrease antigen presenting cell production of il-12 47, 48 and crosslinking of cd46 on t cells, leading to the induction of regulatory cd4 þ t cells and enhanced il-10 levels. 49 these interactions would induce th2-like polarization that would favor b cell maturation, provide lifelong mv antibody memory, and protect against mv reinfection. this polarization, however, would also depress apc activation and th1-like responses to new pathogens. mv suppresses pbmc proliferation to mitogens after mv resolution, and this continues for several weeks (fig. 49.4b ). 20,31 il-2 supplementation can improve, but not fully restore, this responsiveness. this suggests that defective il-2 expression is in part responsible for this proliferative defect. 50 cell cycle arrest in g1 after in vitro infection with mv is a recognized cause of hyporesponsiveness to mitogens. 42,51e53 mv rna can persist in pbmcs for months after mv resolution 54, 55 and may reduce mitogen proliferation, although this has not been established. the receptor used by wildtype mv to infect cells, cd150, is a dual function co-receptor for lymphocyte activation, and enhances ifn-g expression. 56e58 however, mv binding to cd150 can also downregulate receptor expression. 59,60 t cell signaling through the mv glycoprotein complex of h and f1-f2 in the membranes of virions or mv-infected cells 61e65 may also contribute to immunosuppression. this inhibitory signal prevents t cell s-phase entry for several days, and is independent of cell death, membrane fusion, soluble inhibitor production, or t cell infection. 52,61,62,65e67 thus, there is a delay in cell cycle progression and an accumulation of t cells in the g0/g1 phase. 52, 66, 67 the mechanism by which h/f1-f2 suppresses mitogen-induced proliferation is unknown, but it is associated with mv-induced interference of t cell activation of phosphoinositide 3-kinase (pi3k) in t cells, or il-2 receptor ligation. 68 il-2 added to mv-treated cells activates signal transducer and activator of transcription 3 (stat3) but fails to activate akt kinase, which is required for cell cycle progression. 69 the modulatory effects of mv with glycoprotein complexes, and the downstream consequences of this interaction, have recently been summarized. 10,68e70 while the relevance of these processes to the in vivo suppression of t cell lymphoproliferation remains to be identified. the combination of the established mechanisms leading to post-mv infection immunosuppression, and those that remain to be elucidated, cause, in select individuals, severe and on occasion, fatal secondary infection with other microbes. a key epidemiologic factor in measles-related deaths is vitamin a deficiency. in the developing world, the world health organization recommends vitamin a supplementation. studies have demonstrated improved outcomes and suggest an effect on the mucosal barrier and also on improved t cell function though by an undefined mechanism. three world-wide (pandemic) outbreaks of influenza virus (iv) occurred in the 20th century, in 1918, 1957, and 1968. they are now known to represent three different antigenic subtypes of influenza a virus: h1n1, h2n2, and h3n2, respectively. 71 the 1918 "spanish flu" epidemic claimed an estimated 50 million lives and 20%e40% of the worldwide population became ill; approximately 675,000 americans died during this epidemic. 72 since then, iv infections have caused more than 20,000 deaths yearly in each of the 20 epidemics from 1957 to 1991, 73 and greater than 40,000 deaths occurred in each of the more recent epidemics, thus, iv is a formidable pathogen. 74 while influenza-related mortality can in part be attributed to direct effects on the respiratory system, many of the deaths associated with iv infection are caused by increases in susceptibility to secondary bacterial pneumonia. 75 in fact, it has been suggested that the major mortality and morbidity resulting from iv infection may be caused by secondary bacterial infection that is associated with the inhibition of neutrophil function. 73 the bacteria commonly causing pneumonia in the most severely ill influenza patients are streptococcus(s.) pneumoniae, staphylococcus aureus, and haemophilus influenzae. together, iv infection itself, and secondary bacterial pneumonia, were the most common causes of infectious death in the united states in 2002. 76 research, clinical, and epidemiological studies show that there is a positive correlation between the increase in morbidity and mortality during influenza epidemics and pandemics, and an increase in secondary s. pneumoniae infection. 75, 77, 78 it has been hypothesized that influenza infection alters neutrophil function, thereby reducing the effectiveness of phagocytemediated killing of bacteria. an alternative hypothesis is that the tissue damage caused by iv alters the epithelial surface of the respiratory tract, thereby exposing different surface receptors to which s. pneumoniae adhere, and/or increasing the affinity of s. pneumoniae for its receptors, which may result in increased growth and decreased neutrophil killing of s. pneumoniae in the respiratory tract. support for the former hypothesis comes from reports using both in vitro and in vivo models of influenza infection. 79e90 neutrophils are important in resistance to s. pneumoniae infection independent of an influenza infection. 91 influenza a virus alters the three major properties of the neutrophil that are crucial for bacterial clearance, namely chemotactic responsiveness, phagocytosis, and intracellular killing. this alteration of neutrophil function likely increases the susceptibility of an influenza-infected patient to s. pneumoniae infection because of decreased phagocytosis and killing of these bacteria by neutrophils. 80, 84, 89, 92 in humans, iv was reported to alter cell function by interacting with g proteins. 81, 90, 93 human neutrophils express monomeric and trimeric g proteins that have critical roles in activation and regulation of various signal pathways, leading to chemotaxis and metabolic function. 94, 95 susceptibility to s. pneumoniae in mice is greatest at six days after influenza infection, consistent with the clinical findings. 96e99 influenza-induced tissue damage is also greatest six days after influenza infection, as is adherence of s. pneumoniae to murine influenza-infected tracheas. 100 influenza-induced neutrophil dysfunction is also greatest six days after influenza infection, 101 and murine mortality secondary to s. pneumoniae infection is greatest seven days after infection. although neutrophils accumulate in the lungs of mice infected with influenza by day six, they do not function in resistance to s. pneumoniae. thus, it is likely that bactericidal function of lung neutrophils is suppressed, making these influenza-infected mice susceptible to s. pneumoniae. effective neutrophil phagocytosis and killing of s. pneumoniae is necessary to eliminate s. pneumoniae from the lungs. 102 neutrophils are affected within 30 min of in vitro influenza infection, 83, 84, 87, 89, 103 with some effects seen as rapidly as 5 min after incubation with iv. changes in infected neutrophils include decreased protein phosphorylation, accelerated apoptosis, decreased respiratory burst activity, an altered cytoskeleton, depressed bactericidal capacity identified by release of reactive oxygen species, decreased chemotactic ability, decreased adherence, decreased release of lactoferrin into phagosomes, and inhibition of lysosomeephagosome fusion. 81, 82, 84, 87, 89, 92, 103 in addition, the effects of influenza virus infection on neutrophil function are not limited to the lungs, indicating that these effects are systemic in nature. 89, 90 influenza infection also increases susceptibility to s. pneumoniae by increasing cytokine production (tnf, ifn-g, mcp-1, il-10, il-6) in the lung. 75 il-10 is also elevated in post-influenza pneumococcal pneumonia, leading to increased susceptibility to s. pneumoniae long after an influenza infection has been resolved. 104 susceptibility remains at least 14 days after a primary influenza infection, and is thought to be mediated by il-10 inhibition of neutrophil function, resulting in increased bacterial growth in the lungs leading to mortality. 104 of note, viral influenza neuraminidase increases s. pneumoniae adherence in the lungs by cleaving sialic acid residues, and exposing receptors to which s. pneumoniae can adhere. 78 thus, both neutrophildependent and -independent mechanisms cause increased susceptibility to secondary s. pneumoniae infection after a primary influenza infection. novel therapies that can restore neutrophil function caused by iv infection may be helpful. 73, 75 human immune deficiency virus: long-term immunosuppression hiv is a double-stranded, enveloped rna retrovirus from the genus lentivirus within the subfamily retroviridae, with a tropism for human cd4 þ cells, including t cells and macrophages. 105 two hiv types have been identified, hiv-1 and hiv-2, and both cause similar but not identical human disease. the hiv genome contains three structural genes (gag, pol, and env) and six regulatory genes (tat, rev, nef, vif, vpr, and vpu). these proteins, their function, and their role in forming the viral particle have recently been summarized. 105 the env protein is cleaved to produce two envelope proteins, gp120 and gp41, which are involved in hiv binding to cd4 and the chemokine receptors cxcr4 and ccr5 on cell surfaces. 105 tat, nef, and rev proteins play a role in downregulating classical mhc class i molecules on the surface of hiv-infected t cells. 106e108 in the case of tat, hla-c and hla-e are spared. 109 the classical hla presenting molecule hla-c on antigen presenting cells is not as potent in presenting viral peptides to t cells similar to the non-classical mhc class i molecule hla-e. however, the non-classical mhc class i molecule hla-e, similar to hla-a, hla-b, and hla-c, suppress nk function. this viral strategy to evade immune surveillance serves two functions: (1) prevent cd8 þ t cells from recognizing hiv peptides presented by class i mhc molecules, and (2) prevent nk cell recognition and activation because hla-c and hla-e remain on hiv-infected cells leading to inhibition of nk killing of class i mhc expressing target cells. the nef protein also downregulates cd4 expression on the surface of hiv-infected cells, which is a co-receptor of the t cell receptor participating in t cell activation. 110 this facilitates hiv-infected cell escape from immune surveillance. following hiv gp120 protein binding to cd4 and ccr5 molecules on target cells, hiv-infected cells migrate to the lymph nodes, where initial replication and infection of nearby cd4 þ t cells occurs. 111 during acute hiv infection, gut-associated lymphoid tissues, predominantly memory cd4 þ t cells, are severely depleted. there is high hiv viremia and immune activation. 112, 113 hiv induces t cell lymphopenia through several mechanisms: hiv-induced apoptosis; a viral cytopathic effect; non-specific immune activation-induced apoptosis; and cytotoxicity of hiv-infected cells. 105 autophagy is also induced by hiv env protein in uninfected t cells. 114 in addition, shedding of gp120 molecules by hiv triggers a series of events that cause the adaptive immune system to become less effective by altering the normal balance of immunoregulatory th1 and th2 t cells. impaired function of hiv-infected macrophages and dendritic cells contributes to the failure of effective innate and adaptive immune responses to secondary infection. the acute and latency phase of hiv infection is shown in fig. 49 .5. 115, 116 without combined antiretroviral therapy (carv), cd4 þ t cell counts progressively decrease, and the host usually succumbs to infections with opportunistic organisms that occur because of hiv-induced secondary immune deficiency. specific anti-hiv, cd4 þ , and cd8 þ t cells, and neutralizing anti-hiv antibodies develop, however, these responses are eventually overcome by viral escape strategies. 105 patients can present with constitutional symptoms, such as fever, weight loss, diarrhea, lymphadenopathy, secondary opportunistic infections, and viral skin infections, heralding the presence of an immunocompromised immune system. peripheral cd4 þ t cell counts of <200 cells/ml herald the development of aids. this t cell depletion predisposes patients to develop opportunistic infections including, but not limited to, cytomegalovirus, herpes simplex, varicella zoster virus, pneumocystis jirovecii pneumonia, histoplasmosis, toxoplasmosis, coccidioidomycosis, cryptosporidium, nocardia, mycobacterium avium complex, salmonella, and toxoplasma gondii. if hiv-infected patients do not receive antiretroviral treatment, repeated infections with these opportunistic secondary infections ultimately lead to death. thus, hiv infection exhausts the adaptive immune system, leading to chronic depletion of cd4 þ t cells and immune dysfunction of the multiple effector immune responses that are required to prevent secondary bacterial, viral, fungal, and protozoan infections. human t-lymphotropic viruses (htlv) are complex type c retroviruses with a capsid that contains two simple rna strands with associated reverse transcriptase and integrase enzymes which are essential for insertion of the virus into the host genome. 117 several types of htlv have been identified (htlv-1, htlv-2, htlv-3, htlv-4). while hiv-1 was previously called htlv-3, htlv-3 is a different virus. 117 similar to other retroviruses, htlv-1 contains gag, pro/pol and env genes that have structural and functional roles. in addition, the px region codes for regulatory proteins (such as transactivator protein, tax, and the helix basic zipper protein, hbz) essential for viral transcription, and inhibition of signal transduction pathways, such as nfkb (leading to decreased socs1), and ap-1. this leads to htlv-1 inducing cytotoxic t cells that can kill virus-infected cells. 118 the htlv-1 tax gene can decrease th1-like antiviral signaling pathways both by modulating the suppressor of cytokine signaling 1 (socs1) 119 and also through the aryl hydrocarbon receptor protein (aip) that binds to interferon regulatory factor 7 (irf7), thus decreasing type i interferon (ifn-a/b) expression. 120 htlv-1 modifies the behavior of cd4 þ t cells and alters their cytokine production. htlv-1 is clinically associated with adult t cell leukemia/lymphoma (atl), and tropical spastic paraparesis/htlv-1-associated myelopathy (pet/ham) 121 and can cause autoimmune disease, such as rheumatoid arthritis, systemic lupus erythematosis, and sjögren's syndrome. 117e120 the development of the htlv-1 induced autoimmunity is thought to rely on molecular mimicry, and htlv-1 induced t cell immunosuppression is caused by direct infection of cd4 þ t cells. cd4 þ t cells that have altered function and cytokine production. tax is the primary inducer of clonal infected t cell expansion, and genetic instability. 117 tax expression promotes t cell activation, proliferation, and resistance to apoptosis. 122 clinically, patients infected with htlv1 have increased infections and increased autoimmunity, particularly rheumatoid arthritis and sjogren syndrome. the autoimmunity correlates somewhat with viral load, however, there are immunologic mechanisms that probably facilitate breaks in tolerance leading to autoimmunity. in addition, natural killer (nk) cells from individuals with pet/ham have decreased expression of the activating receptor nkp30. 123 this likely results in decreased nk cell activation, leading to decreased ability of nk cells to kill virusinfected cells. htlv infection is clinically associated with an increased risk of disseminated strongyloidiasis, 124 schistosomiasis, 125 likely due to high levels of ifn-g, decreasing the production of th2-like cytokines (il-4, il-5, il-13) and ige essential to contain these parasitic infections. taken together, htlv induces cytotoxic t cells to kill virus-infected cells, alters cd4 þ t cell function and cytokine production and it decreases nk cell activation leading to susceptibility to subsequent disseminated parasitic infections. many viral infections have been associated with bm failure or hyperproliferative syndromes. table 49 .2 summarizes some of the human viral infections that cause self-resolving or persistent bm suppression. 126 however, the specific pathogenic mechanisms that underlie the reported virally induced bm manifestations have not been fully characterized. nonetheless, many different viruses generate the same pathological condition. this suggests that a common underlying mechanism(s), specifically virologic or immunologic are responsible for the clinical pathologic outcome. certain virus can also lead to different pathological manifestations in different patients heralding a genetic basis for aberrant immune activation in bm failure. there are 4 different mechanisms by which viruses can affect hematopoietic stem and progenitor cells (hspc). these mechanisms include: (1) direct viral infection, (2) viral recognition by hspcs and indirect effects, (3) inflammatory mediators, and (4) changes in the bm microenvironment. taken together, many types of viruses can affect hematopoiesis acutely (parvovirus b19, dengue), transiently, permanently, chronically (cmv, hiv), systemically (hiv), locally (influenza), directly, or indirectly, causing disruption of the hematopoietic process. 126 parasites that hijack immune responses to other microbes leishmaniasis: temporary immune suppression vl is fatal if not treated, and is caused by leishmania donovani, leishmania infantum, and leishmania chagasi. 129, 130 immunity to leishmaniasis is mediated by the cellular and humoral arms of the mammalian immune system: the innate system (by neutrophils, macrophages, and dendritic cells) and by adaptive (t cells) responses. 131 sand-fly bites cause minimal tissue damage, but the bite promotes neutrophil recruitment. 132, 133 parasites can survive for a time within neutrophils, and eventually they parasitize neutrophils. viable parasites are engulfed by macrophages or dendritic cells. inside the macrophages, promastigotes change into amastigotes and they reproduce by binary fission, ultimately rupturing the macrophage and releasing amastigotes into the blood. leishmania modulates the normal antimicrobial mechanisms of the macrophages. they increase membrane fluidity and thereby disrupt lipid rafts and apc antigen presentation as well as lysosomal fusion. 134 leishmania-infected apcs interact with t cells, and together they express cytokines and chemokines that begin to "hijack" the immune system, which enables these parasites to survive. 135 cell-mediated immunity is the principle host defense against leishmania species. t h 1-like cells primed mainly by apcs and il-12 expression are major effector responses to this microbe. 136e139 the dichotomy between t h 1-like protection (ifn-g, il-2, and tnf) and th2like disease progression (il-10, il-4) in mice has been shown to be essential in cl infection, 140 although this paradigm is less clear in humans infected with vl. 141, 142 the immunopathology of vl is complex, and involves cellular and genetic factors that confer disease susceptibility, versus resistance to leishmania species. 143 persistent vl correlates with a chronic th2-like (il-10, il-4, il-5, and il-13) response to l. donovani 142 and increased serum il-10 expression. 144e147 il-10 is crucial in establishing and maintaining t h 2-dependent chronic suppression of th1-like mediated immunity in cl. 148e150 and inhibits amastigote killing. 150,151 il-10 also inhibits the production of ifn-g. 152 cellular immunity impairment correlates with active disease progression secondary to il-10 expression, independent of ifn-g levels. 153 in murine cl, cd4 þ cd25 þ foxp3 þ natural tregs are a major il-10 source and are crucial for parasite survival. 154 however, il-10 is also derived from non-tregs. 146, 155 this suggests that any therapeutic approach to treat leishmania infection will require species-specific manipulation of the immune responses made to these parasites. for example, the potent t h 2-like cytokine il-4 is critical in cl, but not in vl. 141 thus, modulating il-4 expression may be helpful in vc, but not in the treatment of cl. 156 resolution of human and murine vl depends on the production of th1-like cytokines. 139,140,147,157e160 il-12 and ifn-g are crucial in controlling parasite growth and development. 157, 161 while il-10 suppresses host immunity and helps parasite survival, it is the antidote to inflammation that reduces tissue damage caused by exaggerated inflammation. 162 since ifn-g -producing cells can also produce small amounts of il-10, this may act as a negative feedback in controlling tissue damage 142, 146, 163, 164 and supporting immune memory. tnf stimulates ifn-g -induced expression of nitric oxide by apcs that can kill vl parasites. 165e170 th17-like cells that produce il-17 are pro-inflammatory, and stimulate il-6, tnfa, and chemokine expression. however, l. donovani strongly induces il-17 and il-22, and enhances the secretion of these cytokines that protect this parasite. 171 cd8 þ cytotoxic t cells are also important in controlling cl and vl. 172 cd4 þ and cd8 þ t cells, and their pro-inflammatory cytokines, are required to control vl parasite proliferation and leishmaniacidal activity. 138,161,173e176 however, in canine vl, antibodies are insufficient in providing protection in the absence of -proinflammatory t cell responses. 132 thus, in addition to protective t cell responses, other t cell subsets, including tregs and th17 cells, confer either susceptibility or resistance to animal verses human vc. other cells may also be important in the resolution of these infections, thus further investigation is required in designing vaccines or treatments for these diseases. greater than 20% of cl patients have reported secondary bacterial infections. 177 s. aureus, coagulase-negative staphylococcus, escherichia coli, proteus, and klebsiella have been cultured from lesions (listed in decreasing frequency). however, the incidence of these infections is higher in ulcerated compared to non-ulcerated lesions. thus, both disruption of the skin, and possibly immunosuppression caused by cl, likely play roles in the development of secondary bacterial infection in cl, leading to severe or lethal bacterial infection. 177 increased treg numbers in cl have been linked to activation/reactivation of latent microbial infections (mycobacterium tuberculosis, toxoplasma, herpes viruses) that cause significant morbidity and mortality, possibly as a result of immunosuppression and other factors, although how activation/reactivation of dormant infections occurs remains unknown. 178 thus, leishmania infection causes secondary infections, and activation/reactivation of dormant infections that can lead to severe or lethal outcomes through multiple mechanisms. 178 l. donovani infection can also clinically resemble and cause hemaphagocytic lymphohistiocytosis (hlh) and successful treatment of leishmaniasis can lead to the complete resolution of hlh symptoms. 179, 180 these reports open the question as to whether hlh patients should be prescreened and aggressively treated for visceral leishmania infection prior to instituting stem cell transplantation for hlh when from endemic areas. 179 vl has been associated with significant secondary infections. these may be confounded by hlh where neutropenia is common. infections ranging from sepsis to urinary tract infections occur in a significant subset of patients with vl. the most common pathogens causing sepsis are s. aureus, klebsialla pneumonia, and pseudomonas aeruginosa. common laboratory features include neutropenia, eosinopenia and leukopenia. co-infection with hiv alters the clinical presentation with more lung and gastrointestinal involvement. taken together, the innate response (professional apcs) is altered by leishmaniasis infection, leading to polarized adaptive immune responses that support or block persistent infection by leishmania organisms. this subverts or supports effective immunity to these parasites, and in patients with persistent infection, the th2-like/treg bias may predispose patients infected with leishmania species to develop or reactivate lethal secondary superinfections, and the development of hlh-like clinical symptoms. malarial organisms are parasites with a major public health impact world-wide. there were an estimated 219 million cases of malaria (range 154e289 million) and 660,000 deaths (range 610,000e971,000) in 2010. 181 plasmodium species cause malaria and can induce immunosuppression in infected individuals 182 resulting in increased susceptibility to secondary infections, such as non-typhoidal salmonella, 183 herpes zoster virus, 184 hepatitis b virus, 185 moloney leukemia virus, 186 and epstein-barr virus reactivation. 187 efficacy of heterologous vaccines can also be suppressed in malaria-infected patients, 188 further documenting the immunosuppressive effects of malaria infection. malaria parasites inhibit dc maturation 189 via uptake of malaria pigment hemozoin (hz) from parasitized rbcs. 190 dc inhibition reduces t cell expansion, cytokine production, and migration into b cell follicles. 191 the effect on dc changes occurs during the different phases of this infection. although dc function is impaired immediately following the initial burst of parasitemia, t and b cell responses to heterologous antigens change dynamically during the course of infection. 191 t cell proliferation, and effector function and migration are suppressed, and b cells do not expand or produce antibodies. hz, prevents the formation of stable, long-lasting cellecell contacts between t cells and dcs impairing costimulatory activity. 192 nonetheless, there is also a t cells functional defect. 188 taken together, impairment of both innate and adaptive signaling induced by malarial infection/hz pigment skews the immune response toward tolerance instead of immunity, and this subversion of effective immunity leads to secondary infection with other organisms as a consequence of this parasitic infection. invasive infections in patients with malaria are due to s. pneumoniae, h. influenzae type b, s. aureus, e. coli and other gram-negative bacteria. risk factors include younger age, recent malaria infection, severe anemia, splenomegaly, hiv-co-infection and severe malnutrition. some of this susceptibility seems to reflect functional asplenia and impaired barrier function. bordetella pertussis: temporary immunosuppression b. pertussis, the causative agent of whooping cough, is an infection that can be fatal in infants, but in older children, adolescents, and adults usually causes a chronic cough of varying severity that generally persists long after clearance of the infection. this bacterial infection of the airways is an important cause of infant death world-wide, and continues to be a public health concern even in countries with high vaccination coverage. while estimates of the incidence of new cases of pertussis vary, 193, 194 pertussis infection world-wide varies between 16 million and 50 million cases yearly, 95% of which are in developing countries. 193, 194 in addition, between 195,000 and 300,000 deaths, mostly of young infants who were either unvaccinated or incompletely vaccinated, occur yearly. 193, 194 for several decades, infant immunization programs using pertussis vaccines of high efficiency have been highly successful in preventing severe pertussis in infants. however, pertussis continues to be a significant health issue world-wide. following an incubation period of 9e10 days (range 6e20 days) patients develop catarrhal symptoms including cough, and over 1e2 weeks develop coughing paroxysms that end in the characteristic "whoop" associated with this infection. although the cause of the characteristic "whoop" associated with b. pertussis infection remains unresolved, a significant body of knowledge has accumulated to explain how b. pertussis induces immune suppression, evasion, and modulation, thereby leading to a poor clinical outcome. fig. 49 . 6 195 gives an overview of several b. pertussis virulence factors that have immunomodulatory properties, the cells that they target, and the mechanisms thought to be involved in the induction of immune dysregulation caused by this infection. it has been shown that b. pertussis-derived filamentous hemagglutinin (fha) is the major surface structure that mediates adherence of b. pertussis to host cells, primarily to cilia of airway ciliated epithelium. however, multiple factors likely contribute to this binding process, 196 including secreted adenylate cyclase toxin (act), which enhances fha adherence to lung epithelial cells. 197 fha also has immunosuppressive and modulatory activities by its ability to induce fha-specific tregs that secrete il-10 and suppress t h 1-like responses to b. pertussis. 198 anti-fha antibodies can also reduce phagocytosis of these bacteria by human neutrophils. 199 act, a secreted toxin, targets host phagocytic cells by binding complement receptor 3 (cr3; cd11b/cd18). 200, 201 act-deficient mutants of b. pertussis are more efficiently phagocytosed by human neutrophils, 202 and in mice, lower bacterial loads are found in the respiratory tract. 203 secreted act upregulates major histocompatibility complex class ii mhc and co-stimulatory molecule expression on dendritic cells (dcs). act also prevents maturity, and thereby decreases their proinflammatory cytokine production. 204e206 tracheal cytotoxin (tct) is also expressed at relatively high levels by b. pertussis, and is a disaccharide-tetrapeptide fragment of peptidoglycan. purified tct, in synergy with lipopolysaccharide (lps), damages ciliated airway epithelial cells through production of il-1a and nitric oxide, 207 causing deleterious effects on neutrophils. 208 pertussis toxin (pt), uniquely produced by b. pertussis, is another secreted toxin expressed by this bacterium. pt adp ribosylates several heterotrimeric g proteins in mammalian cells, has long been known to disrupt signaling pathways with a wide range of downstream effects, 209 and can cause immunosuppression. pt causes lymphocytosis. 210 pt-deficient mutant strains show reduce levels of airway infection 24 h after inoculation because pt delays neutrophil recruitment and influx to the airways, 203, 211, 212 reduces anti-b. pertussis antibody-induced bacterial clearance, 211 depletes airway macrophages 213 enhancing infection. 214 since adp ribosylation of airway macrophage g proteins by pt has been shown to be long-lasting, and correlates with active infection-promoting longevity of this microbe. 212 this evidence supports the concept that the effects of pt on host cells in the airway may be particularly long-lived. pt also exerts multiple suppressive effects on the immune system beyond innate immune cells, including suppression of serum antibody responses to b. pertussis antigens following infection, 214e216 reduction of major histocompatibility complex class ii expression on human monocytes, 217 and modulation of dendritic cell expression of surface markers. 218 b. pertussis infection causes immunomodulation of toll-like receptor (tlr4), 219 which recognizes the lipopolysaccharide found on b. pertussis and gram-negative bacteria. 220,221 tlr4 signaling triggered by b. pertussis induces il-10, which can inhibit inflammatory responses, and limit airway inflammation, 220 and can synergize with act to induce il-10. 206 in addition, tlr4-dependent responses induced by b. pertussis drive lower levels of inflammatory cytokines. 222 thus, pt promotes b. pertussis infection by multiple mechanisms through its effects on innate immunity in the initial stages of disease in naïve individuals. this reduces adaptive immune responses during the initial infection, and promotes re-infection in partially immune individuals. the consequences of these suppressive effects on both the innate and adaptive immune responses induced by b. pertussis infection can cause secondary infection, typically pneumonia, which is the major cause of fatal outcome from this infection. 223 infants are at highest risk of all complications related to b. pertussis infection but all ages have relatively high rates of secondary complications. approximately 50% of infants exhibit apnea of greater or lesser duration and a quarter develop pneumonia. roughly 5% have otitis media and 1% develop seizures. in adults, the most common secondary effect is pneumonia occurring in about 5%. distinguishing patients with secondary immune deficiency disease caused by a primary infection in an individual with an "intact" immune system from patients with an underlying pidd is often a challenge. recognizing primary infections that can cause secondary immunodeficiencies is an important factor in discrimination between these two groups of patients. clinical immunologists need to always consider whether an acute, serious infection is causing a secondary primary infection in an 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moloney leukaemia virus by malarial infection exposure to holoendemic malaria results in elevated epstein-barr virus loads in children impairment of the immune response to vaccination after acute malaria dendritic cells can initiate protective immune responses against malaria phagocytosis of the malarial pigment, hemozoin, impairs expression of major histocompatibility complex class ii antigen, cd54, and cd11c in human monocytes suppression of adaptive immunity to heterologous antigens during plasmodium infection through hemozoin-induced failure of dendritic cell function malaria impairs t cell clustering and immune priming despite normal signal 1 from dendritic cells cdc -pertussis: pertussis in other countries immunomodulation in the pathogenesis of bordetella pertussis infection and bordetella bronchiseptica adherence to cilia is mediated by multiple adhesin factors and blocked by surfactant protein a adenylate cyclase influences filamentous haemagglutininmediated attachment of bordetella pertussis to epithelial alveolar cells pathogen-specific t regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective t helper type 1 responses by bordetella pertussis phagocytosis of bordetella pertussis incubated with convalescent serum bordetella adenylate cyclase toxin: a swift saboteur of host defense pore-forming and enzymatic activities of bordetella pertussis adenylate cyclase toxin synergize in promoting lysis of monocytes influence of cr3 (cd11b/cd18) expression on phagocytosis of bordetella pertussis by human neutrophils pertussis toxin and adenylate cyclase toxin provide a one-two punch for establishment of bordetella pertussis infection of the respiratory tract pertussis toxin and the adenylate cyclase toxin from bordetella pertussis activate human monocyte-derived dendritic cells and dominantly inhibit cytokine production through a camp-dependent pathway adenylate cyclase toxin from bordetella pertussis synergizes with lipopolysaccharide to promote innate interleukin-10 production and enhances the induction of th2 and regulatory t cells bordetella type iii secretion and adenylate cyclase toxin synergize to drive dendritic cells into a semimature state synergistic epithelial responses to endotoxin and a naturally occurring muramyl peptide effect of tracheal cytotoxin from bordetella pertussis on human neutrophil function in vitro pertussis toxin in the analysis of receptor mechanisms biological activities of crystalline pertussigen from bordetella pertussis pertussis toxin inhibits neutrophil recruitment to delay antibody-mediated clearance of bordetella pertussis pertussis toxin plays an early role in respiratory tract colonization by liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications pertussis toxin targets airway macrophages to promote bordetella pertussis infection of the respiratory tract suppression of serum antibody responses by pertussis toxin after respiratory tract colonization by bordetella pertussis and identification of an immunodominant lipoprotein homologous and heterologous protection after single intranasal administration of live attenuated recombinant bordetella pertussis bordetella pertussis infection of primary human monocytes alters hla-dr influence of pertussis toxin on cd1a isoform expression in human dendritic cells toll-like receptor 4-mediated innate il-10 activates antigen-specific regulatory t cells and confers resistance to bordetella pertussis by inhibiting inflammatory pathology host genetics of bordetella pertussis infection in mice: significance of toll-like receptor 4 in genetic susceptibility and pathobiology comparative toll-like receptor 4-mediated innate host defense to bordetella infection cytokine storms in infectious diseases pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology new fronts emerge in the influenza cytokine storm molecular pathogenesis of viral hemorrhagic fever immune-mediated cytokine storm and its role in severe dengue virus-induced pathogenesis, vaccine development, and diagnosis of novel h7n9 avian influenza a virus in humans: a systemic literature review cytokine storm combined with humoral immune response defect in fatal hemorrhagic fever with renal syndrome case lipoxin a4, a 5-lipoxygenase pathway metabolite, modulates immune response during acute respiratory tularemia molecular pathogenesis of ehrlichia chaffeensis infection evidence that lipopolisaccharide may contribute to the cytokine storm and cellular activation in patients with visceral leishmaniasis key: cord-019100-rce6kyu4 authors: heymann, peter w.; zambrano, juan c.; rakes, gary p. title: virus-induced wheezing in children: respiratory syncytial virus (rsv) and rhinovirus date: 1998-02-01 journal: immunol allergy clin north am doi: 10.1016/s0889-8561(05)70345-3 sha: doc_id: 19100 cord_uid: rce6kyu4 the strong association between infantile wheezing and respiratory tract infections caused by the respiratory syncytial virus (rsv) has been well established. in studies of older children, rhinovirus becomes the major virus associated with asthma. these relationships are outlined in the box on page 36. in the past, this relationship was more difficult to appreciate, because rhinovirus does not always grow well in culture. in addition, the linkage between asthma and atopy during childhood has raised the question whether viral infections alone can precipitate exacerbations of asthma. use of the polymerase chain reaction (pcr) to measure viral nucleic acid material has provided the opportunity to study virus-induced wheezing among children in greater detail, and investigations of experimental rhinovirus infections in adults have demonstrated how this virus can augment both the early and late phase manifestations of airway hyperreactivity. this article reviews recent advances that have enhanced our understanding of virus-induced wheezing, along with new information indicating that interactions between viral infections and allergic inflammation may be critical to the pathogenesis of acute symptoms. a. early childhood wheezing (1st 2 years of life): 1. predisposing risk factors: prematurity; lower lung function at birth; maternal smoking during and following pregnancy; frequent exposure to viral infections at day care. 2. major risk factors for acute attacks: viral infections, especially rsv; tobacco smoke exposure. 1. predisposing risk factors: genetic predisposition (maternal asthma and atopy is a stronger influence than asthma and atopy in the father); atopic dermatitis and/or food hypersensitivity: increased exposure to aeroallergens early in life. 2. major risk factors for acute attacks: viral infections, especially rhinovirus; exposure to aeroallergens (dust, pollen, weed, mold allergens); tobacco smoke exposure. the interaction between rhinovirus infections and allergic inflammation becomes significantly associated with wheezing after the age of 2 years. b. development of asthma: *datafrom information and literature cited in references 11, 17, 23, 35 , and 48. a vast majority of children become infected with rsv during their first 2 years of life, and approximately 90% of children have a serologic response to this virus by the age of z. 31 in keeping with this, rsv is the most common viral respiratory tract pathogen isolated from infants who wheeze. many of the attacks that lead to emergency room visits and hospitalizations occur within the first 6 months of life, when infants are more likely to experience their initial infection.", **, 31 current information indicates that rsv infections during infancy are not, by themselves, responsible for the development of a~thma.3~ it has become clear, for example, that most infants who wheeze (approximately 60% to 70%) become symptom-free as they grow older.", 4x nevertheless, up to one third of the children who wheeze with rsv are at risk for recurrent and persistent wheezing, and there is considerable interest in research that will improve our ability to identify those children early in life. it has become apparent, for example, that infants whose parents have allergic symptoms or asthma, especially the mother, have a greater risk for developing asthma and that predisposing events that may influence this process may even begin in utero.zu, 35 infants with atopic dermatitis or those who become sensitized to food allergens early in life also have an increased risk for developing allergic respiratory tract symptoms, including asthma, as they grow ~l d e r .~, '~, 3u among infants who die from serious rsv disease, a vigorous perivascular lymphocyte response has been observed in the lungs at however, a different pathogenic process also has been observed during clinical trials of a formalin-inactivated rsv vaccine; this process led to serious pulmonary disease as well as peripheral blood eosinophilia in over half of the vaccine recipients when they acquired a subsequent rsv infection? pulmonary eosinophilia, characteristic of lung inflammation in asthma, also was noted in autopsy specimens from some of the recipients who died.33 mechanisms for the development of this eosinophilic response in infants are not clear. in previous studies, an ige mechanism for wheezing was proposed for some infants based on the detection of ige antibody specific for rsv antigen in their nasopharyngeal secretion^.^^ increased levels of histamine in secretions from the same patients also were reported. in addition, elevations in nasal eosinophil cationic protein (ecp) were observed by the same investigators in a separate study of rsv infected infants.'* at present, however, it is not clear that ige to rsv has a positive predictive value for the development of asthma;* and high levels of nasal ecp and ige to rsv have not yet been measured in the same patients. thus, the functionality of ige to rsv in the pathogenesis of infantile wheezing and its relevance to the development of asthma is uncertain. under certain circumstances, there is evidence that rsv infections may be capable of inducing a th, lymphocyte response leading to the development of eosinophil inflammation. in mice, th, responses to the capsular rsv-attachment antigen (g protein) have been demonstrated, leading to the production of il-5 when lymphocytes from sensitized mice are stimulated in vitro with rsv-g. these mice also developed pulmonary eosinophilia following subsequent intranasal infection with rsv.4v,4y the quantities of il-4 or ige antibody produced by these animals is not significant, and the eosinophil response in lungs also has been produced in b-cell-deficient mice, suggesting a mechanism independent of ige antibody. this eosinophil process, however, requires prior immunization (sensitization) and may not explain the pathogenesis of rsv-induced wheezing in humans when they experience initial infections during early infancy. other studies have examined the elaboration of cytokines (le., il-11) that may be important in the pathogenesis of airway inflammation caused by rsv, as well as parainfluenza virus.13 taken together, our understanding of infantile wheezing induced by rsv has increased substantially. however, research leading to new treatments (vaccines and pharmacologic agents) to counter the adverse effects of rsv and the identification of wheezing infants who are predisposed to developing asthma remain important problems. in studies of school-age children with asthma, common cold viruses were associated with 80% to 85% of wheezing exacerbations in a community-based study of asthmatic children aged 9 to 11 yearsz6 using pcr analysis, rhinovirus accounted for two thirds of the viruses isolated. inspection of symptom score charts revealed sudden falls in peak flow values within 2 days of the onset of cold symptoms, followed by recovery to baseline during the subsequent 2 to 3 weeks. in another study, a strong relationship between rhinovirus infection and asthma was observed in children treated in a pediatric emergency department for acute wheezing." rhinovirus was the dominant virus cultured in nasal aspirates from wheezing children after the age of 2 years. from this age on, rsv was not significantly associated with wheezing, whereas the combination of allergen-specific ige antibody and a positive culture for rhinovirus was particularly striking (odds ratio for wheezing = 10.8). when nasal washes from another study of children seen in the emergency room were tested using pcr techniques, rhinovirus was again the most common virus associated with wheezing after the age of 2.43 once again, the combination of allergen-specific ige antibody and viral infection increased the risk for wheezing (odds ratio = 16.4). even more striking among the wheezing children was a strong relationship between a positive pcr test for rhinovirus and the presence of eosinophils or ecp in nasal secretions. although school-age children experience approximately six to eight colds per year as compared with two to three colds per year for adults, recent pcr data also indicate that rhinovirus infections were associated with 38% of asthma exacerbations in adult individuals 19 to 46 years old.3y other respiratory tract pathogens have been isolated from children and adults during asthma exacerbations. coronavirus, which has been identified as a causative agent in 10% to 20% of common colds, also has been implicated in attacks of asthma.41 less frequently, influenza and adenovirus have been detected. although mycoplasma pneumoniae has been observed in up to 20% of community-acquired episodes of pneumonia, this organism probably is an infrequent cause of asthma exacerbations.36 thus, evidence from several studies indicate that rhinovirus is the major viral pathogen linked to attacks of asthma in children and adults. this information has stimulated additional studies, primarily experimental infections in adults, to explore how rhinovirus alters lower airways function. because of the importance of rhinovirus as a cause of common cold symptoms, there is considerable information characterizing the structure of rhinovirus and its interactions with the intercellular adhesion molecule-1 (icam-1) receptor. the human rhinoviruses represent a large genus within the class of picornaviruses (small rna viruses). the genus contains up to 100 distinct serotypes showing antigenic diversity. 44 the molecular structure of rhinovirus (type 14) has been characterized in great detail. this virus is a small (30 mm) nonenveloped particle within an icosahedral protein capsule composed of 60 protomeric units with four capsid proteins (vp 1, vp 2, vp 3, and vp 4). x-ray diffraction studies have shown canyons on the capsid surface that contain recognition binding sites for icam-1.44 the receptor icam-1 belongs to the immunoglobulin super-group of proteins and is expressed on the luminal surface of epithelial and endothelial cells. its natural ligand is lfa-1 (lymphocyte function associated antigen), which is widely expressed on leukocytes including eosinophils. its upregulation can be induced by a number of cytokines (i.e., ifn-?) and is associated with the recruitment of leukocytes to the sites of inflammation. icam-1 is also the host receptor used by the vast majority (goo/,) of rhinovirus serotypes for cell attachment and entry.sn it has been suggested that its up-regulation in allergic inflammation may predispose allergic individuals to higher rates of rhinovirus infection, leading to further increases in icam-1 expression and inflammatory cell infiltration^.^ its proposed up-regulation in asthma also may increase the probability of a rhinovirus infection and the capacity of this virus to provoke an asthma attack. standard methods for detecting rhinovirus have included viral culture, serology, and immunofluorescent techniques. many epidemiologic studies of the common cold and initial studies of experimental rhinovirus infections relied on cultures for detection. recovery rates of rhinovirus in nasal secretions from children experiencing natural colds have varied from 10% to 3oy0.~' however, rhinovirus does not grow optimally in culture, and positive results in population based studies may underestimate the prevalence of rhinovirus infections. more recently, pcr methods that detect rhinovirus rna have been compared with cultures in children with common cold symptoms. the results demonstrated a detection rate up to threefold greater using pcr analysis.z8 however, during experimental colds, rhinovirus can be recovered in culture in up to 90% of nasal washes obtained from patients at the height of symptoms (2 to 3 days following inoculation).ir this raises the possibility that the lower detection rates for rhinovirus using cultures from individuals during natural colds may result from the delay in obtaining samples and abatement of viral shedding once the infection is in progress. several investigations have examined rhinovirus infections and the pathogenesis of common cold symptoms in nonallergic individuals as outlined in the box below. these studies have explored the effects of rhinovirus on the upper and lower respiratory tract and have established a framework for more recent studies of rhinovirus infections in allergic individuals. the timing of viral shedding in relation to cold symptoms has been carefully examined following experimental rhinovirus inoculation of adult volunteers.ir viral replication in the nasopharynx was noted to increase 1 day after inoculation and peaked at 48 hours. cold symptoms including rhinitis, pharyngitis, and bronchitis began within 24 to 48 hours and then peaked by day 3 or 4. in a study of ct scans from adults, sinus involvement with membrane thickening and mucous hypersecretion was evident in 87% of patients within 2 to 4 days after experimental rhinovirus inoc~lation.'~ this indicates that rhinovirus colds are likely to encompass a viral rhinosinusitis, not just rhinitis. during experimental infections, no significant cytopathic changes have been found in either ciliated or nonciliated nasal epithelial in addition, no destruction of the nasal mucosa or the inferior turbinate was observed by scanning electron microscopy. implications of these findings are that the production of cold symptoms with rhinovirus are caused by mechanisms other than tissue destruction. these findings are in marked contrast to epithelial cell damage caused by adenovirus and influenza a and b virus in cell cultures. 57 in situ hybridization has been used to localize sites and determine the extent of rhinovirus replication. in experimentally infected volunteers, rhinovirus replicated in a small proportion (patchy involvement) of epithelial cells distributed in the nasal epithelium and in nonciliated cells in the nasopharynx." following the onset of cold symptoms, the number of neutrophils in the epithelium and subepithelial cell layer were noted to increase by day 2 during an experimental infection.5h this was followed by an influx of neutrophils into nasal secretions leading to a purulent discharge in experimentally infected volunteers 4 to 5 days after the onset of symptoms. this change was not accompanied by associated changes in bacterial pathogens, suggesting that rhinovirus itself may be involved in initiating this neutrophil response. although the number of ivmdhocvtes observed in the nasal mucosa did not change significantly, a mild lymphocyte infiltration was noted after a period of 2 to 4 i~ even though the local immunopathologic and pathophysiologic processes of colds induced by rhinovirus are not fully understood, recent information about mediators and cytokines is likely to contribute to knowledge of the pathogenesis of events leading to symptoms. generally, levels of histamine in nasal secretions do not change significantly during acute experimental rhinovirus infections in nonallergic individuals.'*, 38 however, increased concentrations of kinins, which act as potent vasoactive substances, have been detected in nasal secretions of adults given rhinovirus experimentally; and such symptoms as sore throat, nasal congestion, and rhinorrhea have been observed in healthy volunteers after administering bradykinin intranasally.3r, 42 increased levels of il-8, a potent leukocyte chemoattractant, have been measured in nasal secretions from subjects with wild-type rhinovirus infection^.^^ increases in the production of other proinflammatory cytokines (i.e., il-11, gm-csf, ifn-y, and tnf-a) also have been reported?, i5fz9 the capacity of rhinovirus to cause infection and replicate in the lower respiratory tract has been difficult to confirm. in part, this derives from difficulty in obtaining specimens that are free of contamination. in studies of adults with allergic rhinitis who were infected experimentally with rhinovirus (type 16), virus was not isolated either from fluid or cells obtained by bronchoalveolar lavage (bal)." by comparison, virus always was found in high titers in nasal washes. explanations for the preferential replication of rhinovirus in the upper airways has been attributed to its temperature sensitivity in culture, which may tend to favor its replication in the upper a i r~a y s . 4~ more specifically, reducing the temperature of incubation of rhinovirus in culture from 37â°c to 33â°c greatly enhances the efficacy of virus isolation. while studies continue to document rhinovirus replication and infection in the lower airways, investigations focused on inflammatory pathways involving mediators and cytokines will continue to be important in defining the immune response stimulated by this virus. in contrast, there is less doubt that other respiratory viruses (adenovirus, influenza virus, rsv, and parainfluenza virus) can infect the lower airways. several studies have compared the severity of upper respiratory tract symptoms and lower airway responsiveness in allergic and nonallergic adults following experimental rhinovirus inoculation. in studies of experimental viral rhinitis by bardin and colleagues, no difference between atopic patients and controls was observed with respect to shedding of rhinovirus or symptom scores.' a particularly interesting finding was that the severity of colds experienced by nonallergic individuals was increased in those who lacked preinoculation neutralizing antibodies in their sera to the rhinovirus serotype used for inoculation. this was not observed in atopic individuals, who developed severe colds and had a significant increase in nasal wash albumin in spite of the presence of neutralizing antibody. doyle and colleagues found little evidence to support an increased susceptibility of atopic subjects to rhinovirus.lu however, atopic patients did experience an earlier onset of sneezing and nasal congestion. these investigators examined peripheral blood lymphocyte responses during experimental infection. a consistent feature was the persistence of t-cell activation to rhinovirus in atopic subjects up to 3 weeks after i n f e~t i o n .~~ skoner and others from this group noted that experimental rhinovirus (type 39) infection induced significant increases in total serum ige, leukocyte histamine release, and platelet aggregation during the acute phase of infection in adults with allergic r h i n i t i~.~~ however, the acute phase rise in total serum ige could not be correlated to a rise in specific ige antibody to allergens tested, and there was no correlation between the rise in serum ige and enhanced release of histamine from leukocytes stimulated with anti-ige antibody. similarly, total ige levels in atopics given a rhinovirus infection did not correlate with the severity of rhinitis in studies by others.' thus, the functional significance of the rise in total ige during infection is unclear. no changes in plasma histamine or in histamine in nasal secretions have been observed in nonatopic individuals during rhinovirus infections.'2, 38 however, increased histamine secretion was seen more frequently during the initial days of rhinitis in nasal washes from allergic adults following rhinovirus inoculati~n.~~ in studies of bronchial reactivity, increased airway hyperresponsiveness demonstrated by spirometry and methacholine challenges were observed in some but not all asthmatic subjects during experimental infection with rhinovirus (type 39).*' these changes were not observed in nonallergic subjects. in a more recent study, ragweed-allergic patients with rhinitis developed increased lower airway responsiveness to inhaled histamine and ragweed allergen during an acute infection with rhinovirus (type 16).32 of interest in this study by lemanske and colleagues was that their patients also had an increased likelihood of developing late phase bronchoconstrictor responses following allergen challenge. these changes remained persistent up to 4 weeks after the experimental infection. overall, these observations suggest an important relationship between rhinovirus infection and the potentiation of allergic inflammation in the airways. the effects of bronchoprovocation with ragweed allergen during rhinovirus infection were examined in individuals with allergic rhinitis by calhoun and colleague^.^ using a segmental allergen bronchoprovocation method, the investigators detected increased levels of histamine in bal samples, both immediately and 48 hours after antigen challenge. during the infection, tryptase from mast cells was initially detected in bal samples; however, a significant increase in histamine, but not tryptase, during the late phase response suggested that basophils rather than mast cells may be the cell source of histamine at 48 hours. the most striking observation during the acute infection was an augmented recruitment of eosinophils into the airways 48 hours after allergen challenge. moreover, these effects persisted and were apparent up to 1 month after rhinovirus inoculation. these data indicate that rhinovirus infection in the upper respiratory tract can significantly augment both immediate and late phase cell and mediator responses in the lower airways of allergic individuals. in another study of adult volunteers, including six allergic asthmatics, an increase in lower airways responsiveness to histamine was noted during the acute phase of rhinovirus infection.= this was accompanied by increases in submucosal lymphocytes followed by a fall in lymphocyte numbers in the epithelium and submucosa detected in bronchial biopsy specimens during the convalescent period. an increase in epithelial eosinophils also was noted during the cold, and the rise in eosinophil numbers persisted into convalescence in the asthmatic, but not in the normal subjects. increased sensitivity to histamine in the asthmatics also persisted in the convalescent period. taken together, these studies have demonstrated a relationship between rhinovirus upper respiratory tract infections and augmented late phase bronchial responsiveness associated with eosinophil recruitment into the lungs. more detailed studies directly linking infections to the development of this eosinophil response and studies to define the mechanisms leading to this reaction are of great interest. recent studies provide evidence for rhinovirus activation of t cells through a monocyte-dependent mechanism that also promoted eosinophil survival in vitro.lh a link between virus infections and allergic eosinophil inflammation has also been implicated in a mouse model, demonstrating that a bystander cd4 + th, cellular response to ovalbumin can switch virus peptidespecific cd8 + t cells in the lung to produce il-5, leading to eosinophil infiltration following virus peptide challenge.$ epidemiologic studies have shown a correlation between the seasonality of viral upper respiratory tract infections-particularly rsv and rhinovirus-and asthma exacerbations. in the northern hemisphere, rsv infections are most common during the midwinter months (december through february). these are months when infants are more likely to be seen in clinics, emergency rooms, and the hospital for acute attacks of wheezing (fig. 1).11,22 in a time/trend analysis, the seasonal patterns of respiratory tract infections and hospital admissions were evaluated for older children and a particularly strong correlation was found between the seasonal pattern of upper respiratory tract infections, particularly rhinovirus, and hospital admissions for asthma among children. upper respiratory tract infections and admissions for asthma also are more frequent during periods of school attendance and less frequent during school holidays.*7, 51 consistent with this, peak admissions to the pediatric emergency room for wheezing at the university of virginia are observed annually in the fall and spring months when rhinovirus infections are most common combined with increased exposure to tree and grass pollens in the spring and ragweed, alternaria, and higher levels of household dust mite allergen during the fall (fig. 2) . this visiting pattern already becomes apparent for wheezing exacerbations in young children, aged 2 and 3, when the diagnosis of asthma often is considered (figure 2 ). an awareness of the combined seasonal influences of allergen exposures and viral infection can be very helpful in designing treatment plans for individual patients. these treatment plans should include allergen avoidance and emphasize seasonal requirements for daily antiinflammatory medications. physicians also should be aware that these seasonal influences are likely to vary geographically. evidence that infections with rhinovirus may augment the late phase eosino-phi1 response in asthmatic individuals provides a rationale for using steroids to minimize the severity and persistence of airway inflammation and decrease the reliance of patients on inhaled bronchodilators during colds. when children with asthma develop a cold, peak flow tests can be useful to monitor changes in lower airway function. morning peak flow tests should be checked with the onset of cold symptoms and for the duration of the infection, with particular attention focused on changes that may occur during the first 2 to 4 days. recent studies indicate that inhaled steroids may benefit asthmatic children during 45, 55 those who are already using inhaled steroids daily may benefit from an increased dose for 2 to 3 weeks. should symptoms worsen, the need for systemic steroids should be considered with the patient's physician. important questions remain about the capacity of viral respiratory tract pathogens to augment inflammatory pathways in the lungs of asthmatic children. more research is needed to learn whether the examples of pulmonary eosinophilia stimulated by rsv antigens (i.e., the attachment g protein) in animals and humans represent an immune response similar to parasite induced eosinophilia or whether this is a process characteristic of allergic inflammation in an individual who is genetically predisposed to develop allergic respiratory symptoms and asthma. the absence of cytopathic changes in the respiratory tract epithelium during rhinovirus colds, the patchy involvement of infected epithelial cells, and the lack of direct evidence for virus replication and infection in the lower airways suggest mechanisms involving the enhanced production of proinflammatory cytokines in the pathogenesis of inflammation induced by this virus. candidate cytokines such as tnf-a, ifn-y, il-5, il-8, il-11, and gm-csf currently are being investigated with respect to their cell sources and production during rhinovirus infections in allergic and nonallergic patients. in addition, efforts to develop reagents to block the binding of rhinovirus to icam-1, the major receptor used by rhinovirus to infect cells, also represent a therapeutic approach for preventing episodes of asthma induced by this overall, our understanding for the capacity of viral infections to precipitate wheezing attacks in infants and children has improved significantly during the last decade. the need for new information and strategies to treat virus-induced asthma exacerbations is now becoming more important as the prevalence of asthma continues to increase. acknowledgment manuscript. the authors are grateful to richard leahy for his assistance in the preparation of this amplified rhinovirus colds in atopic subjects lower airways inflammatory response during rhinovirus colds. int arch allergy immunol prognosis of asthma in children a common cold virus, rhinovirus 16, potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects icam-1 on epithelial cells in allergic subjects: a hallmark of 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for rhinoviruses school holidays and admissions with asthma increased levels of il-8 in the nasal aspirates of children with virus-associated asthma the development of respiratory syncytial virusspecific ige and the release of histamine in nasopharyngeal secretions after infection the relationships of rsv-specific immunoglobulin e antibody responses in infancy, recurrent wheezing, and pulmonary function at age 7-8 years treatment of acute, episodic asthma in preschool children using intermittent high dose inhaled steroids at home et a1 histopathologic examination and enumeration of polymorphonuclear leukocytes in the nasal mucosa during experimental rhinovirus colds respiratory virus infection of monolayer cultures of human nasal epithelial cells bronchiolitis-state of the art key: cord-003926-ycdaw2vh authors: maslow, joel n. title: zika vaccine development—current progress and challenges for the future date: 2019-07-14 journal: trop med infect dis doi: 10.3390/tropicalmed4030104 sha: doc_id: 3926 cord_uid: ycdaw2vh zika virus is an emergent pathogen that gained significant importance during the epidemic in south and central america as unusual and alarming complications of infection were recognized. although initially considered a self-limited benign infection, a panoply of neurologic complications were recognized including a guillain–barré-like syndrome and in-utero fetal infection causing microcephaly, blindness, and other congenital neurologic complications. numerous zika virus vaccines were developed, with nine different vaccines representing five different platforms entered into clinical trials, one progressing to phase ii. here we review the current landscape and challenges confronting zika virus vaccine development. the zika virus, discovered in uganda in 1947 [1] , was shown to be endemic through sub-saharan africa and tropical areas of southeastern asia in studies through the second half of the 20th century [2] . isolated outbreaks occurred in yap island in 2007 [3] and on french polynesia in 2014 [4] . starting in mid-2015, zika virus infection achieved epidemic status, spreading rapidly through south america, central america, and the caribbean islands [5] . it was soon recognized that zika virus infection occurring during pregnancy caused microcephaly and other congenital disorders in the developing fetus, the latter being the primary reason for the world health organization (who) labelling zika as an international threat in early 2016. beginning in late 2015, numerous academic labs and pharmaceutical companies initiated work to develop a vaccine against zika, however, by the time the first vaccines entered clinical trials, the zika epidemic had started to wane creating significant challenges to vaccine assessment [6] that has engendered discussion of other regulatory pathways to licensure [7] . in this paper, we provide a brief update of current progress in zika virus vaccine development and explore the challenges to vaccine assessment and eventual licensure. zika virus infection presents with a symptom complex consisting of a diffuse maculo-papular rash, fever, asthenia, myalgias, arthralgias, headache, and retroorbital pain. the frequency and degree of symptomatology has, however, varied between studies. a retrospective study immediately following the outbreak on yap island found that only 19% of survey participants reported symptoms consistent with zika virus infection [3] , a figure that has been cited to suggest that zika virus infection is asymptomatic in as many as 80% of individuals. however, of 557 individuals who provided blood samples, representing 16% of households, 38% were symptomatic [3] . a second retrospective study of the larger outbreak in french polynesia [8, 9] , similarly appeared to have a low rate of symptomatology based on a sample of blood donors [10] . however, a subsequent seroprevalence study found that 43% of those with evidence of prior infection had symptoms consistent with zika virus infection [11] . a more recent meta-analysis of 23 studies noted that between 0 and 83% of cases were reported as asymptomatic, although as the authors note, assessment of the prevalence of symptoms was not the goal of many studies [12] . in adults, the most common reported complication of zika virus infection is a guillain-barré-like illness that had a prevalence of 0.24 cases per 1000 cases of infection in the french polynesian epidemic [13] . of interest, a recent meta-analysis has questioned this causal association [14] . other less common reported neurologic complications in adults include meningoencephalitis [15] and an adem illness. in contrast, infection during pregnancy has been associated with fetal microcephaly and a number of other congenital illnesses including visual deficits, hearing disorders, neural calcifications, learning disabilities, and arthrogryposis that may affect as many as 30% children born to mothers infected during pregnancy [16] [17] [18] [19] [20] [21] . additionally, zika can directly invade the placenta and has been associated with prolonged maternal viremia [22] [23] [24] . zika virus can also be transmitted through sexual contact, first reported for a researcher returning from senegal in 2008 [25] . numerous subsequent case reports were published among travelers as part of the epidemic affecting the americas [26] . the frequency of sexual transmission in endemic regions is unknown and could not be differentiated from mosquito-borne transmission. zika virus carriage in the male urogenital tract is common through 90 days post infection and may persist in some to 9 months [27] . in mice, zika virus infection causes testicular atrophy and significantly decreases spermatic function and fertility rates [28] [29] [30] . in one study of a zika virus dna vaccine, the adverse effects in the male reproductive system were prevented by vaccination [30] . the question of whether zika virus can adversely affect human reproductive potential and, if so, whether such effects would be age-related is unknown. females may also excrete zika virus rna for extended times. a prospective study of five women showed that zika rna was detected in vaginal fluid for as long as 6 months and a month or more in three of the five [31] . murine studies showed that zika virus caused infection of the ovaries of non-immunosuppressed c57bl/6 mice and induced a t-cell inflammatory reaction, but without affecting reproductive potential [32] . in contrast to the above human study, female macaques rapidly cleared virus from the genital tract [33] . thus, while zika virus infection is mildly symptomatic and self-limited for the vast majority of individuals, with infrequent neurologic complications in adults, vaccination of the general population may not be warranted. however, vaccination of females at or entering reproductive age and their male partners is prudent. as discussed previously, vaccination during pregnancy is non-ideal due to the time to generate protective immunity and unknown vaccine safety [34] . the one unknown aspect is whether vaccination of males of any age may be beneficial to protect against testicular complications. calls for generalized vaccination programs have, however, been put on hold due to the decreasing incidence of zika infection rates, as discussed in the next section. in late 2015 and through mid-2016, global concern for this spreading epidemic was increasing, however, the experience in the two prior epidemics was predictive of the subsequent epidemiology in the americas. the zika virus outbreaks in yap island and french polynesia were characterized by rapid onset and rapid resolution over a span of 3-4 months [3, 9] . the zika epidemic in the americas was characterized by a longer time to reach peak incidence, taking approximately one year for brazil [35] presumably related to the larger and more varied geography, with attack rates decreasing greater than 100-fold the next year [35] . data published by the pan american health organization (paho) found similar patterns through south and central america (https://www.paho.org/). the almost complete disappearance of zika has created significant barriers to ongoing vaccine studies as attack rates have fallen below those need to meet reasonable sample sizes [6] . the who removed the designation of zika a virus of global concern in 2018 as case rates have fallen. in march 2018, the who and the national institutes of allergy and infectious diseases (niaid) convened a meeting of academics, representatives from industry, and regulators to discuss the approach to zika virus vaccine development in an era of waning incidence [7] . although zika virus remains endemic in asia, africa, and central and south america, current transmission rates, coupled with high background herd immunity, would require an extremely large and logistically difficult study. as population immunity wanes, future outbreaks are likely. the ability to conduct a meaningful future clinical trial may be dependent upon having pre-existing site and regulatory approvals with an established vaccine network to enable rapid response to new reports suggesting increased disease activity. vaccine development against the zika virus began in earnest in late 2015 following the reports of microcephaly in fetuses and infants in brazil. of note, the first demonstration of immunoprotection was as part of a 1953 study to define the ultrastructural characteristics of zika virus, that found intramuscular vaccination of mice with infectious viral filtrates protected against cerebral infection [36] . poland et al. provide a comprehensive listing of almost 40 zika virus vaccines in development as of 2017 [37] . diamond et al. discuss potential immunoreactive epitopes on the zika envelope and provide further information on those vaccines that progressed into clinical trials [38] . the large number of delivery platforms include live attenuated and inactivated whole-viruses; viral-vectored vaccines utilizing adeno-associated virus, vesicular stomatitis virus, measles virus, and dengue or yellow fever chimeric vaccines; dna and mrna vaccines; and peptide and protein subunit vaccines. most have been assessed in animal models utilizing non-human primates and/or lethal challenge experiments involving immunosuppressed mice [37] . as of 2017, six vaccines had advanced to phase i studies [37, 39, 40] . since that time, two additional vaccines have entered into clinical trials (table 1) . a total of three dna vaccines have entered into human testing [41, 42] , including one that has advanced to phase ii (table 1) . gls-5700, a dna vaccine encoding for the zika virus prme genes designed as a consensus based on available zika virus sequences through december 2015 [43] , was the first to enter into clinical trials. in pre-clinical studies, vaccinated mice and non-human primates were shown to develop b and t-cell immune responses against the zika virus envelope and protected against development of neurologic disease and death in immunosuppressed, interferon α, β receptor deficient (ifnar) mice [43] . moreover, histologic sections of brain tissue showed that vaccinated mice were without inflammatory infiltrates evident in non-immunized mice [43] . subsequent studies showed that the vaccine protected against testicular damage, testicular atrophy, spermatozoal damage, and infertility in mice [28, 30] . a phase i study evaluated gls-5700 administered via intradermal injection (id), followed by electroporation (ep) at doses of either 1 or 2 mg per vaccinations followed immediately by electroporation at baseline, 4 weeks and 12 weeks [42] . there were no serious adverse events (sae) reported as part of the study, with the most frequent adverse events related to discomfort, pain, or swelling at the injection site. seroconversion was observed in 83% of individuals after two vaccinations and 100% after three vaccinations with gls-5700 [42] . neutralizing antibodies were detected for 62% of participants in a vero-cell (monkey kidney cell) assay, however, 95% of participants demonstrated the ability to neutralize infection of u87mg neuroblastoma cells [42] . vaccine responses were maintained through a year of follow-up. there was no difference in responses based on dose level. notably, passive transfer of immune serum from vaccinated participants was able to protect 92% of ifnar mice against lethal infection independent of the presence of vero cell neutralizing antibodies [42] . gls-5700 is being evaluated as part of a second double-blind, placebo-controlled clinical trial (nct02887482) performed in puerto rico. analysis of the latter study is in progress. two additional dna vaccines, based on the french polynesian h/pf/2013 strain were developed as chimeras that included the jev prm signal sequence followed by the zika envelope (e) gene (vrc5283) or a similar construct with the terminal 98 amino acids of e, representing the stem and transmembrane regions, exchanged for the analogous jev sequence (vrc5288) [44] . both vaccines were immunogenic for mice and nhps and protected >90% of nhps against viremia at a dose of either 1 or 4 mg given twice [44] . both vaccine candidates were advanced into clinical trials with 4 mg of administered intramuscularly at weeks 0, 4 and 8 with vaccine vrc5288 administered by needle and syringe while vrc5283 was administered either as a single dose or split dose by needle and syringe or as a split dose given by the pharmajet needle-free device [41] . seroconversion was 100% in the group administered vaccine with the needle-free device, less for vaccine administered as split-dose by needle and syringe, and lowest for vaccine given as a single injection with needle and syringe. the vrc5283 vaccine was advanced into phase ii studies in the americas that utilized 140 clinical sites with clinical site selection guided by epidemiologic modeling [38] . long-term follow-up has not been reported. three inactivated vaccines have entered into clinical studies, of which clinical data for only zpiv vaccine has been published [45] . zpiv is a whole inactivated virus vaccine of puerto rican strain prvabc59 [46] . studies in mice showed that a single vaccination given intramuscularly with alum generated antibody titers of approximately 3.7 log10 and fully protected balb/c immunocompetent mice from viremia, whereas unvaccinated mice were unprotected and subcutaneously vaccinated mice were only partially protected against the zika brazil strain [46] . a subsequent study in non-human primates vaccinated twice at four-week intervals with alum generated binding and microneutralization antibody titers of 3.54 and 3.55 log10, respectively, and complete protection against viremia and viruria following challenge with either brazilian or puerto rican strains of zika virus [47] . zpiv safety and immunogenicity was tested in three clinical trials to assess zika vaccine responses relative to dose level, vaccination schedule, or following vaccination with either the yellow fever yf-vax or japanese encephalitis virus (jev) ixaro vaccines to assess responses in a flavivirus-exposed population [45] . there were no vaccine-associated saes reported. the most common adverse events were pain and tenderness at the injection site; no neurologic events were reported. seroconversion was 92% using a cutoff for peak geometric mean titer of 1:10 and 77% using a titer of 1:100. response rates after a single immunization were 11% and 5.5% using cutoffs of 1:10 or 1:100, respectively. vaccine responses were observed through day 57. passive transfer of purified igg derived from 10 zika immunized participants to groups of 5 balb/c mice per participant provided sterilizing immunity against viremia for 82% (41 of 50) of mice overall, with viremia observed for one or more mice per group inoculated with sera from five (50%) individuals [45] . clinical trial data for the other vaccines has not yet been reported as of the date of this monograph, however, pre-clinical data has been reported for three candidate vaccines. an mrna vaccine that incorporates the prm-e genes of a micronesian strain of zika virus was created incorporating with or without four-point mutations in the fusion-loop segment of the dii region of the envelope gene that abolished binding of antibodies directed against the fusion-loop region to reduce the potential risk for antibody-dependent enhancement of infection [48] . immunization of ag129 mice with un-modified lipid nanoparticle-encapsulated vaccines mrna was immunogenic and protective against lethal infection; immunization of c57bl/6 immunocompetent mice followed by treatment with anti-ifnar1 blocking antibody showed protection against viremia in approximately 60% of animals [48] . pizv, an inactivated vaccine derived from puerto rican strain prvabc59 selected as without passage-related mutations, protected against lethal zika virus challenge in ag129 mice when administered with alum adjuvant [49] . a measles-vectored vaccine encoding the zika virus prme was shown to lessen viremia in pregnant ifnar mice and prevented clinical disease in mouse pups [50] . preclinical data for vla-1601 inactivated viral vaccines and the rzikv/d4∆30-713 live virus vaccine has not yet been reported. a number of animal models have been assessed for vaccine development and have been reviewed elsewhere [51] . immunocompetent mice can develop transient viremia following infection and may represent models to test sterilizing immunity. interferon α/β receptor knockout mice (ag129 or ifnar -/-) develop lethal infection following zika virus infection and have been used as a more stringent measure of protection. non-human primates develop a self-limited mild illness associated with viremia and can transmit virus in utero to primate fetus [52] . the logistical difficulties in pursuing standard vaccine evaluation have created significant interest in the possibility of controlled human infection models (chim). the conduct and planning for human challenge trials raises unique medical and ethical considerations. for zika virus trials, one must balance the relative benign and self-limited infection experienced by the vast majority against the risk for less benign complications and transmission risks. as reviewed above, published studies provide estimates that zika virus infection is relatively asymptomatic and self-limited for the majority of individuals, however, methodology in these studies varies widely. in adults, two sets of complications warrant consideration for a proposed chim study. as reviewed above, a guillain-barre-like syndrome occurs in approximately 1 of 5000 zika virus infections [13] , whereas other neurologic complications such as meningoencephalitis, myelitis [15, 53, 54] , and acute disseminated encephalomyelitis [54] are rare. second, and perhaps more important, is the risk of transmission to a sexual partner and the potential for infection during pregnancy. zika virus commonly persists in the male urogenital tract for 3 months, and may persist in some individuals for up to 8 months [27] . some have considered limiting studies to non-pregnant females as zika virus colonization of the female genital tract may be temporally limited. the fact that zika virus is known to cause testicular atrophy in mice [28] [29] [30] , raises yet another as yet theoretical concern for humans. these questions as well as the theoretical potential for vector-borne transmission were debated in detail in late 2016 with the conclusion that the benefits of a human challenge infection did not outweigh the risks [55] , however, this analysis was performed just as the initial epidemic wave in the americas was ending. the group published a follow-up in 2018 [56] . despite the recognition that conducting a placebo-controlled vaccine trial had become significantly more difficult due to declining case rates, the group's conclusion was essentially unchanged. to address safety concerns, there has been work to develop attenuated viral strains deleted for potential neurotropic regions [57] with a goal to prevent viremia. whether the attenuated viral strain (rzikv/d4∆30-713) being tested in a phase i study will serve as putative challenge strain is as yet undetermined. in summary, zika vaccine development continues with multiple candidate vaccines in clinical trials. because of the significant decline in incidence, evaluation of vaccine efficacy is increasingly difficult. there has been renewed interest in animal model and human infection models of infection. the author is an employee of geneone life science, inc., a developer of dna-based vaccines including a vaccine against the zika virus. walter reed army institute of research niaid national institutes of allergy and infectious disease vrc vaccine research center zika virus: (i). isolations and serological specificity zika virus: following the path of dengue and chikungunya? lancet zika virus outbreak on yap island, federated states of micronesia european centre for disease prevention and control. rapid risk assessment: zika virus infection outbreak, french polynesia zika virus in the americas-yet another arbovirus threat steep drop in zika cases undermines vaccine trial demonstrating vaccine effectiveness during a waning epidemic: a who/nih meeting report on approaches to development and licensure of zika vaccine candidates bilan de l'épidémie à virus zika survenue en polynésie française entre octobre 2013 et mars 2014. de la description de l'épidémie aux connaissances acquises après l'évènement potential for zika virus transmission through blood transfusion demonstrated during an outbreak in french polynesia zika virus seroprevalence prevalence of asymptomatic zika virus infection: a systematic review guillain-barre syndrome outbreak associated with zika virus infection in french polynesia: a case-control study zika virus infection and risk of guillain-barre syndrome: a meta-analysis leparc-goffart, i.; et al. zika virus associated with meningoencephalitis congenital cerebral malformations and dysfunction in fetuses and newborns following the 2013 to 2014 zika virus epidemic in french polynesia zika virus infection in pregnant women in rio de janeiro european centre for disease prevention and control. rapid risk assessment: zika virus epidemic in the americas: potential association with microcephaly and guillain-barre syndrome birth defects among fetuses and infants of us women with evidence of possible zika virus infection during pregnancy hearing loss in infants with microcephaly and evidence of congenital zika virus infection-brazil risk factors associated with the ophthalmoscopic findings identifed in infants with presumed zika virus congenital infection zika virus infection with prolonged maternal viremia and fetal brain abnormalities prolonged detection of zika virus rna in pregnant women zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg? probable non-vector-borne transmission of zika virus frequent zika virus sexual transmission and prolonged viral rna shedding in an immunodeficient mouse model zika virus shedding in semen of symptomatic infected men zika-induced male infertility in mice is potentially reversible and preventable by deoxyribonucleic acid immunization zika virus infection damages the testes in mice dna vaccination protects mice against zika virus-induced damage to the testes prolonged shedding of zika virus rna in vaginal secretions zika virus causes acute infection and inflammation in the ovary of mice without apparent defects in fertility zika virus persistence in the central nervous system and lymph nodes of rhesus monkeys vaccine development for emerging virulent infectious diseases pan american health organization/world health organization. zika-epidemiologic report brazil comparison by electron microscopy of the ntaya and zika viruses development of vaccines against zika virus zika virus vaccine development: progress in the face of new challenges vaccines for emerging infectious diseases: lessonso from mers coronavirus and zika virus an update on zika vaccine developments. expert rev safety, tolerability, and immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase 1 clinical trials safety and immunogenicity of an anti-zika virus dna vaccine-preliminary report in vivo protection against zikv infection and pathogenesis through passive antibody transfer and active immunization with a prmenv dna vaccine rapid development of a dna vaccine for zika virus preliminary aggregate safety and immunogenicity results from three trials of a purified inactivated zika virus vaccine candidate: phase 1, randomised, double-blind, placebo-controlled clinical trials vaccine protection against zika virus from brazil protective efficacy of multiple vaccine platforms against 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cord-016020-awanrm9u authors: fox, julie d.; tilley, peter a. title: respiratory pathogens date: 2007 journal: molecular pathology in clinical practice doi: 10.1007/978-0-387-33227-7_41 sha: doc_id: 16020 cord_uid: awanrm9u respiratory tract infections are among the most common presenting complaints of patients in both hospital and community settings. they are a considerable burden in terms of both patient morbidity and public health interventions. laboratory diagnosis of respiratory tract infections should provide guidance in therapy and prognosis, as well as useful epidemiological information reflecting trends in the community. understanding and monitoring such trends facilitates early recognition of new infectious agents in a population. a summary of the common viruses and bacteria causing respiratory tract infections and their clinical relevance is given in tables 41–1 and 41–2, respectively. respiratory tract infections are among the most common presenting complaints of patients in both hospital and community settings. they are a considerable burden in terms of both patient morbidity and public health interventions. laboratory diagnosis of respiratory tract infections should provide guidance in therapy and prognosis, as well as useful epidemiological information reflecting trends in the community. understanding and monitoring such trends facilitates early recognition of new infectious agents in a population. a summary of the common viruses and bacteria causing respiratory tract infections and their clinical relevance is given in tables 41-1 and 41-2, respectively. even with a significant clinical effort and analysis of multiple specimens, current laboratory methods fail to diagnose approximately half of lower respiratory tract infections. in fact, laboratory diagnosis of communityacquired pneumonia (cap) is so poor that current clinical practice guidelines do not recommend testing for all but the most severely affected patients and advise use of empiric therapy. 1 this pragmatic approach fails to address issues of antimicrobial overuse and resistance, public health surveillance, and advancement of medical knowledge. many "atypical" bacteria are known to cause severe respiratory symptoms, but lack of good diagnostic procedures has hampered the measurement of the real impact of such infections in the community. despite vaccination policies, bordetella pertussis infection remains relatively common in children and adults and is associated with chronic cough in adults. 2 mycoplasma pneumoniae, legionella pneumophila, and chlamydophilia (previously chlamydia) pneumoniae are all recognized causes of lower respiratory tract infections, but again, their impact has not been studied in detail. in addition, despite the well-recognized association of viral infections with upper and lower respiratory tract infections, the current diagnostic virology procedures do not provide an answer rapidly enough to with parainfluenza virus type 4, human coronaviruses, rhinoviruses, and some enteroviruses would not ordinarily be identified without rna detection methods. the impact of such infections is only just being realized, and there is probably an underestimation of their clinical importance, particularly for immunocompromised individuals, the elderly, or those with underlying conditions such as asthma. 5, 6 human metapneumovirus has been confirmed as an important cause of severe lower respiratory tract infection. the virus has been circulating for more than 50 years, and studies using molecular assays have confirmed its wide distribution, 7,8 but many laboratories have not been successful in isolating this virus. the recent identification of the agent causing severe acute respiratory syndrome (sars), known as sars-cov, has illustrated the need for expansion of diagnostic testing to encompass new emerging viruses and the limitations of conventional virological laboratory approaches to respiratory pathogen diagnosis. 9, 10 even if it is possible to culture viruses efficiently, isolation and confirmation of the cause of a cytopathic effect can take days to weeks, depending on the pathogen. waiting for a culture-positive result can take many days, during which time the patient may be inappropriately treated and, if hospitalized, infection control measures may not be initiated. also, the use of primary primate cells in culture (which gives the best yields of influenza and parainfluenza viruses) is unlikely to be sustainable in the long term. when available, monoclonal antibodies are useful in direct virus-specific antigen detection tests, and these can be used for rapid diagnosis. many laboratories are able to provide diagnostic testing for influenza, parainfluenza (types 1-3), respiratory syncytial virus (rsv), and respiratory adenoviruses. a respiratory specimen containing cells is necessary for sensitive detection of viruses by immunofluorescence or other antigen-detection methods. good-quality diagnostic samples (often lavage or aspirate samples) can usually be obtained from young, immunocompetent, hospitalized individuals, but in other circumstances the ideal sample may not be available. delays in transportation may reduce specimen quality and compromise assay results. the most-difficult (and least-efficient) specimens for diagnostic testing are swab samples containing minimal cellular material taken from largely asymptomatic individuals in the community. smears from these samples can be difficult to interpret in a direct antigen test and thus culture of the sample is usually required for pathogen identification. bacterial antigen detection by a direct fluorescent antibody (dfa) test is similarly compromised by poor sensitivity and has the added concern of artifacts leading to false-positive results, particularly for b. pertussis and l. pneumophila. serological assays of an antibody response to infection are available for some respiratory pathogens. although useful for retrospective evidence of infection in a community, the results are not timely enough for patient management. for some cell-associated or intracellular bacteria and viruses, antibody responses develop slowly, if they develop at all, and convalescent sera taken many weeks after disease onset are required to make a definitive diagnosis (e.g., for c. pneumoniae, legionella species, rsv). for other infections such as influenza, antibody responses are brisk, but frequent reinfection reduces the igm response and convalescent sera are required to demonstrate changing titers. the limitations of conventional testing are well recognized for viral 11, 12 and bacterial 13,14 pathogens, and some of the key differences between nucleic acid and culture or direct antigen testing methods are summarized in table 41 -3. utilization of rapid viral diagnostic procedures, such as may be provided by molecular amplification methods, could help to reduce the emergence of antibiotic-resistant bacteria. one study demonstrated that a 52% reduction in antibiotic use was possible using molecular methods for viral diagnosis. 15 despite the obvious economic burden of cap, we do not have accurate data on how many of these infections are viral in origin or caused by the atypical bacteria for which routine diagnostic testing is not readily available. the identification of emerging human viral infections (such as h5n1 influenza and sars-cov) has heightened awareness of the gaps in respiratory pathogen diagnosis. nucleic acid detection assays are likely to be utilized more widely to identify novel emerging pathogens that could result in worldwide outbreaks. molecular amplification assays are being used successfully for the identification of organisms associated with pneumonia. 16 the potential for nosocomial spread of respiratory pathogens is well recognized for patients admitted to the hospital with rsv, parainfluenza, or other infections, serving as a reservoir for transmission to vulnerable patients and leading to possible outbreaks. 17 previously, available diagnostic methods were not sensitive enough to identify sources of outbreaks, but the advent of molecular amplification methods has allowed even environmental sampling to be helpful in confirming outbreak sources and linking clustered cases of infection. 18 samples used for detection of respiratory pathogens include swabs (usually nasopharyngeal or throat), aspirates (nasopharyngeal or tracheal), sputum (usually from individuals presenting with pneumonia), or bronchoalveolar lavage specimens. for infections involving the entire respiratory tract, nasopharyngeal specimens are practical for diagnosis. for other infections, which are more focal, melting-curve analysis of pcr products using the intercalating dye sybr green differentiates between specific and nonspecific products without further manipulation of the pcr products. specific sequence information required for "catch-all" approach, which may be advantageous when novel design; triage of testing can be difficult; pathogens need to be identified generic primers may be used to identify novel pathogens sensitivity exquisite sensitivity but can be prone to generally less sensitive than nucleic acid detection methods cross-contamination problems specificity careful handling required to avoid careful handling required to avoid contamination, but contamination (common problem); less-common problem than for molecular methods; dfa primer/probe design crucial subject to over interpretation strain typing most definitive method limited serotyping (e.g., influenza, legionella) automation automated extraction equipment becoming difficult to automate available; automated detection commonplace safety inactivated before analysis, but antimicrobial isolates useful for antimicrobial sensitivity testing and sensitivity information requires knowledge of phenotyping, but specialized safety requirements needed for genotypic mutation culture of category 3/4 pathogens quality assurance proficiency and validation of methods not well culture depends critically on cell line or medium quality; established maintaining quality can be difficult lower respiratory tract specimens are required (e.g., for legionella). sample preparation is a critical step for the detection and analysis of organisms. numerous methods, from simple boiling to sophisticated automated protocols, are available for disruption of the organism and purification of the nucleic acids. many studies have demonstrated inhibitors in respiratory specimens, making some form of extraction (with or without freezing) necessary to avoid frequent false negative results. commercial kits for preparation of samples are available and should reduce interlaboratory variation in results. simultaneous extraction of rna and dna facilitates assays for both viruses and bacteria. molecular techniques for the detection and analysis of pathogens associated with respiratory infection provide specific diagnoses for individual cases and for outbreaks. currently, fda-cleared molecular tests are not available for the detection of respiratory pathogens, with the exception of mycobacterium tuberculosis (see chapter 43). molecular tests are performed using either validated laboratorydeveloped procedures or commercial testing reagents. published diagnostic methods for detection of respiratory pathogen dna or rna directly from clinical specimens utilize target amplification procedures such as polymerase chain reaction (pcr) or nucleic acid sequence-based amplification (nasba).although direct detection methods based on nucleic acid hybridization would be theoretically possible, the amount of target nucleic acid in specimens may be minimal and such methods would lack sensitivity compared to amplification methods, unless the organism was propagated before analysis. thus, the molecular amplification procedures reported for direct detection of respiratory pathogens in clinical samples include pcr (e.g., reference 19 and figure 41 assays have utilized bacterial ribosomal rna (rrna; e.g., reference 22 ). for cellular samples tested for respiratory pathogens, targeting messenger rna (mrna) or genomic antisense rna may enhance diagnostic sensitivity. a variety of formats have been utilized for the detection of amplified products. procedures that separate target amplification from the detection phase (agarose gel analysis or endpoint hybridization) are well established 24, 25 and may allow multiple targets to be analyzed in a single reaction, providing added typing information. for ease of use and incorporation into diagnostic laboratories, most laboratory-developed assays for detection of respiratory pathogens utilize real-time amplification methods in which the amplification and detection steps are combined. some methods use intercalating dyes with the analysis of pcr product melting temperatures (e.g., as described previously 26 and illustrated in figure 41 -1), whereas others use fluorogenic primers or probes (e.g., taqman, hybridization format, and molecular beacons 19, [21] [22] [23] 27 ) to ensure the specificity of the reaction. figure 41 -2 illustrates a real-time rt-pcr assay for sars-cov using probe-specific detection of amplified products (one fluorescence measurement per cycle). the range of real-time pcr or rt-pcr and nasba methodologies used for respiratory targets is diverse given the fact that assays are laboratory developed. 28, 29 the difficulty with diagnosis of respiratory infections is the wide range of pathogens with similar presentations. the nucleic acid technologies utilized currently in the majority of diagnostic laboratories are real-time pcr single-target assays. in some cases, generic primers can be designed to pick up several related pathogens. such generic primers may be based on conserved protein coding sequences (such as those essential for enzyme function) or noncoding regions for which variation is limited because of the need for maintenance of secondary structure. the use of primer sets to pick up genera or even families of organisms has shown promise in limited studies, including analysis of legionella and mycobacteria. generic assays have the ability to detect many related organisms and may be used to characterize previously undescribed species in respiratory infection. limited multiplex procedures have been reported for detection of related organisms using real-time pcr or nasba procedures, but such assays are difficult to set up and control. for many respiratory pathogens, there is sufficient variation that multiplex approaches have been developed to detect, for example, all possible respiratory adenovirus, 19 influenza, 20 or parainfluenza 21 types. a simple, dual-labeled multiplex nasba assay is shown in figure 41 -5 that uses separate primer sets and specific molecular beacon probes for parainfluenza types 2 and 3 (hpiv2 and hpiv3) in the same reaction mix. each probe is labeled with a different fluorophore, allowing detection and differentiation of both viruses in a single reaction. an ambitious multiplex nested rt-pcr procedure with gel analysis of the amplicons detects influenza a, b, and c viruses, rsv (a and b subtypes) , and adenoviruses in a single assay. 30 the procedure, while complex to set up and validate, was reported to have good specificity and better sensitivity than antigen/culture procedures. interpretation and validation of a negative result are important parts of diagnostic tests based on nucleic acid amplification. some assays incorporate an internal control system to distinguish true-negative from false-negative results. the internal control may amplify with the pathogen-specific primers but result in an amplicon with a different size or internal sequence from the pathogen amplicon. alternatively, the internal control may be an external sequence spiked into the reaction (heterologous control) and amplified with a primer set different from the pathogen primers. in the example shown in figure 41 -3, amplification of the rna heterologous control is consistent across many clinical samples and ensures that there are no gross inhibitors present in the reactions. for cellular samples obtained for detection of respiratory pathogens, the internal control reaction can utilize human dna, rrna, or mrna detection. such approaches have the added value of assessing for sample collection and integrity, as well as amplification inhibitors. the relative merits of commercial versus laboratorydeveloped tests depend on the laboratory facilities, the technical expertise available, and the clinical need for expanded diagnosis. commercial testing reagents provide quality controls and procedure standardization that facilitate clinical studies. 24, 32 many companies are focusing on providing analyte-specific reagents (asrs) for respiratory pathogen assays. asrs will provide the laboratory with quality-controlled primers and probes, while allowing them the flexibility to test for currently known circulating pathogens or according to a local testing algorithm. microarray-based detection of multiplexed pcr products also has been reported. 31 classically, typing of bacteria or viruses has used serological techniques that rely on antibody-antigen interactions. one benefit of approaches based on dna or rna detection is the more-detailed, quantitative assessment of the relationship between organisms, providing valuable data relevant to outbreak investigations and community health. variation at the nucleic acid sequence level is not necessarily reflected in altered protein sequence or function; thus, additional sequence variation information may not correlate with conventional typing methods. restriction fragment length polymorphism (rflp) analysis by pulsed field gel electrophoresis (pfge), either with or without blot hybridization, has been utilized for analysis of complex dna genomes from a variety of respiratory pathogens. rflp analysis also has been applied to pcr or rt-pcr products from respiratory bacteria and viruses. in general, such methods can provide resolution down to the subtype level and have proven useful in outbreak investigation, as illustrated in figure 41-6 for b. pertussis isolates. the difficulty with gel-based typing assays, such as pfge, is standardizing results and sharing data between laboratories. amplified fragment length polymorphism (aflp) analysis represents an alternative method with better discriminatory power and portability, but this approach has not been used extensively for respiratory isolates to date. for respiratory viruses, other methods have been used for typing, including heteroduplex mobility assay (hma), single-strand conformation polymorphism (sscp), and rflp analysis of amplified pcr products. in general, hma is considered technically complex but has the capacity to distinguish viral quasispecies with >3% nucleotide differences. sscp and rflp, while technically easier, generally can resolve viruses only to the subtype level, and rflp has the added constraint of assessing only sequence differences in restriction sites. the use of sequencing to assess the relationship among viruses is well established, and molecular phylogenetic knowledge is expanding, allowing modeling of viral populations and prediction of new outbreaks. 33 the level of resolution using primary sequence is at one genome, and point mutations can be identified. sometimes this provides more information than originally sought and creates problems in interpretation; while in other circumstances even small sequence variations can confer important changes in viral transmissibility and disease outcome. identification of emerging viruses, which may have been recently introduced into the human population (e.g., sars-cov and influenza a h5n1 types), is critical to public health. analysis of such novel viruses has relied heavily on sequencing of isolates or amplicons. 9, 10, 34 human influenza a viruses are associated with enhanced morbidity and mortality compared with influenza b or influenza c viruses. differences in pathogenicity for subtypes of influenza a also have been reported; for example, h3n2 is associated with more severe infection than h1n1. such types and subtypes of influenza a can circulate independently, and their identification is important for assessment of current vaccine efficacy. reassortment of the two predominant influenza subtypes infecting humans in recent times has been reported, and analysis of main hemagglutinin (ha) types has been undertaken by a range of molecular and nonmolecular methods. 35 (1997-1998 and 2003-2004 34,38 ) emphasize the need for detailed surveillance of influenza viruses and vigilance in identification of emerging viruses of importance to public health. detailed analysis of avian h5n1 viruses that have infected humans to date have confirmed that all genes are of avian origin and are associated with minimal or very inefficient humanto-human spread. the potential for reassorted viruses that could more easily spread among humans is clear, and molecular methods are now an important part of influenza surveillance. recent studies of human metapneumovirus have identified two main lineages, with sequence diversity within each group (figure 41-8 40 ) , thus displaying a similar pattern to rsv isolates that are classified into two major groups,a and b. 39 further studies will confirm whether this distinction is associated with differences in virulence. sequence analysis for typing of bacteria has been slower to develop than that for viruses but has been utilized for investigation of some atypical bacteria associated with outbreaks of respiratory infection (e.g., legionella 41 ). due to the problem of recombination, characterization of a single bacterial gene often does not reflect the organism as a whole. multilocus sequence typing is a strategy that addresses this and appears useful for analysis of b. pertussis. 42 microarray hybridization methods have been used to identify and differentiate related pathogens. 31 such an approach was useful in first identifying the agent of sars as a coronavirus. 43 molecular methods provide additional information about the virulence and type of infectious organism, as illustrated by recent experience with sars-cov and influenza types. molecular tests have advantages over conventional procedures, but the sensitivity of molecular amplification methods can lead to problems with interpretation of results. for many organisms, a gold standard method is not available that accurately reflects the enhanced sensitivity of molecular methods, as has been seen with pcr testing for b. pertussis or c. pneumoniae in clinical samples. studies confirm that pcr tests are very sensitive, and pcr-positive individuals may be culture negative or asymptomatic, so that results must always be interpreted in the clinical context. inhibitors of amplification are common in respiratory specimens, so a negative result must be interpreted in the context of the nucleic acid extraction method and the control results to monitor for nucleic acid degradation and amplification inhibition. when assays for the detection of respiratory pathogens are designed, primers and probes should not cross-react with normal respiratory flora or other respiratory pathogens. the triage of molecular testing for respiratory infection diagnosis is difficult. currently, a single respiratory pathogen test detects only one or a few related pathogens. also, bacterial testing and viral testing are not combined. thus, many molecular tests must be used to screen for all appropriate pathogens, which increases testing costs. thus, a laboratory that embarks on using molecular methods for the diagnosis of respiratory infections may require a complex testing algorithm. one approach is use a multiplex amplification procedure to identify multiple pathogens in a single assay, with certain assays now commercially available. 24, 32 unfortunately, such tests tend to be expensive and, if developed by the laboratory, are very difficult to control and ensure equal sensitivity and specificity for all pathogens. thus, despite the potential for replacement of many culture and antigen procedures with nucleic acid amplification assays,such a molecular diagnostic revolution has not yet happened. the exception is for new pathogens when nucleic acid amplification and detection methods are clearly far superior to alternatives (e.g., metapneumovirus, sars-cov) or for testing of samples that are suboptimal for routine procedures (e.g., in surveillance situations). respiratory infections are currently underdiagnosed, despite the fact that accurate pathogen identification is important to ensure appropriate patient management and monitor infectious trends in the community. the major stumbling blocks in the diagnosis and investigation of respiratory infections are the complexity of testing algorithms and the number of potential targets that cause both upper and lower respiratory tract symptoms. real-time pcr methods have vastly improved the sensitivity for detection and recognition of some difficult-to-culture organisms, and will likely become standard practice in the clinical laboratory in the next few years. there is, however, a limit to how many organisms can be "multiplexed" in a single test. microarray hybridization of randomly amplified pcr products from respiratory cultures and clinical samples has shown some success. 31 if the promise of early experiments is maintained when applied to large-scale clinical studies, this could answer some of the technical problems surrounding the use of multiplex systems. microarray hybridization, while not currently as convenient as realtime pcr detection methods, potentially has the benefit of being able to resolve complex product mixtures and provide clinically valuable information. the use of molecular methods for typing and outbreak investigation of respiratory pathogens of public health importance is well established and is likely to expand. future directions will incorporate the use of microarray systems for respiratory pathogen detection and analysis to allow crossing of the barriers between conventional virology and bacteriology (and mycology/parasitology). once microarray systems have been developed and validated, the costs of this enhanced technology may be reduced and justifiable. identification of novel viruses, which have presumably only recently been introduced to humans, has reinforced the need for careful surveillance of emerging respiratory pathogens and institution of appropriate infection control measures. lessons should be learned from the continuous sensitive surveillance and typing of organisms such as influenza and b. pertussis to direct the use and efficacy of available vaccines. molecular techniques developed for detection and analysis of the microbes responsible for respiratory infections will be vital to our understanding of pathogenic mechanisms, appropriate management, and prevention of outbreaks in the future. gel-based typing procedures (pfge, hma, sscp) slowly will be replaced by sequencebased alternatives (e.g., multilocus sequence typing or mlst), which are more amenable to standardization and sharing of data among laboratories. phylogenetic tree illustrating the relationships among fusion gene sequences for 12 human metapneumovirus strains (from 2002). af371337 is the genbank accession number for the prototype human metapneumovirus strain, and hmpv 35 is a canadian strain canadian guidelines for the initial management of community-acquired pneumonia: an evidence-based update by the canadian infectious diseases society and the canadian thoracic society. the canadian community-acquired pneumonia working group bordetella pertussis in the aetiology of chronic cough in adults. diagnostic methods and clinic clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study respiratory tract viral infections in inner-city asthmatic adults polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia human metapneumovirus infections in young and elderly adults human metapneumovirus as a cause of community-acquired respiratory illness identification of severe acute respiratory syndrome in canada identification of a novel coronavirus in patients with severe acute respiratory syndrome clinical assessment of a generic dna amplification assay for the identification of respiratory adenovirus infections a comparison of nested polymerase chain reaction and immunofluorescence for the diagnosis of respiratory infections in children with bronchiolitis, and the implications for a cohorting strategy detection of bordetella pertussis in a clinical laboratory by culture, polymerase chain reaction, and direct fluorescent antibody staining; accuracy, and cost polymerase chain reaction as a sensitive and rapid method for specific detection of mycoplasma pneumoniae in clinical samples cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients nucleic acid amplification tests for the diagnosis of pneumonia molecular epidemiology of two consecutive outbreaks of parainfluenza 3 in a bone marrow transplant unit detection of adenoviruses (adv) in culture-negative environmental samples by pcr during an adv-associated respiratory disease outbreak multiplexed, real-time pcr for quantitative detection of human adenovirus rapid detection of influenza a and b viruses in clinical specimens by light cycler real time rt-pcr development and evaluation of nuclisens basic kit nasba for diagnosis of parainfluenza virus infection with "end-point" and "real-time" detection detection of mycoplasma pneumoniae by real-time nucleic acid sequence-based amplification development and evaluation of nucleic acid sequence based amplification (nasba) for diagnosis of enterovirus infections using the nuclisens basic kit rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (hexaplex) multiplex pcr for typing and subtyping influenza and respiratory syncytial viruses multiplex real-time pcr assay for detection of influenza and human respiratory syncytial viruses development and clinical evaluation of an internally controlled, single-tube multiplex real-time pcr assay for detection of legionella pneumophila and other legionella species real-time pcr in virology nasba and other transcription-based amplification methods for research and diagnostic microbiology simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay microarray-based detection and genotyping of viral pathogens evaluation of the prodesse hexaplex multiplex pcr assay for direct detection of seven respiratory viruses in clinical specimens the application of molecular phylogenetics to the analysis of viral genome diversity and evolution molecular aspects of avian influenza (h5n1) viruses isolated from humans molecular diagnosis of influenza influenza virus types and subtypes detection by single step single tube multiplex reverse transcription-polymerase chain reaction (rt-pcr) and agarose gel electrophoresis influenza ah1n2 viruses, united kingdom, 2001-02 influenza season molecular changes associated with the transmission of avian influenza a h5n1 and h9n2 viruses to humans circulation patterns of genetically distinct group a and b strains of human respiratory syncytial virus in a community human metapneumovirus infections in hospitalized children detection of legionella species in respiratory specimens using pcr with sequencing confirmation multilocus sequence typing of bordetella pertussis based on surface protein genes characterization of a novel coronavirus associated with severe acute respiratory syndrome key: cord-020560-jnemlabp authors: tewari, hemant; nangia, vivek title: severe tropical infections date: 2012-03-09 journal: icu protocols doi: 10.1007/978-81-322-0535-7_51 sha: doc_id: 20560 cord_uid: jnemlabp severe infection with multiorgan involvement is one of the most common cause of icu admission in tropical countries. close monitoring and supportive therapy is the mainstay of treatment in most of these infections, but some of them have specific therapies. rapid identification of treatable infection is imperative for a better outcome. severe infection with multiorgan involvement is one of the most common cause of icu admission in tropical countries. close monitoring and supportive therapy is the mainstay of treatment in most of these infections, but some of them have speci fi c therapies. rapid identi fi cation of treatable infection is imperative for a better outcome. fluid resuscitation is the mainstay of initial management in most of the tropical • infections as they present late, have predominant diarrheal component, and are usually dehydrated. close monitoring for volume overload and pulmonary edema should be done. • recent trial conducted in africa on a pediatric population with severe sepsis • have shown fl uid loading may be detrimental in this population. patients presenting with encephalopathic syndromes need airway assessment • and assisted ventilation. while resuscitation is going on, send investigations: • complete blood count-neutropenia is a common feature in many tropical infections. leptospirosis typically has leukocytosis. imaging: chest x-ray, echo, ultrasound of abdomen, and ct scan (when • indicated). step 5: start general supportive care and speci fi c organ support (see chap. 79 ) many tropical infections are self-limiting. close monitoring and general organ • support in the initial days or weeks of viremia or parasitemia will salvage many patients. step 6: initiate empirical therapy based on initial presentation speci fi c therapy is available only for a few tropical infections. • depending on the clinical presentation and endemicity of a particular infection • in the geographical region, an educated guess for initial therapy has to be decided till de fi nitive investigations are available. usually, intravenous ceftriaxone, 2 g iv twice daily, to cover typhoid fever and • leptospirosis is started if mp and dual antigen is negative. in patients with shock and mods, broad-spectrum antibiotics should be started • immediately. de-escalate antibiotics once speci fi c infection is identi fi ed. step 7: start speci fi c treatment once the diagnosis is con fi rmed dengue • a protocol for intravenous fl uid therapy has been developed by the world -health organization (who). an initial bolus of 5% dextrose in normal saline or ringer lactate (20 ml/kg of body weight) is infused over 15 min, followed by continuous infusion (10-20 ml/kg/h, depending on the clinical response) until vital signs and urine output normalize. crystalloids are equally effective as colloids in fl uid resuscitation. -normalization of the hematocrit is an important goal of early fl uid repletion. -however, a normal or low hematocrit may be misleading in patients with overt bleeding and severe hypovolemia. close clinical observation is essential, even after normal blood volume is restored, because patients can develop shock for 1-2 days after initial fl uid resuscitation, which represents the period of increased vascular permeability in dengue hemorrhagic fever. plasmodium falciparum infections acquired in areas without chloroquineresistant strains, patients should be treated with oral chloroquine. a chloroquine dose of 600 mg base (=1,000 mg salt) should be given initially, followed by 300 mg base (=500 mg salt) at 6, 24, and 48 h after the initial dose for a total chloroquine dose of 1,500 mg base (=2,500 mg salt). for chloroquine-resistant strains, treatment options are as follows: -quinine sulfate: quinine has a rapid onset of action and, in combination • with tetracycline, doxycycline, or clindamycin, it has been shown to be a very ef fi cacious treatment option for p. falciparum infections acquired in regions with chloroquine-resistant strains. artemisinin derivatives clear parasites very rapidly, are now a key compo-• nent of malaria treatment worldwide, and have been shown to reduce mortality in severe malaria compared with parenteral quinine. artemisinin-based combination therapies, including artesunate-me fl oquine, artemether-lumefantrine, artesunate-amodiaquine, and dihydroartemisinin-piperaquine, are highly ef fi cacious. under the cdc protocol, intravenous artesunate is administered in four • equal doses of 2.4 mg/kg of body weight over a period of 3 days. the dosing schedule recommended by the who entails doses every 12 h on day 1 and then once daily. up to 7 days of therapy may occasionally be indicated in very ill patients. • fluid boluses signi fi cantly increased 48-h mortality in critically ill children with impaired perfusion in the resource-limited settings in africa intravenous artesunate for the treatment of severe malaria three major studies regarding the use of intravenous artesunate are reviewed. several international studies comparing intravenous quinine and artesunate conclude that artesunate has the highest treatment success dengue haemorrhagic fever: diagnosis, treatment, prevention and control world health organization. guidelines for prevention and control of leptospirosis. geneva: world health organization world health organization. guidelines for malaria geneva: world health organization epidemic of leptospirosis: an icu experience leptospirosis is an important infection with high mortality when associated with organ dysfunction. the poor prognostic factors are preponderance of male sex, alcohol dependence, age group more than 50 years, mods, acute respiratory distress syndrome (ards), presence of acidosis, and need for mechanical ventilation the hospital for tropical diseases is dedicated to the prevention, diagnosis, and treatment of tropical diseases and travel-related infections. an extensive repository on guidelines for many tropical infections key: cord-023767-rcv4pl0d authors: o’ryan, miguel l.; nataro, james p.; cleary, thomas g. title: microorganisms responsible for neonatal diarrhea date: 2009-05-19 journal: infectious diseases of the fetus and newborn infant doi: 10.1016/b0-72-160537-0/50022-0 sha: doc_id: 23767 cord_uid: rcv4pl0d nan at the beginning of the 1st century, diari..eal disease continues to be a significant cause of morbidity and mortality worldwide. during the period of 1986 to 2000, an estimated 1.4 billion children younger than 5 years suffered an episode of acute diarrhea every year in developing countries; among these, 123.6 million required outpatient medical care, and 9 million required hospitalization. approximately 2 million diarrhea-associated deaths occurred in this age group annually, primarily in the most impoverished areas of the world.' these estimates are somewhat lower than the more than 3 million annual deaths from diarrhea reported in the prior 10 years? indicating progress in prevention and treatment of acute diarrhea. in the united states, approximately 400 childhood deaths per year were reported during the late 1 9 8 0~,~*~ although the actual number may be higher: accurate incidence rates for acute diarrhea in neonates from different populations are not readily available. the relative sparing of the newborn probably results from low exposure to enteropathogens and protection associated with brea~t-feeding.~-' after the first few months of life, increasing interaction with other individuals and the environment, including introduction of artificial feeding, increases the risk of exposure to enteropathogens. for most pathogens, the incidence of acute diarrhea peaks in children between 6 months and 4 years old? neonatal diarrhea is more common in underdeveloped areas, where low educational levels, crowding, and poor standards of medical care, environmental sanitation, and personal hygiene favor early contact with enteropathogens. as the incidence of neonatal gastroenteritis rises, there is a proportional increase in neonatal deaths because medical care for the poor often is inadequate.'0*" for very low birth weight infants (<1500g), the death rate from diarrhea is 100-fold greater than for higher-birth-weight infants. 12 this chapter discusses the pathogenesis, diagnosis, treatment, and prevention of gastroenteritis based on the available knowledge about pathogens that can cause neonatal diarrhea. pathogens that rarely or never cause acute diarrhea in neonates are not discussed. after an overview of host defense mechanisms and protective factors in human milk, the remainder of the chapter is devoted to specific pathogens that cause inflammatory or noninflammatory diarrhea. the neonate is a host that is uniquely susceptible to enteric infections. neonates have not had the opportunity to develop local or systemic immune responses, and in the first few days of life, they have not acquired the highly important enteric flora that protects the normal adult gastrointestinal tract.i3-" still less is known about the barrier effect of the neonate's gastric acidity," intestinal mucus,z0 or each of which provides protection against gastrointestinal tract infections in older infants, children, and adults. the gastric acid barrier appears to be least effective during the first months of life. the average gastric ph level of the newborn is high (ph 4 to 7; mean, 6).23,24 although the ph falls to low levels by the end of the first day of life (ph 2 to 3),23 it subsequently rises again; by 7 to 10 days of life, the hydrochloric acid output of the neonatal stomach is far less than that of older infants and ~hildren.~~.'~ the buffering action of frequent milk feedings and the short gastric emptying interpose additional factors in the neonate that would be expected to permit viable ingested organisms to reach the small intestine. the intestinal epithelium serves as a nutrient absorptive machine, barrier to pathogen entry, and regulator of inflammation. intestinal epithelial cells have receptors for bacterial products and produce chemokines (e.g., interleukm [ il]-8, monocyte chemotactic protein type 1 [ mcp-1 1, granulocyte macrophage-cell stimulating factor [ gm-csf] ) and proinflammatory cytokines (e.g., il-6, tumor necrosis factor-a [tnf-a], il-1) in response to invasion by enteropathogens." the gut epithelium orchestrates the immune response. however, in the newborn, phagocytic, chemotactic, and complement functions are immature. b and t lymphocyte functions are impaired, resulting in a preferential igm production in response to antigenic stimulation. igg is actively transferred from mother to infant across the placenta at about 32 weeks' gestation and peaks by about 37 weeks; premature neonates, especially those born before 28 weeks' gestation, are deficient in these maternally derived serum antib~dies.~' h e 2 6 -2 9 the importance of breast-feeding infants for the prevention of diarrheal disease has long been e m p h a s i~e d .~~~~* -~~ published studies reporting the association between breastfeeding and diarrhea are extensive and suggest that infants who are breast-fed suffer fewer episodes of diarrhea than those who are not. this protection is greatest during a child's first 3 months of life and declines with increasing age, during the period of weaning, partial breast-feeding confers protection that is intermediate between that gained by infants who are exclusively breast-fed and that by those who are exclusively bottle-fed. a striking demonstration of the protection afforded by breast-feeding of newborns has been provided by mata and urrutiai3 in their studies of a population of infants born in a rural guatemalan village. despite extremely poor sanitation and the demonstration of fecal organisms in the colostrum and milk of almost one third of diarrheal disease did not occur in any newborns. the incidence of diarrhea rose significantly only after these infants reached 4 to 6 months old, at which time solids and other fluids were used to supplement the human milk feedings. at that time, escherichia coli and gram-negative anaerobes (e.g., bacteroides) were found to colonize the intestinal tract.i3 in contrast, urban infants of a similar ethnic background who were partly or totally artificially fed frequently acquired diarrheal disease caused by enteropathogenic e. coli (epec) . multiple mechanisms by which breast-feeding protects against diarrhea have been postulated. breast-feeding confers protection by active components in milk and by decreased exposure to organisms present on or in contaminated bottles, food, or water. many protective components have been identified in human milk and generally are classified as belonging to the major categories of cells, antibody, antiinflammatory factors, and glycoconjugates and other nonantibody f a~t o r s .~~-~' examples of milk antibodies are summarized in table 20-1. for any given pathogen, multiple milk factors may help protect the infant. human milk typically targets a major pathogenic mechanism using multiple, redundant strategies. redundancy of milk protective factors and targeting of complex virulence machinery have created a formidable barrier to enteropathogens. despite the fact that pathogens can rapidly divide and mutate, milk continues to protect infants. for example, human milk has secretory antibodies to shigellu virulence antigens and lipopoly-saccharide^,^^.^^ neutral glycolipid gb3 to bind shiga and lactoferrin to disrupt and degrade the surface-expressed virulence antigen^.^^-^^ in a similar way, milk contains antibodies directed toward the surface expressed virulence antigens of epec,5' oligosaccharides that block cell attachment?9 and lactoferrin that disrupts and degrades the surface expressed epec antigens6' human milk can initiate and maintain the growth of bifidobacterium and low ph in the feces of newborn infants, creating an environment antagonistic to the growth of e. ~o l i . ' the protective effect of human milk antibodies against enteropathogen-specific disease has been described for vibrio cholerae,62 campylobacter j e j~n i , ~~ epec,59 enterotoxigenic e. coli (etec),64965 shigella,66'67 and giardia lamblia68,69 and for bovine milk concentrate against etec,70 rota~irus,~' and shigella. 72 in 1933, the nonlactose carbohydrate fraction of human milk was found to consist mainly of oligosa~charides.~~ in 1960, montreuil and mullet74 determined that up to 2.4% of colostrum and up to 1.3% of mature milk are oligosaccharides. human milk contains a larger quantity of the oligosaccharides than does milk from other mammals, and its composition is singularly complex.75 the metabolic fate of the oligosaccharides is of interest. only water, lactose, and lipids are present in greater amounts than the oligosaccharides. despite the fact that substantial energy must be expended by the mother to synthesize the many hundreds of different milk oligosaccharides, the infant does not use them as food. most of the oligosaccharides pass through the gut undigested.76377 it is thought that they are present primarily to serve as receptor analogues that misdirect enteropathogen attachment factors away from gut epithelial carbohydrate receptors. likewise, enteropathogens use the oligosaccharide portion of glycolipids and glycoproteins as targets for attachment of whole bacteria and toxins. evidence is emerging that these glycoconjugates may have an important role in protection of the breast-fed infant from disease. 48 human milk protects suckling mice from the heat-stable enterotoxin (st) of e. coli; on the basis of its chemical stability and physical properties, the protective factor has been deduced to be a neutral fucosyloligosaccharide. 79~80 experiments have shown that epec attachment to hep-2 cells can be inhibited by purified oligosaccharide fractions from human milk.59 oligosaccharides also may be relevant to protection from norwalk virus and other caliciviruses, because these viruses attach to human abo, lewis, and secretor blood group antigens.80'81 human milk contains large amounts of these carbohydrates. the ganglioside fraction in human milk has been shown to inhibit the action of heat-labile toxin (lt) and cholera toxin on ileal loops more effectively than secretory iga.82s83 lactadherin in human milk has been shown to bind rotavirus and to inhibit viral replication in vitro and in v~v o . ~~ a study of infants in mexico showed that lactadherin in human milk protected infants from symptoms of rotavirus infection.72 e. coli organisms promptly colonize the lower intestinal tracts of healthy infants in their first few days of life85-88 and constitute the predominant aerobic coliform fecal flora throughout life in humans and in many animals. the concept that this species might cause enteric disease was first suggested in the late 19th and early 20th centuries, when several veterinary workers described the association of diarrhea (i.e., in 1905, m0r0'~ observed that bacterium (now escherichia) coli was found more often in the small intestines of children with diarrhea than in children without diarrhea. adam96*97 confirmed these findings and noted the similarity with asiatic cholera and calf scours. he further extended these observations by suggesting that e. coli strains from patients with diarrhea could be distinguished from normal coliform flora by certain sugar fermentation patterns. although he called these disease-producing organisms dyspepsicoli and introduced the important concept that e. coli could cause enteric disease, biochemical reactions have not proved to be a reliable means of distinguishing nonpathogenic from pathogenic e. coli strains. there are now at least six recognized enteric pathotypes of e. ~oli.'~ the pathotypes can be distinguished clinically, epidemiologically, and pathogenetically (table 20 -2) .98104 etec organisms are defined by their ability to secrete the lt or the st enterotoxin, or both. lt is closely related to cholera toxin and similarly acts by means of intestinal adenylate c y c l a~e , '~~~'~~ prostaglandin s y n t h e~i s , '~~~'~~ and possibly platelet activating f a c t~r . '~~' '~~ st (particularly the variant sta) causes secretion by specifically activating intestinal mucosal guanylate cyclase.' "-' l3 the stb toxin causes noncyclic, nucleotide-mediated bicarbonate secretion and appears to be important only in animals.1'4-"6 enteroinvasive e. coli (eiec) has the capacity to invade the intestinal mucosa, thereby causing an inflammatory enteritis much like shigellosi~.'~~~''~ epec elicits diarrhea by a signal transduction m e~h a n i s m~~~'~~~~~'~'~~ which is accompanied by a characteristic attaching-and-effacing histopathologic lesion in the small intestine.12' enterohemorrhagic e. coli (ehec) also induces an attaching-and-effacing lesion, but in the colon?' ehec also secretes shiga toxin, which gives rise to the sequela of hemolytic-uremic syndrome (hus). diffusely adherent e. coli122 executes a signal transduction effect, which is accompanied by the induction of long cellular processes. 123 enteroaggregative e. coli (eaec) adheres to the intestinal mucosa and elaborates enterotoxins and a major problem in the recognition of etec, eiec, epec, and ehec strains of e. coli is that they are indistinguishable from normal coliform flora of the intestinal tract by the usual bacteriologic methods. serotyping is of value in recognizing epec serotypes'26 and eiec, because these organisms tend to fall into a limited number of specific serogroups (see table 20 -2).'263'27 eiec invasiveness is confirmed by inoculating fresh isolates into guinea pig conjunctivae, as described by sereny. 12' the ability of organisms to produce enterotoxins (lt or st) is encoded by a transmissible plasmid that can be lost by one strain of e. coli or transferred to a previously unrecognized although the enterotoxin plasmids appear to prefer certain serogroups (different from epec or invasive serogro~ps),'~~ etec is not expected to be strictly limited to a particular set of serogroups. instead, these strains can be recognized only by examining for the enterotoxin. this is done in ligated animal or by enzyme-linked immunosorbent assay (elisa)'36 for lt or in suckling mice for st. 137, 138 specific dna probes also are available for lt and st.98 whether there are other mechanisms involved in the ability of the versatile e. coli species to cause enteric disease, such as by producing other types of enterotoxins"' or by fimbriate adherence traits a l~n e , '~~. '~' remains to be elucidated. cytoto~ns.98~103,125 in tissue although early work on the recognition of e. coli as a potential enteric pathogen focused on biochemical or serologic distinctions, there followed a shift in emphasis to the enterotoxins produced by previously recognized and entirely "new" strains of e. coli. beginning in the mid-1950s with work by de and colleague^'^^^'^^ in calcutta, e. coli strains from patients with diarrhea were found to cause a fluid secretory response in ligated rabbit ileal loops analogous to that seen with v; cholerue. work by taylor and associate^'^"^^ showed that the viable e. coli strains were not required to produce this secretory response and that this enterotoxin production correlated poorly with classically recognized epec serotypes. in sho paulo, trabulsi'& made similar observations with e. coli isolated from children with diarrhea, and several veterinary workers demonstrated that etec was associated with diarrhea in piglets and cal~es.'~~-'~o a similar pattern was described in 1971 with acute undifferentiated diarrhea in adults in bengal from whom e. coli could be isolated from the upper small bowel only during acute i l l n e~s . '~' "~~ these strains of e. coli produced a nondialyzable, lt, ammonium sulfate-precipitable enterotoxin.'53 analogous to the usually short-lived diarrheal illnesses of e. coli reported by several workers, a short-lived course of the secretory response to e. coli culture filtrates compared with the secretory response of cholera toxin was de~cribed."~ however, like responses to cholera toxin, secretory responses to e. coli were associated with activation of intestinal mucosal adenylate cyclase that paralleled the fluid secretory r e~p o n s e . '~~. '~~ the two types of enterotoxins produced by e. have been found to be plasmid-encoded traits that are potentially separable from each other and from the equally important plasmid-encoded adherence traits for patho-st causes an immediate and reversible secretory whereas the effects of lt (e.g., cholera toxin) follow a lag period necessitated by its intracellular site of a~t i o n . '~~' '~' '~~ only lt appears to cause fluid secretion by activating adenylate cyclase, which is accomplished by toxininduced adp-ribosylation of the gsa signaling p r~t e i n .~' "~~ the activation of adenylate cyclase by lt and by cholera toxin is highly promiscuous, occurring in many cell types and resulting in development of nonintestinal tissue culture assay systems such as the chinese hamster ovary (cho) cell assay'34 and y1 adrenal cell assay.'35 the antigenic similarity of lt and cholera toxin and their apparent binding to the monosialoganglioside gm, have enabled development of elisas for detection of lt and cholera t~x i n . '~~, '~' -'~~ st is a much smaller molecule and is distinct antigenically from lt and cholera t~x i n . '~~, '~'~'~' al though it fails to alter camp levels, st increases intracellular intestinal mucosal cyclic guanosine monophosphate (cgmp) concentrations and specifically activates plasma membrane-associated intestinal guanylate cy~lase."'-"~ like camp analogues, cgmp analogues cause intestinal secretion that mimics the response to st."' the receptor for sta responds to an endogenous ligand called guunylin, of which sta is a structural homologue.'" because the capacity to produce an enterotoxin may be transmissible between different organisms by a plasmid or even a bacteri~phage,''~-'~' interstrain gene transfer genesis. [129] [130] [131] 160 may be expected to be responsible for occasional toxigenic non-e. coli. enterotoxigenic klebsiella and citrobacter strains have been associated with diarrhea in a few reports, often in the same patients with etec.'65,'66 likewise, certain strains of salmonella appear to produce an lt, cho cellpositive toxin that may play a similar role in the pathogenesis of the watery, noninflammatory diarrhea sometimes seen with salmonella enteritidis i n f e c t i~n . '~~"~ at least equally important as enterotoxigenicity for e. coli to cause disease is the ability of these organisms to colonize the upper small bowel, where the enterotoxin produced has its greatest effect. a separable, plasmid-encoded colonization trait was first recognized in porcine e. coli. veterinary workers demonstrated that the fimbriate k-88 surface antigen was necessary for etec to cause disease in piglets.16' an autosomal dominant allele appears to be responsible for the specific intestinal receptor in piglets. in elegant studies by gibbons and c o -~o r k e r s , '~~ the homozygous recessive piglets lacked the receptor for k-88 and were resistant to scours caused by etec. at least 15 analogous colonization factors have been described for human e. coli isolate^^^"^'"^^ against which local iga antibody may be produced. these antigens potentially may be useful in vaccine development. data on the epidemiology and transmission of etec remain scanty for the neonatal period. in the past 2 decades, these strains have been recognized among adults with endemic, cholera-like diarrhea in calcutta, india, and in dacca, banglade~h,''~*'~' and among travelers to areas such as mexico and central a f r i~a . '~~-'~~ the isolation of etec is uncommon in sporadic diarrheal illnesses in temperate climates where sanitation facilities are good and where winter viral patterns of diarrhea predominate. etec is commonly isolated from infants and children with acute watery summer diarrhea in areas where sanitary facilities are less than optimal.35-165*'75-'87 these include areas such as afria,165 ~~~il,35,175,181,186,187 gentir~a,'~~ bengal,'78''79 mexico,'8o and native american reservations in the southwestern united state^."^"'^ in a multicenter study of acute diarrhea in 3640 infants and children in china, india, mexico, myanmar, and pakistan, 16% of cases (versus 5% of 3279 controls) had etec.le4 a case-control study from northwestern spain showed a highly significant association of etec with 26.5% of neonatal diarrhea, often acquired in the ho~pital.''~ although all types of etec (lt and/or st producers) are associated with cholera-like, non inflammatory, watery diarrhea in adults in these areas, they probably constitute the major cause (along with rotaviruses) of dehydrating diarrhea in infants and young children in these areas. in this setting, peaks of illnesses tend to occur in the summer or rainy season, and dehydrating illnesses may be life threatening, especially in infants and young ~h i l d r e n . 3~~'~'~'~ humans are probably the major reservoirs for the human strains of etec, and contaminated food and water probably constitute the principal vector^.''^"^^ although antitoxic immunity to lt and asymptomatic infection with ltproducing e. coli tends to increase with age, st is poorly immunogenic, and st-producing e. coli continues to be associated with symptomatic illnesses into adulthood in endemic area~.l'~>l'~ the association of etec with outbreaks of diarrhea in newborn nurseries is well documented. ryder and colleagues'g0 isolated an st-producing e. coli from 72% of infants with diarrhea, from the environment, and in one instance, from an infant's formula during a 7-month period in a prolonged outbreak in .a special care nursery in texas. another st-producing e. coli outbreak was reported in 1976 by gross and a~sociates'~~ from a maternity hospital in scotland. etec and epec were significantly associated with diarrhea among infants younger than 1 year in bang1ade~h.l~ ' an outbreak of diarrhea in a newborn special care nursery that was associated with enterotoxigenic organisms that were not limited to the same serotype or even the same species has been reported.lg3 the short-lived etec, klebsiella, and citrobacter species in this outbreak raised the possibility that each infant's indigenous bowel flora might become transiently toxigenic, possibly by receiving the lt genome from a plasmid or even a bacteriophage. the clinical manifestations of etec diarrhea tend to be mild and self-limited, except in small or undernourished infants, in whom dehydration may constitute a major threat to life. in many parts of the developing world, acute diarrheal illnesses are the leading recognized causes of death. there is some suggestion that the diarrheal illnesses associated with st-producing etec may be particularly severe.'79 most probably the best definition of the clinical manifestations of etec infection comes from volunteer studies with adults. ingestion of 10' to 10'' human etec isolates that produce lt and st or st alone resulted in a 30% to 80% attack rate of mild to moderate diarrheal illnesses within 12 to 56 hours that lasted 1 to 3 days.'17 these illnesses, typical for traveler's diarrhea, were manifested by malaise, anorexia, abdominal cramps, and sometimes explosive diarrhea. nausea and vomiting occur relatively infrequently, and up to one third of patients may have a low-grade fever. although illnesses usually resolve spontaneously within 1 to 5 days, they occasionally may persist for 1 week or longer. the diarrhea is noninflammatory, without fecal leukocytes or blood. in outbreaks in infants and neonates, the duration has been in the same range (1 to 11 days), with a mean of approximately 4 days. as in cholera, the pathologic changes associated with etec infection are minimal. from animal experiments in which thiry-vella loops were infected with these organisms and at a time when the secretory and adenylate cyclase responses were present, there was only a mild discharge of mucus from goblet cells and otherwise no significant pathologic change in the intestinal tract.lo6 unless terminal complications of severe hypotension ensue, etec organisms rarely disseminate beyond the intestinal tract. like cholera, etec diarrhea is typically limited to being an intraluminal infection. the preliminary diagnosis of etec diarrhea can be suspected by the epidemiologic setting and the noninflammatory nature of stool specimens, which reveal few or no leukocytes. although the ability of e. coli to produce enterotoxins may be lost or transmitted to other strains, there is a tendency for the enterotoxin plasmids to occur among certain predominant serotypes, as shown in table 20 -2."' these serotypes differ from epec or invasive serotypes, but their demonstration does not prove that they are enterotoxigenic. the only definitive way to identify etec is to demonstrate the enterotoxin itself by a specific gene probe for the toxin codon, by a bioassay such as tissue culture or ileal loop assays for lt or the suckling mouse assay for st, or in the case of lt, by immunoassay such as elisa. however, even these sensitive bioassays are limited by the unavailability of any selective media for detecting etec by culture. even though substantial improvements have been made in enterotoxin assay (particularly for lt), the necessary random selection of e. coli from a relatively nonselective stool culture plate resulted in a sensitivity of only 43% of epidemiologically incriminated cases in an outbreak when 5 to 10 isolates were randomly picked and tested for enter~toxigenicity."~ by also examining paired serum samples for antibody against lt, only 36% demonstrated significant serum antibody titer rises, for a total sensitivity of etec isolation or serum antibody titer rises of only 64%. some have suggested that isolates may be pooled for lt or st assay. the capacity to prove with radiolabeled or enzyme-tagged oligonucleotide gene sequences for the enterotoxins (lt or st) further facilitates the identification of enterotoxigenic organisms.'94s195 a novel method of combining immunomagnetic separation (using antibodycoated magnetic beads) followed by dna or polymerase chain reaction (pcr) probing may enhance the sensitivity of screenin fecal or food specimens for etec or other the mainstay of treatment of any diarrheal illness is rehydration."' this principle especially pertains to etec diarrhea, which is an intraluminal infection. the glucose absorptive mechanism remains intact in e. coli enterotoxininduced secretion, much as it does in cholera, a concept that has resulted in the major advance of oral glucose-electrolyte therapy. this regimen can usually provide fully adequate rehydration in infants and children able to tolerate oral fluids, replacing the need for parented rehydration in most cases .199,200 its use is particularly critical in rural areas and developing nations, where early application before dehydration becomes severe may be lifesaving. the standard world health organization solution contains 3.5 g nacl, 2.5 g nahco,, 1.5 g kcl, and 20 g glucose per liter of clean or boiled drinking water.i9' this corresponds to the following concentrations: 90 mmoyl of sodium, 20 mmovl of potassium, 30 mmoyl of bicarbonate, 80 mmol/l of chloride, and 1 10 mmol/l of glucose. a variety of recipes for homemade preparations have been described?" but unless the cost is prohibitive, the premade standard solution is preferred. each 4 ounces of this solution should be followed by 2 ounces of plain water. if there is concern about hypertonicity, especially in small infants in whom a high intake and constant direct supervision of feeding cannot be ensured, the concentration of salt can be reduced.'02 a reduced osmolality solution with 60 mmol/l of sodium and 84 mmoyl of glucose and a total osmolality of 224 (instead of 311) mosm/kg has been found to reduce stool output by 28% and illness duration by 18% in a multicenter trial involving 447 children in four countries.203 commercially available rehydration solutions are increasingly available ~orldwide.'~' pathogens. lf6,197 the role of antimicrobial agents in the treatment or prevention of etec is controversial. this infection usually resolves within 3 to 5 days in the absence of antibacterial therapy.198 moreover, there is concern about the potential for coexistence of enterotoxigenicity and antibiotic resistance on the same plasmid, and co-transfer of multiple antibiotic resistance and enterotoxigenicity has been well d~cumented."~ widespread use of prophylactic antibiotics in areas where antimicrobial resistance is common has the potential for selecting for rather than against enterotoxigenic organisms. the prevention and control of etec infections would be similar to those discussed under epec serotypes. the use of breast-feeding should be encouraged. eiec causes diarrhea by means of shigella-like intestinal epithelial invasion (discussed later).117s11' the somatic antigens of these invasive strains have been identified and seem to fall into 1 of 10 recognized 0 groups (see table 20 -2). most, if not all, of these bacteria share cell wall antigens with one or another of the various shigelza serotypes and produce positive reactions with antisera against the cross-reacting antigen."* however, not all strains of e. coli belonging to the 10 serogroups associated with dysentery-lke illness are pathogenic, because a large (140 mda) invasive plasmid is also required.205 additional biologic tests, including the guinea pig conjunctivitis (sereny) test or a gene probe for the plasmid, are used to confirm the property of inva~iveness."~ although an outbreak of foodborne eiec diarrhea has been well documented among adults who ate an imported cheese,"' little is known about the epidemiology and transmission of this organism, especially in newborns and infants. whether the infectious dose may be as low as it is for shigella is unknown; however, studies of adult volunteers suggest that attack rates may be somewhat lower after ingestion of even large numbers of eiec than would be expected with shigella. the outbreak of eiec diarrhea resulted in a dysentery-like syndrome with an inflammatory exudate in stool and invasion and disruption of colonic mucosa."' descriptions of extensive and severe ileocolitis in infants dying with e. coli diarrhea indicate that neonatal disease also can be caused by invasive strains capable of mimicking the pathologic features of shigellosis. 206 the immunofluorescent demonstration of e. coli together with an acute inflammatory infiltrate2" in the intestinal tissue of infants tends to support this impression, although it has been suggested that the organisms may have invaded the bowel wall in the postmortem ~e r i 0 d . l '~ there is still little direct evidence concerning the role of invasive strains of e. coli in the cause of neonatal diarrhea.175 the infrequency with which newborns manifest a dysentery-like syndrome makes it unlikely that this pathogen is responsible for a very large proportion of the diarrheal disease that occurs during the first month of life. the diagnosis should be suspected in infants who have an inflammatory diarrhea as evidenced by fecal polymorphonuclear neutrophils or even bloody dysenteric syndromes from whom no other invasive pathogens, such as campylobacter, shigella, salmonella, vibrio, or yersinia, can be isolated. in this instance, it may be appropriate to have the fecal e. coli isolated and serotyped or tested for invasiveness in the sereny test. plasmid pattern analysis and chromosomal restriction endonuclease digestion pattern analysis by pulsed-field gel electrophoresis have been used to evaluate strains involved in outbreaks. 208 the management and prevention of eiec diarrhea should be similar to those for acute shigella or other e. coli enteric infections. the serologic distinction of e. coli strains associated with epidemic and sporadic infantile diarrhea was first suggested by goldschmidt in 1933209 and confirmed by dulaney and michelson in 1935. 205 these researchers found that certain strains of e. coli associated with institutional outbreaks of diarrhea would agglutinate with antisera on slides. in 1943, bra?" isolated a serologically homogeneous strain of e. coli (subsequently identified as serogroup 0111) from 95% of infants with summer diarrhea in england. he subsequently summarized a larger experience with this organism isolated from only 4% of asymptomatic controls but from 88% of infants with diarrhea, one half of which was hospital this strain (initially called e. coli-gomez by varela in 1946) also was associated with infantile diarrhea in a second type of e. coli (called beta by giles in 1948 and subsequently identified as 055) was associated with an outbreak of infantile diarrhea in aberdeen, s~o t l a n d .~'~*~'~ from this early work primarily with epidemic diarrhea in infants has developed an elaborate serotyping system for certain e. coli strains that were clearly associated with infantile these strains first were called enteropathogenic e. coli by neter and colleagues219 in 1955, and the association with particular serotypes can still be observed.220 as shown in table 20 -1, these organisms are distinct from the enterotoxigenic or enteroinvasive organisms or those that inhabit the normal gastrointestinal tract. they exhibit localized adherence to hep-2 cells, a phenotype that has been suggested to be useful for diagnosis and pathogenesis research. 'i9 epec is an important cause of diarrhea in infants in developing or transitional c~u n t r i e s .~"~~' -~~~ outbreaks have become rare in the united states and other industrialized countries, but they still 0ccur.2~~ some have attributed the rarity of this recognition of illness in part to the declining severity of diarrheal disease caused by epec within the past 30 years, resulting in fewer cultures being obtained from infants with relatively mild symptom^.^^^^^^ however, several other variables influence the apparent incidence of this disease in the community. a problem arises with false-positive epec on the basis of the nonspecific cross-reactions seen with improper shortening of the serotyping p r o c e d~r e .~~~,~~~ because of their complexity and relatively low yield, neither slide agglutination nor hep-2 cell adherence or dna probe tests are provided as part of the routine identification of enteric pathogens by most clinical bacteriology laboratories. failure to recognize the presence of epec in fecal specimens is the inevitable consequence. the apparent incidence of epec gastroenteritis also varies with the epidemiologic circumstances under which stool cultures are obtained. the prevalence of enteropathogenic strains is higher among infants from whom cultures are obtained during a community epidemic compared with those obtained during sporadic diarrheal disease. neither reflects the incidence of epec infection among infants involved in a nursery outbreak or hospital epidemic. epec gastroenteritis is a worldwide problem, and socioeconomic conditions play a significant role in determining the incidence of this disease in different populations.22s for instance, it is unusual for newborn infants born in a rural environment to manifest diarrheal disease caused by epec; most infections of the gastrointestinal tract in these infants occur after the first 6 months of conversely, among infants born in large cities, the attack rate of epec is high during the first 3 months of life. this age distribution reflects in large part the frequency with which epec causes crossinfection outbreaks among nursery populations'9'~230~237; however, a predominance of epec in infants in the first 3 months of life also has been described in community epidemic^^^'-^^^ and among sporadic cases of diarrhea acquired outside the h~s p i t a l . '~' -~~~ the disparity in the incidence of neonatal epec infection between rural and urban populations has been ascribed to two factors: the trend away from breast-feeding among mothers in industrialized societies and the crowding together of susceptible newborns in nurseries in countries in which hospital deliveries predominate over home d e l i~e r i e s . 5 '~~~'~~ although the predominant serogroup can vary from year to year,239,242,243,z46.249~250 the same strains have been prevalent during the past 40 years in great britain?51 puerto r~c o ?~~ guatemala: panama, 205 newfoundland,240 indonesia,244 thailand,254 and south when living conditions are poor and overcrowding of susceptible infants exists, there is a rise in the incidence of neonatal diarrhea in general257 and epec gastroenteritis in p a r t i c~l a r .~'~~~~~~~~~ a h igher incidence of asymptomatic family carriers is found in such situations.238b239 newborn infants can acquire epec during the first days of life by one of several routes: (1) organisms from the mother ingested at the time of birth; ( 2 ) bacteria from other infants or toddlers with diarrheal disease or from asymptomatic adults colonized with the organism, commonly transmitted on the hands of nursery personnel or parents; (3) airborne or droplet infection; (4) fomites; or (5) organisms present in formulas or solid food supplements.259 only the first two routes have been shown conclusively to be of any real significance in the transmission of disease or the propagation of epidemics. most neonates acquire epec at the time of delivery through ingestion of organisms residing in the maternal birth canal or rectum. stool cultures taken from women before, during, or shortly after delivery have shown that 10% to 15% carry epec at some time during this period.85~s6~88~260~26' use of fluorescent antibody techniquesz6' or cultures during a community outbreak of epec gastroenteritis" revealed twice this number of persons excreting the organism. virtually none of the women carrying pathogenic strains of e. coli had symptoms referable to the gastrointestinal tract. many of the mothers whose stools contain epec transmit these organisms to their infant~,8~*~' resulting in an asymptomatic infection rate of 2% to 5% among newborns cultured at random in nursery surveys.85~86~19'~262 these results must be considered conservative and are probably an artifact of the sampling technique. one study using 150 0 antisera to identify as many e. coli as possible in fecal cultures showed a correlation between the coliform flora in 66% of motherinfant pairs?63 of particular interest was the observation that the 0 groups of e. coli isolated from the infants' mucus immediately after delivery correlated with those subsequently recovered from their stools, supporting the contention that these organisms were acquired orally at the time of birth. in mothers whose stools contained the same 0 group as their offspring, the mean time from rupture of membranes to delivery was about 2 hours longer than in those whose infants did not acquire the same serogroups, suggesting that ascending colonization before birth also can play a role in determining the newborn's fecal flora. the contours of the epidemiologic curves in nurse$38264-269 and communi@38-240 outbreaks are in keeping with a contact mode of spread. transmission of organisms from infant to infant takes place by way of the fecal-oral route in almost all cases, most likely on the hands of persons attending to their care.86*267,269,270 ill infants represent the greatest risk to those around them because of the large numbers of organisms found in their stools271-274 and crossinfection also has been initiated by infants who were healthy at the time of their admission to the nursery.264,2723278-280 a newborn exposed to epec is likely to acquire enteric infection if contact with a person excreting the organism is intimate and prolonged, as in a hospital or family setting. stool culture surveys taken during outbreaks have shown that between 20% and 50% of term neonates residing in the nursery carry epec in their intestinal tracts.'02,230,231'm despite descriptions of nursery outbreaks in which virtually every neonate or low-birth-weight infant became infected,26232643281 there is ample evidence that exposure to pathogenic strains of e. coli does not necessarily result in greater likelihood of illness for premature infants than for term infants.261,272*279,282 any increased prevalence of cross-infections that may exist among premature infants can be explained more readily by the prolonged hospital stays, their increased handling, and the clustering of infants born in different institutions than by a particular susceptibility to epec based on immature defense mechanisms. the most extensive studies on the epidemiology of gastroenteritis related to e. coli have dealt with events that took place during outbreaks in newborn nurseries. unfortunately, investigations of this sort frequently regard the epidemic as an isolated phenomenon and ignore the strong interdependence that exists between community-and hospital-acquired ~~~~~~2 8 0 , 2 8 3 , 2 8 4 n ot surprisingly, the direction of spread is most often from the reservoir of disease within the community to the hospital. when the original source of a nursery outbreak can be established, frequently it is an infant born of a carrier mother who recently acquired her epec infection from a toddler living in the home. cross-infection epidemics also can be initiated by infected newborns who have been admitted directly into a clean nursery unit from the surrounding d i s t r i~t~~~~~~~*~~~ or have been transferred from a nearby hospital. 27832803286 after a nursery epidemic has begun, it generally follows one of two major patterns. some are explosive, with rapid involvement of all susceptible infants and a duration that seldom exceeds 2 or 3 months.264*265,276*287 the case-fatality rate in these epidemics may be very high. other nursery outbreaks have an insidious onset with a few mild, unrecognized cases; the patients may not even develop illness until after discharge from the hospital. during the next few days to weeks, neonates with an increased number of loose stools are reported by the nurses; shortly thereafter, the appearance of the first severely ill infants makes it apparent that an epidemic has begun. unless oral antimicrobial therapy is instituted (see "therapy"), nursery outbreaks like these may continue for months266-269 or with cycles of illness followed by periods of relative quiescence. this pattern can be caused by multiple strains (of different phage or antibiogram types) sequentially introduced into the nursery?78*288.289 the nursery can be a source of infection for the community. the release of infants who are in the incubation stages of their illness or are convalescent carriers about to relapse may lead to secondary cases of diarrheal disease among young siblings living in widely scattered areas.238,239,243 these children further disseminate infection to neighboring households, involving playmates of their own age, young infants, and mothers.23832393242 as the sickest of these contact cases are admitted to different hospitals, they contaminate new susceptible persons, completing the cycle and compounding the outbreak. this feedback mechanism has proved to be a means of spreading infantile gastroenteritis through entire ~o~n t i e s , 2~~~~~~,~~~ and even provinces.240 one major epidemic of diarrhea related to epec 01 11:b4 that occurred in the metropolitan chicago and northwestern indiana region during the winter of 1961 involved more than 1300 children and 29 community hospitals during a period of 9 months.240,291 almost all of the patients were younger than 2 years old, and 10% were younger than 1 month, producing an age-specific attack rate of nearly 4% of neonates in the community. the importance of the hospital as a source of cross-infection in this epidemic was demonstrated through interviews with patients' families, indicating that a minimum of 40% of infants had direct or indirect contact with a hospital shortly before the onset of their illness. it has been suggested, but not proved, that asymptomatic carriers of epec in close contact with a newborn infant, such as nursery personnel or family members, might play an important role in its t r a n s m i~s i o n .~~~'~"~~~ stool culture surveys have shown that at any one time about 1% of and 1% to 5% of young who are free of illness harbor epec strains. higher percentages have been recorded during community epidemics?38*239s243 be cause this intestinal carriage is transitory:383280 the number of individuals who excrete epec at one time or another during the year is far higher than the 1% figure recorded for single specimens.2803293 nursery personnel feed, bathe, and diaper a constantly changing population of newborns, about 2% to 5% of whom excrete epec.238*280 despite this constant exposure, intestinal carriage among nursery workers is surprisingly low. even during outbreaks of diarrheal illness, when dissemination of organisms is most intense, less than 5% of the hospital personnel in direct contact with infected neonates are themselves excreting pathogenic strains of e. coli.29',294,295 although adult asymptomatic carriers generally excrete fewer organisms than patients with acute illness large numbers of pathogenic bacteria may nevertheless exist in their stools?42s274 however, no nursery outbreak and few family cases24o have been traced to a symptomless carrier. instead, passive transfer of bacteria from infant to infant by the hands of personnel appears to be of primary importance in these outbreaks. cities,238.239,242 epec can be recovered from the throat or nose of 5% to 80% of infants with diarrheal illness275p2949295 and from about 1% of asymptomatic the throat and nasal mucosa may represent a portal of entry or a source of transmission for epec. environmental studies have shown that epec is distributed readily and widely in the vicinity of an infant with active diarrheal disease, often within 1 day of admission to the ~a r d . '~' , '~~ massive numbers of organisms are shed in the diarrheal stool or vomitus of infected e. coli organisms may survive 2 to 4 weeks in dust243.296 and can be found in the nursery air when the bedding or diapers of infected infants are disturbed during routine nursing procedure^^^^^^^ or on floors, walls, cupboards, and nursery equipment such as scales, hand towels, bassinets, incubators, and oxygen tents of other infant^?^,'^^,'^^ documentation of the presence of epec in nursery air and dust does not establish the importance of this route as a source of cross-infection. one study presented evidence of the respiratory transmission of epec; however, even in the cases described, the investigators pointed out that fecal-oral transmission could not be completely ruled additional clinical and experimental data are required to clarify the significance of droplet and environmental infection. coliform organisms have also been isolated in significant numbers from human mi1k,46~297~298 prebottled infant f0rmulas,2~~ and formulas prepared in the home.292 epec in particular has been found in stool cultures obtained from donors of human milk and workers in a nursery formula room.26o in one instance, epec 01 11:b4 was isolated from a donor, and subsequently, the same serogroup was recovered in massive amounts in almost pure culture from her milk.260 pathogenic strains of e. coli have also been isolated from raw cow's milk3'' and from drinking ~a t e r .~" likewise, epec has been isolated from flies during an epidemic, but this fact has not been shown to be of epidemiologic significance.211'220 infection of the newborn infant with epec takes place exclusively by the oral route. attempts to induce disease in adult volunteers by rectal instillation of infected material have been unsuc~essful.~~ there are no reports of disease occurring after transplacental invasion of the fetal bloodstream by enteropathogenic or nonenteropathogenic strains of e. coli. ascending intrauterine infection after prolonged rupture of the membranes has been reported only once; the neonate in this case suffered only from mild diarrhea.86 bacterial cultures of the meconium and feces of newborns indicate that enteropathogenic strains of e. coli can colonize effectively the intestinal tract in the first days of although e. coli may disappear completely from stools of breast-fed children during the ensuing weeks, this disappearance is believed to be related to factors present in the human milk rather than the gastric secretions.5~302~303 the use of breast-feeding or expressed human milk has even been effective in terminating nursery epidemics caused by epec 0 11 1:b4, probably by reducing the incidence of crossinfections among infants.3033304 although dose-effect studies have not been performed among newborns, severe diarrhea has occurred after ingestion of 10' epec organisms by very young the high incidence of cross-infection outbreaks in newborn nurseries suggests that a far lower inoculum can often effect spread in this setting. the role of circulating immunity in the prevention of gastrointestinal tract disease related to epec has not been clearly established. virtually 100% of maternal sera have been found to contain hemaggl~tinating,2'~~~''~~'~ or bacteriostatic2'0~3'2 antibodies against epec. the passive transfer of these antibodies across the placenta is extremely inefficient. titers in blood of newborn infants are, on average, 4 to 100 times lower than those in the corresponding maternal sera. group-specific hemagglutinating antibodies against the 0 antigen of epec are present in 10% to 20% of cord blood samp1es,219~307~308 whereas b a~t e r i c i d a l~'~~~~~ or bacterio-static3" activity against these organisms can be found much more frequently. tests for bacterial agglutination, which are relatively insensitive, are positive in only a small percentage of neonate^.'^^'^" the importance of circulating antibodies in the susceptibility of infants to epec infection is unknown. experiments with suckling mice have failed to demonstrate any effect of humoral immunity on the establishment or course of duration of intestinal colonization with e. coli 0127 in mothers or their infants.313 similar observations have been made in epidemiologic studies among premature human infants using enteropathogenic (01 27:b8)310 and nonenteropathogenic (04:h5)269 strains of e. coli as the indicator organisms. in a cohort of 63 mothers and their infants followed from birth to 3 months old, cooper and associate^'^ were able to show a far higher incidence of clinical epec disease in infants of epec-negative mothers than in infants born of mothers with epec isolated from stool cultures. this finding suggested to the investigators the possibility that mothers harboring epec in their gastrointestinal tracts transfer specific antibodies to their infants that confer some protection during the first weeks of life. protection against enteric infections in humans often correlates more closely with levels of local secretory than serum antibodies. although it is known that colonization of newborns with e. coli leads to the production of coproantibodies against the ingested the clinical significance of this intestinal immunity is uncertain. the previously mentioned experiment with mice showed no effect of active intestinal immunity on enteric col~nization.~'~ in human infants, the frequency of bacteriologic and clinical relapse related to epec of the same and the capacity of one strain of epec to superinfect a patient already harboring a different train^^^,^^^,^^^ also cast some doubt on the ability of mucosal antibodies to inhibit or alter the course of intestinal infection. studies of the protective effects of orally administered epec vaccines could help to resolve these question^.'^' the mechanism by which epec causes diarrhea involves a complex array of plasmid and chromosomally encoded traits. epec serotypes usually do not make one of the recognized enterotoxins (lt or st) as usually measured in tissue culture or animal r n~d e l s ,~'~~~'~ nor do these serotypes cause a typical invasive colitis or produce a positive sereny test only uncommonly do epec strains invade the bloodstream or disseminate.288 nevertheless, epec strains that test negative in these tests are capable of causing diarrhea; inocula of 10'' e. coli 0142 or 0127 organisms caused diarrhea in 8 of 10 adult volunteers.320 some epec strains may secrete weak enterot~xins,~~''~'~ but the consensus opinion is that the attaching and effacing lesion constitutes the critical secretory virulence pheno-clinical pathologic reports reveal the characteristic attachin and effacing lesion in the small intestine of infected infants? the lesion is manifested by intimate (about 10 nm) apposition of the epec to the enterocytes plasma membrane, with dissolution of the normal brush border and rearrangement of the cyto~keleton.'''~~~~ in some instances, the bacteria are observed to rise up on pedestal-like structures, which are diagnostic of the infection.i2' villus blunting, crypt hypertrophy, histiocytic infiltration in the lamina propria, and a reduction in the brush border enzymes may also be ~bserved.~'~'~'~ two major epec virulence factors have been described; strains with both factors are designated as typical epec.98*99*3'3 one such factor is the locus of enterocyte effacement (lee), a type 111 secretion system encoded by the lee chromosomal pathogenicity i~land.~'~-~'* the lee secretion apparatus injects proteins directly from the cytoplasm of the infecting bacterium into the cytoplasm of the target enter~cytes.~'~ the injected proteins constitute cytoskeletal toxins, which together elicit the close apposition of the bacterium to the cell, cause the effacement of microvilli, and most likely give rise to the net secretory one critical secreted protein, called towinterleukin-1 receptor (tir),"' inserts into the plasma membrane of the epithelial cell, where it serves as the receptor for a lee-encoded epec outer membrane protein called intimin.'" animals infected with attaching and effacin pathogens mount antibody responses to intimin and t i r ! 9 and both are considered potential immunogens. the lack of protection from epec reinfection suggests that natural antibody responses to tir and intimin are not protective. the second major virulence factor of typical epec is the bundle-forming pilus (bfp),330 which is encoded on a partially conserved 60 mda virulence plasmid called epec adherence factor bfp, a member of the type iv pilus family, mediates aggregation of the bacteria to each other and probably to enterocytes themselves, thereby facilitating mucosal colonization.332 a bfp mutant was shown to be attenuated in adult volunteers.333 the principal pathologic lesion with epec is the focal destructive adherence of the organism, effacing the microvillous brush border with villus blunting, crypt hypertrophy, histiocytic infiltration of the lamina propria, and reduced brush border enzymes. rothbaum and colleagues324 described similar findings with dissolution of the glycocalyx and flattened microvilli with the nontoxigenic epec strain 0119:b14. there has been a wide range of pathologic findings reported in infants dying of epec gastroenteritis. most newborns dying with diarrheal disease caused by epec show no morphologic changes of the gastrointestinal tract by gross or microscopic examination of tiss~es.~'~'~'' bra?" described such "meager" changes in the intestinal tract that "the impression received was that the term gastroenteritis is incorrect." at the other extreme, extensive and severe involvement of the intestinal tract, although distinctly unusual among neonates with epec diarrhea, has been discussed in several reviews of the pathologic anatomy of this disease.247v3 19,334 changes virtually identical to those found in infants dying with necrotizing enterocolitis have been reported.334 drucker and c o -~o r k e r s~'~ found that among 17 infants who were dying of epec diarrhea, "intestinal gangrene, and/or perforation, andlor peritonitis were present in five, and intestinal pneumatosis in five." the reasons for such wide discrepancies in epec disease pathology are not clear. the severity of intestinal lesions at the time of death does not correlate with the birth weight of the patient, the age of onset of illness, the serogroup of the infecting strain, or the prior administration of oral or systemic antimicrobial agents. the suggestion that the intensity of inflammatory changes may depend on the duration of the diarrhea3'' cannot be corroborated in autopsy s t~d i e s~'~*~"~~~ or small intestinal it is difficult to reconcile such a thesis with the observation that a wide range of intestinal findings can be seen at autopsy among newborns infected by a single serotype of epec during an epidemic. the nonspecific pathologic picture described by some researchers includes capillary congestion and edema of the bowel wall and an increase in the number of eosinophils, plasma cells, macrophages, and mononuclear cells in the mucosa and submucosa.262,319,335 villous patterns are generally well preserved, although some flattening and broadening of the villi are seen in the more severe cases. almost complete absence of villi and failure of regeneration of small bowel mucosa have been reported in an extreme case.338 edema in and around the myenteric plexuses of auerbach, a common associated finding, has been suggested as a cause of the gastrointestinal tract dilatation often seen at autopsy in infants with epec infection^.^^^'^^^'^^^ in general, the distal small intestine shows the most marked alterations; however, the reported pathologic findings may be found at all levels of the intestinal tract. several complications of epec infection have been reported. candidal esophagitis accounted for significant morbidity in two series collected before'" and the antibiotic era. oral thrush has been seen in 50% of epecinfected infants treated with oral or systemic antib i o t i c~. '~~,~~"~~ some degree of fatty metamorphosis of the liver has been reported by several investigators"0i2'5 335; however, these changes are nonspecific and probably result from the poor caloric intake associated with persistent diarrhea or vomiting. some degree of bronchopneumonia, probably a terminal event in most cases, exists in a large proportion of newborns dying of epec i n f e~t i o n .~" " '~'~~~ in one reported series of infant cases, epec was demonstrated by immunofluorescent staining in the bronchi, alveoli, and interalveolar septa. mesenteric lymph nodes are often swollen and congested with reactive germinal centers in the lymphoid f o l l i~l e s . 2 '~~~~, '~~ severe lymphoid depletion, unrelated to the duration or severity of the antecedent illness, also has been de~cribed.'~~ the kidneys frequently show tubular epithelial toxic changes. various degrees of tubular degeneration and cloudy swelling of convoluted tubules are common finding^.^'^,'^^,^^^ renal vein thrombosis or cortical necrosis may be observed in infants with disseminated intravascular coagulation in the terminal phases of the illness. the heart is grossly normal in most instances but may show minimal vacuolar changes of nonspecific toxic myocarditis on microscopic examinati0n.3~~'~~' candidal abscesses of the heart339 and kidneys'85,335,339 have been described. with the exception of mild congestion of the pia arachnoid vessels and some edema of the meninges, examination of the central nervous system reveals few changes?153262 despite the observation of braf l1 that "inflammation of the middle ear [is] exceptional," strains of epec have been isolated from a significant number of specimens of the middle ear in case series in which dissection of the temporal bone has been performed.2093215 exposure of newborns to epec may be followed by one of several possible consequences: no infection, infection without illness, illness with gastroenteritis of variable severity and duration, and rarely, septicemia with or without metastatic foci of infection accompanying gastroenteritis. when infants are exposed to epec, a significant number become colonized as temporary st00185,888231 or pharyngeal238 carriers with no signs of clinical disease. although l a~r e l l~~' showed that the percentage of asymptomatic infections rises steadily as age increases, this observation has not been confirmed by other investigator^.^'^.^^^ similarly, the suggestion that prematurity per se is associated with a low incidence of inapparent epec infection has been documented in several clinical but refuted in others.252,279 most neonates who acquire infection with epec eventually show some clinical evidence of gastroenteritis. the incubation period is quite variable. its duration has been calculated mostly from evidence in outbreaks in newborn nurseries, where the time of first exposure can be clearly defined in terms of birth or admission dates. in these circumstances, almost all infants show signs of illness between 2 and 12 days after exposure, and most cases show signs within the first 7 days.215,231,264 in some naturally and experi-menta1306 infections with heavy exposure, the incubation period may be as short as 24 hours; the stated upper limit is 20 days.2323343 the first positive stool culture and the earliest recognizable clinical signs of disease occur simultaneously in most although colonization may precede symptoms by 7 to 14 days.265,2663344 th e gastroenteritis associated with epec infection in the newborn is notable for its marked variation in clinical pattern. clinical manifestations vary from mild illness manifest only by transient anorexia and failure to gain weight to a sudden explosive fulminating diarrhea causing death within 12 hours of onset. prematurity, underlying disease, and congenital anomalies often are associated with the more severe forms of illness.214,233,345,346 experienced clinicians have observed that the severity of epec gastroenteritis has declined markedly during the past 3 decades.225 the onset of illness usually is insidious, with vague signs of reluctance to feed, lethargy, spitting up of formula, mild abdominal distention, or even weight loss that may occur for 1 or 2 days before the first loose stool is passed. diarrhea usually begins abruptly. it may be continuous and violent, or in milder infections, it may run an intermittent course with 1 or more days of normal stools followed by 1 or more days of diarrhea. emesis sometimes is a prominent and persistent early finding. stools are loose and bright yellow initially, later becoming watery, mucoid, and green. flecks or streaks of blood, which are commonly seen with enterocolitis caused by salmonella, campylobacter, or shigella, are rarely a feature of epec diarrheal disease. a characteristic seminal smell may pervade the environment of infants infected with epec 0 1 1 1:b34,232,262, 347 and an odor variously described as "pungent," "musty," or "fetid" often surrounds patients excreting other strains in their stool^.^^','^^ because the buttocks are repeatedly covered with liquid stools, excoriation of the perianal skin can be an early and persistent problem. fever is an inconstant feature, and when it occurs, the patient's temperature rarely rises above 39" c. convulsions occur infrequently; their occurrence should alert the clinician to the possible presence of electrolyte disturbances, particularly hypernatremia. prolonged hematochezia, distention, edema, and jaundice are ominous signs and suggest an unfavorable p r o g n o~i s .~'~,~~~*~*~ m ost infants receiving antimicrobial agents orally show a cessation of diarrhea, tolerate oral feedings, and resume weight gain within 3 to 7 days after therapy has been those with mild illness who receive no treatment can continue to have intermittent loose stools for 1 to 3 weeks. in one outbreak related to epec 0142:k86, more than one third of the untreated or inappropriately treated infants had diarrhea for more than 14 days in the absence of a recognized enteric pathogen on repeated culturing.267 recurrence of diarrhea and vomiting after a period of initial improvement is characteristic of epec e n t e r i t i~. '~'~~~~~~~ though seen most often in newborns who have been treated inadequately or not treated at all, clinical relapses also occur after appropriate therapy. occasionally, the signs of illness during a relapse can be more severe than those accompanying the initial attack of illness.215,232,285 not all clinical relapses result from persistent infection. a significant number of relapses, particularly those that consistently follow attempts at reinstitution of formula fee ding^?^^.^^^ are caused by disaccharide intolerance rather than bacterial proliferation. intestinal superinfections, caused by another serotype of epecz8393479348 or by completely different enteric pathogens, such as salmonella or shigella,245 also can delay the resolution of symptoms. rarely, infants suffer a "relapse" caused by an organism from the same 0 group as the original strain but differing in its h antigen. unless complete serotyping is performed on all epec isolates, such an event easily could be dismissed as being a recurrence rather than a superinfection with a new ~r g a n i s m . '~~*~~~ antimicrobial agents to which the infecting organisms are susceptible often may not eradicate epec:45,265,267 which may persist for weeks264,283,344 or months349 after the acute illness has subsided. although reinfection cannot always be excluded, a significant number of infants are discharged from the hospital with positive rectal dehydration is the most common and serious complication of gastroenteritis caused by epec or a toxin-producing e. coli. virtually all deaths directly attributable to the intestinal infection are caused by disturbances in fluids and electrolytes. when stools are frequent in number, large in volume, and violent in release, as they often are in severe infections with abrupt onset, a neonate can lose up to 15% of body weight in a few h o~r s .~~~,~~~ rarely, fluid excretion into the lumen of the bowel proceeds so rapidly that reduction of circulating blood volume and shock may intervene before passage of even a single loose before the discovery of the etiologic agent, epidemic diarrhea of the newborn was also known by the term cholera infantum. mild disease, particularly when aggravated by poor fluid intake, can lead to a subtle but serious deterioration of an infant's metabolic status. sometimes, a week or more of illness elapses before it becomes apparent that an infant with borderline acidosis and dehydration who seemed to be responding to oral fluids alone requires parenteral therapy for impr~vement?~~ it is incumbent on the clinician caring for small infants with gastroenteritis to follow them closely, with particular attention to serial weights, until full recovery can be confirmed. there are few other complications, with the possible exception of aspiration pneumonia, directly related to epec gastroenteritis. protracted diarrhea and nutritional failure may occur as a consequence of functional damage to the small intestinal mucosa, with secondary intolerance to dietary necrotizing enterocolitis, which occasionally results in perforation of the bowel and peritonitis, has not been causally related to infection with epec.247,264,266 a review of most of the large clinical series describing epec disease in infants who ranged in age from neonates to children aged 2 years revealed only three proven instances of ba~teremia:~~**~~ one possible urinary tract infection:65 and one documented case of meningitis in an infant of unspecified age.351 focal infections among neonates were limited to several cases of otitis and a subcutaneous abscess294 from which epec was isolated. additional complications include interstitial pneumonia,319 gastrointestinal bleeding with or without disseminated intravascular coagulatio11,3~.~~~ and methemoglobinemia caused by a mutant of epec 0127:b8 that was capable of generating large quantities of nitrite from proteins present in the gastrointestinal tract.353 the gold standard of epec diagnostics is identification in the stool of e. coli carrying genes for bfp and lee. identification of these genes can be accomplished by molecular methods (discussed later), but lack of access to these methods has led many labs to rely on surrogate markers, such as serotyping." classic epec has been recovered from the vomitus, stool, or bowel contents of infected newborns. isolation from bile233 and the upper respiratory t r a~t~~~~~~*~~~ ha s been described in those instances in which a specific search has been made. less commonly, epec is isolated from ascetic fluid'" or purulent exudates209*2157294* , occasionally, the organism has been recovered from blood c u l t~r e s ?~~,~~~ urine:65 and cerebrospinal fluid. stool cultures generally are more reliable than rectal swabs in detecting the presence of enteric pathogens, although a properly obtained swab should be adequate to demonstrate epec in most cases.2'7,296*354 specimens should be obtained as early in the course of the illness as possible because organisms are present in virtually pure culture during the acute phase of the enteritis but diminish in numbers during convalescence. because of the preponderance of epec in diarrheal stools, two cultures are adequate for isolation of these pathogens in almost all cases of active disease. studies using fluorescent antibody methods for identification of epec in stool specimens have demonstrated that during the incubation period of the illness, during convalescence, and among asymptomatic carriers of epec, organisms can be excreted in such small numbers that they escape detection by standard bacteriologic methods in a significant proportion of as many as 3 to 10 specimens may be required to detect epec using methods that identify individual epec isolates in the ~t 0 0 1 . 8~~~ after a stool specimen is received, it should be plated as quickly as possible onto noninhibiting media or placed in a preservative medium if it is to be held for longer periods. deep freezing of specimens preserves viable epec when a prolonged delay in isolation is necessary?" no selective media, biochemical reactions, or colonial variations permit differentiation of pathogenic and nonpathogenic strains. certain features may aid in the recognition of two important serogroups. cultures of serogroups 0 1 11:b4 and 055:b5, unlike many other coliforms, are sticky or stringy when picked with a wire loop and are rarely hemolytic on blood whereas 0 1 11:b4 colonies emit a distinctive evanescent odor commonly described as "~e m i n a l . ' '~~~,~~~ this unusual odor first led b r a y to suspect that specific strains of e. coli might be responsible for infantile gastroenteritis. because serotyping is simpler than molecular detection and because epec have long been known to belong to certain highly characteristic serotypes, serotyping can be used to identify likely epec strains, especially in outbreaks?16 e. coli, like other enterobacteriaceae members, possesses cell wall somatic antigens (o), envelope or capsular antigens (k), and if motile, flagellar antigens (h). many of the 0 groups may be further divided into two or more subgroups (a, b, c), and the k antigens are divisible into at least three varieties (b, l, a) on the basis of their physical behavior. organisms that do not possess flagellar antigens are nonmotile (designated nm). the epec b capsular surface antigen prevents agglutination by antibodies directed against the underlying 0 antigen. heating at 100°c for 1 hour inactivates the agglutinability and antigenicity of the b antigen. slide agglutination tests with polyvalent 0 or ob antiserum may be performed on suspensions of colonies typical of e. coli that have been isolated from infants with diarrhea, especially in nursery outbreaks. however, because of numerous false-positive "cross-reactions:' the 0 and k (or b) type must be confirmed by titration with the specific a n t i~e r a .~~~ the presence of epec does not prove that epec is the cause of diarrhea in an individual patient. mixed cultures with two or three serotypes of epec have been demonstrated in 1% to 10% of patients.244*245*352 this need not mean that two or three serotypes are causative agents. secondary infection with hospital-acquired strains can occur during convalesand some infants may have been asymptomatic carriers of one serotype at the time that another produced diarrheal disease. a similar explanation may pertain to mixed infections with epec and salmonella or shigella.217*2203358 nelson245 reported the presence of these pathogens in combination with epec in 14% of infants who were cultured as part of an antibiotic therapy trial. salmonella and shigella that had not been identified on cultures obtained at admission were isolated only after institution of oral therapy with neomycin. the investigator postulated that the alteration in bowel flora brought about by the neomycin facilitated the growth of these organisms, which had previously been suppressed and obscured by coliform overthe importance of seeking all enteric pathogens in primary and follow-up cultures of infantile diarrhea is apparent, particularly when the specimen originates from a patient in a newborn nursery or infants' ward. although epec gastroenteritis was once considered to be synonymous with "summer diarrhea," community outbreaks have occurred as frequently, if not more frequently, in the colder seasons.133,160*172 it has been suggested that the increased incidence at that time of year might be related to the heightened chance of contact between infants and toddlers cence,l 73,281,283,380 that is bound to occur when children remain indoors in close contact.z94 nursery epidemics, which depend on the chance introduction and dissemination of epec within a relatively homogeneous population and stable environment, demonstrate no seasonal prevalence. average relative humidity, temperature, and hours of daylight have no significant effect in determining whether an outbreak will follow the introduction of enteropathogenic strains of e. coli into a ward of infants.243 there are no clinical studies of the variations in peripheral leukocyte count, urine, or cerebrospinal fluid in neonatal enteritis caused by epec. microscopic examination of stools of infants with acute diarrheal illness caused by these organisms usually has revealed an absence of fecal polymorphonuclear l e~k o c y t e s~'~~~~~~~~~*~~~ although data on fecal lactoferrin in human volunteers suggest that an inflammatory process may be important in epec diarrhea.360,361 stool ph can be neutral, acid, or alkaline.617341 serologic methods have not proved to be useful in attempting to establish a retrospective diagnosis of epec infection in neonates. rising or significantly elevated agglutinin titers rarely could be demonstrated in early investigation^^'^"'^^^'; hemagglutinating antibodies showed a significant response in no more than 10% to 20% of cases.245,297 fluorescent antibody techniques have shown promise for preliminary identification of epec in acute infantile diarrhea. this method is specific, with few false-positive results, and it is more sensitive than conventional plating and isolation t e c h n i q~e s .~~~,~~' .~~~ the rapidity with which determinations can be performed makes them ideally suited for screening ill infants and possible carriers in determining the extent and progression of a n~r s e r f '~,~~~ or om mu nit$^^^^'' outbreak. because immunofluorescence does not depend on the viability of organisms and is not affected by antibiotics that suppress growth on culture plates, it can be used to advantage in following bacteriologic responses and relapses in patients receiving oral the rap^.^","^ the use of fluorescent antibody techniques offers many advantages in the surveillance and epidemiologic control of epec gastroenteritis. immunofluorescent methods should supplement but not replace standard bacteriologic and serologic methods for identification of enteric pathogens. specific gene probes and pcr primers for the bfp adhesin, the intimin-encoding gene (eue) and for a cryptic plasmid locus (eaf) are a~ailable.~~ detection of bfp or eaf are superior to detection of eue, because many non-epec, including nonpathogens, carry the eae gene.98b364 pcr and gene probe analysis can be performed directly on the stools of suspect infants. however, confirmation of infection by the identification of the organism in pure culture should be pursued. before widespread use of molecular methods, the hep-2 cell adherence assay was proposed for epec diagnosis."' the presence of a focal or localized adherence (la) pattern on the surface of hep-2 or hela cells after 3-hour coincubation is a highly sensitive and specific test for detection of epec. 365 the requirement for cell culture and expertise in reading this assay limits its utility to the research setting. an elisa for the bfp has been described but is not readily available?66 the capacity of la + epec to polymerize f-actin can be detected in tissue culture cells stained with rhodamine-labeled phall~idin.~~' this fluorescence-actin staining (fas) test is cumbersome and impractical for routine clinical use. the mortality rate recorded previously in epidemics of epec gastroenteritis is impressive for its variability. during the 1930s and 1940s, when organisms later recognized as classic enteropathogenic serotypes were infecting infants, the case-fatality ratio among neonates was about 50%. 2099210 during the 1950s and 1960s, many nursery epidemics still claimed about one of every four infected infants, but several outbreaks involving the same serotypes under similar epidemiologic circumstances had fatality rates of less than 30h.23424131 in th e 1970s, reports appeared in the literature of a nursery epidemic with a 40% neonatal mortality rate285 and of an extensive outbreak in a nursery for premature infants with 4% fatalities265; another report stated that among "243 consecutive infants admitted to the hospital for epec diarrheal disease, none died of diarrheal disease per se."368 a significant proportion of the infants who died during or shortly after an episode of gastroenteritis already were compromised by preexisting disease233,283,330 or by congenital m a l f~r m a t i o n s~'~,~~'~~~~ at the time they acquired their illness. these underlying pathologic conditions appear to exert a strongly unfavorable influence, probably by reducing the infant's ability to respond to the added stresses imposed by the gastrointestinal tract infection. although prematurity is often mentioned as a factor predisposing to a fatal outcome, the overall mortality rate among premature infants with epec gastroenteritis has not differed significantly over the years from that recorded for term the management of epec gastroenteritis should be directed primarily toward prevention or correction of problems caused by loss of fluids and electr01ytes.i~~ most neonates have a relatively mild illness that can be treated with oral rehydration. infants who appear toxic, those with voluminous diarrhea and persistent vomiting, and those with increasing weight loss should be hospitalized for observation and treatment with parenteral fluids and careful maintenance of fluid and electrolyte balance and possibly with antimicrobial therapy. clinical studies suggest that slow nasogastric infusion of an elemental diet can be valuable in treating infants who have intractable diarrhea that is unresponsive to standard modes of therapy. 369 there is no evidence that the use of proprietary formulas containing kaolin or pectin is effective in reducing the number of diarrheal stools in neonates with gastroenteritis. attempts to suppress the growth of enteric pathogens by feeding lactobacillus to the infant in the form of yogurt, powder, or granules have not been shown to be of value.370 a trial of cholestyramine in 15 newborns with epec gastroenteritis had no effect on the duration or severity of the diarrhea.265 the use of atropine-like drugs, paregoric, or loperamide to reduce intestinal motility or cramping should be avoided. inhibition of peristalsis interferes with an efficient protective mechanism designed to rid the body of intestinal pathogens and may lead to fluid retention in the lumen of the bowel that may be sufficient to mask depletion of extracellular fluid and electrolytes. the value of antimicrobial therapy in management of neonatal epec gastroenteritis, if any, is uncertain. there are no adequately controlled studies defining the benefits of any antibiotic in eliminating epec from the gastrointestinal tract, reducing the risk of cross-infection in community or nursery outbreaks, or modifymg the severity of the illness. proponents of the use of antimicrobial agents have based their claims for efficacy on anecdotal observations or comparative studies.245 nonetheless, several clinical investigations have provided sufficient information to guide the physician faced with the dilemma of deciding whether to treat an individual infant or an entire nursery population suffering from epec diarrheal disease. it should be emphasized, however, that these guidelines must be considered tentative until rigidly controlled, double-blind studies have established the efficacy of antibiotics on a more rational and scientific basis. oral therapy with n e o m y~i n ,~'~'~~' ~olistin,'~~or chloram-phenic01~~~ appears to be effective in rapidly reducing the number of susceptible epec organisms in the stool of infected infants. studies comparing the responses of infants treated orally with ne~mycin?~' gentamicin:65 p~l p y x i n :~~ or kanamy~in'~' with the responses of infants receiving supportive therapy alone have shown that complete eradication of epec occurs more rapidly in those receiving an antimicrobial agent. in most cases, stool cultures are free of epec 2 to 4 days after the start of therapy.2459363 bacteriologic failure, defined as continued isolation of organisms during or after a course of an antimicrobial agent, can be expected to occur in 15% to 30% of patients?45s265 such relapses generally are not associated with a recurrence of ~y m p t~m~.~~i *~~~*~~~ the effectiveness of oral antimicrobial therapy in reducing the duration of epec excretion serves to diminish environmental contamination and the spread of pathogenic organisms from one infant to another. breaking the chain of fecal-oral transmission by administering antimicrobial agents simultaneously to all carriers of epec and their immediate contacts in the nursery has appeared to be valuable in terminating outbreaks that have failed to respond to more conservative m e a s~r e s .~'~,~~,~~~ the apparent reduction in morbidity and mortality associated with oral administration of neomycin,230.233,234 colistin,246.267.285 p o l y m y x i r~,~~~ or gentamicin2& during nursery epidemics has led to the impression that these drugs also exert a beneficial clinical effect in severely or moderately ill infants. reports describing bacteriologic:65 or histopathol~gic~'~ evidence of tissue invasion by epec have persuaded some investigators to suggest the use of parenteral rather than oral drug therapy in debilitated or malnourished infants. on the basis of these data, there appears to be sufficient evidence to recommend oral administration of nonabsorbable antibiotics in the treatment of severely or moderately ill newborns with epec gastroenteritis. the drug most frequently used for initial therapy is neomycin sulfate in a dosage of 100 mg/kg/day administered orally every 8 hours in three divided doses.24s in communities in which neomycin-resistant epec has been prevalent, treatment with colistin sulfate or polymyxin b in a dosage of 15 to 20 mglkglday orally and divided into three equal doses may be appropriate. however, it is rarely necessary to use this approach. treatment should be continued only until stool cultures become negative for epec.245 because of the unavoidable delay before cultures can be reported, most infants receive therapy for 3 to 5 days. if fluorescent antibody testing of rectal swab specimens is available, therapy can be discontinued as soon as epec no longer is identified in smears; this takes no more than 48 hours in more than 90% of cases.245 after diarrhea and vomiting have stopped and the infant tolerates formula feedings, shows a steady weight gain, and appears clinically well, discharge with outpatient follow-up is indicated. bacteriologic relapses do not require therapy unless they are associated with illness or high epidemiologic risks to other young infants in the household. because the infecting organisms in these recurrences generally continue to show in vitro susceptibility to the original drug, it should be reinstituted pending bacteriologic re~ults.2~~ when clinical judgment suggests that a neonate may be suffering from bacterial sepsis and epec diarrheal disease, parenteral antimicrobial therapy is indicated after appropriate cultures have been obtained. the routine use of systemic therapy in severe cases of epec enteritis is not appropriate on the basis of current clinical experience. antimicrobial susceptibility patterns of epec are an important determinant of the success of therapy in infections with these organism^.^^',^^'^^^ these patterns are unpredictable, depending on the ecologic pressures exerted by local antibiotic and on the incidence of transmissible resistance factors in the enteric flora of the particular population served by an i n s t i t~t i o n .~~~"~~ for these reasons, variations in susceptibility patterns are apparent in different n~r s e r i e s~~~, '~~ and even from time to time within the same institution.247,248,250 sudden changes in clinical response may even occur during the course of a single epidemic as drugsusceptible strains of epec are replaced by strains with multidrug r e~i s t a n c e .~~~'~~' ,~~' because differences can exist in the susceptibilities of different epec serogroups to various antimicrobial agents, regional susceptibility patterns should be reported on the basis of ob group or serotype rather than for epec as a whole.250 knowledge of the resistance pattern in one's area may help in the initial choice of antimicrobial therapy. the prevention of hospital outbreaks of epec gastroenteritis is best accomplished by careful attention to infection control policies for a nursery. all infants hospitalized with diarrhea should have a bacteriologic evaluation. if the laboratory is equipped and staffed to perform fluorescent antibody testing, infants transferred from another institution to a newborn, premature, or intensive care nursery and all infants with gastroenteritis on admission during an outbreak of epec diarrhea or in a highly endemic area can be held in an observation area for 1 or 2 hours until the results of the fluorescent antibody test or pcr are received. because of the difficulty in diagnosing epec infection, reference laboratories, such as those at the centers for disease control and prevention (cdc), should be notified when an outbreak is suspected. infants suspected to be excreting epec, even if healthy in appearance, then can be separated from others and given oral therapy until the test results are negative. some experts have suggested that when the rapid results obtainable with fluorescent antibody procedures are not available, all infants admitted with diarrhea in a setting where epec is common may be treated as if they were excreting epec or some other enteric pathogen until contrary proof is obtained.372 stool cultures should be obtained at admission, and contact precautions should be enforced among all who come into contact with the infant. additional epidemiologic studies are needed to establish the advantages of careful isolation and nursing techniques, particularly in smaller community hospitals in which the number of infants in a "gastroenteritis ward may be small. the use of prophylactic antibiotics has been shown to be of no value and can select for increased r e~i s t a n c e .~~~"~~ unfortunately, it can be difficult to keep a nursery continuously free of epec. specific procedures have been suggested for handling a suspected outbreak of bacterial enteritis in a newborn nursery or infant care ~n i t .~~~l~~~*~~~ evidence indicating that a significant proportion of e. coli enteritis may be caused by nontypeable strains has required some modification of these earlier recommendations. the following infection control measures may be appropriate: 1. the unit is closed, when possible, to all new admissions. 2. cultures for enteric pathogens are obtained from nursing personnel assigned to the unit at the time of the outbreak. 3. stool specimens obtained from all infants in the nursery can be screened by the fluorescent antibody or another technique and cultured. identification of a classic enteropathogenic serotype provides a useful epidemiologic marker; however, failure to isolate one of these strains does not eliminate the possibility of illness caused by a nontypeable epec. 4. antimicrobial therapy with oral neomycin or colistin can be considered for all infants with a positive fluorescent antibody test or culture result. the initial drug of choice depends on local patterns of susceptibility. depending on the results of susceptibility tests, subsequent therapy may require modification. 5. if an identifiable epec strain is isolated, second and third stool specimens from all infants in the unit are reexamined by the fluorescent antibody technique or culture at 48-hour intervals. if this is not practical, exposed infants should be carefully followed. 6. early discharge for healthy, mature, uninfected infants is advocated. 7. an epidemiologic investigation should be performed to seek the factor or factors responsible for the outbreak. a surveillance system may be established for all those in contact with the nursery, including physicians and other health care personnel, housekeeping personnel, and postpartum mothers with evidence of enteric disease. a telephone, mail, or home survey may be conducted on all infants who were residing in the involved unit during the 2 weeks before the outbreak. 8. when all patients and contacts are discharged and control of the outbreak is achieved, a thorough terminal disinfection of the involved nursery is mandatory. above all, personnel and parents should pay scrupulous attention to hand hygiene when handling infants.38' since a multistate outbreak of enterohemorrhagic colitis was associated with e. coli 0157:h7,382 shiga toxin-producing e. coli (stec) have been recognized as emerging gastrointestinal pathogens in most of the industrialized world. a particularly virulent subset of stec, ehec, causes frequent and severe outbreaks of gastrointestinal the most virulent ehec belong to serotype 0157:h7. ehec has a bovine reservoir and is transmitted by undercooked meat, unpasteurized milk, and contaminated vegetables such as lettuce, alfalfa sprouts, and radish sprouts (as occurred in more than 9000 schoolchildren in japan).3849387 it also spreads directly from person to the clinical syndrome is that of bloody, noninflammatory (sometimes voluminous) diarrhea that is distinct from febrile dysentery with fecal leukocytes seen in shigellosis or eiec infection^.^^ most cases of ehec infections have been recognized in outbreaks of bloody diarrhea or hus in daycare centers, schools, nursing homes, and c o m m~n i t i e s .~~~-~~~ although ehec infections often involve infants and young children, the frequency of this infection in neonates remains unclear; animal studies suggest that receptors for the shiga toxin may be developmentally regulated and that susceptibility to disease may be age related. 391 the capacity of ehec to cause disease is related to the phage-encoded capacity of the organism to produce a vero cell cytotoxin, subsequently shown to be one of the shiga toxins.392-394 shiga toxin 1 is neutralized by antiserum against shiga toxin, whereas shiga toxin 2, although biologically similar, is not neutralized by anti-shiga toxin.395,396 like shiga toxin made by shigella dysenteriae, both e. coli shiga toxins act by inhibiting protein synthesis by cleaving an adenosine residue from position 4324 in the 28s ribosomal rna (rrna) to prevent elongation factor-1-dependent aminoacyl transfer rna (trna) from binding to the 60s rrna.3923393 the virulence of ehec also may be determined in part by a 60-mda plasmid that encodes for a fimbrial adhesin in 0157 and 026.397,398 this phenotype is mediated by the lee pathogenicity island, which is highly homologous to the island present in epec strains. 328 ehec and other stec infections should be suspected in neonates who have bloody diarrhea or who may have been exposed in the course of an outbreak among older individuals. because most cases are caused by ingestion of contaminated food, neonates have a degree of epidemiologic protection from the illness. diagnosis of stec diarrhea is made by isolation and identification of the pathogen in the feces. e. coli 0157:h7 does not ferment sorbitol, and this biochemical trait is commonly used in the detection of this s e r~t y p e .~~. '~~ because some nonpathogenic e. coli share this characteristic, confirmation of the serotype by slide agglutination is required. these techniques can be performed in most clinical laboratories. however, detection of non-0 157 serotypes is problematic and relies on detection of the shiga toxin; available methods include shiga toxin elisa, latex agglutination, and molecular method^.^^,^^^ these should be performed by a reference laboratory. hus in infants is not necessarily caused by stec infection. even in older patients, however, the stool is typically negative for stec at the time the that hus develops.400340' serum and fecal detection of cytotoxin has been performed in such patients, but no diagnostic modality is definitive once hus has s~pervened!~~,~~~ antimicrobial therapy should not be administered to patients who may have stec infection, although their role in inducing hus remains c o n t r o~e r s i a l .~~~'~~~ management of the diarrhea and possible sequelae is supportive, with proper emphasis on fluid and electrolyte replacement. aggressive rehydration is helpful in minimizing the frequency of serious sequelae. the hep-2 adherence assay is useful for the detection of epec, which exhibit a classic la pattern."' two other adherence patterns can be discerned in this assay: aggregative (aa) and diffuse (da). these two patterns have been suggested to define additional pathotypes of diarrheogenic e. coli." strains exhibiting the aa pattern (i.e., eaec) are common pathogens of infants.lz5 eaec cause diarrhea by colonization of the intestinal mucosa and elaboration of enterotoxins and c y t o t o~i n s .~~~~~ many strains can be shown to elicit secretion of inflammatory cytokines in vitro, which may contribute to growth retardation associated with prolonged otherwise asymptomatic colonization.io3 several virulence factors in eaec are under the control of the virulence gene activator aggr.404 presence of the aggr regulator or its effector genes has been proposed as a means of detecting truly virulent eaec strains (called typical eaec),404,405 and an empirical gene probe long used for eaec detection has been shown to correspond to one gene under aggr the mode of transmission of eaec has not been well established. in adult volunteer studies, the infectious dose is high (> lo8 colony-forming units [ cfu] ), suggesting that in adults at least, person-to-person transmission is unlikely.408.m several outbreaks have been linked to consumption of contaminated f~o d . "~~,~'~ the largest of these outbreaks involved almost 2700 schoolchildren in japan4"; a contaminated school lunch was the implicated source of the outbreak. some studies have demonstrated contamination of condiments or milk, which could represent vehicles of foodborne transmission. several nursery outbreaks of eaec have been 0bserved,4~'~~'~ although in no case has the mechanism of transmission been established. the fist reported nursery outbreak involved 19 infants in nis, serbia, in 1995. because these infants did not ingest milk from a common source, it is presumed that horizontal transmission by environmental contamination or hands of health care personnel was possible. most of the infants were full term and previously well, and they were housed in two separate nursery rooms. the earliest epidemiologic studies of eaec implicated this organism as a cause of endemic diarrhea in developing c o~n t r i e s .~'~-~'~ in this setting, eaec as defined by the m pattern of adherence to hep-2 cells can be found in upward of 30% of the population at any one time>l7 newer molecular diagnostic modalities have revised this figure downward, although the organism remains highly prevalent in many areas. several studies from the indian subcontinent implicated eaec among the most frequent enteric pathogen^.^'^.^'^.^^^ other sites reproducibly reporting high incidence rates include and bra~il."~'*~~' there is evidence that eaec may be emerging in incidence. a study from spo paulo, brazil, implicated eaec as the prevalent e. coli pathotypes in infants4i9; epec had previously been shown to be the most common pathogen in this community. many other sites in developing countries of africa:" asia,4°5~422 and south america4" have described high endemic rates. several studies have suggested that eaec is also a common cause of infant diarrhea in industrialized c~u n t r i e s . "~~~~~~ using molecular diagnostic methods, a large prospective study in the united kingdom implicated eaec as the second most common enteric bacterial pathogen after cumpylob~cter.~~~ a similar study from switzerland found eaec to be the most common bacterial enter~pathogen.~'~ studies from the united states also have demonstrated a high rate of eaec diarrhea in infants; using molecular diagnostic methods, eaec was implicated in 11% and 8% of outpatient and inpatient diarrhea cohorts, respectively, compared with less than 2% of asymptomatic control infants (p < .05). 427 although epidemiologic studies have shown that eaec can cause diarrhea in all age groups, several studies suggest that the infection is particularly common in infants younger than 12 months 01d.405*420 descriptions from outbreaks and volunteer studies suggest that eaec diarrhea is watery in character with mucus but without blood or frank pus.4o834o93412 patients typically are afebrile. several epidemiologic studies have suggested that many infants may have bloody diarrhea,4i6 but fecal leukocytes are uncommon. the earliest reports of eaec infection suggested that this pathogen may be particularly associated with persistent diarrhea (>14 days).414-416 however, later studies suggest that persistent diarrhea may occur in only a subset of infected infants!" in the serbian outbreak of 19 infected infants, the mean duration of diarrhea was 5.2 days4''; diarrhea persisted more than 14 days in only three patients. infants in this outbreak had frequent, green, odorless stools. in three cases, the stools had mucus, but none had visible blood. eleven babies developed temperatures in excess of 38oc; only one had vomiting. despite a lack of clinical evidence suggesting inflammatory enteritis, several clinical studies have suggested that eaec is associated with subclinical inflammation, including the shedding of fecal cytokines and la~toferrin.'~~.~'~ studies in fortaleza, brazil, suggest that children asymptomatically excreting eaec may exhibit growth shortfalls compared with uninfected peers.lo3 a study from germany reported an association between eaec isolation and infant colic in infants without diarrhea.4z5 this observation has not been repeated. eaec should be considered in the differential diagnosis of persistent diarrhea and failure to thrive in infants. diagnosis of eaec requires identification of the organism in the patient's feces. the hep-2 adherence assay can be used for this purpose"'; some reports suggest that the adherence phenotype can be observed using formalin-fixed cell^^^'^^^' thereby obviating the need to cultivate eukaryotic cells for each assay. pcr and gene probe for typical eaec are available. successful antibiotic therapy has been reported using fluoroquinolones in adult although preliminary studies suggest that a~ithrornycin~~~ or r i f a~i m i n~~~ also may be effective. therapy in infected infants should be guided by the results of susceptibility testing, as eaec frequently is antibiotic re~istant.~" additional e. coli pathotypes have been described, including diffusely adherent e. coli (daec), 434 and cytodetaching e. c 0 1 i .~~~ daec has been specifically associated with diarrhea outside of infancy, as infants may have some degree of inherent resistance to infection. 436 cytodetaching e. coli represent organisms that secrete the e. coli hem~lysin.~~' it is not clear whether these latter organisms are true enteric pathogens. there are differences in invasiveness of salmonella strains related to serotype. s. typhi, s. choleraesuis, salmonella heidelberg,439p440 and salmonella dub1inm1 are particularly invasive, with bacteremia and extraintestinal focal infections occurring frequently. salmonella species possess genes closely related to those for the shigella invasion plasmid anti ensthese genes are probably essential to intestinal infection. virulence plasmids, which increase invasiveness in some serotypes, have been recognized, although the precise mechanisms of virulence remain to be elucidated; resistance to complement-mediated bacteriolysis by inhibition of insertion of the terminal c5b-9 membrane attack complex into the outer membrane may be laboratory studies have demonstrated dramatic strain-related difference in the ability of s. typhimurium t o evoke fluid secretion, to invade intestinal mucosa, and to disseminate beyond the production of an enterotoxin immunologically related to cholera toxin by about two thirds of salmonella strains may be related to the watery diarrhea often seen. 447 part because of the properties of their lipopolysaccharide~~~~~~~~ persistence of the organism within phagolysosomes of phagocytic cells may occur with any species of salmonella. it is not completely clear how the organisms have adapted to survive in the harsh intracellular environment, but their survival has major clinical significance. it accounts for relapses after therapy. it explains the inadequacy of some antimicrobial agents that do not penetrate phagolysosomes. it perhaps is the reason for prolonged febrile courses that occur even in the face of appropriate therapy. although humoral immunity and cell-mediated immunity are stimulated during salmonella infections, it is believed that cell-mediated immunity plays a greater role in eradication of the ba~teria.4'~ t cell activation of macrophages appears to be important in killing intracellular salmonella. 454 defective interferon-y production by monocytes of newborns in response to s. typhimurium lipopolysaccharide may explain in part the unusual susceptibility of infants to salmonella infection. 455 studies in mice suggest that helper t cell (th1) responses in peyer's patches and mesenteric lymph nodes may be central to protection of the intestinal m~c o s a .~~~ humans who lack the il-12 receptor and therefore have impaired th1 responses and interferon-y production are at increased risk for salmonella infe~tion.~~' in typhoid fever, presence of an envelope antigen, vi, is known to enhance virulence. patients who develop classic enteric fever have positive stool cultures in the first few days after ingestion of the organism and again late in the course after a period of bacteremia. this course reflects early colonization of the gut, penetration of gut epithelium with infection of mesenteric lymph nodes, and reseeding of the gut during a subsequent bacteremic pha~e.4~' studies of s. typhimurium in monkeys suggest similar initial steps in pathogenesis (e.g., colonization of gut, penetration of gut epithelium, infection of mesenteric lymph nodes) but failure of the organism to cause a detectable level of ba~teremia.4~~ although both salmonella and shigella invade intestinal mucosa, the resultant pathologic changes are different. shigella multiplies within and kills enterocytes with production of ulcerations and a brisk inflammatory response, whereas salmonella passes through the mucosa and multiplies within the lamina propria, where the organisms are ingested by phagocytes; consequently, ulcer formation is less striking,446 although villus tip cells are sometimes sloughed. acute crypt abscesses can be seen in the stomach and small intestine, but the most dramatic changes occur in the colon, where acute diffuse inflammation with mucosal edema and crypt abscesses are the most consistent findings.460v461 with s. typhi there also is hyperplasia of peyer's patches in the ileum, with ulceration of overlying tissues. salmonella strains, with the exception of s. typhi, are well adapted to a variety of animal hosts; human infection often can be traced to infected meat, contaminated milk, or contact with a specific animal. half of commercial poultry samples are contaminated with salmonella. 462 definition of the serotype causing infection can sometimes suggest the likely source. for example, s. dublin is closely associated with cattle; human cases occur with a higher-than-predicted frequency in people who drink raw milk.@' for s. typhimurium, which is the most common serotype and accounts for more than one third of all reported human cases, a single source has not been established, although there is an association with cattle. despite the 1975 ban by the u.s. food and drug administration (fda) on interstate commercial distribution of small turtles, these animals continue to be associated with infection, as illustrated by a series of cases in puerto ~i~~.~~~ various pet reptiles are an important source of a variety of unusual salmonella serotypes such as salmonella marina, salmonella chameleon, salmonella arizonae, salmonella java, salmonella stanley, salmonella poona, salmonella jangwain, salmonella tilene, salmonella pomona, salmonella miami, salmonella manhattan, salmonella litchfield, salmonella rubislaw, and salmonella w a~s e n a a r .~~-~~ salmonella organisms are hardy and capable of prolonged survival; organisms have been documented to survive in flour for nearly a year?67 salmonella tennessee has been shown to remain viable for many hours on non-nutritive surfaces (i.e., glass, 48 hours; stainless steel, 68 hours; enameled surface, 114 hours; rubber mattress, 119 hours; linen, 192 hours; and rubber tabletop, 192 infection with salmonella is, like most enteric infections, more common in young children than in adults. the frequency of infection is far greater in the first 4 years of life; roughly equal numbers of cases are reported during each decade beyond 4 years of age. although the peak incidence occurs in the second through sixth months of life, infection in the neonate is relatively common. researchers at the cdc have estimated the incidence of salmonella infection in the first month of life at nearly 75 cases per 100,000 infants?69 adult volunteer studies suggest that large numbers of salmonella ( lo5 to lo9) need to be ingested to cause di~ease.4~' however, it is likely that lower doses cause illness in infants. the occurrence of nursery ~u t b r e a k s~"~~~~~ and intrafamilial spread497 suggests that organisms are easily spread from person to person; this pattern is typical of low-inoculum diseases transmitted by the fecal-oral route. the neonate with salmonella infection infrequently acquires the organism from his or her mother during delivery. although the index case in an outbreak can often be traced to a mother,474-477,495 subsequent cases result from contaminated objects in the nursery e n~i r o n m e n t~~"~~~ serving as a reservoir coming in contact with hands of attending p e r~o n n e l .~~.~'~ the mother of an index case may be symptomatic479~480i500~501 or asymptomatic with preclinical infecti0n,4'~ convalescent infedon,477,48 1,502 or chronic carriage. 503 the risk of the newborn becoming infected once salmonella is introduced into a nursery has been reported to be as high as 20% to 27%,487,493 but the frequency of infection may be lower because isolated cases without a subsequent epidemic are unlikely to be reported. gastric acidity is an important barrier to salmonella infection. patients with anatomic or functional achlorhydria are at increased risk of developing salmonellosis.504~505 the hyp~chlorhydria'~ and rapid gastric emptying typical of early lifez8 may in part explain the susceptibility of infants to salmonella. premature and low-birth-weight infants appear to be at higher risk of acquiring salmonella infection than term whether this reflects increased exposure because of prolonged hospital stays or increased susceptibility on the basis of intestinal or immune function is unclear. contaminated food or water is often the source of salmonella infection in older patients; the limited diet of the infant makes contaminated food a less likely source of infection. although human milk?06-508 raw milk?09 powdered milk,510-512 formula:93 and cereal5i3 have been implicated in transmission to infants, more often fomites, such as delivery room resu~citators,4~' rectal thermometer~,4'~>~'~ oropharyngeal suction device^,^^^'^^' water baths for heating formula?17 soap dispenser^,^" " clean" medicine airconditioning mattresses, radiant and serve as reservoirs. one unusual outbreak involving 394 premature and 122 term infants was traced to faulty plumbing, which caused massive contamination of environment and personnel.493 after salmonella enters a nursery, it is difficult to eradicate. epidemics lasting 6 to 7 week^,'@^,^^' 17 weeks,& 6 months,485b490 1 year:' " and 27 to 30 months487b493 have been reported. spread to nearby pediatric wards has the incubation period in nursery outbreaks has varied widely in several studies where careful attention has been paid to this variable. in one outbreak of salmonella oranienburg involving 35 newborns, 97% of cases occurred within 4 days of in an outbreak of s. typhimurium, each of the ill infants presented within 6 days of birth.477 these incubation periods are similar to those reported for salmonella newport in older children and adults, 95% of whom have been reported to be ill within 8 days of e~p o s u r e .~'~'~~' conversely, one outbreak of salmonella nienstedten involving newborns was characterized by incubation periods of 7 to 18 days.488 the usual incubation period associated with fecal-oral nursery transmission is not found with congenital typhoid. during pregnancy, typhoid fever is associated with bacteremic infection of the fetus. the congenitally infected infants are symptomatic at birth. they are usually born during the second to fourth week of untreated maternal illness.522 usually, the mother is a carrier; fecal-oral transmission of s. typhi can occur with delayed illness in the newborn.523 several major clinical syndromes occur with nontyphoidal salmonella infection in young infants. colonization without illness may be the most common outcome of ingestion of salmonella by the neonate. such colonization usually is detected when an outbreak is under investigation. most infected infants who become ill have abrupt onset of loose, green, mucus-containing stools, or they have bloody diarrhea; an elevated temperature is also a common finding in salmonella gastroenteritis in the first months of life.44o grossly bloody stools are found in the minority of patients, although grossly bloody stools can occur in the first 24 hours of life. hematochezia is more typically associated with noninfectious causes (e.g., swallowed maternal blood, intestinal ischemia, hemorrhagic diseases, anorectal fissures) at this early age.524 there appear to be major differences in presentation related to the serotype of s. enteritidis causing infection. for example, in one epidemic of s. oranienb~rg~'~ involving 46 newborns, 76% had grossly bloody stools, 11% were febrile, 26% had mucus in their stools, and only 11% were healthy. in a series of s. newport infections involving 11 premature infants;74 90% of infants with gastroenteritis had blood in their stools, 10% had fever, 10% had mucus in their stools, and 9% were asymptomatic. in an outbreak of s. typhim~rium~~' involving 11 ill and 5 healthy infants, none had bloody stools; all of the symptomatic infants were febrile and usually had loose, green stools. of 26 infants infected by salmonella virchow, 42% were asymptomatic; the rest had mild diarrhea!82 seals and colleagues488 described 12 infants with s. nienstedten, all of whom had watery diarrhea and low-grade fever; none had bloody stools. in a large outbreak in zimbabwe of s. heidelberg infection reported by bannerman,485 38% of 100 infants were asymptomatic, 42% had diarrhea, 16% had fever, 15% had pneumonia, and 2% developed meningitis. an outbreak of salmonella worthington was characterized primarily by diarrhea, fever, and jaundice, although 3 of 18 infants developed meningitis and 17% died. 515 in dramatic contrast to these series, none of 27 infants with positive stool cultures for s. tennessee had an illness in a nursery found to be contaminated with that organism.469 a few infants with salmonella gastroenteritis have developed necrotizing e n t e r o c o l i t i~, 4~~~~~~ but it is not clear whether salmonella was the cause. although gastroenteritis is usually self-limited, chronic diarrhea has sometimes been attributed to s a l r n~n e l l a .~~~~~~~ whether chronic diarrhea is caused by salmonella is uncertain. although some infants develop carbohydrate intolerance after a bout of salmonella and salmonella is typically listed as one of the causes of postinfectious protracted diarrhea,529 it is difficult to be sure that the relationship is causal. the prolonged excretion of salmonella after a bout of gastroenteritis may sometimes cause non-specific chronic diarrhea to be erroneously attributed to salmonella. major extraintestinal complications of salmonella infection may develop in the neonate who becomes bacteremic. extraintestinal spread may develop in infants who initially present with diarrhea and in some who have no gastrointestinal tract signs. bacteremia appears to be more common in the neonate than in the older a study of more than 800 children with salmonella infection showed that extraintestinal infection occurred significantly more often (8.7% versus 3.6%) in the first 3 months of life.531 several retrospective studies suggest that infants in the first month of life may have a risk of bacteremia as high as 30% to 50%. 440 one retrospective suggests that the risk is not increased in infancy and estimates that the risk of bacteremia in childhood salmonella gastroenteritis is between 8.5% and 15.6%. prospective studies of infants in the first year of life suggest that the risk of bacteremia is 1.8% to 6.0%.532*533 although selection biases in these studies limit the reliability of these estimates, the risk is substantial. the salmonella species isolated from infants include some serotypes that appear to be more invasive in the first 2 months of life than in older children or healthy adults (s. newport, s. agona, s. blockley, s. derby, s. enteritidis, s. heidelberg, s. infantis, s. javiana, s. saint-paul, and s. typhimurium) and serotypes that are aggressive in every age group (s. choleraesuis and s. dublin). other serotypes appear more likely to cause bacteremia in adults (s. typhi, s. paratyphi a, and s. paratyphi b). 530 virtually any salmonella serotype can cause bacteremic disease in neonates. a few infants with salmonella gastroenteritis have died with e. coli or pseudomonas aeruginosa sepsis;94 but the role of salmonella in these cases is unclear. unlike the situation in older children in whom bacteremic salmonellosis often is associated with underlying medical conditions, bacteremia may occur in infants who have no immunocompromising conditions.534 salmonella bacteremia is often not suspected clinically because the syndrome is not usually d i s t i n~t i v e . 4~~~~~ even afebrile, well-appearing children with salmonella gastroenteritis have been documented to have bacteremia that persists for several days.535 although infants with bacteremia may have spontaneous resolution without therapy:36 a sufficient number develop complications to warrant empirical antimicrobial therapy when bacteremia is suspected. the frequency of complications is highest in the first month of life. meningitis is the most feared complication of bacteremic salmonella disease. between 50% and 75% of all cases of nontyphoidal salmonella meningitis occur in the first 4 months of life.537 the serotypes associated with neonatal meningitis (s. typhimurium, s. heidelberg, s. enteritidis, s. saint-paul, s. newport, and s. panama)497 are serotypes frequently associated with bacteremia. meningitis has a high mortality rate, in part because of the high relapse rates. relapse has been reported in up to 64% of ca~es.5~' in some studies, more than 90% of patients with meningitis have died,539 although more typically, 30% to 60% of infants die.540*541 the survivors suffer the expected complications of gram-negative neonatal meningitis, including hydrocephalus, seizures, ventriculitis, abscess formation, subdural empyema, and permanent neurologic impairment. neurologic sequelae have included retardation, hemiparesis, epilepsy, visual impairment, and a t h e t o~i s .~~~ in large nursery outbreaks, it is common to find infants whose course is complicated by pneum~nia?'~ osteo-myeliti~,5">~~~ or septic arthriti~.4'~,~'~ othe r rare complications of salmonellosis include p e r i~a r d i t i s ,~~ p y e l i t i~,~~ peritonitis:77 otitis media:77 mas ti ti^,^^^ chole~ystitis,~~' endophthalmiti~,~~~ cutaneous abscesses,492 and infected cephal~hematoma?~' other focal infections seen in older children and adults, such as endocarditis and infected aortic aneurysms, rarely or never have been reported in neonates.537,549 altho ugh the mortality rate in two reviews of nursery outbreaks was 3.7% to 7.0%,"959496 in some series, it reached 18%. 485 enteric fever, most often related to s. typhi but also occurring with s. paratyphi a, s. paratyphi b, s. paratyphi c, and other salmonella species, is reported much less commonly in infants than in older patients. infected infants develop typical findings of neonatal sepsis and meningitis. current data suggest that mortality is about 30y0.~~' in utero infection with s. typhi has been described. typhoid f e~e r~" *~~' and nontyphoidal salmonella infections552 during pregnancy put women at risk of aborting the fetus. premature labor usually occurs during the second to the fourth week of maternal typhoid if the woman is untreated.522 in a survey of typhoid fever in pregnancy during the preantibiotic era, 24 of 60 women with well-documented cases delivered prematurely, with resultant fetal death; the rest delivered at term, although only 17 infants survived. 553 the outlook for carrying the pregnancy to term and delivering a healthy infant appears to have improved dramatically during the antibiotic era. however, one of seven women with typhoid in a series still delivered a dead fetus with extensive liver necrosis. 554 in the preantibiotic era, about 14% of pregnant women with typhoid fever died.555 with appropriate antimicrobial therapy, pregnancy does not appear to put the woman at increased risk of death. despite these welldescribed cases, typhoid fever is rare early in life. of 1500 cases of typhoid fever that osler and m~c r a e~~~ reported, only 2 were in the first year of life. in areas where typhoid fever is still endemic, systematic search for infants with enteric fever has failed to find many cases. the few infections with s. typhi documented in children in the first year of life often present as a brief nondescript "viral syndrome" or as p n e~r n o n i t i s ?~~*~~' fever, diarrhea, cough, vomiting, rash, and splenomegaly may occur; the fever may be high, and the duration of illness may be many weeks.522 the current practice of early discharge of newborn infants, although potentially decreasing the risk of exposure, can make recognition of a nursery outbreak difficult. diagnosis of neonatal salmonellosis should trigger an investigation for other cases. other than diarrhea, signs of neonatal salmonella infection are similar to the nonspecific findings seen in most neonatal infections. lethargy, poor feeding, pallor, jaundice, apnea, respiratory distress, weight loss, and fever are common. enlarged liver and spleen are common in those neonates with positive blood cultures. laboratory studies are required to establish the diagnosis because the clinical picture is not distinct. the fecal leukocyte examination reveals polymorphonuclear leukocytes in 36% to 82%359. 559 of persons with salmonella infection, but it has not been evaluated in neonates. obviously, the presence of fecal leukocytes is consistent with colitis of any cause and therefore is a nonspecific finding. routine stool cultures usually detect salmonella if two or three different enteric media (i.e., macconkey's, eosin-methylene blue, salmonella-shigella, tergitol 7, xylose-lysine-deoxycholate, brilliant green, or bismuth sulfite agar) are used. stool, rather than rectal swab material, is preferable for culture, particularly if the aim of culture is to detect carriers.560 on the infrequent occasions when proctoscopy is performed, mucosal edema, hyperemia, friability, and hemorrhages may be seen.*' infants who are bacteremic often do not appear sufficiently toxic to raise the suspicion of b a~t e r e m i a .~~~ blood cultures should be obtained as a routine part of evaluation of neonates with suspected or documented salmonella infection. ill neonates with salmonella infection should have a cerebrospinal fluid examination performed. bone marrow cultures also may be indicated when enteric fever is suspected. there are no consistent abnormalities in the white blood cell count. serologic studies are not helpful in establishing the diagnosis, although antibodies to and flagellar antigens487 develop in many infected newborns. if an outbreak of salmonellosis is suspected, further characterization of the organism is imperative?64 determination of somatic and flagellar antigens to characterize the specific serotype may be critical to investigation of an outbreak. when the serotype found during investigation of an outbreak is a common one (e.g., s. typhimurium), antimicrobial resistance testing475,565 and use of molecular techniques such as plasmid chara~terization~~~ can be helpful in determining whether a single-strain, common-source outbreak is in progress. and ampicillin or amoxicillin versus placebo.572 in contrast to these studies, data suggest that there may be a role for quinolone antibiotics in adults and ~h i l d r e n ,~~~,~~~ but these drugs are not approved for use in neonates, and resistance has been en~ountered.~'~ because these studies have few data as to the risk-benefit ratio of therapy in the neonate, it is uncertain whether they should influence treatment decisions in neonates. studies that have included a small number of neonates suggest little benefit from antimicrobial therapy.477*487*56835773578 however, because bacteremia is common in neonates, antimicrobial therapy for infants younger than 3 months who have salmonella gastroenteritis often is recommended,532v533v561 especially if the infant appears toxic. premature infants and those who have other significant debilitating conditions also should probably be treated. the duration of therapy is debatable but should probably be no more than 3 to 5 days if the infant is not seriously ill and if blood cultures are sterile. if toxicity, clinical deterioration, or documented bacteremia complicates gastroenteritis, prolonged treatment is indicated. even with antimicrobial therapy, some infants develop complications. the relatively low risk of extraintestinal dissemination must be balanced against the well-documented risk of prolonging the carrier state. for infants who develop chronic diarrhea and malnutrition, hyperalimentation may be required; the role of antimicrobial agents in this setting is unclear. the infant with typhoid fever should be treated with an antimicrobial agent; relapses sometimes occur after therapy. colonized healthy infants discovered by stool cultures during evaluation of an outbreak ought to be isolated but probably should not receive antimicrobial therapy. such infants should be discharged from the nursery as early as possible and followed carefully as outpatients. antimicrobial treatment of neonates who have documented extraintestinal dissemination must be prolonged. bacteremia without localization is generally treated with at least a 10-day course of therapy. therapy for salmonella meningitis must be given for at least 4 weeks to lessen the risk of relapse. about three fourths of patients who have relapses have been treated for three weeks or less?37 similar to meningitis, treatment for osteomyelitis must be prolonged to be adequate. although cures have been reported with 3 weeks of therapy, 4 to 6 weeks of therapy is recommended. in vitro susceptibility data for salmonella isolates must be interpreted with caution. the aminoglycosides show good in vitro activity but poor clinical efficacy, perhaps because of the low ph of the phagolysosome. aminoglycosides have poor activity in an acid environment. the stability of some drugs in this acid environment also may explain in vitro and in vivo disparities. the intracellular localization and survival of salmonella within phagocytic cells also presumably explains the relapses encountered with virtually every regimen. resistance to antibiotics has long been a problem with salmonella i n f e c t i~n .~~,~~~,~~' there has been a steady increase in resistance to salmonella in the united states over the last 20 years.581 with the emergence of typhimurium type dt 104, resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline has increased from 0.6% in 1979 and 1980 to 34% in 1996. 582 resistance plasmids have been selected and transmitted, partly because therapy has been given for mild illness that should not have been treated566 and partly because of use of antibiotics in animal feeds. resistance to chloramphenicol and ampicillin has made trimethoprim-sulfamethoxazole increasingly important for the treatment of salmonella infection in those patients who require therapy. however, with increasing resistance to all three of these agents in asia?83 the middle e~r o p e ,~~~,~'~ ar gentina,580 and north america,5793588,589 the third-generation cephalosporins and quinolones represent drugs of choice for invasive salmonellosis. the quinolones currently are not approved for persons younger than 18 years. cefotaxime, ceftriaxone, and cefoperazone represent acceptable alternative drugs for typhoidal and nontyphoidal salmonellosis when resistance is e n c o~n t e r e d .~~"~~~ because the second-generation cephalosporins, such as cefuroxime, are less active in vitro than the third-generation cephalosporins and are not consistently clinically effective, they should not be data suggest that cefoperazone may sterilize blood and cause patients with typhoid fever to become afebrile more rapidly than with chl~ramphenicol,~~~ perhaps because cefoperazone is excreted into bile in high concent r a t i o n~.~~~ the third-generation cephalosporins may have higher cure and lower relapse rates than ampicillin or chloramphenicol in children with salmonella meningitis. 595 the doses of ampicillin, chloramphenicol, or cefotaxime used in infants with gastroenteritis pending results of blood cultures are the same as those used in treatment of sepsis. because of the risk of gray baby syndrome, chloramphenicol should not be used in neonates unless other effective agents are not available. trimethoprim-sulfamethoxazole, although useful in older children and adults, is not used in neonates because of the risk of kernicterus. nosocomial infection with strains of salmonella resistant to multiple antibiotics, including third-generation cephalosporins, has emerged as a problem in south america. 580 nonantibiotic interventions are important in the control of salmonella infections. limited data suggest that intravenous immune globulin (igiv) (500 mg/kg on days 1,2,3, and 8 of therapy) along with antibiotic therapy may decrease the risk of bacteremia and death in preterm infants with salmonella ga~troenteritis.~~~ early recognition and intervention in nursery outbreaks of salmonella are crucial to control. when a neonate develops salmonellosis, a search for other infants who have been in the same nursery should be undertaken. when two or more cases are recognized, environmental cultures, cultures of all infants, cohorting and contact isolation of infected infants, rigorous enforcement of hand hygiene, early discharge of infected infants, and thorough cleaning of all possible fomites in the nursery and delivery rooms are important elements of control. if cases continue to occur, the nursery should be closed to further admissions. cultures of nursery personnel are likely to be helpful in the unusual situation of an s. typhi outbreak in which a chronic carrier may be among the caretakers. culture of health care personnel during outbreaks of salmonellosis caused by other salmonella species is debatable, although often recommended. data suggest that nurses infected with salmonella rarely infect patients in the hospital setting.597 the fact that nursing personnel are sometimes found to be colonized during nursery outmay be a result rather than a cause of those epidemics. the potential role of vaccines in control of neonatal disease is minimal. for the vast number of non-s. ryphi serotypes, there is no prospect for an immunization strategy. multiple doses of the commercially available oral live attenuated vaccine (ty2la; vivotif, berna), has been shown in chilean schoolchildren to reduce typhoid fever cases by more than 70%.598,599 however, the vaccine is not recommended for persons younger than 6 years, in part because immunogenicity of ty2la is age dependent; children younger than 24 months fail to respond with development of immunity!" vi capsular polysaccharide vaccine is available for children older than 2 years and is effective in a single dose. whether some degree of protection of infants could the virulence of shigellae has been studied extensively since their recognition as major pathogens at the beginning of the 20th century. the major determinants of virulence are encoded by a 120-to 140-mda p l a~m i d .~~~.~~~ this plasmid, which is found in all virulent shigellae, encodes the synthesis of proteins that are required for invasion of mammalian cells and for the vigorous inflammatory response that is characteristic of the d i s e a~e .~~*~~ shigellae that have lost this plasmid, have deletions of genetic material from the region involved in synthesis of these proteins, or have the plasmid inserted into the chromosome lose the ability to invade eukaryotic cells and become aviru1ent6o7; maintenance of the plasmid can be detected in the clinical microbiology lab by ability to bind congo red. the ability to invade cells is the basic pathogenic property shared by all ~h i g e l l a e~~'~~~~ and by the shigella-like invasive e. coli, which also possesses the shigella virulence plasmid.205~605~606~610~611 in the laboratory, shigella invasiveness is studied in tissue culture (hela cell invasion), in animal intestine, or in rabbit or guinea pig eye, where instillation of the organism causes keratoconjunctivitis (sereny test)."' animal model studies have shown that bacteria penetrate and kill colonic mucosal cells and then elicit a brisk inflammatory response. in addition to the virulence plasmid, several chromosomal loci enhance virulence.612v613 this has been best studied in s. flexneri in which multiple virulence-enhancing regions of the chromosome have been defined.604s612-614 the specific gene products of some of the chromosomal loci are not known; one chromosomal virulence segment encodes for synthesis of the 0 repeat units of lipopolysaccharide. intact lipopolysaccharide is necessary but not sufficient to cause virulence.6129615 at least two cell-damaging cytotoxins that also are chromosomally encoded are produced by shigellae. one of these toxins (shiga toxin) is made in large quantities by s. dysenteriae serotype 1 (the shiga bacillus) and is made infrequently by other shigellae.616 shiga toxin is a major virulence factor in s. dysenteriae, enhancing virulence at the colonic mucosa and also giving rise to sequelae similar to those caused by stec (discussed earlier). this toxin kills cells by interfering with peptide elongation during protein ~y n t h e s i s .~'~-~'~ additional toxins may also be secreted by shigellae, although their roles in virulence are not established.620 although much of the epidemiology of shigellosis is predictable based on its infectious dose, certain elements are unexplained. shigellae, like other organisms transmitted by the fecal-oral route, are commonly spread by food and water, but the low infecting inoculum allows person-to-person spread. because of this low inoculum, shigella is one of the few enteric pathogens that can infect swimmers. 621 the dose required to cause illness in adult volunteers is as low as 10 organisms for s. dysenteriae serotype 1,6" about 200 organisms for s. f l e~n e r i ,~~~ and 500 organisms for s. ~o n n e i .~'~ personto-person transmission of infection probably explains the continuing occurrence of shigella in the developed world. enteropathogens that require large inocula and hence are best spread by food or drinking water are less common in industrialized societies because of sewage disposal facilities, water treatment, and food-handling practices. in the united states, daycare centers currently serve as a major focus for acquisition of shigell~sis.~'~ numerous outbreaks of shigellosis related to crowding, poor sanitation, and the low dose required for diseases have occurred in this setting. given the ease of transmission, it is not surprising that the peak incidence of disease is in the first 4 years of life. it is, however, paradoxical that symptomatic infection is uncommon in the first year of life.626-629 the best data on the age-related incidence of shigellosis come from mata'~~'~ prospective studies of guatemalan infants. in these studies, stool cultures were performed weekly on a group of children followed from birth to 3 years old. the rate of infection was more than 60-fold lower in the first 6 months of life than (fig. 20-1) . 626 the same age-related incidence has been described in the united states629 and in a rural egyptian village. 628 this anomaly has been explained by the salutary effects of brea~t-feeding.~~'-~~' however, it is likely that breast-feeding alone does not explain the resistance of infants to shigellosis. a review of three large case series633-635 suggests that about 1.6% (35 of 2225) of shigellosis cases occur in infants in the neonatal period. the largest series of neonatal ~higellosis~~~ suggests that the course, complications, and etiologic serogroups are different in neonates than in older children. although newborns are routinely contaminated by maternal feces, neonatal shigellosis is rare. other aspects of the epidemiology of shigellosis elude simple explanation. the seasonality (summer-fall peak in the united states, rainy season peak in the tropics) is not well explained. the geographic variation in species causing infection likewise is not well understood. in the united states, most shigella infections are caused by s. sonnei or, less commonly, s. flexneri. in most of the developing world, the relative importance of these two species is reversed, and other shigella serotypes, especially s. dysenteriae serotype 1, are identified more frequently. as hygiene improves, the proportion of s. sonnei increases and that of s. flexneri decreases.636 data from bangladesh suggest that s. dysenteriae is less common in neonates, but s. sonnei and s. boydii are more c0mmon.6~~ there appear to be some important differences in the relative frequencies of various complications of shigella infection related to age. some of these differences and estimates are based on data that are undoubtedly compromised by reporting biases. s. dysenteriae serotype 1 characteristically causes a more severe illness than other shigellae with more complications, including pseudomembranous colitis, hemolysis, and hus. however, illnesses caused by various shigella serotypes usually are indistinguishable from each other and conventionally are discussed together. the incubation period of shigellosis is related to the number of organisms ingested, but in general, it is between 12 and 48 hours. volunteer studies have shown that after ingestion, illness may be delayed for a week or more. neonatal shigellosis seems to have a similar incubation period. more than one half of the neonatal cases occur within 3 days of birth, consistent with fecal-oral transmission during parturition. mothers of infected neonates are sometimes carriers, although more typically they are symptomatic during the perinatal period. intrauterine infection is rare. in the older child, the initial signs are usually high fever, abdominal pain, vomiting, toxicity, and large-volume watery stools; diarrhea may be bloody or may become bloody. painful defecation and severe, crampy abdominal pain associated with frequent passage of small-volume stools with gross blood and mucus are characteristic findings in older children or adults who develop severe colitis. many children, however, never develop bloody diarrhea. adult volunteer studies have demonstrated that variations in presentation and course are not related to the dose ingested because some patients develop colitis with dysentery but others develop only watery diarrhea after ingestion of the same i n o c u l~m .~~~ the neonate with shigellosis may have a mild diarrheal syndrome or a severe ~o l i t i s .~~~~~~~-~~ fever in neonates is usually low grade (<102" f) if the course is uncomplicated. the neonate has less bloody diarrhea, more dehydration, more bacteremia, and a greater likelihood of death than the older ~h i l d . 6~~ physical examination of the neonate may show signs of toxicity and dehydration, although fever, abdominal tenderness, and rectal findings are less striking than in the older complications of shigellosis are common.646 although the illness is self-limited in the normal host, resolution may be delayed for a week or more. in neonates and malnourished children, chronic diarrhea may follow a bout of shigello~is.6~',~~ between 10% and 35% of hospitalized children with shigella have convulsions before or during the course of usually, the seizures are brief, generalized, and associated with high fever. seizures are uncommon in the first 6 months of life, although neonates have been described with seizures.6399649 the cerebrospinal fluid generally reveals normal values in these children, but a few have mild cerebrospinal fluid pleocytosis. the neurologic outcome generally is good even with focal or prolonged seizures, but fatalities do occasionally occur, often associated with toxic encephalpa thy.^^' although the seizures had been postulated to result from the neurotoxicity of shiga toxin, this explanation was proved to be incorrect because most shigellae make little or no shiga toxin and the strains isolated from children with neurologic symptoms do not produce shiga t~x i n .~'~,~~' hemolysis with or without development of uremia is a complication primarily of s. dysenteriae serotype 1 infection. 652 sepsis during the course of shigellosis may be caused by the shigella itself or by other gut flora that gain access to the bloodstream through damaged mucosa.632'653*654 the risk of sepsis is higher in the first year of life, particularly in neo-nates,632.637-639,649,656 in malnourished children, and in those with s. dysenteriae serotype 1 infection.654 sepsis occurs in up to 12% of neonates with given the infrequency of neonatal shigellosis, it is striking that 9% of reported cases of shigella sepsis have involved infants in the first month of life.657 one of the infants with ba~teremia~~' reportedly had no discernible illness. disseminated intravascular coagulation may develop in those patients whose course is complicated by sepsis. meningitis has been described in a septic neonate. colonic perforation has occurred in n e o n a t e~, 6~"~~~ older children,@' and adults.661 although this complication of toxic megacolon is rare, it appears to be more common in neonates than in older individuals. bronchopneumonia may complicate the course of shigellosis, but shigellae are rarely isolated from lungs or tracheal secretions."2 the syndrome of sudden death in the setting of extreme toxicity with hyperpyrexia and convulsions but without dehydration or sepsis (i.e., ekiri ~yndrome)~~'"~ is rare in neonates. in the nonbacteremic child, other extraintestinal foci of infection, including ~a g i n a~~~. "~ and eye,"' rarely occur. reiter's syndrome, which rarely complicates the illness in children, has not been reported in neonates. although infection is less common in infants than in toddlers, case fatality rates are highest in infant^.^^'^^' the mortality rate in newborns appears to be about twice that of older children.632 in industrialized societies, less than 1% of children with shigellosis die, whereas in developing countries, up to 30% die. these differences in mortality rates are related to n~t r i t i o n . 6~~ availability of medical care, antibiotic resistance of many shigellae, the frequency of sepsis, and the higher frequency of s examination of stool for leukocytes as an indication of colitis is useful in support of the clinical suspicion of shigellosis. the white blood cell count and differential count also are used as supporting evidence for the diagnosis. leukemoid reactions (white blood cells > 50,000/mm3) occur in almost 15% of children with s. dysenteriae serotype 1 but in less than 2% of children with other ~h i g e l l a e .~~~ leukemoid reactions are more frequent in infants than in older ~hildren.6~' even when the total white blood cell count is not dramatically elevated, there may be a striking left shift. almost 30% of children with shigellosis have greater than 25% bands on the differential cell few reports address the white blood cell count in newborns, but those that do suggest that normal or low rather than elevated counts are more common. although serum and fecal antibodies develop to lipopolysaccharides and the virulence plasmid-associated polypeptide~,6~~ serologic studies are not useful in the diagnosis of shigellosis. pcr can identify shigella and eiec in feces.678 colonoscopy typically shows inflammatory changes that are most severe in the distal segments of therapy because dehydration is particularly common in neonatal shigellosis, attention to correction of fluid and electrolyte disturbances is always the first concern when the illness is suspected. although debate continues over the indications for antimicrobial therapy in the patient with shigellosis, the benefits of therapy generally appear to outweigh the risks. the chief disadvantages of antimicrobial therapy include cost, drug toxicity, and emergence of antibiotic-resistant shigellae. because of the self-limited nature of shigellosis, it has been argued that less severe illness should not be treated. however, children can feel quite ill during the typical bout of shigellosis, and appropriate antimicrobial therapy shortens the duration of illness and eliminates shigellae from stool, decreasing secondary spread. complications are probably decreased by antibiotics. given the high mortality rates of neonatal shigellosis, therapy should not be withheld. the empirical choice of an antimicrobial agent is dictated by susceptibility data for strains circulating in the community at the time the patient's infection occurs. multiresistant shigellae complicate the choice of empirical therapy before availability of susceptibility data for the patient's isolate. plasmid-encoded resistance (r factors) for multiple antibiotics has been observed frequently in s. dysenteriae serotype 1 outbreaks682 and with other ~higellae.~'~.~'~ antimicrobial resistance patterns fluctuate from year to year in a given locale.686 however, despite the guesswork involved, early preemptive therapy is indicated when an illness is strongly suggestive of shigellosis. in vitro susceptibility does not always adequately predict therapeutic responses. cefa~lor,6~~ furazolidone,688 ~ephalexin,6'~ amo~icillin,6~' kanam~cin,6~' and ~e f a m a n d o l e~~~ all are relatively ineffective agents. the optimal duration of therapy is debatable. studies in children older than 2 years and in adults suggest that singledose regimens may be as effective in relieving symptoms as courses given for 5 days. the single-dose regimens generally are not as effective in eliminating shigellae from the feces as are the longer courses. a third-generation cephalosporin, such as ceftriaxone, may be the best empirical choice. optimal doses for newborns with shigellosis have not been established. trimethoprim at a dose of lomg/kg/day (maximum, 160 mg/day) and sulfamethoxazole at a dose of 50 mg/kg/day (maximum, 800 mg/day) in two divided doses for a total of 5 days are recommended for the older child if the organism is s~s c e p t i b l e . 6~~-~~~ if the condition of the infant does not permit orally administration, the drug usually is divided into three doses given intravenously over 1 ampicillin at a dose of 100 mg/kg/day in four divided doses taken orally for 5 days may be used if the strain is susceptible. 676 for the rare newborn who acquires shigellosis, appropriate therapy often is delayed until susceptibility data are available. this occurs because shigellosis is so rare in newborns that it is almost never the presumptive diagnosis of the child with watery or bloody diarrhea. although a sulfonamide is as efficacious as ampicillin when the infecting strain is sus~eptible,6~~ sulfonamides are avoided in neonates because of concern about the potential risk of kernicterus. the risk of empirical ampicillin therapy is that shigellae are frequently resistant to the drug; 50% of shigellae currently circulating in the united states are ampicillin resistant.6963697 for the neonate infected with ampicillin-resistant shigella, there are few data on which to base a recommendation. ceftriaxone is generally active against shigellae, but in the neonate, this drug can displace bilirubin-binding sites and elicit clinically significant cholestasis. data on children and adults suggest that clinical improvement occurs with c e f t r i a x~n e .~~~*~~~ quinolones, such as ciprofloxacin and ofloxacin, have been shown to be effective agents for treating s h i g e l l o s i~~~~~~~~ in adults, but they are not approved for use in children younger than 18 years. other drugs sometimes used to treat diarrhea pose special risks to the infant with shigellosis. the antimotility agents, in addition to their intoxication risk, may pose a special danger in dysentery. in adults, diphenoxylate hydrochloride with atropine has been shown to prolong fever and excretion of the ~rganism.~" the response to appropriate antibiotic therapy is generally gratifying. improvement is often obvious in less than 24 hours. complete resolution of diarrhea may not occur until a week or more after the start of treatment. in those who have severe colitis or those infected by s. dysenteriae serotype 1, the response to treatment is somewhat delayed. for most of the developing world, the best strategy for prevention of shigellosis during infancy is prolonged breastfeeding. specific antibodies in milk appear to prevent symptomatic shigellosis6'68; nonspecific modification of gut flora and the lack of bacterial contamination of human milk also may be important. breast-feeding, even when other foods are consumed, decreases the risk of shigellosis; children who continue to consume human milk into the third year of life are still partially protected from in the united states, the best means of preventing infection in the infant is good hand hygiene when an older sibling or parent develops diarrhea. even in unsanitary environments, secondary spread of shigellae can be dramatically decreased by hand hygiene after defecation and before meals.704 spread of shigellae in the hospital nursery can presumably be prevented by the use of contact isolation for infants with diarrhea and attention to thorough hand hygiene. although nursery personnel have acquired shigellosis from infected newborns,685 further transmission to other infants in the nursery, although is rare. in contrast to salmonella, large outbreaks of nosocomial shigellosis in neonates are rare. unfortunately, good hygiene is a particularly difficult problem in daycare centers. the gathering of susceptible children, breakdown in hand hygiene, failure to use different personnel for food preparation and diaper changing, and difficulty controlling the behavior of toddlers all contribute to daycare-focused outbreaks of shigellosis. immunization strategies have been studied since the turn of the 20th century, but no satisfactory immunization has been developed. even if immunizations are improved, a role in managing neonates seems unlikely. campylobacter was first recognized in an aborted sheep fetus in the early 19o0s7o6 and was named vibrio fetus by smith and taylor in 1919. 707 this organism subsequently was identified as a major venereally transmitted cause of abortion and sterility and as a cause of scours in cattle, sheep, and goats.70s3709 it was not until 1947, when it was isolated from the blood culture of a pregnant woman who subsequently aborted at 6 months' gestation, that the significance of campylobacter as a relatively rare cause of bacteremia and perinatal infections in humans was a~preciated.~l'-~'~ during the 1970s, campylobacter was recognized to be an opportunistic pathogen in debilitated in 1963, v fetus and related organisms were separated from the vibrios (such as v cholerae and v parahaemolyticus) and placed in a new genus, campylobacter (greek word for "curved rod"). 715 since 1973, several campylobacter species have been recognized as a common cause of e n t e r i t i~~l~.~~~ and, in some cases, extraintestinal infections. the genus campylobacter contains 15 species, most of which are recognized as animal and human pathogens. the most commonly considered causes of human disease are campylobacter fetus, campylobacter jejuni, campylobacter coli, campylobacter lari, and campylobacter upsaliensis (table 20 -5),'30-732 although campylobacter mucosalis has been isolated from stool of children with diarrhea.733 dna hybridization studies have shown that these species are distinct, sharing less than 35% dna homology under stringent hybridization ~o n d i t i o n s .~~~,~~~ helicobacter pylori was originally named campylobacter pylori, but because of differences in dna, it was reclassified and is no longer considered in the campylobacter genus. strains of c. fetus are divided into two subspecies: c. fetus subsp. fetus and c. fetus subsp. venerealis. the first subspecies causes sporadic abortion in cattle and sheep7369737; in by far the most common syndrome caused by a campylobacter species is enteritis. c. jejuni and c. coli cause gastroenteritis and generally are referred to collectively as c. jejuni, although dna hybridization studies show them to be different. in the laboratory, c. jejuni can be differentiated from c. coli because it is capable of hydrolyzing hippurate, whereas c. coli is not. most isolates that are associated with diarrhea (61% to 100%) are identified as c. jejuni,751-754 and in some cases, individuals have been shown to be simultaneously infected with c. jejuni and c. ~o l i . ~~~ because of the fastidious nature of c. jejuni, which is difficult to isolate from fecal flora, its widespread occurrence was not recognized until 1973.716-732 previously called related vibrios by this organism had been associated with bloody diarrhea and colitis in infants and adults only when it had been associated with a recognized b a~t e r e m i a .~~~-~~~ in the late 1970s, development of selective fecal culture methods for c. jejuni enabled its recognition worldwide as one of the most common causes of enteritis in persons of all ages. it is an uncommon infection in neonates who generally develop gastroenteritis when i n f e~t e d .~'~-~~~-~~' bacteremia with c. jejuni enteritis also is uncommon.718~759,76l*769*772-778 maternal symptoms considered to be related to c. jejuni infection generally are mild and include fever (75%) and diarrhea (30%). in contrast to the serious disease in newborns that is caused by c. fetus, neonatal infections with although meningitis occurs in rare third trimester infection related to c. fetus or c. jejuni may results in abortion or stillbirth. pathogenesis c. fetus does not produce recognized enterotoxins or cytotoxins and does not appear to be locally invasive by the sereny instead, these infections may be associated with penetration of the organism through a relatively intact intestinal mucosa to the reticuloendothelial system and blo~dstream.~'~ whether this reflects a capacity to resist serum factors or to multiply intracellularly remains to be determined. c. jejuni is capable of producing illness by several mechanisms. these organisms have been shown to produce an lt enterotoxin and a c y t o t o x i r~.~~~~~~~ this enterotoxin is known to be a heat-labile protein with a molecular mass of 60 to 70 mda.7793782 it shares functional and immunologic properties with cholera toxin and e. coli lt. c. jejuni and c. coli also elaborate a cytotoxin that is toxic for a number of mammalian cells.783-785 the toxin is heat labile, trypsin sensitive, and not neutralized by immune sera to shiga toxin or the cytotoxin of clostridium dificile. the role of these toxins as virulence factors in diarrheal disease remains unpr~ved.'~~,~~ several animal models have been tested for use in the study of this pathogen.786 potential models for the study of c. jejuni enteritis include dogs, which may acquire symptomatic infection787; 3-to 8-day old ~h i c k s~~' -~~; chicken embryo cells, which are readily invaded by c. jejuni7"; rhesus monkeys791; and rabbits by means of the removable intestinal tie adult rabbit technique. an established small mammal model that mimics human disease in the absence of previous treatment or surgical procedure has not been successful in adult mice.792 an infant mouse mode1793,794 and a hamster of diarrhea appear promising. c. jejuni is negative in the sereny test for invasivenes~,~~~ and most investigators report no fluid accumulation in ligated rabbit ileal loops. the pathologic findings of c. fetus infection in the perinatal period include placental necrosis7'' and, in the neonate, widespread endothelial proliferation, intravascular fibrin deposition, perivascular inflammation, and hemorrhagic necrosis in the brain. 797 the tendency for intravascular location and hepatosplenomegaly in adults infcctcd with c. fetus has been the pathologic findings in infants and children infected with c. fetus can include an acute inflammatory process in the colon or rectum, as evidenced by the tendency for patients to have bloody diarrhea with numerous fecal leukocytes. 798 there also can be crypt abscess formation and an ulcerative colitis or pseudomembranous colitis-like or a hemorrhagic jejunitis or ileitis.717b725,801,802 mesenteric lymphadenitis, ileocolitis and acute appendicitis also have been described. infection with campylobacter species occurs after ingestion of contaminated food, including unpasteurized milk, poultry, and contaminated water.'26,803-812 m any farm animals and pets, such as chickens,813 dogs,814s815 and cats (especially young animals), are potential sources. the intrafamilial spread of infection in h o~s e h o l d s ,~~~,~~~ the occurrence of outbreaks in and the apparent laboratory acquisition of c. jejuni818 all suggest that c. jejuni infection may occur after person-to-person transmission of the organism. outbreaks of c. jejuni in the child daycare setting are not common. volunteer studies8i9 have shown a variable range in the infecting dose, with many volunteers developing no illness. the report of illness after ingestion of lo6 organisms in a glass of milk728 and production of illness in a single volunteer by 500 organisms8i9 substantiate the variation in individual susceptibility. the potential for low-inoculum disease has significant implications for the importance of strict enteric precautions when infected persons are hospitalized, particularly in maternity and nursery areas. when diarrhea in neonates caused by c. jejuni has been r e p~r t e d ,~~~-~~' maternal-infant transmission during labor has generally been documented.758~763*765~768p770~771 the lior serotyping system, restriction length polymorphism, and pulse-field gel electrophoresis have been used to confirm the identity of the infant and maternal isolates. most mothers gave no history of diarrhea during pregnancy.7623763*765,766 outbreaks have occurred in neonatal intensive care units because of person-to-person spread.8z0 the frequency of asymptomatic carriage of c. jejuni ranges from 0% to 1.3%7'6,717 to as high as 13% to 85%.716,717,732,821-823 in a cohort study in mexico, 66% of all infections related to c. jejuni were asymptomatic.732 infected children, if untreated, can be expected to excrete the organisms for 3 or 4 weeks; however, more than 80% are culture negative after 5 ~e e k s .~~~,~~~a~ ymptomatic excreters pose a significant risk in the neonatal period, in which acquisition from an infected mother can be clinically important.718,760s762,766 c. jejuni has increasingly been recognized as a cause of watery and inflammatory diarrhea in temperate and tropical climates throughout the world. it has been isolated from 2% to 11% of all fecal cultures from patients with diarrheal illnesses in various parts of the world.716-724,728*824-829 there is a tendency for c. jejuni enteritis to occur in the summer in countries with temperate climates. 825 the reservoir of campylobacter is the gastrointestinal tract of domestic and wild birds and animals. it infects sheep, cattle, goats, antelope, swine, chickens, domestic turkeys, and pet dogs. c. fetus often is carried asymptomatically in the intestinal or biliary tracts of sheep and cattle. during the course of a bacteremic illness in pregnant animals, c. fetus organisms, which have a high affinity for placental tissue, invade the uterus and multiply in the immunologically immature fetus. the infected fetuses generally are aborted. whether this organism is acquired by humans from animals or is carried asymptomatically for long periods in humans, who may then transmit the organism through sexual contact as appears to occur in animals, is unclear. it is believed that this subspecies rarely is found in the human intestine and that it is not a cause of human enteriti~?'~ c. fetus infections predominantly occur in older men with a history of farm or animal exposure and in pregnant women in their third trimester.710~71 1,716,717 symptomatically or asymptomatically infected women may have recurrent abortions or premature deliveries and are the source of organisms associated with life-threatening perinatal infections of the fetus or newborn infant.710,739-7483830 in several instances of neonatal sepsis and meningitis, c. fetus was isolated from culture of maternal cervix or vagina.712,747s795 a n osocomial nursery outbreak has been associated with carriage in some healthy infants.83' other outbreaks have been associated with meningitis 832, 833 cervical cultures have remained positive in women who have had recurrent abortions and whose husbands have antibody titer elevations. 738 the most commonly incriminated reservoir of c. jejuni is poultry.808,s12,834,835 m ost chickens in several different geographic locations had a large number (mean, 4 x 106/g) of c. jejuni in the lower intestinal tract or feces. this occurred in some instances despite the use of tetracycline, to which the campylobacter was susceptible in vitro, in the chicken feed.828 the internal cavities of chickens remain positive for carnpylobacter even after they have been cleaned, packaged, and fr0zen.8~~ however, unlike salmonella, c. jejuni organisms that survive usually do not multiply to high concen-tration~?~' domestic puppies or kittens w i t h c. jejuni diarrhea also can provide a source for spread, especially to infants or c. jejuni enteritis also has been associated in a number of outbreaks with consumption of unpasteurized in retrospect, the first reported human cases of c. jejuni enteritis were probably in a milk-borne outbreak reported in 1 946. 842 because campylobacter infections of the udder are not seen, milk is probably contaminated from fecal shedding of the organism. these organisms are killed by adequate heating. fecally contaminated water is a potential vehicle for c. jejuni infections.843 several phenotypic and genotypic methods have been used for distinguishing c. jejuni strains from animals and humans involved in epidemics. 844 clinical manifestations of infection caused by campylobacter depend on the species involved (see table 20 -5). human infections with c. fetus are rare and generally are limited to bacteremia in patients with predisposing condition^^^^.^^^ or to bacteremia or uterine infections with prolonged fever and pneumonitis that lasts for several weeks in women during the third trimester of pregnancy. unless appropriately treated, symptoms usually resolve only after abortion or delivery of an infected infant?10j12,739-748*750 these infected neonates, who are often premature, develop signs suggesting sepsis, including fever, cough, respiratory distress, vomiting, diarrhea, cyanosis, convulsions, and jaundice. the condition typically progresses to meningitis, which may be rapidly fatal or may result in serious neurologic ~equelae.~" additional systemic manifestations include pericarditis, pneumonia, peritonitis, salpingitis, septic arthritis, and abscesses.823 c. jejuni infection typically involves the gastrointestinal tract, producing watery diarrhea or a dysentery-like illness with fever and abdominal pain and stools that contain blood and ~u c u s .~~~~~~'~~' " ' older infants and children generally are affected, but neonates with diarrhea have been reported. infection in neonates generally is not clinically apparent or is mild. stools can contain blood, mucus, and pus712p721*762*763; fever often is ab~ent.7~~"~' the illness usually responds to appropriate antimicrobial which shortens the period of fecal shedding.845 extraintestinal infections related to c. jejuni other than bacteremia are rare but include cholecystitis,846 urinary tract and meningitis.761 bacteremia is a complication of gastrointestinal infe~tion,8~' especially in malnourished children. 849 meningitis that appears to occur secondary to intestinal infection also has been reported in premature infants who have had intraventricular needle aspirations for neonatal hydrocephalus.'i2 complications in older children and adults that have been associated with c. jejuni enteritis include reiter's syndrome, 850 guillain-barre ~y n d r o m e , 8~~~~~* and reactive persistent c. jejuni infections have been described in patients infected with human immunodeficiency extraintestinal manifestations generally occur in patients who are immunosuppressed or at the extremes of age.'14 campybbacter zari has caused chronic diarrhea and bacteremia in a neonate!% most important in the diagnosis of campylobacter infection is a high index of suspicion based on clinical grounds. c. fetus and c. jejuni are fastidious and may be overlooked on routine fecal cultures. isolation of campylobacter from blood or other sterile body sites does not represent the same problem as isolation from stool. growth occurs with standard blood culture media, but it may be slow. in the case of c. fetus infecting the bloodstream or central nervous system, blood culture flasks should be blindly subcultured and held for at least 7 days or the organism may not be detected because of slow or inapparent the diagnosis of c. fetus infection should be considered when there is an unexplained febrile illness in the third trimester of pregnancy or in the event of recurrent abortion, prematurity, or neonatal sepsis with or without meningitis. a high index of suspicion and prompt, appropriate antimicrobial therapy may prevent the potentially serious neonatal complications that may follow maternal c. fetus infection. campylobacter is distinguished from the vibrio organisms by its characteristics of carbohydrate nonfermentation and by its different nucleotide base omp position.^^^^^^^-^^^*^^^ campylobacter is 0.2 to 0.5 fm wide and 0.5 to 8.0 long. it is a fastidious, microaerophilic, curved, motile gram-negative bacillus that has a single polar flagellum and is oxidase and catalase positive, except for c. upsaliensis, which is generally catalase negative or weakly positive. c. jejuni and c. fetus are separated by growth temperature (c. fetus grows best at 25' c but can be cultured at 37' c; c. jejuni grows best at 42' c) and by nalidixic acid and cephalosporin susceptibilities, because c. jejuni is susceptible to nalidixic acid and resistant to cephalosporins. c. jejuni grows best in a microaerobic environment of 5% oxygen and 10% carbon dioxide at 42' c. it grows on a variety of media, including brucella and mueller-hinton agars, but optimal isolation requires the addition of selective and nutritional supplements. growth at 42' c in the presence of cephalosporins is used to culture selectively for c. jejuni from fecal specimens. in a study of six media, charcoal-based selective media and a modified charcoal cefoperazone deoxycholate agar were the most selective for identification of campylobacter species. extending the incubation time from 48 to 72 hours led to an increase in the isolation rate regardless of the medium its typical darting motility may provide a clue to identification, even in fresh fecal specimens, when viewed by phase-contrast microscopy. 72 when the organism has been cultured, it is presumptively identified by motility and by its curved, sometimes sea gulllike appearance on carbolfuchsin stain. polymorphonuclear leukocytes are usually found in stools when bloody diarrhea occurs and indicate the occurrence of ~o l i t i s .~~~*~~* to avoid potentially serious c. jejuni infection in the newborn infant, careful histories of any diarrheal illnesses in the family should be obtained, and pregnant women with any enteric illness should have cultures for this and other enteric pathogens. detection of c. jejuni and c. coli by pcr has been reported859 and in the future may be useful for the rapid and reliable identification of this organism. the differential diagnosis of c, fetus infections include the numerous agents that cause neonatal sepsis or meningitis, especially gram-negative bacilli. diagnostic considerations for inflammatory or bloody enteritis include necrotizing enterocolitis, allergic proctitis, and salmonella; rarely shigellu, and other infectious agents occur. agglutination, complement fixation, bactericidal, immunofluorescence, and elisa tests have been used for serologic diagnosis of c. jejuni infection and to study the immune response, but these assays are of limited value in establishing the diagnosis during an acute infection.731 the prognosis is grave in newborn infants with sepsis or meningitis caused by c. fetus. in infants with c. jejuni gastroenteritis, limited data suggest that appropriate, early antimicrobial therapy results in improvement and rapid clearance of the organism from stool.845 campylobacter species are often resistant to p-lactams, including ampicillin and cephalosporins.860v861 mo st strains are susceptible to erythromycin, gentamicin, tetracycline, chloramphenicol, and the newer quinolones, although resistance to these agents has been r e p~r t e d .~~' ,~~~ it appears that a parenteral aminoglycoside is the drug of choice for c. fetus infections, pending in vitro susceptibility studies. in the case of central nervous system involvement, cefotaxime and chloramphenicol are potential alternative drugs. depending on in vitro susceptibilities, which vary somewhat with locale, erythromycin is the drug of choice for treating c. jejuni e n t e r i t i~.~~~~~"~~' ' if erythromycin therapy is initiated within the first 4 days of illness, a reduction in excretion of the organism and resolution of symptoms occur.845 although data regarding treatment of asymptomatic or convalescent carriers are not available, it seems appropriate to treat colonized pregnant women in the third trimester of pregnancy when there is a risk of perinatal or neonatal infection. the failure of prophylactic parenteral gentamicin in a premature infant has been documented, followed by successful resolution of symptoms and fecal shedding with erythromycin. because there appears to be an increased risk of toxicity with erythromycin estolate during pregnancy and other forms of erythromycin should probably be used in these settings. azithromycin appears to be effective if the organism is susceptible.865 strains that are erythromycin resistant often are resistant to azithromycin.866 cumpylobucter tends to have higher minimal inhibitory concentrations for clarithromycin than for a~ithromycin.~~~ furazolidone has been used in children and ciprofloxacin in nonpregnant patients older than 17 years. contact precautions should be employed during any acute diarrheal illness and until the diarrhea has subsided. hand hygiene after handling raw poultry and washing cutting boards and utensils with soap and water after contact with raw poultry may decrease risk of infection. pasteurization of milk and chlorination of water are critically important. infected food handlers and hospital employees who are asymptomatic pose no known hazard for disease transmission if proper personal hygiene measures are maintained. ingestion of human milk that contains anti<. jejuni antibodies has been shown to protect infants from diarrhea due to c. j e j~n i .~~,~~~ c. dificile is a spore-forming, gram-positive, anaerobic bacillus that produces two toxins. in the presence of antibiotic pressure, c. dificile colonic overgrowth and toxin production occur. the virulence properties of c. dificile are related to production of an enterotoxin that causes fluid secretion (toxin a) and a cytotoxin detectable by its cytopathic effects in tissue culture (toxin b).869*870 both the usual manifestations of c. dificile disease in older children and adults include watery d&rhea, abdominal pain and tenderness, nausea, vomiting, and low-grade fever. grossly bloody diarrhea is unusual, although occult fecal blood is common. leukocytosis is present during severe illness. diarrhea usually begins 4 to 9 days into a course of antimicrobial therapy but may be delayed until several weeks after completion of the therapeutic course. usually, the illness is mild and self-limited if the offending drug is discontinued. severe colitis with pseudomembranes is less common now than in previous years because the risk of diarrhea developing during antimicrobial therapy is recognized and the antimicrobial agent typically is stopped. it is unclear whether this organism causes disease in newborns. one study from a newborn intensive care unit suggests that toxin a in stools is associated with an increased frequency of abnormal stools.893 endoscopic findings of pseudomembranes and hyperemic, friable rectal mucosa suggest the diagnosis of pseudomembranous colitis. pseudomembranes are not always present in c. dificile colitis; mild cases are often described as nonspecific colitis. several noninvasive techniques are used to establish the diagnosis, including enzyme immunoassay (eia) for toxin detection and pcr.893-896 isolation of c. dificile from stool does not distinguish between toxigenic and nontoxigenic isolates. if c. dificile is isolated, testing for toxin by cell culture or eia should be performed to confirm the presence of a toxigenic strain. there are multiple commercially available eias that detect either toxin a or both toxins a and b.893-895 these assays are sensitive and easy to perform. other assays are available for epidemiologic investigation of outbreaks of disease due to c. d i f i~i l e .~~~ in older children and adults, the diagnosis is confirmed by culture of c. dificile and demonstration of toxin in feces. in neonates, these data are inadequate to prove that an illness is related to c. dificile. when the clinical picture is consistent, the stool studies are positive for c. dificile and no other cause for illness is found, a diagnosis of "possible" c. dificile is made. a favorable response to eradication of c. dificile is supportive evidence that the diagnosis is c0rrect.8~~ because of the uncertainty implicit in the ambiguity of neonatal diagnostic criteria, other diagnoses must be considered. when the decision is made that a neonate's illness might be related to c. difjcile, the initial approach should include fluid and electrolyte therapy and discontinuation of the offending antimicrobial agent. if the illness persists or worsens or if the patient has severe diarrhea, specific therapy with r n e t r o n i d a z~l e~~~*~~~ should be instituted. metronidazole is considered to be the treatment of choice for most patients with c. difjcile ~olitis.8~' rarely is there a need to consider orally administered vancomycin or bacitracin in after initiation of therapy, signs of illness generally resolve within several days, titers decrease, and fecal toxins disappear eventually. recurrence of colitis after discontinuation of metronidazole or vancomycin has been documented in 10% to 20% of adults.g01 relapses are treated with a second course of metronidazole or vancomycin. drugs that decrease intestinal motility should not be administered. neutralizing antibody against c. dificile otoxin has been demonstrated in human colostrum.' secretory component of siga binds to toxin a to inhibit its binding to receptors 903 data show that there are nonantibody factors present in milk that interfere with the action of toxin b in addition to secretory iga directed at toxin a. 904 breast-feeding appears to decrease the frequency of colonization by c. d i f j~i l e .~'~ in addition to standard precautions, contact precautions are recommended for the duration of illness. meticulous hand hygiene techniques, proper handling of contaminated waste and fomites, and limiting the use of antimicrobial agents are the best available methods for control of c. dificile infection. b! cholerue is a gram-negative, curved bacillus with a polar flagellum. of the many serotypes, only enterotoxin-producing organisms of serotype 01 and 0139 cause epidemics. b! cholerue 01 is divided into two serotypes, inaba and ogawa, and two biotypes, classic and el tor; the latter is the predominant biotype. nontoxigenic 0 1 strains and non-01 strains of v cholerae can cause diarrhea and sepsis but do not cause outbreaks?06~90a pathogenesis b! cholerue 0 group 1 is the classic example of an enteropathogen whose virulence is caused by enterotoxin production. cholera toxin is an 84-mda protein whose five b subunits cause toxin binding to the enterocyte membrane ganglioside gm, and whose a subunit causes adenosine diphosphate ribosylation of a guanosine triphosphate-binding regulatory subunit of adenylate cy~lase."~~~'~ the elevated camp levels that result from stimulation of enterocytes by cholera toxin cause secretion of salt and water with concomitant inhibition of absorption. two other toxins are also encoded within the virulence cassette that encodes cholera toxin. these toxins, zona occludens toxin (zot) and accessory cholera toxin (ace), are consistently found in illness-causing strains of 0 1 and 0139 but not usually in v; cholerae organisms that are less virulent. since 1960, v cholerae 01, biotype el tor, has spread from india and southeast asia to africa, the middle east, southern europe, and the southern, western, and central pacific islands in the aquatic environment. the usual reported vehicles of transmission have included contaminated water or ice; contaminated food, particularly raw or undercooked shellfish; moist grains held at ambient temperature; and raw or partially dried fish. the usual mode of infection is ingestion of contaminated food or water. boiling water or treating it with chlorine or iodine and adequate cooking of food kill the organism.907 asymptomatic infection of family contacts is common but direct person-to-person transmission of disease has not been documented. persons with low gastric acidity are at increased risk for cholera infection. cholera acquired during pregnancy, particularly in the third trimester, is associated with a high incidence of fetal miscarriage can be attributed to a fetal acidosis and hypoxemia resulting from the marked metabolic and circulatory changes that this disease induces in the mother. it is not surprising that the likelihood of delivering a stillborn child is closely correlated with the severity of the maternal illness. the inability to culture v; cholerue from stillborn infants of infected mothers, together with the usual absence of bacteremia in cholera, suggests that transplacental fetal infection is not a cause of intrauterine death. neonatal cholera is a rare disease. this generalization also applies to the new 0139 strains, although mild9'* and severe forms of illness have rarely been described in newborns. 919 among 242 neonates admitted to a cholera research hospital in dacca, bangladesh, there were 25 infants ill with ~holera.~" even infants born to mothers with active diarrheal disease may escape infection, despite evidence that rice-water stools, almost certain to be ingested during the birth process, may contain as many as lo9 organism~/ml.~~~ the reason for this apparently low attack rate among newborns is not certain; however, it probably can be attributed in large part to the protection conferred by breast-feedingg2' human milk contains antibodies62 and receptor-like glycoprotein that inhibit adherence of v choleraeu and gangliosides that bind cholera toxin.65 the role of transplacentally acquired vibriocidal maternal antibodies has not been determined.922 because v cholerae causes neither bacteremia nor intestinal invasion, protection against illness is more likely to be a function of mucosal rather than serum additional factors that may reduce the incidence of neonatal cholera include the large inoculum required for infection925 and the limited exposure of the newborn to the contaminated food and water.229 clinicians should request that appropriate cultures be performed for stool specimens from persons suspected of having cholera. the specimen is plated on thiosulfate citrate bile salts sucrose agar directly or after enrichment in alkaline peptone water. isolates of v cholerae should be confirmed at a state health department and then sent to the cdc for testing for production of cholera toxin. a fourfold rise in vibriocidal antibody titers between acute and convalescent serum samples or a fourfold decline in titers between early and late (>2 months) convalescent serum specimens can confirm the diagnosis. probes have been developed to test for cholera toxin.926*927 the most important modality of therapy is administration of oral or parented rehydration therapy to correct dehydration and electrolyte imbalance and maintain h y d r a t i~n .~~ antimicrobial therapy can eradicate vibrios, reduce the duration of diarrhea, and reduce requirements for fluid replacement. one cholera vaccine, which is administered parenterally, is licensed in the united states but is of very limited value. several experimental oral vaccines are being t e~t e d ? *~-~~' i.: enterocolitica is a major cause of enteritis in much of the industrialized enteritis due to this organism primarily occurs in infants and young children, and infections in the united states are reported to be more common in the north than in the s o~t h .~~~-~~~ h i m als, especially swine, have been shown to serve as the reservoir for y. enterocolitica. a history of recent exposure to chitterlings (i.e., pig intestine) is common. transmission has also occurred after ingestion of contaminated milk and infusion of contaminated blood p r o d~c t s .~~~,~~~ virulence of y. enterocolitica is related primarily to a virulence plasmid, which is closely related to the virulence plasmids of yersinia pseudotuberculosis and yersinia p e s t i~.~~'~~~~ an st enterotoxin, which is closely related to the st of etec,943 may also be important. infection with y. enterocolitica is recognized as one of the causes of bacterial gastroenteritis in young children, but knowledge of neonatal infection with this organism is fragmentary. even in large series, isolation of yersinia from newborns is rare?313932, 944 the youngest infants whose clinical course has been described in detail were 11 days to several months old at the onset of their illness.932,944-952 there were no features of the gastroenteritis to distinguish it from that caused by other invasive enteric pathogens such as shigella or salmonella. infants presented with watery diarrhea or with stools containing mucus with streaks of blood. sepsis was common in these infants particularly in the first 3 months of life when 28% of enteritis was complicated by sepsis.948,949,953,954 fever is not a consistent finding in children with bacteremia, and meningitis is rare. in older children, fever and right lower quadrant pain mimicking appendicitis are often found.940 diagnosis y enterocolitica can be recovered from throat swabs, mesenteric lymph nodes, peritoneal fluid, blood, and stool. because laboratory identification of organisms from stool requires special techniques, laboratory personnel should be notified when yersinia is suspected. because avirulent environmental isolates occur, biotyping and serotyping are useful in assessing the clinical relevance of isolates. pcr has been used to detect pathogenic strain^.^^^'^^^ the effect of antimicrobial therapy on the outcome of gastrointestinal infection is uncertain. it has been recommended that antibiotics be reserved for sepsis or prolonged and severe gastroenteritisg3'; however, there are no prospective studies comparing the efficacy of various antimicrobial agents with each other or with supportive therapy alone. most strains of y. enterocolitica are susceptible to trimethoprimsulfamethoxazole, the aminoglycosides, piperacillin, imipenem, third-generation cephalosporins, amoxicillin-clavulanate potassium, and chloramphenicol, and resistant to amoxicillin, ampicillin, carbenicillin, ticarcillin, and m a c r o l i d e~.~~~-~~~ therapy in individual cases should be guided by in vitro susceptibility testing, although cefotaxime has been successfully used in bacteremic infants?54 aerornonas hydrophila is widely distributed in animals and the environment. although wound infection, pneumonia, and sepsis (especially in immunocompromised hosts) represent typical aeromonas infections, gastroenteritis increasingly is being recognized. the organism is a gramnegative, oxidase-positive, facultatively anaerobic bacillus belonging to the family vibrionaceae. like other members of this family, it produces an enter~toxin~~' that causes fluid secretion in rabbit ileal loops.%' some strains cause fluid accumulation in the suckling mouse model,"' whereas other strains are i n~a s i v e~~~ or cytotoxic. 964 the enterotoxin is not immunologically related to cholera toxin or the heat lt of although volunteer studies and studies with monkeys have failed to provide supportive evidence for enteropathog e n i~i t y ?~~,~~ there is good reason to believe that a. hydrophila does cause diarrhea in children. the earliest description of aeromonas causing diarrhea was an outbreak that occurred in a neonatal unit. 968 although several studies have failed to show an association with diarrhea,%9-974 most studies have found more aeromonas isolates among children with gastroenteritis than among ~o n t r o l s ?~~-~~~ part of the controversy may be caused by strain differences; some strains possess virulence traits related to production of gastroenteritis, whereas others do not. 970, 977 the diarrhea described in children is a disease of summer, primarily affecting children in the first 2 years of life. in one study, 7 (13%) of 55 cases of aeromonas detected during a 20-month period occurred in infants younger than 1 month. typically, watery diarrhea with no fever has been described; although there are descriptions of watery diarrhea with fever?78 however, in 22%, a dysentery-like illness occurred. dysentery-like illness has been described in the neonate. 979 in one third of children, diarrhea has been reported to last for more than 2 ~eeks.9~' there may be species-related differences in clinical features of aeromonas-associated gastroenteritis in children. 980 organisms that were formerly classified as a. hydrophila are now sometimes labeled as aeromonas sobria or aeromonas ~a v i a e .~~'~~~' fever and abdominal pain appear to be particularly common with a. sobria. one series of a. hydrophila isolates from newborns in dallas showed more blood cultures than stool cultures positive for a e r o m o n a~.~~~ diagnosis of enteric infection associated with aeromonas often is not made because this organism is not routinely sought in stool cultures. when the organism is suspected, the laboratory should be notified so that oxidase testing can be performed. the organism is usually susceptible to aztreonam, imipenem, meropenem, third-generation cephalosporins, trimethoprim-sulfamethoxazole, and chloramphenicol.q84~986 plesiomonas shigelloides is a gram-negative, facultative anaerobic bacillus that, like aeromonas, is a member of the vibrionaceae family. it is widely disseminated in the environment; outbreaks of disease are usually related to ingestion of contaminated water or seafood. 987 although it has been associated with outbreaks of diarrheal disease988 and has been found more commonly in ill than well controls, the role of i? shigelloides in diarrheal disease has remained contro~ersial.~~~ if it is a true enteropathogen, the mechanism by which it causes disease is ~n c l e a r . 9~~'~~' the role of this organism in neonatal diarrhea has not been extensively investigated. infections of neonates have been r e p~r t e d ?~' .~~~ but most cases of enteric disease currently reported in the united states are in adults.987 typical illness consists of watery diarrhea and cramps; sometimes, fever, bloody stools, and emesis occur and last for 3 to 42 days. diagnosis is not usually made by clinical microbiology laboratory testing because, as with aeromonas, coliforms can be confused with l ? shigelloides unless an oxidase test is performed. 996 the true frequency of infection is unknown. the organism has antibiotic susceptibilities similar to those of a e r~m o n a s .~~~'~~~ proving that an organism causes diarrhea is difficult, particularly when it may be present in large numbers in stools of healthy persons. bacteria that have been associated with acute gastroenteritis may be considered causative when the following criteria are met: 1. a single specific strain of the organism should be found as the predominant organism in most affected infants by different investigators in outbreaks of enteric disease in different communities. 2. this strain should be isolated in a significantly lower percentage and in smaller numbers from stool specimens of healthy infants. 3. available methods must be used to exclude other recognized enteropathogens, including viruses and parasites, enterotoxigenic agents, and fastidious organisms such as ca mpylobacter. 4. demonstration of effective specific antimicrobial therapy and specific antibody responses and, ultimately, production of experimental disease in volunteers are helpful in establishing the identity of a microorganism as a pathogen. optimally, the putative pathogen should have virulence traits that can be demonstrated in model systems. most bacteria that have been suggested as occasional causes of gastroenteritis in neonates fail to fulfill one or more of these criteria. their role in the cause of diarrheal disease is questionable. this is particularly true of microorganisms described in early reports in which the possibility of infection with more recently recognized agents could not be excluded. much of the clinical, bacteriologic, and epidemiologic data collected earlier linking unusual enteropathogens to infantile diarrhea must be reevaluated in light of current knowledge and methodology. several reports of acute gastroenteritis believed to have been caused by klebsiella suggest that, rather than playing an etiologic role, these organisms had probably proliferated within an already inflamed b~w e l .~~~-' '~' the recovery of klebsiella-enterobacter in pure culture from diarrheal stools has led several investigators to suggest that these bacteria may occasionally play a causative role in infantile gastroenteritis and enterocoliti~.'~~~~'"~ ingestion of infant formula contaminated with enterobacter sakazakii has been associated with development of bloody diarrhea and sepsis.'oo8 however, klebsiella species also may be isolated in pure culture from stools of newborns with no enteric s y m p t o m~. '~~~~'~' ' in one study, certain capsular types of klebsiella were more often isolated from infants with diarrheal disease than from normal infants.ioo2 later work has shown that klebsiella pneumoniae, enterobacter cloacae, and citrobacter species are capable of isolation of citrobacter species, such as those of klebsiella species, describe associations with enteric illnesses in up to 7% of cases.1014-1016 there is inadequate evidence to define the roles of klebsiella, enterobacter, and citrobacter species as etiologic agents of enteric illnesses. listeria monocytogenes, one of the classic causes of neonatal sepsis and meningitis (see chapter 14), has been linked to outbreaks of febrile diarrheal disease in immunocompetent adults and ~h i l d r e n . '~'~~'~~' seventy-two percent of ill individuals have had fever.ioz2 outbreaks have been related to ingestion of contaminated foods. listeria has rarely been described as a cause of neonatal gastroenteriti~.'~~~-~~~~ infection with enterotoxin-producing bacteroides fragilis has been associated with mild watery diarrhea.'027 these infections have a peak incidence in 2-to 3-year-old infants.1028 these toxin-producing organisms cannot be detected in routine hospital laboratories. a variety of organisms has been isolated from infant stools during episodes of diarrhea. most of these reports have failed to associate illness with specific organisms in a way that has stood the test of time. for example, i? aeruginosa 1029-1034 and have been associated with diarrhea, but proteus 1017, [1035] [1036] [1037] [1038] [1039] [1040] [1041] there are few convincing data suggesting that either is a true enteropathogen. these organisms generally are recovered as frequently from healthy infants as from infants with diarrheal disease, suggesting that their presence in stool cultures is significant.273,1042-1046 an association between providencia and neonatal enteritis has been substantiated largely by anecdotal reports of nursery outbreaks.215.27131016~1@'7 these bacteria are rarely isolated from infants with sporadic or community-acquired diarrheal disease.'042~'0m"04b~1050 candida albicans usually is acquired during passage through the birth canal and is considered a normal, although minor, component of the fecal flora of the neonate (see chapter 33).'05' intestinal overgrowth of these organisms frequently accompanies infantile gastroenteriti~,2"~~~~~'~~~*'~~~ particularly after antimicrobial therapy.233,247,'0521055 the upper small gut may become colonized with candida in malnourished children with diarrhea1056; whether the presence of the organism is cause or effect is unclear. stool cultures obtained from infants with diarrheal disease are therefore inconclusive, and although candida enteritis has been reported in adults,'057 the importance of this organism as a primary cause of neonatal gastroenteritis has been difficult to prove. clinical descriptions of nursery epidemics of candidal enteritis are poorly documented, generally preceding the recognition of epec and rotaviruses as a cause of neonatal diarrhea. even well studied cases of intestinal involvement add little in the way of substantive proof because secondary invasion of candida has been shown to be a complication of coliform enteritis.21 1,2333247 producing e n~e r o~o~n s~1 3 9~~~~1 6 7~1 7 5~1 9 3 ' " 0 1 2~1 0 1 3 reports of although diarrhea has sometimes been described as a finding in neonatal disseminated candidiasis, more typically, gastrointestinal tract involvement with disseminated candida is associated with abdominal distention and bloody stools mimicking necrotizing enterocoliti~.~~~"~~~-~~~~ typically, affected infants are premature and have courses complicated by antibiotic administration, intravascular catheter use, and surgical procedures during the first several weeks of life. a trial of oral anticandidal therapy may be helpful in neonates suffering from diarrhea in the presence of oral or cutaneous candidiasis. if the therapy is appropriate, a response should be forthcoming within 2 to 5 days. diarrhea sometimes occurs as a manifestation of systemic infection. patients with staphylococcal toxic shock syndrome, for example, often have diarrhea. loose stools sometimes occur in sepsis, but it is unclear whether the diarrhea is a cause or an effect. the organisms isolated from blood cultures in a group of bangladeshi infants and children with diarrhea included staphylococcus aureus, haemophilus inpuenzae, streptococcus pneumoniae, r aeruginosa, and various gramnegative enteric bacilli.'062 it is unknown whether the bacteriology of sepsis associated with diarrhea is similar in the well-nourished infants seen in industrialized countries. acute diarrhea associated with intestinal parasites is infrequent during the neonatal period. in areas with high endemicity, infection of the newborn is likely to be associated with inadequate maternal and delivery care, insufficient environmental sanitation, and poor personal hygiene standards. the occurrence of symptomatic intestinal parasitic infection during the first month of life requires acquisition of the parasite during the first days or weeks; the incubation period for e. histolytica and g. larnblia is 1 to 4 weeks, and for cryptosporidium parvum, it is 7 to 14 days. the newborn can be infected during delivery by contact with maternal feces,1o63 in the hospital through contact with the mother or personnel, or in the household through contact with infected individuals in close contact with the child. contaminated water can be an important source of infection for g. lamblia and c. parvum. organisms formerly identified as e. histolytica have been reclassified into two species that are morphologically identical but genetically distinct: e. histolytica and e. dispar. the former can cause acute nonbloody and bloody diarrhea, necrotizing enterocolitis, ameboma, and liver abscess, and the latter is a noninvasive parasite that does not cause disease. early acquisition of disease tends to be more severe in young infants; rarely, amebic liver abscess and rapidly fatal colitis have been reported in infant^."^^-'^^^ for example, a 19-dayold child from india who presented with 10 to 12 episodes of watery and mucous diarrhea, lethargy, jaundice, and mildly elevated liver enzymes has been described; the child recovered completely after 10 days of intravenous o m i d a~o l e . '~~~ however, asymptomatic colonization of neonates with various species of ameba is common in areas of high endemi~ity."~~ diagnosis can be established by stool examination for cysts and trophozoites and by serologic studies.'072 through the use of pcr, isoenzyme analysis, and antigen detection assays, e. histolytica and e. dispar can be differentiated.10733'074 serum antibody assays may be helpful in establishing the diagnosis of amebic dysentery and extraintestinal amebiasis with liver involvement. the efficacy of treatment with metronidazole for colitis or liver abscess has not been established for the newborn period, although this therapy has been used with success.1o65 patients with colitis or liver abscess caused by e. histolytica are treated also with iodoquinol, as are asymptomatic carriers. g. lamblia is a binucleate, flagellated protozoan parasite with trophozoite and cyst stages. it is spread by the fecal-oral route through ingestion of cysts. child-care center outbreaks reflecting person-to-person spread have demonstrated high i n f e~t i v i t y . "~~~'~~~ foodborne transmission and waterborne transmission also occur. infection is often asymptomatic or mildly symptomatic; cases of severe symptomatic infection during the immediate newborn period have not been reported. symptoms in giardiasis are related to the age of the patient, with diarrhea, vomiting, anorexia, and failure to thrive typical in the youngest children. seroprevalence studies have demonstrated evidence of past or current infection in 40% of peruvian children by the age of 6 months.io8' in a study of lactating bangladeshi mothers and their infants, 82% of women and 42% of infants excreted giardia once during the study; in some infants, this occurred before they were 3 months old.'o82 of these infected infants, 86% had diarrhea, suggesting that the early exposure to the parasite resulted in disease. in a prospective study of diarrhea conducted in mexico, infants frequently were infected with giardia from birth to 2 months, with a crude incidence rate of first giardia infection of 1.4 infections per child-year in this age group.6 the symptom status of these children was not reported but this study strongly suggests that g. lamblia may be more common than currently recognized among newborns living in developing areas. the diagnosis of giardiasis can be made on the basis of demonstration of antigen by eia or by microscopy of feces, duodenal fluid or, less frequently, duodenal b i~p~y . ' '~~~' '~' breast-feeding is believed to protect against symptomatic g i a r d i a~i s .~-~~* '~~~ this protection may be mediated by cellular and humoral i1nrnunity6~~"~~*"~~ and nonspecifically by the antigiardial effects of unsaturated fatty acids.iob6 giardia infections causing severe diarrhea may respond to metronidazole or furazolidone.'080 c. parvum is a coccidian protozoon related to toxoplasma gondii, lsospora belli, and plasmodium species. 1087s'088 the life cycle involves ingestion of thick-walled oocysts; release of sporozoites, which penetrate intestinal epithelium; and development of merozoites. there is asexual and sexual reproduction, with the latter resulting in formation of new oocysts that can be passed in stools. cryptosporidium species are ubiquitous. infection often occurs in persons traveling to endemic areas.'o89 because cryptosporidium infects a wide variety of animal species, there is often a history of animal contact among infected individ~als."~' person-to-person spread, particularly in household c~n t a c t s '~~'~' '~~ and daycare center^,"^^"'^^ is well documented and suggests that the organism is highly infectious. waterborne outbreaks of cryptosporidiosis occur and can be of massive proportion^."^^ the clinical manifestations of illness in immunocompetent persons resemble those of giardia infection but are somewhat shorter in d~ration'"~; asymptomatic carriage is rare. symptoms and signs include watery diarrhea, abdominal pain, myalgia, fever, and weight loss.1089*1090~1095~'096"098~'09 infection in the first month of life has been described.""~"" because symptoms resolve before excretion of oocysts ceases, a newborn whose mother has been ill with cryptosporidiosis in the month before delivery might be at risk even if the mother is asymptomatic at the time of the child's birth."'* with the increasing frequency of human immunodeficiency virus infection, it is likely that women with symptomatic cryptosporidiosis occasionally will deliver an infant who will become infected. infants infected early in life may develop chronic diarrhea and maln~trition."'~ the diagnosis of cryptosporidiosis is most typically made by examination of fecal smears using the giemsa stain, ziehl-neelsen stain, auramine-rhodamine stain, sheather's sugar flotation, an immunofluorescence procedure, a modified concentration-sugar flotation method, or an eia.' io4,1 lo5 nitazoxanide is effective therapy of immunocompetent adults and children ill with cryptosporidiosis.'io6 because illness is usually self-limited in the normal host, attention to fluid, electrolyte, and nutritional status usually suffices. enteric isolation of hospitalized infants with this illness is appropriate because of the high infectivity. several studies suggest that the risk of infection early in life may be decreased by breast-feeding. ' '"j viruses that infect the intestinal mucosa and cause primarily gastroenteritis are referred to as enteric viruses; they should not be confused with enteroviruses, members of picornaviridae family that are associated primarily with systemic illnesses. enteric viruses include rotaviruses, enteric adenoviruses, human caliciviruses, and astroviruses. other viruses such as coronaviruses, breda viruses, pestiviruses, parvoviruses, toroviruses, and picobirnaviruses have been sporadically associated with acute diarrhea but are currently considered of uncertain relevance. extensive reviews on the role of enteric viruses in childhood diarrhea can be found elsewhere."08-"" all four enteric viruses could conceivably infect the newborn, but the extent of exposure and clinical manifestations are largely unknown for astrovirus, enteric adenovirus, and human caliciviruses. rotavirus is the most extensively studied enteric virus. neonatal rotavirus infections have similar virologic and clinical characteristics to infection in older children, although some differences exist. rotavirus is a 75-nm, nonenveloped virus composed of three concentric protein shells: a segmented genome (1 1 segments), an rna-dependent polymerase, and enzymes required for messenger rna synthesis are located within the inner core. each segment codes for at least one viral protein (vp). the vp can be part of the structure of the virus, or it may be a nonstructural protein (nsp) required for replication, viral assembly, budding, determination of host range, or viral pathogenesis."" six distinct rotavirus groups (a through f) have been identified serologically based on common group of which three (a, b, and c) have been identified in humans.'io8 because group a rotaviruses represent more than 95% of isolated strains in humans worldwide, further discussion focuses on this group. group a rotaviruses are subclassified into serotypes based on neutralization epitopes located on the outer capsid. both rotavirus surface proteins, vp4 and vp7, can induce production of neutralizing antibodies.111431115 at least 10 vp7 types (g serotypes: gi to g6, g8 to g10, and g12) and nine vp4 types (p serotypes: pia, plb, p2a, p3, p3b, p4, p5, p8, and p12) have been detected among human r o t a v i r u~e s .~~~~-~~~~ by sequencing the vp4-coding gene, eight genomic p types (genotypes) have been identified that correspond to one or more of the described p antigenic types (genotype 8 to antigenic type pla, 4 to plb, 6 to p2a, 9 to p3,13 to p3b, 10 to p4,3 to p5, and 11 to p8)."" combining g antigenic with p antigenic and genetic typing, a specific rotavirus strain can be identified p antigenic type (p genetic type), g type. as an example, the human neonatal m37 strain is described as p2a[6], gi. from newborn nurseries, some of which seem to be endemic to the newborn units with high rates of asymptomatic infe~tion,"~~-"~' and less commonly, outbreaks of symptomatic infection.iiz8 these findings suggest that specific conditions of the newborn environment (e.g., child, nursery, personnel) may increase the possibility of reassortments between human strains; such strains may persist in these settings possibly through constant transmission involving asymptomatic newborns, adults, and contaminated surfaces. rotavirus primarily infects mature enterocytes located in the mid and upper villous e p i t h e l i~m .~~~~-"~~ lactase, which is present only on the brush border of the differentiated epithelial cells at these sites, may act as a combined receptor and uncoating enzyme for the virus, permitting transfer of the particles into the cell.1137 perhaps for this reason, infection is limited to the mature columnar enterocytes; crypt cells and crypt-derived cuboidal cells, which lack a brush border, appear to be resistant to rotaviral i n f e~t i o n . "~~' "~~ this concept also may explain why rotavirus infection is less common in infants younger than 32 weeks' gestational age than in more mature infants1139; between 26 and 34 weeks' gestational age, lactase activity is approximately 30% of that found in term infants. 1140 the upper small intestine is most commonly involved, although lesions may extend to the distal ileum and rarely to the ~0 l 0 n .~~~~7~~~~ interaction between intestinal cell and rotavirus structural and nonstructural proteins occurs, resulting in death of infected villous enter~cytes."~~ once infected, the villous enterocyte is sloughed, resulting in an altered mucosal architecture that becomes stunted and flattened. the gross appearance of the bowel is usually normal; however, under the dissecting microscope, scattered focal lesions of the mucosal surface are apparent in most cases. light microscopy also shows patchy changes in villous morphology, compatible with a process of infection, inflammation, and accelerated mucosal renewal. the villi take on a shortened and blunt appearance as tall columnar cells are shed and replaced by less mature cuboidal entero-ischemia may also play a role in the loss and stunting of villi"45 and activation of the enteric nervous system; active secretion of fluid and electrolytes may be another pathogenic mechanism.1146 during the recovery phase, the enteroblastic cells mature and reconstruct the villous structure. because of the loss of mature enterocytes on the tips of the villi, the surface area of the intestine is reduced. diarrhea that occurs may be a result of this decrease in surface area, disruption in epithelial integrity, transient disaccharidase deficiency, or altered countercurrent mechanisms and net secretion of water and electrolytes.1'33~1140~1142~1146~1147~1148 nsp4 has been found to induce age-dependent diarrhea in cd1 mice by triggering calcium-dependent chloride and water secretion.1149 the potential role of this "viral enterotoxin" in human disease is not yet clear.1150,1151 infants with asymptomatic rotavirus infections in the nursery are less likely than uninfected nursery mates to experience severe rotavirus infection later in life1152-1153; this finding suggested protective immunity and supported vaccine development. most studies have indicated that serum and intestinal antirotavirus antibody levels are correlated with protection against i n f e~t i o n "~~-"~~ although this correlation has not been ~n i v e r s a l .~~~~-~~~~ breast-feeding protects against rotavirus disease during the first year of life,57 probably including newborns. 1146 the high prevalence of antirotaviral antibodies in colostrum and human milk has been demonstrated by numerous investigators in widely diverse geographic areas.8 maternal rotavirus infection or immunization is accompanied by the appearance of specific antibodies in milk, probably through stimulation of the enteromammary immune between 90% and 100% of women examined in london, bangladesh, guatemala, costa rica, and the united states had antirotaviral iga antibodies in their milk for up to 2 years of rotavirus-specific igg antibodies have been found during the first few postpartum days in about one third of human milk samples a~s a y e d ,~~@~"~~ whereas i@ antibodies were detectable in about one half.1167 glycoproteins in human milk have been shown to prevent rotavirus infection in vitro and in an animal model."70 the concentration of one milk glycoprotein, lactadherin, was found to be significantly higher in human milk ingested by cytes.l 133, 1135, 1144 infants who developed asymptomatic rotavirus infection than in milk ingested by infants who developed symptomatic infe~tion.4~ rotaviruses probably infect neonates more commonly than previously recognized, although most infections seem to be asymptomatic or mildly symptomatic. 1128" 1 3 0 9 1 1 3 1~1 172-1 in a prospective study, the prevalence of rotavirus infection among neonatal intensive care unit patients was 18.4%. rotavirus has a mean incubation period of 2 days, with a range of 1 to 3 days in children and in experimentally infected adults. fecal excretion of virus often begins a day or so before illness and maximal excretion usually occurs during the third and fourth days, and generally diminishes by the end of the first week, although low concentrations of virus have been detected in neonates for up to 8 weeks.1140, [1186] [1187] [1188] [1189] rotavirus infections are markedly seasonal (autumn and winter) in many areas of the world, although in some countries seasonality is less striking; the reason for this is u n~l e a r .~~~" '~~ in nurseries in which persisting endemic infection has permitted long-term surveillance of large numbers of neonates, rotavirus excretion can follow the seasonal pattern of the community but can also show no seasonal it is not clear how units in which infection remains endemic for months or years differ from those with a low incidence of rotavirus. some nurseries are free of rotavirus infection' or minimally a f f e~t e d~~"~" whereas others have rotavirus diarrheal disease throughout the year or in outbreaks that involve 10% to 40% of low birth weight does not seem to be an important factor in determining the attack rate among infants at risk but may be important in rn0rta1ity.i~~~ infants in premature or special-care nurseries, despite their prolonged stays and the increased handling necessary for their care, do not demonstrate a higher susceptibility to infection; data regarding shedding of the virus are inconsi~tent!~*~~~~ after infection is introduced into a nursery, rotavirus probably will spread steadily and remain endemic until the nursery is closed to new admissions or nursing practices permit interruption of the cycle.1205 exactly how the virus is introduced and transmitted is uncertain, although limited observations and experience with other types of enteric disease in maternity units suggest several possibilities. the early appearance of virus in stools of some neonates indicates that infection probably was acquired at delivery. virus particles can be detected on the first45v11s6 or second"98 day of life in a significant number of infected infants. by day 3 or 4, most infected infants who will shed virus, with or without signs of illness, are doing so.117431186,1198 the large numbers of virus particles e x~r e t e d "~~,~~~~ suggest a fairly large and early oral inoculum. it is unlikely that contamination from any source other than maternal feces could provide an inoculum large enough to cause infection by the second day. transfer of particles from infant to infant on the hands of nursing and medical staff is probably the most important means of viral spread. with 10' to 10" viral particles usually present in 1 g of stool, the hands of personnel easily could become contaminated after infection is introduced into a nursery. there are numerous reports of nosocomial and daycare center rotavirus gastroenteritis outbreaks that attest to the ease with which this agent spreads through a hospital or institutional setting.'io8 admission of a symptomatic child usually is the initiating event, although transfer of a neonate with inapparent infection from one ward to another also has been incriminated. the most important factors influencing the incidence of rotavirus diarrhea in a nursery are the proximity to other newborns and the frequency of handling.1187 during a 4-month study, infants cared for by nursing staff and kept in communal nurseries experienced three epidemics of diarrhea with attack rates between 20% and 50%. during the same period, only 2% of infants rooming in with their mothers became ill, even though they had frequent contact with adult relatives and siblings. there is no clear evidence of airborne or droplet infection originating in the upper respiratory tract or spread by aerosolization of diarrheal fluid while diapers are changed. indirect evidence of airborne transmission includes the high infection rate in closed settings, the isolation of the virus from respiratory secretions,izo6 and the experimental observation of transmission by aerosol droplets in mice.'207 however, the respiratory isolation achieved by placin an evidence indicates that transplacental or ascending intrauterine infection occurs. transmission of virus through contaminated fomites, formula, or food is possible but has not been documented in newborns. rotavirus particles have not been found in human milk or c o l o~t r u m .~~~~~~~~ exposure of a newborn to rotavirus can result in asymptomatic infection or cause mild or severe gastro-outbreaks with high attack rates as measured by rotavirus excretion have been described but the extent of symptomatic infection severe rotavirus infection is seldom reported during the newborn period1203 but the extent of underreporting of severe disease, especially in the less developed areas of the world, has not been evaluated. it has been hypothesized that asymptomatic infections during the newborn period are the result of naturally attenuated strains circulating in this environment. rna electrophoretic patterns of rotaviruses found in certain nurseries have shown uniform and it has been suggested that these strains may be attenuated. the presence of unusual antigenic types such as the p2a[6] type within nurseries also suggests "less virulent strains." at least 10 rotavirus strains were documented to co-circulate in a tertiary care center during a 2-month period12" and in a different setting the same rotavirus strains by electropherotype produced asymptomatic infection in neonates and symptomatic infection in older infants.1183 newborns within a nursery exposed to a given rotavirus strain can develop symptomatic or asymptomatic infection. 130~1 2 1 0~1 2 i because newborns routinely have frequent relatively loose stools, it is possible that mild diarrhea episodes caused by rotavirus are being wrongly labeled as asymptomatic episodes. no clinical feature is pathognomonic of rotaviral gastroenteritis. early signs of illness, such as lethargy, irritability, vomiting, and poor feeding, usually are followed in a few hours by the passage of watery yellow or green stools free of blood but sometimes containing mucus.1187,1212-1214 diarrhea usually decreases by the second day of illness and is much infant in a closed incubator is not fully protective.118 s no enteritisel 129. 1130.1 173.1 179,1196,l 197,1201,1208 varies. 1 175.1 177.1 186.1 198,1203 improved by the third or fourth day. occasionally, intestinal fluid loss and poor weight gain may continue for 1 or 2 weeks, particularly in low-birth-weight infants. 1175 although reducing substances frequently are present in early fecal ~a m p l e s "~~~'~~~' 176~1187 this finding is not necessarily abnormal in neonates, particularly those who are breast-fed.1215 nevertheless, infants with prolonged diarrhea should be investigated for monosaccharide or disaccharide malabsorption or intolerance to cow's milk protein or in a prospective 49% of newborns with gastrointestinal symptoms in a neonatal intensive care unit had rotavirus detected in their stools. frequent stooling (present in 60%), bloody mucoid stool (42%), and watery stools (24%) were risk factors for a rotavirus infection. bloody mucoid stools, intestinal dilatation, and abdominal distention were significantly more common in preterm infants, but severe outcomes such as necrotizing enterocolitis and death did not differ among infected term and preterm infants. longitudinal studies in newborn nurseries and investigations of outbreaks among neonates rarely describe a severe adverse outcome or death.1139,1168,1187 because these infants are under constant observation, early detection of excessive fluid losses and the availability of immediate medical care are probably major factors in determining outcome. rotavirus gastroenteritis causes almost 400,000 deaths of infants every year,i2i7 concentrated largely in the poorest regions of the world. it is likely that in places where hospital-based care is uncommon, rotavirus causes neonatal deaths secondary to dehydration. group a rotavirus has been associated with a wide array of diseases in infants and children; reye syndromes, encephalitis-aseptic meningitis, sudden infant death syndrome, inflammatory bowel disease, and kawasaki syndrome have been described but not systematically studied. 1108 case reports and small case series have associated neonatal rotavirus infection with necrotizing e n t e r o c~l i t i s .~~'~"~~~ rotavirus infection may play a role in a small proportion of cases of necrotizing enterocolitis, although it probably represents one of many potential triggering factors. a significant association between neonatal rotavirus infection and bradycardia-apnea episodes was detected in one prospective study.1220 the possible association between natural rotavirus infection and i n t u s s u~c e p t i o n~~~~~~~~~ gained support after the association was made between the human-simian reassortant vaccine and intussusception in infants older than 2 months (attributable risk = 1: 10,000). 1224 intussusception is extremely uncommon in the newborn; it is highly unlikely that rotavirus triggers this disease in neonates. there are many methods used for detection of rotavirus in stool specimens, including electron microscopy, immune electron microscopy, elisa, latex agglutination, gel electrophoresis, culture of the virus, and reverse transcriptasepolymerase chain reaction. elisa and latex agglutination currently are the most widely used diagnostic techniques for detection of rotavirus in clinical samples. many commercial kits are available that differ in specificity and ~e n s i t i v i t y . '~~~-'~~~ in general, latex agglutination assays are more rapid than elisas but are less sensitive. the sensitivity and specificity of the commercially available elisas surpass 90%. checking of the elisa by another method such as gel electrophoresis or pcr amplification may be desirable if there is concern about false-positive results. fecal material for detection of rotavirus infection should be obtained during the acute phase of illness. whole-stool samples are preferred, although suspensions of rectal swab specimens have been adequate for detection of rotavirus by rotavirus are relatively resistant to environmental temperatures, even tropical temperatures, 1232 although 4°c is desirable for short-term storage and -70°c for prolonged storage.'io8 excretion of viral particles may precede signs of illness by several days' 190; maximal excretion by older infants and children usually occurs 3 to 4 days after onset of symptoms.1233 neonates can shed virus for 1 to 2 weeks after onset of symptoms. the primary goal of therapy is restoration and maintenance of fluid and electrolyte balance. despite the documented defect in carbohydrate digestion with rotavirus diarrhea, rehydration often can be accomplished with glucoseelectrolyte or sucrose-electrolyte solutions given orally .199,1234-1236 intravenous fluids may be needed in neonates who are severely dehydrated, who have ileus, or who refuse to feed. persistent or recurrent diarrhea after introduction of milkbased formulas or human milk warrants investigation for secondary carbohydrate or milk protein i n t~l e r a n c e . '~~~~~~" disaccharidase levels and xylose absorption return to normal within a few days1144 to weeks after infe~tion."~~ intractable diarrhea related to severe morphologic and enzymatic changes of the bowel mucosa is possible although rare in the newborn; it may require an elemental diet or parenteral nutrition. efficacy of anti-rotavirus antibodies (e.g., hyperimmune colostrum, antibody-supplemented formula, human serum immunoglobulin) and of probiotics has been p~s t u l a t e d , '~~~~'~~' although not convincingly shown1242; the widespread clinical use of these measures seems remote. one study suggests that use of lactobacillus during the diarrheal episode may decrease the duration of rotavirus-associated hospital stays, especially when used early in the course of the disease, although more studies are needed before recommending widespread use. 1241 hand hygiene before and after contact with each infant remains the single most important means of preventing the spread of infection. because rotavirus is often excreted several days before illness is recognized, isolation of an infant with diarrhea may be too late to prevent cross-infection unless all nursing personnel and medical staff have adhered to this fundamental precaution. infants who develop gastroenteritis should be moved out of the nursery area if adequate facilities are available and the infant's condition permits transfer. the use of an incubator is of value in reducing transmission of disease only by serving as a reminder that proper hand-hygiene and glove techniques are required, but is of little value as a physical barrier to the spread of encouraging rooming-in of infants with their mothers has been shown to be helpful in preventing or containing nursery epidemics.'243 temporary closure of the nursery may be required for clinically significant outbreaks that cannot be controlled with other measures.1128 development of rotavirus vaccines began in the early 1980s. candidate vaccines included bovine and rhesus monkey elisa. 1230, 1231 attenuated strains, human attenuated strains, and bovinehuman and rhesus-human reassortant strains."" in august 1998, the first licensed rotavirus vaccine, rotashield, an oral formulation of a simian-human quadrivalent reassortant vaccine, was recommended for use in children when they were 2, 4, and 6 months old. after approximately 500,000 children were vaccinated with more than 1 million doses, a significantly increased risk of intussusception was observed among vaccinated children, with an overall odds ratio of 1.8.iz4 use of this vaccine was terminated. two new vaccine candidates are undergoing phase i11 clinical trials: a "pentavalent" bovine-human reassortant vaccine including g types gl-g4 and p type pla[8] and a monovalent human attenuated pla[ 8]g1 vaccine. the epidemiology of rotavirus infection will change significantly if one or both candidates become widely available in the future. the impact on neonatal infection will depend on the effect of herd immunity in decreasing circulation of rotavirus strains. stools from breast-fed neonates are typically watery and yellow, green, or brown. the frequency of stooling can vary from one every other day to eight evacuations per day. in an active, healthy infant who is feeding well, has no vomiting, and has a soft abdomen, these varied patterns of stooling are not a cause for concern. physicians need to consider the child's previous frequency and consistency of stools and establish a diagnosis of acute diarrhea on an individual basis. close follow-up of weight increase in infants with nonformed stools can help confirm the clinical impression. a normal weight gain should direct medical action away from stool exams or treatment. diarrhea during the neonatal period is a clinical manifestation of a wide variety of disorders (table 20-6). the most common initiating factor is a primary infection of the gastrointestinal tract that is mild to moderate in severity, chapter 20 self-limited, and responsive to supportive measures. acute diarrhea can also be an initial manifestation of a systemic infection, including bacterial and viral neonatal sepsis. infants with moderate to severe diarrhea require close monitoring until the etiologic diagnosis and the clinical evolution are clarified. there are noninfectious diseases leading to chronic intractable diarrhea that may result in severe nutritional disturbances or even death unless the specific underlying condition is identified and treated appropriately. the differential diagnosis of a diarrheal illness requires a careful clinical examination to determine whether the child has a localized or a systemic process. lethargy, abnormalities in body temperature, hypothermia or hyperthermia, decreased feeding, abdominal distention, vomiting, pallor, respiratory distress, apnea, cyanosis, hernodynamic instability, hypotension, hepatomegaly or splenomegaly, coagulation or bleeding disorders, petechiae, and exanthemas should lead to an intense laboratory investigation directed at systemic viral or bacterial infection. if the process is deemed a localized intestinal infection, initial evaluation can be focused on differentiating an inflammatory-invasive pathogen from those that cause a noninflammatory process. for this, stool examination for fecal leukocytes, red blood cells, and lactoferrin can be a helpful indicator of the former. inflammatory diarrhea can be caused by shigella, salmonella, carnpylobacter, v parahaemolyticus, k: enterocolitica, eiec, eaec, c. difjcile, necrotizing enterocolitis, antibioticassociated colitis, and allergic colitis (i.e., milk or soy intolerance). noninflammatory causes of diarrhea include etec, epec, rotaviruses, enteric adenoviruses, calicivirus, astrovirus, g. larnblia and cryptosporidium. although supportive fluid therapy is mandatory for all types of diarrhea, the brief examination for fecal leukocytes and red blood cells can direct the diagnostic and therapeutic approach. pathogens such as shigella, salmonella, and ehec can cause watery or bloody diarrhea, depending on the specific host-pathogen interaction and the pathogenic mechanisms involved. some of the noninfectious diseases responsible for neonatal diarrhea are listed in table 20 the united states and panama food based oral rehydration salt solutions for acute childhood diarrhoea international study group on reduced osmolality ors solution. multicentre evaluation of reduced-osmolality oral rehydration salts solution antimicrobial resistance and enterotoxin production among isolates of escherichia coli in the far east high-molecular-weight plasmid correlates with escherichia coli invasiveness shigella guanabara, tip0 serologico destacado do grupo b ceylonensis-dispar a study of specific e. coli infections occurring in a unit for surgical neonates molecular characterization of strains of enteroinvasive escherichia coli 0143 untersuchungen zur kiologie der durchfallserkrankungen des sauglings a study of e. coli mutable from an outbreak of diarrhea in the new-born isolation of antigenically homogenous strains of bart. coli neopolitanum from summer diarrhea of infants slide agglutination of bacterium coli var. neopolitanum in summer diarrhea a complete somatic antigen common to sahone~la adelaide, escherichia coli-gomez and escherichia coli 0 1 ll:b4 an outbreak of infantile gastroenteritis in aberdeen epidemic gastroenteritis of infants in aberdeen during 1947 escherichia strains from infantile epidemic gastroenteritis identification of enterobacteriaceae association of escherichia coli sero-345. gastroenteritis due to escherichia coli enteropathogenic escherichia coli serotype ol11:hnt isolated from preterm neonates in nairobi, kenya bray's discovery of pathogenic esch. coli as a cause of infantile gastroenteritis cross-infection in infantile gastroenteritis acute intestinal infections of nondysenteric etiology protracted diarrhea of infancy treated by intravenous 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enteritis fluorescent antibody techniques for salmonella and other enteric pathogens colistin suppression of escherichia coli in stools. i. control of a nosocomial outbreak of diarrhea caused by neomycin-resistant escherichia coli olll:b4 genetic and phenotypic analysis of escherichia coli with enteropathogenic characteristics isolated from seattle children comparison of two assay methods for patterns of adherence to hep-2 cells of escherichia coli from patients with diarrhea an elisa for the detection of localized adherent classic enteropathogenic escherichia coli serogroups actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic escherichia coli yearbook of pediatrics use of an oral elemental diet in infants with severe intractable diarrhea controlled trial of orally administered lactobacilli in acute infantile diarrhea use of antibiotics in acute gastroenteritis among infants in hospital incidence of antibiotic resistance and r-factors among gram-negative bacteria isolated from the neonatal intestine antibiotic resistance of fecal escherichia coli. a comparison of samples from children of low and high socioeconomic groups transferable antibiotic resistance in enterobacteriaceae: relationship to the problems of treatment and control of coliform enteritis changing pattern of the antimicrobial susceptib of escherichia coli in neonatal infections resistance to antimicrobial drugs-a worldwide calamity discovery and development of new antibiotics: the problem of antibiotic resistance american academy of pediatrics and american college of obstetricians and gynecologists antibacterial effectiveness of routine handwashing mdtistate outbreak of escherichia coli 0157:h7 infections from hamburgers. mmwr morb mortal wkly rep 42258 the emergence of escherichia coli 0152h7 infection in the united states outbreaks of enterohemorrhagic escherichia coli 0157:h7 infection by two different genotype strains in japan a nationwide case-control study of escherichia coli 0157h7 infection in the united states environmental and food safety aspects of escherichia coli 0157:h7 infections in cattle epidemiology and spectrum of disease of escherichia coli 0157 hemolytic-uremic syndrome associated with escherichia coli 0157:h7 enteric infections-united states transmission of escherichia coli 0157:h7 infection in minnesota child day-care facilities an outbreak of diarrhea and hemolytic uremic syndrome from escherichia coli 0157:h7 in freshpressed apple cider pathogenesis of shigella diarrhea: evidence for a developmentally regulated glycolipid receptor for shigatoxin involved in the fluid secretory response of rabbit small intestine pathogenesis of shiga toxin-induced hemolytic uremic syndrome shiga-toxin-converting bacteriophages two distinct toxins active on vero cells from escherichia coli 0157 antigenic heterogeneity of escherichia coli verotoxins a plasmid of enterohemorrhagic escherichia coli 0157:h7 is required for expression of a new h b r i a l antigen and for adhesion to epithelial cells a dna probe to identlfy enterohemorrhagic escherichia coli of 0157:h7 and other serotypes that cause hemorrhagic colitis and hemolytic uremic syndrome methods for detection of stec in humans. an overview shiga toxin-producing escherichia coli in children with diarrhea: a prospective point-of-care study escherichia coli 0157:h7: clinical, diagnostic, and epidemiological aspects of human infection the risk of the hemolyticuremic syndrome after antibiotic treatment of escherichia coli 0157:h7 infections risk of hemolytic uremic syndrome after antibiotic treatment of escherichia coli 0157h7 enteritis: a meta-analysis enteroaggregative escherichia coli typical enteroaggregative escherichia coli are the most prevalent pathotypes causing diarrhea in mongolian children a sensitive and specific dna probe to identify enteroaggregative escherichia coli, a recently discovered diarrheal pathogen the export of coat protein from enteroaggregative escherichia coli by a specific atp-binding cassette transporter system pathogenicity of enteroadherent escherichia coli in adult volunteers heterogeneity of enteroaggregative escherichia coli virulence demonstrated in volunteers laboratory investigation of enteroaggregative escherichia coli 0 untypeab1e:hlo associated with a massive outbreak of gastrointestinal illness enteroaggregative escherichia coli and outbreaks of gastroenteritis in uk enteroaggregative e. coli associated with an outbreak of diarrhea in a neonatal nursery ward identification of a protein with toxigenic activity produced by enteroaggregative escherichia coli. abstracts of the general meeting of the enteroaggregative escherichia coli associated with persistent diarrhea in a cohort of rural children in india enteroaggregative escherichia coli and salmonella associated with non-dysenteric persistent diarrhea association of escherichia coli hep-2 adherence patterns with type and duration of diarrhoea persistent diarrhea in northeast brazil: etiologies and interactions with malnutrition use of pcr to identify enteroaggregative escherichia coli as an important cause of acute diarrhoea among children living in calcutta, india hep-2-adherent escherichia coli strains associated with acute infantile diarrhea, siio paulo age-specific prevalence of escherichia coli with localized and aggregative adherence in venezuelan infants with acute diarrhea heterogeneous virulence of enteroaggregative escherichia coli strains isolated from children in southwest nigeria adherence of non-enteropathogenic escherichia coli to hela cells prevalence of enteroaggregative escherichia coli among children with and without diarrhea in switzerland enteroaggregative and enterotoxigenic escherichia coli among isolates from patients with diarrhea in austria acute and chronic diarrhoea and abdominal colic associated with enteroaggregative escherichia coli in young children living in western europe a study of infectious intestinal disease in england microbiological findings in cases and controls unpublished observations markers of enteric inflammation in enteroaggregative escherichia coli diarrhea in travelers detection of enteroaggregative escherichia coli with formalin-preserved hep-2 cells improved detection of enteroaggregative escherichia coli using formalin-fixed hep-2 cells successful treatment of diarrheal disease associated with enteroaggregative escherichia coli in adults infected with human immunodeficiency virus azithromycin found to be comparable to levofloxacin for the treatment of u s travelers with acute diarrhea acquired in mexico rifaximin versus ciprofloxacin for the treatment of traveler's diarrhea: a randomized, double-blind clinical trial molecular characterization of a fimbrial adhesin, f1845, mediating diffuse adherences of diarrheaassociated escherichia coli to hep-2 cells diffuse and 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secretion, mucosal invasion, and morphologic reaction in the rabbit ileum probing for enterotoxigenicity among the salmonellae: an evaluation of biological assays pathogenesis of experimental salmonellosis: inhibition of protein synthesis by cytotoxin cytotoxin production by salmonella strains: quantitative analysis and partial characterization electron microscope studies of experimental salmonella infection. i. penetration into the intestinal epithelium by s. typhimurium bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes: antagonism of granule cationic proteins by lipopolysaccharide role of charge and hydrophobic interaction in the action of the bactericidallpermeability increasing protein of neutrophils on gram-negative bacteria resistance to intraceuular infection host parasite relations in mouse typhoid enhancement in vitro of the low interferon-gamma production of leukocytes from human newborn infants generation of gamma interferon 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salmonellosis outbreak due to powdered infant formula contaminated with lactose-fermenting salmonella virchow an epidemic among infants caused by s. muenchen rectal thermometer-mediated crossinfection with s. wadnvorth in a pediatric ward transmission of s. worthington by oropharyngeal suction in hospital neonatal unit an outbreak of salmonella enteritidis in a maternity and neonatal intensive care unit plasmid profiles and salmonella epidemiology etude clinique et bacttriologique d'une tpidemie de salmonellose en milieu hospitalier (s. oranienburg) a protracted hospitalassociated outbreak of salmonellosis due to a multiple antibioticresistant strain of s. indiana an extensive outbreak of gastroenteritis caused by s. newport a large outbreak of foodborne salmonellosis on the navajo nation indian reservation: epidemiology and secondary transmission typhoid fever in children typhoid carriage in pregnancy with infection of neonate salmonella infection presenting as hematochezia on the 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s. typhi and s. paratyphi in children younger than two years typhoid and paratyphoid fever in 192 hospitalized children in thailand fecal leukocytes in enteric infections efficiency of cultures of rectal swabs and fecal specimens in detecting salmonella carriers: correlation with numbers of salmonella excreted treatment of salmonella gastroenteritis in infants. the significance of bacteremia antibody response to the somatic antigen of s. newport in premature infants production of 0 and h agglutinins by a newborn infant infected with s. saint-paul an outbreak of salmonellosis associated with a fatality in a healthy child a large dose and severe illness molecular and epidemiological study of salmonella clinical isolates effect of antibiotic therapy in acute salmonellosis on the fecal excretion of salmonellae effect of antibiotic treatment on duration of excretion of s. typhimurium by children. bmw 21343 a controlled trial comparing trimethoprimlsulfamethoxazole, ampicillin, and no 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pathogens: enterobacteriaceae, pseudomonas, alcaligenes and aeromonas the intestinal flora in the etiology of infantile infectious diarrhea the bacteriological considerations of infantile enteritis in sydney beobachtungen iiber die atiologie der gastroenterocolitiden des sauglings-und kindesalters. iv. untersuchung der rolle der proteus morgani-stamme group d streptococci in the faeces of healthy infants and of infants with neonatal diarrhea neonatal enteritis due to providencia organisms infection with the providence type of paracolon bacillus in a residential nursery providence group of organisms in the aetiology ofjuvenile diarrhoea microbial flora of stomach and small intestine in infantile gastroenteritis diarrhoea associated with candida spp.: incidence and seasonal variation prevalence of candida species in nigerian children with diarrhea infantile diarrhea and malnutrition associated with candida in a developing community role of candida in indirect pathogenesis of antibiotic 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thailand review of g and p typing results from a global collection of rotavirus strains: implications for vaccine development neonatal rotavirus infection in bangladesh strain characterization and risk factors for nosocomial infection distinct population of rotaviruses circulating among neonates and older infants epidemiology of rotavirus in india detection and characterization of rotavirus g and p types from children participating in a rotavirus vaccine trial in belen an outbreak of diarrhea in a neonatal medium care unit caused by a novel strain of rotavirus: investigation using both epidemiologival and microbiological methods characterization of rotavirus infection in a hospital neonatal unit in pretoria, south africa neonatal rotavirus infection in belem, northern brazil: nosocomial transmission of a p[6] g2 strain detection and characterization of rotaviruses in hospitalized neonates in blantyre, malawi importance of a new virus in acute sporadic enteritis in children virus 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neutralizing antibody cord blood and breast milk antibodies in neonatal rotavirus infection epidemiology of human rotavirus types 1 and 2 as studied by enzyme-linked immunosorbent assay transfer of anti-rotaviral antibodies h-om mothers to their infants effects of antibodies, trypsin, and trypsin inhibitors on susceptibility of neonates to rotavirus infection antibodies to seven rotavirus serotypes in cord sera, maternal sera, and colostrum of german women rotavirus-inhibitory activity in serial milk samples from mexican women and rotavirus infections in their children during their first year of life secretory antibody directed against rotavirus in human milk-measurement by means of enzymelinked immunosorbent assay human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis clinical immunity after neonatal rotavirus infection. a prospective longitudinal study in young children stool viruses in babies in glasgow. 2. investigation of normal newborns in 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seven kits for detection of rotavirus in fecal specimens with a sensitive, specific enzyme immunoassay detection of rotavirus in stool specimens with monoclonal and polyclonal antibody-based assay systems evaluation of seven immunoassays for detection of rotavirus in pediatric stool samples comparative efficacy of commercial immunoassays for the diagnosis of rotavirus gastroenteritis during the course of infection comparison of direct electron microscopy, immune electron microscopy, and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritis viruses in children enzyme-linked immunosorbent assay (elisa) for detection of human reovirus-like agent of infantile gastroenteritis rotavirus particles can survive storage in ambient tropical temperatures for more than 2 months epidemiological aspects of rotavirus infection in hospitalized venezuelan children with gastroenteritis practice parameter: the management of acute gastroenteritis in young children oral hydration in 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childhood. i, 11 faecal excretion of oligosaccharides and other carbohydrates in normal neonates report of 130 patients diagnosed under 3 months of age over a 20 year period schwachman's syndrome. a review of 21 cases physiologic deficiency of pancreatic amylase in infancy: a factor in iatrogenic diarrhea enterokinase and trypsin activities in pancreatic insufficiency and diseases of the small intestine a new syndrome of bile acid deficiency-a possible synthetic defect disorders of the serum lipoproteins. i. lipoprotein deficiency states acrodermatitis enteropathica: defective metabolism of unsaturated fatty acids acrodermatitis enteropathica without hypozincemia congenital chloride diarrhea prostaglandin synthetase inhibitor in an infant with congenital chloride diarrhea primary hypomagnesemia with secondary hypocalcemia, diarrhea and insensitivity to parathyroid hormone congenital adrenal hyperplasia with disturbed electrolyte regulation watery diarrhoea with a vasoactive intestinal peptide-producing ganglioneuroblastoma chronic diarrhea of infancy: nonbeta islet cell hyperplasia wolman's disease in an infant neonatal megaloblastic anemia due to inherited transcobalamin i1 deficiency in 2 siblings tricho-hepato-enteric syndrome: further delineation of a distinct syndrome with neonatal hemochromatosis phenotype, intractable diarrhea, and hair anomalies circumvention of defective neutral amino acid transport in hartnup disease using tryptophan ethyl ester congenital na+ diarrhea: a new type of secretory diarrhea cow's milk allergy: manifestations, diagnosis and management colitis, persistent diarrhea, and soy protein intolerance milk-and soy-induced enterocolitis of infancy. clinical features and standardization of challenge regional enteritis in early infancy bloody diarrhea in the newborn infant of a mother with ulcerative colitis ulcerative colitis in children under one year: a twenty-year review intractable diarrhoea of infancy acquired immunodeficiency syndrome in infants the irritable colon of childhood (chronic nonspecific diarrhea syndrome) gut transit time and lactose malabsorption during phototherapy. i, 11 neonatal diagnosis of familial dysautonomia familial enteropathy: a syndrome of protracted diarrhea from birth, failure to thrive, and hypoplastic villous atrophy lethal familial protracted diarrhea infantile gastroenteritis due to water with high sulfate content diarrhea, red diapers, and child abuse management of children with infection-associated persistent diarrhea key: cord-016782-aods92rf authors: lessenger, james e. title: diseases from animals, poultry, and fish date: 2006 journal: agricultural medicine doi: 10.1007/0-387-30105-4_27 sha: doc_id: 16782 cord_uid: aods92rf nan a key problem is the lack of foot protection so that the unprotected feet of workers come in contact with feces of the animals. the fecal-hand route of transmission is also critical. perhaps the most insidious and difficult to control is the consumption of raw poultry and meat products by workers in farms and processing plants. many people in agriculture are living on subsistence or below-subsistence wages and consume products off the processing lines. many of these products are not fully processed and may contact pathogens that have not been killed through cooking or irradiation (see chapter 2) (table 27 .1) (3, 4) . the improper handling of manure is a major source of disease, including the use of manure on food crops, the discharge of manure into community water sources, and the spread of manure onto areas where children play. in canada, an outbreak of escherichia coli o157:h7 was traced to organic growers who contaminated their produce with cow manure containing e. coli. also in canada, an outbreak of citrobacter freundii infections was associated with parsley originating from an organic garden in which pig manure was used. other documented infections of humans from manure-contaminated foods includes listeria monocytogenes in cabbage contaminated by sheep waste, cryptosporidium spread by municipal water contaminated by cattle, salmonella hartford in food prepared by contaminated water from a shallow well polluted with poultry manure, and pleisomonas shigelloides infection associated with well-water contaminated by poultry manure (5) . 368 j.e. lessenger guan and holley (5) , and weber and rutala (6) . workers, visitors, inspectors, veterinarians, and people who live on or adjacent to farms, ranches, feedlots, processing plants, and other agricultural endeavors are at risk for contracting diseases from animals, poultry, or fish. one needs only to follow the animals from the farm to the feedlots, slaughter house, processing and sorting lines, and packaging plants to appreciate the large number of people who are at risk due to contact with animals and animal products. physicians and other health care professionals are also at risk as they visit farms and plants for inspections or orientations (6) . methods of preventing the transmission of infectious material from animals and poultry to agricultural workers mirror in many ways the safety techniques for protection from chemicals, trauma and other hazards (see chapter 6) . the methods are summarized in table 27 .2. key to the prevention of the transmission of animal disease to humans is the proper processing of food products. this includes proper cook times and temperatures, adequate refrigeration, and appropriate transportation, processing, and stocking in stores. personal protective equipment includes hats or head coverings and protective coats or uniforms that can be laundered and left at the plant or farm. boots should also be cleaned and left at the farm or plant. especially in poultry operations, protective particulate masks may be necessary. in some 27 . diseases from animals, poultry, and fish 369 protective physical barriers in farm, ranch, or plant design allow for the raising or processing of food products without actual contact of humans with the animals or products. built-in barriers, changing rooms, boot baths, and hand-free handling techniques allow for the safe and efficient handling of food. in british chicken hatcheries, an aggressive combination of egg sanitization and handling methods was successful in decreasing zoonotic infections and diseases spread through flocks. procedures included: 1. design changes in incubators 2. whole building ventilation systems 3. control of dust, fluff, and aerosol production 4. disinfection of surfaces and equipment 5. improved handling of wastes (7). policies and procedures to limit or prevent physical contact with animals, feces, or urine prevent transmission. rules prohibiting the consumption of food products on farms and ranches or on production lines are especially important. not only can the production food product be infectious to workers, but food brought in by workers can become contaminated, which mandates eating areas for workers away from the livestock (7) . aggressive veterinary monitoring of livestock can detect early evidence of disease outbreaks in herds. similarly, public health monitoring of disease in humans can detect and appropriately treat epidemics of food-borne disease in humans and trace the source to the food-processing breakdown that caused the disease. hazard analysis of critical control points (haccp) is crucial to the prevention of infections in herds. low cost, ease of performance, and rapidity of results are the key criteria for the tests, and are sometimes more important than the performance characteristics of sensitivity, specificity, and reproducibility. field test kits are available for bacterial, protozoa, antibiotic residue, and other parameters of animal health (8, 9, 10) . medical monitoring can detect early disease and prevent its spread to other employees, the food product, and family members. pre-placement medical monitoring can identify people who are susceptible to infection, for example people with diabetes or immune diseases. in parts of the world where bovine tuberculosis is common, tb skin test monitoring can detect early infections and allow early treatment (8, 9) . immunizations are expensive, unavailable in many parts of the world, and only recommended for areas of high infectivity or occupations of high risk such as veterinarians. three critical immunizations are tetanus, rabies, and influenza (see chapter 25) . vaccines against salmonella, shigella, and other pathogens are in development or testing. training and education in proper handling techniques are important. proper ways of herding, handling, and caring for animals and poultry can prevent infection and the transmission of infectious material. see chapter 5 for details of education and training. research and the development of new techniques to prevent transmission are critical. for example, airborne dust has been discovered to be a carrier of pathogens in broiler breeder pullets (chicken pens). the use of an electrostatic space charge system has decreased the particle concentration and, in the process, decreased the potential of disease transmission to other chickens and to poultry workers (11) . hygiene, both in the person and in the workplace, is essential in preventing the transmission of disease. for example, in many german piggeries workers must shower and change clothing when they enter and leave the buildings. this technique prevents the infection of the pigs with outside pathogens, the transfer of pathogens from one piggery to another, and the transfer of pathogens to the home environment. especially important are the cleaning of machinery and the timely cleaning of animal and poultry urine and feces. not only can urine and feces be infectious but they can attract insects that can spread pathogens. as in medicine, the most important hygiene procedure is aggressive hand washing for all persons handling food products. in louisiana, for example, alligator farmers must wear rubber boots and waders to protect themselves from pathogens (but not from bites, which can go right through the protective ensembles). each day, the pens must be flushed and hosed off to remove the wastes that could harbor pathogens dangerous to the alligator colonies and workers. governmental regulations and oversight are important in providing standardization and systemization of methods and procedures to reduce the risk of infection to agricultural workers. good regulations and oversight are evidence-based and consistent with sound agricultural methods (see chapter 4). it is not enough to have rules, regulations, equipment and techniques to prevent the spread of pathogens from animals and poultry to workers. fair and consistent supervision by knowledgeable managers is critical to see that the proper equipment and supplies are used and that handling and hygiene rules and regulations are carried out. game are mammals killed or captured in the field for human consumption or for their hides, including elk, boars, bison, and deer. production animals include cattle, pigs, goats, sheep, horses, dogs, deer, and other animals grown in small to large farms and ranches for human consumption. typically the animals are slaughtered and dressed in various cuts made from the different parts of the animal. in addition, many animals are raised and kept as pets. rabies is a common viral infection in children who live in rural areas and in people who handle un-immunized mammalian animals. the prophylaxis for rabies is discussed in chapter 31. with the exception of four cases where the disease was treated with intensive therapy, the disease is considered universally fatal. therefore, immunizations and prophylaxis are critical. during may and june 2003, the first cluster of human monkeypox cases in the united states was reported. most patients with this febrile, vesicular rash illness presumably acquired the infection from prairie dogs. monkeypox virus was demonstrated by using polymerase chain reaction in two prairie dogs in which pathologic studies showed necrotizing bronchopneumonia, conjunctivitis, and tongue ulceration. immunohistochemical assays for orthopoxviruses demonstrated abundant viral antigens in surface epithelial cells of lesions in conjunctiva and tongue, with lesser amounts in adjacent macrophages, fibroblasts, and connective tissues. viral antigens in the lung were abundant in bronchial epithelial cells, macrophages, and fibroblasts. virus isolation and electron microscopy demonstrated active viral replication in lungs and tongue. both respiratory and direct mucocutaneous exposures are potentially important routes of transmission of monkeypox virus among rodents and to humans. prairie dogs can be studied for insights into transmission, pathogenesis, and vaccine and treatment trials, because they are susceptible to severe monkeypox infection (12) . chronic wasting disease (cwd) in north american deer and elk has been associated with creutzfeldt-jakob disease (cjd) in 3 hunters who killed, prepared, and ate their own game. an absolute association was not established, but further monitoring is ongoing (see chapter 29) . creutzfeldt-jakob disease does not appear to be a problem with workers who raise cattle or dairy cows (13) . champylobacter chlamydophila abortus is a well recognized pathogen causing abortions in cattle and goats. a recent report from germany cites a case where a pregnant woman became infected from farm animals and aborted. this rare zoonotic infection underlines the insidious and widespread problem of zoonotic infections on farms (14, 15) . campylobacter jejuni and c. coli have recently become recognized as common bacterial causes of diarrhea. infection can occur at any age. sources of infection are typically mammalian and avian hosts. the usual incubation period of campylobacter enteritis is 2 to 5 days. fever, diarrhea and abdominal pain are the most common clinical features. the stools frequently contain mucus and, a few days after the onset of symptoms, frank blood. significant vomiting and dehydration are uncommon. a rapid presumptive laboratory diagnosis may be made during the acute phase of the illness by direct phase-contrast microscopy of stools. isolation of the organism from stools requires culture in a selective medium containing antibiotics and incubation under reduced oxygen tension at 42˚c. the organism persists in the stools of untreated patients for up to 7 weeks following the onset of symptoms. erythromycin may produce a rapid clinical and bacteriologic cure and should be used to treat moderately to severely ill patients as well as patients with compromised host defenses (14) . salmonellosis is one of the most important public health disease problems, affecting more people and animals than any other single disease in agriculture. in canada, for example, there were 7,138 cases of food-borne salmonellosis in humans during 2003. the native habitat of members of the genus salmonella is the intestinal tract of warm-blooded and many coldblooded vertebrates. in humans, the incubation period is 6 to 48 hours and produces headache, malaise, nausea, fever, vomiting, abdominal pain, and diarrhea (with and without blood). salmonella is also capable of invading the intestinal mucosa, entering the blood stream, and causing septicemia, shock, and death. the diagnosis is made through the clinical presentation and confirmation with blood and stool cultures and serology. treatment is first started empirically pending culture results and then adjusted if necessary. multi-drug resistant s. typhimurium bacteria have been documented to be present in milk after pasteurization (16, 17) . listeria monocytogenes is a zoonotic food-born pathogen that is responsible for 28% of food-related deaths in the united states annually and that is a major cause of food recalls worldwide. agricultural exposure is through drinking unpasteurized milk or direct contact with the animal or manure. the disease pattern is similar to salmonella (18). tuberculosis (tb) continues to be a worldwide infectious problem for humans. while human-to-human infection is of greatest concern, one infected dairy herd can infect hundreds, if not thousands, of people. potentially, tuberculosis can infect any mammal, although production cattle, especially dairy cattle, are at greatest risk. complicating efforts to combat the disease is the fact that deer, badgers, elk and other wild species have been found to harbor the mycobacterium. in england, badgers were found to be spreading the infection to herds of cattle. also, in england and ireland, herds of sheep were found to be infected. in new zealand, wild brush tail possums (trichosurus vulpecula) were discovered to be the main source of infection in livestock, including deer herds. in tanzania, tuberculosis-infected herds were found more often in small, pastoral farms that have little veterinary monitoring, as opposed to the large, commercial enterprises (19) (20) (21) (22) . in a los angeles zoo, tb was found in two asian elephants, three rocky mountain goats, and one black rhinoceros. an investigation found no active cases of tuberculosis in humans; however, tuberculin skin-test conversions in humans were associated with training the elephants and attending an elephant necropsy (23) . human-to-animal transmission of tb has been documented. in an exotic animal farm in illinois, three elephants died of mycobacterium tuberculosis and a fourth tested culture-positive. twenty-two handlers were screened for tb; eleven had positive reactions to intradermal injection with purified protein derivative. one had a smear-negative, culture-positive active tb. dna comparisons by is6110 and tbn12 typing showed that the isolates from the four elephants and the handler with active tb were the same strain, thus documenting that the infection of the elephants came from the handler (24) . mycobacterium (tuberculosis) can infect agricultural workers in a number of ways: 1. human-to-human contact with co-workers through the inhalation of respiratory droplets 2. drinking contaminated, unpasteurized milk 3. direct contact with infected animals 4. direct contact with the secretions of infected animals such as respiratory droplets, milk, manure, urine, semen, and vaginal secretions 5. direct contact or inhalations of respiratory droplets during necropsy, slaughter, or processing of meat or dairy products (20) (21) (22) (23) (24) . the clinical presentation is that of weight-loss, night sweats, a chronic cough, and hemoptysis. asymptomatic workers are typically discovered through public health surveys. diagnosis is through the purified protein derivative (ppd) skin test, smears of respiratory secretions demonstrating acid-fast bodies, cultures of respiratory secretions and other body fluids, radiographs demonstrating caseating granulomas, and other typical findings. treatment is by multidrug therapy, complicated by regional drug resistance patterns (20) (21) (22) (23) (24) . giardia infections have been associated with contaminated sewage and water in agricultural environments, producing gastroenteritis. in the sierra foothills of california, cattle drink water contaminated by infected beavers. beaver-and cattle-contaminated water is then consumed by unsuspecting tourists who develop crampy abdominal pain, fevers, and a profuse bloody diarrhea. the giardia infections are easily treated with metronidazole (5). fowl are birds that grow in the wild. nearly every bird found in the wild can be prepared for human consumption. poultry are birds grown in farm environments for human consumption. common poultry include: chickens, turkeys, ducks, pigeons, game hens, geese, doves, and peacocks. avian influenza a (h5n1) first infected humans in 1997, in hong kong. the virus was transmitted directly from birds to humans. eighteen people were admitted to hospitals, and 6 died. in 2003, 2 cases of avian influenza a (h5n1) infection occurred among members of a hong kong family, 3 of whom had traveled to mainland china. one person died. how or where these 2 people became infected was not determined. influenza a has the potential to cross species and has been implicated in the 3 flu pandemics in the 20th century (1918, 1957 and 1968 ). pandemics occur when 3 conditions are met: 1. the emergence of influenza a virus with a hemagglutinin subtype is completely different from that of strains circulating in humans for many preceding years. 2. there is a high proportion of susceptible people in the community (i.e., a population with low antibody titers to the new strain). 3. efficient person-to-person transmissibility of the new virus is possible with accompanying human disease (25) (26) (27) . the reported signs and symptoms of avian influenza in humans include: 1. typical flu-like symptoms such as fever, cough, sore throat, and muscle aches 2. eye infections 3. pneumonia 4. acute respiratory distress syndrome (ards) 5. multiple organ failure 6. lymphopenia 7. elevated liver enzyme levels 8. abnormal clotting profiles. physicians are advised to isolate the patient, initiate droplet precautions, and contact their local medical officer for further discussions if an epidemiological link is suspected. the world health organization (who) is moving to rapidly produce a new influenza vaccine capable of protecting people against the h5n1 strain of avian influenza a. preliminary genetic tests conducted in cdc laboratories in atlanta, london, and hong kong suggest that the h5n1 strain is resistant to amantadine and rimantadine but is believed to be susceptible to neuraminidase inhibitors. the who has recommended urgent, rapid culling of infected and exposed bird populations to eliminate the reservoir of the h5n1 strain. in addition, who has discouraged the practice of marketing live poultry directly to consumers in areas currently experiencing outbreaks of avian influenza a (h5n1). some countries have introduced trade restrictions to protect animal health. however, available data do not suggest that processed poultry products (i.e., refrigerated or frozen carcasses and products derived from them) or eggs from affected areas pose a public health risk. the virus is killed by cooking (25) (26) (27) . newcastle disease is caused by virulent strains of apmv. death rates among naive bird populations can exceed 50%. the virus responsible for newcastle disease has been known to cause conjunctivitis and upper respiratory infections in humans since the 1940s. the disease is self-limiting and does not have any permanent consequences (28) . in 2002, wisconsin public health officials were notified of two cases of febrile illness in workers at a commercial turkey breeder farm. a high prevalence of west nile virus antibody was found among workers and turkeys. an associated high incidence of febrile illness among farm workers also was observed. possible non-mosquito transmission among birds and subsequent infection of humans was postulated, but the mode of transmission was unknown (29) . avian tuberculosis was diagnosed in two mature rheas on different ratite farms over a 2-year period. both birds died after weight loss and development of granulomas in the lungs of one bird and bilaterally in the cubcutis cranial to the shoulder in the other. smears and cultures of the granulomas were positive for acid-fast bacilli and tuberculosis (30) . chlamydophila (chlamydia) psittaci, c. trachomatis, and c. pneumoniae can be passed from birds of all species to humans. wild pigeons and pheasants have been demonstrated to be a source. wild birds in captivity, pets (usually cockatiels, parakeets, parrots, and macaws), and production animals can infect workers, and there are reports of customs and health inspection workers becoming infected. infection is through contact with feces, urine, and oral secretions (31) . mild infection produces a tracheobronchitis with flu-like symptoms of cough, congestion, myalgias, fatigue, and fever. in severe infections, untreated workers, and immunocompromised workers, pneumonia, sepsis, shock, and death can occur. radiographs reveal a lobar infiltrate (31) . diagnosis is by detection of the 16s rrna gene of c. psittasi in sputum with a pcr analysis, and a typical radiographic appearance and culture. tetracyclines and erythromycin are effective for treatment. prevention is through close monitoring and culling flocks and pet birds and personal protection equipment (32). raising poultry at home is common in low-income countries. studies demonstrate that proximity to free-range domestic poultry increases children's risk of infection with diarrhea-causing organisms such as campylobacter jejuni. corralling might reduce the risk, but research on the socioeconomic acceptability of corralling is lacking. many people report that home-grown poultry and eggs taste better and are more nutritious. they enjoy living around animals and want to teach their children about raising animals. to prevent theft, some residents shut their birds in provisional enclosures at night but allege that birds are healthier, happier, and produce better meat and eggs when let loose by day. many rural peoples view bird feces in the house and yard as dirty, but few see a connection to illness. residents consider chicks and ducklings more innocuous than adult birds and are more likely to allow them inside the house and permit children to play with them. additional food and water costs with corralling are a significant obstacle for some. adequate space and corral hygiene must also be addressed to make this intervention viable. developing a secure, acceptable, and affordable corral remains a challenge for rural populations (33, 34) . although approximately 95% of disease caused by non-typhoidal salmonella is transmitted by food-borne vehicles, four documented salmonella outbreaks in the 1990s have been traced to contact with young poultry. no environmental studies of source hatcheries were completed. a case-control study was performed by comparing culture-confirmed salmonella infantis in michigan residents, identified between may and july 1999, with two age-and neighborhood-matched controls. eighty environmental and bird tissue samples were collected from an implicated hatchery; all salmonella isolates underwent pulsed-field gel electrophoresis (pfge) analysis. the study included 19 case-patients sharing the same pfge subtype and 37 matched controls. within 5 days before illness onset, 74% of case-patients resided in households raising young poultry compared with 16% of controls (matched or 19.5; 95% ci 2.9, 378.1). eight hatchery samples yielded s. infantis with pfge subtypes matching the patients' isolates. this investigation identified birds from a single hatchery as the source of human illness and confirmed the link by matching pfge patterns from humans, birds and the hatchery environment. subsequent public health interventions reduced, but did not eliminate, transmission of poultry-associated salmonellosis. five additional pfge-linked cases were identified in spring 2000, necessitating quarantine of the hatchery for depopulation, cleaning and disinfection (35) . fish farming, or aquaculture, for fish and shellfish is becoming more common and more internationalized with every passing year. in the united states, more than half the seafood consumption is imported, much of it from fish farming. the world's seafood trade is very complex, and if is often difficult or impossible to determine where the seafood is raised or harvested. for example, the united states imports salmon from switzerland and panama though neither country is known for large salmon fisheries (36) . in general, farmed fish is as safe and nutritious as wild-caught species, but there are public health hazards associated with ignorance, abuse, and neglect of aquaculture technology. numerous small fish ponds increase the shoreline of ponds causing higher densities of mosquito larvae and cercaria, which can increase the incidence and prevalence of lymphatic filariasis and schistosomiasis. especially dangerous is the use of human waste draining to fertilize or create ponds. technology abuse includes the misuse of therapeutic drugs, chemicals, fertilizers and natural fish habitat areas. technology neglect includes the failure to pay attention to mosquito habitats and the concomitant increase in malaria, as well as the propagation of other organisms (36) . human exposure can be through direct skin contact with fish or the consumption of contaminated fish or shellfish products or contaminated water. the main pathogens acquired topically from fish (through spine puncture or open wounds) are aeromonas hydrophila, edwardsiella tarda, erysipelothrix rhusiopathiae, mycobacterium marinum, streptococcus iniae, vibrio vulnificus, and vibrio damsela. s. iniae has recently emerged as a public health hazard associated with aquaculture, and m. marinum often infects home aquarium hobbyists. common zoonoses contracted through the consumption of contaminated products or water include salmonella, leptospirosis, yersiniosis, and tuberculosis (37) . salmonellae species have been found associated with all of the poikilothermic vertebrate species studied, as well as the mollusks and crustaceans (38) . leptospirosis does occur in the poikilothermic vertebrates, as evidenced by positive serological reactions and by the isolation of pathogenic leptospiral serovars. the finding of leptospirosis species in fish, mollosks and other aquatic species are of special importance in view of the increased worldwide interest in aquaculture farming. since 1975, 24 of the 101 (23.7%) reported human cases of leptospirosis in hawaii have been associated with aquaculture industries (taro farms, prawn farms and watercress farms) (39) . species of yersinia are a particular problem in fish and in people involved in fish farming. workers who wade in fish ponds or drink drainage water are especially at risk. yersinia enterocolitica has been demonstrated to be a causative agent in acute diarrhea illness in humans after workers become infected through the feces-hand-oral route (19) . tuberculosis has also been reported in freshwater and marine fish species (piscine tuberculosis), especially in those grown on fish farms. mycobacterium marinum and m. celonae have been demonstrated in fish farms (30) . turtles, lizards, snakes, green iguanas (iguana iguana), alligators, and crocodiles are grown from eggs in farms for their hides and meat. some species are also grown for sale as pets. salmonella infections in persons who had contact with reptiles usually cause gastroenteritis but can result in invasive illness, including septicemia and meningitis, especially in infants and immunocompromised persons. for decades, reptiles have been known to be a source for salmonellosis; however, numerous reptile owners remain unaware that reptile contact places them and other household members, including children, at greater risk for infection. (40) captive reptiles (such as iguanas) are routinely identified as reservoirs of salmonella and the number of reports about reptile-associated salmonellosis is increasing. in germany and austria, salmonella was detected in 54.1% of fecal reptile samples cultured. the percentage of salmonella-positive samples was significantly lower in turtles as compared with lizards and snakes, as salmonella was only detected in one sample from a single turtle out of 38 turtles investigated. in all, 42 different salmonella serovars were found. all isolated salmonella belonged to the species enterica, predominantly to the subspecies i (n = 46) and iiib (n = 30) but also to subspecies ii (n = 3), iiia (n = 6), and iv (n = 2). all isolates were sensitive to the antimicrobials examined. a significantly higher percentage of salmonella-positive reptiles was detected in the group of owners who purchased reptiles in comparison with pure breeders. the high percentage of salmonella in reptiles in the study confirms the risk for the transmission of the infection to humans (41) . zoonoses as a risk when associating with livestock or animal products social and environmental risk factors in the emergence of infectious diseases public health implications related to spread of pathogens in manure from livestock and poultry operations occupational bio hazards: current issues pathogen survival in swine manure environments and transmission of human enteric illness: a review zoonotic infections an approach to reduction of salmonella infection in broiler chicken flocks through intensive sampling and identification of cross-contamination hazards in commercial hatcheries maff statuatory incident reports in surveillance, prevention, and control of human salmonella typhimurium infection surveillance and control of emerging zoonoses testing to fulfill haccp (hazard analysis critical control points) requirements: principles and examples effect of an electrostatic space charge system on airborne dust and subsequent potential transmission of microorganisms to broiler breeder pullets by airborne dust veterinary monkeypox virus working group. monkeypox transmission and pathogenesis in prairie dogs wild game feasts and fatal degenerative neurological illness abortion in humans caused by chlamydophila abortus chlamydophila abortus infection in a pregnant woman associated with indirect contact with infected goats human salmonellosis associated with exotic pets multidrugresistant salmonella typhimurium infection from milk contaminated after pasteurization dairy farm reservoir of listeria monocytogenes sporadic and epidemic strains a study of the foodborne pathogens: campylobacter, listeria and yersinia, in faeces from slaughter-age cattle and sheep in australia factors influencing the incidence and scale of bovine tuberculosis in cattle in southwest england diagnostic strategies and outcomes on three new zealand deer farms with severe outbreaks of bovine tuberculosis prevalence of bovine tuberculosis in cattle in different farming systems in the eastern zone of tanzania human exposure following mycobacterium tuberculosis infection of multiple animal species in a metropolitan zoo mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants avian influenza: recent developments human health implications of avian influenza viruses and paramyxoviruses avian influenza outbreak: update phylogenetic relationships among virulent newcastle disease virus isolates from the 2002-2003 outbreak in california and other recent outbreaks in north america center for disease control and prevention. west nile virus infection among turkey breeder farm workers: wisconsin tuberculosis in farmed rheas (rhea americana) a flu like syndrome a woman with a lobar infiltrate due to psittacosis detected by polymerase chain reaction campylobacter, from obscurity to celebrity campylobacter enteritis human salmonellosis associated with young poultry from a contaminated hatchery in michigan and the resulting public health interventions public, animal, and environmental health implications of aquaculture topically acquired bacterial zoonoses from fish: a review epidemiologic aspects of salmonellosis in reptiles, amphibians, mollusks and crustaceans-a review leptospirosis in poikilothermic vertebrates. a review reptile-associated salmonellosis-selected states salmonella enterica in reptiles of german and austrian origin systemic infection with alaria americana (trematoda) amphibians include frogs, toads, newts, and salamanders that are caught in the wild or grown on farms for use as food or as pets. frogs are caught in the wild and grown in farms for their meat, primarily frog legs. eating inadequately cooked frog legs can lead to an infection of alaria americana, a trematode. increasing evidence suggests that amphibians can pose risks for salmonellosis in humans (42) . key: cord-251991-ghbpga1s authors: harcourt, jennifer l.; caidi, hayat; anderson, larry j.; haynes, lia m. title: evaluation of the calu-3 cell line as a model of in vitro respiratory syncytial virus infection() date: 2011-03-31 journal: j virol methods doi: 10.1016/j.jviromet.2011.03.027 sha: doc_id: 251991 cord_uid: ghbpga1s respiratory syncytial virus (rsv) replication is primarily limited to the upper respiratory tract epithelium and primary, differentiated normal human bronchial epithelial cells (nhbe) have, therefore, been considered a good system for in vitro analysis of lung tissue response to respiratory virus infection and virus–host interactions. however, nhbe cells are expensive, difficult to culture, and vary with the source patient. an alternate approach is to use a continuous cell line that has features of bronchial epithelial cells such as calu-3, an epithelial cell line derived from human lung adenocarcinoma, as an in vitro model of respiratory virus infection. the results show that calu-3 fully polarize when grown on permeable supports as liquid-covered cultures. polarized calu-3 are susceptible to rsv infection and release infectious virus primarily from the apical surface, consistent with studies in nhbe cells. the data demonstrate that polarized calu-3 may serve as a useful in vitro model to study host responses to rsv infection. respiratory syncytial virus (rsv) is the major cause of lower respiratory tract illness in infants and children worldwide, and infection can also result in serious disease in elderly and immunecompromised patients (falsey et al., 1995; hall et al., 1991; panitch, 2001; shay et al., 1999) . rsv enters the respiratory tract primarily through fomite or hand-to-nasal epithelium transmission following contact with infectious secretions (hall and douglas, 1981; hall et al., 1980) . infection is usually limited to the upper respiratory tract, and progression into the lower respiratory tract can result in serious complications of infection, including hospitalization for pneumonia and bronchiolitis. in humans, similar to other paramyxoviruses, rsv infection and replication primarily occurs in ciliated airway epithelial cells (wright et al., 1997) . as infection progresses, perivascular and peribronchiolar mononuclear cell infiltration is followed by necrosis of the airway epithelium (aherne et al., 1970; downham et al., 1975; ferris et al., 1973) . the mechanisms of cellular responses to rsv infection have been studied extensively in vitro in a variety of immortalized epithelial cell lines grown in monolayer cultures, including but not limited to vero, hep-2, a549, and ଝ the findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of cdc. * corresponding author at: national center for immunization and respiratory dismadin-darby canine kidney (mdck) cells (kwilas et al., 2009; li et al., 2010; lupfer and pastey, 2010; roberts et al., 1995; stark et al., 1996; swedan et al., 2009; wright et al., 2005) . in contrast to observations made in rsv infected non-polarized epithelial cell lines, the in vivo response to rsv infection is directional. one ex vivo model, differentiated, polarized cell cultures of primary normal human bronchial epithelial cells (nhbe), has provided some insights into the mechanism of rsv infection and the cellular response to infection. this model system more closely mimics the airway epithelium structure than monolayer-cultured cells, and likely provides a better in vitro model of rsv infection and the associated cellular responses. in nhbe cells, rsv primarily infects ciliated lumenal columnar cells, and infectious virus is released primarily from the apical surface of nhbe (zhang et al., 2002) . these data from the polarized, differentiated nhbe model demonstrate the insights that can be gained using cell systems that more closely mimic the airway epithelium. however, nhbe cell systems are time-and labor-intense, costly to maintain, and require access to primary lung tissue samples. additionally they require substantial expertise in isolating and culturing epithelial cells from the lung tissue, and significant amount of time to first polarize and then differentiate at an air-liquid interface. given these limitations, an alternative to nhbe cells as an in vitro model of polarized airway epithelium cells was identified and evaluated. calu-3 cells originate from a human bronchus adenocarcinoma of a mixed phenotype, believed to be derived from submucosal gland serous cells (yoshikawa et al., 2009) . when grown on trans-well inserts, calu-3 polarize into apical and basofig. 1 . calu-3 susceptibility to rsv infection. calu-3 cells were cultured as monolayers and infected with rsv strain a2 at moi 1, 5, 10 or 20 (a). at days 4 and 7 post-infection, the presence of rsv-f message rna was analyzed by one-step rt-pcr (a). tight junction formation and rsv g protein gene expression at 7 days post-infection (moi = 10) was evaluated by immunofluorescence assay (b). tight junction formation was evaluated by staining cells with anti-zona occludens -1 (zo-1) antibody (novus biologics) and 594-alexafluor conjugated anti-ms igg (invitrogen; red), rsv-g protein expression was evaluated by staining cells with 488-alexafluor conjugated anti-rsv-g protein monoclonal antibody 131-2g (green), and nuclei were stained with dapi (blue). stained cells were visualized using a zeiss axioimager microscope at 10× magnification. lateral surfaces, but do not differentiate into multiple cell types and layers as observed with cultured nhbe (grainger et al., 2006) . polarized calu-3 are susceptible to infection by respiratory viruses, including influenza virus a, rhinovirus and severe acute respiratory syndrome -associated coronavirus (sars-cov) (grantham et al., 2009; saedisomeolia et al., 2009; yoshikawa et al., 2009 yoshikawa et al., , 2010 . the studies presented here evaluate polarized calu-3 as an in vitro model of rsv infection. to determine whether rsv infects and replicates in non-ciliated respiratory epithelial cells, monolayers of non-polarized calu-3 were infected with rsv strain a2 at a multiplicity of infection (moi) between 1 and 20, and viral rna expression was determined. rsv-f viral mrna was detectable at both 4 and 7 days post-infection by rt-pcr, demonstrating that calu-3 cells can be infected by rsv (fig. 1a) . the expression of rsv surface proteins attachment, g, and fusion, f, was examined by immunofluorescence assay using anti-rsv-g (131-2g) and anti-rsv-f (131-2a) monoclonal antibodies. at seven days post-infection, there was little evidence of syncytia formation and rsv g (fig. 1b) and rsv f proteins were detected in a few small clusters of cells within the calu-3 monolayer (fig. 1b) . interestingly, tight junction formation, as indicated by immunos-taining for zona occludens (zo)-1 (fig. 1b) , was maintained during 7 days of infection. rsv surface protein expression co-localized with tight junctions (fig. 1b) , consistent with the lack of any obvious cytopathology in differentiated, rsv-infected nhbe cells (zhang et al., 2002) . polarized calu-3 cultures may be generated by growing cells at an air-liquid interface (ali) or as liquid-covered cultures (lcc). calu-3 cells were evaluated in liquid-covered culture (lcc), in which they are primarily non-ciliated (grainger et al., 2006) . the polarization of calu-3 in lcc is dependent on several factors including growth medium, cell density and trans-well membrane pore size and composition. for these studies, calu-3 were seeded at a density of 6.0 × 10 5 cells/cm 2 onto polyester trans-well inserts (0.33 cm) with 3 pores (corning) in eagles modified essential medium (emem, gibco) supplemented with 10% fetal bovine serum (emem-10% fbs, complete medium), and polarization was demonstrated by trans-epithelial electrical resistance (teer) development and by a passive sodium fluorescein equilibration assay. following seeding into trans-well culture, the development of teer by calu-3 cultures was monitored using an epithelial voltohmmeter (world precision instruments). the resistance of calu-3 fig. 2 . directional release of rsv from polarized calu-3. apical and basolateral compartment media of trans-well cultured calu-3 was replaced every 3-4 days. resistance development was confirmed by a passive sodium fluorescein equilibration assay as previously described (a, (geys et al., 2006 (geys et al., , 2007 ), and is representative of 3 independent experiments. resistance development of calu-3 infected with rsv-a2 (moi = 1) at the apical surface (b), and apical (c) and basolateral (d) release of infectious virus were monitored for 6 weeks post-infection. for resistance development, each value represents the median ± sem measurements from 3 individual wells (3 measurements/insert) and is presented as cm 2 . the amount of infectious virus released from the apical (c) and basolateral (d) media of calu-3 infected at gradually increased after seeding into trans-wells, and peaked between 1500 and 2100 cm 2 by 21 days post-seeding (data not shown). calu-3 polarization was confirmed by measuring the passive diffusion of sodium fluorescein through the cell monolayer as previously described (geys et al., 2006 (geys et al., , 2007 . briefly, polarized cells were washed with non-fluorescent buffer, and sodium fluorescein (1 mg/ml) was added to the apical compartment and non-fluorescent buffer to the basolateral compartment of cells. after an hour incubation, the amount of dye that diffused into the basolateral compartment was determined by spectrophotometric analysis of the basolateral sample compared to a sodium fluorescein standard curve. polarized calu-3 cultures do not allow equilibration of sodium fluorescein between apical and basolateral compartments. as the measurable resistance of polarized cells increased, the percent of sodium fluorescein that diffused into the basolateral compartment declined from 25% of the initial amount of dye added to the apical compartment when cells were non-polarized, to less than 1% when teer was ≥1200 cm 2 ( fig. 2a) . calu-3 were considered to be completely polarized once the teer was ≥1200 cm 2 and the amount of sodium fluorescein that diffused into the basolateral compartment was less than 1% of the initial amount in the apical compartment, the threshold of polarization (geys et al., 2006 (geys et al., , 2007 . polarized cells were infected at either the apical or basolateral surface with rsv strain a2 at an moi of 1, based on the concentration of cells initially seeded into the trans-well inserts. apical infections were performed by overlaying virus on the apical surface of polarized cells for 2 h at 37 • c. for basolateral surface infections, trans-well inserts were inverted and virus was applied directly to the basolateral surface and incubated in similar conditions. for both infections, inoculum was removed after incubation, wells that were infected at the basolateral surface were re-inverted into their original locations, and growth medium was added to all wells. at the indicated times post-infection, teer was measured, apical and basolateral media were collected, and virus was quantitated by standard plaque assay on vero cell monolayers as previously described (sullender, 1995) . there was little variability in the teer of calu-3 prior to apical infection (data not shown), but greater variability between individual trans-wells in the teer of rsv-a2 -infected and mock-infected calu-3 following infection (fig. 2b ). this decline in the stability of teer measurements was observed in both mock-and rsv-a2 -infected calu-3, and occurred following infection at either the apical (fig. 2b ) or basolateral (data not shown) surface. the fluctuations in resistance observed following infection may in part be due to a disruption in the polarized state of cells caused by the infection process. despite resistance fluctuations following rsv infection, the overall teer of polarized calu-3 was not significantly decreased compared to mock-infected cells for at least 6 weeks post-infection, suggesting that apical or basolateral rsv infection of polarized calu-3 does not significantly alter tight junction formation. these observations are consistent with rsv-infection in calu-3 monolayer cultures (fig. 1b) , and with rsv infection of polarized, differentiated nhbe (zhang et al., 2002) . in vitro and ex vivo studies have shown that rsv is released apically from infected, polarized epithelial cells (batonick et al., 2008; brock et al., 2003; roberts et al., 1995; wright et al., 2005; zhang et al., 2002) . virus release into the apical compartment of calu-3 infected at the apical surface was detectable as early as 4 days post-infection (fig. 2c) . while the amount of virus release varied between time points and between individual samples at each time point, infectious virus was detectable for at least six weeks the apical surface was evaluated by plaque assay on vero cells from three individual samples, and is presented in pfu/ml for each sample. the lower limit of detection for the plaque assays is indicated with a dashed line. data is representative of 3 independent experiments. fig. 3 . anti-rsv-f antibody neutralizes rsv infection of polarized calu-3. resistance development of calu-3 infected at the apical surface (a), and apical (b) and basolateral (c) release of infectious virus from apically-infected calu-3 (moi = 1) were monitored for 8 weeks post-infection. prior to infection, virus was incubated with anti-rsv-f (143-6c) or anti-rsv-n (130-12h) antibody at either 50 g/ml or 5 g/ml for 1 h at 37 • c. each teer value represents the median ± sem measurements from 4 individual wells (3 measurements/insert) and is presented as cm 2 . the amount of infectious virus released from the apical (b) and basolateral (c) media of calu-3 infected at the apical surface was evaluated by plaque assay on vero cells from four individual samples, and is presented in pfu/ml for each sample. no virus was detectable in basolateral media (c) at day 7 pi. *statistically different values between rsv-a2 -infected wells and wells which were infected with antibody-preadsorbed virus, as determined by two-tailed unpaired analysis, when p < 0.05. post-infection (day 42 pi, fig. 2c ). this persistent rsv infection of polarized calu-3 is consistent with findings in rsv-infected differentiated nhbe cultures, in which infection was detectable for at least 36 days post-infection (zhang et al., 2002) . similar to apical infection, virus release after basolateral infection was detected as early as 4 days post-infection, primarily from the apical sur-face, and was detectable for at least six weeks post-infection (data not shown). after either apical or basolateral infection, virus was detected in the basolateral media of cells only after day 35 pi, corresponding with decreased teer in infected calu-3 (fig. 2d , and data not shown). the detection of virus in the basolateral media may be due to basolateral release of virus or to the mixing of apical and basolateral media in cultures due to decreased polarization. whether cells were infected at the apical or basolateral surface, greater than 90% of the virus release occurred into the apical compartment, demonstrating directionality in the response of calu-3 to rsv infection. in order to determine whether rsv entry into calu-3 was mediated by rsv-f surface protein, anti-rsv-f or anti-rsv-n monoclonal antibody (mab 143-6c and mab 130-12h, respectively) were incubated with virus for 1 h at 37 • c, prior to infecting polarized calu-3 cells. there was sample-to-sample variability in the amount of infectious virus released from infected cells at each time point examined. infection of calu-3 was inhibited by the neutralizing anti-rsv-f antibody (mab 143-6c), a murine mab that recognizes the same epitope as palivizumab (anderson et al., 1988; johnson et al., 1997) , at 50 g/ml but not 5 g/ml while a control anti-rsv-n mab failed to inhibit infection at either 50 or 5 g/ml (fig. 3) . the anti-rsv-f mab completely neutralized apical (fig. 3b ) and basolateral (fig. 3c ) release of rsv at 50 g/ml at day 7 (p < 0.05) and at day 49 pi (p = 0.07). treatment with a control anti-rsv n mab did not significantly neutralize apical or basolateral rsv infection at the timepoints examined ( fig. 3b and c). consistent with protection from rsv infection, treatment with 50 g/ml anti-rsv-f mab, but not of anti-rsv-n mab, protected against the accelerated decline in polarization observed in rsv-a2 infected cells (fig. 3a) . the teer of anti-f mab treated calu-3 was comparable to mock-infected controls as late as day 56 pi. the data presented in this report demonstrate that calu-3 cells are susceptible to rsv infection when cultured as monolayers. infected cells expressed viral message and viral protein, but did not form obvious cytopathology, and infection did not appear to alter tight junction formation. the lack of obvious cytopathology and limited viral spread following rsv infection of calu-3 is in contrast to observations made in non-polarized epithelial cell lines used to study rsv infection, including hep-2, where infection becomes widespread across the monolayer of cells. however, these findings are consistent with those made in cultured nhbe (zhang et al., 2002) and in human adenoidal epithelial cells (hae) and human adenoid organ culture (wright et al., 2005) , in which rsv spread is restricted and few cells appear to be infected with rsv. consistent with previous studies in polarized mdck (roberts et al., 1995) and in differentiated nhbe, polarized calu-3 released infectious virus primarily from the apical surface, and infection was persistent, detectable for at least 6 weeks post-infection. persistent rsv infection of cells in culture has been reported for at least 36 days post-infection of nhbe cells (zhang et al., 2002) . the persistent infection and release of infectious virus for at least 6 weeks post-infection of calu-3 cells is consistent with observations of persistent infection in ex vivo, cultured nhbe, in animal models of rsv infection, and in some rsv-infected individuals (couch et al., 1997; dakhama et al., 1997; hegele et al., 1994; isaia et al., 1985; mejias et al., 2008; sikkel et al., 2008; zhang et al., 2002) . finally, consistent with previous reports in other cell lines, rsv infection in calu-3 could be inhibited by an antibody directed against the attachment protein of rsv. although calu-3 do not differentiate, the results indicate that polarized calu-3 respond to infection similar to polarized, differentiated nhbe cells, and more closely reflect in situ rsv infection than non-polarized epithelial cells. calu-3 are more readily available than nhbe cells, polarize into stable cultures more rapidly than nhbe cells, and demonstrate restricted, persistent infection and directional viral maturation similar to nhbe 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wish to thank dr. don latner for assistance with the immunofluorescence assays, and ms. elisabeth blanchard for assistance with cell maintenance. key: cord-017040-4zajnrsf authors: rihana, nancy; sampson, mindy title: skin infections date: 2019-08-11 journal: infections in neutropenic cancer patients doi: 10.1007/978-3-030-21859-1_5 sha: doc_id: 17040 cord_uid: 4zajnrsf cutaneous infections are common in immunocompromised patients. neutropenia predisposes patients to fungal, bacterial and viral infections. antibacterial antifungal and antiviral prophylaxis have caused a significant reduction in some of these infections. there are two main types of cutaneous infections : primary cutaneous infections and cutaneous manifestations of a disseminated infection. in the latter, skin lesions may be the window to disseminated bloodstream infection and the first and only evidence of a disseminated life threatening infection. the diagnosis may be at your fingertips; therefore a thorough skin exam is the clue. however, it’s also important to know the characteristic lesions associated with different infections. it will help expedite diagnosis so appropriate treatment is initiated promptly in neutropenic patients, which can be lifesaving. in a retrospective study of 43 neutropenic febrile patients with cutaneous lesions, fungal infections were the most frequent, and nodular lesions on the lower extremities were the most prevalent (naorungroj and aiempanakit, j am acad dermatol 74:ab166, 2016). skin biopsy for pathological study and culture remains the gold standard and should be obtained early to confirm the suspected diagnosis. in these immunocompromised patients the inflammatory response is altered by either the primary disease or its treatment. therefore, routine pathogens may present in an atypical fashion, with diminished or absent induration, erythema, or pustulation in response to bacterial resulting cutaneous infection without typical cellulitis (urabe, clin infect dis 39:s53–s55, 2004). skin lesions are evaluated not only by morphology, but also in the context of the clinical setting and biopsy result. the skin biopsy is inexpensive, relatively noninvasive and without contraindication, and may avoid the need for more invasive procedures such an open lung biopsy (grossman, et al., cutaneous manifestations of infection in the immunocompromised host. springer science+business media, llc, new york, 2012). in addition to antimicrobial therapy, surgery should not be postponed in the face of progressive skin and soft tissue infection in this population (brzozowski and ross, j hand surg br 22:679–680, 1997). in addition to antimicrobial therapy, surgery should not be postponed in the face of progressive skin and soft tissue infection in this population (brzozowski and ross, j hand surg br 22:679-680, 1997). keywords ecthyma gangrenosum · bullous cellulitis · fulminant necrotizing infections · spontaneous clostridial myonecrosis · purplish discoloration · crepitation · necrotizing enterocolitis · subcutaneous nodules · disseminated cutaneous mycobacterium · knife-cut sign · herpetic whitlow · disseminated hz neutropenia is one of the major risk factors for gram-negative cellulitis. we will review the main gram negative pathogens and their cutaneous manifestations in this population. pseudomonas aeruginosa, usually a nosocomial pathogen has emerged as a common cause of infection in immunocompromised patients; most often in neutropenic leukemics during chemotherapy. the routine use of anti-pseudomona antimicrobial prophylaxis in cancer patients with prolonged neutropenia has reduced the overall incidence of pseudomonal infections. however the emergence fluroquinolone resistant gram negatives including pseudomonas remains an important threat in neutropenic patients. pseudomonas can invade through areas of micro erosions especially over intravenous, urinary catheters, decubital ulcerations and thermic damage [1] . cutaneous lesions occur in 30% of cases of pseudomonas bacteremia [2] . the site of origin of the pseudomonas is most commonly the respiratory or genitourinary tract. p. aeruginosa infection can also be acquired from a humid environment including showers, sinks, and flower vases. the dermatologic manifestations of pseudomonas sepsis include ecthyma gangrenosum, hemorrhagic bullae, necrotizing/gangrenous or bullous cellulitis, painful vesicular lesions, and small papules on the trunk resembling rose spots of typhoid fever, grouped petechiae, erysipelas-like lesions with hyperesthesia, erythematous or violaceous subcutaneous painful nodules, and necrotizing or malignant external otitis [3] . ecthyma gangrenosum (eg) has classically been considered a pathognomonic sign of pseudomonas aeruginosa septicemia, although it can be reported with multiple other gram-negative bacterial, fungal, and viral infections in the immunocompromised host. it can occur as single or multiple lesions. most cases of ecthyma gangrenosum have been associated with septicemia, but it can also occur in the absence of pseudomonas bacteremia. the most common sites for ecthyma gangrenosum are the gluteal, perineal area, axillary and extremities [4] . it begins as an area of erythema and edema that progresses to hemorrhagic bullae that rupture, evolving into a painless central blackish gray necrotic area (eschar) surrounded by an erythematous halo. it grows up to several centimeters in diameter over 12-24 h . the necrosis may extend as deep as muscle. pus is minimal. lesions at various stages of development may be present at different sites in the same patient [3] . ulcerations are sensitive on palpation [1] . it is assumed that necrosis of the skin is caused by pseudomonas elastase which destroys elastic lamina of the blood vessels and allows liberation of the pathogen into the subcutaneous tissues [5] (fig. 1) . the pathologic hallmark of ec is vasculitis sparing the intima and without thrombosis which distinguishes it from other forms of bacterial vasculitis in which septic intraluminal thrombi attach to bacteria and invade the endothelium [4] . e. coli, is a common cause of cellulitis in the immunocompromised host that is impossible to distinguish from streptococcal cellulitis. it is rapidly progressive and limb threatening if appropriate antibiotic are not started promptly [3, 6] . e. coli may also produce abscesses anywhere in the body but mainly cause perirectal phlegmon in neutropenic patients. ecthyma gangrenosum described above, is considered pathognomonic for pseudomonas aeruginosa but has also been described with multiple other gramnegative organisms including e. coli, e. cloacae, klebsiella, serratia, citrobacter, morganella, and stenotrophomonas [3, [7] [8] [9] [10] [11] aeromonas species are gram-negative, non-sporulating facultative anaerobic bacillus found in brackish or fresh water lakes, streams and soils. they have also been recovered from chlorinated tap water, including hospital water supplies. it may be isolated as well from stools of asymptomatic carriers. among patients with cancer, aeromonas septicemia is more likely to occur in leukemic patients and its associated with water related activities in only 10% cases but is nosocomially acquired in 60% [12] . about 20-30% of infections are associated with skin lesions. in immunosuppressed patients, aeromonas can cause various infections that are likely to be fatal including severe cellulitis, fulminant necrotizing infections, and ecthyma gangrenosum. myonecrosis and gas production have occurred with aeromonas and can simulate clostridial gas gangrene [3] . aeromonas infections of freshwater traumatic wounds after water related activities such as fishing or swimming cause a rapidly progressive cellulitis that develops within 8-48 h after trauma followed by suppuration and necrosis around the wound, myonecrosis and sometimes osteomyelitis. it often requires surgical debridement in addition to antibiotic therapy [4] . chromobacterium violaceum is a rare but frequently fatal infection. its an anaerobic gram-negative rod that is commonly found in soil and water in tropical and sub-tropical climates between latitudes 35° north and 35° south. cutaneous infection is rare and usually occurs with exposure of broken or injured skin to contaminated muddy or stagnant water or soil in patients with neutrophil dysfunction or hiv infection. infection in the skin tends to present with cellulitis, pustules, ulcers, or abscesses accompanied by severe systemic symptoms [3] . over the course of the last several decades, the frequency with which gram-positive bacteria have been isolated from neutropenic patients with cancer has increased [13] . we will review the main gram positive pathogens that lead to cutaneous involvement in neutropenic patients. bacillus cereus commonly presents in a neutropenic patient as a single painful vesicle, pustule, or bulla on a digit or extremities with rapidly spreading cellulitis during the spring and summer. the bulla may become necrotic and develop a black eschar. it's usually associated with systemic toxicity [3] . gram stain of the aspirate smear demostrates large gram-positive rods, which may be mistaken for clostridium infection and treated with penicillin. b. cereus and b subtilus cause most of the infections. about half of these infections arise at sites of indwelling intravascular catheters [4] . occasionally widely disseminated blood stream infections may occur including endocarditis and brain abscess [14] [15] [16] . clostridium species account for 30% of anaerobic bacteremias in all cancer patients [17] . it tends to cause a fulminant infection in neutropenic patients. sixty percent of the isolated cases are secondary to clostridium perfringens and 30% by clostridium septicum. clostridium perfringens is usually part of the colonic flora while c. septicum is typically found in soil and animals, but is not normal human flora [3] . forty percent of the clostridium infections are polymicrobial, which indicates the gastrointestinal tract as the source of these infections [17] . two types of cutaneous manifestation have been described in neutropenic patients: spontaneous, non-traumatic gas gangrene, and spreading cellulitis [4] . (a) spontaneous clostridial myonecrosis (gas gangrene) most commonly associated with a silent colon carcinoma, an underlying hematologic tumor or neutrophil dysfunction [3] . its is clinically characterized by the sudden onset of excruciating pain in the involved site-usually the leg esp. if associated with abdominal involvement. the swelling is rapidly progressive, associated with purplish/ bronze discoloration and blister formation-usually hemorrhagic. involvement of underlying muscle is always more extensive than the evident skin involvement. the blister serosanguinous fluid will contain gpr, but inflammatory cells are infrequent [4] . pathology: there is cell lysis and gas formation in connective tissue and muscle with minimal inflammation [4] . the diagnosis of clostridial myonecrosis requires a high index of suspicion, since the infection spreads rapidly and death may occur within 24-48 h. gram stain of a bulla allows for a timely diagnosis. anaerobic cultures should be sent to determine appropriate antibiotic use. imaging with x-ray or computed tomography (ct) scan can demonstrate soft tissue gas (a late finding) and help determine the extent of infection [3] . (b) spreading cellulitis with clostridial septicemia is a fulminant infection in neutropenic patients. a small area of purplish discoloration develops, often on the flank or abdominal wall. the lesion will expand rapidly over several hours, and more lesions appear in other areas. as it progresses, the lesions turn into brownish to blackish color with blister formation and crepitation. clostridium can be captured in the fluid, but without inflammatory cells [4] . clostridium septicum has been associated with necrotizing enterocolitis (typhlitis), which typically is a fulminant infection of the intestines in neutropenic patients [18] . corynebacterium jeikeium (c. jeikeium) corynebacterium species are part of the normal skin flora. the most common sites of colonization by c. jeikeium are the perineum, rectal, inguinal and axillary areas. corynebacteria species rarely cause infection. infections due to c. jeikeium are mainly seen in immunosuppressed patients, especially those with neutropenia and indwelling catheters [19] . primary cutaneous infection with c. jeikeium occurs at breaks in the skin barrier due to bone marrow biopsy, intravascular catheter insertion, groin or perirectal areas (anal fissures) which serve as a portal of entry into the bloodstream, leading to septicemia. primary skin lesions typically present as cellulitis or wound infection [20] . secondary skin and soft tissue infections with c. jeikeium develop in almost 30-50% of neutropenic patient with c jeikeium bacteremia [4] . it may present as single to multiple, nontender, noninflamed 2 × 2 cm subcutaneous nodules that do not spontaneously drain but are purulent upon surgical drainage [19] ; bright red, non-blanching papules with satellite petechiae and central necrosis or pustulation on the trunk and/or extremities [20] . staphylococci species are frequent colonizers of normal skin but commonly infect immunocompromised individuals. leukopenic patients are at greatest risk for infection. common infections include impetigo, furuncles, carbuncles, ecthyma, folliculitis, cellulitis etc. in immunocompromised patient, vesicle or bullous eruption can be seen [3] . streptococcal infections in bone marrow transplant (bmt) recipients and acute leukemia patients are serious and may be clinically atypical [4] . group a or group b streptococcus cutaneous manifestations include erysipelas/cellulitis in only 25%, the rest of the cases are infections of wounds or ulcers, myositis, necrotizing fasciitis, and toxic shock syndrome. viridans group streptococci (vgs) are part of the normal microbial flora of humans, risk factors for invasive disease is mucositis and neutropenia. a case control study of vgs sepsis was done at the university of texas m. d. anderson cancer center in houston, where controls were randomly selected from patients with other gram-positive septicemia. flushing of the face and a rash occurred in nearly 60% of these patients but were uncommon in the control group. the rash was usually erythematous maculopapular, beginning on the trunk and progressing to the face and extremities compatible with toxic shock syndrome picture. in 25% of the cases, the rash resulted in desquamation of the palms and soles 1-2 weeks later. ten percent of the patients developed ards, shock, or renal failure and died despite more than 4 days of vancomycin therapy. the patients with vsg had a higher rate of oral mucositis, bmt, and severe neutropenia <100, on antacid or h2 antagonist therapy, and parenteral nutrition [4, 21] . mycobacterial cutaneous infection occurs as a result of direct inoculation from an exogenous source, or through contiguous or hematogenous spread. cutaneous mycobacteria can exhibit a large spectrum of morphological presentation. infection may be caused by mycobacterium tuberculosis (mtb), mycobacterium avium intracellulare complex (mac), and other non-tuberculous mycobacteria (ntm) including rapidly growing mycobacteria (rgm), although mtb is commonly seen in hiv patients those undergoing solid organ transplant. tuberculosis in neutropenic patients is one of the most serious opportunistic infections encountered. cutaneous miliary tuberculosis presents as erythematous to brown papules, which can become vesicular or pustular. the tiny vesicles or pustules rupture and form a central crust on the papule. removal of the crust leaves a minute but sharply defined umbilication [3] . pathology results are characterized by the absence of a granulomatous response, giant cells, and true caseating granulomas [6] . an acid-fast stain (if the diagnosis is considered and the stain requested) usually shows numerous acid-fast bacilli (afb) seen quite easily [3] . ntm are ubiquitous in the environment and reside in soil and water. of the over 150 ntm species, only ∼25 are known to cause disease in humans [22] . skin and soft tissue infections are most commonly caused by mycobacterium marinum and mycobacterium ulcerans, which are both slowly growing mycobacteria [23] . in the immunocompromised, the typical history of previous trauma or surgery is absent, thus cutaneous mycobacteria infection are most probably the result of hematogenic dissemination, resulting in multiple skin lesions. this clinical entity is well described in patients with t cell mediated immunodeficiency such as aids, solid organ and bone marrow transplant patients, but infrequently in patients with hematological malignancies, either with or without neutropenia [24] . case studies described highlight the variety of immune system dysfunction (t-cell, humoral, or granulocyte-related) which combined may have predisposed to the disseminated ntm in neutropenic patients [13, 25] . chronic indwelling central venous catheter-related infection is one of the most common risk factor for disseminated nontuberculous mycobacterial infection [26] . the lesions are usually more extensive in the immunosuppressed population, nonspecific and heterogeneous. they range from panniculitis (fig. 2) , single to widespread nodules, sinus tracts, nonhealing ulcers, subcutaneous abscesses, or erythematous plaques (fig. 3 ) [3] . mycobacterium malmoense, a slow-growing mycobacteria may cause cutaneous lesions that were described in a 75 year old neutropenic man with myelodysplastic syndrome, who was also receiving corticosteroids. he developed papulokeratotic pinkish nodules lesions in a sporotrichoid distribution on the arm that later ulcerated and some became necrotic [27] . mycobacterium avium-intracellulare complex (mac), is a common opportunistic pathogen in aids patients and patients with cell medicated immunodeficiency mainly, it can also cause infections in neutropenic patients. the clinical manifestations include widespread erythematous tender nodules on the upper, and lower extremities, chest and abdomen that can ulcerate (fig. 2) . the infection is usually associated with bacteremia and cavitary lung lesions may also be present. skin biopsy demonstrates areas of dermal and subcutaneous necrosis with numerous acid-fast bacilli [3] . mycobacterium fortuitum, mycobacterium abscessus, and mycobacterium chelonae are collectively referred to as rapidly growing mycobacteria (rgm). these environmental pathogens may cause skin infections that usually occur following trauma or surgical procedures and injections, which is usually not present in immunocompromised patients. m. fortuitum infection is more common in immunocompetent patients, while m. chelonae and m abscessus more often infects immunocompromised patients [3] . disseminated cutaneous mycobacterium chelonae infection in neutropenic patients can cause neutropenic fever and multiple dusky red to purple, tender, subcutaneous nodules on the face, arms, and legs, resulting in firm purple ulcerating nodules. within weeks, the nodules can developed larger plaques that can ulcerate [25] . it is essential to maintain a high index of suspicion for atypical mycobacterial infection, as acid-fast organisms may be difficult to identify on histologic sections, and many of these fastidious pathogens are challenging to grow in culture, often requiring weeks for the culture to turn positive, and in some cases requiring specific media. polymerase chain reaction (pcr) is an emerging technique, which may aid in the diagnosis of mycobacterial infections. speciation of the offending organism is crucial, as the antibiotic susceptibility profiles differ among the organisms and are essential to achieving therapeutic success [3] . neutrophils are not the primary defense against viral infections. however hematologic malignancy is the most prevalent background of neutropenic patients with cutaneous lesions 95.3% based on a retrospective review from 2009-2014 [28] . in this population, the combination of dysfuction of different parts of the immune system provides sufficient opportunity for viruses such as herpesiviridae to reactivate and cause disseminated infection [13] . infections caused by herpes simplex virus (hsv) are exceedingly common inpatient with acute leukemia and post bone marrow transplant [4] . they are divided into primary and secondary (recurrent) forms, which are selflimited in normal hosts. in the majority of immunocompromised hosts, the hsv infection is not primary but rather reactivation of latent hsv. most herpetic infections involve the oral mucosa, lips, and nares. genital lesions are less common [4] . any periorificial ulceration in the immunocompromised host should be considered herpes simplex until proven otherwise. symptoms such as paresthesia, burning sensation or pruritus precede the lesions. they begin as vesicles that rupture spontaneously, leaving ulcerations that enlarge, coalesce and become encrusted. infection often follows a chronic course in immunosuppressed patients, resulting in larger, deeper, painful necrotic erosive lesions known as phagedena. superinfection by bacteria or fungi, esp. by staphylococcus aureus may occur, which may distort the initial appearance, resulting in misdiagnosis. healing typically occurs within 1 week in normal hosts, but may take up to 5 weeks in immunocompromised patients [3, 4] . intraoral hsv can present as ulcerations with polycyclic borders, linear fissures on the tongue known as herpetic geometric glossitis. the break of mucosal integrity provides a portal of invasion by both pathogenic and normal microbial flora inhabiting the mouth. other variant of atypical herpes simplex virus infection is deep linear fissures in the skin folds (inframammary, infra-abdominal, inguinal, or vulvar) termed the "knife-cut sign", and bilateral mutidigit herpetic whitlow (fig. 4) reported in a patient after receiving chemotherapy for chronic lymphocytic leukemia (cll) [3] . both oral and intravenous acyclovir has been effective in preventing hsv infection in patients with acute leukemia and bmt recipients. herpes zoster (hz) represents reactivation of the varicella-zoster (vz) virus. following the primary varicella infection (chicken pox), the virus remains dormant in a dorsal root ganglion or a cranial nerve ganglion. reactivation occurs as herpes zoster with cutaneous vesicles and dermatomal pain. the skin lesions of vz in the immunocompromised host occur in three forms: (a) dermatomal hz (which may be less than 3 contiguous dermatomes), (b) disseminated hz and (c) chronic hz or recurrent hz. in patients with malignant disease, the incidence of hz is further increased: highest in lymphoreticular disorders, hodgkin's disease, then non-hodgkin's lymphomas, followed by solid tumors, particularly small cell carcinoma of the lung [29] (fig. 5 ). cmv (hhv-5) is a common human viral infection affecting 40-100% of adults worldwide. acute infections are often asymptomatic, but once the infection is acquired, there is a lifelong latency along with the risk of intermittent reactivation. cmv disease is due to reactivation of latent virus following iatrogenic immunosuppression in organ transplantation, and cancer chemotherapy. skin manifestations of cmv are rare in any setting, and very nonspecific (exanthematous, maculopapular, or morbilliform eruptions) and therefore diagnosis is often delayed. the cardinal manifestation of cmv infection in the skin is a chronic painful ulcer of the anal, perianal, or anogenital area. although uncommon, oral manifestations of cmv infection have included painful erosions or ulcers of the tongue, buccal mucosa, and pharynx. skin biopsy will confirm the diagnosis by demonstrating the characteristic large intranuclear inclusions with a surrounding halo, or "owl's eye" characteristic of cmv. the diagnosis is confirmed with immunoperoxidase stain [3] . there are many fungi including yeast and mold, which are of medical importance, particularly in the immunocompromised population. patients with hematologic malignancies and those receiving chemotherapy are at highest risk [30] . fungal infections have many clinical presentations including dermatologic manifestations. these infections can be localized after local trauma, progress to invasive disease or dissemination, or result from hematogenous infection. superficial cutaneous candida infections such as intertrigo and vaginitis present similarly to immunocompetent patients [30] . however, they may be more common in patients who require systemic antibiotics or steroids during their oncologic treatment. most superficial infections can be treated with topical anti-fungal [31] . disseminated candidiasis can occur in neutropenic and non-neutropenic populations. the portal of entry is typically via gastrointestinal translocation or introduction from a central line. in patients with hematologic malignancies and stem-cell transplantation, non-albicans species predominate including c. glabrata, c. krusei and c. tropicalis [32] [33] [34] [35] [36] . in solid tumors, approximately half of disseminated candidiasis is secondary to c. albicans [32] . the epidemiology of these infections is likely related to the choice of fungal prophylaxis and resistance patterns of nonalbicans species [34] . the frequency of cutaneous manifestations in the setting of candidemia is reported to occur in 11-44% of cases [34] . the typical skin lesions are 5-10 mm pink papules, which rarely lead to eschar formation or skin necrosis. there are often numerous papules involving the trunk and proximal extremities [30] . some lesions may be purpuric, especially if thrombocytopenia is also present [34] . mortality associated with candidemia has been reported to be over 50%, therefore prompt recognition of skin lesions and initiation of empiric treatment is important [35] . diagnostics can be challenging, positive blood cultures occur in less than 50% of the cases. it is important to consider obtaining tissue for histopathology and culture or using novel non-culture diagnostics. histology typically shows fungal elements including pseudohyphae, hyphae and yeast within the dermis or blood vessels [34] . an echinocandin such as micafungin is recommended as empiric treatment in most patients [31] (fig. 6 ). skin manifestations occur in up to 70% of patients with fusariosis and can present as a localized or disseminated infection [36] . neutropenic patients with hematologic malignancies are at high risk for these mold infections [37] . an important clinical presentation of localized fusarium infection in the neutropenic population is fungal paronychia, which presents as erythema and swelling of the periungal skin [30] . fungal paronychia should be suspected in neutropenic patients who have underlying onychomycosis, develop an eschar, and do not improve on systemic antibiotics [30] . infection may be preceded by minor trauma [38] . local infections can lead to fungemia and disseminated infection. disseminated fusarium infection typically presents as multiple erythematous papules or nodules, which can develop central necrosis or targetoid appearance. they often present at different stages of evolution and are widely distributed on the trunk and extremities [36] . skin biopsy with histopathology and culture are helpful in securing a diagnosis. histology will show ballooning branching septated hyphae, which can invade capillaries and blood vessels [39] . skin lesions may appear before systemic symptoms develop or blood cultures become positive [30] . unlike other disseminated mold infections, blood cultures are often positive in disseminated fusariosis and should be collected when fusarium is suspected [37] . empiric treatment with liposomal amphotericin b is recommended [40] . more details about therapy please refer to the chapter titled fungal infections (figs. 7, 8, and 9 ). although aspergillus is one of the most common etiologies of invasive fungal infections in patients with underlying malignancies, dermatologic manifestations occurs in less than 5% of cases [41, 42] . cutaneous involvement can present as a localized infection by direct skin inoculation or via hematogenous dissemination. blood cultures are typically negative in disseminated infection therefore it is recommended to obtain tissue biopsy with culture and histopathology. skin manifestations are similar to fusarium infections. primary or localized lesions can present as cellulitis with focal erythema and swelling that can progress leading to the formation of bullae, necrotic ulcers and eschars. invasive fungal sinusitis secondary to aspergillus sp. can also extend to the skin leading to facial cellulitis; this is covered in further detail in the chapter about fungal infections. disseminated infection typically presents as scattered erythematous papules or nodules [30, 41] . histology will show septated narrow hyphae with 45° branching and club-shaped pseudohyphae [43] . treatment with fig. 7 paronychia and cellulitis secondary to fusarium sp. in a patient with acute myelogenous leukemia and prolonged neutropenia triazoles such as voriconazole is the preferred anti-fungal unless there is a contraindication to this class and then liposomal amphotericin b would be recommended [44] . the most common zygomycetes to cause clinical disease are rhizopus, mucor, rhizomucor, lichtheimia, and cunninghamella spp. [45] . in patients with underlying malignancy, the primary site of mucormycosis is cutaneous in 12% of cases [46] . unlike other fungal infections, cutaneous mucormycosis most often occurs by direct inoculation rather than dissemination. skin lesions may begin as a small erythematous macule but typically develop a black necrotic eschar with surrounding erythema and swelling. if there is concern for disseminated disease it is important to look for metastatic skin lesions [47] . patients with rhinocerebral mucormycosis may present with orbital or facial cellulitis with a classic black necrotic eschar [45] . obtaining tissue histopathology with culture is essential for diagnosis. histologic examination will show broad non septate hyphae branching at 90° [43] . there may be vascular invasion leading to thrombosis and infarction [47] . liposomal amphotericin b is treatment of choice [48] . there are several other fungal infections that are less common but also important to consider in the evaluation of a cancer patient with a rash. geographic location and previous travel can increase risk for endemic fungal infections. histoplasmosis is endemic in the ohio river valley and parts of central america. cutaneous lesions typically present in cases of disseminated disease and often mimic other dermatologic infections due its' ability to manifest with many skin findings including papules, plaques, nodules, ulcers or pustules. histopathology typically shows granulomas with lymphohistiocytic infiltrates and yeast within or outside macrophages [49] . blastomycosis can be found in the ohio and mississippi river valleys and in the southeastern united states [50] . skin lesions begin as an erythematous papule and evolve into a scaling or vegetative plaque. histopathology may show pseudoepitheliomatous hyperplasia with neutrophilic abscesses or noncaseating granulomas. tissue culture will reveal a thick-walled yeast with broad-based budding [51, 52] . coccidiomycosis is found in the southwestern united states and can present with cutaneous findings when dissemination or reactivation occurs. like histoplasmosis, disseminated coccidomycosis can present in many cutaneous forms including papules, plaques, nodules and ulcers. histopathologic examination can show granulomatous or suppurative inflammation with numerous eosinophils. coccidiomycosis can also result in reactive skin eruptions such as erythema nodosum, sweet's syndrome, interstitial granulomatous dermatitis, and exanthems. these presentations typically occur in the setting of pulmonary coccidiomycosis. histopathology for reactive eruptions is variable depending on the skin manifestation and will have sterile tissue culture [53] . cryptococcus spp. are encapsulated yeast, which are ubiquitous in the environment, often being found in the soil and pigeon droppings. these infections are commonly reported in the hiv population however they can occur in other immunocompromising conditions including malignancies. like many other cutaneous fungal infections, skin lesions are polymorphous. cutaneous findings can represent primary infection but most often occur in the setting of disseminated disease. therefore it is important to look for other organ involvement when cutaneous cryptococcal infection is diagnosed [54] . obtaining histopathology with tissue culture is essential for diagnosis, using stains such as mucicarmine and alcian blue to identify the capsule [55] . although it is always important to consider infectious dermatologic etiologies in cancer patients who present with a rash, there are also several non-infectious skin diseases that are important to recognize. cancers themselves can cause unique skin pathology however there are also paraneoplastic and inflammatory skin disorders that can occur secondary to malignancies. obtaining a thorough history and review of medications can assist with determining the diagnosis but skin biopsy is usually necessary given many diseases have similar findings on clinical examination. leukemia cutis (lc) is an extra-medullary presentation of leukemia which is due to the invasion of malignant cells into the skin [56] . lc is most commonly associated with aml but can also occur with other underlying myeloid disorders and rarely presents without bone marrow involvement. typical skin findings are erythematous or violaceous papules, nodules or plaques. the lesions have also been described as rubbery or shiny and may present with bullae [57, 58] . they can be pruritic but are usually not painful. skin biopsy with immunohistochemical analysis is essential for diagnosis [59] . histopathology will show leukemic cells, typically myeloblasts, invading the dermis. prognosis is typically poor and may represent progression of underlying malignancy [58] (fig. 10) . sweet's syndrome (ss) is characterized by the development of fever, a painful rash, and leukocytosis [58] . ss can occur in the presence of an underlying malignancy such as aml, cml, mds or myelofibrosis. it is also known to be drug-induced and can occur secondary to infections, classically it is described after an upper respiratory tract infection [60] . the skin lesions are often erythematous or violaceous papules, nodules or plaques which are tender. vesicles or bullae can develop due to edema of the upper dermis [60] . a unique finding in ss is pathergy. cutaneous findings may appear after trauma including venipuncture or biopsy [61] . histopathology shows neutrophilic infiltrates of the dermal papillae without evidence of leukoclastic vasculitis. unlike leukemia cutis, the neutrophils that infiltrate the dermis are typically mature [60, 62] . when considering ss it is important to rule out an underlying infection; an important differential diagnosis is pyoderma gangrenosum. treatment of the underlying malignancy is necessary but steroids can also be utilized [63] (fig. 11 ). pyoderma gangrenosum is an ulcerative neutrophilic dermatosis. pg is associated with many inflammatory and autoimmune conditions including both hematologic malignancies and solid tumors [64] . it is typically a diagnosis of exclusion but is characterized by a rapidly developing, painful ulceration with undermined borders. like sweet's syndrome it may exhibit pathergy [65] . the base of the ulcer is typically necrotic with a surrounding erythematous or violaceous halo [66] . the bullous subtype is characterized by bullae formation preceding the ulceration and is commonly associated with hematologic malignancies [58] . skin biopsy from the border of the ulcer should be obtained although it is difficult to diagnose pg based on pathology due to changing histology with the evolution of the ulcer. examination may reveal dense neutrophilic infiltrates with leukoclastic vasculitis or necrosis [67] . treatment with steroids usually results in rapid improvement [66] . paraneoplastic vasculitis is most commonly associated with mds and other hematologic malignancies [68] . it is characterized by non-blanchable palpable purpura which may be painful or pruritic [58, 69] . biopsy is essential for diagnosis and will show necrotizing leukocytoclastic vasculitis with fibrinoid necrosis and neutrophil infiltration of the vessel wall [70] . the presentation of vasculitis may precede the diagnosis of cancer but can also present at all stages of disease [68] . treatment of the underlying malignancy can result in resolution of the vasculitis [58] . many medications that patients have exposure to during the treatment of malignancies can lead to rashes and skin pathology. it is important to monitor for allergic drug rashes which usually resolve with discontinuation of the inciting medication. steven-johnson syndrome (sjs) and toxic epidermal necrolysis (ten) are both drug reactions that have a high mortality rate due to epidermal sloughing. sjs and ten may begin with a prodrome which includes high fever. skin findings may begin with erythematous macules or targetoid lesions progressing to the development of bullae and erosions [71] . nikolsky sign is often positive. many patients will also have mucosal involvement [72] . the two conditions are distinguished from each other based on body surface involvement; sjs involves <10% and ten involves >30% with an overlap of disease characterization between 10-30% [71] . many drugs have been linked to the development of sjs or ten, particularly antibiotics. sulfonamide antibiotics are most commonly associated with sjs or ten however all antibiotics should be considered as an etiology [71, 73] (figs. 12 and 13 ). toxic erythema of chemotherapy is a nonallergic reaction that is caused by many chemotherapeutic agents. toxic erythema of chemotherapy presents with painful erythema and edema which can be present on the hands and feet or intertriginous areas. it is most commonly associated with methotrexate, cytarabine, anthracyclines, 5-fluorouracil, and taxanes [58] . multikinase inhibitors such as sorafenib can cause a hand-foot-skin reaction which is characterized by painful hyperkeratotic plaques which develop in areas of friction such as the fingertips and joints [58, 74] . tyrosine kinase inhibitors including imatinib or dasatinib are also commonly associated with cutaneous reactions which can include a maculopapular rash and facial edema [58, 75] . description pathogen pustules staphylococcus aureus, bacillus, corynebacterium, gram negative bacteria (mainly ecoli, citrobacter, chromobacterium) subcutaneous nodules staphylococcus aureus, corynebacterium jk, pseudomonas, e coli, stenotrophomonas. mycobacteria. aspergillosis, fusarium. candidiasis, coccidiomycosis, cryptococcus, histoplasmosis. ulcers pseudomonas, other gram negative bacilli (including but not limited to e coli, serratia, stenotrophomonas, citrobacter, chromobacterium), bacillus. mycobacteria. aspergillosis, candidiasis, mucormycosis, coccidiomycosis, histoplasmosis. hsv/hzv/cmv vesicle or bullae bacillus cereus, clostridium, staphylococcus, pseudomonas, other gram negative bacilli candidiasis, mucormycosis, fusarium [3] acral hemorrhagic bullae clostridium ecthyma gangrenosum in a patient with acute leukemia infections caused by pseudomonas aeruginosa cutaneous manifestations of infection in the immunocompromised host dermatologic manifestations of infections in neutropenic patients the role of the elastase of pseudomonas aeruginosa in experimental infection upper limb escherichia coli cellulitis in the immunocompromised hemorrhagic bullae in association with enterobacter cloacae septicemia skin lesions associated with e. coli sepsis in a patient with acute leukemia. henry ford gangrenous ulcer and septicemia due to citrobacter necrotizing skin ulceration in antibiotic-induced agranulocytosis fulminant stenotrophomonas maltophilia soft tissue infection in immunocompromised patients: an out break transmitted via tap water bacteremia caused by aeromonas species hospitalized cancer patients infections in immunocompromised patients. i. pathogenesis, etiology, and diagnosis fatal bacillus cereus endocarditis masquerading as an anthrax-like infection in a patient with acute lymphoblastic leukemia: case report successful non-surgical treatment of brain abscess and necrotizing fasciitis caused by bacillus cereus gas gangrene-like infection with bacillus cereus in a lymphoma patient clostridial bacteremia in cancer patients clostridial species in the pathogenesis of necrotizing enterocolitis in patients with neutropenia corynebacterium cdc group jk (corynebacterium jeikeium) sepsis in haematological patients: a report of three cases and a systematic literature review cutaneous manifestations of corynebacterium jeikeium sepsis septicemia and shock syndrome due to viridans streptococci: a case -control study of predisposing factors diagnosis of nontuberculous mycobacterial infections disseminated cutaneous mycobacterium chelonae infection in a patient with acute myeloid leukemia infection caused by mycobacterium chelonae: a diagnostic and therapeutic problem in the neutropenic patient disseminated cutaneous mycobacterium chelonae infection in a patient with acute myeloid leukemia nontuberculous mycobacterial infection in hematopoietic stem cell and solid organ transplant patients cutaneous infection caused by mycobacterium malmoense in a patient with myelodysplastic syndrome a study of cutaneous manifestations among febrile neutropenic patients: a five-year retrospective review in a single tertiary university hospital in southern thailand herpes zoster in patients with small-cell carcimona of the lung receiving combined modality treatment cutaneous fungal infections in the oncology patient: recognition and management clinical practice guideline for the management of candidiasis: 2016 update by the infectious diseases society of america epidemiology and outcomes of candidemia in 2019 patients: data from the prospective antifungal therapy alliance registry candidemia in patients with hematologic malignancies in the era of new antifungal agents (2001-2007): stable incidence but changing epidemiology of a still frequently lethal infection acute disseminated candidiasis with skin lesions: a systematic review associated clinical characteristics of patients with candidemia among different candida species cutaneous infection by fusarium species in healthy and immunocompromised hosts: implications for diagnosis and management fusarium infections in immunocompromised patients fusarium infections of the skin disseminated fusariosis with cutaneous involvement in hematologic malignancies: report of six cases with high mortality rate the sanford guide to antimicrobial therapy cutaneous invasive aspergillosis: retrospective multicenter study of the french invasive-aspergillosis registry and literature review invasive aspergillosis. disease spectrum, treatment practices, and outcomes. i3 aspergillus study group an overview of mucormycosis practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the infectious diseases society of america epidemiology and clinical manifestations of mucormycosis epidemiology and outcome of zygomycosis: a review of 929 reported cases disseminated zygomycosis: report of four cases and review mucormycosis: its contemporary face and management strategies skin lesions in histoplasmosis blastomycosis: the great pretender can also be an opportunist. initial clinical diagnosis and underlying diseases in 123 patients treasure island: statpearls dermatoscopic and clinicopathologic findings of cutaneous blastomycosis coccidioidomycosis: a review and update primary cutaneous cryptococcosis in immunocompetent and immunocompromised hosts cryptococcosis diagnosis and treatment: what do we know now cutaneous manifestations in leukemia patients clinical characteristics of 75 patients with leukemia cutis the skin as a window to the blood: cutaneous manifestations of myeloid malignancies histologic and immunohistologic characterization of skin localization of myeloid disorders: a study of 173 cases sweet's syndromedoublehyphena comprehensive review of an acute febrile neutrophilic dermatosis concurrence of sweet's syndrome, pathergy phenomenon and erythema nodosum-like lesions sweet's syndrome: histological and immunohistochemical study of 15 cases sweet's syndrome: a revisit for hematologists and oncologists underlying systemic diseases in pyoderma gangrenosum: a systematic review and meta-analysis pathophysiology of pyoderma gangrenosum (pg): an updated review pyoderma gangrenosum: clinicopathologic correlation and proposed diagnostic criteria pyoderma gangrenosum: a review the spectrum of paraneoplastic cutaneous vasculitis in a defined population: incidence and clinical features vasculitis associated with malignancy. experience with 13 patients and literature review cutaneous vasculitis as a paraneoplastic syndrome in adults stevens-johnson syndrome and toxic epidermal necrolysis: an analysis of triggers and implications for improving prevention toxic epidermal necrolysis: part i. introduction, history, classification, clinical features, systemic manifestations, etiology, and immunopathogenesis medication use and the risk of stevens-johnson syndrome or toxic epidermal necrolysis cutaneous adverse effects of targeted therapies: part i: inhibitors of the cellular membrane dasatinib and nilotinib: a review of adverse cutaneous reactions with emphasis on our clinical experience key: cord-018555-3lta1tbp authors: overstreet, robin m.; lotz, jeffrey m. title: host–symbiont relationships: understanding the change from guest to pest date: 2016-01-06 journal: the rasputin effect: when commensals and symbionts become parasitic doi: 10.1007/978-3-319-28170-4_2 sha: doc_id: 18555 cord_uid: 3lta1tbp the several meanings for the term “symbiosis” create confusion, which can be avoided when the author provides details of the interrelationships between the symbiotic organism and the “host” so that a reader can clearly understand what definition is implied in each case. for example, we, as opposed to many other mentioned readers, consider a symbiont as an organism living in an association with another regardless of whether it causes a pathologic response or not, but from our title, the reader may incorrectly infer that we consider a parasite to be different from a symbiont. a symbiont is an organism that uses another organism as a habitat. this chapter discusses the primary associations and associated conflicts involving the terminology. it also provides both differentiation between and conflicting views regarding the interpretation of the terms “infect” and “infest,” “infection” and “disease,” and other terms. many seemingly harmless symbionts of a wide array of taxonomic groups are triggered to become pathogenic or virulent, and we provide several examples of the provoking (stimulating) triggers, with the understanding that in most cases, the conditions for the triggered activities are much more complex and complicated than presented. examples of triggers follow: environmental ones like temperature, toxic chemicals (dose), chemotherapeutics, dietary changes, and geographic habits; internal ones like host site, host resistance or susceptibility, and host modifications; and combinations of these and other conditions. we provide examples involving multiple triggers for organisms associated with termites, for an endemic virus being affected by multiple factors and having multiple effects on its commercial penaeid shrimp hosts, and for contrasting variables associated with two exotic viruses in wild and cultured commercial penaeid shrimps with an emphasis on hypothesizing how the pathogenicity developed in these two viruses. the chapter ends by trying to answer the question of why would a symbiont become pathogenic in some hosts and not in others from an evolutionary perspective. it uses two hypotheses to explain the increased virulence. when reading an article on symbiosis, most readers assume they know the definition of all the associated words in the document. this is not the case; even the term "symbiosis" is defined differently by different authors in different fields, by those in different geographic areas, and by those taught by different mentors. the term "symbiosis" was originally used by the german de bary (1879) to mean "living together." his meaning referred to all situations where either similar or unlike organisms or species live together in an intimate association. this is typically thought to include "commensalism," "mutualism," and "parasitism." we prefer to include all such associations under the general term "symbiosis" but realize that our title does not comply, and many readers prefer to use a more specific term for symbiosis and other associations. in the preface of his book (1970) , the eminent parasitologist william trager quoted "the conflict in nature between dissimilar kinds of organisms has been popularly expressed in phrases like 'struggle for existence' and 'survival of the fittest.' yet few people realize that mutual cooperation between different kinds of organisms-symbiosis-is just as important, and that the 'fittest' may be the one that most helps another to survive." for purposes of this series, we restrict "symbiont" to organisms that use other organisms as habitat. therefore, symbiont is a division of nature similar to "terrestrial" or "marine." the symbiont is characterized by where it lives rather than the quality of the relationship. for our purposes, we divide up the world into two kinds of organisms, free living and not free living. this accords us the advantage of comparing the quality of the relationship between free-living organisms and their habitat with that between host and symbiont. the symbiont is dependent upon the host as any inhabitant is on its habitat. we consider symbionts to include bacteriophages (viruses that infect bacteria), bacteria, viruses, "protozoans," and metazoans and symbiosis to include commensalism, mutualism, parasitism, and other relationships. moreover, all symbionts, even metazoans, observed best with a microscope will be considered microbes. more important, we will examine in this series a shift in the interaction among a host and some stage in the life cycle of the symbiont, whether it be free living, facultative, or obligate. we will emphasize that some symbiotic relationships can change from harmless to something that becomes far less favorable for the host. symbiosis this term as defined above is usually thought of as an association (mutualism, commensalism, or parasitism) between organisms of different species involving a unilateral or bilateral exchange of material or energy. the symbiont, or symbiote, is any member of a pair of organisms involved in this symbiotic relationship, with the larger member usually designated as the host. barrows (2011) provided a classification of symbiosis separating nonsocial symbiosis from social symbiosis, and others classify various terms differently. most of these terms and classifications will not be treated in this chapter. commensalism a symbiotic relationship in which one of two partner species benefits and the other shows no apparent beneficial or harmful effect. mutualism a symbiotic relationship in which two or more partners gain reciprocal benefits, usually mutual ones. parasitism a symbiotic relationship in which a symbiont lives all or part of its life in or on a living host, usually benefiting while harming the host in some way and usually having a higher reproductive potential than the host. noble et al. (1989) define it as an association between two different species of organisms in which the dependence of the parasite on its host is metabolic and involves mutual exchange of substances; this dependence is the result of a loss of genetic information by the parasite. there are several atypical kinds of parasites. an accidental one infects an unusual or unnatural host; a commensal one derives its substances from the food of its host; and an erratic, or aberrant, one infects an unusual site. a facultative parasite is usually parasitic but is capable of an independent existence, while an obligate one cannot lead an independent, nonparasitic existence. the late gerald schmidt's definitions are listed by roberts and janovy (2009) . he considered a parasite the "raison d'être" for parasitologists, a parasitologist as a quaint person who seeks truth in strange places and one who sits on one stool while staring at another, and parasitology as a study of the most common mode of life on earth. many parasites have complicated life cycles with multiple hosts and with a variety of stages, not all of which harm or depend on the host. one can glean that students find it difficult to define a parasite. this series will treat a variety of facultative and other relationships. predatory association a relationship in which a predator obtains its living by preying on other animals, usually consuming all or part of it, usually killing it. this relationship can be symbiotic when a predator feeds on one or a few species or when it is relatively small and not harming its host. for example, a trematode (all trematodes are considered parasites) may live in the lumen of a host without causing any harm; that trematode is occasionally called a micropredator. other terms that will be causing confusion in this series are infection and infestation. american parasitologists usually define an internal association as an infection, whether harm is caused or not or the organism is small or large. if the association is external, it is referred to as an infestation, and the symbiont is called an ectosymbiont or an ectoparasite. for an internal association to be called an infection, microbiologists usually restrict the agents to viruses, bacteria, and fungi. consequently, some authors restrict an infestation to a metazoan parasite (e.g., maggenti et al. 2005) . barrows (2011) provided three meanings for an infestation: (1) a parasite's colonization, utilization, or both of the host; (2) a host being colonized, utilized, or both by parasites; and (3) and environment being colonized, utilized, or both by pests. other authors consider an infestation to refer to a population rather than to individuals. still, others use the word "infestation" to suggest an action and the term "infection" to suggest a condition or a state. microorganisms such as oral bacteria that live naturally in the mouth or elsewhere in a body are not considered infections or infectious agents by many microbiologists, but the organisms are symbionts. another important term for this series is disease. in medical cases, this often refers to dose. a well-nourished person infected with the american hookworm (necator americanus) with a normal hemoglobin and five eggs per milligram of feces, perhaps resulting from up to 50 adult worms, has an "infection" but without a corresponding disease caused by the loss of blood or other detrimental signs. an egg count of 20 per mg in a healthy person or 5 in one anemic with iron or protein deficiency usually indicates the threshold of disease. massive infections with disease can produce as many as 50 eggs per mg of feces (beaver et al. 1984) . lightner and redman (1998) discussed this term when dealing with shrimp infections. they said "in veterinary and human pathology, the terms 'disease' and 'syndrome' have a range of definitions. dorland's medical dictionary (1968) defines disease as 'a definite morbid process, often with a characteristic train of symptoms' and syndrome as 'a combination of symptoms [signs] resulting from a single cause or so commonly occurring together as to constitute a distinct clinical entity.' a simpler definition of disease is 'any alteration from the normal state of health.' the latter definition permits the inclusion of alterations in health that may result in subtle conditions in which poor health or reduced resistance to stress are the only signs of disease, as well as those disease syndromes on the other extreme, that may be accompanied by catastrophic losses." an important, prudent course of action for an author is to carefully define a term or description that is being treated in any manuscript. a reader, on the other hand, may have serious problems understanding what is being said without that definition. terms being used in symbiosis are especially difficult because research conducted to define an entire relationship seldom exists. several glossaries are careful not to include definitions of either infections or infestations, and others are careful not to define a parasite! many terms used for defining ecology of parasites such as "incidence," "prevalence," "intensity," "mean intensity," "component population," and many others are used differently by different people, and we strongly recommend the article by bush et al. (1997) as a standard or at least a source of discussion. usually, a combination of factors triggers a harmful condition, but only one or two of these factors are known in any detail for most cases. furthermore, most of the examples below treat only one or two of these triggering factors. differentiating the triggering factors into those from the external environment and those originating from the host is, for the most part, impossible to characterize because the two are typically entwined. moreover, genetic triggers that involve expression such as upregulation, downregulation, and gene knockdown may each necessitate a variety of triggers for expression. triggers causing a harmless symbiont to transform into a harmful one can also involve the initiation, interruption, or inhibition of biochemical pathways, complicated actions that sometimes incorporate a cascade of reactions. this chapter will not treat the details of mechanisms but rather present examples of what appear like results of cause-and-effect. as indicated above, the headings indicate only some of the triggers in each case, with an emphasis on the primary one. temperature often regulates disease in marine animals to benefit stocks of all parties. for example, the english sole, parophrys vetulus, arrives into its estuarine nursery area in the northern oregon coast from january to april, grows dramatically in the upper estuary during the summer, and then migrates offshore in the autumn, never to return inshore. when inshore, the sole gets infected by the microsporidian glugea stephani that develops in the intestine at temperatures !15 c. this agent appears to cause mortalities from september through november, but this period is when most of the fish routinely migrate offshore where the temperature remains less than 10 c, a temperature that was shown experimentally to inhibit the microsporidian agent's development, which ultimately results in uninfective spores within a few months. consequently, the parasite seldom harms the host stock. however, approximately 10 % of the sole stock remained in the upper estuary where an estimated 80 % of those fish had a rapidly developing infection and most probably died and decomposed or were eaten, dispersing the spores. those fish plus the overwintering infected fish and few reservoir starry flounder juveniles (platichthys stellatus) (a reservoir host is a primary host that harbors the pathogen but typically exhibits no ill effect and serves as a source of infection) provided infective spores for next year-class of sole (olson 1981; overstreet 1982) . temperature also has a great bearing on many other pathogenic triggers. for example, the coccidian calyptospora funduli commonly infects the liver, pancreas, and occasionally other tissues in the gulf killifish, fundulus grandis, in the coastal gulf of mexico. the fish becomes infected by feeding on either the infected intermediate host palaemonetes pugio (the common daggerblade grass shrimp) or related species. infections are synchronous, meaning that all the developing stages are the same. early developing stages and mature oocyst stages are rarely seen in the same fish. in some cases, over 95 % of the liver can be infected without causing any notable severe harm to the host. when heavily infected and lightly or noninfected killifish were maintained in outdoor raceways and freezing occurred, the heavily infected fish all selectively died. presumably, that was because the liver serves as a storage reservoir for necessary glycogen, vitamins, minerals, and other necessary nutritional resources. those resources found in 5-10 % of the liver were presumably not enough to satisfy the needs of the fish during stressful low-temperature conditions. a series of experimental infections conducted at about 22, 10, and 7 c for periods between 5 and 20 days in length (solangi et al. 1982) showed that both low-temperature treatments inhibited all developmental stages. when the fish were returned from the low temperatures to 22 c, development of all the stages resumed except in fish exposed to 7 c for 20 days. in those infections, many of the coccidian organisms were atrophied or disintegrated within their parasitophorous vacuoles, and necrosis also occurred in some of the pancreatic tissue. in any event, decreased parasitism during inhibition was not linked with a leucocytic inflammatory response. when fish remained at a constant temperature of about 24 c, an inflammatory response to the organism commenced at day 18, intensified at day 20, and diminished by about day 30 (hawkins et al. 1981, solangi and . in other words, inflammation associated with infections in warm water begins as gamonts developed and ends after formation of the oocyst wall. consequently, infections that occur during cold winters can be either helpful or harmful to the host, depending on the stage of the parasite, the extent of low-temperature exposure, the age of the fish, and other variables. atypical temperatures, such as warm water associated with power plants, can cause infections of a specific parasite during periods when the hosts are more likely to be consumed by predators, more susceptible to disease, or more susceptible to interactions among parasites that can occur and result in unusual pathogenic conditions. as also suggested above, temperature can have an effect on development of a parasite and can occasionally result in a harmful effect. the ascaridoid nematode contracaecum multipapillatum in a coastal lagoon at celestún, state of yucatán, mexico, infects as definitive hosts the olivaceous cormorant and the great egret. the intermediate host for this avian nematode is the mayan cichlid (cichlasoma urophthalmus). the juvenile in the fish host for all members of the genus contracaecum is usually a third stage; however, in the warm area of mexico, many of the juveniles had developed into the fourth stage. when these fourthstage juveniles were fed to a kitten, they developed into adults in the intestine, often associated with hemorrhaging small ulcers (vidal-martínez et al. 1994 ); they did not develop or cause a pathogenic response in rats, ducks, or chickens. people commonly eat the cichlid in mexico and, consequently, are potential hosts. when deardorff and overstreet (1980b) fed third-stage juveniles, the typical stage occurring in fish, to day-old chicks and ducklings and to mammals, they neither developed nor survived; however, when the third-stage juvenile was surgically inserted within tied off semipermeable dialysis tubing into the abdominal cavity of the animals, the worms did mature. the gnathostomatid nematode echinocephalus sinensis matures in the intestine of the eagle ray, which acquires the third-stage juveniles by eating the infected oyster crassostrea gigas. infections in this commercial oyster were most prevalent and had the highest intensity in hong kong between july and september. kittens, monkeys, and puppies were fed the infective stage from oysters during every month, but those worms collected only from the warm months of august to october infected the mammals (ko 1976) . the worms penetrated the wall of the stomach and intestine, migrated to and lodged in various tissues, and often killed the hosts. to test this temperature-triggering condition, ko (1977) obtained oysters during the infective season and acclimated them to 5, 15, about 24, 28, and 33 c. worms from these different temperature groups were fed to kittens, and infections were most abundant in the 33 c group and were less so in the 28 c group; only one kitten became infected from the 24 c group, and no kitten from the 5 or 15 c group became infected. since people eat raw oysters, the nematode constitutes a potential public health risk. under normal environmental conditions, parasites seldom harm their hosts. when the habitat is a series of aquaculture ponds, several conditions are modified. the pond design usually accommodates a heavy density of snail intermediate hosts near the shallow shore where fish fry occur, and the fish stock inhabiting the middle of the ponds creates an abundance of prey for fish-eating birds. for example, primarily in mississippi but also less so in adjacent states, the channel catfish, ictalurus punctatus, is reared, comprising a multimillion dollar industry. in the natural environment, catfishes, including their madtom host relatives, do not occur as juveniles in habitats where the host marsh ramshorn snail, planorbella trivolvis, and american white pelican, pelecanus erythrorhynchos, occur and defecate in any abundance. consequently, infections of the metacercaria (larval stage acquired from a cercaria shed by the ramshorn snail) of the pathogenic diplostomoid trematode bolbophorus damnificus are relatively rare in those natural waters. a total of five different diplostomoid trematode species infecting the catfish were able to kill it in aquaculture environments. two of these developed from cercariae shed from p. trivolvis that ultimately mature in the american white pelican; two were from worms shed from the ash gyro, gyraulus parvus, that mature in the doublecrested cormorant; and the fifth species is one that matures in seagulls. the most harmful species, b. damnificus, can be present in the catfish with infections of less than 40 metacercariae without harming it. heavier infections seem to have an effect on the kidney of the fish aiding in their mortalities. in the shallow water where large numbers of infected snails occur, thousands of cercariae can penetrate the young catfish and kill it quickly, even before developing into metacercariae. this is in contrast to two other trematodes, bursacetabulus pelecanus and austrodiplostomum compactum, which both infected the nerve cord, brain, optic nerve, and eye and at least in experimental infections can occur as several hundred individuals without killing the host (overstreet and curran 2004) . the trigger for mortality regarding b. damnificus can be more complicated than pure numbers. in experimental infections with fingerling channel catfish exposed to a sublethal infection of trematode cercariae per fish, they died when additionally exposed to the bacterium edwardsiella ictaluri, the agent of enteric septicemia of catfish. controls given either the cercariae or nothing did not die. those given 7.5 â 10 5 colonyforming units per ml of bacteria for 30 min singularly without cercarial exposure started dying at day 7, and, by day 21, the percent cumulative mortality leveled at about 46 compared with 84 % for the group given both the cercariae and bacteria. then at day 28 when the metacercariae fully developed, groups consisting of the remaining fish given only the trematode and of the negative controls were exposed to the bacteria. both groups starting dying at day 7, but, by day 21, there was no significant difference in the percent cumulative mortality of about 18 % (labrie et al. 2004) . when contaminating forage fish that occur in the ponds in addition to the commercial catfish became exposed to the cercariae of b. damnificus, they did not become infected. on the other hand, there is an unnamed species of bolbophorus which also infects both the same snail and pelican, concurrently with b. damnificus. however, it does not use the catfish as the second intermediate host, but it uses mosquitofish and sunfish, which it can kill when in high numbers (overstreet et al. 2002) . also of note is the finding that channel catfish exposed to the e. ictaluri bacterium 1 day prior to being exposed to the pathogenic ciliate ichthyophthirius multifiliis had 71 % mortality compared with 27, 29, and 0 % for those respectively given only the bacterium or the ciliate or given neither. the bacterium could be detected by pcr in the gills, brain, liver, and kidney of the fish whether infected with the ciliate or not, but, by day 8, the bacterium no longer persisted in the bacteria-only group except in the kidney (shoemaker et al. 2012) . a few free-living amebas are well known because they can infect humans and occasionally cause fatalities (see overstreet 2013) . best known is naegleria fowleri because it causes fatal "primary amebic meningoencephalitis (pam)," but it is restricted to freshwater, and victims are typically those who swim for extended periods underwater in open bodies with a silty-muddy substratum. the swimmers' nasal mucosa becomes weakened to the point that the ameba can penetrate the membrane and enter the central nervous system (cns) along the olfactory nerve. because that site contains little cellular inflammatory response but provides a good medium for amebic growth, the organism replicates rapidly, usually before the disease can be diagnosed and treated. marine free-living species of the genus acanthamoeba do not grow as rapidly and, depending on the species, invade different sites. they enter the skin, lower respiratory tract, or nasopharynx, and the vegetative trophozoite can reach the cns through the circulatory system. these acanthamebic infections may take weeks or months to cause death, and often infections occur in patients that are immunocompromised (unlike the otherwise healthy hosts that become ill due to pam) but are still difficult to diagnose. six different ameba species have also been associated with painful amebic keratitis, a difficult to treat corneal disease. even though recognized in 1973, amebic keratitis was not common until 1985 when contact lenses became popular; these lenses typically are maintained overnight in a saline solution that can become a source of contamination. toxic waste products can trigger a reduction in host resistance, resulting in susceptibility to rapidly reproducing agents that would normally be held in check or not able to produce infections. the myxosporidian henneguya gambusi infects the western mosquitofish, gambusia affinis, and is rare, not previously considered pathogenic, and not known from any other fish (parker et al. 1971) . we have also seen it in mosquitofish but only from a location in mississippi receiving wastes from a timber treatment facility, involving the wood preservative chromated copper arsenate, a mixture of chromium, copper, and arsenic (as copper(ii) arsenate) (overstreet 1997) . presumably, the mosquitofish becomes infected from being in close contact with actinospores shed from a tubificid oligochaete. a small percentage of the few fish in the contaminated creek exhibited mass infection (figs. 2.1 and 2.2), and it was typically histozoic with plasmodia throughout the skeletal muscle mixed with tissue debris not associated with an obvious inflammatory response rather than in pseudocysts located in the epidermis, corium, and subdermal connective tissue as originally described. a few of those fish from the wild had a severely pathogenic infection, with mature spores liberated from the plasmodia and spread into adjacent muscles, replacing most tissues throughout the body (e.g., dyková and lom 2007) . however, when we collected 50 fish from this station and 50 from another station containing tubificids and then maintained them in aquaria in our laboratory and fed them commercial flakes daily, over half the fish from the contaminated location became moribund or died within 2 months. no fish from the other location died or appeared unhealthy. the moribund fish were sectioned or examined fresh and demonstrated the myxosporidian with its 10 by 6 μm spores 1 mass infection of the myxosporidian henneguya gambusi triggered within the skeletal muscle of the western mosquitofish, gambusia affinis, from a location in coastal mississippi that contains heavy metal contamination. vegetative and mature stages in plasmodia located between and within muscle fibers, with mature triggered spores being liberated and destroying muscle and showing no plasmalemma separating spores from fibers invading most tissues throughout the samples. representatives of live fish from both groups did not exhibit infection in section, and none of the remaining fish exhibited infection when critically examined as fresh. the contamination apparently reduced the host's protective immune response which would normally keep a low infection in check. light infections had to be present in about half the fish from the contaminated location when brought into the laboratory, even though not evident in fresh or sectioned material. under normal conditions, spores of all histozoic species occur in small to large pseudocysts, the latter containing millions of spores. when the plasmodium is extremely small or when few vegetative or spore individuals are present, an infection is difficult to see, and polymerase chain reaction (pcr) probes are necessary to detect infections. in fact, the only person we have seen skilled enough to routinely detect minimal infections is iva dyková of the academy of sciences of the czech republic. another example of a pathogenic agent in the western mosquitofish deals with a free-living organism rather than a known parasite. the free-living organism, the ciliate tetrahymena corlissi, has been reported from fish in aquaria and hatcheries, causing pathological alterations and mortality by hoffman et al. (1975) . they were unable to experimentally infect fish with the ciliate and suggested that infection resulted from a wound or stress. we (overstreet et al. 1995) sampled the mosquitofish from an outlet canal from an integrated pulp and paper mill, 2.5 km upriver from the canal where water entering from a dam kept mill effluent from flowing upstream, and three downstream locations. two of 99 fish from upstream, but none from the other locations, had a proliferation of the ciliate in the head and in the musculature, brachial chamber, and pericardial sac; that location also had fish with a significantly higher prevalence of macrophage aggregates in the spleen, a good indicator of stress. after publication, we learned of a long submerged pipe emptying toxic wastes upstream from our locations and originating from a nonrelated facility located several km from the river and apparently promoting the ciliate infection. some ciliate specimens measured larger than those reported by hoffman et al. (1975) , but sequencing of presumed free-living ciliates infecting various aquatic hosts should allow identifications and experimentation to determine details of the mechanisms that shift a free-living organism to become a pathogenic one. for parasites or diseases of a host organism to be used as monitors of environmental health or biological activities, both the host and the symbionts or pathological responses need to fit several criteria (overstreet 1997) . chemotherapeutics can also serve as toxins. a cdc report (u.s. department of health and human services, centers for disease control and prevention 2013) estimated that about 100 million people worldwide possess a chronic infection with the nematode strongyloides stercoralis, and prevalence of the infection in refugee populations ranged from 11 to 69 % by serosurveys. this species has the unusual ability to replicate and auto-infect its human host, persisting for decades. strongyloides hyperinfection syndrome may be triggered many years after migration of a prior refugee to a nonendemic locality, with large numbers of the parasite infiltrating internal organs, resulting in fatality rates exceeding 50 %. the syndrome is generally induced when an individual is placed on corticosteroids, although other immunosuppressive conditions such as cancer and transplant chemotherapeutic immunosuppression may also trigger the hyperinfection. a change in diet as well as some chemotherapeutic compounds can serve as a trigger and induce a parasite located in one internal habitat such as the intestine, blood, visceral organ, or muscle to migrate to more sensitive tissue or be released from a cyst or an encapsulation and migrate or replicate. free-living or symbiotic metazoans, protozoans, algae, fungi, and bacteria can get into abnormal sites (locations in host) or habitats and develop into pathological agents. some cases are rare and others are common. a rare case consisted of finding the diatom amphora sp., which normally is a free-living alga, in the white shrimp, litopenaeus setiferus, presumably after the shrimp's carapace had been punctured or abraded. the diatom occurred in abundance as clusters in the hemolymph, plus some individuals were present in the gills associated with a melanistic response. the shrimp died. to test the effect of the related amphora coffeaeformis on shrimp, overstreet and safford (1980) injected cultures into shrimp resulting in melanistic responses, but no extensive replication occurred as had been observed in the shrimp killed with amphora sp. a more common opportunist is the fungus fusarium solani, which also developed in penaeid shrimp. farfantepenaeus californiensis, a species of penaeid initially being considered for aquaculture, is a highly susceptible species to this fungus. when experimentally infected with a cultured conidial suspension of this free-living species (hose et al. 1984) , all shrimp became infected within 14 days, tissue destruction and a strong but often unsuccessful hemocytic inflammatory response occurred, and over half of the shrimp died within 24 days. biochemical and hemocytic parameters of the hemolymph changed significantly as the infection developed when compared with noninfected shrimp. when farfantepenaeus aztecus or litopenaeus setiferus was held in seawater with macroconidia spores cultured from an infection from farfantepenaeus californiensis or injected with those spores, resistance to infection occurred. a strong hemocytic encapsulation and melanization of the macroconidia and lysis of the conidiospores occurred in the gills by 28 h. if spore dosage was excessive, 3.2 â 10 6 or greater, all brown shrimp died within 24 h from the macroconidia and hyphae blocking the distal portion of the gill lamellae (solangi and lightner 1976) . in litopenaeus vannamei, the primary cultured penaeid today, naturally infected individuals in culture are moderately susceptible to infection and the spore aggregate, often grossly apparent in the distal portion of the eyestocks; penaeus monodon, which used to be the primary commercially cultured species, is relatively resistant (lightner 1996) . for pathogenicity by free-living algae and fungi, the triggering factor seems to be a wound or being compromised by other infectious agents or toxicants, but, as pointed out, the host species is also critical. one or more ciliates identified as or presumed to be the scuticociliate orchitophrya stellarum has an extensive host and geographic range in europe, australia, and north america as determined by morphological and sometimes molecular techniques. it is considered a facultative parasite that can live indefinitely outside the host when cultured with bacteria and tissue detritus or yeast but with different size and morphology when compared with material in male sea stars. cultured specimens enter male but not female sea stars, probably through the gonopores, where it enters the testes and feeds on sperm of fully mature individuals only (stickle et al. 2007 ). it has been reported from several sea star species and may have been inadvertently introduced into the northeast pacific region in the late 1980s (boom, in bates et al. 2010) . recently, small et al. (2013) determined using its (internal transcribed spacer) sequences and pcr (polymerase chain reaction) analyses that the histophagous ciliate infecting the blue crab, callinectes sapidus, in research facilities in virginia and previously thought to be mesanophrys chesapeakensis was actually o. stellarum. the same or similar infectious ciliate was probably responsible for histophagous disease in wild and captive blue crabs in mississippi (shields and overstreet 2007) , penaeid shrimps in mississippi, and the wild lined shore crab, pachygrapsus crassipes, from carpinteria salt marsh, california, that we examined for ryan f. hechinger, university of california, santa barbara. this infection spreads in the partially closed circulatory system of the decapod (mcgaw 2005) from the hemolymph to muscle, visceral organs, and other tissues (figs. 2.3, 2.4, 2.5, and 2.6), usually killing the host. it has been assumed to enter into wounds of its hosts and now shown by miller et al. (2013) to cause rapidly developing fatal infections in the blue crab inoculated with the ciliate or exposed to ciliates after experimental autonomy. when exposed to ciliates and not wounded, the crab seldom died. for comparisons, the fiddler crab uca minax was inoculated with doses of either 10 or over 500 ciliates per crab, and crabs with 10 sometimes established infections, but those with the higher doses developed them rapidly. the infections developed at 10-15 c, and ciliates were attracted to blue crab serum over other nutrient sources, suggesting the facultative nature of a blue crab parasite. viridans streptococci typically occur harmlessly in the mouth. these bacteria can be differentiated from streptococcus pneumoniae using several tests. moreover, they lack either the polysaccharide-based capsule typical of s. pneumoniae or the lancefield antigens, based on the carbohydrate composition of bacterial antigens found on the cell walls in beta-hemolytic bacteria of the pyogenic, or pus-producing, members of the genus. some may be involved in other mouth or gingival infections as pericoronitis or inflammation of the gums around molar teeth. however, if they are introduced into the bloodstream from surgical or other lesions, they have the potential of causing endocarditis, especially in individuals with damaged heart valves. viridans streptococci have the unique ability to synthesize dextrans from glucose, allowing them to adhere to fibrinplatelet aggregates at damaged heart valves. often there occurs an indirect site-associated triggering mechanism for parasite establishment, with a corresponding associated ability to enhance or decrease pathogenic effects. the bothriocephalid anantrum tortum, a long, up to over 15 cm, cestode, twists either singly or in groups of up to eight, within the intestine of its relatively small fish host, synodus foetens (inshore lizardfish). it can occur near the pyloric ceca, along the intestine, or near the anus of the samples, measuring up to 300 mm long. overstreet (1968) detected a 99 % level of significance over a 2-year period between the monthly prevalence of worms located near the anus and water temperature. salinity was held constant, and an inverse sine transformation was applied to the percentage data. this anal location favors loss of the worm during warm weather, possibly assisting probability of best completing life cycle, but to be expelled, the lizardfish appears to have needed a several-mm-long packet of prey fish scales with a diameter much greater than that of the intestine. the carnivorous lizardfish can eat fishes and shrimps larger than itself. consequently, fish prey with large scales tends to be advantageous for the lizardfish to rid itself of the parasite. sometimes, a fortuitous site creates a trigger for pathogenicity. for example, a diver accidently punctured his hand while reloading his spear gun. later, unconcerned about his wound, he was filleting a jackfish, and a female of the nematode philometra sp. invaded the wound and attached deeply. after several unsuccessful attempts to remove the worm, the pain-inflicting infection required surgery (deardorff et al. 1986 ). not to be overlooked is the obvious matter of dose. this obvious matter is treated in discussions and is important for all agents from viruses to metazoans. in the case of metazoans, a host can come into contact with an especially high, unnatural dose of infective agents such as thousands of cercariae killing an intermediate host well before the corresponding metacercariae develop (e.g., overstreet and curran 2004) . intermediate hosts under specific conditions can be heavily infected simultaneously with several parasitic species. some parasites such as microphallid trematodes infect a variety of birds and mammals. therefore, a bird could acquire a harmful infection by feeding on individual heavily infected intermediate hosts, or source communities (bush et al. 1993 ). the host species is also critical for many protozoans that are acquired by feeding on an intermediate host in which replication or maturation of a stage is necessary. for example, whereas a few killifish species are natural hosts of calyptospora funduli, several related atheriniform fishes can be experimentally infected, but no nonatheriniform could be infected (fournie and overstreet 1993) . those experimentally infected demonstrated a variety of abnormalities, including asynchronous development, degeneration of early developmental stages, formation of macrophage aggregates, and a granulomatous inflammatory response, especially one exhibiting liver destruction in fundulus olivaceus and rivulus marmoratus. of course, feeding behavior, environmental conditions, and geographic isolation can also serve as barriers to infection in fish in addition to innate immune barriers. when introduced species become established in a system, they can be resistant to harmful factors in the environment, or they can be susceptible to extensive predation, diseases, and parasites. species of the ascaridoid genus goezia typically embed in an encapsulated ulcer in the wall as well as free in the lumen of the stomach of their natural fish hosts. these associations are described by deardorff and overstreet (1980a) for the nematode species goezia pelagia in the cobia, rachycentron canadum, and the atlantic spadefish, chaetodipterus faber, as well as for goezia minuta in the gafftopsail catfish, bagre marinus; the hardhead catfish, ariopsis felis; and the inshore lizardfish, synodus foetens. in none of these hosts does there appear to be any significant harm associated with the presence of the nematode (deardorff and overstreet 1980a) . however, when the introduced host species such as the blue tilapia, oreochromis aureus; striped bass, morone saxatilis; and hybrid striped bass become infected with goezia sinamora, the nematode produces massive fibrotic nodules in the fish stomach, and the nematode can also cause mortality. goezia sinamora was implicated in mortality of hatcheryreared striped bass and tilapia introduced into a series of lakes in florida, including lake parker [gaines and rogers 1972 , see correction by deardorff and overstreet (1980a) ]. in the tilapia, this roundworm has been observed to migrate through the intestinal wall, causing extensive lesions, in addition to forming nodules in the stomach. when the largemouth bass, micropterus salmoides, and other native species of fish were exposed to this nematode, the resulting infections contained relatively few of the roundworms and demonstrated no conspicuous harmful effect (deardorff and overstreet 1980a ). several books and chapters [such as those by overstreet (1983) , barnard and behnke (1990) , lewis et al. (2002) , moore (2002) , lefèvre et al. (2009) , adamo (2012), adamo and webster (2013)] have reported on parasite infections resulting in virulence, evolutionary, and behavioral changes in intermediate hosts such that the intermediate host has a better opportunity to be preyed upon by the appropriate definitive host than by chance alone. whereas these changes are not necessarily triggers that make an agent more pathogenic, they often are changes that reflect the point at which the undeveloped, noninfective agent becomes infective to the definitive host and pathogenic to the intermediate host. a good example is the microphallid trematode levinseniella byrdi, which infects the intestinal ceca of a few bird species such as the seaside sparrow, clapper rail, willet, and semipalmated sandpiper. these birds acquire the infection from one of a few talitrid amphipods such as uhlorchestia uhleri from salt marshes of texas to north carolina and uhlorchestia spartinophilia in a similar habitat from cape canaveral, florida, to central maine. in the gulf of mexico, the metacercaria also occurs in the amphipod species orchestia grillus and chelorchestia forceps (bousfield and heard 1986) . the dramatic modification in the amphipod usually occurs after about a month, once the metacercaria becomes infective. at this point, the host amphipod turns from greenish or grayish to a translucent or bright orange (fig. 2.7) . the infected amphipod also unusually slows down its movements and, in contrast to its uninfected, negatively phototactic cohorts, does not always hide under wracks of dead, dissociated leaves and stems of spartina grass or other of their dietary debris shelters. apparently, the amphipod carotenoids become unbound from protein of the infected host, allowing the bird host to be preferentially attracted to the now brightly colored amphipods containing infective metacercariae (bousfield and heard 1986) . johnson et al. (2009) experimentally manipulated a few tidal salt marsh creeks in plum island estuary, massachusetts, by nutrient fertilizer enrichment and exclusion of the killifish fundulus heteroclitus (mummichog), a primary predator in the system. interaction of the two treatments reduced abundance of the common uhlorchestia spartinophilia, and apparently infected amphipods moved from the marsh edge to the adjoining creek-wall habitats during 1 year, resulting in 97 % of the amphipods clinging on the creek walls and exhibiting the bright orange. a subsample of the colored amphipods was confirmed to be infected, and they were seen being fed upon by the semipalmated sandpiper and seaside sparrow. in regard to the above host modification, other microphallid species in those amphipods did not turn their hosts orange or noticeably modify their behavior. a similar situation has been noted to occur where levinseniella tasmaniae but not other microphallids induced an orangish color in the amphipod austrochiltonia australis in tasmania (smith 1981) . that is not to say all species of levinseniella induce coloration and other modifications in their amphipod hosts since levinseniella tridigitata does not modify gammarus aequicauda (see thomas et al. 1996) and levinseniella carteretensis (or levinseniella hunteri) does not induce a color change in any of the five talitrids, including u. uhleri, when experimentally infected (bousfield and heard 1986, heard personal communication) . another trigger involves photoreception. the snail host typically sheds large quantities of cercariae of levinseniella tasmaniae infective for the amphipod host when under light conditions, whether natural or experimentally reversed (smith 1981) . and yet, cercariae of other species are shed during dark or other physical conditions. almost all symbiotic relationships, including those discussed above, involve a combination of triggering factors. those factors and host-symbiont relationships involving termites are particularly well studied. we, ourselves, have examined in fig. 2.7 specimens of the amphipod orchestia grillus in coastal mississippi showing on the top an uninfected one and on the bottom a transformed orangish one that is infected with the microphallid levinseniella byrdi, a trematode that infects the intestinal ceca of a few bird species. the transformation triggers phenotypic and behavioral changes specifically attracting infective specimens to predatory birds in which the trematode matures considerable detail the host-symbiont relationships affecting the outcome of pathogenic viruses in populations of commercial penaeid shrimp. we will address in this section some of the symbiotic relationships and interrelationships that can involve a shift from harmless to harmful relative to three host groups. the relationships between termites or related insects and their symbionts are numerous and provide examples of an abundance of triggers, including diet, to control the various relationships. some of these involving termites will be discussed elsewhere by david bignell (volume 2, chapter 6 of this series). investigations by l. r. cleveland (e.g., 1926) were designed to better understand symbiosis using termites and their intestinal flagellates. he was well aware that bacteria and yeasts were involved in the relationships and that there was a fine line separating where one symbiotic association ended and the other began. for example, when one agent in a so-called mutualistic relationship could survive without the host, it nevertheless also can become a parasite living off the host. this situation, however, is complicated and difficult to assess. he tried to remove one or more flagellate microorganisms from a host without harm to the host so that the association could be manipulated. he also thought it best to consider all components, which he defined differently than we do (he considered a commensal association to be one when neither party was benefited nor injured), of an association as symbionts. depending on a specific termite host, he could void the flagellates with starvation, a high temperature not lethal to the host, a specific level of moisture, and oxygen under pressure. for example, by oxygenating the large pacific coast termite (zootermopsis nevadensis) for 7 h at 1.5 atm, two (leidyopsis sphaerica and trichonympha campanula) of the four flagellates survived, and the host lived "indefinitely." additionally, starving the termite at the above conditions for 6 days left only one flagellate (l. sphaerica), showing that both l. sphaerica and the termite do together constitute a necessary and mutualistically beneficial symbiotic relationship. without any flagellates but with a diet of wood, the termite lived for about 3 weeks. reintroducing either l. sphaerica or t. campanula allowed the termite to experience its normal much longer longevity (usually 60-70 days). cleveland realized that the presence of the other two flagellates (trichomonas termopsidis and streblomastix strix, both having a poorly understood symbiosis with epibiotic bacteria) in the termite starved for 8 days would help the termite survive for about 10 weeks but were not necessary symbionts; he realized that bacteria played a role in digesting the diet. the "primitive" termite mastotermes darwiniensis from australia was studied by li et al. (2003) , and it had in its hindgut six flagellates, none of which can yet be cultured. historically, these flagellates were considered to use their digestive enzymes to digest cellulose for the benefit of the termite. however, they determined using pcr technology that the main endoglucanase activity in the flagellates appears to originate from termite cellulases produced in the salivary glands. at least two of the flagellates possess their own endoglucanase genes, which are expressed but without significant enzyme activity in their nutritive vacuole. after millions of years of evolution, these flagellates, suggested by li et al. (2003) , are heading for a secondary loss of their own endoglucanases to exclusive use of the termite cellulases. feeding on the symbionts still seems to be an important nutritional component of the termite diet. some termites, such as those which are soil-feeders, depend entirely on bacterial symbionts (bignell et al. 1980 ). this association, often involved with coprophagy, feeding on fecal pellets containing termite bacteria that digest the cellulose, will be treated by d. e. bignell elsewhere in this series (volume 2, chapter 6). other termites, such as members of the macrotermitinae, depend on a relationship with mutualistic fungal symbionts of the genus termitomyces, which form fungal combs in the nests. these and attine ants can produce nests that often are thousands of liters in volume, able to persist for decades, and contain millions of sterile helper individuals usually resulting as offspring from a single queen. those termites are major decomposers of the old world tropics, and the ants are dominant herbivores of the new world tropics. the fungal symbionts of the termites can produce sexual fruiting bodies allowing their horizontal acquisition, but those of the attine ants rarely fruit and are typically propagated clonally and vertically by the dispersing queens. the life cycles of the fungus are again shown to help trigger different symbiotic relationships. aanen et al. (2002) present phylogenies of the termites (about 330 species in 11 genera) and their fungal symbionts which number about 40 and are shared by different termite species. they show significant congruence between the termite and fungal phylogenies because the interactions at higher taxonomic levels show considerable specificity. they also considered the trait of biparental colony founding to constrain evolution of vertical symbiont transmission in termites, where the male survives and mates repeatedly with the female for life but not in ants where males die after a single mating. the formosan subterranean termite pest (coptotermes formosanus) builds a large nest with spongelike networks of intricate feces-lined tunnels (carton material). fungal pathogenic agents are used, usually unsuccessfully, to kill the termite. studies by chouvenc et al. (2013) have shown that environmental conditions within termite nests promote the growth of actinobacteria, whose presence in turn seems to protect the termite colony against fungal entomopathogens, including metarhizium anisopliae. in other words, the actinobacteria, which represented a nonnutritional exosymbiosis in the termite, was a defensive mutualism that increased survival of the termite and was additive to the termite's individual immunity and social defensive capacity, which in turn increased survival of the termite. baculovirus penaei, commonly called bp or bp (pvsnpv), has a widespread distribution in cultured and wild penaeids, and it can cause severe epizootics in larval, post-larval, and juvenile stages. it is an enveloped, polyhedrosis, rod-shaped, double-stranded, intranuclear, dna virus infecting epithelial cells of either the anterior midgut and midgut gland, or the hepatopancreas (hp; r-cells, f-cells, and b-cells, most commonly found near the base where the hp tubules join the anterior midgut). the e-cell populations (stem cells) at the distal portion of the hepatopancreatic tubules produce cells so quickly as to assure noninfected new cells (figs. 2.8, 2.9, and 2.10) (overstreet et al. 1988 ). baculoviral infections of bp can involve nearly 90 % of the shrimp hp during mysis and early postlarvae stages without necessarily causing death. a number of triggering factors can tip these acutely infected shrimp into mortality. the virus is one out of 25 or so that infects penaeid shrimp and one of a few thousand known from invertebrates. because it has a proteinaceous tetrahedral occlusion body, infections can be detected and followed with a light microscope; because of these bodies, it was the first species reported and characterized from a shrimp (summers 1977) . the virus has a simple direct life cycle, although unknown means allow the agent to remain dormant or in a reservoir host. it can infect an acceptable penaeid either from free or occluded virions (fig. 2.8 ) in the surrounding water or through a carrier host. experimental infections are best accomplished by feeding the virus concentrated in a rotifer to protozoeal or early-stage mysis larvae or concentrated in a brine shrimp to infect late-stage mysis or early postlarvae (overstreet et al. 1988 ). depending on a number of factors, the virus replicates in the host alimentary cells and is most prevalent and infective at about day 3 after being fed. the nucleus of those cells enlarges, and the polyhedra and associated virions rupture into the lumen of the host midgut. we have observed as many as 24 relatively large tetrahedral bodies (about 13 μm on a side) or 100 small distinct bodies in experimental infections. the infective agent is then passed out through the intestine or it can be released when the infected larva is eaten by an appropriate predator. unlike some insect baculoviruses that are used to kill agricultural pests, the bp virion remains active in the polyhedron for less than 2 weeks rather than for 80 or so years. on the other hand, bp maintained in an ultralow freezer remained active for at least three and a half years. natural infections of bp in the brown shrimp, farfantepenaeus aztecus, were monitored monthly between january 1989 and november 2000 with seasonal the monitoring took place in the northern gulf of mexico in or off mississippi waters, but shrimp from florida were also examined. during the mid-1970s, infections occurred in wild populations of the pink shrimp, farfantepenaeus duorarum, but when we monitored all commercial penaeid shrimps for those infections, the virus primarily infected the brown shrimp and only occasionally infected the pink or white shrimp, litopenaeus setiferus, which exhibited the otherwise rare infection. during the monitoring, we tried to use 4-cm long shrimp because they exhibited the highest prevalence and intensity of infection. infection usually occurred only about 3 or 4 months of the year between about april and june, with the highest prevalence occurring in 1998 when we detected values of 56, 10, and 4 %. values reaching 40 and 36 % occurred in may 1993 and 1997, respectively . typically, the prevalence reached about 10-25 % during the remaining years, and infections were almost always absent in fall and winter. rarely was the intensity of infection great, as usually observed in experimental infections with larvae and early postlarvae. the outbreak dynamics probably resulted from a variety of factors, including genetics (primarily brown shrimp), the interaction between the life cycle of the shrimp and the presence of the virus, the age of shrimp when initially infected, and resistance to infection, which occurred primarily in larger shrimp. these factors will be discussed below in more detail such as with infections and problems in aquaculture hatcheries. strains there are at least four strains of bp as differentiated with molecular probes. each seems to be restricted to specific penaeid shrimps. one strain occurs in some commercially important shrimp in the northern gulf of mexico; another occurs in noncommercially important shrimp (trachypenaeus spp.) in the northern gulf of mexico; another occurs in melicertus marginatus in hawaii; and the last occurs in litopenaeus vannamei in ecuador. we have used for our experimental research the latter strain originally obtained and frozen from ecuadorian aquaculture facilities by biologist james brock. there is also a series of strains of mbv (monodon baculovirus), a baculovirus that produces spherical polyhedral bodies but which have many similarities to bp. we attempted cross infections with bp for all but the hawaiian strain, using presumably susceptible penaeid postlarvae without success. free and occluded virions in addition to the virions occurring in the tetrahedron, which occasionally does not contain any virions, free ones lined up along the abnormally-distorted internal surface of the nuclear membrane. the free viral particles were collected from a freeze-thaw preparation either by filtration through a 0.45 μm filter or from processing of the preparation by relatively low-speed centrifugation to prepare a cell-free supernatant, and when next exposed to shrimp larvae, they produced infections. these subsequent infections did not appear to be more virulent than those initiated from virions incorporated in the tetrahedron. however, the free virions of some insect baculoviruses produce secondary infections in the hemocoel, rather than gut cells, and those are considerably more virulent (kelly 1982) . dose dose of bp is difficult to determine because there is no appropriate crustacean cell line available to culture the virus. however, we conducted a replicated relative study using the same standard viral stock suspension and determined a clear dose response involving both prevalence of bp and mortality of shrimp (overstreet 1994) . environmental factors temperature and probably other factors affect both the production of free virus and the relationship between the virus and its shrimp host. when a homogenate of bp-infected cells is placed in seawater (32 ppt), the virus becomes completely inactivated between 7 and 14 days when maintained at 22 c. however, when maintained at 5 c, some virions remained active after 14 days. a low temperature also delayed or inhibited infections in larvae and presumably adults. virions were inactivated by a 10-min exposure to temperatures of 60-90 c. a variety, but not all, of toxicants can probably affect the relationship between the viral agent and shrimp host. couch (1976) reported an increase in prevalence of infection in pink shrimp exposed to low levels of aroclor 1254 ® . we tried to duplicate his experiments using brown shrimp and 3 ppb aroclor 1254 and also 2 ppm nickel but with unsuccessful outcomes. we later discovered from couch (personal communication) that the aroclor 1254 he used was probably unknowingly contaminated with another toxin (overstreet 1994) . studies were conducted by treating free bp in a variety of ways and then feeding it to larvae of l. vannamei as a bioassay (overstreet et al. 1988 ). unlike insect baculoviruses, all of the bp virions that we tested were inactivated when desiccated for 48 h. tests conducted for possible treatments in aquaculture also showed that the virus was completely inactivated by a 30-min exposure to ph 3; ph 11 extended the pre-patency period of infection, but it did not inactivate the virus. ultraviolet irradiation for 40 min at a wavelength of 254 nm when the virus was 5 cm from the light source also inactivated the virus (leblanc and overstreet 1991a). the virus was also completely inactivated by a chlorine (in the form of calcium hypochlorite) concentration of 200 mg/l when treated for 1 h and by 1600 mg/l when treated for a time period as short as 20 s (leblanc and overstreet 1991b). other than for sterilizing aquariums, these methods other than desiccation and steam cleaning seemed impractical for an aquaculture facility. research host shrimp to conduct experimental infections, we used mostly litopenaeus vannamei cultured at the gulf coast research laboratory consortium either at oceanic institute (oi) in hawaii or at gcrl in ocean springs. our early studies with larvae required antibiotics to counter bacterial infections, which otherwise often reduced a larval stock by 50 % and would have been considered an acceptable loss in commercial hatcheries at that time. even with antibiotics, we obtained bp infections with associated catastrophic mortalities when the shrimp larvae were exposed to the virus. after hybridization crosses of shrimp and introducing special wild brood stock, the result was creation of shrimp stocks with improved resistance to bp, and we noted as a bonus our having created crosses exhibiting resistance to other microbial agents. the bp prevalence could be variable, but mortality was reduced or eliminated. in a few cases, we obtained shrimp from commercial hatcheries or other research facilities and had to use antibiotics. host resistance the genetic families of litopenaeus vannamei produced at oi gradually became more resistant to bp. in some cases, it was easy to obtain 100 % prevalence but without the associated mortalities observed using other stocks of shrimp. middlebrooks et al. (1989) examined lectin levels in the hemolymph of monthly samples of wild farfantepenaeus aztecus, and the levels were found to vary by season and among individuals, suggesting that levels could serve as indicators of health or immune status. high titers of activity for lectin agglutination occurred in september, and low levels occurred in april. those observations of positive bp infection results when lectin levels were low corresponded with the time when bp first became apparent in the wild shrimp population, and polyhedra subsequently were no longer apparent when lectin levels were high. age and growth the age of litopenaeus vannamei influences bp infections in a variety of ways. stuck and overstreet (1994) exposed pathogen-free shrimp ages mysis 2-3 through 25-day-old postlarvae (pl) from different sources, each with a single viral exposure, and cultured them for 15-21 days. all groups pl9 or younger became heavily infected within 2-5 days, some experiencing high mortalities compared with controls. surviving postlarvae had reduced growth as determined by dry weight. one of these groups was cultured for an additional 49 days, and the smaller postlarvae that survived the infection appeared similar to the controls after 4 weeks by which time viral prevalence had decreased. exposure of the virus to older postlarvae produced a high prevalence of infection but with little effect on either survival or growth. when 13-to 14-day-old postlarvae in similar size groups of previously infected and noninfected individuals were starved for 10 days, less than 2 % of the infected postlarvae survived the 10-day period compared with 52 % of the noninfected ones. leblanc and overstreet (1990) exposed infected bp to groups of l. vannamei 3, 39, 63, 120, 157, 325, and 454 days after reaching postlarvae. the postlarvae exposed when 3-days-old (pl3) exhibited infections in 77 % of those examined at 5 days postinfection (pi) and in 100 % of the same group when examined at day 9 compared with 0 % prevalence in the controls; 14 % of the shrimp that were exposed when postlarvae for 39 days (pl39) had developed an infection at day 9 pi when 20 % of those exposed as pl63s exhibited infections; and the prevalence in the latter group increased to 42 % at day 14. with the exception of one shrimp exposed when a pl120, none developed an observable infection at 19 days, and only 2 of 20 of the exposed pl157 group examined at day 16 exhibited an infection. no control became infected. other experiments also showed this decrease in prevalence as well as intensity of infection and mortality with age. in general, most postlarvae 12 days old or older did not become infected, and those that became infected did not die. this age corresponds with the age that most postlarvae leave the offshore waters and settle on the substratum in the estuarine area where the infective agents occur. energy the nutritional condition of litopenaeus vannamei can be assessed biochemically in terms of available energy reserve. the principal energy storage materials in the penaeid shrimp are lipids and proteins, with carbohydrates considered to be a minor energy reserve. triacylglycerol (tag), an ester derived from glycerol, and three fatty acids serve as the primary classes of lipid used for energy storage. in patent infections of bp conducted by stuck et al. (1996) , the polyhedra first appeared in the hp 18-24-h postexposure with a maximal infection usually occurring at 72 h (also see hammer et al. 1998) ; however, progression of the infection after the initial appearance of the polyhedra was variable among shrimp potentially corresponding with different levels of body lipid used for energy storage. we measured preinfection and postinfection tag and protein levels during a series of experimental bp exposures using shrimp from a variety of sources with different inherent protein and tag levels. mysis-stage or early post-larval protein levels measured either preexposure or postexposure to bp showed no consistent relationship with bp infections. however, a viral prevalence of 86-100 % occurred at 72 h when initial tag levels measured above 3.5 μg/mg in contrast to 35 % or less in shrimp when initial tag levels <2.0 μg/mg. tag levels in mysis stage-1 larva rose rapidly from 1 to about 11 μg/mg in 2 days for both infected and negative control populations, then dipped to about 2 μg/mg at day 9, and subsequently again rose in both populations but was about 3 μg/mg higher in the infected shrimp at day 15. most shrimp died before the end of a week. two other experiments starting with 9-day postlarvae (pl9s) were conducted when initial tag levels were either about 1 or 7 μg/mg, and substantial mortalities did not occur in either. all individuals in the infected groups became infected, at about day 10 in the initial low tag group and at day 3 in the initial high group. in the initial low tag group, the tag level of the infected group stayed about the same at day 20, but that of the control groups increased to about 4 μg/mg. in contrast, in the initial high tag experiment in which the tag level for both infected and control groups dipped from 7 to about 1.5 μg/mg at day 3, the level increased to about 10 μg/mg at day 20 and about 15 μg/mg at day 25. stress caused by starving postlarvae (pl18s) reduced tag but not protein reserves. detection of the bp infection was delayed for 30 h when the group to be infected was starved for 48 h compared with continuously fed controls, but the prevalence of infection in both groups increased rapidly to similar high prevalence values above 80 % between 72 and 192 h pi. genetics offspring of male and female crosses of high-growth and low-growth "families" of a well-defined population at gcrl consortium at oi, hawaii, were fed bp as 15-day-old postlarvae (pl15s) (alcivar-warren et al. 1997) . the high â high-growth and low â high-growth (female â male) postlarvae, respectively, had a 77 and 85 % survival rate at 18 days postexposure compared with 19 and 24 % survival for the low â low-growth and high â low-growth offspring. all but the high â low-growth offspring, with a 68 % prevalence of infection, had an 88-100 % prevalence at day 4. the low â low-growth cross of 3-month-old shrimp fed the virus ihhnv (infectious hypodermal and hematopoietic necrosis virus, which has been classified as penaeus stylirostris densovirus) exhibited the highest prevalence (48 %) at 30 days compared with the lowest (6 %) in the high â low-growth cross. random amplified polymorphic dna polymorphisms for the four crosses showed no clear relationship between the prevalence values of ihhnv and bp. even with similar mtdna haplotypes included in the initial crosses, the offspring of those crosses exhibited major differences in both steadystate levels and patterns of expression of mitochondrial 12s rrna at various early developmental stages of the resulting offspring of the different crosses. even though specific genetic markers and differences could not be associated with specific differences in susceptibility to different infections, shrimp growth, or regulation of gene expression, the genetics of the shrimp play an important role in triggering infections and mortality from infections. occur concurrently with other disease-causing viruses producing a synergistic effect. we (krol et al. 1990 ) found such a virus infecting the anterior midgut epithelium and the r-and f-cells from the hp in larval specimens of litopenaeus vannamei. the occurrence of the reo-like virus was observed only in bp-infected shrimp; however, shrimp with bp seldom had the reo-like virus. other agents often associated with stress also occur in shrimp (overstreet 1994) . susceptibility to viral infections appears to be enhanced by crowding of host individuals. we determined that the prevalence and intensity of infections in shrimp crowded in commercial bait tanks were higher than in those collected from the wild shrimp population that was used to stock the bait tanks. in summary for the endemic virus bp, there are a variety of susceptibility factors dealing with the environment, virus, and host, including the nutritional and molting state of the host, which can trigger a relatively harmless infection to develop into a severely pathogenic condition. in nature, this condition is seldom recognized because only microscopic-sized larvae and early postlarvae usually die from the infection, dead animals cannot be seen because they are small or readily eaten, and the opportunity for the agent and the larvae to come into close proximity is relatively rare. in aquaculture, the virus and larvae can and do come into contact. hatcheries in the multibillion-dollar shrimp industry have prompted research to solve the previous major threat that collapsed hatcheries in the usa and elsewhere in the western hemisphere during the early days of the industry when brood stock was obtained from the wild. the related mbv in the eastern hemisphere also caused a similar major problem. now that the industry can rear most penaeid species; detect bp and other infectious agents with pcr, gene probes, and other methods in routine monitoring; produce its own brood stock relative to disease resistance and growth potential; and accommodate ambient temperatures, infections of bp in aquaculture are rare. if they do occur, an entire spawn can be destroyed and disposed and the system decontaminated, and there would be a time loss of only a few days before a new spawn could be produced. pathogens of shrimp aquaculture with the exception of baculovirus species, most or all the pathogenic penaeid viruses are not native to the shrimp species being cultured or perhaps to any commercial shrimp. consequently, the cultured species may be highly susceptible to and die from one or more of the viral agents. most of the presently known 25 or so shrimp viruses cause mortality in subadult animals that have been reared for a few months. the high cost of feed and labor plus the relatively long animal growing period results in a high economic cost. therefore, it is necessary to incorporate the costs of an inability to quickly replace the entire infected pond-reared adult or subadult stock as can be done for larvae in hatcheries infected with bp infections. production losses associated with the exotic viruses white spot syndrome virus (wssv) and taura syndrome virus (tsv) provide excellent examples for examining the emergence of pathogenicity in aquaculture on a large scale. the history of the shrimp aquaculture industry is a case study of the emergence of severe pathogens in hosts where none was known previously. for about two decades after the industry began in the 1970s, few viral pathogens were known. beginning in the early 1990s, several severely pathogenic viral diseases emerged. we will consider two of the pathogens in some detail as they inform our understanding of the appearance of highly pathogenic symbionts through host switching. white spot syndrome virus (wssv) was first reported in 1992 from taiwan. the virus belongs to the order baculovirales. although once considered a member of the nudiviridae or baculoviridae, wssv is more closely related to the recently erected family hytrosaviridae (wu et al. 2009; wang and jehle 2009) . wssv is now considered a member of the genus whispovirus in the family nimaviridae. the virus is a rod-shaped, enveloped virion and among the largest viruses known at 120-150-nm wide by 270-290-nm long. the genome is double-stranded dna of 295 kbp. the host range is among the widest of crustacean viruses and infects all decapod crustaceans that have been tested. taura syndrome virus (tsv) was first observed from ecuador also in 1992. the virus belongs to the order picornavirales and is a member of the family dicistroviridae and the genus aparavirus. the virion is icosahedral, nonenveloped, and 32 nm in diameter. the genome contains positive sense, single-stranded rna of 10 kb. the host range is restricted mostly to members of the genus penaeus sensu lato, although recently overstreet et al. (2009) reported experimental infections with replication in at least some symbiotic barnacles. pathogenicity varies with host species being most virulent in litopenaeus vannamei and less so in other species of penaeid shrimp . although it is possible that the viruses were symbionts of wild shrimp prior to being detected in shrimp aquaculture, we wish to explore the hypothesis that they emerged by host transfer from insects or other terrestrial arthropods during the development and expansion of shrimp aquaculture. roekring et al. (2002) perhaps first suggested this tantalizing hypothesis in their study of three shrimp parvoviruses whose closest known relatives are parasites of insects. host switching is a common mechanism for the emergence of pathogenic pathogens. for example, in humans, 75 % of today's emerging human viral diseases are zoonotic, e.g., hiv-1 and hiv-2, influenza virus, ebola virus, hantaviruses, nipah virus, zika virus, and the sars coronavirus that causes severe acute respiratory syndrome, and many of the rest of human infectious diseases have their closest relatives residing in nonhuman animal hosts (parrish et al. 2008; pearce-duvet 2006) . most theories of the emergence of new diseases through host switching recognize several steps: cross-species transmission, establishment, and spread (parrish et al. 2008; domingo 2010; antia et al. 2003 ). the first step in the process is an increase in the number of contact events between the recipient host and the donor host. the increase in contacts is usually due to an ecological change or disturbance. as the number of contacts between the donor and recipient hosts increases, there may be a concomitant increase in successful transfer of the pathogen. in the case of shrimp viruses, we suggest below that there was an increase in contact between penaeid shrimp and other arthropods during the early 1990s, and that facilitated cross-species transmission and the emergence of pathogens in penaeids. although cross-species transmission may occur, permanent establishment within the new host requires enough within-species transmission to maintain the pathogen in the new host. it is unknown how many times the ancestor of these two symbionts was transmitted to penaeid shrimp only to be eliminated because shrimpto-shrimp transmission was not sufficient. however, at some point, either prior to cross-species transmission or after initial colonization, a mutation of the ancestor resulted in penaeid shrimp being highly susceptible to them, resulting in the maintenance of the infection locally in shrimp aquaculture. a third set of factors allows for the pandemic spread. sharp and hahn (2010) review the successful establishments of hiv from primates to humans. although there may have been as many as seven successful transmissions, most are restricted to africa, and only one of the seven establishments of hiv (hiv-1-m) is responsible for the pandemic of hiv in the 1980s (sharp and hahn 2010; pepin 2011) . one or more factors other than trans-species transmission allowed for the amplification and spread after a transmission. in the case of hiv, it is thought that the increased use of vaccination in colonial africa, chance, and increased world travel may have been the contributing factors (pepin 2011) . in the case of shrimp viruses, it may be that the greater number and higher shrimp densities in aquaculture settings coupled with the long-distance transport of live and fresh frozen shrimp has allowed worldwide viral spread after cross-species transmission. the rapid expansion of the shrimp aquaculture industry in the 1980s and 1990s brought with it conditions conducive to increasing the rate of contact between shrimp and terrestrial arthropods, the majority of which are insects. according to fao statistics (fao 2009a, b) in the late 1970s, most of the penaeid shrimp were coming from capture fisheries (fig. 2.11) . however, between 1992 and 1993 production of penaeid shrimp from aquaculture surpassed 50 % of all penaeid shrimp production worldwide (fig. 2.12 ). this local peak occurred near the time that the two viruses appeared in shrimp aquaculture. subsequently, culture production declined as a percentage of total penaeid production and remained below 50 % throughout the 1990s. however, the first decade of the twenty-first century saw a substantial increase and by 2010 the contribution from aquaculture to world's commercial shrimp increased supply by nearly 75 %. clearly, in the early 1990s, when viral pathogens emerged, there were twice as many penaeid shrimp in the market place as there had been a decade before. although this does not mean that the total number of shrimp in the world doubled (not all of the shrimp in the world were captured), it does suggest that aquaculture substantially increased the total number of penaeid shrimp in the world and continues to do so. it is also clear that the increased number of aquacultured shrimp were at higher densities than wild shrimp. this increased population size and density are conducive to the spread of infectious diseases. perhaps more important for the cross-species transmission of the viruses from insect to shrimp is the habitat of those new shrimp. for the most part, they were in earthen ponds in the coastal zone. however, many of the ponds were located a distance from the coastline, and seawater was pumped over a considerable distance to provide the necessary culture medium. the point is that there may be more shrimp that were likely to have contacted terrestrial insects and other terrestrial arthropods, after the expansion of shrimp aquaculture than before it. the second point in building a case for the origin of the two shrimp viruses from insects or other terrestrial arthropods is that the hosts of the viruses most closely related to shrimp viruses are insect or terrestrial arthropod hosts. roekring fig. 2 .11 total world's supply of penaeid shrimp in metric tonnes. production from the capture fishery is light bars. production from aquaculture is in dark bars. source fao statistics (fao 2009a, b) et al. (2002) indicate that shrimp parvoviruses do not form a single clade but are distributed among clades that include insect parvoviruses. roekring et al. (2002) suggested that viral transfers between crustaceans and insects occurred. most members of the dicistroviridae are found in insects (baker and schroeder 2008) . guo et al. (2013) noted that the exceptions are tsv and the recentlydescribed mud crab dicistrovirus-1 (mcdv-1). mcdv-1 groups with tsv which could indicate that tsv in shrimp aquaculture arose from other crustaceans. it should be noted that the first reports of tsv were from the new world in the 1990s and that mcdv-1 was described from asia long after tsv was transferred from the new world into asia in 2000, suggesting that mcdv-1 arose after tsv was introduced into asia. similarly, wssv as a member of the order baculovirales has as its closest relatives symbionts of insects. in particular, jehle et al. (2013) group wssv most closely with two insect viruses causing salivary gland hypertrophy, musca domestica salivary gland hypertrophy virus (mdsghv) and glossina pallidipes salivary gland hypertrophy virus (gpsghv). we have provided a number of examples and triggers of the switch from one quality of a symbiotic relationship to another, specifically from commensal to pathogen. examples of triggers include environmental ones like temperature, toxic chemicals (dose), chemotherapeutics, dietary changes, and geographic habits; internal ones include host physical site, host resistance or susceptibility, host modifications, and host switching; and combinations of these and other conditions. fig. 2. 12 proportion of world's penaeid shrimp from aquaculture. note that the proportion of penaeid shrimp production saw a local peak in the early 1990s above 0.5 and then declined during the emergence of taura syndrome virus (tsv) and white spot syndrome virus (wssv). source fao statistics (fao 2009a, b) the question arises, "is becoming a pathogen an adaptation for a symbiont?" in an evolutionary context, which is where the concept of adaptation lies, why would a symbiont switch to being pathogenic, either at some stage in its life cycle or in some hosts at the same stage but not in others? in some cases, symbionts are pathogenic as a clear adaptation, e.g., the levinseniella byrdi, referenced above causes damage or even death of the intermediate host to complete its life cycle. in particular, l. byrdi causes a phenotypic change in the host that increases the host's chances of becoming a prey. in other cases, it is not so clear. facultative symbionts, such as entomophagous nematodes, are both free living and symbiotic (sudhaus 2008) . the adaptation of being symbiotic is that it increases the habitat breadth of the symbiont, and inhabitation of a living organism removes the organism from competition with other organisms encountered living freely in the habitat. thereby, the relationship allows the symbiont an escape from competition. so that may be the reason for a life history strategy in which both symbiotic and free-living phases exist. however, we are most interested in answering the question of why would a symbiont become pathogenic in some hosts and not in others from an evolutionary perspective. there is a body of theory that addresses virulence evolution in symbiotic organisms. traditionally, the virulence of a pathogen was thought to be a reflection of a recent host-symbiont association and that over time there would result a diminishment in virulence of the pathogen (may and anderson 1983; anderson and may 1992; ewald 1994) . it was argued that no symbiont would benefit from killing its host (destroying its habitat and thereby destroying itself). however, this thinking fails to recognize that natural selection maximizes reproductive success of the organism. reproductive success is a composite of births (new infections) and survival (loss of infection) not just survival. more recent theoretical studies suggest the evolution of an intermediate level of virulence. the new conclusion results from the recognition that there is often a trade-off between pathogen transmission (births) and virulence (pathogen survival) (anderson and may 1982; may and anderson 1983; antia et al. 1994; frank 1996; koella and restif 2001) . one indicator of reproductive success of a symbiont is the basic reproduction number (r 0 ), which is the mean number of new infections produced by a single infection (anderson and may 1992; diekmann et al. 1990; lotz et al. 2003) . it is calculated as the mean life span of an infection times the number of transmissions (equivalent to births) that would occur over that infectious period (mollison 1995) . it is very much the population growth rate of an infection and is analogous to the net reproductive rate (r 0 ) of free-living organisms. r 0 for both free-living and symbiotic organisms indicates population growth and therefore reproductive success (fitness). and as such, it should be maximized by natural selection. for simplicity, we can represent transmission as β and life span as the reciprocal of the virulence α or pathogen-induced mortality. in this case, r 0 ¼ β α and r 0 increases as the infected hosts live longer (α decreases), and the infection is highly transmissible (β increases). however, there often occurs a trade-off between transmission and virulence. for example, in the case of tsv, the greater the load or intensity of the virus in a shrimp host, the greater the transmission (β increases). however, the greater the viral intensity, the greater the chance of mortality of the host (α increases) (lotz 2010) . therefore, the trade-off is mediated by viral intensity. as a result, a balance exists between infectivity and virulence, and the maximum r 0 is obtained at intermediate levels of virulence (fig. 2.13 ). this much of the theory assumes that the source of new infections is living infected hosts and that the death of the host ends infectivity (ewald 1994; hochberg 1998) . what if transmission occurs not so much from living infected hosts but from hosts after they die? this is likely to be the case for several shrimp pathogens. soto and lotz (2001) and lotz et al. (2003) demonstrated for wssv and for tsv that transmission from dead infected shrimp is considerably greater than from living infected shrimp. this indicates that the transmissibility of a shrimp pathogen does not end with the life of the shrimp. if transmission occurs from dead infected hosts, then r 0 is determined by the time to death of an infected host and the length of time that a dead infected host remains infectious. the infectious time of a dead shrimp depends on two factors, the ingestion of dead shrimp by other shrimp and the decay of the carcass (soto and lotz 2001; lotz et al. 2003) . the rate at which dead shrimp infectivity declines, whether by cannibalism or by carcass decay, is unrelated to the viral load; however, transmission rate (β) is. in this case, the load does not affect the time of infectivity of a dead infected shrimp, and the trade-off between load and time of infectivity disappears. in fact, increased virulence in live infected hosts causes a high pathogen load in dead animals and therefore increased infectivity of dead hosts without graph of relationship between two sources of infection (or two habitats) or two hosts assessed as pathogen-induced mortality. curve a is the relationship between r 0 virulence when the source is a living host; b is the relationship between r 0 and virulence when the source is a dead host. which curve obtained depends on the relative contribution of both living and dead hosts to the overall r 0 affecting the time of infectivity. so as virulence increases, r 0 also increases, and we expect virulence to increase over time if transmission is from dead shrimp ( fig. 2.13 ). lotz (2010) did not consider the outcome if both living and dead hosts contribute; however, overall r 0 is β a α þ β d δ , where β a is the transmission rate from a living host, β d is the transmission rate from dead hosts, α is the virulence of a pathogen to the living hosts, and δ is the infectivity decay of a dead infected host. the relative contributions of β a α and β a δ to r 0 are what will determine the final virulence and whether or not virulence will increase or decrease over evolutionary time. although the above reasoning and fig. 2 .13 have been applied specifically to tsv and living and dead shrimp, they apply more generally to any two states that contribute to an overall r 0 . the two states instead can be two habitats or two species of host. the state (habitat or species) that contributes the greatest to r 0 will dominate setting the optimal virulence. for species of symbiont that have both free-living and symbiotic relationships, the free-living habitat will contribute to the net reproductive rate (r 0 ), and the symbiotic habitat will contribute to the basic reproduction number (r 0 ). the two r 0 s contribute to the overall growth (and thereby fitness) of the symbiont. if the two states represent different host species, then the host species that is responsible for the greatest contribution to the basic reproduction number will predominate. the conclusion is that what happens in the lesser contributing state will not be as important, and therefore very high virulence could be obtained in a host with little contribution to fitness of the symbiont. the observed virulence is a coincidental by-product of the adaptation to other habitats (brown et al. 2012; adiba et al. 2010) . in particular, for some human bacterial pathogens, it has been postulated that bacterial pathogenicity evolved from antipredator selection by free-living bacteria and their predatory protists (brüssow 2007; adiba et al. 2010; brown et al. 2012; erken et al. 2013) . we have covered two evolutionary hypotheses to explain increased virulence of a symbiont: one contributes directly to the fitness of the symbiont, and the other is a coincidental outcome of selection for a trait important in another habitat. from grazing resistance to pathogenesis: the coincidental evolution of virulence factors genetic susceptibility of cultured shrimp (penaeus vannamei) to infectious hypodermal and hematopoietic necrosis virus and baculovirus penaei: possible relationship with growth status and metabolic gene expression coevolution of hosts and parasites infectious diseases of humans: dynamics and control within-host population dynamics and the evolution and maintenance of microparasite virulence the role of evolution in the emergence of infectious diseases the use of rna-dependent rna polymerase for the taxonomic assignment of picorna-like viruses (order picornavirales) infecting apis mellifera l. populations impact of temperature on an emerging parasitic association between a sperm-feeding scuticociliate and northeast pacific sea stars clinical parasitology, 9th edn distribution and abundance of bacteria in the gut of a soil-feeding termite procubitermes aburiensis (termitidae, termitinae) systematics, distribution ecology, and some host-parasite relationships of uhlorchestia uhleri (shoemaker) and u. spartinophila, new species (crustacea: amphipoda), endemic to salt marshes of the atlantic coast of north america evolution of virulence in opportunistic pathogens: generalism, plasticity, and control bacteria between protists and phages: from antipredation strategies to the evolution of pathogenicity intermediate hosts as source communities parasitology meets ecology on its own terms: margolis et al. revisited extended disease resistance emerging from the faecal nest of a subterranean termite symbiosis among animals with special reference to termites and their intestinal flagellates attempts to increase baculovirus prevalence in shrimp by chemical exposure taxonomy and biology of species of goezia (nematoda: anisakidae) from fishes, including three new species contracaecum multipapillatum (¼c. robustum) from fishes and birds in the northern gulf of mexico piscine adult nematode invading an open lesion in a human hand on the definition and the computation of the basic reproduction ratio r 0 in models for infectious diseases in heterogeneous populations mechanisms of viral emergence histopathology of protistan and myxozoan infections in fishes: an atlas the rise of pathogens: predation as a factor driving the evolution of human pathogens in the environment evolution of infectious disease global capture production host specificity of calyptospora funduli (apicomplexa: calyptosporidae) in atheriniform fishes models of parasite virulence fish mortalities associated with goezia sp. (nematoda: ascaridoidae) in central florida pathogenicity and complete genome sequence analysis of the mud crab dicistrovirus-1 infectivity and pathogenicity of baculovirus penaei (bp) in cultured larval and postlarval pacific white shrimp, penaeus vannamei, related to the stage of viral development ultrastructural effects of the coccidium, eimeria funduli duszynski, solangi and overstreet, 1979 on the liver of killifishes establishing genetic correlations involving parasite virulence disease of freshwater fishes caused by tetrahymena corlissi thompson, 1955, and a key for identification of holotrich ciliates of freshwater fishes observations on the pathogenesis of the imperfect fungus, fusarium solani, in the california brown shrimp, penaeus californiensis phylogeny and evolution of hytrosaviridae large-scale manipulations reveal that top-down and bottom-up controls interact to alter habitat utilization by saltmarsh fauna baculovirus replication experimental infection of mammals with larval echinocephalus sinensis (nematoda: gnathostomatidae) from oysters (crassostrea gigas) effects of temperature acclimation on infection of echinocephalus sinensis (nematoda: gnathostomatidae) from oysters to kittens coevolution of parasite virulence and host life history reo-like virus in white shrimp penaeus vannamei (crustacea: decapoda): co-occurrence with baculovirus penaei in experimental infections effect of sublethal exposure to the trematode bolbophorus spp. on the severity of enteric septicemia of catfish in channel catfish fingerlings prevalence of baculovirus penaei in experimentally infected white shrimp (penaeus vannamei) relative to age effect of desiccation, ph, heat, and ultraviolet irradiation on viability of baculovirus penaei efficacy of calcium hypochlorite as a disinfectant against the shrimp virus baculovirus penaei invasion of the body snatchers: the diversity and evolution of manipulative strategies in host-parasite interactions the behavioural ecology of 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temperature on the development of the microsporidan glugea stephani in english sole (parophrys vetulus) parasites of the inshore lizardfish, synodus foetens, from south florida, including a description of a new genus of cestoda abiotic factors affecting marine parasitism metazoan symbionts of crustaceans bp (baculovirus penaei) in penaeid shrimps parasitological data as monitors of environmental health infectious diseases: selected entries from the encyclopedia of sustainability science and technology defeating diplostomoid dangers in usa catfish aquaculture diatoms in the gills of the commercial white shrimp experimental infections with baculovirus penaei in the white shrimp, penaeus vannamei (crustacea: decapoda), as a bioassay the western mosquitofish as an environmental sentinel: parasites and histological lesions susceptibility to taura syndrome virus of some penaeid shrimp species native to the gulf of mexico and southeastern united states digenea: bolbophoridae) from the channel catfish ictalurus punctatus and american white pelican pelecanus erythrorhynchos in the usa based on life-cycle and molecular data parasitic crustaceans as vectors of viruses, with an emphasis on three penaeid viruses two new myxosporida, henneguya gambusi sp. n. and myxosoma pharyngeus sp. n., in the mosquitofish cross-species viral transmission and the emergence of new epidemic diseases the origin of human pathogens: evaluating the role of agriculture and domestic animals in the evolution of human disease the origin of aids comparison of penaeid shrimp and insect parvoviruses suggests that viral transfers may occur between two distantly related arthropod groups the evolution of hiv-1 and the origin of aids the blue crab, callinectes sapidus. maryland sea grant effect of ichthyophthirius multifiliis parasitism on the survival, hematology and bacterial load in channel catfish previously exposed to edwardsiella ictaluri discovery of an opportunistic starfish pathogen, ochitophrya stellarum, in captive blue crabs, callinectes sapidus the trematode fauna of a brackish coastal lagoon in tasmania cellular inflammatory response of penaeus aztecus and p. setiferus to the pathogenic fungus, fusarium sp., isolated from the california brown shrimp, p. californiensis biology and pathogenesis of the coccidium eimeria funduli infecting killifishes effect of low temperature on development of the coccidium eimeria funduli in the gulf killifish epidemiological parameters of white spot syndrome virus (wssv) infections in litopenaeus vannamei and l. setiferus physiology of the ciliate orchitophrya stellarum and its experimental infection of leptasterias spp effect of baculovirus penaei on growth and survival of experimentally infected postlarvae of the pacific white shrimp, penaeus vannamei relationship between bp (baculovirus penaei) and energy reserves in larval and postlarval pacific white shrimp evolution of insect parasitism in rhabditid and diplogastrid nematodes characterization of shrimp baculovirus pattern of infection of gammarus aequicauda (amphipoda) with metacercariae of levinseniella tridigitata (trematoda: microphallidae) intestinal parasite guidelines for domestic medical examination for newly arrived refugees experimental infection of contracaecum multipapillatum (nematoda: anisakinae) from mexico in the domestic cat nudiviruses and other large, double-stranded circular dna viruses of invertebrates: new insights on an old topic whole-proteome phylogeny of large dsdna virus families by an alignment-free method acknowledgments we thank janet wright, jean jovonovich, and kim overstreet for assistance. some funding was provided by a grant from bp exploration & production, inc. key: cord-018061-jy3km0fr authors: al kassaa, imad title: antiviral probiotics: a new concept in medical sciences date: 2016-12-02 journal: new insights on antiviral probiotics doi: 10.1007/978-3-319-49688-7_1 sha: doc_id: 18061 cord_uid: jy3km0fr in recent decades, probiotics have shown beneficial effects on animal and human health. probiotics can protect the host against several health threats, including infectious diseases. before 1995, researchers believed that the effect of probiotics was only on gut microbiota which can restore the gut flora and thus prevent pathogenic bacteria from triggering gastroenteritis. recent studies have shown that the immunomodulatory activity is the most important mechanism of action of probiotics. from this information, researchers started to evaluate the effect of some immunobiotics, not only on pathogenic bacteria but also on viruses, including enteric and respiratory viruses. several studies have confirmed the potential antiviral activity of some probiotics due to the immunomodulatory effect. these studies were conducted on humans (clinical trials) and in animal models. in this chapter, probiotics with antiviral effect against respiratory and enteric viruses will be presented and discussed, as well as their mechanisms of action. antimicrobial respiratory infections and gastroenteritis constitute the major causes of mortality and morbidity worldwide, both in developing and developed countries [ 1 ] . despite the widespread adoption of vaccines strategies, some pathogens remain a threat to public health worldwide. the us national institutes of health (nih; bethesda, md, usa) declared the emergence of 16 new infectious diseases, six of which have been considered reemerging infections [ 2 ] . in the united states of america (usa), mortality caused by infectious diseases (ids) was amounted to 170,000 deaths in 2000 [ 3 ] . an increase in immunocompromised patients plays a crucial role in the emergence and/or reemergence of ids; therefore, post-infection complications can lead to death. public health is faced with two major obstacles to eradicating ids: (1) antibiotic therapies, which have been saving infected patient for several years. unfortunately, the rapid emergence of resistant bacteria is occurring worldwide, endangering the effi cacy of antibiotics, which have transformed medicine and saved millions of lives [ 4 ] . (2) the lack of antiviral agents against infectious viruses, which leads to a high treatment level between populations even in the presence of some vaccines covering a few virus types [ 5 ] . several strategies have been developed to overcome this crisis, e.g., (i) the use of bacteriophages as antibacterial agents [ 4 ] , (ii) the extraction and purifi cation of antimicrobial peptides [ 6 ] , and (iii) the prevention of ids by using vaccines and/or recombinant vaccine strategies [ 7 ] . preventing infectious diseases occurring seems to be the perfect method of avoiding id complications, since all of the abovementioned strategies have inconveniences such as side effects and stability in the host. immune system boosting is the essential key factor in id prevention. dietary balance in meals, administration of supplements such as fi ber, and probiotics are three methods to enhance and stimulate the immune system, thus protecting the mucosa against the entry of pathogens. probiotics have demonstrated their capacity to stimulate and modulate the immune system [ 8 ] . in addition to the antibacterial activity of probiotics, some strains showed an effective antiviral activity which can be a solution to the lack of antiviral agents [ 9 ] . in this chapter, we focus on probiotics which have been shown to be effective as antiviral agents against respiratory and enteric viruses. in addition, we give details of some clinical trials and both in vitro and in vivo experiments which have confi rmed this effi cacy. lactic acid bacteria (lab) can be found in many ecosystems, including human and animal fl ora. lab, as well as their metabolites such as bacteriocins, are generally recognized as safe (gras) [ 10 ] . the antibacterial activity of probiotics has been confi rmed in a large number of research studies. this activity may occur through several mechanisms: (1) pathogens exclusion: probiotic strains have a high affi nity for adhesion to epithelial cells. thus, probiotics will saturate the receptors and exert a barrier effect against the pathogens involved in infections. (2) nutrient competition: probiotic strains can ingest many essential molecules, and consequently pathogens cannot grow in this ecosystem. (3) production of antimicrobial compounds, such as lactic acid, hydrogen peroxide, bacteriocin-like inhibitory substances (bliss), nrps, and bacteriocins [ 11 -13 ] (fig. 1.1 ). in addition to food applications, the use of lab is growing, in particular as probiotics for controlling, for example, gastroenteritis, infl ammatory pathologies of the digestive tract [ 14 , 15 ] and to stimulate the local and systemic immune response [ 16 , 17 ] . in recent decades, some probiotics have shown an antiviral activity and several mechanisms have been demonstrated. in respiratory tract infections (rtis), the majority of probiotics can inhibit the most important respiratory viruses (rvs) by immunomodulatory mechanisms [ 18 ] (fig. 1.2 ) . this antiviral mechanism might be explained due to the entry routes of rvs. rvs infect the mucosal cells of the rt, and for this reason, probiotic strains and their antimicrobial compounds cannot directly shows the antiviral mechanisms of some probiotic strains used against viral respiratory infections. although there is a difference between the probiotic colonization ecosystem and the target rv ecosystem, several studies have showed that there is a relationship between gut microbiota and other tissues. probiotics can inhibit viruses and/or help the immune system defend itself against rvs. first, the rvs interact with the respiratory epithelium, which generates an innate immune response by activating the ifn signaling and other proinfl ammatory cytokines. once cytokines have been secreted, macrophages and nk cells will be recruited to phagocytize and kill both viruses and viral-infected cells. to trigger a specifi c immune response, the immune system needs proinfl ammatory cytokines, energy, and some cofactor elements. hence, probiotics can provide some elements to boost the immune response: a . probiotics interact with the gut epithelium and are recognized by intestinal dcs (idcs); this interaction results in the production of il-12 and ifnγ by idcs, which can modulate both the respiratory and gut immune response. b . secretion of ifnγ and il-12 by intestinal dcs; these two proinfl ammatory cytokines have dual functions: ifnγ and il-12 can circulate in the bloodstream to reach the respiratory epithelium and therefore help alveolar macrophages and nk cells eliminate rvs. c . the proinfl ammatory cytokines (ifnγ and il-12) secreted in the gut ecosystem after colonization of some probiotic strains help the immune system to generate a specifi c th1/th17 immune response; the number of cd4+ and cd8+ increases and becomes more effi cient. in addition, cd4+ will secrete il-17, which enhances the innate immune response. d . some probiotic strains, via induction of ifnγ and il-17 production, can stimulate the overexpression of innate immunity-related genes such as the overexpression of tlr7, even in the lung. this overexpression of tlr7 amplifi es the innate immune responses. e . probiotics can help b lymphocytes differentiate and become plasma cells, which can secrete specifi c siga. in our case, some studies showed the impact of some probiotics in increasing siga in lung tissues. however, until now there is no explanation of the real mechanisms of how intestinal probiotics can help secretion of siga which are specifi c to elimination of rvs. this effect can be explained by the capacity of some probiotics to enhance cytokine production, which can improve the rapid differentiation of b lymphocytes to plasma cells in lung tissues. interact with viruses by physical contact. probiotic strains can fi nally reduce or eradicate virus infectivity by immunomodulatory activity, which has led scientists to call them "immunobiotics" [ 19 ] . in this chapter, the majority of anti-rv probiotics will be mentioned with other information, such as the source of probiotic strain, target virus, experimental model, and mode of antiviral activity (table 1 .1 ). lactobacillus is the most studied genus of anti-rvs probiotics, followed by the bifi dobacterium genus. it was reported that lactobacillus plantarum l-137 ( l. plantarum l-137) isolated from fermented foods showed proinfl ammatory activity which can decrease the titer of infl uenza virus type a (iva-h1n1) in mouse lungs [ 20 ] . another strain, l. plantarum yu, isolated from japanese fermented foods, showed anti-h1n1 activity by activating the th1 immune response [ 22 ] . recently, several studies have reported that l. plantarum species reduce the signs of infl uenza-like symptoms and even increase body weight and survival rate in a mouse model [ 21 , 23 , 24 ] . in addition, mice infected by a lethal pneumovirus survived when they were protected using a combination of two lactobacilli strains, l. plantarum ncimb 8826 isolated from human saliva and l. reuteri f275 isolated from the human gastrointestinal tract (git) [ 25 ] . it has not been reported that l. plantarum probiotic strains were assessed in a human experimental model, which may be due to the undesirable acid or metabolites secreted by this species. l. rhamnosus strains are the most important probiotics for human applications. furthermore, the majority of l. rhamnosus strains are immunobiotics, due to their ability to stimulate and enhance the host immune system. in animal experiments, l. rhamnosus gg (lgg), a famous probiotic strain, was evaluated and showed an anti-infl uenza virus activity on intranasal and oral administration [ 26 , 27 ] . in mice infected with respiratory syncytial virus (rsv), heat-killed l. rhamnosus crl1505 and crl1506 showed a good inhibitory effect and increased the body weight of mice [ 34 ] . these two strains were considered good immunobiotics in rsv infection due to their ifn-α stimulation, which decreases the viral load in mouse lungs [ 34 ] . the majority of clinical trials in children were performed using lgg [ 28 , 29 ] . lgg reduces the number of upper and lower viral rtis in children, reduces the days of absence from daycare and decreases antibiotic use [ 28 ] . the administration route of lgg in clinical trials in children was often by drinking probiotic-inoculated milk [ 28 , 30 -32 ] . the antiviral activity of lgg was also assessed in adults and the elderly. several clinical trials were conducted in order to improve the benefi cial effect of this strain in the treatment and prevention of viral infections. kekkonen et al. studied 141 marathon runners (22-69 years old) who drank two bottles of milk daily. the probiotic group drank a milk inoculated with lgg (4.10 10 cfus). the results showed that the ingestion of lgg did not decrease rti episodes or the severity of symptoms [ 33 ] . however, the combination of lgg with bifi dobacterium animalis ssp. lactis bb12 ( b. animalis ssp. lactis bb12) decreased the duration and symptom severity of urtis signifi cantly [ 62 ] . [ 60 ] lgg, l. rhamnosus lactobacillus casei ( l. casei ) is a benefi cial bacterium found naturally in both the mouth and intestines of humans. l. casei may be found in "raw or fermented dairy and fresh or fermented plant products" [ 63 ] . l. casei shirota (lcs) is the major probiotic strain among this species. this strain has been isolated from mouth fl ora [ 64 ] . the intranasal administration of lcs in h1n1-infected mice showed a decrease in the viral titer in a nasal wash. moreover, lcs increases the secretion of antiviral cytokines such as interferon alpha (ifn-α). furthermore, lcs stimulates the innate immune response [ 35 ] . lcs has shown an immunomodulatory activity against rvs. however, in clinical trials, in particular in the elderly group, lcs has shown insignifi cant results in comparison with the placebo group [ 65 , 66 ] . another probiotic strain, l. casei dn-114,001, showed good antiviral activity in clinical trials. l. casei dn-114,001 was evaluated in children, adults, and the elderly in separate studies. in children clinical trials, l. casei dn-114,001 decreased the symptoms and duration of rtis signifi cantly [ 36 , 37 ] . in the adult and elderly groups, the administration of l. casei dn-114,001 decreased the duration of rtis and common infectious diseases (cids) [ 38 -40 , 67 ] . l. paracasei , in particular l. paracasei ssp. paracasei (lpp), was evaluated for its antimicrobial activity in animal models [ 24 , 41 ] . after oral administration in mice, the lpp 06tca19 and lpp 06tca22 strains, isolated from fermented camel milk, showed a signifi cant decrease in tnf-α in bronchoalveolar lavage fl uid (balf). this effect led to an increase in the mice's survival and a decrease in the macrophage and neutrophil concentrations in balf [ 41 ] . l. fermentum is a species which can be found in human and animal fl ora [ 68 ] . this species is usually used as a probiotic in humans. in rtis, l. fermentum was evaluated in both human clinical trials, in particular in children and adults [ 45 , 46 ] , and in animal models in order to investigate the mechanism of viral inhibition [ 42 , 43 ] . l. fermentum -1 and l. fermentum cjl-112 were assessed in h1n1-infected mice. the results have shown an important reduction in the viral load, with high stimulation of iga and il-12 secretion which allows an increase in the mice's survival [ 42 , 43 ] . l. fermentum cect5716 was evaluated only in human clinical trials [ 44 , 45 ] . two hundred and fi fteen healthy infants (6 months old) took 2.10 8 cfus/daily with galactooligosaccharides as prebiotics. this trial showed a signifi cant decrease in the incidence of urtis and lrtis in infants [ 44 ] . l. fermentum vri003 and l. fermentum pcc are two probiotic strains which showed a signifi cant decrease in the duration of rti symptoms in healthy, physically active adults. however, these two strains did not reduce the incidence of rtis [ 46 , 47 ] . l. acidophilus is a famous probiotic strain used in pharmaceutical supplements [ 69 ] . a few studies evaluated the antiviral activity of l. acidophilus , because this species is usually used for gastrointestinal problems [ 47 , 60 ] . all antiviral clinical trials used in humans were conducted using a combination formula with other probiotic strains, while one animal experiment was conducted using l. acidophilus l-92, isolated from a healthy japanese volunteer, which showed an anti-ifv a (h1n1) activity by increasing active nk cells in lungs. moreover, l. acidophilus l-92 showed an increase in ifn-α secretion [ 48 ] . l. salivarius resides in the mouth and small intestine. it mainly plays a role in protection against several kinds of pathogens [ 70 ] . sixty-six endurance athletes participated in a clinical trial; 33 of them have taken 2.10 10 cfus of l. salivarius probiotic strains daily for 16 weeks, while the control group ( n =33) took a placebo. the results showed a nonsignifi cant change in comparison with the placebo group [ 49 ] . these results can lead to the conclusion that the infl uence of probiotics in rtis could be directly related to the species or to a new characteristic presented in a specifi c strain. l. brevis kb-290 (isolated from a traditional japanese pickle called "suguki"), l. gasseri tmc0356 (isolated from the intestine of healthy adults), l. pentosus s-pt84 (isolated from kyoto pickles), and l. pentosus b240 (isolated from fermented tea leaves) are probiotic strains evaluated in mouse model experiments [ 27 , 50 -52 ] . these strains showed a strong anti-ifv activity, in particular against the h1n1 strain. the anti-h1n1 activity of l. brevis kb-290 was reported to increase ifn-α and iga secretion in mouse lungs after oral administration of this strain [ 50 ] . the oral administration of l. gasseri tmc0356 in intranasally h1n1-infected mice showed a positive effect on infl uenza symptoms. l. gasseri tmc0356 can decrease the viral titers by interacting with the intestinal immunity system, in particular in peyer's patch, resulting in high production of il-12, il-6, ifn-c, and iga [ 27 ] . kiso et al. reported that the oral administration of l. pentosus b240 increased protection in mice against a lethal dose of h1n1. the primary mechanism of this effect was by upregulation of antiviral genes such as egr1 (a critical regulator of host infl ammatory chemokines) and rsad2 (an interferon-stimulated gene (isg)) [ 52 ] . izumo et al. reported a new antiviral activity mechanism in a mouse experimental model infected by the h1n1 strain. they showed that the antiviral activity was created by activation of lung nk cells after intranasal administration of l. pentosus s-pt84 in balb/c mice. moreover, this strain can increase the production of ifn-α and iga and decrease the allergic reaction by modulating the th1/th2 balance [ 51 ] . bifi dobacterium is a very important bacterium in animal and human fl ora. this genus has benefi cial effects for the host, and it was used for the fi rst time as a commercial probiotic [ 71 ] . bifi dobacteria promote good digestion, boost the immune system, and inhibit almost all intestinal pathogens [ 72 ] . in viral rtis, the bifi dobacterial strains were used in combination in several human clinical trials to assess antiviral activity and investigate the mechanism of such activity [ 55 , 60 ] . the combination was conducted using lactobacilli probiotic strains [ 28 , 58 , 60 ] . in a few studies, the antiviral mechanisms of bifi dobacteria were investigated in animal models, in particular mouse experiments. bifi dobacterium longum ( b. longum ) bb536, isolated from japanese healthy infants, showed an anti-ifv a/pr/8/34 (h1n1) activity after oral administration for 2 weeks before infection. this antiviral activity was created by decreasing proinfl ammatory cytokines, such as ifnγ and il-6, in balb/c mice. furthermore, this strain showed the capability to signifi cantly decrease the symptom score and body weight loss [ 53 ] . in another balb/c mouse experiment, wu et al. reported that the administration of a bifi co® strains mixture, in particular b. longum , led to upregulation of the expression of several genes involved in antiviral responses, such as tlr7, myd88, irak4, traf6, and nf-kb [ 54 ] . in a double-blind, placebo-controlled study, 109 healthy newborns aged 1 month participated; 55 candidates were subjected to 10 9 cfus/day of b. animalis ssp. lactis bb12 and the control group ( n =54) received control tablets as a placebo. taipale et al. reported that the b. animalis ssp. lactis bb12 strain reduced the number of viral rti episodes, while there was no effect on the occurrence of acute otitis [ 55 ] . several studies investigated the combination of b. animalis ssp. lactis bb12 with other probiotic strains to determine the possibility of the strongest antiviral activity. the combination of l. reuteri atcc dsm 1793 (isolated from peruvian mother's milk) with b. animalis ssp. lactis bb12 was evaluated in a double-blind, placebocontrolled, randomized trial of 201 healthy infants aged between 4 and 10 months. this combination reduced the viral rti symptoms, fever, and antibiotic consumption [ 59 ] . rautava et al. showed that the combination of b. animalis ssp. lactis bb12 and the lgg strain reduced antibiotic consumptions. moreover, this combination reduced the incidence of acute otitis media in the fi rst 7 months of life [ 58 ] . in another double-blind randomized controlled trial, a combination of b. bifi dum with l. acidophilus probiotic strains (infl oran, bern, switzerland) was administered to 80 healthy children aged between 8 and 13 years old. reduced viral rti symptoms and a decrease in the school absence rate were the main outcomes of this combination [ 60 ] . b. animalis ssp. lactis b1-04 reduced viral urti episodes in a clinical trial of 460 physically active adults (18-60 years old) [ 56 ] . lehtoranta et al. reported the effi cacy of a combination of lgg, l. rhamnosus lc705, b. breve 99, and propionibacterium freudenreichii js in nasopharynx bocavirus infection, in particular in otitis-prone patients [ 61 ] . this study was a randomized, double-blind, placebocontrolled trial on 269 otitis-prone children (aged 9 months to 5.6 years) with 6 months of probiotic intervention. the authors showed the specifi c antiviral activity of this combination against bocavirus but not picornavirus. moreover, this combination seems to be effective in children, but not in the elderly [ 61 ] . each of the abovementioned probiotic strains seems to have one or many specifi c antiviral mechanisms. furthermore, the viral specifi city is directly related to the strain used or the combination of several probiotic strains in the same or different genus types. moreover, the antiviral effect of probiotics by immunomodulatory mechanisms depends on the immune system status, which can be explained in the study conducted by lehtoranta et al., who showed that the combination of four probiotic strains worked very well in children but not in the elderly [ 61 ] . lactobacillus and bifi dobacterium genera have the strongest antiviral activity against respiratory viruses, in particular against infl uenza virus type a. this antiviral activity depends on the strain's specifi city and the situation of the host immune system. clinical trials (cts) are the most used evaluation method in these studies. the alleviation of symptoms was measured in these cts in order to investigate the impact of suggested probiotic strains against rvs. the main mechanism of such probiotics is immunomodulation. the use of probiotics in respiratory infections leads to the activation of many signals for innate immunity and the production of iga antibodies in respiratory tissue. anti-infl ammatory probiotics in respiratory viral infections are not welcome, since they block the immune responses against the virus. however, in respiratory infl ammation, probiotics that stimulate the production of anti-infl ammatory (il-10, tgfβ) cytokines play a crucial role in suppressing infl ammation. we recommend avoiding antibiotic treatment for respiratory infections except in the case of a confi rmed bacterial infection. although the antibiotic treatment may prevent respiratory bacterial superinfection, this antibiotic therapy eradicates the microbiota, in particular gram-positive probiotic strains. anti-rov probiotics should be evaluated in depth in germ-free mice and/or antibiotic-treated mice in order to determine the complete mechanism of such probiotics. furthermore, the molecules responsible for this immunomodulatory activity should be investigated and purifi ed for in vivo experiments. in 1907, elie metchnikoff observed the healthy effect of fermented dairy products in his population. recently, researchers have confi rmed the benefi cial effects of fermented foods, in particular the role of lactic acid bacteria (lab), such as lactobacillus , bifi dobacterium and other lab, which have probiotic properties [ 73 ] . gram-positive lactobacillus and bifi dobacterium bacteria are not occasional contaminants, but they are two genera that colonize the primary microbiota of humans. the colonization, development, and maturation of the newborn's gastrointestinal tract that begins immediately at birth and continues for 2 years is modulated by numerous factors, including mode of delivery, feeding regime, maternal diet/weight, probiotic and prebiotic use, and antibiotic exposure pre-, peri-, and postnatally [ 74 ] . this microbiota plays a major role in the host's defense against pathogens [ 75 ] . this microbiota can maintain the health of the gut ecosystem and preserve defensive readiness against enteric pathogens and sometimes prevent enteric chronic diseases [ 76 ] . microbial concentrations are distributed along the digestive tract, with 10 3 bacterial cells/mm 3 in duodenum and stomach, 10 2 to 10 3 bacterial cells/mm 3 in the fasting ileus and distal ileum, and 10 10 to 10 12 bacterial cells/mm 3 in the colon [ 77 ] . unfortunately, due to an incorrect feeding regime, humans are losing the primary microbiota which is related to an increase in diseases, including infectious diseases. to restore this primary microbiota, several health organizations suggested using probiotics as a dietary supplement. recent studies have demonstrated the symbiotic relationship between intestinal microorganisms that benefi ts their human host. intestinal microorganisms, referred to as intestinal microbiota, have several mechanisms of maintaining gut health. the intestinal microbiota degrades indigestible dietary substances, such as fi ber, and converts it into an energy source for gut cells and the immune system [ 78 , 79 ] . the other role of intestinal microbiota is to develop the gut immune system. hooper et al. showed that germ-free infected mice have severely compromised immune responses and a reduction in the level of secretory immunoglobulin a (iga) and the number of intestinal t cells compared with wild-type mice [ 80 , 81 ] . in addition, intestinal microbiota can protect the host against pathogens by inducing intestinal epithelial cells to secrete antimicrobial proteins, such as angionin and c-type lectin regiiiγ [ 82 , 83 ] . the abo groups or "blood group antigens" in human red cells were discovered by karl landsteiner in 1900 [ 84 ] . subsequently, this kind of antigens, called histoblood group antigens (hbgas), has been found in other tissues and biological fl uids, such as gut and saliva [ 85 ] . some hosts lack the function of the fut1 gene and are called "nonsecretory hosts" [ 86 ] . the biological role of hbgas has not yet been completely defi ned. in infectious diseases, the presence of a and b hbgas can inhibit the in vitro motility of carcinoma cells, and their absence is associated with an unfavorable prognosis [ 86 ] . returning to infectious diseases, hbgas play a crucial role in bacterial and viral pathogenesis. specifi c strains of pathogens bind to carbohydrates of the hbg family. several studies have shown that a large number of pathogens bind hbg as the fi rst step of pathogenesis, such as uropathogenic strain of e. coli r45, s. pneumoniae , s. aureus , salmonella typhimirium , and campylobacter jejuni [ 87 -90 ] . the hbgas may not only provide an attachment receptor to pathogens, since they may be present on the pathogens themselves. gram-negative bacteria can present this type of antigen (in some strains may can be on the lps molecules) [ 91 ] ; humoral immune responses can thus be generated. for example, e. coli 086 presents b hbga; thus, an anti-b response will be evoked in a and o hbga individuals. thus, b group individuals were more susceptible to infection caused by e. coli 086 [ 92 ] . in this chapter, the role of hbga in viral infection will be discussed. the gut microbiota contains a large number of microbes, forming the biggest ecosystem in humans and animals [ 73 ] . gastrointestinal infections have a great impact on public health both in developing and developed countries [ 93 ] . viruses are the most frequent causative agent involved in gastroenteritis, in particular in infants and children [ 94 ] . the sources of enteric viruses (envs) are usually contaminated food and water through ingestion by the orofecal route [ 95 ] . after the occurrence of infection, treatment of the symptoms is the only way to prevent infection complications. in addition to antipyretic drugs, rehydration therapy is the most common treatment for viral gastroenteritis. the absence of specifi c antiviral agents against envs requires scientists to fi nd an alternative which can prevent or help in the treatment of such infections [ 5 ] . the defi nition of envs is viruses that are able to replicate in the intestinal epithelium, even if only several types are the causing of gastroenteritis [ 96 ] . noroviruses (novs), rotaviruses (rovs), arboviruses (avs), enteric adenoviruses (advs), and enteroviruses (evs) are the viruses most frequently responsible for gastrointestinal infections worldwide [ 97 ] . reoviruses are one of the many envs that replicate in the intestinal tract, but they are generally asymptomatic. another type of enteric virus, such as poliovirus, can cause severe disease after dissemination to peripheral tissues [ 97 ] . certain retroviruses, including mouse mammary tumor virus (mmtv), can be transmitted orally from an infected mother through her milk, after which they infect the gastrointestinal tract [ 98 ] . does "gut microbiota" enhance or inhibit viral gastroenteritis? upon envs ingestion, viruses will "communicate" with gut microbiota resident in the intestinal lumen, which vary from one host to another. the result of this interaction seems to be dependent on the composition of the microbiota. in some hosts, good microbiota (a high number of commensal bacteria) is an inconvenience; while in other hosts, a complex microbiota (containing a large variety of microbial genera and species) is benefi cial in defending against envs and preventing viral gastroenteritis. this hypothesis is supported by several studies that have demonstrated that the intestinal microbiota is important and plays various roles in reducing infection by enteric viruses [ 97 ] . the role of commensal bacteria in the persistence of enteric viral infections has previously been shown in a series of recent studies published in 2011, using poliovirus, reovirus and mouse mammary tumor virus (mmtv) as env models [ 99 -101 ] . the replication of poliovirus in the intestine was reduced in antibiotic-treated mice, while the reconstitution of the intestinal microbiota restored poliovirus infection [ 100 ] . moreover, kuss et al. showed that intraperitoneal infection with poliovirus was independent of the presence or absence of intestinal microbiota, which highlights the important role of the microbiota in reducing poliovirus infection [ 100 ] . in a recent study [ 101 ] , showed that the use of broad-spectrum antibiotics in a mouse model infected with rotavirus decreased the infectivity of the latter. they reported that viral antigens were reduced in feces, and there was delayed shedding of viruses compared with the control mice group [ 101 ] . in another study, murine norovirus (munov) was used to investigate the importance of intestinal microbiota in viral infectivity [ 102 -104 ] . jones et al. showed that depletion of intestinal microbiota reduced the replication of munov in the distal ileum, mesenteric lymph nodes, and colon compared with the control group [ 103 ] . the munov was reduced in feces shedding in antibiotic-treated mice compared with colonized mice as shown by kernbauer et al. [ 104 ] . a recent study conducted by baldridge et al. showed that the use of a broad-spectrum antibiotic in mice did not help munov to establish persistent infection, while the transplantation of intestinal microbiota from another healthy mouse rescued the infectivity of this virus. moreover, they reported that systemic infection with the munov was independent of the presence or absence of gut microbiota as shown by poliovirus infection [ 102 ] . virion stabilization and promotion of virus attachment are the two direct mechanisms by which the intestinal microbiota enhance env infections [ 100 , 105 ] . these fi ndings were investigated using an in vivo poliovirus model (in mice) and an in vitro model (cell culture testing). kuss et al. studied the stability of the poliovirus virion in the presence or absence of intestinal microbiota. they found that the isolation of poliovirus (before progeny virion production) from colonized mice was more viable and resistant to high temperatures and became bleach resistant. these results were also seen when the poliovirus was incubated with dead gram-negative bacteria [ 100 ] . robinson et al. conducted an in-depth study of the mechanisms of intestinal microbiota. they found that surface compounds of gram-negative bacteria played a crucial role in poliovirus stability. the authors showed that the bacterial lps bound the viral protein 1 (vp1) at threonine 99. the lps-bound virus increases the thermostability and chlorine resistance as well as decreasing the viral genome release [ 105 ] . moreover, the pretreatment of poliovirus particles with gramnegative bacteria/or lps molecules promotes poliovirus attachment to the host cells [ 105 ] . the poliovirus receptor (pvr) seems to be an important element in the abovementioned host cell attachment. the authors showed that poliovirus cannot attach to the permissive cells which were pretreated with anti-pvr antibodies. this result was independent of the presence of lps binding to poliovirus. moreover, non-pvr-expressing cells also showed the same results [ 105 ] . using another viral model and after a long history of in vitro culture diffi culties, b cells seem to be permissive cells for munov [ 103 ] . tan and jiang showed that human and munov required commensal bacteria to infect human b cells. these fi ndings were supported by the reduction of norovirus (nov) infectivity when infected stool was fi ltered with a 0.22 μm fi lter. the infectivity was rescued when live/dead commensal bacteria were added to the stool [ 106 ] . the authors showed that lps molecules were not the bacterial compound that played the cofactor role in norovirus infection. norovirus and b cells were incubated with lps, and the results showed that the lps did not initiate the norovirus infection. moreover, the authors showed that the histo-blood group antigen (hbga) glycan was a cofactor of the norovirus infection [ 106 ] . a recent study showed that a variety of commensal bacteria express these glycans and can bind norovirus in a virus-strain-specifi c manner [ 107 ] . the majority of gram-negative bacteria express this kind of glycan, as shown in several recent studies [ 103 , 108 ] . as shown in fig. 1.3 , the gut microbiota can inhibit and/or reduce the antiviral immune response via an indirect mechanism. the gut microbiota can sometimes create a tolerogenic microenvironment that helps the virus infect cells and suppress antiviral antibody production, and sometimes it can block virus-induced ifn signaling [ 97 ] . the gut microbiota, in particular gram-negative bacteria , induces a tolerogenic microenvironment that allows persistent enteric virus infection [ 109 , 110 ] . briefl y, the capacity of enteric viruses to bind gram-negative bacteria by lps-vp (viral protein) binding can skew the immune response. the story begins when the enteric virus binds the microbiota lps. the lps will be recognized by the tlr4, which induces the production of il-6. the b cells have il-6r; when the il-6 binds to the il-6r of the b cells, the b cells produce il-10, which is an anti-infl ammatory cytokine. this action blocks the antiviral immunity response and leads to viral persistence. this information is supported by several studies using an mmtv and norovirus model in solenocyte and b-cell culturing, respectively [ 99 , 111 , 112 ] . in another study, the norovirus infection occurred in germ-free mice which were also defi cient in production of il-10. this study supports and confi rms that the production of il-10 by the presence of gut microbiota, in particular gram-negative bacteria , was the essential key to viral persistence through the creation of a tolerogenic microenvironment [ 113 , 114 ] . (ii) viral antibody production [ 101 ] in the case of rotavirus infection, showed that the fecal and serum iga titer was higher in germ-free mice compared with the control mice group. this data suggests that gut microbiota suppress the antiviral humoral response. in contrast to the rotavirus case, the munov infection of antibiotic-treated mice reduced the serum igg titer after 35 days of infection compared with the colonized mice group [ 101 ] . these fi ndings will be investigated in depth to identify exactly which bacterial compound is responsible for this mechanism and to determine if this interaction occurred in a virus-strain-specifi c manner. several studies have reported that the ifnλ, which is considered to be in type iii of ifns, activates the same intracellular signaling pathway and many of the same biological activities as other ifn types, including antiviral activity, in a wide variety of target cells [ 115 ] . baldridge et al. reported the importance of ifn type i, ii, and iii responses in reducing munov infection as well as viral persistence. tlr4 is required for bacterial regulation of viral persistence. therefore, the presence of lps/gram-negative bacteria were dispensable in this regulation [ 102 ] . the advantage here is the capacity of probiotics to colonize the gut ecosystem, which is the target of envs: a . some probiotic strains can colonize the gut ecosystem and then form a carbohydrate biofi lm which probably saturates host iec receptors as well as viral receptors. b. probiotics protect the host iecs against damage and lesions. several studies have confi rmed that some probiotic strains play a crucial role in tissue restoration, especially by inducing mucin secretion by iecs and strengthening cell tight junctions. c. the immunomodulatory effect is the principal mechanism of antiviral probiotics (avpr). these probiotics can stimulate the secretion of proinfl ammatory cytokines, especially from dcs such as il-6, il-12, and ifnγ. in addition, avpr can boost innate immune cells, such as macrophages and nk cells. the latter also produce ifn-α, which is an antiviral cytokine. d. avpr help the immune system to react with more rapid specifi c responses. the th2 response is essential for b lymphocytes to be able to differentiate into plasma cells with specifi c siga secretion. e. avpr can inhibit or decrease viral infectivity and spreading by superproduction of mucin and by changing the morphology of villi, which can skew viral attachment. f . tlr3 is the ppr of viral mamps, especially for rna viruses. hence, the overexpression of tlr3 induced by some avpr can amplify the innate immune response by catching a large number of viral particles. g . some avpr interact physically with viral particles. indeed, several studies have showed that some probiotic strains can bind or trap viral particles on their cell wall. moreover, these trapped viruses lose some pathogenic characteristics and consequently lose cell infectivity. h . finally, avpr can play an indirect role in preventing and/or decreasing viral infection, especially against enteric viruses, by excluding the colonization of gram-negative bacteria (see fig. 1.3 ) . moreover, a recent study showed that the type iii ifn response was essential in reducing munov infectivity in the colon [ 116 ] . briefl y, envs are recognized by a variety of tlrs, such as tlr3, tlr9, etc. these tlrs stimulate the ifn production by the b cells or other secretory cells. the ifns, in particular type iii ifnγ, bind to the ifn receptor present on the enterocytes, which can reduce the viral persistence. several studies have reported that commensal bacteria, in particular gram-negative bacteria recognized by tlr4, bind to the envs and then the immune system will be skewed. thus, the tlr4s inhibit the production of ifnγ, allowing viral persistence. this data was confi rmed using munovs as an env model [ 102 , 116 -118 ] . pott et al. reported that ifnγ also controls rotavirus infection in mice; thus, it will be interesting to determine whether this response is similarly regulated by the interactions between the enteric virus and commensal bacteria [ 119 ] . in addition to the immunomodulatory effect of probiotics, these benefi cial bacteria have several mechanisms to defend gut pathogens and infections. in the previous part, the studies have shown that the composition of the intestinal microbiota can help envs to persist and sometimes amplifi es their infectivity. remarkably, the presence of gram-negative bacteria in the microbiota is essential to save the infectivity of envs. therefore, changing the intestinal microbiota composition seems to be effective in preventing or inhibiting enteric viral infection. otherwise, a high percentage of gram-positive bacteria may be a solution in viral gastroenteritis treatment and/or prevention. from this hypothesis, the importance of gram-positive bacteria, in particular lactic acid bacteria (labs) -which is considered to be grasin preventing and even treating this type of infection will be discussed in this part. the implantation of probiotics in the digestive tube is clearly benefi cial, since they have the ability to form a biofi lm on the enterocytes and prevent the adhesion and proliferation of other bacteria such as gram-negatives [ 120 ] . moreover, as shown in fig. 1.3 , probiotics can exclude commensal and pathogenic bacteria by several mechanisms such as the immunomodulatory effect (immunobiotic action), reduction of ph, production of antimicrobial compounds (hydrogen peroxide, lactic acid, nrps, bacteriocins, etc.), trophic competition, and biofi lm formation (receptor competing) [ 121 ] . anti-env probiotics (aenps) are divided into two categories according to the direct or indirect antiviral mechanisms. in this section, direct mechanisms will be discussed in detail. the probiotics with antiviral effects, called further antiviral/anti-env probiotics, can inhibit viral infections with several direct mechanisms. the antiviral compound secreted by these probiotics will be discussed in chap. 4 . in this section, the immunomodulation and physical interaction will be presented and discussed (table 1 .2 ). the microbiota diversity and composition are directly related to the incidence of gastroenteritis, including viral infection [ 163 ] . for example, the presence of bifi dobacteria genera in the fi rst months in the gut microbiota of infants prevents the majority of intestinal infections [ 164 ] . therefore, almost all intestinal probiotics can play a crucial role in preventing or treating viral gastroenteritis by indirect mechanisms. these probiotics reduce the "viral infection cofactor," which is the lps and hbga molecules present in gram-negative bacteria and some commensal bacteria, respectively [ 108 ] . otherwise, orally administered probiotics can change the composition of the gut microbiota by increasing the number of probiotic cells and decreasing commensal and gram-negative bacteria . the meaning of direct mechanism is when the envs interact directly with probiotic cells and/or their metabolic compounds. as shown in fig. 1.4 , probiotics can interact and inhibit envs by several mechanisms. indeed, it is depending to the specifi city probiotic strain and viral type. before talking about the direct mechanism or direct interaction of these probiotics, the viral infection steps should be presented. in general, envs can infect target cells by fi ve steps called the viral replication cycle. the viral replication cycle starts by viral attachment to host cells (1), followed by penetration and uncoating (2), viroplasm formation (3), and fi nishing with virus particle maturation (4) and release (5) [ 165 ] . each env has its own specifi city in infection mechanisms and/or the replication cycle. for this reason, the following information will discuss the direct mechanism of probiotics regarding the type of env. rovs are the major cause of diarrhea and acute gastroenteritis in infants and young children [ 165 ] . rovs are naked viruses containing dsrna. the rov virion or particle consists of three protein layers called a triple-layered particle (tlp) [ 166 ] . the viral protein (vp) and nonstructural protein (nsp) are the two main viral proteins found in rovs. for tlp, the main protein forming the external layer is vp7, with vp4 which forms the viral spike. vp6 forms the second layer of the rov particle. thus, the vp6 layer constitutes the double-layered particle (dlp) of the rov. kam et al. (2014) showed that, in actively transcribing dlp, the middle vp6 layer order decreased, while the number of cores increased. thus, the transcribed mrnas released from these cores translated later to the viral protein (vp and nsp) in host cells [ 167 ] . the rov replication cycle starts with the attachment to the host cells mediated by vp4 and vp7 molecules which play a role in the penetration and uncoating of rov. the third step consists of the synthesis of ssrna (mrna), which is mediated by vp1, vp3, and vp2 molecules. viroplasm formation (viral protein (nsp2, nsp5) and viral rna interact with each other to form cytoplasmic inclusion bodies), rna packaging, minus ssrna synthesis (rna replication), and dlp formation constitutes the fourth step. finally, rov will be released from host cells after maturation of virus particles (from dlps to tlps) [ 165 , 168 ] . fig. 1.3 ) in the gut ecosystem, which were considered a cofactor in some enteric virus infections. thus, proinfl ammatory probiotics (which induce a proinfl ammatory response) are welcome in viral gastroenteritis because they can trigger proinfl ammatory immunity to eliminate envs. probiotic strains capable of binding host cells very well and then creating a microenvironment which prevents many kinds of commensal and pathogenic bacteria from proliferating, including gram-negative bacteria. probiotics have a stronger capacity to adhere to host cells than gram-negative bacteria (probably because of the high hydrophobicity of their cell walls), which can decrease the number of gram-negative bacteria. probiotics can act in different ways: a. biofi lm formation: this biofi lm can protect host cells against other commensal bacteria, because this biofi lm covers the majority of host cell receptors. b. by the immunomodulatory effect, probiotics can stimulate the innate immune response, especially of phagocytes. c. at the same time, probiotics induce the secretion of antimicrobial peptides (amps) such as β-defensins and cathelicidins which target commensal bacteria. however, there is no explanation of the resistance of some probiotic strains against these amps. d. overproduction of mucin can also prevent commensal bacteria adhesion. e. the co-aggregation capacity of probiotic strains leads to the trapping of other microbes, as well as commensal or gram-negative bacteria . f. probiotics can secrete several enzymes to compete with other commensal bacteria for nutrients present in the gut ecosystem. in addition, the majority of probiotic strains possess arginine dehydrogenase, which is important in this mechanism. g. probiotic strains can secrete a variety of antimicrobial substances, such as hydrogen peroxide, lactic acid, non-ribosomal peptide synthetase (nrps), bacteriocins, and bacteriocin-like inhibitory substances (blis). several studies have shown the effectiveness of probiotics in the treatment and prevention of acute diarrhea including rov infections. human, murine, and porcine rotaviruses were used in these studies. the majority of investigations were based on the symptoms, such as duration of diarrhea, duration of hospitalization, virus shedding in feces, and sometimes immunomodulation. a few studies conducted an indepth investigation of the mechanism of action of some probiotics, in particular the interaction between virus-probiotic-host cells. lactobacillus and bifi dobacterium strains were the most studied genera in rotavirus infections. lactobacillus rhamnosus gg (lgg) is the best studied probiotic which showed a signifi cant reduction of diarrhea duration and rotavirus infectivity [ 122 , 123 ] . effects of various probiotic strains on rotaviruses have been conducted using double-blind placebo-controlled randomized trials since 1991 [ 124 , 125 , 134 ] . guandalini et al. showed that lgg administration reduced the diarrhea duration in neonatal patients with rotavirus infection [ 124 ] . in 49 children, the administration of 10 10 -10 11 cfus/ml of lgg twice daily for 5 days reduced the duration of acute diarrhea from 2.7 to 1.8 days, accompanied by an increase of iga-specifi c responses [ 126 ] . in other rcts, lgg reduced the duration of diarrhea caused by rotavirus gastroenteritis and improved the health recovery of infected children [ 127 -132 ] . the l. reuteri sd 2222 strain was administered in patients aged 6-36 months with watery diarrhea caused by rotavirus. this strain showed a strongly reduction of diarrhea duration up to 5 days [ 134 ] . saavedra et al., shornikova et al., and sugita and togawa showed the anti-rotaviral activity in clinical trials of the following probiotic strains: streptococcus thermophilus ( s. thermophiles ), l. reuteri dsm 12246, and l. acidophilus la5 [ 135 -137 ] . another study showed that lgg strains and l. casei shirota (lcs) have an antiviral activity against rotaviruses and transmissible gastroenteritis virus (tgev). the lgg strain showed the strongest activity, because of their strongest attachment capability to different cell lines. in addition to the attachment effect, the induction of reactive oxygen species (ros) release seems to play a role in such activity [ 138 ] . teran et al. conducted a randomized single-blind controlled trial (rsbct) in 75 bolivian children aged from 28 days to 24 months. a 1-gram mix of probiotic strains was administered to the probiotic group ( n =25) for 5 days. the mix contained the following strains: l. acidophilus , l. rhamnosus , b. longum , and saccharomyces boulardii ( s. boulardii ) . the second group ( n =25) was given nitazoxanide (an antiparasitic agent) at the dose of 15 mg/kg. the third group ( n =25) was subjected to the normal protocol of rehydration. the results showed that the duration of diarrhea was reduced to 48 h compared with 54 h and 79 h for the nitazoxanide and rehydration groups, respectively [ 162 ] . moreover, a study conducted by grandy et al. in rdbpc trial showed the effectiveness of probiotic strains against rotavirus infections using s. boulardii alone and s. boulardii with a mixture of probiotic strains. the results showed that the two probiotic prepa-rations reduced the infection symptoms ( p = 0.0042) [ 122 ] . l. reuteri , called probio-16, showed antiviral activity against porcine rotavirus; this activity was poorly demonstrated [ 139 ] . l. reuteri dsm 17938 was evaluated in rcts on 74 children with rotavirus infection, the results showed a decrease in the number of patients with acute diarrhea [ 140 ] . simakachorn [ 142 ] . in a murine infected model, lgg has decreased both the barrier permeability in murine intestine and epithelium vacuolation in the jejunum. furthermore, lgg was able to reduce the duration of acute diarrhea, and fi nally lgg was able to stimulate the secretion of iga [ 143 , 144 ] . l. casei dn-114,001 was administered in germ-free suckling rats infected further by rotavirus. the results showed that l. casei dn-114,001 changed the morphology of the intestinal villi and decreased intestinal cell lesions [ 145 ] . l. reuteri dsm 17938 was also evaluated in normal mice infected by rotavirus. the results showed that l. reuteri dsm 17938 has decreased the intestinal cell lesions and consequently reduced the duration of acute diarrhea [ 146 ] . recently, mao et al. studied the effect of lgg on the intestinal physiology, morphology and primary immune-specifi c responses of weaned piglets infected by the porcine rotavirus. this study showed that lgg administration in the weaned piglets group enhances specifi c immune responses by increasing rotavirus-specifi c iga secretion. in addition, lgg decreased the nsp4 (rotavirus enterotoxin) -consid-ered an intracellular receptor essential for dlp particles to interact with viroplasms and modulate intracellular ca2+ and rna replication [ 165 ] -in the jejunal mucosa induced by rotavirus infection [ 147 ] . the production of mucin 1 and mucin 2 and morphological improvement of the jejunal mucosa were evaluated in the presence of lgg. the results showed that lgg enhanced the production of mucin and recuperated the integrity of both the villus and the tight junction by stimulation of occlusion and other gene expression assisting the morphological jejunal defense against rotavirus [ 147 ] . e. coli nissle (ecn) -gram-negative probiotic strain -was evaluated alone or in combination with lgg in neonatal gnotobiotic piglets. the viral shedding titer was lower using ecn in comparison with lgg, lgg+ecn, and without probiotic strains. this result was correlated with the reduction of the specifi c iga responses in the small intestine in ecn colonized piglets. the in vitro investigation using mononuclear cell culture, ecn, showed stimulation effects on the production of antiinfl ammatory cytokines such as il-6 and il-10 [ 148 ] . these fi ndings support the hypothesis conducted by stephanie karst in 2016 which showed that gram-negative bacteria improve viral infection by various direct and indirect mechanisms [ 97 ] . yang et al. evaluated the impact of dietary rice bran (rb) on the human rotavirus vaccine (hrov) in vaccinated gnotobiotic pigs. they found that the rb-supplemented diet enhanced the vaccination responses in gnotobiotic pigs. in addition, the levels of ifnγ production from cd4 + and cd8 + were increased in intestinal and systemic lymphoid tissues [ 169 ] . in 2015, the authors showed that rb plays a role as a prebiotic for some probiotic strains. the lgg+ecn colonized gnotobiotic pigs were supplemented with rb daily, followed by human rotavirus (hurov) orally challenged. the rb showed a prebiotic effect promoting the growth of lgg and ecn in the gut. moreover, rb-fed pigs had a lower mitotic index and villus width. the rb and/or probiotic strains increased immunomodulation by enhancing the secretion of ifnγ and hurov-ab [ 150 ] . l. ruminis species have shown antiviral activity for the fi rst time against the human rotavirus wa strain. l. ruminis spm0211 showed an anti-hurov activity which was explained by an immunomodulatory effect enhancing the type i ifns immune response [ 149 ] . to fi nish the last investigation of the antiviral mechanism of lgg, a new experimental model was developed in order to understand the benefi cial interaction between pathogens and probiotics. an ex vivo experiment called intestinal organoid (derived from lgr5+ stem cells) was conducted by aoki-yoshida et al. [ 151 ] . the lgg strains showed an increase in tlr3 gene expression -tlr3 is the essential key in innate immune responses following the recognition of rotavirus -in murine intestine both in in vivo and ex vivo experiments, without alteration of other tlr gene expressions. moreover, lgg increased the mrna levels of interferon-α (ifn-α) and a neutrophil chemokine (cxcl1). furthermore, other probiotic strains, b. bifi dum and l. paracasei , failed to increase the tlr3 mrna levels ex vivo [ 151 ] . these fi ndings confi rm the hypothesis about the specifi city of probiotic strains against viruses. thus the antiviral activity occurred in a "virus-strainspecifi c manner." bifi dobacterial probiotic strains were also evaluated against rovs using in vitro and in vivo experiments. b. longum spm1205 and spm1206 showed antiviral activity against the hurov wa strain in an infected neonatal mouse model and caco-2 cells. the two bifi dobacterial strains showed an immunomodulatory effect on the type i ifns immune responses [ 152 ] . a complete genome sequence of b. longum subsp. infantis cect 7210 was conducted in 2015 by [ 170 ] . this strain had previously showed, in a study conducted by muñoz et al. [ 153 ] , a direct effect on rotavirus strains in both in vitro (ma-104 and ht-29 cell culture) and in vivo (mcn mouse model) experiments. the immunomodulatory mechanism was the main effect of this strain [ 153 ] . after complete sequencing of the b. longum subsp. infantis cect 7210 strain, they reported that there were 360 more elements (genes) in this strain compared with the complete genome sequence of b. longum 157f [ 170 ] . thus, more in-depth research must be conducted on this strain to identify the detailed mechanism of antiviral activity, and more specifi cally the anti-hrov activity. noroviruses (novs) are naked rna viruses belonging to the calicivirus family. novs are transmitted via the fecal-oral route and cause gastrointestinal disease with vomiting and acute diarrhea lasting 24-48 h [ 57 ] . novs cause 267 million infections each year and over 200,000 deaths, mostly in infants and the elderly [ 171 , 172 ] . novs need host receptors to start the infection cycle. debbink et al. reported that hbga (see sec. i-b2) is a diverse family of carbohydrates expressed in mucosal surfaces, which are the main receptors of novs, in particular for the gii.4 genotype considered to cause the majority of human nov infection because they can bind to a, b, and o secretors which are the majority (80 %) of the population [ 57 ] . the expression of hbgas depend on the fut2 gene which codes for an enzyme called fucosyltransferase. the gi.1 genotype (norwalk virus) cannot infect patients with a nonfunctional fut2 gene (called a "nonsecretory host"). however, some nov strains are capable of binding other receptors such as lewis carbohydrates [ 173 , 174 ] . the immune responses are very important to blockade novs infection and viral spreading. the iga genogroup-specifi c secretion is the main humoral immune response against novs [ 175 ] . the cd4 + th1 response is essential in the cellular immune response against novs which increases ifnγ and il-2 production [ 176 ] . the development of antiviral treatments and vaccines to fi ght nov infection has been hindered because of their extreme genetic diversity. recently, the uncultivable nature of novs has been resolved by using a b-cell model. thereby, the pathogenesis and replication cycle have been understood deeply in cell cultures and animal models [ 177 ] . the prevention strategies seem to be most effective mainly in infants and the elderly. to prevent and treat hunovs, several researchers have worked on the role of probiotics in such infection. the probiotic effectiveness in nov infections was evaluated using both in vitro and in vivo experiments and clinical trials. lcs introduced in fermented milk alleviated fever in nov-infected elderly patients. the probiotic group ( n =39) showed fast recuperation compared with the control group. moreover, the acetic acid concentration in feces has increased, and thereby bifi dobacterium and lactobacillus genera became dominant [ 154 ] . takeda et al. reported that the administration of lcs improves the natural killer (nk) cell activity by producing the il-12 by macrophages in response to lcs [ 155 ] . lactococcus lactis ssp. lactis lm0230 ( l. lactis ssp. lactis lm0230) -probiotic strains -were evaluated for antiviral activity against feline calicivirus (fcv), a hunov surrogate. this strain, "bacterial cell suspension (bcs)" and its metabolites "bacterial growth medium cell-free fi ltrate (bgmf)" were added to crandell-reese feline kidney (crfk) cells line. the results showed that crfk pretreated by bcs and bgmf caused nonsignifi cant decreases in the fcv titer. the pretreatment of fcv by bcs resulted in a decreased fcv titer after 24 h. the co-incubation of fcv and bcs in crfk cells showed 100 % virus titer reduction (7.5 log tcid 50 /0.1 ml) [ 156 ] . the effect of bgmf will be discussed in chap. 4 . in order to investigate the physical interaction between probiotic cells and nov particles, rubio-del-campo et al. used a p-particles model designed from the c-terminal protruding p-domain of the nov vp1 capsid protein. the p-particles exhibit the same surface conformation of viruslike particles (vlps), and therefore these p-particles can bind to the hbgas. in this study, 11 probiotic strains were tested: e. coli nissle 1917 l. lactis mg1363, l. acidophilus la-5, l. bulgaricus atcc11842t, l. plantarum 299v, l. plantarum 299v adh-(an isogenic derivative of 299v strain with decreased adhesion capacities), l. casei 431 atcc55544, l. casei bl23 cect5275, l. casei vsl#3, lgg atcc53103, and l. rhamnosus hn001. the norwalk virus (gi.1) and gii.4 (hunov) were used in these experiments. the results showed that the probiotic strains possessed the capacity to bind to both gi.1 and gii.4 p-particles. furthermore, l. rhamnosus , l. casei bl23 cect5275, l. casei vsl#3 showed the highest binding effect of both p-particles. as unexpected results, the e. coli nissle 1917 -gram-negative probiotic -showed the poorest binding capacity to gi.1 and gii.4, although other studies showed that gram-negative bacteria can bind enteroviruses via lps molecules or hbgas [ 97 , 107 ] . in contrast, in ht-29 culture cells, e. coli nissle 1917 was more effi cient in nov p-particles blocking, resulting in low host cell binding, while the other probiotic strains showed a low inhibition effect. the low adhesion capacity of probiotic strains to host cells did not affect p-particles binding; this suggestion was confi rmed by the l. plantarum 299v adh-(probiotic strain with low attachment capacity) which showed high gi.1 p-particles binding compared with l. plantarum 99v (normal attachment capacity). in order to investigate the interaction between probiotic strains and nov p-particles in more depth, an exclusion assay (ht-29 cells incubated with bacteria followed by p-particles challenge) and displacement test (ht-29 cells incubated with p-particles followed by bacterial challenge) were performed. the results showed that the probiotic strains enhanced the nov p-particle attachment of monolayer surfaces. these results are not clear, since they disagree with other studies. the probable hypothesis is that the attached probiotic strains can bind to the nov p-particles on their peptidoglycans (teichoic acid), which can lead to higher p-particle retention on the ht-29 surfaces [ 157 ] . a recent study has evaluated an engineered probiotic strain of l. paracasei which can produce the 3d8 scfv protein (an antiviral protein that can penetrate into host cells and hydrolyze nucleic acid molecules) against munov. the results showed that l. paracasei 3d8 scfv retained its cell-penetrating effect, and therefore the intracellular nucleic acids have been hydrolyzed. the pretreatment of raw264.7 cells with this engineered probiotic strain prevented the cell apoptosis caused by munov infection. moreover, l. paracasei 3d8 scfv has decreased mrna expression of the viral capsid protein (vp1) [ 158 ] . recently, b. adolescentis showed an antiviral activity against munov as a hunov surrogate. the results showed that the inhibition did not occur in the viral binding step. using vlps as model, b. adolescentis decreased the attachment of hunov gi.1 vlps to both caco-2 and ht-29 cells, while no effect was shown in the presence of gii.4 vlps [ 159 ] . astroviruses are nonenveloped viruses with positive-sense ssrna. the astroviridae family consists of two genera, mamastrovirus (mastv) and avastrovirus (aastv), based on mammalian and avian species, respectively [ 178 ] . astroviruses can infect a wide variety of mammalian species, such as cats [ 179 ] , dogs [ 180 ] , mice [ 181 ] , sheep [ 182 ] , and cattle [ 183 ] . these mammals are always in direct contact with humans. hastvs are one of the most important causes of acute gastroenteritis in newborn and infant patients [ 184 ] . cross-species transmission is frequent, in particular in poultry as avian species [ 185 ] and between pigs, cats, and humans as mammalian species [ 186 ] . thus, the zoonotic potential of these viruses is high, and future nonhuman-to-human transmissions are likely to occur [ 178 ] . some authors have speculated that probiotics, which may interfere with the biological cycle of enteric viruses at many different stages, may be useful as a measure to prevent and/ or treat intestinal viral infections [ 187 , 188 ] . e. faecium ncimb 10415 is the fi rst probiotic strain authorized by the european union (eu) as a probiotic feed additive for animals, including piglets. e. faecium ncimb 10415 has shown an immunomodulatory effect in several studies [ 189 ] . transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus, causes 100 % mortality in newborn piglets after severe gastroenteritis. tgev can also infect respiratory tissues in some cases [ 190 ] . chai et al. showed the antiviral activity of e. faecium ncimb 10415 against tgev using in vitro swine testicle (st) cell lines. they showed that this strain has a double antiviral mechanism. first, the strain can trap virus particles on its cell wall and consequently prevent infection. the second mechanism is the stimulation of eukaryotic cells that produce no, il-6, and il-8 [ 160 ] . in addition to gastrointestinal infections, enteroviruses can cause extraintestinal infections. via the orofecal route, coxsackievirus type a strain 16 (ca16) and enterovirus 71 (ev71) cause hand, foot, and mouth disease (hfmd) [ 191 ] . this viral infection results in morbidity and mortality in several regions, including asia pacifi c and europe [ 192 ] . hfmd can lead to neurological complications and cardiopulmonary dysfunction resulting from acute ev71 infection [ 193 ] . liu et al. evaluated a bivalent vaccine against ev71, which has completed the phase iii clinical trials [ 161 , 194 ] . since ca16 and ev71 act by the orofecal route, ang yin et al. evaluated the impact of colonization of the probiotic strain on hfmd using in vitro human skeletal muscle and colon cell lines. the authors showed that the use of l. reuteri protectis (atcc 55730) [ 195 ] , decreased the viral load. moreover, this antiviral activity is dose-dependent. the authors suggested that l. reuteri protectis interacted physically with ca6, ca16, and ev71 and impaired viral entry to eukaryotic cells. this antiviral activity seems to be virus probiotic strain specifi c, since no antiviral effect was shown using coxsackievirus b strain 2 (target virus) in the presence of another probiotic strain lcs [ 161 ] . probiotics exhibit direct and indirect mechanisms in eradicating enteric viruses. the effectiveness of probiotics in the gut ecosystem is more relevant, since they interact with viral infections by several mechanisms, including immunomodulation, which is almost the only mechanism available for probiotics in respiratory infections. the impact of enteric viruses can be decreased by changing the microbiota composition. otherwise, hbga and lps are molecules that can be presented by gramnegative bacteria and are considered a secondary receptor for enteric viruses such as novs and rovs. for this reason, using probiotics can change the microbiota to gram-positive dominant fl ora, which blocks the gram-negative cofactor of viral infection. furthermore, the physical interaction of probiotics has been confi rmed in several studies which confi rm the capacity of some probiotic strains to trap viruses. the use of antibiotics in viral gastroenteritis is a double-edged sword. broadspectrum antibiotic therapy kills probiotic strains or inhibits their multiplication. in contrast, using anti-gram-negative antibiotics such as polymyxin b or other nonbroad-spectrum antibiotics can be a crucial factor in blocking the viral cycle. moreover, using probiotic strains with antibiotic resistance should be taken into consideration when treating viral gastroenteritis to keep probiotics live and eradicate gram-negative resident fl ora. the antibiotic resistance of commercial probiotic strains can be found in a review conducted by sharma et al. 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diarrhea: a multicenter european trial a human lactobacillus strain (lactobacillus casei sp strain gg) promotes recovery from acute diarrhea in children lactic acid bacteria in the treatment of acute rotavirus gastroenteritis a placebocontrolled trial of lactobacillus gg to prevent diarrhea in undernourished peruvian children lactobacillus gg and acute diarrhoea in young children in the tropics management of acute diarrhoea with low osmolarity oral rehydration solutions and lactobacillus strain gg lactobacillus gg promotes recovery from acute nonbloody diarrhea in pakistan effi cacy of lactobacillus gg in aboriginal children with acute diarrhoeal disease: a randomised clinical trial lactobacillus casei strain gg in the treatment of infants with acute watery diarrhea: a randomized, double-blind porcine small intestinal epithelial cell line (ipec-j2) of rotavirus infection as a new model for the study of innate immune responses to rotaviruses and probiotics lactobacillus reuteri as a therapeutic agent in acute diarrhea in young children feeding of bifi dobacterium bifidum and streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus bacteriotherapy with lactobacillus reuteri in rotavirus gastroenteritis effi cacy of lactobacillus preparation biolactis powder in children with rotavirus enteritis lactic acid bacteria effi ciently protect human and animal intestinal epithelial and immune cells from enteric virus infection bile tolerant lactobacillus reuteri isolated from pig feces inhibits enteric bacterial pathogens and porcine rotavirus randomised clinical trial: lactobacillus reuteri dsm 17938 vs. placebo in children with acute diarrhoea--a doubleblind study clinical evaluation of the addition of lyophilized, heat-killed lactobacillus acidophilus lb to oral rehydration therapy in the treatment of acute diarrhea in children towards a human rotavirus disease model effective prophylaxis against rotavirus diarrhea using a 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pigs complete genome sequence of bifi dobacterium longum subsp. infantis strain cect 7210, a probiotic strain active against rotavirus infections viral shape-shifting: norovirus evasion of the human immune system systematic literature review of role of noroviruses in sporadic gastroenteritis qualitative and quantitative analysis of the binding of gii.4 norovirus variants onto human blood group antigens human susceptibility and resistance to norwalk virus infection serological correlate of protection against norovirus-induced gastroenteritis heterotypic humoral and cellular immune responses following norwalk virus infection advances in norovirus biology detection of astroviruses in feces of a cat with diarrhea molecular characterisation of calicivirus and astrovirus in puppies with enteritis detection of astroviruses in gut contents of nude and normal mice. brief report complete genomic sequences of astroviruses from sheep and turkey: comparison with related viruses isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves astrovirus infection in children circulation and phylogenetic relationship of chicken and turkey-origin astroviruses detected in domestic ducks ( anas platyrhynchos domesticus) genomic analysis of two orf2 segments of new porcine astrovirus isolates and their close relationship with human astroviruses probiotics and gastrointestinal infections prevention and treatment of enteric viral infections: possible benefi ts of probiotic bacteria enterococcus faecium ncimb 10415 does not protect interleukin-10 knock-out mice from chronic gut infl ammation transgenic mice secreting coronavirus neutralizing antibodies into the milk information essor sjrpd of r analysis and, whitley essor rj, editors. a practice guide to clinical virology epidemiological and clinical characteristics of children who died from hand, foot and mouth disease in vietnam update of enterovirus 71 infection: epidemiology, pathogenesis and vaccine evaluation of monovalent and bivalent vaccines against lethal enterovirus 71 and coxsackievirus a16 infection in newborn mice removal of antibiotic resistance gene-carrying plasmids from lactobacillus reuteri atcc 55730 and characterization of the resulting daughter strain, l. reuteri dsm 17938 antibiotic resistance among commercially available probiotics key: cord-022252-9yiuuye3 authors: mims, cedric a.; dimmock, nigel j.; nash, anthony; stephen, john title: mechanisms of cell and tissue damage date: 2013-11-17 journal: mims' pathogenesis of infectious disease doi: 10.1016/b978-0-12-498262-8.50015-1 sha: doc_id: 22252 cord_uid: 9yiuuye3 nan the impact on the host of microbial damage depends very much on the tissue involved. damage to muscle in the shoulder or stomach wall, for instance, may not be serious, but in the heart the very existence of the host depends on a strong muscle contraction continuing to occur every second or so, and here the effect of minor functional changes may be catastrophic. the central nervous system is particularly vulnerable to slight damage. the passage of nerve impulses requires normal function in the neuronal cell membrane, and viruses especially have important effects on cell membranes. also a degree of cellular or tissue oedema that is tolerable in most tissues may have serious consequences if it occurs in the brain, enclosed in that more or less rigid box, the skull. therefore, encephalitis and meningitis tend to cause more severe illness than might be expected from the histological changes themselves. oedema is a serious matter also in the lung. oedema fluid or inflammatory cell exudates appear first in the space between the alveolar capillary and the alveolar wall, decreasing the efficiency of gaseous exchanges. respiratory function is more drastically impaired when fluid or cells accumulate in the alveolar air space.* the effect of tissue damage is much less in the case of organs such as the liver, pancreas or kidney, which have considerable functional reserves. more than two-thirds of the liver must be removed before there are signs of liver dysfunction. cell damage has profound effects if it is the endothelial cells of small blood vessels that are involved. the resulting circulatory changes may lead to anoxia or necrosis in the tissues supplied by these vessels. here too, the site of vascular lesions may be critical, effects on organs such as the brain or heart having a greater impact on the host, as discussed above. rickettsiae characteristically grow in vascular endothelium, and this is an important mechanism of disease production. by a combination of direct and immunopathological factors there is endothelial swelling, thrombosis, infarcts, haemorrhage and tissue anoxia. this is especially notable in the skin, and forms the basis for the striking rashes in typhus and the spotted fevers. these skin rashes, although important for the physician are less important for the patient than similar lesions in the central nervous system or heart. it is damage to cerebral vessels that accounts for the cerebral disturbances in typhus; involvement of pulmonary vessels causes pneumonitis, and involvement of myocardial vessels causes myocardial oedema. in q fever, rickettsiae sometimes localise in the endocardium, and this causes serious complications. sometimes an infectious agent damages an organ, and loss of function in this organ leads to a series of secondary disease features. the signs of liver dysfunction are an accepted result of infections of the liver, just as paralysis or coma is an accepted result of infection of the central nervous system. diabetes may turn out to be caused by infection of the islets of langerhans in the pancreas. coxsackie and other virus infections of the islets of langerhans can certainly cause diabetes in experimental animals, and coxsackieviruses have been associated with juvenile diabetes in man. there are many diseases of unknown aetiology for which an infectious origin has been suggested. sometimes it is fairly well established that an infectious agent can at least be one of the causes of the disease, but in most instances it is no more than a hypothesis, with little or no good evidence. for conditions as common and as serious as multiple sclerosis, cancer and rheumatoid arthritis it would be of immense importance if a microorganism were incriminated, since this would give the opportunity to prevent the disease by vaccination. accordingly, there is a temptation to accept or publicise new reports even though the evidence is weak or the observations poorly controlled. as if to warn us about this and remind us of possibilities from environmental toxins, parkinson's disease, a chronic neurological condition in which there is a loss of neurons in a sharply defined region of the brain (substantia nigra), can be caused by exposure to the chemical mptp. one example which raises the possibility that subtle cns disturbances may be caused by viruses is experimental infection with borna disease virus. this virus was used to infect tree shrews (tupaia glis) which are primitive primates. there is little overt disease, but afterwards the male is no longer able to enact the ritual courtship behaviour which (as students well know), is an essential preliminary to mating in all primates, and the frustrated male usually ends up bitten by the female. thus it can be said that infection with borna disease virus renders the male psychologically sterile. presumably the virus in some way alters the functioning of neurons concerned in this particular pathway. all other behavioural and physiological aspects appear normal. borna disease virus is not known to occur in man, but speculation about an analogous human situation is fuelled by the finding of borna disease virus-specific antibodies in patients with psychiatric/ behavioural disorders. in an entirely different clinical context, infection of a particular strain of rats with borna disease virus causes immense obesity, the underlying physiological basis of which is not understood. since the aetiology of such diseases raises interesting problems in pathogenesis, the present state of affairs is summarised in table 8 .1, which includes some of the human diseases whose infectious origin is probable, possible, conceivable, or inconceivable. causal connections between infection and disease states are particularly difficult to establish when the disease appears a long time after infection. it was not too difficult to prove and accept that the encephalitis that occasionally occurs during or immediately after measles was due to measles virus. but it was hard to accept that a very rare type of encephalitis (subacute sclerosing panencephalitis or sspe), occurring up to 10 years after apparently complete recovery from measles, was also due to measles virus and this was only established after careful studies and the eventual difficult isolation of a mutant form of measles virus from brain cells. 'slow' infections, in which the first signs of disease appear a long time after infection are now an accepted part of our outlook. the disease kuru occurred in new guinea and was transmitted from person to person by cannibalism. the incubation period of the disease in man appears to be 12-15 years, and was caused by a nonisolatable infectious agent that grew in the brain. this was established when the same disease appeared in monkeys several years after the injection of material from the brain of kuru patients. a similar agent called scrapie (see ch. 7) infects sheep, mice and other animals and also has an incubation period representing a large portion of the life span of the host. in both kuru and sspe the agent was eventually shown to be present in the brains of patients. so far this has only been demonstrated indirectly as, despite strenuous efforts, the causative agent has yet to be isolated. if in a slow infection the microorganism that initiated the pathological process is no longer present by the time the disease becomes manifest, then the problem of establishing a causal relationship will be much greater. this may possibly turn out to be true for diseases like multiple sclerosis and rheumatoid arthritis. liver cancer in humans and leukaemia in mice, cats, humans and cattle can be caused by slow type virus infections. cancer or leukaemia appears as a late and occasional sequel to infection. the virus, its antigens or fragments of its nucleic acid are detectable in malignant cells. one important factor that often controls the speed of an infectious process and the type of host response, is the rate of multiplication of a microorganism.* different infectious agents show doubling times * every infection is a race between the spread and multiplication of the microbe and the generation of an antimicrobial response by the host. a day or two's delay in this response may let the microbe reach the critical levels of growth that give tissue damage and disease. varying from 20 min to 2 weeks, and some of these are listed in table 8 .2. often the rate of multiplication in the infected host, in the presence of antimicrobial and other limiting factors and when many bacteria are obliged to multipy inside phagocytic cells, is much less than the optimal rate in artificial culture. clearly a microorganism with a doubling time of a day or two will tend to cause a more slowly evolving infection and disease than one that doubles in an hour or less. it is uncommon for an infectious agent to cause exactly the same disease in all those infected. its nature and severity will depend on infecting dose and route, and on the host's age, sex, nutritional status, genetic background, and so on. many infections are asymptomatic in more than 90% of individuals, clinically characterised disease occurring in only an occasional unfortunate host, as 'the tip of the iceberg'.* asymptomatically infected individuals are important because they are not identified, move normally in the community, and play an important part in transmission. this chapter deals with demonstrable cell and tissue damage or dysfunction in infectious diseases. but one of the earliest indications of illness is malaise, 'not feeling very well'. this is distinct from fever or a specific complaint such as a sore throat and although it is difficult to define and impossible to measure, we all know the feeling. it can precede the onset of more specific signs and symptoms, or accompany them. sometimes it is the only indication that an infection is taking place. almost nothing is known of the basis for this feeling. toxins', of course, have been invoked and the earliest response to pyrogens (see pp. 298-300) before body temperature has actually risen, may play a part. interferons may have something to do with it because pure preparations of human a or ß interferons cause malaise and often headaches and muscle aches after injection into normal individuals. if interferon is eventually recognised as an important antimicrobial force we may have to regard these side effects as unfortunate but acceptable. soluble mediators of immune and inflammatory responses, such as interleukin-1 (see glossary) or other cytokines may also play a part. in some infectious diseases weakness and debility are prominent during convalescence. this can be especially notable following influenza and hepatitis, but its basis is as mysterious as in the case of malaise. the infections that matter are those causing pathological changes and disease. before giving an account of the mechanisms by which these changes are produced, it is important to remember that many infectious agents cause little or no damage in the host. indeed, it is of some advantage to the microorganism to cause minimal host damage, as discussed in ch. 1. virus infections as often as not fall into this category. thus, although infection with rabies or measles viruses nearly always causes disease, there are many enterovirus, reovirus and myxovirus infections that are regularly asymptomatic. even viruses that are named for their association with disease (poliomyelitis, influenza, japanese b encephalitis) often give an antibody response as the only sign of infection in the host. tissue damage is too slight to cause detectable illness. there is also a tendency for persistent viruses to cause no more than minor or delayed cellular damage during their persistence in the body, even if the same virus has a more cytopathic effect during an acute infection, e.g. adenoviruses, herpes simplex (see ch. 10). a few viruses are remarkable because they cause no pathological changes at all in the cell, even during a productive infection in which infectious virus particles are produced. for instance, mouse cells infected with lcm or leukaemia virus show no pathological changes. a mouse congenially infected with lcm virus shows a high degree of immune tolerance, and all tissues in the body are infected. throughout the life of the animal, virus and viral antigens are produced in the cerebellum, liver, retina etc. without discernible effect on cell function. but sometimes there are important functional changes in infected cells which lead to a pathological result. for example, the virus infects growth hormoneproducing cells in the anterior pituitary. although the cells appear perfectly healthy, the output of growth hormone is reduced, and as a result of this, suckling mice fail to gain weight normally and are runted. when bacteria invade tissues, they almost inevitably cause some damage, and this is also true for fungi and protozoa. the extent of direct damage, however, is sometimes slight. this is true for treponema pallidum, perhaps because the lipopolysaccharide-protein components that might have induced inflammatory responses, are not exposed on the surface of the bacteria. it produces no toxins, does not cause fever, and attaches to cells in vitro without harmful effects. leprosy and tubercle bacilli eventually damage and kill the macrophages in which they replicate, but pathological changes are to a large extent caused by indirect mechanisms (see below). in patients with untreated lepromatous leprosy, the bacteria in the skin invade blood vessels, and large numbers of bacteria, many of them free, may be found in the blood. in spite of the continued presence of up to 10 5 bacteria m l -1 of blood there are no signs or symptoms of septicaemia or toxaemia. mycobacterium leprae can be regarded as a very successful parasite that induces very little host response in these patients, even when the bloodstream is invaded. the resident bacteria inhabiting the skin and intestines of man and animals do not invade tissues and are normally harmless; indeed, as discussed in ch. 1, they may benefit the host. bacteria such as meningococci and pneumococci, whose names imply pathogenicity, spend most of their time as harmless inhabitants of the normal human nasopharynx: only occasionally do they have the opportunity to invade tissues and give rise to meningitis or pneumonia. cell and tissue damage are sometimes due to the direct local action of the microorganism. however, it is not at all clear how viruses cause the death of cells. many virus infections result in a shutdown of rna synthesis (transcription), protein synthesis (translation) and dna synthesis in the host cell, but usually these are too slow to account for the death of the cell. after all, cells like neurons never synthesise dna, and the half life of most proteins and even rnas is at least several hours. a possible alternative mechanism is the alteration of the differential permeability of the plasma membrane. this is important as the cell has a high internal k + concentration and low n a + concentration, while the reverse is true of body fluids. viruses do alter membrane permeability, but the unresolved question is whether or not this is responsible for the death of the cell or is merely an after-effect. it now appears that at least some virus infections (hiv, adenovirus, influenza virus, sindbis virus) cause the cells to commit suicide by a mechanism called 'programmed cell death' or 'apoptosis'. this is the natural process by which the body controls cell numbers and rids itself of superfluous or redundant cells during development. a familiar example is a tadpole 'losing' its tail. cells do not disintegrate but round up, and are then removed by phagocytes. a possible example of apoptosis is seen in the common cold when caused by the rhino virus group of picorna viruses. rhinoviruses infect nasal epithelial cells, and at an early stage the cells round up, fall off the mucosal surface and are carried away, often with their cilia still beating, in a stream of fluid induced by the infection.* this leaves areas of raw mucosa, with the exposed underlying tissues inflamed, oedematous, and susceptible to infection by the normally harmless resident bacteria. the gross pathology of cells infected with viruses appears generally nonspecific, very like that induced in cells by the toxins of diphtheria bacilli and streptococci, or by physical and chemical agents. the most common and potentially reversible change, the oedema seen as 'cloudy swelling' by routine histology, is associated with membrane permeability changes. changes in the endoplasmic reticulum, mitochondria and polyribosomes are seen by electron microscopy at this stage. later the nuclear chromatin moves to the edge of the nucleus ('margination' of chromatin) and becomes condensed (pycnosis), but the cell has already died by this time. there are two more characteristic types of morphological change produced by certain viruses, and these were recognised by histologists more than 50 years ago. the first are inclusion bodies, parts of the cell with altered staining behaviour which develop during infection. they often represent either cell organelles or virus factories in which viral proteins and/or nucleic acids are being synthesised and assembled. herpes group viruses form intranuclear inclusions, rabies and poxviruses intracytoplasmic inclusions, and measles virus both intranuclear and intracytoplasmic inclusions. the second characteristic morphological change caused by viruses is the formation of multinucleate giant cells. this occurs, for instance when hiv 'fusion' proteins (gpl20-gp41) present in nascent virus particles budding from an infected cell attach to cd4 receptors in the plasma membranes of neighbouring cells; membranes then fuse and multinucleate cells are formed. it also happens in measles and certain herpes virus infections. before leaving the subject of direct damage by viruses, one supreme example will be given. here the direct damage is of such a magnitude that the susceptible host dies a mere six hours after infection. if rift valley fever virus, an arthropod-borne virus infecting cattle, sheep and man in africa, is injected in very large doses intravenously into mice, the injected virus passes straight through the kupffer cells and endothelial cells lining liver sinusoids (see ch. 5) and infects nearly all hepatic cells. hepatic cells show nuclear inclusions within an hour, and necrosis by four hours. as the single cycle of growth in hepatic cells is completed, massive liver necrosis takes place, and mice die only six hours after initial infection. the host defences in the form of local lymph nodes, local tissue phagocytes etc. are completely overcome by the intravenous route of injection, and by the inability of kupffer cells to prevent infection of hepatic cells. direct damage by the replicating virus destroys hepatic cells long before immune or interferon responses have an opportunity to control the infection. this is the summit of virulence. the experimental situation is artificial but it illustrates direct and lethal damage to host tissues after all host defence mechanisms have been overwhelmed. most viruses, rickettsiae and chlamydia damage the cells in which they replicate, and it is possible that some of this damage is due to the action of toxic microbial products. this action, however, is confined to the infected cell, and toxic microbial products are not liberated to damage other cells. mycoplasma (see table a .2) can grow in special cell-free media, but in the infected individual they generally multiply while attached to the surface of host cells. as studied in culture and on the respiratory epithelium they 'burrow' down between cells, inhibit the beat of cilia and cause cell necrosis and detachment. the mechanism is not clear. if a complete lawn of mycoplasma covers the surface of the host cell, some effect on the health of the cell is to be expected, but it is possible that toxic materials are produced or are present on the surface of the mycoplasma. dental caries provides an interesting example of direct pathological action. colonisation of the tooth surface by streptococcus mutans leads to plaque formation, and the bacteria held in the plaque utilise dietary sugar and produce acid (see p. 35). locally produced acid decalcifies the tooth to give caries. caries, arguably the commonest infectious disease of western man, might logically be controlled by removing plaque, withholding dietary sugar, or vaccinating against streptococcus mutans. however, fluoride in the water supply or in toothpaste has been the method of choice, and has been very successful. it acts by making teeth more resistant to acid. bacteria generally damage the cells in which they replicate, and these are mostly phagocytic cells (see ch. 4). listeria, brucella and mycobacteria are specialists at intracellular growth, and the infected phagocyte is slowly destroyed as increasing numbers of bacteria are produced in it. bacteria such as staphylococci and streptococci grow primarily in extracellular fluids, but they are ingested by phagocytic cells, and virulent strains of bacteria in particular have the ability to destroy the phagocyte in which they find themselves, even growing in the phagocytes, as described in ch. 4. many bacteria cause extensive tissue damage by the liberation of toxins into extracellular fluids. various toxins have been identified and characterised. most act locally, but a few cause pathological changes after spreading systemically through the body. this is a huge and growing part of our subject and we need to define the term toxin, a task which is more difficult than one might think. an attempt was made by bonventre who in 1970 defined toxins as a 'special class' of poisons which differ from, for example, cyanide or mercury by virtue of their microbial origin, protein structure, high molecular weight, and antigenicity. this view is too embracing, because it includes proteins of doubtful significance in disease, and also too restrictive, because it excludes nonprotein toxic complexes such as endotoxin. another suggestion is that toxin must include all naturally occurring substances (of plant, animal, bacterial or whatever origin) which when introduced into a foreign host are adverse to the well-being or life of the victim. this, too, is unsatisfactory because some substances-potent toxins within the scope of this definition-are being used in some contexts as therapeutic agents! perhaps it is pointless to strive for an all-embracing definition, although the obvious differences between bacterial and fungal toxins warrant the continued use of the appropriate prefix. for example, bacterial toxins are usually of high molecular weight and hence antigenic, whereas fungal toxins tend to be low molecular weight and not antigenic. the problem of definition is compounded because there are substances (aggressins) which help to establish an infective focus as well as those whose action is uniquely or largely responsible for the disease syndrome. also there are substances known to be produced by bacteria in vitro, whose properties on a priori grounds make them potential determinants of disease, but which have not been shown to play a role in vivo. we will concentrate on those toxins known to be, or likely to be, involved in some aspect of disease causation. it is not difficult to demonstrate the production of'toxins' by bacteria in culture, as judged by some bioassays. primary consideration will be given to those substances which are produced under ecologically significant conditions (i.e. in the natural host or relevant animal model) and cause (also in biologically relevant systems) damage to cells or tissues thereby contributing to disease. exotoxins are produced and then either secreted by, or released upon lysis from, both gram-positive and gram-negative bacteria. they are proteins, some of which are enzymes. when liberated locally they can cause local cell and tissue damage. those that damage phagocytic cells and are therefore particularly useful to the microorganism have been described in ch. 4. those that promote the spread of bacteria in tissues have been referred to in ch. 5. a description of some of the more interesting exotoxins follows. proteases and hyaluronidases, which help the spread of bacteria through tissues have already been mentioned in ch. 5. here we consider toxins which act on extracellular substances and are responsible for many of the main features of the diseases caused by the infecting organism. pseudomonas aerwgmosa-elastase, and one of at least six proteases of legionella pneumophila, both induce fibrinopurulent exudation in the rat lung (a model for p. aeragmosa-induced pneumonia in human cystic fibrosis) and the guinea-pig lung (a model for legionnaires' disease) respectively. these characteristics almost certainly arise from the release of oligopeptides from extracellular matrix components of the host which are chemotactic for leucocytes and fibroblasts. the l. pneumophila protease is the same major secretory protein (the 38 000 m r zinc metalloprotease) already considered in ch. 4 in relation to survival within macrophages. staphylococcal exfoliatin is important in staphylococcal 'scalded skin syndrome' (ssss), a disease of newborn babies. the disease is characterised by a region of erythema which usually begins around the mouth and, in 1-2 days, extends over the whole body. during this period, small yellowish exudative lesions often appear. the most striking feature of the disease, however, is that the epidermis, although apparently healthy, can be displaced and wrinkled like the skin of a ripe peach by the slightest pressure. soon large areas of the epidermis become lifted by a layer of serous fluid and peel at the slightest touch. large areas of the body rapidly become denuded in this way and the symptoms resemble those of massive scalding. while the toxin causes cleavage of desmosomes (specialised cell membrane thickenings through which cells are attached to each other) in the stratum granulosum, it may also act intracellularly. despite numerous attempts to characterise the biological activity of exfoliatin, the genetically predicted serine protease and/or lipase activity has never been demonstrated. some enterotoxigenic e. coli elaborate families of low molecular weight heat stable (st) peptides as well as heat labile (lt; cholera-like) toxin. sts bind to a receptor which then activates a tightly coupled membranebound guanylate cyclase in gut cells, resulting in the transmission of a signal to the inside of the cell, thereby elevating cgmp, or some other second message. as described later in the section on diarrhoea this gives rise to efflux of ions, and hence water, from enterocytes. we know that these toxins are able to traverse membranes since their targets are intracellular; we still know very little about the details of how they achieve this. these proteins have common features: one component (usually described as the b fragment or subunit) binds to and interacts with a cell receptor and promotes the uptake of an active component (a fragment or subunit) in which resides the biological activity which confers toxicity. there are several ways in which one can classify these toxins. (1) biologically-some kill cells (cytotoxic toxins) whereas others (sometimes called cytotonic toxins) 'deregulate' cells. (2) biochemically-some belong to a well defined biochemical group recognised by their ability to cleave nad + into nicotinamide and adp-ribose moieties. they are designated adp-ribosyl (adpr) transferases since they transfer adpr to different target proteins; this group straddles groups 1 and 3. (3) genetically-the third possibility reflects the genetic origin of these toxins: (i) production from a single gene of a single peptide which undergoes post-translational modification into a and b fragments which are covalently linked; (ii) production from separate genes of a and b subunits which noncovalently associate into stable complexes; (iii) production from separate genes of different proteins which do not associate into stable complexes but which must act in concert to express toxicity. these are known as binary toxins; again, group 3 straddles group 1. examples of each of these groups will be given illustrating how they work, their importance in disease, and surprisingly (!) how some of the most toxic substances known to man are being used or may be used for therapeutic purposes. diphtheria toxin (dt). corynebacterium diphtheriae organisms multiply on the epithelial surfaces of the body (nose, throat, skin) but do not penetrate deeply into underlying tissues. the infection on the body surface causes necrosis of mucosal cells with an inflammatory exudate and the formation of a thick 'membrane' (hence the name c. diphtheriae: gr., diphthera = membrane) and if the infection spreads into the larynx there may be respiratory obstruction. the toxin probably assists colonisation of the throat or skin by killing epithelial cells and polymorphs. active immunisation with diphtheria toxoid has made diphtheria a clinical rarity in developed countries. dt is disseminated from the infection site and has important actions, especially on the heart and nervous system. dt is encoded by lysogenic phage ß but its expression is controlled by the host bacterium under conditions of iron stress. it is synthesised and processed as shown in fig. 8 .1. there is intense interest in seeking to understand the mechanisms of uptake of the active moieties of diphtheria and other toxins whose target is intracellular. this is driven by the desire to understand fundamental mechanisms in cell biology and to develop selective cytotoxic therapies' in clinical medicine. the mechanism of internalisation of the active a under the acidic conditions of the endosome, some dt-a is released by reduction, dt-b undergoes conformational change and interacts with the endosomal membrane resulting in the opening of cation channels. the latter are not absolutely necessary, but their formation is associated with 10 times greater uptake of dt-a. in contrast, most other toxins which enter the cytosol appear to do so by more complicated routes which initially involving endocytosis. shiga toxin (and ricin, a toxic plant lectin believed to have been located in the tip of an umbrella and used to poison a bulgarian spy!) are not translocated across the endosomal membrane but are transported to the trans-golgi network and all the way back to the endoplasmic reticulum. somewhere in the exocytotic pathway conditions exist for the translocation of shiga toxin (sht) into the cytoplasm. sht acts by modifying 28s ribosomal rrna. for cholera toxin, it used to be thought that a l ( fig. 8 .5) moved down through the central hole of the ring-shaped complex of ct-b which was anchored to the membrane by five ganglioside gm1 binding sites. structural work on this family of proteins shows that this is not physically possible. the finding that the c-terminal sequence lysine-aspartateglutamate-leucine (the kdel* motif) in the a subunit of cholera toxin and related sequences in e. coli heat labile toxin and pseudomonas exotoxin a raises the possibility that transport to the endoplasmic reticulum may also be necessary for these a subunits to reach the cytoplasm. a new and exciting development is beginning. for decades, attempts have been made to make immunotoxins by coupling native toxins to antibodies specific to some surface antigen on tumour cells, with little practical success, as yet. however, scientists have now genetically engineered dt by substituting that portion of the dt structural gene encoding the native toxin receptor-binding domain with modified cdna encoding one of a variety of cytokines and growth factors including il-2, il-4, il-6, egf and α-msh. the resultant fusion toxin bears a new 'cellular address', but retains all of the other biological properties of the native dt molecule as well as a three-dimensional structure nearly identical with native dt. the il-2 receptor-binding domain fusion toxin has undergone phase i/ii clinical trials for the treatment of il-2 receptor positive haematological malignancies (and other il-2 related conditions) and has been shown to be safe and well tolerated. such constructs are potent against a murine model of il-2 receptor positive lymphoma, and the hope is that a durable remission of disease will be achieved in humans. (an era of new 'magic bullets'?) p. aeruginosa exotoxin a. p. aeruginosa, already alluded to above, is common in soil and water and can occasionally be isolated from the faeces of normal, healthy individuals. it is virtually harmless for healthy adults, but its ability to multiply in almost any moist environment and its resistance to many antibiotics have made the bacterium a major cause of * the kdel motif is normally found in proteins which having been processed in the golgi are trapped by the endoplasmic reticulum which recognises the kdel motif. this prevents the protein being lost to the cell via exocytotic trafficking and results in its translocation into the cytoplasm. the schema shows a round of peptide elongation and illustrates the key role played by two enzymes, ef1 and ef2. dta and pea each adp-ribosylates diphthamide (a modified histidine) in ef2-gtp which can no longer translocate the newly elongated peptide from the a site to the p site. sht a fragment is a specific n-glycosidase which cleaves an adenine residue from near the 3'-prime end of the 28s ribosomal rna. this depurination results in failure of ef1-dependent binding of aminoacyl-trna to site a and hence inhibits protein synthesis. poliovirus achieves selective inhibition of host protein synthesis at an earlier stage than is depicted here. host mrna is first modified (capped) then bound to the small ribosomal subunit; poliovirus mrna is not capped. the function of a cap-binding protein, which recognises and binds host mrna to the ribosome, is inhibited by a poliovirus virion protein thereby allowing differential translation of virus messenger rna. ef-ια: nucleotide-binding protein. hospital-acquired infection, particularly among patients with impaired host defence mechanisms such as those with chronic illness, genetic immunodeficiencies, those under treatment with immunosuppressive drugs, or patients suffering from extensive burns. p. aeruginosa causes localised infection in the urinary tract, respiratory tract, burns and wound infections. in severely debilitated patients these localised infections may develop into general septicaemia, with mortality in such cases approaching 100%. the mechanisms of pathogenicity of p. aeruginosa are complicated and unclear because (unlike c. diphtheriae) the organism elaborates several potentially toxic extracellular products, including a phospholipase, several proteases, lipase, haemolysin, enterotoxin, lipopolysaccharide endotoxin, elastase, exoenzyme s (pes), and exotoxin a (pea); evidence definitely implicates the last three. elastase has already been mentioned above (p. 207). pes is without question a virulence determinant, but at present its biological mode of action is not wholly understood. pea is in many ways like dt. it inhibits protein synthesis by cleavage of nad + and subsequent adp-ribosylation of ef2 at the same diphthamide residue, is synthesised predominantly during the decline phase of cell growth, and its production appears to be dependent upon the concentration of iron in the medium. its m r is 66 580, and it can be activated in cell-free systems by proteolysis, or by reduction with thiols in the presence of urea and other chaotropic agents. all the enzymic activity of the toxin resides in a proteolytically derived fragment (m r approximately 26000). however, dissimilarities exist between the two toxins. firstly, the active a fragment resides in the cooh-terminal portion of the molecule, whereas the diphtheria a fragment is at the nh 2 -terminal end. secondly, the b fragment of pea recognises a different receptor to that of dt, and there are no sequence homologies or serological crossreactivity between the two toxins. shiga toxin. this toxin is a subunit protein with an ab 5 type structure (1 a subunit; b, an oligomer of 5 subunits). certain biotypes of e. coli make vero toxins (vts) or shiga-like toxins (slts) which are identical (slt1) or near identical (slt2) with shiga toxin. the role of shiga toxin in bacillary dysentery is discussed below. slt-producing strains of e. coli are responsible for haemorrhagic colitis and haemolytic urea syndrome. very recent crystallographic studies have shown a remarkable similarity in organisational structure of the b subunits of shiga toxin and those of the cholera toxin family discussed below, despite the near complete absence of sequence homology. the b subunits attach to a ganglioside structure gb3 or gb4 (cholera toxin attaches to gm1) on susceptible cell surfaces. sht-a fragment is a specific n-glycosidase which cleaves an adenine residue from near the 3'-prime end of 28s ribosomal rna. this depurination results in failure of efl-dependent binding of aminoacyl-trna to site a and hence inhibits protein synthesis ( fig. 8.3 ). this is based on studies on cultured cells, and how such cytotoxic activity gives rise to the enterotoxic activity-watery diarrhoea seen in cases of dysentery preceding the bloody mode of action of actin-adp-ribosylating toxins. c. botulinum c2 toxin component 211 binds to the cell membrane followed by c2i. the latter is internalised and upsets the equilibrium between polymerisation and depolymerisation of actin. adp-ribosylation of actin inhibits its polymerisation and turns g-actin into a capping protein which binds to the fast growing (concave) ends of actin filaments. capping of the concave ends increases the critical concentration for actin polymerisation. since the slow-growing (pointed) ends of actin filaments are free, depolymerisation of actin occurs at these ends. released actin is substrate for the toxin and will be withdrawn from the treadmilling pool of actin by adpribosylation, i.e. trapped. both reactions will finally induce the breakdown of the microfilament network. (taken with permission of authors (k. aktorieseia/.) and publisher (springer-verlag gmbh & co. kg, heidelberg, germany) from botulinum c2 toxin. clostridium botulinum c2 toxin is not a neurotoxin but belongs to a family of cytotoxins {clostridium perfringens iota toxin, clostridium spiroforme toxin, clostridium difficile adp ribosyltransferase) whose common features are that they are binary toxins and that they target cytoskeletal actin. of these c. botulinum c2 is the best studied at present. component c2i is the equivalent of the a subunit and c2ii, the b subunit. they are produced in parallel although different strains produce different ratios. more c2 is produced during sporulation than in the vegetative phase of growth. c2ii is activated by proteolysis to produce a 74 000 m r protein which binds to cell surfaces and thereby exposes a binding site for c2i (m r ca. 45 000) either on the cell surface or on attached c2ii. once inside, c2i adp-ribosylates monomeric actin and not polymerised f actin. the substrate specificity is high. the toxin will modify a specific arginine residue present in certain isoforms of actin. neither subunit is toxic on its own, but together the ld 50 dose for a mouse is ca. 5-50 ng. (other examples of such 'binary' activity include anthrax toxin and staphylococcal leucocidin). the two components may be injected into different sites or by different routes but toxic effects always occur at the site of injection of c2ii. this mode of action, summarised in fig. 8 .4, is believed to underlie the biological manifestations of toxicity which, in rats, include hypotension, haemorrhaging in and fluid accumulation around the lungs as a result of vascular permeability changes. the toxin is currently being used as a tool in cell biology to study cytoskeletal systems and dissect mechanisms of cell signal transduction. cholera and related toxins. there is a growing number of toxins in this category. easily the most studied is cholera toxin (ct), a major research area for three decades, which was originally stimulated by the desire to make a more effective vaccine against cholera. ironically, effective immunity is best mediated by antibacterial antibodies which prevent colonisation of the gut with v. cholerae rather than antitoxin-neutralising antibodies. cholera toxin is an ab 5 subunit toxin ( fig. 8.5 ). enterotoxigenic e. coli (etec) strains produce two heat labile toxins: lti and ltii. ltii exists as two minor variants, ltiia and ltiib, the latter requiring trypsin treatment for full activation in the y-1 adrenal cell assay. the b subunits of ct, ltiia and ltiib recognise and are bound to cell surface gangliosides gm1, gdlb and gdi a respectively and mediate the translocation of a l subunits into the cytoplasm; lti binds to either gm1 or a glycoprotein. the same reaction occurs as was described above for dt: nad + is cleaved into nicotinamide and adp-ribose (adpr) moieties, with two important differences: (1) adpr transferase is greatly enhanced by a group of proteins (both membrane bound and soluble) of ca. 20 000 m r designated adpribosylation factors (arfs; of as yet unknown physiological significance); (2) adpr is transferred to the a s subunit of the g s regulator of adenylate cyclase as illustrated in fig. 8 .6. this results in the elevation of camp levels in the cell with the consequences described below in the section on diarrhoea. bordetella pertussis toxin (pertussigen) and adenylate cyclasehaemolysin (ac-hly). whooping cough (pertussis) is a severe respiratory tract infection characterised by prolonged paroxysmal coughing, attacks of which continue long after infection has cleared. the disease is capable of striking all ages but is particularly prevalent and severe in young children, where hospitalisation is required in about 10% of cases. the causative agent of pertussis, bordetella pertussis, is transmitted aerially from the respiratory tract of an infected individual to that of a susceptible host. the organism attaches (probably via its filamentous haemagglutinin, pili, and a 69 000 m r outer membrane protein) to, and colonises the mucosal surface between the cilia, and multiplies there during the incubation period of the disease which is commonly around seven days. the infection then manifests as a slight fever and catarrh which is often indistinguishable from a common cold. however, one to two weeks later bouts of uncontrollable coughing begin. it is this paroxysmal coughing, along with the notorious 'whoop' as the child attempts to draw breath, which characterises the disease. the paroxysmal coughing stage often lasts for several weeks and no treatment fully effective in controlling the symptoms. the only proven means of controlling whooping cough is vaccination but, in the uk at least, sporadic reports of vaccine-induced brain damage in infants has diminished public acceptance of the vaccine. it should be noted that permanent encephalopathy (brain damage) is a recognised though rare consequence of whooping cough infection. much current work is being devoted to producing immunogenic, completely nontoxic preparations of pertussis toxin by genetic manipulation of t h e gene encoding t h e s i s u b u n i t ( fig. 8 .5). in clinical trials in italy, such engineered vaccines h a v e been shown to be both safe a n d effective a s j u d g e d by antibody titres to pertussis toxin. several toxic substances h a v e been isolated from bordetella pertussis including a h e a t labile toxin, tracheal cytotoxin, endotoxin, adenylate cyclase-haemolysin (ac-hly), a n d pertussis toxin. t h e latter h a s m a n y biological activities-histamine sensitisation, leucocytosis promotion, islet (a) the production of cyclic amp by adenyl cyclase. cyclic amp is an important second messenger invoked in the intracellular amplification of many cellular responses to external signals including hormones. the nature of the physiological response reflects the biochemical differentiation of the cell responding to the stimulus. for example, in gut cells the response would be altered ion transport and hence fluid secretion; in muscle cells it would be glycogen breakdown in response to the call for more energy. the production of camp is controlled both positively and negatively at two different levels. the central cycle represents a normal membranebound hormone receptor and the heterotrimeric (aß7) g protein regulator complex which is activated upon binding of hormone to the receptor. there are two receptors, each with regulatory g proteins: one system responds to stimulatory and the other responds to inhibitory stimuli; only one system is shown. in gut cells these receptors would be on the basolateral (nonluminal) side of enterocytes enabling responses to stimuli from the circulation. (b) the second level of control involves endogenous gtpase properties of both stimulatory (a s ) and the inhibitory (a a ) subunits of the g protein regulator which may be outlined as follows. stimulation: agonist stimulation of its receptor results in the dissociation of the a s ß7 g protein from the receptor and of the subunits from each other, and the binding of gtp to a s . a s -gtp will productively interact with the catalytic unit of adenyl cyclase (not shown) and stimulate the production of camp from atp. endogenous gtpase activity in a s results in a delayed conversion of a s -gtp to inactive a s -gdp which can no longer stimulate the cyclase but allows the reassociation of the trimeric g protein and loss of gdp. inhibition: antagonist stimulation of its receptor results in an analogous situation for inhibiting cyclase activity via c^: inhibitory cti-gtp inactivates the cyclase; cti-gdp does not inactivate the cyclase. (c) the level of camp may be affected by physiological stimuli (which stimulate removal and modification of the g-protein complex) or by perturbation of the normal regulatory cycle as illustrated, by cholera toxin (ct) or pertussis toxin (ptx). (d) cholera toxin acts first by interacting with its receptor resulting in the internalisation of the active a l subunit. a l adp-ribosylates a s -gtp which promotes continued dissociation of the heterotrimer; also the endogenous gtpase is no longer functional hence the stimulation of the cyclase continues. lt toxins act in a similar manner. (e) pertussigen acts first by interacting with its receptor resulting in the internalisation of the active si subunit. si adp-ribosylates the ai-gdpß7-heterotrimer which can no longer associate with the receptor (or lose gdp) to undergo another cycle of gtp activation; activated cyclase can no longer be turned off. (adapted, with kind permission of author (gierschik, p.) and publisher (springer-verlag gmbh & co. kg, heidelberg, germany) from figure 4 in 'adp-ribosylation of signal-transducing guanine nucleotide-binding proteins by pertussis toxin', current topics in microbiology andlmmunology (1992) 175,78, edited by klaus aktories. activation and adjuvanticity-and is believed to be the most important but by no means the only virulence determinant of b. pertussis: it is called pertussigen and is the basis of the vaccine against whooping cough. pertussigen is a complex subunit toxin whose biochemical mode of action is identical to that of ct except that the target is the aj subunit of the gj regulator of adenylate cyclase (see fig. 8 .6). it is not yet clear how such biochemical activity relates to the clinical syndrome. ac-hly has two functional domains. one confers adenylate cyclase activity and another haemolytic activity. its haemolytic activity arises from its pore-forming properties in cell membranes. pore formation is probably responsible for the translocation of the adenylate cyclase portion of the complex. ac-hly is also known as cyclolysin and belongs to the group of toxins whose common feature is the presence of an array of a nine amino acid repeats; they have been designated rtx (repeats in toxin) toxins. the prototype is the haemolysin of e. coli which is important in extraintestinal infections caused by this organism. other toxins of this group include the haemolysin from proteus vulgaris, and the leukotoxin from pasteurella haemolytica. anthrax toxin. anthrax is a disease of animals, particularly sheep and cattle, and to a lesser extent man, caused by infection with bacillus anthracis. infection takes place following the ingestion of spores, the inhalation of spores, or by the entry of spores through abraded skin. the spores germinate and then the bacteria form a toxin that increases vascular permeability and gives rise to local oedema and haemorrhage. infection of the skin in man leads to the formation of a lesion (malignant pustule; a black eschar, hence b. anthracis; gr. anthrakos = coal) consisting of a necrotic centre surrounded by vesicles, blood-stained fluid and a zone of oedema and induration. in severe infections there is septicaemia with toxic signs, loss of fluid into tissues, with widespread oedema and eventually death. anthrax toxin, the discovery of which by smith and keppie in the early 1950s was a major milestone in our subject, consists of three components, none of which are toxic by themselves: factor i (oedema factor or ef), factor ii (protective antigen or pa) and factor iii (lethal factor or lf). the toxin is largely responsible for local lesions, kills phagocytes, enters the circulation and accounts for the toxicity, oedema and death caused by b. anthracis. virulent strains also have a capsular polypeptide composed of d-glutamic acid, which inhibits opsonisation and phagocytosis (see ch. 4). anthrax in man occurs mainly in those whose work brings them into contact with infected animals. it is not a common disease in the uk, and the usual source of infection is imported bones, hides, skins, bristles, wool and hair, or imported fertilisers made from the blood and bones of infected animals. most of what has just been described was discovered more than three decades ago but more recently new insights have been obtained as to the biochemical basis of the mode of action of anthrax toxin. ef was shown to be an inactive form of adenylate cyclase which is activated by calmodulin; pa and lf were not active. however combinations of pa and ef (but not pa and lf) caused an elevation of camp. thus anthrax toxin (at least the pa/ef combination) is a binary toxin. the response observed is more rapid and greater than that induced by cholera toxin. in contrast to cholera toxin, the anthrax complex is the cyclase, acts without a lag phase and its effects are instantly reversible upon washing toxin-treated cells. this suggests that the enzyme is either rapidly degraded after internalisation, or that it is not completely internalised but wsfhiw ii camp atp fig. 8 .7 mode of action of anthrax toxin. factor ii (pa) interacts with the cell membrane. after proteolytic cleavage sites are exposed which bind factor i (ef) and facilitate internalisation of factor i (ef). this internalisation takes place via an endocytic step, with factor i being released into the cytosol. factor i must be rapidly inactivated since washing toxin-treated cells results in a rapid loss of adenylate cyclase activity. factor i interacts with calmodulin (cal) to become an active adenylate cyclase enzyme. interaction of factor i with factor ii and subsequent internalisation is blocked by prior binding of factor iii (lf) to factor ii. associated with the cell membrane in a manner allowing it to be readily removed. these facts together with the observations that lf would block the activity of ef are summarised in fig. 8 .7. this explains how lf blocks the effect of ef. they either compete for the same sites on pa, in which case the explanation is self-evident, or they bind to separate sites on pa which are situated such that occupancy of one occludes access to the other. one can extend these observations made in vitro to the in vivo situation. the model provides a basis for understanding the protective function of pa. antibodies to pa could either block the initial attachment to the cell membrane of target organs in vivo, or prevent the binding of ef to pa, or both. the fact that camp is known to be a potent secretagogue could explain how the oedematous reaction occurs when pa and ef are injected into test animals, i.e. ef is, after activation, an oedema factor. moreover, in biological tests, addition of lf (factor iii) depresses oedema production as would be predicted by this model. the observation that pa was probably fixed first may now be placed on a firmer basis since binding of pa is a prerequisite for the expression of ef (and by implication) lf activities. the case for the view that, in experimental anthrax in guinea-pigs, animals died (as do humans) of secondary shock, is also placed on a firmer basis with perhaps one important reservation. smith and coworkers claimed that factor iii (lf) decreased oedema but increased lethality; the former but not the latter observation is explicable by the model which is therefore too simplistic to explain the effects of the holotoxin. it is likely that lf exerts its activity together with pa on other cells in vivo. it has in fact been shown that lf in combination with pa is cytolytic for macrophages and macrophage-like cell lines-the probable basis of the earlier observation that anthrax toxin kills phagocytes and perhaps justification for talking about a plurality of anthrax binary toxins. clostridium tetani spores germinate in an infected wound and produce their toxin. spores are ubiquitous in faeces and soil, require the reduced oxygen tension for germination provided locally in the wound by foreign bodies (splinters, fragments of earth or clothing) or by tissue necrosis as seen in most wounds, the uterus after septic abortion, or the umbilical stump of the newborn.* the site of infection may be a contaminated splinter just as well as an automobile or battle injury. all strains of clostridium tetani produce the same toxin. it also reaches the central nervous system by travelling up other peripheral nerves following bloodborne dissemination of the toxin through the body. the motor nerves in the brain stem are short and therefore the cranial nerves are among the first to be affected, causing spasms of eye muscles and jaw (lockjaw). there is also an increase in tonus of muscles round the site of infection, followed by tonic spasms. in generalised tetanus there is interference with respiratory movements, and without skilled treatment the mortality rate is about 50%. botulism* caused by clostridium botulinum, a widespread saprophyte present in soil and vegetable materials. c. botulinum contaminates food, particularly inadequately preserved meat or vegetables, and produces a powerful neurotoxin. the toxin is destroyed at 80°c after 30 min-of great importance to the canning industry-and there are at least seven antigenically distinct serotypes (a-g) produced by different strains of bacteria but which have a pharmacologically similar mode of action. it is absorbed from the intestine and acts on the peripheral nervous system, interfering with the release of acetylcholine at cholinergic synapses of neuromuscular junctions. somewhere between 12 and 36 hours after ingestion there are clinical signs suggesting an acute neurological disorder, with vertigo, cranial nerve palsies and finally death a few days later with respiratory failure. 1 * botulus (latin) = sausage. in 1793 a large sausage was eaten by 13 people in wildbad in germany; all became ill, and six died. the disease was subsequently referred to as botulism. t in recent years a less typical form of botulism has been described in small infants. the spores, present in honey applied to rubber teats, appear to colonise the gut, so that the toxin is produced in vivo after ingestion. tetanus and botulinum toxins are two of the most toxic substances known to man: 1 g of botulinum toxin will kill 10 10 mice. a great deal is now known about the genetics of both these toxins. tetanus toxin and botulinum toxin type g are encoded by plasmids, botulinum toxins types c and d are encoded by phages that infect clostridia, and botulinum type a is encoded in the bacterial genome; the genes have been sequenced. both toxins are synthesised as single peptides and are released from bacteria upon lysis. they are proteolytically activated by endogenous proteases to yield dichain derivatives consisting of -s -s -linked l (m r 50 000) and h (m r 100 000) chains. the recently adopted nomenclature reflects our current understanding of their peptide structures ( fig. 8.8 ). very recently, huge strides have been made in our knowledge of the mode of action of these toxins ( fig. 8.9 ). there is very little overall sequence homology between bonts and tetx; this could account for their immunological characteristics and the animal types normally affected. however, there is one structural feature common to all these neurotoxins located in the middle of the light chain and that is a zincbinding motif. in fact these neurotoxins are zinc endopeptidases. their targets are protein constituents of the synaptic fusion machinery responsible for the exocytotic release of neurotransmitters. the intracellular target cleaved by bonts b, d, and f and tetx is synaptobrevin (vamp, vesicle-associated membrane protein) present in small synaptic vesicles. bonts a and e cleave snap-25 (synaptosomal associated protein of 25 000m r ) a highly conserved protein known to be involved in exocytosis. bont c acts on syntaxin, a synaptic membrane protein also involved in exocytosis. however, some puzzles remain to be resolved. tetanus and botulinum toxins enter neuronal tissue preferentially at motoneuronal endplates, but the nature of the 'physiologically relevant ectoacceptors' is still not known. it remains unclear why botulinum toxin acts directly at the site of uptake and not, as observed with tetanus toxin, in the central nervous system, although a considerable amount of botulinum toxin (like tetanus toxin) is retrogradely transported. botulinum toxin blocks the release of acetyl choline at neuromuscular junctions to cause flaccid paralysis. likewise, one might ask why tetanus toxin fails to act at the motoneuronal junction at concentrations which would completely block release of neurotransmitter from gabaergic synapses. to reach inhibitory interneurons-its principal site of action-tetanus toxin must leave α-motoneurons after the primary uptake step, traverse the synaptic cleft to interneurons, leave those again in order to become finally internalised again from presynaptic membranes-a route identical to that of several neurotropic viruses. it acts by blocking the release of inhibitory transmitters (glycine or gab a) resulting in a failure to relax the affected muscle-pathophysiological 'tetanus'. only in rare cases does it act peripherally like botulinum toxin. the most toxic substances known to man are now being used as therapeutic agents to treat focal dystonias such as neck twists and eye squints, or eyelid closure and, more recently, some childhood palsies. the preparations are made from bont a and consist of toxin-haemagglutinin complex-the form in which the toxin is usually produced by the organism. this complex is less toxic than purified neurotoxin when administered parenterally but relatively more toxic when given orally; the haemagglutinin apparently protects the neurotoxin from proteolytic degradation in the gut. the effects of bont in relieving muscle spasms are not permanent but last for several months. treatment has to be repeated. to date the successes significantly outweigh the failures and no long-term adverse effects have been reported. antibody to the toxin has not so far been detected in the sera of the majority of patients. some toxins destroy membranes by virtue of their proteolytic activities, and some by their ability to degrade lipid components, while others are pore-forming or detergent-like in their mode of action. in addition to their action on protein components of lung connective tissue referred to above, pseudomonas aeragmosa-elastase and the zinc metalloprotease of legionella pneumophila are believed to destroy cell membranes by their proteolytic activity. this is the probable reason for the haemorrhage associated with lung infections caused by these pathogens, i.e. effects on type i alveolar epithelial and endothelial cells. clostridium perfringens α-toxin. a large number of bacterial enzymes are phospholipases some of which, but by no means all, are important toxins. the best example is the α-toxin of clostridium perfringens, the organism most commonly associated with gas gangrene. it is strictly anaerobic and occurs as a normal inhabitant in the large intestines of man and animals; its spores are ubiquitous in soil, dust and air. c. perfringens does not multiply in healthy tissues, but grows rapidly when it reaches devitalised and therefore anaerobic tissues. this could be after contamination of a natural wound with soil or dust, particularly on battlefields or in automobile accidents, or after contamination of a surgical operation site with clostridia from the patient's own bowels or skin. after abortions, particularly in the old days before antibiotics, intestinal clostridia often gained access to necrotic or devitalised tissues in the uterus and set up life-threatening infections. invasion of the blood was common and soon resulted in death, the clostridia localising and growing in internal organs such as the liver after death. c. perfringens has various enzymes that enable it to break down connective tissue materials, including collagen and hyaluronidase, thereby facilitating spread of the infection along tissue planes. most of the enzymes are toxic (a) reflex arc (top). mechanism for inhibiting the antagonists to a muscle contracting in response to stretch. muscles are reciprocally innervated with sensory and motor neurons, although for clarity this is shown only for the protagonist muscle. on stretch, the stretch receptors generate an impulse which is transmitted along the afferent sensory (s) neuron of the protagonist (p) muscle. this sp neuron enters the spinal cord by the dorsal root and synapses with the motor neuron supplying the protagonist muscle (mp) and with an interneuron (i) which in turn synapses with the motor neuron supplying the antagonist muscle (ma); the efferent motor neurons leave the spinal cord by the ventral root. at the sp/mp synapse an excitatory transmitter is released which induces an impulse in mp which leads to contraction of protagonist muscle. however, excitation of i causes release of an inhibitory transmitter at the i/ma synapse which leads to relaxation of the antagonist muscle. note that the basic reflex arc has been shown for simplicity but tetx acts mainly on voluntary muscles. (b) a simplified version of the biochemical events occurring in synapses (lower left). excitatory and inhibitory synapses, neurotransmitter release and action. gly, glycine; r, receptors of neuro transmitters; x, hitherto uncharacterised (candidates include glutamate, dopamine, atp, substance p, and somatostatin). to host cells and tissues, but α-toxin is easily the most important one. it is dermonecrotic, haemolytic (a feature seen mainly in tissues close to the focus of infection but sometimes responsible for large-scale intravascular haemolysis in infected patients), causes turbidity in lipoprotein-rich solutions and is lethal. while it is still true that these activities are all due to one molecular species, they are not different expressions of the one enzymic activity. historically, c. perfringens α-toxin was the first bacterial toxin to be characterised as an enzyme: it is a phospholipase c (plc) which removes the head group, phosphoryl choline, from phosphatidyl choline and from sphingomyelin. it is of undoubted importance in gas gangrene. toxoid prepared by formalin-treated toxin will protect sheep against infection caused by c. perfringens. however, one might ask why all enzymes with such biochemical specificity are not equally toxic or important in determining virulence; there are several reasons which can be put forward. by the combined use of monoclonal antibodies and molecular genetic analyses we now know that there are at least two functional domains in this c. perfringens α-toxin. if one compares c. perfringens α-toxin with the phosphatidyl choline-preferring, nontoxic phospholipase of bacillus cereus, one finds that two-thirds of the n-terminal sequence shows homology with the entire sequence of b. cereus plc. this portion of c. perfringens α-toxin retains its plc activity but not its haemolytic and lethal activities. the c-terminal part is not haemolytic, not enzymatically active and not cytotoxic for mouse lymphocytes, but is necessary for conferring toxicity on the n-terminal part of the protein. in fact, the c-terminus is a potent immunogen that will solidly protect mice -and hopefully man-against experimental infection with c. perfringens. surprisingly, the c-terminal domain of the nontoxic c. bifermentans enzyme shows sequence similarity with that of its c. perfringens α-toxin counterpart. the nontoxicity of this enzyme is ascribed to its comparatively much lower turnover rate, i.e. it is a much less efficient enzyme. recently, attention has been drawn to the distinct possibility that while haemolysis may be initially induced by the plc activity of c. perfringens α-toxin, it is possible that actual disruption of the membrane is due to the activation by the toxin of other phospholipases (phosphatidylinosotol-4,5-biphosphate (pip2) specific phospholipase and phospholipase d) present in the erythrocyte membrane. some now believe (c) sites of neurotoxin action (lower right). the predominant site of action of tetx is the intermotor neuron synapse; the exocytotic machine is interfered with by the endopeptidase action of tetx on vamp. bont acts at the neuromuscular junction inhibiting the release of acetyl choline (ach) by its proteolytic action on vamp (types b, d and f), or snap (types a and e), or syntaxin (type c). (amplified from figs 18 and 19, 'bacterial toxins', 2nd edition, by j. stephen and r. a. pietrowski (1986), pp. 60 and 62, van nostrand reinhold (uk).) that the basis of toxicity is not simply a cytolytic one, but rather a consequence of its ability, in sublytic doses, to release inosotol triphosphate (ip3) and activate the arachidonic acid cascade. there are other pathogenic clostridia that cause gas gangrene and produce similar toxins. infected tissues show inflammation, oedema and necrosis, not necessarily with the formation of gas, and the illness can be mild or very severe according to the extent of bacterial spread and the nature and quantity of toxins that are formed and absorbed. since the bacteria grow and produce their toxins only in devitalised tissues, the most important form of treatment is to remove such tissues. clostridia are strictly anaerobic, and exposure of the patient to hyperbaric oxygen (pure oxygen at 2-3 atmospheres in a pressure chamber) has been found useful in addition to chemotherapy. staphylococcal ß-toxin. staphylococcal ß-haemolysin is known to be produced in vivo. in ch. 4 studies with isogenic mutants were described which indicate that it is important in killing neutrophils. it probably has the narrowest substrate specificity among the phospholipases, and is a hotr-cold haemolysin: lysis of erythrocytes occurs only on cooling after incubation at 37°c. the phenomenon, though of doubtful significance in vivo, has attracted attention and generated speculation about its mechanism. perhaps the most likely explanation is that, when cooled below their phase-transition temperature, the remaining phospholipids undergo quasi-crystalline formation, thereby generating intramembranous stresses incompatible with structural integrity. these proteins (made by some 19 species, not all of which are pathogens) have been called oxygen-labile haemolysins because they are reversibly inactivated on standing in air, and their most studied property is their ability to lyse red cells. sh-activation of crude preparations is necessary for the expression of haemolytic activity but they are also active towards a variety of cell types. thiol-activated cytolysins share the following properties: they are cross-neutralised by hyperimmune sera, lyse a wide range of species of erythrocyte, exhibit similar ph and temperature optima for cytolytic activity, are lethal and cardiotoxic, and lose activity on incubation with erythrocyte ghosts. they are inactivated irreversibly by small amounts of cholesterol. interaction with cholesterol is the key primary event in their interaction with susceptible membranes which leads to the impairment of the latter; cholesterol plays no further part in the subsequent damage process. a schematic outline of how they damage cell membranes is shown in fig. 8.10 . several facts have recently reopened considerable interest in this group. first, the recognition of what these substances do to host defence cells when presented in sublethal doses. for example, pneumolysin from s. pneumoniae inhibits the respiratory burst in neutrophils, inhibits antibody synthesis in b cells and activates the classical complement pathway in the absence of antibodies. since complement is an important defence mechanism against the pneumococcus in vivo this could lead to depletion of complement levels and abrogate protection. in fact, immunisation of mice against pneumolysin affords some protection against challenge infection. second, in addition to its role in promoting invasive bacteraemia, pneumolysin has also been implicated in causing sensorineural deafness associated with meningitis caused by the pneumococcus; this perception is based on very recent work with a guinea-pig other pore-forming toxins t h e r t x toxins h a v e a l r e a d y b e e n referred to above in connection w i t h b. pertussis a c -h l y . b u t t h e r e a r e o t h e r pore-forming toxins. staphylococcal δ-toxin. this toxin acts in a manner similar to that of the sh-activated cytolysins, with an important difference: there is no initial specific binding. δ-toxin initially forms small pores and then islands of membrane or large micelles; this gives rise to its perceived detergent-like properties. there is a family of closely related δ-toxins which inhibit the growth of gonococci. it is not often that one can ascribe a positive function to a toxin which is beneficial for the organism producing it. in this case 8-toxin(s) could have important ecological significance in the mixed culture situation that is characteristic of the real microbial world. of great interest is the synergy that δ-toxin displays. sublytic amounts of δ-toxin causes release of cell constituents without lysis. but, only 0.01 haemolytic units of staphylococcal ß-toxin will cause lysis of cells in the presence of 0.004 lytic units of δ-toxin. this synergistic interaction could be the way in which staphylococcal toxins, which rarely exert their lethal effects in the majority of infections, exercise important cytolytic effects. of less obvious significance is the fact that δ-toxin is a poor antigen; for a long time its antigenicity was controversial. if δ-toxin were to prove of crucial importance as a cytolytic potentiator, then this could also partly explain why natural acquired immunity to staphylococcal infection is either non-existent or sufficiently low as to be easily overcome. staphylococcal a-and 7-toxins. staphylococci produce a range of toxins several of which we have already met. the α-toxin is considered as the main cytolysin produced by s. aureus. like streptolysin-0 and staphylococcal δ-toxin, it is secreted as a water-soluble protein and undergoes self-induced oligomerisation on cell membranes to form pores. it has recently been suggested that transmembrane channels formed by the hexameric form allow the penetration of the monomeric form which interferes with certain steps in translation. in systemic staphylococcal infections death is most probably due to the potent α-toxin but in localised pyogenic infections-such as mastitis in cattle, goats, rabbits and mice-its role is most likely one of killing phagocytes or conferring survivability on intracellular bacteria. 7-toxin is a binary toxin with a haemolytic action on rabbit erythrocytes. there is evidence that α, β and 7 haemolysins and leucocidin are produced in vivo during natural and experimental infections. this is based on the presence of antibodies to these haemolysins in subjects undergoing staphylococcal infection and the extraction of toxin from peritoneal abscesses in mice. however, the precise roles that these or any other staphylococcal toxins play in localised infections is not clear. atrophie rhinitis is a disease of pigs in which the principal clinical sign is atrophy of the nasal turbinate bones and shortening of the snout. scientists have isolated and purified a toxin believed to be important in the causation of this disease. this is a remarkable toxin on at least two counts. it shows an incredible predilection for the target tissue in that it causes turbinate atrophy in gnotobiotic pigs, even when injected intraperitoneally; it also causes necrosis of spleen and thymus. perhaps even more surprising is the fact that it is a potent mitogen, hardly the kind of activity that one would associate with atrophy! one can only assume that it upsets the dynamic balance between the laying down and resorption of mesenchymal bone tissue by osteoblasts and osteoclasts respectively. the remainder of this section deals with other important (or potentially important) toxins which have not already been referred to in earlier chapters or in the section above. several staphylococcal toxins have already been referred to above. staphylococci are ubiquitous and responsible for a large group of diseases which range from the relatively harmless skin pimple through abscesses, impetigo, food poisoning, osteomyelitis, mastitis, primary pneumonia, staphylococcal scalded skin syndrome (ssss) and toxic shock syndrome (tss) to fatal septicaemias. in the case of ssss a toxin is known and is described on p. 208. toxic shock syndrome toxin (tsst-1). tss is seen characteristically in menstruating women whose tampons harbour multiplying staphylococci. it is due to a toxin called toxic shock syndrome toxin 1 (tsst-1; one of the so-called staphylococcal enterotoxins, see next paragraph). toxic shock syndrome is characterised by sudden onset of fever, vomiting, diarrhoea, an erythematous rash followed by peeling of the skin, hypotensive shock, impairment of renal and hepatic functions and occasionally death. the main symptoms of the disease have been reproduced in rabbits by implanting chamber-enclosed tss-strains in the rabbit uterus or peritoneum or by injection of tsst-1 into rabbits. complex changes are observed including: haemorrhage in kidney and liver; congestion and haematomas in the lungs; leakage of blood into the thymus; and fluid in the pericardial sac and in the gut lumen. these effects in rabbits are very similar to those seen in humans and would certainly explain the shock and diarrhoeal syndrome so characteristic of the disease. all of this indicates that the primary effect of tsst-1 is on the integrity of capillary walls. the lethal effect of tsst-1 is enhanced considerably by endotoxin (see below). such synergy is also known for streptococcal erythrogenic toxin and lps, but the mechanism(s) involved in this complex synergy is not clear. although relatively uncommon in the uk, staphylococcal food poisoning accounts for over 40% of all cases of food poisoning in the united states. the disease is characterised by vomiting and diarrhoea commencing 1-6 h after consumption of contaminated food, especially dairy produce. symptoms usually last no longer than 24 h. like botulism, staphylococcal food poisoning is commonly caused by the ingestion of food containing preformed toxins, known collectively as the staphylococcal enterotoxins. they are serotypically heterogeneous single-chain globular proteins, of molecular weight between 28 and 35 kda, secreted by certain strains of s. aureus. one of these, serotype f is identical to tsst-1 and staphylococcal pyrogenic exotoxin c. these toxins are not classical enterotoxins acting on intestinal cells but 'neurotoxins' activating receptors on the abdominal viscera, the stimulus reaching the vomiting centre via the vagus nerve. several of the staphylococcal toxins referred to above (tss-1 and enterotoxins), and also the erythrogenic toxins a and c of streptococcus pyogenes (see below) have an additional important action. they are among the most powerful t cell mitogens known, acting at picomolar concentrations. they bind to mhc class ii molecules on antigenpresenting cells and interact with the vß chain of the t cell receptor (see chs 6 and 7). this causes proliferation and cytokine release by the entire subset of t cells bearing that particular vß chain-2-20% of all t cells are affected, whereas only 0.001-0.01% of t cells respond to a given regular antigen. this represents an important interference with a coordinated immune response, and the widespread polyclonal activation and cytokine release can be regarded as a microbial strategy, a 'diversion' of host immune defences. in addition, if the superantigen reacts with developing t cells (with the correct vß chain) in the thymus, these cells are deleted. it seems probable, therefore, that this is a more important biological function of these toxins than the one responsible for the characteristic disease, which may be no more than an 'accidental' phenomenon. it turns out that similar molecules are formed by mycoplasma and by certain retroviruses (e.g. the mis antigen of mouse mammary tumour virus). scarlet fever is one of the many conditions caused by streptococcal infection and may accompany a streptococcal sore throat or occasionally a streptococcal wound infection. there is a generalised erythematous rash together with fever and a sore throat. the rash is due to an erythrogenic toxin produced by certain strains of streptococcus pyogenes. erythrogenic toxin is a low molecular weight protein produced in a complex form with hyaluronic acid, which acts as a carrier. the protein consists of two parts. one is heat labile, carries the determinants of immunological specificity which give rise to three serotypes, a, b and c, and is responsible for primary toxicity manifestations, including pyrogenicity, low lethality, cytotoxic effects on cultured spleen macrophages, and suppression of the reticuloendothelial and immune systems. the second part is heat stable, antigenically common to types a, b and c, and is responsible for secondary toxicity; that is, hypersensitivity effects, including skin hyperreactivity (the basis of the rash), myocardial necrosis, enhancement of pyrogenicity and lethality, and enhanced host response to other injurious agents. thus, it is not possible to define the biological activity of 'erythrogenic' toxins without considering the immunological state of the host. an individual may suffer no reddening of the skin upon intradermal injection of erythrogenic toxin (negative dick test) because a high neutralising titre of antibody blocks the primary toxiphore or because of a lack of hypersensitivity to the common antigen. streptolysins o and s are dealt with in ch. 4. there is a plethora of toxins produced by numerous clostridial pathogens important in both human and veterinary medicine. the clostridial genus comprises a large number of toxigenic species, some of which are known to produce several toxins, extracellular enzymes, and other factors which are as yet recognised only by a letter of the greek alphabet. all the α-toxins are dermonecrotic and the others have haemolytic, enzymatic properties. toxin production in vitro is used as the basis of typing clostridia, and the picture is complex. no attempt will be made to describe every disease in man or animals associated with clostridia; only those are selected which best serve to illustrate the involvement of some recognisable toxins. in the case of sheep diseases-lamb dysentery, struck, enterotoxaemia, black disease, braxy, black quarter-some of the best evidence for implicating relevant toxins comes from field studies using multivalent vaccines (based on toxoids of these toxins). clostridium perfringens type c causes pig bei in man, essentially due to the production of ß-toxin. this is a rare disease in developed societies but a public health hazard in papua new guinea. four factors are responsible: the ubiquity of c. perfringens type c in the soil and faeces of man and pigs; the relatively low immunogenicity of ß-toxoids in young children, the group most at risk; the high carbohydrate, low protein nature of the staple diet; and the sporadic consumption of large quantities of pork on occasions of celebration. the latter dietary change promotes a proliferation of clostridia in the intestine which may lead to intestinal gangrene and death. ß-toxin damages the mucosa, reduces mobility of villi and causes more bacteria to become attached to the villi. more toxin is absorbed and the mucosa and underlying the intestinal wall become necrotic, leading to death in many cases. the influence of diet is additionally important in t h a t low protein diets cause decreased secretion of pancreatic proteolytic enzymes and sweet potato contains a trypsin inhibitor. these conditions promote the survival of ß-toxin which is highly sensitive to proteolytic inactivation. immunisation with ß-toxoid preparations has dramatically lowered the incidence of this fatal disease in children. gas gangrene in man may be caused by several bacterial species separately or in concert. these include clostridium perfringens type a, c. novyi types a and b, and c. septicum; c. perfringens and its a-toxin have already been discussed. far less is known about c. novyi and c. septicum and their toxins in gas gangrene in man. much more is known about the role these organisms play in diseases of animals, and multivalent vaccines confer a very high degree of immunity (particularly to sheep) against several clinically identifiable but separate diseases. a few of these diseases are described below. clostridium perfringens type b causes lamb dysentery. this is an acute, fatal disease of young lambs occurring during the first week of life and caused by absorption of toxin(s) generated by c. perfringens type b in the small intestine. clostridium perfringens type c causes struck in sheep, a disease occurring in the romney marshes of kent, but rare in other areas in the world. the pathological changes observed differ markedly from other enterotoxaemias and include enteritis. clostridium perfringens type d enterotoxaemia in sheep is another acute fatal disease. the most constant lesion is subendocardial haemorrhage around the mitral valve. c. perfringens type d e-toxin or its protoxin are recoverable from intestinal contents. clostridium novyi type b causes black disease of sheep or infectious necrotic hepatitis. this is an acute infectious disease of sheep (occasionally cattle) caused by the absorption of the α-toxin elaborated by the organism in necrotic foci in the liver, and is nearly always associated with invasion of the liver by immature liver flukes. how c novyi gets to the liver in the first place is not known but it is readily demonstrable in livers of normal sheep in areas where the disease is prevalent. experimental reproduction in guinea-pigs of a similar disease is possible by the combined action of c. novyi spores and liver fluke infestation. clostridium novyi type d causes a rapidly fatal disease in cattle (redwater disease) similar to, and regarded by some as an atypical manifestation of, black disease. the characteristic lesions include jaundice, various haemorrhagic manifestations, and anaemic infarcts in the liver; active liver fluke infestation may or may not be present. in culture this organism produces ß-toxin, which explains the haemoglobinuria, but no a-toxin. clostridium septicum causes braxy in sheep. the role of c. septicum in this acute, fatal disease is assumed because of its association with the characteristic haemorrhagic inflammatory lesion in the abomasum. the disease has not been reproduced experimentally with c. septicum but can be prevented by immunisation with sterile toxoids derived from this organism. clostridium chauvoei causes black quarter in sheep and cattle. this is a gas gangrene type infection of muscles and associated connective tissues in cattle and sheep; c. chauvoei is also the causative agent of parturient gas gangrene in sheep. the initial stimulus which activates the infection in cattle is not known, since the disease is hardly ever associated with any overt wounding. washed spores alone do not cause disease when injected, but do in conjunction with a tissue-necrotising agent. in sheep, wounding caused by parturition, castration, tailing, shearing, vaccination, as well as accidental damage will create a focus within which c. chauvoei can multiply. there are numerous examples now known of synergistic reactions between toxins of the same or different species. bacillus cereus makes a phosphatidyl choline-preferring phospholipase and a sphingomyelinase which are separately nontoxic; in concert they are haemolytic and termed cereolysin a-b. the examples of staphylococcal a-and δ-toxins and of staphylococcal tsst-1 and endotoxin, have already been alluded to above. streptococcal erythrogenic toxins increase sensitivity to other streptococcal factors (for example, streptolysin-o) and also to gramnegative endotoxin. the susceptibility of rabbits to endotoxin is increased 100 000 times, and adult cynomolgus monkeys die within 24 h, when injection of low levels of steptococcal toxin is followed 3 h later by an otherwise sublethal dose of endotoxin. this may be because streptococcal toxin creates a state of hypersusceptibility to a wide variety of stressful agents. perhaps, in view of the high incidence of exposure of man and his domestic animals to streptococci, we should actively investigate possible toxin-mediated synergy in mixed infections involving streptococci. another entirely different type of example is that of the increase in toxicities of staphylococcal a-and 7-toxins, diphtheria toxin and endotoxin for neonatal ferrets preinfected with influenza virus. increases were 14-, 3-, 219-and 84-fold respectively. no increase in viral replication was observed. neonates died suddenly without clinical symptoms as in human babies dying from sudden infant death syndrome (sids). pathological examination showed inflammation of the upper respiratory tract, lung oedema and collapse, and early bronchopneumonia in animals receiving the dual challenge but not those receiving either toxin or virus on their own. thus, some bacterial toxins in conjunction with influenza virus could be one of the several causes of sids. many fungi contain substances that are harmful when taken by mouth, and there are two diseases that result from the ingestion of food containing preformed fungal toxins. as with c. botulinum, the disease is caused without the need for infection. aspergillus flavus infects ground nuts (monkey nuts) and produces a very powerful toxin (aflatoxin). contaminated (badly stored) ground nuts used to prepare animal feeds caused the death of thousands of turkeys and pigs in the uk in 1960 and the survivors of intoxication nearly all developed liver cancer. human disease has not yet been associated with this toxin. clavicepspurpurae is a rust fungus affecting rye, and it produces toxins (ergotamine especially) that give rise to ergot poisoning when contaminated grain is eaten. mushrooms and toadstools have long been recognised as sources of poisons and hallucinogens. unlike the toxins already discussed in this chapter, there is a group of toxins which are distinct structural components and are not released into the surrounding medium in any quantity except upon death and lysis of the bacteria. several protein toxins (e.g. clostridium difficile toxin a, tetanus toxin) are released during the decline phase of batch culture-probably on autolysis-and these are classified as exotoxins. here we deal with toxins which are known to comprise well-recognised structural entities which on a priori grounds must have key functions in the organism: they are found in the outer membranes of gram-negative organisms. there are two chemically distinct types of toxin considered: lipopolysaccharide (endotoxin; lps) and protein. the bulk of this section is taken up with endotoxin. many pathogenic organisms, however, are pathogenic by virtue of possessing various types of surface structure important in conferring virulence. these include, for example, adhesins which are important in colonising body surfaces or a variety of surface molecules (which may or may not be inside capsules) which render them resistant to phagocytosis. but the majority of adhesins and anti-phagocytic determinants are themselves nontoxic. the gram-negative bacterial cell wall is subject to considerable variations in both the composition of lps and in the number and nature of the proteins found in the outer cell membrane. apart from the examples given in ch. 7 in relation to the gonococcus, such phenotypic variation in lps has rarely been examined in the context of pathogenicity. however, the examination of cell-bound proteins of yersinia pestis from organisms grown in vivo led to the discovery of a toxin lethal for mice and guinea-pigs. plague is one of the most deadly diseases of man and has, over several thousands of years, claimed millions of lives. in the fourteenth century, 'the black death' wiped out a quarter of the population of europe before spreading through the middle east and asia. fortunately, however, the last 60 years or so have seen a drastic decrease in outbreaks of plague, though the threat of another epidemic is still with us. the causative organism of plague, yersinia pestis, is primarily a parasite of rodents in which it is endemic in many areas of the world. only when man comes into close proximity with infected rodents do outbreaks of human plague occur. the disease is spread from rat to rat and from rat to man by fleas. rodents in the terminal stages of infection with y. pestis suffer massive bacteraemia and so when a flea sucks the blood of an infected animal it swallows large numbers of bacteria. when the rodent eventually dies, the flea leaves the corpse and awaits a new host. in the meantime, however, y. pestis multiplies rapidly in the alimentary tract of the insect, often completely blocking the proventriculus. the flea becomes voraciously hungry and feeds on any suitable host, which sooner or later is man. however, because the flea's alimentary canal is blocked by bacteria it cannot feed efficiently and usually succeeds in imbibing blood only to regurgitate it, now contaminated with y. pestis, back into the wound. during the next 2-8 days, organisms become localised and multiply in the regional lymph nodes, causing considerable swelling. these swellings, which are often in the axillary and inguinal regions because the most common sites for flea-bites are the arms or legs, are referred to as primary buboes (greek boubon = groin); hence the name bubonic plague. secondary buboes may develop, followed by necrosis of the lymphoid tissue and vascular damage giving rise to haemorrhaging in many organs and tissues. these pathological changes are accompanied by prostration, high fever and delirium, followed in the terminal stages of the disease by shock and death. it is also possible for the disease to be transmitted from human to human by the aerosol route: exhaled organisms from heavily infected patients are capable of infecting others giving rise to pneumonic plague. the principal features of human plague can be reproduced in guineapigs and mice. monkeys show shock-like signs only during the terminal period of 6-10 h, when they become quiet, progressively weak, prostrate and hypothermic; for the previous 2-4 days infected animals are lively and vigorous. in the terminal stages blood pressure drops rapidly but there is no evidence of oligaemia (low blood volume) caused by haemorrhage, or of oedema, suggesting that vascular collapse must be associated with a vasodilatory factor(s), resulting in pooling of blood. in this respect monkeys differ from humans and guinea-pigs. the symptoms of plague-high fever and vascular damage -are characteristic of intoxication with endotoxin. however, it is extremely unlikely that endotoxin alone is the main toxin involved in plague. it is much more likely to act in conjunction with one or more other potentially toxic fractions from y. pestis. plague murine toxin is a protein which, although highly lethal for mice and rats, is relatively nontoxic for guinea-pigs, rabbits, dogs and monkeys. a completely separate guinea-pig toxin complex exists comprising at least two cell wall/membrane protein components, one of which will kill mice, although both are needed to kill guinea-pigs. however, the nature of the toxin or toxins of y. pestis and their role in the human disease syndrome are still far from clear. the classical approach to such a problem is to prepare a specific toxoid and to determine whether injection of this confers immunity to the disease. needless to say, the severity of human plague renders such experiments impossible. until such questions can be answered, we are left to argue whether, in the context of plague, man resembles a mouse, a monkey or a guinea-pig. endoxotins are part of the outer membrane of gram-negative bacteria. it has been known for many years that the cells (alive or dead) or cell extracts of a wide variety of gram-negative bacteria are toxic to man and animals. the literature on this subject is vast, sometimes confusing and often controversial; here we can give no more than a brief outline. some of the diseases in which endotoxin may play an important role include typhoid fever, tularaemia, plague and brucellosis, and a variety of hospital-acquired infections caused by opportunistic gram-negative pathogens which include escherichia coli, proteus, pseudomonas aeruginosa, enterobacter, serratia and klebsiella. in addition, endotoxin has been intensively studied as a possible causative agent of shock arising from post-operative sepsis or other forms of traumatic injury in which the normal flora of the gut is often the source of endotoxin. the toxins we have considered so far have been protein (or at least part protein) in nature but, in contrast, endotoxin is a complex lipopolysaccharide. it is also much more heat stable than protein toxins and much less easily toxoided. in addition to lethality, endotoxin displays a bewildering array of biological effects. the complex nature of the multi-layered gram-negative bacterial envelope is shown in fig. 8 .12 (see also fig. 4.3) . the outer membrane is composed of a bimolecular leaflet arrangement as are other membranes but has a different composition from the cytoplasmic membrane. the lipopolysaccharide (lps) is unique in nature, only found in gramnegative bacteria, and is, or contains within it, what we designate endotoxin. immunoelectron microscopy indicates that lps exists in the outer leaflet of the membrane and extends outward up to 300 nm; it is on, rather than in, the cell. thus it is evident that the term endotoxin is a misnomer which derives from the era when toxins were considered to be either exotoxins, which were synthesised and secreted by the viable organism, or endotoxins, which were intracellular and released only upon lysis. moreover, extraction with edta shows that approximately 50% of lps is held noncovalently linked in the membrane. extraction with a variety of different solvents yields material which is highly heterogenous and of apparent molecular weight 1-20 x 10 6 . however, treatment with pyridine or addition of detergents reduces the polydispersity. the endotoxic glycolipid from the rough mutant of salmonella minnesota, r 595, has an m r of 5900 for the basic unit, from which complex aggregate structures are derived. lipopolysaccharide consists of three regions: polysaccharide side chains, core polysaccharide, and lipid a which consists of a di-glucosamine backbone to which long chain fatty acids are linked (fig. 8.12 ). the relationship of this type of molecule to the outer membrane is also shown in fig. 8 .12. the long chain fatty acids interdigitate between the phospholipids in the outer leaflet and may also be linked (or interact) with lipoproteins, which in turn may or may not be covalently anchored to the rigid peptidoglycan (pg). the polysaccharide side chains project outwards. this structure is not invariant. for example, many organisms when first isolated give rise to colonies with a smooth appearance on agar but on subculture produce colonies with a rough appearance. in general, 'smooth' strains of pathogenic species are more virulent than rough strains. this s->r conversion is accompanied by a loss of region i side chains, which contain the deoxy and dideoxy sugars found in these lps complexes. in addition to these somewhat drastic changes involving loss of side chains, it is possible to induce major compositional changes by manipulating the growth rate of these organisms in a chemostat. thus the lps of salmonella enteriditis, when grown with a mean generation time of 20 min is nearly totally deficient in tyvelose (a dideoxy sugar), possesses 85% of the galactose and 150% of the glucose contents of lps obtained when the generation time is 50 min. these genotypic s organisms exhibit an r-phenotype in terms of their vastly reduced o-agglutinability (see below); such observations are potentially very important in the context of the in vivo phenotype and pathogenicity, since it is well known that the growth rate of salmonella typhimurium in mice is 10-20 times lower than in vitro. examples have already been given in ch. 7 of changes in lps structures in vivo in relation to neisseria gonorrheae and neisseria meningitidis. the extent to which lipid a is common between different genera is uncertain, but it is not likely to vary tremendously. the core polysaccharide structure is the same or very similar within groups of the enterobacteriaciae: thus polysaccharides from salmonellae are similar to each other, but differ from those of e. coli strains. however, within a group such as the salmonellae, there is a wide variation in the composition and detailed structures of the side chains, a fact which is exploited in the kauffman-white scheme for classifying salmonellae, giving rise to several thousand serotypes. the side chains carry the o-somatic antigen specificities of which there are far more than can readily be accounted for on the basis of the known number of sugars involved in the basic repeating units. in the side chains are found a range of depxy and dideoxy sugars. the general principles governing the relationship between the various chemotypes and serotypes are now well understood; the multiplicity of antibody specificities evoked may be explained in terms of antibodies which can recognise different aspects of one three-dimensional structure. lipid a is the primary toxiphore, but the polysaccharide plays an important part in conferring solubility upon, and optimising the size of micellar aggregates of lps, hence affecting biological activity. however, the immune status of the test animal may affect toxicity: as normal animals produce antibodies to the antigenic determinants on the surface of normal gut organisms (including o-somatic antigens), some of the biological effects of endotoxin may be mediated by hypersensitivity mechanisms. the most powerful evidence that lipid a is the primary toxiphore comes from studies on smooth (s) and rough (r) mutants whose biosynthetic capabilities are blocked at various points. this established that neither the o-side chains, nor the core polysaccharide are necessary for endotoxicity. pure lipid a can be made toxic by complexing it to a hydrophilic carrier like bovine serum albumin. one can make synthetic lipid a preparations which are toxic and assume space-filling configurations, which make it easy to see how they could fit into and be part of a bimolecular leaflet arrangement in the outer membrane. the range of biological properties of endotoxin is quite bewildering and the mode(s) of action very complicated. included among those effects which might play a role in gram-negative bacterial infections, are abortion, pyrogenicity, tolerance (not immune tolerance), the schwartzmann phenomenon, hypotension and shock, and lethality, but the precise part played by lps in these phenomena in gram-negative infections is far from clear. lps causes the release of vasoactive substances, activates the alternative pathway of the complement cascade, and also activates factor xii (hageman factor), the first step of the coagulation cascade, which sometimes results in disseminated intravascular coagulation (p. 258). many, perhaps nearly all, the actions of lps are due to the stimulation of cytokine release from macrophages and other cells. there is an effect on the circulation, leading ultimately to vascular collapse. the vascular regions most affected differ from species to species; in man and sheep the main changes are found in the lungs. lps has powerful immunological actions, which is surely no accident; as well as activating the complement system, it induces il-1 production and is a potent b cell mitogen. man is one of the most sensitive of all species to the pyrogenic action of endotoxin. a dose of 2 n g per kg of body weight injected intravenously into man causes the release of an endogenous pyrogen (interleukin-1, see glossary) and tumour necrosis factor (tnf) from macrophages, which act on the hypothalamus to give an elevation of body temperature within an hour. it is possible that the pyrogenic action of lps helps to generate fever in gram-negative bacterial infections, but lps is not the only bacterial factor capable of inducing a febrile response. in spite of all these toxic actions, there have been suggestions that some of the responses to lps (by macrophages, polymorphs) could be advantageous to the host, possibly assisting in the recognition and destruction of bacteria. could it be that host responses to lps are, like the complement or the clotting systems, useful in moderation but harmful in excess? there are reports that when animals with less vigorous responses to lps are infected they suffer fewer symptoms, but permit greater growth of bacteria. very large numbers of gram-negative bacteria are normally present in the intestines (see ch. 2), their continued death and exit in the faeces being balanced by multiplication in the lumen. there is a continuous, inevitable low-grade absorption of endotoxin from the intestine.* absorbed (endogenous) endotoxin enters the portal circulation and is taken * in addition, various antigens are absorbed in small quantities from the intestine, and in normal individuals antibodies are formed against various food proteins and to some extent against resident intestinal bacteria (see ch. 2). kupffer cells remove any antigen-antibody complexes formed locally in the intestine and prevent them from entering the systemic circulation. up and degraded by reticuloendothelial cells, mainly kupffer cells in the liver. continuous exposure to endotoxin probably has profound effects on the immune system and on the histology of the intestinal mucosa, stimulating development of the immune system in the immature individual, but there are no obvious pathogenic consequences. normal people have low levels of antibody to endotoxin as a result of this continuous exposure. the sick individual may be much more susceptible to endogenous endotoxin, perhaps because of defects in removal by kupffer cells. after trauma or after genito-urinary instrumentation endotoxin is detectable in peripheral blood by the limulus test,* but this leads to no particular signs or symptoms. when large amounts of endotoxin enter the blood there are profound effects on blood vessels with peripheral vascular pooling, a drastic fall in blood pressure, collapse and sometimes death. thus, if enough endotoxin enters the blood during massive gramnegative bacterial sepsis, the vasomotor action of endotoxin becomes important and shock intervenes.! in experimental animals endotoxin also causes vasodilation and haemorrhage into the intestinal mucosa, and sometimes haemorrhage into the placenta with abortion, but these actions do not appear to be important in all gram-negative bacterial infections. to summarise, endotoxin, although studied so carefully and for so long, has not yet been shown to play a definitive role as a toxin in the pathogenesis of any infectious disease. however, there is a growing body of opinion that seeks to implicate endotoxin in the inflammatory response induced in meningitis caused by gram-negative bacteria. but, in spite of its effects on various host defence systems including polymorphs, lymphocytes, macrophages, complement, and on endothelial cells and platelets, its overall role in infection is still not clear. it can, however, cause shock when gram-negative bacteria invade the blood. it is for this reason that considerable effort in recent years has gone into the development of antilipid a antibodies for use as therapeutic agents to combat shock in such situations; the success rate is only partial and the expense enormous. for that reason several groups are seeking to exploit the wealth of chemical and biophysical information available on lps in attempts to develop synthetic derivatives which would neutralise the biological activity of lipid a. we await the outcome of such research. however, the characteristics of the o-antigen polysaccharide are sometimes important in determining virulence (pp. 241-2). * a sensitive test for endotoxin based on the ability of endotoxin to induce gelation of a lysate obtained from the blood cells of the horseshoe crab, limulus polyphemus. considerable space has been given to toxins because they are being intensively investigated as possible virulence determinants. the account illustrates the complexity of host-microbe interactions when analysed at the molecular level. most toxins are liberated from the microbial cell and can be studied with greater facility than many of the more elusive determinants of pathogenicity. but remember that microbes that replicate inside host cells are less likely to form powerful toxins because they cannot afford to damage at too early a stage the cell in which they are multiplying. thus, toxins are not prominent products in intracellular infections due to mycobacteria, brucella, rickettsiae, or chlamydia, and viruses do not form toxins. although a single molecule of a toxin like diphtheria toxin is enough to kill a cell, other toxins may do no more than impair cell function when present in sublethal concentrations. this can lead, for instance, to defective function in immune or phagocytic cells. low concentrations of the streptococcal streptolysins* will inhibit leucocyte chemotaxis. the ability to form toxins, whether encoded by plasmids or the microbial genome, is subject to selective forces. if toxin production puts a microorganism at a serious disadvantage it will tend to disappear. if it is advantageous it will be maintained, and will spread through the microbial population, just as the genetic changes that confer resistance to antimicrobial drugs are selected for when these drugs are widely used. it is therefore not unreasonable to ask how many of the well-known toxins are actually useful to the microbe as well as being important in causing disease in the host (table 8. 3). however, microbes that multiply extracellularly must produce a variety of enzymes and other molecules involved in nutrition, adherence to substrate, and so on. in the case of free-living microbes these substances, as well as substances that damage or interfere with competing organisms, are of major importance. probably they cannot all be discarded when the parasitic mode of life is adopted. many will have a toxic action. yet, for the infecting microbe, these substances remain as unfortunate necessities, of no particular advantage and perhaps a disadvantage, in the parasitic way of life. in infectious diseases there is nearly always a certain amount of direct microbial damage to host tissues, as discussed above. host cells are destroyed or blood vessels injured as a direct result of the action of * the streptolysins are responsible for ß-haemolysis. most haemolysins will kill phagocytes. ) . inflammatory materials are liberated from necrotic cells, whatever the cause of the necrosis. also many bacteria themselves liberate inflammatory products and certain viruses cause living infected cells to release inflammatory mediators. therefore it is not always clear how much of the inflammation is directly microbial rather than host in origin.* but inevitably the host (see ch. 3) generates inflammatory and other tissue responses, and these responses sometimes account for the greater part of the tissue changes. pathological changes can then be regarded as occurring indirectly as a result of these responses to the infection. inflammation causes redness, swelling, pain and sometimes loss of function of the affected part (see ch. 6) and is generally a major cause of the signs and symptoms of disease. indirect damage attributable to the host immune response is discussed separately below. in most diseases direct and indirect types of damage both make a contribution to pathological changes, but in a given disease one or the other may be the most important. in a staphylococcal abscess the bacteria produce inflammatory materials, but they also kill infiltrating polymorphs whose lysosomal enzymes are thereby liberated and induce further inflammation. this type of indirect nonimmunological damage is sometimes important in streptococcal infections. virulent streptococci produce various toxins that damage phagocytes, and also bear on their surfaces substances that impede phagocytosis (see ch. 4). nevertheless, with the help of antibody, all streptococci are eventually phagocytosed and killed and the infection terminated. unlike the staphylococci, however, killed group a streptococci pose a digestive problem for phagocytic cells. the peptidoglycan component of the streptococcal cell wall is very resistant to digestion by lysosomal enzymes. when streptococci are injected into the skin of a rabbit, for instance, streptococcal peptidoglycans persist in macrophages for as long as 146 days. hence macrophages laden with indigestible streptococcal cell walls tend to accumulate in sites of infection. lysosomal enzymes, including collagenase, leak from these macrophages, causing local destruction of collagen fibres and the connective tissue matrix. macrophages secrete many other substances some of which may contribute to cell and tissue damage (see also p. 84). many macrophages eventually die or form giant cells, sometimes giving rise to granulomatous lesions (see p. 292). in this way persistent streptococcal materials sometimes cause chronic inflammatory lesions in the infected host. an additional immunopathological contribution to the lesions is to be expected if the host is sensitised to peptidoglycan components. other pathogenic microorganisms that are digested with difficulty by phagocytes include listeria, shigella, candida albicans and, of course, mycobacteria, but the importance of this in the pathogenesis of disease is not generally clear. the expression of the immune response necessarily involves a certain amount of inflammation, cell infiltration, lymph node swelling, even tissue destruction, as described in ch. 6. such changes caused by the immune response are classed as immunopathological. sometimes they are very severe, leading to serious disease or death, but at other times they play a minimal part in the pathogenesis of disease. with the possible exception of certain vertically transmitted virus infections and the transmissible 'prion' dementias (see ch. 10), there are signs of an immune response in all infections. therefore it is to be expected that there will nearly always be some contribution of the immune response to pathological changes.* often the immunological contribution is small, but sometimes it forms a major part of the disease. for instance, in tuberculosis the pathological picture is dominated by the operation of a strong and persistent cmi response to the invading bacillus. in the classical tubercle a central zone of bacilli with large mononuclear and giant cells, often with some necrosis, is surrounded by fibroblasts and lymphocytes. mononuclear infiltrations, giant cells and granulomatous lesions (see p. 292) are characteristic pathological features of tuberculosis. there are no recognised toxins formed by tubercle bacilli, and there seems to be no single antigen or other component that accounts for virulence. bacterial glycolipids (e.g. 'cord factor'), resistance to h 2 0 2 (see pp. 80-81) and ability to utilise host fe (see p. 352) have been correlated with pathogenicity, and inhibition of phagosome-lysosome fusion in macrophages (see pp. 94-95) by release of unidentified bacterial components would also contribute to pathogenicity. however, none of these factors is by itself absolutely necessary for virulence, which in such a complex, ancient parasite is likely to be multifactorial. as a gene library for m. tuberculosis is slowly built up there will be opportunities for clearer definition of virulence determinants. when macrophages are killed by intracellular mycobacteria the lysosomal enzymes and other materials released from the degenerating cell contribute to chronic inflammation as in the case of the streptococcal lesions referred to above. * a number of different microbial antigens are produced during most infections (see ch. 6) and the possible immunological reactions are therefore numerous. for instance, at least 18 types of circulating malarial antigen are found in heavily infected individuals. the mere enlargement of lymphoid organs during infectious diseases is a morphological change that can often be regarded as pathological. the lymph node swelling seen in glandular fever, for instance, is an immunopathological feature of the disease, and the same can be said of the striking enlargement of the spleen caused by chronic malaria and other infections in the condition known as tropical splenomegaly. as often as not the relative importance of direct microbial damage as opposed to immune and nonimmune inflammatory reactions have not yet been determined, but the picture is clearer in most of the examples given below. in one important human disease, pathological changes are certainly immunopathological in nature, but not enough is known about it to classify the type of reaction (see table 8 .4). this disease is rheumatic fever, which follows group a streptococcal infections of the throat. it is the commonest form of heart disease in many developing countries. antibodies formed against a streptococcal cell wall or membrane component also react with the patient's heart muscle or valves, and myocarditis develops a few weeks later. many strains of streptococci have antigens that cross-react with the heart, and repeated infections with different streptococci cause recurrent attacks of rheumatic fever. there is genetic predisposition to the disease, based either on a particular antigen present in the heart of the patient or on a particular type of antibody response. chorea, a disease of the central nervous system, is a rare complication of streptococcal infection and antistreptococcal antibodies have been shown to react with neurons in the caudate and subthalamic nuclei of the brain. a number of microorganisms have antigens similar to host tissue components (p. 173) so that in the course of responding immunologically to such infections the host is vulnerable to autoimmune damage (see ankylosing spondylitis, p. 200). the antibodies to host components such as dna, igg, myofibrils, erythrocytes etc. that are seen in trypanosomiasis, mycoplasma pneumoniae, and eb virus infections appear to result from polyclonal activation of b cells (see p. 183). it is not clear how important these autoimmune responses are in pathogenesis, but they reflect fundamental disturbances in immunoregulation. four types of immunopathology can be distinguished according to the classification of allergic reactions by coombs and gell, and microbial immunopathology will be described under these headings (see table 8 .4). these depend on the reactions of antigens with reaginic (ige) antibodies attached to mast cells, resulting in the release of histamine, leukotrienes (see p. 68) and heparin from mast cells, and the activation of serotonin and plasma kinins. if the antigen-antibody interaction takes place on a large enough scale in the tissue, the histamine that is released can give rise to anaphylactic shock, the exact features depending on the sensitivity and particular reaction of the species of animal to histamine. guinea-pigs suffer from bronchospasm and asphyxia, and in man there are similar symptoms, sometimes with a fall in blood pressure and shock. this type of immunopathology, although accounting for anaphylactic reactions to horse serum or to penicillin, is not important in infectious diseases. when the antigen-ige antibody interaction takes place at the body surface there are local inflammatory events, giving rise to urticaria in the skin, and hayfever or asthma in the respiratory tract. this local type of anaphylaxis may play a part in the pathogenesis of virus infections of the upper respiratory tract (e.g. common cold, respiratory syncytial virus infections of infants), or in skin rashes in infectious diseases. type 1 reactions are common in helminth infections perhaps because ige antibodies have an important role in protection against these parasites. a dramatic type 1 reaction can follow rupture of a hydatid cyst of echinococcus granulosus (the dog tapeworm). slow leakage of worm antigens means that mast cells are sensitised with specific ige antibody, and the sudden release of antigen can cause life-threatening anaphylaxis. when the larvae of ascaris lumbricoides pass through the lung on their journey from blood to intestine, they can give rise to igemediated respiratory symptoms, with infiltration of eosinophils. reactions of this type occur when antibody combines with antigen on the surface of a tissue cell, activates the complement sequence or triggers cytotoxicity by k cells (nk cells or phagocytes with fc receptors). k (killer) cell cytolysis is referred to as antibody-dependent cellular cytotoxicity (adcc). the antibody-coated cell is destroyed. as discussed in ch. 6, the same reaction on the surface of a microorganism (e.g. enveloped virus) constitutes an important part of antimicrobial defences, often leading to the destruction of the microorganism. cells infected with viruses and bearing viral antigens on their surface are destroyed in a similar way. clearly the antibody-mediated destruction of infected cells means tissue damage, and it perhaps accounts for some of the liver necrosis in hepatitis b, for instance, and probably in yellow fever. infected cells can also be destroyed by sensitised lymphocytes or nk cells independently of antibody (see below). in certain infections antibodies are formed against host erythrocytes and these cells are particularly sensitive to lysis. the haemolysis in malaria is caused by antibodies to parasite-derived antigens that have attached to red cells, rather than by autoantibodies to red cells themselves. in pneumonia due to mycoplasma pneumoniae (atypical pneumonia), antibodies (cold agglutinins) are formed against normal human group o erythrocytes. haemolytic anaemia is occasionally seen, and there is reticulocytosis (see glossary) in 64% of patients. the lesions in the lungs are perhaps based on cell-mediated immunopathological reactions. the combination of antibody with antigen is an important event, initiating inflammatory phenomena that are inevitably involved in the expression of the immune response. in the infected host, these inflammatory phenomena are most of the time of great antimicrobial value (see ch. 6). but there are nevertheless immunopathological features of the infection, and immune complex reactions sometimes do a great deal of damage in the infected individual. the mechanisms by which antigenantibody reactions cause inflammation and tissue damage are outlined in fig. 8.13 . iga immune complexes are less harmful. antigens absorbed from the intestine can combine locally with iga antibody and the complex then enters the blood, to be filtered out in the liver and excreted harmlessly in bile (see p. 147). when the antigen-antibody reaction takes place in extravascular tissues, there is inflammation and oedema with infiltration of polymorphs. if soluble antigen is injected intradermally into an individual with large amounts of circulating igg antibody, the antigen-antibody reaction takes place in the walls of skin blood vessels, and causes an inflammatory response. the extravasating poly morphs degenerate and their lysosomal enzymes cause extensive vascular damage. this is the classical arthus response. antigen-antibody reactions in tissues are not usually as serious as this, and milder inflammatory sequelae are more common as in the case of allergic alveolitis (see below), or the red zone seen round the borders of a smallpox vaccination site after seven or eight days. in the latter example circulating antibodies pass through vessel walls, meet vaccinia virus antigen in the dermal tissues and an inflammatory response is generated. a similar mild response can be induced experimentally to cause a reaction known as cutaneous anaphylaxis (see glossary), the test antigen being injected into the skin and reacting with blood-borne antibody. the resulting inflammation is detected by the visible local leakage of plasma proteins from blood vessels, circulating plasma proteins having been coloured by the intravenous injection of evans' blue. when the antigen-antibody reaction takes place in the blood to give circulating immune complexes, the sequelae depend to a large extent on size and on the relative proportions of antigen and antibody. if there is a large excess of antibody, each antigen molecule is covered with antibody and is removed rapidly by reticuloendothelial cells, which have receptors for the fc portion of the antibody molecule (see ch. 4). when equal amounts of antigen and antibody combine, lattice structures are produced, and these form large aggregates whose size ensures that they are also rapidly removed by reticuloendothelial cells. if, however, complexes are formed in antigen excess, the poorly coated antigen molecules are not removed by reticuloendothelial cells. they continue to circulate in the blood and have the opportunity to localise in small blood vessels elsewhere in the body. the mechanism is not clear, but complexes are deposited in the glomeruli of the kidneys, the choroid plexuses, joints and ciliary body of the eye. factors may include local high blood pressure and turbulent flow (glomeruli), or filtering function of vessels involved (choroid plexus, ciliary body). in the glomeruli the complexes pass through the endothelial windows (fig. 8 .14) and come to lie beneath the basement membrane. the smallest-sized complexes pass through the basement membrane and seem to enter the urine. this is probably the normal mechanism of disposal of such complexes from the body. immune complexes are formed in many, perhaps most, acute infectious diseases. microbial antigens commonly circulate in the blood in viral, bacterial, fungal, protozoal, rickettsial etc. infections. when the immune response has been generated and the first trickle of specific antibody enters the blood, immune complexes are formed in antigen excess. this is generally a transitional stage soon giving rise to antibody excess, as more and more antibody enters the blood and the infection is terminated. sometimes the localisation of immune complexes and complement in kidney glomeruli is associated with a local inflammatory response.* there is an infiltration of polymorphs, swelling of the glomerular basement membrane, loss of albumin, even red blood cells, in the urine and the patient has acute glomerulonephritis. this is seen following streptococcal infections, mainly in children (see below). as complexes cease to be formed the changes are reversed, and complete recovery is the rule. repeated attacks or persistent deposition of complexes leads to irreversible damage, often with proliferation of epithelial cells following the seepage of fibrin into the urinary space. under certain circumstances complexes continue to be formed in the blood and deposited subendothelially for long periods. this happens in certain persistent microbial infections in which microbial antigens are continuously released into the blood but antibody responses are only minimal or of poor quality (see below). complexes are deposited in glomeruli over the course of weeks, months or even years. the normal mechanisms for removal are inadequate. the deposits, particularly larger complexes containing high molecular weight antigens or antibodies (igm) are held up at the basement membrane and accumulate in the subendothelial space together with the complement components. as deposition continues, they gradually move through to the mesangial space ( fig. 8.14) where they form larger aggregates. mesangial cells, * see also footnote p. 255; cells in kidney glomeruli, in joint synovium and in choroid plexuses bear fc or c3b receptors. this would favour localisation in these tissues. of cell and tissue damage one of whose functions is to deal with such materials, enlarge, multiply and extend into the subepithelial space. if these changes are gradual there are no inflammatory changes, but the structure of the basement membrane alters, allowing proteins to leak through into the urine. later the filtering function of the glomerulus becomes progressively impaired. in the first place the glomerular capillary is narrowed by the mesangial cell intrusion. also, the filtering area is itself blocked by the mesangial cell intrusion, by the accumulation of complexes (fig. 8.14) , and by alterations in the structure of the basement membrane. the foot processes of epithelial cells tend to fuse and further interfere with filtration. the pathological processes continue, some glomeruli ceasing to produce urine, and the individual has chronic glomerulonephritis. circulating immune complex deposition in joints leads to joint swelling and inflammation but in choroid plexuses there are no apparent pathological sequelae. circulating immune complexes are also deposited in the walls of small blood vessels in the skin and elsewhere, where they may induce inflammatory changes.* the prodromal rashes seen in exanthematous virus infections and in hepatitis b are probably caused in this way. if the vascular changes are more marked they give rise to the condition called erythema nodosum, in which there are tender red nodules in the skin, with deposits of antigen, antibody and complement in vessel walls. erythema nodosum is seen following streptococcal infections and during the treatment of patients with leprosy. when small arteries are severely affected, for instance in some patients with hepatitis b, this gives rise to periarteritis nodosa. immune complex glomerulonephritis occurs as an indirect immunopathological sequel to a variety of infections. first there are certain virus infections of animals. the antibodies formed in virus infections generally neutralise any free virus particles, thus terminating the infection (see ch. 6), but the infection must persist if antigen is to continue to be released into the blood and immune complexes formed over long periods. non-neutralising antibodies help promote virus persistence because they combine specifically with virus particles, fail to render them noninfectious, and at the same time block the action of any good neutralising antibodies that may be present. immune complexes in antigen excess are formed in the blood when the persistent virus or its antigens circulates in the plasma and reacts with antibody which is present in relatively small amounts. virus infections with these characteristics are * it is not clear how inflammation is caused. complement activation would presumably take place while complexes were circulating in the blood. perhaps the complexes bind more antibody after they have localised, or alternatively it is possible that free antigen circulates in the first place, localises, and later binds antibody to generate mediators of inflammation. included in table 8 .5. in each instance complexes are deposited in kidney glomeruli and sometimes in other blood vessels as described above. in some there are few if any pathological changes (ldv and leukaemia viruses in mice) probably because there is a slow rate of immune complex deposition, whereas in others glomerulonephritis (lcm virus in mice, adv in mink) or vasculitis (adv in mink) is severe. a persistent virus infection that induces a feeble immune response forms an ideal background for the development of immune complex glomerulonephritis, but there are no known viral examples in man. there are one or two other microorganisms that occasionally cause this type of glomerulonephritis and it is seen, for instance, in chronic quartan malaria and sometimes in infective endocarditis. in both these examples microbial antigens circulate in the blood for long periods. but immune complex deposition does not necessarily lead to the development of glomerulonephritis, and immune complexes are detectable in the glomeruli of most normal mice and monkeys. even in persistent virus infections the rate of deposition may be too slow to cause pathological changes as with ldv and leukaemia virus infections of mice (see table 8 .5). during the acute stage of hepatitis b in man, when antibodies are first formed against excess circulating viral antigen (hepatitis b surface antigen), immune complexes are formed and deposited in glomeruli. but the deposition is short-lived and there is no glomerulonephritis. persistent carriers of the antigen do not generally develop glomerulonephritis, because their antibody is usually directed against the 'core' antigen (nephrotic syndrome in secondary syphilis) unknown causative agents man + + + + of chronic glomerulonephritis of the virus particle, rather than against the large amounts of circulating hepatitis b surface antigen. kidney failure in man is commonly due to chronic glomerulonephritis, and this is known to be mostly of the immune complex type, but the antigens, if they are microbial, have not yet been identified. immune complex glomerulonephritis occurs in man as an important complication of streptococcal infection, but this is usually acute in nature with inflammation of glomeruli, as referred to above. antibodies formed against an unknown component of the streptococcus react with circulating streptococcal antigen, perhaps also with a circulating host antigen, and immune complexes are deposited in glomeruli. streptococcal antibodies cross-reacting with the glomerular basement membrane may contribute to the picture. deposition of complexes continues after the infection is terminated, and glomerulonephritis develops a week or two later. the streptococcal infection may be of the throat or skin, and streptococcus pyogenes types 12 and 49 are frequently involved. when certain antigens are inhaled by sensitised individuals and the antigen reaches the terminal divisions of the lung, there is a local antigen-antibody reaction with formation of immune complexes. the resulting inflammation and cell infiltration causes wheezing and respiratory distress, and the condition is called allergic alveolitis. persistent inhalation of the specific antigen leads to chronic pathological changes with fibrosis and respiratory disease. exposure to the antigen must be by inhalation; when the same antigen is injected intradermally, there is an arthus type reaction (see p. 252). there are a number of microorganisms that cause allergic alveolitis. most of these are fungi. a disease called farmer's lung occurs in farm workers repeatedly exposed to mouldy hay containing the actinomycete micromonospora faeni. cows suffer from the same condition. a fungus contaminating the bark of the maple tree causes a similar disease (maple bark stripper's disease) in workers in the usa employed in the extraction of maple syrup. the mild respiratory symptoms occasionally reported after respiratory exposure of sensitised individuals to tuberculosis doubtless have the same immunopathological basis. in addition to their local effects, antigen-antibody complexes generate systemic reactions. for instance, the fever that occurs at the end of the incubation period of many virus infections is probably attributable to a large-scale interaction of antibodies with viral antigen, although extensive cmi reactions can also cause fever. the febrile response is mediated by endogenous pyrogen (interleukin-1) and tumour necrosis factor (tnf) liberated from polymorphs and macrophages, as described on p. 298. probably the characteristic subjective sensations of illness and some of the 'toxic' features of virus diseases are also caused by immune reactions and liberation of cytokines. systemic immune complex reactions taking place during infectious diseases very occasionally give rise to a serious condition known as disseminated intravascular coagulation. this is seen sometimes in severe generalised infections such as gram-negative septicaemia, meningococcal septicaemia, plague, yellow fever and other haemorrhagic arthropod-borne virus diseases. immune complex reactions activate the enzymes of the coagulation cascade ( fig. 8.13 ), leading to histamine release and increased vascular permeability. fibrin is formed and is deposited in blood vessels in the kidneys, lungs, adrenals and pituitary. this causes multiple thromboses with infarcts, and there are also scattered haemorrhages because of the depletion of platelets, prothrombin, fibrinogen etc. systemic immune complex reactions were once thought to form the basis for dengue haemorrhagic fever. this disease is seen in parts of the world where dengue is endemic, individuals immune to one type of dengue becoming infected with a related strain of virus. they are not protected against the second virus, although it shows immunological cross-reactions with the first one. indeed the dengue-specific antibodies enhance infection of susceptible mononuclear cells, so that larger amounts of viral antigen are produced (see p. 160). it was thought that after virus replication, viral antigens in the blood reacted massively with antibody to cause an often lethal disease with haemorrhages, shock and vascular collapse. however, it has proved difficult to demonstrate this pathophysiological sequence, and the role of circulating immune complexes and platelet depletion remains unclear. perhaps in this and in some of the other viral haemorrhagic fevers the virus multiplies in capillary endothelial cells. disease seems due to cytokines liberated from infected mononuclear cells. immune complex immunopathology is probable in various other infectious diseases. for instance, the occurrence of fever, polyarthritis, skin rashes and kidney damage (proteinuria) in meningococcal meningitis and gonococcal septicaemia indicates immune complex deposition. circulating immune complexes are present in these conditions. certain african arthropod-borne viruses with exotic names (chikungunya, o'nyong-nyong) cause illnesses characterised by fever, arthralgia and itchy rashes, and this too sounds as if it is immune complex in origin. immune complexes perhaps play a part in the oedema and vasculitis of trypanosomiasis and in the rashes of secondary syphilis. sensitive immunological techniques are available for the detection of circulating complexes and for the identification of the antigens and antibodies in deposited complexes. the full application of these techniques will perhaps solve the problem of the aetiology of chronic glomerulonephritis in man. the mere expression of a cmi response involves inflammation, lymphocyte infiltration, macrophage accumulation and macrophage activation as described in ch. 6, and can therefore by itself cause pathological changes. the cmi response to infection dominates the pathological picture in tuberculosis, with mononuclear infiltration, degeneration of parasitised macrophages, and the formation of giant cells as central features. these features of the tissue response result in the formation of granulomas (see glossary) which reflect chronic infection and accompanying inflammation. there is a ding-dong battle as the host attempts to contain and control infection with a microorganism that is hard to eliminate. the granulomas represent chronic cmi responses to antigens released locally. various other chronic microbial and parasitic diseases have granulomas as characteristic pathological features. these include chlamydial (lymphogranuloma inguinale), bacterial (syphilis, leprosy, actinomycosis), and fungal infections (coccidiomycosis). antigens that are disposed of with difficulty in the body are more likely to be important inducers of granulomas. thus, although mannan is the dominant antigen of candida albicans, glucan is more resistant to breakdown in macrophages and is responsible for chronic inflammatory responses. the lymphocytes and macrophages that accumulate in cmi responses also cause pathological changes by destroying host cells. cells infected with viruses and bearing viral antigens on their surface are targets for cmi responses as described in chs 6 and 9. infected cells, even if they are perfectly healthy, are destroyed by the direct action of sensitised t lymphocytes, which are demonstrable in many viral infections. in spite of the fact that the in vitro test system so clearly displays the immunopathological potential of cytotoxic t cells, this is not easy to evaluate in the infected host. it may contribute to the tissue damage seen, for instance, in hepatitis b infection and in many herpes and pox virus infections. antigens from trypanosoma cruzi are known to be adsorbed to uninfected host cells, raising the possibility of autoimmune damage in chagas' disease, caused by this parasite.* it is also becoming * chagas' disease, common in brazil, affects 12 million people, and is transmitted by blood-sucking bugs. after spreading through the body during the acute infection, the parasitaemia falls to a low level and there is no clinical disease. years later a poorly understood chronic disease appears, involving heart and intestinal tract, which contain only small numbers of the parasite but show a loss of autonomic ganglion cells. an autoimmune mechanism is possible (see p. 173), because a monoclonal antibody to t. cruzi has been obtained that cross-reacts with mammalian neurons. clear that cells infected with certain protozoa (e.g. theileria parva in bovine lymphocytes) have parasite antigens on their surface and are susceptible to this type of destruction. little is known about intracellular bacteria. the most clearly worked out example of type 4 (cmi) immunopathology is seen in lcm virus infection of adult mice. when virus is injected intracerebrally into adult mice it grows in the meninges, ependyma and choroid plexus epithelium, but the infected cells do not show the slightest sign of damage or dysfunction. after 7-10 days, however, the mouse develops severe meningitis with submeningeal and subependymal oedema, and dies. the illness can be completely prevented by adequate immunosuppression, and the lesions are attributable to the mouse's own vigorous cd8 + t cell response to infected cells. these cells present processed lcm viral peptides on their surface in conjunction with mhc i proteins, and sensitised cd8 + t cells, after entering the cerebrospinal fluid and encountering the infected cells, generate the inflammatory response and interference with normal neural function that cause the disease. the same cells destroy infected tissue cells in vitro, but tissue destruction is not a feature of the neurological disease. in this disease the cd8 + t cells probably act by liberating inflammatory cytokines. it may be noted that the brain is uniquely vulnerable to inflammation and oedema, as pointed out earlier in this chapter. the infected mouse shows the same type of lesions in scattered foci of infection in the liver and elsewhere, but they are not a cause of sickness or death. lcm infection of mice is a classical example of immunopathology in which death itself is entirely due to the cell-mediated immune response of the infected individual. this response, although apparently irrelevant and harmful, is nevertheless an 'attempt' to do the right thing. it has been shown that immune t cells effectively inhibit lcm viral growth in infected organs. however, a response that in most extraneural sites would be useful and appropriate turns out to be self-destructive when it takes place in the central nervous system. another type of t cell-mediated immune pathology is illustrated by influenza virus infection of the mouse. when inoculated intranasally, the virus infects the lungs and causes a fatal pneumonia in which the airspaces fill up with fluid and cells. the reaction is massive and the lungs almost double in weight. effectively the animal drowns. the cause is an influx of virus-specific cd8 + t cells. normally when an appropriate number of t cells had entered the lungs, the t cells would issue a feedback response to prevent such over-accumulation, but it is thought that influenza virus infects the t cells and inhibits this control process, so that the lungs are eventually overwhelmed. the action of the virus is subtle as it does not multiply in or kill the infected t cells, and it is presumed that it undergoes limited gene expression. one human virus infection in which a strong cmi contribution to pathology seems probable is measles. children with thymic aplasia show a general failure to develop t lymphocytes and cell-mediated immunity, but have normal antibody responses to most antigens. they suffer a fatal disease if they are infected with measles virus. instead of the limited extent of virus growth and disease seen in the respiratory tract in normal children, there is inexorable multiplication of virus in the lung, in spite of antibody formation, giving rise to giant cell pneumonia. this indicates that the cmi response is essential for the control of virus growth. in addition there is a total absence of the typical measles rash, and this further indicates that the cmi response is also essential for the production of the skin lesions. there is evidence that cell-mediated immune responses also make a contribution to the rashes in poxvirus infections. sometimes in infectious diseases there are prominent pathological changes which are not attributable to the direct action of microbes or their toxins, nor to inflammation or immunopathology. the stress changes mediated by adrenal cortical hormones come into this category. stress is a general term used to describe various noxious influences, and includes cold, heat, starvation, injury, psychological stress and infection. an infectious disease is an important stress, and corticosteroids are secreted in large amounts in severe infections (see also ch. 11). they generally tend to inhibit the development of pathological changes, but also have pronounced effects on lymphoid tissues, causing thymic involution and lymphocyte destruction. these can be regarded as pathological changes caused by stress. it was the very small size of the thymus gland as seen in children dying with various diseases, especially infectious diseases, that for many years contributed to the neglect of this important organ, and delayed appreciation of its vital role in the development of the immune system. appreciation of the effects of stress on infectious diseases and the immune response in particular has led to the establishment of the science of neuroimmunology. properly controlled experiments are difficult to mount but there is a persuasive and growing body of evidence which shows that the nervous system affects the functioning of the immune system. the pathways of this communication are still poorly understood. work on mycobacterium bovis grew out of observations from the turn of the century that stress appears to increase the death rate in children with tb. in one type of experiment mice were stressed by being kept in a restraining device where movement was virtually impossible. this resulted in the reduction of expression of mhc class ii antigens on macrophages, which correlated with increased susceptibility to infection. similarly stressing mice infected with influenza virus caused several immunosuppressive events including reduction of inflammatory cells in the lung, and decreased production of interleukin-2. suppression of antibody responses is found in people suffering a type of stress familiar to students-examinations! the best responses to hepatitis b vaccine in students immunised on the third day of their examinations were found in those who reported the least stress. finally, in a double blind trial at the common cold research unit in england with five different respiratory viruses, it was ascertained in human volunteers that stress gave a small but statistically significant increased likelihood of an individual developing clinical disease. pathological changes are sometimes caused in an even more indirect way as in the following example. yellow fever is a virus infection transmitted by mosquitoes and in its severest form is characterised by devastating liver lesions. there is massive mid-zonal liver necrosis following the extensive growth of virus in liver cells, resulting in the jaundice that gives the disease its name. destruction of the liver also leads to a decrease in the rate of formation of the blood coagulation factor, prothrombin, and infected human beings or monkeys show prolonged coagulation and bleeding times. haemorrhagic phenomena are therefore characteristic of severe yellow fever, including haemorrhage into the stomach and intestine. in the stomach the appearance of blood is altered by acid, and the vomiting of altered blood gave yellow fever another of its names, 'black vomit disease'. haemorrhagic phenomena in infectious diseases can be due to direct microbial damage to blood vessels, as in certain rickettsial infections (see p. 127) or in the virus infection responsible for haemorrhagic disease of deer. they may also be due to immunological damage to vessels as in the arthus response or immune complex vasculitis, to any type of severe inflammation, and to the indirect mechanism illustrated above. finally there are a few infectious diseases in which platelets are depleted, sometimes as a result of their combination with immune complexes plus complement, giving thrombocytopenia and a haemorrhagic tendency (see also disseminated intravascular coagulation, p. 258). thrombocytopenic purpura is occasionally seen in congenital rubella and in certain other severe generalised infections. infection during pregnancy can lead to foetal damage or death not just because the foetus is infected (pp. 302-3), but also because of infection and damage to the placenta. this is another type of indirect pathological action. placental damage may contribute to foetal death during rubella and cytomegalovirus infections in pregnant women. certain viruses undoubtedly cause tumours (leukaemia viruses, human papillomaviruses, several herpes viruses in animals) and this is to be regarded as a late pathological consequence of infection. as was discussed in ch. 7 the tumour virus genome can be integrated into the host cell genome whether a tumour is produced or not, so that the virus becomes a part of the genetic constitution of the host. sometimes the host cell is transformed by the virus and converted into a tumour cell, the virus either introducing a transforming gene into the cell, activating expression of a pre-existing cellular gene, or inactivating the cell's own fail-safe tumour suppressor gene. the transforming genes of dna tumour viruses generally code for t antigens which are necessary for transformation, and the transforming genes of rna tumour viruses are known as one genes.* transformation has been extensively studied in vitro, and the features of the transformed cell described (changed surface and social activity, freedom from the usual growth restraints). simultaneous infection with two different microorganisms would be expected to occur at times, merely by chance, especially in children. on the other hand, a given infection generates antimicrobial responses such as as interferon production and macrophage activation which would make a second infection less likely. dual infections are commonest when local defences have been damaged by the first invader. the pathological results are made much more severe because there is a second infectious agent present. this can be considered as another mechanism of pathogenicity. classical instances involve the respiratory tract. the destruction of ciliated epithelium in the lung by viruses such as influenza or measles allows normally nonpathogenic resident bacteria of the nose and throat, such as the pneumococcus or haemophilus influenzae, to invade the lung and cause secondary pneumonia. if these bacteria enter the lung under normal circumstances, they are destroyed by alveolar macrophages or removed by the mucociliary escalator. in at least one instance the initial virus infection appears to act by interfering with the function of alveolar macrophages. mice infected with parainfluenza 1 (sendai) virus show greatly increased susceptibility to infection with haemophilus influenzae, and this is largely due to the fact that alveolar macrophages infected with virus show a poor ability to phagocytose and kill the bacteria. specialised respiratory pathogens such as influenza, measles, parainfluenza or rhinoviruses damage the naso-* one genes (oncogenes) are also present in host cells, where they play a role in normal growth and differentiation, often coding for recognised growth factors (e.g. human platelet-derived growth factor). they can be activated and the cell transformed when tumour viruses with the necessary 'promoters' are brought into the cell. the one genes of the rna tumour viruses themselves originate from cellular oncogenes which were taken up into the genome of infecting viruses during their evolutionary history. pharyngeal mucosa and can lead in the same way to secondary bacterial infection, with nasal catarrh, sinusitis, otitis media or mastoiditis. the normal microbial flora of the mouth, nasopharynx or intestine are always ready to cause trouble if host resistance is lowered, but under normal circumstances they hinder rather than help other infecting microorganisms (see ch. 2). one interesting example of exacerbation of infection occurs in mice dually infected with influenza virus and microorganisms such as streptococcus aureus or serratia marcescens. under these conditions animals suffer a more severe viral infection. this results from the need to proteolytically cleave the viral haemagglutinin protein which is done by a cellular enzyme. if the appropriate protease is in short supply or lacking completely, virions are formed but they are not infectious. under these circumstances the haemagglutinin can be cleaved extracellularly by microbial proteases with resulting increased amounts of infectious virus and disease. as a final example of dual infections, microorganisms that cause immunosuppression can activate certain pre-existing chronic infections. in measles, for instance, there is a temporary general depression of cmi; tuberculin-positive individuals become tuberculin negative, and in patients with tuberculosis the disease is exacerbated. in aids (see p. 175) immunosuppression by hiv activates a variety of pre-existing persistent infections. diarrhoea deserves a separate section, since it is one of the commonest types of illness in developing countries and a major cause of death in childhood. particularly in infants, who have a very high turnover of water relative to their size, the loss of fluid and salt soon leads to life-threatening illness. it is estimated that on a global scale diarrhoea is responsible for 3-6 million deaths per year in children under five years old. in villages in west africa and guatemala the average 2-3-year-old child has diarrhoea for about two months in each year.* diarrhoea also interacts with malnutrition and can cause stunted growth, defective immune responses and susceptibility to other infections (pp. 343-5). diarrhoea is also a common affliction of travellers from developed countries, * diarrhoea on a massive scale is not always confined to developing countries. there was a major outbreak of cryptosporidium infection in milwaukee, usa, in 1993 with more than 400 000 cases; 285 of these were diagnosed in the laboratory and they suffered watery diarrhoea (mean 12 stools a day) for a mean of nine days. the small (4-5 μιη) oocysts, probably from cattle, had entered lake michigan, and then reached the community water supply because of inadequate filtration and coagulation treatment. and business deals, athletic successes and holiday pleasures can be forfeited on the toilet seats of foreign lands. the most reliable prophylaxis is to 'cook it, peel it, or forget it'. most attacks of diarrhoea are self-limiting. fluid and electrolyte replacement is a simple, highly effective, life-saving treatment that can be used without determining the cause of the diarrhoea. oral rehydration therapy (orf) means giving a suitable amount of salt and sugar in clean water and this is something that can be done by the mother. diarrhoea means the passage of liquid faeces,* or faeces that take the shape of the receptacle rather than have their own shape. this could arise because of increased rate of propulsion by intestinal muscles, giving less time for reabsorption of water in the large bowel, or because there was an increase in the amount of fluid held or produced in the intestine. in many types of infectious diarrhoea the exact mechanism is not known. diarrhoea, on the one hand, can be regarded as a microbial device for promoting the shedding and spreading of the infection in the community, or on the other hand as a host device to hasten expulsion of the infectious agent. diarrhoea is a superb mechanism for the dissemination of infected faeces (see p. 52) and there is no doubt that strains of microbes are selected for their diarrhoea-producing powers. the advantages to the host of prompt expulsion of the infectious agent was illustrated when volunteers infected with shigella flexneri were given lomotil, a drug that inhibits peristalsis. they were more likely to develop fever and had more difficulty in eliminating the pathogen. in recent years significant strides have been made in our understanding of the pathophysiology of diarrhoeal disease. first, a quick resume of the normal structure and function of gut before attempting to understand the processes whereby it may be perturbed. the main function of the gut is the active inward transport of ions and nutrient solutes which is followed by the passive movement of water ( fig. 8.15 ). the driving force is the na + /k + atpase situated in the basolateral membrane of enterocytes on the villus (fig. 8.16 ) which maintains a low intracellular [na" 1 "] thus creating the electrochemical gradient favourable for na + entry and, a high regional [na + ] in the intercellular spaces; cl~ follows na + . a similar situation exists in crypt cells: na + /k + atpase drives secretion. the key difference is the location of the carrier systems responsible for the facilitated entry of the actively transported species. in villus cells the carriers are present in the brush border, whereas in crypt cells they are located in the basal membrane: this is responsible for the vectorial aspects of ion/fluid traffic in villus/crypt assemblies. however, it is clear that several factors in addition to enterocytes are involved in * liquid faeces are not abnormal in all species. the domestic cow experiences life-long diarrhoea, but presumably does not suffer from it. (a) two methods of n a + cotransport are shown involving a glucose-linked symport and two coupled antiports; the latter results in the cotransport of cl~. the coupled antiports are functionally linked via h + and hc0 3~, the relative concentrations of which are a reflection of metabolic activity. these processes occur within the same cells but are shown separately for clarity. the driving force for n a + uptake is the low na + concentration maintained by the na + /k + pump (atpase) which creates the electrochemical gradient which promotes the inward movement of na + ; cl~ follows n a + by diffusion. water is drawn osmotically across the epithelium paracellularly (i.e. across tight junctions) and/or transcellularly, the former pathway accounting for approximately 80% of fluid movement. (b) secretion is the result of the coupled entry of n a + and cl~ across the basolateral membrane. n a + is recycled by the na + /k + pump and cl~ exits by diffusing down an electrochemical gradient and across the undifferentiated crypt cell apical membrane; n a + follows cl~ and water follows passively. if by whatever means, the level of nacl were to increase in such cells (as for example in rotavirus infection of neonatal mice, see below) then one could perceive how this could give rise to a cl~ driven hypersecretion of water. note, (i) the driving force results from the same mechanism that powers absorption i.e. the n a + / k + pump located in the basolateral membrane; it is the location of the 'port' 'diffusion' systems that determines the vectorial aspects of ion movement, (ii) the tight junctions are less tight in the crypts than villi. (iii) the apical membrane of the crypt cell is undifferentiated and only acquires micro villi during ascent into villous regions, φ : na + /k + pump; o: sym-, anti-port or diffusion channel. r e g u l a t i n g fluid t r a n s p o r t in t h e gut; t h e s e include t h e enteric n e r v o u s s y s t e m a n d t h e a n a t o m y of t h e microcirculation. t h e l a t t e r plays a profoundly i m p o r t a n t role in t h e u p t a k e of fluid. this is i l l u s t r a t e d in fig. 8 .16, which shows t h e existence of zones of g r a d e d osmotic fig. 8 .16 schematic representation of a villus. note the central arterial vessel (av) which arborizes at the tip into a capillary bed drained by a subepithelial venous return (vr). movement of sodium into vr creates a concentration gradient between vr and av causing absorption of water from av and surrounding tissue. this results in a progressive increase in the osmolarity of incoming blood moving into the tip region through to vr. tip osmolarity is about three times higher than normal. this counter current system has been demonstrated in man and can be inferred in mice from the morphology of red blood cells which changes during ascent of the same vessel from base to tip regions of villi. the shaded areas indicate a vertical increase in osmolarity. left crypt: represents normal physiological secretion. right crypt: represents hyper secretion. ens, enteric nervous system, depicted schematically and not anatomically. potential. at the tips of villi in adult human gut, osmolalities range from 700 to 800mosmkg _ 1 h 2 0 , which would generate huge osmotic forces. thus, current perceptions are that enterocytes are responsible for generating this gradient and the blood supply acts as a countercurrent multiplier which amplifies the gradient in a manner analogous to the loops of henle in the kidney. the hypertonic zone has been demonstrated directly in whole villi of infant mice in terms of the changing morphology of erythrocytes: in the lower regions of villi they show characteristic discoid morphology, whereas in the upper region they are crenated, indicating a hyperosmotic environment. the hypertonicity is dissipated if the blood flow is too slow and washed out if too fast. it is the villus unit rather than enterocytes by themselves which is responsible for fluid uptake. another consequence of the microcirculatory anatomy is that villus tip regions are relatively hypoxic. in addition, neonatal brush borders contain disaccharidases (principally lactase) which break down nonabsorbable disaccharides (e.g. lactose) into constituent absorbable monosaccharides. villus tips and crypts are regarded as the anatomical sites of physiological absorption and secretion respectively. fluid transport is a bidirectional process in the healthy animal with net absorption in health and net secretion in disease. the balance between absorption and secretion is poised at different points throughout the intestinal tract reflecting differences in both structure and function. proximal small intestine is relatively leaky; in contrast the colon is a powerfully absorptive organ. finally, crypts are the principal sites of cell regeneration, replacing cells which migrate up the epithelial escalator. the epithelium is renewed in approximately 3-5 days. at villus tips senescent cells are shed. diarrhoeal disease can result from interference with almost any one, or combination of these systems. diarrhoea-producing microbes are listed in table 8 .6. some examples are considered in detail below. rotaviruses are known to invade intestinal epithelial cells and cause diarrhoea in man, foals, dogs, pigs, mice etc. extensive multiplication takes place and very large amounts of virus (10 11 particles g _ 1 ) are shed in faeces. the conventional wisdom is that tips of villi especially are affected, leading to reduced absorption of fluid from the lumen. in addition destruction of enterocytes leads to a loss in lactase resulting in an accumulation of lactose in the gut causing an osmotic flux of fluid into the intestine. a major study of rota virus-induced diarrhoea in neonatal mice provides a different model of this important disease of children. oral infection of baby mice with a murine strain of rotavirus resulted in virus replication in enterocytes of the small intestine. before there had been detectable virus replication, villi became ischaemic, enterocytes severely damaged and villi shortened. in gut enterocytes this virus was demonstrably not cytopathic; the cell damage and villus responses were almost identical to that seen in experimentally induced ischaemia in physiological experiments. at the peak of virus replication, which was coincident with maximum shortening of villi, the gut was still absorptive, glucose transport intact and sufficient lactase activity remained to deal with the normal lactose load delivered to the stomach. after this the gut became secretory and diarrhoea clinically evident. villi were rapidly regenerated, but hyperaemia and diarrhoea persisted until the regeneration of the hypertonic zones as judged by the morphology of the red blood cells. peak diarrhoea coincided with the resynthesis of new villi (as judged by thymidine kinase levels, increased mitotic activity and morphometric analyses), and increased levels of intracellular nacl in the zone of cells where cell division was most active. transiently high levels of nacl have also been observed in dividing cells in culture. since ischaemic and hyperaemic villi were occasionally seen in control villi it may well be the case that the pathological changes reflect an exaggerated synchronised response of basic circulatory control mechanisms, which are part of the normal homeostatic mechanisms of villus physiology. thus, the pathophysiology of rotavirus-induced diarrhoea in neonatal mice may be summarised as follows: the reduction of red blood cells flowing through villi in the early stages of infection instigates hypoxia and hence atrophy of villi. the degree of atrophy in this infection is not associated with lack of absorption. the ensuing villus repair processes induce hypersecretion. the increase in blood flow throughout the remaining course of the infection reduces the hypertonicity of villi which impairs water absorption and thus prolongs diarrhoea. the preceding description of the self-limiting diarrhoea induced by rotavirus in neonatal mice is probably applicable to many diarrhoeas. however, the observed pathology may be different according to age, species, or the inducing pathogen. for example, in rotavirus-infected lambs, villus atrophy and crypt hypertrophy occur (the latter indicative of crypt cell division) but as in mice, infected lambs are not lactose intolerant. in rotavirus-infected piglets, crypt hypertrophy occurs but villus atrophy is severe, the animals are lactose intolerant and mortality high; a similar situation exists for the coronavirus, transmissible gastroenteritis (tge) virus. the latter has often been used as the model for infantile diarrhoea but the question is whether human infants are more like piglets or lambs. clinical studies have shown that recovery from mild, acute gastroenteritis of rota virus origin occurs within two weeks irrespective of the carbohydrate ingested. clearly, the severity of disease and the clinical outcome will depend on the extent of vertical' villus/ crypt involvement and the regions of intestine infected. when villus erosion is severe, then lactose may cause an 'osmotic' purge or be fermented by intestinal bacteria to short chain fatty acids which stimulate secretion in the colon. astroviruses, norwalk virus, certain coronaviruses, certain adenoviruses and probably toroviruses all cause gastroenteritic disease by infecting enterocytes. however, parvoviruses cause severe intestinal disease in dogs by virtue of their predilection for the mitotically active crypt cells; this causes the near complete erosion of villi similar to that seen after exposure to sublethal doses of irradiation. the classic paradigm for bacterial watery diarrhoea is cholera caused by vibrio cholerae in the small intestine. v. cholerae attaches to enterocytes of the proximal small bowel and is capable of producing at least three toxins: classical cholera toxin (ct); a toxin which disrupts the zonula occludens tight junction (designated zot); and another less well defined, auxilliary cholera enterotoxin (designated ace). as a result of toxic action, water and electrolytes are lost through the intact epithelial cells into the small intestine. as the multiplying bacteria increase in numbers and more and more epithelial cells are affected, the absorptive capacity of the colon is overwhelmed and there is profuse watery diarrhoea, as much as l l h -1 in severe cases.* the massive loss of isotonic fluid with excess of sodium bicarbonate and potassium leads to hypovolaemic shock, acidosis and haemoconcentration. anuria develops, and the collapsed, lethargic patient may die in 12-24 h. lives are saved by replacing the lost water and salts. the effect of toxin on an intestinal epithelial cell is long lasting, but the patient recovers as affected cells are shed and replaced in the normal fashion. the infection is particularly severe in children who easily develop low levels of plasma potassium. however, on a global scale this greatly feared disease, cholera, is only responsible for less than 1% of the total deaths due to diarrhoea. administration of as little as 5 μg of purified ct alone to a healthy volunteer will reproduce the hugely dramatic, potentially lethal, purge of near isotonic fluid emphasising the central importance of ct in the causation of disease. the effect of zot has been demonstrated by electron microscopy in human biopsies, but the relative importance of zot and ace in human disease has not been quantified. how does ct work? as already outlined (pp. 216-19) the a subunit of this toxin is an adpribosyl transferase whose target is the a s subunit of the regulatory g protein which governs the expression of adenylate cyclase and hence the production of camp. this in turn results in the perturbation of the transport systems leading to a net efflux of ions and hence water. things are slightly more complicated, because elevation of camp occurs in villus tip cells (hence affecting absorption) but not in crypt cells. probably a signal is generated in intoxicated tip enterocytes which is transmitted to the crypt region by the ens causing release of neurotransmitters (e.g. serotonin, acetyl choline, and vasoactive peptide) which act on enterochromaffin (secretory) cells and on intestinal smooth muscle thereby augmenting crypt secretion and increasing peristalsis. studies have been made of sequential histological changes in human biopsies, taken from a series of patients from the onset of symptoms to recovery. it was clear that fluid secretion was not the result of massive desquamation of the epithelium. histological changes included 'engorgement of the capillaries' (i.e. interference with the blood supply), 'dilation of lacteals, vacuolation of enterocytes, exhaustion atrophy of enterocytes' (remember this is a noninvasive pathogen), 'accelerated shedding of cells, and increased mitotic activity'. most of these changes are similar to those described above for experimental murine rotavirus at post-peak diarrhoea and it seems that the range of histological reactions that can occur in the small intestine are limited so that qualitatively the picture is similar in different diarrhoeas. despite the undoubted importance of ct in the causation of the disease, and the potent antigenicity of ct, it is now recognised that protective immunity is very largely antibacterial. it is stopping effective colonisation which is important rather than neutralisation of the toxin. this has been partially achieved by using killed whole cell vaccines. several attempts have been made in the laboratory to genetically manipulate virulent strains (in practice this means deleting or inactivating the known toxin genes) such that the attenuated strain will colonise the gut and stimulate local immune responses and thereby prevent colonisation of the gut by virulent strains. to date, attenuated strains have been developed which fulfil these criteria but induce a mild transient diarrhoea which has prevented their adoption into vaccination programmes. however the recent emergence of v. cholerae strain 0139 (a new, third type) in india and bangladesh is a reminder that today's solution may not be adequate for tomorrow's problems. current vaccines are ineffective against this new strain. the picture withi£. coli is complicated since there are several biotypes of this pathogen which include: etec (enterotoxigenic e. coli, the principal cause of travellers' diarrhoea), epec (enteropathogenic e. coli), eiec (enteroinvasive e. coli), and ehec (enterohaemorrhagic e. coli) variants (see ch. 2). the situation with etec is very similar to v. cholerae as far as heat labile toxin (lt, which is very closely related to ct) is concerned. it is additionally complicated by the fact that some strains of etec also make heat stable toxins (sts), which are nonantigenic low molecular weight peptides which activate membrane-bound guanylate cyclase which causes the production of cgmp which is the functional equivalent of camp. the 'classical' epec lesion is the formation of a characteristic pedestal-like lesion in the brush border of enterocytes with only limited invasion of the mucosa. just how it causes diarrhoea is not wholly clear. eiec is almost indistinguishable from shigella, and causes similar disease. ehec strains, which belong mainly (but not exclusively) to serogroup 0157:h7, cause a spectrum of intestinal disease ranging from asymptomatic carriage through mild diarrhoea to severe haemorrhagic colitis. they also cause extraintestinal infections such as haemolytic uraemic syndrome (hus). the initial lesion seen in the gut is similar to that caused by epec but ehec do not remain localised there: they multiply in the lamina propria and glandular crypts. while there is no conclusive proof of the involvement of slt in the pathogenesis of ehec diarrhoea there is a correlation between slt production and the severity of diarrhoeal disease or hus caused by this organism. salmonella spp. cause acute gastroenteritis and systemic typhoid disease in man, as well as other important infections in domestic animals. virulence in salmonella, especially in their natural hosts (see table 8 .7, footnote) isxomplex and mediated by numerous genes, some of which are present in otherwise nonpathogenic organisms such as e. coli. presumably all these bacteria share nutritional and environmental problems, whether inside or outside a host, that call for such genes. those restricted to salmonella are more likely to be critical in vivo. the clinical features of salmonella-induced diarrhoea (gastroenteritis) in humans and systemic typhoid infections are quite different. for example, gastroenteritis may follow 8-36 h after ingestion of contaminated food, whereas typhoid follows an incubation period of 10-20 days. diarrhoea, (which is usually watery, but may be severe, and sometimes bloody) is the predominating feature of gastroenteritis, whereas in adults, constipation is seen in the early clinical stages of typhoid; diarrhoea may occur much later. although fever may occur in gastroenteritis, in typhoid this may be so severe as to cause delirium. current perceptions are that gastroenteritis results from the initial interactions of salmonella (any one of many serotypes but frequently typhimurium or enteriditis, and/or their products) with the gut mucosa, whereas typhoid fever is produced by s. typhi organisms which translocate the mucosa, survive within macrophages, multiply and release endotoxin which triggers the highly complex endotoxin cascade. gastroenteritis is usually self-limiting, whereas in untreated typhoid mortality can be as high as 10%. the pathophysiology of fluid secretion caused by salmonellae is highly complex and as yet incompletely understood. the only two biological parameters which have been shown to correlate with induction of fluid secretion in a rabbit model (the best available small laboratory animal for human gastroenteritis) is the ability to invade the gut mucosa, and the induction of a leucocyte (mainly polymorphonuclear cell) influx into the mucosa and lumen of the gut. fluid secretion does not correlate per se with the ability to make an enterotoxin: avirulent as well as virulent strains make a cholera-like toxin though neither appear to release the toxin readily. the latter is therefore either not primarily involved, or is involved only after release from bacteria by means other than normal protein secretion mechanisms. these questions are as yet unresolved and the reader is referred to the reference list for a fuller discussion of these complex matters. currently, the major diarrhoeagenic pathogen of the small intestine in the developed world is campylobacter jejuni. the latter are bacteria present in wild birds, chickens and in the faeces of up to 10% of healthy cows in the uk. milk becomes contaminated and those who drink raw (unpasteurised) infected milk develop diarrhoea, sometimes with dysentery (blood and pus in the stools) and fever. the incubation period is 2-7 days and diarrhoea occurs after bacterial replication in the upper small intestine (jejunum). however, we are only at the beginning of understanding the detailed mechanisms and determinants responsible for disease causation by this pathogen. giardia lamblia is a noninvasive protozoan pathogen of the human small bowel which causes a spectrum of infection ranging from asymptomatic carriage through acute watery diarrhoea to chronic diarrhoea and malabsorption. giardia has a simple life cycle, existing in two forms: the multiplying trophozoite which infects mammalian hosts to cause disease and the environmentally resistant cyst. after ingestion, excystation is triggered by the ph in the stomach, and the trophozoite colonises the small bowel; the putative adherence factors have been described in ch. 2. the precise mechanisms involved in subsequent stages responsible for diarrhoea, malabsorption and cystation are less well understood. entamoeba histolytica causes lysis of target cells apparently by direct contact with the cell membrane. this pathogen produces under in vitro conditions a spectacular array of potential (but as yet unproven) virulence determinants including: proteases that round up cells, poreforming proteins, collagenases and oligosaccharidases and neurotransmitter-like compounds; the latter can induce intestinal fluid secretion. some of these factors have been implicated as the determinants responsible for liver abscess formation. not all diarrhoeal disease is the result of small bowel dysfunction. two important pathogens of the colon will be considered-clostridium difficile and shigella dysenteriae. c. difficile causes a spectrum of disease varied hepatitis a there are more than 1000 serotypes of salmonella, distinct from salmonella typhi and salmonella paratyphi. they are primarily parasites of animals, ranging from pythons to elephants, and their importance for man is their great tendency to colonise domestic animals. pigs and poultry are commonly affected, and human disease follows the consumption of contaminated meat or eggs. b other campylobacters cause sepsis, abortion and enteritis in animals. ranging from asymptomatic carriage through mild antibiotic-associated diarrhoea (aad) to fatal pseudomembranous colitis (pmc). normal flora play a major part-by means which are still not completely understood-in suppressing outgrowth of resident c. difficile spores. disruption of the normal flora, by antibiotic treatment for example, results in vegetative growth and production of several toxins of which c. difficile toxin a seems to be the most important in disease. this toxin is responsible for the secretory and inflammatory response in the intestine, and epithelial disruption. it probably acts by disrupting tight junctions between cells, and by causing chemotactic and other responses in polymorphs. diarrhoea could be due to the destruction of the 'absorptive epithelium' and failure to cope with the normal fluid load delivered by the small intestine, or an active type of secretion caused by replacement of the damaged epithelium, or both. the second example is that of dysentery caused by shigellae spp., in particular s. dysenteriae and s. flexneri. we have already dealt with the capacity of shigellae to invade (ch. 2). invasiveness is a vital part of the virulence armoury of shigellae spp.; non-invasive mutants are a virulent. the pathogen invades the gut via m cells in peyer's patches and thence adjacent microvillus-bearing colonocytes. what is the role played by shiga toxin? mutants of s. dysenteriae devoid of the ability to make shiga toxin still retained the ability to cause lethal fulminant dysentery (low volume fluid production, pus cells, mucus) in macaque monkeys, and they invaded, multiplied within and rapidly killed hela cells. the major difference between the mutants and the toxin-positive wild type was that the latter caused a more severe disease with more haemorrhage, giving rise to blood in the stools, and, in some instances greater destruction of mucosal tissues. evidently the production of shiga toxin is not a sine qua non for causing bacillary dysentery: it exacerbates the disease. although much research has been focused on toxins, their mode of action, and their role in disease, it is useful to compare different types of intestinal infection and to refer to the concept of food poisoning. types of intestinal infection are set out in table 8 .7. food poisoning is a loosely used term, and usually refers to illnesses caused by preformed toxins in food, or sometimes to illnesses that come on within a day or so after eating contaminated food. food may be contaminated with plant poisons, fungal poisons (e.g. poisoning due to amanita phalloides), fish poisons,* heavy metals, as well as with bacterial toxins or bacteria. sourcebook of bacterial toxins the virology and immunology of lymphocytic choriomeningitis virus infection * ingestion of scombroid fish (mackerel etc.) containing large amounts of histamine or similar substances leads to headache, flushing, nausea and vomiting within an hour response of man to infection with vibrio cholerae. 1. clinical, serologic and bacteriologic responses to a known inoculum epitopes of streptococcal m proteins shared with cardiac myosin bacterial infections of respiratory and gastrointestinal mucosae pathogenesis and immunology of treponema pallidum t-lymphocyte stimulation by microbial antigens lessons from diarrhoeal diseases, demography to molecular pharmacology clostridium botulinum toxins: a general review of involvement in disease, structure, mode of action and preparation for clinical use disseminated intravascular coagulation: a review campylobacterpyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium typhoid fever: pathogenesis and immunologie control comparison of the alpha-toxin genes of clostridium perfringens type a and c strains: evidence for extragenic regulation of transcription pathogenic mechanism ofneisseriagonorrhoeae: observations on damage to human fallopian tubes in organ cultures by gonococci of colony type i or type 4 rift valley fever virus in mice vi: histological changes in the liver in relation to virus multiplication viral aetiology of diseases of obscure origin campylobacter jejuni. current status and future trends cytolytic pore-forming proteins and peptides: is there a common structural motif? treatment of spasmodic torticollis with local injections of botulinum toxin pathogenesis of diseases caused by entamoeba histolytica: studies of adherence, secreted toxins and contactdependent cytolysis pituitary dwarfism in mice persistently infected with lymphocytic choriomeningitis virus tetanus and botulinum-b neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc experimental salmonella typhimurium induced gastroenteritis bacterial phospholipases c the development of respiratory syncytial virus-specific ige and the release of histamine in naso-pharyngeal secretions after infection immune complexes in human diseases key: cord-022592-g7rmzsv5 authors: wynn, james l.; wong, hector r. title: pathophysiology of neonatal sepsis date: 2016-07-06 journal: fetal and neonatal physiology doi: 10.1016/b978-0-323-35214-7.00152-9 sha: doc_id: 22592 cord_uid: g7rmzsv5 nan a successful immune response is critically necessary to eradicate infectious challenges and prevent dissemination of the infection in the host. however, if inflammation is not limited and becomes generalized, it can result in the constellation of signs and symptoms of a systemic inflammatory response syndrome (sirs). if the infection is not contained, the spread of the pathogen from its local origin through the blood may result in systemic endothelial activation and precipitate sepsis, severe sepsis, and septic shock. progression of sepsis to shock may lead to multiple organ dysfunction syndrome (mods) and ultimately death. host immunity is divided into innate and adaptive immune systems for purposes of discussion and teaching but there is a great deal of interaction between the two systems. innate immunity is rapid, largely nonspecific, and composed of barriers, phagocytic cells, the complement system, and other soluble components of inflammation. after breech of a barrier, cellular elements of the innate immune response are the first line of defense against the development and progression of infection. adaptive immunity, which is antigen specific, is long lived, and often takes several days to develop, provides immunologic specificity and memory. these systems work together to protect the host from pathogenic challenge but may also precipitate host injury through aberrant responses. the outcome of infection is dependent on at least four major factors: (1) the pathogen, (2) the pathogen load, (3) the site of infection, and (4) the host response. less is known about the host response in neonates compared with adults for a number of reasons, the principal one being a highly variable definition of disease. our understanding of the pathophysiology of sepsis is largely from investigations in adult populations, including both humans and animals. there is clear evidence from both preclinical models of sepsis and humans that neonates manifest different host immune responses as compared with adults. [1] [2] [3] [4] even in comparison with children, neonates manifest a unique host immune response to septic shock. 5 thus neonatal-specific clinical investigations, particularly in very preterm infants, are required to improve both survival and long-term outcomes for these populations. a better understanding of the pathophysiology will uncover new opportunities for interventional studies ultimately aimed at improving outcomes. to this end, in this chapter we explore the pathophysiology of sepsis in the neonate, with special attention paid to the immunobiology of sepsis. adult and pediatric intensivists currently use generally accepted definitions for sepsis for goal-based therapeutic interventions. [6] [7] [8] [9] these definitions are critical to facilitate epidemiologic studies, to accurately determine disease prevalence, to select patients for clinical trials, and ultimately to improve the delivery of care. the generally accepted pediatric definition for sepsis, established in 2005, was intended for all children (<18 years old), including term neonates (≥37 weeks' completed gestation). 6 preterm neonates (<37 weeks' completed gestation) were specifically excluded from the pediatric generally accepted definitions, and neonatal-perinatal subspecialists were not represented among the pediatric consensus experts. to investigate whether the pediatric generally accepted definitions for sirs and sepsis applied to term infants, hofer and colleagues 10 retrospectively examined 476 term neonates and found that the generally accepted definitions applied to only 53% of cases of culturepositive early-onset sepsis. neonatal sepsis has been inconsistently defined on the basis of a variety of clinical and laboratory criteria, which makes the study of this condition very difficult. 11 diagnostic challenges and uncertain disease epidemiology necessarily result from a variable definition of disease. the lack of a generally accepted definition for neonatal sepsis remains a significant hindrance towards improving outcomes and accurately describing disease pathophysiology. thus working definitions for the sepsis continuum, specific for preterm and term neonates, are needed to provide a uniform basis for clinicians and researchers to study and diagnose severe sepsis. the addition of immune biomarker-based staging of disease to clinical sign staging is highly likely to increase the accuracy of patient classification for future multicenter clinical trials that will test novel interventions. sepsis or serious infection within the first 4 weeks of life kills more than 1 million newborns globally every year. 12, 13 the incidence of neonatal sepsis is variable (from less than 1% to more than 35% of live births) on the basis of gestational age and time of onset (early-onset sepsis [<72 hours after birth] or late-onset sepsis [≥72 hours after birth]). [14] [15] [16] [17] [18] [19] [20] preterm neonates have the greatest sepsis incidence and mortality rates among all agegroups [21] [22] [23] [24] [25] [26] (figure 152-1) . risk factors for developing sepsis in neonates, particularly the very premature, have been well described. 14, 15, [27] [28] [29] [30] [31] [32] [33] prematurity, low birth weight (especially infants weighing less than 1,000 g), male sex, a maternal vaginal culture positive for group b streptococcus (gbs), prolonged rupture of membranes, maternal intrapartum fever, and chorioamnionitis are strongly associated with an increased risk for early-onset sepsis. 30 chorioamnionitis is associated with the greatest risk for subsequent clinical or culture-proven sepsis. 29 recent studies demonstrate the risk for sepsis in newborn infants born to women with clinical chorioamnionitis is strongly dependent on gestational age, with minimal risk in neonates aged 35 weeks or older and greater risk with increasing degrees of prematurity. [34] [35] [36] [37] [38] [39] [40] [41] the risk for neonatal sepsis conferred by maternal gbs colonization 29 is significantly reduced with adequate intrapartum antibiotic prophylaxis. 42 section xxvi pathophysiology of neonatal diseases ated with sepsis-like syndromes (e.g., echovirus, enterovirus, parechovirus, coxsackie virus, adenovirus, parainfluenza virus, rhinovirus, and coronavirus). 50, [63] [64] [65] physical barriers, including skin and mucosal surfaces, are the first point of contact between the host and potential pathogens. thus a successful immune defense in addition to epithelial barrier function is critical to prevent the development of local infection. multiple immune elements are present to prevent attachment and propagation of pathogens while simultaneously permitting the presence of commensal organisms required for homeostasis. vernix enhances skin barrier function in late-preterm and term neonates. vernix is a complex material comprising water (80.5%), lipids (10.3%), and proteins (9.1%) produced by fetal sebaceous glands during the last trimester 66 and is largely absent in preterm neonates born before 28 weeks' gestation. vernix provides a barrier to water loss, improves temperature control, and serves as a shield containing antioxidants and innate immune factors such as antimicrobial proteins and peptides (apps). 67 the apps on the surface of the newborn's skin (and replete in the amniotic fluid [68] [69] [70] ) are capable of killing/inactivating common neonatal pathogens, including gbs, e. coli, and candida species. 71 erythema toxicum is an immune-mediated manifestation that results from bacterial colonization of the skin occurring shortly after birth. 72, 73 this common cutaneous immune response is less common in preterm infants than in term infants, highlighting the impact of developmental age on host immune capabilities. 74 in contrast to the moist mucosal surfaces of the respiratory and gastrointestinal (gi) tracts, the skin is arid, which further reduces the chances for microbial invasion. the outermost layer of the skin, the stratum corneum, prevents microbial invasion, maintains temperature, and reduces the risk for dehydration through prevention of transcutaneous water loss. 75 the immature and incompletely developed stratum corneum of preterm newborns takes at least 1 to 2 weeks after birth to become fully functional 76 and may take up to 8 weeks in the extremely preterm neonate, significantly increasing the risk for barrier dysfunction. 77 disruption of the cutaneous barrier by trauma (e.g., placement of an intravenous catheter or heel stick) or chemical burn allows microorganisms to enter the subcutaneous tissue, increasing the likelihood of their establishing a local infection ( figure 152 -2). the likelihood of a microbial breach of the cutaneous barrier rises in the presence of intravenous catheters, which are essential for critical care. emollients, aimed at enhancing the barrier function of preterm newborn skin, increase the risk for nosocomial infection and their use is not recommended. 78 mucosal barriers contain multiple components that serve to prevent infection, including acidic ph, mucus, cilia, proteolytic enzymes, apps, opsonins such as surfactant proteins, sentinel immune cells such as macrophages, dendritic cells, polymorphonuclear neutrophils (pmns), and t cells, as well as commensal organisms. 79 like the skin, the gi mucosa is quickly colonized after birth and contains a significant repository of microorganisms. [80] [81] [82] gi barrier integrity, paramount for prevention of spread of microorganisms out of the intestinal compartment, is dependent on the interaction between commensal organisms and host epithelium. interleukin (il)-17, produced by type 3 intestinal innate lymphoid cells in the presence of the microbiota, drives granulocytosis and may protect the neonatal host from infectious challenge. 83 a loss of intestinal barrier integrity likely plays a role in the development of necrotizing enterocolitis (nec) and late-onset sepsis. 84, 85 prolonged antibiotic treatment, hypoxia, and remote infection are factors known to despite the efficacy of this intervention, the incidence of invasive gbs disease in african american neonates is still more than twice that in white babies, 43 and the incidence of escherichia coli sepsis may be rising in very-low-birth-weight (vlbw) neonates. 44 vaginal delivery in the presence of maternal active primary herpes simplex virus significantly increases the risk for a neonatal herpes simplex virus infection, which has a fulminant course and high mortality. [45] [46] [47] preexisting maternal immunodeficiency or sepsis also increases the risk for sepsis in the neonate. 48 in addition, care practices after birth, such as intubation, mechanical ventilation, and placement of central venous lines, increase the risk for the development of sepsis. 49 a number of pathogens have been associated with sepsis in the neonatal period. the predominant cause is bacterial; however, certain viral infections are associated with a fulminant course and significant mortality. [50] [51] [52] in a large (n = 104,676), multicenter study of vlbw infants (<1500 g), gram-positive organisms accounted for 34% of pathogens causing early-onset sepsis and 61% of those causing late-onset sepsis. in contrast, gram-negative organisms were responsible for 58% of early-onset sepsis and 26% of late-onset sepsis. 53 candida species accounted for 3% of cases of early-onset sepsis and 11% of cases of late-onset sepsis. infection by gram-negative organisms, particularly pseudomonas species, carries a higher risk for fulminant course and death than infection by other pathogen groups. 14, 15, 49, [54] [55] [56] gram-positive causes of sepsis are dominated by gbs and coagulase-negative staphylococci (cons). 15, 57 although the high mortality rate for gbs has been well described (especially among infants born prematurely), mortality rates associated with cons are significantly lower. 15, 16 fungi may also be associated with fulminant neonatal sepsis and predominantly affect vlbw infants. 15, 58, 59 independent predictors of in-hospital neonatal mortality after late-onset sepsis were pseudomonas infection (adjusted odds ratio [or], 14.31; 95% confidence interval [ci], 3.87% to 53.0%) and fungemia (or, 5.69; 95% ci, 2.48% to 13.01%). 60 the limited sensitivity of current methods to identify causative organisms is partially due to an inability to take a large sample of blood from newborn infants with suspected sepsis. 61 blood culture-negative ("clinical") sepsis is estimated to occur at a nearly 10-fold greater rate than blood culture-positive sepsis. 62 in some of these infants, sepsis may also be due to novel viral pathogens associy e a r s 3 0 -3 9 y e a r s 4 0 -4 9 y e a r s 5 0 -5 9 y e a r s 6 0 -6 9 y e a r s 7 0 -7 9 y e a r s 8 0 -8 9 y e a r s the production of mucus and mucociliary clearance of pathogens and debris. 95 premature neonates have relatively more goblet cells than do maturer neonates, leading to a decrease in mucociliary clearance. respiratory mucosal function can be impaired by surfactant and saliva deficiency, altered mucus production, and mechanical ventilation. ventilation is associated with decreased mucociliary clearance, airway irritation, and parenchymal lung injury (see figure 152 -2). intubation is also associated with the progressive accumulation of colonizing bacteria and bacterial endotoxin in respiratory fluids, with concomitant mobilization of endotoxin-modulating apps to the airway. 96 neonates with surfactant deficiency lack apps such as surfactant proteins a and d, which are also absent in commercially available surfactant preparations. 97 there is an age-dependent maturation in the ability of respiratory epithelium to elaborate apps (cathelicidin and β-defensins), such that the respiratory epithelium of preterm newborns mounts a deficient app response. 98 disrupt or injure the neonatal intestinal barrier (see figure 152 -2). [86] [87] [88] under these circumstances, the gut may become the motor of systemic inflammation. 89 mechanistically, paneth cells and intestinal lymphoid cells may release excessive amounts of il-17, which, in turn, plays a critical role in the development of sirs. 90 many interventions aimed at reducing the frequency of sepsis in neonates via enhancement of mucosal barrier integrity have been evaluated. neither probiotics nor glutamine supplementation has reduced the incidence of neonatal sepsis. 91 in contrast, human milk feeding is associated with a reduction in the risk for sepsis 92 and nec 93, 94 and is strongly encouraged, especially in preterm infants. respiratory mucosa is defended in utero by amniotic fluid and pulmonary apps, surfactant proteins a and d, alveolar macrophages, and pmns, among other immune elements. the surface and submucosal gland epithelium of the conducting airways is a constitutive primary participant in innate immunity through c the cell surface and in endosomes, whereas rlrs and nlrs detect pathogens only intracellularly. the discovery that tlr4 was integral for a robust lipopolysaccharide (lps)-mediated inflammatory response after gram-negative sepsis may be why tlrs have been more thoroughly investigated in the setting of sepsis than other prrs. 100 each of the 10 known tlrs in humans, present on and within multiple cell types, recognizes extracellular and intracellular pathogens via specific pamps. 101, 102 multiple tlrs may be activated in concert by intact or partial microorganisms and in turn activate multiple second-messenger pathways simultaneously. 102, 103 lps is the prototypic mediator of systemic inflammation and generates many of the clinical findings of sepsis and septic shock, including mods and death. 104 lps signals through tlr4 in conjunction with the adaptor proteins cd14 and myeloid differentiation factor 2. 105 in adults a reduction in mortality and improvement in hemodynamics were demonstrated when the level of serum lps was reduced. 106 the level of lps is elevated in blood from infected neonates and those with nec even in the absence of gram-negative bacteremia. 84 high levels of circulating endotoxin found during sepsis and nec are associated with multiorgan failure, thrombocytopenia, neutropenia, and death. 84 administration of anti-lps antibodies to a small number of these deficiencies as well as those related to cellular function in combination with invasive procedures lead to a reduction in respiratory barrier function that increases the risk for sepsis. once the local barrier function has been compromised, pathogen recognition by local immune sentinel cells is the first step towards the development of an immune response (figure 152-3) . elegant sensing mechanisms have evolved to facilitate detection of potentially pathogenic microorganisms. multiple classes of pathogen recognition receptors (prrs) have been discovered that serve as detectors of pathogen-associated molecular patterns (pamps), including cell wall and membrane components, flagellum, nucleic acids, and carbohydrates. 99 a litany of prr classes have been discovered, including the toll-like receptors (tlrs), nod-like receptors (nlrs), retinoic acid-inducible protein i like receptors (rlrs), peptidoglycan recognition proteins, β 2 -integrins, and c-type lectin receptors. the tlrs, β 2integrins, and c-type lectin receptors detect pathogens both on neonates with sepsis (n = 16) with serum endotoxin present reduced the time to recovery but not mortality as compared with the values in placebo-treated neonates. 107 reduction of serum lps levels by exchange transfusion in infected neonates (n = 10) was associated with improved survival. 108 bacterial cell wall components (such as lipoteichoic acid) signal primarily through tlr1, tlr2, and tlr6, flagellin signals through tlr5, and cpg double-stranded dna signals through tlr9. common viral pamps such as double-stranded rna or single-stranded rna signal through tlr3, and tlr7 and tlr8, respectively. agonist-tlr binding results in a signaling cascade of intracellular second-messenger proteins ultimately leading to production of cytokines and chemokines, as well as activation of other antimicrobial effector mechanisms. 101 signaling through tlrs typically leads to the production of nuclear factor κb (nf-κb)-dependent inflammatory cytokines and chemokines, whereas signaling through toll/il-1 receptor-domain-containing adapter inducing interferon (ifn)-β (trif) induces production of type i ifns, as well as nf-κb-related inflammatory cytokines. in neonates of all gestational ages, up-regulation of tlr2 and tlr4 messenger rna (mrna) occurs during gram-positive and gram-negative infection, respectively. 109 dysregulation or overexpression of tlr4 is involved in the development of nec in experimental animal models, 110 implicating the importance of tlrs in the initial immune response to pathogens and their role in neonatal sepsis. other important intracellular prrs include nlrs and rlrs. for nlrs, multiple cytosolic proteins are able to act as pamp sensors (e.g., nlrp1, nlrp3, and nlrc4) and coalesce with adaptor proteins and procaspase 1 to form a multimeric protein complex termed the inflammasome. 111 the formation of the inflammasome results in the conversion of procaspase to active caspase 1, which cleaves the inactive precursor proteins il-1β and il-18 to their active forms. 111 rlrs are cytoplasmic rna helicases that, like tlr3, sense double-stranded rna of viral origin and induce type i ifn production and nf-κb activation. 102 to date, the impact of rlr and nlr signaling has not been specifically examined in neonates with sepsis. in addition to its roles in leukocyte function (adhesion, phagocytosis, migration, and activation) and complement binding, complement receptor 3 (cr3, also known as mac-1 and cd11b-cd18) functions as a pathogen sensor on the surface of phagocytes. cr3 binds lps, as well as a broad range of other microbial products, in cooperation with or independently of cd14, leading to up-regulation of inducible nitric oxide (no) synthase and no production. 112 diminished expression of l-selectin and cr3 on stimulated neonatal pmns impairs activation and accumulation at sites of inflammation. 99, 113, 114 decreased expression of l-selectin and cr3 persists for at least the first month of life in term infants, possibly contributing to an increased risk for infection. 115 the expression of cr3 (cd11b) may be reduced further in preterm neonates as compared with term neonates. 116 in umbilical cord blood from neonates of less than 30 weeks' gestation, pmn cr3 content was similar to levels found in patients with type 1 leukocyte adhesion deficiency (failure to express cd18). 113, 114 thus decreased leukocyte cr3 surface expression increases the likelihood of suboptimal pathogen detection and cellular activation, particularly in the preterm neonate. c-type lectin receptors are prrs that recognize bacterial, viral, fungal, and parasitic carbohydrate moieties. c-type lectin receptors may be expressed on the cell surface (e.g., macrophage mannose receptor, mincle receptor, dectin 1, and dectin 2) or secreted as soluble proteins (e.g., mannose-binding lectin [mbl], (which is also named mannan-binding protein or mannanbinding lectin) as one of the acute-phase reactants. once bound to its carbohydrate ligand, mbl initiates activation of complement via the lectin pathway to promote opsonization and phagocytic clearance of pathogens. plasma mbl concentrations are low at birth (especially in preterm infants) but rise steadily throughout infancy and childhood. 117 low levels of mbl are associated with the increased incidence of sepsis in neonates. [118] [119] [120] in addition to decreased concentrations at birth, certain genetic polymorphisms of mbl (namely, mbl2), have been associated with an increased risk for infection in some, 121 but not all, studies. 122-124 m-ficolin activates the complement system in a manner similar to mbl and its level is elevated in neonates with sepsis. 125 prr stimulation results in rapid inflammatory mediator transcription and translation directed at cellular activation and clearance of pathogenic organisms 126 (see figure 152 -3). during sepsis and septic shock, multiple proinflammatory cytokines have been identified, including il-1β, il-6, il-8 (cxcl8), il-12, il-18, ifn-γ, and tumor necrosis factor (tnf)-α. 127 compared with adults with sepsis, neonates with sepsis produce less il-1β, tnf-α, ifnγ, and il-12. [128] [129] [130] [131] [132] [133] the decreased cytokine production is due in part to decreased production of important intracellular mediators of tlr signaling, including myeloid differentiation factor 88, ifn regulatory factor 5, and p38, which exhibit gestational age-specific decrements. 134 recent studies have demonstrated impaired inflammasome activation and mature il-1β production by neonatal mononuclear cells. 121, 135 in a comprehensive study (>140 analytes) of serum from neonates evaluated for late-onset sepsis, il-18 emerged as a predictive biomarker to differentiate infected neonates from uninfected neonates. 136 il-18 reduces pmn apoptosis, 137 drives ifn-γ production, 138 and induces production of tnf-α, il-1β, and cxcl8. 139 il-18 primes pmns for degranulation with production of reactive oxygen intermediates on subsequent stimulation. 140 dysregulation of many of these functions linked to il-18 are seen in sepsis and septic shock. increased il-18 levels have been demonstrated in premature neonates with brain injury 141 and also in an experimental model of nec, [142] [143] [144] highlighting activation pathways common with those in ischemia and inflammation. excessive levels of il-1β, tnf-α, il-6, cxcl8, il-10, and il-18, such as those seen with advanced-stage nec, severe sepsis, or septic shock, correlate with poor survival. 84, [145] [146] [147] [148] altered cytokine levels (increased il-10 and il-6 levels and decreased ccl5 levels) may identify those neonates at highest risk for the development of sepsisassociated disseminated intravascular coagulation (dic). 149 proinflammatory cytokine production leads to activation of endothelial cells, including increased expression of cell adhesion molecules that facilitate leukocyte recruitment and diapedesis ( figure 152-4) . up-regulation of cell adhesion molecules (soluble intercellular adhesion molecule, vascular cell adhesion molecule, l-selectin, p-selectin, e-selectins, and cd11b-cd18) during sepsis facilitates rolling and extravascular migration of leukocytes. [150] [151] [152] [153] decreased production of l-selectin and expression of c3 in pmns and monocytes derived from neonates may impair accumulation at sites of inflammation. 113, 114 chemokine gradients produced by endothelial cells and local macrophages are necessary for effective and specific leukocyte attraction and accumulation (see figure 152 -4). without adequate leukocyte recruitment, there is increased risk for propagation from a localized to a systemic infection. although poor cellular chemotaxis in the neonate has been observed, it is not likely a result of reduced serum concentrations of chemokines as baseline levels are similar in preterm and term neonates as compared with adults. 154 suboptimal cellular chemotaxis may be related to other mechanisms, such as poor complement receptor translocation. 162 the role of hmgb-1 and rage signaling in human neonates with sepsis has not been well characterized but has been shown to be involved in the pathophysiology of nec in an experimental model. 163 significantly lower soluble rage levels were found in human fetuses that mounted robust inflammatory responses and hmgb-1 levels correlated significantly with the levels of il-6 and s100β calcium-binding protein in the fetal circulation. 164 other specific damage-associated molecular patterns, including heat shock proteins and uric acid, may also stimulate tlrs, regulate pmn function, and serve as immune adjuvants. heat shock protein production in infected neonates has not been evaluated but polymorphisms in heat shock proteins increase the risk for acute renal failure in preterm neonates. 165 the levels of heat shock proteins are significantly elevated in infected adults and children. 166 elevated heat shock protein 60 and heat shock protein 70 level measured within 24 hours of pediatric intensive care unit admission were associated with septic shock and there was a strong trend towards increased mortality. 167, 168 uric acid can increase cytokine production, pmn recruitment, and dendritic cell stimulation 169 and may also serve as an antioxidant. 170 the level of uric acid is reduced in the serum of neonates with sepsis as compared with control neonates. 171 in addition to facilitating leukocyte attraction, proinflammatory stimuli result in production of vasoactive substances that decrease or increase vascular tone and alter vascular permeability (see figure 152 -4). these include platelet-activating factor, thromboxane, leukotrienes, no, histamine, bradykinin, up-regulation after stimulation, 99 deficiencies in another downstream signaling process, 155 or inhibition by bacterial products. 156 the levels of a wide variety of chemokines are increased during sepsis, including cxcl10 (ip-10), ccl5 (rantes), ccl2 (monocyte chemoattractant protein 1), ccl3 (macrophage inflammatory protein 1α), and cxcl8. 157 the levels of other chemoattractive molecules also increase with sepsis, including complement proteins c3a and c5a, apps, including cathelicidins and defensins, and components of invading bacteria themselves. 127, 136 the importance of chemoattractive substances in the pathogenesis of severe sepsis is highlighted by studies showing that cxcl8 can be used as a stratifying factor for survival in children, 158 and c5a is implicated in sepsis-associated organ dysfunction in adults. 104 chemokine investigations in infected neonates revealed that cxcl10 is a sensitive early marker of infection, 157 and low ccl5 levels may predict development of dic. 149 damage-associated molecular patterns (or alarmins), such as intracellular proteins or mediators released by dying or damaged cells, may also active prrs. for example, the damage-associated molecular pattern high-mobility group box 1 (hmgb-1) is involved in the progression of sepsis to septic shock in adults. 104, 159 macrophages or endothelial cells stimulated with lps or tnf-α produce hmgb-1, which signals through tlr2, tlr4, and receptor for advanced glycation end products (rage). 160 hmgb-1 results in cytokine production, activation of coagulation, and pmn recruitment. 159,161 hmgb-1 mediates disruption of epithelial junctions within the gut via the induction of reactive nitrogen intermediates, leading to increased bacterial a twin study which assessed the frequency of infections among monoygotic and dizygotic prematurely born twins concluded that 49.0% (p = .002) of the variance in susceptibility to lateonset sepsis was due to genetic factors alone. 215 the impact of genetics in the host response is also underscored by the increased risk for death from infection seen with african american race or male sex among low-birth-weight infants. 216 an ethnically unique single nucleotide polymorphism in the tlr4 promoter region was significantly associated with gram-negative bacterial infections in preterm infants. 217 several recent studies in newborn infants have demonstrated an association between small variations in dna, specifically single nucleotide polymorphisms, and infection development and outcomes. 122, [218] [219] [220] [221] [222] because tlrs play an essential role in recognition and response to pathogens, alterations in their expression, structure, signaling pathways, and function can have consequences for host defense. polymorphisms or mutations in tlrs are associated with increased risk for infection in adults [223] [224] [225] [226] and children 210, 227, 228 but are less well characterized in neonates. after confounders had been controlled for, the presence of a tlr4 single nucleotide polymorphism was associated with a three-fold increase in the risk for gram-negative infections in vlbw infants. 222 polymorphisms in the tlr2, tlr5, il10, and pla2g2a (which encodes pla 2 ) genes were associated with the development of neonatal sepsis. 218 modifications in expression or function of costimulatory molecules necessary for tlr activation are also associated with an increased risk for infection. for example, the levels of lpsbinding protein (lbp; which binds intravascular lps) and the lps coreceptor cd14 are both increased during neonatal sepsis. 211, 229, 230 furthermore, genetic variations in these proteins have been associated with increased risk for sepsis in adults. [231] [232] [233] genetic polymorphisms in myeloid differentiation factor 2, a small protein involved in lps signaling through tlr4, increase the risk for organ dysfunction and sepsis in adults 234 but the significance in neonates is unknown. polymorphisms in post-tlr activation intracellular signaling molecules, including myeloid differentiation factor 88, 235 il-1 receptor-associated kinase 4, 236 and nf-κb essential modulator, 237 are associated with invasive bacterial infection in older populations. additional genetic polymorphisms in intracellular second-messenger inflammatory signaling systems with impact on neonatal sepsis risk and progression are likely to be uncovered with the implementation of biobanking and mining of stored samples. mutations have been identified in nlrs that are involved in the pathogenesis of crohn's disease (nod2) 238 and neonatalonset multisystem inflammatory disease (nlrp3). 239 rlr mutations have been identified but have unknown clinical significance. 240 no mutations in specific domains of nlrs have been found in neonates with sepsis or nec. 220, [231] [232] [233] [241] [242] [243] [244] [245] [246] [247] [248] [249] [250] [251] [252] the importance of nlrs in listeria monocytogenes infections in neonates is unknown. complement is an important component of early innate immunity that facilitates killing of bacteria through opsonization and direct microbicidal activity. complement components also possess chemotactic or anaphylactic activity that increases leukocyte aggregation and local vascular permeability. furthermore, complement reciprocally activates a number of other important processes, such as coagulation, proinflammatory and prostaglandins. 172, 173 these substances are produced predominantly by host endothelium and mast cells. activated pmns produce phospholipase a 2 (pla 2 ), the level of which is increased in the serum of neonates with sepsis 174 and leads to generation of vasoactive substances, including prostaglandins and leukotriene. thromboxane produced by activated platelets and endothelin 1 produced by activated endothelium 175 are potent vasoconstrictors that participate in the development of pulmonary hypertension. [176] [177] [178] [179] overproduction of cytokines and vasoactive substances is associated with circulatory alterations and organ failure seen in severe sepsis and septic shock ( figure 152-5) . 6, [180] [181] [182] [183] if the pathogen is not contained locally and inflammatory homeostasis is not restored, sirs may develop, and lead to mods and death (see figure 152 -5). 184 the traditional paradigm for understanding the host response to sepsis consists of an intense proinflammatory response, or sirs, temporally followed by a compensatory antiinflammatory response syndrome. this paradigm has been challenged by the failure of multiple antiinflammatory strategies to improve sepsis outcomes in adults. 185 new data in adults and children demonstrate simultaneous proinflammatory/antiinflammatory responses where the magnitude of either response may determine outcome. 186, 187 near simultaneous increases in antiinflammatory cytokine production (transforming growth factor β, il-4, il-10, il-11, and il-13) occur in neonates during infection, countering the actions of proinflammatory cytokines. 127, 188, 189 these mediators blunt the activation and recruitment of phagocytic cells, reduce fever, modify coagulation factor expression, and decrease production of reactive oxygen and nitrogen intermediates, no, and other vasoactive mediators. [190] [191] [192] [193] [194] [195] soluble cytokine and receptor antagonists produced during sepsis also modulate proinflammatory mediator action. elevation of the levels of tnf receptor 2 (which regulates the concentration of tnf-α), soluble il-6 receptor, soluble il-2, and il-1 receptor antagonist have been documented in neonatal sepsis with resolution after effective treatment. 189, 196, 197 the role of these regulatory cytokine inhibitors in the immune response to neonatal sepsis and septic shock has been incompletely characterized. soluble rage competes with cell-bound rage for the binding of hmgb-1 and other rage ligands. 198 soluble rage has antiinflammatory effects and its level is elevated in adults during sepsis. 199 furthermore, soluble rage improved survival and reduced inflammation when given to infected adult rodents. 200 serum soluble triggering receptor expressed on myeloid cells 1 may reduce inflammatory signaling for triggering receptor expressed on myeloid cells 1, and predict mortality in preterm neonates. 201 micrornas may regulate inflammation at the level of gene expression via several putative mechanisms. 202 several pilot studies in rodents and humans have demonstrated regulatory functions for microrna in neonates. [203] [204] [205] [206] [207] [208] the impact of regulatory micrornas and their effects on the host inflammatory response in neonates with sepsis are unclear. endogenous cortisol is induced by proinflammatory cytokines and attenuates the intensity of sirs associated with severe sepsis and septic shock. 209 the use of cortisol in adults with sepsis has been controversial. 210, 211 cortisol production in newborn infants is significantly increased early in shock. 212 however, very preterm neonates may have relative adrenal insufficiency that may contribute to hemodynamic instability and hypotension. cortisol replacement may be critical in these infants and deserves further study. 213 it is important to note, however, that in children with septic shock, adjunctive corticosteroid therapy is associated with repression of gene programs corresponding to the adaptive immune system. 214 complement-mediated activation of leukocytes during sepsis occurs via up-regulated cell surface receptors (complement receptor 1 [cd35] and cr3). 257, 258 c3b and c5a facilitate opsonization (primarily c3b), redistribute blood flow, and increase inflammation, platelet aggregation, and release of reactive oxygen intermediates (primarily c5a). 259, 260 c5a-mediated local leukocyte activation also results in increased cytokine production with subsequent up-regulation of adhesion molecules on vascular endothelium and increased cell recruitment to the site of infection. 261 data in adults link elevated c5a levels with multiple facets of sepsis-associated disease, such as dic, cardiac dysfunction, increased proinflammatory cytokine levels, sirs, apoptosis of adrenal medullary cells leading to adrenal cytokine production, and leukocyte activation. 104 contrary to its name, the alternative pathway is the primary mechanism of amplification of complement activation after c3 convertase assembly (which cleaves c3 to c3a and c3b). dysregulation of complement activation may contribute to adverse effects in individuals with severe sepsis or septic shock. neonates, particularly the very premature, exhibit decreased basal levels of complement proteins and function for both the alternative pathway and the classical pathway. 253, 254 moreover, as compared with adults, neonates exhibit gestational age-related degrees of depressed complement-mediated opsonic capabilities. 255 as such, complement-mediated opsonization is poor in premature neonates and limited in term neonates. 255 nization and pathogen clearance may help explain the lack of efficacy of intravenous immunoglobulin to prevent sepsis or death from sepsis in neonates. [279] [280] [281] [282] [283] [284] [285] apps are the most phylogenetically ancient means of innate immune defense against microbial invasion. present in nearly every organism, including bacteria, plants, insects, nonmammalian vertebrates, and mammals, these small, often cationic peptides are capable of killing microbes of multiple types, including viruses, bacteria, parasites, and fungi, largely by disruption of the pathogen membrane. 286 constitutive expression of apps occurs in humans on barrier areas with consistent microbial exposure such as skin and mucosa. after microbial stimulation, both release of preformed apps and inducible expression are thought to contribute to early host defense. 287 importantly, there is no evidence for the development of microbial resistance to apps that target fundamental components of the microbial cell wall. some apps can bind and neutralize microbial components such as endotoxin, precluding engagement with tlrs and other prrs, and diminish inflammation. many apps can potentially reduce the intensity of the inflammatory response associated with the presence of bacterial toxins. [288] [289] [290] because endotoxemia is an important contributor to neonatal mods and death with sepsis and nec, 84 lps-binding/blocking strategies, including use of synthetic apps, may have a significant positive impact on outcomes. 288, 291 bactericidal/permeability-increasing protein (bpi) is a 55-kda protein present in the respiratory tract, pmn primary granules, and plasma. bpi exerts selective cytotoxic, antiendotoxic, and opsonic activity against gram-negative bacteria. 288 plasma bpi concentrations were higher in critically ill children with sepsis syndrome or organ system failure than in critically ill children without sepsis syndrome or organ system failure, and bpi levels positively correlated with the pediatric risk for death score. 265 pmns from term neonates are deficient in bpi, potentially contributing to the increased risk for infection. 292 whereas term neonates demonstrate up-regulation of plasma bpi during infection, premature neonates showed a decreased ability to mobilize bpi on stimulation, 293 which may contribute to their risk for infection with gram-negative bacteria. polymorphisms in bpi increase the risk for gram-negative sepsis in children, but the impact of these polymorphisms in neonates is unknown. 294 compared with pmns from adults, pmns from term neonates produce similar quantities of defensins but reduced quantities of bpi and elastase. 292, 295, 296 recombinant bpi (rbpi 21 ) treatment was associated with improved functional outcome, reduced amputation, but no difference in mortality in a multicenter study of children with severe systemic meningococcal disease. 297 lactoferrin is the major whey protein in mammalian milk (in particularly high concentrations in colostrum) and is important in innate immune host defenses. lactoferrin is present in tears and saliva and has antimicrobial activity both via binding iron and by direct membrane disruption activity via a portion of its amino-terminal lactoferricin. 298 lactoferrin is also an alarmin (e.g., hmgb-1 or il-33), capable of activating leukocytes, binding endotoxin, and modifying the host response by acting as a transcription factor that regulates mrna decay. 299, 300 bovine lactoferrin has been shown to reduce the incidence of bacterial and fungal sepsis 301, 302 and nec in preterm infants. 303 lysozyme is present in tears, tracheal aspirates, skin, and pmn primary and secondary granules and contributes to degradation of peptidoglycan in bacterial cell walls. secretory pla 2 can destroy gram-positive bacteria through hydrolysis of their membrane lipids. 174 pmn elastase is a serine protease released by activated pmns with microbicidal function and is believed to play a role in the inflammatory damage seen with pmn recruitment, particularly in the lung. 116, 136 cathelicidin and the insufficiency, and pmn dysfunction. 104 septic shock in adult humans was associated with extensive complement activation, c-reactive protein-dependent loss of c5a receptor on neutrophils, and the appearance of circulating c5a receptor in serum, which correlated with a poor outcome. 262 deficiencies in c5a receptor found in term neonates as compared with adults may limit the ability to respond to c5a and therefore increase the likelihood of infection. 240 the expression of c5a receptor on preterm pmns is unknown. the extent to which c5a or other complement proteins play a role in the development of disease in septic neonates remains to be determined. complement regulatory proteins modify the effects of complement and prevent potential damage due to overactivation. in particular, cd59 blocks c9 polymerization and target lysis, cd55 destabilizes cd35 and c3 and c5 convertases, and cd35 accelerates the deactivation of c3b. 263 dysregulation of complement activation can lead to a vicious activation cycle that results in excessive cellular stimulation, cytokine production, endothelial cell activation, and local tissue damage promulgating sirs and septic shock (see figure 152 -5). 264 in addition to the initial inflammatory response including complement activation, molecular detection of pamps promotes il-1β and il-6 production, which in turn increases the production of multiple other innate proteins that possess valuable immune function and serve to reduce pathogen load. 265 acutephase reactant proteins, produced predominantly in the liver, include c-reactive protein (opsonin), serum amyloid a (cellular recruitment), lactoferrin (reduces the level of available iron/ antimicrobial peptide lactoferricin), procalcitonin (unknown function), haptoglobin, fibronectin (opsonic function), pentraxin 3 (binds c1q and activates the classical complement pathway), mbl, and lps-binding protein. 127, 211, 229, [265] [266] [267] [268] [269] [270] acutephase reactant proteins have been studied in neonates with sepsis primarily to assess them for diagnostic utility rather than immunologic function. in particular, elevated plasma concentrations of c-reactive protein and lps-binding protein are often associated with early-onset sepsis. 229, 271 the levels of il-10 and c-reactive protein were significantly higher in preterm infants who did not survive sepsis, pneumonia, or nec. 272 a lack of sustained increase in the production of c-reactive protein and serum amyloid a during sepsis has also been associated with a fulminant course. 273 the fetus receives antibodies from the mother via active placental transfer, with a significant increase beginning around 20 weeks' gestation. as a result of a shorter period of gestation, preterm neonates have lower igg subclass levels as compared with term neonates, particularly igg1 and igg2 subclasses. 274 preterm neonates (24 to 32 weeks' gestation) with low igg levels (serum total igg levels below 400 mg/dl at birth) were at increased risk for development of late-onset sepsis but not death compared to those with levels above 400 mg/dl. however, igg titers and opsonic activity to cons were not predictive of late-onset cons sepsis. 275 reliance on other means of innate immune defense likely provides the premature neonate with alternative microbial control mechanisms. despite the presence of maternally derived immunoglobulin and acute-phase reactant proteins, neonates exhibit impaired opsonizing activity compared with adults, which likely increases the risk for progression of infection. 276 complement plays a critical role in immunoglobulin-mediated opsonization and effector cell phagocytosis. 277 although immunoglobulin has many putative beneficial immunologic functions, most of these have not been demonstrated or examined in preterm infants. 278 the dependence on complement for effective immunoglobulin-based opso-thrombocytopenia in neonates, 322 which is attributed to reduced megakaryopoiesis in the setting of consumption with clot formation. 323 decreased platelet function in preterm neonates with sepsis further increases the risk for bleeding. 324 in extremely low-birth-weight infants, platelets are hyporeactive for the first few days after birth, complicating the ability of the immune system to contain a microbiologic threat and increasing the risk for hemorrhage. 325 clotting can lead to propagation of inflammation via thrombin-induced production of platelet-activating factor. pmns activated by platelet-activating factor or platelet tlr4 may then contribute to further endothelial injury and dysfunction, leading to the development of a vicious clottinginflammation-clotting cycle. activated platelets may be consumed in clot formation and/or may also be removed from the circulation by the liver, 326 potentially resulting in thrombocytopenia, particularly during gram-negative and fungal infections. 196, 322, 327 systemic activation of coagulation is associated with consumption of clotting factors and increased risk for bleeding, prolonged proinflammatory responses, and dic. 128, 149, 328 this finding is consistent with the elevated serum levels of il-6 52 and high frequency of dic seen with disseminated herpes simplex virus infection. 329 in adult mice, protease-activated receptor 1 plays a major role in orchestrating the interplay between coagulation and inflammation. 330 protease-activated receptor 1 may modify the endothelial response during neonatal sepsis and thus is a target for therapeutic intervention. recent studies have shown the critical importance of vascular endothelial activation in the early recognition and containment of microbial invasion. in transgenic mice, it was shown that pulmonary endothelial cells sense blood-borne bacteria and their products, 156 whereas alveolar macrophages patrol the air spaces. 331 these data illustrate the role of endothelium and help to explain in part the occurrence of acute respiratory distress syndrome (ards) and persistent pulmonary hypertension of the newborn associated with severe sepsis in the absence of a primary pulmonary infectious focus. expression of tlrs allows endothelium to become activated in the presence of microbial components, leading to production of cytokines, chemokines, and adhesion molecules (e.g., vascular cell adhesion molecule, intercellular cell adhesion molecule, l-selectin, p-selectin, and e-selectin). these substances are all necessary to attract immune cells (primarily pmns) to the site of infection and to facilitate pathogen containment. [150] [151] [152] [153] 156 vasoactive substances released from activated leukocytes, platelets, and endothelial cells include platelet-activating factor, thromboxanes, leukotrienes, no, histamine, bradykinin, and prostaglandins. 172, 173 the balance of no and endothelin 1, a vasoconstrictor, may be disrupted with endothelial damage, favoring the constrictive effects of endothelin 1 and leading to ischemia and injury. 175 this phenomenon may explain in part why no inhibitors increased mortality in adults with septic shock. 301 stimulated endothelium can be a doubleedged sword, however, because excessive activation can lead to systemic overproduction of cytokines and vasoactive substances (including no). endothelial cell apoptosis, detachment from the lamina, and alterations in vascular tone combine to promote capillary leak, leading to hypovolemia, shock, and organ failure 156,302,303 (see figure 152 -5). release of myeloperoxidase from pmns may also injure surrounding endothelium. 332 activated or damaged endothelium establishes a prothrombotic environment that can result in local microvascular occlusion 314 or progress to dic. 333 the glucocorticoid receptor is the target for cortisol, the primary endogenous glucocorticoid in humans, produced in the zona fasciculata of the adrenal glands. endothelial glucocorticoid defensins are other apps that possess antimicrobial properties. 304 cathelicidin is present in the amniotic fluid, vernix, skin, saliva, respiratory tract, and leukocytes. α-defensins are cysteine-rich 4-kda peptides found in amniotic fluid, vernix, spleen, cornea, thymus, paneth cells, and leukocytes. β-defensins are found in the skin, gi tract, urinary system, reproductive organs (placenta, uterus, testes, kidney), respiratory tract, breast milk, mammary gland, and thymus. in addition to microbicidal action, apps have a wide range of immunomodulatory effects on multiple cell types from both the innate immune system and the adaptive immune system. 287, 305, 306 these immunomodulatory effects include altered cytokine and chemokine production, improved cellular chemotaxis and recruitment, improved cell function (maturation, activation, phagocytosis, reactive oxygen intermediate production), enhancement of wound healing (neovascularization, mitogenesis), and decreased apoptosis. the cytosolic granules of pmn are rich in apps, including α-defensins, lactoferrin, lysozyme, cathelicidin, soluble pla 2 , and bpi. gestational age-related decreases in the umbilical cord blood concentration of several apps (cathelicidin, bpi, calprotectin, soluble pla 2 , α-defensins) in comparison with maternal serum levels have been drescibed. 307 plasma app deficiencies may contribute to the increased risk for infection associated with prematurity, and their absence may increase the risk for endotoxemia. compared with term neonates, preterm neonates showed lower human β-defensin 2 levels in umbilical cord blood. 308 up-regulation of apps (defensins) occurs in blood of infected adults 309 and children (defensins, lactoferrin). 310 the effect of sepsis on the production of plasma apps in neonates has not been investigated in detail. the development of a procoagulant state in the surrounding microvasculature allows the trapping of invading pathogens and prevents further dissemination (see figure 152 -5). in general, the intrinsic pathway amplifies coagulation after initiation by the extrinsic pathway. 311 reduced levels of vitamin k-dependent factors (factors ii, vii, ix, and x), reduced thrombin generation, reduced consumption of platelets with formation of microthrombi, and reduced levels of counterregulatory elements (inhibitors) increase the risk for bleeding in infants and children. 312 during sepsis, a microvascular procoagulant state develops via stimulation of phagocytes, platelets, and endothelium, resulting in expression of tissue factor. 313, 314 tissue factormediated activation of the coagulation cascade results in activation of thrombin-antithrombin complex, plasminogen activator inhibitor type 1, and plasmin-α 2 -antiplasmin complex, 315 as well as inactivation of protein s and depletion of the anticoagulant proteins antithrombin iii and protein c. [316] [317] [318] decreased activated protein c levels were associated with increased risk for death from sepsis in preterm neonates. 319 a randomized controlled trial of activated protein c revealed no change in mortality among pediatric patients with sepsis, but term infants younger than 60 days old experienced increased bleeding. 320 the coagulation cascade is intimately tied to inflammation and complement activation. 104 cytokine production increases expression of endothelial tissue plasminogen activator inhibitor type 1. plasminogen activator inhibitor type 1 inhibits fibrinolysis by inhibiting the conversion of plasminogen to plasmin, which in turn is important for the breakdown of fibrin. deposition of fibrin in small vessels leads to inadequate tissue perfusion and organ failure. 321 increased plasminogen activator inhibitor type excessive local inflammation and tissue damage. 350 high early levels of circulating free pmn-derived dna produced by nets are associated with mods and death. 351 nets contain destructive proteases capable of killing bacteria even after the pmn has died. 352 formation of nets is reduced in pmns from preterm neonates and nearly absent in term neonates 353 but may occur with sustained cellular stimulation. 354 net formation may result in collateral damage to surrounding tissues when the target microbe is too large to be effectively phagocytosed (e.g., fungal hyphae). 355 the contribution of net production to detrimental outcomes in infected neonates is unknown but excessive net formation with collateral tissue injury may contribute to the poor outcomes seen in preterm neonates with fungal infections. 356 rapid depletion of bone marrow pmn reserves during infection, particularly in neonates, 357 can lead to neutropenia, with consequent impaired antimicrobial defenses and significantly increased risk for death. 358 in a multivariate analysis, neutropenia and metabolic acidosis were associated with fatal neonatal sepsis. 359 neutropenia is particularly common in gram-negative sepsis in neonates. 360 release of immature pmn forms (bands), which exhibit greater dysfunction than mature pmns, 361 may further predispose to adverse outcomes. murine neonates with experimental sepsis exhibit delayed emergency myelopoiesis (a process by which the host repopulates peripheral myeloid cells lost early during sepsis), that is independent of trif and myeloid differentiation factor 88. 362 interventions aimed at addressing reduced pmn numbers in neonates have included provision of mature pmns 363 and prophylaxis or treatment with colonystimulating factors (granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor). despite strong biologic plausibility, these interventions have been unsuccessful at reducing the neonatal infectious burden. [364] [365] [366] in a metaanalysis, treatment with colony-stimulating factor therapy (granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor) in a subgroup (n = 97) of neutropenic neonates (absolute neutrophil count less than 1700/µl) with culture-positive sepsis (largely gram-negative and gbs) significantly reduced the risk for death (relative risk, 0.34; 95% ci, 0.12 to 0.92). 365 therefore, stimulation of granulopoiesis may be beneficial under these specific circumstances, although further studies focused on this subpopulation and outcomes are needed. irreversible aggregation and accumulation of newborn pmns in the vascular space after stimulation leads to decreased diapedesis, rapid depletion of bone marrow reserves, vascular crowding, 367 and increased likelihood of microvascular occlusion. 368 neonatal pmn deformation compared with adult pmn deformation is reduced at the baseline, which increases the risk for occlusion. 367 furthermore, low blood pressure/flow states seen during septic shock further exacerbate existing microvascular ischemia. 295 in combination, these deficiencies increase the propensity for systemic spread of infection, and set the stage for microvascular occlusion. many other cells besides pmns are involved in the development of an immune response to infection. monocytes, macrophages, and dendritic cells amplify cellular recruitment through production of inflammatory mediators, activation of endothelium, phagocytosis and killing of pathogens, and antigen presentation to t and b cells of the adaptive immune system. the primary functions of monocytes are the synthesis of crucial inflammatory proteins 369 and antigen presentation to naïve cd4 + t cells. 370 the patterns of cytokine production can promote the differentiation of naïve cd4 + t cells into distinct subtypes of t cells that serve important roles in the clearance of pathogens. for example, t-helper 1 (t h 1) cells are produced from naïve cd4 + t cells after exposure to ifn-γ and il-12, and support cellmediated immunity against intracellular pathogens through production of ifn-γ, tnf-α, and lymphotoxin. t-helper 2(t h 2) cells receptor is a critical negative regulator of inducible no synthase expression and nf-κb activation, 334 demonstrating a protective role of the endothelium during sepsis. studies have revealed a potential role of plasma angiopoietin during pediatric septic shock. 335 the level of angiopoietin 1, which protects against vascular leak, was reduced, whereas the level of angiopoietin 2, which promotes vascular permeability, was elevated, highlighting a novel potential therapeutic opportunity to reduce endorgan injury. the roles for endothelial glucocorticoid receptor and angiopoietin 1 in neonatal sepsis are unknown. the role of endothelium activation during sepsis and septic shock in neonates, particularly in premature neonates, has been less well investigated. toxins from gbs have been shown to damage pulmonary endothelium 336 and likely participate in pulmonary complications associated with gbs pneumonia such as ards and the development of persistent pulmonary hypertension of the newborn. 337 the levels of the adhesion molecules e-selectin and p-selectin, expressed and secreted by activated endothelium, are increased in the serum of neonates with sepsis 136 and likely reflect significant endothelial activation. endothelial tlr4 activation impaired intestinal perfusion in an experimental model of nec, via endothelial no synthasenitrite-no signaling. 338 the pmn is the primary effector of innate immune cellular defense. endothelial cells produce activating cytokines and chemokine gradients that recruit circulating pmns to the site of infection. expression of cell adhesion molecules by pmns and endothelium allows cells to roll and extravasate into surrounding tissues. activated pmns phagocytose and kill pathogens via oxygen-dependent and oxygen-independent mechanisms. il-1β is produced by activated pmns largely via an nlrp3-asccaspase 1-dependent* mechanism that amplifies the recruitment of additional pmns from the bone marrow to the site of infection. 339 activated pmns may release reactive nitrogen species, reactive oxygen species, and proteolytic enzymes via activation of membrane-associated nadph oxidase. these reactive intermediates and enzymes can lead to destruction of nonphagocytosed bacteria but can also cause local tissue destruction, including neonatal endothelial and lung injury, as well as surfactant inactivation, 116, 340 and thus play a role in progression from sepsis to mods. neonatal pmns exhibit quantitative and qualitative deficits as compared with adult pmns. 295, 341 respiratory burst activity is suppressed in pmns during neonatal sepsis and may contribute to poor microbicidal activity. [342] [343] [344] compared with adult pmns, neonatal pmns exhibit delayed apoptosis, 345, 346 as well as sustained capacity for activation (cd11b up-regulation) and cytotoxic function (reactive oxygen intermediate production) that contributes to tissue damage. 347 reduced apoptosis with prolonged survival of pmns may result in improved bacterial clearance but may also paradoxically increase the risk for sustained pmn-mediated tissue damage. increased serum pmn elastase, urokinase plasminogen activator, and urokinase plasminogen activator receptor levels are found at the time of presentation in infected neonates. 136 with pmn death, dna (chromatin), histones, and apps are expelled into the environment and serve to trap bacteria (neutrophil extracellular traps [nets]). 348 the formation of nets can occur after activation of platelet tlr4 349 and may lead to *asc, apoptosis-associated speck-like protein containing a carboxyterminal card. eosinophils phagocytose antigen-antibody complexes and release cytokines, chemokines, cytotoxic molecules, apps, and other substances (prostaglandins, thromboxanes, leukotrienes) when stimulated. 387 eosinophilia is commonly observed in neonates with sepsis due to candida sp. 388 and bacteria, 389 and is seen in infants with nec. 387 in infants of less than 26 weeks' gestation, eosinophilia (absolute eosinophil count more than 1000/mm 3 ) may predict bacterial sepsis. 389 eosinophilia in premature infants is not associated with production of ige. 390 studies have demonstrated an integral role for eosinophils in adult intestinal integrity and revealed a novel innate bactericidal nonphagocytic function via extracellular catapulting of mitochondrial dna nets with associated bound toxic proteins. 391 the precise role of eosinophils in the neonatal immune response to sepsis and in maintenance of intestinal integrity has yet to be determined. mast cells play a role in the response to pathogen invasion as a part of the innate cellular immune system via production of histamines (which promote vasodilation and up-regulation of p-selectin), cytokines, pmn recruitment, bacterial phagocytosis, and antigen presentation. 392, 393 mast cell involvement was demonstrated in infants with erythema toxicum, where mast cell recruitment, degranulation, and expression of apps occurs. 394 adult rodents deficient in mast cells exhibit impaired pmn influx, 395 impaired clearance of enteric organisms, and decreased sepsis survival. 396 mast cell production of histamine likely contributes to the vasodilation associated with sepsis and septic shock. like eosinophils and pmns, mast cells are capable of killing bacteria via generation of extracellular traps in adults. 397 this means of immune protection has not been investigated in neonates. mast cells may also alter adaptive immune function by patterning the t h 2 immunophenotype seen in the neonate and therefore contribute to the increased risk for infection. immature dendritic cells exposed to histamine during maturation (with lps) exhibit altered t-cell polarizing activity with predominance towards the t h 2 phenotype via increased production of il-10 and decreased production of il-2. 398 furthermore, mast cells from neonates were shown to secrete significantly more histamine after stimulation as compared with adults, 399 which may contribute to the development of shock. 400 the role of natural killer (nk) cells in neonatal bacterial sepsis is incompletely defined. nk cell numbers increase with increasing gestational age, 401 furthermore, a reduced percentage of nk cells present at birth may be a risk factor for late-onset sepsis in preterm infants. 402 it is noteworthy that the numbers of circulating nk cells are not significantly different in neonates with or without infection 370, 403 ; however, the numbers of circulating nk cells are decreased in newborn infants with shock. 404 the mechanisms used by nk cells to destroy bacteria include secretion of apps (defensins), direct contact and lysis, antibody-dependent cellular cytotoxicity, and ifn-γ production. 405 in neonates with bacterial sepsis, nk cells are activated, as evidenced by up-regulation of cd69. 2, 406 despite activation, nk cell cytotoxicity is deficient in infants with sepsis and recurrent infections. 370, 405 although neonatal macrophages exhibit impaired baseline activation in response to ifn-γ, 341 nk cell-mediated production of ifn-γ can enhance their phagocytic capability. further studies are necessary to more clearly define the role of nk cells in neonatal bacterial sepsis. cd71 + ter119 + (erythroid) cells may contribute to the increased susceptibility of the neonate to infection by reducing the inflammatory response associated with bacterial colonization of the gut. for example, ex vivo tnf-α production by stimulated adult effector cells was reduced in the presence of murine neonatal splenic cd71 + erythroid cells via an arginase 2-dependnent mechanism. 407 the cd71 + erythroid population represents a large portion of murine fetal liver, neonatal spleen/bone marrow, and adult bone marrow. [408] [409] [410] furthermore, the murine neonatal spleen contains large numbers of colony-forming progenitor cells for 2 to 3 weeks after birth. 411 of note and in stark contrast arise in the presence of il-2 and il-4, produce il-4, il-5, and il-13, down-regulate t h 1 responses, and support humoral immunity, as well as defense against extracellular parasites. a third subset of t h cells, t-helper 17 cells, are generated in the presence of transforming growth factor β, il-6, il-21, and il-23. these cells produce il-17 and il-22, which are important for defense against extracellular bacteria and fungi. neonatal mononuclear cells exhibit a bias away from t h 1 cell-polarizing activity because of increased il-6 and low tnf-α production. 371 this may be beneficial because of mobilization of antiinfective proteins/ peptides that serve to protect the newborn during microbial colonization 266 and development of immune tolerance. 341 the adverse consequence is a reduced ability to respond to infection with microorganisms; particularly intracellular pathogens such as listeria sp. 372 and mycobacteria. 373 preterm infants (<30 weeks) may have greater attenuation of tnf-α and il-6 secretion compared with term infants and adults. 277 there is decreased monocytic recruitment to areas of inflammation during sepsis because of decreased chemotactic ability. 374 although the levels of peripheral monocytes decrease early during sepsis (between 60 nd 120 hours), secondary to extravasation and differentiation into macrophages, sepsis-related elevation of macrophage colony-stimulating factor 375 results in a late increase in the number of peripheral monocytes (>120 hours). 376 in addition to altered cytokine production and suboptimal recruitment, monocyte phagocytic function is reduced during sepsis. 377 antigen presentation to naïve cd4 + t cells is an important immune function performed by monocytes. the decreased antigen-presenting function in monocytes from newborn infants is in part due to decreased mhc class 2 molecule expression 378 and decreased expression of costimulatory molecules, including cd86 and cd40. 379 monocytes leave the bloodstream, enter the tissues, and differentiate into macrophages and dendritic cells. monocytes and macrophages are closely related to pmns (common myeloid progenitor) and can kill pathogens by similar means. circulating monocytes differentiate into macrophages after exposure to maturing cytokines, and exit the bloodstream into tissues. important substances produced by stimulated monocytes/macrophages include complement components, cytokines (both proinflammatory and antiinflammatory), coagulation factors, and extracellular matrix proteins. 369 located just below epithelial borders, macrophages encounter pathogens immediately after entry. macrophages are avidly phagocytic and generate apps to reduce bacterial burden, such as lactoferrin, defensins, transferrin, and lysozyme. in addition, macrophages play an important role in the amplification of the immune response through the production of cytokines and chemokines, as well as in antigen presentation to naïve cd4 + t cells. macrophages are poorly responsive to several tlr agonists. 380 dendritic cells are antigen-presenting cells that function as a liaison between the innate immune system and the adaptive immune system through induction of antigen-specific t cellmediated immunity. dendritic cells from newborn infants exhibit a reduced antigen-presenting function when compared with adult cells 379 and require increased stimulation for activation. 381 evaluations of neonatal dendritic cell function suggest a tendency towards poor up-regulation of costimulatory molecules (cd80/cd86) and activation markers (cd83), poor stimulation of t-cell proliferation, and a tendency towards the induction of immune tolerance. 382 although preterm and term infants and adults have similar numbers of "plasmacytoid" dendritic cells in their blood, the capacity to produce ifn-α on tlr9 challenge was significantly decreased in preterm neonates and may increase susceptibility to viral infections. 383 dendritic cells in umbilical cord blood can effectively induce cytotoxic lymphocyte responses. 384 depletion of dendritic cells has been reported in adult animals 385 and adult patients 386 with sepsis; their role in the immune response to neonatal sepsis is not well characterized. pmn and γδ t cells. 432 these data show that neonatal t cells are activated and are capable of playing a role in the host response to bacterial sepsis in vivo. neonatal lymphocyte function is skewed towards t h 2 responses, setting the stage for immune tolerance (t h 2) rather than immune priming for infection (t h 1). 417 newborn infants must overcome that immune modulation in order to mount effective responses to specific infectious challenges and respond to vaccination. examples of the impact of this immunopolarization include decreased ifn-γ production by cd4 + and cd8 + t cells as compared with production in children and adults. 433, 434 the likely significance of decreased ifn-γ production is a reduction in activation of other immune cells, such as macrophages. reports of lymphocyte function in infected newborns are very limited. expansion of lymphocytes after antigenic stimulation is important for development of sustained immunity. decreased lymphocyte proliferative responses have been shown during the first 8 weeks of life in vlbw neonates, 421 and may predispose the premature neonate to development of late-onset sepsis. for example, t-lymphocyte function was depressed in infected newborn infants, and especially in those with multiorgan failure, versus healthy term or growing preterm infants. 435 similarly, production of lymphocyte-associated cytokines after stimulation of umbilical cord blood peripheral blood mono nuclear cells with gbs was significantly deficient in preterm and term infants compared with adults. 277 cytomegalovirus infection in utero leads to the expansion and differentiation of mature cytomegalovirusspecific cd8 + t cells, which have characteristics similar to adult cd8 + t cells. 436 these cells showed potent perforin-dependent cytolytic activity and produce antiviral cytokines, highlighting the potential for adult-like immunocompetence of neonatal t cells under specific circumstances. an important location for effective lymphocytic function during systemic bacterial infection is the spleen. the marginal zone of the spleen facilitates the clearance of bacteria, particularly encapsulated organisms, from the bloodstream. these functions are accomplished via the interaction of multiple leukocytes, including macrophages, dendritic cells, b cells, and t cells, within follicles of the spleen. the neonatal splenic marginal zone is immature, owing to a lack of antecedent antigen exposure and is virtually devoid of cd21 + b cells. 412 as a result of this functional asplenia, there is decreased clearance of pathogens from the blood and potential for a more fulminant course with bacteremia. 437, 438 b cells are critically important in the adult host response to sepsis. data suggest antibody-independent and antibodydependent roles for b cells in the outcome of sepsis. 416 studies deciphering the role of b cells in neonatal sepsis are very limited, and thus the role b cells play in the neonatal host response is unclear. after gbs meningitis, the level of igm was increased, suggesting b cells from neonates can respond to pathogenic challenge. 439 premature neonates with perinatal infection or nosocomial infection may show signs of humoral immunoparalysis, manifested by decreased igm/igg production ex vivo as compared with production in their healthy age-matched counterparts. 440 sepsis in early life did not reduce serum antibody titers in preterm infants after heptavalent pneumococcal conjugate vaccine exposure but was associated with a reduced opsonization titer to a single serotype, suggesting the capacity to respond to vaccination or other immune challenge may be altered. 441 in the setting of reduced classic adaptive immune function seen in early life as compared with the function in adults, innate lymphoid populations (which lack b cell receptor and t cell receptor) may play a significant role in protecting the neonate from infectious challenge. [442] [443] [444] [445] [446] [447] [448] examples of innate lymphoid cells include γδ t cells, intestinal lymphoid cells, invariant nk t cells, mucosa-associated invariant t cells, and b1 cells. to the lymphoid and reticuloendothelial system roles of the spleen in the healthy adult, the spleen is normally a major site of erythropoiesis during fetal and neonatal life, to support rapid fetal and postnatal growth in the setting of significantly reduced erythroid reservoirs as compared with the adult reservoirs. 410, 412, 413 a lack of effect on neonatal murine survival to polymicrobial sepsis after adoptive transfer or diminution of cd71 + erythroid splenocytes suggests that the impact of these cells on neonatal infection risk and progression may be limited. 414 the contribution of the adaptive immune system in the neonatal host response to sepsis is uncertain. the 5-to 7-day interval required for development of an adaptive immune responsenamely, the selection and amplification of specific clones of lymphocytes (b cells and t cells) that results in immunologic memory-argues against a central role for adaptive immunity in the protective response to early neonatal bacterial sepsis. as a result, the neonate is thought to largely depend on innate immunity for protection from infection during the first days of life. in adults, absence or dysfunction of the adaptive immune system has a profound impact on survival in preclinical models. 415 b cells (and in particular b-cell cytokine production) and not t cells were shown to be important in the early host response to experimental sepsis. 416 studies using neonatal mice lacking an adaptive immune system showed no difference in polymicrobial sepsis survival as compared with survival of wild-type mice with an intact adaptive immune system. 3 furthermore, there are many quantitative and qualitative differences in lymphocytes from neonates compared with lymphocytes from adults, 417 each with a respective proposed clinical impact. 87 as these findings illustrate, the contribution of adaptive immunity for protection and response against sepsis, and in particular which components are protective, is unclear in the most immature and requires further investigation. peripheral blood examination has yielded inconsistent changes in the percentage, number, and type of circulating lymphocytes during sepsis. 403, [418] [419] [420] [421] [422] [423] moreover, changes related to the timing of sepsis onset (early-onset or late-onset sepsis) and prematurity have been incompletely characterized. t regulatory cells are abundant and potent at birth, facilitating inhibition of t h 1 cell immunity, 424 and perhaps mediating a state of immunologic tolerance. 425 although the numbers of splenic t regulatory cells are increased in murine neonates and adults with sepsis, depletion of t regulatory cells had no effect on survival of murine adults. 2, 426 alterations in the number or function of t regulatory cells in human neonatal sepsis have not been reported. examination of peripheral blood to identify markers of sepsis has yielded a number of lymphocyte cell-surface molecules whose levels increase during sepsis. activation of neonatal t cells is evidenced by increased cd45ro expression (present on t cells after antigenic stimulation) at the time of sepsis diagnosis, 419, 427, 428 and with congenital infection, 429 although changes in number may take several days to occur after stimulation. 430 other markers of lymphocyte activation may be found at different time points during the course of infection. for example, expression of the activation marker cd69 is increased on t cells (cd4 + ) early in the infectious process, whereas cd25 and cd45ro expression persists for several days. 406 increased expression of cd4 + t-cell carcinoembryonic antigen-related cell adhesion molecule 1 (cd66a) in preterm infants with lateonset sepsis may contribute to sepsis-associated immune suppression. 431 hla-dr expression is increased on multiple cell types during neonatal sepsis. 406 in contrast to adults, a large portion of neonatal t cells produce cxcl8, which activates of genes for innate immune and metabolic pathways with decreased levels of adaptive immune transcripts. 467 using a proteomics approach, ng and colleagues 468 identified proapolipoprotein cii and a desarginine variant of serum amyloid a as promising biomarkers for late-onset sepsis and nec in preterm infants. 468 it is very likely that implementation of unbiased "omic" approaches will reveal critical age-appropriate pathways and opportunities for therapeutic interventions aimed at improving neonatal sepsis outcomes. sepsis that leads to shock and organ failure carries the worst prognosis. sirs contributes to the development of organ failure in neonates (see figure 152 -5). 52, 148, 181, 469 persistent decreases in capillary perfusion are associated with mods and death in adults. 470 lethargy, shock, and birth weight less than 1500 g were independent predictors of sepsis-related death. 471 in neonates, impairment of the cardiovascular system, manifested by poor perfusion or hypotension, is invariably associated with septic shock. sustained poor organ perfusion in neonatal sepsis and septic shock due to cardiovascular dysfunction is associated with mods affecting the kidney, 472,473 liver, 474 gut, 475 and central nervous system 476 (see figure 152 -5) . the mechanism of organ failure may be decreased oxygen utilization associated with mitochondrial dysfunction rather than poor oxygen delivery to tissue. 477, 478 on the basis of available evidence, it has been speculated that the prolonged sirs associated with severe sepsis and shock leads to organ failure via a cessation of energy-consuming processes. 479, 480 development of severe nec is also associated with severe sepsis, shock, mods, and death. 84, 481 the need for intubation or initiation of vasoactive medications, and hypoglycemia, thrombocytopenia, increased prothrombin time, or excessive bleeding as presenting laboratory signs of sepsis are risk factors for sepsis-related death. 359, 475, 482 independent predictors of in-hospital late-onset sepsis death during the birth hospitalization were the presence of congenital anomalies (or, 4.12; 95% ci, 1.60 to 10.60), neuromuscular comorbidities (or, 3.34; 95% ci, 1.66 to 6.73), and secondary pulmonary hypertension with/without cor pulmonale (or, 23.48; 95% ci, 5.96 to 92.49), 60 underscoring the impact of organ-level comorbidities that increase neonatal sepsis mortality. the most common organ dysfunction associated with sepsis is cardiovascular dysfunction. cardiovascular dysfunction associated with sepsis may lead to shock that is a composite of hypovolemic, cardiogenic, and distributive shock. distributive shock is related to endothelial no production that leads to excessive vasodilation. cardiogenic shock may be related to mitochondrial death (induced by reactive nitrogen and reactive oxygen intermediates) with subsequent myocardial dysfunction. abnormalities in peripheral vasoregulation and myocardial dysfunction may play a larger role in hemodynamic derangements in pediatric patients, especially infants and neonates. in children, a non-hyperdynamic state with reduced cardiac output and increased systemic vascular resistance is most commonly observed in the setting of sepsis. [483] [484] [485] [486] [487] the hemodynamic presentation in neonates is much more variable 484 and is complicated by an unclear association between a normal blood pressure and adequate systemic blood flow. 488, 489 microcirculatory flow is impaired in term neonates even with mild to moderate severity of infection. 490 preterm neonates with sepsis have relatively high left and right cardiac outputs and low systemic vascular resistances. however, a decrease in right or left ventricular output of more than 50% has been associated with increased mortality in neonatal sepsis. 491 an elevated left ventricular output mechanistic investigations that fully explore the role of these newly discovered populations in the neonatal host response to sepsis are likely to uncover novel therapeutic opportunities. systems biology and the use of "omic" approaches have the potential to produce significant insights into the pathogenesis of sepsis. genomic and proteomic approaches have yielded important data associated with septic shock in older populations. [449] [450] [451] [452] [453] [454] [455] [456] [457] the use of these modern techniques in the study of neonatal inflammation and response to pathogen challenge has only just begun. 136, 198, 458, 459 the ability to profile genome-wide expression has significantly enhanced our understanding of the complexity of the host immune response to sepsis in children. 5, 449, 450, 453, 460 for example, genome-wide expression profiling revealed zinc homeostasis as an important feature of pediatric sepsis. 450, 453, 461 prophylactic zinc supplementation reduced bacterial load and mortality in a murine model of peritoneal sepsis. 462 however, oral zinc supplementation did not alter mortality in neonates with probable sepsis. 463 in a study of pediatric patients who met the criteria for septic shock, 6 a unique whole-blood transcriptomic response was found in neonates as compared with infants, toddlers, and school-age children. neonates manifested the largest number of uniquely regulated genes, representing both innate and adaptive immune system pathways, and showed a predominance of down-regulated transcripts representing the adaptive immune system. 5 the number of up-regulated genes increased in proportion with developmental age. investigation of the murine circulating leukocyte transcriptome revealed significant differences in the host immune response to sepsis across the age spectrum (neonate, young adult, elderly), despite similar increases in mortality among the neonates and elderly mice as compared with young adult mice. 1 these data underscore the impact of developmental age on the host immune response and suggest that therapeutics, which may have efficacy in older populations, may be ineffective in or possibly detrimental to neonates. because the transition to extrauterine life is associated with dramatic changes in physiology, the whole-blood transcriptome is likely to be quite different between both uninfected infants and in the host response to sepsis by timing after birth. indeed, an unsupervised analysis of the whole-blood genome-wide transcriptome on prospectively collected peripheral blood samples from infants evaluated for sepsis revealed the major node of separation between groups (infected or uninfected) was the timing of evaluation relative to birth (early, lass than 3 days or late, more than 3 days). 464 principal component analyses revealed significant differences between patients with early or late sepsis despite the presence of similar key immunologic pathway aberrations in both groups. this study highlights both the uninfected state and the host responses to sepsis are significantly affected by timing relative to birth. a study of vlbw infants with blood culture-proven late-onset sepsis (59% cons) identified a 554-gene signature associated with sepsis, with increased expression of the tnf-α network, including matrix metalloproteinase 8 and cd177 among the most commonly up-regulated genes. 465 elevated matrix metalloproteinase 8 mrna expression and activity in septic shock correlated with decreased survival and increased organ failure in pediatric patients. matrix metalloproteinase 8 is a direct activator of nf-κb. 466 inhibition (genetic or pharmacologic) of matrix metalloproteinase 8 leads to improved survival and a blunted inflammatory profile in a murine model of sepsis. most recently, a 52-gene network was uncovered and validated that accurately identified infected infants, who exhibited increased expression phosphorylation, ubiquitination, sumoylation) may occur after sepsis. 126, 517 these dna alterations may modify transcription factor access of gene-specific promoter regions, ultimately leading to short-term and long-term changes in gene expression and immune function. the dna methylation pattern in the promoter region of the calca gene varied in different types of bacterial sepsis in preterm infants, suggesting its potential use as an epigenetic biomarker. 518 trained immunity, the term coined to describe an adaptive innate immune response, may also be a positive or negative consequence of sepsis in early life. 519 mechanisms that underlie trained immunity are beginning to emerge, and include dna methylation and modification of energy utilization pathways. 520, 521 nonspecific vaccine benefits and resistance to subsequent pathogen challenge after innate immune priming or previous infection are likely manifestations of trained immunity in neonates. 3, 513, 522 the cell types, extent, and duration of trained immunity-based modifications in neonates with sepsis have not been studied. myeloid suppressor cells manifest immunosuppressive activity with sepsis 523 and were recently described in neonates. myeloid suppressor cells are present at high frequency at birth and decline in number with postnatal age. they inhibit t-cell proliferative responses and ifn-γ production. 524 reactivation of viral infection that may contribute to morbidity and mortality has been demonstrated in infected adults. 525 the impact of this phenomenon in neonates is unknown. acute hypoxic respiratory failure, ards, and acute lung injury are common pulmonary complications associated with severe sepsis. destruction of the alveolar capillary membrane leads to refractory hypoxemia. after direct or indirect insults to the lung, alveolar macrophages produce chemokines that mitigate pmn influx to lung parenchyma. activated pmns release reactive oxygen and reactive nitrogen intermediates that damage endothelial and epithelial barriers, leading to leakage of protein-rich edema fluid into the air spaces. other pulmonary complications with severe sepsis may include secondary surfactant deficiency, 526 primary or secondary pneumonia, 527 and reactive pulmonary hypertension. 528, 529 infants with sepsis and persistent pulmonary hypertension of the newborn may require inhaled no in addition to optimized ventilation strategies such as highfrequency oscillatory ventilation. 530 if oxygenation or tissue perfusion remains severely compromised despite optimal medical management, extracorporeal membrane oxygenation should be considered in neonates weighing more than 2 kg without contraindications. 531,532 the detrimental neurodevelopmental long-term impact of sepsis has been demonstrated in multiple studies and has been reviewed in detail. [533] [534] [535] [536] [537] central nervous system injury is predominantly white-matter injury (loss of pre-oligodendrocytes), manifested by focal cystic periventricular leukomalacia, diffuse necrosis, or a combination of these entities. 538, 539 central nervous system injury is, in part, mediated by inflammation with or without direct pathogen invasion. 141, 540, 541 the impact of sepsis on central nervous system injury is intensified with lower gestational ages, highlighting the detrimental effects of sepsis on the developing brain. 539 the importance of evaluating the preterm infant for disseminated infection that may include meningitis cannot be overemphasized. a complete evaluation, including cultures of blood, urine and cerebrospinal fluid, is uncommon, 356 although one third of the cases of culture-positive meningitis in vlbw infants are associated with negative concurrent blood cultures. 542 clinically apparent seizures may occur in 25% of vlbw preterm infants with meningitis. 542 low-voltage background pattern, sleep-wake cycling, and seizure activity on but normal ejection fraction in preterm neonates with septic shock suggests that septic shock in preterm neonates is predominantly due to vasoregulatory failure. neonatal sepsis may or may not be associated with left ventricular diastolic dysfunction; however, cardiac injury as manifested by elevated levels of cardiac troponin t may complicate the clinical picture. 492, 493 abnormal peripheral vasoregulation with or without myocardial dysfunction is the primary mechanism for the hypotension accompanying septic shock in the neonate. 494 therefore, infected neonates may present with hypotension and adequate perfusion (warm shock) or inadequate perfusion (cold shock). myocardial dysfunction can lead to ventricular wall stretch that in turn elevates b-type natriuretic peptide levels. b-type natriuretic peptide levels are elevated in children with septic shock, 495 and increased levels have utility as prognostic indicators of death. 496 plasma no level is elevated in neonates with sepsis and shock compared wit those with shock alone. 182 elevated serum lactate level (>3 mmol/l) distinguished nonsurvivors from survivors in a pediatric study that included neonates. 497 after severe sepsis or septic shock, there is an increased risk for subsequent infection and death in children and adults. this phenomenon is termed immunoparalysis and is associated with reduced mhc class 2 expression and tnf-α production by mononuclear cells after endotoxin stimulation. in addition to altered monocytic responses, there is significant loss of lymphoid cd4 + t and b cells via caspase-dependent apoptotic pathways. 415, 498 whether by clonal selection, apoptosis, or elevated endogenous glucocorticoid levels, [499] [500] [501] lymphocyte loss may lead to a state of immune compromise after the acute phase of sepsis. 412, 499, [501] [502] [503] [504] [505] new data suggests that il-7 may play an important role in promoting t-cell activation and prevention of apoptosis. 506 the importance of immunoparalysis has been convincingly demonstrated in infected adults 507-510 and children. 511 however, the clinical impact in the preterm neonate in whom adaptive immune function is less well developed is uncertain. 512, 513 in examinations of peripheral blood and postmortem spleens from infected adults, there is significant loss of b and cd4 + t lymphocytes and dendritic cells, 386, 498 resulting in decreased antigen presentation, antibody production, and macrophage activation. 514 circulating peripheral absolute lymphocyte counts can drop significantly in adults with sepsis but this phenomena is also seen in critically ill adults who are not infected. 477 sustained lymphopenia significantly increases the risk for secondary infection, mods, and death in children. 505 extensive loss of lymphocytes (both b and t lymphocytes) has been described in postmortem specimens from the thymus and spleen in infected preterm and term infants. 412, 499, [501] [502] [503] [504] the number and the size of the follicles in the spleen decreased significantly and the total number of cells decreased by more than three times; similar changes were found in lymph nodes. 478 however, these histopathologic splenic findings are in contradiction to earlier reports where no differences were described in infected and uninfected infants. 412 splenomegaly may occur in infants with late-onset sepsis and may be due to splenic congestion in the absence of hyperplasia of white pulp. 502 the mechanisms responsible for immune alterations after sepsis are beginning to emerge. the intensity of the inflammatory response may be modified by neural-based mechanisms. 515 t cell-secreted acetylcholine acts on macrophages to reduce production of tnf, il-1, il-18, hmgb-1, and other cytokines. 516 the role of vagal tone in the neonatal host response to sepsis is unclear. discovery and characterization of the impact of epigeneticmediated immune system functional alterations after sepsis is an area of intense research. dna methylation and posttranslational modification of histone proteins (methylation, acetylation, prevented hmgb-1 level elevation, and was associated with longer survival times. 559 increased plasma nitrite and nitrate concentrations are associated with the development of multiple organ failure in pediatric patients with sepsis 560,561 but have not been investigated in neonates. the incidence of neonatal sepsis remains high and outcomes remain poor despite considerable technologic advances in the field of neonatology. much remains to be learned about the impact of developmental age on the host response to sepsis and what facets are critically important. important considerations for future investigations include the development and implementation of a generally accepted definition for neonatal sepsis, the use of homogeneous systems (only neonatal components) for human ex vivo studies, and transgenic approaches in preclinical models, alongside observational studies in humans to ensure meaningful findings. complete reference list is available at www.expertconsult.com. the amplitude-integrated electroencephalogram may be helpful to predict neurologic outcome in infants with sepsis or meningitis. 543 significantly lower resistance, vasodilatation, and higher blood flow were noted in all the cerebral arteries of infants with sepsis. increase in cerebral blood flow velocity was correlated with elevated il-6 concentrations. 544 alterations in blood flow in preterm infants, in addition to factors associated with sepsis, such as respiratory distress, hypercarbia, hypotension, and patent ductus arteriosus, contribute to the risk for intracerebral hemorrhage. endocrine abnormalities may include altered thyroid function 545 and adrenal insufficiency associated with refractory hypotension. 546 inadequate adrenocortical responses are associated with increased mortality. 547, 548 cortisol production in the neonate is significantly increased early in septic shock. 212 however, very preterm neonates can have relative adrenal insufficiency that may contribute to hemodynamic instability and hypotension. hydrocortisone has not been evaluated in large prospective randomized clinical trials for the treatment of septic shock in the neonate but it has been shown to increase blood pressure, decrease heart rate, and decrease vasoactive medication requirements in preterm and term neonates in addition to its cytokine-suppressing effects. [549] [550] [551] if hydrocortisone treatment is considered, the obtaining of a pretreatment serum cortisol level is prudent in order to differentiate contributing causes of hypotension. sepsis was the most common cause (78%) of acute kidney injury in term neonates and was associated with high mortality (37%) (n = 49). 552 the frequency of acute renal failure (defined as a blood urea nitrogen level greater than 20mg/dl) in infected neonates was 26% and oliguria occurred in 15% of acute kidney failure cases. 553 acute kidney injury in preterm neonates is associated with high mortality. 554 hepatic injury and dysfunction are frequent associations with severe sepsis. the mechanisms include reduced hepatic perfusion associated with septic shock and mitochondrial energy failure. reductions in coagulation and complement factors, acute-phase reactant proteins, and increases in the levels of transaminases and bilirubin are commonly seen, especially in association with decreased perfusion states. energy expenditure and oxygen consumption are increased during sepsis, 555 and decreased mitochondrial oxidative function precipitated by hypoxia and the presence of reactive oxygen intermediates may lead to impaired growth, caloric deficiency, and energy failure. 556, 557 sepsis that leads to mods carries a dismal prognosis. inadequate cardiac output and microcirculatory failure, which may be combined with formation of microthrombi and dic, can lead to poor perfusion to the kidney, 472,473 liver, 474 gut, 475 and central nervous system. 52, 148, 181, 469, 476 recent studies suggest that the mechanism of organ failure in sepsis may relate to decreased oxygen utilization associated with mitochondrial dysfunction rather than poor oxygen delivery to tissues. 479, 480 mitochondrial dysfunction can initiate activation of cell death pathways, including apoptosis, pyroptosis, necrosis, and netosis (i.e. cell death mediated by nets). damage-associated molecular patterns (including nucleosomes and microparticles) created by activation of these cell death programs further amplify the host inflammatory response. free radicals play an important role in the inflammatory process of sepsis. 558 in a sepsis model in neonatal piglets, edaravone, a novel free radical scavenger, increased mean arterial pressure and cardiac output, lowered heart rate, reduced hydroperoxide, nitrite, and nitrate levels, delayed the tnf-α surge, 37 . jackson gl, rawiki p, sendelbach d, et al: hospital course and short-term outcomes of term and late preterm neonates following exposure to prolonged rupture of membranes and/or chorioamnionitis. pediatr infect dis j 31 (1) protective immunity and defects in the neonatal and elderly immune response to sepsis increased mortality and altered immunity in neonatal sepsis produced by generalized peritonitis defective innate immunity predisposes murine neonates to poor sepsis outcome but is reversed by tlr agonists the host response to sepsis and 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protease-activated receptor-1: key player in the sepsis coagulation-inflammation crosstalk increased numbers of macrophages in tracheal aspirates in premature infants with funisitis neutrophil-derived microparticles induce myeloperoxidase-mediated damage of vascular endothelial cells coagulation dysfunction in sepsis and multiple organ system failure endothelial glucocorticoid receptor is required for protection against sepsis admission angiopoietin levels in children with septic shock group b streptococcal beta-hemolysin promotes injury of lung microvascular endothelial cells pathophysiologic mechanisms of persistent pulmonary hypertension of the newborn endothelial tlr4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via enos-no-nitrite signaling neutrophil-derived il-1β is sufficient for abscess formation in immunity against staphylococcus aureus in mice correlation between susceptibility of infants to infections and interaction with neutrophils 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potential prognostic marker for posttraumatic development of inflammatory second hit and sepsis novel cell death program leads to neutrophil extracellular traps impaired neutrophil extracellular trap (net) formation: a novel innate immune deficiency of human neonates delayed but functional neutrophil extracellular trap formation in neonates neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens outcomes following candiduria in extremely low birth weight infants exhaustion of mature marrow neutrophils in neonates with sepsis neutrophil storage pool depletion in neonates with sepsis and neutropenia evaluation of risk factors for fatal neonatal sepsis can neutrophil responses in very low birth weight infants predict the organisms responsible for late-onset bacterial or fungal sepsis? the leukocyte left shift in clinical and experimental neonatal sepsis delayed emergency myelopoiesis following polymicrobial sepsis in neonates granulocyte transfusions for neonates with confirmed or suspected sepsis and neutropaenia granulocyte-macrophage colony stimulating factor administered as prophylaxis for reduction of sepsis in extremely preterm, small for gestational age neonates (the programs trial): a single-blind, multicentre, randomised controlled trial for treating or preventing neonatal infections a multicenter, randomized, placebocontrolled trial of prophylactic recombinant granulocyte-colony stimulating factor in preterm neonates with neutropenia passive deformability of mature, immature, and active neutrophils in healthy and septicemic neonates irreversible neutrophil aggregation. a mechanism of decreased newborn neutrophil chemotactic response secretory products of macrophages evaluation of natural killer cells as diagnostic markers of early onset neonatal sepsis: comparison with c-reactive protein and interleukin-8 innate immunity of the human newborn is polarized toward a high ratio of il-6/tnf-alpha production in vitro and in vivo down-regulation of th1 responses in human neonates human deficiencies in type-1 cytokine receptors reveal the essential role of type-1 cytokines in immunity to intracellular bacteria decreased mononuclear and polymorphonuclear chemotaxis in human newborns, infants, and young children association between serum macrophage colony-stimulating factor levels and monocyte and thrombocyte counts in healthy, hypoxic, and septic term neonates neonatal blood cell count in health and disease. ii. values for lymphocytes, monocytes, and eosinophils monocyte phagocytosis as a reliable parameter for predicting early-onset sepsis in very low birthweight infants phenotype of fetal monocytes and b lymphocytes during the third trimester of pregnancy defective antigen-presenting cell function in human neonates molecular mechanisms underlying antiinflammatory phenotype of neonatal splenic macrophages efficient maturation and cytokine production of neonatal dcs requires combined proinflammatory signals differential responses of cord and adult blood-derived dendritic cells to dying cells preterm neonates display altered plasmacytoid dendritic cell function and morphology efficient priming of antigen-specific cytotoxic t lymphocytes by human cord blood dendritic cells cd11c+ dendritic cells are required for survival in murine polymicrobial sepsis depletion of dendritic cells, but not macrophages, in patients with sepsis evaluation of eosinophilia in hospitalized preterm infants perinatal hematological profile of newborn infants with candida antenatal infections eosinophilia in newborn infants eosinophilia in premature neonates. phase 2 of a biphasic granulopoietic response catapult-like release of mitochondrial dna by eosinophils contributes to antibacterial defense mast cells in innate immunity mast cells and neutrophils release il-17 through extracellular trap formation in psoriasis urticaria neonatorum: accumulation of tryptase-expressing mast cells in the skin lesions of newborns with erythema toxicum mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through tnf-alpha critical protective role of mast cells in a model of acute septic peritonitis phagocytosisindependent antimicrobial activity of mast cells by means of extracellular trap formation histamine regulates cytokine production in maturing dendritic cells, resulting in altered t cell polarization mast cells from the umbilical cord matrix and basophils from cord blood molecular mechanism of mast cell mediated innate defense against endothelin and snake venom sarafotoxin nk cell increase in neonates from the preterm to the full-term period of gestation reduced nk cell percentage at birth is associated with late onset infection in very preterm neonates alterations in lymphocyte phenotype of infected preterm newborns natural killer cell cytotoxicity is deficient in newborns with sepsis and recurrent infections multiple leucocyte activation markers to detect neonatal infection immunosuppressive cd71+ erythroid cells compromise neonatal host defence against infection transferrin receptor 1 is differentially required in lymphocyte development transferrin therapy ameliorates disease in beta-thalassemic mice ineffective erythropoiesis in stat5a −/− 5b −/− mice due to decreased survival of early erythroblasts roles of spleen and liver in development of the murine hematopoietic system immaturity of the human splenic marginal zone in infancy. possible contribution to the deficient infant immune response fetal and neonatal development of human spleen: an immunohistological study neonatal cd71 + erythroid cells do not modify murine sepsis mortality caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte b cells enhance early innate immune responses during bacterial sepsis neonatal adaptive immunity comes of age lymphocyte subpopulations in full-term septic neonates neonates with culture proven sepsis have lower amounts and percentage of cd45ra+ t cells pre-inflammatory mediators and lymphocyte subpopulations in preterm neonates with sepsis analysis of lymphocyte proliferative response subpopulations in very low birth weight infants and during the first 8 weeks of life hla-dr+, t lymphocytes and cd25+ cells in eutrophic full-term neonates with staphylococcal septicemia peripheral blood cells with expression of the interleukin-2 receptor (cd25+) in severely infected fullterm neonates cord blood cd4 + cd25 + -derived t regulatory cell lines express foxp3 protein and manifest potent suppressor function regulatory t cells mediate maternal tolerance to the fetus increased natural cd4+cd25+ regulatory t cells and their suppressor activity do not contribute to mortality in murine polymicrobial sepsis transient increase in cd45ro expression on t lymphocytes in infected newborns surface activation markers of t lymphocytes: role in the detection of infection in neonates can expression of cd45ro, a t-cell surface molecule, be used to detect congenital infection? increased cd4 + t cell co-inhibitory immune receptor ceacam1 in neonatal sepsis and soluble-ceacam1 in meningococcal sepsis: a role in sepsis-associated immune suppression? interleukin-8 (cxcl8) production is a signatory t cell effector function of human newborn infants increase of cord blood cytokineproducing t cells in intrauterine infection age-related changes in intracellular th1/th2 cytokine production, immunoproliferative t lymphocyte response and natural killer cell activity in newborns, children and adults identification of t-lymphocyte function in healthy vs. septic preterms and its relation to candidal infections in the hospital setting mature cd8 + t lymphocyte response to viral infection during fetal life asplenic-hyposplenic overwhelming sepsis: postsplenectomy sepsis revisited hyposplenism: a comprehensive review. part i: basic concepts and causes demonstration of circulating group b streptococcal immune complexes in neonates with meningitis in vitro immunoglobulin response of fetal b-cells is influenced by perinatal infections and antibiotic treatment: a study in preterm infants blood stream infection is associated with altered heptavalent pneumococcal conjugate vaccine immune responses in very low birth weight infants shades of grey-the blurring view of innate and adaptive immunity a critical role of t-cell receptor gamma/delta cells in antibacterial protection in mice early in life in vivo and in vitro activation and expansion of gammadelta t cells during listeria monocytogenes infection in humans gamma delta t cells respond directly to pathogen-associated molecular patterns influence of perinatal risk factors on cd3+/tcr αβ and cd3+/tcr γδ lymphocytes in cord blood of preterm neonates tcr usage and functional capabilities of human gamma delta t cells at birth il-23 activates innate lymphoid cells to promote neonatal intestinal pathology identification of pediatric septic shock subclasses based on genome-wide expression profiling genomic expression profiling across the pediatric systemic inflammatory response syndrome, sepsis, and septic shock spectrum divergence of canonical danger signals: the genome-level expression patterns of human mononuclear cells subjected to heat shock or lipopolysaccharide validating the genomic signature of pediatric septic shock genome-level longitudinal expression of signaling pathways and gene networks in pediatric septic shock gene-expression profiling of grampositive and gram-negative sepsis in critically ill patients the use of gene-expression profiling to identify candidate genes in human sepsis gene-expression profiling of peripheral blood mononuclear cells in sepsis recent progress of proteomics in critical illness proteomic profiling of the amniotic fluid to detect inflammation, infection, and neonatal sepsis optimized dna extraction from neonatal dried blood spots: application in methylome profiling validation of a gene expressionbased subclassification strategy for pediatric septic shock genome-level expression profiles in pediatric septic shock indicate a role for altered zinc homeostasis in poor outcome prophylactic zinc supplementation reduces bacterial load and improves survival in a murine model of sepsis oral zinc supplementation for reducing mortality in probable neonatal sepsis: a double blind randomized placebo controlled trial post-natal age is a critical determinant of the neonatal host response to sepsis genome-wide expression profiles in very low birth weight infants with neonatal sepsis a novel role for matrix metalloproteinase-8 in sepsis identification of a human neonatal immune-metabolic network associated with bacterial infection host-response biomarkers for diagnosis of late-onset septicemia and necrotizing enterocolitis in preterm infants fetal thymic involution: a sonographic marker of the fetal inflammatory response syndrome persistent microcirculatory alterations are associated with organ failure and death in patients with septic shock risk factors and predictors of mortality in culture proven neonatal sepsis anatomical study of the kidneys of newborn infants dying after a septic state renal failure, comorbidity and mortality in preterm infants liver perfusion in sepsis, septic shock, and multiorgan failure neonatal sepsis in karachi: factors determining outcome and mortality association of septic shock caused by early-onset group b streptococcal sepsis and periventricular leukomalacia in the preterm infant accelerated lymphocyte death in sepsis occurs by both the death receptor and mitochondrial pathways pathology of lymphoid organs in low birth weight infants subjected to antigen-related diseases: a morphological and morphometric study advances in pathogenesis and management of sepsis molecular biology of inflammation and sepsis: a primer multisystem organ failure and capillary leak syndrome in severe necrotizing enterocolitis of very low birth weight infants clinical and laboratory factors that predict death in very low birth weight infants presenting with late-onset sepsis heart failure in pediatric septic shock: utilizing inotropic support circulatory shock in children: an overview hemodynamic support in fluidrefractory pediatric septic shock clinical practice parameters for hemodynamic support of pediatric and neonatal patients in septic shock distinct hemodynamic patterns of septic shock at presentation to pediatric intensive care which inotrope for which baby? relationship between blood pressure and cardiac output in preterm infants requiring mechanical ventilation microcirculatory changes in term newborns with suspected infection: an observational prospective study hemodynamics in preterm infants with late-onset sepsis echocardiogram done early in neonatal sepsis: what does it add? el-arman mm: myocardial dysfunction in neonatal sepsis: a tissue doppler imaging study diagnosis and treatment of neonatal hypotension outside the transitional period elevation of brain natriuretic peptide levels in children with septic shock brain natriuretic peptide for prediction of mortality in patients with sepsis: a systematic review and meta-analysis predictors of mortality and multiple organ failure in children with sepsis sepsis-induced apoptosis causes progressive profound depletion of b and cd4+ t lymphocytes in humans acute thymus involution in infancy and childhood: a reliable marker for duration of acute illness thymus size and its relationship to perinatal events the grade of acute thymus involution in neonates correlates with the duration of acute illness and with the percentage of lymphocytes in peripheral blood smear. pathological study clinicopathological differences between early-onset and late-onset sepsis and pneumonia in very low birth weight infants spleen depletion in neonatal sepsis and chorioamnionitis acute thymic involution in fetuses and neonates with chorioamnionitis prolonged lymphopenia, lymphoid depletion, and hypoprolactinemia in children with nosocomial sepsis and multiple organ failure immunosuppression in sepsis: a novel understanding of the disorder and a new therapeutic approach prevention of lymphocyte apoptosisa potential treatment of sepsis? mutations in genes required for t-cell development: il7r, cd45, il2rg, jak3, rag1, rag2, artemis, and ada and severe combined immunodeficiency: huge review early circulating lymphocyte apoptosis in human septic shock is associated with poor outcome immunosuppression in patients who die of sepsis and multiple organ failure immunoparalysis and adverse outcomes from critical illness very low birth weight neonates who survive early-onset sepsis do not have an increased risk of developing late-onset sepsis early sepsis does not increase the risk of late sepsis in very low birth weight neonates the pathophysiology and treatment of sepsis nicotinic acetylcholine receptor α7 subunit is an essential regulator of inflammation neural reflexes in inflammation and immunity epigenetic regulation of immune cell functions during post-septic immunosuppression dna methylation pattern of calca in preterm neonates with bacterial sepsis as a putative epigenetic biomarker a prime time for trained immunity: innate immune memory in newborns and infants epigenetic programming of monocyteto-macrophage differentiation and trained innate immunity mtor-and hif-1alpha-mediated aerobic glycolysis as metabolic basis for trained immunity nonspecific effects of neonatal and infant vaccination: public-health, immunological and conceptual challenges myd88-dependent expansion of an immature gr-1 + cd11b + population induces t cell suppression and th2 polarization in sepsis myeloid derived suppressor cells are present at high frequency in neonates and suppress in vitro t cell responses reactivation of multiple viruses in patients with sepsis surfactant-replacement therapy for respiratory distress in the preterm and term neonate diminished inducible nitric oxide synthase expression in fulminant early-onset neonatal pneumonia the paradox of adult respiratory distress syndrome in neonates bench-to-bedside review: developmental influences on the mechanisms, treatment and outcomes of cardiovascular dysfunction in neonatal versus adult sepsis inhaled nitric oxide and persistent pulmonary hypertension of the newborn the diseases treated with ecmo: focus on pphn extracorporeal membrane oxygenation and sepsis neurodevelopment of extremely preterm infants who had necrotizing enterocolitis with or without late bacteremia neonatal infection and 5-year neurodevelopmental outcome of very preterm infants impact of sepsis on neurodevelopmental outcome in a swiss national cohort of extremely premature infants infection-induced inflammation and cerebral injury in preterm infants postnatal sepsis, necrotizing entercolitis, and the critical role of systemic inflammation in white matter injury in premature infants postnatal infection is associated with widespread abnormalities of brain development in premature newborns adverse neurodevelopment in preterm infants with postnatal sepsis or necrotizing enterocolitis is mediated by white matter abnormalities on magnetic resonance imaging at term association between high cytokine levels with white matter injury in preterm infants with sepsis the relationship of csf and plasma cytokine levels to cerebral white matter injury in the premature newborn to tap or not to tap: high likelihood of meningitis without sepsis among very low birth weight infants the prognostic value of amplitude integrated eeg in neonatal sepsis and/or meningitis cerebral blood flow velocity in earlyonset neonatal sepsis and its clinical significance serum cortisol and thyroid hormone levels in neonates with sepsis refractory hypotension in preterm infants with adrenocortical insufficiency hydrocortisone administration for the treatment of refractory hypotension in critically ill newborns circulating adrenocorticotropic hormone (acth) and cortisol concentrations in normal, appropriate-forgestational-age newborns versus those with sepsis and respiratory distress: cortisol response to low-dose and standard-dose acth tests hemodynamic changes after low-dosage hydrocortisone administration in vasopressor-treated preterm and term neonates cardiovascular effects of hydrocortisone in preterm infants with pressor-resistant hypotension dose-related inhibition of proinflammatory cytokine release from neutrophils of the newborn by dexamethasone, betamethasone, and hydrocortisone the main etiologies of acute kidney injury in the newborns hospitalized in the neonatal intensive care unit acute renal failure in neonatal sepsis acute kidney injury in preterm infants admitted to a neonatal intensive care unit effect of sepsis syndrome on neonatal oxygen consumption and energy expenditure nitric oxide inhibits neonatal hepatocyte oxidative metabolism impaired energy metabolism during neonatal sepsis: the effects of glutamine bench-to-bedside review: neonatal sepsis -redox processes in pathogenesis edaravone, a novel free radical scavenger, reduces high-mobility group box 1 and prolongs survival in a neonatal sepsis model inflammatory cytokine and nitric oxide responses in pediatric sepsis and organ failure plasma nitrite and nitrate concentrations and multiple organ failure in pediatric sepsis key: cord-016966-b23o5roz authors: verhoef, jan; snippe, harm title: immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites date: 2005 journal: principles of immunopharmacology doi: 10.1007/3-7643-7408-x_7 sha: doc_id: 16966 cord_uid: b23o5roz despite the introduction of effective health measurements, vaccination and antimicrobial therapy infectious diseases continue to threaten human life. the reasons are numerous and diverse: antibiotic resistance, hospital-invading pathogens, new emerging infectious diseases, bioterrorism, biological warfare. this chapter is an introduction to several aspects of infectious diseases viewed from the host as well as from the pathogen (bacterium, virus and parasite). furthermore the basic principles of innate and adaptive immune responses, especially in debilitated patients, are described. detailed information is given on the pathogenesis of septic shock, aids and vaccination strategies. in the middle of the 19th century,it became clear that micro-organisms could cause disease. effective treatment, however, was not possible at that time; prevention and spread of infectious diseases depended solely on proper hygienic means.at the beginning of the 20th century, passive and active vaccination procedures were developed against a number of these pathogenic micro-organisms in order to prevent the diseases in question (rabies, diphtheria, tetanus, etc.) and due to the discovery of antimicrobial chemicals (ehrlich) and antibiotics (fleming), the threat of infectious diseases seemed to be minimized. largescale vaccination programs against childhood diseases (diphtheria, whooping cough and polio) started in the early fifties, giving hope to finally eradicate these diseases from the planet. this approach was successful for smallpox (1980) ; however, new infectious diseases emerged (e.g., legionella, hiv, helicobacter, sars, etc.). new vaccines and antibiotics are needed. furthermore, due to the intensive medical treatment with antibiotics and immunosuppressive drugs,hospital infections are a growing problem. bacteria hitherto deemed harmless are causing opportunistic infections in immunocompromised patients.the pathogens develop multiple resistances to antibiotics and sometimes no effective antibiotics are available to treat those patients. to make the story evermore serious, man is surrounded and populated by a large number of different non-pathogenic micro-organisms. in the normal -healthy -situation, there is a balance between the offensive capabilities of micro-organisms and the defences of the human body. the body's defences are based on vital non-specific and specific immunological defence mechanisms. an infection means that the microorganism has succeeded in penetrating those lines of defence, signaling a partial or complete breakdown of the body's defence system. the body's first line of defence comprises the intact cell layers of skin and mucous membrane, which form a physical barrier. the skin's low ph level and bactericidal fatty acids enhance the protection provided by this physical barrier.the defence in the respiratory tract and the gastrointestinal tract is mucous, the 'ciliary elevator' of the epithelium, and the motility of the small intestine. the presence of normal microbial flora (colonization resistance) in the intestine also plays a role in protection against colonization. the most important humoral natural resistance factors are complement, lysozyme, interferon, and a number of cytokines. lysozyme, which is found in almost all body fluids, degrades sections of the cell wall of gram-positive and -in combination with complement -gram-negative bacteria. this causes the otherwise sturdy cell wall to leak and the bacterium to burst. interferons are glycoproteins which may inhibit the replication of viruses. within several hours after the onset of a virus infection, interferons are produced in the infected cell and help protect the neighbouring unaffected cells against infection.this protection is brief, but high concentrations of inter-ferons are produced at a time when the primary immunological response is relatively ineffective. cytokines, such as il-2 (interleukin-2), gm-csf (granulocyte-macrophage colony-stimulating factor), and tnf-α (tumor necrosis factor α), stimulate non-specifically the proliferation, maturation, and immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites jan verhoef and harm snippe a7 function of the cells involved in defence (see chapter a.4). innate immune cells recognize microbes by toll-like receptors (see section pathogenesis of shock), giving rise to the above production of cytokines in the early phase of the response. micro-organisms that succeed in penetrating the first line of defence are ingested, killed, and degraded by phagocytic cells (leukocytes, monocytes, macrophages), which are attracted to a microbial infection through chemotaxis. the ingestion by phagocytic cells of the micro-organism is enhanced by serum proteins (opsonins),such as antibodies and the c3b component of complement, for which these phagocytes have a receptor.after ingestion, the particle is surrounded by the membrane of the phagocyte, forming a vacuole known as a phagosome .the phagosome then fuses with some of the countless lysosomes in the phagocyte,thus allowing the lysosomal microbicidal agents and enzymes to do their work.in the case of leukocytes,the formation of toxic oxygen radicals greatly contributes to the killing and elimination of the ingested micro-organism ( fig. 1 ) (see chapter a.6). a special role in cellular natural resistance is reserved for the nk (natural killer) cells, which display considerable cytotoxic activity against virusinfected cells. this nk activity is stimulated by inter-ferons and,at a very early stage in the infection,serves to reinforce the non-specific defence mechanism. in the specific immune response, elements of the natural defence mechanism are directed against a specific enemy. depending on the micro-organism, either the cellular defence mechanism (tuberculosis) or the humoral antibody-dependent defence mechanism (influenza) is of primary importance. in many cases, a joint cellular and humoral response is needed to provide an effective defence (typhus). both t lymphocytes and macrophages play a role in cellular defence. during the first contact with an antigen, macrophages process the antigen and present its protein fragments (t-cell epitopes) to t cells, which then proliferate and remain present for years in the body as memory cells. when a second encounter occurs, t cells produce lymphokines, which activate the macrophages. these activated macrophages grow larger, produce more and better degrading enzymes, and are now able to eliminate micro-organisms which otherwise would have survived intracellularly (tuberculosis, typhoid fever). macrophages from non-immune animals are not able to eliminate these micro-organisms. five different classes of antibodies can be distinguished in man, namely, igg, iga, igm, igd, and ige. they differ from one another in size, charge, amino acid composition, and glycosylation (see chapter a.3, c.2). in principle, the structure of the antibodies is the same, i.e., 2 heavy and 2 light chains: it is the variable part of these chains which recognizes the micro-organism.the biological function (see below) is determined by the constant part (fc) of the heavy chain.with the exception of igd, all these antibodies are important in antimicrobial activity. -iga,which is found in all external secretions,reacts with the surface of micro-organisms, preventing them from adhering to sensitive cells and mucous membranes. -igg neutralizes microbial toxins. -igg, igm, and c3b serve as opsonins, which promote phagocytosis. -igg, igm, and to a lesser extent iga activate the complement system after binding to the microorganism.activation products c3a and c5a ensure that the phagocytes are attracted to the inflammatory response. schematic representation of the progressive steps of phagocytic endocytosis. -ige is of importance in parasite infections. at the site of the infection,mast cells,bearing specific ige, release large quantities of vasoactive amines, which cause the contraction of smooth muscle tissue and increase the permeability of the blood vessels. in the intestine, this results in worms being detached and eliminated. several non-invasive bacteria, i.e., those that do not invade the body, cause disease through the production of exotoxins (tetanus, diphtheria, cholera). the immune system neutralizes the toxin with the aid of antibodies (igg, igm). if the individual has not been inoculated, the toxin will act on certain cells in the body directly through a receptor. this bond is very strong (i.e., has a high affinity), and is difficult to break by the administration of antibodies. in practice, if there are clinical symptoms of the disease, then large doses of antitoxins must be administered.if one is trying to prevent the development of the disease, then the presence of small quantities of specific antibodies (igg) is sufficient. the adherence of bacteria to cells is effectively blocked by iga. oral vaccination against cholera, for example,is aimed at obtaining sufficient specific iga in the intestine, so that no colonization of this bacterium can take place, and the cholera toxin can no longer adhere to its receptor. in general, defence against invasive bacteria is provided by antibodies (igg, igm) that are directed against bacterial surface antigens. in many cases, these bacteria have a capsule which interferes with effective phagocytosis. antibodies against these capsule antigens neutralize the interference, with subsequent elimination of the bacteria by phagocytes. antibodies (igm, igg, iga) in combined action with complement kill bacteria by producing holes in the cell wall of the bacterium. although intracellular bacteria (tuberculosis, leprosy, listeriosis, brucellosis, legionellosis, and salmonellosis) are ingested by macrophages, they are able to survive and multiply. in these cases, cellular immu-nity alone provides the defence, since antibodies are not effective. only activated macrophages are capable of killing and degrading these bacteria. antibodies neutralize viruses (fig. 2) directly and/or indirectly by destroying infected cells that carry the virus antigen on their surface. the mechanisms of this defence resemble those of humoral defence against bacterial surfaces. the antibodydependent cellular cytotoxicity reaction is specific for the defence against viruses. cells which carry on the surface an antigen encoded by the virus are attacked by cytotoxic k cells, bearing antibodies which fit the antigen on the target cell (k cells have fc receptors for igg). not only humoral, but also cellular immunity plays an important role in virus infections. people with a genetic t-cell deficiency are highly susceptible to virus infections. in cellular defence, it is primarily the virus-infected cells which are attacked and eliminated. cytotoxic t cells recognize mhc-1-presented t-cell epitopes on the surface of virus-infected cells and kill them. the fungi responsible for human diseases can be divided into two major groups on the basis of their growth forms or on the type of infection they cause. pathogens exist as branched filamentous forms or as yeasts, although some show both growth forms. the filamentous types (trichophyton) form a 'mycelium'. in asexual reproduction, the fungus is dispersed by means of spores; the spores are a common cause of infection after inhalation. in yeast-like types (cryptococcus), the characteristic form is the single cell, which reproduces by division or budding.dimorphic types (histoplasma) form a mycelium outside, but occur as yeast cells inside the body. candida shows the reverse condition and forms a mycelium within the body. in superficial mycoses, the fungus grows on the body surface, for example skin, hair, and nails (epidermophyton, trichophyton), the disease is mild, and the pathogen is spread by direct contact. in deep mycoses (aspergillus, candida, cryptococcus, histoplasma), internal organs are involved and the disease can be life-threatening and is often the result of opportunistic growth in individuals with impaired immunocompetence. many of the fungi that cause disease are free-living organisms and are acquired by inhalation or by entry through wounds. some exist as part of the normal body flora (candida) and are innocuous unless the body's defences are compromised in some way. the filamentous forms grow extracellularly, while yeasts can survive and multiply within phagocytic cells. neutrophils kill yeasts by means of both intraand extracellular factors. some yeasts (cryptococcus neoformans) form a thick polysaccharide capsule in order to prevent phagocytic uptake. in addition, many cell-wall components of yeasts cause suppression of cell-mediated immune responses.the role of humoral and cellular immunity in controlling infections caused by fungi is not yet well defined, but cellular immunity is the cornerstone of host defence against (some) fungal infections.as a consequence, hiv infection, which affects the cellular arm of the immune system, results in previously uncommon infections such as those caused by c. neoformans. the immunological defence systems against parasites are considerably more complex than those against bacteria and viruses. this is due to various factors. in the first place, each parasite has its own life cycle, consisting of various stages with specific antigen compositions.moreover,parasites are able to avoid the host defence system (mimicry), to combat it (immunosuppression), or to mislead it (antigenic variation). both humoral and cellular immunity are important for the defence against parasites growing intercellularly, as we have seen in the case of bacteria and viruses. antibody concentrations (igm, igg, ige) are often elevated. ige also plays a special role in the removal of parasites (especially worm infections) from the intestine (see above). in gram-negative (fig. 3) bacterial infections, the interaction between bacterial endotoxin and various host-cell systems has been implicated in the pathogenesis of septic shock. in particular, the release of tnf-α (also called cachectin) and interleukin-1 (il-1), after the activation of host cells by endotoxin, induces hemodynamic shock. several lines of evidence support the current hypothesis that the monocyte-macrophage is the principal cellular mediator of endotoxicity. first, c3h/hej mice carrying a single gene defect are nonresponsive to lipopolysaccharide (lps). the transfer of macro-phages of a closely related lps-sensitive strain makes the mice responsive. second, when the host is challenged with endotoxin, soluble factors are produced by macrophages that mediate fever and an acutephase response. these factors include the proinflammatory cytokines, il-1, il-6, il-8, and tnf-α. together, tnf-α and il-1 stimulate endothelial cells to produce and express proteins on their membrane that have adhesive properties for leukocytes, promoting the migration and passage of polymorphonuclear leuko-cytes (pmns) from blood vessels through the endothelial layer, leading to pmn influx into the tissue. adhesion molecules that mediate the binding of pmns appear on the endothelium after an inflammatory stimulus, followed by molecules that are specific for adhesion of monocytes or lymphocytes, which may be why neutrophils enter before mononuclear cells. molecules that are currently known to be involved in leukocyte-endothelium interactions belong to three structural groups: the immunoglobulin gene superfamily, the integrin family, and the selectin family. concomitant with cytokine release, lps induces the activation of pmns,macrophages,and many other cells,resulting in the release of toxic oxygen radicals, which lead to tissue damage.at the same time, membrane-associated phospholipases are activated and products of the arachidonic-acid cascade are released through the cyclooxygenase and/or lipooxygenase pathways (see chapter a.6). plateletactivating factor (paf) is also generated, partly in response to the same signals.all these products contribute to a generalized inflammatory state with influx of pmns, capillary-leak syndrome, disturbances in blood coagulation, and myocardial suppression. endotoxin and tnf-α also produce multiple abnormalities in coagulation and fibrinolysis, leading to microvascular clotting and diffuse intravascular coagulation.they also induce endothelial cells to produce plasminogen activator and il-6, which is an important modulator of the production of acutephase proteins by the liver. interestingly, despite having important structural differences, tnf-α and il-1 have multiple overlapping and few distinct biological activities,act synergistically,and mimic the whole spectrum of toxicity caused by lps (see chapter a.4).il-8 is an important chemoattractant and activator of neutrophils and is crucial in the early stages of inflammation. infusion of endotoxin in healthy humans leads to an early and transient increase in plasma levels of tnf-α (detectable after 30 min, peaking after 90-120 min, and undetectable after 4-6 h), which coincides with the development of clinical symptoms and pathophysiological responses encountered in gramnegative septicemia. tnf-α, il-1, il-6, and il-8 levels are also increased in patients with sepsis syndrome, with high levels of these cytokines being correlated with severity of disease. all these observations support the concept that endotoxin largely acts by initiating an inflammatory response through the activation of monocytes-macrophages and the subsequent release of cytokines. it also activates the complement system (leading to the generation of c5a, which induces aggregation of pmns and pulmonary vasoconstriction) and factor xii of the intrinsic coagulation pathway (hageman factor). finally, it induces the release of endorphins, which are also involved in the complex interactions of the inflammatory response in endotoxic septic shock. gram-positive bacteria are frequently and increasingly cultured from blood obtained from patients in shock. unlike the pathophysiology of shock caused by gram-negative bacteria,not much is known about the sequence of events that controls the signaling of monocytes and macrophages that leads to the release of cytokines. cell-wall components, such as peptidoglycan and teichoic acid, are clearly important in the activation of these cells. exotoxins,however,may also play a role in the pathogenesis of gram-positive bacterial shock. cd14 is a cell surface glycoprotein that functions as a binding receptor for lps.however its membrane anchoring by a glycosylphosphatidyl inositol (gpi) linkage suggests little signaling and suggests the existence of additional coreceptors. recent studies indicate that innate immune cells recognize conserved pathogen-associated molecular patterns (pamps), including lps, through toll-like receptors (tlrs) (fig. 4) . this family of proteins, that resemble the antimicrobial toll proteins of drosophila, has been identified in humans and mice. tlr4 was identified as the missing link in lps-induced cell signal transduction and responsiveness that is associated with md-2 and cd14. the tlr family members are coupled to a signaling adapter protein (myd88) and form differential dimers that may explain the discrete responses to tlr ligands such as lipoprotens, heat shock proteins, unmethylated cpg dna, viral dsrna and bacterial flagellin. intracellular signaling involves several kinases depending on the tlrs involved and includes the map kinase and nf-κb pathways leading to a cellular response. recently other protein families have been identified via genetic screening that also participate in direct recognition of pathogens. a new protein was found to be involved in resistance to gram-positive bacterial infections and recognizes the cell wall component peptidoglycan. the human immunodeficiency virus (hiv) is a retrovirus that infects cells bearing the cd4 antigen, such as t-helper cells (th), macrophages, and dendritic cells.the cd4 molecule,together with other receptor molecules, like chemokine receptor 5, acts as a binding site for the gp120 envelope glycoprotein of the virus. in an attempt to respond to hiv antigens and concomitant secondary microbial infections, these cells are activated, thus inducing the replication of hiv in the infected cd4 t cells, which are finally destroyed. in contrast, hiv-1 infection of macro-phages is self-sustained and results in an inexorable growth of chronic active inflammatory processes in 112 immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites schematic illustration of cell activation through toll-like receptors (tlrs). many tissue compartments including the central nervous system. infected cells bear the fusion protein gp41 and may therefore fuse with other infected cells.this helps the virus to spread and accounts for the multinucleated cells seen in lymph nodes and brain. as a result of the decreased numbers of cd4positive t-helper cells and defects in antigen presentation, depressed immune responses in these patients are observed. during the progression of the disease, opportunistic infections by otherwise harmless micro-organisms can occur. these include candida albicans oesophagitis, mucocutaneous herpes simplex, toxoplasma in the central nervous system, and pneumonia caused by toxoplasma and pneumocystis carinii; kaposi's sarcoma also occurs frequently in these patients.this has been linked to the presence of a previously unknown type of herpes virus (hhv-8).this immune deficiency syndrome is called 'acquired immune deficiency syndrome' (aids). it has been suggested that infected monocytes/macro-phages carry the hiv virus into the brain where it replicates in microglia and infiltrating macrophages. as a consequence many aids patients develop cognitive and motor brain impairments. however, the picture is complicated by the various persistent infections already present in these patients, which give rise to their own pathology in the brain. these include toxoplasma gondii, cryptococcus neoformans and jc virus. so far a cure for hiv infection has not been achieved. the main effort in the prevention of hiv infection lies in mass public education programmes. treatment of infected individuals is possible but expensive. at this moment a triple therapy is being prescribed in the western countries (two reverse transcriptase inhibitors and one protease inhibitor, fig. 5 ), each of which interfere with specific steps in the process of hiv replication. one major problem that has arisen is the increasing resistance to these drugs. blocking of the chemokine receptor 5, a recently described co-receptor on cd4 cells for hiv, may be an alternative treatment for infected persons. this notion is supported by a recent finding that a homozygous defect in this chemokine receptor accounts for resistance of multiple-exposed individuals to hiv-1 infection. preparing his vaccines, koch employed killed germs (cholera) as a vaccine. since diphtheria and tetanus cause disease by means of toxins, the next logical step in the development of vaccines was the use of detoxified toxins to induce protection against these diseases (diphtheria, von behring and tetanus, kitasato).von behring and kitasato were the first to demonstrate that the source of the protective activity induced by vaccines was present in blood serum. von behring was also the first to prove that protective immunity could be passed on via serum. the development of new vaccines had its ups (yellow fever) and downs (tuberculosis). with the arrival of antibiotics, all work on new bacterial vaccines was suspended or severely curtailed, although some researchers continued to work on viral vaccines, such as rubella, measles, polio, and mumps. since it has proved difficult to consistently develop new antibiotics to combat antibiotic-resistant bacteria, interest in vaccines has gradually increased over the last 15 years (see chapter c.1). today, thanks to new insights into the immune system and modern molecular biological and chemical techniques of analysis and synthesis, it is possible to produce well-defined vaccines.these contain only those determinants of the pathogenic microorganism which induce protection (epitopes). these epitopes are usually short peptide or oligosaccharide chains, which can be produced synthetically or by means of recombinant dna techniques. the immunogenicity of these products can be enhanced by coupling them to a carrier (tetanus toxoid, liposomes) and/or by adding an adjuvant (a substance which strengthens the immune response non-specifically). the recombinant dna technique can also be used to obtain attenuated strains of micro-organisms, which are fully immunogenic and thus provide protection,but which are no longer virulent. one example of this is the development of a new cholera vaccine based on a bacterium which has all the characteristics of a virulent strain,except the toxin.the bacterium has retained all its adherence factors, which allow it to adhere to the intestinal mucosa; the length of time it spends in the intestine is sufficient to stimulate the local immune system.the newest trend in vaccinology is immunization by introducing plasmid dna into the host.success has been attained by this method for hepatitis b vaccination. not only are new vaccines being developed, but it is also possible to heighten natural resistance for longer or shorter periods. various interleukins (il-2, gm-csf) and interferons are being studied in order to use them to combat infectious diseases. monoclonal antibodies (antibodies with one specificity) directed against the endotoxin of gram-negative bacteria are now being administered to patients with severe gram-negative sepsis (serum therapy). more work is still necessary, however, to refine this technique, as the therapeutic effect is still limited. as outlined above for a number of bacteria and viruses, effective vaccines have been developed and applied worldwide.the eradication of smallpox (variola major) virus in the seventies of the last century was a milestone for the world health organization. the next goal of the who is to eradicate the poliovirus in the coming years. major problems to be dealt with are the distribution of these vaccines, the costs involved, the registration and the compliance of the vaccinees and molecular techniques to trace the final bug. meanwhile new unexpected microbiological threats come into focus. hospital infections caused by multiple resistant micro-organisms due to the abundant use of antibiotics and exchange of genetic material between micro-organisms impose major problems on patients and healthcare workers. new antibiotics and/or vaccines should be developed and new strategies employed to contain these infections. due to crowding and high mobility of the world population, old and new pathogens, e.g., influenza and sars, threaten our society. on top of this terrorists might intentionally use micro-organisms (smallpox, anthrax, plague etc.), or bacterial toxins (botulism) to cause death and disease in humans or animals in a civilian setting.the recognition that an event was caused by a biological weapon presents a severe challenge to be prepared for such an attack, especially for medical care providers and public health officials. strategies to combat bioterrorism have to be worked out but with the experience of 100 years of combating microorganisms with hygiene measures, vaccination, antibiotic and anti-viral treatment, there must be a way out. despite the introduction of effective health measurements, vaccination and antimicrobial therapy infectious diseases continue to threaten human life. the reasons are numerous and diverse: antibiotic resistance, hospital-invading pathogens, new emerging infectious diseases, bioterrorism, biological warfare. this chapter is an introduction to several aspects of infectious diseases viewed from the host as well as from the pathogen (bacterium, virus and parasite). furthermore the basic principles of innate and adap-tive immune responses, especially in debilitated patients, are described. detailed information is given on the pathogenesis of septic shock,aids and vaccination strategies. medical microbiology a history of immunology the beast in man: microbes and macrobes as intimate enemies. part ii.the battle of bugs the immune system in health and disease microbiology we thank dr. c.p.m. van kessel of the eijkman-winkler center for the design and layout of the artwork. key: cord-017503-g2n4d0wi authors: manson, david title: diagnostic imaging of neonatal pneumonia date: 2010-05-18 journal: radiological imaging of the neonatal chest doi: 10.1007/978-3-540-33749-2_7 sha: doc_id: 17503 cord_uid: g2n4d0wi respiratory infections remain a significant and formidable threat to the health and well being of the neonate despite potent antibiotics, increasingly sophisticated laboratory detection methods and technologically advanced neonatal intensive care nurseries. although the clinical and radiological definitions of pneumonia are variable throughout medical and governmental literature, quoted incidence rates for neonatal pneumonia range between 1.5–5.0 per 1,000 live births (keyserling 1997; webber et el 1990). respiratory infections remain a significant and fi formidable threat to the health and well being of the neonate despite potent antibiotics, increasingly sophisticated laboratory detection methods and technologically advanced neonatal intensive care nurseries. although the clinical and radiological definitions of pneumonia are variable throughout fi medical and governmental literature, quoted incidence rates for neonatal pneumonia range between 1.5-5.0 per 1,000 live births (keyserling 1997; webber et el 1990) . mortality rates from pneumonia are even more difficult to obtain and to interpret. the medical lit-fi erature quotes neonatal pneumonia mortality rates between 5% and 20% (whitsett et al. 1999) . recent reports from the centers for disease control quote mortality rates from neonatal pneumonia of 3.2/1,000 live births in the united states, corresponding to 1.1% of all neonatal deaths (document lwck 7 2003) , http://www.cdc.gov/nhcs/data/dvs/lwck7_2003pdf. the incidence and mortality in developing countries are, not surprisingly, higher, with stated mortality rates estimated at between 0.75-1.2 million neonatal deaths from pneumonia annually, accounting for 10% of all global neonatal mortality (duke 2005) . approximately 10% of all neonatal intensive care unit (nicu) patients will have at least one episode of pneumonia (whitsett et al. 1999) . it is estimated that 17% of very low birth weight infants will have at least one episode of a nosocomial infection (thompson et al. 1992) of which 30% will present as a respiratory tract infection (hemming et al. 1976 ). ironically, it would seem that the increasing sophistication of neonatal care and highly specialised nursery units may actually contribute to the incidence of neonatal pneumonia and sepsis by permitting the care of increasingly premature and sick neonates who may have previously succumbed. the use of highly invasive monitoring and therapeutic equipment has life-saving potential, yet they can introduce a significant iatrogenic infection potential. fi the above-quoted incidence and mortality rates demonstrate that pulmonary infection is a significant risk to the neonate, and especially to the fi premature infant. the reasons for this are multifactorial. the full term neonate is considered immunologically "competent", in that most can respond appropriately to antigenic stimulation. the absolute number of t-cells is similar in the neonate to the adult as long as thymic function is normal during foetal life (roberton 1996) . however, there are other considerations which render the neonate relatively more susceptible to infection. studies have documented reduced leukocyte adherence and chemotaxis, as well as complement deficiencies that fi result in reduced phagocytosis and intracellular killing (roberton 1996; speck et al. 1979 ). surface iga is absent, and serum igg can be deficient in fi early preterm infants for whom sufficient time for fi normal maternal igg transplacental transfer has not occurred. even if sufficient transplacental igg trans-fi fer has occurred, this supply of igg has a limited life span of only several weeks resulting in a physiologically normal, transient hypogammaglobulinemia in the fi rst few months of life. furthermore, while the fi neonate can initially respond to antigenic stimulation with an endogenous igm response, conversion of this response to a more mature igg response is delayed. these deficiencies frequently result in a fi neonate with a limited capability to control and/or limit the spread of invasive organisms. as well, the physical environment of both the foetus and the neonate play significant roles in exposing fi them to potential pathogens. the environment of the foetus inside intact maternal amniotic membranes is generally considered partially protective from external sources of infection. therefore, one of the most common causes of neonatal sepsis is premature rupture of maternal membranes. nevertheless, neonatal sepsis can occur even in the presence of intact maternal membranes (kirkpatrick and mueller 1998; speck et al. 1979) . congenital infections can occur through transplacental spread of a variety of organisms. during and after birth, the neonate is physically exposed to potentially pathogenic organisms which may colonise the maternal vaginal canal, or may be actively infecting the mother. the neonatal nursery provides a large source of invasive monitoring and therapeutic instruments, all of which can iatrogenically introduce organisms into the infant. nursery personnel and family members inadvertently spread nosocomial infections even if strict antiseptic technique is maintained. it is not surprising, therefore, that, since the lungs provide a "front line" exposure between the neonate and its environment, neonatal pneumonia remains a signifi-fi cant problem in the nicu. unfortunately, the clinical signs and symptoms of infection that the neonate may manifest are frequently protean and non-specific. the infant may fi simply demonstrate listlessness and/or decreased physical activity. feeding intolerance or pallor may be the only initial clinical manifestations. either apnoea or tachypnea may be present and either tachycardia or bradycardia may occur. the child may be febrile or even hypothermic. laboratory tests may be equally non-specifi c, demonstrating either an increased or fi a decreased total white cell count. it is, therefore, common for the clinician to perform a chest radiograph to help determine if pulmonary infection is the potential cause of some of these clinically observed changes. the chest radiograph, in this situation, can become useful to localise the problem to a pulmonary aetiology, even if the radiographic changes are not sufficiently specifi fi c to diagnose pulmonary infection fi as the cause of the infant's symptoms. given the fulminant potential for some etiologic pathogens which cause neonatal pneumonia, any abnormality on the chest radiograph which may suggest a pulmonary infection warrants the initiation of broad spectrum antibiotic coverage (dennehy 1987; kirkpatrick and mueller 1998; speck et al. 1979 ). when taken in isolation, the study of the radiological manifestations of neonatal pneumonia is disappointing. there are few definitive correlative studies fi in the literature analysing the various radiological findings of pulmonary infection and comparing fi them with other causes of respiratory compromise, let alone comparing them to potential etiologic microbial agents. most studies concur that the radiological findings alone are non-specifi fi c, such that it fi is almost impossible to determine a causative organism by their radiographic manifestations (burko 1962; currarino and silverman 1957; harris 1963; roberton 1996; wiesenberg 1973) . furthermore, many of these neonates do not suffer from pneumonia in isolation, but may also have complicating features such as hyaline membrane disease, meconium or amniotic fl uid aspiration, persistent fl pulmonary hypertension, transient tachypnea of the newborn, secondary ards, patency of the ductus arteriosus, or a variety of other causes of neonatal respiratory distress. in one of the few studies looking specifically at radiological patterns in neonatal fi pneumonia, haney et al. (1984) reviewed autopsy records of all neonates who died over a 6-year period and in whom an autopsy documented pathological changes of pneumonia as the only significant ab-fi normality. a review of their immediate pre-mortem chest radiographs revealed that the majority of cases demonstrated bilateral air space disease. unfortunately, a pattern indistinguishable from hyaline membrane disease was seen in 13%, and a pattern indistinguishable from transient tachypnea of the newborn was seen in 17%. a few features have been described which may be helpful in identifying pulmonary infection as the source of respiratory distress. some authors have postulated that one fi nding fi that may help to differentiate pneumonia from hyaline membrane disease is the presence of increased lung volumes combined with air-space disease in the non-intubated neonate (harris 1963; wiesenberg 1973) . hyaline membrane disease tends to cause diffusely small lungs from surfactant defi ciency, fi while infection may result in over-inflation of re-fl cruited airspaces. unfortunately, only 17% of the population described by haney et al. demonstrated increased lung volumes, and most of these children were intubated on their pre-mortem examination. others have suggested that air-space disease in the presence of a pleural effusion is more suggestive of bacterial pneumonia than of other causes of neonatal respiratory distress, especially when group b streptococcus is the etiologic agent (haney et al. 1984; leonidas et al. 1977; payne et al. 1988) . the presence of pneumatoceles may also suggest a bacterial aetiology, a finding which is not exclu-fi sive to staphylococcal pneumonia (papageorgiou et al. 1973; wiesenberg 1973) . as well, a diffuse, bilateral, alveolar pattern that develops in the first fi 4-6 h of life is characteristic, although not specific, fi for early neonatal sepsis and pneumonia, again classically seen when group b streptococcus is the etiologic agent. ancillary non-pulmonary chest radiographic fi ndings may be helpful in suggesting fi a diagnosis. air-space disease in conjunction with periostitis or osteomyelitic lesions may suggest congenital syphilis, while a diffusely interstitial reticulonodular pattern in conjunction with characteristic metaphyseal lucencies suggests a congenital viral aetiology. a diffuse interstitial pattern alone is nonspecifi c, but if associated hepatosplenomegaly and fi intracerebral calcifi cations are present, cmv pneu-fi monitis becomes a likely aetiology. although the initial chest radiograph may be non-specific, serial fi chest radiographs can be extremely useful, especially in differentiating the rapidly resolving pattern of transient tachypnea of the newborn from the more persistent pattern of neonatal pneumonia. as well, serial examinations are frequently used to follow the response to therapeutic interventions such as antibiotic administration. it is best to review the major aetiologic organisms and their respective radiographic patterns according to the initial source of neonatal infection. these are commonly divided into those agents causing transplacental infection, agents acquired perinatally and those acquired postnatally or nosocomially. transplacentally transmitted infections, conforming to the traditionally taught pneumonic of "torch" (or "crotsh") are, fortunately, quite rare. the pulmonary manifestations of these particular infections are even less common. while many perinatally acquired infections gain route to the foetus/neonate via aspiration or inhalation, transplacental infections enter the foetus hematogenously, via the umbilical cord. most infants, therefore, tend to manifest systemic and multi-organ disease rather than a primary pneumonitis. it is, therefore, not surprising that the medical literature is generally deficient in reviews of the fi radiological manifestations of pneumonia in infants with transplacentally acquired infections. the most common of these disorders appears to be the fairly ubiquitous cytomegalovirus (cmv), whose presence is well documented in all ages, races and socio-economic levels throughout both the developed as well as developing countries. fortunately, estimates of foetal infection rates are very low. approximately 1% of all newborns demonstrate a serologic response to transplacentally acquired cmv. however, 90% of these infants are asymptomatic and demonstrate no sequelae of the infection. when clinically evident infection does occur, the primary manifestations are usually systemic, including intrauterine growth retardation, hepatosplenomegaly and thrombocytopenia. the most signifi cant primary organ of in-fi volvement is the central nervous system, producing microcephaly, intracranial calcifications and/or sen-fi sori-neural hearing loss. congenital cmv pneumonitis is a rare manifestation, occurring only in 1-2% of cmv infected newborns (dworsky 1982; stagno 1980) . it is signifi cantly more common in infants fi who acquire the infection from other sources such as transvaginal exposure, maternal breast milk, or neonatal blood transfusions (dworsky 1982; stagno 1981; stagno et al 1980; whitsett et al. 1999) . although the radiographic manifestations of congenital cmv pneumonitis have not undergone statistical scrutiny, it is commonly accepted that this infection manifests as a diffuse reticulonodular, non-specific, fi viral interstitial pattern (dennehy 1987; whitsett et al. 1999; wiesenberg 1973) , similar to many viral pneumonitides ( fig. 7 .1). other, less common, transplacentally acquired pneumonitides include rubella, syphilis, listeria monocytogenes and tuberculosis. in general, maternal infection rates with tuberculosis and syphilis are increasing in both developing and industrialised countries. this can be partially explained by the widespread increase in migration rates into industrialised countries from countries in which infections rates are relatively high. as well, the hiv world-wide epidemic has permitted many of these organisms to propagate through immunosuppressed hosts. congenital infection rates from syphilis were increasing in the 1980s and 1990s in predominantly urban geo-graphic foci. this trend appeared to peak in the early 1990s with over 4,000 reported cases to the center for disease control in the united states, decreasing to approximately 2,000 cases in 1996 (sanchez and wendel 1997) . congenital syphilitic pneumonia is an uncommon manifestation of congenital syphilis, seen in only approximately 5-25% of cases of congenital syphilis (sanchez and wendel 1997) . it is commonly referred to as "pneumonia alba", due to the pathologic whitish plaque-like appearance of the areas of consolidation. radiologically, it usually appears as a diffuse process (fig. 7. 2), but may manifest larger patches of air-space disease corresponding to mononuclear organizing infiltrates ( fi roberton 1996). one helpful sign on a chest radiograph is the presence of osseous lesions such as diffuse long bone periostitis, a radiographic sign which is more commonly seen in congenital syphilis than pulmonary consolidation. listeria monocytogenes is a gram-positive organism which can be acquired transplacentally or perinatally and frequently presents as a pneumonitis. maternal infection is usually within 2-3 weeks of delivery with a non-specific fl fi u-like illness. the fl illness in the neonate is clinically similar to group b streptococcus, demonstrating an "early" onset variety which presents in the first 72 h of life, and a fi "late" onset form that becomes manifest after 7 days of life. at least 50% of those with the "early" onset form demonstrate respiratory tract involvement (bortolussi and schlech 2001) . the predominant radiographic pattern described is fairly nonfig. 7 .1. newborn with documented cmv pneumonia. there is a non-specific diffuse interstitial and predominantly re-fi ticular pattern, which is typical of viral pneumonitides newborn infant born at 35 weeks of gestation to a mother treated during a previous pregnancy for congenital syphilis. the radiograph demonstrates a diffuse and bilateral pneumonitis, and the child had clinical fi ndings of fi pneumonia. associated bone changes are barely visible in the humeri, but are more apparent on other bone films. the fi infant responded well to appropriate antibiotics specifi c. in a comprehensive review of 55 cases of fi neonatal listeriosis, 39 of which underwent chest radiographic examination, two equally common patterns were described (willich 1967) . the first fi is of a "bronchopneumonic" pattern of streaky and confl uent opacities, and the second is a diffuse, fi fl ne fi interstitial pattern. it is postulated that some of the coarser interstitial densities correlate with multifocal granulomas in medium and smaller airways. these radiographic manifestations are remarkably similar to group b streptococcal pneumonia acquired perinatally as described below (whitsett et al. 1999; wiesenberg 1973) . congenital tuberculous infection is a rare disorder, having been reported in less than 300 cases in the medical literature (starke 1997) . it occurs secondary to disseminated maternal infection, which produces placental caseating granulomas. pulmonary manifestations are uncommon as the usual primary site of infection is the liver from umbilical cord seeding. however, the patency of the ductus venosus and foramen ovale can result in disseminated infection relatively easily. neonatal tuberculosis may also occur from aspiration of infected amniotic fluid, from ingestion of infected breast milk, or from fl inspiration of maternal respiratory droplets. respiratory distress is a fairly common manifestation of neonatal tuberculosis, seen in approximately 72% of cases (starke and smith 2001) . parenchymal consolidation and adenopathy are common radiographic manifestations, although up to 50% of neonatal cases with radiographic findings demonstrate fi a miliary pattern (starke and smith 2001) . although the pneumonic "torch" includes toxoplasmosis and herpes, the former uncommonly causes pneumonitis, and the latter is more appropriately considered under perinatally acquired infections. cases reports of placental infections with influenza a ( fl arvin and maldonado 2001), varicella (keyserling 1997) , adenovirus (abzug and levin 1991) and echovirus (cheeseman et al. 1977) are described, but are exceedingly rare. perinatally acquired infections can be clinically categorised into those which are acquired via as-cending infection from the vaginal tract, those acquired transvaginally during the birth process and those acquired nosocomially in the neonatal period. ascending infections from the maternal vaginal tract are the usual cause of chorioamnionitis. it is estimated that maternal chorioamnionitis complicates an approximate 1-10% of all pregnancies in industrialised countries (belady et al. 1997) , and is probably much more common in underdeveloped countries due to substandard maternal health care. predisposing factors to chorioamnionitis include premature rupture of membranes of greater than 24 h, foetal instrumentation, increased number of vaginal examinations before birth, and prolonged labour. although the organisms causing foetal sepsis are polymicrobial, nearly half of all infections are attributable to either group b streptococcus or e. coli (belady et al. 1997) . it is postulated (wiesenberg 1973 ) that most organisms causing neonatal pneumonia gain entry to the infant during the birth process as the foetus takes its fi rst gasping efforts at breathing. this fi may occur earlier during the course of labour in the asphyxiated infant who may swallow and/or aspirate in response to non-specific stressful events. fi it is for this reason that clinical signs or symptoms of maternal chorioamnionitis warrant the use of maternal perinatal intravenous antibiotics, which have been shown to significantly decrease the risk fi of sepsis and pneumonia in the neonate (belady et al. 1997) . there appear to be two separate clinical syndromes for neonatal sepsis and/or pneumonia which are significantly different with respect to fi symptomatology and outcome. those infants with pneumonia or sepsis presenting within the first fi 48 h of life tend to have a more acute and severe clinical picture of hypotension, shock, disseminated intravascular coagulation and multi-organ failure. mortality rates in this "early" onset form vary between 30%-50% (bohin and field 1994; kirkpatrick and mueller 1998; speck et al. 1979; whitsett et al. 1999) , especially when the offending organism is group b streptococcus. those infants presenting after 48 h tend to have a less fulminant course with mortality rates of less than 5% (bohin and field 1994) . as well, the clinical symptoms tend to be less drastic, presenting with more isolated respiratory difficulty or less fi severe systemic manifestations. unfortunately, the radiographic manifestations of the various etiologic agents carry very poor specifi cities. as noted previously, multiple studies fi have documented the non-specificity of the radio-fi graphic patterns of neonatal pneumonia (ablow et al. 1976; burko 1962; currarino and silverman 1957; haney et al. 1984; harris 1963; leonidas et al. 1977; lilien et al. 1977) . this holds true both in regards to differentiating between the various etiologic microbial agents, as well as to differentiating pneumonia itself from other cause of respiratory distress such as transient tachypnea of the newborn (ttn), hyaline membrane disease (hmd), and meconium aspiration. the findings which have been fi postulated as helpful in differentiating infection from other causes of respiratory distress include the presence of a pleural effusion (leonidas et al. 1977) , cardiomegaly (hubbell et al. 1988) , and pulmonary over-inflation, the latter of which is fl postulated to help only in differentiating group b streptococcal pneumonia from hmd (ablow et al. 1976 ). the most common radiographic manifestation of neonatal pneumonia is a bilateral coarse pattern of perihilar reticular densities which may also involve scattered areas of air space disease (wiesenberg 1973) (fig. 7.3) . isolated lobar pneu-monia is uncommon in this age group (ablow et al. 1976; currarino and silverman 1957; haney et al. 1984; harris 1963; wiesenberg 1973) , likely related both to the aspirated route of entry as well as to the inability of the neonate to control infection locally. the radiographic differentiation of pneumonia from other processes becomes even more difficult fi in the preterm infant. lilien et al. (1977) reviewed the radiographic pattern of early onset group b streptococcal pneumonia in 73 infants, of which 86% were premature. a significantly larger portion fi of those preterm infants with a radiographic pattern of hyaline membrane disease (hmd) actually had both hmd and group b streptococcal pneumonia than those who had hmd alone. nevertheless, ablow et al. (1976) reviewed the radiographic patterns of a smaller number of preterm infants and found that half of those who died of fulminant early onset group b streptococcal sepsis demonstrated a radiographic pattern that could not be differentiated from hyaline membrane disease. they note however that the "overall volume of the lungs is usually increased" in neonatal pneumonia. leonidas et al. (1977) in their review of 67 infants of all gestational ages hospitalised for respiratory distress, found that the pattern of parenchymal lung disease was just as likely to be "typical" for pneumonia as it was to be "typical" for hyaline membrane disease. in their study, the presence of cardiomegaly or pleural effusions was more likely to represent neonatal sepsis. group b streptococcal sepsis is one of the most common causes of neonatal sepsis. as such, there is more literature published regarding this particular agent than regarding most others. the radiographic manifestations initially described by ablow et al. (1976) were essentially those of hyaline membrane disease. they described a "fi ne, diffuse granular pattern" fi in 50% of infants who died of "early" onset, fatal group b streptococcal infection (fig. 7.4) , with the remainder of fatal cases demonstrating either similar fi ndings or more focal, lower lobe opacifi fi cation. fi non-fatal cases tended to have a more heterogeneous pattern of mixed interstitial and air space changes. fig. 7.3 . chest radiograph of a 1-week-old premature infant born at 26 weeks of gestational age, with documented pseudomonas pneumonia. bilateral air-space changes are noted on a background of diffuse interstitial changes a subsequent study suggested that cardiomegaly and/or pleural effusions may help to differentiate group b streptococcal infection from hyaline membrane disease (leonidas 1977) , while admitting that there are many other causes for the presence of these fi ndings. fi there is a curious association between group b streptococcal pneumonia and the presence of an ipsilateral diaphragmatic hernia, especially when the hernia is right sided. suggested mechanisms for the presence of this association have included a primary abnormality of lung compliance, secondary effects of mechanical ventilation on the infected lung, or direct local effects of the organism itself (potter et al. 1995) . whatever the mechanism, persistent ventilatory requirements or radiographic abnormalities in neonates after the treatment of group b streptococcal pneumonia should alert the radiologist to the possibility of an associated diaphragmatic hernia. other perinatal bacterial infections such as pseudomonas, e. coli, klebsiella and other streptococci have received little attention in the literature with respect to specifi c radiographic patterns. fi perinatal viral infections such as herpes, varicella, rsv and adenovirus tend to be signifi cantly less fi common than the previously described bacterial causes of neonatal sepsis. neonatal herpes infection can be acquired transplacentally, during birth, or even postnatally. the majority of neonates acquire the virus transvaginally from a mother who is actively shedding the virus. only a small minority of infected women are actually shedding the virus during labour (kohl 1997) . as well, only a minority of infants exposed will become clinically infected. as a result, neonatal herpetic pneumonia is an uncommon disorder, affecting approximately 1 in 7,000 live births in the united states (kohl 1997) . this rate, however, is increasing with some studies reporting a ten-fold increase over the past 20 years (kohl 1997) . although pulmonary infection occurs in only 5-25% of infected newborns, it tends to produce a fulminant and progressive course (dominguez et al 1984; hubbell et al 1988; kohl 1997) . the described radiographic fi ndings are similar to most viral pulmo-fi nary infections, starting as bilateral interstitial perihilar reticular densities (fig. 7 .5) that can be initially quite subtle. confluent alveolar changes occur as the fl infection spreads, progressing to diffuse pulmonary opacitifi cation that may have accompanying pleural fi effusions (dominguez et al. 1984; hubbell et al. 1988) . varicella in the neonatal period is a rare disease. it is estimated that approximately 3,000 cases of maternal varicella occur in the united states annually (keyserling 1997) . transplacental infection is extremely rare, but can result in a congenital varicella syndrome, characterised primarily by limb and fig. 7.4 . one-week-old infant with typical non-specific fi changes of diffuse air-space disease from group b streptococcal pneumonia fig. 7 .5. twelve-day-old infant with severe interstitial pneumonitis and diffuse anasarca from viral sepsis secondary to herpes cns malformations. respiratory infection can be acquired by the infant in the neonatal period from a mother who is actively shedding the virus. in order for the mother to be actively shedding the virus, maternal infection must have occurred within 3 weeks of delivery. the severity of neonatal disease acquired from a prenatally infected mother varies with the time of delivery. maternal shedding is most active in the first few days of appearance of the rash. fi at this time, maternal antibody response is still developing, and little signifi cant antibody crosses the fi placenta. birth in this time period, therefore, results in a more severely infected neonate, with fatality rates quoted at between 20-40% (albritton 1998) . administration of varicella-zoster immunoglobulin (vzig) in this period has been shown to ameliorate the severity of neonatal infection (keyserling 1997) . as well, it should be remembered that neonatal infection not uncommonly occurs via nosocomial or familial exposure. neonatal varicella pneumonia is a severe complication of disseminated varicella infection and is a major cause of neonatal mortality from this infection. there is, however, a paucity of published reports concerning the radiographic manifestations of neonatal varicella pneumonia. the classically described radiographic manifestation in older individuals is that of a diffuse interstitial reticulonodular pattern (fig. 7.6) , which characteristically appears a little more nodular than reticular (albritton 1998) . case reports of neonatal pulmonary infections from adenovirus (abzug and levin 1991) , rsv (berkovich and taranko 1964; keyserling 1997; meissner et al. 1984 ), parainfluenza ( fl meissner et al. 1984 ) and enteroviruses (keyserling 1997) have been published which provide only anecdotal descriptions of the radiographic patterns of these viruses in the neonate. most describe bilateral perihilar "infiltrates" as the predominant radio-fi graphic pattern. human metapneumovirus has recently been implicated as a relatively common cause of bronchiolitis in infants and children. it appears to be less common than rsv, yet 10% of all cases are in children less than 1 month of age. it appears to clinically act in a fashion similar to other respiratory viruses, in that younger children and those with respiratory co-morbidities are more severely affected. the radiographic fi ndings described fi are similar to other viral causes of bronchiolitis ( foulongne et al. 2006) . chlamydial pneumonia is caused by chlamydia trachomatis, an obligate intracellular parasite which is a common, sexually transmitted infection. approximately 4 million new cases of maternal chlamydial fig. 7.6 . this infant died at 12 days of life after developing disseminated varicella from a mother who manifested active skin lesions 1 week before delivery infection are reported in the united states annually to the centers for disease control ( hammerschlag 1994) . approximately 30% of infants born to infected mothers will have positive nasopharyngeal cultures, but only 30% of these will develop pneumonia. clinically, the infant typically demonstrates an initial conjunctivitis between 5-14 days after birth. this tends to resolve and the pulmonary infection only becomes manifest after 4-12 weeks of age. clinical manifestations are mild, and fever is characteristically absent (hammerschlag 1994) . the radiographic manifestations are typically non-specific, fi but the pattern described is that of hyperinflation fl with bilateral diffuse reticular perihilar infiltrates fi (fig. 7.7) (hammerschlag 1994; harrison et al 1978; hess 1993; radkowski et al. 1981; rettig 1988) . interestingly, stagno et al. (1981) reviewed a series of infants with pneumonia caused by cmv, chlamydia, ureaplasma and pneumocystis and found the radiographic patterns to be indistinguishable. chlamydial infection is generally mild and even untreated infants usually improve over 4-8 weeks (rettig 1988) . there is some evidence, however, that these children demonstrate long-term obstructive changes on pulmonary function tests, with a significantly greater incidence of physician fi diagnosed asthma in later childhood (weiss et al. 1986 ). ureaplasma urealyticum is a micro-organism which is similar to the mycoplasma species in that it is a unicellular organism without a cell wall. asymptomatic colonisation of the maternal genital tract with ureaplasma urealyticum is common, affecting over half of all pregnant women. it has, however, recently been proposed that it has a pathogenic potential in neonates (dworsky and stagno 1981; wang et al. 1997) . a significant as-fi sociation and causation has been established between maternal colonisation with u. urealyticum and chorioamnionitis, spontaneous abortion and early neonatal death (dworsky and stagno 1981; wang et al. 1997) . there is an increasing volume of literature demonstrating an association between neonatal pneumonia and the isolation of this organism from endotracheal aspirates, pleural fluid, fl lung tissue and/or blood, especially in pre-term infants (pinna et al. 2006 ). in addition, the radiographic changes of ureaplasma infection were evaluated in one study (crouse et al. 1993) , where it was found that abnormalities were diagnosed by an appropriately blinded radiologist twice as frequently in ureaplasma infected babies, than in those who were culture negative. unfortunately, the radiographic findings, which correlated with tra-fi cheal aspirate isolation of ureaplasma, were broad and non-specific. the radiographic fi fi ndings, which fi were taken to be indicative of ureaplasma infection, included any radiographic manifestation of bronchopulmonary dysplasia (bpd), as well as a series of non-specific fi fi ndings of mixed interstitial and fi air space changes ( fig. 7.8) . the study did confirm fi that radiographic changes do occur in the presence of ureaplasma infection; however, the relative frequency or specificity of the fi fi ndings were, unfor-fi tunately, not addressed. they concluded, however, that radiographic manifestations of typical type iii or iv bpd are associated with ureaplasma infection, especially when these changes are seen at a chronological age that is slightly earlier (2 weeks postnatally) than expected from the usual fi ndings fi of bpd in neonates (crouse et al 1993) . interestingly, there is strong evidence which demonstrates a signifi cantly higher incidence of chronic lung dis-fi ease in infants who previously demonstrated culture-proven ureaplasma urealyticum pneumonitis (wang et al 1997; pinna et al 2006) . the global hiv epidemic warrants comment on the neonatal manifestations of this particular infection. although there has been a progressive defig. 7.7 . two-week-old with extensive interstitial and alveolar changes from chlamydia pneumonia. the young age of this infant is atypical, most cases presenting after 1 month of life cline in incidence of vertically and perinatally acquired infection from hiv in recent years due to a combination of educational programs and routine use of anti-retroviral therapies in developed countries, these interventions have not been as widely available in developing countries. as a result, 90% of worldwide perinatally acquired hiv infection is now seen in africa alone (graham 2003) . the clinical presentation of hiv infection in the neonatal period is still somewhat uncommon, and most neonates with hiv are relatively asymptomatic for the fi rst few months of life ( fi marquis and bardeguez 1994) . as hiv testing may be inaccurate in the neonatal period, prophylaxis against pneumocystis jiroveci (previously known as pneumocystis carinii) is started when hiv-exposed infants are approximately 6 weeks old (krist et al 2002) . those few cases that manifest early respiratory symptoms usually do so from infection with an opportunistic organism, most commonly due to p. jiroveci. the described radiographic pattern is that of a fine in-fi terstitial diffuse pattern that rapidly progresses to diffuse bilateral air-space disease (marquis and bardeguez 1994) . early presentation of changes of tuberculous disease or cmv pneumonitis in either the neonatal or infantile period should also raise the suspicion of underlying hiv infection ( marquis and bardeguez 1994; graham 2003) . although the medical care of sick and premature infants has improved to a remarkable extent in recent decades, the problem of nosocomial spread of infection remains a signifi cant cause of morbidity and fi mortality in neonatal intensive care units. the topic is very broad, encompassing all infections acquired from any source while still in the nicu. this includes fungal complications related to the administration of broad-spectrum antibiotics. as mentioned previously, at least one study has documented an incidence rate of 17% of all low birth weight infants who will acquire at least one nosocomial infection during their stay in the nursery (thompson et al. 1992) . hemming et al. (1976) reviewed all nosocomially acquired infections in a 3-year period and discovered that 30% of them resulted in pulmonary infection. in that study, the most common pathogens discovered were staphylococcus aureus (47%) and gram-negative enteric bacilli (45%) (fig. 7.9) . a more recent review (thompson et al. 1992) found streptococcus epidermidis to be the most common organism responsible for secondary infection in infants with birth weights less than 750 g. interestingly, aside from low birth weight, the other significant risk factor for acquisition of a nosocomial fi infection was prolonged ventilation. should instigate an early and aggressive response to diagnosis and treatment of a potential pneumonitis. an aggressive approach is especially needed when some of the potential causes of pneumonia in these infants are fungal in origin. laboratory identifi cation of fungal organisms is frequently diffi fi cult fi and often delayed. in a review of fungal infections in very low birth weight infants (baley et al. 1984 ), a mean of 33 days was required to diagnose the presence of a fungal infection. clinically evident respiratory deterioration was present in all ten infants, and eight of these demonstrated worsening pulmonary infi ltrates. the chest radiograph, therefore, becomes fi an integral part of the early clinical investigation of these infants. a systematic review of previous films fi must be performed to permit recognition of new changes superimposed on the complex chronic abnormalities that are frequently present. neonatal pneumonia remains a significant risk to fi the health and well being of the newborn, despite contemporary advances in the quality and complexity of medical care. ironically, the risk of iatrogenic infection is rising with the level of sophistication of neonatal medicine. both the clinical and radiographic appearances of many of these infections are disappointingly non-specific. the role of the appro-fi priate interpretation of diagnostic images in these children with multi-system disease becomes critical in those cases for which the radiographic pattern is suffi ciently specifi fi c to be diagnostic. in cases with fi non-specifi c radiographic manifestations, the pae-fi diatric imager has a critical role, not only in helping to identify a pulmonary site of disease, but also in following the childs' response to therapeutic interventions. one key to the diagnosis of a nosocomially acquired respiratory tract infection appears to be the presence of deteriorating radiographic changes after an initial period of stability or improvement. the radiographic pattern of deterioration may be non-specifi c, but the presence of any deterioration fi neonatal adenovirus infection: four patients and review of the literature kendig's disorders of the respiratory tract in children infectious diseases of the fetus and newborn infant intra-amniotic infection and premature rupture of the membranes acute respiratory illness in the premature nursery associated with respiratory syncytial virus infections the epidemiology of neonatal respiratory disease listeriosis considerations in the roentgen diagnosis of pneumonia in children fatal neonatal pneumonia caused by echovirus type 9 radiographic changes associated with tracheal isolation of ureaplasma urealyticum from neonates roentgen diagnosis of pulmonary disease of the newborn infant respiratory infections in the newborn neonatal herpes simplex pneumonia: radiographic findings neonatal pneumonia in developing countries newer agents causing pneumonitis in early infancy human metapumovirus infection in young children hospitalized with respiratory tract disease hiv and respiratory infection in children chlamydia trachomatis in children radiographic findings in neonatal pneumonia the newborn with respiratory distress: some roentgenographic features chlamydia trachomatis infant pneumonitis: comparison with matched controls and other infant pneumonitis nosocomial infections in a newborn intensive-care unit. results of forty-one months of surveillance chlamydia in the neonate neonatal herpes simplex pneumonitis other viral agents of perinatal importance: varicella, parvovirus, respiratory syncytial virus, and enterovirus respiratory disorders in the newborn neonatal herpes simplex virus infection mangement of newborns exposed to maternal hiv infection radiographic findings in early onset neonatal group b streptococcal fi septicemia significance of radiofi graphic findings in early-onset group b streptococcal infi fection imaging of hiv infection in the prenatal and postnatal period a simultaneous outbreak of respiratory syncytial virus and parainfl uenza virus type 3 in a newborn nursery klebsiella pneumonia with pneumatocele formation in a newborn infant correlation of clinical and pathologic fi ndings in early onset neonatal group fi b streptococcal infection with disease severity and prediction of outcome neonatal radiology. acquired diaphragmatic hernia with group b streptococcal pneumonia current opinion infectious diseases chlamydia pneumonia in infants: radiology in 125 cases perinatal infections with chlamydia trachomatis neonatal respiratory disorders syphilis in pregnancy neonatal infections pneumocystis carinii pneumonitis in young immunocompetent infants infant pneufi monitis associated with cytomegalovirus, chlamydia, pneumocystis, and ureaplasma: a prospective study tuberculosis: an old disease but a new threat to the mother, fetus, and neonate infectious diseases of the fetus and newborn infant nosocomial bacterial infections in very low birth weight infants ureaplasma urealyticum infections in the perinatal period neonatal pneumonia pulmonary assessment of children after chlamydial pneumonia of infancy acute respiratory disorders neonatal pneumonia and pulmonary hemorrhage the roentgenological appearance of pulmonary listeriosis key: cord-018461-lq1m9h41 authors: elgazzar, abdelhamid h.; elmonayeri, magda title: inflammation date: 2014-06-27 journal: the pathophysiologic basis of nuclear medicine doi: 10.1007/978-3-319-06112-2_4 sha: doc_id: 18461 cord_uid: lq1m9h41 inflammation was described as early as 3000 bc in an egyptian papyrus [1] and is still a common problem despite continuous advancements in prevention and treatment methods. the delineation of the site and extent of inflammation are crucial to the clinical management of infection and for monitoring the response to therapy [2]. infl ammation was described as early as 3000 bc in an egyptian papyrus [ 1 ] and is still a common problem despite continuous advancements in prevention and treatment methods. the delineation of the site and extent of infl ammation are crucial to the clinical management of infection and for monitoring the response to therapy [ 2 ] . this issue is relevant to nuclear medicine, since physiological along with morphological imaging has an important role in achieving this goal. infl ammation is a complex tissue reaction to injury. injury may be caused not only by living microbes, i.e., bacteria, viruses, or fungi, leading to infection, but also by injurious chemical, physical, immunological, or radiation agents: • physical agents -trauma -heat • chemical agents -chemotherapy -industrial accidents • immunological agents -antigen-antibody reactions • radiation -radiation therapy -nontherapeutic radiation exposure infl ammation is fundamentally a protective reaction against the cause of cell injury as well as the consequence of such injury. however, infl ammation is potentially harmful and may even be life threatening. since most of the essential components of the infl ammatory process are found in the circulation, infl ammation occurs only in vascularized tissue. infl ammation is generally considered a nonspecifi c response, because it happens in the same way regardless of the stimulus and the number of exposures to the stimulus [ 2 ] . this is different from the immune system, which has memory, and the antigens are specifi c and induce a specifi c response. infl ammation may be classifi ed as acute or chronic. acute infl ammation is the immediate or early response to injury and is of relatively short duration. it lasts for minutes, hours, or at most a few days. chronic infl ammation, on the other hand, is of longer duration and may last from weeks to years [ 3 ] . the distinction between acute and chronic infl ammation, however, depends not only on the duration of the process but also on other pathological and clinical features. acute infl ammation continues only until the threat to the host has been eliminated, which usually takes 8-10 days, although this is variable. infl ammation is generally considered to be chronic when it persists for longer than 2 weeks [ 2 ] . many regional and systemic changes accompany acute infl ammation and are mediated by certain chemicals produced endogenously called chemical mediators and are behind the spread of the acute infl ammatory response following injury to a small area of tissue into uninjured sites. these chemical mediators include mediators released from cells such as histamine and prostaglandins and others in plasma which are released by some systems contained in the plasma which are the four enzymatic cascade systems: the complement, the kinins, the coagulation factors, and the fi brinolytic system which produce several infl ammatory mediators [ 4 -6 ] . table 4 .1 summarizes the main chemical mediators of infl ammation. acute infl ammation is characterized by the following major regional components: local vascular changes 1. vasodilation following transient vasoconstriction is one of the most important changes that accompany acute infl ammation, and it persists until the end of the process. it involves fi rst the arterioles and then results in the opening of new capillary beds in the area. 2. increased vascular permeability due to: • contraction of endothelial cells with widening of intercellular gaps • direct endothelial injury, resulting in endothelial cell necrosis and detachment • leukocyte-mediated endothelial injury: leukocytes adhere to the endothelium, which becomes activated, thereby releasing toxic oxygen species and proteolytic enzymes and causing endothelial injury. increased permeability of the microvasculature, along with the other changes described, leads to leakage with formation of "exudate," an infl ammatory extravascular fl uid with a high protein content, much cellular debris, and a specifi c gravity above 1.020. this is the hallmark of acute infl ammation, which may also be called exudative infl ammation. it indicates signifi cant alteration in the normal permeability of the small blood vessels in the region of injury. the two components of exudate, fl uid and protein, serve good purposes. fluid increase helps to dilute the toxins. protein increase includes globulins that provide protective antibodies, while fi brin helps to limit the spread of bacteria and promotes healing. exudate varies in composition. in early or mild infl ammation, it may be watery (serous exudate) with low plasma protein content and few leukocytes. in more advanced infl ammation, the exudate becomes thick and clotted (fi brinous exudate). when large numbers of leukocytes accumulate (fig. 4.3 ) , the exudate consists of pus and is called suppurative, while if it contains erythrocytes due to bleeding, it is referred to as hemorrhagic. pus, accordingly, is a variant of exudate that is particularly rich in leukocytes, mostly neutrophils and parenchymal cell debris. exudate should be differentiated from "transudate," which is a fl uid with low protein facilitates phagocytosis of bacteria by macrophages (opsonization of bacteria) kinin system bradykinin included in the system is the most important vascular permeability factor, also a mediator for pain which is a major feature of acute infl ammation coagulation factors responsible for the conversion of soluble fi brinogen into fi brin, a major component of the acute infl ammatory exudate fibrinolytic system plasmin included in the fi brinolytic system is responsible for the lysis of fi brin into fi brin degradation products, which have a local effect on vascular permeability concentration and a specifi c gravity of less than 1.012. transudation is associated with normal endothelial permeability [ 3 , 5 ] . local cellular events 1. margination after stasis develops, leukocytes will be peripherally oriented along the vascular endothelium, a process called leukocytic margination ( fig. 4.1 ). leukocytes emigrate from the microcirculation and accumulate at the site of injury. once outside the blood vessel, the cells migrate at varying rates of speed in interstitial tissue toward a chemotactic stimulus in the infl ammatory focus. through chemoreceptors at multiple locations on their plasma membranes, the cells are able to detect where the highest concentrations of chemotactic factors are and to migrate in their direction. granulocytes, including the eosinophils, basophils, and some lymphocytes, respond to such stimuli and aggregate at the site of infl ammation. the primary chemotactic factors include bacterial products, complement components c5a and c3a, kallikrein and plasminogen activators, products of fi brin degradation, prostaglandins, and fi brinopeptides. histamine is not a chemotactic factor but facilitates the process. some bacterial toxins, this defense mechanism is particularly important in bacterial infections. the polymorphonuclear leukocytes and macrophages ingest debris and foreign particles. acute infl ammation has several possible local sequelae. these include resolution, suppuration (formation of pus), organization, and progression to chronic infl ammation. resolution means complete restoration of tissues to normal. organization of tissues refers to their replacement by granula-tion tissue with formation of large amounts of fi brin, growth of new capillaries into fi brin, migration of macrophages into the zone, and proliferation of fi broblasts resulting in fi brosis and consequently exudate becoming organized. acute infl ammation may progress to a chronic form characterized by reduction of the number of polymorphonuclear leukocytes but proliferation of fi broblasts with collagen production. commonly, chronic infl ammation may be primary with no preceding acute infl ammatory reaction. chronic infl ammation, whether following acute infl ammation or not, is characterized by a proliferative (fi broblastic) rather than an exudative response with predominantly mononuclear a b cell infi ltration (macrophages, lymphocytes, and plasma cells) (fig. 4.3b ) . vascular permeability is also abnormal, but to a lesser extent than in acute infl ammation with formation of new capillaries. abscess is defi ned as a collection of pus in tissues, organs, or confi ned spaces, usually caused by bacterial infection. the fi rst phase of abscess formation is cellulitis, characterized by hyperemia, leukocytosis, and edema, without cellular necrosis or suppuration. this stage is also called phlegmon. it may be followed in some organisms by necrosis and liquefaction and walling off of the pus, which results in abscess formation that can be present with both acute and chronic infl ammation. three major systemic changes are associated with infl ammation: leukocytosis, fever, and an increase in plasma proteins. leukocytosis is an increased production of leukocytes due to stimulation by several products of infl ammation such as complement component c3a and colony-stimulating factors. a febrile response is due to the pyrogens. the increase in plasma proteins is due to the stimulation of the liver by some products of infl ammation, leading to increased synthesis of certain proteins referred to as acute-phase reactants which include c-reactive protein, fi brinogen, and haptoglobin and are anti-infl ammatory [ 2 ] . healing of tissue after injury is closely linked to infl ammation since it starts by acute infl ammation. healing may lead to restoration of normal structure and function of the injured tissue (resolution) or to the formation of a scar consisting of collagen (repair) when resolution cannot be achieved because the tissue is severely injured or cannot regenerate. in either case, acute infl ammation occurs fi rst and for this reason is considered the defensive phase of healing. healing (resolution and repair) occurs in two overlapping phases, reconstruction and maturation, and may take as long as 2 years. the reconstructive phase starts 3-4 days after injury, continues for approximately 2 weeks, and is characterized by fi broblasts followed by collagen synthesis. the maturation phase is characterized by cell differentiation, scar formation, and remodeling of the scar; it begins several weeks after injury and may take up to 2 years to complete. pathophysiology of major soft tissue infl ammation an abdominal abscess may be formed in an abdominal organ or within the abdomen outside the organs. there are several types of abdominal infection: abscess; cellulitis (phlegmon), i.e., early infl ammation of the soft tissue prior to or without formation of an abscess; and peritonitis. abscesses fall into three categories: the organisms causing abscesses may reach the tissue by direct implantation such as penetrating trauma, may spread from contiguous infection, through hematogenous or lymphatic routes from a distant site, or through migration of resistant fl ora into an adjacent, normally sterile area such as in perforation of an abdominal viscus. factors predisposing to abscess formation include impaired host defense mechanisms; trauma/surgery; obstruction of urinary, biliary, or respiratory passages; foreign bodies; chemical or immunological irritation; and ischemia. abdominal surgery (particularly of the colon, appendix, and biliary tree) and trauma are the most common; less common are appendicitis, diverticulitis, and pelvic infl ammatory disease. the formation of fi brin in the abdominal cavity is a common pathophysiological pathway for abdominal abscess formation due to diminished fi brin degradation. hyaluronan-based agents were found to reduce adhesion formation after surgery and reduce abscess formation in experimental peritonitis. possible mechanisms of action of hyaluronan include modulation of the infl ammatory response and enhanced fi brinolysis [ 7 ] . low ph, large bacterial inocula, poor perfusion, the presence of hemoglobin, and large amounts of fi brin (which impedes antibiotic penetration) make the abscess a cloistered environment that is penetrated poorly by many antimicrobial therapies [ 8 , 9 ] . therefore, management of these infections requires prompt recognition, early localization, and effective drainage, as well as appropriate antimicrobial use. once the diagnosis is made and the abscess is localized, treatment should begin promptly. percutaneous or open surgical drainage should be used. broad-spectrum antibiotics should be given until culture and sensitivity data are obtained. localization is crucial since, for example, percutaneous drainage is inappropriate for abscesses in certain locations such as the posterior subphrenic space or in the porta hepatis. in the liver, abscesses occur in the right lobe in approximately 95 % of the cases, and in 70 % of cases, the liver abscesses are solitary [ 10 ] . accumulation of leukocytes in the abscess is the pathophysiological basis for using labeled white blood cells for abscess imaging. in the acute phase, migration of leukocytes is vigorous. later, the migration rate slows, and the cell type changes from predominantly neutrophils to mononuclear cells (lymphocytes, plasma cells, and macrophages). this pathophysiological change associated with the chronic state explains the better diagnostic accuracy of labeled leukocyte scans in acute as opposed to chronic abscesses. infl ammatory bowel disease (ibd) is an idiopathic disease, probably involving an immune reaction of the body to its own intestinal tract. the two major types of ibd are ulcerative colitis (uc) and crohn's disease (cd). crohn's disease is also referred to as regional enteritis, terminal ileitis, or granulomatous ileocolitis. ibd is a dis-ease of industrialized nations and observed most commonly in northern europe and north america. incidence among whites is approximately four times that of other races, slightly greater in females and higher in ashkenazi jews (those who have immigrated from northern europe) than in other groups. the risk of developing uc is higher in nonsmokers and former smokers than in current smokers. incidence peaks in the second and third decades of life. a second smaller peak occurs in patients aged 55-65 years. cd and uc can occur in childhood, although the incidence is much lower in children younger than 15 years with some differences in presentation and more negative effect on quality of life in younger age group [ 11 ] . the etiology of ibd is unsettled. suspected factors include environmental, infectious, genetic, autoimmune, and host factors. a great deal of research has been performed to discover potential genes linked to ibd. one of the early linkages discovered was on chromosome 16 ( ibd1 gene), which led to the identifi cation of the nod2 gene (now called card15 ) as the fi rst gene clearly associated with ibd (as a susceptibility gene for crohn's disease). studies have also provided strong support for ibd susceptibility genes on chromosomes 5 (5q31) and 6 (6p21 and 19p). none of these mechanisms have been implicated as the primary cause, but they are postulated as potential causes. the lymphocyte population in persons with ibd is polyclonal, making the search for a single precipitating cause diffi cult. the trigger for the activation of the immune response has not been defi ned. however, possible triggers include a pathogenic organism (unidentifi ed yet), an immune response to an intraluminal antigen (e.g., cow's milk protein), or an autoimmune process with immune response to an intraluminal antigen and a similar antigen present on intestinal epithelial cells. in any case, activation of the immune system leads to infl ammation of the intestinal tract, both acute and chronic [ 12 -19 ] . the pathophysiology of ibd is still incompletely understood and is under active investigation, but the common end pathway is infl ammation of the mucosal lining of the intestinal tract, causing ulceration, edema, bleeding, and fl uid and electrolyte loss. the infl ammation of the intestinal mucosa includes both acute infl ammation with neutrophilic infi ltration and chronic with mononuclear cell infi ltration (lymphocytic and histiocytic) [ 20 ] . in uc, infl ammation always begins in the rectum, extends proximally a certain distance, and then abruptly stops. a clear demarcation exists between involved and uninvolved mucosa. the rectum is always involved in uc, and no "skip areas" are present. uc primarily involves the mucosa and the submucosa, with formation of crypt abscesses and mucosal ulceration. the mucosa typically appears granular and friable. in more severe cases, pseudopolyps form, consisting of areas of hyperplastic growth with swollen mucosa surrounded by infl amed mucosa with shallow ulcers. in severe uc, infl ammation and necrosis can in rare cases extend below the lamina propria to involve the submucosa and the circular and longitudinal muscles. uc remains confi ned to the rectum in approximately 25 % of cases. in the remainder of cases, uc spreads proximally and contiguously. pancolitis occurs in 10 % of patients. the small intestine is essentially not involved, except when the distal terminal ileum is infl amed in a superfi cial manner, referred to as backwash ileitis. even with less than total colonic involvement, the disease is strikingly and uniformly continuous. as the disease becomes chronic, the colon becomes a rigid foreshortened tube that lacks its usual haustral markings, leading to the lead pipe appearance observed on barium enema. the skip areas (normal areas of the bowel interspersed with diseased areas) observed in cd of the colon do not occur in uc. cd, on the other hand, consists of segmental involvement by a nonspecifi c granulomatous infl ammatory process. the most important pathological feature is the involvement of all layers of the bowel, not just the mucosa and the submucosa, as is characteristic of uc. furthermore, cd is discontinuous, with skip areas interspersed between one or more involved areas. late in the disease, the mucosa develops a cobblestone appearance, which results from deep longitudinal ulcerations interlaced with intervening normal mucosa. the three major patterns of involvement in cd are (1) disease in the ileum and cecum, occurring in 40 % of patients; (2) disease confi ned to the small intestine, occurring in 30 % of patients; and (3) disease confi ned to the colon, occurring in 25 % of patients. rectal spar-ing is a typical but not constant feature of cd. however, anorectal complications (e.g., fi stulas, abscesses) are common. much less commonly, cd involves the more proximal parts of the gi tract, including the mouth, tongue, esophagus, stomach, and duodenum. cd causes three patterns of involvement: (1) infl ammatory disease, (2) strictures, and (3) fi stulas. the incidence of gallstones and kidney stones is increased in cd because of malabsorption of fat and bile salts. gallstones are formed because of increased cholesterol concentration in the bile, caused by a reduced bile salt pool. patients who have cd with ileal disease or resection also are likely to form calcium oxalate kidney stones. with the fat malabsorption, unabsorbed long-chain fatty acids bind calcium in the lumen. oxalate in the lumen normally is bound to calcium. calcium oxalate is poorly soluble and poorly absorbed; however, if calcium is bound to malabsorbed fatty acids, oxalate combines with sodium to form sodium oxalate, which is soluble and is absorbed in the colon (enteric hyperoxaluria). the development of calcium oxalate stones in cd requires an intact colon to absorb oxalate. patients with ileostomies do not develop calcium oxalate stones. extraintestinal manifestations of ibd include iritis, episcleritis, arthritis, and skin involvement, as well as pericholangitis and sclerosing cholangitis. the most common causes of death in ibd are peritonitis with sepsis, malignancy, thromboembolic disease, and complications of surgery. malnutrition and chronic anemia are observed in long-standing cd. children with cd or uc can exhibit growth retardation. patients with uc most commonly present with bloody diarrhea, whereas patients with cd usually present with non-bloody diarrhea. abdominal pain and cramping, fever, and weight loss occur in more severe cases. the presentation of cd is generally more insidious than that of uc. uc and cd are generally diagnosed using clinical, endoscopic, and histologic criteria. however, no single fi nding is absolutely diagnostic for one disease or the other. furthermore, approximately 20 % of patients have a clinical picture that falls between cd and uc; they are said to have indeterminate colitis. accordingly, imaging may be needed for the detection and for evaluation of the disease activity during its course. the chest is a common site of various types of infection, acute and chronic. such infections are frequent in the elderly and in immunosuppressed patients, including cancer patients. common infl ammatory conditions relevant to nuclear medicine include pneumonia, sarcoidosis, diffuse interstitial fi brosis, and pneumocystis ( jiroveci ) carinii pneumonia (see also chap. 12 ). sarcoidosis is an infl ammatory condition of uncertain etiology characterized by the presence of noncaseating granulomas involving multiple organs. the disease is now recognized as a member of a large family of granulomatous disorders and has been reported from all parts of the world. current evidence points to genetic predisposition and exposure to yet unknown transmissible agent(s) and/or environmental factors as etiological agents [ 21 ] . the lung is most commonly and usually the fi rst site of involvement, and the infl ammatory processes extend through the lymphatics to the hilar and mediastinal nodes [ 22 ] . the lung is involved in more than 90 % of cases. pulmonary sarcoidosis starts as diffuse interstitial alveolitis, followed by the characteristic granulomas. granulomas are present in the alveolar septa as well as in the walls of the bronchi and pulmonary arteries and veins. the center of the granuloma contains epithelioid cells derived from mononuclear phagocytes, multinucleated giant cells, and macrophages. lymphocytes, macrophages, monocytes, and fi broblasts are present at the periphery of the granuloma [ 23 ] . sarcoidosis represents a challenge to clinical investigation because of its unpredictable course, uncertain response to therapy, and diversity of potential organ involvement and clinical presentations [ 24 ] . the diagnosis is based on a compatible clinical and/or radiological picture, histopathological evidence of noncaseating granulomas in tissue biopsy specimens, and exclusion of other diseases capable of producing similar clinical or histopathological appearances. even microscopically, the noncaseating granulomas are not specifi c [ 21 ] . infection by mycobacterial species other than mycobacterium tuberculosis frequently leads to the production of noncaseating granulomas [ 25 ] . the condition is underdiagnosed in some areas. however, owing to the increasing awareness, it is being diagnosed more frequently than a few decades ago [ 26 ] . the disease runs a benign course with spontaneous remission of the activity though some degree of residual pulmonary function abnormality persists. only a minority of patients develop complicated disease that may lead to blindness, renal failure, liver failure, and heart involvement. corticosteroids remain the mainstay of treatment. treatment under close clinical monitoring should be tailored to suit the needs of the individual patient hence the need to evaluate disease activity [ 26 ] . advanced age, the presence of pulmonary symptoms, the presence of parenchymal lesions on chest radiograph, a previous history of treatment with corticosteroids, and the presence of extrathoracic involvements at the time of detection are possible prognostic factors in patients with sarcoidosis [ 27 ] . the mode of onset and the extent of the disease are also related to prognosis. an acute onset with erythema nodosum or asymptomatic bilateral hilar lymphadenopathy usually heralds a self-limiting course, whereas an insidious onset, especially with multiple extrathoracic lesions, may be followed by relentless, progressive fi brosis of the lungs and other organs pneumocystis carinii ( jiroveci ) pneumonia (pcp) is a condition that may be endemic or epidemic. it is caused by pneumocystis carinii , which was considered as a protozoon and recently as a fungus. the condition is common in premature infants, debilitated children, and in other immunocompromised conditions, particularly the acquired immune defi ciency syndrome (aids), but it is also seen in congenital immunodeficiency and in patients who are receiving chemotherapy and corticosteroids. it remains a signifi cant cause of morbidity and mortality in human immunodefi ciency virus and nonhuman immunodefi ciency virus-associated immunosuppressed patients [ 28 ] . it is the most common infection in aids patients, and it remains an important cause of morbidity and mortality [ 29 ] . the introduction of highly active antiretroviral therapy in industrialized nations however has led to dramatic declines in the incidence of aidsassociated complications, including pcp. in the developing countries, no decline has occurred [ 30 ] . transmission is usually airborne. the pathological changes are predominantly in the lungs with an infl ammatory reaction consisting of plasma cells of variable amount, monocytes, and histiocytes. this disease has also been reported in immunocompetent patients, and in this case the presentation is more closely resembling the disease of immunocompromised patients other than aids patients [ 31 , 32 ] . the diagnosis is currently established through identifi cation of the organisms in bronchial secretions obtained by bronchoalveolar lavage or bronchial washings [ 33 ] . gallium-67 is an important imaging modality that helps in the diagnosis and evaluation of the activity of the disease. interstitial pulmonary fi brosis, a sometimes fatal condition, is characterized by parenchymal infl ammation and interstitial fi brosis. the pathological changes start with alveolitis; this is followed by derangement of the alveolar-capillary units, leading to the end stage of fi brosis. there is a correlation between the infl ammatory activity and the amount of gallium-67 activity in the lungs [ 34 ] . urinary tract infection (uti) is common particularly in children. there are two main varieties of acute renal infection: pyelitis, which is confi ned to the renal pelvis, and pyelonephritis, where the renal parenchyma is also involved. it is not always possible to differentiate between the two conditions on clinical grounds. the pathology of acute pyelitis is not very well understood. the importance of the condition, however, lies in the fact that recurrent subclinical attacks are believed to be signifi cant in the pathogenesis of chronic pyelonephritis [ 35 ] . the number of patients with chronic kidney disease and consequent end-stage renal disease is rising worldwide [ 36 ] . end-stage kidney disease , defi ned as that requiring dialysis or receipt of a transplant or that which may lead to death from chronic kidney failure, generally affects less than 1 % of the population [ 37 ] . among today's challenges is to identify those at greatest risk for endstage renal disease and intervene effectively to prevent progression of early chronic kidney disease and conditions leading to chronic disease [ 37 ] . rarely, uncomplicated acute pyelonephritis causes suppuration and renal scarring. however, urinary infections in patients with renal calculi, obstructed urinary tract, neurogenic bladder, or diabetes are frequently much more destructive and have ongoing sequelae [ 38 ] . acute pyelonephritis is a common medical problem. the diagnosis and management of this condition is complex. patients initially diagnosed with pyelonephritis typically exhibit symptoms and laboratory evidence suggesting infected urine, with signs referable to upper urinary tract infection. however, no consistent set of signs and symptoms are sensitive and specifi c for this diagnosis. symptoms of acute pyelonephritis generally develop rapidly over a few hours or a day. symptoms of lower uti may or may not be present. these include dysuria; urinary frequency, hesitancy, and urgency; gross hematuria; and suprapubic discomfort, heaviness, pain, or pressure. additionally, fl ank pain and tenderness, unilateral or sometimes bilateral, are present. fever is not always present. when present, it is not unusual for the temperature to exceed 103 °f (39.4 °c). rigor, chills, malaise, and weakness may be present. anorexia, nausea, vomiting, and diarrhea may also be present. most patients have signifi cant leukocytosis, pyuria with leukocyte casts in the urine, and bacteria on a gram stain of unspun urine. many conditions and clinical situations are associated with an increased risk of pyelonephritis. table 4 pyelonephritis is signifi cantly more common in females (higher in white than in black) compared to males. approximately 10-30 % of women develop a symptomatic uti at some point in their lives. acute pyelonephritis is a bacterial infection of the kidney with acute infl ammation of the pyelocaliceal lining and renal parenchyma centrifugally along medullary rays. this can occur by more than one way. most often it occurs because of ascending infection from the lower urinary tract (fig. 4.4 ) . the initial colonization of the walls of the ureter is in areas of turbulent fl ow which leads to paralysis of peristalsis. dilation and functional obstruction result in subsequent pyelonephritis. another way is by direct refl ux of bacteria. hematogenous spread to the kidney by gram-positive and less likely by gram-negative organisms is the third way that can occur. this has become less prevalent since the advent of rapid use of antibiotics. little or no evidence supports lymphatic spread. grossly, the kidney is enlarged and edematous. the cut surface may show small abscesses in the cortex, and more often there are wedgeshaped purulent areas streaking upward from the medulla, with normal areas of the kidney tissue intervening in between infected zones (fig. 4.5 ) . frequently, the pelvis and calyces are infl amed and dilated. in severe infection, renal papillary necrosis may be present. microscopically, there is intense infl ammation, with infi ltration of polymorphonuclear leukocytes throughout the interstitial tissue and abscess formation. there is destruction of the tubules, but the glomeruli and blood vessels are often unaffected. the disease remains essentially focal in character, with areas of normal tissue. following treatment and chronic pyelonephritis is a chronic condition affecting the pelvis and parenchyma and resulting from recurrent or persistent renal infection. it occurs almost exclusively in patients with major anatomic anomalies, including urinary tract obstruction, struvite calculi, renal dysplasia, or, most commonly, vesicoureteral reflux (vur) in young children. grossly, the kidney shows normal areas alternating with zones of scarring. wedgeshaped scars can be seen on the subcapsular surface of the kidney. the appearance differs, depending on the presence or absence of obstruction. chronic pyelonephritis in the presence of intra-or extrarenal obstruction shows dilation of the pelvocalyceal system and sometimes peripelvic fibrosis. if no obstruction is present, the pelvic change is in the form of peripelvic fibrosis rather than dilation ( fig. 4.6 ) . microscopically, the scarred areas show changes in the interstitium and tubules. the interstitial tissue shows infiltration by predominantly lymphocytes and plasma cells. the tubules become atrophic and may collapse (fig. 4.7 ) . the glomeruli may be normal in some cases, while in others periglomerular fibrosis is present. osteomyelitis indicates an infection involving the cortical bone as well as the marrow (see chap. 6 ). it is classified into many types based on several pathological and clinical factors [ 42 -49 ] including route of infection, patient age, etiology, and onset. hematogenous osteomyelitis most commonly affects children, and the metaphyses of long bones are the most common sites. nonhematogenous osteomyelitis, on the other hand, occurs as a result of penetrating trauma, spread of a contiguous soft tissue infection, or inoculation, as in drug addicts [ 48 -54 ] . many organisms have been encountered in the pathogenesis of osteomyelitis, particularly gram-positive organisms, the most common being staphylococcus aureus [ 44 -46 ] . like many other pathological conditions of bone, infections cause reactive new bone formation which -among other factors, particularly increased blood flow -is the principle reason for the accumulation of it is diffi cult to draw the line between acute and chronic osteomyelitis. chronic osteomyelitis has been defi ned as a skeletal infection with a duration as short as 5 days or as long as 6 weeks. it is characterized by less marked infl ammatory cell infi ltrates than acute infection and may exhibit a variable amount of necrotic tissue. acute septic arthritis is a medical emergency, since it may result in destruction of the articular cartilage and permanent disability if treatment is delayed [ 55 ] . see chap. 6 for more details on skeletal infl ammations. fuo describes an illness of several episodes of fever exceeding 38.3 ºc or at least 3-week duration, with no diagnosis after an appropriate inpatient or outpatient evaluation. there are many causes of fever of unknown origin. infection accounts for only about 25 % of these causes. many radioisotopes have been used to detect and localize infection (see table 4 .3 ). several mechanisms explain the uptake of these radiotracers at the site of infection: 1. increased vascular permeability 111 in and 99m tc human polyclonal igg 111 in monoclonal igm antibody 111 in and 99m tc liposomes 111 in biotin and streptavidin -99m tc nanocolloids56 111 in chloride -67 ga citrate 2. migration of wbcs to the site of infection 111 in-and 99m tc-labeled leukocytes -99m tc anti-wbc antibodies 3. binding to proteins at the site of infection, i.e., 67 ga citrate (lactoferrin and other ironcontaining proteins) 4. binding to wbcs at the site of infection -chemotactic peptides -interleukins 5. binding to bacteria -99m tc-labeled ciprofl oxacin antibiotic -67 ga citrate 6. metabolic trapping, i.e., f-18 fl uorodeoxyglucose since there are limitations to the radiopharmaceuticals available for imaging infection, the search continues for better agents with ideal properties [ 56 -59 ] . they should: 1. be easy to prepare 2. have low cost and wide availability 3. ensure rapid detection and localization of infections (< 3 h) 4. have low toxicity and produce no immune response 5. clear rapidly from the blood with no significant uptake in the liver, spleen, gi tract, bone, kidneys, bone marrow, or muscle 6. clear rapidly from the background 7. have high specifi city and sensitivity and be able to differentiate infection from other causes of infl ammation and tumors 8. be able to differentiate acute from chronic infection gallium-67 has been used for many years to detect infl ammation. the multiple mechanisms of uptake of gallium by infl ammatory tissue include the following: 1. increased vascular permeability 2. gallium-67-binding substances at the site of infl ammation • transferrin (due to leakage of plasma proteins) • lactoferrin (secreted with lysosomal contents of stimulated or dead neutrophils) • siderophores produced by bacteria 3. leukocytes: direct uptake 4. bacteria: direct uptake sfakianakis et al. [ 60 ] found that 111 in leukocyte imaging accuracy was best for relatively acute infections (less than 2 weeks) but yielded a 27 % false-negative rate among patients with prolonged infections. on the other hand, 67 ga imaging had its highest sensitivity in long-standing processes, with false-negative results of 19 % in relatively acute infections of less than 1-week duration. in a comparative study of rabbits with experimental abscesses, bitar et al. [ 61 ] found that 111 in leukocytes were clearly superior to gallium for imaging early abscesses. furthermore, they found that the accumulation of 111 in leukocytes in experimental subcutaneous abscesses was inversely proportional to the age of the abscess. in abscesses 1-2 h, 6-8 h, 24 h, and 7 days old, 10.4, 5.2, 3, and 0.73 % of the injected dose, respectively, was accumulated. 67 ga uptake, on the other hand, was not signifi cantly affected by abscess age (table 4.4 ) . in abscesses 7 days old, 67 ga accumulated to a greater extent than did 111 in-labeled leukocytes. thus, bitar et al., based on animal studies, and stakianakis et al. came independently to the conclusion that 111 in-labeled wbcs are more suitable for acute infections of short duration, while 67 ga labeling is better for infections of longer duration. in rats, mcafee et al. [ 62 ] showed that as many as 10 % of circulating neutrophils accumulate daily at focal sites of infl ammation. this high propensity of white blood cells to migrate to an abscess makes positive identifi cation of the abscess likely on an 111 in wbc image. the authors also showed abscess-to-muscle ratios of 3,000 to 1 with 111 in wbcs at 24 h compared with 72 to 1 with 67 ga and 7 to 1 with 111 in chloride. accordingly, a small dose of only 500 μci of 111 in leukocytes is suffi cient for positive identifi cation and localization of abscesses on an image. in 67 ga imaging, a higher dose of approximately 5 mci is needed. there is a higher radiation dose to the spleen from 500 μci of 111 in wbc but radiation doses to gonads, marrow, and the whole body are higher with 5 mci of 67 ga. 99m tc hmpao-labeled wbcs could provide fast diagnosis and localization of the abdomen (within 2-4 h). physiological bowel activity, however, is found in 7 % at 2 h and in 28 % of patients imaged with this agent at 4 h. leukocytes labeled with 111 in or 99m tc hmpao are superior to those labeled with 67 ga for acute infections in terms of sensitivity and specifi city [ 63 , 64 ] . in a recent systematic review of the published studies in humans cited in pubmed written in english, french, german, italian, and spanish, it was again found that labeled leukocyte is a sensitive method to localize abdominal abscesses and can guide dedicated us and ct investigations to improve their diagnostic potential [ 65 ] . table 4 .5 lists the main advantages and disadvantages of the major radiopharmaceuticals used for infl ammation imaging. several monoclonal antibodies are used to detect infections. these antibodies are mainly directed against receptors on infl ammatory cells. labeled antigranulocyte agents most commonly used are intact murine immunoglobulin g (igg) antibodies against normal cross-reactive antigen-95 (anti-nca-95, 99m tc-bw250/183, 99m tc-besilesomab [scintimun®]) and the murine tc anti-nca-90 fab fragments can recognize a specifi c cross-reacting antigen (nca-90) (the surface antigenic glycoprotein) on granulocytes, promyelocytes, and myelocytes [ 66 -68 ] . leukoscan uptake at the site of infection is explained partly by the migration of circulating antibody-labeled granulocytes to the site of infection. uptake is also explained by the fact that the greater proportion of the labeled antibody fragment is in a free soluble form which can easily cross capillary membranes, binding to the leukocyte once in situ. this mechanism is favored by the increased capillary permeability at the site of infection. an important advantage of leukoscan is the 5 min preparation time compared with the 2 h 30 min required by a specialized team for labeling leukocytes. despite the fact that leukoscan involves the i.v. injection of mouse proteins, no anaphylactic or other hypersensitivity reactions were observed. 99m tc ciprofl oxacin ( infecton ) is also being used to image infection. ciprofl oxacin is a broad-spectrum fl uoroquinolone antibiotic that inhibits dna gyrase and/or topoisomerase iv of bacteria. patients receive 99m tc ciprofl oxacin 10 mci, and images are obtained at 1, at 3-4, and, occasionally, at 24 h postinjection. 99m tc ciprofl oxacin may be useful in distinguishing infection from infl ammation. early images of noninfectious rheumatologic infl ammatory conditions were positive, but activity decreased with time [ 69 ] . 111 in-and 99m tc-labeled chemotactic peptide analogs have been used for detecting and localizing infections. imaging can be performed at less than 3 h postinjection, which compares favorably with the 18-24 h or more for most other agents [ 54 ] . labeled liposomes have been used for scintigraphic imaging of infection and infl ammation [ 70 ] . boerman et al. [ 71 ] used 111 in-labeled sterically stabilized liposomes (long circulating) in rats and showed that the clearance of this agent is similar to that of 111 in igg. the uptake in abscess was twice as high as that of igg and the abscess was visualized as early as 1 h post injection. 99m tc nanocolloid has also been tried but has not gained wide acceptance. f-18 fl uorodeoxyglucose (fdg-pet) has emerged as an important diagnostic agent for infectious and noninfectious soft tissue and skeletal infl ammations including infl ammatory bowel disease, fevers of unknown origin, rheumatologic disorders, tuberculosis infection, fungal infection, pneumonia, abscess, postarthroplasy infections, chronic and vertebral osteomyelitis, sarcoidosis, and chemotherapy-induced pneumonitis [ 72 -74 ] . infl ammatory conditions show high fdg uptake which is related to increased glucose metabolism that is produced by stimulated infl ammatory cells, macrophage proliferation, and healing [ 75 ] . while uptake of fdg continues to increase at malignant sites for several hours, as can be shown by an incremental increase of the standardized uptake values (suv), infl ammatory lesions peak at approximately 60 min, and their suv either stabilize or decline thereafter. this difference in the behavior of fdg in malignant versus infl ammatory cells can be explained best by the varying levels of enzymes that degrade deoxyglucose-6-phosphate in the respective cells. glucose-6-phosphatase dephosphorylates intracellular fdg-6-phosphate, allowing it to leave the cell. it has been shown that most tumor cells have low levels of this enzyme, while its expression is high in the mononuclear cells [ 76 -85 ] . for this reason, imaging at 2 time points after administration of fdg may prove to be important in differentiating between these two common disorders. diagnosis and localization of infection by clinical and laboratory methods is often diffi cult. the results frequently are nonspecifi c and imaging may be needed. imaging of infection may be achieved by either nuclear medicine or other strictly morphological methods. several nuclear medicine modalities are used to diagnose and localize soft tissue and skeletal infections. these include 111 in-labeled white blood cells, 67 ga citrate, igg polyclonal antibodies labeled with 111 in or 99m tc, monoclonal antibodies such as antigranulocyte antibodies, 99m tc hmpaolabeled white blood cells, 99m tc nanocolloid, 99m tc-dmsa, 99m tc-glucoheptonate, 99m tc-mdp multiphase bone scan, 111 in-labeled chemotactic peptide analogs, and f-18-fdg. x-ray, ct, mri, and ultrasonography are other modalities useful in the diagnosis and localization of both soft tissue and skeletal infl ammations. these studies are complementary to the physiological modalities of nuclear medicine. the strategy for imaging soft tissue infections depends on the pathophysiological and clinical features, including whether localizing signs and symptoms are present and the location and duration of the suspected infection. abdominal abscess: rapid and accurate diagnosis of an abdominal abscess is crucial. the mortality from untreated abscesses approaches 40 % and may reach 100 % in some series. the mortality among patients treated reaches 11 % [ 86 -93 ] . delayed diagnosis is associated with higher mortality in spite of treatment. if localizing signs suggest abdominal infection, morphological modalities, predominantly ultrasound ( fig. 4.8 ) and ct (fig. 4.9 ) , may be used fi rst, depending on the location of suspected infection in the abdomen. standard radiographs have low sensitivity, although when seen, fi ndings are specifi c. the advantages of these modalities are numerous, but most importantly, they provide quick results and adequate anatomic details. these studies can be used to guide needle aspiration and abscess drainage. ultrasound can be used portably for critically ill patients. one of the major limitations of these modalities is the inability to differentiate infected from noninfected tissue abnormalities, particularly in early stages of infection (phlegmon) before formation of abscesses. the diagnostic accuracy of these morphological modalities may be compromised in cancer patients, and the evaluation of studies that use these techniques may be diffi cult. this is because the interpretation of these modalities depends on the presence of normal anatomical markers, which may be altered or obliterated by either the cancer treatment or the cancer itself [ 94 ] . for example, both ct and mri are often of little value in distinguishing posttreatment scarring from recurrent tumor. when the results of the morphological modalities are inconclusive, nuclear medicine techniques may be used to detect abdominal infections. the ability to image the entire body is the major advantage of nuclear medicine modalities ( fig. 4.10 ) . hence, radionuclide techniques are often used in cases with no localizing signs. in one study, 16 % of patients suspected of having abdominal infection in fact had extraabdominal infections as seen on 111 in leukocyte scans [ 95 ] . accordingly, negative morphological modalities, when used fi rst, may be followed by whole-body nuclear imaging. labeled wbc studies are the most specifi c for acute infections (figs. 4.11 and 4.12 ) . ga-67 is more suitable for infection of longer duration (fig. 4.13 ). 99m tc hmpao-labeled wbcs frequently are used in critically ill patients [ 39 ] after us and/or ct have yielded inconclusive results. it is worthy of note that 99m tc hmpao-labeled wbcs provide quicker results than 67 ga-or 111 in-labeled wbcs. minoja et al. [ 96 ] reported a sensitivity of 95 %, a specifi city of 91 %, and an accuracy of 94 % for 99m tc-labeled wbc scanning in intensive care unit patients with occult infections. gallium-67 scan has been reported to have better diagnostic specifi city than the c-reactive protein test for abdominal infections [ 97 ] . infl ammatory bowel disease: upright chest radiography and abdominal series, barium enema, and upper gi ct scanning, mri, and ultrasonography are the main imaging modalities used for the diagnosis. ct scanning and ultrasonography are best for demonstrating complications such as intra-abdominal abscesses and fi stulas. evaluation of the extent of the disease and disease activity is often diffi cult. a wide variety of approaches depicting the different stages of the infl ammatory response have been developed. nonspecifi c radiolabeled compounds, such as 67ga citrate and radiolabeled polyclonal a b fig. 4.9 representative images of ct scans of the abdomen illustrating ( a ) periappendicular abscess ( arrow ) and ( b ) hepatic abscess ( arrow ) human immunoglobulin, accumulate in infl ammatory foci due to enhanced vascular permeability. specifi c accumulation of radiolabeled compounds in infl ammatory lesions results from binding to activated endothelium (e.g., radiolabeled anti-e-selectin), the enhanced infl ux of leukocytes (e.g., radiolabeled autologous leukocytes, antigranulocyte antibodies, or cytokines), the enhanced glucose uptake by activated leukocytes (18f-fl uorodeoxyglucose), or direct binding to microorganisms (e.g., radiolabeled ciprofl oxacin or antimicrobial peptides). scintigraphy using autologous leukocytes, labeled with 111 in or 99mtc, is still considered the "gold standard" nuclear medicine technique for the imaging of infection and infl ammation, but the range of radiolabeled compounds available for this indica-tion is still expanding. recently, positron emission tomography with 18f-fl uorodeoxyglucose has been shown to delineate various infectious and infl ammatory disorders with high sensitivity . in a study [ 98 ] , gallium, magnetic resonance imaging (mri), and pet-fdg were compared for their ability to detect disease activity. pet-fdg showed more than twice as many lesions in the abdomen of patients with crohn's disease as did gallium. not all lesions on mri were fdg positive, suggesting they might represent areas of prior infl ammation. the role of the chest x-ray cannot be overemphasized. the chest x-ray should be used as the initial imaging modality for most chest pathologies. in-111 wbc in-labeled leukocyte scan obtained in a patient with a 10-day history of fever and no localizing signs. anterior and posterior images reveal physiological uptake in the bone marrow, liver, and spleen with no abnormal accumulation of labeled cells in many instances, however, an additional modality is needed to evaluate certain chest conditions including infections. although ct often clearly depicts chest pathology including infections, 67 ga still is commonly used in such cases. 111 in leukocytes have limited utility for chest infections. siemon et al. [ 99 ] studied 67 ga imaging in a variety of pulmonary disorders and found excellent sensitivity and specifi city (table 4 .6 ). gallium-67 has also been widely used in aids patients to detect pcp (fig. 4.14 ) . it is highly sensitive and correlates with the response to therapy. in a study comparing 67 ga, bronchial washing, and transbronchial biopsy in 19 patients with pcp and aids, 67 ga and bronchial washing were 100 % sensitive compared with 81 % for transbronchial biopsy [ 101 ] . 67 ga is also valuable in idiopathic pulmonary fi brosis, sarcoidosis, and amiodarone toxicity [ 102 , 103 ] . it is also useful in monitoring response to therapy of other infections including tuberculosis (fig. 4.15 ). 111 in wbc imaging is less helpful, as the specifi city of abnormal pulmonary uptake (either focal or diffuse) is very low. noninfectious problems that cause abnormal uptake include congestive heart failure, atelectasis, pulmonary embolism, ards, and idiopathic conditions [ 104 ] . the ct scan has good sensitivity and specifi city in the diagnosis of renal infections. ultrasound has been used frequently to evaluate the kidneys with suspected infections, even though it is not sensitive. it is used primarily to screen for obstruction or abscess when resolution of uti is slower than expected with treatment. the sensitivity of us has been shown to be less than 60 % [ 105 ] and is signifi cantly inferior to that of cortical scintigraphy (sensitivity of 86 % and specifi city of 81 % using 99mtc-glucoheptonate). positive ultrasonography can obviate the need for dmsa; however, because of a large number of false-negative results with reported sensitivities of 42-58 % and underestimation of the pyelonephritis lesions, ultrasonography cannot replace 99mtc-dmsa [ 106 ] . to date 99mtc-dmsa is considered the most sensitive method for the detection of acute pyelonephritis in children ( fig. 4.16 ). it also permits the photopenic area to be calculated as the infl ammatory volume which correlates with the severity of infection and the possibility of scar formation even though some of the defects detected might be too small to be clinically signifi cant. currently us is recommended as the initial imaging modalities by the american academy of pediatrics and the national institute for health and clinical excellence (nice) in atypical and recurrent uti in pediatric age group [ 107 , 108 ] . the pathophysiological basis of the ability of doppler sonography in detecting acute pyelonephritis is the fact that in the acute phase of pyelonephritis the, focal decrease of renal perfusion due to edema causes vascular compression, intravascular granulocyte anterior posterior from [ 100 ] aggregation, or both, leading to capillary and arteriolar occlusion facilitating the detection of these hypovascular areas [ 109 ] . several imaging techniques are being utilized for the detection of osteomyelitis including the standard radiograph, computerized tomography, magnetic resonance imaging, and several nuclear medicine modalities. the choice of modality depends on the clinical presentation, particularly its duration, the site of suspected infection, and whether the site of suspected infection has been affected by previous pathology. the pathophysiology of skeletal infl ammations and relevant scintigraphic considerations are discussed in detail in chap. 5 , on the musculoskeletal system. when no localizing clinical signs are present, which is common in cancer and immunosup-pressed patients, nuclear medicine procedures are often the imaging modalities chosen. the ability to screen the entire body is particularly important for many such cases. the optimal choice of radiotracer again depends on the duration of infection (fig. 4.17 ) . 111 in-labeled white blood cells are the most specifi c for acute infections, but false-positive results have been reported with some tumors, swallowed infected sputum, gi bleeding, and sterile infl ammation. false-negative results have been reported in infections present for more than 2 weeks. more rarely, such false-negative results occur for infections present for only 1 week. gallium-67 is less specifi c than labeled wbcs, as it is taken up by many tumors and by sterile infl ammation. several radiolabeled antibody preparations and a radiolabeled antibacterial agent have been introduced and evaluated, but none of these have been used widely. labeled antibody scintigraphy uses antigranulocyte agents, most commonly intact murine immunoglobulin g (igg) antibodies against normal fig. 4 .14 gallium-67 images of an aids patient with a 5-week history of fever. images show diffuse uptake in both lungs illustrating the typical pattern of gallium-67 in pcp cross-reactive antigen-95 (anti-nca-95, 99m tc-bw250/183, 99m tc-besilesomab [scintimun ® ]) and the murine fab fragment of the igg antibody directed against the glycoprotein cross-reactive antigen-90 (anti-nca-90, 99m tc-sulesomab, leukoscan ® ). 99m tc-igg scintigraphy is a highly sensitive technique for the recognition of infec-tion but has a low specifi city pet-fdg has now taken the place occupied by citrate of gallium-67. visualization of infl ammatory lesions does not just rely on the presence of immune cells, but uptake requires the activation of these immune cells. fdg-pet reveals infectious and noninfectious infl ammatory diseases as well as malignant a fig. 4.15 ( a , b ) gallium-67 studies of a patient with tuberculosis. initial study ( a ) showing abnormal uptake of the right lung ( arrows ) which disappeared on follow-up study ( b ) 3 months after starting therapy, indicating excellent response to treatment diseases; all are causes of fever of unknown origin. recent studies support the use of fdg-pet in the patient with fuo [ 72 , 73 , 110 ] . fdg is sensitive and its short half-life does not delay the performance of any additional radionuclide studies that might be needed. various chronic infectious diseases that are frequent clinical challenges are better diagnosed with the use of pet, particularly when this imaging is combined with ct. for noninfectious infl ammatory diseases, fdg-pet has proved particularly helpful for the diagnosis and management of large vessels arteritis and infl ammatory bowel disease [ 74 , 111 , 112 ] . correlation with morphological modalities after successful radionuclide localization of infection can be of great help. for example, this correlation provides anatomical information prior to surgical interventions. morphological modalities are useful in the management of infl ammatory diseases particularly if localizing signs are present. they have the very important advantages of better spatial resolution than nuclear medicine modalities. x-rays, ct, mri, and us usually yield fast results but unfortunately may not distinguish infected from noninfected tissue. many morphological and functional imaging modalities are available to help diagnose and localize infl ammation of the soft tissue and bone. it is clear that no single technique is ideal in all situations. the choice depends on several factors, including whether localizing signs are present, the site of possible infection, whether anatomy is normal or altered by surgery or trauma, the duration of symptoms and signs, and the presence of other underlying diseases such as cancer. for physicians, understanding the pathophysiological changes is crucial for deciding on an appropriate diagnostic strategy. understanding pathophysiological changes also helps the nuclear physician to recognize and explain the scintigraphic patterns of infl ammatory conditions (table 4 .7 summarizes common examples). further evaluation of pet in diagnosis, localizing, and follow-up of infl ammations is a current interest. the discovery of new radiopharmaceuticals that will be ideal for more specifi c imaging of infl ammation is an important topic for future research. locally increased accumulation of several radiotracers, increased fl ow and blood pool activity on bone scan; hyperemic pattern on delayed bone images may be present with soft tissue infection increased vascular permeability increased migration of wbcs, increased accumulation of 67 infl ammation: basic principles and clinical correlates infl ammation robbins and cotzan, pathologic basis of disease biological mediators of acute infl ammation pathogenesis and mechanisms of infl ammation and pain: an overview infl ammatory cytokine production by immunological and foreign body multinucleated giant cells pathophysiology of intra-abdominal adhesion and abscess formation, and the effect of hyaluronan intra-abdominal infections management of severe abdominal infections comparison of infl ammatory bowel disease at younger and older age etiopathogenesis and aggravating factors of ulcerative colitis environmental risk factors in paediatric infl ammatory bowel diseases: a population based case control study anti-interleukin-12 antibody for active crohn's disease anti-interleukin-12 antibody for active crohn's disease physiological basis for novel drug therapies used to treat the infl ammatory bowel diseases i. pathophysiological basis and prospects for probiotic therapy in infl ammatory bowel disease dietary risk factors for infl ammatory bowel disease: a multicenter casecontrol study in japan cigarette smoking and age at diagnosis of infl ammatory bowel disease the ibd international genetics consortium provides further evidence for linkage to ibd4 and shows gene-environment interaction the vasculature and infl ammatory bowel disease: contribution to pathogenesis and clinical pathology granulomatous infections: etiology and classifi cation early onset sarcoidosis: not a benign disease pulmonary defense mechanisms clinical insights and basic science correlates in sarcoidosis diagnosis and treatment of disease caused by nontuberculous mycobacteria sarcoidosis: global scenario & indian perspective clinical courses and prognoses of pulmonary sarcoidosis pneumocystis carinii infection. update and review pneumonia associated with hiv infection current epidemiology of pneumocystis pneumonia pneumocystis carinii pneumonia in a patient without a predisposing illness: case report and review pneumocystis jirovecii in general population pcr diagnosis of pneumocystis carinii on sputum and bronchoalveolar lavage samples in immunocompromised patients the role of gallium-67 imaging in the clinical evaluation of pulmonary disorders. in: loken mk (ed) pulmonary nuclear medicine pathogenesis of pyelonephritis in the kidney chronic kidney disease: the global challenge chronic kidney disease in the general population the natural history of urinary infection in adults diagnosis and treatment of uncomplicated urinary tract infection complicated urinary tract infections bacterial osteomyelitis in adults: evolving considerations in diagnosis and treatment a clinical staging system of adult osteomyelitis imaging in the diagnosis of musculoskeletal infections in children a practical guide to the diagnosis and management of bone and joint infections the radiology of osteomyelitis the three types of acute hematogenous osteomyelitis: a clinical and vascular study imaging skeletal infections: evolving considerations. in: feeman lm (ed) nuclear medicine annual multimodality imaging of osteomyelitis pyogenic vertebral osteomyelitis: analysis of 20 cases and review contiguous discitis and osteomyelitis in children infectious spondylitis in children. the convergence of discitis and vertebral osteomyelitis vertebral osteomyelitis caused by group b streptococci [streptococcus agalactiae] secondary to urinary tract infection pyogenic and tuberculous spondylodiskitis technetium phosphate bone scan in the diagnosis of septic arthritis in childhood radionuclide imaging of infection radiopharmaceuticals for the study of infl ammatory process. a review radionuclide detection of occult infection: current strategies chemotactic peptides: new locomotion for imaging of infection? comparison of scintigraphy with in-111 leukocytes and ga-67 in the diagnosis of occult sepsis accumulation of in-111 labeled neutrophils and gallium-67 citrate in rabbit abscesses technique of leukocyte harvesting and labeling: problems and prospectives the utility of tc-99m hmpao leukocytes for imaging infection indium-111 labeled leukocytes for the detection of infection: current status the labeledleukocyte scan in the study of abdominal abscesses besilesomab for imaging infl ammation and infection in peripheral bone in adults with suspected osteomyelitis intact versus fragmented 99mtc-monoclonal antibody imaging of infection in patients with septically loosened total knee arthroplasty clinically proven radiopharmaceuticals for infection imaging: mechanisms and applications accumulation of 99m tc-ciprofl oxacin in staphylococcus aureus and pseudomonas aeruginosa infl ammatory joint disease: a comparison of liposome scanning, bone scanning and radiography sterically stabilized liposomes labeled with in-111 to image focal infection clinical value of [18f]fl uorodeoxyglucose positron emission tomography for patients with fever of unknown origin fever of unknown origin: prospective comparison of fdg imaging with a double-head coincidence camera and gallium-67 citrate spet clinical value of fdg pet in patients with fever of unknown origin and patients suspected of focal infection or infl ammation fluorine-18 deoxyglucose uptake in sarcoidosis measured with positron emission tomography fluorine-18-fl uorodeoxyglucose uptake in pneumonia kinetic analysis of infl ammation and cancer with dynamic 18f-fdg pet dual time point fl uorine-18 fl uorodeoxyglucose positron emission tomography: a potential method to differentiate malignancy from infl ammation and normal tissue in the head and neck fdg-pet is an effective alternative to wbc imaging in diagnosing and excluding orthopedic infections critical role of fdg-pet imaging in the management of patients with suspected infection in diverse settings evaluation of 18f-fdg pet imaging in diagnosis of disseminated mycobacterium avium complex (dmac) in aids patients combined 18f-fdg and 11c-acetate pet imaging in diagnosis of pulmonary tuberculosis high accumulation of fl uorine-18-fl uorodeoxyglucose in turpentine-induced infl ammatory tissue the interaction among glucose transport, hexokinase and glucose 6-phosphatase with respect to 3h-2-deoxyglucose retention in murine tumor models diagnosis of intraabdominal infections: clinical fi ndings and imaging pyogenic liver abscesses with escherichia coli: etiology, clinical course, outcome, and prognostic factors the morbidity of penetrating colon injury retroperitoneal abscesses -analysis of a series of 66 cases quality of life, morbidity, and mortality after surgical intensive care: a follow-up study of patients treated for abdominal sepsis in the surgical intensive care unit population-based study of the epidemiology of and the risk factors for pyogenic liver abscess diagnostic and therapeutic diffi culties in retroperitoneal abscess percutaneous drainage of abdominal abscess intraabdominal infection in patients with abdominal trauma radionuclide imaging of infection in the immunocompromised host abdominal abscess detection: gallium, in-111 and tc-99m labeled leukocytes and polyclonal and monoclonal antibodies the clinical use of tc-99m labeled wbc scintigraphy in clinically ill surgical and trauma patients with occult sepsis c-reactive protein and gallium scintigraphy in patients after abdominal surgery antigranulocytescintigraphy and hydro-mri in the determination of bowel wall infl ammation in crohn's disease. the 47th annual meeting of the society of nuclear medicine the use of ga-67 in pulmonary disorders imaging of infl ammation by pet. conventional scintigraphy and other imaging techniques acquired immunodefi ciency syndrome: ga-67 citrate imaging utility of gallium-67 scintigraphy and bronchial washings in the diagnosis and treatment of pneumocystis carinii pneumonia in patients with the acquired immune defi ciency syndrome gallium scintigraphy in the detection of amiodarone lung toxicity pulmonary uptake in indium-111 leukocyte imaging: clinical signifi cance in patients with suspected occult infections role of scintigraphy in urinary tract infection a review of renal scarring in children power doppler sonography and acute pyelonephritis in children: comparison with tc-99m dmsa scintigraphy urinary tract infection: clinical practice guideline for the diagnosis and management of the initial uti in febrile infants and children 2 to 24 months different guidelines for imaging after fi rst uti in febrile infants: yield, cost, and radiation the role of power doppler ultrasonography in the diagnosis of acute pyelonephritis value of 18f-fdg-pet/ct in patients with fever of unknown origin and unexplained prolonged infl ammatory syndrome: a single centre analysis experience pet/ct in the evaluation of infl ammatory bowel disease: studies in patients before and after treatment key: cord-010255-gvkc2hjd authors: chrystie, i.l.; totterdell, b.m.; banatvala, j.e. title: asymptomatic endemic rotavirus infections in the newborn date: 1978-06-03 journal: lancet doi: 10.1016/s0140-6736(78)90967-4 sha: doc_id: 10255 cord_uid: gvkc2hjd between may 1, 1976, and may 14, 1977, 343 (32·5%) of 1056 5-day-old babies in newborn nurseries excreted rotaviruses. the infection-rate was highest during winter (49%). 76% of infected babies at this time were bottle-fed. 41% of neonates excreted low amounts of virus (10(8) particles/g fæces); older children tended to excrete >10(10) particles/g fæces. infected breast-fed babies excreted less virus than those who were bottle-fed. stools of breast-fed babies often contained clumps of complete "smooth" rotavirus particles. when the newborn nurseries were transferred to a newly built hospital wing, infection appeared in the new wards, including those admitting only new patients, within a short period. infection was either mild (8%) or symptomless (92%), and even babies with symptoms required no treatment. in solid myeloma tumours but also for metastatic spread of myeloma. this notion is not in conflict with recent knowledge of the heterogeneity, development, and migratory habits of b-cell subpopulations. 18, 39 there is no doubt about the presence of circulating tumorigenic cells in mice bearing myeloma tumours. however, the exact identity of these cells is not known. the simultaneous detection of malignant change and idiotypic structures in circulating lymphocytes is difficult. nevertheless, lymphocytoid cells bearing some tumour-cell markers have been demonstrated. the number of lymphocytes whose sig was altered by tumour r.n.a.3' is probably much greater than the number of tumorigenic cells estimated in other experiments. 26 although it is highly likely that sig-id+ neoplastic stem cells circulate in human myeloma, the bulk of the sig-id+ lymphocyte population may be partially differentiated and therefore relatively benign. in addition, many circulating tumorigenic cells could be in a non-growth phase or equivalent state, and probably very few of those which are in the cell cycle successfully initiate a focus of tumour because of immunological inhibition and/or ecotactic preference. definitive experiments are needed to detect and isolate myeloma cfu-c stem cells from human peripheral blood, and attempts should be made to propagate these in nude mice. further characterisation of peripheral lymphocytes from myeloma patients must be undertaken because of their possible value in diagnosis and therapy. rotaviruses are the commonest cause of acute nonbacterial gastroenteritis in infancy and childhood,',' and a common cause of severe diarrhoeal disease in newborn calves3 and piglets.4 rotavirus infection is world-wide and iri children admitted to hospital is most common between 6 months and 2 years of age;5 virus is seldom detected in the stools of symptomless age-matched controls. in temperate climates, infection is most frequent in winter.2 after our first report of rotaviruses in the stools of newborn babies with mild diarrhoea we found that 76 of 144 (52%) newborn babies excreted rotaviruses. ' 7 although the babies were infected as early as the third day of life, virus excretion was most frequent among 5-9 day-old babies, who showed few if any of the symptoms of infection found in older children.' this paper describes a 12-month study of the incidence of infection, the amount of virus excreted by breast-fed and bottle-fed babies, and the pattern of virus spread during the transfer of maternity wards to quarters in a newly built hospital wing. stools from 5-day-old babies were examined for virus particles by electronmicroscopy. during the first 6 weeks of the study, the maternity wards (mary 1 and mary 3a and b) were located in the old part of the hospital (south wing). in mid-june, patients were transferred to new wards (haydon and mary) in the new north wing, and the old wards were subsequently cleaned and reopened in early august under new names (garland, wrigley, and bowes) for mothers and babies of uncomplicated deliveries. garland and bowes were next to each other on the same floor and wrigley was one floor below. some patients were transferred from haydon and mary to bowes, but garland and wrigley accepted only new patients. mary ward in the new north wing was used for only 2 months to provide additional accommodation while wards in the old south wing were being cleaned. in general, babies remained with their mothers during the day but at night were accommodated in the nurseries. between may 1, 1976, and may 14, 1977, stools from 1056 babies were examined, of which 343 (32.5%) &ccedil;ontained rotaviruses. bacteriophages but no other virus particles (astroyiruses, caliciviruses, coronaviruses, adenoviruses, small round virus particles) were detected, in contrast with our findings among older children. the number of rotavirus particles detected varied considerably : of 104 consecutive rotavirus-positive infants studied during the winter months, 41% excreted 107-108 particles/g of fasces, but the stools of 20% of the infants contained more than 101o/g which is what we usually find in rotavirus-infected children with acute gastroenteritis. infection had a seasonal variation, reaching a peak during the winter of 1976-77 and the lowest level in summer, 1976 ( fig. 1) in haydon ward rotavirus infection among newborn babies was mild or more often symptomless. only 15 of 189 (8%) babies in this ward who excreted rotavirus passed frequent loose or offensive stools or vomited and there was no obvious correlation between the amount of virus excreted and the presence of gastrointestinal symptoms. such symptoms were noted in 5 (2%) of the 254 rotavirus-negative children in the ward. in other reports of rotavirus infection,8-10 the proportion of newborns with diarrhoea was variable, but usually greater than in our series. however, the significance of this is difficult to assess since newborn babies, although apparently in good health, pass a variable and often frequent number of stools of varying consistency. the assessment of reports of "diarrhoea", "loose stools" or "frequent stools" is thus difficult particularly if, as in our study, assessment is made retrospectively by examining feeding-charts or case-notes. although we may have underestimated the frequency of diarrhoeal episodes, it is certain that no infant was sufficiently ill to require treatment. it is not known why rotavirus infection is mild or symptomless in the newborn human. it is often severe in newborn calves and piglets, particularly if they are colostrum-deprived," and unlike humans, these animals do not acquire antibodies transplacentally. rotavirus antibodies present in the sera of almost all human adults may protect the newborn after transplacental transmission. although it may appear surprising that serum antibody rather than secretory antibody protects against infection localised in the small intestine, vaccine-induced circulating antibodies protect the gut against cholera12 and the respiratory tract against influenza." however, although newborn infants are protected from disease, they are not protected from infection: 33% in our study excreted virus, occasionally in very large amounts. symptomless infection with excretion of large quantities of virus also occurs with cytomegalovirusi4 and hepatitis-b virus. holmes has suggested that the intestinal brush-border enzyme, lactase, acts as the host-cell receptor for rotavirus. 16 in-vitro trypsin treatment enhances the in-vitro infectivity of porcinel7 and bovine18,19 rotaviruses. however, there is no evidence that human neonates are deficient in these enzymes. it is possible that the rotavirus strains circulating in our nurseries were avirulent. pig rotaviruses may vary in virulence.2o however, the demonstrations of mild or symptomless infection in newborn infants but frank diarrhoea in older children in the u.k., australia,'o and india2i do not support this hypothesis. furthermore, in our hospital there is concurrent rotavirus infection among symptomless newborn infants and among older children with gastroenteritis in the ward only one floor above the newborn nurseries. breast-fed babies excreted rotavirus significantly less frequently and, when infected, generally shed less virus than bottle-fed babies. breast-fed babies may be protected by maternal secretory antibodies in colostrum 22, 23 and breast milk24 during the newborn period, or by nonspecific antiviral factors distinct from antibody or interferon in breast milk. 25 in a rural bangladesh environment heavily contaminated with rotaviruses admission to hospital with rotavirus-induced diarrhoeal disease is rare before the age of 6 months.26 how rotaviruses induce diarrhoea in humans is not clearly understood but the newborn's gastrointestinal tract may have physiological characteristics not found in older children which allow symptomless infection. discovery of the mechanism by which young infants are protected might provide an alternative to vaccination. although it is uncertain how infection was introduced into the newborn nurseries, transmission of infection by medical or nursing staff from infected older children in the general wards seems likely. studies in toronto have shown that rotavirus infection, a common cause of hospital-acquired gastroenteritis among older children, was probably transmitted by medical staff. 27 we found infection occasionally in infants in the newborn specialcare nursery. premature babies also had symptomless infections but in this unit many are in incubators and active measures are taken to prevent cross-infection, so rotavirus spread is likely to be rare. we are now investigating whether our newborn rotavirus-infected infants are "immunised" as a result of infection. follow-up studies on 20 babies who excreted rotavirus neonatally have shown that 1 had rotavirusinduced diarrhoea when aged 14 this type appears to be the predominant serotype associated with symptomatic disease in most parts of the world. we thank the medical officers and nursing staff of the maternity wards at st. thomas's hospital for their cooperation. proc. staff meet. mayo clin h&aelig;mat semin. h&aelig;mat archs intern acute diarrhoea in childhood in acute diarrhoea in childhood oxford, and &dagger;hospital for sick children, great ormond street, london summary urinary iron excretion after single intramuscular (i.m.) bolus injections or 12 h subcutaneous (s.c.) infusions of desferrioxamine (d.f.) was determined in sixteen homozygous &bgr key: cord-007176-61e9obb3 authors: jackson, george gee; muldoon, robert lee title: viroses causing common respiratory infections in man. iii. respiratory syncytial viroses and coronavimses date: 1973-11-17 journal: j infect dis doi: 10.1093/infdis/128.5.674 sha: doc_id: 7176 cord_uid: 61e9obb3 nan 1. size. rs virus was estimated, from sucrose density gradient centrifugation studies, to be 90-120 nm in diameter [2] ; viral particles in infected cells measured 65 nm by electron microscopy. particles negatively stained with phosphotungstic acid measured 120-300 nm [23] . some viral particles were as large as 860 nm [25] . structure. in ultrathin sections of infected tissue culture, electron microscopy revealed viruslike particles in vacuoles or invaginations within the cytoplasm [14] , but complete particles were not found in the cell cytoplasm [21] . the enveloping membrane has been described as fringed and the nucleoprotein strand as having a herring-bone appearance, with a mean diameter of 13 cf antibody formed by infants during an rs infection has a higher antigen requirement than antibody present in sera of adults or than that found as maternal antibody [113] . neutralizing secretory antibody occurs in one-half or more of patients with lower respiratory disease and in 10%-20 % of those with milder infections [97, 116] . b. secondary. a rise in cf and neutralizing antibody was observed after reinfection in the presence of pre-existing serum antibody [18] . the cf antibody response in adults was always weak compared with that in children [65] . a. infection of tissue culture. isolates were made from 0.1-0.2 ml of specimen inoculated onto a monolayer consisting of 100,000-300,000 hep-2 cells. eagle's basal medium, containing 5 %inactivated chicken serum, is a suitable maintenance medium that should be changed every three to four days. stationary incubation at 36 c is satisfactory. inoculation of a culture of chang liver cells, four to six days old, maintained with a medium of eight parts eagle's basal medium, two parts inactivated horse serum, and 0.2 parts l-glutamine is satisfactory. the medium is changed every four days. virus can be propagated in kb cells in a medium consisting of eagle's basal medium with 2 %chicken serum. preferred cell line. the most sensitive cell cultures are hep-2, monkey kidney, human amnion, and human kidney, in decreasing order [5, 44] . i. the cytopathic effect begins with small syncytial areas randomly distributed early in infection. within one to four days, the entire cell sheet may be involved, with syncytial areas enlarging and becoming more numerous [2] . the time of appearance of cpe depends, within limits, on the number of serial passages of the virus [2] but is nearly always demonstrable within five days. ii. cytology. eosinophilic cytoplasmic inclusions are commonly found in infected cells, especially in the syncytia [15] . these inclusions are devoid of dna, rna, virions, and demonstrated specific antigens. chromosomal abnormalities are not observed. in infected vero cells, dense intracytoplasmic inclusions have diameters ranging from 90 to 130 nm [115] . iii. plaque formation. small macroscopic plaques developed after incubation for seven to nine days in hep-2 cells overlayed with agar [30] . four conditional-lethal, temperature-sensitive mutants of rs have been isolated and shown to be genetically stable. one of the mutants produced a typical, nonsyncytial plaques [93]. iv. hemadsorption. none demonstrated. no data available. one-day-old mice inoculated intracerebrally or intraperitoneally [1, 2] , weanling hamsters, rabbits, guinea pigs, mice, weighing 10 g, and young adult rats inoculated via the previously mentioned routes were refractory to infection [1] . a. natural infection 1. clinical. infection with rs virus has been observed every year since its recognition. it occurs in rather sharp epidemics, recurring at intervals of nine to 14 months, usually in the fall or spring. illness may be gradual or abrupt after an incubation period of three to five days. the symptoms associated with infection in children are cough (97 %), fever (93 %), rhinitis (57 %), pharyngitis (47%), malaise (38 %), vomiting (30 %), anorexia (27%), lymphadenopathy (22%), otitis media (17%), conjunctivitis (13 %), and abdominal pain (7 %) [13, 29, 49] . in an analysis of symptoms produced by infections with rs and by influenza a viruses, the two diseases could not be differentiated on a clinical basis [112] . in infants, as many as 60% the first isolations of virus were made one to two days before the onset of symptoms. isolates were more frequent and positive over a longer period from subjects with illness than from those who were infected but without symptoms. 3. immunity. all adults tested possessed detectable levels of neutralizing antibody to rs virus before challenge, but the titer of naturally acquired antibody had no significant effect on subsequent rs infection of volunteers and was poorly correlated with development of mild clinical illnesses. resistance to infection and illness appeared to be related to the level of nasal antibody but not to the level of serum antibody [121] . infection elicited an increase in the titer of serum antibody by cf and neutralization. immunity upon rechallenge was not tested. illnesses from infection are more severe in children than in adults [8, 10] . the cf test, with 8 units of antigen, will detect 90 %of infections among individuals older than six months and is as sensitive as neutralization. below six months, the cf test detects only 20 %of infections. the neutralization test is more sensitive than cf when serum from infants is used, but rises in neutralizing antibody have been detected in only half of the virus-positive infections in this age group. the use of unheated serum or the addition of antibody-free fresh serum increases the sensitivity of tests for neutralizing antibody [42, 60]. antiserum was produced in guinea pigs by three weekly ip inoculations of 1 ml of infected tissue culture harvest [2] . rabbits given three weekly iv inoculations of 1 ml each followed by two weekly im inoculations of 1 ml of infected tissue fluid combined with 2 ml of a mixture of mycobacterium butyricum, paraffin oil, and arlacel, produced cf and neutralizing antibody [2] . in adult rabbits, an alternative procedure is to give three injections at two-week intervals, the first two consisting of 8 ml of virus and adjuvant given im, and the third injection of virus alone administered iv [5] . respiratory syncytial virus is considered to be a paramyxovirus on the basis of its size, appearance by electron microscopy, and sensitivity to ether; it differs from other paramyxoviruses in that it has no known hemagglutinin. although minor antigenic variants have been found, they are not well discriminated by antibody in human sera. because there has been no sequential drift in the antigenic character of the prevalent strain, rs virus is considered to be a single type. epidemiologically, respiratory syncytial virus is very important, because it causes annual epidemics of acute respiratory diseases affecting infants, children, and adults. infection spreads rapidly from person to person and characteristically occurs as a discrete outbreak of acute respiratory illness in the winter or early spring. in infants and children, especially during the first six months of life, respiratory syncytial virus is the most important cause of bronchiolitis and a major cause of pneumonia. serum antibody acquired by transplacental passage does not provide immunity against infection and might possibly augment the local respiratory disease by an immunopathologic process. the virus may replicate in the middle ear, but its role in otitis is unproven. croup is an infrequent manifestation. pneumonia, as determined radiographically, is frequent, usually bilateral, and multilobar; it may be lobar with secondary bacterial infection. the pathologic lesion is one of necrosis of the epithelial mucosa of the trachea and bronchi and an interstitial inflammation. in adults, infection with rs virus usually causes upper respiratory symptoms; however, because of the prevalence of infection, it is an important cause of exacerbations of bronchitis, pneumonia, and "flu," requiring the hospitalization of adults. in some years, virus infection has given rise to an increase in secondary pneumonia due to diplococcus pneumoniae. infection is followed by an increase in serum cf and neutralizing antibody; also by secretory neutralizing antibody in the nasopharyngeal and tracheal secretions. primary infection does not establish complete immunity, and reinfection is common at all ages. inactivated vaccines of the type and potency previously produced have been detrimental because they have failed to prevent infections and they have induced a more severe disease with exaggerated pneumonia. attenuated live virus vaccines have not yet been successful, nor has any effective chemotherapy been developed. takano a virus isolated from wild cottontail rabbits was shown to possess chemical, physical, and biologic characteristics of the paramyxovirus family. although formation of syncytia was characteristic of its growth in tissue cultures, no antigenic relationship was detected by cf or neutralization tests with any known member of the paramyxovirus family [1] . isolates shown to be antigenically related to human rs virus were recovered from cattle with bronchopneumonia [2] . cytologic examination of bhk2 1 cells infected with bovine rs virus revealed intranuclear and intracytoplasmic viral components [4] . an a type particle with a diameter of 65 nm has been described in the cytoplasm of such cells [3] . the viral envelope was added as the virion passed through the cytoplasmic membrane in a budding process [4] . a. physical properties characterization 1. size. by gradocol filtration, the size of the virion was calculated to be 89 nm [2] . by electron microscopy, the usual diameter was measured as 80-160 nm [5] . 2. structure. the virions are pleomorphic. most particles are covered with projections (spikes) more densely packed than those seen on influenza viruses. these spikes are attached to the virion-by narrow stalks with a thickening (90-110 a) at the distal end [5, 11] . the internal component is a hollow, threadlike structure with a diameter of 70 nm and a definite structural pattern [21] . 3. heat stability. infectivity was destroyed at 56 c within 10 min. there was no loss of titer after 2 hr at 37 c or 10 days at 4 c. rates of thermal inactivation are dependent on the amount of particle aggregation. aggregation is dependent on the concentration of serum in the medium [ a. group antigen common cf antigens have been observed in known members of the coronavirus family, except in avian bronchitis virus. serologic data are still incomplete, but some observed interrelations are shown in table 1. table 1 . antigenic mosaic of coronavirus as determined by neutralization (n) and cf tests with animal serum [20, 25] . antigens produced in tissue culture or mouse brain cf antigens have been prepared from harvests of infected tissue culture and mouse brain, but attempts to prepare antigens from organ cultures have not been successful [2] . strains oc38 and oc43 cross-react, as shown by neutralization tests in mice or monkey-kidney cell culture. strains 229e and lp cross-react in neutralization tests but not to identical titers, indicating a close but not completely similar antigenic mosaic. a one-way cross exists between 229£ and oc32; antisera against the latter and b814 do not neutralize other coronaviruses. avian bronchitis virus reacts only with homologous virus (see table i ). in immunodiffusion tests, the number of detectable lines of precipitation varies from one (strain b814) to four (strain oc43). this may be a result of the procedure used for production of antibody [25] . c. antigenicity i. animals. in animals, specific antibody is elicited by the initial series of inoculations of cell-culture harvests. in neutralization tests with animal serum, no antigenic relationship has been detected between strains 229e and oc38-43 [20] . a. primary response. initial infection results in an increase of specific neutralizing antibody. about 50% of volunteers challenged with the oc strains of coronaviruses developed cf rises to mhv [20] . b. secondary response. the antigenic primacy of the initially infecting strain, heterologous interrelationships, and anamnestic responses are still to be worked out. [16] . coronaviruses of avian origin have phenotypic differences in the susceptible avian host cell range [4] and grow to a limited degree in nonavian tissues [32] . murine coronavirus has grown adequately only in tissues from mice [20] . coronavirus of rats has isolation propagation grown in only one of several cell lines studied [24] . coronaviruses of swine grow only in tissues of porcine origin [37] . a. infection of tissue culture. for growth of explants, a medium of 2 ml of eagle's medium with 0.2 % (wt/vol) bovine plasma albumin and incubation at 33 c in a humidified atmosphere containing 5 %(vol/vol) co 2 in air is satisfactory [5] . a ph of 7.0 is required for growth since inactivation is accelerated at ph 7.7 and 6.7 [25] . i. preferred cell line. human tracheal organ cultures. ii. growth cycle. after 1 hr at 33 c, only 18%of l132 cells wereinfected with strains 229e. virus structures were detected 6-8 hr later [17] .· infection of wi-38 cells with strain 229e resulted in a reorganization of the cytoplasm, as determined by electron microscopy. new viral structures were observed 6-12 hr after infection. clusters of virus were observed in intracytoplasmic vacuoles, called cisternae, 24-36 hr after challenge. strain oc43 in wi-38 matures in intracytoplasmic vesicles similar to those observed with strain 229e.. budding, such as that described for strain 229e was not observed [30] , and the budding process described for coronavirus is into cytoplasmic vesicles rather than from the plasma membrane, as has been observed with myxoviruses [6, 8, 28] . iii i. cpe. the cpe that gradually developed in human diploid cells gave them a stringy appearance. some intracytoplasmic vacuoles were observed [2] . strain b814 caused no cpe and could be detected only by electron microscopy or by interference with echovirus type 11 [1] . ii. cytology. no inclusions have been observed [2] . fluorescent antibody has been used for identification of viral antigen [15] . morphology, as observed by electron microscopy, is characteristic [5, 37] . the use of specific antisera, in combination with electron microscopy, can facilitate recognition of the virus in culture harvests [38] . iii. plaques. at 33 c, with a methyl cellulose overlay, plaques were formed in wi-38 cell cultures [8] . plaques can be produced in l132 cells infected with the 229e strain [17] . [4, 12, 24] . after four to five passages in tissue culture, strains oc38 and oc43 were administered intracranially to suckling mice. on the first passage in mice, illness, characterized by tremors, rigidity, and lethargy, was observed on days 11-15 after challenge. by the fourth passage in mice, these viruses were lethal for mice within 48-60 hr after challenge [9] . there is marked host specificity of different strains. a. natural infection 1. clinical. the use of explants of human embryonic nasal epithelium or trachea has resulted in the isolation of coronaviruses. serology has shown them to be associated with acute respiratory diseases of man. the exact importance of these viruses with accompanying epidemiologic data is unavailable because of the variability of strains and the difficult techniques required to establish diagnosis of the infection. in most studies, coronaviruses usually caused infections in the period from january through march [18, 33, 34] . only about one-half of naturally occurring infections cause clinical illness [26] . spread appears to be preferentially within families. in one study, secondary cases occurred in 17 of 26 families [22] . serum neutralizing antibody does not influence the occurrence of reinfection [33] . acquisition of infection with avian bronchitis virus from chickens is suggested by studies in poultry handlers [13] . nonsusceptible cells infection in man sponses occurred in 10%-20 % of these and other volunteers infected with coronaviruses [29] . strain 229e was recovered from sick as well as healthy volunteers in each of the four challenge passages [7] . volunteers challenged with the oc or b816 strain often showed cf rises to strains 229e and lp [20] . 3. immunity. incomplete. c. prevention 1. vaccine. none. none. diagnosis is based on electron microscopic examination of cell explants or, with some strains, detection of cpe in monolayers after serial blind passage. b. serology rises in the titer of cf antibody against strain 229e and hal antibody against strain oc43 are the most practical tests. neutralization is the serologic test of choice for specific identification. commercially unavailable. prevention laboratory diagnosis coronaviruses are a distinct group of viruses with common morphology and various degrees of antigenic similarity. the number of different coronaviruses that infect man and their exact interrelations are still unknown. their etiologic role in respiratory diseases has been established. also, they are associated with hepatitis in mice and avian bronchitis. data suggest that coronaviruses cause comment 3 %-4 % of acute respiratory illnesses in humans. the clinical syndrome is usually that of a common cold. asymptomatic infections also occur, possibly because of partial immunity to reinfection. coronavirus infections are most prevalent in the winter months and may occur in epidemic fashion, with the same strain being geographically widespread. recurrent epidemics of the same type do not seem to occur in sequential years. preliminary serologic investigations indicate that certain strains may infect children preferentially, whereas others are prevalent in adults. an alternative explanation of the findings is the emergence of new epidemic strains with disappearance of older strains for a period. transmission of the virus within families is frequent, and acquisition may be possible from poultry and other sources. no effective vaccines or chemotherapy have been developed. recovery of cytopathogenic agent from chimpanzees with coryza recovery from infants with respiratory illnesses of a virus related to chimpanzee coryza agent (cca). i. isolation, properties,and characterization recovery from infants with respiratory illness of virus related to chimpanzee coryza agent (cca). ii. epidemiologic aspects of infiltration in infants and young children association of the chimpanzee coryza agent with acute respiratory disease in children growth characteristic of chimpanzee coryza agent in tissue cultures role of respiratory syncytial virus in bronchiolitis, pneumonia, and minor respiratory disease. i. virus recovery and other observations during 1960 outbreak studies of acute respiratory illness caused by respiratory syncytial virus. i. laboratory findings in 109 cases respiratory syncytial virus infection in adult volunteers. ii. correlation of illness and clinical observations an outbreak of febrile illness and pneumonia associated with respiratory syncytial virus infection respiratory syncytial virus infection in adult volunteers. iii. prediction of illness and clinical observations studies on acute respiratory syncytial virus. 2. epidemiology and assessment of importance role of respiratory syncytial virus in bronchiolitis, pneumonia, and pharyngitis with bronchitis in children. ii. serologic studies over a 34 month period studies on acute respiratory illnesses caused by respiratory syncytial virus. 3. clinical and laboratory findings morphology and development of respiratory syncytial virus in cell culture growth and serologic characteristics of respiratory syncytial virus respiratory syncytial virus experimental cytial virus antigens by agar gel diffusion and immunoelectrophoresis interferon and respiratory syncytial virus speculation on pathogenesis in death from respiratory syncytial virus infection the late detection of respiratory syncytial virus in cells of respiratory tract by immunofluorescence double infection with rs virus and influenza virus infections in children. clinical comparison of overlapping outbreaks of influenza a2-hong kong-68 and respiratory syncytial virus infections differentiation of actively and passively acquired complementfixing antibodies in infants with respiratory syncytial virus infection the use of cough-nasal swabs in the rapid diagnosis of respiratory syncytial virus infection by the fluorescent antibody technique morphogenesis of respiratory syncytial virus in a green monkey kidney cell line (vero) respiratory syncytial virus neutralizing activity in nasopharyngeal secretions rsv infections and infant deaths respiratory syncytial virus tissue culture immunofluorescence as a laboratory aid rapid diagnosis of respiratory syncytial virus infection in children by the immunofluorescent technique experimental respiratory syncytial virus infection of adults. possible mechanisms of resistance to infection and illness recovery of a new syncytium virus from a cottontail rabbit a respiratory syncytial virus of bovine origin cultivation of a novel type of common cold virus in organ culture a new virus isolated from the human respiratory tract a new virus cultivated only in organ culture of human ciliated epithelium immunofluorescence of avian infectious bronchitis virus in primary chicken embryo kidney, liver, lung, and fibroblast cell cultures the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229e) effects of a "new" human respiratory virus in volunteers growth and intracellular development of a new respiratory virus growth in suckling mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease recovery in tracheal organ cultures of novel viruses from patients with respiratory disease direct electron microscopy of organ culture for detection and characterization of viruses neutralization of infectious bronchitis virus by human serum cultivation of "difficult" viruses from patients with common colds intracellular avian infectious bronchitis virus: detection by fluorescent antibody techniques in nonavian kidney cell cultures sensitivity of l132 cells to some "new" respiratory viruses the propagation of "coronaviruses" in tissue culture isolation from man of "avian infectious bronchitis virus-like" viruses (coronaviruses) similar to 229e virus with some epidemiological observations some characteristics of hemagglutinin of certain strains of "ibv-like" virus antigenic relationships among the coronaviruses of man and between human and animal coronaviruses symposium on the biology of large rna viruses community-wide outbreak of infection with 229e-like coronavirus in tecumseh, michigan seroepidemiologic studies of coronavirus infection in adults and children rat coronavirus (rcv); a prevalent naturally occurring pneumotropic virus of rats coronaviruses of man seroepidemiologic survey of coronavirus (strain 0c43) related infections in a children's population presence of neutralizing antibody against the 229e strain of coronavirus in the sera of residents of sendai electron microscopic studies of coronavirus coronavirus antibody titres in sera of healthy adults and experimentally infected volunteers intracellular development and mechanism of hemadsorption of a human coronavirus oc43 studies with human coronaviruses. ii. some properties of strains 229e and jackson and muldoon oc43 replication of avian infectious bronchitis virus in african green monkey kidney cell line vero virologic studies of acute respiratory disease in young adults. v. coronavirus 229e infections during six years of surveillance coronavirus infections in working adults: eight year study with 229e and oc43 protein composition of coronavirus oc43 hemadsorption by coronavirus strain oc43 characteristics of a coronavirus (strain 67n) of pigs detection of coronavirus strain 692 by immune electron microscopy of five subjects from whom coronaviruses were isolated, all developed a serologic response [3] .b. serologic. measurement of the cf antibody response was found to be twice as sensitive an index of infection as virus isolation. the cf antibody tends to be transitory, whereas titers of neutralizing antibody remain elevated for a longer period of time [13] . the observed prevalence of infection varies widely in different years [29, 33] .during a spring outbreak of respiratory disease in 1967, the infection rate in a community was 34 % [22] . from observations in a children's home, it was estimated (based on serology) that 19% of respiratory illness in a single season was caused by coronavirus strain oc43 [26] . among groups of adults during two of four winters when there were high rates of respiratory disease and infrequent virus recovery, infection with 229b occurred in 10%-24% of those with upper respiratory tract disease [18] .in three separate studies, each covering a seven-or eight-year period, coronavirus oc43 accounted for 3 % of 1,328 illnesses observed in children [26] ; coronaviruses 229b and oc43 accounted for 4 % of colds in an 'adult industrial population [34] , and 15%-35 % of students were infected during three seasons of high prevalence [33] .a serologic survey of adults and children showed that infection in infants below one year of age was infrequent. infection with oc38 and/ or oc43 occurred principally in the preschool years, whereas infection with 229b occurred later [23] . data from sera collected in 1966indicate that 30 % of adults and 15%-20 % of children had neutralizing antibody specific for strain 229e [7] . in a study of sera collected since 1967, 50 %-80 % of the population were found to have neutralizing antibody against 229e at a dilution of 1:20, as measured by plaque reduction in l132 cells. in cf tests (serum diluted 1: 20) between 50 %and 98 %of those tested had antibody [25] . sera collected from 139 adults between january 1969 and october 1970 showed that 8.6% had a neutralizing antibody titer 2:: 1: 8 against strain 229b [27] . 1. challenge. in an early study, harvest medium from human embryonic trachea containing strain b814 was used as inoculum. illness was observed in five of 11 volunteers [1] . after tissue and organ-culture passage, strain 229e was passed four times in volunteers and produced illness in each passage [7] . later, six coronaviruses isolated from persons with illness were given to volunteers. all six produced colds. heterologous antibody rekey: cord-024188-d7tnku8z authors: nissen, michael d.; lambert, stephen b.; whiley, david m.; sloots, theo p. title: respiratory infections date: 2010-03-27 journal: pcr for clinical microbiology doi: 10.1007/978-90-481-9039-3_5 sha: doc_id: 24188 cord_uid: d7tnku8z until recently, conventional culture techniques and immunofluorecence assays were considered the gold standard for the detection of respiratory viruses, even though results are mostly available too late or lacked specificity and sensitivity. these methods are now widely replaced with appropriate dnaand rna-based amplification techniques, in particular real time pcr amplification, for the detection of an extended number of agents responsible for acute respiratory infections. real-time pcr offers rapid results, efficiencies in work flow and a reduced risk of false positive results due to contamination. as a result, better patient management or reduction of unnecessary antibiotic administration will be possible leading to enhanced efficiencies in health care. in applying molecular methods to diagnostic use, the laboratory can optimise its diagnostic strategy by applying a combination of real-time amplification tests for respiratory viruses and the non-viral respiratory bacterial pathogens. however this must be done within a context of resource availability, technical expertise available and clinical utility. it seems certain that molecular microbiology will continue to develop, leading to further applications in diagnostic technology, thereby improving our understanding of disease processes and enhancing our knowledge of the pathogens responsible. they account for about two million deaths each year [22] and rank first among causes of disability-adjusted life-years (dalys) lost in developing countries (94.6 million, 6.3% of total [43] ). the populations most at risk for developing a fatal respiratory disease are the very young, the elderly, and the immunocompromised. while upper respiratory infections (uris) are very frequent but seldom lifethreatening, lower respiratory infections (lris) are responsible for more severe illnesses such as influenza, pneumonia, tuberculosis, and bronchiolitis that are the leading contributors to aris' mortality ( fig. 5.1) . pneumonia, with a global burden of 5,000 childhood deaths every day, is a tangible threat that needs to be dealt with accordingly. the incidence of aris in children aged younger than 5 years is estimated to be 0.29 and 0.05 episodes per child-year in developing and industrialized countries, respectively, which translates into 151 million and 5 million new episodes each year, respectively [28] . most cases occur in india (43 million), china (21 million), pakistan (10 million), bangladesh, indonesia and nigeria (56 million each). pneumonia is responsible for about 21% of all deaths in children aged younger than 5 years, leading to estimates that of every 1,000 children born alive, 12-20 die from pneumonia before their fifth birthday [43] . the main aetiological agents responsible for aris in children include streptococcus pneumoniae, haemophilus influenzae type b (hib), staphylococcus a wide variety of well known and newly identified agents cause respiratory illness and disease in humans. it is not possible to differentiate with certainty the aetiological agent in an infection based on clinical symptoms alone. in pre-school aged children, for example, illnesses due to respiratory viruses have seasonal variations and differences in the presence of fever and other symptoms, the likelihood of household transmission, and the impact they have in terms of medical visits and disruption to family life. but none of these features in isolation or combination is sufficiently specific to link illness to a pathogen with certainty. diagnosis in individual mild illnesses may not alter management, but can prevent unnecessary hospitalisation, antibiotic therapy, or further invasive investigation. laboratory confirmation of the cause of infections has been made more sensitive and rapid through the use of pcr technology. pcr has taught us that the constellation and severity of symptoms can cluster with particular infectious agents. for example, recent findings from the new vaccine surveillance network in the united states show that despite respiratory syncytial virus (rsv), parainfluenza viruses (pivs), and human coronaviruses (hcovs) all being common in early childhood; rsv and pivs are more common causes of hospital admission with acute febrile and respiratory illness than hcovs [33, 41] . despite such clustering, in individual illnesses it can be said that even viruses more typically associated with severe childhood illness can cause milder symptoms, modified by immune or possibly genetic factors, and that severe disease, whilst more commonly caused by a small group of well-studied viruses, can result from infection due to any virus. viruses that are typically considered to cause infrequent or mild disease, such as influenza c virus and piv-4, may cause more significant illness in vulnerable populations, including the young and immunocompromised. influenza, the most studied of respiratory viruses, provides broad insights for other common myxovirus and paramyxovirus respiratory agents. a review of healthy adult human volunteer studies showed that viral shedding increased sharply between 0.5 and 1 day after influenza virus challenge, peaking on day two; shedding can be detected 24 to 28 h before clinical onset, and has a mean duration of 4.8 days; two-thirds of subjects had symptomatic infection, and total symptom scores peaked on day three [11] . the natural history of infection may differ in the elderly and children. for example, pre-symptomatic influenza virus shedding has been seen for 6 days before clinical onset and mean duration of virus isolation from hospitalised children not receiving an antiviral was 6.8 days [29] . respiratory viruses can be transmitted through a number of modes: direct contact and fomites, large droplet, and airborne small particles. the importance of each of these modes depends on the virus in question, the site of infection, and the environment. for example, the eyes and nose appear to be much more important routes of infection for rsv than the mouth. modern molecular methods have resulted in the identification of previously unknown viruses from specimens collected from the respiratory tract. testing for new viruses along with known viruses, including rhinoviruses [6] by pcr, is filling the diagnostic void in respiratory illness and infection, and has improved our understanding of the epidemiology of such illnesses. in all but tropical climates there are a group of respiratory viruses that occur more frequently in the non-summer months, often peaking in winter; these viruses include influenza viruses, rsv, human metapneumovirus (hmpv), pivs, and hcovs ( fig. 5.2 ). human rhinoviruses (hrvs) are the most commonly identified group of viruses in both community-managed and more severe respiratory illness in children and older age-groups, having a year round presence but being more common in the spring and autumn months [6] . whilst it is clear rhinoviruses are a major pathogenic group, there is still uncertainty about the predictive value of a positive molecular test for a picornavirus, particularly from children. in tropical settings, influenza and other respiratory viruses can have a high background year-long presence [38] . respiratory viruses circulate freely in all populations, but moderate to severe illness tends to disproportionately affect certain groups. infections due to common viruses that result in disease severe enough to warrant laboratory testing, notification, or hospitalisation occur in the young, the very old, or both, such as with rsv and influenza [9, 14] . in spite of the inclusion of the live attenuated measles vaccine in the expanded program of immunization (epi), measles virus was still responsible in 2002 for some 213,000 deaths worldwide, essentially due to insufficient vaccine coverage [27] . the situation has fortunately been substantially improved lately, but the leading cause of serious respiratory illness in young children is respiratory syncytial virus (rsv), the agent of infantile bronchiolitis, which is associated with substantial morbidity and mortality [21] . parainfluenza viruses (piv-1, piv-2 and piv-3), especially piv-3, are second in incidence immediately after rsv. all children by the age of 2 years have had at least one episode of piv and/or rsv illness. in addition, both viruses can cause severe disease in the elderly, especially in patients with a chronic respiratory or cardiac condition [18] . although the disease burden due to these pathogens has not been accurately quantified in developing countries, extrapolation from known figures in industrialised countries, such as 125,000 reported cases of rsv per year in the usa, leads to the impressive global estimates of 64 million cases and 160,000 deaths per year from rsv infection worldwide. rsv was identified in 15-40% of pneumonia or bronchiolitis cases admitted to hospital in developing countries, followed by influenza viruses, parainfluenza viruses, human metapneumovirus and adenovirus [40] . the elderly also are at risk for severe rsv disease, and 14,000-60,000 rsv-related hospitalisations of the elderly are reported to occur annually in the usa [14] . human metapneumovirus, a member of the paramyxoviridae, is a recognised cause of a large fraction of severe aris in infant, elderly and immunocompromised populations [15] . other viruses that cause respiratory infections are coronaviruses, adenoviruses and rhinoviruses. recently discovered coronaviruses hcov-hku1 and hcov-nl63 are significant pathogens that contribute to the hospitalisation of children for ari [24, 35] . among other members of the coronaviridae are human coronaviruses hcov-229e and hcov-oc43, agents of the common cold. another recently identified coronavirus is that of the severe acute respiratory syndrome (sars), sars-cov, which emerged in southern china in late 2002 and spread in the spring of 2003 to some 30 countries within asia, europe and north america. in the elderly, influenza-related pneumonia remains a leading cause of infectious disease-related deaths. the threat of an avian influenza pandemic has been looming ever since the emergence in 1997 in hong kong of the h5n1 avian influenza virus, the new h5n1 variant is highly pathogenic for poultry and wild birds and can lethally infect cats and humans. at this time, however, it still is not possible to predict which virus is going to eventually cause a pandemic and when it is going to happen, but the preparation of pandemic influenza vaccines is being actively pursued, generating broad new knowledge on how to improve seasonal influenza vaccine immunogenicity. this has proven to be of the utmost importance with the recent emergence of influenza virus a h1n1 (2009) ("human swine influenza") in mexico and the usa in 2009. regarding influenza virus, the average global burden of inter-pandemic influenza may be on the order of 1 billion cases per year, leading to 300,000-500,000 deaths worldwide. however, the substantial reduction in ari mortality observed in developing countries that have implemented simple case management, including provision of antibiotics to children with ari, suggests that bacterial pneumonia contributes to a large proportion of deaths in these populations. available data suggest that dual infections with viral and bacterial pathogens may be quite common, as seen by the fact that, in the industrialized world, epidemics of rsv and/or influenza coincide with epidemics of s.pneumoniae year after year [32] . while influenza virus is the most commonly met pathogen in this context, other respiratory viruses, including rsv, measles virus, parainfluenza viruses, or adenoviruses may also predispose to secondary bacterial infections. several different bacterial species may be implicated, including h. influenzae, staphylococcus aureus, streptococcus pyogenes, mycoplasma pneumoniae, and, most importantly of all, s. pneumoniae [25] . half or more of the flu-associated mortality in the 1918-1919 spanish flu epidemic is believed to have resulted from pneumococcal super-infections. the same is true for developing countries. as an example, the observation was made in south africa that children vaccinated with the 7-valent conjugate pneumococcal vaccine showed 31% reduction in virus-associated pneumonias requiring hospitalisation, strongly emphasising the presumed importance of dual infections involving s. pneumonia [13] . dual infection seems to increase the severity of the disease and to result in higher mortality. this might be due to inhibition of pulmonary antibacterial defenses during recovery from viral infectiions. streptococcus pneumoniae (pneumococcus) was identified in 30-50% of bacterial pneumonia cases in developing countries in the 1990s, followed by haemophilus influenzae type b (hib; 10-30% of cases), then staphylococcus aureus and klebsiella pneumoniae [28] . non-typable h. influenzae (nthi), and non-typhoid salmonella spp. have also been implicated in some but not all studies. other organisms, such as mycoplasma pneumoniae, chlamydia spp., pseudomonas spp. and escherichia coli also can cause pneumonia. the most common syndromes associated with m. pneumoniae infections are acute bronchitis, pharyngitis and otitis, but 10% of infected children develop pneumonia [39] . the introduction of hib conjugate vaccines has resulted in a truly remarkable decline in hib disease where the vaccine has been introduced. however, the vaccine is not yet routinely made available to a majority of children worldwide. as a result, 400,000 deaths are still estimated to occur from hib disease each year [12] . in view of their safety and remarkable efficacy, the who has recommended the global implementation of the hib conjugate vaccines. s. pneumoniae is estimated to cause more than one-third of the 2 million deaths due to aris, especially in developing countries where the bacterium is one of the most important bacterial pathogens of infancy and early childhood [45] . virtually every child in the world is colonised with one or more strains of pneumococcus and becomes a nasopharyngeal carrier during their first few years of life. many children will go on to develop otitis media, and a few will eventually develop invasive pneumococcal disease including bacteraemic pneumonia and/or meningitis. the introduction of the conjugate pneumococcal vaccine in routine infant immunization should have a major impact on pneumonia in children less than 5 years of age worldwide, as already documented in the usa [26] . tuberculosis (tb) continues to be a leading cause of deaths worldwide, with an estimated one third of humanity infected and about 1.7 million deaths each year, a global toll of 4,650 lives daily. the emergence of mycobacterium tuberculosis (mtb) strains carrying drug-resistance mutations against first-line dugs (mdr-tb) and, more recently, against both first-and second-line drugs (xdr-tb), shows that it will most probably be impossible to contain the tb pandemic with drugs alone. more than one hundred new tb vaccine candidates have been tested in animal models and some have moved into clinical trials. testing such a wide variety of vaccine types using different strategies will obviously require time and a lot of coordination, especially as surrogate markers of protection still remain mostly unknown at this time. finally, it should be emphasized that nosocomial or hospital-acquired pneumonia is a major public health problem: pneumonia is the second most common type of all nosocomial infections, with an associated case fatality rate of 20-50%. infectious fungal respiratory diseases can be divided into those that occur opportunistically in immunosuppressed patients and those that occur in generally healthy individuals. fungi which affect immunosuppressed individuals are frequently pneumocystis jiroveci, and species of aspergillus and candida as well as cryptococcus neoformans [5] , while organisms such as histoplasma capsulatum, coccidioides immitis and blastomyces dermatitidis are frequent pathogens in healthy individuals in certain endemic regions. the causative fungi of respiratory infections vary with the population selected and the geographical region. the majority of fungal infections in lungtransplant recipients involve aspergillus spp., followed by candida, pneumocystis, cryptococcus, geographically restricted agents and newly emerging fungal pathogens. aspergillus infection remains the main fungal complication in lung-transplantation recipients. there are various fungal agents responsible for pulmonary fungal infection in patients with haematological malignancies, but aspergillus spp. and other molds such as zygomycetes or fusarium spp. represent the most frequently isolated microorganisms [19] . less commonly, pneumonia could be due to candida spp., cryptococcus spp. or pneumocystis jirovecii [17, 20] . although invasive aspergillosis is an uncommon complication of haematopoietic stem cell transplants (hsct) and solid organ transplants (sot), it continues to be associated with poor outcomes [5] . invasive pulmonary aspergillosis is rare in patients with chronic obstructive lung disease and is commonly associated with high doses of corticosteroids and multiple broad-spectrum antibiotics [19] . candida infection is predominant in patients with non-haematologic malignant tumours and in non-lung sot recipients. candida albicans and candida parapsilosis were the predominant isolates of pulmonary candidiasis in ventilated preterm infants with a birth weight of less than 1,250 g; the incidence rate of pulmonary candidiasis during the first month of life was 8.6% (20/233 cases) [17] . in intensive care units, c. albicans was also the most frequently isolated fungal species in all sites (68.9%). isolation of fungi allowed a diagnosis of fungal infection in 121 patients (7.7%) [47] . cryptococcus infection occurred in both immunosuppressed and immunocompetent individuals. pulmonary cryptococcosis is usually the primary site of a disseminated or central-nervous-system cryptococcal infection that may be fatal [46] . traditionally, capsule-deficient cryptococcus neoformans was considered to have low virulence. however, a recent study showed that the presentations and outcomes did not differ significantly between patients with proven pulmonary cryptococcosis caused by capsule-deficient cr. neoformans and six patients with pulmonary cryptococcosis caused by capsule-intact cr. neoformans [46] . the predisposing factors for fungal respiratory infections are increasing with the emergence of new immunosuppressive treatment. the increase of fungal associated respiratory tract infections may predominantly be attributed to the development of invasive diagnostic tools and the use of new methods for the identification of isolates, such as molecular techniques. recent advances in molecular biology have greatly improved the detection of viral respiratory pathogens. yet, even with the most sensitive molecular techniques, only 40-60% of infections are consistently diagnosed. this suggests that additional respiratory viruses are likely to exist. in fact, since 2001, seven previously undescribed viruses have been identified by analysis of clinical specimens from the human respiratory tract (table 5 .1). these new viral agents were detected by novel molecular methods such as virus discovery based on cdna-aflp (amplified fragment length polymorphism) (vidisca), pan-viral dna microarrays and high throughput sequencing [4] . more random pcr [2, 16] broadly, the advent of these new technologies has greatly stimulated efforts to identify novel viruses in the respiratory tract and in other human disease states. of the viruses discovered over the last 7 years, human metapneumovirus (hmpv) and the newly emerging human coronaviruses (hcov) are considered causative agents of respiratory disease. however, to date, sars coronavirus has been restricted geographically and has only been associated with limited and sporadic outbreaks. recently, human bocavirus (hbov) and the new human polyomaviruses kiv, wuv and merkel cell polyomavirus (mcv) were detected in respiratory secretions, and although an association with the respiratory tract has been postulated, it still remains to be proven [2, 8, 16 ]. hmpv infection is associated with a broad spectrum of clinical signs in patients of all age groups, and is the cause of upper and lower respiratory tract infection in infants and young children [30] . it is second only to rsv as a significant cause of bronchiolitis in early childhood and children are most likely to be hospitalised with severe disease. studies have linked hmpv with acute otitis media and asthma exacerbations in children and with exacerbations of both asthma and chronic obstructive pulmonary disease (copd) in adults. however, severe disease may occur in all patients with underlying medical conditions such as cardiopulmonary disease, the elderly and immunocompromised subjects. in these subjects the virus can cause prolonged and serious infections, particularly severe lung disease including fatality [10] (fig. 5.3) . the first reports of sars coronavirus infection were published in 2003, and the causative agent was subsequently characterised as a novel human coronavirus [23] . the sars epidemic was halted by a highly effective global public health response coordinated by the world health organization, and there is no further evidence that sars cov is currently circulating in humans. however, the sars outbreak focused renewed attention on coronaviruses generally, resulting in the discovery of two more new human coronaviruses, nl63 and hku1. hcov-nl63 was first detected in 2004 in a child from the netherlands with bronchiolitis, shown to be a cause of severe lower respiratory tract infection (lrti) in young children, and an agent of laryngotracheitis (croup) [37] . hcov-hku1 was detected in 2005 in an adult with chronic pulmonary disease in hong kong [44] and was subsequently shown to be globally distributed. hku1 infection presents with common respiratory symptoms as well as a more severe clinical presentation including bronchiolitis and pneumonia [24] . in healthy adults hcov nl63 and hku1 infections are generally not life threatening. this suggests that these coronaviruses, like 229e and oc43, only cause more-severe disease in young children, elderly persons, and the immunocompromised. they may be detected in 1 to 10% of patients with acute respiratory tract infections, and co-detection of these viruses with other respiratory viruses is common [37] . human bocavirus was first described in 2005 with a prevalence of 3.1% in swedish children with lrti [1] and subsequently in australia with 5.2% prevalence in children with arti during winter [31] . although a positive association of hbov with arti was suggested, the results remain inconclusive, because a high prevalence of other respiratory viruses was found in hbov-positive patients. to confirm the role of hbov as a respiratory pathogen, more extensive studies including matched control populations need to be performed. one such study using control subjects [3] proposed hbov as a cause of acute wheezing in children. hbov has been frequently detected in immunosuppressed adults but only rarely in immunocompetent adult subjects. however, it is uncertain if the presence of hbov in these subjects is the result of re-infection, viral persistence or reactivation. recently, three new human polyomaviruses, kiv, wuv and mcv were detected in specimens of patients with arti [2, 8, 16] . allander et al. [2] reported a prevalence of 1% for kiv in nasopharyngeal aspirates (npa) collected from a swedish population and gaynor et al. [16] showed a prevalence of 3% and 0.6% for wuv in respiratory samples from australia and the usa respectively. more recently, although originally found in merkel cell carcinoma, mcv was also found in 5.9% of npa collected from australian subjects with arti [8] . since these first reports, kiv, wuv and mcv have been detected in a number of geographic locations, suggesting a global presence for these viruses. one striking feature of early findings concerning kiv and wuv is their high rate of co-detection with other respiratory viruses. a co-detection rate of 74% has been observed for kiv and rates ranging from 68% to 79% for wuv [7] . so, even though an aetiological role in childhood respiratory disease has been proposed, it is difficult to assess a pathogenic role for these viruses unless observations are compared with those for matched control populations. further studies will need to be completed before the role of kiv and wuv as respiratory pathogens can be confirmed, and it remains possible that these viruses are not involved in respiratory disease, but that their presence in the respiratory tract simply reflects their mode of transmission. laboratory diagnosis of respiratory virus infections requires specimens containing cells from the respiratory tract collected early in the clinical illness. the most appropriate specimens are npas and bronchoalveolar lavage (bal). where the generation of aerosols may pose an infection risk to collection personnel, nose and throat swabs are a viable alternative. recent studies however, have shown that swabs are not as effective as npas for the detection of adenovirus and respiratory syncytial virus. limited data is available suggesting that flocked swabs are superior to traditional swabs for detecting respiratory virus infections. collection swabs should be dry and not contain bacterial transport medium as these often contain substances that are inhibitory to pcr reactions. in the case of influenza virus detection, the cdc recommends that viral transport medium (vtm) is added to swabs to assist in virus preservation. if vtm is to be added to dry swabs, they should be vortexed to release cells in to the medium and transferred to a sterile vial for transportation to the laboratory. vtm cannot be added to swab receptacles as they will invariably leak in transit and pose an infection risk. all respiratory specimen types should be transported to the laboratory at 4 â�¢ c. some npas and sputum samples received in the laboratory are very mucoid. these may either be diluted in vtm or digested with sputasol to facilitate the extraction process prior to molecular analysis. it is preferable to use an extraction method that will extract both rna and dna with equal efficiency as additional testing for agents such as bordetella pertussis and mycoplasma pneumoniae are often requested if a viral cause cannot be found. lung tissues are often collected at autopsy for either culture or molecular viral studies. the specimen requirements for both molecular and culture based respiratory virus detection methods are similar and therefore laboratories can choose the detection method that suits their role. all respiratory specimens must be handled with appropriate personal protection equipment as specified for pc2 laboratories and opened and aliquotted only in a class ii biological safety cabinet. specimens should be stored at 4 â�¢ c until they are processed. c. bletchly (b) molecular diagnostic unit, microbiology division, pathology queensland central laboratory, brisbane, qld 4029, australia e-mail: cheryl_bletchly@health.qld.gov.au traditional laboratory respiratory virus diagnosis involved virus culture with, or without immunofluorescence staining with specific antibodies. culture is highly sensitive if the appropriate cell lines are utilised and once a virus is isolated it can be further characterised and amplified if required. viral culture provides the added advantage that it will only detect infectious virus and not persistent nucleic acid. it has the disadvantages however of being expensive due to the requirement to maintain cells lines and tissue culture media and the requirement for expertise in sterile technique and interpretation of cytopathic effect (cpe) and fluorescence staining. direct immunofluorescence assay (dfa) of respiratory tract cells provides rapid results but does involve a series of manual manipulations and washes along with interpretation by highly skilled personnel (fig. 5.4) . the laboratory's primary role will most often dictate their method of choice for respiratory virus detection. diagnostic laboratories require rapid result turn-around and will most likely opt for molecular detection which can be readily adapted to high throughput batching and multiplexing. most molecular assays can be reported within 24 h of sample receipt whereas culture methods will take from 1 to 14 days and often much longer for confirmation. direct antigen tests are available commercially for some respiratory viruses but lack sensitivity (approximately 70%) although the positive predictive value is high. the expense and false negative rate of direct antigen tests need to be balanced with their rapidity and ease of use. negative direct antigen results should be confirmed by a more sensitive assay such as pcr. public health laboratories will most likely employ viral culture to enable viral isolate characterisation and the ability to detect unsuspected viruses. despite the popularity of molecular detection methods for respiratory viruses very few well validated commercial assays are available for the wide range of respiratory pathogens that are of clinical significance. most laboratories utilise "in-house" methods in combination with a rigorous quality assurance programmes. cell culture and direct immunofluorescent assay (dfa) staining using monoclonal antibodies were previously the most commonly used laboratory techniques for detecting respiratory viruses. although still used widely in the united states, these traditional techniques have gradually been superseded by highly sensitive and rapid reverse transcriptase polymerase chain reaction (rt-pcr) assays, with most laboratories in australia now using rt-pcr methods. additional advantages of rt-pcr detection of respiratory viruses include results that are not significantly affected by a loss of viral viability during specimen transport or storage, and that rt-pcr does not require the presence of intact, infected cells within the specimen. the pcr revolution has also been further stimulated through improvements in the technology, including the advent of real-time pcr and multiplex pcr methods, both of which reduce staff hands-on time, decrease result turnaround-times, increase through-put and are more user friendly compared to conventional pcr techniques. standard quality control practices should be implemented when testing for respiratory viruses by pcr, including the use of a suitable positive control, negative control, extraction control and inhibition control. sequence-related issues (discussed in more detail in chapter 8) are also very relevant to respiratory viruses. consideration needs to be given to the type of probe used when designing real-time pcr methods for respiratory viruses. the main issue is that respiratory viruses, particularly the rna viruses, show considerable genetic variation. for this reason, it is often difficult to identify a sufficiently large and conserved region to accommodate two hybridisation probes and so the single-probe taqman format is more commonly utilised for respiratory virus detection. on the other hand, we have also observed problems using the smaller minor-groove binder (mgb) taqman probes. the issue stems from mgb taqman probes being more susceptible to single nucleotide polymorphisms than standard taqman probes. for example, we have found that some rsv strains provided poor fluorescent signal, barely above the background negative signal, in an rsv mgb assay [42] . cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a third human polyomavirus human bocavirus and acute wheezing in children virus discovery by sequence-independent genome amplification fungal infections in primary immunodeficiencies frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection merkel cell polyomavirus in respiratory specimens: a possible route of transmission? vaccine preventable diseases and vaccination coverage in australia human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease time lines of infection and disease in human influenza: a review of volunteer challenge studies haemophilus influenzae vaccines efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in the gambia: randomised, double-blind, placebo-controlled trial respiratory syncytial virus infection in elderly and high-risk adults human metapneumovirus infection in young children hospitalized with respiratory tract disease identification of a novel polyomavirus from patients with acute respiratory tract infections congenital candida pneumonia and sepsis: a case report and review of the literature respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden invasion of the alveolar-capillary barrier by aspergillus spp.: therapeutic and diagnostic implications for immunocompromised patients with invasive pulmonary aspergillosis cryptococcosis: an emerging respiratory mycosis respiratory synsytial virus and parainfluenza virus vaccines human vaccine research and development: an over-view a novel coronavirus associated with severe acute respiratory syndrome coronavirus hku1 and other coronavirus infections in hong kong insights into the interaction between influenza virus and pneumococcus impact of pediatric vaccination with pneumococcal conjugate vaccine on the risk of bacteremic pneumococcal pneumonia in adults epidemiology and etiology of childhood pneumonia viral shedding in children with influenza virus infections treated with neuraminidase inhibitors evidence of human coronavirus hku1 and human bocavirus in australian children seasonality of invasive pneumococcal disease: temporal relation to documented influenza and respiratory syncytial viral circulation coronavirus infection and hospitalizations for acute respiratory illness in young children a newly discovered human pneumovirus isolated from young children with respiratory tract disease human coronavirus nl63, a new respiratory virus human coronavirus nl63 infection is associated with croup influenza in tropical regions community outbreak of mycoplasma pneumoniae infection: school-based cluster of neurologic disease associated with household transmission of respiratory illness respiratory syncytial virus infection in tropical and developing countries parainfluenza virus infection of young children: estimates of the population-based burden of hospitalization sequence variation can affect the performance of minor groove binder taqman probes in viral diagnostic assays estimates of world-wide distribution of child deaths from acute respiratory infections characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia make any mother and child count pulmonary cryptococcosis in non-aids patients fungal infections in the icu key: cord-018319-tylkbh4h authors: chemaly, roy f.; rathod, dhanesh b.; couch, robert title: respiratory viruses date: 2011-01-04 journal: principles and practice of cancer infectious diseases doi: 10.1007/978-1-60761-644-3_32 sha: doc_id: 18319 cord_uid: tylkbh4h the respiratory viruses as a group are the most common cause of an acute infectious illness in developed societies. the immunocompromised state of many cancer patients constitutes the basis for the frequent failure of the host to promote a normal and rapid recovery from an acute respiratory viral infection and results in a more severe and prolonged infection that causes significant morbidity and mortality in these patients. those respiratory viruses that are most prevalent and most prone to produce lower respiratory illnesses and pneumonia in healthy hosts, rsv, influenza viruses, and parainfluenza viruses, are those most likely to cause severe illness and pneumonia leading to hospitalization in immunocompromised persons. however, viruses less prone to produce a lower respiratory illness but that are highly prevalent, such as rhinoviruses, may frequently be associated with severe illness. the limited availability of antivirals and vaccines for the acute respiratory viruses means that these infections will continue to be important for many years and dictate a need for utilizing infection control procedures as much as possible, particularly in hospitals and institutions, so as to minimize spread. efforts to develop specific vaccines are important as their use could prevent as well as reduce exposure of cancer patients to these viruses. development of specific antivirals is important for use in immunocompromised patients as normal recovery mechanisms may be seriously impaired. community respiratory virus infections were once primarily considered to be infections of children and generally nonserious. however, over the past two decades, it has become clear that these viruses can cause serious infections that require medical attention, particularly in infant, elderly, and immunocompromised patients. historically, the most common causes of respiratory infections in cancer patients were thought to be opportunistic bacteria and fungi, but newer diagnostic methods have revealed that respiratory viruses can cause serious morbidity and mortality in such patients, including leukemia patients and hematopoietic stem cell transplant (hsct) recipients. many viruses are known to cause respiratory tract infections, but the most common in hospitalized cancer patients are influenza viruses, respiratory syncytial virus (rsv), and parainfluenza viruses (piv) [1, 2] . however, all respiratory viruses can cause respiratory infections in cancer patients including rhinoviruses, enteroviruses, coronaviruses, human metapneumoviruses (hmpvs), adenoviruses, as well as cytomegaloviruses, herpes simplex, and varicella zoster viruses. in this chapter, we discuss the common clinical presentations, diagnostic methods, treatments, and prevention measures for respiratory virus infections in general and in cancer patients in particular (table 32. of these cases, most were in hsct recipients (45%). the summer [2] [3] [4] . rsv is a member of the paramyxoviridae family and the genus pneumovirus. it is an enveloped singlestranded rna virus approximately 150-300 nm in diameter that is contained in a helical nucleocapsid surrounded by a lipid envelope. this envelope bears two glycoproteins: the g protein, which attaches to the cell surface, and the f (fusion) protein, which enables the internal components to enter the cell after fusion of host and viral membranes and to initiate replication. both glycoproteins are integral to pathogenesis [2, 3, 5] . there are two groups of rsv viruses, groups a and b [2] [3] [4] [5] . rsv infections can present with a wide array of upper or lower respiratory symptoms. the incubation period is 4-6 days in adults. in infants and young children, the primary infection starts in the upper respiratory tract, with rhinorrhea, low-grade fever, and cough; it may progress to lower respiratory infection (lri) (bronchiolitis or pneumonia), at which stage most patients seek medical attention [3, 6] . it is also likely to involve the sinuses and the middle ear. rsv is known to cause apnea in infants, although the mechanism of this action remains unknown. older children and adults typically present with upper respiratory tract symptoms, but may also have constitutional symptoms such as fever and malaise [3, 7] . rsv upper respiratory infections (uris) can also progress to the lower respiratory tract, particularly in institutionalized adults and those with severe combined immunodeficiency, leukemia, hsct, and in lung transplant recipients [2, 8, 9] . these patients may present with wheezing and shortness of breath, with or without underlying comorbidities or known hyperactive airways. almost 35% of elderly patients with rsv infections report wheezing [8] [9] [10] [11] . patients with weakened immune systems because of malignancy, chemotherapy, steroid use, hsct, or solid organ transplantation are at high risk for developing severe illnesses. they also tend to have a longer duration of infection, with a varied presentation [12, 13] . leukemia patients and solid organ and stem cell transplant recipients are particularly at risk, with the latter being at high risk for pneumonia and death prior to engraftment [11] . rsv infections begin as uri, but progress to lri in 30-60% of hsct recipients, leading to respiratory failure with significant morbidity and mortality; some early studies described a mortality rate of 70-100% [14, 15] . in hsct recipients, the risk factors for progression to lri [16] [17] [18] [19] . torres et al. [20] found that a high apache ii score and not giving aerosolized ribavirin treatment were independent predictors of progression to pneumonia in leukemia patients; those with uri who were treated with aerosolized ribavirin were less likely than untreated patients to develop pneumonia (68 vs. 96%, p < 0.01) and die with rsv infection (6 vs. 36%, p = 0.1). the overall mortality rate in the study was 10% [20] . solid organ transplantation patients with rsv infection may present with dyspnea, cough, fever, and wheezing which progress to pneumonia in more than 70% of patients [21] . in lung transplant recipients, a higher frequency of rsv pneumonia has been reported, but the mortality rate is low; the mortality rate is also low in kidney transplant recipients [21] . in pediatric liver transplant patients, pohl et al. reported a mortality rate of 17% for rsv pneumonia with early infection onset and preexisting lung disease as predictors of severe disease [22] . in children, genetic polymorphisms in cytokine and chemokine-related genes (interleukin [il]-4, il-8, il-10, il-13, and ccr5) and genes related to potential virus-cell surface interactions or cell signaling (tlr-4, cx3cr1, sp-a, and sp-d) have been associated with severe rsv infections [23] . data on hiv patients with rsv infections are scarce; however, a cohort study by miller et al. [24] performed in the winter of 1994-1995 found no evidence of influenza, rsv, parainfluenza, adenovirus, or enterovirus infections in the bronchoscopic alveolar lavage fluids of 44 hiv-1-positive patients. rsv can be diagnosed on the basis of the clinical presentation in infants with lri during an outbreak period [3] . rsv infections in adults cannot be clinically differentiated from other viral infections that cause upper respiratory symptoms. for a specific diagnosis, rsv must be detected in respiratory secretions. nasal aspirates or washes are most likely to give a positive rsv test in young children. if a nasal aspirate or wash cannot be obtained, a nasopharyngeal swab or throat swab may be used. because immunocompromised or intubated patients are more likely to develop an lri due to rsv, tracheal aspiration or bronchoalveolar lavage should be performed [8, 25] . the gold standard for diagnosing rsv remains the identification of virus causing typical syncytia in cultures of hep-2 cells [25] . viral cultures may take 2 days (shell vial cultures) and up to 2 weeks (routine cultures) to become positive, making isolation less relevant in most clinical settings. other available methods include antigen detection by enzyme-linked immunosorbent assay (elisa), immunoflourescence assay, and polymerase chain reaction (pcr) tests. fan et al. [26] reported a sensitivity of 65-95% for rsv detection using rapid antigen testing with elisa. pcr tests have been shown to be more sensitive than direct antigen detection [27, 28] . for most immunocompromised patients, rsv treatment is focused on reducing symptom severity and preventing progression to lris. nonsteroidal anti-inflammatory drugs and antihistamines have been used in patients with a uri. ribavirin, a nucleoside analog, has in vitro activity against rsv and has been approved by the united states food and drug administration for the treatment of rsv infections in children. two schedules of aerosolized ribavirin have been used in immunocompromised patients: continuous and intermittent. on the continuous schedule, a daily dose of 6 g (concentration, 20 mg/ml) is delivered over 18 h via a small particle aerosol generator unit, administered via a face mask in a tent. on the intermittent schedule, a concentration of 60 mg/ml is delivered over 3 h every 8 h. aerosolized ribavirin may be beneficial in certain adults and children with lris. its early use has been shown in some retrospective studies to reduce morbidity and mortality in hsct recipients [3, 29] and patients with hematologic malignancies, particularly when the infection is treated early on [30]. however, its effectiveness in solid organ transplantation patients remains unknown [31] . an rsv-specific monoclonal antibody (palivizumab) did not demonstrate a therapeutic benefit in a major study conducted in children [32] . although the combination of ribavirin and intravenous immunoglobulin (ivig) or palivizumab has not been evaluated in a randomized trial, it is sometimes used in severely ill patients with rsv pneumonia, especially hsct recipients, given that they have high mortality rates from this infection [3, 11, 14] . in patients at risk for progression to lri, aerosolized ribavirin should be considered at an early stage. a recent trial demonstrated that both the intermittent and continuous schedules of aerosolized ribavirin were effective at preventing progression to lris in 91% and 80% of patients with rsv uris and hematologic malignancies (including hsct recipients), respectively [33]. rsv infection may be acquired nosocomially, thus specific infection control measures should be implemented when dealing with patients with known or suspected infections. briefly, patients should be isolated in private rooms when possible, and appropriate personnel protective equipments should be used (i.e., disposable gloves, masks, and gowns) [34] . no licensed vaccine is available for rsv; however, two agents, rsv-ivig and palivizumab, have been used to prevent rsv infection. rsv-ivig has been studied in children younger than 24 months with severe lung disease and those who were born prematurely [35], but it was removed from the market in 2003. a randomized double-blind placebo-controlled study in children found that palivizumab recipients had a 45% relative reduction in rsv hospitalizations ( p = 0.003). twenty-one children (3.3%) died in the palivizumab group vs. 27 (4.2%) in the placebo group with no deaths attributable to palivizumab [36] . palivizumab is easier to administer than regular ivig, which must be given over 4-6 h, and does not interfere with other vaccinations. however, rsv-ivig and ivig may provide additional protection against other respiratory viruses as well [35, 37, 38]. influenza viruses infect both the upper and lower respiratory tracts. outbreaks are common every winter, although the severity of the disease varies considerably. influenza viruses belong to the orthomyxoviridae family, with influenza types a, b, and c constituting one genus. these rna viruses are enveloped and measure about 80-120 nm in diameter. the designation of the viruses into types is based on the stable antigenic characteristics of the nucleoprotein and the matrix protein antigens. influenza a and b viruses have surface glycoproteins known as hemagglutinins (ha) and neuraminidase (na). three hemagglutinin subtypes (h1, h2, and h3) and two neuraminidase subtypes (n1 and n2) have been described for the influenza a viruses that infect humans. major antigenic changes in the glycoprotein (basis for new subtypes) are called antigenic shift and can cause pandemics; minor antigenic changes occur frequently, are called antigenic drift, and cause the annual epidemics. influenza b has only exhibited minor antigenic drift [3, 39] . the 2009 pandemic influenza a (h1n1) contains segments present in north american swine for years, but is a new reassortant virus that acquired m and na gene segments from a eurasian adamantane-resistant swine influenza virus [40] . influenza typically has a short incubation period and an abrupt onset of symptoms, such as headache, fever, chills, myalgia, and malaise, along with respiratory symptoms of runny nose, cough, and sore throat. it can also present as a febrile uri or with constitutional manifestations only or with few-to-no respiratory symptoms. influenza can progress to pneumonia in otherwise healthy persons, but particularly in patients with comorbidities such as lung diseases, heart disease, diabetes mellitus, renal diseases, and hemoglobinopathies, in immunocompromised individuals, residents of nursing homes or chronic care facilities, and in individuals over 65 years of age [41] . primary pneumonia occurs when the influenza virus directly involves the lung and should be suspected when clinical symptoms progress to high fever, dyspnea, and hypoxemia [42] . influenza virus infections also affect the epithelium of the tracheobronchial tree, leading to impaired defense and a secondary bacterial pneumonia. influenza infections can increase morbidity and mortality rates in cancer patients [2] . in a study of leukemia patients with influenza infections, 39% of patients developed pneumonia [43], with cough and dyspnea being the most common manifestations. half of the patients had lymphopenia [43] . the incidence of influenza infection in hsct recipients is reported as 0.4% [44], with a mortality rate up to 38% [43], mainly due to respiratory failure. in one study [44] , 17% of patients who developed influenza after hsct presented with pneumonia; of those who presented with uris, 14% experienced progression to lri. risk factors associated with progression to lri were lymphopenia and days from transplantation (i.e., pneumonia developed more commonly among those infected earlier after transplantation); whereas use of systemic steroids and autologous stem cell transplantation appeared to be protective. x-ray findings include a diffuse interstitial pattern or focal pulmonary infiltrates [45] . among patients with solid organ transplantation, lung transplant recipients are at the highest risk for influenza virus infection [46] . the initial presentation in these patients may not always be in the respiratory tract, as illness can present with nonspecific gastrointestinal symptoms; however, patients who required hospitalization always presented with pneumonia [47] . progression to bronchiolitis obliterans, which is a characteristic of chronic lung rejection after infection, was also reported. in patients postrenal transplantation, influenza pneumonia is often acute, with high fever, cough, dyspnea, cyanosis, leukopenia, and thrombocytopenia [48] . influenza infection can only be diagnosed clinically in persons exhibiting the classic syndrome during an epidemic. however, because other viruses can produce the same syndrome and influenza infection can produce other respiratory syndromes, a confirmatory test detecting the virus or viral antigens in nasal washes, throat swabs, respiratory tract secretions, or bronchoalveolar lavage specimens is needed in sporadic cases and in immunocompromised patients. viral culture remains the gold standard for diagnosis, but can take up to 72 h to yield results [49] . sputum and nasal washes or nose swabs are superior to throat swabs for diagnosis [50] . rapid antigen testing using immunoflourescence assays, enzyme immunoassays, and pcr-based testing are used frequently in clinical settings [51] . the results of these tests can be obtained in hours and can have good sensitivity (72-95%) and specificity (76-84%) if obtained early from those with more severe illnesses [52] . pcr-based assays are more sensitive than rapid antigen testing for diagnosing influenza a and b, but are not often used because of availability and cost [51] . samples should be obtained within 24-48 h of the appearance of symptoms for the most accurate results. two classes of drugs are available to treat influenza infections [53] : the neuraminidase inhibitors, zanamivir and oseltamivir, which are active against influenza a and b viruses; and the m2 inhibitors, amantadine and rimantadine, which are only active against influenza a viruses [54] . amantadines are not effective against influenza b and resistance has been reported for 2009 novel h1n1 virus; resistance to oseltamivir has increased for the seasonal h1n1 virus (table 32. 2) [55, 56] . therapy should be initiated as early as possible, preferably within 48 h after symptom onset [57] . zanamivir and oseltamivir have been used extensively to treat both influenza a and b; both were shown to reduce the mean duration of symptoms by 1 day when used within 48 h of symptom onset [58] . both neuraminidase inhibitors have been shown to significantly decrease the incidence of complications associated with influenza such as development of pneumonia when compared to placebo [59] . the most common side effects of oseltamivir are nausea and vomiting, although other toxicities have been reported. central nervous system side effects such as anxiety, insomnia, impaired thinking, confusion, lightheadedness, and hallucinations have been reported with amantadine use. newer ni inhibitors including parenteral preparations are under development [60] . the mainstay of influenza prophylaxis in the general population is the administration of influenza vaccine. annual vaccination has been recommended for many years for people at high risk for complications, including those older than 65 years, residents of nursing homes or other facilities, adults and children with chronic pulmonary or cardiovascular conditions, adults and children hospitalized during the previous year, women in the second and the third trimesters of pregnancy, immunocompromised patients, healthcare workers, and family member of those at high risk prior to the onset of influenza season [41] . the american committee on immunization practices has recently recommended vaccination for all persons for whom there is no contraindication [61] . currently, there are two types of vaccines available, an intramuscular (inactivated virus vaccine) and an intranasal (attenuated virus vaccine). the intranasal form is to be used only in healthy individuals between the ages of 2 and 49 years; it should not be used to vaccinate immunocompromised patients [62] . individuals with a significant allergy to eggs or those with acute febrile illness should not be given vaccine [63] . the advisory committee on immunization practices recommends that immunocompromised individuals be vaccinated prior to influenza season and receive daily chemoprophylaxis with an antiviral medication during community outbreaks [41] . influenza chemoprophylaxis may be used in patients at high risk for complications if the vaccine is contraindicated or not likely to be completely protective. antiviral drugs should be administered to patients within the first 6 months of hsct, those with documented graft-versus-host disease, unvaccinated healthcare workers who care for immunocompromised individuals, and residents of long-term care institutions during outbreak periods [64] . the american society for blood and marrow transplantation (asbmt) 2009 guidelines [65] for prevention of infection in hsct recipients recommend annual inactivated influenza vaccine before the beginning of the season and before stem cell transplant. the vaccine may be given 4-6 months after hsct. they also recommended prophylaxis and preemptive treatment during community and nosocomial outbreaks of influenza a for hsct recipients regardless of the vaccination status in those who are within 24 months of the transplant or in those with more than 24 months posttransplant, but have gvhd and/or are on immunosuppression. the drug to be used for chemoprophylaxis depends on the susceptibility pattern of the outbreak virus. piv are enveloped, single-stranded rna viruses that belong to the paramyxoviridae family. the envelope contains two glycoproteins, one with both ha and neuraminidase activity and the other with fusion activity. there are five types that share certain antigens with other members of the paramyxoviridae family [3, 66] . of the five, piv-3 is the most prevalent, with most adults demonstrating the presence of antibodies [67] . piv-3 infections may be epidemic in the spring and summer, but may also occur through the year; piv-3 is associated with pneumonia and bronchiolitis in infants. piv-1 and 2 are associated with croup in children which occurs as outbreaks every 2 years in the fall; piv-4a and 4b cause only mild illnesses [67, 68] . piv infections have a wide spectrum of presentations, from a simple uri to a life-threatening complication such as pneumonia. the incubation period is usually 2-6 days. young children may present with coryza, sore throat, hoarseness, and cough with chest x-rays revealing interstitial infiltrates. in adults, most infections are mild, but in immunocompromised individuals, the infection can progress to a lower respiratory tract illness including pneumonia and cause prolonged illness and even death [3, 67, 69] . immunocompromised individuals are at risk for a piv infection that can progress to pneumonia. a study at our institution found a higher rate of progression among leukemia patients than among hsct recipients [70] . piv usually presents as an uri. in a large study in hsct recipients over several years, piv infections were documented in 7.1% of cases, with 78% being communityacquired [71] . patients who have undergone hsct are at particular risk for developing severe piv-associated pneumonia [71] [72] [73] [74] ; wendt et al. [72] reported a mortality rate up to 30%. coinfections (aspergillus fumigatus being the most common) and mechanical ventilation were found to be significant risk factors for piv pneumonia-associated mortality in one study [71] . other factors associated with progression include neutropenia within 1 month prior to infection, an apache ii score higher than 15, and pulmonary coinfection; this study also found a mortality rate around 20%, with no difference between those treated and those not treated with aerosolized ribavirin [70] . patients who have undergone solid organ transplantation do not appear to be at increased risk for developing severe piv illness, but only a few studies have been reported [75, 76] . one of these studies in lung transplant patients found piv infections in 11% of patients, all of whom developed pneumonia, but the majority (74%) were treated with aerosolized ribavirin and all but one recovered [75] . except for croup in young children, the clinical pattern of piv infection is similar to that of other respiratory viruses and cannot be distinguished on the basis of symptoms alone. during a community outbreak, a presumptive diagnosis can be made; however, confirmation in laboratory tests may be appropriate in immunocompromised individuals. the virus can be detected in respiratory tract secretions, nasal washes, nasal swabs, throat swabs, and bronchoalveolar lavage specimens. viral culture remains the gold standard for diagnosis, but can take days to yield a result [3, 77] . rapid antigen detection by immunofluorescence or elisa is most commonly used and can have a sensitivity of as high as 75-95% [78] . recent pcr-based assays have sensitivities up to 100%, with high specificity [26, 79] . management of piv infection is mostly supportive as no piv-directed antiviral therapy has been licensed by the u.s. food and drug administration. ribavirin has been shown to be active against the virus in vitro and in animal models and has been used occasionally to treat immunocompromised patients with severe piv infections [80] . one case series reported a decrease in piv viral loads and clinical improvement after aerosolized ribavirin treatment in children with severe immunodeficiency [81] . nichols et al. [71] reported that aerosolized ribavirin did not reduce viral shedding or mortality rates in hsct recipients after the infection had progressed to the lower respiratory tract. our data also showed no apparent benefit of aerosolized ribavirin on the mortality rate in hsct recipients and leukemia patients [70] . on the other hand, a combination of methylprednisolone and intravenous or oral ribavirin has, apparently, been used successfully to treat piv pneumonia in a hsct and a heart transplant recipient, respectively [82, 83] . currently, no licensed vaccine is available for the prevention of piv infection. hence, infection control measures play an important role in containing the spread of the infection. patients with suspected or confirmed infections should be isolated, and personnel protective equipment should be used. human adenoviruses are dna viruses belonging to the mastadenovirus genus of the adenoviridae family which measure about 70-80 nm in diameter [3, 84] . there are at least 51 known human serotypes divided into subgenera a to f based on the dna genome and pattern of hemagglutination [84] . adenovirus infections are reported most frequently in infants and children and can occur throughout the year; however, they may cause serious infections in immunocompromised patients. after a primary infection in childhood, adenoviruses establish latency in adenoidal tissues along with lifelong persistence of specific antibodies [84, 85] . adenovirus infections are transmitted by either inhalation of aerosolized virus, inoculation of the virus into conjunctival sac, or through the fecal-oral route [3, 84] . subgroup a, type 31 and various types from subgroups b and c have been associated with pneumonia and hepatitis [86] . serotypes 4, 7, 14, and 21 are associated with outbreaks of acute respiratory disease in military recruits, mostly in winter and spring [3] . types 1, 2, 5, and 6 are most common in children and present as an acute upper respiratory tract illness which can progress to lower respiratory disease; types 3 and 7 are less common, but can cause severe disease. in adults, infections due to adenovirus are characterized by sore throat and gradual onset of fever. cough, coryza, and regional lymphadenopathy are commonly seen. the most common clinical symptoms besides respiratory symptoms are fever and diarrhea [3] . adenovirus infections are common after hsct and can occur as a localized illness or as part of a disseminated disease. it has been associated with delayed engraftment and graft failure. infections due to adenovirus in hsct recipients have a reported incidence of 0.5-3% [87, 88] and are more commonly reported in allogeneic hsct recipients than in autologous transplant recipients (6 vs. 0.92%) [87] ; however, the mortality rate can be as high as 75% in both groups [87, 89] . some reports also suggest that the incidence of adenovirus infection in patients after hsct may be rising due to transplantation practices [90, 91] . disseminated infection may occur without respiratory tract symptoms and disease can develop in almost any organ causing gastrointestinal disease, hepatitis, nephritis, pneumonia, conjunctivitis, thrombotic thrombocytopenic purpura, pancreatitis, or hemorrhagic cystitis. viremia may be present, but is not detected in all cases of disseminated disease [92] . adenovirus is known to be fatal even in the absence of any respiratory tract involvement; however, if pneumonia is present, the mortality has been reported to be higher (80 vs. 50%) [87] . coinfections with aspergillus spp. and bacteria such as nocardia, legionella spp., and mycobacterium tuberculous are frequently seen in this patient population [90, 93] . risk factors for adenovirus infections include gvhd, unrelated donor, total body irradiation, t-cell depletion, younger age (<7 years old), chronic disease, and recent transplantation [90, 91, 94, 95] ; the degree of t-cell depletion and posttransplant suppression of t-cell function are the most important ones [92] . adenovirus types 5 and 21 are associated with severe infections in hsct recipients [95] . there are a few reports of adenovirus infections in solid organ transplant patients in whom the virus involved the donor organ and led to pneumonia, hepatitis, hemorrhagic cystitis, nephritis, enterocolitis, or disseminated disease. in patients with previous liver transplantation, adenovirus pneumonia had a reported prevalence of 1.5% with a mortality rate of 66% [96] . serotype 5 is known to be associated with hepatitis [96, 97] , whereas serotypes 1 and 2 are more commonly associated with pneumonia. in lung transplant recipients, one study found adenovirus infection to be an early complication following surgery with a prevalence rate of 1.3% [98] . progressive adenovirus infections are known to be associated with graft loss, progression to bronchiolitis obliterans, or death [99] . adenovirus infection should be suspected in cases of acute respiratory disease in military recruits or during outbreaks. in most cases, infection caused by the virus cannot be differentiated from those caused by other respiratory viruses from the clinical presentation alone [3] . a definitive diagnosis can be established by viral culture or the detection of specific viral antigens. viral culture remains the gold standard for identification of adenovirus. nasopharyngeal aspirate or swab, throat swab, sputum samples, or bronchoalveolar lavage can be used, depending on the site of the infection. a cytopathic effect is seen in human cell lines such as hela (cervix), a549 (lung), hek (human embryonic kidney), and hep-2 (larynx) by strains of adenovirus except for types 40 and 41. adenovirus 40 and 41 grow well in hek 293 cells. adenovirus-specific enzyme immunoassay (elisa) or immunofluorescence assay can be used to detect the presence of virus in clinical samples. these rapid tests suffer from a sensitivity of only about 50%; pcr-based assays can detect adenovirus dna from a variety of clinical specimens and has better sensitivity [100, 101] . viral load quantification is a useful tool to measure prognosis and monitor clinical response [102, 103] . viral loads higher than 1 ã� 10 6 copies/ml have been associated with an increased likelihood of death [104, 105] . there have been no randomized clinical trials for the treatment of adenovirus infection in immunocompromised patients. most patients are managed using symptom-based treatment and supportive therapy. cidofovir is currently being used in immunocompromised patients since it has been shown to decrease plasma viral loads in hsct recipients [106] . although cidofovir is active against all strains of adenovirus in vitro, only retrospective data are available on the efficacy of cidofovir in hsct [106] [107] [108] [109] and solid organ transplant recipients [110] . nephrotoxicity is commonly encountered with this drug and it should be used with caution. there are two accepted regimens: 5 mg/kg every 1-2 weeks, or 1 mg/kg 3 times per week, with the latter being associated with less nephrotoxicity [110] . orally active ether lipid-ester prodrugs of cidofovir (s)-hpmpa are under development with some promising results in in vitro experiments and phase i trials [111] . when tested against five adenovirus serotypes, they were shown to be more active than the unmodified parent compounds [111] . intravenous ribavirin has been used in a few reported cases, but results were conflicting [112] [113] [114] . finally, another treatment option using adenovirus-specific donor t-cells infusion has been shown to be feasible and effective in protecting children from complications due to adenovirus infection and causing a significant decrease in viral loads [115] . currently, there are no vaccines available for adenovirus infection other than the oral partially attenuated vaccines contained in enteric-coated capsules with use restricted to the military [116] . only routine infection control practices are recommended for civilian populations where special precautions must be taken for contact and droplet exposure. in hsct recipients at high risk from adenovirus infection, weekly pcr surveillance of viremia and preemptive treatment with cidofovir can be used [92, 117] . rhinoviruses are members of the picornaviridae family. they are nonenveloped, single-stranded rna viruses that measure about 15-30 nm. the capsid of the virus is icosahedral and contains 60 copies of four polypeptides each. a canyon on the viral surface contains the attachment site for the host-cell receptor, with most rhinoviruses using this site to attach to the intercellular adhesion molecule-1 receptor expressed on the surface of host cells [3, 118] . more than 100 serotypes of the virus have been isolated, making a vaccine unlikely in the near future [3, 119, 120] . rhinoviruses are proven to cause 15-40% of common colds in adults [3] . each year, adults experience 2 or 3 colds, whereas children may experience 8-12 [3, 120] . children are the major reservoir for rhinoviruses with infection rates decreasing with age. although infections occur throughout the year, peaks may occur in the fall and spring. this infection is primarily due to the deposition of the virus on the nasal mucosa. this can occur via self-inoculation or contact with infected secretions such as small-and large-particle aerosols (respiratory droplets) [3, 118, 120] . individuals with rhinovirus infections may be asymptomatic. when symptoms occur, they are typically those of the common cold: most commonly rhinorrhea and sneezing, which are associated with nasal congestion. infections due to rhinovirus have an incubation period of 1-4 days. adults characteristically experience sneezing, nasal obstruction and discharge along with cough, and a sore or scratchy throat. sinuses are commonly involved so that the illness is rhinosinusitis. symptoms may last for 4-9 days and usually resolve with no complications. fever is not usually associated with adult illness [3, 121] . children, on the other hand, may experience fever, cough, and nasal discharge and obstruction. in addition, the duration of symptoms may be longer. although bronchitis, bronchiolitis, and bronchopneumonia have been reported in children, rhinovirus is not usually a major cause of lower respiratory illness [3, 122] . however, they are an important cause of exacerbations of asthma and chronic obstructive pulmonary disease in children and adults and of lri in the elderly [3, 119] . a retrospective study in adults with rhinovirus infections who had undergone hsct found that 32% of patients developed and eventually died of pneumonia [123] . one patient was found to have a coinfection with aspergillus spp. on autopsy; most of the other patients had interstitial pneumonitis and/or acute respiratory distress syndrome [123] . a study performed at another institution reported that 55% of hsct recipients with rhinovirus infections developed pneumonia and 33% died [124] . in a study of community-acquired pneumonia in immunocompromised patients (after hsct or solid organ transplantation, with hiv infection, or receiving steroids or chemotherapy), rhinovirus was responsible for about 12% of cases, with a mortality rate of 18% [125] . these findings suggest that rhinovirus may cause more severe complications in immunocompromised patients than previously thought. many viruses cause common cold symptoms, and a definitive diagnosis cannot be made only on the basis of the presenting symptoms but rhinoviruses are most commonly associated with colds. a definitive diagnosis can be made by isolating the virus from nasal washes or other nasal secretion specimens in tissue culture. newer diagnostic methods such as real-time reverse-transcription pcr (rt-pcr) have been used; however, these tests are not frequently performed given the self-limited nature of most infections. no specific antiviral treatment is available for rhinovirus infections. most cases are managed with supportive care using antihistamines, decongestants, and nonsteroidal antiinflammatory drugs. given the number of rhinovirus serotypes known to cause infection, an effective vaccine is unlikely to be developed in the near future. infection control measures such as hand washing and isolating patients who are known or suspected to have rhinovirus infections can help contain the spread of the virus. hmpv belongs to the subfamily pneumovirinae of the paramyxoviridae family. the virus was discovered in 2001 and is genetically similar to rsv. hmpv is an enveloped rna virus that is known to cause uris and lris in all age groups [126] . although hmpv infections are more common in children, a recent study revealed an infection rate of 4.5% in adults with acute upper respiratory illness; 11% of patients required hospitalization [127] . hmpv usually causes a mild infection of short duration (about 3-5 days) and is self-limiting. the incubation period is 4-6 days [128] . the most common presenting symptoms in adults are cough, nasal congestion, rhinorrhea, dyspnea, hoarseness, and wheezing [127] . children often present with cough, rhinitis, fever, and wheezing [129, 130] . a recent study of patients with hematologic malignancies revealed a 9% incidence of hmpv infection. nine of the 22 patients who had undergone hsct had pneumonia; three of these patients died [131] . another study in hsct recipients detected the presence of hmpv in 3% (in 5 out of 163) of bronchoalveolar lavage specimens. these 5 patients presented with fever, cough, nasal congestion, and sore throat within the first 40 days after transplantation. the infection progressed to respiratory failure, pulmonary hemorrhage, and culture-negative septic shock. the mortality rate was 80% (4 of 5 patients) [132] . hmpv's growth in culture is slow and unreliable, which makes this method of diagnosis impractical. the presenting symptoms are similar to those of other infections that cause an acute respiratory illness, making clinical diagnosis impossible. pcr-based methods have been used to diagnose the infection in some centers. no specific antiviral therapy exists for hmpv infections. in vitro studies have demonstrated that ribavirin has activity against the virus [133] ; however, no clinical studies have been reported. immune serum globulins may neutralize the virus as demonstrated in one in vitro study [133] . anecdotally, a lung transplant recipient with respiratory failure secondary to hmpv pneumonia was treated successfully with aerosolized ribavirin [134] . the use of general preventive measures for patients with known or suspected hmpv infections can help reduce the rate of transmission. no vaccine is currently available for this virus. coronaviruses are single-stranded rna viruses that measure about 80-160 nm in diameter. they are enveloped with clubshaped projections, giving them a crown-like appearancehence the name [3, 135] . coronaviruses can cause diarrhea in infants and may play a role in demyelinating diseases of the central nervous system [3, 136] . coronaviruses are difficult to grow in vitro; some strains can only be grown in human tracheal organ cultures. the major antigenic types that cause diseases in humans are 229e, oc43, hk, and nl63 viruses which cause common colds and may also cause lower respiratory tract illnesses in young children, and sars-cov, which causes a severe acute respiratory syndrome [3, 137, 138] . coronaviruses are found in all tropical, subtropical, and temperate climates. most infections occur in the late fall, winter, and early spring [3, 139] . cyclical outbreaks of these infections may occur every 2-4 years. respiratory infections due to hcov-229e and hcov-oc43 strains probably spread in a manner similar to rhinovirus, i.e., via direct contact with infected secretions or aerosol droplets [3] . the clinical manifestations of coronavirus infections are similar to those of rhinovirus infections. the most common symptoms are rhinorrhea, throat congestion, and fever. the middle ear may also be affected, leading to effusions and acute otitis media, especially in children [3, 135] . the incubation period is 1-3 days and the duration of the illness is shorter than that of rhinovirus infections, a mean of 6-7 days. the subtype sars-cov has a slightly longer incubation period of 4-5 days. in immunocompromised adults, coronaviruses can cause lower respiratory tract infections [3, 140] . a recent case series found that the infection rate among immunocompromised patients was significantly higher when compared to immunocompetent patients (8.8 vs. 4 .5%) [141] . no large studies have been conducted in cancer patients or other immunocompromised individuals, but a few cases have been reported. folz and elkordy [140] reported a patient who had undergone autologous hsct and was diagnosed with a coronavirus lri. the patient developed a fever, sore throat, cough, and severe hypoxia and was treated successfully using supportive measures. kumar et al. [142] described a patient who developed a fatal severe acute respiratory syndrome (sars) after liver transplantation. epidemiologically, these infections should be suspected during the late fall or winter or during an outbreak. however, no practical method is available to confirm the infection except pcr-based tests. these are used to diagnose coronavirus infections at some institutions [3, 143] . similar to rhinovirus, no specific antiviral therapy is available for coronavirus. most patients respond well to supportive treatment with nonsteroidal anti-inflammatory drugs, decongestants and antihistamines. general preventive measures must be used around patients with coronavirus infections; hand washing, disposing of infected material carefully, and proper disinfection can help prevent the spread of the virus. no vaccine against the virus has been developed. enteroviruses are single-stranded rna viruses that can multiply in the gastrointestinal tract. they belong to the picornaviridae family, but are relatively stable at a low ph. the virus is surrounded by an icosahedral capsid comprising four viral proteins. it has no lipid envelope and is not susceptible to alcohol, ether, or detergents. poliovirus is the prototype of this group, but nonpolio enteroviruses have a wide spectrum of manifestations including acute respiratory illnesses [3, 144] . enterovirus infections are more common in developing countries and socioeconomically depressed areas and are associated with poor hygiene and sanitation. they can occur throughout the year, but the peak occurs in the summer and fall [3, 145] . they can be transmitted by direct contact with feces during activities such as cleaning and diaper handling. the clinical picture of enterovirus infections is similar to that of rhinovirus infections; no specific symptoms are associated with the virus. immunocompromised patients have been reported to develop central nervous system infections, chronic disseminated infections, and a dermatomyositis-like syndrome with enterovirus infection. in a study of respiratory tract infections in hematologic malignancy patients, 3% of patients had enterovirus infections; most (66%) were hsct recipients with 33% of these developing pneumonia [131] . a study from spain reported on four hsct recipients who developed enterovirus infections, with a mortality rate of 75% (3 of 4) [146] . these findings demonstrate that immunocompromised patients may be at risk for lower respiratory tract infections and death from an enterovirus infection [3, 131, 146] . most enteroviruses can be isolated in cell cultures from nasopharyngeal or throat swabs. pcr-based testing is used to amplify the viral rna from throat swabs, cerebrospinal fluid, and tissues. for diagnosis, isolation of the virus from the throat is more clinically significant than from stool because this virus has a shorter duration of shedding from the throat. most enterovirus infections are self-limiting and do not require specific treatment. intensive care may be required for central nervous system, cardiac, and hepatic infections. ivigs have been used in some patients with severe infections, but no specific antiviral therapies are available [3, 147] . general infection control measures must be undertaken around patients with enterovirus infections. special attention must be paid to hand hygiene. material in contact with or soiled by feces should be handled carefully and discarded with proper precautions. the respiratory viruses as a group are the most common cause of an acute infectious illness in developed societies. the variety of viruses that can cause infection and illness in all age groups and their presence in high frequencies throughout the year describe the risk of exposure of all persons at all times including immunocompromised persons residing in the community. the immunocompromised state of many cancer patients constitutes the basis for the frequent failure of the host to promote a normal and rapid recovery from an acute respiratory viral infection and results in a more severe and prolonged infection that causes significant morbidity and mortality in these patients. those respiratory viruses that are most prevalent and most prone to produce lower respiratory illnesses and pneumonia in healthy hosts, rsv, influenza viruses and piv, are those most likely to cause severe illness and pneumonia leading to hospitalization in immunocompromised persons. however, viruses less prone to produce a lower respiratory illness but that are highly prevalent, such as rhinoviruses, may frequently be associated with severe illness. although not generally considered respiratory viruses, the herpes viruses are known to produce respiratory infection and disease, sometimes severe, in immunocompromised patients (table 32 .1). a historically important virus, measles virus, is now rarely encountered because of widespread vaccination. the limited availability of antivirals and vaccines for the acute respiratory viruses means that these infections will continue to be important for many years and dictate a need for utilizing infection control procedures as much as possible, particularly in hospitals and institutions, so as to minimize spread. efforts to develop specific vaccines are important as their use could prevent as well as reduce exposure of cancer patients to these viruses. development of specific antivirals is important for use in immunocompromised patients as normal recovery mechanisms may be seriously impaired. community respiratory virus infections in immunocompromised patients with cancer common community respiratory viruses in patients with cancer: more than just "common colds harrison's principles of internal medicine circulation patterns of genetically distinct group a and b strains of human respiratory syncytial virus in a community respiratory syncytial virus and parainfluenza virus risk of primary infection and reinfection with respiratory syncytial virus 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recipient treated with intravenous ribavirin polymerase chain reactionbased detection of rhinovirus, respiratory syncytial virus, and coronavirus in otitis media with effusion persistent infection of neural cell lines by human coronaviruses identification of residues critical for the human coronavirus 229e receptor function of human aminopeptidase n human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses surveillance of communityacquired viral infections due to respiratory viruses in rhone-alpes (france) during winter 1994 to 1995 coronavirus pneumonia following autologous bone marrow transplantation for breast cancer genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients severe acute respiratory syndrome (sars) in a liver transplant recipient and guidelines for donor sars screening community study using a polymerase chain reaction panel to determine the prevalence of common respiratory viruses in asthmatic and nonasthmatic children typing of human enteroviruses by partial sequencing of vp1 enteroviral disease in the united states pulmonary enterovirus infections in stem cell transplant recipients successful treatment of echovirus meningoencephalitis and myositis-fasciitis with intravenous immune globulin therapy in a patient with x-linked agammaglobulinemia key: cord-010570-ytv7dwr0 authors: casadevall, arturo; scharff, matthew d. title: return to the past: the case for antibody-based therapies in infectious diseases date: 1995-07-17 journal: clin infect dis doi: 10.1093/clinids/21.1.150 sha: doc_id: 10570 cord_uid: ytv7dwr0 in the preantibiotic era, passive antibody administration (serum therapy) was useful for the treatment of many infectious diseases. the introduction of antimicrobial chemotherapy in the 1940s led to the rapid abandonment of many forms of passive antibody therapy. chemotherapy was more effective and less toxic than antibody therapy. in this last decade of the 20th century the efficacy of antimicrobial chemotherapy is diminishing because of the rapidly escalating number of immunocompromised individuals, the emergence of new pathogens, the reemergence of old pathogens, and widespread development of resistance to antimicrobial drugs. this diminishment in the effectiveness of chemotherapy has been paralleled by advances in monoclonal antibody technology that have made feasible the generation of human antibodies. this combination of factors makes passive antibody therapy an option worthy of serious consideration. we propose that for every pathogen there exists an antibody that will modify the infection to the benefit of the host. such antibodies are potential antimicrobial agents. antibody-based therapies have significant advantages and disadvantages relative to standard chemotherapy. the reintroduction of antibody-based therapy would require major changes in the practices of infectious disease specialists. the antibiotic era is about 60 years old. as we approach the 21st century, the progress made in controlling infections is threatened by the growing numbers of immunosuppressed individuals; the emergence of new pathogens; the reemergence of old pathogens; and the widespread resistance of organisms to antimicrobial drugs. each development poses its own set of challenges; in combination, these developments are already undermining the efficacy of antimicrobial chemotherapy. there is a worldwide epidemic of immunosuppression due to malnutrition, aids, medical therapies for cancer and autoimmune diseases, and organ transplantation. immunosuppressed hosts provide ecological niches for low-virulence microorganisms [1] . in addition, antimicrobial drugs are less effective and sometimes are unable to eradicate infection in individuals with impaired immunity. classical pathogens such as mycobacterium tuberculosis and treponema pallidum have reemerged and are difficult to treat in immunosuppressed individuals [2, 3] . the increasing frequency of drug-resistant organisms is an equally alarming development, and resistance may be developing faster than new antibiotics can be introduced [4] . for example, in one new york city hospital, the percentage of vancomycinsusceptible enterococcus faecium decreased from 86% to 26% in 2 years [5] . the problem of resistance is not limited to bacteria; there are numerous reports of fluconazole-resistant candida albicans [6] . we briefly review the use of antibody-based therapy in the early 20th century and make the case for reintroducing passive antibody administration for the treatment ofinfectious diseases. advances in monoclonal antibody (mab) technology provide new opportunities for developing antibody-based therapies. in the case of pathogens for which antimicrobial chemotherapy already exists, antibody-based therapies could be used with existing chemotherapy to improve outcome and possibly reduce the development of resistance. in the case of pathogens for which no effective chemotherapy exists, antibody-based therapies could be used for primary therapy. given the diminishing efficacy of existing antimicrobials because of widespread resistance and the difficulties of treating infections in immunosuppressed individuals, the reintroduction of antibody-based therapies is an option that should be given serious consideration. this reintroduction would require major changes in the practice of infectious disease medicine. the administration of immune serum (usually animal) for the treatment of infections was called serum therapy and was introduced in the 1890s for the treatment of diphtheria. by the 1930s, serum therapy was widely used for the treatment of table 1 . infectious diseases that were treated with antibody-based therapies in the preantibiotic era. bacterial and viral infections (table 1) . for example, in the late 1930s, 86% of patients with type i pneumococcal pneumonia admitted to boston city hospital were treated with type-specific serum [36] . from 1927 to 1932 at bellevue hospital (new york), > 3,000 patients with erysipelas were treated with serum [37] (figure 1). the efficacy of serum therapy varied with the type or severity of infection. several large controlled studies revealed that type-specific serum reduced the mortality ofpneumococcal pneumonia [7] . serum therapy also appears to have significantly reduced mortality due to meningococcal meningitis in some epidemics [7, 38] . serum therapy reduced mortality due to haemophilus irif/uenzae meningitis, but the effect was small [8] [9] [10] . serum therapy for erysipelas reduced mortality in comparison to historical controls [37, 39] . serum therapy reduced mortality in diphtheria, and antibody therapy continues to be used today to treat this disease [40] . the efficacy of serum therapy for whooping cough, anthrax, dysentery (shigella dysenteriae), and gas gangrene was uncertain. human convalescent serum was effective for prophylaxis ofmeasles, which had a mortality rate of 6%-7% in some populations [29] [30] [31] . the effectiveness of serum in the pre-paralytic stage of poliomyelitis was uncertain [31, 32] . no consistently effective sera were developed against many pathogens, including staphylococcus [41] , mycobacterium, and salmonella species [42] . the historical use of serum therapy provides lessons for the effective use and development of antibody-based therapies. accurate microbiological diagnosis was essential for successful treatment, and serum therapy was most effective when used for prophylaxis or for therapy early in the course of infection. note. this is not a complete list. [7] [7] [8] [9] [10] nevertheless, it was possible to treat established bacterial infections such as meningococcal meningitis and pneumococcal pneumonia if antibody was administered shortly after symptoms began [7] . antibodies functioned as direct antibacterial agents (e.g., pneumococcal antisera) or antitoxins (e.g., diphtherial antisera). nonphysiological and nontherapeutic animal models of infection allowed investigators to successfully identify clinically useful antibody reagents [7] . a considerable amount of basic immunologic and microbiological research was required to develop each serum, and many fundamental discoveries were made in the search for better sera. (the contributions of pneumococcal research to basic science are discussed in [43] .) sulfonamides were introduced in 1935 and rapidly became the standard therapy for many infections [44] . because antimicrobial chemotherapy had significant advantages over serum therapy, the latter was largely abandoned. systemic administration of animal sera caused fevers, chills, and allergic reactions [23, 45] ; "serum sickness," a self-limited syndrome characterized by rash, proteinuria, and arthralgias, occurred in 10%-50% of patients because of immune complex disease. in addition, strain typing was necessary for choosing pneumococcal antisera; there was significant lot-to-lot variation in serum efficacy [46] ; and dosing was based on clinical experience. inadequate dosage, delays in treatment, errors in typing, mixed infections, and complicated infections (e.g., empyema) could result in failure of serum therapy [47] . serum was expensive because of the costs of animal husbandry, antibody purification, refrigeration, and reliance on mouse protection tests for standardization. chemotherapy was less toxic and more effective than serum therapy. treatment with type-specific serum reduced the mortality of pneumococcal pneumonia from 30%-40% to 10%-20% [7] , whereas the mortality rate among patients treated with sulfonamide was 7% [48] . serum therapy reduced the mortality rate of erysipelas from 10% to 7% [37] , whereas the mortality rate for patients treated with sulfonamides was 1%-2% [49] . for meningococcal meningitis, the efficacy of serum depended on the epidemic. in the mid-1930s, waghelstein [50] reported that the mortality rate for patients with meningococcal meningitis who were treated at sydenham hospital (baltimore, md) with sulfonamide was 15.3% and for those treated with serum it was 27%. in controlled trials, the superiority of chemotherapy over serum therapy was lessevident. for example, the mortality among patients with meningococcal meningitis who were treated early with adequate serum therapy was 16.7% vs. 11.6% for sulfanilamide therapy [50] . the mortality among patients with pneumococcal pneumonia treated with antimicrobial drugs or serum was 11% and 16.7%, respectively, but there was no difference in the mortality rates among younger .. literature sent on request. is used at bellevue hospital. it is supplied in packages of one therapeutic dose of approximately ten cubic centimeters. the data cited were published in [37] . patients, and younger patients who received serum recovered faster than those who received antimicrobials [51] . in the early days of the antibiotic era there was interest in using combination therapies with antibiotics and serum. sulfonamides and serum had a synergistic or additive effect against streptococcus pneumoniae [52] [53] [54] [55] , streptococci [56] , and meningococci [57] . the finding that sulfonamides made pneumococci more susceptible to antibody-mediated phagocytosis supported the idea of combination therapy [58] . it was similarly recognized that sulfonamides, unlike antibody, did not neutralize bacterial toxins or modify the course of disease caused by diphtheria or tetanus toxins [59] . combination therapy was recommended for scarlet fever [60] , pneumococcal pneumonia [44, 54, 61] , whooping cough [20] and meningitis due to pneumococcus [8, 62] , meningococcus [8, 50, 63, 64] , or h. influenzae [8] . however, the side effects of serum made the potential benefits of combination therapy marginal [50] . in summary, serum therapy was abandoned because of toxicity, difficulty in administration, narrow specificity, lot-to-lot variation, and expense. chemotherapy was less toxic and easier to use, lots were more consistent, and it was more effective in eradicating infection. in addition, there was no need for strain typing with chemotherapy. however, it is ironic that the broad antimicrobial activity of these drugs used for chemotherapy might have contributed to widespread resistance, and today susceptibility testing is essential to the selection of appropriate antimicrobial drugs. although antibodies are no longer used directly as antibacterial drugs, they continue to be used in infectious diseases (for recent reviews see [65] [66] [67] ). the majority of antibody preparations are now human. antibody administration reduces infection in individuals with immunodeficiencies [65] [66] [67] [68] and is effective for postexposure prophylaxis against measles, hepatitis a and b, rabies, and varicella viruses. specific antibody is used for the treatment of botulism [22] , diphtheria [40] , tetanus [22] , snake bites [69] , and spider stings [70] . passive antibody administration has been used for prophylaxis and/or therapy of several viral infections, including cytomegalovirus (cmv) [71, 72] , rotavirus [73] , lassa virus [74] , varicella [75] , enterovirus [76, 77] , and parvovirus b19 infections [78] . in recent years, there has been renewed interest in the use of antibody preparations to prevent infection in high-risk groups. intravenous administration of polyclonal antibody has been reported to reduce the number of infections in patients with aids [79] [80] [81] , patients in surgical intensive care units [82] , organ transplant recipients [83] , and neonates [84, 85] (for critical reviews and commentary on this practice see [86] [87] [88] [89] ). antibody therapy is used for a variety of illnesses that may or may not be infectious, including autoimmune thrombocytopenia, kawasaki disease, and autoimmune neuromuscular diseases [65, 66] . passive antibody administration is used for prevention of rh-hemolytic disease [90] . equine lymphocyte antiserum [91] and murine mabs to lymphocytes are used to prevent organ rejection [92] . digitalis-binding antibodies are useful for the treatment of digoxin toxicity [93] . thus, antibody therapy is still widely used in medicine, but its role in the treatment of infections is limited largely to viral and toxin neutralization and replacement therapy in patients with immunoglobulin deficiencies. in reconsidering the antibody option it is clear that heterologous antibody (i.e., serum therapy) is a poor choice because of toxicity. human immune sera have fewer side effects, but there are concerns about availability, potency, and consistency. a major disadvantage of all immune sera is that specific antibodies make up a small minority of the antibody present. there is significant variation in pathogen-specific opsonic activity of commercially available immunoglobulin preparations [94] . there is also concern about the potential of human sera to transmit infectious agents [95] . the discovery of hybridoma technology in 1975 [96] and recent advances in mab technology, including the generation of humanized antibodies [97] , make mab-based therapies a more attractive therapeutic option. in contrast to polyclonal sera, mabs are homogeneous and reproducible reagents that can be generated in large amounts. generation of mabs from mice and rats is still easier and more efficient than production of human mabs. however, several technologies are available for the humanization of murine mabs and generation of human mabs [97] . mouse-human chimeric antibodies can be constructed by linking the genes expressing the mouse variable region to human constant region genes [97] . the result is a molecule that is mostly human and has a longer half-life than the murine precursor [98] . as an alternative, the antigen binding regions of murine mabs can be grafted into human antibody frameworks by molecular techniques [97] , resulting in molecules that are almost completely human in origin. other strategies to generate human antibodies are the transformation of human b cells, the generation ofrecombinant antibody libraries from human b cells, and the use of transgenic mice that have the human immunoglobulin locus. recent setbacks for mab-based therapies: historical perspective clinical trials of antiendotoxin mabs for gram-negative sepsis have produced inconclusive results [99, 100] . when the difficulties encountered in developing antiendotoxin mab strategies are considered in the context of the development of serum therapy, they do not seem unusual or unexpected. for example, passive protection with pneumococcal antisera was demonstrated in 1891 by the klemperers [101] , but 30-40 years elapsed before serum therapy for pneumococcal pneumonia was widely accepted. for pneumococcal antiserum therapy to be consistently successful, several developments were necessary. these included the appreciation of antigenic variation in pneumococci [102] , the need for type-specific sera [103] , the development of rapid in vitro assays to establish antigenic type (i.e., capsular swelling and agglutination reactions) [104] , the development of methods to standardize potency (mouse protection test) [46] , and improved purification techniques that would reduce the toxicity of serum. for meningococcal antisera the situation was reversed; flexner's serum significantly reduced mortality in early epidemics of meningitis [38] but was subsequently less effective, possibly as a result of antigenic changes in neisseria meningitidis [7] . research into better meningococcal antisera was then hampered by the lack of useful animal models, loss of strain virulence in vitro, and poor understanding of the antigenic diversity of n meningitidis [105, 106] . a lesson from the preantibiotic era is that a considerable amount of basic research on the immunology and pathogenesis of each infection is usually necessary before antibody therapy can be developed that will be successful in clinical practice. spectrum ofactivity. antibodies can modify bacterial, fungal, parasitic, and viral infections and are a class of biological agents with broad antimicrobial activity against diverse pathogens. antibody-based therapies are usually pathogen-specific and have the theoretical advantage that they should not affect the normal flora of the patient or select for resistance in nontargeted microbes. nevertheless, narrow specificity is a disadvantage because mixed infections may not be treated by a single antibody preparation. pneumonia due to s. pneumoniae with more than one serotype was recognized as a reason for the failure of type-specific serum therapy [107] . one solution is to use cocktails of mabs active against common antigenic types. cocktails may be designed to include mabs to multiple serotypes and mabs with multiple isotypes to enhance antibody effector function. pathogen-specific drugs (such as mabs) are less attractive to the pharmaceutical industry because the narrow specificity reduces the potential market for the drug. however, the increasing incidence of resistant organisms resulting from the use of broad-spectrum antibiotic regimens could make pathogen-specific drugs attractive to drug companies. precedents for the development of pathogen-specific drugs already exist in the area of antiviral drug research. mechanisms ofaction. antibodies mediate protection by a variety of mechanisms. direct antibody mechanisms of action include inhibition of attachment, agglutination (and immobili-cm 1995;21 (july) zation), viral neutralization, toxin neutralization, antibodydirected cellular cytotoxicity, complement activation, and opsonization [108] . antibody therapy has the potential to enhance immune function in immunosuppressed hosts. however, since the mechanism of action of many antibodies involves promoting microbial clearance through nonspecific cellular immunity, antibody-based therapies may be less effective in individuals with defective macrophage, neutrophil, and natural killer cell function. in this regard, it is encouraging that antibodies are effective against pseudomonas aeruginosa in neutropenic mice [109] and that they can reduce the number of infections in pediatric patients with aids [79] . antibodies are usually considered to be "protective" effector molecules of the immune system. however, not all antibody responses to pathogens are protective and some may be deleterious to the host. for example, some viral-specific antibodies are capable of enhancing infection [110] . thus, antibody molecules being considered for clinical development will require extensive testing in vitro and in vivo. studies of the mechanisms of antibody action are important for understanding the mode of protection and for designing clinical trials. pharmacokinetics. the pharmacokinetics of human igg suggests several useful characteristics for its role as an antiinfective agent. these include a long half-life (~20 days [111]), good tissue penetration [112] , and, depending on the isotype, the ability to either cross the placenta to provide antibody protection to fetuses and newborns [113] or to be excluded from the placenta if fetal toxicity is a concern. heterologous mabs (i.e., murine mabs) have shorter half-lives in humans, but chimeric and humanized mabs have longer half-lives than their murine precursors. mabs with a longer half-life can, in principle, be engineered by altering the regions of the constant domain that regulate clearance. a disadvantage of antibody-based therapies is the need for systemic administration. oral administration is unlikely to be effective, with the possible exception of therapy for enteric pathogens [73, 114] . the blood-brain barrier is a potential obstacle for antibody therapy for infections of the brain. however, in infections of the brain in which there is inflammation, there is increased antibody penetration, and intravenous administration of antibodies appears to have been successfully used for therapy of meningococcal meningitis [115] . antibodybased therapies can also be administered directly into the subarachnoid space, as has been done for the treatment ofmeningococcal and h. influenzae meningitis [8, 38] . enhanced penetration of the brain can be achieved by modifying the charge of the molecule [116] or by linking it to carrier proteins that cross the blood-brain barrier [117] . antibody therapy for brain infections may be feasible if the blood-brain barrier is leaky, if antibody is administered directly into the subarachnoid space, or if antibodies with greater brain penetration are used. additional research into the pharmacokinetics of antibodies in infection is likely to be required for optimal use of antibody-based therapies. studies of antibody binding to tumors have revealed complex pharmacokinetics [112] . there may be problems with antibody penetration into abscesses as have been found with tumors. toxicity. immunoglobulins are generally safe drugs [67] . nevertheless, serious side effects have been reported with highdose (0.5-2 g/kg) antibody therapy, including rare cases of renal failure [118] , aseptic meningitis [119] , and thromboembolic events [120, 121] . high-dose immunoglobulin therapy is unlikely to be required in antiinfective mab therapy. in the past, serum therapy was effective against various pathogens despite the fact that immune sera contained only small amounts of specific antibody. mabs have significantly higher specific activity than polyclonal preparations. for example, 0.7 mg of two anti-tetanus toxin human mabs provides the same activity as 100-170 mg of immune globulin [122] . thus, the toxicities described for high-dose immunoglobulin therapy may not be relevant in antiinfective therapies with mabs. until fully human mabs are available, rodent, mousehuman chimeric, or humanized mabs are therapeutic options. for over a decade, murine mabs directed against t cells have been used to prevent organ graft rejection in humans [92] . a murine igm to endotoxin (e5) was tested in 247 patients with sepsis and appeared to be a safe treatment [123] . although administration of murine mabs is generally well tolerated in humans, allergic reactions occur in 1%-2% of patients [123] and most patients develop antibody responses to the murine mabs that may interfere with their therapeutic function [43, 124] . experience with human-mouse chimeric antibodies and humanized mabs is accumulating rapidly as clinical trials with several compounds progress. a chimeric anti-cd4 mab for the treatment of rheumatoid arthritis has been well tolerated [125] , but approximately half the patients had infusion-related side effects (headaches, nausea, fever, and chills), which were diminished by slowing the infusion rate [125] . mouse-human chimeric and humanized mabs are less immunogenic than their murine precursors [98, 126] , but antiidiotypic responses occur after repeated treatments [127] . overall, the experience with chimeric and humanized mabs suggests that they are relatively safe compounds [98, [125] [126] [127] [128] . antigenic variation and antibody resistance. the efficacy of antibody-based therapies may be diminished by antigenic changes in the pathogen. this could be minimized by antigenic surveillance systems, as is presently done for influenza virus. antibody-resistant mutants can be generated in the laboratory [129] , and it is likely that the same can occur in patients. mechanisms of antibody resistance include mutations that change the antigenic site and protease production. antibody use may select for antigenically distinct variants. evidence for the horizontal transfer of iga protease genes exists in neisseria gonorrhoeae and it is conceivable that widespread antibody use would result in selection of protease-producing strains for many pathogens [130] . thus, large-scale use of antibody-based therapies may result in rapid emergence of antibody resistance in a manner analogous to that of antibiotic resistance. however, one advantage of antibody-based therapies is the versatility of antibody compounds. for example, emergence of an antibodyresistant strain could be countered by a new antibody directed toward the mutated epitope or another antigenic target. emergence of protease-producing strains could be countered by designing antibodies without protease cleavage sites (although these may then be recognized as foreign and be immunogenic) or by addition of antiprotease mabs. selection of antibodyresistant organisms could be minimized by using cocktails of mabs directed at multiple antigenic targets or by using a combination of antibodies and antimicrobials. cost. cost effectiveness is a significant concern that is likely to be a major obstacle for the development of passive antibody therapies. antibody prophylaxis for cmv infections can cost $4,000 to $9,000 per patient [71] . the cost effectiveness of antibody therapy for prevention of infection in leukemic patients has been questioned [88, 131] . however, in selected populations such as premature infants, antibody therapy may be cost effective [85] . furthermore, the cost of a drug when first introduced may not reflect its long-term costs given the potential for improvements in technology and production. mabs are presently made in tissue culture, so the cost of production is high. mabs can be made in bacteria or yeast, and less costly means of production might be developed [97] . in the past, serum therapy was used despite its high cost because it was believed to be effective. new antibody-based therapies are likely to be used if they prove to be effective. for pathogens such as s. pneumoniae, n. meningitidis, and h infiuenzae, there is a large body of experimental and clinical evidence to support the development of passive antibody therapy. however, for many pathogens, antibody immunity has not been proven to be important. for example, cryptococcus neoformans is a fungus for which the importance of antibody immunity is uncertain. however, administration of preformed antibodies can modify the course of cryptococcal infection in various animal models [132] [133] [134] [135] [136] , and the combination of antibody and amphotericin b is a more effective treatment than the use of either agent alone [137] [138] [139] . studies with mabs have shown that there are protective, nonprotective, and deleterious antibodies to the c. neoformans capsule polysaccharide [136] . heavy chain isotype [136, 140] and epitope specificity [141] are important determinants of protective efficacy. the existence of protective and nonprotective antibodies against c. neoformans, a fungus for which antibody immunity mayor may not be important, provides a paradigm that may be applicable to other pathogens. we propose the hypothesis that for every pathogen there exists an antibody that will modify the course of infection to the benefit of the host; such antibodies are candidates for development as antimicrobial drugs. our proposal includes the use of antibodies for intracellular pathogens; for example, anti-bodies may exist that could prevent cell entry of intracellular bacteria and/or shift the intracellular location of such bacteria by promoting internalization through fe receptors. an mab that inhibits intracellular toxoplasma gondii infection has recently been described [142] . in addition, antibodies have been described that can neutralize viruses intracellularly [143] or penetrate the nuclear membrane [144] . our proposal is not limited to those pathogens for which antibody immunity has been demonstrated to be protective. conclusions on the importance of antibody immunity are usually based on observations made with polyclonal sera (i.e., passive protection or correlation of immunity with the presence of antibody). since polyclonal sera might contain protective antibodies, nonprotective antibodies, and disease-enhancing antibodies, the existence of protective antibodies cannot be ruled out on the basis of absence of protection in experiments involving passive transfer of polyclonal sera or immunizations. conversely, experiments that demonstrate that polyclonal sera can mediate protection indicate the existence of protective antibodies. for pathogens for which polyclonal antibody has shown no protection, the question of whether useful antibodies exist must be reevaluated with mabs since the presence of nonprotective or deleterious antibodies in polyclonal sera could create confounding variables. a corollary of our proposal is that antibody-based therapies may be developed against pathogens for which antibody immunity is not considered to be important. infections that are difficult to treat and that can be modified by antibody immunity provide a logical starting point for the development of antibody-based therapies. antibodies have historically been more effective in prophylaxis than in therapy. antibody-based therapies have traditionally been most effective in infections where viral and toxin neutralization modifies the course of the disease. however, most of the historical experience was gained with polyclonal preparations of uncertain composition and mayor may not be applicable to mab preparations with high activity. opportunistic infections. infections with low-virulence organisms in immunosuppressed individuals are often difficult to treat and are sometimes incurable. since the problem is deficient immunity, antibody therapy is an attractive option because antibodies can enhance immune function. table 2 lists examples of opportunistic pathogens for which antibody can modify the course of infection. antibiotic resistance. drug-resistant organisms are targets for development of antibody-based therapies. the spread of penicillin-resistant s.pneumoniae, methicillin-resistant staphylococcus aureus, and multiply-drug-resistant e. faecium has limited the useful antibiotic arsenal against these pathogens [4, 6, 153] . for pneumococcus, antibody therapy has been shown to be useful [7] . antibody may be useful against staphylococcal infection [154, 155] . for e.faecium, the role of antibody immu-ern 1995;21 (july) table 2 . opportunistic pathogens for which experimental data suggest a potential role for antibody-based therapies. nity is uncertain, but some patients make opsonic antibodies, which might be protective [156] . significant work has already been done in the development of pseudomonal antibodies for resistant gram-negative organisms (table 2). the addition of antibody-based therapies to chemotherapy may slow the development of resistance and improve outcome. toxin neutralization. the ability to neutralize toxins is a unique characteristic ofantibody-based therapies. antitoxin antibodies are useful for the treatment of diphtheria, tetanus, botulism, snake bites, and black widow bites (see above). recent attempts to develop antibody therapy for gram-negative sepsis have focused on mabs to neutralize endotoxin [123, 157, 158] and cytokines [159] . pseudomonas aeruginosa sepsis, combination therapy with mabs to endotoxin and tumor necrosis factor was superior to therapy with a single mab [160] . examples ofpotential targets for antibody-based therapy include the toxins and proteases associated with toxic shock syndromes [161, 162] . combination therapy with antibodies to exotoxins could improve outcome, but cocktails of mabs may be necessary because of toxin heterogeneity [162] . intravenous immunoglobulin administration has been associated with dramatic clinical improvement in a patient with streptococcus pyogenes toxic shock syndrome [163] . antivirals. virus neutralization is an established property of antibodies. in the preantibiotic era, serum was used for prophylaxis ofmeasles, chickenpox, mumps, and poliomyelitis. recently, the potential of antibody therapy against many viruses, including hepatitis b [164] , hiv [165] , respiratory syncytial virus [166] , cmv [150] , and parvovirus [167] , has received considerable attention. newly identified viral illnesses are targets for antibody drug research. combination ofantibody therapy and chemotherapy. antibody-based therapies are unlikely to be used alone unless they are the only available therapy or are being used for prophylaxis of infection. combinations of antibody therapy and chemotherapy offer theoretical advantages. antibodies promote microbial killing directly by a complement-mediated lytic process or indirectly by enhancing nonspecific immune mechanisms. table 3 lists examples of bacterial, fungal, and viral pathogens for which combination therapy has shown promise. combination therapy could reduce the amount of either agent required to table 3 . pathogens for which the combination of chemotherapy and antibody based therapy has shown some promise. pathogen chemotherapy antibody [57, 168] neisseria meningitidis sulfanilamide horse immune sera [55] streptococcus pneumoniae rabbit immune sera [8, 9] haemophilus infiuenzae sulfonamide rabbit, horse immune sera [169] staphylococcus aureus sparftoxacin human igg1 mab [172] p. aeruginosa human igm mab [173] p. aeruginosa murine e5 lps antibodies [170] p. aeruginosa human gamma globulin [172] escherichia coli murine e5 lps antibodies [174] shistosoma mansoni praziquantel rabbit immune sera [175] candida albicans amphotericin b human gamma globulin [137] cryptococcus neoformans amphotericin b rabbit immune sera [138, 139] c. neoformans murine igg1 mab [176] c. neoformans murine igg1 mab [177] lassa virus ribavirin monkey immune sera [72, 178] cytomegalovirus ganciclovir murine immune sera [179] herpes simplex virus adenine arabinoside rabbit and human sera [180] herpes simplex virus acycloguanosine human immune globulin note. this is not a complete list. achieve a therapeutic effect. for example, some antibiotics are quite toxic, and the ability to reduce doses could lessen side effects. in a similar vein, the addition of chemotherapy to antibody therapy could reduce the amount of antibody necessary to achieve a therapeutic effect, which would lessen the cost of therapy. the availability of broad-spectrum antibiotics has diminished the need for making exact microbiological diagnoses. for example, gram-negative sepsis or presumed fungal infections can be treated with empirical therapy without identifying the pathogen. this situation is in contrast to the practice of infectious diseases in the 1930s when identification of the pathogen (and its serotype) was necessary for choosing the correct antiserum. for antigenically diverse pathogens such as s. pneumoniae, rapid protocols were developed for typing strains. the present practice of infectious diseases is not unlike a gambling strategy where the probability of infection by a given microbe is matched to the likelihood of activity by the available antibiotics. this has resulted in great emphasis on broad-spectrum antibiotic "coverage" and less emphasis on making an exact microbiological diagnosis. although the relative merits of this practice are beyond the scope of this article, widespread antimicrobial resistance is decreasing the effectiveness of existing antibiotics, and increased caution is warranted when designing broad-spectrum combinations. a 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monoclonal antibody for acute rejection of cadaveric renal transplants treatment of life-threatening digitalis intoxication with digoxin-specific fab antibody fragments: experience in 26 cases opsonic activity of commercially available standard intravenous immunoglobulin preparations human immunoglobulins for intravenous use and hepatitis c viral transmission continuous cultures of fused cells secreting antibody of predefined specificity genetically engineered antibodies: progress and prospects mouse/human chimeric monoclonal antibody in man: kinetics and immune response anti-endotoxin monoclonal antibodies introduction of new technology into critical care practice: a history ofha-la human monoclonal antibody against endotoxin versuche tiber immunisirung und heilung bei der pneumokokkeninfektion a biologic classification of pneumococci by means of immunity reactions weitere untersuchungen uber pneumokokken-heilsera. arb a d k the reliability of sputum typing and its relation to serum therapy the meningococcus (neisseria intracellularis) serums, antitoxin, and drugs in the treatment of meningococcus meningitis the management of the pneumonias principles and practice of infectious diseases therapeutic effects of a human antiflagella monoclonal antibody in a neutropenic murine model of pseudomonas aeruginosa pneumonia immune enhancement of viral infection metabolism of immunoglobulins immunologic and pharmacologic concepts of monoclonal antibodies placental transfer of human igg subclasses treatment of chronic cryptosporidial infection with orally administered human serum immune globulin intravenous treatment of meningococcic meningitis with meningococcus antitoxin blood-brain barrier transport of cationized immunoglobulin g: enhanced delivery compared to native protein anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier acute renal failure after intravenous immunoglobulin therapy aseptic meningitis associated with high-dose intravenous immunoglobulin therapy: frequency and risk factors high-dose weekly intravenous immunoglobulin to prevent infections in patients undergoing autologous bone marrow transplantation or severe myelosuppressive therapy: a study of the american bone marrow transplant group high-dose intravenous immunoglobulin and serum viscosity: risk of precipitating thromboembolic events immunotherapy with human monoclonal antibodies: fragment a specificity of polyclonal and monoclonal antibodies is crucial for full protection against tetanus toxin a controlled clinical trial of e5 murine monoclonal igm antibody to endotoxin in the treatment of gram-negative sepsis human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy chimeric cd4 monoclonal antibody cm-t412 as a therapeutic approach to rheumatoid arthritis human mouse chimeric cd7 monoclonal antibody (sdzchh380) for the prophylaxis of kidney transplant rejection humanised monoclonal antibody therapy for rheumatoid arthritis remission induction in non-hodgkin lymphoma with reshaped human monoclonal antibody campath-lh antibody-resistant mutants of borrelia burgdorferi: in vitro selection and characterization mosaic-like organization ofiga protease genes in neisseria gonorrhoeae generated by horizontal genetic exchange in vivo cost effectiveness of prophylactic intravenous immune globulin in chronic lymphocytic leukemia passive immunization in murine cryptococcosis passive immunization against cryptococcus neoformans protection of mice against experimental cryptococcosis by anti-cryptococcus neoformans monoclonal antibody passive immunization against cryptococcus neoformans with an isotype-switch family of monoclonal antibodies reactive with cryptococcal polysaccharide protective murine monoclonal antibodies to cryptococcus neoformans serum protein enhancement of antibiotic therapy in cryptococcosis improved amphotericin b activity by a monoclonal anti-cryptococcus neoformans antibody: study during murine cryptococco sis and mechanisms of action therapeutic efficacy of monoclonal antibodies to cryptococcus neoformans glueuronoxylomannan alone and in combination with amphotericin b isotype switching from igg3 to igg 1 converts a non-protective murine antibody to c. neoformans into a protective antibody protective and nonprotective monoclonal antibodies to cryptococcus neoformans originating from one b cell toxoplasma gondii: a monoclonal antibody that inhibits intracellular replication intracellular neutralization of virus by immunoglobulin a antibodies a subgroup of murine monoclonal anti-deoxyribonucleic acid antibodies traverse the cytoplasm and enter the nucleus in a time-and temperature-dependent manner passive immunoprophylaxis with specific monoclonal antibody confers partial protection against pneumocystis carinii pneumonitis in animal models anti-cryptosporidium parvum antibodies inhibit infectivity in vitro and in vivo production, characterization, and antibody specificity of a mouse monoclonal antibody reactive with cryptococcus neoformans capsular polysaccharide autoantibody to heat-shock protein 90 can mediate protection against systemic candidosis mills 1. prevention and treatment of experimental herpes simplex virus encephalitis with human immune serum globulin human monoclonal antibodies neutralizing cytomegalovirus (cmv) for prophylaxis of cmv disease: report of a phase i trial in bone marrow transplant recipients polyclonal and monoclonal antibody therapy for experimental pseudomonas aeruginosa pneumonia vitro and in vivo activity of polyclonal and monoclonal human immunoglobulins g, m, and a against pseudomonas aeruginosa lipopolysaccharide confronting drug-resistant pneumococci immunoglobulin g enhances c3 degradation on coagulase-negative staphylococci passive local immunotherapy of experimental staphylococcal pneumonia with human intravenous immunoglobulin roles of antibodies and complement in phagocytic killing of enterococci treatment of gram-negative bacteremia and shock with human antiserum to a mutant escherichia coli phase i study of a murine monoclonal anti-lipid a antibody in bacteremic and nonbacteremic patients therapy with antibody to tumor necrosis factor in sepsis the efficacy of combination immunotherapy in experimental pseudomonas sepsis microbiology of toxic shock syndrome: overview association of phenotypic and genotypic characteristics of invasive streptococcus pyogenes isolates with clinical components of streptococcal toxic shock syndrome intravenous immunoglobulin therapy for toxic shock syndrome human combinatorial antibody libraries to hepatitis b surface antigen passive immunization for the prevention and treatment ofhiv infection human monoclonal fab fragments derived from a combinatorial library bind to respiratory syncytial virus f glycoprotein and neutralize infectivity efficacy of human immunoglobulin and penicillin g in treatment of experimental group b streptococcal infection protection of mice against meningococcus infection by polyvalent antimeningococcic serum combined protective action of human gamma globulin and antibiotics when administered simultaneously in experimental staphylococcal infections synergism between human gamma globulin and chioroamphenicol in the treatment of experimental bacterial infections combined serum and sulphanilamide in the treatment of streptococcal infections in mice monoclonal antibodies for treatment of gram-negative infections efficacy of anti-endotoxin monoclonal antibody e5 alone or in combination with ciprofloxacin in neutropenic rats with pseudomonas sepsis praziquantelinduced exposure of schistosoma mansoni alkaline phosphatase: drugantibody synergy which acts preferentially against female worms. parasite immuno11994 effects of immunoglobulin g and low-dose amphotericin b on candida albicans infections in burned mice combination of 5-flucytosine and capsule-binding monoclonal antibody in therapy of cryptococcus neoformans infection enhanced treatment of lassa fever by immune plasma combined with ribavirin in cynomolgus monkeys -3-dihydroxy-2-propoxymethyl) guanine prevents death but not immunity in murine cytomegalovirus-infected normal and immunosuppressed balb/c mice combined effects of acycloguanosine and humoral antibodies in experimental encephalitis due to herpesvirus hominis synergistic antiviral effects of adenine arabinoside and humoral antibodies in experimental encephalitis due to herpesvirus hominis unequalled but unobtainable key: cord-022176-hprwqi4n authors: löscher, thomas; prüfer-krämer, luise title: emerging and re-emerging infectious diseases date: 2009-07-28 journal: modern infectious disease epidemiology doi: 10.1007/978-0-387-93835-6_3 sha: doc_id: 22176 cord_uid: hprwqi4n emerging infectious diseases (eids) are characterized by a new or an increased occurrence within the last few decades. they include the following categories emerging diagnosis of infectious diseases: old diseases that are newly classified as infectious diseases because of the discovery of a responsible infectious agent. europe including great britain as well as in india, china, and japan. emerging vector-borne disease events concentrated in densely populated subtropical and tropical regions mostly in india, indonesia, china, sub-saharan africa, and central america (see figs. 3.3, 3.4 , and 3.5). the identification of new infectious agents in old diseases with unknown etiology is still the basis in many epidemiological studies. such newly detected bacteria and viruses in the last few decades are listed in table 3 .1. since the detection of helicobacter pylori in 1983, this infection has been identified as the causative agent in 90% of b-gastritis cases. the risk of duodenal ulcer is increased by 4-25-fold in patients with helicobacter-associated gastritis. who declared h. pylori as a carcinogen of first order because of its potential to enhance the risk of stomach carcinoma and malt lymphoma in long-term infection. in highprevalence regions for h. pylori, the frequency of stomach carcinoma is significantly higher compared to low-endemic areas (correa et al. 1990 ). the identification of h. pylori facilitates curative treatment of most associated diseases in individuals. but the most important epidemiological effect on associated diseases is attributed to increased hygienic standards in industrialized countries with a substantial reduction of h. pylori prevalences in younger age cohorts. transmission of h. pylori occurs mainly in childhood. in western developed countries the overall prevalence is around 30%, higher in older age groups due to a cohort effect, and this increases with low socioeconomic status (rothenbacher et al. 1989 ). in countries with low hygienic standards the prevalences are still high in younger age groups and reach 90% in developing countries. in developed countries, migrant subpopulations from less-developed regions show significantly higher prevalences in comparison to the nonmigrant population (mégraud 1993 ). since the early 20th century, a characteristic expanding skin lesion, erythema migrans (em), and an arthritis associated with previous tick bites were known. borrelia for many decades. increased outdoor activities facilitated contacts between humans and ticks in the 1970s and the 1980s and increased transmission of borrelia to humans at the northeastern coast of north america, leading to the discovery of borrelia burgdorferi in 1981 by willy burgdorfer. three different stages of the disease that describe the stage of infection and the involvement of different organ systems are known: stage 1, early localized infection; stage 2, early but disseminated infection; and stage 3, late stage with persistent infection. lyme disease is endemic at the east coast and in minnesota in the united states, in eastern and central europe, and russia. seroprevalence rates that reflect about 50% of nonclinical infections vary between 2 and 18% in the general population in germany (hassler et al. 1992; weiland et al. 1992) . in high-risk groups like forest workers in germany the prevalences reach 25-29% (robert koch institute 2001a). in ticks (ixodes) the prevalences are between 2 and 30% depending on the geographical area and the testing method used [immunofluorescence test, ift and polymerase chain reaction (pcr)]. in most studies the main risk factors of infection are age (children: 4-9 years, adults 35-60 years), outdoor activities, skin contacts with bushes and grass, and the presence of ticks in domestic animals (robert koch institute 2001b) . the probability of infection (seroconversion) after a tick bite in germany is 3-6% and the probability of a clinical disease is 0.3-1.4%. the probability that the bite of an infectious tick leads to infection in the host is 20-30%. this depends on the time duration that the tick is feeding on the human body. since the detection of the etiologic infectious agent and the subsequent development of laboratory diagnostic tests in the 1980s, the number of reported cases of lyme disease has increased from 0 to 16,000 per year, indicating that it is an "emerging diagnosis." the reported numbers vary depending on the reproduction of the hosting rodents for ticks as well as the contacts between humans and nature (spach et al. 1993) . ticks may live for several years and their survival, reproduction rate, and activity are directly affected by changes in seasonal climate through induced changes in vegetation zones and biodiversity, hence causing local alterations of the tick's habitat and in the occurrence of animals that are carriers of different pathogens (like small rodents). several studies in europe have shown that in recent decades the tick ixodes ricinus, transmitting lyme borreliosis and tick-borne encephalitis (tbe), has spread into higher latitudes (e.g., sweden) and altitudes (e.g., czech republic, austria), and has become more abundant in many places. such variations have been shown to be associated with recent variations in climate. as a result, new risk areas of both diseases have recently been reported from the czech republic. climate change in europe seems likely to facilitate the spread of lyme borreliosis and tbe into higher latitudes and altitudes, and to contribute to extended and more intense transmission seasons. currently, the most effective adaptive strategies available are tbe vaccination of risk populations and preventive information to the general public (danielova et al. 2004; lindgren et al. 2006; materna et al. 2005 ). an effective vaccine was licensed for b. burgdorferi in 1999. in europe, where different variants of borrelia are present (mostly b. afzelii and b. garinii), this vaccine is not protective. trivalent vaccines for europe are in clinical trials. in recent years, norovirus infections are increasingly recognized as the cause of large outbreaks of diarrheal diseases in the general population, school classes, nursing homes, hospitals, and cruise ships in western countries with peaks in colder seasons (winter epidemics) (centers of disease control 2006; verhoef et al. 2008; robert koch institute 2008a) . this is a typical example for emerging diagnosis due to increasing availability of routine pcr testing for these viruses in stool samples. noroviruses (family caliciviridae) are a group of related, single-stranded rna viruses first described in an outbreak of gastroenteritis in a school at norwalk, ohio, in 1968. five genogroups are known. immunity seems to be strain specific and lasts only for limited periods, so individuals are likely to get the infection repeatedly throughout their life. it is estimated that noroviruses are the cause of about 50% of all food-borne outbreaks of gastroenteritis. for several years there has been an ongoing epidemic in several european countries due to drift variants of a new genotype (gg ii.4jamboree) previously unknown to this nonimmune population (robert koch institute 2008a). as a result of an analysis of 232 outbreaks in the united states between 1997 and 2000, direct contamination of food by a food handler was the most common cause (57%), person-to-person transmission was less prevalent (16%), and even less frequently waterborne transmission could be proved (3%) (centers for disease control 2006). vomiting is a frequent symptom of norovirus enteritis and may result in infectious droplets or aerosols causing airborne or contact transmission. this may explain the difficulty to stop outbreaks in hospitals, nursing homes, and similar settings despite precautions to prevent fecal-oral transmission. also on cruise ships, person-to-person transmission is most likely in those closed settings, and drinking tap water is a risk factor as well (verhoef et al. 2008 ). searching for an agent which causes large outbreaks of enterically transmitted non-a hepatitis in asia and other parts of the world, the hepatitis e virus (hev) was first described in 1983 and cloned and sequenced in 1990 (reyes et al. 1990 ). meanwhile, hev has been shown to be a zoonotic virus circulating in pigs and other animals. it is implicated in about 50% of sporadic cases of acute hepatitis in developing countries and associated with a high case fatality rate in the third trimester of pregnancy (10-25%). hev is a major cause of large epidemics in asia, and to a lesser extent in africa and latin america, typically promoted through postmonsoon flooding with contamination of drinking water by human and animal feces. recent data show hev also to circulate in european countries and to be associated with severe and fatal disease not only during pregnancy but also in the elderly and in patients with chronic liver conditions. in patients with solid organ transplants, hev may even cause chronic hepatitis and liver cirrhosis (kamar et al. 2008) . a recombinant hev vaccine candidate has demonstrated a high protection rate of approximately 95% during clinical trials in nepal (shrestha et al. 2007 ). for 30 years, specific human papillomaviruses have been linked to certain human cancers and have been identified as causative agents of malignant proliferations. in the 1980s the detection of papillomavirus dna from cervical carcinoma biopsies were published, showing that hpv types 16 and 18 are the most frequent (dürst et al. 1983; boshart et al. 1984) . the relation of hpv infections and cancer is further discussed in chapter 23. definition: only infections that are newly discovered in humans are listed in this chapter: hiv, new variant of creutzfeldt-jakob disease (vcjd), hemorrhagic uremic syndrome (hus) caused by enterohemorrhagic escherichia coli, viral hemorrhagic fevers like hanta, lassa, ebola, and marburg fever, nipah virus encephalitis, monkeypox, human ehrlichiosis, severe acute respiratory syndrome (coronavirus infection, sars), and avian influenza (h5n1) (see fig. 3 .1 and table 3 .2). these infections mostly have their origin in zoonotic wildlife (e.g., avian influenza, monkeypox, hantavirus, nipah virus, and filoviruses) or livestock (e.g., vcjd). factors promoting the spread of these infections in humans are contacts with wildlife, mass food production of animal origin, and globalization (migration, transportation of goods and vectors) (see fig. 3 .2). in addition, new strains or variants of well-known pathogens have emerged showing increased or altered virulence such as clostridium difficile ribotype 027 or staphylococcus aureus strains expressing the panton-valentine leukocidin (see also chapter 22). the epidemiology of hiv is treated in chapter 18 and that of avian influenza and new influenza h1n1 in chapter 16. in the year 1995, 3 years after the peak of the bse epidemic in the united kingdom, with an annual incidence rate in cows of 6.636 per million bovines aged over 24 months, the first mortalities in humans with a new variant of creutzfeldt-jakob disease were observed in the united kingdom. until 2007, smaller incidence rates of bse cases had been reported by 21 other european countries in indigenous bovines and up to more than 43,000 per million in 2004 in ireland. from 1999, bse started to increase in switzerland and portugal, from 2004 in spain and in recent years has spread to eastern european countries (organisation mondiale de la santé animale 2008). the infectious agent is a self-replicating protein, a "prion." the source of infection for cows is infectious animal flour. the transmission to humans occurs through oral intake of cow products, most likely undercooked meat and nerval tissues as well as transplants of cornea, dura mater, contaminated surgical instruments, or the treatment with hypophyseal hormones extracted from animal tissues. after a statuary ban on the feeding of protein derived from ruminants to any ruminant and the export ban of all cow products from england, the epidemic of bse in cows and the occurrence of human infections decreased in the united kingdom since 2004. by june 2008 the total number of deaths in definite/probable cases of vcjd in the united kingdom was 163 (the national creutzfeldt-jakob disease surveillance unit 2008). only a few numbers of vcjd were reported from other european countries and the united states (who 2008). nipah virus encephalitis was first observed in 1997/98 in malaysia. the disease was transmitted by pigs to laborers in slaughterhouses and showed a lethality of 40%. the infectious agent was detected in 1999 (chua et al. 2000; lam and chua 2002) . since then, several outbreaks of nipah virus infections have been observed in asian countries: singapore in 1999, india 2001 , and bangladesh since 2003 (who 2004a harit et al. 2006) . the virus has been isolated repeatedly from various species of fruit bats, which seem to be the natural reservoir (yob et al. 2001 ). west nile is a mosquito-borne flavivirus that was first isolated from a woman with a febrile illness in uganda in 1937. from the 1950s, west nile fever endemicity and epidemics started being reported from africa and the middle east. severe neurological symptoms were thought to be rare. more recent epidemics in northern africa, eastern europe, and russia suggested a higher prevalence of meningoencephalitis with case fatality rates of 4-13%. in 1999, west nile virus was identified as the cause of an epidemic of encephalitis at the east coast of the united states (nash et al. 1999) . a seroepidemiological household-based survey showed that the first outbreak consisted of about 8,000 infections of which about 1,700 developed fever and less than 1% experienced neurological disease ). since then, epidemics occur during summer months in north america each year, with an estimated 35,000 febrile illnesses and over 1,200 encephalitis or meningitis cases in the united states in 2007 (centers for disease control 2008). age above 50 years is the main risk factor for developing severe disease. the virus is transmitted mainly by culex mosquitoes, but also by sandflies, ceratopogonids, and ticks, with birds as reservoir hosts and incidental hosts such as cats, dogs, and horses. efforts are made to reduce the transmitting mosquito population and to prevent mosquito bites through personal protection as well as to prevent transmission through blood donations by screening (centers of disease control 2008). the first case of sars occurred in guangdong (china) in november of 2002, leading to an outbreak with 7082 cases in china and hong kong (8096 cases worldwide) until july 2003. the case fatality rate was 9.6%. a new coronavirus (sars-cov) was identified as the causative agent (drosten et al. 2003) , being transmitted first by infected semidomesticated animals such as the palm civet and subsequently from human to human. some cases were exported to other countries, causing smaller outbreaks there, canada being the most affected country outside asia with 251 cases, before control of transmission was effective. eight thousand and ninety-six cases were reported worldwide, until july 2003, then further transmission stopped (besides one more case of laboratory transmission in 2004), indicating an efficient international cooperation in disease control (who 2004b) . recently, sars-cov has been found in horseshoe bats, which seem to be the natural reservoir of the virus. about 150,000-200,000 cases of hemorrhagic fever with renal syndrome (hfrs) caused by hantaviruses are reported annually worldwide, with more than half in china, many from russia and korea, and numerous cases from japan, finland, sweden, bulgaria, greece, hungary, france, and the balkan with different death rates depending on the responsible virus, ranging from 0.1% in puumala to 5-10% in hantaan infections (schmaljohn and hjelle 1997) . hantaviruses are transmitted from rodent to rodent through body fluids and excreta. only occasionally do humans get infected. different types of hantaviruses are circulating in europe and the eastern hemisphere, predominantly puumala virus, dobrava virus, and tula virus, adapted to different mouse species. depending on the virus type the case fatality rate is between 1 and 50%. as an example, the annual rate of reported cases in germany was about 100 cases per year from 2001 onward. this started to change in 2005 with 448 reported cases and rose dramatically to 1687 cases in 2007. that year, hantavirus infections were among the five most reported viral infections in germany. reasons for the rise in human infections were an increase in the hosting rodent population due to a very mild winter 2006/2007 and an early start of warm temperatures in spring which led to favorable nutritional situations for the mice influencing their population dynamics. in addition, favorable climatic conditions enhanced the outdoor behaviors of humans facilitating transmission in rural areas (robert koch institute 2008b; hofmann et al. 2008) . since 1993, a previously unknown group of hantaviruses (sin nombre, new york, black creek canal, bayou-in the united states and canada; andes, in south america) emerged in the americas as a cause of hantavirus pulmonary syndrome (hps), an acute respiratory disease with high case fatality rates (approx. 35%), causing a new, significant public health concern. a total of 465 cases had been reported until march 2007 in 32 states, most of them in the western part of the united states (centers for disease control 2007). lassa virus was detected for the first time in 1969 during an outbreak affecting nurses in a missionary hospital in lassa, nigeria. however, the disease had previously been described in the 1950s. lassa virus is enzootic in a common peridomestic rodent in west africa, the multimammate rat mastomys natalensis, which is chronically infected and sheds the virus in urine and saliva. human infection through direct or indirect contact with rats or their excretions is rather common in some west african countries and estimates from seroepidemiological and clinical studies suggest that there are several hundred thousand cases annually. however, only a minority of infections seems to progress to severe hemorrhagic disease with a case fatality rate of 5-30% in hospitalized cases. the virus can be transmitted by close person-to-person contact and nosocomial spread has been observed under poor hygienic conditions. marburg and ebola viruses, which were first detected during outbreaks in 1967 and 1975, respectively, have so far been observed only during several limited outbreaks and a few isolated cases in certain countries of sub-saharan africa. however, very high case fatality rates (25-90%), the occurrence of outbreaks that were difficult to control in resource-poor settings, and the obscure origin of these viruses have attracted considerable public interest worldwide. recently, evidence was found for both marburg and ebola viruses to occur in certain species of bats that probably constitute the natural reservoir of these filoviruses (towner et al. 2007 ). although the disease burden of these viral hemorrhagic fevers is low, they gained considerable international attention due to -their high case fatality rates, -the risk of person-to-person transmission, -several imported cases to industrialized countries, and -fears of abuse of these agents for bioterrorism. as a consequence, considerable resources have been invested, even in nonendemic countries, in the setting up of task forces and high containment facilities for both laboratory diagnostic services and treatment of patients using barrier nursing. this highly virulent strain of c. difficile expresses both cytotoxins a and b and, in addition, the binary toxin cdt, an adp-ribosyltransferase. due to a deletion in the regulatory tcdc gene, the synthesis rates of toxin a and b are increased by 16-and 23-fold, respectively. this strain was detected in 2000 for the first time in pittsburgh, usa. since then it has spread to canada, and in 2003 it reached europe causing multiple outbreaks in hospitals and nursing homes (warny et al. 2005) . c. difficile 027-associated colitis has shown high case fatality rates (10-22%) and an increased relapse rate. containment of outbreaks in hospitals and other institutions necessitates isolation of patients or cohorts and strict hygienic measures. during recent decades, a large variety of well known infectious diseases has shown regional or global re-emergence with considerable public health relevance (table 3. 3). globally, tuberculosis is probably the most important re-emerging infectious disease. in developing countries, tb infection still is extremely common and, in the wake of the hiv pandemic, the percentage of those developing overt disease has increased dramatically. worldwide, tb is the most common opportunistic infection in patients with aids. the significance of tb and hiv/tb coinfection is reviewed in chapters 16 and 18. the re-emergence of some infectious diseases is closely related to the lack or the breakdown of basic infrastructures as seen in periurban slums and in refugee camps in developing countries, or as a consequence of war, breakdown of the civil society, or natural or man-made disasters. cholera is a formidable example for both re-emergence and epidemic spread under those conditions. another important group of re-emerging infectious diseases is caused by various vector-borne infections, such as malaria, dengue fever, and yellow fever. these major vector-borne diseases are treated in more detail in chapter 21. in addition, there are a variety of re-emerging infections transmitted by arthropod vectors such as various arboviral diseases and some protozoal diseases other than malaria (i.e., leishmaniasis, human african trypanosomiasis). the reasons for the emergence of several vector-borne diseases are rather variable and may range from climatic factors (e.g., global warming, rainfall), lack or breakdown of control, to changes in agriculture and farming and in human behavior (e.g., outdoor activities). these factors are usually quite specific for each of these diseases and largely depend on the specific ecology of the agent, its vectors, and reservoirs. cholera, an acute diarrheal infection transmitted by fecally contaminated water and food, had been endemic for centuries in the ganges and brahmaputra deltas in the 19th century before it started to spread to the rest of the world. since 1817, six pandemics caused by the classical biotype of vibrio cholerae were recorded that killed millions of people across europe, africa, and the americas. it has been a major driving force for the improvement of sanitation and safe water supply. the seventh pandemic was caused by the el tor biotype, first isolated from pilgrims at the el tor quarantine station in sinai in 1906. it started in 1961 in south asia, reached africa in 1971, and is still ongoing. after more than hundred years, cholera spread to the americas in 1991, and beginning in peru, a large epidemic hit numerous latin american countries with 1.4 million cases and more than 10,000 fatalities reported within 6 years. out of the 139 serogroups of v. cholerae, only o1 and o139 can cause epidemics. the serogroup o139, first identified in bangladesh in 1992, possesses the same virulence factors as o1 and creates a similar clinical picture. currently, the presence of o139 has been detected only in southeast and east asia, but it is still unclear whether v. cholerae o139 will extend to other regions. since 2005, the re-emergence of cholera has been noted in parallel with the everincreasing size of vulnerable populations living in unsanitary conditions. cholera remains a global threat to public health and one of the key indicators of social development. while the disease is no longer an issue in countries where minimum hygiene standards are met, it remains a threat in almost every developing country. the number of cholera cases reported to the who during 2006 rose dramatically, reaching the level of the late 1990s. a total of 236,896 cases were notified from 52 countries, including 6,311 deaths, an overall increase of 79% compared with the number of cases reported in 2005. this increased number of cases is the result of several major outbreaks that occurred in countries where cases had not been reported for several years such as sudan and angola. it is estimated that only a small proportion of cases -less than 10% -are reported. the true burden of disease is therefore grossly underestimated. the absence or the shortage of safe water and sufficient sanitation combined with a generally poor environmental status are the main causes of spread of the disease. typical at-risk areas include periurban slums where basic infrastructure is not available, as well as camps for internally displaced people or refugees where minimum requirements of clean water and sanitation are not met. however, it is important to stress that the belief that cholera epidemics are caused by dead bodies after disasters, whether natural or manmade, is false. on the other hand, the consequences of a disaster-such as disruption of water and sanitation systems or massive displacement of population to inadequate and overcrowded camps-will increase the risk of transmission. chikungunya virus, an arbovirus belonging to the alphavirus group, is transmitted by various mosquitoes. the virus was first isolated in tanzania in 1952 and since then has caused smaller epidemics in sub-saharan africa and parts of asia with low public health impact. in 2005, the largest epidemic ever recorded started in east africa, spread to réunion and some other islands of the indian ocean, and then spread further to asia, with more than 1.5 million cases in india alone so far. characteristics of the disease are high fever and a debilitating polyarthritis, mainly of the small joints that can persist for months in some patients. now, for the first time, severe and fatal cases have been observed that may be due to certain mutations of the epidemic strain (parola et al. 2006) . the asian tiger mosquito aedes albopictus has proved to be an extremely effective vector in recent epidemics causing high transmission rates in big cities and leading to epidemics with high public health impact. this southeast asian mosquito species has been shipped by transport of used tires and plants harboring water contaminated with larvae to other continents and, since 1990, ae. albopictus has successfully spread in italy and other parts of southern europe. in august 2007, an outbreak of chikungunya fever occurred in northern italy with more than 200 confirmed cases. the index case was a visitor from india who fell ill while visiting relatives in one of the villages and further transmission was facilitated by an abundant mosquito population during that time, as a consequence of seasonal synchronicity (rezza et al. 2007 ). ross river virus (rrv) is another arbovirus of the alphavirus group that causes an acute disease with or without fever and/or rash. most patients experience arthritis or arthralgia primarily affecting the wrist, knee, ankle, and small joints of the extremities (epidemic polyarthritis). about one-quarter of patients have rheumatic symptoms that persist for up to a year. the disease can cause incapacity and inability to work for months. it is the most common arboviral disease in australia with an average of almost 5,000 notified cases per year. rrv is transmitted by various mosquito species and circulates in a primary mosquito-mammal cycle involving kangaroos, wallabies, bats, and rodents. a human-mosquito cycle may be present in explosive outbreaks which occur irregularly during the summer months in australia and parts of oceania. heavy rainfalls as well as increasing travel and outdoor activities are considered as important factors contributing to the emergence of rrv epidemics. this flavivirus is transmitted by certain culex mosquitoes and is a leading cause of viral encephalitis in asia with 30,000-50,000 clinical cases reported annually. it occurs from the islands of the western pacific in the east to the pakistani border in the west, and from korea in the north to papua new guinea in the south. only 1 in 50-200 infections will lead to encephalitis, which is, however, often severe with fatality rates of 5-30% and with a high incidence of neurological sequelae. despite the availability of effective vaccines, je causes large epidemics and has spread to new areas during recent decades (e.g., india, sri lanka, pakistan, torres strait islands, and isolated cases in northern australia). je is particularly common in areas where flooded rice fields attract water fowl and other birds as the natural reservoir and provide abundant breeding sites for mosquitoes such as culex tritaeniorhynchus, which transmit the virus to humans. pigs act as important amplifying hosts, and therefore je distribution is very significantly linked to irrigated rice production combined with pig rearing. because of the critical role of pigs, je presence in muslim countries is low. crimean-congo virus is a bunyavirus causing an acute febrile disease often with extensive hepatitis resulting in jaundice in some cases. about one-quarter of patients present hemorrhages that can be severe. fatality rates of 7.5-50% have been reported in hospitalized patients. cchf is transmitted by hyalomma ticks to a wide range of domestic and wild animals including birds. human infection is acquired by tick bites or crushing infected ticks, and also by contact with blood or tissue from infected animals that usually do not become ill but do develop viremia. in addition, nosocomial transmission is possible and is usually related to extensive blood exposure or needle sticks. human cases have been reported from more than 30 countries in africa, asia, southeastern europe, and the middle east. in recent years, an increase in the number of cases during tick seasons has been observed in several countries such as russia, south africa, kosovo, and greece. in turkey, where before 2002 no human cchf cases had been observed, a total of 2,508 confirmed cases, including 133 deaths, were reported between 2002 and june 2008. the emergence of cchf has been associated with factors such as climatic features (temperature, humidity, etc.), changes of vector population, geographical conditions, flora, wildlife, and the animal husbandry sector. rvf is a mosquito-borne bunyavirus infection occurring in many parts of sub-saharan africa. it infects primarily sheep, cattle, and goats, and is maintained in nature by transovarial transmission in floodwater aedes mosquitoes. it has been shown that infected eggs remain dormant in the dambos (i.e., depressions) of east africa and hatch after heavy rains and initiate mosquito-livestock-mosquito transmission giving rise to large epizootics. remote sensing via satellite can predict the likelihood of rvf transmission by detecting both the ecological changes associated with heavy rainfall and the depressions from which the floodwater mosquitoes emerge. transmission to humans is also possible from direct and aerosol exposure to blood and amniotic fluids of livestock. most human infections manifest themselves as uncomplicated febrile illness, but severe hemorrhagic disease, encephalitis, or retinal vasculitis is possible. in 1977, rvf has been transported, probably by infected camels to egypt, where it caused major epidemics with several hundred thousand infections of humans. it has been suggested that introduction of rvf may be a risk to other potentially receptive areas such as parts of asia and the americas. floods occurring during the el niño phenomenon of 1997 in east africa subsequently gave rise to large epidemics and further spread to the arabian peninsula. most recent epidemics occurred in 2006 and 2007 following heavy rainfalls in kenya, somalia, and sudan, causing several hundred deaths. besides mosquito control, epidemics are best prevented by vaccination of livestock. leishmaniasis, a protozoal transmitted by sandflies, has shown a sharp increase in the number of recorded cases and spread to new endemic regions over the last decade. presently, 88 countries are affected with an estimated 12 million cases worldwide. there are about 1.5 million new cases of cutaneous and mucocutaneous leishmaniasis, a nonfatal but debilitating disease with 90% of cases occurring in afghanistan, brazil, bolivia, iran, peru, saudi arabia, and syria. the incidence of visceral leishmaniasis (vl), a disease with a high fatality rate when untreated, is estimated at around 500,000 per year. the situation is further aggravated by emerging drug resistance (table 3 .4) and the deadly synergy of vl/hiv coinfection. epidemics usually affect the poorest part of the population and have occurred recently in bangladesh, brazil, india, nepal, and sudan. for many years, the public health impact of the leishmaniases has been grossly underestimated. they seriously hamper socioeconomic progress and epidemics have significantly delayed the implementation of numerous development programs. the spread of leishmaniasis is associated with factors favoring the vector such as deforestation, building of dams, new irrigation schemes, and climate changes, but also with urbanization, migration of nonimmune people to endemic areas, poverty, malnutrition, and the breakdown of public health. antimicrobial resistance of epidemiological relevance has emerged as a major problem in the treatment of many infectious diseases (table 3 .4). resistance is no longer a problem that predominantly affects the chemotherapy of bacterial infections. it became increasingly important in parasitic and fungal diseases, and despite the short history of antiviral chemotherapy, it already plays a prominent role in the treatment of hiv infection and other viral diseases. resistance is also a problem in some of the emerging infections and will further complicate their treatment and control. resistance of bacterial pathogens has become a common feature in nosocomial infections, especially in the icu and in surgical wards. currently, the number one problem in most hospitals is s. aureus resistant to methicillin (mrsa, see chapter 22). however, common problems of resistance also extend to other major bacterial pathogens such as enterococci, various gram-negative enteric bacilli, and pseudomonas species. resistance has developed not only to standard antibiotics (e.g., penicillins, cephalosporins, aminoglycosides, macrolides, or quinolones) but also to second-line antibiotics including carbapenems, glycopeptides, and newer quinolones. however, there is considerable geographic variation. in 2006, the european antimicrobial resistance surveillance system (earss), a network of national surveillance systems, reported vancomycin-resistant rates among enterococci ranging from none in iceland, norway, romania, bulgaria, denmark, and hungary to 42% of enterococcus faecium strains in greece (earss 2006) . a surveillance study conducted in the united states hospitals from 1995 to 2002 showed that 9% of nosocomial bloodstream infections were caused by enterococci and that 2% of e. faecalis isolates and 60% of e. faecium isolates were vancomycin resistant (wisplinghof et al. 2004) . rates and spectrum of antibacterial resistance of e. coli and other gram-negative enteric bacilli may differ considerably from one hospital to the other. in some important pathogens of hospital-related infections such as klebsiella, enterobacter, and pseudomonas species, resistance to almost all available antimicrobials has been observed. this may complicate the choice of an effective initial chemotherapy considerably. therefore, each hospital has to monitor the epidemiological situation of resistance regularly, at least for the most important bacteria causing nosocomial infections, such as staphylococci, enterococci, gram-negative enteric bacilli, and pseudomonas. even in community-acquired infections, there has been a considerable increase in resistance problems. at present, approximately 15% of pneumococcal isolates in the united states are resistant to penicillin, and 20% exhibit intermediate resistance. the rate of resistance is lower in countries that, by tradition, are conservative in their antibiotic use (e.g., netherlands, germany) and higher in countries where use is more liberal (e.g., france). in hong kong and korea, resistance rates approach 80%. in addition, about one-quarter of all pneumococcal isolates in the united states are resistant to macrolides. this rate is even higher in strains highly resistant to penicillin, and increasingly there is multiresistance against other antibiotics such as cephalosporins. the prevalence of meningococci with reduced susceptibility to penicillin has been increasing, and high-level resistance has been reported in some countries (e.g., spain, united kingdom). although high-dose penicillin is effective in infections with strains of intermediate resistance, most national and international guidelines recommend broad-spectrum cephalosporins such as ceftriaxone as first-line drugs. however, in most developing countries, penicillin and chloramphenicol are the only affordable drugs. in recent years, certain strains of community-acquired s. aureus with resistance to methicillin (cmrsa) have been observed which produce a toxin (panton-valentine leukocidin) that is cytolytic to pmns, macrophages, and monocytes, and which are an emerging cause of community-acquired cases and outbreaks of necrotic lesions involving the skin or the mucosa, and in some patients also of necrotic hemorrhagic pneumonia with a high case fatality (vandenesch et al. 2003) . development of resistance is mainly determined by two factors: -the genetic potential of a certain pathogen, i.e., mobile elements such as plasmids, transposons, or bacteriophages, genes coding for resistance, and mutation rate. -the selection pressure caused by the therapeutic or the para-therapeutic application of antimicrobial drugs. in the hospital these factors are supported by -microbial strains that are highly adapted to this environment (e.g., rapid colonization of patients, resistance to disinfectants), -an increasing percentage of patients who are highly susceptible to infections due to old age, multimorbidity, immunosuppression, extended surgery, and invasive procedures, and -the frequent use of broad-spectrum antibiotics or combinations of antimicrobial drugs. another source of resistant bacteria has been identified in mass animal production and the use of antimicrobials as growth promoters (e.g., the glycopeptide avoparcin, the streptogramin virginiamycin) or as mass treatment in the therapy or the prevention of infections. the inadequate use of antimicrobial drugs is also an important factor responsible for the development of resistance in community-acquired infections. this is especially true in developing countries where only a limited spectrum of antibiotics is available, where shortage of drugs often leads to treatment that is underdosed or too short, and where uncontrolled sale and use of antibiotics is commonplace. as a consequence, resistance of gonococci is extremely frequent in southeast asia, and resistance of salmonella typhi, shigella, and campylobacter to standard antibiotics is common. some of the still effective second-line antibiotics have to be given parenterally or are not available because they are too costly. a typical example of the consequences of insufficient chemotherapy due to lack of compliance and/or unavailability of drugs is the alarming increase in multiresistance and extreme resistance in tb (see chapter 16). resistance is also a problem in parasitic diseases such as malaria (see chapter 21), leishmaniasis, or african trypanosomiasis. plasmodium falciparum developed resistance against all major antimalarial drugs as soon as they were used on a broad scale. resistance had contributed significantly to the increase in malaria-associated morbidity and mortality observed in many endemic areas (wongsichranalai et al. 2002) . a recent report on failures of the new artemisinin combination treatment for p. falciparum malaria at the thai-cambodian border supports fears of the development of resistance to this most promising class of drug at present (dondrop et al. 2009 ). resistance against antiviral drugs has developed almost from the beginning of antiviral chemotherapy (table 3 .4). in the treatment of hiv infection, the risk of development of resistance has been drastically reduced by the combination of several drugs with different mechanisms of action (see chapter 18). however, drug resistance remains the achilles' heel of the highly active antiretroviral therapy (haart) and may be at a considerable risk of expanding haart to the developing world. today, we have to realize that as we develop antimicrobial drugs, microbes will develop strategies of counterattack. antimicrobial resistance occurs at an alarming rate among all classes of pathogens. even in rich countries it causes real clinical problems in managing infections that were easily treatable just a few years ago. in life-threatening infections such as sepsis, nosocomial infections, or falciparum malaria, there is a substantial risk that the initial chemotherapy might not be effective. in addition, the delay caused by inadequate treatment might favor transmission to other people and support the spread of resistant pathogens (e.g., multiresistant tb). last but not the least, surveillance and control and the necessity to use expensive second-line drugs or combinations of antimicrobials are enormous cost factors. for developing countries this is a major limitation in the treatment and control of infections caused by resistant agents. so, in many ways, emerging resistance contributes to the emergence of infectious diseases. despite the availability of effective strategies for treatment and prevention, infectious diseases have remained a major cause of morbidity and mortality worldwide. however, the problems associated with infections are due to considerable changes. in industrialized countries the mortality caused by infectious diseases has decreased tremendously during more than 100 years. however, during recent years, both mortality and morbidity associated with infections are increasing again. ironically, this is closely associated with the advances in medicine which have contributed to profound changes in the spectrum of both patients and their infections. advanced age, underlying conditions, and an altered immune response are common features in the seriously infected hospital patient today. immunosuppressive therapy is frequently used to treat neoplastic and inflammatory diseases or to prevent the rejection of transplants. some infections, most notably hiv/aids, cause immunosuppression by itself. in the compromised patient, infections are generally more severe or may be caused by opportunistic pathogens that will not harm the immunocompetent host. antimicrobial treatment is often less effective in these patients and tends to be further complicated by antimicrobial resistance which may manifest itself or develop at a higher frequency in the immunocompromised patient. an increasing percentage of infections are hospital acquired or otherwise health care associated. it is estimated that nosocomial infections affect 1.7 million patients and contribute to approximately 100,000 deaths in us hospitals annually (klevens et al. 2007 ). considering the rising number of elderly and immunocompromised patients, a further increase in severe infections can be predicted. in developing countries, the significance of infectious diseases has remained high for ages and despite the advances in medicine. until now, infections are by far the leading cause of both disability-adjusted life years and life years lost. the reasons are obvious and mostly related to poverty and lack of development causing poor and unhealthy living conditions, inadequate health systems, and lack of resources for prevention and treatment. this is, of course, just an integral part of the general socioeconomic problems of developing countries. however, poor health conditions per se are an important obstacle to development, and infections such as hiv/aids in sub-saharan africa can be a major cause of lack of development, increasing poverty, and political instability. generally, the situation of many developing countries has not improved during the last two decades, and the gap between the first and the third world has increased. however, most of the mortality and morbidity associated with infectious diseases is avoidable. as laid down in the millennium goals, a major task of the world community will be to counteract the imbalance between the industrialized and the developing countries and to find strategies to ensure participation in the progress of modern medicine for all. developing countries also carry the main burden of diseases caused by newly emerging and re-emerging infections (table 3. 2 and 3.3) . however, the consequences of economical and political crises on emerging infectious diseases are obvious in industrialized countries also-such as the return of diphtheria or the increase in tb and multiresistant tb after the breakdown of the former soviet union. today, all countries worldwide are affected by emerging infections as well as by emerging antimicrobial resistance. in the age of globalization, travel and transport of people, animals, and goods of all kinds have increased tremendously. as a consequence, infectious agents may travel over long distances and at high speed. this is clearly evident with influenza pandemics or outbreaks such as the sars epidemic or with imported cases of viral hemorrhagic fever transmissible from person to person. the spread of antimicrobial resistance or the re-emergence of tb seems to be less spectacular, but the consequences may be at least as important in the long run. management and control of emerging and re-emerging infectious diseases can be very different from disease to disease and has to allow for all relevant factors of the populations at risk and of the specific disease including the ecology of the agent, its vectors, and reservoirs. however, some basic principles apply to all situations: -surveillance -information and communication -preemptive planning and preparedness -provision and implementation of • adequate treatment • adequate control and prevention -international cooperation active and passive surveillance systems with rapid reporting and analysis of data are essential for the early detection of outbreaks, changes in epidemiology, and other events of public health concern (see chapters 8 and 9). however, many resourcepoor countries do not have functional surveillance systems. in addition, reporting of infectious diseases may be neglected or delayed because of fears of stigma, international sanctions including trade and travel restrictions, or interference with tourism. classical examples are plague and cholera, but also recent examples such as the bse/vcjd crisis in the united kingdom or sars originating from china showed undue delays between first occurrence of cases and information to the public. although, in outbreaks of new and unknown diseases it may be difficult, or even impossible, to predict or assess the magnitude of the problem and the potential consequences, timely and adequate information and communication is not only obligatory, according to international regulations, but also the best strategy to avoid rumors, misbeliefs, panic, or disregard. in recent years, many countries have installed national plans of action for important epidemiological scenarios and outbreaks such as pandemic influenza, bioterrorism, import of viral hemorrhagic fevers transmissible from person to person, sars, and comparable diseases or outbreaks. all member states of the world health assembly that have so far not been able to install functional surveillance and/or pre-emptive planning are obliged to do so within a maximum of 5 years after their ratification of the new international health regulations (who 2005) . preparedness not only means surveillance and planning but also has to include the provision of facilities to adequately treat and, if necessary, to isolate patients with infectious diseases of public health importance and relevant epidemic potential and/or at risk of transmission to other persons including health-care workers. task forces and high containment facilities for both laboratory diagnostic services and treatment of patients using barrier nursing have been set up in several countries. however, all health facilities of a certain level such as general hospitals should be prepared by their organization and structure to treat patients with infections of public health relevance such as multiresistant tb under appropriate isolation and barrier nursing conditions. this also applies to hospitals in resource-poor countries. adequate training of health-care workers and strict management have been effective to control outbreaks of highly contagious infections within rural african hospitals lacking sophisticated technical equipments (cdc 1998) . strategies for control and prevention may be quite different for various emerging infections. effective vaccinations are available only for some infections and are usually lacking for newly emerging infections (table 3 .5). for the majority of emerging infections, control and prevention have to rely on information, education and exposure prophylaxis, interruption of transmission by vector control and control of reservoir hosts (e.g., rodents), and case finding with early diagnosis and treatment. for diseases and outbreaks caused by infections of public health relevance that are transmissible from person to person, containment procedures including isolation and treatment of patients under condition of barrier nursing as well as tracking and surveillance of contacts are warranted by national and international health regulations. here, international cooperation is essential to successfully contain outbreaks and epidemics such as the sars epidemic in 2003. despite dramatic progress in their treatment and prevention, infectious diseases are still of enormous global significance with tremendous economic and political implications. emerging and re-emerging infectious diseases as well as emerging antimicrobial resistance are major challenges to all countries worldwide. for the management of current and future problems, it will be most important to counteract the imbalance between the industrialized world, new economies, and developing countries, and to adequately and timely react to new threats on a global scale. a new type of papillomavirus dna, its presence in 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estimating health care-associated infections and deaths in u.s. hospitals nipah virus encephalitis outbreak in malaysia lyme borreliosis in europe: influences of climate and climate change, epidemiology, ecology and adaptation measures. who regional office for europe altitudinal distribution limit of the tick ixodes ricinus shifted considerably towards higher altitudes in central europe: results of three years monitoring in the krkonose mts epidemiology of helicobacter pylori infection 1999: results of a household-based seroepidemiological survey outbreak of west nile virus infection novel chikungunya virus variant in travelers returning from indian ocean islands isolation of a cdna from the virus responsible for enterically transmitted non-a, non-b hepatitis infection with chikungunya virus in italy: an outbreak in a temperate region waldarbeiter-studie berlin-brandenburg 2000 zu zeckenübertragenen und andere zoonosen risikofaktoren für lyme-borreliose: ergebnisse einer studie in einem brandenburger landkreis übertrifft die infektionszahlen der vorjahre zahl der hantavirus-erkrankungen erreichte 2007 in deutschland einen neuen höchststand prevalence and determinants of helicobacter pylori infection in preschool children: a population-based study from germany hantaviruses: a global disease problem safety and efficacy of a recombinant hepatitis e vaccine tick-borne diseases in the united states the national creutzfeld-jakob disease surveillance unit (ncjdsu) marburg virus infection detected in a common african bat community-acquired methicillin-resistant staphylococcus aureus carrying panton-valentine leukocidin genes: worldwide emergence multiple exposures during a norovirus outbreak on a river-cruise sailing through europe toxin production by an emerging strain of clostridium difficile associated with outbreaks of severe disease in north america and europe prevalence of borrelia burgdorferi antibodies in hamburg blood donors nipah virus outbreaks in bangladesh revision of the international health regulations nosocomial bloodstream infections in us hospitals: analysis of 24 179 cases from a prospective nationwide surveillance study large outbreak of norovirus: the baker who should have known better epidemiology of drugresistant malaria nipah virus infection in bats (order chiroptera) in peninsular malaysia key: cord-020700-iko8gy1e authors: calvo, cristina; aguado, isabel; garcía-garcía, maría luz; ruiz-chercoles, esther; díaz-martinez, eloisa; albañil, rosa maría; campelo, olga; olivas, antonio; muñóz-gonzalez, luisa; pozo, francisco; fernandez-arroyo, rosa; fernandez-rincón, adelaida; calderon, ana; casas, inmaculada title: respiratory viral infections in a cohort of children during the first year of life and their role in the development of wheezing() date: 2017-07-06 journal: an pediatr (engl ed) doi: 10.1016/j.anpede.2016.08.008 sha: doc_id: 20700 cord_uid: iko8gy1e introduction: it is known that infants with viral respiratory infections severe enough to require hospital admission have a high risk of developing recurrent wheezing. few data have been published on unselected populations. the main aim of this study was to analyse symptomatic and asymptomatic respiratory viral infections during the first year of life in a cohort of infants, recruited at birth, and the development of recurrent wheezing. patients and methods: a total of 302 newborns were recruited. a nasopharyngeal aspirate was taken when the patients had a respiratory infection, as well as in the visits for vaccination at 2, 4, 6, and 12 months. rt-nested pcr assays were performed to detect 16 viruses. results: a total of 1293 samples were analysed (1005 healthy controls and 288 respiratory infections). samples taken during routine check-ups were positive in 30.8% of cases, while those with respiratory infection were positive in 77.8%, p < .001 (or: 3, 95% ci: 2.4–3.8). a total of 239 (79%) infants had at least 1 positive respiratory viral infection detected. the most frequent virus (71%) was rhinovirus (rv). recurrent wheezing was found in 27 (11%) children during their first year of life (1.2 episodes, sd 2.9). recurrent wheezing was present in 58.3% of patients admitted to hospital during their first viral infection, vs. 8.6% of infants when the first infection was mild or who had asymptomatic viral detection, p < .001 (or: 2.18; 95% ci: 1.05–4.5). conclusions: in our series, severe respiratory infections leading to hospitalisation in the first months of life are risk factors for developing wheezing, but not in the case of mild rv infections. results: a total of 1293 samples were analysed (1005 healthy controls and 288 respiratory infections). samples taken during routine check-ups were positive in 30.8% of cases, while those with respiratory infection were positive in 77.8%, p < .001 (or: 3, 95% ci: 2.4---3.8). a total of 239 (79%) infants had at least 1 positive respiratory viral infection detected. the most frequent virus (71%) was rhinovirus (rv). recurrent wheezing was found in 27 (11%) children during their first year of life (1.2 episodes, sd 2.9). recurrent wheezing was present in 58.3% of patients admitted to hospital during their first viral infection, vs. 8.6% of infants when the first infection was mild or who had asymptomatic viral detection, p < .001 (or: 2.18; 95% ci: 1.05---4.5). conclusions: in our series, severe respiratory infections leading to hospitalisation in the first months of life are risk factors for developing wheezing, but not in the case of mild rv infections. © 2016 asociación española de pediatría. published by elsevier españa, s.l.u. all rights reserved. infecciones respiratorias; infecciones virales; infecciones asintomáticas; rinovirus; sibilancias; lactantes infecciones virales respiratorias en una cohorte de niños durante el primer año de vida y su papel en el desarrollo de sibilancias resumen introducción: las infecciones respiratorias virales que requieren hospitalización parecen conferir riesgo de desarrollar sibilancias recurrentes, pero existen pocos datos publicados en poblaciones no seleccionadas por tener factores de riesgo. nuestro objetivo principal fue analizar si las infecciones respiratorias virales sintomáticas y asintomáticas, de diferente gravedad, durante el primer año de vida en una cohorte de recién nacidos, suponen un mayor riesgo de sibilancias recurrentes. pacientes y métodos: se incluyeron 302 recién nacidos. se recogió aspirado nasofaríngeo a los niños cuando presentaron una infección respiratoria y de forma periódica en los controles de salud (2, 4, 6 y 12 meses). se estudiaron 16 virus respiratorios mediante reacción en cadena de polimerasa (pcr). resultados: se analizaron 1.293 muestras (1.005 controles de salud y 288 infecciones respiratorias). el 30,8% de las muestras tomadas en los controles de salud fueron positivas, frente a un 77,8% en las infecciones respiratorias, p < 0,001 (or: 3, ic 95%: 2,4-3,8). un total de 239 (79%) lactantes tuvieron al menos una detección viral positiva durante el primer año de vida. el virus más frecuentemente identificado (71%) fue el rinovirus (rv). en 27 lactantes (11%) se detectaron sibilancias recurrentes durante su primer año de vida (2,9 de: 1,2 episodios). el 58,3% de los lactantes cuya primera infección respiratoria requirió hospitalización desarrollaron sibilancias de repetición, frente al 8,6% de los niños cuya primera infección fue leve o asintomática, acute respiratory infections (aris) are a major reason for health care use in infants and constitute a substantial economic burden. 1,2 although infections in this age group are most frequently of a viral aetiology, 3,4 inappropriate antibiotic use continues to be widespread. techniques based on the polymerase chain reaction (pcr) have made the aetiological diagnosis of aris possible in children, and made us aware of the high prevalence of asymptomatic viral infections. 5, 6 respiratory syncytial virus (rsv), human metapneumovirus (hmpv) and influenza virus (flu) have been clearly identified as respiratory pathogens, and more recent studies have been analysing the role of other viruses such rhinovirus (rv) and human bocavirus (hbov), although the high prevalence of coinfection with other respiratory viruses or their detection in asymptomatic patients pose challenges to the interpretation of these positive results. 7, 8 it is known that children with severe rsv infection that require hospitalisation are at high risk of developing asthma in the long term, even as late as age 18 years, as sigurs et al. demonstrated. 9 there is also evidence of an increased risk of recurrent wheezing up to age 5 years in infants hospitalised due to bronchiolitis associated to hmpv. 10 there are fewer studies in children that have not been admitted to hospital, among which we would like to mention the classic studies by stein et al., who analysed an unselected cohort and found that milder infections by rsv also increased the risk of developing wheezing. 11 in recent years, the role of rv as a long-term predictor of future asthma has also been investigated. a study of particular interest on the subject is that of lemanske et al., 12 whose cohort of infants that had a rv infection with wheezing in the first year of life were at increased risk of having developed wheezing by age 3 years, with an odds ratio of 10. most of the studies that assess the risk of developing asthma or wheezing have been conducted in selected high-risk populations. 13, 14 furthermore, little is known about mild viral respiratory infections managed in outpatient settings in relation to the future development of wheezing, not to mention asymptomatic viral infections, although it is generally assumed that risk increases with increasing severity. the objective of our prospective study was to analyse asymptomatic and symptomatic infections of varying severity in a cohort of newborns during the first year of life and assess their role in the development of recurrent wheezing. our secondary objective was to analyse the aetiology and clinical characteristics of these infections. we conducted a prospective cohort study in newborns to analyse the association between past viral infections of varying severity and the development of recurrent wheezing. we requested the participation of all newborns delivered in four health care centres in the área sur health zone of madrid during their first neonatal visit (7---14 days of life). the parents or guardians signed an informed consent form and completed a questionnaire of epidemiological data (history of asthma and atopy in the family, exposure to tobacco smoke, pregnancy and delivery data, number of siblings). nasopharyngeal aspirate (npa) specimens were collected during this visit and routine checkups performed at ages 2, 4, 6 and 12 months. furthermore, if the infants developed respiratory symptoms, they were evaluated by a paediatrician, with collection of an additional npa sample. we considered children with specimens collected during routine well-child checkups healthy controls, even if they presented with isolated rhinorrhoea. we defined asymptomatic infection as detection of a virus in the npa of one of these patients. we defined clinically significant respiratory infection as a case that met the clinical criteria for respiratory infection presented in table 1 . we defined ''first detected viral infection'' as pcr detecting a respiratory virus in a sample for the first time in the life of a child, whether the infection was asymptomatic, symptomatic and managed at the outpatient level, or symptomatic and requiring hospital admission. when a child met the criteria for respiratory infection described in table 1 , the paediatrician conducted an evaluation, a clinical data form was completed, and a npa table 1 in the absence of respiratory distress or wheezing. we defined bronchiolitis as the first episode of respiratory infection that manifested with respiratory distress and wheezing. subsequent episodes of similar characteristics were classified as recurrent wheezing. episodes manifesting with inspiratory dyspnoea and wheezing were classified as laryngotracheobronchitis. laryngitis was defined as inspiratory dyspnoea without wheezing. we defined pneumonia as evidence of focal infiltrates or consolidation in chest radiography in the absence of wheezing. we classified all infections requiring hospital admission as severe, and the rest as mild. when the requisite criteria were met, a npa sample was collected and kept at 4 • c to await transportation to the influenza and respiratory viruses laboratory of the centro nacional de microbiología (national centre of microbiology). in hospitalised patients, the npa sample was collected between 7 and 8 am of the morning following admission and kept refrigerated until it was transported. three rt-nested pcr assays were used to test for 16 respiratory viruses. reverse transcription (rt) and the first amplification reaction were performed with onestep rt-pcr kits (qiagen). influenza a, b and c were detected by a previously described pcr assay. 15 a second multiplex pcr assay was used to detect parainfluenza virus (piv) 1 through 4, coronavirus 229e and oc43, enterovirus and rv. 16 the presence of rsv-a and b, hmpv, hbov and adenovirus (adv) was assessed with a third rt-nested pcr assay using the brq method. 3 we have expressed discrete data as percentages and continuous data as mean and standard deviation. we compared clinical and laboratory characteristics using the student's t, mann---whitney u, chi-square and fisher exact tests. we defined statistical significance as a p-value of less than .05 in any of the tests. the statistical analysis was performed with the statistical package for the social sciences (spss) version 13.0. we recruited a total of 302 infants at birth, of which 55.3% were male (167), 80% born to spanish parents, and 14% of latin-american ancestry. thirty-five percent of infants were exposed to tobacco smoke; 36% of mothers and 37% of fathers had asthma and/or atopy. only 2 patients were born preterm at a gestational age of 33 weeks. all infants but 7 completed the yearlong followup. those 7 patients did not attend the 12-month checkup, but we contacted their paediatricians to obtain information about their clinical outcomes, so they were ultimately included in the analysis. a total of 1,239 samples were collected, 1,005 during routine visits and 288 when a respiratory infection was suspected. of the samples collected during the well-child checkups, 30.8% (310) tested positive, while viruses were detected in 77.8% of respiratory infection cases (224/288), p < .001 (odds ratio, 3; 95% confidence interval, 2.4---3.8). the frequency of viral detection in asymptomatic children (healthy controls) increased proportionally with age from 20.8% in newborns aged 15 days to up to 42% in infants aged 12 months. the virus identified most frequently was rv, found in 82% (254) of cases. other viruses were found in a small proportion of cases: adv (9.7%), piv (4.2%), hbov (3.2%), hmpv (2.9%), flu (2.2%) and rsv (1.6%) (fig. 1 ). of the 302 infants included in the study, 239 (79%) had at least one positive viral detection in the first year of life (fig. 2) . the virus detected most frequently was rv (181 samples, 71%), followed by piv (6.3%), adv (5%) and rsv (6%). coinfections were only detected in 21 cases (8.8%). the mean age at detection of the first viral infection was 3.4 months (sd, 2.8). the first detected viral infection was associated with upper respiratory tract infections in 45 patients (20%), bronchiolitis in 19 (8.5%) while remaining 159 samples (70%) corresponded to asymptomatic patients and had been collected during routine well-child checkups. we analysed the outcomes of infants that had at least one positive specimen, and found that 27 (11%) developed recurrent wheezing in the first year of life (mean ± sd, 2.9 ± 1.2 episodes; range, 2---5) (fig. 3) . rhinovirus was the first virus detected in 18 patients (66%), of who 14 (52%) were asymptomatic at the time of the specimen collection (routine checkup). the mean age of infants that developed recurrent wheezing with their first positive viral detection was 2 months (sd, 1.3; all but three were at least 3 months old). ten of the 27 infants with episodes of bronchiolitis required admission (37%). the most frequently involved virus was rv, both in mild bronchiolitis cases managed at the outpatient level (11/17, 69%) and in severe cases that required hospitalisation (7/10, 70%). recurrent wheezing episodes (at least one following the initial bronchiolitis) occurred in 58.3% of infants in who the first viral detection corresponded to a severe infection that required admission, compared to 8.6% of infants whose first detection corresponded to a mild infection managed at the outpatient level or an asymptomatic infection (p < .001; odds ratio, 2.18; 95% ci, 1.05---4.5). a total of 5 infants received montelukast or budesonide during the followup (18%, 3 admitted to hospital and 2 managed at outpatient level at first positive viral detection). there were no differences in the family history of asthma or atopy between children with and without recurrent wheezing. there were 288 episodes of infection managed in outpatient settings that corresponded to 160 infants (53% of the total sample) who had between 1 and 6 episodes each (mean ± sd, 1.6 ± 1). the most frequent symptoms were: fever in 90 cases (38%), rhinorrhoea in 224 (96%), cough in 223 (96%), respiratory distress in 51 (21%) and nonspecific symptoms in 35 (15%). the most frequent diagnoses were upper respiratory tract infection (147, 51%) and bronchiolitis (48, 16%), with a lesser incidence of recurrent wheezing, laryngitis and pneumonia. only 2 patients had infiltrates on radiologic examination, and both required hospital admission during the followup. the viruses detected in these infections were rv in 127 cases (55.8%), followed by piv (13%), rsv (12.5%), hmpv (8.9%) and flu (8%) (fig. 2) . the causative agent of upper respiratory tract infection was rv in 61% of cases, followed by piv (13%) and adv (11%). when it came to bronchiolitis, rv was detected in 53%, rsv in 25% and hmpv in 8% of cases. we analysed a total of 19 hospital admissions corresponding to 14 infants (14/302; 4.6% of the cohort; 5.8% of infants with a positive viral detection). five patients were admitted more than twice in the first year of life. fifty-eight percent of admitted patients (11) were female. the mean age of admission was 3.6 months (sd, 2.5 months; range, 6 days---8 months). forty-two percent presented with fever (mean, 38.6 • c; sd, 0.9 • c) lasting 3.5 days (sd, 2). sixty-eight percent (13/19 episodes) required oxygen supplementation for a mean of 5 days (sd, 3). twenty-six percent (5/19) had infiltrates detected by radiology. the diagnoses were bronchiolitis in 12 cases (63%), wheezing episode in 3 (15%), upper respiratory tract infection in 3 and pneumonia in 1. only 3 infants received antibiotics. respiratory syncytial virus was detected in 42% of the cases and rv in 52%. viral coinfection was detected in 4 cases. a considerable number of infants test positive for at least one respiratory virus in the first year of life, and we observed a percentage of 79% in our cohort. more than half of the cohort experienced at least one symptomatic infection treated at the outpatient level, with a mean of 1.6 symptomatic infections a year (range, 1---6), while approximately 5% required hospital admission. rhinovirus was the virus detected most frequently in symptomatic (43%) as well as asymptomatic (25%) infants. eleven percent of the infants with positive virology results went on to develop recurrent wheezing in the first year of life, but the risk was much higher in patients that required admission due to bronchiolitis (58%; odds ratio, 2.18) compared to patients whose first infection was mild or asymptomatic. our data are consistent with the findings of the study conducted by rhedin et al., who analysed a group of paediatric outpatients with respiratory infections and a group of asymptomatic controls. rhedin et al. detected respiratory viruses in 72.3% of cases (n = 151) and 35.4% of controls (n = 74) (p = .001). rhinovirus was the virus identified most frequently in both cases and controls (47.9 and 21.5%, respectively). 6 although infections by rv are common, a recent study that used nucleotide sequencing demonstrated that the prolonged presence of rhinovirus rna in the respiratory tract following an upper respiratory tract infection is rare in healthy infants (<5%). in these infants, the detection of rv rna in repeat samples at least 30 days apart most likely corresponded to new infections. 17 another study found that the detection of the same virus strain more than 2 weeks apart was unusual. 18 in our study, we found that rv was detected in a significant proportion of infants that were classified as healthy (routine vaccination visit), probably because we did not take isolated rhinorrhoea into account, and this is a frequent symptom in children that does not contraindicate vaccination. however, it is possible that rhinorrhoea is actually a presenting symptom in mild rv infections. unfortunately, we have no data on the percentage of infants considered healthy controls that had rhinorrhoea as the sole symptom. this is consistent with the hypothesis of jartti et al. according to which rv always causes an actual infection, which may or may not be clinically relevant. 5 although there are authors like jansen et al., 19 who found a higher rv load in children with acute symptomatic infections compared to asymptomatic children, or utokaparch et al., 20 who detected significantly higher rv loads in infections of the lower respiratory tract, there are others, like takeyama et al., 21 who have not found a correlation between viral load and disease severity in rv infections. at any rate, the clinical significance of viral loads in respiratory samples is not well established, and we did not measure viral loads in our patients. we also ought to highlight the small percentage of coinfections found in our cohort (8.8%), as other case series, including previous studies by our research group, found a percentages of coinfection of up to 46% in hospitalised children with rhinovirus infections. 22 we believe that the age of our patients (a substantial proportion of the specimens were collected from infants aged less than 6 months) and our decision to include mild and asymptomatic infections with positive virology may account for the low proportion of coinfection. in a cohort study of 140 newborns with a design similar to our own, van der gugten et al. 23 found that rv infections manifesting with wheezing in the first year of life were a risk factor for developing recurrent wheezing in the future. although they did not assess the severity of acute episodes, mild infections without wheezing were not associated with an increased risk of recurrent wheezing. these authors reached the conclusions we have reached based on our cohort, in which infections by rv or rsv that were sufficiently severe to cause bronchiolitis requiring hospital admission were a risk factor for developing wheezing as early as the first year of life. the followup of our cohort in the immediate future will allow us to learn whether the risk of wheezing continues to be higher in early childhood. as lemanske et al. suggested, 12 the severity of infection is a risk factor for the development of asthma and recurrent wheezing. various studies have demonstrated that severe bronchiolitis associated to rv infection increase the risk of asthma, but as we commented above, these studies were conducted in selected populations. 24---26 our case series, which analysed symptomatic and asymptomatic infections and severity at an early age (less than 3 months in most first episodes) in an unselected population contributes a broader picture on the subject. although our study has strengths, such as its unselected sample and testing for all respiratory viruses (as opposed to only the most prevalent) and for the presence of asymptomatic infection, we ought to comment on certain methodological aspects. we did not collect weekly samples to study viral shedding and its duration, and we also did not perform genotyping of rv. the duration of followup was short, and the number of patients with recurrent wheezing small. nevertheless, we believe that the association found in the study is worth considering, and that we will be able to reach broader conclusions in upcoming years. until then, we can state that respiratory infections in the first months of life that are severe enough to require hospitalisation are a risk factor for the development of recurrent wheezing. this study was partially funded by the the economic burden of noninfluenza-related viral respiratory tract infection in the united states viruses associated with acute respiratory infections and influenza-like illness among outpatients from the influenza incidence surveillance project detection of new respiratory viruses in infants hospitalized with bronchiolitis. a three year prospective study respiratory syncytial virus-associated hospitalizations among children less than 24 months of age indentification of respiratory viruses in asymptomatic subjects: aymptomatic respiratory viral infections clinical utility of pcr for common viruses in acute respiratory illness human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection high incidence of human bocavirus infection in children in spain asthma and allergy patterns over 18 years after severe rsv bronchiolitis in the first year of life human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age 5. pediatr pulmonol respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years rhinovirus illnesses during infancy predict subsequent childhood wheezing wheezing rhinovirus illnesses in early life predict asthma development in high-risk children rhinovirus induced wheezing in infancy ----the first sign of childhood asthma? simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays duration of rhinovirus shedding in the upper respiratory tract in the first year of life serial viral infections in infants with recurrent respiratory illnesses frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values the relationship between respiratory viral loads and diagnosis in children presenting to a pediatric hospital emergency department rhinovirus load and disease severity in children with lower respiratory tract infections role of rhinovirus c respiratory infections in sick and healthy children in spain human rhinovirus and wheezing: short and long-term associations in children response to infections in patients with asthma and atopic disease: an epiphenomenon or reflection of host susceptibility? rhinovirus-induced bronchiolitis and asthma development rhinovirus bronchiolitis and recurrent wheezing: 1-year follow-up key: cord-000457-e50a0suk authors: rhim, jung-woo; lee, kyung-yil; youn, you-sook; kang, jin-han; kim, ji-chang title: epidemiological and clinical characteristics of childhood pandemic 2009 h1n1 virus infection: an observational cohort study date: 2011-08-24 journal: bmc infect dis doi: 10.1186/1471-2334-11-225 sha: doc_id: 457 cord_uid: e50a0suk background: there was a pandemic influenza around the world in 2009 including south korea since last pandemic occurred four decades ago. we aimed to evaluate the epidemiological and clinical characteristics of this infection in childhood. methods: we evaluated the epidemiologic characteristics of all the subjects infected with the 2009 h1n1 influenza a virus (2,971 patients, ≤ 15 years of age), and the clinical and laboratory findings of the inpatients (217 patients, 80 had pneumonia) between 1 september 2009 and 31 january 2010 in a single hospital throughout the epidemic. results: the age distribution of all the subjects was relatively even. over 90% of cases occurred during a two-month period. two hundred and five patients (94.5%) received oseltamivir within 48 h of fever onset, and 97% of inpatients defervesced within 48 h of medication. the group with pneumonia included more males than females, and had higher leukocytes counts with lower lymphocyte differentials than the group without pneumonia. the white blood cell count and lymphocyte differential were associated with the severity of pneumonia. corticosteroid treatment for severe pneumonia patients was highly effective in preventing disease progression. conclusion: children of all ages affected with even rates of infection, but males were predominant in pneumonia patients. pneumonia patients showed lymphopenia and its severity was associated with the severity of illness. our results suggest that the mechanism of lung injury in 2009 h1n1 virus infection may be associated with the host immune response. although influenza virus infection has been a major global concern since the pandemic 1918 'spanish flu', there have been no pandemic influenzas for near four decades after the 1968 'hong kong flu'. the pandemic 2009 h1n1 influenza a (2009 h1n1) virus infection was reported first in mexico in february 2009, and then the virus spread rapidly worldwide, including in south korea [1] . it has been reported that the majority of h1n1 patients were children and young adults and the mortality rate was not higher than that of seasonal influenza [2] [3] [4] . the majority of patients affected by the 2009 h1n1 virus infection recovered uneventfully, but some previously healthy patients developed a rapidly progressive pneumonia, leading to acute respiratory distress syndrome (ards), multi-organ failure, and death [5] [6] [7] . with this enigma, the pathogenesis of acute lung injury (pneumonia) in influenza infections remains unknown [1, 8, 9] . in south korea, the first patient with 2009 h1n1 virus infection was reported in may, 2009. the number of patients gradually increased until mid-october, when the number of patients was overwhelming for a month, and then gradually decreased, with few people becoming infected after february, 2010. owing to sensational reports of childhood fatality in the mass media and a new diagnostic tool, the real-time reverse transcriptase-polymerase chain reaction (rt-pcr), we had the opportunity to evaluate patients with 2009 h1n1 virus infection from the onset of their illness. in addition, during the study period we observed a dramatic effect of early treatment with corticosteroids and oseltamivir for patients with severe pneumonia including rapidly progressive pneumonia [9, 10] . in this study, we evaluated the epidemiological, clinical and laboratory features of children with 2009 h1n1 virus infections in a single hospital throughout the epidemic. we retrospectively evaluated all patients with 2009 h1n1 virus infection during the pandemic (2,971 patients) for epidemiologic characteristics, and for clinical characteristics, we reviewed the medical records and chest radiographic findings of 217 children admitted to the catholic university of korea, daejeon st mary's hospital between 1 september 2009 and 31 january 2010. the diagnosis of patients depended on positive results for the 2009 h1n1 virus rt-pcr (accupower ™ in korea, bioneer, alameda, ca, usa) through throat swabs. although indications for admission were not clearly defined in this study, the majority of the inpatients were those who were suspected to have severe disease such as pneumonia and to have risk factors for severe disease such as infants and bronchial asthma. however, it might be possible that excessive concern of parents on fatality of this infection in part affected on admission of the uncomplicated cases. among the 217 inpatients, we selected 80 patients with pneumonia and 137 patients without pneumonia, based on the chest radiographs. the chest radiographic patterns of admitted patients were reviewed and classified by two pediatricians (ky lee and jw rhim) and one pediatric radiologist (jc kim). the patients with chest radiographic patterns that showed increased nodular densities along the bronchial trees unilaterally or bilaterally, were designated the bronchopneumonia group (49 patients). patients with a distinctive large patch of infiltration, segmental or lobar consolidations were designated the segmental/lobar pneumonia group and regarded as having a severe pneumonia (31 patients). the first day of fever was regarded as the first day of illness. among pneumonia patients, we tried corticosteroid treatment for 17 patients with severe pneumonia. as for indication of corticosteroids (methyprednisolone, mp or prednisolone), the subjects had severe respiratory distress with hypoxemia at presentation (12 cases) or during hospitalization (5 cases) requiring o 2 therapy (9,10). we compared the clinical and laboratory characteristics of the different groups. the study was approved by the institutional review board of the catholic university of korea, daejeon st mary's hospital. statistical analyses were performed using the statistical package for the social sciences for windows version 12.0 (spss, chicago, il, usa). continuous variables are reported as means ± standard deviations. statistical significance was assessed using the student's t-test and the paired t-test for continuous variables, and using the χ 2 test for categorical variables. a p value of < 0.05 was considered significant for the statistical tests. during the study period, 5,975 children (aged 2 months-15 years) with influenza-like illness were seen at our hospital. among them, the 2,971 patients were positive by rt-pcr, and 217 patients were admitted to the isolation wards. the hospitalization rate was 7.3%. the mean age of the patients (2,971 cases) was 7.6 ± 4.1 years of age and the male-to-female ratio was 1.1:1 (1,581/1,390). the age distribution of the patients is shown in figure 1 . children of all age groups except infants had a relatively even rate of infection ( figure 1 , gray bars). the numbers of new patients each week is shown in figure 2 . there was an explosive pattern, with over three quarters of the cases occurring during a single month (the 43rd-47th weeks of 2009; 18 october to 14 november). the age distribution and the weekly case rate of the patients admitted to hospital (217 cases) are also shown on figure 1 and 2 (black bars). these demonstrated similar patterns to those of the total patient cohort. for hospitalization rates, younger children (0-4 years, 9.8%, 75/762) showed higher admission rates than older children (5-9 years, 8.6%, 104/1216 and 10-15 years, 3.8%, 38/993). in the 217 hospitalized children, the mean age was 6.2 ± 3.7 years, and the male-to-female ratio was 1.6:1 (132/85). almost all the patients had a high fever (97.2%) and cough (92.6%) suggestive of severe infection such as pneumonia at the time of admission. most of the patients were previously healthy although some had underlying diseases and only 17 patients had underlying disorders (7.8%), including 11 chronic pulmonary diseases (10 patients with bronchial asthma and one with past history of brochopulmonary dysplasia), 4 neuromuscular disorders (two with epilepsy and two with cerebral palsy), one with chronic liver disease, and one with nephrotic syndrome. the outcomes for these patients were uneventful, except for two asthmatic patients who developed a mild pneumonia. all inpatients received oseltamivir of the recommended doses for body weight and a broad-spectrum antibiotic (ampicillin/sulbactam). two hundred and five patients (94.5%) received oseltamivir within 48 h of fever onset. the mean duration of fever before admission (including the day of admission) was 2.0 ± 0.8 d and 167 patients (77%) defervesced on the next day. only seven patients had fever that persisted > 48 h after oseltamivir treatment. during hospitalization, five patients showed progressive pneumonia despite early antiviral therapy. according to the initial chest radiographs, 80 patients had pneumonia and were divided into two groups: the bronchopneumonia group (49 patients) and the segmental/lobar pneumonia group (31 patients). in pneumonia patients, pneumonic infiltrations appeared within 48 h of fever onset in 68 patients (85%). when we analyzed the inpatients according to age (0-4 years, 75 patients; 5-9 years, 104 patients; and 10-15 years, 38 patients), the rates of pneumonia in each age group were 36.0% (27/75), 45.2% (47/104) and 8.8% (6/38), respectively. the severe pneumonia (segmental/lobar type) was predominant in the older age groups (14.8% (4/27), 48.9% (23/47) and 50% (3/6), respectively). among the 80 pneumonia patients, 17 patients showed severe respiratory distress with hypoxemia at presentation (12 cases) and during their hospital stay (5 cases). arterial blood gas analysis was done in 15 patients, and 12 patients showed hypoxemia (po 2 < 60 mmhg in the room air). these patients received additional corticosteroids as soon as possible when indicated; 12 patients received intravenous mp (10 mg/kg/day, divided two doses, at presentation, 5 mg/kg/day at next day and then tapered off within a week) and 5 patients received oral prednisolone (1 mg/kg/day, divided 3 doses, for 3 days tapered off within a week). six patients received early mp with osetalmivir which is recommended early use, before the positive rt-pcr results. interestingly, all patients except one (age 4 years) were among the 5-9 years age group, and male patients were predominant (13/17). we performed serial chest radiographs of some cases in these patients. a 6-year-old male patient complained of one day fever and cough, and his initial chest radiograph showed few pulmonary infiltrations. on the following morning, he complained of severe dyspnea and showed chest radiographic infiltrations on right lower lung and left hilar regions. he had received two doses of oseltamivir before the mp treatment ( figure 3a -c). a 7 year-old male patient was presented with a day fever and severe cough and had a rapidly progressive pneumonia in which initial patchy infiltrates on left upper lobe progressed to total left lung consolidation within 12 h after admission. he was given three doses of oseltamivir before the mp treatment ( figure 4a-g) . these two patients showed dramatic improvements in their clinical symptoms and radiographic findings within 24 h after mp treatment. a 7-year-old male patient with lobar pneumonia was treated with oseltamivir, mp and intravenous immunoglobulin (ivig) treatment. he was admitted with fever, cough and progressive dyspnea of 2 days, and after mp (10 mg/ kg/day, divided 2 doses) infusion, he showed persistent dyspnea and slightly aggravated pneumonic consolidation ( figure 5c ). on the following morning, high-dose ivig (1 g/kg) was infused for 6 hours. at the time of termination of ivig, his clinical symptoms disappeared and a dramatic improvement of radiographic findings was observed within 6 hours after ivig termination ( figure 5a-f) . there was no adverse or rebound reaction in any patient treated with corticosteroids. the clinical symptoms of all patients improved within a day and their pneumonic infiltrations, regardless of severity, ceased immediately after corticosteroid treatment and disappeared within several days without adverse reactions. extrapulmonary manifestations (complications) of h1n1 infection were observed as four cases of febrile seizure, two cases of urticaria, one case of hepatitis and one case of myositis, and no cases of encephalopathy. when we compared the patients with and without pneumonia, and the patients with segmental/lobar pneumonia versus those with bronchopneumonia, there were significant differences in certain parameters between the groups. compared with the group without pneumonia, the males were over-represented in the pneumonia group (p = 0.001) and longer hospitalizations (p < 0.001), higher values for hemoglobin (p = 0.03), wbc (p < 0.001) and crp (p < 0.001) and lower values for lymphocyte differential (p < 0.001) ( table 1 ). the patients with the more severe type of pneumonia (segmental/lobar pneumonia) showed a higher mean age (p = 0.001) and leukocyte count (p = 0.01) and lower lymphocyte differential values (p < 0.001) compared with the group with bronchopneumonia (table 2 ). in addition, the severe pneumonia patients who received corticosteroids (17 cases) had the highest leukocyte counts and crp levels, and the lowest lymphocyte differentials (11 800 ± 3600/mm 3 , 3.0 ± 3.1 mg/dl, and 8.8 ± 6.3%, respectively) [10] . in this study, we found that children of all ages except infants had a relatively even rate of infection with the 2009 h1n1 influenza virus. this is in agreement with other studies showing that few children and young adults have immunity against a new viral infection [2, 11, 12] . our study of all 2009 h1n1 virus-infected children, in a single hospital, throughout the epidemic may have epidemiological implications, along with other studies based on data from admitted patients or gathered during a portion of the pandemic period [11] [12] [13] [14] . it has been reported that younger children (< 4-5 years old) with h1n1 virus infection tended to be admitted to hospital more frequently than older children or adults, and our results are compatible with these studies [2, 3, 6] . it has also been reported that certain risk factors are related to the likelihood of developing severe illness, including a younger age and particular underlying diseases, including obesity [6, 7, 15, 16] . in this study, we did not analyze obesity and young age as risk factors, and only 17 patients (7.8%) had underlying diseases. although the hospitalization rate was higher among the younger children (0-4 years of age), in this study, pneumonia and severe pneumonia were more prevalent in the 5-9-year-old group than in younger children. the explosive increase in the infection rate during two months (mid-october to mid-december) of the study period may be a typical pattern of spread of an acute respiratory viral infectious disease that has a short incubation period (1-3 days). this pattern is similar to those reported in other regions of korea and other countries [3, 4, 17] . vaccination against the 2009 h1n1 influenza virus in korea started on 18 november, 2009 for school-aged children, on 7 december, 2009 for children aged 6 months to 5 years and on 21 december, 2009 for adults with risk factors, which was after the peak of the epidemic. the rapid disappearance of the spread of infection might be the effect of vaccination and the strengthened individual hygiene, but the epidemiologic pattern suggests that an unknown herd immunity against a new viral infection might also be responsible [3, 17] . it is reported that patients with 2009 h1n1 virus infections had a relative leukopenia with lymphopenia [18, 19] . however addition to this finding, we found in the present study that the patients with pneumonia had a higher wbc count with lower lymphocyte differential, and the more severely affected patients had the highest wbc with the lowest lymphocyte differential in the early stages of the infection (within two days of fever onset). previous human studies of h5n1 virus infections have revealed that a lower lymphocyte count was associated with a poor outcome [20] , and mice infected with influenza viruses showed a lymphopenia and the h5n1 subtype was associated with marked lymphopenia with total lymphoid depletion [21] . therefore peripheral lymphocyte may be associated with the pathogenesis of acute lung injury in influenza virus infections [9] . altered crp and esr values were not prominent in 2009 h1n1 virus infections, but higher crp values were associated with a more severe illness. the male-to-female ratio was 1.1:1 among all patients, and 1.6:1 among the admitted patients. furthermore, male patients were predominant among those with pneumonia (3:1, 60/80) and those with respiratory distress who received corticosteroids (3.3:1, 13/17). other epidemiologic studies on children have reported a male predominance [16] , but some studies on inpatients have reported a female predominance [4, 7] . these findings suggest that genetic factors and possibly environmental factors are regarding the pathogenesis of lung injury in influenza virus infections, it has been believed that the viruses from upper respiratory tract spread to lower lung tissues and elicit the cytopathic reaction. however, some clinical and experimental studies have suggested that the innate and/ or cell-mediated immune reaction (t cell) with excessive production of cytokines of the host may also be responsible for the lung injury in influenza virus infection [9, [22] [23] [24] [25] . we experienced no intensive care patient in this series despite large subjects of the study, and it may be, at least in part, explained by a rapid use of corticosteroids on the patients with severe pneumonia [9, 10] . because this pandemic occurred over 40 years after last pandemic (hong kong flu), there have been no controlled-clinical trials for the efficacy of corticosteroids on influenza virus infections, although yearly seasonal influenza and small cases of sporadic h5n1 avian influenza virus infection have occurred during inter-pandemic period. the beneficial effect of corticosteroids in pneumonia caused by influenza virus infections may have resulted from reduction of systemic inflammation caused by immune cells and cytokines [26, 27] . our treatment policy which needs to be proven by controlled clinical studies in coming pandemics or in other influenza virus infections, may help to reduce the morbidity and possibly prevent the progression to fatal pneumonia [9, 10] . we have reviewed the rationale of our corticosteroid treatment and the host immune responses to viral insults in influenza virus infections, and proposed a new concept for the pathogenesis of acute lung injury in influenza virus infections, using a 'protein homeostasis system' of the host [9] . it has been reported that antiviral therapy (oseltamivir) is effective in the acute stages of influenza infection, including h1n1 virus infection in humans and experimental animals [4, 28] . in agreement with this, we found that the majority of patients (97%) defervesced within 48 h after medication and most pneumonic infiltrations in pneumonia patients had improved at discharge. there are some limitations to this study. compared with other studies, we had many uncomplicated inpatients owing to flexibility of our admission policy. because we did not perform extensive microbiological testing, such as viral cultures, paired-sample serologic studies and pcr for other pathogens, we cannot rule out the possibility of coinfection with other respiratory pathogens. in pandemic 2009 h1n1 virus infections, children of all ages were evenly affected, and males were predominant in pneumonia patients. early antiviral treatment was very effective in inducing rapid defervescence for the patients. the patients with 2009 h1n1 infections showed lymphopenia, and its severity was associated with the severity of the illness in the early stages. together this finding, the rapid improvement in clinical signs and the prompt resolution of severe 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lymphopenia and monocytosis may be considered as a surrogate marker of pandemic influenza a (h1n1) swine-origin influenza a (h1n1) in indian children human infection with highly pathogenic avian influenza virus (h5n1) in northern vietnam depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h5n1) virus isolated from humans fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia effect of antilymphocyte serum on influenza virus infection in mice (38047) effect of low-and highpassage influenza virus infection in normal and nude mice delayed clearance of viral load and marked cytoline activation in severe cases of pandemic h1n1 2009 influenza virus infection hydrocortisone infusion for severe community-acquired pneumonia. a preliminary randomized study pro: the illegitimate crusade against corticosteroids for severe h1n1 pneumonia treatment options for 2009 h1n1 influenza: evaluation of the published evidence there was no funding for this study. we thank drs. sung-ku kim, kyung-won bang, hyun-oh kim, ji-jung lee and ja-young hwang for patient care during the pandemic, and drs. jae-wook lee and joon-sung lee for careful review of this paper. the authors declare that they have no competing interests. all authors read and approved the final manuscript. kyl had primary responsibility for concept, design of the study and writing the manuscript; jwr participated in the preliminary data collection, data analysis and writing the manuscript; ysy participated in patient care and data analysis; jhk contributed to the interpretation of the data and editing of the manuscript and supervised the design and execution of the study; jck participated in reading of chest radiograph of the patients key: cord-016690-3gsq724l authors: li, hongjun title: hiv/aids related respiratory diseases date: 2013-09-30 journal: radiology of hiv/aids doi: 10.1007/978-94-007-7823-8_17 sha: doc_id: 16690 cord_uid: 3gsq724l lungs are the most commonly involved organ by hiv/aids related diseases, and pulmonary infections are the main reasons for the increasing death rate from aids. pathogens of hiv related pulmonary infections include parasites, fungi, mycobacteria, viruses, bacteria and toxoplasma gondii. according to international reports, pathogens have different geographical distribution, which is also closely related to the socioeconomic status of the region to produce varied aids related diseases spectra. for instance, in the united states, pneumocystis carnii pneumonia (pcp), tuberculosis and recurrent bacterial pneumonia (at least twice within 1 year) occur frequently in hiv infected patients. an international report published 10 years ago indicated that pcp is the most common and serious pulmonary opportunistic infections in hiv infected patients. now its incidence has dropped with the application of antiretroviral treatment and preventive measures. pcp will continue to occur initially in patients who are aware of their hiv infection. in addition, hiv related viral and parasitic infections have been reported both domestically and internationally. in this section, the clinical manifestations and imaging findings of hiv related pulmonary infections are analyzed and discussed, which provide effective diagnosis basis, so as to reduce the incidence of hiv-related pulmonary infections. lungs are the most commonly involved organ by hiv/aids related diseases, and pulmonary infections are the main reasons for the increasing death rate from aids. pathogens of hiv related pulmonary infections include parasites, fungi, mycobacteria, viruses, bacteria and toxoplasma gondii. according to international reports, pathogens have different geographical distribution, which is also closely related to the socioeconomic status of the region to produce varied aids related diseases spectra. for instance, in the united states, pneumocystis carnii pneumonia (pcp), tuberculosis and recurrent bacterial pneumonia (at least twice within 1 year) occur frequently in hiv infected patients. an international report published 10 years ago indicated that pcp is the most common and serious pulmonary opportunistic infections in hiv infected patients. now its incidence has dropped with the application of antiretroviral treatment and preventive measures. pcp will continue to occur initially in patients who are aware of their hiv infection. in addition, hiv related viral and parasitic infections have been reported both domestically and internationally. in this section, the clinical manifestations and imaging fi ndings of hiv related pulmonary infections are analyzed and discussed, which provide effective diagnosis basis, so as to reduce the incidence of hiv-related pulmonary infections. pneumocystis has been believed to be a kind of protozoon. recently, based on its ultrastructure and ribosomal rna phylogenetic analysis, pneumocystis is now believed to be a kind of fungus, with high affi nity to the lung tissues. due to the compromised immunity, 95 % aids patients sustain different types of pulmonary infections, of which pcp is the most common life-threatening opportunistic infection with an incidence rate of about 60-85 %. about 90-95 % patients suffering from aids complicated by pcp are adolescents and adults with their cd4 t cell counts being less than 200/ μl. clinical manifestations of typical pcp are fever, cough (dry cough without phlegm), dyspnea, chest distress and shortness of breath. dyspnea is shown as progressive difficulty in breathing, which initially occurs after physical activities and develops into diffi culty breathing even in resting state. pcp is commonly accompanied by weight loss, fatigue, anemia, general upset and lymphadenectasis. all these symptoms are non-specifi c, but patients often report subjective feelings of severe symptoms while physical signs are mild. by auscultation, the lungs are normal or with slightly dry, moist rales. these are the clinical fi ndings characteristic to aids complicated by pcp. in most patients with pcp, the serum ldh level increases but it is non-specifi c. in cases of aids complicated by pcp, the blood po2 reduces, commonly being lower than 70 mmhg in patients in the middle and advanced stages. the diagnostic imaging for pcp includes chest x-ray and ct scanning. due to the low resolution of chest x-ray, its demonstrations are negative for pcp patients in the early stage or only include thickened pulmonary markings and decreased pulmonary transparency. however, ct scanning demonstrates tiny lesions or more detailed changes in lungs. especially with the application of hrct, the detection rate of pcp lesions has been greatly improved. it has been internationally reported that nearly 10 % of pcp patients show negative fi ndings by the chest x-ray but with abnormal fi ndings by hrct. due to the rapid progression of pcp as well as its complex pathological changes, ct scanning demonstrations are diverse with specifi city. according to different pulmonary ct scanning demonstrations in different stages of the illness, pcp is divided into early stage (exudative and infi ltrative stage), middle stage (fusion and parenchymal stage) and advanced stage (absorption or fi brosis stage). the early typical manifestations include intrapulmonary multiple miliary nodules, mainly distributed in both middle and lower lung fi elds. it may be accompanied by enlarged hilar shadow, which should be differentiated from acute miliary tuberculosis. the middle stage is a period of infi ltration. as the disease progresses, miliary and patchy shadows fuse and expand into a dense infi ltrative shadow with even density, showing a diffuse ground glass liked change. the typical manifestations include bilaterally symmetric foci with the hilus as the center. the foci infi ltrates from the hilus to bilateral pulmonary interstitium, progressing from the both middle lungs to both lower lungs. hrct can more clearly demonstrate the foci, showing a map liked or gravel road liked appearance, with clearly demonstration of gas containing bronchus penetrating the foci. the pulmonary apex is involved later. the exterior stripe of the lung fi eld has increased transparency, showing typical willow leaf sign or moon bow sign which is the manifestation of compensatory pulmonary emphysema. during the late compensatory repair period, the intrapulmonary lesions are mainly parenchymal changes and fi brosis, with large fl aky high density shadows as well as cords liked and reticular changes. pneumothorax, mediastinal emphysema, pneumatocele, pleural effusion and other complications may occur, with an incidence of pneumatocele in about 10-20 % patients. the autopsy grossly demonstrates swelling of the lung tissue, and the alveoli are fi lled with large quantity foamy liquid. the pathological changes mainly manifested as interstitial pneumonia, with early manifestations of increased permeability of the capillary wall basement membrane in the alveolar walls, which leads to fl uid exudation. the pneumocystis carinii proliferate in large quantity and adhere to cause degeneration of the type i alveolar epithelial cells and shedding of the basement membrane. vascular congestion, edema as well as infi ltration of lymphocytes, plasma cells and mononuclear cells can be found in the pulmonary interstitium. due to the extensive existence of aspergillus in natural world, sputum smear positive often fails to defi ne its invasive infection. in the cases with aspergillus infection, hyphae can be found in the sputum. fungal infections often occur in patients with cd4 t cell count below 100/μl, of which the most common pulmonary infection is aspergillus infection, followed by penicillium marneffei infection. pulmonary infections caused by candida albicans and histoplasma are rarely found. the incidence of cryptococcal pulmonary infection is still in a disagreement, which is increasing recently. there are also some common endemic fungal infections, such as the most commonly found fungal infections of aids complicated by penicillium infections in guangxi zhuang autonomous region and hong kong, china. aspergillus has an extensive existence in the natural world. aids complicated by aspergillus infection is related to the application of corticosteroid hormone or broad-spectrum antibiotics, which occurs commonly in the advanced/critical stage of aids. the cases of pulmonary fungal infections, with fi ndings of hyphae (aspergillus or candida) or yeast in cytoplasm (histoplasma capsulatum) in tissue sections and simultaneous fi ndings of histiocytic reactions including the infi ltration of neutrophilic granulocyte and the necrosis of histocytes, can be diagnosed as having invasive fungal infection. candida albicans is yeast liked fungus, which is widespread in the natural world. it can parasitize in the mocous of skin, oral cavity, intestinal tract and vagina of the human being. candida albicans cannot cause disease in immunocompetent people but is pathogenic in immunocompromised population. after its invasion into the tissues, it turns into mycelia and multiplies in large quantity with great toxicity. it also has the ability to fi ght against phagocytosis. clinically, its infection is characterized by a chronic onset and clinical symptoms of low and moderate grade fever but rarely high fever, cough, shortness of breath, cyanosis, irritation or dysphoria. the pulmonary signs include weakened breathing sounds by auscultation and obvious moist rales of lungs. the serious cases may have symptoms of systemic poisoning. the illness is prolonged and repeated during its whole progression. by diagnostic imaging demonstrations, it can be divided into the following types: (1) bronchitis type, with chest x-ray demonstrations of increased pulmonary markings in lower fi elds of both lungs; (2) pneumonia type, commonly with accompanying extrapulmonary lesions. the lesions are mainly located in the middle and lower lung fi elds and lesions in the lower lung fi eld are more common. the apex is generally not involved. the lesions are recurring one after another. a small number of patients may sustain complications of exudative pleurisy. (3) disseminated type, with miliary shadows, diffuse nodular shadows or multiple small abscesses. the lesions often involve the middle and lower lungs. chest x-ray demonstrates thickened pulmonary markings and accompanying spots, small fl akes and large fl akes of parenchyma shadows, in manifestations of bronchial pneumonia. in some serious cases, the foci may fuse together and enlarge to involve the entire lobe. ct scanning demonstrates pulmonary nodules and few have ground glass liked changes of the lungs. pathological changes include acute infl ammatory lesions in the lungs, alveolar exudation and infi ltration of monocytes, lymphocytes and neutrophils. acute disseminated lesions often cause multiple small abscesses, central caseous necrosis, spores and hyphae in and around the lesions. histoplasma capsulatum belongs to moniliales family, deuteromycetes class and fungal kingdom, whose growth requires organic nitrogen. it is often isolated from the soil with abundant contents of birds or bats faeces and spreads along with chickens, birds, dogs, cats, and mice. when the conidia and mycelial fragments of histoplasmosis are inhaled, most can be expelled by the defense mechanism of the human body. granulomas may form, but in immunocompromised patients, it may cause disseminated histoplasmosis. when the cd4 t cell count in aids patients is less than 150/μl, histoplasma capsulatum infection of lungs may occur. histoplasma capsulatum pneumonia has a higher incidence in south america, africa and india. in the slight cases, the clinical manifestations are similar to symptoms of the cold, with low-grade fever, cough, and general upset. in the serious cases, there are symptoms of infl uenza, including chills, high fever, cough, chest pain, dyspnea, fatigue and poor appetite. in the cases of acute cavity, thin-walled cavity may form within a month. complications may be pericarditis, arthritis, skin nodules, rash fi brous mediastinitis and mediastinal granuloma. diagnostic imaging demonstrations are non-specifi c, with scattering pulmonary acinus exudation, multiple nodules in a diameter of about 3 mm with accompanying thickened septa, and formation of granulomas with accompanying calcifi cation. it should be differentiated from bacterial pneumonia, tuberculosis and other pulmonary fungal infections by laboratory tests to defi ne the diagnosis. the specifi city of the glycogen antigen detection of histoplasma capsulatum is up to 98 %. mucor spreads through the respiratory tract. it commonly invades the blood vessels, especially arteries. it reproduces locally or causes thrombosis and embolism. clinical manifestations are high fever, cough, sputum, shortness of breath, chest distress, chest pain and hemoptysis (pulmonary artery involvement). the diagnostic imaging demonstrates fl akes infl ammatory foci, with manifestations of pulmonary cavity and pulmonary infarction. the pathological changes are hemorrhagic infarction of local tissue, pneumonia and exudation of neutrophils. hemorrhagic infarction of local tissue may be related to hyphae induced minor arteries lesions. it is estimated that one third of the world population was/is infected with tubercle bacillus and 9 % of them are aids patients. the who reported that there are 88,000 newly infected patients of tb each year and 8.4 % of them are caused by aids. it is estimated that each year in 1,000 hiv infected patients, 35-162 sustain active tb, and there is a great risk of active tb progressed from the latent tuberculosis infection. hiv infected patients with tuberculosis are commonly young and middle aged adults, with more male patients than female patients. tuberculosis can occur at any stage of aids and at any level of cd4 t cell counts. it has been internationally reported that hiv infection complicated by tb has no specifi c imaging demonstrations. it has an acute onset, with an incidence of acute onset 2.5 times as high as that in non-hiv infected patients. the lesions are morphologically diverse, which are different from non-hiv infected patients with tb. hiv infection complicated by tb has commonly an acute onset, while tb in non-hiv infected patients is commonly secondary to other lesions, with cavities, fi brosis, pleural thickening and calcifi cation. a study conducted in china has demonstrated that for aids complicated by tb, the acute cases mainly have military and exudative lesions, with an incidence of 33 and 49 % respectively; while the incidence of chronic cases including cavity, fi brosis and calcifi cation is declining, being 11, 11 and 2 respectively. a later occurrence of tuberculosis in hiv infected patients indicates a more seriously immunocompromised immunity, with less typical clinical manifestations and imaging demonstrations. when the cd4 t cell count level is above 350-400/μl, the systemic symptoms are fever, chills, night sweating, fatigue, poor appetite and weight loss. respiratory symptoms are cough, expectoration, hemoptysis, chest pain and dyspnea. it manifests as primary tuberculosis, with its foci distributing in the middle and lower lungs, involving multiple lobes and segments. when the cd4 t cell count decrease, the impact of tb increase including the occurrence of extrapulmonary tuberculosis and disseminated disease. when the cd4 t cell count drops below 200/μl, pulmonary tuberculosis manifests as acute onset (such as miliary tuberculosis) or extrapulmonary tuberculosis (such as ileocecal tuberculosis) and peripheral lymph nodes tuberculosis. its difference from the clinical manifestations of non-hiv infected patients is as the following: (1) more common pulmonary infi ltration with multiple involvements and rare cavities; (2) higher incidence of dissemination (87-96 %) commonly along with blood fl ow and higher incidence of extrapulmonary tuberculosis (60-70 %); (3) more common lymph node tuberculosis, such as hilar, mediastinal and extrapleural lymphadenectasis; (4) lower positive rate of tuberculin test (ppd); (5) more patients with no expectoration, with sputum smear for acid-fast bacilli staining is negative; (6) higher incidence of resistant strains, high recurrence rate, and higher mortality (table 17 .1 ). foci in the cases with aids complicated by pulmonary tuberculosis are change quickly. after anti-tb treatment, the lesions are absorbed quickly. those receiving no anti-tb therapy, the foci tend to fuse together to form a mass or diffusely distribute. bacterial septicemia often occurs in aids patients. many opportunistic pathogens can cause respiratory infections, including bacterial bronchitis, pneumonia and pleuritis. the incidence rate of bacterial pneumonia (bp) is 3-5 %. bp has a larger range of impact on hiv infected patients than on non-hiv infected groups. repeated episode of bp is considered to be the fi rst manifestations of latent hiv infection. therefore, for those individuals who have recurrent pneumonia without other risk factors, they should be alert to hiv infection. the common pathogenic bacteria include streptococcus pneumoniae, staphylococcus aureus, rhodococcus equi, haemophilus and pseudomonas aeruginosa. as non-hiv infected patients, the most common pathogens of pneumonia are streptococcus pneumoniae and haemophilus infl uenzae. legionella and klebsiella are also common. many factors, such as the reduced t lymphocytes in hiv infected patients, manufacturing disorders of neutrophils, mononuclear cells and cytokines, and dysfunctional b lymphocytes, provide chances for opportunistic bacterial infections. in addition, the application of broad-spectrum antibiotics also increases the chance of opportunistic infections. bp can occur in any stage of hiv and at any level of cd4 t cell count. when the cd4 t cell count decreases, the incidence of bp also increases. the clinical manifestations of hiv infected patients are the same as non-hiv infected patients, with acute onset (3-5 days) , high fever (39-40 °c) , chills, chest pain, dyspnea, cough, purulent sputum (bloody or rusty). being different from non-hiv infection, pulmonary infection in hiv-infected patients is often recurrent. the imaging demonstrations of hiv/aids related bacterial pneumonia are similar to those in non-hiv infected patients. most cases of streptococcal pneumonia and haemophilus pneumonia have unilateral, confi ned and partially fused foci with accompanying pleural effusion. the imaging demonstrations include thickened and deranged pulmonary markings, alveolar fi lling of infl ammatory exudates with the progression of the illness, large fl aks infl ammatory infi ltration shadows or parenchymal shadows, bronchial gas fi lling phase in the parenchymal shadows. the lesions distribute along the pulmonary segments or lobes, rarely with accompanying pleural effusion. during the absorption period, the density of the parenchymal shadows gradually reduces and the scope narrows down. there may be cavities in some individual cases. but in most cases it is completely absent after poor effi cacy and more side effects favorable effi cacy and less side effects 3-4 weeks. lesions absorption are slow in elderly patients and recurrent patients, which is diffi cult to be completely absorbed and often develop into organic pneumonia. rhodococcus equi was initially discovered in 1923 and nominated as corynebacterium equi. after structure analysis of the cell wall, it was found to be different from corynebacterium, and therefore it is classifi ed into rhodococcus. rhodococcus equi is generally considered as the pathogens of horses, pigs and cattle. human rhodococcus equi infection is rare. but in recent years, due to an increase in patients with immunodefi ciency syndrome, reports of rhodococcus equi induced human respiratory infection and sepsis are increasing. rhodococcus equi is an intracellular facultative parasitic bacterium. its optimum temperature for growth is 30 °c, and suitable temperature for its growth is 10-40 °c. the acid-fast staining for rhodococcus equi shows uncertain results. due to its various morphology, it is commonly mistaken as diphtheroids bacilli, bacillus or micrococcus. on sheep blood agar plate, the bacterium can have synergistic hemolysis with staphylococcus aureus, mononuclear listeria and corynebacterium pseudotuberculosis. toxicity mechanisms of rhodococcus equi has been recently discovered the existence of toxic plasmid, which provides a new idea for the full understanding of its pathogenesis. clinical symptoms are usually cough, orange red sputum, high fever and other symptoms. e marchiori et al. in 2005 studied fi ve cases of aids complicated by rhodococcus equi pulmonary infection. all the patients had a case history of cough and fever history for 1-2 months with accompanying shortness of breath and chest pain. li et al. in 2009 [ 106 ] studied a group of 13 cases. all patients had fever, with a body temperature being 38-40 °c, cough, orange red sputum. the typical clinical manifestations of the disease are fever, dyspnea and chest pain. other symptoms such as weight loss, diarrhea and joint pain are not representative. based on the course of the disease, the diagnostic imaging demonstrations of rhodococcus equi pulmonary infection can be divided into early stage, showing round liked fl aky blurry shadows surrounding unilateral hilum that has blurry boundary; middle stage (parenchymal change), showing central sphere liked high density shadow surrounding unilateral hilum, in parenchymal changes and with clear boundary; advanced stage (necrosis) showing secondary cavity of the pulmonary mass, possibly with hydropneumothorax and pleurisy. the imaging demonstrations are characteristic, but lack of specifi city. and it should be differentiated from pulmonary tumors. the pathological changes include the most commonly chronic suppurative bronchopneumonia and extensive pulmonary abscesses. the histopathology demonstrates massive bleeding in alveolar space, large quantity erythrocytes, intact cellular wall and large quantity epithelial cells. the predominant pathological changes may also be fi broblasts, with parenchymal changes of lung tissue and thickened alveolar septa. accumulating piles of strip liked purple rhodococcus equi can be found by pas staining. common pathogenic viruses of the opportunistic pulmonary infections in hiv infected patients are cytomegalovirus (cmv) and infl uenza virus. cmv is the most common pathogen of hiv/aids related pulmonary infection. by autopsy, 49-82 % patients with hiv/aids have cmv infection, only second to pneumocystis carinii pneumonia. moskowitz et al. [ 32 ] reported that among the direct causes of death in aids patients, 19 % is due to pulmonary cytomegalovirus infection. because of the lack of typical clinical manifestations and sensitive examinations for its early diagnosis, the defi nitive diagnosis rate of cytomegalovirus pneumonia is only 13-24 % before autopsy. the clinical manifestations of cmv infection are non-specifi c. the systemic symptoms are fever, soreness of joints and muscles. respiratory symptoms are paroxysmal dry cough, progressive shortness of breath, diffi culty in breathing during activities. pulmonary cmv infection may develop secondary fungal infection or be complicated by bacterial, fungal, and pneumocystis carinii infections. the cytomegalovirus can widely spread in the organs and tissues of the infected patients, and the infections can directly lead to the damage of infected host cells. in addition, the virus can also cause pathogenic effects via immune pathological mechanism. some scholars classifi ed cmv pneumonia into diffuse, miliary necrosis and cytomegalic. diffuse and cytomegalic cmv pneumonia are often accompanied by diffuse alveolar damage (dad), which is more common in the diffuse type of cmv pneumonia but less common in cytomegalic type of cmv pneumonia. the pathological basis of diffuse small nodules in lungs is hemorrhagic necrosis. sometimes cmv infection is concurrent with other infections in the lungs, and even co-infects one cell. pulmonary parenchymal changes indicate bacterial and fungal infections, such as fi ndings of inclusion bodies in the cells, commonly known as eagle's eye sign. the imaging demonstrations of cytomegalovirus pneumonia are diverse. some studies summarize that the lungs commonly have manifestations of diffuse interstitial infi ltration or alveolar infi ltration, with ground glass liked changes, pulmonary parenchymal changes, grid liked changes, thickend bronchial wall, bronchiectasis, pulmonary nodules or masses. the principal changes include the early lesions of ground glass liked changes and advanced lesions of pulmonary masses. lymphoid interstitial pneumonia is the abnormal hyperplasia of the pulmonary lymphoid tissue. its occurrence is related to autoimmune diseases, and is believed to be a direct response of the lungs to hiv. the clinical manifestations are recurrent infections, poor appetite, hepatomegaly and splenomegaly, and arrested development. the diagnostic imaging demonstrates no characteristic changes by ct scanning, with thickened bronchial wall, diffuse central lobular nodules or bronchiectasis, grid liked and cords liked shadows in uneven thickness. the pathological changes include accumulating lymphocytes and plasma cells that are mixed to infi ltrate the pulmonary interstitium and expand to surrounding areas of the bronchi. toxoplasma pneumonia is caused by the infection of the intracellular parasite, toxoplasma gondii. ludlam et al. in 1963 fi rstly proposed the concept of pulmonary toxoplasmosis, which was believed to cause atypical pneumonia [ 107 ] . the clinical manifestations are cough and expectoration. in the serious cases, dyspnea and cyanosis can occur. in the chronic cases, there are long term low grade fever, cough, weight loss and enlarged lymph nodes. the diagnostic imaging demonstrates bronchopneumonia, interstitial pneumonia and pleurisy. (1) bronchial pneumonia is also known as lobular pneumonia, with scattered patchy and blurry density shadows. (2) interstitial pneumonia has typical manifestations of reticular and nodular shadows. (3) pleurisy is rare, showing pleural effusion, limited diaphragmatic activity. the imaging demonstrations are non-specifi c, which can be defi ned in combination with the etiologic examinations. the pathological changes are congestion and edema of the surrounding connective tissue of the alveolar wall and bronchial walls, widened pulmonary interstitium, small quantity serous fi brin exudation from alveoli and pulmonary interstitium, and infi ltration of macrophages and lymphocytes. toxoplasma cysts and tachyzoites may be found in pulmonary interstitium and macrophages as well as alveolar epithelium. kaposi's sarcoma, a vascular tumor, was discovered in 1872, and is also known as multiple hemorrhagic sarcomas, multiple vascular sarcomas, or multiple pigmented sarcomas. kaposi's sarcoma is believed to be the defi ning tumor of aids. outbreak of ks occurred in male homosexuals in europe and the united states. data show that in about 30 % caucasian homosexuals, kaposi's sarcoma is a major complication of in hiv/aids patients. it has been confi rmed that, though kaposi's sarcoma has strong invasion, the disease itself has little impact on the mortality of aids. the cause of death in majority of the patients is still opportunistic infections. the clinical manifestations include face and neck lesions in dark red to purple red plaques. the plaques do not fade away when pressed, with surrounding brown ecchymosis. it commonly involves multiple organs including lungs, spleen, oral cavity, lymph nodes, gastrointestinal tract and liver. the lungs are the major target of invasion. the diagnostic imaging demonstrates hilar lymphadenectasis and its surrounding nodular infi ltration, bilateral interstitial changes, and pleural effusion that are its typical x-ray demonstration. early pathological changes are similar to those of common angioma; with gathering of capillaries into groups containing histocytes engulfed hemosiderin and orderly arranged vascular endothelial cells. it further progression see active proliferation of endothelial cells and fi broblasts, increased nuclear mitosis with anaplasia, and scattered lymphocytes and histocytes between blood vessels. in the advanced stage, occlusion and necrosis of the vascular lumen can be found. irregular lumen and fi ssures of the new capillaries can be commonly found in the tumor, fi lled with blood and common hemorrhage. in china, ks is relatively rare. its defi nitive diagnosis can be made by pathological examination. other hiv/aids related malignancies include burkitt's lymphoma, non-hodgkin's lymphoma, hodgkin's lymphoma and lung cancer. in summary, hiv/aids related pulmonary infections are important diseases in the disease spectrum of hiv/aids imaging. the diagnostic imaging is irreplaceable examinations for pulmonary infections. early prevention and correct diagnosis are the keys to improve the quality of life and prolong the lives of patients. the complexity and multiplicity of hiv/aids related pulmonary diseases present challenges for the clinicians. firstly, hiv/aids related diseases should be optimally classifi ed. each type should has a disease spectrum, which can be used for exclusion in combination with immunological indices to make the diagnosis and differential diagnosis. the diagnosis of hiv/ aids related pulmonary infections should be made in combination with case history and laboratory tests for targeting individualized diagnosis to serve clinical practice. carnii pneumonia (pcp) the pathogen is the trophozoites and cysts produced by pneumocystis carinii, principally living in the lungs. pneumocystis carinii was used to being categorized as as protozoon, but recently, it is believed to be belonged to fungus according to its ultrastructure and pneumocystis ribosomal rna phylogenetic analysis. the main infection route of pcp is airborne transmission and reactivation of in vivo latent pneumocystis carinii. infl ammatory and immune responses of the host include phagocytosis of pneumocystis carinii by the alveolar macrophages, infi ltration of lymphocytes in peribronchial and vascular area, proliferation of type ii alveolar cells, local and systemic increase of antibody. by naked eyes observation, there are extensive and diffuse invasion of lungs, which is soft like waterlogged sponge and in milky white with black spots. the fi lled foamy substance in the alveoli and bronchioles is a mixture of necrotic fungus and immunoglobulin. the alveolar septum has infi ltration of plasma cells and lymphocytes, resulting in thickened alveolar septa up to 5-20 times as the normal thickness that occupy 3/4 of the entire lung volume. the cysts are fi rstly located in the macrophage cytoplasm of the alveolar septa. subsequently, the alveolar cells containing cysts sheds off into the alveolar space. after the rupture of the cystic wall, sporozoite is discharged to turn into free trophozoites, which gains its access into the alveolar space. the alveolar exudates include plasma cells, lymphocytes and histocytes ( fig. 17 .1a-c ). the clinical symptoms include dry cough, shortness of breath and an indoor hypoxia. about 95 % aids patients have multi-pathogens induced pulmonary infections. the most signifi cant laboratory abnormality in most pcp patients is hypoxemia. based on correlation between pcp and arterial oxygen partial difference, hypoxemia is divided into three degrees. the slight cases at indoor conditions have their pao 2 being above 70 mmhg, or alveolar-arterial oxygen pressure difference being less than 35 mmhg, or both. the moderate and severe cases have their pao 2 being usually less than 70 mmhg, or alveolar-arterial oxygen pressure difference being above 35 mmhg, or both. the most common manifestations of aids complicated by pcp are progressive subacute onset of dyspnea, fever, dry cough and chest distress, the symptoms aggravating in a few days or weeks. pulmonary examination is usually negative in slight cases. as the disease aggravates, the cases show shortness of breath, cyanosis, tachycardia, and diffuse dry rales. pneumocystis carinii infection accounts for 60-85 % of aids patients, which is one of the major causes of death in aids patients. the diagnostic imaging examinations include chest x-ray, ct scanning and nuclear medicine examination. (1) chest x-ray is the conventional examination for screening. early lesions tend to be missed for the diagnosis due to the limited resolution or atypical lung lesions. (2) ct scanning with high resolution is superior to chest x-ray. (3) nuclear medicine examination demonstrates increased uptake of the isotope-labeled monoclonal antibodies in the lung tissues of the pcp patients. (1) by tracheal suction or lung tissue biopsy, the detection rate of pneumocystis carinii is up to 90 %. by tissue section staining, abundant protozoa can be found in intra-alveolar foamy eosinophil substance mass (by methenamine silver nitrate staining, the dark brown round or oval shaped cysts can be found in a diameter of 6-8 μm out of the cells). (2) by elisa, pneumocystis igg antibody can be detected and by latex particle agglutination test, the protozoa antigen can be detected. (3) molecular biology techniques, such as pcr can be applied for early diagnosis. the following examinations are non-specifi c, but can be used to assess the severity of pcp and its progression. (1) by arterial blood gas analysis, the patients may show reduced blood oxygen saturation and respiratory alkalosis. (2) by serum enzyme spectrum analysis, the patients may show increased ldh. (3) it can be detected to have increased alveolar-arterial oxygen partial pressure difference. in the early stage (exudation period), alveolar fl uids exudate, with diffuse granular shadows in the bilateral lung fi elds extend from the hilum to the surrounding. in the middle stage (infi ltration and fusion period), the intrapulmonary lesions fuse, with ground glass liked or cloudy shadows that are bilaterally symmetric like butterfl y wings. in the middle and advanced stages (parenchymal changes period), the lung tissues show parenchymal changes, with high density shadows and accompanying air bronchogram. the lung periphery shows stripes of transparent shadows. in the advanced stage (pulmonary fi brosis period), the pulmonary interstitium is thickened in dense cords liked appearance, with interval irregular patchy shadows. the pulmonary ventilation improves and the lung periphery shows dense parenchymal shadows with emphysema, pneumomediastinum and pneumothorax. in the early stage (exudation period), the lesions radiatus develop from the hilum to lung fi eld. in the early stage, the diffuse exudative lesions distribute as pulmonary acinus, with changes similar to pulmonary interstitial changes. it was believed to be interstitial pneumonia. however, acute pcp is actually exudation of alveoli and spaces containing a male patient aged 31 years was confi rmatively diagnosed as having aids by the cdc. he complained of dyspnea, cyanosis and wheezing for 3 weeks, with obviously decreased oxygen saturation. his cd4 t cell count was 45/μl. a female patient aged 37 years was confi rmatively diagnosed as having aids by the cdc. she complained of dyspnea, cyanosis and wheezing for 8 weeks, with obviously decreased oxygen saturation. her cd4 t cell count was 45/ μl. patients with acquired immunodefi ciency. the early symptoms include fever, dry cough and shortness of breath. the advanced symptoms are serious dyspnea, cyanosis, progressive hypoxemia and respiratory failure. by pulmonary examinations, scattered dry and moist rales can be heard. trophozoites of pneumocystis cysts can be found by liquid giemsa staining. slight and moderate interstitial infl ammation responses mainly involve lymphocytes and alveolar macrophages. the detection of cysts containing sporozoites is the basis to defi ne the diagnosis. chest x-ray chest x-ray demonstrations of pcp can be classifi ed into four types. (1) early pulmonary interstitial infi ltration and diffuse miliary alveolar exudation; (2) in the middle stage, there are alveolar exudates, with fusion and parenchymal changes; (3) in the middle-advanced stage, diffuse parenchymal changes; (4) pulmonary interstitial fi brosis and lung cavity or lung bulla, as well as pneumothorax and emphysema. for the cases with negative or atypical fi ndings by chest x-ray, ct scanning with high resolution should be performed. ct scanning demonstrates early lesions of multiple symmetric diffuse miliary nodal shadows, which have clear boundaries. in the middle stage, there are thin cloudy shadows or ground glass liked density shadows. in the middleadvanced stage, the lung tissues show parenchymal shadows, with trachea-bronchial sign. in the outer strip of the lung, a transparent area in shape of willow leaf can be demonstrated. in the advanced stage, fi brous cords liked shadows are demonstrated some lung tissues with compensatory emphysema and even pulmonary pseudocysts. the intake of the isotope-labeled monoclonal antibody by lung tissues of pcp patients increases. hiv/aids related pcp should be differentiated from bacterial pneumonia, pulmonary tuberculosis, viral pneumonia, fungal pneumonia, ards, and lymphocytic interstitial pneumonia (lip). bacterial pneumonia has more focal lesions but less diffuse lesions. pulmonary mycobacterium tuberculosis infection has manifestations of military pulmonary tuberculosis by chest x-ray, which is diffi cult to be differentiated from early pcp. hiv/ aids related pcp shows miliary nodules, which further fuse into cloudy or ground glass liked shadows or parenchymal changes. the lesions are commonly symmetrical, with the hilus as the center. the clinical manifestations include fever, dry cough or accompanying diffi culty breathing, and even cyanosis. but in the cases of pulmonary mycobacterium tuberculosis infections, most show miliary nodules, which further fuse into large nodules or mass. after about 2 weeks treatment in the early stage, the military nodules in both lungs can be absent, with common clinical symptom of high fever. correlation studies of miliary tuberculosis and peripheral blood cd4 t cell count have demonstrated that the general incidence of miliary tuberculosis is low, only 6-9 %, but it is the main manifestation of hiv/aids related pulmonary miliary tuberculosis. generally, when cd4 t cell count is below 200/μl, the incidence of cavity lesions is 29 %, noncavity lesions 58 %, complicated by pleural effusion 11 % and lymphadenectasis 20 %. when cd4 t cell count is between 200 and 390/μl, the incidence of cavity lesions and non-cavity lesions each accounts for 44 %, complicated pleural effusion 11 % and lymphadenectasis 14 %. when cd4 t cell count above 400/μl, the manifestation is commonly pneumonia type, in fl aky shadows or parenchymal shadows in just one pulmonary segment. the incidence of cavity lesions is 63 %, non-cavity lesions 33 %, complicated by pleural effusion 3 % and no lymphadenectasis. chest x-ray demonstrates cytomegalovirus pneumonia negative in 1/3 patients. the foci are commonly bilateral, with reticular particles in 33 % patients, alveolar foci in 22 % patients, nodular foci in 11 % patients, complicated by cavity in 11 % patients, cysts in 6 % patients, pleural effusion in 33 % patients and lymphadenectasis in 11 % patients. the incidence of diffuse foci in the cases of cryptococcus neoformans pneumonia is 76 %, interstitial foci or mixed foci 76 %, alveolar foci 19 %, nodular foci 5 %, lymphadenectasis 11 % and pleural effusion 5 %. hiv/aids related pcp is more likely to occur in children with aids, which presents diffi culty for its differentiation form lymphoid interstitial pneumonia. however, lymphoid interstitial pneumonia commonly has a chronic onset, with commonly manifestations of cough and dry rales. systemic lymphadenectasis and enlargement of salivary glands can also be found. by lung tissues biopsy, ebv-dna1 can be detected, which provides basis for their differentiation. pneumocystis, a unicellular organism, is the pathogen of pneumocystis carinii pneumonia. pneumocystis carinii pneumonia is one of common opportunistic infections in aids patients, which is also the leading cause of death in aids patients. in the initial episode of pcp, most patients have a cd4 t cell count of less than 100/μl. diagnostic imaging demonstrates bilaterally symmetrical ground glass liked shadows, which can be diffusely distributed and tend to mainly involve the periphery of the hilus or the middle and lower lung fi elds. hrct scanning is commonly applied to assess early pcp that is demonstrated negative by chest x-ray. hrct scanning demonstrates bilaterally symmetric patchy or fused ground glass liked shadows. the pathological basis of ground glass liked shadows and parenchymal areas refl ect that the acinus is fi lled by the foamy exudates, which are composed of surface active substances, cellulose and cell debris. all of the ground glass liked shadows, overlapping septa and the intralobular linear shadows are in gravel road liked manifestation. the septa and intralobular linear shadows demonstrate pulmonary interstitial edema or cellular infi ltration. in the middle-advanced stage of pcp, there are manifestations of small pulmonary nodules, pulmonary parenchymal changes, thickened interlobular septa, intralobular linear shadows, mass like lesions, pleural effusion, and lymphadenectasis. the cysts tend to mainly involve the upper lobes, which can be unilateral or bilateral pulmonary cysts, pneumothorax, mild or severe interstitial fi brosis and traction bronchiectasis. hrct scanning demonstrations of pcp are non-specifi c. its diagnosis should be in combination with hivph13 and etiological examinations. bacterial infections mycobacterium tuberculosis is still an important pathogen for pulmonary infection in hiv positive patients. since the mid-1980s, the main cause of the increasing incidence of tuberculosis is the prevalence of hiv infection. the incidence of tuberculosis in aids patients is 200-500 times higher than the general population. hiv infection is the most dangerous factor for progression of latent tuberculosis into active tuberculosis. tubercle bacillus belongs to mycobacterium family of mycobacterium genus, which is divided into types of human, bovine and murine. the main cause of human tuberculosis is human mycobacterium tuberculosis, which is known as acid-fast bacilli. tubercle bacillus wall is the complex containing high molecular weight fatty acids, lipids, proteins and polysaccharides, which are related to its pathogenicity and immune responses. lipid can cause the infi ltration of human monocytes, epithelial cells and lymphocytes to form tuberculous nodules. its protein contents can cause allergic reactions, and infi ltration of neutrophils and mononuclear cells. polysaccharides participate in certain immune responses (such as agglutination). these pathogenic factors lay the foundation for the occurrence of tuberculosis in aids patients. human immunity, allergic responses as well as the number and pathogenicity of tubercle bacilli are closely related to the quality, range, spreading rate and the progression of tuberculosis. its pathological changes are characterized by exudation, infi ltration, proliferation and hyperplasia, degenerative necrosis (caseous necrosis) and cavity formation. the manifestations include congestion, edema and infi ltration of leukocytes. the exudative lesions occur in early stage of tuberculosis infl ammation or when the lesions deteriorate. it can also be found in the serosa tuberculosis. there is neutrophilic granulocytes in the exudative lesions, which are gradually substituted by monocytes (phagocytes). the engulfed tubercle bacilli can be found in the large mononuclear cells. the exudative lesions are absorbed and dissipated through the phagocytosis of the mononuclear-phagocyte system, even with no scar. when large mononuclear cells engulf and digest tubercle bacilli, the phospholipid of the bacteria render the large mononuclear cells to enlarge and be fl at, similar to epithelial cells, which is known as epithelioid cells. these epithelioid cells gather into groups, with central langhans giant cells that pass the messages of the bacteria antigens to lymphocytes. surrounding the langhans giant cells, there are often many lymphocytes to form typical tuberculous nodules, which are characteristic lesions of tuberculosis. this is why it is called tuberculosis. in the tuberculous nodules, tubercule bacilli are usually undetectable. proliferation based lesions often occur in the cases with less bacteria invasion and when human cells mediated immunity is predominant. degeneration often occurs on the basis of the exudative or proliferative lesions. tubercle bacilli overcome macrophages and then continually proliferate in large quantity. after the cells become cloudy and swelling, the foci show fatty degeneration, dissolved into fragments, until the occurrence of necrosis. after the death of infl ammatory cells, proteolytic enzymes are released to dissolve the tissues that results in necrosis, which is coagulative necrosis. by naked eyes observation, they are yellowish gray, with loose and brittle quality like caseous. therefore it is known as caseous necrosis. microscopic examination demonstrates an area of solid and eosin staining red necrotic tissues with no tuberculosis. tubercle bacilli in the foci of caseous necrosis proliferate in large quantity to cause liquefaction, which is related to infi ltration of neutrophile granulocytes and large monocytes. part of liquefi ed caseous necrotic substances can be absorbed and part can be discharged by the bronchus to form cavities. otherwise, it may cause intrapulmonary spreading along with bronchi. the small caseous necrosis or proliferative lesions can be shrunk and absorbed after treatment, with only residues of slight fi brous scars. due to the compromised immunity in aids patients, the lesions rarely show fi ber tissues proliferation, but form cords liked scar. calcifi cation rarely occurs. if the necrotic lesions erode the blood vessels, tubercle bacilli can cause systemic miliary tuberculosis along with blood fl ow, including brain, bones and kidneys. large quantity sputum containing tubercle bacilli gains its access into the gastrointestinal tract. it can also cause intestinal tuberculosis and peritoneal tuberculosis. pulmonary tuberculosis can cause tuberculosis pleurisy via direct spreading to the pleura ( fig. 17 .19a-c ). clinically, it is a chronic progression, with rare acute onset. the clinical symptoms are commonly systemic, with fever and fatigue. the respiratory symptoms include cough and hemoptysis. pulmonary tb can be divided into primary and secondary, with the initial episode commonly being primary (type i). the residual bacteria after primary infection can cause secondary infection (type ii-iv) when the immunity is compromised via spreading along blood fl ow or direct spreading. it is common in hiv positive children. most cases are asymptomatic, sometimes with symptoms of low grade fever, mild cough, sweating, rapid heartbeat, and poor appetite. hiv/aids related miliary tuberculosis is one of the major manifestations of pulmonary tuberculosis, which is more common. the onset of acute miliary tuberculosis is rapid, with symptoms of chills and high fever with a body temperature up to 40 °c, mostly remittent fever or continuous fever. there may be decreased leukocytes count and accelerated sedimentation rate. the progression of subacute and chronic hematogenous disseminated pulmonary tuberculosis is relatively slow. infi ltrative pulmonary tuberculosis in aids patients commonly occurs in both middle and lower lung fi elds, with fl aky and fl occulent foci or parenchymal changes in lobes or segments. caseous lesions are rare. the early stage of infi ltrative pulmonary tuberculosis is commonly asymptomatic, with later occurrence of fever, cough, night sweating, chest pain, weight loss, expectoration and hemoptysis. this type of pulmonary tb rarely occurs in aids patients. in non-aids patients, chest x-ray demonstrates three major changes, namely cavity, fi brosis, and bronchial dissemination. in the aids patients, the pulmonary manifestations include single or multiple nodular shadows with clear boundaries. tuberculous pleuritis is an exudative infl ammation caused by the direct invasion of tubercle bacillus from the primary lesion near the pleura into the pleura, or hematogenous dissemination via the lymphatic vessels to the pleura. the routes for occurrence of tuberculous pleurisy include: (1) the bacteria in the hilar lymph tuberculosis counterfl ow to the pleura along lymph vessels. (2) tb lesions adjacent to pleura rupture to cause direct access of the tubercle bacilli or products of tuberculosis infection into the pleural cavity. (3) acute or subacute hematogenous disseminated tuberculosis causes pleuritis. (4) due to the increased allergic responses, the pleura highly respond to tuberculosis toxins to cause exudation. (5) thoracic tuberculosis and rib tuberculosis rupture into the pleural cavity. clinically, pleuritis can be divided into three types, dry pleuritis, exudative pleuritis and tuberculous empyema (rare). the common clinical manifestations are fever, cough with accompanying chest pain of the affected side and shortness of breath. (1) sputum smear examination is simple to manipulate, with high accuracy rate. the fi ndings of the tubercle bacilli can defi ne the diagnosis. it still is the golden criteria for the diagnosis of pulmonary tuberculosis. (2) sputum tubercle bacilli culture has high reliability. tubercle bacilli drug sensitivity test can be performed but requires 6-8 weeks to obtain the results. therefore, its application is limited. (1) tuberculin purifi ed protein derivative (ppd) test is commonly used. its positive result is one of the evidence confi rming a past history of tb infection. (2) bactec test can be performed to detect the metabolites of mycobacterium tuberculosis. generally, mycobacterium can be detected in 2 weeks. the quantity of mycobacteria can affect the period required for test results. (3) pcr has poor specifi city but high sensitivity of up to 98-100 %. both can be applied to observe the enlarged lymph nodes in the chest and mediastinum. in addition, they can be applied to obtain specimens for biopsy, which facilitates the diagnosis and differential diagnosis. diagnostic imaging examinations include chest x-ray and ct scanning. chest x-ray can demonstrate the location, quality and range of the lesions. it can also help to assess the therapeutic effi cacy. ct scanning can demonstrate small or hidden lesions, with a high resolution. primary pulmonary tuberculosis, also known as primary syndrome, is rare in adult aids patients. chest x-ray demonstrates intrapulmonary patchy or large fl aky parenchymal changes, hilar and mediastinal lymphadenectasis in connection to irregular cords liked shadows (located between intrapulmonary lesion and the hilum). lymph node tuberculosis is demonstrated to have mediastinal lymphadenectasis that sometimes fuse into mass. in aids patients, simple mediastinal lymph node tuberculosis is more common than primary syndrome. tuberculosis (1) the acute cases are demonstrated to have diffused miliary nodules in both lungs with even distribution, even size and even density. (2) the subacute and chronic cases are demonstrated to have nodules in both lungs, with uneven distribution, uneven size and uneven density. sometimes calcifi cation occurs in the nodules, with fi brous cords and thickened pleura. infi ltrative pulmonary tuberculosis are demonstrated to have patchy parenchymal changes in the middle and lower lung fi elds as well as parenchymal changes, cavities and fi brous cords liked foci in the segments and lobes. it can also occur in the upper lung fi elds, commonly with accompanying mediastinal and hilar lymph node tuberculosis. it commonly occurs in the advanced stage of aids,, with manifestations of pulmonary interstitial fi brosis and formation of cavities. this type of pulmonary tuberculosis is less common. it rarely occurs, mostly in the early stage of aids. it is rare in the middle and advanced stages of aids. dry pleuritis has manifestations of blunt costophrenic angle and limited diaphragm mobility. exudative pleuritis is manifested as small quantity pleural effusion and thickened pleura, commonly with encapsulated effusion of the lateral pleura. calcifi cation is rare. a male patient aged 28 years was confi rmatively diagnosed as having aids by the cdc. he complained of dull chest pain, dyspnea, fever, night sweating, fatigue and anorexia. his cd4 t cell count was 65/μl. a female patient aged 36 years was confi rmatively diagnosed as having aids by the cdc. she complained of dull chest pain, dyspnea, fever, night sweating and fatigue. her cd4 t cell count was 85/μl. a male patient aged 41 years was confi rmatively diagnosed as having aids by the cdc. he was infected hiv via blood transfusion, with complaints of cervical lymph node tuberculosis, ascites and abdominal infection, fungal stomatitis, biliary stones with infection and severe anemia. his cd4 t cell count was 33/μl. a female patient aged 39 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and night sweating. her cd4 t cell count was 73/μl. a female patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. she was hospitalized due to complaints of chest distress, cough and expectoration for 2 months, with after noon low grade fever and weight loss. on admission, she was confi rmed hiv positive and a cd4 t cell count of 120/μl. a female patient aged 37 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and chest pain for 2 months, with acid-fast bacilli positive in the pleural fl uid culture. her cd4 t cell count was 71/μl. a female patient aged 40 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and chest pain for 2 months, with acid-fast bacilli positive in the pleural fl uid culture. her cd4 t cell count was 891/μl. a female patient aged 34 years was confi rmatively diagnosed as having aids by the cdc. she complained of fever and chest pain for 2 months, with acid-fast bacilli positive in the pleural fl uid culture. her cd4 t cell count was 51/μl. cough expectoration, chest pain, dyspnea, fever, night sweating, fatigue, anorexia, lymphadenectasis and rapid progression of the conditions. ppd skin test with a resulted diameter of more than 5 mm should be considered as tuberculosis infection. but its positive rate remains low. the culture of sputum and bronchoalveolar lavage fl uid can detect the pathogens. nucleic acid analysis or dna probe technique, pcr and chromatography can be applied to detect tubercle bacilli. the commonly used diagnostic imaging examinations include chest x-ray and ct scanning. the main demonstrations include (1) intrapulmonary and extrapulmonary lymphadenectasis; (2) miliary tuberculosis manifestations; (3) infi ltrative (pneumonia type) pulmonary tuberculosis; (4) pulmonary interstitial fi brosis, cavity, pulmonary emphysema, nodules, emphysema, and bronchiectasis with accompanying infections. in the cases with their cd4 t cell count being above 400/μl, the imaging demonstrations are similar to those of non-hiv/aids patients with pulmonary tuberculosis. in the cases with their cd4 t cell count being above 400/μl, the manifestations are intrapulmonary large fl aky parenchymal changes, surrounding satellite lesions as well as mediastinal and hilar lymphadenectasis. sometimes the manifestations may be only mediastinal lymphadenectasis and their fusion into mass. in the cases with cd4 t cell count being above 200/μl, there may be accompanying extrapulmonary tuberculosis, such as tuberculous peritonitis, bone tuberculosis, brain tuberculosis and splenic tuberculosis. in the cases with their cd4 t cell count being lower than 100/μl, the manifestations are mostly miliary tuberculosis. hiv/aids related tuberculosis should be principally differentiated from pneumocystis carinii pneumonia, fungal infections, other pneumonia and lung cancer. hiv/aids related tuberculosis should be differentiated from pcp. pcp is mainly manifested as multiple lesions with hilum as the center to extending symmetrically to outside of the lungs. in the advanced stage, pcp has main lesions of pulmonary interstitial fi brosis, with less accompanying mediastinal and hilar lymphadenectasis. the laboratory tests can facilitate to defi ne the diagnose. hiv/aids related tuberculosis should be differentiated from fungal infections. fungal infections are relatively less common than tuberculosis. the imaging fi ndings of hiv/aids related pulmonary fungal infections are diverse, with manifestations of miliary, fl aky fl occulent liked, parenchymal, mass, and interstitial changes. in general, the diffuse lesions are mainly interstitial changes, while confi rmed lesions, compared to tb lesions, are more likely to have thick walled cavities. satellite lesions of fungal infections are less than those of tuberculosis, with less accompanying mediastinal and hilar lymphadenectasis. sometimes laboratory tests are necessary to defi ne the diagnosis. hiv/aids related tuberculosis should be differentiated from non-tuberculosis mycobacteria pneumonia. their imaging fi ndings are similar to each other, which presents challenges for their differential diagnosis. molecular biology examinations play an important role in the differentiation. hiv/aids related tuberculosis should be differentiated from other pneumonia. non-bacterial pneumonia (mycoplasma, viral and allergic) often shows patchy shadows, which are similar to the manifestations of early infi ltrative pulmonary tuberculosis. when bacterial pneumonia shows lobar lesions, it may be confused with tuberculous caseous pneumonia, which should also be differentiated for the diagnosis. symptoms of mycoplasma pneumonia are mild, with imaging fi ndings being always inconsistency with the clinical symptoms, which usually subside within 2-3 weeks. in the cases of allergic pneumonia, eosinophils in the blood increase, with intrapulmonary mobile shadows, which are the basis for their differentiation. bacterial pneumonia can have acute onset, with chills, high fever, rust colored sputum, and streptococcus pneumoniae positive. recovery is rapid after antibiotic treatment and all these symptoms can subside within 1 month. hiv/aids related tuberculosis should be differentiated from pulmonary abscesses. in the cases of infi ltrative pulmonary tuberculosis with cavities, it should be differentiated from pulmonary abscess. especially, tuberculosis with cavities in the apical segment of inferior lobe should be differentiated from acute pulmonary abscess. chronic fi brous cavity tuberculosis should be differentiated from chronic pulmonary abscess. the key points for the differentiation are tubercle bacilli positive by sputum culture in the cases of tb, while tubercle bacilli negative by sputum culture in the cases of abscesses. pulmonary abscess has an acute onset, with increased leukocytes and neutrophils as well as favorable therapeutic effi cacy of antibiotics. but sometimes tuberculosis with cavity may develop into bacterial infection, with undetectable tubercle bacillus by sputum culture. hiv/aids related tuberculosis should be differentiated from lung cancer. the central type of lung cancer has nodular shadow in the hilum or hilar and mediastinal lymph node metastasis, which should be differentiated from lymphatic tuberculosis. the peripheral type of lung cancer has small fl aky infi ltration and nodules in the periphery of the lungs, which should be differentiated from tuberculoma or tuberculosis infi ltrative lesions. lung cancers occur commonly in people aged above 40 years. the central type mainly is squamous carcinoma and the cases often have a history of long term smoking, with symptoms of no fever but diffi culty breathing or chest distress as well as gradually increasing chest pain. there are also symptoms of irritated cough with blood phlegm and progressive weight loss. the cases with supraclavicular metastasis have palpable harden lymph nodes. the intrapulmonary nodules can lobulated with fi ne spikes, no satellite lesions, generally no calcifi cation and possible vacuole sign. the peripheral type of lung cancer shows pleura invagination sign. tuberculin test often shows negative in the cases of lung, positive or weakly positive in the cases with tb, and negative or weak positive in aids patients. hiv infection is known to be the main factor for the development of latent tuberculosis into active tuberculosis. immunosuppression are similar to those in the cases with primary tuberculosis, with characteristic abnormal manifestations of hilar and/or mediastinal lymphadenectasis and parenchymal changes of the air chambers. ct scanning demonstrates enlarged nodules with low density. enhanced scanning demonstrates marginal enhancement of the lymph nodes. the incidence of military tuberculosis in aids patients is increasing due to reduced thymic t lymphocytes in aids patients and the defects of delayed allergic responses, which result in the formation of granulomas and impaired functions to kill bacilli and confi ne the lesions. nontuberculous mycobacteria (ntm) refer to the mycobacteria except for mycobacterium tuberculosis complex (human, cattle, african and vole) and mycobacterium leprae. the most commonly known nomination is nontuberculous mycobacteria (ntm). more than 100 kinds of ntm have been found so far. according to berger manual of systematic bacteriology, ntm is divided into two categories, rapid growth type and slow growth type. ntm are widely spread in nature, such as soil, dust, fl owing water and raw milk. under a microscope, ntm is morphologically similar to tubercle bacilli, with red stained fi ndings by acid-fast staining. according to the growth of ntm in solid medium, the runyon classifi cation divides ntm into the following four groups, light chromogenic bacteria; dark chromogenic bacteria that can cause cervical lymphnoditis in children, intrapulmonary or extrapulmonary infections and abrasive abscess; non-chromogenic bacteria including mycobacterium avium complex, intracellular mycobacteria that can cause pulmonary infections, lymphnoditis, arthritis and meningitis; rapid growth bacteria including mycobacterium fortuitum, mycobacterium, mycobacterium abscessus that can cause pulmonary diseases and skin infections. immunocompromised populations, such as hiv infected patients, patients with neoplasms, patients with long-term use adrenocortical hormone or immunosuppressive agents, are more susceptible to disseminated ntm infection. immunocompetent people may have mycobacterium kansasii and mycobacterium avium infections. it was reported in the united states that the occurrence of mycobacterium avium complex infection in hiv positive patients is up to more than 95 %. the pathological changes of ntm infections are similar to those of tuberculosis. ntm lymphnoditis is pathologically characterized by granulomatous infl ammation. tuberculous nodules formed by epithelioid cells and langhans giant cells are rare, with no accompanying central caseous necrosis. due to the weak pathogenicity of ntm, the pathological changes are slight, but there is difference in the pathological changes of ntm infections in terms of location, type and host. cavities are common in the cases with pulmonary ntm infection, commonly being multiple or multilocular thin wall cavities. the pleuron is rarely involved, with non-specifi c pathological changes of infl ammation but with large quantity pathogens of ntm. patients often have a history of chronic obstructive pulmonary disease, tuberculosis, silicosis, pulmonary abscess, bronchiectasis, cystic fi brosis, diabetes, ulcer as well as use of hormone or immunosuppressive agents. its occurrence is more common in males than in females. the symptoms include cough, expectoration, hemoptysis, chest pain, diffi culty breathing, low grade fever, weight loss and fatigue. the symptoms are nonspecifi c and the conditions progress slowly. for patients with suspected diagnosis of pulmonary ntm infection, sputum smear for acid-fast staining, sputum culture and bronchial lavage specimen culture can be performed. the positive fi ndings should be identifi ed with two to three times repeated culture. the same fi nding of ntm can defi ne the diagnosis. pathological biopsy can be performed for the diagnosis of ntm lymphnoditis. using 16s-23 srdna gene spacer sequence (igs) of ntm for pcr-restriction fragment length polymorphism analysis (pcr-rflp), ntm species can be identifi ed, which is more accurate, faster and simpler than the conventional morphological and biochemical examinations. mycobacterium tuberculosis and ntm have common antigen. ppd skin test produces cross-reaction, but there are still differences between mycobacterium tuberculosis and ntm. ppd-t of the mycobacterium tuberculosis and ppd-ntm of ntm are simultaneously obtained for mantoux skin tests. the induration diameter of ppd-t in ntm patients is generally within 15 mm. for the cases with the induration diameter of ppd-ntm skin test being 5 mm larger or over 25 % larger than that of ppd-t skin test, ntm infection can be confi rmed. both are the most commonly used imaging examinations. a female patient aged 26 years was confi rmatively diagnosed as having aids by the cdc. she complained of cough and chest distress for half a month, fever for 10 days, 1 day after cesarean section and fi nding of hiv positive for 1 day. her cd4 t cell count was 54/μl, with treponema pallidum antibody negative and ppd test negative. imaging demonstrations include various lesions such as infi ltration, cavity, nodules, fi brous caseation and extensive fi ber contraction in unilateral or bilateral lungs. the incidence of cavity is up to 80 %, being singular or multiple. cavities caused by intracellular mycobacterium are mostly found in the pleura, with thin wall and less surrounding exudates. the incidence of non-tuberculous mycobacterial infections in aids patients is high. mac infection is usually caused by the initial exposure rather than the reactivation of latent pathogens. in patients with complications of mac related lung diseases, most of the imaging fi ndings are normal. the most common manifestation is mediastinal or hilar lymphadenectasis. and the pulmonary symptoms are similar to those of tuberculosis. in the cases with multiple patchy parenchymal changes, cavities can be found, as well as nodules with blurry boundaries, pleural effusion and rarely found miliary nodules. sputum or bronchoalveolar lavage fl uid culture positive, clinical symptoms, imaging fi ndings, and response to treatment can defi ne the diagnosis. staphylococcus aureus is a gram positive coccus and is coagulase positive staphylococcus. the pathogenic substances of staphylococcus aureus mainly are toxins and enzymes, such as hemolytic toxins, leukocidin and enterotoxin, which play a role in hemolysis, necrosis, killing leukocytes and vascular spasm. the staphylococcus aureus coagulase is the main reason for suppurative infection. pneumonia caused by inhaled staphylococcus aureus through the respiratory tract often shows lesions in the large lobes or extensive fusion of bronchopneumonia lesions. bronchial and alveolar rupture allows gas to enter the pulmonary interstitium, which is communicated with the bronchi. in the cases of bronchiolar blockage by necrotic tissues or pus, the one-way valve effect is formed to cause tension pulmonary emphysema. in the cases with superfi cial pulmonary emphysema with excessively high tension, it ruptures to form pneumothorax or pyopneumothorax, as well as bronchooleural fi stula ( fig. 17.45a, b ) . the symptoms include chills, persistent high fever, cough, expectoration, chest pain and other symptoms. there is no sign in the early period. symptoms are scattered moist rales in both lungs, being in consistency to severe toxic symptoms and respiratory symptoms. yellow purulent sputum is the typical characteristics of staphylococcus aureus pneumonia. in the cases with larger lesions or fusion of lesions, signs of parenchymal changes, pneumothorax or pyopneumothorax can be found. in the sputum or pleural fl uid smears examinations, the bacteria with a concentration being no less than 107 cfa/ml is the pathogen, the bacteria with a concentration being 105-107 cfa/ml is the suspected pathogen, and the bacteria with a concentration being less than 105 cfa/ml is the contaminated bacteria. there are increased wbc count and neutrophils, leftward migration of the nucleus and possibly no increase of wbc count in aids patients. immunofl uorescence, enzyme-linked immunosorbent assay and counter immunoelectrophoresis can be performed to detect serum antigen or antibody of the pathogenic bacteria, which can defi ne the diagnosis. polymerase chain reaction has certain signifi cance in pathogen detection. the protected bronchoscopic specimen (pbs) and bronchoalveolar lavage (bal) can be applied to collect the specimen, which has reduced chances of specimens contamination by oral bacteria. biphasic tv monitors guided pulmonary puncture and suction for pulmonary tissues examinations can be performed to detect the real pathogenic bacteria. both are the most commonly used imaging examinations. the diagnostic imaging demonstrates staphylococcus aureus pneumonia as lesions in the inner zone of both middle lower lungs. there are singular or multiple parenchymal changes in patchy or lobar distribution that may fuse into large fl akes. it may be complicated by cavity and pulmonary emphysema, with surrounding compensatory emphysema. a male patient aged 37 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever with a body temperature of about 39 °c and chest pain for 2 months. his cd4 t cell count was 31/μl. chills, fever, cough, expectoration, chest pain and other symptoms commonly; hemoptysis and dyspnea rarely the patients may be found with fever appearance, rarely shortness of breath and cyanosis. in the serious cases, the body temperature can be as high as 39-40 °c and blood pressure decreases, with signs of shock. by chest examinations, decreased ipsilateral respiratory motion; increased or decreased fremitus, dull sound in percussion; bronchial breathing sounds or moist rales by auscultation; rarely pleural friction and weakened breathing sounds. increased wbc count and neutrophils, possible the nucleus left shift; no increase or even decrease of wbc count in aids patients; staphylococcus aureus positive by blood culture. sputum or pleural fl uid smears examinations for pathogenic bacteria culture is positive, and antibiotic sensitivity test is positive. by chest x-ray and ct scanning, the most common demonstrations are lesions of bronchial pneumonia. the fi ndings of pulmonary emphysema and cavities can facilitate the diagnose. the lesions should be differentiated from infi ltrative parenchymal bronchioloalveolar carcinoma. the smaller lesions should be differentiated from pulmonary infarction. the large lesions should be differentiated from obstructive pneumonia. it is diffi cult to identify the types of common bacterial pneumonia simply by chest x-ray and ct scanning. in combination to the laboratory tests, the diagnosis can be defi ned. the incidence of pyogenic bacterial infection is increasing in aids patients, which is caused by their weakened cellular and humoral immunity. the manifestations of these most common bacterial infections are similar to those of non-hiv infected patients by chest x-ray. bacterial pneumonia and purulent bronchitis are the most common causes of pulmonary infections in aids patients. particularly, they are frequently found in patients with a history of intravenous drug abuse and smokers. they are histologically characterized by infl ammations of the bronchi and bronchioles as well as infl ammatory exudates and mucus in the airway lumens. ct scans facilitates the diagnosis of bronchiolitis and early bronchial pneumonia. the demonstrations are characterized by (1) small centrilobular nodular shadows, which is the cross sectional demonstration of bronchioles fi lled with infl ammatory substances and its surrounding infl ammations; rhodococcus equi infection is one of the zoonotic diseases, which commonly occurs in the grazing areas. patients with t lymphocyte immunodefi ciency caused by aids and other factors are especially susceptible to the infection. rhodococcus equi was fi rstly discovered in 1923 and was nominated as corynebacterium equi. after structure analysis of the cell wall, it was found that the bacterium is quite different from corynebacterium, and therefore classifi ed as rhodococcus. rhodococcus equi infection in human is rare. but in recent years, due to an increase of patients with immunodefi ciency syndrome, reports on rhodococcus equi caused human respiratory infections and sepsis are increasing. in the past, the toxicity mechanism of rhodococcus equi was mostly speculated. until recently, the damage process of toxic plasmid to human tissue is discovered, which presents a new way for the study of the pathogenesis of rhodococcus equi infections. rhodococcus equi is one of the facultative parasites in the cells and its optimum growth temperature is 30 °c, with a suitable growth temperature of 10-40 °c. acid-fast staining of rhodococcus equi shows uncertain results. due to its morphological diversity, it is often mistaken as diphtheroid bacillus, bacillus or micrococcus. in sheep blood agar, rhodococcus equi can have synergistic hemolysis with staphylococcus aureus, listeria monocytogenes and corynebacterium pseudotuberculosis, which is a characteristic manifestation of rhodococcus equi. the most common pathological changes in rhodococcus equi infection are chronic purulent bronchitis and extensive lung abscess. imaging often demonstrates subacute pneumonia, commonly with cavities. the clinical manifestations are poor appetite, drowsiness, fever and shortness of breath. studies by e marchiori et al. [ 30 ] in 2005 revealed that all the 5 cases of aids complicated by rhodococcus equi pulmonary infection have cough and fever lasting for 1-2 months, with accompanying shortness of breath and chest pain. all the 13 cases, studied by li et al. in 2011 [ 106 ] , have fever with a body temperature up to 38-40 °c and cough. in addition, there are also expectoration with orange red sputum in 10 cases, hemoptysis in 4 cases, dyspnea in 11 cases, moist rales of lungs in 13 cases, emaciation in 6 cases, poor appetite in 6 cases, diarrhea in 2 cases, joint pain in 1 case, oral candidiasis infections in 13 cases, oral herpes in 4 cases, chest pain in 4 cases, no obvious symptoms in 1 case and hepatitis b in 3 cases. typical clinical manifestations of this disease are fever, cough, dyspnea and chest pain, while others such as emaciation, diarrhea and joint pain are not representative symptoms. identifi cation of the bacteria various specimens were inoculated on blood plates at a temperature of 35 °c for 18-24 culture, with bacteria growth of 18 strains. they are biologically characterized by a diameter of about 0.5 mm, non-transparent and slight yellowish colonies. after 48-72 h, the colonies expand to 1-2 mm, which can be emulsifi ed in mucous fl uid liked state. most of the colonies produce orange and orange red pigments, which can be cultured in ordinary agar. histopathological fi ndings are typical for rhodococcus equi infection. h&e staining demonstrates mainly bleeding in the alveolar space, large quantity epithelial cells, possibly predominant fi broblasts, parenchymal changes of lung tissue and thickened alveolar septa. pas staining demonstrates scattered or clustered rhodococcus equi in pink or purplish red. both are the most commonly used imaging examinations. biphasic tv monitor guided lung puncture can be performed to suck lung tissues for biopsy, based on which the real pathogenic bacteria can be detected. the typical demonstrations include central hilar sphere liked shadow with increased density in unilateral lung, accounted for 70 %. there are also manifestations of exudative infi ltration and large fl aky or spherical mass shadows in the right or left hilar area. the lesions are in patchy or fl aky appearance, radiating from the hilum to the lung fi eld with blurry boundaries. a male patient aged 30 years was confi rmatively diagnosed as having aids by the cdc. he complained of recurrent fever, cough and chest pain for 13 days; and was found hiv positive for 8 days. he was also a carrier of hepatitis c virus, with symptoms of fever with no known causes, cough, chest distress and weak limbs. by examinations, he was found to have complexion of chronic conditions; many moist and dry rales by cardiopulmonary auscultation. he had a past history of hiv positive for 8 years, with drug abuse and extramarital affairs. the history of present illness includes fever, paroxysmal cough with a little whitish yellow thick sputum since may 8, 2008 . he also had subjective paroxysmal dull pain in the left chest, and hemoptysis once which was bright red with blood clot in a volume of about 80 ml. his cd4 t cell count was 10/μl. by sputum culture, rhodococcus equi was found positive. after receiving antibiotic treatment, his conditions improved and he was discharged from the hospital. catalase test of the strain is positive, which is confi rmed as rhodococcus equi by api coryne system. examinations h&e staining demonstrates mainly bleeding in the alveolar space, large quantity epithelial cells or mainly fi broblasts, parenchymal changes of lung tissue and thickened alveolar septa. pas staining and masson staining demonstrate scattered or clustered distribution of rod rhodococcus equi in pink or purplish red. aids complicated by pulmonary rhodococcus equi infection shows subacute infl ammation. firstly, there are exudates in the surrounding area of unilateral hilum as well as sphere shaped mass shadows which is centrally dense and peripherally blurry, with its apex pointing to the hilum. the lesions can be complicated by cavities and fl uid level, with thick wall of the cavities. as the disease progresses, the abscess cavities have increased tension, with gradually thinner abscesss cavity walls and uneven wall thickness, even showing pleural effusion. these are its characteristic imaging fi ndings. it often needs to be differentiated from pneumocystis carinii pneumonia, tuberculosis, staphylococcus aureus pneumonia, central type lung cancer and other diseases. imaging fi ndings of pneumocystis carinii pneumonia usually are ground glass liked changes in the lung fi eld, with parenchymal changes and centrilobular nodules. tuberculosis often shows miliary tuberculosis, with lymphadenectasis, large tubercles and parenchymal changes. these characteristic pathological and imaging fi ndings of staphylococcus aureus pneumonia are similar to those of bronchial pneumonia (lobular pneumonia). the lesions are nodules with blurry boundaries in a diameter of 4-10 mm. the disease progresses rapidly, while pulmonary rhodococcus equi infection has a chronic progression. hrct scanning demonstrates staphylococcus aureus pneumonia as centrilobular nodules and branch linear shadows (tree buds sign), which can be found in 40 % patients, with 15-30 % will develop into commonly singular pulmonary abscess. chest ct scanning demonstrates round liked abscess cavity with thick wall and liquid level in it. the inner wall of the abscess cavity is often irregular, with various changes within short period. the central type of lung cancer is commonly demonstrated as round liked shadow of unilateral hilum with rough boundary. lobulation or bronchial stenosis sometimes occurs. however, aids complicated by rhodococcus equi pneumonia is demonstrated as sphere liked mass in the hilum; mostly sphere liked increased density shadow with hilum as the center. the shadow is centrally dense and peripherally blurry, with no bronchial stenosis. rhodococcus equi was fi rstly discovered in 1923 and was nominated as corynebacterium equi. after structure analysis of the cell wall, it was found that the bacterium is quite different from corynebacterium, and therefore classifi ed as rhodococcus. rhodococcus equi is generally believed to be the pathogen for horses, pigs and cattle. [ 106 ] showed a ct4 t cell count of lower than 49/μl. all the results are in consistency. in conclusion, aids complicated by pulmonary rhodococcus equi infection is mainly subacute infl ammation. there are exudation around the unilateral hilum as well as centrally dense and peripherally blurry sphere shaped mass shadows, with secondary cavities and parenchyma changes and even pleural effusion. all of these are characteristic imaging demonstrations. most cases of hiv positive complicated by respiratory or pulmonary diseases are caused by aspergillus fumigatus. aspergillus fumigatus belongs to filamentous fungi, which is a common opportunistic fungus and has a wide distribution in the nature. as conditional pathogenic bacteria, it can parasitize in the human skin and upper respiratory tract. human has certain resistance to aspergillus so it commonly fails to cause diseases. in immunocompromised aids patients, the pathogenic bacteria can pass through the defects in the skin and mucous membrane into the blood flow to infect the tissues and organs. aspergillus commonly violates bronchus and lung, with involvements of rhinal sinuses, external auditory canal, eye and skin. otherwise, it disseminates to organs of the body along with blood fl ow. the early lesions are diffuse infi ltrative and exudative changes. and advanced lesions are necrosis, pyogenesis or granuloma. large quantity hyphae can be found in the lesions. the hyphae penetrate the blood vessels to cause vasculitis, perivascular infl ammation and thrombosis. and thrombosis can cause ischemia and necrosis of the tissue. according to the pathological changes and imaging fi ndings, it can be divided into three major types: vascular invasion type, bronchopneumonia type and allergic bronchopulmonary aspergillosis type. (1) the vascular invasion type is the result caused by toxins released in the process of aspergillus spreading extensively from the primary focus to the lungs. vascular infi ltration of the pulmonary parenchyma and coagulative necrosis are believed to be the cause of vascular occlusion and pulmonary infarction. (2) bronchopneumonia type is acute bronchitis caused by inhalation of aspergillus spores. in the cases of hyphae invasion into the lung tissues, extensive infi ltrative pneumonia or focal granuloma are resulted in. it can also cause necrosis, pyogenesis and multiple small abscesses. spherical pulmonary aspergillosis is often secondary to bronchiectasis, tuberculosis, carcinous cavity and other lung diseases. mycelia multiply and gather in the cavities of the lungs to form a spherical mass with fi brin and mucosal cells, which are called aspergillar glomera, which do not invade the lung tissue. (3) allergic bronchopulmonary aspergillosis type is the proliferation and germination of inhaled aspergillus spore in the airway, often showing obvious related mucosal lesions and eventually resulting in bronchiectasis ( fig. 17.64a-c ) . the cases with acute onset have symptoms of high fever or irregular fever, cough, shortness of breath and green purulent sputum. the cases with a chronic onset have symptoms of repeated cough and hemoptysis, which are similar to those of tuberculosis. the pulmonary signs are not obvious, with occasional fi ndings of moist rales. most cases are asymptomatic and sometimes there are fever, cough, shortness of breath, and mucous purulent sputum. the main symptoms are persistent fever, cough and chest pain. in the serious cases, there is dyspnea. by microscopic examination of sputum, aspergillus hyphae can be found. the culture for aspergillus fumigatus is positive. serum ige is commonly above 2,500 μg/l. skin test for aspergillus antigen is positive. serum anti-aspergillus antigen igg antibody precipitin is positive. puncture of lungs and pleura for biopsy facilitates the diagnosis of pulmonary fungal infections. both are the most commonly used imaging examinations. hiv/aids related aspergillus bronchopneumonia is commonly demonstrated to have increased pulmonary markings, diffuse patchy blurry shadows and mass shadows in both lungs. spherical pulmonary aspergillosis is commonly demonstrated to have sphere liked aspergillar glomera suspending in the cavities to form a crescent shaped transparent area, in characteristic meniscus sign, rolling ball sign and fi ngertip sign. meniscus sign is nominated due to a meniscus liked space between the aspergillar glomera growing in the cavity and the cavity wall. rolling ball sign means that the aspergillar glomera moves along with the changes of posture. fingertip sign indicates that the substance formed by aspergillar glomera in dilated bronchi is in a fi nger shape, sometimes in v shape sign and y shape sign. invasive lesions refer to lesions invading or destroying lung structures, such as pneumonia, parenchymal changes and necrosis. a female patient aged 28 years was confi rmatively diagnosed as having aids by the cdc. she complained of cough and chest distress, with increased eosinophilic granulocytes. her cd4 t cell count was 45/μl. a male patient aged 36 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever or irregular fever, cough and shortness of breath. his cd4 t cell count was 96/μl. a male patient aged 38 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever or irregular fever, cough and shortness of breath. his cd4 t cell count was 76/μl. a male patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. he was hospitalized due to complaints of fever for 1 week, chest distress and shortness of breath for 1 day. on admission, he was confi rmed as hiv positive, with a cd4 t cell count of 9/μl. by physical examinations, he was in poor physical conditions, respiratory rate 27/min, lips cyanotic, coarse breathing sounds of both lungs with small quantity dry rales. his conditions progressed rapidly and death occurred due to respiratory failure after 3 days. lung and pleura puncture for biopsy can detect the growth of aspergillus hyphae. sputum culture can detect aspergillus hyphae, with fi ndings of aspergillus fumigatus positive. in the cases with allergic bronchopulmonary aspergillosis, the serum ige is above 2,500 μg/l. skin test of aspergillus antigen is positive. the serum anti-aspergillus antigen igg antibody precipitin is positive. characteristic ct scanning demonstrations of hiv/aids related parasitic aspergillar glomera include pulmonary cavities or cavity lesions with spherical contents, smooth boundaries of the spherical contents with even density, lunate shaped or ring shaped transparent shadows between cavity or cavity walls and the contents, migration of the contents with the body postures. according to the pathological and imaging demonstrations, it can be divided into three major types: in the early stage, ct scanning demonstrates soft tissue density nodules or light ground glass liked halo sign around the mass, which is the evidence for the diagnosis of the invasion type pulmonary aspergillosis. air cresent sign refers to round pulmonary infi ltration with accompanying central necrosis and surrounding lunate or ring shaped cavity. other noncharacteristic ct scanning demonstrations include multiple lobular parenchyma lesion shadows or lobular fusion shadows, parenchyma lesion shadows in the lobes, segments and subsegments, nodular or mass shadows and thin/thick wall cavities or low density areas in the mass shadows. it is demonstrated to have parenchymal lesions around the airway or/and central small nodules in the lower lobes. the parenchymal lesions prove the occurrence of mycotic bronchopneumonia. the most common imaging fi nding is the thickened bronchial wall. central bronchiectasis is its characteristic demonstration. in the cases of dilated bronchi containing sputum bolt or mucus, it shows fi ngertip shaped or toothpaste shaped shadow, which should be considered as its characteristic demonstration. hiv/aids related pulmonary aspergillosis should be differentiated from congenital bronchial atresia. most cases of the congenital bronchial atresia are atresia at the proximal pulmonary segment of the bronchi, often with a clearly defi ned mass. in the typical cases, there are bronchial branches and more branches in fi ngertip shape, pointing to the pulmonary hilum. confi ned pulmonary air retention in the pulmonary lobe and segment of the atresic bronchi is the important evidence for the diagnosis of congenital bronchial atresia. hiv/aids related pulmonary aspergillosis should be differentiated from allergic bronchial-pulmonary aspergillosis. in the cases of allergic bronchial-pulmonary aspergillosis have no clearly defi ned mass, with demonstrations of v shaped, y shaped, grapes shaped or fi ngertip shaped shadows with clearly defi ned boundaries, which are characteristic in those patients with bronchial asthma or a case history of exposure to dusts containing fungi. there is also increased proportion of eosinophilic granulocytes in the peripheral blood. detection of aspergillus in phlegm can defi ne the diagnosis. hiv/aids related pulmonary aspergillosis should be differentiated from central lung cancer. central lung cancer also can cause mucus impaction of the distal bronchi, with manifestations of bronchial arctia and/or truncation, and the surrounding soft tissue mass shadows. hiv/aids related pulmonary aspergillosis should be differentiated from pulmonary cavities and abscesses induced by dissolved tuberculoma, secondary pulmonary tb, chronic lung abscess and peripheral lung cancer as well as cystic bronchiectasis. except aspergilloma, spheric morphology caused by other causes is commonly irregular. the cavity contents cannot migrate with body postures, which is the key point for the differential diagnosis. hiv/aids related pulmonary aspergillosis can be caused by many pathogenic bacteria and aspergillus fumigatus is the most common one. the infection is often caused by inhaled aspergillus fumigatus in the environment. vascular invasion type of pulmonary aspergillosis usually has multiple lesions and nodular changes. generally in pathology, the center of nodule presents typical pale color; commonly with fi brous ring surrounding the nodules resulted from hemorrhage and/or lung parenchymal changes. histologically, they are characterized by coagulative necrosis of the lung tissues, infi ltration of large quantity hyphae in the necrotic tissue, pulmonary vascular infi ltration, but usually without responses of vasculitis and thrombosis. the enzymes released by neutrophile granulocytes can cause the separation of necrotic tissue from its adjacent lung tissues to form necrotic mass in the cavities. airway invasion type of pulmonary aspergillosis, also called aspergillus bronchopneumonia, accounts for 15-30 % of invasive aspergillosis. the most common imaging fi ndings are unilateral/bilateral fl aky parenchymal changes, centrilobular small nodules and branches liked linear shadows (tree buds sign). histologically, it is characterized by necrosis and infi ltration of neutrophil granulocytes. the lesions surround the bronchiole and the bronchiole. the invasion of the pulmonary artery can cause bleeding of the adjacent pulmonary parenchyma. allergic bronchopulmonary aspergillosis rarely has lesions, with no unknown pathogenesis, which is generally believed to be related to type i and type ii allergic reactions. it usually shows obvious asthma related mucosal lesions. hyphae generated by aspergillus fumigatus can induce the production of mucus and additional mucosal lesions, eventually leading to bronchiectasis. dilatate bronchial lumen is fi lled with mucus or with absence of epithelium, which is replaced by a granulomatous infl ammatory infi ltration. the most common imaging manifestations are migratory fl occulent, branched y shaped and v shaped (fi ngertip sign) shadows in the pulmonary parenchyma, which are related to the infi ltration of eosinophils. pathologically, bronchial cystic dilatation in the pulmonary segment and sub-segment occurs, with large quantity eosinophils in the bronchial mucus and scattered broken aspergillus hyphae. in combination with the case history, the diagnosis can be defi ned. compromised immunity is an important cause of cryptococcosis, especially in patients with aids or abnormal lymphoproliferative diseases. cryptococcus neoformans, a single phase mould, exists widely in the natural world. the cryptococcus has a diameter of less than 10 μm, which can be inhaled into the human body via respiratory tract. under the impact of a high concentration carbon dioxide, it forms a clearly defi ned protective layer composed of polysaccharide capsule to antagonize the defense mechanisms of the host. thus, lung infection occurs after its inhalation in immunocompetent people, which is commonly asymptomatic. of cryptococcus, inhalation of cryptococcus by aids patients can lead to hilar lymphadenopathy, as well as singular or multiple subpleural small nodules, being similar to those in the cases of mycobacterium tuberculosis infection. in the early stage of cryptococcal infection, only a mild infl ammatory reaction or diffuse infi ltrative exudative changes occur. but in the advanced stage, necrosis, suppuration or granuloma is formed. large quantity hyphae can be found in the focus. in the cases with hyphae penetrating the blood vessels, vasculitis, perivascular infl ammation and thrombosis occur. and thrombosis leads to ischemia and necrosis of the tissue (fig. 17.77 ). pulmonary cryptococcus infection in aids patients often is extensively disseminating, with symptoms of fever, cough, diffi culty breathing, expectoration, chest pain caused by pleuritis, and even acute respiratory distress syndrome (ards). hiv/aids related pulmonary cryptococcus infection has no characteristic imaging demonstrations. chest x-ray and ct scanning show multiple morphology of the lesions. in the slight cases, there are thickened pulmonary markings in both lower lungs or isolated nodular shadows, and occasionally cavities. in the cases of acute interstitial infl ammation, there are diffuse infi ltrative or miliary foci, with infi ltration, nodules or exudation in any lobe which is more common in bilateral middle and lower lungs, in unilateral lung or confi ned to one lobe. the foci may be isolated huge spherical or multiple nodular, without obvious surrounding infl ammatory responses, similar to those of tubercles or tumors. otherwise, they are diffuse miliary shadows or fl aky infi ltrative shadows. a male patient aged 60 years was confi rmatively diagnosed as having aids by the cdc. he was hospitalized on 2009-2-19 due to headache and vomiting for more than 10 days. in the csf, cryptococcus was found. by blood and sputum culture, cryptoccocus was detected. the diagnosis was cryptococcal meningitis, cryptococcal pneumonia and cryptococcal sepsis. after receiving amphotericin b antifungal treatment and dehydration, headache and vomitting were relieved. but chest ct scanning reexamination demonstrated increased pulmonary lesions, which was considered as tuberculosis. on 2009-4-1, he was given herv anti-tuberculosis treatment. twenty days ago he sustained weakened lower limbs, which gradually aggravated and completely paralyzed in the recent 1 week, his cd4 t cell count was 34/μl. hiv/aids related pulmonary cryptococcus infections should be differentiated from tuberculosis, primary or metastatic lung cancer. tuberculosis mostly is secondary tuberculosis, which is caused by repeated infections of tubercule bacillus. lesions show fl aky or fl occulent shadows in the two upper lungs, with blurry boundaries. the wrapped necrotic foci by fi bers develop into nodules. it can also show miliary shadows, mostly with mediastinal lymphadenectasis. it should also be differentiated from primary or metastatic lung cancer. cryptococcus is a relatively common pathogen of pulmonary fungal infection, and mostly develops in aids patients. usually, it is a disseminated disease, with common involvement of the central nervous system and the lungs. in immunocompetent patients, the nodular granuloma caused by the pathogen is similar to those of other pulmonary fungal infections. in patients with serious immunosuppression, wide tissue infi ltration of the pathogens may occur in the lungs. liked shadows, parenchymal changes of air cavity and miliary nodules. the pathogenic fungi can be found mainly in the pulmonary interstitium. imaging fi ndings include singular or multiple nodules or masses, parenchymal changes of lung lobes and lung segments with clear or unclear boundaries in size of 1-10 cm. there may be also miliary lesions, lymphadenectasis and cavity shadows. candida is an opportunistic pathogen, which widely exists in nature. candida albicans parasitize in the oral cavity, laryngopharynx, upper respiratory tracts, vaginal and intestinal mucosa of human being. pulmonary and bronchial moniliasis is commonly caused by candida albicans which has the strongest pathogenicity. after its invasion into the tissues, candida transforms into hyphae and multiplies in a large quantity, with strong toxicity and ability to fi ght against phagocytosis. aids patients may have disseminated pathological changes. only when the immunity is compromised, the pathogen invades into the bronchus or lungs to cause diseases. therefore, pulmonary candida infection is commonly secondary. candidosis can cause acute infl ammation in bronchus and lungs, mainly exudation of neutrophils, which can be divided into two types: bronchitis type and pneumonia type. the pathological changes in the early stage are acute suppurative infl ammation, accompanied with the formation of abscesses. by the naked eyes observation, they are large fl aky parenchymal changes, with central grayish white coagulative necrosis. under a microscope, the lesions are large fl aky caseous necrosis, accompanied with the formation of abscesses, and surrounding infi ltration of hyphae and phagocytes. in the advanced, there are caseous necrosis, formation of cavities, fi brosis and granuloma. symptoms in aids patients are mild, with frequent cough, with a small amount of white mucous phlegm or thick phlegm, no fever or low grade fever; scattered spots of white membranes in the mucosa of oral cavity, throat and bronchus. dry rales can be heard occasionally in both lungs. in aids patients, the manifestations are mostly acute pneumonia or sepsis, with chills, fever, cough, expectoration of white mucous jelly liked phlegm or thick phlegm often with blood or necrotic tissue. the thick sputum, candidal hyphae and shedding cell debris can be condensed into small colloid clumps, with yeast smell. other symptoms include even haemoptysis and diffi culty breathing. dry and moist rales can be heard in lungs. symptoms may be diffi culty breathing, rhinocnesmus, runny nose and sneezing. wheezing rales can be heard in both lungs. chest x-ray chest x-ray demonstrates nodular shadows and fl aky parenchyma changes in unilateral or bilateral lungs. sometimes there is miliary infection. ct scanning demonstrates most lesions in the middle and lower lung fi elds, with rare involvement of the apex. there are thickened lung markings or diffuse small fl aky/patchy shadows, some of which can fuse into large fl aky dense shadows, with blurry boundaries. nodules, due to bleeding around it, may be surrounded by ground glass liked shadows, which is necrotic bronchopneumonia, usually accompanied with a large quantity neutrophils. cough expectoration with white mucous phlegm or thick phlegm, hemoptysis and shortness of breath. examinations of the oral cavity and the throat demonstrate spots liked white membrane covering the surface, and dry and moist rales in the lungs. successive cultures of phlegm, lung tissue, pleural fl uid or cerebrospinal fl uid repeatedly demonstrate the same strain of candida, or direct microscopic fi ndings of large quantity pseudohyphae or hyphae and groups of spores can defi ne the diagnosis. it is demonstrated to have thickened and deranged lung markings in double lung, diffuse small fl aky/patchy shadows, fusion of some small shadows into large fl aky dense shadows, with blurry boundaries, enlarged hilum and blurry structures. the conditions progress rapidly, with repeated lesions occurrence. hiv/aids related pulmonary candida infection should be differentiated from bacterial pneumonia. bacterial pneumonia often has symptoms such as high fever, cough, expectoration, chest pain and shortness of breath. ct scanning demonstrates fl occulent infi ltrative shadows or parenchyma changes and cavities. the pathogen can be detected in the sputum or chest liquid. hiv/aids related pulmonary candida infection should be differentiated from virus pneumonia. viral pneumonia fi rstly causes upper respiratory tract infection, which spread downward to cause pulmonary infl ammation. the demonstrations a male patient aged 40 years was confi rmatively diagnosed as having aids by the cdc. he complained of high fever, cough, expectoration, chest pain and shortness of breath, with pulmonary parenchymal changes sign and moist rales. his cd4 t cell count was 18/μl. include ground glass liked changes in the lung fi elds or mass shadows. the defi nitive diagnosis should be based on throat swabs, virus isolation from the sputum and serum specifi c antibodies test. hiv/aids related pulmonary candida infection should be differentiated from pulmonary tuberculosis. in the early stage, the symptoms and signs include irritative dry cough, expectoration, hemoptysis and cavities in lungs. (detailed manifestations of tuberculosis see the section about tuberculosis in this chapter) its diagnosis mainly should be based on chest x-ray and fi ndings of tubercule bacillus in sputum or other specimens, or tuberculosis specifi c pathological changes. hiv/aids related pulmonary candida albicans is a widespread dimorphism bacteria. the oval shaped budding yeasts and hyphae both can be found in the tissues. candidiasis is a common disease in aids patients. chest x-ray demonstrates unilateral or bilateral patchy parenchymal changes of the air cavity and nodules with unclear boundaries. miliary lesions are common. hrct demonstrates multiple nodular shadows in both lungs, often accompanied with parenchymal changes. its defi nitive diagnosis should be based on the fi ndings of candida albicans in the tissues. penicillium marneffei (pm) is a newly found penicillium in 1956, which is a special strain with a distribution in south east asia and southern china. rhizomys is its natural host. in 1973, disalvo et al. [ 12 ] reported the fi rst case of natural human pm infection. in 1984, the fi rst case of human pm infection in china was reported in guangxi zhuang autonomous zone. pm is an opportunistic pathogen and immunocompromised people are susceptible to its infection. its spreading is along with soil contaminated by rhizomys feces to invade human body via the respiratory tract, the digestive tract and skin defects. pm infection is believed to be one of the most common opportunistic infections in aids patients in southeast asia, which has an increasing incidence. pm is the only dimorphic fungus in hyphomycetes penicillium, which is a special strain of penicillium. at different culture temperatures, it shows conversion of biphasic forms: fungal phase at 25 °c and yeast phase at 37 °c. the fungal phase is the hyphae of many cells, with certain biological morphology, such as penicillus, conidiophore, chain liked conidiospore and chains between spores. the yeast phase shows unicellular or bicellular form. in the growth process of pm, large quantity bright rosy or dark rosy pigments are produced, which is characteristically pm. the pigment of yeast phase is secondary metabolites of cells with strong hydrophobicity, which can promote the adhesion of conidiums in fungal phase and cells in yeast phase to the alveolar macrophages and other cells surface in the human body. the pigment monoclonal antibody (mab) can interrupt the pathological process of adhesion. in addition, this pigment can determine the expression of cluster-encoding genes mbr through diffusion and penetration of drugs to the cell membrane, thus preventing the penetration of hydrophilic antifungal drugs, such as fl uconazole. that is to say, it improves the natural antifungal resistance level of pm. the soluble components of the pigment in fungal phase can trigger the generation of anti-conidium antibody (only igg) in animals to prevent its spreading in the body. the phenomenon proves that, in terms of tissue invasion, fungal phase is less powerful than yeast phase. conidium in fungal phase is the carrier of pathogen while the cells in yeast phase are the real pathogenic factors. when pm spreads to the target organ along with the blood fl ow, it is engulfed by mononuclear phagocytes. in the cases of replication itself and further spreading, reactive proliferation of phagocytes is caused. mononuclear phagocyte system has strong defense ability. in the cytoplasm of proliferated mononuclear phagocytes, various amounts of pm can be found. pm mainly invades into the body via the respiratory tract, digestive tract and skin defects. in immunocompetent people, local abscesses form in the invasion site, which is characterized by the thick mucus fl uid, with mainly necrosis and liquefaction. vascular reactions and exudation of neutrophil leukocytes and body fl uids is less than abscesses induced by common purulent bacteria. the clinical manifestation is confi ned suppurative infl ammation. when the immunity is compromised, due to the insuffi ciency of immunologic factors, it is diffi cult for the immune cells to restrict and digest the engulfed pathogens, which leads to confi ned suppurative reaction. therefore, it often presents with diffuse lesions. the pulmonary lesions are principally interstitial exudative infl ammation. the typical penicillium marneffei disease has acute or subacute onset, along with fever, chills and shivers, cough and expectoration, hemoptysis, shortness of breath, abdominal pain, diarrhea, bloody stool, fatigue, central necrotic papula mainly in the head and face and scattering in the trunk and extremities as well as hepatosplenomegaly. bone marrow smear and pas staining can be performed to detect the pathogen. blood, bone marrow, pleuroperitoneal fl uid, phlegm and skin defect tissue are collected for the culture at double temperatures with sabouraud'broth medium. at the temperature of 25 °c, the colony is in dark red with villous surface, with surrounding red wine liked pigments to gradually spreading into the medium. biopsy of lymph nodes and skin defects with pas staining and wright & gimsa staining can be performed. wbc count, hemochrome, platelets, ast and cd4 t cell count. ct scanning and routine chest x-ray are the diagnostic imaging examinations of choice, which can facilitate to understand the size, morphology, location, quantity and density of the lesions. it demonstrates multiple small nodular shadows in the lungs, multiple honeycomb liked cavities in both lungs and mediastinal lymphadenectasis. abdominal scanning demonstrates different degrees of hepatic, splenic and retroperitoneal lymphadenectasis, which can fuse into a huge mass. routine chest x-ray it demonstrates thickened, deranged and blurry pulmonary markings, small cavities, military nodular shadows, mass liked shadows, spots and patchy shadows, ground glass liked changes, pleuritis and pleural effusion. a female patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. her husband had a history of drug abuse and she complained of abdominal pain and fever for more than 2 months, with accompanying face rash and diarrhea. her cd4 t cell count was 5/μl. by examinations, she sustained skin palpula, abdominal tenderness, central concave skin rashes on the face, neck, and upper limbs. there was a palpable mass in the upper left abdomen, hard and tenderness, in a size of 12 × 12 cm. more than 2 months before her admission, she had persistent dull abdominal pain that is commonly in the upper left abdomen, with accompanying fever and face skin rashes that is gradually increasing and spreads to the neck and upper limbs. she also had hepatosplenomegaly, abdominal aortic lymphadenectasis and ascites. a female patient aged 51 years was confi rmatively diagnosed as having aids by the cdc. she had an unhealthy sexual life, with complaints of fever and cough for more than 1 month. her cd4 t cell count was 7/μl. a male patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. he had been found to be hiv positive for 5 years, with complaints of irregular fever, cough, fatigue and dizziness for 10 days to be hospitalized. by examinations, his cd4 t cell count was 40/μl, hiv positive, subcultivation of strains demonstrated typical biphasic penicillium. by fungus culture, typical penicillus was found. by bone marrow smear, the round corpuscles mainly located in the macrophages. a male patient aged 32 years was confi rmatively diagnosed as having aids by the cdc. he had a history of extramarital affair, with complaints of fever, cough, chest distress, shortness of breath, fatigue, poor appetite, poor sleep, weight loss and shortness of breath after activities for more than 3 months. his cd4 t cell count was 23/μl. chest x-ray and ct scanning both demonstrate multiple nodules in different sizes and cavity shadows. the cavities cluster into honeycomb liked changes with uneven thickness of the walls, clear boundaries and surrounding infl ammatory exudates. some of the nodules infuse into mass dense shadows. lesions in both lungs have a symmetrical or asymmetric distribution, with no characteristic leions. mediastinal lymph nodes are obviously enlarged. sputum smear for direct microscopy demonstrates candida. pms are found by fi brobronchoscopy lavage smear and biopsy. (1) candida skin test shows positive. (2) fluorescent antibody test for pms is performed in procedures of direct smear, fungal colony culture and histopathological examination of the tissue sections. metabolites test of pm and pcr can be performed to determine the gene sequences of pm for the early diagnosis. hiv/aids related penicillium marneffei pneumonia should be differentiated from bronchiectasis and blood borne staphylococcus aureus pulmonary abscess. although the cases of bronchiectasis are demonstrated to have clustering round shadows on the cross sections, the wall is thinner and even, accompanying to the spots liked vascular shadows. some lesions have typical railway liked bronchial dilation signs. it is demonstrated to have multiple small cavity lesions in both lungs in line with the evenly distributing blood borne lesions. the lesions are rarely clustering. the wall of the cavities is thick and even with surrounding marginal exudates and blurry boundaries. with the steady increasing of hiv infections, reports on the complication of pm disease have also been increasing recently. the disease can be localized but mostly disseminated, with the involvement of lungs, liver, skin, lymph nodes and other tissues and organs. therefore, it is known as penicilliosis marneffei or disseminated penicillium marneffei infection in literature reports. due to the insuffi cient knowledge about the disease, diagnosis is delayed or missed. in thailand, penicilliosis marneffei has been the indicator disease of aids. about 20 % aids patients are infected by pm and 70 % have necrotic papula, which is characteristically disseminated penicillium marneffei infection. ct scanning demonstrates hiv/aids complicated by disseminated penicillium marneffei infection as fl aky parenchyma shadows in the lungs, clustering of cavities and nodular shadows, mediastinal lymphadenectasis and pleural infl ammation responses, which can facilitate its clinical diagnosis. mucormycosis is a rare kind of conditional fungal disease, with its pathogen, mucor, distributing widely in the natural world. it generally fails to cause diseases, but can cause systematic infections in immunocompromised people. mucor often invades into the human body via the nose to cause paranasal sinuses and orbital infections, which can further invade into the brain to cause meningitis and frontal abscesses. pulmonary mucormycosis is only second to the nasal-cerebral infection. it can spread via the respiratory tract to cause pulmonary infections. in addition, there are also skin and gastrointestinal mucormycosis as well as disseminated mucormycosis. the pathological changes of hiv/aids related pulmonary mucormycosis are mainly hemorrhagic necrotic infl ammation. the defense mechanism of immunocompetent people is to kill the fungal spores with the phagocytosis of macrophages and oxidation killing mechanism. in immunocompromised or immunodefi cient patients, the macrophages are often too weak to restrain the engulfed spores from germinating. therefore, the disease occurs. vascular vessels are susceptible to the invasion of mucor, especially the arteries. mucor can locally multiply to cause the formation of blood clots and embolization, and disseminate to other organs along with the blood fl ow. the main lesions are hemorrhage and necrosis of local tissues and exudation of neutrophils. the lesions of hemorrhage and necrosis are possibly related to the arteriole lesions caused by hyphae. the clinical manifestation of hiv/aids related pulmonary mucormycosis is a nonspecifi c pneumonia. the most common symptoms reported in literatures are persistent high fever, cough, hemoptysis, chest pain and diffi culty breathing. it has a rapid progression, with a high mortality rate of 65-96 %. lung lesions are hemorrhagic infarction or pneumonia, which can cause high fever, cough, expectoration, shortness of breath, chest distress, chest pain, hemoptysis (pulmonary artery involved) and other symptoms. moist rales can be heard in both lungs and pleural rubs can be heard in the cases of pleura involvement. 1. assisted by bronchofi broscopy, lung biopsy can be performed. 2. histological examinations include bronchial lavage fl uid examination, exploratory thoracotomy and puncture of lung tissues for biopsy. 3. chest x-ray and ct scanning are conventional effective examinations. the lesions are frequently found in the dorsal and medial segments of both lungs. early exudation shows miliary shadows, cavity shadows with no wall or with thin wall and small bronchiectasis shadows. their further progression may cause fusion infi ltration, parenchymal changes, nodules, masses, thick-walled cavities and pleural effusion, with accompanying mediastinal lymphadenectasis. 1. the fi ndings of mucor or their hyphae by sputum and bronchofi broscopic biopsy. 2. the diagnostic imaging demonstrates lesions commonly in the dorsal and medial segments of both lungs. there are diffuse scattering miliary shadows, cavity shadows with no wall or with thin walls and small bronchiectasis shadows. they further progress into fusion infi ltration, parenchymal changes, nodules, masses, thick-walled cavities and pleural effusion. 3. often with accompanying mediastinal lymphadenectasis. chest x-ray of pulmonary mucormycosis demonstrates progressive infi ltrated parenchymal changes, or masses, nodules, cavities and pleural effusion. it needs to be differentiated from miliary tuberculosis. hiv/aids related miliary tuberculosis are commonly demonstrated as chronic blood borne disseminated tuberculosis, with lesions distributing symmetrically in both lungs. its long term progression causes fusion into masses. otherwise, it can be cured by anti-tb therapies. the early lesions are no-wall cavities or thin wall cavities and small bronchioectasis shadows, based on which the differential diagnosis can be made. almost all cases of hiv/aids related pulmonary mucormycosis are found in immunocompromised patients. chest x-ray demonstrates parenchyma changes and isolated or multiple nodules or masses. the parenchyma changes are patchy or fuse, with unilateral or bilateral distribution. about 20 % patients have pleural effusion and less than 10 % patients have hilar or mediastinal lymphadenosis. ct scanning demonstrates singular or multiple nodules or masses, commonly with clustering or honeycomb liked cavities. cytomegalovirus pneumonia has extensive pathological changes in the lungs. pathologically, it shows interstitial pneumonia, with the lesions randomly blood borne distributing in the lungs. the distribution can be diffuse, panlobular or focal. the target cells of pathological changes include alveolar cells and macrophages. diffused pulmonary interstitial edema and fi brosis as well as alveolar swelling, focal necrosis, bleeding and hyperplasia occur after cmv infections to cause hypoxemia. gross observation of fresh specimens demonstrates pulmonary surface edema and fl aky blooding spots. fixed specimens demonstrate brown hard lung tissues. under a microscope, pulmonary interstitial congestion as well as infi ltration of lymphocytes and mononuclear cells can be found, with the involved epithelial cells enlarged. in the pulmonary interstitium and alveoli, there are intranuclear inclusions, cytoplasmic inclusions and fl uid containing abundant proteins. the classical intranuclear inclusions can be found in the cells, purplish red or purplish blue, round or oval, with surrounding halos in eagle eyes sign. atypical cytomegalic inclusions in cells are slender, long and round liked with abundant cytoplasm and accentric nucleolus, which are blurry, unclear and atypical ( fig. 17 .97a-e ). immunohistochemitry demonstrates hiv p24 antigen positive. the systemic symptoms of cmv infection include fever, joint and muscle soreness and pain, abdominal distension and orthostatic hypotension. the respiratory symptoms include paroxysmal dry cough, diffi culty breathing, cyanosis and three depressions sign. according to the imaging fi ndings of cmvp, cmv pneumonia can be classifi ed as diffuse, miliary and mass types, among which the diffuse type is the most common. cytomegalovirus can be separated from respiratory secretions culture and urine culture by using human embryonic fi broblasts. by urine sediment smear, giant cell with inclusions can be found. by using immunofl uorescence, indirect hemagglutination inhibition and complement fi xing test, the antibody titer can be found increased. indirect immunofl uorescence test and immunoenzymic staining test can be applied to detect the anti-cmv-igg and igm antibody. in addition, enzyme-linked immuno sorbent assay (elisa) can also be performed to detect the anti-cmv-igg and igm antibody. cmv-igm antibody positive indicates a recent infection, which has diagnostic value. a single serum cmv-igg antibody positive indicates a previous infection. and during the acute and recovery phases, double serum cmv-igg antibody titer being no less than four times increase has diagnostic value, indicating a recent infection. pcr can be applied to quantitatively determine the viral load in the whole blood, blood plasma, leukocytes, urine, bronchoalveolar lavage fl uid (balf), cerebrospinal fl uid and the tissue specimens, which is believed to be the best way for the diagnosis of invasive cmv infection. chest x-ray is the most commonly used examination. chest ct scanning is superior to chest x-ray in terms of resolution and detection rate of the lesions. pulmonary demonstrations by cmvp include diffuse interstitial infi ltration and alveolar infi ltration to form reticular shadows, nodules and parenchymal changes. a baby boy aged 6 months was confi rmatively diagnosed as having aids by the cdc. he was infected via vertical transmission from mother to child, with recurrent cough after being born and the most recent cough for 4 days as well as wheezing cough in throat for 1 day before he was hospitalized. he had a past history of premature birth, with primary apnea and bronchial pneumonia and was hospitalized for treatment. later, he was admitted for three times due to cough, which was diagnosed as interstitial pneumonia. by examinations, wbc 16.3 × 10 9 /l, lym lymphocyte count 11.7 × 10 9 /l, cmv-ab weak positive, blood sedimentation 11 mm/h, and tuberculosis antibody negative. after treated by broad-spectrum antibiotic therapy, the therapeutic effi cacy is unfavorable. cytomegalovirus (cmv) infection is an important cause of pneumonia in patients with compromised immunity. imaging fi ndings include nodular shadows with blurry boundaries and bilateral fl aky parenchymal changes of the lungs. the nodules tend to be bilaterally symmetric or asymmetric, with centrilobular distribution. histopathological manifestations are nodular alveolar hemorrhage, necrosis and infl ammatory lesions, and diffused alveolar lesions. the nodules tend to have a centrilobular distribution, indicating occurrence of bronchiolitis. in aids patients, if the diameter of nodules is under 10 cm, it is most likely to be viral infection. the size of the nodules can facilitate the differential diagnosis of pulmonary infections. herpes simplex viral pneumonia (hsvp) often occurs in the upper respiratory tract, and rarely in the lower respiratory tract. human herpes simplex virus can be divided into two types, namely herpes simplex virus type i (hsv-i) and herpes simplex virus type ii (hsv-ii). herpes simplex viral pneumonia mostly occurs in patients with immunodefi ciency. herpes simplex viral pneumonia can be caused by hsv-i and hsv-ii, both of which have a nucleocapsid with 20 surfaces. the thickness of the nucleocapsid is about 100 nm, which is composed of 162 capsomeres. the nucleocapsid contains the core of the virus dna. the virion gains the phospholipid rich viral envelope when it passes through the nuclear envelope. the nucleocapsid gemmates after it passes through the nuclear membrane and is released to the cell surface. the nucleocapsid can also be released outside the cells or gains its access into the neighbour cells for further reproduction. herpes simplex virus replicates itself in the cell nucleus to produce histopathologic changes of herpes virus replication, with visible cowdry a type intranuclear inclusions. the pathogenesis process of herpes simplex virus infection in the body can be divided into fi ve stages: initial skin mucosa infection, acute ganglia infection, latent infections, re-activation, and recurrent infections in susceptible hosts. patients infected by herpes simplex virus can produce igm, igg and iga antibodies to fi ght directly against virus protein, which may play a role in changing the severity of the infection. interferon also participates in the control of herpes simplex infection by inhibiting the virus or regulating the defense mechanism. genetic factors may be also related to the herpes virus infection. cellular immunity can confi ne the infection. herpes virus cannot reproduce in the alveolar macrophages of human body, which is also the reason why herpes virus is less than cytomegalovirus in lungs. currently, it is believed that herpes simplex virus is an important pathogen of respiratory infections, especially in immunocompromised patients. localized herpes simplex viral pneumonia occurs due to the direct spreading of virus in the upper and lower respiratory tract. diffuse herpes simplex viral pneumonia is caused by the virus spread from the reproductive organs lesions or oral lesions (most possibly blood borne). viremia caused by hsv-i or hsv-ii has been reported, and both are related to diffused infections. but in patients without herpes simplex viral infection in skin mucosa, herpes simplex viral pneumonia can also occur. herpes simplex viral pneumonia is caused by the direct spreading of the virus from the upper and lower respiratory tract. diffuse herpes simplex viral pneumonia is cause by the spreading of the virus from the reproductive organs lesions or oral lesions (most possibly blood borne). viremia cause by either hsv-i and hsv-ii have been reported, both of which are related to diffuse infections. in such cases, the lung tissues may have infl ammatory infi ltration, lung parenchyma necrosis, bleeding, cellular swelling and round, diffuse interstitial pneumonia. and in most cases, there are accompanying cellular changes of herpes virus infection such as the intranuclear eosinophilic inclusions, necrotic herpes simplex viral trachitis. herpes simplex viral bronchitis has demonstrations of mucosa erythema, edema, exudation and ulcer, with coverage of the surface by fi brous purulent membranous secretions. the common initial clinical symptoms of herpes simplex viral pneumonia are shortness of breath, cough, and fever with a body temperature being higher than 38.5 °c, decreased wbc count, hypoxemia, respiratory dysfunctioning and azotemia. hsv pneumonia may be accompanied by mucocutaneous lesions by hsv, which show earlier than those of pneumonia. there may be concurrent fungus, cytomegalovirus or bacteria infection. herpes simplex viral tracheobronchitis may show tracheal or bronchial spasm or stenosis. etiological examinations hsv can be detected in tracheobronchial secretions, bronchoalveolar lavage fl uid and lung tissues. early sampling should be performed under the guidance of a bronchofi broscope. tissue culture is the most sensitive and specifi c method for the diagnosis, which can also be used for the classifi cation of the virus. papanicolaou (pap) or tzank test is a fast and cheap method for cellular diagnosis. elisa can be used to detect herpes simplex virus, with a sensitivity of up to 95 % and a high specifi city. chest x-ray demonstrations are less valuable for the differential diagnosis. pulmonary ct scanning can be applied for the differential diagnosis. herpes simplex viral pneumonia includes three types, namely local, multiple or diffuse interstitial infi ltration. in the early stage, typical hilar or diffuse interstitial shadows with increased density can be found, with thickened bronchial wall. as the disease progresses, cloudy or patchy alveolar tamponade and fusion can be found. chest x-ray may demonstrate negative for herpes simplex viral trachitis and bronchitis. herpes simplex viral pneumonia should be differentiated from bacterial pneumonia, cmv pneumonia, and infl uenza pneumonia. hiv/aids related herpes simplex viral pneumonia is mostly demonstrated by multiple signs, including small nodules, ground glass liked shadows and patchy parenchymal changes. the nodules are in centrilobular distribution, mostly with accompanying branches liked shadows (tree buds sign). chest x-ray demonstrates diffuse lung parenchymal changes. imaging fi nding are parenchymal change areas with bilaterally blurry boundaries. generally, the nodules have a diameter of 2-10 mm. ct scanning with high resolution demonstrates nodules with surrouding ground glass liked density lesions. lymphoid/lymphocytic interstitial pneumonia (lip) is more common in children with aids. the cdc in the united states has defi ned lip in children under the age of 13 years as the diagnostic indicator of aids. the predictive diagnostic criteria include chest x-ray demonstration reticular nodular changes in pulmonary interstitium of both lungs for no less than 2 months, undetectable pathogens and no responses to the antibiotic therapy. currently, hiv/aids related lymphocytic interstitial pneumonia is considered to be related to hiv and epstein-barr virus, human t cell leukemia-lymphoma type i virus (htlv-i) and hiv-i. the infection of the above viruses causes pulmonary lymphatic hyperplasia and other systemic diseases. about 22-75 % children with hiv infection sustain lip, and 3 % in adults. most cases of non-hiv infected patients with lymphoid interstitial pneumonia are women, at average age of 56 years old, more commonly in the age group of 40-50 years old and above 70 years old. the pathological manifestations are infi ltration of small and mature lymphocytes as well as plasma cells in alveolar septum and the alveolus, extensive interstitial fi brosis and non-caseous granuloma. it is characterized by diffuse infi ltration of lymphocytes, plasmocytes and histocytes in the pulmonary interstitium. the lymph follicle with germinal center is more common. hyperplasia occurs in type ii alveolar epithelium, and the macrophage increases in the alveolar cavity. there are rare or mild intraalveoli organization and macrophage aggregation. staining of the immune globulin light chain demonstrates poly-clone b cells. the clinical symptoms are in progressive development, with cough and suffocation, rare hemoptysis and sjogren syndrome commonly in mouth and eyes. by examinations, the signs have slight difference between adults and children. in children, there are lymphadenectasis, hepatosplenomegaly, enlargement of parotid gland, clubbing fi ngers and wheezing sound. in adults, there are lymphadenectasis, slight fi ne bubbling rales, as well as hepatosplenomegaly and enlargement of parotid gland in 1/3 patients. 1. peripheral hemogram demonstrates increased lymphocytes and eosinophilic granulocytes. 2. myelogram demonstrates increased lymphocytes, plasmocytes and eosinophils. 3. blood biochemical examination demonstrates increased immune globulin, predominantly lgm. 4. blood gas analysis demonstrates hyoxemia. 5. pathogenic examinations by bronchofi broscopy, bronchial alveolar lavage and biopsy can defi ne the diagnosis. 6. pulmonary function examinations demonstrate restrictive ventilatory disorder, lower lung compliance and impaired diffusion function. impaired diffusion function is a more sensitive indicator in monitoring the progress of the disease. 7. chest x-ray is the most commonly used imaging examination, while chest ct scanning is commonly applied for the differential diagnosis. chest x-ray demonstrates hiv/aids related lymphoid interstitial pneumonia as reticular or reticular nodular shadows of lung markings in both lungs. hrct demonstrates bilateral diffuse ground glass liked density shadows. perivascular thin-walled pneumatocele is common. pneumatocele induced by lip is commonly found in the middle lung fi eld, which probably is due to the valve effects caused by infi ltration of cells around bronchioles. manifestations of pneumatocele, together with ground glass liked density shadows, highly indicate lip. centrilobular and subpleural small nodules and thickened intralobular septa can be occasionally found. dysfunctional diffusion is a more sensitive indicator in monitoring the progress of the disease. hiv/aids related lymphoid interstitial pneumonia should be differentiated from allergic pneumonia, carcinomatous lymphangitis and pneumocystis carinii cysts. ct scanning with high resolution demonstrates characteristic lesions of hiv/aids related lymphoid interstitial pneumonia, including intralobular linear shadows and honeycomb liked changes, with common involvement of the subpleural area and the basal lung. its characteristic manifestations are clustering gas containing thin-walled cyst in a diameter of 2-10 mm, with clear cyst wall. shared wall between cysts is its characteristic demonstration. the surrounding ground glass liked density indicates infl ammation. intralobular linear shadows indicate interstitial fi brosis. pulmonary toxoplasmosis (pt) is caused by the toxoplasma parasitizing in cells. ludlam et al. fi rstly proposed the concept of pulmonary toxoplasmosis in 1963 [ 107 ] , arguing that toxoplasma can cause atypical pneumonia. later, there are some pathological reports about pulmonary toxoplasmosis or disseminated toxoplasmosis with lung involvement. in recent years, due to the global prevalence of aids, the incidence of pulmonary toxoplasmosis is increasing, with most cases being disseminated toxoplasmosis with lung involvement. pt has been one of the important opportunistic infections in patients with immunosuppression, especially aids patients. hiv/aids related pulmonary toxoplasmosis is a zoonotic disease, with cats as its main transmission source, followed by pigs and sheep. people are infected by intake the water or food contaminated by cats' feces or without cooked meat. immunocompromised aids patients are susceptible to this disease, and its occurrence is rare in immunocompetent people. after its invasion into the human body, the sporozoite in the cystozygote and intracystic cystozoite overfl ows to penetrate the intestinal wall mucosa and spread to the whole body tissues along with blood or lymph fl ow. the brain, heart, lymph nodes, and lung are the most vulnerable tissues and organs for the infection. any abnormality in the process of defense mechanism can cause impaired immune functions to eliminate the toxoplasma, which ultimately causes systemic and pulmonary infections. pulmonary toxoplasma infection may also be caused by the blood borne spreading of reactivated toxoplasma infection in other body parts, with no exclusion of reactivated pulmonary infection or primary pulmonary infection. ludlam et al. generally nominated toxoplasmosis as atypical pneumonia [ 107 ] . catterall et al. divided toxoplasmosis into three types: necrosis, infl ammatory infi ltration and toxoplasma invasion [ 109 ] . it can also be classifi ed as type a: subclinical or occult infection; type b: interstitial and atypical pneumonia; type c: necrotic pneumonia; type d: lobar pneumonia; and type e: granulomas pneumonia (toxoplasmoma). by naked eyes observation, the involved lungs are solid, with congestion and red brown section. the pleura have bleeding spots, with moderate peribronchial lymphadenectasis. under a light microscope, there is exudation of serous fl uids in alveolar cavities, occasional formation of transparent membrane or fi brin purulent exudation, infi ltration of small quantity neutrophils, proliferation and shedding of alveolar wall cells, and trophozoite and/or cysts of toxoplasma in epithelial cells and macrophages. the pulmonary interstitium may have infi ltration of lymphocytes and plasmocytes as well as visible fi broblasts and macrophages. the granuloma changes are also found in the lung tissues, with central stripes or localized necrosis and surrounding lymphocytes and small quantity multinucleated giant cells. it is diffi cult to fi nd toxoplasma in granuloma, but it can be found in the normal tissues around or near the granuloma. almost all cases of aids complicated by pulmonary toxoplasmosis are caused by disseminated toxoplasmosis with pulmonary involvement. it is commonly diffuse pulmonary infl ammation with serious symptoms, including high fever, cough, cyanosis, breathing diffi culty, possible occurrence of skin rashes, lymphadenectasis and meningitis. the chronic cases may have long-term low grade fever, cough, and weight loss. direct light microscopy of the specimen smear and enprint such as blood, cerebrospinal fl uid, bone marrow, anterior aqueous humor, phlegm, urine, saliva, and other osmotic solutions, as well as lymph nodes, muscle tissue or other living tissues can be performed for pathogen examinations. sabin proposed that the staining test have high sensitivity and specifi city according to the fi ndings that mixture of fresh toxoplasma with normal serum can be stained deep by alkaline methylene blue staining, while its mixture with immune serum can be stained light or blank by the same staining. other assays including indirect fl uorescent antibody, indirect blood coagulation, and complement fi xation test can provide valuable reference for the diagnosis. toxoplasm is tested in pathological eaminations that can provide valuable reference for the diagnosis. chest x-ray is the most commonly used diagnostic imaging examination. and chest ct scanning can be applied for the differential diagnosis. imaging fi ndings of hiv/aids related pulmonary toxoplasmosis can be divided into four types: bronchial pneumonia, interstitial pneumonia, pleuritis and complication of cardiovascular disease. the type of bronchial pneumonia is also known as lobular pneumonia, with thickened pulmonary markings that distribute along with the bronchi in the middle and lower lung fi elds, scattered patchy shadows with uneven density and blurry boundaries, fusion of some shadows into large fl aky shadow and widened hilar shadow. the type of interstitial pneumonia is demonstrated as reticular and nodular shadows. the interstitial lesions widen the space between the bronchiole and the alveolar wall, with stripes and fl occulent shadows. the type of pleuritis is rare, with signs of pleural effusion. the type of complication of cardiovascular disease is demonstrated as heart failure (acute pulmonary edema), with signs of pericardial effusion. a male patient aged 39 years was confi rmatively diagnosed as having aids by the cdc. he complained of cough and fever. his cd4 t cell count was 29/μl. by direct light microscopy, toxoplasma tachyzoites can be found in the specimens such as blood, cerebrospinal fl uid, bone marrow, anterior aqueous humor, phlegm, urine, saliva, and other osmotic solutions, as well as lymph nodes, muscle tissues or other living tissues. the fl uorescent antibody and complement fi xation test are positive. the biopsy tissue culture and inoculation test are positive. in the lesions and their surrounding tissues of interstitial e c d fig. 17 . 105 (continued) pneumonia, necrotic bronchitis or granuloma, toxoplasma can be found. the diagnostic imaging demonstrates any one type of pulmonary toxoplasmosis, including bronchial pneumonia, interstitial pneumonia, pleuritis and cardiovascular disease, can be used as the evidence for the diagnosis of pulmonary toxoplasmosis. the type of bronchial pneumonia is also known as lobular pneumonia, with thickened pulmonary markings with a distribution in both middle and lower lung fi elds along with the bronchi, scattered patchy shadows with uneven density and blurry boundaries, fusion of some patchy shadows into large fl aky shadow and widened hilum. the type of interstitial pneumonia has typical demonstrations of reticular and nodular shadows. the interstitial lesions widen the space between the bronchiole and the alveolar wall, with strip and fl occulent shadows. the type of pleuritis is rare, with signs of pleural effusion. the type of cardiovascular disease may have signs of heart failure (acute pulmonary edema), and signs of pericardial effusion. hiv/aids related pulmonary toxoplasmosis should be clinically differentiated from infectious mononucleosis and mycoplasma pneumonia. with primary pulmonary lesions. pulmonary low malignant b cell lymphoma is the most common primary pulmonary lymphoma which is derived from mucosa related lymphoid tissue. the manifestations include slowly decreased alveolar transparency. pulmonary high malignant b cell lymphoma is extremely rare, which often occurs with singular lesion and primary disease such as immunodefi ciency. hiv/aids related malignant lymphoma is mostly caused by compromised immunity. hiv/aids related hodgkin's lymphoma is relatively rare. there are also reports about hiv/aids related t cell lymphoma with pulmonary involvement. hiv/aids related malignant lymphomas are mostly highly malignant large cells lymphoma. cerebral lymphoma is one of the defi ning diseases of aids. it has been reported that the clinical incidence of pulmonary infi ltration by malignant lymphoma is 10-20 %, but 29-50 % by autopsy. in the early stage, it is commonly asymptomatic. with its progression, symptoms of dry cough, suffocation, and small quantity clear phlegm occur. mediastinal lymphadenosis includes lymphadenectasis to compress the trachea by, blood vessels and nerves and lead to breathing diffi culty, superior vena caval obstruction syndrome, and hoarse voice. the pulmonary parenchyma lesions include reticular structure in the lungs. the clinical symptoms are cough, expectoration, suffocation and breathing diffi culty. a male patient aged 43 years was confi rmatively diagnosed as having aids by the cdc. he complained of chest distress and cough for more than 1 month. his cd4 t cell count was 56/μl. in patients with hiv/aids related kaposi's sarcoma, its serologic positive rate is 100 %. kaposi's sarcoma cells can produce il-6, during which il-6 plays a role as an autocrine factor to maintain the cell growth, paracrine cytokines, stim-ulate proliferation of other interstitial cells and induct the vascular growth. therefore, kaposi's sarcoma is a kind of tumor with abundant blood vessels. before the application of harrt, the incidence of kaposi's sarcoma in male homosexuals is 21 %. after the clinical application of harrt treatment, the incidence is decreasing. in addition to hhv-8, some studies indicated that most patients with kaposi's sarcoma have hia-dr5 alleles, suggesting a possible relationship between kaposi's sarcoma and the heredity. there is no obvious difference between hiv/aids related kaposi's sarcoma and classic kaposi's sarcoma in pathological changes. early pathological manifestations are chronic infl ammation or granulomatous infl ammation, with formation of new vascular and lymphatic vessels and accompanying edema and bleeding. the fi ndings of large and protruding endothelial cells in granuloma tissue with accompanying erythrocytic exudation and hemosiderin particles have great signifi cance for the early diagnosis. the pathological changes in the advanced stage are signifi cant proliferation of the endothelial cells, and proliferation of fi broblasts around capillaries. in the advanced stage, the lesions are often accompanied by extensive connective tissue hyperplasia, which presents diffi culty for its differentiation from common sarcoma. when it is diffi cult to defi ne the diagnosis by light microscopy, immunohistochemical examinations can be used to defi ne the diagnosis. the pathological changes are characterized by lesions confi ned to the epithelial lamina propria, gathering of spindle cells with mild heteromorphism around many lacuna vasorum with irregular lumen, erythrocytic exudation and hemosiderin sedimentation. the atypical lacuna vasorum can be compressed by proliferative spindle cells to be absent. vascular endothelial cells and peripheral spindle cells may have mitotic phase in the advanced stage, with increased heteromorphism cells. the infl ammatory cells are mainly plasma cells, with acidophilic corpuscles and pas staining positive, which can assist the pathological diagnosis. pulmonary kaposi's sarcoma in aids patients rarely has symptoms. it is commonly concurrent with pulmonary opportunistic infections, with symptoms of cough, diffi culty breathing and fever. other symptoms are related with the location of the tumors. the involvement of trachea or bronchi can cause luminal stenosis. the mediastinal tumor can compress and obstruct lymph vessels to cause pulmonary edema or a large quantity pleural fl uids, which result in respiratory diffi culty, and even respiratory failure. (1) sampling by bronchoscopy or endoscopy to prepare pathological section. (2) chest x-ray demonstrates its typical manifestation of pleural effusion. dr demonstrates enlarged and deranged hilum in both lungs in bird nest liked appearance. there is light density fl aky shadows in the both lower lungs. ct scanning demonstrates multiple rounds liked nodular shadows in the middle and lower lung fi elds of both lungs with clear boundaries. there are also mediastinal and hilar lymphadenectasis, with common involvement of the pleura and bilateral pleural effusion in a small quantity. a male patient aged 33 years was confi rmatively diagnosed as having aids by the cdc. he had been detected as hiv positive for 5 months, with complaints of recurrent cough and nausea for 10 days and was hospitalized on jan. 7, 2004. the transmission route was unknown because he denied histories of intraveneous drug abuse, paid blood donation, blood transfusion and unhealthy sexual behaviors. five months ago, he was diagnosed as aids in the stage of aids in our hospital, and hospitalized to treat pcp, with a cd4 t cell count of 17/μl. his symptoms were quickly relieved after pcp treatment and he continued the antiviral therapy for almost 5 months after being discharged. by physical examinations, he was in poor spiritual condition, a light blue nodule in size of 0.5 × 0.5 cm in the left upper chest wall with medium hardness, palpable lymph nodes in size of 1.0 × 2.0 in the opisthotic area and inguen, no tenderness and being movable. by the digital rectal examination, a palpable prominent nodule with wide base at 4 cm 7 points away from the anus, with fl exible texture and smooth surface. by the auxiliary examinations, wbc 3.9 × 10 9 /l, neμt 48.3 %, lym 34.9 %, mon 7.4 %, eos 9.0 %, rbc 3.27 × 10 12 /l, hgb 126 g/l, plt 210 × 10 9 /l, routine urine test normal, blood sedimentation 16 mm/h. by hepatitis b examinations, hbsag, anti-hbe and anti-hbe positive. his cd4 t cell count was 91/μl. by abdominal b ultrasound, multiple low echo nodules in the abdominal cavity, the largest in size of 1.2 × 1.0 cm, which are suspected to be enlarged lymph nodes. on jan. 14, he received inguinal lymph node biopsy, with pathological report of kaposi's sarcoma. during the treatment and following up, the involvement of lungs, digestive tract, lymph nodes and skin is suspected, with the diagnosis of phase ii kaposi's sarcoma and chemotherapy was recommended. reexamination by chest x-ray demonstrated normal cardiopulmonary phrenic. abdominal b ultrasound failed to fi nd enlarged lymph nodes. ct scanning demonstrated shrinkage of lesions in both lungs and mediastinal lymph nodes, with only palpable soybean sized submandibular lymph node. by examinations after chemotherapy, cd4 t cell count 67/μl, viral load 63,000 copies/ml. the patients had multimorphological erythema drug eruptions, which was suspected as drug allergies of chemotherapy, which were absent after symptomatic treatment. the following ups so far show no recurrence of kaposi's sarcoma, with his cd4 t cell count fl uctuating around 400/μl. he can work as usual. a male patient aged 36 years was confi rmatively diagnosed as having aids by the cdc. he had a history of homosexual behaviors, with complaints of fever and cough for 2 months as well as chest distress for more than 20 days. since, july 2010, fever with a body temperature of about 37.5-37.8 °c occurs, with cough, yellowish bloody sputum, and dark purplish patchy skin rashes. by examinations, his anti-treponema pallidum antibody positive, multiple dark purplish patchy skin rashes on the face, eyelid, lower jaw, hairline, chest and abdomen with skin surface desquamation, palpable bilateral cervical lymphadenectasis and the largest in size of 10 × 19 mm. by laboratory tests, wbc 5.98 × 10 9 /l, n 78.74 %, rbc 2.22 × 10 12 /l, hgb 71 g/l, plt 204 × 10 9 /l, cd4 12/μl. a male patient aged 27 years was confi rmatively diagnosed as having aids by the cdc. he complained of cough for more than 2 months, chest distress for more than 1 month, and bloody sputum for half a month and was hospitalized. he had a history of homosexual behaviors. by examinations on admission, multiple round purplish blue skin rashes nodules on the limbs. his cd4 t cell count was 9/μl. demonstrations can also be fl aky fl occulent areas with blurry density or parenchymal density areas along with bronchi. 3. lung puncture for histopathological biopsy demonstrates irregular vascular lumen in the dermis, proliferation of endothelial cell with accompanying heteromorphism. in some cases, there are tumor masses composed of spindle cells and epithelial cells. other sarcoma and vascular tumor hiv/aids related kaposi's sarcoma should be differentiated from other sarcoma and vascular tumor. ks invasion of the digestive mucosa can cause bleeding and upper gastrointestinal symptoms. the pathological lesions can be diagnosed by upper gastrointestinal endoscopy or biopsy. in the cases of no fever and exclusion of infections, the typical imaging demonstrations and bronchoscopy fi ndings can defi ne the diagnosis of pulmonary ks. hiv/aids related kaposi's sarcoma should be differentiated from pneumocystis carinii pneumonia. the lesions of pcp are mostly symmetric ground glass liked density shadows extending outwards from the hilum in both lungs. in the middle and advanced stages, nodules, fi brosis and cavities occur, rarely with pleural effusion. lung cancer commonly refers to the cancer in lung parenchyma, usually does not include those mesodermal tumors originating from other pleura, or other malignancies like carcinoid, malignant lymphoma, or metastatic malignancies for other body parts. therefore, the following lung cancer we are discussing about refers to the malignancies originating from bronchial, or bronchiolar epithelial cells, accounting for 90-95 % of the lung parenchyma malignancies. the cause of lung cancer is still not completely known. data have indicated that the risk factors of lung cancer include smoking (including second-hand smoke), asbestos, radon, arsenic, ionizing radiation, halogen alkenes, polycyclic aromatic compounds and nickel. long-term smoking can cause proliferation of the bronchial mucosal epithelial cells and proliferation of squamous epithelium to induce squamous epithelium carcinoma or undifferentiated small cell carcinoma. non-smokers can also develop lung cancer, but adenocarcinoma is more common among them. long-term exposure to radioactive substances, like uranium and radium, and its derivatives; carcinogenic hydrocarbons, like arsenic, chromium, nickel, copper, tin, ferri, coal tar, bitumen oil, petroleum, asbestos and mustard gas, all can induce lung cancer, which is commonly squamous carcinoma and undifferentiated small cell carcinoma. some chronic pulmonary diseases, such as tuberculosis, silicosis and pneumoconiosis, can concurrent with lung cancer. in the cases with these chronic pulmonary diseases, the incidence of cancer is higher than the general population. in addition,bronchopulmonary chronic infl ammation and pulmonary fi brous scar lesions may cause metaplasia or hyperplasia of squamous epithelium during their healing processes, based on which some cases can develop into cancer. the internal factors of the human body include family heredity, compromised immunity, metabolism and endocrine dysfunction. the lung cancers distribute more in the right lung than in the left lung, more in the upper lobe than in the lower lobe. its locations range from the major bronchus to the bronchioles. the central type of lung cancer has its origination from the major bronchial lobes and locates adjacent to the pulmonary hilum. the peripheral type of lung cancer has its origination from the lower parts of pulmonary segment bronchi and locates in the peripheral areas in the lungs. in the growth process of the lung cancer, it causes the extension and dilation of the bronchial walls, and penetrates the bronchial walls to invade the adjacent lung tissues and form masses. meanwhile it intrudes into the bronchi to cause luminal stenosis or obstruction. with its further progression and dissemination, it spreads from the lungs and directly extends into the chest walls, mediastinum, heart, major vessels and other adjacent organs and tissues. lung cancer can also transfer to other parts of the body along with blood and lymph fl ows or disseminates to other pulmonary lobes via the respiratory tract. the growth rate and transferring paths of lung cancer depend on its histological types, differentiation degree and other biological characteristics. lung cancer has no special symptoms in the early period, only has the common symptoms with common respiratory diseases, including cough, bloody sputum, low grade fever, chest pain and chest distress. therefore, it is often misdiagnosed. (1) face and neck edema; (2) hoarse voice is the most common symptom; (3) shortness of breath. lung cancer tends to occur distant metastases in the early stage. in the cases with metastatic lesions to the brain, the patients sustain persistent headache and blurry vision. in the cases with metastatic lesions to the bone, bone destruction may occur to cause fracture. (1) restrictive wheezing sound, mostly occurring in the inspiratory phase and recurring after cough; (2) hoarse voice, caused by lymph nodes transferring to compress and invade the recurrent laryngeal nerve; (3) superior vena cava syndrome, caused by the compresses or invasion to the superior vena cava by the mass and venous obstruction, with edema in the head, face, neck, and upper limbs, varicose veins and edema in the upper chest, and accompanying dizziness, chest distress, shortness of breath and other symptoms; (4) horner's syndrome, with enophthalmos of the affected side, blepharoptosis, shrinkage of the pupils, eye fi ssure stenosis, increased skin temperature in the upper chest of the affected side and no sweating due to compression or invasion of the apical cancer to the cervical sympathetic ganglia; (5) should and arm pain, which is radial burning pain in the shoulder and upper limbs of the affected side due to compression or invasion of apical cancer to the brachial plexus nerve; (6) phrenic nerve paralysis, with symptoms of shortness of breath and chest distress due to invasion to the phrenic nerve; (7) dysphagia, caused by compressed esophagus by mediastinal lymphadenectasis; and diffi culty breathing caused by compressed trachea by mediastinal lymphadenectasis; (8) pericardial effusion, shortness of breath, arrhythmia and heart dysfunctions due to pericardial invasion; (9) pleural metastasis, with chest pain and cancerous pleural effusion; (10) lung cancer metastasis, spreading of lung cancer along with blood fl ow to the bone, liver, brain, kidney, adrenal gland and subcutaneous tissues. intrapulmonary metastasis is also common. metastasis to different locations shows different symptoms and signs. (11) extrapulmonary signs, commonly including joint pain or joint hypertrophy, clubbing fi ngers and mental disorders. the diagnostic imaging is the most commonly used and an important examination for the diagnosis of lung cancer. it can facilitate to fi nd some specifi c manifestations in the lesions, which provide clues for the diagnosis of lung cancer. it is also the main basis for the staging of lung cancer, but fails to defi ne the qualitative diagnosis. chest x-ray is the main examination for the diagnosis of lung cancer. anteroposterior and lateral chest x-ray are used for preliminary screening. chest ct is the diagnostic imaging examination of the choice for the diagnosis of lung cancer. for the central type of lung cancer in the early stage, there are direct signs to defi ne the diagnosis. in the early stage, thin layer scanning with a layer thickness of 1.5-4 mm can be performed to observe the bronchial changes. mr imaging can demonstrate intraluminal nodules, luminal thickness and luminal stenosis of the bronchi from the transverse, coronal, and sagittal perspectives. mr imaging demonstrates favorably cancer in the lesions of obstructive pneumonia, and masses covered by the hilum. pet/ct scanning can be used for the screening of lung cancer metastasis and assessing the therapeutic effi cacy after treatment. dsa is used for infusion chemotherapy of bronchial artery in the cases of primary lung cancer. bronchoscopy is an important examination for the diagnosis of lung cancer. the pathological changes of the endothelium and the lumen of bronchi can be directly observed by using bronchoscopy. for the cases with caner or cancerous infi ltration by bronchoscopy, sampling of the tissues under the guidance of bronchoscopy for biopsy can be performed. otherwise, bronchial secretions can be suctioned under the guidance of bronchoscopy for cytological examinations to defi ne the diagnosis and the histological classifi cation. in most cases of primary lung cancer, the shed cancer cells can be found in the sputum, which can also facilitate to defi ne its histological classifi cation. after several examinations and short-term exploratory therapies, the qualitative diagnosis cannot be defi ned and the possibility of lung cancer cannot be excluded. therefore, exploratory thoracotomy can be performed if the patient's physical conditions permit. chest x-ray early lesions are confi ned within the bronchi, causing valve ventilatory disorder and changes of obstructive emphysema. the manifestations include restrictive pulmonary gas increase and sparse lung markings. in the cases with certain degree of bronchostenosis due to unfavorable discharge of the secretions, obstructive pneumonia occurs, showing patchy blurry shadows. in the cases with complete blockage of the bronchi, obstructive atelectasis occurs, showing decreased pulmonary volume, increased density and migration of the mediastinum to the affected side. obstructive pulmonary bronchiectasis has demonstrations of intrapulmonary cords liked shadows. lung a male patient aged 41 years was confi rmatively diagnosed as having aids by the cdc. he complained of repeated cough for more than 1 month and reported to have a history of unhealthy sexual behaviors. his cd4 t cell count was 65/ μl. cancer in the middle and advanced stages are mainly manifested as hilar mass and atelectasis. the mass has a high density with clear boundary. however, the cancer cannot be observed due to its common immersion in the large fl aky obstructive pneumonia lesion or large quantity pleural effusion. atelectasis is commonly manifested as shrinkage of pulmonary segments or shrinkage of unilateral lung, with high density. the shadow of atelectasis widens at the hilum to show prominent mass. in the cases of central type lung cancer in the right upper lobe, a transverse s shape is at the hilum (commonly known as pancoast cancer). early diagnosis of central type lung cancer by plain chest x-ray only shows some indirect pulmonary manifestations caused by bronchial obstruction. and these indirect signs are not characteristically lung cancer. in the cases of local obstructive emphysema, these indirect signs can be caused by foreign substances in the bronchi or early infl ammation. obstructive pneumonia is diffi cult to be differentiated from common pneumonia. obstructive atelectasis needs to be differentiated from many other conditions. (1) pathological changes in the bronchial lumen including polypoid, nodular or fl at papula masses. benign tumor has smooth boundary and malignant tumor has unsmooth boundary, commonly with wider base and thickened lumen wall. even the slight bronchial changes caused by the central type of lung cancer can be demonstrated by thin layer ct scanning, including slightly thickened bronchial wall, intraluminal small nodules and lumen stenosis or obstruction. in the middle and advanced stages, the direct signs of the central type lung cancer include thickened bronchial wall, irregular or unsmooth lining of the bronchial lumen. bronchial obstruction is suddenly truncation or gradual thinning of the lumen to obstruction. (2) hilar mass locates adjacent or around the bronchi, with smooth or arch shaped boundary. the indirect signs of the central type lung cancer in the middle and advanced stages include secondary changes to bronchial stenosis. obstructive pneumonia is manifested as patchy blurry shadows or parenchymal changes of the pulmonary segments/lobes, and decreased lung volume. mr imaging demonstrates the tumor as long t1 and long t2 signals. in the cases of central type lung cancer with secondary obstructive atelectasis and obstructive pneumonia, enhanced t1 demonstrates the tumor in the lesion of pulmonary atelectasis and obstructive pneumonia. the signal of atelectasis is higher than that of the tumor. pet/ct scanning can demonstrates increased and thick stained metabolites of metastatic lesions or residual lesions, which has a diagnostic sensitivity of above 90 %, and a reported specifi city of 80-90 %. in addition, it can be applied for the clinical consideration of it hilar, mediastinal lymph node metastasis and extrathoracic distant metastasis, which is an important method to decide clinical stages before lung cancer therapy. but pet has false negative diagnosis in the diagnosis of lung cancer with decreased metabolites, especially the alveolar cell carcinoma. for the diagnosis of pneumonia and pulmonary tuberculosis, it also has false positive results. dsa can demonstrate the blood supply of the tumors. miliary tuberculosis is more common in young adults, with obvious symptoms of systemic toxicity. anti-tuberculosis drug therapy can relieve the symptoms, with gradually absorbed lesions. (3) chest x-ray demonstrates hilar lymph node tuberculosis as mass shadows in the hilum of lung, which may be misdiagnosed as the central type lung cancer. hilar lymph node tuberculosis is more common in teenagers, commonly with symptoms of tuberculosis infection but rarely hemoptysis. lung cancer can be concurrent with pulmonary tuberculosis. (1) bronchial pneumonia; obstructive pneumonia induced by early lung cancer can be misdiagnosed as bronchial pneumonia. bronchial pneumonia has an acute onset with more obvious symptoms of infection. chest x-ray demonstrates patchy or spots shadows, with blurry boundaries and uneven density. the lesions are not confi ned within one segment or one lobe. (2) pulmonary abscesses; central necrosis and liquefaction of the lung cancer results in cancerous cavities. by chest x-ray, the central type lung cancer can be misdiagnosed as pulmonary abscesses. in the acute period, a pulmonary abscess has obvious symptoms of infection, with large quantity purulent sputum. chest x-ray demonstrates thin cavity wall, smooth inner wall, liquid level and infl ammatory changes in the surrounding lung tissues or pleura. pulmonary benign tumors including hamartomas, fi broma and chondroma have slow growth. chest x-ray demonstrates round liked mass shadow, with homogeneous density without lobation. joint united nations programme on hiv/aids (unaids) surveillance for aidsdefi ning opportunistic illnesses, 1992-1997 imaging and pathology of hiv related cytomegalovirus pneumonia roentgenographic patterns of pneumocystis carinii pneumonia in 104 patients with radiology distinction of pyogenic pulmonary infection from pneumocystis carinii pneumonia in aids 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at risk for human immunodefi ciency virus type 1 infection. implications for rational empiric antibiotic therapy the intracellular bacterium rhodococcus equi requires mac21 to mammalian cells virulence of rhodococcus equi isolates from patients with and without aids association between large plasmid and 15 to 17 kilo dalton antigens in virulent rhodococcus equi rhodococcus equi plasmids:isolation and partial characterization rhodococcus equi pneumonia in aids: high-resolution ct fi ndings in fi ve patients cytomegalovirus pneumonitis in patients with aids: fi ndings in an autopsy series immediate causes of death in acquired immunodefi ciency syndrome the causes of death in patients with humanimmunodefi ciency virus infection: aclinical and pathologic study with emphasis on the role of pμlmonary diseases cytomegalovirus pneumonitis: spectrum of parenchymal ct fi ndings with pathologic correlation in 21 aids patients rhodococcus equi -an increasingly recognized opportunistic pathogen a highly representative two hybrid genomic library for the yeast yarrowia lipolvtica rhodococcus equi infection in hiv infected patients rhodococcus equi infection in patients with and without human immunodefi ciency virus infection radiological fi ndings in nine aids patients with rhodococcus equi pneumonia comparison of nucleic acid amplifi cation, serology, and microbiologic culture for diagnosis of rhodococcus equi pneumonia in foals cytokine modulation alters pulmonary clearance of rhodococcus equi and development of granulomatous pneumonia diseases of the lung: radiologic and pathologic correlations pneumonia: high-resolution ct fi ndings in 114 patients radiologic distinction of pyogenic pulmonary infection from pneumocystis carinii pneumonia in aids patients high-resolution ct in the evaluation of clinically suspected pneumocystis carinii pneumonia in aids patients with normal, equivocal, or nonspecifi c radiographic fi ndings pneumocystis carinii pneumonia: spectrum of parenchymal ct fi ndings pulmonia in patient with aids discontinuation of prophylaxis for pneumo-cystis carinii pneumonia in hiv-infected patients treated with highly active antiretroviral therapy carcinoma of the lung in hiv-positive patients: fi ndings on chest radiographs and ct scans pulmonary complications of hiv-associated malignancies pneumocystis carinii infection and aids comparison of histologic stains in the diagnosis of pnemocystis carinii tuberculosis in patients with human immunodefi ciency virus infection comparative histopathological study of pulmonary tuberculosis in human immunodeficiency virus-infected and non-infected patients pulmonary tuberculosis in aids patients: transient chest radiographic worsening after initiation of antiretroviral therapy active pulmonary tuberculosis in patients with aids: spectrum of radiographic fi ndings (including a normal appearance) effect of hiv status on chest radiographic and ct fi ndings in patients with tuberculosis pulmonary mycobacterium avium complex disease without dissemination in hiv-infected patients mycobacterium avium complex infection in the acquired immunodefi ciency syndrome pulmonary disease due to infection by mycobacterium avium complex in patients with aids cd4 t lymphocyte count and the radiographic presentation of pulmonary tuberculosis. a study of the relationship between these factors in patients with human immunodefi ciency virus infection tuberculosis in the aids era mycobacterial pseudotumors of lymph node mycobacterial spindle cell pseudotumor of lymph node managenent of the hiv-infected patient tuberculosis in the aids era cytomegalovirus pneumonia in aids patients atypical cytomegalic cells are diagnostic for cytomegaloviral infection in aids cytomegalovirus as a primary pulmonary pathogen in aids infections with cryptococcus neoformans in the acquired immunodefi ciency syndrome endemic fungal pneumonia in immunocompromised patients disseminated histoplasmosis in adis: fi ndings on chest radiographs opportunistic fungal pneumonia pulmonary aspergillosis in the acquired immunodeficiency syndrome coccidioidomycosis during human immunodefi ciency virus infection. a review of 77 patients cryptococcal pulmonary infection in patients with aids: radiographic appearance pulmonary aspergillosis in patients with aids. clinical and radiographic correlations cryptococcal pneumonia in aids: is cryptococcal meningitis preceded by clinically recognizable pneumonia pulmonary manifestations of disseminated cryptococcosis in patients with clinical and bacteriological aspects of nocardiasis lung abscess in patients with aids focal mycobacterial lymphadenitis following initiation of protease-inhibitor therapy in patients with advanced hiv-1 disease rhodococcus equi endobronchial mass with lung abscess in a patient with primary pulmonary aids-related lymphoma: radiographic and ct fi ndings the expanding challenge of hiv-associated malignancies the pulmonary manifestations of aids-related non-hodgkin's lymphoma varied appearance of aids-related lymphoma in the chest lymphoma versus aids intrathoracic kaposi's sarcoma in women with intrathoracic kaposi's sarcoma. ct fi ndings distribution of human herpesvirus 8 dna in tumorous and nontumorous tissue of patients with acquired immunodefi ciency syndrome with and without kaposi's sarcoma kaposi's sarcoma in lymphnodes concurrent with hodgkin's disease the natural history of kaposi's sarcoma in the acquired immunodefi ciency syndrome pulmonary toxoplasmosis in patients in-fected with human immunodefi ciency virus: a french national survey diagnostic imaging, preautopsy imaging and autopsy fi ndings of 8 aids cases analysis on the imaging features of aids with pulmonary fungal infection liver injury in hiv-1-infected patients receiving non-nucleosides reverse transcriptase inhibitors-based antiretroviral therapy mri demonstrations of aids complicated by toxoplasma gondii infection in cervical spinal cord: with 3 cases reports diagnostic imaging in aids in china: current status and clinical application imaging and pathological fi ndings of aids complicated by pulmonary rhodococcus equi infection magnetic resonance spectroscopy in the diagnosis of cognitive impairment in aids patients ct image demonstrations of hiv-seropositive tuberculosis and their relationship with cd4+ t-lymphocyte count positron emission tomography of 18fdg uptake in localized pulmonary infl ammation penicilliosisde rhizomys sinensis accumulation of nuclear mitochondrial dna in the frontal cortex cells of patients with hivassociated neurocognitive disorders imaging fi ndings of aids complicated with pulmonary rhodococcus equi infection and correlated with pathology pulmonary toxoplasmosis? pathogenesis and prevention of rhodococcus equi infection pulmonary toxoplasmosis a female patient aged 35 years was confi rmatively diagnosed as having aids by the cdc. she complained of cough, fever and no sputum. her cd4 t cell count was 45/ μl. filariform larvae of strongyloides stercoralis invade into skin or mucosa and reach the lungs through lymphatic vessels or venous system and the right heart. they develop into schistosomula in 3-30 days. a few schistosomula develop mature in the lungs or bronchi. most schistosomula penetrate the pulmonary capillaries into alveoli to cause a series of respiratory symptoms. in the cases of serious infection and in patients with compromised immunity, disseminated lesions occur in lungs and other organs. hiv/aids related pulmonary strongyloidiasis can have manifestations of local small bleeding spots, pimples, migratory linear or strips urticaria on skin, as well as manifestations of allergic bronchitis, lobular pneumonia or asthma. aids patient may have severe diffuse infection and systemic infection, with symptoms of fever, severe cough, expectoration, hemoptysis, shortness of breath, breathing diffi culty, and asthma. 1. laboratory tests 2. the pathological examinations include biopsy tissue culture and inoculation test. 3. chest x-ray is the most commonly used examination. there are demonstrations of small spots and fl aky shadows, thickened hilar shadow and thickened pulmonary markings. mitchell reported in 1992 that interstitial or alveolar infi ltration in both lungs accounts for 62 %, nodular shadows in both lungs 15 %, hilar or mediastinal lymphadenectasis 26 %, pleural fl uids 42 %, septal line 25 %, mediastinal lymphadenectasis and ascites are the important clues for the diagnosis. 1. the clinical manifestations of hiv/aids related pulmonary strongyloidiasis are non-specifi c. its diagnosis depends on the etiological examinations. the fi ndings of strongyloides sterocralis in the patient's sputum and feces can defi ne the diagnosis. increases to 20 × 10 9 ⁄l; eosinophils granulocytes 25-30 %, or even as high as 70-80 %; the serum total lge level increases by 50 %; 90 % cases with blood serum lgg and lge of fi lariform larvae antigen positive. in the cases with femal strongyloides stercoralis parasitizing in the bronchial epithelium, rhabditiform larva, fi lariform larva, schistosomula, adult strongyloides stercoralis and the eggs can be found in fresh phlegm, which can defi ne the diagnosis. 3. pathological examinations including biopsy tissue culture and inoculation test are positive. 4. by chest x-ray, the lungs have small spots and fl akes of shadows, thickened hilar shadow and thickened pulmonary markings. hiv/aids related pulmonary strongyloidiasis should be differentiated from hiv/aids related pulmonary toxoplasmosis, infectious mononucleosis and mycoplasma pneumonia. pulmonary infi ltration of hiv/aids related malignant lymphoma commonly has three types: primary pulmonary lymphoma, which is rare and accounts for 0. 3. the diagnostic imaging examinations are the most commonly used examinations for the diagnosis. the imaging demonstrations of hiv/aids related malignant lymphomas include: 1. mediastinal lymphadenectasis is the most commonly found pulmonary manifestations of malignant lymphoma. the lesions are mainly located in anterior and middle mediastinum, in asymmetric wave liked or lobulated mass. it occurs unilaterally or bilaterally, isolated or fusion. 2. the incidence of pulmonary parenchymal lesions is 20-30 %. chest x-ray demonstrates mediastinal lymph nodes extending directly into the lungs, which is susceptible to confusion with pneumonia. they are demonstrated as round shadows in the lung fi elds or distribute in the whole lung fi elds. chest x-ray demonstrates the lymphatic spread of lesions as military nodules in different sizes or isolated intrapulmonary nodules or cavities, commonly accompanying with mediastinal hilar lymphadenectasis. in the cases with its occurrence secondary to endobronchial membrance, obstructive pneumonia or atelectasis can be caused. some patients may have diffuse pulmonary interstitial changes. pulmonary infi ltration by non-hodgkin's lymphoma can also be divided into four types: (1) nodular type; (2) pneumoniaalveolar type; (3) bronchial-vascular-lymphatic type, which can be further divided into the central bronchialvascular type, and diffuse lymphatic type; (4) diffuse lymphatic type can have lesions of reticular or reticular nodular infi ltration and its progression into patchy changes. 3. miliary-blood borne spreading type is rare. 4. the pleural lesions is mainly pleural effusion, with bloody or serous pleural fl uid. a male patient aged 41 years was confi rmatively diagnosed as having aids by the cdc. he complained of chest distress and chest pain for more than 2 months. his cd4 t cell count was 36/μl. (1) tuberculoma is diffi cult to be differentiated from the peripheral type of lung cancer. tuberculoma is more common in young adults, with a long term course of illness. the lesions are commonly found in the apical posterior segment of the upper lobe or dorsal segment of the lower lobe. chest x-ray demonstrates the lesions with uneven density and satellite lesions. (2) miliary tuberculosis is diffi cult to be differentiated from diffuse bronchioloalveolar carcinoma. key: cord-017140-k4lzwfge authors: andersen, bjørg marit title: protection of upper respiratory tract, mouth and eyes date: 2018-09-25 journal: prevention and control of infections in hospitals doi: 10.1007/978-3-319-99921-0_13 sha: doc_id: 17140 cord_uid: k4lzwfge pathogenic bacteria and viruses may invade via upper and lower respiratory tract and via eye mucosa. when an infected person coughs or sneezes heavily, small, invisible droplets with the infective agent may reach a good distance from the source. by using the right form of protection at the right time, infection and disease are prevented. the present chapter is focused on the protection against airborne infections. • contact with infectious patients, according to the isolation procedures. • when performing sterile procedures. • when in close contact with patients who are in an infection-prone situation, for example, during operations and patients with compromised immune system. • contact with wounds and tissues and/or in direct contact with sterile equipment used invasively or when present during ongoing invasive procedures. • self-protection against splashes/aerosol of biological material (trachea suction, vomiting, cough, diarrhoea, secretions, diathermy, etc.). • patient with suspected contagious respiratory infection-during transport, examination, treatment, etc.; use a face mask-also on the patient-to protect others and the environment from contamination. • when cleaning and disinfecting contaminated rooms like isolates and when handling used patient equipment/machinery with organic material (ventilator, cpap, etc.) and used patient textiles, infectious waste and bio-organic waste. • during work with plumbing and construction with increased risk of soil, splatter, aerosols or dust particle clouds, such as working with instrument channels in the patient rooms and surgical departments, ventilation systems, sinks, sewer, water leakages with fungal growth, etc. • caps should always be used when putting on masks to protect hair. hospital management should ensure an infection control programme that informs all employees about the standards of hygiene and infection control at the hospital. furthermore, to provide resources to the acquisition, stock reserves and logistics of adequate personal protective equipment (ppe), also for emergency situations [5, 8] . department management is responsible for training, use and control of face masks, respirators and eye protection and that the equipment and written guidelines are available [5] . each user is responsible for the proper use of ppe at the right time and in accordance with current guidelines. the following are available in all relevant departments/posts: • guidelines-written-for the use of a face mask/respirator/eye protection. • surgical masks: put a date on the box when it opens. only surgical masks of good quality are used. other face masks-thin and of poor quality-fastened behind ears should never be used in the healthcare system because of no protective effect. • respiratory protection-p3 mask-with and without valve, separately packed. put the date on the box. • surgical face masks with visor. • face shield. • goggles. single-use or multi-use goggles that can be disinfected and autoclaved between uses. • cap/hood/head or neck protection (phantom hood, operation caps)-always when using masks. • access to good hand hygiene-hand disinfectant-at the place where the ppe is used. when airways, mouth and eyes are protected, the hair and head should also be covered simultaneously. many people have long hair that can come in contact with the patient, bedding or equipment which can lead to transmission of infection to other patients, in addition to themselves becoming a carrier. nb! always use cap/hood/head protection when using surgical mask or other ppe. these may be single-use or multi-use. single-use devices are thrown away immediately after use. surgical mask with visor may protect against direct spills and splashes. a complete face shield protects against direct splashing. the multi-use equipment (check with infection control personnel) is soaked in chloramine 5% 1 h (or household chlorine) before laundered in soapy water or washed in the instrument washing machine by more than 85 °c or autoclaved. it is stated on the package if the goggles may be autoclaved. quality-controlled surgical face mask is disposable and used for: putting on • disinfect your hands. • put on the cap-thin operating hood-that collects all hair. pull it over your ears. • disinfect your hands. • take the mask from the surgical mask box, and take only one mask. the box must have the date of opening. all boxes that have been exposed to infection should be discarded afterwards. • put the face mask over your mouth and nose with a drawstring at the back of the head/top and the other on the neck. • adapt the face mask that has metal string over the nose-so that it fits tightly and comfortably around the mouth and nose. • replace the face mask between each patient/situation/procedure or after 2-6 h if you are with the same patient or when it is wet on the inside. avoid changing the mask if this can cause more contamination and risk, for instance, during surgery. surgical masks are usually not changed during surgery. • never go with a face mask under the chin or around your neck! it is usually heavily contaminated by mouth and nose secretion after use and by splatter from the patient. • perform hand hygiene. • first take hold of the string in the neck and loose this while you bend forward. the lower part of the mask then falls away from the face. then gently loosen the string on the head, and gently put the mask into the waste container. • face masks should not come in contact with hair or clothes. • dispose in regular waste during normal use or infectious waste if infection. • perform hand hygiene afterwards. • grip the cap back and carefully pull it off while bending forward, and put it into the waste container. • perform hand hygiene afterwards. filtering half masks and guidelines for use of respiratory protection should be available at relevant clinical departments. p3 mask is used by the surgical team and during all sterile procedures: in the case of operative treatment of patients with special types of airborne infection such as tuberculosis, etc., see above. if there is an open breathing valve on the p3 mask, surgical mask must be used outside the p3 mask. p3 mask with covered breathing valve can be used instead. p2 mask or surgical mask is put on the patient with defined or suspected airborne infection (e.g. tuberculosis, varicella, etc.) during transport, and stay outside isolation units. putting on • disinfect your hands. • put on the cap-thin operating hood-that collects all hair. pull it over your ears. • disinfect your hands. • take the p3 mask from the surgical mask box, and take only one mask. each mask is usually separately wrapped. the box must have the date of opening. all boxes that have been exposed for infection should be discarded afterwards. • put the respirator over your mouth and nose with a drawstring at the back of the head/top and the other on the neck. the cap hood underneath makes it easier to put the mask on-it does not slip. • adapt the face mask that has metal string over the nose-so that it fits tightly and comfortably around the mouth and nose. • test tightness by blowing vigorously or breathing in; leaks are then sensed on the sides of the mask. • change the p3 mask after 3-6 h or longer or if it is wet on the inside. • avoid change if this may lead to risk of infection. take it off very carefully, as the p3 mask may be used in a serious contagious situation and may be contaminated on the outside. • disinfect your hands. • grasp the band/string posteriorly, bend forwards and remove the mask gently without coming into contact with the clothing, skin or hair. do not touch the mask directly. • loosen the band in the neck first so that the mask falls forwardly away from the face. then carefully loosen the band on the head. • put the mask gently into infectious waste bin. • perform hand hygiene. • grip the cap back, bend forwards and carefully pull it off without coming into contact with the skin or clothes and place the mask in infectious waste bin. • perform hand hygiene afterwards. eye protection is always used when there is a risk of splashing of human biological material to the eyes and for protection against highly infectious diseases. in the event of a risk of severe airborne disease, wear tight protective goggles where you can use regular glasses on the inside. multi-use goggles may be reused after 1 h of treatment in 5% chloramine bath (or household chlorine 10,000 ppm) with subsequent soapy water and rinsing. putting on • perform hand hygiene. • put on the cap-thin operating hood-that collects all hair. pull it over your ears. • in case of severe, dangerous infection, put goggles on outside of a phantom cap that is sitting outside a surgery cap and a p3 mask; see strict isolation. • disinfect your hands. • goggles are retrieved from the box, usually separately wrapped. the box must have the date of opening. all boxes that have been exposed for infection should be discarded afterwards. • put the glasses or shield over the eyes with a rubber band on the occiput. the operation hood under prevents it from slipping. • adapt the goggles-so that it fits tightly and comfortably all around the eyes. • they can be used as long as they are needed. take it off very carefully, as the goggles/face shields may be contaminated on the outside. • disinfect your hands. • grasp the band/string posteriorly, bend forwards and remove the goggles/face shield gently without coming into contact with the clothing, skin or hair. do not touch the devices directly. • if multi-use: put it carefully into the container with 5% chloramine. if single-use: put it into the infectious waste bin. • perform hand hygiene. • grip the cap back, bend forwards and carefully pull it off without coming into contact with the skin or clothes, and place it in infectious waste bin. • perform hand hygiene afterwards. "the employer must ensure that protective equipment made available to the worker, meets requirements of regulations on construction, design and manufacture of personal protective equipment" [5] . this should be in accordance with official regulations for the use of personal protective equipment [5, 9] . already in roman times, it was pointed out by doctor galen that "when many get sick and die at once, we must look for a common cause, the air we breathe". [1, [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . most cases of these serious, life-threatening diseases may be transmitted via air, droplets and re-aerosols, from patients, carriers and the environment. an adult breathes in at rest 5-8 litres of air per minute, at medium heavy work 30-40 l and at great exertion 70-100 litres of air per minute. in small rooms and with a high air contamination of infectious agents, some contaminants will be drawn into the respiratory tract. it has been demonstrated that when a person coughs or sneezes, drops, droplets and droplet nuclei from the mouth and nose may reach up to 9 m from the source [2] . surgical masks and respiratory protection (filter masks) are defined as "equipment that can help prevent the spread of microbes from one person to another" [2, 3] . the difference between surgical mask and respiratory protection is that surgical masks primarily protect the patient and sterile area/equipment against mouth and nose secretion from healthcare professionals, while respiratory protection protects healthcare workers and others from airborne infections from nearby sources of infection [3-5, 14, 23] . however, surgical masks are not approved as protection against airborne infections: [5, 14, 24, 25] "harmful microorganisms (bacteria, viruses, fungi) or components of microorganisms (e.g. endotoxins) may occur in air, either in dust, smoke or aerosols, or even finer distributed as droplet nuclei where all liquid has dried in. surgical masks only protects against splash and drop, not against airborne infection. therefore, to protect against airborne infection, wear respiratory protection. in most situations, a filtering half mask will provide a good protection. particle filter class p2 protects against most spores of fungi. at risk of exposure to airborne viruses and bacteria, especially tuberculosis, particle filter p3 must be used. in particularly dangerous situations, during prolonged work or if carrying a beard, should special breathing systems be used" [5] (fig. 13.1 ). many human pathogenic bacteria and viruses invade via upper and lower respiratory tract [1] . some viruses may also invade via eye mucosa, such as influenza, hepatitis b and c and hiv. carl schiøtz, a norwegian professor in hygiene and infection control, wrote in the textbook of hygiene in 1937 (80 years ago) that the most important measures against communicable diseases are the following: (1) isolation of the sources of infection (home or hospital), (2) disinfection of the exposed rooms and equipment, (3) vaccination if vaccine-preventable disease, (4) quarantine, (5) food hygiene and (6) insect eradication [26] . he noticed that airborne infection was considered to be "the most important form of transmission of infections at our latitude" [26] . schiøtz meant that drops from the respiratory tracts were large and heavy and went 1.5-2 m away, while saliva droplets went in "up to 20 m distance from high-speaking people and they could stay floating in the air for several hours" [26] . this was important for the spread of "flu, colds, pneumonia, pest-pneumonia, tuberculosis, pertussis, measles, small-pox, chickenpox, scarlet fever, rubella, diphtheria, poliomyelitis, epidemic cerebrospinal meningitis and several other diseases" [26] . he also focused on inhalation of dust containing microbes [26] . the introduction of antibiotics in post-war times and the sharp reduction of lung tuberculosis caused many to forget that it was something that was infected via air. tuberculosis has been "recognized" as an airborne infection at least in 100 years, although some still believe that short-term exposure to the patient does not lead to infection. this perception has led to multiple outbreaks of multiresistant tuberculosis, including in the united states [27] . airborne transmission is most often downgraded by the health authorities to contact and droplet transmission-traditionally within 1 m from the patient. this applies to healthcare professionals who are going to treat patients with severe, deadly infections where surgical masks are estimated "good enough" [10-16, 23-25, 28-31] . droplet transmission is a definition that may put healthcare personnel at risk during dangerous situations since it is still a "form of contact transmission" [17] . the cdc definition from 2007 is upgraded since 2004 but is still very vague and controversial; see the following quotations [17] : droplet transmission is generated when "an infected person coughs, sneezes, or talks -or during procedures such as suctioning, endotracheal intubation, cough induction by chest physiotherapy and cardiopulmonary resuscitation. ---the maximum distance for droplet transmission is currently unresolved,---historically, the area of defined risk has been a distance of ≤3 feet around the patient----investigations during the global sars outbreaks of 2003 suggest that droplets --could reach persons located 6 feet or more from their source. ----thus, a distance of ≤3 feet around the patient is best viewed as an example of what is meant by 'a short distance from a patient' ----it may be prudent to don a mask when within 6 to 10 feet of the patient or upon entry into the patient's room, especially when exposure to emerging or highly virulent pathogens is likely--" [17] . "droplet size is another variable under discussion ---defined as being >5 μm in size. droplet nuclei, particles arising from desiccation of suspended droplets, have been associated with airborne transmission and defined as ≤5 μm in size-----particle dynamics have demonstrated that a range of droplet sizes, including those with diameters of 30μm or greater, can remain suspended in the air" [17] . the worst example until now is the recommendation of "contact and droplet transmission within 1 m" with the use of surgical masks (within 1 m from the patient), by who, the first phase (3 months) of the sars epidemic in 2003, where 90% of those who became ill during this first phase were health professionals [28] . the same was done during the avian influenza epidemic in 2005 and mostly throughout the influenza pandemic in 2009 [29] . who and cdc recommended both "contact and droplet transmission" measures during the first 6 months of the ebola epidemic in 2014 [1, 30] . during some of the most serious global epidemics that have happened in the last 80 years, healthcare personnel were exposed to infection without proper protective equipment and had the highest death rate associated with work-related infection [10-17, 30, 31] . during the sars outbreak in toronto, canada, 169 health personnel were infected with nosocomial sars, and 3 of these died [14, 31] . infection control personnel in toronto insisted that sars was primarily transmitted through large droplets-within 1 m-and that there was lack of evidence-based documentation for airborne infection! [31] despite the fact that health professionals requested it, the respiratory protection (n95) was not handed over to the staff, and the outbreak continued for a long time [31] . a state investigation commission came to the following bottom line conclusion: safety comes first and reasonable measures to reduce the risk of healthcare professionals do not have to wait for scientific evidence [31] . in the event of outbreaks of less severe airborne infections such as common flu, rsv, mycoplasma, adenovirus, metapneumovirus, etc., it is important to protect healthcare professionals from infection. the purpose is that infected personnel should not be "vectors" of infections in the hospital. in addition, a large outbreak among the staff may cause that patients with life-threatening diseases like heart attack, trauma, etc. do not get the necessary treatment. despite all the controversies surrounding airborne transmission, a number of international guidelines for the use of respiratory and face protection equipment have been developed under varying epidemiological conditions and experiences over the past 100 years. recent surveys and experiences show that schiøtz and other researchers had correct facts and guidelines according to airborne infections. secrets from the respiratory tract can go far off 9 m or more, for example, when coughing and sneezing, and pathogenic viruses and bacteria are detected in the air in rooms with infected patients [2, 14, 17, 18, 20, 21, 26, [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] . airborne mrsa and other multiresistant bacteria may be a greater problem than previously thought [17, 18, 20, 42] . even clostridium difficile spores may become airborne under certain conditions [43] . today, it is also known that most microbes are strong, surviving organisms outside the body [1, 44] . they can also survive on the outside of the protective equipment and even re-aerosol from these and even penetrate a wet mask [1, 4, [44] [45] [46] [47] [48] . respirators (half masks) are more expensive (about 4 usd) than surgical mask (<0.1 usd). because of a shortage during large outbreaks, it has been attempted to decontaminate respirators for reuse. however, this is not effective, is not recommended and cannot be done with disposable equipment [49] . most infectious agents are spread through contact, blood, drops, droplets, drop nuclei (aerosol), airborne on dust particles and often in several ways simultaneously [12, 13, 17, 23, [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [50] [51] [52] . transmission of microbes from the respiratory tract to the air occurs by breathing, speech, cough, sneezing, laughing, singing or other aerosol-generating procedures. sneezing usually leads to greater amount of microbes transferred to air than coughing [51] . how long the contaminant stays alive in the air depends on the agent, temperature, humidity, virus dose response, particle size and presence of other material [50] [51] [52] [53] [54] [55] [56] . some larger drops are deposited close to the patient, and smaller dropsdroplet nuclei-pass into a floating phase in the air and are falling slowly over time, such as influenza a virus [2, [50] [51] [52] [53] [54] [55] [56] . transmission of microbes via small particles and droplet nuclei from influenza patients is not adequately controlled by the use of surgical mask [50] [51] [52] . airborne transmission always includes contact transmission and often re-aerosols from the environment [18-21, 39, 42-44, 55-61] . transmission to the air from the skin occurs via the release of millions of skin particles where approximately 10% carry bacteria or virus from the skin or wound. re-aerosols from infectious particles whirled up in the air, for instance, during bed-making, dry mopping of contaminated floor surfaces, from contaminated areas in the room during air currents or when the door is opened or closed quickly [62] . re-aerosols of pathogenic microbes may be important because of a long survival in the environment, for days, months and years [1, 44] . influenza a virus survives on paper, textiles and equipment for more than 3 weeks and can become airborne [55] [56] [57] [58] [59] [60] [61] . influenza a virus is very contagious via aerosols and small airborne droplets [56] . microbial load in the air depends on cleaning and air exchange [5, [10] [11] [12] [13] [14] 63] . proper cleaning and ventilation with a positive pressure of filtered air reduce the load in the operating units or during protective isolation. decontamination and cleaning of rooms, textiles and surfaces and a good air exchange with a negative air pressure are important during isolation of patients with infections. air exchange it has been demonstrated that after aerosol-generating procedures such as bronchoscopy, airway suction and intubation, five fresh air changes in the room can greatly reduce air contamination, provided that the source of infection is gone [13] . respiratory protection prevents people from touching their nose or mouth in an infectious situation and coughing on their own uniforms and thereby exposing themselves to others and to infection [6, 7] . correct use of respiratory protection most microbes survive for hours to days on the outside of a respiratory protection as shown for bacteriophage, influenza virus and coronavirus [45] [46] [47] . this is the reason why respiratory protection should not be reused and why it is not advisable to touch the outside of the mask. during the sars outbreak in canada in 2003, the staff reinfected themselves by handling the mask incorrectly and reusing equipment without decontamination [28] . among 1441 hospital personnel in china, there was a significantly lower incidence of respiratory tract pathogenic microbes among personnel who used respiratory protection with filter n95 (p = 0.02) than among personnel using face mask (p < 0.01) compared to controls [64] . face mask, covering the nose and mouth, has been used since the spanish flu in 1919. it was actually developed to protect patients from postoperative infections from the airway flora of the operating team and protect the team against blood and tissue splash from the patient. there is no standard definition of surgical masks, adaptation, leak test or quality of filter [5, 13, 14] . surgical mask is not approved by the occupational health authorities [3-5, 24, 25] . nevertheless, there are extensive use and purchase of face masks, especially in the national emergency preparedness plans. the combination of good hand hygiene and the use of surgical mask within 36 h after the onset of influenza in an index case may however reduce spread of the flu in the household [65] [66] [67] . volunteer subjects infected with seasonal flu breathed out nearly nine times more virus in small particles, <5 μm, than in larger one, >5 μm, after the onset of the flu [50] . the use of surgical masks reduced particularly the larger particles and showed a 3.4-fold reduction of virus released to the air [50] . using surgical face masks can reduce infection and also had some suppressing effect on the spread of infection by sars [10] . surgical face mask protects the nose and mouth from splashing and saliva particles but does not protect effectively against bacteria and viruses that are, respectively, from 0.2 to 8 μm (0.0002-0.008 mm) and from 20 to 300 nm (0.02-0.3 μm) in diameter. there is no safe protection against aerosol or droplet nuclei, either through the mask or from the sides of the mask [5, 13, 24, 25, 68] . it is not certain that a mask may stop microparticles of the blood that can be formed by certain procedures, like centrifugation accidents, spray from dental treatment or surgery, etc. [36, 68] . in such cases, also eye protection should be used. persons close to sterile areas can contaminate the area by direct drops of saliva and aerosols that are spilled by speech, coughing and sneezing [2, 13] . a good face mask captures these droplets from the respiratory tract and prevents deposition thereof. it catches drops both ways to some extent, but when the surgical mask is wet, it can become less effective, with some growth of bacteria. it is usually effective for at least 2-3 h. face masks with visors protect to some extent both nasal/oral and eye mucosa against infected blood and tissue particles/droplets. airborne microparticles of aerosol, drop nuclei and blood drop-bearing bacteria and viruses can be formed during activities of or around the patient (coughing, sneezing, speech, excessive bodily activity, bed-making, dust cleaning etc.). respiratory protection such as p3 mask or n95 has shown protective effect in highly severe infections such as sars, tuberculosis and pandemic influenza [5, 9-16, 20-23, 56, 64] . respirators are not designed for children or people with beards, and therefore does not provide full protection for these [5, 23] . respiratory protection is filtering half mask, looks almost like surgical mask and is often called filtering face piece (ffp) respirators or dust filter [5] . they are of different quality (ffp1-ffp3) and capture microbes in both ways. the filter is hepafilter/polypropylene filter with static charge to increase the filter power. ffp2 is equivalent to the us n95 masks that are widely used around the world. the european standard is en149: 2001 which is equivalent to ffp3 and has a somewhat higher level of protection [13] . procedures involving large load on the environment around a patient with airborne infection are, for example, bronchoscopy, trachea suction, diathermy aerosol, suction and drilling (orthopaedics, dental treatment, etc.). in such cases, both respirators and eye protection should be used. the p3 mask protects the user and can also be used on patients with specific respiratory infection and then without exhalation valve. protection factor is calculated by reducing the degree of pollution in the air. if there is a pollution of 1000 mg/m 3 air, a respirator with a protection factor of 500 may reduce the pollution to a residue of 2 mg/m 3 . that includes inhalation of air through or on the sides of the respiratory protection, which will almost always happen [69] [70] [71] [72] . "protection factors for filtering half masks ffp1, ffp2 and ffp3 matches the protection factors for half mask with respectively filter classes p1, p2 and p3. motor-assisted filtering air purifying respirators (equipment with a turbo unit) have varying protection factor depending on the equipment design" [5] . a wet filtering mask may be permeable for viruses and bacteria. the norwegian directorate of labour inspection indicates protection factors for filtering respirators as follows [5] : lower protection is expected, because it is often leaked due to poor adaptation; factor 10 is for filtering half mask classes 2 and 3, and factor 100 is for full face masks. "protection factor for both supplied air and air purifying respirators with half and full face masks also require a good fit, and this cannot be expected if beard, glasses or the like, in squeeze along the edge of the mask" [5] . requirements for filtering half masks [5, 12, 13, [69] [70] [71] [72] . p3 has the highest protection factor with 50 times cleaner air inside the mask than outside. the mask's overall efficiency depends on the filter quality of the mask, fitting the face shape (leakage) and leakage through any exhalation [5, 12, 13, [69] [70] [71] [72] . fit test is the control and training using special odour tests that the person perceives within the mask if it is not tight enough [5, 12, 13] . in a study of five models of the n95 mask (3m 1860s cup, 3m 1870 flat fold, kimberly-clark pfr95 duckbill, safelife t5000 cup added with iodine and glaxosmithkline actiprotect cup, added with lemon acid), all five types had more than 95% efficiency: −0.8 μm particles of h1n1 influenza virus and corresponding inert particle sizes [69] . filtration efficiency was largely based on particle size [69] . so-called elastomeric masks appear to be more effective than regular filter masks [70] . studies show considerable variation with regard to protection, and for some dangerous, microbial agents, 95% protection may be too low [71, 72] . • turbo equipment (papr = -powered air-purifying respirators) with p3 filter (protection factor 500-2000) -may have problems concerning the disinfection of battery-operated, recycling systems, and is dependent on a good training [5, 12, 13] . • fresh air/pneumatic equipment (protection factor, 100-100 000) is used for particularly risky situations of special personnel in laboratories and when working in areas with severe epidemics. however, there may be serious infection problems regarding disinfection of reuse systems (filters, mechanical/electrical appliances). during non-epidemic times and with low-virulent microbes, respiratory protection (n95 or p3 masks) are not used for other than special types of respiratory infections and pulmonary tuberculosis. this may influence on the state of readiness for major outbreaks such as pandemic influenza. consumption of masks can be very large as during the sars outbreak in 2003, where a canada-based hospital with sars outbreak used up to 18,000 n95 masks each day [13] . the durability of respiratory protection equipment is good-up to 10 years or more in storage. items made with rubber or rubber parts should be checked, and there should be some systemic rotation in the warehouse [49] . at ouh, ullevål, there has been a strategic stockpile system of such equipment with replacement as required [8] . the experience from norway is that the national health authorities have been unprepared for stockpiling of emergency response requirements for ppe, also during the influenza pandemic of 2009 [29] . the market may soon be empty for respiratory protection masks during serious outbreaks such as sars and ebola virus. using several surgical face masks superposed did not have enough protective effect [73] . during the sars epidemic, the chinese healthcare personnel made their own masks of 12 layers of gauze that supposedly would work well. this is used when there is risk in soil and splatter of biological materials and to protect against highly dangerous infection. if there is a risk of transmission of severe respiratory infection, tight-fitting goggles are used. the goggles can be reused after 1 h of treatment in 5% chloramine, followed by washing with soap and water, and they may also be reprocessed and decontaminated in a washing machine at >85 °c. controversial issues concerning airborne infection and protection against "droplet transmission" should be further studied. particle studies; ventilation variations; kinetics; the effect of humidity, temperature and filter types; re-aerosols from dust; environment and ppe equipment; and the survival of microbes in the environment should be better studied. this is the case for most important microbial agents: bacteria, viruses and fungi. respirators should be preferred over surgical masks when there is suspected droplet or airborne transmission. this choice is essentially a misunderstood price and attitude problem. microbiology and infection control. handbook of hygiene and infection control in hospitals violent expiratory events: on coughing and sneezing protection of workers from the risks when working with biological factors on the protection of workers from the risks related to exposure to biological agents at work (seventh individual directive according to article 16 norwegian directorate of labour inspection. respiratory protection controlling the novel a (h1n1) influenza virus: do not touch your face! european centre for disease prevention and control -swine flu guidelines: "cough hygienically" into your sleeve? 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indoor air air and surface contamination patterns of methicillinresistant staphylococcus aureus on eight acute hospital wards the potential for airborne dispersal of clostridium difficile from symptomatic patients virus survival in the environment corona virus survival on healthcare personnel protective equipment survival of surrogate viruses on n95 respirator material persistence of the 2009 pandemic influenza a (h1n1) virus on n95 respirators re-aerosolization of ms2 bacteriophage from an n95 filtering facepiece respirator by simulated coughing evaluation of five decontamination methods for filtering face-piece respirators influenza virus aerosols in human exhaled breath: particle size, culturability, and effect of surgical masks viral kinetics and exhaled droplet size affect indoor transmission dynamics of influenza infection aerosol transmission is an important mode of influenza a virus spread dynamics of airborne influenza a viruses indoors and dependence on humidity mechanisms by ambient humidity mayaffect viruses in aerosols inactivation of influenza a viruses in the environment and modes of transmission: a critical review high infectivity and pathogenity of influenza a virus via aerosol and droplet transmission survival of influenza viruses on environmental surfaces survival of influenza viruses on banknotes association of private isolation rooms with ventilatorassociated acinetobacter baumannii pneumonia in surgical intensive-care unit an evaluation of hospital special-ventilation-room pressures survival of influenza a (h1n1) on materials found in householders: implications for infection control door-opening motion can potentially lead to a transient breakdown in negative-pressure isolation conditions: the importance of vorticity and buoyancy airflows mopping up hospital infection efficacy of face masks and respirators in preventing upper respiratory tract bacterial colonization and co-infection in hospital healthcare workers face masks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial preliminary findings of a randomized trial of nonpharmaceutical interventions to prevent influenza transmission in households face mask use and control of respiratory virus transmission in households aerosol penetration and leakage characteristics of masks used in the healthcare industry challenge of n95 filtering face-piece respirators with viable h1n1 influenza aerosols comparison of performance of three different types of respiratory protection devices do n95 respirators not provide 95% protection level against airborne viruses, and how adequate are surgical masks? performace of an n95 filtering face-piece particulate respirator and a surgical mask during human breathing: two pathways for particle penetration protecting staff against airborne viral particles: in vivo efficiency of laser masks key: cord-018208-sc8j1ate authors: qu, bo; wang, huijuan title: the accuracy of mean-field approximation for susceptible-infected-susceptible epidemic spreading with heterogeneous infection rates date: 2016-11-09 journal: complex networks & their applications v doi: 10.1007/978-3-319-50901-3_40 sha: doc_id: 18208 cord_uid: sc8j1ate the epidemic spreading over a network has been studied for years by applying the mean-field approach in both homogeneous case, where each node may get infected by an infected neighbor with the same rate, and heterogeneous case, where the infection rates between different pairs of nodes are also different. researchers have discussed whether the mean-field approaches could accurately describe the epidemic spreading for the homogeneous cases but not for the heterogeneous cases. in this paper, we explore if and under what conditions the mean-field approach could perform well when the infection rates are heterogeneous. in particular, we employ the susceptible-infected-susceptible (sis) model and compare the average fraction of infected nodes in the metastable state, where the fraction of infected nodes remains stable for a long time, obtained by the continuous-time simulation and the mean-field approximation. we concentrate on an individual-based mean-field approximation called the n-intertwined mean field approximation (nimfa), which is an advanced approach considered the underlying network topology. moreover, for the heterogeneity of the infection rates, we consider not only the independent and identically distributed (i.i.d.) infection rate but also the infection rate correlated with the degree of the two end nodes. we conclude that nimfa is generally more accurate when the prevalence of the epidemic is higher. given the same effective infection rate, nimfa is less accurate when the variance of the i.i.d. infection rate or the correlation between the infection rate and the nodal degree leads to a lower prevalence. moreover, given the same actual prevalence, nimfa performs better in the cases: 1) when the variance of the i.i.d. infection rates is smaller (while the average is unchanged); 2) when the correlation between the infection rate and the nodal degree is positive. our work suggests the conditions when the mean-field approach, in particular nimfa, is more accurate in the approximation of the sis epidemic with heterogeneous infection rates. by considering the system components as nodes and the interactions or relations in between nodes as links, networks have been used to describe the biological, social and communication systems. on such networks or complex systems, viral spreading models have been used to describe processes e.g. epidemic spreading and information propagation [8, 10, 13, 20] . the susceptible-infected-susceptible (sis) model is one of the most studied models. in the sis model, each infected node infects each of its susceptible neighbors with an infection rate β . the infected node can be recovered with a recovery rate δ . both processes are independent poisson processes. the ratio τ β /δ is called effective infection rate, and when τ is larger than the epidemic threshold τ c , the epidemic spreads out with a nonzero fraction of infected nodes in the metastable state. the average fraction of infected nodes y ∞ in the metastable state, ranging in [0, 1], indicates how severe the influence of the virus is: the larger the fraction y ∞ is, the more severely the network is infected. in this paper, we concentrate on deriving the average fraction y ∞ of infected nodes in the metastable state. although the continuous-time markov theory can be used to obtain the exact value of y ∞ , the number of states is too large to be solved in a large network [12] . hence, the derivation of the average fraction y ∞ of infected nodes in the metastable state mostly relies on mean-field theoretical approaches. the first approach to study the sis model in complex networks is a degree-based mean-field (dbmf) theory, also called heterogeneous mean-field (hmf) approximation, proposed by pastor-satorras et al. [14] , which assumes that all nodes with the same degree are statistically equivalent, i.e. the infection probabilities of those nodes are the same. an individual-based mean-field (ibmf) approximations, called the n-intertwined mean-field approximation (nimfa), of the sis model is then introduced [19] with the only assumption that the state of neighboring nodes is statistically independent. nimfa, taking the network topology into account, turns out to be more precise on different types of networks for the classic sis model with the homogeneous infection rates [7] while comparing to the dbmf approximation. however, as discussed in [4, 15, 22] , the infection rates could be heterogeneous, i.e. the infection rates between different pairs of nodes could also be different. the accuracy of nimfa with heterogeneous infection rates has not yet been discussed. in this paper, we explore the influence of the heterogeneous infection rates on the precision of nimfa. in particular, we compare the average fraction y ∞ of infected nodes as a function of the effective infection rate τ computed by nimfa to that obtained by the continuous-time simulations of the exact sis model when the infection rates are heterogeneous but the recovery rate is the same for all nodes. in fact, the effective infection rate τ refers to the average infection rate divided by the recovery rate in the sis model with heterogeneous infection rates. we set the average infection rate to 1 and tune the recovery rate δ to control the effective infection rate τ. we consider both the independent and identically distributed (i.i.d.) and the correlated heterogeneous infection rates in different network topologies. the n-intertwined mean-field approximation (nimfa) is so far one of the most accurate approximations of the sis model that takes into account the influence of the network topology. for the classic sis model with the homogeneous infection rate β and recovery rate δ . the single governing equation for a node i in nimfa is where v i (t) is the infection probability of node i at time t, and a i j = 1 or 0 denotes if there is a link or not between node i and node j. the governing equation (1) can be extended to the heterogeneous case: where β i j = β ji is the infection rate between node i and j. in the steady state, defined by dv (t) where a is the n × n adjacency matrix with elements α i j , i is the n × n identity matrix, diag(v i (t)) is the diagonal matrix with elements v 1 (t), v 2 (t), ...., v n (t) and b is the infection rate matrix with elements β i j . the trivial, i.e. all-zero, solution of (3) indicates the absorbing state where all nodes are susceptible. the non-zero solution of v ∞ in (3), if exists, points to the existence of a metastable state with a non-zero fraction of infected nodes. or else, the metastable state can be figured as 0 or not existing. we are interested in actually the metastable state in this paper. in this paper, we keep the average infection rate to 1 and tune the recovery rate δ to control the effective infection rate τ. in the case of the i.i.d. heterogeneous infection rates, we aim to explore how the heterogeneous infection rates influence the accuracy of nimfa when the variance of the infection rate varies. we choose the infection-rate distribution that is frequently observed in real-world and importantly the variance is tunable with a fixed mean so that we can systematically explore how the accuracy of nimfa changes with the broadness of the i.i.d. infection rate. we consider the log-normal distribution, of which we can keep the mean unchanged and tune the variance in a large range. the log-normal distribution [18] b ∼ log-n(β ; µ, σ ), of which the probability density function (pdf) is, for β > 0 has a power-law tail for a large range of β provided σ is sufficiently large. the log-normal distribution has been widely observed in real-world, where interaction frequencies between nodes are usually considered as infection rates. wang et al. [21] find that by employing the log-normal distributed infection rates, their epidemic model can accurately fit the infection data of 2003 sars; we also find that the infection rates in an airline network follow the log-normal distribution [15] . in [15] , we find that, if the epidemic does not die out, the larger the variance of the i.i.d. infection rate is, the smaller the average fraction y ∞ of infected nodes is. we will show that this conclusion can actually explain the observation about how the accuracy of nimfa changes with the variance of the i.i.d. infection rates at a given effective infection rate τ in this paper. for correlated heterogeneous infection rates, we build a correlated infection-rate scenario and a reference one. in the correlated infection-rate scenario, we assume where d i and d j are the degree of node i and node j respectively, c is selected so that the average infection rate is 1 and α indicates the correlation strength. as discussed in [17] , such a correlation between the infection rate and the nodal degree is motivated by the real-world datasets. in this case, the infection rate of each link is determined by the given network topology and α. for the reference scenario, we shuffle the infection rates from all the links as generated in the first scenario and redistribute them randomly to all the links. in this way, we keep the distribution of infection rates but effectively remove the correlation between the infection rates and nodal degrees. for simplicity, we name this reference scenario as the uncorrelated infection-rate scenario. though the i.i.d. infection rates are also uncorrelated, we can tune the variance of the infection rate in the case of the i.i.d. infection rates while keeping the distribution and the mean of the infection rates. however, in the scenario of uncorrelated infection rates in this paper, the distribution of the infection rate changes with the parameter α, hence the variance of the heterogeneous infection rates cannot be systematically tuned. a positive α > 0 (or negative α < 0), suggests a positive (or negative) correlation between infection rates and nodal degrees. too large or small values of α could not be realistic. for example, [3, 9, 11] suggest that α is around 0.5 or 0.8 in their datasets. hence, we select α = −0.25, −0.5, −1 for the negative correlation and α = 0.25, 0.5, 1 for the positive correlation. different values of α also offer the possibility to explore how nimfa performs with different correlation strengths. in this paper, we aim to understand how the correlation influences the accuracy of nimfa by comparing the average fraction y ∞ of infected nodes obtained by nimfa and the simulations of the exact sis model. in [17] , we explored the influence of the correlation between the infection rate and the nodal degree on the prevalence of epidemic, which can be used to partially explain the conclusions in this paper. as in our previous work [15, 17] , we perform the continuous-time simulations of the sis model. we consider both the scale-free (sf) and erdös-rényi (er) models for different network topologies. the sf model has been used to capture the scale-free nature of degree distribution in real-world networks such as the internet [5] and world wide web [1] : where d min is the smallest degree, d max is the degree cutoff, and λ > 0 is the exponent characterizing the broadness of the distribution [2] . in real-word networks, the exponent λ is usually in the range [2, 3] , thus we confine the exponent λ = 2.5 in this paper. we further employ the smallest degree d min = 2, the natural degree cutoff d max = n 1/(λ −1) as in [6] , and the size n = 1000. hence, the average degree is approximately 4. the distribution of the degree of a random node in er network is binomial: given a network topology and a recovery rate δ , we carry out 100 iterations. in each iteration, the networks are constructed as described above and the infection rates are generated as described in section 2.2 and 2.3. initially, 10% of the nodes are randomly infected. then the infection and recovery processes of sis model are simulated until the system reaches the metastable state where the fraction of infected nodes is nonzero and unchanged for a long time if the epidemic spreads out, or the fraction is zero if the epidemic dies out. the average fraction y ∞ of infected nodes is obtained over 100 iterations (no matter the epidemic dies out or not). in this section, we first explore the accuracy of nimfa with the i.i.d. infection rates, and particularly how nimfa performs when the variance var[b] of the infection rate b varies. then we explore the influence of the correlated infection rates on nimfa. we aim to understand the precision of nimfa under different effective infection rates, different variances of infection rates and different network topologies: we set the average infection rate to 1 and tune the recovery rate δ to control the effective infection rate τ; we change the variance of infection rates which follow the lognormal distribution; we consider both er and sf networks to represent different topologies. for each value of the variance of the infection rate, we obtain the average fraction y ∞ of infected nodes as a function of the effective infection rate τ for nimfa by numerically solving (3) and compare with that by the continuous-time simulations. as shown in fig. 1a , no matter what the variance of the infection rate is, the curve of y ∞ vs. τ obtained by nimfa for er networks is close to that obtained by simulations when the actual prevalence of the epidemic is high, i.e. the effective infection rate τ is large. in order to quantify the difference between the two curves obtained by nimfa and simulations, we define the variable ζ : where y ∞,n (τ) > 0 and y ∞,s (τ) > 0 denote the average fraction of infected nodes obtained by nimfa and simulations respectively. the larger the value of ζ (τ) is, the less accurate nimfa is at the corresponding τ. in fig. 1b , the plot of ζ vs. τ is shown for er networks. we find that, for a given effective infection rate τ, nimfa becomes less accurate when the variance of the i.i.d. heterogeneous infection rates increases. this observation can be to a large extent explained by: 1) our finding in fig. 1a that nimfa is more accurate when the prevalence is higher; 2) that given an effective infection rate τ a smaller variance of the i.i.d. infection rates leads to a higher prevalence [15] . we observe the same in sf networks, and the figures, which can be found in [16] , are not shown here due to the page limit. we further explore how the variance of the infection rates influences the accuracy of nimfa if the actual prevalence y ∞,s (τ) of epidemic is similar. we plot the variable ζ in (6) as a function of the actual average fraction of infected nodes obtained by simulations in fig. 2 . we find that though it is less evident for er networks in fig. 2a , the difference ζ in (6) is actually larger if the variance of the infection rate is larger as shown in fig. 2b for sf networks when the prevalence is the same. hence, the higher heterogeneity, i.e. the larger variance, of the i.i.d. infection rates tends to lower down more the accuracy of nimfa. overall, we conclude that the prevalence of the epidemic mainly affects the accuracy of nimfa, i.e. the higher the prevalence is, the more accurate nimfa tends to be, and given the same prevalence, a larger variance of the i.i.d. infection rates tends to lower down the accuracy of nimfa. in this subsection, we aim to understand how the correlation between the infection rate and the nodal degree as shown in (5) influences the accuracy of nimfa. we first employ er networks as an example and discuss the case when the correlation is positive. afterwards we explore the influence of the negative correlation. as mentioned in section 2.3, we build the scenario of uncorrelated infection rates as a reference to study the influence of the correlation between the infection rate and the nodal degree by shuffling the infection rates from all the links as generated in the scenario of correlated infection rates and redistributing them randomly to all the links. as shown in fig. 3a , we compare the difference ζ between nimfa and simulations in the scenario of uncorrelated and correlated infection rates for both α = 0.25 and α = 1, and find that ζ is smaller in the scenario of correlated infection rates, i.e. nimfa is more accurate at a given effective infection rate τ when the correlation between the infection rate and the nodal degree is positive comparing to the scenario of uncorrelated infection rates. the observations are also consistent with our conclusion that nimfa is more accurate when the prevalence is higher: the positive correlation tends to increase the average fraction of infected nodes [17] , and thus the accuracy of nimfa, when the effective infection rate τ is small; however, when the effective infection rate τ is large, though the positive correlate may lower down a bit the average fraction y ∞ of infected nodes, the prevalence in both scenarios is high, i.e. nimfa is relatively accurate, and the difference of the accuracy of nimfa in the two scenarios is not obvious. as the correlation strength α increases in fig. 3b , the difference ζ decreases at a given τ. that is to say, nimfa tends to be more accurate when the positive correlation becomes stronger. we further consider the influence of the positive correlation on the accuracy of nimfa when the prevalence is the same. the plots of the difference ζ as a function of the average fraction y ∞ of infected nodes are shown in fig. 3c and fig. 3d . given the prevalence of epidemic, the positive correlation is more likely to increase the precision of nimfa and the stronger the correlation is the more accurate nimfa is. we observe the same on sf networks which is though not shown here. regarding to the influence of the negative correlation between the infection rate and the nodal degree on the accuracy of nimfa, we compare the variable ζ in the scenario of correlated and uncorrelated infection-rate scenario with α = −1 for both er and sf networks as shown in fig. 4a . we find that, in general, the negative correlation significantly decreases the accuracy of nimfa when the effective infection rate τ is small but may slightly increase that when τ is large. moreover, nimfa becomes less accurate when the negative correlation is stronger as shown in fig. 4b . as mentioned in section 2.3, the negative correlation tends to decrease the prevalence when the effective infection rate τ is small while increase the prevalence when τ is large. hence, the influence of prevalence on the precision of nimfa could largely explain our observations here. when the prevalence of epidemic is the same, the influence of the negative correlation on nimfa's accuracy is shown in fig. 4c and fig. 4d . we find that, in general, 1) nimfa is less accurate with the negative correlation comparing to the uncorrelated scenario especially when the prevalence is low as shown in fig. 4c ; 2) nimfa becomes even less accurate if the negative correlation becomes stronger as shown in fig. 4d . in this section, we choose the airline network from the real world as an example to illustrate how its heterogeneous infection rates affect the accuracy of nimfa of sis epidemics on the network. in the airline network, the nodes are the airports, the link between two nodes indicates that there's at least one flight between these two airports, and the infection rate along a link is the number of flights between the two airports. we construct this network and its infection rates from the dataset of openflights 1 . as shown in [17] , the airline network possess roughly a power-law degree distribution. the heterogeneous infection rates from the dataset are normalized by the average so that the average is 1. we compare the difference ζ between nimfa and the simulations of the exact sis model in three scenarios: 1) the network is equipped with its normalized original heterogeneous infection rates (correlated) as given in the dataset; 2) the network is equipped with the infection rates in the normalized original dataset but randomly shuffled (uncorrelated); 3) the network is equipped with a constant infection rate (homogeneous) which equals to 1. the original heterogeneous infection rate between a pair of nodes are approximately correlated with the degrees of the two nodes as the relationship (5), and the parameter α ≈ 0.14 indicates a positive correlation [17] . we show the difference ζ as a function of the effective infection rate τ in fig. 5a for the 3 scenarios defined as above. we find that nimfa is generally more accurate when the effective infection rate τ is larger, i.e. the prevalence of epidemic is high. the variable ζ is smaller in the scenario of homogeneous infection rates than uncorrelated infection rates with any effective infection rate. this is because the i.i.d. infection rates with a non-zero variance tends to decrease the prevalence, and thus lower down the accuracy of nimfa at a given effective infection rate τ. nimfa is more accurate with the positive correlation by comparing the difference ζ in the scenario of correlated infection rates and uncorrelated infection rates. furthermore, fig. 5b shows that, given the same actual prevalence, i.e. the average fraction y ∞ of infected nodes obtained by simulations, nifma is more accurate: 1) in the homogeneous scenario than in the uncorrelated scenario; 2) in the correlated scenario than in the uncorrelated scenario. all the observations agree with our previous observations and explanations about how the heterogeneous infection rate influences the accuracy of nimfa in network models. in this paper, we study how the heterogeneous infection rates affect the accuracy of nimfa -an advanced mean-field approximation of sis model that takes the underly network topology into account. by comparing nimfa with the continuous-time simulations of the exact sis model at a give effective infection rate τ, we find that the prevalence of epidemic could largely characterize the accuracy of nimfa which is reflected in two aspects: 1) nifma is generally more accurate when the effective infection rate τ is larger, i.e. the prevalence of epidemic is higher; 2) when the variance of the i.i.d. infection rates or the correlation between the infection rate and the nodal degree decreases the prevalence at a given τ, nimfa tends to become less accurate as well. moreover, we also explore the influence of the heterogeneous infection rates on the accuracy of nimfa at a given prevalence, i.e. when the average fraction y ∞ of infected nodes obtained by simulations is given. regarding to the i.i.d. heterogeneous infection rates, the accuracy of nimfa tends to decrease as the variance of infection rates increases. in the scenario of correlated infection rates, the positive correlation between the nodal degree and the infection rate is more likely to increase the accuracy of nimfa whereas the negative correlation tends to lower down the accuracy especially when the effective infection rate τ is small. note that we discuss the conditions when nimfa is accurate but the cases where nimfa is far from the simulations are still unexplored. our work sheds light on the conditions when we the mean-field approximation of the sis model with heterogeneous infection rates is accurate. internet: diameter of the world-wide web emergence of scaling in random networks the architecture of complex weighted networks slow epidemic extinction in populations with heterogeneous infection rates the fractal properties of internet resilience of the internet to random breakdowns susceptible-infected-susceptible model: a comparison of n-intertwined and heterogeneous mean-field approximations epidemics on interconnected lattices. epl (europhysics letters) 105 statistical analysis of airport network of china epidemics in interconnected small-world networks minimum spanning trees of weighted scale-free networks epidemic processes in complex networks epidemic dynamics and endemic states in complex networks epidemic spreading in scale-free networks sis epidemic spreading with heterogeneous infection rates the accuracy of mean-field approximation for susceptible-infectedsusceptible epidemic spreading sis epidemic spreading with correlated heterogeneous infection rates performance analysis of communications networks and systems virus spread in networks effect of the interconnected network structure on the epidemic threshold modelling the spreading rate of controlled communicable epidemics through an entropy-based thermodynamic model epidemic spreading in weighted networks: an edge-based mean-field solution key: cord-022122-6ssdamhp authors: berry, winter s. title: otitis, sinusitis, and mastoiditis: ear or facial pain following a common cold date: 2018-10-15 journal: introduction to clinical infectious diseases doi: 10.1007/978-3-319-91080-2_4 sha: doc_id: 22122 cord_uid: 6ssdamhp otitis, sinusitis, and mastoiditis represent a range of common upper respiratory tract infections that are more common in children than in adults. the clinical presentation of otitis varies based on the anatomic site of disease. acute otitis media and otitis media with effusion are infections of the middle ear, and otitis externa is infection of the external auditory canal. otitis is one of the most common infections seen in young children. each case represents an opportunity to practice precise diagnostic skills and to practice judicious antibiotic use. sinusitis, while also relatively common, may present with nonspecific signs and symptoms. an accurate diagnosis requires a complete history and careful physical examination to help distinguish between viral rhinosinusitis, a condition that is usually self-limiting, and bacterial rhinosinusitis, a condition that requires treatment with antibiotics. mastoiditis is a less common condition that can occur as a complication of otitis media. prompt recognition and appropriate antibiotic treatment, often in combination with surgical debridement, is required to prevent further spread of the infection and the development of secondary complications. 5 know the common clinical presentations for otitis, sinusitis, and mastoiditis. 5 identify common and uncommon microbiologic causes of otitis, sinusitis, and mastoiditis. 5 understand the distinguishing characteristics for acute, recurrent, and chronic clinical courses of each disease. 5 list the important risk factors for developing severe infections of the paranasal sinuses. 5 outline the approach to diagnosis, including signs and symptoms that warrant laboratory or imaging evaluations. 5 describe the indications for medical and surgical treatment of otitis, sinusitis, and mastoiditis. the term "otitis" encompasses pathology of both the middle and outer ear. it is generally divided into two categoriesotitis media and otitis externa. otitis media can present either as an acute infectious process of the middle ear (acute otitis media) or as a serous noninfectious process (otitis media with effusion). otitis externa is an infectious inflammatory condition of the external auditory canal (eac). approaches to the diagnosis and treatment of acute otitis media have evolved over the last several decades as new immunizations, and more antibiotic choices have become available. otitis media with effusion (ome) -a collection of serous fluid in the middle ear space without signs of acute inflammation. ome is not an infectious process. chronic otitis media with effusion (come) -a collection of serous fluid in the middle ear space that persists for more than 3 months acute otitis externa develops following disruption of the epithelial cell layer of the eac. epithelial breakdown can be caused by excessive moisture that leads to maceration, trauma during insertion of a foreign body, occlusion by a device such as a hearing aid or earplug, or dermatologic conditions involving the eac. excessive moisture as a cause for breakdown or maceration of the skin that lines the eac is very common. the frequency of otitis externa among swimmers underscores the importance of keeping the eac dry and explains why otitis externa is commonly known as "swimmer's ear. " the presenting symptoms of otitis externa include otalgia, decreased hearing, sensation of fullness in the ear canal, pruritus, tenderness on palpation, and movement of the eac or the pinna. otorrhea, adjacent cervical lymph node enlargement, and local cellulitis may develop later in the course of more severe cases. the most common bacterial causes of oe include pseudomonas aeruginosa and staphylococcus aureus [1] . typical physical examination findings include erythema and edema of the eac with debris, cerumen, and purulent material filling the canal. visualization of the tympanic membrane (tm) is often obstructed by the otorrhea and the swelling associated with the inflammation. an unimpaired view of a normal tm, with visible landmarks, is shown in . when the eac is cultured, one must be cognizant that culture results may reflect eac flora rather than a causative organism. first-line antibiotic treatments include topical otic drops of a fluoroquinolone, such as ciprofloxacin with or without topical glucocorticoid drops. if edema of the eac is severe, placement of a wick inside the eac may be necessary to ensure delivery of medication to the more proximal areas of infection. risk of recurrence of oe is increased in individuals with atopic dermatitis, seborrhea, immune compromise, and repeated local trauma to the area when cleaning the ear. a fungal etiology of the infection should be considered when otorrhea is prolonged, especially if topical antibacterials and a wick have been used already. individuals who use hearing aids and those who have had recent bacterial infections are also at risk for fungal disease. the most common fungal etiologies include aspergillus and candida species [1] . the development of aom begins when there is dysfunction of the eustachian tube. under normal conditions, the eustachian tube allows the middle ear to drain to the pharynx and to equalize pressure between the middle ear and the environment. impaired drainage may be present for several reasons. the anatomic position of the shorter eustachian tube in young children maintains a relatively horizontal orientation allowing the drainage to defy gravity. adenoidal hypertrophy and anatomic anomalies of the palate can also block or impair normal drainage. the presence of gastroesophageal reflux disease, allergic rhinitis, and viral upper respiratory infections can all lead to inflammation of the eustachian tube and surrounding tissues resulting in the presence of increased secretions in the middle ear that accumulates because the eustachian tube is not fully patent [2] [3] [4] . in each of these clinical scenarios, negative pressure develops in the eustachian tube and middle ear space. the small number of bacteria normally present in those secretions replicates in the now closed space resulting in acute infection (aom). retained nasopharyngeal secretions are very common in individuals with otherwise uncomplicated viral respiratory infections. a subgroup of these individuals experience secondary bacterial infection because the initial viral infection and its associated inflammation allow for the closed space conditions where the bacteria can thrive. the risk factors for aom include any condition that promotes eustachian tube dysfunction. the relative horizontal position of the eustachian tube during early childhood explains why the peak incidence of aom is between 6 and 18 months of age. additional risk factors include children who develop their first episode of aom prior to 6 months of age, children in daycare, and the presence of atopy, adenoidal hypertrophy, chronic sinusitis, ciliary dysfunction, immunecompromising conditions, and craniofacial anomalies. individuals with trisomy 21 are especially prone to otitis media because their eustachian tubes are short and their pharyngeal muscle tone is weak [2, 3] . an accurate description of the infectious etiologies of aom requires that middle ear fluid is removed during acute infection and submitted for microbiologic testing. the procedure to remove the fluid, tympanocentesis, is no longer performed on a routine basis. most of the literature that describes the microbiologic causes of aom was published before the routine introduction of haemophilus influenzae type b (late 1980s) and heptavalent (2000) and 13-valent (2010) conjugate pneumococcal vaccines. rates of viral, bacterial, and mixed culture results from middle ear effusion vary significantly across those studies with rates of bacterial infection ranging between 50% and 90% of all aom. the most common viral causes include respiratory syncytial virus; parainfluenza viruses, types 1, 2, and 3; influenza a and b viruses; adenovirus; coronaviruses; parechoviruses; and human metapneumovirus. the most common bacterial causes of aom include streptococcus pneumoniae, non-typeable haemophilus influenzae, and moraxella catarrhalis. s. pneumoniae was unequivocally the most common bacterial agent of aom before conjugate pneumococcal vaccine was added to the universal pediatric immunization schedule in 2000. following vaccine introduction, non-typeable h. influenzae became more predominant, and "replacement" pneumococcal serotypes that were not included in the 7-or 13-valent vaccines emerged [2] [3] [4] . non-typeable h. influenzae should be suspected in the clinical setting of aom when purulent conjunctivitis is also present. suspicion that h. influenzae could be the underlying cause of the patient's condition is important, because unlike s. pneumonia, h. influenzae may produce a beta-lactamase, an enzyme that inactivates some of the most common antibiotics used empirically to treat aom, such as amoxicillin. less common bacterial etiologies of aom include streptococcus pyogenes (also known as group a streptococcus, the cause of "strep throat") and staphylococcus aureus. s. pyogenes is more commonly seen among those older than 5 years of age. its presentation is typically quite aggressive leading to perforation of the tm and/or accompanying mastoiditis [2] . s. aureus should be included on the list of possible infecting agents in patients who have tympanostomy tubes, as these medical devices serve as a conduit between the bacteria normally present in the eac and the middle ear. streptococcus agalactiae (also known as group b streptococcus) should be considered as a possible causative organism in aom when the process is identified in neonates and very young infants. the microbiologic culprits responsible for chronic suppurative otitis media include s. aureus, pseudomonas aeruginosa, and s. pneumonia. chronic suppurative otitis media is especially common among individuals from southeast asia, the western pacific, africa, and native americans of the desert southwest [2] . aom is a closed space infection. as the infection progresses, symptoms change from a feeling of fullness to general achiness. the pain intensifies as the pressure in the infected space increases until that pressure is relieved. relief may come spontaneously as serous fluid is reabsorbed or when the pressure exceeds the capacity of the tm and the eardrum ruptures. tympanocentesis can also be used as a controlled technique to remove middle ear fluid and thereby reduce the pressure. aom causes ear pain. the pain in young children can manifest itself as fussiness, sleep disruption, or ear tugging. fever is expected in young children with aom, but uncommon among older children and adults with aom. in cases where the tm ruptures, the pain is relieved, but the patient or parent will note a purulent or bloody ear discharge. a diagnosis of aom is often suspected while obtaining the history of the illness. the diagnosis is confirmed by finding on the physical examination. a thorough otoscopic exam, including pneumatic otoscopy and clear visualization of the tm and eac, is essential. presuming a diagnosis of aom in the absence of a thorough examination leads to overdiagnosis and subsequent overuse of antibiotics. pneumatic otoscopy should always be included to assess the mobility of the tm. efficient pneumatic otoscopy is aided by using a speculum with a tight seal of the eac [4] . classic otoscopy findings of aom include the presence of fluid in middle ear space, outward bulging of the tm secondary to the increased pressure in the middle ear space, loss of visualization of ossicle bony landmarks behind the tm, and erythema or injection of radial vessels on the tm. the middle ear effusion is purulent in aom but must be carefully distinguished from opaque noninfectious effusions secondary to ome. the absence of erythema of the tm suggests a diagnosis of ome, not aom. a bulging tm with decreased mobility on pneumatic otoscopy is the most specific exam finding for bacterial aom [4] . the presence of purulent otorrhea in the eac is seen if a tm perforation has occurred before the time of examination. this finding must be distinguished from the edema and erythema that occur along the eac during otitis externa. bullae may be present on the tm in conjunction with other signs of acute inflammation. the findings are consistent with a form of aom termed bullous myringitis. the presence of bullae does not suggest a specific microbiologic cause nor does it change the clinical approach to the condition. in summary, the clinical diagnosis of aom is made when there is an acute onset of symptoms, the presence of a middle ear effusion, and objective signs of acute middle ear inflammation such as a red bulging eardrum that does not move during pneumatic otoscopy [2] [3] [4] . the diagnosis of aom does not involve any laboratory evaluation or radiographic imaging unless there is evidence for a significant complication or severe or persistent disease. tympanocentesis with culture of the middle ear effusion is the gold standard for diagnosing the etiologic agent of aom, but is only necessary in a small subset of cases. when necessary, referral to an otolaryngologist is considered the best practice for those who are not certified and experienced in performing the procedure. the inclination to collect samples from the eac for microbiologic cultures, even in cases where there is frank otorrhea, is generally discouraged since the laboratory results often reflect eac flora rather than identifying the true causative organism(s). the differential diagnosis for aom includes otalgia secondary to ome, referred pain from dental or pharyngeal disease, local herpes zoster infection, otitis externa, adjacent soft tissue infection, or eustachian tube dysfunction secondary to alternative causes. recommendations for the treatment of aom have evolved over the past several decades. depending on the details of the clinical circumstance, current recommendations now permit a clinical observation period as an alternative to immediate antibiotic therapy for many cases. this approach is supported by the knowledge that many cases are caused by respiratory viruses and by evidence demonstrating that a reasonable proportion resolve without intervention. ultimately, the approach used should carefully weigh patient-specific factors such as age, severity of presenting symptoms, risk factors for severe infection, history of aom, and the patient's availability to follow up. safety-net antibiotic prescriptions (snap) and wait-and-see prescription (wasp) are prescriptions provided at the time of diagnosis allowing patients and families to employ watchful waiting, with the option to fill an antibiotic prescription if the symptoms persist or worsen over the next 48-72 h [2] [3] [4] . treatment approach algorithms that account for age, examination findings, and illness severity at the time of presentation are shown in . analgesic should be used regularly early in treatment course to provide symptomatic relief of otalgia [2, 4] . the first-line antibiotic choices for the treatment of aom are shown in 7 call out box 4.1 [4] . high-dose amoxicillin (80-90 mg/kg/day) is recommended to overcome concerns about adequate coverage against penicillin nonsusceptible s. pneumonia. recommendations regarding duration of antibiotic therapy are age dependent. except for intramuscular ceftriaxone, a 10-day course is recommended for children less than 2 years of age, whereas a 7-day course can be considered for children between 2 and 5 years of age. a 5-to 7-day course is usually adequate starting at 6 years of age through adulthood [4] . antibiotic therapy for the treatment of csom requires administration of topical fluoroquinolone antibiotic drops with or without glucocorticoid drops. the medications are able to reach the middle ear space by crossing through the perforation in the tm. fluoroquinolones are used because their spectrum of activity includes the most common etiologic agents including p. aeruginosa. complications of aom encompass spread of infection to structures adjacent to the middle ear and include mastoiditis, petrositis, venous sinus thrombosis, and central nervous system infection including brain abscess. patients under treatment or observation for aom should be reevaluated for the persistence or worsening of symptoms 48-72 h after the initial assessment. if symptoms are improving at 48-72 h, short-term follow-up is not usually necessary. tm abnormalities seen on physical examination can persist for up to 12 weeks. 3rd-generation oral cephalosporin such as cefdinir or intramuscular ceftriaxone for 1, 2, or 3 days 5 ongoing emesis or adherence concerns: intramuscular ceftriaxone for 1, 2, or 3 days sinusitis refers to an infection of one or more of the paranasal sinus cavities. acute upper respiratory viral infections that cause the common cold all involve the paranasal sinuses. the vast majority of these infections are self-limiting and do not require treatment with antibiotics. acute bacterial rhinosinusitis is a more precise name for the condition caused by bacterial pathogens, and like aom, it typically occurs when drainage is impaired secondary to the inflammation associated with a recent viral infection. fortunately, only a small number of "colds" become complicated by acute bacterial sinusitis. while viruses and bacteria account for most infections of the paranasal sinuses, molds can cause some of the more severe disease. immune-compromised patients are particularly vulnerable to these disfiguring, often fatal mold infections. leading to inflammation of the paranasal sinuses lasting fewer than 30 days but recurring 3 or more times in a 6-month period or 4 or more times during a 12-month period. each infection responds well to treatment with antibiotics, and the patient experiences at least 10 symptom-free days in between episodes. chronic sinusitis -inflammation of the paranasal sinuses for more than 90 days with persistent symptoms. this condition is most commonly associated with noninfectious processes such as environmental allergies, reactions to environmental pollutants, and gastroesophageal reflux disease. patients with cystic fibrosis and ciliary dyskinesia are especially prone to chronic sinusitis secondary to noninfectious and infectious triggers. the anatomy of the paranasal sinuses changes from birth, not becoming fully aerated until adulthood [5, 6] . the ethmoid sinuses, present at birth, grow proportionally with the child. maxillary sinuses, also present at birth, reach adult size by 4 years of age. the sphenoid sinuses begin to develop around 2 years of age, are functional by age 5, and fully mature by age 12. finally, the frontal sinuses are first evident by age 6-8 years, not fully maturing until early adulthood. acute bacterial rhinosinusitis is incited by predisposing factors such as viral upper respiratory infections, allergic rhinitis, environmental pollutants, nasal septum anomalies, other craniofacial anomalies, adenoidal hypertrophy, masses, polyps, or the presence of a foreign body. risk factors include smoking, second-hand smoke exposure, and extensive dental disease. all of these conditions impede mucociliary clearance of sinus secretions. since the sinus cavities are not sterile sites, stasis of secretions increases the likelihood that resident bacterial flora will overgrow resulting in acute bacterial rhinosinusitis [5, 6] . acute bacterial rhinosinusitis is most common between 4 and 7 years of age. children in this age group are old enough to have relatively developed sinuses, yet young enough to experience frequent predisposing viral upper respiratory infections. these upper respiratory infections impede usual sinus clearance. respiratory viral infections are even more common among children less than 2 years of age, but these younger children have less developed sinuses and wider ostia that facilitate drainage. in addition, younger children will often present earlier with aom. treatment of the aom effectively treats any emerging sinus disease, therefore eliminating the opportunity for the development of acute bacterial sinusitis. a second peak of abrs occurs during adulthood between 45 and 64 years of age. when the infections become recurrent, it is important to maintain a level of suspicion for the presence of contributing factors such as an anatomic blockage (polyps or a foreign body), gastroesophageal reflux disease, allergic disease, a humoral immune deficiency, cystic fibrosis, ciliary dysmotility, or any other condition that might lead to ongoing sinus inflammation and impaired sinus drainage [5, 6] . the optimal manner in which to determine the precise microbiology of acute bacterial rhinosinusitis is to obtain sinus fluid during the acute infection. existing data, obtained decades ago, are based largely on studies where needle aspirates were performed on infected maxillary sinuses and the fluid sent to the microbiology laboratory for culture. the technique is modestly invasive and now seldom performed outside of academic settings, clinical trials, and severe or particularly enigmatic cases [7] . not unexpectedly, the common causes of sinus infections are nearly identical to the list of pathogens that cause aom since both processes arise from bacteria that normally inhabit the human upper respiratory tract at low concentrations. the most common bacterial agents of abrs include s. pneumoniae, non-typeable h. influenzae, and m. catarrhalis. s. pneumoniae and non-typeable h. influenzae are equally common in the post-pneumococcal vaccine era with each accounting for approximately 30% of cases. infection with m. catarrhalis is less frequent at an estimated 10% of total cases [5, 6] . formal sinus aspirate studies have reported that approximately 30% of nasal sinus aspirates are culture negative for bacteria [7] . less common causes of abrs include s. aureus and s. pyogenes. anaerobic bacteria have also been implicated either as a primary pathogen or as co-pathogens in several adult studies including prevotella, bacteroides, and peptostreptococcus species. anaerobic infection should be suspected if there is evidence of extension from dental disease into the sinuses. the initial presentation of sinusitis varies. a careful history of the clinical course of the symptoms over time, the severity of symptoms, and the presence of fever are key details needed to help distinguish abrs from the more common viral upper respiratory infections. symptoms more suggestive of abrs include fever, headache, facial pain and swelling, and halitosis, while cough, nasal congestion, and sore throat are typically seen during bacterial and viral infections [6] [7 call out box 4.2]. a good general rule of thumb: if more than two mucous membranes are involved during a respiratory infection, the cause is viral, not bacterial. bacterial infections of the upper respiratory tract tend to be localized. viral infections can involve the eyes, ears, nose, sinuses, mouth, and pharynx simultaneously. the color of the nasal discharge is sometimes used to justify treatment with antibiotics. viral upper respiratory tract infections usually begin with symptoms of clear, watery, nasal discharge. as the infection and associated inflammation proceed, the discharge becomes thicker and often takes on a yellow-green color. this change is expected during a viral infection and does not herald the presence of a bacterial process. the change in color and consistency is secondary to innate immune responses to the virus, with recruitment of inflammatory cells, including neutrophils. neutrophil myeloperoxidase allows for some of the oxygenfree radicals to be converted to hypochlorite. it is the presence of this halogenated compound that renders the color change, not the presence of bacteria. acute bacterial infections trigger the same innate immune inflammatory pathways, explaining why the discharge seen during a bacterial infection also has a yellow-green color. while the color and consistency of the nasal discharge is not helpful in distinguishing viral from bacterial sinusitis, several characteristics of the infection are. . table 4 .1 lists some of the ways in which the viral and bacterial sinus infections can be differentiated from one another on clinical grounds. physical examination findings associated with abrs include erythema and edema of the nasal turbinates, mucopurulent rhinorrhea, and the presence of postnasal drip. facial pain and sinus tenderness to palpation are less reliable finding in children, but are quite useful indicators in adolescents and adults. none of the physical examination findings are completely specific to either a bacterial or viral infection. the clues obtained during the history of present illness are often the most important key. taking the history of present illness and physical examination findings into account, reliable diagnostic criteria for abrs have been developed [5, 6] imaging is rarely useful in the evaluation of uncomplicated bacterial rhinosinusitis. plain radiographs or computerized tomography imaging of the sinuses undertaken in the absence of compelling history and severe clinical course is likely to demonstrate misleading results. mucosal thickening, sinus opacification, and air fluid levels may be present, but none of these findings are specific for bacterial underlying etiology. sinus abnormalities are typically present on imaging in those who have simple viral upper respiratory infections [5] . in contrast, if bacterial rhinosinusitis is present and complications or spread to adjacent structures is suspected, imaging is always indicated. computerized tomography imaging of the sinuses and surrounding structures is first line to define the sinus anatomy and bony structures. if there is suspicion that the process has spread intracranially, magnetic resonance imaging of the brain should be considered. laboratory evaluation, as with imaging, is not warranted during the evaluation of uncomplicated abrs. however, if the patient is ill appearing, is immunocompromised, and fails antibiotic treatment, or local extension of the infection is suspected, a sinus aspirate may be warranted. when obtained, the sample is typically sent for gram stain, aerobic and anaerobic cultures with susceptibility testing. the presence of more than 10,000 colony forming units of a bacterium is considered clinically significant [5] . . progression of symptoms over time when severity peaks on day 3 or 4 of the illness, followed by gradually improvement with resolution by day 7-10 when symptoms persist for more 10 days when initial symptoms are severe and the patient has an ill appearance for 3-4 consecutive days when severity peaks on days 3-4, followed by gradual improvement, but on days 6-7, the symptoms worsen when new-onset fever, headache, or facial pain occurs on day 7 or later of the illness fever when fever is absent or low grade only when fever is present only on days 1 and 2 of the illness when any fever persists for more than 5 days when fever greater than 39 â°c persists for 3 days or more when there is new onset of fever more than 7 days into the illness when nasal discharge is initially thin and clear, becoming thick and yellow-green mid-illness then resolves when nasal discharge is initially thin and clear, becoming thick and yellow-green, and persisting for more than 3 days when purulent rhinorrhea is unilateral history and physical examination must demonstrate the presence of sinus inflammation: 5 persistent disease: symptoms lasting 10-30 days without improvement (10-30 days is acute disease, 30-90 days is subacute disease, 90 days or longer is chronic disease) 5 or 5 severe disease: severe symptoms at initial presentation with ill appearance, fevers higher than 39 â°c, and persistent purulent rhinorrhea for 3-4 days 5 or 5 worsening disease: symptoms showing improvement on days 6-7 of the illness followed by clinical worsening, including the presence of fever, cough, headache, and recurrence of nasal symptoms the differential diagnosis of bacterial rhinosinusitis includes viral upper respiratory infection(s), allergic rhinitis, a local reaction to environmental pollutants, the presence of a nasal foreign body, and adenoidal hypertrophy or infection. if the chief complaint is a prolonged cough illness, infection with bordetella pertussis should also be considered. treatment of bacterial rhinosinusitis is dependent on the clinical course of the illness. subacute, recurrent, and chronic bacterial rhinosinusitis all warrant prompt antibiotic treatment. in contrast, "persistent" abrs may be followed with clinical observation for up to 3 days. if symptoms continue or worsen during the observation period, antibiotic therapy is initiated. an observation course must be discussed in detail with patient and indications for returning to care must be explained [5, 6] . first-line antibiotic treatment options for abrs include either amoxicillin or amoxicillin with clavulanic acid. the use of amoxicillin alone raises concerns regarding coverage of beta-lactamase-producing organisms such as non-typeable h. influenza, moraxella catarrhalis, s. aureus, and most oral anaerobes. however, rates of clinical failure with amoxicillin alone are low, and amoxicillin boasts a long history of safety and tolerability. amoxicillin-clavulanate provides broader antimicrobial coverage but can cause gastrointestinal side effects and is less palatable in suspension formulations used for young children. therefore, either medication can be considered first line in the treatment of uncomplicated bacterial rhinosinusitis. certain conditions increase the risk for treatment failure on amoxicillin alone and warrant amoxicillin-clavulanate as the preferred first-line treatment. included in this group are patients with immune-compromising conditions, those who are incompletely immunized, patients who have received antibiotics during the last 4 weeks or recently required hospitalization, and those with chronic bacterial rhinosinusitis [6] . if multidrug-resistant s. pneumoniae is suspected or confirmed as the etiologic pathogen, a third-generation cephalosporin (cefdinir, cefixime, or ceftriaxone) or quinolone class antibiotic, such as levofloxacin, may be necessary [6] . patients with non-type 1 penicillin hypersensitivity will most likely tolerate advanced-generation cephalosporins without allergic manifestations. those with type 1 penicillin allergy warrant treatment with a non-penicillin alternative such as levofloxacin, clindamycin, or linezolid [5] . if the illness severity warrants inpatient treatment, intravenous antibiotic regimens with antimicrobial spectra of activity that encompass all of the usual suspects include ampicillin-sulbactam, cefotaxime, ceftriaxone, or levofloxacin. in circumstances where intravenous treatment failure is suspected, multidrug-resistant s. pneumoniae should be considered as a potential cause while also referring the patient for surgical consultation to determine if a drainage procedure might facilitate improvement. while awaiting surgical advice, vancomycin can be added to the antibiotic regimen to broaden coverage that includes highly resistant s. pneumoniae and methicillin-resistant s. aureus. intravenous metronidazole can also be considered to include coverage for the most resistant anaerobes including b. fragilis [5] . adjunctive therapies to treat nasal symptoms, such as intranasal steroids, saline irrigation or lavage, mucolytics, decongestants, and antihistamines, lack a robust evidence basis, but remain common practices by patients and providers. complications of bacterial sinusitis occur when the infection extends to adjacent structures. localized spread of sinus disease may result in periorbital or orbital cellulitis. bony involvement may include the formation of a subperiosteal abscess. orbital extension should be suspected when there is local tissue swelling and edema in the periorbital area. intracranial extension can include septic cavernous sinus thrombosis, meningitis, brain abscess, or osteomyelitis of the frontal bone. intracranial disease should be considered if the patient develops persistent and severe headache, mental status changes, focal neurologic examination findings, or persistent emesis. case 1 a 7-year-old boy presents to the office for a 14-day history of illness that began with cough, rhinorrhea, and complaints of a sore throat. symptoms gradually worsened for the first few days, but began to improve on day 6. his parents assumed this had been "a typical cold." on day 7 of illness, the boy's symptoms worsened. his cough and rhinorrhea persisted, but secretions became thick and purulent again. he developed new facial pain, fevers to 39 â°c, and general malaise. the day prior to the current office visit, the patient began to complain of right eye pain. on physical examination, he appears tired. the right eyelid and surrounding skin is red and swollen. his extraocular movements are intact, but mastoiditis is a suppurative infection of the mastoid air cells that most often presents as a secondary complication of otitis media. its presentation and progression can be acute and require hospitalization and urgent surgical intervention. routine vaccination against s. pneumoniae has reduced the incidence of aom and led to a decrease in the frequency of mastoiditis in the past decade. the mastoid sinuses are networks of air cells divided by bony septae located in the posterior portion of both temporal bones. the space connects to the middle ear by a bony passage that is present at birth and increases in size until approximately 2 years of age. as the bony passages grow, they become lined with epithelium contiguous to the middle ear space. this continuity of epithelium allows for the possibility for infections of the middle ear to spread to the mastoid. the mastoids are bordered anteromedially by the middle ear and ossicles, the facial nerve, the bony portion of the eac, the jugular vein, and the internal carotid artery and medially by the inner ear. the sigmoid sinuses are immediately posterior to the mastoids. the cranial fossa sits above, and the soft tissue and muscles of the lateral neck sit below the mastoids on both sides [8, 9] . mastoiditis is invariably preceded by inflammation and effusion in the middle ear space. the presence of the effusion creates increased pressure between the middle ear and the mastoid air cells. untreated spread of infection from the middle ear to the mastoid air cells results in resorption of the thin dividing bony septae and osteomyelitis of surrounding temporal bone. as mastoiditis is a secondary complication of aom, the two infectious processes share the same set of risk factors. those at risk for severe mastoiditis include patients with immunodeficiencies, functional or anatomic asplenia, and chronic heart or lung disease. patients who are underimmunized or unimmunized against s. pneumoniae are also at increased risk. individuals with cochlear implants are at risk for hardware-based infections [8] . the most common causes of acute mastoiditis include s. pneumoniae, s. pyogenes, and s. aureus (including methicillin-resistant strains). less common etiologies include p. aeruginosa and other gram-negative bacteria, non-typeable h. influenzae, and resident anaerobes of the oropharynx. mycobacterium tuberculosis is an unusual cause call out box 4.4 chemosis is a term used to describe edema of the bulbar conjunctivae. it is usually easiest to appreciate at the lim bus, where the bulbar conjunctiva is elevated above the plane of the cornea. chemosis is a nonspecific finding, but does herald the presence of significant eye irritation. 5 what characteristics of this presentation help you distinguish between viral and bacterial sinusitis? 5 how would you classify his sinusitis? 5 would you prescribe antibiotics? 5 is any other evaluation warranted? why or why not? the child's early illness included symptoms consistent with a " common cold" of viral etiology. careful consider-ation of the clinical course shows that he developed new and more severe symptoms at the time one would expect a typical viral upper respiratory infection to resolve. the new symptoms of fever, headache, and purulent rhinorrhea suggest the evolution of acute bacterial rhinosinusitis (abrs). in addition, his symptoms have persisted for 14 days. his presentation is best classified as "worsening" acute bacterial rhinosinusitis. this classification warrants initiation of antibiotic therapy. in addition, his report of new-onset eye pain and the findings of periorbital inflammation and discomfort with ocular movement raise suspicion for early extension of the infection to the orbital space. these findings should prompt consideration of inpatient antibiotic therapy and sinus and orbital computerized tomography imaging to evaluate for the presence of intraorbital infection. of mastoiditis but should be considered as a possibility when the illness is diagnosed in individuals who live or have lived in tb endemic areas of the world. chronic mastoiditis is most frequently caused by p. aeruginosa. other pathogens to consider when the infection presents as a chronic process include other gram-negative bacilli, s. aureus, and anaerobes [8] . acute mastoiditis presents with abrupt onset of fever, otalgia, and a red, swollen postauricular area. the pathophysiology dictates that these symptoms follow a recent middle ear infection. if the initial aom was treated with antibiotics, there may be an interval of improvement followed by an abrupt worsening of symptoms. the physical examination reveals an abnormal middle ear consistent with aom. otorrhea is present in approximately half of the cases because the tm has perforated under the pressure of the infected space. the affected ear will show edema and erythema of the posterior auricular area. the pinna begins to protrude due to edematous displacement. as the infection progresses, fluctuance may develop in the postauricular area over the mastoid air cells. chronic mastoiditis is preceded by long-standing middle ear disease. fevers and postauricular erythema and swelling become evident as the infection progresses. chronic mastoiditis should be suspected when a patient experiences longstanding tm perforation with chronic otorrhea. due to the chronicity of the process, the patient may also complain of hearing loss [8, 9] . the diagnosis of mastoiditis is based on the classic clinical signs and symptoms. once the diagnosis is established, obtaining fluid for microbiologic culture is important to help guide definitive treatment. samples can be obtained via tympanocentesis or, more commonly, from a mastoid sample collected during surgical debridement. biologic samples should be sent for gram stain and aerobic and anaerobic cultures with susceptibility testing. acid-fast cultures should be requested in cases where tuberculosis is a possibility [8] . imaging should be used in cases where mastoiditis has been diagnosed clinically as the findings will help to guide decisions regarding surgical care. in the absence of objective physical examination findings of mastoiditis, the presence of opacifications in the mastoid air cells is nonspecific since the finding is also common in uncomplicated serous otitis media and during uncomplicated aom [10] . when mastoiditis is clinically suspected, computerized tomography scanning (ct) is preferred to evaluate the bony structures. classic ct findings of mastoiditis include mastoid air cell opacifications, resorption of the bony septae, and coalescence of air cells. ct may also be indicated in patients with aom that has been unresponsive to antibiotic therapy to rule out the development of secondary mastoiditis. if intracranial or soft tissue complications are suspected, magnetic resonance imaging (mri) is preferred [8] . symptoms suggestive of an intracranial complication include focal deficits on neurologic examination, hearing changes, vertigo, meningeal signs, or altered mental status. scalp infection, periauricular cellulitis, extension of otitis externa, and perichondritis of the auricle may all present with posterior auricular erythema and edema. parotid swelling secondary to mumps may displace the pinna, but the direction of deviation is superiorly rather than inferiorly seen with mastoiditis [8] . the differential diagnosis of abnormal findings of the mastoid air cells by ct in the absence of objective physical examination findings of mastoiditis includes underaerated or sclerotic air cells due to prior aom and ome. all cases of mastoiditis are potential candidates for surgical intervention, and all cases require treatment with antibiotics. antibiotic alone may be appropriate if the illness presents with focal erythema and edema, but without fluctuance or signs of adjacent spread. antibiotic therapy in combination with surgical intervention is necessary if fluctuance is present, there is a history of chronic otorrhea, or the patient has developed focal neurologic signs and symptoms such as vomiting, nystagmus, vertigo, or other signs of intracranial disease. surgical intervention may be fairly straightforward with tympanocentesis or placement of tympanostomy tubes or quite extensive with open debridement of the infected mastoid tissue and adjacent structures. the initial antibiotic therapy should be administered parenterally. the typical duration of therapy is a minimum of 3 weeks. depending on the clinical response, the antimicrobial susceptibilities, and the likelihood of adherence, some patients may complete therapy with oral antibiotics. the firstline empiric choice antibiotic for the treatment of acute mastoiditis is typically a medication in the penicillin or cephalosporin class. if the initial presentation is severe, vancomycin is also utilized to ensure coverage against methicillinresistant s. aureus and highly resistant s. pneumoniae strains. chronic mastoiditis is treated empirically with a modified penicillin in combination with a î²-lactamase inhibitor, such as ampicillin plus sulbactam or piperacillin plus tazobactam in combination with gentamicin. this combination provides coverage against, p. aeruginosa, and other gram-negative bacilli and anaerobes. in patients with recurrent aom and a strong suspicion of p. aeruginosa, piperacillin-tazobactam, ceftazidime, or cefepime can be used along with clindamycin [8] . the prognosis of mastoiditis is good if the disease is diagnosed and treated before a more serious complication develops. increasing rates of morbidity and mortality are seen when the infection results in septic thrombosis of the cavernous sinus and/or spread to the temporal lobe of the brain [11] . otitis, sinusitis, and mastoiditis represent a spectrum of otolaryngologic infections that cause signs and symptoms overlapping with the ubiquitous viral upper respiratory infection or "common cold. " clinical guidelines for each stress careful consideration of diagnostic criteria, vigilance for the development of complications, the most appropriate antibiotic options, and when necessary, appropriate surgical interventions. clinical practice guideline: acute otitis externa otitis media clinical practice guideline the diagnosis and management of acute otitis media johns hopkins harriet lane continuity clinic curriculum clinical practice guideline for the diagnosis and management of acute bacterial sinusitis in children aged 1 to 18 years idsa clinical practice guideline for acute bacterial rhinosinusitis in children and adults acute maxillary sinusitis in children in: feigin r, cherry j, editors. textbook of pediatric infectious diseases incidental mastoid opacification in children on mri prognostic effect of pre-and postadmission antibiotic treatment in paediatric acute mastoiditis pediatric otolaryngology. aap please refer to the supplementary information section for answers to these exercises.match the clinical scenario with the most likely pathogen. each pathogen may be used once, more than once, or not at all. as many as 20% of mastoiditis cases are associated with complications. infections caused by s. pyogenes and s. aureus can be particularly aggressive. interestingly, patients who have recently been treated with antibiotics for aom have some of the highest rates of complications. this suggests that causative bacteria are particularly pathogenic or have become resistant to more conservative treatments [11] . known complications involve contiguous spread of the infection to adjacent structures and include subperiosteal abscesses, bezold's abscesses (infection of the sternocleidomastoid and trapezius muscle attachments), facial nerve paralysis, meningitis, subdural empyema, brain abscess, venous sinus thrombosis, labyrinthitis, temporal bone osteomyelitis, cerebrospinal fluid otorrhea, or conductive hearing loss secondary to destruction of the bony ossicles. progressive disease can lead to systemic infection with bacteremia and distal septic emboli. a rare but classic complication known as gradenigo's syndrome is diagnosed when there is petrositis with associated otitis media, ipsilateral medial rectus palsy, eye pain, and possible additional cranial nerve abnormalities [8] . case a 2-year-old boy presents with redness around his ear. two weeks prior, he had a "cold" with mild symptoms of rhinorrhea and cough. seven days into his illness, he developed fevers and began tugging on his right ear. his provider diagnosed him with right aom and treated him with a 10-day course of amoxicillin. two days into his course of amoxicillin, he seemed to improve and his fevers resolved. two days after he completed his antibiotic course, his fevers returned and his mother noticed redness and swelling behind his right ear. on physical examination, the boy appeared fussy and slightly ill. he had clear rhinorrhea. his left eac and tm were unremarkable. his right tm showed dullness and erythema with visible pus behind it. his right eac was mildly erythematous and edematous. manipulation of the right pinna and palpation of the posterior auricular area were quite painful. the postauricular area was erythematous and edematous, but without fluctuance. the right pinna was slightly displaced anteriorly. key: cord-017867-8cn4c6cu authors: collántes-fernández, esther; fort, marcelo c.; ortega-mora, luis m.; schares, gereon title: trichomonas date: 2017-11-08 journal: parasitic protozoa of farm animals and pets doi: 10.1007/978-3-319-70132-5_14 sha: doc_id: 17867 cord_uid: 8cn4c6cu the most widely known trichomonad in veterinary medicine is tritrichomonas foetus. it is the etiologic agent of bovine tritrichomonosis, a sexually transmitted disease in extensively managed herds throughout many geographic regions worldwide. the same trichomonad species is also regarded as the causative agent of chronic diarrhea in the domestic cat, although more recent studies observed molecular differences between bovineand feline-derived t. foetus. trichomonosis in cats has a worldwide distribution and is mainly present among cats from high-density housing environments. other trichomonads are found as inhabitants of the gastrointestinal tract in birds, such as trichomonas gallinae. particularly, columbiformes, falconiformes, strigiformes, and wild passeriformes can be severely affected by avian trichomonads. diagnosis of trichomonosis is often complicated by the fragility of the parasite. to ensure valid test results, it is essential to collect and handle specimens in the right way prior to analysis. cultivation tests, the specific amplification of parasites, or a combination of both test methods is the most efficient and most commonly used way to diagnose trichomonosis in animals. bovine tritrichomonosis is mainly controlled by the identification and withdrawal of infected animals from bovine herds. the control of feline and avian trichomonosis relies mainly on preventive measures. in veterinary medicine the most widely known trichomonad is tritrichomonas foetus. it is located in the urogenital tract of cattle and considered the etiologic agent of bovine tritrichomonosis, a sexually transmitted disease throughout many geographic regions worldwide (bondurant 2005; ondrak 2016 ). the same trichomonad species was initially described in 1999 and finally confirmed in 2003 to be the causative agent of chronic diarrhea in the domestic cat (gookin et al. 1999; levy et al. 2003) . trichomonosis in cats has a worldwide distribution and is mainly present among cats from high-density housing environments such as catteries, shelters, or breeding facilities (yao and köster 2015) . other trichomonads in animals, for example, tritrichomonas suis in swine-presumably genetically identical to t. foetus of bovine origin-are commensals and rarely involved in disease. t. suis was once thought to cause atrophic rhinitis in pigs, further studies were not able to establish a causal relationship, and t. suis is now considered a harmless nasal and gastrointestinal commensal in swine (bondurant and honigberg 1994) . isolates of t. suis that reside in the stomach, caecum, and nasal cavity of pigs are not of clinical significance to their porcine hosts (fitzgerald et al. 1958; hibler et al. 1960; pakandl 1994; mostegl et al. 2011) . trichomonads are occasionally observed in the feces from dogs with diarrhea . the parasite was also detected by culture in the feces of 17.2% of puppies from french breeding kennels, which indicates that t. foetus may be a common parasite in dogs (grellet et al. 2010 ). however, molecular identity of the enteric trichomonads observed in these dogs was not investigated, and the relevance for dogs is not clear. more studies are required to determine the prevalence and clinical significance of t. foetus infection in dogs, since the finding could be also attributed to opportunistic overgrowth of the commensal, pentatrichomonas hominis. in human medicine, the most studied and relevant is trichomonas vaginalis that affects over 150 million people worldwide and is the most common non-viral sexually transmitted disease (van der pol 2007) . other trichomonads are found as inhabitants of the gastrointestinal tract in birds such as tetratrichomonas gallinarum and trichomonas gallinae. particularly, columbiformes, falconiformes, strigiformes, and wild passeriformes can be severely affected by avian trichomonads, whereas the majority of infections in galliformes and anatiformes are subclinical although severe infections are occasionally reported (amin et al. 2014) . other examples of trichomonads found as inhabitants of the gastrointestinal tract are trichomonas muris of mice and pentatrichomonas hominis of a variety of vertebrate species (bondurant and honigberg 1994) . a conspicuous pelta-axostyle complex, and the recurrent flagella are often associated with a lamellar undulating membrane underlain by a striated costal fiber (adl et al. 2005) . the number of free flagella characterizes each genus of the family trichomonadidae. thus, the genus tritrichomonas is characterized by having three free flagella, whereas the genera tetratrichomonas and pentatrichomonas possess four and five flagella, respectively. among the various species of trichomonads thus far identified, only a number of them are regarded as pathogens (bondurant and honigberg 1994) . bovine t. foetus isolated from the urogenital tract of cattle and feline isolates found in the gastrointestinal tract of the domestic cat are morphologically indistinguishable. however, there appears to be no association between t. foetus infection in cats and reported exposure to cattle ). there is an ongoing debate whether t. foetus from cattle and cats should be placed into separate species. a molecular separation of feline and bovine isolates of t. foetus based on a number of gene loci-summarized by yao and köster (2015) -seems to be possible, but on the transcriptomic level, a separation remains difficult (reinmann et al. 2012; slapeta et al. 2010 slapeta et al. , 2012 sun et al. 2012; morin-adeline et al. 2014 , 2015b . there is evidence that t. foetus isolates from cats and cattle show differences in ph tolerance (sect. 14.2.2), and t. foetus from cats are able to survive a passage through the alimentary tract of slugs (morin-adeline et al. 2015a ; van der saag et al. 2011 ). thus, some authors believe that feline t. foetus represents a different species, and a change in name-to t. blagburni-has been proposed (walden et al. 2013) . it has been hypothesized that feline t. foetus has extended its host range into the bovine reproductive tract (morin-adeline et al. 2015a). the picture becomes even more complicated because t. foetus isolated from cattle seems to be morphologically and genetically identical to tritrichomonas suis, that is, a commensal observed in the nasal cavity, stomach, cecum, and colon of the domestic pig (felleisen 1998; hampl et al. 2001; tachezy et al. 2002; reinmann et al. 2012; slapeta et al. 2012; sun et al. 2012) . consequently, it was assumed that t. suis and t. foetus belong to the same species (tachezy et al. 2002; lun et al. 2005; frey and müller 2012; yao and köster 2015) . however, more recent epidemiological studies suggest that cross-species transmission from pigs to cattle on the same farm-e.g., by exposure to t. foetuscontaminated pig feces-is unlikely to occur (mueller et al. 2015) . t. foetus has a trophozoite stage, with a pyriform or ovoid appearance, and a size ranging from 8 to 18 μm in length and 4 to 9 μm in width (bondurant and honigberg 1994) (fig. 14.1) . the trophozoite has several structures with locomotor function such as flagella and the undulating membrane. the flagella originate from the basal bodies or kinetosomes-located in the apical pole of the cell. three of the flagella are of similar length to each other and are directed forward, while the fourth flagellum, called the recurrent flagellum, is directed toward the posterior part of the body, associated with it by an undulating membrane, and continues as a free flagellum beyond the posterior end of the undulating membrane (taylor et al. 1994; benchimol 2004) . the internal organelles of t. foetus are similar to those of other trichomonads. the cytoplasm contains a series of support elements, including the pelta-axostyle complex, the costal fiber bordering the recurrent flagellum, and the parabasal apparatus. these elements together with the flagellum make up the cytoskeleton (benchimol 2005) . the axostyle originates in the same area of the birth of the flagellum-surrounded at this point by a chromatin ring-and is directed toward the back of the parasite, making prominence at the posterior end. the pelta is a semilunar structure, very little developed, located in the anterior part of the axostyle. both structures form the pelta-axostyle complex-composed of groups of connected microtubules-forming a kind of slit that houses the nucleus and parabasal bodies (benchimol 2004 (benchimol , 2005 . in t. foetus, the axostyle has two functions; it serves as a support organelle and participates in the processes of cell division, allowing the constriction of the nuclei during the karyokinesis (ribeiro et al. 2002) . the costa is a rigid structure that sits on the inner margin of the undulating membrane and serves as a support. the parabasal filaments are also filaments striped perpendicularly, and their mission seems to support the parabasal body-i.e., the golgi complex. the parabasal filaments and the parabasal body constitute the parabasal apparatus, located in the anterior part of the cell (benchimol 2004 (benchimol , 2005 . t. foetus has a simple anterior nucleus and hydrogenosomes, which appear as electro-dense corpuscles that act as functional substitutes for mitochondria. other cellular components that can be observed in the cytoplasm are free ribosomes, polysomes, glycogen granules, vesicles, and vacuoles related to processes of endocytosis, digestion, and transport (benchimol 2004 (benchimol , 2005 . under unfavorable conditions, such as a low concentration of nutrients, the presence of certain drugs such as griseofulvin, or abrupt changes in temperature, trophozoites internalize the flagellum and acquire a form of pseudocyst, which is not surrounded by a manifest cell wall (pereira-neves and benchimol 2009) . as regards nutrition, trichomonads lack a cytostome; they are able to capture food through the cell surface by means of pinocytosis and phagocytosis, with the resulting formation of food vacuoles of different size. like other trichomonads that inhabit body cavities, t. foetus feeds mainly on bacteria, whose proliferation depends on the environment conditions where the parasite is based (petrin et al. 1998) . from the metabolic point of view, t. foetus is unable to de novo synthesize purine and pyrimidine nucleotides, as well as complex phosphoglycerides or cholesterol. the parasite obtains its energy through the anaerobic catabolism of carbohydrates, although the trichomonads are able to survive in the presence of oxygen. as already noted, trichomonads lack mitochondria but possess hydrogenosomes that produce molecular hydrogen in anaerobiosis and reduce oxygen. in this way, the parasite manages to keep the ph of its environment close to neutrality favoring its own development (kleydman et al. 2004 ). the reproduction of t. foetus-like that of all trichomonads-is asexual (petrin et al. 1998 ). the parasite divides by longitudinal binary fission in which the nuclear membrane persists-a type of mitosis referred to as cryptopleuromitosis. in addition, when compared to the trophozoite form, pseudocysts present a different mitosis model, since they first divide the nuclei without dividing their cytoplasm, leading to the formation of multinucleated polymastigotes that persist if the cells are maintained under conditions of stress. when the environmental conditions are again favorable, flagella are externalized, and the new flagellated trophozoites emerge from the multinucleated cells (pereira-neves and benchimol 2009). t. gallinae is the only trichomonad species with a clear pathogenic potential for birds (bondurant and honigberg 1994; amin et al. 2014) . t. gallinarum is commonly found in the large intestine of gallinaceous and anseriform birds, yet its role in causing disease either in naturally infected chickens and turkeys or via experimental infection is under discussion (amin et al. 2014) . t. gallinae trophozoites have an ovoidal to pyriform shape with a size of about 7-11 μm. they are provided with four free anterior flagella and a fifth recurrent one, which does not become free at the posterior pole as it extends for only two-thirds of the body length (tasca and de carli 2003; mehlhorn et al. 2009 ). trophozoites of t. gallinarum appear mostly pear shaped and range in size from 6 to 15 μm (clark et al. 2003) . they also have four free anterior flagella and a fifth recurrent one, which becomes free at the posterior pole. another difference to t. gallinae is the occurrence of a sphere of lacunes of the endoplasmic reticulum surrounding in a regular distance the nucleus with its typical perinuclear membranes. furthermore, the food vacuoles appear to be very large (mehlhorn et al. 2009 ). bovine t. foetus is located in the genital tract of its natural hosts, bos taurus taurus and bos taurus indicus cattle (skirrow and bondurant 1988; bondurant 2005; sager et al. 2007 ). the preferred location of the parasite in the bull is the preputial cavity-concentrating mainly in the penile mucosa and adjacent areas of the posterior preputial mucosa-specifically on the surface of the stratified squamous epithelium of the penis and the proximal foreskin in the fornix area parsonson et al. 1974; parker et al. 1999 ). this epithelium undergoes numerous folds-resulting in a greater development of crypts-where t. foetus can develop properly by providing a suitable microenvironment for facultative or microaerophilic anaerobic microorganisms (rhyan et al. 1999) . infection may persist for the life of the bull, in spite of the presence of a measurable humoral immune response in the preputial cavity (rhyan et al. 1999; campero et al. 1990; flower et al. 1983 ). in the female, once the infection has occurred, the parasite colonizes the surface of the entire genital system-vagina, cervix, endometrium, and oviduct-in a period of 2 weeks (parsonson et al. 1976) . as observed in natural infections, the parasite is preferentially concentrated in the folds of the cervix (bondurant 1997). the infection is self-limiting, and the parasite disappears simultaneously from all areas of the genital tract after a period of at least 90-95 days (parsonson et al. 1976; rae et al. 2004; bondurant 2005) . in experimental infections of nonpregnant heifers, t. foetus infection is typically cleared from the uterus and vagina between weeks 6 and 12 following infection (parsonson et al. 1976; anderson et al. 1996; bondurant et al. 1993; skirrow and bondurant 1990a) . a very small proportion of cows in infected herds-a fraction less than 1%-have been shown to remain infected throughout pregnancy and into the following breeding season. fortunately, such carrier cows are rare (bondurant 2005) . under natural conditions, t. foetus is transmitted directly from an infected animal to a healthy animal, almost exclusively through natural mating (bondurant 2005) . the bulls become infected during the mating of infected cows, remaining asymptomatic carriers (fig. 14. 2). very rarely, however, the parasite can be transmitted by other routes, for example, mechanically during the practice of artificial insemination or vaginal examination, if contaminated material is used-e.g., using the same glass rod or insemination pipette for different cows or not properly disinfected specula (murname 1959; goodger and skirrow 1986) . mechanical transmission seems to be possible through a healthy bull-i.e., from an infected cow to a receptive cow-if the time between two services does not exceed 20 min goodger and skirrow 1986; bondurant 2005; ondrak 2016 ). t. foetus was shown to be able to survive in cryopreserved semen and may be present in semen if it is contaminated with preputial fluid during manual collection (blackshaw and beattie 1955) . given the resistance of this parasite in fresh, pure, or diluted semen, refrigerated and even cryopreserved, there is the possibility of transmission through artificial insemination with contaminated semen (bondurant 2005) . because bulls tend to mount each other, feces in the preputial cavity is commonly found. this fecal material may contain non-t. foetus trichomonads, such as pentatrichomonas hominis and any number of tetratrichomonas species that have been shown to be nonpathogenic (taylor et al. 1994; campero et al. 2003; hayes et al. 2003) . the opportunity for transmission of t. foetus between males is regarded as very limited. feline t. foetus appears to be host adapted, i.e., adapted to the intestinal tract of cats. after experimental orogastric infection of kittens, feline t. foetus has been demonstrated to colonize the lumen of the ileum, caecum, colon, and rectum 203 days after infection (gookin et al. 2001) . in naturally infected cats, massive numbers of trichomonads can be observed at the surface of the colonic epithelium and within the colonic crypts (yaeger and gookin 2005) . although the presence of t. foetus in the uterus of a cat with pyometra has been described, it has been speculated that the in the bull, the parasite is located in the preputial mucosa in the female, the parasite is located in the os cervix infected female parasite transmission during mating parasite could have accidentally accessed the genital area through contact with contaminated feces (dahlgren et al. 2007 ). however, colonization of the reproductive tract in both male and female cats from breeding grounds with a high prevalence of feline tritrichomonosis has not been observed (gray et al. 2010 ). feline t. foetus infection occurs by direct fecal-oral transmission. infected cats are shedding trophozoites with their feces, and transmission occurs when two or more cats share the same litter box (fig. 14. 3). trophozoites would adhere to the hair of the animals and could be ingested during grooming tolbert and gookin 2009 ). the viability of the parasite in the environment is limited though it can withstand several days at room temperature facilitating its transmission (hale et al. 2009 ). t. foetus-contaminated food and less likely water may be also a relevant route for transmission. further, shedding of viable t. foetus has been demonstrated in some slug species, which were fed cat food spiked with trophozoites of a feline t. foetus isolate, suggesting that invertebrates like slugs could play a role as mechanical vectors (van der saag et al. 2011). the rock pigeon-columba livia-was regarded as the primary host of t. gallinae and has been considered responsible for the worldwide distribution of this protozoal infection (stabler 1954; harmon et al. 1987) . other species within the columbiformes, falconiformes, strigiformes, and, most recently, different passeriformes have been recognized as potential hosts (forrester and foster 2008; robinson et al. 2010 gallinaceous birds like turkeys and chickens (levine and brandly 1939) . the preferred site for t. gallinae is the upper digestive tract including the mouth, pharynx, esophagus, and crop, with the parasite rarely found posterior to the proventriculus (cauthen 1936) . transmission by direct contact seems to be the most efficient route to establish an infection-e.g., via the crop milk from infected parent birds to the nestlings during feeding (stabler 1954) . in adult pigeons, the infection can occur during courtship while raptors can be infected from prey animals carrying the parasite. the infection of turkeys and chickens happens mainly via drinking water contaminated by pigeons (bondurant and honigberg 1994) . trichomonas gallinae is unable to form true cysts, even though cyst-like stages-pseudocysts-have been reported (tasca and de carli 2003; mehlhorn et al. 2009 ). these pseudocysts may provide another route of transmission and an environmentally resistant stage during unfavorable conditions. tetratrichomonas gallinarum locates in the intestinal tract of different poultry species including chickens, turkeys, guinea fowl, quails, ducks, and geese and can be transmitted via consumption of contaminated food (bondurant and honigberg 1994) . pseudocysts of t. gallinarum have been reported in vivo and in vitro possibly protecting the parasite during fecal-oral transmission (mehlhorn et al. 2009 ). once infected, the male acts as an asymptomatic carrier throughout his life parsonson et al. 1974; parker et al. 1999) . minor histological changes are observed with increased accumulations of neutrophils followed by an infiltrate of lymphocytes and plasma cells penetrating into the intraepithelial area and coalescing in the subepithelium to form lymphoid nodules (rhyan et al. 1999; bondurant 2005) . in the female, 2 weeks after infection, t. foetus may have colonized the different parts of the genital tract (parsonson et al. 1976) . the preferred location is in the cervix and cervicovaginal mucus, but the number of parasites varies throughout the estrus cycle, being higher in the days prior to estrus. the establishment of the parasite in the genital tract of the female does not seem to interfere with the fertilization nor with the early development of the embryo (bielanski et al. 2004 ). in heifers experimentally infected with t. foetus, conceptus deaths peaked at 50-70 days of gestation (parsonson et al. 1976 ). occasional abortions of fetuses older than 4-month gestational age are reported, but typically losses occur 2 months earlier (bondurant 2005) . the infection in the female is usually self-limiting, disappearing between 2 and 4 months after the loss of the conceptus. the immunity that develops is not permanent and usually lasts for about 6 months; after 6 months, the female is again susceptible to infection. carrier cows-these are cows that remain infected for at least 10 months-seem to fail to develop a protective immune reaction against t. foetus. notable lesions in the maternal endometrium and fetal envelopes have been described only at the time of fetal loss (parsonson et al. 1976 ). the mechanisms of pathogenic actions that underlie the loss of the embryo or fetus are not known with accuracy and may include (1) the direct mechanical action of the parasite, (2) the adverse effects of enzymes secreted by the parasite, and (3) the alteration of the intrauterine environment mainly by antiparasitic inflammatory reactions of an infected dam (reviewed by bondurant (2005) and campero and cobo (2006) ). the increase in the number of microorganisms in the female genital tract occurs slowly and probably does not produce any relevant damage until this number exceeds a certain threshold. this fact would explain the long period of time between infection and loss of the conceptus. few studies have examined the interaction of feline t. foetus with intestinal epithelium (recently reviewed by yao and köster (2015) and tolbert and gookin (2016) ). recent studies examining t. foetus infection in a co-culture model with monolayers of porcine intestinal epithelial cells suggest that adhesion to the intestinal epithelium occurs by means of specific receptor-ligand interactions (tolbert et al. 2013) . pathogenesis of t. foetus on the intestinal epithelial cells has been suggested to be both contact-dependent and contact-independent. in the former, a cytopathic effect is mainly exerted via apoptosis induced by cell-associated proteases, whereas extracellular proteases are the major players in contact-independent cytotoxicity. extracellular proteases may also play a role in evading complement killing. the severity of the disease depends on the susceptibility of the infected birds together with the pathogenic potential of the incriminated strain and the stage of infection (cooper and petty 1988; cole and friend 1999) . the severity of pathologic lesions of t. gallinae in the upper digestive tract varies from a mild inflammation of the mucosa to caseous areas that block the esophageal lumen (stabler 1954 ). some virulent strains are able to create diphtheritic membranes-associated with fibrinous lesions in internal organs such as the liver, lungs, and peritoneum-resulting in high mortality (narcisi et al. 1991) . strains of moderate virulence are often associated with caseous abscesses in the upper digestive tract and oropharyngeal region, whereas no appreciable lesions are produced by avirulent strains (cole and friend 1999) . in vitro, t. gallinae proliferation has been associated with a disintegration of the cell monolayer, and genetically different t. gallinae isolates caused diverse magnitudes of cytopathic effects on different cell lines (amin et al. 2012a ). however, little is known concerning the mechanism by which t. gallinae causes pathological changes in its hosts. proteolytic proteins secreted by the parasite have been identified as contributing to the detachment of a cell monolayer (amin et al. 2012b) . various studies investigated the pathogenicity of t. gallinarum either in naturally infected chickens and turkeys or via experimental infection, with contradicting outcomes as reviewed by amin et al. (2014) . recent studies have shown that in vitro, t. gallinarum has no destructive effect on cells and, in vivo, did neither produce clinical signs nor macroscopic or microscopic lesions in turkeys and specified pathogen-free chickens (amin et al. 2011 ). the clinical effects produced by the disease occur only in female cattle, causing early abortion and temporary infertility (reviewed by bondurant (1997 bondurant ( , 2005 bondurant ( , 2007 , yule et al. (1989a) , rae and crews (2006) ). in males, t. foetus infection is asymptomatic and affects neither semen quality nor sexual behavior, but bulls can shed the organism indefinitely rhyan et al. 1999) . the parasite multiplication causes inflammation of the endometrium, cervical, and vaginal mucous membranes in cows or heifers following the infection at breeding (parsonson et al. 1976; rhyan et al. 1988; anderson et al. 1996) . consequently, signs of mild vaginitis, cervicitis, or endometritis, such as mucopurulent vaginal discharge, may be observed, although generally there are no overt signs. conception apparently proceeds normally, but almost all conceptuses are lost at some time early in gestation with early fetal death and resorption but also abortion-with a peak loss at 70-90 days (parsonson et al. 1976; bielanski et al. 2004) . infection can result in fetal maceration and pyometra. the consequence is infertility (parsonson et al. 1976; bondurant 1985; ball et al. 1987; anderson et al. 1996) . abortions of fetuses typically occur around 2 months of gestational age. abortions of fetuses older than 4 months of gestational age due to trichomonosis have been occasionally reported. if the affected cow undergoes early fetal loss, it may cycle regularly without showing any signs but a prolonged interestrous interval (bondurant 1985) . pyometra occurs in less than 5% of infected cows and is followed-as the corpus luteum of pregnancy is maintained-by a large purulent response (rhyan et al. 1988) ; pyometra is probably a result of bacterial contamination that occurs at the time of fetal loss, when the cervix is likely to relax sufficiently to admit contamination from outside the environment (rhyan et al. 1995a) . cows that are infected with t. foetus typically clear the infection within a few months, i.e., after three cycles (parsonson et al. 1976 ). immunity, however, is not permanent, and the cow will be subject to reinfection and embryonic death in subsequent breeding periods, and, as mentioned earlier, some infected cows may carry infections into the next breeding season (skirrow 1987; mancebo et al. 1995) . in an infected herd, bovine tritrichomonosis is associated with lowered fertility. the usual signs in the herd include return to estrus 1-3 months after breeding. at pregnancy exam time, a number of early pregnancies and open cows are observed. the period of infertility may last for another 2-6 months as a result of the infection. other clinical features of the disease in the herd include many services per conception, poor pregnancy rates, long calving intervals, and calf crop reduction. in addition, the calving season is spread out causing batches of calves of different ages with a wide variation in weaning weights (clark et al. 1983a; mccool et al. 1988; rae 1989; collantes-fernandez et al. 2014 ). t. foetus is recognized as an important cause of diarrhea in domestic cats. typical clinical signs in natural infections are chronic or intermittent large bowel diarrhea, which can vary from subclinical to intractable (reviewed by gookin et al. (1999) , gookin et al. (2001 ), foster et al. (2004 , manning (2010) , yao and köster (2015) ). the feces are described as yellow-green in color, gassy, and malodorous with typical signs of colitis including fresh blood, mucus, fecal incontinence, tenesmus, and flatulence. the consistency of the feces can vary from liquid to semi-formed or cow pat (stockdale et al. 2009 ). severe cases may be accompanied by marked inflammation of the anal region, fecal incontinence, and rectal prolapse (gookin et al. 1999; foster et al. 2004; tolbert and gookin 2009; bell et al. 2010 ). in addition, some infected cats have been reported showing systemic signs including anorexia, depression, vomiting, and weight loss (stockdale et al. 2009 ). mortality is extremely rare and only reported in kittens and is presumably caused by endotoxic shock because of deep lesions in the colonic mucosa (holliday et al. 2009 ). the majority of infected cats maintain good body condition and appetite without signs of systemic illness (gookin et al. 1999 (gookin et al. , 2001 tolbert and gookin 2009 ). the long-term prognosis for t. foetus-infected cats is usually good and most will eventually overcome the infection. remission could take between 4 months and 3 years, with irregular episodes of diarrhea of variable length (gookin et al. 1999 . no abnormalities are routinely noted on hematology and serum biochemistry profile of some cats, though they remain infected and continue shedding the organism despite clinical improvement, i.e. these cats represent asymptomatic carriers. (manning 2010) . some positive cats were also infected with other pathogens, like cryptosporidium spp., giardia spp., coccidian, or feline immunodeficiency virus (gookin et al. 1999; stockdale et al. 2009 ); concurrent infections may contribute and increase susceptibility and vulnerability to intestinal disease in infected cats. in birds the two trichomonad species t. gallinarum and t. gallinae are commonly found (reviewed by amin et al. (2014) ). t. gallinarum parasitizes the large intestine of gallinaceous and anseriform birds. t. gallinarum induces usually a latent infection in the absence of clinical signs and lesions, and it is not clear whether t. gallinarum should be regarded a primary pathogen (amin et al. 2011; friedhoff et al. 1991) . however, the presence of t. gallinarum may aggravate primary diseasese.g., caused by histomonas meleagridis-and coinfections have been observed (grabensteiner and hess 2006) . t. gallinae is of veterinary and economic importance, as it causes avian trichomonosis, a disease with important medical and commercial implications, which is known as pigeon canker, canker, roup, or gelber knopf (amin et al. 2014) . t. gallinae is located in the upper digestive tract of pigeons, causing lesions (bondurant and honigberg 1994). the disease is characterized by greenish fluid and caseous lesions-whitish-yellowish fibrinous material-on the oropharyngeal membranes that can block the lumen of the esophagus impairing drinking and feeding. clinical signs associated with avian trichomonosis are loss of appetite, vomiting, ruffled feathers, diarrhea, dysphagia, dyspnea, weight loss, increased thirst, inability to stand or to maintain balance, and a pendulous crop (narcisi et al. 1991) . death may occur within 3 weeks of infection. infected birds can also remain asymptomatic due to the infection with avirulent strains of trichomonads or a lower susceptibility as seen in older birds. avian trichomonosis may also affect domestic fowl; in earlier studies severe outbreaks have been recorded in chickens and turkeys, but we are not aware on recent cases (hawn 1937 in cattle, the prescribed test for international trade is the identification of t. foetus by culture or pcr-world organization for animal health (oie), oie terrestrial manual. the oie terrestrial manual provides protocols for sampling, sample transportation, transport medium, culture media, culture conditions, and how to read out the culture test. in addition, the oie terrestrial manual also provides recommendations for pcr analyses, which can be applied in combination either with or after culture as an ancillary test or-more often-direct as the primary test to examine bovine samples-i.e., preputial material, uterine or vaginal secretions, or abomasal content of aborted fetuses. protocols to diagnose bovine tritrichomonosis have been described in detail previously (sager et al. 2007 ). to diagnose t. foetus infection in cattle, pcr tests may have a higher or at least the same sensitivity as culture tests but have several advantages, because parasites in the sample do not need to be viable and pcr results are rapidly available in contrast to results of culture tests. on the other hand, culture tests are superior to pcr because of their relative easiness (yao 2013) . in cats, both cultivation and pcr tests are regarded as optimal methods for a sensitive and specific detection of t. foetus in fecal samples, although currently pcr is regarded as the gold standard assay for diagnosis of feline t. foetus infection since detection is independent from parasite viability (gookin et al. 2002 manning 2010; yao and köster 2015) . under optimized conditions, pcr is the method of choice when samples have to be shipped-e.g., from practitioner to a veterinary laboratory. similar to t. foetus in cattle, the success of cultivation tests is largely dependent on the viability of t. foetus in the sample (hale et al. 2009 ). in birds infected by t. gallinae, an immediate diagnosis by direct microscopy of material collected via swabbing the oral cavity during clinical examination or necropsy is possible. also, t. gallinarum can be observed in fecal material collected from birds-e.g., by swabbing cloacae. however, although the direct detection by light microscopy is fast and inexpensive, it is regarded as insensitive, and low numbers of parasites may not be detected (amin et al. 2014) . also in birds the use of cultivation for the detection of trichomonads is clearly superior in sensitivity as compared to direct microscopy (cooper and petty 1988; bunbury et al. 2005) . in fresh samples it is possible to diagnose the infection with trichomonads by a direct light microscopical examination. optimal is a 200 to 400-fold magnification. it is advantageous to pre-warm slides, to retain motility of trichomonads. a drop of physiological saline is added to the slide, mixed with a nearly equal volume of material collected, and mounted with a coverslip. it is possible to apply conventional light microscopy-either using a conventional up-light or an inverted microscope-, phase-contrast microscopy, or darkfield microscopy. in phase-contrast microscopy, it is easier to see flagella. in dark-field microscopy, trichomonads appear as small rolling luminescent footballs. in conventional light microscopy, trichomonads are identified by their characteristic movement, which is described as rolling and jerky. they are flashing, due to their rolling movements. the presence of multiple anterior flagella and the characteristic refractile undulating membrane can be observed. however, one disadvantage of direct microscopy is that at the resolution of a conventional laboratory microscope, the exact number of anterior flagella cannot be determined. the specificity of direct microscopy is very limited. the identification of the trichomonad species observed by this method is not possible, and confirmatory pcr analyses are necessary. for inexperienced examiners it might be difficult to differentiate trichomonads from other intestinal parasites-e.g., giardia spp. in cat feces (yao and köster 2015) . also the intestinal commensal trichomonad pentatrichomonas hominis might be misinterpreted as t. foetus. another disadvantage of direct microscopy is its low sensitivity. a sample is only positive, if it contains a sufficient number of parasites per milliliter specimen. for example, in cats, the fecal examination by direct microscopy is reported to have a diagnostic sensitivity of only about 14%, and also in bovine tritrichomonosis, direct examination is estimated to be 25% less sensitive than culture diagnosis sager et al. 2007 ). the advantage of direct microscopy as diagnostic tool is its speed and the low cost of examination. this is the reason why direct microscopy is often used by practitioners for the examination of t. foetus in cats and t. gallinae infection in birds (forrester and foster 2008; amin et al. 2014 ). staining of trichomonads is possible using a number of stains, including giemsa, silver, iron hematoxylin, malachite green, methylene blue, papanicolaou, and acridine orange (amin et al. 2011) . a fast and inexpensive staining protocol-giemsa or diff-quick and iodine-has been reported (lun and gajadhar 1999) . single parasites might be easier to inspect; however, the chance to find small numbers of parasites in a sample might decrease because parasites can no longer be identified by their characteristic movement or flashing essential for parasite identification in low concentrated samples. in tissues, the use of hematoxylin-eosin (he) and periodic acid-schiff (pas) stains was proven to be advantageous for identification of the flagellates, especially in organs that contained only a few protozoal cells (amin et al. 2011 (amin et al. , 2014 . immunohistochemical techniques and in situ hybridization have been successfully applied to demonstrate trichomonads in histological sections of cat or bird tissues, respectively (rhyan et al. 1995b; yaeger and gookin 2005; liebhart et al. 2006; mostegl et al. 2012) . immunohistochemical detection using monoclonal antibodies against t. foetus has been shown to be a valuable tool (hodgson et al. 1990) . a protocol for immunohistochemical detection using the monoclonal antibody mab 34.7c4.4 is provided in the oie terrestrial manual (www.oie.int/international-standard-setting/terrestrial-manual). staining is also applied to confirm positive cultures by morphological criteria. in vitro culture can be performed by incubating the samples at 25-37 °c in a growth medium. if parasites are present, their numbers will multiply in the culture over time, increasing the likelihood of their detection. a large number of culture systems for trichomonads and especially t. foetus have been developed and published. presumably the first culture system for an axenic cultivation of t. foetus isolated from an aborted fetus-i.e., cultivation without bacteria or other living organismswas reported by a german microbiologist (witte 1933) . from that time on, numerous reports of further cultivation protocols have been published. until now diagnostic culturing is of outmost importance for sensitive diagnosis of bovine tritrichomonosis. also in the diagnosis of tritrichomonosis of cats, cultivation has been widely used for epidemiological studies or diagnostic purposes (tables 14.2 and 14.3). in bovine tritrichomonosis cultivation became an important diagnostic tool, because parasite numbers in bovine samples-e.g., preputial smegma or cervico-vaginal mucus-are usually too low to be detected by direct microscopy and a multiplication of parasites after a few days of cultivation increases the chance to find infected bulls. the number of organisms in preputial secretions has been estimated and ranges from less than 200/ml up to more than 80,000/ml (skirrow and bondurant 1988) . in bovine tritrichomonosis, diagnostic sensitivity of a single culture test on infected bulls has been estimated to range between 70 and 100% (skirrow et al. 1985; schönmann et al. 1994; parker et al. 1999 parker et al. , 2003a . in a large field study, including 2832 mature bulls from 124 beef herds in argentina, bayesian estimation revealed a diagnostic sensitivity and specificity of 72.0% (59-87%) and 95.4% (94-96%), respectively (perez et al. 2006) . a repeated testing of bulls-e.g., three times with intervals of several days-has been shown to increase the diagnostic sensitivity of the cell culture test close to 100%. of 29 samples collected from 5 experimentally infected bulls with resting periods of 2-4 days between samplings, 24 (83%) were determined as positive (mukhufhi et al. 2003) . in another study, consecutive testing over a period of more than 7 months resulted in the determination of an infection rate of 100% in 15 bulls (clark et al. 1971) . for bulls from herds in which t. foetus is endemic, two to four tests per bull may be required to ensure that the bull is not infected (parker et al. 1999) . a sexual rest of bulls for a minimum of about 1-2 weeks prior to sampling increases sensitivity (yule et al. 1989a) . also for the analysis of females, i.e., after sampling of cervico-vaginal mucus, sensitivity of cultivation is superior to direct microscopic examination (simmons and laws 1957; skirrow and bondurant 1988) . in female cattle diagnostic sensitivity of culture tests has been reported in the range of 56 and 95% (kimsey et al. 1980; goodger and skirrow 1986; skirrow and bondurant 1988; parsonson et al. 1976 ). the infection in females is usually cleared within 3 months, and it is often difficult to isolate organisms from female cattle in the late stage of their infection. in cats, the culture method is reported to have a detection limit of about 2 × 10 2 trophozoites and a diagnostic sensitivity from 26.4 to 58.8% (hale et al. 2009; gookin et al. 2004) . to achieve optimal test sensitivity, it is essential to retain as long as possible viability of trichomonads after sampling. the number of viable organisms decreases progressively after sampling (todorovic and mcnutt 1967; tedesco et al. 1979; reece et al. 1983; skirrow et al. 1985; kittel et al. 1998; bryan et al. 1999; parker et al. 1999 ). immediate cultivation is ideal but rarely possible. a 1-day delay is estimated to cause a loss of diagnostic sensitivity of 10% (sager et al. 2007 ). sampling the parasite into transportation media providing nutrients has been shown to be essential for the survival of trichomonads, especially if time between sampling and starting cultivation is exceeding 2 days (kimsey et al. 1980; hale et al. 2009 ). an earlier study showed that physiological saline with 5% fetal serum or lactate ringer's solution was effective (kimsey et al. 1980) . a thyoglycolate transport medium was also shown to be suitable; however, sensitivity of the subsequent cell culture test was slightly lower than after transport using inpouch tf medium (biomed diagnostics, white city, or, usa). today the medium used for later cultivation is often also used as transportation medium-i.e., inpouch tf or diamond's medium (bryan et al. 1999 ). an alternative is the direct sampling into a commercially available transport and culture kit-inpouch tf-containing a selective medium, a medium optimized for t. foetus, and a medium repressing the growth of the contaminating bacterial flora. this commercial transport and culture kit is recommended not only for sampling in cattle but also for sampling in cats or birds (thomas et al. 1990; bondurant 1997; gookin et al. 2004; hale et al. 2009; yao and köster 2015) . according to the oie terrestrial manual (www.oie.int/international-standardsetting/terrestrial-manual), bovine samples-after being added to transport mediashould be protected from exposure to daylight and extremes of temperature, which should remain above 5 °c and below 38 °c (bryan et al. 1999) . for cat fecal samples, a storage for 1 to 24 h at room temperature (23-25 °c) was superior to a 4 °c storage for the same period of time as shown in experiments performed with fecal samples spiked with different t. foetus concentrations (2 × 10 2 -2 × 10 4 t. foetus per gram of feces) (hale et al. 2009 ). several culture media have been found suitable for the cultivation of trichomonads. overviews on media have been provided in the oie terrestrial manual, www. oie.int/international-standard-setting/terrestrial-manual/, and in several reviews (skirrow and bondurant 1988; sager et al. 2007 ). currently, the most widely used system is inpouch tf, a commercial transport and cultivation kit (yao 2013) . as noncommercial medium the so-called diamond's medium is widely used, also in epidemiological studies (tables 14.2 and 14 .3). modified diamond's medium is a trypticase-yeast extract-maltose medium which in most studies was used modified by the addition of heat-inactivated serum-e.g., of 5% heat-inactivated horse or lamb serum (diamond 1957; skirrow and bondurant 1988; sager et al. 2007 ). both the use of modified diamond's medium and the inpouch tf kit are recommended for diagnosis of bovine tritrichomonosis by the oie terrestrial manual (www.oie.int/international-standard-setting/terrestrial-manual, accessed 22. febr. 2017). the inpouch tf kit seems superior to diamond's medium to detect t. foetus infection in bulls (schönmann et al. 1994; appell et al. 1993; mendoza-ibarra et al. 2012; yao 2013) . as regards the cultivating of cat samples, in one study, the inpouch tf kit was found to be superior to modified diamond's medium ; however, in a recent study, comparison of both systems revealed a higher sensitivity when modified diamond's medium was used-atcc medium 719 (hale et al. 2009) . a retrospective analysis of data revealed no statistical significant differences between cultivation with modified diamond's medium and inpouch tf. a modified plastridge medium containing antibiotics and antifungal agents as well as heat-inactivated bovine serum was recommended for initial cultivation of trichomonads and can be applied combined with modified diamond's medium for subsequent procedures-e.g., sub-cultivation (reece et al. 1983; skirrow and bondurant 1988; sager et al. 2007 ). in studies conducted in argentina, a commercially available modified plastridge medium has been applied (mardones et al. 2008) . the preparation of modified diamond's medium and test vials, as well as sample processing and reading the results of the culture test, is described in the oie terrestrial manual for bovine samples; many of the recommendations also apply for processing cat and avian samples (www.oie.int/international-standard-setting/terrestrial-manual, accessed 22. febr. 2017). samples collected via preputial scraping-e.g., vigorously by brush, insemination pipette, or aspiration, usually about 0.5-1 ml-can be inoculated directly on top of the medium of a test tube, into the transportation medium, or into the upper chamber of the inpouch tf kit. in contrast, samples collected by preputial washing need to be centrifuged and the supernatant discarded in order to reduce volume. reading the cell culture tests is performed by microscopic detection of the trichomonads. to increase specificity of the culture test, it is recommended to confirm observed parasites by pcr (campero et al. 2003) . for cats, voided feces sampled directly from the litter box, rectal swabs obtained from rectal mucous membranes, or feces collected by manual extraction with the aid of fecal loops or by a colon flush technique can be the starting point for trichomonads cultivation (yao and köster 2015; manning 2010; tolbert and gookin 2009 ). in case of t. gallinae-infected birds, a cotton-tipped applicator moistened with sterile saline is used to swab the oral cavity, and swabs are added to inpouch tf culture devices or other commercial or noncommercial culture media (forrester and foster 2008; rogers et al. 2016; girard et al. 2014; krone et al. 2005) ). microscopic detection of culture-growing organisms can be done by light microscopy, on a wet mount slide prepared directly from the culture or through the plastic wall of the inpouch tf kit using a plastic clip provided by the supplier. the motile organisms may be seen under a standard microscope using a 200-fold or higher magnification. an inverted microscope may be useful for examining culture flasks containing culture medium. culture media should be inspected by microscopic examination at regular daily intervals-from day 1 to day 7 after inoculation (bryan et al. 1999; lun et al. 2000) . during the first 4-72 h of culturing, there might be an initial increase of parasite numbers, subsequently followed by a decrease. organisms may be identified on the basis of characteristic morphological features (oie terrestrial manual, www. oie.int/international-standard-setting/terrestrial-manual, accessed 22. febr. 2017). it has been shown for cattle and cats that without test confirmation-e.g., by using specific pcr or sometimes by using staining or electron microscopy-the diagnostic specificity of the culture method remains well under 100%. therefore, a subsequent pcr analysis of culture-positive samples has been recommended to avoid false-positive findings (parker et al. 2001; campero et al. 2003; ceplecha et al. 2013) . intestinal trichomonads were observed in virgin bull samples submitted for confirmation of inpouch tf culture diagnosis or culture diagnosis using sutherland medium michi et al. 2016) . the medium of inpouch tf-feline is thought to be highly specific to t. foetus, and the morphologically similar flagellates p. hominis and giardia spp. should not survive longer in this medium than 24 h . however, the inpouch tf-feline medium seems to be not entirely selective as p. hominis could be successfully cultivated and identified after 3 days following inoculation of inpouch tf-feline medium using cat feces (ceplecha et al. 2013 ). dna detection has become one of the most important methods for the diagnosis of infections with trichomonads in cattle and cats. the major advantage of dna detection by pcr is that it is independent of parasite viability and contaminating microbes that may inhibit trichomonad cultivation. correspondingly, sensitivity of pcr is often reported to be higher than cultivation and direct microscopical examination (recently reviewed by yao (2013)). however, pcr analysis has also a number of disadvantages since due to its sensitivity it is prone to carry-over and crosscontamination. in addition, samples may contain inhibitory components that may reduce the sensitivity of pcr or even disable amplification. each lab should validate the entire diagnostic process-including dna extraction and pcr amplification-prior to carry out pcr detection as laboratory-specific conditions, equipment, or consumables may have an impact on the outcome of the diagnostic process (hoorfar et al. 2004; conraths and schares 2006) . preputial material for dna extraction is generally collected by sheath scraping combined with aspiration or by sheath washing. material obtained by scraping may contain blood or fecal contaminations from outside the preputium, and material collected by preputial washing may contain urine. blood, fecal components, and urine may act as pcr inhibitors, and, therefore, it is necessary to minimize or avoid such contaminations. analytical sensitivities of pcr protocols are high and usually sufficient to detect the dna of a single parasite (table 14 .1). in field samples, however, analytical sensitivity might be much lower and has been reported to be around 100 organisms per sample (mukhufhi et al. 2003) . in an assessment of diagnostic sensitivity carried out with spiked cat feces, ten organisms per 200 mg of feces were ho et al. (1994) detected in 90% of nested pcr tests, and 100 organisms per 200 mg of feces were detected in 100% of nested pcr tests (gookin et al. 2002) . it has been shown that also in pcr diagnosis the likelihood to detect t. foetus decreases with time between sampling and analysis. in one study it was reported that diagnostic sensitivity in pcr detection declined from 90% when samples were stored for 6 h to 31% when they were stored for 5 days (mukhufhi et al. 2003) . it has been hypothesized that hydrolases secreted by trichomonads are responsible for this effect (thomford et al. 1996; sager et al. 2007 ). adding the dna-stabilizing agent guanidinium thiocyanate-guscn in a final concentration of 200 mmol/lor a commercial lysis buffer for sample collection known to preserve dna for several months at room temperature to the transport medium did not improve results (mukhufhi et al. 2003; mendoza-ibarra et al. 2012) . fecal samples from cats should be submitted within 24 h after sampling at room temperature or 4 °c. to prevent amplification of carry-over contaminants, protocols that incorporate dutp instead of dttp as a nucleotide and allow to apply the uracil-dna glycosylase (udg) system have been reported (longo et al. 1990; felleisen et al. 1998 ). contaminating amplicons carried over from previous pcrs can be removed by this system before a pcr amplification. possible disadvantages of the udg system are that it might become necessary to optimize the reaction mixture used for pcr-e.g., the mgcl 2 concentration-and a standard pcr buffer may not work . it is essential to monitor the presence of a potential inhibitor in each individual sample since preputial material, cervico-vaginal mucus, and fecal samples contain pcr-inhibiting components. there are different possibilities available. the tfr3/ tfr4 pcr protocol includes an artificial internal control dna carrying tfr3 and tfr4 sequences-based on pbluescript ks+ dna-and generates control amplicons, via pcr amplification and composite primers (table 14 .1) sager et al. 2007 ). internal controls of unrelated dnas have been integrated into a 5′ nuclease assay-real-time pcr assays with taqman probes. in this type of assay it is possible to incorporate an unrelated dna into the sample prior to dna purification, which is later amplified in a multiplex assay along with the parasite dna but detected by a probe carrying a fluorophore different than that of the parasite-specific probe. one commercially available t. foetus real-time pcr includes such a control system (vetmax™-gold trich detection kit, life technologies). this principle of inhibition control is becoming more and more popular, also for in-house assay. there are many ways, by which dna from preputial smegma, cervico-vaginal mucus, fecal samples, oropharyngeal swabs, culture material, and others types of samples can be extracted. often commercial kits but also in-house methods have been applied. also non-purified but heat-treated samples were used with success (mcmillen and lew 2006) . however, the use of unpurified dna is prone to inhibition and is not generally recommended. it has been shown that inhibiting components could be successfully removed from preputial smegma by 5% chelex ® -100 and 0.05% agar solution (chen and li 2001) . however, in another study, the use of chelex ® -100 caused significantly lower detection rates (mendoza-ibarra et al. 2012 ). the majority of published diagnostic pcrs for t. foetus are targeting rrnacoding genes (rdna) and their flanking regions (table 14 .1). these regions include the 18s rrna gene, the internal transcribed spacer (its)1 region, the 5.8s-rrna gene, the its2 region, and the 28s rrna gene. one of the first diagnostic pcrs established-the tfr3/tfr4 pcr-is widely used. the tfr3 primer targets the 3' end of the 18s rrna gene and the tfr4 primer the 5′ end of the 28s rrna gene . although this pcr assay is often referred to as being specific for t. foetus, also dna of t. mobilensis-an intestinal parasite of squirrel monkey-or t. suis is amplified (table 14 .1). the rrna gene sequences have been widely used for phylogenetic studies in parabasalia to which trichomonads belong. other genes coding for cysteine proteinases-cp1, cp2, and cp4-cp9-and cytosolic malate dehydrogenase 1 (mdh1) have been used to differentiate t. foetus isolates from cattle and cat or to characterize new strains of t. foetus (kleina et al. 2004; cepicka et al. 2005 cepicka et al. , 2006 gaspar da silva et al. 2007; kolisko et al. 2008; sun et al. 2012; slapeta et al. 2012; casteriano et al. 2016 ). in t. gallinae further genes were used to define lineages (recently reviewed in amin et al. (2014) ). because 18s rrna genes show limited differences between trichomonads, endpoint assays have been applied using primers capable to amplify dna of several trichomonad species simultaneously (felleisen 1997) . in these pcrs, species diagnosis was achieved in a second step, either by pcr-rflp, by determination of the precise size of amplification products, or by single-strand conformation polymorphism (sscp) (hayes et al. 2003; huby-chilton et al. 2009 ). the tfr3/tfr4 pcr protocol has been modified by using the tfr3/tfr4 primer pair for external amplification followed by an internal newly designed primer pair in a single-tube nested pcr (gookin et al. 2002) . the tfr3/tfr4 pcr protocol has been also modified into a sybr®-based real-time pcr assay (mueller et al. 2015) . a 5' nuclease assay-i.e., a real-time pcr applying a taqman probe-based on rrna gene sequences has been established to detect t. foetus, t. suis, and t. mobilensis (mcmillen and lew 2006) . in this assay a 57 bp region of the 5.8s rrna gene region is amplified (table 14 .1). as mentioned earlier in this section, a commercialized 5′' nuclease assay is available (vetmax™-gold trich detection kit) which has been used in epidemiological studies on t. foetus of cattle in southern africa (casteriano et al. 2016 ). serological and other antibody detection tests have been established. however, they are of no importance for the diagnosis of trichomonosis. these tests include: • agglutination and hemolytic tests. in t. foetus-infected cows, antibodies appear in the cervicovaginal mucus about 6 weeks after infection and persist for several months (pierce 1947) . a mucus agglutination test detected 32% (57 of 178) of cows in naturally infected herds, and no cows from clean herds tested positive (pierce 1949) . the mucus agglutination test was shown to be specific as no cross-reactions with campylobacter fetus or brucella abortus has been observed. however, the reliability of the test was strongly influenced by the type of mucus. the mucus agglutination test was regarded as herd test (pierce 1949 ). • a serum agglutination tests, similar to the mucus agglutination test, did not show results that correlated well with the t. foetus infection status of cows (kerr 1944 ). • a hemolytic assay has been established which showed in female cattle a diagnostic specificity of 96% and a diagnostic sensitivity of 94% . however, only 43% of chronically infected bulls tested positive when this test was applied . • indirect elisas to detect parasite-specific antibodies. an indirect elisa (ielisa) based on the tf1.17 surface antigen of t. foetus showed promising results when tested with cervico-vaginal mucus of heifers (ikeda et al. 1995) . tf1.17 surface antigen-specific igg1, iga, and igm antibodies in the smegma of bulls naturally infected with t. foetus-as determined and measured by elisawere observed concurrently with t. foetus-positive smegma cultures (rhyan et al. 1999) ; to the best of our knowledge, this approach was not further elaborated. in vaccination studies, igg1, igg2, iga, and ige responses were monitored in the preputial secretions or in sera of bulls by using a whole t. foetus cell antigen preparation for elisa (cobo et al. 2009 ). • an ielisa coated with whole t. foetus parasites and fixed with ethanol was used to determine an isotype-specific antibody response in the reproductive tract secretions and sera of t. foetus-infected heifers (skirrow and bondurant 1990a) . in cervical and vaginal secretions, parasite-specific iga and igg1 antibodies predominated 7-12 weeks after infection, while in serum, parasite-specific igg1 and igg2 antibodies were detected. interestingly, elevated antibody levels were observed after reinfection using this ielisa (skirrow and bondurant 1990a) . a similar ielisa with immobilized whole t. foetus parasites was used to monitor the t. foetus-antibody response in immunized heifers naturally challenged by being bred with a naturally infected bull (cobo et al. 2002) . the presented serological tests have not been used or validated for routine diagnostic purposes. an ielisa has been also used under experimental conditions to detect antibodies against t. gallinarum and t. gallinae in poultry (amin et al. 2011) . for this ielisa, the plates were coated with t. gallinarum parasites in carbonate buffer per well. analogous to the tuberculin test, a tricin test has been developed to identify t. foetus-infected cattle or herds (kerr 1944) . the antigen used for an intradermal tricin test has been prepared by fixation of cultured t. foetus parasites using trichloroacetic acid. skin reactions-i.e., an increase in skin thickness-were read 30-60 min after the application of the antigen. this test was regarded to represent a herd test (kerr 1944) . attempts to develop a sensitive and specific diagnostic antigen test for detecting t. foetus antigen in cervico-vaginal mucus have failed (yule et al. 1989b ). however, immunohistochemical techniques have been successfully applied to demonstrate trichomonads in histological sections (rhyan et al. 1995b; yaeger and gookin 2005) . for immunohistochemical detection, monoclonal antibodies developed to characterize t. foetus antigens revealed to be valuable tools (hodgson et al. 1990) . a protocol for immunohistochemical detection by using the monoclonal antibody mab 34.7c4.4 is provided in the oie terrestrial manual (www.oie.int/international-standard-setting/ terrestrial-manual, accessed, 22. febr. 2017). a tentative diagnosis of trichomonosis as a cause of reproductive failure in a herd is based on the clinical history, signs of early abortion, repeated returns to service, or irregular estrous cycles. confirmation of infection depends on the demonstration of the organism in placental fluid, stomach contents of the aborted fetus, uterine washings, pyometra discharge, vaginal mucus, or preputial smegma. in infected herds, the most reliable material for diagnosis is preputial scrapings (kittel et al. 1998; mukhufhi et al. 2003; parker et al. 1999; schönmann et al. 1994) . bulls are the main reservoir for the parasite. control programs focus on identifying and culling infected bulls and nonpregnant cows carrying the parasite. prevention of transmission of the disease through culling practices relies on the ability to identify infected animals accurately. advances in cell culture and polymerase chain reaction (pcr) have increased the ability to detect the disease in bulls. however, the collection of an adequate sample is of outmost importance, as this step affects sensitivity, specificity, and repeatability. the low repeatability observed with most sample collection techniques can cause false-negative results. the most efficient method of sampling vaginal and preputial secretions is insertion of an insemination/infusion pipette inside the vaginal fornix or preputial cavity and performing short strokes while concurrently aspirating secretions (cobo et al. 2007 ). vagina or preputial cavity can be washed with pbs to recover more organisms, although this usually dilutes the sample. alternatively, preputial secretions can be collected by scraping with a plastic or metal brush, with no significant differences in culture sensitivity compared to using a pipette (tedesco et al. 1979; parker et al. 1999) . routine herd diagnosis is optimally performed on bulls and not on females, because bulls remain permanently infected while in most female cattle infection is only transient. in addition, sampling of bulls reduces costs as preputial samples have to be taken only from a smaller number of bulls. diagnosis in the bull involves collecting, transporting, and culturing the sample in special growth media and tentatively identifying the organism by microscopic examination. finally a confirmation of detection using pcr has to be carried out to confirm a t. foetus infection (fig. 14.4) . if samples are processed by pcr, the use of pooled direct preputial samples is possible. however, this strategy required repeated sampling to optimize sensitivity (garcia guerra et al. 2013) . optimally, all bulls belonging to the same herd should be sampled. in those herds in which periodic sanitary control is carried out without previous history of venereal diseases and with good pregnancy rates, at least two consecutive samplings of all bulls should be performed, with a minimum of 1 or 2 weeks time between them (skirrow et al. 1985; kimsey et al. 1980) . consecutive testing is necessary due to the low diagnostic sensitivity of an individual test. if both samplings revealed negative results, the herd can be considered tritrichomonosis free. some recommendations have to be taken into account at the time of sampling: • it is generally recommended to allow a sexual rest of about at least 1-2 weeks before taking a preputial sample to allow a repopulation of microorganisms in the preputial cavity and increase diagnostic sensitivity. • it is important to have adequate facilities to perform preputial sampling particularly when sampling a large number of bulls to avoid accidents and injuries. • the bulls should remain in a pen, without access to water approximately 12 h prior to sampling. this will prevent urination during sampling. • the external preputial area must be cleaned with disposable paper towels without soap or disinfectants, and-if necessary-preputial hairs should be trimmed to prevent contamination. the presence of t. foetus seems to be confined to the preputial cavity, and t. foetus is localized in the preputial secretions and does not invade the epithelium of the penis or prepuce . t. foetus could not be cultured from the epididymis, ampulla, seminal vesicle, pelvic urethra, or testis. macroscopic and microscopic examination of the genital tracts of infected bulls did not reveal any lesions populated by t. foetus . a number of techniques for collecting preputial samples from bulls have been described (inta, 2014, técnicas de muestreo para el diagnóstico de enfermedades venéreas en bovinos, https://www.youtube.com/watch?v=g-1strwahka, accessed 21. febr. 2017; navajo technical college, 2012, veterinary technicians perform trich testing, https://www.youtube.com/watch?v=lp8fpdvdoce, accessed 21. febr. 2017). in all these protocols, it is important to avoid contaminations, as this may introduce intestinal protozoa or pcr-inhibiting contaminants. samples can be collected from bulls by scraping the preputial and penile mucosa with an artificial insemination pipette or scraper, by preputial lavage, or by washing the artificial vagina after semen collection (cobo et al. 2007; tedesco et al. 1979; parker et al. 1999) . the latter technique is not recommended as its sensitivity may be lower (ostrowsky et al. 1974; parker et al. 1999) . the collection of the preputial smegma can be carried out by using different devices: • a cassou insemination pipette (cassou straws, imv technologies, l'aigle, france) covered by a plastic sheath. inside the preputial cavity, the pipette is pulled forward through the plastic sheath to expose the tip and moved back and forward in short strokes adjacent to the glans penis, especially near the fornix, while aspirating and massaging the glands penis to release greater amounts of smegma into the pipette. a new plastic sheath is used for each bull (cobo et al. 2007 ). • a sterile, dry, plastic insemination/infusion pipette of 43 cm length with a 10-15 ml syringe attached to one end is placed into the preputial fornix. the pipette is scraped vigorously across the preputial epithelium without aspiration, and then negative pressure is applied with the syringe to collect preputial smegma. the negative pressure is released before removing the pipette from the sheath, to avoid aspiration of urine or other contaminants. after removing the pipette from the sheath, the sample is placed immediately into a transport or culture medium. a new syringe and pipette are used for each bull (rodning et al. 2008 ). • a plastic or metal scraping instrument of approximately 70 cm in length, having at the anterior end a grooved surface of approximately 10 cm in length by 0.8 cm in diameter, which is used to perform scraping of the preputial mucosa. the scraper is introduced into the preputial mucosa by performing 20-30 movements back and forward. it is then carefully withdrawn from the preputial cavity, avoiding contamination with the external part of the foreskin (terzolo et al. 1992) . the aspiration method for the collection of preputial secretions for cultural examination continues to be used although a specially designed scraping instrument was reported to possess advantages clark et al. 1971; stuka and katai 1969) . • a gauze sponge, which to our knowledge is rarely used. in this procedure, the penis is extended by electrostimulation with a rectal probe. once extended, a 16-ply gauze sponge is used to wipe around the glans and down the penile shaft and exposed preputial mucosa two to three times. recently, it has been demonstrated that sponge sampling of t. foetus from bulls is a valid method (dewell et al. 2016 ). the method facilitates easier collection once the penis is extended and is potentially safer for the veterinarian and the bull. additionally, the gauze sponge method might be slightly more sensitive than the pipette method (dewell et al. 2016 ). after collection the material has to be inoculated in corresponding transport or culture media and should arrive at the laboratory within 24-36 h; during transportation, the sample should be protected from exposure to daylight and extremes of temperature. initially, the uterus was regarded as the definitive and persistent site of infection while the vagina was considered to be a relatively unreliable source of t. foetus for diagnosis (bartlett 1947 ). subsequently, however, the persistence of t. foetus in the vagina and/or cervix for periods up to 95 days post-infection was shown, and it was established that samples collected from vagina and the uterine cervix allow a reliable and accurate diagnosis (parsonson et al. 1976 ). more recent studies of naturally infected cows reconfirmed that the cervix belongs to the preferred site of parasite location (skirrow and bondurant 1990b) . the numbers of parasites present in the cervical-vaginal mucus fluctuate during the estrus cycle, and the largest numbers are seen a few days before estrus. the time a cow remains infected was significantly longer for cows experiencing their first, mean 20.3 weeks, than for those experiencing either their second, mean 9.8 weeks, or their third period of infection, mean 11 weeks. however, the rate of isolation of t. foetus from samples of vaginal mucus collected from cows remained similar-mean 83.5%-irrespective of the period of infection or whether the cows showed normal fertility, infertility, or abortion (clark et al. 1986 ). the efficiency of diagnosis in cows increased with temporal proximity between the initial infection and the time of sampling (clark et al. 1986 ). it is important to note that the detection of positive females is of value for the initial detection of the infection in a herd. however, it is not useful for a subsequent disease control because cows usually clear their infection and generally become immune, at least for the actual breeding season (bondurant 1997; fitzgerald 1986 ). sampling should be performed when females with conception failures are observed or at the time of rectal palpation in nonpregnant females. culture sensitivity is lower in cows than in bulls. however, it is important to point out that the optimal period for sampling is near the end of the service period (skirrow and bondurant 1990b; terzolo et al. 1992) . uterine and vaginal secretions can be collected in cows that have aborted, in those that have not been pregnant, or heifers. to perform the extraction of cervico-vaginal mucus, a sterile, dry, plastic 43 cm insemination pipette with a 10 ml syringe attached to one end or a cassou insemination pipette (44 cm long × 0.64 cm outer diameter × 0.32 cm inner diameter; cassou straws, imv technologies, l'aigle, france) can be used. opening the lips of vulva without having to fix the cervix rectally, the pipette is inserted in a dorsal-cranial direction into the vagina. after the pipette has been inserted, slight anteroposterior and circular movements are executed, performing aspiration simultaneously. the vacuum generated is usually sufficient to extract the cervical-vaginal mucus, which will vary in quantity and consistency depending on the moment of the estrous cycle in which it is extracted. in a low percentage of animals, the volume of mucus extracted may be scarce. in this case it is possible to introduce 5 ml of phosphate-buffered saline solution by performing a wash with subsequent extraction. the use of a "screwhead scraper rod" for collecting of samples from the cervico-vaginal mucosa proved to be a practical method and calls for further comparative evaluation with other standard methods in use (abbitt and ball 1978) . apart from other extraction techniques, also the bartlett glass pipette procedure, which is rarely used because it is complicated and the equipment often inaccessible, has been described (hammond and bartlett 1943) . when abortion occurs, t. fetus can be isolated from placental fluids or cotyledons. however, the high degree of contamination of this material limits its use. isolations can be made from samples taken from the fetal mouth. swabbing the mucosa of the tongue and roof of the mouth has been recommended (case and keefer 1938) . nevertheless, the place where t. foetus is most consistently isolated is the abomasal fluid. the sample can be taken with sterile syringe and needle. once the abomasum content has been extracted, it can be sent to the laboratory in the same syringe or can be inoculated in transport or cultured medium. the diagnosis of feline trichomonosis has been reviewed comprehensively (tolbert and gookin 2009; manning 2010; yao and köster 2015) . t. foetus infection is suspected in cats with recent-less than 6 months lasting-clinical signs of chronic large bowel diarrhea, in young, purebred cats, from densely housed origin. routine coprological methods, like flotation-sedimentation or sodium acetate-acetic acidformalin concentration (saf), destroy or fix trichomonads, respectively, and fixation causes the loss of their characteristic movement which makes them hard to be recognized. currently, the preferred diagnostic methods for feline trichomonosis include visualization of the organism in direct smears or culture or t. foetus dna detection by pcr (gookin et al. 2001 (gookin et al. , 2002 levy et al. 2003; foster et al. 2004 ). histopathological examinations of colon, cecum, and ileum samples are not routinely used but can be helpful to diagnose a feline tritrichomonosis. it is important to mention that parasite shedding in feces is erratic throughout the course of infection being occasionally so low that it cannot be detected by diagnostic techniques. consequently, it is advisable to resample cats showing clinical signs that have been tested negative or to increase diagnostic sensitivity by using more than one test method-e.g., culture and pcr (gookin et al. 2002) . cats should not receive any antibiotics within several days prior to or at the time of testing. samples consist of fresh voided feces taken directly from the litter box, rectal swabs, and manual collection with the aid of fecal loops or by a colon flush technique (revised in manning (2010) , yao and köster (2015) , tolbert and gookin (2009) a diagnostic approach based on direct fecal smears, which may reveal motile trophozoites, can be employed during examination at pet clinics. samples are suspended in saline and examined immediately under a cover slip at 200 to 400-fold magnification using a light or, preferably, a phase-contrast microscope (fig. 14.5 ). although direct smears represent a cheap, quick, and readily available technique, the sensitivity of microscopic examination of a direct fecal smear is low. the diagnostic sensitivity of a direct smear using samples from naturally infected cats has been shown to be only 14% in one study, but sensitivity can be increased by analyzing multiple fecal smears . other challenge associated with this diagnostic procedure is the skill of the practitioner in identifying motile trichomonads. t. foetus cannot precisely be distinguished by microscopical examination from p. hominis and is often misdiagnosed as giardia spp. (tolbert and gookin 2009; manning 2010; yao and köster 2015) . a video clip demonstrating the classic jerky motility has been provided by the north carolina state university, college of veterinary medicine website (https://www.youtube.com/watch?v=af06jlbcf8e; accessed 22. febr. 2017). the presence of giardia spp. can be confirmed by a fecal enzyme-linked immunosorbent assay for giardia-specific antigen. differentiation of giardia spp. and t. foetus is crucial to avoid a useless initiation of tritrichomonosis therapy using potentially neurotoxic ronidazole. in vitro culture of t. foetus can be started by incubating the sample feces in a suitable growth medium (gookin et al. 2001 . diagnostic kits-for example, the inpouch tf-feline test kit-can be an attractive option for the clinical practice due to the commercial availability, a shelf life of a year, and the simplicity of use. sample submission requirement for in vitro culture diagnostic includes the collection of fresh feces-roughly the size of a lima bean-and the use of transportgrowth media, inpouch tf or modified diamond's media. it is recommended to dilute the fecal samples in pbs or physiological saline-to about 1 to 20 v/v-to create a homogenous mixture with a semiliquid consistency to be used as the inoculum for the culture medium, reducing the bacteria burden in the sample (arranz-solis et al. 2016). furthermore, it is crucial for a reliable diagnosis of feline trichomonosis to keep the time delay from sample collection to processing as short as possible, given the environmental fragility of t. foetus trophozoites . in order to maximize the diagnostic sensitivity, cat feces should be inoculated into the culture within a 6-h period after voiding and to be submitted at room temperature-23 to 25 °c-to a specialized diagnostic laboratory within 24 h (hale et al. 2009 ). false-negative findings can be due to refrigeration or delayed processing of feces. after culture initiation, samples are then incubated at room temperature or at 37 °c; incubation at room temperature made more robust and long-lived cultures . cultures are examined microscopically periodically up to 7 days-normally every 24-48 h-on a wet mount slide prepared directly from the culture or through the plastic wall of the inpouch tf kit. most of the microscopically positive culture samples are detected 72 h post-incubation. it has to be mentioned that the diagnosis by culture is more difficult for cats than for bovines due to the nature of the feces sample. as feces are directly cultured in enriched media, bacterial and/or fungal contamination is more likely to arise. bacterial contamination severely impairs the culture of t. foetus and is able to reduce the sensitivity of the culture method. incubation at 37 °c shows a quicker positive result but also more bacterial overgrowth potentially inhibiting the growth of t. foetus. if the commercial inpouch tf-feline test kit is used, an accumulation of gas within the pouches due to an overgrowth of fecal flora is a commonly observed problem. moreover, t. foetus and related parasites are hard to distinguish with light microscopy because of the similar morphology. culture-positive samples require confirmation by pcr because also other trichomonads like p. hominis are able to grow (ceplecha et al. 2013) . molecular diagnosis is becoming more widely available than culturing the organism. under optimized conditions, a direct diagnosis by pcr-i.e., without a prior cell culture test-offers a highly specific and a sensitive diagnostic alternative also able to detect nonviable parasites. moreover, pcr is the method of choice to confirm positive results by culture. it is important to monitor a potential pcr inhibition in each individual sample. the choice of an appropriate dna extraction technique greatly influences the reliability and sensitivity of pcr (stauffer et al. 2008; hale et al. 2009 ). several methods have been successful employed for feline trichomonosis diagnosis, such as a modified procedure using the commercial qiaamp dna stool minikit (qiagen, hilden, germany), boom's method, or zr fecal dna kit zr (zymo research, orange, ca, usa), which was able to detect 10 t. foetus organisms per 100 mg feces in 100% of pcr reactions (stauffer et al. 2008 ). diagnostic t. foetus pcr using primers tfr3/tfr4 and a single-tube nested pcr using primers tfits-f/tfits-r in combination with primers tfr3/tfr4 are widely used for feline trichomonosis diagnosis (table 14 .1) ). pcr amplification of dna extracted from feces samples appears to be more sensitive than the inpouch tf culture, and a high level of agreement has been described between the culture and pcr detection (gookin et al. 2002; hosein et al. 2013; arranz-solis et al. 2016) . finally, colonic histopathology can be used as a diagnostic tool. colon, cecum, and ileum samples are collected during necropsy, surgery, or endoscopy. on histologic examination, large numbers of teardrop-to crescent-shaped trichomonads can be found associated with the colonic mucosal surface and less commonly within colonic crypt lumens. histologic features include mild to moderate lymphoplasmacytic colitis with crypt micro-abscesses, increased mitotic activity, loss of goblet cells, and attenuation of superficial colonic mucosa (yaeger and gookin 2005) . the probability of diagnosing a t. foetus infection based on histopathology increases with the number of submitted samples-a minimum of six colon samples is required to have a larger than or equal to 95% confidence of detecting t. foetus in at least one sample (yaeger and gookin 2005) . recently, a fluorescence in situ hybridization assay (fish) and a chromogenic in situ hybridization (cish) technique have been described to allow the localization and identification of t. foetus in formalin-fixed and paraffin-embedded samples, respectively (gookin et al. 2010b) . the trichomonad t. gallinae of birds is of veterinary importance, while tetratrichomonas gallinarum is a commensal that can become important under certain circumstances-e.g., concurrent infections with other pathogens (recently reviewed in amin et al. (2014) ). diagnostic techniques described for t. foetus can be also applied to the diagnosis of bird trichomonosis, including direct microscopy, cultivation, and pcr detection. for direct microscopy, sample material can be collected by swabbing the oral cavity, t. gallinae, or cloacae, t. gallinarum, and is mounted either diluted or undiluted on glass slides. glass slides need to be examined immediately to observe motile protozoa (forrester and foster 2008; amin et al. 2014) . as mentioned, staining of trichomonads is possible and may facilitate detection (amin et al. 2011 ). similar to bovine and feline trichomonosis, the sensitivity of direct microscopy is low, and situations in which the parasite load is low may likely result in falsenegative results. the use of samples to initiate cultures allows the amplification of the number of parasites making this procedure more sensitive than the direct microscopic examination of samples. correspondingly, in a study of pigeons, only about half of the samples positive in culture revealed a positive result in wet mount microscopy (bunbury et al. 2005) . in case of t. gallinae-infected birds, material collected via swabbing from the oral cavity of the animal is added to a inpouch tf culture device or other commercial or noncommercial culture media forrester and foster 2008; rogers et al. 2016; girard et al. 2014; krone et al. 2005) . for cultivation, specimens are usually incubated at 37 °c for several days and examined microscopically every 24 h. although birds have a higher body temperature, 37 °c seems to be the optimal temperature for the cultivation of t. gallinarum (de carli and tasca 2002; de carli et al. 1996; amin et al. 2010) . for pcr, a number of primer pairs have been reported that target the its1-5.8srdna-its2 genomic region but which are not species-specific and able to detect virtually all trichomonad species. most commonly used are primers tfr1 and tfr2 (felleisen 1997) . a nested pcr, targeting the 18s rrna gene, its1, and 5.8s rrna gene, was established but was not species-specific, amplifying also t. gallinae (frey et al. 2009; grahn et al. 2005) . another nested pcr has been developed for the amplification of the its1-5.8s rdna-its2 genomic region of trichomonads used to detect t. gallinae-specific dna from esophageal lesions of finches sampled during an epidemic of finch mortality (robinson et al. 2010; cepicka et al. 2005) . the analytic specificity of this pcr has not been reported. amplification primers specifically designed for the identification of t. gallinarum yielded also a pcr product of specific dna of t. gallinae of identical size (grabensteiner and hess 2006 ). an indirect elisa has only been used under experimental conditions to detect antibodies against t. gallinarum and t. gallinae in poultry (amin et al. 2011) . because birds are often latent carriers of trichomonads, there is hope that serological analyses in bird populations might be able to identify carrier birds and may help to better understand persistence and spread of these parasites (amin et al. 2014 ). bovine tritrichomonosis is a major problem worldwide, affecting a large proportion of herds in north and south america, in parts of europe, africa, asia, and australia (de oliveira et al. 2015; yao 2013; perez et al. 2006; mendoza-ibarra et al. 2012 , 2013 madoroba et al. 2011; yang et al. 2012; guven et al. 2013; mccool et al. 1988 ). bovine tritrichomonosis is considered endemic in herds managed under extensive conditions and using natural service for breeding (bondurant 2005) . the economic impact of tritrichomonosis infection has to be regarded severe in particular regions of the world. the calf crop in affected beef and dairy herds can be reduced up to 50% in beef operations (rae 1989) . economic losses are variable and depend on the percentage of bulls infected and the susceptibility of the cows in the herd. further losses, in addition to calf crop losses, include an extended breeding season due to an increased number of repeat breeders. due to later calving, the growing periods for calves might be shortened, and there are batches of calves of different ages with a wide variation in weaning weights. summarizing these losses, it was estimated that tritrichomonosis in a herd caused a 35% decrease in economic return per cow in an infected herd (rae 1989) . economic losses in a case of bovine tritrichomonosis in a large californian dairy herd were calculated at 665 us$ per infected cow (goodger and skirrow 1986) . the number of tritrichomonosis reports has drastically reduced in regions or production systems in which artificial insemination is the predominant mode of breeding and in which comingling of herds is avoided-for example, in the european dairy industry. a recent survey conducted in switzerland involving 1362 preputial samples from bulls and 60 abomasal fluid samples of aborted fetuses from beef and dairy herds revealed no t. foetus-positive finding (bernasconi et al. 2014 ). however, also in areas assumed to be largely free of bovine tritrichomonosis, a reestablishment of the infection in herds, especially in beef herds farmed under extensive, pastoral systems, is possible as shown by findings from spain (mendoza-ibarra et al. 2012; mendoza-ibarra et al. 2013) . knowledge on risk factors is important for the implementation of effective measures to control tritrichomonosis. results from risk factor studies were used to model effects of vaccination against tritrichomonosis in beef herds. a number of potential herdlevel risk factors were assessed, including "no. of cows," "no. of young bulls," "trichomonad testing yes/no," "no. of trichomonad tests," "shared grazing," "previous diagnosis of trichomonosis," or "duration of breeding season" (villarroel et al. 2004) . t. foetus isolated from cattle and cats and t. suis from pigs are genetically very similar or in case of t. foetus from cattle and t. suis even indistinguishable. nevertheless, there is no evidence that there are links between life cycles of t. foetus in cattle and cats or between life cycles of feline or bovine t. foetus and porcine t. suis. most likely these parasites have evolved separately, and despite their genetic similarity, t. foetus of bovine and feline origin and porcine t. suis show biological traits which differ considerably. as stated in sect. 14.1.2, tritrichomonosis is an almost exclusively venereal transmitted disease in cattle and affects predominantly adult animals (sager et al. 2007; ondrak 2016) . t. foetus is transmitted during coitus, mainly from an infected bull to an uninfected dam or vice versa (ondrak 2016) . single mating with an infected bull may result in a 95% infection rate among susceptible heifers (parsonson et al. 1976 ), but in general a transmission rate of 30-70% is assumed (bondurant 2005) . a mechanical transmission either by uninfected bulls or by contaminated equipment or cryopreserved semen seems to be possible, but the relative importance of these routes of transmission is minor (ondrak 2016; clark et al. 1977; murname 1959; goodger and skirrow 1986; blackshaw and beattie 1955; clark et al. 1971; skirrow and bondurant 1988) . the following risk factors for tritrichomonosis in individual animals have been identified: • carrier state and age as a risk factor of infection: infection in bulls is reported to persist for more than 3 years and may persist for life (rhyan et al. 1999; campero et al. 1990; flower et al. 1983; bondurant 2005) . several studies established that the likelihood of bulls being infected seems to increase with age (skirrow et al. 1985; mccool et al. 1988; bondurant et al. 1990; rae et al. 1999 rae et al. , 2004 mendoza-ibarra et al. 2012) (table 14 .1). in one experiment using bulls-3 to 6 years of age-all bulls more than 4 years old became infected after three to six services while only one of two young bulls, 2-3 years of age, became infected after nine services . bulls less than 4 years of age are rarely carriers of t. foetus (bondurant 1985; perez et al. 1992; ondrak 2016; kimsey et al. 1980; skirrow et al. 1985; bondurant et al. 1990 ). the reason for this finding is not completely understood. old bulls may have had a higher number of sexual contacts than young bulls. an often mentioned other possible reason is the more pronounced invaginations in the penile and preputial epithelium of older bulls-i.e., the crypts of these epithelia are becoming deeper and increase in number with the age of the bull (bondurant 1985; skirrow et al. 1985; mccool et al. 1988; perez et al. 1992; ondrak 2016) . however, the hypothesis that anatomical changes are the cause for older bulls found to be infected more often was recently questioned because no age-related statistically significant differences were observed in the surface architecture of the penile and preputial epithelium of bulls (ondrak 2016; strickland et al. 2014 ). in female cattle, age seems not to be associated with the likelihood of infection; however, there is evidence that repeated exposure induces resistance to infection (simmons and laws 1957; clark et al. 1986; skirrow and bondurant 1990b) . while mature bulls seem to remain infected for life, most cows are able to clear the infection after a few months-usually after 1 to 3 monthsrarely longer (parsonson et al. , 1976 skirrow and bondurant 1990b; bondurant 2005) . several studies of infected cows indicate that the os cervix is the preferred site of multiplication and persistence (skirrow and bondurant 1990b) . initial multiplication of t. foetus after infection seems to be followed by a decline of parasite numbers until next estrus . the numbers of parasites present in the cervicovaginal mucus seem to fluctuate during the estrus cycle, and the largest numbers are seen a few days before estrus . in two infected heifers that have been followed over time after experimental infection, t. foetus was not always observed in vaginal mucus by microscopic examinations or culture isolation suggesting fluctuations in vaginal parasite concentration; however, only in one heifer, there was a coincidence between the detection of t. foetus and the time of estrus (simmons and laws 1957) . not all cows are able to clear infection. carrier dams have been reported-e.g., two chronically infected dams were observed in one australian study 16 and 22 months after initial infection (alexander 1953) . in a californian dairy herd, infected cows were found positive 9 weeks after pregnancy or 63 days after parturition (skirrow 1987; goodger and skirrow 1986) . in a more recent study from argentina, several heifers remained infected up to 300 days after breeding which underlines the importance of these carrier state heifers for persistence of infection in affected herds (mancebo et al. 1995) . • herd management practices: the risk of bulls of being tested t. foetus positive increased when the number of bulls used per unit was higher than 10 or the bull-to-cow ratio per unit was lower than 1 to 25. the higher number of bulls and lower bull-to-cow ratios are typical management practices in large herds to increase conception rates (rae et al. 2004) . it was hypothesized that by these practices the number of potential sexual contacts per bull and the probability for an individual bull to become positive are increased (rae et al. 2004 ). • breed disposition: the possibility of a breed predisposition was discussed based on study results suggesting a higher prevalence of infection in particular breeds (bondurant et al. 1990; perez et al. 1992; rae et al. 1999 rae et al. , 2004 skirrow et al. 1985; abbitt and meyerholz 1979) . a number of epidemiological studies addressed this question ( et al. 1990 ). another study in costa rica also found a strong association between the risk of positive findings and the bos taurus taurus breed-as compared to bos taurus indicus pure or hybrid breeds (perez et al. 1992) ; an additional study also showed an increased risk in angus, charolais, hereford, or simmental breeds relative to b. taurus indicus (rae et al. 2004 ) (table 14. 2). however, findings suggesting breed disposition should be interpreted with care; studies might have been biased by uneven study designs or by not paying enough attention to the differences in the way herds of particular breeds were operated (ondrak 2016) . it was hypothesized that prevalence differences could be due to the increased number of matings accomplished by b. taurus taurus bulls as compared to b. taurus indicus bulls in the same period of time, thus increasing the risk of exposure to infection (perez et al. 1992) . however, there are also some studies that did not observe differences in prevalence of infection between b. taurus taurus and b. taurus indicus (dennett et al. 1974 ). nevertheless, a breed disposition and genetically based predisposition should not be completely ruled out. further studies are needed to elucidate the reason why some of the infected dams are becoming carrier of t. foetus-being still infected after pregnancy-while others immediately eliminate infection during the first 2 to 3 months after loss of the conceptus (alexander 1953; goodger and skirrow 1986; skirrow 1987; mancebo et al. 1995) . it is possible that like in other diseases there are genetic determinants influencing a predisposition to acquire infection and to develop immunity against the pathogen. • other individual-level risk factors: one study observed that the probability of positive findings is lower in bulls in service-i.e., in sexually active bulls (perez et al. 1992) (table 14. 2). this is in accord with previous findings, which suggested that a depletion of the preputial t. foetus population might occur because of intense sexual activity. it also supports recommendations of a sexual rest of at least 1 to 2 weeks before sampling bulls in order to improve the likelihood of accurately identifying t. foetus-positive bulls (clark et al. 1983a; bondurant 1985; yule et al. 1989a; ondrak 2016) . variations in sexual activity between different seasons may also explain fluctuating differences in the proportion of positive findings in bulls during a year (molina et al. 2013; szonyi et al. 2012 ). in addition, there are a number of herd-level risk factor studies-case-control and cross-sectional studies-that have been carried out to elucidate in more detail management practices and other factors increasing the risk of herds to acquire tritrichomonosis (table 14. 2) (mardones et al. 2008; molina et al. 2013; mccool et al. 1988; perez et al. 1992; mendoza-ibarra et al. 2012 , 2013 rae et al. 2004; gay et al. 1996; jin et al. 2014; bondurant et al. 1990; szonyi et al. 2012 ). many of the identified explanatory variables are related to predictors or risk factors in favor to increase the likelihood of venereal transmission of t. foetus in herds and between herds: • transmission between herds: risk factors were identified which characterized the likelihood to acquire tritrichomonosis from other herds. "allotments neighboring a positive herd(s)"; "allotment type, open/public vs. private"; and "mingling with neighboring herd(s)" were recently identified as risk factors in t. foetus-positive beef herds in wyoming, usa (jin et al. 2014 ). these findings confirm previous observations in beef herds from idaho, which identified "commingling on blm-bureau of lands management-allotment; fs, us forest service, allotment; or on any public land allotment" as an important predictor of positive herds (gay et al. 1996) . also in argentina "shared livestock with others" was identified as a significant risk factor for herds testing positive, that is, having positive bulls (mardones et al. 2008) . in summary, these findings suggest that any type of mingling herds and also fence-line contact with other herds represent important risk factors and should be avoided by farmers (jin et al. 2014 ). • transmission within herds: other predictors characterized the likelihood of transmission within a herd. obviously, herd size plays a role. a study on beef herds in idaho, usa, identified "total cattle grazed on fs-us forest serviceallotment more than 844" as a risk factor, and a study from florida, usa, observed that herds with more than or equal to 500 cows had a higher risk of being positive (gay et al. 1996; rae et al. 2004) . the latter study also identified particular herd management practices increasing the individual risk of bulls to test positive; therefore, most likely, herd size is a confounder explained by man-agement practices in large herds in favor for t. foetus transmission, like "no. of bulls used per unit larger than or equal to 10" or "bull-to-cow ratio per unit smaller than 1 to 25" as discussed earlier (rae et al. 2004 ). the management practice "rotation of bulls within a herd" has been identified in an argentinian study to most likely favor to perpetuate transmission of infection within a herd (mardones et al. 2008) . the same may apply to an observation that among "rearing beef herds"-i.e., herds that rear cattle until the weight of 150-250 kg-the prevalence of positive herds was higher as compared to "full-cycle herds," herds with breeding, rearing, and fattening. this observation was explained by hypothesizing a higher proportion of reproductively active animals resulting in an increased spreading of disease in "rearing beef herds" compared to "full-cycle herds" (mardones et al. 2008) . the risk factors related to a potential perpetuation/acceleration of transmission inside herds cannot be easily used to give recommendations with respect to better management practices, because these risk factors are either unspecific or difficult to change. herd-level predictors, possibly related to effects of tritrichomonosis include infertility, early fetal death, and rarely, abortion. in epidemiological studies "pregnancy rate in cows higher than or equal to 90%" and reporting "abortion" were found associated with t. foetus-positive beef herds in argentina (mardones et al. 2008 ). an "increased number in repeat breeder cows" was identified as a predictor for positive herds in extensively managed beef cattle in spain (mendoza-ibarra et al. 2012) . additionally, in a few studies, spatial clustering of t. foetus-positive herds was observed, and in one of these studies, it was also observed that high-risk clusters for t. foetus correlated to high-risk clusters for bovine genital campylobacteriosis (molina et al. 2013; szonyi et al. 2012) . the observed spatial clustering remained unexplained in these studies. although the typical clinical sign in natural infection is chronic or intermittent diarrhea, for many cats, no diarrhea was reported in 6 months preceding diagnosis (xenoulis et al. 2013; kuehner et al. 2011) . chronic t. foetus-associated diarrhea in most cats is likely to resolve spontaneously within 2 years of onset, and chronic infection with t. foetus-without clinical signs-after resolution of diarrhea appears to be common (foster et al. 2004 ). in experimentally infected kittens, t. foetus infections was long lasting and that, in later phases of the infection, the presence of t. foetus in feces was not always associated with clinical signs-such as abnormal consistency of feces (gookin et al. 2001) . thus, in addition to disease-infected cats, cats with asymptomatic infection are able to transmit infection to other cats. as mentioned in sect. 14.1.1, feline t. foetus tolerates a broader ph range than t. foetus from cattle (morin-adeline et al. 2015a). like for t. foetus from cattle, no encysted stages are known for t. foetus from cats. however, the formation of pseudocysts, which may support survival in the environment, has been reported for t. foetus (pereira-neves and benchimol 2009 ). in addition, it has been shown that feline t. foetus is able to survive outside of its host for at least 30 min on dry cat food and 180 min in drinking water or urine (rosypal et al. 2012) . other studies showed that t. foetus can survive in cat feces for several hours or even days or up to at least 5 days in wet cat food (hale et al. 2009; van der saag et al. 2011) . even the survival of a passage through the alimentary tract of slugs (limax maximus, limacus flavus) was demonstrated, suggesting that slugs could transmit t. foetus over short distance (van der saag et al. 2011) . the predominant mode of infection is the fecal-oral route, and most likely a close contact of cats favors the spread of transmission. several epidemiological studies are reported, providing data on putative risk factors for t. foetus infection in individual cats or catteries, cat shelters, and breeding centers (table 14 .3). most of the studies were small-scale studies, including only low numbers of individual cats and catteries, shelters, or breeding centers. some of the studies were restricted to cats from shows or pedigree cats. some studies were restricted to diarrheic cats or cats with a history of chronic diarrhea. therefore, studies listed in table 14 .3 were stratified into (1) studies including only healthy or healthy and diseased cats and (2) studies including almost only diseased cats-i.e., cats with diarrhea or a history of diarrhea. in addition to studies listed in table 14 .3, a large number of case reports and small-scale studies are available-recently reviewed by yao and köster (2015) -which are not mentioned in the following because these studies provided no statistical evidence on putative risk factors. as indicated in table 14 .3, some studies were included after performing own statistical analyses based on data extracted from the reports. individual risk factors for tritrichomonosis in cats include: • diarrhea, abnormal fecal consistency, and history of diarrhea. in a number of studies including only healthy or healthy and diseased cats, it was observed that positivity for t. foetus was associated with a "previous history of diarrhea," "history of diarrhea in the past 6 months," "presence of chronic diarrhea," and "abnormal fecal consistency" (table 14. 3) (kuehner et al. 2011; tysnes et al. 2011; doi et al. 2012) . these findings confirm that t. foetus is an important cause of diarrhea in cats. the likelihood of a cat to test t. foetus positive was also increasing if there was a "history of another cat in the house with diarrhea in the past 6 months" or a "history of diarrhea in cattery in the past 6 months," which suggested that individual positive cats were only part of a larger problem in a cattery, cat shelter, or breeding center (kuehner et al. 2011; hosein et al. 2013 ). • age. with a few exceptions, it is now accepted that t. foetus associated with large bowel diarrhea is mainly a disease of young cats. several studies revealed that cats about 1 year old or younger are more often t. foetus positive than elder cats (table 14. 3). these observations were made in studies including only healthy or healthy and diseased cats as well as in studies including almost only diseased cats (profizi et al. 2013; kuehner et al. 2011; queen et al. 2012 ; galián paris et al. 2014 ). these epidemiological findings confirm the results of follow-up studies of cats with t. foetus infections, which clearly showed that clinical signs spontaneously resolve within 2 years of onset and that it is difficult to detect t. foetus in cats after several weeks of infection (gookin et al. 2001; foster et al. 2004 ). meta-analysis on available data confirmed this view (yao and köster 2015) . the reasons why especially young cats are affected by t. foetus diarrhea are not sufficiently understood. possible explanations are a more intense contact of kittens to their mothers or to larger numbers of other cats in breeding centers, private households, or cat shelters, which may favor transmission of t. foetus and may also influence the dose by which kittens become infected. immunity against feline t. foetus is poorly understood; recently the possibility that parasite-specific secretory iga mediates immunity has been discussed (yao and köster 2015) . histological examinations in infected cats revealed influx of lymphocytes, plasma cells, and neutrophils into the subepithelial lamina propria (yaeger and gookin 2005) . the inflammatory response might not only be involved in pathogenesis but may also be involved in the control of infection (tolbert and gookin 2016 ). • breed. a number of studies identified an increased risk of infection in purebred, or pedigree cats, or particular breeds (hosein et al. 2013; stockdale et al. 2009; steiner et al. 2007; gunn-moore et al. 2007; paris et al. 2014 ). these observations remain largely unexplained. most likely the conditions under which purebred or pedigree cats are reared are responsible for the increased risk. purebred cats from breeding centers represent densely housed populations, and due to the frequent, close, and direct contact, the risk of infection might be higher than in mixed breed cats reared in less dense populations (yao and köster 2015; arranz-solis et al. 2016 ). • other risk factors. one other risk factor identified was "attendance to cat shows," which might be a confounding predictor; the predictor could be related to the increased risk of purebred or pedigree cats (hosein et al. 2013) . there is only a single study report on food-related predictors. in a canadian study, "fed a raw food diet" has been identified as a risk factor (hosein et al. 2013 ). • other concurrent infections and impaired immune system. in experimental t. foetus infection with cats which shed naturally cryptosporidium parvum oocysts and cryptosporidium non-infected cats those with c. parvum infection had an earlier onset, more severe diarrhea, and increased number of trichomonads on direct fecal examination, as compared to non-infected cats (gookin et al. 2001 ). in epidemiological studies none of the concurrent infections examined-giardia sp., cryptosporidium sp., coccidia, clostridium perfringens, feline infectious peritonitis, and coronavirus-revealed a statistical association either to infection or disease on the individual animal level (table 14. 3). nevertheless, it has been discussed whether t. foetus alone can cause clinical signs without an impaired or immature immune system, concurrent infection, or other factors resulting in alterations in the colonic microflora (manning 2010) . in catteries, cat shelters, or breeding centers, t. foetus may cause outbreaks of persistent large bowel disease (holliday et al. 2009 ). there is only a single study on risk factors associated with occurrence of trichomonosis in catteries (table 14. 3). similar to the findings in individual cats, the analysis on the cattery level also identified an association between t. foetus and abnormal fecal consistency or diarrhea; "loose stools or diarrhea in any cats within the past 6 months" was significantly associated with t. foetus-positive findings . a second putative risk factor identified in this study was related to the cat population density in catteries; if the "square feet of facility available per cat was low," this was associated with positive findings . the observation that "coinfection with coccidia" was associated with t. foetus-positive findings may suggest that there are common risk factors in favor of mixed infections of coccidian parasites with t. foetus . in epidemiological studies none of the concurrent infections examined revealed a statistical association either to infection or disease on the individual animal level. as outlined in sect. 14.2.1, t. gallinae affects a large number of avian species where it mainly parasitizes the oropharyngeal membranes-sinuses, mouth, throat, and esophagus-causing a disease characterized by greenish fluid and caseous lesions, of whitish-yellowish fibrinous material, on the oropharyngeal membranes (reviewed by amin et al. (2010) ). t. gallinae infection seems not to be host species-specific. however, studies on parasite diversity suggest that there are subtypes more commonly found in certain bird species (gerhold et al. 2008; grabensteiner et al. 2010; sansano-maestre et al. 2009 ). t. gallinae is most common among domestic pigeons and wild doves, and these species may represent a reservoir also for other bird species. accordingly, rock pigeons-columba liva-were regarded as source for the worldwide distribution of t. gallinae (stabler 1954; harmon et al. 1987) . most pigeons harbor this protozoan but rarely show clinical disease (stabler 1954) . hawks, falcons, and owls may become infected, most likely via predation of other infected birds (rogers et al. 2016) . in recent years, severe outbreaks of avian trichomonosis caused by t. gallinae have been recorded initially in wild finches, later in passeriformes, canaries, and psittacines in europe and north america since 2005 (reviewed by amin et al. (2010) ). trichomonosis is also reported in several other bird species, including corvids in california, usa (anderson et al. 2009 ). t. gallinae has a low tenacity in the environment and is regarded as unable to survive a gastric passage, and droppings of birds are regarded as free of t. gallinae (stabler 1954) . t. gallinae has no cyst stage. however, as mentioned for t. foetus (sect. 14.1.1), the formation of pseudocysts has been reported (tasca and de carli 2003) . the importance of pseudocysts in the transmission of t. gallinae-e.g., via contaminated drinking water-is not sufficiently understood. survival times in tap water and in carcasses are regarded as limited; a survival time of 8-48 h in carcasses has been reported (erwin et al. 2000) . the following important facts about transmission of t. gallinae need to be taken into account: • transmission by crop milk or direct contact: prevalences in pigeons can be very high, and pigeons are regarded as endemically infected, often without clinical signs. feeding on infectious crop milk is regarded as an important route of infection for nestlings (stabler 1954) . in other bird species, feeding nestlings by regurgitation might also be an important route of transmission. close contacte.g., during courtship-is also regarded as a way by which the infection is spread between adult birds (stabler 1954) . in southeastern usa, trichomonosis was diagnosed more often in the spring and summer months than in autumn and winter months, which might be related to the times of courtship and feeding nestlings (gerhold et al. 2007 ). • transmission by drinking water: it is believed that infection of turkeys and chickens is caused by t. gallinae-contaminated drinking water (stabler 1954) . drinking water is also a likely source of infection for other bird species. • effect of weather events on outbreaks of avian trichomonosis: t. gallinaeassociated finch mortality usually peaked in summer and autumn, but a correlation with climatic events has not been observed (neimanis et al. 2010; robinson et al. 2010; lawson et al. 2011) . a coincidence of the emergence of avian trichomonosis with high temperature and low rainfall has been reported (simpson and molenaar 2006; bunbury et al. 2007 ). it has been hypothesized that as a consequence of the dry and hot weather, numbers and volumes of water sources decline, which may favor, due to higher densities of birds aggregating at limited water sources, the transmission of t. gallinae ). • transmission by predation: pigeons and doves may also serve as a source of infection for raptors (boal et al. 1998; krone et al. 2005) . although t. gallinae is a labile protozoan which does not remain viable for a prolonged period and is rapidly killed by desiccation, survival time seems to be long enough to be transmitted also to the nestlings of raptors (krone et al. 2005 ). in contrast to t. gallinae, t. gallinarum is mainly found as a commensal in the intestine of many domestic bird species, including chicken, turkey, guinea fowl, duck, and goose (friedhoff 1982; bondurant and honigberg 1994) . as a parasite of the intestine, t. gallinarum is transmitted via consumption of contaminated food or drinking water. cloacal as well as oral experimental infection of chickens and turkeys is possible. due to the widespread use of artificial insemination, bovine tritrichomonosis has almost disappeared in dairy cattle industries, like in western european countries, in the usa, or in canada. however, the disease is still present in many areas of the world were cattle are raised on pastures and natural mating is used. prevention and control measures are based on the distinctive epidemiologic features of bovine tritrichomonosis, a sexually transmitted disease where infected bulls are asymptomatic carriers and represent a permanent source of infection while in heifers and cows infection is typically transient (clark et al. 1971 parsonson et al. 1974; skirrow and bondurant 1990b) . bovine tritrichomonosis belongs to the oie-listed diseases (http://www.oie.int/ en/animal-health-in-the-world/oie-listed-diseases-2017; accessed 22. febr. 2017). t. foetus as a causative agent of this venereal transmitted disease may be present in semen if the semen has been contaminated with preputial fluid during manual collection (bondurant 2005) . recommendations for the importation of cattle and bulls for breeding can be found in the terrestrial animal health code. in particular, emphasis has been placed in the measures applied to bull semen donor health status in order to avoid dissemination and transmission of the disease (http://www.oie.int/ index.php?id=169&l=0&htmfile=chapitre_trichomonosis.htm; accessed 22. febr. 2017). in dairy herds and in some beef herds, artificial insemination is a very useful measure to reduce and eliminate infection. for bulls destined for artificial insemination, quarantine and periodic testing are required. also, in semen trade, it is of great value to know the country of origin, the reproductive history of the bull, and the tests performed by the artificial insemination center (eaglesome and garcia 1997) . in some areas of the world, as in the eu, specific regulations are applied to bovine semen trade and to regulate the sanitary conditions of the collection center and the animals. specifically, bulls selected for entry into artificial insemination should be tested in quarantine before admission to the center, and regular testing of animals in service is included as basic measures to avoid the presence and dissemination of bovine tritrichomonosis (european directive 88/407/eec of 14th june 1988 and european directive 2003/43/ec of 26th may 2003). in other countries, different policies have been established to control the infection. in the usa, state regulations have endeavored to control the endemic disease as to minimize economic losses by testing bulls. only the importation of t. foetusfree bulls is permitted for reproductive purposes while positive bulls are culled (yao 2013) . in the province of la pampa, argentina, with a bovine census close to four million heads, more than 80% of bulls are tested twice each year, and positive bulls were culled. by this measure, a significant decrease in the number of infected herds and animals has been achieved in this region in the last 10 years (fort et al. 2016) . recently, an online tool has been developed in the usa-trich consult; www. trichconsult.org, accessed 22. febr. 2017-that uses a series of questions to assess the t. foetus status and management practices of a herd. based on the responses to the questions, this page provides feedback to users allowing them to evaluate the importance of implementing suggested control strategies (ondrak 2016) . at present, a consensus exists concerning the most relevant measures as to how to prevent and control bovine tritrichomonosis (reviewed in ball et al. (1987 ), mccool et al. (1988 , bondurant (2005) , rae and crews (2006) , campero and gottstein (2007) , yao (2013) , ondrak (2016) ). in herds where it is not possible to introduce artificial insemination and where natural mating is the normal practice, as is the case in many regions where extensive beef cattle are raised, the following measures to prevent the entrance of the infection are recommended. • quarantine and testing of replacement bulls. replacement should be done by virgin animals or bulls acquired only from disease-free herds with records of excellence in reproductive performance. all the bulls must be tested during quarantine before entrance in the herd. if the bull comes from a tritrichomonosis-free herd, the analysis of two samples of preputial smegma with 1-2 weeks of interval during the quarantine is recommended. three or more samples must be analyzed if bulls are provided from an area where tritrichomonosis is known to be endemic (campero and gottstein 2007; yao 2013; ondrak 2016) . the measure includes the prohibition of the use of communal, shared, or rented bulls, unless their herd of origin and individual health status are known. • avoid communal pastures and keep fences in good conditions. these measures will help to control some of the two most important risk factors influencing disease transmission (gay et al. 1996; mardones et al. 2008; jin et al. 2014 ). • no introduction of cows or heifers of unknown health status during the breeding season. similar to bulls, heifers and cows must be acquired only from disease-free herds with records of excellence in reproductive performance. in argentina several heifers were found infected up to 300 days after breeding; such heifers may represent carrier cows able to introduce infection into naïve herds (mancebo et al. 1995) . in addition to the preventive measures outlined in sect. 14.3, the following control measures are recommended to reduce the impact and eliminate the disease in case that bovine tritrichomonosis has been diagnosed in a herd or the herd is located in a tritrichomonosis endemic area: • testing of bulls before the breeding season and culling of infected bulls. efforts to control the disease focus on using diagnostic tests with a high sensitivity, low cost, and time efficacy. it is recommended to sample the animal twice or three times before the breeding season and every time new bulls are introduced into herds (bondurant 2005; campero and gottstein 2007; yao 2013) . once new cases are not detected, annual testing is recommended to verify the non-infected status of the herd. however, testing and culling policies alone, although effective in improving reproductive efficiency, do not allow the elimination of the disease since other putative risk factors associated with the disease are usually present in the management of beef herds (yao 2013; collantes-fernandez et al. 2014 ). • average age reduction of bulls and replacement of all bulls after four breeding seasons. replacement with negative-tested young bulls reduces the prevalence since 3-year-old bulls seem not to be as susceptible as older bulls in natural service (clark et al. 1971; christensen et al. 1977) . a strong association of infection rate with age has been reported in several studies (rae et al. 1999; mendoza-ibarra et al. 2012 ). • pregnancy examination. it is mandatory to know the reproductive performance and the magnitude of the infertility problem in the herd. all open and aborted cows should be culled or segregated in high-risk sub-herds or groups (campero and gottstein 2007) . • use of commercial vaccines. in the presence of a high prevalence of infection in an area, vaccination of all heifers and cows is a good measure to improve reproductive efficiency especially when risk factors associated with infection cannot be avoided-e.g., the use of communal pastures. commercially available vaccines in the americas offer an improvement in reproductive efficacy (kvasnicka et al. 1989 (kvasnicka et al. , 1992 . • segregation of the herd in low-and high-risk sub-herds or groups. in the low-risk sub-herds, only dams that have recently calved are pregnant for more than 5 months, and virgin females must be included. these must be serviced preferably by virgin bulls or by bulls with two negative examinations and coming from negative herds. in order to follow the effect of the infection, the same group of females should be serviced with the same male until the disease is controlled (campero and gottstein 2007) . • limiting of breeding season. the breeding season should be restricted to less than 4 months to reduce the duration of the possible transmission period as much as possible. in addition, with a shortened breeding season, it becomes easier to monitor reproductive performance. with respect to vaccination against bovine tritrichomonosis, the main objective is to eliminate a t. foetus infection from the reproductive tract before fetal loss occurs without necessarily avoiding parasite colonization of the epithelium during the first 40 days post-infection (bondurant et al. 1993; bondurant 2005) . the mucosal immune response in the genital area seems to be the main protective mechanism which is characterized by a local response with the presence of iga and igg1 and a mild systemic response characterized by the presence of igg2 and igg1 (skirrow and bondurant 1990a; anderson et al. 1996; corbeil et al. 2008) . as a rule, cows immunized with t. foetus have a humoral response pattern similar to that described for natural infections (herr et al. 1991; bondurant et al. 1993 ). however, genital iga levels appear to depend on the type of antigen, adjuvant, and route of administration employed. as to our knowledge, commercial vaccines against bovine tritrichomonosis exist only in the americas but not in europe. one available inactivated vaccine is prepared from whole organisms and can be acquired in a monovalent formulation-trich guard®-but also in a polyvalent formulation combined with campylobacter foetus subspecies venerealis and leptospira, trich guard v5-l®. in addition, in argentina, an alternative inactivated vaccine-tricovac, tandil biological laboratory-containing an oily adjuvant with a concentration of 5 × 10 7 trophozoites of t. foetus is available. in several studies on heifers using whole-parasite-based vaccines, a reduction in the number of infected females, a shorter time of genital infection, and a higher percentage of pregnant females in comparison with control animals have been reported (kvasnicka et al. 1989 (kvasnicka et al. , 1992 herr et al. 1991; gault et al. 1995; anderson et al. 1996; cobo et al. 2002 cobo et al. , 2004 . in addition, a lower number of services, 1.44 vs. 1.73 in non-vaccinated animals, p = 0.16; higher percentage of pregnant animals at the first service, 66.7 vs. 33.3% pregnancies, p < 0.05; and a reduction of embryo/fetal losses of 56.4% were observed (kvasnicka et al. 1992; hudson et al. 1993) . subunit vaccines have also been developed (clark et al. 1983b; bondurant et al. 1993; corbeil et al. 1998) . a trial with an experimental vaccine formulated with membrane antigens of t. foetus was conducted; cows were immunized with this vaccine and subsequently challenged by the vaginal route ). the vaccine used was able to generate a specific humoral immune response and shorten the period of infection in the vaccinated animals compared to the controls. a greater efficacy of a t. foetus membrane vaccine compared to a whole cell vaccine was observed; the animals had a shorter duration of infection, a higher pregnancy rate, and a lower rate of fetal mortality (cobo et al. 2002) . additional work has been done to identify t. foetus surface antigens such as tf1.17 and tf190 (voyich et al. 2001) . the application of the former in experimentally challenged heifers evidenced a rapid shortening of infection and a specific iga production in genital secretions (anderson et al. 1996; corbeil et al. 1998) . vaccine-induced immunity to t. foetus has not been studied in depth in bulls and is only mentioned in some earlier studies (clark et al. 1983b (clark et al. , 1984 soto and parma 1989; campero et al. 1990; herr et al. 1991) . in bulls older than 5 years, the whole cell vaccine lacked a preventive or curative effect (clark et al. 1983b ). therefore, commercial trich guard ® vaccine is not indicated for bulls (herr et al. 1991; bondurant 1997) . prevention of feline trichomonosis is based on interrupting the fecal-oral route of transmission, particularly in catteries, shelters, and other dense cat populations. since the viability of the parasite in the environment is limited, strict cleaning and disinfection measures are sensible measures to be implemented (hale et al. 2009 ). additionally, contamination of food and water by t. foetus is also possible; consequently, regular replacement of water and food and cleaning and disinfection of watering troughs and food bowls should be recommended. due to the suggested role of some invertebrates to function as mechanical vectors, the avoidance of their presence in the litter box area and food and drinking areas may help to prevent infection transmission ( van der saag et al. 2011) . finally, limiting contact between infected and non-infected cats will help to interrupt transmission of t. foetus in the population. measures to prevent t. gallinae outbreaks in wild as well as in captive birds are focused on actions to reduce sources of infection. one of the major aims would be to prevent attracting birds to feeding places. if feeding is necessary, such places should fulfill minimum requirements with regard to sanitary conditions, like regular changing of food and disinfection as suggested in a recent review (amin et al. 2014 ). since t. gallinae seems to require a wet environment to persist, drying of buildings and housing facilities following washing will support to control trichomonad infections. the prevention of the infection of prey birds, like pigeons nesting near urban areas, is necessary to prevent infection of predator birds. due to the loss of habitat, predators have replaced their traditional prey mainly by urban pigeons (boal et al. 1998; höfle et al. 2000; estes and mannan 2003; krone et al. 2005 ). in the past various imidazoles were used to treat bulls, but none was both safe and effective, and drug resistant strains were found (reviewed in bondurant (2005) , rae and crews (2006) ). specifically, ipronidazole is probably the most effective drug, but due to its low ph, it frequently causes sterile abscesses at the injection sites, and resistances have also been observed (skirrow et al. 1985) . a systemic treatment using drugs like metronidazole or dimetridazole produces adverse side effects and resistant populations of trichomonads (campero et al. 1987) . currently, there is no approved treatment for cattle infected with t. foetus because of concerns regarding toxic residues in meat (bondurant 1997). therapies traditionally used for treatment of protozoa are not successful for feline trichomonosis (reviewed in manning (2010) , yao and köster (2015) ). currently, ronidazole has been the most effective drug used to date and is recommended at 20-30 mg/kg orally once daily for 14 days (gookin et al. 2006) . relapse of diarrhea is common, but cats can continue to carry the organism, and resistant strains of t. foetus to ronidazole have also been documented (gookin et al. 2010a ). in addition, neurological toxicity in cats treated with ronidazole in the range of 30-50 mg/kg has been reported (rosado et al. 2007) . it is therefore important that owners are informed of the potential side effects. ronidazole is not registered for veterinary use, and informed consent is necessary prior to its use in cats, and it should only be prescribed in confirmed cases. the drugs of choice for the treatment of avian trichomonosis are nitroimidazoles (metronidazole, dimetridazole, ronidazole, and carnidazole) (reviewed by amin et al. 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tritrichomonas foetus on reproductive efficiency in beef herds antibody responses of cattle immunized with the tf190 adhesin of tritrichomonas foetus a new species of tritrichomonas (sarcomastigophora: trichomonida) from the domestic cat (felis catus) bakterienfreie züchtung von trichomonaden und dem uterus des rindes in einfachen nährböden. zentralblatt für bakteriologie, parasitenkunde, infektionskrankheiten und hygiene intestinal tritrichomonas foetus infection in cats: a retrospective study of 104 cases histologic features associated with tritrichomonas foetus-induced colitis in domestic cats survey of nine abortifacient infectious agents in aborted bovine fetuses from dairy farms in beijing, china, by pcr diagnosis of tritrichomonas foetus-infected bulls, an ultimate approach to eradicate bovine trichomoniasis in us cattle tritrichomonas foetus infection, a cause of chronic diarrhea in the domestic cat bovine trichomoniasis development and preliminary assessment of a polyclonal antibody-based enzyme immunoassay for the detection of tritrichomonas foetus antigen in breeding cattle key: cord-016499-5iqpl23p authors: mackay, ian m.; arden, katherine e. title: rhinoviruses date: 2014-02-27 journal: viral infections of humans doi: 10.1007/978-1-4899-7448-8_29 sha: doc_id: 16499 cord_uid: 5iqpl23p picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide. hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under 5 years and occur in all seasons. aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing and with significant direct and indirect healthcare expenditure. ari incidence is highest in the first 2 years of life, with up to thirteen episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month. picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide [ 1 ] . hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under 5 years and occur in all seasons [ 2 , 3 ] . aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing [ 4 ] and with signifi cant direct and indirect healthcare expenditure [ 5 , 6 ] . ari incidence is highest in the fi rst 2 years of life, with up to 13 episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month [ 3 , 7 -9 ] . in preschool-aged children, nearly 50 % of general practitioner visits are for ari [ 10 ] , many of which are self-limiting. aris can often be managed in the community with supportive care from parents, but complications can arise that require a medical visit for management of asthma, otitis media, or sinusitis [ 11 ] . hrvs replicate in nasal cells, sinus cells, bronchial epithelial cells (becs) [ 12 , 13 ] , and smooth muscle cells [ 14 ] but not in monocytes [ 15 ] or dendritic cells (dcs) [ 16 ] . the infl ammatory immune response they trigger very soon after infection has its greatest impact in the young, the elderly, those with asthma or chronic obstructive pulmonary disease (copd), and in the immunocompromised. first infections usually elicit a stronger response. antiviral interventions have been under development for decades; to date most have met with varying degrees of failure or unacceptability. vaccines have been considered unachievable because of the large number of diverse and distinct viral types. there are 100 classically defi ned and recognized hrv serotypes grouped into two species, hrv-a and hrv-b, and a recently defi ned third species, hrv-c, containing more than 60 genotypes identifi ed and characterized entirely by molecular means. their cousins, the four enterovirus species (ev-a, ev-b, ev-c, and ev-d), are also found in the airways at times. most systematic and mechanistic studies of hrv etiology and pathogenesis have been informed by studies in adults, mostly prior to the discovery of hrv-cs. adults exhibit reduced symptoms from hrv infections because of prior exposure and the resultant protective immune memory which that imparts (see sect. 7.3 ). furthermore, many modern studies (1) draw conclusions about lower respiratory tract (lrt) disease using urt specimens and (2) infrequently sample, doing so across small cross sections of time. these limitations have hampered attempts to associate virus detection and disease. current thinking is that hrv-cs may be key players in asthma exacerbations although our inability to culture them routinely has hindered our progress in understanding their role. the impact of the hrvs has been underestimated for decades, and the concept of the hrvs as a very large assemblage of genetically, immunogenically, antigenically, and temporally distinct and stable viral entities remains rare; they are more commonly considered a single variable virus, a view that science does not support. the disease most commonly associated with the airways and resulting from hrv infection is the common cold, a selflimiting coryzal illness [ 17 -19 ] . the term dates back to ancient greece, but evidence that the syndrome and asthma, another disease most frequently due to hrv infection, has been with us since ancient times can be viewed in writings on the ebers papyrus, a medical document written in the sixteenth century bc [ 20 , 21 ] . in 1930 the common cold was considered either to be due to exposure to the elements or to infection by bacteria [ 22 ] . it was later understood to be largely due to something in bacteria-free fi ltrates, and so the search for viral causes began [ 23 , 24 ] . the common cold unit (ccu) was established in salisbury, uk, to seek solutions to the mysteries of the common cold, mostly through adult volunteer infection studies and careful systematic science [ 23 ] . the ccu functioned for 44 years (1946-1990) , and it was here in 1953 that the fi rst in vitro culture of an hrv was achieved using lung tissue from a particular embryo ( fig. 29 .1 ) [ 25 , 26 ] . propagation failed once this tissue was expended [ 22 , 33 ] . once hrv isolation was possible, viral serotyping developed and culture techniques were further refi ned. this leads to an international effort to characterize and name the hrvs [ 27 -30 ] . in 2006 renewed interest in hrv research was triggered by the description of a distinct clade of hrv types [ 31 ] found using molecular typing. the resultant fl urry of hrv research raised questions about many earlier paradigms of rhinovirology and of the role of established respiratory viruses in aris. the novel clade was proposed as a new species, hrv-c, which was taxonomically confi rmed in 2009 [ 32 -34 ] . prior to the discovery of the hrv-cs, the genus rhinovirus had been abolished and the hrv-a and hrv-b species assigned to the genus enterovirus within the family picornaviridae [ 35 ] . the hrv-cs have been assigned a new naming scheme based on genetic sequence in the absence of antigenic or serological data. while the sequencing of all serotyped hrv genomes was completed in 2009, few of the hrv-cs or apparently novel hrv-as or hrv-bs have been similarly characterized, so the full spectrum of hrv genomes, the rhinovirome, remains incomplete. in this chapter we have described individual serotyped hrvs as the "classical" types, a type being the description for a single, genetically stable, stand-alone hrv. methods for epidemiologic analysis the original clinical defi nition of an hrv infection was written using data from cell and tissue culture and adult human infection studies. after 1953 in vitro isolation methods employed a virus interference test to more easily determine successful isolation; cultures suspected of infection with an uncharacterized hrv prevented infection by another, readily titratable virus [ 36 ] . later, price (1956 ; the jh strain) and then pelon and co-workers (1957; 2,060 strain) developed culture systems that permitted hrv replication to be more easily identifi ed [ 37 , 38 ] . the early hrvs were initially classifi ed as echoviruses (echo 28; later hrv-1) [ 39 ] . at the same time, propagation of the hgp (hrv-2) strain resulted from using increased acidity, lowered cultivation temperatures, and constant motion (rotation) [ 40 , 41 ] . despite the challenges [ 42 ] , virus isolation was a more sensitive indicator of infection than an antibody rise in paired sera [ 43 ] . it was found that several cell lines and methods were required to encompass virus concentrations ranging from 10 1 to 10 5 tcid 50 /ml [ 44 -47 ] and growth differences among the different virus types. additionally, cell age after plating (<72 h), inoculum volume (relevant to the culture vessel), medium ph (6.8-7. 3), and cell density were important factors for the reproducible appearance of hrv-induced plaques and for higher virus yields [ 48 -51 ] . the hrvs can grow at temperatures above 35 °c (some prefer that under certain conditions) [ 52 ] , but rolling at 33 °c, preceded by a 2-4-h stationary incubation period [ 41 ] , has historically provided the highest yield and fastest in vitro hrv growth [ 36 , 50 , 53 , 54 ] . serodiagnosis grew increasingly impractical as the number of serotypes increased [ 49 , 55 ] . however, antibody-based methods were essential for type-specifi c neutralization of infection [ 56 ] from which early epidemiology data were derived and around which the hrv nomenclature system evolved in 1967 [ 28 ] . the fi rst classical strains were officially named in 1967 [ 57 ] , the last in 1987 [ 30 ] . today we know that cell culture-based methods are unreliable for accurately representing respiratory virus epidemiology; although enhanced by immunofl uorescence, they are still used [ 58 ] . the hrv-cs have not been successfully cultured in any cell lines or primary cell culture, although many attempts have been described [ 32 , 59 -62 ] . in 2011 hrv-c15 and w23 (another hrv-c) were shown to grow using organ culture [ 63 ] . sinus tissue hosted increasing levels of viral rna, as did adenoid, tonsil, and nasal polyp tissue, but much less effectively, as measured by in situ hybridization [ 63 ] . the sinus organ culture system also allowed testing of the fi rst reverse engineered hrv-c (pc15) [ 63 ] . isolation identifi ed hrvs in ~23 % of adults with aris, associated with 0.5 illnesses per year [ 64 ] . because culture is ineffi cient and subjective and requires expertise, even for the culturable hrv types, it is becoming an art lost to clinical laboratories the world over. it is unsurprising that pcr-based methods now prevail, providing a much improved understanding of the nature and scope of hrv infections. the virological and immunobiological cost of this improvement is a paucity of low passage "wild" hrv isolates to work with; thus, many research fi ndings from recent years have employed easy to grow highly passaged and adapted hrv isolates. the impact of virus adaptation on the reliability of data from use of such viruses is unknown. pcr-based assays have dramatically increased the frequency of hrv detection [ 65 -70 ] . the improved sensitivity and reduced turnaround time have shown that hrvs, as a group, are usually the predominant viruses in ari cases [ 71 -73 ] . with reliable detection levels that extend from as few as 10 2 tcid 50 /sample to well above clinically relevant loads, pcr can detect virus levels which are commonly shed during all stages of experimental infection studies [ 74 , 75 ] . the common understanding of the systemic [ 76 -78 ] or symptomatic [ 79 , 80 ] context of hrv detections was established during the era of culture detection, and pcr has challenged these paradigms by detecting virus more often than culture. hrvs are sometimes found in "healthy controls"; however, it is likely that with more thoughtful defi nitions of "healthy," these detections would reduce. it is not uncommon to experience a feeling that one is "coming down" with something that never develops further. this is likely due to a transient infection or reinfection by an hrv or other respiratory virus that is eliminated quickly by the host response. it is possible to correlate viral nucleic acid load at the sampling site with disease severity; however, this is made diffi cult by the highly variable sampling effi ciency of respiratory tract specimens which only permit the generation of reliable quantitative pcr (qpcr) data if serial specimens are available [ 81 ] . the 5′ untranslated region (utr; figs. 29.2 and 29.3 ) is the most common target for diagnostic oligonucleotides since the fi rst hrv rt-pcr in 1988 [ 82 ] , and the region has retained relevance for virus detection by its adaptation to reverse transcriptase real-time methods (rt-rtpcr) [ 53 , 65 , 66 , 69 , 74 -76 , 79 , 83 -103 ] . the 5′utr is comprised of a number of conserved sequence "islands" (fig. 29 .2 ) that permit the robust detection of the majority of hrvs and those "respiratory evs" which can be regularly detected in the respiratory tract [ 104 , 105 ] . the detection of respiratory evs in no way detracts from the importance of supporting clinical decision making using these assays. however, repositioning [ 68 ] . the pcr primers of broadly reactive conventional rt-pcr [ 82 , 113 ] and rt-rtpcr [ 75 , 114 ] assays are shown these primers or changing the method of employing them [ 106 -109 ] may undermine assay performance, as evidenced by predicted hybridization mismatches, uncommonly low detection frequencies [ 110 ] , and by comparison of multiple primer sets using the same specimens [ 111 ] . the addition of an oligoprobe rtpcr method increases amplicon detection sensitivity and specifi city, identifying 100-fold fewer tcid 50 /ml or 10 fold fewer genome copies than agarose gel detection of amplicon [ 75 , 79 , 112 ] . other molecular tools, capable of detecting multiple targets, have evolved in recent years [ 58 , 70 , 115 -121 ] , and some have gone on to be approved for clinical laboratory use [ 122 ] . microarrays can detect thousands of viral targets, but are expensive for routine use (usd30-300 per sample) and not sensitive enough to avoid a pre-hybridization pcr amplifi cation when using clinical specimens. at their most robust, microarrays, like pcr, rely on the existence of conserved regions of sequence to detect unknown viruses allowing them to detect previously unknown hrv types [ 123 ] . highthroughput or "deep" sequencing platforms have become less expensive and more readily available, and they have succeeded in fi nding new diversity within the hrv species [ 124 ] . the experiments remain costly so have not yet found a place for regular screening tasks and remain coupled to a need for pre-pcr steps. rapid protein-or virion-based assays are not (yet) adequately sensitive [ 125 , 126 ] . because of the high number of hrvs and the high frequency of infections, genotyping methods have become an essential accompaniment for understanding hrv epidemiology. nucleotide sequencing of the vp1, 5′utr+vp4+vp2 (called hereafter vp4/vp2), or 5′utr region has replaced traditional serological methods, because of its speed and need for fewer specialized reagents compared to serotyping. vp1 yields the most comprehensive subgenomic genotyping information and is essential for the minimal defi nition of a new hrv type [ 127 ] . the vp4/vp2 region (fig. 29. 3 ) is considered easier to use because it encompasses suffi cient genetic diversity to confi rm the identity of a clinical hrv type while also providing broad enough sensitivity to amplify the ~160 hrvs from a challenging biological substrate, clinical specimens [ 128 ] . screening of airway specimens for hrvs is not routine [ 111 ] due to factors including cost and the perceived low clinical relevance of detection. genotyping is mostly relegated to research facilities. because of this, hrv molecular epidemiology studies tend to be smaller and focused on a specifi c disease or research question. most in-depth molecular studies of hrv replication have focused on a single hrv type. generally, it is presumed that results can be extrapolated to the other hrv types and to the in vivo situation. hrvs replicate in the cytoplasm (fig. 29 .4 ) [ 129 ] with membrane-associated replication structures containing double-stranded rna (dsrna) replicative intermediates (ri) which are formed in cells 4 h after infection [ 52 , 130 ] . single-stranded infectious rna forms after ris start to accumulate [ 130 ] . genomic rna (plus strand) is the template for complementary minus strand synthesis which in turn is the template for new genomic plus strands that become incorporated into virions [ 131 ] . virions are synthesized from 4 to 7 h after infection and reach maximum release levels at 10-18 h [ 131 ] . hrv replication in epithelial cells may shut off host cell transcriptional activity via direct cleavage of transcription factors and nuclear pore complex components. protease 2a (2a pro ) of hrv-b2 may directly cleave eukaryotic initiation factor 4g (eif4g) when bound to eif4e [ 132 , 133 ] . the eifs have key roles in initiation and rate control of host cell translation [ 132 ] . host cellular protein production is virtually replaced by hrv-b14 proteins after only 6 h of infection [ 134 ] . hrv-b14-infected cells also display reduced nuclear importing and degraded nuclear pore complex (npc) components [ 135 ] . this may represent another hrv strategy for limiting the host response by preventing or reducing key signaling pathway molecules (e.g., irf-3, stat1, nf-κb) and shutting down host cell protein synthesis. protease 3c (3c pro ) from hrv-a16 targets the nucleus and can disrupt active and passive nucleocytoplasmic transport [ 129 , 136 ] . recombinant 2a pro protein from hrv-a16,hrv-a89, hrv-b4, hrv-b14, hrv-c2, and hrv-c6 exhibited differing specifi cities and kinetics against eif4g as well as npc components demonstrating functional diversity between hrv types [ 137 ] . this fi nding underscores the functional diversity within the hrv species and the risk of extrapolating too greatly from the study of single hrv types. it is apparent from a wealth of immunobiological data that hrvs still effi ciently trigger a proinfl ammatory immune response that has considerable clinical impact among at-risk groups, and that their putative interruption of host cell machinery does little to hinder this. the virion encapsulates an approximately 7 kb positive sense rna genome (fig. 29. 3 ), which tends to be more adenine and uracil (a+u) rich than the ev genome [ 139 ] . in particular, a+u more frequently occupies the third or "wobble" genome replication in association with membranes produces the viral polyprotein which is co-and posttranslationally processed by 2a pro and 3c pro into the proteins ( p1 -p3 ) and structural peptides ( vp1 -vp4 ; vp2 and vp4 derive from the vp0 precursor protein) that assemble into protomers, pentamers, and fi nally capsids. nonstructural proteins are also released in these cleavages as well as through autoproteolytic cleavage. mature hrv virions packaged with an ssrna genome escape by cell lysis (adapted with permission from arden et al. [ 138 ] ) codon position. the single rna "gene" acts as messenger rna to encode the single multi-domain, proteolytically processed "polyprotein." the coding region is bracketed by utrs which perform regulatory functions necessary for genome duplication [ 140 ] . these are very similar genomic, transcriptional, and translational features to those of their close cousins, the evs. most of the information currently required for virus identifi cation by the international committee on taxonomy of viruses (ictv) can be found through analysis of the genetic features of hrvs ( fig. 29.3 ). there are 158 complete hrv polyproteins on the genbank database ( fig. 29 .5 ). the fi rst complete hrv genome sequence (hrv-b14) was described in 1984 [ 141 ] followed by hrv-a2 in 1985 [ 142 ] and hrv-a1b in 1988 [ 143 ] (fig. 29 [ 145 ] . sequencing of the vp4/vp2 region was completed for all classical strains in 2002 [ 146 ] , and the complete set of 1d regions were available in 2004 [ 147 ] . currently there are at least 50 named hrv-c vp1 regions the current spectrum of 168 complete hrv complete polyprotein amino acid sequences available on the genbank database. the alignment was conducted using mafft within geneious pro v5.6 [ 148 ] . the phylogenetic and molecular evolutionary analyses were con-ducted using mega version 5 (poisson model, 500 bootstraps with consensus support shown at the nodes where space permitted [ 149 ] ) (reprinted with permission from miller and mackay [ 150 ]) available and 20 complete hrv-c genomes. many more genomes are appearing as part of the rhinovirus consortium's efforts to complete and study the rhinovirome using highthroughput sequencing technologies to genetically characterize hrvs from their combined clinical specimen stores ( http://www.international-rhinovirus-consortium.org/ ). many 5′utr and vp4/vp2 sequences reside on the genbank database, most of which are labeled using in-house laboratory schemes rather than an approved nomenclature. analysis of the full-length genomes supports the use of 5′utr, vp1, and vp4/vp2 subgenomic regions for useful representation of hrv species and types [ 144 , 147 ] . recombination, the process of genetic exchange which results in a chimeric genome [ 151 ] , can only be detected in mature viruses after the fact, and it must therefore be inferred indirectly through genomic analysis and comparison. predictions of infrequent recombination among the hrvs [ 83 ] have been made based on examination of the available set of hrv coding and noncoding regions [ 152 ] . intensive analyses reported that recombination is not a driving force for the evolution of hrv types [ 144 , 153 , 154 ] . some discrepancies are likely because of the different number of sequences used, the different origins of the viruses used for sequencing, and the analysis methods employed. hrv-c evolution seems to have been more affected by prior recombination, than is apparent for members of hrv-a or hrv-b. this is similar to the ev species but with far fewer predicted recombination events than for ev evolution [ 114 , 151 , 155 , 156 ] . most of the recombination proposed to have affected the hrvs occurred between hrv-c and hrv-a and is often found within the 5′utr or at the 5′utr/vp4 junction [ 83 , 157 , 158 ] but rarely in coding sequence (2a [ 158 ] or 3c [ 83 ] ). the high sequence diversity among the individual hrv polyprotein coding sequences may keep recombination events to a minimum in order to retain viral fi tness [ 158 ] . the ability of hrvs to recombine in practice awaits empirical evidence; the extent of recombination among all hrv or ev types and the frequency with which viable recombinants arise are entirely unquantifi ed. the 28-30 nm hrv virion has been visualized for only a handful of hrv-a and hrv-b types (including hrv-a1a, hrv-a2, hrv-b3, hrv-b14, and hrv-a16), but no hrv-c structures have been empirically determined to date. the fi rst, hrv-b14, was described in 1985 [ 159 ] followed by hrv-a1a in 1989 [ 160 ] , hrv-a16 in 1993 [ 161 ] , hrv-a3 in 1996 [ 162 ] , and hrv-a2 in 2000 [ 163 ] . hrv-c structure has only been predicted using computer modeling, but their basic structure seems to be that expected of an hrv ( fig. 29.6 ) [ 33 ] . the hrv capsid shell is composed of 60 protomers, each comprising one copy of the viral proteins vp4, vp3, vp2, and vp1. vp1, vp2, and vp3 (each ~30 kda) are to some extent exposed on the capsid surface, whereas vp4 (~7 kda) is internalized and associated with viral rna. five protomers come together at a point around a fi vefold axis, and this cluster is called the pentamer. the fi vefold axis is circumscribed by a cleft referred to as the "canyon." vp1, vp2, and vp3 are each formed by a convoluted set of protein sheets and loops [ 159 ] . the loops protrude beyond the external capsid surface and contain discontinuous antigenic sites. of the hrv types studied, four neutralizing antibody immunogenic (nim) regions have been identifi ed on hrv-b14 and hrv-a16: nim-1a (located in vp1), nim-1b (vp1), nim-ii (vp2 and vp1), and nim-iii (vp3 and vp1) [ 159 ] . antigenic sites identifi ed on hrv-a2 are called a, b, and c [ 164 ] . the scope and location of antigenic and immunogenic moieties among the hrv-cs is unknown. using known receptor binding sequence as a guide for computer modeling ( fig. 29 .6 ), it has been predicted that when discovered, the receptor for the hrv-cs will differ from the major and minor receptors defi ned for the hrv-as and hrv-bs [ 33 ] . the three hrv species within the genus enterovirus are a genetically, immunogenically, and antigenically diverse assemblage of >160 viral types (table 29 .1 ). this accounts for the combination of hrv-a1a and -a1b, exclusion of hrv-87, which is actually ev-d68 despite confusion over acid liability [ 169 -171 ] and combination of hrv-hanks which is actually hrv-a21 [ 147 ] . serological studies indicate that some hrv-a and hrv-b types may not be distinct enough to deserve a unique identity [ 147 ] . species within the genus share >70 % amino acid (aa) identity in the polyprotein and in 2c+3cd and >60 % aa identity in p1 ( fig. 29 .3 ) as well as their host cell receptors, a limited natural host range, a genome base composition (g+c) that varies by no more than 2.5 %, and a similar compatibility of proteolytic processing, replication, encapsidation, and genetic recombination [ 172 ] . a variant of the same hrv type shares 87-88 % aa identity or more in vp1 [ 129 ] . much of the nongenetic criteria remain undefi ned for the hrv-cs. in 2008 the genera enterovirus and rhinovirus were offi cially combined, retaining the former genus name enterovirus with the human enterovirus c as the prototype species. a genus in the order picornavirales , family picornaviridae , is at least 58 % different in its amino acid identity from any other genus. in 2009 a proposal establishing the species human rhinovirus c was ratifi ed by the ictv. formal hrv-c numbering commenced in 2010, and type numbers were initially assigned based on the date of submission of relevant sequences to genbank (hrv-c1, formerly nat001; hrv-c2, f. nat045; hrv-c3, f. qpm; hrv-c4, f. c024, etc.; table 29 .1 ) [ 127 ] . a clinical detection of an hrv-c can be considered a novel type principally based on its vp1 sequence or provisionally ("c_pat," table 29 .1 ) based on vp4/vp2 [ 146 ] and could be confi rmed as a variant of a previously characterized hrv-c by identity thresholds to either region. the 5′utr can be and still is used [ 173 , 174 ] for hrv genotyping, but it is a more problematic region than vp1 or vp4/vp2 because of the recombination activity that affects this region, especially among the hrv-cs [ 175 ] . this is presented as phylogenetic intermingling of some hrv-a and hrv-c types [ 114 ] . nonetheless, careful application of sequence identity thresholds when comparing clinical sequences to the genbank database (≥96 % identity required before assigning a clinical detection to a particular type) succeeds in characterizing hrv species and types [ 9 ] . there are currently 50 types within hrv-c (which includes the types once grouped together under hrv-"a2," hrv-x, and hrv-ny clades), 78 hrv-a types, and 25 hrv-bs. the most up-to-date information on current taxonomic trends can be found at the ictv picornaviridae study group website ( http://www.picornastudygroup.com/ ). ( c ) hrv-c3 versus hrv-a2 simplot data projected onto the hrv-c3 pentamer. the domains of interest are mostly shown within a single asymmetric unit. ( d ) a minor group pentamer (hrv-a2, gray ) including antigenic sites (sites a -c , green ) and very-low-density-lipoprotein receptor (vldlr) footprint ( red ) [ 167 ] . attachment of the vldl-r involves adjacent vp1 molecules. magnifi ed vp1 area represents one half of a vldl-r footprint [ 168 ] . amino acid substitutions ( arrowed ) contributed to the differences between minor group sites b and c (adapted with permission from mcerlean et al. [ 33 ] ) historically a key feature distinguishing the hrvs from the evs was the instability of the hrv capsid in the presence of acid and their lower preferred laboratory propagation temperature (33-34 °c versus 37 °c for evs). over time hrvs have been subclassifi ed in different ways. the fi rst was based on tissue tropism and host range. hrvs that preferred growth using monkey cells were called "m" strains and those (the majority) that grew only in human cell cultures, "h" strains [ 56 , 176 -180 ] . these two groups correlate with receptor usage [ 131 ] (table 29 .1 ) and possibly with the titer of the inoculum employed [ 181 ] . in 1962 it was proposed to abandon this terminology in favor of a sequential numbering system [ 177 ] . picornaviruses recognize a variety of cellular receptors [ 169 , 182 , 183 ] . hrv types are also subdivided into major and minor groups defi ned by use of one of the two main receptor molecules [ 184 , 185 ] . the capsid of the majority of classical hrvs ( n = 89) [ 184 ] interacts with the amino-terminal domain of the 90 kda intercellular adhesion molecule (icam-1; cd54) [ 186 -189 ] . receptor binding destabilizes the hrv capsid, probably by dislodging the "pocket factor," and initiates uncoating [ 164 , 182 , 190 ] . icam-1 interacts with its receptor, leukocyte function antigen-1 (lfa-1), and plays a role in recruitment and migration of immune effector cells [ 191 ] . the minor group [ 184 ] of classical viruses employ members of the low-density lipoprotein receptor (ldlr) family to attach to cells [ 167 ] . binding of vldl-r occurs outside of the canyon employing a different destabilizing and uncoating mechanism. heparan sulfate may act as a receptor under specifi c conditions [ 183 , 192 , 193 ] . in 1990 andries et al. defi ned, and laine et al. refi ned, two "antiviral groups" (a and b) based on their susceptibility to a panel of antiviral molecules [ 165 , 194 ] . these groupings refl ected the nature of the amino acid (and hence nucleotide) sequence of the region interacting with the antiviral molecules. these antiviral groups can also be visualized using phylogeny [ 194 ] . when sequences from other subgenomic regions, including p1, 2c, and 3cd, were examined by phylogeny, the species were found, in most cases, to inversely correlate with antiviral grouping labels (table 29 .1 ). m and h indicate early cell tropism-based classifi cation (monkey, human) abandoned in favor of a sequential numbering system [ 177 ] . hrv types were later divided into the major and minor groups defi ned by receptor tropism [ 184 , 185 ] . receptor-designated minor group hrv types are underlined, and major group types are shown in bold. antiviral groups (a and b) are labeled [ 165 , 194 ] . hrv-a8 and hrv-a95 are also likely the same serotype [ 147 ] . a full list of genetically close serotype pairings was presented by ledford et al. [ 147 ] hrv-c nomenclature was defi ned in 2010 and currently includes a number of p rovisionally a ssigned t ypes (pat) which are confi rmed once preliminary vp4/vp2 data can be confi rmed with vp1 sequence and the provisional number removed (e.g., c_pat1 to c_pat13 have already been reassigned) today, sequencing and phylogeny play a central role in species classifi cation within the genus, and together, they are surrogates for the important biological classifi cation criteria [ 146 , 147 , 165 , 195 -197 ] . for the hrv-cs, fi rst described as the "hrv-a2" clade (not to be confused with the single virus, hrv-a2, this naming scheme appeared after the hrv-c clade's name was proposed) of viruses in 2006 [ 31 ] , sequencing of 5′utr and vp4/vp2 has provided the bulk of hrv information from clinical studies. while culture in primary sinus tissue has been reported [ 63 ] , no receptor is yet defi ned. hrvs are the most numerous and frequently detected of all the "respiratory viruses," so-called because of their predominant detection in and tropism for the human urt or lrt ( fig. 29 .7 ). the circulation of hrvs varies with population age, underlying disease, immunocompromise, over time, and across distance. circulation is infl uenced by the nature, strength, distinctiveness, and memory of the immune response hrvs trigger and by the nature and prevalence of other concurrently circulating respiratory, and perhaps nonrespiratory, viruses. with the recent discovery of the unculturable hrv-cs came the realization that previous hrv epidemiology was only reliable if conducted by one or more suitably broad-spectrum hrv pcr assays [ 111 ] ; hence, prior to 1988, detection of the full spectrum of ≥160 hrvs did not occur. after 1988, the ability to detect all types very much depended on the nature of the pcr primers and detection methods used. the great number of distinct hrv types has burdened the search for answers to epidemiology-related questions. however, as for other important respiratory viruses including human respiratory syncytial virus (hrsv) and the infl uenza viruses (ifvs), the virus types within a species show evidence of being both distinct and discrete viruses that are independently recognized by their host and consequently independently infect their hosts. each hrv type is also genetically stable [ 144 ] . the hrv species circulate variably from year to year with evidence of epidemics of distinct types. a prospective longitudinal cohort study over 6 months examined hrv frequency and diversity in 272 specimens from 18 healthy children (0-7 years of age) [ 198 ] . a median of three hrvs and a maximum of six were detected per child. a similar outcome resulted from an australian cohort study [ 9 ] . genotyping reveals more of the hrv diversity at a single site than culture ever could with molecular studies fi nding between 34 and 70 distinct hrvs at a single location [ 9 , 128 , 199 ] . the number of additional hrv cases that occur in children outside of specifi cally defi ned symptomatic periods remain to be defi ned, with current studies indicating that a much higher number of hrv infections may occur. more comprehensive investigation of hrv type and illness will be undertaken during analysis of data from the australianbased observational research in childhood infectious diseases (orchid) study ( http://clinicaltrials.gov/show/ nct01304914 ). interestingly, the hrv-bs are often underrepresented, even when accounting for the smaller number of known hrv-b types [ 128 ] . a number of studies have not found any robust patterns between the circulating hrv types or species and clinical outcome, but the majority of studies seeking this information are short and sample infrequently, limiting their ability to fi nd the patterns they seek [ 128 ] . studies into the relative sensitivities of nasopharyngeal aspirates (npa) and swab sampling methods produce differing results, but generally, if seeking the best diagnostic yield for as many respiratory viruses as possible (i.e., seeking a laboratory diagnosis to support clinical decision making), npas are the sample of optimal choice. one study reported similar clinical sensitivities between swabs and npas for human coronaviruses (hcovs), ifvs, and hrsv, but reduced sensitivities using swabs for hrvs, human adenoviruses (hadvs), human metapneumovirus (hmpv), or parainfl uenza viruses (hpivs) [ 201 ] . a second study reported no difference in sensitivities for hrvs, hadvs, and hpivs but a reduced sensitivity for hrsv and ifvs when using swabs [ 202 ] . nasopharyngeal washes also yield more viral culture success than either nasal or pharyngeal swabs. nonetheless, many studies use nasal swabs as the sample of choice because they allow self-collection and involve much less discomfort than npas, and pcr has meant that infectious virus is not required, only viral nucleic acid which relaxes some limitations imposed by the need for rapid, careful, temperaturecontrolled, and expensive transport requirements [ 64 , 203 , 204 ] . bronchoalveolar lavage samples are best for seeking lrt etiologies, especially in adults where nasal wash viral loads can be low compared to those in children, but this is an invasive method with some risk attached [ 205 ] . hrvs infect all people, all around the globe. spread of hrvs is most obvious and frequent from child to child and from child to parent [ 206 ] . in populations of mixed age, the majority of hrv detections occur in children [ 128 ] . among 272 specimens from 18 healthy children, over a third (37 %) were hrv positive. children less than 5 years of age (44 % of whom were hrv positive) were shown to have more hrv infections and a wider diversity of hrv types than children more than 5 years old (28 % hrv positive) [ 198 ] . healthy adults in the military [ 54 , 207 ] , at university [ 208 ] , at home [ 209 -212 ] , and in the workplace [ 209 ] have also featured prominently in historical, culturebased, and volunteer infection studies and heavily infl uenced our view of hrv infection outcomes [ 64 , 206 ] . although studies of children in hospital-based populations usually report more signifi cant clinical outcomes (relating to the lrt) [ 213 ] than community-based studies, these data are still broadly applicable. hospital populations originate from the community and refl ect the more serious and perhaps fi rst exposures to the virus. hospital-based populations defi ne the potential of a virus to cause severe clinical outcomes. disease at this end of the spectrum has the strongest infl uence on future prioritization of therapeutic research and developments [ 214 ] . modern air travel contributes to the rapid spread of respiratory viruses as seen in their often frequent detection among travelers [ 215 ] including those with febrile illnesses [ 216 ] . apart from children, hrvs are found with the great clinical impact in the elderly (described as 60-90 years of age) with 50 % of aris positive for an hrv, sometimes with a greater burden of disease than ifvs [ 217 ] . those with asthma or copd are also affected by the ari triggering exacerbations of wheezing illness (see sect. 8.2 ). it is thought that this is not a different type of infection but rather a different response to infection by the host. wheezing can also result from infection in atopic people who do not have underlying asthma or copd. hrvs cause signifi cant impact in the immunocompromised, and this group is the only population to date that has been found to host truly persistent hrv infections (see sect. 5.7 ). because the hrvs are the largest group of viruses to infect humans, it is not surprising that they confuse differential diagnoses during pandemics and have key roles in co-detections and asymptomatic disease. the study of hrvs is the study of all respiratory viruses; while each can be considered in isolation, this will likely be detrimental to a greater understanding of respiratory virus pathogenesis. hrvs circulate throughout the year but usually with a bimodal peak in temperate locations in both hemispheres. the highest peaks, mostly defi ned using adult populations, are in the autumn (fall) and spring [ 64 , 66 , 211 ] (and, peculiarly, on a monday [ 218 ] ). the major winter dip in hrv prevalence closely coincides with the peaks of other respiratory viruses, particularly ifvs [ 219 ] and hrsv [ 66 ] . one hypothesis states that a miasma exists in the school classroom, of particular relevance to those who suffer asthma exacerbations, and this miasma maintains immune stimulation, which subsequently wanes among school children during holidays, to be challenged anew upon return to school [ 220 ] . it is clear that an interplay or interference takes place between viruses at the population level, particularly evident among rna viruses. there is a correlation between spiking spring and autumnal hrv case numbers and an asthma exacerbation "season" 10-24 days after return to school from holidays, in a range of climates [ 220 -223 ] . this was particularly obvious among asthma hospitalizations of children (5-15 years of age) in ontario, canada, which peaked at weeks 37-39 across a decade [ 223 ] . upon investigation, hrvs were the most prevalent of the viruses found in a 1-year analysis of emergency room presentations in ontario [ 223 ] . hrvs also predominate during "hay fever season" [ 172 ] . although a defi ned seasonality is not always found in the tropics [ 224 ] , this may sometimes be due to testing that does not include hrvs [ 222 , 225 ] or only some hrvs [ 226 ] . all the hrv types continue to circulate today, including those named in the earliest of the nomenclature assignments. at a single site during 12-24 months, 70 or more types can co-circulate [ 8 , 9 ] [ 174 ], dropping [ 198 , 227 ] if the study time frame at the site is shortened. a recurring hrv type, defi ned using molecular tools, accounted for 1.6 % of any virus detected in a birth cohort followed for 12 months [ 8 ] and, in another cohort, occurred twice in two children, within a 6-month period [ 198 ] . within a given year and across different years, it is apparent that hrv species exchange predominance [ 9 , 36 , 60 , 227 -229 ] . no evidence exists to satisfactorily explain this; however, herd immunity may be a factor. the use of cell and tissue culture underestimated the frequency of multiple infections in patients, most likely because the dominant virus out-replicated any others, or due to viral load differences, specimen quality issues, differing cell tropisms, or the triggering of an antiviral state by the fi rst virus. when the majority of respiratory viruses are sought using pcr techniques, multiple virus-positive specimens can comprise a third of those tested [ 230 ] , dropping to around a fi fth of ari episodes when fewer viruses are sought [ 217 ] . there is sometimes an emphasis on the high number of hrv cases that are identifi ed in the presence of another virus, and including hrv testing does raise the frequency of pathogen detection above one per sample [ 231 ] . coinfections, or, more correctly for pcr-based studies, co-detections (since pcr cannot determine infectivity), have been found to either increase [ 71 , 232 -236 ] or have no impact on the clinical outcome in their host [ 237 -241 ] , and so the issue of clinical relevance of co-detections is still uncertain. in extreme cases, half of all hrv detections can be found concurrently with another virus. on the surface, this is a signifi cant fraction, and yet 80 % or more of hrsv, hmpv, ev, and ifv detections and 71 % of hcov-nl63 detections can be found in the company of another virus [ 242 ] . other studies fi nd different, but still higher proportions of co-detections involving non-hrvs [ 217 ] . whether co-detections represent a particular synergism between the involved viruses, a differential capability to manipulate the host immune response, a sign of innocuousness for the most frequently involved virus [ 243 ] , or a chance due to overlapping seasons remains unclear. it is clear, however, that co-detections are not an anomaly or an error due to "overly sensitive" pcr tests; they are evidence of further biological complexity that, until recently, remained hidden from us. recent studies have shown that the initial impression of hrvs being overrepresented in these cases was incorrect. closer analysis of viral co-detections has revealed patterns [ 231 , 244 ] . these became clear when codetections were examined bidirectionally, not just how many hrvs were positive for virus x but also how many of virus x cases were positive for an hrv. whether in a hospital or a community setting, hrvs more often occur as the sole virus detected in aris [ 9 , 244 ] . considering their ubiquity, it is interesting that relatively low numbers of concurrent detections occur [ 245 , 246 ] , supporting the concept that hrvs have a direct role in the clinical outcome of their infection [ 247 ] . the hrv partnership with host immunity may be a mutualistic one, inadvertently imparting an advantage to the host by protecting against more cytopathic respiratory viral pathogens, while the host provides a vessel for hrv replication and transmission. studies of single respiratory viruses without being in the context of the respiratory virome are of limited value in drawing conclusions about clinical impact. much of the longitudinal epidemiology data previously relied upon to form assessments of hrv signifi cance was acquired using culture-based techniques. with improved and more comprehensive testing, patterns can be seen among the interactions of hrvs and other respiratory viruses. virus interference is a type of virus-virus interaction (vvi) that has been known for decades. vvi has recently been categorized into types [ 248 ] . at the population level, it has been noted that during trials of live attenuated ifv (laiv) vaccines, an interferon (ifn) response was triggered that protected vaccinees against off-target viruses for 7 days postvaccination [ 249 ] . this 1970 study went so far as to suggest such effects could be maintained for a prolonged period using a regime of consecutive schedule vaccinations, each separated by 7 days or more, during times of a prolonged epidemic [ 249 ] . a similar effect was produced using live ev vaccines (lev) to replace pathogenic ev types and interrupt outbreaks [ 250 ] . orally administered levs succeeded in their principal task but also reduced the incidence of aris during epidemics by 50 % overall [ 250 ] . this shows that immune activation in the gastrointestinal system generates an anatomically distinct protective effect and there may be a similar effect on the gut's infl ammatory status after respiratory virus infection. in contrast to the laiv results, the offtarget protective effect was reversed in a study using a trivalent inactivated ifv vaccine [ 251 ] . the mechanism underneath these opposing outcomes is unclear. during the heyday (1960s) of tissue culture for virus studies, a common biological assay for infection with hrv involved attempted infection of the culture with an enterovirus (ev) or hpiv-1 [ 252 , 253 ] . failure of the superinfecting virus to grow heralded the likely presence of a noncytopathogenic hrv. virus interference has been used to measure ifn in specimens through its inhibition of hrv growth [ 254 ] . more recently hrv-hadv dual pcr-positive cases were found less often than expected and harbored lower viral loads of hrv than did specimens from cases of sole hrv infections [ 255 ] . signifi cantly, the majority of these instances of vvi involve rna viruses [ 244 ] . it has been shown that dual infections of peripheral blood mononuclear cells (pbmcs) with viruses other than hrsv (including hrvs) induced immune responses similar to those of single infections, but coinfections including an hrsv resulted in reduced ifn-γ responses [ 71 ] . vvis are affected by the ability of each to moderate the host response against them. virus interference has also been identifi ed in virus positives as a series of patterns among respiratory specimens tested for up to 17 respiratory viruses (fig. 29.8 ) [ 9 , 244 ] . statistical analyses supported that many of the co-detections occurred in patterns, in particular that fewer co-detections involved an hrv than would have been expected by chance alone ( p ≤ 0.05). for some period, rna virus infection, especially the hrv group, may render the host less likely to be infected by other viruses and, by extrapolating to the community level, help constrict the epidemic periods of other viruses by reducing the number of fully susceptible hosts. virus interference as a feature of respiratory virus epidemiology can also be seen in results of other studies [ 256 ] . during an 8-week period that spanned peak 2009 h1n1 pandemic infl uenza season in wisconsin, it was infl uenza a virus (ifav) that seemed to dominate hrv in children with asthma who were sampled weekly [ 236 ] . whether this refl ects all ifv-hrv interactions or just those involving a novel ifv such as 2009 h1n is unclear. it was found that pbmcs from these children exhibited normal immune responses [ 236 ] . reports of subjects with continuous and extended (greater than 2-3 weeks) periods of hrv positivity [ 3 , 257 ] increased as pcr methods replaced cell culture for hrv detection. this had only rarely been recorded using culture [ 54 ] . hrv rna has been detected days prior to symptoms commencing and for as long as 5 or more weeks after they cease [ 3 , 258 -261 ] . studies that only defi ne the period between aris in children as that time when specimens are rt-pcr negative [ 3 ] will not detect overlapping serial infections (fig. 29.9 ). epidemiology that incorporates hrv typing generally does not fi nd chronic shedding [ 204 ] . hrv shedding normally ceases within 11-21 days, after signs and symptoms have stopped [ 3 , 9 , 44 , 75 , 85 , 260 ] . thus, the perception of persistence is probably due to serial or overlapping infections by multiple untyped strains [ 8 , 54 , 210 , 262 ] . few studies [ 263 ] have suitably addressed persistence in hrv infections involving healthy subjects since pre-and post-sampling clinical data are rarely described [ 80 , 264 ] . to date, true persistence-an ongoing detection of a single confi rmed hrv type-has been limited to individuals with underlying immunosuppression or immune dysfunction [ 260 ] . hrv-cs were detected more than three times longer in immunocompromised young patients than in immunocompetent children, with a mean of 16 versus 53 days [ 265 ] . multiple detection of the same hrv type (100 % identical hrv-1a sequence in each patient over time) extended to 4 months in hematopoietic stem cell transplant recipients. the proof of causality is as diffi cult to achieve as the proof of innocuousness when it comes to respiratory viruses and aris. the defi nition of "well" subjects prior to or at the time of sampling or inoculation is sometimes not clear, especially for young children who cannot reliably report symptoms [ 3 , 96 , 204 ] . often parents notice a symptomatic illness before an infection is detected in the laboratory [ 3 ] , supporting the importance of diaries in longitudinal home-based community studies. nonetheless, even with the support of telephone interviews and home visits, milder cold symptoms may be missed. it is not uncommon for an asymptomatic control to subsequently become symptomatic or have been symptomatic before sampling [ 8 , 266 ] . some studies employ sensitive symptom scoring systems [ 267 ] , but the criteria for being symptomatic are usually designed to describe and clearly discriminate overt or more "severe" illnesses, those with obvious and measurable signs. strict defi nitions help improve patient management and the commencement or better direction of treatment or cohorting. however, in research studies the arbitrary degree of severity required for reporting a symptomatic event often overlooks very simple changes in host biology due to a virus's replication. these changes to the norm are mild but nonetheless represent disease (a disorder of structure or function that produces specifi c symptoms or that affects a specifi c location and is not simply a direct result of physical injury) in the literal sense. such minor or short-lived, often unrecorded [ 3 ] , indications of infection include sinus pain, headache, sore throat, earache, watery eyes, fatigue, muscle aches and pains, and mood changes. within families, hrvs are frequently transmitted from vsig activated "shields up" a b children who are usually symptomatic [ 204 ] . infants frequently exposed to other children have more asymptomatic viral infections [ 8 ] . among infected adult family members, asymptomatic infections are more likely [ 204 ] . among older parents, whether their children live at home or not, asymptomatic infections are more frequent following hrv challenge than among adults without children or in younger parents [ 268 ] . in a study of viral species in age-stratifi ed cases and controls, signifi cantly lower viral loads were found in those without the required symptoms [ 269 ] . qpcr may prove useful to determine viral load cutoffs to address this issue in the future, although the respiratory tract is a diffi cult tissue for qpcr [ 200 ] . the high sensitivity of pcr-based methods has raised concerns over the clinical relevance of a virus-positive result [ 269 ] . it is clear that a proportion, around fi ve to 28 % of study-defi ned asymptomatic control populations [ 90 , 91 , 269 ] , are virus positive using sensitive pcr-based methods. this may vary up to nearly 50 % of cases when stratifi ed by age, virus, and season or when including highrisk populations [ 8 , 269 ] . every respiratory virus, even ifvs and hrsv, can be found in cases without symptoms at the time of specimen collection even after specifi c inoculation of adults [ 137 , 269 , 270 ] . this is a complex and incomplete story in need of more research, and so it is frustrating that positivity in asymptomatic people is often used to rank viral importance. better data are required from asymptomatic controls for any conclusion to be drawn about causality [ 266 ] , but this requirement often disregards the memory of a normal functioning protective host immunity. it is the host response that defi nes the degree of clinical severity for the infl ammatory disease that is the hallmark of an ari [ 271 ] . it is well known that previous exposure to a virus affords protection from the full clinical spectrum of disease upon repeat exposure to that virus. it should come as no surprise then that hrvs, which usually cause brief infection anyway, could well produce only minor signs and symptoms upon reinfection. the unique and extremely personal infection history of each member of a control group cannot be determined unless they are part of a longitudinal cohort. so, what do cohort studies, supported by comprehensive pcr-based testing, tell us about asymptomatic virus infections? some cohort studies do not look in asymptomatic children, seeking samples only at times of symptomatic illness [ 66 , 246 , 272 ] . a birth cohort of children enrolled and sampled when ill and every 6 months for 24 months identifi ed hrvs 14-28 % of infants and toddlers who had no nasal symptoms (defi ned solely by the presence of rhinorrhea) [ 273 ] . the childhood origins of asthma (coast) birth cohort followed 285 infants at high risk for allergies and asthma for 12 months and identifi ed hrv infections as preceding (mean age of fi rst detection, 4 months) those of hrsv (mean age at least 6 months), and hrvs were found in 35 % of asymptomatic versus 61 % of moderately to severely ill patients; the most frequently symptomatic children also had the greatest proportion of asymptomatic infections [ 8 ] . in a study of 58 children with asthma sampled weekly for 5 weeks during each of two peak hrv seasons, nearly two-thirds who were virus positive but not sensitized to at least one allergen showed no asthma symptoms, and nearly half showed no ari symptoms; in the children who were sensitized, less than one-third showed no asthma symptoms, and only a fi fth had no ari symptoms [ 227 ] . a convenience population of 15 healthy children (1-9 years old) without asthma were followed during at least three seasons, and picornaviruses were detected in 5 % of 740 specimens (21 % of infections) not associated with symptoms, the impact of hrv typing and of sampling based only on symptoms. the example provided here diagrammatically represents a single, hypothetical monitoring period, starting at time = 0, for a single individual. the period of potentially detectable hrv is indicated by an open box. if sampling occurred at each time point ( 0 -6 ) and hrv positives were genotyped, it would be apparent that three different strains infected the individual, although discerning hrv-x from hrv-z at time point 3 would require a molecular cloning approach. illness, in different forms, may have continued over the entire period depending on the symptoms required/recorded and the period of time represented by the monitoring period. in this case a clinical diagnosis may record only a single symptomatic episode. genotyping may not be performed, and sampling may be intermittent, and so association between viral type or species and disease is impossible. in the study examples indicated by ( a ) start and fi nish sampling or ( b ) symptomatic sampling, ( asterisks mark sampling times in fi lled bars), the laboratory data would have made only one or two identifi cations, respectively. in the third example, ( c ) frequent sampling of this type has previously led to conclusions of hrv persistence or chronic shedding; when combined with genotyping, it becomes apparent that different hrv types are present although 9 of the 25 infections came from households with an infected sibling [ 3 ] . in summary, there is clear evidence for the presence of hrvs in asymptomatic controls. a precise proportion cannot yet be defi ned. some study controls show signs of a "lead-in" period where rna positivity precedes an ari defi ned on follow-up, while others may have been defi ned as symptomatic if more symptoms had been accounted for. mechanisms and routes of transmission hrvs have been found at extra-respiratory sites. viremia was determined in the blood of children with lrt infection or pericarditis [ 274 , 275 ] , and hrv-c was more commonly associated with viremia than was hrv-a, supporting possible increased pathogenicity [ 274 ] . blood was also positive for hrv rna and infectious virus from infants at necropsy [ 276 , 277 ] , and hrv rna was detected in the plasma of children with asthma, bronchiolitis, or common cold [ 76 ] . an hrv was once isolated from feces [ 203 ] , and more recently higher than expected loads of hrvs were detected in fecal specimens from children with suspected meningitis and fever of unknown origin [ 77 ] , with gastroenteritis [ 278 ] , and in a child with pericarditis [ 275 ] . nonetheless, the nasopharynx is still considered the main site of focal virus production [ 279 ] , regardless of inoculation route [ 280 ] , and most studies of transmission routes have centered on the urt. in contrast to ifv and hrsv, hrv infection involves less destruction of tissue. ciliated epithelial cells are sloughed off in proportion to the severity of an hrv ari, but this damage is minimal and does not occur during the viral incubation period or with subclinical infections [ 137 , 281 ] . the incubation period between infection and onset of virus shedding into nasal secretions is 1-4 days with shed viral titers peaking in adults between days 2 and 10 [ 44 , 282 ] . the time until successful hrv transmission among adults in a childless family setting is usually 5-8 days and requires the donor to be shedding at least 10 3 tcid 50 at some stage, to have recoverable virus on the hands and in the nares, enough shared time, and a moderate to severe ari [ 283 ] . the lungs have been shown to host replicating hrv [ 260 ] , and the reader of such reports may be left with the perception that detection of hrv replication in the lrt explains all lrt symptoms. however, relatively few studies seek or identify true hrv replication in the lrt. while the overwhelming majority of lrt cases detect hrv from the urt, a correlation between urt positivity and lrt disease does exist [ 284 ] . it is well known from experimental inoculation studies that hrv infection can result from inoculation of the conjunctival sac after virus is moved through the nasolacrimal duct [ 280 ] . in these studies virus was commonly delivered by aerosol or intranasal instillation of 0.25 ml to 5 ml of suspension [ 43 -46 , 280 , 285 -287 ] . in the laboratory, hrvs can retain infectivity for hours to days on suitable, nonporous solid surfaces, especially if the inoculum remains damp [ 47 , 287 ] , which supports direct self-inoculation especially in the family setting and indirect inoculation via fomites [ 288 ] . in a trial to defi ne the movement of virus from a contaminated donor to a recipient via multiple surfaces or by hand-to-hand contact, 13 % (donor to objects to recipient) and 6 % (donor to recipient fi ngers) of the virus recoverable from the donor's fi ngertips were recoverable from the recipients' [ 289 ] . even under observation, eye rubbing (0.37 h −1 -2.5 h −1 ) and nosepicking (0.33 h −1 -5.3 h −1 ) occur frequently [ 47 , 290 ] , suggesting self-inoculation could outpace personal hygiene, particularly in the young. it was once thought strange that aris were so common, but isolation rates for the expected viruses were so low [ 36 , 291 ] . with a better understanding of the importance of preexisting antibody (something common among the predominantly adult volunteers used by many studies), the discovery of a third, unculturable species of hrv (still causing aris but impossible to isolate or detect using antibody-based systems for which no reagents existed), and a vastly improved diagnostic sensitivity, this is much less confounding. in the past, household cross infection, determined by ari, was low, about fi ve exposures to infected members required for infection [ 17 ] despite viral loads in nasal washings peaking at 1.6 × 10 5 tcid 50 /ml [ 44 ] . experimental transmission was also reportedly ineffi cient [ 45 ] . in contrast, "naturally" close-quartered military populations, interacting over 1-4 weeks, experienced rapid spread of hrvs to >50 % of the group [ 54 ] . the use of pcr recently clarifi ed this discrepancy, confi rming that frequent transmission in families is more common than culture-based studies had identifi ed, often resulting in asymptomatic infection among older siblings and parents [ 204 ] . pcr has helped defi ne the scope of viral rna, if not actual infectious virus, survival, and spread. transmission studies require infectious hrv, and so the hrv-cs do not contribute to the historical data. under crowded or intimate conditions and with more severe colds, transmission reaches 38-100 % [ 283 , 292 ] . in some studies, both large-and small-particle aerosols proved ineffi cient, supported by a low isolation rate from saliva (39 % compared to 65 % of hand washes and 50 % of nasal swabs) [ 44 , 47 , 293 ] and from only 8.3 % of participants exposed to large-particle aerosols [ 293 ] . in other human donor-recipient model studies however, aerosol proved to be the main transmission route among antibody-free adults [ 46 , 282 ] . the discrepancy may have been due to insuffi ciently long or intense exposure in the earlier aerosol experiments [ 45 , 267 ] . apart from particle size, spread of virus by aerosol is affected by existing nasal obstruction which can divert secretions from the nares to contaminate saliva, the presumptive source of virus in coughs and sneezes [ 44 ] . when exposed to 10 liters of a small-particle aerosol, 10 1 tcid 50 of hrv-15 was associated with fever and prominent tracheobronchitis in antibody-free (<1:2) adult volunteers but not when delivered via nasal drops or a coarse aerosol [ 46 ] . it has also been found that simple breathing releases hrv rna (the same type was also identifi ed from nasal mucous) from at least a third of adults and children with symptomatic aris and infectious hrv could be isolated from a fi fth [ 294 , 295 ] . it is apparent that hrvs accumulate at sites with heavy human traffi c, potentially forming a secondary source of infection. hrv rna can be detected from 32 % of ~47-hourold fi lters placed to sample air in offi ce buildings [ 296 ] . in aircraft, high effi ciency particulate air (hepa) fi lters have been found to harbor hrv rna more than 10 days after they were removed for servicing [ 297 ] . hrv infections trigger a vigorous proinfl ammatory immune response that is thought to drive the symptoms experienced as illness [ 271 , 298 , 299 ] , but they do not seem to actively prevent or interfere with the host's immune response the way most other viruses have evolved to do. there may be a role for repeated challenge by hrvs and other respiratory viruses leading to infl ammation and tissue remodeling. the host response to hrv infection can be broadly broken into the innate (very fast, encoded in the germ line, nonadaptive) and adaptive (slower to develop, reliant on t cells, b cells, and the generation of antibody) responses. while the innate system is "always watching," it is signifi cantly amplifi ed by virus infection. the adaptive response is initiated by the host's fi rst infection with a particular virus and then functions to limit subsequent infections through the production of neutralizing antibodies and amplifi cation of existing cell-mediated immunity. after virus-receptor binding and internalization, the earliest host cell immune response to an hrv infection is elicited by the innate immune system (fig. 29.10 ). epithelial cells represent the front line against hrv invasion although alveolar macrophages and dcs are better equipped to respond [ 300 ] and do so despite not hosting hrv replication directly [ 16 ] . virus detection is mediated by pattern recognition receptors (prrs) that have evolved to recognize conserved molecular structures shared among diverse pathogens. internal-or surface-mounted prrs include sentinels that specifi cally recognize picornavirus rna and protein and, in doing so, trigger an immune circuit that results in the production of ifns and subsequently hundreds of ifn-stimulated gene products. the innate response to viral infection hinges on inducing two type i ifns (initially ifn-ß then ifn-α), secreted cytokines that produce antiviral, antiproliferative, and immunomodulatory outcomes [ 301 ] . the type iii ifns (ifn-λ1 or il-29, ifn-λ2 or il-28a, and ifn-λ3 or il-28b) are also produced in response to viral infection in a range of cells, although their receptor is not as widespread [ 302 ] . the type ii ifn, ifn-γ, is produced by activated t cells and natural killer cells rather than in direct response to virus [ 303 ] . detection of viral components triggers protein signaling cascades that regulate ifn synthesis through the activation of viral stress-inducible genes (vsigs) [ 301 , 304 ] . these are sometimes expressed constitutively but upregulated after ifn induction following hrv infection [ 305 ] . released ifn-ß binds to the ifn-α/ifn-ß receptor in an autocrine (the same cell) and paracrine (neighboring cells) manner, starting a positive feedback loop for type i ifn production, the "second wave." vsigs include the antiviral proteins protein kinase r (pkr), 2′5′oas/rnasel, and the mx proteins [ 306 ] . ifn-α upregulates expression of mxa, 2′4′-oas, and pkr [ 307 ] . the mx pathway is also induced after virus infection but is not constitutively expressed [ 307 ] . depending on the sentinel system stimulated, there are different pathways to vsig activation. those vsigs with antiviral properties (e.g., mxa, pkr, 2′5′oas/rnasel) inhibit different stages of virus replication and strengthen an antiviral state in the host. while this state is well known, the nature of its induction by different respiratory viruses and the impact of induction upon the replication of other respiratory viruses are topics for considerable ongoing research. one pathway to ifn induction relies on the ifn-upregulated cytosolic sentinels retinoic acid inducible gene rig-i-like receptors (rlrs) rig-i (specifi c for ifav and others) and melanoma differentiation-associated gene 5 (mda5, specifi c for picornaviruses and others) [ 306 , 308 ] . these rna helicases recognize either rna with a 5′-triphosphate or distinct dsrnas, which results in activation of nf-κb leading to "classical" type i ifn induction [ 301 , 306 ] . studies into the innate response to hrv infection have been limited to the use of a very few easily cultured types. it is presumed that the result can be extrapolated to most if not all types. this is yet to be tested. rig-i is degraded by hrv-a16 [ 309 ] , ifn regulatory factor (irf)-3 homodimerization is interfered with hrv-b14 which limits ifn-β induction [ 310 , 311 ] , and mda5 is degraded by hrv-a1a but not hrv-a16 [ 312 ] . another pathway for recognizing hrv infection involves the toll-like receptors (tlrs), transmembrane prrs that terminate in an intracellular signaling region. the endosomally localized tlr3, tlr7, tlr8, and tlr9 recognize nucleic acids and are also involved in innate antiviral responses. tlr7 and tlr8 identify g/u-rich ssrna from endocytosed viruses, while tlr9 recognizes unmethylated cpg dna present in dna viruses [ 301 , 318 ] . tlr2 and tlr4 are found on the cell surface and recognize hrv or hrsv proteins, respectively [ 318 , 319 ] , and tlr3 recognizes dsrna. tlrs operate mainly, but not exclusively, in plasmacytoid dc [ 301 ] . the particular tlr that notifi es of an hrv incursion may depend on the method of virus approach [ 319 ] . tlr7 activation can reduce 2′5′oas and mxa mrna expression and ip10 protein in adolescents with asthma compared to healthy controls [ 320 ] . tlr3 activation did not result in a similar disparity [ 320 ] . it has been suggested that hrvs may have evolved with humans to such an extent that their symbiotic relationship serves to help train the human immune system [ 321 ] . intriguingly, within the hrv species, there are differences in the type and level of host response induced [ 322 ] which may refl ect receptor usage, route of entry and cell type infected, hrv species, or the degree of laboratory-adapted virus used during in vitro studies. after initial hrv infection, the innate response results in production of proinfl ammatory cytokines, vasoactive peptides, and chemokines that attract leukocytes, granulocytes, dcs, and monocytes (table 29. 2 ) [ 321 , 323 , 324 ] . the t-lymphocyte response to viral intrusion can be broadly categorized as t h -1-like and t h-2-like. other t-cell subsets exist, but most work in relation to hrv has been conducted on the earliest defi ned subsets. the t h -1 cellular response is important in managing cellular immunity and producing interleukin (il)-2 and ifn-γ. the t h -2 cellular response manages humoral immunity and stimulates b cells via il4 (initiating production of ige), il5 (infl uencing eosinophils), and il13 (crucial component of allergen-induced asthma). these two t-cell responses act in concert with epithelialderived chemokines (e.g., eotaxin) to promote the recruitment and activation of eosinophils and mast cells, contributing to chronic airway infl ammation and the hyperresponsiveness of airways to a variety of nonspecifi c stimuli [ 325 ] . t h-2 lymphocytes, opposing t h-1 lymphocytes, contribute to an allergic infl ammatory cascade, akin to what occurs to rid humans of parasites [ 326 ] . the t h-1 response can also be repressed by binding of microrna, which leads to an altered balance favoring a t h-2 state in mice and probably in humans [ 327 ] . regulatory t cells (t reg ) suppress allergic infl ammatory pathways and are therefore fundamental in protecting the airway from allergen sensitization [ 326 ] . considerable immunobiological research has focused on asthma exacerbation, with which hrvs are intimately involved. although upregulated by hrv infection, the t h -1 response is comparatively defi cient in people with asthma [ 328 , 329 ] . this is problematic as an increased t h -1-like cytokine response, deduced from higher sputum mrna ifn-γ/il5 values, speeds clearance of hrv and symptom amelioration [ 85 ] . one possible cause of the t h -1 defi ciency in people with asthma is inadequate maturation of type i and iii ifn responses due to reduced exposure to infections early in life [ 330 ] . the "hygiene hypothesis" [ 331 , 332 ] posits a pathway for an asthma etiology described [ 325 ] in terms of the young, unchallenged immune system, dependent on infections to stimulate the development of its t h-1-like functions. one theory suggests that hrvs play a central role in developing that effi cacious antiviral immunity, particularly in infancy, via their frequent, usually mild self-limiting infections [ 333 ] . genome-wide expression analysis of becs from healthy and asthmatic adult subjects after hrv-a1a infection revealed some signifi cant differences that were found between cell types and response to infection [ 61 ] . these included immune response genes (il1b, il6, il8, il1f9, il24) and airway remodeling genes (loxl2, mmp10, fn1) and an overall proinfl ammatory response and metabolic slowdown consistent with proteolytic cleavage of transcription factors by some hrvs [ 133 , 334 -336 ] in the infected cells. this study further noted some similarities to gene expression changes observed in brushings from people with mild asthma after allergen exposure and in bal cells from subjects with corticosteroidresistant asthma [ 61 ] . overall, hrv replication and the host transcriptional response to it were similar in normal or asthmatic bec cells [ 61 ] . this indicated, at least in adults, that something beyond the epithelial cell is an important contributor to more severe clinical outcomes in asthma. the application of inactivated hrv-b14 was found to promote release of il10 from monocytes (an immunosuppressive cytokine) and to inhibit the stimulation of il12 (drives t h -1 development) [ 337 ] . however, neither il10 nor il12 was signifi cantly induced in asthmatic adult volunteers in response to hrv-a16 compared to healthy subjects [ 338 ] . while ifn-α was detected after transfection of dcs with hrv-b14 ssrna, low tnf-α and il12 levels were also noted [ 16 ] . it was posited that the reduced il12 could indicate negatively affected local immunity possibly predisposing to secondary infections [ 337 ] . infection of stromal lung cells by hrv-b14 triggered exaggerated levels of the pleiotropic il11 (an il6type cytokine), akin to those triggered by hrsv, which were also detected in nasal secretions from children with wheezing [ 339 ] . other cytokine changes have been identifi ed in atopic adult volunteers challenged with hrv-a16. g-csf and il8 (chemo-attractant for neutrophils) levels rose in the urt (as examined by protein detection in nasal lavage) and lrt (mrna detection in sputum) with concomitant rises in blood and nasal neutrophil numbers [ 85 , 203 , 340 ] . the nasal epithelial cells of atopic individuals, especially in season, express more icam-1 than those of nonatopic adults [ 341 ] as do normal subjects infected by the major group hrv-b14 [ 341 ] . by contrast, ifn-γ and il8, which appear later postinfection, downregulate icam-1 expression in infected cells [ 191 ] and encourage infi ltration of neutrophils [ 342 ] , respectively. changes in icam-1 levels may modify participates in creation of an antiviral state; produced by and infl uences the maturation of dcs il-1β proinfl ammatory properties; enhances adhesion molecule expression including icam-1; induces il-2 receptor gm-csf a granulocyte and monocyte growth factor il-2 stimulates growth and differentiation of t and b lymphocytes and cytotoxic activity of nk cells and monocytes il-4 t h 2 differentiation, promotes ige synthesis il-6 activation, differentiation, and proliferation effects on t and b lymphocytes; induces c-reactive protein stimulating pyrexia il-8/cxcl-8 neutrophil chemoattractant resulting in neutrophilic, monocytic, and lymphocytic recruitment and degranulation activity il-10 anti-infl ammatory factor produced by monocytes that acts by inhibiting proinfl ammatory cytokines il-1, il-6, and tnf-α irf7 a master hub, regulating antiviral immunity ip10/cxcl10 chemoattractant for activated t h 1 and nk cells tnf-α proinfl ammatory activity similar to il-1 β; activates neutrophils; induces vascular permeability mpc-1 a monocyte attractant bradykinin potent infl ammatory mediator, increases vascular permeability tslp 3 an il-7-like cytokine that activates myeloid dcs to induce naive t cells into t h 2 cells producing il-4, il-13, and tnf-α; induced by hrv in the presence of il-4 bec bronchial epithelial cells, dc dendritic cell, irf interferon regulatory factor, ifn-γ inducible cytokine protein, nk natural killer, pbmc peripheral blood mononuclear cells, il interleukin, tnf tissue necrosis factor, tslp thymic stromal lymphopoietin t-lymphocyte-mediated cytotoxic or t h interactions with hrv-infected cells, upregulating receptor expression and encouraging eosinophil and t-cell infi ltration into the lower airways of asthmatic individuals [ 187 , 343 ] . before an hrv can enter a cell, it must pass through a defensive barrier of secreted anti-hrv antibody, mostly iga. the ease with which this passage occurs is proportional to the progression of clinical disease. healthy adult volunteers were found to develop iga by at least 3 days to 2 weeks after inoculation-about the same time as serum antibody-and retain peak levels for at least 8 weeks [ 178 , 344 -346 ] , falling faster than serum levels [ 347 , 348 ] . there is also some evidence for a degree of nasal immune memory [ 347 ] . volunteers with pre-study serum antibody could still be infected in some studies [ 46 , 210 , 292 ] , but not in others [ 349 ] . infection is more clear in volunteers without preexisting nasal antibody to experimental challenge virus; they become infected, exhibit more severe ari, and shed more virus for longer [ 292 , 347 ] . iga does not seem to modify illness severity or virus shedding, but high levels prevent reinfection by the initiating virus type. low levels or absence of iga does not prevent reinfection by the same hrv type, which may manifest as symptomatic or asymptomatic disease [ 349 ] . older children, adolescents, and adults have greater amounts of hrv-neutralizing antibody than young children [ 42 ] , accompanying a trend toward decreasing numbers of symptomatic aris with increasing age [ 218 , 350 ] . this feature raises an issue: did the use of older subjects in many common cold studies underplay the pathogenic potential of the hrvs because protective or partially cross-protective antibodies moderated the impact of infection? consequently, quantifying levels of type-specifi c serum antibody became routine practice prior to some studies. adult volunteer studies determined that no infections resulted if preexisting neutralizing antibody titers ≥1:16 existed; as levels grew from 0, so did levels of resistance to infection [ 43 , 210 ] . adults were protected by serum titers of 1:3-1:8 [ 209 , 210 ] . the trend was interrupted by adults in the 20-39 year age group, presumably because they had begun families and their young children acquired and amplifi ed currently circulating types from the community and transmitted them into a household that was either immune naïve or lacking suffi cient antibody or cell-mediated memory for protection [ 351 ] . traditional vaccine strategies were quickly ruled out as a prophylactic intervention for hrv illness because of the extensive antigenic variability that is a hallmark of the genus enterovirus [ 178 , 325 ] . however, if it were possible to identify "master" strains [ 352 ] that exhibit suffi cient antigenic cross-reactivity to induce broad heterotypic responses against many other hrv strains, then an effective vaccine could still be possible. in fact, boosting host immunity to an hrv type by repeat infection does heighten immunity to one or more other types [ 44 , 353 ] . the highest of these heterotypic antibody titers develop against those types with the highest preexisting antibody levels [ 352 ] . the fi rst description of a unifying hrv numbering system recounted the appearance of minor serological cross-reactions, which were removed by modifi cation of the technique [ 57 ] . subsequently, cross-reactions were better defi ned during experimental inoculation when multiple hrv immunogens and antigens were used to deduce the extent of heterotypic responses [ 56 , 210 , 352 , 354 ] . less promising for hrv vaccinology was the description of antigenic variation within hrv types which suggested that immunity to one variant of the type might not protect against infection by other variants [ 355 , 356 ] . the "prime strain" is a specifi c antigenic variant of a prototype hrv type that is neutralized to a lesser extent by antisera from the prototype, while yielding antisera that effectively neutralize both itself and the prototype [ 357 ] . another form of this cross-neutralization is ascribed to the "intertypes," which are hrv isolates that share a lower-level serological relationship with a pair of hrv strains, which themselves share neutralizing reactivity, e.g., hrv-a36 and hrv-a58 [ 358 ] . the low-level reciprocal neutralizing activity was not equivalent in both directions; anti-hrv-a36 sera had a higher titer for hrv-a58 than anti-hrv-a58 sera did for hrv-a36 [ 358 ] . over 40 strains were linked directly by such one-or two-way cross-reactions or indirectly through two or more strains. hrv-a67 and hrv-a28 are linked via hrv-a11, hrv-a13, and hrv-a32 (anti-a11 serum neutralizes hrv-a28, anti-a13 neutralizes hrv-a11, and anti-a32 neutralizes both hrv-a13 and hrva-a67 [ 358 , 359 ] ). a surrogate molecular method which provided insight into these interrelationships, perhaps expanding upon them to identify useful patterns for vaccine immunology purposes, would be most welcome. in summary, heterotypic immunity and hrv intertypes might be exploitable features of hrv immunobiology that could confer maximum protection upon the host from the minimum number of hrv types [ 358 ] . hrvs circulate in great numbers, and any specifi c roles for distinct hrv types in initiating disease remain to be defi ned. the relatively inconsequential common cold is the most frequent manifestation of viral infection in humans, with 30 to >80 % of colds positive for an hrv [ 69 , 360 , 361 ] . furthermore, aris due to hrv infection can exacerbate or result in a much greater burden of disease in those with asthma, copd, or cystic fi brosis. other complications include otitis media, pharyngitis, and wheeze in atopic people without asthma. the role of viruses in the origin of some of these diseases or their exacerbation is still unresolved. the lrt disease may mask the urt nature of the infection, favoring clinical diagnosis of an lrt illness. interestingly, during the 2009 h1n1 pandemic, much of the parentinitiated healthcare visits from a birth cohort in the united states were not due to pandemic virus but hrv and hrsv [ 362 ] . there is no known natural murine rhinovirus on which to base a small animal model of hrv infection, and mice are not natural hosts for hrvs. a recently developed model of airway disease using mengovirus (a picornavirus infecting rodents) may yield valuable in vivo airway infection and infl ammation data [ 363 ] . hrvs are often detected in neonates and infants with lrt signs and symptoms because the very young have narrow, immature airways and are more signifi cantly affected by airway swelling, excessive secretions, and smooth muscle contraction [ 364 ] . this may also be due to the relatively naive immunity of very young children. much of the more severe disease in hrv-positive children occurs in the youngest of them. some key examples are addressed below. for the common cold, as for any illness, accurate epidemiology and burden of disease data underpin the prioritization of preventing, treating, and further researching the etiological agent. to assign funds for researching the agent, health policy makers also need to understand how effi cacious and costeffective the development of an intervention will be [ 365 ] . the host immune response to hrv replication is the main cause of the signs (quantifi able fever, rhinorrhea) and symptoms (feeling of fever, myalgia, headache, fatigue, and mood change) of a cold that the host experiences [ 321 , 366 , 367 ] . a feature of common colds is increased vascular permeability which, enhanced by kinins, results in increased plasma protein (albumin and immunoglobulin [ig] g) levels in mucus, approaching the levels in serum [ 368 ] . histamine levels do not rise in nasal secretions of otherwise healthy cold sufferers [ 368 ] . during the resolving phase of the ari, glandular proteins (lysozyme, siga) predominate [ 346 ] . the common cold syndrome is also described as rhinosinusitis (the agglomeration of rhinitis and sinusitis since they frequently clinically coexist) [ 369 , 370 ] . this consists of nasal discharge or rhinorrhea, nasal obstruction, sore throat, sinus pain, headache, sneezing, watery eyes, cough, fever, fatigue, muscle aches and pains, and mood changes [ 367 , 371 ] . these are caused directly or indirectly by viral infection; cough is the result of vagus nerve irritation by mucus; sneeze results from trigeminal nerve irritation; sore throat is likely due to the action of prostaglandins and bradykinins; and fever, psychological effects, fatigue, and myalgia are mediated by cytokines [ 367 ] . hypertrophic adenoids have also been found to have a high proportion of viral, especially hrv, occupation regardless of host symptomatic state [ 372 ] . observation of natural culture-confi rmed hrv colds in adults noted that cough usually started by day 1 and was more persistent up to 9 days later [ 264 , 373 ] . rhinorrhea, sneezing, and sore throat were reported by half or more of patients and headache by at least a quarter of cases [ 207 , 373 ] . as neutrophils accumulate at the site of primary urt infection, the myeloperoxidase in their azurophilic granules creates the yellow-green coloration of nasal mucus that was once considered a sign of bacterial superinfection [ 271 , 367 ] . a common cold caused by an hrv cannot be clinically distinguished from one that caused by any of the other respiratory viruses [ 207 , 371 ] . as is likely for a single hrv type, once the host has been infected by an hmpv, hpiv, ifv, etc., a secondary exposure to that same virus type will produce less severe clinical outcomes due to pre-primed host immunity. asthma is a clinical diagnosis made on the basis of patient history, physical examination, assessment of airway obstruction or reversibility, and response to bronchodilators [ 374 ] . it is a complex chronic respiratory disease involving airway infl ammation, airfl ow obstruction, and airway hyperresponsiveness, which manifests as recurrent reversible attacks with deteriorating asthma control that are generated by interactions between infectious agents and other environmental and genetic factors that remain incompletely characterized [ 375 ] . the mechanistic role for hrvs in asthma inception and exacerbation is not yet defi ned [ 364 , 376 ] but is being revealed as the extremely complex interplay between infl ammation due to virus versus that due to atopy is explored [ 315 ] . possible virus-host interactions include (i) severe hrv infection of healthy infants which may result in subsequent development of asthma; (ii) hrvs may trigger asthma in children with a genetic predisposition toward atopy; (iii) repeated mild infections may protect against more asthmogenic/cytopathic viruses or the overdevelopment of the t h 2type response; and (iv) hrvs may simply exacerbate that which already exists [ 377 ] . it is unclear if the risk of atopic asthma during infancy is increased by aris which affect the development of the immune system, or whether aris lead to asthma development in children with a genetic predisposition to more severe responses to infection [ 325 , 343 , 378 ] , or a mix of both. in children with asthma, viruses have been detected in at least 77 % of exacerbations (65 % picornaviruses, probably hrvs [ 379 ] ) and in 50 % of adults [ 380 ] . acute wheezing episodes (including bronchiolitis and acute asthma) are a frequent, epidemic, and seasonal lrt manifestation of urt respiratory virus infection of children from all ages, especially during the fi rst year of life [ 214 , 381 -385 ] . bacteria are not major factors in wheezing exacerbations [ 386 ] . wheezing is blamed for high socioeconomic and healthcare costs, overuse of antibiotics, being the primary cause of hospitalization among children, and, rarely, for death [ 109 , 387 , 388 ] . traditionally hrsv infection has most often been the virus causally associated with expiratory wheezing, wheezy bronchitis, or asthma exacerbations because of the virus's well-known ability to infect the lrt, its more frequent detection in some studies [ 386 ] , and the low perceived likelihood of urt viruses such as hrvs replicating in the warmer lrt. nonetheless, periods of epidemic wheezing in the absence of high rates of hrsv detection are common [ 383 , 389 ] . hrvs even predominated in some culture-based studies of wheeze [ 384 , 390 ] . the coast study used sampling criteria that were intentionally designed to investigate the role of hrsv in illness, but instead indicated that hrvs were the most important predictor of subsequent wheezing in early childhood, and this is supported worldwide [ 224 , 391 , 392 ] . the asthmatic airway is characterized by an infi ltration of eosinophils and th2-type t cells (th2 cells) [ 393 ] . in those with an atopic background, eosinophilia was more common, and the virus isolation rate was higher than in the nonatopic group [ 394 ] . the cytokine and eosinophil activation profi les for hrsv-induced wheezing differ from those induced by hrv in which il5 is signifi cantly higher in serum and nasal aspirates than for hrsv [ 118 ] . ip10 was the only cytokine signifi cantly elevated in all symptomatic wheezing groups [ 118 ] . signifi cantly higher rates of hrv detection with more obvious lrt symptoms are more common in children with asthma than in non-asthmatic populations [ 380 , 388 , 395 , 396 ] . exacerbations of asthma are often preceded by a symptomatic rather than asymptomatic hrv infection [ 17 , 379 , 388 , 397 -399 ] although, in some instances, an exacerbation is the only sign of infection [ 400 ] . reduced peak expiratory volume in children is especially associated with detection of respiratory picornaviruses [ 379 ] . severe "wheezy bronchitis," a historical term describing an acute illness with preceding ari and characterized by cough, wheezing, breathlessness, and mucous production, was more often positive for a virus than mild disease [ 394 ] . even the use of culture found that hrvs predominated in both urt and lrt (sputum containing becs) or combined respiratory tract samples [ 394 ] . bacteria were often present with ifv, but not with hrvs [ 394 ] . the airway epithelial cells form a physical barrier in addition to their roles in immune surveillance and regulatory control [ 393 ] . however, the asthmatic bronchial epithelium is compromised by incomplete tight junctions that are more sensitive to airborne pollutants [ 401 ] and most likely to allergens and respiratory viral infections. this is further specifically disturbed by hrv infection which reduces expression levels of tight and adherens junction proteins [ 402 ] . in those with asthma, the presence of an hrv can induce illness that, while often more severe than in non-asthmatics, has been associated with signifi cantly different hrv load or duration of hrv rna detection in people with asthma compared to those without [ 403 ] . hrv-c types are often detected in more serious clinical outcomes than hrv-a or -b [ 265 ] although hospitalizations may be fewer for hrv-cs than the other species [ 404 ] . aom is diagnosed by middle ear effusion (otorrhea) with simultaneous signs and symptoms of ari including fever, earache, rhinitis, cough, sore throat, chest wheeze, nocturnal restlessness, irritability, poor appetite, diarrhea, and vomiting. transient abnormal (negative) ear pressure upon tympanometry occurs in two-thirds to three quarters of uncomplicated colds among healthy children [ 405 , 406 ] . aom is a frequent reason for outpatient antibiotic therapy which can reduce the time to resolution of symptoms in infants and has been attributed to reducing the overall hospital burden of aom [ 407 -412 ] . since a longitudinal day-care study in 1982, the association between aom and viral urt infection has been coalescing, and it is now clear that aom often occurs with or shortly after a viral ari, most frequently in the young and occurring more often during winter than summer [ 413 , 414 ] . the use of infl uenza vaccines reduced aom occurrence by a third during an epidemic period [ 415 ] , but the use of pneumococcal vaccine did not reduce the occurrence of aom overall, just that relatively small fraction (6 %) due to the target bacteria [ 416 ] . the isolation by culture and pcr detection of viruses from middle ear fl uids and the refractory nature of some aom cases to antibiotic therapies confi rmed that viruses play an important role in this illness [ 409 , 414 , 417 ] . studies relying on underperforming culture-based techniques underestimated the role for viral aris [ 414 , 418 ] , but other studies using pcr techniques and including hrvs found them to be the most frequently detected virus in middle ear fl uids and nasopharyngeal secretions [ 409 , 417 ] . the use of pcr has identifi ed respiratory viruses, most often hrvs, in nasal secretions of 50-70 % of children with aom [ 66 , 413 ] . because virus is often detected in the nasopharynx at the same time as the middle ear fl uid, the question of the relevance of a pcr positive is a valid one [ 419 ] . picornaviruses have been detected in 30 % of nasopharyngeal swabs taken during cold season from aom-prone infants and young children, and large quantities of hrv rna have been detected by in situ hybridization of adenoid tissues from 65 % of children with recurrent aom and/or adenoid hyperplasia [ 259 , 420 ] . in a cohort of children followed from 2 to 24 months and using culture-rt-pcr, hrvs in the urt were the second most frequent pathogens associated with aom, after hrsv [ 66 ] . viruses, most often hrvs (30.8 % of aom with ari), were also detected concurrently with non-ari periods associated with aom episodes (14 % of aom without ari) [ 418 ] suggesting that aom may be the only manifestation of some hrv aris, just as wheezing sometimes is. in the united states, 180 subjects were enrolled and followed in a birth cohort until the fi rst aom episode or between 6 and 12 months of age 362 ]. hrvs accounted for 55 % of viruses detected and 69 % of specimens with a single virus detected. this dominance was maintained even through the 2009 h1n1 infl uenza pandemic [ 362 ] . in the day-care aom study mentioned above, primary acquisition of streptococcus pneumoniae or haemophilus infl uenzae had minimal importance as an initiation factor for aom with effusion, but nasopharyngeal colonization was important [ 413 ] . animal studies have shown that virusbacteria interactions have a role in nasopharyngeal colonization and aom development [ 414 ] . positive correlation has been made between hrv detection in aom-prone children and moraxella catarrhalis infection as well as a tendency toward the copresence of streptococcus pneumoniae [ 259 ] . the presence of hrv-b14 was shown to increase adherence of s. pneumoniae in human tracheal epithelial cell cultures [ 421 ] . it is believed that these three bacterial pathogens can colonize without symptoms until a viral ari shifts the balance toward a cytokine-mediated infl ammatory state [ 419 ] . other diseases in which hrvs are often detected this disorder of older patients encompasses emphysema (alveolar destruction) and chronic bronchitis (large airway infl ammation with chronic mucous production) and describes a long-term obstruction to airfl ow in the lung (compared to asthma which is a reversible obstruction with normal fl ow between exacerbations). while bacteria are found in half of all exacerbations, antibiotic therapies have often yielded poor outcomes [ 422 ] . hrv infections result in more copd exacerbations (~66 % of cases [ 92 ] ) than any other virus identifi ed to date [ 422 , 423 ] . an experimental human model of hrv infection in copd provided preliminary evidence that hrvs cause exacerbations [ 286 ] . viral culture associated symptomatic hrv infections with exacerbations among chronic bronchitics, including cases of isolation from sputum (lrt sample) in the absence of hrv in the urt [ 424 ] . adding the measurement of an infl ammatory marker in the serum, like il-6, further improves the speed of predicting an infectious etiology for exacerbations of copd [ 425 ] . pneumonia is a disease that often occurs early in life, is responsible for millions of deaths each year [ 426 ] , and is caused by viral and/or bacterial infections. a diagnosis of pneumonia requires a radiologically confi rmed infl ammatory infi ltration of the lung tissue. childhood communityacquired pneumonia (cap) is common in developing countries [ 427 ] . cap also complicates existing chronic medical conditions and takes advantage of immunosenesence [ 428 ] . the role of hrvs in contributing to the development of bacterial pneumonia is likely underestimated [ 426 , 429 ] . determining an etiology is confounded by the rarity of obtaining lrt specimens, by short-term studies, and by the complex milieu of viruses and bacteria involved. less invasive sampling of the urt permits more routine sampling and screening, and so convenience and reduced risk have led to the detection of putative pathogens in the urt with the general assumption that they account for lrt disease, especially in children under the age of 5 years [ 430 ] . pneumonia studies are complicated by the lack of a suitable control group; sputum is not produced from the healthy lower airway and needle aspiration, while a gold standard is also a hospital procedure with some risk [ 431 ] . studies that are comprehensive and use sensitive molecular testing are also rare for the study of cap etiology. when used for cap investigations, pcr methods almost double the microbiological diagnoses over conventional culture and serology techniques, especially improving the identifi cation of mixed infections and fastidious viruses [ 432 ] . rapid diagnosis aids management and helps make decisions about treatment, while prolonged searching for an etiological agent leads to further invasive testing [ 256 , 433 ] . at least a quarter of clinical cap cases remain unsupported by microbiological fi ndings [ 241 , 432 ] . infections causing pneumonia vary with age and vaccination status [ 433 ] . viruses can be detected in up to 90 % of infants (1-12 months of age) with pneumonia, and these cases follow a seasonal pattern [ 427 , 433 ] . bacteria can also be detected in over 90 % of infants and older children, the elderly, and those with severe cap [ 432 , 434 ] . studies that predated the use of pcr pronounced hrsv, followed by hrvs, the major viral contributors to cap, with viruses comprising 27-72 % of childhood pneumonia cases [ 429 , 434 ] . in the pcr age, the role of hrvs has received increasing attention, and they are increasingly the major viral group detected from both urt and lrt (sputum) specimens of children with cap. this holds true even when studies extend across 1 or more years, which presumably would account for seasonal variation in virus prevalence [ 241 , 256 , 426 , 434 ] . it is suspected that viruses such as hrv prepare the way for subsequent bacterial infection in some direct or indirect fashion [ 65 , 259 , 420 , 435 ] . there are laboratory data which support this [ 436 ] as well as observational data showing a high proportion of hrvbacterial co-detections [ 256 , 437 ] . mixed infections including viruses are a possible cause of antibacterial treatment failure and sometimes a puzzle for physicians. mixed infections occur frequently in lrt diseases such as pneumonia, which is not surprising since new techniques make it clear that the lungs are not the sterile environments we once thought [ 427 , 434 , 438 , 439 ] . viral-bacterial coinfections can comprise 15 % of patients, while viral-viral (2-30 %) and bacterial-bacterial (1-7 %) are much less common [ 230 , 241 , 256 , 434 , 437 ] . hrsv or hrv is often co-detected with s. pneumoniae in urt samples [ 256 , 437 , 440 ] . hrv detections dominate in younger children with pneumonia during peak hrv seasons, although frequently in co-detections with other viruses [ 230 ] . acute bronchitis (less than 4-week duration in children) is defi ned as a sudden cough that often results from large airway infection and frequently involves viruses. croup or laryngotracheobronchitis (viral or recurrent [ 441 ] ) is a common lrt illness in children that includes the trachea and larynx as well as the larger airways, resulting in a barking cough. patients with croup most often have a viral infection with some role for hrvs, although the extent of this is unclear [ 441 , 442 ] . despite testing, a third of cases remain without a viral etiology [ 441 ] . tracheobronchitis resulted from some hrv-a15 infection of volunteers [ 46 ] . chest pain and cough have been reported in half or more of adults with hrv infection [ 207 ] as well as in children and adults with hrvs detected during exacerbations of bronchitis, with or without an associated ari [ 443 , 444 ] . bronchiolitis occurs seasonally, especially in winter, in infants (1-12 months of age), affecting the small peripheral bronchioles. winter is the peak season for hrsv circulation, but not usually for hrv. bronchiolitis is a clinical diagnosis encompassing various disease entities and is most often reported in association with detection of hrsv, a winter virus [ 445 , 446 ] . however, hrvs make up the majority of hrsv-negative bronchiolitis cases [ 128 ] , and hrvs are co-detected with hrsv for which hospitalization is prolonged compared to cases positive for either virus alone [ 447 ] . those children positive for an hrv during a clinically diagnosed bout of bronchiolitis have a signifi cantly higher risk of recurrent wheezing in the subsequent year than those in whom another virus is detected [ 446 ] . hrvs were reported in over fi vefold more cases of bronchiolitis than hrsv among patients in a 2-year prospective cohort of very low birth weight infants in buenos aires, argentina [ 448 ] . after a viral ari, some proportion of infections may be complicated by sinusitis (infl ammation of the sinus mucosa), the extent of which may be underestimated in children if the ari is mild and unattended by parents [ 11 ] . symptoms may include sinus pain, headache, facial pain, discolored nasal discharge, postnasal drip, cough, sore throat, malaise, and sometimes fever (more so in children) [ 367 , 449 ] . the precise role for viruses and bacteria in sinusitis is still unclear [ 450 ] . sinusitis is a common comorbidity in those with asthma [ 451 ] . the in situ presence of hrv-b14 rna in maxillary sinus epithelium was reported in seven of 14 adults with acute sinusitis [ 452 ] . hrvs were also detected by pcr in half of adults with acute maxillary sinusitis; half of the hrv positives were negative for any bacteria [ 65 ] . the common cold is often associated with computed tomographically confi rmed sinus cavity occlusion or abnormality in adults with self-diagnosed aris [ 367 , 453 ] . magnetic resonance imaging identifi ed reversible abnormalities of the paranasal sinuses in a third of healthy adult volunteers following challenge with hrv-a39 [ 454 ] . further evidence of the tropism of hrvs for sinus tissue comes from it being, so far, the only successful host for in vitro hrv-c replication [ 63 ] . culture-and serology-based testing has shown that virus infections in cystic fi brosis (cf) patients occur with the same prevalence as the general community, but the consequences of infection are more obvious or severe. these include deterioration of lung function, cough, increased expectoration and weight loss, and a synergistic increase in bacterial growth or acquisition of new bacterial infections [ 107 , 455 -458 ] . the mechanism behind the acquisition of new bacteria is still unknown and not always observed [ 459 ] , but may involve a reduction in the host's immune response or viral damage to the respiratory epithelium. there is circumstantial evidence that hrv infections have been associated with respiratory exacerbations in cystic fi brosis patients [ 459 , 460 ] , albeit in very low numbers by nonmolecular studies [ 461 ] and without a significantly different clinical outcome from non-hrv aris in these patients [ 107 ] . molecular methods have not yet been applied regularly, thoroughly, and systematically, but they generally fi nd hrvs to be prominent among cf children with ari-associated respiratory exacerbation and involved in mixed viral-bacterial infections [ 459 ] . hand washing and disinfectant wipes have been shown to be effective methods of interrupting transfer from fomites to the nose or to conjunctivae [ 87 , 288 , 293 ] . however, with eye rubbing, face touching, and nose-picking occurring frequently [ 47 , 290 ] , self-inoculation often outpaces personal hygiene, particularly in the young. hand disinfection is frequently recommended for prevention of hrv infection but has not been supported by controlled clinical trials in a natural setting [ 462 ] despite good results in experimental tests [ 463 ] . ethanol-containing disinfectants were more effective than simple hand washing with soap and water for removal of hrv-a39 inoculum, as assessed by culture, and the inclusion of organic acids afforded a residual antiviral effect [ 463 -465 ] . however, continual hand washing with extra ingredients resulted in skin irritation [ 462 ] . the experimental testing [ 463 , 464 ] may have been biased by short study periods, the absence of a mucus carrier to mimic natural surface deposition and overly stringent control over virus application/hand disinfection compared with the natural study. additionally, the natural setting study used pcr [ 462 ] which detects hrvs more often than culture. the disparity between outcomes may also refl ect the contribution of airborne hrv transmission. because of the absence of a vaccine or specifi c antiviral, the most popular method of intervention in uncomplicated hrv aris is treatment of the symptoms. this is achieved using analgesics, decongestants, antihistamines, and antitussives. due to a lack of studies, data are limited on the effectiveness of over-the-counter common cold medications for children [ 466 ] . anticholinergic agents have proven useful to reduce rhinorrhea [ 467 ] . for controlling symptoms in those with exacerbated asthma, most of which do not require hospitalization, bronchodilators and oral corticosteroids are the main treatments [ 468 ] . the interruption of proinfl ammatory immune responses or specifi c signaling pathways using steroids, or other novel therapeutics, may prove to be a more robust approach for treating hrv infections; they have not been successful for hrsv [ 325 ] . when initiated early in the illness, a combination of antiviral (ifn-α2b) and anti-infl ammatory (chlorpheniramine) components showed promise for interrupting nasal viral replication and symptoms [ 469 ] . antiviral agents (table 29. 3 ) require early application to effectively precede the pathogenic immune response to hrv infection [ 325 ] , but they often fail to reproduce their in vitro successes in vivo. most antirhinoviral drugs are based on capsid-binding agents (fig. 29.11 ) . additionally, oral delivery can complicate drug safety because this route increases the risk of systemic side effects compared to a nasal or topical route, but these risks must be considered alongside the disease to be treated; drug side effects are disproportionately severe compared to a common cold than to a severe asthma exacerbation. a systemic route is benefi cial if an effect is sought on hrv replication sites that are otherwise inaccessible, such as those not associated with respiratory tract illness [ 470 ] . the recent discovery of the new species, hrv-c, has shone a bright light on how little was known about the hrvs. the hrv-cs and also the newly discovered hrv-as and hrv-bs are fastidious in culture, with a single report of hrv-c growth in primary sinus tissue, and the identity of a cellular receptor still unknown. thus, it is diffi cult to proceed in many areas, including basic virology, seroepidemiology, immunobiology, and antiviral testing. determination of the receptors for these new hrvs would aid the search for a more accessible culture system. there would be great interest in a vaccine for some or all of the hrvs, but with increasing evidence of the interactions between hrvs, their hosts, and other respiratory viruses, it may not be wise to interfere before we fully understand what the impact of losing a constantly circulating hrv challenge would be. antivirals specifi cally targeting the hrvs may be a better bet, but routine hrv testing and genotyping will fi rst need to be more widespread as surveillance for antiviral resistance will be an important component of monitoring the success of any intervention. studies to determine whether there are differences in clinical and immunobiological impact between the many different types are lacking but would greatly improve our ability to plan future routine testing, understand all the clinical responses to the diverse hrvs and to outbreaks of ari, and improve hrv epidemiology. it is interesting to note that the hrv-bs are signifi cantly underrepresented 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effi cacy of intranasal pirodavir (r77975) in experimental rhinovirus infection combating enterovirus replication: state-of-the-art on antiviral research rhinovirus chemotherapy a comparison of the anti-rhinoviral drug binding pocket in hrv14 and hrv1a a new oral rhinovirus inhibitor bta798 human rhinovirus 3c protease as a potential target for the development of antiviral agents in vitro resistance studies of rupintrivir, a novel inhibitor of human rhinovirus 3c protease effi cacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection inhibitory effects of tiotropium on rhinovirus infection in human airway epithelial cells levofl oxacin inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells pellino-1 selectively regulates epithelial cell responses to rhinovirus azithromycin induces antiviral responses in bronchial epithelial cells suggested reading newly identifi ed human rhinoviruses: molecular methods heat up the cold viruses do rhinoviruses reduce the probability of viral codetection during acute respiratory tract infections? human rhinoviruses: the cold wars resume proposals for the classifi cation of human rhinovirus species c into genotypically-assigned types cold wars: the fi ght against the common cold acknowledgments we wish to sincerely thank the following for valuable discussions: john upham, anne chang, danielle wurzel, michael nissen, ron turner james gern, and stephen b. liggett. we are grateful for the extreme patience of corin and ronan mackay, slightly less so for their frequent provision of our fi rsthand experience in hrv clinical symptoms. key: cord-009773-pbm2vs5h authors: trigg, c. j.; nicholson, k. g.; wang, j. h.; ireland, d. c.; jordan, s.; duddle, j. m.; hamilton, s.; davies, r. j. title: bronchial inflammation and the common cold: a comparison of atopic and non‐atopic individuals date: 2006-04-27 journal: clin exp allergy doi: 10.1111/j.1365-2222.1996.tb00593.x sha: doc_id: 9773 cord_uid: pbm2vs5h background cold virus infections are associated with asthma attacks and with increased bronchial responsiveness even in normal subjects. possible mechanisms include epithelial damage, interaction with adhesion molecules or with t‐helper cell subsets. objective to determine whether colds increase lower airway inflammation, comparing atopic with non‐atopic normal subjects. methods thirty healthy volunteers (15 atopic) took part. basehne tests included viral serology. microbiological culture and polymerase chain reaction for rhinovirus infection (hrv‐pcr), histamine bronchial provocation and bronchoscopy. twenty subjects (eight atopic) underwent repeat tests when they developed a cold. results forced expiratory volume in one second (fev(1)) was significantly lower during colds (‐0.19l [95% confidence mterval ‐0.10, ‐0.29], p= 0,0004) and there was a significant increase in bronchial responsiveness (+0.62 doublings of the dose‐response slope [+0.24, +1.00], p=0.003). eight subjects (two atopic) had a diagnosed viral infection: two hrv. three coronavirus (hcv), one hrv + hcv, one parainfluenza iii(pi) and one respiratory syncytial virus (rsv) (also haemophilus influenzae). in biopsies, during colds, total eosinophils (eg1(+)) increased significantly (geometric mean 6.73‐fold [1.12,40.46], p=o.04). activated eosinophils (eg2(+)) only increased significantly in the subgroup without diagnosed viral infection and particularly in atopic rhinitics. t‐suppressor (cd8(+)) cells also increased significantly (median +178.3 cells mm(2), p= 0.004). epithelial expression of intercellular adhesion molecule‐1 (icam‐1) expression increased in four atopic rhinitics during colds. bronchial washings showed a significant increase in neutrophils (gm 1.53‐fold [1.04,2.25], p= 0.02). conclusion lower airway inflammation was present in atopic and non‐atopic normal subjects with colds. atopic subjects differed in that they were less likely to have positive virological tests and were more likely to show activated eosinophilia in the lower airway, despite a similar spectrum of symptoms. attacks with upper respiratory tract viral infections, a majority of which are rhinoviral [1] [2] [3] [4] [5] . until recently. there is increasing evidence of an association of asthma rhinovirus (hrv) infections were difficult to diagnose genome common to many if not all serotypes, allowing improved diagnosis of hrv infection. two studies using this technique have found particularly high identification rates for hrv in asthma exacerbations [6.7] . several studies have shown that colds may increase asthma severity, using indirect measurements, such as peak expiratory fiow (pefr), bronchial responsiveness and the late asthmatic response to allergen [8] [9] [10] . however, there is little information on the direct effect of cold virus infection on the immunopathology of the lower respiratory tract. bronchial biopsies in influenza and mycoplasma have shown inflammatory features consistent with asthma [11, 12] . nasal biopsies in experimental rhinovirus infection have not shown evidence of an inflammatory infiltrate [13] and it is suggested that mediators such as histamine and bradykinin may cause upper airway symptoms [14] . in nasal epithelial cell cultures, rhinoviruses have a negligible cytopathological effect, compared with the destruction of the monolayer caused by influenza a and adenovirus [15] . nevertheless, nasal epithelial cell shedding is increased in vivo in experimental rhinovirus infection [16] . given the high rates of rhinovirus infection in association with asthma exacerbations, it is intriguing to note that the receptor for major rhinovirus serotypes is the intercellular adhesion molecule, icam-1 [17] . its ligands include integrins present on inflammatory cells and binding to icam-1 is essential to migration of inflammatory cells into tissues [17, 18] . icam-1 is constitutively expressed by vascular endothelial cells but is also upregulated on bronchial epithelial cells in asthma [19, 20] , suggesting a mechanism for inflammatory infiltration of the epithelium. viral infection may upregulate tcam-1: by a direct efl'ect as shown in cultured tumour cell fines [21] or indirectly via expansion of th| t-helper cell clones producing interferon--7 [22.23] . the aim of this study was to examine bronchial epithelial tissue in naturally acquired cold virus infections, in order to determine whether there are differences between the lower airway infiammatory response and icam-1 expression in atopic and nonatopic subjects. we decided to study naturally acquired colds, using respiratory symptoms as the diagnostic endpoint, as experimental infection with a specific rhinovirus serotype may not necessarily result in the spectrum of symptoms and inflammatory changes which occur in common colds. we anticipated ^60% identification of viral infections, with a predominance of rhinovirus infections as in previous studies [6.7] . asthmatics were not studied as we felt that it would be inappropriate to bronchoscope such subjects during an exacerbation. atopic subjects show mild bronchial infiammatory changes [24] which may be exacerbated by environmental factors, without unacceptable risk. thirty normal individuals aged between 18 and 45 years gave written informed consent to take part in the study, which was approved by the district research ethics committee. none of the subjects had a history of asthma nor chronic cough, wheeze or dyspnoea. subjects were excluded if they had smoked in the preceding 2 years or if they had a past history of more than 2 packyears of smoking. fifteen subjects were selected for atopy, on the basis of one or more positive skin-prick tests to a screen of five common inhalant allergens. all had normal spirometry forced expiratory volume in one second (fevi) greater than 70% predicted for age and height [25] . subjects were recruited between november 1992 and march 1993 at a time when they had not had a cold for at least 4 weeks. screening investigations included a brief respiratory symptom questionnaire, medical examination, skin-prick tests to five common inhalant allergens, simple spirometry, histamine bronchial provocation test and a 7-day diary card of respiratory symptoms and peak expiratory flow. subjects were reviewed after 7 days and excluded from the study if their diary cards revealed greater than 15% diurnal peak fiow variability or chronic lower respiratory symptoms. blood was taken for platelet count, clotting screen and viral serological tests. nose and throat swabs were taken for hrv-pcr. bronchoscopy was performed 2-3 days later. bronchial washings were taken for microbiological culture and hrv-pcr and bronchial biopsies for immunopathological studies. subjects were reviewed regularly at monthly intervals and asked to attend for review at the onset ofa cold with lower respiratory symptoms (cough, wheeze or dyspnoea). diary card symptom scores were reviewed in order to confirm the diagnosis of a cold. diagnosis was based on the recording of cough together with at least one other symptom (nasal blockage, rhinorrhoea, wheeze or dyspnoea), provided that at least one symptom was rated grade 2 (moderate) or more [7] . symptom scores were confirmed at interview with the doctor. clinical examination was performed in order to exclude serious respiratory disease. histamine bronchial provocation test, polymerase chain reaction (pcr) on nose and throat swabs and viral serology were repeated, followed by bronchoscopy 2-3 days later, when bronchial washings and biopsies were taken and processed as before. bronchial biopsy sites were randomized so that g) 1996 blackwell science ltd. clinical and experimental allergy. 26, 665-676 half of the subjects underwent biopsy from the right tipper and half the right lower lobe at basehne, the alternate site being biopsied during the acute cold. diary cards of respiratory symptoms and peak fiow were recorded for 4 weeks, commencing at the onset of cold symptoms. convalescent viral serology was repeated at the end of the month. questionnaire and diary cards subjects completed a written questionnaire on respiratory symptoms. subjects were excluded if they reported wheeze or breathlessness, in the absence ofa cold, at any time during the preceding 12 months. diary cards were completed for a week at baseline, recording symptoms of cough, wheeze, breathlessness, nasal blockage and runny nose, twice daily. symptoms were graded 0-3 (nil, mild, moderate, severe). peak expiratory flow was recorded on rising and retiring to sleep, using a mini-wright meter. subjects were instructed to record the best of three reproducible readings. individuals with > 15% diurnal peak flow variation and those with chronic cough, wheeze or dyspnoea were excluded. diary cards were recommenced at the onset of a cold and recordings of peak flow and symptoms continued for i month. spirometry and bronchial provocation testing fevi was recorded after resting for 15-20 mln as the best of three repeatable measurements using a vitalograph dry bellows spirometer (vitalograph ltd, buckingham, uk). histamine bronchial provocation testing was performed according to a standard tidal breathing method [26] , provided that resting fevi was greater than 70% predicted value for age and height [25] , a devilbiss 646 nebulizer driven by air at a how rate of 6lmin ' (output 0.755 nil in 2 min) was used. challenge was terminated when the fev, had fallen by 20% compared with the lowest value after control solution (coca's solution) or at the maximum concentration (16 mg/ml"' histamine). dose-response slope (sl) [27] was calculated as the maximum percentage fall in fev, divided by the cumulative dose of histamine delivered. this parameter was used in place of provocation concentration as many subjects were only minimally responsive to histamine, and did not bronchoconstrict by 20% at the maximum dose. skin-prick tests were performed using skin-testing solutions (alk) dermatophagoides pteronyssinus, mixed grass pollen. aspergillus fumigatus, cat fur, dog hair, plus positive and negative controls (histamine 1 mg/ ml"' and 0.9% sahne). subjects were classified as atopic on the basis of one or more positive skin-prick tests, defined by a skin weal at least 2 mm in diameter and 1 mm greater in diameter than the negative control. bronchoscopy subjects attended for bronchoscopy at 0s.30h. having fasted from midnight. following intramuscular pre-medication (50-75 mg pethidine, 12.5mg prochlopererazine. 0.6mg atropine), 10% lignocaine solution was applied to the fauces, 4% lignocaine solution to the vocal cords and 2% to airways. an olympus bf20 bronchoscope was passed and bronchial washing taken from the right middle lobe, using two 20 ml aliquots of normal saline. cup forceps were used to take biopsies from right upper or lower lobe scgmental/subsegmcntal orifices, according to the randomization sequence. virological and microbiological investigations nasal swabs were taken from the anterior nares and throat swabs passed firmly over the pharynx and tonsils. swabs were placed into 0.5 ml transport medium containing nutrient broth (10% fetal calf serum, penicillin, streptomycin and amphotericin b). swabs and 0.5 ml aliquots of bronchial washings were stored at -70 c. specimens were subsequently analysed using a seminested reverse transcriptase (rt) pcr for human rhinoviruses (hrv) that incorporated a touchdown reaction cycle, as previously described by ireland et al. [7] . the primers and probes used had the following sequences: rt lopcr'cggacacccaaagtag3' pcr + 5'gcacttctgtttcccc3' hrv 2opcr-5'ggcagccacgcaggct3' pcr + 5'gcacttctgtttcccc3' cultured human rhinoviruses of several serotypes were used as positive controls and virus transport medium was used as negative control for each assay. the appearance of a 202 base pair amplification product was taken to indicate hrv infection. serum samples (loml) were taken at baseline and in the acute and convalescent phase of subsequent infection and stored at -2o'c. sera were tested for complement fixing antibodies to adenovirus, influenza a and b, respiratory syncytial virus (rsv), parainfiuenza viruses 1-3, mycoplasma pncumoniac and chlamydia psiitaci. an enzyme-linked immunosorbent assay (elisa) was used to detect rises in antibody levels to coronaviruses 229e and oc43. sera were tested at a dilution of 1 : ioo and a consistent ratio of > 1.3 between absorbance values of convalescent and acute samples was taken as indicating recent infection. bronchial washings were sent at baseline and in the acute phase of infection for standard microscopy and culture for bacterial pathogens. virological cultures were not there were no significant differences between atopic and non-atopic subjects. performed as our co-workers [7] previously found no improvement in diagnostic yield when culture procedures were added to serology and hrv-pcr. a study of children [6] showed a higher rate of picornavirus identifications in asthma exacerbations but the rate of confirmed hrv infection was similar. the excess of picornaviruses may have been unconfirmed hrvs or other enteroviruses. a higher prevalence of hrv infection may be explained by the different age group studied. all samples were processed and analysed without knowledge of subject, viral infection status or symptoms, in order to avoid bias in the interpretation of results. one biopsy was fixed in carnoy's solution, processed in a shandon citadel 1000 automatic tissue processor (shandon southern products ltd, runcorn. uk). embedded in paraffin wax and cut to 3//:m sections. sections were then stained with monoclonal antibodies egl (total eosinophils) and eg2 (activated eosinophils) (pharmacia. milton keynes, uk) using a streptavidin immunoperoxidase method. two biopsies were embedded in optimum cutting compound (agar, stanstead, uk), snap-frozen in liquid nitrogen and stored at -7o'c. cryostat sections were cut at 4//m and stained using monoclonal antibodies for cd3 (total). cd4 (helper). cds (suppressor) t cells and cd25 (interleukin-2 receptor positive cells) (dako ltd, high wyeombe. uk) using the avidin biotin complex (abc) technique. total cell counts were made on each biopsy section and expressed per mm"^ area of tissue. areas were measured using computerized image analysis (kompira ltd, strathclyde, uk). sections were also stained for the intercellular adhesion molecule, icam-1 (cd54) (serotec, oxford, uk) using the abc technique. epithelial expression of icam-1 was graded on a scale of 0-3 by colour intensity and distribution of staining. specimens were processed immediately on lavage fluid chilled to 4''c. aliquots (150//l) were cytocentrifuged at 500 rpm for 2 min. specimens were air dried, then stained with diffquick reagents for differentia! counts. a diffferential cell count was made by counting 300 cells in consecutive high power fields. data were tested for a normal distribution (normal probability plot and the shapiro-wilk test) and logtransformed if appropriate. parametric tests were applied to pefr, fev], log-transformed histamine dose-response slope and log-transformed eosinophil and neutrophil data. lung function and histamine challenge data during colds were compared with the mean baseline value, using a paired r-test. paired /-tests were used to compare baseline and acute phase eosinophil and neutrophil data. two sample /-tests were used to compare changes in eosinophils. neutrophils and bronchial responsiveness during colds, between atopic and nonatopic subjects, between viral diagnostic groups and between symptom groups. for non-normally distributed data (t cells, cd25 -i-cells). wilcoxon's signed rank test was used to compare baseline and acute phase results and the mann-whitney {/-test to compare changes in t-cell infiltration during colds between atopic and non-atopic subjects, between viral diagnostic groups and between symptom groups. fevi. forced expiratory volume in one second. sl. slope of the bronchia! provocation dose response curve. 95% cl 95yo conftdence interval of the mean. where the confidence interval includes zero, the difference is non-significant at the 5% level. * significant change within group: p < 0-05 (paired /-test). ** significant change within group: p < 0 01 (paired /-test). thirty subjects, mean age 26 years (range 20-41 years) took part (table 1) . sixteen were male and 14 female. fifteen were atopic, of whom eight were rhinities, six being grass pollen-sensitive and two house dust mite sensitive. mean fev| was 113vo predicted (range 95-136vo predicted) and mean pefr variation (amplitude as a percentage of mean) was 1.91% (range -3.8-8.5%). three subjeets had bronchial hyperresponsiveness, reacting with >20% fall in ffv, to concentrations of histamine lower than 2mg/ml '. there were no significant differences in age. sex ratios, lung function nor bronchial responsiveness between atopic and nonatopic groups. twenty subjects returned with symptoms of a cold between november 1992 and august 1993. none ofthe pollen-sensitive rhinitics presented with colds during the pollen season. two subjects defaulted from follow-up. eight other subjects did not experience symptoms of a cold during foilow-up. viral identifications were made in eight (40%) ofthe symptomatic subjects. there were two rhinovirus (hrv) infections, three coronavirus (hcv) infections (two of serotype oc43 and one 229e), one with dual infection (pcr positive for hrv and serology positive for hcv 229e), one parainfluenza iii and one respiratory syncytial virus (rsv) infection. none ofthe subjects had positive hrv-pcr at baseline nor high titres on viral serology. a bacterial pathogen was identified in only one case: haemophilus influenzae was isolated from the bronchial washings in the subject who also had rising rsv titres. in addition to considering the efiect of atopy., we also considered the effect of positive virological diagnosis on symptoms, lung function, bronchial responsiveness and immunohistology. the aim of this analysis was to determine whether there were any gross differences between subjects with and without positive virological tests which could be accounted for by infection, albeit by a heterogeneous group of pathogens. although there tnay be differences between the different viral infections, numbers of subjects with any specific infection such as hrv were too few to consider as a separate subgroup in this study. all 20 subjects complained of cough and rhinorrhoea. nine subjects complained of wheezing, five of whom had positive virological tests. nine subjects complained of breathlessness, five of these having positive virological tests. there were no significant differences in symptom severity between those with and without positive virologica! tests. eight atopic and 12 non-atopic subjects experienced cold symptoms. only two of the eight atopic subjects had positive virological tests compared with six ofthe 12 non-atopic subjects. only two atopic subjects complained of wheeze, compared with seven non-atopic subjects. only one atopic subject complained of breathlessness, compared with eight non-atopic subjects. symptom severity was significantly greater for breathlessness in the non-atopic group (median score 1 compared with 0., mann-whitney (;-test, fev i was significantly lower during colds with a mean reduction of0.19l(95% ci -0.10, -0.29l, p= 0.0004) ( table 2 ). pefr and peak flow variation showed no significant change. there was a small but significant increase in bronchial responsiveness, shown by a mean increase of 0.62 doublings of the slope (ds) of the dose response curve (95% ci + 0.24,+ i.00 ds, /»-0.003). table 2 shows the mean changes in fev, and sl in atopic and non-atopic subjects and in those with and egl and eg2-total and activated eosinophils respectively. cd3, cd4 and cd8-total, helper and suppressor t cells, respectively. il-2rinterleukin-2 receptor positive cells. there were no significant difierences between the numbers of inflammatory cells present in the atopic and non-atopic subjects' bronchial rnucosa at baseline. numbers of t-suppressor (cd8"^) eells were greater in the atopic subjects (* borderline significance; p = 0 055, mann-whitney-f/ test). without positive viral diagnoses. there was a greater increase in bronchial responsiveness in non-atopic subjects and those without diagnosed viral infection but the difference between groups was not significant. comparison of infiammatory cell infiltration of the bronchial mucosa in aiopic and non-atopic subjects at baseline (table 3 ) there were no significant differences between atopic and non-atopic subjects when ceil counts were compared at baseline. however, there was a greater number of t cells in the atopic group which approached the 5% level of significatice (mann-whitney f/-test. p^ 0.055) only for cd8 positive t cells. there was a wide scatter of inflammatory cell numbers between subjects some of which may be accounted for by technical factors such as sampling and processing and also by pathophysiological factors. the scatter of results was no more marked than in other studies in the literature. eosinophil staining is shown in fig. i sixteen subjects had satisfactory bronchial biopsy samples for eg2 staining at each bronchoscopy. seven of these showed an increase in activated (eg2) eosinophil infiltration during colds. there was no significant increase in eg2 infiltration during colds. tceil immunostaining is shown in fig. 2 . fourteen subjects had satisfactory bronchial biopsy samples for t-ccll staining. one ot these did not have a satisfactory section for total t-cell staining (cd3). t-cell numbers were not normally distributed and non-parametric statistics were used to analyse the data. there was a significant increase in cd8 (t-suppressor) cells from a median of 93.6 cells mm "^-271.8 ceils mm " (wilcoxon's signed rank test, p -0.004). there was also an increase in il-2r positive cells, although numbers were very small (increase from median of 5.4 cells mm "-12.8 cells mm ". p = 0.059). figure 3 shows the differential cell counts in bronchial washings. seventeen subjects had satisfactory bronchial cytology specimens for differential cell count. there were no significant changes in macrophages, lymphocytes, eosinophils or epithelial cells. however, there was a significant increase in the percentage of neutrophils, from a geometric mean of 6.9% to 10.4%. (paired /-test: geometric mean 1.53-fold increase, confidence interval 1.04 2.25-fold, p = 0.02.) the effect of atopy and viral diagnosis on changes in infiammatory cell infiltration of the bronchial mucosa is shown in table 4 . the table shows the change from baseline in numbers of inflammatory cells in atopic and non-atopic subjects and in those with and without positive viral diagnoses. there was no difference in change in egl infiltration between atopic and non-atopic subjects, nor between those with and without diagnosed viral infection. however, there were diflerences between changes in eg2 infiltration in the subgroups (graphically represented in fig. 4 ). only two of the eight subjects with diagnosed viral infection showed an increase in eg2 infiltration compared with five of the eight subject without diagnosed infection. those without diagnosed viral infection showed a significant increase in eg2 infihration (paired /-test, p = 0.04). the only subject with diagnosed infection to demonstrate a large increase in eg2 infiltration was the subject who had dual infection with rsv and haemophilus influenzae. this subject was also an atopic (grass pollen allergic) rhinitic. there was a significant increase in eg2 cells in those without diagnosed infection compared with those with diagnosed infection (two sample /-testing, p -0.04) atopic subjects were more likely to show an increase in eg2 infiltration than non-atopic subjects. the difference between changes from baseline in the atopic and non-atopic subjects was significant on two sample /-testing (p = 0.05) as there was a reduction in eg2 numbers in a majority of the nonatopic subjects. three of the four atopic subjects who showed an increase in eg2 eosinophils were rhinitics. there were no significant differences between atopic and non-atopic groups nor between those with and without diagnosed viral infection for t cells. there was a greater increase in neutrophils in bronchial washings in those with diagnosed viral infection but this increase was not significantly difterent from the change in those without diagnosed infection. table 5 illustrates the association between lower respiratory tract symptoms and bronchial infiammatory cell infiltration. the table shows fig. 3 . neutrophil count in bronchial lavage specimens. these data were log-normally distributed. statistical analysis as in pig. 1. the majority of samples (13/17) showed an increase in neutrophilia during acute coryzal symptoms. although the mean increase was small, it was statistically significant. table 3 . -fold changes with confidence intervals (cl) which do not incltide ihe value 1.00 (no change) indicate a significant increase or decrease in numbers of cells. * significant change from baseline within tirotip (p < 0-05). paired /-test, (wilcoxon signed rank test for t cells). ^ borderline significant change from baseline within group {p =-0'059). wilcoxon signed rank test. * significant dilterence between atopic, non-atopic or viral diagnosis groups {p < 0 05). comparison of changes: two sample /-test. in the groups of subjects who did or did not report symptoms of wheeze and dyspnoea during their colds. subjects who wheezed during their colds showed an increase in egl. cd8 and 1l-2r ' cells in brotichial mucosa but this was not significantly different in comparison with the change iti those who did not wheeze. there were no significant differences between symptom subgroups for eg2, cd3 * and cd4"^ positive cells in bronchial tnucosa nor for neutrophils in bronchial washitigs. subjects with dyspnoea during their colds showed a significant increase in cds ' cells in comparison with the non-dyspnoeic subgroup (median increase + 203.6 cells mm -compared with +43.9 cellsmm ". mann-whitney tz-test p^0.02). these subjects also showed an increase in il-2r^ cells which did not differ significantly frotn the non-dyspnoeic subgroup. there was no significant association of dyspnoea with changes in other infiamtnatory cells, although there was an increase of neutrophils in bronchial washings in the non-dyspnoeic subgroup. thirteen subjects had satisfactory bronchial biopsy samples for icam-1 staining. icam-l staining of the epithehum was only found in four of seven atopic subjects and none of the six non-atopic subjects. icam-l expression was upregulated during colds in all of the four atopic subjects: on a grading scale of 0-3. two subjects increased expression from i to 2. one from 0 to 1 and one from 0 to 2. only one of these four subjects had an identified viral infection, this being the subject with rsv infection together with haemophilus influerizae (grade 0 1). all the atopie subjects for whom biopsy tissue was available were rhinities. sections were available for five of the eight subjects with viral infection (one hrv. two hcv. one hrv/hcv, one rsv/7/. injlucuzac]. this study was designed in order to determine the effect of the common cold on lower airway inflarnmation. comparing the response in atopie and non-atopic subjects. the study has shown the presence of bronchial inflammation in healthy adult subjects complaining of symptoms of the comtnon cold. we demonstrated two distinct groups among such subjects: those with virologically proven infections and those without. a relative rise in neutrophils was a characteristic of the former group, whereas activated eosinophilic infiltration was present in the latter. despite such differences, symptom scores were similar in the two groups. atopy and rhinitis were factors associated with the development of activated eosinophilic inflammation in the bronchial mucosa. the majority of subjects showed reduced fevi and increases in airways responsiveness, neutrophils. total egl ^ eosinophils and cd8 ^ suppressor t-lymphocytes. regardless of atopy or the ptesence of viral infection. while perception of symptoms of the common cold by subject or observer eould have influenced the results of lung function and bronchial provocation testing, staining and counting of biopsy and iavage specimens was performed without knowledge of subject, eoryzal symptoms or infection status. our ilndings indicate that many subjects with symptotns traditionally thought to indicate upper respiratory tract infection have evidence of lower respiratory tract inflammation. it seems likely that total eosinophils and neutrophils increased as part of an acute granulocyte infiltrate, whereas cd8 ' t-suppressor cells may be involved in a killer cell response to viral infection: their role in those subjects in whom we were unable to identify a pathogen is less clear. numbers of subjects with hrv infection were too few to consider separately. however, our methodology for hrv rt-pcr has previously been well validated [7] , results in adult asthtna exacerbations comparing well with those of a study in children using similar techniques [6] . in the latter study there was a higher rate of picornaviruses identified by pcr but the confirmed rate of hrv was similar. the excess of picornaviruses may have been accounted for by hrv or enterovirus infections, the former being more likely. differences between the two studies may reflect the age groups involved. bronchial immunopathology in the hrv cases was not distinctive, despite lcam-1 being the receptor for major hrv serotypes [17] . icam-1 expression in bronchial epithelium was limited to four atopic rhinilic subjects. we have not previously been able to show bronchial epithelial icam-1 expression in non-asthmatics, whereas icam-1 is constitutively expressed by the vascular endothelium. however, our previous study compared asthmatics with non-atopic non-rhinitics [20] , thus, the findings of this study suggest that icam-1 may be up-regulated in lower airway epithelium in allergic rhinitis as well as in asthma. icam-i was only expressed in diagnosed viral infection in one atopic rhinitic subject with the combination of rsv together with haemophilus influenzae infection. this was a 23-year-old heterosexual who had never smoked. he had no exceptional symptoms and made a full and rapid recovery. bronchial lavage demonstrated high neutrophilia (64%). although this is a small study, we believe that our findings suggest a tendency, often suspected in clinical practice, for allergic subjects to report allergic inflammation as a "cold". this could explain the low diagnostic rate of viral infection particular to the atopic group, despite a combination of pcr and serological diagnostic techniques which have given a high yield of viral diagnoses in previous studies [7] , it is interesting that there were no significant difl'erences in symptoms among atopic and non-atopic subjects save for breathlessness. which was, paradoxically, more common among the non-atopic subjects. lower respiratory tract symptoms were quite common among these subjects with uncomplicated colds, providing a clinical correlation with the inflammatory infiltrate, especially with cd8'^ t cells. other investigators have found slight differences in symptomatology in atopic subjects with experimental hrv infections: symptoms tending to be earlier in onset [28] and more severe in those with evidence of preceding hrv infection [29] , other studies have shown that markers of allergic inflammation can be detected at distant sites in atopic subjects. djukanovic et al. [24] showed greater numbers of inflammatory cells in the bronchial mucosa of atopic rhinitics than in non-atopic normals. in contrast, we found no difference between atopic and non-atopic subjects at baseline, but only half of our atopic subjects had rhinitis. other studies from our laboratory have shown constitutive up-regulation of cytokines such as granulocyte macrophage-colony stimulating factor (gm-csf) in the nasal mucosa of atopic non-rhinitics with eczema [30] . thus, it is perhaps not surprising that activated eosinophils can increase in the bronchial mucosa in atopic rhinitics with episodes of respiratory symptoms. previous studies of the immunopathology of the common cold have concentrated on experimental hrv infection, examining mainly nasal secretions and peripheral blood. there have been few studies of bronchial pathology. halperin et al. [35] isolated hrv from the lower airway in experimental infection, using a bronchoscopic technique which minimised contamination from the upper respiratory tract. calhoun et al. [36] found that subjects experimentally infected with hrv16 showed an increase in eosinophils in bronchoa[veolar lavage specimens following allergen challenge. there was an amplified response in atopic rhinitic subjects, consistent with the increase in allergen responsiveness among similar subjects in a previous study [[0] . other studies of colds have produced conflicting data. neutrophilia has been consistently found in nasal secretions [16.31] , together with shed epithelial cells [16] . levandowski [32] showed a reduction in cd4 + and to a lesser extent, cd8 ^ t cells in peripheral blood after hrv25 infection and suggested that this might indicate recruitment of such cells to the site of inflammation. however, the same investigators found only neutrophils and monocytes in nasal secretions [31] . others have not been able to find an increase in inflammatory cells in nasal biopsies [13] and suggested tbat the symptoms of common cold infection might be caused by mediators such as histamine and kinins [14.33] . hsia et al. [22] showed an increase in production of interleukin-2 and interferon--j by peripheral blood mononuclear cells, inversely related to severity of the cold. they also showed an increase in natural killer (nk) cell-mediated cytotoxicity and antigen-stimulated blastogenesis. skoner et al. [34] showed increases in peripheral blood total leucocytes and lymphocytes, especially, cd3, cd4, cds and cd3 ' dr^; but a decrease in nk cell activity and lymphoproliferative response to rv39, particularly in nonatopic subjects. there were subtle differences in the peripheral blood response between atopic and nonatopic subjects with a tendency to a later response in the atopic allergic group. such peripheral blood changes indicate a systemic immune response to hrv. our findings indicate that healthy non-asthmatic subjects with symptoms of the common cold can develop bronchial inflammation, including an infiltrate of eosinophils. neutrophils and cd8^ t-suppressor cells. atopic rhinitic subjects were most likely to show allergic inflammation with activated eosinophilia and were least likely to have a diagnosed viral g) 1996 blackwell science ltd, clinical and e.xperimenlal allergy. 26. 665-676 infection. therefore, symptoms typical of the common cold may be caused by airway inflammation in the absence of viral infection. exposure to aeroallergens and possibly environmental pollutants should be considered as alternative triggers of cold symptoms. routine antibiotics in hospital management of acule asthma the association of viral and bacterial respiratory infections with exacerbations of wheezing in young astlimalic children respiratory viral infection and wheezy bronchitis in childhood viral and bacterial infections in adults with chronic asthma virul infections as a precipitant of wheeze in children: combined home and hospital study use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms improved detection of rhinoviruses in nasal and throat swabs by semi-nested rt-pcr mechanisms of bronchial hyper reactivity in normal subjects after upper respiratory tract infection effect of atopy on the natural history of symptoms, peak expiratory flow and bronchial hyperresponsiveness in 7 and 8-year-old children with cough and wheeze rhinovirus upper respiratory tract infection increases airway hyperreactivity and late asthmatic reactions bronchotracheal response in human influenza bronchial epithelium in humans recently recovering from respiratory infections caused by influenza a or mycoplasma nasal biopsies in human rhinovirus 16 infection: an immunohistochemical sttidy kinins are generated during experimental rhinoviral colds respiratory virus infection of monolayer culttires of human nasal epithelial cells shedding of infected epithelial cells in rhinovirus colds rothlein r ct al, a cell adhesion molecule icam-i is the major surface receptor for rhinoviruses intercelliilar adhesion moiecule-1 (icam-1) in the pathogenesis of asthma hla-dr and icam-1 expression on bronchial epithelial cells in asthma and chronic bronchitis the expression of intercellular adhesion moiccule-1 and the ,:*l-integrins in asthma selective induction of intercellular adhesion molecule-1 by interferon-gamma in human airway epithelial cells peripheral blood mononuclear cell interleukin-2 and intcrferon-gamma production, cytotoxicity and antigenstimulated blastogenesis during experimental rhinovirus infection induction by ll-i and interferon-gamma: tissue distribution, biochemistry and function of a natural adherence molecule (icam-1) quantitication of mast cells and eosinophils in the bronchial mucosa of symptomatic atopic asthmatics and healthy control subjects using immunohistochemistry changes in the normal maximal expiratory flow-volume curve with growth and aging a general practice survey of bronchial hyperresponsiveness and its relation to symptoms, sex. age, atopy and smoking analysis of dose-response curves to methacholine theory and practice of histological techniques use of avidin-biotin complex (abc) in immunoperoxidase techniques, a comparison between abc and unlabellcd (pap) techniques rhinovirus 39 infection in allergic and nonailergic subjects amplified rhinovirus colds in atopic subjects synthesis of tnf-tt, il-8 and gm-csf by cultured nasal epithelial cells from atopic and non-atopic non-rhinitic subjects and the effect of exposure for 6 h to nitrogen dioxide (n02) pathogenesis of lower respiratory tract symptoms in experimental rhinovirus infection experimental rhinovirus 16 infection potentiates airway inflammation only in allergic subjects nasal secretion leukocyte populations determined by flow cytometry during acute rhinovirus infection acute-phase decrease of t-lymphocyte subsets in rhinovirus infection analysis of nasal secretions during experimental rhinovirus upper respiratory infections eflfect oi' rhinovirus 39 infection on cellular immune parameters in allergic and non-allergic subjects cjt was supported by a bursary from the joint research board of st bartholomew's hospital and a travel grant from the robert malcolm trust. dci was supported by a gram from the cystic fibrosis trust. we thank the volunteers for their co-operation, julie kent, moises calderon and ray sapsford for technical assistance and janice thomas for statistica! advice. key: cord-022337-f3a349cb authors: busse, william w.; dick, elliot c.; lemanske, robert f.; gern, james e. title: infections date: 2007-05-09 journal: asthma doi: 10.1016/b978-012079027-2/50112-x sha: doc_id: 22337 cord_uid: f3a349cb wheezing with respiratory infections is extremely common in early childhood. it is estimated that the prevalence of wheezing during the first five years of life varies from 30–60%. in the majority of children who experience wheezing with respiratory infections, these episodes of wheezing become less frequent as the child grows older. however, determining whether the initial episode of wheezing with a viral respiratory illness is an important factor in the eventual development of asthma is an important question. although a significant body of information suggests an association between respiratory tract illnesses in early life and the later development of airway dysfunction, this relationship is difficult to establish and indicates the complexity of factors that surround the development of bronchial hyperresponsiveness and eventual expression of asthma. a similarly important issue to resolve is the relationship between respiratory infections and the pathogenesis of airway hyperresponsiveness. it is apparent that viral, not bacterial, upper respiratory infections (uris) trigger asthma attacks. with the use of more sensitive techniques to identify respiratory viruses, the relationship between respiratory infections, particularly viral uris, and asthma has become even more convincing and important. these episodes of wheezing become less frequent as the child grows older. however, a question still remains as to whether the initial episode of wheezing with a viral respiratory illness is an important factor in the eventual development of asthma. although a significant body of information suggests an association between respiratory tract illnesses in early life and the later development of airway dysfunction, this relationship is difficult to establish and indicates the complexity of factors that surround the development of bronchial hyperresponsiveness and eventual expression of asthma. furthermore, the source of subjects for study, i.e. follow-up of hospitalized patients vs. outpatients, contributes to the difficulty of understanding this problem. eisen and bacap found that children hospitalized for bronchiolitis prior to age 2 years had an increased risk for asthma. rooney and williams'^ also evaluated, retrospectively, the records of infants hospitalized for bronchiolitis at 18 months or younger; allergic manifestations and a family history of asthma were more frequent in children who eventually experienced one or more episodes of wheezing. finally, mcconnochie and roghmann^ identified 77 patients who had bronchiolitis at 25 months or younger and compared their outcome to children without a history of bronchiolitis. when these children were evaluated approximately 7 years later, only upper respiratory allergy, bronchiolitis and passive smoking exposure were found to be independent predictors of wheezing following bronchiolitis. consequently, it is apparent that the final conclusions on the relationship between respiratory infections in infancy and later asthma must consider a host of influences, including parental smoking, underlying airway responsiveness and gender. a similarly important issue to resolve is the relationship between respiratory infections and the pathogenesis of airway hyperresponsiveness. t o evaluate the effect of bronchiohtis on airway responsiveness, sims et al.^ identified 8-year-old children who had respiratory syncytial virus (rsv) respiratory infections and quantitated bronchial 'lability' by exercise tests. compared with appropriate controls, the fall in the peak flow with exercise was greater in children who had bronchiolitis; however, airway reactivity to exercise was not different between children with or without subsequent episodes of wheezing. since other variables confounded their study, sims et al.^ could not prove that respiratory infections led to the later development of asthma. other efforts have been made to ascertain if viral lower respiratory tract infections (lris) in early life cause persistent pulmonary function abnormalities. pullan and hey^ evaluated 130 children admitted to hospital during the first 5 years of life with rsv lris; 42% of the hospitalized children had future episodes of wheezing, while only 19% of control subjects experienced similar airway symptoms. however, few patients (6.2% vs. 4.5% of controls) had troublesome respiratory symptoms by 10 years of age. furthermore, although a three-fold increase in bronchial responsiveness was found in the children with bronchiolitis, atopy was not increased. analogous conclusions were reached by weiss et al^ when they assessed the outcome of an antecedent acute respiratory illness on airway responsiveness and atopy in young adults. airway responsiveness, evaluated by eucapnic hyperventilation to subfreezing air, was increased in children with a previous history of either croup or bronchiohtis, or greater than two acute lower respiratory illnesses. the possibility has also been raised that a predisposition to wheezing in infancy depends more on intrinsic airway structure than atopy.^ this position is supported by the high degree of airway responsiveness found in infancy in physiological evaluations^^~^^ and the incidence of wheezing with respiratory infections.^^ however, it is difficult to precisely assess airway responsiveness in young children due to limitation of lung size and other age-related factors. to help clarify the relationship between premorbid lung function and wheezing with respiratory illnesses, martinez et al}^ conducted a prospective study of respiratory illness in infancy and childhood. lung function values were determined prior to any lris. included in these measurements were tidal expiratory patterns, specifically the time to peak tidal expiratory flow (tme) divided by total expiratory time ( t e ) , or the tme/te ratio; morris and lane^^ had shown that decreasing tme/te ratios correlated with lower lung function in patients with progressive chronic obstructive lung disease. of the infants studied by martinez et al,^^ 36 developed an lri and 24 wheezed with at least one of these infections. there was no diflerence in preinfection lung function between those infants who did not have an lri and those with an infection but no wheezing (table 30 .1). however, infants who wheezed with the respiratory infection had diminished tme/te values and reduced expiratory system conductance when measured prior to wheezing with the infection. these data suggest that alterations in lung function are compatible with reduced airway conductance or a slow respiratory system time constant that precedes and predicts wheezing with respiratory infections in infants. furthermore, it appears that a given child's response to infection is determined not only by the infection but also by pre-existing lung function. taussig et al}^ also noted that lower levels of lung function predispose to wheezing with lri, as opposed to the infection per se. the precise nature of this predisposition remains to be defined but may lie in airway geometry, airway-parenchymal interaction, or mucosal and smooth muscle response. furthermore, this pulmonary-structural predisposition may be enhanced by an exaggerated ige response to viral infection,^^'^^ resulting in more inflammation and severe wheezing with hospitalization. since the majority of infants who develop wheezing with lris do not wheeze throughout life,^^ it is likely that pulmonary function abnormalities that favour wheezing with viral infections are modified with the growth and development of the lung. long-term outcome then seems to be more closely linked to the persistence of ongoing airway damage or bronchospasm associated with the development of atopy and true clinical asthma. further, and possibly definitive, insight into the relationship between wheezing with early respiratory infections and the later development of asthma has come from a unique they identified a number of factors that affect wheezing before the age of 3 years and their relationship to wheezing at 6 years of age. the study population has previously been reported^"^ and consists of newborns enrolled between 1980 and 1984 with follow-up information at 3 and 6 years of age. key assessments in infancy included cord-serum i g e levels, pulmonary function testing before any lower respiratory tract illness had occurred, measurement of serum i g e at 9 months of age, and a questionnaire completed by the children's parents when the child was 1 year old. the children were classified into four groups: no wheezing, transient wheezing, late-onset wheezing or persistent wheezing (table 30 .2, opposite). at 6 years of age, serum ige, pulmonary function testing and allergy skin testing were repeated and these factors were assessed in relationship to their history of wheezing. at 6 years of age, 20% of the children had at least one lower respiratory illness with wheezing during the first 3 years of life, but with no wheezing at age 6 years. these children had diminished airway function before the age of 1 year and, at 6 years of age, were more likely to have mothers who smoked but not mothers with asthma and did not have evidence of atopy, i.e. elevated serum i g e or skin test reactivity. in addition, 15% had no wheezing before the age of 3 years but had wheezing at age 6 years and 13.7% had wheezing both before 3 years of age and at 6 years of age. those with late-onset wheezing and persistent wheezing were more likely to have mothers with a history of asthma, elevated serum i g e levels and diminished lung function at 6 years of age (tables 30.2 -m g g <u g^ <u 0 g ^ qj on rg g i n c ( n (j <-i h i j 0 <u b o o <u < ! oj g toe g ' n (u <u (u w g o <u § 1 g <u 2 la o <u ^ •^ co g 0 co a 0 u vh o i n o d v gh g c3 th (u n <u (u vh > g 0 g <u 4-1 g c/3 g o co ' v h a. a o u a c« "g u g :3 co ' -' a ^ 0 u vh o c/^ o g n (u <u •»-> g co <u g 0 co "v1 a. a number of conclusions can be drawn from this prospective longitudinal study. most infants who wheeze early in life have a transient condition and this predilection to wheezing with respiratory infection is associated with diminished lung function; these children are not at enhanced risk for asthma or allergic disease later in life. the groups of particular interest regarding the role of respiratory infections and development of asthma are those infants with either persistent or late-onset wheezing. children with asthma at age 6 years appear to have a predisposition to asthma, as indicated by a maternal history of asthma and elevated serum i g e levels in the first years of life. moreover, these children have reduced lung function by 6 years of age. the role of respiratory infections in the actual development of asthma in those with a predisposition to asthma is less clear; that is, do respiratory infections have any relationship to the subsequent development of asthma? this issue remains to be fully elucidated. insight to factors that may determine a host's response to viral infections and its subsequent effects on lung function has been gained by animal studies. sorkness et al?^ infected neonatal rats with sendai virus. at 7 and 13-16 weeks afer infection, the rats that had been infected had lower p02 values and increased lung resistance when compared with controls. furthermore, the infected animals were more sensitive to aerosolized methacholine. lung histology in the infected animals revealed increased numbers of bronchiolar mast cells and eosinophils. in subsequent studies, these investigators have found some rat strains, i.e. brown norway, are more susceptible to the effects of the virus and more likely to have persistently altered lung function. such studies in an animal model are compatible with the observations by martinez et al?^ and indicate that many factors determine the eventual influence of respiratory infections on airway function, i.e. wheezing and bronchial hyperresponsiveness, including a predisposition to produce ige antibodies. however, what needs to be clarified are the mechanisms by which respiratory viruses interact with ige-dependent factors and promote an asthma-like picture. for decades clinicians associated asthma exacerbations with respiratory infections. with prospective studies, it became apparent that viral, not bacterial, upper respiratory infections (uris) triggered these asthma attacks. with the use of more sensitive techniques to identify respiratory viruses, the relationship between respiratory infections, particularly viral uris, and asthma has become even more convincing and important. in early studies, mcintosh and coworkers^^ prospectively studied 32 young children, aged 1-5 years, with severe asthma. the aetiology of each uri was carefully evaluated and confirmed by culture and serological tests for viral antibody titres. over 2 years, there were 102 confirmed viral respiratory infections and 139 episodes of wheezing; 58 episodes (42%) of wheezing occurred in relationship to viral respiratory infections, of which rsv was the most prevalent and likely to provoke asthma. respiratory bacteria, haemophilus influen:(ae. streptococcus pneumoniae, j?-haemolytic streptococcus and staphylococcus aureus, were also cultured but their presence did not correlate with an asthma attack. a prospective outpatient study from the university of wisconsin evaluated 16 children, aged 3-11 years, with histories of four or more asthma attacks associated with respiratory illnesses during the previous year.^^ detailed clinical records profiled each child's asthma severity, which was further substantiated with biweekly examinations. during an asthma exacerbation or apparent uri, additional bacteria and virus cultures were collected and asthma symptoms carefully quantitated. the 16 children experienced 61 episodes of asthma; 42 occurred in conjunction with a symptomatic respiratory infection and 24 were confirmed to be of viral aetiology by culture and/or serum haemagglutination titres. in this study, rhinovirus was the most frequently identified virus in association with wheezing. some patients had episodes of asymptomatic viral infection, but asthma was not worsened. only one episode of wheezing coincided with a bacterial infection. to extend these observations and to further determine if there was a relationship between different respiratory infections and wheezing, mertsola et al?^ identified 54 patients, aged 1-6 years, who had recurrent attacks of wheezing. over a 3-month observation period, these patients experienced 115 episodes of upper or lower respiratory symptoms. of these episodes, laboratory evidence of a viral or mycoplasma infection was found in 45%. more relevant to our discussion was the observation that wheezing occurred in 58% of the laboratory-confirmed viral respiratory infections. moreover, of the children with wheezing with respiratory infections, one-third were admitted to hospital because of severe dyspnoea. finally, of the agents isolated, coronavirus and rhinovirus were the most frequent isolates. there is also evidence that the viruses which cause wheezing with respiratory tract infections may be age dependent; for example, infants wheeze with rsv while older children have exacerbations of asthma with rhinovirus.^^ to extend these observations. duff and colleagues^^ examined the relationship of viral infections, passive smoke exposure and ige antibody to inhaled allergens in infants and children who were treated for acute episodes of wheezing in an emergency department. the investigators identified 99 subjects, aged 2 months to 16 years of age, and 57 control patients, aged 6 months to 16 years of age, who were seen in a paediatric emergency room. in acutely wheezing children less than 2 years of age, viruses were detected by culture of nasal washings in 70% of the patients; the most commonly identified virus was rsv. positive virus cultures for rsv were noted in only 20% of the appropriate age-matched controls. in contrast, nasal washes yielded positive cultures in only 31% of children over 2 years of age who presented with acute wheezing. however, the most frequently identified respiratory virus in this age group was rhinovirus. when these investigators evaluated risk factors associated with wheezing during the viral infections, interesting and insightful correlates were noted (table 30 .4). for children less than 2 years of age, coexisting allergic diseases were not a risk factor. rather, passive exposure to cigarette smoke, as indicated by urinary cotinine values ^1 0 jug/ml, was a risk factor for wheezing. the profile in children over 2 years of age with wheezing and viral uris was quite different. in these children, the presence of allergy, i.e. positive radioallergosorbant test (rast) values, and not passive cigarette smoke exposure, was the dominant risk factor for wheezing with viral respiratory infections. observations by duff et al}^ confirm previous information that rsv is the major viral infection causing wheezing in infants, whereas rhinovirus is the causative respiratory infection associated with wheezing in older children. moreover, wheezing in infants is not increased by the presence of allergic disease but rather made more probable if cigarette smoke exposure is present. in the older child, allergies are a major risk factor for wheezing with rhinovirus; these data also imply that a unique relationship may exist between rhinovirus and allergy, and this interaction may influence the development of wheezing with this particular respiratory virus. the detection of respiratory viruses, particularly rhinovirus, by culture is difficult because of the fastidiousness of viruses in culture. consequently, determination of the association between rhinovirus infections and provocation of asthma using virus culture may give an underestimation of the true frequency between colds and asthma. johnston ef al}^ developed a polymerase chain reaction (pcr) assay to detect picornavirus infections. this approach has an increased sensitivity and thus greater likelihood to detect causative viruses in relationship to clinical symptoms of a cold and eventual influence on asthma. using pcr technology, along with culture, johnston and colleagues^^ studied the association between upper and lower respiratory viral infections and acute exacerbations of asthma in school-age children; 108 children, 9-11 years of age, were followed over a 13-month period. children or their parents recorded the peak expiratory flow rate twice daily and daily upper and lower respiratory tract symptoms were scored as to severity. with the onset of respiratory symptoms, or a significant fall in peak expiratory flow values, the child was visited by an investigator and nasal aspirates were obtained for virus detection. there were a number of important observations by johnston and colleagues in this large study. first, there was a seasonal pattern to the appearance of respiratory symptoms and changes in peak flow values of the asthma patients; the months of n o v e m b e r -december and april appeared to be the most frequent times for colds and asthma exacerbations. second, respiratory viruses were detected in approximately 80% of episodes with respiratory tract symptoms or falls in the peak flow (table 30 .5). third, rhinovirus was the most frequently detected microorganism and constituted over 60% of the viruses detected (table 30, 6) . fourth, the severity of asthma symptoms in many of these subjects was severe. from these observations, and those of others, it is apparent that rhinovirus, the cause of the common cold, is the respiratory infection most likely to exacerbate wheezing in individuals with existing asthma. it is possible that this relationship simply reflects the fact that rhinovirus is the most common cause of infectious respiratory symptoms, i.e. the common cold. however, this association may also mean in contrast to observations in children, the relationship between respiratory infections and episodes of wheezing is not as striking in adults. when both children and adults were evaluated, respiratory viruses were more frequently identified during episodes of asthma in children under 10 years of age compared with adults. eight adult subjects evaluated by minor et alp had only three documented virus infections with increased wheezing. although an explanation for such findings is not clear, it is likely that asthma flares were more diflficult to sharply delineate and the pattern of wheezing often appeared chronic rather than episodic in adults. similarly, when hudgel and coworkers^^ evaluated 19 adult asthma patients over a 15-month period, 76 episodes of asthma were recorded but only eight could be documented with a viral url although clinically apparent viral respiratory infections precipitate asthma in adults, the relative frequency appears less than in children. in another study, huhti et al?^ evaluated adults admitted to hospital with asthma exacerbations. the viral detection rate was 19.0%, with rsv, parainfluenza and influenza the more frequent isolates. using virus culture and pcr techniques, nicholson and coworkers^^ evaluated the relationship of symptomatic colds and asthma exacerbations in 138 adult asthmatic subjects. these investigators found symptomatic colds were reported in 80% of episodes with symptoms of wheeze, chest tightness or breathlessness. in 24%) of laboratoryproved episodes of non-bacterial respiratory infections, there was a reduction in peak flow ^ 50 litre/min. infections with rhinoviruses, coronaviruses, influenza, rsv, parainfluenza and chlamydia were all associated with objective measures of airflow limitation. the findings of nicholson et al. are further evidence of the importance of respiratory viruses, particularly rhinovirus, to asthma exacerbations, and that these episodes can be severe and occur in adults. therefore, evidence exists to underscore the importance of viral respiratory infections to asthma exacerbations in patients of all ages. there is little evidence that bacterial respiratory infections, other than sinusitis, provoke asthma. berman et al?^ performed transtracheal aspirates on 27 adult patients with asthma during an infectious exacerbation. bacteria cultured from transtracheal aspirates were sparse and, more importantly, did not correlate with clinical illness. moreover, transtracheal aspirates from normal subjects, without respiratory infections, yielded a similar degree of bacterial colonization. a common complication of colds is the development of sinusitis, which has been proposed as a factor in the worsening of asthma. although the association between paranasal sinusitis and bronchial asthma has long been observed, it is conceivable that sinusitis and asthma may coexist as complications of the same respiratory infection. however, some feel that an aetiological relationship exists between sinusitis and asthma.^"^'-^^ although these observations and associations are intriguing and clinically important, additional studies are needed to clarify how sinusitis may influence asthma. a number of mechanisms have been proposed to explain how viral respiratory infections provoke asthma (table 30 .7). when evaluating these possibilities, it is important to realize that the effect respiratory viruses have on airway function will depend upon many factors, including the subject's gender, environmental factors such as cigarette smoke exposure, age, baseline lung function and atopic status, along with the particular respiratory virus. however, there is now convincing evidence that rhinoviruses are the predominant respiratory infection provoking wheezing in patients with asthma and the existence of allergic disease appears to be an important risk factor. therefore, it is proposed that respiratory viruses, particularly rhinoviruses, may interact with those factors that contribute to or determine existing allergic airway disease and enhance existing bronchial inflammation. the end-result of the eflects of the virus on underlying altered epithelial function altered autonomic nervous system regulation of airway smooth muscle function enhanced allergic inflammation enhanced eosinophilic recruitment eosinophilic infiltration of the airway generation of pro-inflammatory cytokines enhanced inflammatory mediator release altered j5-adrenergic function sinopulmonary reflex production of virus-specific ige antibodies allergic airway disease will be an enhanced likelihood for wheezing. although other factors likely participate in this process, the following discussion examines how respiratory viruses either influence ige-dependent events or promote the development of allergic inflammation. welliver et al}'^ tested 79 children, all less than 12 months of age and with documented rsv infection, for the presence of ige-specific antibody to the infecting virus. their clinical patterns of illness were divided into four groups: (a) upper respiratory tract illness; (b) pneumonia without wheezing; (c) pneumonia and wheezing; and (d) wheezing (bronchiolitis). nasal secretions from each patient were measured for ige-specific antibody to rsv, and rsv ige antibody titres were compared with the patient's clinical illness. i g e titres to rsv were highest in patients with evidence of airway obstruction, e.g. pneumonia/wheezing or bronchiolitis. parainfluenza virus-specific ige responses were also examined in individuals with upper respiratory illness alone or bronchiolitis by welliver et al}^ parainfluenza-specific ige antibody were detected in 8 of 12 with bronchiolitis but only 1 of 10 with upper respiratory disease alone. these studies suggest that some respiratory viruses stimulate ige-specific antibody responses and the degree to which this occurs is associated with the clinical manifestations of upper vs. lower airway disease. to further evaluate the significance of virus-specific i g e antibody, welliver et al?^ prospectively monitored 38 infants for 48 months after an initial episode of bronchiolitis. only 20% of infants with undetectable titres of rsv ige had subsequent episodes of documented wheezing. in contrast, 70% of those children with high rsv i g e antibody titres continued to experience wheezing when evaluated 4 years later. a portion of these same subjects was evaluated at 7-8 years of age^'^ and underwent skin testing to common inhalant allergens, pulmonary function testing and methacholine provocation to determine bronchial responsiveness. for the entire study groups, rsv i g e responses were evaluated in relationship to family history of asthma, passive smoke exposure, number of recurrent wheezing episodes, number of positive skin tests and the degree of airway responsiveness to methacholine. the investigators found that an association exists between rsv ige responses and the development of any wheezing following bronchiolitis; the nature, however, of the association between rsv i g e antibodies and any recurrent wheezing is unclear. therefore, the consequence of an initial i g e response to the infecting virus with regard to long-term influence on airway function and the development of asthma is unresolved. although rhinoviruses have emerged as the most common respiratory infection causing asthma exacerbations,^^ there are a number of paradoxes to this relationship. first, rhinoviruses are infections that appear localized to the upper airway; identification of rhinovirus by culture in the lower airway is unusuap^ and pneumonic infiltrates rare. second, even though rhinoviruses infect airway epithelium, the inflammatory response is often unimpressive. for example, fraenkel and coworkers^^ performed nasal biopsies before and after an experimental rhinovirus infection. in both normal and atopic groups, there were no significant changes in inflammatory cells during the cold or the convalescent period compared with baseline values. thus, immunohistochemical analysis of rhinovirus-infected upper airways does not reveal inflammation. third, rhinovirus infections tend to be self-limited and later sequelae are rare. therefore, the mechanisms of lower airway dysfunction are not readily explained by an overzealous inflammatory airway response to this particular virus. the frequent association of asthma with respiratory infections, and in particular rhinovirus illnesses, may relate to the frequency with which rhinoviruses are the cause of colds. this frequency, however, does not explain the apparently serious lower airway events that are associated with rhinovirus infections in subjects with asthma. a major goal of current research is to establish how this particular class of respiratory viruses affects lower airways so profoundly and how these effects can last for weeks beyond the acute respiratory illness. in an initial series of experiments to evaluate the effects of respiratory infection on airway responsiveness, we infected subjects with rhinovirus experimentally and determined its effect on a variety of airway functions."^^ the patients selected for study were evaluated on three separate occasions: at baseline, during acute infection and during recovery. of particular interest was the effect of the infection on the immediate and latephase allergic response (lar) to inhaled antigen. all 10 patients had a rhinovirus respiratory infection at time of study. during the acute rhinovirus respiratory infection, airway responsiveness to histamine significantly increased over baseline values. likewise, acute airway reactivity to inhaled antigen was increased. the change in airway responsiveness to histamine and antigen was similar, suggesting that the effects of the respiratory infection on responsiveness to antigen related to alterations in non-specific bronchial reactivity. prior to rhinovirus inoculation, only 1 of 10 patients had an lar to inhaled antigen. however, during the acute respiratory infection, 8 of 10 patients experienced late-phase airway obstruction to inhaled antigen challenge. furthermore, when evaluated during the recovery period (4 weeks after rhinovirus inoculation), five of the seven patients available for testing still had lars to inhaled antigen. these observations indicate that rhinovirus respiratory infection not only increased airway responsiveness but also changed the pattern of the airway response to inhaled antigen and promoted the development of allergic inflammation, as indicated by the development of the lar. the recovery pattern of airway hyperresponsiveness following the viral respiratory infection was also evaluated. although increased airway responsiveness was still detected 4 weeks after rhinovirus inoculation, there was a trend towards recovery in both the reaction to inhaled histamine and the response to antigen. however, as already discussed, the increased frequency of lars to antigen was still noted 4 weeks after the viral infection, suggesting that a viral respiratory infection has a greater, and possibly more lasting, effect on factors that participate in the development of lars. virus-associated airway hyperresponsiveness is a multifactorial process involving a complex interplay of ige-dependent reactions, epithelial activation or damage, autonomic nervous system dysfunction and, of particular interest and relevance to our discussion, enhanced allergic inflammation."^^ in ige-mediated reactions, the tissue response, be it the skin, nose or airway, is influenced by i g e sensitization of mast cells and basophils, release of bronchospastic and inflammatory mediators from sensitized cells, and the response of the target organ, which in asthma is bronchial smooth muscle. t o evaluate the effects of respiratory viruses on a facet of the immediate hypersensitivity response, i.e. mediator release, basophil histamine secretion was determined. in an in vitro model system, peripheral blood mononuclear cells were incubated with influenza a virus and the effect of this exposure upon ige-dependent basophil histamine release and leukotriene (lt)c4 was measured. following incubation with virus, both histamine and ltc4 secretion was enhanced. t o define the role of tcells in this process, t-cells were removed by magnetic head separation with anti-cd3 antibody. in the absence of t-cells, virus incubation did not enhance histamine release."^^ furthermore, analysis of virus-treated peripheral blood mononuclear cells revealed that the virus caused the generation of interferon (ifn)-y. these findings suggest that t-cells, and their cytokine products such as ifn-y, may play an integral role in the processes by which respiratory viruses enhance basophil histamine release, and are likely to affect other cell functions. the use of bronchoscopy with lavage and biopsy has provided direct assessment and analysis of airway mediators and the histology of inflammatory events in asthma. we have used fibreoptic bronchoscopy to perform segmental bronchoprovocation with antigen and bronchoalveolar lavage (bal) to assess immediate and late responses of the airway to antigen and to study the effects of a rhinovirus illness on these allergic events."^"^ t o conduct these experiments, a bronchoscope is introduced into the airways, bronchial segments are identified and an allergen is administered directly on to the airway mucosa. the immediate response to antigen is characterized by a brisk rise in mast cell mediators. when lavage of the previously allergen-challenged segments is performed 48 h later, there is a highly cellular, eosinophil-rich response detected. based on our previous observations that a rhinovirus 16 infection increases bronchial responsiveness and lars to inhaled antigen, we used segmental antigen challenge and lavage to test our hypothesis that the rhinovirus upper respiratory illness may enhance the lower airways allergic inflammatory response to antigen. histamine is released into the airway immediately following antigen challenge and represents mast cell activation. we found histamine release into the airway immediately following local antigen challenge to be significantly potentiated by rhinovirus infection (table 30 .8). these observations indicate that an acute rhinovirus illness can enhance airway mast cell mediator release. these findings are similar to earlier reports and indicate an effect of the infection on mast cell activation."^"^ interestingly, and relevant to earlier observations that rhinovirus effects on airway function are long-lasting, enhanced histamine release to antigen was still present 6 weeks after the acute infection; at this time, the individuals were symptom-free. our findings suggest that viral effects on allergic responses can persist for weeks beyond the acute illness and that such persistence can explain the long-lasting effects on airway function in asthma from a cold. when lavage is performed 48 h after antigen challenge, there is a characteristic and often dramatic influx of eosinophils into the lavage fluid. we found significant enhancement of eosinophil recruitment to the airway 48 h after antigen when the subjects had an acute rhinovirus infection (fig. 30.1) . like the observations with enhanced and persistent histamine release, not only was a greater eosinophil recruitment to antigen challenge noted during the acute infection, but this respiratory virus effect persisted into the recovery period 6 weeks later. these findings suggest a possible explanation for the increase in lars to antigen noted during acute rhinovirus infection; if allergen exposure during rhinovirus infection is associated with enhanced eosinophil recruitment to the airway, allergic inflammation and an lar are more likely to occur. fraenkel and colleagues'^^ provide additional insight into the mechanisms by which rhinovirus infections promote airway inflammation and the likelihood for increased asthma during colds. both normal and asthmatic subjects were recruited for their study. the investigators obtained bronchial mucosal biopsies before and during an acute experimental rhinovirus infection and evaluated the changes in inflammatory cell infiltrate due to the virus and airway responsiveness. an increase in the airway response to histamine during the cold was associated with increases in submucosal lymphocytes. further, there was an increase in epithelial eosinophils during the experimental cold ( fig. 30.2) . in asthmatic compared with normal subjects, the increase in mucosal eosinophils persisted into the convalescent period. although the changes in various features of immunohistology were not dramatic, they did follow a pattern that is consistent with our findings of enhanced eosinophil recruitment to the airways and were correlated with alterations in lung physiosiveness. collectively, there is evidence from airway physiology that rhinovirus infections enhance bronchial responsiveness and histological features of allergic inflammation, i.e. there is a greater likelihood for lars to occur. furthermore, from studies that directly sample the lower airway, there is evidence of increased eosinophil recruitment and mucosal eosinophilia at the time of rhinovirus infection, and these abnormalities persist for weeks beyond the acute illness. all these findings indicate that rhinovirus infections can perpetuate features of allergic inflammation and may thus explain an increase in the likelihood of asthma. the questions now remaining to be answered concern the mechanisms by which rhinovirus infections enhance allergic airway inflammation. using the nasal mucosal response to rhinovirus as a surrogate of the airway response to rhinovirus, fraenkel et al?^ biopsied nasal tissue during an acute rhinovirus infection. mast cells, eosinophils, lymphocytes and neutrophils were identified by appropriate monoclonal antibodies. there were no significant changes in the numbers of inflammatory cells present during the cold or the convalescent period compared with baseline values. although these observations contrast with biopsy findings in the lower airway,'^^ changes in airway 0.048 0028 100-, histology were not dramatic. these findings suggest that other mechanisms, i.e. enhanced mediator release or, more likely, cytokine generation, are responsible for changes in airway function during a cold. in our study with segmental antigen challenge,"^-^ the lavage fluid was analysed for tumour necrosis factor (tnf)-a. lavage samples obtained 48 h after antigen challenge had a significant rise in tnf-a; these changes were noted in both normal controls and allergic subjects. o u r findings raise the possibility that rhinovirus infection causes secretion of tnf-a and if enhanced secretion of cytokines were to occur in the allergic subject, features of allergic inflammation are more likely to occur. these findings also raise the possibility that rhinovirus can directly stimulate cytokine production. linden et a/.^^ measured ifn-y, interleukin (il)-1)s, granulocyte-macrophage colony-stimulating factor (gm-csf), il-4 and il-6 in nasal secretions of subjects with allergic rhinitis. during an experimental coronavirus cold, there was an increase in ifn-y but not the other cytokines. the increase in ifn-y is expected and represents an antiviral response to the virus. however, ifn-y can have pro-inflammatory effects, i.e. promotion of adhesion protein expression and enhanced survival of eosinophils."^^ moreover, these observations indicate that rhinovirus infections can stimulate cytokine production in vivo. t o extend these observations, live rhinovirus was incubated with human mononuclear cells and lung macrophages. gern et al.^'^ found that these cells took up the virus, but it did not replicate inside the cells. none the less, rhinovirus was able to activate monocytes and airway macrophages and cause secretion of tnf-a. these observations indicate that rhinovirus can cause synthesis and release of pro-inflammatory cytokines, which may occur independent of cell infection. to identify the mechanisms by which rhinovirus promotes allergic inflammation, it is essential that the immune response to this virus be established so that the factors, and cells, involved in the promotion of allergic inflammation are identified. in determining the effect of rhinovirus on the immune response, it has been established that the receptor for the majority (90%) of rhinovirus strains is intercellular adhesion molecule (icam)-!."^^ the other 10% of rhinovirus strains use the low-density lipoprotein receptor.^^ interaction of rhinovirus with these receptors is the first step in the host's response to this virus. with this information, we have found that a major rhinovirus, rhinovirus 16, binds to mononuclear cells via the icam-1 receptor, with binding occurring predominantly to monocytes but not lymphocytes. when rhinovirus 16 was incubated with a mixture of monocytes and lymphocytes, the following events occurred: there was a large increase in the number of cd3 "^ cells that express the early activation marker cd69; when the cd3 "^ /cd69 "^ cells were isolated and analysed, they were found to have increased expression of mrna for ifn-y. these in vitro observations suggest that rhinovirus activates monocytes to generate cytokines that stimulate lymphocytes. once activated, these lymphocytes (cds "^/cd69 "^) generate a variety of cytokines, including ifn-y. although ifn-y is normally important in antiviral activity, it also possesses a number of pro-inflammatory functions, including increased expression of endothelial and epithelial icam-1 (more receptors for rhinovirus) and the promotion of eosinophil survival. therefore, in addition to activation of a small number of rhinovirus-specific lymphocytes, rhinovirus can cause a 'non-specific' activation of lymphocytes through the generation of cytokines from monocytes; these cytokines can, in turn, promote either viral elimination or inflammation. perhaps the sequelae of this particular response are more likely if allergic inflammation already exists. to extend this possibility, einarsson and coworkers^^ evaluated the effect of respiratory viruses on stromal cell production of il-11. il-11 is an extremely cationic cytokine and has the capacity to induce airway hyperresponsiveness in the murine lung. these investigators found that rhinoviruses, along with rsv and parainfluenza, were potent stimulators of il-11. in contrast, cytomegalovirus and adenovirus were weaker activators and are viruses not associated with asthma exacerbations. furthermore, increased levels of il-11 were found in nasal secretions of children with uris and were more likely to appear in the nasal aspirates of those children with reactive airway disease. these observations are another example of the mechanism by which respiratory viruses can enhance airway inflammation through the generation of specific cytokines and thus increase the likelihood of asthma during a cold. using a mouse model, coyle et al?^ characterized the immune response to virus peptides and the resulting cytokine patterns. these investigators found that bystander cd4"^ th2 immune responses to ovalbumin switched peptide-specific cds"^ t-cells in the lung to il-5 producers. when these il-5-producing cds"^ t-cells were challenged, via the airways, with virus peptide, a significant eosinophil infiltration was induced. furthermore, in vitro studies indicated that il-4 could switch virus-specific cd8~^ t-cells to il-5 production. these results demonstrate a network by which allergic eosinophilic inflammation can occur during a respiratory viral infection and how this mechanism would impair cd8~^ t-cell responses (less ifn-y production) and thus delay virus clearance. such observations indicate not only a network to promote allergic inflammation but also a mechanism by which virus particles may persist for extended periods. our studies in humans, and those of coyle et al}^ in the mouse, suggest a possible link between eosinophils and airway changes during viral respiratory infections. when garofalo et alp sampled nasolaryngeal samples from children with rsv respiratory illnesses, the eosinophil protein, eosinophil cationic protein, was significantly increased in samples from children with bronchiolitis when compared with subjects with upper airway disease or rsv pneumonia but no wheezing. these data suggest that eosinophil activation by rsv may cause airway injury and the presence of wheezing. to extend these findings with in vitro experiments, kimpen et al}"^ incubated isolated human eosinophils with rsv. rsv directly activated eosinophils to release superoxide and ltc4. furthermore, incubation of eosinophils with rsv primed the cell for further activation and generation of inflammatory mediators. both observations indicate that an airway-active virus, in this case rsv, can interact with eosinophils and cause them to release proinflammatory mediators. as indicated by the authors, these effects of the virus could play a role in the pathogenesis of rsv bronchiolitis and may also be important for the development of more long-term bronchial hyperresponsiveness associated with viral infections. it is also possible that other respiratory viruses, such as rhino virus, may have similar effects during a cold. the mechanisms involved in the development of airway hyperresponsiveness, airway obstruction and recurrent wheezing with viral respiratory infections have yet to be fully appreciated. none the less, current evidence indicates that viruses, or products of virusinfected cells, influence the inflammatory property and potential of many cells. precisely how these effects of the virus translate into increased airway injury, responsiveness and obstruction will require further work. as the mechanisms of these interactions are established, so will improved understanding of asthma pathogenesis and treatment. the significance of early wheezing lung function, pre-and post-natal smoke exposure, and wheezing in the first year of life the relationship of acute bronchiohtis to bronchial asthma: a 4-to-14 year follow-up the relationship between proven viral bronchiolitis and subsequent wheezing bronchiolitis as a possible cause of wheezing in childhood study of 8-year-old children with a history of respiratory syncytial virus bronchiolitis in infancy wheezing, asthma, and pulmonary dysfunction 10 years after infection with respiratory syncytial virus in infancy the relationship of respiratory infections in early childhood to the occurrence 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respiratory tract during naturally acquired respiratory syncytial virus infection activation of human eosinophils in vitro by respiratory syncytial virus acknowledgements support for this chapter has come from grants nih hl 44098, ai-26609, ko8-01828 and general clinical research grant rr-03186. key: cord-001542-f089bs8r authors: lai, kang yiu; ng, wing yiu george; cheng, fan fanny title: human ebola virus infection in west africa: a review of available therapeutic agents that target different steps of the life cycle of ebola virus date: 2014-11-28 journal: infect dis poverty doi: 10.1186/2049-9957-3-43 sha: doc_id: 1542 cord_uid: f089bs8r the recent outbreak of the human zaire ebolavirus (ebov) epidemic is spiraling out of control in west africa. human ebov hemorrhagic fever has a case fatality rate of up to 90%. the ebov is classified as a biosafety level 4 pathogen and is considered a category a agent of bioterrorism by centers for disease control and prevention, with no approved therapies and vaccines available for its treatment apart from supportive care. although several promising therapeutic agents and vaccines against ebov are undergoing the phase i human trial, the current epidemic might be outpacing the speed at which drugs and vaccines can be produced. like all viruses, the ebov largely relies on host cell factors and physiological processes for its entry, replication, and egress. we have reviewed currently available therapeutic agents that have been shown to be effective in suppressing the proliferation of the ebov in cell cultures or animal studies. most of the therapeutic agents in this review are directed against non-mutable targets of the host, which is independent of viral mutation. these medications are approved by the food and drug administration (fda) for the treatment of other diseases. they are available and stockpileable for immediate use. they may also have a complementary role to those therapeutic agents under development that are directed against the mutable targets of the ebov. electronic supplementary material: the online version of this article (doi:10.1186/2049-9957-3-43) contains supplementary material, which is available to authorized users. the recent outbreak of the human zaire ebolavirus (ebov) infection starting in west african countries has resulted in 15,351 infected patients, as of 18 th of november 2014. a total of 5,459 deaths have been reported in six affected countries (guinea, liberia, mali, sierra leone, spain, and the united states of america) and two previously affected countries (nigeria and senegal) [1] . apart from supportive care, neither a licensed vaccine nor a specific therapy is available for the treatment of the human ebov infection [2] . the world health organization (who) has considered that it is ethically acceptable to offer unproven interventions that have shown promising results in laboratory and animal models, but have not yet been evaluated for safety and efficacy in humans as potential sources of treatment or prevention [3] . several promising therapeutic agents have been identified for the treatment and immunization of the ebov. these may include monoclonal antibody (mabs)-based therapies (e.g. zmapp), anti-sense phosphorodiamidate morpholino oligomers (pmo avi-6002), lipid nanoparticle small interfering rna (lnp-sirna: tkm-ebola), and an ebov glycoprotein-based vaccine using live-attenuated recombinant vesicular stomatitis virus (rvsv-ebogp) or a chimpanzee adenovirus (rchad-ebogp)-based vector. human trial results of these agents would not be available until next year. moreover, existing supplies of all these experimental medications and vaccines for compassionate use are either extremely limited or exhausted [4] [5] [6] . to combat such an unprecedented global public-health crisis before these experimental agents are available, alternative available interventions that can target different steps in the replication cycle of the ebov should be explored in the management of the human ebov infection as contingency preparation for the international dissemination of the ebov outbreak in west africa. we have reviewed currently available therapeutic agents that have shown to be effective in suppressing the proliferation of the ebov in cell cultures or animal studies. we propose a therapeutic regimen to supplement the current supportive therapy aiming to reduce viral load, the most important factor in the determination of mortality. through viral load suppression, we may be able to prolong a patient's survival in order to provide a better chance for the patient to develop natural immune defense against the ebov. the ebov is an enveloped filamentous rna virus belonging to the family filoviridae. the 19-kb linear, non-segmented, negative-sense, single-stranded rna genome of the ebov encodes seven structural proteins and two non-structural proteins in the following order within the genome: 3′ non-coding region (leader), nucleoprotein (np), virion protein 35 (vp35), vp40, 3 glycoproteins (sgp/ssgp/gp1,2), vp30, vp24, rnadependent rna-polymerase protein (l-polymerase), and 5′ non-coding region [7] . the ebov genome encodes one transmembrane protein gp1,2 (gp1-gp2) and two secreted non-structural proteins: secretary glycoprotein (sgp) and small soluble glycoprotein (ssgp). a small soluble delta peptide (δ-peptide) is secreted from ebov-infected cells after the carboxylterminal cleavage of sgp [8] . gp1,2 is produced through transcriptional rna editing as a precursor for 676 amino acid polyprotein (gp0), which is post-translationally cleaved by furin into two disulfide-linked subunits; a surface subunit, gp1; and a membrane-spanning subunit, gp2. gp1 contains the receptor-binding domain (rbd) for host cell attachment and a mucin-like domain to protect the rbd from humoral and cell-mediated immunity. the rbd responsible for receptor binding, viral entry, and cellular tropism is covered by a heavily glycosylated "glycan cap". the transmembrane gp2 contains a helical heptad-repeat region, transmembrane anchor, and a 4-residue cytoplasmic tail. the gp2 drives fusion of the viral membrane with the endosomal membrane of the target cell. this gp1-gp2 heterodimer then assembles as a trimer on the viral surface. this homotrimeric gp1,2 complex forms the spike on the envelope membrane of the mature viral particles. during processing, gp1,2 are unstable, and an abundant amount of a soluble non-virion form of gp1 and a scanty amount of gp1,2 are released into the circulation [9] [10] [11] [12] . the virusassociated gp1,2 and not the other soluble glycoproteins released during the virus infection are responsible for primary target cell activation [13] . the highly glycosylated mucin-like region of gp1 is cytotoxic to the host cells [14] . the shedding of souble gp1,2-like protein due to cleavage of ebov glycoprotein on the surface of ebov-infected cells by tumor necrosis factor-alpha converting enzyme (tace) can activate non-infected dendritic cells and macrophages to induce cytokine dysregulation and endothelial cell dysfunction [15] . the gp2 of the ebov is able to counter the interferon (ifn)-inducible antiviral protein tetherin which restricts the vp40-dependent budding of the progeny viral particles from infected cells [16] [17] [18] . the sgp is produced from non-edited mrna species through furin cleavage from a precursor pre-sgp. the sgp shares the n-terminal 295 amino acids with gp1, but differs in the carboxyl terminus by 69 amino acids. the sgp is released into the circulation in the form of homodimers in antiparallel orientation [19] to evade an antibody-associated innate immune response [20, 21] . the sgp has an antiinflammatory function and impairs the transmigration and activation of neutrophils [22, 23] . while the gp1,2 in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function, sgp protects the endothelial cell against cytokine-induced barrier dysfunction. the sgp constitutes at greater than 80% of the total gp synthesized during infection. hence, the hypersecretion of the sgp may protect the ebov against host humoral immune defense and the host endothelial cell against cytokine-induced cytotoxicity during the early phase of the ebov infection [15, 24, 25] . δ-peptide released in ebov-infected cells joins cathepsins and integrins to inhibit further entry of the ebov in a dose-dependent manner to prevent superinfection of ebov-infected cells. δ-peptide inhibits entry of both marburgviruses and the ebov, indicating that they might interfere with a common pathway used by filoviruses to gain entry into target cells [26] . the ssgp of a yet undefined function is produced through transcriptional editing and secreted in the form of a disulfide-linked homodimer that is exclusively n-glycosylated. while ssgp appears to share similar structural properties with sgp, it does not appear to have the same anti-inflammatory function as sgp [22, 23, 27] . the ebov, being a rna virus with limited coding capacity, has utilized the host's unique metabolic pathway for its viral entry, replication, and egress. the entry of the ebov into cells is initiated by interaction of the viral gp1 with host cell surface t-cell immunoglobulin and mucin domain 1 (tim-1) receptors. upon receptor binding, the ebov is internalized into endosomes primarily via macropinocytosis [28] [29] [30] . within the acidified endosome compartment of the host cell, the heavily glycosylated gp1 is cleaved to a smaller 19-kda fusogenic form by the low ph-dependent cellular proteases cathepsin l (catl) and b (catb), exposing residues in the receptor binding site. this allows the binding of gp1 to cholesterol transporter niemann-pick c1 (npc1), a step in the late endosome phase essential for virus-host membrane fusion and viral entry [31] [32] [33] [34] . cells where the npc1 function has been biochemically disrupted or cells lacking npc1 showed resistance to the ebov infection. cells from subjects with npc1 disease were resistant to the ebov because of defects in the npc1 protein [35] [36] [37] [38] . after complete fusion of the viral and host endosomal membranes via conformational change in gp2, viral rna and its associated proteins are released into the host cell cytoplasm [39] . once inside the cytoplasm of the host cell, the ebov suppresses the innate immune response via vp35 and vp24 proteins [40] , and hijacks transcription and translation for robust genome replication and the production of new virions. the ribonucleoprotein (rnp) complex that mediates transcription and replication of the ebov genome comprises np, vp35, vp30, and l protein [41] [42] [43] [44] . vp30 is essential in the initiation of the ebov transcription, but is not required for viral replication. however, dynamic phosphorylation of vp30 is an important mechanism to regulate the balance between the transcription and replication processes in the ebov replication cycle [45] [46] [47] . this unique property of vp30 allows the development of a genetically stable vp30 deleted ebov vaccine with protective efficacy in the mice and guinea pig models [48] . the matrix proteins vp40 and vp24 associated with the viral lipid coat are important for virus structure and stability. both matrix proteins vp24 and vp40 contribute to the regulation of viral genome replication and transcription [49] and the budding of the virus [50] [51] [52] , an important step prior to viral egress [53, 54] . this distinct replication cycle of the ebov serves as an attractive target for the development of therapeutic agents against the ebov (see figure 1 and table 1 ). human ebov hemorrhagic fever, characterized by uncontrolled viral replication together with immune and vascular dysregulation, has a case fatality rate of up to 90% [7] . type i alpha/beta interferons (ifn-α/β), encoded by a single ifn-β and 13 homologous ifn-α genes in humans, represent an essential element of host defense against virus infections, including the ebov [55] . the human ebov infection is associated with robust ifn-α production-with plasma concentrations of ifn-α that greatly (60-to 100-fold) exceed those observed in other viral infections-but limited ifn-β production [56] . the upon receptor binding of ebov gp 1 with host tim-1 receptor, ebov is internalized into endosome via macropinocytosis. within the acidified endosome compartment of the host cell, under the action of the low ph-dependent cellular proteases cathepsins, the receptor binding site of gp 1 to cholesterol transporter niemann-pick c1 (npc1) is exposed. this results in conformational change in gp 2 , leading to complete fusion of the viral and host endosomal membranes in the late endosome and the release of viral rna and its associated proteins into the host cell cytoplasm. ebov then hijacks transcription and translation for robust genome replication and viral protein production under the action of ribonucleoprotein polymerase complex (rnp polymerase). the accumulation of gp 1,2 in the endoplasmic reticulum leads to endoplasmic reticulum overload response (er-overload) which, in turn, induces cytokine dysregulation via the activation of nuclear factor kappa b (nfκb) through the production of reactive oxygen species (ros). new virions are released through atp-dependent budding and egress from host cell membrane. currently available therapeutic agents that target the different steps of the ebov life cycle are described in table 1. ebov, protected from the host interferon response by its encoded vp35 and vp24 proteins [40, [57] [58] [59] , produced a heavy viral load [60] [61] [62] , cytopathic damages [14, 63, 64] , and cytokine dysregulation in humans [65] [66] [67] [68] . the efficient productive replication of the ebov inside monocyte and macrophages leads to a massive release of proinflammatory cytokines/chemokines and reactive oxygen species (ros) [13, 15, 65, 66, [69] [70] [71] , which in turn leads to diffuse endothelial cell dysfunction [72] [73] [74] [75] [76] , disseminated intravascular coagulation [77] [78] [79] , and vasomotor collapse [80] [81] [82] . the infection of the antigen presenting dendritic cells [83] [84] [85] [86] and profound bystander apoptosis of lymphocytes [63, [87] [88] [89] impairs the development of adaptive immunity [90, 91] and ebov-specific cd8+ t [92] [93] [94] , as well as cd4+ t cells [95] that are important for the clearance of, and protection from, the ebov infection. infected monocyte-derived dendritic cells were impaired in the secretion of pro-inflammatory cytokines, the up-regulation of co-stimulatory molecules, and the stimulation of t cells [96] . numbers of cd4+ and cd8+ t cells are substantially reduced in fatal human and nonhuman primate (nhp) infections before death [63, 88, 97] . immune evasion by the glycoproteins of the ebola virus: implications on passive immunization and vaccine development the ebov is able to counteract both humoral and cellmediated immunity through its gp1,2 and sgp [11, 98] . the overexpression of mature gp1,2 on the plasma membrane results in the masking of antigenic epitopes on gp1,2 itself and the shielding of mhc-i and integrin β, leading to evasion of antiviral immunity. steric shielding of surface epitopes by the heavily glycosylated gp impairs the recognition and killing of ebov-infected cells by the natural killer and cytotoxic cd8+ t cell during an acute viral infection. it may also contribute to the persistent infection in the natural reservoir host to perpetuate the spread of the ebov [99] [100] [101] . the sgp can evade host antibody-mediated response through "antigenic subversion" by eliciting non-neutralizing antibodies that cross-react with gp1,2. thus, the massive secretion of sgp by the ebov may prevent effective neutralization of the virus during an ebov infection and reduce the effectiveness of vaccines that rely upon neutralizing antibody responses against gp1,2 [20, 21] . some of the antibodies against gp1 may lead to enhancement of infectivity of the ebov via interaction with complement component c1q, a phenomenon known as the antibody-dependent enhancement. the ebov initiates infection by binding its gp1 to its specific human receptor sites on the surface of human cells. the interaction of c1q enhances binding between the virus-antibody complex and the c1q ligands on the cell surface, promoting interaction between the ebov and its receptor. these infectivity-enhancing antibodies were virus species specific and were primarily correlated with immunoglobulin igg2a and igm levels, but not with igg1 levels [102, 103] . the presence of infectivity-enhancing antibodies against gp1,2 in the ebov infection raises concerns about the effectiveness of gp-based ebov vaccines, and the use of passive prophylaxis or treatment with gp-based antibodies [104, 105] . antibodies against gp1 of the ebov can be neutralizing, enhancing, or non-neutralizing and non-enhancing. neutralizing antibodies are produced in infection by the ebov at a relatively low frequency [106] . some anti-ebov antibodies are known to be neutralizing in vitro but not protective in vivo, whereas other antibodies are known to be protective in animal models in vivo, but not neutralizing in vitro [107] . investigations of anti-gp antibodies against the ebov showed that non-neutralizing antibodies induce interferon-inducible transmembrane proteins (ifitmp) production to restrict entry of ebov. favipiravir suppress viral rna polymerase. inhibit na + /k + -atpase that are important in the budding and egress of encapsulated ebov. ouabain digoxin digitoxin anti-oxidants suppress ros-dependent nfκb activation and cytokine dysregulation induced by gp 1,2 -induced er-overload. high dose n-acetylcysteine infusion 1 chloroquine, amiodarone, dronedarone and toremifene administration is associated with an increased risk of qt prolongation and torsades de pointes. 2 verapamil should be avoided in patient with hypotension. recognized gp epitopes in the sgp or non-essential mucin-like domain of gp1, while neutralizing antibodies were specific to rbd in gp1 or conformation-dependent epitopes at the base of the gp1,2 spike where gp1 meets gp2. two neutralizing antibodies (kz52 and jp3k11) against ebov-that recognize conformation-dependent epitopes comprising residues in gp1 and gp2-were identified to have quite distinct mechanisms of neutralization. kz52 is a human recombinant igg1 neutralizing antibody derived from a human survivor of a natural ebov infection during the 1995 outbreak in kikwit, democratic republic of congo. kz52 has impaired recognition for the sgp and binding was dependent on the presence of gp2 residues which are not present in the sgp. kz52 is able to inhibit cathepsin cleavage of gp1,2. jp3k11, a monkey derived neutralizing monoclonal antibody against ebov, recognized the cleaved, fusion-active form of gp [108] . 16 f6 is a mice derived monoclonal igg1 antibody that neutralizes sudan ebov by preventing the conformational changes in gp1,2 required for membrane fusion. both 16 f6 and kz52 recognize gp1-gp2-bridging epitopes at the base of the gp1,2 trimer, indicating that this overlapping epitope may be one of the key sites for neutralization of the ebov, and is thus a target for immunotherapy and a key goal of vaccine design [109] . antibody subclass may be another important factor in protection against the ebov. igg2 isotype may offer more effective protection against ebov [110, 111] . although fully protecting guinea pigs from infection, kz52 fails to slow viral replication and protect nhps from the ebov infection [112] . in contrast, rvsv-ebogp [113] [114] [115] [116] and rchad-ebogp [117] [118] [119] [120] based vaccination have demonstrated both prophylactic and post-exposure protection in nhps [121] . this was previously attributed to the protective action of ebov-specific cd4+ and cd8+ t-cell response induced by these vaccines in limiting infection and the inability of kz52 to completely block all entries of the ebov into cells and its subsequent explosive replication [112] . rchad-ebogpbased vaccination is able to generate potent humoral and cell-mediated responses. significant antibody titers are detectable at 48 weeks post vaccination [122, 123] . cd8+ cellmediated immunity has been shown to play a critical role in protection against the ebov infection in nhps in rchad-ebogp-based vaccination [124] . on the other hand, humoral rather than the cell-mediated response contributes to protection against the ebov infection in nhps in rvsv-ebogp-based vaccination [125, 126] . candidate vaccines expressing the ebov gp or np protect rodents and nhps from the lethal ebov infection [127] [128] [129] . humoral and cell-mediated immune responses are working together to provide protection against the lethal ebov infection. either response alone may be able to limit virus replication but both arms of the immune response are required to clear the infection [97, 130] . vp proteins (vp24, vp30, vp35, and vp40) are poor inducers of cell-mediated immunity and are inaccessible to the protective effect of vp-induced neutralizing antibodies because they are not found on the surface of virions or infected cells [131] . however, the genetic sites of these internal proteins are susceptible to sirna and pmo interference. tkm-ebola (a sirna targeting l-polymerase, vp24, and vp35) can be administered intravenously or subcutaneously in a lyophilized lipid nanoparticle formulation. tkm-ebola offers post-exposure protection against the ebov infection in nhps. the fda has approved an "expanded access" program for the use of tkm-ebola in patients with confirmed or suspected infections [132, 133] . anti-sense phosphorodiamidate morpholino oligomers avi-6002 effectively reduce viral load, diminish virallyinduced pathology, and improve survival of nhps with the ebov infection by targeting vp24 and vp35 mrna. through judicious placement of positive charges on the drug backbone, the drug is able to bind to a negative charge on the virus even if binding at one or more drugvirus base pairs are lost through mutation. this integration of dual targeting and charge complementation significantly lowers the likelihood of drug resistance through viral mutagenesis [134, 135] . currently available therapeutic agents that are effective in targeting the ebov infection in cell or animal studies may include convalescent plasma, favipiravir, chloroquine, amiodarone, dronedarone, verapamil, clomiphene, toremifene, ifn-β, na + /k + exchangers, na + /k + -atpase pump inhibitors, and antioxidants. except for convalescent plasma and favipiravir, most of the therapeutic agents under review are acting against the non-mutable targets of the host cells which participate in the replication cycle of the ebov. they may also have a complementary role to conventional therapy in the management of the current ebov outbreak in west african countries (see table 1 ). the who issued a consensus statement that the use of whole blood therapies and convalescent blood serum needs to be considered as a matter of priority in the recent ebov outbreak in west african countries [2] . the development of neutralizing antibodies and t-cell responses are important for recovery from the ebov infection [97, 136] . patients who are able to mount an immune response to the ebov will begin to recover in seven to ten days and start a period of prolonged convalescence [137] . in survivors, early and increasing levels of igg, directed mainly against the np and the vp40, were followed by the clearance of circulating viral antigen and activation of cytotoxic t cells. in contrast, fatal infection was characterized by impaired humoral responses, with absent specific igg and barely detectable igm [63] . convalescent blood has been shown to improve survival of ebov-infected patients during the outbreak in kikwit in 1995 [138] . immunity against ebov gp is sufficient to protect individuals against infection, and several vaccines based on ebov gp are under development including recombinant adenovirus, parainfluenza virus, venezuelan equine encephalitis virus, vesicular stomatitis virus, and virus-like particles [139] . neutralizing human monoclonal antibodies is able to protect mouse and guinea pigs from lethal ebov. however, the protection was achieved only by treatment shortly before or after viral infection [140] [141] [142] . the ebov can rapidly mutate to produce antibody-escape mutants. hence, antibody therapy may require hyperimmune polyclonal serum or a panel of monoclonal antibodies of different epitope specificities to be successful [143, 144] . these studies have laid the foundation for subsequent clinical research on the development of monoclonal antibodies [145] [146] [147] [148] and utilization of a monoclonal antibody cocktail such as mb-003 [149] , zmab [150] , and zmapp [151] in the treatment of the ebov infection in nhps. it is interesting to note that all three monoclonal antibody cocktails include one antibody that binds to or close to the glycan cap and that two of the three monoclonal antibody cocktails include at least one antibody that binds the gp1/ gp2 interface, indicating that these two regions may be especially important in protection against ebov [148] . the treatment window of monoclonal antibody therapy can be extended by the co-administration of adenovirus-vectored interferon therapy. in a guinea pig model, monoclonal antibodies combined with adenovirus-vectored interferon given three days after infection resulted in 100% survival, a significant improvement over either treatment alone [152] . a subsequent study showed that such a combination therapy is capable of saving 100% of ebov-infected nhps when initiated after the presence of detectable viremia along with symptoms [153] . (2) favipiravir (t-705; 6-fluoro-3-hydroxy-2pyrazinecarboxamide) favipiravir is a broad-spectrum inhibitor of viral rna polymerase that is able to inhibit the replication of many rna viruses. it is registered in japan for the treatment of influenza virus infection [154, 155] . favipiravir is able to suppress the replication of the ebov in cell culture. favipiravir, initiated at day 6 after ebov infection, induced rapid virus clearance, reduced the biochemical parameters of disease severity, and prevented a lethal outcome in 100% of mice lacking the type i interferon receptor [156] . oral favipiravir taken twice daily for 14 days is able to give 100% protection against an aerosol ebov infection in an immune-deficient mice model [157, 158] . the survival benefit was suboptimal in nhps. only one of the six animals tested survived. studies using dosages that are two to five times higher and have duration longer than shown in influenza studies are being conducted for the human ebov infection [5] . bcx4430, a synthetic adenosine analogue with a viral rna polymerase inhibitor function, is active against the ebov and marburg virus in rodent and cell culture. bcx4430 completely protects nhps from the marburg virus infection when administered as late as 48 hours after infection [159, 160] . the antimalarial drug chloroquine is able to increase endosomal ph. an acidic endosomal environment is important for the ph-dependent activation of cysteine proteases catb and catl, the proteases responsible for the cleavage of ebov gp1,2 essential for endosomal virus-host membrane fusion [35, 39, [161] [162] [163] . however, proteolytic processing of the ebov glycoprotein has been demonstrated to be not critical for ebov replication in cell culture [164] or nhps [165] . a recent study using a catb and catl deficient mouse model for the study of the ebov infection demonstrates that catb and catl activity is not absolutely required for ebov replication. the ebov glycoprotein cleavage seems to be mediated through a broader spectrum of proteases making therapeutic approaches targeting limited proteases unlikely to be beneficial to combat ebov infections [166] . a broad-spectrum small molecule that targets the catl cleavage of the ebov and inhibits the entry of a wide variety of viruses has recently been identified. it has been examined for the potential to develop into a potent broad-spectrum antiviral medication [167] . multiple cationic amphiphiles including amiodarone, dronedarone, verapamil, clomiphene, and toremifene have been identified as potent inhibitors of the entry of the ebov in an npc1-dependent fashion [38, 168] . amiodarone used for the treatment of atrial fibrillation and ventricular cardiac arrhythmia can induce lipidosis with features similar to niemann-pick c disease [169] . amiodarone and dronedarone, having basic pka and high water solubility at acidic ph, accumulates within late endosomal compartments, blocking fluid-phase endocytosis, proteolysis and lipid trafficking, and inducing a niemann-pick c-like phenotype. in contrast to the niemann-pick type-c disease, they are not alleviated by cholesterol removal [170, 171] . amiodarone, at concentrations that are routinely reached in human serum during anti-arrhythmic therapy (1.5-2.5 μg/ml), is a potent inhibitor of filovirus cell entry through late endosomes (ic50 0.25 μg/ml for ebov), when induced as a niemann-pick c-like phenotype. significant inhibition is observed in most endothelial and epithelial cells (e.g. macrophage, monocyte, vascular endothelial cell), except for primary hepatocyte and fibroblast. the inhibitory effect of amiodarone on the entry of the ebov was dose-dependent and reversible upon removal of the drug. prolonged exposure to amiodarone will not lead to a compensatory change in the host cell. a similar inhibitory property is observed with the amiodarone-related agent dronedarone and the l-type calcium channel blocker verapamil [38, 168, 172, 173] . both clomiphene and toremifene have anti-ebov activity in both the vero e6 (interferon-deficient african green monkey kidney epithelial cells) and hepg2 (human hepatocellular carcinoma) cell lines. the anti-ebov activity of clomiphene and toremifene is dependent not on its estrogen receptor antagonistic action but upon the ability of both drugs to induce a niemann-pick c-like phenotype to inhibit viral entry at late endosome. clomiphene and toremifene do not disrupt the interaction between primed gp1 and npc1, but mediate the entry block indirectly through npc1 by targeting other endosomal/lysosomal proteins involved in the cholesterol uptake pathway whose functions may be regulated by npc1. clomiphene and toremifene at 60 mg/kg every other day have been shown to result in a 90% and 50% survival rate, respectively, in ebov-infected mice compared with 100% mortality in the control group in an in vivo murine ebov infection model. they are effective in both male and female mice [38, 174] . however, the therapeutic dose against ebov cannot be achieved with the oral clomiphene dose used for inducing ovulation in humans [175] [176] [177] . the therapeutic dose against ebov with tolerable side effects can be achieved with toremifene at an oral dose used in the human trial for the treatment of advanced carcinoma of the breast [178] [179] [180] [181] . toremifene is well absorbed and >99.5% bound to plasma protein. toremifene undergoes extensive liver metabolism and enterohepatic recirculation. the majority of the toremifene dose is excreted as metabolites in feces. the long terminal half-life of oral toremifene may be due to both plasma protein binding and enterohepatic recirculation [182, 183] . interferon-induced transmembrane proteins (ifitms) are expressed basally in the absence of ifn induction in both primary tissues and cell lines [184] . an ifitm is able to inhibit the entry of viruses to the host cell cytoplasm; permit endocytosis, but prevent subsequent viral fusion; and release viral contents into the cytosol. the human ifitm locus is located on chromosome 11 and composed of four functional genes: ifitm1, ifitm2, ifitm3, and ifitm5. ifitm4p is a pseudogene. viruses that are restricted by ifitm proteins tend to fuse with host cell membranes in a late endosome or lysosome that precedes the induction of type i ifn in infected cells. viral escape from restriction by ifitm proteins could be more challenging than for antagonizing inhibitory factors that function at later stages of the virus life cycle because the opportunity for de novo synthesis of viral inhibitors is not available. all four human ifitm proteins are induced robustly by both type i and type ii ifns. ifitm1 is active against multiple viruses, including the ebov and hepatitis c viruses [185] [186] [187] . ifnβ is able to induce interferon-inducible transmembrane protein production to restrict entry of the ebov [188] . early post-exposure treatment with ifn-β significantly increased survival time of rhesus macaques infected with a lethal dose of the ebov, although ifn-β alone failed to alter the mortality rate. ifn-β treatment was associated with a trend towards lower plasma and tissue viral burden and pro-inflammatory cytokines production [56] . amiloride and its derivatives are used as potassiumsparing diuretics to treat hypertension and congestive heart failure. apart from inhibiting epithelial na + channel and cellular na + /k + exchangers, these drugs could also affect the function of other less well-defined ionexchangers (na + /ca 2+ and na + /mg 2+ ), and disturb the equilibrium of other ions, such as mn 2+ [189] [190] [191] [192] . the entry of the ebov into host cells is the first step of infection and a crucial determinant of pathogenicity. upon receptor binding between gp1 and host tim-1 receptors, the ebov is internalized into endosomes primarily via the macropinocytic pathway. amiloride is able to inhibit the uptake of many viruses that utilize the macropinocytic pathway for host cell entry [193] [194] [195] [196] . amiloride at non-cytotoxic dosages leads to potent dosedependent inhibition of the entry and infection of the ebov [197, 198] . amiloride can lead to dose-dependent inhibition of rna synthesis. this may be due to a direct blockage of a nucleotide entry tunnel or catalytic site, or due to its effect on the equilibrium of mg 2+ and mn 2+ that are essential co-factors for polymerase activity and nucleotide insertion [199, 200] . these novel antiviral mechanisms of amiloride may uncover new targets for drug discovery against the ebov. adenosine triphosphate (atp) is essential in multiple steps in the replication cycle of many viruses. na + /k + -atpase pump is located in the plasma membrane of all animal cells to maintain the cell membrane potential. budding of enveloped viruses is a complex phenomenon that requires concerted actions of many viral and host components. atp may affect multiple steps in the budding process [201] . atp is required for the assembly and maturation of a number of enveloped viruses such as the influenza virus, vaccinia virus, retrovirus, and herpes simplex virus. the na + /k + -atpase pump inhibitors, ouabain, lanatoside c, strophanthidin, and digoxin are able to inhibit the replication of the influenza virus, newcastle disease virus, and vesicular stomatitis virus through an interferonindependent mechanism [202] . digoxin and lanatoside c have been shown to inhibit vaccinia virus replication at non-cytotoxic doses [203] . ouabain has shown antiviral activity against the influenza virus [204] , herpes simplex virus [205] , sendai virus [206] , murine leukemic virus [207] , cytomegalovirus porcine reproductive and respiratory syndrome virus [208] , and human cytomegalovirus virus [209] . one common feature shared by these viruses is that they all possess a lipid envelope. the ebov is an enveloped filamentous rna virus. the secondary matrix protein vp24-apart from its role in the evasion of host immune response, nucleocapsid formation, and regulation of replication-has an important role in viral budding and egress. na + /k + -atpase atp1a1 is detected to have a close interaction with vp24 of ebov during replication. ouabain, at a non-cytotoxic concentration of 20nm, is able to suppress the replication of the ebov in human mrc-5 cells [210, 211] . among the three cardiac glycosides that may include digoxin, digitoxin, and ouabain, only digoxin is commonly used in clinical practice. ouabain, because of its poor oral availability, is used primarily as a research tool. further research should be conducted to investigate whether digoxin and other na + /k + -atpase inhibitors might play a role in the management of the ebov or other enveloped virus infections. the virus-associated glycoprotein gp1,2 is responsible for the activation of human macrophages [13] . the highly glycosylated mucin-like region of the gp1 subunit of gp1,2 is cytotoxic to the host cells [14] . the mucin-like region in gp1 leads to an accumulation of gp1,2 at the endoplasmic reticulum, induces endoplasmic reticulum stress [212] , and activates nuclear factor kappa b (nf-κb) [213] . mutations of the ebov that lead to an enhanced accumulation of gp1,2 in the endoplasmic reticulum were significantly more cytotoxic than wild-type virus [214] . in human cells, the accumulation of protein in the endoplasmic reticulum will lead to endoplasmic reticulum overload response (eroverload) which activates nf-κb through the production of ros [215] . as a major transcription factor for antiviral and immune stimulatory activities, nf-κb is thought to play an important role in the induction of pro-inflammatory molecules, such as interleukin-1β (il-1β), and tumor necrosis factor α (tnf-α), upon cellular responses against a virus infection [216] . the cytokine dysregulation of the ebov involves massive ros, nf-κb, tnf-α, and il-1β activation [65, 66] . the effectiveness of antioxidant therapy for the ebov infection indicates the importance of ros in the pathogenesis of the ebov [217] . the activation of nf-κb by er-overload is ros-dependent [218] . nf-κb-induced cytokine dysregulation of novel h1n1 pneumonia has been shown to be suppressible by high-dose n-acetylcysteine (nac) antioxidant therapy at 100 mg/kg continuous infusion daily [219] . given the poor oral availability of nac in the range of 6% to 10% in humans [220] , a therapeutic dose of nac equivalent to the intravenous route can hardly be delivered by oral preparation. nac is a category b drug for pregnancy and is affordable, with a wide therapeutic window. nac has an established safety profile even in high doses and prolonged use in humans [221] [222] [223] . cytokine dysregulation is a common feature in the ebov infection and is associated with an enhanced mortality [65] [66] [67] [68] . antiviral medications directed against the mutable viral determinants of the ebov cannot directly prevent cytokine dysregulation. the early endothelial vascular damage characteristic of the ebov infection is not a direct effect of virus-induced cytolysis of endothelial cells, but is due to cytokine dysregulation resulting from massive release of proinflammatory cytokines/chemokines and ros by infected macrophage and monocytes [70] [71] [72] . lymphocytes are resistant to the ebov infection. cytokine dysregulation may also contribute to the diffuse bystander apoptosis of lymphocytes [63, [87] [88] [89] . with the safety profile of nac, if the therapeutic efficacy of a high-dose nac antioxidant therapy to manage ebov-induced cytokine dysregulation is confirmed, it may revamp the future management of the ebov infection. there is a desperate need for a viable treatment regimen in africa to engender hope and encourage people with symptoms and their close contacts to seek medical treatment, so as to limit the spread of the disease. this also helps to recruit and maintain adequate medical staff who are at high risk of contracting the disease. a proposed regimen against the human ebov infection based on available medications and information from in vivo animal testing and in vitro cell culture is attached (see tables 2 and 3 ). this regimen contains a cocktail of currently available medications that can target the different steps in the replication cycle of the ebov aiming to suppress viral proliferation. it has been shown that viral load is major contribution to survival in both human and animal studies [60] [61] [62] 136] . through viral load suppression, we may be able to prolong a patient's survival in order to allow the development of natural body immune defense against the ebov. the ebov has undergone a rapid mutation during its spread through humans [224] [225] [226] . the ebov is an rna virus the replication of which is highly error prone with nearly one viral mutation occurring during each cycle of replication. this extremely high mutation rate leads to significant genetic and antigenic diversity that allows the ebov population to evolve resistance to antiviral medications and vaccines [227, 228] . a combination therapy has been used in the treatment of rna virus infections, such as the human immunodeficiency virus (hiv) [229, 230] and hepatitis c [231, 232] to minimize the development of drug resistance. given the broad cell tropism and high replication rate of the ebov due to the potent suppression of both innate and adaptive immune responses of the host, patients with the ebov infection have an extremely high viral load. the selective pressure in the presence of the high mutation rate and viral load during the human ebov infection make the evolution of the ebov viral strains resistant to a single drug inevitable. the currently available medications in the proposed regimen-which is a treatment regimen containing a cocktail of antiviral medications targeting the different steps of the ebov replication in order to achieve maximal suppression of viral replication and to prevent the rapid development of resistance to favipiravir, the only drug in the regimen that is directed against a mutable target of the ebov-has been shown to reduce the replication of the ebov. [233] [234] [235] . the current ebov vaccine (rvsv-ebogp and rchad-ebogp) and therapeutic agents (zmapp, tkm-ebola, pmo avi-6002, and favipiravir) under development are directed against the mutable targets of the ebov, and their effectiveness is limited by viral mutation. the ebov, being a rna virus with limited coding capacity, has utilized the host's unique metabolic pathway for its viral entry, replication, and egress. most of the therapeutic agents in this current review are directed against nonmutable targets of the host which is independent of viral mutation. these medications are fda-approved for the treatment of other diseases. they are available and stockpileable for immediate use. they may also have a complementary role to those therapeutic agents under development that are directed against the mutable targets of the ebov. the primary target of the ebov is the mononuclear phagocytic system. the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells during the advanced stage of the disease [6, 236, 237] . the ebov may produce a viral load of up to 10 10 virions per ml serum in terminally ill patients [80] . oral amiodarone prophylaxis, by inducing a niemann-pick c-like phenotype in the cells of the mononuclear phagocytic system, may prevent viral entry into these cells during needle stick injury. through protection of the mononuclear system by our prophylaxis and cocktail therapy, we hope to offer a better chance of survival to these patients by allowing them to develop a natural body immune defense against the ebov infection. the liver, containing the largest number of fixed tissue macrophages (kupffer cells), as part of the reticuloendothelial immune defense system of the body, is a major target for the ebov infection [238, 239] . the ebov replicates to high titer in the liver [240] . hepatic apoptosis may play a role in the pathogenesis of the ebov infection [88] . toremifene is added to the treatment regimen for hepatic protection because amiodarone does not exert inhibitory action against the ebov in hepatocyte. however, both amiodarone and toremifene can increase qtc and the risk of torsades de pointes. therefore electrocardiogram should be carefully monitored if both drugs are to be used. amiodarone, favipiravir, and toremifene are available and stockpileable in oral preparations. these properties are advantageous in outbreak situations and contingency planning of a potential ebov epidemic or pandemic. the avoidance of intravenous administration will prevent needle stick injury in healthcare workers caring for the infected patients. ifn-β may have potential as an adjunctive postexposure therapy for high-risk exposure, such as needle stick injury, by inducing ifitm1 to limit entry of the ebov. post-exposure ifn-β treatment was associated with a trend towards lower plasma and tissue viral burden and pro-inflammatory cytokines production [56] . the reduction in viral load and cytokine dysregulation coupled with optimal supportive therapy may improve the chance of survival of the host to allow the development of natural immunity to control the underlying ebov infection. ifitm1 is active against multiple viruses, including the ebov [185, 188] and hepatitis c 1 ml of blood may contain 10 9 to 10 10 virions in terminally ill patient. prophylactic amiodarone therapy may protect macrophage, monocyte and endothelial cells immediately from ebov during needle stick injury and accidental exposure and allow time for the consideration of ifn-β, toremifene, favipiravir and convalescent blood serum therapy. 2 amiodarone is unable to protect hepatocyte from ebov infection. 3 both amiodarone and toremifene can increase the risk of qt prolongation and torsades de pointes. 4 the recommended dosage for treatment of human ebov infection may be 2 to 5 times higher than influenza studies. please confirm the recommended dose with the drug company. 5 n-acetylcysteine intravenous infusion at 100 mg/kg/day to control cytokine dysregulation (e.g. add 5 g of intravenous preparation of n-acetylcysteine into each liter of intravenous replacement fluid). [186, 187, 241, 242] . interferon induced ifitm1 plays an important role in the treatment of human hcv infection by inhibiting entry of hcv into the host cell [243] . six million international units (miu) of ifn-β intravenous administration is as effective as a three miu twice-daily regimen for treatment of the hcv infection [244] , but has lesser side effects that require discontinuation of the medication [245, 246] . as the aim of ifn-β therapy in our regimen for post needle stick prophylaxis against the ebov infection is to induce ifitm1 to limit viral entry, the dose of ifn-β for the post needle stick prophylaxis [247, 248] or induction therapy [249, 250] for hcv infection in humans is chosen. once infection is fully established, ifn-β are replaced by convalescent blood serum and high-dose nac infusion for providing passive humoral immunity and for the control of ros-dependent nf-κb-induced cytokine dysregulation respectively. the ebov is classified as biosafety level 4 pathogen and is classified by centers for disease control and prevention as a category a agent of bioterrorism with no approved therapies and vaccines for its treatment but carrying a high potential for large-scale dissemination. recent political, economic, military, and religious turbulence around the world raises concerns that the ebov might be used as an agent of bioterrorism [251] [252] [253] . the recent ebov epidemic is spiraling out of control in west africa. the containment measures that worked in the past, such as isolating those who are infected and tracing their contacts, have failed due to an exponential rise in infected patients. although the short-term (threeand six-week) probability of international spread outside the african region is small, the risk of the extension of the outbreak to other african countries followed by international dissemination on a longer time scale is not negligible, indicating that this public health emergency has the potential to grow to extraordinarily destructive dimensions [254, 255] . although several promising therapeutic agents and vaccines against the ebov are undergoing the phase i human trial, the current epidemic might be outpacing the speed at which drugs and vaccines can be produced [5] . to combat such an unprecedented global public-health crisis before these experimental agents are available, alternative available interventions capable of managing the enhanced viral replication and cytokine dysregulation of the human ebov infection should be explored and stockpiled as contingency preparation for the worst-case scenario of an impending human ebov pandemic [256] . like all viruses, the ebov largely relies on host cell factors and physiological processes for its entry, replication, and egress which, in turn, lead to cytopathic damage, cytokine dysregulation, and death of the host. these non-mutable key steps inside the host may be novel targets for future therapeutic strategies against these rapidly mutating viruses. if the efficacy of amiloride, digoxin, amiodarone, and high-dose nac antioxidant therapy against the human ebov infection is confirmed, the availability and affordability of these stockpileable oral regimen are for those workers who are already on amiodarone prophylaxis with a loading dose of amiodarone 600 mg p.o. twice daily for 8 days followed by maintenance amiodarone 600 mg p.o. daily. electrocardiogram and thyroid function should be monitored. 2 monitor for side effect of thrombocytopenia and proteinuria. 3 intravenous dosage of ifn-β that are used for human hepatitis c virus infection to induce ifitm1 to limit viral entry. 4 intravenous regimen is for those workers who have not been on amiodarone prophylaxis and agreed for the insertion of a central venous line for drug administration. intravenous amiodarone should be administered via central venous line to avoid phlebitis. the dosage for treatment of frequently recurring ventricular fibrillation and hemodynamically unstable ventricular tachycardia is recommended because it can achieve therapeutic drug level immediately after the first dose of amiodarone. agents make them ideal medications in pandemic situation and in countries with limited resources. they may have a complementary role to other antiviral medications to prevent the emergence of resistant strains. this may also signify a major breakthrough in future management of the ebov infection. additional file 1: multilingual abstracts in the six official working languages of the united nations. the authors declare that they have no competing interests. authors' contributions kyl and gwyn contributed to the conception, drafting, and writing of the paper. kyl, gwyn and fc revised the draft paper. all authors read and approved the revised paper. world health organization ebola response roadmap situation reports world health organization statement on the who consultation on potential ebola therapies and vaccines world health organization: ethical 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two-parameter model filoviruses": a real pandemic threat? submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution key: cord-003598-m2fsrwvw authors: elbahesh, husni; gerlach, thomas; saletti, giulietta; rimmelzwaan, guus f. title: response modifiers: tweaking the immune response against influenza a virus date: 2019-04-12 journal: front immunol doi: 10.3389/fimmu.2019.00809 sha: doc_id: 3598 cord_uid: m2fsrwvw despite causing pandemics and yearly epidemics that result in significant morbidity and mortality, our arsenal of options to treat influenza a virus (iav) infections remains limited and is challenged by the virus itself. while vaccination is the preferred intervention strategy against influenza, its efficacy is reduced in the elderly and infants who are most susceptible to severe and/or fatal infections. in addition, antigenic variation of iav complicates the production of efficacious vaccines. similarly, effectiveness of currently used antiviral drugs is jeopardized by the development of resistance to these drugs. like many viruses, iav is reliant on host factors and signaling-pathways for its replication, which could potentially offer alternative options to treat infections. while host-factors have long been recognized as attractive therapeutic candidates against other viruses, only recently they have been targeted for development as iav antivirals. future strategies to combat iav infections will most likely include approaches that alter host-virus interactions on the one hand or dampen harmful host immune responses on the other, with the use of biological response modifiers (brms). in principle, brms are biologically active agents including antibodies, small peptides, and/or other (small) molecules that can influence the immune response. brms are already being used in the clinic to treat malignancies and autoimmune diseases. repurposing such agents would allow for accelerated use against severe and potentially fatal iav infections. in this review, we will address the potential therapeutic use of different brm classes to modulate the immune response induced after iav infections. influenza viruses (ivs) are responsible for significant morbidity and mortality in the human population with ∼500,000 annual deaths worldwide. ivs can cause severe acute respiratory disease especially in high-risk populations like children, the elderly and the immunocompromised. while both influenza a and b viruses (iav and ibv, respectively) cause annual epidemics, the majority of severe human infections are caused by iav. ivs have segmented negative-sense single-stranded rna genomes. the lack of proof-reading activity of the viral rna-dependent rna polymerase (rdrp) and successive replication can lead to the accumulation of nucleotide mutations which drive antigenic drift. in addition, the segmented nature of their genome allows genetic reassortment between iv's to take place, which can produce novel strains that have acquired alternative antigenically distinct hemagglutinin, also known as antigenic shift. both antigenic drift and antigenic shift contribute to the iv's ability to evade pre-existing host immunity induced by previous infections. early recognition and responses to iv infection are largely mediated by innate immune sensors expressed by its primary target, the alveolar epithelial cells (1, 2). recognition of ivs is mediated by pattern recognition receptors (prrs) that include toll like receptors (tlrs), retinoinc acid inducible gene-i (rig-i), and nucleotide oligomerization domain (nod)-like receptor family pyrin domain containing 3 (nlrp3); all of which can recognize viral rnas during various stages of the infection cycle (3-5). activation of these sensors triggers signaling cascades that lead to the production of interferons as well as pro-inflammatory cytokines and chemokines ultimately resulting in an antiviral state within the surrounding cells/tissue (6). accordingly, ivs have multiple mechanisms to evade these responses mediated by the viral nonstructural 1 protein (ns1), polymerase basic 1 protein (pb1), polymerase basic 2 protein (pb2), polymerase acidic (pa) and nucleoprotein (np) [ in otherwise healthy individuals, iav infections are mild and the ensuing pro-and anti-inflammatory responses are balanced. in contrast, a "cytokine storm" is typically associated with severe infections including those caused by highly pathogenic iv strains. during a cytokine storm, chemokine and cytokine responses are dysregulated in both intensity and kinetics resulting in excessive damage to the host due to infiltration of inflammatory immune cells. acute lung injury (ali) caused by this inflammatory response is typically characterized by significant damage or destruction of the respiratory epithelium leading to acute respiratory distress syndrome (ards) (7, 8). clinical treatment options for severe influenza virus infections remain limited and relying heavily on the administration of antiviral neuraminidase inhibitors (nais) and supportive critical care (9). however, nais have not been effective in patients with severe h7n9 or h5n1 infections and there is evidence that fatal outcomes are associated with development of antiviral resistance in patients (10-12). while virus-targeted therapies remain the standard approach, iv's mutability and adaptation to current antivirals has highlighted the need for new therapeutic options that target host factors that regulate iv infections and resulting immune responses. in either approach, the focus is to prevent or limit damage to the lung epithelium due to exaggerated or dysregulated immune cell responses. biological response modifiers (brms) can alter the immune response thereby offering an additional therapeutic approach to treating severe infections. in this review, we highlight several studies that have shown the viability of brms as potential treatment options. for clarity, brms are categorized based on the type of biological agent (table 1) . therapeutic antibodies iav infections and some vaccines elicit broadly-neutralizing antibodies (abs) that target the viral ha-stem. however, their abundance and immune-subdominance is overshadowed by abs targeting the ha-head domain. the effectiveness of these hastem abs against a broad range of iav subtypes, makes them an attractive target not only for vaccine development but also as antivirals. indeed, several ha-stem specific human monoclonal abs are now being evaluated in clinical trials [reviewed in davidson (34) ]. mhaa4549a, medi8852, and vis410 are human monoclonal abs that have been shown to control viral replication and improve symptoms of human patients in phase 2 clinical trials (13-15). while virus-specific abs aim to reduce antigenic load, abs to host targets aim at limiting the secondary wave of cytokines and reduce prolonged damaging cellular infiltration during severe infections. host-target directed antibodies have been utilized to target key regulators of this inflammatory wave and could potentially be used to dampen these overt responses. angiopoietin-like 4 (angptl4) is a soluble angiogenicregulating protein. following proteolytic cleavage, the cterminal portion (cangptl4) is involved in integrin-dependent wound repair and can regulate vascular permeability (35, 36) . angptl4 was significantly elevated in lung biopsies from iav-induced pneumonia patients (16). in mouse studies, neutralizing anti-angptl4 abs reduced pulmonary tissue leakiness significantly accelerating lung recovery and improved lung tissue integrity (16). neutrophil infiltration into the alveolar space occurs within 1 day following iav infections (37) . neutrophil extracellular traps (nets) released during iav-induced pneumonia into the alveolar space caused alveolar damage (38) . the complement protein c5a was shown to induce nets release and administration of anti-c5a abs (ifx-1) reduced h7n9-induced ali due to reduced infiltration of lung macrophages and neutrophils as well as reduction of viral load in african green monkeys (17, 39). tumor necrosis factor alpha (tnfα) is a key cytokine for controlling severe iav infections. it regulates two main antiviral functions: the induction of (i) the nfkb pathway, which ultimately controls expression of several inflammatory cytokines and (ii) apoptosis through multiple signaling cascades (40, 41) . tnf upregulation during iav infections correlates with infection severity, especially following highly pathogenic iav-infections (42) (43) (44) . mice treated with anti-tnf abs showed reduced disease burden; however, the authors of that study reported no effect on viral replication (20). tnf-related apoptosis inducing ligand (trail) can trigger apoptosis in iav-infected cells. iav-infected human epithelial cells are sensitized to trail-mediated apoptosis while peripheral blood mononuclear cells upregulate trail expression. moreover, administration of monoclonal abs against trail increases survival rate following iav infections in mouse studies (18, 19). antimicrobial peptides (amps) are host proteins that have direct antibacterial and antiviral activities and can modulate immune responses to infections. while the literature is largely focused on the antibacterial aspects of amps, several studies have highlighted the antiviral potential of amps against several viruses including ivs [reviewed in hsieh and hartshorn (45) anti-angptl4 -reduced pulmonary tissue leakiness, significantly accelerated lung recovery and improved lung tissue integrity in mice. -mouse-adapted laboratory iav (h1n1) c5a ifx-1 antibody -reduced viral load and virus-induced ali due to reduced infiltration of lung macrophages and neutrophils in iav-infected african green monkeys. -highly-pathogenic avian iav (h7n9) trail anti-trail -increased survival rate following iav infections in mouse studies. -improved symptoms and increased survival of iav infected mice. ed survival after h1n1 or h5n1 mouse infections. -1957 pandemic iav (h2n2); mouse-adapted laboratory iav (h1n1); 2009 pandemic iav (h1n1) (31) (32) (33) and albericio and kruger (46)]. ll-37 is a human cathelicidin derived amp that is found predominantly in neutrophils and its expression can also be induced in epithelial cells and macrophages (47) . aerosol administration of either human ll-37 or its mouse counterpart mcramp led to reduced morbidity and mortality to similar levels as the neuraminidase inhibitor zanamivir that is used for the treatment of human influenza patients (21). both cellular and viral fadd-like il-1β-converting enzymeinhibitory protein (cflip and vflip, respectively) protect cells from death receptor mediated apoptosis. kα2 is a vflip-derived peptide that consists of 10 amino acids from the α2 helix of the kaposi's sarcoma herpes virus (kshv) death effector domain 1 protein. a synthetic version of this peptide, tat-kα2, was generated by fusing kα2 to a portion of the hiv tat protein (22, 48) . in mouse challenge studies, intranasal administration of tat-kα2 at the time of infection with highly pathogenic avian h5n1 virus resulted in protection of the treated mice. no replicating virus was detected in the lungs at either 3 or 5 days after infection suggesting complete protection from infection (22). it should be noted that this effect is largely due to direct destabilization of the virions by the tat-kα2 peptide and it is likely that infection in treated mice was not established; the efficacy of this amp has not been determined during an established infection and warrants further investigation. host kinases regulate not only iav entry and replication but also initiate antiviral signaling cascades that regulate expression of pro-inflammatory chemokines and cytokines during infections and present viable targets for intervention (24, (49) (50) (51) (52) (53) (54) (55) (56) (57) (58) . iav infection has been shown to upregulate c-jun n-terminal kinases 1 and 2 (jnk1/jnk2). these kinases directly regulate the induction of pro-inflammatory responses. iav-induced jnk1/jnk2 activation mediates production of chemokines and cytokines including tnf-α, interferon β (ifn-β), and interleukin 6 (il-6) (24) . in vivo inhibition of jnk1/jnk2 resulted in reduced levels of pro-inflammatory cytokines and reduced viral titers (23, 24). the mitogen activated protein kinase (mapk), p38, regulates viral entry and replication (55, 59) . furthermore, p38 regulates ifn stimulated gene (isg) gene expression and ultimately cytokine production via stat1 phosphorylation (25) . using either of two specific p38 inhibitors (sb 202190 or sb 203580), mice were protected from lethal h5n1 infection exhibiting reduced mortality and pro-inflammatory responses (25) . activation of another mapk, mek, is required for efficient iav replication and its inhibition results in viral ribonucleoprotein (vrnp) retention and reduced titers of progeny virus (26, 60, 61) . importantly, treatment of mice with the clinically approved mek inhibitor (ci-1040) showed reduced lung viral load and mortality of mice following infection with a lethal dose of pandemic h1n1 iav; interestingly, this inhibitor significantly out-performed the clinically recommended oseltamivir in these studies (26) . another central regulator of immune responses at the epithelium as well as immune cells is the nf-κb signaling pathway. accordingly, iav has evolved several mechanisms to modulate this pathway to counteract antiviral responses including directly targeting the ikb kinase (ikk) (62, 63) . sc75741 is a potent nfkb inhibitor that functions by reducing the ability of the p65 subunit of the nfkb complex to bind dna; thereby limiting its transcription-regulating functions (64, 65) . in vivo administration of sc75741 at 4 days after lethal infection with either h5n1 or h7n7 avian viruses resulted in significant protection with most mice surviving and showing little to no clinical symptoms; similar results were obtained by prophylactic administration (27) . g-protein coupled receptor kinase 2 (grk2) is best known for its phosphorylation of gpcrs in cardiac tissue resulting in recruitment of β-arrestin to facilitate rapid receptor internalization and lysozomal degradation (66) . recent phosphoproteomic studies identified grk2 as a potentially proviral host protein for iav that plays a major role in virion uncoating (28) . although in vivo inhibition of grk2 using paroxetine led to a significant reduction in upper respiratory tract viral load and to a modest reduction in lower respiratory tract titers at 4 days post infection, this inhibition was not protective from lethal infections (28) . however, it is possible that the route of administration (intraperitoneal vs. intranasal) and dosing regimen influenced the results. sphingosin kinases (sphk) are lipid kinases that mediate conversion of sphingosine to bioactive lipid sphingosine 1phosphate (s1p) (67), a known modulator of central apoptotic pathways (68) . iav infections leads to increased expression and activation of sphk1 and sphk2 (29) and in vitro inhibition of sphk1 was shown to decrease iav rna synthesis via suppression of nfkb activation (69) . treatment of mice with specific inhibitors to either sphk1 or sphk2 or a pan-sphk inhibitor led to prolonged survival of mice following lethal iav infection (29) . peroxisome proliferator-activated receptors (pparα, pparβ, and pparγ) regulate metabolic homeostasis and are important mediators of the inflammatory response. several ppar agonists have been investigated for efficacy during iav infections with varying results. gemfibrozil (pparα agonist) not only improved symptoms when administered 4 days after infections with an h2n2 virus, but also increased survival of iav infected mice (31) . prophylactic treatment of h1n1-infected mice with pioglitazone (pparγ agonist) resulted in increased survival (32) . combined activation of pparγ and its downstream target ampk improved survival of mice infected with pandemic iav strains (33) . protease activated receptor (pars) link protease activity to inflammatory cellular responses (70) . par1 expression is upregulated in the mouse airways following iav infections (71) . intranasal administration of a par1 antagonist (sch79797) at the time of infection with various iav strains including highly pathogenic avian h5n1 and pandemic h1n1 viruses led to increased survival and a decrease in inflammatory responses. moreover, this effect was also observed when sch79797 was administered 48-72 h after infection (30) . the use of statins, angiotensin ii receptor blockers (arbs) and angiotensin converting enzyme inhibitors (acei) has been proposed to regulate the iav-induced cytokine storm in severe infections (72, 73) . retrospective studies conducted separately in mexico, netherlands, uk and usa reported an association of reduced iav-related pneumonia and lower case fatality due to lower respiratory tract iav infections with statin treatment (74) (75) (76) (77) . however, this association was contested in two additional studies that found no benefit of statin treatment on iavinduced disease burden (78, 79) . this uncertainty regarding the iav therapeutic potential of these widely used compounds warrants further investigations at the basic science level and in clinical trials. the continuous accumulation of adaptive mutations and the introduction of novel viruses in the human population continue to pose a threat to public health, especially to individuals at high risk to influenza. the emergence of strains resistant to existing classes of antiviral drugs and reduced vaccine effectiveness highlights the need for the development of alternative intervention strategies. therefore, therapeutic approaches that can diminish the potential for drug-resistance while being effective against multiple iav subtypes/strains are highly desirable. targeting host cell factors meets these criteria and is more likely to avoid overtly robust immune responses thereby reducing disease severity and improve patient outcome (figure 1) . a large effort has been made in recent years to identify host proteins to serve as intervention targets against iv infections. several genetic and proteomic screens have identified several promising hits with potential roles in the iv replication cycle (80) (81) (82) (83) (84) (85) (86) (87) (88) (89) (90) . in addition to these genome-wide screens, viral and host protein interactions can be mapped into networks that can also be used to identify host factors critical for iv replication (91, 92) . interestingly, meta-analysis of some these studies shows limited overlap in the genes/proteins identified as required host factors (87, (93) (94) (95) . this is likely due to study-specific variations in iv types/strains and cell-lines used, inclusion/exclusion criteria, limited hit-validations and methods used to "knock-down/out" these genes. local microenvironment within a given tissue can dictate the quality and intensity of an immune response. inhibition or activation of critical signaling pathways expressed in both respiratory tract epithelial and immune cells by brms can have opposite and unintended consequences. as discussed above, trail regulates immune cell-mediated apoptosis of infected cells and several studies have shown that blocking trail signaling by genomic deletion or depletion by monoclonal antibody administration can improve infection outcome in iavinfected mice. indeed inhibition of trail signaling in alveolar macrophages and other monocytes limits their ability to induce apoptosis in alveolar cells, prevents lung tissue damage and promotes survival (19, 96, 97) . however, cd8+ t cells from trail−/− mice are less able to protect mice from severe infections, consistent with impaired trail-mediated effector functions of cd8+ t cells (18). similarly, opposing beneficial and detrimental outcomes have also been observed in studies using bcl-2 inhibitors to treat iav infections (98, 99) . brm delivery should be guided by immune system "compartmentalization" to ensure they elicit balanced immune responses. ideally, mucosal delivery deposits brms that reduce viral titers at the site of iav replication; however, systemic delivery of certain brms might be required to dampen dysregulated responses. this not only depends on the brms used but also on the timing of their administration. moreover, the duration of treatment with brms must be considered because sustained inhibition of certain inflammatory responses can result in an immune status that increases susceptibility to secondary opportunistic infections. repurposing of clinically approved drugs could potentially be used as brms for the treatment of severe iav infectious and should be explored (86, 89, 90) . considering that susceptibility to severe iav infections is influenced by host genetics and hostspecific immune responses, selection of therapeutic brms should be carried out using in vivo model systems that are representative of the immune status spectrum and underlying conditions of high-risk influenza patients (young, immunocompromised, nonnaive, obese, pregnant, or aged). using these model systems will increase the likelihood of identifying brms with clinically relevant antiviral and immunomodulatory potentials. he, tg, gs, and gr conceptualized and composed the manuscript. gr and he oversaw all aspects of the manuscript preparation. this work was funded by the alexander von humboldt foundation in the framework of the alexander von humboldt professorship endowed by the german federal ministry of education and research. we apologize to any investigators whose relevant work was not 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virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice antiviral properties of chemical inhibitors of cellular anti-apoptotic bcl-2 proteins key: cord-006841-3u56erru authors: einsele, hermann; bertz, hartmut; beyer, jörg; kiehl, michael g.; runde, volker; kolb, hans-jochen; holler, ernst; beck, robert; schwerdfeger, rainer; schumacher, ulrike; hebart, holger; martin, hans; kienast, joachim; ullmann, andrew j.; maschmeyer, georg; krüger, william; niederwieser, dietger; link, hartmut; schmidt, christian a.; oettle, helmut; klingebiel, thomas title: infectious complications after allogeneic stem cell transplantation: epidemiology and interventional therapy strategies: guidelines of the infectious diseases working party (agiho) of the german society of hematology and oncology (dgho) date: 2003-09-10 journal: ann hematol doi: 10.1007/s00277-003-0772-4 sha: doc_id: 6841 cord_uid: 3u56erru the risk of infection after allogeneic stem cell transplantation is determined by the underlying disease, the intensity of previous treatments and complications that may have occurred during that time, but above all, the risk of infection is determined by the selected transplantation modality (e.g. hla-match between the stem cell donor and recipient, t cell depletion of the graft, and others). in comparison with patients treated with high-dose chemotherapy and autologous stem cell transplantation, patients undergoing allogeneic stem cell transplantation are at a much higher risk of infection even after hematopoietic reconstitution, due to the delayed recovery of t and b cell functions. the rate at which immune function recovers after hematopoietic reconstitution greatly influences the incidence and type of post-transplant infectious complications. infection-associated mortality, for example, is significantly higher following engraftment than during the short neutropenic period that immediately follows transplantation. early post-transplantation period (pre-engraftment) febrile episodes in the early phase after allogeneic stem cell transplantation are in the vast majority of cases caused by infections [21] . after full conditioning regimens, almost all patients develop severe neutropenia and almost all patients develop neutropenic fever as an early clinical sign of infection. however, other symptoms of infection may masked by severe neutropenia. therefore, the source of infection can only rarely be identified on clinical grounds or with the help of imaging techniques. the few, non-infectious causes of neutropenic fever during the early post-transplantation period include transfusion of blood products, administration of immunoglobulins, drug-induced fever (e.g. cytosinarabinoside, amphotericin b, bleomycin, g-csf), allergies, and acute gvhd reaction, which can cause fever within days after transplantation. the risk of developing an infection in the early posttransplantation period is mainly determined by the duration and the severity of neutropenia. other risk factors for infectious complications are extensive mucosal damage as a result of the conditioning treatment, bacterial colonization, local fungal and viral infections, reactivation of infections that have been acquired during previous neutropenic periods, and finally the use of central venous catheters. the number of stem cells in the graft and the type of gvhd prophylaxis are factors which determine the rate of hematopoeitic reconstitution and may therefore also influence incidence and severity of infections during the early post-transplantation period. bacterial and fungal infections after allogeneic transplantation in neutropenia often take a life-threatening course. bacterial pathogens account for about 90% of infections during this phase. bacteremias are documented in 16-31% of patients after allogeneic transplantation, the majority (65-75%) being caused by gram-positive pathogens. infections due to gram-negative bacteria are less frequent. gram-negative infections, however, are generally associated with a significantly higher morbidity and mortality. the most frequent gram-positive pathogens are coagulase-negative staphylococci, corynebacteria, and alphahemolytic streptococci. gram-positive infections are mainly associated with central venous catheters and most frequent in patients with severe mucositis. particularly bacteremias with viridans streptococci such as streptococcus mitis are associated with a toxic shock syndrome or ards in 10% of affected patients and result in high mortality. in contrast, gram-negative pathogens are believed to enter the bloodstream via damaged mucosa of the gastrointestinal tract in patients with severe gastrointestinal mucosal damage. viral infections frequently occur in the early period after transplantation. in seropositive patients without adequate antiviral prophylaxis, hsv infections can be documented in more than 70% of patients. however, since in most centers aciclovir prophylaxis is routinely given to all patients after allogeneic stem cell transplantation, disseminated hsv infections rarely occur. in recent years, an increase in infections with respiratory viruses such as respiratory syncytial virus (rsv), parainfluenza virus, influenza virus, adenovirus, and rhinovirus have been reported [22] . after an initial infection of the upper respiratory tract, these viruses can subsequently lead to interstitial pneumonia causing substantial mortality. diagnostic procedures in patients with neutropenic fever initial diagnostic procedures follow the guidelines that have been described in the manuscript "antimicrobial therapy of unexplained fever in neutropenic patients" [10] . s176 microbiological diagnostic procedures as indicated by the symptoms of infection: if evidence of microorganisms has been documented in blood, urine, or csf culture, a surveillance culture should be obtained after treatment to document the efficacy of microbiological eradication. since conventional chest x-ray is insensitive, and has only a low negative predictive value for detecting pulmonary infiltrates in neutropenic patients, spiral or high-resolution computed tomography of the lungs should be obtained early to establish the cause of fever in neutropenic patients and particularly in those not responding to the initial therapy [8] . antimicrobial therapy in patients with neutropenic fever after allogeneic stem cell transplantation when an empiric antibacterial therapy is selected in patients with neutropenic fever, the local hospital resistance of pathogens must be considered. fever of more than 38.3c or fever of 38.0c lasting for an hour, or that reoccurs within 24 hours, should result in immediate broad-spectrum antibacterial treatment. microbiologic identification of an underlying pathogen is only possibly in about one third of all patients. therefore, it has become accepted practice to initiate broad-spectrum antimicrobial treatment on the basis of clinical or radiological signs or symptoms. in order to avoid ineffective empirical regimens, only combination treatments with documented broad-spectrum activity against enterobacteriaceae, pseudomonas aeruginosa, staphylococci, and streptococci should be used. clinical trials that investigated single agent regimens in patients with neutropenic fever included only few patients after allogeneic stem cell transplantation. therefore, the efficiency of single agent treatment e.g. with cefepim, ceftazidime, or a carbapenem, has not been sufficiently validated to date in the allogeneic setting and can not be recommended. for example, patients with severe mucositis should not receive single agent ceftazidime, because of the risk of a bacteremia with viridans streptococci. in these latter patients, the initial empiric treatment regimen should contain an antibiotic proven to be effective against streptococci and staphylococci, such as a broad-spectrum penicillin in combination with a b-lactamase inhibitor or a glycopeptide [15] . in the case of skin infections or venous catheter infections, addition of vancomycin or teicoplanin to the initial empiric regimen should be considered. however, administration of glycopeptide antibiotics should be discontinued early after a few days, if no multi-resistant gram-positive bacteria have been identified. if aminoglycosides are administered as part of the initial empiric regimen, regular surveillance of serum drug levels is required. to avoid excessive nephrotoxicity from additional nephrotoxic medication (e.g. ciclosporin, aciclovir etc.), daily surveillance of serum creatinine levels is mandatory, if aminoglycosides are used. modification of empiric antimicrobial regimens in patients with neutropenic fever after allogeneic stem cell transplantation when the causative agent of an infection has been identified, antibacterial therapy should be adapted according to the resistance pattern of the pathogen. however, narrowing the spectrum of the initial empiric treatment should be avoided, as identification of a pathogen does not exclude the presence of a polymicrobial infection. in the absence of a clinical response within 72-96 hours, the initial empiric regimen also requires modification. if steroids have been used over prolonged periods, or if steroids are given at a dose of >2 mg/kg/day, systemic antimycotic treatment should be administered as part of the second-line treatment. antimycotic treatment is also recommended as part of the first or second-line treatment as soon as pulmonary infiltrates occur. antibiotic treatment may be discontinued if all of the following conditions are met: 1. afebrile for at least 48 hours 2. negative cultures 3. imaging techniques without evidence of an infection 4. no clinical evidence of an infection 5. neutrophil count above 1000/ml it is mandatory to perform further microbiologic screening if the patient continues to be febrile. if infections have been microbiologically proven, it is advisable to repeat the initial diagnostic procedures, in s177 order to document the microbiological response (e.g. blood cultures, csf cultures, urine cultures, stool cultures, bronchial secretions in case of ventilated patients, smears). reduction of the antimicrobial spectrum (e.g. by discontinuation of aminoglycosides) can be acceptable in individual patients, depending on clinical response and the occurrence of drug toxicity. patients after allogeneic stem cell transplantation are at the highest risk of developing localized as well as systemic fungal infections. in this patient population, the incidence of life-threatening systemic mycoses can be as high as 15%, or more. some of the risk factors that contribute to this high incidence are listed in table 1 [19] . depending on the local epidemiological environment, candida and aspergillus species are the most frequent pathogens of systemic fungal infections in patients after allogeneic stem cell transplantation. fever that is unresponsive to broad-spectrum antibiotic treatment is frequently the first, and only, symptom of a systemic fungal infection. in the case of pulmonary aspergillus infections, pleuritic chest pain, cough, or hemoptysis may also occur. blood cultures may sometimes be positive for candida species, but rarely for aspergillus species. aspergillus spp. that is found in clinical specimens from neutropenic patients may indicate a systemic infection with this pathogen [24] . however, the sensitivity of screening for systemic aspergillus infections by culturing techniques is low. the significance of bronchoalveolar lavage in the diagnosis of pulmonary fungal infections is therefore still disputed. when fungi are found in bal specimens, it may be difficult to distinguish between contamination with fungi from the oropharynx and true invasive pulmonary infection. even in invasive pulmonary aspergillosis, cultures from bal are often negative. unfortunately, all serological procedures that have been established for detection of systemic fungal infections so far, have a low sensitivity and often also a low specificity for the detection of systemic mycoses. new serologic techniques may improve this situation. a new elisa assay for the detection of galactomannan in serum as well as the polymerase chain reaction (pcr) for the identification of fungispecific dna are currently being evaluated [4, 5, 12, 18] . in recent years, imaging techniques have also been increasingly used for the diagnosis of systemic fungal infections. if a systemic fungal infection is clinically suspected, imaging techniques should be used early. especially with the help of computed tomography, characteristic findings of invasive fungal infection may be present often before such alterations are seen with conventional radiological examinations [8] . in the rare case of hepatosplenic candidiasis, characteristic changes may also be identified by ultrasound sonography of the liver and spleen. because of its broad anti-fungal activity, intravenous amphotericin b deoxycholate is still the current standard in the treatment of patients with suspected or documented fungal infections after allogeneic stem cell transplantation. empiric treatment should be initiated as soon as the presence of a systemic fungal infection is suspected. the recommended dose of amphotericin b for empirical therapy is 0.5-0.7 mg/kg/day. this dosage is also used as a therapeutic dose in documented invasive candida infection. a higher dose of 1-1.5 mg/kg/day should be given, in case of a pulmonary infiltrate or if an invasive pulmonary aspergillosis is suspected. antifungal treatment has to be continued until neutrophil recovery and disappearance of all signs of an acute infection are achieved. recently, new lipid formulations of amphotericin b have been approved for the treatment of systemic fungal infections. in several clinical trials, the new amphotericin b formulations proved to be equipotent to conventional amphotericin b deoxycholate and could be administered at higher dosages of 3-4 mg/kg/day. the advantage of these liposomal formulations compared to conventional amphotericin b desoxycholate, is the much lower rate of acute side effects, especially of nephrotoxicity [20] . however, the excessive costs of these preparations limit their clinical usefulness. it is therefore recommended to change to a lipid formulation of amphotericin b, in case of severe clinical nephrotoxicity (e.g. creatinine >2.5 mg/ dl), intolerance, or inefficacy of amphotericin b desoxycholate. new azoles with broad antimycotic activity or new generations of drugs such as the echinocandines or pneumocandins, are currently being investigated in clinical trials [9] and might become alternatives to amphotericin b for the treatment of systemic fungal infections in recipients of an allogeneic stem cell graft in the future. intermediate post-transplantation period (from hematopoietic reconstitution to day + 100 after transplantation) after engraftment, a severe combined quantitative and functional deficiency in the t and b lymphocyte compartment persists despite full hematopoietic reconstitution. if t cell depletion has been used, or if incompatibility in the major histocompatibility antigens between the recipient and the donor had to be accepted, these immunodeficiencies are prominent for prolonged periods after transplantation. these deficiencies manifest as disorders in t helper cell function, immunoglobulin synthesis, but also in an impaired cytotoxic t cell response. in spite of normalization of cell counts, disturbances of granulocyte functions also persist, e.g. impairment of chemotaxis and phagocytosis. in 74% of all allogeneic stem cell transplantation patients, infections develop after day +50. in the majority of patients, these infections are triggered by viruses such as cmv or other viral infections such as hhv6, rsv, adenovirus, vzv, and ebv. in 14% of patients, bacteremias can be documented after hematopoietic engraftment. the mortality rate as a result of bacteremias in allogeneic stem cell recipients is comparable in the periods before and after engraftment. the spectrum of pathogens in patients with documented bacteremias shows that gram-positive pathogens (47% staphylococci) are responsible for about 75% of all infections. in contrast to the microbiologically documented infections in patients before hematopoietic engraftment of only 5-10%, the focus of bacterial infection can be identified in more than 50% of patients after hematopoietic engraftment [21, 23] . catheter infections are responsible in more than 30% of bacteremias during the postengraftment period. chills occurring within the first hour after intravenous drug administration may be the first sign of a catheter infection. other frequent sources of infection during the intermediate post transplant period, are pneumonias, especially caused by streptococcus pneumoniae, klebsiella species, and pseudomonas aeruginosa. cases of pyogenic arthritides with salmonella eneritidis and staphylococcus aureus have also been reported. late infections after allogeneic stem cell transplantation (after day + 100 following transplantation) in the late post transplant period, immune reconstitution is usually advanced, particularly in patients who have received a transplant from an hla-identical family donor. these patients often show full hematopoietic reconstitution and early immune reconstitution. if no gvhd occurs, immunosuppressive treatment is usually discontinued. in these patients who do not demonstrate a graft-versus-host reaction, who require no immunosuppressive therapy, who usually have a cd4-count of >200 per ml blood, and whose serum immunoglobulins levels are in the normal range, infectious complications rarely occur. these patients can be considered as immunocompetent and they are no longer at an increased risk of opportunistic pathogens, so that no intensive antimicrobial therapy is required. however, depending on a number of clinical risk factors, chronic gvhd may occur in 30% of patients, or more, which is characterized by a severe combined cellular and humoral immunodeficiency. due to mucosal damage, functional deficiencies of granulocytes (especially impaired chemotaxis), functional asplenia and qualitative as well as quantitative t and b cell deficiencies, a significantly increased susceptibility to infections must be assumed in patients with chronic gvhd. in these patients, bacterial infections of the upper and lower respiratory tract constitute a main cause of death. life-threatening infections are typically caused by encapsulated bacteria such as streptococcus pneumoniae or haemophilus influenzae. sinusitis, otitis media, and pharyngitis are common manifestations of such infections in this late post-transplantation period. patients who present at least one of the above mentioned risk factors, should receive immediate antibacterial treatment at the earliest signs of infection. if several of the above mentioned risk factors are present, antibiotic prophylaxis with an oral penicillin or a macrolide antibiotic is recommended. if fever occurs in a patient later than 100 days after allogeneic stem cell transplantation, the upper and lower respiratory tract (bronchitis, pneumonia, sinusitis), and bacteremias have to be considered as specific foci of infections. particularly pneumonias and bacteremias constitute the majority of life-threatening infections, which occur in 20% of all patients after day + 50 after allogeneic stem cell transplantation [14] . in a recent analysis, pulmonary infections were documented after day + 100 in approximately 50% of patients with cgvhd, in 21% of patients without cgvhd, and in only 2% of patients after autologous stem cell transplantation [9] . the incidence of infections is particularly high in patients who received a graft from an unrelated donor. frequently, herpes zoster or less often visceral manifestations of a vzv infection are identified (see chapter on varicella infection). an important pathogen of interstitial pneumonias in the late phase after allogeneic stem cell transplantation is pneumocystis carinii. without specific prophylaxis, about 30% of patients with chronic gvhd develop pneumocystis carinii pneumonia (pcp). late infections after stem cell transplantations constitute an important factor for morbidity and can take a fatal course in 4-15% of patients. a. viral infections the evident increase of infections caused by respiratory viruses can be explained by more intensive screening, improved culturing methods and transplantation procedures with prolonged and intensified immunosuppression. respiratory viruses may be acquired prior to sct, so that clinical manifestations can already develop within the first weeks after transplantation. often, infections result from direct contact with infected family members, doctors, or nurses. in this clinical setting, rsv is most often identified, followed by rhinoviruses, parainfluenza virus type 1 and 3, and influenza virus type a [22] (for diagnostic procedures see table 2 .) adenovirus infections can either be caused by primary infections, reinfections, or by virus reactivation. excretion of adenoviruses in urine and throat washings has been documented in 3.8-20.9% of patients after allogeneic stem cell transplantation. adenovirus diseases, however, occur in only 0.95-6.5% of patients [6] . the intensification of immunosuppression or t cell depletion after stem cell transplantation has also led to an increase of adenovirus infections. adenoviruses can first be detected by culture techniques around day 44 (day 13-199) after transplantation, demonstrating a predominance of serotypes 11, 34, and 35. clinical manifestations of adenovirus infections in patients after allogeneic stem cell transplantation that have been reported so far include pneumonia, hepatitis, cystitis, diarrhea, and also disseminated disease (for diagnostic procedures see table 3 ). the mortality rate of these infections is about 60%. there have been reports about successful treatment of adenovirus infections with cidofovir or ribavirin. successful treatment of rsv infection has been reported with either inhalative or intravenous ribavirin and/or monoclonal antibodies. herpes simplex virus infection after allogeneic stem cell transplantation is associated with a high morbidity and leads to a substantial degree of oral mucositis, especially during the early post transplant period. before the introduction of aciclovir prophylaxis, 80% of the seropositive patients excreted hsv within the first 50 days (median second and third week) after transplantation, mainly due to reactivation of persisting virus. aciclovir prophylaxis from day 0 to day 30 reduced the hsv reactivation rate by 75% within the first 100 days after transplantation. clinical manifestations of the hsv infection after stem cell transplantation include skin manifestations, urogenital infections, esophagitis, keratitis, and infrequently, also pneumonias, hepatitides, or encephalitides (see also table 4 ). diagnostic measures should be initiated depending on the manifestation and may either consist of virus culture from throat washings, urine, skin lesions, or mucosal swabs, or antigen detection, or viral dna pcr (especially in csf specimens) ( table 4) . treatment of hsv infections with aciclovir (see also table 4 ) is very effective. aciclovir-resistant herpes simplex virus isolates have been identified in only about 6% of patients treated with aciclovir, in about 2% during primary and in about 9% during secondary prophylaxis. it is important to note that the documentation of persisting excretion of hsv during treatment with aciclovir treatment does not necessarily imply resistance to this drug. therefore, if possible, a sensitivity test should be performed. infections with aciclovir-resistant hsv can be accompanied by local or disseminated manifestations [11] . the treatment of choice in acyclovir-resistant hsv infections is foscarnet. prior to the introduction of aciclovir prophylaxis, vzv reactivations developed in 30-50% of adults and in 25% of children within the first 6 months (median 5 months) after allogeneic stem cell transplantation. acute and chronic gvhd were identified as the major risk factors for reactivation. eighty-four percent of vzv infections in adult patients manifest as localized herpes-zoster. disseminated or visceral vzv infections occur in only 13-25% of patients. if dissemination or visceral involvement occurs, the mortality of such vzv is high. during aciclovir prophylaxis until at least day +30, vzv infections can only be documented in approximately 16% of allogeneic stem cell transplant recipients. visceral manifestations may even precede cutaneous manifestations. in recent years, antiviral treatment has significantly improved the prognosis of vzv infections. intravenous administration of aciclovir (310 mg/kg) within the first 24-48 hours after clinical manifestation shortens the duration of skin manifestations and often also prevents dissemination as well as the incidence of post-herpetic neuralgia [13] . the mortality rate associated with vzv pneumonia, however, is still high. if a vzv infection occurs within the first 9-12 months after stem cell transplantation, treatment is indicated. this also applies for a longer period in patients who receive immunosuppression for acute or chronic gvhd. infections with cytomegalovirus (cmv) are one of the main causes of infection-associated mortality after allogeneic stem cell transplantation. if no antiviral prophylaxis is administered, cmv infection occurs between 30 and 100 days after stem cell transplantation (for diagnostic procedures see table 5 ). cmv infection occurs in approximately 60-70% of cmv-seropositive patients or seronegative patients who receive transplants from a seropositive donor. manifestations of cmv disease are pneumonia, gastroenteritis, hepatitis or retinitis. the mortality of cmv disease has not decreased, in spite of a combination treatment with ganciclovir and cmv hyperimmunoglobulin. despite the fact that an initial response can be documented in about 60-80% of patients, only 31% of patients survive a cmv-induced interstitial pneumonia for more than 3 months. in recent years, antiviral prophylaxis and preemptive therapy based on highly sensitive screening tools have succeeded in substantially reducing the incidence of cmv disease in the early phase after stem cell transplantation (to 2-4%). however, both prophylaxis and early intervention have led to a late increase in cmv infections after day +100 post transplantation. especially in patients with chronic gvhd, the incidence of late cmv disease, which occurs a median 156 days after transplantation, has increased. due to the high mortality of cmv disease, intensive efforts are being made to prevent its occurrence. in cmvseronegative patients who receive a transplant from a seronegative stem cell donor, this can be achieved by transfusion of cmv-seronegative and leukocyte-depleted blood products. the probability of developing a cmv infection can thus be reduced to 3-6%, and the occurrence of cmv disease can be reduced to 0.5-1.5%. in seropositive patients, several different strategies exist. first cmv reactivation and the development of a cmv disease may be prevented by prophylactic administration of antiviral agents. since there is a higher incidence of secondary bacterial and mainly fungal infections due to secondary neutropenia and possible ganciclovir-associated immunosuppression, ganciclovir prophylaxis does not show any advantage in respect to survival despite the marked reduction in the rate of cmv infection and disease. an alternative strategy is early intervention, which means that ganciclovir treatment is initiated when cmv is first identified in clinical specimens (bronchoalveolar lavage, throat washings, blood, or urine). a significant reduction in the incidence of cmv disease as well as in cmv-associated mortality was achieved with this strategy. however, in approximately 30-35% of patients, ganciclovir-associated secondary neutropenia developed. since culture methods demonstrate only a low sensitivity, cmv diseases may occur in 12-13% of patients even before the virus is detected by conventional culture assays. today, due to the widespread application of more sensitive screening tools (pcr, antigenemia), virus infection can be detected and treated earlier. this reduces the incidence of cmv disease to 3-6% of patients [3] . the treatment strategies of cmv disease are presented in tables 6 and 7 . after allogeneic stem cell transplantation, the incidence of ebv-associated lymphoproliferative disorders in hlaidentical family donors is only around 0.45%, but may increase to about 1.4% in family donors who are not fully hla-matched [25] . when t cell depletion methods are performed, the incidence of ebv-associated lymphoproliferative disorders increases substantially. with t cell depletion in combination with stem cell grafts from unrelated donors, the incidence of ebv-associated lymphoproliferative disorder increases even further, to an incidence as high as 29% [25] . ebv-associated lymphoproliferative syndrome is associated with very high amounts of ebv-dna in the peripheral blood. ebv reactivation in the absence of lymphoproliferative disease is observed more frequently. however the association of ebv reactivation with symptomatic disease has not been systematically evaluated. donor lymphocyte infusions have been the only treatment of ebv-associated lymphoproliferative syndrome after allogeneic stem cell transplantation so far. more recently, rituximab, an anti-cd20 antibody, has been successfully administered in a small number of patients. since ebvassociated lymphoproliferative disorders have a high proliferation rate and require immediate treatment, donor lymphocyte infusions have to be initiated promptly in order to be successful. thus, monitoring ebv-dna load in the peripheral blood of patients who received t-cell depleted grafts, grafts from unrelated and/or mismatched donors, or who have other risk factors is essential. if the viral load increases significantly, it is advisable to initiate treatment without delay (for diagnostic and therapeutic approach see table 8 ). donor lymphocyte infusion ebv-specific t cell lines/clones anti-cd20 antibodies (e.g. rituximab) before the introduction of pcp prophylaxis, the incidence of pneumocystis carinii pneumonia in allogeneic stem cell transplant recipients amounted to approximately 6.8% [17] . seventy-five percent of patients with pcp develop dyspnea, cough, and fever as clinical signs of the infection. in 58% of patients, conventional chest radiography shows bilateral infiltrations, typically with sparing of the periphery of the lungs. in 15% of the patients, however, conventional chest x-rays may be normal or may show only minimal abnormalities. in a retrospective analysis, the immunofluorescence staining of bronchoalveolar lavage specimens was identified as an accurate method of diagnosis of pneumocystis carinii pneumonia in the majority of patients. pcr is increasingly used as diagnostic procedure for pcp, but this has not yet become a standard procedure. pneumocystis carinii pneumonia is treated with high dosages of trimethoprim-sulfamethoxazole (tmp/smx) (20/100 mg/kg/ p.o. or i.v. in 3 or 4 divided doses per day) for 2-3 weeks. alternatively, it may be possible to administer trimethoprim 20 mg/kg/day in four doses combined with dapson 1100 mg p.o. for 3 weeks or pentamidin (pentacarinate) 3-4 mg/kg daily by the intravenous route. another alternative is atovaquone (meprone) 2750 mg p.o. for 3 weeks. despite high-dose tmp/smx treatment or intravenous administration of pentamidin, the mortality rate for pneumocystis carinii pneumonia is 89% within the first 6 months in patients after allogeneic stem cell transplantation, and still as high as 40%, if pcp occurs more than 6 months after stem cell transplantation. c. toxoplasmosis toxoplasmosis can occur as early as day +30 after allogeneic stem cell transplantation. it presents as pneumonia, perimyocarditis, encephalitis with focal-neurological signs or convulsions as well as chorioretinitis. the incidence in a large retrospective analysis at the fhcrc in seattle amounted to 0.3% in 4312 examined patients, with a local seroprevalence of 17% [16] . in areas with a higher seroprevalence for toxoplasma gondii (e.g. in france with a 70% seroprevalence), the incidence of toxoplasmosis after allogeneic stem cell transplantation is as high as 2-3% [2] . risk factors for the toxoplasma infections are the serostatus of the patient and the extent of immunosup-pression. particularly in areas with a high seroprevalence, determination of toxoplasma serostatus prior to allogeneic stem cell transplantation should be mandatory. if a toxoplasma infection is clinically suspected, serum, liquor, and bronchoalveolar lavage specimens should be screened, to see if toxoplasma can be detected. however, sensitivity of morphological methods is limited. if the clinical suspicion is high, computed tomography of the chest as well as a ct/mri scan of the cns should be performed. in some centers, qualitative and quantitative pcr techniques are being evaluated in respect of their usefulness in screening for clinical infections via the demonstration of toxoplasma dna. however, despite first encouraging results in high risk patients, this technique is not yet part of routine diagnostic standards at present. the treatment of choice in toxoplasma infections after allogeneic stem cell transplantation consists of sulfadiazin (and pyrimethamin) for 3-6 weeks. dosage: -sulfadiazin: 4-8 g/day in 4 separate doses p.o. (~100-150 mg/kg kg), -pyrimethamin: 2100 mg p.o. as loading dose on day 1, then 50-75 (100) mg/day (~1 mg/kg) -folinic acid: 10-15 mg/day p.o. as a supplement to reduce hematological toxicity. in case of chorioretinitis or increased intracranial pressure, additional corticosteroid therapy is recommended. alternatives for sulfadiazin if sulfonamides are not tolerated: -clindamycin: 4600 mg/day p.o. or i.v. in combination with pyrimethamin (see above) in case of cerebral and ocular toxoplasmosis successful treatment has been reported with: successful modification of cytomegalovirus disease in allogeneic marrow transplant recipients toxoplasmosis in bone marrowtransplant recipients: report of seven cases and review pcrmonitoring after bmt to reduce the incidence of cmv disease and the duration and side effects of antiviral therapy detection and identification of fungal pathogens in blood by using molecular probes prediction of invasive pulmonary aspergillosis from colonisation of lower respiratory tract before rnarrow transplantation increasing incidence of adenovirus disease in bone marrow transplant recipients risk factors for developing ebvrelated b cell-lymphoproliferative disorders (blpd) after non-hla-identical bmt in children pneumonia in febrile neutropenic patients and in bone marrow and blood stem-cell transplant recipients: use of high-resolution computed tomography antifungal agents in the 1990s. current status and future developments antimicrobial therapy of unexplained fever in neutropenic patients aciclovir-resistant herpes simplex causing pneumonia after marrow transplantation comparison of an enzyme immunoassay and a latex agglutination system for the diagnosis of invasive aspergillosis in bone marrow transplant recipients a prognostic score for postherpetic neuralgia in ambulatory patients late infections after allogeneic bone marrow transplantations: comparison of incidence in related and unrelated donor transplant recipients fever in immunocompromised patients toxoplasma gondii infection in marrow transplant recipients: a 20-year experience pneumocystis carinii pneumonitis following bone marrow transplantation detection of antigen in sera of patients with invasive aspergillosis: intra-and interlaboratory reproducibility. the dutch interuniversity working party for invasive epidemiology of aspergillus infections in a large cohort of patients undergoing bone marrow transplantation liposomal amphotericin b for empirical therapy in patients with persistent fever and neutropenia. national institute of allergy and infectious diseases mycoses study group infection in the bone marrow transplant recipient community respiratory virus infections among hospitalized adult bone marrow transplant recipients unique risk factors for bacteraemia in allogeneic bone marrow transplant recipients before and after engraftment significance of isolation of aspergillus from the respiratory tract in diagnosis of invasive pulmonary aspergillosis. results from a three-year prospective study epstein-barr virus lymphoproliferation after bone marrow transplantation key: cord-022254-8y5sq72c authors: nathanson, neal; gonzalez-scarano, francisco title: immunosuppression and virus infection of rodents date: 2012-12-02 journal: viral and mycoplasmal of laboratory rodents doi: 10.1016/b978-0-12-095785-9.50036-6 sha: doc_id: 22254 cord_uid: 8y5sq72c nan immunosuppression is a standard part of the technical armamentarium used in experimental immunobiology and viral pathogenesis. over the past 20 years, an increasing number of methods have been developed to produce selective suppression of different elements of the host defensive response. macrophages, b cells, t cells, and their subsets may be deleted; alternatively their effector molecules, such as immunoglobulins or interferons, may be inactivated. one potential consequence of the use of immunosuppression is the activation or potentiation of latent or intercurrent viral infections which may complicate and invalidate experimental studies. on the other hand, the deliberate use w. ñ. holds a javits award (ns 20904) and f. g-s. holds a tida (ns 00717) and a harry weaver neuroscience fellowship from the national multiple sclerosis society. of immunosuppression complemented by immunoreplacement has enhanced our understanding of the role of various host defenses in acute and persistent viral infection. these two themes are the subject of this brief overview. more detailed reviews are listed in the bibliography (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) . unwanted complications of immunosuppression are rarely published, so that the literature represents only the tip of the iceberg. a representative example was provided by an experience in our own laboratory (17) (18) (19) . an investigator at the johns hopkins university was developing treatment regimes for the maintenance of longterm immunological tolerance. while attempting to suppress the response of adult rats to sheep erythrocytes with cyclophosphamide, an alkylating agent which has a broad immunosuppressive activity, he noted that a small proportion of the animals developed hindlimb paralysis. pathologic examination disclosed an unusual hemorrhagic lesion of the spinal cord. when homogenates of affected tissues were injected into suckling rats, a lethal disease was transmitted, with a more severe form of the same hemorrhagic encephalopathy. a parvovirus was isolated and identified as the causal agent; the isolate was called the her (hemorrhagic encephalopathy of rats) strain of rat virus, a previously described parvovirus of rodents. the her strain failed to cause disease when adult rats were infected intracerebrally. however, when potentiated by cyclophosphamide-induced immunosuppression, the infection progressed to paralysis in about 10% of the animals. it was never determined whether the original episode represented activation of a persistent latent infection or potentiation of an acute infection which happened to occur concomitantly with the administration of cyclophosphamide. many although, from the earliest days of animal virology it had been recognized that immunity protected against reinfection, it has only been 15 years since it was first shown that the immune response also plays a key role in recovery from primary viral infection. the initial experiments utilized methods, such as x-irradiation, cyclophosphamide, or anti-thymocyte serum, which caused temporary abrogation of both humoral and cell-mediated immunity (3-6). the hallmarks of potentiated infection were: 1) no alteration in virus replication during the first week or so (suppression-insensitive phase of infection); 2) a failure to clear virus during the second week (antigen-sensitive phase of infection); 3) replication to higher tissue titers than in immunocompetent animals; 4) enhancement of virusinduced pathology and increased mortality; 5) increased tissue titers of interferon. all of these observations (20-23, 30-35) supported the old concept that there was a race between the immune response and virus replication; anything which retarded immune induction would favor the virus. furthermore, immunosuppression had no direct effect on the rate of viral replication but only on immune defenses. the major limitation of the early studies of immunosuppression was the lack of specificity of the methods employed (3-6, 36-38). two complementary approaches have been taken to refine the experimental dissection of various components of the immune response: specific types of suppression (deletion techniques) and reconstitution with specific immune components (addition techniques). each approach has its advantages and limitations. deletion methods may be incomplete or only partially specific; addition methods may fail to mimic the physiologic response of intact animals and constitute therapy rather than reconstitution. a. specific immunodeletion t cell deletion may be accomplished with neonatal thymectomy; anti-thymocyte serum; adult thymectomy, x-irradiation, and bone marrow reconstitution (atxbm); or through the use of homozygous nude (nu/nu) mice. however, such animals lack helper t cells and therefore cannot mount a normal b cell response. furthermore, it is clear that different effector lymphocytes, such as cytotoxic t lymphocytes (39), nk cells (40), and adcc-mediating k cells (41), can all play roles in anti-viral defense. this underlines the importance of specific deletion methods. the definition of t cell subjects and markers for them should make it possible to delete effector subsets of t cells, such as cytolytic t cells, with consequently improved specificity (42). one example of specific deletion is the use of anti-mu antiserum to delete igm bearing b cells from neonatal animals, thus blocking the b cell arm of the immune response (43-46). when applied to a model of experimental rabies in the mouse, there was a failure to generate circulating antibody, with definite potentiation of infection; t cell populations and responses were intact (43). administration of anti-interferon antiserum is another method · of. considerable specificity. there is a large literature (1, 2, 8, 9) documenting the efficacy of interferon as therapy in ongoing acute experimental virus infection in normal animals. however, it was never clear from such therapeutic trials whether interferon played a vital role in the recovery from infection in infected but otherwise unmanipulated animals. the application of anti-interferon antiserum has now provided convincing evidence that, at least in some experimental infections, neutralization of circulating interferon potentiates the virus (47-53). one action of interferon is to increase the activity of natural killer (nk) lymphocytes, which in turn can provide a nonspecific antiviral defense by attacking virus-infected targets (40, 54-57). antiserum against asíalo gm1, a neutral glycosphingolipid present on the surface of nk cells, can specifically deplete this lymphocyte population. treatment with anti-asialo gm1 antiserum has been shown to potentiate viral infection (58,59). complement plays an important ancillary role as a host defense, since in conjunction with specific antiviral antibody, it can lyse either virions or virus-infected cells (60). decomplementation with cobra venom transiently lowers complement levels and partially potentiates viral infection (43,61). other methods for specific deletion include reduction in macrophages with silica, anti-macrophage antiserum, or other treatments (62) (63) (64) ; or the use of cyclosporin which appears to retard t cell activation (65) (66) (67) (68) . each of these methods has certain drawbacks such as partial efficacy, short duration, unwanted side effects, or inadequate specificity. originally, reconstitution was accomplished with relatively primitive additions, such as polyclonal antiserum or unfractionated spleen, lymph node, or thymus cells. those early experiments bolstered the conclusions from the suppression experiments and added conviction to hypotheses regarding the role of both b and t cells in the host defense against infectious agents (3-5, 69-74). the advent of monoclonal antibodies (11,75) has made it possible to refine this approach and examine the role of antibodies directed against individual virus proteins. from these more recent experiments (76-85) have come a number of significant conclusions: 1) antibodies against individual epitopes on viral proteins confer surprisingly effective protection against infection, in comparison to polyclonal antisera. this finding alone has major implications for the future development of vaccines and suggests that it may be realistic to develop oligopeptide vaccines directed against immunodominant epitopes. 2) as expected, there is a general correlation between neutralizing activity determined in cell culture assays and the protective efficacy of monoclonal antibodies. however, in some systems selected non-neutralizing monoclonal antibodies are also protective (86) . this may relate to the expression on virus-infected cells of viral epitopes which are not exposed on mature virions. 3) monoclonal antibodies also permit the examination of the effect of other variables (87) , such as the immunoglobulin isotope, on protection. the recent development of methods for the cloning of t cells and the culture of t cell lines (88) (89) (90) (91) (92) , has made it possible to study the effect of specific t cell subsets upon virus infection (93) (94) (95) (96) . again, although the data are still limited, it is clear that certain effector subsets, such as cytolytic t cells, are capable of clearing virus or (in the case of virus-induced immunopathology) of mediating disease. it is now well established that certain viral diseases are mediated, not by a direct cytolytic effect of the agent, but by the immune response to the viral antigens. among such diseases, lymphocytic choriomeningitis (lcm) of mice is the classical prototype, while acute hepatitis b is the most familiar human example (97). the salient aspects of such syndromes are that: 1) immunosuppression prevents disease but does not reduce virus tissue titers; 2) the virus is capable of replication and release from host cells without cytolysis (there are exceptions to this feature); 3) under certain conditions the virus can persist in infected cells or animals, often with little cellular or tissue destruction. the essential features of lcm are worth recapitulating, since they illustrate the role of experimental suppression and immune reconstitution in documenting the ability of a virus to initiate lesions indirectly (12-15, 98-101). in adult mice, lcm virus, injected by the intracerebral route, causes an acute choriomeningitis with convulsions, resulting in death about seven days after infection. nonspecific immunosuppressive treatments, such as x-irradiation or cyclophosphamide, will prevent this disease and also prevent the underlying lesions. nude mice and atxbm mice are likewise protected, while "b less" mice treated with anti-mu antiserum are susceptible. virus titers in suppressed and protected animals are high and, in fact, these mice become longterm virus carriers. reconstitution experiments (98, 99, 102) show that antiviral antiserum will not reconstitute disease, while immune t cells (102), or cytolytic t cell clones (94,95), will induce acute disease when transferred into syngeneic recipients. studies of the arenaviruses (of which lcm is the prototype) have extended these classical studies in several directions (15). arenaviruses, in addition to causing inflammatory lesions, can also cause acute tissue destruction of specific organs, such as the cerebellum or liver (103, 105). persistent viremia is usually accompanied by continuous synthesis of antiviral antibodies and the resulting immune complexes, over a period of months to years, produce chronic and eventually fatal glomerulonephritis (106, 107) . persistently infected animals can be cleared of their virus by adoptive immunization with virus-specific immune t cells (108) , and this can be accomplished by cloned (110); the post-infectious demyelination caused by canine distemper virus (cdv); rabies early death syndrome (111) (112) (113) ; dengue hemorrhagic fever (101); respiratory syncytial virus pneumonia; possibly some of the hemorrhagic fever-shock syndromes caused by arenaviruses (argentine hemorrhagic fever, bolivian hemorrhagic fever, lassa fever); and possibly hemorrhagic fever renal syndrome (hfrs) caused by hantaviruses, such as korean hemorrhagic fever virus (114) . one important concept that has emerged from the experimental studies described above is the dual role of the immune response. the immune response can both act as a host defense and cause disease, and the mechanisms involved are identical. thus, the attack on virus-infected target cells, by either antibody and complement or by cytolytic t cells, simultaneously destroys host tissue and removes the sites of viral replication. likewise, the binding of antibody to free infectious virions, both neutralizes and forms immune complexes which potentially can cause vasculitis and nephritis. the kinetics of virus replication and immune induction may together determine which of these effects predominates. thus, if the immune response is sufficiently brisk, the number of infected cells available as targets is limited and the destruction of host tissue is minimal. conversely, if many cells are infected, their removal by immune attack can produce severe or lethal lesions. these concepts have been amply documented in studies of lcmv infection of mice. for instance, it is possible to immunize against lcmv infection and thereby protect against an immunopathology (6,115,116 ) . however, if mice are inadequtely immunized, instead of protection there occurs an accelerated form of the immunopathology. likewise, incomplete post-exposure (post-infection) immunization in experimental rabies can cause "early death" in some animals, which die before any of their infected but unimmunized counterparts (111) (112) (113) . the dual role of the immune response is not merely an immunological curiosity, since it is seen very clearly in human hepatitis b (97). here, some infected subjects develop a brisk immune response and clear their infection rapidly, with minimal or no clinical disease; others fail to develop an adequate response against the hbs antigen, become persistent virus carriers, but are clinically healthy; only those persons who have extensive infection prior to an immune response to hbs antigen, develop severe hepatitis in the process of clearing the infection. the proposal that virus infection could initiate an auto-immune response has been suggested repeatedly in the past. only recently, however, have experimental data evolved to support this hypothesis (16). the advent of b cell hybridoma technology has made it possible to determine whether specific antibodies to viral proteins might also recognize epitopes on self proteins (sometimes called molecular mimicry). it now appears that, if a sufficient number of antibodies against any virus are examined, a small but finite number will also react with epitopes present in one or multiple organs (117) (118) (119) (120) (121) (122) (123) (124) . furthermore, infections of mice with certain viruses, such as reovirus, result in an autoimmune disease which can be prevented by immunosuppression (118, 125) . results of this type make it plausible that an acute or persistent virus infection could give rise to an autoimmune process. recently, a possible example of virus-initiated autoimmunity has been reported in a murine model of virusinduced demyelination (126) . rats infected with mouse hepatitis virus (mhv) developed demyelinating lesions about one month later. some of these animals had spinal cord lesions resembling experimental allergic encephalitis (eae). when spleen or lymph node cells from such rats were cultured in vitro with myelin basic protein (bp), they proliferated as measured by thymidine incorporation. these antigendriven cells, when transferred into a syngeneic (but not an allogeneic) recipient, induced eae, in about one week. several controls indicated that mhv was not being transferred with the cells, and the one week interval was too short for virus-induced demyelination. an inescapable conclusion is that every attempt must be made to utilize animals obtained from sources which monitor for viruses and which are relatively virus-free· perhaps a greater problem is the need to isolate such animals from enzootic agents in the experimenters 1 own facility. our recent experience in a university research facility is that these are very real problems. a barrier facility is probably necessary in most institutions to protect newly introduced animals from rapidly acquiring infections. in addition, minibarriers provided by the use of isolators and filter top cages are important adjuncts. also, the institution must monitor constantly to determine whether enzootic agents can be excluded from clean rodents imported into the facility. finally, it is likely that certain institutions fail to meet these criteria; such institutions are probably inappropriate settings for many immunological and viral studies conducted in animals. adverse effects of interferon in virus infection, autoimmune diseases and acquired immunodeficiency selective effects of antimacrophage serum, silica, and antilymphocyte serum on pathogenesis of herpes virus infection of young adult mice modifications by sodium aurothio-malate of the expression of virulence by defined strains of semliki forest virus use of silica to identify host mechanisms involved in suppression of established friend virus leukemia beneficial effect of cyclosporin a on the lymphocytic choriomengitis virus infection in mice modulation by cyclosporin a of murine natural resistance against herpes simplex virus infection. i. interference with the susceptibility to herpes simplex virus infection cyclosporin a-usefulness, risks, and mechanism of action cyclophilin: a specific cytosolic binding protein for cyclosphorin a suppressor t cells for delayed-type hypersensitivity to japanese encephalitis virus recovery from lethal herpes simplex virus type 1 infection is mediated by cytotoxic t lymphocytes. infect, and immun mechanism of recovery from systemic herpes simplex virus infection. i. comparative effectiveness of antibody and reconstitution of immune spleen cells on immunosuppressed mice an adoptive cell transfer system for the evaluation of immunity to herpes simplex virus in mice e2 glycoprotein of semliki forest virus can protect mice from lethal encephalitis protection against lethal challenge of balb/c mice by passive transfer of monoclonal antibodies to five glycoproteins of herpes simplex virus type 2 use of monoclonal antibodies for analysis of antibodydependent immunity to ocular herpes simplex virus type 1 non-neutralizing monoclonal antibodies can prevent lethal alpha virus encephalitis inductin of subacute murine measles encephalitis by monoclonal antibody to virus haemagglutinin cloning of functional t lymphocytes l!n_: concepts in viral pathogenesis specificity of in vitro cytotoxic clones directed againt vesicular stomatitis virus biology of clone cytotoxic t lymphocytes specific for lymphocytic choriomeningitis virus. i. generation and recognition of virus strains and h-2b mutants characterization of the murine th response to influenza virus hemagglutinin: evidence for three major specificities production and characterization of t cell clones specific for mouse hepatitis virus stain jhm: in vivo and in vitro analysis protection of mice from fatal herpes simplex virus type 1 infection by adoptive transfer of cloned virusspecific and h-2-restricted cytotoxic t lymphocytes neonatal rats: protective effect of immunosuppression with anti-lymphoid serum the effect of neonatal thymectomy on tamiami, virus-induced central nervous system disease virus-induced immune complex formation and disease: definition, regulation effect of immunosuppression on chronic lcm virus virus elimination in acute lymphocytic chorimeningitis virus infection. correlation with virus-specific delayed-type hypersensitivity rather than cytotoxicity virus-induced demyelination in: concepts in viral pathogenesis pathogenesis of borna disease in rats: immune-mediated viral ophthalmoencephalopathy causing blindness and behavioral abnormalities acute rabies death mediated by antibody immunopathologic aspects of infection with lagos bat virus of the rabies serogroup dual role of the immune response in street rabies virus infection of mice hemorrhagic fever viruses the timing of the immune response in relation to virus growth determines the outcome of the lcm infection immune responses to lcm virus infection ±η_ vivo and in vitro. mechanisms of immune-mediated disease infection with vaccinia favors the selection of hybridomas synthesizing autoantibodies against intermediate filaments, one of them cross-reacting with virus hemagglutinin virus-induced diabetes mellitus: autoimmunity and polyendocrine disease prevented by immunosuppression autoimmunity induced by syngeneic membranes carrying irreversibly adsorbed paramyxovirus. infect, and immun molecular mimicry in virus infection: cross reaction of measles virus phosphoprotein or of herpes simplex virus protein with human intermediate filaments cellular proteins reactive with monoclonal antibodies directed against simian virus 40 t-antigen virus-induced autoimmunity: monoclonal antibodies that react with endocrine tissues three monoclonal antibodies against measles virus f protein cross-react with cellular stress proteins virus-induced autoimmunity: monoclonal antibodies that react with endocrine tissues virus-induced diabetes mellitus. xx. polyendocrinopathy and autoimmunity adoptive transfer of eae-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis key: cord-021465-2pj26fmv authors: perdue, michael l.; seal, bruce s. title: impact of avian viruses date: 2007-05-09 journal: viral ecology doi: 10.1016/b978-012362675-2/50016-1 sha: doc_id: 21465 cord_uid: 2pj26fmv nan in considering the ecology of the avian viruses and their impact on life on earth, it may be useful to first consider the host itself. the class aves first diverged from the reptiles between 150 and 300 million years ago, depending on which current paleontological interpretations one accepts. of the vertebrate classes, they are most often compared with the reptiles from which they evolved and with mammals because of their common warm-blooded nature. this shared feature with mammals is probably the most influential with respect to ecology, since a virus adapted to warm-blooded physiology would not fare well in the cold-blooded world, and vice versa. along these lines, our recent experience is that some virologists think that a bird is a bird, and that if a given virus replicates in one, it will replicate in them all. this is, of course, far from the truth and perhaps should be a starting point for discussing avian virus ecology. according to fossil records, the class aves emerged from the extinctions of the late cretaceous period 65,000,000 years ago, somewhat bottlenecked, as did the class mammalia, but since that time they have undergone parallel evolution with mammals and are equally diverse in their own right. certainly some viruses, such as avian paramyxovirus 1 (of which newcastle disease virus is the prototype), are infectious for numerous orders of birds. there are also other important avian virus strains such as the gallid herpesvirus 1 (infectious laryngotracheitis) and the avihepadnaviridae, which appear to be exclusively confined to a single bird family or even genus. it is clear that birds, because of their close association with powered flight, do present a more homogeneous anatomy and physiology than do the class mammalia (feduccia, 1996) . whether this affects or specifies the molecular nature of viruses that infect birds is not really known. this feature, however, almost certainly uniquely impacts the natural distribution and ecology of the viruses that inhabit the flying birds. a virus infecting or persisting in an arctic tern could potentially be translocated up to 12,000 miles in a few weeks. viruses such as some avian orthomyxovirus or avian paramyxovirus strains, which may exhibit subclinical infections, might be shared among a migrating flock of waterfowl and persist for indefinite periods as the flocks move from lake to lake. along the way the virus might be shared with other birds crossing flight paths. so birds do present unique environments for transmission of viruses. the commercial practices of humans have further provided unique opportunities for transmission not normally seen for birds. the order galliformes in particular, which includes domestic and wild fowl; pheasants, quail, and turkeys, has been unquestionably affected. it would be quite safe to say that humans have both determined and upset the ecological balance among members of this order and the viruses that affect them. due to continuous breeding practices, live-virus vaccination regimes, and by housing tens of thousands of birds in a single enclosure, situations never encountered in natural settings are created. whether this has affected the virus-host ecological balance in other orders of undomesticated birds as well is not known; but it would seem highly likely. one of the most important features of a host-parasite relationship, of course, is the host's immune defense. while there are similarities shared with respect to the immune system, particularly the dual (humoral and cell-mediated) nature, there are marked differences between cold-and warm-blooded vertebrates and additional significant differences between birds and mammals (eerola et al., 1987) . the discovery of processing and maturation of immunoglobulin-producing lymphocytes was made as a result of characterization of an avian-specific organ, the bursa of fabricius (ratcliffe, 1989) . although functional equivalents exist in mammals, the bursa is a distinct and wholly avian-specific organ. since several viruses are known to affect this organ specifically, it should be considered a unique ecological niche. a second significant difference in the immune system of birds is in the apparent genetic content responsible for specifying the avian major histocompatibility complex (mhc). the chicken mhc appears considerably more simple (providing the oxymoron: a simpler complex) than the mammalian mhc (kaufman and wallny, 1996) . several alleles have arisen and considerable recombination documented in the mammalian mhcs that thus far have been studied. these are responsible for producing a great variety of class i-, ii-, and iii-type proteins used in recognition and presentation of antigen. in the chicken, some mhc haplotypes produce only one type of class i protein and there is no evidence for recombination at all (kaufman and wallny, 1996) . this has led to speculation that the relationship with the avian pathogens has evolved significantly differently from the mammals. one result of this difference may be the occurrence and frequency of either resistance or sensitivity to specific viral infections encountered in chickens (see below). thus, the class ayes presents several unique features that might ultimately affect the ecology of viruses that infect them. the classification of birds has presented a significant challenge to systematists. the number of species has actually decreased over the years because of the clearer genetic relationships that have emerged. conversely, additional species have been defined as recognition of convergent evolution has become clearer. currently, new species are identified at a rate of about two per year. while there remains some disagreement among specialists, most accept the current classification of 30 orders, 174 families, 2044 genera, and 9020+ species (gill, 1990 ). molecular analysis of avian genes for phylogenetic studies is still in its early stages. restriction fragment length polymorphism (rflp) analysis and sequence analysis of 12s mitochondria dna has yielded some molecular phylogenetic information but not enough to gain any insight regarding the viruses of birds (hedges et al., 1996; cooper and penny, 1997; mindell et al., 1997) . roughly speaking, the flightless orders represented by ostriches, rheas, cassowaries, and kiwis are thought to be the most ancient, while the bewildering array of members of the order passeriformes (representing 60% of known species and 40% of known families) are thought to be the most recent (gill, 1990) . trying to determine how long viruses have been associated with various avian groups is of course as impossible as it is with virus-host relationships in any other classes. orthomyxoviruses, paramyxoviruses, and coronaviruses have recently been isolated from ostriches, rheas, and emus, indicating no absolute barriers in these more ancient birds. the class aves is of course distributed worldwide. in addition, there are several hundred species that migrate, sometimes in spectacular fashion. biogeographers have divided the earth into six distinct faunal regions, which correspond somewhat with the major continental areas (welty, 1982) . these include the nearctic (north america and greenland), palearctic (asia, europe, and north africa), ethiopian (central and southern africa), oriental (india, southeast asia), australasian (australia, new guinea), and neotropical (south and central america). each area contains its own characteristic birds. in the northern hemisphere (nearctic and palearctic), most species are migratory, which is not the case in the other areas. the neotropical has the richest and most abundant bird life, and the southern hemisphere has by far the most families represented, as well as the most families peculiar to a given region. each year, billions of land birds and waterfowl in north america and asia head south to south america and africa, respectively, carrying their viruses with them. the size and scale of these geographic relocations are unmatched by any other land vertebrates. the sea mammals are the only other comparable migrating vertebrates, and they surely cross paths with the birds. in the most interesting putative contacts, purely avian-origin type a orthomyxovirus of at least two different subtypes were isolated from dead and dying seals off the coast of new england in 1980 (webster et al., 1981b hinshaw et al., 1984) . this represents viral ecology at its most forceful, being effected between two warm-blooded vertebrate orders during their natural migration and geographic interaction. if one takes a broad look at the virus families associated with avian hosts (table i) , it is actually easier to list the families of viruses that infect vertebrates but that do not yet have a clear avian member. these are the iridoviridae, arenoviridae, african swine fever-like viruses, and filoviridae. a similar comparison of families that do not contain a mammalian member yields only the birnaviridae. so, in one sense, the mammalian host might be considered more virus-friendly, rather than the alternative. still, many investigators think of bird tissues, in particular the embryo, as being an ideal medium for identifying new viruses. if just from an experimental and practical, rather than a natural, point of view, the avian host has played a tremendous role in our understanding of viral ecology. many of the most important findings in virology have been made utilizing the chicken embryo and in chicken cell cultures. the egg also continues to provide an abundant and important substrate for the production of veterinary and human vaccines. if we think of ecology as the study of organisms and their relationship with their environment to include all other organisms, we most certainly encounter a unique relationship with viruses. unlike higher pathogens, viruses really do not mate, communicate, or colonize (except in the very broadest stretches of the imagination); they simply parasitize and replicate. "we try and make them more familiar by defining higher organism-type genetic alterations as "evolution," when in truth the viruses may simply be adapting to the evolutionary pressures encountered by, or within, its host. obviously, the host is central to the viruses' lifestyle and must be considered a major part of virus ecology. in this sense, information gathered on various viruses as they replicate in embryos or embryo cells, in the absence of immune pressure, should only be considered "natural" for those viruses that are transmitted vertically. the embryo or cell culture then can only provide a window on the natural ecology of the organism in which the immune system is not a factor. in ecological terms, only the natural state of the virus-host relationship becomes important. viruses being parasites, these relationships more often than not eventually result in a disease state. it is not possible to distinguish the molecular biology of avian viruses from that of non-avian viruses on the basis of features unique to the avian system. however, there are some important distinctions with which viruses must deal. one dramatic difference between birds and mammals is body temperature. on average, birds operate at 3--4~ higher than mammals, and most can readily regulate their temperature, some dropping body temperature as much as 10~ during the night (gill, 1990) . while this certainly could influence such things as rates of viral enzyme activity, polymerase fidelity, or protein stability, there are no situations documented in which a particular virus group is restricted to class aves solely on the basis of body temperature. it is possible to distinguish some important receptor-specific distinctions unique to avian systems. one example is found in the influenza type a viruses. the glycosidic linkages associated with sialic acid residues on the avian versus mammalian cell surface serves to restrict the strains of viruses able to replicate. influenza virus hemagglutinin (ha) proteins bind to sialic acid residues on the surface of the host cell. although there is no absolute barrier, those viruses that replicate well in avian cells have a receptor binding pocket on the surface of the ha that has a preference for the o~-2,3 siallyl-sugar linkages abundant in bird tissues. those viruses that replicate well in mammalian cells have a preference for 0~-2,6 linkages more prevalent in mammalian cells (rogers and paulson, 1983; murphy and webster, 1996) . there are probably many more receptor specificities associated with other avian virus infections, particularly among the herpesviruses, where host range is narrowly dictated. another example of virus-specified tissue tropism unique to birds would be infectious bursal disease virus (ibdv; genus avibirnavirus), which has "evolved" an affinity for the bursa, a bird-specific organ. the other two known genera of birnaviridae infect fish and invertebrates. since there are as yet no mammalian members, it is tempting to speculate that ibdv is bird specific because of its evolved affinity for the bursa. air sacs are also uniquely avian structures. the bird lung is a fixed tissue incapable of expansion like mammalian lungs. the air sacs are a series of extensions of the respiratory system that expand with the musculature of the body cavity and allow the large-scale rapid oxygen transfer needed for powered flight. the air sac system is extensive throughout the bird, even encroaching into bone tissue; some birds have as many as nine distinct air sacs. many viruses replicate and cause disease in this unique organ system, and there are examples of apparent preferences by some viruses for air sac tissue over other respiratory tissue. finally, feathers, the most notable and prominent distinguishing feature of the class aves, provide a unique niche for at least two three viruses-avipox, marek's disease, and psittacine beak-and-feather disease virus --which can replicate in and are spread from feather follicles (biggs, 1985; tripathy and reed, 1997) . as mentioned previously, the immune system of the intact bird is a critical feature in the establishment of ecological relationships between many viruses and hosts. as the immune system plays a major role in the relationships, they will be covered, but there are some extraordinary examples that deserve their own mention. marek's disease, caused by an alphaherpesvirus, is most commonly associated with lymphomas developing relatively early in the life of a chicken. the initial infection in md is a respiratory infection probably initiated in macrophages. a typical field infection would then progress through the following phases in a bird: latency is established primarily in lymphoid cells, and mostly in activated t lymphocytes. in susceptible birds, the latent state progresses to a second round of lytic infections at multiple sites in the bird. interestingly, at this point the feather follicle epithelium is the only site where complete virus replication occurs and becomes a significant source of environmental infectious virus (calnek and witter, 1997) . concomitant and permanent immunosuppression occurs in the affected bird. the disease then progresses into a lymphoproliferative phase in susceptible birds that can range in severity depending on the virus strain and breed of chickens. some breeds have shown natural resistance to this progression, and, although the mechanism of resistance is not completely understood, it involves primarily lymphoid tissue and is dictated mostly by genes involved in the immune response (venugopal and payne, 1995) . it should be clear from this scenario that the immune system in this host-parasite relationship plays a critical role in the ecology of marek's disease virus. infectious bursal disease virus, as mentioned earlier, is a virus that replicates exclusively in lymphoid tissue. the virus can be detected replicating in the bursa of fabricius and within circulating lymphocytes as early as 4 hours after infection of a chicken. it causes acute degeneration of various lymphoid tissues within the first day of infection and results in a severe, albeit age-dependent, depression in the humoral immune response, being most dramatic in very young birds. interestingly, the infection does not suppress b-cell responses to the viral antigens themselves; in fact, there is stimulation of proliferation of b cells committed to anti-ibdv antibody production (mcferran, 1993) . one may only speculate as to what role, if any, this may play in the viral ecology or replicative cycle, but the immune system is once again a major participant in this host-parasite relationship. finally, the effects of vaccination programs on the ecology of avian viruses cannot be overemphasized. in one sense, we have an ongoing experiment where humans control the type of viruses to which certain species of birds are exposed. since live viruses generally yield much better immune responses, they are employed most often. in the case of the single-stranded rna viruses, noted for their ability to rapidly mutate and avoid the immune system, this has the effect of artificially challenging the immune system, creating selection pressure between host and parasite that would not normally occur. this may be effective in the short run, protecting against disease, but the long-term effects are unknown. table i are some of the most important features of relationships of various viruses with their avian hosts as well as the wide variety of relationships that exist. it is not feasible to list all the interesting attributes for each virus-host relationship, but the table presents a variety of relationships that will be covered in more detail in the following sections. as such, the table should not be taken as the final word on each member virus. for example, in the case of turkey meningoencephalitis virus, a flavivirus, reduced egg production is listed under pathogenesis. reduction in egg production is a common feature in many virus infections of poultry, and while listing it under pathogenesis somewhat stretches the meaning of that word, this clinical sign is one of the most important aspects of that disease in turkeys. although there is variation in the economic or ecological impact of various viral groups from year to year and among geographic sites, the "top ten" list of virus groups exhibiting routine significant impact on commercial poultry worldwide (not necessarily in order of impact) are paramyxoviruses (newcastle disease); coronaviruses (infectious bronchitis); herpesviruses (infectious laryngotracheitis; marek's disease; duck enteritis); reoviruses (viral arthritis); picornaviruses (avian encephalomyelitis); adenoviruses (egg drop syndrome); retroviruses (lymphoid leukosis); orthomyxoviruses (avian influenza); poxviruses (fowlpox); and birnaviruses (infectious bursal disease). the circoviruses (chick anemia) could likely be included in the above list, except it is not yet known to what extent the viral infection alone influences morbidity and mortality (see below). what may not be obvious from table i is that if one searches for viruses in a given avian species one will likely find them. in some of the virus families listed, investigators were forced to clearly separate the disease-causing virus in question from accompanying "contaminant" viruses, which may or may not have influenced the original disease manifestation. there appear to be many viruses of birds that in certain ecological conditions and in certain species may be considered "normal flora" and are not associated with disease. these, obviously, are less interesting to any funding agencies and consequently do not receive much research attention. no one really knows to what extent their transmission and persistence in avian populations affect their own ecology or that of their hosts. the office international des epizooties (oie), the principal world organization for animal health, provides listings of the most serious infectious diseases of animals (oie, 1996) and divides them into two groups: list a diseases, which "includes those diseases that spread rapidly, the scope of which extends beyond national borders" and "have particularly serious socioeconomic or public health consequences"; and list b diseases, which include those "that are considered to be of socioeconomic and/or public health importance within countries." of the avian viral diseases listed above, only highly pathogenic avian influenza and velogenic newcastle disease are in list a; marek's disease, infectious bursal disease, infectious bronchitis, duck enteritis, and infectious laryngotracheitis are in list b. the only avian disease in oie's list and not on our top 10 list is duck hepatitis, which is a complex of diseases caused by at least three virus families, and generally limited to country-specific origins. the ecological impact of viruses of birds ultimately interests us as homo sapiens, perhaps only to the extent that we are affected. in this respect, there is no question that the major impact thus far has been on raising birds as a food source for our species. ecologically speaking, this impact could have very significant consequences when one considers that poultry provide the most widely used protein source in the world. thus, we will consider for the most part how viruses affect this food source. the most significant and widespread infections of wild birds will be discussed as they are encountered relative to commercial and domestic birds. the most imminent and significant human public health concerns with regard to bird viruses appear to be twofold: the potential relationships with type a orthomyxoviruses that have become at least partially adapted in a totally nonpathogenic state to some avian orders. there is compelling evidence that these viruses may also replicate in pigs and re-assort with pig and/or human strains of influ-enza, yielding new variants capable of replicating and causing disease in humans. they also find their way into commercial poultry, sometimes with devastating consequences. with the recent documented transmission of a lethal avian influenza virus from commercial poultry to humans, these ecological relationships take on new significance. 2. the presence of a large reservoir of arboviruses in wild birds, some of which, when transmitted by invertebrate vectors to mammals, cause disease. beyond these two examples, other relationships are less directly important to human public health. other avian-origin viruses are capable of replicating in and sometimes causing mild disease in humans, but there is obviously not room in one chapter to cover each in detail. infectious laryngotracheitis (ilt) is a respiratory disease almost exclusively of chickens. infections in turkeys and pheasants have been reported, but surveys have yielded no wild bird reservoir or other domestic poultry reservoir (cranshaw and boycott, 1982) . based on this and the knowledge that ilt apparently exhibits little antigenic heterogeneity, it has been proposed that through proper husbandry practices and appropriate vaccination techniques the disease could be eliminated from commercial poultry (bagust and johnson, 1998) . the virus is a member of the alphaherpesvirinae subfamily and is identified taxonomically as gallid herpesvirus i. the disease is almost exclusively respiratory, with no systemic involvement. the severity of disease can vary from significant mortality (70%) in young birds to an inapparent infection of adult birds. there are age-dependent effects on the pathognomonic signs, and there do appear to be strain-specific virulence differences. however, different isolates do not exhibit sufficient genetic heterogeneity thus far to identify specific virulence factors (bagust and guy, 1997) . the most interesting aspect of ilt is its capacity for persistence in infected birds and flocks showing no disease signs. this persistence is most likely due to establishment of the latent state and recrudescence. numerous studies have demonstrated re-isolation of virus many months after initial infection, and one more recent study demonstrated reactivation of latent virus due to stress factors (hughes et al., 1989) . this latency achieved by herpesviruses could certainly be considered a unique ecological state, and most herpesvirus infections in birds are associated with its establishment. duck plague, also known as duck viral enteritis (dve), is caused by an alphaherpesvirus classified as anatid herpesvirus-1, which infects free-living and domestic ducks, geese, and swans (sandhu and leibovitz, 1997) . the disease is acute and often associated with high morbidity and mortality. the virus has a worldwide distribution and has caused numerous documented outbreaks in free-living anatids. the first documented north american outbreak was in commercial ducks on long island, new york in 1967 (leibovitz and hwang, 1968) , and since that time sporadic reappearance of the virus in commercial and wild populations has occurred. major outbreaks in free-living birds along the mississippi flyway and a large epornitic in south dakota in 1973 killed tens of thousands of wild ducks and geese (brand, 1987) . vertical transmission and recrudescence of latent virus has been established experimentally in mallard ducks but has not been demonstrated in wild waterfowl. species susceptibility may vary among various waterfowl, although more than 30 species have been shown to be naturally or experimentally infected and virulence differences among dve strains have been demonstrated. the other notable avian herpesvirus infection occurs in pigeons. the virus is taxonomically designated as columbid herpesvirus i and is antigenically indistinguishable from natural isolates taken from wild falcons and owls (vindevogel and duchatel, 1997) . the causative virus is antigenically distinct from ilt, mdv, herpesvirus of turkeys (hvt), and the anatid herpesvirus i. the disease associated with infection by columbid herpesvirus i is a major cause of growth retardation and bad performance in homing pigeons, though mortality is generally low. this virus, like ilt, becomes latent, reappears, and is shed in asymptomatic birds and flocks. other antigenically distinct herpesviruses have also been isolated from cormorants, quail, and storks. kaleta (1990) has proposed the division of these various herpesviruses into eight antigenic groups. the host specificity for each group varies, but in general they appear to be strictly adapted to the host of origin (as exemplified by the gallid herpesvirus i). gallid herpesvirus ii and hvt will be discussed in the following section. the impact of transmissible neoplastic diseases of poultry has been quite variable over the years. prior to vaccination, losses to marek's disease were often devastating, and even in marketable flocks condemnations due to lymphomas at processing plants could reach 2%. lymphomas caused by mdv and retroviruses are still the most common viral neoplastic diseases of poultry, and a recent increase in mortality and evolution of more virulent mdv strains indicates that the impact of these viruses will continue to be felt (witter, 1996) . marek's disease is caused by a herpesvirus that has two very similar relatives: a second nononcogenic serotype and the herpesvirus of turkeys (hvt). these are sometimes classified as serotypes 1-3, respectively, of the gallid herpesvirus ii strain. they share common antigens that distinguish them from the nononcogenic herpesviruses but can be distinguished on the basis of antigenic differences (calnek and witter, 1997) . only the oncogenic mdvs (serotype 1) cause significant problems in commercial poultry. the herpesvirus of turkeys is ubiquitous among commercial flocks and quite prevalent in wild turkeys but has not been directly associated with disease. the serotype 2 strains were originally thought to be nononcogenic apathogenic mdv isolates until they were serologically distinguished from the original isolates. the type 2 serotypes are associated with subclinical infections in chickens, although not as prevalent as turkey strains. the hvt isolate is a very interesting and important isolate, as it was used (and is still used) in vaccine formulations against marek's disease in chicken flocks. it has been very effective at protecting flocks against lymphomas and thus correctly billed as the first anticancer vaccine. marek's disease is relatively well controlled by using mixtures of primarily serotype 2 and 3 isolates in vaccine formulations. however, this vaccination program may ultimately extract a price. shown in figure 1 is a diagrammatic representation of the evolution of virulence in mdv strains associated with changing vaccine formulations. the extent to which these formulations have influenced the evolution of virulence is not proven, but certainly the association is undeniable. the 1990s have brought an increase in incidence of marek's disease cases, and the presence of these acutely virulent strains raises concerns for the future. what is clear is the role of humans in the generation of these strains. there is evidence that the commercial housing practices developed in the 1950s and 1960s resulted in generation of strains that were oncogenic and that this rapid evolution of virulence in the last 40 years is probably due to human control of commercial bird populations. the avian retroviruses have one of the most interesting histories of all of the avian viruses. the first transmissible lymphomas were demonstrated in 1908 by ellermann and bang (1908) and the first cell-free transmissible solid tumor by peyton rous three years later (rous, 1911) . the etiologic agents of both of these diseases were later shown to be members of what is now known as the avian leukosis virus-avian sarcoma virus complex (alv-asv) of related retroviruses. scientists investigating this interesting group of viruses have garnered more nobel prizes (six) than with any other group. because of their relative simplicity in genetic content and their close association with the genetic character of their host, they have provided a bountiful model for the study of oncogenesis. these are rna-containing viruses that replicate via an intermediate dna stage that is most often incorporated into the host genome. the integrated viral genomes serve as templates for production of new progeny genomic rna molecules and the mrnas needed to make new viral proteins (coffin, 1996) . in the process, these viruses transform the host cell into a tumor cell. there is a significant array of viral subtypes and relationships that exist between the host and avian retroviruses and numerous reviews that may be consulted (crittenden, 1981; swanstrom and vogt, 1990) . there are several viral subgroups, based on antigenic differences alone, in the surface envelope glycoproteins. viral subtypes have also been grouped on the basis of whether the viruses rapidly induce neoplasia because of the presence of a viral oncogene, or whether they induce slow development of tumors due to their integration into the host genome and subsequent activation of cellular oncogenes. in commercial poultry, the predominant problems are caused by the latter, the slow-inducing lymphoid leukosis viruses (llvs). the sarcoma-, myeloblastosis-, and erythroblastosis-inducing strains cause only sporadic problems. additionally, it has been calculated that losses due to llv because of poultry performance decreases are actually greater than those due to the lymphomas. still, these viruses continue to cause condemnations at slaughter and must be distinguished from the more rapidly forming mdv-induced lymphomas. two other oncogenic avian retroviruses pose significant problems and possess much wider host ranges than the llv-sarcoma complex. the relatively new subgroup "j" has been characterized in europe as causing significant myelocytoma and endothelioma. in contrast to the llvs, subgroup j viruses have a high tropism for cells of the myelomonocytic series but a very low tropism for bursal cells (arshad et al., 1997) . a related subgroup j virus has been identified in broiler-breeder flocks in the united states as well . additionally, of note are those retroviruses of the reticuloendotheliosis virus (rev) group. infection with members of this group only rarely results in disease, and then disease most commonly occurs only in the hosts from which rev was first isolated: turkeys. still the rev group is predicted to have significant potential for causing problems in poultry (witter and johnson, 1985) , and the wide host range of the virus group has prompted speculation regarding potential as a public health risk. there are, in fact, data demonstrating antibodies to the virus in human and other mammalian sera . coronaviruses contain a large positive-sense rna genome of approximately 30 kb. members of this virus family infect both mammals and birds (cavanagh et al., 1994) . infectious bronchitis, caused by a coronavirus, infectious bronchitis virus (ibv), is one of the major poultry viral diseases of worldwide economic importance. it is also one of the most rapidly spreading avian respiratory diseases known (mcmartin, 1993) . the virus is antigenically variable owing to a high mutation rate in the surface glycoprotein s gene. new antigenic variants of ibv continue to be isolated from various geographic regions. thus far, it has not been possible to determine the number of distinct ibv subtypes, but there are at least six (siddell, 1995) . strains can differ in their virulence and tissue tropism, but in general the disease is a rapid-onset respiratory distress that can cause significant mortality in young birds. in established flocks, the infection is often associated with growth retardation and reduction in egg production that may be exacerbated by other respiratory pathogens. estimates of a 10 to 20% loss in market value have been made for a typical outbreak in a flock. vaccination is employed to control the disease, and an interesting related feature is the demonstration that vaccine strains can undergo recombination with wild-type strains. consequently, new ibv antigenic variants of the s1 gene emerge with characteristics of both viruses (cavanagh et al., 1992; wang et al., 1993) . ibv is also an excellent example of a virus exquisitely adapted to its host, as it is not found naturally in any other reservoir. chickens seem to be the only reservoir, and the virus is capable of persisting in some manner within populations and later being transmitted to naive flocks. evidence suggests the mode is primarily airborne transmission. in controlled studies, birds in houses 115 feet away from an experimentally "seeded" house were infected via aerosol transmission (mcmartin, 1993) , and circumstantial data from natural outbreaks suggest transmission over distances of several hundred yards (cumming, 1970) . coronaviral enteritis of turkeys, also known as bluecomb disease, may also be species specific since chickens, pheasants, and quail do not exhibit any disease following inoculation with a strain of virus that is virulent for turkeys (hofstad et al., 1970; larsen, 1979) . coronaviruses have been isolated from ratites with enteric disease in zoological collections, but the relationship of these isolates with the turkey coronavirus (tcv) has not been investigated (frank and carpenter, 1992; kennedy and brenneman, 1995) . transmission of tcv appears to be restricted to the fecal-oral route, although cell-free lysates of the bursa from infected birds can be used to transmit the agent orally. among turkey flocks, the primary mode of transmission of coronaviral enteritis is via contaminated personnel and equipment. however, since tcv is excreted in fecal material and is very stable, it is conceivable that wild birds may serve as vectors for the agent. morbidity is usually as high as 100%, with mortality becoming as high as 50% in an infected turkey flock. mortality among poults may be much higher with increased numbers of secondary bacterial gastrointestinal infections contributing to severity of disease. turkey coronavirus also shares features reported for mammalian hemagglutinating coronaviruses and is closely related by sequence and antigenic crossreactivity to bovine coronavirus and a human coronavirus isolate (verbeek and tijssen, 1991) . this close relationship suggests recent interspecies transmission of the coronaviruses. while no etiologic agent has been pinpointed, a bovine origin coronavirus has been implicated in an emerging enteric disease of turkeys called poult enteritis mortality syndrome (pems) (barnes and guy, 1997) . the arthropod-bome viruses (arboviruses) present a unique challenge to evaluating ecological virus-host relationships. this grouping includes members from several virus families, six of which have members isolated from birds: togaviridae, flaviviridae, reoviridae, arenoviridae, bunyaviridae, and rhabdoviridae. of these, only the togaviridae and flaviviridae have strains that have caused documented disease in commercial poultry and game birds. although difficult to assess in feral populations, isolates from the remaining virus families as well as several isolates from the togaviridae and flaviviridae are not associated with any pathology in birds. of the togaviridae, the genus alphavirus contains the encephalitic strains causing eastern equine encephalitis (eee), western equine encephalitis (wee), and a closely related wild bird isolate --the highlands j virus (hjv). pheasants have been the primary targets of significant outbreaks of eee. signs of infection are primarily but not exclusively neural, and mortality rates have reached 80% in some natural outbreaks eleazer and hill, 1994) . economically significant outbreaks of disease due to eee virus in turkeys have occurred in wisconsin, with severity of the disease decreasing with increasing bird age. significant outbreaks have also been recorded in chukar partridges, ducks, and chickens. wee has been only rarely associated with disease in avian species, and the closely related hj virus appears to be the eastern united states equivalent to wee. hjv has caused severe neuropathogenic outbreaks in chukar partridges and has been associated with infections in turkey flocks resulting in acute reduction in egg production . of course, these encephalitic viruses also cause disease in humans and horses. another interesting alphavirus, ockelbo virus, related to sindbis virus, has been implicated in causing arthralgia and rash in humans following its isolation from mosquitos collected during the outbreak. these viruses are transmitted by mosquitos among bird populations, which may act as the vector for transmission between humans and avian species. ockelbo virus, therefore, is apparently maintained in an enzootic cycle involving birds and mosquitos with transmission to other hosts such as humans. antibodies to ockelbo virus, either experimentally or naturally infected, have been detected in passeriformes, galliformes, and anseriformes (lundstrom et al., 1992; lundstrom and niklasson, 1996) . viremia in the absence of disease resulting from infection with ockelbo has also been demonstrated in these bird groups. given the widespread occurrence of antibodies to these encephalitic alphaviruses, it seems logical to conclude that birds in many cases act as "natural" and reservoir hosts. the only flavivirus thus far associated with disease in birds is the israel turkey meningoencephalitis virus. infected birds exhibit neurological dysfunction and occasional significant mortality. this virus has also been identified in turkeys in south africa. double-stranded segmented rna viruses are unique and intriguing. the distinguished virologist dr. wolfgang k. joklik, when asked what in the world he thought they meant in the grand scheme of biology, replied (and i paraphrase), "they represent an evolutionary step forward in the establishment of the ideal genetic material." the reoviruses have fascinated virologists for some time. they have an unusual and complex replication strategy, are able to undergo reassortment of their genes, and are curious in that they retain their infecting subviral cores as their rna replication template. reoviruses are also quite stable outside the host, remaining viable for up to a year at room temperature (nibert et al., 1996) . the mammalian strains have a wide host range but are found without any associated clinical signs, the origin of the name reo (respiratory and enteric orphan) telling most of the story. in poultry, several antigenic subtypes have been identified, and the virus can be classified based on serotyping and virulence, but there is no unified typing scheme as yet (kawamura and tsubahara, 1960; robertson and wilcox, 1986) . the most severe and common pathology associated with reoviruses is arthritis-tenosynovitis, although associations with other clinical syndromes including respiratory and enteric disorders have been described (rosenberger and olson, 1997) . often, the clinical states are influenced by the presence of other pathogens. arthritis is a significant problem in birds but primarily only in young chicks and turkey poults. other reoviridae in birds include several members of the genera orbivirus (arboviruses; see below) and rotavirus, which have been associated with a runting-stunting syndrome in chickens. the birnaviridae are a relatively recently characterized family of viruses that contain two double-stranded segments of rna and have no mammalian members thus far (ictv, 1995) . they were difficult to classify for many years because of their cell-associated nature and slow replication in cell culture systems. currently, two serotypes (1 and 2) are accepted, with significant antigenic variation and numerous proposed subtypes within each serotype (mcferran et al., 1980; jackwood and saif, 1987) . chickens are the only animals known to develop disease and lesions when naturally infected by avibirnaviruses. both serotypes are distributed worldwide, but serotype 1 strains are the only ones associated with pathogenicity and immunosuppression. the bursa is the primary target organ, and strains of differing virulence have been identified. the disease is most significantly manifested when young birds are infected and permanent immunosuppression results. this sets the stage for subsequent severe viral and bacterial infections later in life. interestingly, ibd is an example where maternal antibodies derived by either vaccinations or natural infections provide significant immune protection. highly virulent strains of these viruses exist in various countries worldwide, and there are indications that new virulent variants do arise in the face of immune pressure (chettle et al., 1989b) . avian adenoviruses are double-stranded dna viruses containing 30-40 genes. these viruses can be loosely divided into three groups based on antigenic relationships of internal proteins (mcferran, 1997) , although the three groups have not been officially recognized by the ictv. the avian group i adenoviruses include at least 12 serologically distinct types, all of which have been isolated from mildly or asymptomatic poultry. the type species is known as the celo (chick embryo lethal orphan), or phelps strain, or f1 (fowl) strain. there are also numerous isolates from other avian species plus electron microscopy and immunological evidence that the type i aviadenoviruses are widely distributed in birds. by themselves, these viruses present no particular clinical problems in commercial poultry, but they are thought to cause significant problems in mixed infections with immunosuppressive viruses such as ibdv and chick infectious anemia virus (ciav). a virus virtually indistinguishable from the celo strain known as quail bronchitis virus (qbv) can be devastating in commercial quail operations, causing as high as 80% mortality (dubose and grumbles, 1959; montreal, 1992) . virus isolations and significant antibody levels in wild quail suggest that the virus may present disease problems in nature (king et al., 1981) , although there are no documented cases or epizootics. the group ii aviadenoviruses include turkey hemorrhagic enteritis virus (hev), pheasant marble spleen disease virus (msdv), and the avian adenovirus splenomegaly (aas) of chickens. these three agents are serologically indistinguishable but induce differing clinical manifestations in the different species. infections caused by this virus group appear to target lymphoid tissue, often resulting in immunosuppression, and there are strain-specific differences in virulence among the various group ii isolates (pierson and domermuth, 1997) . hev reached epidemic proportions in the 1960s and still causes significant problems in turkeyproducing states in the united states and elsewhere. msdv is a significant pathogen in confinement pheasant operations, causing significant economic losses. aas is virtually ubiquitous among chicken flocks in the united states, in which case mild respiratory signs and splenomegaly are occasionally associated. the syndrome is similar to the disease state observed in pheasants, but in general aas virus causes no significant problems among chickens. evidence indicates that these group ii avian adenoviruses are limited to the order galliformes, and that wild bird populations (even wild turkeys) are unaffected. the more interesting group iii adenoviruses first appeared in 1976 associated with a distinct clinical manifestation called egg drop syndrome (eds), in which egg production decreases and thin-shelled or shell-less eggs are produced. the disease has caused significant egg production losses mostly in the eurasian and australian-pacific poultry markets. a virus named eds76, which has been found associated with this syndrome, may have been originally introduced via a contaminated vaccine, but it seems clear now that sporadic cases are initiated by introduction of the causative virus from domestic and wild waterfowl, mostly ducks and geese. when endemic in flocks, the virus is transmitted vertically and exhibits a latent phase, which is reactivated in laying hens, usually after egg production begins. the virus initially appears to replicate in lymphoid tissues but rapidly moves to the oviduct, where replication causes inflammation and production of aberrant eggs. unlike other avian adenoviruses, the group iii strains do not replicate in the intestinal mucosa. it is likely that eds76 virus or very similar strains are present ubiquitously in wild ducks and geese, but rarely appear to be associated with disease (mcferran, 1997) . the avipoxviruses are widely distributed throughout the class aves, having been isolated from some 60 species representing 20 avian taxonomic families. the avipoxviruses are responsible for economically important disease problems in commercial poultry and aviaries (tripathy, 1993) . they may cause a slowly developing cutaneous disease with low mortality or, conversely, significant mortality, and generalized infections when in the diphtheritic form on mucosal surfaces of the respiratory tract and associated areas. these large dna viruses replicate in the cytoplasm of the cell, where they form characteristic inclusion bodies within rapidly proliferating nodular lesions. the poxviruses may be transmitted by mechanical means such as introduction from poultry workers into abrasions in the skin of uninfected poultry. there is also real evidence for transmission of the disease by mosquitos and other vectors such as mites in close conditions, where the number of diseased birds is high. the avipoxviruses can also apparently establish a latent state and be naturally or chemically induced to reactivate (kirmse, 1967b) . cutaneous lesions containing infectious virus persistent for more than a year have also been documented in wild birds (kirmse, 1967a) . variant strains of avipoxviruses have since been isolated from previously vaccinated flocks that were experiencing significant mortality from the diphtheritic form of the disease (tripathy and reed, 1997) . this suggests that, similar to marek's disease, vaccination against poxviruses may ultimately lead to escape of more virulent forms of the viruses. the avipoxviruses are also considered one of the most promising dna virus vectors for delivery of recombinant poultry vaccines, and poxvirus-vectored vaccines against newcastle disease and avian influenza have been licensed. avian encephalomyelitis is primarily a disease of young chicks caused by a picornavirus. the disease was quite economically important prior to initiation of live-virus vaccination. the host range is very limited (order galliformes), and there is only one virus serotype. the natural isolate is enterotropic and is transmitted horizontally (orally) and vertically. there is a gradient of pathology dependent on the age of infection of young chicks. pre-immune chicks from non-immune parents will generally die if infected within 1 to 2 days of hatch. if infected between 2 and 8 days of age, they may live but exhibit significant nervous involvement (encephalomyelitis). if infected beyond that age, they may exhibit enteric pathology but not neural signs. at adulthood or full immunocompetence, they may be infected but refractory to any clinical signs. so the immune status of the host in this case is very important in affecting the course of the viral replication (calnek, 1997) . another avian picornavirus, distinct from aev, is the avian nephritis virus (shirai et al., 1991) . this virus is similar to aev in that it is primarily a problem in very young birds and has a very limited host range. as the name implies, a distinct clinical syndrome is associated with the agents, but it is has not yet been determined how extensive infections are in commercial poultry. finally, another picornavirus, duck hepatitis 1 (dhv-1), has caused significant problems in com-mercial duck operations. there are still unknowns with regard to the overall importance of dhv-1, and there are additional virus-induced types of hepatitis disease with at least two others often encountered. the most significant cost involved in raising birds for food sources is feeding them. consequently, diseases that affect the feed-protein conversion ratio will directly affect the cost of marketable birds. as such, enteric disease, even when nonlethal or mild in nature, can affect the performance of food-source birds. in recent years, investigators have identified rotaviruses in chickens, astroviruses in turkeys, and enterovirus-like particles in several avian species associated with enteric diseases. many of these have been associated with significant pathogenesis, particularly in mixed infections where immunosuppression occurs, with subsequent decrease in marketability due to weight loss (barnes, 1997) . the virus designated chicken infectious anemia virus (ciav) is a singlestranded circular dna-containing virus, tentatively classified with two similar agents in a new family: circoviridae. the agent has only recently been recognized, purified, and characterized, and its unequivocal role in economically important disease is not yet fully established. purified virus inoculated into young chicks results in severe anemia and immunosuppression, but inoculation of 2-to 3-week old birds results in little if any pathology (yuasa and imai, 1986) . the virus has been associated with adult anemic conditions, however, in conjunction with other viral and bacterial infections and may thus play a significant role. the agent is widespread in commercial poultry flocks worldwide, and infection with the virus has been statistically related to a decrease in overall growth and performance (chettle et al., 1989a) . the extent to which two other tentative members of the family circoviridae --psittacine beak-and-feather disease virus (bfdv) and porcine circovirus-are related to ciav is under question, and these stated taxonomic relationships may differ in the future. bfdv has proven to be a significant pathogen in aviaries and commercial in psittacine birds to the extent that vaccines are now routinely administered. recent evidence also suggests that circovirus particles can be detected by em and isolated from several other wild and captive bird species. consequently, the circoviridae may emerge as a more significant disease-causing agent in the near future. the caliciviridae is a family of small single-strand positive-sense rna viruses with a polyadenylated genome. virus-like particles resembling caliciviruses have been isolated from a variety of wild-bird and captive-raised species. a chicken calicivirus has been replicated in cell culture and caused apparent gastrointestinal disease in specific pathogen-free day-old chicks (cubitt and barrett, 1985) . intestinal contents of goldfinches with hemorrhagic enteritis have been found to contain calicivirus-like particles and gastrointestinal disease associated with caliciviruses has been reported in guinea fowl and pheasants (gough et al., 1985) . caliciviruses were originally isolated from marine mammals and have caused disease in both domestic and feral swine due to consumption of uncooked garbage containing seafood. interestingly, caliciviruses have been detected in pelagic birds, such as the white tern (poet et al., 1994) . calicivirus isolations such as these lead to the speculation that wild sea birds may be important in the transmission of these agents across large areas or to other animals. parvoviruses are the smallest of the dna-containing viruses, carrying a singlestranded genome of about 5000 nucleotides. they cause or are associated with three infections in birds (kisary, 1993) . in wild and domestic geese and muscovy ducks, derzy's disease refers to a syndrome that had been variously called goose influenza, goose hepatitis, gosling enteritis, and infectious myocarditis in different countries. this collection of names gives some indication of the variety of signs associated with derzy's disease. the virus, which can be transmitted horizontally and vertically, induces differing pathologies depending on the age of the bird and, in the case of hatchlings, the level of maternal antibodies. an interesting pathology often associated with infection of older birds is the virtually complete loss of feathers. the extreme stability of the parvoviruses makes control of this disease difficult in commercial operations worldwide, and the disease can result in 100% mortality in young hatchlings. in chickens and young turkeys, parvoviruses have been associated with runting stunting syndrome (rss), as have other agents. experimental inoculations have indicated that the associated viruses, which are distinct from the goose viruses, can cause significant pathology, but the extent to which these viruses participate in the rss is not yet established (trampel et al., 1983; decaesstecker et al., 1986) . another parvovirus, the avian adeno-associated virus is almost always found associated with adenovirus infections in poultry. the avian adeno-associated viruses are grouped with their mammalian counterparts in the dependovirus genus, although they are serologically unrelated. the avian dependovirus appears to contribute to disease only in the sense that it can affect multiplication of the associated adenoviruses (yates and piela, 1993) . newcastle disease virus (ndv), classified as avian paramyxovirus-1, contains a single-strand negative-sense rna genome of 15 kb containing coding sequences for six genes. the virus infects all bird species tested to date, including flee-living and domestic species. one panzootic outbreak of the disease is thought to have originated in asia, with subsequent spread to europe, with outbreaks of disease first reported in poultry during the 1920s in java, indonesia, and newcastle-upon-tyne, england. worldwide dissemination of the disease, particularly during the 1960s and 1970s, has been attributed to increased international trade of both commercial poultry and pet birds. this led to development of inactivated and live-virus vaccines for commercial poultry. the transmission of infectious ndv may occur by either ingestion or inhalation, and this knowledge is the basis for mass-application vaccination procedures during poultry production. isolates of ndv are grouped into three main pathotypes depending on the severity of disease that they cause (alexander and parsons, 1974) . mildly virulent "lentogenic" viruses may cause unnoticeable infections in adult chickens or a mild respiratory distress and are used extensively as live-virus vaccines. "mesogenic" ndvs are of intermediate virulence and cause respiratory distress, with mild infections of various organs detectable only by histopathology. highly virulent viruses that cause severe morbidity and mortality are termed "velogenic." velogenic viruses can manifest themselves as neurotropic or viscerotropic forms of newcastle disease with extensive systemic replication throughout a bird. virulent forms of ndv can replicate within cultures of most avian and mammalian cell types without the addition of trypsin, while lentogens require added proteases for replication in cell culture. the presence of dibasic amino acids at the proteolytic cleavage site (pcs) of the viral fusion protein and the ability of cellular proteases to cleave the fusion protein of various pathotypes specify the molecular basis for ndv virulence. fewer basic amino acids are present in the fusion protein cleavage site of lentogenic ndv than is the case for mesogenic or velogenic isolates. the presence of the increased number of dibasic amino acids in the fusion protein pcs sequence of ndv allows for systemic replication of these more pathogenic viruses in the host (nagai et al., 1976) . although the principal concern with ndv is its effect on poultry production, it may have severe consequences in other flee-living avian species. major outbreaks of newcastle disease have occurred in north american cormorants during the summers of 1990 and 1992, and again in 1997. outbreaks during 1990 and 1992 occurred in the north central united states and south central canada, while in 1997 newcastle disease occurred among cormorants of the western united states and canada. mortality in young nestlings in some areas was as high as 80 to 90%, and the affected birds had characteristic neurotropic lesions (heckert et al., 1996) . during the 1992 outbreak in cormorants, an unvaccinated north dakota turkey flock became infected with ndv, resulting in high mortality. using nucleotide sequence analysis, the virus causing disease in turkeys was proven to be the same virus isolated from afflicted cormorants in the central united states and canada. the cormorant virus was also phylogenetically related to other known ndv isolates of psittacine origin (seal et al., 1995) that had caused a major outbreak in southern california poultry during the early 1970s that resulted in a depopulation of two million chickens (utterback and schwartz, 1973) . illegal importation of pet bird species into the united states continues to be a source of highly virulent ndv and certainly may play a role in spread of viruses that threaten commercial poultry worldwide (panigrahy et al., 1993) . what role free-living birds play in spread of ndv is unclear. although persistent infections of chickens by ndv do not appear to occur, the virus' persistence in poultry flocks may result from virus reintroduced from wild populations. also, different bird species vary in their level of susceptibility to ndv. ducks, geese, and certain psittacine birds do not exhibit signs of disease when infected with highly virulent ndv, while other psittacine species may have high mortality. persistent infections have been demonstrated in various psittacine birds, with virus isolations noted up to a year following experimental infection of parrots with velogenic ndv chicken isolates (erickson et al., 1978) . although psittacine birds have been directly linked as a source of ndv highly virulent for gallinaceous birds, such as chickens and pheasants, no studies have shown direct isolation of ndv from feral psittacines. newcastle disease occurred in racing and show pigeons during the 1980s in both western europe and north america. the outbreaks in western europe were linked to contaminated feed, and disease was controlled in both areas via vaccination with mildly virulent ndv commonly used for poultry. the virus isolated from these birds varied somewhat from traditionally virulent ndv in having only one set of dibasic amino acids in the fusion protein cleavage site. increased virulence for poultry subsequently occurred only after passage in chickens (collins et al.. 1994) . these examples demonstrate the fact that variant forms of ndv may arise in different bird species and that free-living birds may be a constant source ot viruses that affect both domestic and wild birds. vaccination programs against ndv for the most part have been effective al controlling the virus in commercial poultry. however, all of the vaccine strains available today, while effective against lethal disease, share less sequence identit3 with currently circulating disease strains. this is effectively illustrated in figure 2 , which may also be considered a useful example for other rna-containin~ viruses. the phylogenetic tree demonstrates the relationship between currenl 2 . phylogenetic relationships among newcastle disease virus isolates. nucleotide sequences from the fusion protein gene coding for the cleavage site from several newcastle disease virus isolates were aligned and analyzed by parsimony analysis. all the viruses listed are intermediately virulent mesogens or highly virulent velogenic isolates when inoculated into chickens, with the exception of the two vaccine viruses and the chicken/92 field isolate. the data demonstrate that field isolates from chickens related to vaccine virus are isolated from poultry. the chicken/72 virus caused the last major outbreak of newcastle disease in u.s. poultry and was epidemiologically linked to a pet bird. it is genetically related to a parrot isolate and viruses that caused disease in pigeons during the 1980s. viruses isolated from cormorants in 1992 infected an unvaccinated turkey flock. these viruses appear to be circulating among cormorants, since closely related viruses were isolated again in 1997. no viruses related to the virulent chicken viruses from 1948 have been isolated since that time, indicating these virus types may no longer be circulating among birds. all the recent virulent newcastle disease viruses were isolated from pet or exotic birds and chickens infected by free-living birds. these viruses are related to virulent viruses originally isolated during the 1930s. avian influenza (ai) presents one of the most interesting ecological relationships between birds and their viruses. its ecology has been reviewed in depth hinshaw et al., 1980) , but ai presents such a complete picture of a virus with multiple impacts on several species that it must be included in this chapter. the type a orthomyxoviruses are essentially bird viruses. the infections in birds can range from clinically inapparent with minimal serologic response, to a devastating systemic disease that can result in 100% mortality within a matter of days. there are 15 identified subtypes of ai viruses based on the hemagglutinin (ha) protein antigenic structure and nine subtypes based on the neuraminidase (na) structure. these two genes code for the predominant surface glycoproteins, which are embedded in the lipid bilayer of the viral envelope. these are only 2 of 10 genes coded for by the virus. the virus' genetic material is contained on eight separate negative-sensed single-stranded rna segments ranging from 890 to 2450 nucleotides in length. all of the ha and na viral subtypes have been identified in feral waterbirds, and it has been proposed that these birds act as a "natural" reservoir for the virus slemons and easterday, 1977) . determination of the phylogenetic relationships of several genes from several subtypes of aiv collected from wild birds indicated that the general rate of evolution in the avian reservoir is low compared to the rate of evolution observed in human and other mammalian strains of type a orthomyxoviruses . other recent studies, however, have shown that the mutation rate of the ha gene and the ns gene even in wild birds approaches that seen in human strains . measurement of clinical signs among infected migrating waterbirds is, of course, a difficult task. thus far, only a single instance of severe clinical signs has been associated with free-living birds --a lethal outbreak in terns in south africa in 1961 caused by an h5n 1 strain (becker, 1966) . given the recently measured mutation rates, and the appearance of this severe lethality in a wild bird population, it must be considered that aivs do continue to evolve in feral birds (indeed 15 subtypes have arisen already!). there is no reason to believe that the present count of 15 subtypes will be the final tally. while some have proposed that the aiv strain in wild birds causes no disease problems as a result of "evolutionary stasis," without question the virus has dramatic effects when it leaves this proposed reservoir. in addition to the fixed influenza populations in pigs and horses, avian strains can directly infect mammals and have been identified in whales, seals, and mink (lang et al., 1981; berg et al., 1990) . the dramatic association with seals in 1980 and 1982 mentioned earlier clearly points out the potential impact of aivs. this avian reservoir then serves as a sort of base for the viral "biological invasions" that are a part of interspecies transmission. ecologically and economically, the transmission of these aiv subtypes to commercial poultry has perhaps yielded the most important impact. the situation is most clearly played out in minnesota each year. due to the large number of lakes in the state, migratory waterfowl, primarily anseriformes (ducks, geese), frequent the area in large numbers seasonably. nearby commercial turkey operations suffer infections nearly every year, particularly in the cooler fall months, caused by different influenza subtypes of different virulence (halvorson et al., 1985) . in severe years, the associated costs due to turkey mortality, as well as performance and egg production losses, can reach several millions of dollars (poss and halvorson, 1986) . in chickens, introduction of influenza viruses from waterfowl has been more devastating. in the northeastern united states, the h5 subtype caused the first large-scale outbreak of highly pathogenic ai in numerous flocks in 1983. twentythree million birds were destroyed at a cost of more than $65,000,000 in order to contain the infection. clinically, the disease was indistinguishable from the disease originally described as "fowl plague" in 1927 in europe (eckroade and silverman, 1986) . today we know that all of the classical "fowl plague"-type outbreaks have been associated exclusively with only two subtypes: h5 and h7. evolution to virulence in these subtypes has been closely associated with accumulation of basic amino acids at the proteolytic cleavage site of the hemagglutinin protein (bosch et al., 1981; webster and rott, 1987) . in order for aiv strains to be infectious, the ha protein must be cleaved into ha1 and ha2 subunits, which subsequently allows structural rearrangement and exposure of a protein sequence needed to fuse the viral envelope with the plasma membrane of the target cell (klenk et al., 1975; skehel et al., 1995) . isolates of low pathogenicity lack multiple basic amino acids and are only cleaved by trypsin-like extracellular proteases. these proteases are abundant on the mucosal surfaces of the respiratory tract and gut, so the viruses replicate unrestricted at these sites. the highly pathogenic (hp) forms, however, have additional basic amino acids, which are recognized by the intracellular furin-like proteases ubiquitous in bird tissues (rott et al., 1995) . thus, in birds, these viruses are able to escape the respiratory tract and infect a wider variety of internal tissues and organs. this is one of the most important of the virulence factors (webster and rott, 1987) . only two subtypes have been shown to accumulate these basic amino acids: h7 and h5. these subtypes appear to do so by either base substitution or by insertion events (wood et al., 1993; perdue et al., 1997) . nucleotide insertion events appear to be the most common mechanism, and table ii and figure 3 illustrate what is presently known about occurrence of these hp virus isolates in commercial poultry. the hp isolates, with only two exceptions, have additional inserted basic amino acids within the well-conserved region surrounding the cleavage site. from the data in table ii and figure 3 , it may be easily surmised that both the number of hp isolates and the number of basic amino acids at the cleavage site are increasing. athe amino acids surrounding the hemagglutinin protein cleavage sites of a representative member of each of the 15 subtypes of avian influenza viruses are shown above. subtypes 7, 10, and 15, which cluster together in a phylogenetic tree, all have five amino acids between the conserved proline and arginine; the remaining subtypes have four. highly pathogenic isolates (subtypes h5 and h7) more often than not, have insertions of arginines and lysines, increasing the length of the cleavage site and making the hemagglutinin accessible to ubiquitous proteases. this has the effect of greatly increasing the tissue distribution and virulence of the virus. table ii for further explanation. the avian influenza viruses are distributed worldwide. in addition to the united states, outbreaks of highly pathogenic ai have occurred in mexico, canada, europe, asia, and australia. in most cases, there is some sort of connection between the affected flocks and nearby waterfowl. the extent to which the reservoir of viruses exists in waterfowl is not really known. surveys have shown that, in addition to the viruses being widespread, the various subtypes increase and decrease in prevalence over successive years. the order charadriiformes (shorebirds and allies), in addition to anseriiformes, has been shown to carry aiv strains (kawaoka et al., 1988) , and the extent to which aivs are carried in other orders has really only been superficially explored. molecular and phylogenetic evidence have clearly shown that these segmented viruses share various genes back and forth within the populations of circulating virus strains . they are also capable of donating genes through genetic reassortment to viruses in swine and those genes ultimately end up in humans (webster et al., 1993) . add this capacity to the known (albeit rare) transmission of purely avian influenza strains directly into mammals and one creates a potential ecological bonanza for the virus, and potential nightmares for new hosts. as advanced as our study of viruses has become, there are still a variety of unknowns with regard to the extent to which avian viruses infect other vertebrates. it would not be unreasonable to suggest that, because of their mobility and longevity of existence, that the class aves may have played a critical role in distribution of viruses to other classes. in the case of the avian orthomyxoviruses, phylogenetic evidence points to recent introduction of these viruses from birds to mammals. following introduction, the natural mutation and evolution of the virus produced strains that now appear to be adapted to their new hosts. there are swine, equine, and human strains of type a influenza viruses that do not replicate well outside their respective host species. if birds are indeed responsible for us coming down with the flu today, we know of no reason why they might not have participated over the eons in the establishment of other purely mammalian diseases. of course, one could easily argue the opposite relationship, but, intuitively, birds do present an ideal mobile vector, particularly for the enterically transmitted viruses. in addition to the diseases discussed here, there is a growing group of emerging diseases or diseases of as yet unknown etiology in commercial poultry (saif, 1997) . many of them have virus particles clearly associated with the syndrome, but only limited research and characterization has been carried out thus far. it is probably safe to assume that the future will continue to bring new avian diseases and bring to light new or undiscovered avian viruses. the relationships existing among birds, insects, and viruses is one area that should be explored in more detail by increased research efforts. in free-living birds, the large number of apparently innocuous infections with bunyaviridae, togaviridae, and flaviviridae suggest that birds may act as an ecological reservoir for maintenance of these virus populations. from a virocentric point of view (if there is such a thing), having a potentially highly mobile population of susceptible hosts on which transmission vectors may feed would be advantageous. it would provide a mechanism by which to transfer viruses over large geographical distances where they could then be transmitted by new related or unrelated vectors, thus increasing host range. of course, one runs the risk here of assuming that the virus would be ecologically improved by extending its replicative capacity in new hosts. this may not be a valid assumption. for example, if variola virus or human poliovirus type 1 had been exploiting this strategy, they made a bad choice in extending their host range into humans! from one human vantage point, birds provide a useful monitor for detection of arboviral diseases in a given area. sentinel chickens are actually in use today along the east coast of the united states to gauge the extent to which some mosquito populations are carrying eastern equine encephalitis and st. louis encephalitis viruses. the future is perhaps the most important aspect to consider. what viruses other than the orthomyxoviruses may someday establish a similar relationship where transmission to a mammalian intermediate or even direct transmission may allow introduction into the human population? one fundamental unanswered question is whether birds, acting as reservoirs for arboviruses, should be considered an important ecological niche for those viruses. there is no evidence of which we are aware to indicate either yes or no. there have been no documented transmissions of arboviruses from birds to mammals without the insect vector in nature, however, so at this point wild birds could be considered dead-end hosts for the arboviruses. in commercial poultry during outbreaks of the alphavirus eeev, there is no evidence of infection of workers in close contact with infected birds. if, however, a bird provides the necessary reservoir of eeev that infected the human, then this bird becomes very important. but there are no data that as yet identify the bird as the required reservoir for disease transmission to human. newcastle disease virus provides an interesting new disease problem in humans that has only arisen as a result of increased vaccination of birds. a live-virus vaccine is available for control of ndv that is administered by aerosol spray. in a small number of cases where precautions have not been taken, vaccinators can contract a conjunctivitis caused by the vaccine. this outcome has been seen in the heavy poultry-producing areas of northern georgia, on a common enough basis now, to be easily recognized by ophthalmologists. there is usually no seroconversion to the virus in these cases, and they are not particularly serious infections. there is also suggestive evidence in the form of measured seroconversion that poultry workers occasionally may be subclinically infected with avian coronaviruses and avian retroviruses. the evidence for direct infection of humans with avian influenza viruses is growing. human strains have classically included only three subtypes (h1-3). until recently, the only other strains shown to cause disease in humans were ot the h7 subtype. conjunctivitis caused by purely avian h7 subtype viruses has been reported on two occasions (kurtz et al., 1996; webster et al., 1981a) . the most disturbing set of events, however, was the more recent highly publicized influenza outbreak in hong kong. a purely avian h5 subtype virus was isolated from some 18 patients, 6 of whom died, from may through december of 1997. the index case, a 3-year-old child, in may followed a highly pathogenic outbreak of avian influenza that had just previously occurred in chickens in hong kong and its environs. the child had been exposed to ill birds at a day care center, and it was clear that the recovered virus was essentially the same as that recovered during the chicken outbreak. considerable efforts were made to determine whether the virus was a contaminant and whether it was replicating in the child. the evidence strongly suggested that it was indeed replicating (subbarao et al., 1998) . subsequent to this event, in november-december the remaining confirmed cases were reported and an intense effort was mounted to determine exactly how this virus was transmitted. it is clear now that the h5n1 viruses infecting humans in hong kong 1997 were all of avian origin . there is limited serologic suggestion of human-to-human transmission but no genetic evidence of adaptation to humans. this case was touted as a premier test run for the next predicted influenza pandemic. whether this outbreak represents a simple dead-end zoonotic transmission or whether these purely avian influenza viruses can become fixed in the human population is a matter for conjecture. the avian h5 and h7 influenza strains have been the only natural influenza strains thus far to unequivocally cause systemic lethal infection in any species owing to the unique structure of their ha protein (see fig. 3 ). a major fear is that by adding such ha subtypes to a population of mammalian viruses a whole new class of virulent strains might be created. our experience in working with numerous highly pathogenic and nonpathogenic strains has been one of very little or no evidence for human infection in the 25 years that scientists and technicians have spent working with hundreds of different stains. but our experience has also been that, when one attempts to confine influenza viruses by setting biological rules for them, they usually find a way to break them. if ecology is the study of the relationship of organisms to their environment, for bird viruses, the active, pertinent, environment is always within the bird and its tissues. while one cannot discount the effects of the environment outside the host, there are no positive effects on an avian virus life cycle outside the host of which we are aware. that is, there are no activation events outside the avian host of which we are aware; only events of inactivation. certainly, avian viruses, as all viruses, have evolved survival strategies to allow passage from host to host, and some are much more refractory to environmental inactivation than others. but without really knowing which, if any, "evolutionary direction" viruses are taking, it is impossible to determine whether a large herpesvirus of 150 genes is ecologically more fit than the enterovirus with five genes! the relationship of birds and insects in the transmission and life cycle of the arboviruses is poorly understood. until this recent transmission of h5n1 influenza strains to humans, this relationship appeared to be the only significant one in which there is interaction among a genetically unaltered virus, an avian host, and a non-avian host. as mentioned earlier, other documented cases of avian-to-mammalian viral transmission are newcastle disease infections of humans and influenza a infections of mammals and humans. there is also serological evidence for avian retrovirus and avian coronavirus infections. in the case of newcastle disease, the documented infections are mostly minor conjunctivitis. in the case of influenza, discussed earlier, the avian viruses most often acquire rna segments from other sources before they are established in a new host. thus, in general, the impact of avian viruses on public health might currently be considered small, but clearly the potential exists for significant future impact. a major interesting question arises when evaluating virulence of avian-origin viruses. conventional wisdom seems to be suggesting that virus entry into naive host populations is more likely to result in a general decrease in virulence as the virus adapts to the new host (morse, 1994) . this may be true under truly "natural" conditions; but what happens under human-dictated conditions such as vaccination and by defining the host population through breeding and housing? in the case of marek's disease, we see an example of virulence increasing in the face of continued vaccination against the virus (fig. 1) . whether this is occurring as a result of the vaccination or a combination of factors, the fact is that the population of marek's disease herpesviruses in nature is becoming more virulent. the vaccination programs for newcastle disease utilize vaccines whose genetic compositions are becoming farther and farther removed from the virulent strains circulating in wild fowl (fig. 2) . this may not bode well for future control of virulent strains in commercial poultry. in the case of avian influenza viruses, we have seen the number of highly virulent outbreaks increase in recent years, often following prior circulation of a nonpathogenic precursor, and we have seen concomitant changes in genetic structure related to virulence (fig. 3) . again there are unknowns in this system, but the fact remains that we are seeing more virulence as the nonpathogenic aiv subtypes replicate in commercial poultry settings. thus, this naive commercial population, unlike the waterfowl hosts to which the virus has adapted, appears to allow generation of heretofore unencountered virulence phenotypes. similar scenarios appear to be playing out as more virulent infectious bronchitis strains and infectious bursal disease strains appear worldwide. one must be cautious, then, in working strictly under the previously mentioned virulence-decrease paradigms for mammalian viruses. these have been suggested mostly by the experiences with artificial introduction of rabbit papilloma viruses in australia or where new introductions occur as human encroachment ensues (such as in the recent cases of ebola virus or monkey poxvirus infections). in these cases, it was proposed that virus transmission results first in high virulence for the host followed by subsequent adaptation and attenuation. finally, it may be dangerous to attempt to confine avian viruses to some of our more "logical" inferences based on darwinian evolution. one paraphrased definition of evolution is "moving from a prior more primitive (or less fit) state to a current more advanced (or more fit) state." it is difficult, for us at least, to say that any avian viruses are following such a progression. additionally, other than use as molecular biological vectors, viruses in general have not been shown to provide anything positive to the ecology of any other organisms. the future may yet 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seals b). characterization of an influenza a virus from seals evolution and ecology of influenza a viruses influenza: a model of an emerging virus disease increased virulence of marek's disease virus field isolates epidemiology of reticuloendotheliosis virus in broiler breeder flocks deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza a viruses of h5 and h7 subtypes avian adenovirus-associated virus pathogenicity and antigenicity of eleven isolates of chicken anemia agent (caa) key: cord-267671-ys43n672 authors: whary, mark t.; baumgarth, nicole; fox, james g.; barthold, stephen w. title: biology and diseases of mice date: 2015-07-10 journal: laboratory animal medicine doi: 10.1016/b978-0-12-409527-4.00003-1 sha: doc_id: 267671 cord_uid: ys43n672 today’s laboratory mouse, mus musculus, has its origins as the ‘house mouse’ of north america and europe. beginning with mice bred by mouse fanciers, laboratory stocks (outbred) derived from m. musculus musculus from eastern europe and m. m. domesticus from western europe were developed into inbred strains. since the mid-1980s, additional strains have been developed from asian mice (m. m. castaneus from thailand and m. m. molossinus from japan) and from m. spretus which originated from the western mediterranean region. laboratory animal medicine development of the 'modern' laboratory mouse. research use of mice has grown exponentially during the past and current century with the recognition of the power of the mouse for gene and comparative mapping and have made the laboratory mouse, in genetic terms, the most thoroughly characterized mammal on earth (silver, 1995; lyon et al., 1996; morse, 2007a) . the current ability to create highly sophisticated, genetically engineered mice by inserting transgenes or targeted mutations into endogenous genes has also made the laboratory mouse the most widely and heavily used experimental animal. historical reviews have documented the origins of the laboratory mouse, which extend thousands of years into antiquity (keeler, 1931; morse, 1978; silver, 1995) . the laboratory mouse belongs within the genus mus, subfamily murinae, family muridae, superfamily muroidea, order rodentia, and within the m. musculus clade collectively called the 'house mouse' (lundrigan et al., 2002) . anatomic features of molar teeth and cranial bones were traditionally used by zoologists to identify over 100 different species within the genus, and to differentiate them from other murids. because of considerable phenotypic variation within a single mus species, this approach has proven to be inaccurate, and given way to contemporary genetic analysis. the native range of the genus mus is eurasia and north africa. members of this genus are generally classified as aboriginal, consisting of species that live independent of humans, or commensal, which includes taxa that have coevolved and geographically radiated with human civilization since the dawn of agriculture 12,000 years before present (bp). this close association with human agrarian society gave rise to the genus name, derived from sanskrit, mush: to steal. the commensal group is known as the 'house mouse' clade, consisting of several subspecies of mus musculus, including m. m. domesticus, m. m. musculus, m. m. castaneus, m. m. bactrianus , and a lesser known lineage, m. m. gentilulus (prager et al., 1998) . the japanese house mouse, m. m. molossinus, is a natural hybrid of m. m. musculus and m. m. castaneus. the progenitor of the m. musculus clade arose in the northern indian subcontinent and diverged into genetically isolated and distinct species or subspecies due to geographic barriers (mountain ranges). there is debate whether these taxa are species or subspecies, and some have referred to them as 'incipient species,' but their genetic divergence is now blurring as they colonize the world and hybridize. the native ranges of these taxa are important for understanding the origins of various laboratory mice, whose genomes are mosaics derived from m.m. domesticus (~60%), m.m. musculus (~30%), and m.m. castaneus (~10%) (wade and daly, 2005; wade et al., 2002) . it is now apparent that the m.m. musculus and m.m. castaneus contributions to the laboratory mouse genome were primarily derived from m.m. molossinus japanese fancy mice (takada et al., 2013) . mus m. domesticus is indigenous to western europe and southwest asia, m.m. musculus to eastern europe and northern asia, m.m. castaneus to southeast asia, and m.m. molossinus to japan and the korean peninsula. the cohabitation of humans with commensal mice gave rise to captive breeding for coat color and behavioral variants in china over 3000 years bp. by the 1700s, mouse 'fanciers' in asia had created many varieties of fancy mice, as did european fanciers, who subsequently acquired asian stocks, particularly japanese fancy mice (m.m. molossinus) , to mix with european (m.m. domesticus) fancy mouse varieties. this genetic mixing for fancy variants was also occurring in the united states, and these mouse lines contributed to many of the major laboratory mice used today. meanwhile, the european colonial expansion era contributed to the worldwide dissemination of m.m. domesticus, which now occupies every continent of the world. it is well documented that wild-caught m.m. domesticus also contributed to the genetic composition of fancy and laboratory mice on multiple occasions. despite their diverse genetic origins and phenotypic differences, most laboratory mouse strains are closely related, since many were derived from a genetically mixed but small number of fancy mice from a single mouse breeder (abbie lathrop's granby mouse farm, massachusetts) at the beginning of the 20th century. most inbred laboratory mice share a common maternal mitochondrial genome derived from m.m. domesticus (ferris et al., 1982; yu et al., 2009) , and a common y chromosome contributed by m.m. musculus (bishop et al., 1985) through its contribution to the genome of m.m. molossinus (nagamine et al., 1992) . thus, the most inclusive name that can be assigned to the genetically mosaic laboratory mouse is m. musculus, the over-arching name for the entire commensal clade. there are exceptions, however. c57bl/6 mice contain minor genetic elements derived from m. spretus (hardies et al., 2000) , and a number of wild aboriginal species that are not members of the m. musculus clade, including m. spretus, m. caroli, and others, have been established as inbred lines of mice. genetic mapping in mice began in the early 1900s with a focus on inheritance of coat color. the first autosomal genes, albino and pink-eyed dilution, were linked in 1915 (haldane et al., 1915) . extensive linkage maps and an impressive array of inbred strains are now available to expedite genetic research (table 3 .1) (lyon et al., 1996) . mice have 20 pairs of telocentric chromosomes that are differentiated by their size and patterns of transverse bands. the chromosomes are designated by arabic numbers in order of decreasing size. during the 1970s, chromosome rearrangements were used to assign known genetic linkage groups -identified by roman numerals -to specific chromosomes and for determining locus order with respect to the centromere. genes can be located physically on chromosomes by fluorescent in situ hybridization (fish). development of quantitative trait loci (qtl) methodology for mapping genes and the similarity between mouse and human genomes have made the mouse invaluable for identifying genes and underlying complex traits that are inherent to the most common human genetic diseases (moore and nagle, 2000) . for more information on comparative genomics, see chapter 35 animal models in biomedical research, subsection c. one of the most thoroughly studied genetic systems of the mouse is the histocompatibility complex. histocompatibility (h) loci control expression of cell surface molecules that modulate critical immune responses, such as the recognition of foreign tissue. for example, the time, onset, and speed of skin graft rejection are controlled by two groups of h loci. the major group is located in the major histocompatibility complex (mhc, h2) on chromosome 17. the h2 complex contains several loci, including k, d, l, i-a, and i-e. inbred strains of mice, being homozygous, each have unique sets of h2 alleles, termed h2 haplotypes. for example, the balb h2 haplotype is h2 d and the c57bl h2 haplotype is h2 b . the international immunogenetics (imgt) information system provides details on h2 haplotypes for various inbred mice (www.imgt.org/imgtrepertoiremhc/polymorphism/ haplotypes/mouse/mhc/mu_haplotypes.html). minor h loci groups are scattered throughout the genome and are responsible for delayed graft rejection. genes associated with the h2 complex also control other immunological functions, such as cell-cell interactions in primary immune responses and the level of response to a given antigen. immune-mediated responses to infectious agents such as viruses and complement activity are influenced directly or indirectly by the h2 complex (stuart, 2010) . non-mhc or minor histocompatibility systems also are under active study (roopenian et al., 2000) . mouse genomics have accelerated tremendously in the last two decades, heralded by the development of a robust physical map and high-quality genome sequence of the c57bl/6j mouse in 2002 by the international mouse genome sequencing consortium (waterston et al., 2001) . the mouse genomes project/wellcome trust sanger institute is extending this effort to include the genomic sequences of 17 key mouse strains. completed and evolving sequence data are available through the european nucleotide archive (www.ebi.ac.uk/ena/home). the burgeoning numbers of inbred mouse strains, natural mutants, induced mutants, transgenic lines, and targeted mutant lines of mice are cataloged in the mouse genome informatics (mgi) database: http://www.informatics.jax.org/mgihome). the growing number of mutant mice has fostered the development of a number of mouse repositories, from which specific mice can be located and acquired. in the united states, there are four regional national institutes of health (nih)-supported mutant mouse regional resource centers (http://www.mmrrc.org), which link to international repositories in europe, japan, china, australia, and canada, as well as additional resource programs in the united states through the international mouse strain resource (imsr; http://www.informatics.jax.org/ imsr/index.jsp) for depositing, archiving, and distributing mutant mouse and embryonic stem cell lines to the scientific community. in addition to numerous mutant mice produced independently by scientists in various academic institutions, three major targeted gene knockout programs, all utilizing c57bl/6n embryonic stem cells, are under way internationally, and funded by the nih, the european community, and genome canada (collins et al., 2007; skarnes et al., 2011) . these include the knock out mouse project (komp; http://www.knockoutmouse.org), the european conditional mouse mutagenesis program (eucomm; http://www.eucomm.org), and the north american conditional mouse mutagenesis project (norcomm; http://norcomm.phenogenomics.ca/index. htm). these mouse lines will be available through laboratory animal medicine three distribution centers: the german resource center for genome research (rzpd; http://www.rzpd.de), the komp repository (https://komp.org), and the canadian mouse consortium (cmc; http://www.mousecanada. ca/index.htm). the repositories are all linked to the imsr, and provide access to mice, germplasm, genomic detail, and phenotypic data. genetic, genomic, and biological data are also available through the international mouse phenotyping consortium (impc; www.mousephenotype. org) and the mouse genome database (mgd; http://www. informatics.jax.org) (eppig et al., 2012) . inbreeding is a fundamental genetic tool applied to the laboratory mouse and detailed information is available on the web (table 3 .2). the first inbred strain (dba) was developed by c.c. little in 1909, with the subsequent creation of over 1000 inbred strains and stocks of mice (festing, 1996) . genetic origins, basic characteristics, references, and breeding performance of inbred strains of mice are available through michael festing's online version of inbred strain characteristics (http:// www.informatics.jax.org/external/festing/mouse/ strains.shtml). overviews of genetic manipulation for the creation of different types of mice are available (lyon et al., 1996; silver, 1995) . inbred mouse lines are termed strains, and are achieved by 20 or more brother × sister (filial; f) generations (table 3 .3). mice within an inbred strain, for practical purposes, are genetically identical (syngeneic or isogenic) to other mice of the same strain and sex. because of residual heterozygosity, a strain is not fully inbred until after 60 f generations. most commonly used inbred mouse strains represent 200 or more f generations, providing a high degree of experimental reproducibility. the mouse genome is not static, so when branches of an inbred strain are separated, spontaneous mutations, residual heterozygosity, and retroelement integrations result in genetic differences. therefore, if branches of an inbred strain are separated before f 40 , if branches have separated for 100 generations, or if genetic differences arise, the different branches become substrains. the same holds true if branches of a substrain diverge, resulting in substrains of the inbred substrain. when two inbred mouse strains are crossed, the f 1 hybrids are genetically identical to one another (isogenic), but maximally heterozygous (with chromosomes of each chromosomal pair separately contributed by each parental strain), whereas f 2 hybrids are maximally genetically diverse from one another (with chromosomes of both chromosomal pairs containing a mixture of contributions from each parental strain). with each subsequent f generation, mice once again approach inbred status. this technique is used for creating recombinant inbred (ri) strains. ri strains are sets of inbred strains of mice derived from crossing two inbred strains, and developed by singlepair random matings of sibling mice from the f 2 generation, thereby creating separate breeding lines. each line created is maintained separately, and then propagated by brother-sister matings for 20 generations, with each line becoming a separate inbred strain, but belonging to a set of ri strains. ri mice are useful for mapping phenotypic or quantitative traits that differ between the progenitor strains (bailey, 1971) . ri sets are generally limited to two parental strains. an ongoing international effort has been undertaken to increase allelic diversity among ri strains by creating the collaborative cross (cc) in which a panel of ri strains are being generated mixing the genomes from eight disparately related inbred (octo-parental) mouse strains, including a/j, c57bl/6j, 129s1/svimj, nonobese diabetic (nod)/shiltj, nzo/ hlltj, cast/eij, pwk/phj, and wsb/eij. these eight strains capture nearly 90% of the known genetic variation present among laboratory mice. future applications of the cc will utilize ri intercrosses of pairs of ri cc lines (threadgill and churchill, 2012; welsh et al., 2012) . recombinant congenic strains are sets of inbred strains derived in a manner similar to that for ri sets, except that one or more backcrosses to one parental strain (designated the background strain) are made after the f 1 generation, before inbreeding is begun. the other parental strain is designated as the donor strain. the proportion of background and donor genomes is determined by the number of backcrosses preceding inbreeding (demant and hart, 1986) . advanced intercross lines (ails) are another type of ri lines. they are made by producing an f 2 generation between two inbred strains and then, in each subsequent generation, intercrossing mice but avoiding sibling matings. the purpose is to increase the possibility of recombination between tightly linked genes. when a mutation arises spontaneously or is induced within an inbred strain, that mutant mouse becomes co-isogenic with the parental inbred strain, being virtually identical except for the single mutant allele. frequently, a mutation that arose in one inbred strain may be desired within the genetic background of another inbred strain. this can be accomplished by backcrossing, in which an f 1 hybrid is created by mating the donor mutant strain to the desired background strain, with subsequent matings to the background strain while retaining the mutant locus. after 10 backcross generations (n generations), the mutant mouse line is now congenic to the background inbred strain. backcrossing to create congenic strains of mice has been used extensively when targeted mutations have been induced in 129 embryonic stem cells, with backcrossing onto c57bl/6 inbred mice. congenic mice are never co-isogenic, as the preserved locus in a congenic mouse is invariably surrounded by flanking dna, which may significantly influence phenotype (linder, 2006) . in contrast to inbred mice, outbred mice are genetically heterogeneous and are maintained by breeding systems that intentionally minimize inbreeding. outbred mice are called stocks, which are defined as a closed population (for at least four generations) of genetically variable mice that are bred to maintain maximal heterozygosity. outbred mice may be used when high genetic heterogeneity is desired or for experiments requiring large numbers of mice. outbreeding can be achieved only in a large breeding population using a systematic breeding scheme, or randomized selection of breeders from the population. a small breeding population or passage through the genetic 'bottleneck' of rederivation to improve health status will reduce genetic heterogeneity and lead eventually to some degree of inbreeding. in a population of 25 breeding pairs, e.g., heterozygosity will decrease at 1% per generation with standard randomization techniques. random breeding involves the statistically random selection of breeders by using a random numbers table or computer program. an outbreeding program that is easy to manage is the circular pair mating system, in which each pair is mated only once. conceptually, cages are visualized in a circle, and each cage contains one breeding pair in the nth generation. another 'circular' set of cages serves as the breeding nucleus for the n + 1 generation. each mated pair in the nth generation contributes one female and one male to the n + 1 generation. outbreeding is accomplished by assigning the female and male derived from each nth generation cage to different cages in the n + 1 generation. most outbred mouse stocks are of 'swiss' origin, derived from nine mice imported to the united states in 1926, and are therefore quite homogeneous genetically (chia et al., 2005) . various lines of these mice have been maintained at different institutions, giving rise to numerous closely related stocks. although considered outbred, they have a high degree of homozygosity, exemplified by the fact that many swiss mouse stocks are blind due to the homozygous recessive rd1 allele (serfilippi et al., 2004b) . it is preferable to ensure genetic heterogeneity by intercrossing multiple inbred strains to achieve heterogeneity with known genetic input. in that regard, the diversity outbred mouse has been developed, which is a heterogeneous stock derived from the same eight founder inbred strains of the cc . additional types of inbred mice are utilized in research, including consomic and conplastic strains. consomic strains, also known as chromosome substitution strains, are inbred mice that are congenic for entire chromosomes, and are useful for studying polygenic traits (singer et al., 2004) . conplastic mice are inbred mice that are congenic for different mitochondrial genomes (mtdna) contributed by other inbred strains, other subspecies, or other species of mus (yu et al., 2009 ). in addition to spontaneously occurring mutations that are maintained as co-isogenic strains (such as the c57bl/6 beige mouse), mutant lines of mice have been created by radiation mutagenesis, chemical mutagenesis, or transgenesis. radiation was one of the earlier methods for in vivo mutagenesis (silver, 1995) , but in vitro radiation of embryonic stem (es) cells is also performed (thomas et al., 1998) . chemical mutagenesis involves in vivo treatment of male mice or in vitro treatment of es cells with mutagenic chemicals such as ethylmethanesulphonate (ems) or n-ethyl-n-nitrosourea (enu) , which induce point mutations in dna (o'brien and frankel, 2003; justice et al., 1999 justice et al., , 2000 . technically, a transgenic mouse is any mouse in which foreign dna has been integrated into its genome, regardless of method. however, the term transgenic commonly refers to mice that are genetically altered by additive transgenesis through microinjection of foreign dna into the pronucleus of a fertilized egg. each ensuing embryo results in a genetically different founder mouse, since the transgene is integrated in random sites of the genome of each founder mouse. since the injected dna is not homologous to the mouse genome and is not an allele, transgenic founders are hemizygous (rather than heterozygous) for the transgene until the mice carrying the transgene are bred into homozygosity for the transgene. transgenes typically integrate as tandem repeats, copy numbers affect phenotype of each founder, and may be lost in subsequent generations, thereby changing the phenotype of the mouse line (tinkle and jay, 2002) . transgenes are often constructed with an upstream promoter, which confers widespread (ubiquitous) or tissuespecific expression of the cdna, so that the transgene expression pattern reflects the expression pattern of the promoter. transcriptional regulation of the transgene can be inducible by drug-dependent regulatory control, such as the widely used tetracycline (tet) regulatory system, in which treatment of mice with tetracycline or doxycycline induces up-or down-regulation of the transgene (jaisser, 2000) . es cells are used for the less efficient integration of genetic material by homologous dna recombination, but allow large-scale screening of es cell clones for transformation. integration can be achieved in a random fashion by gene trapping, or by targeted mutation. both methods involve homologous dna recombination. gene trapping is a high-throughput approach that randomly introduces insertional mutations within the genome. vectors contain a gene trapping cassette with a promoter-less reporter gene and/or selectable genetic marker flanked by an upstream 3′ splice site and a downstream termination sequence. when inserted into an laboratory animal medicine intron of an expressed gene, the gene trap is transcribed from the endogenous promoter of that gene. gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a dna tag for the rapid identification of the disrupted gene (skarnes et al., 2011) . targeted gene mutations are achieved by homologous recombination of specific sites within the genome of es cells. homologous sequences flank the upstream and downstream regions of the targeted gene, and the construct between the flanking sequences may inactivate (knock out) or replace (knock in) a gene, and typically contains a reporter gene to track the integration. a variation on this approach is site-specific recombinase (ssr) technology. two of the most common recombinases are cre from the coliphage p1 and flp from saccharomyces cerevisiae. cre and flp mediate recombination between target sites, termed loxp and frt, respectively. for example, cre loxp target sites are engineered to flank the gene target, which can be used in different ways to achieve different outcomes (conditional mutations), depending upon the orientation and location of the flanking loxp sites. if the loxp sites are oriented in opposite directions, cre recombinase mediates inversion of the floxed segment. if the loxp sites are on different chromosomes (trans), cre recombinase mediates a chromosomal translocation. if the loxp sites are oriented in the same direction on the same chromosome (cis), cre recombinase mediates deletion of the floxed segment. once the floxed mutation is created in es cells, the transformed es cells are developed into a mouse with the conditional mutation. the conditional mutant mouse is then genetically crossed with a cre transgenic mouse, in which cre recombinase is under the control of a ubiquitous or tissue-specific promoter. wherever and whenever cre is expressed, cre recombinase will recognize and recombine the loxp sites. this approach can include insertion of reporter genes and selectable markers, and can be under the control of inducible gene expression systems (http:// www.eucomm.org/docs/protocols/mouse_protocol_1_ sanger) (nagy, 2000) . es cells are pluripotent with the full genetic capacity to develop into mice when implanted into the blastocyst of a developing embryo. interest in 'embryonal carcinomas' (teratomas) that arose in relatively high frequency in the testes of 129 mice and early gene transfer experiments in the late 1970s and early 1980s led to the development of es cell lines derived from several different 129 strains. this early emphasis on teratomas prompted creation of 'better' 129 mouse lines that were more prone to development of testicular teratomas, resulting in genetic corruption of the 129 mouse (simpson et al., 1997; threadgill et al., 1997) . this realization gave rise to the need to revise 129 mouse nomenclature (festing et al., 1999) . this was necessary because genetic variation significantly impacts homologous recombination in order to match genome sequence of the es cell line with the mouse from which it was derived. es cells can be created from any mouse strain or hybrid, but 129 es cell lines have been commonly used. recent international knockout mouse program efforts use c57bl/6n es cells. transformed es cells are microinjected into the inner cell mass of recipient blastocysts, which are then implanted into the uteri of pseudopregnant surrogate mothers. the pups that are born are composed of a mixture of cells derived from recipient blastocysts and the transformed es cells (chimeras). the goal is for male chimeric progeny to produce spermatozoa of es cell origin (containing the mutation), in order to create f 1 progeny by mating the chimera with the desired background strain (http://www.eucomm.org/docs/protocols/mouse_protocol_1_sanger). for this reason, most es cell lines are xy, which favors 129 male chimerism. if the es cells are of 129 (or other) strain origin, the chimeras are often bred to a desired background mouse strain (commonly c57bl/6) and backcrossed for n10 generations, thereby creating congenic inbred mouse lines. recent international knockout mouse efforts utilize c57bl/6n es cells, so that chimeric males are bred directly with c57bl/6 mice, thereby creating co-isogenic lines. the latter approach saves time and money, and creates a more genetically refined mutant mouse. an alternate approach is to allow es cells to aggregate with a developing embryo to form blastocysts in culture (aggregation chimera), then implant the chimeric blastocysts (tanaka et al., 2001) . rna interference (rnai), which functions through short double-stranded rna (dsrna), has also been utilized to produce transgenic mice, known as gene knockdown mice (gao and zhang, 2007; peng et al., 2006) . the dsrna is enzymatically processed into small molecules, termed small interfering rna (sirna), which find homologous target mrnas, resulting in interference. this phenomenon is believed to be a self-defense mechanism against viral infection. in order to adapt this approach to generation of transgenic mice, small hairpin rna (shrna) can be expressed in the same way as other transgenes in mice, resulting in processing of the shrna into sirna with gene-silencing effects. constructs are introduced into mouse es cells by electroporation or lentiviral infection. this method can be embellished conditionally, as with other transgenes. although rnai knockdown mice are genetically stable, rnai-mediated transgenesis is never complete, has variable tissue expression, and cannot induce point mutations (peng et al., 2006) . recent advances in engineered endonuclease (ee) technology, including zinc finger nucleases (zfns), laboratory animal medicine transcription activator-like effector nucleases (talens), and rna-guided endonucleases (rgens), have revolutionized the field of transgenics (sung et al., 2014; wijshake et al., 2014; gaj et al., 2013) . zfns and talens consist of engineered proteins that target dna fused to the nonspecific endonuclease, fok1 (cathomen and joung, 2008; joung and sander, 2013) . zfns are comprised of three to six tandem zinc finger proteins, each of which targets a specific 3 bp nucleotide sequence. paired zfns are generated, with each half of the pair targeting opposite dna strands, allowing dimerization of fok1 which is required for introduction of double-stranded breaks (dsbs) in the dna of interest (cathomen and joung, 2008) . talens function similarly, but are composed of tandem repeats of 33-35 amino acids, each with nucleotide specificity occurring in two hypervariable amino acids, the 'repeat variable di-residue (rvd)', at positions 12 and 13 (joung and sander, 2013) . in contrast to zfns and talens, clustered regularly interspaced short palindromic repeats (crisprs) paired with crispr-associated (crispr/cas) systems are rgen systems that target specific dna sequences. cas proteins, rather than fok1, produce dsb (hsu et al., 2014) . dsb generated by ee are repaired by host cells by either nonhomologous end joining (nhej) or, less commonly, by homologous recombination (hr). nhej is an error-prone repair system and results in insertions or deletions (indels) with a relatively high frequency, which can result in gene disruption. hr is a less common repair pathway, but certain manipulations of the engineered nucleases can increase hr efficiency. for example, nucleases can be engineered to generate a break in a single strand of dna rather than inducing dsb, and the resulting nickases increase the incidence of hr with high fidelity (gaj et al., 2013; wijshake et al., 2014) . hr allows for the introduction of donor dna to generate knock-ins, specific point mutations, or for the generation of larger modifications such as insertions of loxp sites (brown et al., 2013; wijshake et al., 2014) . vectors encoding the ee can be injected into mouse embryos by pronuclear injection of dna, intracytoplasmic injection of rna, or transfection of mouse es cells (sung et al., 2014; wijshake et al., 2014) . one advantage of ee technologies over more traditional transgenic methods is the ability to target dna and induce mutations in any background strain of mouse negating the need to backcross onto the desired strain. multiple genes can be targeted with crisprs simultaneously, thus avoiding the need to cross single knockout animals (zhou et al., 2014) . in addition, it is possible to obtain bi-allelic mutations in some cases, allowing for the generation of functional gene knockout animals in a single generation (zhou et al., 2014; wijshake et al., 2014) . vectors for generating ee are available through plasmid repositories; websites are available to assist in identifying appropriate dna sequences to target; and multiple websites post protocols for generating the various types of engineered endonucleases (xie et al., 2014; sander et al., 2010; bae et al., 2014; reyon et al., 2011; herscovitch et al., 2012; wolfson, 2013) . crisprs tend to be particularly cost effective and easy to design, with minimal restrictions for targeting specific dna sequences. there are currently more than 1000 separate outbred stocks and traditional inbred strains, often with multiple substrains (table 3 .4). in addition, there are thousands of induced mutant strains. therefore, it is critical that strain or stock designations be complete and accurate to avoid semantic and genetic confusion, and to ensure reproducibility of research results. as an example of substrain variation that makes precise nomenclature important, cba/j mice are homozygous for the retinal degeneration allele (rd1), whereas cba/caj mice do not carry this allele. the international committee on standardized genetic nomenclature for mice and rats, established in the early 1950s, is responsible for genetic nomenclature rules. the rules are available online at the mgi website (http:// www.informatics.jax.org/mgihome/nomen). inbred mouse strains are designated by a series of capital letters and/or numbers, which often provide a shorthand description of the origin and history of the strain. the c57bl/6j mouse serves as an example. the inbred strain c57bl originated from abbie lathrop's female 57 (and male 52) at the cold spring harbor laboratory (c), and was the black (bl) line from this female. early in their history, inbred c57bl mice split into major substrains, e.g., c57bl/6 and c57bl/10. substrains are identified by appending a forward slash (/) after the inbred strain name. since 1950, uniform international nomenclature has been built upon these historical names, so that substrains of an inbred strain are now designated using lab codes that are registered in the international laboratory code registry maintained at the institute for laboratory animal research (ilar) of the national academies (dels. nas.edu/global/ilar/lab-codes). laboratory codes are composed of one to five letters that identify an institute, laboratory, or investigator. each lab code starts with an uppercase letter, followed by lowercase letters if more than one letter is used (such as n, j, jci, crl, and tac). the j in c57bl/6j means it is a substrain maintained at the jackson laboratory (j). another common substrain of c57bl/6 mice is c57bl/6n, which is maintained at nih (n). substrains can be cumulative, reflecting the genetic history of the mouse strain. for example, there are a number of c57bl/6j substrains (such as c57bl/6jjci and c57bl/6jjmsslc), and a number of c57bl/6n substrains (such as c57bl/6njci, c57bl/6ncrlcrlj, dba/2j inbred strain named for its characteristic coat color genes (using their original gene symbols), dilute (d), brown (b), and nonagouti (a); it is the second of two sublines separated before 20 generations of brother × sister breeding and is the subline maintained at the jackson laboratory (j) c3h/hesn-ash/+ co-isogenic segregating inbred mutant strain carrying the ashen (ash) mutation, which arose on c3h/hesn c57bl/6j-tyrc-2j /+ co-isogenic segregating inbred mutant strain carrying the albino 2j mutant allele of the cloned tyrosinase gene (tyr) aej/gnj-a e /a w-j inbred strain segregating for two alleles at the agouti gene congenic inbred strain in which the b haplotype at the h2 complex was transferred from c57bl/6j (b6) to the akr background b6.cba-d4mit25-d4mit80 congenic strain in which the chromosomal segment between d4mit25 and d4mit80 was transferred from cba to b6 b6.cg m lepr db /++ congenic inbred strain in which the linked mutant genes misty (m) and diabetes (lepr db ) were transferred from multiple, mixed, or unknown genetic backgrounds to b6 and are carried in coupling, i.e., on the same chromosome b6.cg-m +/+ lepr db congenic inbred strain in which the m and lepr db mutations are carried in repulsion bxd-1/ty recombinant inbred (ri) strain number 1 in a set of ri strains derived from a c57bl/6j (b) female mated to a dba/2j (d) male and made by taylor (ty) recombinant congenic (rc) strain number 1 in a set made by crossing the balb/c (c) and sts (s) strains, backcrossing one or two times to balb/c and then inbreeding as with ri strains and c57bl/6ntac). significant differences may exist among these substrains (mekada et al., 2009 ). thus, a string of substrain designations indicate the genetic progression of the substrain, which can be identified when reading the entire strain name. this nomenclature is highly nuanced, as c57bl/6ncrlcrlj mice, whose last letter is a lowercase j, are not a substrain maintained at the jackson laboratory (j), but rather at charles river japan (crlj), underscoring the importance of upper-and lowercase lettering in rodent nomenclature. balb/c mice are another popular inbred strain with numerous substrains. like the '6' in c57bl/6, the 'c' that follows laboratory animal medicine of the mutational event as a superscript. for example, cftr tm1unc is a targeted mutation (tm), first line (1) congenic mice are often derived from 129 es cells, backcrossed onto a background strain, such as c57bl/6. under such circumstances, when the backcross generation is at n10, the '.' symbol is used between the background inbred strain and the donor strain (e.g., c57bl/6n.129p2/olahsd-abc tm1zzz , abbreviated as b6.129-abc tm1zzz . when backcrossing is incomplete but at the n5 generation, the mouse is an incipient congenic, designated with a ';' in lieu of a '.': b6;129-abc tm1zzz . if the background strain is mixed genetic origin, it is designated stock.129-abc tm1zzz . if the donor strain is mixed origin, it is designated 'cg'. for example, b6.cg-abc tm1zzz outbred stock that meets specific criteria is designated by placing the lab code before the stock symbol, separated by a full colon (':'). for example, hsd:icr designates an icr (swiss) outbred stock maintained by harlan sprague dawley (hsd). the above overview covers the nomenclature of commonly encountered types of mice. there are numerous additional specifications for nomenclature of mice. details are available at the mgi website (http://www. informatics.jax.org/mgihome/nomen). optimum housing conditions and husbandry practices for research mice should be guided by program requirements to ensure biosecurity, occupational health, efficient use of equipment, labor and financial resources, behavioral needs of mice, and investigator needs for consistent colony maintenance, including standardized husbandry practices and nutrition. the emerging interest in the mouse microbiome in combination with the immune competency of diverse genetically engineered mouse strains demands high standards of mouse care. mouse colonies are optimally maintained as specificpathogen-free (spf) which obligates veterinary and facility management to exclude specific organisms. housing options for spf immunocompetent mice typically include static or individually ventilated microisolator cages, which differ significantly in cost and labor required to maintain. severely immunodeficient strains such as nod.cg-prkdcscid il2rgtm1wjl/szj (nsg) mice require staff training, caging systems and husbandry practices that minimize risk for opportunistic infections the '/' in balb/c is a lowercase letter because of historical precedent. subsequent substrains follow accepted nomenclature, e.g., balb/cbyj and balb/cann. hybrids of two inbred strains are often used in research, and are particularly common with engineered mutations that are created in 129-derived es cells, followed by intercrossing the 129 chimeric mice with c57bl/6 or other background strains of mouse. when an f 1 hybrid is created, the female partner is listed first, e.g., a c57bl/6j × 129s2/svpas hybrid would be designated: c57bl/6j129s2/svpasf1. ri strain sets that are derived from two parental inbred strains are identified by an x between the two parental strains followed by a hyphen designating the specific ri line, e.g., c57bl/6jxdba/2j-1, c57bl/6jxdba/2j-2, etc. cc ri strains do not use the x between the parental strains because they are derived from eight parental strains, so they are designated cc-1, cc-2, etc. in order to simplify the complexity of this nomenclature, abbreviations are used for common inbred strains and substrains of mice (table 3 .4), but it is important to include the full genetic nomenclature in publications. using the abbreviated nomenclature, c57bl/6j129s2/svpasf1 mice would be b6129f1 and c57bl/6jxdba/2j-1 ri mice would be bxd-1. parental order is an important consideration in nomenclature, as a b6129 mouse is genetically different from a 129b6 mouse due to mitochondrial dna (from the female) and y chromosome (from the male) differences. mutant genes are designated by a brief abbreviation for the mutation (e.g., bg for beige which arose at the jackson laboratory, j). the symbol for the parent gene is noted in italics, starting with an uppercase letter (e.g., lyst) and the mutant allele is designated in superscript (e.g., lyst bgj ). thus, the beige mutation arose in c57bl/6j mice, so that c57bl/6j beige mice, which are co-isogenic with c57bl/6j mice, are designated c57bl/6j-lyst bgj . a transgenic strain is designated by the strain and substrain name, followed by a symbol for the transgene. transgene symbols take the form tg(yyy)#zzz, where 'tg' indicates transgenic, yyy defines the transgene as a brief description of the inserted dna (such as a gene symbol), '#' is the assigned number in the series of events generated using a given construct, and 'zzz' is the lab code. for example, fvb/n-tg(mmtv-erb2)1led mice are inbred fvb/n mice in which the rat erb2 gene was introduced under control of the mouse mammary tumor virus (mmtv) ltr promoter (mmtv-erb2), the first line (1) created in the laboratory of phil leder (lab code led). when a transgene causes an insertional mutation in an identified endogenous gene, the mutant allele of the gene is designated by using the gene symbol and an abbreviation for the transgene as a superscript (-abc tg1zzz ). a targeted mutation, or knockout, is designated by the mutated gene with the identification laboratory animal medicine (foreman et al., 2011) . barrier practices and microisolator techniques may include autoclaved or irradiated feed and bedding, autoclaved or acidified water, cage-tocage transfer of mice using disinfected forceps, positive displacement change hoods, and verified sanitation of caging and equipment through tunnel or rack washers to prevent fomite transmission of infectious agents (compton et al., 2012) . in addition to husbandry staff, it is critical to maintenance of colony health status that investigators who handle cages are also trained in these techniques. the microenvironment for mice is the cage which will vary in design, size, and composition. vendors often successfully house production colonies in open-top cages to expedite detection of pathogen transmission should a break occur. end-users usually prefer filter-top microisolator cages which prevent (at least) gross contamination between cages by fecal contamination and aerosolized debris. the objective is to keep mice in an uncrowded, socially compatible, low-odor, dry and clean environment. ambient temperature should minimize any confounding impact on the animal model and energy expenditure for the mice, while also being suitable for staff and investigators. shoebox static cages made of polycarbonate, polypropylene, or polystyrene plastic (in order of decreasing cost and durability) with filtered microisolator tops continue to be used for housing and breeding mice. older cage designs are being rapidly supplanted by individually ventilated caging systems that promote the advantages of increasing housing capacity, decreasing labor costs, and mitigating exposure of mice to noxious gases such as ammonia and exposure of humans to allergens. as more advanced caging systems are developed, the level of biosecurity may be increased but at the cost of increased health surveillance efforts to detect the source of an infectious outbreak (shek, 2008) . disposable, recyclable polyethylene caging is a recent innovation, particularly for facilities not equipped with a cage wash facility. animal care programs should carefully consider the necessity for housing mice on wire-mesh flooring because of injury risk to limbs and thermoregulation issues in neonates and hairless mice which are more difficult to maintain without nesting material. solidbottom cages should contain sanitary bedding, such as hardwood chips, paper products, or ground corn cob. criteria for selecting bedding vary with experimental and husbandry needs. it may be preferable to irradiate or autoclave bedding, but if this is not done, the bedding should be used only after its origin and microbial content have been evaluated (table 3 .5). germfree and gnotobiotic mice require positive pressure isolators, most usually flexible film, with additional protection provided by sterile air through high-efficiency particulate air (hepa) filters. this equipment can be negatively pressurized when the objective is to contain known or unknown pathogens. animal care programs should establish enrichment policies which for mice should include social housing when mice are compatible and experiments do not require single housing. species-specific behaviors are encouraged by nesting material and hiding places such as tubes or shacks. nutrient requirements for the mouse are influenced by genetic background, disease status, growth rate, pregnancy, lactation, and environmental factors such as ambient temperature. the best current estimate of nutritional requirements is shown in table 3 .6. nutritional requirements for laboratory mice are also published periodically by the national research council and have been reviewed by knapka and coworkers (knapka et al., 1974; knapka, 1983) . feed intake and weight gain data are used to estimate the nutritional needs of a particular stock or strain. mice consume about 3-5 g of feed per day after weaning, and maintain this intake throughout life. outbred mice tend to gain weight faster than inbred mice and are heavier at maturity (figs. 3.1 and 3.2). diet is often neglected as a variable in animal-related research. diet can influence responses to drugs, chemicals, or other factors and lead to biased research results. therefore, diet must provide a balance of essential kraft (1980) . knapka (1983) . b linoleic acid: 0.6% is adequate. c john and bell (1976) . d theuer (1971) . e knapka et al. (1974 ). f nutrition (1977 . g hurley and bell (1974) . h pleasants et al. (1973) . nutrients, and contaminants must be kept to a minimum (see also chapter 29). natural-product commercial diets for mice are usually satisfactory for breeding and maintenance. animal care programs should avoid using fresh produce, grains, fish meal, or other supplements to minimize exposure of colonies to pathogens or harmful chemicals such as pesticide residues or phytoestrogens (guerrero-bosagna et al., 2008) . mouse diets can be purchased as open-formula, fixedformula, constant nutrition, and closed-formula which laboratory animal medicine are designed to reduce variation in experimental data attributable to diet (reviewed in barnard et al. (2009) ). diets are supplied in standard, irradiated, or autoclavable formulations. irradiated diets will be virtually free of live microorganisms but have the risk of residual, radio-resistant bacteria. autoclavable diets are higher in heat-labile nutrient content. many programs use sterilized mouse chow exclusively to minimize risk of opportunistic infections. because commercial diets vary in nutrient content, diets should be selected for optimal maintenance of adult mice or for growth and reproduction in breeding colonies. mice should have continuous access to potable water even if a high-moisture diet is fed. water is needed for lubrication of dry food and for hydration. adult mice drink 6-7 ml of water per day. decreased water intake will decrease food consumption. water imbalance may occur immediately post weaning and weanlings on automatic watering systems need extra attention. water intake will decrease in sick mice. therefore, dosing mice with medicated water requires careful assessment of hydration and clinical or experimental efficacy of the compound administered. the main reference used to update this section of the 3rd edition is volume iii; normative biology, husbandry and models in the mouse in biomedical research, 2nd edition, aclam series published by academic press. normative data on the mouse are presented in table 3 .7, and clinical chemistry reference ranges are summarized in table 3 .8. mice have a relatively large surface area per gram of body weight. this results in dramatic physiologic changes in response to fluctuations in the ambient temperature (t a ). the mouse responds to cold exposure, e.g., by nonshivering thermogenesis. a resting mouse acclimated to cold can generate heat equivalent to triple the basal metabolic rate, a change that is greater than for any other animal. a mouse must generate about 46 kcal/m 2 per 24 h to maintain body temperature for each 1°c drop in t a below the thermoneutral zone. mice cannot tolerate nocturnal cooling as well as larger animals that have a greater heat sink. therefore, it is not advisable to conserve energy in animal quarters at night by lowering t a . because of the ratio of evaporative surface to body mass, the mouse has a greater sensitivity than most mammals to water loss. its biological half-time for turnover of water (1.1 days) is more rapid than for larger mammals. water conservation is enhanced by cooling of expired air in the nasal passages and by highly efficient concentration of urine. the conservation of water can preempt thermal stability. if the mouse had to depend on the evaporation of body water to prevent elevations of body temperature, it would go into shock from dehydration. the mouse has no sweat glands, it cannot pant, and its ability to salivate is severely limited. mice can partially compensate for changes in t a increases from 20°c to 35°c. it adapts to moderate but persistent increases in environmental temperature by a persistent increase in body temperature, a persistent decrease in metabolic rate, and increased blood flow to the ears to increase heat loss. its primary means of cooling in the wild is behavioral -retreat into a burrow. in the confinement of a cage, truck, or plane, mice do not survive well in heat and begin to die at an ambient temperature of 37°c or higher. thus, the mouse is not a true endotherm. in fact, the neonatal mouse is ectothermic and does not have well-developed temperature control before 20 days of age. the thermoneutral zone for mice varies with strain and with conditioning but is about 29.6-30.5°c, narrower than that of any other mammal measured thus far. thermoneutrality should not be equated with comfort or physiological economy. recent data have suggested that mice housed under routine vivarium conditions are chronically cold-stressed. mice maintained at 21°c were shown to expend more energy compared with mice housed at intermediate (26°c) and a higher temperature (31°c) with an increase in glucose utilization and activation of brown adipose tissue (david et al., 2013) . in contrast, other studies report that mice in a t a range of 21-25°c grow faster, have larger litters, and have more viable pups than those maintained in the thermoneutral zone. the respiratory tract has three main portions: the anterior respiratory tract consists of nostrils, nasal cavities, and nasopharnyx; the intermediate section consists of larynx, trachea, and bronchi, all of which have cartilaginous support; and the posterior portion of the respiratory tract consists of the lungs. the left lung is a single lobe. the right lung is divided into four lobes: superior, middle, inferior, and postcaval (cook, 1983) (fig. 3.3 loeb and quimby (1999) . a mouse at rest uses about 3.5 ml o 2 /g/h, which is about 22 times more o 2 /g/h than is used by an elephant. to accommodate for this high metabolic rate, the mouse has a high alveolar p o 2 ; a rapid respiratory rate; a short air passage; a moderately high erythrocyte (rbc) concentration; high rbc hemoglobin and carbonic anhydrase concentrations; a high blood o 2 capacity; a slight shift in the o 2 -dissociation curve, enabling o 2 to be unloaded in the tissue capillaries at a high p o 2 ; a more pronounced bohr effect, i.e., the hemoglobin affinity for o 2 with changes in ph is more pronounced; a high capillary density; and a high blood sugar concentration. the kidneys, ureters, urinary bladder, and urethra form the urinary system. the paired kidneys lie against the dorsal body wall of the abdomen on either side of the midline. the right kidney is normally located anterior to the left kidney. kidneys from males of many inbred strains are consistently heavier than kidneys from females. the glomeruli of mice are small, about 74 μm in diameter, or about half the size of glomeruli in rats. there are, however, 4.8 times as many glomeruli in the mouse, and the filtering surface per gram of tissue is twice that of the rat. mice excrete only a drop or two of urine at a time, and it is highly concentrated (table 3 .8). the high concentration is made possible by long loops of henle and by the organization of giant vascular bundles (vasa recta) associated with the loops of henle in the medulla. the mouse can concentrate urine to 4300 mosm/l, whereas humans can concentrate to a maximum of 1160 mosm/l. mice normally excrete large amounts of protein in the urine. taurine is always present in mouse urine, whereas tryptophan is always absent. creatinine is also excreted in mouse urine, a trait in which mice differ from other mammals. the creatinine/creatine ratio for fasting mice is about 1:1.4. mice excrete much more allantoin than uric acid. the submaxillary salivary gland, a mixed gland in most animals, secretes only one type of saliva (seromucoid) in the mouse. the tubular portion of the gastrointestinal (gi) tract consists of esophagus, stomach, small intestine, cecum, and colon. the esophagus of the mouse is lined by a thick cornified squamous epithelium, making gavage a relatively simple procedure. the proximal portion of the stomach is also keratinized, whereas the distal part of the stomach is glandular. gastric secretion continues whether or not food is present. the gastrointestinal flora consists of (at least) 1000 species of bacteria that begin to colonize the alimentary canal selectively shortly after birth. the ceca of normal mice contain up to 10 11 bacteria/g of feces. the bacteria throughout the gastrointestinal tract form a complex ecosystem that provides beneficial effects, such as an increase in resistance to certain intestinal pathogens, production of essential vitamins, and homeostasis of important physiological functions. gnotobiotic animals colonized with known microbiota have been used to great advantage as models for biomedical research (see chapter 39). for certain studies, it is desirable to colonize germfree mice with a defined microbiota. in the mid-1960s, schaedler was the first to colonize germfree mice with selected bacteria isolated from normal mice (schaedler and orcutt, 1983) . he subsequently supplied animal breeders with this group of microorganisms. these defined bacteria included aerobic bacteria and some less oxygen-sensitive anaerobic organisms. the so-called extremely oxygen-sensitive (eos) fusiform bacteria, which make up the majority of the normal microbiota of rodents, were not included, because of technical difficulties in isolation and cultivation. of the defined microbiotas later used for gnotobiotic studies, the one known as the 'schaedler flora' was the most popular. in 1978, the national cancer institute (nci) decided to revise the schaedler flora, or 'cocktail' consisting of eight bacteria, in order to standardize the microbiota used to colonize germfree rodents. the new defined microbiota, now known as the 'altered schaedler flora' (asf), consisted of four members of the original schaedler flora (two lactobacilli, bacteroides distasonis, and the eos fusiform bacterium), a spiral-shaped bacterium, and three new fusiform eos bacteria. studies have quantified the regional colonization of the asf strains along the gastrointestinal tract (sarma-rupavtarm et al., 2004) (fig. 3.4) . individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant lactobacillus murinus asf361 present at 10 5 -10 7 cells/g of tissue in the upper gastrointestinal tract and obligate anaerobic asf strains being predominant in the cecal and colonic flora at 10 8 -10 10 cells/g of tissue. it is difficult to monitor a gnotobiotic mouse colony with a defined microbiota. it is necessary to demonstrate that microorganisms of the specified microbiota are present and that adventitious microorganisms are absent. in the past, monitoring relied on bacterial morphology, limited evaluation of biochemical traits, and growth characteristics. with the advent of polymerase chain reaction (pcr) technology, the eight asf strains were identified taxonomically by 16s rrna sequence analysis . three strains were previously identified as lactobacillus acidophilus (strain asf 360), l. salivarius (strain asf 361), and bacteroides distasonis (strain asf 519), based on phenotypic criteria. 16s rrna analysis and genome sequencing indicated that each of the strains differed from its presumptive identity (wannemuehler et al., 2014) . the 16s rrna sequence of strain asf 361 is essentially identical to the 16s rrna sequences of the type strains of l. murinus and l. animalis (both isolated laboratory animal medicine from mice), and all of these strains probably belong to a single species. strain asf 360 is a novel lactobacillus that clusters with l. acidophilus and l. lactis. strain asf 519 is a parabacteroides sp. the spiral-shaped strain, strain asf 457, is in the flexistipes phylum, exhibits sequence identity with rodent isolates of robertson, and has been formally named, mucispirillum schaedleri (robertson et al., 2005) . the remaining four asf strains, which are eos fusiform bacteria, group phylogenetically with the low-g + c content gram-positive bacteria (firmicutes, bacillus-clostridium group) (asf 492 -eubacterium plexicaudatium; asf 500 -firmicutes bacterium; asf 502 and asf 356 -clostridium sp.) (fig. 3.5) . the 16s rrna sequence information was determined by dewhirst et al. (1999) and draft genome sequences for each member of asf were recently published (wannemuehler et al., 2014) . this genetic data will permit detailed analysis of the interactions of asf organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms. the lymphatic system consists of lymph vessels, thymus, lymph nodes, spleen, solitary peripheral nodes ( fig. 3.6) , and intestinal peyer's patches. mouse lymph nodes are numerous but typically are small, reaching only a few millimeters. the typical lymph node is beanshaped and consists of a cortex and a medulla. the cortex is divided into b lymphocyte domains, called primary follicles, and t lymphocyte domains, known s2 i1 i2 i3 i4 i5 i6 c1 c2 l1 l2 l3 e1 s1 s2 i1 i2 i3 i4 i5 i6 c1 c2 l1 l2 l3 e1 s1 s2 i1 i2 i3 i4 i5 i6 c1 c2 l1 l2 l3 e1 s1 s2 i1 i2 i3 i4 i5 i6 c1 c2 l1 l2 section of the gi tract bacteroides sp. asf519 the sections are taken from the esophagus (esop.) (section e1), stomach (sections s1 and s2), small intestine (sections i1 to i6), ileocecal junction and apical cecum (sections c1 and c2, respectively), and colon (sections l1 to l3). from sarma-rupavtarm et al. (2004). as the diffuse cortex. the mouse does not have palatine or pharyngeal tonsils. the spleen lies adjacent to the greater curvature of the stomach. different strains of mice have varying degrees of accessory splenic tissue. age, strain, sex, and health status can affect the size, shape, and appearance of the spleen. male spleens, e.g., may be 50% larger than those of females. most lymphocytes enter and leave the spleen in the bloodstream. the so-called white pulp of the spleen is organized along the central arteriole and is subdivided into t-and b-cell zones. the periarteriolar sheath is composed mainly of cd4 + and cd8 + t cells, and lymph follicles, which often contain germinal centers, are located at the periphery. the red pulp consists of sinusoids and hemoreticular tissue. cellular and humoral components of immunity are distributed to the bloodstream and tissues by efferent lymphatic vessels and lymphatic ducts, which empty into the venous system. the thymus is a bilobed lymphoid organ lying in the anterior mediastinum. it reaches maximum size around the time of sexual maturity and involutes between 35 and 80 days of age. the thymus plays a major role in maturation and differentiation of t lymphocytes. this function is not complete in newborn mice. thymectomy is routinely performed in immunological research for experimental manipulation of the immune system. thymectomy of newborn mice causes a decrease in circulating lymphocytes and marked impairment of certain immune responses, particularly cellular immune responses. thymectomy in adult mice produces no immediate effect, but several months later mice may develop a progressive decline of circulating lymphocytes and impaired cellular immune responses. the mutant athymic nude mouse is a powerful experimental tool in the study of the thymus in immune regulation (fogh, 1982) . the mucosa-associated lymph tissue (malt) contains more lymphoid cells and produces greater amounts of immunoglobulin than both the spleen and the lymph nodes. the term malt designates all peripheral lymphoid tissues connecting to cavities communicating with the external milieu. they include the peyer's patches, the cecal lymphoid tissue, and the lymphoid tissue in upper and lower respiratory tract, as well as the respiratory and genitourinary system. lymphatics drain these lymphoid-rich areas, thus providing a direct link with lymph nodes and the bloodstream. bone marrow and splenic red pulp produce erythrocytic, granulocytic, and megakaryocytic precursors over the life of the mouse. bone marrow is located in the protected matrix of cancellous bone and is sustained by reticular tissue rich in blood vessels and adipose cells (pastoret et al., 1998) . normal hematologic values are listed in table 3 .7. bone marrow-derived mononuclear phagocytes remove particulate antigens and act as antigen-presenting cells for lymphocytes. tissue macrophages, which often function in a similar way, are found in many tissues, including peripheral lymphoid tissues, lung, liver, intestine, and skin. the cardiovascular system of mice is reviewed extensively by hoyt et al. in the 2nd edition of volume iii; normative biology, husbandry and models in the aclam series the mouse in biomedical research (hoyt, 2007) . the heart consists of four chambers, the thin-walled atria and the thick-walled ventricles ( fig. 3.7) . mice conditioned to a recording apparatus have mean systolic blood pressures ranging from 84 to 105 mmhg. an increase in body temperature does not lead to an increase in blood pressure. heart rate, cardiac output, and the width of cardiac myofibers are related to the size of the animal. heart rates from 310 to 840/min have been recorded for mice, and there are wide variations in rates and blood pressure among strains. the skeleton is composed of two parts: the axial skeleton, which consists of the skull, vertebrae, ribs, and sternum, and the appendicular skeleton, which consists of the pectoral and pelvic girdles and the paired limbs. the normal vertebral formula for the mouse is c7t13l6s4c28, with some variations among strains, especially in the thoracic and lumbar regions. normal mouse dentition consists of an incisor and three molars in each quadrant. these develop and erupt in sequence from front to rear. the third molar is the smallest tooth in both jaws; the upper and lower third molar may be missing in wild mice and in some inbred strains. the incisors grow continuously and are worn down during mastication. the mouse brain has a typical mammalian structure as documented by a detailed study of the neuroanatomy of the c57bl/6j mouse (sidman et al., 1971) . more recently, gene expression patterns have been used to study the functional anatomy of the mouse brain (bohland et al., 2010) . use of wild-type and genetically modified mice in behavior, learning, and memory paradigms has exponentially increased over the last decade. the male reproductive organs consist of paired testes, urethra, penis, prostate and associated ducts and glands ( fig. 3.8) . the female reproductive organs consist of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands ( fig. 3.9 ). the clitoral glands are homologous to the male preputial glands and secrete a sebaceous substance through ducts entering the lateral wall of the clitoral fossa. the female mouse normally has five pairs of mammary glands, three in the cervicothoracic region and two in the inguinoabdominal region ( fig. 3 .10). the mammary glands are often not appreciated for how far they extend over the cervical, axillary, and inguinoabdominal flank regions which become the following section summarizes normal reproduction in the mouse. the reader is referred to a more comprehensive text in the aclam series (pritchett and taft, 2007) and online resources such as the jackson laboratories publication of the biology of the laboratory mouse (http://jaxmice.jax.org/jaxnotes/509/509j.html). external influences, such as noise, vibration, diet, light cycle, and cage density, and intrinsic factors, such as health status, genetics, and parity impact reproductive success by directly or indirectly influencing the hypothalamic-pituitary axis for hormonal control of ovarian and testicular function. genotype also dramatically affects the reproductive performance of the mouse. coincident with the explosion in the number of mouse strains, each with unique induced or spontaneous mutations, a sound breeding program must include training of care staff to recognize anticipated and unanticipated breeding performance and strain or stock characteristics. in the new age of genomics, older methods of confirming genetic purity of mouse lines are being replaced with formal genetic monitoring by comparing strain-specific panels of single-nucleotide polymorphisms (snps). follicle-stimulating hormone promotes gametogenesis in both sexes. luteinizing hormone promotes the secretion of estrogen and progesterone in the female and androgen in the male. prolactin promotes lactation and development of the ovary during pregnancy. these gonadal hormones also ensure proper maintenance of the reproductive tract and modulate behavior to promote successful mating. the hypophysis is usually responsive to hormonal influence by day 6 in the male and day 12 in the female. ovarian follicle development begins at 3 weeks of age and matures by 30 days. rising levels of gonadotropins evoke signs of sexual maturity at about the same age. in the female, estrogen-dependent changes such as cornification of vaginal epithelium at the vaginal opening can occur as early as 24-28 days. puberty is slightly later in the male (up to 2 weeks). sexual maturation varies among strains and stocks of mice and is subject to seasonal and environmental influences. mating behavior and the ability to conceive and carry fetuses to parturition are under complex hormonal control mediated by the anterior pituitary. the mouse is polyestrous and cycles every 4-5 days. in the first two phases (proestrus and estrus), active epithelial growth in the genital tract culminates in ovulation. degenerative epithelial changes occur during the third phase, followed by diestrus, a period of quiescence or slow cell growth. the cycle can be followed by changes in the vaginal epithelium that are often used to determine optimum receptivity of the female for mating and fertilization (table 3 .9). patency of the vaginal orifice and swelling of the vulva are useful signs of proestrus and estrus ( fig. 3.11) . irregularities of the estrous cycle occur during aging. seasonal and dietary factors, such as estrogenic substances found in a variety of feeds, and genetic backgrounds also influence estrous cycles. estrus is routinely observed in mice at about 14-24 h after parturition (postpartum estrus). however, cornification of the vagina is not complete, and fertile matings are not as frequent compared with normal estrus. mice are spontaneous ovulators. ovulation does not accompany every estrus, and estrus may not coincide with every ovulation, because estrus is dependent on gonadal hormones, whereas ovulation is responsive to gonadotropin. the cyclicity of estrus and ovulation is controlled by the diurnal rhythm of the photoperiod. mating, estrus, and ovulation most often occur during the dark phase of the photoperiod. reversing the timing cook (1983) . laboratory animal medicine of the light-dark cycle reverses the time of estrus, ovulation, and mating. pheromones (table 3 .10) and social environment also affect the estrous cycle. for example, estrus may be suppressed in group-housed female mice and reentry into estrus can be synchronized by exposure to pheromones in male mouse urine ('whitten effect'). once exposed to male urine, most female mice will be in estrus within 3 days with a second estrus in about 11 days. hence, estrus can be synchronized by group-housing females prior to pairing with males. in contrast, pheromones from a strange male mouse, particularly of a different strain, may prevent implantation or pseudopregnancy in recently bred females and is known as the 'bruce effect'. see section ii.c on behavior for more detail on the effect of pheromones on mouse reproductive behavior. mating is normally detected by formation of a vaginal plug (a mixture of the secretions of the vesicular and coagulating glands of the male) whose prevalence is highly strain dependent. the plug usually fills the vagina from cervix to vulva (fig. 3.11 ). plug detection is often coupled with vaginal cytology to evaluate fertility and conception. when the cervix and vagina are stimulated physically during estrus, prolactin is released from the anterior pituitary to enable the corpus luteum to secrete progesterone. secretion continues for about 13 days. if fertilization has occurred, the placenta takes over progesterone production. if fertilization does not occur, a pseudopregnant period ensues, during which estrus and ovulation do not occur. fertilization usually takes place (1) testis, (2) head of epididymitis, (3) caudal epididymitis, (4) vas deferens, (5) testicular vein, (6) ampullary gland, (7) seminal vesicle, (8) anterior prostate, (9) ureter, (10) bladder, (11) ventral prostate, (11') dorsal prostate, (12) urethra, (13) bulbourethral muscle, (14) ischiocavernosus, (15) bulbourethral gland, (16) diverticulum of bulbourethral gland, (17) penis, (18) preputial gland, (19) glans penis, (20) prepuce, (21) testicular artery, and (22) vas deferens artery. adapted from (komarek, 2007) . fallopian tube, (3) uterine horn, (4) endometrium, (5) cervix, (6) vagina, (7) vaginal vestibulum, (8) clitoris, (9) clitoral gland, (10) urethra, (11) bladder, (12) medial ligament of bladder, (13) lateral ligament of bladder, (14) left ureter, (14') right ureter, (15) mesovarium, (16) mesometrium, (17) ovarian artery, (18) uterine horn artery, and (19) ovarian artery and vein. adapted from (komarek, 2007) . adapted from komarek (2007) . in the ampulla or the upper portion of the oviduct. ova can be fertilized to produce normal embryos for 10-12 h after ovulation. gestation is usually 19-21 days. because of postpartum estrus, lactation and gestation can occur simultaneously. lactation can delay gestation because of delayed implantation. this may cause prolongation of gestation for up to 12-13 days in certain inbred strains. the effective reproductive life of some inbred strains approaches 2 years where optimum environmental conditions are maintained, but litter size usually decreases as the female ages. therefore, females are usually retired by 6 months of age. average litter size is strain dependent and commonly ranges from 1 to 12 pups. maternal care can account for about 70% of the variation in body weight of neonatal mice. nursing females usually lactate for 3 weeks. milk production increases up to 12 days postpartum and then declines until weaning at 21 days. interestingly, oxytocin is required for nursing but is not essential for parturition or reproductive behavior (nishimori et al., 1996) . some transmission of humoral immunity from dam to progeny occurs in utero, but the majority of antibody is transferred through colostrum. transmission of passive immunity by colostral antibodies has been demonstrated to a wide variety of antigens, including viruses, bacteria, and parasites. antibodies continue to be secreted in the milk throughout lactation. decay of maternally acquired immunity occurs within several months after weaning. loss of maternal immunity increases susceptibility to infection and warrants continued care of weaned mice under barrier conditions. mice are socially gregarious animals with strong family bonds who communicate through complex olfactory, auditory, tactile, and visual signals. wild mice aggregate into groups called demes with low exchange of individuals between different groups. each deme consists of kinrelated members with a high degree of natural inbreeding, higher mutation rates compared to other mammals, and a wide range of developmental flexibility based on early life experience, which all contribute to their remarkably successful environmental adaptability. the deme is composed of a dominant breeding male, a hierarchy of females, subordinate males, and juveniles. wild mice occupy territories measuring just a few square meters when food is abundant to several square kilometers. mice are crepuscular (active during the twilight hours of dawn and dusk), strongly territorial, and omnivorous. coprophagy contributes to approximately one-third of their ingesta as an essential nutritional activity. aside from territoriality, social interactions, breeding, burrowing (when conducive substrates are available), and nest building are major activities. in managing laboratory mice, it is important to understand the complex behavioral biology of their free-living counterparts (latham and mason, 2004) . chemo-olfactory communication is mediated through extremely diverse chemical factors that trigger innate (non-learned) social responses among conspecifics, known as pheromones (table 3 .10). pheromones have been traditionally divided into two broad categories: releaser pheromones, which elicit an immediate behavioral response, and primer pheromones, which mediate a slowly developing and longer-lasting endocrine response. this original definition of pheromone categories has been expanded to another category, termed signaler pheromones, which convey individual or group identity, as well as mediating parent-offspring recognition and mate choice. the biology and genetics of pheromone signaling is being extensively studied in the mouse as a model of mammalian pheromone communication (brennan and zufall, 2006; rodriguez and boehm, 2009) . mouse pheromones are excreted in the urine, as well as plantar, salivary, lacrimal, preputial, and mammary glands. in the urine, major urinary proteins (mups), small peptides, mhc class i peptides, volatile chemicals, and sex hormones all contribute to chemosignals that communicate dominance, kinship, diversity, and gender. wild mice possess a great deal of individual variations of roberts et al. (2012) . c ferrero et al. (2013) . laboratory animal medicine these elements, providing a 'bar code' that distinguishes individuals. inbreeding of laboratory mice has reduced individual variation, but each inbred strain possesses a characteristic array of signals, and to a certain extent, unique signals exist among individuals within a strain (sharrow et al., 2001; sturm et al., 2013) . pheromones are detected by sensory neurons in the vomeronasal organ, the olfactory epithelium, and the lesser known septal organ of masera within the olfactory epithelium, and the gruenberg ganglion, which is located at the anterior end of the nasal cavity (breer et al., 2006; chamero et al., 2012; liberles and buck, 2006; restrepo et al., 2004) . neuronal signals are transmitted to the ganglion layer of the olfactory bulb, and thence to the brain. mups are important components of chemosensory communication in mice, and also an important occupational hazard to human handlers. chromosome 4 contains a cluster of 21 mup genes, plus a number of pseudogenes. mups are small soluble proteins known as lipocalins, which bind small organic chemicals (pheromones) with high affinity, and function as pheromone transporters and stabilizers (thereby contributing to slow release), but also act as protein pheromones themselves. they are synthesized in the liver and excreted in the urine, as well as nasal mucosa, lacrimal glands, and salivary glands. their endogenous role on metabolic activity is not yet understood. male mice excrete significantly more mups in the urine than females. one wellcharacterized mup is 'darcin', named after fitzwilliam darcy, the romantic hero in pride and prejudice. as its name implies, it is a female attractant. mups also act as kairomones, which function as chemical signals between species. for example, cat and rat mups invoke fear in mice. mups are important in the laboratory animal management context, as they are excreted in copious amounts (1-5 mg/ml in urine) and are potent allergens for humans, particularly mus m 1 (ag1 or ma1), which is encoded by the mup 17 gene (sharrow et al., 2001) . chemosensory communication has numerous behavioral effects that influence mouse social interactions. one of the most studied behavioral effects is the bruce effect, or pregnancy block, which is a complex physiologic response in which recently conceived females resorb fetuses during early pregnancy in the presence of an unrelated male, particularly a dominant male. the continued presence of the original mate protects the female from this effect (bruce, 1959) . the vandenbergh effect results in acceleration of puberty of juvenile females in response to male urine (vandenbergh, 1973) . the lee-boot effect occurs among group-housed females that are isolated from males, in which there is suppression of estrus cyclicity (van der lee and boot, 1955) . the whitten effect results in synchronization of estrus among a group of females in response to a male (whitten et al., 1968) . the lee-boot and whitten effects are utilized in the laboratory to assist in induction of synchronized timed pregnancy, but the bruce effect can have deleterious consequences on breeding colonies when foreign males are introduced to a breeding colony, as pheromone communication can occur in the absence of direct contact. the above effects are well-defined pheromone-driven behavioral responses, but chemosensory communication has a myriad of other effects. estrus, pregnant, or lactating females also accelerate puberty among juvenile females. females use odor cues to avoid parasite-laden males, males prefer odors of estrus females, and estrus females prefer odors of dominant males. mice have strong mating and social preferences based upon mhc proteins, which indicate genetic relatedness. maternal recognition of young is also mhc-related, and pups prefer nest odors of maternal and sibling pups based upon mhc relatedness. male aggression against unrelated males is also a strong mhc-related phenomenon. mhc haplotypes determine not only mhc proteins in the urine, and mhc-specific olfactory receptors, but also the composition of volatile chemicals in the urine (kelliher and wersinger, 2009) . the complexity of social communication extends to auditory stimuli as well. male mice utilize ultrasonic 'birdsong' to vocally communicate and attract females. mouse vocalization patterns are largely genetically innate and unique to each strain of mouse, but they can also be modified, or learned, to a limited extent (arriaga et al., 2012) . the behavioral biology of the mouse is highly complex, and depends upon genetic, physiologic, social, and environmental variables, which all impact on how laboratory mice can best be managed in captivity. it is clear that this rich complexity cannot be fully addressed under laboratory conditions, but that does not mean that basic needs, such as nest building, burrowing, foraging, and olfactory environments, cannot be provided. for example, intermale aggression, which is particularly apparent in some strains of mice such as balb/c and swiss-origin stocks and strains, can be minimized by maintaining males from infancy as sibling groups, since adult siblings tend not be aggressive to one another. this sibling bond, however, can be easily broken by short-term separation. environmental enrichment often features provision of plastic houses, which may make vivarium managers feel good, but maximal enrichment can be provided by provision of nesting material, which includes structural scaffolding, such as crinkled cardboard, which facilitates construction of three-dimensional nests. mouse nests are replete with 'appeasement' pheromones, thereby contributing to harmony within the cage, whereas introduction of dirty bedding has the opposite effect. frequent cage changing, including removal of established nests, is highly stressful and disruptive to social harmony within a cage. provision of appropriate and adequate amounts of bedding material that is conducive to burrowing is desirable. it is important to remember that mice are socially gregarious, and that mouse welfare is optimally enriched by other mice within a socially harmonious deme (latham and mason, 2004; van loo et al., 2003) . a laboratory mouse ethogram, defined as an operationalized list of mouse behaviors, arranged by their adaptive meaning to the animal, is available on the web: www. mousebehavior.org. behavioral phenotyping, particularly of transgenic mice, is used extensively in genomic research. a wide variety of standardized test batteries and approaches are used, depending upon the focus of research (reviewed in crawley 2008) . initial behavioral evaluations include general health, body weight, body temperature, appearance of the fur and whiskers, and neurological reflexes assessment. specific tests include observations of home cage behaviors, righting reflex, acoustic startle, eye blink, pupil constriction, vibrissae reflex, pinna reflex, digiscan open field locomotion, rotarod motor coordination, hanging wire, footprint pathway, visual cliff, auditory threshold, pain threshold, and olfactory acuity. novel and complex environmental enrichment in animal housing conditions facilitates enhanced sensory and cognitive stimulation as well as physical activity. environmental enrichment and exercise have beneficial effects such as cognitive enhancement, delayed disease onset, enhanced cellular plasticity, and associated molecular processes in animal models of brain disorders (pang and hannan, 2013) . the immune system of the mouse is very similar to that of humans. the availability of inbred mouse strains, in which each individual animal expresses identical mhc alleles so that tissues and cells can be transplanted without tissue rejection, greatly simplifies and indeed enables functional analyses of immune system components not possible with any other outbred mammalian species. in addition, the ability to genetically manipulate the mouse genome, adding to, altering, and deleting existing genes, enables unprecedented in vivo analysis of immune cell functions. it is for these reasons that the mouse is the primary animal model for immunology research. the immune system is an unusual organ system in that it consists of both solid tissues and various migrating cell populations. the bone marrow and thymus are considered primary lymphoid organs, as sites of hematopoiesis and b-and t-lymphocyte development, respectively. lymph nodes, spleen, and intestinal peyer's patches are considered secondary lymphoid tissues, as sites of immune response initiation. lymph nodes and spleen are analyzed frequently for studies of immune responses and as organs for immune cell isolation. tertiary lymphoid tissue sites are those that form in other solid organs in response to an insult or microbial exposure. among them are the lymphoid cell aggregates of the gastrointestinal and respiratory tract, also called 'gut-associated lymphoid tissue' (galt) and bronchusassociated lymphoid tissues (balt). leukocytes are classified as belonging to the innate or adaptive immune system. the innate immune system responds rapidly to an antigen insult via recognition of pathogen-associated molecular patterns (pamps), such as lipopolysaccharide, bacterial flagellin, single (s)-and double-stranded (ds) rna, and non-methylated dna, via extracellular or intracellular pattern recognition receptors (prrs). receptors include the toll-like receptors (tlrs), such as tlr4 (recognizing lps), tlr3/7 (ss and dsrna) and tlr9 (dna), nod-like receptors (nod1/2), and rig-like receptors (rig-i, mda-5) among others (takeuchi and akira, 2010) . cells of the innate immune system are monocytes/macrophages, granulocytes and dendritic cells as well as innate-like lymphocyte populations (ilc) 1, 2 and 3, which include natural killer (nk) cells (spits et al., 2013) . cells of the adaptive immune system (t and b lymphocytes) express a highly antigen-specific receptor that has arisen through gene rearrangement (t-cell and b-cell receptors, respectively). b cells of the b-1 lineage and γδ t cells are regarded as innate-like cells, as they express a rearranged antigen receptor but seem to respond in an innate-like manner. leukocytes are identified and classified by sets of monoclonal antibodies (mab) against uniquely expressed surface receptors, typically measured by flow cytometry. identification of a unique receptor by one or more mab of the same specificity leads to the assignment of a receptor name, as a 'cluster of differentiation (cd)'. for example, t cells are differentiated into two subsets based on their expression of either cd4 or cd8. cd4 + t cells (t helper cells) recognize peptides presented in mhc class ii and promote b-lymphocyte activation and activate and regulate cellular immune responses via secretion of differing cytokines (see below). cd8 + t cells recognize antigenic peptides presented in mhc class i and serve as cytotoxic cells during the cell-mediated immune response where they can destroy infected cells (e.g., against cells containing infectious agents). the major function of b cells is to respond to an encounter with an antigen/pathogen with the production of highly antigen-specific immunoglobulins (ig; antibodies), which can bind to and inactivate pathogens and toxins. activation of b cells can lead to their differentiation to plasma cells, which produce large amounts of ig. five laboratory animal medicine classes or ig 'isotypes' can be distinguished, which differ in effector function: igm, igg, iga, ige, and igd. the latter is expressed only on the surface of b cells in mice. the igg class, the most abundant antibody class in the serum, is further divided into subtypes: igg 1 , igg 2a/c , igg 2b , and igg 3 . polymorphisms exist on the ig locus such that some strains of mice produce the igg2a subtype (e.g., balb/c), whereas others produce igg2c (e.g., c57bl/6) (zhang et al., 2012) . additional allelic polymorphisms of the locus also exist. for example, balb/c and 129sv mice express the igh-a allotype, whereas c57bl/6 mice express the igh-b allotype. recombinant inbred strains of mice exist for both balb/c and c57bl/6, which harbor the reciprocal igh locus (i.e., igh-b for balb/c and igh-a for c57bl/6 mice). these mice are useful tools for tracking b cells following adaptive cell transfer via allotype-specific mab (see below). immunoglobulin isotype production varies according to the type of immunogen used to evoke the response. igm is secreted short term after initial exposure to an antigen, followed by the other ig isotypes. in viral and intracellular bacterial infections, igg 2a/c is dominant, whereas in extracellular bacterial infections igg 1 dominates the response. igg 2b and igg 3 are usually induced to carbohydrate or lipid antigens. ige is linked to parasitic infections and to allergy. serum antibodies specific for an immunogen can often be measured for the life of the animal. while serum iga levels are low, iga is the highest produced ig in mice. iga production, however, occurs in plasma cells lodged in the lamina propria of mucosal tissues, from where the iga is actively transported in dimeric form onto the luminal surface of mucosal tissues as 'secretory' iga (brandtzaeg, 2009 ). cytokines are secreted signaling molecules involved in cell-cell communication in a complex biological system (table 3 .11). these include the large family of interleukins (ils, currently il-1 to il-37), tumor necrosis factors (tnfs), interferons (type i, ii, and iii) and growth factors such as granulocyte-macrophage colonystimulating factor (gm-csf) and stem cell factor (scf). cytokine secretion often occurs in response to recognition of antigen via prr or tcr. because of their importance in modulating immunity to antigenic stimuli, mice with specific deletions or overexpression of individual cytokines have been made and have contributed to a detailed understanding of many of their often pleotropic functions (akdis et al., 2011) . chemokines are a similarly large group of small, secreted molecules that regulate cell trafficking to sites of antigen encounter but also facilitate cell-cell contact by acting as chemoattractants. chemokines are grouped according to the number of cysteines and disulfide bonds in the molecule into c-x-c-, c-c, c, and cx 3 cl chemokine ligands (l) and receptors (r) and designated accordingly as cxcr1-7/cxcl1-16 and ccr1-10/ccl 1-28 (allen et al., 2007) . immune responses must be coordinated to provide the most appropriate effector functions for the type of pathogen/antigen encountered. immune effector responses differ depending on the life cycle (facultative or obligate intracellular, extracellular, localized, systemic, etc.) and antigen types displayed by the encountered antigen/ pathogen, because this affects the type of prr engaged and activated. prr engagement leads to cytokine and chemokine responses by the first responders, i.e., epithelial cells, local macrophage populations and other innate cells. the type of cytokines and chemokines produced then dictates the types of cells recruited to the site of infection and their subsequent differentiation and functions. the prr engagement also leads to antigen uptake, activation and migration of dendritic cells (dcs) from the site of insult to the regional lymph nodes, where dcs present antigen peptides on mhc molecules to t cells. in addition, the dcs secrete cytokines induced by the initial prr activation, which cause the differentiation of cd4 t cells towards a particular effector response. for example, secretion of il-12 in response to activation of tlr2 or 4 will result in the induction of interferongamma (ifn-γ) production by cd4 t cells, whereas il-6 and tgf-β production by dc will induce cd4 t cells to secrete il-17 (kara et al., 2014) . because the dc translates signals from prr at the site of infection into differentiation signals for t cells in the lymph tissues, these cells are regarded as a 'bridge' between the innate and adaptive immune systems. the specific ig isotype secreted in response to a pathogen depends to a large degree on the type of cytokine produced by cd4 t cells that provide 't-cell help' for b cells. t cells that interact with b cells are identified as a discrete subset termed 't follicular helper cells (t fh )' and it is their cytokine profile that directs b cells to secrete a particular ig isotype (kara et al., 2014) . the classic t h 1/t h 2 dichotomy outlined above was in part shaped by the observation that ifn-γ production will lead to switching of b cells to secrete igg2a/c, whereas production of il-4 leads to the secretion of igg1. interestingly, it appears that the cytokine profile induced by the effector t-cell population is mirrored by the innate immune response. innate-like lymphocytes also have effector phenotypes that correspond to those of cd4 t cells and are induced by the same signals and transcriptional regulators (spits et al., 2013) and the same appears to be true also for macrophages and other innate immune cells (sica and mantovani, 2012) . while initial studies identified two particular antagonistic effector response types (termed t h 1 and t h 2 and classified by t-cell production of ifn-γ and il-4, respectively), more recent studies now demonstrate a much wider array of effector responses in which innate and adaptive immunity acts together to reinforce an immune response phenotype as well as modulate its size by induction of t regulatory cells (t regs ) that generate inhibitory cytokines (kara et al., 2014; sica and mantovani, 2012; spits et al., 2013) . the use of cytokine-deficient and reporter mice that enabled the identification of cytokine-producing cells via expression of a fluorescent reporter was particularly valuable for the development of this more nuanced view of the quality of immune responses. spontaneous mouse models of immune deficiencies have been used extensively in research. their use, plus the expanding number of knockout, transgenic, and dominant negative mouse mutants, has advanced understanding of human immune deficiency diseases as well as basic understanding of the immune system (table 3 .12). interbreeding of multiple immune-deficient mice has allowed the development of 'humanized' mice in which immune cells of the mouse are replaced with those of humans. while many challenges remain to fully replenish mice with components of the human immune system, the use of immune-deficient nod/severe combined immunodeficient (scid)/il-2rγ -/recipients for transfer of human peripheral blood lymphocytes, cordblood or bone marrow-derived cd34+ stem cells with human liver and thymus (blt-mice) is yielding promising results (akkina, 2013) . investigators using genetically engineered mice are constantly reminded that phenotypic analysis of these animals must be done cautiously because the immune system may be profoundly affected and in ways that are not always anticipated. this may make it difficult to determine whether a given gene product is directly involved or may be secondary to a more global dysregulation of the immune system. as with other biological systems, compensation mechanisms also may mask the phenotype. experimental approaches are being increasingly used to refine the knockout technology by restricting a specific genetic deficiency to a particular tissue of interest using the cre-lox system, in which tissue-specific or temporal restricted expression of the cre recombinase induces the deletion of a 'floxed' gene (mak et al., 2001) . transgenic mice are available that restrict cre expression to various hematopoietic cells or tissue or drive cre recombination following injection of tamoxifen. other approaches are the generation of 'bone marrow irradiation' chimeras. here, inbred wild-type mice or mice deficient in certain immune cells (table 3 .12) are lethally irradiated by exposure to a gamma-irradiation source to deplete the hematopoietic stem cells. these are then replaced by transfer of bone marrow cells to the irradiated mice. reconstitution of the hematopoietic system is usually achieved within about 6 weeks, during which time mice are provided with antibiotic-containing drinking water to avoid infections of these temporarily immune-compromised animals. transfer of bone marrow from a congenic knockout restricts the genetic defect to the hematopoietic system. a mix of bone marrow from two sources is also often used to generate tissue-specific knockouts. for example, mixing a bone marrow from t-cell-deficient mice (75%) with that of a gene knockout (25%) generates 'mixed bone marrow chimeras' in which all t cells only develop from the knockout, thus lack the gene of interest, whereas most of the other cells t from the wild-type source, effectively constraining the genetic defect to the t-cell population. sets of congenic mice with defined allotypic differences are often used to confirm the source of individual cells. such markers include the gene locus cd45.1/cdc45.2 or cd90.1/cd90.2 (thy1.1/thy1.2). alternatively, cells may express a fluorescent transgene, such as green-fluorescent protein (gfp). identification is usually performed by flow cytometry, or less commonly by immunofluorescence or immunohistochemistry. generation of bone marrow chimeras circumvents the time-consuming breeding of cre recombinase-expressing flx/flx mice. however, numerous controls are needed to exclude off-target effects due to irradiation damage. repeat injection of antibodies targeting specific cell populations is another rapid approach that avoids the potential for irradiation damage and allows short-term depletion of individual cell subsets. its main disadvantage is the need to identify mab that bind to surface receptors uniquely expressed by a cell subset of interest and the verification of the efficacy of the depletion. frequently used is antibody treatment for the short-term depletion of t-cell subsets using mab against cd4 or cd8 as well as individual cytokines. contemporary knowledge about diseases of laboratory mice has developed primarily from examining the effects of disease on traditional strains and stocks. the widespread use of genetically engineered mice is likely to modify current concepts because of novel or unpredictable interactions among genetic alterations, the genetic backgrounds on which they are expressed, and exogenous factors, such as infectious agents. because the number of combinations is extraordinarily high, clinical and laboratory diagnosticians should be alert to the potential for altered disease expression in genetically engineered mice and not be misled by unexpected signs, lesions, and epizootiology. many microbial agents have the potential to cause disease in mice or interfere with mouse-based research. housing and husbandry in microbiologically sheltered environments are designed to reduce the risks of disruptive infection, especially among immunologically dysfunctional mice, but must be accompanied by effective microbiological surveillance. surveillance should encompass resident mice and mouse products (serum, cell lines, transplantable tumors) procured from external sources. because surveillance strategies will vary with research needs and operating conditions, it is prudent to consult a number of sources, such as the federation of european laboratory animal science associations (felasa) (nicklas et al., 2002) and commercial laboratories, for guidance. detailed discussion of microbial quality control is provided in chapter 10. there are also recommendations regarding specific agents in following sections. diagnostic methods involve gross and microscopic pathology, parasitology, microbial isolation and culture, serology, and pcr. serology is particularly important for viral surveillance, and now relies principally on enzyme-linked immunosorbent assay (elisa), multiplex fluorescent immunoassay (mfi) for simultaneous detection of antibodies to multiple agents (hsu et al., 2007) , indirect fluorescent antibody (ifa) assay, or hemagglutination inhibition (hai), with the latter two methods generally used for confirmation (livingston and riley, 2003; pritchett-corning et al., 2009) . mouse antibody production (map) testing has been historically used for testing biological materials for contamination by infectious agents. pcr panels for murine infectious agents are now commercially available and have cost and time-saving advantages as well as improved assay sensitivity and specificity. beyond the classic bacterial and viral murine infections, pcr assays are now available for endo-and ectoparasites (see chapter 11). etiology mousepox is caused by ectromelia virus (ectv), an orthopoxvirus that is antigenically and genetically closely related to a number of other poxviruses, including vaccinia, variola, and cowpox viruses. the original isolate of ectv, known as the hampstead strain, was discovered by j. marchal in 1930 (marchal, 1930 as the cause of epizootic disease among laboratory mice in england. the disease featured amputation of extremities, which marchal termed ectromelia (from the greek, ectro, amputation and melia, limb). other strains of the virus include moscow, nih-79, washington university, st. louis 69, beijing 70, ishibahsi i-iii, and naval (nav) strains, which vary in virulence, but are essentially indistinguishable genetically and serologically, suggesting a common origin. virus can be isolated from infected tissues by inoculation of cell cultures (bs-c-1, hela, l cells) or embryonated eggs. the natural host (and original source of infection of laboratory mice) of ectv remains unknown. clinical signs the expression of clinical signs reflects an interplay among virus-related factors, including virus strain, dose and portal of entry, and host-related factors, including age, genotype, immunological competence, and gender (brownstein et al., 1991a) . during natural epizootics, it was observed that a, bc, dba/1, dba/2, and cba strains developed acute fatal infections, whereas c57bl/6 mice were resistant to severe disease (briody, 1966) . experimental studies have shown that all strains of mice are susceptible to infection, but balb/c, a, dba/2, and c3h/he mice were highly susceptible, akr and sjl mice were moderately susceptible, and c57bl/6 mice were highly resistant to lethal infection (bhatt and jacoby, 1987c; wallace and buller, 1985) . the mechanisms of genetic resistance are not fully understood but appear to reflect multiple genes, some of which appear to be expressed through lymphoreticular cells, including nk cells (brownstein et al., 1991a; jacoby et al., 1989) . the nuances of cytokine and cellular immune responses to ectv infection have received recent attention (reviewed in buller and fenner (2007) and esteban and buller (2005) ). outbreaks among susceptible mice are often volatile, with variable morbidity and high mortality in susceptible strains of mice. clinical signs such as ruffled fur or prostration may occur for only a few hours before death. mice that survive acute infection may develop chronic disease characterized by a focal or generalized rash anywhere on the body ( fig. 3 .12). conjunctivitis also may occur. skin lesions usually recede within several weeks, but hairless scars may remain. additionally, severe viral infection of the feet and tail during the rash syndrome can lead to necrosis and amputation. epizootiology mousepox is not a common disease. outbreaks occur sporadically and recent outbreaks have been traced to the importation of contaminated mice or mouse products. for example, contaminated mouse serum was responsible for recent outbreaks in the united states (dick et al., 1996; . natural exposure is thought to occur through direct contact and skin abrasions. cage-to-cage transmission is low and can be virtually nil if filter-topped cages are used (bhatt and jacoby, 1987b) . ectromelia virus is highly stable at room temperature, especially under dry conditions, leading to the potential for prolonged environmental contamination in infected colonies (bhatt and jacoby, 1987d) . aerogenic exposure is not a major factor in natural outbreaks, and arthropod-borne transmission does not appear to occur. virus-free progeny can be obtained laboratory animal medicine from immune dams (bhatt and jacoby, 1987b) . however, intrauterine infection and fetal deaths, albeit rare, have been reported. natural transmission is facilitated by intermediately resistant mice, which survive long enough to develop skin lesions that can shed virus for relatively long periods of time. the risks for transmission are further increased by persistence of infectious virus in excreta and exfoliated scabs. although virus excretion typically lasts for about 3 weeks, virus has been found in scabs and/or feces for up to 16 weeks. resistant mouse strains also are dangerous because they can shed virus during subclinical infections. however, infections in resistant mice tend to be short-lived. highly susceptible mice are a relatively small hazard for dissemination of infection, if properly discarded, because they die before virus shedding becomes prominent. thus, juxtaposition of resistant or intermediately resistant infected mice with highly susceptible mice can provoke explosive outbreaks. infant and aged mice are usually more susceptible to lethal infection than young adult mice. maternal immunity among enzootically infected breeding mice may perpetuate infection by protecting young mice from death, but not from infection. such mice may subsequently transmit infection by contact exposure. pathology the classic descriptions of ectv pathogenesis by fenner remain timely, including the frequently cited and reproduced figure summarizing the pathogenesis of infection ( fig. 3 .13) (fenner, 1948b) . interest in smallpox has renewed the interest in ectv as a model of host response to infection (esteban and buller, 2005) . ectv multiplies in the cell cytoplasm and produces two types of inclusion bodies. the a type (marchal body) is well demarcated and acidophilic in histological sections. it is found primarily in epithelial cells of skin ( fig. 3 .14) or mucous membranes and can also be found in intestinal mucosa. the b type of inclusion is basophilic and can be found in all ectromelia-infected cells. however, it is difficult to visualize unless cells are stained intensely with hematoxylin. ectv antigen can be readily visualized by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections (esteban and buller, 2005; jacoby and bhatt, 1987) . following skin invasion, viral multiplication occurs in the draining lymph node and a primary viremia ensues. splenic and hepatic involvement begin within 3-4 days, whereupon larger quantities of virus are disseminated in blood to the skin. this sequence takes approximately 1 week and, unless mice die of acute hepatosplenic infection, ends with the development of a primary skin lesion at the original site of viral entry. the primary lesion is due to the development of antiviral cellular immunity. severe hepatocellular necrosis occurs in susceptible mice during acute stages of mousepox. white spots indicative of necrosis develop throughout the liver ( fig. 3.15 ). in nonfatal cases, regeneration begins at the margins of necrotic areas, but inflammation is variable. splenic necrosis in acute disease commonly precedes hepatic necrosis but is equally or more severe. necrosis and scarring of red and white pulp can produce a macroscopic 'mosaic' pattern of white and red-brown the primary skin lesion, which occurs 6-10 days after exposure, is a localized swelling that enlarges from inflammatory edema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop 2-3 days later as the result of viremia. they are often multiple and widespread and can be associated with conjunctivitis, with blepharitis, and, in severe cases, with buccal and lingual ulcers. the skin lesions also can ulcerate and scab before scarring. diagnosis mousepox can be diagnosed from clinical signs, lesions, serological tests, and demonstration of virus or viral antigen in tissues. observation of characteristic intracytoplasmic eosinophilic inclusions aids detection of infection. several serological tests are available to detect mousepox. historically, the standard test was hai, using vaccinia antigen as a source of hemagglutinin. elisa is more sensitive and specific and has replaced hai for serological monitoring among nonvaccinated mice (buller et al., 1983) . ectv infection also can be detected by ifa (buller et al., 1983) and pcr. serological differentiation of mousepox from vaccinia infection in vaccinated mice is based on the lack of hemagglutinin in the vaccine strain of virus. thus, serum from vaccinated mice may react by elisa but should not react by hai. differential diagnosis mousepox must be differentiated from other infectious diseases associated with high morbidity and high mortality. these include sendai pneumonia, mouse hepatitis, and tyzzer's disease. the latter two can be expressed by acute necrosis in parenchymal organs, but they can be differentiated by morphological, serological, and virological criteria. the skin lesions of chronic mousepox must be differentiated from other skin diseases caused by opportunistic or pathogenic bacteria, ascariasis, and bite wounds. prevention and control mousepox is a dangerous disease because of its virulence for susceptible mice. therefore, infected colonies should be quarantined immediately. depopulation has been used as a primary means for control, but confirmation of infection should be obtained before exposed mice are destroyed. tissues, supplies, instruments, or other items that have had potential contact with infected mice should be disinfected by heat or chemicals such as formalin, sodium hypochlorite, or chlorine dioxide. materials should be autoclaved or, preferably, incinerated. disinfected rooms should be challenged with susceptible sentinel animals that are observed for clinical signs and tested for seroconversion after several weeks. depopulation and disinfection must be carried out vigorously. because modern housing and husbandry methods based on the use of microbarrier caging are effective for containing infection, testing and culling properly isolated mice is a potential alternative, especially for irreplaceable breeding mice. such mice can be quarantined along with cessation of breeding to permit resolution of infection (bhatt and jacoby, 1987b) . sequential testing with contact-exposed sentinels should be employed with this option. additionally, maternal immunity from fully recovered dams can protect mice from infection, thereby enhancing opportunities to derive virus-free mice from previously infected dams, with the caveat that progeny will be transiently seropositive with maternally derived antibody. vaccination can control or prevent clinically apparent mousepox. the hemagglutinin-deficient strain of vaccinia virus (ihd-t) is used to scarify skin on the dorsum of the tail. 'takes' should occur in previously uninfected mice by 6-10 days, but not in infected mice (bhatt and jacoby, 1987a) (fig. 3.17 ). infected mice should be quarantined separately or eliminated. vaccination may not prevent infection, although infection in vaccinated mice is often transient. furthermore, vaccinia virus can be shed from scarification sites for at least several days. therefore, other preventive measures, such as strict controls on the entry of mice or mouse products, combined with periodic serological monitoring, should not be relaxed until diagnostic testing has confirmed the elimination of vaccinia and ectromelia virus. additionally, seroconversion evoked by vaccination must be taken into account in serological monitoring of vaccinated colonies. finally, vaccinia virus is a human pathogen, so vaccination procedures should include personnel protective measures to prevent exposure. research complications the primary threat from mousepox is mortality in susceptible mice. the loss of time, animals, and financial resources can be substantial. (shellam, 2007, 96) mice are naturally susceptible to two herpesviruses from the subfamily betaherpesvirinae and in the genus muromegalovirus, the two species murid herpesvirus 1 (of which one of the members is mouse cytomegalovirus (mcmv)) and murid herpesvirus 3 (of which one of the members is mouse thymic virus (mtv)). they are species-specific viruses and distinct from each other and from other rodent herpesviruses. mctv has received considerable attention as a model of human cmv infection. etiology mouse cytomegalovirus (mcmv) is a mouse-specific betaherpesvirus. it can, however, replicate in cell cultures from several species, including mouse (fibroblasts and 3t3 cells), hamster, rabbit, sheep, and nonhuman primate. cocultivation may be required to rescue latent virus. clinical signs mcmv causes subclinical infection in adult immunocompetent mice, but experimental inoculation of neonates can cause lethal disease due to multisystemic necrosis and inflammation. epizootiology the prevalence of mcmv in laboratory mice is probably uncommon but undefined, since infection is clinically silent and serological surveillance is not widely practiced. wild mice are commonly infected and serve as a natural reservoir for infection, which implies that the entry of virus into a modern vivarium is most likely to occur from contaminated animal products. persistence is a central feature of nonlethal infection. persistently infected mice excrete virus in saliva, urine, and tears for many months, resulting in horizontal transmission through mouse-to-mouse contact. virus also can infect prostate, testicle, and pancreas, implicating other modes of excretion. vertical transmission does not appear to be a common factor in natural infection. further, maternal immunity protects sucklings from infection. pathology mouse cytomegalovirus can replicate in many tissues, and viremia commonly occurs. lesions are not remarkable during natural infection and may be limited to occasional enlarged cells (megalocytosis) containing eosinophilic intranuclear and/or cytoplasmic inclusions associated with lymphoplasmacytic interstitial inflammation, especially in the cervical salivary glands. susceptibility to experimental infection varies with age, dose, route, virus strain, and host genotype. infection can occur in young and adult mice. however, the pathogenicity of mcmv for mice decreases with age. neonates are highly susceptible to lethal infection, but resistance to disease develops by the time mice are weaned. immunodeficient mice, however, remain susceptible to pathogenic infection as adults. persistent infection often affects the salivary glands and pancreas. the persistence of salivary gland infection appears to be dose dependent. there is experimental evidence that mcmv can produce latent infection of b cells, probably t cells as well as aforementioned tissues. persistent infection may lead to immune complex glomerulonephritis. latent persistent infection can be reactivated by lymphoproliferative stimuli and by immunosuppression. diagnosis mcmv antigens appear to be weak stimuli for humoral antibody production, which is consistent with the fact that cellular immunity is critical for protection against infection. neutralizing antibody titers are low during acute infection and difficult to find during chronic infection. serology and pcr-based diagnosis are available, but neither is widely used because of assumptions that infection has a very low prevalence. detection of enlarged cells with intranuclear inclusions, especially in salivary glands, is diagnostic, if they are present. in situ hybridization can be used as an adjunct to routine histopathology. differential diagnosis mcmv infection must be differentiated from infection with mtv. the latter virus can produce necrosis of thymic and peripheral lymphoid tissue when infant mice are experimentally inoculated. lytic lesions of lymphoid tissues are not a hallmark of mcmv. the viruses can also be distinguished from each other serologically. sialoadenitis with inclusions can occur during infection with mouse polyoma virus. like mcmv, mtv infects the salivary gland as its primary target organ. prevention and control control measures for mcmv have not been established, because it has not been considered an important infection of laboratory mice. cage-to-cage transmission has not been demonstrated, but horizontal infection from contaminated saliva must be considered. the exclusion of wild mice is essential. research complications mcmv can suppress immune responses. apart from the potential for interfering with immunology research, it can exacerbate the pathogenicity of opportunistic organisms such as pseudomonas aeruginosa. etiology mouse thymic virus (mtv) is a herpesvirus (murid herpesvirus 3) that is antigenically distinct from mcmv. no suitable in vitro method for cultivation has been developed; therefore, viral propagation depends on mouse inoculation. clinical signs natural infections are subclinical. epizootiology the prevalence of mtv is thought to be low. mice can be infected at any age, although lesions develop only in mice infected perinatally. mice infected as infants or adults can develop persistent infection of the salivary glands lasting several months or more. excretion of virus in saliva is considered the primary factor in transmission. seroconversion occurs in adults but does not eliminate infection. infection in neonates may not elicit seroconversion, rendering such mice serologically negative carriers. the mode of infection is obscure, but virus is excreted in saliva, suggesting that transmission from infected dams to neonatal mice occurs by ingestion. mtv also has been isolated from the mammary tissue of a lactating mouse, suggesting the potential for transmission during nursing. prenatal transmission has not been found. pathology mtv causes severe, diffuse necrosis of the thymus and lymphoid tissue with tropism for cd4 + t cells in mice inoculated within approximately 1 week after birth. the severity of thymic and lymph node necrosis can be mouse strain-dependent. grossly, the thymus is smaller than normal. infected thymocytes display mtv-positive intranuclear inclusions. necrosis is followed by granulomatous inflammation and syncytium formation. reconstitution of lymphoid organs takes 3-8 weeks. diagnosis thymic necrosis associated with intranuclear viral inclusions is the hallmark lesion. viral antigen can be detected by immunohistochemistry. serologic detection is effective, but generally not utilized, and is potentially negative in neonatally exposed mice. suspicion of infection in seronegative mice can be tested by inoculation of virus-free neonatal mice with homogenates of salivary gland or with saliva. inoculated mice should be examined for thymic necrosis 10-14 days later. pcr or the mouse antibody production (map) test can also be used to detect infection. differential diagnosis reduction of thymus mass can occur in severe mouse coronavirus infection, during epizootic diarrhea of infant mice, or following stress. prevention and control because mtv induces persistent salivary infection, rederivation or restocking should be considered if infection cannot be tolerated as a research variable. research complications mtv transiently suppresses cellular and humoral immune responses because of its destructive effects on neonatal t lymphocytes. parvoviruses are among the most common viral infections in contemporary laboratory mouse populations (livingston et al., 2002) (pritchett-corning et al., 2009) , and pose major challenges to both detection and control. the mouse parvoviruses are composed of two antigenically and genetically distinct but related groups, including minute virus of mice (mvm) and mouse parvovirus (mpv), with each group containing a number of strains. the international committee on taxonomy of viruses classifies mpv, mvm, and several other rodent parvoviruses into one genus, protoparvovirus, and species, rodent protoparvovirus 1, but these viruses will be treated separately herein. etiology minute virus of mice (mvm) is a small (5-kb) single-stranded dna virus. the prototypic strain is designated mvmp. an allotropic variant with immunosuppressive properties in vitro is named mvmi, and additional laboratory animal medicine named strains include mvmc and mvmm. the genome encodes two nonstructural proteins, ns-1 and ns-2, which are highly conserved among the rodent parvoviruses and account for prominent cross-reactivity in serological assays that utilize whole virus antigen. the viral capsid proteins, vp-1 and vp-2, are virus-specific and form the basis for serological differentiation of mvm from mpv. mvm has a broad in vitro host range. it replicates in monolayer cultures of mouse fibroblasts (a9 cells), c6 rat glial cells, sv40 (simian virus 40)-transformed human newborn kidney (324k cells), t-cell lymphomas (el4), and rat or mouse embryo cells, producing cytopathic effects that can include the development of intranuclear inclusions. clinical signs natural mvm infections are subclinical. neonatal mice of some inbred strains are experimentally susceptible to lethal renal and/or intestinal hemorrhage during mvmi infection, but this syndrome has not been reported in natural outbreaks. experimental inoculation of adult c.b-17-prkdc scid (scid) mice with mvmi results in lethal infection (lamana et al., 2001; segovia et al., 1999) , and similar severe illness has been noted in naturally infected b-cell-deficient nod. cg-h2h4-igh6 null mice (naugler et al., 2001) . epizootiology mvm is a common virus that naturally infects laboratory mice, but appears to be less common than mpv (besselsen et al., 2006; livingston et al., 2002) . mvm is moderately contagious for mice, its only known natural host. virus can infect the gastrointestinal tract and is excreted in feces and urine. the resistance of rodent parvoviruses to environmental inactivation increases the risks of transmission after virus is excreted. therefore, contamination of caging, bedding, food, and clothing must be considered a risk for the spread of infection. transmission occurs by oronasal exposure, but viral contamination of biologicals used for experimental inoculation, such as transplantable tumors, also can be a source of infection. continuous contact exposure to infected animals or soiled bedding usually induces a humoral immune response within 3 weeks, but limited exposure may delay seroconversion. young mice in enzootically infected colonies are protected by maternal antibody, but actively acquired immunity develops from infection sustained after the decay of maternal immunity. mvm, in contrast to mpv, is not thought to cause persistent infection; infection in immunocompetent adult mice usually lasts less than 3 weeks (smith, 1983; smith and paturzo, 1988 ). infection appears to last less than 1 month, even in oronasally inoculated neonatal mice, but immunodeficient mice may be persistently infected. there is no evidence that mvm is transmitted in utero. pathology natural infections or experimental inoculation of adult mice appears to be nonpathogenic. contact-exposed neonates have been reported to develop cerebellar lesions, but these are very rare. experimental infection of neonatal balb/c, swr, sjl, cba, and c3h mice with mvmi can cause renal hemorrhage and infarction (brownstein et al., 1991b) . dba/2 mice also developed intestinal hemorrhages and accelerated involution of hepatic hematopoiesis. c57bl/6 neonates are resistant to vascular disease. this lesion has been attributed to viral infection of endothelium. infection of immunodeficient mice, including scid and b-cell-deficient mice, results in lethal damage to granulomacrophagic, megakaryocytic, and erythrocytic hematopoietic tissue with severe leukopenia (lamana et al., 2001; naugler et al., 2001; segovia et al., 1999) . intranuclear viral inclusions and viral antigen have been observed in splenic mononuclear cells of b-cell deficient mice (naugler et al., 2001) . diagnosis serology is the primary method of detecting infection, which utilizes recombinant mvm and mpv major capsid viral proteins (vp2) as antigens, which discriminate between the two groups of mouse parvoviruses. in contrast, the conserved nonstructural protein, ns1 can be used to detect antibody to both groups, but is less sensitive than vp2 assays (livingston et al., 2002) . mvm infection also can be detected by pcr, in situ hybridization, and immunohistochemistry. pcr assays can be used to detect mvm-or mpv-specific vp2 or all rodent parvovirus group specific ns1 exons (besselsen, 1998; besselsen et al., 2006) . mvm can be isolated from the spleen, kidney, intestine, and other tissues by inoculation of the c6 rat glial cell line. it also can be detected by the mouse antibody production test. prevention and control because mvm does not persist in immunocompetent mice, control and elimination should exploit quarantine combined with thorough disinfection of the environment, because parvoviruses are resistant to environmental inactivation. mpv has been shown to be successfully eliminated by a cage-bycage test (serology and fecal pcr) and cull approach, although there are no published reports confirming the success of this strategy for eliminating mvm (macy et al., 2011) . cesarean rederivation or embryo transfer may also be used to rederive virus-free progeny. prevention of mvm infection depends on strict barrier husbandry and regular surveillance of mice and mouse products destined for use in vivo. research complications mvm contamination of transplantable neoplasms can occur; therefore, infection can be introduced to a colony through inoculation of contaminated cell lines. failure to establish long-term cell cultures from infected mice or a low incidence of tumor 'takes' should alert researchers to the possibility of mvm contamination. mvmi has the potential to inhibit the generation of cytotoxic t cells in mixed lymphocyte cultures. etiology mouse parvovirus (mpv) is among the more common viruses detected within contemporary laboratory animal medicine mouse colonies, and is more common than mvm (livingston et al., 2002; livingston and riley, 2003; pritchett-corning et al., 2009) . mpv was initially isolated following its detection as a lymphocytotropic contaminant in in vitro assays for cellular immunity. the virus grew lytically in a cd8 + t-cell clone designated l3 and inhibited the proliferation of cloned t cells stimulated with antigen or interleukin 2 (il-2) (mckisic et al., 1993) . molecular analysis of mpv indicates that regions encoding the ns proteins are similar to those of mvm (and other rodent parvoviruses). however, they differ significantly in regions encoding the capsid proteins, accounting for their antigenic specificity. the prototype isolate was first called an 'orphan' parvovirus of mice because its biology and significance were obscure, but it has subsequently been named mouse parvovirus (mpv). immortalized t cells (l3) are the only cells found thus far to support replication of mpv. there are three genetically distinct variants of mpv, including mpv-1, mpv-2, and mpv-3. mpv-1 includes a number of closely related variants, including mpv-1a, mpv-1b, and mpv-1c. in addition, a hamster parvovirus isolate is closely related to mpv-3, which is infectious to mice and likely to be of mouse origin (besselsen et al., 2006; christie et al., 2010) . clinical signs mpv infection is clinically silent in infant mice and adult immunocompetent or immunodeficient mice (besselsen et al., 2007) . immunologic perturbations are the most likely signs of infection (mckisic et al., 1993) . epizootiology mpv causes persistent infection in infant and adult mice, a property that differentiates it from mvm. in situ hybridization has identified the small intestine as a site of viral entry and early replication, but respiratory infection cannot be excluded. experimental studies following inoculation of neonatal balb/c and c.b-17-prkdc scid (scid) mice revealed that balb/c mice shed high levels of virus for 3 weeks, with transmission to sentinels exposed during the first 2 weeks of infection. thereafter, balb/c mice shed extremely low virus intermittently. in contrast, scid mice shed high levels of virus until weaning, but lower levels at 6 weeks of age, yet they effectively transmitted infection to sentinels at all stages of infection (besselsen et al., 2007) . others have shown that transmission of mpv by sencar mice inoculated as infants was intermittent up to 6 weeks, whereas transmission by mice inoculated as weanlings occurred during the first 2 weeks of infection . transmission to balb/c progeny from infected dams was shown to occur, but embryo transfer rederivation was found to be successful in experimentally infected scid mice (besselsen et al., 2008) . humoral (e.g., passively or maternally acquired) immunity can protect against mpv infection. however, immunity to mvm may not confer cross-immunity to mpv (hansen et al., 1999) . pathology mpv appears to enter through the intestinal mucosa, which is a site of early virus replication ( fig. 3.18 ). acute infection is widespread but mild, involving the lung, kidney, liver, and lymphoid organs. histological lesions are not discernible. lymphocytotropism is a characteristic of acute and persistent mpv infection in infant and adult mice. during acute infection, virus is dispersed within lymph nodes, but during persistent infection virus localizes in germinal centers (fig. 3.19) . diagnosis because infected mice do not manifest signs or lesions and the virus is very difficult to propagate in cell culture, detection and diagnosis rely on serology and molecular methods. serology that utilizes mpv vp2 as antigen is a sensitive and specific assay that differentiates mpv from mvm (livingston et al., 2002) . the map test also can be used to detect parvovirus infections but is relatively time-consuming and expensive. as noted for mvm, pcr for murine parvoviruses, using nucleoprotein gene sequences that are conserved among murine parvoviruses, can be used as a screening test. pcr also can be used to detect mpv-specific sequences in the vp2 gene. although diagnostic pcr is sensitive and specific, it is effective only in actively infected animals. it can be used on feces to detect virus shedding, or applied to tissues, such as mesenteric lymph nodes, obtained at necropsy. differential diagnosis mpv infection must be differentiated from mvm infection. because both viruses are enterotropic and lymphocytotropic, serology and pcr must be used to distinguish between them. prevention and control the persistence of mpv in individual mice, its potential for provoking immune dysfunction, and the resistance of murine parvoviruses to environmental inactivation favor active control and prevention of mpv infection. quarantine of infected rooms is appropriate. elimination (depopulation) of infected mice should be considered if they are an immediate threat to experimental or breeding colonies and can be replaced, but a cage-by-cage test and cull approach has been shown to be successful under natural conditions (macy et al., 2011) . for mice that are not easily replaced, virus persistence in the absence of transplacental transmission favors cesarean rederivation or embryo transfer as relatively rapid options to eliminate infection. control of infection also should include environmental decontamination. chemical disinfection of suspect animal rooms and heat sterilization of caging and other housing equipment are prudent steps. prevention is based on sound serological monitoring of mice and surveillance of biologicals destined for inoculation of mice. with the increasing use of mouse germplasm, it is important to note that mouse sperm, oocytes, ovarian tissue, and preimplantation embryos from enzootically mpvinfected mouse colonies may have a high prevalence of mpv contamination, based upon pcr (agca et al., 2007) . research complications murine parvoviruses can distort biological responses that depend on cell proliferation. for mpv, such effects are seen on immune function and include augmentation or suppression of humoral and cellular immune responses. etiology adenoviruses are nonenveloped dna viruses that produce intranuclear inclusions in vitro and in vivo. two adenovirus species in the genus mastadenovirus have been associated with mice: murine mastadenovirus a (with the representative strain being mav-1 or fl) and murine mastadenovirus b (with the representative strain being mav-2 or k87). both strains replicate in mouse kidney tissue culture but are antigenically distinct. clinical signs mav-1 can cause severe clinical disease after experimental inoculation of infant mice. signs include scruffiness, lethargy, stunted growth, and often death within 10 days. mav-2 virus is enterotropic and is responsible for virtually all naturally occurring infections in contemporary mouse populations. infection is usually subclinical in immunocompetent mice, with the possible exception of transient runting among infant mice. wasting disease can occur in athymic mice infected with mav-1. epizootiology the prevalence of adenovirus infection in mouse colonies is low, particularly mav-1 riley, 2003, pritchett-corning et al., 2009) . transmission occurs by ingestion. adult mice experimentally infected with mav-1 may remain persistently infected and excrete virus in the urine for prolonged periods. adult mice experimentally infected with mav-2 excrete virus in feces for at least 3 weeks but eventually recover. athymic mice can shed mav-2 for at least 6 weeks and episodically for at least 6 months. pathology infection with mav-1 causes multisystemic disease characterized by necrosis. infant mice are especially susceptible to rapidly fatal infection characterized by necrosis of brown fat, myocardium, adrenal cortex, salivary gland, and kidney, with the development of intranuclear inclusions. more mature mice usually develop subclinical infection leading to seroconversion; however, athymic and scid mice can develop intestinal hemorrhage and wasting, with fatal disseminated infection (lenaerts et al., 2005) . infection with mav-2 produces amphophilic, intranuclear inclusions in intestinal epithelium, especially in the distal small intestine (fig. 3.20) . inclusions are easier to detect in infant mice than in adults. infection of c.b-17-prkdc scid mice with mav-2 results in enteric infection, but also hepatic lesions resembling reye's syndrome (pirofski et al., 1991) . diagnosis although mav strains can be isolated in tissue culture, routine diagnosis depends on detection of infection by serological assay and/or demonstration of adenoviral inclusions, most commonly in the intestinal mucosa. cross-neutralization tests have revealed that antiserum to mav-2 neutralizes both strains, but antiserum to mav-1 neutralizes mav-2 weakly at best. therefore, mav-2 antigen should be used for the serological detection of adenovirus infection irrespective of the assay employed. mav also can be detected by pcr. differential diagnosis intranuclear adenoviral inclusions in intestinal epithelium are pathognomonic laboratory animal medicine and differentiate mav-2 infection from other known viral infections of mice. infection may resemble rotavirus infection, with runting and abdominal bloating in infant mice. prevention and control prevention requires serological monitoring of mice and examination for contamination of animal products such as transplantable tumors. because mav-2 infection appears to be transient in individual mice, segregation of infected colonies may be effective for control. however, rederivation coupled with subsequent barrier housing is a more conservative approach. research complications mav infection is unlikely to affect research using immunocompetent mice. however, it has the potential for pathogenicity in immunodeficient mice. mice can incur natural infection with two polyomaviruses: polyoma virus (pyv) and k virus. these viruses belong to the family polyomaviridae. k virus belongs to the genus polyomavirus and the species murine pneumotropic virus, while the classical polyoma virus belongs to the species murine polyomavirus. etiology polyoma virus (pyv) is a small dna virus that derives its name 'polyoma' (many tumors) from its ability to experimentally induce multiple types of tumors in mice experimentally infected as neonates. its primary importance stems from use in murine models of experimental oncogenesis, with natural infection being rare. the transformative activity is mediated by 't' (tumor) antigens, encoded by large t, middle t, and small t genes, with middle t (mt) being considered the major viral oncogene, and as a result has been used extensively in transgenic constructs. clinical signs natural infections in immunocompetent mice are usually subclinical. however, tumor induction, neurological disease, and wasting can occur in naturally exposed immunodeficient mice (mccance et al., 1983; sebesteny et al., 1980) . epizootiology modern husbandry and health care have essentially eliminated natural exposure in laboratory mice. pyv is used for experimental studies and thus can inadvertently be introduced to mouse colonies. inoculation of mice with contaminated biologicals or cell cultures is a potential source of entry and spread. natural transmission occurs via the respiratory route. exposure of neonatal mice results in persistent infection and shedding of the virus in urine, feces, and saliva, thereby contaminating the environment for spread to other mice. infection of adult mice is transient, with minimal virus shedding, although pcr has revealed infection lasting up to 5 months in cba mice inoculated with virus as adults (berke and dalianis, 1993) . maternal antibody is highly effective at preventing infection of newborn mice, but as maternal antibody wanes, mice are partially susceptible, with transient virus shedding. thus, the natural cycle of transmission in enzootically infected populations requires contamination of bedding and nesting material in order to infect and be inefficiently transmitted, which is readily precluded by modern husbandry. intrauterine infection also can occur, and persistent renal infection, contracted neonatally, can be reactivated during pregnancy. as in immunologically immature neonatal mice, pyv infection can persist in adult immunodeficient mice. pathology pyv-induced tumors are essentially a laboratory phenomenon, optimized by virus strain and mouse strain, with akr, c3h, c58, cba, swr, and others being most susceptible, and c57bl/6 being among the most resistant to pyv oncogenesis. intranasal inoculation of neonatal mice results in initial replication in pulmonary respiratory epithelium (gottlieb and villarreal, 2000) followed by viremic dissemination and acute, lethal disease. tumors appear 2-12 months after inoculation of surviving mice. tumors of both epithelial and mesenchymal origin arise in multiple organs, particularly mammary carcinomas, basal cell tumors of the skin, carcinomas of salivary glands, thymomas, and various types of sarcomas. athymic mice can develop cytolytic and inflammatory lesions, followed by multisystemic tumor formation. intranuclear inclusions may be present in cytolytic lesions. demyelinating disease and skeletal tumors have been reported in experimentally . laboratory animal medicine inoculated and naturally exposed athymic mice, and myeloproliferative disease has been reported in experimentally inoculated c57bl/6-scid mice (szomolanyi-tsuda et al., 1994) . diagnosis pyv can be isolated in mouse fibroblast cell lines, but infection is ordinarily detected serologically. additionally, pcr and immunohistochemistry can be used. differential diagnosis wasting in athymic mice can be caused by other infectious agents, including coronaviruses, sendai virus (sv), and pneumocystis. intranuclear inclusions can occur in infections caused by mouse adenovirus, mouse cytomegalovirus, and k virus. prevention and control control depends on elimination of infected mice and material, together with prevention of airborne spread. biological material destined for mouse inoculation should be tested for pyv by the map test or molecular diagnostics. research complications pyv infection can affect experiments by inadvertent contamination of cell lines or transplantable tumors, leading to infection of inoculated mice and the potential for epizootic spread. k virus infection k virus has historical importance, and is apparently absent from contemporary mouse populations (livingston and riley, 2003; pritchett-corning et al., 2009) , but it continues to be tested for, adding to the expense of infectious disease surveillance. oral inoculation of neonatal mice results in initial infection of capillary endothelium in the intestine, followed by viremic spread. vascular endothelium is the primary target in affected tissues, which often include the lung, liver, spleen, and adrenal glands. dyspnea occurs from pulmonary infection because of edema and hemorrhage. infection of immunocompetent adult mice is subclinical and results in a vigorous immune response. however, both adults and infant mice develop persistent infection. the primary organ for persistence is the kidney, with shedding of virus from tubular epithelium, and shedding can be reactivated by immunosuppression (greenlee et al., 1991) . additionally, infection of athymic mice can lead to clinical signs and lesions akin to those described for neonatally inoculated mice. gross lesions are limited to pulmonary hemorrhage and edema. histologically, intranuclear inclusions, which are visualized more easily using immunohistochemistry, are present in vascular endothelium of infected tissues. mild hepatitis with hepatocyte degeneration also may develop. infection can be detected by serology or pcr. prevention and control measures, if ever to be found within a mouse population, are similar to those described for pyv. etiology lactate dehydrogenase-elevating virus (ldv) is a mouse-specific small enveloped rna virus belonging to the family arteriviridae. infected mice are persistently viremic, resulting in increased concentration of several serum enzymes, most notably lactate dehydrogenase (ldh). infection is common among wild mice, but is now rare in contemporary laboratory mouse populations. however, surveys of biologic material indicate that ldv may be a common contaminant of biologic materials (nicklas et al., 1993) . clinical signs infection is subclinical. however, poliomyelitis has occurred in immunosuppressed c58 and akr mice inoculated with ldv, and has recently been observed in icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., 2011) . epizootiology the primary mode of mouse-tomouse transmission is mechanical transfer from aggressive behavior (e.g., bite wounds). inoculation of mice with contaminated animal products such as cell lines, transplantable tumors, or serum is probably the most common source of induced infection. it is important to note, with respect to mechanical transmission, that infection induces lifelong viremia. natural transmission between cagemates or between mother and young is rare even though infected mice may excrete virus in feces, urine, milk, and probably saliva. pathology viremia peaks within 1 day after inoculation, then persists at a diminished level. the elevation of enzyme levels in blood is thought to result primarily from viral interference with clearance functions of the reticuloendothelial system. ldv selectively targets mature f4/f8-positive macrophages, which are continually produced by uninfected progenitor cell populations, thereby maintaining persistent infection. virus also escapes immune clearance by evolution of neutralizing antibody-resistant quasi-species. no lesions are seen in naturally infected mice. the only significant lesion that can arise from experimental infection is poliomyelitis. this syndrome requires a combination of immunosuppression (due to age, genetics or induced means), mouse strain (c58, akr, c3h/fg, and pl), neurotropic ldv strains, and endogenous ecotropic murine leukemia virus. the mouse strain-dependent element is homozygosity for the fv-1 n allele, which permits replication of endogenous n-tropic ectotropic murine leukemia virus. mice develop spongiosis, neuronal necrosis, and astrocytosis of the ventral spinal cord and brain stem, with axonal degeneration of ventral roots. lesions contain both ldv and retrovirus. although this syndrome is largely experimentally induced, a natural outbreak of poliomyelitis has been reported in fv-1 homozygous icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., 2011) . diagnosis plasma ldh levels are elevated, a response that is used to detect and titrate ldv infectivity. of the five isoenzymes of ldh in mouse plasma, only laboratory animal medicine ldh-v is elevated. sjl/j mice in particular show spectacular increases in ldh levels (15-20 times normal), a response controlled by a recessive somatic gene. ldv is detected by measuring ldh levels in mouse plasma before and 4 days after inoculation of specific pathogenfree (spf) mice with suspect material. it is important to use nonhemolyzed samples because hemolysis will produce falsely elevated readings. plasma enzyme levels are measured in conventional units/ml, 1 conventional unit being equivalent to 0.5 international units (iu). normal plasma levels are 400-800 iu, whereas in ldv infection, levels as high as 7000 iu can occur. ldv also interferes with the clearance of other serum enzymes and results in their elevation in serum. in a recent survey, 6000 serum samples were tested by serum ldh enzyme assays, among which 10% were deemed potentially positive. however, pcr revealed that all were false-positives (pritchett-corning et al., 2009) , emphasizing the inaccuracy of traditional enzyme assays. infection provokes a modest humoral antibody response, but it is difficult to detect because of formation of virus-antibody immune complexes. molecular diagnostics also can be used to diagnose infection in mouse tissues and serum and biologic materials. however, inhibitory factors in cells and serum may cause falsenegative results in pcr testing, so appropriate quality control measures are essential if this method is used (lipman and henderson, 2000) . prevention and control transplantable tumors have been a common source of ldv historically. therefore, tumors or cell lines destined for mouse inoculation should be monitored for ldv contamination. although ldv can contaminate tumor cell lines, it does not replicate in the tumor cells. therefore, one can attempt to free tumors of virus by passaging them through athymic nude rats, which are not nonpermissive to ldv but are permissive to xenografts. research complications ldv has numerous potential effects on immunological function. it may reduce autoantibody production, cause transient thymic necrosis and lymphopenia, suppress cell-mediated immune responses, and enhance or suppress tumor growth. etiology the house mouse is the natural host for lymphocytic choriomeningitis virus (lcmv), an old world member of the arenaviridae family that has spread worldwide along with m. musculus. lcmv virions are pleomorphic, containing single-stranded rna, and bud from the cell membrane. disease associated with infection is due to host immune response to the otherwise non-cytolytic virus. its name is derived from the immune-mediated inflammation resulting from the intracerebral inoculation of virus into immunologically competent mice. lcmv is a zoonotic virus that may cause a variety of clinical manifestations in humans, including meningitis. it has been extensively studied as an experimental model of virus-induced immune injury, using a number of closely related strains, including ones that have been selected for their relative neurotropism or viscerotropism. lcmv can be propagated in a variety of mammalian, avian, and even tick cell lines, with minimal cytopathic effect. these characteristics favor its propensity to persistently and silently contaminate biologic products, such as tumor cell lines. clinical signs natural infection in immunocompetent adult mice is usually self-limiting and subclinical. during enzootic infection of a mouse population, lcmv is transmitted in utero from persistently infected dams to their fetuses or to neonates, which are persistently infected and immunologically tolerant to lcmv. since lcmv is non-cytolytic in and of itself is minimally pathogenic, congenitally infected mice grow into adulthood, reproduce, and therefore transmit infection to the next generation. however, with age, immune tolerance breaks down, and mice develop a syndrome known as 'late disease' in which mice will progressively lose weight and die. in utero infection results in a low level of fetal mortality and maternal cannibalism of infected pups. the immune tolerance to lcmv is virus-specific, with the mice capable of eliciting effective immune responses against other agents. clinical signs following experimental inoculation of lcmv vary with age and strain of mouse, route of inoculation, and strain of virus. when virus is inoculated intracerebrally into immunocompetent adult mice, mice develop immune-mediated lymphocytic choriomeningitis, characterized by illness beginning 5-6 days after inoculation. sudden death may result or subacute illness associated with one or more of the following signs may develop: ruffled fur, hunched posture, motionlessness, and neurological deficits. mice suspended by the tail display coarse tremors of the head and extremities, culminating in clonic convulsions and tonic extension of the rear legs. spontaneous convulsions also can occur. animals usually die or recover in several days. a visceral form of infection can occur in adult mice inoculated by peripheral routes with 'viscerotropic' strains. it can be subclinical or lead to clinical signs, including ruffled fur, conjunctivitis, ascites, somnolescence, and death. if mice survive, recovery may take several weeks. surviving mice may have immune exhaustion due to consumption of lymphoid tissue, in contrast to immune tolerance that occurs when mice are infected in utero or as neonates. runting and death from lcmv infection may occur in neonatally infected mice and can lead to transient illness or to death. clinical signs are nonspecific, recovery is slow, and survivors may remain runted. this early form of disease is attributed to endocrine dysfunction caused laboratory animal medicine by lcmv infection. late-onset disease can occur in previously subclinical carrier mice that develop immune complex glomerulonephritis. it is usually the result of prenatal or neonatal infection and occurs in persistently infected mice when they are 9-12 months old. clinical signs are nonspecific and include ruffled fur, hunched posture, weight loss, proteinuria, and ascites. epizootiology lcmv is distributed widely in wild m. musculus throughout the world. among common laboratory species, mice, hamsters, guinea pigs, and nonhuman primates are susceptible to infection, but only the mouse and the hamster are known to transmit virus. lcmv infection is rare in laboratory mice produced and maintained in modern quarters (livingston and riley, 2003; pritchett-corning et al., 2009) . infection is usually introduced through inoculation of virusinfected biologicals, such as transplantable tumors, or by feral mice. wild mice are a natural reservoir of infection and a potential threat to research colonies if they gain entry inadvertently. naturally infected carrier mice can have persistently high concentrations of virus in many organs, thereby facilitating virus excretion in saliva, nasal secretions, and urine. persistently infected neonates usually reach breeding age and can perpetuate infection in a breeding colony. thus introduction of a single lcmv carrier mouse to a breeding colony can eventually result in a high prevalence of persistently infected mice. infection in adult mice, in contrast, is often acute because of the onset of effective immunity, and the spread of virus is halted. horizontal spread of infection is enhanced by close contact, but rapid horizontal spread is not characteristic. mice can transmit lcmv to hamsters, which can remain viremic and viruric for many months, even if they contract infection as adults. infected hamsters can transmit virus to other hamsters and mice and are the primary source of human lcmv infection. persistent infection in immunodeficient mice may carry greater risks for viral excretion and zoonotic transmission. pathology lcmv disease is a prototype for virusinduced, t-lymphocyte-mediated immune injury, noncytolytic endocrine dysfunction, and immune complex disease. however, lesions comparable to experimentally induced disease are rare during natural infection. intracerebral inoculation of virus into immunocompetent adult mice induces nonsuppurative leptomeningitis, choroiditis, and focal perivascular lymphocytic infiltrates. host tissues are damaged during the course of the cellular immune response to the virus. the character of visceral lesions depends on virus strain and mouse strain; the ratio of cytolytic to proliferative responses in lymphoid organs is mouse strain-dependent. in severe infection, nonsuppurative inflammation can occur in many tissues. the severity of accompanying cytolytic lesions seems to parallel the intensity of cellular immunity. liver lesions can include hepatocyte necrosis accompanied by nodular infiltrates of lymphoid cells and kupffer cells, activated sinusoidal endothelium, an occasional granulocyte or megakaryocyte, and fatty metamorphosis. cytolysis, cell proliferation, and fibrinoid necrosis can develop in lymphoid organs. necrosis of cortical thymocytes can lead to thymic involution. lesions of late-onset disease are characterized by formation of immune complexes and associated inflammation. renal glomeruli and the choroid plexus are most severely affected, but complexes may also be trapped in synovial membranes, blood vessel walls, and skin. lymphoid nodules can form in various organs. lesions associated with early deaths in neonatally infected mice have not been thoroughly described but include hepatic necrosis. the lesions of acute and persistent lcmv infection reflect separate immunopathologic processes. in adult mice with acute lcmv infection, virus multiplies in dcs, b cells, and macrophages, whereas t cells are resistant. internal viral epitopes induce humoral immune responses, but surface epitopes elicit cell-mediated immunity and neutralizing antibodies. thus, elimination of virus and virus-associated immunological injury are both t-cell-mediated. this apparent paradox has been explained by the view that prompt cellular immunity limits viral replication and leads to host survival, whereas slower cellular immune responses permit viral spread and increase the number of virus-infected target cells subject to attack once immunity is fully developed. antibody can be detected by 1 week after infection but does not play a significant role in eliciting acute disease. lesions of lcmv infection appear to develop from direct t-cell-mediated damage to virus-infected cells and may involve humoral factors released from immune effector t cells. lcmv also can suppress humoral and cellular immunity in acutely infected mice. persistent infection commonly evolves from exposure early in pregnancy, and virus has been demonstrated in the ovaries of carrier mice. prenatal or neonatal infection induces immunological tolerance to lcmv, which can then replicate to high titer in many tissues. nevertheless, persistently infected mice develop humoral antibody to lcmv. antibody can complex with persistent virus to elicit complement-dependent inflammation in small vessels. immune complex glomerulonephritis exemplifies this process, as noted above. diagnosis lcmv infection can be diagnosed serologically. whereas immunocompetent adult mice will normally seroconvert after exposure, carrier mice may develop poor humoral immune responses. therefore, testing must avoid false-negative results. employment of adult contact sentinel mice is a useful strategy for detecting lcmv infection by seroconversion. tissues, including biologic products and cell lines, can be tested laboratory animal medicine by pcr. a traditional method for detection involved collection of small blood samples from persistently infected live suspects, which are often viremic, and using them to inoculate cultured cells or adult and neonatal mice. intracerebral inoculation of lcmv-positive tissues should elicit neurological signs in adult mice within 10 days, whereas infant mice should remain subclinical. histological examination of brains from affected adults may reveal nonsuppurative inflammation, but lesions may be minimal in mice infected with viscerotropic isolates. immunohistochemistry can be used to detect viral antigen in brains of suckling and adult mice. intraperitoneal inoculation of adult mice may yield short-lived infection with seroconversion, i.e., the map test. virus can be grown and quantified in several continuous cell lines, including mouse neuroblastoma (n-18) cells, bhk-21 cells, and l cells. application of immunofluorescence staining to detect lcmv antigen in inoculated cultured cells yields results more quickly than animal inoculation. of course, all diagnostic procedures involving potential contact with live virus should be carried out under strict containment conditions to avoid infection of laboratory personnel (see chapter 29). the use of in vitro detection has the added advantage, in this regard, of reducing biohazardous exposure and the use of live animals for testing. differential diagnosis neurological signs must be differentiated from those due to mouse hepatitis virus, mouse encephalomyelitis virus, and meningoencephalitis from bacterial infection. trauma, neoplasia, and toxicities also must be ruled out in neurological disease with low prevalence. late-onset disease is associated with characteristic renal lesions, including deposition of viral antigen in tissues. early-onset disease must be differentiated from other causes of early mortality, such as mouse hepatitis virus, ectromelia virus, reovirus 3 infection, tyzzer's disease, or husbandry-related insults. prevention and control adequate safeguards for procurement and testing of animals and animal products are essential to prevent entry. because mouse-to-mouse spread is slow, selective testing and culling for seropositive or carrier mice is possible. if mice are easily replaced, however, depopulation is a safer and more reliable option. valuable stock can be rederived, but progeny must be tested to preclude in utero transmission. because infected hamsters can excrete large quantities of virus, exposed hamsters should be destroyed and hamsters should not be housed with mice. infection of immunodeficient mice poses similar risk. lcmv can be transmitted to human beings, who can contract flu-like illness or severe cns disease. more frequently, human infection is subclinical. the zoonotic potential of lcmv infection makes it especially important to detect and eliminate carrier animals and other potentially contaminated sources, such as cell cultures, transplantable neoplasms, and vaccines to prevent human exposure. serum banking and periodic serological testing of highrisk human populations, such as those working with lcmv experimentally, are recommended. research complications lcmv may stimulate or suppress immunological responses in vivo and in vitro, and it can replicate in cells used as targets or effectors for immunological studies. introduction of immune cells to a carrier animal may elicit an immunopathological response. immune complex disease can complicate longterm experiments and morphological interpretations. illness and death in mice and zoonotic risk to humans are obvious research-related hazards. etiology sv is a paramyxovirus that is antigenically related to human parainfluenza virus 1. viral particles are pleomorphic, contain single-stranded rna, and have a lipid solvent-sensitive envelope that contains glycoproteins with hemagglutinating, neuraminidase, and cell fusion properties. sv grows well on embryonated hens' eggs and in several mammalian cell lines (e.g., monkey kidney, baby hamster kidney , and mouse fibroblast [l]). virus replicates in the cytoplasm and by budding through cell outer membranes. once common in laboratory rodent populations, sv is now rare or absent (livingston and riley, 2003; pritchett-corning et al., 2009) . clinical signs clinically affected adult mice often assume a hunched position and have an erect hair coat. rapid weight loss and dyspnea occur, and there may be chattering sounds and crusting of the eyes. although highly susceptible adults may die, lethal infection is more common in suckling mice. sex differences in susceptibility have not been found. genetically resistant mice usually have subclinical infection. athymic mice and immunodeficient mice are at high risk for development of a wasting syndrome. they develop illness later than their immunocompetent counterparts, since clinical signs in immunocompetent mice are related to immune-mediated destruction of respiratory epithelium. opportunistic infections can complicate the clinical presentation. for example, secondary bacterial infections of the ear can cause vestibular signs. epizootiology sv is transmitted by aerosol and is highly contagious. morbidity in infected colonies is commonly 100%, and mortality can vary from 0% to 100%, partly because strains of mice vary greatly in their susceptibility to lethal sv infection. for example, c57bl/6 mice are highly resistant to clinically apparent infection, whereas dba/2 mice are highly susceptible. aerogenic infection is promoted by high relative humidity and by low air turnover. prenatal infection does not occur. enzootic infection is commonly detected in postweaned mice (5-7 weeks old) and is associated with seroconversion within 7-14 days and the termination of infection. therefore, entrenched infection is perpetuated by the introduction of susceptible animals. there is no evidence for persistent infection in immunocompetent mice, but prolonged infection is common in immunodeficient mice. maternally acquired immunity protects young mice from infection, and actively acquired immunity is thought to be long-lived. rats, hamsters, and guinea pigs also are susceptible to sv infection. therefore, bidirectional cross-infection is a risk during outbreaks. pathology viral replication is nominally restricted to the respiratory tract and peaks by the first week after infection. gross lesions feature partial to complete consolidation of the lungs (fig. 3.21) . individual lobes are meaty and plum-colored, and the cut surface may exude a frothy serosanguinous fluid. pleural adhesions or lung abscesses caused by secondary bacterial infection are seen occasionally, and fluid may accumulate in the pleural and pericardial cavities. sv targets airway epithelium and type ii pneumocytes. type i pneumocytes are less severely affected. histologically, the pattern of pneumonia is influenced by mouse genotype. susceptible mice usually have significant bronchopneumonia and interstitial pneumonia, whereas the interstitial component may be less prominent in resistant mice. typical changes begin with inflammatory edema of bronchiolar lamina propria, which may extend to alveolar ducts, alveoli, and perivascular spaces. necrosis and exfoliation of bronchiolar epithelium ensue, frequently in a segmental pattern (fig. 3.22 ). alveolar epithelium also may desquamate, especially in severe disease, and necrotic cell debris and inflammatory cells can accumulate in airways and alveolar spaces. alveolar septae are usually infiltrated by leukocytes to produce interstitial pneumonia (fig. 3.23) . lymphoid cells also invade peribronchiolar and perivascular spaces. the lymphocytic response to sv infection reflects the fact that cellular immunity contributes both to lesions and to recovery. local immunoglobulin synthesis by infiltrating cells also occurs. the extent of inflammatory cell infiltration corresponds to the level of genetic resistance expressed by the infected host, with clinically susceptible hosts mounting a more florid immune response than resistant hosts. additionally, strain-related differences in the severity of infection may reflect differences in airway mucociliary transport. multinucleated syncytia are occasionally seen in affected sucklings and scid mice, and inclusion bodies have been reported in infected athymic mice. regeneration and repair begin shortly after the lytic phase and are characterized by hyperplasia and squamous metaplasia of bronchial epithelium, which may extend into alveolar septae. proliferation of cuboidal laboratory animal medicine epithelium may give terminal bronchioles an adenomatoid appearance. repair of damaged lungs is relatively complete in surviving mice, but lymphocytic infiltrates, foci of atypical epithelium, and mild scarring can persist. acute phase lesions are prolonged in immunodeficient mice, which can lead to wasting and death. aged mice also have a prolonged recovery phase accompanied by focal pulmonary fibrosis (jacoby et al., 1994) . diagnosis sv is notable for its ability to cause epizootics of acute respiratory distress in adult genetically susceptible strains. serology is an effective means to detect infection in all strains of immunocompetent mice. antibody can be detected by 7 days postinfection and coincides with development of clinical signs related to the immune-mediated necrotizing bronchiolitis and alveolitis. repeated serologic sampling over several weeks can help stage infection within a population. alternatively, sentinel animals can be added to seropositive colonies to detect active infection. irrespective of serologic results, histopathology, immunohistochemistry (which can be performed on formalin-fixed, paraffin-embedded sections), and, where possible, virus isolation should be used to confirm infection. virus can be isolated from the respiratory tract for up to 2 weeks, with peak titers occurring at about 9 days postinfection. nasopharyngeal washings or lung tissue homogenates are most reliable and should be inoculated into embryonated hens' eggs or bhk-21 cell monolayer cultures. sv infection of cultured cells is non-cytolytic, so erythrocyte agglutination or antigen detection methods must be used. rt-pcr also can be used to detect virus in infected lungs. differential diagnosis respiratory infection caused by pneumonia virus of mice (pvm) is generally milder or subclinical. histologically, necrosis of airway epithelium is less severe. bacterial pneumonias of mice, including murine respiratory mycoplasmosis, are sporadic and can be differentiated morphologically and by isolation of causative organisms. because sv pneumonia may predispose the lung to opportunistic bacterial infections, the presence of bacteria should not deter evaluation for a primary viral insult. control and prevention sv infection is self-limiting in surviving immunocompetent mice. suckling mice from immune dams are protected from infection by maternal antibody until after weaning. control and eradication measures must eliminate exposure of susceptible animals, so that infection can 'burn out.' this is most easily accomplished by a quarantine period of 4-6 weeks wherein no new animals are introduced either as adults or through breeding. control also is aided by the fact that sv is highly labile. barrier housing is preferred for prevention and for control of transmission. vaccination with formalin-killed virus can provide short-term protection of valuable mice but is not commonly used for prevention. research complications sv can cause immunosuppression and can inhibit growth of transplantable tumors. this effect has been attributed to virus-induced modification of tumor cell surface membranes. pulmonary changes during sv pneumonia can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other bacteria. they also have been associated with breeding difficulties in mice. this sign is thought to be an indirect effect due to stress, fever, or related changes during acute infection. clinical signs natural pvm infection in mice is subclinical. therefore, its name is clinically misleading, being derived from pneumonic illness that occurred after serial passage of the agent in mice. however, dyspnea, listlessness, and wasting may develop in immunodeficient mice infected with pvm (weir et al., 1988) . pvm is used experimentally as a model to study acute respiratory infection, using highly pathogenic strains of the virus (dyer et al., 2012) . epizootiology pvm causes natural infections of mice, rats, hamsters, and probably other rodents and may be infectious for rabbits. serological data indicate that pvm was once common, but is now relatively uncommon (livingston and riley, 2003; pritchett-corning et al., 2009 ). pvm appears to spread less rapidly than sv. intimate contact between mice is probably required for effective transmission. this characteristic may reflect the fact that environmental inactivation of virus occurs rapidly. infection is acute and self-limiting in immunocompetent mice but may persist in immunodeficient mice. pathology pvm replicates exclusively in the respiratory tract and reaches peak titers in the lung 6-8 days after infection. although pulmonary consolidation can occur in experimentally infected mice, gross lesions are rare during natural infection. histological lesions can occur in the upper and lower respiratory tract. they consist of mild necrotizing rhinitis, necrotizing bronchiolitis, and interstitial pneumonia, which usually occur within 2 weeks after exposure to virus and are largely resolved by 3 weeks. the predominant inflammatory infiltrate is comprised of mononuclear cells, but some neutrophils laboratory animal medicine are usually present. immunohistochemistry on paraffinembedded tissues can be used to detect viral antigen in bronchiolar epithelium, alveolar macrophages, and alveolar epithelium during acute infection. residual lesions include nonsuppurative perivasculitis, which can persist for several weeks after acute infection has ceased. severe progressive pneumonia, with wasting, can occur in immunodeficient mice. it is characterized by generalized pulmonary consolidation that reflects severe interstitial pneumonia with desquamated alveolar pneumocytes and leukocytes filling alveolar spaces (fig. 3.24) . diagnosis diagnosis is based primarily on serological detection that can be supplemented by histopathology, immunohistochemistry, in situ hybridization, and virus isolation. virus replication in bhk-21 cells is detected by immunofluorescence or other antigen detection methods. virus also can be detected in tissues by rt-pcr. differential diagnosis because pvm is antigenically distinct from other murine viruses, serology is the most useful method to separate pvm infection from other respiratory infections of mice. however, in immunodeficient mice, where clinical signs and lesions are typical, it must be differentiated from other pneumonias, especially those due to sv and pneumocystis. additionally, pvm can coexist with and exacerbate pneumocystis infection in immunodeficient mice (bray et al., 1993) . prevention and control pvm infection is acute and self-limiting in immunocompetent mice, but persistent in immunodeficient mice. seropositive mice should be viewed as either immune or in the final stages of acute infection. therefore, control and prevention follows guidelines applicable to sv infection. research complications pvm can exacerbate pneumocystosis, as noted above. (ward et al., 2007) two members of the family reoviridae infect laboratory mice: reovirus per se (species: mammalian orthoreovirus) and murine rotavirus (species: rotavirus a), also known as epizootic diarrhea of infant mice (edim) virus. etiology reoviruses of mammals, although taxonomically considered one type species, have been divided into three cross-reacting prototypic serotypes: reovirus 1, 2 and 3, which can be differentiated by cross-serum neutralization. mice can be infected with any serotype, but reovirus 3 is emphasized because it has been associated with naturally occurring disease. natural infections in mice are usually not caused by pure serotypes, because reoviruses actively recombine. a number of wild-type and laboratory strains have been characterized, and related viruses have been recovered from virtually every mammal tested, as well as birds, reptiles, and insects. the virion contains segmented, double-stranded rna and is relatively heat stable. reoviruses replicate well in bhk-21 cells and other continuous cell lines, as well as in primary monolayer cultures from several mammals. clinical signs clinical disease is rare and age dependent. acute disease affects sucklings at about 2 weeks of age, whereas adults have subclinical infection. signs in sucklings include emaciation, abdominal distension, and oily, matted hair due to steatorrhea. icterus may develop and is most easily discerned as discoloration in the feet, tail, and nose. incoordination, tremors, and paralysis occur just before death. convalescent mice are often partially alopecic and are typically runted. alopecia, runting, and icterus may persist for several weeks, even though infectious virus can no longer be recovered. infants born to immune dams are protected from disease by maternal immunity. epizootiology the prevalence of reovirus 3 infection in contemporary mouse colonies is rare (livingston and riley, 2003; pritchett-corning et al., 2009 ). reoviruses are highly contagious among infant mice and can be transmitted by the oral-fecal or aerosol routes, but mechanical transmission by arthropods has also been documented. additionally, virus may be carried by transplantable neoplasms and transmitted inadvertently by injection. transmission is inefficient among adult mice. there is no evidence that vertical transmission is important or that genetic resistance or gender influence expression of disease. infection in immunocompetent mice appears to be self-limiting, lasting up to several weeks but terminating with the development of host immunity. the course of infection in immunodeficient mice should be considered prolonged, but the duration has not been determined. pathology reovirus 3 can cause severe pantropic infection in infant mice. after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following ingestion, reoviruses gain entry by infecting intestinal epithelial cells (m cells) that cover peyer's patches. virus can be carried to the liver in leukocytes, where it is taken up by kupffer cells prior to infecting hepatocytes. in acute disease, livers may be large and dark, with yellow foci of necrosis. the intestine may be red and distended, and, in infants, intestinal contents may be bright yellow. myocardial necrosis and pulmonary hemorrhages have been reported. myocardial edema and necrosis are especially prominent in papillary muscles of the left ventricle. the brain may be swollen and congested. central nervous system lesions have a vascular distribution, and are most prevalent in the brain stem and cerebral hemispheres. neuronal degeneration and necrosis are followed quickly by meningoencephalitis and satellitosis. severe encephalitis may evoke focal hemorrhage. in the chronic phase, wasting, alopecia, icterus, and hepatosplenomegaly may persist. orally infected suckling mice can develop multifocal hepatocyte necrosis, which may include the accumulation of dense eosinophilic structures resembling councilman bodies. hepatocytomegaly, kupffer cell hyperplasia, and intrasinusoidal infiltrates of mononuclear cells and neutrophilic leukocytes also can develop. in experimentally inoculated mice, necrotic foci can persist in the liver for at least 4 weeks. chronic active hepatitis may develop after acute infection and result in biliary obstruction. acinar cells of the pancreas and salivary glands can undergo degeneration and necrosis. because pancreatic duct epithelium is susceptible to infection, parenchymal lesions in the pancreas may be caused by obstruction rather than by viral invasion of parenchyma. pulmonary hemorrhage and degeneration of skeletal muscles also have been observed. both humoral and cellular immunity seem to participate in host defenses, but it is unclear how host immunity may influence the course of chronic infection. oronasal inoculation of infant mice with reovirus 1 results in a similar distribution but significantly milder lesions compared to reovirus 3. in contrast, reovirus 2 is highly enterotropic, inducing mild enteritis without lesions in other tissues, similar to epizootic diarrhea of infant mice (edim) . diagnosis serology uses reovirus 3 as antigen, which detects seroconversion to all serotypes, and viral rna can be detected by rt-pcr. a presumptive diagnosis of reovirus infection is aided clinically by detection of the oily hair effect, accompanied by jaundice and wasting. the presence histologically of multisystemic necrosis is consistent with severe reovirus 3 infection but should be confirmed by immunohistochemistry or virus isolation. differential diagnosis reovirus infection must be differentiated from other diarrheal diseases of infant mice, including those caused by mouse coronaviruses, edim virus, salmonella spp., or clostridium piliforme. prevention and control although surviving mice appear to recover completely from infection, the potential for a carrier state is unresolved. therefore, it may be necessary, after adequate testing for the continued presence of virus by the use of sentinels, map testing, or other appropriate means, to rederive or replace infected stock. prevention depends on adequate barrier husbandry coupled with adequate serological monitoring. research complications reovirus 3 infection can interfere with research in several ways. infections in breeding colonies can result in high mortality among sucklings from nonimmune dams. virus has been commonly recovered from transplantable neoplasms and is suspected of being oncolytic. the potential exists for interference with hepatic, pancreatic, cardiovascular, or neurological research. etiology rotaviruses are double-stranded, segmented rna viruses that have a wheel-like ultrastructural appearance. edim virus is a group a rotavirus that replicates in differentiated epithelial cells of the small intestine by budding into cisternae of endoplasmic reticulum. currently, only a single antigenic strain is recognized, but antigenically distinct variants may exist. edim virus shares an inner capsid antigen with rotaviruses of rabbits, fowl, nonhuman primates, human beings, and domestic and companion animals. these agents tend to be species-specific under natural conditions and can be differentiated by serum neutralization tests. cultivation of edim virus requires the presence of proteolytic enzymes to cleave an outer capsid polypeptide. clinical signs clinical signs occur in infant mice less than 2 weeks old. this age-related susceptibility also applies to infection in immunodeficient mice. furthermore, clinical signs occur only in offspring of nonimmune dams, because maternal immunity protects infants until they have outgrown susceptibility to clinical disease. the cardinal signs are bloated abdomens with fecal soiling of the perineum, which may extend to the entire pelage in severe cases. despite high morbidity, mortality is low because affected mice continue to nurse. transient weight loss does occur, and there may be a delay in reaching adult weight. recovery from infection usually occurs in about 2 weeks and, once weight is regained, is clinically complete. epizootiology edim virus appears to be infectious only for mice and occurs episodically in mouse colonies, and infection is probably widespread geographically (livingston and riley, 2003; pritchett-corning laboratory animal medicine et al., 2009) . all ages and both sexes can be infected, but genetic resistance and susceptibility have not been determined. the virus is highly contagious and is transmitted by the oral-fecal route. subclinically infected adult mice can shed virus in feces for at least 17 days, an interval that may be extended in immunodeficient mice. after oral inoculation, virus is essentially restricted to the gastrointestinal tract, although small amounts of virus may be present in the liver, spleen, kidney, and blood. nursing dams can contract infection from their litters. transplacental transmission has not been demonstrated. pathology gross lesions occur primarily in the gastrointestinal tract, but thymic involution can result from infection-related stress. the intestine is often distended, flaccid, and filled with gray-green gaseous liquid or mucoid fecal material that soils the pelage. the stomach contains curdled milk, except in terminal cases with anal impaction due to caking of dried feces. virus preferentially infects terminally differentiated enterocytes in the small and large intestines, which accounts for the agerelated susceptibility to disease; the number of such cells decreases as the intestinal tract matures. characteristic histological lesions are often very subtle, but are most easily discerned in the small intestine in mice less than 2 weeks old. they consist of vacuolation of villar epithelial cells with cytoplasmic swelling, which give villi a clubbed appearance (fig. 3.25) . the vacuoles must be differentiated from normal absorption vacuoles in nursing mice. the lamina propria may be edematous, but necrosis and inflammation are not prevalent. diagnosis edim virus infection is readily detected serologically. clinical disease is diagnosed from signs and typical histological lesions in the intestine, which can be confirmed by immunohistochemical or ultrastructural demonstration of virus in the intestine or in intestinal filtrates or smears. rotavirus antigen can be detected in feces by elisa, but certain dietary ingredients can cause false-positive reactions. infection can also be diagnosed by rt-pcr. differential diagnosis edim virus infection must be differentiated from other diarrheal diseases of suckling mice such as intestinal coronavirus (mouse hepatitis virus) infection, reovirus 3 infection, tyzzer's disease, and salmonellosis. the presence of milk in the stomach can be helpful in differentiating edim virus infection from more severe enteric infections, such as those caused by pathogenic coronaviruses, during which cessation of nursing often occurs. the possibility of dual infections must also be considered. thymic necrosis in edim virus-infected mice, although nonspecific, must be differentiated from that due to mouse thymic virus (mtv) infection or other stressors. prevention and control the spread of edim can be controlled effectively by the use of microbarrier cages and good sanitation. because infection appears to be acute and self-limiting, cessation of breeding for 4-6 weeks to allow immunity to build in adults while preventing access to susceptible neonates also is recommended. alternatively, litters with diarrhea can be culled, in combination with the use of microbarrier cages. the duration of infection in immunodeficient mice has not been determined, but it is reasonable to assume that chronic infection occurs. therefore, such animals should be eliminated. litters from immune dams are more resistant to infection. if edim virus is allowed to become enzootic within a colony, clinical signs will disappear within the population, which may be an appropriate management approach in conventional colonies. prevention of edim virus infection depends on maintenance of sanitary barrier housing with adequate serological surveillance. research complications the research complications of edim infection pertain to clinical illness with diarrhea and retarded growth. transient thymic necrosis may perturb immunological responses. infection (barthold, 1997a,b) etiology coronaviruses are large, pleomorphic, enveloped rna viruses with radially arranged peplomers (spikes). in mice, early clinical and laboratory investigations emphasized their potential to induce hepatitis, so their original designation, which is still used actively, is mouse hepatitis virus (mhv). during that time, enteritis in infant mice was recognized as a separate entity caused by an uncharacterized virus, known as lethal intestinal virus of infant mice (livim). subsequent studies revealed that hepatitis-causing mhv and enteritis-causing livim were closely related coronaviruses, now collectively termed mhv. mhv isolates differ in biologic behavior according to their organ tropism into two biotypes: enterotropic strains, which infect primarily the intestinal tract, and polytropic strains, which initially infect the respiratory tract but may progress to multisystemic dissemination, including the liver and brain. these differences are often reflected in their cell tropism in vitro. however, natural isolates may contain features of both biotypes. several prototype polytropic strains have been extensively studied as experimental models of hepatitis and encephalitis. they include jhm (mhv4), mhv-1, mhv-3, mhv-s, and mhv-a59. numerous additional strains have been identified that differ in virulence, tissue tropism, and antigenicity. differentiation by strain, particularly under natural conditions, is irrelevant, since mutation is common among coronaviruses, and even named prototype strains differ significantly depending upon passage history. although mhv isolates and strains share internal antigens (m and n), they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens. mhv shares antigens with the coronaviruses of rats, a finding that has been exploited to develop heterologous antigens for serological tests. mhv also is related to human coronavirus oc43. a number of established cell lines may be used for propagating polytropic mhv strains in vitro. however, field isolates are difficult to maintain in vitro. nctc 1469 mouse liver cells are useful for growing many polytropic strains. mhv can also be grown in mouse macrophages, cells that have been used for genetic studies of resistance and susceptibility to infection. enterotropic strains, because of their tendency to be strictly enterotropic, have been grown in cmt-93 cells derived from a rectal carcinoma in a c57bl mouse, but are generally difficult to propagate in cell culture. irrespective of cellular substrate used for isolation or propagation, syncytium formation is emblematic of mhv infection (fig. 3.26) . clinical signs clinical signs depend primarily on the age, strain, and immunological status of infected mouse and strain and tropism of virus. as with many murine viruses, infection is often clinically silent among immunologically competent mature mice. clinical morbidity is most often associated with suckling mice less than 2 weeks old or with immunodeficient mice. suckling mice infected with enterotropic mhv develop inappetence, diarrhea, and dehydration, often terminating in death (fig. 3.27) . epizootics of enterotropic mhv have been known to result in 100% mortality among neonatal mice in a breeding colony. older mice (2-3 weeks of age) may have ruffled pelage and runting. neurotropic strains such as mhv-jhm may induce flaccid paralysis of the hindlimb, but this sign is rarely encountered alone during natural infection. conjunctivitis, convulsions, and circling may be seen occasionally. enterotropic strains may not cause acute disease in athymic mice when exposed as adults, whereas mildly pathogenic polytropic strains can cause a progressive wasting syndrome that may be accompanied by progressive paralysis. epizootiology mhv infection, despite constant surveillance and preventive programs, continues to be a common threat to laboratory mouse populations (livingston and riley, 2003; pritchett-corning et al., 2009 ). there are no reports of natural transmission from mice to other species, but suckling rats have been found to develop necrotizing rhinitis after intranasal inoculation with mhv-s. mhv is highly contagious, with natural transmission occurring by respiratory or oral routes. mouse appears normal and has a milk-filled stomach. lower mouse is runted and dehydrated and has an empty stomach. from barthold et al. (1982) . enterotropic biotypes predominate in natural infections in contemporary laboratory animal facilities, since they tend to be the most contagious due to copious excretion of virus in feces, whereas polytropic strains generally spread by direct respiratory contact. natural vertical transmission has not been demonstrated. introduction of mhv through injection of contaminated biologicals can be an important factor in epizootics, especially because some isolates infect b lymphocytes and, by implication, hybridomas nonlytically. infection in immunocompetent mice is self-limiting. immune-mediated clearance of virus associated with seroconversion usually begins about a week after infection, and mice recover fully within 3-4 weeks. humoral and cellular immunity participate in host defenses to infection, and t-cell-dependent immunity is an absolute requirement. thus, age-related resistance to mhv correlates with maturation of lymphoreticular tissues, but intestinal proliferative kinetics are critical determinants of disease susceptibility with enterotropic mhv. enzootic infection had been construed to include persistent infection in individual mice. current evidence suggests, however, that enzootic infection results either from the fresh and continuous introduction of immunologically naive or deficient mice or from the recurrent infection of immune mice with mhv variants that arise by natural mutation. mutation is favored by immune pressure in enzootically infected colonies as well as missteps during natural replication, which include copying errors and recombination. thus, mice that have developed immunity to one strain of mhv can remain susceptible to one or more genetically and antigenically divergent strains, resulting in reinfection. this caveat has practical importance for breeding colonies. maternal immunity protects suckling mice against homologous mhv strains but not against antigenically variant strains. however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine. therefore, the susceptibility of young mice to infection increases significantly at weaning. strain differences in resistance and susceptibility to polytropic mhv can be inherited as an autosomal dominant trait. for example, dba/2 mice are highly susceptible to mhv-3 and die acutely even as adults, whereas a/j mice develop resistance to lethal infection shortly after weaning. however, genetic resistance is also virus strain-dependent. therefore, mice resistant to one strain of mhv may be susceptible to another strain. it also is worth noting that the expanded use of genetically altered mice with novel or unanticipated deficits in antiviral responses may alter the outcome of virus-host interactions unpredictably. this pertains to mhv as well as other agents. for example, mhv infection has presented as granulomatous peritonitis and pleuritis in interferongamma (ifn-γ) knockout mice (france et al., 1999) . pathology polytropic strains replicate initially in the nasal mucosa, where necrotizing rhinitis may occur. viremic dissemination can follow if virus gains access to regional blood vessels and lymphatics. thus, viremia leads to secondary infection of vascular endothelium and parenchymal tissues in multiple organs including liver, brain, lymphoid organs, and other sites. mice also may develop central nervous system disease by direct extension of infection from the olfactory mucosa along olfactory tracts. at necropsy, yellow-white foci indicative of necrosis can occur in multiple tissues, with the involvement of the liver as the classical lesion. liver involvement may be accompanied by icterus and peritonitis. histologically, necrosis can be focal or confluent and may be infiltrated by inflammatory cells (fig. 3.28) . syncytia commonly form at the margin of necrotic areas and, in mild infections, may develop in the absence of frank necrosis. syncytia formation is a hallmark of infection in many tissues, including the intestine (fig. 3.26) , lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow and in vascular endothelium in general. although syncytia are transient in immunocompetent mice, they are a persistent feature in chronically infected, immunodeficient mice ( fig. 3.29) . neurotropic variants cause acute necrotizing encephalitis or meningoencephalitis in suckling mice, with demyelination in the brain stem and in peri-ependymal areas secondary to viral invasion of oligodendroglia. convalescent mice may have residual mononuclear cell infiltrates around vessels or as focal lesions in the liver. immunodeficient mice can develop progressive necrotic lesions in the liver and elsewhere. compensatory splenomegaly may occur because of expansion of hematopoietic tissue. enterotropic strains infect primarily the intestine and associated lymphoid tissues, although some may also figure 3.28 necrosis, inflammation, and syncytium in the liver of a mouse infected with mhv. courtesy of s.w. barthold. cause systemic lesions, especially in the liver and brain. the most common sites are terminal ileum, cecum, and proximal colon. the severity of disease is age-related, and dependent upon intestinal proliferative kinetics, similar to edim, with young infants being at highest risk for lethal infection. pathogenic strains can cause lesions ranging from villus attenuation to fulminant necrotizing enterotyphlocolitis, which can kill suckling mice within a few days (fig. 3.27) . the stomach is often empty, and the intestine is filled with watery to mucoid yellowish, sometimes gaseous contents. syncytia are a consistent feature in viable mucosa (fig. 3.30) and not only are formed in intestine but also may be present in mesenteric lymph nodes and endothelium of mesenteric vessels. enterocytes may contain intracytoplasmic inclusions, but they are not diagnostic. surviving mice develop compensatory mucosal hyperplasia, which eventually recedes, but may contribute to clinical signs due to osmotic, secretory, and malabsorptive diarrhea. older mice are equally susceptible to infection, but are resistant to severe disease due to their mature (more rapid) intestinal proliferative kinetics. pathology may be subtle, consisting of transient syncytia without necrotic lesions. in adult mice, syncytia can be found most often in the surface mucosal epithelium of the ascending colon. the exception occurs in immunodeficient mice, such as athymic and scid mice, which can develop chronic proliferative bowel disease of varying severity with mhv antigen in mucosal epithelium (figs. 3.31 and 3.32). this may not always be present, as athymic nude mice exposed as adults may only manifest a few enterocytic syncytia without hyperplasia. diagnosis because mhv infection is often subclinical, serological testing is the most reliable diagnostic tool. many animal resources rely on sentinel mouse protocols for continuous serological surveillance. serology is well established, sensitive, and reliable. neutralization tests are used to differentiate individual virus strains in the research laboratory but are inappropriate for routine use, because of cost, technical complexity, and serologic identification per se does not predict biological behavior, including virulence or tissue tropism. serology also can be used in the context of map testing in which adult mice are inoculated with suspect tissues to elicit seroconversion. rt-pcr protocols to detect virus in tissues or excreta are available. the detection of syncytia augmented, when possible, by immunohistochemistry to laboratory animal medicine detect mhv antigen is a useful and practical means to confirm infection. this strategy should attempt to select mice that are in early stages of infection, because necrosis in infant mice or seroconversion in older mice may reduce the chances of detecting syncytia or viral antigens. the option of using immunodeficient mice as sentinels can be considered, because they sustain prolonged infection. however, they should be securely confined because they also amplify virus loads. if properly controlled, amplification in immunodeficient mice can, however, facilitate subsequent virus isolation in tissue culture. differential diagnosis mhv infection must be differentiated from other infectious diseases that cause diarrheal illness, runting, or death in suckling mice and wasting disease in immunodeficient mice. these include edim, mousepox, reovirus 3 infection, tyzzer's disease, and salmonellosis. neurological signs or demyelinating lesions must be differentiated from mouse encephalomyelitis virus infection or noninfectious cns lesions, such as neoplasms, including polyoma virus-induced tumors in athymic mice. prevention and control control and prevention of mhv infection can be difficult because of the numerous variables that influence its expression. perhaps the most important factor is the duration of infection in individual mice and in mouse colonies. there is evidence that infection in an individual immunocompetent mouse is acute and self-limiting. such mice can be expected to develop immunity and eliminate virus within 30 days. therefore, selective quarantine at the cage (not room) level with the temporary cessation of breeding can be used effectively to eliminate infection. quarantine at the room basis is likely to fail, since mutations arise and continually reinfect the mouse population. additionally, maternally derived immunity can protect infant mice from infection until they are weaned and moved to uncontaminated quarters. careful testing with sentinel mice should be used to assess the effectiveness of quarantine or 'natural rederivation,' as just described. immunodeficient mice, in contrast, are susceptible to chronic infection and viral excretion. mice with unrecognized or unanticipated immune dysfunction or with selective immune dysfunction may impact on mhv infection and its control. such colonies, which may contain highly valuable or irreplaceable mice, may be rescued by cesarean rederivation or embryo transfer if vertical transmission of mhv infection is subsequently ruled out. although rodent coronaviruses are not viable for extended periods in the environment, excreted virus may remain infectious for up to several days, so proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. the prevention of mhv requires procurement of animals from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance program. control of feral mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbor virus (e.g., transplantable neoplasms, cell lines) are also important strategies to prevent adventitious infection. research complications numerous research complications have been attributed to mhv, and the unpredictable outcome of infection in genetically altered mice is likely to lengthen the list. for example, apart from its clinical impact, mhv may stimulate or suppress immune responses, contaminate transplantable neoplasms, and be reactivated by treatment of subclinically infected animals with several classes of drugs, including immunosuppressive agents, and by intercurrent infections. it also can alter tissue enzyme levels. additionally, the ubiquitous threat of mhv infection and uncertainty about its potential effects on a given research project provoke concerns that may exceed its true impact. for example, transient infection with a mild enterotropic strain is unlikely to disrupt systemic immune responses, whereas infection with a polytropic strain may be highly disruptive. this is not to say that subclinical or strictly enterotropic infection should be taken lightly but simply to caution against overreaction in assessing the impact of an outbreak. etiology mice are susceptible to infection by two members of the cardiovirus genus within the theilovirus. emcv has a less selective host range and can infect wild mice, but is not known to infect laboratory mice. tmev is a small, nonenveloped, rna virus that was discovered by max theiler during experimental studies of yellow fever virus in mice. established prototype strains include to (theiler's original), fa, da, and gd vii, the last of which is named after george martine (george's disease), an assistant in theiler's laboratory. tmev is rapidly destroyed by temperatures over 50°c and by alcohol but not by ether. it can be cultivated in vitro in several continuous cell lines, but bhk-21 cells are routinely used for isolation and propagation. tmev is antigenically related to emcv. as with other nonenveloped viruses, tmev is resistant to environmental inactivation, a factor that must be considered in control and prevention of infection. clinical signs the development of clinical disease depends on virus strain, mouse strain, and route of exposure, but natural disease is exceedingly rare (estimated at 0.1-0.01% of infected mice). when clinical signs occur, they are expressed as neurological disease. the characteristic sign is flaccid posterior paralysis, which may be preceded by weakness in the forelimbs or hindlimbs, but in mice that are otherwise alert (fig. 3.33) . some mice may recover, but death frequently ensues, often because of failure to obtain food or water. furthermore, mice that recover from the paralytic syndrome are disposed to a chronic demyelinating phase, which is expressed as a gait disturbance. epizootiology infection occurs primarily in laboratory mice with the exception of the mgh strain, which has been isolated from laboratory rats and is pathogenic in mice and rats after experimental inoculation. the prevalence of tmev in mouse colonies is low, a reflection of the slow rate at which virus is transmitted from mouse to mouse, but it continues to be among the more common viral contaminants of mouse colonies (livingston and riley, 2003; pritchett-corning et al., 2009) . tmev infection is acquired by ingestion and replicates primarily in the intestinal mucosa. enteric infection can persist after the development of host immunity and can result in chronic or intermittent excretion of virus in feces over several months . mice often become infected shortly after weaning, but virus is seldom recovered in mice over 6 months of age. however, neurologic infection can persist in the brain and spinal cord for at least 1 year. immunity to one strain of tmev provides cross-protection to other strains. there are no reports of differences in mice with respect to susceptibility to infection under natural conditions. prenatal transmission has not been found. pathology intestinal tmev infection does not cause lesions, but virus can be detected in enterocytes by immunohistochemistry or in situ hybridization. poliomyelitis-like disease, the syndrome that may be encountered during natural infections, is characterized by acute necrosis of ganglion cells and neurons, neuronophagia, and perivascular inflammation, which occur particularly in the ventral horn of the spinal cord gray matter but also can involve higher centers such as the hippocampus, thalamus, and brain stem. during the subsequent demyelinating phase, mononuclear cell inflammation develops in the leptomeninges and white matter of the spinal cord, accompanied by patchy demyelination. the white-matter lesions are due to immune injury. spontaneous demyelinating myelopathy, affecting the thoracic spinal cord and associated with mev infection, has also been reported in aged mice. virulent strains may cause acute encephalitis after experimental inoculation, whereas less virulent isolates produce acute poliomyelitis followed by chronic demyelinating disease. diagnosis infection is usually detected serologically or by pcr of feces, but virus shedding from infected mice may be intermittent. clinical signs are striking, if they occur, but are too rare to rely on for routine diagnosis. histological lesions in the cns and especially the spinal cord are characteristic when present. differential diagnosis neurotropic variants of mhv may, on occasion, cause similar neurological signs. injury or neoplasia affecting the spinal cord can also produce posterior paralysis. polyoma virus infection in athymic mice can induce tumors or demyelination in the cns, which may result in clinical signs resembling those of tmev infection. prevention and control disease-free stocks were originally developed by foster-nursing infant mice. this technique, cesarean rederivation, or embryo transfer can be used successfully to eliminate infection. in either case, foster mothers should be surveyed in advance to ensure their mev-free status. selective culling can be considered as an option to eliminate infection, because laboratory animal medicine infection spreads slowly. however, the virus is hardy in the environment and resists chemical inactivation, so it may be prudent to depopulate and disinfect rooms if the presence of infection is unacceptable. research complications the principal hazard from tmev for research relates to its potential effects on the cns. noroviruses are nonenveloped rna viruses that belong to the family caliciviridae. they are notoriously resistant to environmental inactivation, and cause significant gastrointestinal morbidity in humans. noroviruses are species-specific, including mnv, which exclusively infects mice. until the discovery of mnv, replication of noroviruses in vitro has not been possible. for this reason, mnv has emerged as an important small animal model of norovirus pathogenesis. mnv was relatively recently discovered in 2003, and subsequent surveillance has revealed that it is the most common adventitious virus infection in laboratory mice (hsu et al., 2005; pritchett-corning et al., 2009) . over 35 mnv isolates have been found in mouse research colonies around the world, which display nearly 90% genetic identity, comprising a single genetic cluster. although genetically homogeneous, significant biological differences exist among mnv strains (thackray et al., 2007) . mnv effectively replicates in macrophages and dendritic cells, including the mouse macrophage-like raw264.7 cell line, as well as a microglial cell line (wobus et al., 2004) . clinical signs clinical signs of infection in immunocompetent mice are usually absent, but infection leads to systemic disease with high mortality in interferon αβγ receptor and stat1 null mice. affected mice have loss of body weight, ruffled fur, and hunched posture (ward et al., 2006) . experimental infection of 129 and c3h mice with mnv-1 caused mild diarrhea (kahan et al., 2011) . epizootiology mnv is transmitted by the fecaloral route, and contaminates the environment as an environmentally resistant virus. for this reason, it can efficiently infect sentinel mice with soiled bedding (manuel et al., 2008) . duration of infection varies with mnv strain, mouse immunocompetence and mouse genotype. experimental studies have revealed that several mnv strains persist in various tissues of c57bl/6j, hsd:icr, and jcl:icr and c.b-17-prkdc scid mice, with fecal shedding for at least 35-60 days (goto et al., 2009; hsu et al., 2006; thackray et al., 2007) . although not clinically ill, rag1 null mice are unable to clear infection . comparative studies with mnv-1 and mnv-3 have shown differences in virus replication and shedding (kahan et al., 2011) . mnv has a tropism for macrophages and dendritic cells, and virus can be detected in the intestine, intestinal lymphoid tissue, liver, and spleen (hsu et al., 2006; kahan et al., 2011; wobus et al., 2006) . pathology naturally and experimentally infected stat1 or ifnγr null mice may develop splenomegaly and multifocal pale spots on the liver. microscopic findings include varying degrees of hepatitis, focal interstitial pneumonia, vasculitis, peritonitis, and pleuritis (karst et al., 2003; ward et al., 2006) . encephalitis, cerebral vasculitis, pneumonia, and hepatitis have also been described in intracerebrally infected stat1 null mice (karst et al., 2003) . infection of immunocompetent mice may be associated with mild inflammation of the intestine, splenic hypertrophy, and lymphoid hyperplasia of spleen and lymph nodes (mumphrey et al., 2007) . diagnosis mnv infection can be detected by serology or rt-pcr. sentinel mouse surveillance, using soiled bedding, is an effective strategy for detecting mnv (manuel et al., 2008) differential diagnosis the mild change in fecal consistency associated with mnv in adult mice may mimic rotavirus, coronavirus, helicobacter spp., citrobacter rodentium, or other enteric diseases. disseminated lesions in stat1 or ifnγr null mice must be differentiated from other polytropic viral diseases in immunodeficient mice, including mhv. prevention and control depopulation and decontamination has been shown to be effective at eliminating mnv from an enzootically infected colony, whereas testand-removal of positive mice was found to be ineffective (kastenmayer et al., 2008) . embryo transfer and cesarean rederivation are also effective (goto et al., 2009; perdue et al., 2007) . neonatal mice are resistant to infection, so that cross-fostering neonates onto uninfected dams is another effective means of rederivation mnv-free mice (artwohl et al., 2008; compton, 2008) . research complications the tropism of mnv for macrophages and dendritic cells is likely to modify immune responses, and mnv infection may interfere with studies involving enteric disease. hantaviruses are rna viruses belonging to the very large bunyaviridae family. they differ from other members of this family by not being arthropod-borne. each hantavirus is antigenically distinct and maintained within single or at most a few rodent or insectivore hosts, but are infectious for other hosts. infection is lifelong, and virus is transmitted by shedding of virus in urine, feces, and saliva. several hantaviruses are zoonotic and may cause severe disease in humans. although there is overlap, hantaviruses in asia and europe cause hemorrhagic fever with renal syndrome (hfrs) in humans, a multisystem disease with significant renal involvement, and hantaviruses that are endemic in the americas cause laboratory animal medicine hantavirus pulmonary syndrome (hps) in humans, which is a multisystem disease with pulmonary involvement. among the better-known old world hfrs hantaviruses are hantaan, seoul, puumala, and dobrava-belgrade viruses. sin nombre virus is the best known new world phs hantavirus, among many others. most notably from the perspective of laboratory animal medicine, the norway rat, rattus norvegicus, serves as a reservoir host for hantavirus in the wild, but infection has also been associated with laboratory rats. in addition to being endemic in wild rats in asia, it has been found to be endemic in wild rats in the eastern united states and associated with human cases of hfrs (childs et al., 1988; leduc et al., 1984; tsai et al., 1985) . over 120 cases of hantavirus infection have been transmitted to humans from laboratory rats in japan, belgium, and the united kingdom (desmyter et al., 1983; kawamata et al., 1987; lloyd et al., 1984; umenai et al., 1979) . m. musculus is not considered to be a primary reservoir host, but hantavirus infection has been documented serologically in conventional and barrier-maintained laboratory mice and rats in korea (won et al., 2006) , infection of wild m. musculus has been documented in the united states (baek et al., 1988) , and infection of wild mice in europe has been associated with human exposure (diglisic et al., 1994) . hantaviruses are difficult to culture in vitro. infection in rodents is subclinical and is detected by serology or rt-pcr. the main research complication from natural infection is the zoonotic risk and potentially subclinical effects on the immune response associated with viral defenses such as cd8 + t cell (taruishi et al., 2007) and nk function as demonstrated in human studies (braun et al., 2014) . the mouse is host to a number of enveloped rna viruses of the family retroviridae, subfamily orthovirinae, including the two type species (and their variants) mouse mammary tumor virus (mmtv) and murine leukemia virus (mlv). these viruses belong to a diverse assemblage of related mobile dna elements that are integrated into the host genome, and collectively termed 'retroelements', which include retrovirus-related elements and nonviral elements. during cell division, retroelements are transcribed into rna, and subsequently reverse-transcribed into dna copies that become integrated into a new location within the genome. this process utilizes reverse transcriptase, which is encoded by the retroelement. over millennia, retroelements have been repeatedly integrated within the genome in large numbers, comprising approximately 40% of the mammalian genome. various families of mouse retroelements share sequence similarity, despite their random distribution throughout the mouse genome, and the majority of them are truncated, mutated, and methylated to become incapable of infectivity. nevertheless, many of them continue to be mobile within the genome. noninfectious retrovirus-related retroelements include iap, vl30, musd, and etn elements. replication-competent retroviruses represent the pinnacle of the retroelement constellation and are best considered as the most evolutionarily recent members. these include mmtv and mlv. mtv and mlv share similar genetic structure, except that the long terminal repeat (ltr) region of the mmtv genome encodes an additional superantigen (sag). both mmtv and mlv include exogenous viruses, which are horizontally transmitted, replication-competent viruses, and endogenous viruses, which are closely related to exogenous viruses, encoded within the mouse genome, and transmitted by mendelian inheritance. exogenous mmtv and mlv exist in wild mouse populations, but have been eliminated from contemporary laboratory mice. however, they may continue to be used experimentally, including bittner mmtv, and gross, friend, moloney, and rauscher mlvs. in particular, mouse colonies may be purposely infected with mmtv for mammary cancer research and are termed 'mmtv-positive', reflecting their exogenous virus status, even though the mice may also carry endogenous mmtv. the genomes of all inbred strains of mice encode one or more (over 50 in some mouse strains) endogenous mmtv loci, the distribution of which is unique to each inbred strain of mouse. most mmtv genomic loci do not encode infectious virus or are transcriptionally inactive, except for mouse strains (dba, c3h, grs) that carry mtv1 or mtv2 loci. these loci encode infectious virus, which can be visualized as b-type particles by electron microscopy. likewise, all mouse strains carry endogenous mlv loci within their genome, but not all mice carry replication-competent mlv sequences. some endogenous mlvs encode infectious virus, which can be visualized as c-type particles by electron microscopy. mice have often evolved mechanisms to counter the deleterious effects of retroviruses by preventing reentry or replication of virus into other cells. if an endogenous retrovirus is still infectious to other mouse cell targets, it is termed ecotropic, whereas if it is no longer infectious for mouse cells, but can infect cells of other species, it is termed xenotropic. viruses capable of infecting cells of mice as well as other species are termed polytropic. the combinations of endogenous replication-competent mlvs and cell tropism factors are a reflection of selective breeding of mouse strains for susceptibility to various types of cancer. clinical signs mice were originally inbred for specific phenotypes, including mammary tumors and lymphomas. thus, some strains of mice were genetically selected for unique combinations of endogenous mmtv and mlv in concert with susceptibility factors laboratory animal medicine that favored their expression and disease manifestations. in addition, noninfectious retroelements continue to reintegrate randomly within the genome during cell division as retro transposons. these ongoing integrations contribute to genetic drift, spontaneous mutations, and well-recognized mouse strain phenotypes, including the athymic nude allele, the hairless allele, and the rodless retina allele, among others. epizootiology exogenous mmtv and mlv are horizontally transmissible, primarily through the milk of lactating females. endogenous retroviruses and retroelements are inherited through the genome. replicationcompetent endogenous mmtvs and mlvs are also transmissible like their exogenous counterparts, but differ by being integrated within the genome of the mouse. pathology replication-competent mmtv and mlv, regardless of their exogenous or endogenous origin, are usually clinically silent. their ability to cause neoplasia is a reflection of genetic selection for susceptibility factors that are genetically encoded within individual mouse strain genomes. mmtv derives its name from its association with induction of mammary carcinomas in mammary cancer-susceptible strains of mice. mlv is associated with lymphomas, the pattern of which is mouse strain specific. for example, akr mice develop 100% prevalence of thymic lymphoma between 6 and 12 months of age, whereas aging balb/c mice commonly develop multicentric lymphoma. in these strains of mice, multiple endogenous mlvs are coexpressed in tissues and undergo recombination events that allow them to target and transform cells into neoplasia. despite its name, mmtv can induce lymphomas in some strains of mice, such as sjl mice which develop lymphomas arising from enteric lymphoid tissue and mesenteric lymph nodes. diagnosis exogenous retroviruses have been eliminated from contemporary mouse populations, unless purposely introduced for experimental purposes. because endogenous retroviruses and retroelements are encoded within the genome, and reflect the unique genetic composition of each strain of mouse, they are not targets of diagnostic pursuit. differential diagnosis patterns of some types of neoplasia within individual inbred strains of mice are a reflection of their endogenous retroviral integration. prevention and control exogenous retroviruses have been eliminated from laboratory mice by cesarean rederivation and foster nursing. mmtv-s, the 'bittner agent', continues to be purposely maintained in some mouse breeding populations, but can be eliminated by foster-nursing or other means. caution is advised when re-deriving such mouse colonies for other purposes, as elimination of exogenous mmtv will be an unintended consequence. research complications endogenous retroviruses and retroelements influence the life span of individual strains of mice, and random integrations during cell division can give rise to spontaneous mutations and genetic drift. it is estimated that significant mutations may arise due to mobile retroelement integrations every 50 generations. astroviruses are small, nonenveloped, singlestranded rna viruses that have been associated with human gastroenteritis and detected in association with other enteric pathogens. the viral family astroviridae is split into two genuses: avastrovirus for those astroviruses infecting avians and mamastrovirus for those infecting mammals. astrovirus infection has been detected in research mice (muastv) using metagenomic analyses and appears to have a wide geographical, institutional, and host strain distribution. clinical signs none reported. epizootiology pcr screening has found muastv infection in up to 22% of a variety of mouse strains housed in vendor and academic facilities in the united states and japan. the virus has been detected most commonly in immunocompromised mice (nsg, nod-scid, nsg-3gs, c57bl6-timp-3 −/− , and upa-nog), but also in immuncompetent strains (b6j, icr, bash2, and balb/c). both immunodeficient and immunocompetent mice are susceptible to muastv, but adaptive immunity is required to clear the virus. based on human epidemiology indicating children are at highest risk for infection, the virus may preferentially infect young mice. pathology immunodeficient mice showed no sign of pathology based on histopathology. diagnosis pcr data has indicated that muastv causes a systemic, chronic infection in immunocompromised mice, indicating samples from most tissues will be pcr positive. yokoyama et al. (2012) detected high viral load (up to 10 9 genome copies) per fecal pellet from immunocompetent mice. differential diagnosis none, in the absence of lesions and clinical disease. prevention and control because immunocompetent mice clear the infection, quarantine may be successful but lack of routine screening for muastv in laboratory mice will allow for uncontrolled spread of the infection. research complications based on limited surveys, muastv may have a high prevalence in laboratory mice. the impact of infection on both innate and adaptive immune responses warrants further investigation to assess the potential for confounding research data. this section briefly describes the etiology, clinical signs, epizootiology, pathology, diagnosis, differential diagnoses, prevention and control, and research complications of the most common bacterial diseases encountered in research colonies of mice. as sequencing technology becomes more available, the number and genus/species classification of bacteria potentially responsible for infections, in particular, opportunistic infections, will grow (benga et al., 2014) . potential candidates include members of pasteurellacaeae, bordetella hinzii, streptococcus danielae, acinetobacter spp., and others, for which little is currently known about their pathogenic potential. etiology lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, is not a pathogen encountered in research colonies of mice but has been reported to infect wild mice and rats in close contact with infected livestock (collins et al., 2011) . clinical signs none reported but should consider lawsonia as a differential in necropsy cases with gross or histologic evidence of proliferative lower bowel lesions. epizootiology although mice are experimentally susceptible to infection and develop classic lesions of hyperplastic ileitis and typhlocolitis (murakata et al., 2008) , susceptibility varied with mouse strain and source of inoculum from rabbits or swine, suggesting important differences in l. intracellularis strains. pathology lawsonia infection may result in hyperplastic ileitis, typhlitis and/or colitis, and hemorrhagic intestines may be noted (percy and barthold, 2007) . diagnosis lawsonia spp. has been diagnosed using a variety of techniques, including pcr, immunohistochemistry, in situ hybridization, and warthin-starry silver stains. differential diagnosis bacterial infections associated with hyperplastic intestinal epithelium, including c. rodentium and enterohepatic helicobacter species in susceptible (typically immunodeficient) mouse strains. prevention and control species separation from hosts more commonly associated with natural infection (hamsters, ferrets, pigs). research complications none reported. the following section describes infection due to mycoplasma pulmonis and summarizes infections associated with other murine mycoplasmas including m. arthriditis, m. neurolyticum, m. collis, and m. muris. antigenic cross-reactivity among these species, and especially between m. pulmonis and m. arthriditis, mandates that reliable diagnostic strategies incremental to serology (elisa, ifa, mfia) such as culture (often false negative) and pcr be employed to distinguish potentially pathogenic infections. when screening cell lines for opportunistic pathogens, pcr is the most efficient method to discriminate between m. pulmonis and mycplasma contaminants associated with cell culture. etiology m. pulmonis is a pleomorphic, gram-negative bacterium that lacks a cell wall and has a single outer limiting membrane. it causes murine respiratory mycoplasmosis (mrm). clinical signs mice are relatively resistant to florid mrm; thus, subclinical infection is more common. when clinical signs occur, they reflect suppurative rhinitis, otitis media, and chronic pneumonia. affected mice may display inactivity, weight loss, and ruffled hair coat, but the most prominent signs are 'chattering' and dyspnea, due to rhinitis and purulent exudate in nasal passages. otitis media may cause a head tilt, whereas suppurative inflammation in the brain and spinal cord, although rare, can cause flaccid paralysis. experimental infection of the genital tract can cause oophoritis, salpingitis, and metritis, which may lead to infertility or fetal deaths. experimental inoculation of scid mice has caused systemic infection accompanied by severe arthritis (evengard et al., 1994) . epizootiology mrm historically was a common infectious disease of mice, but improved housing, husbandry, and health surveillance have reduced its prevalence dramatically. serologic data from a large diagnostic laboratory indicated m. pulmonis infection affects about 0.01% of conventionally housed mouse colonies in the united states and 0.16% in europe (pritchett-corning et al., 2009) . m. pulmonis infection is contracted by inhalation and can occur in suckling and adult mice. therefore, infection should be considered highly contagious. mice injected with cells harvested from m. pulmonis contaminated cell cultures may develop disease. m. pulmonis can also be transmitted venerally; in utero infection has been demonstrated in rats but not in mice. because transplacental infection occurs in rats, the same route may be possible in mice, particularly immunocompromised strains. concomitant viral pneumonia (sv, mouse coronavirus) or elevated environmental ammonia concentrations may increase susceptibility to mrm. m. pulmonis also infects rats, hamsters, guinea pigs, and rabbits. among these species, only rats are significant reservoirs of infection for mice. pathology m. pulmonis is an extracellular organism that colonizes the apical cell membranes of respiratory epithelium. attachment occurs anywhere from the anterior nasal passages to the alveoli and may be mediated by surface glycoproteins. the organism may injure host cells through competition for metabolites such as carbohydrates and nucleic acids or by release of toxic substances such as peroxides. ciliostasis, reduction in the number of cilia, and ultrastructural changes leading to laboratory animal medicine cell death have also been described. detrimental effects on ciliated epithelium can lead to disrupted mucociliary transport, which exacerbates pulmonary disease. experimental infection of mrm is dose dependent. doses of 10 4 colony-forming units (cfus) or less cause mild, transient disease involving the upper respiratory tract and middle ears, whereas higher doses often lead to acute, lethal pneumonia. additionally, m. pulmonis strains can differ in virulence. survivors of severe infection may develop chronic bronchopneumonia with bronchiectasis and spread infection to other mice. intravenous inoculation of m. pulmonis can cause arthritis in mice, but arthritis is not a significant feature of natural infection. host genotype also is a major factor in the outcome of infection, with resistance being expressed phenotypically through the bactericidal efficiency of alveolar macrophages. strains derived from a c57bl background appear to be resistant to pathogenic infection, whereas balb/c, c3h, dba/2, swr, akr, cba, sjl, and other strains have varying degrees of increased susceptibility (cartner et al., 1996; lai et al., 1993) . the initial lesion of mrm is suppurative rhinitis, which may involve the trachea and major airways. early inflammatory lesions, if not quickly resolved, progress to prominent squamous metaplasia. transient hyperplasia of submucosal glands may occur, and lymphoid infiltration of the submucosa can persist for weeks. syncytia can sometimes be found in nasal passages, in association with purulent exudate (fig. 3.34) . affected mice also develop suppurative otitis media and chronic laryngotracheitis with mucosal hyperplasia and lymphoid cell infiltrates. pulmonary lesions are typified by bronchopneumonia, which spreads from the hilus. lymphoid cells and plasma cells accumulate around bronchi which often contain neutrophils in their lumen. chronic lung disease features suppurative bronchitis, bronchiolitis, and alveolitis (fig. 3.35) . chronicity also increases the prevalence of bronchiectasis and abcessation. diagnosis accurate diagnosis should exploit the complementary use of clinical, serological, microbiological, molecular, and morphological methods. clinical signs are variable but can be characteristic when they occur. serology is sensitive but although antibodies do not clear the infection, seroconversion may be weak or take months and may not accurately differentiate between m. pulmonis infection and m. arthriditis infection (cassell et al., 1981) . therefore samples for culture and pcr of the upper respiratory tract should be obtained to confirm diagnosis. buffered saline or mycoplasma broth should be used to lavage the trachea, larynx, pharynx, and nasal passages. specimens for culture from the genital tract are warranted if this site is suspected. mycoplasma spp. may be difficult to grow, so it is prudent to confirm that the relevant expertise and quality control exist in the diagnostic laboratory. speciation can be accomplished by immunofluorescence or immunoperoxidase staining or by growth inhibition. immunohistochemistry should be considered to supplement basic histopathologic examination. immunofluorescence and immunoperoxidase techniques are available to identify mycoplasma antigens in tissue sections or in cytological preparations of tracheobronchial or genital tract lavages (brunnert et al., 1994) . pcr assays for m. pulmonis at veterinary diagnostic laboratories and pcr kits to screen cell culures for mycoplasma are readily available. differential diagnosis mrm must be differentiated from bronchopneumonia associated with ciliaassociated respiratory (car) bacillus. silver stains may reveal car bacilli adherent to the respiratory epithelium. sv also can cause bronchopneumonia in mice but can be detected by serology and immunohistochemistry. other causes of respiratory infection include pvm, corynebacteriosis and, in immunodeficient mice, pneumocystis murina infection. combined infections with known pathogens or secondary opportunists also must be considered. prevention and control mice mount an effective immune response to m. pulmonis, as measured by their recovery from mild infection and their resistance to infection after active or passive immunization (cartner et al., 1998) . antibodies of various classes are produced locally and systemically, but clearance of the infection has been attributed to innate immune responses (love et al., 2010; sun et al., 2013) . there is some evidence that antibody may facilitate phagocytosis of m. pulmonis. t-cell responses, however, appear to exacerbate m. pulmonis in mice, because immunity cannot be transferred with immune cells. in addition, athymic and neonatally thymectomized mice are not more susceptible than immunocompetent mice to m. pulmonis pneumonia. nude and scid mice develop less severe respiratory disease than immunocompetent mice but infection becomes systemic and they may develop suppurative disease in multiple organs and joints (arthritis). host immunity aside, effective control and prevention of mrm depend primarily on maintenance of mycoplasma-free colonies under barrier conditions supported by careful surveillance for infection by serology, microbiology, pcr, and histopathology. cesarean or embryo rederivation may eliminate infection, although vertical transmission may occur in immunocompromised mice. treatment with tetracycline suppresses clinical disease but does not eliminate infection. earlier interest in developing dna-based vaccines against m. pulmonis has not achieved clinical application (lai et al., 1997) . research complications m. pulmonis can interfere with research by causing clinical disease or death. experiments involving the respiratory tract, such as inhalation toxicology, can be compromised by chronic progressive infection. additionally, affected mice are at greater risk during general anesthesia. m. pulmonis may alter immunological responsiveness. for example, it is mitogenic for t and b lymphocytes and can increase nk cell activity. perhaps one of the most important complications of mycoplasma infection is contamination of cell lines and transplantable tumors. other murine mycoplasmas cell lines are often contaminated with mycoplasma species such as m. arginini, m. hyorhinis, m. orale, or m. fermentans that can distort the results of in vitro assays (garner et al., 2000) . initial evidence of a contamination is often by pcr evidence of mycoplasma at the genus level when cell lines are pcr screened for opportunistic murine pathogens prior to use in mice. other than m. pulmonis, these mycoplasmas are not normally considered mouse pathogens in immunocompetent mice. in contrast, injection of mycoplasma contaminated cells into immunodeficient mice (e.g., xenografts) may result in clinical disease or confounding effects on immune responses (peterson, 2008) . mycoplasma contamination of murine embryonic stem cells has adversely affected germline transmission and postnatal health of chimeric progeny (markoullis et al., 2009) . mycoplasma arthritidis is antigenically related to m. pulmonis. therefore, serological evidence of mycoplasma infection must be supplemented by other diagnostic tests, as outlined above, to differentiate between these agents. differentiation is important because m. arthritidis, though arthritogenic in mice after intravenous inoculation, is nonpathogenic during natural infection. mycoplasma collis has been isolated from the genital tract of mice but does not appear to cause natural disease. mycoplasma neurolyticum is the etiological agent of rolling disease, a rare syndrome which occurs within hours after intravenous inoculation of m. neurolyticum exotoxin. characteristic clinical signs include spasmodic hyperextension of the head and the raising of one foreleg followed by intermittent rolling on the long axis of the body. the rolling becomes more constant, but mice occasionally leap or move rapidly. after 1-2 h of rolling, animals become comatose and usually die within 4 h. all published reports of rolling disease are associated with experimental inoculation of m. neurolyticum or exotoxin. large numbers of organisms are needed to produce disease, and there is no indication that, under natural conditions, organisms replicate in the brain to concentrations required for the induction of these signs. because animals are frequently inoculated with biological materials by parenteral routes, contamination with m. neurolyticum may induce rolling disease inadvertently. diagnosis can be made from the appearance of typical clinical signs, astrocytic swelling, and isolation of the causative organism. clinical signs must be differentiated from rolling associated with pseudomonas-and p. pneumotropica-caused otitis. m. pulmonis has been recovered from the brain of mice but does not seem to cause overt neurological disease. hemotropic mycoplasmas ribosomal rna sequencing has reclassified hemobartonella muris and eperythrozoon coccoides as mycoplasma hemomuris and mycoplasma coccoides, respectively (neimark et al., 2005; percy and barthold, 2007) . distinct from the mycoplasmas just discussed, these agents are trophic for red blood cells and cause anemia and hemolytic disease. these laboratory animal medicine infections could be encountered in wild mice but are rarely found in research mice. diagnosis is by morphologic assessment of blood smears and pcr. clinical signs mice infected with m. coccoides may remain clinically normal or develop febrile, hemolytic anemia and splenomegaly, which can be fatal. hepatocellular degeneration and multifocal necrosis have been recorded in acute infections. hemotropic mycoplasma infections are long-lived and are expressed clinically in one of two ways: acute febrile anemia and latent or subclinical infection that can be reactivated by splenectomy. the carrier state may be lifelong. epizootiology the primary natural vector of m. coccoides, historically, is the mouse louse, polyplax serrata. infection was associated with primitive housing and husbandry conditions that no longer occur in modern vivaria. although the risks for infection have been reduced substantially by modern animal care procedures, m. coccoides can be transmitted to mice from contaminated biological products such as transplantable tumors or blood plasma. diagnosis splenectomy or inoculation of test material into splenectomized mice is the most sensitive means of detecting m. coccoides infection. these procedures provoke mycoplasmemia, usually within 2-4 days. because mycoplasmemia may be transient, blood smears stained by the romanowsky or indirect immunofluorescence procedures of the blood should be prepared every 6 h, beginning at 48 h after splenectomy of index animals or inoculation of test specimens into splenectomized animals to ensure that mycoplasmemia is not missed. prevention and control treatment of m. coccoides infection is not practical. control is based on culling or rederivation of infected stock. if replacement animals are readily available, euthanasia is a more prudent course. suspect biological materials destined for animal inoculation should be screened for mycoplasma contamination by inoculation of splenectomized mice. research complications subclinical infection can be reactivated by irradiation, immunosuppressive therapy, or intercurrent disease. conversely, m. coccoides may potentiate coincident viral infections in mice. this effect has been clearly demonstrated for mouse coronavirus and has been suspected for lymphocytic choriomeningitis virus and ldv. active infection also may suppress interferon production. etiology car bacillus is a slender, gram-negative, non-spore-forming bacillus, which, in rats, produces clinical disease and lesions that closely resemble those of mrm (see chapter 4). clinical signs chronic respiratory disease has been produced in mice by experimental inoculation, but natural clinical disease is rare (griffith et al., 1988; pritchett-corning et al., 2009) . furthermore, putative natural cases were reported in mice that were seropositive for sv and pneumonia virus of mice. therefore, car bacillus may exacerbate respiratory disease as an opportunist rather than as a primary pathogen. on balance, it is assumed that mice contract natural infection, but attributing severe chronic respiratory disease in mice solely to car bacillus should be supported by screening for other respiratory pathogens. epizootiology car bacillus is transmitted by direct contact; dirty bedding transfer to sentinel mice may not reflect colony infection status. pathology lung lesions are typically mild in mice and are similar to respiratory mycoplasmosis. uncomplicated car bacillus infection results in peribronchiole cuffing with lymphocytes and plasma cells. severe bronchiolitis and pneumonia are possible (fig. 3.36) . fatal bronchopneumonia was reported in ob/ob mice (griffith et al., 1988) . diagnosis an elisa for serological screening is routinely used; pcr and histology are used for definitive diagnosis. in active infection, histologic assessment using warthin-starry or similar stains will reveal argyrophilic bacilli adherent to the apical membranes of bronchial respiratory epithelium along with the presence of peribronchial lymphocytes (fig. 3.37) . alternatively, immunohistochemistry assays have also been used successfully to detect infection. recovery of car bacillus requires cell culture or culture in embryonated eggs. differential diagnosis respiratory mycoplasmosis, bordetella (avium, hinzii). prevention and control given car bacillus does not form spores, disinfection of the environment should be effective. treatment using sulfamerazine (500 mg/l) in drinking water may eradicate infection (matsushita and suzuki, 1995) but culling or embryo rederivation is recommended. research complications infection is most often subclinical, but like other infectious agents for mice, may confound studies particularly when mice are immunocompromised (griffith et al., 1988) . etiology the causative agent of transmissible murine colonic hyperplasia, c. rodentium (formerly citrobacter freundii strain 4280), is a nonmotile, gramnegative rod that ferments lactose but does not utilize citrate or does so marginally (barthold, 1980; schauer et al., 1995) . clinical signs c. rodentium infection can be a selflimiting colitis with sterilizing immunity or lead to severe colitis with life-threatening dehydration. clinically apparent infection is characterized by retarded growth, ruffled fur, soft feces or diarrhea, rectal prolapse, and moderate mortality in older suckling or recently weaned mice (barthold et al., 1978) . epizootiology c. rodentium is not detected in the gastrointestinal flora of normal mice, and therefore, there is not a carrier state. it is thought to be introduced by contaminated mice, food, or bedding, from which it spreads by contact or additional fecal contamination. c. rodentium shares several pathogenic mechanisms, such as attaching and effacing lesions mediated by the intimin receptor, with select escherichia coli (reviewed in collins et al. (2014) ). c. rodentium is used experimentally to model colitis caused by enteropathogenic (epec) and enterohemorrhagic e. coli (ehec) in humans (mallick et al., 2012; collins et al., 2014) . host genotype can influence the course and severity of disease (barthold et al., 1977) . for example, dba, nih swiss, and c57bl mice are relatively resistant to mortality, whereas c3h/hej mice are relatively susceptible both as sucklings and as adults. interestingly, c57bl mice obtained from different commercial sources have varying susceptibility to c. rodentium (ostensibly due to the presence or absence of segmented filamentous bacteria). diet also can modulate infection, but specific dietary factors responsible for this effect have not been identified. pathology c. rodentium attaches to the mucosa of the descending colon and displaces the normal flora. attachment is accompanied by effacement of the microvillus border and formation of pedestal-like structures (attaching and effacing lesions) (schauer and falkow, 1993; newman et al., 1999) . colonization results in prominent mucosal hyperplasia, by unknown mechanisms. the characteristic gross finding is severe thickening of the descending colon, which may extend to the transverse colon and lasts for 2-3 weeks in surviving animals ( fig. 3.38) . affected colon segments are rigid and either are empty or contain semiformed feces. histologically, accelerated mitotic activity results in a markedly hyperplastic mucosa, which may be associated with secondary inflammation and ulceration (fig. 3.39 ). lesions subside after several weeks. intestinal repair is rapid and complete in adults but slower in sucklings. diagnosis diagnosis depends on clinical signs, characteristic gross and histological lesions, and isolation of c. rodentium from the gastrointestinal tract or feces. the organism can be cultured on macconkey's agar during early phases of infection, whereas the intestine may be free of c. rodentium during later stages of the disease. c. rodentium also can be detected by molecular hybridization (schauer et al., 1995) . barthold et al. (1978) . diagnosis transmissible murine colonic hyperplasia must be differentiated from other diarrheal diseases of mice, including infections caused by coronavirus, rotavirus, adenovirus, reovirus, salmonella, c. piliforme, and helicobacter spp. prevention and control some success in curtailing epizootics has been achieved by adding antimicrobials to the drinking water (barthold, 1980; silverman et al., 1979) . because c. rodentium may contaminate food, bedding, or water, proper disinfection of such materials is prudent before they are used for susceptible animals. additionally, the employment of microbarrier caging can reduce transmission. surveillance for c. rodentium should be incorporated into quality-assurance programs, and the organism screened for during quarantine of incoming mice from atypical sources. research complications the potential effects on research of colonic hyperplasia as a clinically severe disease are obvious. colonic hyperplasia has been shown to increase the sensitivity of colonic mucosa to chemical carcinogens and to decrease the latent period between administration of carcinogen and the appearance of focal atypical cell growth (barthold and beck, 1980) . c. rodentium infection has been incriminated in immune dysfunction, poor reproductive performance, and failure to thrive in t-cell receptor transgenic mice (maggio-price et al., 1998) . immunocompromised mice infected with c. rodentium will die from sepsis. etiology pseudomonas aeruginosa is a motile, gramnegative rod. clinical signs p. aeruginosa infections are almost always silent, but immunologically compromised animals are prone to septicemia (brownstein, 1978) . p. aeruginosa can, e.g., cause severe or lethal infections in athymic and scid mice. sick mice may have equilibrium disturbances, conjunctivitis, serosanguinous nasal discharge, edema of the head, weight loss, and skin infections. immunosuppressed mice may also develop gastrointestinal ulcers. generalized infection is associated with severe leukopenia, especially neutropenia. neurologic signs are rare, but there are reports of central nervous system infection. chronic proliferative inflammation in the cochlea and vestibular apparatus with dissolution of surrounding bone may cause torticollis. epizootiology p. aeruginosa is not considered a component of the normal flora. however, it is an opportunist that inhabits moist, warm environments such as water and skin. once established in a host, it may be found chronically in the nasopharynx, oropharynx, and gastrointestinal tract, all sites from which additional environmental contamination or direct transmission to susceptible mice can occur. pathology pathogenic infection is most common in immunodeficient mice. organisms enter at the squamocolumnar junction of the upper respiratory tract and, in some cases, the periodontal gingiva. bacteremia is followed by necrosis or abscess formation in the liver, spleen, or other tissues. if otitis media occurs, the tympanic bullae may contain green suppurative exudate. the bowel may be distended with fluid, and gastrointestinal ulceration has been reported. diagnosis infection is diagnosed on the basis of history (e.g., immune dysfunction or recent immunosuppression), clinical signs, lesions, and isolation of p. aeruginosa from affected mice. carrier mice can be detected either by nasal culture or by placing bottles of sterile, nonacidified, nonchlorinated water on cages for 24-48 h and then culturing the sipper tubes. p. aeruginosa can also be cultured from feces. differential diagnosis pseudomoniasis must be differentiated from other bacterial septicemias that may occur in immunodeficient mice. these include, but are not limited to, corynebacteriosis, salmonellosis, colibacillosis, staphylococcosis, and tyzzer's disease. prevention and control infection can be prevented by acidification or hyperchlorination of the drinking water (homberger et al., 1993) . these procedures will not, however, eliminate established infections. entry of infected animals can be prevented by surveillance of commercially procured colonies. maintenance of pseudomonas-free animals usually requires barrierquality housing and husbandry. p. aeruginosa has a long history in the literature of antibiotic resistance and resistance to quaternary amine disinfectants. research complications p. aeruginosa infection is not a substantial threat to immunocompetent mice but can complicate experimental studies by causing fatal septicemia in immunodeficient mice. viral infections that alter host defense mechanisms, such as mcmv may enhance susceptibility to pseudomoniasis. (lindsey et al., 1991a; percy and barthold, 2007) etiology pasteurella pneumotropica is a short, gramnegative rod. clinical signs many early observations concerning the pathogenicity of p. pneumotropica are questionable because they were made on colonies of mice with varying levels of bacterial and viral contamination. infection is usually subclinical. therefore, p. pneumotropica is most properly viewed as an opportunistic pathogen. studies of experimental p. pneumotropica suggest that it may complicate pneumonias due to mycoplasma pulmonis or sv. it has also been associated with suppurative or exudative lesions of the eye, conjunctiva, skin, mammary glands, and other tissues, especially in immunodeficient mice or in mice with a predisposing primary infection. epizootiology p. pneumotropica is a ubiquitous inhabitant of the skin, upper respiratory tract, and gastrointestinal tract of mice. litters from infected dams can become infected during the first week after birth. pathology infections can cause suppurative inflammation, which may include abcessation. dermatitis, conjunctivitis, dacryoadenitis, panophthalmitis, mastitis, and infections of the bulbourethral glands have been attributed to p. pneumotropica. preputial and orbital abscesses also occur, especially in athymic mice (fig. 3.40 ). its role in metritis is unclear, but it has been cultured from the uterus, and there is some evidence that it may cause abortion or infertility. cutaneous lesions can occur without systemic disease. they include suppurative lesions of the skin and subcutaneous tissues of the shoulders and trunk. diagnosis diagnosis requires isolation of the organism on standard bacteriological media. although infection can be detected serologically by elisa (wullenweber-schmidt et al., 1988; boot et al., 1995a, b) , subclinical carriers often do not seroconvert. pcr assays also are available (dole et al., 2010) and have shown that p. pneumotropica did not transmit from infected mice to contact or dirty bedding sentinels (ouellet et al., 2011; dole et al., 2013) . differential diagnosis suppurative lesions in mice may be caused by other bacteria, including staphylococcus, streptococcus, corynebacterium, klebsiella, and mycoplasma. treatment antibiotic sensitivity testing in vitro indicated p. pneumotropica was significantly more sensitive than p. aeruginosa to enrofloxacin (sasaki et al., 2007) . enrofloxacin in the drinking water at 85 mg/kg daily for 7 days eliminated clinical signs and infection in a closed breeding colonic of transgenic mice and after 14 days of treatment there were no detectable carriers when the colony was screened 4 weeks later (matsumiya and lavoie, 2003) . prevention and control because p. pneumotropica is an opportunistic organism, it should be excluded from colonies containing immunodeficient mice and from breeding colonies. achieving this goal will normally require barrier housing supported by sound microbiological monitoring. rederivation should be considered to eliminate infection in circumstances where infection presents a potential threat to animal health or experimentation. research complications clinically severe infection in immunodeficient mice is the major complication. although clinically silent, experimental evidence has shown that p. pneumotropica infection in immunocompetent mice (c57bl/6) stimulated transcription of multiple proinflammatory cytokines for at least 7 days with residual elevation detectable 28 days later (patten et al., 2010) . pioneering studies conducted in the 1990s first linked a novel microaerobic bacterium, helicobacter hepaticus, with chronic active hepatitis and hepatic tumors in a/jcr mice (fox et al., , 2011 ward et al., 1994) . the organism could be visualized by electron microscopy in the bile canaliculi of the liver in susceptible mouse strains (fig. 3.41 ). subsequently, it was associated with inflammatory bowel disease in several murine models (table 3 .13) which were further developed to examine the role of immune cell subsets, such as t regulatory cells, in the pathogenesis of inflammatory bowel disease (ibd) and colon cancer (fig. 3.42 ). helicobacteriosis is laboratory animal medicine now appreciated to be a common infection of laboratory mice. it is caused by a growing list of helicobacter spp. that vary in clinical, pathologic, and epidemiologic significance (whary and fox, 2004; fox et al., 2011) . because recognition and investigation of helicobacteriosis continues to evolve, many important questions about the impact of this infection on mice remain unresolved. h. hepaticus infection is emphasized here, because it is among the most prevalent causes of helicobacteriosis and has been studied more extensively than other murine enterohepatic helicobacter spp. (ehs) (fox et al., , 2011 ward et al., 1994; suerbaum et al., 2003) . however, current information about other murine helicobacters is summarized in the concluding section. etiology helicobacter spp. are gram-negative, microaerophilic, curved to spiral-shaped organisms that have been isolated from the gastrointestinal mucosa of many mammals, including humans and mice whary and fox, 2004) . to date, the genus includes 20 formally named helicobacter spp. assigned on the basis of 16s rrna analysis, complemented by biochemical, molecular, and morphological characteristics. the organisms can be grown on freshly prepared antibiotic impregnated blood agar or in broth supplemented with fetal bovine serum in a microaerobic atmosphere (5% co 2 , 80% n 2 , 10% h 2 ). there are currently 11 formally named helicobacter species have been isolated from laboratory mice, as well as several other novel helicobacter spp. awaiting formal naming. species isolated from mice include h. hepaticus, h. bilis (which also infects rats), h. muridarum, h. rappini, and h. rodentium, h. ganmani, h. mastomyrinus, h. magdeburgensis, and h. typhlonius , each of which cahill et al. (1997) tcrα, β mutants defective t-receptors typhlocolitis chin et al. (2000) scid icr-defined flora b t-and b-cell deficient typhlocolitis shomer et al. (1998 ), shomer et al. (1997 c57bl/il-10 −/−c lacks il-10 typhlocolitis burich et al. (2001) , kullberg et al. (2001) , kullberg et al. (2006) , kullberg et al. (2002) c57blrag2 mice infected with h. bilis also developed ibd (shomer et al., 1997) . c ibd also developed in c57bl/il-10 −/− mice experimentally infected with a novel urease-negative helicobacter spp. now named h. typhlonius (franklin et al., 2001) ; also ibd produced with h. trogontum (whary et al., 2006) and h. cinaedi (shen et al., 2009) . d h. bilis produces ibd (maggio-price et al., 2002 and colon cancer (maggio-price et al., 2006) . have been formally named (except for h. rappini) (fox and lee, 1997; franklin et al., 1999; whary and fox, 2004) . most recently, helicobacter pullorum, a human pathogen, has been isolated from commercial, barriermaintained mice (boutin et al., 2010) . these ehs are most commonly urease-, catalase-, and oxidase-positive. however, h. rodentium, h. typhlonicus, and another novel helicobacter sp. are urease-negative. clinical signs helicobacteriosis in adult immunocompetent mice is usually asymptomatic. liver enzymes are elevated in h. hepaticus-infected a/jcr mice (fox et al., 1996a) . infection of immune-dysregulated mice with h. hepaticus can cause inflammatory bowel disease, which may present as rectal prolapse and/or diarrhea (miller et al., 2014) . epizootiology recent surveys and anecdotal evidence suggest that helicobacteriosis is widespread among conventional and barrier-maintained mouse colonies (shames et al., 1995; fox et al., 1998b; taylor et al., 2007; lofgren et al., 2011) . furthermore, h. hepaticus (and probably other helicobacters) can persist in the gastrointestinal tract, particularly the cecum and colon, and is readily detected in feces. these results indicate that transmission occurs primarily by the fecal-oral route and imply that carrier mice can spread infection chronically in enzootically infected colonies. pathology helicobacter spp. colonize the crypts of the lower bowel, where, depending on host genotype, the organisms can be pathogenic or nonpathogenic. h. hepaticus and h. bilis, e.g., can cause inflammation in the gastrointestinal tract, which is expressed as ibd and colon cancer in immunodeficient mice or typhlitis in a/jcr mice infected with h. hepaticus (ward et al., 1996; knutson et al., 2013; shomer et al., 1997; erdman et al., 2003b; nguyen et al., 2013) . thickening of the cecum and large bowel develops because of proliferative typhlitis, colitis, proctitis, and lower bowel carcinoma. these lesions can occur without coincident hepatitis. indeed, helicobacter spp. induced ibd and colon carcinoma are increasingly popular models to study pathogenesis of the disease in humans (table 3 .13). helicobacter spp. also can cause liver disease. bacterial translocation is thought to occur and results in colonization of the liver and progressive hepatitis. it is characterized by angiocentric nonsuppurative hepatitis and hepatic necrosis (fig. 3.43) . inflammation originates in portal triads and spreads to adjacent hepatic parenchyma. hepatic necrosis also may occur adjacent to intralobular venules, which can contain microthrombi. additionally, phlebitis may affect central veins. this lesion has been linked to the presence of organisms in bile canaliculi by silver stains and electron microscopy. age-related hepatocytic proliferation can develop in infected livers, a response that is more pronounced in male mice than in female mice (fox et al., 1996a) . this lesion may laboratory animal medicine increase susceptibility to hepatomas and hepatocellular carcinomas among aged male a/jcr and b6c3f1 mice from infected colonies. an increased incidence of hepatic hemangiosarcoma also has been noted in h. hepaticusinfected male b6c3f1 mice. in this context, a/jcr, c3h/ hencr, and sjl/ncr mice are susceptible to hepatitis, whereas c57bl/6 mice are resistant (ward et al., 1994) . the finding of severe liver disease and tumor induction in b6c3f1 mice infected with h. hepaticus infers that genetic susceptibility to h. hepaticus-induced neoplasia has a dominant pattern of inheritance. studies with h. hepaticus in recombinant inbred mice also indicate that disease susceptibility has multigenetic properties (hailey et al., 1998; fox and lee, 1997; ihrig et al., 1999; franklin, 2006; hillhouse et al., 2011) . diagnosis rapid generic diagnosis can be accomplished by pcr detection of the highly conserved 16s rrna region of the helicobacter genome in feces or tissues, using suitable oligonucleotide primers (fox et al., 1998a; shames et al., 1995; beckwith et al., 1997) . however, genus-specific pcr does not differentiate among different helicobacter spp. molecular speciation can be accomplished by 16s rrna sequencing, restriction fragment length polymorphism analysis of the pcr product or use of species-specific pcr assays. this procedure requires suitable skill and experience to avoid technological pitfalls and should be performed by qualified laboratories. an igg elisa using the outer membrane protein as the antigen has been proposed for serological diagnosis, but shared antigens among ehs create lack of specificity for the assay. as noted above, helicobacters can be isolated on antibiotic-impregnated blood agar under microaerobic conditions and can then be speciated biochemically, and by helicobacter species-specific pcr. isolation of h. hepaticus and from other helicobacter spp. with spiral to curved morphology from feces should be preceded by passing slurried samples through a 0.45-μm filter before plating. if infection with larger fusiform helicobacters (h. bilis, h. rappini) is suspected, filtration at 0.65 μm is preferred. helicobacters grow slowly and require prolonged incubation of cultures (up to 3 weeks) before they can be deemed negative. signs (rectal prolapse) and lesions (hepatitis, typhlocolitis), depending on host genotype, can be suggestive of infection. histopathological examination should include silver stains, especially of liver, to attempt to visualize spiral or curved organisms (whary and fox, 2004) . differential diagnosis clinically apparent helicobacteriosis must be differentiated from other gastrointestinal or hepatic infections of mice. coronavirus infection, clostridium piliforme, and salmonella spp. can cause enterocolitis and/or hepatitis. c. rodentium also causes colonic hyperplasia, which can present as rectal prolapse. infections caused by other helicobacters of mice h. bilis has been isolated from the livers and intestines of aged mice and experimentally induces ibd in scid mice as does h. hepaticus. h. bilis also experimentally produces lower bowel cancer in immunocompromised mice (nguyen et al., 2013) . helicobacter muridarum colonizes the ileum, cecum, and colon. it appears to be nonpathogenic, although it can colonize the stomach of mice and induce gastritis under certain circumstances. h. 'rappini' has been isolated from the feces of mice without clinical signs. h. rodentium also colonizes the intestine and may be a component of normal flora. a dual infection of h. bilis and h. rodentium was noted in a natural outbreak of ibd in immunocompromised mice (shomer et al., 1998) . a novel urease negative helicobacter, which has been named h. typhlonius, causes ibd in il-10 −/− and scid mice (franklin et al., 1999 (franklin et al., , 2001 fox et al., 1999) . decreased reproductive efficiency has been reported in il10 knockout mice infected with h. rodentium and/or h. typhlonius (sharp et al., 2008) . prevention and control eradication of infection from small numbers of mice, such as quarantine groups, can be achieved by standard rederivation or intensive antibiotic therapy. the best results have been obtained by triple therapy with amoxicillin, metronidazole, and bismuth given for 2 weeks (del carmen martino-cardona et al., 2010) . this strategy requires repeated daily gavage rather than administration in drinking water, but it has successfully eliminated h. hepaticus from naturally infected mice. antibiotic impregnated wafers have been used to eradicate helicobacter spp. in mouse colonies (kerton and warden, 2006) . wide-scale, eradication of enzootic helicobacteriosis can be expensive and time-consuming, without guarantee of success. careful husbandry procedures can limit infection within a colony (whary et al., 2000) . therefore, strategies have to be weighed carefully against risks of enzootic infection for the health and use of mice. in contrast, infection should be avoided in immunodeficient mice, including genetically engineered mice with targeted or serendipitous immune dysfunction. lastly, the outcome of opportunistic helicobacteriosis has not been thoroughly examined. this condition could occur during simultaneous infection with two or more helicobacter species or during combined infection with an intestinal virus (e.g., coronavirus) and helicobacter spp. if highly valuable animals are exposed, antibiotic therapy or rederivation may be warranted. research complications chronic inflammation of the liver and or gastrointestinal tract may be injurious to health. additionally, it may impede the development and assessment of noninfectious disease models, such as ibd models in mice with targeted deletions in t-lymphocyte receptors (fox et al., 2011) . h. hepaticus infections provoke a strong th1 proinflammatory response, which may perturb other immunological responses. h. hepaticus infection also has been incriminated as a cofactor or promoter in the development of hepatic neoplasia in a/ jcr, b6c3f1, ab6f1, b6af1, and carko mice (hailey et al., 1998; fox et al., 1998a; garcia et al., 2008 garcia et al., , 2011 h. salmonellosis (ganaway, 1982; lindsey et al., etiology the genus salmonella contains two species, s. bongori which infects mainly poikilotherms and rarely, humans, and s. enterica which includes approximately 2500 serovars and are a major cause of food-borne illness in humans (fookes et al., 2011) . the salmonella of historical importance in mice that are now rare include s. enterica subsp. enterica serovar typhimurium (aka s. typhimurium) and serovar enteritidis (s. enteritidis). s. enteritidis is a motile, gram-negative rod that rarely ferments lactose. the genomes of many strains have been sequenced. virulence factors carried on pathogenicity islands and plasmids include antimicrobial resistance genes, type iii secretion systems, vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and factors essential for a intracellular life cycle in macrophages (de jong et al., 2012) . pathogenassociated molecular patterns (pamps) unique to salmonella interact with tlrs and nod-like receptors (nlrs) which recruit neutrophils and macrophages leading to inflammasome formation and release of pro-inflammatory il-6, il-1β, tnf-α, and ifn-γ. clinical signs acute infection is especially severe in young mice (casebolt and schoeb, 1988) . it is characterized by anorexia, weight loss, lethargy, dull coat, humped posture, and occasionally conjunctivitis. gastroenteritis is a common sign, but feces may remain formed. subacute infection can produce distended abdomens from hepatomegaly and splenomegaly. chronic disease is expressed as anorexia and weight loss. enzootic salmonellosis in a breeding colony can produce episodic disease with alternating periods of quiescence and high mortality. the latter can be associated with diarrhea, anorexia, weight loss, roughened hair coat, and reduced production. epizootiology s. typhimurium is commonly used experimentally and cross-contamination in a mouse facility is a risk. modern production and husbandry methods have reduced the importance of salmonellosis as a natural infection of mice. however, the organisms are widespread in nature. therefore, cross-infection from other species or from feral mice remains a potential hazard. salmonellas are primarily intestinal microorganisms that can contaminate food and water supplies. infection occurs primarily by ingestion. salmonella have a broad host range and vermin, birds, feral rodents, and human carriers are potential sources of infection. other common laboratory species such as nonhuman primates, dogs, and cats also can serve as carriers. conversely, murine salmonellosis presents a zoonotic hazard to humans. the induction and course of infection are influenced by the virulence and dose of the organism, route of infection, host sex and genetic factors, nutrition, and intercurrent disease. suckling and weanling mice are more susceptible to disease than mature mice. immune deficiency, exposure to heavy metals, and environmental factors such as abnormal ambient temperatures can increase the severity of disease. nutritional iron deficiency has an attenuating effect on salmonella infection in mice, whereas iron overload appears to promote bacterial growth and enhance virulence. resistance to natural infection is increased by the presence of normal gastrointestinal microflora. resistance to infection also can be an inherited trait among inbred strains. among the most important considerations is that mice that recover from acute infection can become subclinical carriers and a chronic source of contamination from fecal shedding. pathology the virulence of s. enteritidis depends on its ability to penetrate intestinal walls, enter lymphatic tissue, multiply, and disseminate. organisms reach peyer's patches within 12 h after inoculation and spread quickly to the mesenteric lymph nodes. bacteremia results in spread to other lymph nodes, spleen, and liver within several days. in chronic infections, organisms persist in the spleen and lymph nodes as well as in the liver and gallbladder and from the latter are discharged into the intestinal contents. bacteria reaching the intestine can reinvade the mucosa and can be shed intermittently in the feces for months. s. enteritidis infection also has been associated with chronic arthritis. acute deaths may occur without gross lesions, but visceral hyperemia, pale livers, and catarrhal enteritis are more common. if mice survive for up to several laboratory animal medicine weeks, the intestine may be distended and reddened, whereas the liver and spleen are enlarged and contain yellow-gray foci of necrosis. affected lymph nodes are also enlarged, red, and focally necrotic. focal inflammation can develop in many organs, including the myocardium (percy and barthold, 2007) . histologic lesions reflect the course of disease and the number of bacteria in affected tissues. during acute infection, necrotic foci are found in the intestine, mesenteric lymph nodes, liver, and spleen. neutrophilic leukocytes and histiocytes accumulate in lymphoid tissues. thrombosis from septic venous embolism may occur, especially in the liver. granulomatous lesions are particularly characteristic of chronic salmonellosis, especially in the liver. diagnosis diagnosis is based on isolation of salmonellas together with documentation of compatible clinical signs and lesions. in mice with systemic disease, bacteria may persist in the liver and spleen for weeks. during acute stages, bacteria can also be isolated from the blood. subclinically infected animals can be detected by fecal culture using selective enrichment media (selenite f broth plus cystine followed by streaking on brilliant green agar). culture of the mesenteric lymph nodes may be more reliable, because fecal shedding can be intermittent. isolates can be speciated with commercial serotyping reagents. alternatively, isolates can be sent to a reference laboratory for confirmation. antibodies to salmonellas can be detected in the serum of infected mice by an agglutination test. however, this method is not entirely reliable, because serological crossreactivity is common even among bacteria of different genera. pcr-based assays are also available. differential diagnosis salmonellosis must be differentiated from other bacterial diseases, including tyzzer's disease, helicobacter spp., pseudomoniasis, corynebacteriosis, c. rodentium, and pasteurellosis. viral infections that cause enteritis or hepatitis must also be considered, especially infections caused by coronavirus, ectromelia virus, and reoviruses. among noninfectious conditions, mesenteric lymphadenopathy is an agingassociated lesion in mice and is not indicative of chronic salmonellosis. prevention and control salmonellosis can be prevented by proper husbandry and sanitation. contact between mice and potential carriers, such as nonhuman primates, dogs, and cats, should be prevented. diets should be cultured periodically to check for inadvertent contamination. contaminated colonies should be replaced to eliminate infection and its zoonotic potential. research complications apart from the clinical manifestations, the zoonotic potential for salmonellosis is a major concern. this includes transmission among laboratory species, but especially between mice and the personnel working with them. i. streptobacillosis (lindsey et al., 1991e; percy and barthold, 2007) etiology streptobacillus moniliformis is a nonmotile, gram-negative, pleomorphic rod that can exist as a nonpathogenic l-phase variant in vivo. however, it can revert to the virulent bacillus form. clinical signs streptobacillosis generally has an acute phase with high mortality, followed by a subacute phase and finally a chronic phase that may persist for months. signs of acute disease include a dull, damp hair coat and keratoconjunctivitis. variable signs include anemia, diarrhea, hemoglobinuria, cyanosis, and emaciation. cutaneous ulceration, arthritis, and gangrenous amputation may occur during chronic infection. the arthritis can leave joints deformed and ankylosed. hindlimb paralysis with urinary bladder distention, incontinence, kyphosis, and priapism may occur if vertebral lesions impinge on motor nerves. breeding mice may have stillbirths or abortions. epizootiology streptobacillosis has historical importance as a disease of rats and mice, but modern husbandry, production, and health surveillance strategies have reduced its impact dramatically (wullenweber, 1995) . subclinical, persistently infected rats are the most likely source of dissemination to mice, but mouse-tomouse transmission then ensues. transmission may occur from aerogenic exposure, bite wounds, or contaminated equipment, feed, or bedding. s. moniliformis is also pathogenic for humans, causing rat bite fever (haverhill fever). pathology during acute disease, necrotic lesions develop in thoracic and abdominal viscera, especially in the liver, spleen, and lymph nodes. histological lesions include necrosis, septic thrombosis of small vessels, acute inflammation, fibrin deposition, and abscesses. chronically infected mice may develop purulent polyarthritis because of the organism's affinity for joints. diagnosis diagnosis depends on clinical and pathological evidence of septicemia and isolation of the organism on blood agar. the organism has been recovered from joint fluid as long as 26 months after infection. isolation from chronic lesions requires serumenriched medium. s. moniliformis as a cause of septic joints in humans has been diagnosed using pcr and electrospray-ionization followed by mass spectrometry (mackey et al., 2014) . differential diagnosis clinical signs must be differentiated from septicemic conditions, including mousepox, tyzzer's disease, corynebacteriosis, salmonellosis, mycoplasmosis, pseudomoniasis, and traumatic lesions. prevention and control control is based on exclusion of wild rodents or carrier animals such as latently infected laboratory rats. bacterins and antibiotic therapy are not adequately effective. the potential laboratory animal medicine for cross-infection is a reason not to house rats and mice in the same room. research complications infection can be disabling or lethal in mice and has zoonotic potential for humans. j. corynebacteriosis (lindsey et al., 1982; weisbroth, 1994; percy and barthold, 2007) etiology corynebacteria are short gram-positive rods. corynebacterium kutscheri is the cause of pseudotuberculosis in mice and rats. corynebacterium bovis has been associated with hyperkeratosis, especially in immunodeficient mice (clifford et al., 1995; scanziani et al., 1998; dole et al., 2013) . clinical signs c. kutscheri infection is often subclinical in otherwise healthy mice. active disease is precipitated by immunosuppression or environmental stresses and is expressed as an acute illness with high mortality or a chronic syndrome with low mortality. clinical signs include inappetence, emaciation, rough hair coat, hunched posture, hyperpnea, nasal and ocular discharge, cutaneous ulceration, and arthritis. c. bovis infection causes hyperkeratotic dermatitis characterized by scaly skin, which is accompanied by alopecia in haired mice. severe infection may cause death. corynebacterial keratoconjunctivitis has been reported in aged c57bl/6 mice (mcwilliams et al., 1993) . epizootiology subclinically infected animals harbor c. kutscheri in the upper alimentary tract, colon, respiratory tract, regional lymph nodes, middle ear, and preputial gland. c. bovis colonizes skin and is shed in feces. therefore, transmission is by direct contact, fecaloral contact, and aerosol. resistance to infection appears to be under genetic control in some mouse strains. rats are susceptible to c. kutscheri, so cross-infection to mice may occur. pathology lesions caused by c. kutscheri develop from hematogenous spread to various internal organs and appear as gray-white nodules in the kidney, liver, lung, and other sites. cervical lymphadenopathy and arthritis of the carpometacarpal and tarsometatarsal joints also may occur. septic, necrotic lesions often contain caseous material or liquefied exudate. histologic lesions are characterized by coagulative or caseous necrosis bordered by intense neutrophilic infiltration. colonies of gram-positive organisms with 'chinese letter' configurations can usually be demonstrated using tissue gram stains of caseous lesions. mucopurulent arthritis of carpal, metacarpal, tarsal, and metatarsal joints are related to bacterial colonization of synovium accompanied by necrosis, cartilage erosion, ulceration, and eventually ankylosing pan arthritis. c. kutscheri is not a primary skin pathogen, but skin ulcers or fistulas follow bacterial embolization and infarction of dermal vessels. subcutaneous abscesses have also been reported. hyperkeratotic dermatitis caused by c. bovis is characterized grossly by skin scaliness and alopecia. microscopically, skin lesions consist of prominent acanthosis and moderate hyperkeratosis accompanied by mild nonsuppurative inflammation (fig. 3.44) . hyperkeratosis is typically more severe in glabrous athymic mice than in haired mice. organisms can be demonstrated in hyperkeratotic layers by gram stain. diagnosis c. kutscheri is usually diagnosed by culture and tissue gram stains on lesions from clinically apparent cases. agglutination serology is available, and immunofluorescence, immunodiffusion, and elisa tests have been reported (boot et al., 1995a) . pcr of skin swabs or feces is a sensitive and specific method for the detection of c. bovis infection in mice (dole et al., 2013) . differential diagnosis the caseous nature of c. kutscheri-induced lesions helps separate them from necrotic changes or abscesses caused by other infectious agents of mice. thus, they can be differentiated from streptococcosis, mycoplasmosis, and other septicemic bacterial infections in which caseous necrosis does not occur. because mice can sustain natural infections with mycobacterium avium, histochemical techniques for acidfast bacilli and appropriate culture methods for mycobacteria should be considered if nodular inflammatory lesions of the lung are detected. diffuse scaling dermatitis in athymic nude mice is classic for c. bovis infection; however, in one case report staphylococcus xylosus was instead isolated in high numbers from the skin lesions (russo et al., 2013) . hyperkeratotic dermatitis caused by laboratory animal medicine c. bovis must be differentiated from scaly skin caused by low humidity in glabrous mice. prevention and control c. kutscheri infection occurs sporadically and infected colonies should be culled or rederived into an spf facility as treatment is not curative and control is difficult. c. bovis can be endemic in athymic nude mouse colonies. prevention and control are difficult because both immunocompetent and athymic mice as well as humans can carry c. bovis on the skin and in the upper respiratory system, respectively. c. bovis readily contaminates the environment as aerosolization within a class ii biosafety cabinet was shown to spread the bacterium during cage-change procedures (burr et al., 2012) . antibiotic treatment has been unrewarding (burr et al., 2011) research complications corynebacteriosis can cause morbidity and mortality, especially among immunodeficient mice. dermatologic disease in suckling mice can be fatal but is less severe and transient in weanling mice. etiology staphylococci are gram-positive organisms that commonly infect skin and mucous membranes of mice and other animals. the two most frequently encountered species are staphylococcus aureus, which can be highly pathogenic, and s. epidermidis, which is generally nonpathogenic. species subtypes are identified by phage typing and biochemistry profiles. pathogenic staphylococci are typically coagulase-positive, although s. xylosus has caused serious infections and is coagulasenegative (gozalo et al., 2010) . clinical signs staphylococcosis causes suppurative conjunctivitis, periorbital and retroorbital abscesses, preputial adenitis, and pyoderma in mice, particularly in immunocompromised strains such as nude mice. some evidence suggests that staphylococci can produce primary cutaneous infections, but they are more likely opportunistic organisms that induce lesions after contamination of skin wounds. eczematous dermatitis develops primarily on the face, ears, neck, shoulders, and forelegs and can progress to ulcerative dermatitis, abscessation (including botryomycotic granulomas), and cellulitis. because lesions are often pruritic, scratching causes additional trauma and autoinoculation. staphylococcal infection in the genital mucosa of males may produce preputial gland abscesses. these occur as firm, raised nodules in the inguinal region or at the base of the penis and may rupture to spread infection to surrounding tissues. male mice also may develop septic balanoposthitis secondary to penile self-mutilation. retrobulbar abscesses caused by s. aureus are frequently noted in athymic mice. sjl mice, which are nk cell deficient, are prone to necrotic dermatitis on the tail secondary to s. xylosus infection. epizootiology staphylococci are ubiquitous and can be carried on the skin and in the nasopharnyx and gastrointestinal tract. they also can be cultured from cages, room surfaces, and personnel. the prevalence of staphylococcal dermatitis appears to be influenced by host genotype, the overall health of the animal, and the degree of environmental contamination with staphylococcus spp. c57bl/6, c3h, dba, and balb/c mice are among the most susceptible strains. age may also influence susceptibility, with young mice being more susceptible than adults. immunodeficient mice (e.g., athymic mice) contaminated with staphylococci often develop abscesses or furunculosis (fig. 3.45 ). as noted above, behavioral dysfunction resulting in selfmutilation, including scratching and trichotillomania, is a likely predisposing factor. once virulent staphylococci contaminate the environment, colonization of the gastrointestinal tract can occur and produce a carrier state. phage typing can help to determine the source of infection. human phage types of staphylococci can infect mice, but the zoonotic importance of this connection is not clear. pathology gross lesions are typified by suppurative, ulcerative and necrotic dermatitis involving the head and neck but may extend to the shoulders and forelegs (percy and barthold, 2007) . superficial or deep abscesses may occur in conjunction with dermatitis or separately, as, e.g., in the external male genitalia. histologically, acute skin infections result in ulceration with neutrophils in the dermis and subcutis. chronic lesions contain lymphocytes, macrophages, and fibroblasts. deep infections appear as coalescing botryomycotic pyogranulomas with necrotic centers containing bacterial colonies. infected athymic mice may develop laboratory animal medicine furunculosis of the muzzle and face accompanied by regional lymphadenitis. diagnosis diagnosis is made by documenting gross and histological lesions, including gram staining of suspect tissues, complemented by isolation of grampositive, coagulase-positive (s. aureus), or coagulasenegative staphylococcus species. differential diagnosis staphylococcosis must be differentiated from other suppurative infections of mice, including pasteurellosis, streptococcosis, corynebacteriosis, and pseudomoniasis. ectoparasitism, fight wounds, and self-mutilation per se should also be considered. prevention, control, and treatment removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. in affected animals, nail trimming can reduce self-inflicted trauma. conditions that facilitate aggressive or self-mutilating behavior should be avoided. research complications staphylococcosis can cause illness and disfigurement in mice. immunodeficient mice are at increased risk. etiology streptococci are ubiquitous commensal gram-positive organisms and in some cases, primary pathogens. pathogenic streptococcal infections in laboratory mice are caused by β-hemolytic organisms in lancefield's group c, but epizootics caused by group a streptococci have occurred, and group g organisms have been isolated occasionally. group d has been reclassified as an enterococcus. alpha-hemolytic streptococci can cause systemic disease in scid mice, and group b streptococcus sp. infection has been reported to cause meningoencephalitis in athymic mice (schenkman et al., 1994) . additionally, streptococcus dysgalactiae subsp. equisimilis has lancefield group g or c antigens and was isolated from visceral abscesses of immunocompetent mice (greenstein et al., 1994) . clinical signs cutaneous infections can cause ulcerative dermatitis over the trunk, which may appear gangrenous, whereas systemic infections may be expressed as conjunctivitis, rough hair coat, hyperpnea, somnolescence, and emaciation. epizootiology mice can carry streptococci subclinically in their upper respiratory tracts. lethal epizootics can occur, but factors leading to clinical disease are unknown, although some infections may be secondary to wound contamination. pathology systemic lesions reflect hematogenous dissemination and include abscessation, endocarditis, splenomegaly, and lymphadenopathy (percy and barthold, 2007) . streptococcal cervical lymphadenitis can lead to fistulous drainage to the neck complicated by ulcerative dermatitis. infection with α-hemolytic streptococci can cause inflammatory lesions affecting kidney and heart. diagnosis diagnosis and differential diagnosis depend on isolation of organisms from infected tissues, combined with histopathologic confirmation. differential diagnosis streptococcosis must be differentiated from other suppurative infections of mice, including staphylococcosis, pasteurellosis, corynebacteriosis, and pseudomoniasis. prevention and control removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. research complications immunodeficient mice are at increased risk for streptococcosis. etiology e. coli is a small gram-negative rod that is a normal inhabitant of the mouse intestine. epizootiology infection is considered nonpathogenic in immunocompetent mice. however, hyperplastic typhlocolitis resembling transmissible murine colonic hyperplasia has been reported in scid mice infected with a non-lactose-fermenting e. coli (waggie et al., 1988; arthur et al., 2012) . clinical signs affected mice develop lethargy and fecal staining. pathology gross lesions consist of segmental thickening of the colon or cecum, which may contain blood-tinged feces. microscopically, affected mucosa is hyperplastic and may be inflamed and eroded. diagnosis diagnosis depends on demonstrating lesions and isolating non-lactose-fermenting e. coli. differential diagnosis this condition must be differentiated from proliferative and inflammatory intestinal disease caused by lawsonia intracellularis, c. rodentium, or enterotropic mouse hepatitis virus, especially in immunodeficient mice. colibacillosis provides an example of the morbidity associated with a nominally innocuous organism when it affects an immunocompromised host. prevention and control removal of affected animals and disinfection of caging and equipment will limit or reduce transmission. research complications clinical illness may develop in immunodeficient mice. historically, klebsiella pneumoniae is a ubiquitous gram-negative organism that is a natural inhabitant of the mouse alimentary tract. most commercial vendors have excluded it from their barriers. it can be pathogenic for the respiratory and urinary tract of mice after experimental inoculation but is not a significant cause of naturally occurring disease. etiology klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. epizootiology k. oxytoca also is purported to be an etiological agent of antibiotic-associated hemorrhagic colitis (aahc) in adult humans and adolescents. in animals, k. oxytoca has been isolated from apparently healthy sentinel rodents being monitored for pathogens in health surveillance programs and from utero-ovarian infections including suppurative endometritis, salpingitis, perioophoritis, and peritonitis in aged b6c3f1 mice (davis et al., 1987; rao et al., 1987) . a model of aahc has been developed in rats by administering amoxicillinclavulanate followed by orally infecting rats with a strain of k. oxytoca cultured from a patient with aahc. studies in humans suggest that k. oxytoca exerts its pathogenicity in part through a cytotoxin. recently, authors have showed that several animal isolates of k. oxytoca, including clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on hep-2 and hela cells, indicating the ability to produce cytotoxin. using mass spectroscopy techniques, they also confirmed tilivalline as the cytotoxin present in animal k. oxytoca strains. tilivalline may serve as a biomarker for k. oxytoca-induced cytotoxicity (darby et al., 2014) . clinical signs k. oxytoca has been cultured from cases of suppurative otitis media, urogenital tract infections, and pneumonia in c3h/hej and nmri-foxn1 (nu) mice (bleich et al., 2008) . additionally, k. oxytoca was recently cultured from three breeding colonies of nod. cg-prkdc scid il2rg tm1wjl /szj (nsg) mice with chronic renal inflammation and ascending urinary tract infections (foreman et al., 2011) . differential diagnosis other bacterial infections capable of causing suppurative lesions, including staphylococci, streptococci, pasteurella sp., and e. coli, among others are considered a differential diagnosis. research complications morbidity and mortality from spontaneous infections can affect ongoing research. etiology clostridium difficile was identified as the etiology of antimicrobial-associated pseudomembranous colitis in humans and currently a considerable cause of morbidity in hospitalized patients who acquire nosocomial infections. in the early 2000s, an increased interest in c. difficile infection (cdi) resulted from the emergence of a hyper-virulent strain (nap1/bi/027) associated with frequent recurrences and more severe clinical disease (abou chakra et al., 2014; mcfarland, 2009; kuijper et al., 2006) . c. difficile has also been implicated in antibioticassociated colitis in syrian hamsters (bartlett et al., 1977) , guinea pigs (lowe et al., 1980) , rabbits (thilsted et al., 1981; ryden et al., 1991) , prairie dogs (muller et al., 1987) , ostriches (frazier et al., 1993) , and horses (diab et al., 2013) . c. difficile is a rod-shaped strict anaerobe. cycloserinecefoxitin-fructose agar (ccfa) is a commonly used selective medium for c. difficile. cultures are incubated under anaerobic conditions at 35-37°c. when grown on blood agar, c. difficile colonies are nonhemolytic and gray, and have a slightly raised umbonate profile with filamentous edges and a ground-glass appearance. colonies grown on blood agar have fluorescence under ultraviolet light. c. difficile forms acid from glucose and fructose, but is negative on lactose, maltose, and sucrose. two closely related exotoxins, toxin a and toxin b, are produced by c. difficile. recent taxonomic classification support placement of c. difficile and its close relatives within the family peptostreptococcaceae. the authors suggested renaming it peptoclostridium difficile (yutin and galperin, 2013) . epizootiology it is estimated that c. difficile spores germinate and establish infection less than 10 h after ingestion. spores rapidly transit through the upper gastrointestinal tract and colonize the colon and cecum. spore shedding begins less than 2 h postingestion. when c57bl mice were challenged with 10 8 cfu of c. difficile spores, severe cdi signs developed and all mice were clinically affected by 48 h postchallenge (chen et al., 2008) . specific methods to control and prevent c. difficile infections in mice have not been described. given the method of transmission of c. difficile and c. perfringens are via ingestion or spores, these clostridia can probably be excluded from mouse colonies by maintaining strict husbandry practices, robust sanitation, and use of autoclaved feed, bedding, cages, and cage accessories. sudden dietary changes should be avoided and antibiotics should be used judiciously to minimize disruption of the normal gut microbiota of mice. diagnosis of c. difficile-associated disease is generally based on detection of cytotoxin using a tissue culture cytotoxicity assay. pcr assays for detection of both c. difficile and its cytotoxins have been developed (eastwood et al., 2009) . there are no published regimens specifically for the treatment of natural c. difficile infections in mice. oral doses given twice daily of 2 mg vancomycin for 7 days to experimentally infected gnotobiotic mice caused a 2-to 3-log decrease in vegetative bacterial cell count and no detectable cytotoxin. bacterial counts and cytotoxin levels returned to previous levels after treatment was discontinued. clinical signs untreated mice are relatively resistant to infection with c. difficile and do not develop fatal infections, although these mice can become asymptomatic carriers that persistently shed low numbers of spores (lawley et al., 2009) . susceptibility of mice to infection must be induced by disrupting the microbiota through antibiotic treatment. brief exposure to environmental laboratory animal medicine spore contamination is sufficient for transmission of c. difficile to naïve but susceptible mice. the cdi transmission model has been used to demonstrate that clindamycin treatment of asymptomatic carriers of c. difficile can inadvertently trigger the excretion of high levels of spores (lawley et al., 2009) . a c57bl mouse model of recurrence/relapse cdi has been reported (sun et al., 2011) . the primary bout of cdi induced little or no protective antibody response against c. difficile toxins and mice continued shedding c. difficile spores. antibiotic treatment of surviving mice induced a second episode of diarrhea. a simultaneous reexposure of mice to c. difficile bacteria or spores elicited a full clinical spectrum of cdi similar to that of the primary infection. immunosuppressive agents resulted in more severe and fulminant recurrent disease. vancomycin treatment only delayed disease recurrence; however, neutralizing polysera against both tcda and tcdb completely protected mice against cdi relapse (sun et al., 2011) . a recent study in c57bl mice demonstrated that antibiotic-mediated alteration of the gut microbiome favors a global metabolic profile, and therefore increases susceptibility to c. difficile clinical diseases (theriot et al., 2014) . c. difficile is not tissue invasive and only toxigenic strains are associated with disease. experimental c. difficile infections include diarrhea, cecitis, polymorphonuclear cell infiltration of the lamina propria, inflammation, pseudomembrane formation, and death. differential diagnosis c. difficile-induced diarrhea is most often associated with antibiotic treatment. other clostridial diseases in mice must be ruled out as well as other enteric pathogens in mice causing diarrhea and mortality. salmonella spp. and c. rodentium should be considered in the differential diagnosis. etiology clostridium perfringens is associated with a number of diseases in domestic animals and humans. c. perfringens is a nonmotile, rod-shaped, encapsulated, anaerobic bacterium measuring 4-8 µm in length and 0.8-1.5 µm in diameter (murray et al., 2002) . c. perfringens grow rapidly on blood agar, and colonies are smooth, round, and grayish in color, and are surrounded by a double zone of hemolysis. c. perfringens is grouped into five types based on the production and secretion of four major toxins. c. perfringens produces a number of other virulence-enhancing toxins and hydrolytic enzymes. the most significant of these is probably enterotoxin, released with the bacterial spore after cell lysis. epizootiology c. perfringens is most likely acquired by the ingestion of spores that originated in the soil or in the intestinal tract of a carrier animal. the organism can be a member of the normal microbiota in human and domestic animals. factors that have been associated with the proliferation of the organism of these species include poor husbandry and sudden dietary changes (quinn et al., 2002) . methods to control and prevent c. perfringens infections have not been evaluated in mice. because the bacterium is most likely acquired by the ingestion of spores, it can probably be excluded from mouse colonies by maintaining good sanitation and sterilizing feed, bedding, cages, and cage accessories. sudden dietary changes have also been associated with proliferation of the organism and should be avoided if possible (quinn et al., 2002) . clinical signs only a few reports in the literature exist describing clinical disease associated with c. perfringens infection in mice (matsushita and matsumoto, 1986; rozengurt and sanchez) . disease has been observed in mice of both sexes, from 2 to 32 days old, and in female mice of breeding age. clinical signs have included hunched posture, ruffled hair coat, enlarged painful abdomen, soft or impacted feces, hindquarter paralysis, and dyspnea. sudden death without premonitory signs has also been reported. the toxin types of c. perfringens isolated from these cases were reported to be non-type a (matsushita and matsumoto, 1986) , type b (rozengurt and sanchez, 1999) , and type d (clapp and graham, 1970) . mucosal necrosis in both the large and small intestine is a consistent finding on microscopic examination of tissues from mice with clinically apparent c. perfringens infections. differential diagnosis c. perfringens produces a number of major and minor toxins. different types of the bacterium produce different toxins which account for different disease outcomes. c. perfringens type a is a constituent of the normal microbiota of the intestine of humans and other animal species. bacterial culture should be obtained from live or recently dead animals, and placed in anaerobic transfer medium for transport to a microbiology laboratory and should be cultured soon after their arrival. a presumptive diagnosis for c. perfringens can be based on the presence of large grampositive rods in fecal smears or in histologic sections of intestines (quinn et al., 2002) . definitive diagnosis is based on toxin identification. mice treated with chlortetracycline hydrochloride in drinking water at a level of 11 mg/l for 2 weeks have eliminated c. perfringens-associated disease (matsushita and matsumoto, 1986) . penicillin g in the diet or changing the diet has also been reported to be effective in disease remission. c. perfringens treatments in domestic species include ampicillin, amoxicillin-clavulanate, tylosin, clindamycin, metronidazole, and bacitracin (marks, 2013; mcgorum et al., 1998) . commercially available bacterins for use in mice were not effective in controlling the disease (clapp and graham, 1970) . research complications clostridia are large, rodshaped, gram-positive anaerobic bacteria. naturally occurring clostridial infection in mice is rare. epizootics of c. perfringens type d infection with high mortality laboratory animal medicine have been reported in a barrier colony where heavy mortality occurred in 2-to 3-week-old suckling mice. clinical signs included scruffy hair coats, paralysis of the hindquarters, and diarrhea or fecal impaction. however, attempts to reproduce the disease experimentally with clostridia isolated from naturally infected animals were unsuccessful. c. perfringens also has been isolated from sporadic cases of necrotizing enteritis in recently weaned mice. clostridium piliforme -tyzzer's disease (fujiwara and ganaway, 1994; ganaway, 1982; ganaway et al., 1971; percy and barthold, 2007) etiology tyzzer's disease is named for ernest tyzzer, who first described it in a colony of japanese waltzing mice. the causative organism, c. piliforme (formerly bacillus piliformis), is a long, thin, gram-negative spore-forming bacterium that appears to require living cells for in vitro growth. it has not been grown successfully on cell-free media, but it can be propagated by inoculation of susceptible vertebrates, in select cell lines, the yolk sac of embryonated eggs, or hepatocyte cell cultures obtained from mice (ganaway et al., 1985; kawamura et al., 1983) . clinical signs clinical disease occurs as unexpected deaths that may be preceded by diarrhea and inactivity. although outbreaks can be explosive and mortality is usually high, morbidity varies. additionally, subclinical infections can occur, accompanied by the development of antibodies to c. piliforme. stresses, such as overcrowding, high temperature and humidity, moist food, and immunosuppression, and young age, may predispose mice to tyzzer's disease. susceptibility and resistance also are influenced by host genotype. it has been shown, e.g., that c57bl/6 mice are more resistant than dba/2 mice to tyzzer's disease (waggie et al., 1981) . resistance to severe infection appears to be due, in part, to b-lymphocyte function. the role of t cells in resistance is not clear, because susceptibility among athymic mice appears to vary (livingston et al., 1996) . however, the involvement of t cells can be inferred by the fact that several interleukins modulate resistance and susceptibility. depletion of neutrophils or nk cells also increases susceptibility to infection. epizootiology current prevalence rates, reservoirs of infection, carrier states, and the mechanism of spread remain speculative. tyzzer's disease occurs in many species of laboratory animals and in domestic and free-living species. some strains appear capable of cross-infecting mice, rats, and hamsters, whereas others have a more restricted host range (franklin et al., 1994) . therefore, the risks for cross-infection depend on the strain causing a given outbreak. although the vegetative form of c. piliforme is unstable, spores can retain infectivity at room temperature for at least 1 year and should be viewed as the primary means of spread. natural infection is probably due to ingestion of organisms, which are subsequently shed in feces. feces-contaminated food and soiled bedding are the most likely sources of environmental contamination. prenatal infection can be induced by intravenous inoculation of pregnant mice, but its importance in the natural transmission of infection has not been determined. pathology infection begins in the gastrointestinal tract, followed by bacteremic spread to the liver and, to a smaller extent, the heart. the lesions are characterized by necrosis in these tissues and in the mesenteric lymph nodes. grossly, segments of the ileum, cecum, and colon may be red and dilated, with watery, fetid contents, whereas the liver, mesenteric lymph nodes, and heart often contain gray-white foci. histologically, intestinal lesions include necrosis of mucosal epithelium, which may be accompanied by acute inflammation and hemorrhage. in the liver, foci of coagulation necrosis are generally distributed along branches of the portal vein, a finding compatible with embolic infection from the intestine. peracute lesions are largely free of inflammation, but neutrophils and lymphocytes may infiltrate less fulminant lesions. myocardial necrosis is sporadic in natural infection. diagnosis tyzzer's disease is diagnosed most directly by the demonstration of characteristic intracellular organisms in tissue sections of liver and intestine. bundles of long, slender rods occur in the cytoplasm of viable cells bordering necrotic foci, especially in the liver (fig. 3 .46) and intestine. they are found more easily during early stages of infection. organisms in tissue sections do not stain well with hematoxylin-eosin stain. silver stains, giemsa stains, or periodic acid-schiff stains are usually required for visualization of the organism. pcr and serologic assays are readily available at diagnostic laboratories. older supplemental procedures included inoculation of cortisonized mice or embryonated eggs laboratory animal medicine with suspect material, followed by histological or immunocytochemical demonstration of organisms in tissues. differential diagnosis the histological detection of organisms is essential for differentiating tyzzer's disease from other infections that can produce similar signs and lesions, especially mousepox, coronaviral hepatitis, reoviral hepatitis, helicobacteriosis, and salmonellosis. it also is important not to misconstrue extracellular rods as c. piliforme. prevention and control barrier housing and husbandry that incorporate sanitation measures to avoid the introduction or buildup of spores in the environment are the bases for control or prevention of tyzzer's disease. if infection occurs, spore formation will make control or elimination by antibiotic therapy problematic. therefore, strict quarantine, followed by replacement of affected or exposed stock, must be considered. rederivation by embryo transfer or cesarean section should take the potential for prenatal transmission of infection into account in housing and testing offspring. thorough decontamination of the environment with an oxidizing disinfectant must be included in any control program. additionally, procurement of food and bedding from suppliers with thorough quality assurance and vermin control programs is essential for both prevention and control. husbandry supplies should be stored in vermin-proof quarters, and the option of heat sterilization of food and bedding should be considered. research complications research complications stem from clinical morbidity and mortality. mice with immune dysfunction are at increased risk. there is recent evidence that infection causes elevations in selected cytokines (van andel et al., 2000) . etiology two mycobacteria are known to be pathogenic for laboratory mice: mycobacterium avium-intracellulare and m. lepraemurium. both are acid-fast, obligate intracellular bacteria. epizootiology mycobacteria are widespread in water and soil. their presence in laboratory mice would indicate a significant break in husbandry practices. infection with m. avium-intracellulare should be considered extremely rare, with the only published report describing an episode in a breeding colony of c57bl/6 mice . the source of the outbreak was presumed to be drinking water. mycobacterium lepraemurium has been isolated from healthy laboratory mice and can persist as a latent infection, but its significance is primarily historical, as a model for human leprosy. it is highly unlikely to encounter this infection in a modern, well-managed mouse colony. clinical signs m. avium-intracellulare infection is typically subclinical but mice have developed granulomatous pneumonia . pathology lesions are classically a chronic granulomatous disease with granulomas, langhans giant cells, and concurrent presence of acid-fast bacteria in various organs including the lungs, liver, spleen and lymph nodes. m. lepraemurium may cause alopecia, thickening of skin, subcutaneous swellings, and ulceration of the skin. disease can lead to death or clinical recovery. gross lesions are characterized by nodules in subcutaneous tissues and in reticuloendothelial tissues and organs (lung, spleen, bone marrow, thymus, and lymph nodes). lesions can also occur in the lung, skeletal muscle, myocardium, kidneys, nerves, and adrenal glands. the histologic hallmark is perivascular granulomatosis with accumulation of large, foamy epitheloid macrophages (lepra cells) packed with acid-fast bacilli. diagnosis acid-fast bacilli in lesions are the hallmark of presumptive diagnosis of mycobacteriosis. definitive diagnosis results from positive culture which takes days to weeks to rule out or positive pcr assays which are more time-efficient but require associated expertise. differential diagnosis other bacterial species that cause granulomatous lesions in mice. research complications natural infection is very rare. etiology proteus mirabilis is a ubiquitous gram-negative organism that can remain latent in the respiratory and intestinal tracts of normal mice (percy and barthold, 2007) . epizootiology proteus mirabilis colonizes the intestinal tract of most humans and is commonly found in research mice unless specifically excluded. clinical signs clinical disease can occur following stress or induced immunosuppression. immunodeficient mice have a heightened susceptibility to pathogenic infection. pathology proteus has been associated with ulcerative lesions in the gastrointestinal tract of immunodeficient mice. infected animals lose weight, develop diarrhea, and die within several weeks. if septicemia develops, suppurative or necrotic lesions, including septic thrombi, may be found in many organs, but the kidney is commonly affected. proteus pyelonephritis is characterized by abscessation and scarring. ascending lesions may occur following urinary stasis, but hematogenous spread cannot be ruled out. proteus mirabilis and pseudomonas aeruginosa have been isolated concomitantly from cases of suppurative nephritis or pyelonephritis. infection in immunodeficient mice is typified by splenomegaly and focal necrotizing hepatitis. pulmonary lesions include edema and macrophage activation. septic thrombi can occur, however, in many tissues. diagnosis culture recovery of proteus mirabilis as a predominant or single isolate confirms an opportunistic local or systemic infection. differential diagnosis gram-negative bacterial infections. research complications natural infections are typically isolated cases. etiology leptospirosis remains one of the most common zoonoses transmissible from rodents (desvars et al., 2011) but is exceedingly rare in laboratory mice. infection with leptospira interrogans serovar ballum has been reported on several occasions (see chapter 29). epizootiology leptospira are gram-negative organisms that, after a septicemic phase, establish persistent infection in the renal tubules and are periodically excreted in the urine. clinical signs natural infection is subclinical and causes no significant lesions. experimental infections can result in severe vascular, hepatic and renal lesions dependent on serovar, mouse strain and immunocompetency. diagnosis diagnosis requires isolation of organisms in kidney culture. serological testing should be used with caution because neonatal exposure can lead to persistent infection without seroconversion. histologic examination of kidney using silver stains can also be attempted. pcr assays are reliable for preliminary diagnosis. differential diagnosis not applicable in research colonies. research complications persistent murine infections associated with active shedding present a zoonotic hazard for humans; therefore, infected mice should be culled. elimination of infection from highly valuable mice requires rederivation. (percy and barthold, 2007) etiology chlamydia trachomatis is an intracellular organism that produces glycogen-positive intracytoplasmic inclusions (elementary bodies). c. trachomatis causes ocular and urogenital disease in humans. however, at least one strain historically referred to as the 'nigg agent' after clara nigg, is most recently classified as chlamydia muridarum and is used experimentally to model human chlamydia infection. epizootiology mice are susceptible to natural infection and experimental infection with c. trachomatis and chlamydophila psittaci, especially immunodeficient mouse strains. clinical signs natural infections are typically subclinical but persistent. pathology c. muridarum is also known as the 'mouse pneumonitis agent' due to severe acute infection which is characterized by ruffled fur, hunched posture, and labored respiration due to interstitial pneumonitis and death in 24 h. mice with more chronic infections may develop progressive emaciation and cyanosis of the ears and tail. experimental infections to model human venereal chlamydia infections will develop hydrosalpinx, cervical, and vaginal infections in female mice and urethritis in male mice. diagnosis chlamydia can be diagnosed by impression smears stained with giemsa or macchiavello stains, cell culture, or inoculation of embryonated eggs. pcr and sequencing can be used to speciate the type of chlamydia. differential diagnosis c. muridarum, c. trachomatis, and c. psittaci are included in the differential diagnosis. research complications chlamydia is a rare spontaneous infection in research mice; its potential significance is low. etiology pneumocystis murina (pm) is a common opportunistic organism of laboratory mice and other mammals. when first described by chagas in 1909, p. carinii was misidentified as trypanosoma cruzi and was considered a protozoan (chagas, 1909) . it was renamed as a new species, p. carinii, when observed in a rat in 1912 (delanoë, p. and delanoë, m. 1912) . p. carinii, however, has now been grouped taxonomically with the fungi based on dna analysis and the homology of p. murina housekeeping genes with those found in fungi (edman et al., 1989; stringer et al., 2002; wakefield et al., 1992) . these dna studies and apparent differences of host susceptibility prompted a new name, p. jiroveci, for pneumocystis isolated from humans (stringer et al., 2002; frenkel, 1999) . p. carinii is now used to name the organism in rats and p. murina, the organism in mice. clinical signs pm infection is subclinical in immunocompetent mice. however, it can be clinically severe in immunodeficient mice, because an adequate complement of functional t lymphocytes is required to suppress infection (roths et al., 1990; shultz and sidman, 1987; walzer et al., 1989; weir et al., 1986) . b cells have also been shown to be critical to clearance of infection and the mechanism appears only partially related to igg and has a more important role in promoting activation and expansion of t cells (lund et al., 2003) . b cells may also protect early hematopoietic progenitor activity during systemic responses to pneumocystis infection (hoyt et al., 2014) . infection proceeds slowly, but relentlessly in immunodeficient mice leading to clinical signs of pneumonia, usually within several months. primary signs include dyspnea and hunched posture, which may laboratory animal medicine be accompanied by wasting and scaly skin. severe cases, such as those that occur in advanced disease in scid mice, may be fatal. epizootiology pm is known to infect a number of mammalian hosts, including ferrets, rats, mice, and humans. pm is a ubiquitous organism that is often present as a latent infection. although firm prevalence data are not available, because detection methods are not simple to apply, infection is assumed to be present in mouse colonies unless ruled out by extensive surveillance. although these organisms appear morphologically similar, there are antigenic and genetic differences among p. murina isolated from different hosts (weinberg and durant, 1994; cushion, 1998) . furthermore, studies indicate that p. carinii isolated from one host species is unable to survive and replicate after inoculation into a different immunodeficient host species (gigliotti et al., 1993b) . pm infection also occurs in human beings, but transmission between rodents and human beings has not been documented. pm is transmitted by aerosol and establishes persistent, quiescent infection in the lungs of immunocompetent mice. prenatal infection has not been demonstrated. pathology pm is normally not pathogenic but can be activated by intercurrent immunosuppression. activation fills the lung with trophic and cystic forms. gross lesions occur in the lungs, which are often rubbery and fail to deflate (fig. 3.47 ). histopathological changes are characterized by interstitial alveolitis with thickening of alveolar septa from proteinaceous exudate and infiltration with mononuclear cells (fig. 3 .48) (roths et al., 1990) . alveolar spaces may contain vacuolated eosinophilic material and macrophages. special stains are required to visualize pm. silver-based stains reveal round or partially flattened 3-to 5-mm cysts in affected parenchyma (fig. 3.49) . in florid cases, alveolar spaces may be filled with cysts, but cysts may be sparse in mild cases. disease can be especially severe when subclinically infected immunodeficient mice are reconstituted with competent immune cells that subsequently promote pneumonitis. diagnosis respiratory distress in immunodeficient mice should elicit consideration of pneumocystosis. pathologic examination of the lung, including silver laboratory animal medicine methenamine staining, is essential to confirm a presumptive clinical diagnosis. past infections of immunocompetent mice also can be detected by elisa (furuta et al., 1985) . pcr can be used to detect active infection (gigliotti et al., 1993a; reddy et al., 1992) and is particularly useful for screening immunodeficient mice. differential diagnosis pneumocystosis must be differentiated from viral pneumonias of mice. it is worth noting, in this regard, that pneumonia virus of mice has been shown to accelerate the development of pneumocystosis in scid mice (bray et al., 1993; roths et al., 1993) . prevention and control pm infection is a significant disease threat to immunodeficient mice. its widespread distribution strongly suggests that susceptible mice should be protected by microbarrier combined, where possible, with macrobarrier housing. husbandry procedures should include proper sterilization of food, water, and housing equipment and the use of hepa-filtered change stations. infected colonies can be rederived by embryo transfer or cesarean methods, because infection does not appear to be transmitted in utero. research complications pneumonia in immunodeficient mice is the major complication of pm infection. trichophyton mentagrophytes is the most common fungal agent of mice. however, infection rarely causes clinical disease. clinical signs include sparse hair coats or well-demarcated crusty lesions, with a chalky surface on the head, tail, and legs (favus or ringworm). skin lesions are composed of exfoliated debris, exudate, mycelia, and arthrospores with underlying dermatitis. invasion of hair shafts is not characteristic. diagnosis depends on effective specimen collection. hairs should be selected from the periphery of the lesion, and hairless skin should be scraped deeply to obtain diagnostic specimens. t. mentagrophytes rarely fluoresces under ultraviolet light, and hyphae must be differentiated from bedding fibers, food particles, and epidermal debris. histological sections should be stained with a silver stain or schiff's reagent to reveal organisms. trichophyton also can be cultured on sabouraud agar. plates are incubated at room temperature (22-30°c), and growth is observed at 5-10 days. ringworm is not easily eradicated from laboratory mice. the use of antifungal agents to treat individual mice is time-consuming, expensive, and variably effective. rederivation is a more prudent course. cages and equipment should be sterilized before reuse. concurrent infection with ectoparasites also must be considered during eradication steps. candida albicans and other systemic mycoses are not important causes of disease in mice, but they can be opportunistic pathogens in immunodeficient mice. etiology giardia muris is a pear-shaped, flagellated organism with an anterior sucking disk. it inhabits the duodenum of young and adult mice, rats, and hamsters. clinical signs infection is often subclinical, unless organisms proliferate extensively, and can cause weight loss, a rough hair coat, sluggish movement, and abdominal distension, usually without diarrhea. additionally, immunodeficient mice may die during heavy infestation. epizootiology the contemporary prevalence of affected mouse colonies is not well documented, but surveys during the 1980s found the rates exceeding 50%. transmission occurs by the fecal-oral route. crossinfection between mice and hamsters after experimental inoculation of organisms has been demonstrated, whereas rats were resistant to isolates from mice and hamsters (kunstyr et al., 1992) . c3h/he mice are particularly susceptible to giardiasis, whereas balb/c and c57bl/10 mice are more resistant. additionally, female mice appear to be more resistant to infection than male mice (daniels and belosevic, 1995) . c57bl/6 females, e.g., have lower trophozoite burdens and for a shorter interval than male mice. females also shed cysts later than male mice. these differences may be related to a more potent humoral immune response to giardia in female mice. pathology gross lesions are limited to the small intestine, which may contain yellow or white watery fluid. histopathology reveals organisms in the lumen that often adhere to microvilli of enterocytes or reside in mucosal crevices or mucus. the crypt/villus ratio may be reduced, and the lamina propria may have elevated numbers of inflammatory cells. diagnosis diagnosis is based on detection of trophozoites in the small intestine or in wet mounts of fecal material. organisms can be recognized in wet preparations by their characteristic rolling and tumbling movements. ellipsoidal cysts with four nuclei also may be detected in feces. infection also can be detected by serology (daniels and belosevic, 1994) and by pcr (mahbubani et al., 1991) . treatment, prevention, and control murine giardiasis can be treated by the addition of 0.1% dimetridazole to drinking water for 14 days. prevention and control depend on proper sanitation and management, including adequate disinfection of contaminated rooms. research complications accelerated cryptal cell turnover and suppression of the immune response to sheep erythrocytes have been observed in infected mice. the potential for severe or lethal infection in immunodeficient mice was noted previously. etiology spironucleus muris is an elongated, pearshaped, bilaterally symmetrical flagellated protozoan that commonly inhabits the duodenum, usually in the crypts of lieberkühn. it is smaller than giardia muris and lacks an anterior sucking disk. clinical signs s. muris infection is usually subclinical in normal adult mice. it is more pathogenic, however, for young, stressed, or immunocompromised mice (kunstyr et al., 1977) . additionally, clinical morbidity may indicate an underlying primary infection with an unrelated organism. clinically affected mice can have a poor hair coat, sluggish behavior, and weight loss. mice at 3-6 weeks of age are at notably higher risk for clinically evident infection. they can develop dehydration, hunched posture, abdominal distension, and diarrhea. severe infections can be lethal. epizootiology transmission occurs by the fecaloral route and can occur between hamsters and mice as well as between mice. it does not appear to be transmitted between mice and rats (schagemann et al., 1990) . the most recent surveys, which are somewhat dated, indicated that prevalence rates exceeded 60% among domestic mouse colonies in the mid-1980s. there is some evidence that inbred strains vary in their susceptibility to infection and their rate of recovery (baker et al., 1998; brett and cox, 1982) . pathology gross findings associated with infection include watery, red-brown, gaseous intestinal contents. however, it is essential to rule out primary or coinfection by other organisms before attributing these lesions to spironucleosis. microscopically, acute disease is associated with distension of crypts and intervillous spaces by pear-shaped trophozoites and inflammatory edema of the lamina propria. organisms can be visualized more easily with periodic acid-schiff staining, which may reveal invasion of organisms between enterocytes and in the lamina propria. chronic infection is associated with lymphoplasmacytic infiltration of the lamina propria and occasional intracryptal inflammatory exudate. diagnosis diagnosis is based on identification of trophozoites in the intestinal tract. they can be distinguished from giardia muris and tritrichomonas muris by their small size, horizontal or zigzag movements, and the absence of a sucking disk or undulating membrane. pcr-based detection also is available (rozario et al., 1996) . it is not clear whether duodenitis is a primary pathogenic effect of s. muris or represents opportunism secondary to a primary bacterial or viral enteritis. therefore, it is prudent to search for underlying or predisposing infections. treatment, prevention, and control treatment consists of adding 0.1% dimetridazole to drinking water for 14 days, as described for giardiasis. prevention and control require good husbandry and sanitation. research complications as with giardiasis, infection can accelerate enterocytic turnover in the small intestine. there is some evidence that infected mice may have activated macrophages that kill tumor cells nonspecifically and that infection can diminish responses to soluble and particulate antigens. additionally, infected mice also have increased sensitivity to irradiation. such effects should, however, be interpreted cautiously in order to rule out intercurrent viral infections. tritrichomoniasis t. muris is a nonpathogenic protozoan that occurs in the cecum, colon, and small intestine of mice, rats, and hamsters. no cysts are formed, and transmission is by ingestion of trophozoites passed in the feces. it can be detected by microscopy or by pcr (viscogliosi et al., 1993) . coccidiosis eimeria falciformis is a pathogenic coccidian that occurs in epithelial cells of the large intestines of mice. it was common in european mice historically but is seldom observed in the united states. heavy infection may cause diarrhea and catarrhal enteritis. klosiella muris causes renal coccidioisis in wild mice but is rare in laboratory mice. mice are infected by ingestion of sporulated sporocysts. sporozoites released from the sporocysts enter the bloodstream and infect endothelial cells lining renal arterioles and glomerular capillaries, where schizogony occurs. mature schizonts rupture into bowman's capsule to release merozoites into the lumen of renal tubules. merozoites can enter epithelial cells lining convoluted tubules, where the sexual phase of the life cycle is completed. sporocysts form in renal tubular epithelium and eventually rupture host cells and are excreted in the urine, but oocysts are not formed. infection is usually nonpathogenic and subclinical. gray spots may occur in heavily affected kidneys and are the result of necrosis, granulomatous inflammation, and focal hyperplasia. destruction of tubular epithelium may impair renal physiology. diagnosis is based on detection of organisms in tissues. prevention and control require proper sanitation and management techniques. there is no effective treatment. cryptosporidiosis cryptosporidium muris is a sporozoan that adheres to the gastric mucosa. it is uncommon in laboratory mice and is only slightly pathogenic. cryptosporidium parvum inhabits the small intestine and is usually nonpathogenic in immunocompetent and athymic mice (ozkul and aydin, 1994; taylor et al., 1999) . athymic mice may develop cholangitis and hepatitis, however, if organisms gain access to the biliary tract. entamoebiasis entamoeba muris is found in the cecum and colon of mice, rats, and hamsters throughout the world. organisms live in the lumen, where they feed on particles of food and bacteria. they are considered nonpathogenic. encephalitozoonosis encephalitozoon cuniculi is a gram-positive microsporidian that infects rabbits, mice, rats, guinea pigs, dogs, nonhuman primates, humans, and other mammals. infection is extremely rare among laboratory mice. the life cycle of the organism is direct, and animals are infected by ingesting spores or by cannibalism. spore cells are disseminated in the blood to the brain and other sites. infection can last more than 1 year, and spores shed in the urine serve as a source of infection. vertical transmission has not been confirmed in mice. e. cuniculi is an obligate intracellular parasite, but infection usually elicits no clinical signs of disease. organisms proliferate in peritoneal macrophages by asexual binary fission. they have a capsule that accepts giemsa and goodpasture stains but is poorly stained by hematoxylin. fulminating infection can cause lymphocytic meningoencephalitis and focal granulomatous hepatitis. in contrast to encephalitozoonosis in rabbits, affected mice do not develop interstitial nephritis. infection is diagnosed by cytological examination of ascitic fluid smears, histopathologic examination of brain tissues stained with goodpasture stain, and elisa serology. no effective treatment has been reported. prevention and control require rigid testing and elimination of infected colonies and cell lines. pcr-based assays may also be useful. toxoplasmosis toxoplasma gondii is a ubiquitous gram-negative coccidian parasite for which the mouse serves as a principal intermediate host. however, the prevalence of natural infection is negligible because laboratory mice no longer have access to sporulated cysts shed by infected cats, which were historically the major source for cross-infection. toxoplasmosis can cause necrosis and granulomatous inflammation in the intestine, mesenteric lymph nodes, eyes, heart, adrenals, spleen, brain, lung, liver, placenta, and muscles. diagnosis is based on elisa serology, histopathology, and pcr. control and prevention depend largely on precluding access of mice to cat feces or to materials contaminated with cat feces. oocytes are very resistant to adverse temperatures, drying, and chemical disinfectants; therefore, thorough cleaning of infected environments is required. b. cestodiasis baker, 2007) etiology hymenolepis (rodentolepis) nana, the dwarf tapeworm, infects mice, rats, and humans although the zoonotic risk has been questioned (macnish et al., 2002) . adults are extremely small (25-40 mm) and have eggs with prominent polar filaments and rostellar hooks (fig. 3.50) . clinical signs young adult mice are most frequently infected. signs and lesions include weight loss and focal enteritis, but clinical disease is rare unless infestation is severe. epizootiology the life cycle may be direct or indirect (r. nana is the only cestode known that does not require an intermediate host). the indirect cycle utilizes arthropods as intermediate hosts. liberated oncospheres penetrate intestinal villi and develop into a cercocystis stage before reemerging into the intestinal lumen 10-12 days later. the scolex attaches to the intestinal mucosa, where the worm grows to adult size in 2 weeks. the cycle from ingestion to patency takes 20-30 days. pathology cysticerci are found in the lamina propria of the small intestine and sporadically in the mesenteric lymph nodes, whereas adults, which have a serrated profile, are found in the lumen. inflammation is not a feature of infection. diagnosis infection can be diagnosed by demonstrating eggs in fecal flotation preparations or by opening the intestine in petri dishes containing warm tap water to facilitate detection of adults. r. nana can be differentiated from another species of rodent tapeworm, h. diminuta, by the fact that r. nana has rostellar hooks and eggs with polar filaments. however, h. diminuta requires an intermediate arthropod host, so it is rarely found in contemporary mouse colonies. treatment, prevention, and control drugs recommended for treatment and elimination include praziquantel (0.05% in the diet for 5 days), albendazole, mebendazole, and thiabendazole. although the benzimidazoles have an excellent activity against cestodes and nematodes in rats, they have not been tested extensively in mice. the potential for successful treatment is high, however, because eggs do not survive well outside the host and because the prevalence of infestation is low in caged mice kept in properly sanitized facilities. because r. nana can directly infect humans, proper precautions should be taken to avoid oral contamination during handling of rodents (see chapter 28). hymenolepis microstoma is found in the bile ducts of rodents and could be confused with r. nana in the mouse. however, the location of the adult as well as the large size of h. microstoma eggs compared with those of r. nana make differential diagnosis relatively simple. the mouse and the rat are intermediate hosts of the cestode taenia taeniaformis. the definitive host is the cat. this parasite should not be found in laboratory mice housed separately from cats. c. nematodiasis (wescott, 1982) syphacia obvelata (mouse pinworm) infestation etiology syphacia obvelata, the common mouse pinworm, is a ubiquitous parasite of wild and laboratory mice. the rat, gerbil, and hamster are also occasionally infected. female worms range from 3.4 to 5.8 mm in length, and male worms are smaller (1.1-1.5 mm). eggs are flattened on one side and have pointed ends (fig. 3.51 ). the nucleus fills the shell and is frequently at a larval stage when eggs are laid. clinical signs infestation is usually subclinical, although heavily infested mice can occasionally sustain intestinal lesions, including rectal prolapse, intussusception, enteritis, and fecal impaction. epizootiology pinworm infestation is one of the most commonly encountered problems in laboratory mice. a national survey revealed that more than 30% of barrier colonies and about 70% of conventional colonies were affected (jacoby and lindsey, 1997; carty, 2008) . syphacia obvelata infestation can occur unexpectedly in commercial barrier murine colonies, resulting in widespread dissemination of the parasite into academic mouse colonies. the epizootiological impact of pinworm infestation is increased by the airborne dissemination of eggs, which can remain infectious even after drying. the life-cycle is direct and completed in 11-15 days. females deposit their eggs on the skin and hairs of the perianal region. ingested eggs liberate larvae in the small intestine and they migrate to the cecum within 24 h. worms remain in the cecum for 10-11 days, where they mature and mate. the females then migrate to the large intestine to deposit their eggs as they leave the host. there is unconfirmed speculation that larvae may reenter the rectum. infestation usually begins in young mice and can recur, but adult mice tend to be more resistant. syphacia infestation often occurs in combination with aspiculuris tetraptera. because the life cycle of syphacia is much shorter than that of aspiculuris, the number of mice that are apt to be infected with s. obvelata is correspondingly greater. there is evidence that resistance to infestation may be mouse strain-specific (derothe et al., 1997) . pathology gross lesions are not prevalent, aside from the presence of adults in the lumen of the intestine. diagnosis infestation is diagnosed by demonstrating reniform-shaped eggs in the perianal area or adult worms in the cecum or large intestine. four-to 5-weekold mice should be examined because the prevalence is higher in this age group than in older mice. because most eggs are deposited outside the gastrointestinal tract, fecal examination is not reliable. eggs are usually detected by pressing cellophane tape to the perineal area and then to a glass slide that is examined by microscopy. aspiculuris tetraptera eggs are not ordinarily found in tape preparations and are easily differentiated from eggs of s. obvelata (see below). adult worms can be found in cecal or colonic contents diluted in a petri dish of warm tap water. they are readily observed with the naked eye or with a dissecting microscope. an elisa also is available to detect serum antibodies to s. obvelata somatic antigens (sato et al., 1995) . pcr assays are increasingly being used to augment traditional diagnostic methods and to discriminate between pinworm species (dole et al., 2011) . pcr panels for pinworm detection using fecal pellets are available from commercial diagnostic laboratories. treatment, prevention, and control pinworm infestation can be treated effectively by a number of regimens, which include the use of anthelmintics such as piperazine, ivermectin, and benzimidazole compounds alone or in combination (klement et al., 1996; le blanc et al., 1993; lipman et al., 1994; flynn et al., 1989; wescott, 1982; zenner, 1998) . because some of the recommended therapies have the potential for toxicity, it is prudent to keep mice under close clinical observation during treatment (davis et al., 1999; skopets et al., 1996; toth et al., 2000) . fenbendazole diets can be fed with 1 week on/1 week off rotation with normal chow although the potential impact on experimental data must be considered (duan et al., 2012; gadad et al., 2010; landin et al., 2009) . prevention of reinfestation requires strict isolation because syphacia eggs become infective as soon as 6 h after they are laid, and they survive for weeks, even in dry conditions. strict sanitation, sterilization of feed and bedding, and periodic anthelmintic treatment are required to control infestation. the use of microbarrier cages can reduce the spread of infective eggs. syphacia muris is the common rat pinworm. it can potentially infest mice but is not found in well-managed colonies. it can be differentiated from s. obvelata because s. muris eggs are smaller. treatment is the same as for pinworms of mice. etiology aspiculuris tetraptera is the other major oxyurid of the mouse and may coinfect mice carrying s. obvelata. females are 2.6-4.7 mm long, and males are slightly smaller. the eggs are ellipsoidal (fig. 3.52) . clinical signs ingested eggs hatch, and larvae reach the middle colon, where they enter crypts and remain for 4-5 days. they move to the proximal colon about 3 weeks after infection of the host. because the life cycle is 10-12 days longer than in s. obvelata, infestations appear in somewhat older mice; heaviest infestation is expected in 5-6 weeks after initial exposure. infection is usually subclinical, but heavy loads can produce signs similar to those discussed for s. obvelata. light to moderate loads do not produce clinical disease. epizootiology as noted under s. obvelata, pinworm infestation is highly prevalent and contagious in laboratory mice. the life cycle is direct and takes approximately 23-25 days. mature females inhabit the large intestine, where they survive from 45 to 50 days and lay their eggs. the eggs are deposited at night and are excreted in a mucous layer, covering fecal pellets. they require 6-7 days at 24°c to become infective and can survive for weeks outside the host. pathology see s. obvelata (section iii, a,5,c). diagnosis aspiculuris tetraptera eggs can be detected in the feces, and adult worms are found in the large intestine. eggs are not deposited in the perianal area; therefore, cellophane tape techniques are not useful. measures for treatment, prevention, and control are similar to those described for s. obvelata. because a. tetraptera takes longer to mature and because eggs are deposited in feces rather than on the host, adult parasites are more amenable to treatment by frequent cage rotations. immune expulsion of parasites and resistance to reinfection are hallmarks of a. tetraptera infection. research complications see s. obvelata (section iii, a,5,c). several species of mites infest laboratory mice. they include myobia musculi, radfordia affinis, myocoptes musculinus, and, less commonly, psorergates simplex. the common murine mites are described below, while less frequently encountered species are listed in table 3 .14. these include the mouse mite trichoecius romboutsi, which resembles myocoptes and ornithonyssus bacoti, the tropical rat mite, which can infect laboratory mice. characteristics of specific infestations are described after a general introductory section. clinical signs mites generally favor the dorsal anterior regions of the body, particularly the top of (jacoby and lindsey, 1997; carty, 2008) reported mite infestations in 40% of colonies. acarids spend their entire lives on the host. populations are limited by factors such as self-grooming, mutual grooming, the presence of hair, and immunological responses, which tend to produce hypersensitivity dermatitis. inherited resistance and susceptibility also affect clinical expression of acariasis. mite populations, e.g., vary widely among different stocks and strains of mice housed under similar conditions. pathology gross lesions include scaly skin, regional hair loss, abrasions, and ulcerations. histologically, hyperkeratosis, acanthosis, and chronic dermatitis may occur. long-standing infestation provokes chronic inflammation, fibrosis, and proliferation of granulation tissue. ulcerative dermatitis associated with acariasis may have an allergic pathogenesis but often results in secondary bacterial infections. lesions resemble allergic acariasis in other species and are associated with mast cell accumulations in the dermis. diagnosis classic methods of detection include direct observation of the hair and skin of dead or anesthetized mice. hairs are parted with pins or sticks and examined with a dissecting microscope. examination of young mice, prior to the onset of immune-mediated equilibrium, is likely to be more productive. alternatively, recently euthanized mice can be placed on a black paper, and double-sided cellophane tape can be used to line the perimeter to contain the parasites. as the carcass cools, parasites will vacate the pelage and crawl onto the paper. sealed petri dishes can also be used. cellophane tape also can be pressed against areas of the pelt of freshly euthanatized mice and examined microscopically. skin scrapings made with a scalpel blade can be macerated in 10% koh/glycerin or immersion oil and examined microscopically. this method has the disadvantage of missing highly motile species and low-level populations of slower moving immature forms. it is important to remember that mite infestations may be mixed, so the identification of one species does not rule out the presence of others. detecting mites in sentinels exposed to dirty bedding from colony animals has been reported to be unreliable (lindstrom et al., 2011) . thus, pcr assays offered by commercial diagnostic laboratories are increasingly being used to augment traditional diagnostic methods and to test individual animals or equipment using a swabbing technique; samples can be pooled to decrease cost (jensen et al., 2013) . gross anatomical features facilitate differentiation of intact mites. myocoptes has an oval profile with heavily chitinized body, pigmented third and fourth legs, and tarsal suckers (fig. 3.54) . myobia and radfordia have a similar elongated profile, with bulges between the legs. myobia has a single tarsal claw on the second pair of legs (fig. 3.55) , whereas radfordia has two claws of unequal size on the terminal tarsal structure of its second pair of legs (fig. 3.56) . histopathological examination of skin is helpful for diagnosing unique forms of acariasis, such as the keratotic cysts associated with psorergates simplex infestation. treatment, prevention, and control ivermectin can be used topically, in drinking water or as a medicated feed and often is the first-choice approach for attempting eradication although cost and potential toxicity are concerns. because of potential differences in laboratory animal medicine blood-brain barrier permeability to ivermectin, pilot treatments should be evaluated. for large facilities, ivermectin medicated feed may be an attractive option (ricart arbona et al., 2010) . for valuable lines of mice, rederivation may be cost-and time-effective. control and prevention programs should be carried out on a colony-wide basis, which includes thorough sanitation of housing space and equipment to remove residual eggs. research complications hypersensitivity dermatitis has the potential to confound immunological studies (jungmann et al., 1996) , especially those involving skin, and has been shown to elevate serum ige (morita et al., 1999) . heavy mite infestations can cause severe skin lesions and have been associated with weight loss, infertility, and premature deaths. chronic acariasis also may provoke secondary amyloidosis due to long-standing dermatitis. myocoptes musculinus this is the most common ectoparasite of the laboratory mouse but frequently occurs in conjunction with myobia musculi. the life cycle includes egg, larva, protonymph, tridonymph, and adult stages. eggs hatch in 5 days and are usually attached to the middle third of the hair shaft. the life cycle may range from 8 to 14 days. transmission requires direct contact, for mice separated by wire screens do not contract infestations from infested hosts. bedding does not seem to serve as a vector. neonates may become infested within 4-5 days of birth, and parasites may live for 8-9 days on dead hosts. myocoptes appears to inhabit larger areas of the body than myobia and tends to displace myobia during heavy infestations. it has some predilection for the skin of the inguinal region, abdominal skin, and back, but it will also infest the head and neck. it is a surface dweller that feeds on superficial epidermis. infestation can cause patchy thinning of the hair, alopecia, or erythema. lesions can be pruritic, but ulceration has not been reported. chronic infestations induce epidermal hyperplasia and nonsuppurative dermatitis. myobia musculi this is a common mite of laboratory mice. the life cycle of myobia can be completed in 23 days and includes an egg stage, first and second larval stages, protonymph, deutonymph, and adult. eggs attach at the base of hair shafts and hatch in 7-8 days. larval forms last about 10 days, followed by nymphal forms on day 11. adults appear by day 15 and lay eggs within 24 h. myobia are thought to feed on skin secretions and interstitial fluid but not on blood. they are transmitted primarily by contact. mite populations increase during new infestations, followed by a decrease to equilibrium in 8-10 weeks. the equilibrated population can be carried in colonies for long periods (up to years). population fluctuations may represent waves of egg hatchings. because mites are thermotactic, they crawl to the end of hair shafts on dead hosts, where they may live for up to 4 days. infestation may result in hypersensitivity dermatitis, to which c57bl mice are highly susceptible. clinical signs vary from ruffled fur and alopecia to pruritic ulcerative dermatitis. therefore, lesions can be exacerbated by self-inflicted trauma. radfordia affinis radfordia is thought to be common in laboratory mice, but it closely resembles myobia and may occur as a mixed infestation. therefore, its true prevalence is conjectural. additionally, its life cycle has not been described. it does not appear to cause clinical morbidity. psorergates simplex this species has not been reported as a naturally occurring infection in well-managed colonies for several decades, but it is unique in that it inhabits hair follicles. its life cycle is unknown, but developmental stages from egg to adult may be found in a single dermal nodule. transmission is by direct contact. invasion of hair follicles leads to development of cyst-like nodules, which appear as small white nodules in the subcutis. histologically, they are invaginated sacs of squamous epithelium, excretory products, and keratinaceous debris. there is usually no inflammatory reaction, but healing may be accompanied by granulomatous inflammation. diagnosis is made by examining the subcuticular surface of the pelt grossly or by histological examination. sac contents also can be expressed by pressure with a scalpel blade or scraped and mounted for microscopic exam. mesostigmoid mites rarely, blood-sucking ornithonyssus bacoti and laelaps echidnina, normally limited to wild rodents, can also infect laboratory rodent colonies (watson, 2008; fox, 1982) . these mites may also transiently bite humans and can transmit zoonotic infections (see chapter 29). unlike the more common rodent fur mites, mesostigmoid mites live off the host and can travel a long distance in search of a blood meal. they access research colonies via contaminated supplies or wild rats and mice gaining access to the facility. polyplax serrata, the mouse louse, is encountered in wild mice but no longer is a significant issue in research colonies. eggs are deposited at the base of hair shafts and nymph stages and adults can be found principally on the dorsum. p. serrata causes pruritus with associated dermatitis, anemia and debilitation and historically is the vector for mycoplasma coccoides. amyloidosis is caused by the deposition of insoluble (polymerized), mis-folded amyloid protein fibrils in organs and/or tissues. primary amyloidosis is a naturally occurring disease in mice, associated with the deposition of amyloid proteins consisting primarily of immunoglobulin light chains. secondary amyloidosis is associated with antecedent and often chronic inflammation. it results from a complex cascade of reactions involving release of multiple cytokines that stimulate amyloid synthesis in the liver (falk and skinner, 2000) . primary amyloidosis is common among aging mice (lipman et al., 1993) but also may occur in young mice of highly susceptible strains such as a and sjl or somewhat older c57bl mice. other strains, such as balb/c and c3h are highly resistant to amyloidosis (percy and barthold, 2007) . secondary amyloidosis is usually associated with chronic inflammatory lesions, including dermatitis resulting from prolonged acariasis. it can be induced experimentally, however, by injection of casein and may occur locally in association with neoplasia or in ovarian corpora lutea in the absence of other disease. in reactive amyloid a (aa) amyloidosis, serum aa (saa) protein forms deposits in mice, domestic and wild animals, and humans that experience chronic inflammation. aa amyloid fibrils are abnormal β-sheet-rich forms of the serum precursor saa, with conformational changes that promote fibril formation. similar to prion diseases, recent findings suggest that aa amyloidosis could be transmissible in mice and other species (murakami et al., 2014) . amyloid fibrils induce a seeding-nucleation process that may lead to development of aa amyloidosis. amyloidosis can shorten the life span of mice and can be accelerated by stress from intercurrent disease. amyloid appears histologically as interstitial deposition of a lightly eosinophilic, acellular material in tissues stained with hematoxylin and eosin. however, it is birefringent after staining with congo red when viewed with polarized light. deposition patterns vary with mouse strain and amyloid type. although virtually any tissue may be affected, the following sites are common: hepatic portal triads, periarteriolar lymphoid sheaths in spleen, renal glomeruli and interstitium (which can lead to papillary necrosis), intestinal lamina propria, myocardium (and in association with atrial thrombosis), nasal submucosa, pulmonary alveolar septa, gonads, endocrine tissues, and great vessels (fig. 3.57) . naturally occurring mineralization of the myocardium and epicardium and other soft tissues is a common finding at necropsy in some inbred strains of mice. although this condition is usually an incidental finding at necropsy, interference with organ function such as the heart cannot be ruled out if lesions are severe. it occurs in balb/c, c3h, and especially dba mice (eaton et al., 1978; brownstein, 1983; brunnert et al., 1999) . it is found in the myocardium of the left ventricle ( fig. 3.58) , in the intraventricular systems, and in skeletal muscle, kidneys, arteries, and lung and may be accompanied by fibrosis and mononuclear inflammatory infiltrates. dba mice also can develop mineralization in the tongue and cornea. dietary, environmental, disease-related, and endocrine-related factors are thought to influence the prevalence of this lesion. ectopic mineralization is associated clinically with skin and vascular connective tissue conditions in humans and mouse models have been developed to study metastatic and dystrophic tissue mineralization (li and uitto, 2013) . pseudoxanthoma elasticum (pxe), a heritable ectopic mineralization disorder in humans, is caused by mutations in the abcc6 gene. knockout abcc6 −/− mice model the histopathologic and ultrastructural features of pxe, notably with mineralization of the vibrissae dermal sheath, serving as a biomarker of tissue mineralization (benga et al., 2014) . other inbred mouse strains, including kk/hlj and 129s1/svimj, also develop vibrissae dermal mineralization and have an snp (rs32756904) in the abcc6 gene associated with low levels of abcc6 protein expression in the liver. dba/2j and c3h/hej mice have the same polymorphism and low abcc6 protein levels; however, these mice only develop tissue mineralization when fed an experimental diet enriched in phosphate and low in magnesium. a reye's-like syndrome has been reported in balb/ cbyj mice (brownstein et al., 1984) . the etiology is unknown; however, antecedent viral infection may be involved. affected mice rapidly become lethargic and then comatose. they also tend to hyperventilate. high mortality ensues within 6-18 h, but some mice may recover. lesions are characterized grossly by swollen, pale liver and kidneys. the major histopathological findings include swollen hepatocytes with fatty change and nuclear swelling among astrocytes in the brain. hepatic lesions resembling changes in reye's syndrome have been reported in scid mice infected with madv-1 (pirofski et al., 1991) . deficiencies (tobin et al., 2007) vitamin deficiencies in mice have not been thoroughly described. unfortunately, much of the information that does exist reflects work carried out 30-50 years ago; thus, the reliability and specificity of some of these syndromes is questionable. vitamin a deficiency may produce tremors, diarrhea, rough hair coat, keratitis, poor growth, abscesses, hemorrhages, and sterility or abortion. vitamin a is recognized for its importance in development of the immune system (ross, 2012) and knockout mouse models have been used to demonstrate genetic polymorphisms in humans that negatively regulate intestinal β-carotene absorption and conversion to retinoids in response to vitamin a requirements for growth and reproduction (von lintig, 2012) . vitamin e deficiency can cause convulsions and heart failure, as well as muscular dystrophy and hyaline degeneration of muscles. two knockout mouse models of severe vitamin e deficiency were independently developed and lack α-tocopherol transfer protein (α-ttp), a gene that controls plasma and tissue α-tocopherol concentrations by exporting α-tocopherol from the liver. ttpa −/− mice have very low to undetectable levels of α-tocopherol and are infertile. the phenotype includes neuronal degeneration associated with progressive ataxia and age-related behavioral defects (yu and schellhorn, 2013) . deficiency of b complex vitamins produces nonspecific signs such as alopecia, decreased feed consumption, poor growth, poor reproduction and lactation, as well as a variety of neurological abnormalities. choline deficiency produces fatty livers and nodular hepatic hyperplasia, as well as myocardial lesions, decreased conception, and decreased viability of litters. folic acid-deficient diets cause marked decreases in red and white cell blood counts and the disappearance of megakaryocytes and nucleated cells from the spleen. pantothenic acid deficiency is characterized by nonspecific signs, such as weight loss, alopecia, achromotrichia, and posterior paralysis, as well as other neurological abnormalities. thiamin deficiency is associated with neurological signs, such as violent convulsions, cartwheel movements, and decreased food consumption. dietary requirements for ascorbic acid have not been shown in mice, and mouse diets are generally not fortified with ascorbic acid. the gulonolactone oxidase knockout mouse (gulo −/− ) on the c57bl/6 background requires vitamin c supplementation although the plasma ascorbate concentration of gulo −/− mice fed a vitamin c-deficient diet is maintained at 15% of wild-type concentrations, suggesting an uncharacterized pathway to generate a small amount of ascorbate (yu and schellhorn, 2013) . the gulo −/− mouse has become the model of choice in studying the role of vitamin c in complex diseases. vitamin c production has been successfully restored in gulo −/− mice using adenovirus vectors, making it possible to robustly manipulate physiological ascorbate concentrations in an inbred mouse. mineral deficiencies have been described only for several elements, and the consequences of the deficiencies are similar to those observed for other species. for example, iodine-deficient diets produce thyroid goiters; magnesium-deficient diets may cause fatal convulsions; manganese deficiency may cause congenital ataxia from abnormal development of the inner ear; and zinc deficiency may cause hair loss on the shoulders and neck, emaciation, decreased liver and kidney catalase activity, and immunosuppression. chronic essential fatty acid deficiency may cause hair loss, dermatitis with scaling and crusting of the skin, and occasional diarrhea. infertility has also been associated with this syndrome. mice have an absolute requirement for a dietary source of linoleic and/or arachidonic acid. (sundberg, 1994; ward, 2000) the significant syndrome of ulcerative dermatitis (ud) is a common idiopathic skin lesion that causes morbidity and early euthanasia losses in c57bl/6 and related lines of mice. significant pruritus leads to skin trauma associated with opportunistic bacterial infection and deep dermal ulcerations. initial signs include alopecia and papular dermatitis, which usually occur over the dorsal trunk (fig. 3.59) . progressive inflammation can be halted, sometimes reversed, by nail trimming and therapy with a wide spectrum of topical or systemic antibiotics, steroids, and other drugs such as vitamin e and aloe, all of which speak to the frustrating search for a primary etiology. treatment should be based on microbiological culture and sensitivity and screening for ectoparasites as hypersensitivity to acariasis has been proposed. seasonal fluctuation in the incidence of disease suggests that environmental factors may play a role. the incidence appears to increase during periods of significant seasonal changes in temperature and humidity, i.e., the onset of winter and early spring. there is some evidence that incidence is related to dietary fat with mice on high fat or ad libitum diets being more susceptible than those on restricted diets (neuhaus et al., 2012) . ileus associated with high mortality has been reported to occur in primiparous female mice during the second week of lactation (kunstyr, 1986) . this disorder has been described as acute intestinal pseudo-obstruction (ipo) in c57bl/6 mice free of known pathogens (feinstein et al., 2008) . lactating mice are either found dead or becoming moribund. segments of the small intestine become distended with fluid contents and histologically there is apoptosis of the villus epithelium of the small intestine and superficial epithelial cells of the large intestine. the enteric nervous system appears morphologically normal but necrotic enterocytes, mucosal erosions, and acute mucosal inflammation are commonly observed. there is no strong evidence for metabolic issues such as hypocalcemia or low blood glucose. the direct cause is unknown but death probably results from sepsis secondary to loss of barrier function reflected in apoptosis of the gut epithelium during peak lactation. environmental variables can affect responses of mice in experimental situations. changes in respiratory epithelial physiology and function from elevated levels of ammonia, effects of temperature and humidity on metabolism, effects of light on eye lesions and retinal function, and effects of noise on neurophysiology are examples of complications that can vary with the form of insult and the strain of mouse employed. mice do not easily acclimatize to sudden and dramatic changes in temperature. therefore, they are susceptible to both hypothermia and hyperthermia. mice also are susceptible to dehydration. poorly functioning automatic watering system valves or water bottles, resulting in spills (hypothermia) or obstructed sipper tubes (dehydration), are a significant cause of husbandry-related morbidity. shipping mice between facilities, irrespective of distance, warrants institutional guidelines to minimize exposure to temperature extremes. reheat coils should be designed to fail in the closed position to avoid overheating holding rooms. ringtail is a condition associated with low relative humidity. clinical signs include annular constriction of the tail and occasionally of the feet or digits, resulting in localized edema that can progress to dry gangrene ( fig. 3 .60). it should be differentiated from dryness and gangrene that may occur in hairless mice exposed to low temperatures and perhaps other environmental or nutritional imbalances. necrosis of legs, feet, or digits also can occur in suckling mice because of disruption of circulation by wraps of stringy nesting material such as cotton wool. corneal opacities can result from acute or chronic keratitis, injury (unilateral) and developmental defects; the latter may occur in combination with inherited microphthalmia in c57 black mice (koch and gowen, 1939) . there is some evidence that the buildup of ammonia in mouse cages may contribute to inflammatory keratitis, because it can be controlled by increasing the frequency of cage cleaning. corneal opacities and anterior polar cataracts are a developmental defect in inbred c57 black mice (pierro and spiggle, 1967) . corneal opacity may be associated with keratolenticular adhesions involving a persistent epithelial stalk of the lens vesicle, which normally disappears around day 11 of gestation (koch and gowen, 1939) . typically noted in runted or cachectic mice soon after weaning, malocclusion of the open-rooted, continually growing incisor teeth is an inherited trait expressed as poorly aligned incisors, especially of the lower incisors causing osteomyelitis, soft tissue abscesses, or necrosis in the lips or oral cavity. the incidence of inherited malocclusion varies with mouse strain (petznek et al., 2002) . malocclusion in older mice may be the result of trauma or oral neoplasia. overgrown molar teeth have been associated with trauma to developing tooth buds. skin lesions can be caused by fighting, tail biting, and overgrooming such as whisker chewing. barbering of facial hair and whiskers in subordinate mice by a dominant cagemate is common and may be solved by removing the dominant, normally haired mouse. hair or whisker chewing (barbering) has long been interpreted to be a manifestation of social dominance. apparent dominant animals retain whiskers, whereas cagemates have 'shaved faces' (fig. 3.61 ). chronic hair chewing can produce histological abnormalities such as poorly formed or pigmented club hairs. once chewing has ceased, many mice regrow previously lost hair in several weeks. both sexes may engage in this activity, and sometimes females may be dominant. barbering of whiskers and fur-plucking behavior in mice has been suggested to model human trichotillomania (compulsive hair plucking) because of similarities including elevated serotonin levels (dufour et al., 2010) , 'barbers' predominately pluck hair from the scalp and around the eyes and the genitals; the behavior is female biased, and begins during puberty and is impacted by genetic background (garner et al., 2004) . fighting is more common in male mice and more aggressive in some strains (sjl, fvb, balb/c) with bite wounds typically located on the head, neck, shoulders, perineal area, and tail. often one mouse in the cage is free of lesions and is the likely aggressor. removal of the unaffected male may end the fighting or simply reorder the dominance order. removing males for breeding and then regrouping them often results in fighting. for programs that produce sentinel mice in-house, castration is an option to reduce aggression in group-housed male sentinels (lofgren et al., 2012) . regional alopecia, especially around the muzzle, may result from abrasion against cage surfaces. improperly diluted disinfectants may also cause regional hair loss. ear tags used for identification may cause pruritis and self-induced trauma. hair removal products or clipping prior to imaging or application of experimental compounds to the skin may cause pruritus and can augment lesions that interfere with test results. dermatophytosis, ectoparasitism, or idiopathic hair loss must be considered in the differential diagnoses for muzzle or body alopecia. (burek et al., 1982; percy and barthold, 2007) common idiopathic lesions in aging mice include cardiomyopathy (with or without mineralization or arteritis), chronic nephropathy (frequently with mineralization), myelofibrosis (fibrotic change in the bone marrow) especially in female mice, melanosis in the meninges, ovarian atrophy (with or without hyaline material), pigment (ceroid-lipofuscin), tubular or stromal hyperplasia, cystic endometrial hyperplasia, testicular tubular degeneration or mineralization, prostate atypical epithelial hyperplasia, gastric glandular epithelial hyperplasia, pancreatic islet cell hyperplasia, dental dysplasia of incisor teeth, pituitary hyperplasia of pars intermedia and pars distalis, cataracts, increased extramedullary hematopoiesis in spleen, and lymphocytic infiltrates or other inflammatory changes in various tissues, including harderian gland, salivary gland, kidney, liver, gall bladder, nasal, trachea, thyroid, periovarian fat, epididymis, and urinary bladder. lymphoma is also very common . spontaneous atrial thrombosis is rare in mice (<1% in 2-year-old mice) and appears to be strain-related, with a high prevalence in rfm mice. it also is more common in aged mice affected by kidney disease and amyloidosis. organizing thrombi will be found usually in an enlarged, hyperemic left atrium and auricle and may be accompanied by amyloidosis. affected mice may display signs of heart failure, particularly severe dyspnea. induction of atrial thrombosis in b6c3f1 mice has been used to assess cardiovascular risk of chemical exposures (yoshizawa et al., 2005) . myocardial and epicardial mineralization is described above (section iii,b,2). periarteritis, also known as arteritis, polyarteritis, or systemic arteritis, impacts older mice and lesions may be observed in multiple tissues, including the spleen, heart, tongue, uterus, testes, kidney, and urinary bladder. the media of the affected vessels is homogenous and intensely eosinophilic with hematoxylin and eosin stain. fibrosis and mononuclear cells infiltrate the vessel wall. experimental coronary arteritis with cardiac hypertrophy has been model in dba/2 and other strains by intraperitoneal administration of mannoprotein-beta-glucan complex isolated from c. albicans (nagi-miura et al., 2006) . hyperplasia of alveolar or bronchial epithelium occurs in old mice and must be differentiated from pulmonary tumors. pulmonary histiocytosis, acidophilic macrophage pneumonia, and acidophilic crystalline pneumonia are synonymous morphologic descriptions of an idiopathic lung lesion that can be incidental or the cause of significant morbidity. incidence varies with mouse strain or stock, with c57bl, 129s4/svjae and swiss mice and older mice in general particularly susceptible. histologically, alveoli and bronchioles are filled with varying quantities of macrophages containing eosinophilic crystalline material . the crystalline material consists of ym1 and/or ym2 chitanases and can be found in other tissues including the upper respiratory tract, stomach, gall bladder, and bone marrow where it is described as hyalinosis (nio et al., 2004) . gastric lesions include crypt dilatation, submucosal fibrosis, adenomatous gastric hyperplasia, mineralization, and erosion or ulceration. gastric ulcers have been induced by cold stress, food restriction (rehm et al., 1987) , chemical injury (yadav et al., 2013) , and gastritis and gastric tumors by helicobacter infection (fox et al., 2003) . germfree mice have reduced muscle tone in the intestinal tract. cecal volvulus is a common cause of death in germfree mice and is caused by rotation of the large, thin-walled cecum. age-associated lesions are common in the livers of mice. cellular and nuclear pleomorphism, including binucleated and multinucleated cells, are detectable by 6 months. mild focal necrosis occurs with or without inflammation, but an association of mild focal hepatitis with a specific infectious disease is often hard to confirm. other geriatric hepatic lesions include biliary hyperplasia with varying degrees of portal hepatitis, hepatocellular vacuolization, amyloid deposition (especially in periportal areas), strangulated or herniated lobes, hemosiderosis, lipofuscinosis, and fibrosis. extramedullary hematopoiesis occurs in young mice and in response to anemia. exocrine pancreatic insufficiency has been reported in cba/j mice. acinar cell atrophy is common but is strain-and sex-dependent. blood-filled mesenteric lymph nodes may occur in aged mice, especially c3h mice. this condition is an incidental finding and should not be confused with infectious lymphadenopathy such as that associated with salmonellosis. aggregates, or nodules of mononuclear cells, are found in many tissues of aged mice, including the salivary gland, thymus, ovary, uterus, mesentery and mediastinum, urinary bladder, and gastrointestinal tract. these nodules should not be mistaken for lymphosarcomas. grossly observable black pigmentation in the spleen of c57bl/6 is normal and is melanosis caused by melanin deposition (weissman, 1967) . the spleen is subject to amyloidosis and hemosiderin deposition. lipofuscin deposition is common, especially in older mice. the thymus undergoes age-associated atrophy. a variety of genetic immunodeficiencies have been described in mice, many of which increase susceptibility to infectious diseases. perhaps the most widely known of these is the athymic nude mouse that lacks a significant hair coat and, more importantly, fails to develop a thymus and thus has a severe deficit of t-cellmediated immune function. additionally, scid mice, which lack both t and b lymphocytes, are used widely and are highly susceptible to opportunistic agents such as pneumocystis murina. specific immune deficits have become excellent models for studying the ontogeny and mechanisms of immune responsiveness (table 3 .12). age-associated osteoporosis or senile osteodystrophy can occur in some mice. it is not associated with severe renal disease or parathyroid hyperplasia. nearly all strains of mice develop some form of osteoarthrosis. it is generally noninflammatory, affects articulating surfaces, and results in secondary bone degeneration. glomerulonephritis is a common kidney lesion of mice. it is more often associated with persistent viral infections or immune disorders rather than with bacterial infections. its prevalence in some strains approaches 100%. nzb and nzb × nzw f 1 hybrid mice, e.g., develop immune complex glomerulonephritis as an autoimmune disease resembling human lupus erythematosus, whereas glomerular disease is relatively mild in nzb mice (nzb mice have a high incidence of autoimmune hemolytic anemia). renal changes occur as early as 4 months of age, but clinical signs and severe disease are not present until 6-9 months. the disease is associated with wasting and proteinuria, and lesions progress until death intervenes. histologically, glomeruli have proteinaceous deposits in the capillaries and mesangium. later, tubular atrophy and proteinaceous casts occur throughout the kidney. immunofluorescence studies show deposits of immunoglobulin and the third component of complement, which lodge as immune complexes with nuclear antigens and antigens of murine leukemia virus in glomerular capillary loops. mice infected with lcmv or with retroviruses can also develop immune complex glomerulonephritis. mice also can develop chronic glomerulopathy characterized by progressive thickening of glomerular basement membrane by pas-positive material that does not stain for amyloid. this lesion can be accompanied by proliferation of mesangial cells; local, regional, or diffuse mononuclear cell infiltration; and fibrosis. advanced cases may lead to renal insufficiency or failure. interstitial nephritis can be caused by bacterial or viral infections but may also be idiopathic. typical lesions include focal, regional, or diffuse interstitial infiltration of tubular parenchyma by mononuclear cells, but glomerular regions also may be involved. severe lesions can be accompanied by fibrosis, distortion of renal parenchyma, and intratubular casts, but not by mineralization. if renal insufficiency or failure ensues, it can lead to ascites. some strains of mice, such as balb/c, can develop polycystic kidney disease, which, if severe, can compromise normal renal function. urinary tract obstruction occurs as an acute or chronic condition in male mice. clinical signs usually include wetting of the perineum from incontinence. in severe or chronic cases, wetting predisposes to cellulitis and ulceration. at necropsy, the bladder is distended, and proteinaceous plugs are often found in the neck of the bladder and proximal urethra. in chronic cases the urine may be cloudy, and calculi may develop in the bladder. additionally, cystitis, urethritis, prostatitis, laboratory animal medicine balanoposthitis, and hydronephrosis may develop. this condition must be differentiated from infectious cystitis or pyelonephritis and from the agonal release of secretions from accessory sex glands, which is not associated with an inflammatory response. hydronephrosis also may occur without urinary tract obstruction. ascending pyelitis occurs in mice secondary to urinary tract infection. parvovarian cysts are observed frequently and may be related to the fact that mouse ovaries are enclosed in membranous pouches. amyloidosis is also common in the ovaries of old mice. cystic endometrial hyperplasia may develop unilaterally or bilaterally and may be segmental. in some strains, the prevalence in mice older than 18 months is 100%. endometrial hyperplasia is often associated with ovarian atrophy. mucometra is relatively common in adult female mice. the primary clinical sign is abdominal distension resembling pregnancy among mice that do not whelp. testicular atrophy, sperm granulomas, and tubular mineralization occur with varying incidence. preputial glands, especially of immunodeficient mice, can become infected with opportunistic or pathogenic bacteria. spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands were described in control b6c3f1 mice from 12 national toxicology program 2-year carcinogenicity and toxicity studies conducted in one of four different laboratories (suwa et al., 2002) . lymphocytic infiltration, inflammation, edema, epithelial hyperplasia, mucinous cyst, mucinous metaplasia, adenoma, adenocarcinoma, granular cell tumor, and glandular atrophy were variously observed in accessory sex glands. accessory adrenal cortical nodules are found in periadrenal and perirenal fat, especially in females. these nodules have little functional significance other than their potential effect on failures of surgical adrenalectomy. lipofuscinosis, subcapsular spindle cell hyperplasia, and cystic dilatation of cortical sinusoids are found in the adrenal cortices of aged mice. some inbred strains have deficiencies of thyrotropic hormone, resulting in thyroid atrophy. thyroid cysts lined by stratified squamous epithelium and generally of ultimo-branchial origin may be seen in old mice. amyloid can be deposited in the thyroid and parathyroid glands as well as in the adrenal glands. spontaneous diabetes mellitus occurs in outbred swiss mice and genetic variants of several strains such as nod mice (lemke et al., 2008) . high levels of estrogen in pregnancy may influence postpartum hair shedding. various endocrine effects on hair growth have also been described. abdominal and thoracic alopecia have been reported in b6c3f 1 mice. symmetrical mineral deposits commonly occur in the thalamus of aged mice. they may also be found in the midbrain, cerebellum, and cerebrum and are particularly common in a/j mice. lipofuscin accumulates in the neurons of old mice. age-associated peripheral neuropathy with demyelination can be found in the nerves of the hindlimbs in c57bl/6 mice. deposits of melanin pigment occur in heavily pigmented strains, especially in the frontal lobe. a number of neurologically mutant mice have been described. they commonly have correlative anatomical malformations or inborn errors of metabolism. a seizure syndrome in fvb mice has been described (goelz et al., 1998) and can be spontaneous or associated with tail tattooing, fur clipping, and fire alarms. mice are most often female with a mean age of 5.8 months (range, 2-16 months) and can exhibit facial grimace, chewing automatism, ptyalism with matting of the fur of the ventral aspect of the neck and/or forelimbs, and clonic convulsions that may progress to tonic convulsions and death. ischemic neuronal necrosis was consistently observed in these mice and is consistent with status epilepticus in humans. unilateral and bilateral microphthalmia and anophthalmia are frequent (as high as 12%) developmental defects in inbred and congenic strains of c57bl mice, especially impacting the right eye and female mice. these conditions may first be recognized due to ocular infections, secondary to inadequate tear drainage. other common findings include central corneal opacities, iridocorneal and corneal-lenticular adhesions, abnormal formation of the iris and ciliary body, cataracts, extrusion of lens cortical material with dispersion throughout the eye, failure of vitreous development, and retinal folding. these syndromes can be reproduced by exposure to alcohol at critical stages of embryogenesis when the optic cup and lens vesicle are developing and impacting normal development of other ocular structures, including the iris, ciliary body, vitreous, and retina . retinal degeneration can occur as either an environmental or a genetic disorder (chang et al., 2007) in mice. nonpigmented mice, both inbred and outbred, can develop retinal degeneration from exposure to light, with the progression of blindness being related to light intensity and duration of exposure. mouse genetics have laboratory animal medicine been shown to be more important than potential light associated tissue injury (serfilippi et al., 2004a) . other strains such as c3h, cba, and fvb are genetically predisposed to retinal degeneration because they carry the rd gene, which leads to retinal degeneration within the first few weeks of life and has been used extensively as a model for retinitis pigmentosa (farber and danciger, 1994) . presence of the rd gene in some mouse strains highlights that impaired vision must be a consideration when selecting strains for behavioral assays that rely on visual clues (garcia et al., 2004) . blindness does not interfere with health or reproduction and blind mice cannot be distinguished from non-blind mice housed in standard caging. cataracts can occur in old mice and have a higher prevalence in certain mutant strains. vestibular syndrome associated with head tilt, circling, or imbalance can result from infectious otitis or from necrotizing vasculitis of unknown etiology affecting small and medium-sized arteries in the vicinity of the middle and inner ears. (jones et al., 1983 (jones et al., -1993 maronpot et al., 1999; percy and barthold, 2007) neoplasms of lymphoid and hematopoietic tissues are estimated to have a spontaneous prevalence of 1-2%. there are, however, some strains of mice that have been specifically inbred and selected for susceptibility to spontaneous tumors. leukemogenesis in mice may involve viruses and chemical or physical agents. viruses associated with lymphopoietic and hematopoietic neoplasia belong to the family retroviridae (type c oncornaviruses) and contain rna-dependent dna polymerase (reverse transcriptase). these viruses are generally noncytopathogenic for infected cells, and mice appear to harbor them as normal components of their genome. although they may be involved in spontaneous leukemia, they are not consistently expressed in this disease. recombinant viruses have recently been discovered that can infect mouse cells and heterologous cells and are associated with spontaneous leukemia development in high leukemia strains such as akr mice. their phenotypic expression is controlled by mouse genotype. endogenous retroviruses are transmitted vertically through the germ line. horizontal transmission is inefficient but can occur by intrauterine infection or through saliva, sputum, urine, feces, or milk. the leukemia induced by a given endogenous virus is usually of a single histopathological type. loss of function in nucleic acid-recognizing, tlr3, tlr7, and tlr9 can result in spontaneous retroviral viremia and acute t-cell lymphoblastic leukemia (yu et al., 2012) . chemical carcinogens, such as polycyclic hydrocarbons, nitrosoureas, and nitrosamines, and physical agents such as x-irradiation can also induce hematological malignancies in mice. the most common hematopoietic malignancy in the mouse is lymphocytic leukemia that originates in the thymus. disease begins with unilateral atrophy and then enlargement of one lobe of thymus as tumor cells proliferate. cells can spread to the other lobe and then to other hematopoietic organs, such as the spleen, bone marrow, liver, and peripheral lymph nodes. clinical signs include dyspnea and ocular protrusion. the latter sign is due to compression of venous blood returning from the head. tumor cells spill into the circulation late in disease. most of these tumors originate from t lymphocytes or lymphoblasts, but there are leukemias of b-lymphocyte or null cell lineage. in the last two syndromes, the lymph nodes and spleen are often involved, but the thymus is generally normal. reticulum cell sarcomas are common in older mice, especially in inbred strains such as c57bl/6 and sjl. primary tumor cell types have been divided into several categories based on morphological and immunohistochemical features. histiocytic sarcomas correspond to the older dunn classification as type a sarcomas and are composed primarily of reticulum cells. the tumor typically causes splenomegaly and nodular lesions in other organs, including the liver, lung, kidney, and the female reproductive tract. follicular center cell lymphomas correspond to dunn type b sarcomas. they originate from b-cell regions (germinal centers) of peripheral lymphoid tissues, including the spleen, lymph nodes, and peyer's patches. typical tumor cells have large vesiculated, folded, or cleaved nuclei and ill-defined cytoplasmic borders. tumors also often contain small lymphocytes. type c reticulum cell tumors often involve one or several lymph nodes rather than assuming a wide distribution. they consist of reticulum cells with a prominent component of well-differentiated lymphocytes. myelogenous leukemia is uncommon in mice and is associated with retrovirus infection. disease begins in the spleen, resulting in marked splenomegaly, but leukemic spread results in involvement of many tissues including the liver, lung, and bone marrow. leukemic cells in various stages of differentiation can be found in peripheral blood. in older animals, affected organs may appear green because of myeloperoxidase activity, giving rise to the term chloroleukemia. the green hue fades on contact with air. affected mice are often clinically anemic and dyspneic. erythroleukemia is rare in mice. the major lesion is massive splenomegaly, which is accompanied by anemia and polycythemia. hepatomegaly can follow, but there is little change in the thymus or lymph nodes. erythroleukemia can be experimentally induced in mice by friend spleen focus-forming virus (sffv) which initially activates the erythropoietin (epo) receptor and the receptor tyrosine kinase sf-stk in erythroid cells, resulting in proliferation, differentiation, and survival. in a second stage, sffv activates the myeloid transcription factor pu.1, blocking erythroid cell differentiation, and in conjunction with the loss of p53 tumor suppressor activity, results in the outgrowth of malignant cells (cmarik and ruscetti, 2010 ). mast cell tumors are also very rare in mice. they are found almost exclusively in old mice and grow slowly. they should not be confused with mast cell hyperplasia observed in the skin following painting with carcinogens or x-irradiation. natural plasma cell tumors are infrequent in the mouse. they can, however, be induced by intraperitoneal inoculation of granulomatogenic agents such as plastic filters, plastic shavings, or a variety of oils, particularly in balb/c mice. mineral oil-induced plasmacytomas in balb/c mice produce large amounts of endogenous retroelements such as ecotropic and polytropic murine leukemia virus and intracisternal a particles. associated inflammation may promote retroelement insertion into cancer genes, thereby promoting tumors (knittel et al., 2014) . similar to other spontaneous cancers, plasmacytoma development in mice is inhibited by innate immune responses of nk cells which when activated by viruses will release γinf (thirion et al., 2014) . mammary tumors can be induced or modulated by a variety of factors, including viruses, chemical carcinogens, radiation, hormones, genetic background, diet, and immune status. certain inbred strains of mice, such as c3h, a, and dba/2, have a high natural prevalence of mammary tumors. other strains, such as balb/c, c57bl, and akr, have a low prevalence. among the most important factors contributing to the development of mammary tumors are mammary tumor viruses. several major variants are known. the primary tumor virus mmtv-s (bittner virus) is highly oncogenic and is transmitted through the milk of nursing females. infected mice typically develop a precursor lesion, the hyperplastic alveolar nodule, which can be serially transplanted. spontaneous mammary tumors metastasize with high frequency, but this property is somewhat mouse strain dependent. metastases go primarily to the lung. some mammary tumors are hormone dependent, some are ovary dependent, and others are pregnancy dependent. ovary-dependent tumors contain estrogen and progesterone receptors, whereas pregnancy-dependent tumors have prolactin receptors. ovariectomy will dramatically reduce the incidence of mammary tumors in c3h mice. if surgery is done in adult mice 2-5 months of age, mammary tumors will develop, but at a later age than normal. grossly, mammary tumors may occur anywhere in the mammary chain. they present as one or more firm, welldelineated masses, which are often lobular and maybe cystic (fig. 3.62) . histologically, mammary tumors have been categorized into three major groups: carcinomas, carcinomas with squamous cell differentiation, and carcinosarcomas. the carcinomas are divided into adenocarcinoma types a, b, c, y, l, and p. most tumors are type a or b. type a consists of adenomas, tubular carcinomas, and alveolar carcinomas. type b tumors have a variable pattern with both well-differentiated and poorly differentiated regions. they may consist of regular cords or sheets of cells or papillomatous areas. these two types are locally invasive and may metastasize to the lungs. type c tumors are rare and are characterized by multiple cysts lined by low cuboidal to squamous epithelial cells, and they have abundant stroma. type y tumors, which are also rare, are characterized by tubular branching of cuboidal epithelium and abundant stroma. adenocarcinomas with a lacelike morphology (types l and p) are hormone dependent and have a branching tubular structure. the control or prevention of mammary neoplasms depends on the fact that some strains of mammary tumor virus are transmitted horizontally, whereas others are transmitted vertically. although horizontally transmitted virus such as mmtv-s can be determined by cesarean rederivation or by foster nursing, endogenous strains of tumor virus may remain. fortunately, these latter tumor viruses have generally low oncogenicity relative to the bittner virus. mammary tumors are increased in frequency in c57bl apc +/− female mice infected with h. hepaticus (rao et al., 2007) . mice develop an assortment of liver changes as they age, including proliferative lesions which can progress from hyperplastic foci to hepatomas to hepatocellular carcinomas. almost all strains of mice have a significant prevalence of hepatic tumors, some of which appear to result from dietary contamination or deficiency and h. hepaticus infections in susceptible strains of mice such as the a/jcr male mouse ward et al., 1994) . the prevalence of spontaneous liver tumors in b6c3f 1 hybrids is increased by feeding choline-deficient diets or when infected with h. hepaticus (hailey et al., 1998) . tumors also can develop in mice exposed to environmental chemicals, many of which are carcinogenic or potentially carcinogenic (hoenerhoff et al., 2009) . spontaneous liver tumors in mice occur grossly as gray to tan nodules or large, poorly demarcated darkred masses. they are usually derived from hepatocytes, whereas cholangiocellular tumors are rare. hepatomas are well circumscribed and well differentiated, but they compress adjacent liver tissue as they develop. hepatocellular carcinomas are usually invasive and display histopathological patterns ranging from medullary to trabecular. large carcinomas also may contain hemorrhage and necrosis. carcinomas also may metastasize to the lungs. primary respiratory tumors of mice occur in relatively high frequency. it has been estimated that more than 95% of these tumors are pulmonary adenomas that arise either from type 2 pneumocytes or from clara cells lining terminal bronchioles. pulmonary adenomas usually appear as distinct whitish nodules that are easily detected by examination of the lung surface. malignant alveologenic tumors are infrequent and consist of adenocarcinomas and squamous cell carcinomas. they invade pulmonary parenchyma and are prone to metastasize. the prevalence of spontaneous respiratory tumors is mouse straindependent. for example, the prevalence is high in aging a strain mice but low in aging c57bl mice. the number of tumors per lung is also higher in susceptible mice. pulmonary tumors often occur as well-defined gray nodules. microscopically, adenomas of alveolar origin consist of dense ribbons of cuboidal to columnar cells with sparse stroma. adenomas of clara cell origin are usually associated with bronchioles. they have a tubular to papillary architecture consisting of columnar cells with basal nuclei. pulmonary adenocarcinomas, though comparatively rare, are locally invasive. they often form papillary structures and have considerable cellular pleomorphism. given the rapid development of mouse strains genetically predisposed to neoplasia, the mouse tumor biology database maintained by jackson laboratory is a valuable centralized resource for the most current tumor descriptions. the database contains information on more than 4400 strains and substrains, 348 tissues and organs, over 42,000 tumor frequency records, and nearly 4800 histopathological images and descriptions. risk factors for recurrence, complications 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modifications by crispr/cas9 mediated genome engineering key: cord-022582-2e9i3m4b authors: potsic, william p.; wetmore, ralph f. title: otolaryngologic disorders date: 2012-03-21 journal: pediatric surgery doi: 10.1016/b978-0-323-02842-4.50055-3 sha: doc_id: 22582 cord_uid: 2e9i3m4b nan the ear is divided into three anatomic and functional areas: the external ear, the middle ear, and the inner ear. the external ear consists of the auricle, external auditory canal, and the lateral surface of the tympanic membrane. the auricle is a complex fibroelastic skeleton that is covered by skin and subcutaneous tissue that directs sound into the external ear canal. the external auditory canal is oval with the long axis in the superior to inferior direction. in neonates, the external canal is almost entirely supported by soft, collapsible cartilage. as the temporal bone grows over several years, the bony portion of the canal enlarges to comprise the inner one third, leaving the outer two thirds supported by firm cartilage. hair and cerumen glands are present in the outer two thirds of the external canal. the ear canal is lined by skin that is continuous with the lateral surface of the tympanic membrane, and it is innervated by cranial nerves v, vii, and x and cervical nerve iii. the tympanic membrane separates the external ear canal from the middle ear. it has three layers: an outer layer of skin; a middle layer of fibrous tissue that is attached to the malleus, the most lateral middle ear ossicle; and an inner layer of mucosa that is continuous with the mucosa lining the middle ear. the fibrous layer is also attached to a thick fibrous annulus that anchors it to the temporal bone. the middle ear is an air-filled space within the temporal bone of the skull that is lined by ciliated, columnar respiratory epithelium. the middle ear communicates with the mastoid air cell system posteriorly and is lined by the same mucosa. it also communicates with the nasopharynx anteriorly through the eustachian tube. the mucociliary transport system of the middle ear moves mucus and debris into the nasopharynx, where it is swallowed. secretory cells are not evenly distributed throughout the middle ear and mastoid complex and are more numerous anteriorly near the eustachian tube. three ossicles are present in the middle ear-the malleus, incus, and stapes-that transmit sound from the vibrating tympanic membrane to the stapes footplate. stapes movement creates a fluid wave in the inner ear that travels to the round window membrane and is dissipated by reciprocal motion to the stapes. there are two striated muscles in the middle ear. the tensor tympani muscle lies along the side of the eustachian tube, and its tendon attaches to the medial surface of the malleus. the stapedius muscle lies along the vertical portion of the facial nerve in the posterosuperior part of the middle ear. its tendon attaches to the head of the stapes. these muscles stiffen the ossicular chain in the presence of sustained loud noise. the facial nerve traverses the middle ear with its horizontal portion lying superior to the stapes. posterior to the stapes, the facial nerve turns inferiorly in a vertical fashion to exit the stylomastoid foramen deep to the tip of the mastoid. the chorda tympani nerve is a branch of the facial nerve that innervates taste to the anterior two thirds of the tongue. it exits the facial nerve in the vertical segment and passes under the posterosuperior surface of the tympanic membrane, crossing the middle ear lateral to the long process of the incus and medial to the malleus. the facial nerve lies within a protective bony canal throughout its course in the middle ear. however, the bony canal may be absent (in the horizontal portion) in as many as 30% of patients. cranial nerve ix supplies sensation to the floor of the middle ear. the inner ear consists of the cochlea, semicircular canals, and vestibule. the cochlea is a coiled fluid-filled tube consisting of 2 3 / 4 turns surrounded by dense bone. it contains the membranes that support the organ of corti and has hair cells that detect the fluid wave from vibration of the stapes footplate. the hair cells create the neural impulses that are transmitted from the auditory nerve (cranial nerve viii) to the brain, providing the sensation of hearing. the three paired semicircular canals (horizontal, superior, and inferior) are also fluid-filled tubes surrounded by dense bone. the semicircular canals each have a hair cell-containing structure (the ampulla) that detects motion. the utricle and saccule of the vestibule also have hair cell structures that detect acceleration. 26 the external ear develops during the sixth week of gestation and is completely developed by the 20th week. six hillocks fuse to form the basic units of the pinna. defects in the fusion of the hillocks lead to preauricular tags and sinuses. the external auditory canal develops from the first pharyngeal cleft. a solid epithelial plug forms during the beginning of the third month of gestation and canalizes in the seventh month to form the external auditory canal. the middle ear canal develops from the first pharyngeal pouch. the ossicles develop from the first and second pharyngeal arches. the inner ear arises from neuroectodermal tissue within the otic placode that forms the otic pit. 26 any combination of anomalies may occur. abnormalities of the development of the ear may create anomalies of the pinna, external auditory canal, middle ear structures, and inner ear. one of the anomalies that involves the external and middle ear is aural atresia (absence of the external auditory canal). absence of the external canal may occur with a deformed or normal external ear. the ossicles may be deformed and are usually fused to each other as well as the bony plate representing the undeveloped tympanic membrane. the facial nerve may also be altered in its course through the temporal bone. reconstruction of the atretic canal, removal of the bony tympanic plate, release of the fused ossicles, and reconstruction of a new eardrum is a complex surgical procedure that may improve hearing. rarely there is incomplete development of the inner ear structures. the most common of these is dysplasia of the cochlea, and it may vary in severity. dysplasia causes sensorineural hearing loss. 6, 14 the examination of the ear should always start with inspection of the outer ear and surrounding structures. deformities of the outer ear structure may suggest the presence of other anomalies, such as a first branchial cleft sinus. a first branchial cleft sinus usually presents below the ear lobe near the angle of the jaw. the sinus tract may connect to the ear canal or, rarely, the middle ear. the external auditory canal and tympanic membrane are best examined with a hand-held otoscope with a bright fiberoptic light source. the largest speculum that comfortably fits in the external canal should be used to maximize visualization and minimize pain. a very small speculum may be inserted deeply, but it might lacerate the ear canal as well as limit visibility of the tympanic membrane. the otoscope permits visualization of the ear canal and tympanic membrane. a translucent tympanic membrane will also permit visualization of the contents of the middle ear. cerumen may be encountered in the ear canal that obstructs the view of the tympanic membrane. removal of cerumen may be performed by using an operating otoscope head and an ear curet. however, the use of a headlight such as the lumiview (welch allyn, skaneateles, ny) or operating microscope permits the use of both hands and superior visualization. care should be taken to secure the child to prevent sudden movement, and the ear curet should be used gently to avoid causing pain and a laceration of the ear canal. examination of a child with an apparent or suspected ear condition often requires objective assessment of hearing by audiometry. current technology and expertise makes it possible to test a child at any age. behavioral audiometry can usually be accurately performed for a child who is older than 6 months of age by sound-field testing. older children are presented with a tone through insert earphones and a range of frequencies between 250 and 8000 hz for ear-specific testing. the hearing thresholds are recorded at each presented frequency; and this represents the air conduction threshold. the sound has to traverse the ear canal, tympanic membrane, and middle ear. the inner ear must respond by creating electrical impulses that are transmitted to the brain. normal thresholds are less than 20 db for children. bone conduction thresholds test the sensorineural component of hearing. a bone oscillator is used to test a range of frequencies by vibrating the skull, which stimulates the inner ear, directly bypassing the external and middle ear. normally, air conduction thresholds require less energy than bone conduction thresholds. if bone conduction thresholds require less sound intensity than air conduction to be heard, the child has a conductive hearing loss. if air conduction and bone conduction thresholds are elevated but the same, the child has a sensorineural hearing loss. most sensorineural hearing loss in children is a result of hair cell dysfunction in the organ of corti. hearing loss may be conductive, sensorineural, or mixed. electrophysical tests such as brainstem auditory evoked response and sound emission tests that measure the intrinsic sounds from the inner ear (otoacoustic emissions) may be employed in young infants and children who cannot participate in behavioral audiometry. a mechanical test of tympanic membrane compliance (tympanometry) may also be used for audiometric assessment. all of these tools are employed by pediatric audiologists. 23 for purposes of describing hearing loss, a threshold between 20 and 40 db is considered mild, 40 to 65 db is moderate, 55 to 70 db is moderately severe, 70 to 90 db is severe, and greater than 90 db is profound. four of 1000 children are born with a hearing loss, and 1 of those children is born with a severe to profound hearing loss. conductive hearing loss may be corrected with otologic surgery. hearing aids and fm systems may be helpful to children with both conductive and sensorineural hearing loss. assistance may be needed through auditory training, speech language therapy, and education to maximally develop communication skills. when a child has a sensorineural hearing loss that is too severe to be helped with hearing aids, a cochlear implant may be considered. a cochlear implant is an electrical device that is implanted under the scalp behind the ear. its processor converts sound to electrical impulses. a cable travels through the mastoid and facial recess to reach the middle ear, and the electrode array is inserted into the scala tympani of the cochlea through an opening that is made in the cochlea. cochlear implants stimulate the neural elements of the cochlea directly and bypass the hair cells. because the vast majority of sensorineural hearing loss in children is due to hair cell dysfunction, nearly all children get sound perception from a cochlear implant. rare conditions such as an absent auditory nerve or an absent cochlea preclude the use of a cochlear implant. a multidisciplinary evaluation by a cochlear implant team is required to evaluate a child and determine family expectations before performing a cochlear implant. a temporal bone computed tomographic (ct) scan and/or magnetic resonance imaging (mri) is performed to assess the cochlea and auditory nerves. children who are born deaf and are younger than the age of 3 years, as well as children who have already developed communication skills, language, and speech before losing their hearing, derive the greatest benefit from cochlear implants. cochlear implantation is approved for children 12 months of age or older by the u.s. food and drug administration. after a cochlear implant is performed, considerable auditory oral training is required to maximize a child's benefit to develop skills of audition, speech, and language. a child who has been deaf and without sound perception for several years is expected to benefit to a lesser degree. 33 otitis media with effusion is the most common chronic condition of the ear during childhood. all children are born with small eustachian tubes that may at times be unable to clear mucus that is secreted in the mastoid and middle ear. fluid may develop in the middle ear during an upper respiratory infection. it usually clears within a few weeks as the upper respiratory tract infection resolves. children with craniofacial anomalies such as cleft palate and down syndrome are also prone to middle ear effusions; there is no medication that is consistently effective in resolving such effusions. persistent effusion may cause a conductive hearing loss in the range of 20 to 40 db. a middle ear effusion may also function as a culture medium and predispose children to recurrent acute suppurative otitis media (asom). when fluid persists in the middle ear for 3 to 4 months, causing a hearing loss or is associated with asom, myringotomy and tympanostomy tube placement is helpful to resolve the hearing loss and reduce the frequency and severity of infection. myringotomy and placement of a tube is performed under general anesthesia using an operating microscope. a small incision is made in any quadrant of the tympanic membrane except the posterosuperior quadrant, where there would be risk of injuring the ossicles. the mucus is suctioned from the ear, and a silastic tube is placed in the myringotomy to provide prolonged ventilation of the middle ear. the tube will usually be extruded and the tympanostomy will heal in 6 months to 1 year. when the ear is no longer ventilated by a tube, the eustachian tube must ventilate the middle ear. if fluid recurs and persists, a repeat procedure may be needed. most children outgrow this problem as their eustachian tube grows. occasionally, adenoid tissue in the nasopharynx may contribute to the persistence of middle ear effusion and may also be removed at the time that a tube is placed. children who have had multiple sets of tubes are candidates for adenoidectomy. acute suppurative otitis media is the most common infection of childhood except for acute upper respiratory tract infections. it is the most common condition for which children seek medical care from their primary care physician. usual pathogens causing asom include streptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis. 25 acute suppurative otitis media usually causes severe deep ear pain, fever, and a conductive hearing loss in the affected ear. the purulence in the middle ear is also present in the mastoid air cells because they are connected. asom is treated with broad-spectrum oral antibiotics; however, there is growing concern that indiscriminant use of antibiotics may result in antibiotic resistance. for this reason, accurate diagnosis by otoscopy should be made before initiating a course of antibiotics. occasionally, asom does not respond as expected to standard antibiotic therapy. when this occurs, culture and sensitivity testing can be obtained by tympanocentesis. after sterilizing the ear canal with alcohol, a 22-gauge spinal needle can be placed through the posterior or anterior inferior quadrant of the tympanic membrane and fluid can be aspirated with a small syringe. complications of asom are uncommon if appropriate antibiotic therapy is used. the conductive hearing loss resolves as the middle ear effusion clears. however, infection may necrose the tympanic membrane, causing a spontaneous perforation. small perforations usually heal in less than 7 days, but larger perforations may persist, cause a conductive hearing loss, and require a tympanoplasty for closure. the ossicular chain may also be disrupted by necrosis of the long process of the incus requiring ossicular reconstruction to restore hearing. acute coalescent mastoiditis occurs when infection erodes the bony mastoid cortex and destroys bony septa within the mastoid. a subperiosteal abscess may also be present. there is usually postauricular erythema and edema over the mastoid area. the auricle is displaced laterally and forward ( fig. 52-1 ). otoscopy reveals forward displacement of the posterior superior skin of the ear canal. in addition to antibiotics, treatment should include a wide field myringotomy from the anterior inferior quadrant to the posterior inferior quadrant, a tympanostomy tube placement for middle ear drainage, and a postauricular mastoidectomy to drain the subperiosteal abscess and the mastoid. facial nerve paralysis may occur from inflammation of that portion of the facial nerve that is exposed in the otolaryngologic disorders middle ear during asom. treatment with parenteral antibiotics, ototopical antibiotic drops applied in the ear canal, and a wide field myringotomy and tympanostomy tube placement almost always result in complete recovery of facial function. facial nerve recovery may take a few weeks to several months. intracranial complications of asom may include meningitis, epidural abscess, brain abscess, otitic hydrocephalus, and lateral sinus thrombosis. meningitis is the most common intracranial complication of asom and may be associated with profound sensorineural hearing loss and loss of vestibular function. treatment of the intracranial complications of asom is focused on appropriate treatment of the intracranial process, in addition to a wide field myringotomy and tympanostomy tube placement in the affected ear. 2 chronic otitis media is a descriptive term that refers to a persistent perforation of the tympanic membrane or the presence of a cholesteatoma of the middle ear. a cholesteatoma is a squamous epithelial-lined cyst that may be congenital or acquired. congenital cholesteatomas are caused by epithelial rests that persist in the middle ear during temporal bone development. they present behind an intact tympanic membrane and appear as a white, smooth mass in the middle ear. they expand over time and are filled with squamous debris and may erode the ossicular chain and extend into the mastoid. acquired cholesteatoma develops from skin entering the middle ear after a tympanic membrane perforation or a retraction pocket from eustachian tube dysfunction. cholesteatomas are usually painless, cause a conductive hearing loss, and, in acquired cases, often present as otorrhea. the otorrhea should be treated with ototopical antibiotic eardrops, but the only treatment of cholesteatomas is complete surgical excision by tympanomastoid surgery and ossicular reconstruction. 27[pp 18-59] the potential complications of cholesteatomas are the same as those for asom. objects stuck deeply into the ear canal such as a cottontipped applicator may perforate the tympanic membrane. this usually causes acute pain, bleeding, and a conductive hearing loss. if the ossicular chain is not disrupted, the vast majority of these perforations will heal spontaneously in about 2 weeks. if the tympanic membrane is perforated and the middle ear is contaminated with water, oral antibiotics should be given. lacerations of the auricle should be cleaned to prevent tattooing and repaired by careful approximation of the skin and soft tissue to restore the contours of the ear. the cartilage itself does not usually need to be sutured. partially or totally avulsed tissue should be replaced. if necrosis of tissue occurs, it can be dã©brided as needed. in severe injuries of the auricle, oral antibiotic treatment is helpful to prevent chondritis and loss of the cartilage framework. blunt trauma to the ear is commonly seen in wrestlers, in children with poor neuromuscular tone, or in children with self-injurious behaviors. blood or serum collects between the periosteum and the auricular cartilage. if the cartilage is fractured, the collection may occur on both sides of the ear. evacuation of the collection is required to restore the contours of the ear, prevent infection, and prevent scarring with formation of a "cauliflower ear." aspiration of the fluid and placement of a mastoid dressing for compression may be tried but is most often unsuccessful. incision and drainage provides for complete evacuation of the blood or serum. cotton dental rolls placed in each side of the auricle and held in place with bolster mattress sutures is the most effective management. the dental rolls should be left in place for 7 to 10 days while the patient also continues with a course of oral antibiotics. no outer dressing is required. 27[pp 106-109] blunt head trauma may disrupt the inner ear membranes causing sensorineural hearing loss and vertigo. no treatment is required, and the injury and symptoms may resolve spontaneously, but the sensorineural hearing loss may persist. severe head trauma may cause fracture of the temporal bone of the skull. temporal bone fractures can be classified as longitudinal, transverse, or mixed ( fig. 52-2 ) but are often complex and do not neatly fit into one category or another. a high-resolution, thin section ct scan of the temporal bone will define the extent of the fracture. the middle ear and mastoid are filled with blood when a fracture is present. the blood causes a conductive hearing loss that resolves when the ear clears. otoscopic evaluation of a child with a temporal bone fracture may reveal a laceration of the ear canal and tympanic membrane. blood is usually present in the ear canal, and the tympanic membrane appears to be dark blue because the middle ear is filled with blood. there is often ecchymosis of the mastoid area (battle's sign). it is important during evaluation of a skull and temporal bone fracture to note and record the function of the facial nerve if the patient is not unconscious. facial nerve paralysis may be immediate or delayed in onset. delayed facial nerve paralysis has a good prognosis for spontaneous recovery. immediate facial paralysis may indicate disruption of the nerve or compression by bone fragments. immediate facial nerve paralysis requires exploration and repair once the patient is stable and sufficiently recovered from any associated trauma. the facial nerve should be decompressed in the mastoid, middle ear, and middle cranial fossa. bone chips impinging on the nerve should be removed, and the nerve should be sutured or grafted if needed. all patients with temporal bone fractures should have an audiogram once their condition has stabilized. if the fracture disarticulates the ossicles, a conductive hearing loss will persist after the blood has cleared from the middle ear and mastoid. fractures of the temporal bone may transverse the cochlea and vestibular apparatus. these fractures usually cause a severe sensorineural hearing loss and loss of vestibular function on the affected side. a concussive injury of the cochlea may also simultaneously be present in the opposite ear in severe head trauma. temporal bone fractures may permit leakage of cerebrospinal fluid (csf) into the middle ear and mastoid. csf may also drain through the lacerated tympanic membrane, causing csf otorrhea. these leaks usually stop spontaneously, but persistent csf otorrhea may require a lumbar drain to reduce the pressure and permit healing. rarely, tympanomastoid exploration is required to close the leak. persistent csf leaks in the ear are associated with meningitis. benign and malignant tumors of the ear are rare. glomus tympanicum tumors and neuromas of the facial nerve may present in the middle ear. also, eosinophilic granuloma and rhabdomyosarcoma may involve the structures of the temporal bone. 5, 13 the nose can be divided into three anatomic sections. the bony vault is the immobile portion of the nose. it consists of the paired nasal bones, the frontal process of the maxillary bone, and the nasal process of the frontal bone. the cartilaginous vault is supported by the upper lateral cartilages and the cartilaginous nasal septum. the nasal lobule is supported by the lower lateral cartilages and the cartilaginous septum. the nasal septum is formed by the quadrilateral cartilage anteriorly. the posterior septum is composed of bone from the vomer, perpendicular plate of the ethmoid, nasal crest of the maxillary bone, and palatine bone. both the internal and external carotid artery systems supply blood to the nose. the roof and lateral wall of the internal nasal cavity are supplied by the anterior and posterior ethmoidal arteries, sphenopalatine artery, and greater palatine artery. the septum is supplied by the anterior and posterior ethmoidal arteries, palatine artery, and the superior labial artery. the convergence of these vessels in the anterior segment of the nose is referred to as kiesselbach's plexus or little's area. venous drainage is accomplished mainly by the ophthalmic, anterior facial, and sphenopalatine veins. the olfactory bulb is positioned high in the roof of the nasal cavity and is responsible for the sense of smell. sensory information is transported by nerves that penetrate the cribriform plate and traverse cranial nerve i (the olfactory nerve) to the brain. smell is also an important component of what is perceived as taste. bony projections, turbinates, form the lateral nasal wall and significantly increase the surface area of the nose, allowing for more efficient humidification and warming of the air to 36â°c. three turbinates are usually present (i.e., inferior, middle, and superior). a supreme turbinate, which is essentially a flap of mucosa, is occasionally present. the turbinates contribute to the turbulent airflow that creates approximately 50% of the total airflow resistance to the lungs. cleaning of air is accomplished through the nasal hairs (vibrissae) and the mucosal surface. anteriorly, the nose is lined with stratified squamous epithelium, which changes to respiratory epithelium immediately anterior to the turbinates. trapped debris is transported in a posterior direction into the nasopharynx by a mucociliary transport mechanism. speech is affected by nasal anatomy and pathologic conditions. hyponasality from nasal obstruction or hypernasality from an excessive air leak can affect voice quality and intelligibility of speech. the nose serves as a drainage port for the paranasal sinuses. the meati are spaces between the lateral aspect of the nasal turbinates and the medial aspects of the lateral nasal wall. each meatus is named for the turbinate that surrounds it. the maxillary, frontal, and anterior ethmoidal sinuses drain into the middle meatus. the posterior ethmoidal sinuses drain into the superior meatus. the sphenoidal sinus drains into an area known as the sphenoethmoidal recess that is located posterior and superior to the superior turbinate. the nasolacrimal duct drains into the inferior meatus. the nasal cavities develop from the nasal pits in the 4-week embryo. these pits deepen and move medially to form the nasal cavity. the oronasal membrane that separates the nose from the mouth resolves in the seventh week to permit communication between the nose and nasopharynx. the paranasal sinuses develop from an outpouching of the lateral nasal walls during the third and fourth months of development. the maxillary and ethmoidal sinuses are present at birth. the frontal and sphenoidal sinuses begin to develop several years after birth. the frontal sinus begins to develop at 7 years of age but is not fully aerated until adulthood. 1 viral rhinosinusitis (the common cold) accounts for the majority of nose and sinus infections. it is caused by many strains of viruses and is a self-limited infection. symptoms of fever, nasal congestion, headache, and clear rhinorrhea usually resolve over 5 to 7 days. treatment is symptomatic. acute bacterial rhinosinusitis may often follow an acute viral upper respiratory tract infection. the most common bacteria causing rhinosinusitis are streptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis. acute rhinosinusitis causes malaise, headache, and nasal congestion. there may also be pain localized to the sinus region or pain on palpation over the maxillary or frontal sinuses. chronic sinus infection may persist after the acute phase and symptoms often last longer than 30 days. the "gold standard" for diagnosing sinusitis is a ct of the sinuses, but a thorough history and nasal examination is usually sufficient to diagnose acute rhinosinusitis. the nasal cavity can be visualized by using a large speculum on an otoscopic head. the posterior nasal cavity can be visualized with either a straight rod endoscope or a flexible fiberoptic nasopharyngoscope. the treatment of rhinosinusitis includes oral antibiotics, short-term use of topical nasal decongestants (e.g., oxymethazoline), and saline nasal sprays. topical nasal corticosteroid sprays may be helpful for the treatment of chronic sinusitis. chronic sinusitis in a child may be exacerbated by gastroesophageal reflux disease, immunodeficiencies, mucociliary dysfunction, and, more commonly, upper respiratory allergy. these predisposing conditions should be managed while treating the sinus infection. if the signs and symptoms of chronic sinus infection persist, a sinus ct is required to evaluate the condition of the sinus mucosa and the drainage pathways. endoscopic sinus surgery may be necessary to open the involved sinuses to provide drainage. chronic inflammation of the nasal and sinus mucosa may lead to nasal and sinus polyp formation that chronically obstructs the nose and sinuses. antrochoanal polyps are large polyps that originate from the walls of the maxillary sinus and extend through the nasal cavity into the nasopharynx. nasal polyps may be removed endoscopically, but a large antrochoanal polyp may require removal through an open maxillary sinus procedure. nasal polyps in a child should always prompt an evaluation for cystic fibrosis. the sinuses surround the orbit so a common complication of acute rhinosinusitis in children is orbital cellulitis with erythema and edema of the eyelids. chemosis (edema of the ocular conjunctiva) is usually absent. however, if a periorbital subperiosteal abscess forms adjacent to an infected sinus, there may be proptosis, chemosis, ophthalmoplegia, and loss of vision. infection in the ethmoidal sinuses most commonly results in this complication. subperiosteal periorbital abscess is demonstrated best by sinus ct. initial treatment should include intravenous antibiotics. endoscopic or external drainage may be required in some cases. intracranial complications of sinusitis include cerebritis, cavernous sinus thrombosis, as well as epidural, subdural, and brain abscess. treatment of intracranial complications or impending intracranial complications requires surgical drainage of the involved sinus and concurrent treatment of the intracranial lesion by a neurosurgeon. 30 fungal sinusitis may occur in immunocompromised children, specifically severe diabetics, children undergoing chemotherapy, and bone marrow transplant recipients. the treatment of fungal sinusitis involves surgical drainage and intravenous antifungal agents. however, a chronic form of fungal sinusitis is allergic fungal sinusitis. these patients usually have other signs of allergy, such as asthma. the treatment of this condition is corticosteroids and dã©bridement of the involved sinuses. the diagnosis is made by sinus ct findings and the presence of eosinophils as well as fungi in the sinus secretions that are removed at the time of surgery. 11 congenital stenosis of the anterior bony aperture causes partial nasal obstruction that may be severe enough to cause difficulty feeding, respiratory distress, and failure to thrive. anterior rhinoscopy demonstrates a very constricted nasal opening bilaterally. ct of the nose shows marked narrowing of the pyriform aperture. neonates are obligate nasal breathers, and severe stenosis must be surgically corrected. because the stenotic segment is very anterior and the remainder of the nasal cavity is normal, removal of the constricting bone with drills is done through a sublabial approach. the nasal openings are stented with 3.0-mm endotracheal tube stents that are sutured in place and removed after a few days. choanal atresia may be unilateral or bilateral. the obstructing tissue is usually a bony plate, but a few cases will have only membranous atresia. unilateral choanal atresia presents as chronic unilateral rhinorrhea. there is no significant respiratory distress. because neonates are obligate nose breathers, bilateral choanal atresia is associated with severe respiratory distress, difficulty feeding, and failure to thrive. the diagnosis is suspected if catheters cannot be passed through the nose and into the pharynx. the obstruction may be visualized with a narrow flexible nasopharyngoscope after the nasal cavity has been suctioned of mucus and the nasal mucosa has been constricted with a nasal decongestant (e.g., oxymetazoline). the diagnosis is best made with ct of the nasal cavity. ct will demonstrate the atresia, define the tissue (bony or membranous), and show the configuration of the entire nasal cavity. choanal atresia may be successfully treated by removing the obstructing tissue transnasally. curets, bone punches, and drills may all be effective to remove the atresia plate. however, when the bony plate is very thick and there is an extremely narrow posterior nasal cavity, a transpalatal repair is more direct. a transpalatal repair provides better access for more effective removal of the bony plate and posterior septum ( fig. 52-3 ). stents fashioned from endotracheal tubes are placed and secured with sutures to the septum. they are removed in several weeks. the stents must be moistened with saline and suctioned several times daily to prevent mucus plugging and acute respiratory distress. transpalatal repair of choanal atresia has a lower incidence of restenosis. 27[pp 196-205] nasal dermoid cysts or sinuses present in the midline of the nasal dorsum ( fig. 52-4 ). they usually appear as a round bump or a pit with hair present in the pit ( fig. 52-5 ). they also may become infected. nasal dermoid sinuses may extend through the nasal bones into the nasofrontal area and have an intracranial component. both ct and mri may be necessary to demonstrate the extent of the dermoid. surgical removal is required to prevent infection and recurrence. this may be done between ages 3 and 5 years if prior infection has not occurred. dermoids confined to the nose are resected completely using a midline incision with an ellipse around the sinus tract. otolaryngologic disorders the tract is followed to its termination, and the nasal bones may need to be separated to reach the end of the tract. 27[pp 188-191] if an intracranial component is present, a combined craniotomy and nasal approach with a neurosurgeon is recommended. a nasal glioma presents as an intranasal mass and may be confused with a nasal polyp. the mass contains dysplastic brain tissue and may have an intracranial connection. ct and mri are important to define the extent of the glioma and intracranial component as well as to plan the surgical approach. an encephalocele presents as a soft compressible mass and may also be confused with a nasal polyp. intranasal encephaloceles extend through a defect in the skull at the cribriform plate. ct and mri define the extent of the encephalocele and are necessary to design the surgical approach. surgical removal often includes a frontal craniotomy. nasal encephaloceles may be associated with csf rhinorrhea and meningitis. an infant may be born with the soft nasal bones and the septum deviated to one side either as a result of a difficult delivery or from persistent intrauterine compression of the nose. the nasal structures can most often be returned to the midline with digital manipulation. if the nasal deformity is partially reduced, the nose usually straightens with growth during the first year to 18 months of age. nasal bone and nasal septal fractures in older children usually occur from a blow to the face during sports. there is usually a brief period of epistaxis and deviation of the nasal dorsum to one side. swelling occurs rapidly, and the degree of the cosmetic deformity or the need for fracture reduction may not be easily determined. at the fourth to sixth day after injury, the edema subsides and the need for reduction can be determined. nasal bone radiographs are of little help in making this judgment, so the need for nasal fracture reduction is usually based solely on clinical examination. effective nasal fracture reduction may be done up to 2 weeks after the injury. closed reduction under general anesthesia is the method of choice. oral antibiotics prevent infection and are essential if nasal packing is used to support the nasal bone. although nasal fracture reduction is not urgent, a septal hematoma from a fractured septum should be excluded by the initial physician seeing the child. a septal hematoma that remains untreated may cause cartilage necrosis and loss of nasal support, with a resulting saddle-nose deformity. treatment of a septal hematoma is with incision and evacuation of the clot. the mucoperichondral flap should then be sutured in place by bolster sutures through the septum. a small rubber band drain should remain in place for 12 to 24 hours, and antibiotics should be given. epistaxis in children usually occurs in little's area of the anterior septum and frequently results from digital trauma (nose picking). the bleeding usually stops with pressure by squeezing the nasal ala. infrequently, cauterization of the vessels under general anesthesia is needed. children may be observed inserting a foreign body into their nose, or they may inform their parents of the event. most children, however, present with a foul-smelling unilateral purulent nasal discharge and deny putting anything into their nose. most nasal foreign bodies are painless and do no harm to the nose except cause a foul nasal discharge. disc batteries, on the other hand, cause very rapid alkali burns of the nasal cavity and pain. batteries must be removed from the nose quickly because the chemical burn occurs in minutes to hours. if extensive tissue necrosis occurs, it may cause a nasal stenosis. removal of a nasal foreign body is aided by decongesting the nasal mucosa and using a headlamp to visualize the foreign body. a variety of forceps or hooks may be used. if the object is deep in the nose, the removal is best performed under general anesthesia. the endotracheal tube prevents aspiration of the object into the tracheobronchial tree if it is pushed back into the nasopharynx. one must remember that multiple foreign bodies may be present. nasal lacerations should be closed with care to match edges and restore the contours of the nose. standard wound closure technique is employed. the nasal mucosa does not need to be sutured unless a large flap is displaced. rhabdomyosarcoma, lymphoma, squamous cell carcinoma, and esthesioneuroblastoma may occur in the nose and sinuses of children. fortunately, these malignant tumors are very rare in children. the treatment of children with malignant tumors of the nose and sinuses usually involves a multidisciplinary, multimodal approach. juvenile nasopharyngeal angiofibroma is a benign tumor of adolescent males that originates from the lateral wall of the nose and nasopharynx. the tumor may completely obstruct the nose and fill the nasopharynx. this type of angiofibroma may also extend intracranially through the base of the skull. patients with these tumors present with nasal obstruction, recurrent epistaxis, and rhinorrhea. the tumor may be seen with a flexible fiberoptic nasopharyngoscope or a rod lens telescope after decongesting the nasal mucosa. it appears as a smooth reddish mass. biopsy of the mass should be avoided because of the potential for severe bleeding. ct and mri define the extent and location of the tumor. mr angiography helps to delineate the blood supply, which may originate from both the internal and external carotid arteries. contrast angiography may be reserved for presurgical planning and embolization of the copious blood supply that is often present. the treatment of juvenile nasopharyngeal angiofibroma is complete surgical resection after preoperative embolization. depending on the material used, the embolization may be effective for days to weeks. a variety of surgical approaches may be used, including endoscopic resection of small tumors. extensive tumors may require a combined midfacial and craniotomy approach. 29 some authors have proposed radiation therapy as the primary treatment of juvenile nasopharyngeal angiofibroma, but many surgeons are concerned about the long-term effects of radiation in children, including the induction of malignant tumors. the boundaries of the oral cavity include the lips anteriorly, the cheeks laterally, and the palate superiorly. the posterior boundary is a plane that extends from the soft palate to the junction of the anterior two thirds and posterior one third of the tongue. the oral cavity is composed of the vestibule, the space between the lips and cheeks and alveolar ridges, and the oral cavity proper. the vestibule and oral cavity proper are separated by the alveolar ridge and teeth. the vestibule is divided in the midline by the labial frenula of the upper and lower lips. the alveolar ridge is contiguous superiorly with the hard palate. the parotid ducts (stensen's ducts) enter the vestibule opposite the second maxillary molars. the submandibular ducts (wharton's ducts) enter the floor of mouth near the lingual frenulum. the palate is formed by a fusion of the primary palate anteriorly and medial growth of the palatal processes that form the secondary palate. the hard palate divides the nasal and oral cavities and is formed by the premaxilla and the horizontal plates of the palatine bones. the soft palate is formed by a muscular aponeurosis of the tensor veli palatini tendon. five muscles insert into this aponeurosis and include the tensor veli palatini, levator veli palatini, palatoglossus, palatopharyngeus, and the musculus uvulae. defects in formation of the hard and/or soft palate result in clefting. the sensory and motor innervation of the palate is through the trigeminal nerve and pharyngeal plexus. the circumvallate papillae divide the tongue into the anterior two thirds that lies in the oral cavity and the posterior one third lying in the oropharynx. the innervation and vascular supply to the two major divisions of the tongue reflect their differences in origin-the anterior two thirds of the tongue being a first branchial arch derivative (trigeminal) whereas the posterior one third being a combination of third and fourth arch derivatives (pharyngeal plexus). the hypoglossal nerve supplies motor innervation to the intrinsic musculature. in addition to the intrinsic tongue musculature, the action of four extrinsic muscles combine to provide mobility. the genioglossus protrudes and depresses, the hyoglossus retracts and depresses, the styloglossus retracts, and the palatoglossus elevates. in addition to the circumvallate papilla, other taste buds on the tongue surface include conical, filiform, fungiform, and foliate papillae. the pharynx is a fibromuscular tube that extends from the skull base to the level of the cricoid cartilage of the larynx and can be divided into three levels. the nasopharynx extends from the skull base to the level of the soft palate, the oropharynx extends from the soft palate to the tongue base, and the hypopharynx extends from the tongue base to the cricoid cartilage. three muscular constrictors combine to form the muscular portion of the pharynx: superior, middle, and inferior constrictors. passavant's ridge is a muscular segment of the superior constrictor that is involved in velopharyngeal closure. lower fibers of the inferior constrictor help to form the upper esophageal sphincter. the motor and sensory innervation of the pharynx is from the glossopharyngeal and vagus nerves via the pharyngeal plexus. a collection of lymphoid tissue within the pharynx forms waldeyer's ring that includes the palatine tonsils, the adenoid (pharyngeal tonsil), and lymphoid follicles lining the lateral and posterior pharyngeal walls. in addition to the acute onset of sore throat, viral pharyngitis typically presents with fever and malaise. signs include erythema of the pharynx and cervical lymphadenopathy. depending on the viral agent, associated symptoms of nasal obstruction and rhinorrhea may also be present. rhinovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, adenovirus, and influenza virus are agents responsible for viral pharyngitis. primary herpetic gingivostomatitis, caused by herpes simplex types 1 or 2, presents as fever, adenopathy, and vesicles and ulcers on the lips, tongue, buccal mucosa, soft palate, and pharyngeal mucosa. herpangina and hand-foot-and-mouth disease are viral infections that involve the oropharynx. epstein-barr virus (ebv) infection (infectious mononucleosis) presents as acute pharyngotonsillitis, fever, generalized adenopathy, malaise, and splenomegaly. although ebv infection is suspected by the appearance of 10% or more atypical lymphocytes on a complete blood cell count and the presence of a positive monospot test, the definitive diagnosis is confirmed by elevated titers of ebv. group a î²-hemolytic streptococcus (gabhs, i.e., s. pyogenes) commonly infects the pharynx. in addition to sore throat, associated symptoms include fever, headache, and abdominal pain. associated signs include pharyngeal erythema, halitosis, tonsillar exudates, and tender adenopathy. diagnosis may be confirmed initially with a rapid streptococcal antigen test. because rapid antigen testing is more sensitive than formal plating on blood agar, a negative test does not need confirmation, but positive rapid streptococcal tests should be confirmed with formal plating. other bacterial pathogens that cause acute pharyngitis include haemophilus influenzae and groups c and g î²-hemolytic streptococci. occasionally, concurrent infection with penicillin-resistant staphylococcus aureus may interfere with treatment of a gabhs infection. 28 although many cases of gabhs infections respond to treatment with penicillin v or amoxicillin, emerging resistance to oropharyngeal pathogens mandates treatment of recalcitrant cases with an antibiotic having known effectiveness against î²-lactamase-producing organisms. in cases in which a lack of compliance is suspected, intramuscular benzathine penicillin or ceftriaxone may be used. acute pharyngitis may also be associated with acute bacterial infections of the nose, nasopharynx, and sinuses. these infections may be caused by a variety of viral and bacterial pathogens; and in addition to sore throat, symptoms include fever, mucopurulent nasal drainage, nasal obstruction, and facial pain. recurrent infection of the pharynx may be either viral or bacterial. gabhs are the most worrisome bacterial organisms because recurrent infection may lead to complications such as scarlet fever, acute rheumatic fever, septic arthritis, and acute glomerulonephritis. in addition to a history of multiple positive cultures for s. pyogenes, elevated antistreptolysin-o (aso) titers may identify patients with chronic infection who are at risk for developing complications. some asymptomatic children may be chronic carriers of gabhs, and elevated aso titers may not be a reliable indicator for distinguishing between an active infection and the carrier state. treatment of recurrent streptococcal infection or the child who is a carrier should include a trial course of an antibiotic shown to reduce carriage (e.g., clindamycin, vancomycin, or rifampin). children with recurrent pharyngotonsillitis unresponsive to medical therapy or those who suffer a complication should be considered for surgical management. whereas treatment of each child should be individualized, suggested guidelines for surgical candidates include seven infections in 1 year, five or more infections per year for 2 years, or three or more infections per year for 3 years. 24 other factors to be considered in employing a surgical option include severity of infection, response to antibiotic therapy, loss of time from school, and need for hospitalization. the pharynx and specifically the tonsils may be the target of chronic infection. affected children complain of chronic throat pain, halitosis, and production of white particles or tonsilliths. signs include erythema of the tonsils, cryptic debris, and chronically enlarged cervical adenopathy. a variety of viral and bacterial agents can be blamed for chronic infection of the pharynx. cultures may or may not be positive in these patients, because surface cultures may be negative while core tissue is positive. antibiotic therapy directed at anaerobes or s. aureus may be helpful in resistant cases. children with infections unresponsive to medical management are candidates for tonsillectomy. localized extension of tonsillar infection may result in peritonsillar cellulitis. the same pathogens that cause acute pharyngotonsillitis are responsible for peritonsillar cellulitis. in addition to severe sore throat, symptoms and signs include drooling, trismus, muffled voice, ipsilateral referred otalgia, and tender lymphadenopathy. the affected tonsil is usually displaced in a medial and inferior position. peritonsillar cellulitis may progress to frank abscess formation (quinsy). early cases of peritonsillar cellulitis may respond to oral antibiotics, such as the penicillins, cephalosporins, erythromycins, or clindamycin. unresponsive cases of cellulitis or abscess should be treated with intravenous antibiotics. in children with suspected abscess formation, a variety of surgical drainage procedures can be performed. needle aspiration or incision and drainage have been shown to be equally effective. 15 in persistent cases or in those children who will require general anesthesia for drainage, consideration should be given to performing a tonsillectomy (quinsy tonsillectomy). signs and symptoms of deep neck space (retropharyngeal/ parapharyngeal) infections that involve the pharynx typically present as fever, drooling, irritability, decreased oral intake, torticollis, and/or trismus. often there is a history of a preceding viral illness. stridor or symptoms of upper airway obstruction may be seen in half of patients. 32 a neck mass or enlarged cervical nodes may be present depending on the location of the infection. usual pathogens include coagulase-positive staphylococci and gabhs. anaerobic bacteria have been found in as many as 50% of cases. 32 complications of deep neck space infections include airway obstruction, bacteremia, rupture of the abscess into the pharynx with aspiration, mediastinal extension of infection, jugular thrombosis, and carotid artery rupture. in suspected cases, the diagnosis of a retropharyngeal/parapharyngeal space infection is confirmed with either contrast medium-enhanced ct or mri. widening of the retropharynx on a lateral neck radiograph suggests a retropharyngeal infection. while ultrasound can detect the presence of an abscess cavity, ct or mri are most helpful in demonstrating the extent of infection and the location of surrounding structures of importance, specifically the great vessels. contrast medium-enhanced ct is particularly useful in distinguishing a phlegmon (cellulitis) from cases of frank suppuration. demonstration of a hypodense region with surrounding rim enhancement has been shown to correlate with an abscess in 92% of cases (fig. 52-6) . the initial management of a deep neck infection should begin with intravenous antibiotics, including oxacillin, clindamycin, cefazolin, î²-lactamase penicillins, or a combination thereof. surgical drainage should be reserved for those children who fail to show clinical improvement or progress to frank abscess formation on ct. the usual approach to surgical drainage is intraoral if the abscess points medial to the great vessels or extraoral if the infection points lateral to the great vessels. complications of deep neck infections should be treated aggressively. mediastinal spread requires prompt surgical drainage in most cases. an infected jugular thrombosis (lemierre's syndrome) can be a source of metastatic spread of infection as septic emboli. signs and symptoms include spiking chills and fever (picket-fence fevers) and a neck mass in spite of appropriate antibiotic therapy. ligature or excision of the infected thrombus may be required to eradicate the infection. in the past decade, the impact of sleep-disordered breathing (sdb) on the health of children has been well described, beginning with the report of normative sleep data by marcus and colleagues. 20 children appear to have briefer but more frequent episodes of partial (hypopnea) and complete (apnea) obstruction. because an apnea of less than 10 seconds may represent several missed breaths in a child, an apnea of any duration is abnormal. in most cases the site of obstruction during sleep is in the pharynx. in contrast to adults with this disorder in whom the pharyngeal impingement is due to adipose tissue surrounding the pharyngeal musculature, the major cause of airway obstruction in children results from adenotonsillar hypertrophy. the apnea index represents the number of apneas in an hour, with a normal value being less than 1 in children. because most children have an increased frequency of partial obstructions compared with adults, a measure of hypopneas may be more significant. a hypopnea is variably described as a reduction in airflow or respiratory otolaryngologic disorders effort or oxygen desaturation or combination thereof. respiratory disturbance index is a measure of both apneas and hypopneas in an hour and may be a better reflection of sdb in children. a respiratory disturbance index greater than 5 is abnormal. upper airway resistance syndrome represents obstructed breathing with normal respiratory indices but with sleep fragmentation and electroencephalographic arousals that indicate disordered sleep. the major group at risk for sdb includes children with adenotonsillar hypertrophy secondary to lymphoid hyperplasia (figs. 52-7 and 52-8). whereas the age of affected children ranges from 2 years through adolescence, the prevalence mirrors the age of greatest lymphoid hyperplasia, 2 to 6 years, the age the tonsils and adenoids are largest in size. other at-risk groups include syndromic children with down syndrome, children with craniofacial disorders, and patients with cleft palate or storage diseases (hunter's, hurler's syndromes). adverse effects of obstructive sleep apnea on children include poor school performance, failure to thrive, facial and dental maldevelopment, and, rarely, severe cardiac impairment, including systemic hypertension, cardiac arrhythmias, and cor pulmonale. daytime symptoms include noisy mouth-breathing, nasal obstruction and congestion, hyponasal speech, and dyspnea on exertion. in contrast to adults, hypersomnolence is uncommon in children because of the lower incidence of gas exchange abnormalities, specifically hypercarbia. children may complain of headaches, seem irritable, and perform poorly in school. nighttime symptoms are more obvious and include snoring, gasping and choking respirations, apnea, coughing, and a variety of other behaviors including sleepwalking, sleeptalking, rocking, head banging, and bruxism. enuresis may appear in children with airway obstruction and then resolve after surgical treatment. in addition to enlarged tonsils, signs include the presence of a posterior pharyngeal flap in cleft palate patients, a craniofacial disorder, adenoid facies, and, rarely, evidence of right-sided heart failure. the diagnosis of sdb is suggested by history and physical examination. confirmation of obstruction and apnea may be made with overnight pulse oximetry and video or audio monitoring of sleep. the "gold standard" in the diagnosis of obstructive sleep apnea remains formal polysomnography, including measures of nasal and oral airflow, chest wall movements, electrocardiography, extraocular muscle movements, and gastric ph monitoring in selected cases. depending on the suspected site of obstruction, adjuvant studies such as a lateral neck radiograph, mri of the head and neck, and flexible upper airway endoscopy might be helpful. the nonsurgical management of sdb consists of weight loss in obese patients and treatment of underlying allergies and gastroesophageal reflux. nasal and dental appliances to maintain airway patency that may be useful in adults are usually poorly tolerated in children. nasal continuous positive airway pressure, the mainstay of treatment in adults, is tolerated in many children and should be considered as a treatment option, especially in patients in whom other therapies have been exhausted or proven ineffective. the initial surgical treatment for most children with sdb remains a tonsillectomy and adenoidectomy, a therapy that is usually curative. in patients with documented sleep apnea or a sleep disorder, both procedures should be utilized even if the tonsils appear small. tonsillectomy and adenoidectomy techniques that have been standard for decades have been supplanted in some institutions by new technology including use of coblation, harmonic scalpel, and the microdebrider. efficacy of these newer techniques over established methods remains unproven. complications after tonsillectomy and adenoidectomy usually consist of respiratory compromise and acute or delayed bleeding. since the advent of modern pediatric anesthesia, respiratory complications such as aspiration with resultant pneumonia and lung abscess are rare. humidification, corticosteroids, and antibiotics have all been shown to improve the postoperative course after tonsil and adenoid surgery. young children are most vulnerable to complications, and in most institutions children younger than 4 years of age are observed overnight for signs of dehydration and respiratory compromise. adjuvant surgery in the management of sdb includes craniofacial repair or posterior flap revision surgery in appropriate patients. midface, mandibular, and hyoid advancement have proved useful in selected patients, along with nasal surgery such as septoplasty, partial inferior turbinectomy, or nasal polypectomy. tracheostomy remains the treatment of last resort in patients who fail to respond to other forms of therapy. ankyloglossia or tongue-tie is a common congenital disorder involving the lingual frenulum ( fig. 52-9 ). neonates with diminished tongue mobility due to a foreshortened frenulum may have problems in sucking and feeding. because the frenulum is thin and relatively avascular in neonates and young infants, it can often be incised as an office procedure. in older children the greatest effect of ankyloglossia is on speech. because the tip of the tongue curls under on extrusion and has limited lateral and superior movement, speech articulation may be affected. surgical treatment in these patients may require a short general anesthetic as the frenulum is thicker and more vascular, requiring surgical correction that includes either simple division with or without a z-plasty repair. macroglossia is uncommon. generalized macroglossia, as seen in association with omphalocele, visceromegaly, and adrenal and renal disorders (beckwith-wiedemann syndrome), with glycogen storage diseases (hunter's and hurler's syndromes) or hypothyroidism, is rare. relative macroglossia can be seen normally on occasion but is most common in down syndrome. the most serious complication of this condition is airway obstruction. in infants, macroglossia should be distinguished from focal enlargement of the tongue seen in patients with a lymphatic malformation or hemangioma. glossoptosis, posterior displacement of a normal-sized tongue, is seen in association with cleft palate and micrognathia in infants afflicted with the pierre robin sequence. infants with airway obstruction secondary to an enlarged or displaced tongue may require a tracheostomy. macroglossia in older children that affects cosmesis, interferes with speech, or causes drooling may be treated with a variety of tongue reduction techniques. epulis is a congenital granular cell tumor that typically presents as a soft, pink submucosal mass on the anterior alveolar ridge of the maxilla (fig. 52-10) . females are otolaryngologic disorders more commonly affected, and symptoms are usually confined to feeding problems. surgical excision is curative. ranula is a pseudocyst located in the floor of the mouth that may occur congenitally or result from intraoral trauma (fig. 52-11) . large ranulas may extend through the mylohyoid musculature and present in the neck as a "plunging ranula." treatment of ranulas is by excision or marsupialization of the pseudocyst, often in conjunction with excision of the sublingual gland. mucoceles are also pseudocysts of minor salivary gland origin and frequently rupture spontaneously. recurrent or symptomatic mucoceles respond to surgical excision. hemangioma is a proliferative endothelial lesion found commonly in the head and neck. their growth characteristics include enlargement during the first year of life, followed by spontaneous resolution. surgical excision or treatment with corticosteroids may be necessary in lesions that cause ulceration and bleeding, airway obstruction, cardiovascular compromise, or platelettrapping coagulopathy (kasabach-merritt syndrome). vascular malformations, including venous, arterial, or arteriovenous malformations, rarely occur in the oral cavity and pharynx and necessitate intervention only if they cause pain, bleeding, ulceration, or heart failure. management of complicated cases is by surgical excision or sclerotherapy for low-flow lesions (venous) and angiographic embolization for high-flow lesions. lymphatic malformation, formerly known as lymphangioma or cystic hygroma, is congenital and usually presents before 2 years of age. histologically, lymphatic malformations consist of multiple dilated lymphatic channels or may contain either capillary or venous elements (venolymphatic malformations). lymphatic malformations can occur anywhere in the neck and may cause extensive cosmetic deformity and functional problems in cases with involvement of the tongue, floor of mouth, mandible, or larynx. surgical resection of lymphatic malformations may be fraught with difficulty because they lack a capsule and are infiltrative. during surgical excision, care should be taken to avoid damaging nearby vital structures, and debulking is an acceptable option to total radical excision in many cases. postoperative suction drains can be helpful in preventing the recurrence of lymphatic drainage under skin flaps. carbon dioxide laser therapy has been employed in superficial lymphatic malformations of the tongue, and sclerotherapy of large cystic lesions may be an option. foregut cysts are true cysts, lined with respiratory epithelium, that present in the floor of mouth and should be distinguished from dermoid cysts, lined with stratified squamous epithelium and skin appendages, which may also be found in this location. a thyroglossal duct cyst may rarely present in the base of the tongue. likewise, aberrant thyroid tissue, lingual thyroid, presents as a purple mass in the tongue base. thyroid tissue in this location is usually hypofunctioning, and affected children require thyroid supplementation. other aberrant rests of tissue, choristomas, consist of gastric, enteric, or neural tissue of normal histology in an abnormal location. second branchial cleft derivatives will rarely present as a cystic mass near the superior pole of the tonsil. their extent and associated tracts can be demonstrated on mri. tornwaldt's cyst is a blind pouch in the nasopharynx that represents a persistence of an embryonic connection between the primitive notochord and the pharynx. other benign nasopharyngeal masses include nasopharyngeal teratomas, dermoid lesions (hairy polyp), and nasopharyngeal encephaloceles. most of these lesions are best evaluated by ct and/or mri to determine their extent and the presence of an intracranial connection. surgical excision is curative in most cases. squamous papillomas are benign slow-growing lesions typically found on the soft palate, uvula, and tonsillar pillars and are the result of infection with serotypes 6, 14, or 22 of the human papillomavirus (hpv). because of concern that these lesions could spread to the larynx or trachea, complete surgical excision is usually recommended. pleomorphic adenoma (mixed tumor) is a benign neoplasm of minor salivary glands with a predilection for the palate, although it may also be found in the lip and buccal mucosa. treatment is with surgical excision. rhabdomyosarcoma, the most frequent soft tissue malignancy of childhood, typically occurs in the 2-to 6-year age group and is derived from embryonic skeletal muscle. 4, 18 in the oral cavity and oropharynx it presents as a rapidly growing mass in the tongue, palate, and uvula or cheek. these tumors metastasize early to local nodes, lung, and bone. surgical therapy is limited to biopsy, excision of small lesions, or surgical salvage of treatment failures. the usual therapy includes a combination of chemotherapy and radiation therapy. lymphoma of the oral cavity and oropharynx typically involves the lymphoid tissue of waldeyer's ring and presents as a mass of the tonsil or in the nasopharynx. 8 the diagnosis may be suspected by evidence of involved adenopathy in the neck but is confirmed by surgical biopsy. treatment is with a combination of chemotherapy and radiation therapy. other rare malignant neoplasms of the oral cavity and pharynx include malignant salivary gland tumors (mucoepidermoid carcinoma) and epidermoid or squamous cell carcinoma. this latter tumor has been reported in organ transplant patients and adolescents who use snuff or chewing tobacco. 19 treatment is usually surgical depending on the site and extent of involvement. with the exception of the hyoid bone, the major structural framework of the larynx consists of cartilage and soft tissue. the hyoid bone lies superior to the larynx and is attached to it by the thyrohyoid membrane and strap muscles. the hyoid bone is derived from the second and third branchial arches. the cartilaginous structures of the larynx are composed of hyaline cartilage, with the exception of the epiglottis, which is composed of elastic cartilage. the cartilaginous structures of the larynx develop from the fourth, fifth, and sixth branchial arches. there are nine laryngeal cartilages, three that are single (thyroid, cricoid, and epiglottis) and six that are paired (arytenoid, cuneiform, and corniculate). the thyroid cartilage consists of two quadrilateral cartilages that form the anterior framework of the larynx. the cricoid cartilage is the only complete cartilaginous structure in the airway and provides posterior stability and a base of support for the cricoarytenoid and cricothyroid joints. the cricothyroid muscles are paired extrinsic laryngeal muscles that serve to tilt the larynx down and forward, tensing the vocal folds. paired intrinsic muscles-the thyroarytenoid, thyroepiglottic, and aryepiglottic muscles-act as a sphincter to close the larynx. the vocalis muscle comprises the internal fibers of the thyroarytenoid muscle and attaches to the vocal ligament. action of this muscle serves to regulate the pitch of the vocal ligament. the other set of paired muscles includes the posterior cricoarytenoid, lateral cricoarytenoid, and interarytenoid muscles. the posterior cricoarytenoid muscles serve to abduct the vocal folds, whereas the cricoarytenoid and interarytenoid muscles adduct the vocal folds. the quadrangular membrane is a connective tissue covering of the superior larynx that ends in a free margin along the vestibular ligament of the false cord. the conus elasticus is a membrane of elastic tissue that extends superiorly from the cricoid cartilage to form the paired vocal ligaments, the supporting structures of the vocal folds. the blood supply of the larynx arises from the superior and inferior laryngeal arteries. the former is a branch of the superior thyroid artery, whereas the latter is a branch from the thyrocervical trunk. the intrinsic muscles of the larynx are innervated by the recurrent laryngeal nerve, which also supplies sensory branches to the inferior larynx. the superior laryngeal nerve has two branches: the external branch innervates the cricothyroid muscle, while the internal branch supplies sensation to the superior larynx. the larynx has multiple functions within the upper airway. during respiration, it regulates airflow by opening during inspiration. the posterior cricoarytenoid muscle contracts with each inspiration to abduct the cords just before activation of the diaphragm. the protective function of the larynx produces two reflexes: cough and closure. cough is important to expel mucus and foreign objects. the closure reflex serves to prevent aspiration of foreign matter. in addition to closure, the larynx elevates during swallowing. both closure and elevation occur simultaneously along with relaxation of the cricopharyngeus muscle during the swallow of a bolus. finally, the larynx plays an important role in speech production by generating sound. vibration of the mucosa covering the vocalis structures produces sound whose pitch and register is altered by changes in tension, length, and mass of the underlying vocalis muscle and ligament. the larynx of an infant sits much higher than that of an adult. the cricoid is located at the level of c4, whereas the tip of the epiglottis is at c1. the close approximation of the epiglottis to the soft palate makes the infant an obligate nose breather. by 2 years of age, the larynx has descended to the level of c5 and reaches the adult level of c6 to c7 by puberty. the glottis of the newborn is 7 mm in the anteroposterior dimension and 4 mm in the lateral dimension. the narrowest area of the infant airway, the subglottis, is approximately 4 mm in diameter. symptoms of acute airway obstruction include dyspnea, cough, vocal changes, dysphagia, and sore throat. dyspnea and rapid or labored breathing are indications of inadequate ventilation and may be triggered by changes in pco 2 and po 2 . a stimulus anywhere in the airway may produce cough. it is difficult to localize the site of the stimulus from the quality of the cough. changes in the child's vocal character such as hoarseness or a muffled or weak cry may help in localizing the area of obstruction. dysphagia for solids and/or liquids is often associated with airway obstruction. depending on the cause of airway obstruction, affected patients may complain of sore throat. the child's overall appearance is the first sign to be assessed in airway obstruction, because airway status often dictates how quickly further evaluation and intervention need to be performed. the level of consciousness should be determined because the unconscious or obtunded patient may need immediate airway management. along with cyanosis in a patient without cyanotic heart disease, the presence of anxiety, restlessness, and diaphoresis are all ominous signs of impending airway compromise. other symptoms of airway obstruction include tachypnea and substernal retractions. the child with airway obstruction is often tachycardic. the presence of bradycardia is a otolaryngologic disorders late indicator of severe hypoxia. the presence of a muffled cry often suggests obstruction at the level of the pharynx, whereas a barking cough is associated with laryngeal inflammation and edema. stertor is a snorting sound whose origin is often in the pharynx. stridor is noise produced by turbulent airflow in the laryngeal or tracheal airway. inspiratory stridor suggests turbulence at or above the glottis. expiratory stridor results from turbulent airflow in the distal trachea or bronchi. biphasic stridor suggests a tracheal source. the degree and loudness of the sound is not always indicative of the severity of obstruction, because stridor can become softer just before complete obstruction. other important signs of airway obstruction include drooling and use of accessory respiratory muscles. in addition to determination of the child's physical status, assessment of the degree of airway obstruction should include an evaluation of the ventilatory status. pulse oximetry provides an immediate record of arterial oxygenation while transcutaneous monitoring of co 2 is a good indicator of ventilation. the lateral neck radiograph remains the best study for the initial evaluation of a child with airway obstruction because it demonstrates the anatomy from the tip of the nose to the thoracic inlet. the anteroposterior view of the neck is also helpful, specifically in defining areas of narrowing, such as a steeple sign associated with subglottic edema. a chest radiograph is also important in the initial assessment to identify foreign bodies or other conditions such as unilateral emphysema, atelectasis, or pneumonia that may account for the child's respiratory compromise. if time permits, a barium swallow or airway fluoroscopy may provide additional information. additional airway evaluation may include a brief flexible endoscopic examination. the nose is first sprayed with a combination of 2% lidocaine and oxymetazoline, and the child is gently restrained. the airway can be examined from the nares to the glottis. attempts to pass a flexible scope through the glottis in a child with airway obstruction should be avoided. likewise, flexible endoscopy should be avoided in a child with supraglottitis because of the possibility of precipitating complete obstruction. children with suspected airway pathology distal to the glottis or those in whom the possibility that flexible endoscopy could compromise the airway should undergo any airway examination in the operating room where rigid endoscopes and other airway equipment is immediately available to secure the airway if necessary. nonsurgical intervention in the child with acute airway obstruction may begin with just observation alone in a high surveillance unit. humidified oxygen administered by face mask will improve po 2 and clearance of secretions. racemic epinephrine administered by nebulizer acts to reduce mucosal edema and is useful in conditions such as laryngotracheobronchitis (infectious croup). because its length of action lasts 30 to 60 minutes, treated patients should be observed for signs of rebound for 4 to 6 hours after administration. corticosteroids have been shown to have value in the management of postintubation croup, adenotonsillar hypertrophy that results from ebv infection, allergic edema, and spasmodic croup. use of corticosteroids in infectious croup and in infants with a subglottic hemangioma remains controversial. 10, 16 other adjuvant therapies include antibiotics and inhalation of helium/oxygen mixture (heliox). although viral agents are often responsible for inflammation in the larynx and trachea, bacterial superinfection is also common. because of the prevalence of penicillin-resistant organisms, broad-spectrum antibiotics, including a higher-generation cephalosporin, penicillinase-resistant penicillin, or î²-lactamase penicillin, are useful in preventing or eradicating infection. heliox is a mixture of gas in which helium is used to replace nitrogen. the advantage of the helium-oxygen mixture is that its low density reduces air turbulence and gas resistance, allowing improved delivery of oxygen in patients with airway obstruction. nonsurgical airway management may include use of nasal or oral airways, endotracheal intubation, and, rarely, transtracheal ventilation. nasal airways of rubber or other synthetic material can be easily inserted into the nose of most children after adequate lubrication with a water-soluble lubricant. their best use is in cases where the pharynx is the site of obstruction. oral airways are not as readily tolerated by children and only serve as a brief solution to an airway problem. during the 1970s, endotracheal intubation with polyvinyl chloride tubes revolutionized the management of supraglottitis, and even today intubation remains the mainstay of initial airway therapy in most children with severe airway obstruction. the size of the endotracheal tube used correlates with the age of the child. the subglottis, the narrowest part of the infant airway, typically admits a 3.5-or 4.0-mm inner diameter tube. the tube used in children older than a year can be roughly estimated by using the following formula: tube size = (16 + age in years) ã· 4. once the airway has been established, the tube should be carefully secured and the child appropriately sedated and/or restrained if necessary to avoid accidental self-extubation. another method of airway management should be considered in children with an unstable cervical spine or in whom oral or neck trauma makes visualization difficult. transtracheal ventilation, insertion of a 16-gauge needle through the cricothyroid membrane for the delivery of oxygen, should be reserved for emergencies and used only until a more stable airway can be obtained. the surgical management of the child with acute airway obstruction should begin with endoscopy. the larynx can be visualized with one of a variety of pediatric laryngoscopes and the airway secured with a rigid pediatric ventilating bronchoscope of appropriate size. once the airway is secured, a more stable form of airway management can be utilized. rarely, in a child with acute airway obstruction, an airway cannot be established, and a cricothyrotomy may need to be performed. as in adults, this procedure avoids some of the risks of bleeding and pneumothorax inherent in a formal emergency tracheostomy. a small endotracheal or tracheostomy tube can be inserted through the incision in the cricothyroid membrane, but conversion should be made to a more stable airway as soon as possible. tracheostomy remains the preferred airway in cases of acute obstruction in which a translaryngeal approach is unsuccessful or must be avoided. the emergent tracheostomy should be avoided if at all possible to lessen complications of bleeding, pneumothorax, pneumomediastinum, subcutaneous emphysema, or damage to surrounding structures. the incidence of these complications can be reduced by careful attention to surgical technique, good lighting, and adequate assistance. laryngomalacia is the most common cause of newborn stridor and is caused by prolapse of the supraglottic structures (arytenoid cartilages, aryepiglottic folds) during inspiration ( fig. 52-12 ). symptoms typically appear at birth or soon thereafter and include inspiratory stridor, feeding difficulties, and, rarely, apnea or signs of severe airway obstruction. gastroesophageal reflux disease tends to worsen symptoms of laryngomalacia. the diagnosis is confirmed by flexible endoscopy of the larynx, and other airway pathology can be excluded with lateral neck, chest, and barium swallow radiography. in most cases, laryngomalacia is self-limited and resolves by 18 months of age. changes in positioning and feeding, treatment of reflux, and, in some neonates, use of monitoring may be necessary. in severe cases, surgical intervention with either a supraglottoplasy (surgical division of the aryepiglottic folds) or a tracheostomy may be necessary. tracheobronchomalacia is defined as collapse of the tracheobronchial airway. it may be congenital or acquired (from long-standing intubation and infection) and may be segmental or involve the entire tracheobronchial tree. depending on the extent and location, symptoms include low-pitched biphasic stridor and signs of respiratory compromise. the diagnosis is usually made with endoscopy, although fluoroscopy of the airway may often demonstrate it. treatment ranges from observation in most cases to airway management with a tracheostomy tube and positive-pressure ventilation in severe cases. vocal fold paralysis is the second most common congenital laryngeal anomaly (after laryngomalacia) and may be unilateral or bilateral. congenital vocal fold paralysis may be caused by neurologic abnormalities (hydrocephalus, arnold-chiari malformation), birth trauma, or rarely in association with neoplasms of the larynx or neck. neonates with bilateral involvement typically present with highpitched inspiratory or biphasic stridor but a good cry. respiratory compromise and feeding difficulties may accompany the stridor. in infants with unilateral involvement, the airway may be adequate although a few infants will show evidence of compromise, especially during feeding. the cry is often hoarse or breathy. acquired vocal fold paralysis may result from trauma or from neoplasms of the chest or neck or may be iatrogenic, typically after surgery of the neck or arch of the aorta. the diagnosis of unilateral or bilateral vocal fold paralysis is confirmed with endoscopy. additional studies in the evaluation of patients with vocal fold paralysis include lateral neck and chest radiography, barium swallow, and ct or mri of the head and neck. most cases of unilateral involvement can be observed, but infants with bilateral vocal fold paralysis often require a tracheostomy. in addition, infants with associated feeding difficulties may necessitate a gastrostomy. in older children (> 4 or 5 years of age) a more permanent solution such as a cordotomy or arytenoidectomy can be considered to improve the glottic airway. congenital subglottic stenosis is the third most common congenital laryngeal anomaly and is defined as a neonatal larynx that fails to admit a 3.5-mm endotracheal tube without a history of prior instrumentation or intubation ( fig. 52-13 ). the underlying abnormality is a cricoid cartilage that is either small or deformed. infants with congenital subglottic stenosis present with inspiratory or biphasic stridor, barking cough, and other symptoms of airway obstruction. the diagnosis is often suggested by narrowing of the subglottis on a lateral neck radiograph and confirmed by endoscopy. treatment depends on the severity of symptoms and ranges from observation to laryngeal reconstruction to tracheostomy. a child with a subglottic hemangioma presents with the onset of progressive stridor during the first few months of life (fig 52-14) . hemangiomas are proliferative endothelial lesions that can form in the submucosa of the posterior subglottis. occasionally, they may involve the subglottis in a circumferential pattern. associated cutaneous hemangiomas may be found in approximately 50% of patients. symptoms are dependent on the amount of airway compromise and include biphasic stridor, barking cough, difficulty feeding, and other symptoms and signs of airway obstruction. the diagnosis may be suggested on a lateral neck radiograph but is confirmed with endoscopy. nonsurgical management of infants with a subglottic hemangioma includes observation or treatment with systemic corticosteroids. surgical therapy includes laser excision, open excision through a laryngofissure, or a tracheostomy. a laryngocele is an air-filled dilatation of the saccule of the larynx that communicates with the laryngeal airway. it may present internally into the posterior superior false cord region or externally through the thyrohyoid membrane. a saccular cyst is fluid filled and protrudes between the true and false vocal folds. the diagnosis of this lesion is confirmed endoscopically, and ct of the larynx is helpful in assessing its extent and if it is fluid or air filled. treatment is with endoscopic marsupialization or excision through a laryngofissure. laryngotracheobronchitis (viral croup) is an inflammation of the subglottic airway caused by a variety of parainfluenza and influenza viral agents. the infection may involve the entire glottis and extend into the trachea and bronchi. affected children fall typically into the 1-to 3-year age group; males are more commonly affected than females. symptoms and signs of viral croup include biphasic stridor, barking cough, and hoarseness, often in association with a prodromal viral upper respiratory tract infection. the diagnosis of croup is made clinically, but endoscopic examination may help to exclude other pathologic processes. care should be taken not to instrument the subglottis, causing more swelling and inflammation and precipitating acute obstruction. lateral neck radiography demonstrates subglottic narrowing, whereas anteroposterior neck films show a "steeple sign," the result of subglottic edema. treatment of viral croup is typically supportive with humidification. use of corticosteroids remains controversial. treatment with nebulized racemic epinephrine in the emergency department or hospital setting often relieves symptoms; however, rebound of signs may occur several hours later and children should be monitored accordingly. severely affected children may require intubation for respiratory failure. a smaller than normal tube should be employed. in rare cases, a tracheostomy may be required if the inflammation fails to resolve. a child younger than 1 year of age with recurrent bouts of "croup" should be suspected of having either congenital subglottic stenosis or a hemangioma. spasmodic croup is the recurrence of croup-like symptoms in a child who is otherwise well. fever is rarely present, and the attacks frequently occur at night. gastroesophageal reflux disease has been suggested as a possible inciting process. treatment of spasmodic croup is usually observant, although corticosteroids or reflux medications may prove beneficial. supraglottitis (epiglottitis) is an infectious disease that involves the supraglottic larynx. in children the typical pathogen is type b haemophilus influenzae (hib). other pathogens have been implicated in adolescent and adult cases. the incidence of supraglottitis in children has diminished markedly since the introduction of the conjugated hib vaccine in the early 1990s. 17 affected children are somewhat older than those seen with croup-in the 3-to 6-year age group. symptoms and signs have a rapid onset, progress quickly to frank airway obstruction, and include stridor, dysphagia, fever, muffled voice, and signs of systemic toxicity. affected children frequently sit and assume the "sniffing" position in an attempt to maximize their airway. intraoral or endoscopic examination should be avoided in suspected patients because of concern for precipitating complete obstruction. lateral neck radiography demonstrates a classic "thumbprinting" of the epiglottis but should only be obtained if facilities are present in close proximity to secure the airway. prompt airway management is essential in children with supraglottitis. the child's airway should be secured in either the emergency department or operating room with team members who include a pediatrician, anesthesiologist, critical care physician, otolaryngologist, or pediatric surgeon or others familiar with the pediatric airway. after inducing the child with general anesthesia, the airway should be intubated. examination of the supraglottis may be made, and cultures of the larynx and blood are obtained. equipment to perform a tracheostomy should be readily available. the child should remain intubated for 24 to 72 hours and should be supported with intravenous fluids and antibiotics that treat antibioticresistant haemophilus (third-generation cephalosporins, chloramphenicol). bacterial tracheitis (membranous croup) often occurs as a complication of another infection, such as measles, varicella, or other viral agents. the most common organisms include s. aureus, gabhs, m. catarrhalis, or h. influenzae. it can occur in any age child and present with stridor, barking cough, and low-grade fever. symptoms and signs then progress to include high fever and increasing obstruction and toxicity. the diagnosis may be suspected by diffuse narrowing of the tracheal air shadow on chest radiograph but is confirmed by endoscopic examination in the operating room. purulent debris and crusts can be removed at this time. cultures of secretions and crusts may be helpful in guiding intravenous antibiotic therapy that should be aimed initially at the usual pathogens. the airway should be secured with an endotracheal tube or, rarely, a tracheostomy. repeat endoscopic examination of the airway may be warranted to continue dã©bridement and to determine the feasibility of extubation. the chronic management of subglottic stenosis and other prolonged airway disorders is discussed in chapter 63. recurrent respiratory papillomatosis is the most common benign neoplasm of the larynx in children. squamous papillomas involve the larynx and occasionally the trachea and lower respiratory tract as exophytic lesions. because of its recurrent nature, recurrent respiratory papillomatosis causes morbidity and, rarely, mortality due to malignant degeneration. patients may be almost any age, but the disease is more aggressive in children. human papillomavirus subtypes 6, 11, 16, and 18 have all been identified within papilloma tissue. the first two subtypes have been associated with genital warts, whereas the latter two have been associated with cervical and laryngeal cancers. the exact mechanism of human papillomavirus infection in the larynx remains unknown. transmission of virus to the child from a mother with genital warts is suspected in many cases, but there is no concrete evidence to support this route of infection. children afflicted with recurrent respiratory papillomatosis present initially with hoarseness but may also have symptoms and signs of airway obstruction, including stridor. lateral neck radiography may suggest laryngeal involvement, but the diagnosis is confirmed by direct laryngoscopy and biopsy (fig. 52-15 ). in addition to the trachea and bronchi, squamous papillomas may also be found in the oral cavity. surgical excision is the mainstay of therapy in patients with recurrent respiratory papillomatosis. in the past, papillomas were excised using the carbon dioxide laser. more recently, the laryngeal microdebrider has become the preferred method of excision in many centers. in aggressive cases with swift recurrence and accompanying airway obstruction, tracheostomy may be necessary for airway management, although tracheostomy has been implicated in the spread of disease to the trachea and lower respiratory tract. medical adjuvant therapy that has been employed with mixed results includes interferon, photodynamic therapy with dihematoporphyrin ether, indole-3-carbinol, or antiviral agents such as cidofovir. other benign laryngeal neoplasms are rare and include connective tissue tumors such as chondromas or fibromas, neurogenic tumors such as neurofibromas, or granular cell tumors and other cell types such as hamartomas or fibrous histiocytomas. malignant tumors of the larynx are also rare and include squamous cell carcinoma and a variety of epithelial and connective tissue malignancies such as spindle cell carcinoma, rhabdomyosarcoma, mucoepidermoid carcinoma, and chondrosarcoma. metastatic tumors and lymphoma may also rarely involve the larynx in children. diagnosis is suspected by the sudden appearance of stridor, hoarseness, and airway obstruction and confirmed by biopsy. treatment is dependent on cell type and may include surgical excision, radiation therapy, and/or chemotherapy. the surgical anatomy and embryology of the neck is discussed in chapter 56. the initial examination of a disease or disorder of the neck begins with a thorough history. a detailed history can often serve to focus the differential diagnosis of a neck disorder. the age of the child is an important otolaryngologic disorders first consideration. the appearance of a neck mass in an infant often suggests a congenital disorder, whereas the sudden appearance of a mass in an adolescent might suggest a malignant process. inflammatory diseases of the neck may occur in any age group but typically mirror the incidence of upper respiratory tract infections in children. growth and temporal relationships are often important clues to a diagnosis. neck masses that grow rapidly suggest either an inflammatory or malignant process, whereas slow-growing masses are typically benign. a history of systemic infection elsewhere in the body or recent travel or exposure to farm animals often points to an infectious origin. a history of trauma to the neck may explain the sudden appearance of a neck mass. likewise, changes in the size of a neck mass with eating may suggest a salivary gland origin. vascular lesions enlarge with straining or crying. finally, systemic symptoms of fever, weight loss, night sweats, or fatigue in association with the sudden development of a neck mass may indicate a malignant process. the physical examination of a child with a neck mass should begin with a comprehensive examination of the entire head and neck. because the vascular, neural, and lymphatic patterns of the head drain into the neck, the source of neck disorders may be found in the head. depending on the differential diagnosis, a physical examination of the entire body, including an assessment of lymph nodes in the groin and axillae and the presence of an enlarged spleen or liver, is essential. palpable lymph nodes in the neck of children are a common finding, but lymph nodes larger than 2 cm fall outside the range of normal hyperplastic nodes and should be either monitored or investigated. the sudden appearance of large nodes in either the posterior cervical or supraclavicular regions may suggest a malignancy. 31 the consistency of a neck mass is also important in narrowing the differential diagnosis. hard masses tend to be associated with either infection or malignancy. fixation of a neck mass to skin or nearby structures is also suggestive of a malignancy. cysts or abscesses tend to have a characteristic feel on palpation. depending on the differential diagnosis after a history and physical examination, radiologic studies may be useful. a lateral neck radiograph may demonstrate an abnormality of the nasopharynx, retropharynx, or cervical spine. likewise, a chest radiograph may identify a malignancy, sarcoidosis, or tuberculosis. infection or a neoplastic process in the sinuses may appear on a sinus series. ct and mri are useful in the evaluation of a neck mass. demonstration of hypodensity on ct suggests an inflammatory or necrotic process. ring enhancement of a hypodense region on a contrast ct scan is indicative of an abscess. mri is excellent for distinguishing fine detail within soft tissue and in the evaluation of vascular lesions of the neck. finally, ultrasound is helpful in distinguishing solid and cystic masses. use of ultrasound preoperatively in patients with a thyroglossal duct cyst is also a simple and economic way to assess the presence of normal thyroid tissue when it is not easily felt. ultrasound and thyroid scanning should be employed in the assessment of any thyroid mass. selected laboratory studies may be helpful in the evaluation of a child with a neck disorder. a complete blood cell count with differential may identify patients with either a malignancy or systemic infection. serologic testing for ebv or cytomegalovirus infection, toxoplasmosis, or cat-scratch disease may be diagnostic. thyroid function testing is essential in any child with a suspected thyroid disorder. finally, collection of urine for catecholamine metabolites (vanillylmandelic acid) may assist in the diagnosis of neuroblastoma. if the diagnosis remains in doubt at this point, incisional or excisional biopsy may be indicated. biopsy provides material for pathologic examination, culture, and other more sophisticated testing if necessary. fine-needle aspiration of a neck mass in children for suspected malignancy is not as reliable as in adults. congenital sinuses and cysts are discussed in chapter 56. viral adenitis is the most common infectious disorder to involve the neck in children. enlarged or hyperplastic lymph nodes are frequently the result of viral upper respiratory tract illnesses. common pathogens include rhinovirus, adenovirus, and enterovirus, but measles, mumps, rubella, varicella, ebv, and cytomegalovirus may also cause lymphadenopathy. the diagnosis is often suspected by other findings in the history or physical examination and can be confirmed by serologic testing. acute human immunodeficiency virus infection may present, as do other viral syndromes, with fever, headache, malaise, gastrointestinal symptoms, and a neck mass. the usual source of bacterial cervical adenitis is the pharynx. causative organisms are often streptococcal or staphylococcal species. patients present with systemic symptoms of fever and malaise in addition to a neck mass that is diffusely swollen, erythematous, and tender. in contrast to viral adenitis, which is frequently bilateral, bacterial infections of the neck are usually unilateral. ct with contrast medium enhancement may be helpful in the evaluation of large infectious neck masses that may contain an abscess cavity. needle aspiration of suspected infectious masses may provide material for culture and decompress the mass. broad-spectrum antibiotic therapy, administered either orally or intravenously, may be curative, although surgical drainage is usually necessary in extensive cases. cat-scratch disease is caused by bartonella henselae infection. the clinical picture includes the sudden appearance of unilateral lymphadenopathy after a scratch from a cat. fever and malaise may be accompanying symptoms in many cases. serologic testing for antibodies to bartonella is diagnostic. cat-scratch disease is usually self-limited, although some benefit has been described with the use of erythromycins and other antibiotics. 21 in the past most mycobacterial infections have been caused by atypical organisms such as mycobacterium avium-intracellulare, m. scrofulaceum, m. bovis, or m. kansasii. in the past decade or so, mycobacterial tuberculosis has made a resurgence as the pathogen responsible for a neck infection. atypical mycobacterial infections present as nontender nodes in the preauricular, intraparotid, or posterior triangle regions. the skin overlying the node typically assumes a violet color, and systemic symptoms are rare. a chest radiograph should be obtained if m. tuberculosis is suspected. the diagnosis is made by obtaining material for acid-fast stain and culture with needle aspiration, surgical drainage, or excision of involved nodes. surgical curettage or total excision is curative for atypical lesions. tuberculosis should be treated with appropriate antituberculin chemotherapy. rarely, the neck may be involved with infections such as tularemia, brucellosis, actinomycosis, plague, histoplasmosis, or toxoplasmosis. inflammatory disorders that may affect the neck include kawasaki syndrome, sarcoidosis, sinus histiocytosis (rosai-dorfman disease), kikuchi-fujimoto disease, and pfapa syndrome (periodic recurrent fever). thyroid malignancies are not uncommon in the adolescent age group, with 10% of thyroid carcinomas occurring in patients younger than 21 years of age. 3 welldifferentiated tumors, usually papillary carcinoma, make up the majority of tumors. follicular, mixed, and medullary tumors occur less commonly. most patients present with a painless midline neck mass. on presentation, cervical adenopathy can be palpated in a majority of patients, a finding that reflects the high incidence of papillary disease that metastasizes via the lymphatics. 9 other important symptoms and signs include a rapid rate of growth, pain, hoarseness, and dysphagia. children who have received prior radiation are at greater risk of thyroid malignancy. the occurrence of thyroid malignancy may be associated with iodine deficiency, hashimoto's thyroiditis, and graves' disease. 9, 22 preoperative assessment should include thyroid nucleotide scanning to distinguish between cold (hypofunctioning) and hot (hyperfunctioning) nodules. up to a third of cold nodules can be malignant, whereas hot nodules are rarely malignant. 12 ultrasonography can distinguish between solid and cystic lesions, and fine-needle aspiration is an alternative to surgical biopsy for diagnosis. surgical management includes near-total or total thyroidectomy, neck dissection if indicated, and postoperative 131 i ablation. lymphoma is a common pediatric malignancy and can present in the neck as painless lymphadenopathy. hodgkin's disease occurs most often in late adolescence and has four histologic subtypes: lymphocyte predominance, nodular sclerosing, mixed cellularity, and lymphocyte depletion. lymphocyte predominance and nodular sclerosing types make up most cases. staging of hodgkin's disease depends on the amount and location of nodal involvement and the presence or absence of systemic or b symptoms (fever, night sweats, weight loss). treatment is with multiple-agent chemotherapy and localized radiation therapy. non-hodgkin's lymphoma can be divided into low-, intermediate-, or high-grade subtypes. high-grade tumors may be further divided into large cell, lymphoblastic, and small cell types. staging of non-hodgkin's lymphoma is by location and extent. treatment is with multiple-agent chemotherapy. langerhans' cell histiocytosis (previously histiocytosis x) includes the disease entities known as eosinophilic granuloma, hand-schã¼ller-christian syndrome, and letterer-siwe disease. the exact nature of this entity remains an enigma: it may represent a neoplasm or a hyperimmune response. 7 symptoms and signs include lymphadenopathy, rashes, otorrhea, oral lesions, and hepatosplenomegaly. diagnosis is dependent on the identification of langerhan's cells on pathologic specimens. treatment ranges from curettage or excision to intralesional or systemic corticosteroids to chemotherapy and radiation therapy. two major categories of neural tumors may be found in the neck. neurofibromatosis is a benign disorder that in some forms (plexiform) may infiltrate surrounding tissues. for this reason, ct and/or mri are vital in the preoperative evaluation of these lesions. surgical resection is the mainstay of treatment. neuroblastoma is a malignancy that develops from neural crest cells and may present as a solitary tumor or as lymphadenopathy. clinical staging determines the mode of therapy that includes surgery, chemotherapy, and radiation therapy. rhabdomyosarcoma rarely presents as a primary tumor in the neck, more often being found as a primary tumor in the orbit, temporal bone, or nasopharynx. the diagnosis is made by biopsy, and patients are staged according to involvement. treatment includes surgery, chemotherapy, and radiation therapy. malignancies of almost any type and location in the body can metastasize to the neck. the most common are thyroid malignancies. in adolescents, carcinomas, especially those arising in the nasopharynx, may spread to the neck lymphatics. anatomy and embryology of the paranasal sinuses otitis media and eustachian tube dysfunction cancer of the thyroid in youth pediatric malignancies neoplasms of the ear and temporal bone congenital malformations of the temporal bone head and neck langerhans' cell histiocytosis otolaryngologic disorders lymphomas of the head and neck thyroid tumors in children corticosteroids in airway management allergic fungal sinusitis: pathophysiology, epidemiology and diagnosis solitary thyroid nodules in 71 children and adolescents glomus tympanicum in infancy grading system for the selection of patients with congenital aural atresia an evidence-based review of the treatment of peritonsillar abscess steroid treatment of laryngotracheitis: a meta-analysis of the evidence from randomized trials childhood epiglottitis in recent years is fetal cellular rhabdomyoma an entity or a differentiated rhabdomyosarcoma? a study of patients with rhabdomyoma of the tongue and sarcoma of the tongue enrolled in the intergroup rhabdomyosarcoma studies i, ii and iii squamous cell carcinoma of the tongue in a nine-year renal transplant survivor: a case report with a discussion of the risk of development of epithelial carcinoma in renal transplant survivors normal polysomnographic values for children and adolescents current knowledge of bartonella species thyroid carcinoma in children and adolescents pediatric audiology efficacy of tonsillectomy for recurrent throat infection in severely affected children: results of parallel randomized and nonrandomized clinical trials first-line treatment of otitis media structure and function of the temporal bone surgical pediatric otolaryngology influence of penicillin-producing staphylococci and the eradication of group a streptococci from the upper respiratory tract by penicillin treatments angiofibroma: changes in staging and treatment regional and intracranial complications of sinuses pediatric neck masses: guidelines for evaluation computed tomography in the evaluation of pediatric neck infections pediatric cochlear implantation key: cord-017518-u2gsa4lg authors: divatia, j. v.; pulinilkunnathil, jacob george; myatra, sheila nainan title: nosocomial infections and ventilator-associated pneumonia in cancer patients date: 2019-07-09 journal: oncologic critical care doi: 10.1007/978-3-319-74588-6_125 sha: doc_id: 17518 cord_uid: u2gsa4lg nosocomial infections or healthcare-acquired infections are a common cause of increased morbidity and mortality among hospitalized patients. cancer patients are at an increased risk for these infections due to their immunosuppressed states. considering these adverse effects on and the socioeconomic burden, efforts should be made to minimize the transmission of these infections and make the hospitals a safer environment. these infection rates can be significantly reduced by the implementing and improving compliance with the “care bundles.” this chapter will address the common nosocomial infections such as ventilator-associated pneumonia (vap), catheter-associated urinary tract infections (cauti), and surgical site infections (ssi), including preventive strategies and care bundles for the same. the term "healthcare-associated infections" (hcais) is commonly used to refer to the whole spectrum of infections that a patient acquires from the healthcare environment including hospitals, intensive care units, hospice, nursing homes, etc. nosocomial infections or hospital-acquired infections (hais) are defined by the centers for disease control and prevention (cdc) as "those infections that were not present in carrier state or incubating state at the time of admission and manifest 48 h after hospital admission" [24] . these infections are often unrelated to the primary cause of hospital admission and can present even after the hospital discharge of the patients [42] . as per the cdc criteria for surveillance, nosocomial infection sites can be of 9 types affecting over 35 infection sites that can be differentiated on the basis of microbiological and clinical criteria [11] . patients in intensive care units (icus) are more vulnerable to nosocomial infections. the extended prevalence of infection in intensive care (epic ii) study showed a prevalence of infections within the icu as high as 51% [82] . nosocomial infections are associated with worse outcomes including increased length of hospital stay, long-term disability, and increased mortality rate, and are associated with increased antibiotic use and antibiotic resistance [42] . due to the multiple risk factors like immunosuppression, disrupted skin and mucosal barriers, recurrent hospital visits, exposure to multiple antibiotics, and the presence of invasive lines and other devices, cancer patients, irrespective of whether they have solid or hematologic malignancies, are at high risk for nosocomial infections [20] . as cancer patients are increasingly being admitted to icus for management of diseaseand treatment-related complications, the incidence of nosocomial infections is also increasing in icus caring for cancer patients [5] . the common nosocomial infections are catheter-related bloodstream infection (crbsi), catheter-associated urinary tract infections (cauti), surgical site infections (ssi), and ventilator-associated pneumonia (vap). this chapter will focus on vap, cauti, and ssi, and central line-related bloodstream infection will be dealt with separately. hospital-acquired infection is common across all parts of the world, with an estimated incidence of 5-10% in developed countries and up to 30% in developing countries [72] . data from the international nosocomial infection control consortium (inicc) suggests that among developing countries, the crbsi rates were 4.1 per 1,000 central venous catheters (cvc)-days, ventilator-associated pneumonia (vap) rates were 12.2 per 1,000 ventilator-days, and the catheter-associated urinary tract infection (cauti) rates were 5.07 per 1000 catheter-days [68] . with increased awareness and constant vigilance, there has been a steady and gradual decrease in the incidence rates of hospital-acquired infections with a 50% reduction in central line-associated bloodstream infections (clabsi) rates and a 17% reduction in surgical site infections (ssi) [13, 68] . table 1 shows the national health safety network (nhsn) and inicc benchmarks for various hospital-acquired infections [23] . although nosocomial infections can be caused by a variety of organisms including bacteria, virus, fungi, and parasites, bacterial infections are the commonest. these agents may be commensals in the patient or may originate from an exogenous source and spread via cross infection. hospital-acquired pathogens are often resistant to most antibiotics (multidrug resistant) or at times extremely drug resistant or pan drug resistant, thereby increasing the treatment costs, antibiotic use, and antibiotic resistance. this is evident from microbiology data demonstrating an increasing incidence of nosocomial infections that are caused by multidrug-resistant bacteria over the years [19, 86] . the common pathogens are gram-negative bacteria including pseudomonas, klebsiella, and acinetobacter and gram-positive bacteria like methicillin-resistant staphylococcus aureus (mrsa), coagulase-negative staphylococci, and enterococci. invasive candidal infections also occur in those with indwelling catheters, lines or contaminated abdominal surgeries [66] . the other common nosocomial organisms are clostridium difficile, vancomycin-resistant enterococci, anaerobes, and enterobacter. nosocomial infections result in an increased mortality and morbidity to the patients with a significant effect on the treatment cost due to the need for higher antibiotics and prolonged icu and hospital length of stay. these infections are responsible for 4-56% of all death causes in neonates in developing countries and 11-25% in the united states [46] . as per the world health organization (who) report, nosocomial infections result in direct financial losses of approximately €7 billion in europe and $6.5 billion in the united states. the risk factors for developing nosocomial infections are: (a) patient factors such as extremes of age, immunosuppression due to malignancy, acquired immunodeficiency syndrome (aids), patients requiring emergency admission to the intensive care unit (icu), duration of stay more than 7 days, chronic illness like renal failure, diabetes mellitus, chronic liver disease, presence of indwelling catheters, ventilation, total parenteral nutrition, trauma, abdominal surgeries, and impaired functional status [44, 45] (b) organizational factors such as the poor environmental hygiene inside the hospital or icu, lack of efficient infection control measures, inadequate manpower such as an inadequate nurse to patient ratio or inadequate waste management staff, and inadequate equipment for patient use (c) iatrogenic factors such as ignorance regarding infection control practices, lack of training in infection control, etc. [41] treatment and prevention of nosocomial infections strategies for the prevention of nosocomial infections as majority of the patient risk factors for developing nosocomial infections cannot be modified, care should be given for focused education and training to the hospital staff, by distribution of education materials regarding healthcareassociated infections and basic infection control policies including identifying the need of isolation, types of isolation, barrier nursing, hand hygiene, etc. [14] an infection control committee should be formed headed by an infection control nurse and hospitalinfection control policies should be laid down. the infection control committee should be entrusted with the responsibility of formulating and implementing "care bundles" for common nosocomial infections that can be adopted from health organizations like cdc, who, etc. and modified as per hospital policy. across the world, implementation of such "bundles of care" and adherence to these bundles have been proven to significantly reduce nosocomial infections, especially in the developing countries [1, 69] . the infection control team should conduct audits and give necessary feedback regarding compliance with hand-washing and other infection control policies. [55] . an antibiotic stewardship program should be initiated with a multidisciplinary team, with members such as an infectious disease specialist, a clinical pharmacist with training in infectious disease, a clinical microbiologist, an information system specialist, and an epidemiologist, with a policy for regulating higher antibiotic prescription. review of practice of antibiotic prescription, ensuring environmental decontamination with surface cleaning, air filtration and decontamination of water source, increasing strength of healthcare personnel (improving nurse to patient ratio and increasing waste management staff), and regular training and feedback to hospital staff are some important measures that can be adopted at an institutional level for reducing the nosocomial infections inside the hospital. the treatment of common nosocomial infections and bundle of cares will be discussed under respective sections in the chapter. previously nosocomial pneumonia was considered as a spectrum of high-risk diseases comprising of ventilator-associated pneumonia, non-ventilator-associated hospital-acquired pneumonia, and healthcare-associated pneumonia. the terminology "healthcare-associated pneumonia (hcap)" was introduced by the infectious diseases society of america (idsa) in 2005 for patients in the community to be considered at high risk for mdr pathogens similar to those associated with hap. these patients, in spite of not being hospitalized, were still considered as high risk in virtue of their interaction with the healthcare system. over years, increasing evidence suggested that this could be a false assumption that also led to inappropriate use of antibiotics [15, 37] . probably this group of patients needs to be considered as a high-risk group when they present to the emergency department with communityacquired pneumonia. hence the current guidelines do not consider hcap as a part of hap [40, 78] . the current idsa guidelines recommend the use of two mutually exclusive termsventilator-associated pneumonia and hospitalacquired pneumoniathereby avoiding the terminology of "non-ventilator-associated hospital-acquired pneumonia." the terminology has been explained in fig. 1 . nosocomial pneumonia (hap and vap) is the most common hospital-acquired infection in the developed world, with a prevalence of 22% [40] . vap contributes almost 50% of all cases of nosocomial pneumonia and is a major cause of increased morbidity and mortality. the attributable mortality rates of vap range from 13% to 15% across both developing and developed countries [57] . although hospital-acquired pneumonia (hap) is generally considered to be less severe than vap, patients who develop complications of hap have mortality rates similar to those of vap. in the icu, data suggests that treatment of vap is the main reason for antibiotic usage, with more than 50% of antibiotic use in icu being for vap [83] . vap also significantly prolongs ventilation days, hospital length of stay, and treatment costs as compared to patients who do not develop vap. ventilator-associated pneumonia (vap) is defined as pneumonia occurring after 48-72 of intubation and ventilation, associated with a new or progressive infiltrate on chest x-ray along with fever, altered leucocyte count, and changes in sputum characteristics for which a definitive causative agent can be found [39] . early vap occurs in the initial days of ventilation (within 48-96 h) and is more likely to be caused by antibiotic-sensitive bacteria. late vap (occurring after 4 days) is likely caused by bacteria which are likely to be multidrug resistant. however, this distinction might not hold true always as patients who are hospitalized for more than 2 days prior to intubation will probably harbor multidrug-resistant bugs [31] . vap is the most common nosocomial infection in patients who are mechanically ventilated with rates of 10% being reported among patients admitted in multiple hospitals across the united states [31, 85] . the international nosocomial infection control consortium (inicc) data from the developing world suggests that the overall vap rate was 12.2 per 1,000 ventilator days with a pooled crude excess mortality of 35.9% [68] . the incidence increases with duration of ventilation. the risk of vap is highest during the initial days of ventilation (3% per day), which gradually decreases over time (2% per day from fifth to tenth day and 1% afterward). older data suggested that most of the vap episodes occurred in the initial part of icu stay itself (early vap) probably because of the increased practices of short-term ventilation in majority of patients [27] . recent studies however suggest the converse with an increase in the late vap rates (as much as 66% of total vap) [32] . the data on vap is difficult to assimilate for surveillance reporting due to the technical issues in diagnosing vap from radiologic criteria alone and in differentiating vap from other conditions such as pulmonary edema or acute respiratory distress syndrome. hence the cdc has laid down a set of epidemiological definitions called ventilator-associated events (vaes). [11] . this is a surveillance system to prevent underreporting of the complications (including vap) occurring in mechanically ventilated patients, irrespective of their origin or mechanism, and should not be used in the clinical management of patients. ventilator-associated events are defined for a period of 2 weeks and require patients to be ventilated for a minimum of 4 days, with at least 2 days of clinical stability, to be assessed for vae. vaes are further classified into ventilatorventilator-associated condition (vac) is defined as 2 days of worsening oxygenation, assessed by an increase in peep requirement more than 3 cm of h2o or an increase infio2 requirement more than 0.2, after an initial clinical stability (of 48 h) or improvement. any vac associated with either a change in temperature or leucocyte count (fever >38 c or hypothermia <36 c, or leukocytosis >12,000/ mm 3 or leukopenia <4,000/mm 3 ) and that requires addition of a new antibiotic for at least 4 days is an infection-related ventilator-associated complication (ivac). an ivac, with a positive microbiological test from respiratory tract specimens, is called possible vap (pvap). a positive microbiological test is defined as a positive microbiological culture in specimens, meeting the threshold of quantitative or semiquantitative culture, without purulent respiratory secretions; or a representative lower respiratory tract sample that is visibly purulent, but the positive culture does not meet the thresholds as per quantitative or semiquantitative criteria; or positive pleural fluid culture or lung tissue culture or a positive test result for legionella or viruses implicated in respiratory diseases. organisms such as candida species, coagulase-negative staphylococcus (cons), and enterococcus species can be considered as positive microbiological test only if isolated from pleural fluid or lung tissue and not from sputum, endotracheal aspirates, bronchoalveolar lavage, or protected specimen brush specimens. positive microbiological test of normal/respiratory flora should be ignored [11] . vap is defined as a pneumonia occurring in a patient on ventilator for at least 2 calendar days before the onset of a vae, with same duration of ventilation, and the patient was on ventilator on the day of the event, or a day prior [11] . a complex interplay between host factors, microbiology of the oropharyngeal flora, and the presence of endotracheal tube is responsible for the development of vap. this is summarized in fig. 2 . after hospitalization and antibiotic administration, the normal flora of the upper respiratory tract is replaced by exogenous aerobic gramnegative flora due to alterations in host defense properties. these organisms colonize in the oropharynx from multiple sources (see table 2 ). the normal cough reflex is hampered by the endotracheal tube, and these pathogens gain access to the lower respiratory tract through micro-aspiration along the endotracheal cuff, aided by the ventilator gas flow. the stomach is an important source of bacterial colonization. change in the acidic ph of the stomach due to drugs favors colonization with these virulent bacteria, and with regurgitation of gastric contents, they pool in the oropharynx and reach the lower respiratory tract by micro-aspirations. the risks are further increased in the absence of adequate cuff seal or with multiple attempts of intubation. these virulent bacteria are usually difficult to treat owing to the thick biofilm that they produce alongside the endotracheal tube. the presence of this biofilm hampers antibiotic penetration and increases antibiotic use and antibiotic resistance [31, 39] . although increased pharyngeal colonization with virulent bacteria, micro-aspiration, and biofilm formation all contribute to the risk of developing vap, it is the host's immune response to these pathogens that determines whether vap will develop or not. immunosuppression is common in critically ill patients due to dysfunction of monocytes and t cells [76] . apart from that, critically ill patients have an over expression of c5a that leads to neutrophil dysfunction and reduced phagocytic activity, again predisposing them to severe infections [31] . other contributory factors to the development of vap include advanced age, emergency intubation for surgery or trauma, severity of illness and organ dysfunction, immunosuppressant drugs, previous antibiotic exposure, presence of nasogastric tubes (resulting in sinusitis), and preexisting illness like diabetes mellitus, chronic lung disease, and chronic renal failure. the etiology for vap varies between icus and hospitals which highlights the importance of knowing local infection and susceptibility patterns. the duration of hospital stays before intubation, length of icu stays, and duration of ventilation are also significant as they determine the nature of flora causing vap (see table 3 ). vap before 4 days (early-onset vap) is often caused by microbes similar to the organisms causing community-acquired pneumonia like streptococcus pneumoniae, haemophilus influenzae, methicillin-sensitive s. aureus (mssa), and susceptible enterobacteriaceae [50] . late-onset vap is usually caused by microbes from the hospital environment. they are usually the aerobic gramnegative bacilli like klebsiella, pseudomonas, acinetobacter, enterobacter, and e. coli, while mrsa is rarely implicated [50] . the odds that they are multidrug resistant is high, and hence these infections are more difficult to treat. this difference between early and late vap may not always be clinically relevant, and there are increasing reports of lack of difference in microbiology and mortality across both groups [21, 26] . patients who are previously exposed to healthcare environment such as those who received antibiotics within the preceding 3 months, those who are currently hospitalized for more than 5 days, those who are on immunosuppressants, or those who have immunosuppressive states such as chronic renal failure, diabetes mellitus, aids, etc. are prone to multidrugresistant infections irrespective of the onset of vap. vap is diagnosed in patients who are being ventilated or was on a ventilator recently and develops signs of infection such as fever or hypothermia, leukocytosis or leukopenia, and worsening in gas exchange along with the appearance of new infiltrates on radiologic imaging and changing nature (increase in purulence) of the tracheobronchial secretions [39] . these signs are highly nonspecific and may be also associated with various noninfectious causes. moreover, the sensitivity and specificity of routine icu x-rays is much lower than the conventional x-rays. interobserver variability in interpreting x-ray findings also affects their accuracy as a diagnostic tool. hence the diagnosis of vap in icu lacks sensitivity and specificity and may result in both over diagnosis or underdiagnosis. however, if the clinical suspicion of pneumonia is high, empiric antibiotics should be administered immediately as delay in antimicrobial treatment leads to increased mortality [38, 49] . to aid in the diagnosis and to rationalize the use of empirical antibiotic therapy for vap, a clinico-radiologic criterion was proposedthe clinical pulmonary infection score (cpis). the cpis (table 4) consists of six clinical and laboratory parameters with scores range from 0 to 12. a score 6 has a sensitivity of 72% and a specificity of 85%, for the presence of vap [60, 89] . although seemingly simple and straightforward, calculation of cpis score also varies substantially from observer to observer, hereby limiting its routine use in clinical trials [89] . the current idsa guidelines suggest the use of clinical criteria rather than cpis score for initiating and stopping of antibiotics [40] . a microbiologic diagnosis can be made by gram staining and culture of the tracheal aspirate or lower respiratory secretions obtained by direct/ non-direct bronchoscopic alveolar lavage (bal). bronchoscopic techniques like bal, mini-bal, and protected specimen brush (psb) specimens provide reliable lower respiratory tract samples, and quantitative cultures of these samples may help to differentiate colonization from true infections. technically it has the advantage of identifying the pathogens correctly, leading to less antibiotic exposure and thereby minimizing antibiotic resistance. however, bronchoscopy and sample collection require expertise and still result in false negative reports. blind sampling requires less expertise and infrastructure and is easy to perform. however, blind sampling is likely to produce false positive results with colonizing organisms, thereby increasing inappropriate antibiotic use and promoting antibiotic resistance. the available data remains conflicting with no benefits of one method over the other with respect to mortality, length of icu stay, and mechanical ventilation days [10, 75] . the idsa recommends blind methods of sample collection, while the european guidelines recommend bronchoscopicdirected methods [40, 78] . the threshold values for cultured specimens recommended by cdc for the diagnosis of pneumonia are mentioned in table 5 . numerous biomarkers for infection/inflammation have been studied in vap including erythrocyte sedimentation rate (esr), c-reactive protein (crp), procalcitonin, pro-adrenomedullin, lps-binding protein, soluble-triggering receptor expressed on myeloid cells (strem-)1, presepsin, etc. other than esr, crp, and procalcitonin, the use of other biomarkers is not widely practiced out of research field [74] . procalcitonin, a precursor hormone of calcitonin, is actively produced by neuroendocrine cells in the lung and intestine on exposure to bacterial endotoxin and inflammatory cytokines. the level peaks at 6 h and may aid in early identification of infections as compared to blood culture. procalcitonin is not useful in cases of viral or fungal infections and in cases of localized bacterial infections. it is also elevated in noninfectious conditions such as burns, major surgery, end-stage renal failure, etc. [84] . procalcitonin testing is expensive and serial measurements make it even more expensive. based on the current data, procalcitonin levels should not be used to rule out an infection or influence the decision of antibiotic initiation. the main role of procalcitonin is in its role as a guide for early stoppage of antibiotics, thereby preventing unwanted exposure to antibiotics [9, 84] . the current guidelines do not recommend the use of these biomarkers over clinical criteria for a diagnosis of vap [40, 78] . idsa recommends coverage for methicillinsensitive staphylococcus aureus and gramnegative bacilli including pseudomonas for patients with suspected vap, pending culture and sensitivity reports [40] . mrsa coverage is not usually required, unless there is an increased risk for mdr organisms such as recent antibiotic exposure, septic shock, acute respiratory distress cpis clinical pulmonary infection score, ards acute respiratory distress syndrome, pao 2 partial pressure of alveolar oxygen, fio 2 fraction of inspired oxygen, cxr chest x-ray table 5 threshold values for cultured specimens used in the diagnosis of pneumonia bronchoalveolar lavage: more than 10 4 colony-forming unit/ml protected specimen brush: more than 10 3 colony-forming unit/ml nondirected bronchoalveolar lavage obtained from (blind) specimens: more than 10 4 colony-forming unit/ml endotracheal aspirate: more than 10 5 cfu/ml open lung biopsy/transthoracic or transbronchial biopsy: more than 10 4 colony-forming unit/g tissue syndrome (ards) prior to the current episode of vap, acute kidney injury requiring renal replacement therapy, or in case of high infection rates of mrsa in the hospital, i.e., >10-20%. in patients without risk factors for gram-negative mdr infection, such as those without any structural lung disease or those who have not received antibiotics in recent past, a single antipseudomonal agent that also covers mssa will be appropriate. only in patients with underlying structural lung disease like bronchiectasis or cystic fibrosis or those having higher risk for mdr infection, dual antipseudomonal coverage should be given [40] . recommended empirical antibiotics are those with antipseudomonal and mssa activity such as ceftazidime, cefepime, piperacillintazobactam, fluoroquinolones such as levofloxacin, carbapenems such as meropenem or imipenem, etc. aminoglycosides are not recommended as monotherapy for vap. in case mrsa is suspected, linezolid or vancomycin may be used. with the increased incidence of infections due to mdr gram-negative pathogens, there has been a resurgence of polymyxins in the treatment of vap. they may be particularly useful in places with increased baseline mdr acinetobacter rates [40] and for empiric therapy in patients with septic shock or high risk such as neutropenic patients or those who have hypersensitivity to beta-lactams. current evidence suggests that different doses or dosage schedules might be required for various bacteria, depending on the pharmacokinetic/pharmacodynamic parameters [28] . the idsa guidelines recommend the addition of nebulized colistin along with intravenous route for managing vap [40] . regarding newer antibiotics, daptomycin is inactivated in the lungs and hence is not recommended for vap. tigecycline monotherapy in the doses as per the label is not recommended for hap or vap [40, 78] . doxycycline and fosfomycin have not been studied for hospital-acquired mrsa as standalone treatments [64, 73] . antibiotics should be changed according to culture and sensitivity reports and may be administered for a total duration of 7 days, or fewer, perhaps guided by procalcitonin levels. the european guidelines for vap recommend a total treatment duration of 7-8 days for all immunocompetent hosts in the absence of complications such as empyema, lung abscess, or necrotizing pneumonia, if initial empiric therapy was adequate and their response to treatment has been good irrespective of the microbiological etiology. patients infected with pseudomonas, carbapenem-resistant enterobacteriaceae, and acinetobacter, those on antibiotics such as tigecycline and colistin, and immunocompromised hosts will require a prolonged duration of treatment [78] . a simplified algorithm for antibiotic selection is shown in fig. 3 guidelines for managing vap and hap were published by the ats and idsa in 2016, while the european respiratory society/european society of intensive care medicine/european society of clinical microbiology and infectious diseases guidelines were published in 2017. while addressing nosocomial pneumonia including hap and vap, both guidelines concur with each other except for a few points. the ats guidelines do not make any new recommendations regarding vap prevention and encourage the use of clinical criteria to decide on initiation and discontinuation of antibiotics rather than use of clinical pulmonary infection score (cpis) score. the ats guidelines recommend the usage of noninvasive or minimally invasive techniques for microbiological investigations of vap. risk factors for mdr pathogens are described by ats as prior hospitalization and organ failure such as septic shock ards and requirement of dialysis prior to vap onset. they recommend combination therapy for target therapy and set a duration of treatment for hap and vap as 7 days. the european guidelines mention prevention strategies for vap but do not make any recommendation on the use of chlorhexidine for selective oropharyngeal decontamination. the european guidelines introduce the concept of "low probability of hap" and suggest the usage of cpis score to identify the same. they endorse invasive sampling over noninvasive sampling, as it might help to avoid overdiagnosis and unnecessary antibiotic exposure. the risk of mdr pathogens is described based upon local prevalence rates of mdr organisms and presence of septic shock. although they recommend 7 days of treatment, they suggested prolonged treatment for selected patients. both guidelines agree on the need of appropriate empiric antibiotic treatment, the need for de-escalation, and the need to minimize antibiotic exposure [40, 54, 78, 79] . the antimicrobial drug concentration in the lung is the most important factor that determines the efficacy of the antibiotic treatment. drugs delivered reach the lung parenchyma by bulk flow, permeation, active transport, and passive diffusion [81] . patient factors such as parenchymal inflammation, volume of distribution, renal function, and drug factors such as water solubility, tissue penetration, molecular weight, inactivation of drug in local site, etc. are important factors in deciding the further efficacy of these drugs. hydrophilic drugs like beta-lactams, aminoglycosides, and colistin attain less concentrations in the lung even after administering of therapeutic dose, while linezolid, fluoroquinolones, and macrolides concentrate well inside the lung. hence the pharmacokinetic (pk) and pharmacodynamic (pd) parameters of drugs should be kept in mind while determining loading dose, dosing frequency, and dosing route. alternate routes such as nebulization may be tried as an additional measure to improve the lung deposition of the antibiotics [36] . the exact time frame of when to expect resolution of symptoms of vap after initiation of treatment is unclear and varies upon the symptoms or signs that are being monitored for resolution. the lack of improvement in clinical condition and/or clinical parameters such as fever, tachypnea, oxygenation, etc. after initiation of treatment can be either due to poor response to treatment/persistence of infection or due to a secondary infection. typically, non-resolving pneumonia is common in elderly patients or in those with comorbidities, underlying immunosuppression, chronic lung disease, or infection with virulent/drug resistant pathogen. treatment factors such as inappropriate initial therapy (either drug or its dose, route, frequency, and duration) are other important factors responsible for vap. workup for non-resolving pneumonia should be undertaken, including microbiological workup for mdr pathogens and imaging for complications such as lung abscess or empyema, while ruling out noninfectious causes of fever and radiologic infiltrates. once an infective etiology is confirmed, optimizing antibiotics as per the pk/pd principles with a hike in antimicrobial coverage will be needed to manage non-resolving pneumonia [51] . infection control programs form the most crucial step in the prevention of vap [69] . vap rates can be reduced by proper decontamination of ventilatory equipment and practice of infection control measures during care of the mechanically ventilated patient. a brief outline of the steps to reduce vap is given below. 1. adherence to the five moments of hand hygiene as recommended by the world health organization (who) [88] . avoiding intubation and re-intubation by the judicious use of noninvasive ventilation and high-flow nasal oxygen helps reduce the risk of development of vap. 2. preferring the use oral route than nasal route for intubation, thereby reducing the chances of nosocomial sinusitis and vap. 3. avoid routine stress ulcer prophylaxis as alteration in gastric ph is associated with increased microbial colonization in the stomach. 4. enteral feeding reduces the gut translocation of endogenous bacteria and reduces bacteremia. caution should be taken to avoid overdistension of the stomach and monitor gastric residual volumes if there are signs of feed intolerance. 5. daily oral hygiene with 0.12-2% chlorhexidine gel reduces the pathological colonization of oral flora. its role is supported by evidence of multiple meta-analyses of randomized controlled trials which are open-labelled trials [47] . role of selective decontamination of the gut is controversial in areas with high antibiotic resistance. elevation of the head end of the bed by 30-45 has shown to reduce vap rates significantly and is recommended by many professional societies [58] . continuous low-pressure suction of the subglottic secretions above the endotracheal cuff is useful [52] . silver-coated endotracheal tubes may prevent bacterial colonization and biofilm formation though the current evidence is weak. [53] iii. vap bundle the term "bundles" in critical care refers to collective group of practice statements, each with high level of evidence in itself; when practiced together, they result in better patient outcome by the consistent delivery of these practices and avoidance of individual preferences or practices [34, 43] . infection surveillance, hand hygiene, semi-recumbent positioning, early extubation, ensuring adequate cuff pressure, and continuous subglottic suctioning have been proven to be simple and efficient methods that help to reduce vap rates significantly [17] . the initial vap bundle suggested by ihi comprised of five components: head end elevation of bed, daily interruption of sedation combined with assessment of the likelihood of weaning, prophylaxis for stress ulcer and deep vein thrombosis, and daily oral care with chlorhexidine [33] . table 6 represents the suggested practice from scottish intensive care society [18] . they differ from the classical institute for healthcare improvement (ihi) vap bundle by not suggesting peptic ulcer prophylaxis or dvt prophylaxis as they have no direct relation with vap rates. similarly, the implementation of a bundle for vap as proposed by the international nosocomial infection control consortium (inicc) also led to substantial reduction in vap rates in multiple countries including india, kuwait, saudi arabia, etc. [1, 2, 56] . the bundle proposed by the inic consortium included the following elements: 1. adherence to guidelines for hand hygiene 2. patient nursing in a semi-recumbent position, with head of the bed elevated at 30-45 3. use of weaning protocols and daily assessment of readiness to wean 4. regular oral care with chlorhexidine 5. minimization of the duration of mechanical ventilation and use of noninvasive ventilation if feasible 6. preferable the orotracheal route instead of nasotracheal route for intubation 7. endotracheal cuff pressure monitoring and attempts to keep it at least 20 cm h2o 8. care of ventilator circuits and removal of condensates from circuits while keeping the ventilator circuit closed 9. avoiding scheduled changes of ventilator circuits and changing them only if they are visibly soiled or malfunctioning 10. prevention against gastric overdistension 11. avoidance of stress ulcer prophylaxis similar to the inicc study, a spanish group reported successful reduction of their vap rates by more than half with the implementation of a vap bundle among icus across the country [3] . the bundle they used is similar to other vap bundles and notably avoided the dvt and peptic ulcer prophylaxis of the ihi recommendation [4] . they had seven mandatory recommendations including staff training in airway management, hand hygiene in airway management, monitoring cuff pressure, chlorhexidine oral care, positioning in bed, striving to reduce ventilator days, and discouraging scheduled changes of ventilator circuits. they also added "highly recommended measures" such as selective decontamination of the digestive tract, subglottic suctioning, and short-course antibiotics for patients intubated with altered sensorium. the data published seemed robust and the reduction in vap rates was sustained and significant. these data suggest that vap bundles are pragmatic, are easy to implement and adhere to, and are effective in reducing the vap rates substantially across the world including developed and developing countries. adapting evidence-based vap bundles that are tailor-made to suit the prevailing practices and hospital policies does not affect the effectiveness of the program [1, 2, 56] . with an increased awareness against vap and with active infection control measures, the incidence of vap is on a declining trend. there has been an increased incidence in hap due to an increased use of noninvasive devices for respiratory support such as noninvasive ventilation and high-flow nasal oxygen [65] . currently hap is one of the leading causes of nosocomial infections that in turn leads to prolonged hospital stay and increased treatment costs. a recent study found that the incidence of hap is 3.63 per 1000 patient days, occurring in both wards and the intensive care units [25] . hap is classified into icu acquired and non-icu acquired hap, with icu acquired hap having an increased incidence of mdr pathogens increased incidence of septic shock, and worse outcomes as compared to non-icu hap [59] . the term "non-icu acquired pneumonia (niap)" has been recently proposed and refers to a specific subset of hap patients who developed pneumonia outside icu and has an estimated incidence of 1.6-3.7 cases per 1000 admissions [62] . hap differs from vap with respect to the microbiology, diagnostic investigations, and mortality. the microbiological diagnosis is by culture of a pathogen identified from a representative sputum sample. there are no data to suggest invasive sampling techniques like bronchoscopy over simple sputum collection; rather some data suggest that they may be harmful and do not improve outcomes [29] . the use of rapid diagnostic methods such as polymerized chain reactionbased technologies in hap has yielded promising results especially in the choice of antibiotics and detection of antibiotic resistance. however further studies are needed to confirm the benefits of such systems over the possible disadvantages such as false diagnosis of colonization [65] . the microbiology of hap differs from vap, and there seems to be an increased incidence of s. pneumoniae and respiratory viruses and a lower incidence of mdr gram-negative pathogens [59] . the ats/idsa guidelines recommend antipseudomonal therapy for most patients with hap. this may lead to unnecessary use of broad spectrum antibiotic therapy [22] . the ers guidelines suggest that antipseudomonal treatment is not necessary for initial empiric treatment of most patients with hap, in the absence of risk factors or septic shock [78] . thus, hap patients need to be treated based on factors such as local epidemiology, surveillance cultures, presence or absence of mdr risk factors, and septic shock [59, 65] . infections involving any part of the urinary system, from the urethra to kidney, are labelled as urinary tract infections. they comprise more than one third of all nosocomial infection. in the intensive care units, uti comprises of 8-21% of all nosocomial infections and is the third most common nosocomial infection occurring in the icu [12] . icu patients require a catheter for reasons such as immobility, strict intake output charting, etc. and retain it for prolonged duration. each catheter day is associated with a 3-7% increase in the risk of acquiring a catheter-associated urinary tract infection (cauti). the cdc guidelines classify cauti as symptomatic urinary tract infection or asymptomatic urinary tract infection as follows: cauti is an infection occurring in a patient with an indwelling catheter of more than 48 h duration before the event, with the catheter remaining in situ or removed 24 h prior at the time of the event. signs and symptoms of infection such as fever, suprapubic tenderness, loin pain, andin those patients without the catheterincreased urinary frequency, urgency, and dysuria and a significant bacteriuria should be present [12] . significant bacteriuria is defined as a urine culture with no more than two species of organisms identified, of which at least one of which is a bacterium which has a colony-forming unit count more than 10 5 cfu/ml [12] . the most common pathogens associated with cauti are enterobacteriaceae. in the setting of icus, candida, enterococcus, pseudomonas, klebsiella, and e. coli become increasingly prevalent and are often drug resistant [16] . cauti as well as other nosocomial infections prolongs hospital stay, increases treatment cost, and increases mortality. as with an endotracheal tube for vap, the indwelling catheter is the main risk factor for uti. other risk factors include female sex, severity of current illness, age greater than 50 years, presence of diabetes mellitus, altered rft with serum creatinine level more than 2 mg/dl, location of catheter insertion, nonadherence to aseptic precautions of catheter care, etc. [16] . laboratory examination will demonstrate pyuria irrespective of symptoms and urine wbc >10 cells/microl. quantitative urine wbc >10 cells/microl has low sensitivity but retains high specificity for the likelihood of getting a positive microbiological culture. a proper sample of urine should be sent for culture when cauti is for obtaining culture results. samples should be collected from the "needleless site" after applying aseptic precautions or with a needle from the aspiration port. the idea of changing the catheter before sample collection has been suggested in some studies but currently cannot be recommended [71] . the empiric antimicrobial therapy for cauti depends on the presentation, i.e., whether they are symptomatic/asymptomatic and also upon the complications if any. antibiotics for asymptomatic bacteriuria do not prevent the progression to symptomatic cauti nor its complications. as the risk of antibiotic resistance is high, patients with asymptomatic bacteriuria are usually not treated with antibiotics except in pregnancy and in patients undergoing surgical procedures of the lower urinary tract [48] . for symptomatic bacteriuria, the choice of antibiotic will depend on patient's risk factors for mdr infection and ongoing antibiotics. a 7-to 14day duration of intravenous antibiotic therapy is generally advocated and can be switched to oral route as per the sensitivity reports if the patient can tolerate oral medications [30] . candiduria is a common occurrence among hospitalized patients, with candida being isolated from almost one third of the total samples among hospitalized patients. candiduria with symptoms and signs of infection including fever, leukocytosis or leukopenia, shock, etc. should be evaluated for disseminated candidiasis. treatment for otherwise asymptomatic candiduria is not required except in the high-risk population such as neutropenia or urinary tract instrumentation. imaging should be obtained in patients with diabetes mellitus or urinary tract abnormalities in case of persistent candiduria as they have a high risk for developing fungal balls. treatment should be guided by the culture and sensitivity reports. fluconazole 200-400 mg daily is recommended for susceptible strains for a total duration of 7-14 days. fluconazole-resistant strains should be treated with amphotericin b (0.3-0.6 mg/kg per day) for 7 days. lipid formulations of amphotericin b do not penetrate the kidney and cannot be used for the treatment of fungal cauti. the data on efficacy of echinocandins is still evolving, and the preliminary data shows clearance of candiduria with micafungin. further data is required for making any recommendation [61] . strategies for reducing cauti include strict aseptic techniques including hand hygiene for insertion and maintenance of catheters and maintaining a closed drainage system, monitoring the insertion of urinary catheters for appropriate indications, development of cauti bundles [35] (table 7) for placement, daily check list, early removal, encouraging other alternatives such as intermittent catheterization, and condom catheter. a recently conducted study in the united states demonstrated that a cauti bundle could reduce the catheter use and cauti rates in non-icu acute care settings [70] . other methods proposed to reduce the incidence of cauti were to use urinary catheters coated with antibiotics or to use urinary catheters with silver impregnated in it. silver compound was found to reduce biofilm formation on the catheter significantly. however, the use of both types of catheter was not associated with a reduction in uti rates or any other meaningful benefit [63] . surgical site infections (ssis) are those occurring at the incision site and/or extending to deeper tissue spaces or adjacent organs within 30 days of a surgery or within 90 days if the procedure involved prosthetic material implants. they are further classified into superficial ssi, deep ssi, and organ/space ssi (table 8 ) [8] . these are the most common healthcareassociated infections in patients undergoing surgery, with an incidence of about 38% [87] . studies have estimated that more than half of these infections are preventable, if appropriate measures were taken [80] . the most common organisms responsible for ssi are staphylococcus aureus (mrsa and mssa), e. coli, coagulase-negative staphylococci (cons), pseudomonas, etc. [77] . the inicc data from developing countries shows a significantly higher incidence of ssi, as compared to the data from developed countries. the incidence rates reported by the inicc group are 2.6% after hip prosthesis, 4.5% after cardiac surgeries, 2.7% in abdominal hysterectomy, 4.1% in other abdominal surgery, and 12.9% after ventricular shunt [67] . patients at risk include those who are elderly patients; those with history of skin or soft tissue infection, recent radiotherapy, diabetes, obesity, alcoholism, and preoperative hypoalbuminemia; those who are current smoker; or those having immunosuppression. other risk factors include emergency procedure, wounds of increasing complexity, prolonged surgeries, contaminated environmental surfaces, lack of strict asepsis in the operating room, inappropriate antibiotic with respect to choice/timing/weight-based dosing, impaired glycemic control, etc. [6] . maintaining strict asepsis during wound handling and timely administration of the correct antibiotics in appropriate doses are the most important factors to prevent ssi. other measures such as hand hygiene, skin antisepsis, avoiding shaving of hair (if necessary, to clip), and use of double gloves and other barrier devices are also recommended by various societies for reducing ssi. surgical antimicrobial prophylaxis (amp) is the administration of a short-course antibiotic to reduce the microbial burden at the time of skin incision. an ideal amp program has to select the antibiotics that are active against the likely pathogens at the surgical site and administer the optimum dose at correct time, so that adequate serum and tissue concentrations are achieved. it is recommended that the full dosa should be administered within 60 min of the surgical incision and re-dosed as per the half-life of the drug or in case of blood loss more than one third of circulating blood volume. cefazolin as a single agent is the recommended drug of choice for cardiothoracic and upper gastrointestinal surgeries, surgery of non-obstructed small bowel, cesarean section, orthopedic surgery, spinal surgery, and neurosurgery. in patients with allergy to cefazolin, aminoglycosides such as gentamycin may be used. infective endocarditis prophylaxis is restricted to specific procedures in patients with few high-risk cardiac conditions. the cdc recommendation for prevention of ssi is outlined in table 9 [7] . table 9 strong recommendations from cdc regarding prevention of ssi bath with water and soap on the night prior to surgery administer amp only if indicated, and amp to be administered in the correct time and correct dose such that optimal bactericidal concentration of the agents is established in the serum and tissues when the incision is made administer amp for all caesarean section procedures use an alcohol-based antiseptic agent for surgical site preparation unless otherwise specified avoid application of all antimicrobial agents including ointments, solutions, or powders to the surgical incision use of antibiotic (triclosan)-coated sutures strict glycemic control in the perioperative period with target blood glucose levels less than 200 mg/dl in all patients irrespective of diabetic status maintain perioperative normothermia optimize tissue oxygenation by maintaining normothermia and euvolumia provide an increased fio 2 during surgery and in the immediate postoperative period after extubation transfuse blood and blood products as per transfusion thresholds avoid additional amp after closure of the surgical incision in the operating room cdc centre for disease control and prevention, amp antimicrobial prophylaxis nosocomial infections increase patient's morbidity, hospital and icu length of stay, treatment costs, and mortality. they are also responsible for increased antibiotic use, leading to antibiotic resistance and outbreaks of multidrug-resistant infections. implementing and enforcing infection control measures is the pivotal step toward curbing the nosocomial infection. increased awareness, health education, and adhering to care bundles have been proved to be efficacious in reducing nosocomial infections. impact of the international nosocomial infection control consortium (inicc)'s multidimensional approach on rates 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in patient safety for four common conditions nosocomial infections in the icu: the growing importance of antibioticresistant pathogens surgical site infections: control., causative pathogens and associated outcomes who|my 5 moments for hand hygiene. who. world health organization ventilator-associated pneumonia: the clinical pulmonary infection score as a surrogate for diagnostics and outcome key: cord-006464-s8rjoyse authors: bauer, michael; kölsch, uwe; krüger, renate; unterwalder, nadine; hameister, karin; kaiser, fabian marc; vignoli, aglaia; rossi, rainer; botella, maria pilar; budisteanu, magdalena; rosello, monica; orellana, carmen; tejada, maria isabel; papuc, sorina mihaela; patat, oliver; julia, sophie; touraine, renaud; gomes, thusari; wenner, kirsten; xu, xiu; afenjar, alexandra; toutain, annick; philip, nicole; jezela-stanek, aleksandra; gortner, ludwig; martinez, francisco; echenne, bernard; wahn, volker; meisel, christian; wieczorek, dagmar; el-chehadeh, salima; van esch, hilde; von bernuth, horst title: infectious and immunologic phenotype of mecp2 duplication syndrome date: 2015-02-27 journal: j clin immunol doi: 10.1007/s10875-015-0129-5 sha: doc_id: 6464 cord_uid: s8rjoyse mecp2 (methyl cpg binding protein 2) duplication causes syndromic intellectual disability. patients often suffer from life-threatening infections, suggesting an additional immunodeficiency. we describe for the first time the detailed infectious and immunological phenotype of mecp2 duplication syndrome. 17/27 analyzed patients suffered from pneumonia, 5/27 from at least one episode of sepsis. encapsulated bacteria (s.pneumoniae, h.influenzae) were frequently isolated. t-cell immunity showed no gross abnormalities in 14/14 patients and ifny-secretion upon cona-stimulation was not decreased in 6/7 patients. in 6/21 patients igg(2)-deficiency was detected – in 4/21 patients accompanied by iga-deficiency, 10/21 patients showed low antibody titers against pneumococci. supra-normal igg(1)-levels were detected in 11/21 patients and supra-normal igg(3)-levels were seen in 8/21 patients – in 6 of the patients as combined elevation of igg(1) and igg(3). three of the four patients with iga/igg(2)-deficiency developed multiple severe infections. upon infections pronounced acute-phase responses were common: 7/10 patients showed crp values above 200 mg/l. our data for the first time show systematically that increased susceptibility to infections in mecp2 duplication syndrome is associated with iga/igg(2)-deficiency, low antibody titers against pneumococci and elevated acute-phase responses. so patients with mecp2 duplication syndrome and low iga/igg(2) may benefit from prophylactic substitution of siga and igg. electronic supplementary material: the online version of this article (doi:10.1007/s10875-015-0129-5) contains supplementary material, which is available to authorized users. mecp2 duplication on chromosome xq28 (#300260) causes a severe form of x-linked intellectual disability (xlid). it was first clinically described as lubs syndrome, the underlying genetic condition was identified in 2005 [1] [2] [3] . up to now more than 200 patients have been described . the exact prevalence of mecp2 duplication syndrome is still unknown, but in synopsis of different screening attempts it is estimated that mecp2 duplication syndrome may explain~1 % of cases of severe xlid [51] . the core phenotype of mecp2 duplication syndrome includes infantile hypotonia, mild dysmorphic features, developmental delay/severe to profound intellectual disabilty and absent to minimal speech [51] . spasticity, ataxia and autism are facultative clinical signs that occur in different combinations and to different degrees. since the description of the first individuals it became evident that besides the striking neurological phenotype patients with mecp2 duplication syndrome are at increased risk for severe infections. about 70-75 % of the affected individuals are reported to develop recurrent infections, especially of the respiratory tract. respiratory infections are the main cause of early death reported among the population [51, 52] . however a detailed description of the infectious phenotype and an investigation of the immunological phenotype based on a large cohort has been missing to date. suspecting an immune dysfunction in patients with mecp2 duplication syndrome we assessed the infectious and immunological phenotype of a cohort of 30 patients. we here provide for the first time a detailed description of the type of infections, the pathogens isolated and the core cellular and humoral immunological phenotype in patients with mecp2 duplication syndrome. patients the current study was conducted in accordance with the helsinki declaration, with informed consent obtained from each patient or the patient's family. samples from the patients were used in this study with approval from the local ethics committee of the charité (approval # ea2/063/12). all patients enrolled in this study show a genetically confirmed duplication of at least mecp2 and irak1. the diagnostics were performed prior to this study and the results were submitted to us by the performing laboratories or geneticists. in patients p1, p2, p8, p9, p10, p11, p12, p13, p14, p17, p19, p20, p21, p25, p27, p28, p29 and p30 the duplication had been detected by use of array-cgh thus revealing the actual size of the duplication on xq28. in patient p15 quantitative pcr had been performed and in patients p3, p4, p5, p6, p7, p18, p23, p24 and p26 multiplexligation probe amplification was used for the diagnosis of mecp2 duplication and single neighbouring genes, e.g., irak1. so in patients p15, p3, p4, p5, p6, p7 and p18, p23, p24 and p26 the actual size of the duplication is unknown. the following patients have been described previously: p1, p2 in bartsch et al. [21] , p3, p4, p5, p6, p7 in echenne et al. [18] , p8 in budisteanu et al. [31] , p9 in jezela-stanek et al. [28] , p10 in mayo et al. [32] , p12 in grasshoff et al. [30] , p13 in bauters et al. [9] , p15 in meins et al. [2] , p17 in madrigal et al. [8] , p20, p21 in xu et al. [43] , and p22, p27 in vignoli et al. [40] . for detailed molecular analyses of these patients see the corresponding publications. patients p11, p14, p16, p18, p19, p23, p24, p25, p26, p28, p29 and p30 have not been published before. for molecular characterization of these patients see supplementary table 1. blood samples of the patients venous punctures were performed in parallel to routine blood tests. the blood samples were sent to our laboratory to ensure that all patients' samples were analyzed with the same methods and with the same laboratory equipment. assessment of the infectious phenotype a detailed questionnaire on the clinical phenotype was completed by the physicians caring for the patients with mecp2 duplication syndrome and sent to two of the authors (mb and hvb) for thorough review. against tetanus toxoid and pneumococci total immunoglobulin levels and immunoglobulin subclasses of all patients' samples in our cohort were measured by eclia on a cobas 6000 (roche, switzerland) in our laboratory. antigen-specific igg antibodies against tetanus-toxoid and pcp as well as anti-pcp-igg 2 of all patients' samples in our cohort were measured by elisa according to manufacturer's protocols (mk013, mk012 and mk010, the binding site) in our laboratory. lymphocyte subsets of patients' edta-blood were stained with fluorescence-labelled monoclonal antibodies against cd14 (rmo52), cd56 (n901), cd16 (3g8), cd4 (sfci12t4d11), cd19 (j3-119), cd8 (sfci21thy2d3), cd3 (uch-t1), cd45 (j33), cd45r0 (uchl1), tcrαβ (ip26a), tcrγδ (immu510) and cd45ra (2h4ldh11ldb9) (all beckman coulter, usa) for 15 min at room temperature and were measured and analyzed after erythrocyte lysis (versa-lyse, beckman coulter) on a navios-facs (beckman coulter, usa) in our laboratory. lymphocyte proliferation assay lymphocyte proliferative responses were tested according to standard protocols in heparinised whole blood and were stimulated with phytohemagglutinin (pha, sigma-aldrich, l1668), plate bound anti-cd3 (3, 10, and 30 μg/ml; clone hit3a, becton dicinson), pokeweed-mitogen (pwm, 1, 5, and 15 μg/ml, sigma-aldrich, l-9379), il-2 (20 ng/ml, #200-02, peprotech, rocky hill/nj, usa) and staphylococcus aureus cowan-1 strain (sac, pansorbin, 1:1000, 1:10,000, merck, darmstadt, germany) for 72 h and with tetanus-toxoid rt50 (40, 20, and 10 lf/ml, staten serum inst. copenhagen), candida albicans antigen (0.025, 0.25, and 2.5 μg/ml; can001-p; biologo, kronshagen, germany) and diphtheria-toxoid (20 lf/ml, chiron-behring) for 120 h. after pulsing with 1 μci [ 3 h]thymidine per well during the last 12 h incorporated [ 3 h]thymidine was measured on a β-scintillation counter (microbeta 1450 trilux, perkinelmer). assessment of ifnγ after stimulation with concanavalina (cona) heparinised blood was stimulated 1:5 diluted in rpmi with 50 μg/ ml cona (sigma-aldrich, #7246) and ifny was assessed by th1/th2-cytokine detection kit (becton dicinson biosciences, #550749) according to the manufacturers protocol. measurement was performed on a facs-navios (beckman coulter). for analysis fcap-array-software (soft flow hungary) was used. the normal range of ifny-secretion upon activation with cona after 4 h of activation was assessed based on the activation of 50 healthy donors (25 male and 25 female). the normal range of ifny-secretion upon activation with cona after 24 h of activation was assessed based on the activation of 10 healthy male donors. cohort, genotypes and infectious phenotype 30 patients with mecp2 duplication syndrome from germany, france, italy, poland, romania, spain, the netherlands, belgium, usa and china were enrolled in the study (28 male patients and two female patients). all patients showed a duplication of mecp2 and neighbouring irak1 (fig. 1 ). patients' age ranged from 3 to 48 years. we had access to the history of 27 of the 30 patients with 343 cumulative patient-years. the occurrence of infections varied in our cohort of patients: in 9/ 27 patients (including the two girls) no severe infections were reported at all. 18/27 patients developed at least one severe infection requiring intravenous antibiotic treatment. pneumonia occurred in 17/27 patients. regarding the type of pneumonia, complete assessment was possible in patient p14 and in this patient all pneumonias were x-ray-proven bronchopneumoniae. 5/27 patients developed at least one episode of sepsis. urinary tract infections were reported in 6/27 patients. recurrent fever of unknown origin occurred in three patients. no meningitis was diagnosed in our cohort. the frequency of severe infections also varied with a maximum of 27 episodes requiring intravenous antibiotic treatment since birth in a 16-year old boy (p1). two patients died of pneumonia during the course of the study (table 1) . of the 25 different pathogens detected in the patients, the majority were bacteria with 84 %. only 3 viruses (12 %) and 1 fungus (4 %) were among the 25 pathogens. of the 21 different bacteria, 7 bacteria were gram-positive (33 %) and 14 bacteria were gram-negative (66 %). thirteen of the 21 isolated bacteria (61.9 %) are capable of building a capsule (a.hydrophila [53] , b.fragilis [54] [55] [56] , e.coli [57] , h.influenzae [57] , k.oxytoca [58] , k.pneumonia [59] , p.mirabilis [60] , p.aeruginosa [59, 61] , s.aureus [62] [63] [64] [65] , s.epidermidis [66, 67] , s.agalactiae [57] , streptococcus group a [68] [69] [70] , s.pneumoniae [57, 64] ). pathogens were isolated in pharyngeal swab, sputum, bronchoalveolar lavage, bronchial secretion/aspirate, tracheal secretion, ear swab, urine, stool, peritoneal liquid and blood. when considering the isolation of pathogens in these biological materials altogether, the pathogens isolated in most patients were s.pneumoniae (5/27) and h.influenzae (5/27) -both are typical encapsulated bacteria [64] . in 4/27 patients e. coli, in 4/27 c.albicans, in 3/27 patients s.aureus, in 3/27 p.aeruginosa, in 2/27 p.mirabilis and in 2/27 streptococcus group a were isolated. s.agalactiae, streptococcus group c, streptococcus group f, s.epidermidis, k.pneumonia, k.oxytoca, m. tuberculosis, o.anthropi, a.hydrophila, c.freundii, b.fragilis, s.enteritidis, s.marcescens and s.plymuthica were each detected in one patient. influenza a, respiratory-syncytial-virus and rhinovirus were also each isolated in only one patient (fig. 2a) . when considering only the isolates in biological material of the respiratory tract, the pathogens isolated in most patients were h. influenzae (4/27) and c.albicans (4/27) followed by s.pneumoniae (3/27) (fig. 2a) . the spectrum of pathogens isolated in blood included s.pneumoniae, h.influenzae, s.aureus, s.epidermidis and c.albicans, each only isolated in one patient (fig. 2b ). pathogens isolated in urine were p.mirabilis (in 2/27 patients), e.coli (in 1/27 patients) and s.pneumoniae (in 1/27 patients) (fig. 2b) . neither infections by pneumocystis jirovecii, nor invasive infections by (supplementary table 3a and supplementary table 3b) . lymphocyte proliferation upon stimulation with mitogens (pha, pwm) was normal in 13 patients tested. stimulation with il-2 showed attenuated responses in 2/13 patients and stimulation with anti-cd3 revealed a lymphocyte response below reference value in 1/13 patients. when stimulating cells of the 13 patients with sac, 3 patients showed reduced lymphocyte response. attenuated lymphocyte proliferation was also observed upon stimulation with candidaantigen (3 of 13 patients tested), diphtheria-antigen (5 of 13 patients tested) and tetanus toxoid antigen (2 of 13 patients tested) (supplementary table 4) . we tested ifny-secretion upon stimulation with cona for 24 h in whole blood of 7 patients. we observed a normal production of ifny in blood that was stimulated soon after the blood withdrawal (n=1). in blood that had travelled, 4 patients showed an ifny-level after stimulation that was within the reference values of healthy controls. the ifny-level of one patient was above the reference value and the ifny-level of one patient was below reference values. so in total in 6/7 patients tested ifny-production upon stimulation with cona was not impaired (fig. 3) . in summary lymphocyte subsets and lymphocyte proliferation assays show no gross abnormalities and ifny-production upon stimulation with cona seems to be normal. of 30 patients enrolled we could assess immunoglobulin levels (igg, iga and igm), and levels of igg subclasses (igg 1 -igg 4 ) and specific antibodies against tetanus and pneumococci in 21 patients. this revealed six patients with igg 2 age, sex, number of infections requiring intravenous antibiotic treatment, occurence of invasive versus non-invasive infections and outcome. order of patients runs from the most often infected to the least often infected deficiencyfour of them with an additional iga-deficiency. one of the four patients with iga/igg 2 -deficiency had globalized low levels also for igg, igg 1, igg 3, igg 4 and igm. additionally igg 2 was in low normal range in 3 further patients. igg 4 was below reference values in 4 patients, all of which also had igg 2 -deficiency. igg 1 was above age-dependent reference values in 11/21 and igg 3 was above age-dependent reference values in 8/21 patients; 6/21 patients showed supra-normal values for igg 1 and igg 3 ( fig. 4a-g) , igg 2 -antibodies against s.pneumoniae were below normal values in 4 patients, in whom also igg 2 was low. additionally s.pneumoniae igg 2antibodies were in the lower range of normal in 6 further patients. so in total 10/21 patients showed low antibody titers against pneumococci (fig. 5) . of the 21 patients in whom we assessed the levels of s.pneumoniae igg 2 -antibodies, 8 patients had received vaccination against pneumococci, 8 patients had not received vaccination and in five patients information on vaccination status was not available. interestingly s.pneumoniae igg 2 -antibodies were low even in the patients with vaccination against pneumococci (fig. 5 ). both pneumococcal polysaccharide vaccines and pneumococcal conjugate vaccines have been used in the 8 patients with pneumococcal vaccination and 7 of the 8 patients have received several pneumococcal vaccinations. the period between last vaccination and assessment of apcp-igg 2 ranged from 1 month up to 7 years and 6 month. it is noteworthy that even in the patient with the short interval of 1 month between last vaccination and ascertainment of antibody titers no appropriate antibody production against s. pneumoniae was determined after three vaccinations (table 2 ). in contrast to this rather common impairement of igg 2 formation against pneumococci iggantibodies against tetanus-toxoid were below age-dependent reference values in only 2/21 patients and low in normal range in 2 further patients, so normal in 19/21 patients (supplementary figure 2 ). in summary 6/21 patients show igg 2 -deficiencyfour of them with an additional iga-deficiency -and half of the patients (10/21) show antibodies against pneumococci either below normal range or in the low range of normal. acute-phase responses as infections in patients with mecp2 duplication syndrome have not only been reported for their high frequency but also for the severity of the single infectious episode we systematically assessed acute-phase responses in these patients. data on acute-phase responses was available for 10 patients. 7/10 patients experienced non-invasive infections with maximal crp values above 200 mg/l. in 4/10 patients crp values above 300 mg/l were reported during non invasive infections. only one patient (p16) showed no elevated crp values upon non-invasive infections. the mean value of all reported maximal crp values during non-invasive infections of all patients was 157,58 mg/l (fig. 6a) . the maximal temperature measured during non-invasive infections ranged from 38.5 to 41.6°c and the mean value measured during non-invasive infections of all patients was 39.75°c (fig. 6b ). 2/10 patients experienced non-invasive infections with leucocyte counts above 30/nl and 8/10 patients showed leucocyte counts above 20/nl during single infectious episodes. mean of all reported maximal leucocyte values was 15.69/nl (fig. 6c) . 5/ 10 patients showed a neutrophil count above 15/nl during a non-invasive infection. the mean value of the maximal neutrophil count for all patients was 10.51/nl (fig. 6d) . taken together these data indicate a strong acute-phase response in patients with mecp2 duplication syndrome. the duplication of mecp2 on chromosome xq28 leads to a rather common syndromic form of xlid (estimated~1 % of x-linked patients). besides the neurological phenotype it was soon noted that many of the patients suffer from recurrent infections [51, 52] . we here describe for the first time, systematically the infectious and immunological phenotype of mecp2 duplication syndrome based on data derived from a standardized study-questionnaire filled out by the physicians together with the parents of 30 patients. our findings show that patients with mecp2 duplication syndrome are at an increased risk for infections by bacteria that are able to build capsules (61.9 % of isolated pathogens); in particular for infections by s.pneumoniae and h.influenzae. in our cohort we have not observed increased susceptibility to mycobacteria, pneumocystis spp, fungi and viruses. we could not identify a correlation between the size of the duplication (genotype) and the infectious or immunological phenotype. the predominance of respiratory and severe invasive infections caused by bacteria that are able to build capsules, suggested an impairment of humoral immunology. igg 2 -deficiency was detected in 6/21 analyzed patients -in 4/6 patients accompanied by iga-deficiency, while 15 out of 21 analyzed patients showed no igg2 or iga deficiency. igg 2deficiency in the 6/21 patients and low antibody titers against pneumococci in 10/21 patients suggest that among patients with mecp2 duplication humoral impairement is more common than in the normal population. 5/6 patients with igg 2deficiency also developed severe infections. as 3/4 individuals with combined iga/igg 2 -deficiency additionally suffered from a very high frequency of infections and developed the most severe infectious phenotype in our cohort there seems at least an association of iga-/igg 2 -deficiency with severe infections. in line with this hypothesis only 2/15 patients without iga or igg 2 deficiency developed recurrent severe infections comparable to the phenotype observed in the patients with iga/igg 2 -deficiency. one of them showed low igg 2 -antibodies against pneumococci even after vaccination indicating an inability to mount an appropriate antibody production although global levels of immunoglobulins are normal in this patient. the other patient (p9) with a severe infectious phenotype despite having an iga-/igg 2 -deficiency showed massively elevated igg (32 g/l). given the fact that this patient has a persistently raised leucocyte count, it seems plausible that he additionally suffers from an unknown chronic inflammatory process. 7 of the 15 patients without iga or igg 2 deficiency developed few infections and 2 of the 15 patients developed no infections. in 4 of the 15 patients without iga or igg2 deficiency the severity of the infectious phenotype could not be assessed as the patient was either still at young age or because the history could not be completed appropriately. the single patient who has to date not developed severe infections despite we further investigated whether patients with mecp2 duplication syndrome showed stronger acute phase responses, which we could confirm in 7/10 patients in terms of elevated crp values above 200 mg/l during non-invasive infections, mainly pneumoniae. this observation raises the question if the genetic alterations in patients with mecp2 duplication syndrome lead to a hyper-inflammatory immune response. because all patients show a duplication on xq28 that includes mecp2 and neighbouring irak1 and given the fact that these two genes were assumed to be the minimal critical region leading to the phenotype of mecp2 duplication syndrome until recently [15, 17, 51] , it seems plausible to hypothesize a role of irak1 in the pathogenesis of strong acute-phase responses. irak1 encodes for a protein in the tlr/il1rsignalling pathway and it could be assumed that its overexpression leads to stronger or constantly triggered nfκbmediated pro-inflammatory signalling and to a hyperinflammatory immune response. in our opinion recently published results suggesting normal tlr-mediated signalling in pbmcs of patients with mecp2/irak1 duplication do not sufficiently falsify this hypothesis as these experiments were performed in pbmcs (without granulocytes) and as it remains unclear whether lps-stimulation was performed with or without serum [44] . so it seems at least possible that the previously described similar production of cytokines upon activation with tlr-agonists in patients and controls in these experiments is due to the insufficient stimulation of cells or a lack of stimulation of granulocytes or both. so to date it remains an open question whether the duplication of irak1 renders patients with mecp2 duplication prone to strong inflammatory responses. it has been published that mecp2 duplication causes a lack of th1-differentiation, elevated levels of cd4+cd45ra+ naïve/ lowered levels of cd4+cd45r0+ memory t-cells and impaired ifnγ-secretion by t-cells in mice and in patients with mecp2 duplication. it was concluded that this lack of th1 differentiation/ ifnγ-secretion is the major cause for the increased susceptibility for infections in patients with mecp2 duplication [44] . these observations are partially in line with ours. also in our cohort 5/14 patients showed elevated levels of cd4+cd45ra+ naïve/ lowered levels of cd4+cd45r0+ memory t-cells. the observed ratio of cd4+cd45ra naïve versus cd4+cd45r0+ memory t-cells in our cohort is >1 in 11/14 patients and < 1 in 3/14 patients. however our data do not fully confirm the hypothesis of an impaired ifnγsecretion as sufficient explanation for the increased susceptibility to infections: 1. upon cona-stimulation we observed a normal ifnγ-production in 5/7 patients, an even stronger ifnγ-production in one patient and only a slightly impaired ifnγ-production in one patient compared to healthy controls (fig. 3) ; 2. if the observed reduction of ifnγ-secretion in patients with mecp2 duplication was causative for severe infections one would expect disseminated and/or invasive infections with atypical mycobacteria and s.enteritidis as described for molecular precisely-defined partial and complete defects in at least 9 genes leading to the impairment of ifnγreceptor signalling or ifnγ-secretion [71] [72] [73] [74] ; 3. conversely during a total of 343 patient years in our study only one patient (p9) developed a single, non-complicated infection by m.tuberculosis, which remained restricted to the lung and was successfully treated by standard therapy. in a patient with a strongly relevant impairement of ifnγ-secretion an infection with m.tuberculosis would have taken a severe course [71] [72] [73] [74] . interestingly however 13/21 patients developed either supra-normal values of igg 1 of igg 3 or of both, igg 1 and igg 3 . this observation suggests a direct or indirect effect of mecp2 duplication on the expression of igg 3 and igg 1 at the γ3/γ1 locus and of iga, igg 2 and igg 4 at the α1/γ2/γ4 locus. it has been shown that ifny stimulates the expression of the igg 2a -isotype and inhibits the production of igg 3 , igg 1 , igg 2b and ige in mice [75] . so a deficiency in ifny-secretion may lead to alterations of immunoglobulin levels in humans with mecp2 duplication although the role of ifny in the regulation of igg 3 is controversial [76, 77] . we did not observe a generally impaired ifny secretion in our cohort. however previously published results on irregularities in ifny secretion should be taken into account as being responsible for altered immunoglobulin levels in patients with mecp2 duplication syndrome [44] . besides the classical encapsulated bacteria s. pneumoniae and h.influenzae several other bacteriae were also isolated in the patients of our cohort, that are indeed able to build capsules but are not clearly linked to capsule-related virulence. so it remains unclear if humoral immunodeficiency is causative also for infections by these facultative-encapsulated pathogens or if additional cellular immune deficits also account for the broad spectrum of observed pathogens in patients with mecp2 duplication syndrome. in summary we here show for the first time systematically that patients with mecp2 duplication syndrome are at increased risk for in particular non-invasive but also for invasive infections with potentially encapsulated bacteria, that this increased susceptibility to infections may be associated with igg 2 -subclass deficiency/ low titers against pneumococci and elevated acute-phase responses, while the precise role of t-cell immunity and in particular the extent of impaired ifnγsecretion and its role for the observed infectious phenotype is still to be defined. based on our observation we hypothesize a multifactorial pathogenesis of infections in patients with mecp2 duplication syndrome: pulmonary infections may be triggered by frequent aspirations due to the severe neurological phenotype and favoured due to humoral immunodeficiency. the observed alterations in the igg subclass composition (high igg 1 /igg 3 and low iga/igg 2 /igg 4 ) may be caused directly by interaction of mecp2 with the respective loci or indirectly by spatially impaired ifnγ-secretion. an increased susceptibility to hyper-inflammation either caused by irak1 duplication or other direct or indirect effects of mecp2 duplication may aggravate infections. based on the compiled immunological and infectious findings we recommend to vaccinate patients with mecp2 duplication against pneumococci and to evaluate post-vaccination titers. if postvaccination titers are not sufficient, several boosts might be required. in patients with mecp2 duplication syndrome, suffering from recurrent infections and with an igg 2 -subclass deficiency and/or low post-vaccination titers against pneumococci prophylactic substitution of igg seems a plausible strategy to prevent infectionseventually in combination with the use of prophylactic antibiotics. xlmr syndrome characterized by multiple respiratory infections, hypertelorism, severe cns deterioration and early death localizes to distal xq28 submicroscopic duplication in xq28 causes increased expression of the mecp2 gene in a boy with severe mental retardation and features of rett syndrome duplication of the mecp2 region is a frequent cause of severe mental retardation and progressive neurological 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staphylococcus aureus capsular polysaccharides encapsulated staphylococcus aureus strains vary in adhesiveness assessed by atomic force microscopy development of a vaccine against staphylococcus aureus capsular polysaccharides are an important immune evasion mechanism for staphylococcus aureus immune evasion by staphylococci the ica locus of staphylococcus epidermidis encodes production of the capsular polysaccharide/adhesin upregulation of capsule enables streptococcus pyogenes to evade immune recognition by antigen-specific antibodies directed to the g-related alpha2-macroglobulin-binding protein grab located on the bacterial surface the importance of the group a streptococcus capsule in the pathogenesis of human infections: a historical perspective thermoregulation of capsule production by streptococcus pyogenes inborn errors of interferon (ifn)-mediated immunity in humans: insights into the respective roles of ifn-alpha/beta, ifn-gamma, and ifn-lambda in host defense revisiting human il-12rβ1 deficiency: a survey of 141 patients from 30 countries mycobacterial disease and impaired ifn-γ immunity in humans with inherited isg15 deficiency primary immunodeficiencies: a rapidly evolving story interferon-gamma and b cell stimulatory factor-1 reciprocally regulate ig isotype production induction of igg3 secretion by interferon gamma: a model for t cell-independent class switching in response to t cellindependent type 2 antigens t-bet regulates t-independent igg2a class switching key: cord-009169-hzxgi1t0 authors: sun, bingwei title: nosocomial infection in china: management status and solutions date: 2016-07-01 journal: am j infect control doi: 10.1016/j.ajic.2016.01.039 sha: doc_id: 9169 cord_uid: hzxgi1t0 nan to the editor: in china, nosocomial infection is a prominent public health concern and is associated with an annual direct economic burden of $1.5-$2.3 billion (¥10-¥15 billion). 1 advances in medical technology, extensive application of novel diagnostic and therapeutic techniques, and increased adoption of traumatic and invasive interventions have drastically altered the source, transmission route, and susceptible population of nosocomial infection. emergence of multidrug-resistant bacteria has increased the proportion of refractory infections and the challenges of infection control. [2] [3] [4] china has reinforced legislation to control nosocomial infection, drawing extensive support from society and medical community. 5 this transition is reflected in the chinese governmental response to the epidemic of middle east respiratory syndrome. in january 2013, 99 out of 120 patients receiving treatments for varicosity at donggang social insurance medical clinic were infected with hepatitis c virus. investigations indicated that this aggressive, multisource, and nosocomial hepatitis c virus infection resulted from repeated syringe use among different patients. 6 in july 2011, 15 cataract patients undergoing surgical treatment at an ophthalmic hospital in the raodu district, linfen, suffered from endophthalmitis. investigation showed that patients were infected with pseudomonas aeruginosa endophthalmitis, which resulted from a shortage of surgical equipment and nonstandard disinfection of the surgical instruments. 7 in march 2009, incidence of neonatal nosocomial infection was reported in a maternal and child care service center in the ji county of tianjin. in a neonatal ward, 5 of the total 6 newborns were dead. investigations revealed a severe nosocomial infection resulting in deaths attributed to negligence by staff with poor infection prevention and control standards. 8 the ongoing incidence of such serious events reflects poor standards of nosocomial infection management. first, inadequate medical sources and imbalance in hospital development are increasingly serious challenges. nearly 50 million of the total 1.3 billion people in china require hospitalization annually because of diseases or trauma. however, the chinese health care system is riddled with long wait lists, shortages, and poor equipment because of funding constraints. the national medical system is not hierarchical in structure. as a result, most patients are traditionally segregated in large-and medium-sized hospitals, and hospital wards have become breeding grounds for microbial pathogens, with increased risk of infection. second, the under-reporting of nosocomial infection and the number of full-time staff experienced in infection control have been highlighted by health administrators during the performance appraisal of health care institutions. in some hospitals, hospital leaders neglect poststandardization training and continuing education of the managers involved in controlling infection. full-time professional staff members fail to undergo training in nosocomial infection and occupational health. in most hospitals, target and prospective monitoring are rare, which results in delayed discovery of high-risk areas. third, hospitals lack expertise and human resources skilled in infection management. senior managers fail to consider qualified personnel. a few health care institutions simply fill available vacancies with retired nurses to ensure the number of required personnel in the department of nosocomial infection. forth, drug-resistant bacteria as a result of antibiotic abuse and unreasonable use are still important factors leading to nosocomial infection. epidemiology studies suggest that the usage rate of antibiotics in hospitalized patients in some regions and hospitals is still >60%, reaching a rate of 74.67%, 9, 10 or even exceeding 90% in the neonatal ward. inadequate catheterization and subsequent care along with poor concept of asepsis are other factors. failure to comply with standard regulations of hand hygiene or even handwashing after examining patients with infections, lack of standardized or complete disinfection of surgical instruments, failure to implement surgical standards, failure to consider oxygen humidifiers as an important source of lower respiratory infection, and inappropriate measures of disinfection and isolation are some of the factors that artificially increase nosocomial infection. a solution for improved nosocomial infection management in china has been proposed. the overall goal of the chinese action plan on prevention and control of nosocomial infection (2012-2015) 11 includes enhanced prevention and control of nosocomial infection, insisting on "scientific prevention and control, standard management, highlighting the key points, and enforcing the implementation." the solutions are designed to improve the related technical criteria, upgrade and implement prevention and control measures, raise the professional skills and capacities, and increase the quality and safety of medicines. the following additional steps are recommended to address the challenges of nosocomial infection: • governments at all levels must invest increasingly to improve hospital conditions. public hospitals should be transformed into not-for-profit organizations in the national health care system. • to disseminate the knowledge of nosocomial infection, the government and health workers should popularize and publicize new techniques and know-hows for infection prevention and control. • to improve environmental hygiene, a national system of sanitation should be created to provide population access to adequate sanitation measures given the current unsanitary conditions in hospitals sustainable care and development and nosocomial infection management. the government and the director of the hospital should emphasize the role of rational design of the wards and hospital layout and balance the costs and benefits in prevention of infection. • the hospital information system (his) enables the control of infection by regulating all of the operational aspects, such as medical, administrative, financial, and legal issues and the corresponding services. the his is the essential technologic backup of the environment and infrastructure in a modernized hospital system. information sources related to nosocomial infection may be enriched using the his and enhance the ability of health care professionals. the his facilitates organizations in nosocomial infection management, official documentation, and ensures data security. • evidence-based medicine (ebm) is intended to optimize decision-making by emphasizing the use of evidence from welldesigned research studies. according to the theory of ebm, the strongest evidence based on meta-analyses, systematic reviews, and randomized controlled trials yields strong recommendations. combined with the data of epidemiologic survey and analysis, ebm may provide useful strategies to improve nosocomial infection management. however, the inadequacy of clinical application of ebm in nosocomial infection management in china needs to be addressed. • a no-pay policy by the government and medical insurance organizations must be implemented rigorously for items of nosocomial infection that are not covered by medical insurance. this policy will encourage awareness of prevention and control measures that reduce the incidence of hospital infection. the quality of infection control is an important indicator balancing quality of management and medical administration. during the early period of the severe acute respiratory syndrome epidemic in 2002, the nosocomial infection rate of medical staff reportedly exceeded 10% because of inadequate disease knowledge. however, this rate does not represent all the data because there were several hospitals or wards with a zero infection rate among the medical staff during the outbreak. the discrepancy is associated with different attitudes toward infection. overall, nosocomial infection management in china and worldwide has a long way to go. to the editor: serratia marcescens has become an important cause of nosocomial infections over the last 2 decades. 1 these bacteria can survive on inanimate surfaces and 2% chlorhexidine solutions for months 2, 3 or in medications, and they can subsequently cause outbreaks. 4, 5 although s marcescens strains are usually reported to be a cause of outbreaks, especially in neonatal intensive care units (icus), 6, 7 there are also several reports about these strains causing pseudo-outbreaks. 8, 9 a pseudo-outbreak is defined as the recovery of the same organism from the cultures of patients who are not infected or colonized with the organism. 9 such pseudooutbreaks increase laboratory costs and may lead to unnecessary treatment. leaving the source of contamination undetermined may also eventually cause nosocomial infections. during a 35-day period, 22 s marcescens strains were isolated from 20 patients (19 blood cultures, 2 tracheal aspirates, and 1 urine sample). after isolating the first 4 isolates from blood cultures, the laboratory informed hospital infection control committee. throughout the environmental investigation, the number of isolates reached 22 before the source was determined. these patients were hospitalized in 2 different icus. nearly all of the examined patients (19/ 20) had a history of admittance to the emergency department (ed) on the same day or 1 day before hospitalization in the icus, and some of the patients were transferred to the ed via a common ambulance delivery system. all of the invasive procedures performed on the patients were also examined. most of the s marcescens instances were isolated from the active surveillance cultures that were taken when the patients entered the icus. in addition to these factors, the patients did not have any signs and symptoms compatible with clinical infection. this situation was therefore considered a survey on current status of hospital infection management in 52 medical institutions (master's thesis) the prevalence of nosocomial infection in intensive care units in europe. results of the european prevalence of infection in intensive care (epic) study. epic international advisory committee effect of selective decontamination on antimicrobial resistance in intensive care units: a systematic review and meta-analysis epidemiology of infections acquired in intensive care units procalcitonin levels in patients with positive blood culture, positive body fluid culture, sepsis, and severe sepsis: a cross-sectional study ministry of health. 99 patients were afflicted with infections of hepatitis c virus (hcv) shanxi provincial government. 15 cataract patients suffered from endophthalmitis neonatal nosocomial infections in maternal and child care service center in the ji county of tianjin analysis on incidence of hospital infection and antibiotic usage rational use of antibiotics and preventive medication ministry of health. action plan on prevention and control of nosocomial infection key: cord-027550-yyqsatqw authors: mammas, ioannis n.; drysdale, simon b.; rath, barbara; theodoridou, maria; papaioannou, georgia; papatheodoropoulou, alexia; koutsounaki, eirini; koutsaftiki, chryssie; kozanidou, eleftheria; achtsidis, vassilis; korovessi, paraskevi; chrousos, george p.; spandidos, demetrios a. title: update on current views and advances on rsv infection (review) date: 2020-06-15 journal: int j mol med doi: 10.3892/ijmm.2020.4641 sha: doc_id: 27550 cord_uid: yyqsatqw respiratory syncytial virus (rsv) infection represents an excellent paradigm of precision medicine in modern paediatrics and several clinical trials are currently performed in the prevention and management of rsv infection. a new taxonomic terminology for rsv was recently adopted, while the diagnostic and omics techniques have revealed new modalities in the early identification of rsv infections and for better understanding of the disease pathogenesis. coordinated clinical and research efforts constitute an important step in limiting rsv global predominance, improving epidemiological surveillance, and advancing neonatal and paediatric care. this review article presents the key messages of the plenary lectures, oral presentations and posters of the '5th workshop on paediatric virology' (sparta, greece, 12th october 2019) organized by the paediatric virology study group, focusing on recent advances in the epidemiology, pathogenesis, diagnosis, prognosis, clinical management and prevention of rsv infection in childhood. precision medicine has evolved in recent years allowing the incorporation of novel taxonomies and stratification of patients, and using standardized clinical endpoints, genetic and other biomarker information (1) . its role in paediatric healthcare involves the selection of targeted diagnostic, therapeutic and prevention strategies matched to precise molecular, epidemiological and clinical profile of each patient; the management of respiratory syncytial virus (rsv) infection represents a good paradigm of precision medicine (2) . rsv is a single-stranded rna virus (figs. 1 and 2), which represents the most frequent viral cause of acute lower respiratory tract infection (alrti) in infants, with a worldwide distribution and seasonal occurrence (2) (3) (4) (5) . it was first isolated in 1956 from nasal secretions of chimpanzees with rhinorrhea and coryza; the novel virus was initially named 'chimpanzee coryza agent' (cca) (6, 7) . in the following year, when cca was also isolated from children with alrti, it gained its final name due to the syncytia observed on electron microscopy; syncytia are formed by fusion of infected host cells with neighboring cells leading to the formation of multi-nucleate enlarged cells. although the formation of syncytia is the hallmark of the cytopathic effect of rsv that is associated with host cellular membrane merging, syncytia are not pathognomonic of rsv (8) . syncytia are also observed in cell culture with several other viruses, such as parainfluenza, hsv-1, hiv and mev. recently, the international committee on taxonomy of viruses (ictv), which authorizes and organizes the classification and naming of viral species, grouped rsv under the genus orthopneumovirus within the family pneumoviridae (9, 10) . bronchiolitis is the most common clinical manifestation of rsv infection in infants and although it is usually self-limiting, in infancy it accounts for a significant number of hospitalizations and paediatric intensive care unit (picu) admissions (3) . despite its association with relatively high morbidity and mortality in premature neonates and in certain paediatric populations with underlying conditions, such as immunodeficiency and congenital heart disease, rsv infection may also lead to hospitalization of previously healthy, full-term infants (11, 12) . rsv-positive bronchiolitis is characterized by airway inflammation and oedema, mucus production and debris leading to airway obstruction and turbulent gas flow. even though various therapeutic interventions have been tried, such as bronchodilators, hypertonic saline and corticosteroids, supportive care remains the mainstay in most settings, with gentle suctioning of nasal secretions, prone position, fluid replacement and oxygen or respiratory support, as necessary. several clinical trials on the management and prevention of rsv-positive bronchiolitis have been recently completed, are underway, or in development (3) . currently, there is rapid expansion of rsv vaccine candidate development and there is hope that one will become available in the near future. of course, the safety of vaccines proposed for primary immunization in an antigen naïve child remains the top priority. this review article summarizes the key messages of the plenary lectures, oral presentations and posters of the '5th workshop on paediatric virology' held in sparta (greece) on october 12th, 2019, which was focused on rsv (table i) understanding the burden of rsv infections in real-time. rsv poses significant disease burden in infants and children worldwide (13) , and the international paediatric community is only beginning to appreciate its global impact in both high and low resource settings (4, 14, 15) . the clinical presentation of an rsv infection depends on the patient's age and individual risk factors (16, 17) . the concept of measuring individual-level differences in disease severity has drawn the attention of both public health stakeholders and regulatory agencies in recent years (18) (19) (20) . the impact of rsv infections of course should be differentiated from the disease caused by influenza and other viral respiratory infections (21, 22) . however, a recent extensive literature review and prospective cohort have shown that in infants and children this cannot be done based on clinical symptoms alone; distinguishing rsv from other viral respiratory infections requires laboratory confirmation (23, 24) . with rsv vaccines and antiviral agents in development, it will be important to: a) diagnose rsv infections in a timely manner; b) differentiate rsv from other forms of acute respiratory infections; and c) communicate the test results back to patients and parents/caregivers along with information on the individual disease risk and severity. the pedsidea programme. the vienna vaccine safety initiative (vivi, https://www.vi-vi.org) is an international non-profit research organization which, in collaboration with academic institutions and public health agencies in europe and the united states, has developed digital tools and programs to improve the quality of care for children and adults with alrti or influenza-like illnesses (ili) (13, 25, 26) . taking a person-centered approach, the vivi disease severity score ('vivi score') is a mobile application enabling healthcare professionals to measure disease severity at the point of care within minutes. it was designed to provide a uniform approach to define ad hoc severity at any given time point, based on extensive literature review as well as who criteria for uncomplicated and complicated ili (27) . in collaboration with the robert koch institute, the vivi score was validated in a cohort of 6,000 children (age 0-18) in berlin, germany and subsequently used in a european pilot project entititled 'partnering for enhanced digital surveillance of influenza-like disease and the effectiveness of antivirals and vaccines' (pedsidea) (27, 28) . since then, the pedsidea programme has been implemented in community clinic networks and adult intensive care units in the unites states for the real-time digital surveillance of influenza and rsv disease incidence and severity at the point of care (29) . the programme is expected to continue monitoring, in great detail, the clinical outcomes of 'natural' rsv and other viral respiratory infections in children and adults, including patients at the extremes of age. understanding the real-world disease burden may help facilitate the study of the effectiveness of novel influenza and rsv antiviral agents and vaccines, once they become available (30) . rsv infection: not for children only. rsv was not recognized as a potentially serious problem in older adults until the 1970s, when outbreaks of the virus occurred in long-term care facilities for the elderly (31) (32) (33) . since then, additional studies in hospitalized adults have suggested that rsv may be an important cause of illness in adults. molecular diagnostics suggest that rsv positive specimens are commonly identified in elderly and high-risk adults, in a frequency similar to that of seasonal influenza (31, 34) . even though a positive rsv respiratory panel does not equate pathogenesis, it has been suggested that rsv may account for as much as 10,000 deaths annually in the united states among individuals above the age of 65 years (31) (32) (33) 35) . this, in addition to the morbidity in infants, has stimulated interest in rsv vaccines and antiviral agents. additional natural history studies are needed to better understand the actual burden of rsv infection among the elderly and high-risk adults. the immune response to rsv infection. maternal antibodies may be able to mitigate rsv disease severity in young infants (17, (36) (37) (38) . it is assumed that the transplacental passage of rsv-specific antibodies occurs predominately during the third trimester of pregnancy. high titers can potentially protect term infants up to four months of age (38) . in premature infants, passive immunity may be 'compromised' (39) , but still has a role to play. the degree to which breast feeding may also contribute to passive immunity and to priming of immune system is currently under investigation (40) . once infection is established, the innate immune system plays a dual role in lowering the viral load and in mounting a secondary immune response. prematurity and other conditions that compromise the immune response may lead to reduced levels of antiviral cytokines, such as the interferons (41) . in infants, reduced signaling by tlrs and altered antigen-presenting cell functions, including low interleukin (il)-12 and enhanced il-6 and il-23 production, coupled with reduced activation of regulatory t cells, may result in an adaptive response that is skewed toward th2 and th17 and away from protective th1 and ctl responses (42) . the potential specific role(s) of certain pattern recognition receptors in humans has/ve been suggested by the fact that certain tlr missense mutations are associated to a phenotype with propensity to wheezing (43) . apart from the individual genetics (44), the stage of lung maturation among term and premature infants also impacts on the th1 to th2 switch. in the case of prematurity, the mucosa prior to alveolarization is being deluged with th2 inflammatory responses, even in the earlier stages of bronchopulmonary dysplasia (45) . impaired th1 activation, coupled with little or no b cell memory, and inhibition of antibody production by ifnγ, produces low-titer, low-affinity antibody (46) . the result may be a poorly protective and dysregulated defense mechanism that leads to bronchiolitis in susceptible infants (43) . later, the host immune response is permanently oriented to the direction of wheezing exacerbations, specifically triggered by rsv (47) . micrornas (mirnas) are involved in post-transcriptional gene regulation and play significant roles in the maintenance of the airway epithelial barrier of the respiratory tract (48) (49) (50) (51) (52) (53) (54) (55) (56) . mirnas have been implicated in the modulation of antiviral defense mounted by host innate and adaptive immunity, involving not only immune effector and inflammatory cells, but also parenchymal cells (50) . respiratory viruses, including rsv, attack, as a primary target, the epithelial cells of the respiratory tract causing an altered expression of distinct mirnas in the airway cells. the human innate immune response inhibits rsv replication early after inoculation, mainly through the action of interferons (53) . multiple mirnas are induced by infection in a cell-type-specific fashion (51) . rsv appears to alter host cell gene expression also through regulation of expression of mirnas related to the interferon response (50, 53) . abnormal expression of mirnas has been detected in both peripheral blood cells and airway epithelial cells in rsv-infected infants (54) . understanding alterations in mirna expression profiles and identifying mirna target genes in relation to the pathogenesis of rsv may help clarify the mechanisms of virus-host interactions, and immune dysfunction leading to airway hyper-reactivity and chronic respiratory diseases, such as asthma (49,50,53,54). there are several methods for the purification, quantification and being able to compare severity over time and/or across cohorts is useful in hospital-based qi programmes but also in multi-centre networks, such as pedsidea understanding the real-world disease burden caused by rsv will facilitate the study of the effectiveness of antivirals and vaccines, once they become available recent epidemiological data indicate that rsv infection is an important illness in elderly and high-risk adults, with a disease burden similar to that of non-pandemic influenza rsv and immune response maternal rsv-specific antibodies transmitted transplacentally during the third trimester of pregnancy are related to rsv disease severity in young infants rsv and mirnas a greater understanding of mirnas may enable them to be used as biomarkers of severe rsv infection and as novel targets for treatment or prophylaxis of rsv infection rsv and thrombocytosis thrombocytosis in rsv-positive bronchiolitis does not require routine prophylactic anti-platelet treatment or further investigations rsv and asthma there is compelling evidence that severe respiratory infection induced by rsv is associated with subsequent development of asthma later in childhood further understanding of the role of rsv in asthma pathogenesis will enable our understanding of the impact of future vaccines against rsv in asthma prevention rsv as a cause of pibo there are only few reports in the literature of children with pibo secondary to rsv as a single infection further research is required in order to investigate the potential impact of rsv co-infection in the severity and worse outcome in children with pibo imaging of rsv infection although imaging cannot diagnose rsv infection, it is important to identify the possible pattern of viral disease, in order to avoid unnecessary administration of antibiotic therapy and predict possible late effects standard radiological techniques, including ct, are unable to distinguish between acute bronchiolitis caused by rsv versus that caused by other respiratory viruses hrct of the lungs may be required to assess possible bronchial thickening and remodeling, the development of bronchiectasis and air-trapping antivirals against rsv ribavirin is currently the only licensed antiviral medication used to treat rsv infection; it has very limited efficacy and multiple toxicities, which means its use is usually reserved for severely immunocompromised children due to ethical and technical constraints human challenge models are only undertaken in adults, but if a product is shown to be efficacious in this setting it allows a faster move to trials in children than traditional trials which often take much longer to do a greater understanding of individual data in newly developed pharmaceutical agents against rsv will potentially lead to future personalized treatment regimens rsv and picu hfnc might have a role as a rescue therapy for children with rsv-positive bronchiolitis admitted to picu to reduce their requirement for high-cost intensive care heliox could be useful in addition to standard medical care in the management of children with rsv-positive bronchiolitis admitted to picu characterization of mirna expression profiles in biofluids, whole blood samples and tissue samples obtained in in vivo studies (55) . further research on mirnas is expected to clarify their value as biomarkers of rsv infections and their sequelae (i.e., recurrent wheezing and asthma). thrombocytosis and rsv infection. the most frequent causes of secondary thrombocytosis in childhood are acute respiratory tract infections (57) (58) (59) (60) (61) . to date, several authors have reported significantly higher mean platelet counts in patients with rsv than in patients with other acute respiratory tract infections (61) (62) (63) (64) . thrombocytosis is more likely to occur in younger patients, who have clinical manifestations of wheezing and dyspnoea (62, 64) . moreover, thrombocytosis has been suggested as an early marker of rsv infection (62) . excessive thrombocytosis has also been detected at an early stage in cases of rsv-positive bronchiolitis (65) . it has been proposed that thrombocytotic patients have a more severe clinical course and longer duration of hospitalization and that the platelet count may be a useful clinical marker associated with alrti severity (61, 64, 66, 67) . conversely, other authors have found that platelet counts do not correlate with disease severity and clinical outcome (68, 69) . routine prophylactic anti-platelet treatment or further investigations are not necessary in children with rsv-positive bronchiolitis and thrombocytosis (61, 68, 70) . there is compelling evidence that infants with severe rsv infection in the early months of life have a subsequent increased risk of developing recurrent wheezing and/or asthma, with a prevalence of up to 30% compared with non-rsv groups (71) (72) (73) (74) (75) . whether this association is causal has been the subject of considerable debate on the potential role of rsv infection in the pathogenesis of asthma as well as the impact of asthma predisposition (genetic, environmental exposure, etc.) on the clinical course of rsv infection. a recent large retrospective cohort analysis of australian children born between 2000 and 2010 suggested that different subgroups of high risk children, who developed rsv disease within the first 2 years of life, continued to be at elevated risk of having a first asthma hospitalization beyond the age of 7 years (76). on the other hand, large epidemiological observational studies demonstrate that the vast majority of infants hospitalized for rsv bronchiolitis do not fit into an 'at-risk' group (atopy, family history, etc.), suggesting that viral or host factors not thought of as classical risk factors, may play a role in disease severity (74, 77) . prospective studies with rsv-immunoprophylaxis (e.g., palivizumab) suggest that long-term effects of rsv prophylaxis appear less efficacious in infants with a family history of atopy. in addition, palivizumab decreases parent-reported recurrent wheeze, however the incidence of physician-diagnosed asthma is similar (78) . a recent single-blind, randomized, placebo-controlled trial showed that rsv prevention in otherwise healthy preterm infants, did not have a major effect on asthma or lung function at the age of 6 years (79). considering the above findings, perhaps a more appropriate conclusion would be that rsv infection is important in the mechanism of wheezing development, at least in the first few years of life (80) . rsv possesses the ability to counteract host defense systems through complex mechanisms that facilitate viral replication. this significant increase in asthma frequency seems to be predominantly related to long-term changes in neuroimmune control of airway tone rather than to allergic sensitization. in contrast to rsv bronchiolitis, atopy has been clearly associated with childhood asthma development after rsv-induced early wheezing (81, 82) . high-risk (parental atopy or asthma) birth cohort studies from wisconsin, united states, and australia have shown that young children suffering from rsv-induced wheezing episodes are at high risk of developing school-age asthma (81, 82) . further prospective, follow-up studies are needed to clarify individual and environmental factors that promote more severe viral illnesses and long-term adverse respiratory outcome of children hospitalized for severe rsv infection. developing a greater understanding of the pathophysiological mechanisms through which rsv causes recurrent wheezing/asthma, will lead to an evidence-based prevention strategy and perhaps reduce the subsequent risk for asthma (71) . the biomarker ccl5 (previously known as rantes, a β-chemoattractant for inflammatory cells including t-lymphocyte subsets), in the nasal epithelium during rsv bronchiolitis, is strongly predictive of physician-diagnosed asthma (83, 87) . furthermore, it has been suggested that prematurely born infants have a predisposition to rsv infection-related respiratory morbidity, including subsequent respiratory dysfunction (44,85). single-nucleotide polymorphisms in genes coding for il-8, il-19, il-20, il-13, mannose-binding lectin, ifng and rantes, have been associated with wheezing following rsv lrti in term-born infants (85) . the site of infection might be another important factor related to asthma risk, thus viral alrti in infancy indicates an increased risk of subsequent asthma, while gastrointestinal infections might be protective (86) . asthma after severe rsv bronchiolitis is positively correlated with maternal asthma, exposure to high levels of dog allergen, aeroallergen sensitization and recurrent wheezing; day care attendance and white race have been associated with decreased asthma risk (87) . several host factors, including respiratory allergy and virus-induced interferon responses, viral virulence factors, individual risk factors (e.g., young age, especially the first 6 months of life, small lung size and genetics), and environmental exposures (e.g., exposure to tobacco smoke, airway microbiome) modify the risk of virus-induced wheezing and promote more severe wheezing illnesses and the risk for progression to asthma (86, 88) . the anti-rsv mab palivizumab decreases the risk of severe rsv-induced illness and subsequent recurrent wheeze in prematurely born infants (89) . further understanding of the role of rsv in asthma pathogenesis may help develop vaccines against rsv as a way of asthma prevention. bronchiolitis obliterans (bo) is a chronic and irreversible lung disease leading to the obstruction and/or obliteration of the small airways (90) . most cases of bo in children are post infectious (pibo) and are mainly associated with adenovirus infections, although other viruses may also be implicated, including measles, influenza, parainfluenza and rsv (91) (92) (93) . an extensive search of the current literature in the context of the workshop demonstrated that rsv is detected in children with pibo with an incidence ranging from 4.3 to 30% (94-103); however, there are only a few reports of children with pibo secondary to rsv as a single infection. this creates skepticism about the aetiological role of rsv in pibo. further research is required to investigate the potential impact of rsv co-infection in the severity and outcome of children with pibo. chest radiography and rsv-positive bronchiolitis. although imaging cannot confirm the diagnosis of rsv infection, it is important to identify the possible pattern of viral disease, in order to avoid unnecessary administration of antibiotic therapy and to predict possible late effects (figs. 3-5) (104). the clinical syndrome of bronchiolitis is commonly diagnosed based on the patient's history and physical examination; chest radiography is not routinely recommended to reach the diagnosis due to recommended restriction of radiation exposure in the paediatric age group (104) (105) (106) . chest imaging, however, may be considered when a child with rsv infection and severe alrti is admitted to intensive care to better understand the extent of lung involvement and atelectasis, which is common in acute rsv infection (106). it is important to note that chest radiographs in children with rsv infection may be entirely normal or reveal non-specific findings, which are also encountered in other viral infections: most commonly, perihilar opacities and hyperinflation, atelectasis and rarely consolidation and bronchial cuffing or air-leak (107) . radiography is commonly obtained to rule out atelectasis and foreign body aspiration. guidelines suggest performing a chest radiograph in the presence of significant respiratory distress or hospitalization (108) . in newborns with rsv infection, the radiological pattern on chest radiography may be a predictor of clinical outcome (109) . however, it is highlighted that chest radiographs should not be routinely performed in children with bronchiolitis to avoid radiation exposure (106). it is also important to emphasize that chest radiograph is not the right way to rule out bacterial infection (106); the correct diagnostic approach for bacterial or ventilator-associated pneumonia in children in the picu is to perform respiratory culture or matrix-assisted laser desorption ionization time-of-flight (maldi-tof) mass spectrometry from sputum/aspirate or bronchoalveolar lavage (bal) specimens. chest ct and rsv-positive bronchiolitis. several studies have revealed that standard radiological techniques, including computed tomography (ct), are frequently unable to distinguish between acute bronchiolitis changes caused by rsv vs. those caused by other respiratory viruses (104, 108) . it is interesting that the radiographic findings, especially in high-resolution computed tomography (hrct), reflect the histopathologic changes that rsv infection provokes: plugging or occlusion of the bronchiolar airway lumens by sloughed necrotic and irregular epithelium and exudate, combined with peri-bronchiolar infiltration and reaction with inflammatory cells and submucosal oedema. the infiltration is a combination of neutrophils entering the airway submucosa and epithelial cell debris in the airway lumens. these cellular accumulations are likely to result in acute obstruction of the distal airways, an outcome much more likely to occur in the extremely narrow bronchioles of infants. because this is combined with the inherent loss of mechanical clearance of these small airways, it likely leads to increased spread of infection, augmented inflammation and clinical signs of wheezing/obstruction (110) . consistent with obstruction, the most common ct findings in rsv pneumonia include centrilobular nodules, ground-glass opacities, air-space consolidation, and peribronchial thickening (111) . these findings have a bilateral, usually asymmetric, central and peripheral distribution. up to 40% of children with bronchiolitis will develop further wheezing episodes in the first five years of life. in very severe or atypical cases, hrct of the lungs may be required to assess the extent of bronchial thickening and remodeling, the development of brochiectases and air-trapping (105) . restricted to the airways. case reports have also described clinical pictures resembling viral encephalitis and/or encephalopathic syndromes with severe sequelae in isolated cases (112) . the mechanism of the spread of the rsv infection to the cns compartment remains unclear (112) . brain magnetic resonance imaging (mri) in infants with cns involvement has shown predominantly non-specific findings similar to those also encountered in other viral and/or limbic system encephalitides (113) . in very rare instances, extra-pulmonary findings in rsv infection have also included acute necrotizing encephalopathy (ane) and acute hepatic failure with encephalopathy (114) . clinicians should have a high suspicion of ane in cases of children with a respiratory infection and acute neurological manifestations. antivirals against rsv. thus far, ribavirin is the only antiviral agent that has ever been licensed for the treatment of rsv infection (115) . however, its efficacy is not proven and due to significant toxicity its use has been primarily restricted to severe cases in immunocompromised patients with severe rsv-positive alrti (116) . several other antiviral candidates have been developed since, but none have been licensed as yet. types of molecules being tested include influenza antivirals, such as baloxavir, cc-42344, vis410, immunoglobulin, hyperimmune plasma, mhaa4549a, pimodivir (jnj-63623872), umifenovir, and ha minibinders, rsv antivirals including presatovir (gs-5806), ziresovir (ak0529), lumicitabine (als-008176), jnj-53718678, jnj-64417184, and edp-938, broad spectrum antivirals such as favipiravir, vh244, remdesivir, and eidd-1931/eidd-2801, as well as host directed strategies including nitazoxanide, eritoran, and diltiazem (117-119). novel molecules disrupt various stages of the virus life cycle, including cell entry, viral replication, and polymerization as well as after virus release through rsv neutralizing anti-or nanobodies (120) . the human challenge models. one method used occasionally in phase 2 clinical testing is human challenge models (115) . this method was used in phase 2a clinical testing of the non-fusion inhibitor edp-938 (clinicaltrials.gov identifier: nct03691623). all participants were inoculated with a known strain of rsv and were then randomized to receive the medication or placebo. the advantage of this methodology is the removal of the variability in exposure with natural infection and the collection of samples at precise, known times after infection, which can aid with the understanding of the biological mechanisms of the infection and development of antiviral agents or vaccines (121, 122) . due to ethical and technical constraints, experimental studies are only undertaken in adults, but if a product is shown to be efficacious at this setting, a faster move to trials in paediatrics takes place than in traditional childhood trials. rsv therapeutics and personalized medicine. the current clinical data indicate that rsv disease dynamics may not be identical in all patients (2, 3, 115, 119) . more research is needed to identify uniform clinical endpoints reflecting how patients function, thrive, and survive (us food and drug administration) and to understand inter-individual differences in disease presentation, with the goal of ultimately selecting the right treatment for the right patient. a greater understanding of individual differences may ultimately lead to future personalized treatment strategies. individualized approaches and a well-standardized methodology to assess disease severity at the time of enrolment, as well as during follow-up visits, will require integration of diagnostic, clinical and laboratory markers at the point of care (115) . individualized targeted treatment will constitute an important step in improving outcomes in patients with rsv infection while minimizing toxicity. a greater understanding of individual data in newly developed pharmaceutical agents against rsv will potentially lead to future personalized treatment regimens. applying such co-ordinated diagnostic, clinical and research efforts constitutes an important step in advancing paediatric care, improving outcomes and limiting global rsv morbidity and mortality. over the last decade, high-flow nasal cannula (hfnc) therapy has emerged as a new method to provide respiratory support in children with rsv-positive bronchiolitis (12, (123) (124) (125) (126) . its main advantages include its ease to set up and the fact that it is well tolerated, leading to better compliance, especially in comparison to other devices of non-invasive ventilation (125, 127) . initially, hfnc was trialed in infants with moderate to severe bronchiolitis admitted to picus, but nowadays its application has expanded to paediatric wards, even to emergency departments, in order to avoid a picu admission (127, 128) . recent data have shown that it does not significantly reduce time on oxygen compared with standard therapy, suggesting that early use of hfnc does not modify the underlying disease process (129) . however, the proportion of children who experi-ence treatment failure is lower in hfnc and many of those who experience treatment failure on standard therapy can be rescued by hfnc. additional studies comparing hfnc with continuous positive airway pressure (cpap) in the picu setting led to the same conclusion (130, 131) . consequently, hfnc may reduce the need for intubation and invasive respiratory support, thus potentially lowering costs and adverse effects of mechanical ventilation, such as ventilator-induced lung injury, infections and exposure to sedatives. in addition to effectiveness, most studies have shown no adverse events with hfnc and have concluded that it is a relative safe method for use even in general wards or emergency departments (123) . few cases of pneumothorax have been reported, abdominal distension has been less significant compared with cpap, and the majority of infants have been able to be fed orally or by nasogastric tube (123, 127) . heliox and rsv-positive bronchiolitis. since rsv-positive bronchiolitis is associated with airway obstruction and turbulent gas flow, its clinical course can be improved by heliox, which facilitates gas flow through high-resistance airways (132) (133) (134) (135) (136) (137) (138) . heliox is a mixture of helium-oxygen, which can be administered by all modes of ventilation in spontaneously breathing patients by face mask, hfnc or cpap, and can be adjusted to specific ventilators in intubated children. current evidence suggests that the addition of heliox may significantly reduce clinical scores evaluating respiratory distress and the respiratory rate, and may enhance co 2 elimination in the first hour after starting treatment in infants with acute refractory rsv bronchiolitis (134, 139) . recently, seliem and sultan (140) reported that heliox results in improvement of oxygenation when used with high flow nasal cannula in infants with acute rsv bronchiolitis, during the initial phase of therapy. the combination of heliox with cpap also seems to be beneficial, as the application of cpap may reduce the fio 2 needed in these infants. however, no benefit has been observed in terms of need for intubation and mechanical ventilation, length of treatment or picu stay. in addition, its application in the emergency department does not change the discharge rate (138, 139) . more clinical trials are needed to define the population that may respond to heliox and its place in the therapeutic regimens of rsv bronchiolitis. passive immunization and palivizumab. the prevention of rsv morbidity and mortality remains a global healthcare priority (115, 141) . according to the world health organization (who), the strategic focus for the prevention of rsv infection in children and adults includes the passive administration of immunoglobulins, as well as active immunization. passive immunization is currently the only option available to infants less than 6 months of age, which can be achieved through administration of antibodies to the infant or through active immunization of the mother during pregnancy. passive immunity wanes fast over time, thus, active immunization is the preferred approach for infants above six months of age, as well as older children and adults, including the elderly (141). to date, there is only one product available for prevention of rsv infection, palivizumab, the monoclonal antibody (mab) that has been shown to reduce hospital admission due to rsv infection in some high-risk infants by up to 80% (142) . it is expensive and, thus, reserved for high risk infants, mainly in high income countries. active immunization against rsv: looking back to the past. while antibodies are costly and transitory in their effect, active vaccination would represent the most cost-effective approach for the prevention of rsv infections and their transmission to high-risk individuals (115, 141) . up to date, several vaccine candidates are in development, but none have reached licensure yet (143, 144) . one of the main barriers for the development of rsv vaccines has been the fact that the majority of severe cases in infants occur within the first three months of life, i.e. at a time when active immunization is not really possible (145) . additional caution has been employed during vaccine design because of the failure of a historical vaccine [formalin inactivated rsv (fi-rsv)], which triggered a severe adverse effect, enhanced respiratory disease (erd) (146) . for more than 50 years live-attenuated vaccine approaches have been unsuccessful because of the difficulty in balancing immunogenicity and vaccine safety. it is worth noting that only live-attenuated vaccines have been tested for active infant immunization. active immunization against rsv: perspectives. recent breakthroughs in determining the structure and antigenic content of the rsv fusion (f) glycoprotein has enhanced interest in vaccine development research (115, 141, 147, 148) . the general approaches to vaccine development include engineered viruses that use knowledge of rsv gene function, naturally attenuated chimeric virus combining genes from rsv-related viruses, viral vectors encoding rsv surface antigens, and nucleic acid vaccines using plasmid dna or messenger rna encoding rsv antigens (149, 150) . as of august 2019, 43 rsv vaccines were in development (151). of these, 21 are in clinical trials in humans; 14 in phase 1, five in phase 2 and two (one just completed) in phase 3. twelve vaccines are in trials in children, four in pregnant women and 10 in older adults (some products are undergoing trials in more than one target population). vaccine types under investigation include live-attenuated/chimeric, particle-based, subunit and recombinant vector vaccines. this highlights the variety and breadth of immunization types and different populations that are being investigated to find an answer to the 60-year-old problem of producing a safe and effective rsv prophylactic agent. the resvax. the most advanced candidate vaccine, resvax, is an rsv fusion protein recombinant nanoparticle with aluminum phosphate as an adjuvant (115, 152, 153) . the new approach to develop this vaccine is based on engineering small particles that carry altered rsv proteins. the nanoparticles sensitize the immune system to the virus so that when a person comes in contact with it the immune system delivers a robust response. the phase 3 clinical trial included more than 4,600 pregnant women examining the efficacy of prevention of rsv disease in infants through maternal immunization. although the trial narrowly missed its primary end point of a reduction in medically attended rsv-positive alrti, it showed a 44% vaccine efficacy against rsv hospitalization, 25% efficacy against all respiratory hospitalizations and 39% efficacy against all-cause severe hypoxaemia (152) . a possible route to licensure is currently being sought with the us food and drug administration (fda) and european licensing agencies, bringing hope of a vaccine that could save the lives of countless young infants worldwide. we would like to thank the participants of the '5th workshop on paediatric virology' (sparta, greece, october 12th, 2019) for their comments, corrections and feedback. we would also like to thank the organizing committee the '24th world congress on advances in oncology' and the '24th international symposium on molecular medicine' for the outstanding hosting of the workshop, as well as all members of the pvsg and the newly founded institute of paediatric virology (ipv) based on the island of euboea for their valuable contribution in the preparation of the manuscript. no funding was received. not applicable. all authors (inm, sbd, br, mt, gp, ap, eik, ck, elk, va, pk, gpc and das) contributed to the conception and design of the study, wrote the original draft, edited and critically revised the manuscript, read and approved the final manuscript. not applicable. not applicable. das is the editor-in-chief for the journal, but had no personal involvement in the reviewing process, or any influence in terms of 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bronchiolitis the history and physics of heliox helium-oxygen mixture: clinical applicability in an intensive care unit the therapeutic use of helium use of heliox delivered via high-flow nasal cannula to treat an infant with coronavirus-related respiratory infection and severe acute air-flow obstruction noninvasive ventilation with helium-oxygen in children heliox inhalation therapy for bronchiolitis in infants heliox delivered by high flow nasal cannula improves oxygenation in infants with respiratory syncytial virus acute bronchiolitis prevention of rsv infection: what is new with the vaccines? immunoprophylaxis against respiratory syncytial virus (rsv) with palivizumab in children: a systematic review and economic evaluation who rsv vaccine consultation expert group: who consultation on respiratory syncytial virus vaccine development report from a world health organization meeting global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis development of respiratory syncytial virus (rsv) vaccines for infants enhancement of respiratory syncytial virus pulmonary pathology in cotton rats by prior intramuscular inoculation of formalin-inactivated virus vaccine development for respiratory syncytial virus vaccines against respiratory syncytial virus: the time has finally come recombinant low-seroprevalent adenoviral vectors ad26 and ad35 expressing the respiratory syncytial virus (rsv) fusion protein induce protective immunity against rsv infection in cotton rats nucleoside modified mrna vaccines for infections diseases will the resvax vaccine be key revenue driver for novavax. market realist key: cord-016109-vbzy11hc authors: damjanovic, v.; taylor, n.; williets, t.; van saene, h. k. f. title: outbreaks of infection in the icu: what’s up at the beginning of the twenty-first century? date: 2011-08-10 journal: infection control in the intensive care unit doi: 10.1007/978-88-470-1601-9_12 sha: doc_id: 16109 cord_uid: vbzy11hc surveillance cultures are the only cultures that allow the distinction between secondary endogenous and exogenous infections. these types of infection are the two known to cause outbreaks. secondary endogenous infections can be controlled by enterally administered antimicrobials and should be integrated into the routine infection control measures. exogenous infections can be controlled by topically applied antimicrobials and hygiene. two recent sets of publications were taken into consideration when preparing our analysis of infectious outbreaks in the intensive care unit (icu). the first concerns the emergence of severe acute respiratory syndrome (sars) and avian flu in 2003, and a spread across the world of a novel influenza caused by swh1n1 in 2009. these viral infections had a major impact on intensive care and are described in chap. 20 . this chapter is dedicated to describing outbreaks caused by bacteria and fungi, with references to secondary infections associated with flu and sars [1, 2] . the second publication concerns the ''international study of the prevalence and outcomes of infection in intensive care units'' published in december 2009 [3] . although this is a point-prevalence study, it provides information about the global epidemiology of infection in icus. unfortunately, it could not give insight into outbreaks of infection in icus, so we searched for specific publications describing such outbreaks. in the second (2005) edition of this book, we analysed the usefulness of molecular techniques in selected outbreaks [4] . the majority of outbreaks occurred in the last decade of the twentieth century. however, reports were usually published several years later. a similar pattern was observed when we analysed outbreaks published in the first decade of the twenty-first century: the actual outbreaks occurred a few years earlier. indeed, the above-mentioned point-prevalence study was conducted on 8 may 2007 but published in december 2009 [3] . therefore, for accuracy, this analysis indicates when outbreaks actually happened and when they were subsequently published. acinetobacter outbreaks were selected to illustrate this point ( fig. 12.1 ). in addition to the reported outbreaks, a number of publications considered many relevant aspects of infection and outbreaks in icu. some of these are included in this chapter. we analysed 97 publications, the majority of which met the definition of an outbreak in neonatal (nicu), paediatric (picu) and adult (aicu) icus and reported since 2000. the main objective of this analysis was to find out whether there were any new features in the outbreaks of infection in icu at the beginning of the new century, including those influenced by new viruses. we searched medline for outbreaks published between january 2000 and september 2009. the search terms used were intensive care unit, adult icu, paediatric icu, neonatal icu and outbreaks. we used the same framework as in the second edition of this book; however, outbreaks were not presented separately per icu type but according to causative organisms, in the following order: methicillin-resistant staphylococcus aureus (mrsa), vancomycin-resistant enterococci (vre), aerobic gramnegative bacilli (agnb), pseudomonas spp., acinetobacter spp. and fungi, together with the selected features searched (table 12 .1). the number of analysed outbreaks is stated, but only selected outbreaks are shown and listed in the references. we retrieved reports on six outbreaks [5] [6] [7] [8] [9] [10] published since 2000; five occurred in aicus and one in an animal icu. reports of two outbreaks were published in 2002 and three in 2004, all occurring between 1997 and 2000. one report published in 2007 did not report the actual time of the outbreak. these outbreaks are summarised briefly according to their countries of origin. a paper from italy published in 2002 reported a unique experience of controlling a mrsa outbreak of 8 months' duration in a medical/surgical aicu in 1998 using enterally administered vancomycin in mechanically ventilated patients [5] . another report from italy, published in 2004, described the identification of a variant of the ''rome clone'' of mrsa responsible for an outbreak in a cardiac surgery icu, which occurred in 1999 in a hospital in rome. this strain had decreased sensitivity to vancomycin and was resistant to many antibiotics [6] . a study from germany published in 2002 described the occurrence of mrsa in icu in terms of endemic and epidemic infections followed from january 1997 to june 2000.this study involved 139 icus, 51 of which (37%) had mrsa infections. outbreaks (three or more mrsa infections within 3 months) were registered in 13 icus, clusters (two mrsa infections within 3 months) in further 12 units and single events in 26 [7] . a publication from spain showed that enterally administered vancomycin can control endemic mrsa in icus without promoting vre. this study was carried out over a 49-month period from july 1996 to 2000 and published in 2004 [8] . in 2007, a report from canada presented a recent outbreak of mrsa carriage in an animal icu. this finding appears important, as the strain responsible for the animal outbreak was indistinguishable from a strain in humans commonly isolated in canada and the usa. infection control measures, including active surveillance of all animals in the icu, were used to control the outbreak. as transmission of mrsa within the unit occurred without infections and did not persist for a prolonged period of time, staff screening was surprisingly not initiated [9] . a paper from china published in 2004 described an mrsa outbreak due to an increased there have been ten outbreaks in aicus published since 2000: eight were caused by vre, one was sensitive to vancomycin and one was sensitive to vancomycin but resistant to linezolid. we selected seven reports and summarised them according to the countries of origin and time of events and publishing. a paper from pakistan published in 2002 was the country's first experience with a vancomycin resistant enterococcus faecium outbreak in the icu and nicu. the outbreak occurred in 2002, lasted 1 month and all but one isolate was of a single clone [11] . all isolates were resistant to gentamicin, ampicillin and tetracycline but sensitive to chloramphenicol. six patients were colonised and four infected, with positive blood cultures; two of each died before specific therapy could be started (50% mortality rate). in 2005, a report from italy described an outbreak of vre colonisation and infection in an icu that lasted 16 months (2001-2002) [12] . fifty-six patients were colonised by e. faecium, and e. faecalis was detected in only two cases. because of the low pathogenicity of vre, the authors questioned whether it was worthwhile to have a specific vre surveillance programme. for the 2004 lowbury lecture, pearman reported the australian experience with vre, which he described as ''from disaster to ongoing control''. this was the first outbreak of vre, which was caused by e. faecium in an icu and hospital wards and lasted 5 months in 2001. a vigilant vre control programme prevented the epidemic strain from becoming endemic in the hospital [13] . an outbreak due to glycopeptide-resistant enterococci (gre) in an icu with simultaneous circulation of two different clones was reported from france in 2008. the outbreak lasted several months in 2003 without infections, but the significant colonisation caused organisational problems in the icu [14] . an outbreak of vre in an icu was reported from china in 2009. the outbreak was caused by e. faecium and lasted 11 months (2006-2007) . a detailed molecular analysis showed that genetically unrelated isolates had transferred vancomycin resistance by conjugation [15] . a paper from korea reported an outbreak of vre in a neurological icu. vre was mainly isolated from urine specimens associated with the presence of a foley catheter. of 52 patients colonised with vre, only two had active infection [16] . in 2009, a report from spain presented an outbreak of linezolid-resistant e. faecalis in an icu and reanimation unit [17] . this was the first report of a clonal outbreak of linezolid-resistant e. faecalis in spain. the strain was sensitive to imipenem, vancomycin, teicoplanin and rifampicin. most patients were exposed to linezolid within a year (2005) (2006) . the use of linezolid began in 2002. the increase in its use continued until 2005 when a mutant was identified by molecular analysis. fourteen reports on outbreaks were retrieved since 2000. eight were caused by klebsiella pneumoniae, four by serratia marcescens, one by enterobacter cloacae and one by simultaneous infection of e. cloacae and s. marcescens. three klebsiella, three serratia and the remaining two were selected for analysis. we discuss pseudomonas and acinetobacter outbreaks separately. an outbreak of klebsiella infection in nicu and picu was published from spain in 2004; this outbreak occurred in 2002-2003 and lasted 1 year [18] . the outbreak was polyclonal. two predominant clones of klebsiella harboured a special gene (shv5) for the beta-lactamase enzyme responsible for multi-drugresistant klebsiella. according to the authors, this type of klebsiella was not reported previously in spain. another clone harbouring two different genes responsible for multidrug resistance but dissimilar from the above was reported. a report from the netherlands published in 2001 described an outbreak of infections with a multi-drug-resistant klebsiella strain [19] associated with contaminated roll boards in operating rooms. this outbreak in 2000 showed how an unusual source of the outbreak can be revealed by systematic surveillance. in 2008, a polyclonal outbreak of extended spectrum beta-lactamase (esbl)-producing k. pneumoniae in an icu of a university hospital in belgium was reported [20] . this was a 2-month outbreak that occurred in 2005 with 18 isolates. there was one predominant clone, two clones with several isolates and four with unique isolates. the cause of the outbreak was not clear but was associated with a dramatic increase in the number of imported carriers during the previous weeks. an outbreak caused by esbl-producing e. cloacae in a cardiothoracic icu was reported from spain in 2007 [21] . the outbreak occurred in 2005, lasted 3 months, and involved seven patients. molecular analysis revealed two clones responsible for the outbreak: one carried a single esbl; the other carried two esbls. both clones showed resistance to quinolones and aminoglycosides. the outbreak was brought under control by the implementation of barrier measures and cephalosporin restrictions. an outbreak was reported from germany in 2002 [22] in both the nicu and picu, lasted from september to november 1998 and involved 15 patients. two epidemic strains were associated with cross-infection in groups of five and ten patients, respectively. two epidemic clones were detected from the surfaces of an icu room, but an original source was not identified. the outbreak was stopped by routine infection-control measures. a report from malaysia in 2004 described an outbreak of serratia infections that lasted 10 days in an aicu [23] . the single outbreak strain was found in insulin and sedative solutions administered to patients. an outbreak of s. marcescens colonisation and infection in a neurological icu that occurred from may 2002 to march 2003 was reported from a dutch university medical centre in 2006 [24] . the outbreak strain was traced to a healthcare worker (hcw) with longterm carriage on the hands. the skin of the hcw's hands was psoriatic. the epidemic ended after the colonised hcw went on leave, with subsequent eradication treatment. a heterogeneous outbreak of e. cloacae and s. marcescens infections in a surgical icu was published by a group of authors from san francisco, usa [25] . the outbreak lasted from december 1997 through january 1998. molecular techniques ruled out a point source or significant cross-contamination as modes of transmission. the authors concluded that patient-related factors, such as respiratory tract colonisation and duration of central line placement might have played a role in this outbreak. several reports have been published on infections caused by multi-drug-resistant pseudomonas spp. in icus since 2000. we retrieved 19 reports; not all were outbreaks, as some were described as endemic infections. in addition, one outbreak was caused by burkholderia cepacia. we selected a few outbreaks that we believed would represent the main problems occurring in icus, such as multidrug resistance, clonality, transmission source and mode and infection severity. in 2000, a publication from norway reported an outbreak of multi-drug-resistant p. aeruginosa associated with increased risk of death [26] . the outbreak occurred from december 1999 to september 2000, was monoclonal and the strain was introduced into the icu early in 1998 and was maintained thereafter. all patients were ventilated. the strain was resistant to carbapenems, quinolones and azlocillin. in 13 infected patients, ten of whom died, pseudomonas was found in one or all specimens, such as respiratory secretions, ventilator tubes, connection tubes and the water catcher of the ventilator system. the bacterium was also isolated from water taps. in addition to enhanced control of infection measures, complete elimination of the outbreak was achieved after water taps were pasteurised and sterile water was used when a solvent was needed. in 2003, french authors published a report on the epidemiology of p. aeruginosa in an icu [27] . although between 1996 and 1997 the prevalence of p. aeruginosa infections reached 30% of all hospital-acquired infections, the authors did not call this an outbreak, despite the fact that this was twice the national prevalence of 15% observed in icus. however, this high prevalence prompted the authors to conduct a prospective epidemiological study from july 1997 to february 1998. we selected this study as a good example of activities necessary to prevent a major outbreak. the authors described how systematic surveillance was carried out (oropharyngeal and rectal swabs on admission and twice weekly afterwards). this practice revealed that during the study period, the overall incidence of p. aeruginosa carriage was 43%: 17% on admission and 26% acquired in the icu. in addition 16/191 (8%) patients developed the infection. the authors also pointed out that intestinal carriage was a prerequisite for colonisation or infection. genotyping analysis of 81 isolates indicated that 70% belonged to genotype 1, 4% to genotype 2 and that remaining isolates were not genetically related. it has also been shown that mechanical ventilation was associated with p. aeruginosa carriage and ineffective antibiotics significantly increased the risk of colonisation and infection in icu. the authors concluded that not only do endogenous sources account for the majority of colonisation or infection due to p. aeruginosa but that exogenous sources may be involved in some instances. in an epidemic setting, the authors' stance was to reinforce standard barrier precautions. however, the main message of this study is the necessity to adopt and pursue preventive measures. in 2008, an outbreak of severe b. cepacia infections in an icu was reported from spain [28] . the outbreak occurred over a period of 18 days in august 2006 when b. cepacia were recovered from different clinical samples associated with bacteraemia in three cases, lower respiratory tract infection in one and urinary tract infection in one. samples of antiseptics, eau de cologne and moisturising milk available on treatment carts were collected and cultured. b. cepacia was isolated not only from three samples of the moisturising body milk that had been applied to the patients but also from two new hermetically closed units. all strains recovered from environmental and clinical samples belonged to the same clone. the cream was withdrawn from all hospital units, and no new cases of b. cepacia developed. the authors concluded that the presence of bacteria in cosmetic products, even within accepted limits, may lead to severe life-threatening infections in severely ill patients. we retrieved 34 publications on acinetobacter outbreaks, 11 of which were not strictly outbreaks, and actually not reported as such, but rather described general epidemiology, antibiotic resistance, infection control or treatment options. most of these problems are dealt with in relevant chapters of this edition. following our approach, we summarise only a few outbreaks, which appeared to offer some new findings or insights. a 2000 report from italy described an outbreak of infusion-related a. baumannii bacteraemia in an eight-bed icu [29] . from 6 june to 15 july 2000, six cases were identified. all patients received parenterally administered solutions prepared by icu nurses, which was subsequently proven to be the source of infection. three patients died from sepsis despite treatment with a combination of meropenem and amikacin, which were shown by laboratory tests to be synergistic. this high mortality rate (50%) was explained by the authors as being due to persistent bacteraemia related to the repeated infusions of contaminated solutions. once aseptic preparation was carried out in the hospital pharmacy, this outbreak was controlled, and further infusion-related nosocomial bacteraemia was prevented. from the usa, a publication in 2001 reported an outbreak of multiresistant acinetobacter colonisation and infection in an icu [30] . the strain was sensitive only to polymyxin. the outbreak lasted an entire year between 1996 and 1997 and involved 57 patients, 27 of whom were infected and 25 colonised. the arrival of a colonised burn patient ([50% total body surface area) from an outside hospital was responsible for the outbreak. although on typing two strains were found, the only identified primary source was the original burn patient. ten deaths resulted from infections (37% of infected patients). the authors claimed that this outbreak served as a model of eradication of multi-drug-resistant organisms, as the acinetobacter was eliminated from all icu patients by multidisciplinary measures that included the following: cohort and contact isolation of all colonised and infected patients; introduction of strict aseptic measures such as hand washing, barrier isolation, equipment and room cleaning; sterilisation of ventilator equipment; and individual dedication of medical equipment to each patient. a paper was published from australia in 2007 regarding carbapenem-resistant a. baumannii [31] . we selected this publication as an illustration of an extensive molecular analysis rather than for a critical review of the outbreak, which occurred in an icu between 1999 and 2000. based on their findings, the authors claim that antibiotic-resistant genes are readily exchanged between co-circulating strains in epidemics of phenotypically indistinguishable organisms. in conclusion, they recommend that epidemiological investigation of major outbreaks should include whole-genome typing as well as analysis of potentially transmissible genes and their vehicles. finally, we found a paper in a journal from kuwait not found by our internet research [32] . the authors reported three different outbreaks of multi-drug-resistant a. baumannii infections involving 24 patients aged 16-75 years that occurred in an icu in the course of 1 year between 2006 and 2007. the outbreak was polyclonal and successfully controlled with tigecycline, to which two causative clones were sensitive. three additional distinct clones were isolated from the environment. due to lack of appropriate surveillance cultures, no explanation was offered for the origin of epidemic clones. subsequently, in a letter to the editor, our interpretation that ''…microbial gut overgrowth increased spontaneous mutation, which led to polyclonality and antibiotic resistance in the critically ill'' was accepted by the authors [33, 34] . thirteen publications were retrieved from medline, five of which described outbreaks of remarkable findings. the remaining papers reported some important aspects of fungal species, colonisation, infection and treatments, predominantly as surveys, and as such were not included in our analysis. outbreaks presented here were caused by uncommon opportunistic fungi. two reports described icu outbreaks caused by hansenula anomala, an opportunistic yeast first reported from a liverpool, uk, nicu in 1986 [35] . in 2001, a report from croatia described an outbreak in a surgical icu [36] . h. anomala was isolated from blood taken from eight patients between 23 august and 6 december 1993. all patients were treated with antifungal therapy; three died from complications of underlying disease. the introduction of strict hygienic measures stopped the spread of infection, but the outbreak ceased with the introduction of a new batch of cotton from another manufacturer, which was used for venipuncture-site disinfection. however, the authors could not find evidence for infection source and transmission route. the second report, from brazil (2005), describes an outbreak in a picu [37] . the authors reported their finding as an outbreak of pichia anomala, a newly introduced name for h. anomala. from october 2002 to january 2004, 17 children developed p. anomala fungemia. the median age was 1.1 year, and the main underlying conditions were congenital malformations and neoplastic disease. the overall mortality rate was 41.2% despite treatment with amphotericin b. during a 2-week period in april 2003, when new cases occurred, surveillance cultures revealed that 67.9% of patients were colonised with yeasts, but no single patient was found to be colonised with p. anomala. thus, no source was found at that time. the outbreak was not controlled until orally administered prophylaxis with nystatin and topical application of an iodoform to venipuncture sites were started. an extraordinary outbreak of invasive gastritis caused by rhizopus microsporus in an adult icu was reported from spain in 2004 [38] . over a 14-week period (between november 1995 and march 1996), gastric mucormycosis was diagnosed in five patients, four of whom were admitted to icu with severe communityacquired pneumonia and one with multiple trauma. the main symptom was upper gastrointestinal haemorrhage. isolated filamentous fungi were identified as r. microsporus var. rhizopodiformis and were detected in gastric aspiration samples and traced to wooden tongue depressors used to prepare medication for oral administration (and given to patients through a nasogastric catheter) and in some tongue depressors stored in unopened boxes unexposed to the icu environment. the outbreak was terminated when contaminated tongue depressors were withdrawn from use. this outbreak was attributable to the 40% mortality rate; wooden material should not be used in the hospital setting. in 2004, an outbreak of three cases of dipodascus capitatus infection in an icu was reported from japan [39] . the index case was pulmonary infection with a fulminant course of fungal infection, which resulted in death, in a patient with acute myelocytic leukaemia who shared a room for at least 1 week with the two other patients, suggesting the possibility of transmission. one of the other two patients died from multiple organ dysfunction. the presence of d. capitatus might have been due to contamination in the respiratory icu. in all cases, d. capitatus was identified in sputum, deep tracheal aspiration samples, blood and urine samples. the authors concluded that d. capitatus should be added to the lengthening list of opportunistic fungal pathogens that can cause infection in immune-compromised patients, with the danger of transmission and potential outbreak. an outbreak of saccharomyces cerevisiae fungemia in an icu was reported from spain in 2005 [40] . during the period from 15 to 30 april, three patients with s. cerevisiae fungemia were identified. the only identified risk factor was treatment with a probiotic containing this yeast. the three patients received the product via nasogastric tube for a mean of 8.5 days before the culture was positive. surveillance cultures for the control patients admitted at the same time did not reveal any carriers. all three patients died from causes unrelated to s. cerevisiae. discontinuation of use of the product for treatment or prevention of clostridium difficile-associated diarrhoea in the unit stopped the outbreak of infection. in conclusion, the authors warned that the use of s. cerevisiae should be carefully reassessed in immune-compromised or critically ill patients. an outbreak is defined as an event where two or more patients in a defined location are infected by identical, often multi-drug-resistant, microorganisms transmitted via the hands of hcw, usually within an arbitrary time period of 2 weeks. there are two different types of infection involved in outbreaks: secondary endogenous and exogenous. outbreaks of secondary endogenous infections are invariably preceded by outbreaks of carriage of abnormal flora, whereas outbreaks of exogenous infections are not preceded by outbreaks of abnormal carriage. these two types of outbreaks each require a different type of management: enterally and topically administered antimicrobials for secondary endogenous and exogenous outbreaks, respectively. ongoing surveillance efforts, i.e. throat and rectal swabs on admission and twice weekly thereafter, to monitor the efficacy of systematic decontamination of the digestive tract (sdd) and to identify the emergence of antimicrobial resistant threats, is an intrinsic component of any decontamination programme. in this sense, a well-designed programme contains an intrinsic degree of protection against antibiotic-resistant organism emergence. surveillance cultures of throat and rectum are more sensitive in detecting resistance than are diagnostic samples [41] . additionally, there is a close relationship between surveillance and diagnostic samples. once a resistant microorganism reaches overgrowth concentrations, i.e. c10 5 /ml saliva and/or gram of faeces, diagnostic samples become positive [8] . in our review, 28 outbreaks were selected to illustrate the situation at the beginning of this century. as a matter of fact, the majority of the outbreaks was related to the previous decade. however, biased or not, our analysis described 19 outbreaks that occurred after 2000 and nine from last century, although the outbreaks were published in this century (fig. 12.1 ). this suggests that some new problems indeed emerged in this century. it is important to record the number of papers retrieved according the causative organisms: mrsa six, vre ten, agnb 14, pseudomonas spp. 19, acinetobacter spp. 23 and fungi 13. perhaps, against our expectation, agnb organisms-in particular, opportunists such as pseudomonas and acinetobacter-prevailed significantly, for which there must be a reason. if we take mrsa as an example, all around the world, this drug-resistant pathogen has been a primary focus for nosocomial infection control and treatment for years. thus, there are fewer outbreaks. an extensive study from germany that involved 139 icus showed that cluster and single mrsa infections were significantly more common than actual outbreaks (38 icus compared with 12, respectively) [7] . to our knowledge, there were no similar studies for vre and agnb, but one would anticipate similar findings and interpretation. on the other hand, opportunistic pathogens such as pseudomonas spp., acinetobacter spp. and fungi often caused unexpected outbreaks, particularly in immunocompromised patients. they originated from external sources and were difficult to treat because of their resistance to multiple antibiotics. our search for specific features relevant to published outbreaks revealed some new, and confirmed some older, trends (table 12. 2). probably the best example of how new viral infections-such as sars-can change the rate of bacterial and fungal infections in icus came from the experience in china [10] . there was a significant increase in the rate of mrsa and candida spp. acquisition in an icu during the sars period. it may be anticipated, therefore, that in the future, sars and influenza viral infections would lead to complex icu outbreaks. we pointed out earlier how using molecular techniques revealed that many outbreaks were due to more than one clone [4] . our analysis confirms this, although the origin of different clones remained obscure in all reports in which polyclonality was detected. however, we recently put forward a hypothesis that microbial gut overgrowth is responsible for increased spontaneous mutation leading to polyclonality and antibiotic resistance [42] . furthermore, extensive use of molecular techniques not only revealed a number of new genes responsible for antibiotic resistance [18] but showed that genetically unrelated organisms readily exchange antibiotic resistance genes [15, 31] . yet further, a new trend is related to the sdd concept. two studies, one from italy and one from spain, reported the use of enterally administered vancomycin to control and prevent, respectively, mrsa outbreaks [5, 8] . this is further evidence that the principle of sdd can be used with antimicrobials directed specifically to the causative organism. as early as 1993 we reported how selective decontamination with nystatin successfully controlled a candida outbreak in an nicu [43] . among older trends, surveillance cultures, or lack of them, are still prominent. even in 2009 there were authors responsible for infection control in hospitals and icus who claimed that ''…surveillance cultures of all patients with potential to develop infection are difficult and very costly…'' [44] . some time ago (1994), we expressed an alternative view in response to an identical attitude [45] . needless to say, lack of surveillance cultures not only delays the recognition of an outbreak and its control but also precludes the understanding of the pathogenesis of the majority of outbreaks. surveillance cultures are also crucial for detecting outbreaks of exogenous pathogenesis, i.e. without carriage. on the other hand, the source of an exogenous outbreak is readily identified with molecular techniques. some of these outbreaks are striking, such as one from this analysis in which acinetobacter-contaminated parentally administered solutions were repeatedly infused to patients, leading to a very high mortality rate of 50% [29] . in conclusion, new trends as well as old confirm what we indicated in the previous edition of this book, which is that to control and prevent icu outbreaks, surveillance cultures and sdd should be integrated in routine infection-control measures. severe acute respiratory syndrome: another challenge for critical care nurses h1n1 influenza is here international study of the prevalence and outcome of infection in intensive care units outbreaks of infection in intensive care units-usefulness of molecular techniques for outbreak analysis enteral vancomycin to control methicillinresistant staphylococcus aureus outbreak in mechanically ventilated patients identification of a variant 'rome clone' of methicillin-resistant staphylococcus aureus with decreased susceptibility to vancomycin, responsible for an outbreak in an intensive care unit occurrence of methicillin-resistant staphylococcus aureus infections in german intensive care units effectiveness and safety of enteral vancomycin to control endemicity of methicillin-resistant staphylococcus aureus in a medical/surgical intensive care unit cluster of methicillin-resistant staphylococcus aureus colonisation in a small animal intensive care unit increase in methicillin-resistant staphylococcus aureus acquisition rate and change in pathogen pattern associated with an outbreak of severe acute respiratory syndrome emergence of vancomycin-resistant enterococcus faecium at a tertiary care hospital in karachi outbreak of vancomycin-resistant enterococcus spp. on an italian general intensive care unit lowbury lecture: the western australian experience with vancomycin-resistant enterococci-from disaster to ongoing control glycopeptide-resistant enterococcus outbreak in an icu with simultaneous circulation of two different clones molecular characterization of outbreak-related strains of vancomycin-resistant enterococcus faecium from an intensive care unit in beijing incidence and risk factors of infections caused by vancomycin-resistant enterococcus colonization in neurosurgical intensive care unit patients nosocomial outbreak of linezolid-resistant enterococcus faecalis infection in a tertiary care hospital outbreak of shv-5 beta-lactamase-producing klebsiella pneumoniae in a neonatal-pediatric intensive care unit in spain outbreak of infection with a multiresistant klebsiella pneumoniae strain associated with contaminated roll boards in operating rooms intensive care unit outbreak of extended-spectrum beta-lactamase-producing klebsiella pneumoniae controlled by cohorting patients and reinforcing infection control measures nosocomial outbreak due to extended-spectrumbeta-lactamase-producing enterobacter cloacae in a cardiothoracic intensive care unit nosocomial neonatal outbreak of serratia marcescens-analysis of pathogens by pulsed field gel electrophoresis and polymerase chain reaction using pulsed-field gel electrophoresis in the molecular investigation of an outbreak of serratia marcescens infection in an intensive care unit outbreak of serratia marcescens colonization and infection traced to a healthcare worker with long-term carriage on the hands a heterogeneous outbreak of enterobacter cloacae and serratia marcescens infections in a surgical intensive care unit an outbreak of multidrug-resistant pseudomonas aeruginosa associated with increased risk of patient death in an intensive care unit epidemiology of pseudomonas aeruginosa and risk factors for carriage acquisition in an intensive care unit mosturizing body milk as a reservoir of burkholderia cepacia: outbreak of nosocomial infection in a multidisciplinary intensive care unit clinical and molecular epidemiology of an outbreak of infusion-related acinetobacter baumannii bacteremia in an intensive care unit eradication of multi-drug resistant acinetobacter from an intensive care unit hospital gene transfer in a polyclonal outbreak of carbapenem-resistant acinetobacter baumannii role of tigecycline in the control of carbapenemresistant acinetobacter baumannii outbreak in an intensive care unit origin of epidemic clones of acinetobacter in the critically ill control of acinetobacter outbreaks in the intensive care unit infection and colonisation of neonates by hansenula anomala hansenula anomala outbreak at a surgical intensive care unit: a search for risk factors an outbreak of pichia anomala fungaemia in a brazilian pediatric intensive care unit outbreak of gastric mucormycosis associated with the use of wooden tongue depressors in critically ill patients an outbreak of dipodascus capitatus infection in the icu: three case reports and review of the literature saccharomyces cerevisiae fungemia: an emerging infectious disease colonization with broadspectrum cephalosporin-resistant gram-negative bacilli in intensive care units during a nonoutbreak period: prevalence, risk factors, and rate of infection microbial gut overgrowth guarantees increased spontaneous mutation leading to polyclonality and antibiotic resistance in the critically ill selective decontamination with nystatin for control of a candida outbreak in a neonatal intensive care unit candida colonisation as a source of candaemia the multiple value of surveillance cultures: an alternative view key: cord-018421-wy3mtafh authors: waghmare, alpana; boeckh, michael title: rhinovirus, coronavirus, enterovirus, and bocavirus after hematopoietic cell transplantation or solid organ transplantation date: 2016-02-15 journal: transplant infections doi: 10.1007/978-3-319-28797-3_32 sha: doc_id: 18421 cord_uid: wy3mtafh respiratory viral infections represent a significant cause of morbidity and mortality in immunocompromised hosts. newer molecular detection assays have allowed for the characterization of several respiratory viruses not previously recognized as having significant clinical impact in the immunocompromised population. human rhinoviruses are the most common respiratory viruses detected in the upper respiratory tract of hematopoietic cell transplant and lung transplant recipients, and evidence on the impact on clinical outcomes is mounting. other respiratory viruses including enteroviruses (evs), coronaviruses (covs), and bocavirus may also contribute to pulmonary disease; however, data is limited in the immunocompromised population. further studies are needed to define the epidemiology, risk factors, and clinical outcomes of these infections; this data will help inform decisions regarding development of antiviral therapy and infection prevention strategies. human rhinoviruses (hrvs), the viruses predominantly associated with the common cold, are highly prevalent in both immunocompetent and immunocompromised individuals. prior to the development of sensitive molecular viral detection assays, infl uenza, respiratory syncytial virus, and parainfl uenza virus were the most common and most concerning respiratory viral pathogens detected in hematopoietic cell transplant (hct) recipients [ 1 ] . due to the development of polymerase chain reaction (pcr) assays for viral detection, hrvs are now known to be the most common viruses detected from respiratory specimens in hct recipients and can account for 25-40% of cases of viral respiratory infections in these patients [ 2 -4 ] (figure 32-1 ). due to their high prevalence and their ability to cause progressive infection, hrvs are also a signifi cant cause of lower respiratory tract infection (lrti) in hct recipients (table 32-1 ) . hrv infection is also common in solid organ transplant (sot) recipients , although the incidence is not known among sot recipients as a whole. in lung transplant recipients , data from older retrospective and prospective studies suggests an incidence of 35-55% among patients with positive respiratory samples [ 5 -7 ] (figure 32-2 ) . in a recent prospective surveillance study of 112 lung transplant recipients, hrvs represented 62% of all positive samples [ 8 ] . among symptomatic lung transplant recipients, hrv represented 34% of all respiratory viruses detected [ 9 ] . hrvs are members of the picornaviridae family and are classifi ed into three species, hrv-a, hrv-b, and hrv-c, based on similarity in genome organization, capsid features, and conserved sequences [ 10 ] . the total number of genotypes continues to grow as new genotypes are characterized; currently at least 160 unique genotypes are described. due to poor growth in traditional viral culture models, hrv-c was only recognized after the development of molecular diagnostic techniques. thus, hrv-c is not a novel species, but rather one that has been circulating unnoticed due to lack of an appropriate diagnostic assay. there are several biologic characteristics of hrv-c that differentiate the species from hrv-a and hrv-b. hrv-a and hrv-b both use icam-1 or ldlr for cell attachment and entry, whereas it appears that hrv-c may utilize a distinct receptor, cadherin-related family member 3, that is associated with asthma susceptibility [ 11 , 12 ] . additionally, hrv-c species are stable at higher temperatures and readily infect upper and lower airways, whereas hrv-a and hrv-b species tend to be more limited to the sinuses and upper airways [ 13 , 14 ] . these biologic characteristics are thought to play a role in variations in clinical outcomes observed among the different species. most immunocompetent patients with hrv present with an afebrile, self-limited syndrome characterized by rhinorrhea, nasal congestion, and malaise, and less frequently sore throat, mild cough, and hoarseness [ 15 -19 ] . hrv may also be associated with exacerbations of sinusitis, chronic bronchitis, and asthma, and with lower respiratory tract syndromes and atypical pneumonias in otherwise healthy people, including the young and the elderly [ 20 , 21 ] . the specifi c mechanisms by which hrvs produce lung diseases are not well understood. hrvs are also implicated in asthma and chronic obstructive pulmonary disease (copd) exacerbations, but again the mechanisms are poorly defi ned. with the widespread availability of pcr diagnostics, data are emerging on the incidence and clinical relevance of hrv infections in immunocompromised patients. early studies relied on culture to detect hrv, a specifi c but insensitive method because the standard viral culture systems are not optimized for hrv detection, especially hrv-c [ 22 ] . for example, a fred hutchinson cancer center surveillance study from 1987 to 1992 detected hrvs in 29 specimens, and only one was from a lower respiratory tract specimen [ 2 ] . a prospective 5-year study at md anderson cancer center cultured specimens specifi cally for hrvs at lower temperatures with roller culture methods, and reported that hrv infections were associated with substantial morbidity and mortality in 7 of 22 (32%) myelosuppressed patients [ 23 ] . in that study, approximately one third of the adult hct recipients who developed symptomatic hrv infections prior to engraftment had progression of upper respiratory tract symptoms to lrti, and all cases with pneumonias were fatal. lung biopsies and autopsies revealed fi ndings consistent with interstitial pneumonitis and/or ards, but no in situ evaluation was performed to defi nitively assess hrv infection. similar reports with evidence of lrti based on radiographic and bal fi ndings continue to be noted [ 24 -26 ] , but it remains unknown if pneumonia is a direct cause of viral invasion of the lung tissue or by host responses in the lung. evidence for in vitro and in vivo replication in lower respiratory tract has been shown in experimental infection, where hrv was isolated from human volunteers after intranasal hrv challenge by in situ hybridization [ 27 ] . the use of rt-pcr continues to provide new information about the frequency of hrv infection. in a study of bal samples from 77 hct recipients that were tested using rt-pcr , hrv was detected in six patients (8%), mortality rate was very high (83%) and two of the six patients showed persistent hrv infection. however, all of the hrv-infected patients had signifi cant coinfections and it was not certain whether hrv infection was the direct cause of poor prognosis [ 25 ] . in a small cohort of patients with hematologic malignancy, lrti was associated with hypoalbuminemia and bacterial copathogens were seen in 25% of patients [ 28 ] . recent studies have shown that immunocompromised adults with hrv demonstrated similar hospital admission rates, intensive care unit admissions, and mortality rates as patients with pandemic h1n1 infl uenza [ 29 ] . several reports have linked hrv infection to severe respiratory failure and even death [ 23 -25 ] . furthermore, recently presented data suggest that lrti associated with hrv leads to a mortality rate comparable to that of rsv, infl uenza virus, and piv [ 30 ] , independent of the presence of co-pathogens. risk factors for mortality following hrv lrti included bone marrow stem cell source, oxygen requirement at time of diagnosis, and steroid use ≥1 mg/kg prior to diagnosis [ 30 ] . other factors that may infl uence clinical severity include the presence of hrv rna in blood, viral load, and hrv species type; however, no data exist in immunocompromised patients to date. hrv viral rna was detected in the sera of 30 (12%) of 243 pediatric patients with severe hrv respiratory infection, with hrv-c being the predominant species [ 31 ] . in healthy pediatric patients, increased respiratory viral load has been associated with hrv lrti and hrvc has been implicated as a more virulent pathogen [ 32 -34 ] . others, however, have shown lack of correlation between hrv-c and oxygen requirement, length of hospitalizations, and coinfections [ 35 ] . the predominance of hrv-c in hct recipients has also been described in small studies, with higher rates of pneumonia in patients with hrv-c detected from the upper respiratory tract [ 36 ] . in a small cohort of patients with hematologic malignancies, the rate of lrti was not different between patients infected with hrv-a, hrv-b, or hrv-c [ 28 ] . the relative risk of hrv-c infection in the immunocompromised population remains unknown, and more research is needed to defi ne the role of strain differences on outcomes. detection and diagnosis of respiratory viral infections prior to transplant is a common clinical concern that has until recently only been evaluated in small cohorts for certain viruses [ 37 -40 ] . in a large, prospective surveillance cohort of allogeneic hct recipients, detection of hrv pretransplant was associated with signifi cantly fewer days alive and out of the hospital, and signifi cantly higher mortality at 100 days posttransplant [ 41 ] . further, larger prospective studies are needed to determine risk factors for posttransplant complications, the role of viral load and symptom burden at the time of transplantation, and the need to potentially delay transplantation for patients with hrv present prior to transplantation. ultimately, the issue of viral causality of disease and evaluation of prophylactic and treatment modalities will need to be addressed. the impact of hrv infection prior to sot is not known. like hct recipients, sot recipients are exposed to highly immunosuppressive regimens that leave them susceptible to respiratory viral infections. lung transplant recipients have the added disadvantage of altered lung immunity due to factors such as impaired ciliary clearance, poor cough refl ex, and abnormal lymphatic drainage. these factors can predispose to lower respiratory tract infections. the impact of hrv on outcomes in lung transplant recipients can range from asymptomatic infection to severe disease. in a pooled analysis of all respiratory viruses detected in lung transplant recipients, viruses were detected fi ve times more frequently when respiratory symptoms were present [ 42 ] . a correlation between higher symptom scores and higher rhinovirus load in the upper respiratory tract has been demonstrated, although even asymptomatic patients can have relatively high viral loads [ 43 ] . the relative rate of progression from upper to lower tract disease for hrv specifi cally is not known, although the effect on lung function has been evaluated in aggregate for all respiratory viruses and suggests a decline in forced expiratory volume (fev1) of −5% to −30% [ 42 ] . for hrv specifi cally, the fev1 loss was similar to that seen in other respiratory viruses [ 8 ] . the correlation between respiratory viral infections and acute rejection, chronic rejection, and bronchiolitis obliterans syndrome (bos) remains somewhat unclear, with several confl icting fi ndings when respiratory viruses were evaluated in aggregate [ 6 -8 ] . a recent large cohort of 250 lung transplant recipients, however, showed an independent association between respiratory viral infections (34% hrv) and chronic lung allograft dysfunction in multivariate models [ 9 ] . this association was infl uenced by time, with more of an effect within a shorter period following respiratory infection. larger, prospective studies investing individual viruses are needed to clearly assess the impact on these outcomes. unlike paramyxoviruses, hrv infection cannot be diagnosed based on characteristic histopathologic changes or changes in cell morphology. in the past, cell culture was used to diagnose hrv infection using multiple cell lines at low temperatures of 33-34 °c, often in rolling tubes. the cell lines utilized for the detection of hrvs may detect enteroviruses; hrv isolates are distinguished from enteroviruses by their lability in acid (loss in viral titer following exposure to a ph of 5). there are no commercially available antigen-detection assays or simple kits for the detection of hrv. rt-pcr has dramatically improved the ability to both detect and characterize hrvs, with current assays at least two to three times more sensitive than conventional culture methods [ 44 ] . some pcr assays are able to distinguish between enteroviruses and hrvs instead of the acid lability assays [ 45 ] . typing of hrvs based on pcr amplifi cation sequence variations in 5′-noncoding region also has been described [ 46 ] . new standardized methods to detect more of the over 100 strains of hrv have now been described [ 47 ] ; however, commercially available multiplex respiratory viral pcr panels contain primer/probe sets that can cross-react between enterovirus and hrv strains. new strains and types of hrv are being detected frequently and more diseases associated with hrv are being described using new and diverse molecular methods. there are no approved antivirals for the treatment of hrv infections. several agents have been evaluated in preclinical and clinical trials for the treatment of hrv infection in immunocompetent hosts, including capsid binding inhibitors, protease inhibitors, and rna synthesis inhibitors [ 48 ] . none of these agents have been evaluated in immunocompromised hosts. given the high prevalence and potential severity of hrv infection in this population, there is a great need for drug development and clinical trials for the prevention and treatment of lrti. outside of transplant recipients, there is a potential need for intervention in other populations such as patients with asthma or copd to prevent disease exacerbation [ 49 , 50 ] . covs are a frequent cause of the common cold, but little is known about the role of covs in immunocompromised patients [ 51 ] (table 32 -1 ). human group 1 (subtypes 229e and nl63) and human group 2 (oc43 and hku1) covs were originally reported as causes of human respiratory illnesses. the availability of more sophisticated diagnostic tools, such as rt-pcr , has facilitated the detection of covs in normal and immunocompromised persons. these improved molecular methods of viral discovery facilitated the recent identifi cation of the novel group 1 and 2 human cov subtypes-nl63 in 2004 [ 52 ] and hku1 in 2005 [ 53 ] . a more accurate clinical epidemiology of cov infection is beginning to emerge. it is now known that all four known subtypes of cov circulate simultaneously [ 54 ] , and that in addition to the common cold, cov is associated with upper respiratory tract infection and lrti in persons with and without underlying conditions [ 55 , 56 ] . in lung transplant recipients, covs appear to be the second most common respiratory viruses after picornaviruses with a detection rate of 13-27% of positive samples [ 5 -7 ] (figure 32-2 ) . in a prospective surveillance cohort of lung transplant recipients, coronaviruses were detected in 13% of all positive samples, again only second to picornaviruses [ 8 ] . two additional covs associated with outbreaks are the severe acute respiratory syndrome-associated cov (sars-cov) and the recently described middle east respiratory syndrome-cov (mers-cov) . the sars outbreak originated in guangdong province in china in 2002 and was characterized by a life-threatening, atypical pneumonia and was spread by close contact with infected humans, mostly to household contacts and health care workers [ 57 ] . sars-cov is not currently circulating in the world with the most recent human cases of infection reported in china in 2004 [ 58 ] . mers-cov fi rst emerged in the arabian peninsula in 2012, and since then travel-associated cases have been found in a number of countries outside the region [ 59 ] . in adults, the fatality rate is estimated to be 40%; in children asymptomatic infection is common but patients with underlying medical conditions are at increased risk [ 60 , 61 ] . there is little data on the incidence of sars-cov and mers-cov in immunocompromised hosts, although immune suppression is considered a risk factor. sars-cov has been described in liver transplant recipients and in patients with myelodysplastic syndrome [ 62 , 63 ] . mers-cov has been described in patients on chronic immunosuppression and in renal transplant recipients with a broad range of clinical presentations [ 64 , 65 ] . other covs have been reported to cause pneumonia in children and immunocompromised patients treated for hematologic malignancies [ 66 -68 ] . the role of coronavirus virus infection prior to transplantation is not known. although most cov infections result in relatively mild upper respiratory tract infection, these viruses have been associated with more severe lrti (e.g., bronchiolitis and pneumonia) in patients who are immunosuppressed, have asthma, or are premature. in one retrospective study carried out over 1 year, cov was detected in six immunocompromised children-fi ve with acute lymphocytic leukemia and one renal transplant recipient [ 54 ] . five patients were febrile at the time coronavirus was present, with fevers lasting 1-7 days. all patients initially presented with rhinorrhea and nasal discharge; two children had cough as a presenting symptom. chest radiographs of only one of the three children were abnormal; lrti based on decreased oxygen saturation, tachypnea, and abnormal chest radiograph was present in only one child with leukemia, who was signifi cantly neutropenic and lymphopenic at the time cov was detected. covs have been associated with lrti in hct recipients with sometimes fatal outcomes [ 66 , 67 , 69 -71 ] . the clinical characteristics of sars-cov and mers-cov infection in hct and sot patients are not well described, and presentation can range from mild symptoms to respiratory failure and death [ 62 -65 , 72 ]. until the advent of rt-pcr , techniques for the detection of cov were limited and the reliable identifi cation of cov was problematic. early detection techniques isolated two subtypes-oc43 and 229e, originally using organ cultures of human embryonic trachea, with morphology determined using negative staining with electron microscopy [ 73 ] . with the advent of molecular detection methods and increased interest in cov detection during the sars outbreak, new strains of covs have been discovered and new rt-pcr assays developed that facilitate further studies of these viruses. based on rt-pcr assays, four strains of non-sars covs (oc43, 229e, nl63, and hku1) appear to cocirculate during the non-summer months in temperate climates, and are associated with symptomatic disease in immunocompromised hosts [ 54 ] . guidance on rt-pcr and serologic assays for the confi rmation on mers-cov can be found on the world health organization website [ 74 ] . there are no approved antivirals for prophylaxis or treatment of cov infections and supportive care remains paramount in managing patients infected with coronaviruses. though several antivirals were used during the sars-cov epidemic, no clear benefi t could be established on systematic review [ 75 ] . oral ribavirin was evaluated in retrospective studies for the treatment of mers-cov in immunocompetent individuals; decreased survival was noted in one study when compared to matched controls [ 76 , 77 ] , however, larger prospective studies are needed to show true effi cacy. shedding of all coronaviruses may persist for up to months, and routine infection control practices are encouraged. evs are part of the picornaviridae family of viruses and can be associated with severe illness in immunocompromised hosts. evs include polioviruses, coxsackieviruses, and echoviruses; these are now all classified into four species: enterovirus a (ev-a), ev-b, ev-c, and ev-d. risk for infection and subsequent poor outcomes appears to be heavily influenced by age, although factors such as sex and socioeconomic status play a role in the general population. ev activity can be either sporadic or epidemic, and several outbreaks have been described. evs are typically found during the summer and early autumn in temperate climates. enterovirus-d68 (ev-d68) was fi rst identifi ed in california in 1962 [ 78 ] and has since been associated with several small outbreaks, both in the us and internationally, from 2009 to 2013 [ 79 -84 ] . in the summer of 2014, several hundred cases of severe respiratory illnesses in children in the united states were found to be associated with ev-d68 infection [ 85 ] , and several additional clusters have been described worldwide [ 86 -96 ] . evs can cause a wide spectrum of illnesses in immunocompetent individuals including asymptomatic infection, poliomyelitis, meningitis, encephalitis, cardiac disease, muscle disease, eye infections, respiratory infections, exanthems, and neonatal disease. the most frequently described manifestation in immunocompromised patients is respiratory disease, although the incidence and spectrum of disease is not known. according to one study, evs can be associated with lower respiratory tract infection and mortality; however, larger studies are needed to establish specifi c risk factors for worse outcomes [ 97 ] . most confi rmed cases of ev-d68 infection have been in children, occurring primarily in patients with underlying lung disease such as asthma or a history of wheezing. ev-d68 was also associated with several cases of acute fl accid paralysis in children during the 2014 outbreak in the united states, although defi nitive causation has not yet been established [ 98 , 99 ] . the impact of ev-d68 infection in immunocompromised hosts is not known; however, the association between ev-d68 and severe illness was described in eight adult immunocompromised patients with presumptive ev-d68 infection including hct recipients [ 100 ] . additionally, one recent report of adults with confi rmed ev-d68 infection included solid organ transplant recipients [ 87 ] . depending on the clinical scenario, evs can be detected from a number of clinical specimens including cerebral spinal fl uid, serum, respiratory specimens, cardiac tissue, and stool. evs may be identifi ed in throat samples as well as fecal specimens and cerebrospinal fl uid. commercial multiplex pcr assays contain primer/probe sets that may cross react between rhinoviruses and enteroviruses. a specifi c ev-d68 rt-pcr has been developed by the cdc and has been made publically available [ 101 ] . there are no approved antivirals approved for the treatment of evs. intravenous immunoglobulin (ivig) has been used in the treatment of neonatal enteroviral sepsis, but the effect on clinical outcomes is highly dependent on the presence of specifi c neutralizing antibodies and timing of administration [ 102 , 103 ] . pleconaril , an oral capsid inhibitor with activity against picornaviruses, has been evaluated in treatment of enteroviral infections including meningitis, neonatal sepsis, and respiratory infections [ 104 -108 ] but is not available for treatment. other capsid binders, protease inhibitors, and polymerase inhibitors are in various stages of development, but none are currently available for treatment of enteroviral infections [ 109 ] . no studies have shown effi cacy in immunocompromised hosts. human bocavirus (hbov) is a newly identifi ed human parvovirus that was originally identifi ed by random pcr amplifi cation/cloning technique on pooled respiratory secretions from hospitalized children with respiratory tract symptoms [ 110 ] . this virus was named "human bocavirus," due to its relatedness to the genome organization of two other parvoviruses, bovine parvovirus and minute virus of canines, in the family parvoviridae. this virus continues to be detected in young children with a winter seasonality [ 111 -113 ] . the relationship of hbov and respiratory disease in immunocompromised patients is not yet clear. preliminary evidence to date demonstrates case reports of disseminated hbov infection with involvement of the respiratory tract, blood, and stool in several patients, sometimes associated with gvhd and prolonged viral shedding in the feces [ 114 , 115 ] . other studies have reported little evidence linking this virus with pulmonary pathology or severe respiratory disease in hct or lung transplant recipients [ 116 -118 ] . further research is necessary to link this virus with the disease in the transplant recipient. no specifi c antiviral therapy is available. respiratory viruses are a signifi cant concern following hct and sot and can be associated with substantial morbidity and mortality, even among viruses traditionally not concerned pathogenic. new, sensitive diagnostic assays allow for routine detection of rhinoviruses, enteroviruses, coronaviruses, and bocavirus, and additional data on the epidemiology, risk factors, outcomes of infection, and the impact of different viral strains are desperately needed. preliminary studies suggest that detection of these viruses prior to transplant may affect outcomes, but additional studies are needed to explore this important clinical area. furthermore, as new antivirals are being developed, it will be important to identify high-risk patients that may benefi t from treatment. finally, a better understanding of these viruses will be able to inform better infection prevention 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acute severe fl accid myelitis associated with enterovirus d68 infection in children enterovirus d68 infection low-level circulation of enterovirus d68-associated acute respiratory infections emergence of enterovirus d68 in denmark clinical characteristics and molecular epidemiology of enterovirus infection in infants <3 months in a referral paediatric hospital of barcelona enterovirus d68 nosocomial outbreak in elderly people upper and lower respiratory tract infections by human enterovirus and rhinovirus in adult patients with hematological malignancies a novel outbreak enterovirus d68 strain associated with acute fl accid myelitis cases in the usa (2012-14): a retrospective cohort study a cluster of acute fl accid paralysis and cranial nerve dysfunction temporally associated with an outbreak of enterovirus d68 in children in colorado clinical disease due to enterovirus d68 in adult hematologic malignancy patients and hematopoietic cell transplant recipients ev-d68) 2014 outbreak strain-specifi c real-time reverse transcription/polymerase chain reaction (rrt-pcr) assay instructions neonatal enterovirus infection: virology, serology, and effects of intravenous immune globulin effect of intravenous immunoglobulin for neonates with severe enteroviral infections with emphasis on the timing of administration enteroviral meningitis: natural history and outcome of pleconaril therapy oral pleconaril treatment of picornavirus-associated viral respiratory illness in adults: effi cacy and tolerability in phase ii clinical trials effi cacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses a randomized, double-blind, placebo-controlled trial of pleconaril for the treatment of neonates with enterovirus sepsis replication and inhibitors of enteroviruses and parechoviruses from the cover: cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infection frequent and prolonged shedding of bocavirus in young children attending daycare human bocavirus 1 primary infection and shedding in infants disseminated bocavirus infection after stem cell transplant persistence of human bocavirus dna in immunocompromised children rare and emerging viral infection in the transplant population human bocavirus: passenger or pathogen in acute respiratory tract infections? absence of human bocavirus in bronchoalveolar lavage fl uid of lung transplant patients key: cord-002043-z1b7pj3s authors: wang, xue-yang; yu, hai-zhong; geng, lei; xu, jia-ping; yu, dong; zhang, shang-zhi; ma, yan; fei, dong-qiong title: comparative transcriptome analysis of bombyx mori (lepidoptera) larval midgut response to bmnpv in susceptible and near-isogenic resistant strains date: 2016-05-11 journal: plos one doi: 10.1371/journal.pone.0155341 sha: doc_id: 2043 cord_uid: z1b7pj3s bombyx mori nucleopolyhedrovirus (bmnpv) is one of the primary pathogens causing severe economic losses in sericulture. however, the molecular mechanism of silkworm resistance to bmnpv remains largely unknown. here, the recurrent parent p50 (susceptible strain) and the near-isogenic line bc9 (resistance strain) were used in a comparative transcriptome study examining the response to infection with bmnpv. a total of 14,300 unigenes were obtained from two different resistant strains; of these, 869 differentially expressed genes (degs) were identified after comparing the four transcriptomes. many degs associated with protein metabolism, cytoskeleton, and apoptosis may be involved in the host response to bmnpv infection. moreover, some immunity related genes were also altered following bmnpv infection. specifically, after removing genetic background and individual immune stress response genes, 22 genes were found to be potentially involved in repressing bmnpv infection. these genes were related to transport, virus replication, intracellular innate immune, and apoptosis. our study provided an overview of the molecular mechanism of silkworm resistance to bmnpv infection and laid a foundation for controlling bmnpv in the future. the silkworm, bombyx mori l. (lepidoptera: bombycidae) has been domesticated for production of cocoons for more than 5000 years. silkworm rearing and the silk industry still play an important role in china, india and many other developing countries. b. mori is also a good model for the study of insect genetics and immunology [1] [2] [3] [4] . bombyx mori nucleopolyhedrovirus (bmnpv) is the principal silkworm pathogen and causes serious economic losses in sericulture every year. among numerous silkworm strains, most are susceptible to bmnpv infection, although a few strains exhibit resistance [5] . the heredity of silkworm resistance against bmnpv infection is a relatively complicated process because resistance is controlled both by major dominant genes and multiple genes of micro-effect [6] . a series of studies have made significant progress in understanding silkworm resistance against bmnpv infection. xu et al. reported that bms3a was potentially involved in resistance to bmnpv infection [7, 8] . b. mori lipase-1, serine protease-2 and alkaline trypsin protein extracted from the digestive juice of larvae midguts showed strong antiviral activity in vitro [9] [10] [11] . using comparative proteomics, arginine kinase was found to be involved in the antiviral process of different resistant strains of silkworm [12] . in our laboratory, a total of 12 proteins that are potentially involved in viral infection were identified using one-and two-dimensional electrophoresis followed by virus overlay assays. these proteins could be categorized into the following groups: endocytosis, intracellular transportation, and host responses [13] . immune responses were found to be synergistically regulated by the toll, janus kinase/signal transducers and activators of the transcription (jak/stat) and immune deficiency (imd) pathways, which could act as an important defense against exogenous pathogenic infection in conjunction with subsistent pathogen recognition receptors and response proteins [14] [15] [16] [17] [18] . however, the molecular mechanisms of silkworm resistance to bmnpv infection are still not fully elucidated. in recent years, the high-throughput nature of next generation sequencing (ngs), using platforms such as illumina hiseq tm 2500 have provided fascinating opportunities in the life sciences and dramatically improved the efficiency and speed of gene discovery, especially in the research of host cell responses to exogenous pathogenic infection [19] . for example, hu et al. obtained numerous differentially expressed genes (degs) involved in metabolism, immunity, and inflammatory responses in microtus fortis following infection with schistosoma japonicum based on comparative transcriptome analysis [20] . diege et al. examined different fish tissues infected with salmon anemia virus (isav) using high-throughput transcriptomics and found a strong correlation between functional modules and viral-segment transcription [16] . ngs technology was also used to explore the molecular mechanism of silkworm resistance against exogenous pathogens. kolliopoulou et al. reported that several genes related to physical barriers, immune response, proteolytic/metabolic enzymes, heat-shock proteins, and hormonal signaling were possibly involved in silkworm resistance against bombyx mori cytoplasmic polyhedrosis virus (bmcpv) infection; although these genes might be induced by the virus in order to increase infectivity [21] . additionally, several candidate genes, such as bmets, bmtoll10-3 and hsp20-1, have been identified in the initial stage of bmnpv infection by analyzing the global transcriptional profile of silkworm cell lines and heads following bmnpv infection [22, 23] . in order to gain a global view of the molecular changes in silkworms during bmnpv infection, we selected near-isogenic line bc9 and recurrent parent p50 for transcriptome sequencing. through comparative analysis of the transcriptomes from these two strains, a total of 869 degs were obtained, which included many genes potentially related to bmnpv-resistance. our results may provide some reliable evidence to clarify the bmnpv-resistance molecular mechanism in silkworm. bmnpv (t3 strain) was maintained in the key laboratory of sericulture, anhui agricultural university, hefei, china. the virus was obtained from the hemolymph of infected larvae and purified by repeated and differential centrifugation according to the protocol developed by rahman et al. [24] . the concentration of the virus (ob/ml) was determined by hemocytometer. the recurrent parent p50 (susceptible strain), the donor parent a35 (resistant strain) and the near-isogenic line bc9 were maintained in our laboratory. the near-isogenic line was constructed according to the protocol used by yao et al. [6] . in brief, the recurrent parent p50 was crossed to the donor parent a35; progeny were repeatedly backcrossed with the recurrent parent for nine generations and each progeny was screened by bmnpv. the first three instar larvae were reared on a fresh artificial diet at 26±1°c, 75±5% relative humidity, and a 12 hours day/night cycle. the rearing temperature for the last two instars was reduced to 24±1°c; other conditions were unchanged. on the first day of fifth instar, all larvae were starved for 24 hours and then fed with 5 μl bmnpv suspended in sterile water (1.0×10 5 ob/ml) per larva orally; the control group was treated with sterile water. bmnpv occlusion bodies (ob) began fast proliferation at approximately 24 hours post inoculation (hpi) [25] ; thus, this time was considered optimal for sample collection. silkworm larvae were dissected and the midgut tissues were removed and then washed in pbs (137 mm nacl, 2.7 mm kcl, 4.3 mm na 2 hpo 4 , and 1.4 mm kh 2 po 4 , ph 7.4) prepared with diethy pyrocarbonate (depc) (sangon, china) treated h 2 o. thirty larvae midguts were mixed together to minimize individual genetic differences. samples were flash-frozen in liquid nitrogen and pulverized, and 100 mg of sample were added directly into a rnaase free microcentrifuge tube containing 1.0 ml trizol reagent (invitrogen, usa) and stored at -80°c for later use. the level of silkworm resistance to bmnpv was tested following the protocol developed by cheng et al. [26] . the fourth instar larvae were inoculated with bmnpv at different concentrations; inoculations were conducted in triplicate. the level of silkworm resistance was calculated using ibm spss statistics 20 (ibm, usa). the silkworm midguts dissolved in trizol reagent were homogenized. total rna of midguts were extracted according to the manufacturer's protocol. concentrations were quantified using a nanodrop 2000 spectrophotometer (thermo scientific, usa). the purity of all rna samples were assessed at an absorbance ratio of a 260/280 and a 260/230 , and the integrity of the rna was confirmed by 1% agarose gel electrophoresis. library construction, illumina sequencing and read assembly fragment interruption, cdna synthesis, addition of adapters, pcr amplification and rna--seq were performed by beijing biomarker technologies (beijing, china). the standard illumina methods and protocols were adopted to prepare and sequence the cdna libraries. nebnext poly(a) mrna magnetic isolation module (neb, usa) was used to enrich mrna, and then the cdna library was constructed using the nebnext mrna library prep master mix set for illumina (neb, usa) and nebnext multiplex oligos for illumina (neb, usa). the size of the library insert fragments was determined by 1.8% agarose gel electrophoresis, and the fragments were quantified using a library quantification kit/illumina ga universal (kapa, usa). suitable fragments were selected as templates and sequenced on an illumina hiseq tm 2500 using paired-end technology. three biological replicates were used to minimize sample differences. in order to obtain clean and high-quality reads for sequence assembly, the raw reads were filtered by removing adaptor sequences, low-quality sequences (reads with ambiguous bases 'n') and reads with 10% > q < 20% bases [27, 28] . the trinity assemble program was used to assemble the clean reads into contigs, which covered more full-length transcripts through a broad range of expression levels [29] . the resultant contigs were added to transcripts based on paired-end information. the longest transcript from alternative splicing transcripts was selected as the unigene. these unigenes were combined to produce the final assembly and used for annotation. to annotate unigenes, different sequences were searched by blastx against the ncbi nonredundant protein (nr) database and other databases, including swiss-prot protein database, the kyoto encyclopedia of genes and genomes (kegg) and the cluster of orthologous groups (cog) database. gene ontology (go) annotations, including molecular functions, biological processes, and cellular components, were obtained using the blast2go program (https://www.blast2go.com/) [30, 31] . all searches were performed with an e-value < 10 −5 . fragments per kilobase of transcript per million fragments mapped (fpkm) was calculated to represent the expression abundance of the unigenes [32] . fpkm may reflect the molar concentration of a transcript by normalizing for rna length and for the total read number. after normalizing genes expression levels, degs were obtained by pair-wise comparison of the four transcriptome libraries using ideg6 software [33] . an fpkm fold change of 1.2 or 0.83 between two libraries was defined as the reference standard, with the benjamini-hochberg false discovery rate (fdr < 0.01) used to adjust the p-values. in order to validate the results from our transcriptome sequencing analysis, the relative expression levels of 15 randomly selected genes were confirmed by reverse transcription quantitative pcr (rt-qpcr). additionally, 9 genes with well reported previously were selected to further validate the genes of interest that might be involved in bmnpv resistance. all the primers are listed in table 1 . rt-qpcr reactions were prepared with the sybr premix ex taq tm kit (takara), following the manufacturer's instruction. reactions were carried out in bio-rad cfx96tm real-time system (bio-rad, usa). the thermal cycling profile consisted of an initial denaturation at 95°c for 30 s, 40 cycles at 95°c for 5 s, and 60°c for 30 s. all samples were performed in triplicate. relative expression levels were calculated using the 2 -44ct method following the protocol of livak et al. [34] . in this study, b. mori ribosomal protein s3 (bmrps3) gene was used as a reference gene. statistical analysis was conducted using the spss software (ibm, www.ibm.com). the lc 50 value was used to evaluate the resistant level of silkworm to bmnpv infection. the lc 50 value of a35 was approximately 26-fold greater than that of bc9 and over 500-fold greater than that of p50. the value of bc9 was 23-fold greater than that of p50 ( table 2 ). transcriptome sequencing is an efficient technology for comparing gene expression levels in different samples, and in our study, it was used to search and analyze degs among p50, bc9 control and treatment groups. a total of four cdna libraries were sequenced: p50-(p50 treated with sterile water), p50+ (p50 infected with bmnpv), bc9-(bc9 treated with sterile water), bc9+ (bc9 infected with bmnpv), with each group created in triplicate. after removing the adaptors and low quality sequences, 144,439,382 sequence reads were obtained ( table 3 ). the gc content of each of the four libraries was approximately 50%, and cycleq30% was greater than 89.91% for each library. thus, the quality and accuracy of the sequencing data were sufficient for further analysis. most of the reads matched silkworm genomic locations. all the unigenes matched previously described sequences with approximately 70% coverage. the length distribution of unigenes had similar patterns among the four libraries, suggesting that there was little bias in the construction of the four cdna libraries (fig 1) . in order to annotate the unigenes, reference sequences were searched using blastx against the ncbi nr database (e-value < 10 −5 ). a total of 12,591 of 13,342 unigenes provided a blast result (s1 table) . s2 table shows the species with the closest match for each unigene. most of table 1 . primers used in rt-qpcr for validation of degs. forward primer reverse primer doi:10.1371/journal.pone.0155341.t001 in order to determine the reliability of the transcriptome sequencing, the relative expression levels of 15 randomly selected genes were analyzed by rt-qpcr (fig 2a) . the results were consistent with the transcriptome data. for example, the gene encoding peptidoglycan-recognition protein was down-regulated in both rna-seq and rt-qpcr analyses, with a similar fold change. the lipase 1 gene was significantly up-regulated in the resistant strain bc9 after infection with bmnpv, which was also consistent with previously reported results [10] . linear regression analysis of the correlation between rt-qpcr and ran-seq ( fig 2b) showed an r 2 (goodness of fit) value of 0.9169 and a corresponding slope of 1.5281, suggesting a strong positive correlation between rt-qpcr and transcriptome data. therefore, the transcriptome data were satisfied for further analysis. to further elucidate which degs had a potential role in antiviral response, a venn diagram was constructed. a total of 285 degs were found to be differentially regulated when comparing p50+ and p50-, of which 122 were up-regulated and 163 were down-regulated. similarly, 193 degs were found to be differentially regulated in the comparison of bc9+ vs. bc9-, with 56 genes up-regulated and 137 down-regulated. in addition, there were 154 degs differentially regulated in the comparison of bc9-and p50-, among which 78 genes were up-regulated and 76 genes were down-regulated (fig 3, table 4 , s3 table) . there were 197, 119, 82 unique degs in p50+ vs. p50-, bc9+ vs. bc9-and bc9-vs. p50-, respectively. go assignments were used to assign a functional classification to these degs. for cellular components, the number of unique degs fell into the macromolecular complex classification was distinct in bc9 and p50 following bmnpv infection. for molecular functions, the number of transporter activity related unique degs was distinct in bc9 and p50 following bmnpv infection. for biological progresses, the number of unique degs involved in metabolism processes and localization was distinct in bc9 and p50 following bmnpv infection (fig 4) . the comparisons of bc9+ vs. bc9-and p50+ vs. p50-identified many degs that might either be involved in silkworm defense against bmnpv or facilitate bmnpv infection. these genes could be divided into three categories: protein metabolism, cytoskeleton, and apoptosis. most of the degs (70%) associated with protein metabolism were down-regulated in bc9 following bmnpv infection. in contrast, 80% of the genes in p50 were up-regulated after bmnpv infection. most of the degs (87.5%) associated with the cytoskeleton were up-regulated in p50 after bmnpv infection, but the number of up-regulated genes (50%) decreased in bc9. a total of 19 degs associated with apoptosis were identified following bmnpv infection. ten of these degs were altered in bc9 after bmnpv infection. the other degs were all overexpressed in p50 following bmnpv infection (table 5) . pathogen infection stimulated both cellular and humoral responses of insects [33] [34] [35] [36] . genes participating in innate immunity pathways were identified and analyzed in regards to their potential role in bmnpv infection (table 6 ). thirty degs were identified, which could be classified into toll pathway, imd pathway, polyphenol oxidase (ppo) pathway, pattern recognition receptor, and antimicrobial peptide. as shown in table 6 , 14 (47%) of these genes were downregulated and 9 (30%) were up-regulated in bc9 after infection with bmnpv, while 17 (57%) were down-regulated and 6 (20%) were up-regulated in p50 after bmnpv infection. after removing the genetic background and strain specific immune stress response genes, a total of 22 degs were identified as possibly being involved in silkworm resistance to bmnpv (fig 3, table 7 ). for bc9-vs. p50-, 13 genes were up-regulated and the rest were down-regulated. however, the 22 genes were all down-regulated in bc9 following bmnpv infection ( fig 5) . some genes, including prostatic acid phosphatase, protease inhibitor 6, actin cytoskeletonregulatory complex protein pan1, and ef-hand domain-containing protein, were further analyzed by rt-qpcr (fig 6) . after bmnpv infection, the expression levels of 4 genes were down-regulation in bc9 and a35 (resistant strain) (fig 6) , which was consistent with the transcriptome data. thus, we deduced that the 22 genes were possibly involved in resistance to bmnpv infection. gene functions fell into the following categories: transport, virus replication, intracellular innate immunity, and apoptosis. despite the confirmation of an association between many genes and proteins and resistance to bmnpv, the molecular mechanism of antiviral activities was still unclear. here, transcriptome sequencing was carried out to identify genes related to bmnpv-resistance in silkworm across the genome. by using the near-isogenic line bc9 (resistant strain) and the recurrent parent p50 (susceptible strain) to study silkworm antiviral mechanisms, some degs responding to bmnpv infection were successfully identified after comparing infected groups and controls in the two strains. based on the go analysis, more degs were found to be involved in metabolic processes in bc9 (17.8%) than in p50 (9.2%) following bmnpv infection (fig 4) , which was consistent with previous reports for sogatella furcifera (hemiptera: delphacidae) and campoletis sonorensis (hymenoptera: ichneumonidae) [35, 36] . approximately twice as many degs related to transporter activity were identified in bc9 (12.2%) than in p50 (6.3%) (fig 4) . down-regulation of transporter related genes, such as lactase-phlorizin hydrolase (lph), b(0, +)-type amino acid transporter 1 (bat1), actin cytoskeleton-regulatory complex protein pan1 (pan1), mfs-type transporter (mfs), and otoferlin, could repress virus infection in host cells [37] [38] [39] [40] [41] [42] . therefore, the increased number of these genes with altered expression levels in bc9 might be related to bmnpv infection. moreover, the number of macromolecular complex genes in bc9 (14%) following bmnpv infection that showed an increase in expression compared with p50 (5.1%) were similar to previous reports [43, 44] . protein metabolism, cytoskeleton, and apoptosis may play an important role in host response to bmnpv infection cellular and metabolic processes will be dramatically changed after viral infection [35] . in our study, several degs that participate in protein metabolism were found to be of interest. for example, solute carrier family 12 is involved in transporting extraordinarily diverse solutes [45] , and cystathionine gamma-lyase participates in hydrolysis of cystathionine [46] . viruses may have to rely on cell proteins to accomplish replication in intercellular regions [47] , therefore, the downregulation of the genes involved in protein metabolism could inhibit the replication of bmnpv in the host cell. we speculated that the down-regulation of these genes affected virus replication. cytoskeleton-dependent intracellular transport is an important strategy for transport of viral particles to different destinations [48] . in this study, some cytoskeleton related genes were found to be of interest, including actin cytoskeleton-regulatory complex protein pan1 and actin-binding protein. these genes related to actin-coupled endocytosis could promote viral transport [49, 50] . we speculated that the down-regulation of the cytoskeleton genes might affect bmnpv transport. apoptosis plays a vital role in regulating cell response in lepidopteran insects during viral infections, where larvae use selective apoptosis and subsequent sloughing of the infected cells in the midgut epithelium to resist virus infection [51, 52] . in this study, some genes related to activation of apoptosis were found to be of interest, including cytochrome c, inhibitor of caspase-activated dnase, amyloid precursor protein and b-cell lymphoma protein 2. the activity of caspase-activated dnase was blocked by hepatitis c virus core at physiological levels, resulting in the inhibition of apoptotic cell death [53] . amyloid precursor protein is a member of several signaling pathways that are involved in abnormal cell cycles, subsequently leading to apoptosis [54] . b-cell lymphoma protein 2 could bind to bh3 domains of various pro-apoptotic regulators to activate apoptosis [55] . the overexpression of cytochrome c in rabies virus showed a decreased pathogenicity in vitro and in vivo [56] . based on their role in apoptosis activation, hosts need to increase the expression level of these genes to promote apoptosis when exposed to a virus; this supposition explains the up-regulation of genes involved in apoptosis in the transcriptome following bmnpv infection. additionally, a significantly higher relative transcriptional level of cytochrome c was detected by rt-qpcr in bc9 and a35 (fig 6) , which indicated the up-regulation of this gene could activate apoptosis to repress further bmnpv infection. however, some genes were down-regulated, including cysteine aspartic acid specific protease 9l, protein kinase a, apoptosis-inducing factor, apoptosis signal-regulating kinase 1, tnf-receptor-associated factor 6, tgf-beta-activated kinase 1, and p90 ribosomal s6 kinase. the inhibition of these genes may strongly impair viral infectivity and virus-induced apoptosis [57] [58] [59] [60] [61] [62] . although some differential expression of immune genes was observed, this might not be considered biologically important due to low expression levels; low expression levels were also found after bmcpv infection [21] . in this study, most immune related genes were down-regulated, with a few exceptions, such as the 2-fold up-regulation of toll-like protein and lysozyme. the expression of several other genes, including 18 wheeler, macrophage mannose receptor 1, and slit homolog 3 protein, were also up-regulated during the bmnpv infection. unfortunately, the relationship between immune genes and bmnpv remains unclear and requires further study. we presumed that the low expression levels of immunity related genes may be associated with the disruption of the immune system by bmnpv, similar to the pathogenicity of human immunodeficiency virus (hiv). based on the venn diagram, 22 genes of interest were identified, all of which were down-regulated in bc9 following bmnpv infection. these genes were grouped based on their functions, as reported in the literature. these groups, including transport, virus replication, intracellular innate immune, and apoptosis, may play an important role in the process of silkworm resistance to bmnpv. several genes related to virus transport, including lactase-phlorizin hydrolase (lph) [37] , b (0,+)-type amino acid transporter 1 (bat1) [38, 46] , actin cytoskeleton-regulatory complex protein pan1 (pan1) [63] , and otoferlin [41, 42] were identified in this study. the expression level of these genes were all down-regulated in bc9 following bmnpv infection (fig 5) . the resistant strain a35, a donor parent, was used to validate our results. the expression level of pan1 was chosen for further testing by rt-qpcr. expression levels of pan1 were down-regulated in both bc9 and a35 following bmnpv infection (fig 6) . the down-regulation of virus transport-related genes could inhibit the transmembrane and intracellular transport of bmnpv thereby preventing further infection. mfs-type transporter (mfs) [39, 40] , a transmembrane facilitator, was induced to increase intracellular monovalent ion concentrations, which led to lysis and cell death after hiv-1 infection. the down-regulation of this gene in silkworm could potentially block bmnpv infection. several genes related to virus replication were found to be of interest, including engrailed nuclear homeoprotein-regulated protein [64] , 1-acyl-sn-glycerol-3-phosphate acyltransferase alpha (asgpa) [65, 66] , alanine aminotransferase 2 (alt2) [67, 68] , u2af domain protein (u2af) [69, 70] , adenylate cyclase type 5 (act5) [71] , ef-hand domain-containing protein (efhp) [72, 73] , prostatic acid phosphatase (pap) [74] , and zinc ribbon domain protein (zrdp) [75] . in this study, the expression level of these genes were all down-regulated in the transcriptome of bc9 following bmnpv infection (fig 5) . in order to validate the results, rt-qpcr was conducted as described above. the expression levels of efhp and pap were lower following bmnpv infection in both bc9 and a35 (fig 6) . the down-regulation of these genes could directly or indirectly participate in repressing bmnpv replication in host cells. of the genes related to apoptosis, the expression level of pyruvate dehydrogenase kinase (pdk) was obviously inhibited after treatment with apoptosis-inducing agents [76] ; such down-regulation might activate apoptosis in response to bmnpv infection. protease inhibitor 6 is a member of the protein superfamily that contains til functional domain. zhao et al. used genome sequences to demonstrate that the expression level of the til superfamily were down-regulated following bmnpv infection [77] , which was consistent with our results (fig 6) . furthermore, peptidoglycan-recognition protein 2, hemolin, facilitated trehalose transporter tret1, integument esterase 2, defense protein precursor, and antimicrobial protein 6tox precursor also showed differential expression following bmnpv infection. the relationship of these genes to bmnpv is an important area for further study. four degs were down-regulated in bc9 following bmnpv infection, including rasresponsive element-binding protein 1, gtpase-activating protein, trypsin alkaline a and peroxidase (fig 5) . previous studies revealed that these proteins play a role in virus infections. mdv1-mir-m4 encoded by marek's disease virus efficiently targeted the 3' untranslated regions of ras-responsive element-binding protein 1 (rreb1) [78] . tbc domain proteins belonging to a gtpase-activating protein were knocked out by double stranded rna interference (rnai), which led to a decrease in the level of transcripts of white spot syndrome virus genes [79] . trypsin in the myocardium was able to trigger acute myocarditis following influenza a virus infection [80] . overexpression and rna silencing studies revealed that peroxidase was involved in the production of hepatitis c virus particles [81] . moreover, rt-qpcr results indicated that the expression levels of all genes were down-regulated in bc9 and a35 following bmnpv infection (fig 6) . therefore, we speculated that the down-regulation of these genes might be involved resistance to bmnpv infection. we hypothesized that the 22 degs discussed above played a role in the process of host response to bmnpv infection. the endocytosis process is triggered when the bmnpv nucleocapsid containing envelope binds to the cytomembrane. vacuolar atp synthase is activated by lph to promote the fusion of the envelope and endosome thereby releasing the nucleocapsid into the cytoplasm. this process can be promoted by pan1 and otoferlin. however, the transmembrane transport channel is an alternative pathway for virus to enter the cytoplasm, a process which can be facilitated by bat1. the released nucleocapsid is transported into the nucleus with the help of the cytoskeleton (efp). once viral dna is released into the nucleus, it will utilize host nucleotides to complete replication. in the final step of replication, viral dna has to rely on host cell amino acids for assembly on the cytoskeleton (table 5 ) [82] . in the cytoplasm, efhp, asgpa, alt2, u2af, act5 and zrdp play an important role in facilitating virus replication, although the exact mechanism is still unclear. moreover, transmembrane protein, mfs, is induced by bmnpv to increase cell volume, leading to lysis and cell death. we speculated that the down-regulation of these genes may affect the entry of virus into host cells and virus replication. the apoptosis process could also be triggered by pdk to inhibit bmnpv from further infecting other cells once bmnpv entered a host cell (fig 7) . taken together, our results provide useful information on silkworm resistance to bmnpv infection. supporting information s1 the genetics and genomics of the silkworm, bombyx mori silkworm genomics-progress and prospects genetic diversity among silkworm (bombyx mori l., lep., bombycidae) germplasms revealed by microsatellites lipopolysaccharide elicits expression of immune-related genes in the silkworm, bombyx mori gene expression profiling of resistant and susceptible bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host gene transcript levels screening of molecular markers for npv resistance in bombyx mori l. 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chromosomal mapping, and functional expression of a novel human alanine aminotransferase activating transcription factor 4 mediates up-regulation of alanine aminotransferase 2 gene expression under metabolic stress genomic functions of u2af in constitutive and regulated splicing a suboptimal src 3' splice site is necessary for efficient replication of rous sarcoma virus herpesvirus quiescence in neuronal cells iv: virus activation induced by pituitary adenylate cyclase-activating polypeptide (pacap) involves the protein kinase a pathway efhc1 interacts with microtubules to regulate cell division and cortical development selective reovirus infection of murine hepatocarcinoma cells during cell division. a model of viral liver infection naturally occurring fragments from two distinct regions of the prostatic acid phosphatase form amyloidogenic enhancers of hiv infection zinc ribbon domain-containing 1) is a host cellular factor that influences hiv-1 replication and disease progression pyruvate dehydrogenase kinase 4 (pdk4) could be involved in a regulatory role in apoptosis and a link between apoptosis and insulin resistance genome-wide identification and immune response analysis of serine protease inhibitor genes in the silkworm, bombyx mori marek's disease virus microrna designated mdv1-pre-mir-m4 targets both cellular and viral genes pmtbc1d20, a rab gtpase-activating protein from the black tiger shrimp, penaeus monodon, is involved in white spot syndrome virus infection ectopic trypsin in the myocardium promotes dilated cardiomyopathy after influenza a virus infection quantitative proteomics identifies the membrane-associated peroxidase gpx8 as a cellular substrate of the hepatitis c virus ns3-4a protease baculovirus infectivity and the actin cytoskeleton key: cord-012484-c9ajmbw2 authors: wahlund, martina; sinha, indranil; broliden, kristina; saghafian-hedengren, shanie; nilsson, anna; berggren, anna title: the feasibility of host transcriptome profiling as a diagnostic tool for microbial etiology in childhood cancer patients with febrile neutropenia date: 2020-07-26 journal: int j mol sci doi: 10.3390/ijms21155305 sha: doc_id: 12484 cord_uid: c9ajmbw2 infection is a common and serious complication of cancer treatment in children that often presents as febrile neutropenia (fn). gene-expression profiling techniques can reveal transcriptional signatures that discriminate between viral, bacterial and asymptomatic infections in otherwise healthy children. here, we examined whether gene-expression profiling was feasible in children with fn who were undergoing cancer treatment. the blood transcriptome of the children (n = 63) was investigated at time of fn diagnosed as viral, bacterial, co-infection or unknown etiology, respectively, and compared to control samples derived from 12 of the patients following the fn episode. rna sequencing was successful in 43 (68%) of the fn episodes. only two genes were significantly differentially expressed in the bacterial versus the control group. significantly up-regulated genes in patients with the other three etiologies versus the control group were enriched with cellular processes related to proliferation and cellular stress response, with no clear enrichment with innate responses to pathogens. among the significantly down-regulated genes, a few clustered into pathways connected to responses to infection. in the present study of children during cancer treatment, the blood transcriptome was not suitable for determining the etiology of fn because of too few circulating immune cells for reliable gene expression analysis. infection is a common and serious complication of cancer treatment in children that often presents as fever with neutropenia, a condition termed febrile neutropenia (fn). because prolonged neutropenia is a risk factor for severe infections, the recommendations for children with that condition is to immediately initiate treatment with empirical broad-spectrum antibiotics [1] . despite that, bacterial bloodstream infections are detected in only 10-30% of fn episodes, the majority of which are of unknown etiology [2] [3] [4] . previous studies detected common respiratory viruses in up to 40-50% of children with fn [3, 5, 6] ; however, the presence of a virus does not always correspond to an acute infection, as respiratory viruses can be detected in asymptomatic children [7] . in addition, some children may suffer from fever induced by their tumor or chemotherapy (drug), which further complicates the interpretation of the etiology of fn. as a consequence, it is challenging to determine when to use antibiotic treatment in children with fn, which typically involves administration of broad-spectrum antibiotics for 5 to 10 days [2, 8] . now when antibiotic resistance is a raising global health problem, there is an urgent need to find better diagnostic tools that can reduce the overuse of broad-spectrum antibiotics. infectious microbes have a number of signature molecules, referred to as pathogen-associated molecular patterns (pamps), that are essential for their survival and pathogenicity. the immune system recognizes pamps through highly conserved pattern-recognition receptors (prrs), that are predominantly expressed by innate immune cells such as the epithelial lining, monocytes, dendritic cells and neutrophils. when pamps are recognized by the immune system, innate immune cells rapidly launch anti-microbial responses tailored to eliminate different categories of pathogens by producing inflammatory cytokines, chemokines and type i interferons (ifns). ultimately, those innate immune responses trigger and shape long-lasting adaptive immunity [9] . gene-expression profiling techniques have initiated a new era in the identification of prognostic and diagnostic biomarkers of infectious disease. aside from pathogen-related factors, host responses to infectious agents can be examined on a genome-wide scale to reveal transcriptional signatures that associate to innate-response signaling pathways [10] . by assessing immune-related gene-expression profiles, hu et al. were able to distinguish children with asymptomatic and symptomatic viral infection, as well as those with bacterial infection [11] . in addition, the study also identified that children with asymptomatic viral infection were indistinguishable from their afebrile uninfected peers. a subsequent study identified a 2-script host-rna signature that could differentiate between viral and bacterial infections with high sensitivity and specificity rate [12] . however, even if the new techniques are promising, they remain on an experimental level; to be implemented as a diagnostic tool in the clinic, further studies on different patient cohorts are necessary. in previous studies of diagnostic gene-expression profiling in febrile children, all participants were immunocompetent [11, 12] . hence, little is known about gene-expression profiles in children whose immune response is altered by cancer treatment. therefore, we asked whether diagnostic gene-expression profiling is feasible in children with fn who are undergoing cancer treatment. we examined the blood transcriptomes of a cohort of children that presented with fn during cancer treatment, which was clinically attributed to viral, bacterial, co-infection or unknown etiology. a total of 63 fn episodes from 45 patients were included in the final analysis ( figure 1 ). the rna samples collected in 43 of the fn episodes (68%) were sufficient for rna-sequencing (rna-seq). the characteristics of the episodes with (n = 43) and without (n = 20) sufficient rna for sequencing are outlined in table 1 . the episodes with insufficient rna for sequencing had lower white blood cell counts (wbcs) and absolute neutrophil counts (ancs) on average than those with sufficient rna for sequencing (p < 0.001 for each comparison). samples from children with hematological malignancies tended to contain insufficient rna for sequencing (p = 0.06). furthermore, episodes with insufficient rna for sequencing had higher c-reactive protein (crp) levels (p < 0.01), and more days with broad-spectrum antibiotic treatment (p ≤ 0.05) compared with episodes with sufficient rna for sequencing. the 43 episodes with sufficient rna for sequencing originated from 35 individuals. the etiology was classified as respiratory virus infection in 15 episodes, unknown in 22 episodes, co-infection in 4 episodes, and bacterial infection in 2 episodes (figure 1 ). twelve control samples were obtained from 12 different individuals. nine of those were control samples obtained after an fn episode with sufficient rna for sequencing (four episodes with viral etiology and five episodes with unknown etiology). the remaining three samples were control samples obtained after an fn episode with insufficient rna for sequencing. the characteristics of the study and control samples are outlined in table 2 . the control samples had higher wbcs and ancs than the samples with viral infection (p < 0.001 and p < 0.001, respectively) or unknown etiology, (p ≤ 0.05, p < 0.001, respectively), and higher the 43 episodes with sufficient rna for sequencing originated from 35 individuals. the etiology was classified as respiratory virus infection in 15 episodes, unknown in 22 episodes, co-infection in 4 episodes, and bacterial infection in 2 episodes (figure 1 ). twelve control samples were obtained from 12 different individuals. nine of those were control samples obtained after an fn episode with sufficient rna for sequencing (four episodes with viral etiology and five episodes with unknown etiology). the remaining three samples were control samples obtained after an fn episode with insufficient rna for sequencing. the characteristics of the study and control samples are outlined in table 2 . the control samples had higher wbcs and ancs than the samples with viral infection (p < 0.001 and p < 0.001, respectively) or unknown etiology, (p ≤ 0.05, p < 0.001, respectively), and higher ancs, but not wbcs than the samples with co-infection (p < 0.01). no other differences were seen among the cohorts. respiratory symptoms were present in 87% and 100% of the episodes with viral or co-infection etiology, respectively, but were less common in the episodes with unknown etiology or bacterial etiology (64% and 50%, respectively) ( table 2) . gastrointestinal-symptoms and local symptom were uncommon in all groups. in one episode, two viruses were detected (rhino and corona). b co-infections consisted of parainfluenza virus detected with clostridium toxin b (feces), rhinovirus detected with pseudomonas aeruginosa (wound), coronavirus detected with staphylococcus aureus (wound), and bocavirus detected with alpha streptococcus (blood). c staphylococcus epidermis and escherichia coli were each detected separately in blood samples. d hematological malignances included; acute lymphoblastic leukemia, acute myeloid leukemia and non-hodgkin lymphoma. e wbc and anc counts were collected the same day as the rna samples f anc below.0,1 was reported from the laboratory as "<0.1". g in two patients the exact number of days with neutropenia was not possibly to calculate because of too infrequent sampling. they are stated as > 30 days. abbreviations: wbc, white blood cells; anc, absolute neutrophil counts; crp, c-reactive protein. to determine whether it is possible to detect pathogen-specific immune responses on the basis of gene expression in immunosuppressed children, we compared the blood transcriptomes of samples with viral infection, bacterial infection, co-infection, or unknown etiology with those of the control samples. the differentially expressed genes identified in the viral-, co-infection and unknown etiology are shown in figure 2a . the up-and down-regulated genes for the comparisons, and similarities and differences amongst them, are visualized in figure 2b . in the samples with viral infection and unknown etiology, the majority of the differentially expressed genes were upregulated; 178 out of 231 and 261 out of 442, respectively. in the samples with co-infection, the majority of the deviating genes expressed was down-regulated; 124 out of 219. when comparing samples with bacterial infection with the control group, only two genes were differentially expressed (data not shown). to determine whether it is possible to detect pathogen-specific immune responses on the basis of gene expression in immunosuppressed children, we compared the blood transcriptomes of samples with viral infection, bacterial infection, co-infection, or unknown etiology with those of the control samples. the differentially expressed genes identified in the viral-, co-infection and unknown etiology are shown in figure 2a . the up-and down-regulated genes for the comparisons, and similarities and differences amongst them, are visualized in figure 2b . in the samples with viral infection and unknown etiology, the majority of the differentially expressed genes were upregulated; 178 out of 231 and 261 out of 442, respectively. in the samples with co-infection, the majority of the deviating genes expressed was down-regulated; 124 out of 219. when comparing samples with bacterial infection with the control group, only two genes were differentially expressed (data not shown). next, we performed pathway analysis of the differentially expressed genes identified in the viral, unknown, and co-infection etiologies. the top five ranked canonical pathways that were up-and down-regulated respectively, in each etiology are outlined in figure 2c . the up-regulated genes in the three etiologies were enriched with cellular processes related to proliferation and cellular stress response, with no clear enrichment with innate responses to pathogens ( figure 2c ). among the down-regulated genes in the three etiologies, few genes clustering into pathways connected to responses to infection, such as ifn in the co-infection etiology and helper t cell activation signaling in samples with viral infection ( figure 2c ). to investigate if there were any group-specific pathways that were activated, a new pathway analysis was performed on group-specific genes (viral, n = 75, unknown n = 265, and co-infection n = 149, figure 2a ) and only few genes clustered into any pathway in samples with viral infection etiology, while the resulting top canonical pathways for samples with unknown and co-infection etiology were identical (data not shown). gene expression was further compared, in a pair-wise manner, among the viral, unknown and co-infection etiologies. the pair-wise comparisons did, however, not reveal any differentially expressed genes. on the other hand, there were 47, 7 and 32 genes that were differentially expressed between the bacterial etiology and the viral, unknown, and co-infection etiologies, respectively, which included the sialic acid-binding immunoglobulin-type lectin (siglec) family of molecules (table s1 ). in this study, we asked whether it is possible to use the pathogen specific innate transcriptome as a diagnostic tool in immunosuppressed children with fn. for this purpose, we examined a relatively large sample set from clinically well-characterized children that presented with fn during the course of cancer treatment. despite the relatively low rna-input requirements, the host transcriptome did not reliably distinguish different types of pathogens in children with fn. to explore gene-expression profiles, it is critical to obtain sufficient amounts of rna for sequencing. insufficient rna has not been a problem in earlier studies on non-immune suppressed patients that used rna sequencing for gene expression profiling [13] . in an earlier study on patients presenting with fn edgeseq was used instead of rnaseq due to the lack of wbc [14] . in the present study, there was insufficient rna for sequencing in 33% of the episodes, likely due to lower wbcs and ancs in the samples with insufficient rna compared with those in the samples with sufficient rna. that finding is of great importance as it implies that in children with the highest risk of severe bacterial infections [15] , due to low levels of circulating leukocytes, gene expression profiling is not feasible. the risk of severe infection is high in patients with an anc of 0.5 × 10 9 /l and increases with anc < 0.1 × 10 9 /l [15, 16] . in the samples with insufficient rna for sequencing in our study, the median wbc was 0.3 × 10 9 /l. furthermore, episodes with insufficient rna for sequencing and anc < 0.1 × 10 9 /l had more days on antibiotics, higher crp, and a trend towards more bacterial infections (15% vs. 5%) compared with the episodes with sufficient rna for sequencing. this clearly demonstrate that host transcriptional profiles are not suitable for the detection of specific microbial infections in high-risk patients with fn. we and other researchers have identified respiratory viruses as a common agent in episodes of fn with rhinovirus, influenza virus and respiratory syncytial virus being the most common detected viruses [3, 17, 18] . it is still unclear, however, if the detected viruses are the causative agents of fn, because pcr techniques can detect viruses from earlier infections, and viruses can also be detected in asymptomatic children [7] . an alternative way to assess etiology might be to examine the host immune response to specific pathogens. in that regard, studies have shown that the immune responses in children with asymptomatic viral infections differ from those children with symptomatic viral infections that display activated innate signaling [11, 19] . in our cohort, no pathogen-specific canonical pathways were activated in the samples with virus infection in comparison with the control samples. the lack of a pathogen-specific response in the samples from patients with viral infections might reflect viral dna/rna from a previous infection that was unrelated to the fn episode. in all patients, there was a possibility that the cause of the fever was tumor and/or drug related. the majority of the patients presented with focal symptoms, that were mainly associated with the respiratory tract, indicating an active infection. therefore, the most likely explanation for the lack of pathogen-specific pathway activation is the low numbers of immune cells in the blood. even in such cases, the local mucosal innate response generated by the infected cells might still be intact. this has indeed been shown in a study comparing host gene expression in blood and nose [20] . therefore, to further address the etiological role of respiratory virus in children with fn, future studies should include gene-expression analyses of secretions representing relevant mucosal sites. when comparing samples with unknown etiology with the control samples, we saw the same pattern of no apparent immune response in the blood. by contrast, in the samples with co-infection (respiratory virus and bacteria), we observed a larger set of downregulated genes and a down-regulated innate immune response. these episodes differed from the unknown and viral episodes as they occurred in children with a high wbc count, despite also having a low anc count. therefore, the gene expression signals are probably generated from other subgroups of immune cells such as lymphocytes and could theoretically be one reason for the differences seen in this group as compared to the others. downregulation of immune cell responses in children suffering from co-infection infections may reflect migration and redistribution of effector cells to the affected tissue, and thus, a corresponding lack of them in the circulation. in addition, neutrophils are key players in the early innate response to invading pathogens and low anc could thus facilitate host colonization by multiple pathogens. whether the dampened gene expression profile in the patients with co-infection is related to cancer-related or treatment-related immunosuppression, redistribution of cells from the circulation to peripheral tissue or to pathogen modulation of the innate immune response remains an open question. when comparing the group with bacteria with the control group, only two genes were differently expressed. this could be due to the small sample size and the rather stringent statistical methods used. the high infection parameters support the bacterial etiology. earlier studies of immunocompetent children with microbiological documented infections identified pathogen-specific host transcriptional profiles that could differentiate between viral and bacterial infections, which has since been called a paradigm shift in how to address etiology in patients with an infection [11] [12] [13] 21] . however, before implementation as diagnostic tools in the clinic, accessible and time effective diagnostic platforms needs to be developed and validated on a large-scale [22] . in addition, even if the findings so far have been reproducible in children of different genetic backgrounds and for various pathogens [21, 23] , these findings still need to be investigated in groups of children with potential impaired immune system at risk for severe infections. thus, further studies should also focus on children treated with immune suppressant drugs, children in low income countries with malnutrition and children in areas with a high incidence of chronic infectious diseases, such as hiv or tuberculosis [22, 24] . in the present study, there were no differences in gene expression detected among the samples with virus infection, co-infection, and unknown etiology. a set of genes correlated with the neutrophil response to bacteria was differentially expressed, between those samples and the samples with bacterial infection, although the latter consisted of only two samples. to our knowledge, only one previous study has investigated the gene expression signals in adult patients presenting with fn. kelly et al. investigated the gene expression profile of 2560 genes related to cancer and immunology using edgeseq [14] . they compared the transcriptome profiles of seven patients with fn to 22 controls with fever of unknown origin and, in addition, metabolomic profiling from the same cohort was performed. they could identify a set of three genes that correlated to a bacterial response; however, it is difficult to make any comparisons to the present study as different methods were applied [14] . importantly, our data also suggest that the heterogeneity of cells (neutropenia) impact gene expression signals. hu et al. investigated the association of gene expression level with total wbc and observed no correlation [11] . however, they did find correlations between the expression levels of some genes and the counts of sub-groups of white blood cells, such as monocytes, neutrophils and lymphocytes [11] . in our study, the wbcs and ancs were low in all of patients because of immunosuppression, and differential counts of monocytes and lymphocytes were not taken because of local routines. in addition, the function of the circulating immune cells might be affected by chemotherapy, making it difficult to draw definite conclusions on gene expression. we acknowledge the following limitations to our study: first, our cohort was heterogeneous in terms of the characteristics of the underlying cancers and the different treatment regimens used, both of which might impact the host immune responses. second, transcriptional profiles investigating infectious etiology is a new technique and has not been used on a large scale before in immune suppressed children. in one-third of the episodes had rna-concentrations that were too low for rna-sequencing and were mirrored by low wbcs and/or ancs. those factors are correlated with an increased risk of bacterial infections that imposed a selection bias against inclusion of cases with bacterial infections. to our knowledge, however, our study is the first to assess gene expression signatures against pathogens in a large number of pediatric oncology cases on immunosuppressive treatment, which are clinically well-defined, to understand if there is a connection between the blood transcriptome and pathogen type in fn. patients were enrolled at the childhood cancer unit at astrid lindgren children's hospital in stockholm between january 2013 and june 2014 [8] . all care takers to children (0-18 years of age), who were undergoing chemotherapy and met the criteria for fn were asked to participate. fn was defined as a body temperature ≥38.5 • c on one occasion or ≥38.0 • c on two occasions at least 60 min apart, combined with an absolute neutrophil count of either ≤0.5 × 10 9 /l on one occasion or ≤1.0 × 10 9 /l with a decline to less than ≤0.5 × 10 9 /l over a subsequent 48 h period. the same individual could be enrolled multiple times if he or she experienced recurrent episodes of fn during the study period, with the prerequisite that the child had been afebrile for more than 72 h and had completed his or her antibiotic treatment for the previous episode of fn. children included within 72 h of fever onset and from whom a complete set of samples were collected (nasopharyngeal aspirate (npa), blood culture, and paxgene tube) were eligible. after discharge from the hospital, each child was asked to leave a convalescence sample at the next scheduled appointment at the hospital, further addressed in the study as control samples. no time limit was set for when the control sample was collected, but it was defined as a control sample only if the child lacked any clinical sign of infection at the time the sample was taken. in addition, in the cases where a respiratory virus was detected, the virus must have been cleared (negative npa) at the time of control sampling. clinical data, including age, gender, type of malignancy, treatment intensity, symptoms of infection, days with antibiotics and days with fever and the duration of hospitalization, were collected from the medical records. in total, blood samples taken during 67 fn episodes met the study criteria and were included in the study. further, four of those samples were excluded because rna was isolated more than 72 h after the onset of fever and the final study cohort consisted of 63 fn episodes representing 45 individuals (figure 1 ). oral and written information regarding the study were provided to caretakers prior to enrollment, and signed consent was obtained from the participants' caretakers. in the cases, the children had sufficient reading and comprehension abilities the children were provided with a simplified version of study information. the study was approved by the regional ethical review board in stockholm; 2008/648-31/4 and 2009/286-32. blood samples were collected and stored at −20 • c in a paxgene blood rna tube (preanalytix, homebrechtikon, switzerland). white blood counts (wbc) and absolute neutrophil counts (anc) were analyzed at the karolinska university laboratory. for the definition of the study criteria fn the anc counts at time of admission was used. wbc/anc counts used as comparison between the study groups was sampled the same day as the rna sample. in addition, npa was collected and directly analyzed with in-house real-time pcrs for the following 16 viruses: adenovirus; bocavirus; coronaviruses nl63/oc43/229e/hku1; enterovirus; influenza virus a, including a(h1n1)pdm09 and b; metapneumovirus; parainfluenza viruses 1-3; respiratory syncytial virus; and rhinovirus [25] . as per clinical routine, blood cultures (1-10 ml) were collected from all children and analyzed at the karolinska university laboratory. control blood samples were collected as described above, and when a child tested positive for a respiratory virus at inclusion, a follow-up npa was collected and analyzed in the same manner as the first sample [25] . rna was extracted using the paxgene®blood rna system kit (preanalytix) according to manufacturer's instruction. the concentration and purity of the extracted rna [rna integrity number (rin)] were measured using a nanodrop nd-1000 spectrophotometer (thermo fisher scientific, ma, usa) and an rna screentape assay (on an agilent 2200 tapestation, agilent technologies, santa clara, ca, usa), respectively, according to the manufacturers' instructions. generally, an rin > 7 indicated good sample quality. patients with confirmed virus or bacterial infections at the time of their fn episode were considered to have a microbiologically documented infection (mdi). patients with a viral mdi were those in whom a respiratory virus was detected by virus-specific pcr. testing for other viruses, such as gastro-intestinal viruses and herpes virus, was guided by clinical symptoms. positive findings for non-respiratory viruses were included as mdis if the treating clinician considered them clinically relevant to the fn episode. bacterial infections were diagnosed on the basis of a positive blood culture. positive blood cultures determined to be either contaminants, or not clinically relevant, by the treating clinician and/or the laboratory were not considered positive findings of mdi. in addition, bacteria cultured from local foci that appeared with other fn symptoms and/or were determined to be of clinical relevance by the treating clinician were considered bacterial mdis. co-infection was defined as coincident viral and bacterial mdis; this was confirmed using the same criteria as described above. patients with unknown fn etiology had no mdi, according to the criteria described above. sequencing libraries were constructed using an illumina truseq stranded kit (san diego, ca, usa). a specific sample preparation protocol was performed that included mrna isolation, rna fragmentation, cdna synthesis, ligation of adapters (barcode and binding spots) and cdna amplification according to the manufacturer's instructions. the index cdna was normalized, samples were combined into a pool, and the pool (containing all specifically marked samples) was sequenced using an illumina nextseq 550 with 75-cycle v2 sequencing, generating 75-bp single-end reads (san diego, ca, usa). the reads were mapped to a human reference genome, grch38 and the mapping data were normalized using deseq2. a false discovery rate (fdr) < 0.05 and a fold change (fc) difference of at least 1.5 were used to identify genes that were differentially expressed between groups with different mdi status and controls. seqmonk (version 1.46.0) was used to perform the data analysis. pathway analysis was assessed by ingenuity pathway analysis software (https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysisand-visualization/qiagen-ipa/; content version: 51963813; release date: 2020-03-11; ingenuity systems, redwood city, ca, usa). circos software package (http://circos.ca/) was used to visualize the genomic data in a circular plot [26] . raw data can be assessed at the geo data base, accession number; gse152341. fisher's exact tests and the mann-whitney u tests were used for group comparisons of categorical and continuous data, respectively. a p-value ≤ 0.05 was considered statistically significant. data were analyzed using graphpad prism (graphpad prism, san diego, ca, usa). blood count levels reported as "<0.1" from the laboratory was uniformly set to 0.01 when calculating statistics. differences in pathogen-specific host transcriptional profiles between bacterial infections and viral infections are suggested as diagnostic tools in the clinic. our results suggest that they are not suitable for determining the etiology of fn in immunosuppressed children during cancer treatment, because children with low wbcs or ancs and, hence, an elevated risk of infection, have too few immune cells in their blood for reliable gene expression analysis. therefore, future studies should investigate the relationship between circulating counts of immune cells and gene expression levels, both in blood and at the local infection site before diagnostic gene expression profiling is implemented in the clinic. guideline for the management of fever and neutropenia in children with cancer and hematopoietic stem-cell transplantation recipients: 2017 update a prospective multicenter study of microbiologically defined infections in pediatric cancer patients with fever and neutropenia: swiss pediatric oncology group 2003 fever and neutropenia study respiratory viruses, a common microbiological finding in neutropenic children with fever a prospective study on the epidemiology of febrile episodes during chemotherapy-induced neutropenia in children with cancer or after hemopoietic stem cell transplantation respiratory viral infections and coinfections in children with cancer, fever and neutropenia: clinical outcome of infections caused by different respiratory viruses nasopharyngeal detection of respiratory viruses in febrile neutropenic children clinical utility of pcr for common viruses in acute respiratory illness frequent respiratory viral infections in children with febrile neutropenia-a prospective follow-up study principles of intracellular viral recognition gene expression signatures diagnose influenza and other symptomatic respiratory viral infections in humans gene expression profiles in febrile children with defined viral and bacterial infection diagnostic test accuracy of a 2-transcript host rna signature for discriminating bacterial vs viral infection in febrile children association of rna biosignatures with bacterial infections in febrile infants aged 60 days or younger integrative omics to detect bacteremia in patients with febrile neutropenia quantitative relationships between circulating leukocytes and infection in patients with acute leukemia febrile neutropenia in paediatric oncology acute respiratory infections in children and adolescents with acute lymphoblastic leukemia frequency and clinical outcome of respiratory viral infections and mixed viral-bacterial infections in children with cancer, fever and neutropenia rhinovirus detection in symptomatic and asymptomatic children: value of host transcriptome analysis host gene expression in nose and blood for the diagnosis of viral respiratory infection a 2-transcript host cell signature distinguishes viral from bacterial diarrhea and it is influenced by the severity of symptoms host-based peripheral blood gene expression analysis for diagnosis of infectious diseases a qpcr expression assay of ifi44l gene differentiates viral from bacterial infections in febrile children the at risk child clinic (arcc): 3 years of health activities in support of the most vulnerable children in beira development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses circos: an information aesthetic for comparative genomics we would like to thank bea, the core facility for bioinformatics and expression analysis, which is supported by the board of research at the karolinska institute and the research committee at the karolinska university hospital. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-001972-1zisomq5 authors: wang, xue; tan, jiying; biswas, santanu; zhao, jiangqin; devadas, krishnakumar; ye, zhiping; hewlett, indira title: pandemic influenza a (h1n1) virus infection increases apoptosis and hiv-1 replication in hiv-1 infected jurkat cells date: 2016-02-02 journal: viruses doi: 10.3390/v8020033 sha: doc_id: 1972 cord_uid: 1zisomq5 influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. it is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (hiv)-1 replication in hiv-1-infected patients. using a lymphoma cell line, jurkat, we examined the in vitro effects of pandemic influenza a (h1n1) virus (ph1n1) infection on cell death and hiv-1 rna production in infected cells. we found that ph1n1 infection increased apoptotic cell death through fas and bax-mediated pathways in hiv-1-infected jurkat cells. infection with ph1n1 virus could promote hiv-1 rna production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated b cells (nf-ĸb), nuclear factor of activated t-cells (nfat) and activator protein 1 (ap-1) through mitogen-activated protein kinases (mapk) pathways and t-cell antigen receptor (tcr)-related pathways. the replication of hiv-1 latent infection could be reactivated by ph1n1 infection through tcr and apoptotic pathways. these data indicate that hiv-1 replication can be activated by ph1n1 virus in hiv-1-infected cells resulting in induction of cell death through apoptotic pathways. it is estimated by the world health organization (who) that approximately 36.9 (34.3~41.9) million persons worldwide are infected with human immunodeficiency virus (hiv), which is the etiologic agent of acquired immunodeficiency syndrome (aids), including 1.7 million in the united states [1] . influenza a virus can cause acute respiratory infection in humans and animals throughout the world, and has continued to be a significant public health threat, leading to substantial global morbidity and mortality and an average of approximately 23,600 deaths annually in the united states alone [2] . the impact of influenza virus on hiv infection has not been well investigated. little is known about influenza virus infection in hiv-positive individual. hiv infection has been shown to be related to worse prognosis of influenza. hiv-infected patients in canada who also had pandemic 2009 influenza a (h1n1) virus (ph1n1) infection had more severe illness than those who did not have the co-infection [3] , and fatality was higher than for patients who were not co-infected in california (usa) [4] . recent reports indicate that adults with aids experience substantially elevated influenza-associated mortality [5, 6] . influenza virus infection has been associated with viremia in human and animal models. viral rna has been detected in blood in severe human ph1n1 infection [7] [8] [9] . encapsidated ph1n1 rna is stable in blood derived matrices [10] and influenza viruses can be transmitted by blood transfusion in ferrets [11] . normally, influenza a virus infection is confined to the airways where the virus replicates in respiratory epithelial cells. cumulated reports indicate that influenza viruses can infect and replicate in blood cells, such as dendritic cells [12] , primary monocytes/macrophages, and t cells [13, 14] . infection with hiv-1 can result in apoptotic cell death through activation of both death receptor-mediated and bax/mitochondrial-mediated apoptotic pathways [15] , which cause a progressive depletion of a select group of immune cells namely the cd4+ t helper cells leading to immunodeficiency. while hiv directly and selectively infects cd4+ t cells, the low levels of infected cells in patients is discordant with the rate of cd4+ t cell decline and argues against the role of direct infection in cd4 loss [15] . a viral protein, neuraminidase (na), derived from the human influenza virus was reported to enhance the level of hiv-1-mediated syncytium formation and hiv-1 replication [16, 17] . however, it is not known whether influenza a virus in blood affects hiv-1 replication, or reactivates hiv-1 replication in hiv-1-infected cells. here, we showed that pandemic influenza a (h1n1) virus infection increased apoptotic cell death and hiv-1 replication in hiv-1 infected jurkat cells. rabbit polyclonal antibodies against bax, bcl-x l (b-cell lymphoma-extra large), caspase-3, caspase-8, cavoelin-1, cd4, cd28, cytochrome c, fadd (fas-associated protein with death domain), fas, flip ((fadd-like il-1β-converting enzyme)-inhibitory protein), p53, zap-70 (zeta-chain-associated protein kinase 70), and gapdh (glyceraldehyde 3-phosphate dehydrogenase were from santa cruz biotechnology (santa cruz, ca, usa). ap-1, erk (extracellular-signal-regulated kinases), jnk (c-jun n-terminal kinases), p38, nfat, and nf-kb p65 were bought from cell signaling technology, inc. (danvers, ma, usa). all other chemicals were from sigma (st. louis, mo, usa). the human jurkat t cell line (clone je6.1) and j1.1, a latently hiv infected cell line cloned by limiting dilution from hiv-infected jurkat cells were obtained from national institutes of health (nih) aids reagent program (germantown, md, usa) and cultured at 37˝c in 5% co 2 in rpmi 1640 medium containing 10% fetal calf serum, 2 mm glutamine, 50 µg/ml penicillin, and 50 µg/ml streptomycin. for hiv-1 infection, jurkat cells (clone je6.1) were seeded at 2ˆ10 5 cells/ml for 24 h, and infected with known amounts of hiv-1 (hiv-1 mn strain, 10 9 copies per 10 6 cells) for 2 h, washed twice with pbs, and cultured for periods of time indicated. quantitative real-time reverse-transcriptase (rt) pcr was used for quantitation of viral rna. viral rna was isolated from 140 µl of culture supernatant by using the qiaamp viral rna mini kit (qiagen inc., valencia, ca, usa) according to the manufacturer's protocol. the primers and taqman probe were designed in the gag capsid (p24) region, which is the variable region among most of the hiv-1 subtype b isolate sequences according to genbank database. the forward primer was 5 1 -gacatcaagcagccatgcaa-3 1 , corresponding to nucleotides 1367-1386, and the reverse primer was 5 1 -ctatcccattctgcagcttcct-3 1 , corresponding to nucleotides 1430-1409. the taqman probe was oligonucleotide 5 1 -attgatggt ctcttttaaca-3 1 , corresponding to nucleotides 1488-1507, coupled with a reporter dye (6-carboxy fluorescein) (fam) at the 5 1 end and a non-fluorescent quencher and a minor groove binder (mgb), which is a tm enhancer, at the 3 1 end. the nucleic acids were amplified and detected in an automated taqman 7500 analyzer by using quantitect™ probe rt-pcr kit (qiagen inc.). the 25-µl pcr mixture consisted of 100 nm primers and 100 nm probe. following three thermal steps at 55˝c for 5 min, at 50˝c for 30 min, and at 95˝c for 10 min. 45 cycles of two-step pcr at 95˝c for 15 s and at 60˝c for 1 min were performed. the data are expressed as copy numbers/ml. known concentrations of hiv-1 (mn) viral rna (serially diluted: 10 8 to 100 copies) were used as templates and quantitative rt-pcr performed to generate a standard curve. each value represents the average concentration of six reactions in triple isolated repeats based on the standard curve. enzchek ® caspase-3 assay kit #1, z-devd-amc substrate from invitrogen (grand island, ny, usa) was used to test caspase-3 activity, which was performed according to the manufacturer's instruction. cell viability was determined by trypan blue exclusion analysis (life technologies, waltham, ma, usa). to generate membrane and cytoplasmic lysates, subcellular protein fractionation kit for cultured cells was used (thermo scientific, rockford, il, usa). lysis of cells generated membrane and cytoplasmic protein extracts. the protocol was performed according to the manufacturer's instructions. normalized portions of each extract (10 µg) were analyzed by western blotting using specific antibodies against proteins from various cellular compartments, including cytoplasmic (cytochrome c) and plasma membrane (caveolin-1) ( figure s1 ). proteins were isolated from the culture of jurkat cells with ripa buffer (1ˆpbs, 1% (v/v) np-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sds, 0.1 mg/ml pmsf, 30 µl/ml aprotinin, 1 mm sodium orthovanadate). equal amounts of protein were boiled in the loading buffer (100 mm tris-hcl, 200 mm dtt, 4% sds, 0.2% bromphenol blue, 20% glycerol) and separated on sds-page and blotted onto polyvinylidene difluoride membranes. for immunoprecipitation (ip), 1 µg of antibody was added to 500 µg of total protein in 500 µl, rotated for 2 h at 4˝c, then incubated with 20 µl of protein a-sucrose beads (santa cruz biotechnology) for another 2 h, spun down at 500ˆg, and washed three times with ripa buffer. the data represented are from three independent experiments. the unpaired student's t test was used for data analyses as indicated, and a value of p < 0.01 was considered very significant (**). it was reported that influenza virus infection could cause severe complications in hiv-1-infected individuals leading to an increased risk of complications and death compared with uninfected individuals [5, 6] . we found that ph1n1 could infect and replicate in jurkat cells and primary cd4+ t cells ( figure s2 ). to examine whether ph1n1 infection increased cell death due to hiv-1 infection, jurkat cells were infected with hiv-1 for three days, followed by incubation in the presence of ph1n1 for another three days. as shown in figure 1a , ph1n1 infection caused increased cell death in hiv-1 infected cells compared with uninfected cells (control). we also tested caspase-3 activities ( figure 1b ,c) and found that more caspase-3 was activated by ph1n1 in hiv-1 infected cells than uninfected cells (control). we also obtained the similar results with using primary cd4 t cells incubated with hiv-1 for seven days and then infected with ph1n1 for another three days ( figure s3a ,b). the total cell lysates were subjected to test caspase-3 activities with enzchek®caspase-3 assay kit #1-z-devd-amc substrate from invitrogen (grand island, ny, usa), which was performed according to the manufacturer's instruction; (c) the total cell lysates with ripa buffer were subjected to western blot analysis to detect caspase-3.the unpaired student's t test was used for data analyses and a value of p < 0.01 was considered very significant (**) relative to control. these data indicated that ph1n1 infection caused cell death through apoptotic signaling pathways in t cells. recently, we found that ph1n1 is able to induce apoptotic cell death in a549 cells [18] . to determine whether ph1n1 can promote death through fas-mediated apoptotic pathway in jurkat cells, total cell lysates from cells infected with hiv-1 followed by infection with ph1n1 for an additional three days were used for ip with anti-fas antibody and western blotting with anti-caspase-8 antibody to evaluate death-inducing signaling complex (disc) formation. as shown in figure 2a , more disc formation was found with the ph1n1/hiv-1 infection relative to hiv-1 infection or ph1n1 alone. figure 2b showed that high level of expression of p53 and bax proteins was detected in jurkat cells infected with ph1n1 infection and hiv-1-infected cells, compared with ph1n1 or hiv-1 alone. these data indicate that pandemic influenza a (h1n1) infection can induce both fas-mediated and bax-mediated apoptotic pathways. a higher level of activation of both apoptotic signaling pathways was found in ph1n1 infected jurkat cells after hiv-1 infection. recent reports indicate that ph1n1 infection was associated with increase in mortality in patients with late and advanced hiv disease [5, 6] and increased cell death was found in ph1n1-infected jurkat cells after hiv-1 infection ( figure 1a) . we determined whether ph1n1 infection could influence hiv-1 replication. as shown in figure 3a , jurkat cells infected with hiv-1 and incubated with ph1n1 for another three days, had higher levels of hiv-1 rna relative to cell with hiv-1 infection alone, suggesting that ph1n1 infection increases hiv-1 replication in co-infected cells in jurkat cells and in cd4+ t cells ( figure s3c ). it has been well-known that some host transcription factors are required for hiv replication, including nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb), nfat, ap-1, which are up-regulated when t-cells become activated [19] [20] [21] . they stimulate viral gene expression through transcription with the long terminal repeat (ltr) of hiv-1 [22] . cells treated with ph1n1 had higher level of nf-kb phosphorylation and increased protein expression of nfat and ap-1 ( figure 3b ) relative to hiv-1 infection alone, suggesting ph1n1 infection can activate host transcription factors required for hiv-1 replication in jurkat cells. the mitogen-activated protein kinases (mapks) are central components of signal transduction pathways activated by diverse extracellular stimuli. several studies have shown that the mapk signal pathway can positively regulate replication of hiv-1, and pathway inhibitors can block hiv-1 replication [23, 24] . our results indicate that ph1n1 infection promotes mapk pathway molecules, such as p38, erk, and jnk phosphorylation ( figure 3c) . these data approve the conclusion that pandemic influenza a (h1n1) infection increases hiv-1 replication in infected cells through activation of nf-kb, nfat, ap-1, and mapk pathways that are necessary for hiv-1 replication. it is well known that cd4 is the receptor of hiv-1 [25, 26] . hiv-1 gp120 binds to the cd4 molecule on the surface of host cells [24] . since hiv-1 entry takes place in lipid rafts of the plasma membrane [27] , we examined whether ph1n1 infection affects cd4 expression on the plasma membrane in jurkat cells infected with hiv-1 and ph1n1. as shown in figure 4a the t cell activation and proliferation control hiv-1 replication and gene expression [28] ; and activation of t cells through involvement of tcr, cd3/cd8, or cd28, can trigger and activate downstream molecules, such as zap-70 and pkc8, and which activate transcription factors, ap-1 nfat and nf-κb [19, 29] , and increase hiv-1 gene expression and replication directed by the hiv-1 ltr [22] . activation events of t cells require association with lipid rafts of the plasma membrane [24, 27] . our results showed that ph1n1 infection increased more zap-70 and pkc8 expression on plasma membranes relative to hiv-1 infection alone ( figure 4b,c) . these data indicate that pandemic influenza a (h1n1) infection can increase accumulation of cd4 protein and induce t cell signaling and activate host transcription factors required for hiv-1 replication. hiv-1 infection is presently readily controlled with combination antiretroviral therapy. the persistence of hiv-1 in the face of potent antiretroviral drugs appears to be largely due to the ability of the virus to establish a state of latent infection in a long-lived population of resting memory cd4+ t cells [30] . this latent reservoir is widely considered to be the major barrier to curing infection and is currently the subject of intense research [30] . to determine whether ph1n1 infection can reactivate hiv-1 latent infection, j1.1 cells were infected with ph1n1 for three or seven days days. as shown in figure 5a , measure with real-time pcr analysis showed that significant high level of hiv-1 rna production was detected in ph1n1 infected cell relative to non-ph1n1 infected controls. cell fractionation showed that ph1n1 infection caused increased accumulation of cd28 and zap-70 proteins in plasma membrane in j1.1 cells ( figure 5b ). western blotting analysis showed that ph1n1 infection increased nfat expression and activated nf-kb signaling ( figure 5c ). ph1n1 infection could activate both apoptotic pathways with an increased expression of pre-apoptotic proteins (such as fas, disc formation, fadd, and bax) and a decreased expression of anti-apoptotic proteins (such as flip and bcl-2) (figure 6 ), suggesting that apoptotic pathways may be also involved in ph1n1-induced reactivation of hiv-1 replication. these data indicate that pandemic influenza a (h1n1) infection is able to reactivate latent hiv-1 infection in vitro. during the 2009 h1n1 influenza a virus pandemic, it was reported that some patients developed severe pneumonia leading to acute respiratory distress syndrome (ards) and multiple organ dysfunction, associated with high (17%-54%) mortality [31, 32] . however, there is little information available on the direct interaction between influenza virus and the host immune system that pertains to severe disease outcomes. it has been reported that pathogenesis due to influenza infections was associated with alterations in the lymphohematopoietic system, leading to the destruction of lymphocytes and histopathological necrosis of lymphoid tissues [33, 34] . there is little information available on whether and how influenza infection could damage the immune system directly and alter its ability to control other infectious pathogens that ultimately are detrimental to the host. our study showed that pandemic influenza a (h1n1) virus infection is able to enhance cell death in hiv-1-infected cells (figure 1) . hiv-1 infection may be associated with a worse prognosis of influenza and adults with aids experience substantially elevated influenza-associated mortality, which has declined with widespread introduction of haart (highly-active anti-retroviral therapy), but not completely eliminated [35] . in hiv patients, well controlled on haart, pandemic influenza a (h1n1) virus infection has resulted in a similar clinical outcome and prognosis to that of non-hiv patients [34] and oseltamivir treatment has been known to shorten the duration of influenza viral shedding in children [36] and adults [37, 38] . cd4 cell count and plasma hiv-1 rna did not differ before and 4-6 weeks after influenza a h1n1 diagnosis in the presence of oseltamivir treatment in the hiv-positive group [37] . however, hiv-1-infected subjects that had progressed to immune deficiency presented an increased mortality by ph1n1 [5] . this information suggests that hiv-1 activation, induced by influenza virus infection, can be controlled by oseltamivir in "normal" hiv-positive persons on haart treatment, and may not be controlled in hiv-infected patients under illness condition [5] . influenza a virus induces apoptotic death in infected epithelial, lymphocyte, and phagocytic cells [18, 33, 34, 39] , and apoptosis is a source of tissue damage during infection [33] . distal lung epithelial cell apoptosis and apoptosis-resulting in necrosis were described as a classical feature of h5n1-or pandemic h1n1-induced ards [40] . epithelial cell apoptosis is generally seen as a major underlying cause of diffuse alveolar damage in many forms of ards [40] . so far, it has been clear that influenza infection can affect both intrinsic and extrinsic apoptotic pathways [18] . it was also reported that after influenza virus infection, some cd4+ cells in the spleen and thymus may express viral proteins [41] , and a population of t cells is susceptible to influenza a virus in infected mice [42, 43] . influenza virus infection has been shown to activate tcr-signaling pathways in jurkat cells [16] and pandemic influenza a (h1n1) virus infection causes apoptotic death in hiv-1-infected jurkat cells (figures 4 and 5b,c) . previously, we reported that pre-apoptotic molecules from apoptotic pathways increase hiv-1 replication, while anti-apoptotic proteins inhibit viral rna yield [23] . consistent with our previous observation, pandemic influenza a (h1n1) virus infection can increase hiv-1 replication with up-regulation of pro-apoptotic molecules, down-regulation of anti-apoptotic proteins, and with an increased activation of t cells through tcr-related signaling pathways ( figures 1c, 2 and 6 ) required for t-cell activation and hiv-1 replication; however the detailed mechanisms by which pandemic influenza a (h1n1) virus infection induces hiv-1 replication needs further study. in conclusion, our results demonstrate that pandemic influenza a (h1n1) virus infection can induce cell death through apoptotic signaling pathways and promote hiv-1 replication through the mapk and tcr-related signaling pathways in hiv-1-infected jurkat cells. pandemic influenza a (h1n1) virus infection is also able to reactivate hiv-1 replication from its state of latent infection through activating apoptosis and tcr-signaling pathways. stimating seasonal influenza-associated deaths in the united states: cdc study confirms variability of flu. cdc. available online canadian critical care trials group h1n1 collaborative. critically ill patients with 2009 influenza a (h1n1) infection in canada california pandemic (h1n1) working group. factors associated with death or hospitalization due to pandemic 2009 influenza a (h1n1) infection in california severe 2009 pandemic influenza a (h1n1) infection and increased mortality in patients with late and advanced hiv disease influenza-related mortality among adults aged 25-54 years with aids in south africa and the united states of america clinical and virological factors associated with viremia in pandemic influenza a/h1n1/2009 virus infection evidence of viremia in 2 cases of severe pandemic influenza a h1n1/09 influenza viral rna detection in blood as a marker to predict disease severity in hematopoietic cell transplant recipients hewlett, i. stability and infectivity of novel pandemic influenza a (h1n1) virus in blood-derived matrices under different storage conditions highly pathogenic avian influenza a virus (h5n1) can be transmitted in ferrets by transfusion high susceptibility of human dendritic cells to avian influenza h5n1 virus infection and protection by ifn-alpha and tlr ligands efficient generation and expansion of antigen-specific cd4+ t cells by recombinant influenza viruses active nf-kappab signalling is a prerequisite for influenza virus infection hiv-1 induced bystander apoptosis syncytium formation and hiv-1 replication are both accentuated by purified influenza and virus-associated neuraminidase neuraminidase from a bacterial source enhances both hiv-1-mediated syncytium formation and the virus binding/entry process novel pandemic influenza a (h1n1) virus infection modulates apoptotic pathways that impact its replication in a549 cells regulation of nf-κb induction by tcr/cd28 nfat and irf proteins regulate transcription of the anti-hiv gene, apobec3g modulation of hiv-1 transcription by cytokines and chemokines akt/nox2/nf-κb signaling pathway is involved in tat-induced hiv-1 long terminal repeat (ltr) transactivation hewlett, i. molecules from apoptotic pathways modulate hiv-1 replication in jurkat cells some mechanisms of flip expression in inhibition of hiv-1 replication in jurkat cells, cd4+ t cells and pbmcs assembly and architecture of hiv the functional roles of lipid rafts in t cell activation, immune diseases and hiv infection and prevention viral entry, lipid rafts and caveosomes hostile takeovers: viral appropriation of the nf-kb pathway hiv reservoirs: pathogenesis and obstacles to viral eradication and cure pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico critically ill patients with 2009 influenza a(h1n1) in mexico apoptosis: a mechanism of cell killing by influenza a and b viruses depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h5n1) virus isolated from humans clinical presentation and prognosis of the 2009 h1n1 influenza a infection in hiv-1-infected patients: a spanish multicenter study pandemic influenza a (h1n1) outbreak among 15 school-aged hiv-1-infected children acute respiratory distress syndrome due to influenza virus a/h1n1v in a patient with hiv/hcv co-infection influenza a h1n1 in hiv-infected adults aryl hydrocarbon receptor activation during influenza virus infection unveils a novel pathway of ifn-gamma production by phagocytic cells early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection influenza h5n1 and h1n1 virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation role of itk signalling in the interaction between influenza a virus and t-cells antigen signal strength during priming determines effector cd4 t cell function and antigen sensitivity during influenza virus challenge the authors wish to acknowledge of mohan haleyurgirisetty and viswanath ragupathy for critical review of this manuscript, and acknowledge of the nih aids reagent repository for the reagents. this work was supported by an internal cber modernizing science grant. the findings and conclusions in this article have not been formally disseminated by the food and drug administration and should not be construed to represent any agency determination or policy.author contributions: x.w. and i.h. conceived and designed the experiments; x.w., j.t., s.b., j.z., and k.d. performed the experiments; and x.w. and z.y. analyzed the data, and x.w. and i.h. wrote the paper. all authors have read and approved the final manuscript. the authors declare no conflict of interest. key: cord-006261-yw5k8qkz authors: heath, gregory w.; macera, caroline a.; nieman, david c. title: exercise and upper respiratory tract infections: is there a relationship? date: 2012-10-23 journal: sports med doi: 10.2165/00007256-199214060-00003 sha: doc_id: 6261 cord_uid: yw5k8qkz nan runners and other exercise enthusiasts are traditionally motivated to participate in sports for competitive or recreational reasons. however, some exerciseparticipants are convinced that exercisewill provide health benefits (nash 1986 ). indeed, regular exercise and physical activity have been established as a means of reducing cardiovascular disease risk and contributing to longevity (paffenbarger et al. 1986; powell et al. 1987 ). in addition, habitual exercisers often report other health-related rewards from endurance exercise such as weight control (heath et a1. 1981) , improved energy levels (hughes 1984) and stress reduction (blumenthal et al. 1980; sinyor et al. 1983 ). many exercisers also believe that exercise improves their resistance to infection and anecdotally report that they have fewer colds and other upper respiratory tract infections (urti) [simon 1987 ]. despite these beliefs, few studies have examined the relationships that exist among exercise and clinical expressions of upper respiratory tract infections. this article seeks to examine the existing studies that have investigated the relationship between exercise and upper respiratory tract infections and to determine ifany such relationship exists. it begins by defining and discussing the specific health outcome of infections and the classification of these diseases. secondly, the epidemiology and natural history are discussed with the clinical and experimental evi-dence of the relationship between upper respiratory tract infections and exercise explored. the suggested immunological and physiological mechanisms of this possible relationship are examined, followed by a review of the epidemiological data linking an association of exercise to upper respiratory tract infections. finally, guidelines for the exercising public are presented as an application of our attempt to answer the question : exercise and upper respiratory tract infections -is there a relationship? upper respiratory tract infections consist of a number of acute illnesses that occur in the upper portion of the respiratory tract. the most common infections include pharyngitis, croup, bacterial tracheitis, epiglottitis and the common cold. since the common cold is the leading acute illness and cause of visits to a physician in the developed world, the common cold is a focus of the article. the term 'cold' carries different meanings for many people; however, it is usually defined as an acute illness that involves nasopharyngitis and catarrh , little or no fever and insignificant systemic symptoms. ruses it is not known which of these modes of spread are most important. for rhinovirus and respiratory syncytial virus close contact with an infected person or infected secretions is necessary, with a number of studies demonstrating that people infected with rhinovirus colds have recoverable infectious virus on their hands (gwaltney 1983; gwaltney & hendley 1978; hendley et al 1973) . furthermore, these studies have shown that rhinovirus may be readily transferred from the contaminated hands of one person to the hands of another who, if susceptible, may acquire infection by touching his nasal or conjunctival mucosa. the aetiology of this group of upper respiratory tract infections is viral in nature. the predominant group of viruses which cause colds are the rhinoviruses followed by the coronaviruses (table i) . the parainfluenza viruses, respiratory syncytial virus and influenza viruses are all epidemic viruses that are associated with colds. however, these viruses cause more serious upper respiratory tract infections which tend to mask the milder colds (hall & mcbride 1989 ). transmission of upper respiratory tract infection-causing viruses may occur through several modes. suspension of viral particles in large droplets produced by a cough or sneeze with direct contact into the eyes or on to the upper respiratory passages is a common mode, usually produced by close contact with the infected individual. virus suspended in the small particle aerosol of a sneeze or cough is capable of travelling greater distances and is another common mode of infection. finally, the virus can be spread by contact with contaminated secretions by hand from contaminated surface to mucous membranes (self-inoculation) [hall & mcbride 1989] . for most of the respiratory vi-when examining the numbers and types of viruses causing upper respiratory tract infections it is clear that this is a rather ubiquitous group of infections. in general, the number of infections acquired per year decreases with age (bader et al 1953; brimblecombe et al. 1958; dingle et al. 1964; fox et al. 1972; gwaltney et al. 1966) . infants and children have the highest incidence with 4 to 8 infections per year. this rate may even double when children are in day care or nursery school. in schoolaged children the incidence is 2 to 6 colds per year (badger et al. 1953; brimblecombe et al. 1958; dingle et al. 1964; fox et al. 1972) . adults acquire 2 to 5 upper respiratory tract infections per year, with women and adults who live in households with children tending to suffer even more colds per year (gwaltney et al 1966) . smoking has been shown to aggravate the signs and symptoms of infection but not to increase the attack rate (gwaltney et al. 1966 (gwaltney et al. , 1967 . exposure to cold or to chilling does not increase the chance of acquiring or aggravating a cold (douglas et al. 1968 ). recent studies have demonstrated that psychological stress is a risk factor for the development of an upper respiratory tract infection (cohen et al. 1991; graham 1986) . finally, in studies exploring the prophylactic use of vitamin c in prevention, the attack rate of upper respiratory tract infections was not diminished alfig. 2 . effect of marathon running on lymphocyte responsiveness to concanavalin a (con a) at pre-exercise andat 30 min and 3h recovery from the marathon. though symptoms were ameliorated (coulehan et al. 1976; karlowski et al. 1975; miller et al. 1977 ). human and animal experimental evidence has demonstrated a relationship between exercise and upper respiratory tract infections (nieman & nehlsen-cannarella 1991 a, 1992 . furthermore, these studies provide insight into potential mechanisms that might explain the relationship between physical activity and infection. (nk) cell activity at 3 effector: target (e: t) ratiosat 1.5, 6 and 21h following a 3h run at 70%~02max. several researchers have reported that various aspects of immune function are depressed following intense, prolonged endurance exercise (nieman & nehlsen-cannarella 1991a) . heavy exertion is a form of physiological stress that causes large increases in epinephrine (adrenaline) and cortisol levels, hormones which have been consistently associated with a suppression of immune function, and rapid perturbations in circulating levels ofleucocyte and lymphocyte subsets. nieman et al. (1989a) and berk et al. (1990) ran 10 seasoned marathoners at their fastest marathon pace on treadmills for 3 hours. cortisol rose 59% abo ve baseline levels after the 3-hour run , remain-ing elevated for 1.5 hours of recovery before falling to normal daytime levels. this increase in cortisol correlated inversely with a 25 to 46% decrease in natural killer (nk) cell activity at 1.5 hours after recovery, which persisted for nearly 6 hours ( fig. 1 ). pedersen et al. (1988 pedersen et al. ( , 1989 pedersen et al. ( , 1990 and kappel et al. (1991) have carefully demonstrated that the post exercise suppression of natural killer cell activity is also related to increased levels of prostaglandins released from monocytes. eskola et al. (1978) and gmunder et al. (1988) have reported a significant decrease in lymphocyte proliferative response for several hours after a marathon (42.2km) [ fig. 2 ]. macneil et al. (1991) have demonstrated that the lymphocyte proliferative response is decreased for at least 2 hours following cycle ergometer exercise, especially following high intensity exercise by athletes. following 1 hour of cycling at 80% v02max by untrained individuals, tvede et al. (1989) found suppression of b lymphocyte function for at least 2 hours because of an inhibitory effect of activated monocytes. have determined that neutrophil killing capacity is decreased in elite athletes engaging in prolonged periods of intensive tra ining in comparison to untrained controls. nieman et al. (1989b) and have reported significantly lower serum complement in long distance runners relative to sedentary controls. a significant decrease in salivary immunoglobulin concentrations following 2 hours of intense cycling or 50km of cross-country ski racing z 20 10 has been described by 2 groups of investigators (mackinnon et al. 1987; tomasi et al. 1982) . israel et al. (1982) have reported that serum immunoglobulins fall 10 to 28% for at least 1 day after athletes run 45 or 75km at high intensity. russian investigators have related that exhaustion of immune reserves can be observed during periods of important competitions, manifested by lowered immunoglobulin levels and suppression of phagocytic activity ofneutrophils (pershin et al. 1985; petrova et al. 1983 petrova et al. , 1985 . results from animal studies have rather consistently supported the view that heavy acute and chronic exertion are related to negative changes in immune function. several researchers have reported that exhaustive single bouts of exercise by both trained and untrained animals, or 6 days to 4.5 months of heavy exercise training, are linked to increased splenic epinephrine and cortisol levels and decreased splenic natural killer and t cell lymphocyte function (ferry et al. 1990; hoffman-goetz et al. 1986; mahan & young 1989; simpson & hoffman-goetz 1990) . thus, both circulating immune cells and those found in secondary lymphoid tissues may have their function suppressed because of the increase in cortisol and catecholamine levels that occur following heavy exertion. further research is needed to better elucidate the clinical significance of exercise induced changes in immune status and function (many of which are transient in nature), and which variables best predict potential changes in host protection. the data at present are not consistent enough between studies to even suggest thresholds for various immune system markers that may indicate increased risk of upper respiratory tract infection. psychological factors may also play an important role in the relationship between exercise and upper respiratory tract infection. exercise is a form of physiological and psychological stress, varying according to the intensity and duration of the training programme. interestingly, the acute re-sports medicine 14 (6) 1992 sponse of the immune system to psychological stressors alone is in many ways similar to those that occur in response to acute exercise (naliboff et al. 1991) . if the exercise training programme is deemed stressful by the athlete, the combined psychological and physiological impact may overwhelm the ability of the immune system to protect the host (nieman & nehlsen-cannarella 1992) . mental stress alone has been related to a wide variety of negative changes in immunity. bereavement, major depression, loneliness, schizophrenia, marital discord and other forms of mental stress have all been associated with suppression of immune function (jemmott & locke 1984; khansari et al. 1990) . a biochemical basis for bidirectional communication between the immune system and neuroendocrine system has been established (blalock 1989) .these systems produce and use many of the same signal molecules in the form of hormones, lymphokines and monokines for inter-and intrasystem communication and regulation. lymphoid organs are innervated by the autonomic nervous system, and lymphocytes have receptors for the various stress hormones. in the other direction, for example, products of leucocytes have been shown to alter neuronal activity in certain areas of the brain. thus, stress of any form may decrease host protection from infection through both autonomic nervous system and hormonal mechanisms. research by graham and associates (1986) , for example, has demonstrated that during a given 6month period, highly stressed individuals have twice as many days with respiratory infection symptoms compared with low-stressed people. cohen et al. (1991) gave nasal drops containing respiratory viruses to 394 subjects and reported that psychological stress was associated in a dose-response manner with an increased risk of acute infectious respiratory illness. although specific research in this area has not yet been conducted, it would seem logical to assume that athletes around the time of competition, when both physiological and psychological stress are high, would be most vulnerable to respiratory infections. another factor that may be important for the risk of respiratory infection in athletes is the involvement of the immune system in the tissue repair process that occurs following strenuous exercise (for a review see neiman & nehlsen-cannarella 1991 b). it has been well established that both heavy acute and chronic exertion are associated with muscle cell damage, local inflammation and the stereotyped sequence of host defence reactions known as the acute phase response (evans & cannon 1991; nieman & nehlsen-cannarella 1991a) . the acute phase response following endurance exercise involves the complement system, neutrophils, macro phages, various cytokines and acute phase proteins, and can last for several days, promoting clearance of damaged tissue and setting the stage for repair and growth. lymphocytes, neutrophils and macrophages are attracted to the injured muscle cells, and invade the area to aid in the process. neutrophils phagocytise tissue debris and release a wide variety of factors that aid in the digestion of adjacent dead tissue cells (smith 1991) . macrophages have surface receptors which allow them to react nonspecifically to a variety of substances, a process enhanced by the presence of opsonins (primarily complement and antibody). macrophages also are a prime source of cytokines that mediate most of the physiological and inflammatory reactions accompanying muscle cell injury. dufaux and order (1989b) have shown that plasma elastase-a j-antitrypsin, neopterin, tumour necrosis factor and soluble interleukin-2 receptor increase during recovery from a 2.5-hour running test, supporting the concept of a functional involvement of polymorphonuclear neutrophils and an activation of macrophages .and t-lymphocytes. dufaux and order (1989a) have also provided evidence for complement activation after 2.5 hours of running. could the active enmeshment of the immune system in the muscle tissue repair and inflammation process mean that protection from respiratory infection is compromised? research to answer this question is certainly warranted, and may greatly increase our understanding as to how and why ath-357 letes appear to be more susceptible to respiratory tract infections during periods of heavy training. few studies have investigated the common belief that moderate physical activity is beneficial in decreasing risk of respiratory tract infections and improving immune function. more research is certainly warranted to investigate this interesting area. since the turn of the century, the influence of exercise training on resistance to infection has been investigated using animal models. cannon and kluger (1984) have reviewed the animal literature and concluded that moderate exercise prior to infection may increase resistance to infection, but that exhaustive exercise after contracting an infection may be detrimental, in accordance with this viewpoint, slubik et al. (1987) have reported that moderate physical exercise preceding irradiation diminishes radiation injury in animals while intensive exercise and stress may aggravate the damage. have shown that 1 hour of cycling at 60%~02max may increase resistance to infection by improving the 'killing capacity' of neutrophils, an effect which persists for at least 6 hours of recovery. given that neutrophils are the body's best phagocyte, these findings suggest that regular episodes of moderate exercise may increase resistance to infection. in a randomised controlled study by nieman et al. (1990) and nehlsen-cannarella et al. (1991) , the effects of walking on immune response and acute respiratory. infection tract symptomatology were measured on a group of sedentary, mildly obese women. the exercise subjects walked 45 minutes per session, 5 times per week, for 15 continuous weeks on a measured course under supervision. subjects recorded health problems in a daily log book using 10 codes supplied by the centers for disease control. exercise subjects experienced half the number of days with respiratory infection symptoms during the 15 weeks compared with the sedentary control group (5.1 â± 1.2 vs 10.8 â± 2.3 days, respecthus, the response of the immune system to exercise may have much to do with the degree of intensity and total exertion load, and corresponding changes in concentrations of cortisol and epinephrine. both of these hormones have been associated with many negative effects on immune function (cavallo et al. 1986; crary et al. 1983; cupps & fauci 1982) . lymphocytes have~-adren ergic receptors, and the presence of epinephrine during exercise increases receptor number on t suppressor/cytotoxic and natural killer cells, which are the major lymphocyte subsets that increase in response to an exercise challenge (field et al. i991; maisel et at. 1990; van tits et at. 1990 ). the degree to which these lymphocyte subsets increase in the peripheral blood as they are recruited from lymphoid tissue pools is highly dependent on the magnitude of change in epinephrine. additionally, the effect on lymphocyte function is also dependent on the change in both epinephrine and cortisol. moderate exercise such as walking does not increase the concentration of epinephrine and cortisol in the blood, resulting in a small lymphocytosis in contrast to intense exercise, in which levels of both hormones increase (nieman 1991) [ fig. 4 ]. thus , it may be argued that moderate exercise induces a small increase in natural killer cells and t cytotoxic/suppressor cells without the potential suppressive effect of epinephrine or cortisol, creating a milieu which may be favourable for host protection. there is only a finite number of lymphocytes that is specific to any particular antigen. theoretically, by recruiting lymphocytes from the periphery, exercise may increase the rate of lymphocyte circulation through the body, improving the potential for interaction of lymphocyte and antigen without the attending negative effects of stress hormones (nieman & nehlsen-cannarella 1992) . 3 ]. moderate exercise training led to a 20% net increase in each of the 3 serum immunoglobulins, and was significantly correlated with fewer respiratory infection symptoms days (nieman & nehlsen-cannarella 1991 b) . moderate exercise training also led to a significant increase in natural killer cell activity which was correlated with a decrease in the duration of respiratory infection symptoms per episode. these results are similar to those of crist et al. (1989) who moderately exercised elderly women for 16 weeks and measured a 33% higher natural killer cell activity at rest, and a heightened increase following maximal testing. laboratory and clinical studies suggest that changes in immune parameters associated with heavy train ing lead to adverse health effects, particularly an increase in the incidence or severity of upper respiratory tract infections. however, anecdotal reports by marathon runners that running increased (or decreased) their incidence of upper respiratory tract infections were not correlated with immune system changes (green et al. 1981) . if the documented changes in immune parameters are associated with upper respiratory tract infections, there are several methods by which epidemiological investigations can be used to evaluate this effect. to use the epidemiological approach, the following must be present: a defined population from which the exposed group and a comparison group can be selected, and a clear operational definition of the outcome of interest. the exposure (training) can be measured in a number of ways that would indicate relative levels of individual exertion. while this is not as accurate as the measurement that would occur in a laboratory setting, there is confidence that measuring levels of the exposure is possible in a population-based setting. however, defining the appropriate control or comparison group is a more difficult problem. among studies involving runners, there have been a variety of methods used to determine the comparison group: faster runners were compared with slower runners of the same road race (peters et al. 1983 ); low mileage runners were compared to high mileage runners over the same time period (heath et al. 1991) ; marathon runners were compared with those who ran shorter races on the same day nieman et ai. 1989c) ; marathon runners were compared with trained nonrunners (nieman et al. 1990b) , or marathon runners were compared with nonrunning friends (peters et al. 1983 ). each of these comparison groups has its advantages and drawbacks. the true measure of the outcome (upper respiratory tract infections) is impossible to ascertain without a clinical examination and a laboratory workup. in lieu of a clinical assessment, the best surrogate measure for upper respiratory tract infection is self-reported symptoms. however, many other disease processes or allergic reactions may present with symptoms similar to those of upper respiratory tract infections. unless these symptoms are severe or proceed to disability, most affected individuals will not be seen by medical personnel, and the symptoms will soon resolve without a medical diagnosis. in spite of these limitations, selfreport is the best choice, and steps can be taken to operationally define upper respiratory tract infection symptomatology so that definitions are consistent across studies. in spite of these measurement difficulties, several studies have explored the relationship between exercise and upper respiratory tract infection symptomatology among population-based samples, usually runners. among these studies, some have supported the suggestion that athletes engaging in marathon type events or very heavy training are at increased risk of respiratory tract infections (heath et al. 1991; nieman et al. 1990b; peters et al. 1983 ), while others have found no difference among exposed and unexposed groups nieman et al. 1989c) ,and still others have found a decrease in infections among the exposed compared with the unexposed (nieman et al. 1990a; schouten et al. 1988 ). peters and bateman (1983) studied the incidence of respiratory tract infections in 1550 randomly selected runners who took part in a 56km race compared with matched controls who did not run. those who ran the race reported more symptoms of respiratory tract infections during the 2week period following the race than those who did not run, and those who ran the faster race times reported more symptoms, indicating a doseresponse relationship. nieman et al. (l990b) studied the incidence of respiratory tract infection in a large group of marathon runners who varied widely in running ability and training habits. those who ran the marathon reported more symptoms during the week following the race than similarly experienced runners who had applied but did not participate in the race for sports medicine 14 (6) 1992 possible confounders, the researchers concluded that, compared with nonrunners, runners experience increased risk for respiratory tract infection during heavy training or following a marathon race event. heath et al. (1991) followed a cohort of 530 runners who self-reported any symptoms of respiratory infections daily for 1 year. the average runner was about 40 years of age, ran 20 miles (32km) per week, and experienced a rate of 1.2 respiratory infections per year. controlling for various confounding variables, the lowest odds ratio for respiratory infection was found among those running less than 10 miles (16km) per week. the odds ratio more than doubled for those running more than 17 miles (27km) per week. the authors concluded that running mileage was a significant risk factor for upper respiratory tract infection symptoms. in another study, no difference (for either men or women) was found in the incidence of upper respiratory tract infection symptomatology during the month after the race for marathon runners compared with those who ran on the same day, but for shorter distances. in this study, the most important determinant for symptoms of upper respiratory tract infection after the race was the presence of upper respiratory tract infection symptoms before the race. these results persisted after controlling for total training mileage and percentage increase in training mileage during the month before the race. nieman et al. (l989c) studied the incidence of upper respiratory tract infection symptoms among participants in 5km, iokm and half-marathon road races. during the 2 months before the race, those running more than 15 miles (24km) per week reported more events than those running fewer miles. there was no increase in symptoms after the road race compared with the week before the road race, indicating that the race did not appear to be associated with an increased risk of acute upper respiratory tract infection. in a study of young adults, schouten et al. (1988) found no difference in the incidence or duration of upper respiratory tract infection symptoms when level of sports activity or maximum aerobic power. they found a slight inverse association between symptoms and level of sports activity for women only. although this study had the advantage of measuring several types of physical activity as well as aerobic power, the upper respiratory tract infection measurement relied on memory recall for the past 6 months, leading to possible misclassification. in a 15-week study of immune response and exercise, 36 mildly obese women were randomly assigned to walking or nonexercising groups (nieman et a1. 1990a) . during this time, the women in the exercise group experienced fewer upper respiratory tract infection symptom days, improved cardiorespiratory fitness, and increased natural killer cell numbers compared with their sedentary controls. this study indicates that intensity of exercise may playa role, and that moderate activity may actually improve immune function and associated upper respiratory tract infection symptoms. because this is a new field it is not surprising that the results of the few studies done to date have not been consistent. while most, but not all, of the published studies have found a relationship between increased training and upper respiratory tract infections, and considering that the type and intensity of exercise has not been fully explored, no definitive conclusions can be drawn. however, on the basis of the available studies, some general guidelines can be provided for future studies. separate comparison groups, a lower intensity running or exercise comparison group as well as a nonexercising or sedentary comparison group should be considered. during the analysis stage the comparison groups could be used to sort out some of the biases present in the previous studies. because reporting behaviour differs between men and women, the sample size should be large enough to allow separate analyses for men and women. a consistent time frame for reporting upper respiratory tract infection symptoms after racing events should be used. the studies noted here have used follow-up times ranging from i to 4 weeks. when using self-report data, every attempt 361 should be made to differentiate symptoms due to allergy from symptoms because of upper respiratory tract infections. because allergic rhinitis may be present (but undiagnosed) the case definition of upper respiratory tract infections should be defined in such a way as to capture most of the events. data should also be collected on duration of the event, and for longer studies, the number of symptom-free days between events should be defined. at present, insufficient evidence exists to recommend precisely what laboratory tests of immune function should be conducted to ascertain when an athlete is at increased risk of an infectious episode due to overtraining and/or psychosocial stress. although some evidence would suggest that low immunoglobulin and complement levels, decreased lymphocyte proliferative response, diminished neutrophil phagocytic activity, depressed natural killer cell activity, low total lymphocyte count and low helper/suppressor t cell ratio are each important markers of increased risk, the exact level at which one or a combination of some or all of these immune components becomes predictive is unknown (nieman & nehlsen-cannarella 1992) . there is an interesting similarity in the metabolic and immunological responses to intense endurance exercise and to an infectious challenge (lewis et a1. 1986; schaefer et a1. 1987) . in both conditions, the number of circulating leucocytes increases, lymphopenia occurs (especially t cells with cells trafficking to peripheral tissues), the lymphocyte responses to phytohaemagglutinin (pha) and concanavalin (con-a) decrease, body core temperature rises, plasma levels of acute phase proteins increase and degranulation of neutrophils develops. since endurance exercise is associated with muscle cell damage and an increased intake of potential pathogens through heightened ventilation, it is logical that in preparation for such a challenge, the immune system receives a signal from the neuroendocrine network that activates the immune system. why then do clinical experience and epidemiological data point toward an increased risk of respiratory infection in some athletes? the mass of evidence favours the view that psychosocial variables play an important role in effecting immunological competence. the net effect of combined psychological and physiological stress from unusually heavy endurance exercise, especially during times of competition, may lead to suppression or down-regulation of the immune system. for those athletes who must exercise intensely for competitive reasons, several precautions can help decrease the risk of sickness. these include spacing vigorous workouts and race events as far apart as possible, eating a well balanced diet, keeping other life stresses to a minimum, avoiding overtraining and chronic fatigue and obtaining adequate sleep. before and after intense race events, the athlete should try to avoid contact with sick people if at all possible. for the fitness enthusiast, the area of concern is not so much the harm that may come from overexertion, but the benefits that may derive from engaging in regular, moderate forms of exercise. at this time, even though investigative evidence suggests improved host protection and immunosurveillance from moderate physical activity, more research is needed to improve our understanding of the workload threshold below or above which exercise becomes protective rather than detrimental. should athletes exercise when they have an upper respiratory tract infection? most clinical authorities in this area recommend that if the athlete has symptoms of a common cold with no constitutional involvement, then regular training may be safely resumed a few days after the resolution of symptoms (roberts 1986; simon 1987) . mild exercise during sickness with the common cold does not appear to be contraindicated. however, if there are symptoms or signs of systemic involvement (e.g. fever, extreme fatigue, muscle aches, swollen lymph glands), then 2 to 4 weeks should be allowed before resumption of more intense training. these precautions are advised because of the well documented relationship between intense. exercise and sports medicine 14 (6) 1992 the risk of developing a viral cardiomyopathy and other severe form of viral infection (sharp 1989) . physical activity and exercise produce a variety of alterations of the immune system, most of which have not been fully investigated. however, the effects of vigorous exercise appear to depress immune function and may compromise host defense against upper respiratory tract infections. because of the complexity of host defence mechanisms and the physiologyof exercise,further research is needed in this area. clinical studies examining the effects of moderate levels of physical activity have shown possible enhanced immune responses with a concomitant impact on the length and severity of an upper respiratory tract infection. therefore, the relationship between exercise and upper respiratory tract infections appears to be 'j' shaped with the most sedentary at greatest risk of upper respiratory tract infections along with the vigorously active, with those engaged in moderate levels of activity manifesting the apparently better host defence. epidemiological studies have generally demonstrated a greater risk of upper respiratory tract infection with vigorous levels of exercise; however, these studies must be considered to be limited because of the lack of adequate nonexercising control groups. further studies are necessary to demonstrate the clinical and public health significance of these relationships. engaging in regular moderate level physical activity is most beneficial for maintaining health and in preventing an initial upper respiratory tract infection. moderate level physical activity coupled with generally good hygiene and avoidance of close contact with individuals known to have an active upper respiratory tract infection appear to be the most commonsense measures for avoiding contracting an upper respiratory tract infection. however, since avoidance of a high risk situation, such as the presence of children, is often difficult, the hygienic measures of frequent handwashing and the thorough washing of potential fomitic surfaces will aid in reducing exposure to the upper respiratory tract infection agent. for those athletes who are required to engage in high intensity training and competition, further steps should be taken to minimise contact with situations where exposure to upper respiratory tract infection agents is high. in addition, training techniques should take into consideration the need for the organism to restore host resistance by 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exercise, training and neutrophil microbicidal activity effect of exercise on complement activity acute inflammation: the underlying mechanism in delayed onset muscle soreness? immune parameters in athletes before and after strenuous exercise mechanisms of b-iymphocyte suppression induced by acute 365 physical exercise catecholamines increase lymphocyte beta 2-adrenergic receptors via a beta 2-adrenergic, spleen-dependent process division of chronic disease control and community intervention, ms-k47, centers for disease control venue: netanya, israel for further information, please contact: hony tenenbaum international congress wingate institute for physical education and sport wingate post office netanya key: cord-003855-so8xl199 authors: ebert, gregor; paradkar, prasad n.; londrigan, sarah l. title: virology downunder, a meeting commentary from the 2019 lorne infection and immunity conference, australia date: 2019-09-02 journal: virol j doi: 10.1186/s12985-019-1217-6 sha: doc_id: 3855 cord_uid: so8xl199 the aim of this article is to summarise the virology content presented at the 9th lorne infection and immunity conference, australia, in february 2019. the broad program included virology as a key theme, and the commentary herein highlights several key virology presentations at the meeting. the lorne infection and immunity conference is one of five scientific meetings held during each month of february in seaside town of lorne, on the great ocean road in victoria (australia). the specific aim of the meeting is to bring together basic, clinical and translational researchers -those who examine microbes and their impact on the innate or adaptive immune response, researchers who study the mechanisms that regulate immune responses, and those who apply this knowledge to preventing and treating infectious and inflammatory diseases. 2019 was the 9th lorne infection and immunity conference, convened by heidi drummer (burnet institute, melbourne, australia) and paul hertzog (hudson institute of medical research, melbourne, australia). the broad program included virology as a key theme, and the commentary herein highlights several key virology presentations at the meeting. the 'infection and inflammation' session of the meeting was opened with a well-received presentation by linfa wang (duke-nus medical school, singapore) entitled 'holy immune balance, batman', about the highly adapted immune response of bats to viral infections. bats have been characterized as an important reservoir of various zoonotic viruses including nipah, sars, mers, marburg and ebola viruses [1] , but remarkably are able to live asymptomatically with otherwise potentially lethal viruses [2] . wang and colleagues are interested in understanding the underlying immune mediated regulatory mechanisms that facilitate a highly effective balance between viral defense and tolerance in bats as viral hosts. during their evolutionary adaption to effective flight, bats not only developed elevated levels of basal alertness reflected in increased metabolic heart rate and body temperature, they also seem to have evolved a highly adapted immune defense against viral infections [3] . remarkably, bats have increased tolerance to viral infections by exhibiting higher basal levels of innate defence regulators but also a dampened innate immune response upon infection, substitutional to increased responsiveness to viral pathogens [4] . the bat innate immune response appears to be 'pre-activated' with higher basal levels of type i interferon expression, in contrast to humans, who are very quick responders to viral infections, but require a lot more dampening of their immune signals afterwards to get back to basal levels. current research also shows the absence of any aim2 mediated inflammasome activation [5] , dampened nlrp3 mediated inflammasome activation [6] and dampened sting activation [7] in bats upon infection, which are all mediators of a robust type i interferon response. overall, wang et al. demonstrated that bats' response to stress in form of viral infections is more targeted and thus potentially more effective by numerous adaptions and modifications of the innate immune system. in 'viruses and their hosts' session, vinod sundaramoorthy (csiro-australian animal health laboratories) discussed a novel defence mechanism in neurons against rabies virus (rabv). rabv is a neurotropic virus, which causes tens of thousands of deaths every year, despite available vaccines [8] . endemic dog rabies results in an ongoing risk to humans in many resource-limited countries, whereas rabies in wildlife is important in north america and europe [9] . requirement for an uninterrupted vaccine cold chain and the high cost of the immunoglobulin component of rabies prophylaxis therapies substantiate the unmet need for novel rabv-specific antivirals [10] . furthermore, the pathogenesis of rabv relating to viral replication in neurons is not fully understood [11] . although most of the infection with rabv manifests as the 'furious' form, where virus silently spreads through the neuronal axon without any damage, in 20 % of cases it can cause axonal damage in peripheral neurons leading to paralysis [12] . using a new in vitro microfluidics model for studying synaptically connected neurons, sudaramoorthy investigated the pathogenesis of different strains of rabies virus, showing distinct mechanisms at play in determining disease outcomes for each strain. future efforts to define a key molecular determinant in the viral replication pathway in neurons may provide a novel therapeutic target. in 2015, an association between zika virus (zikv) infection during pregnancy as a cause of microcephaly and other congenital abnormalities in the developing fetus and newborn, was made [13] . as such, there has been a plethora of recent zikv research focused on understanding the pathogenesis of disease, as well as immunity to the virus and the development of vaccines and effective therapeutics. novel findings pertaining to all of these areas were presented throughout the meeting. developments in understanding the structure of zikv particles was showcased by shee-mei lok (duke-nus, singapore). lok and colleagues have previously solved a thermally stable 3.7 å resolution cryo-electron microscopy structure of zikv [14] , and were able to now show high resolution structures of zikv at various stages of viral assembly. specifically, the organisation of the envelope protein of the virus and changes during assembly and maturation were presented. previously, lok's lab has also published structures of mature and immature dengue virus, providing insight into the viral maturation process [15] . the research from lok and colleagues will have future implications in designing novel therapeutics and vaccines for zikv. efforts to develop a zikv vaccine using virus like particles were presented by julio carrera, working with researchers at the monash university and the university of melbourne. in the 'pathogenesis and prevention of infection' session, rosa coldbeck-shackley working with michael beard at the university of adelaide, australia, and also colleagues at the hudson institute, presented findings on the importance of interferon-epsilon (ifn-ɛ) in the innate immune response to zikv infection. ifn-ɛ is a novel type i ifn, encoded within the type i ifn locus in mice and humans, whose function has recently been characterized [16] . like other type i ifns, it acts via ifn-α receptors 1 and 2, activating interferon stimulated genes (isgs). however, ifn-ɛ is preferentially expressed by epithelial cells of the female reproductive tract in both mice and humans, and in contrast to viral induced type i ifn expression, ifn-ɛ is hormonally regulated. other than mosquito-borne transmission, zikv is also sexually transmitted [17] . this makes the research presented by coldbeck-shackley significant, and concurs with previous studies where ifn-ɛ-deficient mice were more susceptible to infection with sexually transmitted pathogens [16] . thus, ifn-ε appears to be a potent antipathogen and immunoregulatory cytokine that may be important in combating sexually transmitted infections that represent a major global health and socioeconomic burden. also in the 'pathogenesis and prevention of infection' session, allison abendroth (university of sydney) presented 'disarming the killer: targeting of natural killer cells by varicella zoster virus'. varicella zoster virus (vzv), is known to infect numerous immune cell types such as t-cells and dendritic cells [18] . moreover, the virus is able to modulate and manipulate innate and adaptive immune responses to infection to its replicative benefit. vzv infection of dendritic cells dampens type i ifn responses [19] and leads to evasion of cd8 t cell recognition via downregulation of mhc class i expression [20] . in addition, vzv mediated delay in immune responses to infection facilitates establishment of initial primary infection and lifelong latency in neurons. most recently, abendroth and colleagues found that vzv also productively infects natural killer (nk) cells and that nk cells effectively transmit infection to other permissive cell types [21] . the group is now trying to understand, how nk cells, a cell type that normally demonstrates very effective direct and indirect antiviral capacity, is manipulated by vzv infection, leading to impaired cytotoxicity and cytokine responses upon infection and facilitating infection and spread of the virus. led by the observation that patients with impaired nk cell functionality are highly susceptible to severe and life threatening vzv infection, abendroth et al. found limited nk cell activation and efficacy upon vzv infection of target cells and characterised differential modulation of ligands normally recognised by activated nk cells [22] . the exact underlying mechanisms, how vzv infection prevents the release of proinflammatory cytokines during infection of nk cells to impair their general function and whether correlations can be drawn to vzv infection of monocytes and macrophages [23] , is currently under their investigation, abendroth stated. a key highlight at the conclusion of the meeting was a presentation from the victorian infection and immunity network young investigator prize winner, simone park (the university of melbourne). park, working alongside thomas gebhardt and laura mackay, has recently published seminal research investigating skin tissue-resident memory t cells (trm cells). specifically, park has demonstrated how these cells contribute to antiviral immune memory in peripheral tissues, using a herpes simplex model of infection [24] . using an epicutaneous melanoma model, park and colleagues also demonstrate that trm cells play protective role in tumor surveillance [25] , which has important implications for advancing anticancer immunotherapies. the organisers are looking forward to celebrating the 10th lorne infection and immunity meeting in 2020 and invite all researchers with an interest in infectious and inflammatory diseases and associated immune responses to participate. for more information: http://www.lor neinfectionimmunity.org/. viruses in bats and potential spillover to animals and humans bats and viruses: friend or foe? comparative analysis of bat genomes provides insight into the evolution of flight and immunity the egyptian rousette genome reveals unexpected features of bat antiviral immunity unique loss of the pyhin gene family in bats amongst mammals: implications for inflammasome sensing dampened nlrp3-mediated inflammation in bats and implications for a special viral reservoir host dampened sting-dependent interferon activation in bats rabies molecular virology, diagnosis, prevention and treatment status of antiviral therapeutics against rabies virus and related emerging lyssaviruses immunological aspects of rabies: a literature review human rabies: neuropathogenesis, diagnosis, and management increase in reported prevalence of microcephaly in infants born to women living in areas with confirmed zika virus transmission during the first trimester of pregnancy -brazil structure of the thermally stable zika virus immature and mature dengue serotype 1 virus structures provide insight into the maturation process interferon-epsilon protects the female reproductive tract from viral and bacterial infection leparc-goffart i. evidence of sexual transmission of zika virus varicella-zoster virus infection of human dendritic cells and transmission to t cells: implications for virus dissemination in the host impact of varicella-zoster virus on dendritic cell subsets in human skin during natural infection varicella-zoster virus productively infects mature dendritic cells and alters their immune function varicella zoster virus productively infects human natural killer cells and manipulates phenotype varicella -zoster virus and herpes simplex virus 1 differentially modulate nkg2d ligand expression during productive infection infection and functional modulation of human monocytes and macrophages by varicella-zoster virus local proliferation maintains a stable pool of tissue-resident memory t cells after antiviral recall responses tissue-resident memory cd8(+) t cells promote melanoma-immune equilibrium in skin publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions ge, pp and sl all contributed equally in the writing of this manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-015640-zdwmxaz3 authors: tong, c. y. w.; schelenz, s. title: clinical virology in nicu, picu and aicu date: 2011-08-10 journal: infection control in the intensive care unit doi: 10.1007/978-88-470-1601-9_20 sha: doc_id: 15640 cord_uid: zdwmxaz3 viruses are significant causes of nosocomial infections, particularly in intensive care unit (icu) where seriously ill and vulnerable patients are being cared for. four major routes of nosocomial virus transmission in the icu are identified, viz. respiratory, faecal–oral, exposure to blood and body fluid and direct contact with infected patients or through fomites. different infection control measures are available according to the natural history, biology, pathogenesis, epidemiology and mode of transmission of each virus. in this chapter, we discuss some of the important viruses that could be associated with nosocomial infections in the icu. intensivists should work closely with microbiologists, virologists and the laboratory to diagnose such infection early, work proactively to prevent outbreaks and manage viral infections using appropriate strategies. influenza viruses (family orthomyxoviridae) are classified into types a, b and c. annual seasonal outbreaks of influenza are caused by minor antigenic changes (antigenic drift) seen in influenza a and b viruses. major changes in antigenic subtypes (antigenic shift) are only found in influenza a virus and typically involve the emergence of novel hemagglutinin (h) and/or neuraminidase (n) proteins on the viral envelope. pandemic influenza occurs when a new influenza a strain emerges, to which the majority of the world's population has little or no immunity. there were three influenza pandemics in the last century, of which the pandemic in 1918 due to the h1n1 virus was the most severe. the first pandemic of this century occurred in 2009 [1] and was due to another h1n1 variant that emerged through a quadruple reassortment of viral rnas derived from human, avian, eurasian and north american swine influenza sources [2] . the presence of animal influenza subtypes, particularly avian influenza viruses such as h5n1, is of continuous concern, as these could be the source of future pandemics. though with relatively high case-fatality rate, h5n1 avian influenza virus has so far only caused a limited number of human infections in restricted geographical locations with little evidence of human to human spread. however, the 2009 pandemic h1n1 virus proved to be a major burden for icu staff [3] . clinically, influenza infection is characterised by abrupt onset of fever, sore throat, myalgia, cough, headache and malaise. young children may develop croup, pneumonia or middle ear infection. with seasonal influenza, complications are often seen in the elderly, the immunocompromised and those with pre-existing chronic heart or lung disease or diabetes. during the 2009 h1n1 pandemic, children and young adults were more susceptible [4] . overall fatality rate was .5%, but as many as 9-31% of hospitalised patients needed icu admission [5] . severe disease and high mortality rates were seen in pregnant women, patients with underlying medical pulmonary, cardiac, metabolic, neuromuscular illness and severe obesity, and those in whom the diagnosis and admission was delayed [6] [7] [8] . respiratory failures could be caused by viral pneumonia and acute respiratory distress syndrome (ards). in addition, secondary bacterial infection with streptococcus pneumoniae or staphylococcus aureus (often methicillin resistant) were found in 20-24% of icu patients and 26-38% of patients who died [3, 5, 9] . fatal cases were often complicated by multiorgan failure. influenza has a short incubation time of 1-4 days. the virus is transmitted via droplets, and patients are infectious during the prodromal phase and up to 7 days after symptom onset. rapid antigen detection from respiratory secretions is available, but this was found to be insensitive for the 2009 h1n1 pandemic virus [10] . more sensitive and specific real-time polymerase chain reaction (pcr) methods had to be used [11] . due to the infection-control hazards of taking nasopharyngeal aspirates or bronchoalveolar lavage, the use of throat and nasal swabs were advocated. a complete respiratory diagnostic workup needed to be performed to exclude other viral, bacterial and noninfectious causes. a single negative influenza pcr result on an upper respiratory sample did not definitively exclude the diagnosis [12] . in addition, other concurrent or secondary infections had to be considered. protocols needed to be in place to ensure satisfactory triage of patients according to severity [13] . early administration of specific neuraminidase inhibitors, such as oral doses of oseltamivir or inhalation zanamivir, seemed to be beneficial [14] . in more refractory cases, the off-license use of intravenously administered zanamivir or peramivir was tried. extracorporeal membrane oxygenation (ecmo) was found to be useful in very severe cases [12] . the risk of nosocomial transmission to other hospitalised patients and staff is well documented. infected patients should ideally be cared for in a single room or cohorted together. health care workers should be protected through the proper use of personal protective equipments, including respirators or masks, eye protection, gowns/aprons and gloves [15, 16] . high-filtration respirator to ffp3 (europe) or n95/n99 (usa) standard should be used for staff carrying out aerosol-generating procedures after fit testing and training. surgical masks should be adequate for nonaerosol contacts [16] . environmental contamination is an important source of transmission. good hand hygiene can prevent transmission through this route. vaccination is the most specific preventative measure. annual seasonal influenza vaccination to vulnerable individuals and health care workers has been advocated. a specific vaccine against the h1n1 pandemic strain was developed within months of the onset of the outbreak. however, vaccine uptake rates amongst health care workers are usually poor, and more needs to be done to educate both patients and staff. respiratory syncytial virus (rsv) (family paramyxoviridae) is a major cause of lower respiratory tract infections in young children and infants. there are two subtypes, a and b, with varying dominance in different years [17] . the incidence of rsv is seasonal in temperate climates, and hospital admissions usually peak during winter months. prematurity, bronchopulmonary dysplasia and congenital heart disease are associated with a significant risk for admission to high-dependency units or picu. in switzerland, it was estimated that approximately 1-2% of each annual birth cohort required such admission. rsv can also cause significant disease in adults, particularly in immunocompromised individuals such as patients undergoing therapy for haematological malignancies, the elderly and those with chronic pulmonary disease [18] . the most rapid diagnosis of rsv is by direct antigen detection methods such as chromatographic immunoassays. a typical rapid test method is completed within 30 min and can be used as a point of care testing method in emergency rooms and icus. however, these rapid tests lack sensitivity [19] . more recently, many laboratories have begun using multiplex real-time nucleic acid amplification techniques (naat) to diagnose respiratory tract infections, including rsv [20] . although naat is highly sensitive, it is not a rapid testing method. hence, it is desirable to have a mixed strategy of diagnostic approaches, such as an initial rapid direct antigen test followed by retesting of negative samples by naat. nosocomial transmission of rsv in the icu and haemoncology units has frequently been reported. it is important to identify infected patients and to apply prompt and effective infection control measures (table 20. 2). it is recognised that a combination of cohorting patients using dedicated health care staff, contact isolation of patients, strict adherence to hand hygiene; and screening visitors, family members and health care staff for upper respiratory tract infection symptoms significantly reduce the cross-infection rate of rsv. in haemoncology units, the practice of enhanced seasonal infection control programs for rsv has been shown to be effective [21] . the usefulness of wearing masks and goggles is less clear. there is no safe and effective vaccine to prevent rsv infection. however, immunoprophylaxis in the form of rsv immunoglobulin (rsv-ig) or humanised monoclonal antibodies (palivizumab) is available as prophylaxis for some highrisk patients to prevent serious rsv disease or to limit further nosocomial spread. both palivizumab and rsv-ig have been shown to decrease the incidence of rsv hospitalisation and icu admission, although there was no significant reduction in the risk of mechanical ventilation or mortality rate. when given prophylaxis, infants born\35 weeks gestational age and those with chronic lung and congenital heart disease all had a significant reduction in the risk of rsv hospitalisation [22] . treating rsv infection is mainly supportive, including oxygen, ventilation and bronchodilatative drugs. aerosolised ribavirin has often been used in severe cases, with or without gamma globulin i.v. [23] . however, evidence for the clinical efficacy of ribavirin in rsv infection remains inconclusive [24] . the use of aerosolised ribavirin needs to be carefully controlled, as there are potential teratogenic effects on pregnant staff and visitors. others have tried a combination of palivizumab i.v. with or without ribavirin [25] . another paramyxovirus, known as human metapneumovirus (hmpv), shares a similar spectrum of clinical illness as rsv. it is likely that general infection control measures against rsv would also be effective against hmpv. there are four types of human parainfluenza virus (piv) types: piv 1-4 (family paramyxoviridae). infections with piv1 and 2 are seasonal, with a peak in autumn affecting mainly children between 6 months and 6 years of age. clinically, patients often present with croup or a febrile upper respiratory tract infection. in contrast, piv3 is endemic throughout the year and infects mostly young infants in the first 6 month of life and up to 2 years of age. clinically, there is no specific presentation in piv3, but bronchiolitis and pneumonia are not uncommon. in immunocompromised adults, such as stem cell transplant recipients, piv3 is associated with a high mortality rate. such patients often present with severe pneumonia and many require admission to the icu. the diagnosis of piv infection can be confirmed by immunofluorescence antigen detection or naat [26] . nosocomial transmission is often due to piv3 and has been documented in neonatal care and adult haematology units [27] . infection control precautions are the same as for rsv. despite several uncontrolled case series of apparent successful use of intravenously, orally or aerosolised administration of ribavirin to treat piv infections, there is no clear evidence that ribavirin with or without immunoglobulin alters mortality rates from piv3 pneumonia or decreases the duration of viral shedding from the nasopharynx [28] . nevertheless, there may be a role for pre-emptive early therapy with ribavirin to prevent progression of upper airway infection to pneumonia. adenovirus (family adenoviridae) multiplies in the pharynx, conjunctiva or small intestine. clinically, the infection is localised and typically presents with pharyngitis, conjunctivitis or gastroenteritis depending on serotype. however, in young infants and immunocompromised patients such as organ transplant recipients or aids patients, adenovirus can cause severe pneumonia, disseminated infection or haemorrhagic cystitis. the diagnosis can be confirmed by specific antigen detection tests on respiratory or stool samples. viremia and viruria can be confirmed and quantified using real-time pcr. in respiratory infections, the virus spreads via droplets or through contaminated hands or fomites. nosocomial adenovirus infections have been reported and can be a particular problem in neonatal units. it is important to adhere to strict infection control procedures to prevent nosocomial spread (table 20. 2). in vitro, adenovirus is susceptible to antivirals such as cidofovir and ribavirin [29] . use of cidofovir in selected patients may be successful [30] . a respiratory virus that caused a severe acute respiratory syndrome (sars) emerged from southern china in 2002. the virus was subsequently identified as a novel virus from the coronaviridae family and was named sars coronavirus (sars cov) [31] . sars was associated with a high mortality rate, and of the most concern to the international community was the potential in causing nosocomial infections. from a single index case in a hong kong hotel, a series of chains of outbreaks occurred in vietnam, singapore and canada [32] . subsequently, infections were reported in major cities in asia, europe and usa, transmitted through international travel. in total, 8,422 individuals were infected, with 916 deaths around the world. the emergence of sars was the first wake-up call to the medical community regarding the need for comprehensive infection control policies in hospitals and icu. this also led to the general provision of personal protective equipment (ppe) with training and fitting programmes for health care workers in many countries. sars is infectious from the onset of illness and infectiousness correlates with the degree of viral shedding. incidences of superspreaders or superspreading events may have accounted for most of the large-scale transmissions. older age and underlying comorbidity are major risk factors for fatality [33] . viral loads in various anatomical sites also correlate with the severity of symptoms and mortality. shedding of sars cov peaks at day 10 after the onset of symptoms. the disease pathology is characterized by uncontrolled viral replication, with a major proinflammatory response. the optimal therapy for sars is still not clear, as there were no randomized controlled trials conducted. treatment with interferon (ifn)-a, steroid, protease inhibitors (such as lopinavir) together with ribavirin, or convalescent plasma containing neutralising antibody, could all be useful. prophylaxis with ifn or hyperimmunoglobulin may also be considered as postexposure prophylaxis [34] . sars cov is identified as a zoonosis with a natural reservoir in chinese horseshoe bats [35] . its emergence is associated with local culinary practice in southern china, leading to captured palm civets acting as the amplifying host and passing on infection to human. as long as the reservoirs and amplifying hosts coexist, there is a potential for sars to re-emerge. intensivists should always be on the lookout for patients with unexplained severe respiratory infections and consider sars as a possible differential diagnosis. primary varicella zoster virus (vzv) (family herpesviridae) infection causes chickenpox. this is a common self-limiting childhood infection characterised by a mild fever and a generalised vesicular rash. risk factors for severe disease include immunosuppression, smoking and pregnancy. complications include bacterial sepsis, pneumonia, encephalitis, ataxia, toxic shock, necrotising fasciitis and haemorrhagic chickenpox with disseminated coagulopathy and fatality [36] . chickenpox is highly infectious and can be transmitted via inhalation of respiratory secretions or by direct contact. patients are likely to be infective 48 h before the appearance of the rash until the last lesion has crusted over. outbreaks in the icu have frequently been reported [37, 38] . infected patients should be promptly isolated, preferably in negative-pressure rooms. a rapid diagnosis of chickenpox can be made by electron microscopy or immunofluorescence of scrapings from the vesicle base. a person who has had chickenpox does not develop chickenpox again, but the virus may reactivate as zoster/shingles. susceptibility to chickenpox can be determined by testing for the presence of vzv immunoglobulin (ig)g. infected patients need to be isolated immediately, and exposed patients and staff investigated. exposed staff who are susceptible to vzv should be excluded from contact with high-risk patients for 8-21 days postexposure. susceptible individuals at risk of severe disease should receive varicella-zoster immunoglobulin (vzig) prophylaxis, which could be given up to 10 days after exposure. neonates born to mothers who developed chickenpox 7 days before to 7 days after delivery are highly susceptible due to a lack of protective maternal antibodies. in such cases, vzig prophylaxis to the neonate is recommended. the baby should also be isolated. intravenously administered acyclovir should be started promptly at the first sign of illness. most childhood chickenpox does not require treatment. however, in severe cases (e.g. pneumonitis, disseminated disease with visceral involvement and patients requiring hospitalisation), intravenously administered acyclovir (10 mg/kg 8 hourly) is the treatment of choice. treatment of neonates will require a higher dose (20 mg/kg 8 hourly). a live attenuated vaccine against vzv is available. susceptible health care workers should be immunised. rotavirus (family reoviridae) is highly infectious and a significant cause of nosocomial gastroenteritis, particularly in children \5 years of age. patients present with sudden onset of fever, vomiting, abdominal pain and watery diarrhoea. due to the high viral shedding in the faeces, a diagnosis can be easily obtained using antigen-detection enzyme-linked immunosorbent assay (elisa) or electron microscopy. in temperate climates the infection is seasonal with peaks in winter, and hospital outbreaks often coincide with outbreaks in the community. in europe, it was found that 49-63% of paediatric nosocomial gastroenteritis was positive for rotavirus, with an incidence of 1-2.3 per 1,000 hospital days, leading to prolonged hospitalisation between 1.5 and 4.5 days [39] . very sick infants with gastroenteritis may require intensive care and could, in turn, be the source of nosocomial infection in icu. premature and very low birth weight infants (\1,500 g) are particularly at risk, as severe complications such as necrotising enterocolitis and intestinal perforation are commonly reported. a dutch study found that amongst all nosocomially acquired viral infections in nicus, 10% were due to rotavirus, which demonstrates the importance of this infection in the icu setting [40] . nosocomial rotavirus infections in adults have also been reported and occasionally cause serious complications in the elderly and immunosuppressed patients. nosocomial transmission has been previously associated with ungloved nasogastric feeding, contaminated toys, shortage of nurses, overcrowding and high patient turnover. adherence to effective infection control measures (hand hygiene, enteric precautions; table 20 .3), as well as adequate staffing and patient cohorting/ isolation can therefore help prevent or manage an outbreak [41] . the recently developed rotavirus vaccine could substantially reduce the incidence of nosocomial infections [42] . norovirus (family caliciviridae) is the most common cause of nosocomial outbreaks of gastroenteritis. symptoms typically comprise profuse diarrhoea and projectile vomiting. the diagnosis can be confirmed by elisa, rt-pcr or electron microscopy of stool samples. noroviruses are highly infectious and are usually transmitted by direct contact via the faecal-oral route or via oropharyngeal exposure to aerosolised vomit. a number of outbreaks have recently been described in nicus involving mainly premature neonates, some of whom developed necrotising enterocolitis. neonates and immunocompromised patients can shed the virus for a prolonged time over months, which emphasises the need for rigorous adherence to effective infection control measures (table 20. 3). additional measures such as increased hand hygiene and wiping of floors and incubators with agents active against caliciviruses have been proven to be particularly useful in controlling outbreaks in nicu wards [43] . both enteroviruses and parechoviruses (family picornaviridae) have numerous subtypes. enteroviruses include polioviruses, coxsackieviruses, echoviruses and other numbered enteroviruses. there are as many as 14 types of human parechoviruses [44] . parechovirus type 3, in particular, can cause severe infection in young infants [45] . both viruses are significant causes of nosocomial infections, particularly in the nicu. enterovirus outbreaks involving up to 23 neonates have been reported [46] , and an attack rate of 29% was reported. enterovirus infections can present as neonatal sepsis, meningoencephalitis, myocarditis, hepatitis or gastroenteritis. necrotising enterocolitis with pneumatosis intestinalis is a known complication in neonates. some enteroviruses, such as enterovirus 71, can cause severe and fatal illness in older children. parechoviruses can cause meningoencephalitis [47] and a sepsis syndrome in young infants [48] . enteroviruses and parechoviruses are genetically distinct from each other and require a different rt-pcr for diagnosis. sequencing of the gene encoding the vp1 region of the virus has been used to identify outbreak strains. with the global polio eradication programme, poliomyelitis is no longer a common nosocomial infection, although health care workers in the icu who may [41] • hand washing (liquid soap) or decontamination (aqueous antiseptic/alcohol based-hand rub) (a). however, alcohol-based products are known to be less effective against nonenveloped viruses, for which hand washing with soap and water is preferred (b) • wear disposable gloves and aprons when contact with stool or vomitus is likely (b) • isolate symptomatic individuals (particularly with uncontrolled diarrhoea, incontinence, and children) (b) • avoid unnecessary movement of patients to unaffected areas (b) • staff working in affected areas must not work in unaffected areas within 72 h (b) • exclude symptomatic staff members from duty until symptom free for 72 h (b) • if a large number of patients is involved and no further isolation facilities are available, close the unit to new admissions or transfers until 72 h after the last new case (b) • terminal cleaning of the environment, using freshly prepared hypochlorite (1,000 ppm) on hard surfaces (b) • caution visitors and emphasise hand hygiene (b) categorisation of recommendations: a strongly recommended for all hospitals and strongly supported by well-designed experimental or epidemiological studies; b strongly recommended for all hospitals and viewed as effective by experts in the field be in contact with live vaccine poliovirus shedding infants should ensure that they are immunised. rigorous hand washing (table 20. 3) is the most important measure during an outbreak. cohort nursing, source isolation and screening are other measures frequently used (table 20. 3). clearance of the virus by the host is antibody-mediated and many have advocated the use of normal human immunoglobulin (nhig). hepatitis a virus (family picornaviridae) belongs to the same family as enteroviruses and is usually transmitted via the faecal-oral route. nosocomial transmission of hepatitis a virus is well documented. an outbreak in an adult icu (aicu) occurred as a result of inadequate precautions taken while handling bile of a patient not suspected of incubating hepatitis a [49] . most other outbreaks occurred in picus or nicus, with attack rates varying between 15 and 25%. risk factors for outbreaks have been attributed to handling soiled bed pads, nappies or gowns of an index patient, failure to wash hands, and eating in the icu. in the nicu, vertical transmission and blood transfusion have been implicated as the cause of infection in the index case. the effect of nosocomial hepatitis a infection varies from asymptomatic to classic presentation with acute hepatitis. diagnosis is by serological detection of hepatitis-a-specific igm. the use of molecular techniques such as rt-pcr can help identify early infection or in difficult cases, such as those with immunodeficiency. sequencing of pcr products is useful in establishing epidemiological linkage during outbreaks. nhig has been successfully used for postexposure prophylaxis to control outbreaks. there is now increasing evidence that hepatitis a vaccine can be used for prophylaxis if the contact occurs within 14 days from onset of illness in the index case [50] . the most commonly encountered nosocomial blood-borne viruses are hepatitis b virus (hbv), hepatitis c virus (hcv) and human immunodeficiency virus (hiv). the main risks are transmission from patients to health care workers. however, transmissions between patients and from health care workers to patients have been reported. the best way to prevent occupational exposure of blood-borne viruses is to practice universal precautions. blood and body fluids (table 20 .4) from any patient, whether or not there are identifiable risk factors, should be considered as a potential risk. this encourages good and safe practice and helps prevent unnecessary accidents. physical isolation of patients with blood-borne virus infection is generally not necessary unless there is profuse uncontrolled bleeding. infection-control teams and occupational health departments should adopt a proactive approach to educate and prevent sharps injury (table 20 .5). there should also be specific instructions on how to deal with blood and body fluid exposure (table 20 .6). hbv is the most infectious of the three common blood-borne viruses. the risk of transmission depends on the viral load of the source patient. an hbv-infected individual with hepatitis b ''e'' antigen (hbeag) tends to have a high viral load and is therefore more infectious than carriers without hbeag. estimate of infectivity ranges from 2% (hbeag absent) to 40% (hbeag present). all health care workers should be immunised against hbv. exposed health care workers who are susceptible (not immunised or vaccine nonresponders) should receive hepatitis b immunoglobulin for postexposure prophylaxis. a booster dose of vaccine should be given to those exposed individual who had previously been successfully immunised. hcv is probably the commonest blood-borne virus encountered in western countries. in the uk over a 3-year period, 462 incidences of occupational exposure to hcv were reported in comparison with 293 of hiv and 151 of hbv [51] . follow-up studies of health care workers who sustained a percutaneous exposure to blood from a patient known to have hcv infection have reported an average incidence of seroconversion of 1.8% (range 0-7%). no vaccine or postexposure prophylaxis was available to prevent hcv transmission. early diagnosis is essential, as early interferon treatment after seroconversion has a high success rate for eradication [52] . exposed health care workers should be followed up at 6 and the average risk of hiv transmission after percutaneous exposure to hiv-infected blood is about 0.3%. after mucocutaneous exposure, the risk is estimated to be .1%. a case-control study [53] identified four factors with increased risk of transmission: • deep injury; • visible blood on the device that caused the injury; • injury with a needle that has been placed in a source patient's artery or vein; • terminal hiv-related illness in the source patient. this study also showed that the use of zidovudine prophylaxis reduce the risk of transmission by 80%. postexposure prophylaxis (pep) should therefore be offered to all health care workers who have significant exposure to blood or body fluid from a patient known to be at high risk of or to have hiv infection. various pep options are available depending on national recommendations. this should be started as soon as possible after exposure and continued for 4 weeks. viral haemorrhagic fevers (vhfs) are severe and life-threatening diseases caused by a range of viruses. they are either zoonotic or arthropod-borne infections and are often endemic in certain parts of the world. they are often highly infectious through close contact with infected blood and body fluid and therefore pose a significant risk of hospital-acquired infection. as many patients with vhf present with shock and require vigorous supportive treatment, it is a potential problem in the icu. the major viruses of nosocomial concern in this setting are marburg, ebola, rift valley fever, lassa and crimean congo haemorrhagic fever (table 20 .7). the incubation period for these vhfs ranges from 3-21 days. initial symptoms are often nonspecific but may eventually lead to haemorrhage and shock. any febrile patient who has returned from an endemic area of one of the vhf agents or has a history of contact with cases suspected to have vhf within 3 weeks should be considered as at risk. however, malaria should always be excluded. a risk assessment needs to be performed, and any patient known or strongly suspected to be suffering from vhf should be admitted to a high-security infectious disease unit that is designed to manage these patients. while awaiting transfer to a secure unit, such patients should be placed in a negative-pressure room with strict source isolation. specimens for patient management should be processed in a high-security laboratory designated for category 4 pathogens, and the aetiological agent established using pcr, serology and virus culture. all areas and materials in contact with infected patients should be autoclaved, incinerated or treated with hypochlorite (10,000 ppm of available chlorine). if the patient dies, the body should be placed in a sealable body bag sprayed or wiped with hypochlorite. individuals who have been in contact with a case of vhf should be put under surveillance for 3 weeks. the successful i.v. use of ribavirin has been reported in some cases of vhfs (lassa, crimean congo haemorrhagic fever and hantaan). apart from yellow fever, no vaccines are available. shingles or zoster is the result of the reactivation of latent vzv (family herpesviridae) in the dorsal root or cranial nerve ganglia. the clinical presentation is a painful vesicular eruption covering the affected dermatome. the clinical diagnosis can be confirmed rapidly by immunofluorescence, electron microscopy or pcr of the cellular material obtained from a vesicular scraping. the infection is usually self-limiting but can be more severe in immunocompromised patients, in whom it may present over multiple dermatomes or as a disseminated infection. the latter cases should be managed as if they were chickenpox, and respiratory precautions for infection control have to be enforced. patients or health care staff members with classic shingles are contagious from the day the rash appears until the lesions are crusted over. there is some risk of nosocomial transmission if the lesions are on exposed areas of the body or in immunocompromised infected patients. nonimmune (vzv-igg negative) patients or health care staff members with no history of chickenpox are susceptible if they have close contact with shingles and should be managed as described for chickenpox contact. the herpes simplex virus (hsv) (family herpesviridae) consist of two types: hsv-1 and hsv-2. clinically, they most commonly manifest with oral (mainly hsv-1) or genital (mainly hsv-2) ulcerations/vesicles, and reactivation is common, particularly in the icu. other presentations include keratitis, encephalitis, meningitis, herpetic whitlow or neonatal infection. the diagnosis can be confirmed rapidly by immunofluorescence, electron microscopy or pcr of vesicle/ulcer scrapings. in the immunocompromised patient, hsv can cause life-threatening disseminated infection and, early treatment with acyclovir i.v. is recommended. it has also been suggested that occult herpes virus reactivation may increase the mortality risk of icu patients [54] . as the infected lesions contain virus, there is an increased risk of nosocomial transmission until the lesions have crusted over. standard isolation precautions should be in place to reduce transmission (table 20. 2). patients with active lesions should be nursed away from high-risk patients (i.e. immunocompromised, severe eczema, burns, or neonates). as patients can be asymptomatic secretors, health care workers should wear gloves when dealing with mucosal secretions (i.e. saliva) to avoid infections such as herpetic whitlow. infected staff should cover lesions if possible and should not attend those at risk. neonatal herpes is usually transmitted from mother to the child at the time of delivery and may not be noticed until the infant develops the disease. universal precautions, in particularly, hand washing, should always be in place to reduce transmission of infection. to contain or prevent an outbreak, infected cases should be cohorted and nursed by dedicated staff who will not attend noninfected infants. rabies virus (family rhabdoviridae) is usually transmitted to humans following exposure to saliva of a rabid animal (e.g. dog, fox, bat) via a bite or scratch, but only 40% of exposed people develop disease. the virus spreads from the wound to the central nervous system causing fatal encephalitis, and the virus may be present in the patient's saliva, skin, eye, and brain tissue. the diagnosis can be confirmed by demonstrating the virus directly in brain tissue or saliva by rt-pcr or by immunofluorescence detection of antigen in skin biopsies from the nape of the neck. due to the severe and paralysing effect, patients may be admitted to the icu. to date, no case of nosocomial transmission has been reported apart from two patients who received corneal transplants from infected donors. suspected or proven cases should be placed in standard isolation and appropriate precautions taken when dealing with potential infectious secretions (e.g. wearing of mask if dealing with oral secretions). any health care worker with a 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hand hygiene in health-care settings. recommendations of the healthcare infection control practices advisory committee and the hipac/shea/apic/idsa hand hygiene task force healthcare-associated viral gastroenteritis among children in a large pediatric hospital norovirus infections in preterm infants: wide variety of clinical courses parechovirus typing in clinical specimens by nested or semi-nested pcr coupled with sequencing specific association of human parechovirus type 3 with sepsis and fever in young infants, as identified by direct typing of cerebrospinal fluid samples diagnosis of horizontal enterovirus infections in neonates by nested pcr and direct sequence analysis extensive white matter abnormalities associated with neonatal parechovirus (hpev) infection human parechovirus infections in dutch children and the association between serotype and disease severity an outbreak of hepatitis a in an intensive care unit hepatitis a vaccine versus immune globulin for postexposure prophylaxis exposure of healthcare workers in england, wales, and northern ireland to bloodborne viruses between management of acute hepatitis c a case-control study of hiv seroconversion in health care workers after percutaneous exposure. centers for disease control and prevention needlestick surveillance group occult herpes family viral infections are endemic in critically ill surgical patients key: cord-032181-gmcugd8h authors: song, jian-xin; zhu, lin; zhu, chuan-long; hu, jin-hua; sun, zi-jian; xu, xiang; xin, min-you; zhang, qiong-fang; zhang, da-zhi; shang, jia; huang, jia-quan; xu, dong title: main complications of aechb and severe hepatitis b (liver failure) date: 2019-05-21 journal: acute exacerbation of chronic hepatitis b doi: 10.1007/978-94-024-1603-9_2 sha: doc_id: 32181 cord_uid: gmcugd8h this chapter describes the clinical features, and diagnosis of complications in aechb including secondary bacterial infections, coagulation disorder, water electrolyte disorder, hepatorenal syndrome, hepatic encephalopathy, hepatopulmonary syndrome and endotoxemia: 1. patients with severe hepatitis have impaired immunity and are therefore vulnerable to all kinds of infections. after infection, these patients may experience shock, dic and multiple organ failure, all of which seriously affect their prognosis and are major causes of death. concurrent infections consist primarily of infections of the lungs, intestines, biliary tract, and urinary tract, as well as spontaneous bacterial peritonitis and sepsis. 2. severe hepatitis may reduce the synthesis of coagulation factors and enhance their dysfunction and increase anticoagulants and platelet abnormalities, leading to coagulopathy. infection, hepatorenal syndrome and complications can further aggravate coagulopathy, resulting in dic and seriously affecting patient prognosis. 3. hepatorenal syndrome, which is characterized by renal failure, hemodynamic changes in arterial circulation and abnormalities in the endogenous vascular system, is a common clinical complication of end-stage liver disease, and one of the important indicators for the prognosis of patients with severe hepatitis. 4. water electrolyte disorder (water retention, hyponatremia, hypokalemia, hyperkalaemia) and acid-base imbalance are common in patients with severe hepatitis. these internal environment disorders can lead to exacerbation and complication of the illness. 5. hepatic encephalopathy is a neurological and psychiatric anomaly syndrome based on metabolic disorder, and an important prognostic indicator for patients with severe hepatitis. 6. the hepatopulmonary syndrome is an important vascular complication in lungs due to systemic hypoxemia in patients with cirrhosis and portal hypertension. the majority of patients with hps are asymptomatic. long-term oxygen therapy remains the most frequently recommended therapy for symptoms in patients with severe hypoxemia. 7. endotoxemia, an important complication of severe hepatitis, is not only a second hit to the liver, but also leads to other complications including sirs and mods. (nacseld) for survival analysis of prospectively enrolled cirrhosis patients hospitalized with infections, bajaj found that there is 15.8% of nosocomial infection in all 507 patients [1] . domestic data showed significant difference of infection incidence in different times. it is related to raising diagnosis and treatment level. according to the data from shanghai public health clinical center, in 372 patients who were treated as severe hepatitis from january 2007 to december 2009, there were 68 patients obtained nosocomial infection with a rate of 18.3% [2] . the incidences of infection complicated with acute liver failure, acute on chronic liver failure or chronic liver failure are similar. patients with old age or long hospitalization are more easily to get infection. there was rare report about infection in children patients with severe hepatitis. in 2011, godbole et al. from uk reported that 25% in 145 children patients with severe hepatitis were complicated with infection, and mostly are sepsis, respiratory tract infection and urinary infection. most of the infections occurred within 2 weeks of admission, while patients with infection had longer hospitalization [3] . reduced innate immunity the innate immune system is the first line of defense against premier environmental challenges and injury. in liver, it is a complex system that includes nk cells, nkt cells, kcs, neutrophils, eosinophils and complement components. the innate immune response acts much more rapidly compared with adaptive immunity. monocytes and macrophages: the liver plays an important role in defense and immune function. the main cellular components of the innate immune system within the liver are the kupffer cells. kupffer cells represent 80-90% of the tissue macrophages in the human body [4] . in normal condition, kupffer cells in liver help to clear the macromolecular substance such as pathogen, endotoxin, heteroantigen and immune complex to defense infection. in severe hepatitis, due to massive hepatocytes necrosis, the number and function of monocytes/macrophages are impaired, thus the activity of fibronectin, which is opsonin of macrophages, is decreased. therefore, bacterium, endotoxin and other poison from gut directly access into circulation. subrat kumar acharya from india reported that the plasma fibronectin (fn) level in severe hepatitis patients was significantly lower than that in healthy controls (85.6 μg/ml ± 75.8 μg/ml, vs. 295.5 μg/ ml ± 88.5 μg/ml). the fn level was remarkably correlated with incidence of infection and mortality [5] . complement: liver is the organ where complements are mainly produced, such as c2, c3, c4, c5. the complements help to expand phagocytosis of phagocytes by chemotaxis, opsonization or adhesion, as well as help antibody to kill or solute some gram negative bacilli [6] . report from wyke showed that the defect of complement closely correlated with impaired opsonization [7] . defect of c3 or c5 can result to weakened movement of polymorphonuclear leucocyte. in severe hepatitis, the ability of liver to produce complement has been weakened due to massive injury of parenchymal hepatic cells, which leads to decreased activity of complement to 40% of normal condition. meanwhile, serum opsonization to e. coli or saccharomycetes are also diminished. besides, high ammonia level in severe hepatitis also restrains complement activity to impact germicidal effect. the most direct and also the most important result for decreased complement production and reduced opsonization is the susceptibility to infection. it was also reported that complement and the alternative pathway play an crucial role in lps/d-galn-induced fulminant hepatic failure [8] . neutrophils: a majority of patients with severe liver disease have altered function of neutrophil granulocytes [9] . the most common manifestation include abnormal ultrastructure or function of neutrophil granulocytes, as cytoplasm degeneration, organelle reduction, mitochondrial swelling, pyknotic nuclei, etc. decrease filtration and phagocytosis of reticuloendothelial system, as well as impaired chemotaxis of blood cells, making immunity weakened, lead to invasion of bacterium. therefore, patients with severe hepatitis are vulnerable to be infected with bacterium or fungi due to decreased phagocytosis and germicidal effect of neutrophil granulocytes, and impaired adhesion of macrophages and white cells [10] . data from liu h demonstrated pretreatment neutrophil-lymphocyte ratio was associated with the prognosis of patients with hbv-aclf, and elevated nlr predicted poor outcome within 8 weeks [11] . natural killer and natural killer t cells: natural killer (nk) and natural killer t cells (nkt) are important components of the innate immune response. natural killer cells have potent cytolytic activities that are exerted through the death receptor and perforin/granzyme pathways. activated nkt cells have both perforindependent and fas-ligand dependent cytotoxic function that are triggered upon tcr recognition of an antigen [12] . nk cells and nkt cells play an important role in many experimental models of liver injury, such as viral hepatitis, alcoholic liver disease, and autoimmune liver disease [13] . however, their role in aclf has not yet been clearly elucidated, it was reported the median percentage of nk cells in the lymphocytes of patients with acute and fulminant liver failure were significantly lower compared to healthy controls. meanwhile, patients with acute and fulminant liver failure had significantly high and comparable nkt cells compared to control group [14] . the important pathophysiological role of innate immune dysfunction in patients with acute-on-chronic liver failure (aclf) has been investigated in recent years. however, dysregulation of adaptive immunity remains poorly elucidated [15] . patients with severe hepatitis has varying degrees of impaired cellular immunity, manifested as decreased cd4+ cell number and declined cd4+/cd8+ ratio, which is pathogenesis of opportunistic infection. it was reported that there exists a reduction in cd4(+) t lymphocytes in hbv-aclf patients. these cd4(+) t cells predominantly are cd4(+) tconv, and the development of suppressive cd4(+) tregs greatly surpass tconv, which constitutes important characteristics of adaptive immune dysfunction of hbv-aclf [15] . a report from china showed total amount of lymphocytes, cd4(+) t cells, cd8(+) t cells and nk cells in circulation were lower in the hbv-aclf patients compared to the chb patients [16] . hbv-specific cd8(+)t-cell responses are considered to be of great importance in viral control and immune-mediated liver damage [17] . however, cd8(+) t cell has seldom been studied in aclf. ye [18] reported decreased activated cd8(+) t cells may be related to poor outcomes in patients with sh. the frequency of circulating th17 cells increased with disease progression from chb to aclf patients compared to healthy control. th17 cells were also found largely accumulated in the livers of chb patients. the increases in circulating and intrahepatic th17 cells were positively correlated with plasma viral load, serum alanine aminotransferase levels and histological activity index. in addition, the serum concentration of th17-associated cytokines was also augmented in both chb and aclf patients [19] . in process of diagnosis and treatment for severe hepatitis, repeatedly abdominal paracentesis, retention catheterization, venous cannula, hemofiltration, or trachea cannula are usually necessary. unthoroughly sterilization or nonstandard sterile operation will lead to pathogen invading to develop an infection. in addition, artificial liver treatment is also an important cause of infection. it was reported that the incidence of fungal infection in severe hepatitis correlated with the number of artificial liver treatment. application of broad spectrum antibiotics is also a major cause of infection in severe hepatitis. antibiotics inhibit or kill sensitive normal bacterium as well as pathogens, especially normal bacterium colonized in natural orifice, leading to flora disproportionality. this time, nonpathogen could cause infection, or mass produced pathogen become dominant colony to develop infection. it was proved that dosage, exposure time, or varieties of antibiotics used in patients was closely correlated with severity of dysbacteriosis and incidence of sbp [20] . nosocomial origin of infection, longterm of norfloxacin prophylaxis, history of recent infection by multiresistant bacteria and recent use of β-lactams were independent inducements associated with the development of multiresistant infections. bacterial translocation is defined as the migration of viable microorganisms or bacterial products (i.e., bacterial lps, peptidoglycan, and lipopeptides) from theintestinal lumen to the mesenteric lymph nodes and other extraintestinal sites [21] . there are multiple mechanisms which are involved in defective gut functions and altered microbiota in patients with cirrhosis or liver failure. these include small intestine bacterial overgrowth (sibo), increased intestinal permeability, and impaired antimicrobial defense. additionally, decreased bile acids, due to decreased synthesis and defective enterohepatic circulation, contribute to altered gut microbiota [22] . small intestinal bacterial overgrowth (sibo) was defined as ≥10 5 total colonyforming units per milliliter of proximal jejunal aspirations, which presents in 59% of cirrhotic patients and is associated with systemic endotoxemia [23] . in the condition of hepatic failure, due to impaired immunity, bacterium overgrowth and translocation happened, which increased incidence of infection [24] . in vivo study from wang showed phagocytosis of monocytes were prominently and immediately diminished after resection of liver tissue up to 90%, accompanied by decreased oxygen ingestion [24] . mass propagation of e. coli in the lower part of the small intestine and bacterium translocation were all happened 2 h after resection. in rats with ascites in severe liver disease induced by carbon tetrachloride, the bacteria easily transferred from the intestine to extra-intestine, including local lymph nodes and blood circulation. these evidences proved that liver failure prompted the proliferation of intestinal bacterial overgrowth and translocation, which may be the main reason for the endogenous infections in patients with liver failure. another reason for bacterial translocation is that patients with cirrhosis or liver failure display increased intestinal permeability. in patients with severe hepatitis, gastrointestinal mucosa membrane has impaired ability for regeneration and repair, leading to dysfunction of intestinal barrier and declined anti-infection ability. patients with severe hepatitis have weakened regeneration and repair capacity of gastrointestinal mucosa, as well as decreased gastrointestinal barrier function and resistance to infection. the intestinal mucosa often manifested as congestion, edema or erosion. especially when intestine get infection, bacteria can invade to abdominal cavity directly or through slight mucosal defect [25] . besides, ascites often present in severe hepatitis patients, which provide a good medium for bacteria to multiply. patients with severe hepatitis often have ascites combined with portal hypertension. due to lobular structural damage, abnormal construct of liver sinusoids or blood vessels, bacteria directly access into the systemic circulation without filtration and phagocytosis by liver, leading to formation of bacteremia. meanwhile, bacteria in blood circulation may access into the abdominal cavity and cause abdominal infections. hence, patients with severe hepatitis are not only prone to get endogenous infection but also prone to have intestinal endotoxemia. patients with severe hepatitis have decreased bile secretion, changes of bile composition, so that infection are prone to occur in epithelium of bile duct and gallbladder. in patients with severe hepatitis, intestinal edema and cellulitis are obvious, some may develop acute serositis. for patients with portal hypertensive gastrointestinal disease, regeneration and repair capacity of gastrointestinal mucosa are decreased, accordingly, the natural immune barrier function is reduced. once esophageal and gastric venous bleeding occur, gastrointestinal ischemia aggravate, which result in decreased resistance to infection. invasive procedures, complications, prophylactic use of broad-spectrum antibiotics, underlying diseases, long hospital stay, and old age are risk factors for infection in patients with severe hepatitis. elderly patients with other underlying diseases, decreased immune function, more severe primary disease, have a high risk of infection and may predispose to severe infections. in addition, albumin level is closely related to ascites production and sbp occurrence. irrational uses of immunosuppressive agents such as corticosteroids suppress immune function, lead to flora, and promote the formation of drug-resistant strains and pathogenic bacteria. mortality of severe hepatitis is closely related to infection, which directly affects the prognosis of severe hepatitis. to reduce the incidence of infection in patients with severe hepatitis, we should aim at a variety of risk factors, improve patients' immunity, rectify hypoproteinemia, treat the primary disease, prevent complications, strengthen disinfection and isolation, operate with strict aseptic technique and strict indications for invasive procedures, and use antibiotics rationally. once patients with severe hepatitis are infected, the consequences are often serious, as infection easily diffuse, which is difficult to control. after infection, the bacteria produce more toxins, which aggravate liver disease and cause a series of adverse effects, finally lead to severe complications (hepatic encephalopathy, hepatorenal syndrome, and etc.), which is the major cause for death in patients with severe hepatitis. bacterial endotoxin plays a major role in development and prognosis of severe hepatitis when infection happened [26] . endotoxemia may directly or indirectly worsen liver injury. intrahepatic cholestasis positively correlated with endotoxin levels. endotoxin can trigger hepatorenal syndrome. for kidney, endotoxin is a potent renal vasoconstrictor substance, which can make a strong contraction of the renal artery and renal blood flow reduction. in addition, endotoxin can cause kidney capillary thrombosis and acute renal tubular necrosis, or even renal cortex necrosis. endotoxin can activate coagulation factor vii and factor vi, making the intrinsic coagulation system startup, as well as directly damage hepatocytes which release tissue thromboplastin to start the extrinsic coagulation system. meanwhile, endotoxin can also damage the vascular endothelial cells to cause bleeding, especially upper gastrointestinal bleeding. in the event of gastrointestinal bleeding, infections are more prone to happen or primary infection aggravates. in recent years, due to extensive use of broad-spectrum antibiotics, the mortality of severe hepatitis has been significantly reduced. however, infection is still an important cause of death. nosocomial infection control directly affects the prognosis of patients with severe hepatitis, which is an important part for the treatment of severe hepatitis. once the etiology and other evidence of infection appear, patients should be treated with antibiotics. effective prevention and treatment for nosocomial infections are positive to promote recovery and reduce mortality. bacterial translocation (bt) is the key mechanism in the pathogenesis of sbp, which is because of the concurrent failure of defensive mechanisms in liver failure [30] . investigators have demonstrated pronounced impairment of gastrointestinal tract motility in cirrhosis [31] . the disturbance of gut flora microecology was associated with changes in the (ultra)structure of the gastrointestinal tract [32] . meanwhile, reduced cellular and humoral immunity make it easier for the free flow of microorganisms and endotoxins to the mesenteric lymph nodes [33] . besides, it was reported that hypoalbuminemia was related to sbp. a prospective study from runyon reported that the incidence of sbp in patients with ascites protein less than 1.0 g/l was 15%, while the number in patients with ascites protein more than 1.0 g/l was only 2%. after 3 years of follow-up, sbp incidence in patients with ascites protein higher than 1.0 g/l were negligible [34] . the level of ascites protein correlates with opsonin activity, which means ascites protein <1.0 g/l was an independent risk factor for sbp. the clinical manifestations of sbp are diverse. most patients have subtle and insidious onset, which usually manifested as abdominal pain and fever. acute onset may burst chills, fever and abdominal pain. generally the body temperature of patient is around 38 °c, sometimes as high as 40 °c. fever type usually presents as remittent fever. abdominal pain is always around the navel or lower abdomen, paroxysmal or persistent. nausea and vomiting are obvious, sometimes with diarrhea. ascites can occur in patients without ascites, or increased significantly. patients may also have significant bloating, abdominal muscle tension, tenderness, rebound tenderness, diminished or disappeared bowel sounds, and so on, or even shock in severe cases. according to clinical manifestations, sbp can be divided into five types: 1. common type: acute onset, protruding abdominal pain, followed by fever. or irregular fever take place followed by abdominal pain, abdominal tenderness and rebound tenderness, mild to moderate abdominal tension and growing ascites. increased wbc count and nuclear left shift. routine examination of ascites showed acute inflammatory changes. 2. shock: septic shock break out in a few hours to one day after abdominal pain or fever. the clinical manifestations include low temperature, lip cyanosis, abdominal tenderness, and hardly relieved shock. wbc count increases, blood culture is positive. 3. encephalopathy: fever and abdominal pain are often obscure, the early emergence of trance and other neuropsychiatric symptoms rapidly develop into a coma. careful examination of the abdomen in patients with light coma may still find in patients with pain expression. jaundice is deep, liver damage is serious. 4. refractory ascites: ascites progressively increase, which is difficult to subside with invalid diuretic therapy. abdominal distension is obvious, often without pain. carefully check the abdomen may still find slight peritoneal irritation. 5. asymptomatic: symptoms are mild, exhibited as slight bloating, occasional fever. mild tenderness can be found by deep palpation. in addition, a considerable part of the patients showed non-specific symptoms and signs, such as deepened coma, deepening jaundice, oliguria, azotemia or dramatically increased ascites. diagnosis of sbp should be considered if following conditions exist: (1) fever, which can't be explained by other reasons or other parts of infection; (2) abdominal pain, abdominal tenderness or rebound tenderness, but not serious; (3) sudden increased ascites or poor diuretic effect manifested as refractory ascites; (4) sudden onset of septic shock; (5) rapid deterioration of general condition, or deteriorated liver and kidney function in a short term, deepening jaundice, or hepatic encephalopathy. the diagnoses of sbp mainly rely on ascites puncture. easl clinical practice guidelines recommend that a diagnostic paracentesis should be performed in all patients with new onset grade 2 or 3 ascites, and in all patients hospitalized for worsening of ascites or any complication of cirrhosis (level a1) [34] . for patients with severe hepatitis, diagnostic paracentesis indications are: 1. in liver cirrhosis patients with ascites that paracentesis should be performed after admission to determine whether sbp exist. 2. if the following conditions happened during hospitalization, diagnostic paracentesis should be performed: (1) abdominal sighs suggest abdominal infections, such as abdominal pain, rebound tenderness and gastrointestinal symptoms (such as vomiting, diarrhea, intestinal paralysis); (2) systemic infection signs such as fever, leukocytosis, or septic shock; (3) no clear incentive for hepatic encephalopathy, or rapidly emerged renal dysfunction. 3. in patients with ascites and gastrointestinal bleeding, paracentesis should be performed before prophylactic antibiotics. once ascites was acquired, polymorphonuclear cells (polymorphonuclears, pmn) count and ascites culture should be performed. diagnosis of sbp is made according to international guidelines [34, 35] in patients with liver cirrhosis if the ascites polymorphonuclear (pmn) cell count exceeds 250 cells/μl and other forms of peritonitis have been excluded. an ascites fluid polymorphonuclear (pmn) leukocyte count ≥250/mm 3 should be considered as sbp, if pmn >500/mm 3 can be confirmed as sbp. if there is bloody ascites (erythrocytes more than 10,000/mm 3 ), pmn count are as 1/250 of red blood cells. the leukocyte esterase reagent strips (lers) test is based on the esterase activity of the leucocytes. since 2000, 26 studies have examined the validity of using leukocyte esterase reagent strips (lers) in sbp diagnosis. lers appeared to have low sensitivity for sbp. on the other hand, a high negative predictive value (>95% in the majority of the studies)supported the use of lers as a preliminary screening tool for sbp diagnosis [31] . there is bacteria colonization in ascites but no inflammation. the diagnosis is based on positive ascites culture, ascites pmn countless than 250/mm 3 , without evidence of systemic or local infection. bacterascites have two outcomes: a shortterm or transient bacterial ascites (mostly asymptomatic), or the development of sbp (mostly with symptoms). once the diagnosis was established, paracentesis examination should be conducted again after 2-3 days. take appropriate action under the circumstances. if the second sample has a pmnl count >250/mm 3 , treat as for sbp. if the pmnl count is<250/mm 3 and a second set of cultures is positive, treat as for sbp. if the pmnl count is <250/mm 3 and the second set of cultures is negative, no further action is recommended [36] . if ascites culture is positive but ascites pmn <250 /mm 3 and there were signs of abdominal infection, it usually progress to sbp within a few days. these patients should be given appropriate antibiotic therapy. brolin first proposed the concept in 1982. the mechanism: in early stage of severe hepatitis, ascites has not been exist yet, however because of necrosis of hepatocytes, dysfunction of kupffer cell, impairment of liver immune barrier, intestinal bacteria easily invasive into the systemic circulation through liver, causing spontaneous bacteremia, and then sbp. diagnostic criteria: (1) primary disease was severe hepatitis, no ascites was detected by strict examination and ultrasound. (2) clinical manifestation include fever, varying degrees of abdominal pain, diarrhea, diffuse abdominal tenderness and rebound tenderness, increased peripheral blood leukocyte, positive rivalta's test, ascites fluid leukocyte count>500/mm 3 , or pmn > 250/mm 3 . (3) exclude abdominal organ perforation or primary foci. it was used to describe the clinical situation when the ascitic pmnl count is >250/mm 3 but cultures fail to grow any bacteria. however, the term is now considered obsolete. in severe hepatitis with secondary infection, pneumonia is most common. patients with hepatic encephalopathy are susceptible to pulmonary infections as pneumonia since bed-ridden, impaired cough reflex and inadequate ventilation, especially comatose patients with intubation and tracheotomy. inpatients underwent thoracentesis, paracentesis and other invasive procedures, non-specific damage to immune barrier provide conditions for the bacterial invasion. reported in patients with invasive procedures, lung infection risk increased significantly, which demonstrated blood infection is an important way for pulmonary infection. furthermore, there was a significant increase of pulmonary infection in patients with intestinal infections or abdominal infection. pathogen: in recent years, pneumonia was classified as "community acquired pneumonia" (community acquired pneumonia, cap) "and" nosocomial pneumonia (hospital acquired pneumonia, hap). cap is the pneumonia acquired outside hospital [37] . although cap can be caused by a wide variety of micro-organisms, the pneumococcus, mycoplasma pneumoniae and chlamydophila pneumoniae, staphylococcus aureus and certain gram-negative rods are more usual pathogens encountered [38] . hap is the pneumonia that develops 48 h or more after hospital admission and that was not incubating at hospital admission. hap infected with gram-negative bacilli accounts for more than 60%, of which pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli, acinetobacter baumannii, other aeruginosa are common bacteria. the primacy is pseudomonas aeruginosa, while gram-positive cocci accounted for about 20%, mainly are staphylococcus aureus, coagulase-negative staphylococci, viridans and streptococcus pneumoniae. anaerobic is rare. clinical manifestations and diagnosis: clinical manifestations are characterized as fever, cough, expectoration, dyspnea, and cyanosis. diagnostic criteria: 1. chest percussion of dullness, auscultation of rales, along with one of the following conditions: (1) purulent sputum or change of phlegm; (2) positive pathogens in sputum culture. 2. the chest x-ray and/or ct examination revealed new or progressive exudative lesions, and the emergence of one conditions above. urinary tract infection is divided into upper urinary tract infection and lower urinary tract infection. upper urinary tract infections are mainly pyelonephritis, which can be manifested as acute pyelonephritis and chronic pyelonephritis. lower urinary tract infections include urethritis and cystitis. the most common pathogen of urinary tract infection is escherichia coli, followed by enterococcus faecalis, klebsiella pneumoniae and streptococcus agalactiae. urinary tract infections often occur in patients with indwelling catheter, therefore, aseptic urethral catheterization and management and timely replacement of catheterization are effective to prevent such infections [39] . 1. acute pyelonephritis: (1) acute onset; (2) chills, chills; (3) fever; (4) malaise, headache, fatigue; (5) loss of appetite, nausea, vomiting; (6) urinary frequency, urgency, dysuria; (7) low back pain, kidney area discomfort; the (8) tenderness of upper ureter point; (9) tenderness of rib waist point; (10) percussion pain in kidney area or bladder. 2. chronic pyelonephritis: (1) acute onset of acute pyelonephritis with the same, but usually much lighter, even without fever, malaise, headache and other systemic manifestations, urinary frequency, urgency, dysuria and other symptoms are not obvious; (2) edema; (3) hypertension. 3. urocystitis and urethritis: frequent urination, urgency, dysuria, urinary bladder pain. urethral secretions. laboratory tests and diagnosis: diagnosis elements include: (1) tenderness in rib waist point, percussion pain in kidney area. (2) urine leukocytosis, pyuria. (3) urinary sediment smear find bacteria. (4) positive in urine culture. (5) urinary colony counts>10 5 /ml; in patients with urinary frequency and other symptoms, colony counts>10 2 /ml are meaningful; counts 10 3 -10 4 /ml also helpful in diagnosis; (6) one hour urine wbc count>200,000. (7) blood test showed leukocytosis, neutrophil nucleus left. (8) increased esr. biliary tract infection is a common complication in severe hepatitis, which is often in company with cholelithiasis to reinforce each other. hepatitis b virus can directly violate bile duct cells and cause cholecystitis. on this basis, cholelithiasis and secondary bacterial infections are easy to happen and become a major focus, which can cause other parts of infection in severe hepatitis patients. furthermore, severe hepatitis patients often have reduced gastric acid secretion, so that e. coli in duodenum easily reproduce and cause ascending infection. biliary tract infection is usually a mixed infection of aerobic and anaerobic infections. enteric gram-negative bacteria include escherichia coli, klebsiella, enterobacter, proteus and enterococcus. anaerobic bacteroides include clostridium and fusobacterium, in which bacteroides is common, about 80-90%, particularly bacteroides fragilis. common symptoms of biliary tract infection include chills, fever, nausea, vomiting, right hypochondrium pain and tenderness in gallbladder area. clinical performance of concurrent biliary tract infection in severe hepatitis is not always very clear, often unconfirmed by pathogen test. symptoms were epigastric or right upper quadrant pain, radiating to the right shoulder, hours after a heavy meal or a high fat meal. the patient may have severe colic, often accompanied by nausea and vomiting. patients with chronic biliary tract infection have indigestion symptoms as heartburn, belching, acid reflux and bloating, or sometimes fever and right upper quadrant pain. severe hepatitis patients have weakened intestinal resistance, especially for reduced immunoglobulin a secretions, which creates good invasion opportunities for bacterium. in addition, as mentioned above, intestinal flora are prone to happen in severe hepatitis patients, which makes intestinal infection very common [24] . few patients exhibit cellulitis like colitis, which can further result in peritonitis and septicemia, and finally lead to death. pathogens in intestinal infections could be shigella spp., salmonella, campylobacter jejuni, clostridium difficile, and salmonella typhi etc. clinical manifestations are nausea, vomiting, abdominal pain, diarrhea, watery stool or bloody mucopurulent stool. some patients may have fever and tenesmus. depending on the pathogenesis and clinical manifestations, intestinal infections are classified as enterotoxigenic bacterial enteritis and invasive bacterial enteritis. pathogenesis of enterotoxigenic bacterial enteritis is that bacteria adhere but not invade intestinal mucosa. intestinal toxins are secreted in the process of bacteria growth and reproduction, which stimulate small intestinal epithelial cells secreting large amount of water and electrolytes. overuptake of water and electrolyte and retention in intestine makes watery stool, which is called "secretory diarrhea." secretory diarrhea is exhibited as frequent, large amount stool with no pus, usually without pain or tenesmus. it is often accompanied by vomiting, which is prone to bring out dehydration, electrolyte imbalance and acidosis, but less severe systemic toxic symptoms. stool examinations show less erythrocytes or leukocytes. invasive bacterial enteritis refers to pathogenic bacteria adhere and invade the intestinal mucosa and submucosa, causing significant inflammation. different pathogens violate different parts of intestine, small intestine, or colon, and sometimes cause inflammation both of small intestine and colon. the basic clinical manifestations include obvious systemic sepsis, high fever, and even septic shock in severe patients. stool can be mucus bloody or purulent, less amount and more frequency. abdominal pain are often severe, paroxysmal colic. if the lesion invades the rectum and distal colon in particular, there may be tenesmus. sigmoidoscopy examination showed diffuse inflammation and ulceration. if only the small intestine or upper part of colon are invaded, the stool is more moisture, and without tenesmus. stool examination show large amount of leukocytes. although diagnosis of intestinal infection is not difficult, it should be careful to distinguish the site of infection, make sure of pathogens, and pay particular attention to water, electrolyte imbalance and acid-base imbalance. therefore, in addition to routine examinations and stool culture, keeping abreast of the general condition is also important to avoid disturbance of the internal environment which aggravate liver damages. in severe hepatitis, the more severe hepatic damage and immune dysfunction, the higher of incidence of sepsis. bacterium most commonly enters through intestine into the portal vein and then into the systemic circulation, followed by skin, respiratory tract, urinary tract or other intrusion. pathogens are often opportunistic bacteria, in which gram-negative bacteria are more than gram-positive bacteria, especially escherichia coli. clinical manifestations of nosocomial infection concurrent with severe sepsis are not specific, easily masked by the primary disease and complications. in some cases primary focus is not obvious, therefore the diagnosis mainly rely on blood culture. clinical manifestations include: (1) unexplained sudden chills, fever, shock, increased peripheral blood leukocytes or neutrophils; (2) deepening jaundice, an increase in ascites, or appearance of hepatic coma, hepatorenal syndrome in a short term. when complications appear in severe hepatitis patients, sepsis should be alert. the mortality of sepsis in severe hepatitis is high. nosocomial infection not only aggravates liver damages, but also induces hepatic coma, hepatorenal syndrome, upper gastrointestinal bleeding and other serious complications, leading to multiple organ failure. nancy rolando reported mortality of septicemia in patients with liver failure was as high as 59%, in which 98% with septic shock [40] . fungal infections can be classified into endogenous and exogenous infection. according to the source of the fungus, the former belongs to opportunistic pathogens, while the latter is in the environment, being infected through various routes of exposure. fungal infections in severe hepatitis are invasive fungal infection in most occasions, and the majority are nosocomial infection and endogenous pathogenic fungi infection [27] . candida infection is most common, followed by aspergillus, again neoformans and histoplasma monocytogenes. candida albicans is widely present in normal human digestive tract. other fungi such as cryptococcus neoformans, aspergillus are widely found in nature, which can colonize in body surface, or non-enclosed cavity. they also exist in hospital work environments, increasing the chance of nosocomial infection in hospitalized patients. clinically, severe hepatitis with fungal infections are mostly superinfection. the rate of fungal infection in severe hepatitis is increasing in recent years. nancy rolando reported fungal infection was present in 16 of 50 acute severe hepatitis patients (15 candida, 1 aspergillus) and in seven cases was considered the major cause of death [41] . domestic data reported that in a group of patients with liver failure, fungous infection was found in 143 cases in which the rate of nosocomial infections was 86.71%. in 155 separated fungous strains, 90 strains (58.06%) were candida albicans, 17 strains (10.97%) were aspergillus fumigatus and 25 (16.13%) strains were non-candida albicans. the main sites of fungus infection were lungs (94 cases) and oral cavity (53 cases) [29] . for severe hepatitis complicated by fungal infection, the mechanism is complex. there are many influencing factors such as immune dysfunction and reduced defensing ability, besides, application and abuse of broad-spectrum antimicrobial drugs are also related [42] . because of broad-spectrum antibiotics destroy the imbalance between bacteria and fungi in digestive system, which inhibit the growth some gram-negative bacteria that had an anti-fungal effect and some bacteria that are able to synthesize vitamin b family. lack of vitamin b can lead to inhibition of oxidation coenzyme, enabling weakened immunity, which is conducive to fungal growth. research and experience have demonstrated that repeatedly use of glucocorticoid is also an important factor to induce fungal infection in patients with severe hepatitis [28] . and therefore, the use of corticosteroids also need special care of. currently the use of glucocorticoids in patients with severe hepatitis is still controversial. the majority do not advocate glucocorticoids, or consider a short-term use in the early stages of the disease and be removed as soon as possible. otherwise it will cause a large increase of possibility of fungal infection. fungal infections are among the leading causes of death in patients with severe hepatitis. according to an analysis from rolando, among 11 cases of severe hepatitis, seven cases of deaths directly related to fungal infections [41] . a recent domestic research analyzed outcomes of 115 patients with severe hepatitis, it was found that the mortality of patients with fungal infection was significantly higher than those without fungal infections (59.1% compared to 34.8%) [28] . according to statistics, fungal infections often occur eight days after admission (0-24 days) or 5.5 days after broad-spectrum antibiotics usage (1-14 days). symptoms of fungal infection are often covered by severe liver injury related symptoms, while clinical systemic manifestation such as fever is also difficult to identify with the bacterial infection. the site of infection often occurs in mouth, respiratory, digestive or urinary tract. severe immunocompromised persons may appear disseminated infection. oropharyngeal is the site that fungal infections mostly occur, and candida albicans is the most common pathogen, followed by non-candida albicans and aspergillus infection. bedridden patients with severe hepatitis can not properly maintain oral hygiene, which results in change of oral local environmental ph and blood flow. candida albicans easily retains in the mouth, and multiplies, causing oral flora and opportunistic infections. usually general symptoms are mild. patients often have abnormal taste or loss of taste, followed by xerostomia, mucosal burning and other symptoms. candida stomatitis may have pseudomembrane formation which is not easy to peel, accompanied by angular cheilitis, or sometimes manifests as mucosal congestion, erosion or clumps shrink of tongue papillae, thickening of coating on the tongue. there are clear lines between oral pseudomembranous damages. if remove the pseudomembrane it will leave a bright red base, sometimes thick film is like a layer of cheese. take the film directly under microscopic examination shows hyphae and spores. oral fungal infection is often a prelude of deep fungal infection, which should be on the alert. simple oral candida albicans infection is not always accompanied with fever. oral fungal infection is easy to be found, therefore, if oral fungal infection exists with fever and increased leukocytes, it should be paid attention to merger of deep fungal infections such as the lungs or other organs [43] . aspergillus is ubiquitous in nature which can be spread through air flow. the most common pathogen that causes invasive aspergillosis is aspergillus fumigatus, while aspergillus flavus, aspergillus niger and aspergillus soil are less common. inhalated aspergillus spores can reproduce in healthy or immunity weakened people. respiratory tract is an infective route of invasive aspergillosis, accounted for 95%. once tissue infection exists, blood vessels violation and bloodstream invasion are common. invasive aspergillosis infection has three characteristics: tissue necrosis, hemorrhage, dissemination [44] . the mortality of invasive pulmonary aspergillosis accompanied by severe hepatitis is up to 90% [44] . it is lack of specific clinical manifestations. in most patients fever is the first arisen symptom, mostly with middle or low heat, sometimes with sudden high fever. hemoptysis occurs with fever simultaneously or 1-8 days later, often accompanied with purulent tan sputum or bloody sputum, sometimes with a pinhead size of gray-green particles in it. shortness of breath and chest tightness often exist in late stage of infection, as well as dyspnea, cyanosis, hypoxemia, and expectoration of a lot of bright red bloody sputum or blood clots. pulmonary signs appear later, manifests as pulmonary wet or dry rales, occasional pleural friction sound. in some cases there are no pulmonary signs. x-ray examination of invasive pulmonary aspergillosis showed patchy infiltrates and (or) nodular lesions. typical nodules were like cotton, which can occur in unilateral or bilateral lobes. the lesion progresses rapidly to cause expanded infiltrates, and segmental or lobar consolidation. ct examination showed mass shadow, nodules, or exudative lesions. there are two typical imaging performance: (1) a characteristic finding on chest ct is the halo sign: a solid nodule surrounded by a halo of ground-glass attenuation [45] . (2) the formation of voids, which appear in late infection. signs are hollow cavity lesions, the balloon "crescent" sign was seen around necrosis tissue [46, 47] . in order to prevent hepatic encephalopathy, patients can be given high doses of laxatives such as lactulose, which can reduce the intestinal ph value, creating an environment for growth of fungi, thereby increasing the chances of fungal infection of the intestine. in particular, some antibiotics combination increases the incidence of intestinal candidiasis. patients usually have watery or jerry-like diarrhea, with foam or blood. diarrhea is accompanied by bloating, sometimes with vomiting and fever, however, abdominal pain is not obvious. in patients with severe hepatitis, due to decreased intestinal mucosal defense capability, candida may invade the muscle and cause intestinal bleeding, or even perforation. in part of patients, it may even progress to fungal peritonitis, which is similar to clinical manifestations of bacterial peritonitis. in patients with oral candidiasis, if there is dysphagia or pain, especially retrosternal burning, it should be considered that esophagus is invaded. incoordination motor of the upper and lower esophageal can be found by esophageal barium enema. gastroscopy helps to confirm the diagnosis. urinary fungal infection usually involves bladder and kidney, among which candida infection is the most common. however, all pathogenic fungi (such as cryptococcus neoformans, aspergillus species, mucor species, histoplasma, blastomycete, coccidioides) can spread to the urinary system as a systemic or part of disseminated fungal infection, which is more related to usage of broad-spectrum antibiotics and indwelling catheters. clinical manifestations include fever and urinary tract irritation, while some patients were asymptomatic with candidiasis urine. candida and bacterial infections often occur simultaneously. candida infections of the kidney, mostly secondary, are caused by the spread of blood candida. renal cortex and medulla abscesses can occur, which affect renal function in severe cases. patients may have low back pain, abdominal pain, fever and chills, accompanied by urgency, urinary frequency, proteinuria and hematuria. urine tests may find hyphae and fungal spores, culture for candida is positive. candida albicans is common, but now there is a growing trend of candida glabrata [27] . the main pathogen of fungal sepsis is saccharomyces. most patients have high fever, often above 39 °c. the thermal type varies, of which intermittent fever or remittent fever is more common. wbc count and neutrophil are usually increased. disease in patients with fungal sepsis followed by severe hepatitis is deteriorating rapidly, even progressing to shock. fungal septicemia may invade all the tissues and organs. involved organs have corresponding performance, such as fungal pneumonia, oral fungal infections, intestinal and urinary tract infections. disseminated fungal infection often occurs in severe immunocompromised patients who have long-term use of antimicrobial agents. candida, cryptococcus, aspergillus can disseminate along with blood to all of the organs, such as kidneys, lungs, heart, and liver. the condition is dangerous, which often lead to death in a short term. in addition to common bacterial and fungal infections, severe hepatitis can also be complicated by other pathogens, such as viruses, tuberculosis, protozoa and others. cmv (cytomegalovirus, cmv), herpes simplex virus (herpes simplex virus, hsv), or varicella -zoster virus (varicella-herpes and zoster virus, vzv) infections are three of common herpes viruses infections in severe hepatitis. their common characteristics rely on that once the host is infected, the virus can persist for long periods in the host. when the host immunity is weakened, the virus can re-proliferative, which leads to disease resurgence [48] . hsv infections manifest as perioral or external genital herpes, oral and esophageal mucosa inflammation and ulcers, also viremia which leads to pneumonia and encephalitis. common clinical symptoms of herpes simplex are mild, only a few people show fatigue, fever and other symptoms. local manifestations are single or multiple blisters on skin or mucous membrane, with tingling. due to reduced immunity, skin rashes in patients with severe hepatitis perform as varicella-like rash, vaccination herpes, herpes keratoconjunctivitis and disseminated herpes simplex. severe herpes usually manifests as herpes simplex virus encephalitis with high mortality. there are fever, headache, mental disorders, coma and other clinical symptoms, often without skin herpes lesions. the sites of infection are commonly in the frontal and temporal lobes. elevated serum antibodies help confirm the diagnosis. cytomegalovirus infections are common in cirrhosis of the liver [49] . interstitial pneumonia is the most common clinical manifestation in severe hepatitis concurrent with cmv infection. chest ct examinations mainly perform as diffuse interstitial or alveolar infiltrations, very few case show as nodular shadows, occasionally as pleural effusion [50] . pulmonary consolidation reminds complicated bacterial or fungal infections. pathology manifestations show alveolar interstitial edema, with varying degrees of fibrosis, lymphocyte infiltration and epithelial cell proliferation. blood examination shows leukopenia. because viral pneumonia shares a certain similarity in clinical manifestations, diagnosis mainly depends on pathologic examination. pathogenic examinations for cmv often use methods below: (1) detect of cmv inclusion body cells and viral particles: eosinophilic nuclear inclusions giant cells are found in respiratory secretions and bronchoscopy lung biopsy specimens. respiratory secretions, saliva, urine, cervical secretions, liver or lung biopsy specimens were inoculated to human embryonic fibroblast cell culture medium, where cytomegalovirus was separated. (2) immunological methods: cmv antigen from secretions was detected by fluorescent antibody assay, or elisa, which is conducive to the early diagnosis. serum antibodies can also be detected by complement combined experiment, in which acute and convalescent serum antibody titer more than 4 times are positive. (3) molecular biology methods: pcr technology and nucleic acid hybridization, which helps to make distinction between a variety of different subtypes of the virus. most of vzv infections in patients with severe hepatitis are due to latent virus reactivated. in patients who have had chickenpox, there is a small amount of virus lurking in the spinal cord dorsal root ganglia or cranial nerve sensory ganglia. when severe hepatitis happened, latent virus is reactivated in ganglia due to decreased immunity. the activated viruses spread along with sensory nerve axons to downstream disposal areas, proliferate and cause shingles. in early time, there is paresthesia, itching, and pain in local skin. and then rashes and herpes break out, chaining into a strip, which distribute in denomination or trunk, unilateral, with duration of about three weeks or several months. part of patients with severe hepatitis show severe disseminated herpes zoster. varicella-like rashes appear in a few days, often accompanied by fever, which may be complicated by lung, brain damage with a high mortality rate. latent tuberculosis can burst to tuberculosis and extrapulmonary tuberculosis when cellular immune function gets weakened. under normal host immune function, lymphocytes, macrophages and common langerhans cell may promote granuloma formation and make infection localized. when host immunity is dysfunctional, tissue reaction is very small or even disappeared, leading to mycobacterium growing rather than formation of granulomas, nor any effective defense against infection. severe hepatitis patients with m. tuberculosis infection may develop acute miliary tuberculosis that manifest as fever, cough, expectoration, bloody sputum, chest pain, and shortness of breath in addition to deteriorated liver function. moreover, tuberculosis easily spread in patients with severe hepatitis, with poor anti-tuberculosis efficacy. common extrapulmonary tuberculosis can be lymphatic tuberculosis, intestinal tuberculosis, bone tuberculosis, renal tuberculosis, epididymal tuberculosis, genitourinary tuberculosis, nervous system tuberculosis, tuberculous meningitis [51] . in patients with severe hepatitis concurrent with tuberculous mycobacterial infections, tuberculin reaction is negative in about half of patients, especially in those applied with glucocorticoid. diagnosis relies on sputum acid-fast staining or pcr, but the diagnostic yield is not high. interferon gamma release assays (igras), including quantiferon ® -tb gold in-tube, and the t-spot tb, have been extensively used for the auxiliary diagnosis of tuberculosis infection in adults. igras detect circulating t-cells responsive to specific mycobacterium tuberculosis antigens, which are absent in bcg and other non-tuberculosis mycobacteria, and exhibited similar sensitivity and higher specificity than tst in adults [52, 53] . however, these igra tests are also affected by host immune status [54] . in addition, the decision regarding whether to treat ltbi should be dependent not only on igras results but also on clinical histories. ntm generally causes local wound infections. however, in severe hepatitis patients with impaired immune function, non-tuberculous mycobacteria can invade the lungs, causing tuberculosis-like diseases, but rarely causes hematogenous dissemination. histological examination of the lesion is mainly characterized by epithelioid cell granulomas and foam cell-like balls of tissue proliferation, detection of non-tuberculous mycobacteria [55] . protozoa and worms, such as toxoplasma gondii, giardia lamblia, cryptosporidium and stercoralis, can also infect patients with severe hepatitis, especially those with immunosuppression drugs or combined with cancer. main lesions of toxoplasmosis manifest as lymphadenopathy, hepatosplenomegaly, encephalitis and pneumonia [56] . clinical manifestations of giardia lamblia infection are chronic diarrhea and malabsorption, also fever and cholecystitis [57] . pathological changes are deformation of small intestine villi and lymphoid hyperplasia. parasites present in small intestinal surface and gallbladder, and the detection of parasites from the stool and duodenal drainage fluid that is eligible confirmed. strongyloides stercoralis is a weak virulent worm, there is few clinical stercoralis infection [58] . but this worm infection in patients with severe hepatitis is a serious threat, even causing death. clinical manifestations include long-term nausea, vomiting, diarrhea, bloating, intestinal paralysis, dehydration, electrolyte imbalance, edema, weight loss and difficulty breathing in cases with extensive lung lesions. all patients have hypoproteinemia and anemia. patients with increased eosinophils have good prognosis, whereas eosinophils reduction often is a dangerous signal. varieties of complications, such as system or local infections, coagulopathy, hepatic encephalopathy, hepatorenal syndrome, hepatopulmonary syndrome, acid-base imbalance and water-electrolyte imbalance, are main causes of hbv-associated mortality during deterioration of liver function of aechb. reducing and effective treatments of these complications are still nodules in therapy of severe phase of aechb. infection is one of usual complications of aechb, which shows 18-52% morbidity in civil china. the most common localities are respiratory system, especially lungs, and abdomen, which shows the incidence of spontaneous bacterial peritonitis (sbp) as much as 49%. others include urinary tract and bloodstream. gram-negative bacteria are the predominant causes including aerobic gram-negative bacilli, escherichia coli, klebsiella, enterococcus and anaerobic bacteroides fragilis, etc. however in recent years, gram-positive bacterial infections are found increasing in patients with aechb, including pneumococcus and other streptococcus, but staphylococcus aureus infection is relatively infrequent. fungal infections are usually secondary to bacterial infection. candida, aspergillus, sporotrichosis, histoplasmosis and coccidioidomycosis infection, especially candida albicans, are most frequent in occurrence. but, infections of cryptococcus and mucormycosis are relatively rare. beyond of bacterial and fungal infection, other infectious pathogens in aechb are virus, mycobacterium tuberculosis, mycoplasma, chlamydia, parasites and protozoa. bacterial infection is the most frequent complication in aechb. infections are more likely occurred in abdomen, including abdominal cavity, biliary tract, gastrointestinal tract, or other parts like respiratory tract, and urinary tract. keeping oral and perineal asepsis, unobstructed respiratory tract and urine tract, anti-bedsore care for patients with limited mobility, rational use of antibiotics, avoiding long-term use of broad-spectrum antibiotics, strict controlling usage of glucocorticoid, aseptic practices in invasive operation, parenteral nutrition and protecting the intestinal mucosa are all necessities to prevention of bacterial infection in aechb. empirical use of antimicrobial agents could be determined by the localities of infection without antibiotic susceptibility testing results. gram-negative infections are more frequent in peritoneal or biliary tract, in which cephalosporin or quinolone could be the first choice. penicillin and vancomycin could be considered in pulmonary infection. azithromycin and quinolone could be adopted in urinary tract infection. metronidazole or tinidazole could be used in anaerobic infection. broad-spectrum antibiotics can be used in serious infection, such as ceftriaxone, cefoperazone, cefepime, and carbapenems, but the secondary infections need to be highly concerned. initial antibiotics should be adjusted according to antibiotic susceptibility testing results as soon as possible. non-specific immune enhancer agents, such as thymosin, are well used in treatment of infection during aechb, but it currently still lack of evidence-based support. the incidence of fungal infection ranks secondly in aechb associated infection. respiratory tract, gastrointestinal tracts and genitourinary system are the major sites. keeping wards dry and ventilated can help to reduce environmental fungal growth. oral and perineal cleaning and regularly dental check are necessary. if oral fungal spots are found, alkaline mouthwash and nystatin with glycerol can be used in treatment. for patients with limited mobility, anti-bedsore care is terribly necessary. rational use of antibiotics, especially avoiding long-term usage of broadspectrum antibiotics, can prevent secondary fungal infection. glucocorticoid should be used strictly according to indication. during invasive procedures, aseptic operations are obligated. regular check of sputum, lungs, urine and feces will facilitate early diagnosis. empiric antifungal treatment is generally not recommended, but to patients with aechb with aids, especially with count of peripheral blood cd4 + t cell-less than 200/μl or oropharyngeal candidiasis found, the sulfamethoxazole (smz-tmp) should be chosen to prevent pneumocystis pneumonia. fluconazole should be considered when moderate amount of candida albicans has been indicated by sputum culture. empirical treatment. if candida albicans infection is highly susceptible, the initial treatment choice is fluconazole (200-400 mg/days). but if aspergillus infection is preferred, amphotericin b liposome, itraconazole, caspofungin or voriconazole could be considered as the initial treatment, in which process liver and kidney function should be intensively monitored. to patients with cryptococcal encephalopathy combining severe liver damage, fluconazole or flucytosine combined with intrathecal injection of amphotericin b treatment could be implemented. evidence based treatment. initial antifungal strategy should be adjusted according to antibiotic susceptibility testing results. for certain invasive pulmonary aspergillosis, combination treatment of several different types of antifungal agents should be considered under monitoring of liver and kidney function. however, for pulmonary mucormycosis infection, the combination of amphotericin b and flucytosine is the only effective strategy. amphotericin b is a type of polyene antifungals, mainly for aspergillus, candida, cryptococcus, histoplasma infection, but is invalid in aspergillus terreus or ringworm fungus infection. intravenous or intrathecal injection of amphotericin b should be administrated because of non-digestive absorbance. the recommended initial dose of intravenous administration is 1-5 mg/days, and gradually increases to 0.5-1 mg/kg.days. the infusion track needs to be dark and process needs not less than 6 h. the initial dose of intrathecal injection is 0.1 mg/days and gradually increases to 0.5-1 mg/days. amphotericin b has an extra toxicity to liver and kidney, and also causing hypokalemia. intensive monitoring of liver and kidney function and serum potassium levels is necessary during treatment, and be sure largely avoiding combination with other agents with liver and kidney toxicity. itraconazole is one of triazole antifungal agents, mainly for aspergillus, candida, cryptococcus and histoplasma infection, but it is invalid in fungi fusarium or zygomycetes infection. the intravenous dose for the initial 2 days is 400 mg/days, administrated by twice, and then followed by 200 mg/days for 2 weeks. the oral doses of 200 mg bid could be subsequent. the total curative course could be determined by the improvement of symptoms and largely absorption of radiographic lesions of infection. the monitoring of liver function is necessary and recommended, especially in long duration treatment. furthermore, combination treatment with other hepatotoxic drugs should be avoided. fluorocytosine is one of bacteriostatics, mainly for cryptococcosis and candida infection, frequently based on the combination with amphotericin b. the daily dose for adults is 2.5 g with intravenous dripping speed of 4-10 ml/min. a half dose should be conducted in renal insufficiency. the inhibited administration includes severe liver or kidney dysfunction and allergy to fluorocytosine, and also cautious treatment for pregnant or breastfeeding women. no combination treatment of fluorocytosine with cytarabine or bone marrow suppression agents is recommended. fluconazole is a triazole antifungal, mainly for candida albicans or cryptococcus infection, but not as good in candida glabrata infection, totally invalid in aspergillus or candida krusei infection. the recommended daily dose for adult is 200-400 mg, and the initial dose should be doubled. voriconazole belongs to triazole antifungals, mainly for candida, cryptococcus, aspergillus, fusarium and histoplasma capsulatum infection, especially used for invasive aspergillosis and invasive fluconazole-resistant candida infection, but is invalid for mucor or rhizopus infection. the recommended intravenous initial dose in adult is 6 mg/kg, q12h, with infusion rate of 3 mg/kg within 1-2 h. the maintenance dose from the second day is 4 mg/kg, q12h. to patients without tolerance to normal dose, it could be reduced to 3 mg/kg, q12h. the reduction of daily dose could not be necessary in mild to moderate liver dysfunction. but intravenous administration should be avoided in patients with severe renal dysfunction. the side effects of voriconazole include transient visual disturbances, mental disorders, thrombocytopenia and so on. caspofungin belongs to echinocandin antifungals with antibiotic spectrum of pathogenic aspergillus, candida and pneumocystis, without in cryptococcus neoformans, fusarium and mucor. it is mainly used for invasive aspergillosis. the initial dose for adults is 70 mgqd, and with subsequent 50 mg qd. the infusion time is no less than 1 h. no fixation curative course is suggested. caspofungin should be avoided for patients with severe liver function, because of highly hepatic distribution and metabolic pathways during treatment. liver is the largest solid organ in the body, and it plays a central role in the clotting process, except for general function such as metabolism, detoxification and choleresis [59] . a majority of the coagulation factors are synthesized almost exclusively in the liver. in the pathogenesis of severe hepatitis, massive hepatic necrosis leading to reduced production and dysfunction of coagulation factor. in addition, the increased levels of anticoagulation and platelet abnormalities also contribute to occurrence of coagulopathy, which were further exacerbated by severe complications such as spontaneous bacterial peritonitis (sbp) and hepatorenal syndrome (hrs). coagulation abnormalities, even disseminated intravascular coagulation (dic) often occur in those patients with severe hepatitis. therefore, the changes in coagulation factors were usually used to evaluate prognosis of severe hepatitis [60] . the normal procedure of coagulation includes two independent coagulation process, namely intrinsic and extrinsic pathway, which initiated by coagulation factor xii, and factor viia/tissue factor, respectively. the two pathways reach a confluence at the points of factor xa and va. wherein factor xa activates prothrombin to thrombin in the presence of ca 2+ and factor va bound to membrane surfaces, and then thrombin converts fibrinogen to fibrin. thus, blood becomes clotted and the "y" shaped pathway was established [61] . simultaneously, there are some anticoagulation systems existing in our body, which prevents the formation of thrombus, then keeps normal circulating of blood flow. it also participates in maintaining of normal permeability of the blood vessel [62] . among anticoagulation systems, the most important one is the fibrinolytic system, whose basic process can divided into two stages, i.e. plasminogen activation and fibrin degradation [63] . plasminogen activators include tissue-type plasminogen activator (tpa) and urokinase-type plasminogen activator (upa), and the principal function of these two plasminogen activators is to convert plasminogen to plasmin, then plasmin cleaves fibrin into soluble small peptides, namely fibrin degradation products [64] . moreover, the process of fibrinolysis was affected by fibrinolysis inhibitors, such as plasminogen activator inhibitor (pai) and alpha2antiplasmin (α2-ap). those fibrinolysis inhibitors can either inhibit plasminogen activation, or reduce the function of plasmin [65] . thus, there is a dynamic balance between coagulation and anti-coagulation systems under physiological conditions ( fig. 2 .1) [66] . many factors involved in coagulation process, i.e. clotting factors, thrombin inhibitor, fibrinolytic system and so on, are synthesized almost exclusively in the liver. thus, liver plays a pivotal role in remaining the balance between hemorrhage and coagulation under physiological conditions (table. 2.1) [67] . it is the important basis for pathogenesis of coagulopathy that massive hepatocyte necrosis occurs in severe hepatitis patients, resulting in reduced production and dysfunction of those blood clotting factors [68] . coagulation abnormalities, such as decreased production and dysfunction of coagulation factors, are commonly found in severe hepatitis, there are at least 14 types of factors participated in the coagulation process, including 12 classical coagulation factors, namely factor i/fibrinogen, factor ii/prothrombin, factor iii/tissue factor, factor iv/calcium ions, as well as factor v, vii, viii, ix, x, xi, xii, and factor xiii. some bradykinin factors, such as rk (prekallikrein) and hmwk (high molecular weight kininogen) are also associated with coagulation. among above factors, the others belong to proteins excepting factor iv. plasma coagulation factors are synthesized exclusively in the liver, excepting factor iii/tissue factor, factor iv, factor vi/activated factor v, factor viii and factor viiia [69] . massive hepatic necrosis leads to the decreased production of clotting factor in patients with severe chronic hepatitis. moreover, the coagulation factors are sensitive indicators for clinical evaluation of severe hepatitis. it is common that the serum levels of factor v, vii, ix, x, xi and prothrombin decreased in the patients with severe hepatitis. on the contrary, clotting factor viii is synthesized, and then excreted increasingly by the mononuclear phagocytic cells with stimulation of various inflammatory cytokines in the patients with severe hepatitis [70] . anorexia and antibiotics overuse lead to obstacles in assimilation, absorption, and application of vitamin k in these patients. vitamin k dependent coagulation factors includes factor ii, vii, ix, and x. the function of γ-hydroxylation was weaken by the deficiency of vitamin k and damage of hydroxylase, then the abnormal clotting factors without γ-hydroxy glutamate were synthesized. finally, these abnormal clotting factors lead to dysfunction of the vitamin k dependent coagulation factors [71] . coagulation factor vii eliminated significantly in the initial stage of liver injury was usually used as an early diagnosis index [72] . in addition, coagulation factors ii and x, then factor ix decreased markedly, along with exacerbation of liver injury. the deficiency of vitamin k induced by obstructive jaundice, malabsorption syndrome, can be rescued after intravenous injection of vitamin k, which is different from coagulopathy caused by liver injury. the plasma level of fibrinogen is within normal range in patients with compensatory cirrhosis. however, patients with severe hepatitis or liver failure have a significant reduction in the level of fibrinogen, and then develop dysfibrinogenemia [73] . fibrinogen, as the main hydrolysis substrate of thrombin, is the crucial factor in the coagulation process. thus, decreased levels or abnormal structure of fibrinogen leads to coagulation abnormalities in patients with severe hepatitis [74] . there are two clotting factors associated with thrombin except for fibrinogen, i.e. factor v and factor xiii. factor xiii induce a crosslink of sfm (soluble fibrin monomer), resulting in the formation of insoluble fibrin [75] . it reports that the levels of circulating coagulation factor xiii were decreased in 30% of patients with liver disease, resulting in fibrin decline or abnormality. these patients with liver disease will receive a bad prognosis if the plasma level of fibrin was at level below 35% of normal concentration [76] . it is uncommon that the plasma level of plasma factor v decreased markedly in patients with severe hepatitis, except for those complicated with dic or hyperfibrinolysis. the low level of factor v cannot induce the formation of the enzyme-substrate complex, which delay the activation of prothrombin [77] . contact factors include classical coagulation factor xii and xi, as well as some bradykinin factors, i.e. pk and hmwk. these factors contact with vascular intima damage or abnormal surface in blood vessel walls, then active the intrinsic coagulation pathway [78] . moreover, the contact factors can connect with bradykinin, fibrinolysis and complement system. in addition, coagulation factor xiii also activates fibrinolysis system as the initiating factor. factor xii deficiency does not cause excess bleeding, but induce thrombus due to its decreasing activation of fibrinolytic system [79] . there is a dynamic balance between pro-and anti-coagulation systems in physiological conditions, which keep normal blood circulation in the body. the anticoagulation system includes physiological anti-coagulation factors (e.g. antiprothrombin-iii, protein c, and so on) and fibrinolysis factors (e.g. plasminogen, α-2 plasmin inhibitor, and so on) [80] . at-iii, with a half-life period of 2.8 days, synthesized mainly in hepatocytes and partly in endotheliocyte, is responsible for about 70% of anticoagulation in plasma. it is the main reason for low plasma level of at-iii that decreased production and increased consumption of at-iii caused by hepatocytes necrosis in patients with severe hepatitis. the activity of at-iii obviously decreased in severe hepatitis [81] . therefore, heparin, thrombin, activated coagulation factors (e.g. factor x, ix, xi, xii) cannot be inhibited, due to rare at-iii combining with them [82] . there is a negative correlation between at-iii: a (the activity of at-iii) and pt (prothrombin time), which indicate that the level of at-iii: a decreased obviously along with exacerbation of liver injury [83] . the plasma levels of pro-and anti-coagulant factors are low in patients with liver injury. when the necrotic tissue and cytolysis are released in the blood, the balance of pro-and anti-coagulant is destroyed. finally the depletion of at-iii by massive activated coagulation factors will lead to dic [84] . massive hepatocytes necrosis, vitamin k deficiency, and protein c without γ-hydroxyglutamic acid result in blocking activation of protein c. thus, va, viiia and pai cannot be degraded, and the plasma level of them will rise up, due to reduction of activated protein c (apc) in the patients with severe hepatitis [85] . plasminogen synthesis will decreased by about 70% in the liver when sever hepatitis occurs. as the activators of plasminogen, tpa and upa are synthesized by vascular endothelial cells, and their production will increase after vascular endothelial cells initiated with virus, immunocomplex or endotoxin in the patients with severe hepatitis b [86] . with exacerbation of liver injury, pai synthesis decreased significantly in the liver. the main physiological activity of pai is to inhibit tpa induced plasminogen activation. therefore, the activity of tpa will increase with reduction of pai synthesis, resulting in promoting the conversion of plasminogen into plasmin [87] . plasmin, as a kind of powerful proteolytic enzyme, can hydrolyse fibrinogen into fdps, degrade coagulation factors, and inhibit platelet aggregation, resulting in the aggravation of bleeding [88] . hyperfibrinolysis can be caused by congenital or acquired reason, and it commonly leads to a rapid depletion of coagulation and anticoagulation factors, especially in those patients with dic [89] . synthesis and secretion of tpa and upa markedly increase, while pai, namely tpa or upa inhibitor, has been a decrease in the plasma of severe hepatitis patients, resulting in hyperfibrinolysis [90] . at the same time, mononuclear phagocyte system cannot degrade plasminogen activator, also leading to hyperfibrinolysis. it is not necessarily accompanied by hemorrhagic tendency, although there are many risk factors that can cause hyperfibrinolysis, even hyperfibrinolysis occurred in severe hepatitis [91] . in addition, fdps inhibit fibrin monomer polymerize function, and also block platelet aggregation, then further worse the deficiency of hemostasis and coagulation, finally leading to the aggravation of bleeding tendency [92] . studies have shown that low-level endogenous small molecule heparin in patients with chronic hepatitis may be associated with many factors, such as increased mastocyte, decreased production of heparinase in the liver and reduced activity of ph4 (platelet factor4) [93] . when the disease progress to cirrhosis or chronic severe hepatitis, pt was significantly prolonged, while endogenous heparin wasn't increased markedly, indicating that low-level endogenous heparin has little effect on pt elongation and hemorrhagic tendency [94] . however, if the patient is undergoing the following condition together, the endogenous small molecule heparin will increase significantly. (1) esophageal variceal bleeding with serious infections (such as abdominal infection, biliary tract infection and pulmonary infection) or portal hypertension. (2) combining with the significant increased white blood cell count. (3) percentage of neutrophils >75%. the level of endogenous small molecule heparin in plasmin will decrease after those infections were cured. taken together, these data indicate that increased level of endogenous small molecule heparin in blood circulation was closely related to severe hemorrhagic tendency when combining with infection [95, 96] . platelet significantly promotes blood coagulation through connecting with many coagulation factors (such as fibrinogen, factor v, factor xi, factor xiii and so on). α-granule includes fibrinogen, factor xiii, and some platelet factor such as platelet factor2 (pf2) and platelet factor3 (pf3), which promote coagulation activation process of factor xii and factor xi can be accelerated by activated platelets [97] . it is estimated that pf3 provided by platelets could accelerate activating of thrombin by twenty thousand times. xa and factor v could not be inhibited by at-iii and heparin if they were linked with pf3. when platelets aggregated and formed platelet plugs, the process of coagulation was initiated at this site, and then platelets reveal a large number of phospholipid surfaces, which were helpful for activating of factor x and fibrinogen [98] . various platelet factors were released from α-granule after platelet aggregating, and then hemostyptic fibre was produced increasingly. those hemostyptic fibre finally produced blood clot formation after capturing the other hemocytes. thus, platelet plugs would progress independently of platelet disintegrating gradually [99] . two aspects of platelet abnormalities consist of depletion and dysfunction. in patients with chronic hepatitis and cirrhosis, the occurrence of decreased platelet level is usual through hypersplenism accompanied with other hemocyte decreasing [100] . the ratio of which reaches 37-77%. its incidence rate in severe hepatitis and explosive hepatic failure also reaches approximately by 50%. when severe hepatitis occur, myelosuppression, decreased megacaryocyte replication leading short-life platelet, lacking of hemopoietic material such as vitamin b, folate and so on, all of these can initiate thrombocytopenia [101] . other reasons leading to thrombocytopenia contain low expression and metabolic disturbance of thrombopoietin (tpo), as well as platelet antibody production. researches show that the serum level of tpo related positively to platelet expression [102] . we next studied platelet associated immunoglobulin (paig) and its sorts such as paigg, paiga, paigm etc., with chronic hepatitis. in line with previous reports, we found that serum levels of paig and paigg negatively correlated with blood platelet count, corroborating the crucial role of the paig-mediated autoimmunization in controlling thrombocytopenia in viral hepatitis [103] . various factors impact platelet function forwardly or passively. increased expression of oxide and prostacyclin, two kinds of platelet inhibitor derived by endothelium, may inhibit platelet activation in vivo [104] . on the other hand, increased serum level of von willebrand factor (vwf) can promote platelet adhering and aggregation in patients with cirrhosis. when severe hepatitis occur, 66.7% and 77.8% of patients have decreased levels of platelet adhesion rate and platelet aggregation rate (par), respectively. in addition, reduced effectiveness of pf3 and abnormal clot contraction occur in patients with severe hepatitis by an incidence of 65.6% and 77.8%, severally [105] . patients with terminal liver disease significantly exert hemorrhagic tendency, especially in the digestive tract [106] . the latest report indicated that basic laboratory examinations for coagulation function testing in common use at present, such as pt, aptt, international normalized ratio (inr) etc., have little correlation with occurrence of gastrointestinal bleeding in these patients, thereby revealing the importance to search and pay close attention to those complicating disease upregulating bleeding risk, such as bacterial infection, renal failure, hemodynamic change after portal hypertension, dysfunction of endotheliocyte as well as macrophagocyte and so on [107] . it is common to see renal failure occurrence in advanced hepatopathy. when it happened, acquired platelet dysfunction, abnormal activation of platelet and vascular wall, anemia and so on, all of them significantly promote hemorrhage [108] . as another severe complication, bacterial infection is also very important and common [109] . when tumor necrosis factor (tnf) were injected into healthy individuals, we found that endotoxin have an important role to exert its function directly in clotting cascade reaction. early researches indicated that the body can express endogenous heparin-like substance through stimulation by endotoxin in patients with cirrhosis [110] . furthermore, some studies revealed the relevance of endotoxin with prothrombin fragment, indicating that infection promoted occurrence of dic-like status in laboratory examination in cirrhosis [111] . coagulation system became weaker in patients with chronic hepatitis. it can hardly mediate factors due to the relative deficiency of pro-and anticoagulation factors. when the balance was destroyed, it may tend to hemorrhage or thrombosis depending on related risk factors which were in the ascendant. to assess abnormality of blood coagulation in patients with liver disease, we should be in consideration of hypercoagulability, one state may easily be overlooked. otherwise, it will be unfair. current literatures indicated that, unlike previous concept that the body of patients initiated anticoagulation state automatically when infected by the hepatitis virus. various clinical evidences revealed that hepatitis virus infecting cannot inhibit thrombus forming. furthermore, it may increase dangerous to thrombus forming especially in the portal system, for existence of individuals having hereditary mutation to promote thrombus forming [112] . slower bloodstream, abnormal fibrinolysis initiated by blood stasis, and decreased activity of anticoagulant accelerate formation of venous thrombosis. moreover, the changes of platelet phospholipids membrane activity were also helpful to the formation of thrombosis in patients with chronic liver disease [113] . it is reported that the incidence of periphery deep venous thrombosis and pulmonary embolism was 0.5 and 1.0% in patients with cirrhosis, respectively [114] . the rate of portal vein thrombosis is about 1% in patients with compensated cirrhosis, but it reaches 8-25% in the candidates waiting for liver transplantation [115] . for mutations (such as leiden mutation of factor v, g20210a mutation of prothrombin, c677 mutation of methylenetetrahydrofolate reductase) or the existence of antiphospholipid antibody syndrome, the hypercoagulable state will be represented in patients with liver disease [116] . the hypercoagulable state represents diverse modes in the body of patients with chronic hepatitis. among them, thrombosis is more traditional and common. the mechanism of dic complicated with severe hepatitis may contain several aspects as follows. 1. with attacks from endotoxin, virus and immune complex etc., endotheliocyte was injured, and then it activated plasmakinin system and complement system, leading to the aggravation and participation of dic progress. damaged endotheliocyte can simultaneously activate factor vii, intrinsic coagulation pathway and platelets, and participate in micro-thrombosis with platelets adhesion and aggregation beneath endothelium [117, 118] . 2. in patients with severe hepatitis, massive necrotic hepatocytes activated extrinsic coagulation system with the releasing of various tissue thromboplastin-like substances [119] . 3. hepatocellular necrosis or dysfunction decreased expression of anti-coagulation factors such as at-iii, pc, protein s and so on, and then it enhanced activation of thrombin and plasmin [120, 121] . 4. impaired function of the mononuclear phagocyte system. mononuclear phagocytes can express activated tissue factor with the releasing of tnf, il-1 and platelet activating factor (paf) on its surface after stimulation by endotoxin, inflammatory cytokines and complement activation. tnf and il-1 can decrease the activation of protein c through its function to increase the expression of plasminogen activator and plasminogen activator inhibitor-1 and to inhibit the production of thrombomodulin (tm) [122] . activated clotting factor and other factors with promoting coagulation can lead to the occurrence and aggravation of dic, since it cannot be promptly removed [123] . 5. the onset and enhancement of fibrinolysis. massive clotting factors and platelets were depleted in the extensive formation in vivo microthrombus. thrombin promoted the conversion of fibrinogen into fibrous protein [124] . simultaneously, it activated fragments which dropped in the formation of activated factor xiii, factor xa and factor xiia. all of them can activate plasmin, and it enhanced fibrinolysis with tpa, one factor released by damaged blood vessel endothelium [125] . fibrinogen and fibrous protein were degraded after plasminogen activation to generate the corresponding fdps, one factor inhibit blood coagulation and also block platelet aggregation [126] . taken together, the above process exacerbates bleeding initiated by coagulation factors depletion and platelet deficiency. coagulopathy, characterized by prolongation of blood clotting time, tendency of hemorrhage, or even dic, were commonly found in severe hepatitis patients. it may cause uncontrolled external or internal hemorrhage. as common clinical symptoms, the external hemorrhages include gingivale or nasal mucosal bleeding, skin petechiae, the punctures or injection-site ecchymosis, and so on. the internal hemorrhages include esophagogastric varices, intracranial, subcutaneous, and muscle interval bleeding. except for esophagogastric varices hemorrhage, the other internal hemorrhages rarely occur in those patients. in addition, massive hemorrhage from esophagogastric varices is a serious medical emergency, can potentially cause death and cardiac arrest if proper medical treatment is not received quickly [127] . when dic occur, a wide range of thrombogenesis in microvasculature can cause circulatory collapse, characterized by low blood pressure and shock. microcirculatory dysfunction occur after microthrombosis producing, resulting in hypofunction of multiple organs (e.g. kidney, liver, lung and pancreas), which perform from dysfunction to failure, with illness progressing [88] . crushed by the fibrous protein in the vessels, red blood cell destruction can lead to intravascular hemolysis. at the early stage of disease, it shows hypercoagulability. the blood in the needle is easy to coagulate, when getting blood sampling from the vein. after that, it comes with the stage of consumed hypocoagulation. the consuming of a great number of blood coagulation factors leads to significant tendency of hemorrhage [107] . it is difficult to stop bleeding in many parts of the body, including visceral organs, operative sites, injection sites, puncture sites, and mini-invasiva sites [128] . when the third stage, namely secondary fibrinolytic stage comes, a great number of the blood coagulation factors have already been used up, resulting in severe bleeding under the condition of low coagulation. shock, acidosis and mods make the patient's condition continue deteriorating, and are also the main reasons for death [129] . the latest study indicates that thrombosis is also a noticeable state in cirrhosis and end-stage liver disease. portal thrombosis and peripheral vein thrombosis (e.g. deep vein thrombosis and lung embolism) are commonly seen. the deep vein thrombosis is more danger than lung embolism. anyway, the incidence rate of thrombosis in cirrhosis and end-stage liver disease is still very low. the clinical symptoms depend on the embolizing position after the thrombosis occurs [130] . presently blood clotting factors test is the most maturely and frequently used test in the term of blood coagulation function in the world. the indicators including prothrombin time (pt), international normalized ratio (inr) and prothrombin time activity (pta) have always been chosen in the patients with severe hepatitis. prothrombin time (pt) reflects whether there are anticoagulant substances in the extrinsic blood coagulation system and blood circulation or not. the elongation of prothrombin time (pt) presents the declined activity of several blood clotting factors including factorii (fii), factorv (fv), factor vii (fvii), factorx (fx) or the existence of anticoagulant substances. in severe hepatitis patients, the incidence rate of prothrombin time (pt) elongated can reaches to 90%, thus it is regarded as a sensitive and frequently used indicator in the term of liver function [131] . another index international normalized ratio (inr), a reckoned ratio calculated from prothrombin time (pt) and international sensitivity index (isi), making prothrombin time (pt) between different laboratories and different reagents comparable, is an international general indicator. the guides about acute-on-chronic liver failure and acute liver failure take the index, inr ≥ 1.5, as one of the most significant diagnostic criteria in the american association for the study of liver diseases (aasld) and the asian pacific association for the study of liver (apasl). international normalized ratio (inr) can also be used as a monitor on blood coagulation function [132] . hepaplastin test (hpt), reflecting not only blood coagulation mechanism in hepatitis patients but also the function of hepatocytes to synthetize vitamin k dependent clotting factors including factorii (fii), factor vii (fvii), factorix (fix), factorx (fx) synthetically, is a test about liver reserve function; but this indicator can't reflect the change of factorv (fv). when severe hepatitis occur, the function of liver to synthetize above-mentioned clotting factors declines and the time of hepaplastin test (hpt) elongates. with illness progressing, hpt continues elongating. survivors undergoing effective therapies can have gradual recovery in the time of hepaplastin test (hpt). so this test is regarded as the specific test of liver diseases or the optimal indicator reflecting liver reserve function [133] . the severity of liver damage is positively correlated with the decline degree of prothrombin time activity (pta): the more severe damage occurs in hepatocytes, the more significantly prothrombin time activity (pta) will decline. therefore prothrombin time activity (pta) <40% and total bilirubin (tbil)>171 μmol/l have always been used as the main laboratory indicators to diagnose severe hepatitis domestically. pta <40% is also regarded as diagnostic criterion of blood coagulation dysfunction in the guideline of acute-on-chronic liver failure in the asian pacific association for the study of liver (apasl) [134] . in severe hepatitis, total bilirubin (tbil), total cholesterol, prothrombin time activity (pta) and complications (e.g. rectory hyponatremia, hepatic encephalopathy, hepatorenal syndromes and so on) are all independent risk factors to evaluate prognosis. the lack of any factors including factori (fi), factorii (fii), factorv (fv), factorvii (fvii), factor x (fx), can lead to the decline of prothrombin time activity (pta). moreover the half-life time of those factors are extremely short, factorii (fii) 50 -80 h, factorv (fv) 12 -24 h, factorv (fv) 2 -6 h, factorv (fv) 48-60 h, respectively. it means that when hepatocytes suffer from severe serious damage and necrosis in severe hepatitis, prothrombin time activity (pta) will have dramatic decline just in a few days. as a result, prothrombin time activity (pta), characterized by significant advantages in evaluating patients condition and judging prognosis over other laboratory indicators, has been widely used. elongation of aptt prompts the lack of any clotting factor belonging to intrinsic coagulation system or the existence of anticoagulant substances. the incidence of the elongation of aptt reaches to 80-100% in severe hepatitis patients [135] . fxii:c reflects liver synthetic function. fvii, characterized by the shortest halftime 6-8 h, is the first one to be affected when facing liver synthetic dysfunction. on the contrary, fv:c, characterized by a relatively long half-time, is one of the latest factors to be affected and is correlated with the degree of liver damage. it prompts severe liver failure, bad prognosis and even easy death when plasma levels of fv:c under 20%. some literatures report that fv:c and pta can be used as significant prognostic factors in liver failure and significant screening indicators in liver transplantation. however, the indicators-fxii:c and fv:c-are not listed as routine examination items. they are just selected on the condition of illness demand [136, 137] . the main test about anti-clotting factors is the determination of antithrombin iii activity (at-iii:a). in the state of pathosis, the decline of antithrombin iii activity (at-iii:a) is not parallel with the decline of antithrombin iii (at-iii) content, namely the depletion of antithrombin iii activity (at-iii:a) more apparent. owe to this, it has more clinical value to determine antithrombin iii activity (at-iii:a) rather than antithrombin iii (at-iii) content. moreover, anti-clotting factors tests include the determination of protein c activity (pc:a) as well [138] . serum levels of fdps are very low in normal people. significantly elevating of fdps indicates the existence of hyperfibrinolysis and reflects the occurring of dic indirectly. there are many assay methods including immunization fi test (namely latex particle agglutination test, normal titer<1:8), fdps flocculation test, radial immunodiffusion staphylococcal clumping test, indirect hemagglutination inhibition test, enzyme-linked immunosorbent assay (elisa) and so on. if the serum levels of fdps elevate, it indicates acute dic may occur [92] . plasma protamine paracoagulation test (3p test) and ethanol gel test (egt) reflect the soluble fibrin complexes in plasma. soluble fibrin complexes, combination of fdps and fibrin monomer, can't be solidified by thrombase. but protamine is able to make the complexes isolate and then fibrin monomers separate out again. the paracoagulation test means self-polymerization between fibrin monomers and fdps, then forming macroscopic flocks. ethanol gel test (egt) has the same principle with plasma protamine paracoagulation test (3p test), whereas the former has a lower positive rate. the two methods may have false negative results and false positive results. in contrast, egt has a relatively lower sensitivity, while 3p test has a relatively lower specificity. for example, relative small molecular mass of the shreds of fdps may lead to negative result using 3p test. so it is more valuable to compare the two indicators simultaneously [139, 140] . euglobulin, a protein (including fibrinogen, plasminogen and other activins except for fibrinolysis inhibitor) separating out from plasma in acid circumstances, can be used to determine whether levels of plasminogen activators increase or not [141] . when hyperfibrinolysis occurs, plasma levels of plasminogen decline, plasma levels of plasmin increase, and euglobulin suffer from accelerated dissolution by a great number of plasmin. the normal value of euglobulin lysis time (elt) is above 2 h. that is to say dissolution within 2 h means the occurrence of hyperfibrinolysis. domestic population data report the positive rate of elt test reaches 25-42.9%, when dic occur [142] . furthermore there are other tests about fibrinolysis including tissue-type plasminogen activator test (t-pa), plasminogen activator inhibitor test (pai), plasminogen antigen test (plg:ag), plasmin activity test (pl:a), α2-plasmin inhibitor test (α2-pi) and so on [143, 144] . blood platelet count reflects the absolute number of platelet in peripheral blood circulation. according to the reports at home and abroad, platelet count have a significant decline in patients with chronic severe hepatitis. moreover, studies on domestic population find that platelet count ranges from 68 × 10 9 /l to 130 × 10 9 /l in peripheral blood of severe hepatitis patients. some studies have already compared the platelet count among early stage, typical stage and late stage in severe hepatitis, and they have turned out to be 130 × 10 9 /l, 109 × 10 9 /l and 87 × 10 9 /l respectively. as a result, it indicates that platelet count is positively correlated with the severity of hepatitis [145] . except for platelet count, other routine indicators including mean platelet volume (mpv), plateletcrit (pct) and platelet distribution width (pdw) also have significant reference value. when severe hepatitis occurs, the above-mentioned three indicators will be dramatically lower, and they have the tendency to continue declining with illness progressing. what's more, platelet quality tests include platelet aggregation rate, platelet factor 3 validity tests and blood clot retraction test (table 2. 2) [146] . diagnosis of blood hypercoagulability in the early stage of dic relies on several molecular marks including plasma thrombinogen segment 1 + 2 (f1 + 2), thrombinantithrombin complex (tat) and d-dimer, due to no significant changes in general laboratory tests. this stage is characterized by the elevated levels of those three molecular marks, and levels will increase more significantly with the occurrence of typical dic symptoms. dynamic monitoring of above-mentioned indicators is helpful for early diagnosis of dic [147] . the stages are mainly characterized by the decline of blood clotting factors (including factorv (fv), factorvii (fvii), factorxii (fxii), factorix (fix), fac-torx (fx)), platelet count and plasminogen, and increasing of fibrinolytic the elevating levels of fibrin(−ogen) degradation products (fdps) and d-dimer; the shortening of euglobulin lysis time (elt) and the positive reaction of 3p test [148, 149] . with the occurrence of dic, there will be a wide range of blood coagulation and highly-activated fibrinolysis in the patient's body [150] . what's more, abnormal increased soluble fibrin monomer and fdp fragments will exist in plasma [151] . the level of soluble fibrin monomer complex (sfmc), a complex combined fibrin monomer with fdp, is determined by 3p test. the 3p test shows positive with the occurrence of secondary fibrinolysis, whereas it shows negative with the occurrence of primary fibrinolysis. this means 3p test is negative when there is no blood coagulation. domestic population data report that the positive rate of 3p tests reach 50-60%. however, the test can't be used as the ideal diagnostic indicator for dic, owing to many affected factors. false positive reactions are mainly found in the following conditions: gastrointestinal bleeding, massive hemoptysis, malignant carcinoma or blood sampling reserved improperly, whereas false negative reactions are usually found at the late period of the stage of secondary fibrinolysis [152, 153] . as mentioned above, plasma thrombinogen segment 1 + 2 test (f1 + 2), thrombinantithrombin complex test (tat) directly reflects production of intracorporeal thrombase, which increased in the early stage of dic. plasmin degrades the crosslinked fibrin to release fibrin degradation products and expose the d-dimer antigen, which reflects production of intracorporeal plasmin. d-dimer will have an apparent increase with the occurrence of secondary fibrinolysis, but d-dimer test shows negative when primary hyperfibrinolysis occurs. monitoring the above molecular markers dynamically is helpful to estimate therapeutic efficacy and guide treatment [154] . as mentioned above, clinical manifestations of blood coagulopathy in patients with severe hepatitis are lack of specificity. the most common manifestation is bleeding, not only little hemorrhage from superficial sites, but also massive hemorrhage from internal sites, such as esophagogastric varices [155] . the diagnosis of severe hepatitis complicated with blood coagulopathy is mainly based on the results of laboratory tests and clinical manifestations [156] . according to current guidelines, basic diagnose conditions of dic contain the following points [157] . 1. severe or multiple bleeding tendency. 2. microcirculation collapse or shock which is difficult to explain using protopathy. 3. a wide range of embolism in skin and mucosa, focal ischemic necrosis and ulcer, or unexplained dysfunction of kidney, lung, brain. 4. anticoagulant therapy is effective. if severe hepatitis b patients have one of the above-mentioned points except for (1) and exhibit blood coagulating easily or prothrombin time (pt) shortening over 3 s simultaneously, it can be considered as the early stage of dic in which the tendency of bleeding is not obvious. in addition, if severe hepatitis b patients have two of the above-mentioned four points, dic can be considered as the preliminary clinical diagnosis. furthermore, it can be definitely diagnosed when combined with the aforementioned items of laboratory tests ( table 2. 3). firstly, we should use anti-viral therapy as a mainly method to treat primary disease of severe hepatitis b [158] . then, we must eliminate the incentive, maintain the balance of water and electrolyte, and correct hypoxia and acidosis. it is very important to focus on massive upper gastrointestinal hemorrhage and disseminated intravascular coagulation when treating coagulation disorders [159] . gastrointestinal bleeding, including esophageal varices bleeding and non-bleeding esophageal varices, were correlated with coagulation dysfunction and portal hypertension. prevention is still focused on improving the coagulation function, including adequate vitamin k1 supplements, coagulation factors, fibrinogen, fresh plasma or platelets supplements. it is particularly critical to control diet in patients with severe hepatitis. in which, light and easily digestible diet was recommended, but rough, hard and too greasy food was prohibited. for these patients, appropriate antacids can be used to protect the gastric mucosa [160] . 1. general treatment: bed rest is necessary, meanwhile vital signs were closely monitored. the patients can eat liquid diet when bleeding mildly or having no active bleeding. however, abrosia is required when the patients have a heavy bleeding. 2. fluid resuscitation: firstly, intravenous access should be established rapidly in patients with gastrointestinal bleeding, then, the patients were given intravenous infusion of normal saline, lactated ringer's solution, plasma, whole blood or plasma substitute [161] . 3. vaso-active agents: vaso-active agents such as dopamine and alamin may be given to maintain normal blood pressure, if blood circulation is still not stable after fluid resuscitation [162] . 4. hemostatic: if mucosal bleeding was caused by portal hypertension, oral administration of norepinephrine and ice normal saline can promote mucosal vascular contraction and hemostasis. in addition, oral administration of yunnan baiyao may be effective [163] . 5. acid suppression therapy: the proton pump inhibitors, such as omeprazole, pantoprazole and esomeprazole, are commonly used to inhibit gastric acid secretion [164] . 6. reduction of portal pressure: patients with severe hepatitis complicated by gastrointestinal bleeding often accompanied with portal hypertension, so it may be considered to give them drugs to decrease portal pressure. especially in patients with portal hypertension gastropathy, reduction of portal pressure is more important than acid suppression therapy [165] . 7. compression hemostasis via using three-chamber double-balloon catheter: after the above treatment, if there is still active bleeding in patients with bleeding esophageal varices, it can be considered to use three-chamber double-balloon catheter [166] . 8. endoscopic hemostasis: if the above treatments have no effect in upper gastrointestinal bleeding, endoscopic hemostasis, including endoscopic hemostatic agents spray, endoscopic ligation or endoscopic injection sclerotherapy may be used [167] . 9. others: surgery or interventional therapy may be considered, if internal medicine therapy is ineffective [168] . firstly, we should treat original disease, then improve coagulative function in those patients. in addition, prevention and control of infection, correction of electrolyte disturbance, avoiding the hemorrhage and reduction of allergies and transfusion reactions should be considered [169] . the key is early diagnosis and early treatment. 1. anticoagulant drugs: low molecular weight heparin is the most commonly used drug in earlier stage of dic. it is recommended to periodic test blood routine and coagulation, and dynamically observe coagulation status during medicine therapy. others such as dextran, anti-platelet aggregation ticlopidine, salvia injection, urokinase may be effective, but it should be support by more evidence-based medicine [156] . 2. plasma and blood products: during the process of dic formation, the patients should be transfused with fresh plasma, each 10-20 ml/kg. when they developed the stage of secondary fibrinolysis, prothrombin complex containing coagulation factors, cryoprecipitate and platelets can be supplemented due to a large consumption of coagulation factors [170] . 3. others: such as hemodialysis, anti-fibrinolytic 6-aminocaproic acid and tranexamic acid should be supported by more evidence-based medicine. hepatorenal syndrome (hrs) is a common and serious complication occurring in patients with decompensated cirrhosis and liver failure, who have overt circulatory dysfunction. the 1-year incidence of hrs in patients with ascites is about 20% [171] . hrs may predict a poor prognosis in spite of it being functional reversible [172, 173] . the clinical characteristics of hrs were first described in 1963 [174] . in 1979, hrs was defined by a group of international investigators as a progressive renal dysfunction that occurred in severe liver diseases, with features of prerenal failure (low urine sodium concentration and hyperosmolar urine) but without any improvement following volume expansion [175] . the international ascites club (iac) developed hrs definition in 1996, as a syndrome that occurs in patients with cirrhosis, portal hypertension and advanced liver failure, characterized by impaired renal function with marked abnormalities in the arterial circulation and activity of endogenous vasoactive systems [176] . hrs is a potentially reversible syndrome that occurs mainly in patients with liver cirrhosis, ascites and all kinds of liver failure [172] . it's clinical features include impaired renal function, marked changes in cardiovascular function and over activity of the renin-angiotensin systems and the sympathetic nervous. progressive hrs with severe renal vasoconstriction is able to cause a decrease of gfr [177] . clinically, hrs can be divided into two types (1 and 2). type-1, so-called acute hrs is a rapid progressive form of renal failure defined by doubling of the initial serum creatinine concentrations to a level higher than 220 mmol/l (2.5 mg/dl) or a 50% reduction of the first 24 h creatinine clearance to <20 ml/min within 2 weeks [178] . it appears mainly in patients with acute liver failure, but often develops after a precipitating event, such as bacterial infections (especially spontaneous bacterial peritonitis, sbp). the prognosis of type 1 is poor with the median survival about 1 month [177] [178] [179] . type-2, so-called chronic hrs is a steady or slowly progressive form of renal failure defined by serum creatinine from 133 to 226 mmol/l or from 1.5 to 2.5 mg/ dl [175] . type-2 hrs is mostly related to refractory ascites. survival of patients with type-2 hrs is generally around 6-7 months, which is better than that of patients with type-1 hrs but shorter than that of non-azotaemic cirrhotic patients with ascites. patients with type-2 hrs tend to develop type-1 hrs while infections or other trigger events occurred [171, [177] [178] [179] ]. portal hypertension is the essential factor of haemodynamic changes, which resulted from the development of cirrhosis associated with distortion, compression and even obliteration of the hepatic sinus and vessels [175] . in patients with portal hypertension, bacterial translocation is increased and intrahepatic hypercontractile stellate cells activated [180, 181] . this overall increased resistance to portal hypertensional causes increased local production of various vasodilators such as nitric oxide, leading to splanchnic vasodilation [182, 183] . there are several other factors contributing to the splanchnic vasodilation, including hyporesponsiveness of the splanchnic vessels and mesenteric vascular hyperplasia [181] . in addition, portal hypertension per se can cause renal vasoconstriction by activating sympathetic nervous. for example, when tips is used to reduce portal hypertension and improve renal blood flow, a corresponding reduction in sympathetic nervous activity has been observed [184, 185] . as a result of splanchnic vasodilation, blood is accumulated in the splanchnic vascular bed just like a splanchnic steal syndrome [186] . the combined effect leads to reduction in the effective arterial blood volume (relative hypovolemia) causing a relative inadequacy in the systemic circulation, which triggers a hyperdynamic circulation in these patients [187, 188] . vasodilatation induces activation of neurohumoral systems including the reninangiotensin-aldosterone system (rass); sympathetic nervous system (sns); and non-osmotic release of antidiuretic hormone (adh) [189] . relative hypovolemia initially causes sodium and water retention, increases intravascular volume, and simultaneously increases cardiac output. as cirrhosis progresses, vasodilatation aggravates, which activated vasoconstrictive systems, causing renal vasoconstriction and decreased renal blood flow [181, 190] . local release of potent vasodilators such as nitric oxide (no) leads to splanchnic visceral vasodilation, as well as enables the splanchnic circulation against a variety of vasopressor agents, including norepinephrine, vasopressin, angiotensin ii and endothelin [191] . the resistance of the splanchnic circulation to these vasopressor agents makes the control of arterial pressure during cirrhosis dependent on the extra-splanchnic effects produced by the endogenous vasoconstrictor systems. the role of vasoconstrictors in maintaining haemodynamic stability becomes pivotal as arterial vasodilatation increases during cirrhosis, which makes clear why cirrhotic patients with hrs are prone to develop renal, hepatic and cerebral vasoconstriction [189] . the reduction of effective arterial blood volume leads to the compensatory activation of various vasoconstrictor systems. normally, the kidneys increase the production of renal vasodilators including prostaglandins and kallikrein to maintain blood flow. however, renal vasodilator production is generally reduced in patients with cirrhosis, thus contributing to renal vasoconstriction. this type of renal hypoperfusion further increases the production of various intrarenal vasoconstrictors such as angiotensin ii and endothelin, causing further decline of renal haemodynamics and renal function, occasionally accompanied by glomerular ischaemia and mesangial constriction [178] . when blood pressure fluctuates, renal auto-regulation regulatory mechanisms initiate to make sure that the kidneys receive a relatively constant blood supply. when the critical threshold is below 65 mmhg, renal blood flow is proportional to renal perfusion pressure which, in turn, is dependent on mean arterial pressure. in cirrhosis, with the development of liver disease in patients of cirrhosis, the renal auto-regulation curve gradually shifts to the right -which means as liver disease advances, the renal blood flow gradually decreases for each given renal perfusion pressure [178] . furthermore, lumbar sympathetic blockade increases renal blood flow in patients with hrs, suggesting that the renal sympathetic activity is involved in this outgoing hepatorenal arm. insufficient cardiac output is considered one of the leading causes for renal hypoperfusion in patients with hrs in recent years. despite control of infection, the cardiac output of cirrhotic patients with sbp who developed progressive renal failure was lower than that in similar sbp patients without renal failure. similarly, when patients with non-azotaemic cirrhotic patients who developed hrs are compared with similar patients who did not, it is observed that low cardiac output and high plasma renin activity (pra) were independent predictors of hrs. in addition, in patients developing hrs, the evolvement of circulatory dysfunction leading to arterial hypotension and renal failure occurs in the setting of a continued decline in cardiac output and increase in pra. therefore, effective hypovolaemia occurs when cardiac output decreases, resulting in renal hypoperfusion and hrs [192] . to summarize, the principal mechanisms leading to renal vasoconstriction include systemic circulation changes, accompanying portal hypertension which are characterized by decreased peripheral vascular resistance with subsequent vasodilatation, hyperkinetic circulation and the activation of compensatory mechanisms, i.e., the sns, raas, and adh. with the progression of cirrhosis, the combined effective of all the above factors will result in the gradual deterioration in renal function. any event that leads to a sudden deterioration in hemodynamics can cause a rapid renal dysfunction, precipitating type 1 hrs [193] . the diagnostic criteria for hrs have been first defined by iac in 1996 [176] . the main findings include reduced glomerular filtration (creatinine clearance) less than 40 ml/min or serum creatinine increased more than 135 μmol/l after excluding the other causes of renal dysfunction. however, estimation of renal function by using creatinine clearance is not reliable, because these patients have lower levels of serum creatinine and higher renal tubular creatinine secretion compared with filtered creatinine. furthermore, it is often incomplete for the collection of a 24 h urine. iac developed the new definition and diagnostic criteria for hrs in 2005, which (1) excludes creatinine clearance because of its inaccuracy of renal function estimation and the complicity to perform; (2) includes renal failure at the time of combined bacterial infection (but absence of septic shock), indicating that hrs can be diagnosed before antibiotic treatment; (3) determines by using albumin for plasma volume expansion better than saline. (4) excludes minor diagnostic criteria (urinary indices) because of its poor sensitivity and specificity for the diagnosis [178, 189, 194] . the diagnostic criteria of hrs for patients with liver cirrhosis are as follows: 1. cirrhosis with ascites; 2. serum creatinine >133 mmol/l (1.5 mg/dl); 3. no improvement in serum creatinine (decrease to a level of ≤133 mmol/l or 1.5 mg/dl) after at least 2 days of diuretic withdrawal and volume expansion with albumin. the recommended dose of albumin is 1 g/kg body weight/day up to a maximum of 100 g/day; 4. absence of shock; 5. no current or recent treatment with nephrotoxic drugs; 6. absence of parenchymal kidney disease as indicated by proteinuria >500 mg/ day, microhematuria (>50 red blood cells/high power field) and/or abnormal renal ultrasonography [177, 194] . there are some other causes of renal failure in patients with cirrhosis that were not included, such as membranoproliferative glomerulonephritis and/or iga nephropathy in patients with chronic liver diseases. these chronic forms of kidney disease can cause acute rises in serum creatinine. determining whether it is a potential kidney disease or hrs that causes a sudden increase in serum creatinine in patients with cirrhosis and chronic kidney disease could be difficult [195] . naturally, liver transplantation is the only rational solution in cases of advanced liver disease while it is also the treatment of choice for both type-1 and type-2 hrs. as calcineurin inhibitors (ciclosporin and tacrolimus) may induced gfr impairment, it is recommended to delay their administration until a partial recovery of renal function is recorded, usually 48-72 h after transplantation [177] . clinically, the haemodynamic associated with hrs as well as neurohormonal abnormalities fade away within one month of transplantation, and the patients recover their ability to excrete sodium and free water. compared with patients without hrs, patients with hrs tend to have more complications, take more days in intensive care units and have higher in-hospital mortality rates after liver transplantation. nevertheless, their 3-year survival rate is acceptable (60% vs. 70-80% in liver transplant patients without hrs [196] . the primary limitation of liver transplantation is that most patients with type-1 hrs die before transplantation due to the shortage of donor liver and their extremely short survival time. reference to the model of end-stage liver disease (meld, including scr, tbil, inr) for organ prioritisation has partially addressed this problem, since patients with hrs have a high priority on the waiting list. in addition, the use of vasoconstrictors and albumin in the treatment of type-1 hrs can improve survival rate of these patients, and increase the likelihood of their transplantation [177] . in patients with advanced liver disease and the renal insufficiency, simultaneous liver and kidney transplant (slkt) is taking into consideration. however the reversibility of renal function in some patients when they receive slkt should be taken into account. therefore, to ensure allocation of transplants only to those truly in need, the transplant community proposed an evaluation algorithm in 2006, whose purpose is to determine the presence of kidney disease with structural damage (preferably on biopsy) before giving slkt. in the case of chronic kidney disease, a decreased creatinine clearance at 30 ml/minute or less is considered an indication of slkt. slkt should not be performed for the patients with simple hrs, but for the patients with hrs who become dialysis dependent and without any recovery after 6 to 8 weeks of dialysis [179, 189] . vasoconstrictors combined with albumin are the first line of therapy for type-1 hrs patients. it was recognized long time ago that the effective plasma volume was reduced when patients of advanced liver diseases complicated with hrs, and this led to many attempts to improve the patients' renal function by expanding their plasma volume, including a large dose of albumin or saline perfusion. with the advent of safer compounds including terlipressin, a vasopressin analogue with longer activity, and the α2-agonist midodrine combined with octreotide the analogue, vasoconstrictors is widely used in the patients with hrs. these vasoconstrictors are able to ameliorate vasodilatation while increasing effective arterial blood volume, improving renal vasoconstriction and improving renal flow. in order to further increase effective blood volume, vasoconstrictors have been used in conjunction with intravenous albumin. the clinical results from 12 uncontrolled studies including 258 patients with hrs (type-1, 240) showed that a total of 60% were observed complete response (mostly defined as a decrease in scr to 1.5 mg/dl). interestingly, once the treatment is stopped, hrs relapses only in a few "responders" [177, 189] . there were several randomized controlled trials (rcts) published, suggesting that terlipressin was associated with an increase in gfr compared with albumin alone or with a placebo. the rate of hrs reversal in the terlipressin group was higher than that in the control group (46% vs. 11%). as survival rate was not improved in the two largest rcts, liver transplantation is still the preferred treatment for hrs, but terlipressin seems to serve as a "bridging" treatment. two recent small, open-label rcts suggested that the incidence of hrs reversal and the rate of side effects showed no significant difference between the two groups of norepinephrine and terlipressin [189, 197, 198] . the initial dose of terlipressin recommended in many studies ranged from 0.5 to 1 mg per 4-6 h [199, 200] . if the creatinine level did not decrease by 25% on the third day, the dose could be increased to 2 mg every 4 h or 12 mg/days by continuous intravenous infusion, respectively. in some studies, the daily dose of albumin was generally 20-40 g by a load of 1 g/kg body weight. some mentioned central venous pressure to establish albumin doses and to prevent body fluid from overloading. this treatment was maintained until hrs is reversed, but did not exceed 2 weeks [177] . about 20% of patients relapsed after the treatment withdrawal. however, these patients should be given repeated treatment with terlipressin, which is often effective [177] . several studies have evaluated the role of transjugular intrahepatic portosystemic stent-shunt (tips) in hrs. these studies show that tips help decreasing in scr in most patients, even in a minority of organic renal failure, but it is slower compared to those obtained using terlipressin combined with albumin [201] . recrudescence of hrs is rare provided the shunt remains patent, while hepatic encephalopathy often comes. it is worth noting that the vast majority of patients included in these studies suffered from alcoholic cirrhosis, many of whom have active alcoholism, and therefore the improvement observed may be caused by the improvement in an acute-on-chronic process. in addition, since all these studies excluded patients with a child-pugh score ≥ 12, resulting in a lack of data, the efficacy of tips should be further explored in rcts [189] . extracorporeal albumin dialysis molecular adsorbent recirculation system (mars) is designed for making clearance of water-soluble cytokines (i.e., il-6) and albumin-bound toxins (i.e., bile acids) which is implicated in the pathogenesis of hrs. two studies showed that mars was ineffective in improving survival rate and emic haemodynamics in type-1 hrs [193, 202] . another clinical observation including 32 patients with type-1 hrs reported a rate of complete renal response of 28% [201] . extracorporeal albumin dialysis (ecad) reduces serum creatinine levels, but it is not clear whether this effect is due to a real improvement of renal function or merely a filtration process. several studies demonstrated that patients' systemic haemodynamics improved after ecad, manifested as an increase in systemic vascular resistances and arterial pressure, as well as a decrease in cardiac output, pra and levels of norepinephrine. however, there were too few studies on the effect of ecad on survival in type-1 hrs patients to draw any definitive conclusions [203, 204] . in addition, ecad is very expensive and therefore not suitable for wide and rapid clinical application [177] . the treatment of type-2 hrs should take into account the survival rate as well as controlling the ascites. both hrs-1 and hrs-2 are indications for the tips treatment. the therapeutic effect of tips is excellent for its better controlled of complications of portal hypertension compared with other treatments. tips have been reported not only to improve renal function in patients with type-1 hrs but also to treat refractory ascites in patients with type-2 hrs [205] [206] [207] . the contraindications to the creation of tips are shown in the followings [195] . • contraindications to placement of a tips: there were only a few studies evaluated the role of tips in type-2 hrs and the number of cases was quite low. in most patients, tips could decrease scr, even in some with organic renal failure [208] . hrs recurrence is rare as long as the shunt remains patent, but hepatic encephalopathy often occurs [189] . nine patients were followed-up for 1 month after the treatment of tips in a study, eight cases were found with decreased scr decreased and notably controlled ascites. four patients died, two of them died within 1 month, the other two died at 12 months and 14 months respectively. the remaining five patients survived for a long time. although tips can be used in improving refractory ascites which often contributes to type-2 hrs, data on the effect of tips on survival are still insufficient. therefore, the efficacy of tips should be further explored in randomized controlled trials (rcts) [189] . the information about combining albumin and vasoconstrictive agents treated in type-2 hrs is limited. only a few patients with type-2 hrs have been specifically treated with terlipressin and albumin. in one clinical study, 39 patients with hrs-2 were assigned to receive this treatment and 21 of them achieved improvement of renal function. however after the treatment withdrawal, 11 hrs patients showed relapsed during the follow-up. the most common side effects during terlipressin therapy are cardiovascular and ischemic and reported as an incidence of nearly 12%. the high recurrence rate of hrs after terlipressin and albumin treatment discontinuation suggests that they are less effective in treating type-2 hrs compared to type-1hrs [209] . prevention of hrs is important because it develops at a constant frequency in cases of spontaneous peritonitis (sbp) and advanced liver disease [210, 211] . it becomes possible to prevent hrs if sbp is diagnosed and treated promptly [212] . according to current data, using albumin in combination with antibiotics for the treatment of patients with sbp seems to be warranted but only for those with jaundice or renal dysfunction. the prophylactic use of antibiotics in cirrhosis with gastrointestinal bleeding also seems to be necessary, because the use of antibiotics contributes to reducing incidence of infection and rebleeding whereas improving survival rate. furthermore, the incidence of hrs in sbp patients decreases by albumin administration, and prevention of hrs can also be related to increased survival. the recommended dose of albumin is 1.5 g/kg body weight on the first day then 1 g/kg body weight on the third day, a maximum of 150 g and 100 g, respectively. albumin administration is strongly recommended in sbp patients with serum bilirubin levels higher than 68.4 mmol/l (4 mg/dl) or serum creatinine more than 88.4 mmol/l (1 mg/dl). a placebo-controlled rct that enrolled the patients with low (<1.5 g/l) ascites protein who also had advanced liver diseases or "renal dysfunction"(defined as scr ≥ 1.2 mg/dl or blood urea nitrogen≥25 mg/dl, or serum sodium level ≤ 130 meq/l) suggested that oral norfloxacin contributed to a reduced hrs incidence within 1 year (28% vs. 41%) and an improvement in survival at the end of 3 months [213] . norfloxacin may ameliorate or prevent vasodilatation by reducing bacterial translocation and overt infections, as well as suppressing plasma renin activity, thereby prevent these patients from developing hrs. the concept that the severity of the clinical course of patients with cirrhosis complicated with serious bacterial infection is related to the degree of an impairment of circulatory function, which has led to new and effective approaches in the prevention and treatment of these complications. in patients with severe hepatitis, multiple causes may lead to disorders of internal environment, mostly manifesting fluid and electrolyte imbalance as well as acidbase imbalance, usually resulting in deterioration, greater complexity and even death. accurately recognizing the occurrence of severe hepatitis with complications such as fluid and electrolyte imbalance and/or acid-base imbalance, and therefore giving appropriate treatment to maintain balance of internal environment, is of great importance for improving prognosis of the patients [214] . water is the major component of human body. electrolytes are substances that dissociate in solution to form charged particles, orions. body fluid comprise mainly of water and electrolytes and electrolytes comprise mainly of na + , k + , ca 2+ , mg 2+ , cl − , hco 3 − , hpo 4 2− and so 4 2− . the primary function of electrolyte include: (1) to maintain osmotic pressure and acid-base balance of body fluids; (2) maintain nerve, muscle, cardiac cells resting potential, involved in the formation of action potentials; (3) involved in metabolism and physiological activities [215] . body fluid include intracellular fluid and extracellular fluid, the latter can be divided into plasma and interstitial fluid. intracellular and extracellular fluid differ in ion components. na + is major cation in extracellular fluid and its main anions are cl − and hco 3 − . k + is major cation in intracellular fluid and its major anion is hpo 4 2− . the total number of ions in body fluids is called osmolality, its unit is mosm/l. if the osmolality on both sides of a semipermeable membrane is not equal, water moves toward the side with the higher osmolality. this phenomenon is called osmosis [216] . osmosis of water can be opposed by applying a pressure across the semipermeable membrane in the direction opposite to that of the osmosis. the amount of pressure required to oppose the osmosis is defined to be osmotic pressure. in spite of various solute concentrations are different in extracellular and intracellular fluid, the osmotic pressure remain equal. normal plasma osmotic pressure is 280-310 mosm/l. steady osmotic pressure is the basic guarantee to maintain the fluid balance across the cell membrane. multiple mechanisms in nervous and hormonal system are involved in the regulation of body fluid and electrolyte balance [217] . (1) there exists sensation of thirst in central nervous system, which plays an important role in regulating body water. (2) there is a powerful feedback system for regulating plasma osmolarity and sodium concentration that operates by altering renal excretion of water independently of the rate of solute excretion. a primary effector of this feedback is called antidiuretic hormone (adh) which plays an important role in regulation of renal concentration and dilution to maintain the body fluid homeostasis. (3) reninangiotensin-aldosterone system (raas) is an important regulator of sodium reabsorption and potassium secretion by the renal tubules. human body fluid environment must be suitable ph value for maintaining normal metabolism and physiological function, under normal conditions, human body take in acidic and basic food and drinking water, produce acids and bases during metabolism and eliminate acidic or basic substances by the kidneys and lungs. in the plasma, the normal ph value ranges from 7.35 to 7.45 with an average value at 7.40. the regulation of the acid-base balance is accomplished by the buffer system of the body fluid, the respiration of the lungs and the excretion of the kidney [218] . (1) blood buffer system is composed by a weak acid and its corresponding buffer base, includes bicarbonate buffer system, phosphate buffer system, plasma protein buffer system, hemoglobin and oxygen synthetic hemoglobin buffer system. (2) the role of the lung in acid-base balance is to adjust the concentration of plasma carbonic acid by changing the amount of co 2 , so that the ratio of hco 3 − and h 2 co 3 in the plasma is close to normal, so that the ph is relatively constant. (3) the major role of the kidneys in maintaining acid-base balance is to conserve circulating stores of bicarbonate and to excrete h + . the kidneys maintain ph by increasing urinary excretion of h′ and conserving plasma hco 3 − when the blood is too acidic, or increasing urinary excretion of hco 3 − , and decreasing urinary excretion of h + when the blood is too alkaline. patients with severe hepatitis are prone to develop water retention, with the main manifestation of seroperitoneum (ascites) as well as body weight gain. with the acatharsia of water becoming more serious, oliguria and edema of lower extremities occur. sbp (spontaneous bacterial peritonitis) can also occur, which is manifested with symptoms such as fever and abdominalgia. several factors contribute to ascites include an increase in capillary pressure due to portal hypertension, obstruction of venous and lymph flow through the liver, decrease in colloidal osmotic pressure due to impaired synthesis of albumin by the liver, salt and water retention by the kidney [219] . some theories have been used to explain the increased salt and water retention by the kidney. because of vasodilation or an actual loss of fluid into the peritoneal cavity, the effective blood volume maybe reduced, which may in turn decrease the renal blood flow leading to a lower glomerular filtration rate (gfr) and an activated rennin-angiotensin-aldosterone system (raas). the diagnosis of water retention depends on typical clinical symptoms such as ascites, pleural effusion, and edema of lower extremities, when the body begins to excess water, blood pressure is increased, which leads to many complications such as congestive heart failure and pulmonary edema. hyponatremia is a common complication in severe hepatitis patients, and always incorporate with the retention of water and sodium, but the total sodium can be decreased, normal or even increased, that is dilutional hyponatremia. in laboratory test, serum sodium is below 135.0 mmol/l. the mechanism of hyponatremia probably depends on the following factors: (1) the decreased function of adh inactivation in liver brings adh increase, enhancing the reabsorption of water in renal tubule, which causes the formation of water retention. this is the main cause of dilutional hyponatremia. (2) water retention brings about volume extension, causing the aldosterone secretion decrease, which leads to the sodium egestion increase in urine. (3) some severe hepatitis patients frequently vomit, and can not eat, bringing about a major loss of body fluid and electrolyte. (4) severe hepatitis patients, serum albumin reduces, combining with the factors such as poor appetite, anorexia, fasting or limit sodium, bring about a state of low permeability in the cell, which causes the extracellular na + moving into the cell. (5) iatrogenic factors, exhaust potassium diuretic such as hydrochlorothiazide and furosemide and spironolactone have a strong role in the excretion of sodium, so a large number diuretic is liable to hyponatremia in ascites patients, and in the treatment of cerebral edema, a large infusion of mannitol may cause hyponatremia either [220] . hyponatremia due to the osmotic pressure of extracellular fluid decreased, water moves to the cells, causing cell edema, especially brain edema. so the symptoms of nerve system are the main manifestation in hyponatremia patients [221] . generally dilutional hyponatremia in severe hepatitis patients develops slowly and progressively, and the symptoms are often covered by primary disease symptoms, like weak, feeble, nausea, vomiting, lethargy, significant body weight increase, pale and moist skin and sometimes saliva, tears increase. improper treatment will bring about a sharp serum sodium decrease in the short term, such as serum sodium rapidly decreasing to below 125 mmol/l, acute hyponatremia syndrome comes, the manifestation include convulsions, coma, hypotension, pulse narrowing, tachycardia, oliguria even respiratory arrest and death. if cerebral hernia happens, corresponding nerve location signs will follow. hypokalemia refers to the condition in which the concentration of potassium (k + ) in serum is less than 3.5 mmol/l. it could occur during the whole period in severe hepatitis patients and is more common in early metaphase of disease. hypokalemia can be the result from one or more following medical conditions: (1) insufficient intake of potassium due to the poor appetite or anorexia in the patients with severe hepatitis. (2) frequent vomiting leads to excessive loss of stomach acid, which causes alkalosis and extracellular potassium is transferred into cells. (3) the reduce of effective circulating blood volume can cause to high aldosterone levels and excessive urinary losses of potassium. (4) the decreased function of aldosterone inactivation in liver brings aldosterone increase, enhancing urinary losses of potassium. (5) some medications such as diuretics can also cause urinary losses of potassium. the clinical syndromes of hypokalemia are related to the degree of the shortage of intra/extracellular potassium and disorders of other electrolytes and acid-base, but more depends on how soon it occurs. in the early time it shows muscle weakness, first in limbs, then develops to the torso and respiratory muscle. deficiency of potassium also can lead to weak peristalsis, poor appetite, sick and constipation in mild hypokalemia but abdominal distention and paralytic ileus in severe situation. in cardiac syndromes, it mainly presents atrioventricular block and arrhythmia which including premature ventricular contraction or atrial premature beats, sinus bradycardia, paroxysmal auricular tachycardia or junctional tachycardia, even ventricular fibrillation. in hypokalemia state an increasing shift of potassium from extracellular fluids into cells and an obligate loss of potassium from kidney can cause metabolic alkalosis and abnormal acidic urine. long-term hypokalemia also can lead to hypokalemic nephropathy with proteinuria and cylindruria syndrome. the changes of ecg [222] : in the early stage, flattened t wave and an obvious u wave, st-segment depression can be found, qu interval is widen. in severe situation, a wide pr interval, low voltage, wide qrs interval and ventricular arrhythmia can occur. hyperkalemia refers to the condition in which the concentration of potassium (k + ) in serum is higher than 5.5 mmol/l. it is more common in the middle and late period of severe hepatitis. the mechanism of hyperkalemia: (1) the most usual way lead to hyperkalemia is oliguria or uroschesis which are caused by renal dysfunction among the patients have severe hepatitis with hepatorenal syndrome. (2) metabolic acidosis and na + -k + -atp enzyme inactive lead to a shift of potassium out of cells also contribute to develop hyperkalemia. (3) long-term and high dose potassium-sparing diuretics applied during the treatment lead to hyperkalemia is not rare, and easy to get sudden death in patients. hyperkalemia mainly influences myocardium and skeleton muscle. the most dangerous situation is fatal arrhythmia. when the concentration of k + is higher than 5.5-6.5 mmol/l, there are peaked t waves. when it is over 7-8 mmol/l, pr interval is widen and p wave is flattened even vanish. when it is up to 9-10 mmol/l, t waves and qrs complex can evolve to sinusoidal shape and cardiac arrest. hyperkalemia is a common critical and severe symptom in clinic. when it happens, all of the potassium-sparing diuretics and potassium uptake should be stopped. at meantime, the treatment against the toxicities to myocardium and skeleton muscle should be taken to accelerate a shift into cells and potassium excreting. there also can happen hypocalcemia, which shows neuromuscular excitability, cardiac electrical instability and instable emotion. the concentration of calcium in serum under 2 mmol/l is significant in diagnosis. hypomagnesemia can be found too. it mainly presents similar symptoms as hypocalcemia such as weakness, muscle cramps, increased irritability, tetany and chvostek positive. hypomagnesemia can cause cardiac arrhythmia, when it occurs, the concentration of magnesium is less than 0.75 mmol/l, and it should be urgently treated. severe hepatitis patients prone to acid-base imbalance [223] , mainly to alkalemia. the main types of acid-base imbalance include respiratory alkalosis, metabolic alkalosis, respiratory alkalosis plus metabolic alkalosis, secondly include respiratory alkalosis plus metabolic acidosis, triple acid-base disorders (tabd), metabolic alkalosis plus metabolic acidosis [224] . history and clinical manifestations provides important clues for the judgment of acid-base imbalance. the result of blood gas monitoring is the decisive basis for judging the type of acid-base imbalance. serum electrolyte examination is an important reference. anion gap (ag) has important diagnostic value in determining the type of acidbase imbalance [225] . ag = na + -(cl − + hco 3 − ) is a simple formula for the value between the number of cations and anions in serum. its normal value was 12 ± 4 mmol/l. ag can not only help diagnose "potential" metabolic acidosis and to distinguish different types of metabolic acidosis, can also help determine special types of mixed acidosis, and has its unique role in the judgment of tabd. sometimes the indicators of blood gas analysis are normal, the calculation of ag value become the only evidence of diagnosis of metabolic acidosis. in addition, ag value can be used as reference for correction of acid-base imbalance. in severe hepatitis, the change of ag values can be used as an indicator to estimate the complications and prognosis. clinical observations indicate: ag value significantly increased often suggestive of severe infection, kidney dysfunction or severe bleeding, and the prognosis is poor. respiratory alkalosis refers to arterial paco 2 decrease and ph > 7.45 as well as compensatory decrease of blood hco 3 − . respiratory alkalosis occurred in early stage of severe hepatitis. in severe hepatitis, respiratory alkalosis related to hyperventilation: accumulated ammonia and other vasoactive peptides excited respiratory center, ascites and pleural fluid increase respiratory rate, hypoxemia excited respiratory center. compensatory mechanisms: co 2 reduction, breathing shallow and slow, so that co 2 retention, h 2 co 3 compensatory rise; when last longer, reduce renal row h + , hco 3 − excretion increased, hco 3 − /h 2 co 3 equilibrium at a low level. most patients have the performance of shortness of breath and heart rate increase. can have vertigo, hand, foot and mouth numbness, muscle tremor, hand and foot convulsions. convulsions associated with low calcium. dysfunction of nervous system is related to the damage of brain function and cerebral blood flow decrease. respiratory alkalosis diagnosis relies on the following: (1) ph is normal when fully compensated, increased underdecompensation. (2) paco 2 lower (typically <35 mmhg or 4.67kpa). (3) hco 3 − compensatory decline. (4) ag value may have a slight increase. (5) blood cl − may increase. metabolic alkalosis refers to the type of acid-base imbalance characterized by an increase hco 3 − in extracellular liquid. the inappropriate application of basic drugs, potassium-sparing diuretics, dehydrating agents, hormones can often induce or aggravate metabolic alkalosis. severe gastrointestinal symptoms, anorexia, vomiting or diarrhea are also the reasons for the occurrence of metabolic alkalosis. compensatory mechanisms: when alkaline substances increased in body, buffer system instantly transfer strong base into weak base, increase hco 3 − consumption, h 2 co 3 increased. inhibit respiratory center, decrease pulmonary ventilation, co 2 retention, hco 3 − compensatory increase. renal carbonic anhydrase activity decreased and h + formation and excretion decreased, nahco 3 reabsorption is also reduced, so hco 3 − /h 2 co 3 compensatory restore to 20:1, ph value is normal. patients with mild metabolic alkalosis usually have no obvious symptoms. many disorders can occur in severe metabolic alkalosis. (1) functional changes in the central nervous system, the patient may have irritability, confusion, delirium, consciousness disorders. (2) slow and shallow breathing, hypoxemia. brain tissue is particularly sensitive to hypoxia, thus neurological symptoms first appeared. (3) hypocalcemia and neuromuscular stress increased, the performance of tendon hyperreflexia, face and muscle twitching, and limbs twitching. (4) hypokalemia can cause neuromuscular symptoms and arrhythmias. according to ph value, paco 2, hco 3 − , level of k + and cl − , effective circulating blood volume and performance of primary disease, diagnosis of metabolic alkalosis is no difficult to make. metabolic alkalosis should be divided into two categories based on the urinary level of cl − . (1) chloride positive metabolic alkalosis: supplement sodium chloride can correct the alkalosis. it indicates that the body has cl − deficiency, urinary cl − < 10 mmol/l. (2) chlorine negative metabolic alkalosis: alkalosis can not be corrected by supplement sodium chloride, urinary cl − >20 mmol/l. respiratory alkalosis plus metabolic alkalosis tend to occur on the early stage of severe hepatitis. in most cases, there is no obvious complication, more often metabolic alkalosis happens on the basis of respiratory alkalosis, or the other way around. due to respiratory and metabolic factors are inclined to alkaline change, name as reduce paco 2 and elevated plasma hco 3 − , there is no mutual compensation between them, so it is easy to present as severe decompensation and poor prognosis. main point of diagnosis: (1) ph value of blood increase significantly. (2) paco 2 decrease. (3) hco 3 − increases, the value should be greater than 0.5×(40-paco 2 ) + 2.5. (4) hypokalemia and hypochloremia are common phenomenon. respiratory alkalosis plus metabolic acidosis is relatively rare. metabolic acidosis can be divided into 3 types: (1) value of ag is normal (high chlorine acidosis), commonly seen at long-term diarrhea, combined with renal tubular acidosis and a large amount of physiological saline input in patients or water intoxication. (2) high ag value type (normal blood chlorine acidosis), regularly present in combination of hepatorenal syndrome, lactic acidosis and patients with ketoacidosis. (3) a hybrid type (high ag merged high blood chlorine), mainly in patients with severe diarrhea following lactic acid or ketoacidosis. when respiratory alkalosis plus metabolic acidosis happens, paco 2 and plasma concentration of hco 3 − are higher than scope of compensation to each other. its characteristics as follows: (1) the range of blood ph change is not large, normal, slightly higher or slightly lower. (2) paco 2 reduce to less than 1.5 × hco 3 − + 6 or hco 3 − < 24-(40-paco 2 ) × 0.5-2.5. (3) ag values can be normal or elevated, the latter is more common. if the elevated blood cl − value is equal to the hco 3 − decrease, ag value normal metabolic acidosis type can be diagnosed; the cases that the rising value of ag is equal to the decline of hco 3 − values can be diagnosis as ag increased metabolic acidosis type. on the third occasion, the increase value of ag is equal to the sum of hco 3 − and cl − drop, the diagnosis is mixed metabolic acidosis. for this type of offset mixed acid-base balance disorders, treatment should be moderate, the measure of the metabolic factors correcting should be precede to respiratory factors, avoid paco 2 quickly returning to normal in the process of treatment, which would lead to blood ph drop rapidly and acidosis more worse. to patients with severe hepatitis, the harm of alkalosis is greater than acidosis, thus the blood ph should be kept slight acidic in a normal state. generally, the target of alkali supplement can be arterial blood ph value recovered to 7.20. respiratory alkalosis tabd refers to respiratory alkalosis, metabolic acidosis and metabolic alkalosis three primary imbalances coexist in the same patient, which is one sort of serious acid-base imbalance, mostly develops in the late stages of severe hepatitis, fatality rate is high. respiratory alkalosis tabd characteristics as follows: (1) blood ph value depends on the relative severity of these three primary imbalances, which can be normal, or slightly high generally. (2) reduce paco 2 , its value is less than 1.5xhco 3 − + 6. (3) hco 3 − can raise, normal or lower. (4) value of ag rise significant, and extent of ag raise is greater than the hco 3 − lower. (5) cl − often lower than normal. the occurrence of metabolic alkalosis plus metabolic acidosis in patients with severe hepatitis is not uncommon. usually it is accompanied with existing lactic acidosis or ketoacidosis, and the patient may manifest frequent vomiting. since the causes for raising and lowering hco 3 − coexist, they tend to cancel one another. the ph and hco 3 − concentration can be normal, increased or decreased, depending on the relative severity of the two kinds of imbalances. severe hepatitis can also be accompanied by metabolic acidosis, metabolic acidosis plus respiratory acidosis, tabd of respiratory acidosis, and etc. pure metabolic acidosis refers to arterial blood ph < 7.35 and compensatory decline of paco 2 due to primary decrease of hco 3 − . typical manifestation is known as kussmaul breathing, characterized by deeper and faster breathing, as well as obvious contraction of respiratory muscle, and also ketone-smelled exhaled breath. the patients often flush, companied by increased heart rate and decreased blood pressure. there may be reduced or disappeared tendon reflexes, confusion or stupor. due to respiratory and metabolic factors both towards to acidic changes, there is no respiratory compensation for decrease in hco 3 − , nor renal compensation for increase in paco 2 , hence presenting severe decompensated status. the resulting distinct decrease in ph and vicious circle are the characteristics for metabolic acidosis plus respiratory acidosis. the characteristics for tabd of respiratory acidosis include significantly increased paco 2 , elevated hco 3 − , ag > 16 mmol/l, and significantly decreased cl − . the incidence of the above types of acid-base imbalance is very low in patients with severe hepatitis. once it happens, it should be actively treated with corresponding methods, so that the blood ph quickly restores to the safety range. during therapeutic process against various pure acid-base imbalance, interactions among various treatments need to be taken into consideration, in order to avoiding the possibility that the treatment for one type of acid-base imbalance causes or aggravates another type. in summary, water-electrolyte imbalance and acid-base imbalance have a relatively high morbidity in patients at various stages of severe hepatitis. this often results in deterioration and complication of the disease, evermore, the death of the patient. therefore, the functions of heart, lung, kidney, blood circulation as well as changes of body weight in the patient need to be intently monitored. regular detection of k + , na + , cl − , carbon dioxide combining power (co 2 cp), blood urea nitrogen, creatinine, ph, data about arterial blood gas analysis, and also detailed records of patient's input and output are demanded. during the process of diagnosis and treatment, careful analysis of the history, clinical manifestations and laboratory examination are necessary to achieve correct diagnosis, early prevention and prompt treatment. in normal condition, proper amount fluids can make a lubrication action on organs in peritoneal cavity. but in those patients who have severe hepatitis, especially with cirrhotic portal hypertension, too many fluids over 200 ml can lead to ascites. the ascites can be categorized into uncomplicated ascites and refractory ascites. there is no infection in uncomplicated ascites and won't lead to hepatorenal syndrome, but refractory ascites is in the contrast. the refractory ascites includes diureticresistant and diuretic-intractable ascites. the diuretic-resistant ascites shows no response to diuretic and diuretic-intractable ascites limits the application of diuretic due to the complications induced by diuretic. 100 mg antisterone per day as an initial dose can be given to those patients with moderate ascites, if it goes to no satisfied effect, 100 mg can be added after every 7 days till the maximum dose to 400 mg/d. if the patients show a hyperkalemia or aldosterone antagonist-resistant, nicorol can be combined and with an increasing dose from 40 to 160 mg/d gradually. the patients without edema losing weight should be less than 0.5 kg and those with edema should be less than 1 kg per day to avoid electrolyte disturbance or hepatorenal syndrome during the whole treatment period. diuretics should be withdrawn on the patients with severe hepatic encephalopathy, severe hyponatremia, progressive renal failure or severe muscle spasm. the patients should only take the minimum dose of diuretics to maintain the state after the syndrome controlled, or withdraw when it is necessary. due to the poor outcome and living quality, the median survival time of refractory patient is half year. so liver transplantation can be considered in the patients with refractory ascites induced by cirrhosis, which required more cautious before make a diagnosis. generally, if the patients meet the following conditions when they are receiving 400 mg/days antisterone and 160 mg/days nicorol treatment over 1 week and sodium uptake limited in 90 mmol/days, refractory ascites can be diagnosed. (1) losing weight less than 0.8 kg/days over 4 days (2) sodium uptake is more than elimination (3) grade 2-3 ascites is arisen again after 4-week long treatment (4) hepatic encephalopathy, hepatorenal syndrome and severe electrolyte disorder induced by diuretics are shown up. refractory ascites patients without complications can be treated with abdominocentesis, but it will possibly induce circulation failure, and increases the risk of hepatic coma and hemorrhage. so low rate infusion of albumin with abdominocentesis is combined to avoid circulation failure, meantime diuretics also need to give after abdominocentesis. aldosterone antagonist combined with nicorol is a suitable strategy: the dosage of antisterone is increased from 100 to 400 mg/days and nicorol from 40 to 160 mg/days gradually. the goal of this strategy is to maintain the situation without ascites under the minimum dosage. but when severe hepatic encephalopathy and electrolyte disorder show up, which means serum sodium concentration is less than 120 mmol/l, serum potassium concentration is less than 3.0 mmol/l, nicorol should be withdrawn, if serum potassium concentration is more than 6.0 mmol/l, antisterone should be withdrawn. to those patients who need abdominocentesis repeatedly, transjugular intrahepatic portosystemic shunt (tips) can be considered, but the risk of hepatic encephalopathy will be higher and the outcome is poor. so the patients with severe liver and renal failure, cardiorespiratory function failure or active infection should be in cautious. the mean survival time of refractory ascites patients complicated with hepatorenal syndrome is 3 months, preventive antibiotics combined with albumin is an option for these patients. to those patients who have already showed hepatorenal syndrome, terlipressin combined with albumin could be useful. meantime, abdominocentesis, tips or artificial liver supporting treatment can improve patients' living quality in short term, but long-term outcome won't be good, so liver transplantation should be execute as soon as possible. in general, medicine can improve the symptoms in short term but with poor outcome, liver transplantation is more meaningful. patients with hypovolemic hyponatremia can have a supplement with sodium and decrease the dosage of diuretics, patients with hypervolemic hyponatremia can restrict fluids uptake (less than 1000 ml/d) and combined with vasopressin v2 receptor blocker or antidiuretic hormone receptor blocker. currently, vaptans, tolvaptans, conivaptan and satavaptan have already applied in clinical practice. there was research showed that vaptans could improve 45-82% patients' symptoms significantly after patients took it for 1 week to 1 month, and main side reaction was thirsty. the patients complicated with hepatic encephalopathy should be used vaptans with cautious due to its high risk in dehydration and hypernatremia. meantime, vaptans is metabolized by cyp4a, so rifampin, barbital and phenytoin can decrease its effect, and ketoconazole, clarithromycin can increase its plasma concentration. tolvaptans can give some relief but increase the risk of hemorrhage. satavaptan will decrease patients' survival rate. so the proper treat period and side reactions of these drugs in long-term using need to make clear. potassium uptake should be withdrawn immediately after hyperkalemia occurs, emergency treatment for detoxicating potassium should be taken to protect cardiac. the treatment depends on the plasma concentration of potassium. the treatment for patients with fulminant hepatitis b with cirrhosis complicated with hyperkalemia is to restrict the uptake of potassium, improve the microcirculation, correct renal filtration decrease induced by hepatorenal syndrome and increase the elimination of potassium. there are also some patients have abnormal distribution of potassium due to hypoxia, acidosis, catabolism, and deficiency of energy supplies, which leads to intracellular potassium is transferred into extracellular. the treatment for these patients is to correct hypoxia and acidosis, high glucose, insulin and atp are administered to boost glycogen synthesis to transfer potassium from extracellular to intracellular. peritoneal dialysis and plasmapheresis can be given to the patients with intractable hyperkalemia. hypokalemia can present during the whole period of fulminant hepatitis b, it will occur more often on the early and middle stage. long-term inappetency and abdominal distension lead to insufficient potassium uptake. nausea, vomiting and diarrhea lead to increase of potassium losing. renal filtration rate decreasing and aldosterone increasing lead to potassium elimination increase. complicated with alkalosis and anabolism increase also can cause hypokalemia. the treatment for hypokalemia should focus on comprehensive therapy, correcting alkalosis, increasing potassium supply, improving microcirculation. disturbance of acid-base balance occurs quit often in hepatitis b patients, especially with alkalosis. during the early stage, it can present in pure respiratory alkalosis, also can complicated with metabolic alkalosis. during the middle and late stage, both of above symptoms and metabolic acidosis occurs concurrently. treating idiopathy and correcting hyperventilation is a treatment for respiratory alkalosis. arginine hydrochloride injection is used to treat metabolic alkalosis to avoid secondary metabolic alkalosis. the general regulation is prefer acid to base, till ph value of arterial blood back to 7.2. in prevention of disturbance of acid-base balance, the effective strategy is to correct hypokalemia and hypochloremia, control vomiting. meantime, controlling infection, endotoxemia and upper gastrointestinal hemorrhage are necessary. hepatic encephalopathy (he) due to metabolic disturbance is a complex neuropsychiatric syndrome caused by severe liver dysfunction or disorder and is one of the common complications and causes of death in severe liver diseases. patients with he mainly present with neuronal or mental abnormalities and disturbance of consciousness, even coma and death. the clinical manifestations and the severity of the disease vary because of its complex pathogenesis. hepatic encephalopathy is the result of acute and chronic hepatic failure caused by cirrhosis or various kinds of portosystemic shunt (pss) created. a diagnosis of he can be made after excluding encephalon diseases. the syndrome is caused by metabolic disorders and is potentially reversible. he clinical features differ due to the wide degree and range of neuropsychiatric symptoms that vary from subtle abnormalities detected only by intelligence tests or electrophysiological methods geared for detecting personality changes to abnormal behavior, intellectual impairment, and even different degrees of consciousness disorders. he was previously known as hepatic coma, but that is only one of the worst severe signs of he and does not represent all types of he. in 2003, the world congress of gastroenterology (wcog) suggested that based on the cause he can be divided into three types (a, b, and c) [226, 227] . type a: type a is acute liver failure-related he and the symptoms occur within 2 weeks. in subacute liver failure-related he, the symptoms of he occur within 2-12 weeks with or without predisposing factors. type b: patients with type b he have obvious pss and normal liver histology without associated intrinsic liver disease. these clinical manifestations are similar to those in patients with he and cirrhosis. the pss may be spontaneous or caused by surgical or interventional procedures [228] . common causes of pss include congenital vascular malformation, intrahepatic or extrahepatic portal vein obstruction (including trauma, carcinoid, and bone marrow hyperplastic disease caused by a high coagulation state due to portal vein branch embolization and thrombosis) and generation of portal hypertension by oppression of lymphoma, metastatic tumors, and bile duct carcinoma. type c: type c he is related to chronic liver diseases, with cirrhosis being the most common type, and is generally accompanied by portal hypertension and pss. type c he is mainly caused by liver function failure, rather than by pss. according to the clinical manifestations, duration and characteristics, type c can be divided into three types: episodic he, persistent he, and minimal he [226] . episodic he, related to chronic hepatic disease, is defined as a disturbance of consciousness and cognitive change in a short time and can be alleviated by spontaneous remission or drug treatment in the short term, which cannot be explained by a relevant preexisting mental disorder. episodic he can be divided into three types according to the presence of known risk factors: (1) incentive type: there is a clear history of predisposing factors; (2) spontaneous type: there is no history of predisposing factors. (3) recurrent type: he attacks more than two times within a year. persistent he related to chronic hepatic disease is defined as an occurrence of continuous neural mental abnormality, including cognitive decline, disturbance of consciousness, coma and even death. persistent he can be further divided into three types according to the severity of the disturbance in the patient's self-control and self-discipline: 1, mildest type, namely west haven level 1; 2, severe type: namely west haven level 2-4; and 3, therapeutic resistance type: medication can alleviate he quickly, but withdrawal can aggravate he rapidly. patients with minimal he, with normal clinical manifestations and routine biochemical tests, have mild cognitive and psychomotor deficits detected by neuropsychology and neural physiology tests, and these patients usually have a history of chronic hepatic disease [229] . the prevalence of minimal he in patients with cirrhosis is 30-80%. patients with minimal he with reduced physical and mental ability have gained more and more attention recently because they have a high risk of accidents when engaged in occupations involving mechanical, or driving work. the pathogenesis of he has not been fully elucidated so far, and many theories have been put forward. it is generally believed that he is caused by acute and chronic liver failure and/or pss. when toxic substances absorbed by the intestines cannot be detoxified and cleared by (or through) the liver, they directly enter into the systemic circulation and pass through the blood-brain barrier to reach the brain tissue and cause central nervous system dysfunction. a variety of the risk factors mentioned above can result in he. hyperammonemia is still recognized as one of the most important factors, especially in he related to chronic liver disease, liver cirrhosis and/or pss. according to the ammonia intoxication theory several factors including false neurotransmitters, such as γ-aminobutyric acid/benzodiazepine (gaba/bz) receptor complex, an imbalance in the ratio of branched chain amino acids to aromatic amino acids, brain cell edema, astrocyte dysfunction, mercaptan, short chain fatty acid toxicity and manganese deposition are all involved in the occurrence of he [230] . ammonia intoxication caused by an ammonia metabolism disorder is the most important factor in the pathogenesis of he [231] . ammonia comes mainly from the gut and the generation and absorption of ammonia increase in a serious liver disease when excess ammonia cannot be cleared sufficiently by ornithine cycle due to serious damage to liver parenchyma. when pss occurs, intestinal ammonia directly enters the systemic circulation without liver detoxification, resulting in increased blood ammonia. high levels of blood ammonia can enter the brain through the blood-brain barrier and generate central nervous system toxicity by interfering with cerebral energy metabolism, neurotransmitter and nerve cell membrane ion transport; increasing cerebral edema; and changing gene expression (such as stellate cell glutamate carrier, stellate cell structural protein, glial fibrillary acidic protein, peripheral benzodiazepine receptor and aquaporin-4) and inducing the mitochondrial permeability transition (mpt). the main way of removing ammonia from the brain is through urea cycle. during glutamine synthesis, glutamic acid is formed from ammonia and α-ketoglutaric acid and the glutamic acid combines with ammonia to generate glutamine. this process requires atp and consumes a large amount of α-ketoglutaric acid, which interferes with the brain energy metabolism and causes an energy supply shortage in brain cells. glutamate is an important excitatory neurotransmitter in the brain, and lack of glutamate increases inhibition in the brain. glutamine synthetase is present in astrocytes, where glutamic acid is detoxified to glutamine. glutamine is a strong intracellular osmotic agent, and increases in glutamine can lead to brain cell swelling. reports have identified a strong correlation between the content of glutamine in cerebrospinal fluid (csf) and the degree of he [232] . during he, excess ammonia under the effect of glutamine synthetase, not only reduces the formation of active glutamate but also consumes a lot of energy, leading to the accumulation of glutamine, which increases intracellular osmotic pressure and causes brain cell swelling. swollen astrocytes with impaired function further affect ammonia metabolism, reduce the ability of neurons to efficiently uptake or release extracellular ions and neurotransmitters, and stimulate glial cell synthesis of neurosteroids by upregulating their expression of the peripheral-type bz receptor (translocator protein, 18 kda). neurosteroid is an endogenous bz that can enhance gaba nerve tension and cause symptoms in patients with he [233] (fig. 2.2 shown that the metabolic rate of cerebral ammonia in he patients is increased. increased levels of blood ammonia enter the brain through the blood-brain barrier. brain dysfunction also occurs even if blood ammonia levels appear normal; this partially explains the occurrence of he in the case of normal blood ammonia and invalidates he treatment by simply reducing blood ammonia. in addition, increasing evidence suggests a synergistic effect between blood ammonia and its metabolic disorders with systemic inflammation, nerve steroids, oxidative stress, nitrification stress, manganese poisoning, and gaba/bz [233] . the main inhibitory neurotransmitter in the mammalian brain is gaba. plasma gaba is derived from the conversion of glutamic acid by glutamate decarboxylase in intestinal bacterial. notably, gaba has dual role. on one hand, during liver function failure and pss, the removal of gaba in liver is significantly decreased; on the other hand, gaba can directly enter the systemic circulation bypassing the liver, resulting in increased concentration of gaba in blood. the concentration of gaba in csf and brain tissue increases as more gaba crosses the abnormal blood-brain barrier. in addition, endogenous bz was found in the blood and csf, and the gaba receptor on the membrane surface of the brain's postsynaptic neurons increased significantly in some patients with he and in animal models. this receptor not only combines with gaba but also binds to barbiturates (barb) and bz on different parts of the receptor surface; thus, it has been named the gaba/bz complex receptor or the super receptor complex. when liver function is severely impaired, the binding affinity of this complex receptor to its three ligands is also increased. binding of gaba, barb, or bz with the complex receptor can promote entry of chloride ions from neuronal membrane ion channels into the cytoplasm of postsynaptic neurons, causing membrane hyperpolarization and nerve conduction inhibition. he symptoms were relieved in about 30% of patients treated with a gaba receptor antagonist or bz receptor antagonist, and gaba/bz and ammonia were reported to act synergistically in he. recently, some studies focused on peripheral type bz receptors, which are different from central gaba [234, 235] . some questions, including the source of endogenous bz, and the correlation between the increased degree of gaba or bz and the disease, remain to be answered. therefore, therapy targeted at reducing the blood ammonia concentration in patients with he and significantly reducing the increased gaba nerve tension seems reasonable [236] , but may not be completely effective. treatment effects of reducing ammonia vary, because of the different levels of ammonia in he patients that can be produced by the interaction between various known or unknown factors and the different effects of bz receptor antagonists. this theory is related to the metabolism of aromatic amino acids (aaa), the precursors of true neurotransmitters, including norepinephrine and dopamine. due to the reduction in the liver's detoxification function or formation of pss, the amines (phenylethylamine and tyramine) produced in the intestine cannot be cleared completely, resulting in elevated concentrations of these amines in the systemic circulation and increased levels in the brain through the blood-brain barrier. under the effect of β-hydroxylase, phenethanolamine and β-hydroxytyramine (β-dopamine) are generated from phenylethylamine and tyramine, respectively and are similar to norepinephrine and dopamine in chemical structure. these amines can be taken up, stored and released by adrenergic neurons in the brainstem reticular structure. phenethanolamine and β-hydroxytyramine are called false neurotransmitters because of their low physiological effects on the postsynaptic membrane, which is about 1/10 of norepinephrine. when these false neurotransmitters accumulate in the nerve synapse, they can outcompete or replace normal neurotransmitters, resulting in a disorder of nerve conduction. it was reported that plasma aaa (such as phenylalanine, tyrosine, and tryptophan) increased and branched-chain amino acids (bcaa, such as valine, leucine, isoleucine) decreased in patients with decompensated liver cirrhosis, leading to an imbalance of amino acid metabolism. aaa are decomposed and metabolized in the liver, and liver failure decreases aaa decomposition resulting in an elevated concentration of aaa in the plasma. insulin can promote bcaas entering muscle, which is then broken down and metabolized in the skeletal muscle instead of the liver. insulin inactivation is decreased in patients with liver failure, promoting a large number of bcaas entering the muscle tissue and decreasing the concentration of bcaas in plasma. finally, the bcaa/aaa ratio is reduced from a normal 3-3.5:1 to 1:1 or lower. the above process reduces the bcaa concentration, but increases the aaa concentration, leading to an increase in synthesis of false neurotransmitters and reduction of the normal neurotransmitter [237] [238] [239] . the epidemiological data suggests that manganese poisoning and he extrapyramidal have common clinical symptoms. the liver is an important organ for manganese excretion. the concentration of blood manganese can be increased when liver function is affected, during pss, or when excretion of bile is reduced. manganese content in plasma was sharply increased in more than 80% of patients with acute hepatitis and liver cirrhosis and the density of globus pallidus increased in the brain basal ganglia of he patients (partially 2-7 times higher by mri). based on histological results, the above changes were caused by manganese deposition, which disappears after liver transplantation. it has been suggested that manganese deposition may cause dopamine dysfunction. deposition of manganese not only cause direct brain injury, it can influence the function of 5-hydroxytryptamine (5-ht), norepinephrine and gaba neurotransmitters; impair astrocyte function; and have a synergistic effect with ammonia. however, there is no reliable correlation between the concentration of serum manganese and he severity, which may be due to the chronic deposition of manganese [240] . the characteristic change in mri imaging as the deposition of manganese remains to be verified. the effectiveness of manganese removal to improve the symptoms and neurological signs of patients with he needs further validation. the synergistic toxic effects between toxins (ammonia and mercaptan) and short chain fatty acids [241] , the 5-ht hypothesis, the effect of helicobacter pylori urease, opioids, endotoxin, tumor necrosis factor, melatonin, and hepatitis b virus termed additional theories of he syndrome. this theory also suggests the same hypothesis mentioned in the above theories. due to the extensive amount of liver cell damage caused by acute liver failure in type a he, the residual liver cells cannot effectively remove toxins leading to central nervous system dysfunction. type a he, known as non-ammonia encephalopathy, is endogenous he without clear causative agents. simple type b he is rare in mainland china; the liver can clear limited metabolic toxins in patients with chronic liver failure or pss, but once these toxins exceed the compensatory capacity of the liver, type c he occurs. the occurrence of type c he is largely related to the following risk factors, which are the most important factors in the prevention and treatment of he. patients with chronic liver failure or pss are less tolerant to the protein found in food, especially animal protein. a large amount of ammonia and aaa are produced by the decomposition of intestinal bacteria, which can induce he. oral ammonium salts, urea, and methionine can induce he by increasing the absorption of nitrogenous substances and elevating blood ammonia. intestinal production of ammonia can be increased by hemorrhage in the intestine (100 ml of blood contains 15-20 g protein). at the same time, because of the lack of isoleucine in the blood, after digestion and absorption of a hemorrhage, extra blood leucine and valine increase bcaa decomposition by enhancing the activity of bcaa dehydrogenase, thereby exacerbating the imbalance in the bcaa/aaa ratio. loss of blood volume, cerebral ischemia and hypoxia also increase the sensitivity of the central nervous system to ammonia and other toxic substances [230] . infections such as spontaneous peritonitis, pneumonia, and urinary tract infection can increase tissue decomposition and production of ammonia. secondary sepsis or sirs induce he through tnf-α, il-1, il-6 and other inflammatory factors, exacerbates oxidative stress, and increases the blood-brain barrier permeability of ammonia and other toxic molecules to liver and brain [242] . studies have shown that sirs is directly related to the deterioration of he in patients with liver cirrhosis, and its extent and mortality increase with the deterioration of sirs [243] . similarly, sirs is a common factor in triggering chronic liver failure characterized by he and renal failure. in a study of patients with liver cirrhosis, artificially-induced hyperammonemia by oral administration of glutamine may have worsened the results of psycho-mental testing in 10 cases of sepsis patients; while brain toxicity was not obvious after the inflammation was relieved, the observation of decreased cytokine levels indicated that infection and induced inflammatory mediators enhanced brain toxicity of hyperammonemia. accordingly, some researchers suggested that sirs could be an independent pathogenesis of he rather than a risk factor [243] . hyponatremia can affect the intracellular osmotic pressure and lead to brain edema, which induces he. hypokalemia is often associated with metabolic alkalosis [244] . mass use of diuretics or extraction of ascites can also cause alkalosis. ammonia is easily absorbed by the intestinal tract or through the blood-brain barrier inducing he [245] . a variety of reasons can cause pre-renal azotemia such as hypovolemia, anorexia, diarrhea, limiting the amount of liquid, mass use of diuretics, or extraction of ascites. hepatorenal syndrome or other causes can result in renal azotemia. pre-renal azotemia and renal azotemia caused by hepatorenal syndrome or other causes can increase the concentration of ammonia in the blood. several other predisposing factors can contribute to he such as constipation, hypoglycemia, the use of sedatives and proton pump inhibitors, and epilepsy. after the occurrence of constipation and intestinal obstruction, the patient's intestinal mucosa is exposed to ammonia longer thus increasing the absorption of ammonia. hypoglycemia can reduce brain deamination. the binding of sedatives, hypnotics and the brain gaba/bz receptor produce an inhibitory effect on the brain. it was reported that proton pump inhibitors increase the risk of he in patients with cirrhosis in a population study [246] . another study also suggested that epilepsy was associated with an increased risk of he in patients with cirrhosis [247] . patients with type a he often have no obvious anatomical abnormalities in their brains, but 38-50% of patients have brain edema, which may be a secondary change of the disease. hypertrophy and hyperplasia of the original plasma astrocytes in gray matter and subcortical tissue can be found in patients with type c he. patients with longer course of the disease will exhibit brain atrophy (especially in patients with alcoholic cirrhosis) of different degrees, thinning of the cerebral cortex, loss of neurons and nerve fibers, and deep cortical sheet necrosis, even the cerebellum and the base may also be involved. the majority of patients with cirrhosis may have different degrees of he at some stage in the course of the disease. the incidence of he in patients with liver cirrhosis is at least 10-50% in mainland china while the incidence of post-tips (transjugular intrahepatic portosystemic shunt) he is 25-45%. if patients with chronic liver disease have he, the outcome is poor; the one year survival rate is lower than 50% and the 3 year survival rate is less than 25% [248, 249] . the incidence of mild he is 39.9% in mainland china in patients with liver cirrhosis, 24.8% in patients with child-pugh a, 39.4% in patients with child-pugh b, and 56.1% in patients with child-pugh c. the incidence of mild he is not significantly associated with cirrhosis; however, with the increased degree of decompensated liver cirrhosis, the incidence of mild he increase. several studies have found that the incidence of depression and anxiety in patients also increased, with the increase of liver function damage, the incidence also increased, and the outcome is poor [250, 251] . the clinical manifestations of he vary, because of the difference in the nature of underlying disease, the degree of liver cell damage, the speed of injury and incentives. they are not specific to he compared with other metabolic encephalopathies. early pathological changes of he are mild he. the neuropsychological and intelligence tests detect mild form of he, which exhibit no clear clinical symptoms and often develop symptomatic he. the main clinical manifestations seen in acute liver failure induced by type a he are rapid-onset jaundice, bleeding, decrease in prothrombin, and eventually, change in mental status that can start as mild confusion but progress to coma and even death. type c he is characterized by chronic recurrent episodes of changes in personality and behavior [252] , stupor and coma, which is often accompanied by increased muscle tone, hyperreflexia, hepatic flap, ankle clonus or positive babinski sign and nervous system abnormalities. most patients in the early stages relapse, but then their symptoms become persistent. he often has a variety of risk factors such as consuming a high-protein diet or discontinuing treatment of he. patients with type c he not only have the clinical manifestations of encephalopathy, they also have chronic liver injury, cirrhosis and other clinical manifestations [226] . observation of encephalopathy dynamic changes is beneficial for early diagnosis, treatment and analysis of treatment efficacy. he can be graded and quantified according to the degree of disturbance of consciousness, nervous system performance and eeg changes. according to the 2009 edition of the "consensus on the diagnosis and treatment of hepatic encephalopathy" in china, he is divided into 0-4 periods, but each period can be overlapping or distinct but each period can be overlapping (table 2 .4). at present, scholars have stressed that the occurrence of he is a continuous progression of the disease and should be viewed as a continuum of a wide range of neuropsychiatric abnormalities, rather than isolated clinical stages. according to the traditional west haven criteria diagnosing grade 1 he is based on clinical signs and physician assessments, resulting in diagnostic criteria confusion [253] . (fig. 2.3) . covert he is diagnosed by a variety of neuropsychological and intelligence tests; the evaluation of overt he widely uses the modified west haven semi-quantitative grading table for the analysis of patients with neuropsychiatric state (table 2 .5), the glasgow coma scale for the analysis of the degree of consciousness of patients, and the simple he severity rating scale for the disease in addition to abnormal liver function (such as increased bilirubin, enzyme bile separation, and decreased prothrombin activity) commonly used auxiliary examinations for he diagnosis include: determination of ammonia, amino acid analysis of plasma and csf, psychological intelligence test, neurophysiological test, electroencephalogram and neuroimaging. the normal level of fasting venous ammonia is 6-35 μg/l (serum) or 47-65 μg/l (whole blood) and arterial ammonia concentration may be 0.5-2 times that of venous ammonia. generally, the determination of arterial ammonia is common in clinical practice than intravenous determination; however, if venous blood has been transported on ice box and detected in a timely manner after proper collection, the result is expected to be as effective as arterial detection. ammonia levels are increased in type b and c he, but are normal in type a he. thus, he cannot be ruled out based on having a normal ammonia level. the increased level of ammonia was reported to be associated with the degree of type a he, but significant overlaps in different clinical stages of patients were also found [255, 256] . therefore, ammonia detection is not routinely recommended in the diagnosis of he. notably, we need to rule out falsely elevated levels of ammonia caused by lab error, renal failure, complete parenteral nutrition, gastrointestinal bleeding, the use of steroid hormones and other extrahepatic factors. the fischer ratio (bcaa/aaa) is used as a marker of he, the plasma bcaa levels decrease while aaa levels increase; resulting bcaa/aaa: < 1 (normal >3). it was reported that the concentration of glutamate in csf in he patients is increased compared to healthy controls. the concentrations of phenylalanine and tyramine in csf were also significantly increased, and the level of phenylalanine was closely related to the degree of he [257] . recently, it was reported that h-nuclear magnetic resonance spectroscopy could select biomarkers for these diseases, such as in patients with he [258] , but this is not commonly used clinically because the the characteristic manifestations of cognitive dysfunction in patients with covert he are lack of attention, working memory problems, and deficits in executive function. therefore, various intelligence tests are used to assess the subtle changes in a patient's cognitive or precise movement, which is important for the diagnosis of covert he, but not for overt he. [259] . at present, computer-aided psychological tests such as information and communication technology (ict), cognitive drug research test (cdr), and critical flicker fusion test (cff) are not influenced by the factors mentioned above and easily operated, which can be used as an alternative choice for pen and paper tests. ict with sensitivity 87% and specificity 77% was one of the most commonly used tests to diagnose minimal he. cff was originally used to detect the critical flicker frequency of alert patients, reflecting brain conduction dysfunction. based on a spanish study of 217 cases, including patients with cirrhosis and healthy controls, cff was a sensitive method to diagnose covert he with sensitive, simple and reliable advantages [260, 261] . because the diagnosis of minimal he has just started, the related diagnostic value still needs to be further evaluated. an abnormal eeg is often observed before biochemical abnormalities or mental abnormalities [262] . the main abnormalities by eeg are slowed rhythm, sporadic or universal θ wave (4-7 times/s) and the occasional α wave (1-3 times/s). with the deepening of consciousness, symmetrical δ-wave and three-phase waves appear on both sides simultaneously. this change usually occurs on both sides of the forehead and the top, gradually moving backwards. although these eeg changes are not specific to he and can appear in uremic encephalopathy and other metabolic encephalopathy, the severity of changes have a good correlation with clinical stages of he. computer analysis of eeg frequency distribution, such as artificial neural network-expert system (aness) and short epoch dominant activity cluster analysis (sedaca), is more objective and valuable in diagnosing minimal he than conventional eeg [263] . there are many kinds of evoked potential tests, including visual evoked potential (vep), brainstem auditory evoked potential (baep), somatosensory evoked potential (ssep) and endogenous event evoked potential (event-related potentials, erps) p300, of which the p300 is the most sensitive test for the diagnosis of he. compared with intelligence tests, neurophysiologic tests, independent of age and education background, are more objective. however, they are only used in clinical studies and are limited by equipment, and professional operation. based on cerebral ct and mri, brain edema can be found in patients with type a he while brain atrophy in the frontal cortex, and the t1-weighted signal enhancement in the globus pallidus can also be found, which may be associated with manganese deposition. detected by h-magnetic resonance spectroscopy (h-mrs), the metabolic changes of he patients in the brain include increased levels of glutamate and glutamine, and decreased levels of inositol, taurine and choline [263] . using fluid attenuation inversion recovery (flair) and diffusion weighted imaging (dwi) techniques, diffuse t2-weighted signal enhancement is found in the hemisphere white matter and corticospinal tracts, which may be associated with cerebral ischemia. however, the sensitivity and specificity of the above-mentioned imaging abnormalities remain unknown, and the correlation with he staging is not clear. therefore, the main significance of cranial nerve imaging is to exclude cerebrovascular accident, intracranial tumors and other diseases, rather than diagnose he. there is no gold standard diagnostic criteria of he, and diagnosis is mainly based on the exclusion of other diseases. but one should consider the following five factors [232]: several forms of hepatic diseases may lead to different kinds of he. type a he is caused by acute hepatic failure, but without chronic hepatic disease. type b is caused by pss, but without any history of hepatic diseases. type c is caused by serious hepatic diseases and/or widespread pss, such as cirrhosis, liver cancer, post-tips and so on. psychiatric symptoms can be found such as change of mood and personality, dementia, behavior disorder and disorientation. drowsiness alternating with excitability, hypermyotonia, asterixis, ankle clonus, insanity and coma are physical signs could be present in progressed patients. some patients may lack related physical signs and psychiatric symptoms, but have deficits in ability of learning, understanding, concentration, and quick verbal response. upper gastrointestinal hemorrhage, ascites tapping, excessive diuresis, high protein diet, medicine (such as sedatives) and infection could lead to he. previous he symptoms could be helpful for the diagnosis. type a usually does not have any risk factors. metabolic encephalopathy includes ketoacidosis, hypoglycemia, uremia, pulmonary encephalopathy, serious electrolyte disturbances and toxic encephalopathy. nervous system diseases include intracranial hemorrhage, infection or tumors, mental diseases and excessive use of sedatives [264] . but one should also watch out for the coexistence of he in these situations. an overt he should be considered if (1), (3), (4), and (5) coexist; and covert he is based on (2), (3), (4), and (5) [228] . then, based on the degree of neuropsychiatric symptoms, determine the stage of he, or for he classification refer to the west have semi-quantitative classification table or ishen scores. the flow chart of diagnosis is shown in fig. 2.4. he is a complex metabolic disorder caused by many factors and comprehensive measures should be taken to cure it from different aspects. according to the clinical type, inducements and the severity of the disease, different plans of treatment should be designed for he. at present, the treatment of overt he generally includes the following aspects: (1) supportive treatment; (2) identification of possible concurrent encephalopathy and removal of other precipitants; (3) cause of treatment; and (4) empirical treatment (fig. 2.5 ). the point of nutritional therapy is to promote anabolism, inhibit catabolism, and maintain a positive nitrogen balance, rather than simply limiting protein intake. to reduce the source of ammonia, it has been suggested that patients with he should limit protein intake. in critically ill patients, it has been suggested that they should stop all protein intake and, after the disease improves, gradually increase protein intake to the maximum clinical tolerance. these recommendations are now being questioned because most cirrhotic patients are malnourished and all long-term protein-restricted diets increase the severity of malnutrition. in addition, a negative nitrogen balance increases mobilization of skeletal muscle, resulting in a reduction in ammonia metabolism that may increase blood ammonia levels. recent studies have shown that normal ingestion of protein 1.2 g/(kg • d) can also improve the health-related quality of life (hrqol), especially in mhe [265] ; and have no adverse effects on the recovery of blood ammonia and he fig. 2.4 the flow chart of diagnosis of he compared with the restricted protein intake. according to the guidelines of the european society of enteral nutrition in 2009, the intake of protein should be guided by the following principles: patients with acute phase he on the first day should be put on a prohibited protein diet and given glucose to ensure energy supply and those who cannot eat food may be fed through a nasogastric tube without short-term fasting; patients with chronic he do not need to fast and their intake of protein should be 1-1.5 g/(kg • d); oral or intravenous use of bcaa and essential amino acid preparations can be administered to adjust the balance of aaa/bcaa, promote the balance of nitrogen, and also reduce the risk of he recurrence [266] ; probiotics and prebiotics can enhance the body's tolerance to protein; plant protein is superior to animal protein because it contains methionine, has less aaa, and more bcaa, but it also contains cellulose, which is conducive to maintain the normal flora in the colon and acidize the intestinal tract, shortening the transit time of the colon and reducing absorption of ammonia. the above points need further verification. additional supportive treatments include: maintaining adequate hydration, electrolytes and acid-base balance; ensuring an energy supply of 30-35 kal/(kg • d), which should be composed of 50-60% sugar, 20-30% protein, and 10-20% fat; administering appropriate vitamins and trace elements; treating for hypokalemia, hyperkalemia, hyponatremia, hypocalcemia, hypomagnesemia and metabolic alkalosis as needed; strengthening the basis of treatment with the appropriate infusion of fresh plasma or albumin, increased plasma colloid osmotic pressure; treating for hypoxemia and cerebral edema; and preventing and treating any bleeding and bacterial infection. type c he has a variety of precipitents. actively finding and eliminating triggers can effectively prevent the development of he, such as esophageal variceal bleeding that can develop into he. active hemostasis, anemia correction, and removal of intestinal blood are also conducive to controlling he. in addition, active control of infection, correction of water and electrolyte imbalance, elimination of constipation and improving renal function are essential to control he. anesthetics, painkillers, sedatives, sleeping pills, and other drugs should be strictly controlled. patients with mania or convulsions can reduce the use of diazepam and scopolamine, and the frequency of administration can also be reduced for promethazine, chlorpheniramine, and other antihistamines. toxic substances causing he mainly come from the intestine. thus, in order to prevent and control he, it is very important to clean the intestinal tract to reduce the generation and absorption of ammonia and other toxic substances. saline or weak acidic solution enemas (such as a dilute acetic acid solution), or oral or nasal feeding of 25% magnesium sulfate (30-60 ml) can be used to clear intestinal hemorrhage, intestinal impaction, and other toxic substances. an enema composed of non-absorbable lactulose (300-500 ml) plus water (500 ml) is also useful, especially when applied for type b he. a recent clinical trial suggested that polyethylene glycol was more effective than the current standard first-line therapy in treating these patients [267] . another two studies showed that polyethylene glycol was more effective than the standard lactulose therapy in treating patients with acute he by cirrhosis [268, 269] . other available drugs include pear liquors, mannitol, rhubarb, and so on, but excessive use of these substances may lead to dehydration and aggravate he. non-absorbable disaccharides include lactulose and lactitol. lactulose, a kind of synthetic ketone disaccharide, cannot be broken down in the stomach and small intestine due to a lack of enzymes that can break down galactose in the digestive tract. after entering the colon, lactulose can be broken down into acetic acid and lactic acid with the help of gut bacteria, leading to a reduction in the colonic ph and inhibition of the absorption of ammonia in the intestine. these non-absorbent disaccharides are decomposed into organic particles in the intestinal tract, which can increase the osmotic pressure of the intestine, and their acidic products stimulate the intestinal wall and can slightly promote intestinal excretion. these non-absorbent disaccharides, acting as prebiotics in the intestine, can inhibit the growth of bacteria, which can produce ammonia and urea, finally reducing the production of ammonia and reversing low-grade cerebral edema when combined with rifaximin [270] . however, probiotics can benefit patients in the long-term [271] . oral or nasal feeding (15-30 ml, 2 or 3 times daily) was recommended to adjust the daily defecation appropriately, about 2-3 times daily. main adverse reactions include abdominal discomfort, abdominal distension, abdominal pain, loss of appetite, nausea, vomiting, and diarrhea. lactulose can even be used in patients with diabetes or lactose intolerance when the purity of non-absorbable disaccharide was high (≥ 98%), but is not used in patients with intestinal obstruction. numerous randomized controlled studies showed that lactulose or lactitol can significantly alleviate overt he and improve the patient's cognitive function and quality of life [272, 273] . lactulose is still the first-line therapy of anti-he, although its effect on improving the survival rate of patients is uncertain. antimicrobial agents can be used as a substitute for non-absorbable disaccharides in treating acute and chronic he. in the past, oral aminoglycoside antibiotics, such as neomycin, which are rarely orally ingested, were used to inhibit the overgrowth of bacteria in the intestine. however, recent randomized placebo-controlled studies have shown that neomycin may not benefit patients with he compared with placebo-treated patients and that long-term use of neomycin may lead to increased ear and renal toxicity risk and impair the function of small intestinal mucosa [274] . metronidazole can inhibit anaerobic bacteria in the intestine and alleviate he, but long-term use may lead to disruption in the intestinal flora, gastrointestinal discomfort, or neurotoxicity. rifaximin, a derivative of rifamycin with a broad-spectrum, has a potent inhibitory effect on intestinal bacterial growth, is a minimally-absorbed oral antibiotic and only plays a role in the gastrointestinal part. administration of rifaximin (550 mg, twice a day) can significantly prevent the occurrence of he compared with placebo-treated patients [275] [276] [277] ; rifaximin was equivalent to or better than lactulose and neomycin in treating patients with chronic he [278] . a study indicated that rifaximin-α in combination with lactulose was a cost-effective therapy for patients who had experienced at least two prior overt he episodes [270] , and this therapy could also improve the driving ability of patients with covert he without toxicity to the auditory nerve and renal function [274, 277] . thus, rifaximin has been recommended by the us food and drug administration (fda) for the prevention of recurrent he. the efficacy of rifaximin and relation between longterm use of rifaximin and intestinal flora in the treatment of he needs to be further investigated. however, a recent study in mice showed that rifaximin beneficially alters intestinal ammonia generation by regulating intestinal glutaminase expression in mhe [277] . the study may provide a new opportunity to study intestinal flora in the treatment of he. microecologics with bifidobacterium and lactobacillus can regulate intestinal flora structure to inhibit the growth of bacteria that produce ammonia and urease. in combination with prebiotics, microecologics can reduce the production and absorption of intestinal ammonia and other toxic substances [279, 280] . in a recent openlabel study, 190 patients with cirrhosis were randomized to three groups and treated with lactulose (30-60 ml daily), probiotic capsules, or with both drugs. after a month of treatment, patients with he showed better results in the neuropsychological test, p300 auditory evoked potentials, and blood ammonia. however, there was no difference in the therapeutic effect among the three groups [281, 282] . clinicians commonly use sodium glutamate, potassium glutamate, arginine hydrochloride and potassium magnesium aspartate, but the exact efficacy is highly controversial at present and effective drug reduced ammonia is described below. (a) l-ornithine-l-aspartate lola, a dipeptide, can lower blood ammonia by promoting ammonia consumption and the synthesis of urea, glutamic acid and glutamine in brain, liver and kidney [283, 284] . ornithine, a substrate of the ornithine urea cycle, can increase activity of carbamyl phosphate synthetase and ornithine carbamyl transferase, and promote urea synthesis. n-methyl-d-aspartate (nmda) is a substrate of glutamine synthesis, and the conversion of glutamic acid to glutamine in the body can remove blood ammonia [285] . nmda is also involved in nucleic acid synthesis in liver cells and indirectly improves the metabolism of the krebs cycle process in liver cells to facilitate the repair of liver cells. clinical studies show that, compared with a placebo group, 20 g/days lola intravenously could noticeably reduce fasting blood ammonia (fnh3), postprandial blood ammonia, and improve the mental status of patients with he [286] . patients with oral lola also had improved he examination results for the digital connection test, the flapping tremor, and eeg results [287] . in addition, glycerol phenylbutyrate (gpb) can safely reduce the incidence of he as well as ammonia in patients with cirrhosis and he. the results showed that gpb had therapeutic potential in this population [288] . zinc is an important cofactor in urea cycle enzyme catalysis. a study in he patients showed that serum zinc concentration is reduced, and showed a negative correlation with the blood ammonia concentration; the serum ammonia level is much lower after zinc supplementation in patients, and he can be improved in some patients. a new study suggests that antioxidant and zinc supplementation can improve mhe in patients with liver cirrhosis [289] . oral zinc preparation can also reduce absorption of divalent cations such as manganese in the intestine; however, it has not been determined if zinc has a positive therapeutic effect on he. (c) sodium benzoate sodium benzoate can lower the blood ammonia concentration by activating the urea cycle for ammonia detoxification and promoting urinary ammonia. randomized controlled studies showed that sodium benzoate had the same efficacy as lactulose in treating patients with he. the recommended sodium benzoate dose is 5 g twice a day; nevertheless, few patients can tolerate this dose because of its high gastrointestinal side effects [232] . a recent study showed that tranilast could protect patients from thioacetamide-induced acute liver injury and alleviate he [290] . endogenous bz analogues combine with the inhibitory neurotransmitter gaba receptor to depress the action on the cns, and is one of the occurring hallmarks of he pathogenesis. a large-scale clinical study on 560 he cases showed that the improvement rate in brain function in treatment and control groups were 15% and 3%, respectively [291] . the study showed that treatment of he with receptor antagonists such as fluorine marcie is feasible. a meta-analysis which included 13 casecontrol studies of 805 patients show that fluorine marcie can noticeably improve he, but didn't show any long-term benefits or improve patient survival rate. so fluorine marcie should only be considered for he patients who had used bz. although the reduction of dopamine neurotransmitter activity is also one of the pathogenesis, the application of bromocriptine, levodopa, has been unable to bring more benefits besides partly improving symptoms of patients. oral or intravenous infusion with a bcaa-based amino acid mixture can theoretically correct an imbalance in amino acid metabolism and control false neurotransmitter formation in the brain [292] . a meta-analysis which included five studies showed that intravenous bcaa did not reduce the mortality rate of he. three studies with bcaa did not reduce the mortality rate of he; however, two larger studies (randomized controlled study about patients with liver cirrhosis in 174 cases and 622 cases, respectively) show that the application of bcaa not only reduced the occurrence of he and liver failure, but also improved the nutritional status, liver function and survival rate in patients. another study showed that bcaa could stimulate liver cell regeneration thus reducing the occurrence of liver failure. supplementation with a bcaa-rich amino acid mixture showed improved restoration of the patients' positive nitrogen balance, and increased the patient's susceptibility to protein food, improving cerebral perfusion [293] . considerable progress has been made to understand treatment of he, studies of basic and clinical research are underway using newly discovered treatment strategies, such as toll-like receptor 4 antagonists (with the ability to reduce systemic inflammation and oxidative stress) as well as non-steroidal anti-inflammatory drugs (ibuprofen) [286, 294] , nmda antagonists, anticholinesterase. however, research using gene therapy should not be ignored [295] . after tips, lola can significantly reduce the increase of venous ammonia concentration in patients with he [296] . and the positive dietary intervention can significantly reduce the incidence of he [297] . for patients with refractory he, embolization of pss is a safe and effective treatment strategy [298] . improving liver function antiviral treatment with nucleos(t)ide analogues can reduce or eliminate liver inflammation and necrosis, promote the regeneration of liver cells, and help restore the functions of hepatic metabolism and detoxification in chronic liver failure caused by hepatitis b virus. artificial liver support systems can be divided into three types including nonbiological type, biological type and mixed type. the non-biological liver support system is the most widely used type, and consists of hemodialysis, hemofiltration, plasma exchange, blood perfusion, plasma adsorption, and the molecular adsorption recirculation system (mars) [299] . the artificial liver support system can replace the partial function of the liver, remove the poison accumulated in the body, create conditions that allow for the regeneration of liver cells and provide enough time to wait for liver transplantation for patients with he. an artificial liver support system can be used to treat acute and chronic he, but patients with overt stage 2 he should be especially careful with plasma exchange. liver transplantation remains the only promising therapy for patients with an acute liver failure or endstage liver disease. liver transplantation is an effective means for all kinds of persistent and severe he; however, in patients with he there is a significant increase in mortality among patients awaiting liver transplantation [300] . recently, it has been reported that cognitive function was not fully recovered after liver transplantation in some patients with severe he [301] . the key point in improving the prognosis of he is early recognition and timely treatment. active treatment should be given when diagnosing covert he. theoretically, for patients with serious pss, interventional therapy, surgery or permanently/temporarily and partially/totally blocking the pss can improve the patient's symptoms. the use of this therapy should be carefully weighed because it can increase the risk of gastrointestinal bleeding in case of portal hypertension. covert he has gained an increasing amount of attention in recent years. patients with covert he do not exhibit obvious signs and symptoms; however, their quality of life is reduced because of reduced operational ability or sleep disorders. without treatment, covert he will progress to overt he over time. the population with high risk should be examined and treated early, especially those engaged in potentially dangerous occupations. the following solutions can be referred to: (a) adjusting dietary structure (vegetable protein is the main intake); (b) oral administration of lactulose (15-30 ml, 2-3 times daily); (c) oral administration of rifaximin (550 mg, twice a day); (d) oral administration of lola (6 g, 3 times a day); (e) oral administration of baaa; and (f) oral administration of probiotic preparations [271, 275, 289 ]. although medical technology has made great progress and the research into he is also increasing in recent years, the pathogenesis of he is still unclear. due to a lack of specific methods, combination treatment is still the main therapy for he. it is generally believed that the onset of he may be a result of the synergistic effects of many factors. therefore, it is difficult to implement and draw convincing conclusions from randomized controlled trials with a single intervention targeting a specific pathogenesis and risk factor. ongoing issues remain, such as standardizing the research design of he treatment and evaluating the efficacy of he treatment more scientifically and objectively. some clinical studies may bring new hope for he treatment by new ongoing strategies of targeted systemic inflammation, oxidative stress, and neurosteroids. in addition, the key point in improving the prognosis of he is early recognition and timely treatment. active treatment should be given when diagnosing covert he. it is difficult to popularize the cognitive dysfunction detection methods of latent he. so the key and difficult point is to develop new method of assessments for clinicians in the future. jia shang hepatopulmonary syndrome (hps) is a syndrome of shortness of breath and hypoxemia induced by vasodilation in the lungs of patients with a variety of acute and chronic liver disease. essentially primary liver disease, pulmonary vasodilation and arterial oxygen lack of co-triad constituted. due to abnormal increase of vasodilators、ventilation/ blood flow disproportion and pulmonary hypertension caused by liver disease, the hypoxemia (pao 2 < 9.33 kpa) (70 mmhg) is included in hps. when fluckiger reported a 37-year-old syphilis female patient as early as in 1884, he described cirrhosis, cyanosis and clubbing at the same time, while he was not aware of the intrinsic relationship between these clinical manifestations [302] . in 1935, snell reported decreased arterial oxygen saturation (sao 2 , less than 94%) with abnormal hemoglobin in 38 patients with liver parenchymal lesions and biliary obstruction, and 3 years later, he proposed that such a phenomenon was associated with decreased affinity of o 2 with hemoglobin. in 1956, rydell and hoffbauer reported the detailed clinical diagnostic and treatment process of a 17-year-old male with "juvenile cirrhosis", and found multiple arterial-venous anastomoses in the lungs during autopsy, which he thought contributed to clinical cyanosis mainly. this provided a histological basis for the patient, and people conducted a large amount of studies thereafter. in 1966, berthelot et al. injected opaque glue into the pulmonary vascular beds at the time of biopsy after the patient's death for the first time, and he found abnormal small arterial dilation in the lungs of the patient with cirrhosis, which he termed lung spider nevus. the term hepatopulmonary syndrome (hps) was first proposed by kennedy and knudson in 1977 [303] . after nearly 20 years of studies in a large number, people gradually developed a clear understanding of the mechanisms underlying its pathogenesis. in 1988, eriksson used the term functional hepatopulmonary syndrome for the first time. in 1989, the famous liver disease expert sherlock formally used this diagnostic term in his monograph hepatobiliary diseases, which has been recognized by many scholars [304] . hps can occur in patients of any age groups, and various literature reports show conflicting incidences of hps in patients with cirrhotic portal hypertension, with the average incidence of various chronic liver diseases being about 5-29%. the incidence of cirrhosis in patients is high, and 30-70% of patients can additionally develop mild arterial hypoxia and 12-28% develop arterial hypoxia. in the study by binay on indian cirrhotic populations arising from hepatitis b mainly, the incidence of this disease is relatively low (6.7%) [305] . the differences in incidence were mainly attributable to the different diagnostic criteria adopted. schenk et al. studied the incidence of hps by performing transthoracic contrast echocardiography (ttce), pulmonary function tests and blood gas analysis on 98 patients with cirrhosis patients. the results showed that the incidence of hps patients in whom alveolar-arterial partial pressure of oxygen (aapo 2 ) was used as an indicator of hypoxemia was significantly higher than those in whom arterial partial pressure of oxygen (pao 2 ) was used [306] . when arterial partial pressure of oxygen (pao 2 ) was reduced to reflect hypoxemia, hps incidence was 19% when <80 mmhg and 15% when <70 mmhg, respectively. while when increase in alveolar-arterial partial pressure of oxygen (aapo 2 ) was used to reflect hypoxemia, the incidence of hps was high, with 32% when >15 mmhg and 31% when >20 mmhg, respectively. hps is most common in cirrhosis due to various causes. pulmonary vascular abnormalities and arterial hypoxemia can occur in a variety of acute and chronic liver diseases, and this is true mainly when it comes to cirrhotic patients due to chronic liver diseases, especially cryptogenic liver cirrhosis, alcoholic cirrhosis, hepatitisinduced cirrhosis and primary biliary cirrhosis. besides, hps can also occur in chronic hepatitis, acute severe hepatitis, cholestasis, ɑ-anti-trypsin deficiency [307] , tyrosinemia, wilson disease, and non-cirrhotic portal hypertension (such as idiopathic portal hypertension and schistosomiasis cirrhosis, etc.). arterial hypoxemia can also occur in extrahepatic portal vein occlusion. the observation of these patients suggests that portal hypertension may be the main factor for the pathogenesis of hps. hps can also occur in non-cirrhotic portal hypertension, and even cirrhosis-and portal hypertension-free chronic viral hepatitis. in 2000, binay et al. found that patients with progressive liver failure with hyperdynamic circulation are most likely to suffer from hps, while they did not find the correlation with the severity of liver cirrhosis. hps is, in essence, hypoxemia due to anomaly in pulmonary vascular dilatation and arterial oxygenation when liver disease occurs. arterial hypoxemia occurs as the result of insufficient oxygenation by blood cells in the blood when blood flows through the lungs, or a proportion of blood fail to flow through the alveoli [308] . since primary heart and lung diseases have been excluded when hps occurs, the abnormal pathways that red cells may pass through include: (1) passing through the pleural and hilar bronchial vessels while not reaching the alveoli; (2) blood flows directly into the pulmonary veins due to the high pressure portal system in the mediastinum, thereby bypassing the pulmonary circulation; (3) flowing directly into the pulmonary veins through the expanded alveolar capillaries or the pulmonary-venous fistula. alveolar telangiectasia may be more important to the formation of hypoxemia, and existing study data show that the development of hps is at least associated with the systemic hyperdynamic state, portal hypertension, hepatic encephalopathy, hepatorenal syndrome and pulmonary hypertension [308] . therefore, it is believed that the main causes of hps are systemic metabolism and hemodynamic disorders, and that it is involved in the formation of systemic metabolism and hemodynamic disorders, which is of important pathophysiological significance. 1. the basic pathological change of hps is pulmonary vascular dilatation, which is manifested as: (a) dilation of anterior capillaries in a large number. (b) formation and opening of the pulmonary basilar arterial -venous communicating branches. (c) formation of pleural "spider mole", which is mainly manifested as dilation of anterior capillaries. in autopsies, it was found that the basic pathological changes in patients with liver cirrhosis and other chronic liver diseases were extensive pulmonary vascular dilatation and arteriovenous communicating branches. some people found the pathological changes through vascular shaping, with pleural vasodilation at the basal aspect of the lungs or the formation of subpleural spider nevus. domestic professor gu changhai summarized these pathologic changes in 1997 as arterial dilation within the pulmonary acinus in a pattern of inhomogeneous distribution, thin-walled blood vessels, 60-80 μm in diameter, in the lower lobes of the whole lungs, extensive dilation of pulmonary vascular beds adjacent to the alveolar gas at the anterior capillary level, and significantly expanded pulmonary artery branches and pulmonary capillaries up to 160 μm in diameter. electron microscopy showed thickened pulmonary capillaries, pulmonary arterial walls and the basal layers of small veins. 2. factors that affect the dilation of blood vessels: the mechanisms underlying pulmonary vascular dilatation have not yet fully elucidated, and the possible influencing factors include: (a) increased activity of vascular dilators various acute and chronic liver diseases, liver cell failure and metabolic disorders, particularly reduced inactivation of vasoactive substances which can enter directly into the systemic circulation through abnormal anastomotic collateral vessels, result in disorder of the systemic hemodynamics and increased contents of vasodilators in the blood circulation. just as visceral congestion in patients with portal hypertension, they can act on the intrapulmonary vessels, causing pulmonary vascular dilatation and pulmonary congestion. substances that cause vasodilation include glucagon, prostaglandin, vasoactive intestinal peptide, nitric oxide, angiotensin, bradykinin and endotoxin, etc. (b) reduced vasoconstrictors or decreased sensitivity of intrapulmonary vascular beds to the endogenous vasoconstrictors, such as norepinephrine, endothelin, atrial natriuretic peptide, vasopressin, serotonin and tyrosine, etc. the contents of the substances are not absolutely reduced because maybe their sensitivity is reduced. when chronic liver disease occurs, the anterior communicating branches of the originally closed non-functional capillaries may be opened, and a disorder occurs in the hypoxic pulmonary vascular systolic dysfunction which should have been normal, and it is only 75% of the normal state [309] . (c) neurological factors cirrhotic patients show sympathetic nerve hyperactivity, but after the formation of portal hypertension, their sympathetic nerve function may be damaged, which play an important role. animals with portal hypertension often show abnormal pressure responses and reduced sensitivity of blood vessels to norepinephrine, resulting in increased cardiac output, and dilated pulmonary vascular volumes. besides, hemodynamics within the lungs is also a manifestation of the body's hyperdynamics. (d) decreased reactivity of intrapulmonary blood vessels to hypoxia recent inert gas dispersion tests show that cirrhotic patients with over two spider nevus are manifested as not only liver damage, but also decreased systemic intrapulmonary vascular resistance, decreased reactivity of blood vessels to hypoxia and dilated pulmonary vessels. however, it was also found using pulmonary angiography that in spite of the dilated vessels at the ending of arteries, the responses of vessels to oxygen were almost normal, which did not support this view. (e) intrahepatic angiogenesis or dysplasia may also be one of the factors for the formation of hps. to date, the mechanisms underlying pulmonary vascular dilatation caused by hps is still unclear. however, long-term administration of intrapulmonary vasoactive substances can cause significantly increased intracellular cyclic adenosine monophosphate (camp) and/or cyclic guanosine monophosphate (cgmp), resulting in hypoxic pulmonary vasomotor dysfunction and pulmonary artery dilatation, which may be an important cause of this disease and also pulmonary manifestations of systemic hyperdynamic circulation. due to the significant dilation of the pulmonary capillaries and the anterior capillaries, some of the blood around the capillaries in contact with the alveoli can still undergo exchanges with gases, while the central blood, due to the increased diffusion distance from the alveoli, leads to insufficient gas exchange, resulting in insufficient arterial oxygenation and thus a series of hypoxemic manifestations. to date, the pathogenesis underlying the pathogenesis of hps has not yet been elucidated. in view of the above pathophysiological changes and current studies, it is believed that the disease may be caused by insufficient ventilation, diffusion disorder, ventilation/blood flow imbalance and decreased oxygenated hemoglobin affinity, or the above factors in combination. under normal circumstances, insufficient ventilation due to a variety of reasons causes insufficient oxygen inhaled into the alveoli and reduced blood oxygen exchanges, which can result in hypoxemia [310] , such as chronic bronchitis, foreign bodies in trachea, atelectasis and respiratory muscular paralysis, etc. and the presence of insufficient ventilation in patients with chronic liver disease and cirrhosis or not is still controversial. in 1982, fujiwara studied the lung function in 22 patients with decompensated liver cirrhosis and reported that vital capacity (vc), functional residual capacity (frc) and respiratory reserve volume (evr) in the patients were significantly reduced, that r/t was mildly increased, and that there was no changes in 1 s forced expiratory volume (fev1). therefore, it was believed that mechanical compression and insufficient ventilation due to pulmonary interstitial edema in patients with liver cirrhosis was the main reason for impaired lung function. subsequently, edison et al. studied the pulmonary function of 21 patients with decompensated liver cirrhosis, and found that their vc, maximum ventilation volume (mvv), frc, total lung volume, and r/t were significantly reduced, and they believed that patients with cirrhosis had obvious obstructive and limited insufficient ventilation, which were mainly caused by compression of lung tissue due to increased abdominal pressure, elevated diaphragm and increased chest volume and pressure when patients had additional ascites, and atelectasis [306] . however, decreased fev1 resulted from compression of small trachea due to pulmonary interstitial edema and vasodilation, and early closure of expiration. theoretically, all of the above factors can lead to insufficient ventilation, one of the factors resulting in this disease. this was also substantiated by significantly increased arterial partial pressure of oxygen and decreased co 2 partial pressure in cirrhotic patients with pleural effusion after pleural effusion extraction and recovery from atelectasis. however, there are also some people who do not think that hypoxemia results from insufficient ventilation, but because cirrhotic patients are not complicated by high concentrations of co 2 when their arterial partial pressure of oxygen is decreased [311] . this is likely because when patients have hypoxemia, compensation of hyperventilation causes arterial blood co 2 partial pressure not to increase, and results in decreased paco 2 or even respiratory alkalosis. besides, in some patients without decompensated liver cirrhosis, arterial hypoxemia can also occur. even it has been found that the lung function tests in patients with decompensated liver cirrhosis are normal. therefore, the majority of scholars currently believe that insufficient ventilation is not the main cause of hypoxemia in cirrhotic patients. for patients with hps, the inert gas exclusion technique should be performed to prove that there is a disorder in the diffusion of oxygen, which is determined by the basic pathological changes of hps -pulmonary vasodilatation. pulmonary angiography can show small spider-like to obviously cavernous diffuse vasodilation within the lungs. due to the significant dilation of the pulmonary capillaries and the anterior capillaries, the diffusion distance of the blood flow in central blood vessels and the alveoli is increased, preventing the gases in the alveoli from entering the pulmonary capillaries, thereby affecting the gas exchanges. studies have shown that hypoxemia often occurs in patients with cirrhosis or aggravates during exercises, and it is believed that diffusion disorder or limitation of oxygen occurs in patients. in fact, factors that affect o 2 diffusion do occur in patients with cirrhosis, but they are still not sufficient to explain the apparent hypoxemia. although vascular dilatation occurs at arterial endings in patients with hps, their arterial partial pressure of oxygen can be reduced while inhaling air and increased when they are given oxygen inhalation, which further proves that although diffusion disorder does exist and it plays a role in the formation of this disease, the role is not important. to engage in gas exchanges is the most important biological function of lung tissues, and this gas exchange must be completed when there is an appropriate ventilation/blood flow ratio. under the normal circumstance (normal adult resting state), the most appropriate ventilation/blood flow ratio physiologically is 0.8. changes in the ratio due to any cause can affect the gas exchange, and the imbalanced ventilation/blood flow ratio in hps patients with hypoxemia is mainly because of pulmonary vascular dilatation and arteriovenous shunt [312] . 1. intravascular vascular dilatation: pulmonary vascular dilatation has been confirmed pathologically and by angiography. dilated blood vessels in the lungs leads to gas diffusion disorder. besides, since the oxygen molecules in the air can not be diffused to the central dilated blood for the gas exchange, causing decreased ventilation/blood flow ratio and pulmonary arterial partial pressure of oxygen. this decreased ventilation/blood flow, together with increased amount of reactive cardiac output, shortens the duration of blood that flows through the capillary network and insufficient oxygenation [312] . excessive ventilation can in part enhance patients' pao 2 . if the alveolar oxygen partial pressure is increased at this time, some oxygen molecules can reach the central areas of dilated blood vessels, increasing the arterial partial pressure of oxygen. therefore, it is called the diffusion -perfusion disorder or pulmonary arteriovenous functional shunt rather than the true lung shunt. 2. arterial -venous shunt: intrapulmonary vascular fistula and pleural spider nevus can occur in cirrhosis of the liver and allow the pulmonary arterial blood to circumvent gas exchange and directly flow into the pulmonary vein so that patients may develop hypoxemia. this hypoxemia can not be corrected by oxygen inhalation and represents the true pulmonary shunt, which has been confirmed by pulmonary histopathology, angiography, transthoracic echocardiography and other examinations. it is now believed that pulmonary vascular casting is still the most direct evidence for determining the arterialvenous shunt. this intrapulmonary arterial -venous shunt is the main cause of abnormal ventilation/blood flow ratio and insufficient gas exchange. although pleural spider nevus can also cause arterial -venous shunt, it generally does not suffice to cause significant hypoxemia due to the small amount of shunt. in addition, studies in recent years also show that portal-pulmonary vein shunt in a small amount occurs in some patients with cirrhosis, in whom blood flow circumvents the alveolar gas exchange and enters directly the systemic circulation. this can also cause ventilation/blood flow abnormalities, causing insufficient gas exchange 3. airway closure: in 1971, ruff et al. proved that cirrhotic patients had significantly increased closed volume (cv) and total amount of closed gas (cc) and increased gases trapped in the lower fields of the lungs, resulting in an extremely low ratio of ventilation/blood flow, and they believed these were due to reduced airway ventilation [302, 313] . in 1984, furukawa et al. measured the lung function of 105 patients with liver cirrhosis and did not find abnormalities; however, most patients had flow -volume abnormalities and significantly increased cv, suggesting the closed the airway in advance and decreased ratio of ventilation/blood flow, which might important causes of hypoxemia. 4. decreased affinity of oxygen with hemoglobin: some reports showed that 15 patients with cirrhosis (mostly alcoholic cirrhosis) patients had mild systemic vascular or pulmonary vascular dilatation, normal pao 2 , mild hypocapnia, mild right shift of the oxygenated hemoglobin dissociation curve, normal amount of carbon monoxide diffusion [313] , and mild imbalance of the ventilation/pao 2 blood flow, indicating that the right shift of the oxygen dissociation curve due to decreased affinity of oxygen with hemoglobin in the patients. this was possibly caused by the increased concentration of 2,3-diphosphate glyceride in red blood cells, which, however, is not an important factor in the occurrence of hypoxemia [302] . in summary, hypoxemia can result from many factors, while none of the factors can completely explain the pathogenesis underlying the disease. since the basic pathological changes in patients with hps are intrapulmonary vascular dilatation and opening of arterial -venous communicating branches, together with recent findings, it is suggested that the diffusion disorder of the alveoli and pulmonary capillaries and ventilation/blood flow imbalance may coexist, and are the main cause of hypoxemia in this disease. other factors may aggravate hypoxia and are secondary factors. therefore, it is believed that the disease occurs as result of the above factors. the pathological features of hps are dilation of capillaries in the anterior aspect of the lungs and telangiectasia. autopsies show arterial-venous short circuit within the lungs, vasodilation and thickened pulmonary muscles [314] . at the same time, arterial hypoxemia is common in liver diseases, often attributable to a variety of factors (such as ascites, hepatic pleural effusion, and copd in patients with alcoholism); it shows unique pathophysiological characteristics under specific circumstances of hps. its prominent features are dilation of micro-arteries in the anterior aspect of pulmonary capillaries and true capillaries (the normal diameter of these vessels is 8 to 15 μm, which can reach 15-100 μm when patients rest), with the number of dilated vessels increased macroscopically. some patients show arterial-venous communicating between the pleura and lungs, vascular anastomosis in the liver and the lungs, and thickened walls of small veins and capillaries. pulmonary vascular dilatation is promoted, and mixed venous blood quickly or directly enter the pulmonary veins through the anastomosis in the lungs, leading to oxygenation defects. increased nitric oxide (no) is a key cause for pulmonary vasodilation, and whether other mediators, such as heme oxygenase-derived carbon monoxide, are causes of pulmonary vasodilatation are not yet confirmed. abnormal arterial oxygenation seriously affects the survival of patients, and is an important indicator that determines the timing and risk of liver transplantation as well as an important basis for the grading of severity of hps. causes of deaths associated with hps are often multifactorial and are associated with basic liver diseases, and there are few cases of respiratory failure due to severe hypoxemia. hps is a triad composed by intrapulmonary vascular dilatation and insufficient arterial oxygenation due to primary liver disease, and it is mainly clinically manifested as primary liver disease and pulmonary lesions. hps can occur in various liver diseases, mostly in chronic liver disease, especially cirrhosis caused by various causes, such as cryptogenic cirrhosis, alcoholic cirrhosis, liver cirrhosis, viral cirrhosis, postnecrotic cirrhosis and biliary liver cirrhosis, etc. the most common clinical manifestations include liver palms [314] , spider nevus, jaundice, ascites, hepatosplenomegaly, gastrointestinal bleeding and abnormal liver function, etc., while they are not significantly correlated with hps. some patients with clinically stable liver disease may also develop the clinical manifestation of progressive pulmonary insufficiency. since hps patients have no primary cardiopulmonary diseases, most (80-90%) patients gradually develop respiratory manifestations on the basis of various liver diseases, such as cyanosis, dyspnea, clubbing, orthodeoxidation and platypnea, etc. among them, progressive dyspnea is the most common lung symptoms of hps. binay et al. believed that cyanosis was the only reliable clinical sign, and that platypnea and orthostatic hypoxia are the most characteristic manifestations. pulmonary examinations generally show no obvious positive signs [315] . a small number of patients (about 16-20%) can present complaining dyspnea on exertion in the absence of clinical manifestations of a variety of liver diseases, to which attention should be paid clinically so as to prevent misdiagnosis. the domestic researchers gao zhi et al. reported that 2 patients presented to hospitals with cyanosis, palpitation after exercises and shortness of breath; meanwhile, they found that the patients had clinical manifestations of liver cirrhosis [308] (such as liver palms, spider nevus, hepatosplenomegaly and ascites), which were conducive to the diagnosis of this disease. if liver disease patients have other lung diseases (such as chronic bronchitis, emphysema and pneumonia, and pleural effusion, etc.), then significant respiratory symptoms may occur. therefore, differential diagnosis should be made. data show that the duration of initial dyspnea to the conformed diagnosis in patients with hps average 2-7 years; that is to say, about 18% of patients already have dyspnea at the time confirmed diagnosis. 1. orthodeoxidation: pao 2 is decreased by >10% when patients switch from the supine position to the standing position. 2. platypnea: when patients switch from the supine position to the standing position, they have palpation, chest tightness and shortness of breath, and when patients resume the supine position, the above symptoms are improved [313] . krowka reported that about 80-90% of patients with hps had the above two manifestations because vascular dilatation in the patients was mainly distributed in the middle and low lung fields. when patients switch from the supine position to the standing position, the blood flow in the middle and lower lobes of the lungs is increased under the action of gravity, aggravating hypoxemia [315] . although the two manifestations are not unique to hps, they suggest the significant abnormality in the patients' pulmonary vascular system. if patients with a variety of liver diseases present with the above two manifestations, further examinations are needed for confirmation. patients may present with liver palms, hepatosplenomegaly, spider nevus and ascites; patients show palpitation, chest tightness, shortness of breath when switching from the supine position to the standing position due to hypoxemia. schenk et al. defined the values of pao 2 for the diagnosis of hps, thinking that pao 2 < 70 mmhg suggested a high possibility of hps, and that for pao 2 < 65 mmhg, the diagnosis of hps could be made [306] . pulmonary function tests mainly showed significantly decreased vc, frc, mvv and fev1, but sometimes the total lung volume and fev1 were normal. chest x-ray, ct scan and transthoracic contrast echocardiography (ttce) hps patients are mostly normal on chest radiography or show diffuse small millet shadows predominantly in both lower lobes of the lungs, nodular shadows in both lower lung interstitium, dilated pulmonary arterial trunks, and thickened pulmonary markings, whereas these manifestations have no specific values to the diagnosis of this disease. ct scan shows certain diagnostic values in that it demonstrates distal vasodilation and even pleural blood vessels, and can suggest the presence of hps. arteriovenous communicating occurs in hps patients due to pulmonary vascular dilation, indicating that subclinical pulmonary vascular dilatation and abnormal gas exchanges occur in cirrhotic patients with normal pao 2 [306] . contrast echocardiography: when hps is suspected, transthoracic echocardiography can be used as a preliminary screening to determine whether the intrapulmonary vascular dilation occurs or not. the microbubble contrast material in the right atrium, after intravenous injection of dioxane isotonic saline, will develop images in the left atrium through the dilated vascular beds after 3-6 cardiac cycles, while the microbubbles cannot pass through the normal capillaries (normal capillaries are <8-15 μm in diameter). approximately 40% of patients with cirrhosis had positive changes on contrast echocardiography, while only a small proportion of patients are in line with the diagnosis of hps due to the influence of dilated blood vessels. if contrast echocardiography is positive for liver cirrhosis or portal hypertension patients with hypoxemia and cardiopulmonary diseases in them can be ruled out, then the diagnosis of hps is established. this means is used to confirm the diagnosis of intrapulmonary vasodilatation. the pulmonary vascular abnormalities in hps patients are as follows: (1) diffuse spider nevus images. patients of this type have severe hypoxemia and erectile hypoxia and respond well to inhalation of 100% oxygen; (2) cavernous or spotted arterial dilatation mainly seen in the basal aspect of the lungs. patients during this period respond poorly to 100% oxygen; and (3) intermittent local arterial malformation or communicating branches, isolated earthworm-like or bulk images. in addition to severe hypoxemia and erect hypoxia, patients of this this type respond extremely poorly to the inhalation of 100% oxygen. when hypoxemia is caused by hps and cardiopulmonary diseases concurrently, 99cm tc-maa scan can determine hypoxemia resulting from hps more likely. radiolabeled albumin 99cm tc, which is administered through intravenous injection, is about 20 pm in diameter. when pulmonary vascular shunt occurs, a proportion of the polymerized albumin passes through the lungs and enters the systemic circulation, the intake of albumin by other organs can be simultaneously determined by scintigraphy [308] . therefore, the amount of shunt can be calculated. a study showed that 99 cm tc-maa scan was positive for hps patients with pa02 < 60 mm hg, while the scan was negative for chronic obstructive pulmonary disease (copd) patients with the same degree of hypoxemia, a result indicative of the good specificity of this means. compared with contrast echocardiography, 99cm tc-maa scan, in spite of its low sensitivity, can be used for the diagnosis of hps in patients with copd. pathological examinations are the most reliable means for the diagnosis of hps, whose basic pathological change is pulmonary vasodilation manifested as diffuse anterior capillary dilation or discontinuous formation of arteriovenous branches [312] . in addition, pulmonary perfusion scan and right cardiac catheterization are also valuable for the diagnosis of hps to a certain extent. there is no unified standard for hps diagnosis to date. diagnosis should be based on clinical manifestations plus imaging evidence of pulmonary angiography. (c) the domestic scholars gao zhi et al. thought in 1998 that the diagnosis of this disease should be based on the following manifestations in patients, hepatosplenomegaly, ascites, liver palms, spider nevus, dyspnea on exertion, hypoxia while breathing in the supine and orthostatic positions, increased mesenchyma in the basal aspects of the lungs and vascular markings on chest radiography, patchy or nodular shadows, dilated basilar pulmonary vessels and increased pulmonary vascular branches on ct, severe hypoxemia or not on blood gas analysis, increase in alveolar -arterial oxygen gradient by ≥20 kpa (150 mmhg), and 82% diffusion disorder on pulmonary function tests [316] . in addition, shunt-related examinations should be performed, such as 99cm tc-maa scanning, contrast-enhanced two-dimensional echocardiography and pulmonary angiography, etc., while the last one does not show the same sensitivity as that of the former two because the small blood vessels in the lungs may not develop on angiography. most hps patients have a slow onset of the disease, are difficult to treat, and have a poor long-term prognosis, with a mortality of more than 40% after 3 years. therefore, early diagnosis of the disease and its differential diagnosis are vital to improving the prognosis of patients. first of all, the previous liver and lung diseases in the patients should be ruled out, such as chronic obstructive pulmonary emphysema, pulmonary infection, interstitial pneumonia and silicosis, etc. at the same time, cirrhosis with pulmonary hypertension, infections secondary to pleural effusion, interstitial pulmonary edema, atelectasis and hyperventilation syndrome, etc. need to be ruled out. hps should be differentiated mainly from the following diseases: 1. liver cirrhosis following pulmonary heart disease: this is mainly because pulmonary diseases result in cardiac insufficiency and thus increased pulmonary venous pressure. repeated or long-term existence of liver congestion can lead to central venous hypertrophy and lobular central connective tissue hyperplasia, and further progression of the lesion will lead to the formation of liver cirrhosis following pulmonary heart disease. patients with pulmonary cirrhosis often have a long history of chronic lung disease and signs of cardiac insufficiency, such as edema of lower extremities, palpitation, shortness of breath and other symptoms. this patient has no history of chronic lung disease or edema of lower extremities, making him inconsistent with liver cirrhosis following pulmonary heart disease. 2. left heart insufficiency: both hps and left heart insufficiency can cause severe dyspnea and hypoxemia. a history of liver disease or evidence of chronic liver damage and decreased po 2 can be found in patients with hps, especially such characteristics as orthostatic hypoxemia and intrapulmonary vascular dilatation. patients with left heart insufficiency have a history of heart disease, orthopnoea, pink frothy sputum and moist rales in the lungs, etc. this patient is inconsistent with such manifestations. 3. primary pulmonary hypertension: after inhalation of pure oxygen, hypoxemia in most of patients with hps will be significantly alleviated. the effects of oxygen are poor in patients with hypoxemia [317] , and hps is manifested as orthostatic hypoxemia. the characteristics of hemodynamics of patients with hps are hyperdynamics and normal or decreased pulmonary artery pressure and pulmonary vascular resistance, while those in hypoxemic patients are increased. 4. others: ductus arteriosus, eisenmenger syndrome and pulmonary embolism, etc., need to be differentiated from this disease, and comprehensive judgments should be provided based on other clinical data of the medical history. hps patients can also have the above-mentioned diseases, and careful and meticulous examinations are needed for differentiation. because hps is developed on the basis of original liver disease, the frequency of its occurrence and its severity are mostly associated with the liver cell function of patients, while there are also hps patients in whom chronic liver disease is relatively stable and liver functions are normal. besides, pleural effusion, ascites and infections secondary to pulmonary edema after liver function decompensation can aggravate patients' respiratory function injury. therefore, under the current circumstances in which there are no effective measures for hps, active and effective treatment of primary liver diseases is the basis for the treatment of hps. therapy of primary diseases, including correction of hypoproteinemia, elimination of pleural effusion, improvement of liver function and treatment of complications, etc., can improve tissue oxygenation and improve arterial oxygen saturation. on this basis, the following treatment can be given. oxygen therapy also helps the differential diagnosis of pulmonary shunt: if pao 2 is resumed after oxygen inhalation, then the diagnosis of intrapulmonary vascular dilatation (ipvd) can be made; for patients with partial improvement, pulmonary anatomical shunt and functional shunt may coexist; for patients in whom the oxygen therapy proves inefficacious, pulmonary arteriovenous fistula is a possible diagnosis. it is now believed that once the diagnosis is established, treatment should be given as soon as possible. in the early stage of correcting hypoxemia in patients with mild conditions, even in patients in whom the critical value of hypoxemia (pao 2 , 8-9 kpa (60-67.5 mmhg) is reached and who have ascites, the hemoglobin saturation may still be less than 85% when patients are in activities or even sleep. that is to say, nasal catheter oxygen inhalation at 2-3 l/min is needed so as to improve hypoxemia [318] . with the development of the disease, oxygen flow needs to be gradually increased, and intratrachea oxygen supply can be offered when necessary. during the late stage, patients can receive pressurized oxygen through a ventilator or a hyperbaric oxygen chamber. for patients whose conditions are severe, the efficacy of oxygen therapy alone is not obvious. 2. vasoactive drugs vasoactive drugs for the treatment of patients with hps are most studied; however, since its pathogenesis has not been clarified to date and primary liver disease is difficult to reverse, it is hard to define the clinical efficacy of these drugs. the commonly used drugs include: used aerosolized ephedrine hydrochloride for the treatment of 12 patients with hps, and the preliminary efficacy was significant. the mechanisms were that ephedrine could excite the pulmonary vascular α receptor, resulting in contracted bronchial mucosa and pulmonary capillaries and alleviated bronchial edema, so that the dilated blood vessels within the lungs were contracted and intrapulmonary shunt was reduced. meanwhile, the bronchial β2 receptors were excited and the bronchi were dilated so as to improve the ventilation/blood flow ratio and relieve hypoxia. further studies are merited. (f) others: there have been reports on sympathomimetic drugs (isoproterenol) and β-blockers (propranolol), etc. that improve the symptoms of hps. theoretically, vascular endothelin, estrogen suppressor (tamoxifen) and so on can relieve the spider nevus and pulmonary vascular dilatation in patients with liver cirrhosis and improve their respiratory symptoms, while further studies are needed. no is most studied currently, and there are reports indicating that no synthesis inhibitors can increase pulmonary vascular resistance. alexander et al. used no for the treatment of severe hypoxemia in patients after liver transplantation, and obtained good results. durand et al. also reported that an hps patient was cured by inhaling no, while its mechanisms and clinical efficacy needed to be further confirmed. 3. pulmonary embolism it is generally considered that pulmonary vascular dilatation can vanish after liver transplantation in hps patients who are normal on pulmonary angiography or who have cavernous vessels on imaging [310] ; for patients who show diffuse pulmonary vascular dilation features on pulmonary angiography, embolization is usually not adopted since patients' lesions are extensive and the efficacy is poor; for patients with isolated and severe pulmonary vascular dilation or arterial-venous communicating branches, local pulmonary embolism therapy can yield a satisfactory effect. 4. liver transplantation it is currently considered that liver transplantation is still a possible fundamental measure for the treatment for hps. in the past, it was believed that serious hypoxemia was an absolute contraindication against liver transplantation, while recent studies show that liver transplantation is preferred for patients who have good alveolar gas diffusion function, who can respond well to pure oxygen inhalation and who can undergone oxygenation safely during anesthesia. recent reports further prove that hypoxemia can be cured after liver transplantation [308] . through literature review and case reports, krowka et al. believed that progressive hypoxemia in hps could be used as an indication of liver transplantation. temporary hypoxemia following liver transplantation can be adjusted by using no and taking the head-down supine position and the alternate lateral decubitus position. and for hps patients who fail to respond to the inhalation of pure oxygen, who have direct pulmonary arterial communicating branches on pulmonary angiography and who have severe clinical hypoxia, liver transplantation cannot improve their hypoxic status, has limited efficacy, or even increases intraoperative and postoperative risks. therefore, liver transplantation should not be performed on them. tips was an effective method for the treatment of hps, and its effects of improving symptoms, enhancing oxygenation and reducing intrapulmonary shunt could last up to 4 months. riegler et al. performed tips on an hps patient with diffuse intravascular dilatation who was not suitable for vascular embolization, and the results showed significantly increased pao 2 and significantly improved hypoxemia. however, coley et al. also reported that a patient failed to respond to tips, and therefore, its exact effects remain to be studied. 6. other treatment options one patient with hps was once treated with garlic, and 18 months later, his oxygenation was significantly improved and his symptoms were relieved. there are also patients who receive plasma replacement therapy, which has limited effects on the oxygenation of patients with hps [308] . to sum up, no effective treatment options are currently available for hps. since the basic cause of hps is liver cell failure, the usual cause of patients' deaths is not lung failure, mostly complications such as gastrointestinal bleeding, renal failure, hepatic encephalopathy and sepsis. therefore, we consider that the therapy of primary liver disease is particularly important. oxygen inhalation alone can be given in the early stage of hypoxemia, or conservative treatment can be provided if additional drugs are effective. liver transplantation is the best solution whenever possible. it is generally accepted that liver transplantation is the most promising regimen with confirmed efficacy. if oxygen inhalation is less satisfactory and patients are diagnosed with local intrapulmonary vascular dilatation or arterial -venous fistula by such means as pulmonary angiography, pulmonary embolism should be carried out as soon as possible. for patients with additional obvious portal hypertension, tips treatment can also be given. the interval from chronic liver disease and cirrhosis in patients to the confirmed hps due to such respiratory symptoms as anoxic dyspnea is usually several years or even more than 10 years [average interval, (4.8 ± 2.5) years], and a small number of patients can develop such a disease acutely in the short term. besides, signs of chronic liver disease can be traced in patients complaining breathing difficulties. once hps is established, obvious hypoxemia has occurred already. it confers a poor prognosis, and most patients die within 2-3 years often due to other complications of liver disease [312, 315] . if patients' oxygenation is satisfactory and they have undergone liver transplantation, or with the improvement in liver function, their hypoxemia can be resolved or improved of its own volition with good prognosis. if patients' oxygenation deteriorates severely and they have a very poor prognosis, most of them will die in the short term. hps often progresses slowly. although it is not a direct cause of death in patients with cirrhosis, it can significantly aggravate the disease. therefore, cirrhotic patients, especially those with positive liver palms and spider nevus as well as patients with portal hypertension, should be careful of the possibility of hps. timely detection and symptomatic treatment (such as low flow oxygen inhalation) can improve the prognosis of patients. active and effective treatment of primary liver disease forms the basis for the prevention of this disease. education of common sense should be given to patients with liver diseases so as to avoid factors inducing hps in their life. for patients with liver disease, mild hps should be found as soon as possible and appropriate treatment should be given. jia-quan huang and dong xu lipopolysaccharide (lps) is a constituent of bacteria cell wall which plays an essential role in the pathogenesis of septic shock by generating endogenous mediators such as nitrous oxide, cytokines, superoxide anions, and lipid mediators. despite the recent advances in antibiotic treatment and hemodynamic monitoring, septic shock still remains a serious disorder that is associated with a high mortality rate, to more comprehensive definition of the mechanisms that underlie innate immunity against bacterial pathogens, lps has been extensively studied [319] . the pathophysiological consequences of bacterial sepsis are contributed by the dysregulation of these same mechanisms. before we can hope to design effective anti-sepsis therapies, greater insight into the nature of host interactions with lps is extremely essential. the gram-negative bacterial envelope is composed of two bacterial membranes, outer and inner membrane. the outer membrane consists of the following substances, like lipopolysaccharide (lps), several kinds of outer membrane proteins, lipid a and metal ions. for most gram-negative bacteria, lps is a major component in the outer monolayer of the outer membrane which works like a tight shield. the shield is composed by unique molecules, such as polysaccharide, or long chain of sugar, and lipid a. during the process to evoke the signaling events of lps, lipid a plays a pivotal role. the entire lipid component of lps molecule, however, is required for optimal activity [320] . the basic principles of lps bioactivity are nowadays well understood. endotoxins do not elicit their toxic effects -as we might suspect and as it is known for many proteinous exotoxins which can kill host cells or inhibit cellular functions. rather, lps requires the active response of host cells. according to present knowledge we get, lps interacts with various host cell types through lipid a, those cells include mononuclear cells, thrombocytes, endothelial and smooth muscle cells, and polymorphonuclear granulocytes, among which macrophages/monocytes are of particular importance. through the lps-induced activation, macrophages produce many substances, like bioactive lipids, reactive oxygen species, and in particular, cytokines such as tumor necrosis factor a (tnf), interleukin-1, il-6, il-8, and il-10. it appears that when low levels of mediators are produced, beneficial effects (e.g., induction of resistance to infection, adjuvant activity) are elicited and when high levels of mediators reach the circulation that detrimental effects (e.g., high fever, hypotension, irreversible shock) are induced. however, when the host organism is in a hyperreactive state lps, low mediator concentrations may also become harmful. the hyperreactivity to endotoxin may be caused by exo-toxins, chronic infection, and by growing tumors, and interferon-γ. to function properly, organism requires an immune system that must detect pathogens, from viruses to parasitic worms, and distinguish them from the organism's own healthy tissue. the immune system can be classified into humoral immunity versus cell-mediated immunity or the innate immune system versus the adaptive immune system. when microbes invade organism, the innate response is usually triggered by pattern recognition receptors, which recognize components that are conserved among broad groups of microorganisms, or when injured, damaged, or stressed cells send out alarm signals, many of which (but not all) are recognized by the same receptors as those that recognize pathogens. the innate immune system defenses are nonspecific, meaning that the system responds to pathogens in a generic way. this system does not confer long-lasting immunity against a pathogen. in most organisms, the innate immune system is the dominant system of host defense [321] . they activate innate immune responses by identifying some conserved non-self molecules, so as to protect the host from infection,. bacterial lipopolysaccharide (lps), an endotoxin which is found on the bacterial cell membrane, is considered to be the prototypical pamp. lps is specifically recognized by toll-like receptor (tlr) 4, a recognition receptor of the innate immune system. the interaction of the lipid a moiety of lps with macrophages appears to be especially important because subsequent cellular activation results in the release of systemically active pro-inflammatory molecules, which in turn mediate systemic toxicity. lps has extreme potential in macrophages activated at concentrations of lps as low as 1 pg/ml [322] . host-defense peptides (hdps) could be a possible alternative solution since they possess the antimicrobial, antiseptic, and immunomodulatory properties [323] . endotoxins lipopolysaccharide is released not only from dead gram-negative bacterial, but also from the growing ones. endotoxins are very stable molecules, which are resisted to extreme temperatures and ph values in comparison to proteins. endotoxins are shed largely during cell death as well as growth and division. they are highly heat-stable and are not destroyed under regular sterilizing conditions. endotoxin can be inactivated through exposed at temperature of 250 ° c for more than 30 min or 180 ° c for more than 3 h. acids or alkalis of at least 0.1 m strength can also be used to destroy endotoxin in laboratory scale [324] . gut microbiota is composed of strict anaerobes, facultative anaerobes and aerobes. recent reports suggest the existence of over 35,000 bacterial species in the human gut microbiota. an important characteristic of gut microbes is their heterogeneity [325] . the composition and the frequency of the microbiome changes with the different segments of the elementary tract. the composition is influenced by the environment, consumed diet and host factors. endotoxin to surrounding tissues and organs or blood shift that shift pathway include: (1) via the portal vein, liver into the systemic circulation; (2) through the intestinal tract into the lymphatic system lymphatic; (3) through the intestinal wall into the peritoneal cavity and then absorbed into the bloodstream. under physiological conditions, although a small amount of toxins continued to parenteral shifted via the portal vein into the liver, but it does not cause endotoxemia; mild in gram-negative bacilli infections, although bacteria continue to release to the tissue or blood endotoxin, but it does not give rise to a strong inflammatory response, the above are dependent on the presence of an effective mechanism within the body to remove toxins and detoxification. the liver is the main organ of clearance of endotoxin, and the spleen, also removes toxins. molecules in lps removed include cationic antimicrobial peptides (cationic antimicrobial peptides, cap), acyloxy acyl hydrolase (acyloxyacyl hydrolase, aoah) lipoprotein binding protein and anti-lps antibodies are important endotoxin clearance methods [326, 327] . liver blood endotoxin clearance, primarily through kupffer cells, hepatocytes internal toxin endocytosis achieved, but the specific metabolic process is not entirely clear. mediated by kupffer cells engulf toxins may be scavenger receptor, it may be a molecular weight of 119,000 and 83,000 protein; mediate phagocytosis liver toxin structure may be the lectin-like receptor (lectin-like receptor). endotoxin receptor hepatocyte sinusoidal plasma membrane on the surface: after one to one binding, is taken up within the liver cells endocytosis way to microtubule-dependent vesicular transport through the liver cells, transported to the liver cells bile duct surface to exocytosis into the bile duct, and then discharged into the biliary system through the intestine [328] . splenic macrophages containing approximately 15% of the body to settle within the organization, and macrophages are important endotoxin removal cells. when endotoxin intravenously into the body, except gathering in the liver, a lot of endotoxin can be quickly gathered and taken up into macrophages in the spleen, the spleen and therefore equally important endotoxin removal organs. in addition to its clear role in the performance of its direct effect, but more importantly, spleen macrophages is the precursor cells of kupffer cells in the liver, having a very big impact on removing toxins within the liver [329] . mechanisms for removing toxins in the intestine are related to the intestinal villus tip epithelial cells. under normal circumstances, injected into the intestinal, endotoxin in intestine does not enter intestinal epithelial cells, but after intravenous injection of endotoxin, endotoxin may enter intestinal epithelial cells inside. therefore, endotoxin receptor may identify ways by endotoxin and (or) simple diffusion way into the intestinal epithelial cells. endotoxin way into the intestinal epithelial cells intravenously may have two: (1) displaced from the lamina propria macrophages to intestinal epithelial cells basolateral; and enter intestinal epithelial cells from the side; (2) including lower toxin, intestinal lamina propria macrophages, intestinal epithelial cells release large amounts of no and oxygen free, resulting in intestinal lamina propria microvascular injury, increased permeability, extravasation of endotoxin and ultimately displaced into the outer intestinal epithelial cells. villus tip epithelial cells within a stronger uptake of toxins, thus endotoxin can be started from the crypt, moving along the intestinal villi, and finally to the top of the inner hair cells of the intestinal epithelium. uptake of endotoxin villus tip epithelial cell loss, while the endotoxin into the intestine, which constitutes one of the effective clearance mechanisms of endotoxin [327, 330] . plasma lipoproteins in endotoxin detoxification mechanisms play an important role, in which the lipopolysaccharide binding protein (lipopolysaccharide binding protein, lbp) and high-density lipoprotein (high density lipoprotein, hdl) play a major role. endotoxin into the bloodstream within minutes there were half white blood circulation due to binding to the edge of the pool or the pool is cleared, the remaining residual endotoxin and rapidly bound to plasma lipoprotein is inactivated. in plasma, lipoproteins and endotoxin when hdl plays a major role in its binding of endotoxin to reach more than 50% of the total, hdl, and thus research endotoxin important [331] . cationic antimicrobial peptides are an ancient ingredients in the natural evolution of the immune system, including bactericidal/permeability-increasing protein (bactericidal/permeability-increasing protein, bpi), cathelicidin, lactoferrin, defensins and other substances, with not only the activity against gram-negative bacteria, but also the ability to combine internal toxins. cationic antimicrobial peptides are mainly in regular contact with the pathogen site mammalian skin, digestive tract, respiratory tract, and inherently express or express induced by pathogens and their products in the blood, secretions and neutrophil granules. cationic antimicrobial peptides have two types of three-dimensional structure; one is a α-helix, having such a molecular structure include cathelicidin and lactoferrin; the other is β-fold, including mammals α and β-defensins, etc. [332] aoah aoah is a glycoprotein produced by white blood cells with weight of 5.2-60,000, the large subunit of 50,000 and small subunits of 1.4 million to 20,000, the large and small subunits connected by covalent disulfide bond. aoah as a lipase with hydrolysis for toxin, can selectively hydrolyze the secondary acyl chain on lipid a acyl groups acyloxy. when hydrolyzing endotoxins, both the large and small subunits of aoah play an important role, and both are indispensable. in addition to directly destroying toxin, the deacylated lps after the hydrolysis of endotoxin by aoah is also involved in aoah's detoxification mechanism on endotoxin; the material can accumulate and inhibit endotoxin-induced inflammatory response in the cell. however, due to a limited number of secretion by local infiltration of leukocytes, the internal detoxification of toxins is also limited [333] . when the body respond with endotoxin, one trigger inflammation, on the other hand can be cleared to produce or activate, specific polysaccharide inactivate toxins, including antimicrobial-specific polysaccharide antibody and anti-core polysaccharide antibody. after two antibodies binding with endotoxin, and then with the fc receptors on the cell membrane, inner source of the toxin-mediated, so that the endotoxin inactivated intracellularly. anti-endotoxin antibodies can interfere with toxin within lbp binding, thus preventing lbp endotoxin transport [334] . in the body's defense system, the shift from the inhibition of intestinal endotoxin ingredients include intestinal mucosal mechanical barrier, intestinal mucosal immune barrier, the normal intestinal flora and liver hepatocytes and kupffer cells, in which intestinal mucosal mechanical barrier, intestinal mucosal immune barrier, hepatocytes and kupffer cells play a direct inhibitory effect, while the normal flora plays an indirect inhibition. the intestine is huge "endotoxin library", a special anatomical location determines the intestinal mucosa must be an effective defense barrier. immunological barrier intestinal barrier formed by the epithelial cells of mechanical barrier and secretory iga (siga) and the like components [335] . intestinal epithelial cells and tight junction formation mucosal mechanical barrier, is a significant barrier in the intestine endotoxin translocation defense to maintain its integrity is it to play an important role in defense guarantee. in severe trauma, burns, infection, considerable loss of body fluids, hypovolemia, cause the body ischemia and hypoxia. in order to maintain blood pressure, to ensure that the blood supply to the heart brain and other vital organs, compensatory splanchnic vascular contraction, including gastrointestinal ischemia and hypoxia longer time than other organs, even after shock patients after resuscitation to restore normal hemodynamics, stomach intestinal still in a state of shock occult. therefore, when the intestinal microvascular perfusion recovery, intestinal ischemic/reperfusion injury, epithelial cells produce large amounts of reactive oxygen species and other media, resulting in intestinal epithelial cell apoptosis, destruction of tight junctions between cells, thus rapidly increasing intestinal permeability mechanical barrier function weakens, thus contributing to the intestine of the endotoxin absorbed through the intestinal wall to parenteral tissue displacement [336] . intestinal mucosal intestinal immunology barrier is a defense of the invasion of pathogens and endotoxin important line of defense, siga plays an important role in intestinal mucosal immunity. siga is an important component of the protection of the intestinal mucosa, both to prevent bacteria in the intestine mucosal surface colonization, but also in endotoxin. studies have found that e. coli o157 infection suffered intestinal mucosa, the anti-endotoxin core polysaccharide-specific siga secretion, in convalescent patients has been particularly evident, suggesting siga endotoxin to prevent the transfer of the body has a protective effect. in addition, studies suggest that nitric oxide (nitrogen monoxide, no) preventing endotoxin translocation in intestinal mucosal barrier oxide formed locally. under physiological conditions, nitric oxide synthase (inducible nitric synthase, inos) expression only in the respiratory epithelium, the pregnant uterus and ileal mucosa and a few other parts. induced by endotoxin including but under normal colonic epithelial cells also express inos and catalytic synthesis of no, are formed in the local oxidation barrier to prevent bacterial translocation colon, thus effectively preventing bacterial translocation, also indirectly prevents endotoxin shift [337] . under physiological conditions, intestinal flora forms a relatively balanced microecosystem. flora distribution in the intestine has certain rules: deep close to the intestinal mucosal surface, parasitic anaerobic bifidobacteria and lactobacilli, these anaerobic bacteria are sugar coated, relatively stable, known as membrane flora; middle class bacteria, streptococcus digest, veillonella and excellent bacilli; the surface of e. coli and enterococci, can swim in the intestine, known as cavity flora. the antagonism between the layers flora, mutual cooperation, to maintain a dynamic equilibrium, in which the film anaerobic flora is a very important body's natural defense barrier that can prevent opportunistic pathogens such as e. coli colonization in the mucosa, but also can inhibit the overgrowth of opportunistic pathogens. intestinal flora micro-ecosystem is a very sensitive system, in severe stress or long-term systemic administration of large doses of broad-spectrum antibiotics, etc., the film significantly reduced the number of anaerobic bacteria, e. coli and other bacteria thrive conditions and continuous release of endotoxin to the intestine, since the film flora defense decreased, these opportunistic pathogens to colonize the intestinal mucosa, resulting in intestinal mucosal barrier damage, followed by the occurrence of intestinal bacteria, endotoxin translocation [338] . under normal circumstances, the liver is one of the major barriers preventing endotoxin translocation, via the portal vein into the liver hepatocytes and a small amount of the toxin can be kupffer cell depletion. in conditions such as stress, not only liver cell dysfunction, so the ability to reduce endotoxin detoxification and collaterals between the portal vein and the vena cava, causing an overflow of endotoxin from the liver into the systemic circulation. endotoxin absorbed into the bloodstream, which in turn may increase the intestinal epithelial cells of the intestinal microvascular endothelial cell damage and, in a vicious cycle [339] . when the body's defense system to produce responses in endotoxin, the innate immune system plays a major role. pathogens can be identified conserved receptors called pattern recognition molecules (pathogen-associated molecular patterns, pamps), including endotoxins of gram-negative bacilli, peptidoglycan grampositive cocci, lta and other cell wall composition and gram-negative bacteria, gram-positive bacteria such as dna. although a variety of pattern recognition molecules of different chemical structures, but they have similar characteristics: (1) characteristic structure in which different types of pathogens in a relatively constant conserved; produced by a pathogen, the host body without these molecules; survival or disease-causing pathogen is generally the essential, such as mutations, death or loss of a pathogen will pathogenicity. natural immune system to recognize the receptor molecule called pattern recognition receptors (pattern-recognition receptors, prrs), including cd14, toll-like receptor family (toll-like receptor, tlrs) and scavenger receptors. but in recognition of toxins, some differences exist between the different kinds of cells [340] . macrophages in addition to expressing cd14, tlrs and scavenger receptors and other associated endotoxin receptors on the cell surface, but in the cytoplasm also express the protein molecules nod1 recognizing toxins. cd14, tlrs are key receptors that mediate endotoxin within macrophage activation; and scavenger receptor has relationship with macrophage clearing and inactivating toxins [341] . kupffer cell is the main cell that clears the endotoxin in the liver. under physiological conditions, although there is still a small amount of bacteria and endotoxin via the portal vein into the liver, but kupffer cells will clear. kupffer cells are the most resident macrophages in the liver and are the largest number of resident cells in tissues. there is a theoretical speculation that if kupffer cells and on is very sensitive to endotoxin as other macrophages, the cell will be in constant activation, but in fact when kupffer cell engulfs, removes endotoxin, its itself is not activated by endotoxin, which suggests that in the treatment of endotoxin, kupffer cells have different mechanisms with other macrophages: kupffer cells treats endotoxin mainly depending on its phagocytosis. in the absence of serum, the phagocytic effects of kupffer cell on endotoxin can play a normal; and with the appropriate increase in endotoxin concentration, the phagocytic activity of kupffer cell was enhanced. the effect has something to do with phosphorylation events of two protein tyrosine residue individually weighting 11.8 million and 8.3 million. cd14 is the main receptor that mediates endotoxin activating kupffer cell, scavenger receptor is kupffer cells' important defense of receptor mediated kupffer cell to remove and inactivate endotoxin. there are four stages of the activation of kupffer cells, of which cd14 is the characteristic marker of cellular activation and function change. (1) the stationary phase; the performance of less number of kupffer cells, small shape, a number in the hepatic sinusoids, cd14 staining negative; (2) reaction period: for the local kupffer cells stimulate hyperplasia and systemic mononuclear macrophage intrahepatic accumulation; (3) pre excitation period: kupffer cell phenotype occurred transformation period, expressed cd14 cell membrane receptor, kupffer cell functional changes; (4) the activation period: the performance of nuclear transcription factor nf kb activation, cellular secretion cytokines [342] . the cd14 (cd14 membrane-bound, mcd14) and tlr4 endotoxin were activated by the neutrophil surface to activate the neutrophils by binding with the receptor. in addition to the expression of the high affinity endotoxin receptor cd14, the surface of the neutrophils also expressed the low affinity endotoxin receptor l-and the activated cells were activated by the receptor. in addition, the integrin is considered to be a low affinity endotoxin receptor for the surface of the neutrophils [343] . it is generally believed that the expression of mcd14 was not on the surface of endothelial cells and serum soluble cd14 (soluble cd14 (scd14) is mediated endothelial cell recognition of lps molecules, and tlr4 is involved in lps induced endothelial cell activation. lbp was transported to scd14 by endotoxin, and tlr4 was activated by lps/scd14 and activated endothelial cells in the endothelial cell membrane. scd14 in addition to mediated endothelial cell activation and also mediated by endothelial removal of endotoxin and lps/scd14/lbp form trimers and binding to endothelial cells, following the lps/scd14 endogenise, thus the removal of endotoxin [344] . the epithelial cells of the intestinal mucosa were consistently associated with the bacterial and its products, and the bacteria and its products could stimulate other types of cells and induce inflammatory response, but did not induce intestinal epithelial cell defense, this feature for colonic epithelial cells is particularly important, because if can react to the normal intestinal flora in intestinal epithelial cells, it will cause adverse effects on the body. but this does not mean that intestinal epithelial cells are immune cells, when suffered pathogens and their products invasion, intestinal epithelial cells produce normal response. description: intestinal epithelial cells with normal differentiation of natural bacteria and pathogenic ability, and the recognition system of subcellular localization. there are different endotoxin recognition mechanisms in the intestinal epithelium and the myeloid cells [327] . uncontrolled inflammatory responses (uncontrolled inflammatory response) has a relationship with infection, bacteremia, septicemia, sepsis, systemic inflammatory response comprehensive sign (systemic inflammatory response syndrome (sirs), compensatory anti-inflammatory response syndrome (compensatory antiinflammatory response syndrome, cars) and other related terms used for a long time, but also has an essential difference. out of control including inflammatory reaction syndrome (msas anti-inflammatory response syndrome, mars), a dynamic process of sirs and cars and the mixed antagonistic response syndrome, at present clinical many diseases occurrence and development are closely related. uncontrolled inflammation is a common pathological phenomenon in clinic, which is the important mechanism of the development of the complication after trauma. lps is one of the main factors that induce the uncontrolled inflammatory reaction in the most common. lps receptor on the monocyte/macrophage surface is the he initial factor for the body to recognize and start inflammatory reaction, also is one of the key links for the induction of uncontrolled inflammatory response. the concept of uncontrolled inflammatory response refers to inflammatory disorders then resulting in multiple organ dysfunction syndrome (multiple organ dysfunction syndrome. mods), emphasizes the importance of the of balance inflammatory/anti-inflammatory mechanism in the body, changes the limitations that in the past we only attached importance of the pathogenic effects of inflammatory factors. it is believed that the response of the body to the inflammatory factor is the dominant factor in the development of the whole body inflammatory reaction and mods. this concept is more focused than the previous focus on the dynamic changes of the whole process of inflammation. this can be divided into two types of mods: one is the early stage after the injury, that is, the speed hair style. the main blame is a strong inflammatory reaction induced by proinflammatory factors, and the other is a late phase of the disease, which is "late style", mainly due to the immune paralysis or worse immune disorders caused by cars or mars. the inflammatory is actually a kind of medium disease mainly caused by the chain reaction of cytokine. endotoxin is thought to be one of the most important predisposing factors in the chain reaction and can be referred for chain reaction "trigger" (trigger). endotoxin induced inflammation mechanism is mainly mediated by pamps that can induce cytokines such as il-1, tnf alpha and other active molecules synthesis, the formation of the cytokine network, has a very important role in the occurrence and development of infection. the excessive activation of cytokines can cause septic shock, and is the leading cause of death in patients with bacterial infections. accordingly, prrs plays an important role in innate immunity and inflammation, and it can distinguish the pathogens from self organization through prrs organism, which is characteristic of immune response [345] . inflammatory response syndrome systemic (sirs) is a systemic inflammatory response caused by any pathogenic factor to the body. the concept is first proposed by coris in 1985. 1991 august american college of chest physicians and critical care medicine to present the diagnostic standard of sirs, think to have the following each of the two or more than two, sirs can be established: (1) temperature > 38 deg c or < 36 deg c; ii heart rate 90 beats/min; (3), the breathing frequency > 20 times per minute or arterial blood carbon dioxide into pressure (paco 2 ) < 4.27kpa 32 mmhg; (4) peripheral white blood cell count >12 × 109/l or 4 × 109/l < or immature myeloid cells >10%. what should be paid attention to is that sirs is a common athophysiological state of body with severe inflammatory reactions, and should be differentiated from some abnormal factors such as leukemia or cause increase or reduction of white cells after chemotherapy. although the naming of sirs has been generally concerned, but some scholars have raised objection to the concept, for example sirs has following problems: the sensitivity and specificity of the diagnostic criteria is poor, has the same meaning with the "critical"; can not understand the pathophysiology of the original disease; is difficult to guide clinical trials and practice. the production of sirs can be divided into two cases, the sirs caused by the infection and the non infectious sirs. from the point of view of the clinical development process, sirs can be followed by injury immediately aroused, then known as the "single phase velocity hairstyle; also to start local, and later developed into a systemic sirs, namely after the initial shock is brief period of stability, later gradually intensified when sirs is called" dual phase delayed onset. either of the factors or the clinical development process, the systemic inflammation of the control of the uncontrolled, and ultimately can lead to mods [346] . the intestine is the biggest bacterial and endotoxin warehouse in the body. in severe trauma, systemic infection, intestinal ischemia and liver disease, there may be the occurrence of endotoxin. the main source is due to intestinal gram-negative bacteria in the excessive growth and reproduction, or due to increased intestinal permeability lps entry into the portal vein increased. if hepatic kupffer cell phagocytic function is low, the amount of endotoxin over the liver ability to remove endotoxin can "flood" (spill over) into the body of the loop and the endotoxemia formated. because of the endotoxin from the gut, so it is called intestinal endotoxemia (intestinal endotoxemia, ietm) [347] . hepatitis b patients are often accompanied with ietm. its formation mechanism is: the production and absorption of intestinal endotoxin increased. there is a large number of gram negative bacteria in the body's normal intestinal, so endotoxin in intestinal contents is very high, but the intestinal mucosal epithelial cells have stronger resistance to toxins so that endotoxin is not easy to run through the intestinal mucosa into the blood, even a small amount of endotoxin breaking through the intestinal mucosal barrier into the portal vein, will be swallowed up by the kupffer cells in the liver. severe hepatitis b when the intestinal flora disturbance, endotoxin increased, increased intestinal hyperemia, edema and the permeability of the intestinal mucosa; endotoxin itself can damage the mitochondria and lysosome of intestinal epithelial cells, leading to epithelial cell autolysis; endotoxin can cause intestinal microvascular contraction of the intestinal mucosa, reduce blood, intestinal ischemia, hypoxia, cause the intestinal mucosal barrier function decreased, increased the absorption of endotoxin; severe hepatitis, due to intrahepatic bile acid and bilirubin deposition in kupffer cell phagocytosis was inhibited, resulting in the removal of endotoxin in the endotoxin decreased; through the door body circulation circuit into the systemic circulation, resulting in blood within liver cells to escape kupffer toxin the phagocytosis and clearance, which aggravate endotoxemia; the endotoxin can also pass through the celiac lymphatic system into systemic circulation by thoracic duct. in addition, severe hepatitis patients with sepsis, spontaneous bacterial peritonitis, etc., in the release of endotoxin, so the formation of the endotoxin is exogenous [348] . in viral hepatitis and other basic diseases complicated with ietm and liver function failure are closely related and endotoxin can directly cause arterial vasoconstriction, the organ ischemia; endotoxin can activating endogenous clotting system, coupled with kupffer cell dysfunction, decrease delimination of blood coagulation or fiber soluble substances, easily lead to dic, so as to damage to multiple organs. endotoxin activated phospholipase a2 mediated membrane phospholipid degradation and lipid peroxidation, which is an important part of liver cell damage. nolan has pointed out that the effect of kupffer cell dysfunction induced by intestinal endotoxemia on liver and body, far more than the direct action the endotoxin, and production and release of inflammatory mediators and factors from kupffer cells activated by endotoxin are closely related. the occurrence of ietm affect hepatic energy metabolism, resulting in liver cell damage and necrosis, also caused hepatic microcirculatory disturbance, performing liver hemorrhagic necrosis. on the basis of severe hepatitis, it can accelerate liver function failure [349] . hepatic cellular jaundice the acute and chronic liver function is often accompanied by intrahepatic cholestasis jaundice, and ietm plays an important role in the occurrence of intrahepatic cholestasis.. endotoxin involves in liver cell damage mainly through activation of phospholipase a2, and inhibits the activity of na + − k + − atpase on liver canalicular membrane to make the bile excretion disorder, and then to cause intrahepatic cholestasis in the liver cells. endotoxin can start the peroxidation of liver parenchymal cells mitochondrial membrane lipid so that an increase in the content of oxygen free radical in blood, resulting in the disorder of energy generation, atp was reduced, so that the active uptake, metabolism and secretion of bile acid by liver parenchymal are short of energy, resulting in cholestasis [350] . the liver disease with ietm often causes the coagulation dysfunction, the serious person appears the different degree bleeding, in particular the severe liver disease patient may concurrent dic, endangers the life. violi found that, when liver dysfunction in patients with ietm, the expression of tissue factors on the surface of macrophages and endothelial cell factor induced by endotoxin increased, promoting the synthesis of tumor necrosis factor (tumor necrosis, factor, tnf) to increase, and thrombin generation increasing, activation of coagulation system in about 70% of patients, followed by hyperfibrinolysis, suggesting that ietm in liver dysfunction can be used as the warning signal as the activation of coagulation and fibrinolysis system and activate, and with the increased hepatic lesions, plasminogen activation decreases with endotoxin levels increased, thus plasminogen may decline with endotoxin induced by chronic consumption of dic on the microstructure of ii related factors, new blood can not correct. in fact, before variceal rupture bleeding, patients with liver function severely damaged already have the gastrointestinal mucosa extensive ischemia and erosion, which is the potential causes of bleeding, ietm in the process. and gastric h + at this time can occur abnormal reverse diffusion and stimulate mast cells to release histamine, may lead to mucosal blood vessel dilation and permeability enhanced. as a result, hemorrhage, edema; histamine and directly stimulate the secretion of gastric acid, so that an increase in the number of h + and reverse diffusion, lesions persisted, form a vicious circle [351] . it is important for the formation of ascites in ascites due to the obstruction of the hepatic vascular outflow tract obstruction. the initial vascular response to endotoxin was the rapid obstruction of the hepatic venous outflow tract and increased the portal pressure which may be related to endotoxin induced swelling of kupffer cells, liver cells with microvilli swelling, platelet aggregation and fibrin deposition effect, while others think that is endotoxin of blood vessels of the liver has a direct effect. ietm continuous damage to the liver cells, resulting in albumin synthesis and of hormones such as aldosterone de live function obstacles, thus affecting the renal function, and led to the emergence of the refractory ascites plays an important role in the process [352] . patients with severe liver disease always are complicated with the functional renal failure, hepatorenal syndrome (hrs). the patients with severe liver disease can be associated with the pre-renal azotemia and acute renal tubular necrosis, and there is a certain relationship with ietm. the clinical studies showed that the levels of no 3 --no 2 and endotoxin in serum of patients with liver cirrhosis were significantly higher. at the same time, plasma renin activity, aldosterone and vasopressin levels increased and urinary sodium excretion decreased. the mechanism about ietm induced by hrs is not clear, may be related to the following factors: leukotrienes (leukotrienes, lts) lts can lead to renal vasoconstriction, increased renal vascular resistance, reduced renal blood flow and renal blood redistribution, decreased glomerular filtration rate induced by hrs, in ietm lts generation and release increased obviously. in addition, liver dysfunction, liver's uptake, inactivation and excretory function of lts decline, causing blood concentration increased; the thromboxane a2 (thromboxane a2 txa2)/i2 (prostaglandin i2, prostaglandin pgi2) can contract renal arterioles, decrease glomerular filtration rate, while pgi2 and pge2 (prostaglandin e2, pg e2) is caused by on the role of txa2. in patients with severe hepatitis with ietm, elevated systemic levels of pgi2, reducing the renal vascular resistance, leading to renal vascular resistance txa2, reduce the renal blood flow and glomerular filtration rate, promote the formation of hrs; third, nitric oxide, nitrogen monoxide) nono can through vasodilatation of the systemic, resulting in effective circulating blood volume reduction and evoked hrs; endothelial endothelin (et) et caused renal cortical blood priming hrs; and platelet activating factor (growth factor (paf) endotoxin and platelet activating factor (paf) can lead to a decrease in cirrhotic rats cardiac ejection fraction, reducing blood flow to the kidney, and paf antagonist can improve hemodynamics changes [353] . the mechanism of endotoxin induced hepatic encephalopathy is not clear. we have known that lps can increase the permeability of blood brain barrier, promote intestinal toxic substances through the blood brain barrier (bbb), damage mitochondrial oxidative metabolism in brain cells, reduce oxygen utilization in patients with liver cirrhosis and disrupt energy metabolism of brain cells, induce brain edema. the clinical symptoms of chronic severe hepatitis with endotoxemia included in addition to fatigue, anorexia, tiresome of the oil, nausea, vomiting, yellow skin and sclera and, performance of endotoxemia. endotoxin can cause the release of histamine, 5-hydroxytryptamine (5-ht), prostaglandin, bradykinin, resulting in micro circulation expansion, venous blood volume reduction, decreased blood pressure, inadequate tissue perfusion, hypoxia and acidosis, and main symptoms and signs are: fever, elevated white blood cell count, bleeding tendency, heart failure, renal dysfunction, hepatic injury, nervous system symptoms and shock [354] . improve liver function this is the basic treatment of ietm. liver function improved can strengthen mononuclear phagocyte system function to help the endotoxin removal. it can also decrease portal vein pressure to relieve intestinal congestion, edema, hypoxia, improve the intestinal microenvironment, reduce production and lymph reflux, and lower door shunt. these all contribute to the prevention and treatment of ietm. clean the gut saline is available as enema if severe liver disease, which helps reduce intestinal endotoxin generation and absorption. decompensated cirrhosis is often accompanied by small intestinal bacterial overgrowth and intestinal flora disturbance. thus, the promotion of intestinal flora back to normal state help prevent and treat intestinal endotoxemia. a variety of bifidobacterium, lactobacillus can be selected. a synthetic disaccharide, it is not digested and absorbed in the small intestine, but can be broken down into lactic acid, acetic acid and other small molecules by the bacteria into the colon. such acidification of the intestine reduces the generation and absorption of endotoxin, and promotes the growth of intestinal bacteria, stimulates bowel movements so as to increases tool frequency and so on. in addition, the lactulose may have internal direct inactivation of toxins, prevents activation of macrophages to release cytokines. oral absorption of antibiotics can effectively suppress the generation of intestinal endotoxemia. patients with liver cirrhosis taking oral polymyxin e or neomycin, the level of plasma lps and no 2 -/no 3 -horizontal declines in synchronization. polymyxin b has an internal direct antitoxin effect [355] . it play a role by reducing intestinal absorption of toxins, inactivating toxins and inhibiting those lps-induced media by monocyte macrophages. the new anti-endotoxin therapy including interrupt endotoxin synthesis, binding or neutralizing its activity, preventing its interaction with the host effector cells, or interfering with toxin-mediated signal transduction pathways. therapeutic formulations include endotoxin analogs, antibodies, subunit vaccines, polymyxin combination column, recombinant human protein, small molecule inhibitors of endotoxin synthesis and intracellular signal transduction. bacteriophage producing a piece of short nucleotide sequence which plays the role of an antisense rna, blocking the synthesis of bacterial lps synthase. current clinical studies carried out an experiments in cloning of human anti-endotoxin lipid a light and heavy chain variable region. it laid the foundation for the next screening and expression that recombination between dna of antibodies and phage's succeeded. the clone45 is one kind of anti-polymyxin b (polymyxin b) monoclonal antibody of the igm class. it can play the role of anti-endotoxin shock by imitating the surface antigen structure of lipid a so as to substitute receptor antagonist of lipid a and lps blocking the causative link that the endotoxin induces inflammatory mediators [356] . isolating antibodies having a high affinity of various g-bacteria to prepare a chimeric monoclonal igg1 antibody sdz219-800 which have a therapeutic effect on the human endotoxemia. anti-endotoxin core glycolipid monoclonal antibody (antimonoclonal antibody r595) can prevent and treat the metabolic disorders in peritoneal infection with mods; it plays a significant role in conditioning in high catabolism, and can significantly improve metabolic disorders under the condition of abdominal infection associated with mods. bactericidal/permeability-increasing protein (bpi) bpi is a human endogenous protein, found primarily in neutrophils primary particles. its molecular amino-terminal and carboxy-terminal appear v-shaped structure planar symmetry. many amino acids to form a hydrophobic capsule hold lps's lipid a. it was reported that bpi has an obvious protective effect on intra-abdominal infection induced sepsis, which might be related to its antagonism against endotoxin [357] . reconstructing hdl (high density lipoprotein, hdl) hdl can be used as an endogenous lps scavenging system, binding of bacterial endotoxin with high affinity to form a stable hdl-lps. lps-hdl complexation may contribute to a reduction in endotoxic activities in vivo by preventing lps (lipid a) from generating important transmembrane signals after binding to cells [358] . e5531 is the first generation lipid a analogue, which is derived from the lipid a structure from the endotoxin of rhodobacter capsulatus. it can block lps in cell culture without any endotoxin-like activity. e5531 can protect mice from lethal doses of lps, and viable e. coli infections in combination with antibiotics. in human healthy volunteers who are exposed to intravenous lps. e5531 also blocks the endotoxin response [359] . an anti-cd14 antibody cd14 has a very important role in monocyte-macrophage cell signaling. since epitopes of lps on the cell membrane at the binding site is the same material with soluble cd14, so we can develop a monoclonal antibody interfering with lps binding to cd14 and blocking to pass activation signals from immune effector cell [360] . tyrosine kinase and mitogen-activated protein kinase are involved in lps cellular signal transduction. build anti-endotoxin (lps) single-chain antibody gene and attempt to make it express in e, coli. the scfv gene was successfully constructed and gst scfv fusion protein highly expressed in e. coli was obtained. glucocorticoids, including the synthetic glucocorticoid dexamethasone, are recognized for their anti-inflammatory properties and have the ability to inhibit the production of proinflammatory cytokines such as tnf-α. in intestinal ischemia-reperfusion methylprednisolone pretreatment can prevent endotoxemia. combined lps and dexamethasone treatment at 120 h significantly changed tnf-α [361] . this points that glucocorticosteroids added before or during stimulation of macrophages can prevent tnf release, after which the administration would be invalid. in fact, it is very difficult to use corticosteroids before tnf release. current clinical anti -tnf antibodies and tnf antagonist use exists, and before many scholars have obtained a more satisfactory results of blocking or neutralizing excessive tnf with anti-tnf. although the clinical symptoms improved, yet the survival rate is not higher than expected. the effect of anti--tnf clinical application needs further evaluation. the first proposed concept is sequential organ failure, based on which multiple organ failure (multiple system organ failure, msof or mof) is put forward, and in 1980 diagnostic criteria is developed, but it reflected the end-stage, denied reversibility, ignore the dynamic development from organ dysfunction to failure. therefore, the us accp/sccm proposed to replace the concept with mods. mods emphasized an early phase of organ dysfunction before overt failure occurred. it is defined as "the presence of altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention." [362] organ dysfunction may be relative, or can be absolute; with extension of time, mods can increase or reverse. thus, the term. mods was coined to indicate the wide range of severity and the dynamic nature of this disorder, which contributes to early diagnosis and treatment of patients and is more in line with clinical practice. there are two relatively distinct (although not mutually exclusive) pathways by which mods can develop: in primary mods, there is a direct insult to the organ that becomes dysfunctional. examples of such direct insults include gastric aspiration in the lungs or rhabdomyolysis in the kidney. this direct insult causes: an inflammatory response that is localized, at least in the beginning, to the affected organ. secondary mods is a consequence of trauma or infection in one part of the system that results in the systemic inflammatory response and dysfunction of organs elsewhere [363] . secondary mods means not directly caused by damage, but to experience the "second strike", the first blow can make the immune system in a preactivated state under which inflammation lost control and significant sirs appears, then the following second strike can quickly cause multiple distant organ dysfunction and easily form sepsis with the basis of sirs/cars. this type of mods often develops as the following model: the original cause → stress → immune wpw → sirs/cars → infection → sepsis → mods → mof. the process of severe hepatitis, can cause intestinal damage and massive release of endotoxins into the blood, leading to intestinal endotoxemia (ietm). endotoxin is a powerful trigger for complement activation, and then these complement can activate "cascade effect" (cascade) to release oxygen free radicals, prostaglandins, endorphins, paf, cytokines and other inflammatory mediators to cause cytotoxicity, the error of microcirculation and tissue metabolism, eventually leading to the occurrence of mods. in this case, we emphasize mods originated in continuous, uncontrolled inflammation and factors causing systemic inflammatory response can be induced mods, including bacteria, fungi, parasites, viruses, toxins and other infectious agents. it is a debate of long-century problems that whether endotoxin has effects on hearts. the late 1970s a large number of experiments prove endotoxins has a role of heart. it enables reduction of coronary blood flow, decrease of total coronary vascular resistance, and have meanings in heart failure. studies have shown that endotoxin can damage myocardial mitochondria, muscle paddle net, muscle membrane, contractile proteins etc., leading to cell membrane system damage, energy metabolism. past research in cardiac dysfunction under endotoxin shock did not attract enough attention, for they think that the heart is the final failure among all organs so that clinical treatment value is little. now people's awareness has completely changed that heart dysfunction can occur early in sepsis or septic shock. therefore, early identification and prevention of the occurrence and development of cardiac dysfunction may have some clinical value for treatment of septic shock and other serious complications [364] . clinical gram-negative bacteria sepsis often complicated by adult respiratory distress syndrome, or server development of mods. in this pathological process, the high biological activity of endotoxin plays an important role. endotoxins often involving the lungs firstly, and the pathogenesis of non-injury may be associated with the direct damage on endothelial cell by endotoxin through complement pathway and induction of cytokines [365] . the liver is the major site of endotoxin on clearing and detoxifying and the place where clinical gram-negative bacteria sepsis often can be complicated, and it is also the primary organ suffering attacks by toxins. it is generally believed that toxins in the liver circulating mainly from the gut and liver dysfunction is closely related to the formation of endotoxemia. histological examination revealed the inner toxins damage the liver cells, showing sinusoidal congestion, dieldrin expansion chamber, kupffer cell swelling, endoplasmic reticulum、mitochondria swelling, crest destroy and lysosomal activation etc. [366] . the liver is the body's largest metabolic organ, and many cases of acute liver failure often involve other organ complications, which has become an important factor in determining the prognosis. endotoxins may cause the reduction of liver nutritional blood flow, mitochondrial oxygen metabolism, interfering with sugar metabolic pathways in liver leading to metabolic disorders. various damaging factors (such as gastrointestinal disorders, ischemia, immunocompromised, dysbiosis) promote absorb of intestinal bacteria and toxins and displacement via the portal vein, the lymphatic system into the systemic circulation. on the one hand these infectious agents can directly damage liver cells, or mediate hepatic injury whether by kupffer cell; on the other hand it can induce systemic inflammatory response by the monocyte-macrophage cells to release the media, both leading to organ perfusion disorder, affecting protein synthesis and energy metabolism, eventually resulting in severe sepsis, mods and even death. the effect of endotoxin on kidneys is not clear yet. in the early stage, it can affect the kidneys, decreased its blood flow renal via vasoconstriction. when endotoxemia is complicated by renal failure, the mechanism of glomerular filtration rate decreasing is unclear. the pathophysiology of aki in sepsis is complex and multi-factorial and includes intrarenal hemodynamic changes, endothelial dysfunction, infiltration of inflammatory cells in the renal parenchyma, intraglomerular thrombosis, and obstruction of tubules with necrotic cells and debris [367] . the relationship between endotoxin and dic is quite complicated. dic is considered to be an important incentive of mods, especially patients with severe sepsis with dic having a highly possibility of developing mods, what's more, the prognosis is very poor, and the mechanism is multifaceted. endotoxin can start the endogenous coagulation system directly or via activating factor xii (hageman factor) by damaged endothelial cells, also can act on the monocyte-macrophage cells, stimulate the release of tissue factor to trigger the extrinsic coagulation pathway [368] . in clinical practice it has been noted that serious infections is very possible to be complicated by gastrointestinal failure. the intestine is the biggest reservoir of bacteria in the body and leakage of bacteria or microbial products, notably lps, from the lumen of the gut into the systemic compartment, leads to initiation or amplification of a deleterious inflammatory response and mods [369] . after endotoxins challenging the gastrointestinal mucosa, it initially shows mucosal telangiectasia, interstitial edema and hemorrhage. microcirculation leading to damage of lysosomal and release of proteases in the cell, cell degeneration and necrosis. in addition, mucosal cellular energy metabolism decrease, h + reverse diffusion, prostaglandins and bradykinin further aggravate mucosal damage. at the same time destroy of the gastric mucosal barrier also make a lot of bacteria and endotoxins pass through the gastrointestinal mucosa, migrate to the blood circulation, the lymphatic system and the abdominal cavity etc., leading to systemic multi-system organ damage. clinical characteristics of mods 1. organ failure usually do not result directly from the primary injury. there is a certain time interval from the primary injury to organ failure. 2. not all of infection have bacteriological evidence, and more than 30% of patients and autopsy found no infected lesions. thus, to identify and treat the infection may not be able to improve the patient's survival. 3. mods may have perfectly healthy organ involved, and it is ferocious and rapidly progresses. once happened, it is difficult to depress in the event almost, so often with a high mortality rate. 4. in pathology, mods lacks features, the affected organ only showing acute inflammation, such as inflammatory cell infiltration and so on, and these changes are very inconsistent with severe clinical manifestations, and once restored, patients do not have any clinical sequelae. 5. mods is closely with shock and infection. shock, infection, injury (including trauma and surgery, etc.) are the three main causes of mods. 6. generally the later period of shock will typesetting idc and mods, and the order of occurrence of mods usually is the lungs, liver, kidney, gastrointestinal tract, finally the heart. the characteristic clinical manifestations 1. instability of circulation due to a variety of inflammatory mediators have effects on the cardiovascular system, the circulation is most likely involved. almost all cases, at least in the course of the early and middle will be in highpower type of cycle of "high ranked low resistance". cardiac output up to 10 l/ min or more and low peripheral resistance cause shock and need vasopressors to maintain blood pressure. 2. high metabolic systemic infection and mods are usually accompanied by severe malnutrition. its metabolic mode has three salient features: (1) persistent high metabolism, metabolic rate up to 1.5 times more than normal; (2) abnormalities of energy pathway. in starvation, the body obtain energy mainly through the decomposition of. however, with systemic infection, the body will get energy by breaking down proteins while the use of sugar is limited and fat utilization may increase early, fall later; (3) poor response to exogenous nutrient, supplement of exogenous nutrition can not effectively prevent itself consumption, which suggests that a high metabolism itself has a "mandatory" also known as "autophagy metabolism." high metabolic may have serious consequences. first, protein malnutrition result from it will cause serious damage to the structure and function of the enzyme system of organs; secondly, imbalance of branched-chain amino acids and aromatic amino acid which makes the latter formate into a pseudo-neurotransmitter, then further lead to dysfunction of nerve. 3. hypoxia in tissue cells at present many scholars believe that the high metabolic and circulatory disorders often cause oxygen supply and oxygen demand does not match, so that the tissues of bodies are in a hypoxic state, mainly clinically manifesting "oxygen supply dependency" and "lactic acidosis. ". currently mods still lacks an unified diagnostic criteria, and any one of the diagnostic criteria of mods is difficult to reflect the entire contents of organ dysfunction, so in clinical practice we can select one according to our own specific situation. 1. the main contents from national critical care medicine conference standard in 1995 are: (1) respiratory failure: r > 28/min; pao 2 < 6.7 kpa; pco 2 > 5.89 kpa; pao 2 /fio 2 ≤ 26.7 (200 mmhg); p (aa) do 2 (fio 2 1.0) > 26.7 kpa (200 mmhg); x-ray of chest shows alveolar consolidation ≥1/2 lung (which have more than three or three); (2) renal failure: except prerenal factors, little or no urine, serum creatinine, increased blood urea and nitrogen levels, exceeding more than twice the normal value; (3) heart failure: systolic blood pressure < 80 mmhg (10.7 kpa), sustained more than 1 h; ci <2.6 l/(min · m 2 ); ventricular tachycardia; ventricular fibrillation; degree atrioventricular block; resuscitation after cardiac arrest (with which three or more); (4) liver failure: total bilirubin>34 μmol/l; liver enzymes increased more than 2 times compared with the normal; prothrombin time > 20s; with or without hepatic encephalopathy; (5) dic: platelets 100 × 10 9 /l; prothrombin time and partial thromboplastin time prolong 1.5 times, and fibrin degradation products increase; systemic hemorrhage; (6) brain failure: glasgow score below 8 means coma, and less than 3 points means brain death. 2. the sooner the primary diseases or the primary risk factors are eliminated or controlled, the greater the possibility of organ recovery is. 1. to effectively rescue and debride as soon as possible, prevent infection, prevent ischemia-reperfusion injury, use a variety of supportive care; 2. to reduce stress response, mitigate and shorten high metabolism and the magnitude and duration of glucocorticoid receptor; 3. to pay attention to the patient's breathing and circulation, as soon as possible to correct hypovolemia and hypoxia; 4. 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evolving history pathophysiology of septic acute kidney injury: what do we really know? sepsis, thrombosis and organ dysfunction bile high-mobility group box 1 contributes to gut barrier dysfunction in experimental endotoxemia key: cord-021588-ec7udsmw authors: craighead, john e. title: enteric viral disease date: 2007-05-09 journal: pathology and pathogenesis of human viral disease doi: 10.1016/b978-012195160-3/50033-9 sha: doc_id: 21588 cord_uid: ec7udsmw nan al kapikian learned immune electron microscopy from the talented british investigator june alameda, who had perfected the technique into an art form, as much as a skill. in the early 1970s, kapikian focused on this approach to the search for the elusive viruses believed to be responsible for nonbacterial gastroenteritis. it was not long before he identified the virions of the so-called norwalk agent in a diarrheal stool from an adult volunteer who had been challenged with a fecal sample from a child ill during an outbreak of diarrhea. this virus and its soon-to-be-discovered close relatives (the so-called norwalk-like viruses [nlvs]) proved to be important causes of explosive outbreaks of diarrhea in both children and adults. nlvs were first reported in 1972 (kapikian et al, 1972; kapikian, 1994) . only a year later, ruth bishop and her colleagues (1973) found virions of a different size and appearance in cells of the duodenal mucosa of infants with gastroenteritis. these agents proved to be the prototype for a new genus, the rotaviruses (rvs), members of which are infectious for humans and a wide variety of domestic animals. rotaviruses, classified into group a, are now recognized to be the major cause of severe diarrheal disease in infants and young children worldwide. cholera morbus has plagued and threatened the lives of infants and children since the beginning of recorded history. morbidity is universal, and the resulting mortality, particularly in developing areas of the world, continues to be tragic. overall, as many as a third of the deaths in children under the age of 5 in the less-developed countries of the world are attributed to diarrhea. when pathogenic bacteria are excluded, roughly 80% of the episodes of diarrhea (>3 nonsolid stools per day) are either of unknown etiology or are due to viruses. with the discovery of the enteroviruses (see chapter 1) and the demonstration of their chronic presence in the stools of many of those who are infected, it was thought that the search for the cause of nonbacterial diarrhea would soon be over. but despite the presence of high concentrations of enteroviruses in the stools of both ill and healthy children, it shortly became apparent that these viruses were not common etiologic agents of enteritis, but merely nonpathogenic "passengers'' in our digestive tracts. other viruses such as members of many of the common serotypes of the adenoviruses similarly can often be found in the gut, but they too usually fail to cause disease. as an outgrowth of an enormous amount of laboratory work, it was ultimately concluded that the elusive viruses of childhood enteritis are sufficiently fastidious that they cannot be easily grown in tissue culture and in laboratory animals. thus, in the early 1970s, investigators initiated attempts to identify viral particles in stool extracts using electron microscopy. to accomplish this, the background was stained (so-called negative staining), with the virions in startling contrast, that is, like stars in a dark sky. should the virions be present in sufficient number, and should their morphology be sufficiently distinctive, infection could be established. by adding specific antibody (or serum from a previously infected patient) to the suspension, the virions would clump with the antibody and an antigenic identification of the virus accomplished. this also proved a means for assaying the relative concentration of antibody in the blood of those who were infected. the technique of negative-staining electron microscopy using clinical enteric specimens is an arduous art form that requires exceptional skill and attention to detail, clearly not a characteristic of many of our species. but, this painstaking approach has now yielded evidence to indicate that viruses of at least six families may contribute to enteric illness in children and in adult citizens whose immunity has waned (figure 32.1, table 32 .1). studies in domestic animals have also shown that diarrheal disease of economic importance is caused by members of these same virus families, possibly by strains of virus that are host-specific. with the development of refined rnolecular and immunological tools, more sensitive and less laborious diagnostic approaches fortunately are now replacing negative-staining electron microscopy of stool specimens. a number of antigenically distinct, nonenveloped, round, 27-to 32-nm rna viruses classified into a newly proposed genus of the calcivirus family have been found to cause sporadic outbreaks of transient severe enteritis in both children and adults. the etiological role of these viruses as a cause of intestinal disease was established by demonstrating a temporal association of naturally occurring infections (as demonstrated by stool examination using electron microscopy) with illness and by experimental induction of disease in both human volunteers and experimental animals (hall et al, 1984) . the nlvs are provisionally divided for classification purposes into two distinct groups based on genetic analysis (table 32 .2). the relative clinical importance of the viruses of the various groups listed in table 32. 2 has yet to be established, but they are believed to be responsible for a substantial proportion of the nonbacterial outbreaks of acute vomiting and diarrhea that occur in families and institutions. although survey information is incomplete, infections with nlv are thought to occur worldwide. in developing countries, the majority of children are infected during the first 10 years of life ( figure 32 .2), whereas in north america infection is relatively uncommon in children, and by the fifth decade of life only 50% of the population possess serological evidence of past infection. it is not clear what proportion of these infections are accompanied by clinical illness, and we possess only limited information on the persistence of serum antibodies during convalescence from infection. thus, nlv-related disease may occur more commonly than serological surveys of the population now suggest. outbreaks of illness develop as a result of a contaminated common source, such as a water supply or food, or as a consequence of person-to-person transmission (fankhauser et ah, 1998) . the mean incubation period is approximately 40 hours, and symptoms persist for 12 ( to 24 hours (table 32 .3). the clinical course observed in two volunteers is shown in figure 32 .3) (dolin ei al., 1971) . volunteer studies have yielded important histological and ultrastructural documentation of the profound but relatively transient changes that occur in the mucosa of the small intestine during the course of infections with nlvs (agus ei al, 1973; schreiber ei al, 1973 schreiber ei al, , 1974 dolin ei al, 1975) . unfortunately, biopsy material from these experimental subjects is, by necessity, limited; thus, the extent and distribution of lesions in the digestive tract is unknown. of obvious significance is the reduction in the height of the intestinal villi, accompanied by blunting of the villi and evidence of crypt hypertrophy. the individual lining cells of the small intestine exhibit vacuolization of the cytoplasm with loss of polarity. these findings indicate a marked increase in enterocyte replacement as documented by mitotic counts in the epithelium of the crypts. although cell infiltrates are found in the lamina propria and the core of the villi, inflammation is customarily not a prominent feature of the lesion. ultrastructurally, the mucosal cells remain intact, but intercellular edema reprinted with permission from kapikian (1994) . is prominent and the microvilli of the brush border are significantly shortened as shown by electron microscopy. dilatation of both the endoplasmic reticulum and mitochondria of the mucosal cells is seen, and endocytotic vesicles are evident. ultrastructural studies thus far have not defined the cell site of replication of the virus, most probably because the virions are relatively small, that is, in a size range that would not readily allow one to distinguish them from ribosomes. immunohistochemistry documents a substantial reduction in disaccharides and alkaline phosphatase in the epithelium of the gut. intestinal biopsies obtained from volunteers 5 to 6 days after the ingestion of nlvs (i.e., 2-4 days after clinical recovery) continue to show shortening of villi and crypt hypertrophy, but the inflammatory response in the lamina propria and submucosa appear to be reduced. at this time, there remains a significant reduction in the surface area of the gut, and a striking increase in mitotic activity of the epithelium is seen. rvs are the most important cause of severe and often life-threatening diarrheal disease in infants and young children worldwide (moulton ei al, 1998) . although adults are infected, their symptoms are customarily mild, with asymptomatic infections being common. however, severe infections tend to occur in older persons (hrdy, 1987; wenman ei al, 1979; abbas and denton, 1987; lewis ei al, 1989 ) and in persons with attenuated immunity (kaljot ei al, 1989; dryden and shanson, 1988; eiden ei al, 1985; wood ei al, 1988) , but rvs do not appear to be a major contributing factor to the diarrheal disease that so commonly afflicts those with aids. rvs are members of a genus classified in the reovirus family. the latin roia refers to the spoke-wheel appearance of these nonenveloped 70-nm rna virions in negatively stained electron micrographs. based on antigenic analysis of the virion coat proteins, five groups (a-e) have been established. the majority of human infections are due to viruses of group a in regions of the world where intensive studies have thus far been conducted. overall, the prevalence of group b and c infections is low, and they appear only sporadically in developed countries. widespread epidemics of group b viruses have been reported from the people's republic of china; however, overall, infection by group b and c viruses in china as measured by presence of antibodies in the blood serum of members of the general population remains low, that is, -10% (qui et al, 1986) . because these viruses are highly mutable, stool specimens from individuals may yield a diversity of variants that differ one from another on the basis of minor changes in the amino-acid makeup of the virion coat protein. the relative pathogenicity of these genetically diverse agents is unknown. although rvs from domestic animals have been shown experimentally to infect children, there is little evidence to indicate that animals are a significant reservoir for human infections. in developed countries, rv infections typically occur between the ages of 6 and 24 months. in one study conducted in north america, 40% of children were infected with at least one strain of rv during the first year of life (ward and bernstein, 1994) (figure 32.4) . however, fewer than a third of these infections were symptomatic (wenman et al, 1979) . in developing countries and in persons residing under poor socioeconomic conditions, as many as 30% of children are infected by 6 months of age (moulton et al, 1998) , and many infants experience their first encounter with rvs in the newborn nursery during the first week of life (bishop, 1994) . this high prevalence of infection during early life doubtlessly represents both the common occurrence of rv infections in persons residing in these environments (thus facilitating person-to-person spread) and the lack of acquired secretory iga to the virus in the gut. the clinical features of rv infections -fever, vomiting and diarrhea -are known to all mothers. yet, because symptoms usually are m^ercifuuy short-lived (24 to 48 hr), the maintenance of fluid and electrolyte balance is rarely a problem in otherwise healthy infants (table 32 .4). in developing countries, however, the threat to life is more imposing, particularly since infections so frequently occur among the very young. illness in adults is less severe (table 32 .5), if it occurs at all. indeed, 60 to 80% of infections in adults are asymptomatic. this may reflect age-related resistance (as documented in animals) (ciarlet et al, 1998) or the acquisition of a spectrum of serotype-specific iga antibodies in the gut resulting from previous natural infection. as the years pass, immunity may wane among the elderly, in part due to less frequent encounters with the virus in their home setting. older folks more often develop severe diarrheal illnesses when infected with rvs, and deaths are reported (hrdy 1987) . rvs appear to replicate exclusively in the mucosal lining cells of the villi of the small intestine and not elsewhere in the gut (figures 32.5 and 32.6 ). up to lo^^ virions can be found in a gram of feces during the acute illness! this extraordinarily high concentration of virus in diarrheal stools no doubt accounts in part for the ease of transmissibility of these viruses among close contacts. since the virions are also relatively resistant to environmental stresses, they can persist in water supplies and food. infection occurs exclusively by the oral route. as might be expected, relatively few morphologic studies have been carried out on enteric biopsies from infants and children with documented rv infections. davidson and barnes (1979) examined duodenal biopsies obtained 24 to 120 hours after the onset of symptoms from children 2 to 33 months of age. abnormalities were found in 40% of the biopsies. there was blunting of villi, along with an increase in crypt depth and flattening of the epithelial cells lining the villi. inflammatory cells were present in the lamina propria in 2 of the 17 infants studied. the pathological changes were said to resemble those seen in celiac disease with loss of villi, prominent crypt hypertrophy, and an infiltrate of inflammatory cells. rv particles were found in the enterocytes of these patients by electron microscopy. histochemical evaluation of the gut mucosa reprinted with permission from uhnoo et al. (1986) . "statistically significant (p > 0.05) difference between hospitalized and nonhospitalized patients. reprinted with permission from wenman et al. (1979) . figure 32 .6. reprinted with permission from saif et al. (1978) . experimentally induced rotavirus infection in gnotobiotic piglets. electron micrographs of villous epithelium. note distended cisternae of the rough endoplasmic reticulum and rarefaction of cytoplasm in panel a. the affected cell is situated between two seemingly normal cells. panel b shows cells with shortened irregular microvilli and a break in the viroplasm-vp border (arrow) (see figure 32 .5). reprinted with permission from saif et al. (1978) . the associated changes in the villus lengthicrypt length ratio is shown for infected (solid black) and control (broken line) animals. note the protracted recovery time. reprinted with permission from crouch and woode (1978). showed that disaccharidase concentrations were reduced in the epithelium. repeat biopsies were done on several children during convalescence (3 to 8 weeks), and regeneration of the mucosa was found. as noted above, a wide variety of wild and domesticated mammals and poultry (domermuth and gross, 1985) acquire rv infections naturally. experimental studies in calves (reynolds et al, 1985) , lambs (snodgrass et al, 1977 (snodgrass et al, , 1979 , and piglets crouch and woode, 1978) have provided insight into the location of viral replication in the gut and the associated histologic changes (figure 32.7) . to a large extent, the changes observed in children (davidson and barnes, 1979) were found in these animals. in calves, the lesions tended to be more prominent in the proximal small intestine, whereas in lambs the distal ileum was more severely affected. table 32 .6 summarizes the results of immunohistochemical studies that document viral replication in the gut of lambs after experimental infection per os. in general, the sites of rv replication correlate with the pathological changes found in the intestinal mucosa (snodgrass et al, 1977) . as might be expected, the concentrations of virus in the gut of these piglets were highest before lesions in the mucosa appeared (crouch and woode, 1978) . reprinted with permission from snodgrass et al. (1977) . viruses of several additional families have been implicated in human enteritis, but, in general, the disease is relatively mild and not life threatening (table 32 .1). information concerned with the pathological effects of these viruses on the gut mucosa is lacking. in many studies, the common occurrence of asymptomatic enteric infections often makes it difficult to establish, on epidemiological grounds, an etiological relationship between the infection and disease; yet, under certain circumstances, these viruses may be pathogenic for humans. the enteric adenoviruses types 40 and 41 are recognized causes of enteritis, but they account for fewer than 10% of cases (see chapter 14) (brandt et al, 1985) . in two recent studies, toroviruses were associated with enteritis manifest in children as both vomiting and diarrhea. although not severe, the stools commonly were bloody and disease persisted for several days (koopmans et ah, 1997) . enteritis due to toroviruses also occurs in cattle and horses (weiss and horzinek, 1987; woode et al, 1982; jamieson et al, 1998) . astroviruses and coronaviruses have been implicated as causes of diarrheal disease in humans, but the evidence supporting a cause-and-effect association is weak (phillips et al, 1982; lew et al, 1990) . these latter viruses are etiologically responsible for disease in young domestic animals (mebus et al, 1973; thake et al, 1973; gray et al, 1980; kurtz et al, 1979) (figures 32.8 and 32 .9). figure 32.9 degeneration of mucosal lining cell in the midgut of a lamb infected with an astrovirus. note the changes in the microvilli (arrows) and virions in a lysosome (v). reprinted with permission from gray et al. (1980) . pathological observations provide insight into the physiological basis for enteritis in those infected with enteropathic viruses. at present, relatively few clinical studies have addressed these issues in detail. involvement of the upper intestinal tract (duodenum and jejunum) may account for the vomiting that so frequently occurs in nlv infections. diarrheal disease no doubt is consequent to the profound changes in the intestinal mucosa that occur during the acute illness. reduction in the surface area of the gut mucosa and functional alterations in the individual enterocytes that line villi reduce absorption of fluids and solids beyond the capacity of the colon to compensate. the loss of disaccharidases in the mucosal cells of the small intestine increases the carbohydrate concentrations of the large bowel content, resulting in the generation of fermentation products. an outbreak of rotavirus infection in a geriatric hospital acute infectious nonbacterial gastroenteritis: intestinal histopathology. histologic and enzymatic alterations during illness produced by the norwalk agent in man viral infections of the gastrointestinal tract virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis rotavirus disease, but not infection and development of intestinal histopathological lesions, is age restricted in rabbits serial studies of virus multiplication and intestinal damage in gnotobiotic piglets infected with rotavirus structural and functional abnormalities of the small intestine in infants and young children with rotavirus enteritis transmission of acute infectious nonbacterial gastroenteritis to volunteers by oral administration of stool filtrates viral gastroenteritis induced by the hawaii agent: jejunal histopathology and serologic response hemorrhagic enteritis of turkeys the microbial causes of diarrhoea in patients infected with the human immunodeficiency virus rotavirus rna variation during chronic infection of immunocompromised children molecular epidemiology of "norwalk-like viruses'' in outbreaks of gastroenteritis in the united states ultrastructure of the small intestine in astrovirus-infected lambs prevalence of antibody to the norwalk virus in various countries lesions of gnotobiotic calves experimentally infected with a calcivirus-like (newbury) agent epidemiology of rotaviral infection in adults human torovirus: a new nosocomial gastrointestinal pathogen prevalence of acute enteric viral pathogens in acquired immunodeficiency syndrome patients with diarrhea norwalk and norwalk-like viruses visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis association of torovirus with acute and persistent diarrhea in children astrovirus infection in volunteers six-year retrospective surveillance of gastroenteritis viruses identified at ten electron microscopy centers in the united states and canada. pediatr outbreaks of astrovirus type 1 and rotavirus gastroenteritis in a geriatric inpatient population pathology of neonatal calf diarrhea induced by a coronavirus-like agent pathophysiology of viral diarrhea the protective effectiveness of natural rotavirus infection in an american indian population astrovirus within human small intestinal mucosa antibody against adult diarrhoea rotavirus among healthy adult population in china pathology of natural rotavirus infection in clinically normal calves morphogenesis of porcine rotavirus in porcine kidney cell cultures and intestinal epithelial cells the mucosal lesion of the proximal small intestine in acute infectious nonbacterial gastroenteritis the small intestinal lesion induced by hawaii agent acute infectious nonbacterial gastroenteritis rotavirus infection in lambs: pathogenesis and pathology small intestinal morphology and epithelial cell kinetics in lamb rotavirus infections epithelial cell dynamics in transmissible gastroenteritis of neonatal pigs pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotic pigs clinical features of acute gastroenteritis associated with rotavirus, enteric adenoviruses, and bacteria protection against rotavirus disease after natural rotavirus infection the proposed family, toroviridae: agents of enteric infections rotavirus infection in adults: results of a prospective family study chronic enteric virus infection in two t-cell immunodeficient children studies with an unclassified virus isolated from diarrheic calves key: cord-017537-ztdz4a2s authors: bologna, mauro title: biological agents and bioterrorism date: 2014-09-18 journal: detection of chemical, biological, radiological and nuclear agents for the prevention of terrorism doi: 10.1007/978-94-017-9238-7_1 sha: doc_id: 17537 cord_uid: ztdz4a2s for this very stimulating course, i want to share with you some of my studies and even some of my scientific and phylosophical considerations on biological agents living in the environment and their relations with humans, in the very wide concepts of ecological relationships, parasitism, immunolgical defenses and infectious disease mechanisms. all these concepts must be studied and considered in the event of criminal use of biological agents (bioterrorism) aimed at harming human populations in time and in geographical space. terrorism is the use of violence to condition societies or governments in their political choices. bioterrorism is the use (or menace of use) of biological agents to enact terrorism events and induce generalized fear concerning negative consequences in target populations. (a) use of poison darts/arrows (primitive populations): mostly for hunting, but also for battles against enemies. from here derived many of the advances of toxicology, the science of toxic substances (the word "toxicology" derives from the greek words "toxon" = arch and "farmakon" = poison; toxicon farmakon = poison for arch hunting): (b) from such practice derives the knowledge we have of stricnin, curare, ouabain, aconite, other plant/animal poisons primarily devised for hunting and fi ghting. (c) impingement of darts in decomposing cadavers or putrefaction soil (or manure >> tetanus) before throwing at enemies (new guinea, tribal combats; sciites 400 b.c.) (d) last but not least, the use of fear that humans have of beasts. here comes the example of hannibal (from carthago), leading the ships of prusia i, king of bithynia (west turkey) in a battle of year 184 b.c. against eumene ii (attalides, pergamon); he won that naval battle because he managed to throw canisters full of reptiles at the enemy ships, causing terror and uncoordinated reactions leading to his victory. he therefore used fear as a weapon: snakes were not even harmful (not poisonous), but big was the surprise and reactions were out of control! (e) in recent history, we can see that first world war (also named the war of chemistry) contributed to the development and use of nervine gases , chemical weapons banned everywhere but still existing in some countries. second world war (also named the war of physics) led to the development and use of the atomic bomb. thepeace treaties: geneva protocol ruled against chemical weapons (1925) and was followed later by additions concerning bacteriologic war. not all the states however subscribed it. most states anyway have banned, in time, chemical and bacteriological weapons by 1975. today, how scared should we be of biological and chemical terrorism? well, since these are lethal and cheap weapons, they are of considerable concern, because they may be seen as the atomic bomb of poors and represent remarkable threats to peace in local confl icts and in terroristic attacks worldwide. we should all know more on the subject and do extensive prevention. albert einstein once said "i do not know by what weapons the third world war will be fought, but for sure the fourth will be fought with stones". well, we do not want any more world wars, for sure, period. life began on earth with unicellular beings: primitive bacteria and algae, ∼3.5 billion years ago (archean age). biological evolution of species produced today's forms of life, as we observe them. millions of species co-exist and share the stage (biosphere). humans (we) pretend to have absolute priority, but … share the stage in such a crowded environment on earth means to learn, respect, understand and prevent. on this planet we have millions of different species of live beings, with variable proportions in the biosphere and in different ecosystems: they are all co-existing, interacting and competing for food and survival. this encompasses the very universal phenomena of competition and of parasitism. one of the most recent and precise evaluations of the number of species existing on planet earth (2012), but still very approximate and provisional, (well illustrated and summarized in national geographic, 2013) fi nds evidence of more than 5,000 species of mammals, 10,000 species of birds, 12,000 species of reptiles, 15,000 species of amphibians, 45,000 species of fi sh, 150,000 species of crustaceans, 200,000 species of mollusks, 600,000 species of aracnids, fi ve million species of insects and many, many millions (unestimable indeed) species of bacteria, viruses and other microrganisms. are we many on earth? is there enough work for the immune system of each living multicellular organism to distinguish "self" from possibly harmful "not self"? species of microrganisms ascertained as pathogenic for humans are indeed a very small fraction of the existing species: we come to know them better because we study the diseases connected with them, but we ignore a lot about the great number of other, presumably innocuous species. in general, different species interacting may set a parasitic relationship , in which the larger animal ( the host ) may receive harm (food loss or disease) and the smaller one ( the parasite ) may get advantages (more food, protection). both must preserve their identity and prevent contamination by foreign genetic material (immunologic surveillance, bilaterally). some parasites can even live within the hosts (endoparasitism), like some bacteria and all viruses. three types of interactions may occur between a microrganism and a human host: (a) symbiotic relationship, in which the microrganism and the host both benefi t; (b) commensal relationship, in which the microrganism gains but the host suffers no harm; and (c) a true parasytic relationship, in which the microrganism gains and the host is harmed. symbiosis offers frequently mutual advantages and remains very stable in time. pathogens are a minimal part of existing microrganisms. we humans host some advantageous bacterial populations (intestine, surface germs on the skin, commensal germs on the mucosae): we indeed are also made of the germs living in/on our body. indeed, only one cell out of ten in our body is a human cell: the rest are bacteria (the so-called micro-biome). among these, we count billions of bacteria in the intestines, useful for many functions (vitamin production, competition with pathogens, contribution to metabolism, etc.). the balance between host and parasites depends on two basic forces, an aggressive force by the parasite depending on survival/proliferation/invasion capacity of the parasite itself and a defensive force by the host depending on the immune mechanisms (phagocytosis, cellular and humoral immune reactions). in this balancing of opposite forces the parasitic relationship is played by the contendents. if we have prevalence of parasite, we may have disease (and eventually death) of the host, but if we have prevalence of host defence we may have control (and eventually elimination) of parasites. in the light of recent concern and interest about the potential for biological terrorism (biofarware) there are several diseases and bacterial toxins that must be considered in particular, like anthrax [ 1 , 2 ] , smallpox [ 3 , 4 ] , plague [ 5 ] , botulinum toxin [ 6 ] , and tularemia [ 7 ] . a very detailed discussion of such diseases and other infectious diseases with similar risks in terms of bioterrorism goes beyond the scopes of this concise chapter, but some features of these and other infectious diseases representing important threats in the biofarware fi eld will be mentioned. in this respect, we may distinguish in time diseases which are: smallpox is a highly contagious disease (incubation 10-12 days) caused by the smallpox virus, an orthopoxvirus. it causes death in up to 30 % of infected subjects. indigenous infection has been eradicated (last case, ethyopia, 1990 -who). the main concern for outbreaks of smallpox is today from bioterrorism. smallpox is characterized by severe constitutional symptoms (fever, headache, extreme malaise) and a characteristic pustular rash. treatment is supportive; prevention involves vaccination, which, because of its risks (eczema, encephalitis, etc.), is done selectively. pathogenesis of smallpox demonstrates that the virus is transmitted from person to person by direct contact or inhalation of droplet nuclei. clothing and bed linens can also transmit infection. most contagions are in the fi rst 7-10 days after the skin rash appears. once crusts form, infectivity declines. the virus invades the oropharyngeal and respiratory mucosa, multiplies in regional lymphnodes, causing viremia and localization in small blood vessels of the skin (rash) and rarely in cns (encephalitis). offi cially, smallpox is dead on earth. there are no longer cases detected in the world population since 1990, but can we destroy the samples of smallpox virus existing in some virology laboratories around the world? certainly not [ 3 ] , because we could no longer prepare vaccine doses without live virus samples to start from. and without vaccine, a small amount of wild virus could ignite a wide epidemic killing a large proportion of the human population, since the vaccination is no longer mandatory in any country and a large percentage of young populations have no longer been vaccinated after the early 1990s. poliomyelitis is an acute infection caused by a poliovirus. manifestations include a nonspecifi c minor illness (abortive poliomyelitis), sometimes aseptic meningitis without paralysis (nonparalytic poliomyelitis) and, less often, fl accid weakness of various muscle groups (paralytic poliomyelitis). diagnosis is clinical, although laboratory diagnosis is possible. treatment is supportive. vaccination is available, still mandatory in many countries, although soon legislations may change. childhood vaccination produces immunity in 95 % of recipients. declared cases worldwide have diminished remarkably, but some areas with particularly poor sanitary services or with confl icts preventing health services to operate are recording increased numbers of cases recently (syria, 2013; china 2013). polioviruses have three serotypes. the virus enters the mouth via the fecal-oral route, then enters the lymphoid tissues of the gi tract. if not contained, infection may enter the cns with signifi cant damage in spinal cord and brain, specifi cally to nerves controlling motor and autonomic function (breathing). spreading is through the enteric route. vaccine is live, attenuated virus, able to immunize many contacts respect to the vaccinated subjects (community vaccination strategies; problems in nomad populations). anthrax is caused by bacillus anthracis , toxin producing, encapsulated, aerobic or facultative anaerobic organisms. anthrax, an often fatal disease of animals, is transmitted to humans by contact with infected animals or their products (woolsorter's disease). in humans, infection tipically occurs through the skin. inhalation infection is less common; oropharingeal, meningeal and gi infections are rare. for inhalation and gi infections, nonspecifi c local symptoms are typically followed in several days by severe systemic illness, shock and often death. empyric treatment is with cyprofl oxacin or doxycycline. a vaccine is available (antitoxin). pathogenesis of anthrax takes place since bacillus anthracis readily forms spores when germs encounter dry environment -a condition unfavorable for growth . spores resist destruction and can remain viable in soil, wool, and animal hair for decades. spores germinate and multiply in favourable conditions (wet skin, tissue, blood) and can give human disease by contact (papules, black eschars, contagious also via fomites) ingestion (raw meat > fever, nausea, vomiting, diarrhea), and inhalation (fl u-like illness, respiratory distress, cyanosis, shock, coma). of note is the anthrax bioterrorist attack through mailings (using spores in powder form) that took place in the usa in 2001 (us postal service, washington dc), event that highly sensitized the public to the global theme of bioterroristic attacks. plague is caused by yersinia pestis (formerly named pasteurella pestis ). short bacillus with hairpin shape, infects wild rodents and can infect humans via tick bites. symptoms are either severe pneumonia or massive lymphadenopathy with high fever, often progressing to septicemia. diagnosis is epidemiologic and clinical, confi rmed by culture and serologic testing. treatment is with streptomycin or doxycycline. unfortunately, a vaccine is not available for plague. tularemia is a febrile disease caused by francisella tularensis ; it may resemble typhoid fever: symptoms are a primary local ulcerative lesion, regional lymphadenopathy, profound systemic symptoms, and, occasionally, atypical pneumonia. diagnosis is primarily epidemiologic and clinical and supported by serologic tests. treatment is with streptomycin, gentamycin and other antibiotics. tetanus is an acute poisoning from a neurotoxin produced by clostridium tetani. symptoms are intermittent tonic spasms of voluntary muscles. spasm of the masseters accounts for the name "lockjaw" (trismus). incubation requires 2-10 days. diagnosis is clinical. treatment with immune globulin and intensive support. only unbound toxin can be neutralized. a vaccine is available, with a good extent of preventive protection. botulism is a neuromuscular poisoning due to clostridium botulinum toxin. botulism may occur without infection if toxin is ingested. symptoms are symmetric cranial nerve palsies accompanied by a symmetric descending weakness and fl accid paralysis without sensory defi cits. diagnosis is clinical and by laboratory idenifi cation of toxin. treatment is with antitoxin and support therapies. tbc is a chronic, progressive infection by mycobacterium tuberculosis , often with a long period of latency following initial infection. it occurs most commonly in the lungs, with productive cough, chest pain and dyspnea. diagnosis is most often by sputum culture and smear. tbc can involve any tissue (organ disease). treatment is with multiple antimicrobial drugs. forms of multiresistant tb bacteria are becoming more and more frequent. coronavirus infections in humans most frequently cause common cold symptoms; however in 2002, a relatively new coronavirus caused an outbreak of severe acute respiratory syndrome (sars), which was much more severe than other coronavirus infections. sars is an infl uenza-like disease leading to progressive respiratory insuffi ciency with signifi cant mortality rate. first detected in china (guandong, 2002), the sars epidemic spread to more than 30 countries. in mid-july 2003, there were >8,000 cases with >800 deaths (10 % mortality). then the outbreak subsided and no new cases have been identifi ed from 2004 to 2012. in 2012 a new similar epidemic (sustained by the virus ncov, novel coronavirus) started in middle east (arabia), with an estimated mortality above 40 %. later the ncov epidemic has been named mers (middle east respiratory syndrome) and is being studied as a new zoonosis trasmitted to humans from dromedary camels. studies are currently in progress, with great attention by the international sanitary authorities [ 9 ] . who in 2013 indeed alarmed many countries against the new sars-like coronavirus responsible of mers, that infected at the moment of this writing (december 2013; www.who.int/en ) more than 160 persons (arabia, great britain, france, germany, tunisia, italy, abu dhabi, united arab emirates, etc.) with reduced infective capacity as compared to sars, but still highly lethal and communicable via close contacts (family members). the latest available numbers call for 163 ascertained diagnoses in humans, with 71 deaths (mortality, 43.5 %). updates can be found at the following web sites: www.who.int/en ; www.cdc.gov and (recommendations for clinicians) emergency.cdc.gov , emphasizing the need to consider the novel (ncov) coronavirus when treating patients with a severe respiratory illness who have recently traveled to the arabian peninsula (or close contacts of the travelers). marburg and ebola are fi loviruses that cause hemorrhage, multiple organ failure and high mortality rates. diagnosis is with enzyme-linked immunosorbent assay, pcr or electron microscopy. treatment is supportive. strict isolation and quarantine measures are necessary to contain outbreaks. incubation 5-10 days. marburg virus has been identifi ed in bats and in primates. human to human transmission occurs via skin and mucous membranes contact (humans/primates). filoviruses can affect intestines (nausea, vomiting, diarrhea), respiratory tract (cough, pharingitis), liver (jaudice), cns (delirium, stupor, coma), and cause hemorrhagic phenomena (petechiae, frank bleeding) with high mortality rates (up to 90 % with ebola virus). survivors recover very slowly and may develop long lasting complications (hepatitis, uveitis, orchitis) with only supportive care available: no specifi c antivirals nor vaccines are available fo fi lovirus infections. bunyaviridae contain the genus hantavirus (four serogroups, nine viruses) causing hemorrhagic fevers with renal and pulmonary consequences, starting with fl u-like symptoms and evolving with severe renal and pulmonary consequences. lethal in 10-15 % of cases. lassa fever is an often fatal arenavirus infection occurring mostly in africa. it may involve multiple organs, except cns. treated with ribavirin. no vaccinations are available so far for hantavirus infections. outbreaks of such infections have been recorded in nigeria, liberia, central africa, with some rare imported cases in the usa and in the united kingdom. the animal reservoir of such viruses is in wild african rats ( mastomys natalensis ), frequently found in african houses. direct human to human transmission is documented via urine, feces, saliva or blood. mortality (up to 45 %) can be reduced by prompt ribavirin treatment. universal hygiene precautions, airborne isolation and surveillance of contacts are essential. last but not least … we must mention now infl uenza! flu viruses are in nature among the most rapidly changing (mutating) organisms through their ability to infect a variety of hosts: birds (migrating waterfowl -ducks-, stantial poultry -chickens-), mammals (pigs, felines) and humans. in south east asia (mostly in china, but also in viet-nam, laos, thailand, etc.) it is very common to have mixed farms of pigs, poultry and ducks, attended by humans. every year, new strains appear in se-asia, favoured by the recyprocal passage between migrating birds (mostly fowl), pigs and chickens, with exposure of many humans in farms, markets, rooster fi ghting sports, and food preparation places. a common say in china tells that "anything with four legs (except chairs) and anything that fl ies (except airplanes), can be eaten". with this phylosophy, there is generally a lot to be desired in food safety and in general hygienic prevention in such geographical areas. after the avian fl u h5n1 of 2005-2006, highly lethal but unable to give human to human contagion, new combinations of fl u strains are expected and feared, with high lethality and high human to human transmissibility. on this widely interesting theme for the world diffusion of new virus strains with pandemic potential, i wrote in 2010 together with the colleague virologist aldo lepidi a book entitled "pandemics -virology, pathology and prevention of infl uenza" (bollati boringhieri publisher, turin, italy , 2010) [ 10 ] . in summary, we can see that a continuous surveillance is being devoted worldwide to the appearance of new strains of infl uenza viruses, in order to isolate as soon as possible potentially pandemic new strains and to prepare biological stocks suitable for massive vaccine preparations in due time to prevent the global spreading of potentially lethal new variants of the infl uenza viruses. examples in time recall the cases of the highly lethal pandemics known as "spanish fl u" in 1917-1918 (in excess of 40 million deaths worldwide), "asian fl u" in 1956 (in excess of 100,000 deaths worldwide) and "hong kong fl u" in 1978 (in excess of 700,000 deaths worldwide). the basic question is: when the new pandemic will strike ? sometimes soon, as international experts say. the so called "avian fl u" came close to that, but sometimes in the future new mutations may emerge with the potential of being much worse. in conclusion of this wide although rapid overview of the most frequent or alarming causes of microrganism-related human diseses with potential interest for bioterrorism, i hope to have provided suffi cient matter for discussion and for further future diffusion of medical and microbiological culture that may be useful for prevention and the betterment of human social relationships and for peace promotion. anthrax as a biological weapon: medical and public health management anthrax as a biological weapon, 2002: updated recommendations for management smallpox as a biological weapon: medical and public health management addressing the unthinkable: preparing to face smallpox plague as a biological weapon: medical and public health management. working group on civilian biodefense botulinum toxin as a biological weapon: medical and public health management tularemia as a biological weapon: medical and public health management the merck manual of diagnosis and therapy, 19th edn deadly mers coronavirus not yet a global concern pandemie¸ virologia, patologia e prevenzione dell'infl uenza (pandemics -virology, pathology and prevention of infl uenza) key: cord-010175-p2py9wau authors: winter, harland; chang, tien-lan title: gastrointestinal and nutritional problems in children with immunodeficiency and aids date: 1996-04-01 journal: pediatr clin north am doi: 10.1016/s0031-3955(05)70421-1 sha: doc_id: 10175 cord_uid: p2py9wau nan harland winter, md, and tien-lan chang, md children acquire human immunodeficiency virus (hiv) either perinatally from an infected mother (vertical transmission) or from infected blood or blood products. the number of children infected following a blood transfusion has dropped markedly following the institution of rigorous screening protocols for blood donors in the mid-1980s. by the early 1990s, more than 95% of newly diagnosed hiv-infected children acquired the disease via vertical infection.= the world health organization estimates that more than 10 million people throughout the world are infected with hiv (table 1) . three million of these individuals are women, most of whom are fewer than 40 years of age, whereas 500,000 of them are children. 37 thus, heterosexual transmission of hiv is the most common means of acquiring the infection when viewed from a worldwide perspective. hiv, a single-stranded rna lentivirus, infects cells that express a receptor capable of binding to the envelope glycoprotein (gp), gp 120. t lymphocytes and monocytes or macrophages that are cd4-positive are the primary targets of the virus, but reports suggesting that other cells in the gastrointestinal tract can be infected have led investigators to data from world health organization: the hiv-aids pandemic: 1993 overview. geneva: who/ gpngnp193. 1, 1993. speculate that gastrointestinal symptoms may be related to epithelial cell infection with hiv-1. fox and colleagues9 reported that hiv-1 infection of the gastrointestinal tract was limited to the lymphoid elements of the lamina propria; other investigators believe that, because intestinal epithelial cell line cultures became infected in the laboratory,1y epithelial cells were infected in vivo in hiv-infected adults.'o, the characteristics of the mucosal immune system most likely have a significant role in the pathobiology of hiv-1 disease in children; however, mucosal immune function has not been studied specifically in hiv-infected children and, thus, pediatricians are left to speculate that observations made in the adult hiv-infected population are relevant to children. table 2 summarizes gastrointestinal mucosal immunologic changes that occur in hivinfected individuals. vertical transmission occurs in approximately 30% of hiv-infected pregnant women who do not take antiretroviral therapy during pregnancy. the observations that transmission is increased in women who were symptomatic or who had more advanced aids27 and that zidovudine therapy given during pregnancy reduces perinatal transmission3 suggest that viral burden is an important factor in vertical transmission; however, the effects of maternal nutritional status, micronutrient deficiency, or acute infection on viral replication are difficult to evaluate. in addition, most hiv-infected women in africa, asia, and south america breast-feed their infants. this additional means by which infants can possibly become infected complicates assessment of factors contributing to transmission. in africa, the percentage of postnatal transmission is approximately 50y0.~~ nevertheless, the morbidity and mortality caused by formula feeding in countries where potable water is a premium and safe infant formula is not readily available seem to be greater than the risk of acquiring hiv-1 from breast milk. the current recommendation is for the hiv-exposed infant to have formula feeding if and only if safe the deterioration of the immune system and mucosal immune systems results in cellular and humoral immunoregulatory deficiencies. in the gastrointestinal tract, hiv-infected lymphocytes could migrate from the lymphoid aggregates through the mesenteric nodes, the thoracic duct, and into the circulation. following selection by receptors on high endothelial venules, these infected cells then migrate home to the lamina propria, whereby in situ hybridization isolated hiv-infected cells can be identified (fig. 1 ). most evidence 'supports the hypothesis that deterioration of mucosal immune function results in bacterial overgrowth; increased production of bacterial products, such as endotoxin; activation of mucosal lymphocytes with increased cytokine production; and probable interaction between immunoregulatory elements and epithelial cell function (fig. 2) . although the reasons for early development of lactose intolerance and malabsorption are not known, substances involved in immune regulation also may interact with intestinal epithe-lial cells, resulting in dysfunction. hiv also may have a role in the genesis of intestinal dysfunction, but data are not available. clearly, enteric infections begin to occur at the time when immune function is deteriorating (fig. 3) . the contribution of chronic intestinal infection to immune dysfunction, malabsorption, and malnutrition suggests that all of these factors are interrelated (fig. 4) . one of the more important determinants of survival for the hivinfected child is the health status of the mother. in studies from africa, if an hiv-infected mother is symptomatic or dies, her hiv-infected infant is at increased risk for chronic diarrhea partially because of the resulting reliance on formula.31 chronic diarrhea in the hiv-infected child is an important prognostic variable for predicting malnutrition and death. because of the availability of safe formula in north america and europe, the relationship between maternal health and infant survival is not as obvious. nevertheless, a chronically ill mother has an obvious negative impact on infant growth and development, particularly if no additional support is available, such as respite and day care programs designed to enrich infants' psychosocial development and nutritional status. in hiv-infected children, nausea and vomiting can be caused by infectious diseases, such as helicobacter pylori or cytomegalovirus, medications, or central nervous system disorders. in a child with nausea, anorexia may be the presenting manifestation because she or he is not able to verbalize the sensation. in these individuals, refusal to chew or eat may be caused by gingival disease or painful lesions of candida in the mouth. in many children, an identifiable agent or pathogen may not be found despite a thorough search. some of the therapeutic agents that have been implicated as causes of nausea and vomiting are as follows: altered mental status or developmental delay should alert the clinician to the possibility of central nervous system disease, such as encephalopathy caused by hiv, or pathogens, such as toxoplasmosis. lymphoproliferative disorders in the central nervous system are rare in the pediatric population; however, lymphoma of the gastrointestinal tract can cause splenomegaly resulting in compression of the stomach and early satiety. evaluation of hiv-infected children with anorexia, nausea, or vomiting should begin with a careful history, social history, physical examination, and neurologic evaluation. an upper gastrointestinal radiograph is not reliable enough to establish or rule out mucosal disease. for this reason, endoscopic evaluation is frequently necessary in children with persistent symptoms and normal hepatobiliary and pancreatic tests. mucosal biopsies may identify an enteric pathogen or inflammation that can be treated with a specific agent. if no cause can be found, symptoms can be managed with phenothiazine derivatives, such as triethylperazine maleate (torecan), prochlorperazine (compazine), or promethazine (phenergan). other agents for which anecdotal treatment experience exists in children include: benzquinamide (emete-con); trimethobenzamide hydrochloride (tigan); hydroxyzine (vistaril or atarax); metoclopramide (reglan); cisapride (propulsid); and scopolamine (transdermscop); dronabinal (marinol). if treatment fails to relieve the symptoms, re-evaluation should be considered. difficulty in swallowing (dysphagia) or pain with swallowing (odynophagia) in children can be caused by oral lesions that can be identified by careful inspection of the mouth. stomatitis caused by 2',3'-dideoxycytidine 5'-triphosphate (ddc), herpes simplex or candida is treatable if the diagnosis is established. when oral lesions are present, coexistent esophagitis should be suspected. in contrast, if the mouth is free of lesions, esophagitis cannot be ruled out. candida and cytomegalovirus are the most common infectious agents causing esophagitis. dysphagia and odynophagia in hiv-infected children are more commonly associated with candida than with cytomegalovirus. children who are taking h, antagonists seem to be at increased risk for developing candida esophagitis. medications, such as zidovudine, have been reported to cause esophageal ulceration if, when swallowed, they do not reach the stomach.6 treatment for specific causes of oral or esophageal lesions is summarized in table 3 . although enteric pathogens are frequently identified as the cause the incidence of of diarrhea and weight loss in hiv-infected enteric infection in hiv-infected children seems to be lower,4o and the relationship between diarrhea, enteric pathogens, and growth retardation is not as clearly understood. in figure 4 , the interrelationship between malabsorption, malnutrition, immune deficiency, and enteric infection is depicted. enteric infection results in intestinal injury and malabsorption, which, if not compensated by additional nutrient support, results in nutritional deficiency. the development of malnutrition causes immune deficiency, which is characterized by a defect in t-cell function that is similar to the defect caused by hiv disease. defective t-cell function results in increased susceptibility to enteric infection, and the circle is completed. hiv can interact at any of the stages of this cycle. in theory, intestinal absorption can be altered by modifying enterocyte function through immune modulators. by increasing apoptosis, hiv could cause premature senescence of enterocytes and decrease brushborder expression of disaccharidases and peptidases. some of these same agents, such as the cytokine, tumor necrosis factor-a), are upregulated by hiv infection, affect intermediate metabolism, and cause malnutrition by increasing nutrient requirements. the effects of hiv on the immune system are well known and result in immunocompromise and increased susceptibility to opportunistic infection. similar immunoregulatory abnormalities probably occur in the mucosal immune system, resulting in enteric infection. thus, hiv interacts at many levels to potentiate the development of malabsorption, malnutrition, immune deficiency, and enteric infection. giardia zarnbzia causes watery diarrhea, abdominal distention, and crampy abdominal pain.4, 22, 31 metronidazole or furazolidone is effective therapy and eradicates the organism in more than 70% of infected individuals. giardia zambzia does not occur more frequently in hivinfected children than in the general population, but retreatment may be necessary in the immunocompromised host. crypfosporidium parvum causes an acute, self-limited diarrheal illness in the immunocompetent host, but in the immunodeficient child with hiv disease, the infection causes a secretory diarrhea that is chronic and debilitating. the organism usually can be identified in the stool by immunofluorescent techniques or by kinyoun carbolfuchsin stain.7 in hiv-infected children in the united states, the incidence of cryptosporidiosis is lower than that reported in africa and south america.2, 4, 33 crypfosporidium can infect the small intestine, colon, gallbladder, biliary tract, and pancreatic duct. no therapy is consistently effective in eradicating the organism, but octreotide is reported to decrease stool reports of the beneficial effects of hyperimmune bovine colostrum suggest that this form of passive immunotherapy may be effective in hiv-infected individual^.^^ other enteric parasitic infections, including isospora bezzi and microsporidium, are rarely identified in hiv-infected children; however, blastocysfis hominis, a protozoan whose role as an enteric pathogen is still debated, may be more prevalent in hiv-infected children with diarrhea than in hiv-negative ~h i l d r e n .~ bacteria are an important cause of diarrhea throughout the world and for this reason contribute to the list of identifiable pathogens found in hiv-infected children. in africa, pathogenic strains of escherickia coli were identified in over three fourths of hiv-infected children. 22 the risk for other bacterial enteric infections is not known for hiv-infected children, but the incidence of salmonella, skigella, campylobacter, yersinia, and clostridium difficile do not seem to be increased in hiv-infected children. the incidence of helicobacter pylori may be decreased in hivinfected chi1dren.l the most serious enteric bacterial infection is mycobacterium avium-intrace2lulare, which causes a multisystemic infection involving the lungs, liver, mesenteric lymph nodes, gastrointestinal tract, and bone marrow in the most severely immunocompromised hosts with cd4 counts less than 50 cells/mm3. acidfast bacilli can be identified in the jejunal mucosa or grown from stool or blood. the most common gastrointestinal symptoms of m avium-intracellulare are abdominal pain and diarrhea, and neither responds dramatically to therapeutic intervention. combinations of medications chosen from clarithromycin, ethambutol, ciprofloxacin, amikacin, rifampin, clofazamine, and azithromycin have been tried.14 rotavirus is the viral agent that most frequently causes chronic diarrhea. in the immunocompromised child, rotaviral diarrhea can be severe, persistent, and difficult to distinguish from other agents causing secretory diarrhea. diagnosis is established by identification of rotavirus in the stool using an enzyme-linked immunoassay. enterally administered serum immunoglobulin is effective therapy,i5 but little published data exist on the treatment for rotavirus in hiv-infected children. other viral pathogens, such as adenoviruses, can cause diarrhea but also are associated with systemic infection and fulminant hepatitis. cytomegalovirus usually causes an asymptomatic enteric infection, but some individuals develop focal ulcerations in the colon or jejunum and present with bloody diarrhea and abdominal pain. gastrointestinal bleeding is unusual in hiv-infected children, but, when present, it may be caused by focal ulcerations in the colon, stomach, small intestine, or esophagus from cytomegalovirus-induced disease. merely culturing cytomegalovirus from the intestinal mucosa does not establish a link between diarrhea and the infection. histologic evidence of mucosal injury is necessary. ganciclovir and foscarnet are used to treat cytomegalovirus-induced intestinal disease in children with active symptoms. bone marrow suppression is the main serious side effect. many children with hiv disease develop lactose intolerance earlier than predicted by genetic predisp~sition.'~ nevertheless, these lactoseintolerant children do not seem to have an increased probability for growth retardation or diarrheal disease. the impact of lactose malabsorption on the nutritional health of hiv-infected children is unclear; however, children who have decreased absorption of the carbohydrate d-xylose have an increased incidence of harboring an enteric pathogen. 17 to evaluate hiv-infected children with chronic, nonbloody diarrhea, stool analysis for bacterial, viral, and parasitic infection should be performed. blood and polymorphonuclear leukocytes in the stool are indicative of colitis and should prompt evaluation of the colonic mucosa. if no enteric pathogen is identified, functional tests, such as lactose breath hydrogen and d-xylose absorption, may be useful in guiding nutritional therapy. the most beneficial diagnostic test is an upper endoscopy with biopsy. in addition to routine histology, mucosal biopsies of any focal lesions should be tested for bacterial, fungal, and viral culture and analyzed via electron microscopy. because mycobacterium and cytomegalovirus may not be detectable during endoscopic evaluation, surveillance biopsies of the jejunum should be evaluated by electron microscopy and culture. despite these diagnostic studies, enteric pathogens frequently are not identified in many hiv-infected children with diarrhea. hiv-infected children with abdominal pain should be evaluated for enteric infection, especially if they have diarrhea. fever and abdominal pain are symptoms that can indicate the presence of mycobacterium. association of these symptoms with the ingestion of milk should alert the clinician to the possibility of lactose intolerance, but for many children with lactase deficiency, the relationship is not evident. in addition, pancreatitis in the hiv-infected child is a serious and debilitating illness. not only do these children experience crampy abdominal pain, but the association with meals results in decreased caloric intake and increases the potential for malnutrition. lipase seems to be an early and sensitive marker for pancreatitis in the pediatric population.18 medications such as ddi and ddc are associated with pancreatitis, which may develop following many manths of therapy.39 other medications including pentamidine, trimethoprim-sulfamethoxazole, and dapsone have been implicated as causes of pancreatitis in children. the development of pancreatitis is an ominous event, and in one published study, the mean survival of children with pancreatitis was 8 months following the diagnosis.ls because of the guarded outcome, decisions to perform additional diagnostic tests should be made after much discussion with the health care team. if a dilated pancreatic duct is identified by ultrasonography, the indication for endoscopic retrograde cholangiopancreatography should be based on quality-of-life issues. although strictures of the pancreatic duct could contribute to the symptoms, if therapeutic intervention is not feasible, invasive diagnostic studies should not be performed. although the majority of hiv-infected children have hepatomegaly, few experience severe hepatocellular dysfunction; fibrosis; or cirrhosis that results in coagulopathy, ascites, varices, or hepatic failure. many of the medications used to treat complications of hiv disease cause hepatocellular injury or cholestasis; however, infectious agents, such as hepatitis b, that cause hepatocellular injury by immune mechanisms have milder clinical courses in immunodeficient hosts.z4 preservation of immune function in hiv-infected children could account for the apparent increase in chronic active hepatitis in the pediatric population compared with the incidence in although abnormalities in liver function tests are not diagnostic, they are beneficial as screening procedures. elevated transaminases are caused by infectious agents, medications, or nutritional deficiency and malnutrition. when the transaminases exceed four times normal, viral disease or a drug-induced hepatitis should be suspected. m avium in tracellulare, hepatic pneumocysfis carinii, fungal-induced hepatitis, cytomegalovirus, or extrahepatic biliary tract obstruction cause elevation of alkaline phosphatase. liver biopsy is necessary to identify hepatic pathogens and should be considered in a child presenting with either fever and elevated liver function tests or a focal hepatic lesion. therapeutic intervention is available for some of the viral agents that cause hepatitis, but most infectious disorders in immunodeficient hosts do not respond favorably to treatment. wasting of body mass is one of the more serious manifestations of hiv disease. in adults, the decline in lean body mass correlates with decreased quality of life and eventual death.s, l3 in children with aids, growth failure and failure to thrive have been recognized symptoms from the beginning of the epidemic.28 infants born to hiv-infected mothers seem to weigh less by 3 months of life and to be shorter by 6 months of life when compared with hiv-exposed, but noninfected infants. in long-term survivors more than 8 years of age, lean body mass wasting and short stature are common clinical features. the etiology of these derangements in growth is multifactorial, possibly including deranged metabolism, malabsorption, or decreased nutrient intake. the mechanism for the catabolic process is not known, but futile cycling of energy substrates, protein wasting, or hypermetabolism mediated by cytokines such as tnf, interleukin (1l)-1, il-6, and the interferons may contribute to the problem. the initial assessment of hiv-infected children with failure to thrive is directed at determination of caloric intake, nutrient losses, and metabolic requirements. if caloric intake is diminished, the reason for anorexia should be determined. nausea, abdominal pain, oral lesions, depression, despair, or lack of access to food need to be evaluated by the health care team. nutrient losses caused by diarrhea and malabsorption may contribute to increased nutrient requirements. enhanced metabolic requirements from febrile illnesses, recurrent infection, or from hiv replication may result in weight loss. anti-retroviral therapy can result in weight gain shortly after starting therapy. 23 counseling and oral supplements are the first steps in nutritional treatment for children with weight loss or decreased lean body mass. providing increased calories and protein may reverse the loss, but most children require additional measures of support. although nasogastric tube feeding is simple and effective for short-term management, the adverse effect on quality of life and the increased possibility of sinus disease are limiting factors. in children requiring nutritional supplementation lasting greater than 2 weeks, endoscopic placement of a gastrostomy tube button increases compliance and tolerance. as many as 150% recommended daily allowance for calories may be required to achieve weight gain in hiv-infected children. newly developed one-step gastrostomy buttons permit endoscopic insertion of devices that do not limit activity and provide access for nutritional support. despite providing sufficient nutrition to gain weight, enteral supplementation16 and gastrostomy tube feedings" do not increase lean body mass in hiv-infected children. similarly, appetite stimulants, such as megestrol acetate, a progesterone derivative, and dronabinol, a tetrahydrocannabinol derivative, do not increase lean body mass in adults infected with hiv. promising data in adults suggest that mammalian cell-derived recombinant human growth hormone therapy results in weight gain and anabolism as measured by stool nitrogen, urine nitrogen, and potassium excretion.20 if valid in the pediatric population, growth hormone could prove to be an effective treatment for failure to thrive by increasing lean body mass. anecdotal experience implicates specific vitamin deficiencies as contributing to the nutritional problems of hiv-infected children. in regions in which vitamin deficiency is endemic, it is not surprising to see the problem amplified in hiv-infected children. decreased vitamin a causes diminished t-cell response to mitogens and antigens, atrophy of lymphoid tissue,3â° and is associated with increased maternal-child transmission.z9 supplementation of vitamin a seems to increase cd4 + cells, boost antibody response, and decrease morbidity and mortality from other infectious diseases.8 the effect of vitamin a supplementation on the health of hiv-infected children in the united states is not known. other vitamins, including vitamins d, e, 8, (thiamine), b2 (riboflavin), niacin, b6, bi2, folic acid, c, and carnitine, have been evaluated in various populations of hiv-infected individuals, and although abnormalities can be demonstrated for some vitamins, deficiencies related to the generalized state of malnutrition and not specifically to hiv-induced disease are difficult to prove beyond a reasonable doubt. similarly, deficiencies of iron, zinc, and selenium have been described in hiv-infected individuals. although these minerals have an important role in immunoregulathe redundancy of the immune system to provide protection against infection suggests that by the time the system begins to fail, no single cause can be found to correct the problem. for this reason, supplementation with a single therapeutic nutrient intervention can improve laboratory phenomena, but rarely impacts on a patient's quality of life or immunoregulatory defects. patients with primary immunodeficiency disorders frequently experience gastrointestinal problems in association with other clinical manifestations of systemic disease. the respiratory and gastrointestinal tracts are exposed to the environment and, in response, have developed complex systems to protect their mucosal surfaces from pathogens. antibody production, cell-mediated immune function, complement, and phagocytic function act together to prevent infection and uncontrolled inflammation. in the gastrointestinal tract, enteric pathogens and chronic inflammatory bowel disease are the two major clinical aspects of primary immune deficiency. surprisingly, individuals with identical deficiencies may not experience similar gastrointestinal symptoms. for example, children with immunoglobulin a deficiency may be asymptomatic or may have chronic diarrhea associated with chronic intestinal inflammation disease. in general, children with t-cell defects seem to have a higher incidence of chronic gastrointestinal problems compared with children with antibody defiqiency syndromes, complement defects, or disorders of phagocytic function. table 4 lists the common primary immunodeficiencies together with the gastrointestinal manifestations commonly associated with each disorder. immunodeficient children pose a challenge to clinicians because of the interrelationship between infectious disease, metabolism, gastrointestinal tract function, psychosocial problems, and immune function. the interplay between these factors is not always clear, and frequently the best course of therapy is obscured because of an inability to determine which factors have the greatest impact on child health. to optimize therapeutic intervention, a multidisciplinary health care team must be involved with the management of children and their families. heiicobacter pylari in children with acquired immunodeficiency syndrome disseminated histoplasmosis as the acquired immunodeficiency syndrome-defining illness in an infant intestinal parasites and hiv infection in tanzanian children with chronic diarrhea centers for disease control: zidovudine for the prevention of hiv transmission from mother to infant significance of altered nutritional status in acquired immune deficiency syndrome (aids) esophageal ulceration induced by zidovudine aids, zinc deficiency and thymic hormone failure et a1 vitamin a supplementation and child mortality: a meta-analysis detection of hiv-1 rna in the lamina propria of patients with aids and gastrointestinal disease human immunodeficiency virus infection of enterocytes and mononuclear cells in human jejunal tissue effect of enteral tube feeding on growth of children with symptomatic human immunodeficiency virus infection italian multicentre study: epidemiology and clinical features of pediatric hjv infection: results from an italian multicentric study on 544 children bosy composition studies in patients with the acquired immunodeficiency syndrome recommendations on prophylaxis and therapy for disseminated mycobacterium avium complex disease in patients infected with the human immunodeficiency virus benefit of oral immune globulin therapy in patients with immunodeficiency and chronic diarrhea growth and body composition in children infected with the human immunodeficiency virus-1 malnutrition and carbohydrate malabsorption in children with vertically transmitted human immunodeficiency virus 1 infection et a1 pancreatitis in pediatric human immunodeficiency virus infection hiv replication and persistence in human gastrointestinal cells cultured in vitro anabolic effects of recombinant human growth hormone in patients with wasting associated with human immunodeficiency virus infection human immunodeficiency virus detection in bowel epithelium from patients with gastrointestinal symptoms diarrhea among african children born to human immunodeficiency virus-infected mothers: clinical, microbiologic and epidemiologic features effect of continuous intravenous infusion of zidovudine (azt) in chldren with symptomatic hiv infection hepatic involvement in patients with human immunodeficiency virus infection: discrepancies between aids patients and those with earlier stages of infection pediatric acquired immunodeficiency syndrome: special considerations for developing nations efficacy of octreotide in the management of chronic diarrhea in aids perinatal transmission of the human immunodeficiency virus type 1 to infants of seropositive women in zaire survival in children with perinatally acquired human immunodeficiency virus type 1 infection chlehanaw j d maternal vitamin a deficiency and mother-tochild transmission of hiv-1 altered t cell subset proportions in vitamin a deficient children a prospective study of diarrhea and hiv-1 infection among 429 zairian infants chronic active hepatitis in a child with human immunodeficiency virus infection survival experience of 789 children with acquired immunodeficiency syndrome gastrointestinal symptoms in patients infected with human immunodeficiency virus: relevance of infective agents isolated from gastrointestinal tract et a1 cessation of cryptosporidium-associated diarrhea in an acquired immunodeficiency syndrome patient after treatment with hyperimmune bovine colostrum postnatal transmission of human immunodeficiency virus type 1 from mother to infant. a prospective cohort study in kigali, rwanda statement on breast feeding and hiv. who/ unicef consultative meeting of world health organization: the hiv/aids pandemic: 1993 overview. geneva: who/ gpa/cnp/93 long-term toxicity/activity profile of 2',3'-dideoxyinosine in aids or aids-related complex gastrointestinal dysfunction and disaccharide intolerance in children infected with human immunodeficiency virus key: cord-021399-gs3i7wbe authors: dada, m.a.; lazarus, n.g. title: sudden natural death | infectious diseases date: 2005-11-18 journal: encyclopedia of forensic and legal medicine doi: 10.1016/b0-12-369399-3/00357-8 sha: doc_id: 21399 cord_uid: gs3i7wbe nan a wide range of deaths from natural causes is encountered in the field of forensic medicine. despite the advances in the diagnosis and treatment of infectious diseases, a substantial number of sudden and unexpected deaths are caused by infections. in most medicolegal systems these deaths are subject to a forensic investigation. the world health organization defines sudden death as that occurring within 24 h of the onset of symptoms. some authors variably define sudden death as that occurring within 1, 6, and 12 h of the onset of symptoms. forensic pathologists should be aware of the importance of infectious causes of sudden death in the present era of bioterrorism and emergent and reemergent diseases. genetic engineering has led to the development of highly infectious and virulent strains of microorganisms (e.g., anthrax). emerging infectious diseases are infections whose incidence has increased in recent years and/or threatens to increase in the near future. reemergence refers to the reappearance of a known infection after a period of disappearance or decline. death from infectious agents may occur as a direct consequence of the infection or from complications such as immunosuppression caused by the infection and adverse reactions to therapeutic drugs. sudden death due to infectious disease may be classified by organ system involvement (e.g., cardiac -myocarditis; nervous system -meningitis and encephalitis) or according to the etiological agent (e.g., viral, chlamydial, bacterial, fungal, protozoal, or helminthic) . the common infectious causes of sudden death by organ system are listed in table 1 . the morphological findings at autopsy will depend on the type of organism, the site involved, and the host's response to the organism. microbiological demonstration of an organism does not equate to disease, as a host may be colonized by bacteria or the patient may have an asymptomatic viral infection. the exquisite sensitivity of molecular tests, e.g., polymerase chain reaction, may exacerbate this problem if the results are not correlated with the pathological findings at autopsy. categories of human pathogens include prions; viruses; chlamydiae, rickettsiae, and mycoplasmas; bacteria; fungi; protozoans; and helminths. infection by prions, rickettsiae, and mycoplasmas is not normally associated with sudden and unexpected death. viruses are ubiquitous and cause a spectrum of disease in humans. these may range from asymptomatic infection, severe debilitating illness, to sudden death. viral infections causing sudden death usually involve the cardiac, respiratory, or the central nervous system. morphologic findings in viral infections may include intranuclear and/or intracytoplasmic inclusions, multinucleate giant cells, and tissue necrosis (cytopathic effect). in many cases the diagnosis can only be made on special investigations, e.g., culture, electron microscopy, serology, or molecular testing. viral hemorrhagic fevers such as marburg, lassa, and ebola virus may cause sudden death in children. if there is any suspicion of a viral hemorrhagic fever, special care must be taken to avoid unwarranted exposure to health workers. the local public health officials must be informed and consideration given to limited autopsy examination in consultation with a virologist (e.g., postmortem blood sampling and liver biopsy). cardiac involvement usually takes the form of myocarditis. although many viruses may cause myocarditis (table 2) , coxsackie a and b are responsible for most cases. fulminant coxsackievirus infection may also cause leptomeningitis, florid interstitial pneumonitis, pancreatitis, and focal hepatic necrosis. coxsackie b viruses should also be considered as a cause of sudden infant death. at autopsy, the myocardium is usually mottled and flabby. histology reveals focal infiltrates of inflammatory cells (neutrophils and/or lymphocytes, plasma cells, and macrophages). at least two foci of individual myofiber necrosis associated with 5-10 inflammatory cells are required for the histological diagnosis of myocarditis. focal aggregates of lymphocytes not associated with necrosis may be seen in elderly patients and are not diagnostic of myocarditis. myocardial involvement may be patchy. for adequate histological sampling, it is recommended that at least six sections be taken from various areas of the myocardium, including the left ventricle and nodal tissue. indirect damage to the myocardium may occur as an allergic response to a viral infection and eosinophilia, e.g., in eosinophilic myocarditis. this is a rare cause of sudden death in apparently healthy children due to the cardiac toxicity of eosinophils. studies have shown that persons undergoing severe mental or physical stress may have reduced immunity to viral infections. in the investigation of sudden death in athletes, the diagnosis of viral myocarditis must be considered. enteroviral infection may also play an important role in coronary plaque instability and may precipitate coronary thrombosis, leading to ventricular tachyarrhythmias and sudden death. viral infections of the respiratory system sudden death due to viral involvement of the respiratory system may be due to fulminant viral pneumonitis or bacterial pneumonia complicating an initial viral pneumonitis. viruses implicated include respiratory syncytial virus, human herpesvirus-6, and parainfluenza virus in children, and adenovirus and influenza a and b in adults. microscopically, the findings of a viral pneumonitis are usually nonspecific and include edema and widening of the interstitial septa with a mononuclear cell infiltrate. in some cases, diagnostic viral inclusions may be demonstrated. emergent diseases such as severe acute respiratory syndrome (sars) have a high mortality and may cause death within hours. sars refers to an acute respiratory illness caused by infection with a novel coronavirus currently known as the sars virus. postmortem histopathological evaluations of lung tissue show diffuse alveolar damage consistent with the pathologic manifestations of acute respiratory distress syndrome. there is usually mild interstitial inflammation with scattered alveolar pneumocytes showing cytomegaly, and enlarged nuclei with prominent nucleoli. when faced with the finding of diffuse alveolar damage at autopsy, the pathologist should consider other infective causes such as influenza, para influenza, respiratory syncytial, and adenoviruses, chlamydia, mycoplasma, pneumococcus, legionella, and pneumocystis. sudden death may occur due to direct infection of the nervous system or a complication of a viral infection such as toxoplasmosis in human immunodeficiency virus/acquired immunodeficiency syndrome (hiv/aids). herpes simplex virus-1 encephalitis is usually due to reactivation of latent infection. commonly affected sites include the temporal lobe(s) (medial before lateral), the inferior frontal lobe(s), and the sylvian cortex(es). at autopsy there is widespread and asymmetrical necrosis. in fulminant cases there is prominent hemorrhage and swelling with raised intracranial pressure and brain herniation. histological findings include perivascular cuffing by mononuclear cells (figure 1 ) and, in a small number of cases, intranuclear inclusions may be seen in astrocytes and neurons. in adult hiv infections, sudden death from infective causes may be due to opportunistic infections (e.g., toxoplasmosis) or rupture of mycotic aneurysms. in viral central nervous system infections the brain may appear macroscopically normal, especially in very young, elderly, debilitated, and immunocompromised individuals. specimens should be taken for microbiology and histology. serum and cerebrospinal fluid (csf) should be sent for antibody studies. tissue for histological examination should be taken from normal, obviously abnormal, and transition areas. routine sections should be taken from the cerebral cortex (all four lobes), thalamus, basal ganglia, hippocampus, brainstem, and cerebellum. as poliomyelitis has been described as a cause of sudden death in infants, autopsy protocols in sudden death should include histological examination of spinal cord and dorsal root ganglia. chlamydia pneumoniae may be associated with myocarditis and sudden unexpected death. bacterial infections are responsible for sudden unexpected death in adults and children. in the pediatric population bacterial infections of the respiratory, gastrointestinal, and central nervous system account for the majority of cases of sudden death. bacterial infections of the cardiovascular system bacterial causes of myocarditis include corynebacterium diphtheriae, neisseria meningitidis, and borrelia burgdorferi. in b. burgdorferi, cardiac involvement occurs in 1-8% of cases and death may occur as a result of conduction disturbances. in diphtheritic myocarditis myocardial damage is caused by the release of toxins. bartonella-induced silent myocarditis has been described as a cause of sudden unexpected cardiac death in athletes. granulomatous myocarditis may also lead to sudden death ( table 3 ). the mechanism of death includes arrhythmias, cardiac rupture, coronary occlusion, obstruction to pulmonary blood flow leading to fatal hemorrhage, and impaired myocardial contractility. cardiac tuberculosis is usually an autopsy diagnosis. histological examination of the myocardium shows a nodular, miliary, or diffuse infiltrative pattern. the coronary arteries may show narrowing or complete occlusion due to an intimal or diffuse tuberculous arteritis. it is uncommon to demonstrate acid-fast bacilli within the lesions. molecular tests such as the ligase chain reaction (lcr) and polymerase chain reaction (pcr) may be used to demonstrate the organism. sudden death in infective endocarditis occurs as a result of perforation of a free-wall myocardial abscess or rupture of a valve leaflet. staphylococcus aureus is responsible for 10-20% of cases and is the major cause in intravenous drug abusers. other bacterial causes include haemophilus, actinobacillus, cardiobacterium, eikenella, and kingella (hacek group). negative bacterial cultures may be found in 10% of cases as a result of prior antibiotic therapy. the most common sites of infection are the aortic and mitral valves, except in intravenous drug abusers, where the right-sided valves are primarily affected. tertiary syphilis causing aortitis may cause sudden death from rupture of aortic aneurysms with aortic dissection. the mechanism of death is either blood loss with hypovolemic shock or a fatal cardiac tamponade from intrapericardial rupture. bacterial infections of the respiratory system sudden death from acute epiglottitis occurs from respiratory obstruction caused by swelling of the epiglottic folds, uvula, and vocal cords. the most common cause of acute epiglottitis in developing countries is haemophilus influenzae type b. in countries with established immunization programs, the incidence of h. influenzae epiglottitis has decreased and other bacteria, such as streptococcus, staphylococcus, and pneumococcus, have been implicated as possible causes. postmortem blood cultures are positive in 50-75% of cases. lobar pneumonia ( figure 2 ) and confluent bronchopneumonia are the most frequent cause of sudden death from acute pulmonary disease. some 90-95% of lobar pneumonia is due to streptococcus pneumoniae type 3. bronchopneumonia is caused by staphylococci, streptococci, h. influenzae, pseudomonas aeruginosa, and coliform bacteria. pulmonary tuberculosis may result in hemoptysis, which can cause hypovolemic shock and sudden death. histologically, caseating granulomas are found. acid-fast bacilli are demonstrated using the ziehl-neelsen stain (figure 3) . corynebacterium diphtheriae produces a gray pseudomembrane from the pharynx to the larynx, and this may lead to respiratory obstruction and sudden death. legionnaire's disease is associated with outbreaks of sudden death. the disease is caused by legionella pneumophila, a facultative intracellular organism. it causes severe pneumonia in the elderly, in smokers, and in immunocompromised patients. the organisms may be transmitted via droplet spread from contaminated air-conditioning units and water coolers. the organism may be demonstrated by a modified silver stain (dieterle stain) or by immunofluorescence and culture. pyogenic meningitis may cause sudden death. the causative organism varies according to the age of the patient ( table 4) . the location of the exudates depends on the organism. in h. influenzae it is basally located. in pneumococcal meningitis it occurs over the convexities of the brain in the parasagittal region ( figure 4) . microscopic examination reveals neutrophils filling the subarachnoid space with extension of the inflammation into the leptomeningeal veins in fulminant cases. blood spread is the most common means of entry; however other routes of infection include local extension of infection, e.g., paranasal sinusitis, osteomyelitis, direct implantation, and via the peripheral nervous system. diffuse bacterial meningitis may follow rupture of a brain abscess, which may lead to sudden death. the organisms may be demonstrated by microbiological culture of the csf and examination of gram stains of the csf and brain tissue. bacterial urogenital tract infections fulminant acute bacterial pyelonephritis may lead to septicemia, causing sudden death. at autopsy, the kidneys show tubular necrosis with interstitial suppurative inflammation. renal papillary necrosis may also be present. severe bacterial enterocolitis may lead to sudden death, especially in the young. the pathogenesis of the diarrhea depends on the cause. vibrio cholerae and clostridium perfringens cause diarrhea by ingestion of a preformed toxin that is present in contaminated foods. enteroinvasive organisms such as salmonella, shigella, and enteroinvasive escherichia coli invade and destroy mucosal epithelial cells. death occurs as a result of dehydration and electrolyte imbalance. bleeding peptic ulcers that are caused by helicobacter pylori may be the first indication of an ulcer and account for 25% of ulcer deaths, many of which are sudden and unexpected. fulminant bacterial peritonitis secondary to acute appendicitis, acute salpingitis, ruptured peptic ulcer, diverticulitis, strangulated bowel, and cholecystitis may cause sudden death. primary peritonitis may occur postsplenectomy and in patients with splenic hypoplasia. patients with sickle-cell disease may have anatomical or functional asplenia. the former is due to repeated bouts of infarction leading to autosplenectomy. the latter is due to a defect in opsonization of encapsulated bacteria. massive bilateral adrenal hemorrhage with adrenocortical insufficiency may occur as a result of septicemic shock from overwhelming bacterial infection (waterhouse-friderichsen syndrome). the most common association is with neisseria meningitidis septicemia; however, other virulent organisms, e.g., h. influenzae and pseudomonas species, may also lead to this syndrome. sudden death due to fungal infection may occur in an immunocompromised host such as in hiv/aids. organisms include cryptococcus (meningitis or disseminated disease) and pneumocystis carinii (pneumonia). intravenous drug abusers are susceptible to endocarditis due to fungi such as candida. these patients are prone to fungal thromboembolism, leading to sudden death. sudden death may also be due to a complication of fungal diseases such as fatal subarachnoid hemorrhage complicating actinomycotic meningitis or fatal hemoptysis complicating pulmonary mucormycosis. diagnostic modalities include culture of the organism and the histological demonstration of the organisms in tissue. this may be facilitated by special stains such as the periodic acid-schiff (pas) or grocott's methenamine silver stain. fatal cardiac tamponade may occur with intrapericardial rupture of an amebic liver abscess due to entamoeba histolytica. fatal amebic meningoencephalitis may be caused by naegleria fowleri. the organism enters the arachnoid space through the cribriform plate of the nose. there is meningeal hemorrhage with fibrinoid necrosis of blood vessels. cerebral malaria does not usually cause sudden death. however, it may be the primary cause of sudden death in nonimmune persons. susceptible individuals are tourists, business travelers, and sailors. at autopsy, the brain is swollen and may have a ''slate gray'' color due to the brown-black malarial pigment called hemozoin. histology reveals petechial hemorrhages as well as intravascular parasitized red cells. small perivascular inflammatory foci called malarial or dã¼ rck's granulomas may be present. sudden death in malaria may also be due to rupture of an enlarged spleen. an enlarged spleen is fragile and more vulnerable to rupture. other infections that may lead to splenic rupture and sudden death are infectious mononucleosis and typhoid. sudden death due to cardiac involvement in chagas disease (trypanosoma cruzi) occurs in 5-10% of acute cases. the damage to the myocardium causes fatal ventricular tachycardia. histological examination shows myofiber necrosis with an acute inflammatory reaction. clusters of organisms may be found within dilated myofibers, resulting in intracellular pseudocysts. clinically occult helminthic diseases such as hydatid disease (echinococcus granulosus) and neurocysticercosis (taenia solium) may cause sudden death. in neurocysticercosis death may occur due to epilepsy or raised intracranial pressure. parasitic cysts containing scolices are present, especially in the subarachnoid space, cortical sulci, and cortical gray matter. large multilocular cysts (racemose cysts) may be present in the basilar cisterns near the cerebellopontine angle ( figure 5) . isolated cardiac hydatid cyst is an uncommon manifestation and accounts for fewer than 3% of all hydatid disease. sudden death may be the initial manifestation of the disease. death may be due to involvement of the left ventricular myocardium or to massive pulmonary embolism. all autopsies must be approached using universal precautionary principles. in sudden deaths complete autopsy examination is recommended with appropriate tissue and body fluid sampling for special investigations. autopsy sampling for microbiological investigations is indicated in the following circumstances: sudden unexpected deaths in children and adults, deaths in immunocompromised patients, deaths in patients with clinically suspected infections, and deaths with organ changes of infection. the problems encountered with autopsy microbiological testing are contamination during procurement of the sample because of poor technique or due to the postmortem spread of commensals. to prevent false-positive postmortem blood cultures the following should be observed: the body should be refrigerated as soon as possible; and movement of the body should be limited to decrease the possibility of postmortem bacterial spread. an aseptic technique should be used to collect the sample, which should be stored and transported in the correct medium and temperature. close liaison with the microbiology and virology laboratories is important to guide collection, preservation, transport, and evaluation of specimens. this is particularly important in cases where there are positive cultures with negative histological findings. sampling at multiple sites and determining the antibiotic sensitivities may be helpful in determining the significance of positive cultures. the finding of a ''pure'' as opposed to ''mixed'' culture helps to determine the significance of the findings. the type of organism in relation to the site where it was cultured also helps to differentiate contaminants from significant positive cultures. relevant special techniques should be used by the pathologist in order to improve the diagnostic yield in infectious diseases ( table 5) . in a small group of cases (so-called negative autopsies) no obvious cause of death is apparent after detailed initial external and internal examination. the incidence of negative autopsies is 5%-10%; this figure improves to about 5% when special tests such as postmortem chemistry and microbiology are carried out. infectious agents are not a common cause of sudden death. even in cases with little or no morphological changes, investigation of appropriate autopsy samples by recently developed laboratory techniques may prove invaluable and shed light on the cause of death. children: sudden natural infant and childhood death; sudden natural death: cardiovascular; central nervous system and miscellaneous causes in the year 2000 an estimated 815 000 people died from suicide around the world. this represents an annual global mortality rate of 14.5 per 100 000 population. according to the world health organization (who), suicide is the 13th leading cause of death worldwide. it leads among violent causes of death (e.g., suicide, homicide, traffic deaths). among those aged between 15 and 44 years, suicide is the fourth leading cause of death, and violence against the self is the sixth leading cause of disability. suicidal behavior ranges in degree from merely thinking about ending one's life, through developing a plan to commit suicide and obtaining the means to do so, attempting to kill oneself, to finally carrying out the act of ''completed suicide''. the term ''suicide'' is based on the latin words sui (of oneself) and caedere (to kill). the encyclopaedia britannica defines suicide as: ''the human act of self-inflicting one's own life cessation.'' however, it is often difficult to reconstruct the thoughts of people who commit suicide unless they have made clear statements before their death since all suicidal deaths are not clearly planned. in many legal systems, a death is certified as suicide if murder, accidental death, and natural causes can all be ruled out and if the circumstances are consistent with suicide. this article deals with fatal suicidal behavior. this is the term proposed for suicidal acts that result in death and that directly concern forensic medicine; it does not cover nonfatal suicidal behavior, attempted suicide, or deliberate self-harm, i.e., suicidal actions that do not result in death and which may be referred to psychiatrists. even if it is not always clearly planned, suicide is a result of an act deliberately initiated and performed by a person in expectation of its fatal outcome. suicide is also now a major public health problem, as evidenced by epidemiologic data. according to who, taken as an average for 53 countries for which complete data are available, the age-standardized suicide rate for 2000 was 14.5 per 100 000. the rate for males was 22.9 per 100 000 and for females 6.8 per 100 000. the rate of suicide is almost universally higher among men compared to women by an aggregate ratio of 3.5 to 1. for some countries the most recent data are shown in table 1 . over nearly 30 years , for 39 countries for which complete data are available, the suicide rates seem to have remained quite stable. geographically, changes in suicide rates vary considerably. according to the french national institute on demographic studies (ined; institut national des etudes dã©mographiques), which provides reliable information on suicide mortality, the rates range from 40.1 per 100 000 in the russian federation to 31.6 per 100 000 in hungary, 25.1 per 100 000 in japan, an introduction to neuropathology forensic pathology introduction to medico-legal practice general pathology of infectious diseases demonstration of infectious agents in tissue key: cord-021977-yu0hrg6h authors: pham, phuong-thu t.; danovitch, gabriel m.; pham, phuong-chi t. title: medical management of the kidney transplant recipient: infections and malignant neoplasms date: 2010-12-27 journal: comprehensive clinical nephrology doi: 10.1016/b978-0-323-05876-6.00101-5 sha: doc_id: 21977 cord_uid: yu0hrg6h nan despite prophylactic therapy against common bacterial, viral, and opportunistic pathogens in the perioperative and postoperative period, infections are the second most common cause of death after cardiovascular disease (cvd) in renal transplant recipients. according to the u.s. renal data system (usrds), infections occurred at a rate of 45 per 100 patient-years during the first 3 years after transplantation. 1 the most common infections are bacterial, followed by viral and fungal. parasitic infections are rare. notably, cytomegalovirus (cmv) and herpes simplex virus (hsv) infection rates have decreased since the mid-1990s as a result of effective antiviral prophylaxis; hepatitis b virus (hbv) and hepatitis c virus (hcv) infection rates increased during the same period for unclear reasons. both the type and occurrence of infections in the immunocompromised transplant recipient follow a "timetable pattern" (fig. 101.1 ). 2 although rare, both blood-borne and kidney infections have been transmitted during donation. these include viral infections (e.g., hcv, hbv, human immunodeficiency virus (hiv), cmv, and bk, among others), parasitic infections (malaria, babesia), and bacterial infections (from undiagnosed bacteremia or renal infections). most infections in the first month are due to common bacteria and candida acquired in the hospital setting. except for hsv, other viral infections are uncommon during this period. similar to those that follow any major surgical procedure, most bacterial infections during this period involve wounds, catheters, and drainage sites. aspiration pneumonia and urinary tract infections (utis) are common. infections specific to renal transplant recipients include perinephric fluid collections due to lymphoceles, wound hematomas, or urine leaks; indwelling urinary stents; and utis secondary to urinary tract abnormalities, such as ureteral stricture, vesicoureteral reflux, or neurogenic bladder. most utis are caused by common gram-negative bacteria (escherichia coli, enterobacteriaceae, and pseudomonas) and gram-positive bacteria (enterococcus). preventive measures for utis include early urethral catheter removal and antibiotic prophylaxis. trimethoprim-sulfamethoxazole or ciprofloxacin prophylaxis during the first 3 months after transplantation effectively reduces the frequency of utis to less than 10% and essentially eliminates urosepsis unless anatomic or functional derangement of the urinary tract is present. infections with multidrug-resistant microorganisms have recently emerged as an important cause of morbidity and mortality in organ transplantation. hence, in some centers, the routine use of antibiotic prophylaxis is no longer recommended. although strict aseptic surgical techniques and perioperative use of firstgeneration cephalosporins reduce the incidence of wound infections, infections are still observed, especially in subjects with comorbid conditions such as diabetes mellitus (dm) and obesity. antibiotic-associated clostridium difficile infection (particularly cephalosporins, ciprofloxacin, and amoxicillin-clavulanate) has become a serious epidemiologic problem worldwide. judicious use of antibiotic prophylactic therapy may decrease the incidence of iatrogenic c. difficile infections. whereas most infections during the first month are due to routine bacterial infections, nosocomial outbreaks have also been reported for rarer infections, such as legionella from contaminated hospital water supplies. during months 1 to 6, opportunistic infections secondary to immunosuppression are most common. viral infections, such as cmv, hsv, varicella-zoster virus (vzv), epstein-barr virus (ebv), hbv, and hcv, may occur from exogenous infection or reactivation of latent disease due to the immunosuppressed state. repeated courses of antibiotics and corticosteroid therapy increase the risk of fungal infections, whereas viral infections may not only result from the immunosuppression but may themselves further impair immunity to increase the risk for additional opportunistic infections. opportunistic infections may occur with pneumocystis jiroveci (previously pneumocystis carinii), aspergillus species, listeria monocytogenes, nocardia species, and toxoplasma gondii. trimethoprim-sulfamethoxazole prophylaxis (see early post-transplantation period, outbreaks of donor-transmitted viral infections, such as lymphocytic choriomeningitis and west nile virus, have been reported. lymphocytic choriomeningitis occurs within the first 4 weeks after transplantation and is associated with a greater than 90% mortality rate. 3 in the late posttransplantation period, infections with community-acquired viral pathogens, including vaccine-preventable diseases such as mumps and measles, have reemerged. there is currently no effective antiviral therapy against either infection, and adherence to current guidelines for vaccinations in solid organ transplantation is recommended (discussed later). other emerging or reemerging viral infections include adenovirus, human herpesvirus 6, metapneumovirus, parainfluenza, and respiratory syncytial virus. interestingly, only rare cases due to severe acute respiratory syndrome (sars) coronavirus have been reported. the following sections discuss selected infections in renal transplant recipients. suggested prophylactic therapy is shown in figure 101 cmv infection may be a primary infection in a seronegative recipient (donor seropositive, recipient seronegative), reactivation of endogenous latent virus (donor seropositive or seronegative, recipient seropositive), or superinfection with a new virus strain in a seropositive recipient (donor seropositive, recipient seropositive). primary cmv infection is usually more severe than reactive infection or superinfection. cmv infection occurs primarily after the first month of transplantation and continues to be a significant cause of morbidity in the first 6 months after organ transplantation through both direct and indirect effects. cmv infection may be asymptomatic, presenting as a mononucleosis-like syndrome or influenza-like illness with fever and leukopenia or thrombocytopenia, or a severe systemic disease. hepatitis, esophagitis, gastroenteritis with colonic ulceration, pneumonia, chorioretinitis (associated with retinal hemorrhage), and even otitis 4 may occur. in enterically drained pancreas transplantation, cmv has been reported to cause bleeding ulcer from the duodenal segment. clinical manifestations usually after 6 months, the infection risk can be categorized on the basis of the patient's status. the first category consists of the majority of transplant recipients (70% to 80%), who have satisfactory or good allograft function, relatively low doses of immunosuppression medication, and no history of chronic viral infection. the risk of infection in these patients is similar to that of the general population, with community-acquired respiratory viruses constituting the major infective agents. opportunistic infections are unusual unless environmental exposure has occurred. the second group (approximately 10% of patients) consists of those with chronic viral infection that may include hbv, hcv, cmv, ebv, bk virus, or papillomavirus. in the setting of immunosuppression, such viral infections may lead to the development of progressive liver disease or cirrhosis (hbv, hcv), bk nephropathy, post-transplantation lymphoproliferative disease (ebv), or squamous cell carcinoma (papillomavirus). the third group (approximately 10% of patients) consists of those who experience multiple episodes of rejection requiring repeated exposure to heavy immunosuppression. these patients are the most likely to develop chronic viral infections and superinfection with opportunistic organisms. causative opportunistic pathogens include p. jiroveci, l. monocytogenes, nocardia asteroides, and cryptococcus neoformans and geographically restricted mycoses (coccidioidomycosis, histoplasmosis, blastomycosis, and paracoccidioidomycosis). in these high-risk candidates, lifelong prophylactic therapy with trimethoprim-sulfamethoxazole (80 mg/ 400 mg daily) has been advocated. lifelong antifungal prophylaxis should also be considered and environmental exposure minimized (primarily avoidance of pigeons and areas of active building construction). several uncommon viral infections have recently been reported in both the early and late post-transplantation periods. 3 in the figure 101 .1 timetable of infections.* *geographically focused infections will need to be considered in certain cases, such as malaria, leishmaniasis, trypanosomiasis, and strongyloidiasis. 1 sources of infections specific to recipients of renal transplant: perinephric fluid collections (e.g., lymphoceles, wound hematomas, urine leaks), indwelling urinary stents, or anatomic or functional genitourinary tract abnormalities (e.g., ureteral stricture, vesicoureteric reflux, neurogenic bladder blood, such as cmv dna polymerase chain reaction (pcr) or pp65 antigenemia during surveillance studies. the former assay is highly specific and sensitive for the detection of cmv viremia. the latter is a semiquantitative fluorescent assay in which circulating neutrophils are stained for nonspecific uptake of cmv early antigen (pp65). various prophylactic and preemptive protocols have been developed. oral acyclovir provides effective cmv prophylaxis solely in recipients of seronegative donor organs. oral or intravenous ganciclovir or oral valganciclovir provides superior prophylactic or preemptive therapy against primary cmv infection or cmv reactivation. prophylactic or preemptive therapy should be based on the intensity of immunosuppression (i.e., during antilymphocyte antibody therapy) and the seropositive status of the donor, the recipient, or both. seronegative individuals who receive organs from latently infected seropositive donors are at greatest risk for primary infection and severe cmv disease. a suggested cmv prophylaxis protocol is shown in figure 101 clinical cmv disease is treated with intravenous ganciclovir (5 mg/kg twice daily for 3 weeks, dose adjusted for renal dysfunction) with reduction of immunosuppression, such as withholding of mmf. treatment is continued until clearance of viremia as assessed by pcr or antigenemia. anecdotal reports have suggested that calcineurin inhibitor (cni) to sirolimus switch in conjunction with ganciclovir therapy may be beneficial in patients with apparent ganciclovir-resistant cmv. 8 in patients with gastrointestinal cmv infection, the use of these assays is unreliable, and repeated endoscopy should be considered to assess response to therapy. in patients who have primary infection and respond slowly to therapy, the addition of cmv hyperimmune globulin (150 mg/kg per dose given intravenously every 3 to 4 weeks for 3 months) may be of benefit. 5 in patients with tissue invasive disease, intravenous ganciclovir is recommended with conversion to oral therapy when there is evidence of a good response, followed by a 3-month course of oral ganciclovir or valganciclovir prophylaxis. 5 whereas oral valganciclovir provides good bioavailability and may be effective in mild cmv disease, it is not recommended for the treatment of occur 1 to 4 months after transplantation except for chorioretinitis, which occurs later in the transplant course. 5 quantitative cmv assays of serum in patients with invasive colitis and gastritis or neurologic disease including chorioretinitis are often negative. diagnosis in such cases may require invasive testing and biopsies. cmv infection is associated with immune modulation and dysregulation of helper/suppressor t cells and may be a risk factor for chronic allograft rejection, secondary infection with opportunistic agents (such as p. jiroveci, candida, and aspergillus), reactivation of human herpesvirus hhv-6 and hhv-7, and the development of post-transplantation lymphoproliferative disease. cmv infection is also associated with acceleration of hcv infection and the development of new-onset dm after transplantation. 6 donor and recipient seropositive status and the use of blood products from a cmv-seropositive donor are well-established risk factors for cmv infection. other factors associated with an increased risk of cmv infection include the use of antilymphocyte antibodies, prolonged or repeated course of antilymphocyte preparations, comorbid illnesses, neutropenia, and acute rejection episodes. mycophenolate mofetil (mmf) has been reported to increase the risk for cmv viremia and cmv disease in some studies, especially in patients receiving more than 3 g/day. although the cause-effect of allograft rejection and cmv infection remains conjectural, several studies suggest that one may increase the risk for the other, possibly owing to the release of inflammatory cytokines. prevention of cmv infection, for example, results in a lower incidence of graft rejection. 7 prophylactic therapy begins in the immediate postoperative period. preemptive therapy involves treatment of those who are found to seroconvert by quantitative laboratory assays of the † check glucose-6-phosphate dehydrogenase deficiency before initiation of therapy. ‡ in order of efficacy. § fluconazole is recommended for recipients of combined kidney-pancreas or combined kidney-liver transplants. consider reinstitution of prophylactic therapy for 3 months after acute rejection episodes requiring intensification of immunosuppression. cmv, cytomegalovirus. trimethoprim-sulfamethoxazole (tmp-its routine use reduces or eliminates the incidence of smz)* (80/400 mg) one tablet daily pneumocystis jiroveci, listeria monocytogenes, × 3 months nocardia asteroides, and toxoplasma gondii in renal transplant recipients, tmp-smz reduces the incidence of urinary tract infection from 30%-80% to <5%-10% monthly intravenous or aerosolized replaces tmp-smz for patients with sulfa allergies pentamidine > dapsone † > or atovaquone ‡ nystatin 100,000 units/ml, 4 ml after for fungal prophylaxis meals and before bedtime or fluconazole § 200 mg one tablet daily close monitoring of cyclosporine or tacrolimus levels × 2 months when starting and stopping antifungal agents acyclovir/valganciclovir/ganciclovir for cmv prophylaxis, see figure 101 .3 bk virus is a ubiquitous human virus with a peak incidence of primary infection in children 2 to 5 years of age and a seroprevalence rate of more than 60% to 90% among the adult population worldwide. after primary infection, bk virus preferentially establishes latency within the genitourinary tract and frequently is reactivated in the setting of immunosuppression. in renal transplant recipients, bk virus is associated with a range of clinical syndromes including asymptomatic viruria with or without viremia, ureteral stenosis and obstruction, interstitial nephritis, and bk allograft nephropathy. during the last decade, bk nephropathy has emerged as an important cause of allograft dysfunction after renal transplantation. most series report that 30% to 40% of renal transplant recipients develop bk viruria, 10% to 20% develop bk viremia, and 2% to 5% develop bk nephropathy. the highest prevalence of bk viruria and viremia occurs at 2 to 3 months and 3 to 6 months, respectively. the risk for development of bk viremia increases when urine viral load is greater than 10 4 copies/ml, whereas bk nephropathy is unusual in the absence of bk viremia. bk nephropathy commonly presents with an asymptomatic rise in serum creatinine during the first posttransplantation year. however, bk nephropathy may occur as early as the first week to as late as 6 years after transplantation. diagnosis is made by allograft biopsy, which demonstrates bk viral inclusions in renal tubular cell nuclei and occasionally in glomerular parietal epithelium (fig. 101.4a ). there are variable degrees of interstitial mononuclear inflammation (fig. 101 .4b), often with plasma cells, degenerative changes in tubules, and focal tubulitis, which may mimic acute rejection. bk nephropathy often is associated with very focal and sharply demarcated areas of tubulointerstitial inflammation, corresponding to foci of viral infection. immunohistochemistry (fig. 101.4c ), in situ hybridization, or electron microscopy is required to confirm the diagnosis. bk infection and acute rejection may occur simultaneously, and distinguishing between bk nephropathy and acute rejection or the presence of both can be a diagnostic challenge. in late bk nephropathy, few characteristic intranuclear inclusions are seen, and the histologic changes may be indistinguishable from chronic rejection. a histologic classification system for bk nephropathy based on the degree of active inflammation, acute tubular injury, and tubulointerstitial scarring may have prognostic significance. 10 urine cytology for decoy cells and quantitative determinations of viruria and of viral load in blood have been proposed as surrogate markers for the diagnosis of bk nephropathy. treatment strategies include reduction in immunosuppression that involves reduction or discontinuation of mmf and azathioprine with judicious reduction in cni therapy or other immunosuppressive regimen. switching from tacrolimus to cyclosporine or to sirolimus (rapamycin) has resulted in resolution of bk nephropathy and viremia or viruria in anecdotal case reports. switching from cni to sirolimus may have the added benefit of avoiding the long-term nephrotoxic effect of cni therapy. although no approved antiviral drug is available, adjunctive therapy with leflunomide, cidofovir, quinolones, or intravenous immune globulin (ivig) may be beneficial, especially in patients with progressive allograft dysfunction. quinolones are preferred by some centers because of low cost and ease of administration; leflunomide is used by others because of its potential simultaneous antiviral and immunosuppressive properties. cidofovir is highly concentrated in urine and renal tissue, and the use of low-dose cidofovir in bk nephropathy has been reported to be devoid of nephrotoxicity or serious adverse events. anecdotal established cmv disease, and intravenous ganciclovir is required. cidofovir and foscarnet are alternative therapeutic agents, but in view of their nephrotoxicity and potential synergistic nephrotoxicity with cnis, they are reserved for use when ganciclovirresistant strains are clinically suspected. candida infections are common in transplant recipients; candida albicans and candida tropicalis account for 90% of the infections. dm, high-dose corticosteroids, and broad-spectrum antibacterial therapy predispose patients to mucocutaneous candidal infections such as oral candidiasis, intertriginous candidal infections, esophagitis, vaginitis, and uti. skin infections are treated with nystatin and topical clotrimazole; candidal utis are treated with fluconazole or voriconazole or more rarely with liposomal amphotericin or caspofungin for fluconazole-resistant species (see chapter 53). whenever possible, foreign objects such as bladder catheters, surgical drains (e.g., percutaneous nephrostomy tube), and urinary stents should be promptly removed. the ideal management of asymptomatic candiduria in immunocompromised patients remains uncertain (see chapter 53), but a short course (7 to 10 days) of fluconazole is generally recommended. systemic antifungal therapy is indicated in the presence of any positive blood culture for candida species. 1 if cmv status is unknown, give intravenous dhpg until cmv status is determined. 2 dose adjustment for renal function is necessary. dhpg, 9-(1,3-dihydroxy-2-propoxymethyl) guanine. 3 although low-dose valganciclovir, 450 mg daily, has been shown to be effective, the canadian society of transplantation consensus workshop on cmv management recommends dosing valganciclovir at 900 mg daily for cmv+ recipients of a cmv+ organ (kidney, liver, pancreas, heart). (from reference 9.) prophylaxis protocol 1 acyclovir 400 mg daily (or valganciclovir 450 mg daily) × 3 months cmv dna every 2 weeks × 3 months during antibody treatment, dhpg 2 5.0 mg/kg iv everyday, then following antibody treatment/valganciclovir 900 mg po everyday × 6 months if no antibody treatment: valganciclovir 900 mg everyday for 6 months cmv dna every 2 weeks x 3 months during antibody treatment, dhpg 5.0 mg/kg iv everyday, then following antibody treatment, valganciclovir 900 mg po everyday × 6 months if no antibody treatment: acyclovir 400 mg daily (or valganciclovir 450 mg daily) × 3 months cmv dna every 2 weeks × 3 months during antibody treatment, dhpg 5.0 mg/kg iv everyday, then following antibody treatment, valganciclovir 900 mg po everyday × 6 months if no antibody treatment: acyclovir 400 mg daily (or valganciclovir 450 mg daily) 3 × 3 months cmv dna every 2 weeks × 3 months reports have suggested that ivig may be effective in treating corticosteroid-resistant rejection, 11 and its use may be beneficial in patients with concomitant rejection and bk nephropathy or in those with histopathologic changes that are indistinguishable from those of rejection. despite treatment, 30% to more than 60% of patients with established bk nephropathy developed progressive decline in renal function with graft loss. early diagnosis and intervention may improve prognosis. intensive monitoring of urine and serum for bk by pcr during the first year with preemptive reduction of immunosuppressive therapy may lead to the resolution of viremia and prevent bk nephropathy. in the absence of active viral replication, patients with graft loss due to bk nephropathy can safely undergo retransplantation. active surveillance for bk virus reactivation after transplantation is recommended. suggested guidelines for post-transplantation screening and monitoring for bk replication are shown in figure 101 .5. tuberculosis (tb) infection in the renal transplant recipient varies according to the prevalence in the general population (e.g., the incidence of tb in transplant recipients has been reported to occur in 0% to 1.3% in the united states, compared with 11% in south africa and 11% to 14% in india and pakistan). 12 most tb infection in the transplant recipient results from reactivation of dormant lesions in the setting of immunosuppressive therapy. hence, all renal transplant candidates should have a ppd skin test (tuberculin skin test) placed before transplantation. a positive skin test response or a prior history of tb mandates further evaluation to rule out active disease. isoniazid prophylaxis for a total of 9 months is recommended for those who have a positive skin test response. of interest, most of the patients who develop tb after transplantation had negative ppd skin test results before transplantation. 13 some centers recommend isoniazid prophylactic therapy in selected ppd-negative patients with (1) a history of inadequately treated tb, (2) radiographic evidence of granulomatous disease and no history of adequate treatment, (3) an organ from a ppd-positive donor, or (4) close and prolonged contact with a case of active tb. 13 in patients with a known history of adequately treated tb infection, we advocate the use of isoniazid prophylaxis for the first 9 months after transplantation and during intensification of immunosuppression. others, however, have suggested that isoniazid prophylaxis is not indicated for those patients whose tb had been properly treated. 12 clinical, radiologic, or culture evidence of active tb infection is a contraindication to transplantation. enzyme-linked immunospot (elispot), which detects t cells specific for mycobacterium tuberculosis antigens, is unaffected by bacille calmette-guérin (bcg) vaccination and has become a major advance in tb screening. in some centers, the tuberculin skin test has been replaced by the elispot assays (t-spot.tb assay). a rare but important cause of infection in transplant patients, particularly those from endemic areas such as southeast asia, is strongyloides. in the presence of immunosuppression, a "hyperinfection" syndrome may be observed with parasitic pneumonia (fig. 101.6 ) and gastrointestinal involvement. post-transplantation gastrointestinal complications are common and can arise from a variety of causes. only selected complications are discussed; for a comprehensive review of nystatin "swish and swallow" during the first month after transplantation is recommended. in high-risk candidates, including liver or pancreas transplant recipients and those receiving antilymphocyte antibody therapy, fluconazole prophylactic therapy (3 to 6 months) is warranted. clostridium difficile infection may be asymptomatic or present with diarrhea, intestinal obstruction, or even fulminant pseudomembranous colitis with toxic megacolon and perforation. c. difficile colitis is reported in 3.5% to 16% of transplant recipients. 15 risk factors include young (<5 years) or advanced age, female gender, use of monoclonal antibodies to treat acute rejection episodes, and intra-abdominal graft placement. among transplant recipients receiving antimicrobial therapy, c. difficileassociated diarrhea develops in approximately 50% of patients. 15 in mild cases of c. difficile infection, oral metronidazole is as effective as oral vancomycin and is the preferred first-line treatment. treatment failure, however, requires treatment with oral vancomycin. in severely ill patients with gastrointestinal dysmotility or ileus, in which oral agents may not reach the colonic mucosa, metronidazole should be administered intravenously. severe colonic disease refractory to medical treatment may necessitate colectomy. helicobacter pylori infection is associated with a wide range of gastrointestinal complications including chronic gastritis, duodenal and gastric ulcers, mucosa-associated lymphoid tissue (malt) lymphoma, and gastric carcinoma, both in the general population and in recipients of solid organ transplants. treatment includes a triple-drug regimen consisting of two antibiotics and an acid-suppressive agent such as an h 2 blocker or a proton pump inhibitor. the first-line h. pylori regimen as recommended by the american college of gastroenterology is shown in figure 101 .7. in recipients of orthotopic heart transplants, triple-drug therapy resulted in a lower eradication rate compared with the general population, suggesting that immunosuppression may hinder the clearance of h. pylori. unexplained dyspeptic or reflux symptoms should be investigated further with endoscopy and biopsy to exclude malignant transformation. h. pylori is now recognized as a risk factor for malt lymphoma, which may occur in kidney, liver, and heart transplant recipients. in renal transplant recipients infected with h. pylori, malt lymphoma may be less aggressive than other lymphomas, and the disorder may be cured by eradication of h. pylori. post-transplantation colonic complications, such as diverticulitis and colonic perforation, may be life-threatening and difficult to diagnose because symptoms may be masked by immunosuppressive therapy, particularly in the early postoperative period. diverticulitis complicated by perforation, abscess formation, phlegmon, or fistula has been reported to occur in 1.1% of renal transplant recipients 16 and may be increased in patients with polycystic kidney disease (pkd). early post-transplantation colonic perforations are largely due to high-dose corticosteroids, diverticulitis, cmv colitis, and intestinal ischemia; perforations occurring late or years after transplantation are commonly due to diverticulosis or malignant disease. abdominal symptoms may be absent because of the effects of immunosuppression and may only be suggested by the post-transplantation gastrointestinal complications, readers are referred to chapter 83 and references 13 and 14. mmf commonly causes gastrointestinal side effects, including nausea, vomiting, dyspepsia, anorexia, flatulence, and diarrhea. dose reduction, transient discontinuation of the drug, or dividing the dose into three or four times a day often ameliorates or resolves the symptoms. switching to the enteric-coated formulation of mmf may improve gastrointestinal tolerability in some patients but has not been consistently shown to be better than the original formulation. a large randomized double-blind study using patient-reported outcomes to assess the impact of gastrointestinal symptoms on patients' health-related quality of life and symptom burden is currently under way. sirolimus may cause oral mucocutaneous lesions that can be confused with hsv or cmv infection but are culture negative. drug-related oral ulcers usually resolve after discontinuation of the offending agent. sirolimus, tacrolimus, and cyclosporine have also been suggested to cause diarrhea in some patients. post-transplantation infections of the gastrointestinal tract may be viral, fungal, or bacterial in etiology. viral infections are most commonly caused by cmv and hsv; c. albicans and c. tropicalis are common opportunistic fungal infections. leukoplakia and post-transplantation lymphoproliferative disorder (ptld) may develop in patients with ebv infection (ptld is discussed in a later section). commonly encountered bacterial pathogens include clostridium difficile and helicobacter pylori. cmv can affect any segment of the gastrointestinal tract. patients may present with dysphagia, odynophagia, nausea, vomiting, gastroparesis, abdominal pain, diarrhea, or gastrointestinal bleeding. leukopenia and elevated transaminases are common. persistent or unexplained symptoms of nausea, vomiting, or diarrhea, particularly in the early post-transplantation period or during intensification of immunosuppression, warrant further investigation with upper or lower endoscopies and biopsies. hsv infection results primarily from reactivation of endogenous latent virus, causing clinical infection within the first 1 to 2 months after transplantation. patients commonly present with oral mucocutaneous lesions or gingivostomatitis with or without odynophagia and dysphagia. hsv esophagitis has been noted to occur in patients receiving high-dose corticosteroids and antilymphocyte preparations for acute rejection. limited oral mucocutaneous lesions are treated with oral acyclovir; extensive infections require intravenous acyclovir or ganciclovir. rare cases of hsv hepatitis have been reported. 14 the routine use of acyclovir prophylaxis in the early post-transplantation period is recommended. candida stomatitis and esophagitis are common during the first 6 months after transplantation and are increased in subjects with leukopenia or with severe immunosuppression, diabetes, or concomitant infections. bleeding or perforation with formation of tracheoesophageal fistulas has been reported. prophylactic oral recipients of organ transplants are at increased risk for development of neoplasms compared with the general population. similar to post-transplantation infectious complications, the time to occurrence of different types of malignant neoplasms after transplantation appears to follow a timetable pattern. the israel penn international transplant tumor registry data on the time of appearance of different neoplasms after solid organ transplantation are shown in figure 101 .9. ptld generally occurs early after transplantation; skin cancers occur with increasing frequency with time. the intensity and duration of immunosuppression as well as the ability of these agents to promote replication of various oncogenic viruses are important risk factors. the associations between human papillomaviruses and cervical and vulvar carcinoma, ebv and ptld, hbv and hcv and hepatocellular carcinoma, and hhv-8 and kaposi's sarcoma are well established. figure 101 .10 provides a summary presence of tachypnea and tachycardia. mortality after colonic perforation is high. management includes prompt exterioration of the perforated colon, early and broad-spectrum antimicrobial therapy, and minimization of immunosuppressive therapy. although uncommon, the presence of abdominal pain and gastrointestinal bleeding with unexplained fevers or weight loss should raise the suspicion for gastrointestinal tb. the characteristic endoscopic findings include circular ulcers, small diverticula, and sessile polyps. the presence of caseating granulomas or acid-fast bacilli, or both, confirms the diagnosis. all potential renal transplant candidates should receive immunization for hepatitis b, pneumococcus, and other standard immunizations appropriate for age. up-to-date recommendations for routine adult immunizations are available through the centers for disease control and prevention website (www.cdc.gov/nip/rec/ adult-schedule.pdf). immunizations should ideally be administered at least 4 to 6 weeks before transplantation to achieve optimal immune response and to minimize the possibility of live vaccinederived infection in the post-transplantation period. household members, close contacts, and health care workers should also be fully immunized. live virus or live organism vaccines should be avoided after transplantation. these include measles-mumps-rubella (mmr), live oral poliovirus (which is also contraindicated for household contacts), smallpox (vaccinia), varicella, yellow fever, adenovirus, live oral typhoid (ty21a), bcg, and intranasal influenza vaccine. in addition, exposure to persons who have chickenpox or herpes zoster should be avoided until the lesions have crusted over and no new lesions are appearing. vaccinations using inactivated or killed microorganisms, components, and recombinant moieties are safe for transplant recipients. these include hepatitis a and hepatitis b, intramuscular influenza a and b, pneumococcal, haemophilus influenzae b, inactivated polio virus vaccine, diphtheria-pertussis-tetanus (dpt), and neisseria meningitidis. in general, vaccination should be avoided in the first 6 months after transplantation because of the potential for stimulating the immune response, with a higher chance of graft dysfunction and rejection. in addition, vaccinations within the first 6 months after transplantation are often ineffective because of heavy immunosuppression. for prevention of infection in adult travelers after solid organ transplantation, readers are referred to reference 17. recommended vaccinations before and after transplantation are listed in figure 101 .8. no * standard-dose ppi twice daily (or esomeprazole once daily) + clarithromycin 500 mg twice daily + amoxicillin 1000 mg twice daily for 10-14 days yes † standard-dose ppi twice daily + clarithromycin 500 mg twice daily + metronidazole 500 mg twice daily for 10-14 days yes bismuth subsalicylate 525 mg orally 4 times daily + metronidazole 250 mg orally 4 times daily + tetracycline 500 mg orally four times daily + ranitidine 150 mg orally twice daily (or standard-dose ppi once daily to twice daily) for 10-14 days measles-mumps-rubella x renal cell carcinomas (27%), and breast carcinomas (23%). 21 in an analysis of registry data involving 90 patients with a history of pretransplantation prostate adenocarcinoma (77 renal, 10 heart, and 3 liver transplant recipients), prostate cancer recurrences were found to relate to the stage of disease at initial diagnosis. 22 tumor recurrence rates were 14%, 16%, and 33% for stage i, stage ii, and stage iii diseases, respectively. hence, a longer waiting time may be necessary for more advanced disease. suggested guidelines for tumor-free waiting periods for common pretransplantation malignant neoplasms are shown in figure 101 .12. ptld is the most common post-transplantation malignant neoplasm in children; in adults, it is the second most common malignant neoplasm after skin cancer. ptld has been reported to occur in 1% to 5% of renal transplant recipients. the majority of ptld is non-hodgkin's lymphoma of b-cell origin, and more than 80% to 90% are linked to ebv infection. based on the world health organization classification, ptld can be divided into three distinct morphologic groups: (1) diffuse b-cell hyperplasia, (2) polymorphic ptld (usually monoclonal), and (3) monomorphic ptld that includes high-grade invasive lymphoma of b-or t-lymphocyte centroblasts. diffuse b-cell hyperplasia is usually seen in children and young adults and commonly occurs within the first year after transplantation. polymorphic ptld represents the most common type of ptld in both children and adults and may occur at any time after transplantation. in contrast, monomorphic b-cell ptld is often seen several years after transplantation and may resemble non-hodgkin's lymphoma in the general population. in a retrospective analysis of registry data for 402 recipients of kidney transplants, ptld occurred at a median of 18 months (range, 1 to 310 months) after transplantation. ptld may present with constitutional symptoms such as fevers, night sweats, and weight loss or localized symptoms of the respiratory tract (infection or mass, including tonsillar or even gingival involvement), gastrointestinal tract (diarrhea, pain, perforation, bleeding, mass), or central nervous system (cns) (headache, seizure, confusion). in contrast to lymphomas in the general population, in which lymph nodes are almost always involved, lymph node involvement is absent in more than 80% of patients with ptld. risk factors for ptld include primary ebv infection, younger age, antecedent history of cmv disease, and use of antilymphocyte antibody (e.g., antithymocyte globulin, okt3). a history of pretransplantation malignant disease and fewer hla matches are associated with an increased risk of ptld. cyclosporine and tacrolimus may enhance the development of ebv-associated ptld by directly promoting the survival of ebv-infected b cells, presumably through the inhibition of ebv-transformed cells from apoptosis. 23 reduction or discontinuation of immunosuppressive therapy, particularly antilymphocyte antibody, cyclosporine, tacrolimus, or mmf, is recommended as first-line treatment; prednisone is increased to 10 to 15 mg daily to prevent allograft rejection. sirolimus has a strong antiproliferative effect on ptld-derived b-cell lines, 24 but whether sirolimus may limit b-cell lymphoma growth while simultaneously providing immunosuppression to prevent graft rejection awaits studies. acyclovir or ganciclovir therapy and reduction in immunosuppression are beneficial and may be curative in benign polyclonal b-cell proliferation. the of the incidence of cancers related to infections in transplant recipients. 18 an analysis of the usrds database 19 documented that the cancer rates for most common cancers, such as colon, lung, prostate, stomach, esophagus, pancreas, ovary, and breast, are nearly twofold higher after kidney transplantation compared with the general population. although registry studies have limitations, all transplant recipients should adhere to standard cancer surveillance appropriate for age (fig. 101.11 ). 20 in patients with a history of pre-transplantation malignant neoplasms, close monitoring for recurrences in the post-transplantation period is mandatory. the highest recurrence rates have been observed with multiple myeloma (67%), non-melanoma skin cancers (53%), bladder carcinomas (29%), sarcomas (29%), symptomatic of 375 mg/m 2 ) in patients with ptld (in conjunction with reduction in immunosuppression) have shown promising results. complete remission rates of 30% to 60% have been reported. 24 although the response rates appear to vary substantially among patients and centers, rituximab in conjunction with reduction in immunosuppression is evolving as the treatment of choice for cd20 + ptld. the role of cytokine-based therapy, such as interferon alfa and anti-il-6, remains poorly defined 25 ; increased risk of allograft rejection is seen with anti-il-6 treatment. sirolimus, an immunosuppressant with antiproliferative properties, has been demonstrated to prevent proliferation of b-cell (but not t-cell) ptld-derived tumor cell lines in vitro and in vivo. 26 limited data from nine european transplant centers have shown tumor regression in 15 of 19 patients with ptld who underwent minimization or withdrawal of cnis and sirolimus conversion. 27 factors that adversely affect survival include multiple-versus single-site involvement, increasing age, b-cell predominance, use of antilymphocyte globulin or antithymocyte globulin and okt3, and "early" versus "late" onset (within 6 to 12 months versus more than 12 months). in recipients of renal transplants with ptld restricted to the allograft alone, transplant nephrectomy may improve survival. role of antiviral therapy in b-cell monoclonal malignant transformation is less well defined; 50% to 90% mortality has been reported despite antiviral therapy. surgical resection with or without adjunctive local irradiation has been suggested for localized disease. local irradiation has been advocated as the treatment of choice for ptld involving the central nervous system. in lesions not amenable to surgery or more aggressive monoclonal types of ptld, chemotherapy has been used with favorable results compared with reduction in immunosuppression alone. the most frequently used regimens are chop (cyclophosphamide, doxorubicin [adriamycin], vincristine, and prednisone) and vapec-b (doxorubicin, etoposide, cyclophosphamide, methotrexate, bleomycin, and vincristine). other reported promising novel therapies include promace-cytabom (prednisone orally, doxorubicin, cyclophosphamide, etoposide-cytarabine, bleomycin, vincristine [oncovin], methotrexate). 25 adverse effects of chemotherapy include high mortality rates from sepsis and treatment-related toxicities. rituximab, a chimeric monoclonal antibody with murine variable regions targeting the cd20 antigen and human igg1-κ constant regions, has antitumor activity against cd20-expressing b-cell lymphomas. early experiences with rituximab (two to six weekly doses figure 101 .11 preventive care recommendations for cancer surveillance in renal transplant recipients. 1 as recommended by the american transplant society and the european best practice guidelines on renal transplantation. 2 the american college of preventive medicine recommends regular screening for high-risk individuals but none for low-risk individuals. 3 neoplasms. it has been proposed by experts in the field that immunosuppression dose reduction or withdrawal may permit recovery of the immune system and control the progression of life-threatening malignant neoplasms. the former allows intact immune surveillance against malignant cells. nonetheless, this approach is not without its attendant risk of graft rejection and graft loss. furthermore, little is known as to how much and to what extent immunosuppression reduction or withdrawal might alter the natural history of established post-transplantation malignant neoplasms. in our opinion, cni to sirolimus switch or cni minimization in conjunction with sirolimus may be a viable therapeutic option (the antitumoral effect of sirolimus is discussed later). in patients with metastatic cancer, manipulation of immunosuppression is probably futile, and the risk of rejection and graft loss necessitating a return to dialysis is likely to outweigh the benefit. studies suggest that immunosuppressive agents have different effects on cancer risk after transplantation. the carcinogenic effects of okt3, antithymocyte globulin, cyclosporine, tacrolimus, and azathioprine have been well documented. in contrast to azathioprine, mmf has been shown to have antiproliferative effects and has been suggested to protect against posttransplantation malignant neoplasms. 30, 31 analysis of more than 17,000 adult patients with preexisting dm indicated a significantly higher incidence of malignant transformation in azathioprine-treated than in mmf-treated patients (3.7% versus 2.2%; p < .01). 31 however, whether mmf is protective of posttransplantation malignant neoplasia remains speculative. both preclinical and clinical studies have demonstrated that mtor inhibitors such as sirolimus and everolimus have antiproliferative and antitumor effects. early studies in renal transplant recipients demonstrated a lower incidence of skin cancer with sirolimus-based therapy without cyclosporine or sirolimus maintenance therapy after early cyclosporine withdrawal compared with those who remained on cyclosporine and sirolimus combination therapy. it has been suggested that the protective effect of sirolimus against skin cancer is due to its inhibition of several ultraviolet light-induced mechanisms involved in skin carcinogenesis. the 5-year malignancy data of the rapamune maintenance regimen trial demonstrated a lower incidence of both skin and non-skin cancers at 5 years after transplantation in recipients receiving sirolimus-based therapy and cyclosporine withdrawal at month 3 compared with those receiving sirolimus and cyclosporine combination therapy. 29 sirolimus therapy has also been reported to result in successful clinical and histologic remission of kaposi's sarcoma in renal transplant recipients. 32 although sirolimus appears to provide satisfactory outcomes in certain cancers after transplantation, its use in the management of malignant disease after solid organ transplantation remains to be defined and should be tailored to each individual patient. there is no consensus on the management of immunosuppressive therapy in patients with post-transplantation malignant rates of first infection following kidney transplantation in the united states infection in organ transplant recipients emerging virus in transplantation: there is more to infection after transplant than cmv and ebv sun-exposed areas and 7 times higher in non-sunexposed areas. the use of sirolimus, an inhibitor of mammalian target of rapamycin (mtor)-induced signaling, may delay the onset or reduce the incidence of post-transplantation skin and non-skin malignant neoplasms (discussed under management of post-transplantation malignant neoplasms) risk factors for skin cancer include light skin color, intensity of sun exposure (ultraviolet light exposure), genetic factors, and duration of follow-up after transplantation. in addition, immunosuppression in combination with enhanced sunlight exposure may induce malignant changes in papilloma cancer screening in renal transplant recipients: what is the evidence? evaluation of transplant candidates with pre-existing malignancies prostate cancer prior to solid organ transplantation: the israel penn international transplant tumor registry experience effect of cyclosporine and tacrolimus on the growth of esptein-barr virus-transformed b-cell lines rapamycin inhibits the interleukin 10 transduction pathway and the growth of epstein-barr virus b cell lymphomas prospective study of sequential reduction in immunosuppression, interferon alpha-2, and chemotherapy for posttransplant lymphoproliferative disorder the immunosuppressive macrolide rad inhibits growth of human epstein-barr-virus-transformed b lymphocytes in vitro and in vivo: a potential approach to prevention and treatment of posttransplant lymphoproliferative disorders post-transplant lymphoproliferative disorder-the potential of proliferation signal inhibitors immunosuppressants and skin cancers in transplant patients: focus on rapamycin sirolimus therapy after early cyclosporine withdrawal reduces the risk for cancer in adult renal transplantation the changing pattern of posttransplant malignancies post-transplant de novo malignancies in renal transplant recipients: the past and the present cancers after renal transplantation infection in renal transplant recipient new onset diabetes mellitus after transplantation valacyclovir for the prevention of cytomegalovirus disease after renal transplantation. international valacyclovir cytomegalovirus prophylaxis transplantation study group the use of sirolimus in ganciclovir-resistant cytomegalovirus infections in renal transplant recipients canadian society of transplantation consensus workshop on cytomegalovirus management in solid organ transplantation final report histological patterns of polyomavirus nephropathy: correlation with graft outcome and viral load reversal of steroid-and anti-lymphocyte antibody-resistant rejection using intravenous immunoglobulin (ivig) in renal transplant recipients tuberculosis and renal transplantation mycobacterium tuberculosis gastrointestinal complications of transplant immunosuppression gastrointestinal complications in renal transplant recipients herpes simplex virus hepatitis after renal transplantation prevention of infection in adults travelers after solid organ transplantation incidence of cancers in people with hiv/aids compared with immunosuppressed transplant recipients: a meta-analysis cancer after kidney transplantation in the united states key: cord-003053-5sucu1cg authors: yang, liu; xie, honglang; liu, zhengzhao; chen, yinghua; wang, jinquan; zhang, haitao; ge, yongchun; hu, weixin title: risk factors for infectious complications of anca-associated vasculitis: a cohort study date: 2018-06-14 journal: bmc nephrol doi: 10.1186/s12882-018-0933-2 sha: doc_id: 3053 cord_uid: 5sucu1cg background: severe infections are common complications of immunosuppressive treatment for antineutrophil cytoplasmic antibody (anca)-associated vasculitis (aav) with renal involvement. we investigated the clinical characteristics and risk factors of severe infection in chinese patients with aav after immunosuppressive therapy. methods: a total of 248 patients with a new diagnosis of anca-associated vasculitis were included in this study. the incidence, time, site, and risk factors of severe infection by the induction therapies were analysed. multivariate cox proportional hazards models were used to calculate hazard ratios (hrs) with 95% confidence intervals (ci). results: a total of 103 episodes of severe infection were identified in 86 (34.7%, 86/248) patients during a median follow-up of 15 months. the incidence of infection during induction therapy was 38.5% for corticosteroids (cs), 39.0% for cs+ intravenous cyclophosphamide (iv-cyc), 33.8% for cs+ mycophenolate mofetil and 22.5% for cs + tripterygium glycosides, 76 (73.8%) infection episodes occurred within 6 months, while 66 (64.1%) occurred within 3 months. pneumonia (71.8%, 74/103) was the most frequent type of infection, and the main pathogenic spectrum included bacteria (78.6%), fungi (12.6%), and viruses (8.7%). the risk factors associated with infection were age at the time of diagnosis (hr = 1.003, 95% ci = 1.000–1.006), smoking (hr = 2.338, 95% ci = 1.236–4.424), baseline secrum creatinine (scr) ≥5.74 mg/dl (hr = 2.153, 95% ci = 1.323–3.502), cd4(+) t cell< 281 μl (hr = 1.813, 95% ci = 1.133–2.900), and intravenous cyclophosphamide regimen (hr = 1.951, 95% ci =1.520–2.740). twelve (13.9%) patients died of severe pneumonia. conclusion: the infection rate during induction therapy was high in patients with aav. bacterial pneumonia was the main type of infection encountered. age at the time of diagnosis, smoking, baseline scr ≥5.74 mg/dl, cd4(+) t cell< 281 μl, and iv-cyc therapy were identified as risk factors for infection. antineutrophil cytoplasmic antibody (anca)-associated vasculitis (aav) is a systemic vasculitis syndrome including microscopic polyangiitis (mpa), granulomatosis with polyangiitis (gpa), eosinophilic granulomatosis with polyangitis (egpa) and renal-limited vasculitis (rlv). the diagnosis of aav is based on the presence of clinical manifestations with characteristic histopathological findings and the presence of mpo-anca or pr3-anca [1] [2] [3] [4] [5] [6] [7] . aav may have predominant involvement of the upper respiratory tract, lungs, kidneys, skin, and nervous system. most patients with aav achieved remission after appropriate immunosuppressive therapy with corticosteroids and immunosuppressants, including cyclophosphamide (cyc), mycophenolate mofetil (mmf), and rituximab (rtx) [8] [9] [10] [11] . nevertheless, infection after immunosuppressive therapy contributes to the most common cause of death. the burden of infectious disease in patients with aav has been reported [1] [2] [3] [4] [5] [6] [12] [13] [14] [15] . nonetheless, risk infectors reported so far are inconsistent. in this study, we retrospectively analysed the epidemiological and clinical characteristics of chinese patients with anca-associated vasculitis and discussed major infection episodes occurring during immunosuppressive therapy in a single centre. a total of 248 patients newly diagnosed with aav and renal involvement who met the criteria of the chapel hill consensus conference [7] between january 1, 1998 and december 31, 2013 at the national clinical research center of kidney diseases jinling hospital were included, among whom 194 patients had renal biopsies that showed pauci-immune necrotic and crescentic glomerulonephritis. all patients were anca-positive. patients with secondary vasculitis, including henoch-schonlein purpura, allergy, autoimmune disease, tumour, cryoglobulinemia and infection, were not included. patients with end-stage renal disease (esrd) or who received only non-immunosuppressive treatment for infection at the time of diagnosis of aav were excluded from the study. ethical statement: this study was approved by the institutional review board of our hospital and performed in accordance with the ethical standards laid down in appropriate version of the declaration of helsinki. all patients signed informed consent. all clinical and laboratory data were collected retrospectively at diagnosis and during the follow-up period, including the patients' age, gender, medical history, routine blood analysis, 24-h urine protein excretion, urinary sediment red blood cell count, serum albumin and serum creatinine (scr), liver enzymes, immunoglobulin and t lymphocyte counts, serum ancas, lung involvement, birmingham vasculitis activity score (bvas) [16] , the usage of immunosuppressive agents, methlyprednisone pulse therapy, plasma exchange, and adverse events including major infection. major infections were diagnosed according to common terminology criteria for adverse events (ctcae) v4.0 in addition to clinical and radiological manifestations and microorganism cultures. none of the patients had received any immunosuppressive therapy before diagnosis. patients without contraindication initially received intravenous methylprednisolone pulse therapy (0.5 g, once daily, for 3 consecutive days) after diagnosis of aav. patients with severe manifestations of aav underwent plasma exchange therapy. all patients received oral prednisone at a dose of 0.6-0.8 mg/ kg/day for 4 weeks, which was then tapered by 5 mg each week to 10 mg/day. induction immunosuppressive agents included mmf 1-1.5 g/day orally, monthly intravenous cyclophosphamide (iv-cyc) at 0.75-1.0 g/m 2 body surface area in monthly pulses, tripterygium glycosides (tw, extract from the traditional chinese herb tripterygium wilfordii, which mainly contains triptolide) and multi-target therapy (prednisone, mycophenolate mofetil and tacrolimus) [8] . maintenance therapy included prednisone 5 mg/day combined with mmf and azathioprine. prophylaxis of pneumocystis jirovecii pneumonia (pjp) with smz-co (trimethoprim-sulfamethoxazole 400/80 0.48 g per day) was used in patients whose cd4 + t cell counts were less than 200/μl, and the doses were tapered in patients with renal dysfunction [17] . all immunosuppressive agents, except prednisone, were discontinued in patients with aav who suffered from major infection during the follow-up period. antimicrobial therapy was prescribed according to clinical and radiological manifestations and microbiological characteristics. patients diagnosed with pjp were treated with smz-co and echinocandin together. patients with weight loss were prescribed enteral nutrition. the patients with severe acute kidney injury or acute respiratory distress syndrome (ards) were treated with continuous blood purification. a recorded severe infectious complication was defined as implying the administration of an antimicrobial medication for an observable clinical, microbiological and radiologic suspected infection requiring hospitalization. immediate dialysis was defined as the clinical necessity of renal replacement therapy on admission. the first immunosuppressive agent used in addition to corticosteroids was termed induction therapy. the immunosuppressive regimen used during follow-up was termed the maintenance agent. the diagnosis criteria for deep fungal infection included clinical manifestations, such as fever, cough, diarrhoea or lower urinary tract symptoms, and the detection of fungi in sputum, urine, stool or tissue specimens. cytomegalovirus (cmv) infection was diagnosed by cmv polymerase chain reaction (pcr). the range of quantification of this assay was 600-100,000 copies/ml for cmv. cmv pneumonia was defined as the detection of ground glass opacity by chest x-ray film or computed tomography, the detection of cmv in the bronchoalveolar lavage fluid or lung tissue samples, and clinical signs such as fever, cough, dyspnoea and hypoxemia. the diagnosis of pjp was made clinically or by the identification of pneumocystis from sputum, bronchoalveolar fluid, tracheal secretions or lung tissue by special stains or a non-nested pcr, specifically designed to diagnose pneumonia rather than colonization [18] . ards was defined as the acute onset of hypoxemia (arterial partial pressure of oxygen to fraction of inspired oxygen [p ao2 /f io2 ] ≤ 200 mmhg) with bilateral infiltrates on chest radiographs, without left atrial hypertension. multiple organ dysfunction syndrome (mods) was defined as the simultaneous failure of at least two organs. esrd was defined as egfr < 15 ml/min per 1.73m 2 or requiring renal replacement treatment for > 3 months. the follow-up endpoints included the final date of december 31, 2014, dropping out before the final date, reaching esrd, or death. statistical analysis was performed with spss 20.0 for windows (spss inc., chicago, il, usa). medians and ranges were reported for non-normally distributed data, and means ± standard deviations were reported for normal-distributed data. the kruskal-wallis test was applied for the comparison of non-normal distributed data. differences between means were tested using the student's t-test. a mann-whitney u test was used for nonparametric distributions. chi-squared tests were used for the comparison of categorical data. to address the independent predictive value of factors associated with the rate of infections, the variables with p values of less than 0.1 in univariate analysis as well as those reported in the literature were selected for multivariate analysis using the cox regression model. the group with corticosteroids only was used as a reference group in multivariate analysis. only the time to first severe infection was evaluated. laboratory values and bvas used for modelling were from the time of diagnosis. receiver operating characteristic (roc) curve analysis was performed to determine the cut-offs of scr, haemoglobin, albumin, cd4+ t cells and bvas. all tests were two-tailed, and p-values of < 0.05 were considered significant. confidence intervals (cis) were calculated at the 95% level. this study identified 248 individuals with ages ranging from 14 to 78 years (median 55 years), including 214 cases diagnosed as mpa, 16 cases diagnosed as rlv, 10 cases diagnosed as gpa and 8 cases diagnosed as egpa. seventy-five patients (30.2%) showed lung involvement, 30 (12.1%) had alveolar haemorrhage, and 54 (21.8%) had sinus involvement. mpo-anca was more prevalent, and only 21 cases (8.5%) were pr3-anca-positive. fifty-three patients started immediate dialysis. initial immunosuppressive treatment consisted of pulse methylprednisolone (67.3%), plasma exchange (23.8%), iv-cyc (26.6%), mmf (31.0%) and tw (16.1%). twenty-six percent of patients received only oral corticosteroids (table 1) . forty-two patients (16.5%) received smz-co to prevent pjp, and 29 of them were cyc users. a total of 103 infectious episodes occurred in 86 patients (34.7%) during follow-up for 1~155 months (median 15 months). fifteen cases experienced a second episode of infection, and one patient experienced a third episode. seventy-six episodes (73.8%) of infection occurred during induction therapy (median 1.5 months). twenty-seven episodes (26.2%) occurred during maintenance therapy (median 18 months), and six episodes (5.8%) occurred after 24 months. pulmonary infections (71.8%, 74/103) were the most frequent type of infection, followed by skin (n = 7, 6.8%), digestive tract (n = 3, 2.9%), urinary tract (n = 2, 1.9%) and central nervous system (n = 1, 1.0%) infections. six patients (5.8%) developed sepsis because of their reported infection. the pathogens responsible for infection were confirmed in 87 episodes. the whole pathogen spectrum included bacteria, fungi and viruses. bacterial infection was the most common (n = 57, 66%), especially acinetobacter baumannii, followed by fungal (n = 21, 24%) and viral infections (n = 9, 10%) there were four cmv, seven pjp and 16 unspecified infections (table 2) . the infectious rate of induction therapy with corticosteroids only was 38.5% (25/65), that for cs + iv-cyc was 39.0% (26/66), cs + mmf was 33.8% (26/77) and cs + tw was 22.5% (9/40). the incidence of smoking (36.0% vs. 17.3%, p = 0.000) and diabetes (8.1% vs. 2.5%, p = 0.032) was significantly higher among the infected patients. the cutoff level of scr haemoglobin, albumin, cd4+ t cells, and bvas were determined as 5.74 mg/dl, 7.75 g/dl, 33.95 g/l, 281/ul, and 25.5 respectively based on roc curve analysis. single factor analysis revealed that risk factors for complicated infection in patients with aav included age, smoking, pulmonary involvement, hemoglobulin, albumin, scr level, cd4 + t cell count, bvas, and immunosuppressive therapy with mmf, cyc and tw. in adjusted models for the aav cohort, increased risks of infection were observed in patients who were older at the time of diagnosis (hr = 1.003, 95% ci = 1.000-1.006), smoking (hr = 2.338, 95% ci = 1.236-4.424), with baseline scr ≥5.74 mg/dl (hr = 2.153, 95% ci = 1.323-3.502), cd4 + t cell< 281 μl (hr = 1.813, 95% ci = 1.133-2.900), and users of intravenous cyclophosphamide regimen (hr = 1.951, 95% ci =1.520-2.740) ( table 3) . the exact pathogen was identified in 44 of 82 episodes of pneumonia. bacteria were the most common pathogens (n = 27, 61.4%), especially acinetobacter baumannii (n = 6, 13.6%), staphylococcus aureus (n = 5, 18.5%) and pseudomonas aeruginosa (n = 3, 6.0%). thirteen cases were diagnosed as fungal infections, and most were caused by c. albicans (n = 8, 61.5%). cmv was identified in all four cases with viral pneumonia. the main pulmonary radiologic findings included consolidation (n = 38, 51.4%), diffuse interstitial pneumonia (n = 21, 28.4%) and multiple nodules (n = 13, 17.6%). bacterial pneumonia presented with consolidation (n = 24, 32.4%), nodules (n = 9, 12.2%) and a diffuse reticular pattern (n = 6, 8.1%). cmv pneumonia mainly presented with ground-glass opacities (4, 5.4%), diffuse reticular thickening (n = 3, 4.1%) and nodules (n = 1, 1.4%) on bilateral lungs. fungal pneumonia was characterized by consolidation (n = 14, 18.9%), nodules (n = 4, 5.4%), halo (n = 4, 5.4%) and air crescent sign (n = 2, 2.7%). thirteen cases were complicated by ards, and 10 were complicated by mods. nine patients required mechanical respiration (5 bipap and 4 endotracheal intubation). all 103 episodes were treated with intravenous antibiotics. twelve (11.7%) of 103 patients died and all due to severe pneumonia. the time to death was from one to sixteen months after the initiation of immunosuppressive therapy. none died due to aav (table 4 ). a link between vasculitis and infection has long been suspected. bacterial infections can trigger the production of various autoantibodies, including anca [19] . infection is a major concern in the management of aav and is the most common cause of death, especially in patients with malnutrition or immunosuppressive therapy [1] [2] [3] . immunosuppressive therapy is performed with consideration of the disease activity, which is comprehensively evaluated based on the bvas score [20] . nonetheless, even in patients with severe anca-associated vasculitis, secondary infection, rather than active aav, is the leading cause of death [21] . there still remains no firm conclusion about the burden and characteristics of major infections in patients with aav. we retrospectively reviewed the clinical charts of 248 chinese patients with aav. major infections were reported in 34.6% of our single-centre cohort. approximately 64.1% of these infections developed in the first three months of induction therapy. in the reported studies, corticosteroids contributed to 89% of infections of patients with aav, and the infection rate decreased when the corticosteroids were tapered [1] [2] [3] [4] . corticosteroid treatment leads to an immunocompromised status in patients by inhibiting cytokines, neutrophils, and immunologic response and by exerting anti-inflammatory and immunosuppressive effects [22] . infection is suspected when fever (≥37.3°c) persists for no less than three days and c-reactive protein increases after remission of aav [22] . the evaluation of infection is based on the presence of organ manifestations. identificating methods of causative microorganisms, such as common bacteria, viruses, and fungi, include mycological, histological, and genetic tests [20] . the main areas of infection included the lungs and skin. the lung infection rate was as high as 79.6% in this cohort. most aav patients had impaired renal function, and lung involvement and diffuse alveolar haemorrhage injure the local protective barrier. renal injury also increases the risk of severe infection and is closely associated with a poor outcome [6, 18] . according to the literature, the most common causative pathogens are bacteria, such as streptococcus pneumonia and haemophilus influenza [23, 24] , followed by fungi and viruses. in our cohort, the main bacteria included acinetobacter baumannii, staphylococcus aureus, and pseudomonas aeruginosa (table 2) , and our rates of infections with fungi and viruses were higher and lower, respectively, than those of previous reports [25] . characteristics of aav, such as global inflammation, renal injury, lung involvement, malnutrition, and immunosuppressive therapy, contribute to infections by opportunistic pathogens [26] . cmv, pjp and 13 cases of pneumomycosis developed during induction therapy. it is also possible that a pjp diagnosis may have been [27] [28] [29] . thus, improving the vaccination coverage against streptococcus pneumonia and influenza in high-risk populations could play an important role in pulmonary prevention [30] . the most common computed tomography findings were ground-glass attenuation, reticular pattern, and fibrous bands with infiltration. in cases of bacterial, fungal and viral pneumonia, a consolidation and reticular pattern, patchy consolidation and glass-ground attenuation were most commonly observed, respectively. these characteristics are predominantly seen in pneumonia patients with aav. given the high incidence of infections in patients with aav, risk factors need to be defined in order to increase surveillance and prescribe prophylactic antibiotic therapy. many studies have reported that age, female gender, diabetes, impaired renal function, clinical grade category of rapidly progressive glomerulonephritis (rpgn), lymphopenia and immunosuppressive therapy are risk factors for infection in aav [6, 12-14, 20, 31] . however, there remains no consensus about the infectious risk factors in chinese patients with aav. in this cohort, bvas and the frequency of diabetes in the infectious group were higher than that in the control group, indicating that higher bvas and diabetes are potential risk factors of infection. age at the time of diagnosis (hr = 1.003, 95% ci = 1.000-1.006), smoking (hr = 2.338, 95% ci = 1.236-4.424), baseline scr ≥5.74 mg/dl (hr = 2.153, 95% ci = 1.323-3.502), cd4 + t cell< 281 ul (hr = 1.813, 95% ci = 1.133-2.900), and use of intravenous cyc were independent risk factors of infection. whether or not the use of cyc was a risk factor for developing infection in aav patients remains controversial [9, 10, 14] . masaharu [20] also reported that the use of cyc was a risk factor for developing infection in aav patients, but no difference was observed in renal failure between those with or without infection. on the other hand, cyc showed similar adverse events when compared to rituximab in two randomized controlled trials [9, 10] . in our study, the infection-related mortality (11.7%) was less than that reported in most of the literatures [4, 13, 14, 32] . half of these cases died within the first month after diagnosis. thus, clinicians should consider adaptive immunosuppressive agents to avoid life-threatening infection. there are some limitations in this retrospective study. first, the treatment protocols were not uniform and lack of data on rituximab. only a minority of patients were given smz-co prophylaxis because of the insufficient awareness. none of these patients received prophylaxis for fungal infection. in addition, some cases with pulmonary or central nervous system infection failed to show a definitive pathogen. the frequency and severity of pneumonia should be lowered by prophylactic treatment and early diagnosis. infections can develop during every stage of aav, primarily in the lungs and skin. the pathogens identified in this study mainly consisted of bacteria, candidiasis, cmv and herpes simplex virus, and age at the diagnosis, smoking, baseline scr higher than 5.74 mg/dl, cd4 + t cell< 281 μl, and cyc therapy were independent risk factors for infection in patients with aav. antineutrophil cytoplasmic antibody -associated vasculitis; anca: antineutrophil cytoplasmic antibody; ards: acute respiratory distress syndrome; bvas: birmingham vasculitis activity score cs: corticosteroids; ctcae: common terminology criteria for adverse events; cyc: cyclophosphamide; egpa: eosinophilic granulomatosis with polyangitis; esrd: end-stage renal disease; gpa: granulomatosis with polyangiitis; hrs: hazard ratios mmf: mycophenolate mofetil; mods: multiple organ dysfunction syndrome mpa: microscopic polyangiitis; pcr: polymerase chain reaction; pjp: pneumocystis jirovecii pneumonia; rlv: renal-limited vasculitis; roc: receiver operating characteristic; rtx: rituximab; scr: secrum creatinine risk factors for major infections in wegener granulomatosis: analysis of 113 patients an interdisciplinary approach to the care of patients with wegener's granulomatosis-long-term outcome in 155 patients long-term followup of polyarteritis nodosa, microscopic polyangiitis, and churg-strauss syndrome. analysis of four prospective trials including 278 patients longterm patient survival in anca-associated vasculitis eular recommendations for the management of primary small and medium vessel vasculitis anca-associated renal vasculitis at the end of the twentieth century-a disease of older patients nomenclaure of systemic vasculitides: the proposal of an international consensus conference mycophenolate mofetil versus cyclophosphamide for inducing remission of anca vasculitis with moderate renal involvement rituximab versus cyclophosphamide in anca-associated renal vasculitis rituximab versus cyclophosphamide for anca-associated renal vasculitis rituximab versus azathioprine for maintenance in anca-associated vasculitis frequency, risk factors and prophylaxis of infection in anca-associated vasculitis adverse events and infectious burden, microbes and temporal outline from immunosuppressive therapy in antineutrophil cytoplasmic antibodyassociated vasculitis with native renal function risk factors associated with replapse or infectious complications in japanese patients with microscopic polyangiitis severe infection in antineutrophil cytoplasmic antibody-associated vasculitis development of comprehensive disease assessment in systemic vasculitis prophylaxis for pneumocystis pneumonia in patients with rheumatoid arthritis treated with biologics, based on risk factors found in a retrospective study pneumocystis jirovecii testing by real-time polymerase chain reaction and direct examination among immunocompetent and immunosuppressed patient groups and correlation to disease specificity infections and antineutrophil cytoplasmic antibodies: triggering mechanisms simple, readily available clinical indices predict early and late mortality among patients with anca-associated vasculitis the prevalence and management of antineutrophil cytoplasmic antibody-associated vasculitis in china strategy of infection control in immunosuppressive therapy for anca-associated vasculitis prolonged infections associated with antineutrophil cytoplasmic antibodies specific to proteinase 3 and myeloperoxidase: diagnostic and therapeutic challenge infectious complications of immunosuppressive treatment for anti-neutrophil cytoplasm antibodyrelated vasculitis value of anti-infective chemoprophylaxis in primary systemic vasculitis: what is the evidence? autoimmune inflammatory disorders, systemic corticosteroids and pneumocystis pneumonia: a strategy for prevention influenza vaccination dose not result in an increase in relapses in patients with anca-associated vasculitis wegener's granulomatosis patients show an adequate antibody response to influenza vaccination eular recommendations for vaccination in adult patients with autoimmune inflammatory rheumatic diseases improving the vaccination coverage against influenza and streptococcus pneumonia in the populations at risk: the role of pulmonary care services severitybased treatment for japanese patients with mpo-anca-associated vasculitis: the jmaav study complications of long-term therapy for anca-associated systemic vasculitis we acknowledge dr. le weibo for the assistance of statistic analysis. we appreciated all the participants for their contribution to this study. this study was supported by national key research and development program of china (2016yfc0904100). the dataset used and analysed during the current study is not publicly available due to patient-related confidentiality, but it is available from the corresponding author on reasonable request.author's contributions yl, xhl, lzz, cyh, wjq, gyc, zht, hwx: contributed to the design of study, performed the data collection and interpretation. yl and xhl wrote the first draft. lzz, cyh, wjq, zht, gyc and hwx revised the manuscript. all authors are accountable for all aspects of the work and have approved the final manuscript.ethics approval and consent to participate this study was approved by ethics committee of jinling hospital (nanjing, china). written consent was obtained from each participant. declaration of helsinki was followed while conducting this analysis. not applicable the authors declare that they have no competing of interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.received: 6 october 2017 accepted: 25 may 2018 key: cord-016208-u12ngkpc authors: andersen, bjørg marit title: intensive patient treatment date: 2018-09-25 journal: prevention and control of infections in hospitals doi: 10.1007/978-3-319-99921-0_45 sha: doc_id: 16208 cord_uid: u12ngkpc intensive care units (icus) are treating hospital’s poorest patients that need medical assistance during the most extreme period of their life. intensive patients are treated with extensive invasive procedures, which may cause a risk of hospital infections in 10–30% of the cases. more than half of these infections can be prevented. the patients are often admitted directly from outside the hospital or from abroad with trauma after accidents, serious heart and lung conditions, sepsis and other life-threatening diseases. infection or carrier state of microbes is often unknown on arrival and poses a risk of transmission to other patients, personnel and the environment. patients that are transferred between different healthcare levels and institutions with unknown infection may be a particular risk for other patients. in spite of the serious state of the patients, many icus have few resources and are overcrowded and understaffed, with a lack of competent personnel. icu should have a large enough area and be designed, furnished and staffed for a good, safe and effective infection control. the following chapter is focused on practical measures to reduce the incidence of infections among icu patients. all patients treated in intensive care units (icus) and personnel working in the intensive care units. the hospital's management should provide resources and written guidelines regarding infection control work, proper patient/care ratio, sufficient patient areas, isolation capacity and documented competence. the departmental management should provide competent personnel that follow guidelines. in circumstances that expose patients to increased infection risk, this is reported to the director immediately. the staff follows routines. expertise and practice in intensive medicine must be documented. it should be reported to the management if exposed to or infected by resistant or special microbes, for example, in work at other departments/countries. the patient and relatives/visitors should be informed about the department's routines, hand hygiene and good personal hygiene and cleanliness. intensive care unit (icu) should have a large enough area and furnished for a good, safe and effective infection protection [1] . the state of infection or carrier state is often unknown on arrival and poses a risk of infection to other patients, personnel and the environment [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . patients transferred between healthcare levels and healthcare networks may spread infections [12] [13] [14] [15] [16] . note! comprehensive responsibility! there must be one responsible department/ unit manager who has total responsibility for patients, personnel and the environment-in terms of infection control and patient protection. fragmentary leadership means that no one has direct responsibility for the patient's security and prevention of infection. 1 . hygienic standard should correspond to an operation department-to reduce the number of infections, the use of antibacterial agents and resistance development. employees should carry out a good personal hygiene (see personal hygiene and regimes for isolation, chap. 6 and 6-14). 2. separate patient rooms are recommended. this is due to the many direct openings into sterile tissues, via respirator, intravascular catheters, urinary tract catheter, surgical site and other wounds. all intensive patients should be protected against infections from other patients, personnel and the environment [1, 17] . when sharing a room with a fellow patient, the distance between the patients must be 3-4 m from bed edge to bed side, and no one must have infections. 3. medical technical equipment associated with every intensive bed take a lot of space in the room, generates dust, particles and heat and are easily contaminated with skin particles and bacteria. written cleaning procedures must be available for all equipment. 4. the burden of bacteria, particles and other organic materials from the use of respirators, cough/suctions and particles from opening single-use equipment needs good room ventilation, preferably more than ten air exchanges/hour and control of bacterial numbers in the air, <100 cfu/m 3 . the air should enter the ceiling level and be filtered by 99% effective filter with particle diameter max 5 μm. exhaust air is extracted approximately 15 cm above floor level (opposite side). avoid turbulence, shaking of clothes, abrupt removal of bandages, etc. that increase the amount of bacteria in the air [7] . 5. hand hygiene is routinely enjoined for all personnel. proper use of hand disinfectants and gloves is important. non-sterile gloves often have a rich growth of environmental flora and do not protect the patient. jewellery, piercing and wristwatches are not allowed since this collects microbes and inhibits personal hygiene/hand hygiene. patient and visitor's hand hygiene routines will usually be the same in the hospital [18] . 6. coats and outerwear are taken off outside the unit. 7. glove box for each patient, set date, and throw the box when the treatment ends. 8. water control. avoid splashes and aerosols from the sinks that are often colonized with gram-negative rods, and check the water for legionella and other gram-negative bacteria (see legionella and water control) [8, 9, 19] . (a) cooling systems, water coolers and large water reservoirs are linked to the outbreak of legionella and resistant gram-negative rod bacteria [20] . use sterile water for intensive care patients for oral hygiene. (b) biofilm formation in water systems, on surfaces, etc. is increasing. preventative measures are good such as daily cleaning with soap and water and disinfecting with chlorine or peracetic acid [19] [20] [21] . biofilm can be resistant to chlorine [21] . 9. clean work uniform; change every day or more often if necessary. (a) separate gown for each patient-long arms and cuffs. the gown is changed for each shift or more frequently if contaminated. (b) cap/surgical mask and visor are used by work with respiratory tract (respirator), intravascular catheter, surgical wound, drain, urinary tract catheter. 10. cleanliness and order to reduce biological burden in the environment and to reduce the risk to the patient. good daily cleaning removes possible pathogenic flora fairly quickly. good cleaning depends on good space and order. large amounts of skin waste, hair and microbes are released continuously from people in the room-and fall down as grey "dust" on all surfaces after the floating state in the air-and carry invisible amounts of bacteria. in addition, there may be huge amounts of particle release from all disposable devices that are opened. (a) all patient rooms, service rooms, guard rooms, wardrobes, etc. must be cleaned at least once a day with soap and water. (b) the washbasin is washed and disinfected daily [19] . disinfect mobile phones, glasses, etc. and all other equipment before being brought into the icu. note! persons who perform room cleaning should not work with patients at the same time. this is to avoid cross infection. 11 . medical technical equipment must be cleaned outside daily, when the treatment is completed and when it is going to enter or leave the department. it must also be cleaned inside after infection and after certain periods of use or after each use. all equipment is stored clean and dry, preferably with a clean chapel, on a separate clean storage. 12. sterile equipment is stored clean and dry on a separate storage for sterile equipment with requirements of low bacterial numbers in the air. 13. respiratory treatment: see separate chapters. 14. intravascular treatment: see separate chapters [22] [23] [24] [25] [26] . 15 there is estimated at least two competent intensive care nurses per patient. the icu patient is monitored continuously-in place-at the bed (not via ward room) on a 24-h basis. small changes in the patient can lead to a dramatic worsening of the condition if not detected early enough. understaffing leads to less competent personnel and temporary workers, increased stress in the work situation, reduced hand hygiene and lack of infection control [5] . this increases the risk of serious nosocomial infection and fatal outcome. understaffing and overcrowding will also expose personnel and visitors to increased risk of infection [5] . the icu should be large enough to cover demographic needs, operational activity and specialty in the coverage area [1, 10, 11] . the department should be functional with ample space for each patient, necessary number of isolates, good ventilation, service rooms and storage space. the department must be prepared for good infection control, hand hygiene and a thorough cleaning system [1, 10, 11]. patient groups vary according to type of intensive care unit: surgically intensive, medical intensive, child intensive or a blend of patient categories. • in germany, nosocomial infections were detected in 9.4% of 1860 icu patients, and after discharge, the rate increased to 10.6% [27] . without follow-up afterwards, 12% of icu-associated infections were not recorded [27] . • in belgium, there were 6500 icu patients examined in 2010 [28] . nosocomial pneumonia and ventilator-associated pneumonia (vap) starting in the icu were 13/1000 and 12/1000 patient days, respectively, and icu-associated blood infections were 3.2/1000 patient days. during the period 1997-2010, icu-associated infections increased, while infections related to equipment (respirator, catheter) were reduced [28] . in 2010, at least 31% of adult icu patients had nosocomial infections [29] . • later studies showed that more than 10% of icu patients had infections like vap and catheter-associated infections [30] . among 78, 200 patients with more than 2 days of stay in 525 icus in 6 european countries from 2005 to 2008, it was estimated that 52% of the vap cases and 69% of bloodstream infections could be prevented [30] . • intensive studies in the united states show that 65-70% of catheter-associated blood and urinary tract infections can be prevented and 55% of cases of vap and postoperative wound infections [31] . • the american network study (2013) of catheter-associated intravascular and urinary tract infections, vap and postoperative wound infections showed that these four infection categories caused 70,000 hospital infections per year, and it was found at least 81,000 pathogenic microbes in these patients [32] . nearly 20% of the microbes were multidrug-resistant organisms (mdro) like mrsa, vre, esbl and cre isolates [32] . • in 2010, ca. 300 general intensive beds were registered at 44 hospital icus in norway (five million inhabitants) [33] . in 2013 it was about 15, 200 icu treatments and approximately 60,300 days of intensive care in norway are distributed between local hospitals 28.6%; central hospitals, 34.5%; and regional hospitals, 36.9% [34] . • more than half of the patients were on respiratory treatment, average stay in the icu was 4 days, and 82.8% were discharged from hospital, while 11% died on the icu and 6.2% on other wards [34] . • there is a calculated need for about 11 icu beds per 100,000 inhabitants [35] . calculated from today's approximately 350 intensive beds, this amounts to 5.8/100,000. • in 2015, the prevalence of hospital infections in the norwegian icus was 23% in surgical icu/postoperative ward (218 beds) and 11.5% in medical icu/ward of monitoring (192 beds) [36] . about the same results were recorded in 2013 [36] . • hospital infections and mortality are high in norwegian icus, especially in surgical icus. since postoperative beds are included in the number registered for intensive, the real number of hospital infections is probably much higher. the costs are uncertain but probably amounts to at least 36,000 nkr per day for regular intensive care. the presence of resistant microbes in norwegian icus is unknown but probably an increasing problem. • there are still too few icu beds in norway-maybe only half of what is needed-and this can lead to unnecessary disease and death [35] . every 6th (17%) icu patient died on intensive or afterwards on ward in 2013 [34] . the risk of death in icu is therefore very high. • many entry ports through skin/mucous membranes (trauma, surgical procedure, respirator, etc.). • flat bed rest over time. • reduced immunity to infections, impaired general condition. • many procedures and a lot of handling of the patient. • much instrumentation. • acute care procedures. • h-2 blockers with gastric colonization of gram-negative bacteria from the gut. • aspiration of stomach contents. • short distance between patients-high patient density and overcrowded. • many patients in the same room, open care-"dormitory" [2, 3, 17] . • mixing of potentially infected patients with non-infected patients. • lack of isolates/single rooms and cross infection [1] [2] [3] 37] . • poor cleaning, lack of hygiene measures and lack of follow-up of hygiene guidelines [6, 38] . • environmental contamination [4, 6] . -contaminated water [8, 9] . -biofilm formation with microbe growth in water, on surfaces and equipment [20, 21] . -contaminated textiles. -contaminated/unsterile equipment and liquids [38] . -contaminated patient room [39] . • large consumption of antibacterial agents. -the use of multi-dose vials instead of single-dose. • large nursing load on few nurses, patient/nurse ratio-understaffing [5] . -short-term employed and a high personnel turnover [5] . -lack of knowledge, experience and use of written routines, large number of part-time workers and many "leaders" in the system. • number of days in hospital > 7 days; transferral of the patient between several departments and undergone at least one invasive procedure. • transport between departments and hospitals [15] . • airborne-droplet-borne infection: [40] [41] [42] . see separate chapter. • aerosol from contaminated nebulizer and other respiratory equipment. • aerosol/re-aerosol from change of bed linen, making the bed and other activities [7] . • tracheal suction/coughing. • oral hygiene-lack of [43] . • intravascular treatment, haemodialysis, peritoneal dialysis, etc. [22] [23] [24] [25] [26] 44] . • drainages and urinary tract catheters. • prolonged use of antibiotics and increasing bacterial resistance [42, 45] . • personnel with infection/carrier state-without knowing-"cloud" personnel [46] . • lack of tuberculosis control. the greatest risk of infection is the number of days in the icu and respiratory treatment (see separate chapter concerning vap). the icu patients have often complicating infections that lead to increased use of broad-spectrum antibacterial agents, which in turn often lead to resistant microbes. the patients' microbes become environmental, robust microbes that may be transmitted to the lungs, blood or wounds of room-mates. the accumulation of robust environmental microbes and bacterial-bearing dust (gram-negative rods, staphylococci and fungi), over time, is a great risk that may be removed only by good daily cleaning and environmental control. occasionally, patients bring serious infections or infectious agents to the hospital and the icu; multi-drug resistant organisms (mdro) such as mrsa, vre, esbl, ehec (entero-haemorrhagic e. coli, may be esbl at the same time), cre (carbapenemase-producing/resistant enterobacteriaceae). a number of others are introduced periodically in the department like norovirus and influenzae. the most common infections in icus are caused by s. aureus, e. coli, enterobacter, pseudomonas, klebsiella, serratia, acinetobacter, stenotrophomonas, burkholderia cepacia, enterococcus, clostridium difficile, coagulase-negative staphylococci and candida. during the stay in the icu, the early infections are often with "common" bacteria like staphylococci, and the later infections after longer stay in the icu are caused by more resistant bacteria like pseudomonas and fungi. in sweden, an increased incidence of invasive haemophilus influenzae, nontype b, has been reported, with more than one third being treated at the icu for septic shock [47] . group a streptococci (gas) are epidemic in a number of countries and constitute an important intensive treatment and infection problem. in england, there was registered 14,000 gas cases in 2014-the highest number of the past 50 years of scarlet fever [48] . healthcare professionals can become carriers of gas, and the infection can spread through air [46, 49, 50] . bacteria that are transmitted via direct inhalation, swirling of dust or aerosols are gas, staphylococci, mrsa, meningococci, pneumococci, pertussis bacteria, diphtheria bacteria, ehec, acinetobacter legionella, pseudomonas, clostridium difficile (spores), mycobacterium tuberculosis and other mycobacteria, nocardia and a variety of other bacteria [46, [49] [50] [51] [52] [53] . virus that is nosocomial through the air is respiratory virus (rsv, influenza, parainfluenza, rhinovirus, corona-, adeno-, entero-, parecho-, metapneumovirus), varicella-zoster virus, rubella, morbilli, norovirus, sapovirus, etc. [51] . spores of fungi from aspergillus and similar fungus types are liberated easily into the air and causes severe systemic infections in immunosuppressed patients [51] . antibiotic resistance has increased dramatically over the past 20 years, and a large proportion of these occur in icu departments with severe infectious disease [54] [55] [56] [57] [58] . new types of resistance appear as cre (carbapenem resistant), enterobacteriaceae: xdr (extensive drug resistance), acinetobacter species; and totally resistant (ndm-1 and colistin resistant) gram-negative rods, extremely to totally resistant tuberculosis bacteria, and others that can cause fatal nosocomial progress, also in almost immune-competent patients [59] [60] [61] [62] . these patients are often associated with icu, respiratory treatment, pulmonary disease, antibiotic use and a high bacterial burden in the environment [59] . a specific "clad b" (xdr) strain of a. baumannii is-in the united states-a recent cause of fatal outcome of nosocomial infection in six patients: it is a highly resistant and dangerous (hypervirulent) strain [61] . another multiresistant a. baumannii caused nosocomial infections in more than 19 patients, of which five died in germany [62] . in a study from brazil, the consumption of piperacillin-tazobactam, fluoroquinolones and cephalosporins increased at the hospital [63] . at the icu, multi-resistance was detected in more than 30% of the cases, with increasing meropenem-resistant klebsiella and acinetobacter species. a significant correlation was detected between the proportion of multiresistant bacteria and the consumption of cephalosporins and fluoroquinolones [63] . among 400 hospitalized patients in germany, 18.5% were colonized with fluoroquinolone-resistant e. coli (fqrec), and this was linked to poor clinical outcomes and high mortality [56] . stay in hospital, cancer diagnosis and the use of first-generation cephalosporin or cefepime treatment increased the incidence of new colonization with fqrec [56] . isolation, good hygienic measures and a better antibiotic policy reduce the spread of infection. fluoroquinolone resistance is an increasing problem since these drugs are used extensively both inside and outside hospitals [55] . multiresistant bacteria may increase when using antibiotics in food and beverage production. an alarming occurrence of carbapenemase-producing gram-negative bacteria has been detected in food, for example, in canada [57] . also in norway there are significant problems with resistant bacteria and other unfortunate agents in some food products [64] . resistant microbes in the health system will migrate between health levels, such as either silent colonization or with infection symptoms, "the top of the iceberg". the real problem (the entire iceberg) is only detected by general screening and mapping of the patients [12] [13] [14] [15] . international studies show insufficient knowledge about how to use the testing of resistance against antibiotics, also shown in a norwegian study [65, 66] . today, active screening of faeces/rectum samples (cre, esbl, vre, (mrsa)) may be used in combination with c. difficile detection [67] . this has proven cost effective in icu departments with problems with a. baumannii [68] . chlorhexidine "bathing" of icu patients to reduce infections is discussed, both for adults and children [69] . this measure is probably not so successful concerning the development of resistance as long as it is done so little for general hygiene and infection control in icus. in norway, there is increasing resistance development and outbreaks of resistant microbes such as mrsa, vre and esbl in hospitals, especially in recent years, but is still thought to be a "limited problem" [70]. many icus around the world, including norway, have until recently appeared like large dormitories, with mix of patient categories, no isolates and a strong understaffing in old and run-down areas. the outmost sever ill patients have often had the poorest treatment offer in the icu of the hospitals. "bad design, bad practices, bad bugs" is the starting point for a number of serious infection outbreaks in icus [71] . • space around the patient is needed for different intensive equipment, nursing staff and family. each patient should have a separate room (approximately 25 m 2 patient) to be protected against infection/noise/stress from the environment and other patients. recent studies indicate that patients in separate icu rooms will have fewer hospital infections and thus a lower risk of fatal outcome [72, 73] . • isolates. twenty-five percent of the bed capacity at intensive should be isolates with negative air pressure (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) . the size should be at least 35 m 2 /isolate which includes patient room, sluice and disinfection room/toilet/shower. • service room. the unit must have service rooms/store rooms adapted to the number of intensive beds. the service rooms should be store room for single-use equipment (16 m 2 ), medicine room (16 m 2 ), disinfection room (needed, crosscontamination danger, 16 m 2 ), textile room (needed, danger of cross-contamination, 16 m 2 ) for technical equipment (respirator, infusion pumps, surveillance equipment, racks, etc., 15-20 m 2 ), shower room for patients (4 m 2 ) and waste disposal room (large amounts of waste, 12 m 2 ). • entrance control. a separate sluice for entrance to the icu with wardrobes with washbasins and disinfectants. note: washbasins/sinks/shower rooms. a number of studies have documented bacterial aerosols around the sink/washbasin/shower room. a french multicentre study of 185 washbasins in 13 icus showed that one of three washbasins was contaminated with multiresistant esbl gram-negative bacteria [19] . it was recommended that washbasins should only be used for hand washing and disinfected with chlorine agents daily [19] . washbasins, with a small fall height from opening to bottom, should therefore be placed at least 2 m from the patient to avoid aerosols. 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aureus from a neonatal intensive care unit in oslo active surveillance for carbapenem resistant enterobacteriaceae using stool specimens submitted for testing for clostridium difficile economic value of acinetobacter baumannii screening in the intensive care unit does chlorhexidine bathing in adult intensive care units reduce blood culture contamination? a pragmatic cluster-randomized trial bad design, bad practices, bad bugs: frustration in controlling an outbreak of elizabethkingia meningoseptica in intensive care units single rooms may help to prevent nosocomial blood stream infection and cross-transmission of methicillin-resistant staphylococcus aureus in intensive care units infection acquisition following intensive care unit room privatization key: cord-025155-ow3r3469 authors: lokida, dewi; hadi, usman; lau, chuen-yen; kosasih, herman; liang, c. jason; rusli, musofa; sudarmono, pratiwi; lukman, nurhayati; laras, kanti; asdie, rizka humardewayantie; murniati, dewi; utama, i made susila; mubin, risna halim; karyana, muhammad; gasem, muhammad hussein; alisjahbana, bachti title: underdiagnoses of rickettsia in patients hospitalized with acute fever in indonesia: observational study results date: 2020-05-24 journal: bmc infect dis doi: 10.1186/s12879-020-05057-9 sha: doc_id: 25155 cord_uid: ow3r3469 background: reports of human rickettsial infection in indonesia are limited. this study sought to characterize the epidemiology of human rickettsioses amongst patients hospitalized with fever at 8 tertiary hospitals in indonesia. methods: acute and convalescent blood from 975 hospitalized non-dengue patients was tested for rickettsia igm and igg by elisa. specimens from cases with seroconversion or increasing igm and/or igg titers were tested for rickettsia igm and igg by ifa and rickettsia genomes using primers for rickettsia (r.) sp, r. typhi, and orientia tsutsugamushi. testing was performed retrospectively on stored specimens; results did not inform patient management. results: r. typhi, r. rickettsii, and o. tsutsugamushi igg antibodies were identified in 269/872 (30.8%), 36/634 (5.7%), and 19/504 (3.8%) of samples, respectively. for the 103/975 (10.6%) non-dengue patients diagnosed with acute rickettsial infection, presenting symptoms included nausea (72%), headache (69%), vomiting (43%), lethargy (33%), anorexia (32%), arthralgia (30%), myalgia (28%), chills (28%), epigastric pain (28%), and rash (17%). no acute rickettsioses cases were suspected during hospitalization. discharge diagnoses included typhoid fever (44), dengue fever (20), respiratory infections (7), leptospirosis (6), unknown fever (6), sepsis (5), hepatobiliary infections (3), uti (3), and others (9). fatalities occurred in 7 (6.8%) patients, mostly with co-morbidities. conclusions: rickettsial infections are consistently misdiagnosed, often as leptospirosis, dengue, or salmonella typhi infection. clinicians should include rickettsioses in their differential diagnosis of fever to guide empiric management; laboratories should support evaluation for rickettsial etiologies; and public policy should be implemented to reduce burden of disease. rickettsioses are arthropod-borne zoonoses caused by obligate intracellular bacteria from rickettsia or orientia genera. they include murine typhus, spotted fever, and scrub typhus groups [1] . small mammals serve as reservoirs and arthropods (ticks, fleas, lice, and mites) as vectors. humans are incidental hosts for many pathogenic rickettsiae [2] . human rickettsioses in indonesia are not well characterized. limited reports have found murine typhus in travelers returning from indonesia [3] [4] [5] . in 2004, over 450 travel-associated cases were reported worldwide; a significant proportion were r. typhi from tropical and subtropical areas, r. conorii from southern asia and o. tsutsugamushi from the asia-pacific [6, 7] . an active surveillance study of children in asia showed that 7.6% of indonesian cases were due to rickettsia [8] . other fever studies revealed prevalence of murine typhus, spotted fever, and scrub typhus in northeastern papua to be 5, 1, and 3%, respectively [9] , whereas prevalence of murine typhus in central java was 7% [10] . clinically, rickettsioses are difficult to distinguish from other conditions causing acute fever in endemic areas, especially during the early phase. common presentations include fever, abdominal discomfort, headache, myalgia, and rashes. lung, liver, and kidney involvement may complicate the disease [7] . given the non-specific clinical syndrome and limited access to diagnostics, rickettsioses are likely underdiagnosed in indonesia. underdiagnoses could engender inappropriate management, treatment delays, prolonged hospitalisation, and increased morbidity and mortality [11, 12] . therefore, early diagnosis and empirical therapy of rickettsioses are important. to characterize the epidemiology of rickettsioses in indonesia, we performed rickettsia diagnostic panels on blood from subjects in the acute fever requiring hospitalization (afire) study [13] . presentation of rickettsial infection in subjects that were initially diagnosed with another infection such as dengue, salmonella and leptospirosis were evaluated to identify features that may confound diagnosis of rickettsiosis. patients found to have rickettsial infection by reference laboratory testing were identified from ina-respond's [14] afire observational cohort study conducted in indonesia from 2013 to 2016. it recruited patients presenting to hospital for evaluation of acute fever, at least 1 year old, hospitalized within the past 24 h, and not hospitalized within the past 3 months. study sites were eight tertiary hospitals in seven cities in indonesia: bandung, denpasar, jakarta, makassar, semarang, surabaya and yogyakarta. details of afire have been previously described [13] . subjects were evaluated at enrollment, between 14 and 28 days post-enrollment and 3 months post-enrollment. demographics, clinical data, blood and other clinically indicated specimens were collected during these visits. blood specimens from the first visit were considered "acute" and specimens from the two follow-up visits were considered "convalescent". buffy coat and plasma from blood were stored at − 70°c and tested retrospectively for pathogens approximately 1 year after enrollment. specimens from 1464 subjects were first screened for dengue infection. non-dengue cases were then tested for other pathogens, including rickettsia. details of the diagnostic procedures are shown in fig. 1 . r. typhi igm, r. rickettsii igm, r. typhi igg, and spotted fever group igg were tested using enzyme-linked immunosorbent assay (elisa) (fuller laboratories, san fransisco, ca). igm and igg for scrub typhus were tested using elisa (inbios, seattle, wa). detailed methods for these assays have been described previously [15, 16] . convalescent plasma from 975 patients were tested for igg against r. typhi. due to logistic reasons, only a subset was tested for spotted fever group (n = 634) and scrub typhus (n = 504). if igg was positive, igm and igg from acute and convalescent plasma were tested simultaneously to assess seroconversion, increase or high optical density and index value in paired samples. in these subjects, indirect immunofluorescence assay (ifa) was performed to detect igm and igg reactivity to r. typhi and r. ricketsii (focus diagnostics, ca) following the manufacturer's procedures as previously described [15] . a specimen was considered positive when igm or igg fluorescence was observed in the 1:64 dilution. to determine four-fold increase, acute specimens were diluted by two-fold until igm or igg fluorescence was not observed. the dilution where igm or igg fluorescence was still detected was the end titer of the specimens. the corresponding convalescent specimens was then diluted four-fold of the end titer dilution of the acute specimens. four-fold increase of igm or igg was confirmed when fluorescence was still detected in these diluted convalescent specimens [17] . seroconversion of igm or igg antibodies was confirmed when no fluorescence was detected in 1:64 dilution in acute specimens but was detected in 1:64 dilution in convalescent specimens. acute plasma and buffy coat from subjects with seroconversion or increased igm/igg and from subjects that only had acute specimens were tested using pcr. bacterial dna was extracted using qiaamp dna mini kit (qiagen, hilden, germany). rickettsia sp. were detected by the 17-kd outer membrane protein (17-kd omp) gene of rickettsia sp., while r. typhi was identified by the ompb gene of r. typhi following previously published methods [18, 19] and o. tsutsugamushi by its 47-kd omp gene [20] . specimens positive for 17-kd omp gene of rickettsia sp but not the ompb gene of r. typhi underwent pcr and nested pcr amplification using primer set rompb3503f, rompb4293r, rompb4246r targeting 743-bp sequence of rickettsia sp. ompb gene and followed by dna sequencing to determine rickettsial species [21] . randomly selected samples with positive r. typhi based on qpcr, were confirmed with the same amplification and sequencing method. sequence chromatograms were edited using bioedit 7.2.5. software [22] ; edited sequences were searched for similarity using blastn. phylogenetic analysis was conducted in mega7 and inferred using the neighbor-joining method. the analysis involved 704-bp (nucleotide 3556-4259 of r. typhi ompb gene). 1) when both 17-kd omp gene of rickettsia and ompb gene of r. typhi were detected and/or seroconversion or four-fold increase of r. typhi igm and/or igg by ifa was observed. 2) when the detection of 17-kd omp gene of rickettsia or ompb gene of r. typhi was supported by dna sequencing of the 743-bp sequence of rickettsia sp. ompb gene or sero-conversion or four-fold increase of r typhi igm and/or igg by ifa. spotted fever group was confirmed by the detection of 17-kd omp gene of rickettsia and dna sequencing of the 743-bp sequence of rickettsia sp.ompb gene and /or sero-conversion or four-fold increase titers of spotted fever group igm and igg by ifa. scrub typhus was confirmed by the detection of 47-kd omp gene of o. tsutsugamushi and/or sero-conversion or four-fold increase o. tsutsugamushi igm and/or igg by elisa. data were collected in openclinica v.3.1 (openclinica, llc) and analyzed using stata v.15.1 (statacorp llc). clinical and laboratory profiles of confirmed cases were characterized by descriptive statistics and compared according to the three most commonly attributed diagnoses. proportions were compared between groups using the chi-squared test. the t-test was used to compare means between groups. specimens from 975 of 1464 subjects were evaluated using the rickettsia diagnostic panels. rickettsia was identified as the etiology of febrile illness in 103/975 (10.6%) cases ( fig. 1) . none of these patients were diagnosed with rickettsia upon clinical presentation. one case was clinically diagnosed as rickettsia during hospitalization but was not laboratory confirmed. characteristics of patients with acute rickettsial infection at enrollment are shown in table 1 (1). (fig. 3) . subjects averaged 5 days (range 1-12 days) of fever before hospital admission (table 1) . other reported symptoms included nausea (72%), headache (69%), vomiting (43%), lethargy (33%), anorexia (32%), arthralgia (30%), myalgia (28%), chills (28%), and epigastric pain (28%). the clinical triad of r. typhi infection (fever, headache and rash) was found in 11%. the three most frequent confirmed diagnoses in the study cohort, dengue, typhoid and leptospirosis demonstrated overlap with rickettsial infection. details are shown in table 2 . most subjects presented with normal hematocrit (median 40.8%) and leukocyte count (median 7354/mm 3 ). the majority had low lymphocyte proportion (median 21.9%) and platelets (median 123,426/mm 3 ). mildly increased liver enzymes were found in 77%, with bilirubin increases primarily attributable to direct bilirubin. during hospitalization, no clinically relevant changes were observed. the hematologic profile of r. typhi cases was similar to typhoid, but distinguishable from dengue and leptospirosis. dengue showed lower leukocyte and platelet counts. leptospirosis showed higher leukocyte and neutrophil counts, but lower absolute lymphocyte counts. increased total bilirubin and direct bilirubin were more prevalent than in dengue or typhoid, while increased total and indirect bilirubin were more frequent in leptospirosis. ast above 100 iu was more common in r. typhi cases compared to the three diseases, whereas creatinine > 1.2 mg/dl was more common in leptospirosis. for the 103 rickettsioses patients, discharge diagnoses were: typhoid fever (44), dengue fever (20), leptospirosis (6), respiratory infections (1 upper, and 6 lower), unidentified fever (6), sepsis (6), hepatobiliary infections (3), unidentified viral infections (3), uti (3) and others (one each: hiv, chikungunya, enteritis, meningoencephalitis, and diabetic neuropathy). in all cases of rickettsia initially suspected to be leptospirosis, typhoid fever, chikungunya, or dengue fever, diagnostic assays for those pathogens at the reference laboratory were negative, except in one r. felis case where leptospira pcr was positive and leptospira igm and igg sero-converted, suggesting co-infection. clinicians diagnosed typhoid despite negative or weak positive s. typhi igm rapid tests in 24 presumed typhoid cases; in 4 other cases rapid tests were not performed. in the remaining cases [16] , positive results from the rapid test were not supported as blood culture, pcr and elisa igm tests for salmonella were negative. in these 16 cases, r. typhi was confirmed by pcr and/or serological assays. fifteen dengue diagnoses were not supported by rapid dengue antigen or antibody tests. in contrast, 6 presumed leptospirosis cases had positive rapid tests, but pcr and elisa at the reference laboratory were negative except in the r. felis case above. details of the diagnostic tests to confirm rickettsia infection and to exclude s. typhi, dengue, and leptospira infections are shown in additional file (see additional file: table s1 ). antibiotics were taken prior to hospitalization in 23 subjects, including amoxicillin (8), cephadroxil (4), cotrimoxazole (3), chloramphenicol (2), cefixime (1), spiramycin (1), and a combination of antibiotics (4). antibiotics were given at hospitals in 76 of 90 (84%) subjects with documented treatment data as shown in table 1 . the majority received ceftriaxone (17) , ciprofloxacin (9) and levofloxacin (9), or a combination of antibiotics (30) . the drug of choice for rickettsia infection, doxycycline was given to 2 patients, one in combination with ceftriaxone and one with amoxicillin. 14 (58%) subjects with suspected viral infections received antibiotics at hospital several days after no clinical improvement with symptomatic treatment. the median hospital stay was 6 days (range 1-36). twenty-four subjects (23.3%) recovered with sequelae and 72 (69.9%) recovered without sequelae. seven (6.8%) patients (median 54.7 years, range 36.1-75 years) died. of these, 5 had underlying disease (stroke, hiv and chronic liver disease, hiv and tb, dm, and copd). six deaths were attributed to sepsis; in one hiv positive patient with meningoencephalitis, death was attributed to cardiogenic shock. all patients who died received antibiotics, however none received doxycycline. contribution of rickettsial infection to these deaths could not be ascertained. note: significant (p < 0.05) a between r. typhi and dengue, b between r. typhi and s. typhi, c between r. typhi and leptospira. †one rickettsia felis case is not included our results confirm that rickettsial infections are an important, and often overlooked, cause of fever in hospitalized patients in indonesia. the prevalence of subjects with r. typhi igg was unexpectedly high (30.8%), suggesting significant population exposure. furthermore, acute rickettsial infection was the etiology of acute febrile illness in 10.6% of hospitalized subjects, none of whom were clinically diagnosed or managed as having rickettsial infection during hospitalization. the most common clinical manifestations in our subjects (fever, headache, and nausea/vomiting) have been reported in other studies [23, 24] . however, rash, the hallmark of ricketsial disease diagnosis that usually occurs late (around 5 days of illness in patients with murine typhus) [11] was less common than other reports (17% vs. 12 to 80%, respectively) [24, 25] . this may be attributable to the design of the afire study, which only recorded clinical signs and symptoms of subjects during admission, while other studies monitored them throughout illness. lack of longitudinal monitoring may explain why prevalence of the r. typhi infection clinical triad was lower in our study (11%) than in other studies (49-64%) [26, 27] . several factors may contribute to the misdiagnosis of rickettsioses during hospitalization. first, presentation of rickettsia infection overlaps with that of other infectious etiologies, particularly typhoid fever, as demonstrated in our study and by data from the us cdc [7] . second, clinicians may not include rickettsioses in their differential diagnosis. literature demonstrating the importance of rickettsia infection in the hospital setting is lacking. previous reports were from a few serologically-confirmed patients from three cities in 1976, 1996 [28] , 1997-2000 [9] , 2006 [10] and may not have reached clinicians. third, access to diagnostic tests for rickettsioses is poor and specificity is low for available rapid diagnostics for other pathogens such as s. typhi, dengue virus and leptospira spp. reports from laos also describe difficulties in differentiating these 4 pathogens [29] . prior reports also suggest that rickettsioses are an important etiology of fever in indonesia. a hospital-based study by groen et al. in semarang, central java found rickettsioses in 7 of 118 (6%) suspected dengue cases during 1995-1996 [28] . a fever study by gasem et al. in the same city 10 years later reported 20 rickettsia cases amongst 127 children and adults visiting primary health centers and hospitals. it is unclear if these cases were due to r. typhi [10] . as part of a dengue vaccine study, copeding et al. found that 7% of childhood fever was caused by rickettsia, based on elisa igm [8] . previous studies have typically confirmed rickettsioses by serologic assays only. our study applied a panel of diagnostic assays including molecular assays, elisa, and ifa, and therefore provides additional information about rickettsia subgroups, as well as the acutely infecting species. furthermore, this study demonstrated the occurrence of human rickettsiosis in geographical areas excluded from previous studies (yogyakarta, surabaya, and makassar) and reconfirmed that rickettsioses continue to circulate in semarang, bandung, jakarta, and bali, both in children and adults. although the prevalence of rickettsioses in indonesia was as high as in thailand or malaysia, predominant species differed. in our study, r. typhi was most common, whereas in thailand and malaysia o. tsutsugamushi was more frequent [30, 31] . as o. tsutsugamushi has been found in hosts and vectors from several areas in indonesia and evidence of previous infection was detected in our samples, we likely underdiagnosed scrub typhus. this may be because our study did not include primary health centers, where cases of o. tsutsugamushi may be managed [9] . it is also possible that there are factors, such as exposure or genetic constitution, in our population that predispose to murine typhus [32] . in 70% of subjects initially diagnosed as typhoid fever or dengue fever, diagnoses were unchanged despite subsequent negative or doubtful rapid test results, suggesting that in the absence of comprehensive diagnostic tests, clinicians had no choice but to judge based on the clinical presentations. in 16 cases with positive (≥4) rapid test, the detection of igm may be associated with persistent igm that can be detected more than a year after infection [33] and/or multiple exposure for people living in endemic area [34] . in contrast, the six initially suspected leptospirosis cases had positive leptospira igm, although reference laboratory confirmatory antibody testing and pcr were negative, except in one case with possible co-infection. this suggests that severe r. typhi may clinically resemble leptospirosis (myalgia, abdominal pain, icterus) [29] and clinicians should interpret the results of leptospira antibody tests cautiously. poor specificity of the leptospira igm test, both with rapid testing and elisa, has been reported [35] . leukocyte count may also help differentiate between rickettsiosis and leptospirosis as it is more likely to be leukocytosis in leptospirosis. the case fatality rate of r. typhi infection in our study (6.8%) is higher than previously reported (0.3 to 4%) [28] [29] [30] . our study's higher mortality may be related to presence of comorbidities in 5 of the 7 fatal cases. however, we cannot exclude the possibility of severe r. typhi. complications including meningitis and encephalitis, acute respiratory distress syndrome, acute liver failure, acute renal failure, endocarditis, and multi-organ failure have been reported [36] . diagnostic inaccuracy can result in inappropriate management of patients. ideally, rickettsioses should be quickly identified and doxycycline, the antibiotic of choice, administered. amongst patients with rickettsial infection in the afire study, two subjects initially diagnosed as leptospirosis received doxycycline in combination with other antibiotics. in 58% of suspected viral infection subjects, antibiotics were later given, suggesting that clinicians considered bacterial infections but had difficulty making definitive diagnoses. broad spectrum antibiotics such as ceftriaxone or ciprofloxacin are effective against rickettsioses and are reasonable empiric choices while awaiting laboratory confirmation given the overlap of clinical presentations of rickettsia and other infections. however, unnecessary administration of broad spectrum antibiotics should be discouraged. appropriately targeted treatment could hasten recovery, reduce healthcare utilization, and minimize development of antibiotic resistance [37, 38] . on the other end of the antimicrobial stewardship spectrum, misdiagnosis of rickettsial infection as dengue could result in witholding necessary antibiotics. this is of particular concern in environments where dengue, which is managed supportively, is diagnosed empirically without laboratory confirmation and rickettsia is not considered. policy makers and clinicians should prioritize diagnosis, treatment and prevention of rickettsioses in indonesia. improved detection with subsequent appropriate management could decrease patient morbidity and reduce healthcare costs. ideally, laboratories should be equipped with valid diagnostic assays (pcr for molecular detection during acute illness and ifa as the gold standard for serology). however, pcr and ifa have several disadvantages, including the need for an expensive thermal cycler or a fluorescence microscope, which are often unavailable in endemic resource-limited settings, and experienced technicians [39] . therefore, elisa can be an alternative when both are unavailable [40] . proper empiric management, including administration of appropriate antibiotics, and early diagnostic strategies will minimize disease sequelae. finally, development and implementation of prevention guidelines may also reduce disease burden. our study had several limitations. first, we only enrolled hospitalized patients with fever, and therefore the results cannot be generalized to cases not requiring hospitalization. second, this study was conducted in 7 large indonesian cities, so may not reflect what is seen in more rural areas or other cities. finally, as the parent afire study was not designed as a rickettsia study, we did not specifically collect clinical data or request laboratory tests targeting rickettsial infections. to address this limitation, we retrospectively reviewed medical records for additional data not recorded in case report forms. lastly, we do not know that outcomes would be different if cases had been diagnosed and appropriate targeted treatment provided. in conclusion, our study demonstrates the importance of including rickettsioses in the differential diagnosis for fever in hospitalized patients, developing laboratory capacity and point of care test to rapidly and accurately diagnose rickettsioses, and implementing public policy to reduce disease burden. further studies should be conducted to better characterize the epidemiology of rickettsioses in indonesia and evaluate outcomes when appropriate empiric and targeted therapy are provided. rickettsioses as paradigms of new or j emer infect dis syndromic classification of rickettsioses: an approach 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in indonesia evaluation of serological tests for the diagnosis of rickettsiosis in denmark diagnostic accuracy of the inbios scrub typhus detect elisa for the detection of igm antibodies in chittagong center for disease control and prevantion. rocky mountain spotted fever (rmsf): clinical and laoratory diagnosis. cdc development of quantitative real-time pcr assays to detect rickettsia typhi and rickettsia felis, the causative agents of murine typhus and flea-borne spotted fever rickettsia parkeri in gulf coast ticks, southeastern virginia, usa development of a quantitative real-time polymerase chain reaction assay specific for orientia tsutsugamushi molecular characterization of novel mosquito-borne rickettsia spp. from mosquitoes collected at the demilitarized zone of the republic of korea bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt. nucleic acids symposium series. london: information retrieval ltd guidelines for the diagnosis and treatment of tick-borne rickettsial diseases murine typhus: an unrecognized suburban vectorborne disease murine typhus in returned travelers: a report of thirty-two cases clinical, laboratory, and epidemiologic features of murine typhus in 97 texas children murine typhus in central greece: epidemiological, clinical, laboratory, and therapeuticresponse features of 90 cases serological evidence of human hantavirus infections in indonesia rickettsial infections and fever epidemiology and clinical aspects of rickettsioses in thailand febrile illness in malaysia--an analysis of 1,629 hospitalized patients the role of host factors in the severity of spotted fever and typhus rickettsioses kinetics of the human antibody response against salmonella enterica serovars enteritidis and typhimurium determined by lipopolysaccharide enzyme-linked immunosorbent assay kinetics of the natural, humoral immune response to salmonella enterica serovar typhi in kathmandu limited diagnostic capacities of two commercial assays for the detection of leptospira immunoglobulin m antibodies in laos clinical and laboratory characteristics, epidemiology, and outcomes of murine typhus: a systematic review diagnosis and management of tickborne rickettsial diseases: rocky mountain spotted fever, ehrlichioses, and anaplasmosis. united states: a practical guide for physicians and other health-care and public health profesionals: cdc diagnosis and management of tickborne rickettsial diseases: rocky mountain spotted fever and other spotted fever group rickettsioses, ehrlichioses, and anaplasmosis -united states state of the art of diagnosis of rickettsial diseases: the use of blood specimens for diagnosis of scrub typhus, spotted fever group rickettsiosis, and murine typhus comparison of commercial enzyme-linked immunosorbent assay and immunofluorescence assay for diagnosis of acute rickettsia typhi infections. vector borne zoonotic dis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank our patients and their families for support of this study and nurses and laboratory technicians for collecting and testing the specimens at sites. we thank dr. richards and dr. widjaja for advice on appropriate rickettsial testing and reference laboratory team (gustiani, ungke anton jaya, deni pepi butarbutar, wahyu nawang wulan, yuanita djajadi, rizki amalia sari) for testing the specimens. we also would like to thank antonius arditya pradana and aly diana for technical assistance with the manuscript. received: 2 february 2020 accepted: 28 april 2020 supplementary information accompanies this paper at https://doi.org/10. 1186/s12879-020-05057-9.additional file 1: table s1 . diagnostic tests to confirm rickettsia infection and to exclude s. typhi, dengue, leptospira, and chikungunya infections and diagnostic tests to confirm rickettsia infection in an hiv patient and 31 patients with non-rickettsial clinical diagnoses. this study was conducted by ina-respond, a collaborative research network of nihrd, ministry of health, indonesia, and us-niaid, nih. this project has been funded in whole or in part with federal funds from the niaid, nih, under contract nos. hhsn261200800001e and hhsn261201500003i. niaid collaborators contributed to design of the study; collection, analysis, and interpretation of data; and writing of the manuscript. the content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor does mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate all subjects provided written informed consent before study participation. the study was conducted in accordance with the declaration of helsinki, and the protocol was approved by the not applicable. the authors declare that they have no competing interests.author details key: cord-271122-3fsl5589 authors: wathes, d. claire; oguejiofor, chike f.; thomas, carole; cheng, zhangrui title: importance of viral disease in dairy cow fertility date: 2019-07-24 journal: engineering (beijing) doi: 10.1016/j.eng.2019.07.020 sha: doc_id: 271122 cord_uid: 3fsl5589 many viral diseases are endemic in cattle populations worldwide. the ability of many viruses to cross the placenta and cause abortions and fetal malformations is well understood. there is also significant evidence that viral infections have additional actions in dairy cows, which are reflected in reduced conception rates. these effects are, however, highly dependent on the time at which an individual animal first contracts the disease and are less easy to quantify. this paper reviews the evidence relating to five viruses that can affect fertility, together with their potential mechanisms of action. acute infection with non-cytopathic bovine viral diarrhea virus (bvdv) in mid-gestation increases abortion rates or causes the birth of persistently infected calves. bvdv infections closer to the time of breeding can have direct effects on the ovaries and uterine endometrium, which cause estrous cycle irregularities and early embryo mortality. fertility may also be reduced by bvdv-induced immunosuppression, which increases the susceptibility to bacterial infections. bovine herpesvirus (bhv)-1 is most common in pre-pubertal heifers, and can slow their growth, delay breeding, and increase the age at first calving. previously infected animals subsequently show reduced fertility. although this may be associated with lung damage, ovarian lesions have also been reported. both bhv-1 and bhv-4 remain latent in the host following initial infection and may be reactivated later by stress, for example associated with calving and early lactation. while bhv-4 infection alone may not reduce fertility, it appears to act as a co-factor with established bacterial pathogens such as escherichia coli and trueperella pyogenes to promote the development of endometritis and delay uterine repair mechanisms after calving. both schmallenberg virus (sbv) and bluetongue virus (btv) are transmitted by insect vectors and lead to increased abortion rates and congenital malformations. btv-8 also impairs the development of hatched blastocysts; furthermore, infection around the time of breeding with either virus appears to reduce conception rates. although the reductions in conception rates are often difficult to quantify, they are nevertheless sufficient to cause economic losses, which help to justify the benefits of vaccination and eradication schemes. although viral disease remains a major cause of financial loss to the modern cattle industry, its potential impact on fertility is generally underestimated, and the main mechanisms of action are often unclear. factors including trade globalization, increases in herd size, and environmental change have contributed to the spread of existing pathogens and the introduction of disease into regions and animal populations that were previously free of it [1] . poor fertility and udder health/milk quality remain the two major causes of concern among dairy producers [2] . in terms of fertility, the ability of viruses to cause abortions and fetal malformations has probably received the most attention [3] . the outcome is generally dependent on the stage of pregnancy during which the initial infection occurs. the effects of viral diseases on reproductive performance are, however, much more pervasive and can have many subtle effects through reductions in conception rates and increased risk of culling through failure to conceive in a timely fashion. excluding fertilization failure, approximately 40% of bovine embryos die in the first three weeks after service or insemination, with cows returning to estrus after 21-24 d. a further 10%-20% of embryos are lost between days 24-60 of gestation [4] . in comparison, abortion rates on cattle farms are usually quite low (5%-10%) and have many potential etiologies that are often difficult to diagnose reliably [5] . in addition to the loss of the fetus, an abortion does, however, often have adverse effects on the fate of the dam. depending on the stage of gestation when it occurs, the cow either may need to be rebred (thus increasing her calving interval) or may start the next lactation prematurely. in one study, for example, 4.8% of 7768 holstein heifers aborted. this increased their risk of leaving the herd without completing a first lactation 2.73 times. one third of the animals which did not complete a first lactation either died or had to be culled within 50 d of calving [6] . the present short review focuses on five different viral infections showing a variety of mechanisms that can have an impact on dairy cow fertility. bovine viral diarrhea virus (bvdv) is discussed first and in the most detail, as its effects on fertility have been the most widely studied; thus, more is understood about its potential underlying mechanisms. bvdv is a flaviviridae pestivirus that is endemic in many countries worldwide, with a prevalence of 40%-90% in individual cattle and 28%-66% in cattle herds [7, 8] . it comprises a single-stranded, positive-sense rna genome that is classified by sequence differences as type 1 or 2 (bvdv-1 or bvdv-2). there is also a third type, bvdv-3 (a hobi-like, atypical pestivirus). the virus exists as either non-cytopathogenic (ncp) or cytopathogenic (cp) biotypes, with the ncp biotype causing the majority of field losses [9] . bvdv exhibits vertical transmission from mother to fetus, has a broad tissue tropism, and can infect the host either transiently or persistently [10] . such rna viruses display significant genetic variation, facilitating the emergence of new species [11] . mammalian cells normally produce type i interferons (ifns) in response to viral infection, which then trigger a cascade of antiviral pathways. bvdv causes immunosuppression through its ability to inhibit ifn production, thereby delaying the host's responses and enhancing the ability of the virus to complete its replication cycle [12, 13] . bvdv infection generally occurs via the oronasal route, but direct transmission to the reproductive tract via semen or embryo transfer is also possible [14, 15] . acutely infected animals usually eliminate the virus within 10-14 d, but transmissible virus can persist for much longer in some animals that have apparently recovered [16] . in rare cases, bulls develop a persistent infection of the testes-an immune-privileged site. more commonly, bvdv is detectable by reverse transcription polymerase chain reaction (rt-pcr) in semen for some months after an initial acute infection, although the continued risk of viral transmission appears to be unlikely after nine weeks [15] . fetal infection with ncp bvdv before the development of immune competence (i.e., prior to gestation day 120) results in early embryonic death, later abortion, or the birth of an immunotolerant calf that is persistently infected (pi) [17] . the pi calf can continuously shed virus from all secretions, and is therefore a major source of infection within a herd. the effects of acute bvdv infection vary extensively depending on both biotype and virulence, and this can lead to either avoidance or initiation of apoptotic and innate immune responses. ncp bvdv can dampen innate immune responses in several ways [13, 18] . the virus is first detected by toll-like receptor (tlr)-3 or tlr-7/tlr-8 located in intracellular compartments or by cytoplasmic pattern-recognition receptors (rig-i, ddx58), which detect single-stranded rna. the downstream signaling pathway from tlr-3 involves the ifn regulatory factor (irf)-3 and irf-7, which usually upregulate the transcription of type i ifns. the bvdv protein n pro targets irf-3 toward proteasomal degradation, thus inhibiting downstream signaling and preventing the ifn rise [18] . guanylate-binding protein 4 (gbp4), an ifn-inducible gtpase, can also inhibit this pathway while leaving nf-jb signaling intact [19] . in addition, the secreted bvdv structural protein e rns degrades viral rna through its extracellular function as a ribonuclease [20] . many of the economic losses attributed to bvdv are due to suboptimal fertility, in addition to causing abortion and fetal deformity at later stages of gestation [10, 17] . bvdv-induced immunosuppression increases susceptibility to other diseases, which may then also affect fertility. conception rates fell by up to 44% following experimental infections with bvdv either 9 d before or 4 d after insemination [21] . the review by fray et al. [22] cited many similar results that have been reported following ncp bvdv infection in the field, in spite of the occasional report to the contrary. since then, rüfenacht et al. [23] measured fertility parameters in swiss dairy herds with a high prevalence of bvdv using individual seroconversion measurements to assess the time of likely exposure. infection during the first 45 d of gestation did not influence non-return rates, but infection in mid-gestation was associated with an increased abortion rate from 6.1% to 15.8%. the timing of exposure is clearly critical, as rodning et al. [24] reported that when pi animals were introduced to naïve heifers 50 d prior to the start of breeding, they developed active immunity and there was no adverse effect on reproductive performance. newcomer et al. [25] undertook a meta-analysis of 46 studies to determine the potential benefits of vaccination against bvdv on three reproductive outcomes. vaccinated cows experienced a reduction in both abortion and fetal infection rates of nearly 45% and 85%, respectively, compared with unvaccinated cohorts, while the risk of becoming pregnant was smaller but nevertheless improved by about 5%. it is likely that a change of this magnitude would fail to reach significance in smaller studies due to a lack of statistical power. a variety of mechanisms have been suggested to account for such reductions in fertility via effects on the ovary, uterus, and early embryo. bvdv antigen was detectable in ovaries 60 d after acute infection [26] and in oocytes and follicular cells of pi heifers [27] . animals infected with bvdv develop oophoritis [26] and have impaired ovulation and ovarian steroidogenesis [27] [28] [29] . when heifers were infected with acute ncp bvdv, follicular growth patterns were affected through the subsequent two estrous cycles, including reduced growth of dominant follicles [30] . similarly, when heifers were infected 9 d before a synchronized estrus, luteinizing hormone (lh) pulsatility was decreased, there was a delay from ovulation to the progesterone rise, and subsequent progesterone levels were lower [28, 31] . these results align with studies showing that various types of stress can either delay or inhibit ovulation mechanisms [32, 33] , while both heat stress and intramammary infection can reduce follicular steroidogenesis, disrupt follicular dominance, and reduce the pre-ovulatory lh surge [34] . any acute infection occurring at this critical stage of the estrous cycle is likely to have a similar effect. the uterine endometrium is also recognized as a major site for bvdv infection [17, 29] . bvdv was found in the uterus 7-16 d after infecting heifers with bvdv by either intravenous inoculation or by breeding to a pi bull [14, 35] , while ncp bvdv was isolated from uterocervical mucus 24 d after initial infection [22] . bvdv antigen was also detected in macrophage-like cells of the endometrium in 23% of 65 cows examined in a slaughterhouse survey [36] . there is good evidence for two mechanisms by which the uterine presence of bvdv may have detrimental effects on fertility: first, by predisposing cows toward the development of endometritis; and second, by interference with the establishment of pregnancy. the bovine uterus is colonized with many bacterial species following calving in over 90% of cows [37] . these bacteria should be cleared rapidly using mucosal defense systems and an innate immune response involving endometrial epithelial and stromal cells in addition to professional immune cells [38, 39] . this early innate response is crucial to avoid the development of uterine disease; nevertheless, many dairy cows do develop metritis and/or endometritis (estimated at around 40% and 20% of all animals, respectively) [38] . in cultured bovine endometrial cells, experimental infection with ncp bvdv inhibited a variety of immune pathways normally activated in response to a challenge with bacterial lipopolysaccharide (lps), including downregulation of many interferon-stimulated genes (isgs), which are an important part of uterine defense mechanisms [40, 41] . infection with ncp bvdv was also able to switch endometrial prostaglandin production from prostaglandin (pg)f 2a to pge 2 [42] . pgf 2a is recognized as an immune enhancer, while pge 2 acts as an immune suppressor and is luteotrophic [43, 44] ; therefore, this switch may also reduce the endometrial immune response to bacteria and increase the likelihood of a cow developing a persistent corpus luteum [45] , which is often found in association with uterine disease [46] . maternal recognition of pregnancy in cows is achieved through the production of interferon tau (ifnt) by the trophectoderm of the elongating conceptus [47, 48] , which inhibits the development of endometrial oxytocin receptors, thereby preventing luteolysis [49, 50] . ifnt is a type i ifn that is structurally related to ifn-a and ifn-b but lacks viral responsive elements in its promoter and is therefore not upregulated by viral infection [51] . ifnt does, however, bind to the same ifn-a/ifn-b receptor on the uterine endometrium. together with progesterone, ifnt programs the uterine endometrium to develop a receptive environment for implantation, including upregulation of many isgs [50, 52, 53] . these are likely to have crucial roles in the establishment of pregnancy via modulation of uterine immunity, stromal remodeling, hyperplasia of the endometrial glands, and development of the uterine vasculature [52, 54] . acute infection with ncp bvdv alone has been shown to have a limited influence on endometrial gene expression in vitro [40] . however, infection did interfere with the isg regulatory irf-stat1 and stat2 pathways to inhibit ifnt-induced isg expression including isg15, herc5, usp18 (involved in protein modification via isgylation), ddx58, ifih1 (cytosolic detection of viral rna) and ifit3, mx2, rsad2, and samd9 (immune regulators with antiviral activity) [41] . upregulation of the endometrial isgylation pathway is an important process in early pregnancy that is conserved across mammalian species [55] . therefore, dysregulation of the antiviral ifn response by bvdv can undoubtedly interfere with ifnt signaling in the endometrium, suggesting another mechanism whereby infection in early gestation may reduce conception rates. there has been considerable research on the effects of bvdv on bovine embryos following concern that naïve cows might develop bvdv following embryo-transfer procedures. embryos produced using both in vivo and in vitro techniques have been infected with either ncp or cp virus at all stages from oocyte to hatched blastocyst. the affinity of bvdv for in vivo-derived embryos varied according to the strain of bvdv [56, 57] . uterine inoculation with ncp bvdv-1 in the medium used for embryo transfer on day 7 of a synchronized estrous cycle resulted in 6/10 heifers becoming pregnant 30 d later, but these pregnancies had been lost within the following 30 d [58] . although bvdv replicated efficiently in cumulus cells surrounding bovine oocytes, this did not affect the development of the blastocysts subsequently produced by in vitro fertilization [59] . similarly, when oocytes, zygotes, 8-cell embryos, morulae, and hatched blastocysts were infected with either ncp or cp virus, development was only adversely affected with cp bvdv and when the zona pellucida was not present [60] . in a more recent study, cumulus-oocyte complexes were infected with bvdv-1, bvdv-2, or bvdv-3 at different doses [57] . bvdv-1 had no effect on the embryos that did develop, and bvdv-2 infection actually increased cleavage rates but did not affect blastocyst rates. in both cases, however, the degenerate embryos tested positive. overall, the oocytes infected with bvdv-1 and bvdv-2 developed normally but carried the virus. bvdv-3 (hobi-like virus) reduced both cleavage and blastocyst rates, so would be expected to cause preimplantation embryo loss in vivo. bielanski et al. [35] used semen from a pi bull on superovulated cows, collected day 7 embryos, and transferred washed embryos to clean recipients. although bvdv was detected in the pre-transfer embryos, it did not infect the new host. from this work, it was concluded that the risk of transmission of bvdv to host cows via embryo transfer was minimal providing correct washing procedures were applied, as recommended by international embryo transfer society guidelines [61] . this resulted in low copy numbers of virus, as measured by a sensitive quantitative polymerase chain reaction (qpcr) technique [62] . in summary, acute ncp bvdv infection causes intracellular changes to ovarian and endometrial tissues through combined effects on pathways regulating immunity. these effects can reduce cow fertility by causing estrous cycle irregularities, early embryo mortality, and immunosuppression. infections during midgestation increase abortion rates or may give rise to the birth of pi calves. infectious bovine rhinotracheitis (ibr) is a highly contagious respiratory disease caused by bovine herpesvirus (bhv)-1 that is characterized by acute inflammation of the upper respiratory tract. bhv-1 is a virus of the family herpesviridae and subfamily alphaherpesvirinae. although some countries have achieved ibr eradication [63] , the disease remains endemic in dairy herds in many parts of the world, including britain and ireland [8, 64] . a recent metaanalysis found a pooled prevalence of bhv-1 of 40% in chinese cattle [65] . it is a major contributing factor in calf pneumonia, which remains the most common cause of mortality and morbidity in dairy calves between 1 and 5 months of age [66] . bhv-1 can also cause conjunctivitis, abortions, encephalitis, and generalized systemic infections [5, 63] . after the first infection, the virus is never fully eliminated, remaining latent in nerve cells of the brain. from there, it can be reactivated in times of stress, mediated via increased glucocorticoids [67] [68] [69] . bhv-1 is only one of a diverse range of pathogens that can contribute to bovine respiratory disease (brd) including several other viruses (i.e., bovine respiratory syncytial virus (brsv), parainfluenza iii virus (pi3), bvdv, and corona viruses), bacteria (e.g., mannheimia haemolytica, haemophilus somnus, pasteurella spp., and mycoplasma), and fungal genera (e.g., aspergillus) [70] . numerous epidemiology studies in various countries around the world have determined that up to 46% of calves contract brd [70, 71] . for the calves that survive, there is mounting evidence of longer term consequences of juvenile disease on adult performance [72, 73] . brd-affected animals have reduced growth rates [71, 74] , which in turn delay the age at first breeding and first calving. this is often associated with bronchopneumonic lesions and pleural adhesions [75] . for example, first parity was delayed by a median of six months in heifers that had brd in the first three months of life [76] . bach [74] reported that calves experiencing four episodes of brd before first calving had 1.87 ± 0.14 greater odds of failing to complete their first lactation in comparison with healthy calves. another study found that calving intervals were increased by 12% in mature cows that had experienced severe brd as calves during their first three months [77, 78] . in irish herds with a seasonal calving pattern that were identified as positive by a bulk tank bhv-1 enzyme linked immunosorbent assay (elisa), the three-week calving rate was significantly lower in multiparous cows in comparison with bhv-1 negative herds [79] . two related epidemiology studies in ethiopia found significantly higher rates of uterine infection and retained fetal membranes in cows that were seropositive for bhv-1 [80, 81] . a meta-analysis of over 7500 animals showed an overall decrease in abortion risk of 60% in pregnant cattle vaccinated against bhv-1 [82] . a number of studies have investigated the effects of treating cattle with modified live ibr vaccine around the time of breeding. heifers inoculated at estrus [83] , the day after [84] , or on days 7 or 14 post-breeding [85] developed mild oophoritis characterized by foci of necrosis, a few necrotic follicles, and mononuclear cell accumulation in the corpus luteum. heifers inoculated on days 21 or 28 post-breeding did not have lesions in the corpus luteum, but there were numerous necrotic follicles [85] . such lesions were not found in ovaries from which bhv-1 was not isolated [84] . vaccination at estrus was followed by a reduction in circulating progesterone [84, 86] ; conception rates were also reduced [86, 87] . although this review relates primarily to cows, there is evidence that young bulls exposed to bhv-1 at about six months of age had reduced sperm quality six months later [88] . givens [89] recently reviewed the effects of a number of viral diseases on bulls and the transmission risks of these diseases via semen. in summary, a high proportion of dairy calves experience brd, which is often associated with bhv-1 infection. this slows growth, leading to an increased age at first calving. fertility, risk of culling, and abortion rates are all subsequently increased. information on the direct effects on the reproductive tract is sparse, but there is some evidence that infection can have a direct effect on ovarian function. bhv-4 is a double-stranded dna virus that is highly prevalent in some dairy herds and has been associated with reduced fertility [90, 91] . in common with other herpes viruses, it can remain latent in the host following an initial infection in several cell types including macrophages. this results in a persistent infection [92] , which can be reactivated in vitro by glucocorticoids [93, 94] . there is evidence from measuring seroconversion that it can also be reactivated in vivo during the periparturient period [95] and in association with clinical metritis [96] . like bvdv, bhv-4 can readily infect the uterus and has been associated with metritis and endometritis; however, its role in fertility is somewhat unclear, as it has often also been found in control cows that did not have uterine infection. in addition, tested cows were usually also positive for recognized bacterial pathogens including escherichia coli, trueperella pyogenes, streptococcus spp., and histophilus somni [97] [98] [99] [100] . nevertheless, there is evidence that bhv-4 can be associated with reduced fertility. a comparison between cows requiring one or two inseminations to conceive and those needing more than two inseminations found a higher prevalence of bhv-4 in the cows requiring more inseminations [101] . klamminger et al. [100] also recorded reduced risks of infected animals either being inseminated before 80 d after calving or conceiving within 200 d. unlike ncp bvdv, bhv-4 is cytopathic, and infection can kill endometrial epithelial and stromal cells [102, 103] . accumulating evidence supports the view that bhv-4 can act as a co-factor with established uterine pathogens to promote the development of endometritis [99, 104, 105] . replication of bhv-4 depends on immediate early gene 2 (ie2) transactivation, and it has been shown that this promoter is upregulated by pge 2 , tumor necrosis factor-a (tnf-a), escherichia coli, and lps, all of which are associated with bacterial infection of the endometrium [104, 106] . bhv-4 in turn activates the interleukin (il)-8 gene promoter in endometrial cells [103, 107] . this is a key chemokine that attracts granulocytes to the uterus. in a recent study, tebaldi et al. [108] measured global gene transcription caused by the bhv-4 infection of cultured bovine endometrial stromal cells. in addition to il-8, another main pathway that was activated involved the upregulation of matrix metalloproteinase (mmp)-1. mmps are involved in the remodeling of the postpartum endometrium [109] . they are also important in controlling the balance of immune responses. on the one hand, their proteolytic activity can promote immune cell migration and activate cytokines such as il-1, il-8, tnf-a, and defensins [110] . on the other hand, over-activation of mmps has been associated with many immunopathological outcomes (reviewed in ref. [108] ). in summary, the evidence to date suggests that bhv-4 infections are quite common in dairy cows. the virus on its own probably does not cause clinical uterine disease, but it can be reactivated from latency in the endometrium following calving and then act together with bacterial pathogens to increase the risk of uterine disease by disrupting innate immunity and impairing uterine repair mechanisms. schmallenberg virus (sbv) first emerged in europe in 2011. phylogenetic analysis showed that it belongs to the simbu serogroup of the genus orthobunyavirus [111] . sbv is transmitted by culicoides midges and affects both domestic and wild ruminants including sheep, goats, and cattle. the clinical signs of disease in adult cows are quite mild and include fever, a drop in milk yield, and diarrhea with peak viremia 4-7 d post-infection [112] . sbv can both persist in and cross the placenta to replicate in the fetus itself [113] . depending on the time of exposure, this may result in abortion or severe congenital malformations causing dystocia and the birth of non-viable calves [114, 115] . a case control study on swiss dairy farms found that the abortion rate increased to 6.5% in 2012 when the sbv infection started, in comparison with a rate of 3.7% the year before [116] . while these effects on the fetus are the most obvious sign of disease, there is also evidence for adverse effects on the establishment of pregnancy and/or early embryo development. similar to bvdv, it is possible that sbv infection during early pregnancy may disrupt ifnt production, thus compromising the survival of the conceptus. like bvdv, sbv uses a non-structural protein (in this case nss) that degrades cellular rna polymerase ii, resulting in the inhibition of type i ifn production and an increase in virulence [117] . the impact of the 2011 epidemic on the productivity of dairy cattle in the netherlands and parts of germany was assessed at the herd level in a study by veldhuis et al. [118] , who compared milk production, fertility, and mortality during the epidemic with those from an earlier reference period. in both countries, there was a small but demonstrable decline in fertility parameters during the epidemic, including a significant increase in the number of repeat inseminations required and a decrease of about 5% in the 56-day nonreturn rate (from 61.5% to 55.7%). a further analysis was undertaken based on the effects of sbv on swiss dairy cows [119] . this was analyzed at the individual animal level and similarly found that the number of inseminations per cow was higher during the epidemic for cows showing clinical signs of infection in comparison with non-clinical animals from case and control herds. in this study, the non-return rate was not affected, although this may have been influenced by farms with affected animals stopping their services during the period of active infection. bluetongue virus (btv) is an important orbivirus virus infection of both domestic and wild ruminants. its geographical distribution is primarily dependent on the distribution of culicoides midges, which are the insect vectors [120] . many serotypes of btv exist, including the btv-8 strain, which is currently circulating in europe [121] . in addition to potentially causing high morbidity and mortality and reduced milk production, btv affects reproductive performance in dairy cows [122, 123] . the virus can cross the placenta, and bovine fetuses infected before 130 d of gestation develop fatal malformations of the central nervous system [124] . later studies on btv-8 also found a higher incidence of congenital malformations in newborn calves [122] . cows that were seropositive for btv in a californian study were significantly older at first calving [125] . fetal mortality increased during an outbreak of btv-8 in belgium in 2007 [126] . an early epidemiological study provided evidence for lower conception rates and longer calvingto-conception intervals in cattle [124] . more recently, this was confirmed from data obtained after an outbreak of btv-8 in the netherlands [127] . this study found that infected cows were five times more likely to return to service within 56 d after their first artificial insemination (ai) and required 1.7 times more inseminations. using a different analytical approach, nusinovici et al. [123] provided evidence that french cows infected with btv-8 experienced reduced fertility if they had been inseminated from four weeks before until five weeks after the date of disease detection within the herd. together, these studies provide good evidence that btv-8 infection prevents initial conception and/or has an adverse effect on early embryos. experimental infection of pre-implantation cattle embryos was only cytopathic in embryos with damaged zona pellucidae; there was no evidence of btv transmission to the early embryo in viremic donors [128] . days 8-9 hatched blastocysts were, however, susceptible to btv-8 infection, showing growth arrest and increased apoptosis [129] . there is again evidence that btv has the ability to inhibit ifn synthesis. in this case, viral ns4 protein is able to counteract the host's immune response by downregulating the expression of type i ifn and isgs [130] . as discussed above for bvdv, this may potentially negate the signals normally associated with the maternal recognition of pregnancy. this literature review confirms that many common viral infections of cattle have adverse effects on dairy cow fertility. abortions and fetal abnormalities are easy to quantify, although in many cases the causal factor remains unknown. in contrast, reductions in conception rates are much more difficult to detect reliably. the effects are dependent on the exact stage of the reproductive cycle when the animal becomes infected, and are influenced by herd and season. some viruses can remain latent, and reactivation around calving is likely in association with the metabolic stress of early lactation. others have synergistic actions with other infectious agents, either directly or indirectly by promoting immunosuppression in the host. this may interrupt reproductive processes such as ovulation and implantation as well as predisposing the animals to bacterial infections of the reproductive tract. determination of significant effects on fertility rates in the field is dependent on having significant power in the study to detect potentially small changes. it is also complicated by our inability to capture reliable data on many other factors that influence fertility, such as the previous disease and vaccination history and the current metabolic status of individual cows. in vitro studies using primarily uterine endometrial cells and embryos have provided useful evidence on mechanisms of action. however, very few studies have made a thorough examination of the effects on the reproductive tract of viral infection in vivo. this is understandable, given the costs involved and the practicalities of maintaining infectious cows in containment facilities over a sufficient period of time. despite these limitations, the available data do strongly suggest that viral disease plays a key but currently under-recognized role in reducing cow fertility. given the importance of viral diseases in global cattle production, attempts to eradicate-or at least reduce-the prevalence of such diseases is vital. rigorous quarantine procedures can help prevent the spread of novel diseases between countries. national measures can incentivize farmers to increase their use of regular testing and vaccination. local regulatory organizations must remain vigilant to detect novel viral diseases or variant strains of existing viruses as rapidly as possible after their emergence. disease monitoring may also be facilitated by new technologies, such as a computational approach to pathogen discovery based on bioinformatic analysis of rna sequencing data from whole blood [131] . such measures should pay dividends by improving conception rates and longevity within the dairy herd. potential applications for antiviral therapy and prophylaxis in bovine medicine setting priorities for non-regulatory animal health in ireland: results from an expert policy delphi study and a farmer priority identification survey common, emerging, vector-borne and infrequent 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fertility of dutch dairy cattle and is vertically transmitted to offspring bluetongue virus and embryo transfer in cattle susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype 8 bluetongue virus ns4 protein is an interferon antagonist and a determinant of virus virulence unmapped reads from cattle rnaseq data: a source for missing and misassembled sequences in the reference assemblies and for detection of pathogens in the host the work in the authors' laboratory was funded by contributions from the royal veterinary college, the china scholarship commission, and the commonwealth scholarship commission. the authors thank professor joe brownlie and miss olivia anstaett for their generous provision of bvdv for use in in vitro experiments. rvc manuscript number is pps_01849.compliance with ethics guidelines d. claire wathes, chike f. oguejiofor, carole thomas, and zhangrui cheng declare that they have no conflict of interest or financial conflicts to disclose. key: cord-021596-5s8lksxp authors: colegrove, kathleen m.; burek-huntington, kathy a.; roe, wendi; siebert, ursula title: pinnipediae date: 2018-10-26 journal: pathology of wildlife and zoo animals doi: 10.1016/b978-0-12-805306-5.00023-7 sha: doc_id: 21596 cord_uid: 5s8lksxp this chapter reviews common diseases of pinnipeds, including species in the otariidae (fur seals and sea lions), phocidae (true seals), and odobenidae (walrus) families. much of the knowledge on pathologic conditions of pinnipeds comes from necropsies of stranded animals and those housed in captivity. as such, disease knowledge is biased toward species frequently housed in zoos and aquaria, those that strand more commonly, or those in which free-ranging populations are more easily accessible. though historically systematic evaluations of wild populations have rarely been accomplished, in the past 10 years, with advances in marine mammal medicine and anesthesia, biologists and veterinarians more frequently completed live animal health field investigations to evaluate health and disease in free-ranging pinniped populations. the pinnipeds consist of 33 species of mammals inhabiting marine/terrestrial interfaces around the globe. they are the otariidae (fur seals and sea lions), phocidae (true seals), and odobenidae (walrus) families. common and scientific names of species described in this chapter are presented in supplemental table e1 . much of the knowledge on pathologic conditions of pinnipeds comes from necropsies of stranded animals and those housed in captivity. as such, disease knowledge is biased toward species frequently housed in zoos and aquaria, those that strand more commonly, or those in which free-ranging populations are more easily accessible. pinnipeds generally demonstrate sexual dimorphism with males being larger, possessing a large sagital crest or tusks depending on species. their organ systems broadly resemble those of the terrestrial carnivores from which they evolved, with modifications for their aquatic lifestyle. the most pronounced differences are in the lungs, skin, and vasculature, with minor modifications in the spleen and liver. many adaptations are associated with the physiological demands of prolonged diving, including an increased requirement for oxygen stores, protection from pressure effects, and insulation/thermoregulation at low temperatures. the main differences between terrestrial carnivores and pinnipeds are described below; specific differences between phocids and otariids are listed in supplemental table e2 . pinnipeds have proportionally larger livers and kidneys than terrestrial species, and the small intestine is proportionally longer. the lungs and liver are lobated, with variation in the extent of lobation between species, and the kidneys are reniculate. the heart is broad and flat. vascular adaptions, particularly pronounced in deep diving species, include an enlarged hepatic sinus, caudal vena cava, proximal aorta (aortic bulb) and spleen. the hepatic sinus in species, such as elephant seals is very expansive and should not be confused with an aneurysmal dilation. a muscular sphincter cranial to the hepatic sinus and the thick splenic muscular capsule and trabeculae enhance blood storage. thermoregulatory adaptations include arterio-venous anastomoses in the skin and reproductive organs (ponganis, 2008) . the lungs have prominent fibrous interlobular septae, and smaller airways are reinforced by either cartilage (otariids), smooth muscle (phocids), or a combination of the two (odobenids) (denison and kooyman, 1973) . the reniculate kidneys have a thick capsule and prominent outer cortical veins (stewardson et al., 1999) . pinniped epidermis is thick with no arrector pili muscles, increased numbers of sebaceous glands, and a thick adipose rich hypodermis (ling, 1970) . brown discoloration of teeth occurs with age. walrus upper canines develop into large, ventrally directed tusks. large nerve ganglia can be seen histologically near the adrenal gland capsule of some species and should not be confused with neoplasia. vacuolation of cells within the adrenal zona glomerulosa is a common finding in california sea lions (csls). pinnipeds have a zonary, endotheliochorial placenta. hormone induced morphologic changes are noted in the reproductive tract during different periods of the reproductive cycle and have been reviewed in csls by colegrove et al. (2009c) . accumulations of hemosiderin laden macrophages are found in regions of the uterus associated with previous placental attachment. these "placental scars" can be noted up to 12 months following pregnancy. hyperplasia of subsurface epithelial structures within the ovary can be noted in aged female csls. scattered meningeal aggregates of lymphocytes and plasma cells can be seen in chronic disease (goldstein et al., 2008; silvangi et al., 2005) . in more recent years, occasional cases have been noted with gliosis and neuronal necrosis in the amygdala and only relatively mild or no lesions in the hippocampus, though the cause of these lesions is unknown. also, some csls with da-associated hippocampal lesions may have multifocal lymphohistiocytic encephalitis, though whether this is due to a concurrent infectious process or to da exposure has not been elucidated. cardiac lesions associated with da toxicosis can manifest grossly as regions of myocardial pallor or streaks along the epicardial surface, pericardial effusion, or a globally flaccid heart. histologically, lesions affect the interventricular septum and left ventricle with the earliest lesions appearing at the base of the septum. acute lesions include interstitial edema, rowing of cardiomyocyte nuclei, cardiomyocyte vacuolation, and, less frequently, necrosis. in subacute to chronic lesions, there is myocyte loss and replacement by adipocytes or loose fibrous connective tissue and areas can contain occasional macrophages with cytoplasmic lipofuscin (fig. 23.5) . lesions are suspected to be primarily due to direct toxin reaction with cardiac glutamate receptors causing myocyte apoptosis (silvangi et al., 2005; zabka et al., 2009.) . domoic acid may also cause abortion and premature parturition in affected pinnipeds and the toxin can be detected within amniotic fluid, fetal stomach contents and urine. cerebral edema is rarely identified in fetuses and placental abruption can occur in dams due to seizure activity . abnormal behavior can be noted in pup and yearling csls thought to have been exposed to da in utero, though brain lesions in these animals are often not identified. domoic acid can be detected in feces, serum, urine, and gastric contents using tandem mass spectrometry coupled with liquid chromatographic separation. due to the short half-life, especially in serum, it will only be present in animals very recently exposed to the toxin (fire et al., 2011; silvangi et al., 2005) . additional diseases of vitamin deficiency and toxic conditions are presented in table 23 .1. congenital abnormalities have most often been identified in harbor and northern elephant seals and northern fur seals, with sporadic case reports in other species (colegrove, 2018; spraker and lander, 2010; st. leger and nilson, 2014; trupkiewicz et al., 1997) . in harbor seals, the most common abnormalities identified are hernias, skeletal malformations, and proliferative conditions. in harbor seals there figure 23.3 normal hippocampus from a california sea lion. dg, dentate gyrus; ca1-4, cornu ammonis sectors 1-4. there is parenchymal collapse, neuronal loss throughout the dentate gyrus (dg) and cornu ammonis (ca) sectors 1-4, and gliosis. note the loss of myocytes and replacement by loose fibrous connective tissue and adipocytes, with reactive cardiomyocyte nuclei surrounding lesions. may be functional patency of the foramen ovale and ductus arteriosus up to 6-7 weeks of age, therefore, these should not be considered congenital defects in very young pups without additional evidence of cardiac disease ( dennison et al., 2011) . in elephant seals, hydrocephalus and cardiac abnormalities are most common. low genetic diversity likely plays a role in the high prevalence of congenital defects noted in elephant seals (trupkiewicz et al., 1997) . trauma in pinnipeds can include anthropogenic factors, such as gunshot, vessel strike, and entanglement in marine debris and fishing gear (goldstein et al., 1999; moore et al., 2013) . entanglement in fishing gear, plastic, or metal rings can cause chronic infections and respiratory problems. peracute underwater entrapment or by-catch is a major source of mortality. lesions in affected animals include evidence of a struggle, net marks, or changes suggestive of hypoxia including pulmonary congestion, edema, or emphysema. gas-bubbles may also be detected in multiple tissues (moore et al., 2009) . systematic detailed, postmortem evaluation is essential to diagnosing anthropogenic trauma and criteria have been reviewed by moore et al. (2013) . predation by sharks and killer whales is a recognized major cause of trauma. spiral lacerations and wounds have been associated with gray seal predation with gray seals predating upon harbor seals, other gray seals, and harbor porpoise (fig. 23.6 ) (brownlow et al., 2016) . conspecific trauma can be associated with breeding and crowding on rookeries. ocular disease affecting the lens and cornea is common in pinnipeds, especially those in captivity. in otariids, progressive keratitis is characterized by corneal edema, keratitis, corneal ulceration, pigmentation, vascularization, and in severe cases, stromal thinning and secondary bacterial and fungal infections (fig. 23.7 ). exposure to low salinity water, worthy, g.a., 2001. nutrition and energetics. in: dierauf, l and gulland, f.m.d. (eds) . crc handbook of marine mammal medicine, 2nd edition, crc press, boca raton, fl, zabka, t.s., haulena, m., puschner, b., gulland, f.m., conrad, p.a., lowenstine, l.j., 2006 oxidants (ozone), and concurrent lens disease (lens induced keratitis), and exposure to ultraviolet radiation are also potential factors (colitz et al., 2010a) . cataracts and lens luxation also occur frequently in pinnipeds in captivity ( fig. 23.8 ). exposure to uv light, concurrent or previous keratitis and fighting are risk factors for lens disease (colitz et al., 2010b) . cutaneous microabsesses and ulcerations can occur due to sepsis and vasculitis. folliculitis secondary to bacterial infection has been reported in captive walrus (mulcahy and fravel, 2018) . a skin disease characterized by alopecia, ulceration, and hyperkeratosis was described in northern elephant seals though diagnosis has been less frequent in more recent years (beckmen et al., 1997) . alopecia or abnormal molts can be noted on occasion, most commonly in phocids. this may be related to environmental, endocrine, dietary, or stress mediated processes, however, the pathogenesis has not been elucidated. myocardial interstitial fibrosis is seen occasionally in aged csls, most often in males. this condition has been associated with acute death during anesthetic procedures in several animals. age-related arteriosclerosis can be noted in pinnipeds. myocardial necrosis can be secondary to hypoxia in pinnipeds that develop complications during anesthetic events. valvular endocarditis due to bacterial infection has been reported in csls (kim et al., 2002) . acute death due to cerebral infarction has been reported occasionally in aged pinnipeds and may be related to age-associated arteriosclerosis. few scattered meningeal lymphocytes may be seen incidentally in free-ranging csls. cerebral edema and meningitis are common lesions identified in septic phocid pups. systemic aa amyloidosis has been reported in csls and likely develops secondary to chronic inflammatory conditions. the kidney, thyroid gland, and vessels are most commonly affected. in the kidney, amyloid deposition occurs within the interstitium and glomeruli and often in a distinct band-like region along the corticomedullary junction (colegrove et al. 2009a) . urolithiasis is occasionally diagnosed in pinnipeds, though an underlying cause for urolith development is unknown (colegrove, 2018 ; dennison et al., 2007) . multifocal papillary foci of urothelial hyperplasia are noted occasionally within the urinary bladder of csls and are not related to sea lion ugc (fig. 23.9 ). the cause is unknown and lesions are not typically associated with significant cystitis. spiral bacteria morphologically consistent with helicobacter sp. have been identified in the stomach of csls; however, often they are not associated with gastritis. spiral to filamentous bacteria can be seen within colonic crypts of debilitated csls with dysbiosis. protozoa morphologically compatible with trichomonads rarely can be found within gastric glands of free-ranging csls and also may be a form of dysbiosis. acute perforating proximal duodenal/pyloric ulceration is a condition identified in free-ranging, often malnourished pup and yearling csls. lesions often lead to acute death. histologically, there is inflammation and necrosis at the site of ulceration. ulceration is not thought to be related to parasitism, bacterial infection, or any specific extra-gastrointestinal disease. instead ulcers may develop due to alterations in gastric emptying, abnormal gastrointestinal motility, or acid hypersecretion (zabka et al., 2005) . intestinal volvulus and intussusception have been reported in multiple species and may be related to bacterial enteritis (siebert et al., 2007) . hepatic hemosiderosois is seen frequently in several pinniped species including young northern elephant and harbor seals, hawaiian monk seals, northern fur seals, and csls. mild chronic cholecystitis and portal hepatitis are common findings in wild pinnipeds secondary to trematode infection and trematode-associated pigment accumulation can occur. cystic hyperplasia of the gall bladder is a common finding in older sea lions. this change may or may not have associated inflammation or infection. small, randomly distributed granulomas in hepatic parenchyma can be noted secondary to parasite migration. dental disease, abnormal tooth wear, gingivitis, secondary bacterial infections, and osteomyelitis have been reported in multiple pinniped species (colegrove, 2018) . tusk pulp bacterial infections have been an occasional problem in walruses in captivity secondary to wear. these infections can extend into the overlying dorsal bone causing osteomyelitis and fistulous tracts into the nasal cavity. preventative measures aimed at preventing worn tusks have helped to manage this condition (mulcahy and fravel, 2018) . there have been a number of case reports describing neoplasia in pinnipeds and reviews detailing reported neoplasms have been previously published. among the pinnipeds, neoplasms are most frequently diagnosed in freeranging and captive csls (colegrove, 2018; newman and smith, 2006) . in captive csls, mammary carcinoma, lymphoma, and laryngeal squamous cell carcinoma are among the most common. neoplasia is less common in captive harbor and gray seals, though flower et al. (2014) documented esophageal squamous cell carcinoma in six aged harbor seals possibly associated with regurgitation. uterine leiomyomas can be seen in aged pinnipeds. urogenital carcinoma (ugc) in csls is the most extensively studied neoplastic disease affecting pinnipeds over the past 2 decades and is considered an important wildlife model of carcinogenesis (browning et al., 2015) . a high prevalence of ugc occurs in adult csls stranding along the west coast of north america, with 26% of adult deceased csls affected at one rehabilitation facility over a 15 year period. occasional cases of ugc have been diagnosed in captive csls, most of which were wild-born. carcinogenesis is considered multifactorial with infectious agents, genetic factors, and high levels of organochlorine contaminants all possibly playing a role in tumor development. otarine herpes virus-1 (othv-1), a gammaherpesvirus that is sexually transmitted, has been associated with ugc (buckles et al., 2007) . alterations in p53, the heparanase 2 (hpse2) gene, endogenous hormones, and contaminants that interact with steroid hormone receptor have all been postulated as other potential factors involved in ugc (browning et al., 2015; colegrove et al., 2009b) . sea lions with ugc can have perineal edema, hind limb paresis, abdominal effusion, and penile prolapse with necrosis (gulland et al., 1996a) . tumors originate in the cervix, vagina, penis, or prepuce and often there is widespread metastasis at the time of death. subtly raised plaques, thickening, roughing, or dulling of the mucosa, and/or tan firm masses can be noted within affected urogenital epithelium (figs. 23.10 and 23.11). metastatic tumors often are found in the inguinal and sublumbar lymph nodes as well as many other organs, such as liver, kidney, and lung. hydronephrosis is a common sequela due to ureter compression by sublumbar lymph node masses and can be used to diagnosis ugc antemortem via ultrasound. carcinoma in situ in the reproductive tract is often very difficult to discern grossly. the in situ lesions may be seen incidentally in csls that die from other causes without metastatic disease. as such, it is critically important to routinely perform gross and histologic examination of the entire reproductive tract of male and female csls to identify these tumors (colegrove et al., 2009b; gulland et al., 1996a) . histologically, intraepithelial lesions may be observed multifocally along cervical, vaginal, penile, or preputial epithelium and often large regions of epithelium may be affected. penile or prostatic urethral epithelium is rarely affected. intraepithelial lesions may extend into the uterus along the endometrial surface; however, this is thought to be a form of intraepithelial metastasis and not primary transformation of uterine epithelium. affected genital epithelium is often markedly thickened with dysplastic cells and disordered maturation involving up to the entire thickness of the epithelium ( fig. 23.12 ). increased numbers of mitotic figures can be seen. direct infiltration of the underlying submucosa is difficult to identify, even in animals with widely metastatic disease. multiple distinct morphologic patterns including squamous, cribiform, comedone, or basaloid can be observed in metastases. neoplastic cells are polygonal with large nuclei and there is often a high mitotic rare with bizarre mitotic figures or multinucleated neoplastic cells ( fig. 23.13 ). central necrosis within masses is common and lymphoplasmacytic inflammation is adjacent to both intraepithelial lesions and surrounding metastases. despite the association between ugc and othv-1, intranuclear inclusions are noted infrequently. when present, they are most common in genital intraepithelial lesions in which there is superficial ballooning degeneration ( fig. 23 .14). inclusions are extremely rare in metastatic masses. phocine herpesvirus-1 (phhv-1) is classified in the subfamily alphaherpesvirinae and has been isolated from harbor and gray seals in europe and from both coasts of north america. serosurveys have documented titers in free-ranging phocids in both the southern and northern hemispheres (goldstein et al., 2003) . phhv-1 can be associated with high morbidity and mortality and outbreaks have been documented in rehabilitation facilities (gulland et al., 1997b) . clinical disease and fatal generalized infection are most common in neonates and young pups. horizontal transmission via direct contact and aerosols is most common though vertical transmission has been identified (goldstein et al. 2004) . in pacific harbor seals, multifocal adrenocortical and hepatic necrosis are the most common lesions associated with infection. within and adjacent to areas of necrosis, cells may have smudgy eosinophilic intranuclear inclusions figure 23.10 urogenital carcinoma in the penis of a california sea lion. there is multifocal to coalescing mucosal thickening. there is thickening of the vaginal and cervical mucosa and a polypoid mass in the vagina. transition zone between normal cervical epithelium (left) and carcinoma in situ (right). ( fig. 23.15 ). adrenal necrosis may be associated with mineralization ( fig. 23.16 ). thymic atrophy is often noted in affected pups and lesions secondary to bacterial infection and septicemia, including omphalophlebitis, pneumonia, and meningoencephalitis, may be concurrently identified (gulland et al., 1997b) . oral ulceration and small intestinal crypt dilation and necrosis are rare lesions. in infected european seals, interstitial pneumonia and massive liver necrosis have been reported (borst et al., 1986) . virus isolation, pcr, ihc, and serology (elisa) are used to diagnose infections. otarine herpesvirus-1 (othv-1) is in the subfamily gammaherpesvirinae and genus rhabinovirus. the virus has been amplified via pcr from csls with ugc (see neoplasia section) and is considered a likely factor in tumor development. viral dna can be amplified from the urogen-ital tract and, less frequently, the oral cavity of sea lions with no clinical evidence of ugc and prevalence of virus in the genital tract is higher in adults than in juveniles. sexual transmission is thought to occur and females may transmit the virus to pups during birth (browning et al. 2015; buckles et al., 2007) . the virus has also been associated with a single case of genital carcinoma in a south american fur seal and detected in a steller sea lion without evidence of neoplasia (dagleish et al., 2013) . pox viruses have been identified in skin and mucosal lesions from numerous pinniped species including csls, south american sea lions, stellar sea lions, northern fur seals, and harbor and gray seals. pinniped poxviruses are in the genus parapoxvirus and viruses from atlantic and pacific pinnipeds are phylogenetically distinct (nollens et al., 2006c) . there is widespread exposure of free-ranging pinnipeds and transmission occurs through direct contact, such as from conspecific rubbing or biting behaviors. outbreaks have occurred in rehabilitation facilities, especially in young animals, and lesions may develop during times of stress or concurrent disease (nollens et al., 2006a) . poxvirus lesions appear as raised and firm, sometimes ulcerated nodules along the head, neck, and thorax ( fig. 23.17 ). nodular or plaque-like lesions may also occur along the tongue and oral commissure ( fig. 23.18) . histologically, there is marked epidermal and follicular hyperplasia with ballooning degeneration in the stratum spinosum. moderately sized, round to chunky eosinophilic cytoplasmic inclusions are noted within epidermal and follicular keratinocytes ( fig. 23.19 ). there is variable epidermal necrosis and ulceration and often moderate associated mixed inflammation. dermal fibrosis is occasionally noted (nollens et al., 2006b ). lesions will often spontaneously regress. sealpox is a zoonotic disease that causes nodular cutaneous lesions in people and transmission likely occurs when virus enters broken skin (waltzek et al. 2012) . adenovirus infections have been reported sporadically in csls and recently been associated with an outbreak involving multiple otariids in managed care (britt et al. 1979; inoshima et al., 2013) . viral sequence analysis has determined that california sea lion adenovirus-1 (csladv-1) is unique from canine adenovirus-1 (cav-1) and represents a new member of the genus mastadenovirus (goldstein et al. 2011) . stellar sea lions have antibodies to cav-1 and canine adenovirus-2 (cav-2); however, canine adenoviral antibodies have not been detected in one infected csl (burek et al., 2005) . multifocal necrotizing hepatitis with hepatocellular intranuclear inclusions is the most common lesion reported in csl infections (fig. 23.20) . in 1 case, endothelial infection was identified in the lung, lymph nodes, and eye in the absence of hepatic lesions. ocular lesions included corneal edema, keratitis, and iridocyclitis. several cases of necrotizing enteritis in csls have also been attributed to adenoviral infection; one csl exhibited diarrhea prior to death ( fig. 23.21 ). csladv1 has been amplified via pcr from liver and feces in affected individuals and adenoviral particles have been identified via morbillivirus infection in pinnipeds was first observed in baikal seals in 1987. the infections were likely due to spread of a canine distemper virus (cdv) outbreak in terrestrial mammals (grachev et al., 1989) . in 1988, the first large-scale epizootic of phocine distemper virus (pdv) in harbor seals followed in european waters of the north and baltic seas causing 23,000 deaths, and in 2002 another outbreak occurred in the same region and affected 30,000 seals. several stranding peaks in pinnipeds on the atlantic coast of the united states have also been linked to pdv infections in harbor, harp, hooded, and gray seals but these have never caused significant die-offs as in european waters. positive morbillivirus antibody titers are regularly found in arctic seal species and provide evidence of widespread distribution of pdv in seal populations (duignan et al., 2014; hã¤rkã¶nen et al., 2006) . morbilliviruses are most commonly transmitted through respiratory, nasal, or ocular secretions. clinical findings with pdv include oculonasal discharge, conjunctivitis, keratitis, coughing, dyspnea, diarrhea, abortion, head tremors, convulsions, and increased buoyancy. pathological findings include bronchointerstitial pneumonia, pulmonary atelectasis, congestion, and edema as well as pulmonary, mediastinal and subcutaneous emphysema, nonsuppurative encephalitis, and lymphocyte depletion in different lymphoid tissues ( fig. 23.22 ). intracytoplasmic and intranuclear eosinophilic inclusion bodies can be found in the respiratory epithelium, biliary and pancreatic ducts, renal pelvis, urinary bladder, gastrointestinal epithelium, neurons, and astrocytes (duignan et al., 2014; kennedy, 1998; siebert et al., 2010) . due to viral-induced immunosuppression, pdv is often associated with secondary parasitic (pulmonary nematodiasis), bacterial (e.g., bordetella bronchiseptica) and concurrent viral infections (herpesvirus, influenza virus). the diagnosis of cdv or pdv is accomplished by immunohistochemistry using cross-reactive monoclonal antibodies for the morbillivirus nucleoprotein and by the detection of morbillivirusspecific nucleic acid in tissues by rt-pcr. antibody titers allow assessment of exposure and susceptibility to pdv (duignan et al., 2014; ludes-wehrmeister et al., 2016) . mortality events associated with influenza a have occurred in harbor seals in the united states in 1979/1980 (h7n7) and 1982/1983 (h4n5) , when large numbers of seals were found dead in the northeast (geraci et al., 1982; hinshaw et al., 1984; webster et al., 1981) . in 1991/1992 two additional isolates of influenza subtypes, h4n6 and h3n3, were obtained from harbor seals from the northeast united states (callan et al., 1995) . in europe, harbor seal mortality due to influenza a (h10n7) infection occurred in 2014/2015, starting in waters around sweden and denmark, and subsequently spreading to the wadden sea of germany and the netherlands resulting in large numbers of dead seals (bodewes et al., 2015; zohari et al., 2014) . avian influenza a (h3n8) was also associated with a 2011 harbor seal dieoff in the northeast united states (anthony et al., 2012) . influenza b, believed to be an exclusively human pathogen, was only reported from a single case, a young seal from the dutch wadden sea (osterhaus et al., 2000) . clinical findings of influenza are similar to pdv and include dyspnea, nasal discharge, lethargy, and emphysema. pathological findings include partially collapsed lungs, pulmonary congestion, necrotizing bronchitis and bronchiolitis, hemorrhagic alveolitis, bronchial gland adenitis, and occasionally interstitial pneumonia (fig. 23.23 ). additional concurrent respiratory tract lesions caused by parasitic or bacterial infections are often present (bodewes et al., 2015; geraci et al., 1982; hinshaw et al., 1984; siebert et al., 2010) . diagnosis is accomplished by elisa, immunohistochemistry using influenza a virus nucleopro-tein-specific monoclonal antibody and rt-pcr in lung and throat swabs (bodewes et al., 2015; de boer et al., 1990; vahlenkamp and harder, 2006) . marine caliciviruses are within the vesivirus genus and include over 40 serotypes including san miguel san lion viruses (smsv-1-17) and walrus calicivirus (wcv) (mcclenahan et al., 2009) . isolates and serologic evidence of exposure is documented in multiple species and in both clinically affected and healthy individuals. an unusually wide variety of marine and terrestrial species including fish, birds, and other mammals may be infected, indicating that these viruses have an ability to infect many species of marine animals, some of which may act as reservoirs for others. opal eye perch are a likely natural host. these viruses are indistinguishable from caliciviruses causing swine vesicular exanthema, and feeding swine uncooked garbage containing marine mammals and fish was a likely route of introduction into the swine population. caliciviruses are zoonotic, with rare cases of human vesicular dermatitis and flu-like illness after laboratory and field exposures (smith et al. 1998; waltzek et al., 2012) . gross findings include vesicular dermatitis that progresses to ulceration primarily of the nonhaired surfaces of the flippers (fig. 23.24) . occasionally, necrosis of underlying digits can occur. ulcerative stomatitis and ulcerative and nodular dermatitis of the lips, nasal planum, and chin have also been reported in csls. abortion and premature birth in csls have been attributed to calicivirus based on positive viral isolation and serologic evidence of exposure, but causation has not been proven. lesions begin as spongiosis of the stratum spinosum followed by subcorneal vesicle formation without formation of inclusion bodies infection causes vesicles that may rupture and ulcerate. (moeller, 2003) . the vesicles are very transient and ulcers with secondary bacterial infections are common. diagnosis is by culture or rt-pcr (moeller, 2003; smith et al., 1998) . because of the importance in differentiating these viruses from foreign animal diseases, a real-time reverse transcription pcr has recently been developed that can identify most serotypes and differentiate them from reportable vesicular diseases (mcclenahan et al., 2009) . miscellaneous viral diseases are reported on supplemental table e3 . infection with leptospira sp. is most commonly reported in free-ranging pinnipeds including csls, harbor, and northern elephant seals along the pacific coast of north america (colegrove et al., 2005b; gulland et al., 1996b) . sporadic infections and outbreaks have been reported in captive animals and exposure has been documented serologically in other geographic regions (kik et al. 2006; mackereth et al. 2005) . leptospira interrogens serovar pomona has been implicated in disease in csls. based on the long-term serosurveys utilizing the microscopic agglutination test leptospirosis is thought to be enzootic in the csl population. periodic epizootics occur every 3-5 years and result in large numbers of csls stranding with clinical leptospirosis (gulland et al., 1996b; lloyd-smith et al., 2007) . an asymptomatic, chronic carrier state may provide a mechanism for persistence of the bacterium in the population (prager et al., 2013) . though cases in freeranging csls can occur throughout the year they are most commonly noted between july and december (gulland et al., 1996b) . little is known about the epidemiology of leptospirosis in other pinniped species, where infections occur more sporadically or as small outbreaks in rehabilitation situations (colegrove et al., 2005b) . leptospirosis may also cause abortion (gulland et al., 1996b) . pinnipeds with leptospirosis have clinical signs and serum biochemistry values consistent with renal failure including polydipsia and elevated bun, creatinine, and phosphorus. grossly, kidneys are swollen, pale tan, and exhibit lack of renule and corticomedullary differentiation (fig. 23.25) . histologically, there is subacute to chronic lymphoplasmacytic tubulointerstitial nephritis, typically with a plasma cell heavy inflammatory infiltrate (fig. 23.26) . leptospira-related liver lesions are not noted. in some phocids with infection, significant tubular necrosis is more severe than inflammation suggesting that the animals die in the more acute stage of infection. infection can be confirmed histologically by demonstrating spirochetes with silver stains or ihc. ihc is particularly useful in animals with chronic infections or those treated with antibiotics since the spirochetes may be difficult to visualize; labeled antigen will be present in renal tubular epithelial cells and interstitial macrophages (colegrove et al., 2005b; gulland et al., 1996b) . pcr can also be used for diagnosis. given the zoonotic potential of the bacterium, appropriate personal protective measures should be undertaken when necropsying affected animals (waltzek et al., 2012) mycobacteriosis was first described in a group of captive pinnipeds at a marine park in australia (forshaw and phelps, 1991) , and later classified as caused by a previously undescribed member of the mycobacterium tuberculosis complex, m. pinnipedii (cousins et al., 2003) . since then the disease has been reported in a number of captive otariid species worldwide, most frequently affecting southern sea lions (kriz et al., 2011) . infection in free ranging pinnipeds is confined to the southern hemisphere and transmission between species is thought to be by sub-antarctic fur seals whose range overlaps with eight other otariid species (bastida et al., 1999) . to date, mycobacteriosis due to m. pinnipedii has not been reported for any phocid species; however, the potential host range is broad and transmission from infected fur seals and sea lions has been described for zoo species, domestic cattle, and humans (cousins et al., 2003; kiers et al., 2008; loeffler et al., 2014; moser et al., 2008; thompson et al., 1993; thorel et al., 1998) . the most likely route of infection is aspiration of aerosolized bacteria shed in respiratory secretions of infected pinnipeds. pinnipeds with mycobacteriosis typically present in poor body condition. the majority of clinical infections involve the thoracic organs, supporting inhalation as the dominant route of infection, with two main gross presentations. the first involves granulomatous to pyogranulomatous pleuropneumonia, thoracic lymphadenitis and mediastinitis with pronounced fibrinous or serosanguinous effusion (fig. 23.27 ). in the second type of presentation, there are multifocal caseating granulomas within the lungs and thoracic lymph nodes. several cases of respiratory mycobacteriosis have included concurrent granulomatous lesions in the mesenteric lymph nodes, possibly reflecting spread into the abdominal cavity through swallowing infected respiratory secretions (de amorim et al., 2014; kriz et al., 2011) . meningeal, hepatic, and disseminated granulomas have also been reported (forshaw and phelps, 1991) . histologically, lesions are dominated by aggregates of epithelioid macrophages containing few to many intracytoplasmic acid-fast bacilli admixed with variable numbers of lymphocytes, plasma cells and neutrophils. caseous necrosis of the centers of these granulomas is common; mineralization is generally minimal and multinucleated giant cells are rare. a presumptive diagnosis of mycobacteriosis can be made during post mortem examination based on the presence of acidfast positive bacteria within granulomas. confirmation of m. pinnipedii as the causative agent requires either culture and biochemical assays, which can be slow and laborious, or pcr. population monitoring is best accomplished by necropsy of all dead pinnipeds and monitoring of live animals including lymph node cytology, thoracic radiographs, and bronchoalveolar lavage. klebsiella pneumoniae are gram-negative bacteria in the enterobacteriaceae family. they are ubiquitous in the environment and are common inhabitants of the gastrointestinal or respiratory tract of healthy mammals. historically, most reports of k. pneumoniae in marine mammals were of sporadic respiratory infections or isolation from tissues of healthy individuals. however, in 2002 a mass mortality event caused by k. pneumoniae occurred in new zealand sea lion pups in the sub-antarctic (castinel et al., 2007) . the causal isolate was later identified as a hypermucoviscous phenotype (roe et al., 2015 ), but appears not to be of human origin (castinel et al., 2007) . a similar isolate has also been reported in csls (jang et al., 2010; seguel et al., 2017a) . the clinical presentation of hypermucoviscous k. pneumoniae varies somewhat between host species. affected new zealand sea lion pups have bacteremia and fibrinosuppurative to histiocytic inflammation of the meninges, joints, lymph nodes, respiratory tract, peritoneum, and subcutaneous tissues (fig. 23 .28) (roe et al., 2015) . disease has not been reported in juveniles or adults. pleuritis, pyothorax, pneumonia, and abscesses are the most frequent lesions in csls and appear to occur in all ages (jang et al., 2010; seguel et al., 2017a) , and septicemia and meningoencephalitis have recently been identified. additionally, spraker et al. (2007) describe a syndrome in csls on san miguel island, several different mycoplasma spp. have been isolated from phocids and ottarids. these include m. phocicerebrale, m. phocirhinis, m. phocidea, and m. zalophi. mycoplasma spp. are thought to be part of normal pinniped oral flora and infection is associated with skin wounds, abscesses, polyarthritis, and lymphadenitis, and necrotizing pneumonia in csls and skin wounds in gray and harbor seals (ayling et al., 2011; haulena et al., 2006; lynch et al., 2011) . in utero infection with m. phocicerebrale is associated with myocarditis and pneumonia and has caused abortion in australian fur seal fetuses (lynch et al., 2011) . a mycoplasma sp. was isolated from the respiratory tracts of harbor seals during an influenza outbreak, though the role of the bacteria in respiratory tract disease was undetermined (geraci et al., 1982) . infection is diagnosed via culture or pcr. mycoplasma sp. is the cause of "seal finger" in humans, an important zoonotic disease most often associated with the bite of a pinniped to the extremities or contact with broken skin (waltzek et al., 2012) . several new species in the genus brucella have been recently described in marine mammals. brucella pinnipedialis is the organism isolated from pinnipeds ( foster et al., 2007) . serological studies show worldwide distribution in pinnipeds with a wide variation in prevalence between geographical locations and species, although a lack of consistency in serological assays used makes valid comparisons difficult (hueffer et al., 2013; nymo et al., 2011) . the route of infection for marine mammals has not been elucidated but a fish or invertebrate vector is possible. brucella-infected lungworms can be found in the lungs of pinnipeds though whether the parasites serve as a primary route of transmission or are incidentally infected is unknown (garner et al., 1997) . while zoonotic transmission is a concern for humans who consume seal or whale meat as part of a traditional diet, none of the reported human cases had direct exposure to marine mammals (waltzek et al., 2012) . the majority of b. pinnipedialis infections in pinnipeds are subclinical. however, suppurative and necrotizing placentitis in a northern fur seal was confirmed to be due to brucella spp infection by immunohistochemistry and pcr (duncan et al., 2014) . nocardiosis is most commonly reported in juvenile stranded hooded seals; multiple different nocardia spp. are associated with infection. affected seals stranded along the east coast of the united states and canada and have typically been found outside of their normal range. underlying immunosuppression, environmental stressors, and decreased prey availability secondary to extralimital ranging may increase susceptibility to infection. lesions in affected seals include pyothorax and abscesses or pyogranulomas in the lungs and thoracic lymph nodes. infection is often systemic at death and pyogranulomatous inflammation in the skin, brain, or other tissues can occasionally be observed. infection is diagnosed via culture or pcr, and organisms can be demonstrated using gms and acid fast stains (st. leger et al., 2009) . pseudomonas sp., most commonly p. aeruginosa, can cause severe systemic infections in pinnipeds, most frequently in young animals in rehabilitation centers. pneumonia is most common; however, meningoencephalitis, hepatitis, and splenitis can be seen with septicemia. lesions are characterized by fibrinosuppurative inflammation with necrosis, interlobular edema, and fibrin accumulation in affected lungs. histologically, the bacterium causes vasculitis with characteristic "swarms" of gram-negative bacilli (gaffney et al., 2008) . p. aeruginosa infection has also been associated with hemorrhagic pneumonia in a cluster of deaths in adult free-ranging harbor seals (nollens et al., 2010) . additional bacterial diseases are presented in supplemental table e4 . fungal disease represents a relatively small proportion of infectious diseases in pinnipeds. systemic infections with opportunistic fungi have been reported sporadically and have been reviewed by reidarson et al. (2018) . exposure most often is respiratory exposure but can be due to implantation. distribution of lesions is widely variable but often involves pulmonary tissues with subsequent systemic spread. cytology and histopathology can be used to identify characteristic fungal morphology; however, culture and/or pcr are often needed to differentiate the type and species of fungus. burek (2001) reviewed epidemiology, gross and histopathologic features, and diagnosis of each fungal group. coccidioides immitis, an important human pathogen, is endemic in california and has been reported in csls and rarely in harbor and northern elephant seals. it is typically associated with disturbance of dry soil where it occurs in the mycelial phase and produces highly infectious arthroconidia. transmission is generally through inhalation. in most animals c. immitis causes only mild respiratory disease, but can progress to disseminated fatal systemic infection. animals with coccidioidomycosis are often emaciated and have pyogranulomatous pneumonia, pleuritis, peritonitis, and thoracic and abdominal lymphadenitis (fig. 23.29) . with systemic spread multiple miliary to large pyogranulomas, sometimes with indented necrotic centers, can be seen throughout the body. histologically, there is intense pyogranulomatous inflammation centered on few to many, double contoured, 5-30 âµm diameter immature spherules that mature into 30-200 âµm diameter spherules containing a myriad of endospores. mycelia with production of arthroconidia are rare in tissue. because of the danger of exposure of necropsy personnel and laboratory workers to fungal spores, affected carcasses should be incinerated. cytology, histology, and pcr are the preferred diagnostic methods (huckabone et al. 2015) . in csls fungal pyogranulomas may be grossly difficult to distinguish from ugc masses, though heavy involvement of the lungs and granulomatous inflammation on impression smear cytology can aid in differentiation. fungal dermatitis has been reported sporadically both in captive and free-ranging pinnipeds due to a wide variety of yeasts, non-dermatophylic hyaline molds, and dermatophytes. in addition to previously described predisposing factors, mechanical disruption of the skin and water quality issues (low salinity, high temperatures) can predispose to infection. alopecia, acanthosis, and hyperkeratosis can be associated with candida albicans, fusarium spp. microsporum sp. and trichophyton sp. infections. commonly affected areas include mucocutaneous junctions, nail beds, and the axillae (pollock et al., 2000; reidarson et al., 2018) . in stellar sea lions in alaska, "fungal patches" due to trichophyton sp. distributed across the body are very commonly seen and can be present for extended periods of time. affected skin is thickened with a corrugated, often alopecic, appearance, sometimes in a target pattern with either healed or raw skin in the center (fig. 23.30) . histologically, dermal skin infections cause chronic dermatitis, epidermal hyperplasia, hyperkeratosis, folliculitis, and in cases of dermatophyte infection, there may be intrafollicular or superficial epithelial fungal hyphae. special stains, such as pas or silver stains can aid in identification and characterization of the organisms. ulceration and suppurative dermatitis is generally related to self-trauma and secondary bacterial infection. diagnosis can be made by skin scraping, fungal cultures, pcr; pcr is often needed to differentiate to species (burek, 2001) . there are two general categories of lungworms that infect pinnipeds, small lungworms, the parafilaroides species that includes decorus, gymnus, hispida, normani, measuresae, hydrurgae, and gullandae and the large lungworms otostrongylus circumlitus. they are metastrongyloid nematodes of the filaroididae and crenosomatidae families, respectively. life cycles of marine mammal lungworms are not well understood, though fish species have been shown to be intermediate hosts (measures, 2001 (measures, , 2018 . parafilaroides spp. infect otariids and phocid hosts in both the northern and southern hemisphere and subclinical infections are very common. adult parasites reside in the pulmonary parenchyma, have a thin, ridged cuticle, coelomyarian polymyarian musculature, and shed embryonated eggs into the small airways where they develop into l 1 larvae. l 1 are coughed up, swallowed and shed in the feces. parafilaroides spp. infect all age-classes at different intensities in different species, though infections are often more intense in younger animals. in uncomplicated infections there are small raised nodules along the pleural surface and in the parenchyma adjacent to bronchioles (fig. 23.31 ). parasites are fine and difficult to detect grossly; however, scraping the cut surface of lesions with a knife may enable visualization. histologically, there are cross sections of small nematodes within alveolar spaces and bronchioles. infection in most animals results in minimal lymphocytic interstitial and peribronchiolar inflammation and occasional granulomas may develop around degenerating parasites or larvae (fig. 23.32) . heavy infections and secondary bacterial pneumonia can result in more significant inflammation, especially in young or debilitated animals. mild bronchiole epithelial hyperplasia and lymphocytic tracheitis are also common (measures, 2001) . otostrongylus circumlitus is distributed in a circumpolar region and affects a variety of species. the primary hosts are ringed and harbor seals; infections have been reported in other phocids including gray, elephant, ribbon, and hooded seals (measures, 2001 (measures, , 2018 . after ingestion, larvae migrate to the right ventricle, through the wall of the pulmonary artery and into the lungs. significant infection generally occurs only in young animals. heavy infections may impact diving performance, health and recruitment. adult parasites reside in the bronchi and bronchioles (fig. 23.33) . the head of a worm is embedded in the parenchyma and the tail extends into the lumen. l1 larvae are released into airways. infection induces variable amounts of mucus plugging of airways, mucosal cell hyperplasia, moderate lymphoplasmacytic and eosinophilic peribronchitis and mild chronic arteritis with tunica intima hyperplasia (measures, 2001) . in northern elephant seals reaction to larval parasites during migration in the pulmonary arteries can cause severe disease and death in the prepatent period. clinical signs include harsh lung sounds, depression, neutrophilia, and disseminated intravascular coagulation (dic). grossly, nematodes are found in the right ventricle and pulmonary arteries; they are often associated with extensive hemorrhage in lung parenchyma. histologically, nematodes are present within the vasculature and there is severe neutrophilic and lymphocytic arteritis, thrombosis, and interstitial pneumonia that can be complicated by secondary bacterial infection (fig. 23.34) (gulland et al., 1997a) . the severe reaction in this species suggests a poorly adapted host (measures 2001) . hookworm infections caused by members of the genus uncinaria have been described in a number of otariid and phocid species. both northern and southern fur seals experience significant pup mortality associated with infections. in a recent study, 94% of pups examined had infections and infections were identified as the cause of death in 35% of pups (seguel et al., 2017b) . while only two distinct hookworm species, u. lucasi and u. hamiltoni, have been confirmed to infect pinnipeds, recent studies suggest that there is considerably more species diversity than currently recognized (nadler et al., 2013) . the pinniped hookworm life cycle was largely characterized in northern fur seals in the 1960s and appears to be similar for other pinniped host species. briefly, pups are infected via the transmammary route, with ingestion of l3 larvae in the milk when a pup first suckles. larvae mature within the pup's intestine and eggs are shed in pup feces. larvae develop in the environment and l3 larvae penetrate the skin of a susceptible pinniped host or are transmitted via ingestion. larva then migrates from the intestines or subcutaneous tissues to the host ventral blubber where they lie dormant until, in pregnant females, they migrate into the milk during late pregnancy. adult hookworms survive in the intestinal tract of infected pups from around 6 weeks (lyons et al., 2011) to 8 months (hernandez-orts et al., 2013; katz et al., 2012; lyons et al., 2000) depending on host species. during this time, they attach to the small intestinal mucosa and feed on blood. clinical signs are generally only seen in pups and are associated with blood loss into the intestine from parasiteinduced damage. severely affected pups have a reduced growth rate with severe fibrinohemorrhagic enteritis, anemia, and death. typical gross lesions include pale tissues, nematodes admixed with hemorrhage in the small intestine, and numerous 2-4 mm diameter red foci on the serosal surface of the intestine that correspond to hemorrhagic and inflamed feeding sites (fig. 23.35) . penetration of the wall of the intestine with subsequent peritonitis and bacteremia also occurs (seguel et al., 2017b) . gastric and intestinal roundworm infection is extremely common in free-ranging pinnipeds, with anisakis sp. and contracaecum sp. most commonly reported. within the stomach, infection results in large, "volcanic" ulcers due to embedded parasites in the mucosa and adjacent mucosal hyperplasia and submucosal fibrosis. infection is typically considered subclinical even in cases with very large parasite loads and multiple gastric ulcers (measures, 2018) . heartworm, dirofiliaria immitis, and acanthocheilonema spirocauda, has been observed in multiple seal and sea lion species. infection mirrors the condition in domestic dogs with congested, edematous and hemorrhagic lungs, pulmonary emphysema, interstitial and exudative pneumonia, right ventricular hypertrophy pulmonary catarrhal bronchitis, and hepatic congestion. intimal proliferation of arteries and arterioles is observed microscopically. worms can be found in the right ventricle, pericardial sac, pulmonary artery, and/or lung. identification of microfilaria helps to differentiate between the two parasites; microfilaria of d. immitis are generally larger. not all blood microfilaria signal pathogenic heartworm infection. a. odendhali microfilaria is similar in size to a. spirocauda. a. odendhali infection is generally incidental with adults located in subcutaneous, intramuscular, and cavity spaces without associated cardiovascular disease. captive seals and some sea lions in endemic regions are routinely screened for and placed on heartworm preventative to protect from this infection (measures, 2018) . toxoplasma gondii is an intracellular apicomplexan parasite that infects many warm-blooded species, including people worldwide. serologic evidence of exposure to t. gondii has been reported in a number of free-ranging and captive pinnipeds including artic and subartic seals and walrus with low exposure to felid definitive hosts. parasite-associated disease is most commonly identified in free-ranging csls and monk and harbor seals (miller, 2008) . in csls with t. gondii infection, the most common lesions are necrotizing and lymphohistiocytic meningoencephalitis, myocarditis, and myositis with associated intralesional protozoa (fig. 23.36) . neurologic signs including ataxia, seizures, and abnormal mentation have been observed with infection and concurrent da toxicosis has been noted, illustrating the difficulty distinguishing protozoal disease from da exposure on clinical signs alone. infection has also 586 pathology of wildlife and zoo animals been identified in aborted csl pups, confirming vertical transmission of the parasite. incidental tissue cysts can be occasionally seen within skeletal or cardiac myocytes (carlson-bremer et al., 2015) . in harbor seals, widespread meningoencephalitis is the most common lesion of toxoplasmosis and coinfection with sarcocystis neurona have been identified in a number of seals. in one investigation in the pacific northwest of the united states, t. gondii infections were most commonly caused by type i genotypes (gibson et al., 2011) . in hawaiian monk seals, infection is typically disseminated and very large numbers of organisms are associated with lesions. adipose tissue is often severely affected (barbieri et al., 2016) . pcr and ihc can confirm infection. occasionally, organisms are not observed with ihc in areas of protozoal-compatible inflammation, though t. gondii dna can be amplified by pcr. most commercially available ihc assays employ polyclonal antibodies to t. gondii, therefore, cross reaction with other protozoa is possible . though serologic assays may be used to document exposure and rising titers have been associated with active infection, lack of pinniped specific antibodies often makes defining positive titer ranges difficult. mortality due to sarcocystis neurona, an intracellular apicomplexan parasite, is most commonly identified in free-ranging pinnipeds from the pacific coast of north america, and antibody titers reflecting exposure have been documented in harbor seals and csls (carlson-bremer et al., 2012; greig et al., 2014; miller, 2008) . several cases have also been identified in captive animals (lapointe et al., 1998) . harbor seals are the most commonly reported species to develop severe fatal disease with infection, and in california subadults and adults are primarily affected (barbosa et al., 2015; miller, 2008) . lesions include nonsuppurative meningoencephalitis which is often most severe in the cerebellum and can be associated with numerous organisms (lapointe et al. 1998) (fig. 23.37 ). rosetteform schizonts with occasionally visible residual bodies can be used to differentiate organisms from t. gondii. barbosa et al. (2015) reported a novel genotype, type xiii, associated with increased severity of encephalitis in multiple marine mammal species. while incidental sarcocysts can occasionally be noted in skeletal or cardiac myocytes in multiple species, infected csls can develop a severe polyphasic myositis. multiple muscle regions are affected including the esophagus and diaphragm. grossly, affected muscle may be atrophied and/ or have pale gray to tan streaks (fig. 23.38 ). histologically there is multifocal lymphohistiocytic myositis with myonecrosis, and evidence of regeneration (fig. 23.39 ). rare sarcocysts can be noted in myocytes, but often are not directly associated with lesions, suggesting a possible immune-mediated pathogenesis (carlson-bremer et al., 2012) . some may also develop secondary aspiration pneumonia and/or myoglobinuric nephrosis. a novel sarocystis spp. closely related to s. canis and tentatively called s. pinnipedi has been reported to cause necrotizing hepatitis sporadically in several species including csls, stellar sea lions, monk, and gray seals. ringed seals have been identified with s. canis-like muscle cysts but no apparent hepatic lesions. in affected animals schizonts and merozoites are seen in hepatocytes and kupffer cells (haman et al., 2006; miller, 2008; welsh et al., 2014) . eimeria phoca infection in young harbor seals results in enteritis ranging from mild self-limiting inflammation to severe fatal necrotizing enteritis (van bolhuis et al., 2007) . several novel apicomplexan parasites with closest homology to neospora sp. and evidence of sexual replication in enterocytes have been identified within the intestinal tracts of csls. though infection in csls causes only mild enteritis, one of these novel coccidian species was linked to fatal infection in a neonatal harbor seal, suggesting csls may be a definitive host for a protozoan pathogenic to other species . enteric coccidia have also been noted in elephant seals and south american fur seals. both serologic evidence of exposure to neospora sp. and infection diagnosed via pcr have been reported in several species (miller, 2008; gibson et al. 2011) . a more extensive review of pinniped parasitic concerns is available in supplemental table e5 . acute domoic acid toxicosis, california sea lion, hippocampus. acute neuronal necrosis and neuropil vacuolation within the hippocampus due to domoic acid toxicity. neurons have the classic shrunken "red" necrotic appearance. in acute da toxicosis, neuronal necrosis is most common in the pyramidal cells of the hippocampus, commonly most severe in sectors ca1, ca3 and ca4, the dentate gyrus, and neurons in the amygdala and pyriform lobe. (see . eslide: vm04969 23.e2 domoic acid cardiomyopathy, california sea lion, heart. multifocal vacuolation of cardiac myocytes, rare necrotic myocytes and myocyte loss associated with domoic acid toxicosis. the interstitium is edematous. in chronic lesions, with myocyte loss there is replacement by fibrosis and adipocytes. lesions occur primarily within the interventricular septum and left ventricle. (see fig. 23 .5). eslide: vm04970 chronic domoic acid toxicosis, california sea lion, hippocampus. in chronic domoic acid toxicosis, there is parenchymal collapse, neuronal loss throughout the dentate gyrus and cornu ammonis (ca) sectors 1-4, and gliosis. hippocampal atrophy can be grossly evident. there is also mild lymphocytic perivascular cuffing and meningeal lymphoplasmacytic infiltrates. (see fig. 23 there are no parasites within these sections of lung and they are often found in the right ventricle and pulmonary artery. (see fig. 23 .34). eslide: vm04975 23.e6 gastric ulceration due to ascarid infection, california sea lion, stomach. volcanic ulcer in the stomach of a california sea lion with embedded ascarids, most frequently anisakis or contracaecum sp. inflammation and fibrosis is present within the submucosa and muscularis. infections are commonly considered incidental. also present is a section of trachea with lymph node and lung infected with parafilaroides (see also ss23.04). eslide: vm04980 emergence of fatal avian influenza in new england harbor seals the occurrence of mycoplasma phocicerebrale, mycoplasma phocidae, and mycoplasma phocirhinis in grey and common seals (halichoerus grypus and phoca vitulina) in the united kingdom protozoal-related mortalities in endangered hawaiian monk seals neoonachus schauinslandi a novel sarcocystis neurona genotype xiii is associated with severe encephalitis in an unexpectedly broad range of marine mammals from the northeastern pacific ocean tuberculosis 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causes of mortality in northern fur seals (callorhinus ursinus) on st. paul island, alaska in 2007 gross and microscopic visceral anatomy of the male cape fur seal, arctocephalus pusillus pusillus (pinnipedia : otariidae), with reference to organ size and growth seals, seal trainers, and mycobacterial infection isolation of mycobacterium bovis from baboons, leopards and a sea-lion congenital defects in northern elephant seals stranded along the central california coast fatal enterocolitis in harbor seals (phoca vitulina) caused by infection with eimeria phocae influenza virus infections in mammals marine mammal zoonoses: a review of disease manifestations. zoonoses public health characterization of an influenza a virus from seals sarcocystis canis associated hepatitis in a stellar sea lion (eumetopias jubatus) from normal gastrointestinal anatomy and perforating ulcerative gastroduodenitis in california sea lions (zalophus californianus) characterization of a degenerative cardiomyopathy associated with domoic acid toxicity in california sea lions (zalophus californianus) avian influenza a(h10n7) virus involvement in mass mortality of harbour seals (phoca vitulina key: cord-017622-aqhyt7jg authors: robertson, lucy j.; björkman, camilla; axén, charlotte; fayer, ronald title: cryptosporidiosis in farmed animals date: 2013-08-17 journal: cryptosporidium: parasite and disease doi: 10.1007/978-3-7091-1562-6_4 sha: doc_id: 17622 cord_uid: aqhyt7jg cryptosporidiosis was first identified as a disease of veterinary, rather than human medical, importance, and infection of farmed animals with different species of cryptosporidium continues to be of veterinary clinical concern. this chapter provides insights into cryptosporidium infection in a range of farmed animals – cattle, sheep, goats, pigs, cervids, camelids, rabbits, water buffalo and poultry – presenting not only an updated overview of the infection in these animals, but also information on clinical disease, infection dynamics and zoonotic potential. although extensive data have been accrued on, for example, cryptosporidium parvum infection in calves, and calf cryptosporidiosis continues to be a major veterinary concern especially in temperate regions, there remains a paucity of data for other farmed animals, despite cryptosporidium infection causing significant clinical disease and also, for some species, with the potential for transmission of infection to people, either directly or indirectly. significant clinical disease and also, for some species, with the potential for transmission of infection to people, either directly or indirectly. farmed animals: overview farmed animals, also commonly referred to as livestock or domesticated animals, are those animals that are reared in an agricultural setting in order to produce various commodities -usually food (meat, organs, eggs, dairy products), and/or hair or wool. in some settings farmed animals are also used to supply labour, and the manure of domesticated animals is often used as fertilizer. animals were probably first farmed, that is their breeding and living conditions controlled by their human owners, around 7000-8000 bc during the first transitions from huntergatherer lifestyles to more settled agricultural living. the physiologies, behaviours, lifecycles of farmed animals generally differ quite substantially from those characteristics of the equivalent wild animals, and this difference impacts the interactions of these farmed animals with their parasites. farmed animals are exposed to different stresses than wild animals, are kept at different densities, and their lifecycles regulated to such an extent that a parasite-host interaction in a farmed animal may differ significantly from that in a wild animal. additionally, for infections that are of significant clinical importance, farmers may implement control measures (including treatment or prophylaxis) that alter the infection dynamics. with respect to cryptosporidium infection, for which a satisfactory chemotherapeutic cure or prophylaxis is not yet available, different species infect different species of farmed animal, and may or may not be of clinical relevance. table 4 .1 provides an overview of the farmed animals included in this chapter, the species of cryptosporidium to which they are susceptible and brief notes on the clinical relevance. greater details are provided in the appropriate chapter sections. various categories of animals that are 'farmed', including mink, foxes, guinea pigs etc. are not included, largely because of a lack of information on cryptosporidium in these animals in the domesticated setting. additionally, farmed fish are not included in this chapter. c. parvum a common in pre-weaned calves -acute onset diarrhoea. intestinal location c. bovis common in post-weaned calves -less pathogenic than c. parvum c. andersoni older post-weaned calves, yearlings and adults-some failure to thrive. infects the gastric glands of the abomasum c. ryanae common in post-weaned calves a range of other species has been reported from cattle and other bovines. these seem to be unusual and are apparently of minor clinical significance small ruminants, including sheep (ovis aries) and goats (capra aegagrus hircus) c. parvum a relatively common in pre-weaned lambs, associated with diarrhoea c. xiaoi common in older lambs and sheep, often apparently asymptomatic c. ubiquitum a common in older lambs and sheep, often apparently asymptomatic pigs (sus scrofa domesticus) c. parvum a less common than in bovines and small ruminants; diarrhoea and vomiting c. suis relatively common, mild symptoms c. scrofarum relatively common, mild symptoms deer (cervids), including red deer (cervus elaphus) fallow deer (dama dama), elk/wapiti (cervus canadensis), white-tailed deer (odocoileus virginianus), and reindeer (rangifer tarandus) c. parvum a information on species detected amongst farmed deer is lacking; diarrhoea in young calves, possibly severe, but can also be asymptomatic c. meleagridis a appears to have a wide host range, including farmed poultry (and mammals). mostly infects the intestines and has been associated with generally mild clinical symptoms c. baileyi a a wide avian host range reported, including various farmed poultry species. detected in many different anatomical sites including digestive tract, respiratory tract, and urinary tract. has been associated with high morbidity and mortality (continued) of various cryptosporidium species (see table 4 .1) means that public health may also be affected by infections in farmed animals. infection may be direct, from animal to human, or indirect, via a transmission vehicle. a large number of small outbreaks associated with c. parvum in calves and in veterinarians or veterinary students that have been exposed to calf faeces are documented in the literature (e.g. grinberg et al. 2011; gait et al. 2008; robertson et al. 2006) . in addition, a number of outbreaks have been documented associated with children visiting 'petting farms' or similar venues, where interaction with young animals such as lambs or calves is encouraged. less commonly, transmission between animals such as camels or alpacas and their carers has also been reported. drinking water, and less often food, has been associated with transmission of cryptosporidium infection from animals to human populations, with c. parvum from grazing cattle contaminating water supplies particularly implicated. the high densities of farmed animals in water catchment areas mean that implementation of catchment control measures, including preventing defecation into water courses, may have a significant effect on minimising the risk from this potential transmission pathway. there are various species of farmed bovines, with cattle and zebu (bos taurus and bos indicus, respectively) amongst the most important livestock worldwide. both provide meat, milk and other dairy products, and are also used as draught animals, with an estimated 1.5 billion head globally (3 cattle for every 14 people). a comprehensive overview of cryptosporidium infection in cattle has been published by and the information presented here is largely an update built on this solid basis. domesticated water buffalo (bubalus bubalis) is also important domestic livestock in the bovinae sub-family. domesticated buffalo consist of swamp buffalo and river buffalo. in 2011, the world population of buffalo was estimated to approximately 195 million animals, of which 97 % were in asia (faostat 2012). the first report on bovine cryptosporidiosis was published in 1971, when parasites were identified in an 8-month-old heifer with chronic diarrhoea (panciera et al. 1971) . since then, cryptosporidium infection in cattle has been documented in most countries worldwide. four major cryptosporidium species infect cattle: c. parvum, c. bovis, c. ryanae and c. andersoni (table 4 .2; fayer et al. 2007; feng et al. 2007; santín et al. 2004; langkjaer et al. 2007) . cryptosporidium parvum has a broad host range and apparently has the ability to infect most mammals, including humans and cattle. in contrast, the other three species have almost exclusively been found in cattle. in addition to these four common species, sporadic natural infections with c. felis, c. hominis, c. scrofarum, c. serpentis, c. suis and c. suis-like genotype have been detected in cattle (bornay-llinares et al. 1999; geurden et al. 2006; langkjaer et al. 2007; santín et al. 2004; smith et al. 2005; chen and huang 2012) . the extent to which these findings reflect true infections or accidental carriage, i.e. ingested oocysts that pass intact through the gastrointestinal tract, remains to be clarified. cattle have also been experimentally infected with c. canis, but natural infection has not been reported ). many reports on cryptosporidium in cattle in different countries and settings have been published over the years, showing that cryptosporidium spp. infections are common worldwide. both dairy and beef cattle are infected and the prevalence estimates vary considerably among studies. reported herd level prevalences range from 0 to 100 % (olson et al. 1997; chang'a et al. 2011; maddox-hyttel et al. 2006; santín et al. 2004 ). infected animals have been reported from all age groups but infection is most common in preweaned calves. when calves up to 2 months of age have been investigated in point prevalence surveys, 5-93 % of the calves shed oocysts (table 4.2; maddox-hyttel et al. 2006; santín et al. 2004; uga et al. 2000) . longitudinal studies performed in infected dairy herds showed that all calves in such herds shed oocysts at some time during their first months of life (o'handley et al. 1999; . the overall picture is that there is an age-related pattern in the species distribution. c. parvum is mostly found in preweaned, monogastric calves up to 2 months of age where it is often the most prevalent species, responsible for more than 80 % of cryptosporidium infections (brook et al. 2009; fayer et al. 2007; plutzer and karanis 2007; santín et al. 2004 trotz-williams et al. 2006) . in some areas, however, c. bovis is the dominating species found in preweaned calves (budu-amoako et al. 2012a; wang et al. 2011a; silverlås et al. 2010b ). the prevalence of c. parvum is considerably lower in older calves and young stock, and there are few reports of c. parvum infection in adult cows langkjaer et al. 2007; silverlås et al. 2010b; castro-hermida et al. 2011a; khan et al. 2010; ondrácková et al. 2009; muhid et al. 2011; budu-amoako et al. 2012a, b in older calves and young stock, c. bovis and c. ryanae are the most commonly found species fayer et al. 2007; muhid et al. 2011; langkjaer et al. 2007; silverlås et al. 2010b) . c. andersoni is mainly found in young stock and adult cattle (enemark et al. 2002; wade et al. 2000; fayer et al. 2007; ralston et al. 2003) . older studies, based on microscopy alone, overestimated the c. parvum prevalence in weaned animals because the similarity in oocyst size makes it impossible to differentiate between c. parvum (~5.0 â 4.5 μm), c. bovis (~4.9 â 4.6 μm) and c. ryanae (~3.7 â 3.2 μm). molecular studies have revealed different genetic subtypes within the c. parvum and c. hominis species and dna sequence analysis of the 60 kda glycoprotein gene is commonly used to further characterize the isolates. a number of c. parvum gp60 subtype families, designated iia-iio, have been described. of these, iic and iie are considered anthroponotic, whereas iia and iid are commonly found in both humans and animals. the other subtype families are uncommon and their zoonotic potential has not been determined. in cattle, c. parvum of the iia subtype family is especially common. in addition to iia, iid and occasionally iil (sometimes named iij) subtypes are found (table 4 .3; xiao 2010). whether there is a difference in pathogenicity between subtypes is currently unknown, as most genotyping studies to date have focused on herds with a history of calf diarrhoea. it has been suggested that herd management strategies affect subtype distribution. studies from areas with closed herd management (limited animal movements between herds) have shown a high number of subtypes in the calf population, but only one subtype in each herd (brook et al. 2009; mišic and abe 2007; soba and logar 2008; ). it has also been shown that a unique gp60 subtype can persist over time in a closed dairy herd (björkman and mattsson 2006) . in contrast, only a few subtypes have been identified in areas with more animal movements between herds, but several subtypes could be present in a herd (brook et al. 2009; peng et al. 2003; trotz-williams et al. 2006 ). the information on the distribution of cryptosporidium infection in water buffalo is rather fragmentary. reported prevalences vary between 3 % and 38 % (table 4.4 ). an association between prevalence of infection and age of the animals, with the highest prevalence in young calves, has been found (helmy et al. 2013; maurya et al. 2013; nasir et al. 2009; bhat et al. 2012; díaz de ramírez et al. 2012 ). the first report on cryptosporidium species identification in water buffalo was published in 2005, in which gómez-couso et al. (2005) used molecular tools to characterize cryptosporidium oocysts from an asymptomatic neonatal calf in a dairy buffalo farm in spain. sequence analysis of a fragment of the oocyst wall protein (cowp) gene revealed that the isolate was closely related to the cryptosporidium 'pig' genotype. a few years later, c. parvum was identified in water buffalo calves from italy (cacciò et al. 2007 ) and today c. ryanae, c. bovis and (table 4 .4). most molecular investigations have been done in calves and thus it is not known if the species distribution differs between animals of different age. one of the few studies that include faecal samples from both calves and older animals was done in egyptian smallholder herds (helmy et al. 2013) . c. parvum was most common in calves younger than 3 months but was detected in animals up to 2 years of age. in another study, also from egypt but from another part of the country, c. parvum was only found in calves, whereas none of the sampled cows shed any cryptosporidium oocysts (amer et al. 2013) . when the c. parvum isolates were subtyped by sequence analysis of the gp60 gene, subtype families iid and iia were found, with a majority of iid in both studies. subtype iid is the dominating subtype family also in cattle in egypt (amer et al. 2010; helmy et al. 2013 ). cryptosporidium parvum and c. andersoni are the two species that have been associated with clinical disease in cattle. c. parvum infection is considered a major cause of diarrhoea in young calves (blanchard 2012; radostits et al. 2007 ). calves are often already infected during the first week of life (uga et al. 2000) and clinical cryptosporidiosis is mostly seen in calves up to 6 weeks of age. the most prominent finding is pasty to watery diarrhoea, sometimes accompanied by lethargy, inappetence, fever, dehydration and/or poor condition. the calf most often recovers spontaneously within 1-2 weeks, but there is a large variation between individuals in how they respond to, and recover from, infection. in some cases the infection may be fatal (tzipori et al. 1983; fayer et al. 1998) . a decrease in growth rate may be seen in the weeks after the calves have recovered from the acute phase of the disease (klein et al. 2008 ), but no long-term effects on growth and performance have been reported. the pathogenesis of bovine cryptosporidiosis is not fully understood but the clinical signs are attributed to both malabsorption and an increase in fluid secretion in the ileum and proximal portions of the large intestine. for references and a brief overview see o'handley and olson (2006) . cryptosporidiosis may be seen in individual calves, but frequently it soon develops among the calves into a herd problem. concomitant infection with other pathogens, e.g. rotavirus, coronavirus and enteropathogenic escherichia coli (e. coli f5+) can worsen the clinical signs and prolong the duration of illness (blanchard 2012) . a number of studies have reported an association between c. parvum infection and diarrhoea in young calves. many of these were published at the time when all cryptosporidium oocysts of around 3-5 μm in diameter were considered to be c. parvum. more recent investigations, applying molecular methods to analyse faecal samples from diarrhoeic calves, corroborate these earlier findings. when samples from young calves with diarrhoea were analysed, c. parvum is found to be the dominant species (quílez et al. 2008b; imre et al. 2011; karanis et al. 2010; soba and logar 2008; plutzer and karanis 2007) . interestingly, this dominance of c. parvum in diarrheic calves was also seen in a recent swedish investigation of diarrheic calves ), although c. bovis is the predominant species in randomly selected calves in sweden. only one experimental trial has been performed with c. bovis (fayer et al. 2005 ). three calves under 1-8 weeks of age were orally inoculated with oocysts. this resulted in subclinical infection in 2 of 3 calves. both animals had, however, previously been infected with c. parvum and cross-protective immunity could not be excluded. calves with diarrhoea are significantly more likely to be infected with c. parvum than with c. bovis starkey et al. 2006; kváč et al. 2011) . based on these findings, and based on the fact that c. bovis is not common in calves, but is a widespread subclinical infection in older animals in most countries, c. bovis is commonly considered to be apathogenic to cattle. however, the pathogenic potential deserves further attention as high numbers of c. bovis oocysts in samples from diarrhoeic calves have been reported, even in the absence of c. parvum or other diarrhoeal agents (silverlås et al. 2010a (silverlås et al. , b, 2013 . cryptosporidium ryanae was first described as a separate species by fayer et al. (2008) , and until then it was known as cryptosporidium deer-like genotype. an experimental trial was performed in two colostrum-deprived calves 17-18 days old. both calves started excreting oocysts 11 days after inoculation, but neither of them showed any clinical signs ). there are several reports of the distribution of cryptosporidium deer-like genotype and c. ryanae. most studies found a predominance of the parasite in older calves and young stock. so far no association with clinical disease has been reported. in contrast to the other species, c. andersoni infects the abomasum. it does not cause diarrhoea, but c. andersoni infections have been associated with maldigestion. the infection may cause moderate to severe weight gain impairment in young stock and reduced milk production in cows (anderson 1998; esteban and anderson 1995; lindsay et al. 2000) . a major obstacle from a disease control perspective is the lack of effective means to control cryptosporidium infection and decrease the level of contamination of the environment with oocysts. preventive hygiene measures and good management are currently the most important tools to control cryptosporidiosis. reducing the number of oocysts ingested by neonatal calves may reduce the severity of infection and allow immunity to develop. a common recommendation is to ensure good hygiene in calf facilities and ascertain that all newborn calves ingest an adequate amount of colostrum during their first 24 h of life. sick calves should be housed in a clean, warm, and dry environment and isolated to prevent spreading of the infection to other calves. acutely infected animals may need supportive care with fluid and electrolytes, and milk should be given in small quantities several times daily to optimise digestion and minimise weight loss. over the years, several substances have been tested for potential anticryptosporidial effects with limited success . halofuginone lactate has shown some beneficial effects such as milder clinical signs and reduced oocyst output when used as prophylactic treatment (de waele et al. 2010; silverlås et al. 2009a ). this drug is approved in europe to treat calf cryptosporidiosis. however, the safety margin is narrow and the substance is toxic at only twice the therapeutic dose, so careful dosage is necessary. halofuginone lactate treatment should only be considered in herds with severe diarrhoeal problems strongly associated with c. parvum. when treatment is used, it should always be in conjunction with applying measures to reduce environmental contamination and risk of infection. a recent study investigated if an antibody-biocide fusion consisting of a monoclonal antibody "armed" with membrane-disruptive peptides (biocides) could be used for treatment of cryptosporidiosis in calves (imboden et al. 2012) . calves 36-48 h of age were challenged once with c. parvum oocysts and were simultaneously administered the antibody-biocide fusion mixed with milk replacer. the antibody-biocide fusion treatment was repeated 5-8 times. control calves were given milk replacer with placebo. calves receiving the antibody-biocide fusion had a significantly higher health score and shed fewer oocysts than control calves. these results suggest that this concept might be effective in cattle, but further testing is necessary (imboden et al. 2012) . vaccination is successfully used to control many infectious diseases in livestock. however, it takes weeks for a protective immune response to develop after a vaccine has been administered, and as calves may be exposed to cryptosporidium oocysts immediately after birth, vaccination of newborn calves is unlikely to be successful in preventing cryptosporidiosis. thus it has been suggested that the most feasible approach is likely to involve passive immunisation (innes et al. 2011) . dams are immunised in late gestation and their colostrum is fed to the calves. a recent study investigated antibody responses in calves fed colostrum from heifers vaccinated with a recombinant c. parvum oocyst surface protein (rcp15/60). the calves had measurable quantities of the specific antibody in their serum. however, as the calves were not subsequently challenged with oocysts it remains to be seen whether this immunisation scheme can also prevent symptomatic infection and eliminate oocyst shedding (burton et al. 2011) . an association between cryptosporidium oocyst shedding and diarrhoea in buffalo calves has been reported from investigations performed in egypt, india and venezuela (mohanty and panda 2012; bhat et al. 2012; maurya et al. 2013; el-khodery and osman 2008; díaz de ramírez et al. 2012) suggesting the cryptosporidium infection is part of the calf diarrhoea syndrome in water buffalo, as it is in cattle. species identification was only performed in one of these studies, and c. parvum was the only species that was found (maurya et al. 2013 ). calves begin shedding c. parvum oocysts 2-6 days after infection and shedding continues for 1-13 days tzipori et al. 1983) . during the first 2 weeks an infected calf can shed millions of oocysts uga et al. 2000) resulting in heavy environmental contamination, and efficient dissemination of the parasite within the herd and to the environment. in faecal samples obtained from symptomatic calves naturally infected with c. parvum 10 6 -10 8 oocysts per gram faeces (opg) are often seen ). in herds with established c. parvum infection, most calves are excreting oocysts between 2 and 4 weeks of age (o'handley et al. 1999; uga et al. 2000) . when repeatedly sampled the same 30 calves in a dairy herd from birth to 2 years of age they found c. parvum oocysts in faeces of all individuals before they were 3 weeks old, i.e. a cumulative prevalence of 100 %. c. parvum oocysts were also found in samples collected from a calf at 16 weeks of age and from another at 6 months of age, indicating that oocysts can be shed intermittently over a long period after the initial infection. alternatively, these late-shedding individuals might not have developed a fully protective immunity after the first infection, and rather than this being a sign of prolonged infection, they had acquired new infections. in this study, molecular analyses indicated the same sub-genotype at the gp60 locus. however, this does not necessarily indicate prolonged infection, as re-infection with the same genotype in the environment may occur if the immunity is not protective. it has been suggested that an increase in c. parvum oocyst shedding may occur in adult cows around calving (so called periparturient rise), but to date there have been few reports to support this. in a recent study, however, dams in a suckler beef herd were found to shed low levels of c. parvum oocysts around the time of calving (de waele et al. 2012) . only one experimental infection for each of c. bovis and c. ryanae has been reported so far. regarding c. bovis, one calf shed oocysts from 10 to 28 days after infection and the other only for 1 day (day 12) (fayer et al. 2005 ). for c. ryanae, oocyst shedding started 11 days after inoculation. both infected calves excreted oocysts during 15 and 17 consecutive days, respectively . shorter prepatent periods have been seen for both c. bovis and c. ryanae in natural infections (silverlås et al. 2010b; silverlås and blanco-penedo 2013) . no oocyst excretion rate values were determined from the experimental infections, but in naturally infected calves 300 to 8 â 10 6 opg and 100-835,000 opg have been reported for c. bovis and c. ryanae, respectively (silverlås and blanco-penedo 2013) . young stock and adults may also be infected by the larger c. andersoni (oocyst size~7.4 â 5.5 μm) and may shed oocysts intermittently for many years (olson et al. 2004; ralston et al. 2003) . a periparturient rise in c. andersoni oocyst shedding, seen both as increase in prevalence and in number of oocysts in faeces, has been reported (ralston et al. 2003) . several studies have shown that age is associated with cryptosporidium infection and that young calves have the highest risk of being infected (maddox-hyttel et al. 2006; santín et al. 2004; sturdee et al. 2003; fayer et al. 2007 ). this is also the age group that is most often infected with c. parvum and suffers from clinical cryptosporidiosis. thus, from clinical and zoonotic perspective, knowledge on the epidemiology of cryptosporidiosis in young calves is highly valuable. when potential risk factors for cryptosporidium infection in pre-weaned calves have been explored, the results differ between studies. one factor that recurs in several studies is the type of flooring in the calf housing area. in spain, castro-hermida et al. (2002) found that straw on the floor or earth floors in the calf pens increased the risk for infection compared with cement flooring, and in malaysia calves kept in pens with slatted floors and sand floors had an increased risk compared with those in pens with cement floors (muhid et al. 2011) . a protective effect of cement floors was also reported from the usa (trotz-williams et al. 2008) . it was suggested that the reason for this protective effect of cement floors is that they facilitate thorough cleaning. this assumption corroborates the finding that a low frequency of cleaning of the calf pens increased the risk for infection (castro-hermida et al. 2002) . it is also consistent with the finding that the use of an empty period in the calf pen between introductions of calves was associated with a lower risk for infection in danish dairy herds (maddox-hyttel et al. 2006) . when cows as a cause of infection were investigated, a higher risk of infection was identified in calves that were housed separately from their dams (duranti et al. 2009 ), and a lower risk of infection in dairy calves kept with the cow for more than 6 h after birth (silverlås et al. 2009b) . in one of the few reports to investigate risk factors for infection with different cryptosporidium species in pre-weaned dairy calves to date (szonyi et al. 2012) , risk of infection with c. parvum differed to some extent from that of c. bovis. both c. parvum and c. bovis were more common in the younger calves, but herd size and hay bedding were associated with an increased risk for c. parvum infection, whereas jersey breed was a risk factor for c. bovis infection. experimental cryptosporidium infections in water buffalo have not been reported. however, oocyst shedding dynamics were investigated in naturally infected buffalo calves in a farm located in a tropical dry forest area in venezuela. twenty-five calves were sampled from birth to 12 weeks of age. oocysts were detected from day 5 and 72 % of the calves shed oocysts before they were 30 days (díaz de ramírez et al. 2012) . regarding risk factors for infection, there are some reports of seasonal variations in prevalence (bhat et al. 2012 there are numerous reports of cryptosporidiosis outbreaks in humans after contact with infected calves. these have often involved veterinary students and students at farm schools (see, e.g., gait et al. 2008; grinberg et al. 2011; pohjola et al. 1986; robertson et al. 2006; kiang et al. 2006 ), but also young children have fallen ill after visiting petting zoos or open farms (gormley et al. 2011; smith et al. 2004) . contact with cattle has been identified as a risk factor for disease also in casecontrol studies of sporadic human cryptosporidiosis (hunter et al. 2004; robertson et al. 2002; roy et al. 2004) . altogether, there is plenty of evidence to conclude that cryptosporidium can be transmitted from calves to humans by direct contact or by contaminated equipment. the risk for zoonotic transmission is likely to be highest in herds with cryptosporidium associated calf diarrhoeal problems, where oocyst contamination in the barn can reach high levels and where contact with naïve individuals is most likely to occur. key measures to prevent visitors becoming infected are to ensure good hygiene in the visitor area, providing suitable handwashing facilities and ensure that they are used when workers and visitors leave the premises. c. bovis infections have recently been detected in a few persons living or working on cattle farms (khan et al. 2010; ng et al. 2012) . it is not known if these were active infections and the implication of these findings is thus unclear. as molecular typing methods become more accessible, epidemiological studies can investigate c. parvum gp60 subtype distribution in cattle and human populations in different regions. the reports so far indicate that in many areas the subtypes that are most common in cattle are those most often found in humans. for example, c. parvum iiaa15g2r1 was the predominant subtype in both bovine and human infections in slovenia and portugal (soba and logar 2008; alves et al. 2006) . in new south wales, australia c. parvum iiaa18g3r1 dominated in both calves and people living on cattle farms (ng et al. 2012) , whereas iiaa16g2r1 was the predominant genotype identified in beef cattle and humans in prince edward island, canada (budu-amoako et al. 2012c). further information is provided in the review by xiao (2010) . that the same subtypes are found in cattle and humans might be taken as an indication of zoonotic transmission. however, it is important to note that even when zoonotic c. parvum subtypes are identified in humans, cattle are not necessarily the source of the infection. these zoonotic subtypes can circulate and propagate in the human population in addition to the anthroponotic subtypes. the occasional finding of c. hominis chen and huang 2012) in cattle highlights the fact that cryptosporidiosis may be transmitted not only from cattle to humans, but also from humans to cattle. food-related cryptosporidiosis outbreaks have sometimes been associated with cattle. foodborne transmission was implicated in cases of children who had drunk unpasteurized milk (harper et al. 2002) or cider, made from apples collected in an orchard where calves from an infected herd had grazed (millard et al. 1994 ). other outbreaks in which cattle were suspected as the source involved vegetables that had been sprayed with water that could have been contaminated with cattle faeces. often there was only circumstantial evidence that cattle were the source of contamination, and it was not possible to exclude other potential sources (see e.g. cdc 2011; robertson and chalmers 2013) . outbreaks of cryptosporidiosis associated with drinking water have often been attributed to contamination of water catchments by cattle manure. the evidence implicating cattle has sometimes been substantial (bridgman et al. 1995; smith et al. 1989 ), but for others the evidence was not conclusive. grazing cattle or slaughterhouse effluent contaminating lake michigan were mentioned as two possible sources of cryptosporidium oocysts in the large outbreak in milwaukee, wisconsin in 1993 (mac kenzie et al 1994 , but retrospective analysis of clinical isolates revealed that it was caused by the anthroponotic species c. hominis . this was also the case in the most recent outbreaks in uk (compiled by chalmers 2012) and a large drinking waterborne outbreak in sweden in 2010 (anonymous 2011) . given that pre-weaned calves are the most likely age group to shed c. parvum oocysts, any measure to prevent waterborne zoonotic transmission should be directed towards this age group. protective measures could be to prevent young ruminants from accessing water catchments, and compost or spread calf manure on fields where runoff cannot occur. the manure of older ruminants is generally not a zoonotic concern with respect to cryptosporidium. the seemingly common occurrence of c. parvum in buffalo calves highlights the potential role of water buffalo in zoonotic transmission. thus the same precautions to prevent transmission of the parasite to humans, by direct contact or through food or water, are also applicable to water buffalo. sheep (ovis aries) and goats (capra aegagrus hircus) are important in the global agricultural economy -producing meat, milk and wool -both in developing countries such as india and iran, and industrialised countries such as australia and the united kingdom (de graaf et al. 1999; noordeen et al. 2000; robertson 2009 ). in 2010, the world stocks were approximately one billion sheep and 910 million goats (faostat 2013a). asia has the largest populations of both species, with 42 % and 60 % of the total world populations, followed by africa (faostat 2013a). according to a fao report, over 90 % of the goat population can be found in developing countries (fao 2012) . sheep and goats tend to be managed differently to cattle, with flocks grazing large enclosures rather than being kept indoors. there have been fewer studies on cryptosporidium infection in sheep than in cattle, and even fewer studies have been performed on goats. nevertheless, it is known that these protozoans are economically important parasites in both ruminant species (noordeen et al. 2000; robertson 2009 ). infection and disease was first described in 1974 for sheep (barker and carbonell 1974) and in 1981 for goats (mason et al. 1981) . younger animals are more susceptible to infection than older ones, reflected in high shedding rates and diarrhoeal prevalences in lambs and kids up to 1 month of age, whereas infection in older animals is usually subclinical with lower shedding rates (vieira et al. 1997 ). as for other animals, ovine and caprine cryptosporidium infection can be found throughout the world. the prevalence varies widely between studies, from 0 % to 77 % in sheep and from 0 % to 100 % in goats. all age groups are susceptible, but infection is more common in lambs and kids than in older animals (tables 4.5 and 4.6). study design factors other than age of sampled animals, such as whether only diarrhoeal animals were sampled or not, if a point prevalence study or a longitudinal study was performed and the diagnostic method(s) used, also can affect prevalence data. the effect of using different diagnostic methods is evident in, for example, giadinis et al. (2012; see also tables 4.5 and 4.6) and ryan et al. (2005) , where microscopy resulted in lower prevalences than detected by elisa and pcr, respectively. prevalence and species distribution for studies conducted on sheep dating back to 2007 are summarised in table 4 .5. specific data for studies on sheep published before 2007 can be found in "cryptosporidium and cryptosporidiosis" ; tables 18.9 and 18.10). prevalence rates and species distribution for all identified surveys of goats are summarised in table 4 .6. several species and genotypes have been identified in sheep, and the species distribution varies between studies and with age of the animals. cryptosporidium parvum, c. ubiquitum (previously cryptosporidium cervine/cervid genotype) and c. xiaoi (previously c. bovis-like genotype) are the most common species. sporadic infection with c. hominis, c. suis, c. andersoni, c. fayeri (previously marsupial genotype i), c. scrofarum (previously pig genotype ii), sheep genotype i, and unknown/novel genotypes have been identified (chalmers et al. 2005; giles et al. 2009; karanis et al. 2007; ryan et al. 2005 sweeny et al. 2011b; wang et al. 2010c) . species distribution differs between studies and between age groups within studies. for instance, c. parvum is commonly found in lambs in italy, romania, spain and the uk (díaz et al. 2010a; imre et al. 2013; mueller-doblies et al. 2008; paoletti et al. 2009 ). in other studies, c. ubiquitum or c. xiaoi is the most common species (fiuza et al. 2011a; geurden et al. 2008; robertson et al. 2010; sweeny et al. 2011b) . for example, wang et al. (2010c) identified c. ubiquitum in 90 % of all analysed samples, and the species dominated in all age groups, whereas sweeny et al. (2011b) found c. xiaoi to be the most common (table 4 .5). cryptosporidium bovis has also been reported in sheep (mueller-doblies et al. 2008; soltane et al. 2007; wang et al. 2010c ). whether c. bovis has actually been identified in sheep, or if it is the closely related species c. xiaoi is uncertain. for instance, soltane et al. (2007) reported isolates similar to c. bovis, and mueller-doblies et al. (2008) reported c. bovis, but a blast search of the genbank accession numbers identified these isolates as c. xiaoi. for the isolates reported as c. bovis by yang et al. (2009) , no genbank accession numbers are available, so the true identity of those isolates is uncertain. similarly, no genbank records are available from the study of ryan et al. (2005) reporting the "new bovine b genotype" in sheep. since c. bovis was first identified as the bovine b genotype, this could actually be the "c. bovis-like genotype", i.e. c. xiaoi. cryptosporidium andersoni has been identified in a few naturally infected adult sheep (wang et al. 2010c ), but experimental infection in 4-month-old lambs failed (kváč et al. 2004) . a couple of apparently related surveys from mexico have been published, but because of lack of clarity in the data, they will not be reviewed in this text. two studies from brazil (sevá et al. 2010) and mongolia (burenbaatar et al. 2008 ) failed to identify cryptosporidium in any of the collected samples, but the number of sampled sheep was small -11 and 5 animals, respectively. because of the small number of studies and isolates analysed, it is hard to draw any conclusion about the species distribution in goats. cryptosporidium parvum, c. xiaoi and a novel genotype have been identified in naturally infected goats (table 4 .5). in addition, one report of natural infection with c. hominis is also available (giles et al. 2009 ). the identification of c. xiaoi in a number of samples is in contrast with a failed attempt to infect 36-week-old goats to determine the host range of c. xiaoi (fayer and santín 2009 ). because there is only scant information about cryptosporidiosis in goats, we do not know if an age-related resistance or immunity from a previous cryptosporidium infection could have affected this experiment, as natural infections indicate that c. xiaoi is infectious to goats. two studies from mongolia (burenbaatar et al. 2008 ) and the united kingdom failed to identify cryptosporidium in any of the collected samples, but the number of sampled goats was small -16 and 15 animals, respectively. cryptosporidiosis has been associated with high morbidity and mortality rates in both lambs and goat kids (cacciò et al. 2013; chartier et al. 1995; de graaf et al. 1999; giadinis et al. 2007 giadinis et al. , 2012 johnson et al. 1999; munoz et al. 1996; paraud et al. 2011; vieira et al. 1997) . high mortality has been described both from natural infection and from experimental studies, where infection doses are generally high (chartier et al. 1995; giadinis et al. 2007; paraud et al. 2010) . in fact, it has been stated to be one of the most important pathogens associated with diarrhoeal disease and mortality in neonatal lambs and kids (quílez et al. 2008a) . anorexia and apathy/depression are common symptoms, accompanied by abdominal pain and pasty to watery, yellow and foul-smelling diarrhoea (de graaf et al. 1999; snodgrass et al. 1984) . diarrhoea can last from a few days up to 2 weeks (de graaf et al. 1999) . faecal consistency is correlated with oocyst excretion (de graaf et al. 1999; paraud et al. 2010 paraud et al. , 2011 , and a longer duration of diarrhoea is potentially associated with infection early in life (paraud et al. 2010) . body condition score and growth are affected (de graaf et al. 1999) , probably due to both anorexia and the intestinal damage, that can reduce nutrient uptake for weeks (de graaf et al. 1999; klein et al. 2008) . infection in animals older than 1 month is usually subclinical, and even younger animals can be subclinically infected. however, the infection can still affect production, with reduced body condition score (sweeny et al. 2011a (sweeny et al. , 2012 , reduced growth rate, and reduced carcass weight and dressing percentage at slaughter (sweeny et al. 2011a ). as discussed above for cattle, before molecular methods were developed c. parvum was the only species considered to infect and cause disease in sheep and goats (chartier et al. 1995; de graaf et al. 1999; munoz et al. 1996) . cryptosporidium parvum infection has since been associated with diarrhoea in studies using molecular methods (caccio et al. 2013; díaz et al. 2010a; drumo et al. 2012; imre et al. 2013; mueller-doblies et al. 2008) . however, c. xiaoi has also been associated with mild to severe diarrhoea and mortality (díaz et al. 2010b; navarro-i-martinez et al. 2007; rieux et al. 2013) , and c. ubiquitum too has been found in a few diarrhoeal samples from lambs (díaz et al. 2010a) , indicating that c. parvum is not the only pathogenic species in small ruminants. the prepatent period is 3-4 days in goat kids (paraud et al. 2010 ) and 2-7 days in lambs (de graaf et al. 1999 ). the patent period can last for at least 13 days (paraud et al. 2010 ). shedding peaks a few days to a week into the patent period, and maximum shedding can be as high as 2 â 10 7 opg (rieux et al. 2013) . the length of the patent period and shedding intensity are determined by age, immune status and infection dose (de graaf et al. 1999) . a natural age-related resistance to infection seems to be present. in one study, the prepatent period increased and intensity of shedding decreased in lambs with increasing age at infection (ortega-mora and wright 1994). in another study, one naturally infected group of goat kids started shedding at 17 days of age and excretion peaked at a mean of 23 â 10 4 opg 5-11 days later (rieux et al. 2013) , whereas another group of animals studied by the same authors started shedding at the age of 10 days, with a mean peak of 3 â 10 6 opg 0-4 days later, indicating higher virulence, infection pressure or an age-related higher sensitivity in the latter group. factors such as hygienic conditions, milking practices, herd size (population density), season, climatic zone (within a country), and lambing/kidding season are factors that have been associated with prevalence of infection, prevalence of clinical cryptosporidiosis and intensity of oocyst shedding (alonso-fresan et al. 2009; bomfim et al. 2005; craig et al. 2007; delafosse et al. 2006; giadinis et al. 2012; maurya et al. 2013; noordeen et al. 2000 noordeen et al. , 2001 . however, factors associated with infection and shedding intensity are also impacted by different management systems and climatic conditions; results from small farms in, for example, india cannot be extrapolated to large-scale farming in, for example, the united kingdom. the significance of sheep and goats as reservoirs for zoonotic cryptosporidiosis is unclear (robertson 2009 ). the first case of suspected zoonotic transmission from sheep was described in 1989 (casemore 1989 ), but at that time diagnosis was based solely on microscopy and thus zoonotic transmission cannot be confirmed. however, since the introduction of molecular tools in diagnostics, a number of cases and outbreaks with suspected or confirmed zoonotic transfer from sheep have been described (cacciò et al. 2013; gormley et al. 2011 ). in the uk, where sheep farming is an important industry, a seasonal pattern with spring and autumn peaks of human cryptosporidiosis cases is observed, with the spring peak concurring with the lambing season (anonymous 2002; gormley et al. 2011; mclauchlin et al. 2000; nichols et al. 2006) . lambs in petting zoos seem to be a common infection source (chalmers et al. 2005; elwin et al. 2001; gormley et al. 2011; pritchard et al. 2007 ). the incidence of human cryptosporidiosis, especially due to c. parvum, dropped significantly during the foot-and-mouth outbreak in the spring and summer 2001 (hunter et al. 2003) when >6 million livestock animals (~4.9 million sheep; anonymous 2001) were slaughtered and there were restrictions in animal movements and farm visits, providing further evidence of the importance of zoonotic transmission in this region. in 2012, an outbreak occurred in norwegian schoolchildren visiting a farm raising several animal species. cryptosporidium parvum oocysts of an identical and unusual gp60 subtype were identified in faecal samples from six human patients, two lambs and one goat kid. another human outbreak with the same c. parvum subtype had occurred at the same farm 3 years previously, but at that time very few oocysts were detected in animal faecal samples and molecular analyses were not conducted (lange et al. submitted) . cacciò et al. (2013) described a case where a farmer's son fell ill with cryptosporidiosis, being infected with the same and unusual c. parvum subtype that caused high morbidity and mortality in the farm's lambs (table 4.7) . studies on c. parvum gp60 subtypes have been performed with sheep and goat isolates, and all isolates were found to belong to the iia and iid families (table 4.7) . several studies using multi-locus genotyping (mlg) have found evidence of specific host associated c. parvum populations (drumo et al. 2012; mallon et al. 2003a, b; morrison et al. 2008 ). in the united kingdom, sheep mlgs clustered with human and bovine isolates (mallon et al. 2003a, b) , indicating frequent zoonotic transmission, whereas only one of the 34 mlgs identified in sheep/goats in italy was also identified in human samples (drumo et al. 2012) , indicating a low rate of zoonotic transmission. however, the latter study included very few human isolates. zoonotic transmission is commonly observed with c. parvum, but c. ubiquitum has also been identified in a number of sporadic human cases (cieloszyk et al. 2012; elwin et al. 2012; feltus et al. 2006; leoni et al. 2006; ong et al. 2002; soba et al. 2006; trotz-williams et al. 2006 ). oocysts of this species have been identified in storm water, wastewater, raw water and drinking water (jiang et al. 2005; liu et al. 2011; nichols et al. 2010; van dyke et al. 2012) . in scotland, c. ubiquitum was the third most common species in raw water and the most common species identified in drinking water (nichols et al. 2010) . thus, in areas where c. ubiquitum is common in sheep and goats, this species could be a more important cause of zoonotic infection than c. parvum. in addition, the relatively common presence of this species in water indicates a potential for waterborne outbreaks. natural infection with c. hominis has been reported in one goat and two sheep (giles et al. 2009; ryan et al. 2005 ) and in three lambs following experimental infection (ebeid et al. 2003; giles et al. 2001 ), but since animals are not natural hosts for this species, risk of zoonotic transmission with this species should be negligible compared with the risk of human-to-human transmission. it is important to note that because cryptosporidium infection can be subclinical, the zoonotic potential is not restricted to contact with diarrhoeic cryptosporidium-infected animals (pritchard et al. 2007 ). since domestication around 4900 bc in china, the pig has been an important food source (moeller and crespo 2009) . pigs are farmed worldwide, with the global swine inventory estimated at over 800 million in 2002. because asian countries are major consumers but do not produce sufficient pigs for their needs, there is a significant international trade in live and slaughtered pigs. china has the world's largest pig population, mostly small herds consisting of only a few animals, and is a net importer of pigs. the united states, european union, and canada are major exporters with relatively few but very large production units (moeller and crespo 2009) . the global trend is for fewer producers responsible for larger numbers of pigs, and more concentration within the swine industry. pigs are the primary host for c. suis ) formerly identified as cryptosporidium pig genotype i and for c. scrofarum (kváč et al. 2013 ) formerly identified as cryptosporidium pig genotype ii. farm pigs have also been found infected with c. parvum, c. muris, on one occasion with c. tyzzeri, a species common to mice, and with the novel genotype isolate eire 65.5 (kváč et al. 2013) . cryptosporidiosis occurs in pigs of all ages in 21 countries on 6 continents (table 4 .8). before molecular methods were developed c. parvum was thought to infect 152 species of mammals and to consist of several genotypes. consequently some early studies erroneously reported c. parvum infection in pigs based on the identification of oocysts in faeces by microscopy. subsequent use of molecular methods provided the necessary tools to identify and distinguish species. overall, prevalence data for locations, herds and age groups vary greatly and are not directly comparable because some data represent pooled samples (some from litters, others from fecal slurry), some data originate from single farms while other data come from multiple farms. some surveys have studied individual pigs at various ages, or only those pigs with diarrhoea, or simply specimens submitted to diagnostic laboratories from unspecified locations (table 4 .8). even in comparable populations, such as preweaned pigs in the same country or indifferent countries, data differences are too great to draw any conclusions on prevalence. for example: in australia-reports of 32.7 % versus 6.0 % prevalence (johnston et al. 2008 vs. ryan et al. 2003a ; in the czech republic-reports of 21.8 % versus 5.7 % prevalence (kváč et al. 2009a vs. vitovec et al. 2006 ; or between serbia and spain -reports of 32 versus 0 % prevalence (mišić et al. 2003 vs. quílez et al. 1996a ). some studies found significant association between the presence of a particular species and the pigs' age, with c. suis prominent in piglets and c. scrofarum prominent in weaners (enemark et al. 2003) . in contrast, others found no significant association between species and age or housing conditions (featherstone et al. 2010b ). these prevalence data reflect vast differences in management practices from location to location with too many unknown factors to draw valid conclusions on cause and effect or location within the 49 cited studies in table 4 .8 that reported a prevalence of infection between 0.1 % and 100 %. the only variable repeatedly associated with detection of cryptosporidium is age. most positive samples were from weaners and growers (table 4 .8). generally, prevalence increased until pigs were 10 weeks of age, then gradually declined. the first reports of cryptosporidiosis in pigs found one piglet among 81 herds of nursing piglets with necrotic enteritis, but the significance of this finding was described as unknown (bergeland 1977) and cryptosporidium was found at necropsy in three pigs without clinical signs (kennedy et al. 1977) . although a higher prevalence of diarrhoea was found in cryptosporidium-infected pigs than in uninfected pigs (hamnes et al. 2007) , others found no significant relationship between infection and diarrhoea (quílez et al. 1996b; guselle et al. 2003; maddox-hyttel et al. 2006; vitovec et al. 2006; suárez-luengas et al. 2007 ). cryptosporidium was detected histologically in the microvillus brush border of 5.3 % of 3,491 pigs from 133 farms examined for routine diagnostic evaluation (sanford 1987) . most infected pigs were 6-12 weeks old. organisms were detected in the jejunum, ileum, caecum, and colon, but primarily in microvilli of dome epithelium in the ileum. twenty six percent of cryptosporidium-infected pigs had diarrhoea but most of these also had other primary agents capable of causing diarrhoea. similar observations have been made by others. whereas most infections are asymptomatic or cause only mild, non-specific colitis (higgins 1999) , pigs known to be naturally infected with c. suis or c. scrofarum have not been found with clinical signs of infection while pigs infected with c. parvum or co-infected other enteropathogens such as rotavirus, salmonella, or isospora have had associated diarrhoea and some have died (enemark et al. 2003; núñez et al. 2003; hamnes et al. 2007) . experimental infections with different species have helped to clarify the relationship of species with clinical disease. pigs experimentally infected with c. suis (enemark et al. 2003) or c. scrofarum (kváč et al. 2013) showed no clinical signs. the pathogenicity of c. parvum isolated from calves was demonstrated in early transmission studies to pigs (moon and bemrick 1981; tzipori et al. 1981b tzipori et al. , 1982 argenzio et al. 1990; vitovec and koudela 1992; pereira et al. 2002) . experimental infection with the avian species, c. meleagridis, obtained from a human infection, consistently resulted in oocyst excretion and diarrhoea in pigs, although mucosal changes were milder than those described for c. parvum (akiyoshi et al. 2003) . piglets infected with c. suis had mild or no clinical signs despite excreting large numbers of oocysts, in contrast to those infected with c. parvum that had diarrhoea for a mean duration of 3.5 days and developed inappetence, depression and vomiting (enemark et al. 2003) . developmental stages of cryptosporidium have been observed throughout the intestinal tract. villous atrophy, villous fusion, crypt hyperplasia, and cellular infiltration of the lamina propria have been observed (kennedy et al. 1977; moon and bemrick 1981; tzipori et al. 1982 tzipori et al. , 1994 sanford 1987; vitovec and koudela 1992; argenzio et al. 1990; pereira et al. 2002; enemark et al. 2003; núñez et al. 2003; vitovec et al. 2006) . lesions caused by c. parvum were the most severe, as were clinical signs associated with that species. changes in the location of stages have been noted. in the first days of infection more stages were found in the proximal intestine, but later more stages were found in distal locations (tzipori et al. 1982; vitovec and koudela 1992) . extra-intestinal infections also have been reported in pigs. in two naturally infected piglets, the gall bladder was infected (fleta et al. 1995) . in experimentally immunosuppressed piglets, the gall bladder, bile ducts, and pancreatic ducts were found infected (healey et al. 1997) . infections in the trachea and conjunctiva were detected in experimentally infected normal piglets (heine et al. 1984 ). a survey of faecal slurry from swine finishing operations in ireland found c. suis, c. scrofarum and c. muris and concluded that cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff . hamnes et al. (2007) reported c. suis and c. scrofarum in faeces of suckling pigs in norway and reasoned that farrowing operations were sources of these parasites. additional data on oocyst concentrations, numbers of oocysts excreted, how long oocysts remain infectious under environmental condition, and modes of transmission of cryptosporidium species and genotypes are rare or non-existent. a year-long investigation was conducted at four types of swine operations (finishing, farrowing, nursery and gestation) in georgia, usa (jenkins et al. 2010) . mean oocyst concentrations ranged from 11 to 354 oocysts per ml of lagoon effluent; the nursery had the highest concentration of oocysts and the greatest percentage of viable oocysts (24.2 %), c. suis and c. scrofarum were the dominant species with some c. muris and c. parvum. experimental attempts to transmit c. scrofarum to adult scid mice, adult balb/c mice, mongolian gerbils, southern multimammate mice, yellow-necked mice, and guinea pigs were unsuccessful, suggesting that rodents are an unlikely source of transmission of this species under natural conditions (kváč et al. 2013 ). cryptosporidium suis was detected by immunofluorescence microscopy and rflp analysis of pcr products in stools from an hiv patient in peru (xiao et al. 2002a ). the patient was not severely immunosuppressed and was asymptomatic. he had a dog but reported no contact with other animals or animal faeces, including pigs and pig faeces, so the source and method of transmission are unknown. many countries, including australia, canada, china, korea, norway, russia, sweden, uk, usa and vietnam, have thriving deer farming industries. new zealand, a country where deer are not native, has the world's largest and most advanced deer farming industry. although it is difficult to find estimates on the numbers of deer farmed worldwide, more than one million deer were being farmed in new zealand in 2011 (sources: statistics new zealand and deer industry new zealand http://www.deernz.org/about-deer-industry/nz-deer-industry) -compared with five million dairy cows -and there are over 2,800 deer farmers. more than 90 % of the new zealand deer industry's products are exported, with approximately half of the export going to germany and benelux. species of deer which are commercially farmed varies regionally, but the following species are now being farmed in various parts of the world: red deer (cervus elaphus), wapiti or elk (cervus canadensis), fallow deer (dama dama), sika (cervus nippon), rusa deer (rusa timorensis), and reindeer (rangifer tarandus) (fao http://www.fao.org/docrep/004/x6529e/x6529e02.htm). although farmed deer are an important resource in many countries, much of the published information on cryptosporidium in deer refers to studies on wild or free-ranging cervids (e.g. white-tailed deer in usa, roe deer in spain, caribou in canada, and moose, red deer, roe deer and reindeer in norway; rickard et al. 1999; castro-hermida et al. 2011b; johnson et al 2010; hamnes et al. 2002) . while these studies on free-ranging cervids may give useful information regarding the species or genotype of cryptosporidium that might infect farmed deer (c. ubiquitum, c. parvum), as farmed deer probably differ quite substantially from their wild counterparts regarding exposures and stresses, extrapolation of prevalence data from wild to farmed deer may give an incorrect picture. indeed, a study in poland found that the prevalence of cryptosporidium was significantly higher in wild red deer than farmed red deer (27 % compared with 4.5 %), and mean oocyst concentration was also five times higher in faecal samples from wild red deer (paziewska et al 2007) . however as the sample size was relatively small (52 wild deer, 66 farmed deer) and from only single locations and as age and symptoms were not indicated, it is not possible to determine the reason for these differences. the few studies on the prevalence of cryptosporidium infection in different farmed or domesticated cervids are summarised in table 4 .9. the lack of surveys for cryptosporidium infection in farmed deer is surprising, given the clear association of infection with clinical disease in farmed cervids. some of the first published studies on cryptosporidium infection in cervids are case reports of severe (high mortality) outbreaks among farmed red deer calves. in one outbreak in scotland, uk among 82 artificially reared red deer calves, 56 developed cryptosporidiosis and 20 subsequently died; 80 % of the calves with diarrhoea and 50 % of apparently asymptomatic calves excreted oocysts and post mortem histopathological examination of the intestines demonstrated lesions similar to those seen in other species (tzipori et al. 1981a) . another outbreak among new-born red deer calves in the uk also resulted in high mortality, with calves dying at 24-72 h of age. however, this outbreak was not characterized by diarrhoea, and terminal uraemia was proposed as the symptom leading to death (simpson 1992) . outbreaks of cryptosporidiosis in red deer calves have also been reported from new zealand, again with relatively high mortality (10 out of 10 calves dying within a few days of illness onset in one outbreak, and 7 out of 10 calves dying within 3 days of illness in another outbreak) (orr et al. 1985) . severe subactute enteritis in the small and large intestine were reported in both outbreaks. information from other species of farmed deer is more scanty, but a retrospective study of neonatal mortality in farmed elk (pople et al 2001) identified cryptosporidium infection as one of the most important causes of enteritis leading to death (7 cases out of 11 of infectious enteritis from a total of 111 cases in which 46 had no specific cause of death identified). among these 7 cases, 4 were associated with an outbreak on a single farm (pople et al 2001) . unfortunately, information on the species of cryptosporidium infection associated with clinical disease in deer and cryptosporidium associated with asymptomatic infection is lacking. one outbreak on a scottish farm occurred when deer were put to graze on a pasture that had previously been grazed by a cryptosporidium-infected herd of cattle (angus 1988) , and therefore it seems probable that this might indicate infection with c. parvum; it might be speculated that infection of deer with deer-adapted c. ubiquitum is less likely to cause severe symptoms. published information about infection dynamics in farmed deer is minimal. however, a longitudinal study in asymptomatic farmed red deer in ireland (skerrett and holland 2001) provides some interesting data. asymptomatic low-level (<10 opg) oocyst shedding from adult hinds appeared to continue throughout the year, except during the calving season (may -june), when there was a 2-log increase in oocyst excretion rate; the highest level recorded was 67,590 opg. the authors speculate that this may be related to hormonal or immunological changes, or perhaps alterations in stress levels. the authors note that this preparturient rise in oocyst shedding results in contamination of the environment for the new born calves. however, in this study, although calves became infected, oocyst excretion was low (not exceeding 150 opg, and usually less), and clinical disease was not observed. again, the species of cryptosporidium in these infections is unknown. both c. parvum and c. ubiquitum have zoonotic potential, but there appear to be no documented cases of proven zoonotic transmission from/to deer. those studies (usa and australia) that have investigated deer as sources of contamination in watersheds, have focused on wild deer only and provided contrasting results (cinque et al 2008; jellison et al 2009 camelids are members of the family camelidae, and include the tribe camelini (including dromedaries and bactrian camels), and the tribe lamini (llamas, alpacas, vicuñas, and guanacos). there are two species of camel. approximately 14 million domesticated onehumped dromedaries (camelus dromedarius) are found in middle eastern countries including the sahel and horn of africa, as well as parts of southern asia where they provide people with milk, food, and transportation. nearly two million domesticated two-humped bactrians (camelus bactrianus) are native to the steppes of central asia the gobi and taklamakan deserts in mongolia and china. alpacas (vicugna pacos) and llamas (lama glama) exist only in the domesticated state and are found worldwide. however, both are native to south america and are raised primarily for fibre production although llamas were once used extensively as work animals. the young of both are called crias. camels and dromedaries: in the relatively few studies of dromedaries in northern africa and the middle east, the prevalence of cryptosporidiosis varied greatly (table 4 .10). none of 23 camels from iraq (mahdi and ali 1992) and none of 110 camels on farms in tunisia were found positive for cryptosporidium (soltane et al. 2007) . cryptosporidium was detected in one of four camel calves in egypt (abou-eisha 1994). however, in an abattoir in yazd province in iran, microscopic examination of 300 faecal specimens detected 61 (20.3 %) positive for cryptosporidium and 12 (12 %) positive abomasal mucosa specimens. at an abattoir in isfahan province in central iran, of 63 adult male and 40 adult female dromedary camels examined, 39 (37.9 %) were cryptosporidium-positive (razawi et al. 2009 ). in northwestern iran, of 170 faecal samples from camels 17 (10 %) were positive for cryptosporidium-like oocysts (yakhchali and moradi 2012) . the prevalence was significantly higher (20 %) in calves less than a year old. oocysts have also been recovered from wild and zoo-housed camels. faeces from a 3-year-old bactrian camel in the wild animals rescue centre of henan province in china were found positive for c. andersoni (wang et al. 2008b) . oocysts from a zoo-housed bactrian camel (fayer et al. 1991) were infectious for mice (anderson 1991) and were identified as c. muris (xiao et al. 1999; morgan et al. 2000) ; those from camels in the czech republic were identified as c. andersoni. other zoo-housed camels have been found to be infected with cryptosporidium (abou-eisha 1994; gomez et al. 2000; gracenea et al. 2002) . alpacas and llamas cryptosporidium oocysts have been detected in both these species. however, in california none of 354 llamas from 33 facilities were found positive (rulofson et al. 2001 ) nor were 61 alpacas on two farms in maryland . elsewhere in north america, europe, and australia small numbers of alpacas, llamas and guanacos have been examined and a few have been found positive for cryptosporidium (table 4 .11). most examinations were conducted by microscopy, but those that utilized molecular methods identified only c. parvum (morgan et al. 1998; starkey et al. 2007; o'brien et al. 2008; twomey et al. 2008) . the exception is a study in which a cria was found infected with c. ubiquitum (gomez-couso et al. 2012) . a national survey of 5,163 1-15-day-old alpacas in 105 andean herds in peru, the natural habitat for nearly 80 % of the world's alpacas, found 2 % of the youngest alpacas increasing to 20 % of the oldest alpacas, infected with cryptosporidium spp., with an overall prevalence of 13 % (lopez-urbina et al. 2009 ). more recently in peru, 4.4 % of 274 alpacas from 12 herds were found positive for cryptosporidium spp. (gomez-couso et al. 2012) . herd prevalence was 58.3 % (7/12 herds) for cryptosporidium. the highest prevalence (20 %) was found in the 8-week-old group (gomez-couso et al. 2012) . camels and dromedaries few data are available on the subject of clinical illness associated with cryptosporidiosis in camels. of 170 faecal samples, 17 camels (10 %) were positive for cryptosporidium-like organisms (yakchali and moradi 2012). the prevalence was significantly higher in camel calves (<1 years old) (20 %) than other age groups, in which the diarrhoeic calves had a prevalence of 16 %. alpacas and llamas not all alpacas and llamas infected with cryptosporidium show clinical signs of infection. of 110 healthy crias and their 110 dams 7 % and 8 %, respectively, were found excreting oocysts (burton et al. 2012) . oocysts of c. parvum were detected in 4 of 14 faecal samples from healthy crias and in one sample from a cria with diarrhoea (twomey et al. 2008) . cryptosporidium was observed in a post-operative neonatal llama with diarrhoea, cachexia, dehydration and electrolyte abnormalities (hovda et al. 1990 ). during 8 days that intravenous fluids and nutritional support were provided, these signs were not observed. of 20 cryptosporidium-infected alpaca crias with diarrhoea, 15 exhibited weight loss and 5 had a poor appetite (waitt et al. 2008 ). most were 8-18 days old when examined. additional potential gastrointestinal pathogens were found in 7 of these crias. sixteen crias recovered after supportive therapy that included intravenous rehydration, with partial parenteral administration of nutrients, antimicrobials, oral nutrients, plasma, insulin and other palliative treatments. additional reports of diarrhoea associated with cryptosporidiosis have been reported in alpaca and llama crias (cebra et al. 2003; shapiro et al. 2005; whitehead and anderson 2006; starkey et al. 2007 ). three fatal cases (2 with diarrhoea) of cryptosporidiosis were reported in alpaca crias less than 30 days of age (bidewell and cantell 1998) . at necropsy, intestinal congestion and distension were noted, oocysts were detected in ziehl-neelsen stained smears, and no other significant organisms or toxins were detected. in south america, llama and alpaca husbandry is a vital economic activity and neonatal diarrhoea syndrome (nds) is the most common and costly enteric disease in newborn llamas and alpacas (lopez-urbina et al. 2009 ). however, the role of cryptosporidiosis in nds has not been clearly identified. camels and dromedaries only rare circumstantial data of zoonoses are available and the link is very tenuous. in yazd province in iran, 24 of 100 people in long-term contact with camels were found infected with cryptosporidium spp. (sazmand et al. 2012) . infection was higher in winter than summer (16/50 compared with 8/50). alpacas and llamas in new york, cryptosporidium parvum infection was identified in 5 crias, 3 of their caretakers were confirmed to have cryptosporidiosis, and three others were suspected to have cryptosporidiosis, suggesting zoonotic transmission (starkey et al. 2007 ). rabbit farming (cuniculture) for meat, wool, and fur production occurs in a variety of settings around the world, and mostly involves the european (or common) rabbit (oryctolagus cuniculus). small-scale backyard cuniculture is common in many countries (especially in africa and south america), but commercial operations on a larger scale are found in europe (particularly italy, spain and france) and asia (particularly china and indonesia). in the eu, rabbit meat production was estimated to be around 520,000 tonnes carcass-weight equivalent in 2005 (efsa-ahaw 2005). in addition, rabbits continue to be bred for biomedical purposes -but this type of rabbit breeding will not be considered further in this chapter. production and consumption of rabbit meat is relatively low in north america. different rabbit breeds are used for meat, wool, and fur -with the most commonly used meat breeds being new zealand, californian, florida white and altex, all having good growth rates and desirable reproductive characteristics. much of the information presented in this section is derived from a comprehensive review article from 2010 (robinson and chalmers 2010) . the majority of published prevalence information on cryptosporidium in rabbits refers to studies on wild rabbits. nevertheless, there have been several studies on the occurrence of infection in farmed rabbits and also in laboratory rabbits. the majority of these studies (involving both wild and domestic rabbits) are summarised in robinson and chalmers (2010) . in table 4 .12, selected prevalence studies (rather than case reports) from farmed rabbits only are summarized, including two recent studies from china. additionally, a further three studies from china and referenced in zhang et al. (2012) are not included in table 4 .12 due to inaccessibility of the original publications. zhang et al. (2012) do not provide any details on these studies and it is not certain that they refer to farmed rabbits. although some surveys refer to cryptosporidium parvum, all those studies in which genotyping has been used (including from wild rabbits; e.g. nolan et al. 2010) suggest that the majority of natural infections in rabbits, if not all, are caused by c. cuniculus. nevertheless, experimental infections with other species of cryptosporidium have been established in rabbits, as summarised by robinson and chalmers (2010) . although the majority of surveys do not report symptoms associated with cryptosporidiosis in rabbits, experimental infections in preweaned rabbits have been associated with diarrhoea and high mortality (e.g. as reported by robinson and chalmers 2010; mosier et al 1997) and also as described by pavlásek et al. (1996) in farmed rabbits. however, even asymptomatic infection may result in some pathology, as noted by inman and takeuchi (1979) , who reported blunted villi, a decrease in villus-crypt ratio, and mild oedema in the lamina propria in an apparently asymptomatic adult rabbit. thus, even asymptomatic infection may reduce stock productivity. although no outbreaks of cryptosporidiosis in rabbit farms have been documented in the literature, acute outbreaks of diarrhoea with high mortality rates are frequently observed in rabbits (banerjee et al 1987) . although bacterial agents are frequently considered to be the aetiological agent, it seems probable that some may be due to undiagnosed cryptosporidiosis. for example, the parasitological techniques (direct microscopy and flotation) used for investigating epizootic outbreaks of diarrhoea, characterized by a high morbidity and mortality, in different commercial rabbit farms in mexico (rodríguez-de lara et al 2008) may have been insufficient for detecting cryptosporidium infection, particularly if the operators had little experience in diagnosing this infection. information on the dynamics of cryptosporidium infection in farmed rabbits is mostly lacking, although low oocyst excretion rates were reported in the majority of studies on rabbits in general (not just farmed rabbits). the studies from the czech republic provide some data, but, as the animals were not sampled individually, the data are difficult to interpret, and suggest that the source of infection for young rabbits may be low-level excretion of oocysts from mother rabbits at around parturition (pavlásek et al. 1996) . c. cuniculus is rarely, but sporadically, identified in human infections. in 3030 cryptosporidium-positive faecal samples submitted for routine typing in uk between 2007 and 2008, 37 (1.2 %) were identified as c. cuniculus, with both gp60 va and vb subtype families detected . however, the greatest evidence for c. cuniculus from rabbits having a significant zoonotic potential came from a waterborne outbreak of cryptosporidiosis in england in 2008 affecting 29 people; c. cuniculus, subtype vaa18 was identified in eight patients, a water sample from the implicated supply, and from the colon of a carcass of a rabbit (presumably wild) that was found in a tank at the water treatment works . nevertheless, transmission of cryptosporidium to humans from farmed rabbits has not been recorded, and an investigation exploring associations between farm animals and human patients with cryptosporidiosis did not implicate rabbits as a source of infection . the world stock of birds in production in 2011 was estimated to 22 â 10 9 animals (faostat 2013b). approximately 56 % of the world stock was found in asia, whereas europe, north america and south america had approximately 10-11 % each of the population. the largest group was chickens, with 90 % of the total stock. ducks, turkeys and geese/guinea fowls constituted 6.1 %, 2.1 % and 1.7 % respectively, and other birds (ratites, pigeons etc.) only constituted 0.1 % of the world stock. the main chicken, duck and goose/guinea fowl production is in asia (54 %, 90 % and 91 % within each group respectively), and most of the turkey production in north america (54 %), followed by europe (23 %). for other birds, 50 % of the reported production was located in asia, 41 % in africa and 9 % in europe. chickens (gallus gallus domesticus) are descendants of the red jungle fowl (gallus gallus), with some hybridization with the grey junglefowl (g. sonneratii). broilers are usually kept in intense systems and reach slaughter size at about 6 weeks of age. organically bred broilers and broilers kept on free range grow a bit more slowly. laying hens can produce over 300 eggs in their first production year, but after that production declines rapidly. domesticated ducks (anas platyrhynchos domesticus) are, except for the moscovy duck (cairina moschata), descendants of the mallard (anas platyrhynchos). the majority of domesticated geese (anser anser domesticus) descend from the greylag goose (anser anser), but the breeds chinese goose and african goose are derived from the swan goose (anser cygnoides). ducks and geese are bred for meat, eggs and down, and ducks, to a lesser degree, also for the production of foie gras. the domestic turkey (meleagris gallopavo) is a progeny of the wild turkey, which is found in the wild in the united states http://www.turkeyfed.com.au/turkey_info. php. turkeys are bred for meat production. the breed used is the white broad breasted turkey, introduced into commercial production in the 1950s http://bizfil.com/turkeyraising-primer. as with commercial chicken broiler farming, turkey farming is intense. the poults are extremely fast-growing, and reach approximately 6 kg at 10 weeks of age if given proper nutrition http://bizfil.com/turkey-raising-primer/. the united states has the highest consumption of turkey meat per person, and they are also the largest turkey producer, with 7.32 billion pounds of turkey meat produced in 2011 http://www.agmrc.org/commodities__products/livestock/poultry/turkey. among ratites, mainly ostriches (struthio camelus) are farmed, but rheas (rhea americana) and emus (dromaius novaehollandiae) are also kept for production. ratites are bred for meat, egg, and feather and leather production. farming for feather production began already in the nineteenth century. partridges, such as the grey or english partridge (perdix perdix) and red-legged partridge (alectoris rufa), are gallinaceous birds used as game, and have been introduced in different parts of the world for this purpose. another gallinaceous bird is the helmeted guinea fowl (numida meleagris). they are used for pest control, eating ticks and other insects, and can be kept as an alarm system among other domesticated birds due to their loud and shrieking warning call. the meat is considered a delicacy. the japanese quail (coturnix japonica) is bred for meat and eggs. domestic pigeons (columba livia domestica) are the progeny of the world's oldest domesticated bird, the rock pigeon. pigeons are bred for meat, sporting competitions, homing, as exhibition birds or pets. cryptosporidium infection has been associated with large morbidity and mortality in different bird species (bezuidenhout et al. 1993; hoerr et al. 1986; pages-mante et al. 2007; penrith et al. 1994; ritter et al. 1986; santos et al. 2005 ) and can thus be of great economic importance. avian cryptosporidiosis was first described in chickens (tyzzer 1929) . the infection was subclinical and situated in the caecum. invasive stages looked identical to those of c. parvum, but no oocyst description was made, and no name was proposed. today, three valid species have been identified in poultry. in addition, five genotypes have been identified in wild ducks and geese, and five additional genotypes have been described from other birds. the cryptosporidium oocysts identified by slavin in 1955 were morphologically similar to c. parvum, described in mice in 1912 (tyzzer 1912) , and the infection site was the distal ileum. slavin identified this bird cryptosporidium as a unique species, c. meleagridis. when molecular methods were introduced as a means of species determination, it was verified that c. parvum and c. meleagridis were indeed different species (sreter et al. 2000) . a species with a larger oocyst, first identified in chickens, and infecting the intestine, bursa and cloaca, was described and named c. baileyi ). this species is also involved in respiratory cryptosporidiosis, infecting the epithelium of sinuses, air sacs, nasopharynx, trachea and bronchi (itakura et al. 1984; lindsay et al. 1987) . infection of the conjunctiva (chvala et al. 2006 ) and urinary tract, including the kidneys has also been shown trampel et al. 2000) . a third species, c. galli, infecting the proventriculus of chickens, was described by pavlásek in 1999 , and re-described in 2003 (pavlásek 1999 ryan et al. 2003b) . the species was probably described in finches already in 1990 (blagburn et al. 1990 ) and later the name c. blagburni was proposed ). however, molecular analyses have shown that c. blagburni is the same species already described as c. galli, and thus the latter is considered to be the valid species name. in addition, isolates referred to as goose genotypes i-iv have been identified in canada geese and a duck genotype has been described in a black duck and canada geese. of the other genotypes described in birds (avian genotypes i-iv and the eurasian woodcock genotype), avian genotype ii has been detected in ostriches. two proposed species are today considered as nomen nudum due to lack of sufficient data. cryptosporidium tyzzeri in chickens was described in 1961 (levine 1961 ) and later c. anserinum, found in the large intestine of geese was described (proctor and kemp 1974) . based on 18s rdna phylogeny, c. galli and the woodcock genotype belong to the clade of gastric cryptosporidia together with c. andersoni, c. muris and c. serpentis, whereas c. meleagridis, c. baileyi, goose genotypes i and ii and the duck genotype belong to the intestinal clade . cryptosporidium meleagridis is closely related to the group including c. parvum and c. hominis; c. baileyi is closely related to the snake genotype, goose genotypes i, ii and the duck genotype cluster together and are closely related to c. scrofarum, c. bovis, c. ryanae and the deer genotype. goose genotypes iii-iv and avian genotypes i-iv were not included in the phylogenetic tree. in another publication, goose genotypes i (goose #1, 2, 3, 6 and 8), ii (goose #9) and the duck genotype (goose #5) are closely related, whereas goose genotypes iii (goose #3b) and iv (goose #7) are more distant (jellison et al. 2004) . the avian genotypes are more scattered. avian genotypes i and ii belong to the intestinal clade and are closely related to c. baileyi. genotypes iii and iv belong to the gastric clade, where genotype iii is closely related to the eurasian woodcock genotype and c. serpentis, and genotype iv is closely related to c. galli (ng et al. 2006) . prevalence data based on fecal examination could be affected by the time from sampling to analysis. this has been observed when oocyst numbers in chicken faeces dropped to approximately one third in samples stored for a week from first to second analysis, and where first analysis was performed on the day after sampling (c. axén, unpublished data). it is possible that oocysts die and are quickly degraded by detrimental effects (extreme ph) due to the high ammonium content of bird droppings. a flock prevalence of 41 % (23/56), with 10-60 % within-flock prevalence, was reported for c. baileyi respiratory infection in broilers in the usa (goodwin et al. 1996) . in morocco, cryptosporidium sp. were found in 14 (37 %) of 38 investigated flocks. within-flock prevalence ranged from 14 % to 100 %, and the highest prevalence (52 %) was identified in broilers aged 36-45 days, with no infection prior to 25 days of age (kichou et al. 1996) . diagnosis was based on histopathology. an overall cryptosporidium prevalence of 10.6 % for layer chickens and 3.4 % for broiler chickens was shown in a study of faecal samples from 2015 birds in china (wang et al. 2010a ). the highest prevalence (24.6 %) was found in 31-60-day old laying chickens, whereas prevalences in broiler chickens never exceeded 5 %. dna analysis identified c. baileyi as the major species, with 92/95 investigated samples, and only 3 samples were positive for c. meleagridis (wang et al. 2010a) . in contrast, another recent study identified c. meleagridis as the major species in chickens (baroudi et al. 2013) . the overall cryptosporidium prevalence was 34.4 % by histopathology, and the highest prevalence (46.2 %) was identified in 16-30-day-old chickens, which is in line with the results from kichou et al. (1996) and wang et al. (2010a) . the majority of the birds were infected with c. meleagridis only (n ¼ 25). cryptosporidium baileyi only was detected in four birds and a mixed c. meleagridis/c. baileyi infection was found in one bird. however, these chickens had died from diarrhoea, which could affect the outcome regarding cryptosporidium sp. a morbidity of 5-10 % due to sinusitis was reported for a flock where cryptosporidium sp. could be isolated from diseased poults (glisson et al. 1984) . it was stated that macroscopic post mortem examination of the infraorbital sinuses of healthy birds was normal compared with those of diseased birds, but it was not clearly stated whether cryptosporidium sp. was also identified in the healthy birds and thus the infection prevalence cannot be estimated. goodwin et al (1988b) identified invasive cryptosporidium stages in turkey poults form a farm where the poults suffered from self-limiting diarrheal of unknown aetiology, but no prevalence estimation was given. prevalences of 80 % in 17-day-old poults, 38 % in 24-day-old and 0 % in !60-day-old poults was found by woodmansee et al. (1988) . oocysts were identified as c. meleagridis based on morphology and infection site. a 35.5 % (17/60) prevalence in diarrhoeic or just unthrifty poults was reported in iran (gharagozlou et al. 2006) . prevalence was based on histological examination of intestinal, bursal and cloacal tissues. examination of faeces revealed that only 29 % of the infected birds shed oocysts. infection was identified in 1-7-week-old poults, whereas the 43 uninfected poults all were older than 7 weeks. dna analysis was not performed and oocyst size was not stated in the publication, but based on infection site, host species and symptoms the authors suggested that c. meleagridis was the species responsible. a 10.0 %, 10.5 % and 2.5 % pre-slaughter prevalence respectively (age 4-9 weeks) was detected upon faecal examination of three flocks from the same farm (mcevoy and giddings 2009). one of 59 turkeys was positive at post-slaughter examination (age 14 weeks). upon dna analysis, all six positive samples were identified as c. parvum. in a recent study, a 43.9 % prevalence of c. meleagridis was shown in deceased turkeys, with the highest prevalence (57.9 %) in poults aged 16-30 days (baroudi et al. 2013) . in one study, 73 (57 %) of 128 ducklings and 44 (59 %) of goslings aged 8-35 days were infected with cryptosporidium (richter et al. 1994) . infection was present in both intestinal and respiratory tract, but oocyst morphology was not described. in a study on experimental infection with usutu virus in geese, cryptosporidium developmental stages in tissue samples were an accidental finding. this was further investigated by in situ-hybridization, and cryptosporidium infection was detected in 89 % of conjunctival tissue samples and 88 % of bursal tissue samples. dna analysis revealed presence of c. baileyi (chvala et al. 2006) . c. baileyi was also identified in two ducks in rio de janeiro (huber et al. 2007 ). ratites cryptosporidium infection in ostriches was first described in the early 1990s (allwright and wessels 1993; bezuidenhout et al. 1993; gajadhar 1993 gajadhar , 1994 penrith et al. 1994; penrith and burger 1993) . infection was first identified in faecal samples from 14 (8.5 %) of 165 ostriches imported from africa to canada (gajadhar 1993) . penrith and burger (1993) identified invasive stages in a section of the small intestine of a 4-week old chick that has suffered from rectal prolapse, and allwright and wessels (1993) identified cryptosporidium in histology sections of the bursa, intestine and pancreatic ducts. in 1994, gajadhar et al. characterized the isolated oocysts and investigated host specificity. the oocysts were morphologically similar to those of c. meleagridis, but attempts to infect suckling mice, chickens, turkeys and quail failed, indicating that this was probably another species. in addition, only faecal samples were investigated, so the infection site was not determined (gajadhar 1994) . as this study was conducted before molecular tools were commonly used for cryptosporidium species determination, the true identity of this isolate will remain unknown. a low prevalence, with only 2 (0.6 %) of 336 investigated samples from ostriches aged 2 months-5 years being cryptosporidium positive, was found in greece (ponce gordo et al. 2002) . oocysts were of two sizes, 3.8 â 3.8 μm and 5.7 â 4.8 μm, indicating the presence of two different species. in contrast, in a spanish study a 60 % cryptosporidium prevalence in adult rheas and ostriches was found (ponce gordo et al. 2002) . the authors reported an oocyst diameter of 3-5 μm, which is similar to the description provided by gajadhar (1994) . molecular analysis of the isolates was not performed. oliveira et al. (2008) found 44 % prevalence in 77 ostriches based on microscopy. oocysts were generally morphologically similar to c. baileyi and cryptosporidium avian genotype ii . however, the morphometric variation was so large that the authors suggested that more than one species had been identified (oliveira et al. 2008) , but this was not verified by molecular analysis. an isolate similar to c. baileyi in both oocyst morphology and pcr-rflp banding pattern was described from brazilian ostriches (santos et al. 2005) . the isolate was characterized as a sister taxon to c. baileyi by sequence analysis of the 18s rdna, hsp70 and actin genes (meireles et al. 2006) , and was named cryptosporidium avian genotype ii by another research group (ng et al. 2006) . experimental infection (oral or intratracheal) with the brazilian isolate in chickens failed (meireles et al. 2006) . the avian genotype ii has also been identified in vietnam. on a single ostrich farm 110 (23.7 %) of 464 samples were positive for cryptosporidium oocysts. the highest prevalence as well as the highest shedding intensity (35.2 %) was found in 2-3 month-old animals. of 17 samples used for molecular characterization, all were found to be avian genotype ii (nguyen et al. 2013) . wang et al (2011b) reported cryptosporidium infection in 53 (11.7 %) of 452 investigated ostrich samples. prevalence peaked at the age of 4-8 weeks with 16.2 %. no infection was detected in birds younger than 1 week or older than 12 months. molecular analysis of positive samples identified only c. baileyi. quails and partridges enteric cryptosporidiosis in quails, with oocysts similar to c. meleagridis, was first described in 1986 (hoerr et al. 1986; ritter et al. 1986) . early attempts at experimental infection of quail with c. baileyi isolated from chickens failed lindsay et al. 1986 ), but were later successful (cardozo et al. 2005) . natural infection was first documented in 2001 ). since then, natural c. baileyi infection has been described in two reports (murakami et al. 2002; wang et al. 2012) . one large survey of cryptosporidium infection in quails was performed in china . out of 1,818 faecal samples, 239 (13.1 %) from 29 (61.7 %) farms were positive. infection was most common among 72-100-day old quails (23.6 %). dna analysis revealed c. baileyi in 237 samples and c. meleagridis in two samples. one case of cryptosporidium infection in partridges was described (pages-mante et al. 2007) . pigeons there are a few reports of cryptosporidiosis in pigeons (ozkul and aydin 1994; qi et al. 2011; radfar et al. 2012; rodriguez et al. 1997) . radfar et al. (2012) describe an overall prevalence of 2.9 % in 102 examined adult and nestling birds, with 3.4 % prevalence in adults and 2.3 % prevalence in nestlings. the other articles are case reports (ozkul and aydin 1994; rodriguez et al. 1997 ) and a study on pet birds in general, where c. meleagridis was found in one pigeon (qi et al. 2011 ). respiratory as well as intestinal and bursal cryptosporidium infections cause disease in chickens, but infection without clinical symptoms has also been observed (fletcher et al. 1975; taylor et al. 1994) . in spain, a 90 % morbidity due to respiratory infection in one flock was caused by cryptosporidium sp. weekly mortality rates were 0.9-1.5 % (fernandez et al. 1990 ). infection was detected in the trachea and oesophagus. in another flock investigated in the same study, weight loss was the primary symptom, and bursal cryptosporidiosis was diagnosed (fernandez et al. 1990 ). goodwin et al. (1996) found a correlation between c. baileyi infection of the trachea and severity of tracheitis symptoms, airsacculitis and condemnation of birds. in respiratory cryptosporidiosis, co-infection with other pathogens has been identified in a number of studies. cryptosporidium sp. and concurrent adenovirus infection was identified in a large broiler flock with respiratory disease (dhillon et al. 1981) . in a retrospective study on post mortem diagnoses of respiratory cryptosporidiosis, it was found that co-infection with virus or bacteria was common (goodwin et al. 1988a) . in another study, cryptosporidium sp. and aspergillus or bacteria were detected in the lungs of four layer chickens that died from pneumonia. cryptosporidium were also found in the ureters and kidneys (nakamura and abe 1988) . the effect of cryptosporidium infection alone on development of clinical symptoms in these cases cannot be estimated, but there is probably a synergistic effect of co-infections, increasing the severity. such a synergistic effect of co-infection with infectious bronchitis virus or escherichia coli has been reported (blagburn et al. 1991) . respiratory symptoms were reported from chickens that had been experimentally inoculated intra-tracheally with c. baileyi, whereas infection was successful but caused no symptoms in orally inoculated chickens (lindsay et al. 1988) . c. meleagridis infection was associated with diarrhoea and mortality in one study of algerian chickens (baroudi et al. 2013 ). experimental c. meleagridis infection of chickens has been observed to result in the chickens becoming indolent and having soiled feathers. growth retardation was reported, but compensatory growth occurred after a few weeks (tumova et al. 2002) . turkey was the first animal species in which clinical cryptosporidiosis was described (slavin 1955) . infection was associated with diarrhoea at 10-14 days of age, but other parasites (including histomonas, trichomonas and strongylides) were also detected. experimental infection (crop inoculation) with c. meleagridis produced infection of the ileum, caecum and bursa, but was not associated with clinical symptoms (bermudez et al. 1988 ). the isolate used was from symptomatic poults; however, these were simultaneously infected with reovirus (causing enteritis and hepatitis). co-infection with cryptosporidium and reovirus in turkeys with enteritis and hepatitis, leading to increased mortality, has also been shown in another study (wages and ficken 1989) . the presence of other pathogens in these studies could indicate a low to moderate primary pathogenicity of c. meleagridis. self-limiting diarrhoea (moderate to severe in character), a slower growth rate and growth deformities were reported from one farm where diseased poults were diagnosed with cryptosporidium infection (goodwin et al. 1988b ). other pathogens were not excluded, as was also mentioned by the authors. in dead poults that had suffered from depression and diarrhoea (faeces adhered on the hind part of the body), necropsy revealed lesions in the small intestine. microscopic investigation identified cryptosporidium sp. in the respiratory tract and kidneys, as well as in the gastrointestinal tract (tacconi et al. 2001) . diarrhoea, emaciation, lethargy and reduced growth associated with natural c. meleagridis infection have been reported from iran, but the presence of other pathogens was not excluded (gharagozlou et al. 2006) . of 60 diarrhoeal and/or unthrifty birds, 17 (35.3 %) were identified as cryptosporidium positive by histology, and c. meleagridis was reported based on oocyst morphology. baroudi et al. (2013) identified c. meleagridis in 25 (44 %) of 57 examined turkeys that died from diarrhoea, but infection with other pathogens was not investigated. respiratory cryptosporidiosis has also been described in turkeys (ranck and hoerr 1987; tarwid et al. 1985) . tarwid et al. (1985) identified cryptosporidium sp. in necropsied birds from two outbreaks of colibacillosis. colibacillosis is, according to the authors, a secondary disease in turkeys, and cryptosporidium sp. was identified as the primary pathogen. symptoms were frothy conjunctivitis and increased mortality. necropsy revealed pathological changes such as pericarditis, peritonitis and air-sacculitis in addition to the conjunctivitis that was observed in live birds. thirteen birds with respiratory disease were all positive for cryptosporidium sp. by histology (ranck and hoerr 1987) . microscopy of sinus and/or tracheal exudates revealed oval oocysts in some samples, but oocyst size was not described, and both c. baileyi and c. meleagridis can appear oval (length/width ratios of 1.05-1.79 and 1.00-1.33 respectively ). symptoms such as coughing, rattling, sneezing, frothy eyes and swollen sinuses were reported. other pathogens were present in all but two of the examined birds, and it is unclear whether the infection with cryptosporidium played a primary role in the pathogenesis or not. studies on cryptosporidiosis in turkeys, including clinical symptoms, are summarised in table 4 .13. clinical cryptosporidiosis in ducks and geese seems to be less common and milder (see table 4 .14) than in other poultry. only mild respiratory symptoms resulted from experimental c. baileyi infection (both oral and intratracheal inoculation) in ducks (lindsay et al. 1989 ). respiratory and intestinal infection occurred for both infection routes, but symptoms (sneezing, rales, mild dyspnea) were only present in animals infected by the intratracheal route. mason (1986) described a case of conjunctival cryptosporidiosis. however, since only one of 97 affected ducks was cryptosporidium positive, the author concluded that the parasite was not the cause of the disease. similarly, no symptoms occurred in geese in which cryptosporidium infection was detected in the conjunctivas and bursas (chvala et al. 2006); and richter et al. (1994) noted that enteritis and upper respiratory tract symptoms were equally present in infected and non-infected ducks and geese. mortality was not increased in the positive flocks (richter et al. 1994) . ratites cryptosporidium infection in ostrich chicks has been associated with cloacal and phallus prolapse, leading to high mortality (bezuidenhout et al. 1993; penrith et al. 1994; santos et al. 2005) . bezuidenhout et al (1993) found that prolapsed cloacas were heavily infected, whereas penrith et al. (1994) described heavy infection of both the bursa and cloaca in affected chicks, but healthy chicks were not infected. santos et al (2005) also identified cryptosporidium infection in the rectum, coprodeum, urodeum and bursa of two dead chicks with cloacal prolapse, both originating from a farm with high mortality rates in 7-30-day-old chicks. however, the authors did not associate the problems with the infection, since changed management practices decreased clinical symptoms and mortality, although cryptosporidium infection was still present on the farm. enteritis was indicated by the presence of intestinal invasive stages and rectal prolapse in one chick examined by penrith and burger (1993) . because diarrhoea was not reported it is unknown whether the prolapse was caused by intense bowel movements or something else. cryptosporidium infection has also been associated with pancreatic necrosis (allwright and wessels 1993) . quails and partridges cryptosporidium infection has been shown in both diarrhoea and respiratory disease in quails (guy et al. 1987; hoerr et al. 1986; murakami et al. 2002; ritter et al. 1986 ). hoerr et al. (1986) reported high mortality rates from 5 days of age in quails infected with cryptosporidium sp., and with no bacterial or viral pathogens detected. acute fatal diarrhoea with mortality rates of up to 45 % in 0-17-day-old birds was described by ritter et al (1986) . reovirus was also detected in necropsied birds, but another study reported that experimental infection with reovirus alone did not produce diarrhoea, whereas infection with cryptosporidium sp., either alone or simultaneously with reovirus, resulted in severe diarrhoea and mortality (guy et al. 1987) . a synergistic effect of co-infection was, however, shown, since oocyst shedding was higher and reovirus infection became systemic and liver necrosis occurred (guy et al. 1987) . muramaki et al. (2002) reported a daily mortality rate of 5.7 % in one farm, where birds suffered from upper respiratory tract disease and decreased egg production. respiratory symptoms were head swelling, nasal discharge and increased lacrimation, and necropsy revealed sinusitis, airsacculitis and egg peritonitis. co-infection of cryptosporidium sp., mycoplasma gallisepticum and other bacteria was shown. the authors concluded that m. gallisepticum was the primary pathogen, but that the mixed infections in conjunction with high ammonia concentrations in the air worsened the symptoms. the role of cryptosporidium infection in respiratory disease in quails thus remains unclear. wang et al. (2012) reported that no clinical symptoms were seen in 1,818 sampled quails, of which 239 were cryptosporidium positive. c. meleagridis was the only pathogen identified in an outbreak of diarrhoea and cough in red-legged partridge chicks (pages-mante et al. 2007) . morbidity rates were 60-70 % and mortality more than 50 %, indicating high pathogenicity. invasive stages were identified in both the respiratory and intestinal tract, suggesting that not only c. bailey might be associated with respiratory avian cryptosporidiosis. pigeons diarrhoea associated with cryptosporidiosis in pigeons has been described in four birds (ozkul and aydin 1994; rodriguez et al. 1997) . rodriguez et al. (1997) described a 40 % morbidity of yellow watery diarrhoea, weight loss, dehydration and weakness in a farm with 280 pigeons. mortality was 5 % and necropsy of three birds revealed invasive stages of cryptosporidium in the small intestine, caecum, colon, cloaca, and bursa. no viruses or bacteria could be isolated. ozkul and aydin (1994) identified invasive stages in the small intestine of a pigeon that had been depressed and had evidence of diarrhoea in the form of faeces in its hind feathers. isolates of both c. baileyi and c. meleagridis derived from one domestic bird species have been successfully transmitted to other domestic birds lindsay et al. 1987) . c. galli has not been experimentally transmitted between different domestic birds, but has been shown in finches as well as chickens (blagburn et al. 1990; pavlásek 1999 pavlásek , 2001 ryan et al. 2003b) , and thus has the potential to infect different bird species. the prepatent period of c. baileyi is approximately 4-8 days (hornok et al. 1998; lindsay et al. 1988; rhee et al. 1991; tumova et al. 2002) . however, in the first report on c. baileyi infection in chickens, a prepatent period of up to 24 days was described ). older chicks have a slightly longer prepatent periods than younger ones (lindsay et al. 1988; rhee et al. 1991; taylor et al. 1994; tumova et al. 2002) . the patent period varies more. at oral inoculation of 2-day-old chicks, a patent period of 26 days was seen, whereas it was 11-15 days in chicks inoculated at 14 days of age, 11-12 days in chicks inoculated at 28 days of age and <7 days in chicks inoculated at 42 days of age (lindsay et al. 1988 ). with intratracheal inoculation, the same authors described patent periods of 27, 11-19, 10-11 and <7 days in these age groups (lindsay et al. 1988 ). rhee et al. (1991) and tumova et al. (2002) observed a mean patent period of approximately 14 days. oocyst excretion peaked on day 12 and days 11-17 post inoculation, respectively (rhee et al. 1991; tumova et al. 2002) . taylor et al. (1994) showed shorter patent periods and lower total oocyst output in older than younger chickens. there was also an effect of infection dose, in that oocyst output was higher and declined more slowly with lower infection doses (taylor et al. 1994) . similar observations were made for 1-and 9-week old chicks (sreter et al. 1995) . in that study, the mean patent period for 1-week old chicks was 32 days, but one chicken shed oocysts for 151 days. c. meleagridis was shed in the faeces on days 4-7 post infection in two chickens experimentally infected at 6 weeks of age (woodmansee et al. 1988 ). tumova et al. (2002) infected 7-day-old chicks. oocysts first appeared 3 days later and the patent period lasted for 16-17 days. shedding rates were significantly lower than in chicks inoculated with the same number of c. baileyi oocysts. the prepatent and patent period of c. galli has been described to be 25 and 6 days respectively (pavlásek 2001) , but was later reported as unknown when c. galli was redescribed (ryan et al. 2003b ). the prepatent period of c. meleagridis in turkeys inoculated at 7-11 days of age was 2-4 days (bermudez et al. 1988; sreter et al. 2000; woodmansee et al. 1988) . woodmansee et al. (1988) reported that oocysts were shed for only 4 days; sreter et al. (2000) found the patent period to be 8-10 days, whereas bermudez et al. (1988) reported oocyst shedding and invasive stages still being present at day 21 post inoculation. oocyst shedding rates were moderate (sreter et al. 2000) , and low to moderate (bermudez et al. 1988) . experimental infection with c. baileyi induced mild infection of the bursa ). lindsay et al. (1987) inoculated turkey poults via the intratracheal, oral and intracloacal route. all three experiments caused infection, but only poults inoculated via the trachea developed symptoms (lindsay et al. 1987 ). lindsay et al (1986) described a prepatent period of 5 days and a possible patent period of 9-10 days in experimentally infected muscovy ducks, based on investigation of pooled faecal samples (lindsay et al. 1986 ). oocyst morphology was not described. the intestine, bursa and cloaca were positive for invasive stages, but these tissues can be infected by both c. baileyi and c. meleagridis (table 4 .14). for quails, one study describes a prepatent period of 7 days and a patent period of 21 days for c. baileyi (cardozo et al. 2005) . otherwise, no data are available. only one of the species and genotypes commonly infecting birds -cryptosporidium meleagridis -has, so far, proved to be important in human cryptosporidiosis. this species is the third most common species in human cryptosporidiosis worldwide. in the industrialised world, c. meleagridis infection is usually associated with cryptosporidiosis cases in travellers to asia or africa (elwin et al. 2012; insulander et al. 2013; leoni et al. 2006 ), but autochthonous cases have also been described (elwin et al. 2012; leoni et al. 2006; silverlås et al. 2012) . studies on cryptosporidium prevalence and species distribution in humans in south america have identified c. meleagridis infection at about the same prevalence as c. parvum (cama et al. 2003 (cama et al. , 2008 . although this is a true zoonotic species, there is only one report in which the bird source has been identified, and in that case, chickens and not turkeys were involved (silverlås et al. 2012) . it is not known whether anthroponotic transmission occurs with this species, but it has been indicated by the fact that not all c. meleagridis-infected patients in an epidemiological investigation had had bird or animal contact (elwin et al. 2012) . c. meleagridis has the potential to infect other mammalian species as well, and experimental infection of mice, rats, rabbits, pigs and calves has been reported (akiyoshi et al. 2003; darabus and olariu 2003) . due to the wide host range and the close relationship of c. meleagridis to c. parvum and c. hominis, it has been proposed that this species originated as a mammalian cryptosporidium species, and later adapted to birds (xiao et al. 2002b . one study has identified c. parvum in turkeys (mcevoy and giddings 2009), indicating that this species could play a role in zoonotic transmission. however, only one of 59 birds post-slaughter was positive compared to 2.5-10.5 % of the 5-10-week younger poults, which means risk of transmission via contaminated meat should be very small. the higher prevalence in poults should not pose a risk as long as the flocks are closed to the public. the shedding intensity was not reported, but prevalence indicates that infection rather than just intestinal passage was present. some studies have identified c. parvum, c. hominis and c. hominis-like isolates in canada geese (jellison et al. 2004 (jellison et al. , 2009 zhou et al. 2004 ). the authors conclude that these findings are probably not associated with infection and parasite proliferation, but rather transient carriage. nevertheless, this indicates that domesticated ducks and geese can potentially act as transmission vehicles for these species. infection with c. baileyi has been identified in one immunodeficient patient. diagnosis was based on oocyst morphology and biology -experimental infection of mice failed whereas inoculated chickens developed infection of the intestine, bursa and trachea (ditrich et al. 1991) . since this patient was immunodeficient and no other reports exist, this species should not be considered as a true zoonotic agent. ever since animals were first domesticated, and humans became dependent upon them for the commodities that they supply, particularly food and fibre, the infections that affect the health and productivity of livestock have been a concern. cryptosporidiosis was first identified as a disease of veterinary significance in the 1950s (in turkeys) and then in the early 1970s in calves, but major interest in cryptosporidiosis only developed with the first report of a human cases later that decade, and the recognition that cryptosporidium infection was also of medical importance. since then our knowledge on the veterinary significance of cryptosporidium infection has expanded enormously -particularly in the livestock sector most impacted by cryptosporidiosis -young calves. however, as demonstrated in this chapter, it should not be forgotten almost all farmed animals may be pathologically affected by at least one species of cryptosporidium, often causing clinical disease that in some instances may be fatal. for some cryptosporidium species in some farmed animal species, transmission may be anthropozoonotic. 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cryptosporidiosis screening study in calves in tanzania prophylaxis using paromomycin of natural cryptosporidial infection in neonatal kids prevalence and molecular characterization of cryptosporidium spp. in dairy cattle from farms in china prevalence of cryptosporidium spp. in pigs in shanghai china cryptosporidium infection in domestic geese (anser anser f. domestica) detected by in-situ hybridization two cases of zoonotic cryptosporidiosis in spain by the unusual species cryptosporidium ubiquitum and cryptosporidium felis investigating public health impacts of deer in a protected drinking water supply watershed epidemiology of parasitic protozoan infections in soay sheep the life cycle of cryptosporidium baileyi n. sp. 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(apicomplexa: cryptosporidiidae) in sheep (ovis aries) chronic cryptosporidiosis in a bactrian camel (camelus bactrianus) cryptosporidium parvum infection in bovine neonates: dynamic clinical parasitic and immunologic patterns cryptosporidium canis n. sp. from domestic dogs cryptosporidium bovis n.sp. (apicomplexa: cryptosporidiidae) in cattle (bos taurus) prevalence of species and genotypes of cryptosporidium found in 1-2-year-old dairy cattle in the eastern united states prevalence of cryptosporidium species and genotypes in mature dairy cattle on farms in eastern united states compared with younger cattle from the same locations cryptosporidium ryanae n. sp. (apicomplexa: cryptosporidiidae) in cattle (bos taurus) species of cryptosporidium detected in weaned cattle on cow-calf operations in the united states cryptosporidium species in calves submitted for postmortem examination in england and wales cryptosporidium species infection in pigs in east anglia evidence supporting zoonotic transmission of cryptosporidium spp. in wisconsin wide geographic distribution of cryptosporidium bovis and the deer-like genotype in bovines common occurrence of a unique cryptosporidium ryanae variant in zebu cattle and water buffaloes in the buffer zone of the chitwan national park cryptosporidiosis in chickens from southern spain molecular characterization of cryptosporidium in brazilian sheep cryptosporidium pig genotype ii diagnosed in pigs from the state of rio de janeiro, brazil detection of cryptosporidium oocysts in extra-intestinal tissues of sheep and pigs cryptosporidiosis of the bursa of fabricius of chickens outbreak of cryptosporidiosis among veterinary students cryptosporidium species in imported ostriches and consideration of possible implications for birds in canada host specificity studies and oocyst description of a cryptosporidium sp. isolated from ostriches prevalence and genotyping of cryptosporidium in three cattle husbandry systems in zambia prevalence and molecular characterisation of cryptosporidium and giardia in lambs and goat kids in belgium intestinal cryptosporidiosis in turkeys in iran effect of halofuginone lactate on treatment and prevention of lamb cryptosporidiosis: an extensive field trial comparison of two techniques for diagnosis of cryptosporidiosis in diarrhoeic goat kids and lambs in cyprus experimental infection of a lamb with cryptosporidium parvum genotype 1 cryptosporidium hominis in a goat and a sheep in the uk sinusitis in turkeys associated with respiratory cryptosporidiosis further report on cryptosporidium in barcelona zoo mammals characterisation of a cryptosporidium isolate from water buffalo (bubalus bubalis) by sequencing of a fragment of the cryptosporidium oocyst wall protein gene (cowp) presence and molecular characterisation of giardia and cryptosporidium in alpacas (vicugna pacos) from peru respiratory cryptosporidiosis in chickens diarrhea associated with intestinal cryptosporidiosis in turkeys respiratory coccidiosis (cryptosporidium baileyi) among northern georgia broilers in one company zoonotic cryptosporidiosis from petting farms transmission dynamics of cryptosporidium in primates and herbivores at the barcelona zoo: a long-term study giardia sp. cysts and infectious cryptosporidium parvum oocysts in the feces of migratory canada geese (branta canadensis) retrospective cohort study of an outbreak of cryptosporidiosis caused by a rare cryptosporidium parvum subgenotype biology of cryptosporidium parvum in pigs: from weaning to market experimental reproduction of enteritis in bobwhite quail (colinus virginianus) with cryptosporidium and reovirus prevalence of cryptosporidium and giardia in free-ranging wild cervids in norway occurrence of cryptosporidium and giardia in suckling piglets in norway outbreak of cryptosporidium linked to drinking unpasteurised milk bovine fallopian tube epithelial cells, adult c57bl/6 mice, and non-neonatal pigs as models for cryptosporidiosis experimental tracheal and conjunctival infections with cryptosporidium sp. in pigs prevalence of giardia and cryptosporidium and characterization of cryptosporidium spp. isolated from wildlife, human and agricultural sources in the north saskatchewan river basin in alberta molecular epidemiology of cryptosporidium in livestock animals and humans in the ismailia province of egypt surveillance for cryptosporidiosis fatal cryptosporidiosis in quail assessment of maternal immunity to cryptosporidium baileyi in chickens total parenteral nutrition in a neonatal llama genotypic characterization and phylogenetic analysis of cryptosporidium sp. from domestic animals in brazil foot and mouth disease and cryptosporidiosis: possible interaction between two emerging infectious diseases sporadic cryptosporidiosis case-control study with genotyping antibody fusions reduce onset of experimental cryptosporidium parvum infection in calves molecular characterisation of cryptosporidium isolates from pre-weaned calves in romania: is there an actual risk of zoonotic infections? zoonotic cryptosporidium parvum in romanian newborn lambs (ovis aries) spontaneous cryptosporidiosis in an adult female rabbit developing vaccines to control protozoan parasites in ruminants: dead or alive? molecular epidemiology and clinical manifestations of human cryptosporidiosis in sweden cryptosporidial infection in chickens prevalence of cryptosporidium parvum infections in weaned piglets and fattening porkers in kanagawa prefecture phylogenetic analysis of the hypervariable region of the 18s rrna gene of cryptosporidium oocysts in feces of canada geese (branta canadensis): evidence for five novel genotypes source tracking identifies deer and geese as vectors of human-infectious cryptosporidium genotypes in an urban/suburban watershed concentrations, viability and distribution of cryptosporidium genotypes in lagoons of swine facilities in the southern piedmont and in coastal plain watersheds of georgia distribution of cryptosporidium genotypes in storm event water samples from three watersheds in new york atypical outbreak of caprine cryptosporidiosis in the sultanate of oman prevalence of cryptosporidium genotypes in pre and post-weaned pigs in australia serum biochemistry, serology and parasitology of boreal caribou (rangifer tarandus caribou) in the northwest territories prevalence of cryptosporidium oocysts in livestock in trinidad and tobago molecular characterization of cryptosporidium from animal sources in qinghai province of china first description of cryptosporidium bovis in japan and diagnosis and genotyping of cryptosporidium spp. in diarrheic pre-weaned calves in hokkaido cryptosporidiosis in three pigs prevalence and molecular characterization of bovine cryptosporidium in qazvin province molecular characterization and assessment of zoonotic transmission of cryptosporidium from dairy cattle in west bengal recurrent outbreaks of cryptosporidiosis associated with calves among students at an educational farm programme natural cryptosporidium sp. infection in broiler chickens in morocco effect of cryptosporidium parvum infection on the absorptive capacity and paracellular permeability of the small intestine in neonatal calves preliminary communication on cryptosporidiosis of pigs in viet-nam isolation of cryptosporidium andersoni kawatabi type in a slaughterhouse in the northern island of japan occurrence of cryptosporidium and giardia in wild ducks along the rio grande river valley in southern new mexico failed attempt of cryptosporidium andersoni infection in lambs prevalence and age-related infection of cryptosporidium suis, c. muris and cryptosporidium pig genotype ii in pigs on a farm complex in the czech republic molecular characterization of cryptosporidium isolates from pigs at slaughterhouses in south bohemia molecular characterization of cryptosporidium spp. in pre-weaned dairy calves in the czech republic: absence of c. ryanae and management-associated distribution of c. andersoni, c. bovis and c. parvum subtypes cryptosporidium scrofarum n. sp. (apicomplexa: cryptosporidiidae) in domestic pigs (sus scrofa) prevalence and risk factors associated with intestinal parasites in pigs in chongqing submitted) second outbreak of infection with a rare cryptosporidium parvum genotype among schoolchildren associated with contact with lambs/goat kids at a holiday farm in norway langkjaer rb, vigre h, enemark hl, maddox-hyttel c (2007) molecular and phylogenetic characterization of cryptosporidium and giardia from pigs and cattle in denmark genetic analysis of cryptosporidium from 2414 humans with diarrhoea in england between 1985 and host specificity of cryptosporidium sp. isolated from chickens experimentally induced infections in turkeys with cryptosporidium baileyi isolated from chickens effect of broiler chicken age on susceptibility to experimentally induced cryptosporidium baileyi infection experimental infections in domestic ducks with cryptosporidium baileyi isolated from chickens cryptosporidium andersoni n. sp. (apicomplexa: cryptosporiidae) from cattle, bos taurus molecular identification and distribution of cryptosporidium and giardia duodenalis in raw urban wastewater in harbin, china prevalence of neonatal cryptosporidiosis in andean alpacas (vicugna pacos) in peru a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply cryptosporidium and giardia in different age groups of danish cattle and pigs -occurrence and management associated risk factors cryptosporidiosis among animal handlers and their livestock in basrah molecular characterization of cryptosporidium spp. in native breeds of cattle in kaduna state population structures and the role of genetic exchange in the zoonotic pathogen cryptosporidium parvum multilocus genotyping of cryptosporidium parvum type 2: population genetics and sub-structuring conjunctival cryptosporidiosis in a duck intestinal cryptosporidiosis in a kid goat prevalence and risk factors associated with cryptosporidium spp. infection in young domestic livestock in india cryptosporidium in commercially produced turkeys on-farm and postslaughter molecular epidemiological analysis of cryptosporidium spp. in the united kingdom: results of genotyping cryptosporidium spp. in 1,705 fecal samples from humans and 105 fecal samples from livestock animals biological studies and molecular characterization of a cryptosporidium isolate from ostriches (struthio camelus) molecular characterization of cryptosporidium spp. in dairy calves from the state of são paulo, brazil an outbreak of cryptosporidosis from fresh-pressed apple cider subtype analysis of cryptosporidium parvum isolates from calves on farms around belgrade, serbia and montenegro using the 60 kda glycoprotein gene sequences cryptosporidium infection in nursing, weaning and post-weaned piglets and sows in the belgrade district in: lal r (ed) agricultural sciences -volume 1. encyclopedia of life support systems prevalence of cryptosporidiosis in buffaloes in and around bhubaneswar fecal transmission of calf cryptosporidia between calves and pigs molecular characterization of cryptosporidium from various hosts molecular and biological characterisation of cryptosporidium in pigs molecular and phylogenetic analysis of cryptosporidium muris from various hosts molecular and phylogenetic characterisation of cryptosporidium from birds the population structure of the cryptosporidium parvum population in scotland: a complex picture experimental cryptosporidiosis in adult and neonatal rabbits distribution of cryptosporidium species in sheep in the uk prevalence of land management factors contributing to cryptosporidium sp. infection in pre-weaned and post-weaned calves in role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in spain occurrence of conjunctivitis, sinusitis and upper region tracheitis in japanese quail (coturnix coturnix japonica), possibly caused by mycoplasma gallisepticum accompanied by cryptosporidium sp. infection respiratory (especially pulmonary) and urinary infections of cryptosporidium in layer chickens prevalence of cryptosporidium parvum infection in lahore (pakistan) and its association with diarrhea in dairy calves detection and molecular characterization of cryptosporidium bovis-like isolate from a newborn lamb in spain subtype analysis of cryptosporidium parvum and cryptosporidium hominis isolates from humans and cattle in iran identification of novel cryptosporidium genotypes from avian hosts evidence of cryptosporidium transmission between cattle and humans in northern prevalence and molecular characterization of cryptosporidium in ostriches (struthio camelus) on a farm in central vietnam cryptosporidiosis: a report on the surveillance and epidemiology of cryptosporidium infection in england and wales identification of cryptosporidium species and genotypes in scottish raw and drinking waters during a one-year monitoring period molecular detection of cryptosporidium cuniculus in rabbits in australia prevalence of cryptosporidium infection in goats in selected locations in three agroclimatic zones of sri lanka excretion of cryptosporidium oocysts by goats in relation to age and season in the dry zone of sri lanka coinfection by cryptosporidium parvum and porcine circovirus type 2 in weaned pigs cryptosporidium gp60 genotypes from humans and domesticated animals in australia, north america and europe cryptosporidium infection in birds and mammals and attempted cross transmission studies giardiasis and cryptosporidiosis in ruminants duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea occurrence of cryptosporidium spp. oocysts (apicomplexa, cryptosporidiidae) in ostriches, struthio camelus l., 1758 (aves, struthionidae) reared in north and lowered coastline regions of the state of rio de janeiro giardia and cryptosporidium in canadian farm animals update on cryptosporidium and giardia infections in cattle prevalence and molecular characterization of cryptosporidium spp. in dairy cattle in south bohemia, the czech republic novel cryptosporidium genotypes in sporadic cryptosporidiosis cases: first report of human infections with a cervine genotype cryptosporidiosis in deer calves age-related resistance in ovine cryptosporidiosis: patterns of infection and humoral immune response small-intestinal cryptosporidiosis in a young pigeon an outbreak of disease associated with cryptosporidia on a red-legged partridge (alectoris rufa) game farm cryptosporidial infection in a calf immunoenzymatic analysis and genetic detection of cryptosporidium parvum in lambs from italy evaluation of oral tilmicosin efficacy against severe cryptosporidiosis in neonatal kids under field conditions control of cryptosporidiosis in neonatal goat kids: efficacy of a product containing activated charcoal and wood vinegar liquid (obionekk(r)) in field conditions cryptosporidia: biology diagnosis, host spectrum specificity and the environment findings of cryptosporidia in the stomach of chickens and of exotic and wild birds spontaneous cryptosporidium infection in weaned rabbits (article in czech) distribution of cryptosporidium and giardia spp. in selected species of protected and game mammals from north-eastern poland genetic diversity of cryptosporidium spp. in cattle in michigan: implications for understanding the transmission dynamics a cryptosporidium sp in an ostrich evidence for cryptosporidial infection as a cause of prolapse of the phallus and cloaca in ostrich chicks pathogenesis of human and bovine cryptosporidium parvum in gnotobiotic pigs genotype and subtype analyses of cryptosporidium isolates from cattle in hungary outbreak of cryptosporidiosis among veterinary students parasites from farmed ostriches (struthio camelus) and rheas (rhea americana) in europe a retrospective study of neonatal mortality in farmed elk cryptosporidium parvum infection in orphan lambs on a farm open to the public cryptosporidium anserinum sp. n. (sporozoa) in a domestic goose anser anser l, from iowa cryptosporidium spp. in pet birds: genetic diversity and potential public health significance comparison of oocyst shedding and the serum immune response to cryptosporidium parvum in cattle and pigs prevalence of cryptosporidium infections in pigs in aragón, northeastern spain cryptosporidium genotypes and subtypes in lambs and goat kids in spain cryptosporidium species and subtype analysis from dairy calves in spain biodiversity and prevalence of parasites of domestic pigeons (columba livia domestica) in a selected semiarid zone of south khorasan diseases of the newborn. in: veterinary medicine, 10th edn prevalence and infection pattern of naturally acquired giardiasis and cryptosporidiosis in range beef calves and their dams cryptosporidia in the respiratory tract of turkeys prevalence of cryptosporidium infection in camels (camelus dromedarius) in a slaughterhouse in iran isolation and identification of cryptosporidium from various animals in korea natural infections by cryptosporidium sp. in farm-raised ducks and geese the prevalence of cryptosporidium and giardia spp. in fecal samples from free-ranging, white-tailed deer (odocoileus virginianus) in the southeastern united states molecular characterization of cryptosporidium spp. in pre-weaned kids in a dairy goat farm in western france cryptosporidium and giardia in water buffaloes (bubalus bubalis) of the italian mediterranean bred giardia and cryptosporidium in water buffaloes (bubalus bubalis) intestinal cryptosporidiosis and reovirus isolation from bobwhite quail (colinus virginianus) with enteritis giardia and cryptosporidium infections in sheep and goats: a review of the potential for transmission to humans via environmental contamination foodborne cryptosporidiosis: is there really more in nordic countries? case-control studies of sporadic cryptosporidiosis in melbourne and adelaide a small outbreak of human cryptosporidiosis associated with calves at a dairy farm in norway the zoonotic potential of giardia and cryptosporidium in norwegian sheep: a longitudinal investigation of 6 flocks of lambs the european rabbit (oryctolagus cuniculus), a source of zoonotic cryptosporidiosis intestinal cryptosporidiosis in pigeons (columba livia) studies on the evolution pathology, and immunity of commercial fattening rabbits affected with epizootic outbreaks of diarrhoeas in mexico: a case report risk factors for sporadic cryptosporidiosis among immunocompetent persons in the united states from 1999 to fecal shedding of giardia duodenalis, cryptosporidium parvum, salmonella organisms and escherichia coli o157:h7 from llamas in california identification of a novel cryptosporidium genotype in pigs a redescription of cryptosporidium galli pavlasek, 1999 (apicomplexa: cryptosporidiidae) from birds identification of novel cryptosporidium genotypes from the czech republic cryptosporidium suis n. sp. (apicomplexa: cryptosporidiidae) in pigs (sus scrofa) sheep may not be an important zoonotic reservoir for cryptosporidium and giardia parasites cryptosporidium fayeri n. sp. (apicomplexa: cryptosporidiidae) from the red kangaroo (macropus rufus) porcine neonatal coccidiosis: clinical, pathological, epidemiological and diagnostic features enteric cryptosporidial infection in pigs: 184 cases (1981-1985) livestock prevalence and age-related variation of cryptosporidium species and genotypes in dairy calves a longitudinal study of cryptosporidiosis in dairy cattle from birth, to 2 years of age cryptosporidium infection in ostriches (struthio camelus) in brazil: clinical, morphological and molecular studies prevalence of cryptosporidium infection in goats maintained under semi-extensive feeding conditions in the southeast of spain the prevalence of cryptosporidium species in diarrhoeic lambs in kars province and potential risk factors prevalence of cryptosporidium spp. in camels and involved people in yazd province occurrence and molecular characterization of cryptosporidium spp. isolated from domestic animals in a rural area surrounding atlantic dry forest fragments in teodoro sampaio municipality, state of são paulo highlights of camelid diagnoses from necropsy submissions to the animal health laboratory, university of guelph from the identification of the cryptosporidium ubiquitum in pre-weaned ovines from aba tibetan and qiang autonomous prefecture in china studies on zoonotic cryptosporidiosisparvum in ismailia governorate cryptosporidium spp. in calves and cows from organic and conventional dairy herds systematic review and meta-analyses of the effects of halofuginone against calf cryptosporidiosis prevalence and associated management factors of cryptosporidium shedding in 50 swedish dairy herds cryptosporidium infection in herds with and without calf diarrhoeal problems molecular characterisation of cryptosporidium isolates from swedish dairy cattle in relation to age, diarrhoea and region zoonotic transmission of cryptosporidium meleagridis on an organic swedish farm is there a need for improved cryptosporidium diagnostics in swedish calves? cryptosporidiosis in newborn red deer (cervus elaphus) asymptomatic shedding of cryptosporidium oocysts by red deer cryptosporidium meleagridis (sp. nov.) an outbreak of waterborne cryptosporidiosis caused by posttreatment contamination outbreaks of enteric infections caused by multiple pathogens associated with calves at a farm day camp natural cryptosporidium hominis infections in scottish cattle investigation of farms linked to human patients with cryptosporidiosis in england and wales experimental cryptosporidiosis in germfree lambs genetic classification of cryptosporidium isolates from humans and calves in slovenia molecular characterisation of cryptosporidium isolates from humans in slovenia prevalence of cryptosporidium spp. (eucoccidiorida: cryptosporiidae) in seven species of farm animals in tunisia pcr-rflp analysis of the cryptosporidium oocyst wall protein (cowp) gene discriminates between c. wrairi and c. parvum, and between c. parvum isolates of human and animal origin age-dependent resistance to cryptosporidium baileyi infection in chickens morphologic host specificity and molecular characterization of a hungarian cryptosporidium meleagridis isolate risk factors associated with cryptosporidium infection on dairy farms in a new york state watershed an outbreak of cryptosporidiosis among alpaca crias and their human caregivers survey of zoonoses recorded in scotland between long-term study of cryptosporidium prevalence on a lowland farm in the united kingdom molecular characterization of cryptosporidium isolates from pigs in zaragoza, northeastern spain differentiating human from animal isolates of cryptosporidium parvum cryptosporidium and giardia associated with reduced lamb carcase productivity longitudinal investigation of protozoan parasites in meat lamb farms in southern western australia impacts of naturally acquired protozoa and strongylid nematode infections on growth and faecal attributes in lambs evaluation of factors associated with the risk of infection with cryptosporidium parvum in dairy calves cryptosporidium in market pigs in southern california usa retrospective ultramicroscopic investigation on naturally cryptosporidial-infected commercial turkey poults cryptosporidiosis in the respiratory tract of turkeys in saskatchewan variations in oocyst output associated with cryptosporidium baileyi infections in chickens genotypes and subtypes of cryptosporidium spp. in neonatal calves in northern ireland urinary tract cryptosporidiosis in commercial laying hens genotype and subtype analyses of cryptosporidium isolates from dairy calves and humans in ontario association between management practices and within-herd prevalence of cryptosporidium parvum shedding on dairy farms in southern ontario detection of assemblage a giardia duodenalis and eimeria spp performance and oocyst shedding in broiler chickens orally infected with cryptosporidium baileyi and cryptosporidium meleagridis cryptosporidiosis in two alpaca (lama pacos) holdings in the south-west of england cryptosporidium parvum (sp. nov.), a coccidium found in the small intestine of the common mouse coccidiosis in gallinaceous birds diarrhea in young red deer associated with infection with cryptosporidium experimental infection of piglets with cryptosporidium enterocolitis in piglets caused by cryptosporidium purified from calf faeces experimental cryptosporidiosis in calves: clinical manifestations and pathological findings prevalence of cryptosporidium parvum infection and pattern of oocyst shedding in calves in japan identifying host sources, human health risk and indicators of cryptosporidium and giardia in a canadian watershed influenced by urban and rural activities outbreak of cryptosporidiosis in dairy goats in brazil cryptosporidium parvum in cattle, sheep and pigs in galicia (n.w. spain) incidence an risk factors of cryptosporidium sp. infection in water buffaloes confined in a communal management system in the philippines pathogenesis of intestinal cryptosporidiosis in conventional and gnotobiotic piglets prevalence and pathogenicity of cryptosporidium suis in pre-and post-weaned pigs prevalence of giardia sp. cryptosporidium parvum and cryptosporidium muris (c. andersoni) in 109 dairy herds, in five counties of southeastern new york cryptosporidiosis and turkey viral hepatitis in turkeys cryptosporidiosis in 20 alpaca crias molecular epidemiology spatiotemporal analysis and ecology of sporadic human cryptosporidiosis in australia molecular characterization of the cryptosporidium cervine genotype from a sika deer (cervus nippon temminck) in zhengzhou, china and literature review multilocus phylogenetic analysis of cryptosporidium andersoni (apicomplexa) isolated from a bactrian camel (camelus bactrianus) in china large-scale survey of cryptosporidium spp. in chickens and pekin ducks (anas platyrhynchos) in henan, china: prevalence and molecular characterization prevalence and molecular identification of cryptosporidium spp cervine genotype is the major cryptosporidium genotype in sheep in china prevalence of cryptosporidium baileyi in ostriches characteristics of cryptosporidium transmission in preweaned dairy cattle in henan cryptosporidium spp. in quails (coturnix coturnix japonica) in henan, china: molecular characterization and public health significance neonatal diarrhea in llamas and alpacas prevalence of enteropathogens in suckling and weaned piglets with diarrhoea in southern germany subclinical cryptosporidiosis of turkeys in iowa molecular epidemiology of cryptosporidiosis: an update prevalence of cryptosporidium and giardia infections on two ohio pig farms with different management systems phylogenetic analysis of cryptosporidium parasites based on the small-subunit r-rna gene locus identification of the cryptosporidium pig genotype in a human patient host adaptation and host-parasite co-evolution in cryptosporidium: implications for taxonomy and public health cryptosporidium taxonomy: recent advances and implications for public health prevalence and identity of cryptosporidium spp. in pig slurry prevalence of cryptosporidium-like infection in one-humped camels (camelus dromedarius) of northwestern iran prevalence and molecular characterisation of cryptosporidium and giardia species in pre-weaned sheep in australia prevalence of the cryptosporidium pig genotype ii in pigs from the yangtze river delta infection status of pigs with cryptosporidium parvum cryptosporidium cuniculus and giardia duodenalis in rabbits: genetic diversity and possible zoonotic transmission host-adapted cryptosporidium spp. in canada geese (branta canadensis) prevalence of cryptosporidium species in intensively farmed pigs in ireland key: cord-022103-4zk8i6qb authors: siegel, jane d.; guzman-cottrill, judith a. title: pediatric healthcare epidemiology date: 2017-07-18 journal: principles and practice of pediatric infectious diseases doi: 10.1016/b978-0-323-40181-4.00002-5 sha: doc_id: 22103 cord_uid: 4zk8i6qb nan jane d. siegel the reduction of healthcare-associated infections (hais) is an important component of patient safety programs. five of the 16 hospital national patient safety goals for 2016 of the joint commission (formerly the joint commission on accreditation of healthcare organizations) target prevention of hais. 1 hospitals have learned from high-reliability organizations (e.g., the aviation industry) the importance of adopting changes that include the leadership's commitment to achieving zero patient harm, a fully functional culture of safety throughout the organization, and the widespread deployment of highly effective process improvement tools. 2 involvement of new stakeholders for improving patient safety and outcomes related to hais (e.g., children's hospitals' solutions for patient safety, children's hospital association, individual states' mandatory hai public reporting programs, the centers for medicare and medicaid services, the joint commission) has broadened the arena for hai prevention efforts. knowledge of the complexities of prevention and control of hais in children is critical to many different leaders of children's healthcare facilities. one framework for patient safety in children's hospitals that includes infection prevention and control (ipc) was developed by the ohio children's hospital solutions collaborative and demonstrates the effectiveness of hospitalwide collaboration. 3 as more disciplines in healthcare become engaged in prevention of hais as well as antimicrobial stewardship, it is the responsibility of the healthcare epidemiologist and the ipc staff (infection preventionists, healthcare epidemiologists) to educate the facility leadership on the discipline of ipc. ipc for the pediatric population is a unique discipline that requires understanding of various host factors, sources of infection, routes of transmission, behaviors required for care of infants and children, pathogens and their virulence factors, treatments, preventive therapies, and behavioral theory. although the term nosocomial still applies to infections that are acquired in acute care hospitals, the more general term, healthcare-associated infections (hais), is preferred because much care of high-risk patients, including patients with medical devices (e.g., central venous catheters, ventilators, ventricular shunts, peritoneal dialysis catheters), has shifted to ambulatory settings, rehabilitation or chronic care facilities, and the home; thus, the geographic location of acquisition of the infection often cannot be determined. the principles of transmission of infectious agents in healthcare settings and recommendations for prevention are reviewed in the healthcare and infection control practices advisory committee (hicpac) guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings, 2007 4 and in the management of multidrug resistant organisms in healthcare settings, 2006 document. 5 as new pathogens emerge, epidemiologists will continue to learn more about preventing transmission; therefore, for such pathogens, the most up-to-date guidance posted on the centers for disease control and prevention (cdc) or the world health organization (who) website should be consulted. the experience treating ebola virus disease (evd) in the united states in 2014 is the most recent example of changes in the usual infection prevention paradigm that were required, with emphasis on the hierarchy of controls 6 and donning and doffing of personal protective equipment (ppe) with trained observers. 7 a detailed discussion of hais can be found in chapters 99 and 100. this chapter focuses on the components of an effective pediatric hospital epidemiology program. unique aspects of hais in children are summarized in the following sections. specific risks and pathogens are addressed in several other chapters in this textbook. intensive care units (icus), oncology services, and gastroenterology services caring for patients with short gut syndrome who are dependent on total parenteral nutrition (and lipids) have the highest rates of bacterial and fungal infection associated with central venous catheters. a newer definition of mucosal barrier injury laboratory-confirmed bloodstream infection (mbi-lcbi) currently is used by the national healthcare safety network (nhsn) of the cdc to distinguish bacteremia that represents translocation of gut microorganisms related to mucosal barrier injury in patients with oncologic conditions, hematopoietic stem cell transplantation (hsct), and intestinal failure from bacteremia associated with central venous catheters. 8 hais can result in substantial morbidity and mortality, as well as lifetime physical, neurologic, and developmental disabilities. host (i.e., intrinsic) factors that make children particularly vulnerable to infection include immaturity of the immune system, congenital abnormalities, and congenital or acquired immunodeficiencies. children with congenital anomalies have a high risk of hai if their unusual anatomic features predispose them to contamination of normally sterile sites. moreover, these children require prolonged and repeated hospitalizations, undergo many complex surgical procedures, and have extended exposure to invasive supportive and monitoring equipment. innate deficiencies of the immune system in prematurely born infants, who may be hospitalized for prolonged periods and exposed to intensive monitoring, supportive therapies, and invasive procedures, contribute to the relatively high rates of infection in the neonatal icu (nicu). all components of the immune system are compromised in neonates, and the degree of deficiency is proportional inversely to gestational age (see chapter 9) . the underdeveloped skin of the very low birth weight (<1000 g) infant provides another mode of pathogen entry. populations of immunosuppressed children have expanded with the advent of more intense immunosuppressive therapeutic regimens used for oncologic conditions, hsct, solid-organ transplantation, and rheumatologic conditions and inflammatory bowel disease for which immunosuppressive agents and tumor necrosis factor-α-inhibiting agents (infliximab [remicade] ) and other immune modulators are used. genetic mutations in the genes for the transmembrane conductance regulator (cftr) in children with cystic fibrosis result in thick secretions, chronic endobronchial infections, and gastrointestinal malabsorption. knowledge of the epidemiology of infection of patients with cystic fibrosis and effective methods to prevent patient-to-patient transmission have expanded with the use of newer molecular diagnostic methods, resulting in a 2013 update in the infection prevention and control guideline for cystic fibrosis. 9 fortunately, the population of children with perinatally acquired human immunodeficiency virus (hiv) infection and acquired immunodeficiency syndrome (aids) has decreased dramatically since 1994, but new cases of sexually transmitted hiv infection continue to be diagnosed in teens who receive care in children's hospitals. finally, young infants who have not yet been immunized, or immunosuppressed children who do not respond to vaccines or who lose antibody during disease or treatment (e.g., patients with nephrotic syndrome), have increased susceptibility to vaccine-preventable diseases. the source of many hais is the endogenous flora of the patient. an asymptomatically colonizing pathogen can invade a patient's bloodstream or be transmitted to other patients on the hands of healthcare personnel (hcp) or on shared equipment. other important sources of hais in infants and children include the mother in the case of neonates, toys were implicated in an outbreak of multidrug-resistant pseudomonas aeruginosa in a pediatric oncology unit. 23 although the source of most candida hais is the patient's endogenous flora, horizontal transmission, most likely through hcp hands, has been demonstrated in studies using typing by pulsed gel electrophoresis in the nicu and in a pediatric oncology unit. 24, 25 newer molecular diagnostic methods (e.g., whole genome sequencing) are more sensitive and specific than pulsed gel electrophoresis and have proven to be valuable in identifying outbreaks of a variety of pathogens in both pediatric and adult settings. 26, 27 droplet. infectious respiratory droplets >5 µm in diameter are generated from the respiratory tract by coughing, sneezing, or talking or during such procedures as suctioning, intubation, chest physiotherapy, or pulmonary function testing. transmission of infectious agents by the droplet route requires exposure of mucous membranes to large respiratory droplets within 3 to 6 feet (1 to 2 m) of the infected person. large respiratory droplets do not remain suspended in the air for prolonged periods, and they settle on environmental surfaces. the dynamics of infectious aerosols can be affected by a variety of factors including characteristics of specific strains of bacteria, temperature, humidity, and number of air exchanges in a room. adenovirus, influenza virus, and rhinovirus are transmitted primarily by the droplet route, whereas rsv is transmitted primarily by the contact route. 28 although influenza virus can be transmitted by the airborne route under unusual conditions of reduced air circulation or low absolute humidity, ample evidence indicates that transmission of influenza is prevented by droplet precautions and, in the care of infants, the addition of contact precautions. 29 airborne. droplet nuclei that arise from desiccation of respiratory droplets and are <5 µm in diameter and contain infectious agents remain suspended in the air for prolonged periods and travel long distances on air currents. 4 susceptible persons who have not had face-to-face contact or been in the same room as the source person can inhale such infectious particles. m. tuberculosis, varicella-zoster virus (vzv), and rubeola virus are the agents most frequently transmitted by the airborne route. although transmission of m. tuberculosis by the airborne route can occur rarely from an infant or young child with active tuberculosis, the more frequent source is the adult visitor with active pulmonary tuberculosis that has not yet been diagnosed; thus screening of visiting family members is an important component for control of tuberculosis in pediatric healthcare facilities. 30 some agents (e.g., severe acute respiratory syndrome-coronavirus [sars-cov]) can be transmitted as small-particle aerosols under special circumstances of aerosol-generating procedures (e.g., endotracheal intubation, bronchoscopy); therefore, an n95 or higher respirator is indicated for persons in the same airspace when these procedures are performed, but an airborne infection isolation room (aiir) may not always be required. roy and milton 31 proposed the following classification for aerosol transmission when evaluating routes of sars-cov transmission: 1. obligate: under natural conditions, disease occurs following transmission of the agent only through small-particle aerosols (e.g., tuberculosis). 2. preferential: natural infection results from transmission through multiple routes, but small-particle aerosols are the predominant route (e.g., measles, varicella). 3. opportunistic: agents naturally cause disease through other routes, but under certain environmental conditions they can be transmitted by fine-particle aerosols. this conceptual framework can explain rare occurrences of airborne transmission of agents that are transmitted most frequently by other routes (e.g., smallpox, sars, influenza, noroviruses). concern about airborne transmission of influenza arose during the 2009 influenza a (h1n1) pandemic. however, the conclusion from all published experiences during the pandemic was that droplet transmission is the usual route of transmission, and surgical masks were noninferior to n95 respirators in preventing laboratory-confirmed influenza in hcp. 32, 33 concerns about unknown or possible routes of transmission of agents that can cause severe disease and have no known treatment often result in more extreme prevention strategies. therefore, recommended precautions could change as the epidemiology of emerging agents is defined and these controversial issues are resolved. although no evidence supports airborne transmission of the ebola virus under usual circumstances in the field, the aerosolization of body fluids that contain high titers of ebola virus requires additional protection. 34 invasive monitoring and supportive equipment, blood products, total parenteral nutrition fluids, lipids, infant formula and human milk, hcp, and other contacts, including adult and sibling visitors. maternal infection with neisseria gonorrhoeae, treponema pallidum, hiv, hepatitis b virus, parvovirus b19, mycobacterium tuberculosis, herpes simplex virus, or group b streptococcus, or colonization with multidrug-resistant organisms (mdros), pose substantial threats to the neonate. during perinatal care, procedures such as fetal monitoring using scalp electrodes, fetal transfusion and surgical procedures, umbilical cannulation, and circumcision are potential risk factors for infection. intrinsically contaminated powdered formulas and infant formulas prepared in contaminated blenders or improperly stored or handled have resulted in sporadic and epidemic infections in the nursery (e.g., cronobacter [formerly enterobacter] sakazakii), but such infections have become less frequent since the pathogenesis was defined and contamination reduced. 10 human milk that has been contaminated by maternal flora or by organisms transmitted through breast pumps has caused isolated serious infections and epidemics. the risks of neonatal hepatitis, cytomegalovirus (cmv) infection, and hiv infection from human milk warrant further caution for handling and use of banked breast milk. with increasing numbers of procedures being performed by pediatric interventional radiologists, 11 an understanding of appropriate aseptic technique, as well as prevention and management of infectious complications, by interventional radiologists is important. 12 construction, renovation, demolition, and excavation in and near healthcare facilities are important sources of environmental fungi, (e.g., aspergillus spp., agents of mucormycoses, fusarium spp., scedosporium spp., bipolaris spp.). 13 immunocompromised patients and patients in the pediatric icu (picu) and nicu are at greatest risk for fungal infection, and case fatality rates can be ≥50%, especially if diagnosis and treatment are delayed. practices related to care of infants and young children. several practices must be evaluated with respect to the potentially associated risk of infection. a significant association between reduced levels of nurse staffing and appropriately trained nurses has been demonstrated to increase risk of infection in many studies in both children and adults. 4, 14, 15 theoretical concerns exist that infection risk also will increase in association with the innovative practices of co-bedding of twins and kangaroo care in the nicu because of increased opportunity for skin-to-skin exposure of multiple-gestation infants to each other and to their mothers, respectively. neither the benefits nor the safety of co-bedding multiple-birth infants in the hospital setting has been demonstrated. 16 overall, the infection risk is reduced with kangaroo care, but transmission of tuberculosis and respiratory syncytial virus (rsv) has occurred in kangaroo mother care units in south africa. 17 parents providing kangaroo care should be monitored for the presence of skin infections. antimicrobial selective pressure. exposure to vancomycin and to thirdgeneration cephalosporins contributes substantially to the increase in infections caused by vancomycin-resistant enterococcus (vre) 18 and multidrug-resistant gram-negative bacilli, including extended spectrum β-lactamase (esbl)-producing organisms 19 and carbapenem-resistant enterobacteriaceae 20 (cre) in children. additionally, exposure to thirdgeneration cephalosporins also is a risk factor for the development of invasive candidiasis in low birth weight infants in the nicu. 21 studies of the human microbiome using culture-independent methods have demonstrated the bacterial community diversity on mucosal surfaces and the profound suppressive effect of antimicrobial agents on the population of protective bacteria, firmicutes, thus increasing the risk of colonization and subsequent invasive disease caused by pathogenic bacteria. 22 the principal modes of transmission of infectious agents are direct and indirect contact, droplet, and airborne. 4 contact. most infectious agents are transmitted by the contact route on the hands of hcp or through shared items; many pathogens can be transmitted by more than 1 route. viruses, bacteria, and candida spp. can be transmitted horizontally. toddlers often share waiting rooms, playrooms, toys, books, and other items and therefore have the potential of transmitting pathogens directly and indirectly to one another. contaminated bath part i understanding, controlling, and preventing infectious diseases (nnis), now nhsn icus. hais caused by mdros are associated with increased length of stay, increased morbidity and mortality, and increased cost, in part because of the delay in initiating effective antimicrobial therapy. 43, 44 although the prevalence of specific mdros is lower in pediatric institutions, the same principles of target identification and interventions to control mdros apply in all settings. c. difficile is an important pathogen in children, as it is in adults, especially in children receiving chemotherapy. testing for c. difficile in the first year of life is not advised because of the high asymptomatic colonization rate with toxigenic strains in this age group. candida spp. are the third most frequent pathogens associated with bloodstream infections in us nicus. there is considerable center-tocenter variability in both the incidence of invasive candidiasis and the proportion of candida infections caused by candida non-albicans spp., most of which are resistant to fluconazole. risk factors for candida infections include prolonged length of stay in an icu, use of central venous catheters, intralipids, histamine (h 2 )-blocking agents, and exposure to third-generation cephalosporins. gnb and candida spp. are especially important pathogens for hais in patients with intestinal failure who are receiving total parenteral nutrition, and these organisms can cause repeated episodes of sepsis. the incidence of candida infections had increased in incidence in most picus and nicus during the 1990s, but the rate of c. albicans and non-albicans central line-associated bloodstream infections decreased by 75% in all birth weight categories from 1999 to 2009, 45 likely a result of improved infection control practices, antimicrobial stewardship, and use of fluconazole prophylaxis in the very low birth weight preterm infants. the most recently published clinical practice guidelines of the infectious diseases society of america (idsa) recommend the use of oral or intravenous fluconazole prophylaxis in infants weighing <1000 g at birth in nicus with high rates (>10%) of invasive candidiasis, based on high quality of evidence to support efficacy and safety. 46 additionally, empiric antifungal therapy in preterm infants of ≤1000 g birth weight is associated with improved survival rates without adverse outcomes. 47 the staff members of each nicu first must optimize infection control practices and then assess the remaining risk of candida infections. finally, environmental fungi (e.g., aspergillus, fusarium, scedosporium, bipolaris, agents of mucormycosis) are important sources of infection for severely immunocompromised patients; meticulous attention to the conditions of the internal environment of any facility that provides care for severely immunocompromised patients is required, as well as prevention of possible exposure to construction dust in and around healthcare facilities. 13 with the advent of more effective and less toxic antifungal agents and improved outcomes, it is important to identify promptly the infecting agent by obtaining tissue samples and to determine susceptibility to candidate antifungal agents. prevention remains the mainstay of infection control and requires special considerations in children. the goals of ipc are to prevent the transmission of infectious agents among individual patients or groups of patients, visitors, and hcp who care for them. as new pathogens emerge, new strategies for prevention emerge. the experience treating evd in the us in 2014 and 2015 is the most recent example of changes in the usual infection prevention paradigm that were required, with a renewed emphasis on the 3 tiers of the hierarchy of controls (e.g., engineering, administration, and ppe), donning and doffing of ppe, and use of trained observers. 6, 7 if prevention cannot always be achieved, the strategy of early diagnosis, treatment, and containment is critical. a series of ipc guidelines have been developed and updated at varying intervals by the hicpac/cdc, idsa, society for healthcare epidemiology of america (shea), american academy of pediatrics, association for professionals in infection control and epidemiology, and others to provide evidence-based and rated recommendations for practices that are associated with reduced rates of hais, especially those infections associated with the use of medical devices and surgical procedures. recommended isolation precautions by infectious agent also can be found in the most recent edition of the red book report of the committee on infectious diseases of the american academy of pediatrics. prevention bundles are groups of 3 to 5 evidence-based "best practices" with respect to a process that individually improve care, but when applied together result in substantially greater reduction in infection transmission of microbes between children and hcp is a risk because of the very close contact that occurs during care of infants and young children and is facilitated by overcrowding, understaffing, and too few appropriately trained nurses in pediatric facilities. 4, 14 staffing levels and composition are important components of an effective ipc program. hcp rarely are the source of outbreaks of hais caused by bacteria and fungi, but when they are, certain factors are usually present that increase the risk of transmission (e.g., sinusitis, draining otitis externa, respiratory tract infections, dermatitis, onychomycosis, wearing of artificial nails). [35] [36] [37] persons with direct patient contact who were wearing artificial nails have been implicated in outbreaks of p. aeruginosa and esbl-producing klebsiella pneumoniae in nicus; therefore, the use of artificial nails or extenders is prohibited in persons who have direct contact with high-risk patients. 4 several published studies have shown that infected pediatric hcp, including resident physicians, transmitted bordetella pertussis to other patients and can be the source of other vaccine-preventable infections in healthcare. 38 pathogens associated with hais in children differ from those in adults in that respiratory viruses are more frequently associated with transmission in pediatric healthcare facilities. respiratory viruses (e.g., rsv, parainfluenza, adenovirus, human metapneumovirus) have been implicated in outbreaks in high-risk units. as more respiratory viruses and gastrointestinal pathogens are identified by using highly sensitive molecular methods, epidemiologic studies will be required to define further the risk of transmission in healthcare facilities and the clinical significance of positive antigen detection test results. 40, 41 healthcareassociated outbreaks of varicella, measles, and rotavirus infection now are rare events because of the consistent use of vaccines by children and hcp. the emergence of community-associated mrsa isolates characterized by the unique scc mec type iv element was first observed among infants and children. as rates of colonization with community-associated mrsa at the time of hospital admission increased, so did transmission of community strains, most often usa 300, within the hospital and especially within the nicu, thus making prevention especially challenging. other mdros (e.g., vre, esbls, and cre, especially k. pneumoniae) have emerged as the most challenging healthcare-associated pathogens in both pediatric and adult settings, and otherwise healthy children in the community can be colonized asymptomatically with these mdros. 42 gnb, including esbl and other multidrug-resistant isolates, are more frequent than mrsa and vre in many picus and nicus. patients who are transferred from chronic care facilities may be colonized with mdr gnb at the time of admission to the picu. trends in targeted mdros are tracked in the national nosocomial infections surveillance system 2. oversight of occupational health services related to ipc (e.g., assessment of risk and administration of recommended prophylaxis following exposure to infectious agents, tuberculosis screening, influenza and pertussis vaccination, respiratory protection fit testing, administration of other vaccines as indicated during infectious disease crises such as preexposure smallpox vaccine in 2003 and pandemic influenza a [h1n1] vaccine in 2009) 3. preparedness planning for annual influenza outbreaks, pandemic influenza, sars, middle east respiratory syndrome (mers), bioweapons attacks, and evd 4. adherence monitoring for selected ipc practices 5. oversight of risk assessment and implementation of preventive measures associated with construction, renovation, and other environmental conditions associated with increased infection risk 6. participation in antimicrobial stewardship programs, focusing on prevention of transmission of mdros 7. evaluation of new products and medical devices that could be associated with increased infection risk (e.g., endoscopes, 51 contaminated injectable medications 52 ) and introduction and assessment of performance after implementation of modified products 8. mandatory public reporting of hai rates in states according to enacted legislation 9. increased communication with the public and with local public health departments concerning infection control-related issues 10. participation in local and multicenter reporting and research projects ipc programs must be adequately staffed to perform all the foregoing activities. thus the ratio of 1 infection preventionist to 250 beds that was associated with a 30% reduction in the rates of nosocomial infection in the study on efficacy of nosocomial infection control (senic) performed in the 1970s no longer is sufficient because the complexity of patient populations and responsibilities have increased. many experts recommend that a ratio of 1 infection preventionist to 100 beds is more appropriate for the current workload, but no study has been performed to confirm the effectiveness of that ratio. no information is available on the number of ipc personnel required outside acute care, but it is clear that persons well trained in ipc must be available for all sites where healthcare is delivered. data collected from a member workforce survey conducted in 2015 by the association for professionals in infection control and epidemiology are expected to help determine the optimal number of infection preventionists for different healthcare settings based on the current responsibilities and demographics of infection preventionists. surveillance for hais consists of a systematic method of determining the incidence and distribution of infections acquired by hospitalized patients. the cdc recommends the following: (1) prospective surveillance on a regular basis by trained infection preventionists, using standardized definitions; (2) analysis of infection rates using established epidemiologic and statistical methods (e.g., calculation of rates using appropriate denominators that reflect duration of exposure; use of statistical process control charts for trending rates); (3) regular use of data in decision making; and (4) employment of an effective and trained healthcare epidemiologist who develops ipc strategies and policies and serves as a liaison with the medical community and administration. [53] [54] [55] the cdc has established a set of standard definitions of hais that have been validated and accepted widely with updates posted on the cdc nhsn website. standardization of surveillance methodology has become especially important with the advent of state legislation for mandatory reporting of hai rates to the public. the nhsn now receives, analyzes, and reports data from >17,000 healthcare facilities in the us. a standardized infection ratio (sir) that takes into account differences in risk among healthcare settings, unit types, procedures, and patient populations has been included in summary reports of hai rates since 2009. 56 the centers for medicare and medicaid services and most states use the nhsn data for public reporting of hai rates on their websites. although much effort has been directed toward making these data understandable and useful to consumers, interpretation of rates. adherence to the individual measures within a bundle is readily measured. bundled practices are used most frequently for prevention of device-or procedure-related hais, but they can be applied to prevention of any type of hai. the importance of certain administrative measures for a successful ipc program has been demonstrated. a white paper published by shea summarizes the necessary infrastructure for an effective ipc program in modern times. the paper addresses the expansion of ipc responsibilities from a relatively narrow focus on acute infectious disease events in the acute care hospital, surveillance, and implementation of recommended isolation precautions to a broader set of activities across the continuum of care requiring team work within and beyond individual facilities, usually including large networks. 48 because ipc comprises one component of the institutional culture of safety, it is critical to obtain support from the senior leadership of healthcare organizations to provide necessary fiscal and human resources for a proactive, successful ipc program. critical elements requiring administrative support include access to the following: (1) appropriately trained healthcare epidemiologists and ipc personnel; (2) clinical microbiology laboratory services needed to support infection control outbreak investigations, including ability to perform molecular diagnostic testing; (3) data-mining programs and information technology specialists; (4) multidisciplinary programs to ensure judicious use of antimicrobial agents and control of resistance; (5) development of effective educational information for delivery to hcp, patients, families, and visitors; and (6) local and state health department resources for preparedness. provision of adequate numbers of well-trained infection preventionists and bedside nursing staff is critical for success. an effective ipc program improves safety of patients and hcp and decreases short-term and long-term morbidity, mortality, and healthcare costs. 49 the ipc committee of a facility establishes policies and procedures to prevent or reduce the incidence and costs associated with hais. this committee should be one of the strongest and most accessible committees in the facility; committee composition should be considered carefully and limited to active, authoritative participants who have well-defined committee responsibilities and who represent major groups within the hospital. the chairperson should be a good communicator with expertise in ipc issues, healthcare epidemiology, and clinical pediatric infectious diseases. important functions of the ipc committee are regular review of ipc policies and development of new policies as needed. annual review of all policies is required by the joint commission and can be accomplished optimally by careful review of a few policies each month. with the advent of unannounced inspections, a constant state of readiness is required. the hospital epidemiologist or medical director of the pediatric ipc department usually is a physician with training in pediatric infectious diseases and dedicated expertise in healthcare epidemiology. in multispecialty medical centers where infants and children comprise a small proportion of patients, pediatric infectious disease experts should be consulted for management of pediatric ipc issues and report to the broader ipc leadership. the skillsets, training, and competencies needed for success as a healthcare epidemiologist were summarized in another white paper published by the shea. 50 certification for healthcare epidemiologists has not yet been developed. infection preventionists are specialized professionals with advanced training, and preferably certification, in ipc. although most infection preventionists are registered nurses, other professionals, including microbiologists, medical technologists, pharmacists, and epidemiologists, are successful in this position. pediatric patients should have infection preventionist services provided by professionals with expertise and training in the care of children. in a large, general hospital, at least 1 infection preventionist should be dedicated to ipc services for children. the responsibilities of infection preventionists have expanded greatly and include the following: 1. surveillance and ipc in facilities affiliated with primary acute care hospitals (e.g., ambulatory clinics, day-surgery centers, long-term care facilities, rehabilitation centers, home care) in addition to the primary hospital part i understanding, controlling, and preventing infectious diseases according to 2006 guidelines, if transmission continues after standardized horizontal interventions have been completely implemented. 5 at this time, no formal recommendation has been made to discontinue routine use of contact precautions for patients with asymptomatic colonization with mrsa or vre in an endemic setting; thus each ipc program must determine practice based on local conditions and follow with close auditing and surveillance for potential adverse outcomes. the microbiology laboratory can provide online culture information about individual patients, outbreaks of infection, antibiograms (antibiotic susceptibility patterns of pathogens summarized periodically), and employee infection data. the laboratory also can assist with surveillance cultures and facilitation of molecular typing of isolates during outbreak investigations. rapid diagnostic testing of clinical specimens for identification of respiratory and gastrointestinal tract viruses and b. pertussis is especially important for pediatric facilities. the ipc division and the microbiology laboratory must communicate daily because even requests for cultures or other diagnostic testing from physicians (e.g., m. tuberculosis, neisseria meningitidis, c. difficile) can identify patients early who are infected, are at high risk of infection, or require isolation. if microbiology laboratory work is outsourced, it is important to ensure that the services needed to support effective icp be available, as delineated in a 2013 guideline developed by the idsa and the american society for microbiology. 66 control of unusual infections or outbreaks in the community generally is the responsibility of the local or state public health department; however, the individual facility must be responsible for preventing transmission within that facility. public health agencies can be helpful, particularly in alerting hospitals of community outbreaks so that outpatient and inpatient diagnosis, treatment, necessary isolation, and other preventive measures are implemented promptly to avoid further spread. conversely, designated persons in the hospital must notify public health department personnel of reportable infections to facilitate early diagnosis, treatment, and infection control in the community. benefits of community or regional collaboratives of individual healthcare facilities and local public health departments for prevention of hais, especially those caused by mdros, have been demonstrated, and this collaboration should be encouraged. 4 the rapid increase of mdros is a public health threat. between 20% and 50% of antibiotics prescribed in us hospitals are either inappropriate or unnecessary. 68 in 2014, the president's council of advisors on science and technology submitted a 78-page report to the president on combating antibiotic resistance that raised awareness of antimicrobial resistance to a national level. 69 a national action plan based on this report was released in march 2015, and funding was made available for its implementation. antimicrobial stewardship was defined in a consensus statement by the idsa, shea, and pediatric infectious diseases society in 2012 as "coordinated interventions designed to improve and measure the appropriate use of antibiotic agents by promoting the selection of the optimal antibiotic regimen, including dosing, duration and route of administration." 70 antimicrobial stewardship programs are collaborative partnerships among infection preventionists, healthcare epidemiologists, clinical pharmacists, and microbiologists. hospital administrative support for the infrastructure required for ongoing measurement and reporting of antimicrobial use and other related outcome measures, including feedback to prescribers, is a critical component of a successful antimicrobial stewardship program. an antimicrobial stewardship program can optimize clinical outcomes while decreasing unintended consequences of antimicrobial use, including the emergence of resistant organisms. additionally, use of specific antimicrobial agents can alert the ipc program to the presence of potentially infectious patients (e.g., with pulmonary tuberculosis, mdros). national guidelines exist for developing and implementing an institutional antimicrobial stewardship program, including core components for acute care hospitals and for long-term care facilities. 68, 70 the natinal quality forum and its partners have also developed a playbook that provides additional guidance for implementation of antimicrobial stewardship programs in acute care. the knowledge and skills required for antimicrobial stewardship leaders also have been defined. 50, 71 these data by the public remains difficult, and more research is needed to optimize methods of data display to the public. 57 new york state is the first state to have published an improvement in process and outcomes of central line-associated bloodstream infection rates in nicus following implementation of a public reporting program. 58 although various surveillance methods are used, the basic goals and elements are similar and include using standardized definitions of infection, finding and collecting cases of hais, tabulating data, using appropriate denominators that reflect duration of risk, analyzing and interpreting the data, reporting important deviations from endemic rates (epidemic, outbreaks) to the bedside care providers and to the facility administrators, implementing appropriate control measures, auditing adherence rates for recommended processes, and assessing efficacy of the control measures. medical centers can use different methods of surveillance, as outlined in box 2.1. most experts agree that a combination of methods enhances surveillance and reliability of data, and some combination of clinical chart review and database retrieval is important. whatever data collection systems are used, validation is required. administrative databases created for the purposes of billing should not be used as the sole source to identify hais because of overestimates and underestimates that result from inaccurate coding of hais. 59 use of software designed specifically for ipc data entry and analysis facilitates real-time tracking of trends and timely intervention when clusters are identified. the ipc team should participate in the development and update of electronic medical record systems for a healthcare organization, to ensure that surveillance needs will be met. controversy has surrounded the role of obtaining active surveillance cultures from all patients admitted to an acute care hospital, especially to an icu, to detect asymptomatic colonization with mrsa or vre and then placing those persons on contact precautions in an endemic setting, a practice referred to as a vertical approach. 60, 61 more recently published experiences demonstrate the benefits of a horizontal approach to reduce the risk of transmission of a broader variety of pathogens, 61 and a framework for a less restrictive approach has been published. 62 contributing factors to the benefits of the horizontal approach include the following: (1) widespread implementation of bundled prevention practices, including limiting use of unnecessary medical devices; (2) improved understanding and more consistent implementation of standard precautions, especially hand hygiene; (3) establishment of the safety and efficacy of universal decolonization using chlorhexidine bathing in icus 63, 64 and nicus for infants weighing >1000 g at birth 65 ; (4) improving environmental cleaning; and (5) identified the following potential infection control breaches: (1) use of multidose vials for heparin or saline administration; (2) poor compliance with hand hygiene before and after patient contacts or after touching a possibly contaminated surface; (3) failure to change gloves between patient contacts or after contact with a potentially contaminated surface; (4) failure to disinfect environmental surfaces adequately; (5) unsafe injection practices; (6) failure to disinfect shared equipment between patient uses; (7) lack of a separate area for medication preparation; and (8) failure to have clean and dirty utility rooms clearly separated. 78 two additions were made to standard precautions in 2007: (1) respiratory hygiene or cough etiquette for source containment by people with signs and symptoms of respiratory tract infection and (2) use of a mask by personnel inserting an epidural anesthesia needle or performing a myelogram when prolonged exposure of the puncture site is likely. both components have a strong evidence base. implementation of standard precautions requires the availability of ppe in proximity to all patient care areas. hcp with exudative lesions or weeping dermatitis must avoid direct patient care and handling of patient care equipment. persons having direct patient contact should be able to anticipate exposure incurring risks and steps to take if a highrisk exposure occurs. exposures of concern are as follows: exposures to blood or other potentially infectious material defined as an injury with a contaminated sharp object (e.g., needlestick, scalpel cut); a spill or splash of blood or other potentially infectious material onto nonintact skin (e.g., cuts, hangnails, dermatitis, abrasions, chapped skin) or onto a mucous membrane (e.g., mouth, nose, eye); or blood exposure covering a large area of normal skin. patient-related duties that do not constitute high-risk exposures include handling food trays or furniture, pushing wheelchairs or stretchers, using restrooms or telephones, having personal contact with patients (e.g., giving information, touching intact skin, bathing, giving a back rub, shaking hands), or performing clerical or administrative functions for a patient. if hands or other skin surfaces are exposed to blood or other potentially infectious material, the area should be washed immediately with soap and water for at least 10 seconds and rinsed with running water for at least 10 seconds. for an eye, nose, or mouth splash with blood or body fluids, the area should be irrigated immediately with a large volume of water. if a skin cut, puncture, or lesion is exposed to blood or other potentially infectious material, the area should be washed immediately with soap and water for at least 10 seconds and rinsed with 70% isopropyl alcohol. any exposure incident should be reported immediately to the occupational health department to determine whether blood samples are required from the source patient and the exposed person and if immediate prophylaxis is indicated. all hcp should know where to find the exposure control plan specific to each place of employment, whom to contact, where to go, and what to do if inadvertently exposed to blood or body fluids. important resources include the occupational health department, the emergency department, and the infection control or hospital epidemiology division. the most important recommendation in any accidental exposure is to seek advice and intervention immediately because the efficacy of recommended prophylactic regimens is improved with shorter intervals after exposure, such as for hepatitis b immune globulin administration after exposure to hepatitis b virus or for antiretroviral therapy after percutaneous exposure to hiv. chemoprophylaxis following exposure to hiv-infected material is most effective if it is initiated as soon as possible, but within hours of exposure. 79 the current guidelines recommend using ≥3 drugs for postexposure prophylaxis of hiv independent of the severity of exposure. updates are posted on the cdc website as they are developed. reporting a work-related exposure is required for subsequent medical care and workers' compensation. transmission-based precautions are designed for patients with documented or suspected infection with pathogens for which additional precautions beyond standard precautions are needed to prevent transmission. the 3 categories of transmission-based precautions are contact precautions, droplet precautions, and airborne precautions, and they are based on the likely routes of transmission of specific infectious agents. transmission-based precautions are combined for infectious agents that have more than 1 route of transmission. when used singly or in the effectiveness of antimicrobial stewardship programs in achieving improved patient outcomes is evident in pediatric acute care hospitals, 72, 73 including the nicu, 74, 75 in ambulatory settings, and in long-term care facilities. the area of antimicrobial stewardship, however, requires additional research to establish optimal methods in various pediatric specialty populations. one practice from the cdc get smart program that can be implemented by each prescriber in most settings is the antibiotic "time out" that consists of reviewing patient data at 48 to 72 hours of treatment to determine which of the following is indicated: (1) continue antibiotic treatment; (2) change to a narrower-spectrum agent; (3) change from a parenteral to an oral agent; or (4) shorten or conclude therapy. 68 isolation of patients with potentially transmissible infectious diseases is a strategy proven to prevent transmission of infectious agents in healthcare settings. many published studies, performed in both adult and pediatric settings, provide a strong evidence base for most recommendations for isolation precautions and for limiting outbreaks. however, controversies exist concerning the most clinically and cost-effective measures for preventing certain hais, especially those associated with mdros. as discussed earlier in the section on surveillance, a call has gone out to reconsider recommendations for isolation of patients who are asymptomatically colonized with mrsa or vre, but no definite recommendation has been made by the hicpac/cdc, shea, or association for professionals in infection control and epidemiology. since 1970, the guidelines for isolation developed by cdc have responded to the needs of the evolving us healthcare systems. for example, universal precautions became a required standard in response to the hiv epidemic that emerged in the 1980s and the need to prevent acquisition of bloodborne pathogens (e.g., hiv, hepatitis b and c viruses) by hcp through skin, eye, mucous membrane, or parenteral contact with blood or other potentially infectious materials from persons not known to be or suspected of being infected. universal precautions were modified and have been known as standard precautions since publication of the 1996 guideline for isolation. the federal needlestick safety and prevention act, signed into law in november 2000, authorized the occupational safety and health administration's revision of its bloodborne pathogens standard more explicitly to require the use of safety-engineered sharp devices. 76 evidence and recommendations are provided for the prevention of transmission of mdros such as mrsa, vre, visa, vrsa, and gnb. 4, 5 the components of a protective environment for prevention of environmental fungal infections in hsct recipients are summarized. 4 finally, evidence-based, rated recommendations for administrative measures that are necessary for effective prevention of infection in healthcare settings are provided. the most recent guideline for isolation precautions published in 2007 4 reaffirms standard precautions, a combination of universal precautions and body substance isolation, as the foundation of transmission prevention measures. critical thinking is required for hcp to recognize the importance of body fluids, excretions, and secretions in the transmission of infectious pathogens and take appropriate protective precautions by using ppe (e.g., masks, gowns, gloves, face shields, or goggles) and safety devices when exposure is likely even if an infection is not suspected or known. in addition, these updated guidelines provide recommendations for standard precautions in all settings in which healthcare is delivered (acute care hospitals, ambulatory surgical and medical centers, longterm care facilities, and home health agencies). the components of standard precautions are summarized in table 2 instruct symptomatic persons to cover the mouth or nose when sneezing or coughing; use tissues and dispose in no-touch receptacle; observe hand hygiene after soiling of hands with respiratory secretions; wear a surgical mask if tolerated or maintain spatial separation, >1-2 m (3-6 feet) if possible a during aerosol-generating procedures on patients with suspected or proven infections transmitted by aerosols (e.g., severe acute respiratory syndrome), wear a fit-tested n95 or higher respirator in addition to gloves, gown, and face and eye protection. although targeted contact precautions and universal gowning and gloving are effective for preventing transmission of infectious agents, potential adverse effects in patients placed on contact precautions have been described (e.g., depression, fewer visits from the healthcare team, increased rates of hypoglycemia or hyperglycemia, increased falls). 80 additionally, adherence to contact precautions decreases as the number of patients on contact precautions increases. 81 finally, a simulation study demonstrated contamination of hcp skin and clothing during doffing of gowns and gloves 82 ; this study effectively demonstrated the ppe lessons learned during the sars and evd experiences. evidence supports the importance of applying contact precautions only when indicated, obtaining training on the use of ppe, having effective ppe readily available, and practicing consistent and precise use of ppe. 83 table 2.2 lists the 3 categories of isolation based on routes of transmission and their necessary components. table 2 .3 lists precautions by syndromes, to be used when a patient has an infectious disease and the agent is not yet identified. for infectious agents that are more likely to be transmitted by the droplet route (e.g., pandemic influenza), droplet precautions (with use of surgical mask) are appropriate; however, during an aerosol-generating procedure, n95 or higher respirators are indicated. 84 contaminated environmental surfaces and noncritical medical items have been implicated in transmission of several infectious agents, including vre, c. difficile, acinetobacter spp., mrsa, and rsv in healthcare settings. 4, 85, 86 pathogens on surfaces are transferred to the hands of hcp and are then transferred to patients or items to be shared. occupying a room previously occupied by a patient with a key pathogen is a risk factor for acquiring that pathogen during a hospital stay. most often, the failure to follow recommended procedures for cleaning and disinfection contributes more than does the specific pathogen to the environmental reservoir during outbreaks. education of environmental services personnel combined with direct observation and feedback was associated with a persistent decrease in vre acquisition in a medical icu. use of a standardized cleaning checklist and implementation of monitoring for adherence to recommended environmental cleaning practices are important determinants of success. visual markers (e.g., invisible fluorescein powder) and adenosine triphosphate bioluminescence technologies are self-disinfecting surfaces can be created by altering the structure of the surface material or by incorporating a material that has antimicrobial activity. [85] [86] [87] copper has antimicrobial activity against a wide range of organisms including bacteria and fungi. thus, incorporating copper into high-touch surfaces such as toilet seats, bed rails, door handles, or countertops is a novel infection prevention strategy that has been shown to reduce bacterial colony counts compared with control surfaces in healthcare settings. 89 however, no recommendation for routine use has yet been made. disinfection and sterilization as they relate to ipc have been reviewed, 90 and the hicpac/cdc developed comprehensive guidelines in 2008. 91 cleaning is the removal of all foreign material from surfaces and objects. this process is accomplished using soap and enzymatic products. failure to remove all organic material from items before disinfection and sterilization reduces the effectiveness of these processes. disinfection is a process that eliminates all forms of microbial life except the endospore. disinfection usually requires liquid chemicals. disinfection of an inanimate surface or object is affected adversely by the following: the presence of organic matter; a high level of microbial contamination; use of too dilute germicide; inadequate disinfection time; an object that also useful for monitoring effective environmental cleaning and providing immediate feedback to workers. 87 a program of environmental cleaning should be developed collaboratively by the ipc and environmental services departments. certain infectious agents (e.g., rotavirus, noroviruses, c. difficile) can be resistant to some routinely used hospital disinfectants; thus when ongoing transmission occurs despite appropriate cleaning procedures, a 1 : 10 dilution of 5.25% sodium hypochlorite (household bleach) or other special disinfectants are indicated. "no-touch" automated room decontamination technologies have been developed and added to room turnover procedures in some facilities. ultraviolet light irradiation and hydrogen peroxide vapor systems have been shown to reduce surface contamination with common pathogens and decrease the risk of acquiring hais caused by those pathogens when these systems are added to a terminal cleaning regimen. [85] [86] [87] at specific wavelengths, ultraviolet light breaks the molecular bonds in dna, thus destroying the organisms. ultraviolet technology also has been considered as a method of disinfecting ppe, as a risk mitigation strategy for hcp caring for patients with evd. 88 these technologies supplement, but do not replace, standard cleaning and disinfection because surfaces must be physically cleaned of particulate matter and debris. other disadvantages of these systems are that they cannot be used when people are in the rooms, room turnover is delayed, and the systems are expensive to purchase. no recommendations have been made for routine use or specific indications because research on antimicrobial effectiveness, cost effectiveness, and feasibility of these systems is ongoing. patients with the syndromes or conditions listed may have atypical signs or symptoms (e.g., neonates and adults with pertussis may not have paroxysmal or severe cough). the clinician's index of suspicion should be guided by the prevalence of specific conditions in the community, as well as clinical judgment. c the organisms listed under the column "potential pathogens" are not intended to represent the complete, or even most likely, diagnoses, but rather possible etiologic agents that require additional precautions beyond standard precautions until they can be excluded. influenza season. several children's hospitals provide influenza vaccine or tetanus, diphtheria, and acellular pertussis (tdap) vaccine, or both, to household contacts at no charge, thereby supporting the cocooning strategy endorsed by the advisory committee on immunization practices and the american academy of pediatrics. 96 for patients requiring contact precautions, the use of ppe by visitors is determined by the nature of the interaction with the patient and the likelihood that the visitor will frequent common areas on the patient's unit or interact with other patients and their families. it is important to distinguish parents or guardians from nonhousehold visitors when determining whether the visitor should wear ppe. the risk-benefit decision should weigh not only the specific pathogen in question, but also the effect of parental or guardian ppe on breastfeeding, bonding, and family participation in the child's medical care. for family members who are rooming in with children who have prolonged hospitalizations, restriction of visitation to other patients is emphasized. a shea expert guidance document has been published to summarize the principles to follow to prevent transmission of infectious agents by visitors to patients because few data are available to inform evidence-based recommendations. 97 although most pediatricians encourage visits by siblings in inpatient areas, the medical risk must not outweigh the psychosocial benefit. families favorably regard sibling visitation, and no evidence indicates increased bacterial colonization or subsequent bacterial infection in the neonate or older child who has been visited by siblings. strict guidelines for sibling visitation should be established and enforced in an effort to maximize visitation opportunities and minimize risks of transmission of infectious agents, most frequently viruses. the following recommendations regarding visitation can guide policy development: 1. sibling visitation is encouraged in the well-child nursery and nicu, as well as in areas for care of older children. 2. before visitation, parents should be interviewed by a trained staff nurse concerning the current health status of the sibling. siblings should not be allowed to visit if they are delinquent in recommended vaccines, have fever or symptoms of an acute illness, or are within the incubation period following exposure to a known infectious disease. after the interview, the physician or nurse should place a written consent for sibling visitation in the patient's permanent record and a name tag indicating that the sibling has been approved for visitation for that day. 3. asymptomatic siblings who recently were exposed to varicella but who previously were immunized can be assumed to be immune. 4. the visiting sibling should visit only his or her sibling and not be allowed in playrooms with groups of patients. 5. visitation should be limited to periods of time that ensure adequate screening, observation, and monitoring of visitors by medical and nursing staff members. 6. children should perform hand hygiene before and after contact with the patient or upon entry and departure from the patient's room. 7. during the entire visit, sibling activity should be supervised by parents or another responsible adult. animal-assisted therapy can be of substantial clinical benefit to the child hospitalized for prolonged periods; therefore it is important for healthcare facilities to provide guidance for safe visitation. many zoonoses and infections are attributable to animal exposure (see chapter 89) . most infections result from inoculation of animal flora through a bite or scratch or self-inoculation after contact with the animal, the animal's secretions or excretions, or contaminated environment. although few data support a true evidence-based guideline for animal visitation (including personal pets) in healthcare facilities, updated expert guidance is provided in the shea expert guidance on animals in healthcare facilities: recommendations to minimize potential risk, which includes a review of the literature related to animal-assisted activities. 98 prudent visitation policies should limit visitation to animals that: (1) are domesticated; (2) do not require a water environment; (3) do not bite or scratch; (4) can be brought to the hospital in a carrier or easily walked on a leash; (5) are trained to defecate and urinate outside or in appropriate litter boxes; (6) can be bathed before visitation; and (7) are known to be free of respiratory, dermatologic, and gastrointestinal tract disease. despite the established risk of salmonellosis can harbor microbes in protected cracks, crevices, and hinges; and ph and temperature. sterilization is the eradication of all forms of microbial life, including fungal and bacterial spores. sterilization is achieved by physical and chemical processes such as steam under pressure, dry heat, ethylene oxide, and liquid chemicals. the spaulding classification of patient care equipment as critical, semicritical, and noncritical items with regard to sterilization and disinfection is used by the cdc. critical items require sterilization because they enter sterile body tissues and carry a high risk of causing infection if they are contaminated; semicritical items require disinfection because they may contact mucous membranes and nonintact skin; and noncritical items require routine cleaning because they come in contact only with intact skin. if noncritical items used on patients requiring transmission-based precautions, especially contact precautions, must be shared, these items should be disinfected between uses. guidelines for specific objects and specific disinfectants are published and updated by the cdc. multiple published reports and manufacturers similarly recommend the use and reuse of objects with appropriate sterilization, disinfection, or cleaning recommendations. recommendations in guidelines for reprocessing endoscopes to avoid contamination focus on training of personnel, meticulous manual cleaning, high-level disinfection followed by rinsing and air-drying, and proper storage. 92 however, outbreaks of mdr gnb infections associated with exposure to duodenoscopes used for retrograde cholangiopancreatography that have been reprocessed according to recommendations suggest a need for new endoscope reprocessing technologies. 51, 93 these endoscopes have a complex design with long, narrow channels, crevices that are difficult to access with a cleaning brush, right-angle turns, and heavy microbial contamination following procedures. until new methods are developed, meticulous adherence to recommended processes with enhancements should be followed. medical devices that are designed for single use (e.g., specialized catheters, electrodes, biopsy needles) must be reprocessed by third parties or hospitals according to the guidance issued by the food and drug administration (fda) in august, 2000 with amendments in september, 2006; such reprocessors are considered and regulated as "manufacturers." available data show that single-use devices reprocessed according to the fda regulatory requirements are as safe and effective as new devices. deficiencies in disinfection and sterilization leading to infection have resulted either from failure to adhere to scientifically based guidelines or failures in the disinfection or sterilization processes. when such failures are discovered, an investigation must be completed, including notification of patients and, in some cases, testing for infectious agents. a guidance document for risk assessment and communication to patients in such situations is published. 94 healthcare facility waste is all biologic or nonbiologic waste that is discarded and not intended for further use. medical waste is material generated as a result of use with a patient, such as for diagnosis, immunization, or treatment, and it includes soiled dressings and intravenous tubing. infectious waste is that portion of medical waste that potentially could transmit an infectious disease. microbiologic waste, pathologic waste, contaminated animal carcasses, blood, and sharps are all examples of infectious waste. methods of effective disposal of infectious waste include incineration, steam sterilization, drainage to a sanitary sewer, mechanical disinfection, chemical disinfection, and microwave treatment. state regulations guide the treatment and disposal of regulated medical waste. recommendations are available for developing and maintaining a program within a facility for safe management of medical waste. 95 special visitation policies are required in pediatric units, especially the high-risk units, because acquisition of a seemingly innocuous viral infection in neonates and in children with underlying diseases can result in unnecessary evaluations and empiric therapies for suspected septicemia as well as serious, life-threatening disease. all visitors with signs or symptoms of respiratory or gastrointestinal tract infection should be restricted from visiting patients in healthcare facilities. increased restrictions may be required during a community outbreak (e.g., sars, pandemic influenza, enterovirus d68). during respiratory virus season, the number of visitors can be limited and the age restriction increased. it is preferred for all visitors to be immunized against influenza during part i understanding, controlling, and preventing infectious diseases respiratory viruses, norovirus, and tuberculosis. important preventive procedures for hcp with infants at home or who are pregnant are as follows: (1) consistent training and observance of standard precautions, transmission-based precautions, and especially hand hygiene according to published recommendations; (2) annual influenza and 1-time tdap immunization (unless pregnant, when a tdap immunization during each pregnancy is recommended); (3) routine tuberculosis screening; (4) assurance of immunity or immunization against poliomyelitis, measles, mumps, hepatitis b, and rubella; (5) early medical evaluation for acute infectious illnesses; (6) routine, on-time immunization of infants; and (7) prompt initiation of prescribed prophylaxis or therapy following exposure or development of certain infections. hcp who are, could be, or anticipate becoming pregnant should feel comfortable working in the healthcare workplace. in fact, with standard precautions and appropriate adherence to environmental cleaning and isolation precautions, vigilant hcp can be at less risk than a preschool teacher, childcare provider, or mother of children with many playmates in the home. pathogens of potential concern to pregnant hcp include cytomegalovirus, hepatitis b virus, influenza, measles, mumps, parvovirus b19, rubella, vzv, m. tuberculosis, and, since 2015, zika virus. the causal association between zika virus and microcephaly and other neurodevelopmental abnormalities 109 has led to recommended precautions. although zika virus is more frequently acquired outside of healthcare, pregnant hcp are advised to follow safe injection practices for prevention of exposure to infectious blood. 4 pregnancy is an indication for influenza vaccine to prevent the increased risk of serious disease and hospitalization that occurs in women who develop influenza in the second or third trimester of pregnancy. in 2011, the cdc recommended universal immunization with tdap (if previously not immunized with tdap) for pregnant women after 20 weeks of gestation, and since 2012, the cdc recommends a dose of tdap with each pregnancy. 110 pregnant workers should assume that all patients potentially are infected with cytomegalovirus and other "silent" pathogens and should use hand hygiene and gloves when handling body fluids, secretions, and excretions. table 2 .4 summarizes information about infectious agents that are relevant to the pregnant woman working in healthcare. chapters on each agent may be consulted for more specific information. the risk of hais in pediatric ambulatory settings is substantial, and it usually is associated with lack of adherence to routine ipc practices and procedures, especially disinfection, sterilization, and hand hygiene. respiratory viral agents and m. tuberculosis are noteworthy pathogens transmitted in ambulatory settings. transmission of rsv in an hsct outpatient clinic has been demonstrated using molecular techniques. 111 crowded waiting rooms, toys, furniture, lack of isolation of children, unwell children, contaminated hands, contaminated secretions, and susceptible hcp are only some of the factors that result in sporadic and epidemic illness in outpatient settings. the association of communityassociated mrsa in hcp working in an outpatient hiv clinic with environmental community-associated mrsa contamination of that clinic indicates the potential for transmission in this setting. 112 patientto-patient transmission of burkholderia species and p. aeruginosa in outpatient clinics for patients with cystic fibrosis has been confirmed and prevented by implementing recommended ipc methods. 9 ipc guidelines and policies for pediatric outpatient settings, including office practices, were published by the american academy of pediatrics in 2007, 113 reaffirmed in 2015, and are updated currently. prevention strategies include definition of policies, education, and strict adherence to guidelines. in pediatrics, among the most important interventions are separation of children with respiratory tract illnesses from well children and consistent implementation of respiratory etiquette or cough hygiene. a guideline for ipc for outpatient settings with a checklist and a guideline for outpatient oncology settings can be found on the cdc website. 114 principles and recommendations for safe living after hsct 115 and for patients with cystic fibrosis 9 are valuable contributions to management of infectious risks for specific populations in the ambulatory setting. a guideline based on data and expert consensus opinion for ipc in residential facilities for associated with reptiles (e.g., turtles, iguanas), many reports of outbreaks of invasive disease associated with reptiles continue to be published 99 ; reptiles should be excluded from pet visitation programs, and families should be advised not to have pet reptiles in the home with young infants or immunocompromised persons. exotic animals that are imported should be excluded because of unpredictable behavior and the potential for transmission of unusual pathogens (e.g., monkeypox in the us in 2003). 100, 101 visitation should be limited to short periods and confined to designated areas. visiting pets must have a certificate of immunization from a licensed veterinarian. children should observe hand hygiene after contact with animals. most pediatric facilities restrict pet interaction with severely immunosuppressed patients and patients in icus. occupational health and student health collaboration with the ipc department of a healthcare facility is required by the occupational safety and health administration. hcp are at increased risk of infection in hospitals caring for children because (1) children have a high incidence of infectious diseases, (2) personnel can be susceptible to many pediatric pathogens, (3) pediatric care requires close contact, (4) children lack good personal hygiene, (5) infected children can be asymptomatic, and (6) hcp are exposed to multiple family members who also may be infected. the occupational health department is an educational resource for information on infectious pathogens in the healthcare workplace. in concert with the ipc service, occupational health provides preemployment education and respirator fit testing and annual retraining for all employees regarding routine health maintenance, available recommended and required vaccines, standard and transmission-based precautions, and exposure control plans. screening for tuberculosis at regular intervals, as determined by the facility's risk assessment, can use either tuberculin skin testing or interferon-γ release assays. 102 with new pathogens being isolated, new diseases and their transmission described, and new prophylactic regimens and treatment available, it is mandatory that personnel have an up-to-date working knowledge of ipc and know where and what services, equipment, and therapies are available for hcp. all hcp should be screened by history or serologic testing, or both, to document their immune status to specific agents, and immunization should be provided for the following for all employees who are nonimmune and who do not have contraindications to receiving the vaccine: diphtheria toxoid, hepatitis b virus, influenza (yearly), mumps, poliomyelitis, rubella, rubeola, varicella, and tdap. the 2006 advisory committee on immunization practices recommendation to administer a single dose of tdap to certain hcp was amended in 2011 to have no restriction based on age or time interval since the last td dose. providing vaccines at no cost to hcp increases acceptance. influenza vaccine coverage among hcp has increased over time to 77% overall for the 2014 to 2015 influenza season, with the highest coverage rate of 90% in hcp working in hospitals and the lowest rate of 64% in long-term care settings. 103 although mandatory influenza vaccination programs for all employees in healthcare facilities are endorsed by many professional societies, 104,105 some facilities have had success using novel strategies that include incentives, without a mandate. 106 publications from several large institutions, including children's hospitals, indicate that mandatory programs with only medical and religious exemptions are well received, and only rare employees are terminated for failure to be vaccinated. 107, 108 special concerns of healthcare personnel hcp who have common underlying medical conditions should be able to obtain general information on wellness and screening when needed from the occupational health service. hcp with direct patient contact who have infants <1 year of age at home often are concerned about acquiring infectious agents from patients and transmitting them to their susceptible children. an immune healthcare worker who is exposed to vzv does not become a silent "carrier" of vzv. however, pathogens to which the healthcare worker is partially immune or nonimmune can cause a severe, mild, or asymptomatic infection in the employee that can be transmitted to family members. examples include influenza, pertussis, rsv and other pediatric patients and their families provides practical guidance for settings where high-risk patients live with their families for varying periods of time. 116 ipc challenges now are being addressed in long-term care facilities for children. 117 more data are needed to determine the most effective and least restrictive practices. all references are available online at www.expertconsult.com. ohio children's hospitals' solutions for patient safety: a framework for pediatric patient safety improvement mucosal barrier injury laboratoryconfirmed bloodstream infection: results from a field test of a new national healthcare safety network definition prevention and management of infectious complications of percutaneous interventions review of fungal outbreaks and infection prevention in healthcare settings during construction and renovation nurse staffing and nicu infection rates carbapenem-resistant enterobacteriaceae in pediatric patients: epidemiology and risk factors the effects of antibiotics on the microbiome throughout development and alternative approaches for therapeutic modulation necessary infrastructure of infection prevention and healthcare epidemiology programs: a review guidance for infection prevention and healthcare epidemiology programs: healthcare epidemiologist skills and competencies approaches for preventing healthcareassociated infections: go long or go wide? disinfection and sterilization: an overview references 1. the joint commission. national patient safety goals high-reliability health care: getting there from here ohio children's hospitals' solutions for patient safety: a framework for pediatric patient safety improvement guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings management of multidrug-resistant organisms in healthcare settings addressing infection prevention and control in the first u.s. community hospital to care for patients with ebola virus disease: context for national 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carbapenemase-producing enterobacteriaceae whole genome sequencing in real-time investigation and management of a pseudomonas aeruginosa outbreak on a neonatal intensive care unit modes of transmission of respiratory syncytial virus transmission of influenza a in human beings tuberculosis among adult visitors of children with suspected tuberculosis and employees at a children's hospital airborne transmission of communicable infection: the elusive pathway control of influenza in healthcare settings: early lessons from the 2009 pandemic surgical masks for protection of healthcare personnel against pandemic novel swine-origin influenza a (h1n1)-2009: results from an observational study protecting healthcare personnel from acquiring ebola virus disease how often do asymptomatic healthcare workers cause methicillin-resistant staphylococcus aureus outbreaks? a systematic evaluation outbreak of extended-spectrum betalactamase-producing klebsiella pneumoniae in a neonatal intensive care unit 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translating policy to practice employee designation and health care worker support of an influenza vaccine mandate at a large pediatric tertiary care hospital zika virus centers for disease control and prevention. updated recommendations for use of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (tdap) in pregnant women-advisory committee on immunization practices (acip) molecular characterization of strains of respiratory syncytial virus identified in a hematopoietic stem cell transplant outpatient unit over 2 years: community or nosocomial infection? epidemiology of community-acquired methicillin-resistant staphylococcus aureus skin infections among healthcare workers in an outpatient clinic infection prevention and control in pediatric ambulatory settings guide to infection prevention for outpatient settings: minimum expectations for safe care safe living after hematopoietic cell transplantation shea guideline: infection prevention and control in residential facilities for pediatric patients and their families impact of infection prevention and control initiatives on acute respiratory tract infections in a pediatric long-term care facility key: cord-017030-tzuyo6tx authors: henao-martínez, andrés f.; montoya, josé g. title: infections in heart, lung, and heart-lung transplantation date: 2018-12-08 journal: principles and practice of transplant infectious diseases doi: 10.1007/978-1-4939-9034-4_2 sha: doc_id: 17030 cord_uid: tzuyo6tx half a century has passed since the first orthotopic heart transplant took place. surgical innovations allowed for heart, lung, and heart-lung transplantation to save lives of patients with incurable chronic cardiopulmonary conditions. the complexity of the surgical interventions, chronic host health conditions, and antirejection immunosuppressive medications makes infectious complications common. infections have remained one of the main barriers for successful transplantation and a source of significant morbidity and mortality. recognition of infections and its management in this setting require outstanding clinical skills since transplant recipients may not exhibit classic signs or symptoms of disease, and laboratory work has some pitfalls. the prevention, identification, and management of infectious diseases complications in this population are a priority to undertake to improve the medical outcomes of transplantation. herein, we reviewed the historical aspects, epidemiology, and prophylaxis of infections in heart, lung, and heart-lung transplantation. we also discuss the most prevalent organisms affecting the host and the organ systems involved. louder! louder!" dr. john gibbon jr. used for the first time in 1953 a heart-lung respirator to keep a patient alive while performing heart surgery. dr. norman shumway at stanford developed and perfected the first surgical technique leading to heart transplantation surgery. after dr. christian barnard's first orthotopic heart transplant in december 1967, and dr. shumway first heart transplant in the united states in january 1968, heart transplantation became a standard therapeutic option for life-threatening congestive failure and started to be performed in the hundreds over the next following years at different centers. heart transplant surgery faced complications due in part to rejection and infection. however, the development of more selective immunosuppressive therapy and improvements in prevention, detection, and treatment of infections allowed for heart transplant surgery to increase rapidly worldwide. four thousand and ninety six heart (3529 adults) transplants were reported to the international society of heart and lung transplant registry (ishl) in 2011 [1] . the landscape of infection affecting heart transplant patients has been shaped by different factors: (a) implementation of more selective calcineurin-based immunosuppressive protocols, (b) lessened immunosuppressive induction regimens, (c) the institution of antimicrobial prophylaxis resulting in a significant decrease or delay in the emergence of major infections episodes including p. jirovecii (pcp), nocardia spp., listeria spp., toxoplasma gondii, cytomegalovirus, toxoplasmosis, cytomegalovirus (cmv), herpes simplex virus (cmv), varicella zoster virus (vzv), and invasive fungal infections, (d) introduction of novel diagnostic technology facilitating earlier recognition and treatment of infections, (e) expansion in the criteria to select donors and recipients to include various scenarios dealing with hbv, hcv, and hiv infections [2] , and (f) shift toward predominantly grampositive bacterial infections and multiresistant bacteria in recent years [3] [4] [5] . a stanford team lead by dr. bruce reitz performed a lung transplantation as a combined heart-lung transplant procedure in 1981 [6] . shortly after, thoracic surgeons optimized the single-and double-lung transplant procedures. improvement of surgical techniques, especially bronchial anastomosis and evolution of flush perfusion lung preservation, decreased the perioperative bronchial complications substantially. similarly to heart transplantation, improvements in immunosuppressive regimens, antimicrobial prophylaxis, and graft preservation led to enhancement in survival among lung transplant recipients. in contrast to cardiac, lung transplantation has faced the challenge of infections unique to the transplant of this organ. mold infections of the anastomotic site, host versus graft disease, and serious infections with mycobacterium abscessus, chlamydia spp., bronchiolitis, and burkholderia cepacia complex are among infectious complications rarely observed in other transplant patients [7] . transplantation of thoracic organs has improved the quality of life and prevented the death of thousands of individuals worldwide. graft survival and life expectancy have been markedly improved in these patients due to the introduction of more optimal immunosuppression, antimicrobial prophylaxis, and diagnostic technology allowing the earlier diagnosis and treatment of infection and rejection. finally, further control of infection is likely to result from implementation of new approaches to assess the net state of immunosuppression in these patients. infection was recognized as a major threat to thoracic transplantation from the early inception days [8] . there are several factors predisposing thoracic transplant recipients to infections: (a) factors present before transplantation: age, presence of comorbidities (e.g., chronic kidney disease, diabetes mellitus, cancer, etc.), nutrition status, latent infections, colonization with healthcare-associated organisms, and occult community-acquired infections; (b) factors during the surgery: duration of the transplant procedure, graft injury including ischemic time, colonization or latent infection of the graft, surgical instrumentation (e.g., mechanical ventilation, invasive devices such as catheters, drains, foley catheters, etc.), icu stay, and need for re-interventions; and (c) factors present after transplant: degree of immunosuppression, cmv infection, and rejections ( a total of 4096 heart transplants were performed in 2011. heart transplant recipients have an average age of 54 years and are predominantly man (76%). they have a significant history of smoking (46%) and hypertension (45%) and have cardiomyopathy (54%) followed by coronary artery disease (37%) as the leading causes of transplant [1] . the historical (pediatric and adult transplants between 1982 and 2011) 1-year, 5-year, and 10-year survival rates are 81%, 69%, and 50%, respectively. overall median survival is 11 years, but it increases up to 13 years for those surviving the first year after transplantation. although not associated with increased posttransplant mortality, infections before transplant can affect up to 25% of heart transplant candidates. being bronchitis and soft tissue infections, the more commonly present [9] . despite no major changes in the distribution of causes of death since 1994, infections remained a predominant factor of mortality during the first 3 years after transplant. it contributes with up to almost 20% of causes of death [3] . the global incidence of infections in heart transplant ranges between 30% and 60% and the associated mortality between 4% and 15% [10] . the incidence of infection measured as major infectious episodes per patient has steadily declined from 2.83 in the early 1970s to 0.81 in the early 2000s [3, 8, 11] . the most frequent type of infection is bacterial (44%), followed by viral (42%), fungal including pneumocystis jirovecii (14%), and protozoa (0.6%). unfavorable functional outcomes are observed in patients who developed infections in the first year of transplant, mainly associated with bloodstream, cmv, and lung infections [12] . pulmonary and central nervous system (cns) infections are independent predictors of mortality among heart transplant recipients. reactivation of latent parasitic infections residing in extra-cardiac tissues in the host or transmitted in the transplanted heart is an important consideration. the classic example is the reactivation of trypanosoma cruzi. chagas disease is a vectorborne illness transmitted by triatomine bugs, and it is endemic in latin america. the ethnicity or origin of either the donor or the recipient from these regions should raise the concern for possible reactivation. chagas reactivation was documented in 38.8% of cases in a cohort of brazilian heart transplant recipients, where chagas cardiomyopathy was the second most common indication for transplant (34.9%) [13] . chagas can also reactivate from the transplanted heart procured from a seropositive donor and transplanted into a seronegative recipient. although with a substantial decreased on its prevalence in the most recent eras, toxoplasmosis is another important consideration in this setting. similarly to chagas, toxoplasma gondii-also with a predilection to invade the myocardium-can be transmitted by reactivation of quiescent cysts in the recipient or the transplanted heart [14] . by 2011, 3640 adults received lung transplantation, the highest reported number of procedures up to that date, driven mainly by the increase of double-lung transplants. doublelung transplant is indicated for septic lung diseases (e.g., cystic fibrosis). around 66% of recipients were aged 45-65 years old. the most frequent indications for transplant were copd (34%), followed by interstitial lung disease (ild) (24%), bronchiectasis associated with cystic fibrosis (cf) (17%), and α1at deficiency-related copd (6%) [15] . the overall (from 1994 to 2011) 1-year, 5-year, and 10-year survival rates among lung recipients are 79%, 53%, and 31%, respectively. overall median survival is 5.6 years. lung transplants from cmv seronegative donors have better survival rates than from cmv seropositive donor. thirty-day mortality was led by graft failure (24.7%) and non-cmv infections (19.6%). during the remainder of the year, non-cmv infections were the leading cause of death (35.6%). infection is still prominent as the cause of death following the first year of transplant after bronchiolitis obliterans syndrome (bos)/chronic lung rejection or graft failure [15] . other infections complications historically present among the ten primary causes of death within the first year include sepsis, pneumonia, and fungal infections [16] . high lung allocation score (las) at the time of transplantation is associated with a lower 1-year survival and higher rates of infections among lung transplant recipients [17] . sixty-three adult heart-lung transplantations were reported to the ishl registry in 2011. sixty-six percent of recipients were in the group range from 18 to 49 years old. sixty-three percent of the indications were for congenital heart disease and idiopathic pulmonary arterial hypertension. heartlung transplant for cf was higher in europe and other centers compared to north american. when compared to lung only transplants, short-term survival was worse, but long-term survival was better for the heart-lung transplant recipients. their 1-year, 5-year, and 10-year survival rates were 63%, 44%, and 31%, respectively. the median survival was 3.3 years and 10 years for those surviving the first year. similarly, they have graft failure (27%), technical complications (21.9%), and non-cmv infections (17.8%) as leading causes of death during the first 30 days posttransplant. non-cmv infections (35.1%) were the top cause of death after 1 month and within 1 year of transplant. after the first year, bos/late graft failure and non-cmv infections were the predominant causes of death [15] . among other risk factors for mortality in lung transplantation are cystic fibrosis, nosocomial infections, and mechanical ventilation before transplant [18] . infections in lung transplant recipients are predominantly bacterial (48%), viral (35%), fungal (13%), and mycobacterial (4%) [19] . in 60%, the infection site is pulmonary. risk factors for infection vary by the type of organism. mechanical ventilation (mv) for >5 days immediately following transplant surgery and isolation of staphylococcus aureus (sa) from airway cultures in the recipient were considered risk factors for invasive sa infections in a retrospective study of patients with lung and heart-lung transplants [20] . likewise, risk factors for the development of healthcare-associated infections with gram-negative organisms, aspergillus, legionella, and mrsa (methicillin-resistant staphylococcus aureus), include prolonging mv, renal failure, use of atg (antithymocyte globulin), and recurrent rejections episodes [21] . additionally, α-1-antitrypsin deficiency and repeat transplantation are also risk factors for nosocomial infections. mycobacterium tuberculosis transmission from lung donors with latent infection has been documented in highly endemic areas [22] . colonization with mdr organisms (pseudomonas aeruginosa, burkholderia, acinetobacter, nontuberculous mycobacteria (ntm), and scedosporium) before transplantespecially important in cf patients-can predict the development of challenging infections to treat after transplant [23] . patients should undergo a comprehensive evaluation of potential infectious complications associated with transplantation. a detailed medical history including previous vaccinations, history of past infections, exposures (geographical, occupational, animal, etc.), travel, and foreign-born status among others should be obtained. clinicians shuold perform routine serologies for the detection of pathogen-specific igg for cmv, hsv, ebv (vca), vzv, hepatitis b (hbsag, hbsab, hbcab), hiv, hepatitis c, and syphilis. toxoplasma igg should also be performed in heart and heart-lung transplant candidates. additionally, we recommend to obtain ua, urine culture, cxr, and tuberculin skin test (tst), or a quantiferon assay. in lung and heart-lung transplant candidates, sputum should be cultured for bacterial, fungal, and afb studies. some centers advocate the screening of patients for colonization with mdr (multidrug resistant) bacteria such as mrsa and vre (vancomycin resistant enterococci), which it may have an impact on the type of antibacterial prophylaxis used preoperatively or the empirical antibiotics should sepsis develop in the immediate postoperative period. in potential lung recipients, previous respiratory colonization with mdr pseudomonas, especially in cf patients, should not exclude them from transplant [24] . on the other hand, if colonization with b. cenocepacia (genomovar iii) in cf is present transplant is relatively contraindicated [25, 26] . checking for endemic fungi such as coccidioides immitis or for the parasites trypanosoma cruzi, strongyloides stercoralis, and leishmania spp. is indicated in the presence of the appropriate risk factors [27] [28] [29] [30] [31] . histoplasma capsulatum has reactivated during immunosuppressive therapy [32] . infections after solid organ transplantation (sot) are rare and attributable to transmission from the donor [33] . furthermore, latent histoplasmosis can be present with negative serologies and treatment after transplant carries a good outcome. therefore the role of screening for histoplasmosis is of questionable significance [34] . the type of evaluation may change if the donor is alive or deceased depending on the available time to collect the samples. similarly to recipients, donors should undertake a comprehensive assessment including a complete history, assessment of risk factors, exposures, immunizations, and previous or current infections. donors should be screened for hiv, hepatitis b/c, syphilis, and tuberculosis. furthermore, we recommend to obtain serologies for cmv, ebv, hsv, vzv, and toxoplasma gondii, and for htlv-1/ htlv-2 in endemic areas. in high-risk donors, the use of nucleic acid amplification tests (naat) for hbv, hcv, and hiv should be considered. additionally, blood cultures to document an occult bacteremia are recommended. in lung transplant donors, we recommend obtaining respiratory cultures through bronchoscopy to detect colonizing organisms and target them to prevent invasive infections in the donor. culturing the media of the allograft during acquisition or processing have been advocated to reduce the risk of mycotic aneurysms among kidney transplant recipients, which may apply to other sot [35] . screening of donors for endemic mycosis is not well established. on the other hand, heart transplant donors should be screened for chagas if the donor was born in latin america [29] . finally, it is important to highlight the increase recognition of emerging, unusual viral infections such as west nile virus, lymphocytic choriomeningitis virus, rabies, and different human coronaviruses [34, 36] . testing for those organisms should be done based on individual assessments. immunization should be optimized before transplantation since the recipient will have better chances to mount an adequate immune response [37] . the advisory committee on immunization practices (acip) [38] and the guidelines for immunizations in solid organ transplantation [39] recommend inactivated influenza vaccine annually. tetanus, diphtheria, and acellular pertussis (tdap) should be administered to all adults who have not previously received tdap or have an unknown status. varicella vaccination with two doses in patients without evidence of immunity or a single dose of zoster vaccination, inactivated polio vaccine, hepatitis a/b, hpv (three series through 26 years of age), and meningococcal and pneumococcal vaccines should be administered [38] . it is remarkably important to vaccinate all household members as well. bcg and rabies vaccines can be considered under some extenuating or exposure-related indications. see table 2 .3. education of the patient and the family members is a cornerstone to establishing effective preventive measures. emphasis should be enforced about hand hygiene and food handling. additionally, potential sources of bacteria, fungi (e.g., aspergillus), and toxoplasmosis such as plants and flowers, cleaning pet's litter or cages, eating uncooked meat, acquiring new pets, construction areas, farming, barnyard activities, and smoking marihuana should be avoided. if those recreational or occupational exposures are unavoidable; appropriate gear, such gloves, must be worn. education about possible community exposures is also important. close contacts with persons with fevers or rash potentially infected with vzv, herpes zoster, or influenza should be circumvented as well. patients should cook all meals thoroughly, wash all fruits and vegetables, and shun all unpasteurized products. safe sex practices are recommended. if any foreign travel is planned, seeking evaluation in a specialized travel clinic is advisable. guidelines for the management of surgical antimicrobial prophylaxis list cefazolin (2 g, 3 g for patients with weight >120 kg every 4 h) as the recommended regimen for heart, lung, and heartlung transplantation surgery. clindamycin (900 mg every 6 h) or vancomycin (15 mg/kg) can be substituted as alternative agents in beta-lactam allergic patients [40, 41] . this recommendation can be adjusted individually, based on local hospital surveillance data or previous knowledge of colonizing organisms (e.g., addition of aztreonam, gentamicin, or a single-quinolone dose). however, the widespread use of quinolones may increase the resurgence of antimicrobial resistance. the antibiotic should be administered within 60 min before surgical incision (within 120 min for vancomycin or quinolones) and to be continued for 24-48 h in heart transplants and 48-72 h and no longer than 7 days in lung and heart-lung transplant recipients. recommendation to continue antibacterial prophylaxis until chest and mediastinal tubes are removed lacks sufficient evidence. redosing will depend on the procedure duration and associated blood loss. the recipient does not need treatment if a localized infection was present in the donor, except during meningitis where concomitant bacteremia often coexist. in meningitis and bacteremia, it is prudent to treat the recipient for 2-4 weeks [34] . indications for antifungal prophylaxis in heart transplant recipients are not clear. a systemic review showed no benefit of antifungal therapy to prevent invasive fungal infections in transplants recipients other than liver [42] . although a prospective cohort of heart transplant recipients showed targeted prophylaxis-an echinocandin for a median of 30 days with the presence of at least one risk factor for invasive aspergillosis (ia) (reoperation, cytomegalovirus disease, posttransplantation hemodialysis, and another patient with ia in the program 2 months before or after the procedure)-was highly effective and safe in preventing ia episodes [43] , no consensus exists for universal antifungal prophylaxis in heart transplant recipients. most centers have adopted antifungal prophylaxis including inhaled amphotericin b, oral itraconazole, or iv targeted echinocandin prophylaxis. in lung and lung-heart transplant recipients, fungal prophylaxis should be considered, especially if pretransplantation respiratory cultures either from the donor lung or recipient airways shows aspergillus or candida. one approach is to use inhaled amphotericin b (50 or 100 mg in extubated or intubated patients, respectively) daily until 4 days after transplant and then weekly until hospital discharge in patients with no known colonization [44, 45] . if a mold has been isolated, voriconazole is recommended up to 4 months after transplant. although evidence and efficacy need to be confirmed, combination antifungal prophylaxis therapies is used at some centers [46] . pneumocystis jiroveci prophylaxis is done with trimethoprimsulfamethoxazole (tmp-smx) for 6 months, up to 1 year. some centers extend the pjp prophylaxis to lifelong. tmp-smx also confers protection against toxoplasma, nocardia, and listeria species infections. alternatively, dapsone, inhaled pentamidine, or atovaquone can be used in patients with a history of sulfa allergy. tmp-smx is recommended at many centers for lifelong in toxoplasmosis seronegative recipients of seropositive cardiac donors (toxoplasma d+/r−) [11] . cmv prevention is recommended to all d+/r− and r+ patients. there are two common strategies for cmv prevention: antiviral prophylaxis and preemptive therapy. both approaches possess similar success rate and their advantages and disadvantages [47] . guidelines recommend valganciclovir or intravenous ganciclovir as the preferred antivirals. oral ganciclovir is an option in heart transplant patients, although it possesses a low oral bioavailability and therefore the theoretical risk of increased resistance. often, cmv immune globulin is used as an adjunctive agent. in heart recipients, prophylaxis is recommended for 3-6 months in d+/r− and 3 months in r+. in lung and heart-lung recipients, the duration of prophylaxis is 12 months and 6-12 months in d+/r− and r+ recipients, respectively [48] . in d−/r− patients, otherwise not receiving cmv active agents, antiviral prophylaxis against other herpes viruses, such as hsv and vzv, should be considered. use of oral cmx001 (oral liposomal formulation of cidofovir) in hematopoietic-cell transplants reduced cmv-related events and may have a potential role in preventing cmv in other transplant settings [49] . refer to table 2 .4 for a list of prophylaxis recommendations. this period is characterized more commonly for nosocomial, bacterial infections. thus, the bacterial organisms present are often mdr (e.g., vre, mrsa). in heart transplant recipients, skin and soft tissue infections (ssti), surgical site infection, and mediastinitis are of concern during this period. likewise, lung and lung-heart transplant recipients may develop infections related to previous respiratory colonization (pseudomonas, aspergillus). other significant infections include aspiration pneumonitis, healthcare-and ventilatorassociated pneumonia, catheter-related bloodstream infections (crbsi), nosocomial utis, and clostridium difficile colitis. donor-derived infections during this period can be present and will include hsv, lymphocytic choriomeningitis virus (lcmv), rhabdovirus (rabies), west nile virus (wnv), and hiv. toxoplasma gondii and trypanosoma cruzi are also serious donor-derived infections in heart transplant recipients that can develop within the first 6 months posttransplantation [50] . during this period, reactivation of latent infections usually occurs. hence, bacterial infections such as those caused by nocardia asteroides, listeria monocytogenes, and mycobacteria tuberculosis typically occur. additionally, fungal infections by aspergillus spp., cryptococcus neoformans, and p. jiroveci and parasitic by toxoplasma gondii, leishmania spp., strongyloides, and trypanosoma cruzi can also be seen. viral infections present during this period include herpesviruses (hsv, vzv, cmv, and ebv) and adenovirus. development of infections after 6 months are predominantly community-acquired pneumonia and urinary tract infections. other diseases include aspergillus and mucor species, nocardia, rhodococcus, and late viral infections including cmv, hepatitis b and c, jc polyomavirus infection, posttransplant lymphoproliferative disorder (ptld), hsv encephalitis, and viral community-acquired infections (e.g., coronavirus, west nile virus, influenza). it is important to recognize transplant recipients as a patient population with increased susceptibility to infections and the antibiotic should be administered within 60 min before surgical incision (within 120 min for vancomycin or quinolones) and to be continued for 24-48 h in heart transplants and 48-72 h and no longer than 7 days in lung and heart-lung transplant recipients b doses of valganciclovir, ganciclovir, and other antibiotics may require adjustment for renal function have a low threshold to perform diagnostic workup in the presence of any concerning signs or symptoms. infections monitoring is also done in a structured way when preemptive therapy for cmv is in place (as opposed to universal prophylaxis). protocols vary by the transplant center but, usually, implies a weekly cmv pcr or pp65 ag monitoring [51] . likewise, monitoring of cell-mediated immunity (cmi) using a quantiferon-cmv assay may be useful predicting late-onset cmv disease once cmv prophylaxis has been stopped [52] . cmi also have been monitored for ebv using an enzyme-linked immunospot assay [53] . immunoglobulin g (igg), c3, igg2 levels, and nk cell counts have been proposed as an attempt to identify the risk of infection in heart transplant recipients within the first year [54] . significant drug-drug interactions exist among antimicrobial and immunosuppressive agents. patient medication list should be reviewed carefully. ctp3a4 strong inducers such as nafcillin reduce tacrolimus serum concentrations. in contrast, azoles such as fluconazole can result in increased levels of tacrolimus or cyclosporine. for voriconazole, the dose of tacrolimus needs to be reduced by two-thirds [55] and the cyclosporine dose by 50% [56] . rifamycins can have an opposite drug-drug interaction by decreasing the concentrations of prednisone, cyclosporine, tacrolimus, sirolimus, and mycophenolate mofetil (mmf) [57, 58] . likewise, tacrolimus administration along with quinolones may cause qt prolongation [59] . in heart transplant patients, bacterial infections have similar clinical manifestations commonly observed in other patient populations. however, clinical signs may be subtle or absent (e.g., afebrile). they are the most frequent type of infections in this setting, reaching up to 50% of all infections [3] . the most common are pulmonary infections followed by bacteremias, mediastinal, and skin infections. staphylococcus aureus-predominantly methicillin-resistant-can cause ssti, ventilator-associated pneumonia, mediastinitis, crbsi, other forms of bacteremia, and osteomyelitis. in contrast, coagulase-negative staphylococcus is more commonly associated with crbsi. among gram-negative bacteria, pseudomonas aeruginosa is common, usually of pulmonary origin. escherichia coli is the primary causal organism of utis. extended-spectrum β-lactamase (esbl)producing klebsiella pneumoniae, escherichia coli, klebsiella oxytoca, and citrobacter freundii are also found in 2.2% of heart transplant recipients [60] . nocardia species are well recognized as an opportunistic pathogen in this setting. although relatively rare in heart transplant recipients (frequency <1%), nocardia is only second in frequency in heart transplant after lung transplant recipients [61] [62] [63] . pertinent-independent risk factors associated with the development of this infection in sot include high-dose steroids, history of cmv disease, and high levels of calcineurin inhibitors [62] . with the almost universal prophylaxis with tmp-smx, nocardia infection is less common and often present late, usually after 1 year posttransplant [63] . when they occurred, they affect the lung predominantly, which is the port of entry for disseminated infections and cns invasion. also, it can cause skin nodules and abscesses. listeria monocytogenes can also be seen in heart transplant recipients and can count for a significant proportion of the bacterial meningitis cases in this setting [64] . additionally, myocarditis and myocardial abscesses with this organism have also been documented [65] . mycobacterium tuberculosis and nontuberculous mycobacteria (ntm), although, documented to occur in heart transplantation, are rare in the united states [66, 67] . however, it is important to recognize that the development of tuberculosis (tb) can be more prevalent in some endemic regions and often present with extrapulmonary involvement [68, 69] . legionellosis and rhodococcus equi with mainly pulmonary manifestations (pneumonia, pulmonary infiltrates, or cavitation) are another significant infections among heart transplant recipients [70] . fungal infections excluding pcp represent around 4.0% of all the infections. from them, invasive mold infections (imi) are a significant contribution to morbidity and mortality among heart transplant recipients. the incidence in this population can reach 10 per 1000 person-years, and its associated mortality is approximately 17% [71] . aspergillus represents up to 65% of all imi. its median time of onset is about 46 days, although late presentation (>90 days) has been more recently recognized associated with receipt of sirolimus in conjunction with tacrolimus for refractory rejection or cardiac allograft vasculopathy [72] . the most common clinical presentation for aspergillosis includes fever, cough, and single or multiple pulmonary nodules [73] . extrapulmonary manifestations include spondylodiscitis, infective endocarditis, mediastinitis, endophthalmitis, and brain and cutaneous abscesses [74] [75] [76] [77] [78] . dissemination tends to affect the cns in a good proportion of the cases. mucormycosis is the second most frequent mold affecting heart transplant recipients. mucor, along with other non-aspergillus molds (e.g., scedosporium, ochroconis gallopava), are associated with disseminated infections, cns involvement, and poorer outcomes [79, 80] . pneumocystis jiroveci (pcp)-although with a marked reduction in inci-dence with the introduction of universal prophylaxis-is still a significant pathogen and cases may occur late after heart transplant. cryptococcosis, although infrequent among sot patients, has its higher incidence in heart transplant recipients [81] . usually, its manifestations present late and affect the lungs and the cns predominantly. histoplasmosis and coccidioidomycosis occurred typically in the first year after transplant. antigenuria was the most sensitive diagnostic test in sot for histoplasmosis [82] . finally, candida infections are an important cause of morbidity and mortality as well. rate of colonization is higher than in the general population [83] . candida most commonly causes an oral mucosa infection. although there has been a decline of invasive infections over time, these do occur and typically in the form of bloodstream infections secondary to catheter-related infections, tracheobronchitis, or disseminated disease [84] . additionally, other confined end-organ injuries such as endophthalmitis and esophagitis can also be seen. cmv infection is of critical importance among sot. in heart transplant recipients, cmv has been inconsistently associated with cardiac allograft vasculopathy [85] . furthermore, cmv leads to upregulation of pro-inflammatory cytokines, increase procoagulant response, left ventricular dysfunction, allograft rejection, and an increase of opportunistic infections [86] . the greatest risk for developing cmv disease is cmv-negative recipients of cmvpositive organs (d+/r−), followed by d+/r+ and d−/r+. a clinical report estimated that the rate of infections in heart transplant ranges between 9% and 35%, and disease is present in around 25% of patients [87] . the clinical manifestations are not unique to heart transplant recipients and include a cmv syndrome (fevers, myalgias, arthralgias, malaise, leukopenia, and thrombocytopenia). cmvassociated end-organ injury in this setting includes most frequently pneumonitis and gastrointestinal disease [10] . other manifestations comprise myelosuppression, hepatitis, and pancreatitis. in contrast to the high frequency observed in aids patients, chorioretinitis in heart transplant patients is relatively rare [87] . guidelines on cmv diagnosis and managements are discussed in more detail in chap. 55 and also have been published elsewhere [88] . other herpes viruses are of important consideration as well. ebv-associated t-cell ptlds are more frequent in heart transplant recipients (0.4%) than in other sot patients [89] . ptld is a significant contributor to morbidity and mortality in the pediatric heart transplant population [90] . human t-lymphotropic virus type i (htlv1), human herpes virus (hhv)-6, hhv-7, and hhv-8 might play a role in ebv(−) t-cell ptlds as well. herpes viruses can manifest, as in other hosts, as mucocutaneous lesions for hsv, herpes zoster for vzv, infectious mononucleosis in the case of ebv, kaposi sarcoma for hhv-8, and encephalitis for hhv-6/7. hepatitis, colitis, pneumonitis, and gastrointestinal disease have also been attributed to dissemination with certain herpes viruses. herpes viruses can present with disseminated skin lesions (with or without vesicle formation) and fever of unknown origin. adenovirus has been associated with rejection, ventricular dysfunction, coronary vasculopathy, and the need for retransplantation. the current standard treatment for adenovirus is cidofovir, but outcomes are not optimal [91] . chronic hepatitis without an identifiable cause should prompt testing for hepatitis e virus (hev). chronic hev infection leads to the rapid development of fibrosis. hev testing should be done with rna pcr due to a delay in the antibody response. we recommend decreased immunosuppression and ribavirin therapy for 3 months [92, 93] . other less common manifestation that should be considered under the correct epidemiologic risk factors include htlv-1/ htlv-2-associated myelopathy, rabies, lymphocytic choriomeningitis virus, subacute measles encephalitis, mumps (associated parotitis, orchitis, vestibular neuritis, and allograft involvement), dengue virus, orf virus, human coronavirus, and influenza [36] . cardiac transplant itself is one the predictors for development of toxoplasmosis [94] . other associated risk factors include negative serum status before transplant, diagnosis of cytomegalovirus (cmv) infection, and high-dose prednisone. toxoplasmosis can be transmitted by the donor heart (d+/r−, especially during the first 3 months) or can reactivate from the recipient (>3 months). most of the infections developed during the first 6 months posttransplant and are predominantly primary infections. about 22% of infected patients had a disseminated infection carrying an estimated 17% mortality. toxoplasmosis can manifest otherwise with myocarditis, encephalitis, pneumonitis, or chorioretinitis. diagnosis requires identification of tissue cysts surrounded by an abnormal inflammatory response, detection of toxoplasma dna in body fluids by pcr, or positive toxoplasma-specific immunohistochemistry in affected organs. posttransplant serological tests are not helpful for diagnosis and may be misleading since results may change or not regardless of the presence of toxoplasmosis [95] . the preferred treatment regimen is a combination of pyrimethamine with sulfadiazine [96] . advanced chagasic cardiomyopathy is a primary indication for heart transplantation in some centers [13] . trypanosoma cruzi, the causal organism of chagas disease, can be transmitted up to 75% of the time from infected heart donors (d+/r−) [97] . additionally, chagas disease can reactivate from the donor once immunosuppression is in place (r+). the reactivation rate can range between 22% and 90% in recipients with chronic chagasic cardiomyopathy undergoing heart transplant [98] [99] [100] . additional risk factors for reactivation include rejection episodes, neoplasms, and use of mmf [98] . the mean onset of symptoms is approximately 112 days [101] . once manifested, chagas can present with nonspecific symptoms such as fever, malaise, anorexia, hepatosplenomegaly, and lymphadenopathy. myocarditis, pericarditis, and encephalitis are also seen. reactivation can mimic rejection and exhibits congestive heart failure, av block and skin manifestations such as nodules and panniculitis. increased eosinophil count and anemia can be indirect indicators of reactivation [102] . diagnosis is made with the visualization of circulating trypomastigotes in peripheral blood. additionally, blood and tissue pcr can be used. tissue amastigotes can be seen in biopsy h&e preparations (fig. 2.1) . finally, serologies are a crucial aspect in the diagnosis especially if seroconversion have been documented. in asymptomatic individuals, when the diagnosis of chagas has been established in the donor, monitoring should be instituted with weekly blood t. cruzi pcr and microscopy [29] . preferred antitrypanosomal therapy consists on benznidazole. nifurtimox is an alternative treatment option. posaconazole has anti-parasitic activity but carries high failure rates [103, 104] . gi disease with isospora (cystoisospora) belli, cryptosporidium, cyclospora, and microsporidia has been reported to affect sot recipients. microsporidiosis can manifest with disseminated disease: fever, keratoconjunctivitis, cns involvement, cholangitis, cough, and thoracic/ abdominal pain [94] . other rare parasitic infections affecting heart transplants include leishmaniasis, strongyloidiasis, and free-living amoebas [94, 105] . the rate of surgical site infections (ssi)-sternal wound infections-in patients receiving antimicrobial prophylaxis ranged from 5.8% to 8.8% following heart transplant procedures [41] . heart transplantation itself is an independent risk factor for ssis. other risk factors include age, prophylaxis with ciprofloxacin alone, positive wire cultures, female gender, previous left ventricular assist device (vad) placement, bmi >30 kg/m 2 , previous cardiac procedures, and inotropic support for hemodynamic instability [41, 106] . similarly to other hosts, staphylococcus species are the predominant organism causing sstis. mrsa can reach up to 21% of the cases. gram-positive organisms: vre (e. faecalis), coagulase-negative staphylococci, and other enterococcus species are other etiologic agents. candida and selected gram negatives such as enterobacteriaceae, p. aeruginosa, and stenotrophomonas maltophilia can cause ssis as well [107] . sternal osteomyelitis often complicates deep ssi. additionally, sternal wound infections by ntm and fungi such as aspergillus and scedosporium have been documented [108, 109] . herpes zoster is also an important consideration and source of morbidity. herpes zoster (hz) is found as a complication in 19-22% of the patients with a median time of presentation ranging from 0.73 to 2.10 years [64, 110] . close to half may develop postherpetic neuralgia. multi-dermatome involvement, zoster ophthalmicus, and meningoencephalitis are also described. exposure to mmf is an independent risk factor. conversely, cmv prophylaxis reduces the risk for hz. bloodstream infections (bsis) are a risk factor for mortality among heart transplant recipients. likewise, sot recipient status is an independent risk factor for developing bacteremia [111] . in heart transplant recipients; the rate of bsi ranged between 16% and 24%. the median onset is about 51-191 days, and the sources are in order of frequency: lower respiratory tract, urinary tract, and crbsi. gram-negative bacteria were more commonly isolated. they are in order of appearance e. coli, p. aeruginosa, and k. pneumoniae. more common grampositive bacteria were s. aureus, s. epidermidis, e. faecalis, and l. monocytogenes. directly attributable mortality is 12.2%. among the identifiable independent risk factors to develop bsi are hemodialysis, prolonged intensive care unit stay, and viral infections [112, 113] . infective endocarditis (ie) is seen more frequently among heart transplant recipients than in the general population. with ie occurred, it most commonly involves the mitral and tricuspid valves and staphylococcus aureus and aspergillus are the main etiologic organisms. the main predisposing factors in this setting are believed to be the frequent use of vascular indwelling catheters and the frequency of endomyocardial biopsies [114] . staphylococcus aureus bacteremia in heart transplant recipients ranges from 10% to 38% [11, 115] . the sources of sa bacteremia in sot are crbsi (30%), pneumonia (24%), wound (14%), endocarditis (10%), intra-abdominal infections (9%), bone and joint (7%), cardiac devices (3%), uti (1%), and ssti (1%) [115] . immediately following heart transplant and during the 1st month, patients are more susceptible to develop pneumonia, most of which are healthcare or ventilator associated and therefore caused by nosocomial organisms such as mrsa, pseudomonas aeruginosa, and other gram negatives including acinetobacter and esbl-enterobacteriaceas. pneumonia is one the major contributors to mortality in the early postoperative period. pneumonia-related mortality approaches 15% [116] . after the 1st month, interstitial pneumonia and pneumonitis can develop, and the differential includes herpesviruses (hsv, cmv, vzv) and respiratory syncytial virus (rsv), toxoplasma gondii and pneumocystis jiroveci. pulmonary nodules with or without cavitation can be caused by fungi such as coccidioidomycosis, aspergillosis, mucormycosis, cryptococcosis; bacterial including actinomycosis, tuberculosis, atypical mycobacterial infections, nocardia, rhodococcus equi, and gramnegative bacilli; and noninfectious causes like pulmonary infarction or lymphoproliferative disorders [117, 118] . pulmonary nodules are seen in about 10% of the patients, and the median detection time is about 66 days. the associated symptoms are fever and cough. the most frequent etiology is aspergillus followed by nocardia, and rhodococcus. cmv is an exceedingly rare cause of pulmonary nodules. the diagnostic approach with the higher yield is transthoracic fine needle aspiration followed by bronchoalveolar lavage and transtracheal aspiration [118] . communityacquired pneumonia caused by streptococcus pneumonia, legionella spp., mycoplasma, and influenza is another source of morbidity [10] . mediastinitis is a common complication in this setting. in patients receiving antimicrobial prophylaxis, mediastinitis develops in 3-7% of the patients [107, 119] . a ct scan is usually necessary to determine the extension of the infection. mrsa staphylococcus epidermidis, gram-negative bacteria, and aspergillus fumigatus are frequently found as the causal organisms [120] . antimicrobial therapy should be accompanied by aggressive surgical debridement [121] . there are not distinctive abdominal-pelvic complications among heart transplant recipients. clostridium difficile is a common hospital-related cause of diarrhea associated with the use of antimicrobials. other etiology for diarrhea second-ary to acute gastroenteritis can present in a protracted way in this setting. listeria infection can present as a febrile gastroenteritis illness as well. nontyphoid salmonella infection has been described to complicate the early postoperative period in a center in taiwan [122] . acute cholecystitis can affect heart transplant recipients advocating to have a low threshold to use ultrasound as a screening method [123] . acute pancreatitis with abscess formation has also been described [124] . as pointed above, hepatitis e can present with persistently abnormal liver tests. although less frequent than in kidney transplant recipients, urinary tract infections are an important cause of morbidity. utis are predisposed by foley catheters. the organisms most commonly involved are gram-negative bacteria, enterococcus, and candida. polyomavirus nephropathy by bk virus has been described in heart transplant recipients and might be a contributor to chronic kidney disease [125] . the need for urgent transplantation and multiple transfusions are independently associated with infectious, neurologic complications. its overall mortality can reach 12% [64] . donor-derived meningoencephalitides affecting heart transplant recipients usually manifest within the first 30 days. these infections include west nile virus, arenaviruses (e.g., lcmv), and rabies. wnv can manifest with a guillain-barré-like axonopathy with cerebrospinal fluid (csf) pleocytosis. in addition to meningitis or encephalitis, ataxia, myelitis, optic neuritis, polyradiculitis, and seizures can also be observed [126] . wnv can be also acquired by the recipient in the community or through blood transfusions and present at a later time [127] . other infectious forms of meningitis and encephalitis that can present after the 1st month include listeriosis, streptococcus pneumoniae, trypanosoma cruzi, toxoplasma, hhv-6, and disseminated herpes virus infections (cmv, vzv, hsv, and ebv) [128] [129] [130] . the absence of appropriate primary prophylaxis or monitoring increases their risk. aspergillus causes the majority of brain abscess. additionally toxoplasma, tuberculosis, listeria spp., cryptococcus neoformans, scedosporium spp., and nocardia can also be causative agents [129] . concomitant pulmonary involvement is common, particularly for those whose portal of entry is the respiratory tract. progressive multifocal leukoencephalopathy (pml), a demyelinating disease caused by the reactivation of jc virus, has a usual median onset of 27 months. it carries a marked high case fatality rate and a median survival of 6.4 months in sot [131] . the use of rituximab as an antirejection treatment seems to confer an increased risk for pml [132] . htlv-1-associated myelopathy (ham) has been described as well in sot. bacterial infections are the most common type of infections among lung and lung-heart transplant recipients. the anatomic site most frequently affected is the respiratory tract, usually manifested with pneumonia, sinusitis, or tracheobronchitis. previous colonization, healthcare associated, and procedures related are the primary sources. for patients with cystic fibrosis (cf), knowledge of previous colonization results may provide some diagnostic and therapeutic advantages. pseudomonas aeruginosa is a predominant colonizing pathogen in cf. however, acinetobacter baumannii, burkholderia species, stenotrophomonas maltophilia, achromobacter xylosoxidans, ntm, pandorea, and ralstonia are also observed [23] . furthermore, pathogens that are known to cause nosocomial pneumonia during the 1st month include staphylococcus aureus, pseudomonas aeruginosa, other gram negatives (klebsiella pneumoniae, enterobacter cloacae, serratia marcescens, escherichia coli, acinetobacter species), and anaerobes. gram-positive bacteria are a common source of infections making up to 40% of them [133] . the most common sites affected were the respiratory tract, followed by bacteremia, skin, wound, and catheter related. the pathogens more frequently identified are staphylococcus species (77%), enterococcus species (12%), streptococcus species (6%), pneumococcus (4%), and eubacterium lentum (1%). staphylococcus aureus infection can develop up to 20% of lung recipients. sa commonly causes pneumonia, followed by tracheobronchitis, bacteremia, intrathoracic infections, and sstis [20] . streptococcus pneumoniae is community acquired and present with pneumonia, usually after 6 months posttransplant. pseudomonas aeruginosa has high rates of colonization (up to 40%) and disease (30%) [134] . other significant bacterial infections that may present after the 1st month are mycobacterium tuberculosis, ntm, nocardia, rhodococcus, and legionella. isolation of ntm in lung transplant recipients without evidence of disease is not associated with increased mortality [135] . nocardiosis can occur in about 2% of the lung transplant recipients. the median time of onset ranges from 14.3 to 34.1 months [136, 137] . nocardia asteroides, n. farcinica, n. nova, and n. brasiliensis have been reported. n. farcinica appears to carry worse outcomes. this infection can present as a breakthrough in the presence of trimethoprim-sulfamethoxazole for p. jiroveci prophylaxis, although the isolates may remain susceptible. mortality has been reported to range between 18% and 40%. the native lung is more frequently affected in single-lung transplant recipients. nodules are the more prevalent radio-graphic finding. extrapulmonary involvement affecting the skin and brain can be seen. hypogammaglobulinemia and neutropenia seem to confer additional risk factors for nocardiosis in this setting [137] . fungal infections are frequent complications in lung and lung-heart transplant. they present in about 15-35% and carry an overall mortality close to 60% [138] . aspergillus and candida are the most frequent causative agents. other important fungi include cryptococcus spp., mucormycosis, endemic fungi (histoplasma, coccidioides, and blastomyces spp.), scedosporium spp., fusarium spp., and dematiaceous molds. candida infections are prominent during the 1st month after transplantation. it can be one of the most common causes of bsi in this setting [139] . although colonization of the upper airways and gastrointestinal tract is common, candida additionally can cause mucocutaneous disease, tracheobronchitis, anastomosis site infections, crbsi, and disseminated disease. aspergillus spp. lead as the cause of invasive fungal infections. its attack rate of infection is almost ten times compared to that in other sot patients (estimated incidence of 6% among lung transplant recipients) [140, 141] . a. fumigatus is the most common species, but a. terreus, a. flavus, and a. niger have been described as well. the main predisposing risk factors in this setting are intense immunosuppression, previous colonization with aspergillus spp., airway ischemia, and bos. single-lung transplant possesses the greatest risk to developing an invasive aspergillus infection carrying a higher mortality than double-lung and heart-lung transplant recipients. single-lung recipients are usually older and more likely to have copd as the indication for transplantation [140] . aspergillus infections can present as tracheobronchitis, pneumonia, or disseminated disease. extrapulmonary involvement includes sinusitis, cns or orbits infections, and vertebral osteomyelitis. aids in the diagnosis can include surveillance bronchoscopies (bronchoalveolar lavage stain and culture; biopsy), chest ct and serum/bal galactomannan, beta-d-glucan, and pcr. the presence of pulmonary nodular lesions in invasive infections can carry better outcomes [142] . voriconazole is the treatment of choice. it is important to note that immune reconstitution inflammatory syndrome (iris) can develop at a median of 56 days in 7% of treated lung transplant recipients [143] . in aspergillus tracheobronchitis, nebulized amphotericin b and debridement of the bronchial anastomosis are important adjuvant measures to systemic antifungal therapy [144, 145] . pneumocystis jirovecii pneumonia manifests from 1 to 6 months. its incidence has been reduced dramatically with universal tmp/smx prophylaxis. cryptococcosis with a rate of 2% in lung transplant recipients presents with pulmonary involvement, but dissemination with meningitis can occur. furthermore, cryptococcus skin manifestations like cellulitis and cryptococcus-associated iris have been documented [146, 147] . viral infections are a common cause of morbidity among lung transplant recipients. the most common viruses are (1) cmv among the herpes viruses and (2) community-acquired respiratory viruses. as in other sot recipients, the higher risk to develop cmv infection is among d+/r−, followed by d+/r+, d−/r+, and d−/r−. this last scenario carries less than 5% of risk [48, 148] . lung transplant recipients possess higher risk for cmv than other sot with an estimated incidence of 30-86% [87] . the lung is considered a primary reservoir for cmv latency, and abundant lymphocytic tissue surrounds the transplanted organ. additionally, the use of antilymphocyte antibodies to treat rejection or for immunosuppression and other herpesviruses infections are additional risk factors for cmv disease [149] . interferon (ifn)-γ (+874t/t) polymorphism increases ifn levels and may be a predisposition for cmv disease [150] . cmv is significantly associated with bos, which reduces survival after the first year posttransplant [151] . cmv disease is most commonly manifested by pneumonitis or viral syndrome and less frequently with gastrointestinal disease. among lung transplant recipients, ganciclovir-resistant cmv carries an increased morbidity and mortality [152] . infections with community-acquired respiratory viruses ranged from 7.7% to 64%. these infections are associated with increased risk to develop pneumonia, graft dysfunction manifested by lung function loss, bos, high calcineurin inhibitor blood levels, and increase mortality [153] [154] [155] . these viruses include influenza, parainfluenza, respiratory syncytial virus (rsv), coronaviruses, human rhinovirus, adenovirus, human metapneumoviruses, and bocaviruses. the hospitalization rates are higher for influenza and parainfluenza (50% and 17%, respectively) [154] . symptoms are usually nonspecific. diagnosis often requires detection of viral nucleoprotein antigens in nasopharyngeal swabs or bronchoalveolar lavage (bal) by enzyme immunoassay or fluorescent antibody or the amplification of nucleic acid by pcr. ribavirin may possess activity against paramyxoviruses (rsv, metapneumovirus, and parainfluenza). ribavirin is administered inhaled, orally, or intravenously. oseltamivir or zanamivir is the treatment choice of influenza a or b [156] . adamantanes (amantadine and rimantadine) are not active against influenza b, and there is a marked increase resistance among influenza a strains [156] . similarly to other sot recipients, dna viruses like non-cmv herpesviruses (hsv-1,-2), vzv, hhv-6,-7,-8, and ebv are a source of significant morbidity including but not limited to cmv-negative viral syndrome, rash, pneumonitis, hepatitis, and encephalitis [157] . lastly, polyomavirus such as bk virus (bkv), jc virus (jcv), and simian virus 40 (sv40)-although fre-quently encountered in lung transplant recipients with an unclear causality-may cause worsening renal function or survival [158] . ptld is also a well-recognized complication. a trend toward late ptld presentation (>1 year) has been documented where b symptoms are more predominant as well as extra-graft involvement [159] . as other immunosuppressive states, certain parasitic infections can complicate lung and heart-lung transplants recipients. it is critical to elicit a detailed history and geographic risk factors to determine the risk of acquisition and the potential etiologic agent. toxoplasmosis can result from primary infection or reactivation of previous latent infections. toxoplasmosis can develop in patients with negative epidemiological history for cat ownership or consumption of undercooked meat. in patients with primary toxoplasmosis, nonspecific symptoms such as fever, lymphadenopathy, or organ injury may be present. reactivation can cause encephalitis with or without space-occupying brain lesions, seizures, chorioretinitis, fever of unknown origin, pneumonitis, myocarditis, and rash. although cases of the lung fluke, paragonimus westermani have not been reported in lung transplantation, it can be a potential threat in endemic areas where this organism is endemic. other parasites that can target the lung in immunosuppressive states include echinococcus, schistosoma, and strongyloides stercoralis [160] . strongyloidiasis can present as hyperinfection syndrome [161] . leishmania, although infrequently seen, has been reported among lung and lung-heart recipients [30] . free-living amoebas can affect this population as well. amoebic granulomatous dermatitis and disseminated infection presenting with ulcerative skin lesions, respiratory failure, and seizures have been described in lung transplant recipients [162, 163] . finally, alimentary protozoa, including cryptosporidium, which present with diarrhea and may elevate tacrolimus levels [164] , and microsporidia, which present with unusual manifestations like myositis or granulomatous interstitial nephritis, affects lung transplant recipients [165, 166] . the overall rate of ssis is about 13% with a significant proportion of infections being organ or space occupying (72%), deep incisional (17%), and superficial (10%) [18, 41] . independent risk factors to develop ssi are diabetes, female donor, prolonged ischemic time, and the number of red blood cells transfusion during the perioperative period [167] . ssis are associated with a 35% mortality within the first year of transplantation. the most common organisms found to cause ssi or mediastinitis are p. aeruginosa, candida species, s. aureus (including mrsa), enterococcus, coagulasenegative staphylococci, burkholderia cepacia, e. coli, proteus mirabilis, serratia marcescens, acinetobacter baumannii, enterobacter cloacae, and klebsiella species. there is a correlation in up to 33% of the patients' ssi causative organisms with previous pathogens colonizing recipients' native lungs at the time of the transplant [167] . the median onset is 25 days after lung transplant [167] . although rare, ntm can cause ssi infections among lung transplant recipients. the most frequently encountered are mycobacterium avium complex followed by mycobacterium abscessus and mycobacterium gordonae. ntm ssi infections can be complicated by progressive disseminated disease or requirement of lifelong suppressive therapy [135] . other organisms such as mycoplasma hominis and lactobacillus spp. have also been described. deep infections can affect up to 5% of the patients. sternal osteomyelitis can reach up to 6% of these deep infections. causative organisms for sternal osteomyelitis include pseudomonas aeruginosa, serratia marcescens, and scedosporium. non-sternal osteomyelitis affecting the calcaneus bone has complicated a disseminated infection with aspergillus fumigatus [168] . bloodstream infections (bsis) occur with an estimated rate of 25% among lung transplant recipients. a major proportion of bsis occur in the early posttransplant period. bsis infections are significantly associated with worse survival [139, 169] . the most common organisms encountered are staphylococcus aureus, pseudomonas aeruginosa, and candida [139] . pseudomonas aeruginosa bsi-predominantly present during the transplant hospitalization period and more commonly affecting cf patients-is followed in frequency by burkholderia cepacia and candida albicans. conversely, staphylococcus aureus was the predominant organism after transplantation discharge. in an estimated 70% of bsi, the source was pulmonary, followed in frequency by crbsi, gastrointestinal infection, peritonitis, and uti. a pulmonary source of bacteremia in sot often develops into septic shock [170] . although unusual, cases of aspergillus fumigatus endocarditis have been described following lung transplantation [171] . often patients had cf as the underlying lung disease and a median of 8 ± 6 months presentation. this complication carries a high mortality and often requires a combination of antifungal therapy with valvular replacement surgery. infectious complications related to the chest cavity include mediastinitis, cardiac (pericarditis and myocarditis), lung parenchyma infections (nodular infiltrates, cavitation, or pneumonia), bronchial anastomosis infections, and pleural space infections (bronchopleural fistula and empyema). empyema followed by mediastinitis and pericarditis, in addition to surgical wound infections and sternal osteomyelitis, is the most frequent deep ssi complications affecting the chest cavity. empyema presents in around of 3.6% of cases. it occurs during the first 6 months after transplantation (median 46 ± 39 days) carrying an estimated mortality of 28.6% [172] . most common organisms found are staphylococcus spp., e. coli, enterobacter spp., klebsiella spp., mycoplasma hominis, vre, and candida. furthermore, mycobacterium abscessus was isolated as a rare causative agent of empyema as well [173] . the degree of immunosuppression, reduced renal function, previous sternotomy, and re-exploration due to bleeding are listed as potential risk factors for mediastinitis [119] . there is an increased prevalence of mediastinitis caused by gram negatives and fungi among lung transplant recipients. causative organisms for mediastinitis are similar to ssi and are listed above. infectious pericarditis can be present up to 6% of the patients (isolated organisms include mssa, mycoplasma hominis, and scedosporium prolificans) [167, 174, 175] . due to their high fatal rate, fungal bronchial anastomotic infections are critical to recognize. pneumonia is believed to affect around 21% of lung recipients and 40% of heart-lung recipients. nosocomial organisms cause early pneumonia as in other posttransplant settings. the donor's lung seems to be the primary source for pneumonic infections, although the recipients' upper airways or sinuses are also potential sources. preoperative colonization with gram-negative rods and colonized infected donor bronchus or perfusate are recognized risk factors for pneumonia. likewise, pretransplantation colonizing microorganisms from suppurative lung disease are associated with pneumonia development posttransplant [176] . the most common causal organisms are pseudomonas aeruginosa, staphylococcus aureus, and aspergillus spp. other pathogens include bacteria such as b. cepacia, enterobacter species, s. maltophilia, klebsiella species, s. epidermidis, and e. coli, and fungi such as fusarium spp., cryptococcus neoformans, and paracoccidioides brasiliensis [176] . after the 1st month, pneumonia can present as local infiltrates, diffuse interstitial infiltrates, and nodules with or without cavitation. this type of presentation may aid in the possible causative microorganism. the list of potential pathogens is extensive and includes in addition to the already mentioned nocardia, chlamydia pneumonia, legionella, tb, ntm, pneumocystis jirovecii, rhodococcus, herpesviruses (cmv, hsv, and vzv), respiratory viruses, endemic fungi (e.g., histoplasmosis), mucormycosis, and scedosporium spp. [177] [178] [179] . similarly to other sot, common infectious complications affecting the gastrointestinal or genitourinary tract include clostridium difficile colitis and utis. intra-abdominal com-plication carries an overall increase mortality [180] . frequent gi symptoms presenting posttransplant are diarrhea which can affect almost 30% of lung transplant recipients and abdominal pain. abdominal pain should prompt further investigation for potential intra-abdominal causes. in the pediatric population, the possibility of ptld should be investigated since it carries a high mortality [181] . other described infectious intra-abdominal complications include digestive perforation (seen in 6%) [182] , retroperitoneal abscesses, cholecystitis, perianal abscesses, esophagitis, pancreatitis, pancreatic abscesses, hepatitis, diverticulitis, appendicitis, cmv colitis, megacolon, and colon rupture [180, 183, 184] . in developing countries, persistently abnormal liver enzymes should prompt testing for hev. hev rna should be used for screening. oral ribavirin seems to be safe and effective in this setting [185] . cns symptoms developing during the 1st month following lung or heart-lung transplantation should trigger the concern for donor-derived viral infections. lcmv often is accompanied by csf normal to low glucose, marked elevated protein, and mild pleocytosis [36] . although with unclear benefit, ribavirin has been used. donor-transmitted rabies is an uncommon but neurologic devastating complication that occurs within the first 30 days of transplant. lung transplantation has been described as a potential causal mechanism [186] . other organisms known to cause meningitis in lung transplant recipients are cryptococcus, tuberculosis, wnv, and herpesviruses [187, 188] . diagnosis of wnv in this setting requires nuclear acid amplification due to the unreliability of serologic testing. scedosporium apiospermum infections often cause dissemination including cns abscesses in addition to pulmonary involvement among lung transplant recipients [189] . it is important to differentiate from other molds, since amphotericin b is ineffective against scedosporium spp. in severe cases or refractory disease without an appropriate surgical debridement, the addition of terbinafine to voriconazole may prove to be useful [190] . other recognized organisms causing occupying brain lesions are fusarium, nocardia, aspergillus, toxoplasmosis, cryptococcus neoformans, listeria, and cladophialophora bantiana [191] [192] 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10 cases reveals an association with elevated tacrolimus concentrations myositis due to the microsporidian anncaliia (brachiola) algerae in a lung transplant recipient key diagnostic features of granulomatous interstitial nephritis due to encephalitozoon cuniculi in a lung transplant recipient epidemiology and outcomes of deep surgical site infections following lung transplantation aspergillus fumigatus empyema, arthritis, and calcaneal osteomyelitis in a lung transplant patient successfully treated with posaconazole epidemiology of bloodstream infections in the first year after pediatric lung transplantation bacteremia and septic shock after solid-organ transplantation aspergillus endocarditis in lung transplant recipients: case report and literature review empyema complicating successful lung transplantation mycobacterium abscessus empyema in a lung transplant recipient scedosporium prolificans pericarditis and mycotic aortic aneurysm in a lung transplant recipient receiving 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multiple corneal and solid organ transplantations from a donor infected with rabies virus tuberculous meningitis in a lung transplanted patient impact of rituximab-associated b-cell defects on west nile virus meningoencephalitis in solid organ transplant recipients scedosporium apiospermum (pseudoallescheria boydii) infection in lung transplant recipients combination antifungal therapy in the treatment of scedosporium apiospermum central nervous system infections cladophialophora bantiana brain abscess in a solid-organ transplant recipient: case report and review of the literature disseminated fusarium infection with brain abscesses in a lung transplant recipient central nervous system infections in heart and heart-lung transplant recipients no funding agencies had any role in the preparation, review, or approval of this paper. the views expressed in this paper are those of the authors and do not necessarily represent the views of the university of colorado denver or stanford university. no conflict of interests was reported by andrés f. henao-martínez and josé g. montoya. key: cord-009860-qebenhxz authors: falsey, ann r.; treanor, john j.; betts, robert f.; walsh, edward e. title: viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings date: 2015-04-27 journal: j am geriatr soc doi: 10.1111/j.1532-5415.1992.tb01929.x sha: doc_id: 9860 cord_uid: qebenhxz objective: to prospectively evaluate the incidence and impact of viral respiratory infection in the institutionalized elderly during a winter season. design: prospective descriptive study, without intervention. method: patients with respiratory illnesses were evaluated by a directed history and physical examination. nasopharyngeal secretions for viral culture were obtained, and acute and convalescent serum samples were obtained for analysis. serologic evidence of infection with respiratory syncytial virus (rsv) and parainfluenza were determined by enzyme immunoassay (eia), and influenza by hemagglutination‐inhibition assay and eia. setting: a 591‐bed nursing home. participants: residents with signs or symptoms of acute respiratory illness (nasal congestion, pharyngitis, cough, wheezing, or respiratory difficulty) were eligible for study. results: a viral etiology was documented in 62 out of 149 illnesses (42%). rsv was the most common virus associated with illness; it was documented in 27% of respiratory illnesses, followed by rhinovirus (9%), parainfluenza (6%), and influenza (1%). rsv was associated with significantly more severe disease when compared with rhinovirus. clustering of specific viral infections occurred, suggesting nosocomial transmission. conclusions: viruses are an important cause of acute respiratory infections in the institutionalized elderly during the winter months. cute respiratory tract infections are a major cause of acute morbidity in the united states. the a frequency of respiratory infections is high in young children and appears to decline with advancing age.' however, the impact on the elderly host may be of greater consequence due to an aging respiratory tract, a declining immune system, and multiple medical problems.' residents of long-term-care facilities represent a debilitated subgroup of the elderly population and thus are particularly prone to excess morbidity with respiratory infections. however, with the exception of influenza, comprehensive studies on viral respiratory infections in the institutionalized elderly are lacking. this is despite the fact that each year many residents of nursing homes become ill with respiratory infections not proven to be influenza, either by culture or serology. the purpose of this study was to identify all common respiratory viruses which cause symptomatic dsease during the winter months in nursing home patients, by utilizing both viral cultures and serology, and to assess their clinical impact. respiratory illness (nasal congestion, pharyngitis, cough, wheezing, or respiratory difficulty) were eligible for study. results: awiral &ology was documented in 62 out of 149 illnesses (42%). rsv was the most common virus associated with illness; it was documented in 27% of respiratory illnesses, followed by rhinovirus (9 %) , parainfluenza (6%), and influenza (1 %). rsv was associated with significantly more severe disease when compared with rhinovirus. clustering of specific viral infections occurred, suggesting nosocomial transmission. conclusions: viruses are an important cause of acute respiratory infections in the institutionalized elderly during the winter months. j am geriatr soc 40115-119,1992 health-related facility for more independent residents. each floor of the skilled nursing facility had a separate dining area, whereas residents of the health-related facility ate in a single large dining hall. medical care for residents was provided by three full-time staff physicians. infection control policies at the facility included quarantine of floors with 2 3 documented cases of influenza and amantadine prophylaxis for 2 weeks for residents of such floors. no specific isolation procedures were implemented for non-influenza respiratory illnesses. no attempt was made to influence either individual patient care or existing infection control policy during this study. subjects between december 11, 1989 and march 13, 1990 , the head nurse on each floor identified residents who had signs or symptoms of acute respiratory illness including nasal congestion, pharyngitis, cough, wheezing, or respiratory difficulty with or without fever. an institutionwide illness log was created and reviewed by a study nurse daily from monday through friday. each subject listed in the illness log was then evaluated by a directed history and physical examination. a global seventy of illness score was assigned by the study nurse, using an analog scoring system rangng from 0 to the analog score consisted of a horizontal line with one end representing mildest illness and the other end the most severely ill. this score represented the study nurses' overall assessment of the severity of an illness based on both the patient's symptoms and objective findings. in a separate analysis the analog scores were found to have a high correlation with objective indices of disease (falsey, j med virol, in press). arterial oxygen saturation (sa02) was measured percutaneously, and a nasopharyngeal swab for virus culture and serum were obtained on the initial visit. chest roentgenograms were ._ -obtained and antimicrobials prescribed at the discretion of the patient's staff physician. each patient was reevaluated daily for 3 days and convalescent sera obtained 4-8 weeks after the acute illness. viral cultures nasopharyngeal secretions were inoculated onto cell culture within 4 hours. each sample was placed on hep-2, madin-darby canine kidney (mdck), and human foreskin fibroblast (hff) cell lines. all terminal hep-2 cultures were examined for rsv by a direct immunofluorescent antibody test (genetic systems, seattle, wa). any cell line demonstrating cpe was examined by ifa for parainfluenza virus (piv) 1,2,3, adenovirus, influenza a and 8, and herpes simplex virus (hsv). rsv isolates were subgrouped a or b with group specific monoclonal antibodies.4 rhinovirus isolates were confirmed by acid lability testing. paired sera were analyzed for evidence of recent infection with rsv, influenza, and parainfluenza viruses. serum igg levels to the fusion (f) and attachment (g) glycoproteins of a and b rsv subgroups (designated ga and gb) were determined for all sera by enzyme immunoassay (eia) as previously r e p~r t e d .~ serologic evidence of rsv infection was defined as a ?$-fold rise in igg to any of the rsv antigens. sera was also tested for evidence of influence infection by hemaglutination-inhibition assay (hai) and hemagglutinin-specific eia.6 serum igg levels to parainfluenza virus, types 2 (piv-2) and 3 (piv-3), were determined by enzyme immunoassay using virus infected vero cells as antigen. briefly, vero cells in 96-well microtiter plates were infected with piv-2 or piv-3 (wild isolates) and when cpe was detectable, the cells were fixed in 80% acetone/pbs. paired sera were incubated in duplicate serial 2-fold dilutions in the wells for 1 hour at 37oc. plates were washed and then incubated with horseradish peroxidase conjugated goat anti-human igg for 1 hour. substrate was added and optical density was read on a dynatech spectrophotometer. control wells with uninfected vero cells were used to define background absorption which was substrated from the values of the antigen plates. because heterologous titer rises between piv types 1 and 3 are common and serologic differentiation of infecting type is not possible, specimens were screened using piv-2 and piv-3 antigens. evidence of parainfluenza infection was defined as a 24-fold nse in igg to either piv antigen. during the 3-month study period, 177 respiratory illnesses were identified in 164 residents. of these, 133 residents who experienced 149 illnesses agreed to be studied. eighty-four percent of the study patients were female, and the mean age was 88. most had underlying medical conditions, with neurologic (77%), cardiac (47%), pulmonary (25%), diabetes (12%), and malignancy (7%) being the most frequent. cultures and serology forty-one viruses were isolated during 39 of the 149 illnesses (26%) ( table 1) . rsv was isolated from 18 patients, followed by rhinovirus in 14, herpes simplex (hsv) in six, parainflu-enza-1 in two, and parainfluenza-2 in a single patient. influenza virus was not isolated. two patients had dual infections (hsv/rsv and hsv/rhinovirus). rsv group could be determined for 15 of the 18 rsv isolates. nine of these were group b, and six were group a. one-hundred-six of the 133 (80%) had acute and convalescent sera available for analysis. thirty-three (31 %) showed serologic evidence of rsv infection. forty rsv infections were documented either by culture alone (5), serology alone (22), or both (13). fifteen of the 18 individuals from whom rsv was isolated had acute and convalescent sera available. of these, 13 showed 14-fold rises in rsv titers. the other two patients had acute titers of igg greater than the highest dilution and, therefore, rises could not be detected. in addition, six patients (5.7%) demonstrated 24-fold rises in piv titers (3 to piv-2 and 3 to piv-1). two patients had a 24-fold rise in ha1 titers to influenza which was confirmed by ha-specific eia. overall, a viral etiology of the respiratory infection was documented by culture or serology in 62 of 149 illnesses (42%). epidemiology respiratory illnesses were reported throughout the study period from 12/11/89 to 3/13/ 90, with the majority reported in january (figure 1) . illnesses were noted on all nine floors of the skilled nursing facility and on 18 of 19 floors in the healthrelated facility. rsv was isolated throughout most of the study period with a peak occurrence in mid-january. although rsv infections were geographically scattered throughout the institution, eight and 14 cases occurred on the 7th and 8th floors of the skilled nursing facility, respectively. on the 8th floor, all five of the typeable rsv isolates were group b, while on the 7th floor four of five typeable isolates were group a. it was also noted that in three of the four wings on these two floors, cases of rsv tended to occur in patients residing on one side of the hallway. rhinovirus infections also occurred throughout the study period, although nine of the 14 cases occurred during a 4-day period in december. five of the 14 cases also occurred on the 8th floor, with the remainder scattered widely throughout the institution. all six cases of piv infection occurred during late december or early january. the three piv-1 infections occurred within a 5-day period in patients who lived on the same floor in adjacent rooms. clinical syndromes the clinical features of illness were compared in patients with the two most frequently documented viral infections, rsv and rhinovirus ( table 2 ). the spectrum of illness associated with rsv infection was broad, ranging from mild nasal congestion only to high fever and respiratory distress. the most prominent complaint was nasal congestion (92%), followed by cough (90%) and sputum production (60%). appearance on physical examination was also variable with analog scores ranging from 1 to 7 (mean analog score 3.7 -t-1.5). fifty-four percent had a score of 1 4 , suggesting moderately severe disease. nine had oral temperatures greater than 101of. sixtyfive percent of patients had abnormal lung findings including rales in 4096, wheezing in 35%, and rhonchi in 13%. the mean room air saoz was 95%, and two patients required supplemental oxygen during the acute illness. twelve patients had chest roentgenograms, four of whom had an infiltrate. antibiotics were prescribed in 49% of rsv-infected patients. one patient was hospitalized with pneumonia, and two others died, one during the acute rsv infection, the other 1 month later after a steady decline in health and with an ill-defined pulmonary process. most patients recovered without sequelae, although the functional status of two patients deteriorated after their illness. in contrast, illness associated with rhinovirus infection was generally mild. rhinorrhea was the most frequent complaint (79%). cough was also a prominent symptom (71%), although less common than in the rsv-infected group (90%). sputum production was significantly less frequent with rhinovirus infection compared to rsv (21% vs 60%, p = 0.05 by chi square). on physical examination patients generally did not appear acutely ill. only one person had an analog score >3, and the mean analog score was 1.6 -i-1.5 which was significantly lower than the mean illness score in rsv infection (p < .001 by t test). the most prominent sign was nasal discharge (28%). only two patients (14%) received antibiotics, and all recovered without sequelae. piv infection was documented in six individuals, three with piv-i, and three with piv-2. although the numbers are too small to permit comparison of clinical syndromes to other infecting agents, each type of piv infection appeared to produce a distinct illness. the three individuals with piv-1 had a mean analog score of 3.7. all complained of cough and constitutional symptoms. fever and abnormal chest exams were present in two of three patients, and all three received antibiotics during their illness. piv-2 illnesses were somewhat milder, with a mean analog score of 2.0. all three complained of sore throat, and two were hoarse. pharyngeal erythema was noted in two of three patients, and none had a temperature >99of or received antibiotics. no cases of pneumonia were documented in either group, and all recovered without sequelae. viruses are a major cause of acute respiratory infections in the general population. the groups of viruses responsible for most acute respiratory infections are rhinovirus, coronavirus, influenza, rsv, parainfluenza, and adeno~irus.~ an attempt was made to diagnose each of these pathogens, with the exception of coronavirus, utilizing either culture, serology, or both. several studies of "influenza-like" illnesses in nursing homes have shown that a variety of infectious agents can be found.*-" this study provides evidence of the importance of viruses as a cause of respiratory infection in the nursing home since 42% of acute respiratory illnesses during one winter season were viral in origin. the number and severity of respiratory illnesses during the study period was not unusual compared with previous winter months at this nursing home. however, the season was somewhat atypical since influenza was not isolated, despite its prevalence in other nursing homes in the community. it is possible that high rates of influenza vaccination among staff (37%) and residents (78%) may have influenced influenza infection within the institution." respiratory syncytial virus (rsv), the most common virus isolated in this study, is usually considered a pathogen of infants and young children. while less common in adults, rsv can lead to severe communityacquired pneumonias in elderly or immunocompromised a number of outbreaks of rsv have been reported from chronic care fa~ilities.'~-'~ it is noteworthy that, in our prospective analysis, rsv accounted for 27% of the acute respiratory infections. while it represents only 1 year of study, these data provide further evidence that rsv is an important pathogen in the institutionalized elderly. the clinical syndrome of nasal congestion, cough with expectoration, and fever is consistent with previous reports of rsv infection in the elderly. wheezing, which is common in infants with rsv, was also found in 35% of patients in this study, most of whom did not have preexisting pulmonary disease. one of the prominent features of rsv infection in this study was the wide spectrum of illness it produced, ranging from a mild coryza1 illness to high fever and respiratory distress. rates of pneumonia (5%-67%) and death (0%-20%) varied significantly among previously published outbreaks. factors determining the severity of illness are not completely understood but may be related to the status of the host and to the infecting group or strain of rsvs4 recovery of virus did not appear to be associated with more severe illness as the clinical course of culture-positive cases of rsv did not differ from those with only serologic evidence of infection. cases of rsv infection were widely distributed geographically, occurred over a 3-month period, and were of both a and b groups. the presence of both groups suggests, in contrast with most previous reports, that the present outbreak was not from a single nosocomial source. there was, however, epidemiologic evidence suggesting nosocomial transmission within the institution. clustering of a group infections on the seventh floor and b group infections on the 8th floor, as well as clustering of cases by sides of the hall on three of the four wings, is suggestive of nosocomial spread. rsv is thought to be transmitted by fomites and requires direct contact with respiratory secretions." although documentation of illness among staff members was not attempted during this study, nosocomial transmission of rsv by hospital personnel has been described." therefore, strict handwashing and the use of gowns and gloves, which have been shown to be effective measures for controlling nosocomial rsv infections in hospitalized infants, may be effective in the nursing home as well. rhinovirus was the second most frequent virus isolated in this study. in healthy adults rhinoviral infection is a self-limited illness characterized by sneezing, rhinorrhea, cough, and mild sore throat. 21 there are very limited data on rhinoviral infection in the elderly. analysis of the 14 culture-documented infections in this study indicates rhinovirus produces a relatively benign illness in the elderly as well. infection was characterized by rhinorrhea and cough, but without high fever or signs of lower respiratory tract involvement. generally, the patients did not appear acutely ill, and all recovered without sequelae. unlike other respiratory viruses, such as influenza or adenoviruses, rhinovirus infection produces relatively minor damage to the nasal epithelium and probably none to the tracheal mucosa. 22 since rhinovirus replication is markedly reduced at body temperature, direct invasion of the lower respiratory tract should be unusual at any age. rhinoviruses are transmitted primarily by contact with infected secretions followed by autoinoculation, and transmission can be decreased by handwashing and environmental di~infection.'~, 24 piv is a common cause of croup and bronchitis in young ~hildren.'~ reinfection in young healthy adults typically presents as a uri; however, pneumonia has rarely been described. 26 while piv infection in the elderly has not been well characterized, there have been several outbreaks of piv-1 and -3 infections reported in nursing homes.27, 28 the illness has been characterized by fever, rhinorrhea, pharyngitis, and cough, and pneumonia has been common, ranging from 17% to 29%. although piv infection was uncommon in the present study, we can state that patients with piv-1 infection were all febrile, moderately ill, and received antibiotics. the mode of transmission of piv appears to be direct person-to-person and/or droplet aerosol spread. therefore, infection control measures similar to those used in rsv outbreaks may be useful in piv outbreaks. in summary, viral respiratory tract infections are common in the nursing home during the winter months. both rsv and rhinovirus were prevalent during this study, with rsv being associated with a significantly more severe illness. frequently, the diagnosis of "a viral infection" is one of exclusion and of little use to the clinician. however, diagnosis of these agents is important for several reasons. first, infection control policies for rsv, rhinovirus, and piv are different from those for influenza. secondly, with specific viral diagnosis, the use of antibiotics and amantadine might be decreased, thereby reducing unnecessary side effects. and, finally, as new vaccines become available, the elderly may represent a potential target population. acute respiratory illness in an american community: the tecumseh study the effect of aging on susceptibility to infection studies with different types of visual analog scores for measurement of pain variation in seventy of respiratory syncytial virus with subtype strain-specific serum antibody responses in infants undergoing primary infection with respiratory syncytial virus serum antibody responses in naturally occurring influenza-a virus infection determined by enzyme-linked immunosabsorbent assay, 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and chronically ill respiratory syncytial infections within families control of nosocomial respiratory syncytial viral infections rhinovirus infections in an industrial population. 1. the occurrence of illness principles and practices of infectious disease transmission of rhinovirus colds by self-inoculation interruption of experimental rhinovirus transmission viral respiratory illnesses parainfluenza pneumonia in adults centers for disease control. parainfluenza outbreaks in extended care facilities-united states public health laboratory science communicable disease surveillance centre. parainfluenza infections in the elderly we thank patricia hennessey, rn, for data collection, joanne prives for transcription assistance, and the staff of st. ann's home for their help in conducting the study. key: cord-003357-4qrg6lqu authors: wang, yingchen; dong, tuo; qi, guiyun; qu, lixin; liang, wei; qi, binbin; zhang, zhe; shang, lei; gao, hong; du, xiqiao; lu, bing; guo, yan; liu, zhenwei; yu, huisong; cui, qi; wang, xiaocen; li, ye; guo, weiyuan; qu, zhangyi title: prevalence of common respiratory viral infections and identification of adenovirus in hospitalized adults in harbin, china 2014 to 2017 date: 2018-11-27 journal: front microbiol doi: 10.3389/fmicb.2018.02919 sha: doc_id: 3357 cord_uid: 4qrg6lqu background: respiratory infections pose a great challenge in global health, and the prevalence of viral infection in adult patients has been poorly understood in northeast china. harbin is one of the major cities in northeast china, and more than half of any given year in harbin is occupied by winter. to reveal the viral etiology and seasonality in adult patients from harbin, a 4-year consecutive survey was conducted in harbin, china. methods: from january 2014 to december 2017, specimens were obtained from adult patients admitted to the second affiliated hospital of harbin medical university with lower respiratory tract infections. sputum samples were examined by direct immunofluorescence assays to detect seven common respiratory viruses, including influenza virus (type a and b), parainfluenza virus (type 1 to 3), respiratory syncytial virus and adenovirus. adenovirus positive samples were seeded onto a549 cells to isolate viral strains. phylogenetic analysis was conducted on the highly variable region of adenoviral hexon gene. results: a total of 1,300 hospitalized adult patients with lower respiratory tract infections were enrolled, in which 189 patients (14.5%) were detected as having at least one viral infection. the co-infection rate in this study was 25.9% (49/189). the dominant viral pathogen from 2014 to 2017 was parainfluenza virus, with a detection rate of 7.2%, followed by influenza virus, respiratory syncytial virus and adenovirus. based on the climate seasons determined by daily average temperature, the highest overall viral detection rate was detected in spring (22.0%, 52/236), followed by winter (13.4%, 109/813), autumn (11.4%, 13/114) and summer (10.9%, 15/137). adenovirus type 3 strains with slight variations were isolated from positive cases, which were closely related to the gb strain from the united states, as well as the harbin04b strain isolated locally. conclusion: this study demonstrated that common respiratory viruses were partially responsible for hospitalized lower respiratory tract infections in adult patients from harbin, china, with parainfluenza virus as the dominant viral pathogen. climate seasons could be rational indicators for the seasonality analysis of airborne viral infections. future surveillance on viral mutations would be necessary to reveal the evolutionary history of respiratory viruses. background: respiratory infections pose a great challenge in global health, and the prevalence of viral infection in adult patients has been poorly understood in northeast china. harbin is one of the major cities in northeast china, and more than half of any given year in harbin is occupied by winter. to reveal the viral etiology and seasonality in adult patients from harbin, a 4-year consecutive survey was conducted in harbin, china. methods: from january 2014 to december 2017, specimens were obtained from adult patients admitted to the second affiliated hospital of harbin medical university with lower respiratory tract infections. sputum samples were examined by direct immunofluorescence assays to detect seven common respiratory viruses, including influenza virus (type a and b), parainfluenza virus (type 1 to 3), respiratory syncytial virus and adenovirus. adenovirus positive samples were seeded onto a549 cells to isolate viral strains. phylogenetic analysis was conducted on the highly variable region of adenoviral hexon gene. results: a total of 1,300 hospitalized adult patients with lower respiratory tract infections were enrolled, in which 189 patients (14.5%) were detected as having at least one viral infection. the co-infection rate in this study was 25.9% (49/189). the dominant viral pathogen from 2014 to 2017 was parainfluenza virus, with a detection rate of 7.2%, followed by influenza virus, respiratory syncytial virus and adenovirus. based on the climate seasons determined by daily average temperature, the highest overall viral detection rate was detected in spring (22.0%, 52/236), followed by winter (13.4%, 109/813), autumn (11.4%, 13/114) and summer (10.9%, 15/137). adenovirus type 3 strains with slight variations were isolated from positive cases, which were closely related to the gb strain from the united states, as well as the harbin04b strain isolated locally. lower respiratory tract infections are a persistent public health problem, causing more than two million deaths per year worldwide, with a rate of 36 deaths per 100,000 population (gbd 2016 causes of death collaborators, 2017 . the morbidity and mortality of respiratory infections could be even worse in developing countries, including china. viral infections played an important role in pediatric lower respiratory tract infections, and the corresponding common viral pathogens were influenza a and b virus (iav and ibv), parainfluenza virus (piv, type 1 to 3), respiratory syncytial virus (rsv) and human adenovirus (adv) (pavia, 2011) . these seven viruses were also common in respiratory infections of the adult population in shandong province, china (liu et al., 2015) . to the best of our knowledge, there were no commercial vaccines available for most of these viruses, except for influenza a and b viruses (lambkin-williams et al., 2018) . the etiology of respiratory infection in the adult population has been overlooked, at least in china, although there are plenty of reports on the epidemiology of respiratory viral infection in the pediatric population (wang et al., 2016) . china is a vast country with notable variations in climate characteristics among different regions. due to the differences in socioeconomic, geographic or climate factors, the epidemiological features of viral infection in the respiratory tract show dramatic variation among different study populations (sloan et al., 2011; lu et al., 2013; he et al., 2014; rehder et al., 2015) . harbin is one of the major metropolises in northeast china, hosting more than eight million people. the winter in harbin lasts for more than half a year, while the summer is short, resulting in the length of four seasons being unequal. viral respiratory infections in the pediatric population from harbin were reported by the authors' team in 2009 (zhang et al., 2009) , in which the adult population was not included. a clear picture of the viral etiology in hospitalized adults with respiratory infections ought to be critical for both public health policy makers and clinical practitioners. phylogenetic history of several respiratory viruses isolated from china in recent years have been well established, including the influenza virus yang et al., 2018) , the piv (pan et al., 2017) and the rsv zou et al., 2016) . the information about adenoviral evolutionary history in china was limited to outbreak reports and novel strains identification (lu et al., 2014; li et al., 2018) , leaving a gap for circulating stains among adult patients. human adenovirus is one of the common pathogens in respiratory infections and could be divided into seven species (ismail et al., 2018) . the most recent reported human adenovirus type is type 85 in species d, which was isolated from japan (hashimoto et al., 2018) . human adenovirus species b type 55 (hadv b55) was reported to be responsible for adult pneumonia outbreaks in beijing china during 2011 with the genome identity as 99.87%, comparing to the prototype strain of hadv b55 in 2006 (cheng et al., 2018) . adenoviral infections in children from harbin were found to be adv species b, as reported by the authors, while the viral type among adult patients is still poorly understood. the adenoviral virion was composed of 252 capsomeres, containing 240 hexons and 12 pentons, while each penton was anchored by one fiber (or two fibers in several types) (nemerow et al., 2012) . the knob region in adenoviral fiber was responsible for cellular attachment through host receptors including car, cd46, dsg-1 and sialic acids (baker et al., 2018; lenman et al., 2018) . the rgd loop of penton could be recognized by the host cell's integrin (veesler et al., 2014) . the hexon protein is the major neutralizing antigen of adenoviruses (madisch et al., 2005) , while the hyper variable region (hvr) in hexon protein hosts its type specific epitopes reported by the authors' team (yuan et al., 2009) . experiments on chimeric adenoviral capsids showed that the replacement of hvrs in hexon could alter the viral serotypes (qiu et al., 2012; tian et al., 2018) . the hexon specific cd4 + and cd8 + t cells have been proved to be an effective treatment for human adenoviral infections in clinical settings (zandvliet et al., 2010; feucht et al., 2015) . recent reports indicated that antibodies against fiber knob or penton base may also be involved in the neutralization of adenoviruses tomita et al., 2018) . to the best of our knowledge, the hypervariable region of adenoviral hexon protein plays an essential role in viral type-specific immunity. in this report, the prevalence of common viruses in the lower respiratory tract infection of hospitalized adult patients from harbin, china was explored in hopes of revealing the clinical and pathogenic features of respiratory viruses. adenoviral strains were isolated from positive cases to identify potential molecular variations within its hypervariable regions. from january 2014 to december 2017, sputum samples were collected from 1,300 adult patients hospitalized for lower respiratory tract infections in the second affiliated hospital of harbin medical university. all patients enrolled in this study lived in urban and suburban areas of harbin without travel histories within 3 months before admission and sampling. all patients were older than 18 years and suffering from at least one complaint, including fever with body temperature ≥ 38 • c, cough, expectoration, hemoptysis, chest tightness (chest pain), or dyspnea. the sputum samples were expectorated spontaneously into sterile containers and delivered to the laboratory within 2 h, tested immediately or stored at −80 • c prior to use. patients' clinical data, including symptoms, history of illness, clinical diagnoses and laboratory test results were surveyed through the hospital's health information system. this study had been approved by the ethical review committees of harbin medical university in accordance with the declaration of helsinki. both informed and written consents were obtained from each participant who provided specimens. the presence of common respiratory viruses, including piv type 1 to 3, influenza virus a and b virus (iav and ibv), rsv, and human adv was determined by the d 3 ultra tm dfa respiratory virus screening kit (diagnostic hybrids, inc., athens, oh, united states). virus isolation for the adv-positive specimens was performed using the a549 cell line (provided by the cancer hospital of harbin medical university) following the protocol described previously (smith et al., 1986; huang et al., 2013) . in brief, cells seeded with clinical samples were incubated at 37 • c for 4 days. if there was no observed cytopathic effect, two additional blind passages of the culture were performed to avoid false negative results. the cultures with adv-like cytopathic effects were passaged again to confirm viral presence. the viral dna of human adv was extracted from infected cells using the axyprep multisource genomic dna miniprep kit (axygen biosciences) according to the manufacturer's instructions and eluted in 100 µl elution buffer. the highly variable region in the adenoviral hexon gene was amplified using the following primer pairs: forward, 5 -caattcactcgctccta-3 and reverse, 5 -gtggaaaggcacataacg-3 . all primers were synthesized by comate bioscience co., ltd. pcr was performed with the platinum pcr supermix (invitrogen) following the manufacturer's instructions. a total reaction volume of 50 µl contained 5 µl of 10 × pcr buffer (takara bio premix ex taq tm ), 200 nm of each primer, 200 nm of each dntps, 2 µl of each dna sample, 0.25 µl taq enzyme (takara bio premix ex taq tm ), and sterile water. the cycling conditions were 5 min at 95 • c, followed by 35 cycles of 30 s at 95 • c, 30 s at 55 • c, and 30 s at 72 • c, with a final 5-min extension at 72 • c. dna extracted from the a549 cell culture and sterile water were used as negative controls. the amplicons were bi-directionally sequenced using the sanger sequencing method with an abi prism 3130 genetic analyzer (comate bioscience co., ltd., jilin, china). the daily average air temperature from january 1st, 2014 to december 31st, 2017 in harbin city was kindly provided by the harbin meteorological observatory. historical temperature datasets covering 1981-2010 in harbin were accessible from the china meteorological data service center. climate seasons were determined based on the smoothed daily average temperature curve according to the chinese national standard no: qx/t 152-2012 (administration, 2012) . in brief, spring starts when the daily average temperature permanently rose above 10 • c, while summer comes when it rose above 22 • c permanently. the autumn starts when the daily temperature drops to 22 • c permanently and the winter begins at 10 • c. "permanently" means the daily temperature has remained above or below the threshold for at least five consecutive days. the details can be found in the supplementary table s1. statistical analysis was conducted using the r language (version 3.4.2). the prevalence (detection percentage) of viruses was calculated by dividing the sum of positive cases by the number of cases in total. chi-square tests or fisher's exact tests were selected for comparing the cross tables of categorical variables. the wilcoxon rank-sum or kruskal-wallis tests were chosen for continuous variable comparisons as appropriate. datasets were visualized by an excel spreadsheet. the quality of sequencing data was checked manually via chromas software version 2.4 (technelysium pty ltd., south brisbane, qld, australia), before contigs for each isolate were assembled using ugene (protsyuk et al., 2015) software suite (version 1.9) guided by the published hexon gene sequence of human adenoviruses. similar strains to the isolates in this report were found from the genbank database via the blast search method (boratyn et al., 2013) . fifty-eight nucleotide sequences of the hexon gene covering all seven species of human adv were selected as the reference in this study. before the tree building process, multiple sequences were aligned by muscle (edgar, 2004) software (version 3.6). the unrooted neighbor-joining tree was reconstructed using the kimura 2 parameters distance, whose robustness was tested by the bootstrap method with 1,000 replications implemented in mega (kumar et al., 2018) software (version 10.0.1). the parsimony tree was also built on the same dataset. in total, 1,300 hospitalized adult patients with respiratory infection were enrolled from january 2014 to december 2017 in harbin city. among these patients, 710 were male and 590 were female, as shown in bronchiectasis, 20 with asthma and 51 with other infections. some clinical signs were more frequent among cases with certain diagnoses. in all, 370 (66.5%) pneumonia cases complained of fever, while only 185 (37.3%) cases with bronchitis had fever (p < 0.001). cough was the chief complaint of the bronchial infections (p < 0.001), including bronchitis (56.0%), copd (55.5%) and asthma (60.0%). out of 1,300 enrolled patients and 583 were diagnosed with chronic diseases, including chronic respiratory disease, cancer, diabetes mellitus, chronic cardiac diseases, cerebrovascular disease, chronic kidney disease, and other chronic diseases lasting more than 1 year. among the 1,300 total patients, 189 (14.5%) cases were determined to be positive with at least one of seven viruses, including influenza virus (a and b), piv (type 1, 2, and 3), rsv and adv. one hundred and forty patients were infected with only one virus as the single infection, and 49 patients were co-infected with more than one virus. double infections were observed in 43 cases, while triple infection was found in six cases. as shown in table 2 , the most frequent single infection was piv (59 cases) and the major double infection was combined influenza and parainfluenza viruses (17 cases). the details were accessible from the supplementary table s2 . the median age of the viral positive group was 63 (iqr 54-69) and the negative group was 61 (iqr 51-71). the age difference between the viral infection positive and negative group was not statistically significant (p = 0.730). the viral detection rate varied from 16.6% (108/652) in the middle age group to 11.9% (43/360) in the senior citizen group, but the difference was not statistically significant (p = 0.104). there was no significant difference in the viral infections between genders (p = 0.902). the virus detection rate among the study years varied significantly (p = 0.017) from 18.6% (52/280) in 2015 to 9.7% (29/299) in 2014, as shown in table 1 . the detection rate was not equal among calendar seasons (p = 0.002) or climate seasons (p = 0.003). based on the climate seasons, the highest overall detection rate was in spring (22.0%, 52/236), followed by winter (13.4%, 109/813), autumn (11.4%, 13/114), and summer (10.9%, 15/137). for individual virus positive samples, certain viruses were more frequently detected in spring than in other seasons, including influenza virus (8.9%, 21/236), piv (9.7%, 23/236), and rsv (9.3%, 22/236), as shown in table 3 . adv was more frequently detected in winter, with a detection rate of 1.8% (15/813). piv was the dominant viral pathogen during the study period, accounting for 65.5% (19/29) of the viral infection cases in 2014, 40.3% (21/52) in 2015, 54.7% (29/53) in 2016, and 43.6% (24/55) in 2017. as shown in table 3 , viral co-infections were more not equally distributed among seasons, with the highest ratio in spring at 7.2% (17/236, p = 0.022). flu piv rsv adv cases total 189 overall detected respiratory viral positive cases were plotted against daily average air temperature in figure 1 . the low temperature over days was a strong indicator for respiratory viral infections. positive cases were rare in summer, which began late in june and ended in mid of august. one exception was found in the summer in 2017 which was related to a sudden drop of air temperature from 27.2 • c on 18th july to 18.8 • c on 27th july 2017. the overall percentage of viral infection was significantly connected with clinical diagnoses (p = 0.025), as shown in table 1 . the highest detection rate was observed in the pneumonia group (17.8%, 99/556), followed by bronchitis (13.9%, 69/496), while the lowest detection rate was found in the asthma group (5%, 1/20). chest tightness was the only symptom that showed statistically significant difference (p = 0.011) between the positive (38.1%, 72/189) and negative (48.1%, 534/1111) group of viral infections. the respiratory viral infection did not prolong hospital stay statistically (p = 0.306), although the median days in the hospital for the positive viral infection group was 12 (iqr 9-17), which was shorter than 13 in the negative group (iqr 9-17). overall, the viral detection rate was relatively lower in the chronic diseases group (11.7% vs. 16.9%), with statistical significance (p = 0.008). detection rates varied among different chronic diseases, but the difference was not significant (p = 0.467). the viral co-infection ratio among different diagnosed groups was not significant (p = 0.372). two strains of human adv were isolated from two patients diagnosed with pneumonia, respectively. the harbin17b strain ( came from a male patient aged 69 whose samples were collected in december 2017. the other strain, named harbin17a, was isolated from a male patient aged 24 whose samples were collected in january 2017. based on the evolutionary tree reconstruction of the hexon gene sequencing data, both strains isolated in this study were identified as human adv species b type 3. these two strains were nearly identical, with the closest evolutionary distance to the strain gb (genbank: ay599834; atcc: vr-3) from the united states as well as the harbin04b strain from a previous local epidemic, as shown in figure 2 . the parsimony tree was similar in topology with neighborjoining tree (figure not shown in the article). multiple sequence alignments indicated that, compared with the gb strain, the harbin17a and harbin17b strain shared a variable site in the 482nd codon (gb numbering) of the hexon gene hosting an a to c mutation, resulting in threonine (t) to proline (p) mutation of the amino acid sequences. the harbin04b strain had a threonine on the same site in the hexon protein encoded by the codon acu, which was identical to the harbin17a and harbin17b strain. adult populations with respiratory infections bear heavy burdens of hospitalization due to severe respiratory illness, especially for senior citizens (muller-pebody et al., 2006) . the constantly changing nature of viral pathogens has made the surveillance of these infections critical to public health authorities (zou et al., 2016; li et al., 2017) . the key indicators for viral infections from this report and recent published surveys in china has been summarized in table 4 . the overall detection rate of viral infection among hospitalized adult patients in this report is 14.5%, which was consistent with the result of 16.8% in the age group above 14 years old by a national survey from 2009 to 2013 in china (feng et al., 2014) . the dominant viral pathogen throughout this study period was piv, with a detection rate of 7.2%, followed by influenza virus. the national survey on hospitalized patients with respiratory infection in china from 2009 to 2013 showed that influenza virus had the highest detection rate of 8.0% in adult patients. the detection rate of respiratory viruses in the over 16 years age group was 24.2% in shandong province of china (liu et al., 2015) and was dominated by influenza virus (8.7%) from 2011 to 2013. from 2012 to 2015, the respiratory viral detection rate in hospitalized patients above 14 years old within beijing and shandong china was reported to be 38.9%, in which influenza virus had the highest frequency of 8.0% (yu et al., 2018) . compared with recent surveys in beijing, shandong and other regions in china, this report showed that the piv dominated epidemic seasons from 2014 to 2017 in adult patients in harbin, which should be important for preparing future epidemic control strategies in public health. the viral infection rate within asthma subgroup of this report was 5%, and it was one patient with rsv among 20 asthma patients in total as shown in table 3 . asthma is related to viral infection, while the detection rates varies among different pathogens. a recently published meta-analysis showed that the mean prevalence of rsv, influenza virus, piv and adv was 13.6%, 10%, 5.6%, and 3.8%, respectively (zheng et al., 2018) . the apparently low detection rate within asthma group in this report could be possibly caused by the sampling bias due to the limited subgroup size. air temperature was a major meteorological variable associated with respiratory viral infection (chan et al., 2015) . harbin city is located at 46 degrees north latitude with an annual average temperature of 4.9 degrees celsius. climate seasons are not in equal length in harbin, according to historical statistics from 1981 to 2010. the longest season in harbin is winter, which starts on the 4th of october and lasts 201 days, followed by spring on the 23rd of april with 61 days. starting on the 23rd of june, summers in harbin are relatively short at 53 days, while autumn is 50 days and is the shortest season, starting on the 15th of august. in routine epidemiological analysis, 12 months in a year were usually divided into four seasons equally, which is not the case in harbin and possibly compromises contagious disease seasonality prediction. based on climate seasons defined by the daily average air temperature, influenza, parainfluenza and rsv detected in this report all presented significant seasonal peaks in spring, although similar phenomena were absent within the calendar seasons. sputum samples were acceptable both in viral culture and antigen detection in respiratory infections compared with nasopharyngeal swabs and aspirates (covalciuc et al., 1999) , although they were not frequently collected in recent surveys on lower respiratory tract infected adult patients from china. detection of respiratory virus in sputum samples has been proven effective on a small scale, either by the immunofluorescence method (honkinen et al., 2012) or the pcr method (branche et al., 2014) . this report provided a medium scale test on the validity or effectiveness of respiratory viral detection from sputum samples with 1,300 cases. in this report, two nearly identical strains were isolated from pneumonia patients in harbin, possessing high similarity with the local strain harbin04b and the strain gb. the harbin04b strain was isolated from a pediatric patient in harbin (zhang et al., 2009) . adv strain gb was named vr-3 in the atcc database, which was isolated from an adult patient with common cold in maryland, united states in 1953 (huebner et al., 1954) . the similarity of these strains across asian and american continents through several decades may result from the globalization movement in modern china. it could also be explained by another hypothesis based on the evolution history of adenoviruses. on the geological scale, homo sapiens inherit human adenoviruses from our ancestors within the hominidae, resulting in an evolutionary rate of human adv as low as 2.8e-8 substitution per site per year (hoppe et al., 2015) . considering its 36 kbp genome, we would have to wait a millennium (or 992 years exactly) before a single site mutation within an adenoviral genome occurred naturally. there were a few limitations to this study. first, the 4 years of investigation on a single city in this present report was relatively short and much more localized than other long-term or national surveillance, which may lead to a bias of the viral etiology. second, the enrolled population in this study consisted of adult patients only, while the samples from pediatric patients were not collected simultaneously. the comparison of the viral prevalence status between adult and pediatric populations in this study was difficult. third, the bacterial culture results of the clinical samples were not included in this report, which prevents us from drawing a comprehensive picture on the pathogens associated with lower respiratory tract infections. in this report, 14.5% of lower respiratory tract infections in hospitalized adults from harbin were found to be related with common respiratory viruses. the piv dominated viral infection from 2014 to 2017 with detection rates of 7.2%. climate seasons based on daily temperature records were found to be reliable in seasonality analysis. adv type 3 strains with slight variations were isolated from positive cases. further surveillance would be important to continuously monitoring the viral etiology in adult patients both local and abroad. zq, yw, and td designed this study and prepared the manuscript. gq, yg, and wg collected the specimens. yw, td, and yg conducted the epidemiological investigation. td, lq, wl, bq, zz, ls, hg, xd, bl, zl, hy, qc, xw, and yl performed the laboratory experiments. yw and td analyze the data and made the figures and tables. all authors reviewed the manuscript. division of climatic season designer oncolytic adenovirus: coming of age blast: a more efficient report with usability improvements detection of respiratory viruses in sputum from adults by use of automated multiplex pcr hospitalization incidence, mortality, and seasonality of common respiratory viruses over a period of 15 years in a developed subtropical city comparative genomic analysis of re-emergent human adenovirus type 55 pathogens associated with adult severe 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neutralizing antibodies to adenovirus capsid proteins in human and animal sera comparison of the prevalence of respiratory viruses in patients with acute respiratory infections at different hospital settings in north china molecular modeling and epitopes mapping of human adenovirus type 3 hexon protein combined cd8+ and cd4+ adenovirus hexon-specific t cells associated with viral clearance after stem cell transplantation as treatment for adenovirus infection respiratory viruses in hospitalized children with acute lower respiratory tract infections in harbin regional, age and respiratory-secretion-specific prevalence of respiratory viruses associated with asthma exacerbation: a literature review evolution and transmission of respiratory syncytial group a (rsv-a) viruses in guangdong we gratefully acknowledge the harbin meteorological observatory and the china meteorological data service center for providing air temperature datasets in this report. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02919/full#supplementary-material key: cord-022555-a7ie82fs authors: nan title: digestive system, liver, and abdominal cavity date: 2011-12-05 journal: the cat doi: 10.1016/b978-1-4377-0660-4.00023-5 sha: doc_id: 22555 cord_uid: a7ie82fs nan these complex pathways highlight the need to consider the whole cat and not just the cat's gastrointestinal vomiting can be defined as the ejection of part or all of the contents of the stomach and/or upper intestine through the mouth, usually in a series of involuntary spasmodic movements. the disturbances in gastrointestinal (gi) motility are coordinated with respiratory and abdominal muscle contractions and mediated by the central nervous system (cns). vomiting begins with retching, a series of brief negative intrathoracic pressure pulses that coincide with positive abdominal contractions. these pressure changes occur as a result of repeated herniations of the abdominal esophagus and cardiac portion of the stomach into the esophagus. during retching, food freely moves back and forth in the esophagus, which is now dilated because of the ingesta. ultimately, the diaphragm rapidly moves cranially, resulting in positive intrathoracic pressure that leads to expulsion of these contents. 12 vomiting is such an active process that it seems to involve the whole cat, and so it is little wonder that it concerns owners so much. since vomiting is mediated by the cns with input and influence from just about anywhere in the body, it is important to summarize this physiology so it can be appreciated when managing clinical cases. vomiting results from stimulation of the "vomiting center," which is located in the brainstem; there are four main pathways that stimulate the vomiting center, 12 and these are summarized below and in figure 23 (though not all older cats have grown out of this habit). some extragastrointestinal problems, such as hyperthyroidism and renal disease are more likely to occur in older cats. most texts and references instruct clinicians to distinguish between vomiting and regurgitation, with the latter noted as being quite passive. 3, 11, 12 in practice, it can be hard to make this distinction, because it is the author's experience that cats with esophageal disease can have quite forceful, spasmodic movements when ejecting ingesta by regurgitation-although it is also possible for regurgitation to be a passive process. given that the physiology of vomiting, as described above, results in ingesta being forced to and then evacuated from the esophagus, it is hardly surprising that it can resemble regurgitation. fortunately, regurgitation and esophageal disease do vary from vomiting in other ways! vomiting 3. blood and urine testing 4. imaging (radiography, ultrasonography) 5. biopsy samples 6. treat and manage underlying problem the decision to proceed to steps 4 and 5 is based on the assumption that the prior steps have narrowed down the underlying cause as gastrointestinal, pancreatic, or hepatic in origin. the important aspects of the clinical history are given in chronic kidney disease. the author has found that some cats with dental disease can gorge their food, resulting in vomiting; so, paying attention to the state of the teeth and gums is important. of course, some cats have multiple problems, and correction of dental disease may not resolve vomiting if there is another process. in the examination, it is also important to note consequences of both the underlying process and the vomiting itself; these include the demeanor of the cat, hydration status, and abdominal pain. the physical examination findings, together with the clinical history, help determine the next appropriate steps. well cats that are not continually vomiting and are appropriately hydrated, with no other specific signs, may be treated as outpatients by fasting them for 24 hours, then returning to food with a bland diet, such as plain cooked chicken or commercial, low-residue prescription diets designed for this purpose. follow-up is important to ensure signs do not progress. cats with nonspecific signs may require supportive care with subcutaneous or intravenous fluids and perhaps analgesia (with opioids). if clinical signs do not resolve, the pursuit of a specific diagnosis should be attempted. the practitioner must ask the following important questions: • are ancillary tests appropriate? • is supportive care necessary? • are any medications required? routine serum/plasma biochemistries, hematology, urinalysis, and total thyroxine (t 4 ) (for older cats) testing is not only important to distinguish primary from secondary gastrointestinal disease but to look for consequences of vomiting that may need to be addressed, such as hydration status and electrolyte abnormalities. careful interpretations should be made. severe azotemia, even with hyperphosphatemia, can occur as a result of primary gastrointestinal disease, and the distinction from renal disease usually requires an assessment of urine specific gravity. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity (fpli) tests are useful markers of intestinal and pancreatic disease, 7, 8, 9, 10 but it is important to note that they mostly do not give a precise diagnosis. more detail about the utility of these tests is noted below in the section approach to the cat with diarrhea. is usually preceded by the cat licking its lips, salivating, or making attempts to swallow. regurgitated ingesta is often in a tubelike structure and if undigested can be covered with frothy saliva. partially digested food suggests vomitus, and the presence of bile or digested blood confirms this. it is important to determine if the cat vomits regularly. many owners have seen their cats vomit on a regular basis with no evidence of the cat being unwell, and this is noted frequently in the veterinary literature. 3, 12 hairballs can cause gastric irritation, and it may be that eating quickly also stimulates the peripheral sensory receptors that contribute to vomiting. if a cat does vomit regularly, it is important to assess if the cat is presenting for a change in the vomiting pattern (e.g., frequency or timing in relation to eating) and if the cat is unwell in any way, such as anorexia or weight loss. the pattern of vomiting is important in all cases, because cats presenting with acute gastritis usually have a sudden onset of frequent vomiting compared with those with chronic disease processes that may vomit every few days. the timing in relation to eating can be helpful, because the stomach should empty by 6 to 8 hours after a meal; so, vomiting longer than 8 hours after a meal can suggest motility or retention disorders. the description of the vomitus can be helpful. if bile is present, the pylorus is not obstructed; the presence of blood (digested or fresh) indicates ulceration. hair in the vomitus can indicate hairball gastritis, and the possibility of trichobezoar obstruction should be considered. access to foreign bodies or toxins is an important aspect of the clinical history. has the cat been seen playing with an insect, mouse, or other prey? are there any medications unaccounted for (e.g., a dropped aspirin tablet)? are lilies present in the house? vomiting is the major sign of gastric disease, but given the number of potential organ systems that can be involved, a thorough physical examination should be undertaken. because linear foreign bodies are a common cause of vomiting, all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of string caught there. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see . a thorough examination may reveal specific signs, such as a palpable thyroid nodule and tachycardia in the case of hyperthyroidism or palpably small kidneys with and analgesia, many cats recover uneventfully. one survey assessed that 83% of cats undergoing exploratory laparotomy survived the hospitalization, and although complications occurred in 26% of cats, these were more likely to be associated with the underlying disease process and not surgery or anesthesia. 4 laparoscopy is not readily available in all veterinary clinics. this alternative is less invasive and allows exploration of the abdomen but not as thoroughly as with laparotomy. organs are usually exteriorized for biopsy. there is the possibility of anesthetic complications associated with insufflating the abdomen. endoscopy is the least invasive procedure and is the only alternative that allows examination of the intestinal lumen. this option limits the parts of the gastrointestinal tract that can be biopsied; it does not allow examination or sampling of any other part of the gastrointestinal tract and does not enable full-thickness biopsy samples. one study found that, of cats investigated for gastrointestinal disease, 9 of 33 cats (27%) had no pathology recognized proximal to the jejunum (i.e., the effective length of diagnostic endoscopes would have precluded diagnosis), and other organs were affected in 9 of 10 cats with inflammatory bowel diseases and 7 of 8 cats with intestinal small cell lymphoma. 1 careful case selection for endoscopy from survey ultrasonography can reduce the number of missed diagnoses from endoscopy, but the possibility still remains. the quality of endoscopically obtained biopsy samples varies greatly with the skill of the endoscopist. it has been stated that "it is exceedingly easy to take inadequate tissue samples with a flexible endoscope." 5 in an assessment of endoscopically obtained biopsy samples, two laboratories were compared, one that received samples from any practitioner and the other that received samples only from practitioners trained to take, mount, and submit endoscopy samples. all slides were reviewed by three pathologists who found that, of samples from the first laboratory, 15% of the slides were considered inadequate for diagnosis, 71% were considered questionable, and only 14% were adequate. by comparison, in the second laboratory (with samples from experienced practitioners) 0% of slides were inadequate, 21% were questionable, and 79% were considered adequate for diagnosis. 13 in the case of distinguishing between lymphocytic intestinal infiltrates (commonly known as inflammatory bowel disease) and lymphocytic neoplasia (small cell lymphoma), endoscopically obtained samples can give an incorrect diagnosis. 2 many of these problems can be minimized with experienced operators and careful case selection from prior ultrasonography. radiography is most useful for identifying foreign bodies or signs of intestinal obstruction from other causes. the major findings are noted below in the section intestinal obstruction. contrast radiography can aid the diagnosis for both discrete and linear foreign bodies but should be used with caution, because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium irritates the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. 6 ultrasonography is a useful diagnostic adjunct and helps to detect and characterize localized thickening of the stomach or intestinal wall, lymphadenopathy, radiolucent foreign bodies, and changes in the size and echogenicity of the pancreas, liver, kidneys, or spleen. abdominal effusions can be assessed and sampled. ultrasound-guided fine-needle aspiration can be used to sample masses, bile, or peritoneal fluid. it should be recognized that in most cases of gastrointestinal disease, imaging will not give a definitive diagnosis and biopsy will be required, usually using either endoscopy or laparotomy. ultrasonography can be a considered as a means to "survey the field," assessing • the nature of the underlying disease, such as • thickened intestines with or without discrete layers • lymph node involvement • other organ involvement • the location of disease, for example, • diffuse or focal • proximal duodenum (reachable by endoscope) versus distal ileum these factors may be used to assess the appropriateness of endoscopy versus laparotomy to obtain diagnostic samples. most commonly used antiemetics all control vomiting by different mechanisms and include mirtazapine, metoclopramide, dolasetron/ondansetron, maropitant, and the phenothiazines (tables 23-2 and 23-3) . metoclopramide functions both as an antiemetic and prokinetic in cats, while cisapride functions solely as a prokinetic. mirtazapine, a piperazinoazepine, antagonizes the presynaptic alpha 2 -adrenergic receptor, increasing noradrenergic and serotonergic neurotransmission; the primary mechanism targeted for its use is as an antidepressant in humans. mirtazapine is also a potent antagonist of the postsynaptic serotonergic receptors (5-ht 2 and 5-ht 3 ) and histamine h 1 receptors. because of its antiserotonergic and antihistaminic effects, mirtazapine is used as an entiemetic and appetite stimulant in cats. anorexia is a common clinical problem in ill cats, and in some anorexic or partially anorexic cats the use of an appetite stimulant as adjunctive therapy to nutritional support (i.e. feeding tubes) may be of clinical benefit. prior to the development of mirtazapine, cyproheptadine was used as an appetite stimulant in cats, with variable clinical results. recently, the pharmacokinetics and pharmacodynamics of mirtazapine have been reported in cats. in a group of healthy cats, mirtazapine was found to be an effective appetite stimulant, with a shorter half-life than that reported in humans. the recommended oral dose is 1.88 mg/cat every 48 hours. 55a in humans, age and kidney and liver dysfunction affect mirtazapine metabolism (hepatic cyp 450 enzymes) and clearance (excreted in urine and feces), suggesting that dose adjustment may be necessary. 69a side effects reported in cats treated with mirtazapine include behavior changes (vocalization and interaction), tremors, muscle twitching, and hyperactivity. 9a,55a metoclopramide is both an antiemetic and prokinetic drug that acts peripherally on the gastrointestinal tract and centrally within the central nervous system (cns). at low doses metoclopramide inhibits dopaminergic (d 2 ) transmission, and at higher doses it inhibits serotonergic 5-ht 3 receptors in the chemoreceptor trigger zone (crtz). 15, 23 metoclopramide also acts peripherally as a prokinetic at the level of the gastrointestinal smooth muscle of the stomach and duodenum, triggering gastric emptying and duodenal contractions. multiple mechanisms mediate metoclopramide's prokinetic activity, including augmentation of acetylcholine release and increased smooth muscle sensitivity to cholinergic neurotransmission, which may in part be because of antagonism of dopamine, but more recently, serotonergic 5ht 4 receptor activation has been suggested. 23, 56 metoclopramide has been reported to increase the lower esophageal sphincter tone in humans, 20 although in cats metoclopramide's affect on the lower esophageal sphincter is reported to be weak. 32 adverse central nervous system, extrapyramidal signs occur secondary to dopamine (d 2 ) antagonism, including excitement and behavior changes. extrapyramidal signs are most often seen at the higher doses needed to block 5-ht 3 receptors. because of metoclopramide's prokinetic properties, an intestinal obstruction should be ruled out prior to its use. dopamine is a less important neurotransmitter in the chemoreceptor trigger zone of cats than alpha 2adre nergic and 5-ht 3 -serotonergic receptors, suggesting that d 2 -dopaminergic antagonist may be a less effective antiemetic in cats. clinically metoclopramide commonly controls vomiting in cats, although this clinical response may be secondary to 5-ht 3 antagonism and/ or its prokinetic effects. 32, 44 extrapolated from the short elimination half-life of metoclopramide in dogs (90 minutes), frequent be clinically significant side effects. phenothiazines have the potential to lower the seizure threshold; their use is not recommended in patients with a known seizure history. other cns-associated side effects linked to d 2 antagonism occur at higher doses and produce extrapyramidal signs, including rigidity, tremors, weakness, and restlessness. antagonism of the histaminergic receptors carries the risk of sedation. because of the need for frequent dosing (0.2 to 0.4 mg/ kg subcutaneously every 8 hours) and the risk of hypotension and sedation, the clinical use of phenothiazine antiemetics is limited to hospitalized patients with refractory vomiting and should be avoided in patients who are dehydrated or hypotensive. cisapride is a serotonergic 5-ht 4 agonist that increases propulsive gastrointestinal motility from the lower esophageal sphincter to the colon. cisapride binds serotonergic 5-ht 4 receptors in the myenteric plexus, increasing the release of acetylcholine in gastrointestinal smooth muscle. in dogs cisapride has greater prokinetic activity in the stomach relative to metoclopramide. 29 cisapride has no direct antiemetic effect, although it is indicated in a vomiting cat with colonic dysmotility secondary to megacolon. colonic distention can trigger the vomiting reflex in cats. cisapride induces colonic smooth muscle contractions in cats with megacolon that is dependent on the influx of extracellular calcium and is only partially cholinergic dependent. 30 other potential indications include refractory generalized ileus or gastroesophageal reflux. dosage recommendations based on the pharmacokinetics in healthy cats is 1.5 mg/kg orally every 12 hours. 41 prior to the use of cisapride, an intestinal obstruction should be ruled out because of its strong prokinetic effects. side effects reported in humans are cramping and diarrhea. potentially life-threatening side effects include qt prolongation and ventricular arrhythmias, the primary concern in humans that led to cisapride's removal from the market in the united states. 47 in cats qt prolongation associated with cisapride administration requires 20 times the therapeutic dose. 37 because of the risk of prolongation of the qt interval and ventricular arrhythmias, the concurrent use of cisapride and dolasetron is not recommended. 14 other potential drug interactions associated with cisapride include concurrent therapy with azole antifungals (ketoconazole and itraconazole), because of their inhibition of hepatic cyp3a isoenzyme system and the inhibition of cisapride metabolism. 47 diet trials are commonly used in cats with idiopathic gastrointestinal signs or in cats with suspected or known intermittent dosing or delivery by a constant rate infusion (cri) is necessary. empirical dosing in cats is 0.2 to 0.4 mg/kg subcutaneously or orally every 8 hours or 1 to 2 mg/kg/day as a cri. approximately 25% of metoclopramide is excreted in the urine, thus dose reduction is recommended in cats with underlying renal azotemia. 42 dolasetron and ondansetron are selective serotonin antagonists that inhibit central and peripheral 5-ht 3 receptors. their main antiemetic effect is through antagonism of the peripheral 5-ht 3 receptors in the gastrointestinal tract. in cats 5-ht 3 antagonism of the crzt is also likely important in the antiemetic effect of dolasetron and ondansetron. dolasetron and ondansetron were originally used for vomiting secondary to chemotherapy because of their superior clinical efficacy. the clinical use of dolasetron and ondansetron in cats has not been associated with reported side effects, and experimental studies report minimal toxicity in animals at doses 30 times the antiemetic dose. 15 side effects reported in humans include headaches, elevated liver enzymes, rare hypersensitivity reactions, prolongation of the qt interval, and arrhythmias. 14, 24 dolasetron is commonly used for parenteral administration and ondansetron for oral administration, dictated primarily based on the tablet sizes available and cost. recommended dosing of dolasetron is 0.5 to 1 mg/kg intravenously every 24 hours and ondansetron 0.5 mg/ kg orally every 12 hours. maropitant is a neurokinin-1 (nk-1) receptor antagonist, blocking the binding of substance p to the nk-1 receptors located in the emetic center, crtz, and the enteric plexus. 55 in cats maropitant has been reported to be efficacious in treating xylazine-induced vomiting and motion sickness. 31 recommended dosing in cats is 1 mg/ kg intravenously, subcutaneously or orally every 24 hours for up to 5 days. 31 maropitant is reported to be well tolerated in cats. prochlorperazine and chlorpromazine are considered broad-spectrum antiemetics by antagonism of d 2dopaminergic, histaminergic (h 1 and h 2 ), and cholinergic (muscarinic) receptors within the crtz and, at high doses, the alpha-adrenergic receptors (alpha 1 and alpha 2 ) within the vomiting center. in cats alpha 2 -receptors play a key role in emesis (recall xylazine is the emetic of choice in cats), suggesting cats may be more sensitive to the antiemetic effects of the phenothiazines. prochlorperazine and chlorpromazine produce an antiemetic effect at relatively low doses, thus avoiding profound sedation; although, because of antagonism of the alpha-receptors, vasodilation and hypotension can hyperacidity alone is not considered a common cause for vomiting in cats, but famotidine is effective in treating vomiting in cats associated with gastric ulcers or gastritis. recommended dosage in cats is 0.5 mg/kg every 12 to 24 hours. ranitidine is also a competitive inhibitor of the h 2 receptor associated with gastric parietal cells. in addition, ranitidine increases lower esophageal sphincter tone and functions as a prokinetic agent (increasing gastric emptying and stimulating intestinal motility, including colonic motility), because of its anticholinesterase food hypersensitivities. dietary strategies used to control vomiting in cats focus on either a highly digestible diet or an elimination (novel protein/carbohydrate or hydrolyzed protein) diet. 72 the empirical use of elimination diets in cats is reported to be relatively successful, with approximately 50% of cats with idiopathic gastrointestinal signs responsive to a novel protein/carbohydrate diets within 2 to 3 days. 28 interestingly, traditional diet trials are recommended for a minimum of 8 to 12 weeks, but in this group of diet-responsive cats with chronic gastrointestinal disease, clinical improvement was reported within days. 28 thus if a cat is going to be diet responsive, clinical improvement to a diet trial should be noted relatively early. highly digestible diets enable more effective absorption and assimilation of nutrients in the face of a compromised digestive tract. these diets contain highly digestible proteins and carbohydrates, moderate to low fat, soluble fiber but low concentrations of insoluble fiber, and are supplemented with omega-3 fatty acids. these diets are recommended when food allergy or intolerance is suspected. these diets contain a single highly digestible novel carbohydrate source and novel protein source. alternatively, diets formulated with hydrolyzed proteins can be used as an alternative to novel protein/carbohydrate diets. see tables 23-4 and 23-5 for information on gastrointestinal ulcers. famotidine has no direct antiemetic effect but is a competitive inhibitor of the histamine (h 2 ) receptors associated with the gastric parietal cells. the h 2 -receptor is the dominant receptor involved in gastric acid secretion. h 2receptor antagonism is reported to result in a 70% to 90% reduction in acid production. 13 famotidine is more effective at suppressing gastric acid secretion relative to ranitidine. famotidine is well tolerated, although, with chronic therapy, there is the potential for hypoacidity and gastric bacterial overgrowth. in humans dose reduction is recommended in association with renal dysfunction. 21 famotidine is not an inhibitor of the hepatic microsomal cytochrome p-450 enzyme system, therefore significant drug interactions are not anticipated. activity. 40, 54 significant drug interactions associated with hepatic microsomal cytochrome p-450 enzyme system inhibition are not a clinical concern with ranitidine. 46 an adverse effect to be aware of in cats treated with ranitidine is transient hypotension associated with ranitidine administered as an iv bolus. 19 in humans dose reduction is recommended in patients with renal azotemia. 39 ranitidine is effective in decreasing gastric acid in cats. 22 ranitidine would be a logical choice in a cat with gastrointestinal ulceration and/or atony. the reported dosage recommendation for ranitidine in cats is 3.5 mg/ kg orally every 12 hours or 2.5 mg/kg intravenous every 12 hours. 19 omeprazole omeprazole is a proton pump inhibitor that targets the h + /k + atpase pump on the luminal surface of partial cells. omeprazole is effective at suppressing parietal cell acid secretion, and its effects persist for ≈24 hours after drug withdrawal because of drug accumulation in the parietal cell (by ion trapping). indications for omeprazole therapy are for the treatment and prevention of nonsteroidal antiinflammatory drug (nsaid)-induced ulcers. 9 omeprazole is enteric coated to prevent its degradation by gastric acid; therefore oral formulations should not be crushed. based on human studies, omeprazole is a hepatic microsomal cytochrome p-450 enzyme inhibitor with known drug interactions with diazepam. 2 the extent of clinically significant drug interactions in cats has yet to be studied. omeprazole is reported to be effective in reducing gastric acid secretion in cats. 22 the recommended empirical dosage in cats is 0.5 to 1 mg/kg orally once daily. long-term use in humans 33 and dogs 11 is associated with gastric polyps and parietal cell hyperplasia, respectively, but the effect of long-term use in cats is currently unknown. sucralfate is a disaccharide complexed with aluminum that dissociates to sucrose octasulfate and aluminum hydroxide upon exposure to gastric acid. the sucrose octasulfate spontaneously polymerizes, producing a viscous material capable of binding ulcerative lesions in the gastric mucosa. once bound to the exposed mucosa, it prevents back diffusion of h + , inactivates pepsin, absorbs bile acids, and increases mucosal prostaglandin synthesis, collectively supporting ulcer healing. sucralfate is not systemically absorbed but does prevent the absorption of drugs capable of chelating with aluminum, including fluoroquinolones, tetracyclines, and digoxin. if sucralfate is indicated in a cat being treated concurrently with fluoroquinolones, tetracyclines, or digoxin, the recommendation is to administer the other drug 2 hours prior to the administration of sucralfate to optimize drug absorption. clinical indications for the use of sucralfate in cats are for the treatment of gastric ulcers and esophagitis. 36 dosage recommendation in cats is 250 mg orally every 12 hours. sucralfate can be crushed, suspended in water, and administered as slurry. diet trials are used in some cats with diarrhea if the underlying cause is from known or suspected food hypersensitivities. dietary management includes either a highly digestible diet, an elimination (novel protein/ carbohydrate or hydrolyzed protein) diet (see above for both), or a diet high in fiber. 72 high-fiber diets contain a mixture of both soluble and insoluble fiber that can be beneficial in patients with signs of large bowel diarrhea. insoluble fiber, such as cellulose, functions to increase the bulk of the stool, bind fluid, and regulate intestinal motility. soluble fiber, including fruit and vegetable pectins and beet pulp, functions as a source of butyric acid that can be used by the colonic mucosa and decreases proinflammatory cytokines. 69, 72 cobalamin cobalamin (vitamin b 12 ) is an essential vitamin needed by a number of different enzymes, including key enzymes involved in methionine metabolism and the conversion of methylfolate to tetrahydrofolate needed for dna synthesis. cobalamin and folate are intimately linked, and hypocobalaminemia can lead to a functional deficiency of folate. 57 ingested cobalamin requires intrinsic factor binding for enterocyte absorption at the level of the ileum. hypocobalaminemia is commonly associated with distal small intestine diseases in cats, including inflammatory bowel disease. in addition, low cobalamin has a negative impact on enterocyte function; therefore in many cats with intestinal disease and hypocobalaminemia, cobalamin supplementation is necessary for resolution of clinical signs. 60, 64 quantification of serum cobalamin levels is recommended in cats with clinical signs of small bowel diarrhea, ones suspected to have an infiltrative disease of the small intestine (inflammatory bowel disease or gastrointestinal lymphoma), or ones with pancreatic dysfunction. when hypocobalaminemia is identified, supplementation is recommended mannanoligosaccharides, inulin, chicory, and lactosucrose. 72 reports on the use of prebiotics in cats are limited to their use in healthy cats; healthy cats fed fructooligosaccharides were reported to have a trend toward an increase in fecal concentrations of lactobacilli and a decrease in concentration of c. perfringens and e. coli relative to the controls. 65 to date no reports are available on the use of prebiotics in cats with gastrointestinal disease. probiotics and prebiotics potentially have a supportive role in the treatment of gastrointestinal disease in cats. the important clinical consideration in the use of probiotics as an adjunctive therapy is to ensure the use of live nonpathogenic microorganisms that have been documented to colonize the intestinal tract of cats. gastrointestinal flora co-evolve with their host. gastrointestinal microorganism colonization varies among species and within each individual animal. the distribution of fecal microflora for a given individual is considered unique but stable over time. 68 antimicrobial and antiparasitic therapies for the treatment of feline diarrhea are indicated based on the specific diagnosis of infectious diarrhea, bacterial enteritis, or as adjunctive therapy for inflammatory bowel disease. infectious pathogens more commonly associated with feline diarrhea include bacterial enteropathies (clostridium, campylobacter), protozoal enteropathies (tritrichomonas foetus, giardia spp.), and helminthic enteropathies associated with ascarids, hookworms, whipworms, and tapeworms. only the more common anthelminthic, antimicrobial, and antiprotozoal therapies are discussed below (tables 23-6 and 23-7) . more information about antimicrobials and antiparasitics is found under specific infections in the discussions of infectious enteritis and gastrointestinal parasites. fenbendazole is an anthelmintic used to treat common helminth infections, including ascarids, hookworms, whipworms, and a single species of tapeworm, taenia pisiformis. giardia spp. are also considered susceptible to fenbendazole. fenbendazole binds beta-tubulin subunits of microtubules, interfering with their polymerization. side effects include vomiting and diarrhea, although both are considered rare. fenbendazole is not approved for use in cats in north america but is commonly used clinically, and an empirical dosage of 50 mg/kg (250 µg/cat every 7 days) while the underlying cause of cat's malabsorption is being investigated and at initiation of targeted therapy. probiotics probiotics are ingested live microorganisms intended to benefit the host, specifically to support the microflora environment of the gastrointestinal tract as well as to provide an overall benefit to the body's immune function by immunomodulation. 8, 18, 51 probiotics chemically modify ingesta and intestinal mucus, as well as affect immune cells, enterocytes, and goblet cells within the intestinal mucosa through direct receptor interactions and indirectly through the action of cytokines. the microorganisms commonly used are nonpathogenic bacteria and yeast that have a vital role in gastrointestinal health, including lactobacillus spp., enterococcus faecium, bifidobacterium spp., and saccharomyces spp. for example, lactobacilli synthesize b vitamins, digestive enzymes, and folate coenzymes. 63 clinical indications for the use of probiotics are diverse, including primary gastrointestinal disease, chronic renal disease, and pancreatitis. 71 the rational use of probiotics in the treatment of gastrointestinal diseases include their ability to modulate gastrointestinal flora, minimize colonization by pathogenic bacteria, and decrease the likelihood of bacterial translocation. 17 in healthy cats, lactobacillus acidophilus is reported to reduce fecal clostridium counts. 45 when lactobacillus acidophilus was used adjunctively with antimicrobial therapy, fecal shedding of campylobacter was reduced in cats with campylobacter-induced diarrhea relative to cats treated with antimicrobials alone. 3 specifically, in cats with gastrointestinal disease, available research supports the probiotic enterococcus faecium as clinically beneficial in resolving diarrhea in kittens. 16 relative to the control group, the kittens treated with probiotics had increased fecal bifidobacteria and blood iga concentrations and decreased fecal counts of clostridium perfringens. prebiotics are dietary supplements used to select for the more beneficial enteric flora, support gastrointestinal function, and prevent the overgrowth of pathogenic bacteria, including salmonella, escherichia coli, clostridium, or campylobacter. for a food additive to be considered a prebiotic, it must be nondigestible by the gastrointestinal tract (resistant to gastric acidity, gastrointestinal hydrolysis and absorption), yet fermentable by gastrointestinal microflora to short-chain fatty acids to stimulate the growth of "good" intestinal bacterial. 72 prebiotics include nondigestible oligosaccharidescommonly, oligofructose, fructo-oligosaccharides, pyrantel pamoate is a nicotinic anthelmintic used primarily for the treatment of ascarids, but its spectrum of activity also includes hookworms and the stomach worm, physaloptera spp. pyrantel is toxic to susceptible parasites through its selective action on their nicotinic acetylcholine receptors, resulting in depolarization and spastic paralysis. pyrantel is not approved for use in cats but is considered safe in cats and is commonly used clinically. the dosage recommendation in cats is 5 mg/ kg orally once, repeat in 3 weeks, and finally repeated in 3 months. metronidazole is a nitroimidazole antibiotic with an anaerobic antibacterial spectrum with antiprotozoal activity against giardia spp. in an anaerobic environment, metronidazole is converted to unstable intermediates (nitroso free radicals) that disrupt bacterial dna synthesis. immunomodulatory properties capable of inhibiting cell-mediated immunity have been described for metronidazole, although its immunomodulatory properties are reported at dosages well beyond what is recommended for clinical use, 62 raising questions about the clinical use of metronidazole as an adjunctive therapy for treating inflammatory bowel disease. 34, 43 resistance to metronidazole is considered rare. 43 the most common adverse reaction is gastrointestinal upset, including inappetence, anorexia, nausea, and vomiting. profuse salivation can occur in cats after oral administration of metronidazole base (formulation used in standard tablets), which has lead to the use of metronidazole benzoate (a compounded formulation not approved by the food and drug administration) in some cats because of its better oral palatability. 61 at high doses (>200 mg/ kg/day) benzoic acid is reported to be neurotoxic in cats, but with appropriate clinical dosing of metronidazole benzoate benzoic acid toxicity is unlikely. 6 dose-related metronidazole toxicity in cats results in cerebellovestibular ataxia secondary to gamma-aminobutyric acid (gaba) inhibition at dosages greater than or equal to 58 mg/kg/day 12, 52 ; clinical signs include nystagmus, head tilt, ataxia, seizures, and obtundation. in cats with inflammatory bowel disease, the dosage recommendation for the metronidazole base is 10 to 15 mg/kg/day. metronidazole benzoate contains approximately 60% metronidazole base by weight, translating to an empirical dosage of 20 mg/kg/day of metronidazole benzoate (equivalent to 12.4 mg/kg/day of metronidazole base). 61 little is known about the safety of chronic metronidazole use in cats, but oral metronidazole has been reported to disrupt dna within feline peripheral mononuclear cells following 7 days of therapy. 61 this metronidazole-induced genotoxicity is reversible and is no longer detected 6 days after antibiotic therapy is discontinued. ronidazole is a nitroimidazole antibiotic (similar to metronidazole) and available as a powder-on-feed antibiotic. ronidazole is not approved for use in cats but has immunosuppressive therapies used in cats with inflammatory bowel disease include glucocorticoids, cyclosporine, and chlorambucil (tables 23-8 and 23-9 ). more information on the treatment of inflammatory bowel disease is found elsewhere in this chapter. glucocorticoids are considered first-line therapy in the treatment of cats with inflammatory bowel disease. glucocorticoids bind their intracellular glucocorticoid receptors, modifying the expression of genes with glucocorticoid response elements. immunomodulation is achieved through inhibition of cytokine release and response, including decreasing leukocyte phagocytosis, chemotaxis, and antigen expression. the more common side effects in cats include gastrointestinal ulceration, opportunistic infections (e.g., urinary tract infections), pancreatitis, and diabetes mellitus. cats are less susceptible to iatrogenic hyperadrenocorticism than dogs. initial therapy is usually with oral prednisone or prednisolone. prednisone is a prodrug that is metabolized to its active form prednisolone. cats are reported to be less efficient in the conversion of prednisone to prednisolone 27 ; therefore prednisolone may be preferred in cats, especially in cats refractory to prednisone therapy. been used off-label to effectively treat tritrichomoniasis in naturally and experimentally infected cats (30 mg/kg orally every 12 hours for 14 days). 25 t. foetus reduces nitroimidazoles to their nitroso free radicals. ronidazole has been reported to have better in vitro and 10-fold higher in vivo activity against t. foetus relative to metronidazole. 25, 35, 49 ronidazole resistance is beginning to be reported in t. foetus isolates from cats with diarrhea. 26 side effects include hepatoxicity and neurotoxicity. neurotoxicity is associated with high doses and has been reported in cats. 59 the use of ronidazole is recommended only for confirmed cases of t. foetus, and dosing should not exceed 30 mg/kg once daily in cats, especially in cats at risk for neurotoxicity. ronidazole is not registered for human or veterinary use in the united states; therefore its use in cats requires owner informed consent and client education of the potential human hazards. immunosuppressive therapies are considered the standard of care for cats with gastrointestinal biopsies consistent with inflammatory bowel disease (lymphoplasmacytic or eosinophilic inflammation). the common alternative forms of glucocorticoids can be considered in specific patient populations. in patients with severe malabsorption, injectable dexamethasone may provide improved bioavailability and clinical response. also dexamethasone maybe preferred in patients with a history of heart failure, fluid retention, or hypertension because of its lack of mineralocorticoid activity relative to prednisone/prednisolone. dexamethasone's potency is 4 to 10 times that of prednisolone; therefore a dose reduction is necessary when prescribing dexamethasone (the dexamethasone dose is one seventh that of prednisolone). 4, 10 budesonide is an oral, locally active, highpotency glucocorticoid that is formulated to be released in the distal gastrointestinal tract (based on the ph differential between the proximal and distal small intestine), where it is absorbed and is locally immunomodulating at the level of the enterocyte. the amount of systemically absorbed budesonide is minimized, because 80% to 90% of the budesonide absorbed from the gastrointestinal tract undergoes first-pass metabolism in the liver. some systemic absorption does occur, as evidenced by a blunted adrenocorticotropic hormone (acth) stimulation test in dogs treated with budesonide at 3 mg/m 2 for 30 days. 66, 70 the use of budesonide in cats remains anecdotal, with a suggestive empirical dose of 0.5 to 1 mg/cat/day. initial glucocorticoid therapy for cats with inflammatory bowel disease consists of antiinflammatory (0.5 to 1 mg/kg/day) to immunosuppressive (2 to 4 mg/kg/ day) dosages, with dosages based on the potency of prednisone/prednisolone. the goal of therapy is to achieve clinical remission and slowly taper the dose of glucocorticoids to the lowest dose that will control the cat's clinical signs. 67 some cats may be completely weaned off therapy, while others require long-term lowdose therapy. the tapering of therapy should be slow, with a 25% to 50% dose reduction every 3 to 4 weeks. cyclosporine is considered a second-tier immunosuppressive drug used to treat inflammatory bowel disease in cats. use of cyclosporine in the treatment of diarrhea associated with inflammatory bowel disease in cats is extrapolated from its use in dogs to treat glucocorticoid refractory inflammatory bowel diarrhea. 1 cyclosporine suppresses t-lymphocyte-mediated inflammation in the gastrointestinal tract secondary to suppression of inflammatory cytokines. specifically, cyclosporine attenuates t-lymphocyte activation and proliferation through the inhibition of interleukin-2 (il-2) production. side effects of cyclosporine in cats include dose-dependent inappetence and vomiting, which may occur at the onset of therapy and are generally responsive to dose reduction. other less common side effects reported in cats are opportunistic infections, including toxoplasmosis 5 and hepatoxicity. the microemulsion formulation of cyclosporine has higher oral bioavailability and less variable pharmacokinetics. 58 a suggested initial dosage of cyclosporine is 4 mg/kg every 12 or 24 hours. serum cyclosporine levels can be used to monitor for excessive trough plasma concentration (>400 ng/ml) as determined using a highperformance liquid chromotography (hplc) analytical method. 53 chlorambucil is a slow-acting nitrogen mustard that alkylates and effectively cross links dna, leading to altered protein production. the immunosuppressive effects of chlorambucil are the result of its cytotoxic effect on lymphocytes, similar to other nitrogen mustards. bone marrow suppression is considered mild to moderate and is rapidly reversible. neurotoxicity and myoclonus has been reported in a cat accidently overdosed with chlorambucil. 7 chlorambucil is used as a second-tier drug in cats to treat immune-mediated disorders, in part because of ease of administration and its low risk of myelosuppression. for the treatment of inflammatory bowel disease, the recommended dosing in cats is 2 mg/cat every 48 hours in cats greater than 4 kg and 2 mg/cat every 72 hours in cats less than 4 kg. 50 chlorambucil is commonly used in combination with glucocorticoids in the treatment of immune-mediated diseases, including inflammatory bowel disease, 48 overlooked. awareness about feline esophageal diseases is low, the clinical signs are often not specific, and imaging beyond survey radiographs may be required for diagnosis. the esophagus is composed of four layers (from inner to outer): mucosa, submucosa, muscularis, and adventitia (there is no serosal layer). in the dog, the muscle layer is entirely composed of skeletal muscle, but in cats, the distal third of the esophagus is composed of smooth muscle. the upper esophageal sphincter prevents reflux of esophageal contents into the pharynx and minimizes aerophagia. the lower esophageal sphincter prevents gastroesophageal reflux and relaxes during swallowing to allow food and fluid to enter the stomach. clinical signs of esophageal disease include drooling, dysphagia, pain on swallowing (odynophagia), and, most classically, regurgitation. weight loss may occur secondary to inadequate food intake when disease is severe or chronic. other clinical signs, such as anorexia, cough, dyspnea, and fever, may occur if complications such as aspiration pneumonia or esophageal perforation occur. regurgitation is passive expulsion of food or fluid from the esophagus. the food is undigested and often accompanied by mucus and saliva. mucosal erosions may produce frank blood in the regurgitated material. regurgitation must be differentiated from vomiting (table 23 esophageal disease is uncommon in the cat when compared with dogs, but it is also likely that problems such as esophagitis and esophageal strictures are often salivation, retching, and abdominal contractions. the vomitus consists of partially digested food from the stomach and/or intestines and may be mixed with bilestained fluid. some cats will have both vomiting and regurgitation. expectoration may also be confused with vomiting or regurgitation. expectoration is associated with coughing, but cats that cough excessively may also stimulate vomition so that a careful history is needed to characterize the clinical signs correctly. coughing may also occur in cats that have aspirated as a result of regurgitation. drooling, dysphagia, and odynophagia are most commonly seen with conditions of the oropharynx and/or proximal esophagus. odynophagia is most commonly associated with esophagitis and foreign bodies. dysphagia and regurgitation together most commonly indicate oral or pharyngeal dysfunction; if regurgitation is not accompanied by dysphagia, esophageal dysfunction is likely. 55 regurgitation in cats with esophageal disease is caused by obstruction or muscular dysfunction. causes of obstruction include vascular ring anomaly, foreign object, stricture, and neoplasia. causes of muscular dysfunction include congenital disease, esophagitis, myopathies, neuropathies, and dysautonomia. regurgitation may occur immediately after eating if the lesion is in the proximal esophagus. however, a dilated esophagus provides a reservoir for food and fluid so that regurgitation may not be associated in time with eating. young cats with signs of esophageal disease should be suspected of congenital defects, such as vascular ring anomaly, or a foreign body. adult cats with esophageal disease may have a recent history of general anesthesia, administration of certain oral medications, or ingestion of irritant chemicals. acute onset of clinical signs may suggest a foreign body, while chronic, slowly worsening signs may indicate a stricture or tumor. all cats suspected of esophageal disease should have a minimum database as part of the diagnostic plan (complete blood cell count, serum chemistries, urinalysis, and other tests as indicated by age or concurrent diseases, such as serum total t 4 and blood pressure measurement). an important part of diagnosis is observation of the cat while eating food, to localize the location of the dysfunction. if the cat is unwilling to eat while in the veterinary clinic, the owner can make a video of the cat eating at home for the clinician to view. the general diagnostic approach to regurgitation in cats is found in figure 23 -3. plain and contrast radiography and endoscopy are important diagnostic tools for esophageal disease. fluoroscopy is valuable for the diagnosis of motility disorders, but availability is limited to universities and referral centers because of the cost of equipment. ultrasonography is limited to evaluation of * references 1, 8, 11, 15, 38, 43. the cervical esophagus and a small segment of abdominal esophagus between the cardia of the stomach and the diaphragm. the entire esophagus should be evaluated with cervical and thoracic radiographs. thoracic radiographs may also show evidence of complications such as aspiration pneumonia or esophageal perforation. the normal esophagus is not visualized on plain radiographs, but may be seen if food or fluid are retained or a foreign body or mass is present. radiographic contrast agents useful for esophagrams in cats include liquid or paste barium. a water-soluble iodinated contrast agent (e.g., iohexol, gastrografin) is preferred if there is any risk the esophagus is perforated, because these agents are less irritating and more rapidly reabsorbed. esophagrams are most useful for diagnosis of luminal obstructions, extraluminal compression, mucosal irregularities, and possibly alterations in motility. dilute liquid barium can be administered with a syringe or it may be mixed with canned food, especially if a motility disorder or stricture is suspected. multiple lateral radiographs are taken rapidly, starting within 10 seconds of swallowing the contrast agent. contrast is rapidly cleared from the normal esophagus by peristalsis. if the contrast in the esophagus terminates abruptly, an obstruction is likely. if the contrast is retained throughout the esophagus, muscular dysfunction is suspected. some conditions, such as esophagitis, are difficult to diagnose radiographically, because contrast agents may or may not adhere to ulcerated mucosa. flexible endoscopy is a noninvasive diagnostic tool for esophageal disorders and is often used if plain and contrast radiographs have failed to establish a diagnosis. it is most sensitive for diagnosis of masses, ulcers, perforations, and obstructions. in addition, it is often possible to retrieve foreign bodies using endoscopy as well as to assist with dilatation of strictures or placement of gastrostomy feeding tubes if required. biopsy of the esophageal mucosa is more difficult than biopsy of gastric or intestinal mucosa and is not commonly performed with the exception of mass lesions. esophagitis may result from various causes of inflammation, such as contact irritation from foreign bodies (including trichobezoars lodged in the esophagus), chemical irritants or caustic medications, gastroesophageal reflux, persistent vomiting, hiatal hernia, or general anesthesia. inflammation disrupts the esophageal mucosa and exposes the submucosa. an important part of the treatment plan is identification and treatment of the underlying cause. clinical signs include dysphagia, regurgitation, salivation, and repeated swallowing, although signs may be absent in cats with mild esophagitis. cats with odynophagia may repeatedly extend the head and neck while swallowing. if the esophagitis or underlying disease is severe, weight loss and dehydration may occur secondary to anorexia. if the submucosa and muscularis are damaged, strictures may form as a result of the production of fibrous connective tissue and compromise the esophageal lumen. 54 neoplasia is an important cause of esophageal stricture in humans, but not in cats. most cases have single strictures, but multiple strictures are possible. in two studies, the mean stricture diameter was reported as 5 mm. 26, 32 most strictures are less than 1 cm in length. clinical signs associated with strictures appear 5 to 14 days after the esophageal injury and may be present for weeks before definitive treatment is pursued. regurgitation typically occurs immediately after eating, although if the stricture is long standing, a pouch may form cranial to the lesion where food accumulates. survey radiographs may be normal in cats with esophagitis and strictures, but are useful to rule out other causes for the clinical signs, such as a foreign body, or to detect related problems, such as aspiration pneumonia. in some patients, dilation of the esophagus with fluid or air may be seen. 45 a contrast esophagram may disclose irregularities of the mucosa in cats with severe esophagitis. segmental dilation may occur with severe inflammation. strictures may be diagnosed with an esophagram (figure 23-4) ; however, in some cases, it may be difficult to differentiate a stricture from intramural thickening (e.g., because of neoplasia). endoscopy is useful for diagnosis of esophagitis; findings include mucosal erythema, hemorrhage, and erosions or ulcerations. if gastroesophageal reflux is present, the lesions will be most severe in the distal esophagus, and the lower esophageal sphincter may be dilated. endoscopy is often used for definitive diagnosis of esophageal stricture as well as to visualize the lesion during treatment by bougienage or balloon catheter dilation. strictures appear as a ring of white fibrous tissue that narrows the esophageal lumen. if endoscopy is performed after a barium esophagram, 24 hours should be allowed to elapse between the procedures or the barium will obscure visualization with the endoscope. 19 general anesthesia is an important cause of esophagitis (sometimes leading to stricture formation) in cats, probably because gastroesophageal reflux appears to occur commonly in anesthetized cats.* for example, in a series of seven cats with benign esophageal stricture, recent anesthesia for ovariohysterectomy was the suspected cause in five cases. 1 clinical signs appeared up to 21 days after anesthesia. abnormal esophageal tissue was performed in two cases. the authors noted that the esophageal mucosa may appear grossly normal, but submucosal inflammation may be found on histopathologic examination of biopsies. a consequence of chronic severe gerd in humans is the development of metaplastic columnar epithelium (barrett esophagus) that replaces the normal squamous epithelium. one case series reported on barrett-like esophagus in three cats. 23 two cases were associated with hiatal hernia and one with cardial incompetence. drug-induced esophageal damage and stricture formation is well known in humans and cats (see . in humans over 70 drugs have been implicated, and most are antibacterials or nsaids. 30 implicated drugs in the cat include tetracycline, doxycycline, and clindamycin in tablet or capsule form administered without a food or water bolus. 4, 17, 32, 36, 37 clinical signs (dysphagia, regurgitation, salivation, anorexia) appear 3 to 16 days after drug treatment is started. strictures commonly form in the midcervical esophagus or over the heart base in the thoracic esophagus. doxycycline hyclate is most commonly associated with esophageal strictures in cats, and the principle reason for its irritating properties is an acidic ph. the monohydrate salt of doxycycline is less irritating and is marketed as tablets and a palatable paste licensed for use in dogs and cats in some countries. 48 in humans esophageal ulceration after doxycycline therapy is more common than stricture formation. although the development of strictures in cats would appear to be uncommon, it seems possible the incidence of esophagitis is underestimated, because the clinical signs (e.g., odynophagia, chest pain) may go unrecognized. esophageal transit studies of normal cats have shown that the passage time of dry-swallowed tablets and capsules is often prolonged (longer than 30 seconds). 20, 53 complete entrapment (retention for more than 4 minutes) in the midcervical region occurs commonly. however, a small bolus of food or water is sufficient to ensure immediate passage of the medication into the stomach. 20, 53 the risk of esophageal retention can also be lessened by coating a tablet or capsule with butter or a gel dietary supplement (nutri-cal; vétoquinol, fort worth, tex.). 21 one study determined that tablets or capsules administered using a one-step pill gun with flavored liquid (flavorx pill glide; flavorx, columbia, md.) or a pill delivery treat (greenies pill pockets; nutro products, franklin, tenn.) ensured an average transit time of 60 seconds or less. 6 delayed esophageal transit of medications allows tablets and capsules to disintegrate within the esophagus, exposing the mucosa to irritating chemicals. cats may be at risk of delayed esophageal transit, because they do not typically drink water with medication, and they do not have an upright posture. in addition, medications are often given to sick or dehydrated patients many preanesthetic drugs and induction agents reduce lower esophageal sphincter pressure. 27, 28 other predisposing factors may be intraabdominal surgery and a head-down position on the surgery table. reflux fluid with a ph less than 4 is likely to cause esophageal mucosal damage, as is prolonged contact time. esophageal defense mechanisms include clearance of the reflux fluid by peristalsis and neutralization of the acidic ph by the bicarbonate present in saliva. in a study of 40 kittens less than 15 weeks of age, risk of gastroesophageal reflux during anesthesia was evaluated with use of a laryngeal airway mask versus endotracheal intubation. 47 gastroesophageal reflux was observed in 50% of kittens with use of the laryngeal airway mask but more importantly in 22% of kittens with endotracheal intubation. the reflux episodes occurred shortly after anesthesia induction. in a study of 50 cats anesthetized with thiopentone or propofol, gastroesophageal reflux occurred in 14%. 16 reflux also occurred shortly after anesthesia was induced and lasted for a mean of 23 minutes. it is unknown why esophageal strictures form only in a small number of cats that experience gastroesophageal reflux during anesthesia. gastroesophageal reflux disease (gerd) is a commonly reported cause of esophagitis in humans, but it is rarely reported in cats when not associated with general anesthesia. 24, 33 the true incidence is unknown, and diagnosis may be hampered by scant knowledge about the clinical presentation and diagnosis. clinical signs and diagnostic procedures are as for other causes of esophagitis. in one case series of three cats, diagnosis of gerd was based on clinical signs, contrast radiography, and endoscopic findings. 24 biopsy and histopathology of obtained from a compounding pharmacy in most countries and can only be given orally. h 2 -receptor antagonists are competitive inhibitors that block parietal h 2 receptors and decrease the amount of gastric acid produced. proton pump inhibitors are noncompetitive inhibitors that act on the h + /k + atpase enzyme system at the secretory surface of gastric parietal cells. they are considered superior for decreasing gastric acid secretion and are therefore the first choice, despite their greater cost. 45 a drawback of proton pump inhibitors is that they must be administered orally. sucralfate may be beneficial for reflux esophagitis, because it binds to mucosal erosions in an acid environment and provides a protective barrier. it is given as oral slurry, ideally separate from meals or other medications. antibiotics are not commonly recommended unless aspiration pneumonia is present or the eroded mucosa is at risk of bacterial infection in a patient with severe disease or a compromised immune system. corticosteroids are often recommended for cats with esophagitis to reduce esophageal inflammation and impair the formation of fibrous connective tissue. however, the benefit of corticosteroids in cats with esophagitis has not been investigated and administration must be weighed against potential adverse effects, especially in patients with aspiration pneumonia. treatment of esophageal stricture typically requires dilation with either bougienage or a balloon catheter; both are used with endoscopic visualization under general anesthesia. appropriate analgesia should be provided, because dilating the stricture is painful. it does not appear that placement of a gastrostomy feeding tube is specifically required to recover from dilation procedures, although a tube may be placed in some anorexic cats to ensure nutritional intake and administer oral medications. a bougie is a long, narrow, oblong, mechanical dilator available in various sizes (typically 9-to 12-mm sizes are used in cats) that is gently passed through the stricture, usually over a guide wire. established criteria for selection of bougie diameter and dilation end points are not available. in one study, the initial bougie chosen was approximately the same size as the estimated diameter of the stricture, or no more than 2 mm larger. 8 once the first bougie is passed, subsequent bougies of increasing diameter are employed. two to four bougies of increasing size may be passed in a single session, with the goal of dilating the stricture without causing esophageal tear or perforation. determining when dilation should be stopped is a matter of clinical judgment. the procedure may be repeated as needed to maintain improvement; the total number of procedures required is variable. in one retrospective case series of eight cats treated with bougienage, the median number of procedures was 4.5, and a good outcome was achieved in 75% of the cases. 8 in some cases, the endoscope tip itself has been used for that may be at greater risk of esophageal retention of medication. all oral medication given to cats in tablet or capsule form should be followed with food or a liquid. mild esophagitis will resolve on its own, especially if an underlying cause can be removed or treated. frequent meals of canned food should be provided. cats with moderate to severe esophagitis will require medical therapy, and those with difficulty eating or weight loss may also require gastrostomy tube feeding. esophagostomy or pharyngostomy feeding tubes should be avoided in these patients. treatment is provided to control inflammation and promote healing while reducing gastric acid secretion and increasing lower esophageal sphincter tone. the length of medical treatment will vary from about one week to several weeks, depending on the underlying cause and severity of disease. medications indicated for esophagitis include prokinetics, h 2 -receptor antagonists, proton pump inhibitors, and sucralfate (table 23-11) . prokinetic drugs enhance gastric emptying and increase lower esophageal sphincter tone. metoclopramide also has antiemetic effects, which may be beneficial in patients with chronic vomiting. it can be administered by the subcutaneous (sc) route, an advantage in a vomiting or regurgitating patient. cisapride may be more effective at enhancing both gastric emptying and lower esophageal sphincter tone, but it must be stent placement has recently been described in cats with esophageal strictures with variable results. a 1-year-old cat presented with a 4-week history of dysphagia and regurgitation caused by a single cervical esophageal stricture after treatment with oral clindamycin. 18 guided balloon dilation was performed 6 times over a period of 3 weeks, but stricture formation always recurred. a self-expanding metal stent was placed using endoscopy and fluoroscopy after another dilation procedure. the cat did well eating a canned diet from an elevated position for 10 months, but by 12 months, the cat was no longer able to eat even liquid food and was euthanized. on necropsy, the stent had migrated and was obstructed by swallowed hair. in another case, a biodegradable self-expanding stent was used to successfully treat an 11-year-old cat that presented with a stricture in the cervical esophagus after anesthesia for dentistry. 3 balloon dilation was performed twice, but regurgitation recurred 5 days after the last procedure. the stricture was dilated a third time with a balloon catheter, and a tubular selfexpanding polydioxanone stent was placed with fluoroscopic guidance. the life span of the stent was estimated to be 10 to 12 weeks, sufficient time to allow healing of the esophagus. foreign bodies are less commonly found in the esophagus of the cat than in other gastrointestinal locations. reported foreign bodies include string, needles, fish hooks, and bones. trichobezoars may cause obstruction when they become lodged in the esophagus during vomiting ( figure 23 -5). recurrent esophageal trichobezoars have been infrequently reported in the literature. 12, 51 it is not known if an esophageal motility disorder is the underlying cause for recurrent obstructions. in one case, an esophageal diverticulum developed in association with recurrent trichobezoars. 12 treatment for recurrent trichobezoars includes prokinetic drug therapy (e.g., cisapride), moderate to high-fiber diets, and shaving of long-haired cats. common areas for foreign bodies to lodge include the thoracic inlet, the heart base, and the esophageal hiatus in the diaphragm. 5 obstruction of the esophageal lumen may be complete or partial. clinical signs include acute onset of gagging, salivation, repeated swallowing, dysphagia, and regurgitation. however, chronic esophageal foreign bodies have been reported in cats with dysphagia, intermittent regurgitation, and weight loss over a period of weeks or months. 2 bougienage when bougies or balloon catheters were not available. balloon catheter dilation has become a popular method in recent years. 26, 32, 38 although some clinicians feel this is a safer procedure than bougienage, there is no data in the literature to support this assumption. the catheter can be placed through the endoscope biopsy channel, alongside the endoscope, or with the aid of a preplaced guide wire. as for bougienage, established criteria for selection of balloon diameter and dilation end points are not available, and the clinician's best judgment must be used. various balloon sizes are available; in one study, the size was selected so that the inflated diameter was 4 mm larger than the stricture diameter. 32 the balloon is passed into the stricture with endoscopic guidance. it is then inflated to a predetermined pressure for 1 to 2 minutes to stretch the stricture, usually with saline, but contrast agents may also be used if fluoroscopy is used. as for bougienage, some cases may require more than one dilation procedure (typically two to four). cuffed endotracheal tubes are not appropriate substitutes for balloon catheters. regardless of the method used, after the dilation procedure, the endoscope should be used to look for other strictures and should be passed into the stomach to look for potential causes, such as causes of chronic vomiting. after treatment, medical management to decrease ongoing gastroesophageal reflux, resolve inflammation, and prevent further stricture formation should be instituted (as described previously). most cats are able to eat the day following the dilation procedure. corticosteroid treatment after dilation is controversial, and no controlled studies in animals are available. antibiotics are not routinely recommended. the prognosis for cats undergoing esophageal dilation is generally good based on the ability to eat canned food with minimal episodes of regurgitation. however, published studies show 10% to 30% of cats died or were euthanized despite multiple episodes of dilation, and up to 30% could only be fed liquid diets. 1, 8, 32, 38 even among cats with good outcomes, a return to a dry kibble diet may not be possible. the dilation technique employed may be dictated by the clinician's experience, the equipment available, and the cost. potential complications of both methods include esophageal tear or perforation, hemorrhage, infection, and aspiration. esophageal tears or perforations may lead to pneumothorax or pneumomediastinum. repeated stricture formation is also possible, leaving only less desirable treatment options, such as long-term percutaneous gastrostomy tube feeding or surgery. esophageal surgery is generally avoided whenever possible, because it is difficult and invasive (requiring a thoracotomy), with risk of serious complications, such as failure of anastomosis, necrosis, and stricture formation. closure of incisions in the esophagus is following uncomplicated foreign body removal, the esophagus should be carefully inspected for lesions and bleeding before the endoscope is withdrawn. food and water should be withheld for 24 to 48 hours. supportive care includes fluid therapy and analgesia; a gastrostomy feeding tube may be required in selected cases for nutritional support. broad-spectrum antibiotics are administered to control bacterial infection and therapy for esophagitis should be instituted as described previously. careful follow-up should include evaluation for stricture formation. if an esophageal perforation has occurred, conservative management may be sufficient if the defect is small. a broad-spectrum antibiotic should be administered along with other supportive care, such as fluid therapy and analgesia. feeding through a gastrostomy tube for several days is recommended as well as close monitoring for complications such as pleuritis. large perforations require thoracotomy for surgical repair. megaesophagus is a diffuse hypomotility disorder that may be classified as congenital versus acquired or idiopathic versus secondary to other diseases. it is uncommon in cats compared with dogs. at least two dog breeds have been identified with heritable congenital megaesophagus. a heritable form of megaesophagus has been suggested for cats, particularly for siamese cats, although no detailed studies have been performed. 13, 29 it is often frustrating to determine the underlying cause of acquired megaesophagus. megaesophagus may be a manifestation of neuromuscular diseases, such as dysautonomia or myasthenia gravis (see chapter 27) . megaesophagus may also develop secondary to esophagitis from chronic vomiting or gerd. 24, 43 other uncommon causes of megaesophagus are found in the literature. one case report describes a young cat with megaesophagus secondary to a large nasopharyngeal polyp that extended into the cervical esophagus. 10 megaesophagus resolved once the polyp was removed. in another report, a young cat with diaphragmatic hernia was diagnosed with megaesophagus and gastric dilation. 31 megaesophagus resolved with medical treatment and surgical correction of the diaphragmatic defect. clinical signs are typically those of esophageal dysfunction; regurgitation is the most consistently found sign. regurgitation may not be closely related in time to eating if the esophagus is markedly distended and holds food. cats with long-standing disease may suffer from weight loss or secondary rhinitis. the appetite is typically normal or increased. additional signs may occur if systemic neuromuscular disease is present. aspiration pneumonia may cause fever, dyspnea, and cough. two case reports describe cats with idiopathic cough, mucopurulent nasal discharge, and fever may be found if aspiration has occurred. trauma to the esophagus may cause esophagitis and even esophageal stricture. perforation of the esophagus by the foreign body may lead to pneumothorax, pneumomediastinum, or pyothorax with signs of depression, anorexia, fever, and dyspnea. if the perforation occurs in the cervical esophagus, swelling, cellulitis, and drainage of serous or purulent material may be noted. many foreign bodies are readily diagnosed with survey radiographs, especially if they are radiopaque. other radiographic findings include an esophagus dilated with fluid or air. radiolucent objects may be detected with an esophagram. care must be taken when performing esophagrams on cats that may have an obstruction, because aspiration is a concern. if abnormalities that could be consistent with an esophageal perforation (e.g., periesophageal gas or fluid, pleural effusion) are detected on survey radiographs, an aqueous iodine contrast solution should be used. removal of esophageal foreign bodies should be performed as soon as possible to minimize esophageal trauma and pressure necrosis. endoscopy can be used to confirm the diagnosis and often to remove the object. both rigid and flexible endoscopes may be used along with accessories such as various forceps and foley catheters. care should be taken to remove the object as atraumatically as possible, especially if the object is sharp or pointed. if the object is in the caudal esophagus and it cannot be grasped and removed, an attempt should be made to gently push it into the stomach, where it can be retrieved using laparotomy and gastrotomy. if esophageal perforation has occurred, esophagotomy is recommended and is described elsewhere. 5, 14 removal of fish hooks may require a combination of surgery and endoscopy. 5, 39 a surgical approach to the esophagus is made, but the esophagus is not incised; rather, the portion of the hook protruding through the esophagus is cut and removed, and the endoscope is used to retrieve the remainder. clinical sign is regurgitation, and most patients are underweight. a distended cervical esophagus may be palpated, and secondary aspiration pneumonia may occur. a history of regurgitation since weaning is very suggestive of a vascular ring anomaly, but other causes of regurgitation must be ruled out. survey radiographs show a dilated esophagus cranial to the heart, while the caudal esophagus is usually normal. the bulge of the aortic arch normally seen on a ventrodorsal radiographic view is absent. an esophagram is used to confirm the location of the obstruction and the severity of disease. definitive treatment is surgical repair of the vascular defect (i.e., ligation and transection of the ligamentosum arteriosum). some patients will require nutritional support through gastrostomy tube feeding and treatment for aspiration pneumonia before surgery. early diagnosis and surgical intervention brings the best prognosis for return of normal esophageal function. some affected cats are left with residual esophageal hypomotility, which is managed as for idiopathic megaesophagus. esophageal neoplasia is rare in the cat as in the dog. although parasitic granulomas caused by spirocerca lupi are associated with esophageal neoplasia in dogs, this parasite does not infect cats. both primary and metastatic esophageal tumors can occur in the cat. squamous cell carcinoma is the most common primary esophageal tumor in cats and is often found in the caudal two thirds of the esophagus. 7, 22, 25, 46 affected cats are middle aged or older. clinical signs are typically those associated with esophageal obstruction, such as regurgitation, dysphagia, odynophagia, and salivation. patients with advanced disease may suffer anorexia, depression, and weight loss. on physical examination, an esophageal mass may or may not be palpable. survey and contrast radiographs reveal esophageal dilation, a soft tissue mass, or periesophageal lesions that displace the esophagus. computed tomography is useful to identify periesophageal or intraluminal masses. definitive diagnosis is made with endoscopy and biopsy. mucosal biopsies are difficult to obtain, because the esophageal mucosa is tough; exfoliative cytology may also be helpful. treatment is rarely undertaken, because disease is often advanced at the time of diagnosis, and many patients have complications such as aspiration pneumonia. palliation may be attempted with chemotherapy or radiation, although data on efficacy is unavailable. in general, squamous cell carcinomas in other anatomic locations respond poorly to treatment. surgical resection may be attempted if anastomosis can be accomplished without excessive tension. megaesophagus and chronic vomiting associated with intermittent gastroesophageal intussusception. 35, 50 survey and contrast radiographs may identify a dilated esophagus (figure 23-6), but contrast fluoroscopy is the diagnostic tool of choice when available, because it allows for assessment of peristalsis. care must be taken with contrast studies because of the risk of aspiration. treatment of megaesophagus is largely symptomatic and supportive unless an underlying disorder can be identified and treated. frequent small meals are offered with the cat feeding in an upright position. the upright position should be maintained for at least 10 minutes after eating to allow for gravity-assisted passage of food into the stomach. this is best accomplished by having the owner hold the cat over their shoulder so that the esophagus is in a vertical position. 44 different types of diets should be offered to determine which is best for the individual patient; calorically dense diets may be beneficial for patients with weight loss. prokinetic drugs, such as cisapride, stimulate smooth muscle, but since most of the esophagus is skeletal muscle, the efficacy of such drugs is questionable for treatment of megaesophagus. prokinetic drugs also increase lower esophageal sphincter tone and may increase esophageal transit time, neither of which is desirable in patients with megaesophagus. vascular ring anomalies are congenital malformations of the great vessels that entrap the thoracic esophagus and cause obstruction. the most commonly reported anomaly is persistent right aortic arch. the esophagus is entrapped by the aorta on the right, the ligamentum arteriosum and the pulmonary trunk on the left, and the heart base ventrally. other vascular anomalies are rarely described in cats, such as a double aortic arch described in a siamese cat. 56 onset of clinical signs occurs around the time of weaning to solid food so that most affected cats are presented at less than 6 months of age. the most common surgery. 34 surgery is the treatment of choice for large defects, especially in young cats with congenital disease or cats that have failed medical management. various reconstructive surgical techniques have been described. 14 disorders of the hiatus are rare in cats. hiatal hernia is protrusion of the distal esophagus and stomach through the esophageal hiatus of the diaphragm into the thoracic cavity; the protrusion may be intermittent ("sliding") or persistent. other organs are occasionally involved, such as the omentum. 40 this is distinct from a gastroesophageal intussusception where the stomach is prolapsed into the lumen of the distal esophagus. 35, 49 both congenital and traumatic hiatal hernias have been described in cats. 9, 23, 41, 42, 52 congenital hernias appear to be more common than acquired hernias, and affected cats typically present with clinical signs before 1 year of age. it is suspected that increased inspiratory effort associated with upper airway obstruction, such as a nasopharyngeal polyp, may also lead to development of hiatal hernia. 23 hiatal herniation reduces lower esophageal sphincter pressure. clinical signs associated with hiatal hernia, such as intermittent vomiting and regurgitation, may be because of reflux esophagitis, hypomotility, or obstruction. large hernias and secondary aspiration pneumonia may be associated with respiratory distress. survey radiographs may reveal a gas-filled soft tissue density in the caudal dorsal mediastinum. an esophagram will show the gastroesophageal junction and gastric rugae cranial to the diaphragm (figure 23-7) . both fluoroscopy and endoscopy may be useful for diagnosis but are not typically necessary. the prognosis for cats with hiatal hernia is considered to be good. a trial of medical management (as for reflux esophagitis) for 1 month has been recommended before the stomach is a frequent site for gastrointestinal problems in cats, and the most common gastric problems are described in this chapter. some conditions such as gastric dilatation-volvulus are often reported in dogs but rarely reported in cats. in one report of three feline cases, all were associated with diaphragmatic hernia. 15 gastric parasites, the diagnostic approach to the vomiting cat, the gastric emptying time of normal cats is shorter than that of other mammals. in one study, the gastric emptying half-time for solid food in normal cats was 1.4 to 3.6 hours. 53 this implies prolonged fasting (longer than 8 hours) in preparation for anesthesia and surgery is unnecessary. the main clinical sign of gastric disease is vomiting, but it is important to note that vomiting is also associated with many nongastric problems, including concurrent intestinal disease, such as enteritis or colitis. vomiting patients therefore require a thorough physical examination and diagnostic plan to determine the cause. vomiting must be distinguished from regurgitation, which is primarily associated with esophageal disease (see table 23 -10). vomitus often contains food, hair, refluxed bile, and therapeutics for vomiting are covered elsewhere in this chapter. the anatomy of the feline stomach is similar to that of other mammals having a simple glandular stomach. most of the stomach is situated on the left side of the abdominal cavity. it has five regions, starting from the lower esophageal sphincter: cardia, fundus, body, antrum, and pylorus ( figure 23-8) . the pylorus of the cat is unique compared with other species in that it is narrow and has high resistance in order to maintain a tight seal ( figure 23 involve a wide variety of objects, including linear objects (e.g., dental floss, thread with or without a needle, tinsel, string). the owner may or may not be aware of the ingestion. ingestion of multiple foreign bodies may be seen in cats with pica ( figure 23-12 ). in one case report, a young domestic shorthair cat required gastrotomy for removal of 32 copper pennies. 43 some patients require multiple surgeries, because of repeated foreign body ingestion. 22 in such cases, a behavioral diagnosis should be sought and treatment instituted (see chapter 13) . trichobezoars (large masses of hair) also represent a type of foreign object. both long-and shorthaired cats may be affected. hair is normally ingested during grooming and is eliminated in vomitus and feces. cats lack the strong peristaltic contractions ("housekeeper" contractions) that clear the stomach of undigested contents normally found in other species. this may explain why cats seem to be susceptible to gastric trichobezoars. gastric motility dysfunction is suspected to cause repeated gastric trichobezoars in some cats. intestinal 3, 22 and esophageal 14, 59 obstruction with trichobezoars has also been documented. traditional treatments for cats with recurrent trichobezoars include regular grooming, shaving the hair coat of long-haired cats, flea control, or blood. fresh blood may appear as large or small clots. older blood clots have a brown "coffee ground" appearance. gastric bleeding may also cause melena. other clinical signs may be associated with gastric disease, such as anorexia, weight loss, pain, lethargy, bloating, and nausea. gastritis may be acute or chronic in nature, and this distinction may be useful in assessing the potential cause. for example, cats with acute gastritis may be suspected of foreign body or plant ingestion, drug or toxin exposure (see chapter 31), or dietary indiscretion. cats with chronic gastritis may be suspected of parasitism, helicobacter spp. infection, or dietary intolerance or hypersensitivity (see chapter 17) . chronic lymphocytic plasmacytic gastritis of unknown etiology is also a common cause of chronic vomiting. whenever possible, a specific underlying cause should be sought and treated. patients with sudden onset of vomiting may have an obvious cause in the history (e.g., dietary indiscretion), but in many cases, the cause is not apparent. abdominal radiographs should be taken if foreign body ingestion is possible, especially in a young cat. if the patient is systemically well, further diagnostic testing may be postponed pending response to therapy. treatment for uncomplicated acute gastritis is symptomatic and supportive. clinical signs are expected to resolve in 24 to 48 hours; if signs persist, re-evaluation and further investigation is warranted. subcutaneous fluid therapy using an isotonic balanced electrolyte solution may be used to correct mild fluid deficits (<5%). oral intake of fluids and food should be discontinued for up to 24 hours. a highly digestible diet, either commercial or homemade, is introduced with a gradual transition back to the normal diet over the next several days. antiemetic therapy may be indicated for acute uncomplicated gastritis if the vomiting is frequent or the cat has signs of nausea (see table 23 -3). protectants, such as kaolin and pectin, are difficult to administer to cats and are without proven efficacy. bismuth subsalicylate is controversial; it is considered contraindicated by some experts, because of the cat's sensitivity to salicylates, 39 yet is commonly used in clinical practice. cats ingest foreign bodies less commonly than dogs. in one study of 208 cases of gastrointestinal foreign body ingestion, only 12% were in cats. 22 foreign body ingestion is most likely to be seen in young cats and may a b taken just before surgery to ensure the object has not moved further down the gastrointestinal tract. postoperative management after gastrotomy includes maintenance of hydration and electrolyte balance. hypokalemia is common with anorexia and vomiting and should be treated by supplementation of iv fluids with 20 to 40 meq/l potassium chloride (not to exceed 0.5 meq/kg/hour). refractory vomiting should be treated with an antiemetic. a highly digestible diet can be introduced the day after surgery. in general, the prognosis for recovery is good. in one study, 88% of cats with gastrointestinal foreign bodies survived to discharge. 22 those cats that did not survive had linear foreign bodies of long-standing duration with subsequent peritonitis. helicobacter are spiral or curved gram-negative bacteria that inhabit the glands, parietal cells, and mucus of the gastric antrum and fundus. helicobacter contain large amounts of urease, which alters the ph in the vicinity of the bacteria and allows for colonization of the acidic environment of the stomach. in the early 1980s, the discovery of the association of helicobacter pylori with gastric disease (gastritis, peptic ulcers, and neoplasia) in humans revolutionized treatment of those diseases. since then, helicobacter spp. have been associated with gastric disease in various veterinary species, including cats and dogs. several helicobacter spp. (e.g., h. heilmannii, h. bizzozeronii, h. felis) have been identified in cats, some of which have the potential to infect humans, although transmission is thought to be rare. 19, 48 the prevalence of helicobacter infection in cats varies geographically and may be very high (>40%) in some locations. 1, 27, 37, 47, 58 the importance of helicobacter as a cause of gastric disease is cats is unclear; the bacteria may be found in the stomach of both clinically normal cats and cats with gastritis. the prevalence of helicobacter infection is not higher in cats with gastritis compared with normal cats. 61 determination of the role of helicobacter is also hampered by the paucity of controlled clinical trials that evaluate eradication of gastritis and clinical signs in infected cats. an immune response to infection characterized by gastric lymphoid hyperplasia is common, although the local immune response in cats is generally less severe than the response in humans infected with h. pylori. to date gastrointestinal ulcers have not been associated with helicobacter infection in cats. recent studies have suggested a possible association between helicobacter infection and gastric lymphoma in cats, although more research is needed to confirm the association and understand the pathogenesis. 7, 32 helicobacter spp. may be commensal in most cats, and perhaps loss of tolerance explains the development of gastritis in some individuals. 49 another possibility is that the inflammatory response is normally well managed and disease may treatment of underlying dermatologic disorders, and administration of semisolid petroleum laxatives. more recently, commercial diets have been formulated for control of trichobezoars. cats with recurrent trichobezoars causing illness and suspected motility disorders may benefit from treatment with prokinetic drugs such as cisapride. clinical signs of gastric foreign bodies are variable but typically involve intermittent or persistent vomiting because of gastric outflow obstruction, distention, and mucosal irritation. gastric obstruction may be partial or total. patients with complete obstruction will present with more dramatic signs, including anorexia and depression. the base of the tongue should always be examined, because linear foreign bodies are sometimes anchored either in this location, or they may be lodged in the pylorus, causing intestinal plication. gastric foreign bodies may also be asymptomatic and found incidentally. 5 physical examination may be unremarkable or may reveal dehydration or abdominal pain. if the stomach is markedly distended, the foreign body may be palpable in some patients. survey radiographs are always indicated when foreign body ingestion is suspected. radiopaque foreign bodies may be readily diagnosed, although some, along with radiolucent objects, will require a contrast study for diagnosis ( figure 23-13) . barium is commonly used as a contrast agent, although if gastric perforation is suspected, an aqueous iodinated agent is preferred. ultrasonography is also useful for detection of gastrointestinal foreign bodies. 56 removal of some foreign bodies can be attempted endoscopically, particularly if the object does not have sharp edges and is not too large. successful removal of fish hooks, particularly single-barb hooks, using endoscopy has been described. 35 otherwise, foreign objects are best removed using gastrotomy through a ventral midline laparotomy. a radiograph should always be to know when treatment should be attempted. one expert recommends treating only patients with clinical signs of gastritis that have biopsy-confirmed helicobacter infection with a treatment regimen of amoxicillin (20 mg/kg, every 12 hours, po), clarithromycin (7.5 mg/ kg, every 12 hours, po) and metronidazole (10 mg/kg, every 12 hours, po) for 14 days. 49 a common dilemma would be determining the treatment of choice for patients with lymphoplasmacytic inflammation of the stomach and small intestine and confirmed helicobacter infection. are such patients best treated for inflammatory bowel disease, helicobacter infection, or both? currently, guidelines for determining the best treatment approach are lacking. also, few studies on the efficacy of combination therapy have been conducted in cats. long-term eradication of infection may be difficult, and histopathologic resolution of gastritis may not be possible, which raises the question of whether helicobacter is the true underlying cause. 38, 41 in one study, two cats with clinical gastritis and helicobacter infection were treated with oral metronidazole, amoxicillin, and bismuth subsalicylate for 3 weeks and were also fed a commercial elimination diet. 25 posttreatment gastric biopsies were obtained a mean of 7 weeks after the cessation of treatment. resolution of clinical signs occurred rapidly, and clearance of helicobacter spp. was achieved at that time point, but gastric inflammation persisted in post-treatment biopsies. in another study, 13 cats with asymptomatic helicobacter infection were treated with oral omeprazole, amoxicillin, metronidazole, and clarithromycin for 14 days. 26 treatment failed to eradicate infection in 4 of the cats based on molecular analysis of post-treatment gastric biopsies. it is unclear if treatment failure is because of recrudescence or reinfection. the reader is referred to excellent reviews of helicobacter in cats for more information. 27, 38, 48 chronic gastritis chronic gastritis is common in cats with chronic intermittent vomiting. ollulanus tricuspis is a worm that infects the stomach of cats, causing chronic gastritis, and it is difficult to diagnose (see below, gastrointestinal parasites). the worm is occasionally found on histologic examination of gastric biopsy samples. 9 it is reasonable to treat empirically (fenbendazole 10 mg/kg, once daily, po × 2 days) for this parasite when the cause of gastritis is not apparent. 49 the frequency of vomiting in cats with chronic gastritis is highly variable, ranging from once or twice per week (and not necessarily every week) to more than once daily. most patients are otherwise well, although other clinical signs (inappetence, anorexia, depression, or weight loss) are possible depending on disease severity. results of routine laboratory testing are typically normal but may show neutrophilic leukocytosis, result when there is an abnormality of the immunoregulatory system. 21 the most commonly used methods for diagnosis of helicobacter infection in cats are based on gastric specimens obtained during endoscopy (or laparotomy): exfoliative cytology, histopathologic examination of biopsy specimens, and rapid urease testing of biopsy specimens. 28 however, it is important to note that even when helicobacter organisms are identified, the infection may not be the cause of the patient's clinical signs, and other causes of vomiting should always be evaluated. exfoliative cytology is the least expensive and most easily performed diagnostic test. in one study, it was also the most sensitive diagnostic method when compared with urease testing and histologic examination. 20 brush cytology samples gathered during endoscopy are airdried on microscope slides and stained with wright's stain. the slide is examined at 100× magnification under oil immersion. spiral bacteria are readily seen if present. at least 10 oil-immersion fields on two slides should be examined before determining a specimen is negative for helicobacter-like organisms. 28 since helicobacter produce abundant urease, a rapid urease test (e.g., clotest, ballard medical products, draper, utah) may be used for diagnosis. 38 the kit consists of an agar gel impregnated with urea and a ph indicator. a gastric biopsy sample is applied to the gel, and if urease is present, ammonia will form and change the ph (and thus the color) of the gel. the gel may change color rapidly (within 30 minutes), but 24 hours must elapse before the test can be considered negative. 28 the more rapidly the color changes, the higher the bacterial load. both false-positive and false-negative results are possible with rapid urease testing for various reasons, giving the test a sensitivity of 70% to 90%. 28, 37 histopathologic examination of gastric biopsy samples using hemotoxylin and eosin (h&e) or silver stains is highly sensitive and specific in human studies for detection of helicobacter-like organisms. the organisms are not equally distributed; so, examination of biopsy specimens from multiple sites will increase sensitivity. the bacteria may be seen in mucus on the surface epithelium as well as in the gastric pits, glandular lumen, and parietal cells. organisms may also be seen submucosally within gastric lymphoid follicles. 46 histopathologic examination of biopsy samples also allows for assessment of other abnormalities. mild to severe lymphocytic-plasmacytic or lymphocytic gastritis may be present. in humans combination therapy with antibiotics and antisecretory drugs is recommended to reduce the risk of gastric ulcers and cancer from h. pylori infection. treatment is highly successful at eradicating both clinical signs and histologic changes in the gastric mucosa. since helicobacter infection is common in cats, yet no clear pathogenic role has been established, it is difficult cases. 29 depending on the underlying cause and severity of disease, abdominal pain, anorexia, lethargy, pale mucous membranes, and drooling may also be seen. cats with neoplastic disease may have prolonged clinical signs and are more likely to present with anorexia and weight loss. 29 cats with perforated ulcers may or may not present with signs of shock. diagnosis may be problematic because the clinical signs and physical examination findings are often not specific, even in cats with perforated ulcerations. 8 the causes of gastric ulceration in cats are not well characterized. in dogs the most common cause is the administration of ulcerogenic drugs, particularly nsaids, either alone or in combination with corticosteroids. several cases of nsaid-induced gastroduodenal ulceration or perforation have been reported in cats. 8, 34, 45 additional cases may be reported in the future, because long-term administration of these drugs is gaining in popularity for treatment of chronic diseases such as osteoarthritis. nsaids cause direct mucosal damage and interfere with prostaglandin synthesis. although inhibition of the cox-1 enzyme is thought to be the cause of adverse effects, such as gastric ulceration, even cox-2-selective drugs have been associated with adverse effects, and safety in sick cats is not well evaluated. recently, guidelines for the long-term use of nsaids in cats were published by the international society of feline medicine and the american association of feline practitioners. 51 the recommendations include administering nsaids either with or shortly after food, withholding therapy if inappetence or anorexia develops, determining dose based on lean body weight, and titrating to the lowest effective dose. neoplastic causes of gastric ulceration include systemic mastocytosis, mast cell tumor, lymphosarcoma, adenocarcinoma, and gastrinoma (zollinger-ellison syndrome). cats with chronic renal disease may suffer mucosal damage from uremic toxins and increased gastric acid production secondary to hypergastrinemia (because of decreased renal metabolism of gastrin). 18 hepatic disease is a cause of gastric ulceration in dogs but is uncommonly reported in cats. 23 recent anesthesia and surgery have been implicated as a cause of gastric ulceration and perforation, perhaps through hypovolemia, hypoperfusion, or stress. 8, 29 other non-neoplastic causes reported for gastric or gastroduodenal ulceration in cats include parasites (e.g., ollulanus tricuspis, toxocara cati, aonchotheca putorii, gnathostoma spp.), bacterial infections, toxins, inflammatory bowel disease, and foreign bodies. one case report describes a cat with severe gastric ulceration caused by intoxication with dieffenbachia leaves. 36 in some case reports, the cause for the gastric ulcerations could not be determined. a minimum database should be collected for cats suspected of gastric ulceration, to identify underlying diseases. anemia, usually regenerative, may be present. eosinophilia, or hypoproteinemia. survey and contrast radiographs are often normal. the most common finding on histopathologic examination of biopsy samples is lymphocytic plasmacytic (lp) gastritis ( figure 23-14) . some patients will also have concurrent evidence of lp inflammation in the small intestine, pancreas, and/or liver. such patients will be treated for their concurrent problem; treatment of inflammatory bowel disease, pancreatitis, and cholangiohepatitis is covered elsewhere in this chapter. some cats with chronic lp gastritis respond to treatment for dietary intolerance or hypersensitivity with a limited antigen diet (see chapter 17) . patients with moderate to severe lp gastritis may be best treated with a limited antigen diet and immunosuppressive therapy (prednisolone 1 to 2 mg/kg/day, po tapering to every other day at the lowest dose that controls clinical signs). patients that fail this initial treatment approach may require additional immunosuppressive therapy, such as chlorambucil (see table 23-9) . occasionally, cats with chronic gastritis are diagnosed with eosinophilic inflammation on histopathologic examination of biopsy specimens. treatment is similar to that for lp gastritis, although such patients should be evaluated for evidence of hypereosinophilic syndrome and eosinophilic enteritis. eosinophilic fibrosing gastritis was suspected to be caused by toxoplasmosis in one case report. 33 gastric or gastroduodenal ulcerations are uncommon in the cat compared with the dog and may be caused by a variety of disorders, both gastric and nongastric. 29 classical clinical signs include vomiting, hematemesis, and melena. however, in one review of eight cats, hematemesis and melena were present in less than one third of suturing of the ulcer site as well as collection of biopsy samples for histopathologic examination. the prognosis for recovery was excellent in two studies, particularly for cats with non-neoplastic causes of gastric or gastroduodenal ulceration. 8, 29 in one study of seven cats with perforated gastric or duodenal ulcers, the survival rate was low (14%). 23 disorders of gastric motility are better characterized in dogs than in cats. the most common clinical sign is vomiting of undigested food 8 hours or more after a meal. if outflow obstruction is present, vomiting may be projectile. there may also be a history of recurrent trichobezoars. various disorders are associated with impaired gastric motility, such as chronic gastritis, drug therapy (e.g., anticholinergic and narcotic drugs), dysautonomia, gastric neoplasia, metabolic disorders (e.g., hypokalemia), and temporary postsurgical gastroparesis. in some cases of chronic motility dysfunction, no cause can be identified. outflow obstruction may be caused by neoplasia, foreign bodies, and extragastric masses. pyloric stenosis is infrequently documented in young cats, often siamese cats. 4, 40, 55 since the range of underlying disorders is diverse, the diagnostic approach should allow for detection of both gastric and nongastric disorders. a minimum database (cbc, serum chemistries, urinalysis, feline leukemia virus [felv] and feline immunodeficiency virus [fiv] serology) is used to establish overall health status. radiographs are used to confirm presence of food in the stomach for longer than 8 hours. ultrasonography may detect gastric lesions, such as masses. endoscopy is used to identify outflow obstruction as well as other lesions, such as ulcers, and evidence of gastritis. assessment of gastric emptying using nuclear scintigraphy is the most accurate method but is limited to referral centers. gastric emptying times for liquids, canned food, and dry diets have been established using nuclear scintigraphy. 11, 16, 17 however, emptying times are variable, depending on the amount and type of diet fed as well as the amount of water ingested. even the shape of kibble affects emptying time. 2 radiographic contrast series are widely used, but gastric emptying times are variable for barium in either liquid form or mixed with canned food. contrast radiography using liquid barium (8 to 10 ml/kg) is performed in a fasted patient. radiographs are taken immediately after administration of the barium and again at 15 and 30 minutes, in some cases, also at 1 and 3 hours. liquid barium is expected to enter the duodenum no more than 30 minutes after administration, and the stomach should be completely empty of barium within 3 hours. the clinician should be aware that some cats with gastric motility disorders will have other findings will be dependent on the presence of underlying diseases; for example, azotemia and isosthenuria may indicate renal disease. electrolyte and acidbase abnormalities may be because of chronic vomiting and anorexia. survey and contrast radiographs and ultrasonography are primarily useful to rule out other causes for the clinical signs, such as foreign bodies. cats with perforated ulcers may have evidence of pneumoperitoneum (sometimes severe) on plain radiographs or ultrasonographs, and this is an indication for surgical exploration. 6, 8, 24, 31, 34 evidence of peritonitis on imaging studies should be followed with peritoneal fluid analysis. a definitive diagnosis may be made using endoscopy, which allows direct visualization of lesions and collection of biopsy samples. however, some cats with gastric ulceration present in poor condition, which may preclude the use of endoscopy because of anesthetic risk and risk of ulcer perforation. 29 the location of ulcers is typically pyloroantral or fundic in cats with nonneoplastic disease. 8, 29 areas of erosion may appear pale or hemorrhagic; the mucosa is often friable and bleeds easily. fresh or clotted blood may be seen in the stomach lumen. in some cases, mucosal ulceration must be distinguished from ulcerated tumors. nsaid-induced ulcers are typically found in the antrum and do not have marked mucosal thickening; ulcerated tumors frequently have thickened edges and surrounding mucosa. 49 biopsy samples should be taken at the periphery of the ulcer to avoid perforation. treatment should be directed at any underlying disorder. treatment for nsaid toxicity is described in chapter 31. general supportive measures include fluid therapy and electrolyte replacement; blood transfusion may also be required (see chapter 25) . gastric acid production can be decreased with the use of h 2 -receptor blockers or proton pump inhibitors, and sucralfate is used as a mucosal protectant (see table 23 -5). sucralfate may inhibit absorption of other oral medications and should be given 2 hours apart from other drugs. if vomiting is severe or persistent, antiemetic therapy is warranted (see table 23 -3). analgesia should be provided for painful patients; a good choice is the opioid buprenorphine (see table 6 -1). broad-spectrum antibiotic therapy is indicated for patients with significant mucosal barrier dysfunction, perforation, leukopenia and/or neutrophilia, fever, and melena. surgical intervention is warranted for patients with life-threatening hemorrhage, failure to respond to medical management, or evidence of perforation. 29 the entire abdominal cavity and gastrointestinal tract should be thoroughly explored to locate extragastrointestinal lesions, non-perforated ulcers, and multiple ulcers. in one case series, nonperforated ulcers were detected at laparotomy by association with adhesions or a gastric mass. 29 surgical management includes débridement and months. physical examination findings are nonspecific, although occasionally a gastric mass or gastric thickening may be palpated if the stomach is markedly enlarged. results of routine diagnostic testing are generally nonspecific; anemia may be associated with ulceration. survey or contrast radiography may reveal a mass ( figure 23 -15, a); other findings include delayed gastric emptying, impaired motility, and mucosal ulceration. ultrasonography is also useful for diagnosis and can be used to guide needle aspirates of masses ( figure 23-15 , b). endoscopy allows for visualization of lesions as well as the ability to obtain partial thickness biopsy samples. problems with interpretation of endoscopic biopsy samples include detection of necrosis, inflammation, and ulceration rather than the primary lesion. in dogs some neoplastic lesions are submucosal, making it very difficult to obtain diagnostic samples by endoscopy. therefore several biopsies should be taken and masses should be biopsied multiple times in the same place to sample deeper tissues. the center of ulcerated lesions should not be biopsied. surgical biopsies are more reliable for diagnosis. a normal gastric emptying time with liquid barium. barium can also be mixed with canned food and fed as a meal; retention of barium-containing food in the stomach for more than 8 to 12 hours is abnormal. gastric emptying time may also be established with the use of barium impregnated polyspheres (bips; med i.d. systems, grand rapids, mich.) and radiography. gastric emptying times for bips have been established in healthy fasted and fed cats as well as in sedated cats, 10,52 but the values do not correlate well with scintigraphic studies. 17 a mixture of small (1.5 mm) and large (5 mm) spheres are administered with food, and two to four radiographs are taken over the next 24 hours. the small spheres are intended to mimic liquid transit time and the large spheres solid transit time. however, studies assessing the clinical relevance of this method are lacking. one review concluded that bips are probably sufficiently sensitive to detect grossly delayed gastric emptying. 60 treatment of gastric emptying disorders is directed at identifiable causes. treatment for gastric ulcers, chronic gastritis, and foreign bodies is described elsewhere in this chapter. pyloric stenosis is managed surgically. if no outflow obstruction exists, treatment with prokinetic agents, such as metoclopramide or cisapride, may be beneficial (see table 23 -3). gastric tumors account for less than 1% of malignancies in dogs and cats. 30 benign gastric tumors are even less common than gastric malignancies. gastric smooth muscle hamartoma has been reported in one 11-year-old cat. 50 although adenocarcinoma is the most common gastric cancer of the dog, lymphoma is the most common gastric cancer in the cat. feline gastrointestinal lymphoma occurs as two major types: small cell (lymphocytic) and the more aggressive large cell (lymphoblastic) form. small cell lymphomas are more frequently enteric. 57 in one study of 12 cats with gastric lymphoma, diffuse large b-lymphocyte tumors of immunoblastic nuclear type predominated. 42 gastric lymphoma is not associated with felv, and the role of helicobacter in the development of gastric lymphoma in cats requires investigation. 7 adenocarcinoma, 12,13,54 plasmacytoma, 62 and gastric carcinoid 44 have also been described. the siamese cat may be predisposed to adenocarcinoma. 12, 54 as would be expected, most cats with gastric neoplasia are older cats. as for most gastric diseases, vomiting is the most common clinical sign of neoplasia. the vomitus may contain blood and melena may be present. other clinical signs include anorexia, weight loss, bloating, and depression. perforation of the tumor may occur, leading to pneumoperitoneum or septic peritonitis. clinical signs present gradually and are often present for weeks to surgical resection is the most common treatment for gastric neoplasia other than lymphoma ( figure 23 -16). the prognosis for most patients is poor, typically because of debilitation, concurrent diseases, and recurrent or metastatic disease. 30 the success of chemotherapy for lymphoma depends on cell type, with small cell tumors carrying a better prognosis than large cell tumors. in diarrhea can be defined as increased volume and/or increased frequency of defecation of stools with increased water content. approaches to diarrhea, as for any clinical sign, need to take into account the individual animal. for example, neoplasia is much less likely to occur in a kitten than in a geriatric cat. in many cases, the precise diagnosis of gastrointestinal disease cannot be reached without biopsy samples. the decision to obtain biopsy samples should follow a logical pathway that is appropriate to the cat's condition. these are summarized in figure 23 -17. for example, many cases of acute diarrhea in a well cat can resolve with limited or no intervention, and so do not require a precise diagnosis. the diagnostic steps are 1. signalment and clinical history 2. physical examination 3. fecal assessment 4. blood and urine testing 5. imaging (radiography, ultrasonography) 6. biopsy samples these steps do not include treatment/diet trials or other empiric therapies that are appropriate in many cases. steps 3 and 4 are often undertaken at the same time, and there is no definite order for these steps. they are divided here for reasons of clarity. in a younger cat, where infectious causes are more likely, thorough fecal testing is more important; in an older cat, extragastrointestinal diseases, such as hyperthyroidism, are more likely; so, blood and urine testing is more important, but fecal assessment should not be neglected. the decision to proceed to step 4 (and each subsequent step) should take into account several considerations. the main considerations in assessing and managing a cat with diarrhea are • is there an acute onset or a chronic time course? • are there any dietary changes or indiscretion? 36 consider treatment trials: antibiotics (e.g., amoxicillin/ clavulanate ؉ metronidazole). food trials with novel proteins (e.g., rabbit, kangaroo, venison). • is the cat well or unwell? • is there primary or secondary gastrointestinal disease? • is there small or large bowel diarrhea? the components of the clinical history for cats with diarrhea are detailed in table 23 -12. after establishing the cat's age, breed, vaccination, and deworming history, it is important to establish the duration and nature of the diarrhea. chronic diarrhea is usually defined as greater than 3 weeks in duration and mostly warrants at least some degree of a diagnostic workup, whereas acute diarrhea is often self-limiting in a well cat. a description of the feces helps determine whether the diarrhea is small or large bowel in origin (table 23 -13); this will affect how any investigations might proceed. important questions to ask concern frequency of defecation (and how this compares with the normal state), tenesmus (straining usually indicates large bowel diarrhea, since an irritated colon leads to urgency), volume of feces (smaller volumes are typical of large bowel diarrhea; larger volumes are more typical of small onset and duration of diarrhea acute versus chronic? acute diarrheas are abrupt in onset and of short duration, and generally they are self-limiting. chronic diarrheas persist usually longer than 3 weeks and fail to respond to symptomatic therapy. appearance of diarrhea quantity and quality of the stool (color, consistency, character, presence of blood or mucus)? loose to watery feces that contain fat droplets, undigested food, melena, and variable colors suggests small intestinal disease. the volume is always increased with small intestinal disease. loose to semisolid feces containing excess mucus and fresh blood (hematochezia) indicates large intestinal disease. the volume may be normal to slightly decreased with large intestinal disease. description of defecation process tenesmus (straining) and dyschezia (painful defecation)? these are hallmarks of large intestinal disease (e.g., inflammatory or obstructive lesions of the colon, rectum, or anus). frequency is normal to slightly increased with small bowel disease, but greatly increased with large bowel disease. associated physical signs vomiting, anorexia, weight loss, and dyschezia may help localize the disorder to a specific part of the gastrointestinal tract. clinical signs relating to problems in other organs or body systems should be noted and may suggest a more generalized disease. vomiting may occur as a consequence of small intestinal inflammation in some cats with diarrhea. weight loss may result from decreased caloric intake (anorexia), decreased nutrient assimilation (maldigestion/malabsorption), or excessive caloric loss (protein-losing enteropathy or nephropathy). weight loss is observed uncommonly with large bowel disease. in many cases, the answers to these questions are obvious. for example, a cat may seem well but has had access to lilies (the author has seen diarrhea as a primary presenting sign for this!) or has a palpable abdominal mass. substantial weight loss is an indicator that further investigations are warranted sooner rather than later. if the decision is made for empiric management and outpatient care, it is vital to follow up either by scheduling a recheck visit or calling the client, because simple acute problems can turn into complicated chronic problems. if the diarrhea has been present for less than a week and the cat has no weight loss, dehydration, fever, or palpable abdominal abnormalities, it is appropriate to manage the cat as an outpatient. even in the absence of fecal testing, it is appropriate to deworm the cat (see the section gastrointestinal parasites). the cat should be fasted for 24 hours (12 hours, if less than 4 months old) and then fed a bland diet (such as plain, cooked, skinless chicken, or low-residue prescription diets designed for cats with gastrointestinal problems). it is appropriate to maintain the cat on the low-residue diet for at least 7 to 10 days and then slowly reintroduce the regular diet. fecal assessment is mostly used to assess infectious agents, such as parasite-associated diarrhea, but the importance of assessing feces, even when parasitic or bacterial infections are not suspected, should not be underestimated. gross examination of feces can determine if melena or fresh blood or mucus are present to help distinguish large from small bowel disease when the owner's observations may be misleading. occult fecal blood can be an indicator of gastrointestinal inflammation in cases of subtle disease, and undigested starches and fats can indicate maldigestion or malabsorption. 8 for assessment of feces for parasites, the fecal sample should ideally be fresh (<1 hour old). refrigeration (for no longer than one week) can preserve ova, oocysts, and cysts but not protozoal trophozoites. feces should be assessed by a. to assess for trophozoites bowel), how formed the stool is (from soft stool to cow-pat consistency to liquid tea; usually more watery stool relates to small intestinal disease), color (darker indicates digested blood), and presence of any mucus or blood (presence relates to large bowel). most household toxins, such as plants, cause signs additional to diarrhea such vomiting or neurological signs, 11 but it is important to ascertain if the cat has had access to anything unusual. likewise, it is important to find out if the cat has had any possible exposure to dietary indiscretions; this can include if the cat has been seen with or is known to hunt prey including insects. cockroaches carry pathogenic bacteria 12, 18 and other prey such as birds and rats can carry salmonella; salmonellosis in cats has been dubbed songbird fever. 6 simple causes of self-limiting diarrhea include dietary change (either a new flavor or a new style of food, such as dry food for the first time); so, the owner must also be quizzed if anything new has been offered, either new cat food or treats (such as greasy fish or chicken). although the physical examination will usually determine how unwell a cat is, the owner's impressions are also important, because cats can hide signs from strangers, particularly in a practice setting. lethargy and inappetence are important signs, as ill cats typically do not eat well. the cat's general demeanor can be an indicator of how unwell a cat is and therefore dictate the extent of diagnostic testing required. this can be noted by assessing how interested the cat is in its surroundings or any behavior changes from previous visits, such as if a normally difficult-to-handle cat is placid. body weight should be assessed and, if possible, compared with that of previous visits (even those noted on a clinical record from another veterinarian). the body condition score (bcs) should also be assessed and can be very important when there is no prior weight information. dehydration is usually a sign that a cat needs more involved management. abdominal palpation should be performed to assess pain (where?), any masses (foreign bodies, lymph nodes, or even focally thickened intestines, such as with neoplasia), or turgid intestines. fever often indicates infection but can also reflect neoplasia or other inflammatory changes. a thorough examination of all body systems should always be performed, no matter what a cat presents for. in the case of diarrhea, extragastrointestinal signs can be of vital importance, such as a palpable thyroid and tachycardia suggesting hyperthyroidism. after the clinical history has been taken and the physical examination performed, the veterinarian must make the important decisions of whether any interventions are required and whether the patient should be factors affecting interpretation include whether the growth is a heavy and pure growth of a known pathogen, such as salmonella, campylobacter, yersinia, or clostridium difficile. further information about the relevance of culture and pcr results is contained below in the section infectious enteritis. investigations begin by assessing if the diarrhea is the result of primary gastrointestinal disease or secondary to another process, by performing routine serum/plasma biochemistries, hematology, urinalysis, and total t 4 (for older cats). in most cases of secondary gastrointestinal disease, diarrhea is not usually the primary presenting complaint, but since the approach to investigations and management diverge so much, this is an important step to take. biochemistry and urine tests may also show the consequences of diarrhea, such as dehydration and electrolyte abnormalities. a. can aid in the visualization of internal structures of some protozoa 3. fecal flotation (preferably with centrifugation) a. to find cysts, oocysts, and ova fecal culture should be undertaken with the understanding that bacteria will be cultured; so, interpretation is based on the relevance of the positive culture result. used to evaluate the smear for the presence of trophozoites, such as giardia spp. and tritrichomonas foetus. 1. place peppercorn size amount of feces on a warm slide and mix with a drop of 0.9% saline (smear must not be too thick, because trophozoites will be easily missed). 2. apply coverslip. 3. evaluate systematically for motile organisms using the 10× magnification. 4. confirmation at 40× magnification. adding iodine to a wet mount through the edge of the coverslip can aid in the visualization of internal structures of some protozoa. the direct wet preparation must be examined without any stain for motility first, because staining the preparation kills the organism. methylene blue is useful for identifying trophozoites, particularly those of entamoeba histolytica. this method has little to no diagnostic value for the diagnosis of bacterial-associated diarrhea. used to find cysts, oocysts, and ova in feces. standing (gravitational) flotation methods are easier and quicker but have much poorer sensitivity than centrifugation methods. 2 solutions used in centrifugation flotation methods include zinc sulfate and sheather sugar. 1. weigh out 2 to 5 g of feces. 2. mix feces with approximately 10 ml of flotation solution. 3. pour mixture through a tea strainer into a beaker or fecal cup. 4. pour strained solution into a 15-ml centrifuge tube. 5. fill tube with flotation solution so that a slight positive meniscus forms, being sure not to overfill the tube. 6. place a coverslip on the tube, and put the tube in the centrifuge. 7. make sure the centrifuge is balanced. 8. centrifuge at 1200 rpm (280× g) for 5 minutes. 9. remove the tube and let stand 10 minutes. 10. remove the coverslip, and place it on a glass slide. systematically examine the entire area under the coverslip at 100× magnification (i.e., 10× objective). you may wish to use the 40× objective lens to confirm your diagnosis and make measurements; however, with practice, most parasites can be identified using the 10× objective (100× magnification). (fpli) are useful markers of intestinal and pancreatic disease, [14] [15] [16] [17] but it is important to note that they typically do not give a precise diagnosis. cobalamin and folate are water-soluble vitamins and are readily found in commercial cat foods so that dietary insufficiency is rare, and decreased levels are almost always because of gi disease. these vitamins are taken up by specific receptors in different areas of the small intestine. chronic inflammatory gastrointestinal disease may damage the receptors and lead to decreased serum concentrations of one or both vitamins, provided the disease process is severe and long standing enough to deplete body stores. serum cobalamin and folate concentrations may also be decreased in cats with exocrine pancreatic insufficiency (epi). trypsin-like immunoreactivity is a pancreasspecific marker, and assessment of serum tli is used for diagnosis of epi and pancreatitis in the cat, although the sensitivity of the assay for pancreatitis is low. pli is a marker for pancreatic inflammation and is more sensitive than tli for the diagnosis of pancreatitis. since inflammation of the small intestine may be seen concurrently with pancreatitis, serum tli and pli are useful adjunctive tests in the diagnosis of diarrhea. tli, pli, and cobalamin are stable in serum at room temperature for several days, but folate is unstable so that samples for cobalamin/folate analysis should be frozen (table 23-14) . samples submitted for folate concentration should not be hemolyzed, because red blood cells contain high levels of folate. in addition, folate is light-sensitive, and samples should be wrapped to exclude light. severe lipemia may interfere with common assays for tli and pli. the main utility of these tests are to indicate that further investigation of gastrointestinal disease is warranted. when a cat presents for weight loss with no overt signs of gi disease, decreased cobalamin or folate can indicate that further investigations with imaging and, ultimately, biopsy sampling are warranted. many clients are more willing to proceed with hematology can be normal in some cats, with changes expected, and so should not be used to rule out any condition. it can be useful, for example, if there is a left shift neutrophilia, indicating acute infection, or eosinophilia, reflecting parasitism. monocytosis can suggest chronic disease that was not suggested by the clinical history. in the case of acute onset diarrhea, the cat may be unwell as a consequence of the diarrhea (e.g., from dehydration) and not because of the cause of the diarrhea. if rehydration is required (with intravenous or subcutaneous fluids, depending on severity of illness), then it is important that biochemistry tests are performed before fluid administration so that any diagnostic clues are not lost by alteration of the profile from the fluid therapy. fever and neutrophilia may indicate the need for antibiotic therapy. if infection is suspected, fecal sampling (see step 4) should occur before starting antibiotics. if a cat is unwell from dehydration, then further testing may not be warranted. the clinician should be alert that linear foreign bodies can result in diarrhea (see the section intestinal obstruction). diarrhea of chronic duration (greater than 3 weeks) does require a more thorough investigation at the outset. however, if clinically well, the cat can be managed as an outpatient in the first instance, at least while waiting for results of diagnostic testing. a diet trial with a novel protein is appropriate for a well cat with stable weight. as with any patient managed as an outpatient, follow-up is vital and, in this scenario, includes scheduling revisits. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity invasive diagnostics when a specific marker of the disease in the organ involved has been recognized. caution should be exercised, because either cobalamin or folate may not be reduced with gi disease. in one study of small cell lymphoma, only 78% of cats were hypocobalaminemic, 7 meaning that if this was the only instigating factor to investigate, nearly one fourth of cats would not have been investigated further. also, cobalamin may be reduced in nonalimentary illness. 1 2. to detect hypocobalaminemia that may indicate the need for supplementation for clinical improvement. 13 to recognize pancreatic pathology when fpli is increased. 17 it is important to note that an elevated value gives no indication of the nature of the pancreatic pathology. 4. to make a diagnosis of epi when the ftli is low. 15 it should be noted that epi can result from other pathology that may require further investigations. veterinarians if the patient is new to the practice) are usually helpful. body condition scoring (using a 5-point or 9-point scale) for every cat seen is helpful in recognizing those that are underweight. weight loss often occurs with loss of muscle mass in cats, and muscle mass can be assessed over the ribs and pelvis as well as scapulae and nuchal crest. thickened intestines are also a subjective finding; it is the author's opinion that thickened intestines are actually intestines with increased turgidity, since differences between normal intestines and those with inflammatory infiltrates can be as little as 0.5 mm. perhaps more important during the history taking and physical examination are those signs that can point to extragastrointestinal disease. when confronted with a cat showing weight loss or vomiting or diarrhea (or a combination of signs), the clinician should start with trying to distinguish the signs as being either primary gastrointestinal or secondary signs. examples of clues pointing to extragastrointestinal diseases include tachycardia and palpable thyroid nodule, indicating hyperthyroidism, or polydipsia/polyuria, which has a variety of causes but is not typical of primary intestinal disease. inflammatory bowel disease has traditionally been considered an immune-mediated disease. the local immune system of the intestinal mucosa no doubt plays an important role, but recent work has also shown the importance of the normal bacterial population in perpetuating and, perhaps, even initiating pathology. it is known for certain that ibds are an expression of an overanxious immune response, with a recent study 104 indicating increases in inflammatory (il-6), type-1 immunity (il-12 p40), and immunomodulatory (transforming growth factor [tgf]-beta, il-10) cytokines. other researchers have found an association with bacterial counts (enterobacteriaceae, e. coli, and clostridium spp.) and abnormalities in mucosal architecture, indicating that mucosal bacteria are involved in the etiopathogenesis. 65 we can summarize these theories by saying that ibds are likely to be a consequence of hypersensitivity reactions to antigens from the intestinal lumen (e.g., bacterial, parasitic, or dietary antigens). this hypersensitivity may occur because of failed immunoregulation (suppressive function) of the gut-associated lymphoid tissue (galt). it is known that granulomatous colitis in boxer dogs is associated with infection, 143 and pathogens may well be found in at least some cases of ibd in cats that cause the immune response and subsequent inflammatory infiltrate of the lamina propria typically seen. although not described specifically in cats, chronic intestinal inflammatory change can impair motility. inflammatory bowel disease (ibd) refers to intestinal inflammatory infiltrates of the small or large intestine (or both) of unknown etiology. the term ibd should strictly be applied to mean idiopathic ibd, thus excluding inflammatory enteritis because of food sensitivities, although common usage has led to ibd referring to intestinal inflammatory infiltrates of both known and unknown causes. ibd is not a diagnostic end point but a description of a series of intestinal diseases that have similar histopathology. recent efforts by the world small animal veterinary association (wsava) gastrointestinal standardization group have led to both diagnostic and classification guidelines 35, 168 ,169 that encompass chronicity, nonresponse to symptomatic treatment, no specific cause found, as well as histologic confirmation of non-neoplastic intestinal inflammatory changes. there are no obvious breed or gender predispositions, and although cats of any age can be affected, inflammatory intestinal diseases are more likely to occur in middle-aged to older cats (5 to 10 years of age or older) than in younger cats. presenting clinical signs include vomiting, diarrhea, and weight loss with increased or decreased appetite. these signs can occur in isolation or together. weight loss without vomiting or diarrhea deserves special mention because not only have several studies 38, 58 shown this to be the most common presenting sign for ibd, but many veterinarians do not consider primary intestinal disease without the presence of vomiting or diarrhea. weight loss despite normal to increased caloric intake can represent poor absorption of food because of small intestinal disease, although it can also represent maldigestion associated with exocrine pancreatic insufficiency or increased metabolism associated with hyperthyroidism, or even lack of energy utilization associated with diabetes mellitus. conversely, appetite may be reduced, most likely because of nausea. if the large bowel is affected, signs are typically discomfort when defecating, resulting in frequent small volumes of diarrhea, often with mucus and blood; if the large bowel alone is affected, there may be no weight loss. physical examination findings are often nonspecific, but the most consistent findings for small intestinal disease are weight loss (or being underweight in a cat not seen previously) and palpably "thickened" intestines. noting a cat as underweight can be subjective, and prior recorded weights (even from previous paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma than those with ibd, but this change was also seen in 12% of cats with normal small intestine. 177 inflammatory bowel diseases require histologic findings obtained from biopsy samples for diagnosis, but diagnosis should not be made solely on these findings. the wsava international gastrointestinal standardization group has proposed "an all encompassing definition of inflammatory bowel disease" that comprises clinical criteria, imaging criteria, as well as pathophysiologic criteria. 169 the clinical criteria for the diagnosis of ibd include there are no typical laboratory findings in ibd, and many cats may have entirely normal results from routine biochemical and hematologic investigations. moderate liver enzyme elevations may be seen 5,38,58,66 even in the absence of recognizable hepatic pathology, and this may reflect subclinical secondary hepatic disease, secondary cholestasis, or showering of the liver with inflammatory cells from the small intestine through the portal circulation. 66 other changes can reflect consequences of the intestinal disease, such as azotemia or hemoconcentration reflecting dehydration, 58 or hypokalemia reflecting inappetance. 38 the chronic inflammation may be reflected by neutrophilia, monocytosis, 5, 38, 58, 66 or hyperglobulinaemia. 38 hypocobalaminemia can reflect ileal inflammation, and low serum folate can reflect proximal small intestinal inflammation. 149 typical ultrasonographic findings consistent with ibd are focal or diffuse intestinal wall thickening ( figure 23-19) ; normal wall thickness is less than or equal to 2.8 mm for the duodenum and less than or equal to 3.2 mm for the ileum, 50 and large mesenteric lymph nodes with hypoechoic changes may be seen. one study found that ultrasonographic findings correlated with histologic grade of ibd. 5 there is no clear distinction between ultrasonographic changes from ibd and those from small cell lymphoma. one recent therapeutic trial, follow-up visits are vitally important. many cats with small intestinal disease may show initial improvement simply because of the diet having lower residue, since there is decreased substrate for intestinal bacteria to digest and lower osmotic potential. the corollary of this is that failure of one novel protein diet does not mean that all novel protein diets will fail. when food sensitivities are responsible for gastrointestinal clinical signs in cats, the responsible food ingredient is usually a dietary staple. commonly incriminated ingredients are beef, fish, wheat, and corn gluten. 55 a careful dietary history is therefore important. large bowel inflammation typically improves with higherfiber diets, 38, 58 and attempting a trial with such a diet is certainly appropriate. immune suppressive therapy is the mainstay of ibd treatment, and glucocorticoids, such as prednisolone, are most commonly used. sulfasalazine use for large bowel signs has not been critically evaluated but seems safe and effective. in cats with substantial weight loss or severe clinical signs, such as chronic diarrhea, the author prefers to start with corticosteroid therapy, even if dietary causes have not yet been ruled out. the diet should also be changed to one containing a novel protein, and, if and when clinical signs resolve, an attempt is made to wean the cat from corticosteroid therapy, hopefully to the point of being discontinued. a diet challenge can then be used to confirm the diagnosis of food sensitivity. there are no universal guidelines for doses of corticosteroids. the author prefers the use of orally administered prednisolone to reduce the chance of side effects and will choose the starting dose based on the severity of disease. the starting dose is usually 2 mg/kg, once daily, po (10 mg/cat/day for most cats) starting 10 days after biopsies have been obtained to allow time for the mucosa to heal. if there is an improvement noted after a recheck at 2 weeks, the higher dose is maintained for a further 2 to 4 weeks, at which point, many cats are back to their normal weight and are not exhibiting clinical signs. if this is the case, the corticosteroid dose can be weaned down to 1 mg/kg, po (often 5 mg/cat/day) for several months, with continued rechecks scheduled to assess weight, clinical signs, and diet. the goal is to wean down to the lowest effective dose. if hypocobalaminemia is present, cobalamin supplementation may be required. 144 cobalamin is administered parenterally at 250 µg/cat subcutaneously weekly for 6 weeks, then every second week for 6 weeks, then monthly. owners can be shown how to inject their cats (as practitioners routinely do with diabetics). clinicians often consider the assessment of histologic samples to be out of their hands; however, it is important to work with the pathologist by providing good quality samples and a good clinical history, as well as having an open dialogue if the findings are not within expectations. for example, with lymphocytic/plasmacytic infiltrations, the pathologist has the difficult task of distinguishing diseased from normal tissue in a site that is laden with lymphocytes in the healthy state. once deciding the tissue has pathology, the pathologist's next task is distinguishing inflammatory infiltrate from neoplastic infiltrate with normal, mature lymphocytes (as seen in small cell lymphoma). inflammatory change also results in changes to normal tissue architecture, with thickened villi, edema, or erosion of the epithelium being typical changes. clinicians should expect morphologic descriptions as well as assessments of degree and type of inflammation. these difficulties are further compounded with the recognition that histologic grading of mild, moderate, or severe does not necessarily correlate with severity of clinical signs. this means that a cat with severe clinical signs of weight loss and vomiting or diarrhea may have only mild histologic changes (and vice versa). concurrent inflammation of the pancreas and liver with intestinal inflammation was first described in the mid-1990s, 171 and despite constant reference to this phenomenon at conferences and veterinary websites, there has been little description since then, though one study found 70% of ibd cases had liver inflammation and 30% had pancreatic inflammation. 6 the term "triaditis" has frequently been used, but the author prefers to spell this "tri-iditis" to distinguish it from inflammation of the hepatic portal triads. there has been no assessment of prognosis when the pancreas and/or liver are involved, but the author has found no difference in prognosis. many cases diagnosed with intestinal inflammatory infiltrates have these changes because of dietary sensitivity. in one study, 29% of cats with histologic gastrointestinal changes improved with dietary elimination therapy alone. interestingly, improvement was noted within 4 days compared with the longer duration of 8 weeks often recommended for improvement of dermatologic manifestations of food sensitivities. this careful study made note of the cat's prior diets and likely dietary causes of sensitivities. 55 another study found dietary therapy to be unsuccessful in 52 of 60 cats but no specifics of diets tried are noted. 58 as with any national cancer institute working formulation (nci wf) system. 116 for most veterinarians in practice, the most important distinction is the histologic grade, because low-grade (lymphocytic or small cell) lymphoma has a much better prognosis (and requires different treatment) compared with high-grade (often lymphoblastic) or intermediategrade lymphoma. for the purposes of simplicity and practicality, only small cell lymphoma and high-grade lymphoma will be addressed here. the prognosis and treatment for intermediate-grade intestinal lymphoma should be considered as for high-grade lymphoma. small cell lymphoma was first described in human pathology in 1966. 122 earlier, small lymphocytes were considered end-stage cells without the ability to divide. in cats small cell lymphoma is most commonly associated with the gastrointestinal tract or skin. 163 small cell neoplasia can be a confusing concept, since our traditional ideas of malignant neoplasia focus on rapidly dividing cells. the confusion is compounded by various terms used in the literature, such as lymphocytic lymphoma, low-grade lymphoma, well-differentiated lymphoma, or diffuse lymphoma; another term, epitheliotropic malignant lymphoma predominantly applies to small cell lymphoma, and other papers fail to distinguish these lymphomas from lymphoblastic lymphosarcoma (the traditional, aggressive form). "small cell lymphoma" seems to be most widely used term, though the author prefers "lymphocytic lymphosarcoma," since it is more descriptive. intestinal small cell lymphoma can be considered as a severe lymphocytic intestinal infiltrate, the most common form of which is commonly called ibd. not only is lymphocytic ibd hard to distinguish histologically from lymphocytic lymphosarcoma, but the approaches and treatments are similar. several reports have suggested a relationship between the two conditions in that inflammatory infiltrates may become neoplastic over time. 27, 43, 90 prevalence the true prevalence of intestinal small cell lymphoma is unknown, but several recent studies have indicated similar rates to inflammatory bowel diseases, with kleinschmidt et al noting 10 small cell lymphoma cats compared with 14 with intestinal lymphocytic infiltrates, 74 evans et al reporting 10 cases compared with 12 with ibds 40 , and baral et al diagnosing 8 cases compared with 10 with ibds. 6 traditionally, 90% of feline lymphosarcoma is regarded as intermediate or high grade, 163 but this may not be the case within the gastrointestinal tract. fondacaro et al found 75% of gastrointestinal lymphoma to be lymphocytic 43 ; a more recent paper found some cats seem resistant to conventional therapy. if this is the case, the diagnostic findings should be re-assessed to ensure no steps were missed or findings disregarded; the cat should be reexamined to look for emergence of other signs; and the pathologist who reads the histology should be contacted to recheck the findings. some cases of apparently resistant ibd are actually food sensitive, but it can be difficult to find the incriminating diet source, and commercial diets are not always effective. if underlying infectious causes have been entirely ruled out and the practitioner is certain of the diagnosis of idiopathic disease, immune suppressive therapy can be increased by either increasing the dose of prednisolone or using other agents, such as chlorambucil, typically at 2 mg/cat, po, every second day. it has been suggested that cats with eosinophilic inflammation may be more likely to be refractory to standard therapy. side effects of immunosuppressive therapy are rare but include inducing diabetes mellitus, immune suppression, delayed healing, and gastrointestinal ulceration. reported doses of sulfasalazine to manage large bowel ibd are 10 to 20 mg/kg, po, once daily for 7 to 10 days. 174 because this drug is usually only available as 500 mg tablets, one eighth of a tablet, providing a dose of 62.5 mg, is usually appropriate for most cats. in some countries, it is possible to have a compounding pharmacist formulate the drug into more convenient tablet sizes or as an oral suspension. cats are generally regarded as susceptible to salicylates, and possible side effects include vomiting or diarrhea, or anemia. the exact pharmacodynamics of this drug are not known; so, caution for extended use should be exercised and the drug withdrawn if any possible adverse signs are noted, but there are anecdotal reports of extended use of this drug without adverse consequences. a survey of the online veterinary cancer registry (http://www.vetcancerregistry.com) identified 6% of all submitted feline tumors to be intestinal tumors. approximately 74% of reported feline small intestinal tumors were lymphomas. adenocarcinomas accounted for 17%, and other tumor types reported included mast cell tumors and leiomyosarcomas. 135 "lymphoma in veterinary medicine: no longer a oneword diagnosis" was the title of an editorial in a recent issue of the veterinary clinical pathology journal, 95 and this is nowhere truer than in the feline gastrointestinal tract! a recent study classified 50 cases of feline gastrointestinal lymphoma both histologically and immunophenotypically, and it found eight different categories according to the revised european and american lymphoma/world health organization (real/who) classification system and six categories according to the approximately equal numbers of high-grade and lowgrade gastrointestinal lymphoma. 84 older cats are more at risk of small cell lymphoma, with mean or median ages reported from 9 to 13 years. younger cats with the disease have, however, been recognized. 40, 43, 73, 84 no breed or gender predispositions have been definitively recognized. two larger studies have suggested a skew to males with 28 males compared with 22 females in one report, 43 and 24 males compared with 17 females in the other 73 ; most other studies looking at gender and breed did not clearly distinguish between lymphoblastic and lymphocytic neoplasia. clinically, it is impossible to distinguish cats with ibds from cats with small cell lymphoma. this is hardly surprising when even histologic distinction can be difficult! therefore cats will present with weight loss or vomiting or diarrhea at a similar frequency to those with ibd. weight loss has been recognized as a presenting sign in 82% to 100% of cases, diarrhea in 25% to 60% of cases, and vomiting in 25% to 73% of cases, with various combinations of these signs also possible. other variable signs are lethargy and inappetence or, conversely, polyphagia. 40, 43, 73, 84 these findings can be summarized by stating that cats with gastrointestinal small cell lymphoma can present with any combination of signs relating to the gastrointestinal tract. intestinal small cell lymphoma is typically a diffuse disease, and therefore multiple areas of the alimentary tract are usually affected. in studies where different locations of the small intestine were assessed, the jejunum was most commonly affected (100%), with the ileum frequently affected (93% to 100%), and duodenal pathology slightly less prevalent (83% to 90%). 40, 84 although the numbers of cats assessed in these studies are small, the important fact that the duodenum is not always affected needs to be recognized, which has important implications for how biopsy samples are obtained, because lesions beyond the duodenum are likely to be beyond the reach of an endoscope. further difficulties in precise diagnosis may arise, since non-neoplastic lymphocytic infiltrates (e.g., ibd) are often found in other locations along the intestinal tract. 27, 40, 84 the stomach is also affected in 14% to 40% of small cell lymphoma cases. 40, 43, 84 although not fully assessed, involvement of the colon appears rare. 84 local lymph node involvement is common, being noted in up to 59% of cases. 84 this percentage may be even higher, because many studies assessed lymph node cytology from ultrasound-guided fine-needle aspirates, which may miss spread to the lymph node, because the population of neoplastic lymphocytic cells is indistinguishable from the normal population of lymph node cells. histology is required to assess changes in lymph node architecture. liver involvement is not uncommon but not thoroughly assessed. one study noted liver lymphocytic neoplasia in 8 of 38 cats with small intestinal lymphocytic neoplasia, 73 another found 5 of 15 affected cats in which the liver was biopsied, 84 another noted 2 of 4 cats had liver involvement, 27 and a further study detected neoplasia "in the lymph nodes, liver, or both" in all 10 cats with intestinal small cell lymphoma. 40 the pancreas may also be involved. 73, 84 this may be akin to the noted association of lymphocytic inflammation of intestine, pancreas, and liver 171 that has been dubbed tri-iditis. ultrasound findings may not suggest extragastrointestinal involvement. in the case of liver pathology, ultrasonography may show no changes in as many as 75% to 80% of cases. 40, 84 focal nodular changes and he patomegaly have been recognized as ultrasonographic signs of hepatic small cell lymphoma. 7 both lymphocytic ibd and lymphocytic neoplasia are often recognized simultaneously in the same cat, 40, 84 and numerous authors have suggested that lymphocytic ibd may be a precursor to intestinal lymphoid neoplasia. 90, 125 if this is the case, then antigenic factors, such as bacterial population changes or food sensitivities, could be considered primary initiating factors for small cell lymphoma since they are potential underlying etiologies of ibds. 79 however, neoplasia also requires genetic mutations to occur (often affecting regulation of cell death and cell survival), and these may be initiated by the inciting antigenic factors or the ongoing inflammatory changes. 154 as opposed to other feline lymphoid neoplasia, no association has been made with felv infection. 27, 43, 73, 125 intestinal lymphocytic lymphosarcoma begins in the superficial mucosa and progresses to involve the entire mucosa and submucosa; then advancing in a perivascular pattern into the tunica muscularis, eventually infiltrating all four intestinal tunics. 43 lymph node and other organ (such as liver or pancreas) involvement likely represent metastasis through lymphatics and perhaps hematogenously. more distant metastasis is not reported. serum or plasma biochemistry and hematologic findings are typically nonspecific. however, this testing is important as part of the diagnostic workup to rule out extra-gi disease, such as hyperthyroidism or diabetes mellitus. common biochemistry findings are mild to moderate increase of liver enzymes, such as alanine aminotransferase (alt), aspartate aminotransferase (ast), and/or alkaline phosphatase (alp). 27, 40, 43, 84 as with ibds, these liver enzyme changes may or may not represent overt hepatic disease. 67 albumin may be reduced 43 but is normal in most cases 27, 43, 84 ; azotemia may be present and may be of prerenal origin or represent concurrent renal disease. in one study, 25 of 32 cats were hypocobalaminemic; 1 of 27 cats had low folate, but 10 of 27 had elevated folate; and 12 of 16 cats had increased ftli. 73 hematologically, a mature neutrophilia with or without monocytosis is sometimes present, representing the inflammatory response; lymphopenia may be present as a stress response. anemia may be present and may occur as a result of chronic slow gi blood loss, and in some cases, ulceration, or it may be because of chronic disease; hemoconcentration is also possible, reflecting dehydration. 27, 40, 43, 84 palpable or ultrasonographically visible thickened intestines (30% to 41% of cases) 27, 40, 43, 84 or mesenteric lymph nodes (20% to 50% of cases) 27, 40, 43, 84 are no more or less likely to be present in comparison with ibds. there are no defined ultrasound guidelines for cats with intestinal small cell lymphoma, because most prior papers do not distinguish between small cell and lymphoblastic neoplasia. 54 ,113 a more recent paper found 9 of 15 cats undergoing ultrasound examination had diffuse small intestinal wall thickening, with a mean of 4.3 mm (range, 3.4 to 5.0 mm; median, 4.5 mm), and focal mural thickening of 20 mm was noted in one cat. 84 in many cases, against expectations, intestinal wall layering was preserved. these findings also mean that 5 of 15 cats had ultrasonographically normal intestinal wall thickness (≤2.8 mm for the duodenum and ≤3.2 mm for the ileum). 50 if affected, jejunal lymph nodes may appear as hypoechoic and enlarged; in the same study, 12 of 15 cats had lymph node changes with a mean diameter of 15.9 mm (range, 6.5 to 30 mm; median, 10 mm) 84 compared with the normal diameter of less than or equal to 5.0 mm. 132 none of these findings can definitively distinguish small cell lymphoma from ibds; although one recent paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma (figure 23 -20) than those with ibd, this change was also seen in 12% of cats with a normal small intestine. however, thickening of the muscularis layer together with lymphadenopathy was recognized in 26% of those cats with small cell lymphoma compared with 4% of those with ibd and 2% of cats with no small intestinal pathology. 177 biopsy samples and histopathology are required for definitive diagnosis. an example of jejunal and mesenteric lymph node appearance at laparotomy is shown in figure 23 it is difficult to distinguish between lymphocytic inflammation and small cell lymphocytic neoplasia in any location; some histopathologic features that might help in differentiating the ends of the spectrum may include therefore become known as the fondacaro protocol. this consists of a combination of prednisolone and chlorambucil given orally by the client at home (table 23-15 ). the rationale is that a slow alkylating agent, such as chlorambucil, is more appropriate to use for the slowly dividing, well-differentiated lymphocytes that cause disease. this can be contrasted to the aggressive chemotherapeutic agents required for the rapidly proliferating cells in lymphoblastic neoplasia that is typically associated with lymphosarcoma. reported response rates to this protocol are excellent, with 59% to 76% of cats achieving complete clinical remission, reported median survival times ranging from 20 to 30 months for those cats responding to therapy, and reports of individual cats surviving as long as 76 months. 43, 73, 84 the original reported protocol comprised prednisolone (10 mg/cat, po or 2 mg/kg, po) given daily with chlorambucil pulsed by administration of 15 mg/m 2 for 4 days every 3 weeks. a more recent study 73 dosed prednisolone similarly, but chlorambucil was given as continuous therapy of 2 mg/cat, po every second or third day. no mucosal congestion, edema, or fibrosis in lymphocytic neoplasia, 43 compared with ibd 4. epitheliotropism, or homing of neoplastic t lymphocytes to the mucosal epithelium in lymphocytic neoplasia 27 these features can be seen in figure 23 -22. each of these criteria may be useful but are unlikely to be definitive. further studies that may not be routinely available but which may be helpful are immunophenotyping; most reports have found purely t lymphocytes in most cases of intestinal small cell lymphoma 27, 73, 84, 116 (figure 23-23 ). 2. clonality; the detection of a clonal population of cells, as recently described for intestinal lymphocytic lymphosarcoma, 101 would be closest to providing the basis for definitive diagnosis. effective treatment of feline intestinal small cell lymphoma was brought to light by fondacaro et al 43 and has cat to be weaned off corticosteroids, with chlorambucil continued as monotherapy (as is often the case with humans). iatrogenic diabetes mellitus usually needs to be managed with insulin therapy, at least initially (see chapter 24) . high-grade lymphoma or lymphosarcoma is the traditional style of aggressive, rapidly dividing lymphoid neoplasia that carries a much poorer prognosis than small cell lymphoma. most early studies do not distinguish grade of neoplasia; so, the prevalence of low-grade and high-grade alimentary lymphoma are difficult to assess. several recent studies found a similar prevalence of each, 84 ,116 but the seminal paper describing small cell lymphoma found only 17 cases of lymphoblastic lymphoma compared with 50 cases of small cell lymphoma. 43 this ratio of approximately one high-grade gi lymphoma case for every three low-grade cases more closely approximates the rate found in the author's practice. the reported median ages of affected cats range from 10 to 12 years, but cats as young as 1 year old have been diagnosed. most papers note that males are overrepresented, and siamese cats may also be overrepresented although most affected cats are domestic shorthairs. 43, 49, 90, 116, 176 precise signalment is difficult to determine from the literature, because many papers assess all anatomic locations of lymphoma without necessarily breaking down epidemiologic data for each anatomic site. also, there are few comparisons to a reference population. the association of lymphoma with felv infection is well established and documented 139 and is covered in chapter 28; fiv has also been shown to be lymphomagenic. 12, 139 since the control of felv through vaccination began in the 1980s, nonretroviral-associated lymphoid neoplasia has become more common, and the rates of intestinal lymphoma have, in fact, increased since felv infection rates have decreased. 86 the underlying causes for this increase are not known. the association with inflammation from ibds was noted for small cell lymphomas, and perhaps there is a spectrum from lymphocytic ibd to small cell lymphoma to high-grade lymphoma. that some cats are more likely to have inflammatory changes become neoplastic is suggested by a paper noting higher lymphoma rates in cats with vaccine-associated sarcomas (a neoplastic condition where the role of chronic inflammation is well noted). 89 similar protocols are used in humans with both lowgrade (i.e., lymphocytic) lymphosarcoma and chronic lymphocytic leukemia. 117,151 some studies with humans have indicated that continuous therapy with chlorambucil results in prolonged survival, 64 although metaanalyses have not been able to determine optimum dosing and scheduling of administration of chlorambucil or other alkylating agents in these conditions in humans. 20, 72 although we do not have enough data to critically compare pulsed therapy to continuous dosing, the study assessing continuous dosing appeared to have a lower number of cats completely responding, although those cats that did respond had a longer median survival 73 than those in the studies assessing pulsed chlorambucil dosing. 43, 84 the differences may also relate to the definitions used for complete response. the chlorambucil dose of 2 mg/cat, po every second day (or third day) is often chosen because of the ready availability of 2 mg coated tablets, the breaking of which can expose the owner to these cytotoxic medications. chlorambucil can be compounded into smaller doses, thus allowing daily dosing of 1 mg capsules. the author has used this dose to apparent good effect, but there has been no critical assessment. it is unknown whether involvement of lymph nodes or other organs, such as the liver, affects prognosis. the only study of substantial size to include extra-gi locations found anatomic location was not prognostic for response or survival time. 73 in another study, of the five cats with liver involvement, two cats did not survive more than 5 months, yet the other three lived longer than 2 1 2 years, with two surviving longer than 4 1 2 years. 84 a study of hepatic small cell lymphoma suggests the density of neoplastic lymphocytes may influence survival, and density may relate to the stage of the disease when diagnosis occurs. 7 adverse effects of chlorambucil are rare, but gastrointestinal signs, myelosuppression, and myoclonus have all been reported. gastrointestinal signs, such as vomiting, diarrhea, or inappetence, can be difficult to distinguish from continuation of the gastrointestinal disease diagnosed. these signs are usually self limiting. myelosuppression is also possible with thrombocytopenia reported. 57, 84 monoclonus has been reported on one occasion. 17 it is ideal to check hematologic parameters every 2 months for cats receiving chlorambucil. continuous therapy using lower doses of chlorambucil may be less likely to lead to these adverse effects. high doses of corticosteroids can induce diabetes mellitus, and thus blood glucose should be checked regularly. if diabetes occurs, the author has found that budesonide (1 mg budesonide is generally considered to be equivalent of 5 mg prednisolone) can be substituted for prednisolone, since it has reputed lower systemic effects (though no assessments of this drug's effectiveness in cats have been made). an alternative is for the of distinction of intestinal layering as shown in figure 23 -24. the area of lymphomatous infiltration is hypoechoic, because it contains a uniform cell population without much reactionary fibrous tissue. mesenteric lymphadenomegaly is common (figure 23-25) , as are changes in other organs, such as kidney, liver, or pancreas. ascites may also be seen. 54, 113 although ultrasonographic distinctions predominate, there is considerable overlap between ultrasonographic findings with small cell lymphoma and high-grade lymphoma. the clinician must not lose sight of the fact that microscopic distinctions are required to diagnose either condition. cytologic diagnosis of high-grade lymphoma from fine-needle aspirates (fna) is much more likely than with small cell lymphoma. this is because there is usually a focal lesion, and the neoplastic cells are a monomorphic population of large, immature cells (i.e., that are not normally seen in tissue). sometimes, mixed lymphoid whether the underlying cause is retroviral or chronic inflammation or anything else, the pathogenesis of highgrade intestinal lymphoma, as with small cell lymphoma and other neoplasia, depends on chromosomal changes that affect regulation of cell growth and death, resulting in malignant transformation and clonal expansion of immature lymphocytes. 152 metastasis can occur in one third to two thirds of cases, 90,92 with involvement of mesenteric lymph nodes most commonly noted, but spread to liver, spleen, kidneys, and thorax is also possible. 90 a recent survey of gastrointestinal lymphoma found that most cases (37 of 50) involved the small intestine (including 3 that also involved the stomach and 4 that also involved the large intestine), and 4 of 50 cases involved the large intestine only. 116 cats with high-grade alimentary lymphoma often present similarly to those with other gastrointestinal diseases. typical clinical signs are weight loss, anorexia, lethargy, vomiting, diarrhea, or a combination of these signs. repeated studies have found cats with no vomiting or diarrhea; in one study, 13 of 28 cats had only anorexia or weight loss on presentation. 90 cats with large bowel pathology usually present, as with other causes of colitis, with increased urgency, and small, frequent amounts of diarrhea, often with blood or mucus. cats with large bowel neoplasia of any form can present for constipation caused by intestinal obstruction. palpation of an abdominal mass has been recognized in 59% to 85% of cases, 43, 90 but the corollary of this is that 15% to 41% of cases did not have a palpable mass. it is also important to note that up to 50% of cats with intestinal small cell lymphoma, and a number with ibds, have palpable mesenteric lymph nodes; so, a palpable abdominal mass is not a specific indication of high-grade neoplasia. many cats have palpably thickened bowel loops. 124 hematology and plasma or serum biochemistry findings are also nonspecific. increased liver enzymes may or may not indicate liver involvement. anemia may be recognized and can be non-regenerative, reflecting chronic disease or slow blood loss, or regenerative if there is more substantial blood loss associated with mucosal ulceration. hypoalbuminemia can be because of blood loss or intestinal protein loss. hypercalcemia of malignancy is a possibility but not commonly reported. despite nonspecific signs, laboratory testing is important to rule out extra-gi diseases and help manage consequences of enteric disease, as with small cell lymphoma and ibds. ultrasonography commonly shows a focal intestinal thickening (of 5 to 25 mm) with partial or complete loss although noted as the next most common intestinal neoplasia, after the various forms of lymphoma, adenocarcinoma is seen relatively infrequently in practice. most cats are more than 10 years old, 32,76,126 males may be overrepresented, and several studies have recognized a distinct overrepresentation of siamese cats. 32, 76 three distinct forms have been described 140 : cats typically present with nonspecific signs of gastrointestinal disease but can present with obstructive signs. cats with large bowel neoplasia can present for tenesmus or hematochezia and even constipation, if the lesion is obstructive (or partially obstructive). on physical examination, an abdominal mass is palpable in approximately 50% of cases, but other findings are usually nonspecific. anemia can be found if mucosal ulceration has occurred, but there are no distinctive laboratory findings. lesions can occur anywhere along the intestinal tract. one study of 100 cases found 40% of feline intestinal adenocarcinoma lesions were present at the ileum or ileocolic junction. 32 twenty-five to 50 percent of cases have metastasis at the time of the diagnosis, and this is a poor prognostic indicator. 32, 76, 145 radiology may show a mass lesion or intestinal obstruction, and ultrasonography can localize lesions to an intestinal origin. the ultrasonographic appearance of the proliferative, circumferential, outwardly expansile populations (of immature lymphoblasts and mature lymphocytes) are seen if a germinal lymphoid follicle is aspirated, and precise diagnosis may be difficult if there are a large number of lymphoblasts. 160 fna samples are best obtained with ultrasound guidance. the cytologic sample quality is greatly improved by not aspirating when the needle is visualized in the mass but merely "pecked" into the mass so that the needle is merely acting to finely "core" the mass. on removing the syringe and needle, the hub of the needle is removed before drawing air into the syringe, the hub is replaced, and the sample within the needle is expressed onto a slide. usually, the decision to diagnose by cytology from fna is based on the ultrasonographic appearance of a mass. since there is substantial crossover of ultrasonographic appearance of intestinal masses, laparotomy for excision is often performed with the affected bowel submitted for histology. except when intestinal obstruction has resulted, there is no therapeutic benefit of excising a gastrointestinal lymphoma (which requires excision and anastomosis), but there is minimal room for doubt when a histologic diagnosis is achieved. the response to therapy for high-grade intestinal lymphoma is significantly worse than that for small cell lymphoma. 43, 124 further, response to therapy for highgrade intestinal lymphoma appears to be worse than for lymphoma in other anatomic locations. 142 precise remission rates and survival times are difficult to quantify, because many studies assess lymphoma from multiple locations and do not necessarily differentiate response of gastrointestinal lymphoma or report the grade of lymphoma. with remission rates reported from 18% to 80% 43,92,176 and a median survival time of up to 41 weeks (range, 4 to 120 weeks), 176 it can be said with some certainty that some cats respond to therapy for reasonable durations. multiple authors have noted that the best prognostic indicator is response to an initial treatment cycle, 92,100,176 which should prompt clinicians to encourage owners to start therapy and decide whether to continue based on the cat's response. there are several published chemotherapeutic protocols, 43 ,92,100,142,176 but all follow the same principles of using medications to target specific phases of the cell division cycle (such as l-asparaginase and vincristine) with other medications that interrupt multiple phases of the cell cycle (cyclophosphamide and doxorubicin). targeting the cancer cell in different ways enables more cells to be killed, reduces the toxicity of the individual drug used, and reduces the likelihood of resistance to a specific drug. several authors have noted increased success with the addition of l-asparaginase and doxorubicin to protocols. 92, 162, 176 chemotherapy for lymphoma is covered in more detail in chapter 28, oncology. look promising, but only two cats assessed had gastrointestinal mast cell neoplasia. lomustine was used unsuccessfully in one cat with sclerosing mct; another cat with sclerosing mct received eight treatments of vinblastine and had a survival time of greater than 4 years. 56 adenomatous polyps have been reported in the duodenum 87 and ileum 106 and can result in intussusception. 133 cats of asian ancestry, predominantly siamese, are greatly overrepresented, and most reported cases have been males. 87 cats usually present for vomiting or hematemesis that, surprisingly, can be very acute in onset; complete intestinal obstruction may result. 87, 106, 133 resection is curative, with survival times of more than 4 years reported. 87 eosinophilic sclerosing fibroplasia has recently been described in a series of 25 cases and is not strictly neoplasia. 29 the ulcerating mass lesions that can occur anywhere from the stomach to the colon are often grossly and histologically mistaken for neoplasia. there appears to be no breed predisposition or age predisposition (with ages ranging from 14 weeks to 16 years), but 18 of 25 cases (72%) were castrated males cats compared with 7 of 25 (28%) female spayed cats. eighty-four percent of cats presented for vomiting, 68% presented for weight loss, and 7 of 12 (58%) cats had peripheral eosinophilia. all cases had a palpable abdominal mass. the pyloric sphincter was the most common site, and lesions in this location were mostly considered unresectable. fourteen of 25 cats (56%) had bacterial colonies within microabscesses and necrotic foci within the lesion. the bacteria recognized were predominantly gram-negative rods, but antibiotics did not seem to be clinically effective. the bacteria are suspected to initiate the lesions, having been embedded after foreign body penetration. there are no specific treatment recommendations, but excision, where possible, would be prudent; corticosteroids appear to be helpful adjunctive therapy. survival times are difficult to estimate since many cats were euthanized because neoplasia was suspected and follow-up times were short (up to 6 months) for the remaining cats. 29 there are very few reports of intestinal leiomyosarcomas in cats, 8, 159 which have been reclassified as gastrointestinal stromal tumors. 99 these tumors may be more likely to arise from the ileocecocolic junction. resection, if possible, is usually recommended, with survival times of 3 to 4 months reported before recurrence. the author owns a cat with this tumor where resection was not form is better described than the annular, constrictingband form with minimal outward enlargement. in these cases, sonographically, a solitary segmental intestinal mural mass is present and characterized by circumferential bowel wall thickening with transmural loss of normal sonographic wall layers. the thickening can vary in echogenicity but may be hypoechoic and may be symmetric or asymmetric. there is no definitive distinction, however, from lymphosarcoma, mast cell tumor, smooth muscle origin tumors, or even segmental benign inflammatory bowel disease. 126 surgical resection is the treatment of choice. there seem to be two distinct groups in terms of survival time postresection: 1. short-duration survival (euthanasia or death within 2 weeks of surgery) 2. long-duration survival (mean survival time of 15 months, 76 with a number of cats surviving greater than 2 years) 76, 109 because clean margins improve prognosis, for large bowel adenocarcinoma, subtotal colectomy may be required for complete excision. 145 because of the potential for success after resection, it is recommended to excise unidentified masses at the time of surgery. 145 other forms of intestinal neoplasia are recognized infrequently, and include intestinal mast cell tumors, adenomatous polyps, eosinophilic sclerosing fibroplasia, gastrointestinal stromal tumors (leiomyosarcoma), and hemangiosarcoma. mast cell tumors (mct) are often cited as the third most common form of feline gastrointestinal tumor, 85 but the intestines are a far less common site than cutaneous, splenic, or hepatic mast cell neoplasia. 85, 124 masses are usually segmental nodular thickenings that occur in older cats. 140 the masses are indistinguishable ultrasonographically from other tumors, such as lymphoblastic lymphosarcoma. 126 a recent series of 50 cases described a variant of feline intestinal mast cell tumor, dubbed sclerosing mast cell tumor, for which neoplastic cells form a trabecular pattern with dense stromal collagen. additionally, eosinophilic infiltrates were moderate to marked in most cases. these cases can be confused histologically with eosinophilic enteritis, gastrointestinal stromal tumor, or fibrosarcoma. 56 surgical resection is recommended, but lesions are commonly infiltrative or metastasize widely, and there are few reports of successful treatment. lomustine (dosed 50 to 60 mg/m 2 , po every 4 to 6 weeks) has recently been assessed as adjunctive chemotherapy for mast cell neoplasia in various locations, 123 and results recognized in that many intestinal bacteria can be found in healthy animals. 70 further antibiotic administration can result in increase of other bacteria. 69 fungal causes of diarrhea are usually recognized from histology of biopsy samples. it remains to be seen whether the recent ready availability of pcr panels looking at a number of infectious causes of diarrhea will be beneficial for recognizing pathogens that had previously been misdiagnosed or a hindrance for readily recognizing commensal organisms not necessarily causative of the clinical signs being investigated. the most common viral, bacterial, and mycotic causes of diarrhea in cats are described below. parasitic gastrointestinal diseases are covered later in this chapter. viral causes of diarrhea are not usually specifically diagnosed, since, with the exception of the canine fecal elisa possible, and the cat appears healthy 24 months past diagnosis (figure 23-27 ). cats with intestinal hemangiosarcoma often present with anemia, and the disease appears to be highly metastatic. 33 the intestines appear grossly thickened by dark red tissue. 138 the small and large intestines seem to be affected with similar frequency. removal of macroscopic disease is recommended, but often the full extent of the severity is only recognized at surgery. 33 the prognosis is poor. suspicions of infectious causes of diarrhea should be aroused in younger cats, cats from shelters, or cats with immune suppression. when considering infectious causes of diarrhea, clinicians should assess whether the diarrhea is large bowel or small bowel in origin and correlate this with specific pathogens that are likely to cause clinical signs as shown in table 23 -16. to increase the diagnostic yield of fecal examination for parasitic causes of diarrhea, wet smears and appropriate fecal flotations should be performed on fresh fecal samples (<1 hour old). it is appropriate to administer broad-spectrum anthelminthics, even if fecal tests are negative. bacterial and viral causes of diarrhea should be considered when the cat is systemically unwell with fever. fecal culture should be performed in these circumstances, but the limitations of this testing need to be yersinia enterocolitica current theory of fip pathogenesis involves initial infection with fecv and then mutation to fipcv in small numbers of susceptible individuals. 112,165 routine serologic testing for fecv in cats with diarrhea would neither prove correlation with the clinical signs nor affect how the disease is managed and so is not recommended. cats with fecv diarrhea should be managed with symptomatic therapy of fasting, then reintroducing a bland diet and supportive care with fluid therapy if necessary. other viruses, such as astrovirus, reovirus, rotavirus, and torovirus-like agent, have been recognized to cause diarrhea in cats, but their roles as pathogens are unclear. they are not routinely recognized in practice, since electron microscopy of fecal samples is necessary for diagnosis and is not routinely performed. management is supportive care with appropriate fasting, then reintroduction of bland diets and fluid and electrolyte replacement if necessary. 51 successful identification of a known bacterial pathogen from a fecal sample does not necessarily mean that the agent found is the cause of disease in the cat. although a number of bacterial pathogens have been demonstrated to cause diseases when specific pathogen-free (spf) cats are experimentally infected, 42 these same organisms can be found in healthy cats. 93 the differences between healthy and diarrheic cats that have bacteria found in their feces may relate to virulence factors of the organism, or host factors (local or systemic immunity) of the cat. there is no definitive answer for this quandary. the author's opinion is that • if a diarrheic cat is systemically unwell and has a fever, then feces should be cultured. • if an organism is isolated that is known to cause signs consistent with those the cat is showing, the cat should be treated appropriately. campylobacter diarrhea is usually caused by c. jejuni. clinical signs of infection are poorly documented, but most cats are asymptomatic. younger cats are more likely to have clinical signs and hemorrhagic, mucoid diarrhea has been reported. diagnosis can be from culture of feces or swabs, and the organism is quite hardy; so, it usually survives transport to the laboratory. 28 in individual cases, the organism has not been cultured after antibiotic treatment, 45,46 but it is not definitively proven that antibiotic therapy affects the natural course of the disease. antibiotics that can be used are amoxicillin-clavulanate (15 mg/kg, every 12 hours, po) for parvovirus, routine definitive tests are not available. clinical signs of panleukopenia (feline parvovirus infection) are more likely to occur in kittens, with the highest morbidity and mortality occurring between 3 and 5 months of age. subclinical cases in older (susceptible) cats probably go unrecognized. the organism is very stable in most environments, and infections mostly occur from environmental contact. peracute cases can result in death within 12 hours, with little or no warning signs. acute cases often have fever, depression, and anorexia, with signs beginning approximately 3 to 4 days before presentation. vomiting is usually bile tinged and unrelated to eating. diarrhea does not always occur, and when it does, it is usually later in the course of the illness. leukopenia is not pathognomonic, because this can also occur with acute bacterial infection (e.g., salmonellosis can present identically). 53 commercially available elisa tests for canine parvovirus antigen in feces can detect feline parvovirus; however, shedding may have ceased by the time clinical signs occur, and vaccination can result in positive test results for up to 2 weeks. 111 aggressive fluid therapy, usually at twice maintenance rates, is usually required. broad-spectrum antibiotic coverage is used to prevent or treat secondary bacterial infection from viral injury of intestinal mucosa. parenteral antibiotics are preferred to prevent the possibility of further gastrointestinal irritation. the author recommends calculating iv doses and introducing appropriate amounts of antibiotics to the fluids bag to create a constant rate infusion (cri); cefazolin can be used in this way at 100 mg/kg/24 hours, and betalactam cris are commonly used in human medicine. 127 aminoglycosides or fluoroquinolones can be used concurrently at routine doses if fever persists after 24 hours or the cat is moribund on presentation, but care must be used with these agents. aminoglycosides are potentially nephrotoxic, and fluoroquinolones have been reported to result in cartilage damage in growing animals, although this has not been demonstrated clinically in cats. 134 fluoroquinolone retinal toxicity has been seen in all animals. cats that survive the first week usually recover, and prior infection imparts lifelong immunity. 28 vaccinations are highly effective for disease prevention. feline enteric coronavirus (fecv) mostly causes mild, self-limiting diarrhea and must be distinguished from feline infectious peritonitis coronavirus (fipcv), which is essentially always fatal and for which diarrhea is not a typical sign (but is possible). the most widely accepted or fluoroquinolones, such as enrofloxacin (5 mg/kg, once daily, po) for durations of 14 to 21 days. 44 macrolides, such as erythromycin (10 to 15 mg/kg, every 8 hours, po), are regarded as the drug of choice for humans but can cause gastrointestinal side effects. 93 clostridium difficile has been recognized in up to 5% of diarrheic cats. 93 clinical signs are typically acute onset watery diarrhea and anorexia. diagnosis has been made with detection of toxin a or toxin b in fecal samples using elisa. although these tests have not yet been validated for cats, they may prove to be a useful aid to diagnosis 94 and are available for testing of equine feces at some commercial laboratories. nontoxigenic strains exist; so, positive culture alone does not ensure diagnosis. metronidazole (10 mg/kg, every 12 hours, po) for approximately 7 days is the treatment of choice. 93 clostridium perfringens typically results in large bowel diarrhea with tenesmus, mucus, and hematochezia, but small bowel signs can also be seen. 42 pcr testing for enterotoxin a is commercially available and may prove to be a useful adjunct in diagnosis. antibiotics that can be used include metronidazole (10 mg/kg, every 12 hours, po), tylosin (10 to 20 mg/kg, twice daily, po), or amoxicillin-clavulanate (22 mg/kg, every 12 hours, po) for 7 days. 94 escherichia coli is a ubiquitous organism within the feline intestinal tract, and it would be unusual not to successfully culture e. coli from the feces of both healthy and unwell cats. when e. coli is associated with clinical signs of gastrointestinal disease, it is mostly as an opportunistic pathogen, with overgrowth resulting from changed environmental conditions, such as inflammation from other pathology or another pathogen. there are also specific strains of e. coli that are true pathogens because of virulence factors not present in commensal e. coli; these include enteropathogenic e. coli and enterotoxigenic e. coli, which both induce a watery diarrhea, and enterohemorrhagic e. coli, which produces a diarrheal syndrome with copious bloody discharge and no fever. 77 pcr testing is commercially available to identify pathogenic strains of e. coli 16,148 ; although not offered at routine veterinary laboratories, this testing is available to veterinarians, and laboratories offering these services can readily be found online. diagnosis should also document histologic lesions corresponding to the strain of e. coli identified. 77 there is emerging resistance to e. coli worldwide in all species of animals, including humans. this includes the typical therapies for gram-negative bacteria of beta-lactamenhanced penicillins and fluoroquinolones. 167 a major risk factor is prior antibiotic usage, because commensal organisms are exposed to antibiotics. pcr testing does not enable antibiotic sensitivity testing, and fecal culture may not be able to distinguish pathogenic from nonpathogenic strains; so, sensitivities may not be an accurate reflection of the pathogenic organism. pcr testing for genes that impart resistance to e. coli have recently been described but are not yet commercially available. 34 in some circumstances, supportive care with fluid and electrolyte replacement may be all that is required while the cat's immune system combats the infection. empiric therapy could include beta-lactam-enhanced penicillins (such as amoxicillin-clavulanate at 20 mg/kg, every 12 hours, po), fluoroquinolones (such as enrofloxacin at 5 mg/kg, once daily, po), or cefovecin (8 mg/kg, every 2 weeks, sc), but the clinician must be aware of possible drug resistance. salmonella typhimurium infection is possible from ingestion of infected prey, infected food sources, or from a contaminated environment, including the veterinary hospital. the resulting clinical signs depend on the number of infecting organisms, the immune status of the cat, and the presence of concurrent diseases. infection rates in cats (and humans) have been correlated with seasonal bird migrations, 153 and the illness has been dubbed songbird fever, 52 but there is no distinction between this and other salmonella infections. clinical signs usually begin 3 to 5 days after exposure, starting with fever (often >40° c [104° f]), malaise and anorexia, and progressing to diarrhea, vomiting, and abdominal pain. hematology can show leukopenia with a left shift and nonregenerative anemia, and biochemistry results are usually nonspecific. diagnosis is based on isolation of the organism by culture or identification with pcr, but care should be taken to correlate pathogen identification with clinical signs since, as with most gi pathogens, the organism can be isolated from healthy animals. 147 as with e. coli, antibiotic resistance is widespread, 120 with one united kingdom survey finding the multiple drugresistant strain dt104 to be the most frequent bacteriophage type identified. 115 treatment should be reserved only for those cats showing systemic signs, because routine antibiotic use in treating salmonellosis induces drug-resistant strains and prolongs the convalescent excretion period. 52 antibiotic choice should be based solely on sensitivity findings, since resistances are so widespread and unpredictable. 98 this means that if the organism has been identified by pcr, then culture of feces must also be undertaken. the duration of treatment must be long enough to eliminate fecal excretion of the organism, prevent the chance of relapse, and reduce the chance of resistance developing; up to 28 days has been advocated. 4, 164 these cautions are particularly important because of the zoonotic potential of salmonellosis. linear foreign bodies have traditionally been considered more common than discrete foreign bodies in cats, 10,14,41 but a study from a primary care facility indicated only 33% of foreign body cases were because of linear foreign bodies. 60 the larger case load of linear foreign bodies at referral institutions noted in earlier studies may indicate the abilities of primary care practitioners to recognize and effectively deal with discrete foreign body obstructions. most studies have found that cats with intestinal foreign bodies are generally younger (mean, 1.0 to 2.7 years), with a notable exception being obstruction from trichobezoars where three of five cats in one study were 10 years or older; the greatest risk factor appears to be length of hair coat. 9 no specific breed predispositions have been described but siamese and siamese-related cats have been noted to have oral fixations 13 and so may be expected to be overrepresented with intestinal foreign bodies. clinical signs will vary depending on the type of foreign body (linear or discrete), the position of obstruction, and the time since obstruction. most cats present for anorexia or vomiting. partial obstruction can result in diarrhea (which can be bloody). foreign body obstruction is typically considered an acute condition, with duration of obstruction because of a linear foreign body, measured from the onset of clinical signs to diagnosis, reported to range from 1 to 10 days. 10,41,60 however, one paper demonstrated chronic, intermittent, gastrointestinal disease from a linear foreign body of a 1-month duration 175 demonstrating that partial obstruction can result in a chronic course. physical examination may or may not reveal abdominal pain, palpable abdominal mass (or plication), dehydration, or fever. all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of a linear foreign body. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see figure 3-8) . life-threatening consequences can result from the interactions of local and systemic factors that arise from intestinal obstruction. locally, damage to the mucosa from traction and pressure of the foreign object can cause hemorrhage, ischemia, and necrosis. systemically, hypovolemia, toxemia, and acid-base and electrolyte imbalances can ensue. complete intestinal obstruction by discrete masses results in gas and fluid distention of the lumen proximal other bacterial causes of diarrhea have been reported in cats, such as yersinia enterocolitica, 48 yersinia pseudotuberculosis, 63 clostridium piliforme (tyzzer's disease), 71 and anaerobiospirillum sp. 36 specific diagnosis of these (and other bacterial infections) may be found in the course of investigation. management follows the principles of supportive care and appropriate antibiosis based on sensitivity testing. small intestinal bacterial overgrowth (sibo) has not been specifically described in cats. the criteria defined for dogs is a fasting bacterial count in duodenal juice of greater than 10 5 organisms/ml 11 and is often recognized with other chronic gastrointestinal diseases. 130 healthy cats appear to have at least this number of upper intestinal bacterial with a range of 10 5 to 10 8 /ml recognized. 68 bacterial overgrowth could potentially occur with ileus or intestinal inflammation of any underlying cause. foul-smelling small bowel diarrhea with no specific pathogen recognized may be an indicator of this condition, as could an increase in bacterial metabolites, such as folate. if suspected, it is appropriate to manage with broad-spectrum antibiotics, such as metronidazole (10 to 15 mg/kg, every 12 hours, po) or amoxicillin (10 mg/ kg, every 12 hours, po) for an extended duration such as 21 to 28 days. alterations in intestinal flora have been recognized after such treatment 69 ; however, any advice for this "condition" is entirely empirical. all efforts should be directed at identifying a precise underlying cause. mycotic and other infectious agents are only rarely recognized as intestinal pathogens in cats. diagnosis is made by histologic and microbial analysis of samples obtained at biopsy. possible agents include histoplasma capsulatum, 24 aspergillus spp., candida albicans, 140 and pythium insidiosum. 119 intestinal obstructions arise most commonly as a result of neoplasia in older cats and foreign body ingestion predominantly in younger cats. 10, 41, 60 less common causes include intussusception 83 and granulomatous inflammation (e.g., from fip) 59 ; tapeworm infection, with greater than 30 worms acting as a linear foreign body, has also been reported. 173 other listed causes are volvulus, intestinal torsion, incarceration of bowel in a hernia, adhesions or stricture, intramural abscess or hematoma, and congenital malformations. 140 may not occur if the obstruction is partial or intermittent, or if vomiting results in less fluid present. since most foreign body obstructions in cats are proximal, identifiable dilatation may not be recognized for this reason. 78 linear foreign bodies present further challenges for radiographic recognition; the following typical radiographic signs 129,137 may or may not be present: to the obstruction. most gas accumulation is a result of swallowed air, which is predominantly nitrogen that cannot be absorbed by the intestinal mucosa. gas also arises from bacterial fermentation. fluid accumulates as a result of increased secretions (saliva, bile, and secretions of gastric, pancreatic, and small intestinal origin) and retention of fluid already ingested, and it can be augmented by local hemorrhage. 39 since most intestinal obstructions in cats do not reach the midjejunum, 60 reabsorption of fluids that normally occurs at the jejunum and ileum is impaired. 39 linear foreign bodies, such as string, dental floss, or elastic toys, require proximal anchoring, usually under the tongue or in the pylorus (for example, by part of a toy attached to elastic). peristalsis moves the free end of the "string" through the intestinal tract, resulting in pleats of intestines around the foreign body. as the foreign body is forced against the intestinal mucosa, the mucosa becomes edematous, and even partial penetration affects mucosal integrity, allowing systemic entry of bacteria. intraluminal bacterial populations increase for both discrete and linear foreign bodies as a result of stasis. mucosal permeability can be affected by prolonged luminal distention, allowing entry of bacteria and toxins systemically or into the peritoneal cavity. direct entry of bacteria to the peritoneal cavity, causing septic peritonitis, can result from perforation of the intestinal wall from linear foreign bodies or sharp discrete foreign bodies, such as toothpicks or plastic toys. definitive diagnosis requires identification of the foreign body retrieved at surgery or in some cases, by endoscopy. this may be aided greatly prior to surgery by diagnostic imaging. however, imaging findings, particularly in the case of partial obstructions, may be subtle enough that obstruction of no identifiable cause is recognized or no overt signs are apparent. laboratory findings are not helpful in the precise diagnosis but are important to assess fluid and electrolyte balances that must be corrected. cats rarely help practitioners by ingesting radiopaque objects, but on the rare occasions that they do, these can be observed easily on plain radiographs. nonopaque foreign bodies depend on dilatation of the intestine from gas and fluid accumulation proximal to the obstruction for radiographic recognition (figure 23-28) . one study has suggested that if the jejunal diameter is greater than 2.5 times the length of the cranial end plate of the second lumbar vertebra, then intestinal obstruction is the most likely abnormality. care must be taken that the jejunal and not duodenal diameter is measured and that the radiographs must be positioned strictly lateral, because an oblique view can alter the measurement of the lumbar vertebra. 1 however, dilatation of an obstructed intestine successful treatment of foreign body obstruction requires evacuation or removal of the foreign body as well as correction of any bacteremia or endotoxemia, acid-base or fluid imbalances. discrete foreign body obstruction requires surgery or endoscopy to remove the object. in some specific circumstances, linear foreign body obstruction may be managed conservatively by cutting the anchor point below the tongue and allowing the cat to pass the foreign body by peristalsis. however, the decision to manage a cat conservatively must be done with the cat hospitalized, with fluid therapy and antibiotic coverage and a clear recognition on behalf of the practitioner and the owner that surgery may subsequently be required. cutting a sublingual linear foreign body may be achieved in a conscious cat by applying pressure with the thumb of one hand in the intermandibular space to elevate the tongue and gently grasping it using gauze with the other hand while a second person cuts the line with a suture cutter. there is a chance of a small nick on the sublingual surface. if the cat will not tolerate the procedure, sedation is appropriate. when cutting the line, the nature of the linear foreign body should be assessed (i.e., is it more or less likely to cut mucosa). in one study, 10 19 cats with linear foreign bodies were managed conservatively with 10 cats subsequently requiring surgery. the authors of that paper created guidelines that will be adapted here. conservative management should be attempted if the cat • is presented acutely (within 2 days) after known ingestion of a linear foreign body • has a sublingually fixed linear foreign body that can be cut • has no overt signs of peritonitis • altered gas pattern with luminal gas collecting in small bubbles instead of normal curved tubular columns. this can be subtle when there is only minimal involvement of the intestine but overt when involving the entire small intestine. commashaped gas patterns are more likely to occur with linear foreign bodies. 1 contrast radiography can aid diagnosis for both discrete and linear foreign bodies but should be used with caution because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium is irritating to the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. 137 ultrasonography is a very useful diagnostic tool, particularly for discrete foreign bodies, where, in most cases, there is overt distention of the small intestines with intraluminal fluid apparent (figure 23-31) . this modality has not been extensively assessed as an adjunct to diagnosis of foreign body intestinal obstruction in cats specifically, although there are several papers assessing dogs and small numbers of cats that agree with its utility. 114,156,161 linear foreign bodies are more difficult to assess ultrasonographically, but plicated bowel can be recognized, sometimes with the foreign body seen as a hyperechoic line centrally. 156 si a technique has been described for removal of linear foreign bodies by making a single enterotomy incision proximally and passing a red rubber catheter over the linear foreign body aborally, milking the foreign body within the catheter through the colon for retrieval from the cat's anus by an assistant. 2 this technique is not always effective, because it can be hampered if the foreign body is knotted or does not run smoothly through the red rubber catheter. 102 if the affected bowel segment demonstrates evidence of necrosis or perforation on the mesenteric border of the intestine, resection and anastomosis should be performed. necrosis is indicated by dark discoloration, thin intestinal wall, poor arterial pulsation, poor capillary bleeding, or lack of peristalsis. end-to-end anastomosis can be accomplished using a simple interrupted appositional pattern or a modified simple continuous appositional pattern with the same type of suture material used for enterotomy closure. 14,88 intraabdominal masses causing intestinal obstruction are often presumed to be neoplastic but can also be of infectious origin. resection, where possible, is always recommended, because resection of neoplasia (if no metastasis) can offer a good prognosis, 76, 87, 109, 145 and infectious causes may be managed with adjunctive therapy after definitive diagnosis. intestinal obstruction in older cats is more likely to be secondary to neoplasia. any neoplasia can cause obstruction, but adenocarcinoma 32,76 and adenomatous polyps 88, 106 are reported to cause obstruction more often surgical intervention is mandatory if • clinical signs (e.g., vomiting or anorexia) persist or deterioration occurs with conservative management • the cat has overt signs of peritonitis • the linear foreign body is fixed at the pylorus some authors disagree with attempting conservative management, since a perforated intestine from a linear foreign body reportedly carries a 50% mortality rate, 41, 88 and early surgical intervention is never an incorrect decision. this should be balanced with the observation that cats can carry a linear intestinal foreign body, such as an elastic cord for a 1-month duration without intestinal perforation. 175 however, fishing line, for example, would not be so forgiving! surgery to remove an intestinal foreign body (figures 23-32 and 23-33) should be considered an exploratory laparotomy. that is, the aim of the surgery is not only to remove the foreign body but to assess the entire intestinal tract and abdomen for other foreign bodies or pathology. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction, thus delaying healing and creating the potential for surgical dehiscence. linear foreign bodies require multiple enterotomy incisions, since pulling the object out through a single incision could create iatrogenic intestinal perforation. the anchor point (either sublingual or pylorus by gastrotomy) must be released in the first instance. enterotomy incisions are closed with 5/0 synthetic, monofilament, absorbable suture material, such as polydioxyanone (pds) or equivalent, in either a simple interrupted or simple continuous pattern. 14,88 removal of a discrete foreign body (a piece of leather) at laparotomy. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction. this is the same cat as in the radiology image in figure 23 -28. affected bowel is required, with anastomosis of the healthy tissue. there appears to be no benefit to enteroplication, which can result in significant ileus. there is no benefit to performing resection-anastomosis if the intussusception does reduce manually. 15, 25 the prognosis depends on the underlying disease process and the chronicity of the intussusception, and therefore how debilitated the cat is at presentation, however, prognosis is mostly good, with survival reported in up to 80% of cases, though recurrence can occur in some cats with idiopathic disease, often at different locations of the intestinal tract. 15 constipation is defined as infrequent or difficult defecation associated with retention of feces within the colon and rectum. prolonged constipation results in harder than other types of neoplasia. please refer to the sections on intestinal neoplasia earlier in this chapter for more details. granulomatous inflammation causing a single focal intestinal lesion can lead to obstruction in the same way that neoplastic change can. feline infectious peritonitis (fip) can present as focal lesions, 59 often in the colon or ileocecocolic junction. in the case of fip, the focal lesion is usually an indicator of multisystemic disease; so, resection does not help prognosis. the fungus-like organism, pythium insidiosum has also been reported to cause granulomatous lesions, resulting in intestinal obstruction 119 from large extraluminal masses that are approximately fist sized. resection with adjunctive itraconazole (10 mg/kg) for 2 months after surgery was a successful treatment. intussusception refers to invagination or prolapse of one portion of the intestine into the part of the tract that either precedes or follows it. there is a bimodal age distribution with intussusceptions in older cats, most likely associated with neoplasia (or ibd in some cases) 25 ; underlying causes for younger cats are ill defined and may be idiopathic in many cases, 15,25 but associations with parasitism and, in one case, a linear foreign body, have been made. siamese and burmese cats seem to be overrepresented. the most common locations are the ileocolic region and the jejunum. 15, 25, 83, 110 affected cats present with nonspecific signs of gastrointestinal disease, such as anorexia and lethargy. vomiting is not necessarily a presenting sign; diarrhea may occur. abdominal palpation reveals a mass in most cases. plain and contrast radiography only show evidence of obstruction and usually do not help define that the bowel has intussuscepted. 15, 25, 83, 110 ultrasonography is very useful for diagnosis, because a distinctive pattern of alternating hypoechoic and hyperechoic concentric rings (figure 23-34) is present in transverse sections. 25, 110 sometimes, the target lesion seen can be hard to distinguish from the pathology of other intraabdominal masses, such as lymph nodes, and in these cases, the size of the lesions can help, because the width will always be greater than 11 mm with an intussusception (because the sum of at least four intestinal wall widths cannot be less) and is often greater than16 mm. 110 surgical correction is always required, and manual reduction is typically not possible because there is usually significant venous infarction, edema, and congestion (figure 23 -35) as well as adhesions from fibrin and effusions from the affected bowel. 15, 25 if the intussusception does not reduce manually, resection of the and drier feces that become impacted, and this is known as obstipation. 140 chronic, recurrent constipation and obstipation can result in megacolon, which refers to persistent increased bowel diameter that is not responsive to therapy. megacolon is not a specific disease entity; it may be considered the most advanced stage in the spectrum of chronic constipation. 18 in most cases, constipation can be managed quite simply if the underlying cause is determined and dealt with. a comprehensive list of causes of constipation is noted in table 23 of course, multiple factors can interact. for example, an older cat may have renal disease and so will be dehydrated to some degree and have arthritic hips and so be reticent to squat. the presenting signs of constipation are usually evident to owners and include straining in the litter box and producing hard dry feces, if at all. sometimes, however, owners can misinterpret signs. cats can strain because of lower urinary tract problems, and, if no urine is produced, some owners assume the problem is because of constipation. some constipated cats can intermittently have diarrhea because of direct colonic irritation from hard dry feces and so may present for diarrhea and not constipation. cats can also present for less specific signs, such as anorexia, lethargy, weight loss, and even vomiting. 140,170 vomiting can occur because of colonic receptors stimulating vagal afferent endings, which, in turn, can stimulate the chemoreceptor trigger zone. 61 sometimes owners are concerned that their cat is defecating less, but the cat has just changed its diet to a much lower-residue diet and so is producing less feces. a full dietary history is an important aspect of the initial assessment. physical examination should confirm presence of feces in the colon and assess the degree of impaction. the presence of feces can usually be confirmed by abdominal palpation. in constipated cats, the colon is often palpated as a long firm tube extending cranially; sometimes, feces can be palpated to and around the colic flexure. alternatively, the feces may be palpated as large, discrete fecal concretions (that can sometimes be hard to distinguish from intraabdominal masses such as lymph nodes). if there is any doubt of the presence or degree of fecal impaction, survey abdominal radiographs should be taken. a lateral view taken in a conscious cat should be adequate to confirm the diagnosis. the physical examination should also assess for contributing causes, including musculoskeletal conditions. any recent trauma should be taken into account. the hips and lumbosacral region should be assessed for pain. the degree of flexion and extension of the hips should be gently assessed. the lumbosacral spine can be assessed by running two fingers on either side of the spinous processes. the cat will flinch in painful areas. any arthritic change is magnified in an underweight cat, since there may be less muscle mass and the joints may bear a heavier load. any suspicions of underlying musculoskeletal abnormalities can be confirmed with radiographs. neurologic assessment should also be performed. subtle changes just affecting colonic innervation will not be apparent on physical examination alone. however, an assessment of proprioception, placing reflexes, and gait should at least be performed to assess for lumbosacral spinal cord disease. anorectal abnormalities or lesions should be evaluated. impacted or infected anal sacs can lead to reticence to defecate; and therefore anal sacs should be assessed and expressed. because this is painful for most cats, the cat should be held by an experienced assistant. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb positioned externally. a rectal exam can be performed with a well-lubricated (gloved!) middle finger, feeling over the pelvic rim for masses as well as assessing if the colon closes over (squeezes) the finger. if the colon feels open around the finger, this can be an indicator of impaired colonic innervation but does not imply that this is a permanent change. if there are impacted feces continuing to the anus, rectal examination is not possible until this has been cleared. if a cat finds anal gland expression or rectal examination too painful to tolerate (based on the clinician's judgment), these procedures should be done under sedation. hydration and electrolyte status are also important factors in the constipated cat. chronic renal disease is defined by azotemia (in conjunction with inadequately concentrated urine), which means the cat must be dehydrated to some degree. plasma or serum biochemistry and urinalysis can be used to diagnose renal disease, assess degree of renal disease, or recognize prerenal dehydration. electrolyte changes including hypokalemia and hypocalcemia may also contribute to reduced colonic smooth muscle function. 140 in young to middleaged cats of apparent good health and hydration, blood difficulty defecating and pass hard, dry stools but do not have fecal impaction at the time of examination. after the obstructing feces have been removed, steps must be taken to ensure colonic motility and smooth passing of feces. medical management of constipation traditionally involves laxatives and prokinetic agents. these may not be required in straightforward cases. as long as there is no obstructive lesion, cisapride at 2.5 mg/ cat, every 12 hours, po 82 is very safe and can be instituted with a view to reducing the dose to once daily after 10 to 14 days and discontinuing if signs remain abated. doses of up to 7.5 mg/cat, every 8 hours, po have been reported. cisapride is only available from compounding agencies in most countries. an osmotic or lubricant laxative (table 23 -18) may be used concurrently at reduced doses as necessary. reducing the fecal bulk produced is an important part of long-term management. traditional dietary recommendations are to increase the amount of fiber. 18, 26, 140, 170 increased dietary fiber results in production of shortchain fatty acids, which have been demonstrated to stimulate feline colonic smooth muscle contractions. 128 however, dietary fiber is also classified as a bulk laxative and so, by definition, will increase fecal bulk. in humans dietary fiber has been considered a mainstay of therapy for constipation, but a recent review concluded that many patients with more severe constipation have worsening symptoms when increasing dietary fiber intake. 103 because megacolon is believed to be the end result of chronic dilatation, 140,170 it is the author's firm belief that initial dietary efforts should be directed to reducing fecal bulk and thus introducing a low-residue diet. reduced dry matter intake reduces stool volume, 31 and the author has found that recurrence rates of constipation reduce greatly when cats are transitioned to entirely wet food diets. wet food diets also help ensure adequate water and urine assessments are usually not required at an initial presentation for constipation. in all cases, the same principles of management apply: 1. ensure removal of obstructing feces 2. ensure colonic motility and smooth passage of feces 3. reduce fecal bulk 4. ensure adequate hydration 5. manage underlying problems the first step is to ensure obstructing feces are removed. in simple cases, the cat will evacuate feces after use of a glycerin or sorbitol pediatric rectal suppository. another option is administration of a microenema, such as microlax (mcneil consumer healthcare, fort washington, pa.), which contains 5 ml of sodium lauryl sulfoacetate. these products act to lubricate the colon wall and therefore facilitate the passage of feces. the author prefers to use one or two of these within the consult room to observe the cat defecating (the cat must be provided with a litter tray!). the outside tube should be lubricated with the suppository contents before carefully inserting and then expressing the rest of the contents. there are also stimulant laxatives (containing bisacodyl) and emollient laxatives (containing sodium docusate) that have reportedly been used. 140 if a rectal suppository vial cannot easily be inserted because hardened fecal content obstructs its entry, a more substantive enema will be required (sometimes requiring sedation or anesthesia), and this is covered in the next section on management. some cats present for the abdominal wall with the other hand, but great care must be taken with this maneuver, because the devitalized colon can be perforated more easily. 140,170 enemas as described are painful for the cat, and opioid analgesia is recommended at the time of anesthesia. opioids can reduce peristalsis in humans, 96 but having evacuated the bowel, the pain relief is more important than this transient effect. an alternative to enemas is administration of an oral polyethylene glycol (peg 3350) solution (e.g., colyte, golytely). a nasoesophageal tube is placed and the solution is given as a slow trickle (6 to 10 ml/kg/hour) over 4 to 18 hours. defecation usually results in 6 to 12 hours. in a retrospective study of 9 cats, median time to defecation was 8 hours and the median total dose of peg 3350 was 80 ml/kg. 26a no adverse effects were noted. a cat that has been obstipated needs supportive therapy when discharged. there are no controlled comparisons of the various therapies noted in table 23-21; the author prefers cisapride 2.5 mg, every 12 hours to every 8 hours, po 82 (first thing in the morning, when the owner returns from work, when the owner goes to bed), and lactulose syrup 2 ml/cat, every 12 hours, po. a cat that has been so severely obstipated that an enema under anesthesia is required can be expected to continue these medications lifelong. to reduce fecal bulk and decrease the opportunity for recurrence, low-residue canned foods (or sachets) are preferred for cats that have become obstipated. some cats may benefit from high-fiber diets. as with simple initial episodes, canned food helps maintain adequate hydration, and at home subcutaneous fluids may be used additionally in cats with chronic kidney disease. with repeat episodes or severe obstipation, investigations for an underlying cause should be thorough and include evaluation for colonic mass obstructions. a review of published cases indicated that 96% of cases of megacolon are accounted for by idiopathic megacolon (62%), pelvic canal stenosis (23%), nerve injury (6%), or manx sacral spinal cord deformity (5%). 170 although most cases are idiopathic, an attempt should be made to identify and treat any specific underlying causes. megacolon is not specifically defined in cats. it has been described as "generalized colonic dysfunction manifesting as severe colonic dilation and fecal impaction," or a "severely and irreversibly dilated and hypomotile" colon 140 and "a subjective evaluation of the diameter of the colon, usually based on radiographic assessment." 18 there are specific radiographic guidelines for humans with megacolon, in that a colonic diameter of more than 6.5 cm at the level of the pelvic brim is considered diagnostic. 118 intake and therefore help maintain hydration. however, increased dietary fiber is beneficial for some cats, and trial and error may be required to determine whether a high-fiber or low-residue diet will be of benefit to each individual cat. in one report, 15 cats with recurrent constipation refractory to traditional medical and dietary management were successfully treated with a psylliumenriched dry extruded diet. 48a after 1 month on the diet, 14 cats had no clinical signs of constipation. the remaining cat was clinically normal after 2 months on the diet. improvement was noted in 10 of 15 cats after only 7 days of dietary therapy. measures should be taken to ensure adequate hydration. maintaining adequate hydration is particularly relevant for cats with chronic kidney disease that have impaired ability to conserve water. changing to wet food diets helps increase water intake. some cats with chronic kidney disease may need additional fluid support, such as subcutaneous fluids administered by the owner at home on a regular basis. underlying problems may be minor and simple to manage, such as an anal gland abscess, or more involved, such as reduced pelvic outflow, as a result of prior trauma. arthritis is a common underlying factor in many older cats and may be managed with prudent use of nonsteroidal agents (see chapter 26) . in cases of obstipation, the cat is more likely to be debilitated to some degree; so, laboratory investigations to assess plasma or serum biochemistry parameters as well as hematology and urinalysis are ideal. any hydration deficit or electrolyte abnormalities should be corrected before the anesthesia that is often required to remove the obstructing feces. rectal suppositories and microenemas are usually ineffective in obstipated cats. enemas are often required to remove impacted feces in such circumstances. the enema solution must be warmed and introduced slowly to avoid vomiting. the typical volume required is 5 to 10 ml/kg (so, up to approximately 50 ml/cat). the enema solution can be an isotonic electrolyte solution or tap water, and mild soap can be added (but any soap used must not contain hexachlorophene, which is neurotoxic if absorbed); mineral oil can be used (5 to 10 ml/cat) as a lubricant or docusate as an emollient (5 to 10 ml/cat), but the two agents must not be used together since docusate promotes mucosal absorption. sodium phosphate-containing enemas must not be used, because they can induce severe hypernatremia, hyperphosphatemia, and hypocalcemia in cats. often, the enema solution alone is insufficient to reduce the fecal mass, and manual manipulation of the feces by abdominal palpation is required. sometimes the feces must be broken down by a gloved finger perrectum while the colon is massaged manually through radiographically, in the lateral view, the normal colon should be approximately the same diameter as the length of the body of the second lumbar vertebra. 81 in cats, however, "there are no published guidelines for determining megacolon, so, diagnosis of abnormal colonic dilatation is subjective." 75 however, one author has suggested that "as a rule of thumb, the diameter of the colon should be less than the length of the body of the seventh lumbar vertebra (l7)." 105 this author continues, "enlargement of the diameter of the colon beyond 1 1 2 times the length of the body of l7 is indicative of chronic large bowel dysfunction and an explanation must be sought." 105 a recent paper found that 15 of 20 cats with no gastrointestinal disease had a colon diameter greater than the length of l7; however, no assessment of constipated cats was made. 1 in practice, many cats with megacolon have a colonic diameter far exceeding this guideline ( figure 23 -36). one study of 11 cats with megacolon found the mean diameter of the colon was 2.7 times greater than the length of the seventh lumbar vertebra (median, 2.4; range, 1.8 to 3.3), 107 but in general, objective descriptions of this condition are lacking in the veterinary literature. the definition of megacolon in cats should include functional as well as radiographic guidelines. in the absence of broadly recognized radiographic recommendations, the author proposes that the o'brien rule-ofthumb guidelines 105 (as noted above) be introduced until a more comprehensive study can establish other radiographic diagnostic criteria (or confirm these). the author therefore proposes to define megacolon as dilatation of the colon, to more than 1.5 times the length of the seventh lumbar vertebra, which is refractory to medical and dietary management. practitioners can expect the radiographic assessment of colonic dilatation to exceed this guideline in cats with megacolon and, conversely, there are likely to be cats having colonic distention greater than this amount that will respond to medical and dietary management and can therefore not be defined as having megacolon. by the definition used above, megacolon is refractory to medical and dietary therapy; so, to be defined as having megacolon, a cat may have had several episodes of obstipation managed by enema as well as dietary trials (with both low-residue and high-fiber diets) and medical therapy with cisapride and an osmotic or emollient laxative; yet the cat will still obstruct with feces. in these circumstances, the only possible therapy is subtotal colectomy. subtotal colectomy refers to surgical excision of 95% to 98% of the colon, whether it is grossly diseased or not with preservation of the ileocolic junction (icj). this approach has resulted in a more favorable clinical response than when the icj is also excised. 22, 170 when preserving the icj, it has been noted that, in some rare cases, it can be difficult to join the proximal segment of colon to the distal piece of descending colon because of the tethering effect of the ileocecocolic blood vessels. in these cases, sacrificing these vessels and removing the icj (i.e., total colectomy) is recommended to facilitate approximation of the ileum to the distal colonic segment. 21 a recently described technique using a biofragmentable anastomosis ring, compared with sutured anastomoses, showed no discernible effect on prognosis. 131 prognosis following subtotal colectomy is generally good. a review of multiple papers, totaling over 100 cats that had undergone subtotal colectomy, found the most commonly reported perioperative complication was diarrhea or loose stools immediately after surgery. in the majority of individuals, stool consistency improves without further treatment so that within 1 to 6 weeks of the surgery soft, formed stools are developed. diarrhea can persist in a small number of cases. in the longer term, in some cats, constipation can eventually return, but this can usually be managed by dietary and medical therapies. 172 pathology of the rectum or anus is relatively rare in cats and therefore poorly described in the veterinary literature. consequently, published information is often not referenced, suggesting it expresses the authors' opinions. readers are directed to surgical texts for details and approaches about surgical corrections. the anal sacs are paired cutaneous evaginations situated between the internal and external sphincter muscles. these sacs store secretions from alveolar and sebaceous glands that reside within the sacs. each anal gland has an associated duct that opens to the skin surface just lateral to the anus. 136,155 normal anal gland secretions have only very recently been described 47 and vary markedly; the color can be white, brown, orange, yellow, tan or gray, and consistency can range from watery to thick and creamy, with two thirds of cats having solid portions within the secretion. on microscopic examination, epithelial cells are commonly seen, with most cats having some neutrophils present. bacteria are commonly recognized as are, on some occasions, yeasts. bacteria seen in this study were mainly gram-positive cocci (63%) or gram-negative cocci (30%). gram-negative or grampositive rods were also seen but were rarely the dominant bacterial population. 47 with such a wide range of normal secretions, it is difficult to diagnose any pathology from the nature of the secretion alone. however, blood is infrequently recognized, and neutrophils are typically present in only small numbers in normal secretions. anal sac diseases described in cats include impaction, inflammation (sacculitis), infection, abscessation, and neoplasia (essentially the same as in dogs). 140,155 it has been contended in dogs that sacculitis and abscessation are an extension of impaction. it is not known in dogs or cats what the predisposing causes are, but suggested underlying reasons are loose stools (that are less effective at expressing the sac during defecation), local swelling or edema occluding the duct, and obesity. 155 the author's observations have also indicated that constipation can result in anal sac impaction because of less frequent expulsion of the sac contents; the resultant pain of the anal sac impaction can lead to further constipation, thus establishing a cycle. the retention of secretions may predispose to sacculitis, but impacted anal sacs do not always result in inflammation. abscessation is a likely sequel to sacculitis. 155 cats usually present for licking, scratching, or biting at the perineal area and can present for scooting (or dragging their anus) as dogs do. other presenting signs can be inability to sit or settle, a lump seen by the owner, or a generally unwell state. expression is the only management required for impacted (and not infected) anal sacs. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb externally. this is painful for most cats; so, the cat should be held by an experienced assistant, and it is sometimes not possible without some degree of sedation. with frequent episodes, underlying causes should be investigated. sometimes, trial-and-error diet change to manipulate the nature of the feces to either more (highfiber diets) or less (low-residue diets) bulk help reduce the frequency of episodes. obesity should be managed by reduced caloric intake, but dietary management for this should also take into account the nature of the feces. overt infection may be recognized by pus secretion from the anal sacs, which will have a high numbers of neutrophils. this can be managed by broad-spectrum antibiotics, such as amoxicillin/clavulanate or cephalosporins. a single treatment with a nonsteroidal antiinflammatory drug, such as meloxicam, can be given in animals with appropriate hydration and without other illness. anal sac abscesses often present already open and draining. many heal well by secondary intention with antibiotic treatment until they are closed over; so rechecks are required before the completion of an antibiotic course. large abscesses may require surgical drainage with the insertion of a penrose drain and management as for a cat fight abscess. it must be remembered that wounds in this area are easily re-infected by fecal contamination. recurrent impaction, sacculitis, or infection may require anal sacculectomy (as in dogs). this procedure should be delayed until infection is cleared. the procedure is similar to that performed in dogs. reports of anal sac/gland neoplasia were confined to sporadic case reports, 97,108 until a large case series was recently published. 141 in this study, 64 cases of anal gland carcinoma were recognized at a private diagnostic laboratory during a 12-year period, with submissions from 62 practices. this indicates that, for most practices, this condition will be seen, at most, once every 12 years. affected cats ranged in age from 6 to 17 years (median and mean, 12 years); female (mostly spayed) cats were overrepresented (61% of cases), and siamese cats may have been over-represented (7.8% of cases). the number of siamese cats with anal sac neoplasia was 3 times greater than the number of siamese cats in the laboratory reference population. affected cats presented for dyschezia, recurrent constipation, change in the nature or volume of feces, b). a low-residue wet diet is recommended to reduce fecal bulk during the healing period. rectal prolapse occurs as a result of a disease process that causes chronic straining, such as intestinal conditions that result in diarrhea and tenesmus 2. conditions that result in constipation or other intestinal obstruction 3. lower urinary tract diseases 4. dystocia and/or perineal swelling or ulceration, sometimes with purulent or hemorrhagic discharge. most tumors were originally interpreted as and initially managed as anal sac abscess. presumptive metastasis in liver, lung, or abdominal lymph nodes was recognized by physical examination or radiography in six cats; one cat was hypercalcemic. excision appeared to be curative (with a 3-to 4-year follow-up period) in 3 of 29 cats undergoing surgery for resection or debulking (others had only incisional biopsy performed). for the remaining 36 cats with known postsurgical outcome, median survival was only 3 months, with a 19% 1-year survival rate (with none of these cats surviving to 2 years). 141 atresia ani is a developmental defect of the anal opening or terminal rectum (see figure 41 -4). kittens usually present within days or weeks of birth with abdominal distention, discomfort, tenesmus, restlessness, vomiting, and/or loss of appetite. there are several anatomic variations 22 : • type i: a membrane over the anal opening remains, with the rectum ending as a blind pouch just cranial to the closed anus. • type ii: the anus is closed as in type i, but the rectal pouch is located somewhat cranial to the membrane overlying the anus. • type iii: the rectum ends as a blind pouch cranially within the pelvic canal (rectal atresia), whereas the terminal rectum and anus are normal. • type iv: occurs in females and atresia ani exists with a persistent communication between the rectum and the vagina (rectovaginal fistula). this fistula can occur with a normal anal opening as well. most reported cases have been type iv, 23, 146, 150, 158, 166 and this has also been recognized with concurrent sacrococcygeal agenesis. 3 surgical correction has been described for type ii 157 and type iv 19,91 atresia ani in cats. the reader should consult these references for surgical advice; possible complications include megacolon after prolonged obstruction, postsurgical anal stricture, and fecal incontinence because of sphincter dysfunction. foreign bodies in cats rarely obstruct the gastrointestinal tract distal to the jejunum 60 ; however, large fecal balls resulting from constipation can, additional to constipation or obstipation, cause distention of the anus. this distention can result in inflammation of the anal sphincter with loss of tone ( figure 23-37, a) , which, in the author's experience, is temporary with correction of the underlying cause of constipation. it can take some weeks for the dilated anus to return to normal (figure 23 -37, with antibiotics, such as cephalosporins, and regular cleansing. prolapses are usually classified in three ways. 62 first degree: prolapse of only mucous membrane 2. second degree: prolapse of full rectal wall thickness 3. third degree: prolapse is sufficient to bring mesorectum outside the anus the prolapsed rectum is obvious but must be differentiated from ileocolic intussusceptions, which have been described with neoplasia. 37 this distinction can be made by inserting a thermometer through the anus alongside the prolapsed mass. insertion will not be possible for an intussusception but will be for an anorectal prolapse. the prolapsed tissue must be assessed for viability, and management must include determining and managing the underlying cause as well as management of the prolapse. in simple cases where the mucosa is viable, the prolapse can be reduced with lubrication and gentle pressure. a temporary purse-string suture may be required to prevent recurrence. perineal dermatitis is often confused with gastrointestinal or urogenital disease, because there are often copious sebaceous secretions that can mimic fecal or urinary secretions. perineal dermatitis can result from flea or other allergies but also fecal or urine scalding associated with diarrhea or urinary incontinence, respectively. skin fold dermatitis can also occur in obese cats ( figure 23-38 ). episioplasty has been described to correct this, 121 but the author has found that stringent dieting can result in improvement while managing the skin fold dermatitis these populations. 48 the reported prevalence for each parasite varies greatly with the population studied, the geographic location of the population, and the sensitivity of the diagnostic test used to study that population. 48 the presence or absence of diarrhea is not a reliable predictor of whether a particular cat is infected with or shedding a parasite. 42 in fact, most cats with diarrhea do not harbor enteric protozoa. 48 on the other hand, most cats with diarrhea because of enteric pathogens will shed those organisms, often intermittently. it is important to remember that infection with most gastrointestinal parasites may not cause clinical signs. therefore detection of a pathogenic parasite in a cat with diarrhea does not necessarily prove causation. 48 a search should always be undertaken to identify other causes of diarrhea prior to convicting a cat of having diarrhea because of a particular parasite. in addition, co-infections or the presence of other noninfectious causes of diarrhea can result in more severe diarrhea that is often refractory to treatment for the parasite. treatment will be more rewarding if all potential causes of diarrhea are identified in the patient. enteric parasites with zoonotic potential occur commonly enough that cats, particularly those with diarrhea and who are owned by immunocompromised persons, should be evaluated for those pathogens. 26, 27 the following is a discussion of the most common enteric parasites found in cats. for more on parasite prevention and control, see chapter 8, and for more on zoonotic enteric parasites, see chapter 34. ollulanus tricuspis is an almost microscopic nematode worm infecting the stomach of domestic and wild cats. 5 the worm measures less than 1 mm long. 3 the larvae of o. tricuspis develop and hatch within the uterus of the female worm. they develop to maturity in the stomach of the cat where it is capable of re-infecting the host. 3 the worm is transmitted to other cats that ingest the vomitus of an infected cat. 41 clinical signs shown by infected cats include vomiting, anorexia, and weight loss. 2, 5 histologic findings in infected cats include lymphocytic-plasmacytic gastritis, lymphoid hyperplasia, and mucosal fibrosis. gross lesions may be absent, or the cat may develop nodular gastritis. 41 one report suggested the parasite may have been a contributing factor in the carcinogenesis of a gastric adenocarcinoma in an infected cat. 10 160. a common theme when discussing the prevalence of most gastrointestinal parasites in cats is that they occur more commonly in younger cats and in cats housed in crowded conditions, such as catteries and shelters. it is likely an increased chance for transmission exists in lungs. after further development in the lungs, the parasite migrates up the trachea and is swallowed. adult s. felis and s. planiceps burrow into the wall of the small intestine, while adult s. tumefaciens lives in the colonic mucosa. ova may be shed in the feces or hatch in the intestinal tract. autoinfection occurs if larvae become infective and penetrate the intestinal wall before being shed. ova and larvae that are shed develop into freeliving adult worms. 11 the prepatent period is between 7 and 10 days. 1, 11 clinical signs and diagnosis signs of a strongyloides spp. infection are usually absent. 1, 40 lung migration may cause cough or respiratory distress. the presence of the parasite in the intestinal tract may result in diarrhea and weight loss. 11 strongyloides tumefaciens is associated with the formation of small, worm-filled nodules in the colon. 40 identification of strongyloides spp. larvae using the baermann fecal concentration technique is required to diagnose most infections. unless the infection is heavy, examination of a fresh fecal smear is insensitive for identification of these larvae. 1 the nodules formed by s. tumefaciens infection can be visualized during colonoscopy. histopathology of the biopsied nodules should reveal many adult worms. 40 infection with strongyloides spp. can be treated with fenbendazole, 11 pyrantel pamoate, 40 thiabendazole, 1,11 or ivermectin. 3 to evaluate efficacy, repeat a fecal examination 2 to 3 days after the treatment ends. because of the presence of free-living adult worms in the environment and the ability of larvae to cause infection by penetrating intact skin, prevention is difficult. keeping cats indoors in warm, humid climates may be an owner's only means of preventing infection with strongyloides spp. parasites. infections with trichuris vulpis rarely occur in cats and are considered to be clinically unimportant. 3, 14 the two species of roundworms commonly infecting cats are toxocara cati (figure 23-39) and toxascaris leonina (figure 23-40) . the latter also has the ability to infect dogs. 14 cats are infected with t. cati in several ways. most commonly, infection is by ingestion of contaminated food, water, or infected paratenic hosts such as rodents. transuterine transmission has not been reported. 14 the diagnosis of infection with o. tricuspis is difficult, because ova are not shed in the feces; rather, the vomitus must be examined for worms or larvae. the worms may also appear in gastric mucosal biopsy samples. 41 a report of 131 cats undergoing endoscopic examinations found the parasite in gastric biopsy samples from 4 cats. 5 fenbendazole may be effective in treating infections with o. tricuspis. 41 preparations with febantel may also be expected to successfully treat these infections. transmission can be prevented by appropriately treating infected cats. other cats should not be allowed to ingest infected vomit. this parasite is of no zoonotic concern. physaloptera another parasite rarely inhabiting the stomach in cats is in the genus physaloptera. larger than ollulanus tricuspis, this blood-sucking worm infects cats that have ingested intermediate hosts, such as cockroaches, crickets, or flour beetles. 11 preying on transport hosts, such as mice that have eaten an intermediate host, is another way cats become infected with this parasite. clinical signs of infection with physaloptera spp. include vomiting, anorexia, and melena. a diagnosis of physaloptera infection can be made after identifying the ova in the patient's feces or adult worms in the vomitus. occasionally, the worms may be seen during gastroscopy. the adult worms must be differentiated from ascarids. 11 infection can be treated with ivermectin, pyrantel pamoate, or fenbendazole. 3 because there is no migratory phase of the life cycle, the treatment does not need to be repeated. 11 three species of strongyloides infect cats. strongyloides felis infects cats in india and tropical australia, 1,43 s. tumefaciens is a rare parasite of cats in the southeastern united states, 3 and s. planiceps is found in cats in malaya and japan. 1 strongyloides stercoralis, found in dogs and humans, produces experimental infections in cats, but natural infection with this species has not been observed. 1 feline infection with strongyloides spp. is considered by most to be rare. however, one report from australia identified s. felis in 169 of 504 necropsied cats. 43 infection with strongyloides spp. occurs after ingestion of infective larvae. infection can also take place after the larvae penetrate the skin of the cat. 11 ingested larvae penetrate the intestinal wall and migrate through the diaphragm into the lungs. after cutaneous penetration, the larvae enter the venous circulation and enter the clinical illness because of roundworm infection is uncommon. illness, when it does happen, most often occurs in kittens 26 signs may be mild and can include vomiting, 14 diarrhea, weight loss, poor growth, and a "pot belly." 26 a heavy infection with t. cati can result in catarrhal enteritis. severe infections can lead to intestinal obstruction and, possibly, perforation. 26 much less dramatic changes arise after infection with t. leonina, although enteritis may occur. 14 roundworms are frequently diagnosed with a fecal floatation. the centrifugal floatation technique is more sensitive than the simple fecal floatation technique many hospitals use. 14 occasionally, adult worms will be passed with the feces. the goals of treating roundworms include disease prevention in an individual cat or kitten, prevention of environmental contamination by cats defecating outside, and the prevention of zoonotic infections. many effective and safe anthelmintics are available (table 23-19) . benzimidazoles, such as fenbendazole, act on the parasite's microtubular structure, leading to disintegration of the worm's intestines, muscular layer, and hypodermis. 14 pyrantel in the pamoate formulation is poorly absorbed and causes paralytic parasite death. macrocyclic lactones, such as milbemycin, also lead to paralytic parasite death. these compounds act on the parasite's gamma-aminobutyric acid (gaba)-and glutamate-controlled ion channels. these channels are lacking in tapeworms, accounting for the lack of efficacy against these parasites. 14 lastly, emodepside (a cyclic octadepsipeptide) has been combined with praziquantel in the product profender (bayer animal health). this topical parasiticide has been shown to be both safe and effective. 14 these drugs appear to be so safe that overdosing is almost impossible. 14 kittens can be dewormed starting at two weeks of age and again at 4, 6, 8, 12, and 16 weeks. 26 older kittens and adults can be dewormed every month to 4 months. 14 because of the safety of these drugs, the possibility of false-negative tests and, more importantly, the zoonotic potential of these infections, perhaps all kittens should be dewormed, not just those testing positive. roundworm ova are very hardy and can remain infective for years. 14 they survive sewage treatment and composting, and there is no practical means of decreasing the ova population once the environment is contaminated. thus it is best to attempt to prevent contamination in the first place. when practical, keeping cats indoors allows appropriate control of potentially transmammary infection occurs, but only if the queen is acutely infected late in pregnancy. chronically infected queens do not pass t. cati ova in their milk. 14 after ingestion, t. cati larvae migrate through the small intestinal wall, into the liver, and then to the lungs where they are coughed up and swallowed. these larvae then infect the small intestine. some of the migrating larvae become encysted in the cat's muscle tissue. larvae from ova ingested through the milk tend not to undergo migration and mature directly in the small intestine. 14 the prepatent period is approximately 8 weeks. infection with t. leonina occurs after ingestion of infective ova or an infected paratenic host. unlike t. cati, very few t. leonina larvae migrate through the cat's tissues. most develop in the wall of the small intestine. the prepatent period is 7 to 10 weeks. toxascaris leonina ova can become infective within 8 days of being passed in the feces when the ambient temperature is 27° c but normally require 3 to 4 weeks. 14 lungs, and kidneys. ocular larval migrans results in granulomatous retinitis that is often misdiagnosed as retinoblastoma in older children. 3 this can lead to unnecessary enucleation. toxocara cati appears, however, to be less important than t. canis as an infection in humans. 3 the species of hookworms that infect cats are ancylostoma tubaeforme and ancylostoma braziliense (see figure 23 -39). they are reported to be an uncommon infection in cats. 26, 34 ancylostoma braziliense can also infect dogs. hookworm infections occur after ingesting food or water contaminated with hookworm larvae or eating contaminated fecal material. if the pet cat is allowed outdoors, attempts at preventing hunting may reduce the possibility of infection. keep children's play areas, such as sand boxes, inaccessible to cats when children are not at play. feeding only well-cooked food can prevent infection by contaminated food. finally, empirical, preventative deworming for cats that go outdoors should be performed 3 to 4 times yearly. any less frequently does not lead to an appreciable decrease in the prevalence of the parasite. 7 roundworms easily infect humans who ingest the ova, particularly children. visceral larval migrans occurs after infection with toxocara canis in humans. infection can lead to the formation of nodules in the brain, liver, tapeworm infections are well tolerated by the cat. usually there are no signs of infection other than finding segments on the feces or attached to perianal hair. because both d. latum and spirometra tapeworms absorb vitamin b 12 across the cuticle, megaloblastic anemia is possible, but unlikely. 11 tapeworm infections are diagnosed by identifying the typical appearance of the segments 8 or the egg packets within the segments. 26 the segments of t. taeniaeformis are flat, while those of d. caninum have been described as appearing like a grain of rice. the segments should be handled carefully, because they are friable and rupture may result in exposure of the handler. 8 the operculated ova of d. latum and spirometra spp. must be differentiated from trematode ova. even though tapeworm infections are well tolerated, cats should be treated for reasons of owner discomfort and public health concerns (see table 23 -19) . these infections are easily treated, because drug treatment is highly effective. re-infection must be controlled using preventative measures, especially flea control to prevent re-infection with d. caninum. praziquantel and infected paratenic hosts. the larvae can survive for months in the tissues of paratenic hosts. 14 infection also occurs after larval migration through the skin. in either case, the worm matures in the small intestine. 14 unlike dogs, transmammary infection has not been reported in cats. 3, 14 the prepatent period is between 19 and 28 days, depending on the route of infection. the time to patency after transcutaneous infection is longer than for direct colonization. infective l3 larva develop 2 to 7 days after the ova are passed. 3 developing larvae attach to the mucosa of the small intestine where they ingest copious amounts of blood. because the worms can remove a significant volume of blood from kittens, weakness from iron-deficiency anemia 34 or blood-loss anemia may be noted. 14 melena and diarrhea may also be recognized. signs are uncommon in adult cats. identification and treatment of hookworm infections are similar to that for roundworm infections (see table 23 -19) . hookworm larvae are not as hardy as roundworm eggs. soil contamination may be a temporary problem in areas that experience a hard frost. 3 hookworm larvae will not develop in temperatures less then 15° c or greater than 37° c. frequent, appropriate disposal of feces, cleaning surfaces with a 1% bleach solution, and deterring hunting may prevent infections. migration through the skin of persons coming into contact with the larvae of a. braziliense is the most common cause of cutaneous larval migrans, particularly in the southeastern united states. 3 this is an erythematous, pruritic skin eruption often found on the soles of the feet of infected children. the tapeworms most commonly found in cats are dipylidium caninum and taenia taeniaeformis. diphyllobothrium latum, spirometra spp., and echinococcus multilocularis occasionally infect cats. the latter is important, because it can lead to alveolar echinococcosis in humans. 8 spirometra tapeworms are found in north america (s. mansonoides) and far-east asia (s. mansoni and erinacei), while d. latum prefers temperate climates. 11 unnoticed, but the cat may cough or experience hemoptysis. 3 diagnosis involves demonstration of fluke ova in the feces. although therapy may be unnecessary, 11 praziquantel or epsiprantel are effective in eliminating the intestinal population of the fluke. platynosomum spp. are flukes living in the gall bladder, bile ducts, 46 and pancreatic ducts. 3 these flukes are most prevalent in the southeast united states and caribbean islands 3 and require two intermediate hosts. the first host is a snail, while the second intermediate host is a lizard, toad, gecko, or skink. 46 cats become infected with this fluke after ingesting an infected second intermediate host. the prepatent period for the fluke is 8 weeks. 46 most infections are subclinical. if clinical signs do occur, they may include weight loss, vomiting, diarrhea, icterus, hepatomegaly, or abdominal distention. diagnosis involves identification of ova shed in the feces using a fecal sedimentation method 46 or by finding adult flukes in the gall bladder or bile ducts during abdominal surgery. treatment involves administering praziquantel (20 mg/kg, q24h, po for 3 to 5 days) and/or surgical removal of the flukes. 3 two species of coccidians are the most common to infect cats, isospora felis and isospora rivolta (figure 23-41) . the genus isospora may be renamed cystoisospora. these are epsiprantel are safe and effective. fenbendazole is effective against t. taeniaeformis, but not d. caninum. without controlling exposure to intermediate hosts, tapeworm infections are difficult to eliminate. flea control is imperative in eradicating infections with d. caninum. controlling predation helps prevent ingestion of t. taeniaeformis-infected rodents. infection with d. caninum occurs in young children who are most likely to eat fleas. infection results in only minimal signs of illness. 8 the larval stage of t. taeniaeformis is of little zoonotic importance. 3 although cats are uncommonly infected with echinococcus multilocularis, potentially life-threatening alveolar damage occurs in north american humans infected with this tapeworm. 8 plerocercoids of spirometra spp. can penetrate the mucous membranes or open skin wounds of humans and migrate around the subcutaneous connective tissue, forming nodules, a condition called sparganosis. 3 megaloblastic anemia, as a result of vitamin b 12 deficiency, may occur in humans infected with d. latum or spirometra spp. tapeworms. 11 alaria marcianae flukes reside in the intestinal tract of cats and the mammary glands of lactating queens. miracidia hatch underwater from ova shed in the feces and penetrate the skin of a snail. after further development, cercariae penetrate the skin of leopard frog tadpoles and are able to survive the metamorphosis to the adult frog. 3 if the tadpole is eaten by a snake, bird, or mammal, the parasite enters the host's tissues but does not undergo further development. after a male or nonlactating female cat ingests the infected intermediate host, the parasite penetrates the wall of the small intestine, passes through the diaphragm, and enters the lungs for further development. finally, the parasite is coughed up and swallowed to complete maturation and reproduce in the small intestine. if, however, an infected host is ingested by a lactating queen, the parasite migrates through the tissues to the mammary glands, rather than the lungs. 3 once shed in the milk, the parasites develop into mature adults in the kittens. some of the mesocercariae remain in the mammary glands to infect future litters. clinical signs associated with worms in the small intestine are uncommon. 11 migration through the lungs often goes species-specific obligate intracellular parasites. 4, 13 they are able to survive in the environment for months. 13 a detailed description of the coccidial life cycle can be found elsewhere. 4, 13 simply put, direct transmission is by ingesting oocyst-contaminated food or water or by grooming contaminated body parts. indirect transmission occurs after ingesting a mechanical vector or the infected tissues of paratenic hosts. 39 after ingestion by a cat, the oocyst excysts in the small intestine and enters the enterocyte where further development occurs. 26 the parasite may also migrate through the intestinal wall to form cysts in mesenteric lymph nodes. these cysts may serve as a source for reinfection. 4, 13 the prepatent period is 4 to 11 days 26 and the shed oocyst becomes infective after several days of exposure to warmth and moisture. 13 infection with isospora spp. is usually subclinical. 26 signs, if they occur, range from mild, transient watery diarrhea to severe mucohemorrhagic diarrhea with vomiting and resultant dehydration and weight loss. 4, 13 signs are most commonly recognized in severely infected neonatal kittens, 26 particularly those with concurrent illness, and arise because of small intestinal congestion, mucosal erosion, or villus atrophy. 39 signs may also be noted in immunosuppressed adult cats. 26 isospora species are readily found in fecal floatation or wet-mount examinations. shedding can be intermittent, but most cats with diarrhea caused by coccidial infection shed large numbers of oocsyts. 39 fortunately, in most cats, the diarrhea from isospora spp. infection is self-limiting. 26 in fact, if a kitten is persistently shedding oocysts despite appropriate treatment 13 or the parasite is identified in an adult cat with chronic diarrhea, attempts should be made to identify co-infections or other diseases that may cause diarrhea. 39 anticoccidial drugs are either coccidiostatic or coccidiocidal (table 2320) . coccidiostatic drugs are the most commonly used drugs for individual pet cats. trimethoprim-augmented sulfadiazine (tribrissen; intervet/schering-plough animal health, summit, nj) or another sulfa-containing antibiotic, sulfadimethoxine (albon; pfizer animal health, madison, nj), can be used. supportive care for severely affected kittens, such as parenteral rehydration, should be used as needed. coccidiocidal drugs are often reserved for use in densely populated situations such as catteries or shelters. 26 however, many veterinarians are now using them as a first-line defense against isospora spp. infection. 13 ponazuril (marquis oral paste; bayer animal health, shawnee mission, kan.), formulated for horses, is effective and can be safely administered to cats. for more on the use of ponazuril in cats, see chapter 46. a related drug, diclazuril, is also available and may be administered once at 25 mg/kg po. 13 while not available in north america, toltrazuril (baycox, bayer animal health) may be administered once at 30 mg/kg po or 15 mg/kg po once daily for 3 days. 32a a second course of therapy 10 days later may be required to completely eliminate the oocysts. sanitation is very important, because the oocyst requires several days to become infective. frequent removal of feces, preferably daily, is recommended to prevent re-infection and transmission to other cats. 39 controlling a cat's ability to hunt reduces the chance of ingesting an isospora-infected rodent. control of mechanical vectors, such as cockroaches and flies, is also useful. 13 since a cat can become infected after grooming an infected cat's perineum, consideration should be given to treating all cats in contact with the patient. 39 in addition, catteries and shelters should ensure all food is well cooked, litter boxes are cleaned daily, and surfaces are well cleaned with steam 13 or 10% ammonia. 39 where recurrent isospora spp. infections are a problem, prophylactic treatment of all 2-to 3-week-old kittens with ponazuril should be considered. 13 despite all wellintentioned efforts at hygiene and treatment, isospora spp. infection can still be transmitted to other cats. 13 because these are species-specific parasites, transmission of i. felis and i. rivolta from cats to humans does not occur. the flagellated protozoal parasite, giardia duodenalis, has seven microscopically indistinguishable genotypes or assemblages. 26 assemblages a and b infect humans, while assemblage f is harbored by cats. cats will occasionally harbor assemblages a and b. 39 infection with g. duodenalis occurs after ingesting cystcontaminated feces, by grooming an infected cat or from contaminated fomites. 37 re-infection may occur by selfgrooming. only a small number of cysts need be ingested to establish an infection. in humans as few as 10 cysts are required to cause infection. 37 after ingestion of infective cysts, trophozoites begin to excyst in the stomach. 37 this process is completed in the proximal duodenum. 26 the trophozoites adhere to enterocytes along the length of the small intestine using the ventral suction disk. intermittent shedding of immediately infective cysts begins 5 to 16 days after infection. 28 proteins released during encystment of the trophozoites are detected by the fecal antigen tests. 37 cysts may adhere to the perianal region, facilitating re-infection by self-grooming. 28 occasionally, trophozoites are found in examinations of fresh, watery feces. these do not survive for long and are not infective. 4 the mechanisms of disease induced by g. duodenalis are still unclear. after the trophozoite attaches to the brush border of the enterocyte, the tight junction between cells is disrupted, increasing intestinal permeability. 37 the brush border becomes attenuated, further exacerbating malabsorption of water, electrolytes, and other nutrients. 39 the alteration in intercellular adhesion results in t-lymphocyte activation and mucosal cell injury. 37 infection also promotes mucosal cell apoptosis (preprogrammed cell death). 28 in addition, small intestinal bacterial overgrowth may accompany g. duodenalis infections, resulting in more severe clinical signs. 39 fortunately, most cats infected with g. duodenalis show no clinical signs. 28, 39 the most common sign is acute, transient, small bowel diarrhea 28 without systemic illness, such as fever or vomiting. 39 less commonly, a cat might have profuse, watery malodorous diarrhea 37 with mucus. 39 also possible, but uncommon, is weight loss 26, 28 or abdominal pain. 37 the severity of clinical signs exhibited in an individual cat depends on the age and general health of the cat. 37 cats co-infected with cryptosporidium felis or tritrichomonas foetus may have more severe diarrhea that is more difficult to control, 39 as will the presence of bacterial overgrowth. the diagnosis of g. duodenalis requires demonstration of trophozoites or cysts in a fecal examination, or detection of encystment proteins or giardial dna in a fecal sample. a reliable diagnosis may be difficult to obtain for several reasons. cysts are small, easily missed, and must be differentiated from plant debris or yeast. 37 trophozoites are short lived outside the body and can only be found in very fresh, watery feces or, better yet, in diarrheic feces collected directly from the cat's rectum. 37 shedding of cysts is usually intermittent, and the intensity of shedding varies greatly. 28, 37 because of these difficulties, the absence of the organism in a fecal sample does not eliminate it as the cause of diarrhea. it is often necessary to test multiple fecal samples, using at least two different techniques in order to find the organism. 37, 39 the easiest test to perform is a fecal smear or wet mount examination to identify trophozoites or cysts (figures 23-42 and 23-43 ). the sample examined should be very fresh, warm, diarrheic feces. 39 one drop of feces is placed on a slide along with a drop of 0.9% saline or lugol iodine. 28 trophozoites are identified by their characteristic structure (table 2321) . the motile trophozoites have a motion described as appearing like the back and forth rolling motion of a falling leaf. since lugol iodine stain kills the trophozoite, there will be no motion to detect. 28 this test is not very sensitive; however, with trained examiners, the test has a high specificity. increased sensitivity can be gained by performing a centrifugal flotation using zinc sulfate. the sample should be warm, fresh feces or feces refrigerated for no more than 2 days. 28 the processed sample is examined for the same structures as the wet mount. the sensitivity of examining one sample is 70% 28 and increases as more samples are examined. the sensitivity of looking at three samples is 95% 26, 28 ; therefore the test is not considered negative until three specimens have been found free of the organism. 39 a fecal antigen test that identifies the encystment protein is available. the snap giardia antigen test (idexx laboratories) uses fresh or frozen feces, or feces refrigerated for less than 7 days. 34 since the antigen is continuously shed, this test avoids the problem of intermittent shedding of the whole organism. 28 the sensitivity of the test is 85%, with a specificity of 100%. 35 by combining the antigen test with a zinc sulfate fecal centrifugal flotation, the sensitivity improves to 97.8%. 11 it is unknown how long the antigen remains in the feces after treatment. thus a zinc sulfate centrifugal flotation examination should be used to evaluate therapeutic efficacy. 28, 39 the use of this test in cats without diarrhea is controversial, because these cats are unlikely to shed cysts. the zoonotic significance of a positive antigen test in a cat not shedding cysts is unknown and may cause confusion. 39 polymerase chain reaction detection of giardia dna is available, but the test has not been standardized across all diagnostic laboratories. one needs to ensure the laboratory performing the test has validated it for assemblage f. the test may also be used to identify cats harboring the zoonotic assemblages a and b. the sensitivity of this test is unknown. 39 two commonly available drugs are used most frequently to treat infections with g. duodenalis (see table 23 -20) . fenbendazole may be effective and can be used in pregnant queens 28 and in cats co-infected with roundworms, hookworms, and taenia spp. tapeworms. 39 however, in one small study, only four of eight cats infected with both g. duodenalis and cryptosporidium felis stopped shedding giardia permanently after receiving fenbendazole. 29 febantel, in the combination product drontal plus (bayer animal health), is converted to fenbendazole. when six experimentally infected cats received 56.5 mg/ kg of febantel q24h po for 5 days, four of them stopped shedding g. duodenalis cysts. 39 metronidazole has been the traditional drug used to treat g. duodenalis in pets. 28 the drug is also useful for treating concurrent small intestinal bacterial overgrowth and clostridial infections. 39 the administration of metronidazole may eliminate shedding in 67% of cats. 26 neurologic side effects may occur at the dose recommended for treatment of giardia (see above, therapeutics for vomiting and diarrhea). the use of a giardia vaccine was ineffective in clearing infection by itself. 44 the combination of fenbendazole and metronidazole has been suggested as the initial treatment of choice for g. duodenalis infections. 39 although controlled studies are lacking, they may work synergistically by acting on two different targets within the parasite. 28 febantel would be expected to have the same synergism with metronidazole. drug therapy may not be necessary in cats without diarrhea that are infected with g. duodenalis, 37 because it is uncommon for a cat to carry the assemblages required to infect humans. the veterinarian may be obligated to treat a healthy cat if the owner wants to treat, the owner is immunocompromised, or the goal is eradication of an infection from a multicat home or prevention of parasite transmission to giardia-naïve cats is attempted. 28 what may appear to be treatment failure is more likely to be re-infection. in addition to drug therapy, steps should be taken to prevent re-infection. all cats with diarrhea positive for g. duodenalis should be treated along with their housemates. 37 sanitation is imperative in the fight against re-infection and transmission of g. duodenalis. dispose of old litter pans and scoops and use disposable litter boxes during treatment. when the infection is eliminated, not just controlled, new litter boxes and scoops may be purchased. bathe all cats during treatment to remove cysts from the hair coat. since giardia spp. cysts are susceptible to desiccation, 28 blowdry all cats using a warm air blower, paying particular attention to the perineal area. disinfect bowls, housing, and other utensils with bleach. 28 in addition to antiprotozoal drugs and sanitation, supportive care may become necessary. probiotics and a highly digestible, bland diet may be offered to cats with small bowel diarrhea, while a high-fiber diet may be useful for those few cats with large bowel diarrhea. 39 where required, hydration and electrolyte imbalances must be corrected and antiemetics used to control vomiting. therapy can be evaluated by retesting feces with a zinc sulfate centrifugal flotation examination 1 to 3 days after the end of treatment and again 3 weeks later. a positive test immediately posttreatment is most likely because of therapeutic failure. if the cat is negative immediately after treatment ends, but is positive 3 weeks later, re-infection is likely. 28 since the fecal antigen test may remain positive long after the infection is eradicated, this test is inappropriate for evaluating therapy. 28, 39 re-treatment of fecal flotation-positive, recovered cats may be handled in a manner similar to the positive healthy cat mentioned above. 28 cats with diarrhea that continue to shed cysts may be re-treated for g. duodenalis infection along with dietary modification and empirical treatment for other common intestinal parasites. however, serious consideration should be given to investigation into other potential causes of diarrhea. 28 the giardia vaccine has been found to be ineffective in preventing infection 37 and production has been discontinued. 39 this means prevention of giardia infection involves avoiding exposure, stress and re-infections. providing a clean environment, feeding only processed foods, and controlling potential transport hosts will help reduce the chances of exposure. isolation of cats with diarrhea may be important, too. 39 municipal sanitation control is difficult as the cyst survives for weeks in cool, moist environments. 28 cysts are also able to survive water treatment and can pass through attempts at water filtration. 37 giardiasis is associated with debilitating diarrhea in some humans, particularly those who are immunocompromised. 35 however, cats do not commonly carry the assemblages needed to infect humans. transmission of g. duodenalis from cats to humans is rare and unproven. 28 still, it seems prudent to consider the owner's health when contemplating management of giardial infections in cats. to avoid human health risks, cats with diarrhea that test positive for g. duodenalis should be treated with the goal of controlling the diarrhea. 39 since no treatment for g. duodenalis is completely effective or 100% safe, treatment of positive cats without diarrhea should only begin after a discussion of the benefits and risks of the treatment with the owner. 39 tritrichomonas foetus is best known for causing bovine reproductive infections. it is an obligate anaerobic parasite 26 that also colonizes the lower intestinal tract of cats. there are enough differences between the two isolates that the feline isolate does not cause disease in heifers and vice versa. 39 the parasite depends on the host's normal intestinal flora and secretions for obtaining nutrition. 7 a report from the united states of purebred cats tested at an international cat show found t. foetus in 36 of the 117 cats tested, a prevalence of 31%. 23 this parasite seems to have a higher prevalence in purebred cats than nonpurebred cats. a study of pet cats visiting veterinary hospitals across the united states reported 12 of 32 purebred cats were positive for t. foetus, while only 5 of 141 nonpurebred cats were positive. in this same study, 12 of the 17 positive tests were from purebred cats. 45 a study from the united kingdom of diarrheic fecal samples sent to a veterinary diagnostic laboratory reported similar results. purebred cats represented 14 of the 16 cats testing positive for t. foetus. the u.k. study also found the siamese and bengal breeds each represented 6 of 14 positive cats; only two other breeds tested positive. 25 transmission like most other protozoal parasites, t. foetus is transmitted by ingestion of the parasite, in this case, the trophozoite. unlike most of the other parasites, t. foetus does not form cysts and only survives up to 3 days outside the body in moist feces. 47 a cat becomes infected through the use of a shared litter box with an infected cat. after walking into the box, the parasite is transferred from the infected feces of one cat to the paws of the other. infection then occurs through ingestion of the trophozoites during grooming. 47 after infection, t. foetus colonizes the distal ileum and colon, 15 followed by shedding of infective trophozoites 2 to 7 days later. 19 there are several mechanisms by which t. foetus causes diarrhea. these include alteration of the cat's normal bacterial flora population, increases in local inflammatory cytokine concentrations, production of enzymes, and direct mucosal injury. the resulting injury leads to plasmacytic-lymphocytic 49 and neutrophilic colitis. 37 although most infections involve only the mucosa of the colon, one study reported two of seven cats with diarrhea and t. foetus infections as having trophozoites in deeper layers of the colonic wall. 49 co-infection with cryptosporidium felis 17 or giardia duodenalis 39 can be associated with increased numbers of t. foetus trophozoites and increased severity of diarrhea. signs of infection are most frequent in kittens and young cats, although infections without clinical signs can occur. 39 adult cats, however, may also show signs of t. foetus infection. the most common sign is a foulsmelling large bowel diarrhea with increased frequency of defecation, 39 mucus, blood, 15 and flatulence. 37 the consistency of the diarrhea may wax and wane, but the presence of diarrhea does not. 47 cats with diarrhea are otherwise in good health and maintain their body condition. 15, 39 severe diarrhea can result in anal swelling and fecal incontinence. 39 diarrhea may respond to the use of antibiotics because of changes in the cat's intestinal microbial flora. however, it always returns at the cessation of therapy. 39, 47 many cats experience a spontaneous resolution of the diarrhea within 2 years of diagnosis. 15, 38 since t. foetus causes reproductive infections in heifers and bulls, there is speculation the parasite also infects the reproductive tract in cats. tritrichomonas foetus was found in the uterus of a queen with pyometra. 9 however, in a study of 60 breeding male and female cats from 33 catteries, no cytologic or molecular evidence of t. foetus was found in the reproductive tract. the authors reported colonic infection with t. foetus in 15 of the 60 cats representing 22 of the 33 catteries. 24 detection of the trophozoites in a sample of feces is the most expedient means of diagnosing an infection with t. foetus (figure 23-44 ). an index of suspicion is required, because the clinical presentation of t. foetus infection is often mistaken for infection with giardia duodenalis. if a cat is not responding to treatment for that parasite, consider t. foetus as a cause of the diarrhea. the sample required for the diagnosis of t. foetus is a fresh, nonrefrigerated sample of watery feces. refrigeration kills the trophozoites, and they are not found in normal feces. 34 the sample may be freshly passed diarrhea, feces collected using a wire loop passed into the colon, or collected by a colonic flush using a red rubber catheter and 10 ml of saline. 47 a wet mount or smear examination of the feces should be performed on all cats with diarrhea. examination of multiple samples may be required to find the t. foetus trophozoites with this technique because it is insensitive. 39 the trophozoites must be differentiated from giardia duodenalis based on structural differences and motility patterns (see table 23 -21). the trophozoites of t. foetus can be cultured using the inpouch tf system (biomed diagnostics). this test is more sensitive than the fecal wet mount examination and detects 1000 trophozoites per sample. 15 the number of parasites shed by a cat with diarrhea is high enough to be routinely detected with this method. 18 the test should be performed in-house, because the parasite is unlikely to survive the trip to the laboratory. 47 the test pouch is inoculated with 50 µg of freshly collected feces, about the size of a peppercorn. 18 any more than this increases the chances of bacterial overgrowth. 15 the pouch is incubated at 25° c and examined under the microscope for motile trophozoites every other day for 12 days. the pouch should be tapped gently to dislodge the parasites, which tend to collect along the seams. 15 the test is considered negative if parasites are not found after 12 days. one benefit of this system is that it does not support growth of giardia duodenalis or pentatrichomonas hominis. 18 if a fecal wet mount examination and culture are both negative and infection with t. foetus is still under consideration, a pcr test can be performed. this test detects dna from live or dead trophozoites, but is more expensive than other diagnostic methods. 47 this test is more sensitive than the other two methods and can detect 10 parasites per sample. 16 the sample size is 200 mg of feces not contaminated by litter preserved in 3 to 5 ml of rubbing alcohol shipped at room temperature. 15 trophozoites of t. foetus are sometimes found in colonic biopsy samples adhered to the surface or in the lumen of crypts. 15 the most effective drug for the treatment of t. foetus in cats is ronidazole. 17 the drug has a bitter taste and should be compounded into capsules. veterinary staff and owners should use gloves when handling ronidazole. 15 if a confirmed relapse occurs, another course of treatment may eliminate the parasite. 39 diarrhea may take several weeks to resolve after elimination of the parasite, because significant colitis is often present. 47 effectiveness of treatment can be evaluated by performing fecal pcr tests 2 and 20 weeks after the end of treatment. 15 apparent treatment failures may occur because of re-infection, co-infection with giardia duodenalis or cryptosporidium felis, or the presence of another concurrent diarrhea-causing disorder. a more worrisome cause for treatment failure is a recent report of parasite resistance to ronidazole in two cats. 22 fortunately, diarrhea ultimately resolved in both cats despite the continued presence of the parasite. if the cat retests negative and the diarrhea is not improving after 2 weeks, consider the possibility that another disease may exist. nonspecific treatment for diarrhea is unhelpful 37 and may prolong the duration of diarrhea. 15 diarrhea may respond to antibiotics as they alter the intestinal flora population; however, once treatment is stopped, the diarrhea will return. 47 an important and potentially serious adverse effect of ronidazole administration in cats is a reversible neurotoxicity. onset of signs often begins within 1 week of the onset of therapy and may last between 1 and 4 weeks after cessation of therapy. 38 these signs can include depression, ataxia, seizures, 47 behavioral changes, weakness, hyperesthesia, and trembling. 38 neurotoxicosis usually requires only supportive care along with discontinuation of the drug. the neurologically affected cat should be retested for the parasite, because it may have been eliminated. 38 because of the potential for neurotoxicity, the use of ronidazole should be restricted to cats with confirmed infections with t. foetus. 47 crowded conditions should be avoided, because transmission of t. foetus trophozoites is more efficient in these settings. 39 cats testing positive should be isolated from other cats during treatment. 37 providing a clean environment will help prevent transmission of trophozoites. although there is a report of an infection in one immunocompromised person, transmission of t. foetus trophozoites from cats to healthy humans has not been reported. 39 still, prudence dictates handling feces infected with t. foetus trophozoites carefully. recent genetic evaluations have shown that most feline infections with cryptosporidium spp. are with c. felis; not, as previously thought, with c. parvum. 36 cryptosporidium parvum seems to be limited to farm animals. 4 cryptosporidium felis is an obligate intracellular parasite infecting the small intestine. 4 infective oocysts are ingested from contaminated feces during self-grooming of contaminated body parts and from contaminated food and water. 32, 39 after infection, the parasite attaches to the brush border of the enterocyte. the prepatent period is 3 to 6 days, 39 and the oocysts are infective as soon as they are shed, making this a very contagious disease. 20 like most intestinal parasites, shedding is often intermittent. the pathogenic effects of c. felis infections are not well understood. direct cytotoxicity and inflammation causes villus atrophy and decreased surface area for absorption of water, electrolytes, and other nutrients. 20, 32 apoptosis (preprogrammed cell death) of the mucosal cells may be accelerated, adding to the malabsorption. 20 most infections with c. felis are subclinical. 39 signs, if present, range from a mild, self-limiting small bowel diarrhea 33 to chronic intermittent small bowel diarrhea. 32 severe diarrhea with weight loss and anorexia may also occur. 32, 33 clinically apparent infections are most common in kittens, adult cats with concurrent gastrointestinal diseases, and cats co-infected with giardia duodenalis or tritrichomonas foetus. 39 cats with co-infections may experience more severe clinical signs. 32 a fecal flotation, which should be performed on all cats with diarrhea, may reveal c. felis if there are large numbers of oocysts (figure 23-45) . the fecal floatation test, however, is often negative 39 because of intermittent shedding. the parasite is small and floats in a higher plane than helminth ova; the high-power lens and appropriate adjustment of the microscope stage is required to find the parasite. 32 the small size of the oocyst makes identification difficult, particularly if the examiner is not specifically looking for them. 34 a modified ziehl-neelsen stain of a thin fecal smear may help in the identification of the oocysts. 39 this technique works well in humans with large numbers of oocysts. 33 once signs resolve or the oocyst numbers decline, a single examination of a stained smear becomes insensitive. when only one sample is available, testing for c. felis antigen is a good choice. 34 the prospect microplate assay (alexon biomedical, sunnyvale, calif.) is more sensitive and specific for the diagnosis of c. felis than is the examination of a stained smear. 6 immunofluorescent antibody testing is available from some laboratories. fecal c. felis dna can be detected using pcr testing. this test is available at many veterinary diagnostic laboratories; however, at present, there is no test standardization among laboratories. 39 the clinical and zoonotic significance of a positive pcr test combined with an oocyst negative test is unknown. 39 therefore a positive pcr test in a cat without diarrhea presents a confusing situation for the attending veterinarian with regard to recommendations for the owner. unfortunately, there are no completely effective and safe treatment protocols available for c. felis. 32, 39 a concerted attempt to find other causes of diarrhea should take place prior to convicting a cat of having diarrhea solely from c. felis infection. most reports on therapy for c. felis are uncontrolled and anecdotal. a number of drugs have been discussed. azithromycin for at least 10 days appears safe but produces variable results. 39 paromomycin, an oral aminoglycoside, may be effective. however, one study reported acute renal failure in 4 of 32 cats receiving the drug. deafness also occurred in three of those four cats. 21 nitazoxanide is a drug approved for treating humans with diarrhea caused by cryptosporidium spp. infections. the administration of nitazoxanide to cats at 25 mg/kg q12h po for at least 5 days 39 up to 28 days 32 may be effective. however, nitazoxanide is a gastrointestinal irritant and commonly results in vomiting and foul-smelling diarrhea. co-infections with giardia duodenalis and/or tritrichomonas foetus are more difficult to control. if diarrhea from c. felis infection improves but does not resolve at the end of therapy, the duration of treatment may be prolonged. 39 additional diagnostic testing should also be performed to ensure the only cause of the diarrhea is infection with c. felis. environmental control of c. felis is difficult, because it is extremely hardy. it is resistant to chlorination and most disinfectants. 32 oocysts remain viable at temperatures above freezing up to 65° c. 4 the parasite is difficult to filter and survives treatment at municipal water treatment facilities. 20 steam-cleaned housing and utensils may be beneficial in controlling parasite numbers, and they are susceptible to 5% ammonia solutions; however, the required contact time is 18 hours. 39 cryptosporidium spp. are relatively species specific, and there are no reports of waterborne outbreaks of human cryptosporidiosis associated with c. felis. 32 cryptosporidiosis can cause life-threatening diarrhea in hivpositive persons. 20 fortunately, humans are rarely infected with c. felis. 39 in fact, the zoonotic species most commonly found in humans (often veterinary students), is c. parvum found in young heifers. 4 regardless of a person's health, feces from a cat with diarrhea should be handled carefully. if a cat infected with cryptosporidium spp. is owned by an immunocompromised person, a pcr test may be useful in determining the species of the parasite and its zoonotic risk. like other coccidians, toxoplasma gondii is an obligate intracellular parasite. 12 domestic cats and other felids are the only animals that shed oocysts. any warmblooded animal, including humans, can be infected with this parasite. toxoplasma gondii can be transmitted by ingestion of infective oocysts in fecally contaminated food or water after ingestion of tissue cysts through carnivorism, or by transplacental or trans-mammary transmission of the parasite. the parasite enters into one of two cycles, depending on the host species. the enteroepithelial cycle only occurs in cats and results in shedding of oocysts after sexual reproduction of the parasite. after a cat ingests an infective oocyst or a tissue cyst, the parasite enters the mucosal cells of the small intestine, where it may undergo development and sexual reproduction, after which oocysts are shed. 12 the prepatent period after ingesting an infective oocyst is 19 to 48 days, while shedding after ingesting tissue cysts starts in 3 to 10 days. 4 fecal shedding, which occurs only after initial infection, lasts for 2 to 3 weeks 4,31 and the oocysts become infective 1 to 5 days after they are shed. 12 the extraintestinal cycle occurs in any animal, including cats. after ingestion, the parasite penetrates the cells of the small intestine and rapidly replicates in the enterocytes and associated lymph nodes into tachyzoites. after hematogenous and lymphatic spread, tachyzoites infect cells in all tissues of the body. 4 tissues most commonly infected include the brain, liver, pancreas, and lungs. 30 if a pregnant queen becomes infected, tachyzoites cause placentitis, after which they infect the fetus. 13 in 3 weeks, the host's immune response slows parasite replication, and the resultant bradyzoites form tissue cysts 30 in the brain, striated muscle, and liver, and they remain viable for the life of the animal. 4 immunosuppressive drugs or disease may dull the suppression of parasite division by the host immune system and allow the slowly dividing bradyzoites in tissue cysts to begin rapid division, thereby reactivating the infection with tachyzoites. 30 none of the forms of t. gondii produces a toxin. rapid replication of tachyzoites within a cell leads to rupture of the cell and necrosis of the tissue in which they are located. 12 the most commonly injured tissues are the brain, lungs, liver, and pancreas. prenatal infection leads to more severe illness, because the immature immune system is unable to slow down replication by tachyzoites, allowing continued damage to tissues. prenatal infection is more likely to result in ocular infections, and neonatal death is usually caused by pulmonary or hepatic infection. 30 type ii and iv hypersensitivities may be involved in the pathogenesis of chronic disease from bradyzoites in tissue cysts. 30 kittens infected perinatally can be stillborn or die shortly after birth. they may also suffer from hepatomegaly and ascites, central nervous system signs resulting from encephalitis, respiratory distress, or uveitis. 12, 13 clinical signs of infection in healthy adult cats are uncommon (box 23-2). 31 diarrhea from enteroepithelial development of the parasite is rare. 39 cats that develop clinical disease often have an episodic course with vague signs 30 that depend on the body system affected. onset of illness may be acute or chronic, and the most commonly affected organs include the brain, lungs, liver, heart, pancreas, and the eyes. 13 signs are the result of spread of tachyzoites after initial infection or after reactivation of tissue cysts. cats suffering from uveitis may develop lens luxation and glaucoma. the best way to identify a cat shedding t. gondii oocysts is to demonstrate them with a centrifugal fecal flotation technique using sheather sugar solution. the oocysts are about a quarter of the size of isospora felis oocysts ( figure 23-46 ). 12 oocysts of t. gondii are morphologically indistinguishable from hammondia or besnoitia spp. oocysts. 13 detection of fecal t. gondii dna using a pcr test can be used to definitively differentiate t. gondii oocysts from similar coccidians. 31 it is probably best, however, to assume suspicious oocysts are those of t. gondii until proven otherwise. proving infection with t. gondii is responsible for a cat's systemic illness is also difficult. finding tachyzoites in cytology samples is uncommon. they are most likely to be identified from body cavity effusions. 13 the most common method of identifying an infected cat is by detecting t. gondii-associated immunoglobulins using immunofluorescent antibody or elisa techniques. since cats are infected for life, a seropositive cat has been infected at some point in its life. however, use of serology alone is insufficient to diagnose an active t. gondii infection. serum immunoglobulin m (igm) is produced within 1 to 2 weeks after infection, but increased igm titers may persist for months to years. serum immunoglobulin g (igg) begins to rise later; in some cats, igg may not be detectable for 4 to 6 weeks. 12 by the time igg is detectable, shedding will have ceased. maternally acquired igg persists in kittens for 8 to 12 weeks. 13 a rising igg titer is associated with an active infection, but the degree of increase is not associated with the severity of the clinical signs. if a cat becomes seronegative, it is more likely the titer has fallen below the sensitivity of the test rather than the parasite has been eliminated from the body. 31 because of the vague nature of the clinical signs, many cats are presented later in the course of the disease. by this time, they may have switched from igm to igg production or passed the time of maximal igg production. thus a negative igm titer or a lack of rising igg titer does not rule out t. gondii infection. 30 also, reactivation of tissue cysts is rarely associated with rising igg titers. 31 ultimately, the diagnosis of an active systemic t. gondii infection requires demonstration of an igm titer greater than 1 : 64 or a fourfold increase in igg titers over a 2-to 3-week period along with signs consistent with toxoplasmosis, the exclusion of other disorders that may cause the clinical signs, and response to appropriate anti-t. gondii treatment. 31 although serum igm titers may be increased in otherwise healthy cats, increased igm titers in cerebrospinal fluid or aqueous humor only occurs in cats with active cns or ocular infections. 30 the goals of treating a cat infected with t. gondii are to reduce shedding of oocysts and to control the clinical signs in sick cats. shedding can be reduced by using ponazuril, 13 toltrazuril, or high doses of clindamycin. the drug options for treating a sick cat include clindamycin, trimethoprim-augmented sulfadiazine, or azithromycin for at least 4 weeks (see table 23 -20) . recurrences are more common if the cat is treated for less than 4 weeks. 13, 30 the antifolate drug pyrimethamine may be more effective than trimethoprim, but megaloblastic anemia develops in many cats. supplementation with folinic acid (5 mg/cat, once daily, po) or brewer's yeast (100 mg/kg, once daily, po) may prevent or reverse the anemia. 12 no drug clears all of the tissue cysts; so, cats remain infected for life. if uveitis is also present, use appropriate topical, oral, or parenteral corticosteroids. for a cat with proven t. gondii-associated uveitis alone, a topical ocular glucocorticosteroid is the only required treatment; no antibiotics are necessary unless the uveitis is persistent or recurrent. 31 • wash hands after handling cats, especially if you are pregnant or immunocompromised. • remove fecal material from the home environment daily, since shed oocysts require a minimum of 24 hours to become infective. • do not have immunocompromised persons clean the litter box. if they must clean the litter box, they should wear gloves and wash hands thoroughly when finished. • use litter box liners, and periodically wash the litter box with scalding water and detergent. • wear gloves when gardening, and wash hands thoroughly when finished. • cover children's sandboxes when not in use to avoid fecal contamination by outdoor cats. • only feed cats cooked or commercially processed food. • control potential transport hosts, such as flies and cockroaches, that may bring the organism into the home. • filter or boil water from sources in the environment. • cook meat for human consumption to 80° c for 15 minutes minimum (because of uneven heating, microwave cooking does not kill all t. gondii 12 ). • freeze meat at −12° c for 24 hours. 12 • wear gloves when handling meat, and wash hands thoroughly with soap and water when finished. clinical signs such as malaise, fever, and muscle pain should begin to resolve in 2 to 3 days. 30 if there is no response within 7 days, switch to or add another drug. 31 if there is still no response, search for another condition that may cause the observed clinical signs. however, ocular and cns signs resolve more slowly and thoracic radiographic changes may take weeks to resolve. 30 some cns changes may never completely resolve. cats co-infected with feline immunodeficiency virus (fiv) do not respond to anti-t. gondii treatment as well as fivnegative cats respond. 12 feeding cats commercially processed cat food and avoiding undercooked or raw meat can prevent exposure to t. gondii. controlling hunting reduces access to paratenic hosts with infective tissue cysts. access to mechanical carriers of t. gondii, such as earthworms or cockroaches, should be minimized. human infection with t. gondii is common, more so in warm, humid climates where the prevalence of t. gondii seropositive persons approaches 100%. the number of persons seropositive for t. gondii is estimated to be around 500,000,000 worldwide. 12 infective oocysts are hardy and may remain viable in the environment for up to 18 months. 12 human infection most often occurs after eating raw or undercooked meat infected with tissue cysts or by transplacental infection. 31 seropositive cats are finished shedding and are unlikely to resume shedding even if the infection becomes reactivated. 31 cats found to be shedding oocysts should be quarantined at a veterinary hospital until shedding ends. oocysts of t. gondii have not been found on the hair coat 13 ; so, transmission of toxoplasmosis does not occur after touching a cat. 31 pregnant women infected with t. gondii for the first time, or chronically infected women who are also hiv positive, can transmit the parasite to their unborn child. transplacental infection can result in stillbirths, cns, or ocular disease. 30 more severe fetal disease may occur if the infection happens in the first half of the woman's pregnancy. 12 toxoplasma gondii infection of immunocompetent humans usually results in a self-limiting fever and malaise. 30 steps useful in preventing transmission of t. gondii to humans can be found in box 23-3. pancreatitis refers to inflammation of the pancreas only, with no implication of the underlying cause or pathology. for example, acute necrotizing pancreatitis (anp) with pancreatic auto-digestion, requiring predominantly supportive care by maintaining fluid and electrolyte balances and pain relief, must not be confused with chronic pancreatitis (cp) caused by lymphocytic infiltration, and commonly associated with lymphocytic inflammatory bowel disease (ibd), and often requires corticosteroids to manage. these two conditions (and others) can only be definitively distinguished histologically. in many cases, the clinical signs of cats with acute pancreatitis will resolve with supportive care before a precise diagnosis is reached and will thus remain undiagnosed. there are no formal classifications for feline pancreatitis, but most authors 78, 89, 90 use the terms • acute pancreatitis • acute necrotizing pancreatitis, characterized by severe peri-pancreatic fat necrosis • acute suppurative pancreatitis, characterized by neutrophilic infiltration • chronic pancreatitis, characterized by lymphocytic infiltration the exact prevalence of feline pancreatitis is unknown. necropsy studies from the 1970s to 1990s reported prevalence of feline pancreatitis ranging from 0.45% to 2.4%. 21,67 a more recent study 17 found 67% of 115 cats had evidence of pancreatitis. however, this included pancreatic pathology in 45% of apparently healthy cats, which suggests that mild pathology is unlikely to cause clinical signs. these studies all show lymphocytic pancreatitis to be significantly more prevalent than acute pancreatitis. this may underestimate the true prevalence of acute pancreatitis, since it is understood that no permanent histopathologic changes are present after resolution of acute pancreatitis. 89 it is also possible that studies assessing pathology in necropsy cases do not reflect clinical practice. there are no specific age, breed, or sex predispositions. although one study reported siamese cats to be at increased risk of acute pancreatitis, 33 subsequent studies have recognized the majority of cases are domestic shorthair cats, suggesting no specific breed predispositions. 22, 29, 60, 71 most studies have indicated older cats (8 to 10 years of age) are more likely to be affected, 22, 29, 60, 71 but these studies most likely underrepresent cats with less severe clinical disease for which definitive diagnosis may not be reached and which may be younger. no association has been made with a high-fat diet or obesity. in most cases of both acute and chronic pancreatitis, no specific cause is found, and the disease is primarily considered to be idiopathic. 22, 90 there are, however, some specific underlying causes that are sporadically recognized. these include infections with herpesvirus, 75 calicivirus, 37,49 feline infectious peritonitis (fip), 44 liver fluke 58 and pancreatic fluke, 26, 77 and toxoplasmosis. 20 however, a recent paper found no association between serum feline pancreatic lipase immunoreactivity (fpli) concentrations and toxoplasma gondii serology. 8 pancreatitis has also been recognized subsequent to trauma 81 and organophosphate poisoning. 33 the association of pancreatitis with inflammatory bowel disease and cholangitis is frequently mentioned (triaditis) but poorly described in the literature. 80 one study found 30% of ibd cases to have histologic evidence of pancreatic involvement, 6 and another found fpli concentrations were elevated in 70% of cases with histologically confirmed ibd. 3 it is the author's experience that many cases of pancreatitis recognized with ibd have no specific clinical signs attributable to pancreatitis and should therefore be diagnosed and treated as intestinal disease. diabetes mellitus is a recognized co-morbidity of pancreatitis in cats. a recent study found fpli concentrations were significantly higher in 29 diabetic cats compared with 23 non-diabetics. no association could be made between fpli concentrations and the degree of diabetic control. 23 one study found 5 of 13 cats (38%) histologically diagnosed with hepatic lipidosis were also histologically diagnosed with acute pancreatitis. it is not known if pancreatitis is a cause, consequence, or coincident disease of hepatic lipidosis. for example, anorexia associated with acute pancreatitis could predispose to fatty infiltration of the liver. however, the high rate of concurrent disease has important implications for ensuring cats with pancreatitis receive adequate caloric intake. 1 ongoing or recurrent pancreatitis may lead to pancreatic cysts 10 or exocrine pancreatic insufficiency, 74 which are both covered later in this chapter. although pancreatitis has been experimentally induced in cats, 18, 41, 56 the pathophysiology of spontaneous pancreatitis remains unknown. acute pancreatitis is initiated by an increase in secretion of pancreatic enzymes that leads to inappropriate cellular activation of trypsin and subsequently other digestive zymogens. these activated digestive enzymes lead to local effects including inflammation, hemorrhage, acinar cell necrosis, and peripancreatic fat necrosis. 43, 78, 86 chronic pancreatitis may result from any of several underlying processes: ongoing, low-grade acute pancreatitis episodes may instigate chronicity; chronic pancreatitis, with a predominance of lymphocytic inflammation has been induced experimentally within 5 weeks by narrowing the main pancreatic duct to approximately 25% of its normal diameter 18 ; and the association with ibd 80 may suggest an immune-mediated cause. the clinical signs of pancreatitis in cats are nonspecific. a review of eight prior series totaling 159 cases of acute pancreatitis in cats found anorexia (87% of cases) and lethargy (81%) to be the most common historical findings. 78 vomiting was recognized in 46% of cases, diarrhea in 12%, and weight loss in 47%. physical examination findings were similarly nonspecific with dehydration (54%) being the major finding; fever was recognized in only 25% of cases and abdominal pain in 19%. it is important to note that vomiting and abdominal pain, key features of pancreatitis in dogs, are not consistently recognized in cats. similar, nonspecific findings indistinguishable from ibd are recognized in cats with chronic pancreatitis. 3, 6 diagnosis because the presenting signs and physical examination findings are nonspecific, the diagnosis of pancreatitis can be challenging, requiring not only clinical suspicion but a combination of diagnostic modalities. for the most part, hematology and plasma biochemistry findings are unremarkable, although a combination of findings may increase clinical suspicion. for example, moderate elevations in liver enzymes, bilirubin, and glucose are present in approximately 50% of cases and hypocalcemia in approximately two of three of cases; hypocalcemia infers a poorer prognosis. hypoalbuminemia is seen in approximately one of three of cases and has important implications for fluid therapy. 78 amylase and lipase elevations are not reflective of pancreatitis in cats. 47 feline trypsinlike immunoreactivity (ftli) is the diagnostic test of choice of exocrine pancreatic insufficiency, but elevations in pancreatitis are not seen consistently enough to warrant use of this test for this purpose. 29, 47, 71 the biggest recent advance in feline pancreatic diagnostics has been the characterization of feline pancreatic lipase, 69 leading to the development of a radioimmunoassay for the measurement of feline pancreatic lipase immunoreactivity (fpli). 70 it must be remembered, however, that an increase in fpli only tells the clinician that pancreatic pathology is present, but not the cause of pathology, which may be, for example, neutrophilic or lymphocytic pancreatitis or neoplasia, and it may or may not involve the intestines or liver. fpli should therefore be used as a screening test, with elevated results not suggesting a diagnostic end point. further, the high interassay variability of this test 70 would suggest that mild cases may be missed as shown in one study 24 and that the test may not be appropriate for serial monitoring. fpli is currently available as "spec fpl" from commercial laboratories and has a sensitivity of 79% and a specificity of 82% when 5.4 µg/l is used as the diagnostic cut off 25 compared with 3.5 µg/l, which is the listed reference range high point. in an acutely unwell cat (less than 2 days) with only mild to moderate signs of disease, further diagnostics may not be warranted, and many cats will improve with supportive therapy of balancing fluid and electrolytes, pain relief, and antinausea/vomiting therapy. cats with chronic duration of signs and acutely unwell cats that do not improve with supportive therapy warrant further diagnostics. the underlying disease process cannot be assumed from an elevated fpli; in one study of 63 cases, acute necrotizing pancreatitis could not be distinguished from chronic nonsuppurative pancreatitis by signalment, duration of signs, or clinical findings. 22 the major utility of diagnostic imaging is to rule out other differential diagnoses, such as an intestinal foreign body, and perhaps confirm that the pancreas is affected. radiography is non-specific for diagnosis of pancreatitis, but findings may include decreased abdominal detail (sometimes associated with ascites), soft tissue density in the right cranial quadrant of the abdomen, hepatomegaly, or gas-filled intestines 22,60,64 (see . additionally, thoracic radiographs may show pleural effusion. one study found 5 of 20 cats with pancreatic necrosis had such a change 60 ; the mechanisms resulting in pleural effusion are not precisely defined. ultrasonography has high specificity (>85%) but low sensitivity (<35%) for recognizing pancreatitis in cats, 22, 29, 60, 71 with findings dependent on operator skills, quality of equipment, and severity of lesions. typical findings are hypoechogenicity of the pancreas, which may be enlarged or irregular; hyperechogenicity of the peripancreatic fat; the possible presence of abdominal effusion; and abnormal findings with other organs, such as liver or intestine, may add to the clinical picture 22, 60, 64, . one study indicated that contrast-enhanced doppler ultrasonography can provide further diagnostic insights. 54 a recent study suggested that endosonography may be useful in cases where transabdominal ultrasonography is difficult, for example, because of obesity, hyperechoic mesentery, or excessive intestinal gas. 61 for more than 20 years, computed tomography (ct) has been a commonly used modality to confirm pancreatitis in humans, 55 but this reliability has not been demonstrated in cats, where sensitivity may be as low as 20%. 24, 29 definitive diagnosis of pancreatitis, including differentiation of the inflammatory process, can only be made by cytologic assessment of pancreatic tissue. in most cases, ultrasound-guided fine-needle aspiration (fna) of the pancreas is technically difficult because of the small dimension of the feline pancreas; there appears to be no assessment of feline pancreatic fna findings in the literature. gross inspection of the pancreas and samples for histologic assessment can be obtained during laparotomy 22, 64 (see or laparoscopy. 16, 79 because pancreatitis often occurs concurrently with pathology of other organs, 22 thorough evaluation of the abdomen by ultrasonography or gross inspection is recommended, as are multiple biopsies of, for example, intestines, liver, and mesenteric lymph nodes, where appropriate. clinicians may be reluctant to biopsy the pancreas because of perceived risks of deleterious effects. studies of pancreatic biopsy in healthy cats dispel the concern that the pancreas is unforgiving to mild manipulation and biopsy 16,42a and the author's clinical experience is consistent with these findings. supportive care comprising correction of fluid/ electrolyte imbalances, pain management, and nutritional support are the mainstay of therapy for cats with figure 23-49 gross appearance of pancreas at laparotomy; this was histologically diagnosed as chronic pancreatitis (i.e., lymphocytic infiltration was recognized). gross appearance of pancreas at laparotomy; this pancreas was found to be histologically normal. it does look smaller than is typically seen; pancreatic atrophy can look similar to this, grossly. pancreatitis. 78, 86, 89 specific underlying causes, when diagnosed, should be managed, as should concurrent diseases. follow-up evaluation is determined on a case-by-case basis; reduction or resolution of clinical signs is the main criterion for success of therapy. serial fpli values may be monitored when initial results are extremely high but are of limited value for mild increases because of assay variability. dehydration, acid-base and electrolyte abnormalities should be corrected during the first 12 to 24 hours. hypocalcemia, if present, should be treated with a calcium gluconate infusion of 50 to 150 mg/kg during 12 to 24 hours, with continued assessment of plasma calcium concentrations. plasma transfusions can be considered in cats with hypoalbuminemia. 78, 86, 90 although abdominal pain is not commonly described in cats with pancreatitis, it is likely to be present in most cases and may contribute to anorexia. historical concern about exacerbation of pancreatitis with opioids is no longer accepted, and this class of drugs is considered appropriate. meperidine (1 to 2 mg/kg sc or im) every 1 to 2 hours, butorphanol (0.2 to 0.4 mg/kg sc) every 6 hours, or sustained-release buprenorphine (120 µg/kg sc) every 72 hours are alternatives. 67, 78, 86 the author uses one dose of methadone (0.1 to 0.2 mg/kg sc, im, or iv) initially and places a fentanyl patch for longer-term pain management. the traditional recommendation for management of pancreatitis across all species has been nil per os for several days. this recommendation is appropriate for cats with severe vomiting, but there is no evidence to support this approach in cats that are not vomiting and that are eating normally. further, nutritional support is vital for those cats with concurrent hepatic lipidosis. if the cat is not eating voluntarily, nutritional support by tube feeding is often warranted. 67,78,86 a recent paper found nasogastric tube feeding of cats with pancreatitis was tolerated well and resulted in few clinically significant complications. 42 other reported nutritional strategies for cats with pancreatitis incorporate partial parenteral nutrition (ppn; 8.5% amino acids, 20% lipids), or total parenteral nutrition (tpn; 6% amino acids, 20% lipids, 50% dextrose), or both instead of enteral feeding. 14,39,53 cats do not seem to benefit from feeding of specially formulated low-fat diets; commercially available, veterinary liquefied diets appear to be well tolerated despite their high-fat contents. 86 other therapy may be appropriate in individual cases. all cats with pancreatitis that are vomiting should be treated with antiemetics. examples of drugs that can be used are 5-ht 3 antagonists, such as dolasetron (0.5 to 1.0 mg/kg iv or po, once to twice daily); ondansetron (0.1 to 0.2 mg/kg iv every 6 to 12 hours); and maropitant, an nk 1 -inhibitor (0.5 to 1.0 mg/kg sc once daily). these drugs are covered in detail earlier in this chapter under therapeutics for vomiting and diarrhea. dopaminergic antagonists, such as metoclopramide, are less effective antiemetic agents in cats than the other choices mentioned. 78, 89 in most cases, pancreatitis begins as a sterile process, and antibiotic therapy is controversial. pancreatic necrosis and inflammation may predispose to bacterial colonization of the pancreas as demonstrated in experimental models. 82, 84 this has not been demonstrated in spontaneous disease, and no comparison of outcomes has been made of cats with pancreatitis treated with or without antibiotics. cefotaxime (20 to 80 mg/kg iv, im) has been used to prevent bacterial colonization in experimental models. 83 other broad-spectrum cephalosporins or ampicillin may act similarly. antibiotic considerations are possibly more important for acute pancreatitis than for treatment of chronic disease. cats with demonstrated lymphocytic pancreatitis, with or without concurrent ibd or lymphocytic cholangitis, should be treated with corticosteroids (e.g., prednisolone, 1 to 2 mg/kg once to twice daily) with tapering to the lowest effective dose. there is no justification for use of corticosteroids in cats with acute necrotizing or acute suppurative pancreatitis, or cats for which the cause of pancreatitis has not been diagnosed histologically. use of corticosteroids in cats with pancreatic disease creates a risk of iatrogenic diabetes mellitus. surgical intervention is warranted to relieve any bile duct obstruction that may result or for the débridement of pancreatic abscesses or necrotic tissue; in many cases, cats will survive multiple years after such corrective surgery. 65 pancreatic cysts, pseudocysts, and bladders have been described sporadically in cats.* pancreatic cysts are lined by a single layer of cuboidal epithelium and do not communicate with the pancreatic duct; pseudocysts are enclosed by a wall of fibrous tissue, lacking the epithelial lining characteristic of true cysts and can form secondary to pancreatic inflammation; cystic dilations of the pancreatic duct are referred to as pancreatic bladder. true pancreatic cysts have been described in three cats 9,10,15 ; a congenital pancreatic cyst with associated inflammation was described as an incidental finding in an adult cat 15 ; multiple pancreatic cysts were described in a cat with concurrent polycystic disease in the kidney and liver 9 ; and a another cat had multiple recurrent pancreatic cysts with concurrent mild pancreatic inflammation and atrophy associated a with rapid clinical course resulting in diabetes mellitus. 10 cysts, pseudocysts, and bladders may be identified ultrasonographically or by ct. they may be benign, but the associated pancreatic inflammation and other sequelae, such as diabetes mellitus, may need to be managed. pancreatic bladders may result in biliary obstruction, and surgical correction may be required. pancreatic nodular hyperplasia is recognized quite frequently as an incidental finding in older cats or at necropsy. 45 neoplasia of the exocrine pancreas is rare in cats. its frequency was assessed in the 1970s when one study estimated 12.6 cases per 100,000 patients per year at risk, 52 and another found pancreatic tumors in 5 of 800 feline necropsies. 45 a more recent study recognized, from 15,764 feline admissions over a 20-year study period, only two cats with pancreatic adenomas (0.013% of admissions) and eight with pancreatic adenocarcinomas (0.05% of admissions). 62 adenomas appear as small, solitary or multifocal nodules and are not typically associated with adjacent pancreatic inflammation. they do not cause clinical signs, unless large, when any clinical signs result from the physical size and are usually an incidental finding. 45, 86 few generalities can be made about the presentation for pancreatic adenocarcinoma. the age range is large (4 to 20 years), there is no sex predisposition, and no clear breed predispositions are present. 62, 86 only cytology or histopathology can distinguish pancreatitis from pancreatic carcinoma in cats antemortem, yet it is important to differentiate the two conditions, because, in contrast to adenomas, pancreatic adenocarcinoma is associated with a grave prognosis. the presence of lesions consistent with metastases on radiography or ultrasonography may suggest malignancy, but one study could not distinguish neoplasia from pancreatic nodular hyperplasia ultrasonographically based on the appearance of the pancreas alone 32 (figures 23-53 and 23-54) . pancreatic adenocarcinomas in cats can result in a paraneoplastic dermatologic condition consisting of nonpruritic, symmetric alopecia affecting the face, ventral body, and medial aspect of the limbs of cats. the skin is usually glistening but not fragile, and there can be crusty lesions on the footpads.* the pathogenesis of this dermatologic disease is unknown. in one case, surgical excision of the pancreatic carcinoma resulted in resolution of dermatologic disease, indicating that the process is reversible (although signs recurred as the tumor re-emerged). 73 diabetes mellitus is a recognized complication of pancreatic adenocarcinoma. the mechanism is unknown and may simply be secondary to compression or invasion of islet cells by the tumor. in some cats, diabetes is recognized ahead of pancreatic neoplasia. 31, 40, 62 obstructive jaundice has also been described with pancreatic adenocarcinoma. 13 most cases of pancreatic adenocarcinoma in cats have metastasized by the time of diagnosis, and most reported cases die or are euthanized within 7 days of diagnosis. 62 surgical excision is a potential option if neoplasia is confined to one limb of the pancreas, but recurrence is possible even if there is no evidence of metastasis and excision seems complete at the time of surgery. 73 exocrine pancreatic insufficiency (epi) is a condition caused by insufficient synthesis and secretion of pancreatic digestive enzymes from the exocrine portion of the pancreas. 66 in humans it has been reported that 90% of pancreatic acinar cells must be lost before clinical signs of epi are seen. 19 epi is considered rare in cats but is perhaps being recognized more frequently because of increased awareness. there are less than fifty cases described in the veterinary literature* with one of these papers describing only 16 cases from five institutions, with prevalence described as 0.01% to 0.1% of cats seen over a 15-year period. 74 in contrast to this, the gastrointestinal laboratory at texas a&m university recognized 1342 samples with serum ftli concentrations at or less than 8.0 µg/l, which is diagnostic for epi, out of 84,523 submissions, 66 which equates to 1.6% of cats with known or suspected gastrointestinal disease. all studies indicate a wide age range of cats can be affected, from kittens less than 6 months of age to cats more than 15 years old, with a median age of approximately 7 years. there is no apparent breed predisposition. 66, 68, 74 one paper recognized 10 of 16 (62.5%) cats to be male, 74 and another recognized 15 of 20 (75%) male cats, 68 suggesting a possible sex predisposition. chronic pancreatitis is believed to be the most common cause of epi in cats, 66 acinar atrophy (paa) is recognized as the most common cause of epi in dogs, and has been definitively described in two feline cases 74 and mentioned as a cause for three other cases. 85 other potential causes of epi include disruption of pancreatic enzyme flow at the duodenal papilla following duodenal resection 72 and pancreatic fluke infection (eurytrema procyonis), 2,26 and amyloid deposition and neoplasia are other possible causes of pancreatic cell damage that have not definitively been described in cats. 66 congenital pancreatic hypoplasia or aplasia has not definitively been reported in cats, but reports of epi in cats as young as 3 months of age 63, 74 suggest this possibility. since chronic pancreatitis is a common cause of epi and chronic pancreatitis has a strong association with ibd, many cats may have concurrent lymphocytic pancreatitis and enteritis. 66, 74, 85 therefore cats failing to respond to therapy for epi may require further diagnostics and management of an underlying condition. further, destruction of functional exocrine pancreatic tissue can also affect pancreatic endocrine tissue, resulting in concurrent diabetes mellitus. 35 several studies have indicated that all cats with epi will have weight loss when diagnosed, unless a kitten, in which case ill-thrift is recognized. 68, 74 diarrhea is not necessarily present, being described in 50% to 75% of cats; the nature of feces can vary from voluminous, malodorous stools that can be discolored (yellow or pale), sometimes with steatorrhea, to normal feces in other cats. increased frequency of defecation and the presence of mucus in the feces of some cats can lead to the diarrhea being characterized as large bowel. only about 20% to 30% of cats are polyphagic, some described as having a ravenous appetite; conversely, some cats present with anorexia. vomiting has also been described. since cats with epi often have concurrent disorders, such as ibd, the clinical signs recognized may reflect the concurrent disease and not necessarily epi alone. physical examination findings are similarly nonspecific, with thin/ emaciated body condition being the most common finding. hematologic findings are non-specific, but a mild nonregenerative, normocytic, normochromic anemia may be recognized as well as lymphopenia or neutrophilia. plasma biochemistry results may show a mild to moderate increase in alanine aminotransferase (alt) and a mild increase in alkaline phosphatase in some cats. mild to moderate hyperglycemia may be seen, as may mild hypoglycemia or normoglycemia. 66, 68, 74 hypocobalaminemia is recognized in nearly all cats with epi. 66, 68, 74, 85, 87 this may be because of insufficient production of intrinsic factor, a cobalamin-binding protein only produced by the pancreas in cats and necessary for ileal absorption of cobalamin 27 ; it may also be because of failure of pancreatic enzymes to liberate cobalamin from binding by r protein in the duodenum or small intestinal bacterial overgrowth (sibo), not yet specifically described in cats. 74 folate concentrations may be reduced (because of concurrent intestinal malab sorption), 68 normal, 68, 74 or increased, 74 which may relate to reduced pancreatic bicarbonate secretion, secondary to severe hypocobalaminemia, 59 or associated with sibo. 7 none of these presenting complaints, physical examination findings, or routine testing results are specific to epi. therefore epi requires a degree of clinical suspicion and/or thorough diagnostics to ensure the diagnosis is not missed. a low level of serum ftli is diagnostic for epi. 66, 68, 74 samples can be sent to the gastrointestinal laboratory at texas a&m university from anywhere worldwide (with instructions about sample handling requirements on their website: http://vetmed.tamu.edu/gilab/). the reference range for serum ftli is 12 to 82 µg/l, with concentrations at or less than 8.0 µg/l diagnostic for epi. since the clinical signs and routine laboratory findings are nonspecific for epi, it is ideal to test serum for ftli in any cat with weight loss or ill-thrift. the texas a & m gastrointestinal panel also includes testing for levels of cobalamin, folate, and fpli, ensuring concurrent hypocobalaminemia will not be missed and potentially providing indications of other gastrointestinal disease. conversely, although a low level of serum ftli confirms a diagnosis of epi, it is not necessarily a diagnostic end point, since epi is so often recognized concurrently with other gastrointestinal disease. failure to respond to therapy should prompt the clinician to consider and investigate further for concurrent processes. most cats with epi can be successfully managed with dietary supplementation of pancreatic enzymes. commercial products (e.g., viokase [axcan pharma, birmingham, ala.], pancrezyme [virbac, fort worth, tex.], and creon [abbott laboratories, abbott park, ill.]) are available, and powder is considered more effective than tablets or capsules (some capsules can be opened and the contents sprinkled onto food, like powder). the required dose can vary quite substantially from cat to cat. it is appropriate to start with one teaspoon of powder with food twice daily, and adjustments can be made depending on the response; most cats accept the powder readily if it is mixed thoroughly through canned food, but other flavors (e.g., fish oil or brine from canned tuna) can be used to disguise the taste if necessary. raw pancreas (e.g., from beef or pork) may also be used, with 30 to 60 g twice daily an appropriate starting dose. 66 since most cats with epi are hypocobalaminemic, supplementation by subcutaneous injection is required (oral supplementation is not effective since cobalamin deficiency leads to cobalamin malabsorption). an appropriate dose for most cats is 250 µg, and it is usually given weekly for 6 weeks, then every second week for a further six doses; it is appropriate to continue dosing every month beyond that. owners can be taught to inject their cats at home (as owners of diabetic animals are taught to do with insulin). 66 because some cats may have sibo, antibiotics such as metronidazole (15 to 25 mg/kg po every 12 hours for 14 days) may be warranted. an elevation of folate may arouse suspicion of sibo, but it is appropriate to try antibiotics in a cat failing to respond to enzyme and cobalamin supplementation. concurrent diseases, such as lymphocytic, chronic pancreatitis, or ibd may need to be managed with corticosteroids, or diabetes mellitus with insulin. no studies have assessed specific dietary requirements in cats with epi. most cats respond to appropriate treatment, with a return to normal weight and normal feces. with ongoing therapy, cats can lead normal lives for a full life span. 83 the feline liver is a large, complex organ involved in a variety of essential metabolic, functional, and detoxification processes that can be affected, individually or collectively, by disease or dysfunction. cats have a unique set of liver diseases that occur more commonly in this species compared with the typical diseases that occur in dogs, and these include hepatic lipidosis, feline cholangitis syndrome, and infectious hepatopathies (e.g., fip, flukes, histoplasmosis, toxoplasmosis). 2, 15, 34, 58, 61 nevertheless, these conditions often present with characteristic clinical, laboratory, and histopathologic changes that are necessary for proper diagnosis and management. the goal of this section is to review the interpretation of clinical and laboratory changes that occur in these feline liver diseases, provide an approach for separating the more common diseases by their clinical footprint, and then discuss therapy of each liver disease based on our current level of understanding of hepatoprotectants, antioxidants, and drugs used for specific therapeutic purposes. the clinical signs of liver disease in cats are often vague and nonspecific; however, recognition of certain clinical and laboratory abnormalities and their association with liver disease can greatly aid the diagnostic process. the most common early clinical signs observed in cats with liver disease are anorexia, lethargy, and weight loss, which are signs present in many (if not most!) feline diseases. 2, 15 because these early indicators of disease do not point specifically toward liver disease, a delay in diagnosis will occur unless the clinician carefully considers all possibilities and performs other tests to further evaluate the situation. for example, feline hepatic lipidosis is the most common form of liver disease in cats in the united states, united kingdom, japan, and western europe, occurring with a prevalence of nearly 16% in one study. 2 however, the most common, and often only, clinical sign associated with onset of this condition is anorexia; the signs of serious hepatic disease (especially jaundice and vomiting) do not occur until later (days or weeks) in the course of the disease. 2, 21 recognition that anorexia in a cat, even for a few days, is a risk factor for development of hepatic lipidosis is essential, and this risk is increased in obese cats. 11, 21 further, the clinical signs of liver failure develop much more slowly; many cats with hepatic lipidosis present alert and responsive until much later in the course of the disease, thus delaying onset of appropriate therapy. a similar clinical situation exists for the second most common form of liver disease in cats, feline cholangitis syndrome. 15, 28, 58 this complex of diseases in the cat can be associated with signs ranging from anorexia and lethargy to vomiting and jaundice, and these signs can vary in severity and prevalence. the key point is that except for development of jaundice, there is no constellation of clinical signs that are classic clinical indicators of liver disease in cats. 15, 28 as with many feline diseases, the subtle clinical signs of anorexia, lethargy, or inactivity are often the only signs of illness and should be further investigated. there are few changes that occur in the complete blood count that are specific indicators of primary liver disease in cats. the most common finding is the presence of poikilocytes, which are red blood cells with an irregular shape, speculated to be caused by changes in membrane lipids as a result of liver dysfunction. 14 other abnormalities may occur, such as anemia of chronic disease or neutrophilia, but these findings are nonspecific and occur with variable frequency. perhaps the most important reason for obtaining a hemogram is in icteric cats, because this test is essential to help rule out hemolysis as the cause of the hyperbilirubinemia. the serum chemistry profile can be very helpful, but there are several critical points in interpretation of these values that are important to review. the hepatic transaminases (alanine aminotransferase [alt] and aspartate aminotransferase [ast]) are leakage enzymes but do not discriminate among hepatobiliary disorders, nor do they provide an indicator of severity or disease origin. thus although increases in alt may be noted in cats with liver disease, they are also present in a variety of other systemic infectious, inflammatory, neoplastic, and and protein-losing nephropathies can also cause loss of albumin and affect cholesterol, it is essential to evaluate the cat for these problems when interpreting these results. finally, bilirubin metabolism is a critical function of the liver, but interpretation of hyperbilirubinemia requires a careful consideration of bilirubin disposition. hyperbilirubinemia develps because of one of three possible causes: (1) excessive hemolysis of red blood cells (rbc) (also known as prehepatic icterus)-high bilirubin in the blood stream occurs because of an overload of the mononuclear/phagocyte system with heme pigments from rbc destruction, (2) hepatic parenchymal disease or insufficiency (also known as hepatic icterus)-resulting in lack of normal bilirubin metabolism in hepatocytes and regurgitation of the pigments into the blood stream when they are not taken up into cells and excreted in bile, and (3) disease of gall bladder, biliary tract, or pancreatic duct (also known as posthepatic icterus)resulting in obstruction of the bile ducts or loss of bile into the abdomen (duct or gall bladder rupture and bile peritonitis). 41 the bottom line is that in any cat with hyperbilirubinemia, an assessment of the packed cell volume and rbc morphology should be completed to determine whether icterus is caused by hemolysis. once hemolysis is ruled out, then assessment of primary endocrine diseases, including hyperthyroidism, feline heartworm disease, fip, and neoplasia.* alternatively, the cholestatic membrane-associated enzymes alkaline phosphatase (alp) and gamma glutamyltransferase (ggt) are especially useful for recognizing disorders involving biliary or pancreatic ductal components. unlike the dog, these enzymes will only increase modestly in cats, even in severe disease, and there is no glucocorticosteroid or drug induction of the enzymes to influence interpretation. 14, 37 thus increases in alp in the adult cat represent a release of enzyme from the hepatobiliary tree and should be considered clinically important. both alp and ggt are produced in other tissues than the liver, with the highest ggt activity present in the kidney and pancreas; however, sources other than the liver do not contribute to the activity of these enzymes in health. recent studies of the effects on these enzymes in cats with pancreatitis, cholangitis, extrahepatic bile duct obstruction (ehbdo), and hepatic lipidosis reveal some important characteristics in interpreting increases in these enzymes. 14 first, both alp and ggt are increased in cats with pancreatitis, cholangitis, or ehbdo, because inflammation in the biliary tree also affects the pancreatic ducts (and vice versa, figure 23-55) , and if the fold increases in these enzymes are similar, the diagnosis is likely one of the three. 15 conversely, in cats with hepatic lipidosis (without concurrent inflammatory disease of the biliary or pancreatic duct system), large increases in alp are observed, but ggt will remain normal or only slightly increased. thus if the increase in alp is 5 to 10 times, while ggt is not increased or is only increased 1 to 2 times, then the likely diagnosis is hepatic lipidosis. [14] [15] [16] other than enzymes on the biochemistry panel, which are of limited value for assessing liver function, there are several key tests that can be used to help assess liver function cats with elevated liver enzymes. these five tests found on most routine biochemistry panels are helpful functional indicators: cholesterol, bilirubin, glucose, albumin, and urea nitrogen (bun). however, none are immune to outside influences on their interpretation, including bilirubin and cholesterol, which are the most liver specific. in cats with severe liver disease or failure, bilirubin levels tend to be quite elevated, while bun, albumin, cholesterol, and glucose concentrations tend to be significantly decreased, reflecting inability to metabolize urea (lack of arginine), inability to produce albumin or cholesterol, and abnormal metabolization of glucose. however, these changes represent severe loss of liver function and thus are not sensitive indicators of liver function because the changes occur quite late in the course of the disease. nevertheless, in cats with elevated liver enzymes and clinical signs of liver disease, these values should be carefully assessed. because gi disease of hepatic failure. in nonicteric cats with severe liver disease or in young cats suspected of having a portosystemic shunt, serum bile acids are the more reliable indicator of hepatic insufficiency. 18 the measurement of serum bile acid concentrations, preprandially and postprandially, is the most reliable, readily available, and sensitive test of hepatic function in nonicteric cats. 5, 14 that being said, although increases in bile acids are accurate indicators of hepatic insufficiency, the levels cannot be used to assess severity of disease or the type of dysfunction. further, bile acid assays are most effective when paired samples (preprandial-and postprandial) are compared, because single, fasting, or random bile acid samples can result in a falsenegative (normal) result. however, cats will often not eat in the hospital or when they are sick, and this prevents collection of a postprandial sample. however, this does not invalidate the results, because if the result of the single bile acid sample is abnormal, it does reliably indicate liver dysfunction. an alternative to using serum for testing bile acids in cats is urine bile acid analysis. healthy cats excrete a small percentage of conjugated bile acids in the urine 14 ; however, in cats with liver disorders that cause increased serum bile acids (and especially cholestatic liver diseases) a significant increase in urine bile acid excretion occurs. when urine bile acids (uba) were collected 4 to 8 hours after a meal and measured (normalizing the value with urine creatinine: uba/ucr) and compared with serum bile acids in a study of 54 cats with hepatic disease, 17 cats with nonhepatic disease, and 8 normal cats, the results were highly correlated. 47 the utility of the urine bile acid test is that it does not require a paired sample (postprandial test), and it is not as affected as the serum test is by hemolysis or lipemia of the blood sample. normal cats will have an uba/ucr of less than 4.4 µmol/mg, while values greater than 4.4 are considered evidence of significant hepatic dysfunction. 47 it is well known that the liver plays a central role in coagulation homeostasis and is the single site of synthesis of many coagulation proteins, anticoagulant proteins, and fibrinolytic factors. vitamin k is one of the most common factors found to be inactive or deficient in cats with liver dysfunction, and it is essential for normal functioning of factors ii, vii, ix, and x; protein c and s; and thrombin. insufficient or inactive vitamin k can occur for a variety of reasons, including dietary restriction (e.g., anorexia or diet deficiency), disruption of the enteric microflora that synthesize vitamin k (e.g., chronic antibiotic therapy), diseases causing fat malabsorption (e.g., ibd, exocrine pancreatic insufficiency), ingestion of vitamin k antagonists, or liver dysfunction. 20 for example, in cats with hepatic lipidosis, approximately 25% will have an increased prothrombin time (pt), 35% will have an increased partial thromboplastin time (ptt), but 60% of cats will have increased pivka parenchymal disease versus disease of the biliary tree is completed by evaluating the clinical presentation, laboratory values, and imaging of the biliary tree and abdomen for possible evidence of biliary or pancreatic disease. a urinalysis is also an important part of the minimum database, and it is no different in a sick cat with suspected liver disease. in cats the presence of hyperbilirubinuria is abnormal at any urine concentration, because they do not conjugate bilirubin in their renal tubules. 14 however, like bilirubinemia, presence of bilirubin in the urine can occur because of any of the three possible causes of hyperbilirubinemia: prehepatic, hepatic, and posthepatic; thus further evaluation is necessary once bilirubin is detected. ammonium biurate crystalluria suggests the presence of hyperammonemia, which in the cat is either because of a congenital portosystemic shunt (less common in cats than in dogs) or because of severe, end-stage liver disease resulting in portal hypertension, which is typically caused by cirrhosis or advanced polycystic liver disease. 6, 41 the most common feline liver diseases are hepatic lipidosis and feline cholangitis syndrome, which are two diseases that often result in development of clinical or biochemical icterus. thus because hyperbilirubinemia is a more sensitive indicator of liver function than bile acids or other liver function tests, the need for further testing is moot. however, there will be circumstances when further assessment of liver function is indicated, and for this, serum bile acids, blood ammonia levels, and urine bile acids may be needed. there are several situations where liver function testing may be indicated, but the most common indications for additional testing would be a cat with persistently elevated liver enzymes of unknown origin, a cat that develops urethral obstruction because of urate stones (suggestive of portosystemic shunting) or a cat with possible polycystic liver disease. 5 one of the oldest tests of liver function, because of its association with development of hepatoencephalopathy, is measurement of blood ammonia levels. 38 however, although this test is the only practical way to diagnose hepatoencephalopathy in dogs, the test has a number of limitations, including differences in ammonia levels between arterial and venous (lower) samples and significant sample handling issues (ammonia is labile and results are affected by improper sample handling or lack of immediate measurement) that make its use difficult in practice. 43 in cats hyperammonemia is even less common than in dogs likely because of their highfunctioning urea cycle pathways 14 ; the assays have not been validated for feline blood in most laboratories, and as such, the test is not recommended as the sole indicator uncommon in cats, the most common causes are neoplasia (primarily of the pancreas, but cholangiocarcinomas can occur) or chronic pancreatitis, which can occur concurrently with cholangitis in cats, resulting in both intrahepatic and extrahepatic cholestasis in some cats. 23, 41 the bile ducts are affected in cats with chronic pancreatitis, because the feline biliary system and pancreatic duct system merge at the level of the pancreas to form a single duct that empties into the duodenum. thus in cats with either pancreatitis or biliary disease, recent evidence has shown that the inflammation affects both organs. 54, 59 further, in chronic pancreatitis, either persistent inflammation or development of fibrosis can result in dilation or obstruction of the common bile duct. 33 in cats with chronic ehbdo, the common bile duct will become widely dilated and tortuous, a finding easily seen on abdominal ultrasonography but a problem not easily managed (figures 23-56 and 23-57) . interestingly, the gallbladder is often not enlarged, and may in fact be small in cats with this condition, because the remaining fluid in the gallbladder is white bile (highly concentrated mucinous bile from which the pigment has been resorbed). 41 in addition, variable filling of the gall bladder is a normal phenomenon; thus gallbladder size is not an indicator of ehbdo. (proteins induced by vitamin k antagonists or absence). 20 nevertheless, although pivka is a very sensitive test for abnormalities of vitamin k function, most cats with liver disease that have a normal pt/ptt, but abnormal pivka do not represent clinical evidence of bleeding. in any case, abnormalities in the clotting cascade related to vitamin k deficiency in cats with liver disease are common, whether or not they show evidence of active bleeding. and because the balance of the coagulation system in a cat with liver disease can be disrupted by a procedure that initiates small amounts of bleeding (e.g., a biopsy), all cats with liver disease should be given vitamin k as a precautionary measure before and after invasive procedures, even if the clotting times (pt and ptt) are normal. this may be especially important in cats with hepatic lipidosis, because their vitamin k clotting status is likely to be even more affected by the concurrent anorexia and disruption of enteric microflora. 14 the dose of vitamin k 1 (phytonadione, aquame-phyton [merck, west point, pa.]) used prophylactically is 2.5 mg sc, im, or po q12h for 3 to 5 days, then weekly until recovered. see box 23-4 for a summary of the causes of icterus. cholestasis is the reduction of bile flow, which can occur at any point along the biliary tree; bile production occurs in hepatocytes, and flow is connected to the distal concentrating components (gallbladder and common bile duct) by the bile ductules. thus cholestasis can occur inside the liver's biliary tree (intrahepatic cholestasis) or outside the liver in the gallbladder and common bile duct (extrahepatic cholestasis). intrahepatic cholestasis most often occurs in diseases involving hepatocellular damage, leakage, or swelling, such as infections (e.g., bacterial cholangiohepatitis, toxoplasmosis, fip, or other diseases causing inflammation), infiltrative diseases (e.g., lymphoma), metabolic diseases (e.g., hepatic lipidosis), or diseases causing disruption of architecture (e.g., cirrhosis or severe polycystic disease). 41 intrahepatic cholestasis occurs in zone 1 of the liver lobules (periportal zone); at the level of hepatocytes, canaliculi or bile ductules; and is damaging to cells because of the emulsifying properties of lipid on membrane lipids. however, because the liver has a large reserve capacity, clinical icterus (e.g., jaundice) only occurs in the most severe cases when the liver is affected diffusely. thus severe or persistent intrahepatic cholestasis can serve to perpetuate the inflammation and cell damage if it is not corrected. extrahepatic cholestasis or extrahepatic bile duct obstruction (ehbdo) is less common than intrahepatic cholestasis and is most commonly associated with obstruction of the common bile duct. since gallstones are icterus is the result of cholestasis, and the underlying cause can be either hemolysis or hepatobiliary disease, for which further clinical examination will be needed to determine if rbc destruction or liver disease is occurring. in most hepatobiliary diseases of cats, cholestasis is occurring, but there may be no clinically apparent icterus because the degree of hyperbilirubinemia must be at least 2 to 3 times greater than the normal values to exceed the capacity of the liver to process the excess bilirubin. in cats with hyperbilirubinemia not caused by hemolysis, whether it is clinical or subclinical, there is no need for further evaluation of liver function (e.g., bile acid assays), because bilirubin is a more sensitive indicator of liver function than bile acids. the degree of hyperbilirubinemia does not suggest differentiation of intrahepatic versus extrahepatic cholestasis; however, the presence of acholic feces (white feces) is diagnostic for extrahepatic bile duct obstruction (ehbdo), because lack of stercobilinogen (the brown/black pigment in feces) is only found in cats with complete obstruction of the bile duct. finally, the presence of intrahepatic cholestasis and clinical icterus in a cat indicates a diffuse hepatobiliary disease, such as cholangitis or hepatic lipidosis, as focal liver disease, even if severe, will not cause clinical hyperbilirubinemia because of the tremendous reserve capacity of the liver for bilirubin uptake. portal hypertension is an abnormally high venous pressure in the portal system and is typically caused by increased resistance to portal blood flow. there are potentially three regional causes of portal hypertension: prehepatic (disease in the portal vein itself), hepatic (intrahepatic diseases causing compression or decreased flow), and posthepatic (diseases of the caudal vena cava, right heart or pulmonary vasculature). the most common cause of portal hypertension in the cat is cirrhosis or portal venous thrombosis, because portal vein hypoplasia (formerly known as microvascular dysplasia) is known to occur only in the dog, and the other causes of portal hypertension (budd-chiari syndrome, heartworm caval syndrome, pulmonary hypertension) are rare and more likely to occur in the dog. 41, 44 in any case, the clinically recognizable effects of portal hypertension are development of ascites (unusual in the cat), acquired portosystemic shunting (reported in cats), and development of hepatic encephalopathy (less common in cats than in dogs, because of their profound ability to handle protein wastes). 5, 36, 41 most cats and dogs that develop hepatic encephalopathy (he) secondarily to portal hypertension do so because of reduced liver function (because of portosystemic vascular shunting [pss] or cirrhosis and the acquired shunting that develops). cats can develop another form of chronic he because of hepatic lipidosis, but this is believed to be because of the combination of liver failure and prolonged fasting, resulting in arginine deficiency and impaired ammonia detoxification. 2 portosystemic vascular anomalies, also called portosystemic shunts or portovenous shunts (pss), although less common than in dogs, also occur in cats. these vascular anomalies can be either congenital or acquired, single or multiple in number, and occur as extrahepatic vascular shunts or within the liver itself (intrahepatic shunts). 5 the shunting of blood around the liver is the cause of hepatic atrophy and reduced hepatic function that results in an accumulation of toxins, particularly ammonia that leads to the development of hepatoencephalopathy. the two most common veins that serve as the connection point for the shunting portal venous blood are the caudal vena cava and the azygous. 5 in cats a single, extrahepatic, portocaval shunt is the most commonly reported form, and occurs in 75% of cats with pss. 5 as in dogs, specific breeds of cats may have pss more commonly, and these include domestic shorthair cats, burmese, siamese, persian, and himalayan breeds. 5 in contrast to dogs, males may be more predisposed to pss than females, but the clinical signs relate to the three body systems most affected: the central nervous system, gi tract, and urinary tract. the most common presenting complaints in cats are weight loss or poor/stunted growth, and dull, bizarre or lethargic behavior, especially after eating. signs of gi disease common in dogs, such as vomiting, diarrhea, or inappetence, are less common in cats, but in one report, 75% of cats with pss drooled. 5 finally, cats with pss often present with signs of lower urinary tract disease (e.g., hematuria, stranguria, or even obstruction) because of the development of urate uroliths (which are radiolucent, thus difficult to detect). 5 because the most common signs of he are apathy, listlessness, and decreased mental alertness, they are often not recognized specifically as indicative of brain dysfunction but as part of the constellation of signs of the liver disease. however, with progression of the has not been reported. the clinical presentation is typically nonspecific (the most common signs are vomiting, lethargy, and anorexia), and there are no laboratory changes that are suggestive of hepatic neoplasia. thus the diagnosis must be made by identification of disease, other signs will develop, including ataxia, salivation, stupor, or coma. the best and only practical diagnostic test for he is plasma measurement of ammonia levels. 43 however, as previously noted, the test has many technical issues that make its clinical utility in the practice setting difficult at best, and there are few laboratories that have validated ammonia measurement in the cat. cancer of the liver can occur as a primary disease (table 2322) or as a result of metastasis of neoplastic disease occurring elsewhere and, most typically, the abdominal cavity. the most common neoplastic infiltration of the liver that is not a primary liver tumor is lymphoma (figures 23-58 and 23-59), followed by visceral mastocytosis. 4 as with many other types of cancer, hepatobiliary neoplasia is most common in middle-aged to older cats, and it is relatively rare, with a reported incidence of 1.5% to 2.3%. 4 benign tumors, such as biliary cystadenoma ( figure 23-60) , carry a good prognosis if they are amenable to surgical resection. the incidence of metastatic neoplasia (including lymphoma and mast cell tumors) the most common clinical signs are related to spontaneous rupture of the enlarged and friable liver. affected cats may present with lethargy, anorexia, pale mucous membranes, and a heart murmur secondary to anemia. clinical signs of liver disease are usually absent. hepatomegaly and hypotension may also be found. results of routine laboratory testing (mild to marked increases in alt and globulins while alp and ggt are typically normal) and ultrasonographic examination (hepatomegaly, generalized increase in hepatic parenchymal echogenicity) 4a of the liver may be supportive, but definitive diagnosis relies on histopathologic examination of a liver biopsy. fna of the liver is not helpful because amyloid is rarely detected with this method. hemostasis should be evaluated carefully before any biopsy procedure is planned. the most important differential diagnoses are fip, hepatic lipidosis, and hepatic lymphoma. scintigraphic imaging using i-123 serum amyloid p component has potential as a noninvasive test. 39a there is no specific treatment for amyloidosis in cats, so therapy is primarily supportive care (antioxidants, vitamin k, blood transfusion). attention should be paid to identification and control of any underlying chronic inflammatory disease. unfortunately, the long-term prognosis is poor as most affected cats die of intra-abdominal bleeding. survey abdominal radiography is the simplest and most readily available imaging modality to assess structures in the abdominal cavity. radiographs are most useful to assess liver size, will reveal large hepatic masses, and provide evidence of radiopaque masses or other abnormalities in the abdomen. however, the preferred imaging modality used to assess hepatic structures in cats with suspected liver disease is abdominal ultrasonography (aus). the reasons why ultrasonography is a more useful tool for assessment of the liver in cats are numerous, but because feline liver diseases are primarily diffuse, infiltrative, or metabolic diseases that also affect the biliary tree, ultrasonography is the only imaging tool that will give reliable diagnostic information. this widely available diagnostic tool can be helpful in determining liver size and parenchymal echogenicity, in identifying mass lesions, evaluating the biliary tree and gallbladder, quantifying flow (doppler techniques), and identifying vascular anomalies. 29 as with all diagnostic modalities, the skill and experience of the operator is vital to accurate procurement and interpretation of the images. further, it is important to remember that although ultrasonographic images are extremely useful in the clinical evaluation of a cat with possible liver disease, the images themselves do not represent a histologic diagnosis. structural abnormalities by hepatobiliary imaging and subsequent examination of the tissue either by fna or biopsy techniques. historically, amyloidosis has been recognized as primarily a renal disease, especially in abyssinian cats. more recently, cases of hepatic amyloidosis without renal involvement have been diagnosed in siamese and related breeds, as well as in nonpedigreed cats. 4a,10a,30a the majority of cases have been described in australia, the united kingdom, and europe. amyloid a is deposited in the liver, probably in response to chronic inflammation in another organ. in the siamese breed, a genetic component may contribute. 48a the amyloid a protein occurring in the siamese breed differs from that known in the abyssinian breed. 48a contraindicated in cats, because they may cause a lethal shock reaction. 40 a similar reaction may be seen with penetration of the larger bile ducts or gallbladder with a large-bore biopsy needle, because these tissues have a significant autonomic innervation in the cat that may result in bradycardia and shock following the procedure. 40, 48, 54 it is particularly important to recognize this as a risk in cats with ehdbo or dilated bile ducts, and this risk factor reiterates the need for ultrasound examination of the liver prior to making biopsy decisions. nonetheless, owners should be informed of these potential risks, in addition to the risk of bleeding from biopsy sites in any cat undergoing liver sampling. 7, 48 biopsy techniques liver biopsies, whether they are obtained by needle, laparoscopy, or surgical means, should be taken from a location that represents the primary liver pathology, handled appropriately to ensure accurate interpretation of the sample, and the histopathologic description should be interpreted according to the guidelines set by the wsava standards for clinical and histologic diagnosis of canine and feline liver disease. 42, 61 guidelines for obtaining and handling surgical biopsies of the liver are reviewed elsewhere 27 and will not be further discussed. because needle aspirates/biopsies, tru-cuttype biopsies, and laparoscopic biopsies are commonly used to obtain liver tissue in cats, the benefits and limitations of each of these techniques will be discussed. as a general rule, the more tissue that can be obtained, the better the pathologist's interpretation of the tissue abnormalities will be. for example, most pathologists believe that at least six portal areas are necessary to make a diagnosis of inflammation liver disease in cats. 42 this will require either a 16-or 18-gauge needle size or larger piece of tissue than is obtained with smaller needles or an aspirate. the amount of tissues required to view at least six portal areas is approximately 15 mg, and 5 mg will be required for culture of the tissue. 42 if other analyses of the tissues are considered (e.g., metal analysis), approximately 20 to 40 mg of liver is needed. 42 a typical laparoscopic cup biopsy forceps will provide 45 mg of liver tissue, a 14-g tru-cut-type biopsy needle provides 15 to 20 mg, and an 18-g needle biopsy provides only 3 to 5 mg of liver tissue. 42 thus, depending on the clinical circumstances and considered differentials, the best approach for obtaining the needed tissue must be considered prior to planning the procedure. fine-needle aspiration to obtain liver tissue for cytologic examination is commonly performed in cats with liver disease for good reason. the procedure is inexpensive, easy to do, is relatively low risk, and often requires only sedation to complete. 57 further, samples obtained by this method can be diagnostic for hepatic lipidosis, hepatic lymphoma or other round cell tumors, and in for the most common liver diseases of cats (hepatic lipidosis, feline cholangitis syndrome, and neoplasia/ lymphoma), aus examination provides a useful means of obtaining clinical clues and tissue to support or refute the differentials. for example, in cats with hepatic lipidosis, the liver is quite enlarged and typically diffusely hyperechoic, while in cholangitis or other inflammatory diseases, the liver is more often diffusely hypochoic. 29 however, these sonographic findings are very nonspecific and can easily lead to errors in diagnosis if the tissue is not subsequently sampled for confirmation. 24, 35 thus one of the most important utilities of the aus is the ability to obtain liver tissue (either by aspiration or guided-needle biopsy) and for aspiration of the gallbladder to obtain bile for culture. 24, 49 these techniques alone have made the aus an extremely important diagnostic tool in the evaluation of liver disease in cats. the diagnosis of most liver diseases requires a histopathologic sample of liver tissue, and this is particularly true in the most common feline liver diseases, which tend to be diffuse diseases affecting the entire liver. cats with one of these diffuse diseases can be sampled randomly using any one of these commonly employed techniques: ultrasound-guided fine-needle aspirates (fna), ultrasound-guided needle biopsy, laparoscopic biopsies, or biopsies obtained surgically. some types of neoplasia (particularly round cell tumors) and vacuolar hepatopathies (hepatic lipidosis) can often be diagnosed by cytology using fna techniques. however, differentiation of liver cell tumors (adenomas and carcinomas) and inflammatory diseases of the liver cannot be diagnosed without a larger sample of tissue and histopathologic examination. 30, 50 further, even in cats with classic hepatic lipidosis changes, concurrent diseases such as cholangitis or lymphoma can be missed if only fna techniques are employed. 60 thus it is essential to consider that in many liver diseases the lesions, although typically diffuse, may also have focal components; for example, inflammation may be throughout the liver, but fibrosis will be present only in focal areas. thus the results of fna or tru-cut needle biopsies should always be considered in the light of the clinical, laboratory, and ultrasonographic evidence. prior to scheduling a cat for a biopsy, the risk-tobenefit ratio of performing a liver biopsy should always be considered. this is primarily because heavy sedation or anesthesia will be essential in most cats undergoing a liver fna, and for all cats undergoing a liver biopsy (needle or otherwise). in addition to anesthesia risks, the use of automatic spring-loaded biopsy guns to obtain ultrasound-guided biopsies of liver tissue is equipment, the interested reader is referred to several recent reviews on the subject. 48, 54 to maximize the histopathologic accuracy, biopsies taken at laparoscopy or surgically should be taken from both normal-appearing and abnormal areas in the liver. further, if there is a need to obtain samples from the deeper tissues, the laparoscope can be used to direct a tru-cut needle biopsy to the best location for sampling. one of the major advantages of the laparoscopic technique is that it allows the operator to observe the biopsy sites for excessive bleeding, which is unusual, but if observed can be staunched by using pressure on the site, gelatin coagulation material placement, or electrocautery. with experienced operators, the complication rate for laparoscopy is very low (less than 2%), and most complications were because of anesthesia, bleeding, or air embolism. 48 finally, although not necessary to have direct visualization to obtain an aspirate of gallbladder bile, laparoscopy allows easy sampling of bile for culture, which is important in all cats with suspected inflammatory liver disease or hepatobiliary disease. once a diagnosis of liver disease is made in the cat, specific therapy for the cause (if available) should be instituted; however, for many feline liver diseases, no specific therapy is available, and thus hepatoprotective therapy is used concurrently to aid in the recovery of the liver from the insult. in this section, therapy of two of the most common diseases of the feline liver will be considered, with a special emphasis on nutritional aspects of treatment, nutraceutical therapy, and the unique needs of cats. the most common liver disease of cats is idiopathic hepatic lipidosis (figures 23-61 and 23-62) , a disease that results in liver failure because of a combination of factors including hepatic lipid accumulation, insulin resistance, fasting, and protein (especially arginine) deficiency. 2, 8, 9, 11 thus, unlike many diseases of the liver, the primary focus of therapy and the essential component for recovery is nutritional support. as in any patient with serious liver disease, initial therapy is always aimed at correction of any fluid or electrolyte abnormalities that may exist, because these may be profound if the cat has been vomiting. in addition, normalization of electrolytes is particularly important in cats that have been anorexic for an especially long time (1 to 2 weeks), because refeeding syndrome may be triggered with the initiation of feeding, resulting in sudden drops in potassium, phosphate, and magnesium. 1 although this phenomenon is less common and usually less profound in cats fed enterally versus areas where appropriate, definitive diagnosis of certain infectious diseases (e.g., histoplasmosis). 57 however, even with these relatively straightforward diseases, fna of liver tissue has significant limitations, the most important of which is the failure to accurately identify the primary disease. for example, although it is easy to make a diagnosis of hepatic lipidosis using this technique, a paper recently showed four cats that were incorrectly diagnosed with hepatic lipidosis instead of lymphoma because the fna samples were obtained from areas that did not have lymphoma infiltration. 60 in another study, reviewing the agreement between fna cytologic samples of liver and the histopathologic diagnosis, only 51% of the cases had overall agreement. 50 thus although cytology of fna samples of liver tissue in cats with diffuse hepatic disease remains a useful first step, it is important for the clinician to carefully interpret the results and discuss the potential limitations of this technique with owners. there are several needle biopsy techniques available for sampling liver tissue, but not all are suitable or safe for use in cats. the menghini technique is one such approach that is not suitable for use in cats, because it is a blind procedure using a large-bore needle that cannot be used with ultrasound guidance. 42 the second option among the needle biopsy techniques that is not recommended for cats is the biopsy gun device. tru-cut biopsy guns are operated by a triggering device that can result in the induction of a lethal vagotonic shock reaction in the cat immediately following the procedure. 40 for most ultrasound-guided liver biopsy procedures, either the manual or, preferably, the semiautomatic tru-cut device is recommended for use in obtaining needle biopsies from cats. as a general rule, the tru-cut device will advance into the liver to a depth of 2 cm; so, it is essential to carefully note the amount of liver tissue available during the ultrasound assessment before advancing the needle for tissue collection. properly obtained tru-cut needle biopsies are a valuable technique for obtaining a representative sample of liver tissue 61 ; however, because of the risk for bleeding or liver fracture with any movement, it is essential that cats be anesthetized for this procedure. laparoscopy is an intermediate step between needle biopsy and surgical laparotomy for obtaining liver tissue for histopathology in cats. 48, 54 this technique is becoming more widely used as more specialists are trained for this procedure that allows visualization of tissues to be biopsied without opening the entire abdomen. although this technique does require general anesthesia, the limited degree of invasiveness, the large biopsy sample size, and rapid patient recovery make laparoscopy a valuable tool for obtaining liver tissue, 48 and it can be used to obtain biopsies from the spleen, pancreas, kidneys, lymph nodes, or to aspirate the gallbladder. for a detailed discussion of laparoscopic techniques and lipidosis. the echogenicity of the parenchyma is uniformly increased, which is more apparent when compared with other ultrasonographic images presented in this chapter. additionally, the gall bladder is distended. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease. (courtesy dr. randolph baral.) liver gb figure 23-62 gross appearance of liver from a cat with hepatic lipidosis. note the pale tan and exaggerated reticular pattern. in most cases, the edges appear more rounded than is evident here. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease (same cat as in figure 23-61) . (courtesy dr. randolph baral.) those started on intravenous nutrition, it can be a significant source of morbidity if electrolyte replacement and monitoring are not carefully attended. once the cat is hemodynamically stable, the next step in treatment planning in cats with hepatic lipidosis is re-introduction of nutrition, which must include placement of a feeding tube (box 23-5). however, because many of these cats are extremely ill and are not good candidates for anesthesia, placement of a nasoesophageal (ne) tube to allow initiation of enteral feeding is often the most appropriate step for the first few days. when administering food through a feeding tube, there are several important points: 1. the food should be room temperature (not too hot or cold). 2. the tube should be flushed with water following feeding, to remove any particles of food or medication that may cause the tube to clog. 3. if the cat is volume sensitive, it is important to carefully calculate how much water is used for flushing the tube, because a significant volume of fluid can be infused, creating a potential fluid overload. if the cat is fluid sensitive, the total amount of fluids (amount in the food, amount added to food if blenderized, and amount of flush) must be determined, and the amount of fluid used in flushes or food preparation may have to be reduced. force feeding is to be strongly discouraged in these sick cats for several reasons: • it is highly stressful and will further increase the stress response and insulin resistance phenomena that are perpetuating the hepatic lipidosis. • it can be dangerous to the cat (aspiration) or operator (scratches/biting). • it is rarely able to meet the necessary nutritional goals set for the patient. • it may induce food aversion, a phenomenon unique to cats, but creating a profound aversion to the chosen food that can be lifelong. 12 although ne tubes are excellent choices for short-term feeding of cats unwilling to eat, there are several disadvantages to their long-term use, including the nasal irritation that occurs, the relative ease with which cats can (and will) remove them, and the need to use liquid enteral diets. 62 thus once the cat is deemed stable enough for general anesthesia, a long-term feeding tube solution is needed, and this typically is either an esophageal (e) tube ( figure 23 -63) or percutaneous endoscopic gastrostomy (peg) tube. 22, 62 both feeding options are generally well tolerated methods for providing long-term feeding, but e tubes have the advantage of being placed without the need for any specialized equipment, and if complications occur, they are generally easily addressed, because the most common complications are infection at the tube site or premature removal of the tube by the cat. placement of a peg tube, although relatively easy to learn to place, requires having the appropriate endoscopic equipment, and if complications occur as a result of infection or tube removal, more significant morbidity can result. because there is no advantage to placement of peg tubes easiest to use, and are an acceptable choice in most situations. finally, because many cat stomachs are volume sensitive with initiation of feeding, it is very important to start conservatively with small-volume feeding on a more frequent schedule. with prolonged fasting, the stomach volume of a cat with hepatic lipidosis may be reduced dramatically, preventing normal expansion and limiting intake to as little as 10% of normal. thus to avoid vomiting when feeding, the starting volume may have to be as small as 10 to 15 ml every 2 to 3 hours. a good rule of thumb is to start with estimation of resting energy requirement (rer) (40 to 50 kcal/kg is a good estimate of rer), and then attempt to meet 25% of rer the first day. if no problems are encountered, increase the amount to 50% of rer the second day, and so on, but during this period, keep the frequency as high as possible (feed four to six meals per day) so that the volume remains relatively small at each meal. once full rer has been achieved with multiple meals per day, the frequency of feeding can be gradually reduced to three to four meals per day. most cats will eventually tolerate three meals per day well, and some can tolerate two meals per day, but this is quite variable and should not be attempted during the first weeks of feeding. in general, most cats with hepatic lipidosis will require tube feeding for a minimum of 3 to 6 weeks before they will show interest in food and begin eating again on their own. the tube should be retained until the cat has been eating on his or her own for at least 1 week or longer and can be maintained for a longer duration if it is being used to administer medications, because cats can eat normally with the e tube in place. the other therapeutic considerations for cats diagnosed with hepatic lipidosis are directed toward dealing with the complications of the disease and reducing the oxidative stress on the liver with hepatoprotective therapy (table 2323) . in cats that are vomiting, in cats versus e tubes, placement of e tubes is advocated as the best approach for most practice situations. interested readers are referred to several recent reviews on tube placement for specific details on each method and to chapter 18. 22, 62 diet selection is the next step in treatment planning for cats with hepatic lipidosis. in contrast to the belief that animals in liver failure need lower quantities of protein to reduce the workload on the liver, cats with hepatic lipidosis actually need protein to recover. in fact, the work of biourge and coworkers showed that protein was the essential nutrient in reducing hepatic lipid accumulation, was essential to eliminate the negative nitrogen balance, and also appeared to minimize muscle catabolism. 9 further, diets high in protein can improve insulin sensitivity and assist weight loss in recovery from obesity. 8, 11 conversely, although carbohydrates are a readily available energy source, they are often associated with gastrointestinal distress (diarrhea, abdominal cramping) and hyperglycemia (secondary to the insulin resistance in place as a result of obesity and hepatic lipidosis). 2 thus diets selected for cats with hepatic lipidosis should ideally be high in protein (>40% metabolizable energy [me]) and have lower amounts of carbohydrates (<20% me), with the remaining calories coming from fat. the diets that best fit this profile are the diets formulated for diabetic cats; however, kitten food, many adult cat foods, and some of the enteral recovery diets have this high protein/low carbohydrate profile. many of the intestinal diets are not higher protein and are higher in carbohydrates, and so would not be the ideal choice. the key to using any of the foods that are not designed for use in a feeding tube is to blenderize them (and if necessary, strain the food) so that the food will easily go through the 14-or 16-g feeding tube without clogging it. enteral diets designed for use in feeding tubes are the because the primary starting point of the inflammatory disease in cats is the bile ducts (cholangitis), with inflammation extending to the hepatic parenchyma (cholangiohepatitis) only with time and severity, the term cholangitis syndrome has become the preferred terminology. the disease syndrome has been further classified by the wsava liver diseases group into one of three primary types: neutrophilic or suppurative, chronic lymphoplasmacytic (figures 23-64 and 23-65) , and lymphocytic (non-suppurative). 61 each of the forms appears to behave quite differently clinically as well as in their progression and outcome. in general, cats with the suppurative form of cch typically have an acute onset of illness, which often includes fever, anorexia, and vomiting, and they may become icteric quite quickly (figure 23-66) . 28, 52 the nonsuppurative form of cch (lymphocytic form) tends to be a more chronic condition, with affected cats showing nonspecific signs of illness that may include partial anorexia and lethargy, but the signs may wax and wane or are non-progressive. 28, 52 because of the feline pancreatic and bile duct anatomy, it is common for cats with cch to have pancreatitis and vice versa, and in some cases, cats will also have concurrent ibd; the constellation of the three conditions occurring together is called triaditis. 59 this combination is increasingly recognized in cats, and recent reports suggest from 50% to 85% of cats with one syndrome have all three diseases. 25, 51, 54, 59 at this time, the etiology of each of these syndromes and the pathogenesis is not well understood; however, the enteric microflora are presumed to play an important role in the suppurative form, and immune mechanisms are presumed to be the cause of the chronic inflammation found in the chronic nonsuppurative antiemetic therapy is beneficial, because it is imperative that the cat continues to receive some food, and vomiting will complicate this. metoclopramide is often used in cats because of its ready availability and low cost, but it is a very weak antiemetic in cats and thus may not be the best choice. in most cats, the novel nk-1 receptor antagonist maropitant has been a safe and effective choice. 32 the most commonly used antiemetics in the author's feline practice are maropitant (1 mg/kg iv, sc or through the e tube q24h), ondansetron (0.22 mg/kg iv q8-12h), or dolasetron (0.5 mg/kg iv, sc q24h). in addition to control of vomiting, all cats with hepatic lipidosis should be given vitamin k 1 (2.5 mg/cat po, sc) daily for a week, then weekly until the cat has recovered, and vitamin b 12 (cobalamin) (250 µg/cat sc) weekly for 6 weeks, then monthly until blood values are normal. 46 other vitamins may become deficient, such as some of the b vitamins and vitamin e; however, feeding is likely to rapidly replenish these deficiencies if they exist. this is also likely true of amino acid deficiencies, but supplementation of l-carnitine (250 mg/day po) may be beneficial by improving fatty acid oxidation. 10 finally, hepatoprotectant and antioxidant therapy with s-adenosylmethionine (same) (20 mg/kg po q24h) has been advocated to increase glutathione and may be beneficial in cats with hepatic lipidosis. 13, 19, 56 it is important to note that if same is given through the tube (and thus the tablets must be crushed), the dose must be increased by approximately 50% to allow for the loss of absorption from loss of the enteric coating. because drug metabolism is often impaired in cats with hepatic lipidosis, appetite stimulants, such as mirtazapine, cyproheptadine, and clonazepam, should not be used in cats because dosing and side effects can be unpredictable. benzodiazepine agonist drugs (e.g., diazepam) should be completely avoided in cats with possible lipidosis-induced hepatoencephalopathy, because they will exacerbate the signs and may cause fulminant liver failure. 2, 14 fortunately, most cats with idiopathic hepatic lipidosis that receive immediate and aggressive therapy and feeding for their disease will recover completely. cats that develop hepatic lipidosis secondary to other serious diseases (e.g., lymphoma) have a much lower chance of complete recovery and often die of their disease or its complications. the most common inflammatory liver disease in the cat is a complex syndrome with multiple subgroups of disease previously termed cholangitis/cholan giohepatitis complex (cch) but currently recognized under the terminology feline cholangitis syndrome. 61 this disease is quite variable in both its presentation and severity, and it may occur as a primary process or secondary to/ concurrent with other diseases (e.g., pancreatitis, ibd). ultrasonographic appearance of liver with lymphocytic/plasmacytic inflammation. note the varying echogenicity throughout the hepatic parenchyma; areas of hypoechogenicity likely reflect inflammatory cell infiltration. the gall bladder is distended; its shape is distorted by pressure from the transducer. (courtesy dr. randolph baral.) liver gb [10 mg/kg po q24h]), and if pancreatitis is concurrent, pain control with opioid pain relievers (e.g., buprenorphine 0.05 to 0.1 mg/kg po, sq q8-12h). 52 if culture is not possible, combination therapy with enrofloxacin (4 mg/kg po q24h) and metronidazole (5 mg/kg po q12h) is reasonable. in cats with chronic lymphoplasmacytic forms of cholangitis, management must be tailored to the individual situation and often requires therapy with either immunosuppressive doses of prednisolone (2 to 4 mg/kg po q24h) or chlorambucil (4 mg/m 2 po q2d), along with the hepatoprotectants and cholerectics, and concurrent treatment of other diseases (pancreatitis or ibd) that may be occurring. 52 the lymphocytic or lymphoplasmacytic forms of cholangitis may wax and wane in intensity over time, and may require long-term continuous or intermittent therapy to control the disease. there is no specific diet that is recommended for cats with inflammatory liver disease, but protein restriction should not be initiated unless the cat has clear evidence of severe hepatoencephalopathy. the diet should be selected based on other conditions (such as ibd), for which the diet may be more critical in the management. monitoring of serum chemistry values (especially glucose), clotting times, cobalamin levels, and pli/tli concentrations are recommended every few months, as well as careful monitoring of the cbc for all cats on chlorambucil. in all cats with chronic inflammatory liver disease, prior to initiation of immunosuppressive therapy, a careful assessment of the cat for other possible causes of inflammation should be completed (box 23-6). as in dogs, if a cat with pss can have surgical closure of the shunting vessel (ligation, placement of an ameroid constrictor, intravenous coiling), the long-term forms. however, whether or not these syndromes are related, a continuum of disease or completely different diseases remains undetermined. once a definitive diagnosis is obtained by histopathology of the liver tissue and culture of bile, treatment can be tailored to needs of the cat. cats with the more aggressive suppurative form of cholangitis often require intravenous fluid therapy, antibiotic therapy (based on results of culture whenever possible), and supportive therapy (antiemetics, vitamin k 1 , hepatoprotectants such as same [ important antioxidant and stabilizes membrane functions • n-acetylcysteine-a precursor to glutathione and antioxidant, also improves tissues oxygen delivery • ursodeoxycholic acid (tertiary bile acid)-used to replace hepatotoxic, hydrophobic bile acids and increase bile flow • silymarin (milk thistle)-a free radical scavenger and anti-inflammatory/antifibrotic agent • vitamin e-an antioxidant and antiinflammatory vitamin* although few clinical trials of these nutraceuticals have been performed in feline liver disease, a few studies have recently appeared showing that same, ursodeoxycholic acid, silymarin, and n-acetylcysteine all are hepatoprotective, have few adverse side effects, and may be beneficial in many types of liver disease in cats. 3, 26, 39, 53, 55 feline liver disease is a common problem that requires careful consideration of the presenting complaint, clinicopathologic findings, imaging results, and, if available, histopathologic interpretation to be able to provide an accurate diagnostic and therapeutic plan. a variety of insults can be responsible for liver dysfunction or failure, but hepatic lipidosis and feline cholangitis syndrome remain the most common reasons for cats to present prognosis for function and quality of life is generally very good. 5 however, even if surgical correction is anticipated, and especially if surgical correction is impossible or not completely successful, medical management of he is indicated. see table 23 -24 for the basic therapeutic approach to medical management of cats with he resulting from pss. because hepatocytes, by their position in the body between the gi tract and rest of the body, as well as their critical role in metabolism and detoxification, are uniquely susceptible to oxidative injury and reactive intermediates of metabolism, they must be able to protect themselves. the natural defenses of the liver include superoxide dismutase and glutathione, free-radical scavengers such as vitamin e and ascorbate, and other prosurvival signaling pathways that are controlled by hormones and growth factors. 56 however, in injury or overwhelming infection or inflammation, the natural defenses of the liver can be overwhelmed, and then it is essential for medicines and nutraceutical therapy to be included in the treatment plan to help reduce inflammation and fibrosis, protect against oxidant injury, and enhance bile flow. the cytoprotective agents most commonly used in liver diseases to assist in these processes (table 23 -25) are: • s-adenosylmethionine (same)-a precursor in the synthesis of glutathione and an important methyl donor to dna and proteins, is an with icterus or liver failure. therapy must be tailored to the individual, but nutritional support is critical in the management of hepatic lipidosis, and appropriate supportive therapy with hepatoprotectants may be crucial to treatment success. resorption from that space. effusion accumulation is therefore correlated to increased capillary hydrostatic pressure, widening of the oncotic pressure gradient, increased endothelial permeability, increased interstitial hydrostatic pressure, or loss of effective lymphatic drainage or a combination of these factors. 10, 23, 32 peritonitis of any cause results in vascular dilation, increased capillary permeability, and the migration of inflammatory cells into the peritoneum in response to immunomodulatory mediators. the inflamed peritoneum becomes a freely diffusible membrane, allowing a massive outpouring of fluid and plasma proteins from the circulation. 5, 30 ascites is not commonly seen in practice; one study recognized ascites in only three cats out of 1000 admissions to an american veterinary teaching hospital, 34 but the prevalence may be greater in primary care practice. in that study, dilated cardiomyopathy (dcm) was the most common disease associated with peritoneal effusion; however, dcm was diagnosed in most of these cats before 1987, when taurine deficiency was found to be a primary cause of this form of cardiomyopathy in cats. neoplasia was the most common cause after 1987. 34 feline infectious peritonitis (fip) was by far the most common cause of feline ascites diagnosed over a 10-year period at the feline centre at the university of bristol, comprising 50% of all cats with recognized ascites. 32 cats with ascites usually present with nonspecific clinical signs, such as anorexia or lethargy. the owners may present the cat because they recognize abdominal enlargement ( figure 23-67 ), but in many cases, owners perceive this as weight gain. clinicians should be aware that sudden weight gain in a chronically underweight cat may be because of fluid accumulation (which can be intrathoracic fluid if ascites is not present), particularly if muscle mass seems reduced. ascitic cats presenting subsequent to trauma may have intraabdominal hemorrhage or urinary tract rupture. fever in a young ascitic cat will often suggest fip, and cats with fip may or may not be jaundiced. presence of jugular distention or even a jugular pulse can suggest right-sided heart failure. a palpable fluid thrill can help to distinguish ascites from other causes of abdominal enlargement, such as organomegaly, abdominal masses, bladder distention, abdominal wall weakness, obesity or, occasionally, accumulations of gas within the abdominal cavity 27 (table 23-26) . recognizing a fluid thrill involves gently tapping one side of the abdominal wall with the fingers of one hand while feeling for a sensation of fluid movement the peritoneum is the serous membrane lining the abdominal cavity, as well as covering the organs of the abdomen. it comprises a single layer of squamous mesothelial cells resting on a deeper layer of loose connective tissue. the layer of peritoneum that lines the inner surface of the abdomen is called parietal peritoneum; the abdominal organs are lined by visceral peritoneum. the total surface area of the peritoneum is one to one-and-ahalf times that of the total cutaneous area of the body. 5, 30 the peritoneal cavity contains a small amount of fluid (less than 1 ml/kg body weight) that reduces friction between the abdominal organs as they slide over each other. the fluid is a pure transudate and contains solutes in the same concentration as serum (box 23-7). this fluid is absorbed from the abdominal cavity predominantly through lymphatic vessels lying beneath the mesothelial basement membrane on the surface of the diaphragm. lymphatic drainage occurs predominantly to the sternal lymph nodes. 5, 30 ascites is the abnormal effusion and accumulation of fluid in the abdominal cavity. fluid exchange across the capillary bed is determined by starling forces, that is, the balance between hydrostatic pressure, which causes transudation of fluid out of blood vessels, and the colloid osmotic pressure, which acts to retain fluid within blood vessels. the amount of peritoneal fluid is therefore determined by the balance of these, as well as vascular permeability, with excess fluid drained by the lymphatic system. accumulation of fluid within a body cavity results when the rate of filtration of fluid into a space is greater than the rate of fluid routine laboratory findings are usually nonspecific but may provide clues to the underlying cause of ascites. for example, neutrophilia may point towards septic peritonitis but can also occur with fip; most cats with hemoperitoneum are anemic at presentation 8 ; uroperitoneum often results in azotemia and electrolyte abnormalities; hypoglycemia may reflect sepsis with septic peritonitis, and a recent study recognized 83% of cases of septic peritonitis had ionized hypocalcemia 17 ; elevated liver enzymes may be associated with inflammatory, infectious or neoplastic hepatopathies including fip; elevation of serum globulins occurs in many cats with fip but can also be associated with neoplasia or septic peritonitis; and a finding of hypoalbuminemia (which can cause a pure transudate) should prompt for an assessment of urine protein : creatinine ratio to assess if there is renal protein loss. imaging may be required to confirm the presence of fluid as well as to aid in diagnosis of the underlying cause. radiographic findings can vary greatly depending on the amount of abdominal fluid present and the underlying etiology. loss of normal detail or presence of a "ground glass" appearance to the abdominal cavity is suggestive of the presence of fluid (figures 23-68 and 23-69). very young, thin or dehydrated cats may also have a loss of detail that can mimic the presence of fluid. ultrasonography of the abdomen (figures 23-70 and if a large volume effusion causes discomfort because of abdominal distention, a three-way stopcock may be used so large volumes can be drained from one puncture (figure 23-72) . however, removal of large volumes of ascitic fluid can be detrimental, because it may prevent the subsequent reabsorption of valuable protein and/or red blood cells; the resulting reduction in intraabdominal pressure may encourage further accumulation of fluid; and rapid removal of large volumes can lead to fluid shifts causing cardiovascular collapse. 32 fluid can be collected into ethylenediaminetetraacetic acid (edta) tubes (for total nucleated cell count, packed cell volume, total protein, and cytology), serum tubes (for biochemistry, such as albumin, bilirubin, creatinine, potassium, triglyceride, glucose, lactate, and lipase), sterile tubes for culture, and/or other tubes for effusion-specific tests such as pcr. samples should be prioritized according to the volume of fluid available and to the suspected underlying disease process. 10 initial assessment of fluid retrieved is made on the basis of color and protein concentration, and much information can be gleaned from this simple assessment, even before cell numbers and types are assessed. although this brief, initial assessment is useful to refine the differential diagnoses, a thorough assessment based on underlying etiology and pathophysiology is required for definitive diagnosis and therefore appropriate management (table 23-27) . ascitic fluid, classified according to its pathophysiologic cause, can be divided into transudates, modified transudates, exudates (septic or nonseptic), or effusions (chylous or hemorrhagic). 10,23 can allow the detection of even very small volumes of fluid. it also enables evaluation of the size and structure of intraabdominal organs, such as the liver and spleen, which can help determine the underlying cause of ascites. abdominocentesis confirms the presence of abdominal fluid (in cases of low-volume effusion) and assessment of the fluid is required to diagnose the underlying cause of ascites. most cats tolerate abdominocentesis without sedation and the cat can be held in a standing position or in lateral recumbency (whichever is more comfortable for the cat and familiar to the clinician). the abdomen is clipped and aseptically prepared. a 20-to 22-gauge butterfly needle may be used with a 5-to 10-ml syringe. in cases with low-volume effusion, ultrasonography can help to guide fine needle aspiration from small pockets of abdominal fluid. diagnostic peritoneal lavage can be used if ultrasound-guided aspiration is unsuccessful. for this procedure, 10 to 20 ml/kg of warmed, sterile fluid is infused into the abdomen over 2 to 5 minutes after aseptic preparation of the site. the cat is gently rolled from side to side or allowed to stand; gentle massage of the abdomen also helps distribute the fluid. the fluid is allowed to dwell for a minimum of 2 to 5 minutes before aseptic preparation is repeated before paracentesis. no attempt is made to remove all the fluid. it must be remembered that, since the recovered fluid has been diluted by this procedure, cell counts and biochemical analyses will be affected. 33 transudates are a consequence of altered fluid dynamics. protein-poor transudates (commonly referred to as pure transudates) form predominantly as a result of severe hypoalbuminemia, which causes a lowered colloid osmotic pressure. since there is no change in endothelial or mesothelial permeability, as fluid accumulates, there is no concurrent cell leakage; so, there is a decrease in the cell count through a dilutional effect. consequently, transudative effusions are typically clear and colorless. 10, 23, 32 other pathologic causes of proteinpoor transudates include cirrhosis, lymphatic obstruction, and noncirrhotic portal hypertension (presinusoidal and sinusoidal). since hypoalbuminemia is the most common cause of transudates, serum albumin concentrations must be measured to guide further diagnostics. if the serum albumin concentration is normal (or only minimally decreased), then radiographs, abdominal ultrasonography, and/or echocardiography are indicated to assess cardiac function and for urinary bladder rupture. 10 one review of feline ascitic cases found 24% of effusions were protein-poor transudates, of which 82% were the result of hepatic failure or primary renal disease. 34 modified transudates can result from increased hydrostatic pressure within the postsinusoidal vessels of the liver secondary to right-sided congestive heart failure (e.g., tricuspid insufficiency) or potentially from mass lesions (such as neoplastic masses) obstructing blood flow from the hepatic vein or caudal vena cava into the right side of the heart. the increase in hydrostatic pressure within the vessels of the liver causes a protein-rich fluid to leach out of the liver into the abdominal cavity. since cell membrane permeability does not change, cells do not accumulate in the effusion. 10 modified transudates can also result from increased vascular permeability in the early stages of an inflammatory process, in which case cellularity will be increased. modified transudates were described as the most common type of ascitic effusion identified in cats in one study, with most being resulting from neoplasia and congestive cardiac failure; however, this study partially included cases prior to 1987, when right-sided heart failure associated with dilated cardiomyopathy (dcm) was prevalent. 34 the recognition of the role of taurine deficiency in this condition and the subsequent addition of this amino acid to feline diets now means that right-sided heart failure is only rarely encountered as a cause of ascites in cats. exudates are a consequence of altered mesothelial and/ or endothelial permeability. this permeability results from a cytokine-mediated inflammatory response of any underlying cause (e.g., infectious, neoplastic, immune mediated). exudates have high protein and moderate to high cell concentrations and are classified as nonseptic or septic. exudates are often primarily composed of neutrophils. nondegenerate neutrophils (and the absence of organisms) points to a nonseptic exudate (mostly fip but also neoplasia). fip is the most common cause of exudative effusion in cats and was the most common cause of feline ascites diagnosed over a 10-year period at the feline centre at the university of bristol. 32 the presence of neoplastic cells rules in neoplasia, but the absence of such cells does not rule out this diagnosis since many cases of neoplastic ascites are not associated with exfoliated neoplastic cells. other causes of nonseptic exudates include pancreatitis, lymphocytic cholangitis, and viscus rupture, such as the gall bladder or urinary bladder. degenerate neutrophils typify septic exudates (i.e., septic peritonitis), and their presence should instigate investigation for causes of infection (mostly leakage of gastrointestinal contents). 34 chylous effusions appear as milky or pink opaque fluid, and small mature lymphocytes initially predominate in cell counts. after drainage, more macrophages and nondegenerate neutrophils may be found. chyle is typically classified as an exudate, but its physical characteristics can be consistent with a modified transudate (protein content between 25 and 40 g/l); biochemical analysis of triglyceride and cholesterol levels in the fluid are required to confirm the diagnosis. pseudochylous effusions resemble true chyle both in appearance and cytology but do not contain fat. similar conditions result in both chylous and pseudochylous effusions. chylous abdominal effusions are rarely reported in the cat and only accounted for 7% of cases of ascites in one study. 34 the described causes of chylous ascites in cats are predominantly neoplastic. in a series of nine cats, chylous ascites was associated with nonresectable abdominal neoplasia in four cases (i.e., hemangiosarcoma and paraganglioma), with intestinal and mesenteric lymphoma in two cases and lymphangiosarcoma of the abdominal wall in another. 13 one described case in a 10-year-old cat was thought to be because of fip. 28 figure 23 -71 shows an ultrasonographic image of a cat with chylous abdominal effusion associated with pancreatitis. other potential causes include right-sided congestive cardiac failure, steatitis (inflammation of fat), biliary cirrhosis, and lymphangiectasia. hemoperitoneum in companion animals is categorized as traumatic or spontaneous. traumatic hemoperitoneum is further divided into blunt causes of trauma (i.e., motor vehicle accidents and high-rise falls) and penetrating trauma (i.e., gunshot wounds and bite wounds). 8, 21 inadvertent splenic aspiration, venipuncture, or acute severe hemorrhage should be suspected if the cytology is consistent with peripheral blood including platelets but without erythrophagocytosis or if the blood clots readily. when there is no history of trauma, coagulopathy or spontaneous rupture of a vascular neoplasm should be considered. in one study of 16 feline cases of spontaneous hemoperitoneum, 12 cases (75%) were associated with hepatic pathology such as neoplasia, necrosis, and amyloidosis. 21 in another study of 65 cases of spontaneous hemoperitoneum, 46% (30 of 65) of cats had abdominal neoplasia, and 54% (35 of 65) had non-neoplastic conditions. cats with neoplasia were significantly older and had significantly lower packed cell volumes (pcvs) than cats with non-neoplastic disease. hemangiosarcoma was the most often diagnosed neoplasm (18 of 30, 60%), and the spleen was the most common location for neoplasia (11 of 30, 37%). coagulopathies (8 of 35, 23%) and hepatic necrosis (8 of 35, 23%) were the most common causes of non-neoplastic hemoperitoneum. 8 other nonneoplastic causes of hemoperitoneum include ruptured bladder, hepatic rupture secondary to hepatic amyloidosis, gastric/duodenal ulcer, hepatic hematoma, hepatitis, perinephric pseudocyst, feline infectious peritonitisinduced liver rupture, and feline infectious peritonitisinduced nephritis. 8, 21 the prognosis of cats with spontaneous hemoperitoneum is poor. in two studies, only approximately 12% of cases survived to be discharged from hospital. 8, 21 median survival time for cats that were discharged in one of those studies was 54 days (range, 5 to 1825 days). 8 feline infectious peritonitis (fip) comprised 50% of cats with recognized ascites over a 10-year period at the feline centre at the university of bristol, 32 and, as a rule of thumb, when ascites is recognized in a younger cat, fip should be considered the major rule-out. the abdominal effusion found with fip is typically straw to golden yellow (although the color can be very variable, for example, chyle may be present), may contain fibrin clots, and has a high protein concentration. the total protein content is greater than 35 g/l and often greater than 45 g/l, with globulins comprising 50% or more. 31 one study described an effusion with total protein greater than 80 g/l as 90% specific, 55% sensitive, and having a 0.78 positive predictive value to diagnose fip. 31 the rivalta test evaluates the fluid's globulin content, and was found to be very sensitive but only 80% specific; this test is performed by adding one drop of acetic acid (98%) to 5 ml of distilled water. this fluid is mixed thoroughly, and then one drop of effusion is gently placed on the surface of the mixture. if the drop stays at the top of the fluid or slowly floats to the bottom, the test is considered to be positive. this test can give inaccurate results if inappropriate technique is used or if there is a significant temperature difference between the fluid sample and the acetic acid solution. a positive rivalta test can result from lymphosarcoma, septic, or fip effusions; these can be distinguished by cytology and culture. immunofluorescence staining of coronavirus antigen in macrophages had a positive predictive value of 1.00 but a negative predictive value of 0.57. 15 the potential clinical presentations, diagnosis, and management of fip are covered in detail in chapter 33. one study found neoplasia to be the most common cause of ascites in cats, 34 and neoplasia should be considered the major rule-out in older cats with ascites. the effusion from cats with ascites resulting from neoplasia may be a modified transudate, resulting from compression of hepatic veins or the caudal vena cava, or metastases to the peritoneum; hemorrhage from neoplasia can cause hemoperitoneum; chylous effusions may result from reduced lymphatic drainage or rupture of lymphatic vessels; and raised vascular permeability caused by neoplastic infiltration can result in an exudative effusion. carcinomas, mesotheliomas, and discrete (round) cell neoplasms (e.g., lymphoma, mast cell tumors, malignant histiocytosis) exfoliate cells into effusions more readily than sarcomas, and of these, lymphosarcoma is the most common malignancy of cats. cytology of ascitic fluid reveals neoplastic cells in less than a quarter of cases; so the absence of such cells does not rule out a diagnosis of neoplasia. in these circumstances, the diagnosis may be achieved by ultrasound-guided fine-needle aspiration of affected organs, or even biopsy samples obtained at laparotomy. the specific approaches will depend on the specific neoplasia diagnosed. exudates caused by septic inflammation usually result from bacterial contamination of the peritoneal cavity secondary to gastrointestinal tract leakage or penetrating wounds associated with trauma. gastrointestinal tract leakage may occur as a result of ulceration associated with neoplasia or inflammatory disease or as a result of penetration of a sharp object ingested (such as a toothpick), it can also occur subsequent to prior abdominal surgery. 7, 9, 17, 24 primary septic peritonitis in which no apparent cause can be identified has also been described in cats. 26 septic exudates are usually yellow to tan in color, with yellow particulate matter and are foul-smelling. microscopically, the fluid is characterized by the presence of degenerate neutrophils and bacteria. bacteria are often seen intracellularly within neutrophils. the condition is associated with high morbidity and mortality rates, with survival rates reported between 32% and 80%. 7, 9, 17, 24, 26 the history and clinical signs are often vague and nonspecific but can include abdominal pain, vomiting, lethargy/depression, and anorexia. abdominal pain is an inconsistent finding, being recognized in only 62% of cats in one study 7 and 43% in another. 24 some cats may have an inappropriately low heart rate. 7, 26 hematologic and serum biochemistry findings are also inconsistent; neutrophilia with a left shift may be present, as may neutropenia or a normal neutrophil count. similarly, cats may be hypoglycemic, hyperglycemic, or normoglycemic, and they may be hypoalbuminemic. 7, 24, 26 one study recognized ionized hypocalcemia in 89% of cats with septic peritonitis at the time of diagnosis, 17 and another suggested hyperlactatemia, when present, may be associated with a poorer prognosis. 24 radiographic findings are usually typical of ascites of any cause, but presence of pneumoperitoneum in a cat that has not undergone recent surgery may suggest the presence of gas-forming bacteria or rupture of an abdominal viscus and warrants immediate surgical intervention. ultrasonography does not directly aid the diagnosis of septic peritonitis. 7 exploratory laparotomy to determine and correct an underlying problem, such as full-thickness gastrointestinal perforation (often requiring partial resection) is required, as is copious abdominal lavage with sterile, warmed fluids ( figure 23-73 ). there are no statistically significant survival differences between postsurgical primary closure, open peritoneal drainage, or closed suction drainage postsurgical lavage; however, a trend toward a higher survival rate has been seen in cats treated with primary closure. 24, 26 treatment also involves antibiotics, initially parenterally, based on culture and sensitivity findings. consistent with intestinal contents, most bacteria recognized are gram-negative aerobes, such as e. coli or enterobacter spp., but mixed infections are usually found. 7, 24 anaerobes seem more common in cats with primary septic peritonitis, 26 which perhaps suggests these cases may result from healed over-bite wounds into the abdomen. amoxicillin/ clavulanate would be an appropriate empirical choice of figure 23-73 fulminant peritonitis associated with gastrointestinal perforation. in this case, the effusion volume was low but the high degree of serosal inflammation is evident. antibiotics while awaiting sensitivity results. there are no definitive guidelines for duration of antibiotic treatment; the author uses extended treatment courses of 4 to 6 weeks. supportive care with intravenous fluids to maintain fluid and electrolyte balances is also required perioperatively. bile peritonitis is infrequently reported in cats but has been recognized in association with gunshot 20 or motor vehicle 2 trauma, with biliary obstruction from gall stones 2,22 and subsequent to percutaneous ultrasoundguided cholecystocentesis in a cat with infectious cholangitis. 4 concurrent bacterial infection was recognized in each case; this increases severity of inflammation and worsens the prognosis, although full recovery was achieved in most reported cases. 2, 4, 20 bile peritonitis has the potential to result in small-volume effusions; so, if abdominocentesis does not yield a sample of effusion but bile peritonitis is high on the differential list, then diagnostic peritoneal lavage is appropriate. since repair of or removal of the gall bladder and abdominal lavage are required, exploratory laparotomy is an appropriate means to diagnose this condition. management should be considered as for septic peritonitis of other causes. trauma, including blunt abdominal trauma, urethral catheterization, and bladder expression, is the most common cause of uroperitoneum in cats. 1 it is also recognized as a complication of ureteral surgery. 18 the bladder is the most frequent site of urine leakage after blunt abdominal trauma, whereas the urethra is most commonly injured following catheterization. cats with ruptured bladders may still have a palpable bladder and the ability to urinate. common historical complaints are anuria (53.8%) and vomiting (50%). azotemia is a common finding, and hyperkalemia is seen in around 50% of cases. drainage of urine from the peritoneal cavity seems to improve patient stabilization. morbidity and mortality depended largely on the severity of associated injuries. 1 regardless of the site of injury or the cause of uroabdomen, the first goal of treatment is patient stabilization. isotonic replacement fluids are used for initial resuscitation. treatment of hypovolemic shock, if present, is the first order of fluid therapy. after fluid resuscitation, drainage of urine from the abdomen should be established. continuous passive drainage of the urine is necessary for stabilization and allows effective diuresis to occur. indwelling catheterization of the urinary bladder is recommended to keep the bladder decompressed and reduce urine flow into the abdominal cavity in patients with bladder and proximal urethral injury. if the urethra is traumatized and a catheter cannot be placed, prepubic tube cystostomy may be necessary to achieve temporary urinary diversion. the decision to treat the uroabdomen patient surgically or conservatively should be based on the location and severity of the underlying injury, the condition of the patient at presentation, and the patient's response to initial stabilization. 1, 12 congestive heart failure has become an uncommon cause of ascites in cats since the late 1980s/early 1990s, from which time dilated cardiomyopathy has been largely eradicated. 32, 34 ascites does still result from rightsided congestive heart failure in conditions such as tricuspid insufficiency, 6 arrhythmogenic right ventricular cardiomyopathy, 16 myocardial fibrofatty infiltration, 14 or restrictive cardiomyopathy. 29, 34 concurrent pleural effusion or pulmonary edema is often, but not necessarily, present with cardiac induced ascites. a heart murmur is not necessarily noted. noting a jugular pulse or thrill is helpful diagnostically, if present. the ascitic fluid is typically a modified transudate, but a chylous effusion is also possible. cardiac diseases are covered in chapter 20. in some cases, hepatic lipidosis has been reported to cause ascites, particularly in association with pancreatitis. these cats are often hypoalbuminemic, with the possibility of intravenous fluid therapy contributing to the ascites by raising hydrostatic pressure. 11 other liver diseases which can result in ascites include lymphocytic cholangitis, 19, 25 neutrophilic cholangitis, cirrhosis, 13 necrosis, neoplasia, and suppurative cholangiohepatitis. 34 portosystemic shunts in cats rarely result in ascites, compared with dogs. 3 hypoalbuminemia and hepatic failure result in transudates; portal hypertension and cirrhosis cause higher protein ascites because of raised capillary hydrostatic pressure causing leakage of high protein lymph. hepatopathies are covered in detail elsewhere in this chapter. gold references 1. adamama-moraitou kk, rallis ts, prassinos nn et al: benign esophageal stricture in the dog and cat: a retrospective study of 20 cases chronic oesophageal foreign body in a cat use of a biodegradable self-expanding stent in the management of a benign oesophageal stricture in a cat suspected clindamycinassociated oesophageal injury in cats: five cases feline gastrointestinal foreign bodies a comparative study 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with feline infectious peritonitis restrictive cardiomyopathy in a cat with hypereosinophilic syndrome kirk's current veterinary therapy xii: small animal practice feline infectious peritonitis: a review of clinicopathological changes in 65 cases, and a critical assessment of their diagnostic value differential diagnosis of ascites in cats abdominal paracentesis and diagnostic peritoneal lavage peritoneal effusion in cats: 65 cases (1981-1997) *when present for any length of time, a pure transudate will become modified. this is particularly true of transudates that develop slowly, such as those associated with congestive heart failure or portal hypertension. modified transudates are therefore more common than pure transudates. adapted from tasker s, gunn-moore d: differential diagnosis of ascites in cats, in practice 22:472, 2000. key: cord-002659-566uoozj authors: fujimoto, yousuke; hasegawa, shunji; matsushige, takeshi; wakiguchi, hiroyuki; nakamura, tamaki; hasegawa, hideki; nakajima, noriko; ainai, akira; oga, atsunori; itoh, hiroshi; shirabe, komei; toda, shoichi; atsuta, ryo; morishima, tsuneo; ohga, shouichi title: pulmonary inflammation and cytokine dynamics of bronchoalveolar lavage fluid from a mouse model of bronchial asthma during a(h1n1)pdm09 influenza infection date: 2017-08-22 journal: sci rep doi: 10.1038/s41598-017-08030-w sha: doc_id: 2659 cord_uid: 566uoozj asthmatic patients present more rapid progression of respiratory distress after a(h1n1)pdm09 influenza infection than after seasonal infection. here, we sought to clarify the pathophysiology of early deterioration in asthmatic patients after a(h1n1)pdm09 infection. cytokine levels and virus titres in bronchoalveolar lavage fluid from mice with and without asthma after a(h1n1)pdm09 or seasonal h1n1 infection were examined. in asthma/a(h1n1)pdm09 mice, il-6 and tnf-α levels peaked at 3 days post-infection and were higher than those in all other groups. ifn-γ levels in asthma/a(h1n1)pdm09 mice at 3 days post-infection were higher than in all other mice at any time point, whereas at 7 days post-infection, the levels were lowest in asthma/a(h1n1)pdm09 mice. virus titres in asthma/a(h1n1)pdm09 mice were highest at 3 days post-infection, and decreased by 7 days post-infection, although the levels at this time point were still higher than that in any other group. histopathological examination showed more inflammatory cell infiltration and lung tissue destruction in the asthma/a(h1n1)pdm09 group than in any other group. the distinct cytokine profiles in a(h1n1)pdm09-infected asthmatic mice indicated excessive inflammation and virus replication within a few days after infection. thus, bronchial asthma could be a more exacerbating factor for pandemic influenza infection than for seasonal influenza infection. the incidence of a(h1n1)pdm09 viral infection was significantly higher in children with asthma than in children without asthma 7 . paediatric patients with a(h1n1)pdm09 infection showed milder symptoms than those with seasonal h1n1 infection. however, severe respiratory issues, including pneumonia and acute respiratory distress syndrome (ards), have been reported in children and young adults with a(h1n1)pdm09 infection 5, [8] [9] [10] . bronchial asthma increases the risk of hospital and intensive care admission in infants and children [3] [4] [5] [6] [10] [11] [12] [13] [14] . we previously reported that a(h1n1)pdm09 infection, but not seasonal h1n1 infection, induces severe pulmonary inflammation in a mouse model of asthma 7 days after infection 14, 15 . however, we observed that the duration of the latent period for a(h1n1)pdm09 infection is shorter than 7 days, and patients present earlier progression of pulmonary disease and systemic conditions after infection. since exacerbation was frequently observed in asthmatic patients after viral infection, we suspect that a cytokine storm, including inflammatory cytokines (interleukin [il]-6 and tumour necrosis factor [tnf]-α), anti-inflammatory cytokines (il-10), anti-viral cytokines (interferon [ifn]-γ), and helper t (th) 2 cytokines (il-4 and il-13), may occur in the lung following a(h1n1)pdm09 infection. however, little is known about the early host response against a(h1n1)pdm09 infection in patients with underlying bronchial asthma. in the present study, we investigated the sequential changes in intra-tracheal cytokine production, viral loads, and pulmonary inflammation in a mouse model of bronchial asthma during the first 7 days after a(h1n1)pdm09 or seasonal h1n1 influenza infection. inflammatory cytokine concentrations in bronchoalveolar lavage (bal) fluid. il-6, tnf-α, and il-1β concentrations in bal fluid obtained 2, 3, and 7 days post-infection are shown in fig. 1 . although the mean il-6 levels in asthmatic mice challenged with a(h1n1)pdm09 were low (118.9 pg/ml) at 2 days post-infection, the levels were markedly increased (to 1578.2 pg/ml) at 3 days post-infection, and the levels in all groups remained high at 7 days post-infection. in contrast, il-6 levels in a(h1n1)pdm09-challenged control mice were 198.6 pg/ml at 2 days post-infection, peaked at 463.5 pg/ml by 3 days post-infection, and remained at similar levels at 7 days post-infection. after challenge with influenza a/puerto rico, il-6 levels in asthmatic mice slowly increased to 161.7 pg/ml by 3 days post-infection, and then reached 654.7 pg/ml at 7 days post-infection. in contrast, il-6 levels in control mice increased to 433.2 pg/ml at 3 days post-infection, similar to the levels after a(h1n1)pdm09 infection. il-6 levels in control mice at 3 days post-infection exceeded those of asthmatic mice at this time point (p = 0.015), and peaked to 1696.8 pg/ml at 7 days post-infection. the tnf-α levels in asthmatic/a(h1n1)pdm09 mice increased to 188.5 pg/ml at 3 days post-infection, which was the highest for all groups, and levels remained high at 7 days post-infection (p = 0.33). in contrast, tnf-α levels in the control/a(h1n1)pdm09 group increased to 63.7 pg/ml at 3 days post-infection, and peaked to 110.7 pg/ml by 7 days post-infection (p = 0.12). after seasonal virus infection, tnf-α levels in asthmatic mice increased to only 92.2 pg/ml at 3 days post-infection, which was similar to the levels in control/a(h1n1)pdm09 mice (p = 0.06), and these levels were maintained until 7 days post-infection. in contrast, the levels in control mice increased to 161.4 pg/ml by 3 days post-seasonal virus infection, which were similar to those in asthma/a(h1n1)pdm09 mice (p = 1.00), and these levels were maintained until 7 days post-infection. elevations in il-6 and tnf-α levels in bal fluid were not observed in the two mock-infected groups. the bal il-1β levels in control (145.6 pg/ml) and asthmatic mice (100.7 pg/ml) after seasonal infection were significantly higher than the a(h1n1)pdm09-infected groups (control/a(h1n1)pdm09: 16.5 pg/ml, asthmatic/a(h1n1)pdm09: 45.0 pg/ml), respectively. the early increasing pattern of il-6 and tnf-α levels (but not il-1β) in asthmatic mice after a(h1n1)pdm09 infection (but not control mice), was in contrast to the dynamics observed in a/puerto rico-infected mice. other cytokines in bal fluid. ifn-γ levels in asthmatic/a(h1n1)pdm09 mice significantly increased to 240.4 pg/ml by 3 days post-infection, which was the highest among all mice at this time point (vs. control/a(h1n1)pdm09, p = 0.007; vs. asthma/seasonal, p = 0.002; vs. control/seasonal, p = 0.001), and then levels increased to 651.5 pg/ml at 7 days post-infection (p = 0.001). ifn-γ levels in control mice at 3 days after a(h1n1)pdm09 infection increased to 70.8 pg/ml, which was significantly lower than the levels in asthmatic/a(h1n1)pdm09 mice at 3 days post-infection (p = 0.007). however, ifn-γ levels in control/a(h1n1)pdm09 mice peaked at 1209.4 pg/ml at 7 days post-infection, which were the highest of all the groups. il-10 levels were undetectable in all mice until 3 days post-infection. at 7 days post-infection, the levels in asthmatic/a(h1n1)pdm09 mice increased to 322.1 pg/ml, which was the highest of all the groups (vs. control/a(h1n1)pdm09, p = 0.007; vs. asthma/seasonal, p = 0.0001; vs. control/seasonal, p = 0.005). il-10 levels in control/a(h1n1)pdm09 mice increased to 202.4 pg/ml at 7 days post-infection. these levels were similar to those in control/seasonal mice (185.3 pg/ml, p = 0.44) but higher than those in asthma/seasonal mice (122.9 pg/ml, p = 0.03). after seasonal virus infection, the ifn-γ levels in asthmatic mice increased to 72.8 pg/ml at 3 days post-infection, which were similar to the levels in a/puerto rico-challenged control and asthmatic mice at 3 days post-infection, but were lower than those in asthmatic/a(h1n1)pdm09 mice at 3 days post-infection (p = 0.002). the ifn-γ levels in asthmatic/seasonal mice then increased to 420.6 pg/ml by 7 days post-infection, which were lower than that in any other group at this time point (vs. asthmatic/a(h1n1)pdm09, p = 0.03; vs. control/a(h1n1)pdm09, p = 0.002; and vs. control/seasonal, p = 0.002). ifn-γ levels in control/seasonal mice only increased to 80.7 pg/ml at 3 days post-infection, but then increased to 945.0 pg/ml by 7 days post-infection (p = 0.0001). neither il-10 levels nor ifn-γ levels were elevated in non-infected groups. bal il-13 levels in all asthmatic groups were higher than those in all non-asthmatic control groups, but the differences were not statistically significant. additionally, the levels in the control/a(h1n1)pdm09 and asthma/a(h1n1)pdm09 groups increased from 3 to 7 days post-infection. no significant differences in the levels of il-2, il-4, or il-17a, were observed among the groups, and the levels were all below the detection limits. p < 0.05, † † p < 0.01; control/seasonal vs. asthma/seasonal: || p < 0.05, || || p < 0.01; control/a(h1n1)pdm09 vs. control/seasonal: p < 0.05; asthma/a(h1n1)pdm09 vs. asthma/seasonal: § p < 0.05, § § p < 0.01; asthma/a(h1n1) pdm09 vs. control/seasonal: $$ p < 0.01; and control/a(h1n1)pdm09 vs. asthma/seasonal: ‡ p < 0.05, ‡ ‡ p < 0.01; mock/control or mock/asthma vs. each group: *p < 0.05, **p < 0.01; day 2 vs. day 3, ¶ ¶ p < 0.01; day 3 vs. day 7: fig. 2 . the numbers of total cells, lymphocytes, cd4 + cells, and cd8 + cells in all infected groups were increased at 3 days post-infection, and then decreased at 7 days post-infection. the numbers of cd8 + cells in the asthma/a(h1n1)pdm09 and control/seasonal groups were maintained until 7 days post-infection. the number of lymphocytes in control/seasonal group were higher than that in control/a(h1n1) pdm09 at 3 days post-infection, however, there were no significant differences between control/seasonal and asthma/a(h1n1)pdm09, control/a(h1n1)pdm09 and asthma/a(h1n1)pdm09 groups, respectively. additionally, the numbers of cd4 + cells, neutrophils and eosinophils on the 3 days and cd8 + cells on the 7 days post-infection were higher in the asthmatic/a(h1n1)pdm09 group than other groups respectively, but not statistically. in contrast, the numbers in the control/mock and asthma/mock groups were lower than those in the infected groups. virus titres in bal fluid. the virus titres in bal fluid at 2, 3, and 7 days post-infection are shown in fig. 3 . the mean titres at 2 days post-infection in the a(h1n1)pdm09-infected groups (asthma/a(h1n1)pdm09: 1.9 × 10 5 pfu/ml and control/a(h1n1)pdm09: 1.1 × 10 5 pfu/ml) were higher than those in the seasonal-infected groups (asthma/seasonal: 1.4 × 10 4 pfu/ml, control/seasonal: not detected); however, these differences were not statistically significant. the mean titre in the asthmatic/a(h1n1)pdm09 group at 3 days post-infection (2.2 × 10 7 pfu/ml) was the highest of all groups, and the differences were significant (vs. control/a(h1n1)pdm09, p = 0.001; vs. asthma/seasonal, p = 0.001), with the exception of the control/seasonal group. the virus titre in the asthmatic/a(h1n1)pdm09 group at 7 days post-infection (2.6 × 10 6 pfu/ml) was higher than those in any other groups at this time point (vs. control/a(h1n1)pdm09, p = 0.03; vs. asthma/seasonal, p = 0.001; vs. control/seasonal, p = 0.03). after a(h1n1)pdm09 infection, the titre in asthmatic mice at 3 days post-infection was higher than the titre at 7 days post-infection (p = 0.0002). the virus titres in control mice were also higher at 3 days post-infection than at 7 days post-infection (p = 0.006) after challenge with seasonal h1n1. histopathological findings in the lungs. figure 4 shows the h&e staining of lung tissues from mice at 2 (a), 3 (b) and 7 (c) days post-infection. the degrees of inflammatory cell infiltration and abscess formation in the asthma/a(h1n1)pdm09 group were more remarkable than in the control/a(h1n1)pdm09, asthma/seasonal, and control/seasonal groups on 2, 3 and 7 days post-infection. in addition, on 7 days post-infection, they were most severe in asthma/a(h1n1)pdm09 mice, compared with other mice. nucleoprotein antigen (infa-np) in the lungs of mice after a(h1n1)pdm09 or seasonal infection were observed by immunohistochemistry (fig. 5) . infa-np was detected in the epithelial cells and suspected macrophages in the lungs of asthmatic/a(h1n1)pdm09 (a, day 3) and control/a(h1n1)pdm09 group mice (a, day 2) since the early phase to day 7 (b) after infection, but not in mice from seasonal infected groups. infiltration of various inflammatory cells was noted, mainly in the alveolar walls in all four infected groups and more around the bronchioles in the a(h1n1)pdm09-infected groups, than in the seasonal h1n1-infected groups. only the asthmatic/a(h1n1) pdm09 group showed abscess formation with severe inflammation. the notable findings in the present study were the early peak in both il-6 and tnf-α levels, the high inflammatory cell infiltration in bal fluids, and the severe pulmonary inflammation at 3 days post-infection in asthmatic/a(h1n1)pdm09 mice. the pulmonary cytokine storm at 3 days post-infection in asthma/a(h1n1)pdm09 mice may mirror the rapid exacerbation observed in asthmatic patients 13 . in contrast, the delayed peak in il-10 levels and insufficient surge of ifn-γ levels in a(h1n1)pdm09 mice at 7 days post-infection could lead to ineffective exclusion of the viruses. the early potent inflammation associated with high viral loads in the lungs of asthmatic/a(h1n1)pdm09 mice may corroborate the rapid progression of asthmatic patients during outbreaks of pandemic virus infection. because the dynamics of il-1β was different from those of il-6 and tnf-α levels, il-1β may play other roles after influenza virus infection. in addition, the il-13 levels were increased in only the asthma/a(h1n1)pdm09 group after the infection. il-13 may be involved in pathophysiology of a(h1n1)pdm09 infection in asthmatic children. although il-2, il-4, and il-17a may be involved in the pathogenesis of bronchial asthma and influenza infection, they were undetectable in the bal fluid from mice in this study. this may be explained by the cytokines' short half-lives and/or limited roles in this microenvironment. in asthma/a(h1n1)pdm09 group, the number of cd4 + cells at 3 days and cd8 + cells at 7 days post-infection were higher than those of other groups, respectively. these results show that cd8 + cells may act anti-viral function during influenza infection. we could not recognized whether these were th1 or th2 lymphocytes, however both cd4 + and cd8 + cells may play important roles in pathophysiology of a(h1n1)pdm09-infected asthmatic patients. the histopathological findings in the early phase of infection in asthmatic/a(h1n1)pdm09 mice were severe pneumonia with abscess formation, and were not observed in any other groups (fig. 4) . these results demonstrated that pulmonary inflammation in asthmatic mice is induced beginning in the early phase of a(h1n1)pdm09 infection, which mirrors the finding that a(h1n1)pdm09 infection in asthmatic children induces severe pulmonary complications, including pneumonia, atelectasis, etc., after a shorter incubation period than with seasonal virus infection. after a(h1n1)pdm09 infection, the viral loads in bal fluid from asthmatic mice were higher than those from control mice, which was not typically observed after seasonal infection (fig. 3) . immunostaining of the virus showed many infa-np-positive cells in the lungs of asthmatic/a(h1n1) pdm09 and control/a(h1n1)pdm09 mice since early phase after viral infection, but not in the lungs of mice from seasonal h1n1 groups, as shown in fig. 5 . previous reports demonstrated that a(h1n1)pdm09 viral proteins were detected in damaged type ii pneumocytes, epithelial cells, and infiltrated macrophages in the lung by immunohistochemistry [16] [17] [18] . in addition, at autopsy after a(h1n1)pdm09 infection, acute diffuse alveolar damage was observed 19 . avian influenza viruses preferentially bind to saα2-3 gal, which is expressed on distal bronchioles and type ii pneumocytes in the lower respiratory tract 19 . in contrast, seasonal h1n1 influenza viruses bind to saα2-6 gal, which is expressed on epithelial cells in the upper respiratory tract. a(h1n1)pdm09 virus binds to both saα2-6 gal and saα2-3gal 19, 20 . predominant replication of a(h1n1)pdm09 virus in the lower respiratory tract, compared with that of seasonal influenza virus, could explain the distinct viral loads shown in fig. 3 . these findings suggested that a(h1n1)pdm09 virus may induce severe pneumonia in asthmatic patients, which is much less likely in seasonal influenza-infected asthmatic patients or non-asthmatic patients. we control/seasonal, : asthma/seasonal, : control/mock, : asthma/mock. control/a(h1n1)pdm09 vs. asthma/ a(h1n1)pdm09: † p < 0.05, † † p < 0.01; control/seasonal vs. asthma/seasonal: || p < 0.05, || || p < 0.01; control/ a(h1n1)pdm09 vs. control/seasonal: *p < 0.05; asthma/a(h1n1)pdm09 vs. asthma/seasonal: § p < 0.05, § § p < 0.01; asthma/a(h1n1)pdm09 vs. control/seasonal: $$ p < 0.01; and control/a(h1n1)pdm09 vs. asthma/seasonal: ‡ p < 0.05, ‡ ‡ p < 0.01; mock group vs. each group: *p < 0.05, **p < 0.01; day 2 vs. day 3, ¶ ¶ p < 0.01; day 3 vs. day 7: ## p < 0.01. concluded that severe pulmonary complications are caused not only by the characteristics of the infecting viruses but also by factors in the host defence of asthmatic children during a(h1n1)pdm09 infection. in the present study, cytokine levels in bal fluid ( fig. 1 ) appeared not to be associated with the lung histopathology. the histopathological analyses showed that airway inflammation was augmented in asthmatic mice when compared to control mice infected with either a(h1n1)pdm09 or seasonal influenza (fig. 4) . however, bal fluid cytokine levels showed no paralleled alterations. in fact, inflammatory cytokine levels in the non-infected groups were equivocally low in mice with or without bronchial asthma, which suggested that these histopathological changes without detectable cytokine elevations, were independent of asthma. at 3 days post-infection, ifn-γ levels in bal fluid were significantly higher in the asthmatic/a(h1n1) pdm09 group than in the control/a(h1n1)pdm09 group, in contrast to the pattern at 7 days post-infection. a previous report also showed that ifn-γ levels in asthmatic mice were lower than those in control mice at 7 days after a(h1n1)pdm09 infection 21 . virus titres in the asthmatic/a(h1n1)pdm09 group at both 3 and 7 days post-infection were significantly higher than those in control mice, and the titres of asthmatic/a(h1n1)pdm09 mice at 3 days post-infection were the highest among all groups at both 3 and 7 days post-infection. in contrast, the virus titres of the control/seasonal group were significantly lower than those of the asthmatic/seasonal group at both 3 and 7 days post-infection. ifn-γ levels were reportedly reduced in bronchial asthmatic patients, indicating an alteration in the cytokine milieu, with excess production of th2 cytokines and decreased production of th1 cytokines 22, 23 , and it has been reported that bronchial asthma patients show suppressed innate immunity [24] [25] [26] . ifn-γ is produced by th1 cells, cd8 + t (cytotoxic t) cells, natural killer (nk) cells, and nkt cells 27, 28 , and in our study, ifn-γ levels were elevated against the high virus load in the asthmatic/a(h1n1)pdm09 group at 3 days post-infection, but were not sufficiently augmented at 7 days post-infection. pulmonary inflammation, through not only the ifn-γ pathway but also other inflammatory molecules, might be involved in the exacerbation observed in a(h1n1)pdm09-infected asthma patients. we have some limited reasons for the unexplainable reciprocal pattern of virus titres in a(h1n1)pdm09-and seasonal influenza-infected asthmatic mice. the viral titre may depend on both the specificity of these viruses and the distinctive host defences in asthmatic individuals. however, further investigations are needed to characterize the immune responses against a(h1n1)pdm09 infection in asthmatic patients. in this study, the lung tissues of asthmatic/a(h1n1)pdm09 and control/a(h1n1)pdm09 mice were positive for infa-np antigens, whereas the lung tissues of mice in seasonal groups were not, even though virus titres were detected in the bal fluid of all infected groups. the reasons for this discrepancy may be the lower affinity for the virus receptors present in the lower respiratory tract or some yet unknown properties of the polyclonal antibodies and/or viral strains used, although the reason remains unclear. further research should be directed toward immunohistochemical studies of the upper respiratory tract along with lung function and airway hyperresponsiveness. in conclusion, a(h1n1)pdm09 infection can induce more severe pulmonary inflammation in patients with bronchial asthma than seasonal h1n1 infection, based on the dynamics of early excessive production of inflammatory cytokines and the reciprocal depression of anti-viral cytokines, along with high viral loads in a mouse model of bronchial asthma. sensitization of mice and allergen challenge. balb/c mice (age: 6-8 weeks) were obtained from chiyoda kaihatsu co., ltd. (tokyo, japan) and were sensitized and challenged with grade ii ovalbumin (ova; sigma-aldrich., st. louis, mo, usa), as previously described 14, 15 . all animal procedures were approved by the institutional animal care and use committee of yamaguchi university (no. 29-s01), and all methods were control/seasonal, ▲: asthma/seasonal. data are the mean ± sd of three independent experiments. control/ a(h1n1)pdm09 vs. asthma/a(h1n1)pdm09: † p < 0.05, † † p < 0.01; control/seasonal vs. asthma/seasonal: ||p < 0.05, || ||p < 0.01; asthma/a(h1n1)pdm09 vs. asthma/seasonal: § p < 0.05, § § p < 0.01; day 2 vs. day 3, ¶ ¶ p < 0.01; day 3 vs. day 7: ## p < 0.01. conducted in accordance with the approved guidelines. this study was performed independently of our previous reports 14, 15 . viruses, infection of mice, and preparation of bal fluid. mouse-adapted a(h1n1)pdm09 (strain: a/narita/1/09) or seasonal h1n1 (strain: a/puerto rico) viruses were provided by the national institute of infectious diseases (tokyo, japan). on day 31, influenza virus (concentration: 1 × 10 5 pfu/20 μl) or vehicle (mock-infection) was administered intranasally to mice. then, mice were euthanized at 2, 3, or 7 days post-infection, and samples were collected. bal fluids were collected on day 33, 34, and 38 (2, 3, and 7 days post-infection) with three consecutive 1-ml instillations of phosphate-buffered saline (pbs) at room temperature. the collected bal fluid was centrifuged at 1,500 rpm for 5 min at 4 °c, and the supernatants were stored at −80 °c for estimation of cytokine levels and virus titres. pg/ml, respectively. measurement of cd4 + cells, cd8 + cells, eosinophils, and neutrophils in bal fluid. cell pellets were resuspended in pbs and stained with fluorescein isothiocyanate (fitc)-conjugated anti-cd4 (bd biosciences) and allophycocyanin (apc)-conjugated anti-cd8 (bd biosciences) antibodies; erythrocytes were lysed by the addition of facs lysing solution epidemiology of 2009 pandemic influenza a (h1n1) deaths in the united states factors associated with death or hospitalization due to pandemic 2009 influenza a (h1n1) infection in california fatal cases associated with pandemic influenza a (h1n1) reported in greece does glycosylation as a modifier of original antigenic sin explain the case age distribution and unusual toxicity in pandemic novel h1n1 influenza? hospitalized patients with 2009 h1n1 influenza in the united states risk factors for severe outcomes following 2009 influenza a (h1n1) infection: a global pooled analysis increased h1n1 infection rate in children with asthma pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico acute respiratory distress syndrome induced by a swine 2009 h1n1 variant in mice three children with plastic bronchitis associated with 2009 h1n1 influenza virus infection plastic bronchitis in three children associated with 2009 influenza a(h1n1) virus infection a retrospective cross-sectional study of risk factors and clinical spectrum of children admitted to hospital with pandemic h1n1 influenza as compared to influenza a characteristics of atopic children with pandemic h1n1 influenza viral infection: pandemic h1n1 influenza reveals 'occult' asthma of childhood analysis of bronchoalveolar lavage fluid in a mouse model of bronchial asthma and h1n1 2009 infection cytokine profile of bronchoalveolar lavage fluid from a mouse model of bronchial asthma during seasonal h1n1 infection sudden death of a patient with pandemic influenza (a/h1n1pdm) virus infection by acute respiratory distress syndrome influenza a viruses target type ii pneumocytes in the human lung increased severity of 2009 pandemic influenza a virus subtype h1n1 infection in alveolar type ii cells from patients with pulmonary fibrosis histopathological and immunohistochemical findings of 20 autopsy cases with 2009 h1n1 virus infection receptor-binding specificity of pandemic influenza a (h1n1) 2009 virus determined by carbohydrate microarray the immune profile associated with acute allergic asthma accelerates clearance of influenza virus interferon-gamma: a historical perspective rhinovirus-induced interferon-gamma and airway responsiveness in asthma il-33 is more potent than il-25 in provoking il-13-producing nuocytes (type 2 innate lymphoid cells) and airway contraction influenza enhances caspase-1 in bronchial epithelial cells from asthmatic volunteers and is associated with pathogenesis innate immunity to influenza in chronic airways diseases inhibition of nk cell activity by il-17 allows vaccinia virus to induce severe skin lesions in a mouse model of eczema vaccinatum nk cells and nkt cells in innate defense against viral infections a mutant h3n2 influenza virus uses an alternative activation mechanism in tmprss2 knockout mice by loss of an oligosaccharide in the hemagglutinin stalk region we thank dr. takashi plaque assay. plaque assays were performed as described previously 10, 11 . briefly, madin-darby canine kidney (mdck) cells (lonza, walkersville, md, usa) were maintained at 37 °c in a humidified 5% co 2 chamber under stationary conditions. each well of a 6-well plate was seeded with 1 × 10 6 cells and cultured in α-minimum essential medium (mem; gibco/invitrogen, carlsbad, ca, usa) containing 10% foetal bovine serum (fbs), 100 units/ml penicillin (gibco), and 100 μg/ml streptomycin (gibco). after two washes with serum-free dulbecco's modified eagle's medium (dmem; gibco/invitrogen), the cells were maintained in serum-free dmem at 37 °c for 1 h. then, each well was overlaid with 200 μl of diluted bal (10 −3 , 10 −4 , and 10 −5 dilutions) and incubated at 37 °c for 1 h. after one wash in serum-free dmem, the cells were overlaid with serum-free dmem containing 0.8% agarose (becton, dickinson and company, sparks, md, usa), 0.1% diethylaminoethyl-dextran (sigma-aldrich), and 7 μg/ml trypsin (sigma-aldrich). the cells were cultured at 37 °c for 72 h, fixed in 10% formaldehyde (wako pure chemical industries, ltd., osaka, japan), and then stained with 0.037% methylene blue (wako pure chemical industries, ltd.). each experiment was performed in duplicate.histological and immunohistochemical examination of the lungs. lung tissues were fixed in 10% buffered formalin for 24 h at room temperature and then embedded in paraffin. serial sections (3-µm thick) were cut and stained with hematoxylin and eosin (h&e; muto pure chemicals co., ltd., tokyo, japan). the distribution of viral antigens was examined by immunological staining with a rabbit polyclonal antibody against infa-np, which recognize not only a(h1n1)pdm09 and seasonal h1n1, but also h3n2 influenza 29 . specific antigen-antibody reactions were visualized by 3,3′-diaminobenzidine tetrahydrochloride staining with the envision rabbit/hrp system (dako cytomation). the stained sections were observed by light microscopy to evaluate the degree of pulmonary inflammation and localization of a(h1n1)pdm09-infected cells.statistical analysis. the differences between groups were analysed by the mann-whitney u test. p values less than 0.05 were considered statistically significant. all analyses and calculations were performed using spss version 11.0 (spss inc., chicago, il, usa). yousuke fujimoto, shunji hasegawa, and shouichi ohga were the principal investigators who take primary responsibility for the study. yousuke fujimoto, shunji hasegawa, takeshi matsushige, hiroyuki wakiguchi, and tamaki nakamura performed the mouse experiments. hideki hasegawa, noriko nakajima, akira ainai, atsunori oga, and hiroshi itoh performed the histopathological examinations. komei shirabe, shoichi toda, ryo atsuta, and tsuneo morishima supported this study with helpful discussions. yousuke fujimoto, shunji hasegwa, and shouichi ohga wrote the first draft of the manuscript. competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-021571-7kbq0v9w authors: heath, joan a.; zerr, danielle m. title: infections acquired in the nursery: epidemiology and control date: 2009-05-19 journal: infectious diseases of the fetus and newborn infant doi: 10.1016/b0-72-160537-0/50037-2 sha: doc_id: 21571 cord_uid: 7kbq0v9w nan neonates, especially premature neonates, requiring intensive care support constitute a highly vulnerable population at extreme risk for nosocomial or health care-associated infections. it has been estimated that as many as 6% to 22% of infants who survive 48 or more hours in a high-risk nursery or neonatal intensive care unit (nicu) acquire a nosocomial infection."* although nosocomial infections have long been recognized in nicus, only recently have data on rates been documented in the literature. as technology and treatments have advanced to significantly diminish mortality and morbidity among critically ill neonates, especially infants of very low birth weight (less than 1500 g), this vulnerability has only increased, as a result of both more profound immune system immaturity and more frequent use of invasive interventions that bypass skin and mucous membrane barriers. ' nosocomial infections in neonates carry high attendant morbidity and mortality and health care costs. prevention and control of these infections, although highly desirable, present a formidable challenge to health care professionals. because control over birth weight-the most significant predictor of nosocomial infection risk-is limited, proper nicu customs, environment, and procedures (e.g., hand hygiene, antimicrobial usage, catheter-related practices, skin and cord care, visitation policies, unit design, and staffing) can reduce the risk for infection in the nicu. understanding the epidemiology of nosocomial infections in neonates and methods for their prevention and control is critical to minimizing poor outcomes. this chapter describes the epidemiology, etiology, and clinical characteristics of neonatal nosocomial infections as well as the methods required for effective infection prevention and control. it is well recognized that the immune system of the newborn infant, especially the premature infant, is functionally inferior to that of older infants, children, and adults (see chapter 4). the lineages of the cells that will develop into the immune system are present at the beginning of the second trimester. the major components of the neonatal immune system, including t cells, neutrophils, monocytes, and the complement pathways, are functionally impaired, however, when compared with those in older infants and adults. for example, neonatal neutrophils show decreased chemotaxis, diminished adherence to the endothelium, and impaired phagocyto~is~~~; neonatal complement levels and opsonic capacity also are reduced, particularly in the premature ne0nate.4'~ in addition, neonatal t cell lymphokine production, cytotoxicity, delayed-type hypersensitivity, and help for b cell differentiation all are inferior when measured against those in adults! antigenic naivete may account for many of these differences; however, inherent immaturity also appears to account for certain inequities. for example, neonatal t cells are delayed in their ability to generate antigen-specific memory function after hsv infection, even in comparison with naive adult t cells. ' passively acquired maternal immunoglobulin g (igg) is the sole source of neonatal igg. soon after birth, maternal igg levels begin to fall; weeks later, production of immunoglobulins by the neonate commences. neonatal igg levels reach about 60% of adult levels by 1 year of age.6 unfortunately, because much of the maternal igg is not transferred to the infant until the last 8 to10 weeks' gestation, premature infants start with significantly lower levels of serum igg than in their term counterparts, which persist throughout most of the first months of life. other issues specific to the premature neonate also affect the functional immune system. for instance, the immature gastrointestinal tract (lack of acidity worsened by use of histamine h2 blockers and continuous feedings) and easily damaged skin constitute open potential portals of entry for pathogens or commensals. in addition, like other intensive care unit populations, the nicu population frequently experiences extrinsic breeches of the immune system through use of intravascular catheters as well as other invasive equipment and procedures used to care for critically ill patients. it is generally accepted that colonization with "normal flora" prevents, to some degree, colonization by pathogenic organisms. the neonate begins life essentially sterile. in the healthy term neonate, colonization occurs within the first few days of life. the organisms involved by site are a-hemolytic streptococci in the upper respiratory tract, staphylococcus epidermidis and other coagulase-negative staphylococci (cons) on the skin, and gram-negative bacilli and anaerobes in the gastrointestinal tract. this process of colonization with normal flora is disrupted in infants cared for in an nicu in part because of exposure to the nicu environment, the hands of health care workers (hcws), antimicrobial agents, and invasive procedures. as a result, the microflora of infants in the nicu can be markedly different from that of healthy term infantses9 multiple antimicrobial agent-resistant cons, klebsiella, enterobacter, and citrobacter species colonize the skin and the respiratory and gastrointestinal tracts of a high proportion of nicu neonates by the second week of hospitali~ation.'~-'~ in addition, neonates in the nicu become colonized not only with candida albicans but also with non-albicans candida species and malasse~ia.'~-' ' because colonization of the neonate with pathogenic organisms is a prelude to invasive infection from the same pathogens? measures to prevent such colonization need to be considered. first, as a result of abnormal colonization, infants in the nicu themselves serve as an important reservoir of potential pathogens. second, contamination of the hands of hcws during routine patient care has been well documented.'* thus, careful attention to hand hygiene before and after contact with patients and their environment, as well as decontamination of potential fomites, are crucial measures in preventing spread of colonization and infection. nosocomial infections in healthy term infants are uncommon unless other conditions require that they be cared for in the nicu for several days to weeks. on the other hand, these other conditions are frequent in neonates of very low birth weight (less than 1500 g), who require prolonged nicu care. understanding the epidemiology of nosocomial infections in nicus can be challenging, because reported rates vary dramatically by institution. this variation probably results from use of nonstandard definitions of nosocomial infection and from differences in patient populations, such as mean gestational age, birth weight, and severity of underlying illness, which significantly affect the incidence of nosocomial infection." the national nosocomial infections surveillance (nnis) system is a national surveillance system of the centers for disease control and prevention (cdc) that uses standardized surveillance protocols and the involvement of multiple medical centers to provide benchmark data for the epidemiology of nosocomial nicu infections. using standardized definitions, "is reported in 1996 that 13,179 nosocomial infections occurred between 1986 and 1994 in 10,296 neonates in 99 nicus.~' in this study, rates of intravascular catheterassociated bloodstream infection, the most frequent nosocomial infection, ranged from fewer than 5 infections per 1000 umbilical or central catheter days in infants with a birth weight greater than 1500 g to almost 15 infections per 1000 catheter days in the lowest-birth-weight group (less than another national, multicenter surveillance study, the pediatric prevention network's (ppn) point prevalence survey, was undertaken in 1999 to determine the point prevalence of nosocomial infections in nicus and to define risk factors associated with development of these infections." this study included 827 infants from 29 nicus. of the 827 infants, 94 (1 1.4%) had an active nosocomial infection on the day of the survey. bacteremia accounted for 53% of infections; lower respiratory tract infections, ear-nose-throat infections, and urinary tract infections accounted for 13%, 9%, and 9%, respectively (table 35-1) . in contrast with the nicu setting, the frequency of nosocomial infection in well-baby nurseries has been estimated to be between 0.3% and 1.7%?2-24 in general, non-lifethreatening infections such as conjunctivitis account for a majority of infections in the well-baby population. the remainder of this chapter focuses almost entirely on nosocomial infections in and control measures for the nicu setting. 1000 g). 2/26 (7.7) 2/2 (100) 0 2/5 (40) 115 for purposes of surveillance and tracking, all infections occurring in hospitalized newborns could be considered nosocomial. infections that are manifested in the first few days of life, however, usually are caused by pathogens transmitted vertically from the maternal genital tract. unfortunately, no precise time point perfectly distinguishes maternally acquired neonatal infections from those transmitted within the nicu. nnis has attempted to address this issue by stratifying infections according to whether they are likely to be maternally acquired.20 in 89% of neonates who had an infection thought to be maternally acquired, onset occurred within 48 hours of birth. use of a cutoff period of 48 hours or less to designate maternally acquired infections allowed 15.3% of bacteremias and 14.5% of pneumonias to be considered as originating from a maternal source. maternally acquired bloodstream infections were more likely to be caused by group b streptococci, other streptococci, and escherichia coli, whereas those not maternally acquired usually were caused by coagulase-negative staphylococci.20 in general, nonmaternal routes of transmission of microorganisms to neonates are divided into three categories: contact (from either direct or indirect contact from an infected person or a contaminated source), droplet (from large respiratory droplets that fall out of the air at a maximum distance of 3 feet), and airborne (from droplet nuclei, which can remain suspended in air for long periods and as a result travel longer distances). specific microorganisms can be spread by more than one mechanism; in most instances, however, a single mode of spread predominates. the cdc has developed a system of precautions for the control of nosocomial infections that is based on these modes of transmi~sion.~~ contact transmission of bacteria, viruses, and fungi on the hands of hcws is arguably the most important yet seemingly preventable means of transmission of nosocomial infection. spread of infection by this means can occur either by transmission of the hcw's own colonizing or infecting pathogens or, more often, by transmission of pathogens from one patient to another. that the hands of hcws become contaminated even in touching intact skin of patients has been well demonstrated.'* poor compliance with hand hygiene is another means by which the hands of hcws can spread organisms from one patient to another.26227 furthermore, hands of hcws have been implicated in multiple outbreaks with a variety of different organisms; through experimental studies, a causal link between hand hygiene and nosocomial infection has been established.28 contact transmission by means of fomites also can occur and has been described as a potential mechanism of spread of pathogens in multiple nicu outbreaks. as described later in this chapter, implicated items have included linens, medical devices, soap dispensers, and breast pumps, to name a few. these observations highlight the need for careful attention to disinfecting items shared between infants. spread through large respiratory droplets is an important mode of transmission for pertussis and infections due to neisseria meningitidis, group a streptococci, and certain respiratory viruses, whereas airborne transmission by means of droplet nuclei is relevant for measles, varicella, and pulmonary tuberculosis. for large droplet or droplet nuclei transmission, usually an ill adult, either an hcw or a parent, is the source of infection in an nicu setting. in general, these organisms are rare sources of outbreaks. infusates, medications, and feeding powders or solutions can be intrinsically or extrinsically contaminated and have been reported as the source of outbreaks due to a variety of different pathogens. it is important when possible to mix infusates in a controlled environment (usually the pharmacy), to avoid multiuse sources of medication, and to use bottled or sterilized feeding solutions when breast milk is not available. of course, nosocomial infection also can arise from endogenous sources within the neonate. the "abnormal flora" of the neonate residing in the nicu, however, is determined at least in part by the nicu environment and hcws' hands. with use of molecular techniques, even organisms typically considered to originate solely from normal flora (e.g., cons) have been shown to have clonal spread in the hospital setting, suggesting transmission by means of the hands of hcws.~~,~' as discussed earlier, infants in nicus have intrinsic factors predisposing them to infection, such as an immature immune system and compromised skin or mucous membrane barriers. in addition, multiple extrinsic factors play important roles in the development of infection, such as presence of indwelling catheters, performance of invasive procedures, and administration of certain medications, such as steroids and antimicrobial agents. birth weight is one of the strongest predictors of risk for nosocomial infection. for instance, "is data demonstrate that compared with larger infants, low-birth-weight infants are at higher risk of developing bloodstream infections and ventilator-associated pneumonia, even after correction for central intravascular catheter and ventilator use." similarly, in the ppn's point prevalence survey, infants weighing 1500 g or less at birth were 2.69 (95% confidence interval [ci] 1.75% to 4.14%; p < .001) times more likely to have an infection than those weighing more than 1500g.2' the relationship between birth weight and nosocomial infection is complicated by multiple other factors that accompany low birth weight and also increase risk for nosocomial infection. low birth weight, however, has been shown to be an independent predictor for nosocomial infection, after adjustment for use of vascular catheters, parented alimentation, and mechanical ~entilation.~~ it is likely that birth weight also is a surrogate marker for other unmeasured factors, such as immune system immaturity. central venous catheters (cvcs) increase the risk for development of nosocomial bloodstream infections. in a study by chien and colleagues 19,507 infants admitted to 17 nicus in canada, nosocomial bloodstream infections were found to occur at a rate of 3.1 to 7.2 infections per 1000 catheter days, depending on the type of catheter, versus 2.9 infections per 1000 noncatheter days. other studies have demonstrated that the association between cvcs and bloodstream infection is independent of birth weight.2' mechanisms for cvc-related nosocomial bloodstream infections probably involve colonization of the catheter by means of the catheter hub, colonization of the skin at the insertion site?3 or hematogenous spread of pathogens from distant sites of infection or colonization. bloodstream infections also can result from contaminated intravenous fluids, which have the potential for intrinsic or, especially with use of multiuse vials, extrinsic contamination. factors related to the management of cvcs influence the risk of infection. disconnection of the cvc and the frequency of blood sampling through the catheter increase the frequency of catheter-related infection^.^^ by contrast, administration of a solution with heparin and exit-site antisepsis decreased infection. lower frequency of cvc tubing changes (every 72 hours versus every 24 hours) was associated with increased catheter contamination, suggesting a potential for increased risk of infecti0n.3~ cvc management techniques, including use of antiseptic-impregnated dressings, antimicrobialcoated catheters, and avoidance of scheduled replacement of cvcs, are discussed in the most recent cdc recommendations, summarized in "guidelines for the prevention of intravascular catheter-related infection," published in 2002 and prepared by the hospital infection control practice advisory c~m m i t t e e .~~ it has been suggested that use of peripherally inserted central catheters (piccs) may be associated with a lower rate of infection than for other cvcs. studies based in nicus have yielded conflicting results. in a study by chien and colleagues?' the relative risk of bloodstream infection, after adjustment for differences in infant characteristics and admission illness severity, was 2.5 per 1000 catheter days for umbilical venous catheters, 4.6 for piccs, and 4.3 for broviac catheters, compared with no catheter ( p < .05). another study also documented similar rates of infection for broviac catheters and for piccs.~~ by contrast, a higher rate of infection with broviac catheters than with piccs was suggested by brodie and co-w~rkers.~~ further study of different cvcs in nicu infants is needed to delineate infection risks for individual catheter types. parented alimentation and intralipids have been shown to increase risk of bloodstream infection in premature infants even after adjustment for other covariables such as birth weight and cvc use.38 etiologic agents often associated are cons, candida species, and malassezia species. the pathogenesis of this association remains unclear. potential hypotheses are many. intralipids, for example, could have a direct effect on the immune system, perhaps through inhibition of interleukin-2.39 alternatively, as with any intravenous fluids, parented alimentation has the potential for intrinsic and extrinsic contamination, and intralipids especially may serve as a growth medium for certain bacteria and fungi. finally, total parented alimentation and intralipids delay the normal development of gastrointestinal mucosa because of lack of enteral feeding, encouraging translocation of pathogens across the gastrointestinal mucosa. it is well accepted that mechanical ventilation is an important risk factor for nosocomial lower respiratory tract infection. a large multicenter study of 8263 neonates found that mechanical ventilation was a risk factor for bloodstream infection as well, even after adjustment for a number of covariables such as birth weight, parented nutrition, and umbilical catheterization:' clinically obvious respiratory infection appeared to precede some but not all cases of bloodstream infection associated with mechanical ventilation. the study authors suggested that the increased risk of mechanical ventilation could be attributed to colonization of humidified air, as well as to physical trauma from the endotracheal tube and its suctioning. a number of medications critical to the survival of infants in the nicu increase risk of infection. broad-spectrum antimicrobial agents, especially with prolonged use, are important in the development of colonization with pathogenic micro-organism~.~ the widespread use of broad-spectrum antimicrobial agents has been associated with increased colonization with resistant organisms in many settings, including nicus." in addition to colonization, antimicrobial agents also have been shown to increase risk of infection with resistant bacteria4' and with fungal pathogens!' other medications also appear to play a role in nosocomial infection. for instance, infants who receive corticosteroids after delivery are at approximately 1.3 to 1.6 times higher risk for nosocomial bacteremia in the subsequent 2 to 6 weeks than that observed for infants who do not receive this in addition, colonization and infection with bacterial and fungal pathogens have been shown to increase with the use of h, blocker^.'^"' measures of illness severity have been developed, in part, in an effort to account for variations in birth weight-adjusted mortality scores between nicus. the score for neonatal acute physiology (snap) was developed and validated by richardson and ass0ciates,4~ and the clinical risk index for babies (crib) was developed by the international neonatal network.% these scores are highly predictive of neonatal mortality even within narrow birth weight strata and are predictive of nosocomial infection. thus, in investigating potential risk factors for nosocomial infection, it is important to consider adjusting for illness severity using such measures, in addition to adjusting for other potential confounders. other risk factors related to infection include poor hand hygiene and environmental issues, such as understaffing and overcrowding.47b48 these and related issues are discussed later in this chapter under "prevention and control." nosocomial infections can affect any body site or organ system and manifest in a multitude of different ways. "is and ppn data demonstrated that bloodstream infections are the most common manifestation of nosocomial infection and account for 32% to 53% of infections (table 35-2; see also table 35 -1).20,'' respiratory infections and eye, ear, nose, or throat infections are second and third in frequency, whereas gastrointestinal infections, urinary tract infections, surgical site infections, meningitis, cellulitis, omphalitis, septic arthritis, and osteomyelitis are reported less frequently."*" bloodstream infections are the most common and one of the most potentially serious nosocomial infections that occur in nicu patients. factors discussed earlier, including birth weight, intravascular catheters, mechanical ventilation, use of parented alimentation, and steroids, all have been shown to be associated with an increased risk of bloodstream infection. the most common pathogen associated with nosocomial bloodstream infections is cons (see table 35 -2). staphylococcus aureus, enterococcus, candida species, e. coli, enterobacter species, klebsiella pneurnoniae, and pseudomonas aeruginosa also play important roles and are associated with higher morbidity and mortality rates than those associated with cons?^,^" in one study, the frequency of fulminant sepsis (fatal within 48 hours) was estimated to be 56% (95% ci 38% to 72%) when the bloodstream infection was caused by pseudomonas species, whereas it was only 1% (95% ci 0% to 4%) when infection was caused by cons.^" the difficulty of assigning an etiologic role to cons on the basis of one blood culture that could be contaminated probably accounts for some distortion of the incidence and mortality data related to this organism, and this problem is discussed in detail in chapter 6. pneumonia accounts for 12% to 18% of nicu nosocomial infections" and has been associated with prolonged hospital stay and increased mortality.51 organisms most commonly associated with nosocomial pneumonia include cons, s. aureus, and i? aeruginosa (see table 35 -2). mechanical ventilation and birth weight are important risk factors for nosocomial respiratory infection^.^^ diagnosis of nosocomial respiratory infections requires correlation of microbiologic results with clinical findings and can be challenging in lowbirth-weight infants because of the mostly nonspecific associated signs of illness and often misleading results of radiologic ~tudies.~' eye, ear, nose, and throat infections account for approximately 8% to 21% of infections, depending on birth weight." common etiologic organisms include cons and s. aureus, although gram-negative organisms, such as e. coli, e! aeruginosa, and k. pneumoniae, also can be isolated from these sites (see table 35 -2). conjunctivitis appears to be the most common of these infections, accounting for 54% to 76%, depending on birth weight?' risk factors for neonatal conjunctivitis identified in a study from nigeria included vaginal delivery, asphyxia, and prolonged rupture of membranes.53 in the nnis review, gastrointestinal infections were estimated to account for 5% to 11% of nosocomial infections, depending on birth weight. necrotizing enterocolitis (nec) was the most common presentation.'" nec carries high morbidity and mortality rates. a review of 17 nec epidemics estimated that surgery was required for a mean of 16% (range, 0% to 67%) of infants, and death occurred in a mean of 6% (range, 0% to 8 8 y 0 ) .~~ in controlled studies, identified risk factors for nec have included young chronologic age, low gestational age, low birth weight, and young age at first feeding.54 implication of specific pathogens is complex, requiring careful selection of an appropriate control population and attention to how and where specimens are collected. pathogens associated with nec outbreaks have included pseudornonas species, salmonella species, e. coli, k. pneumoniae, enterobacter cloacae, s. epidermidis, clostridium species, coronavirus, and r~t a v i r u s .~~'~~ the importance of infection control methods such as strict attention to hand hygiene and cohorting patients in the nicu is suggested by the observation that their implementation has been followed by resolution of the outbreak.55 a detailed discussion of the cause of nosocomial sepsis and meningitis is found in the chapter on bacterial sepsis (chapter 6) and chapters describing specific etiologic agents. s. aureus is a colonizing agent in neonates and has been a cause of nosocomial infection and outbreaks in well-baby nurseries and nicus. methicillin-resistant s. aureus (mrsa) has become a serious nosocomial pathogen, and outbreaks have been reported in many areas of hospitals, including n~rseries.~~-~' in addition to the usual manifestations of neonatal nosocomial infection (conjunctivitis, bloodstream infections, and pneumonia), nosocomial s. aureus infections can manifest as skin infection^?^ bone and joint infections,@' parotitis:' staphylococcal scalded skin syndr0me,6~*~~ toxic shock syndrome?6 and disseminated sepsis. the role of the hands of hcws in transmitting and spreading pathogenic organisms among infants was demonstrated with s. aureus in the 1960~."*~~ currently, in a majority of instances, s. aureus transmission is thought to occur by direct contact. thus, it is not surprising that understaffing and overcrowding have been associated with s. aureus outbreaks in n i c u s .~~.~ the potential for airborne transmission, however, has been suggested by the occurrence of "cloud babies? described by eichenwald and colleagues67 in 1960. "cloud" hcws also have been described; in such cases, the point source of an outbreak was determined to be a colonized hcw with a viral respiratory infe~tion.~~'~' in one of these studies, dispersion of s. aureus from the implicated hcw was found to be much higher after experimental infection with rhinovirus.68 more recently, molecular techniques not only have defined outbreaks57 but also have demonstrated that transmission to infants probably occurs from colonized hcws,6* and sometimes from colonized parents. 69 nasal mupirocin ointment has been used to control outbreaks of both methidin-susceptible s. aureus and mrsa.62,70 the pharynx, rather than the anterior nares, however, may be a more common site of colonization in neonates and infants," and eradication of the causative organisms with nasal mupirocin may be more difficult in this site.72 since the early 1980s, cons has been the most common cause of nosocomial infection in the nicu. ' this finding suggests that a portion of cons infections may be preventable by strict adherence to infection control practices. the fact that a hand hygiene campaign was associated with increased hand hygiene compliance and a lower rate of cons-positive cultures supports this ~ontention.'~ enterococcus has been shown to account for 10% of total nosocomial infections in neonates, 6% to 15% of bloodstream infections, 0% to 5% of cases of pneumonia, 17% of urinary tract infections, and 9% of surgical site sepsis and meningitis are common manifestations of enterococcal infection during nicu outbreak^'^,^^; however, polymicrobial bacteremia and nec frequently accompany enterococcal sepsis.77 identified risk factors for enterococcal sepsis, after adjustment for birth weight, include use of a nonumbilical cvc, prolonged presence of a cvc, and bowel resection?' because enterococcus colonizes the gastrointestinal tract and can survive for long periods of time on inanimate surfaces, the patient's environment may become contaminated and, along with the infant, serve as a reservoir for ongoing spread of the organism. the emergence of vancomycin-resistant enterococci (vre) is a concern in all hospital settings, and vre have been the cause of at least one outbreak in the nicu setting7' in the neonate, resistant strains appear to cause clinical syndromes indistinguishable from those due to susceptible enteroco~ci.'~ the conditions promoting vre infection, such as severe underlying disease and use of broad-spectrum antimicrobial agents, especially vancomycin, can be difficult to alter in many nicu settings. guidelines for the prevention and control of vre infection have been published; these focus on infection control tools such as rapid identification of a vrecolonized or vre-infected patient, cohorting, isolation, and barrier precautions. 80 historically, before the recognized importance of hand hygiene and the availability of antimicrobial agents, group a streptococci (gas) were a major cause of puerperal sepsis and fatal neonatal sepsis. although less common now, gas continue to be a cause of well-baby and nicu outbreak^.'^-'^ gas-associated clinical manifestations include severe sepsis and soft tissue infections. one report described a high frequency of "indolent omphalitis"; in this outbreak, the umbilical stump appeared to be an important site of gas colonization and an ongoing reservoir of the organism." routine cord care included daily alcohol application. after multiple attempts, the outbreak finally was interrupted after a 15-day interval during which bacitracin ointment was applied to the umbilical stump in all infants, and affected infants received intramuscular penicillin. molecular techniques have enhanced the ability to define outbreaks, and use of these techniques has suggested that transmission can occur between mother and infant, between hcw and infant, and between infantsprobably indirectly on the hands of hcws.",~~ in one recurring outbreak, inadequate laundry practices appeared to be a contributing factor. 85 nnis data have shown that group b streptococci (gbs) infections account for less than 2% of non-maternally acquired nosocomial bloodstream and pneumonia infections." a number of studies from the 1970s and 1980s demonstrated nosocomial colonization of infants born to gbs-negative ~o m e n .~~-~o these studies suggested a rate of transmission to babies born to seronegative mothers as high as 12% to 270/0.8~,~' a recent case-control study evaluating risk factors for lateonset gbs infection demonstrated that premature birth was a strong predictor?' in that study, 50% of the infants with late-onset gbs infection were born at less than 37 weeks of gestation (compared with 15% of controls), and only 38% of the mothers of these infants were colonized with gbs, suggesting possible nosocomial transmission of gbs during the nicu stay. the hands of hcws are assumed to account for the transmission of most cases of nosocomial gbs infection. breast milk also has been implicated as a potential mode of acquisition, however. in one report, gbs probably was transmitted from breast milk to one set of premature triplets between days 12 and 63 of life.92 two maternal vaginal swabs taken before delivery did not grow gbs, but repeated cultures of the mother's breast milk yielded a pure growth of gbs (greater than 10' colony-forming units [ cfu] /ml) despite no evidence of mastitis. in this report, antimicrobial therapy administered to the mother appeared to eradicate the organism. the enterobacteriaciae family has long been recognized as an important cause of nosocomial infection. neonatal infection can be manifested as sepsis, pneumonia, urinary tract infections, and soft tissue infections; morbidity and mortality rates frequently are enterobacter species, k. pneumoniae, e. coli, and serratia marcescens are the members of the family enterobacteriaciae most commonly encountered in the nicu. enterobacter species have been estimated to account for 3% of bloodstream infections, 8% of cases of pneumonia, and 8% of surgical site infections in the nicu setting (see table 35 -2). outbreaks due to enterobacter species in nicus have been associated with thermometer^?^ a multidose vial of d e x t r o~e~~ intravenous fluids,% and powdered formula?7 as well as with understaffing, overcrowding, and poor hand hygiene practice^.'^ in one outbreak in which contaminated saline was linked to the initial cases, subsequent ongoing transmission was documented, presumably by means of the hands of hcws and the environment." in that study, early gestational age, low birth weight, exposure to personnel with contaminated hands, and e. cloacae colonization of the stool were associated with e. cloacae bacteremia, whereas use of cvcs and mechanical ventilation was not. k. pneumoniae has been estimated to account for a similar proportion of infections in the nicu setting to that identified for enterobacter species. investigations in outbreaks involving klebsiella species have implicated contaminated breast milk,1oo infusion therapy practices,"' intravenous dextrose,io2 cockroaches, 134 di~infectant,"~ incubator humidifier^,'^' thermometers, oxygen saturation probes,io6 and ultrasonography coupling ge1.io8 in a surveillance study of 383 nicu infants in brazil, 50% became colonized with kleb~iella.'~ in this study, colonization was associated with use of a cephalosporin and aminoglycoside combination therapy, as well as with longer duration of the nicu stay. e. coli has been estimated to cause 4% of bloodstream, 14% of gastrointestinal, and 12% of surgical site infections. e. coli also has been responsible for outbreaks of pyelonephritis,io8 ga~troenteritis,'~'*"o and nec. s. marcescens is an opportunistic pathogen that survives in relatively harsh environments. disease due to s. marcescens often is manifested as meningitis, bacteremia, and pneumonia.'" s. marcescens infections have a high potential for morbidity and mortality."2,'13 s. marcescens outbreaks have been associated with, but not limited to, contaminated soap,114 multiuse bottles of theophylline,"5 formula,"' enteral feeding additives,l16 breast pumps,' ",'i8 and transducers from internal monitors.ii6 although point source environmental contamination is important in serratia outbreaks, in many of these outbreaks and in reports in which no point source was identified,"' patient-to-patient spread of the organism by means of the hands of hcws appeared to be an important mechanism of spread.i13 extended-spectrum p-lactamases (esbls) are plasmidmediated resistance factors produced by members of the enterobacteriaceae family. esbls inactivate third-generation cephalosporins and aztreonam. they most commonly occur in k. pneumoniae and e. coli but have increasingly been found in other gram-negative bacilli. colonization with esblproducing organisms has been associated with administration of certain antimicrobials and longer duration of hospitalization, whereas infection has been associated with prior colonization and use of cvcs.i3 that the esbl-containing plasmids can be transmitted to other enterobacteriaceae organisms has been demonstrated in nicu outbreaks in which the implicated plasmid spread from klebsiella species to e. coli, e. cloacae, and citrobacter fieundii.'20s'zl the gastrointestinal tract in neonates and the hands of hcws serve as reservoirs for members of the enterobacteriaceae family. thus, in general, measures aimed at controlling spread of organisms in this family have focused on attention on hand hygiene, cohorting of patient and staff, and observation of isolation precautions.'21"22 i? aeruginosa, an opportunistic pathogen that persists in relatively harsh environments, frequently has been associated with nosocomial infections and outbreaks in the nicu setting. nosocomial i! aeruginosa infections vary in their clinical presentation, but the most common manifestations are respiratory, ear, nose, or throat and bloodstream infections.*' from the ppn data it has been estimated that j? aeruginosa species account for 6.8% of total pathogens, 5% of bloodstream infections, and 15% of respiratory infections.21 l ? aeruginosa infections, particularly bloodstream infections, have been associated with a very high mortality rate.'23 feeding intolerance, prolonged parented alimentation, and long-term intravenous antimicrobial therapy have been identified as risk factors for pseudomonas infe~ti0n.l~~ outbreaks due to i! aeruginosa have been linked with contaminated hand lotion,'24 respiratory therapy solution, 12' a water bath used to thaw fkesh-frozen plasma,'26 a blood gas analy~er,'~' and bathing sources. in one case, neonatal pseudomonas sepsis and meningitis were shown by pulsed-field gel electrophoresis to be associated with shower tubing from a tub used by the infant's mother during labor.i2' of importance, hcws and their contaminated hands also have been linked with pseudomonas infections in the nicu setting. in a study of a new york outbreak, recovery of pseudomonas from the hands of hcws was associated with older age and history of use of artificial nails.12' this and other studies suggest that the risk of transmission of pseudomonas to patients is higher among hcws with onychmycosis or those who wear long artificial or long natural nail^.'^^,'^^ as a result of these and other findings, the cdc revised its 2002 hand hygiene recommendations to include a recommendation against the presence of hcws with artificial fingernails in intensive care units.'31 bordetella pertussis is a rare cause of nosocomial infection in neonates. when b. pertussis infection occurs, parents and hcws typically are the source. a parent was the source of an outbreak involving three neonates and one nurse in a special care nursery in a~stralia.'~' in 1999 in knoxville, tennessee, an outbreak involving six neonates probably was due to transmission of infection by an hcw."' as a result of the tennessee outbreak, 166 infants received erythromycin prophylaxis. subsequently, an increase in infantile hypertrophic pyloric stenosis was noted by local pediatric surgeons. results of a cdc investigation suggested a causal role of erythromycin in the cases of hypertrophic pyloric steno~is.'~~~~" erythromycin remains the recommended agent of choice for prophylaxis after pertussis exposure, but parents should be informed of the risk and signs of hypertrophic pyloric stenosis, and cases associated with erythromycin use should be reported to medwat~h.'~~ newborn infants are particularly prone to infection and disease following exposure to mycobacteriurn tuberculosis. a cluster of multidrug-resistant m. tuberculosis infections was noted in three infants born during a 2-week period in one new york h0spita1.l~~ investigation implicated an hcw who visited the nursery several times during that period. pulmonary and extrapulmonary disease occurred in three infants 4 to15 months after exposure, highlighting the vulnerability of the newborn p~pulation.'~~ tuberculosis screening of hcws, ultraviolet lighting, and a high number of air exchanges appear to be effective methods in preventing nosocomial tuberculosis infe~ti0n.i~' the cdc's "guidelines for preventing the transmission of mycobacteriurn tuberculosis in health-care settings" emphasizes (1) use of engineering controls and personal protective equipment, (2) risk assessments for the development of institutional tuberculosis control plans, (3) early identification and management of individuals with tuberculosis infection and disease, (4) tuberculosis screening programs for hcws, (5) hcw education and training, and (6) evaluation of tuberculosis control prograrns.l3* candida species are an increasingly important cause of nosocomial infection in nicu patients and have been estimated to account for 6.9% of bloodstream infections and 42% of urinary tract infection^.^^^"^ prospective studies have estimated colonization rates with candida to be 12% to 54% in low-birth-weight neonate^,'^"^^-'^' and colonization has been associated with subsequent invasive disease.14' the mortality rate can be high in invasive candidiasis. in one study of 34 patients with fungemia due to candida species, a case-fatality rate of 41% was r e~0 r t e d . i~~ risk factors for fungal infections in neonates are similar to risk factors for bacterial infections; low birth weight and gestational age are important predictors. in addition, a prospective, multicenter study of 2157 infants found that use of a third-generation cephalosporin, presence of a cvc, intravenously administered lipids, and hz blocker therapy were associated with candida colonization after adjusting for length of stay, birth weight of 1000 g or less, and gestational age less than 32 weeksl4 candida parapsilosis appears to be the most frequent species associated with nosocomial candida infection in nicu infants. both cross-contamination and maternal reservoirs are sources of nosocomial candida albicans infection, as demonstrated in studies using molecular typing method^.'^-'^^ malassezia species, lipophilic yeasts, frequently colonize nicu patients. in one french study, 30 of 54 preterm neonates (56%) became colonized with malassezia fit+r.147 malassezia pachydermatis, a zoonotic organism present on the skin and in the ear canals of healthy dogs and cats, also has been associated with nosocomial outbreaks in the nicu setting. 147, 148 in one report, the outbreak appeared to be linked to colonization of hcws' pet dogs.i4' pichia anornala, or hansenula anornala, a yeast found in soil and pigeon droppings, and on plants and fruits, also can colonize the human throat and gastrointestinal tract. in general, it is an unusual cause of nosocomial infection in neonates, but it was the cause of two reported outbreaks in this ~e t t i n g . '~~, '~~ in both reports, carriage on the hands of hcws appeared to be a factor. invasive mold infections are a rare cause of nosocomial infection in neonates, but when they occur, they are associated with high mortality rate. aspergillus infections may manifest as pulmonary, central nervous system, gastrointestinal, or disseminated disease. a cutaneous presentation, with or without subsequent dissemination, appears to be the most common presentation for hospitalized premature infants without underlying immune defi~iency.'~'.'~~ often, skin maceration is the presumed portal of entry. in a series of four patients who died of disseminated aspergillus infection that started cutaneously, a contaminated device used to collect urine from the male infants was impli~ated.'~' similarly, contaminated wooden tongue depressors, used as splints for intravenous and arterial cannulation sites, were associated with cutaneous infection due to rhizopus microsporus in four premature infants.'53 in addition to preterm birth, use of broad-spectrum antimicrobial agents, steroid therapy, and hyperglycemia are thought to be risk factors for mold infection. even zoophilic dermatophytes have been described as a source of nosocomial infection in neonates. in one report, five neonatal cases in one unit were traced to an infected nurse and her cat.154 prolonged therapy for both the nurse and her cat was necessary to clear their infections. although many pathogens can cause nosocomial gastroenteritis, rotavirus is responsible for 95% or more of viral infections in high-risk nurseries, including the nicu." in one longitudinal study, rotavirus infection developed during hospitalization in 95 of 194 neonates (49?40).'~~ in this study, rotavirus was manifested as frequent and watery stools in term infants and as abdominal distention and bloody, mucoid stools in the preterm neonates. a high titer of virus is excreted in stool of infected persons, and the organism is viable on hands and in the environment for relatively prolonged periods of time.'56"57 attention to hand hygiene and disinfection of potential fomites are crucial in preventing spread of infection. this concept is illustrated by the results of one study in which rotavirus infection was associated with ungloved nasogastric tube feeding.157 respiratory viruses including influenza a virus, parainfl uenza virus, coronavirus, respiratory syncytial virus, and aden )virus have been reported to cause nosocomial infections in qicu patient^.'^^-'^' associated clinical findings include rhino rrhea, tachypnea, retractions, nasal flaring, rales, and wheezir g, but illness also can be manifested as apnea, sepsis-like i lness, and gastrointestinal symptoms. 16',16* identified risk f ictors for acquisition vary from study to study but have includl id low birth weight, low gestational age, twin pregnancy, mech anical ventilation, and high crib s~o r e . '~~-'~* contact and d :oplet transmission are the most common modes of sprcad of infection, again highlighting the importance of scrul (dous hand hygiene in delivery of patient care for this popul, tion. numerous nursery and nicu outbreaks of enterovii a1 infection have been r e p~r t e d . '~~"~ in the neonate with e iteroviral infection, clinical manifestations can range fron mild gastroenteritis to a severe and fulminant sepsis-like sync home or meningitis/encephalitis. the latter presentation c m be associated with a high mortality rate.lw in index cas :s, the patient may have acquired disease vertically, with subst quent horizontal spread leading to outbreak^'^^'^^; with othe r viral pathogens, virus can be shed into the stool for prolnged periods, enabling patient-to-patient transmission by the hands of hcws when hand hygiene procedures are impr iperly performed. congenitally acquired cytomegalovirus (cmv) infecti )n is a cause of morbidity and occasionally death, whereas postnatally acquired cmv infection follows a benign col rse in virtually all healthy term infants. postnatal cmv infi ction, however, can cause considerable morbidity and death i n premature infants. 166 hepatitis, neutropenia, thrombocyto penia, sepsis-like syndrome, pneumonitis, and developmi nt of chronic lung disease each have been associated with postnatal acquisition of cmv in premature infant^.'^^''^^ w th the routine use of cmv-seronegative blood products in these neonates, a majority of postnatal cmv infections ap1 ear to be acquired through breast milk.'69 it has been estinated that transmission by this mode occurs in approximate1 y 37% of breast-fed infants of mothers with cmv detec ed in breast milk. '70 in one study, approximately 50% of these infants had clinical features of infection, and 12% pre iented with a sepsis-like syndrome. nosocomial person-to-] ierson transmission has been d o~u m e n t e d , '~'~'~~ but the exl ent to which this occurs is contr~versial.'~~ at present, no p :oven, highly effective method is available for removing cml ' from breast milk without destroying its beneficial compc nents. some data, however, suggest that freezing the breas: milk before use may decrease the cmv titer, thereby liniting subsequent transmission."* in a majority of cases, neonatal herpes simplex virus hsv) infection is acquired vertically from the mother. nursery transmission of hsv infection is rare but has been describe( . [175] [176] [177] in each of these cases, hsv-1 was involved. in one infa it, the source of virus was thought to be a patient's father, wl 10 had active herpes labiali~.'~~ subsequent spread of virus from this first infant to a second infant was thought to have occurred by means of the hands of an hcw. in another report, the source of hsv for the index case, an infant who died of respiratory distress in whom evidence of hsv infection was found at postmortem examination of the brain, was unthe hands of hcws were implicated in the spread of hsv to three subsequent cases, however. in another report, direct spread from an hcw was thought to be responsible for transmission of hsv to three infants over a period of approximately 3 years.'77 studies of adults with herpes labialis suggest a high frequency of recovery of virus from the mouth and the hands (78% and 67%, re~pectively).'~~ in this same study, hsv was shown to survive for 2 to 4 hours on skin, cloth, and plastic. implementing contact precautions for infants with hsv and instructing hcws with active herpes labialis regarding control measures, such as covering the lesion, not touching the lesion, and using strict hand hygiene, are reasonable means to prevent nosocomial transmission of hsv. if there are concerns that an hcw would be unable to comply with control measures or if the hcw has a herpetic whitlow, such persons should be restricted from patient contact. nosocomial transmission of varicella in the nicu setting, although unusual, has been de~cribed.'~~ large-scale outbreaks in nurseries and nicus are rare, most probably because of the high rate of varicella-zoster virus (vzv) immunity in hcws and pregnant women. premature infants born at less than 28 weeks of gestation are unlikely to have received protective levels of vzv igg from their mothers, so their potential risk is significant if an exposure occurs. transmission is most likely to occur from an adult with early, unrecognized symptoms of varicella. in such instances, the potential risk for vzv-seronegative exposed infants and hcws is substantial, especially if the patient in the index case is an hcw.'" for this reason, it is recommended that hcws be screened for prior varicella infection by history, with subsequent immunization as indicated. hepatitis a is a rare cause of nosocomial infection in nicus, but a number of outbreaks in this setting have been r e p~r t e d . '~' . '~~ in most instances, disease in neonates is clinically silent. neonatal cases often are detected only through recognition of the symptomatic secondary adult cases. in one report, disease was acquired by patients in the index cases through blood transfusion from a donor with acute hepatitis of note, the virus subsequently spread to another 11 infants, 22 nurses, and 8 other hcws. overall, hepatitis a affected 20% of the patients and 24% of the nurses. lapses in infection control practices and the prolonged shedding of the virus in infants stool probably contributed to the rapid spread and high attack rate documented in the outbreak. outbreaks such as this one are unlikely because of current blood product practices to eliminate transmissible agents from donor blood. an effective infection control program that focuses on reducing risk on a prospective basis can decrease the incidence of nosocomial infection^.'^"^^ the principal function of such a program is to protect the infant and the hcw from risk of hospital-acquired infection in a manner that is cost-effective. activities crucial to achieving and maintaining this goal include collection and management of critical data relating to surveillance for nosocomial infection, and direct intervention to interrupt the transmission of infectious diseases.22 reducing the incidence of nosocomial infection for neonates must begin with surveillance for these events. surveillance has been defined as "a comprehensive method of measuring outcomes and related processes of care, analyzing the data, and providing information to members of the health care team to assist in improving those outcomes."'86 essential elements of a surveillance program include the following: defining the population and data elements as concisely collecting relevant data using systematic methods consolidating and tabulating data to facilitate evaluation analyzing and interpreting data reporting data to those who can bring about changeia7 surveillance systems necessarily vary, depending on the population; accordingly, a written plan, based on sound epidemiologic principles,ls7 should be in place to track rates of infection over time. because new risks can emerge, such as new interventional technology or drugs, changing patient demographics, and new pathogens and resistance patterns, the plan should be reviewed and updated the joint commission on accreditation of healthcare organizations (jcaho) recommends that hospitals have a written infection control plan that includes a description of prioritized risks; a statement of the goals of the infection control program; a description of the hospital's strategies to minimize, reduce, or eliminate the prioritized risks; and a description of how the strategies will be evaluated. the jcaho further recommends that hospitals identify risks for transmission and acquisition of infectious agents (table 35system provides high-risk nursery-specific data collection methods as well as denominator data and allows external benchmarking of infection rates for this populati~n.'~~,'~' the nnis system defines a nosocomial infection as a localized or systemic process that results from adverse reaction to the presence of an infectious agent(s) or its toxin(s) and that was not present or incubating at the time of admission to the hospital. nnis also recognizes as special situations, and defines as nosocomial, some infections in neonates that result from passage through the birth canal but do not become clinically apparent until several or more days after birth. it does not, however, consider infections that are known or proved to have been acquired transplacentally to be noso-~omial.'~' distinction between maternal and hospital sources of infection is important, although difficult at times, because control measures designed to prevent acquisition from hospital sources will be ineffective in preventing perinatal acquisition of pathogen^.'^' surveillance for infections in healthy newborns also is challenging because of the typically short length of stay. infections can develop after discharge, and these are more difficult for infection control practitioners (icps) to capture. methods for postdischarge surveillance have been developed, but because most neonatal infections that occur following discharge are noninva~ive,'~~ such surveillance has not been widely implemented, because of concerns about the cost-effectiveness of these labor-intensive processes. the ultimate goal of surveillance is to achieve outcome objectives (e.g., decreases in infection rates, morbidity, mortality, or cost).is7 baseline infection rates for an inpatient unit must be established so that the endemic rate of infection can be understood and addressed. in the nicu, concurrent surveillance (initiated while the infant is in the hospital) should be conducted by persons trained to collect and interpret clinical information. typically, such persons are icps working closely with hcws and using various data sources (table 35-4) . using nnis or other accepted definitions, the icp should collect data regarding cases of nosocomial infection in the nicu population as well as population-specific denominator data. denominators must be carefully chosen to represent the population at risk. attempts to stratify risk should take into account both underlying infant-specific risks and those resulting from therapeutic or diagnostic interventions.'86 risk stratification techniques that attempt to control for distribution of risk have included severity of illness score, intensity of care required, and birth weight.74 because the risk for developing nosocomial infection is greater for lower-birthweight infants:' the nnis system breaks down data collection and analysis into birth weight categories (tables 35-5 and 35-6).'912' 94 the use of invasive devices, however, also is an important factor to consider. the appropriate denominator for an infection related to the use of a medical device, such as a cvc-related primary bloodstream infection, according to nnis, would be total device days for the population during the surveillance period. the formula generally used for calculating nosocomial infection rates is (x/y)k, where x equals the number of events (infections) over a specific time period, y equals the population at risk for development of the outcome, and k is a constant and a multiple of 10. rates can be expressed as a percentage (k = loo), although device-related infections usually are expressed as events per 1000 device days (k = 1000). a value should be selected for k that results in a rate greater than because use of invasive devices is such a significant risk factor both for bloodstream infection and ventilatorassociated pneumonia, assessing nicu practices with device use may be warranted. "is provides a benchmark for nicu device utilization broken down into birth weight categories. an nicu device utilization ratio can be calculated using the following formula: in those units with device utilization ratios above the nnis 90th percentile, investigation into the practices surrounding use of invasive devices may be ~a r r a n t e d . '~~ calculating monthly and annual rates to employ as benchmarks can assist in identification of a potential problem in device-related procedures. surveillance data must be arranged and presented in a way that facilitates interpretation, comparison both directed internally and with comparable external benchmarks, and dissemination within the organization. quality improvement tools (e.g., control and run charts) can be useful for these purposes. statistical tools should be used to determine the significance of findings, although statistical significance should always be balanced with the evaluation of clinical ~ignificance."~ external benchmarking through interhospital comparison is a valuable tool for improving quality of are'^,'^^ but should be performed only when surveillance methodologies (e.g., case definitions, case finding, data collection methods, intensity of ~urveillance)'~~ can reasonably be assumed to be consistent between facilities. few overall infection rates in nicus are available, but a small study done in 17 children's hospitals performing nicu nosocomial infection surveillance reported a median nosocomial infection rate of 8.9 infections per 1000 patient days (range, 4.6 to 18.1).19' nnis does not provide a benchmark for overall infection rates within nicus. instead, nnis provides birth weight-stratified device-associated infection rates for umbilical and central intravascular line-associated bloodstream infections. the most recent rates for catheterrelated bloodstream infections (137 to 143 nicus reporting) and ventilator-associated pneumonias (78 to 96 nicus reporting) are summarized in table 3 e~6 . l~~ once arranged and interpreted, nosocomial infection data must be shared with personnel who can effect change and implement infection control interventions. written reports summarizing the data and appropriate control charts should be provided to the facility's infection control committee, unit leaders, and members of the hospital administration on an ongoing basis. the interval between reports is determined by the needs of the institution. in addition to formal written reports, face-to-face reports are appropriate in the event of identification of a serious problem or an outbreak. icps can serve as consultants to assist nicu or neonatology service leaders in addressing infection rate increases or outbreak management. surveillance activities typically identify endemic nosocomial infections (i.e., those infections that represent the usual level of disease within the nursery or nlcu).'99 although the rate can fluctuate over time, in the absence of interventions that successfully reduce risk of infection, the difference rarely is statistically significant. establishing an nicu's endemic infection rate and expected variation around that rate allows the icp to rapidly identify unusual increases in rates that may indicate on outbreak (epidemic) of a particular infection. using baseline surveillance data along with aggregate data from sources such as the "is system allows the icp to develop meaningful threshold rates for initiating outbreak investigation.is8 alternatively, hcws can be the first to sense an increase in infections, which then can be confirmed or refuted by surveillance data?00 even a single case of infection due to an unusual and potentially dangerous pathogen (e.g., salmonella) can constitute the index case for a subsequent outbreak and thus merits rapid and comprehensive investigation. outbreaks may need to be reported to health authorities, depending on local and state requirements as well as the organism involved. numerous studies have described nursery and nicu epidemics caused by a variety of pathogens (table 35-7) , and most such epidemics have required the coordinated efforts of icps, nicu leadership, staff, and hospital administration outbreak investigation and intervention should be approached systematically, applying sound epidemiologic principles. in general, the process should include the follo~ing'~~~~''": for resolution.79, 106,115,136, i 57j6 i, 162,zo 1 -2 12 confirming that an outbreak exists, by comparing the outbreak infection rate with baseline data (or with rates reported in the literature if baseline data are not available), and communicating concerns to stakeholders within the institution (and to those in other agencies if notification of health authorities is necessary) assembling the appropriate personnel to assist in developing a case definition and in planning immediate measures to prevent new cases performing active surveillance using the case definition to search for additional infections and collecting critical data and specimens characterizing cases of infection by person, place, and time, including plotting of an epidemic curve (to facilitate identification of shared risk factors among involved patients, such as invasive devices, proximity to other infected patients or temporal association with infection in such patients, common underlying diagnoses, shared medical or nursing staff, surgery, and medications, including antimicrobial agents) formulating a working hypothesis and testing this hypothesis (if the severity of the problem warrants this level of study, and provided that the institution has and can commit the necessary resources), with use of analytic approaches, including case-control and cohort studies, as appropriate to determine the likely cause of the outbreak instituting and evaluating control measures, which can be implemented anywhere in the foregoing process (more directed measures become possible as more is learned about the outbreak, and efficacy of control measures can be judged on whether the outbreak resolves, as indicated by return of number of cases to endemic levels or by cessation of occurrence of infections) reporting findings to appropriate personnel, including unit staff, hospital administration, and public health authorities (if involved in management of the outbreak), in comprehensive written reports, including summaries of how the outbreak was first recognized, study and analysis methods used, interventions implemented to resolve the epidemic, results, and a discussion of any other important outcomes or surveillance and control measures identified interventions used to control and limit outbreaks usually have consisted of isolation and cohorting of infected or colonized infants to prevent transmission of organisms. transmission-based precautions, a system developed by the cdc, can be used to determine the most effective barrier precautions to use with affected patients. cohorting, or placing infants infected or colonized with the outbreak organism together in geographically segregated areas and assigning dedicated staff and equipment to their care, also has been used successfully to halt outbreaks in nurseries and nicus. in extreme cases, closure of an nicu to admissions has been necessary to bring an outbreak under ~o n t r o l .~~~'~~, '~~ every attempt should be made to identify the source of a nursery outbreak, although this is not always possible. sources implicated in nicu outbreaks have included medications, equipment, and enteral feeding solutions; person-to-person transmission and environmental reservoirs also have been efforts to identify the source may include culturing of specimens from hcws, equipment, and the environment, although careful consideration should be given to the potential benefits before initiating these measures. culture of samples from the environment and equipment, in view of the vast number of objects that could be contaminated, usually is not helpful in identifying the source of an outbreak unless specific case characteristics or microbiologic data strongly suggest the location. culture of specimens obtained from hcws when person-to-person transmission is suspected may be more likely to identify the source of an outbreak, but it must be remembered that an hcw whose culture specimen yields the outbreak organism may have been transiently colonized while working with an affected infant, rather than constituting the source of the infection. management of culture-positive hcws (possible furlough, treatment, and return to work criteria) should be planned in advance of widespread culture surveillance and should involve supervisors of affected employees and occupational health services.'" reported. 102, 106, 157, 201, 202, 213 the most widely accepted guideline for preventing the transmission of infections in hospitals was developed by the cdc. most recently revised in 1996, the system contains two tiers of precautions. the first and most important, standard precautions, was designed for the management of all hospitalized patients regardless of their diagnosis or presumed infection status. the second, transmission-based precautions, is intended for patients documented or suspected to be infected or colonized with highly transmissible or epidemiologically important pathogens for which additional precautions to interrupt transmission are needed.25 standard precautions are designed to reduce the risk of transmission of microorganisms from both recognized and unrecognized sources and are to be followed for the care of all patients, including neonates. they apply to blood; all body fluids, secretions, and excretions except sweat; nonintact skin; and mucous membranes. components of standard precautions include hand hygiene and wearing gloves, gowns, and masks and other forms of eye protection. hand hygiene plays a key role for caregivers in the reduction of nosocomial infection for patient^'^.^'^ and in prevention of nosocomial or health cart+associated infections. hand hygiene should be performed before and after all patient contacts; before donning sterile gloves to perform an invasive procedure; after contact with blood, body fluids or excretions, mucous membranes, nonintact skin, and wound dressings; in moving from a contaminated body site to a clean body site during patient care (ie., from changing a diaper to performing mouth care); after contact with inanimate objects in the immediate vicinity of the patient; after removing gloves; and before eating and after using the re~troom.'~~ when hands are visibly soiled or contaminated with proteinaceous materials, blood, or body fluids, and after using the restroom, hands should be washed with antimicrobial soap and water. soaps containing 2% to 4% chlorhexidine gluconate or 0.3% t r i~l o s a n '~~ are recommended for hand washing in n~rseries.'~~ when hands are not visibly soiled, alcohol-based hand rubs, foams, or gels are an important tool for hand hygiene. compared with washing with soap and water, use of the alcohol-based products is at least as effective against a variety of pathogens and requires less time, and these agents are less damaging to skin. the cdc "guideline for hand hygiene in the health care setting" calls for use of alcohol hand rubs, foams, or gels as the primary method to clean hands, except when hands are visibly soiled.i3l programs that have been successful in improving hand hygiene and decreasing nosocomial infection have used multidisciplinary teams to develop interventions focusing on use of the alcohol rubs in the settin of institutional commitment and support for the initiativetj7~15 hcws should wash hands and forearms to the elbows on arrival in the nursery. a 3-minute scrub has been suggested?l6 but consensus on optimal duration of initial hand hygiene is lacking. at a minimum, the initial wash should be long enough to ensure thorough washing and rinsing of all parts of the hands and forearms. routine hand washing throughout care delivery should consist of wetting the hands, applying product, rubbing all surfaces of the hands and fingers vigorously for at least 15 seconds, rinsing, and patting dry with disposable t0we1s.l~~ wearing hand jewelry has been associated with increased microbial load on hands. whether this results in increased transmission of pathogens is not known. many experts, however, recommend that hand and wrist jewelry not be worn in the in addition, the cdc guideline states that staff who have direct contact with infants in nicus should not wear artificial fingernails or nail extenders."' only natural nails kept less than y.. inch long should be allowed. clean, nonsterile gloves are to be worn whenever contact with blood, body fluids, secretions, excretions, and contaminated items is anticipated. the hcw should change gloves when moving from dirty to clean tasks performed on the same patient, such as after changing a diaper and before suctioning a patient, and whenever they become soiled. because hands can become contaminated during removal of gloves, and because gloves may have tiny, unnoticeable defects, wearing gloves is not a substitute for hand hygiene. hand hygiene must be performed immediately after glove removal. 25 personnel in nurseries including the nicu historically have worn cover gowns for all routine patient contact. the practice has not been found to reduce infection or colonization in neonates and is u n n e c e~s a r y ? '~~~~~ instead, cdc guidelines recommend nonsterile, fluid-resistant gowns to be worn as barrier protection when soiling of clothing is anticipated and in performing procedures likely to result in splashing or spraying of body substance^.^^ possible examples of such procedures in the nicu are placing an arterial line and irrigating a wound. the perinatal guidelines of the american academy of pediatrics and the american college of obstetricians and gynecologists recommend that a longsleeved gown be worn over clothing when a neonate is held outside the bassinette by nursery personnel.'" nonsterile masks, face shields, goggles, and other eye protectors are worn in various combinations to provide barrier protection and should be used during procedures and patient care activities that are likely to generate splashes or sprays of body substances and fl~ids.'~ standard precautions also require that reusable patient care equipment be cleaned and appropriately reprocessed between patients; that soiled linen be handled carefully to prevent contamination of skin, clothing, or the environment; that sharps (i.e., needles, scalpels) be handled carefully to prevent exposure to blood-borne pathogens; and that mouthpieces and other resuscitation devices be used, rather than mouth-to-mouth methods of re~uscitation.'~ in addition to standard precautions, which must be used for every patient, the cdc recommends transmission-based precautions when the patient is known or suspected to be infected or colonized with epidemiologically important or highly transmissible organisms. always used in addition to standard precautions, transmission-based precautions comprise three categories: contact precautions, droplet precautions, and airborne precautions. contact precautions involve the use of barriers to prevent transmission of organisms by direct or indirect contact with the patient or contaminated objects in the patient's immediate e n~i r o n m e n t .~~ sources of indirect contact transmission in nurseries can include patient care equipment such as monitor leads, thermometers, isolettes, breast pumps,le6 toys, and instruments and contaminated hands. 222 the patient requiring contact precautions should be placed in a private room whenever possible but, after consultation with an infection control practitioner, can be cohorted with a patient infected with the same microorganism but no other infection.25 many nurseries, however, have few if any isolation rooms. the american academy of pediatrics states that infected neonates requiring contact precautions can be safely cared for without an isolation room if staffing is adequate to allow appropriate hand hygiene, a 4-to 6-footwide space can be provided between care stations, adequate hand hygiene facilities are available, and staff members are well trained regarding infection transmission modes.'" hcws should wear clean, nonsterile gloves when entering the room or space of a patient requiring contact precautions and should wear a cover gown when their clothing will have contact with the infant, environmental surfaces, or items in the infant's area. a cover gown also should be worn when the infant has excretions or secretions that are not well contained, such as diarrhea or wound drainage, which may escape the diaper or dressing. infant care equipment should be dedicated to the patient if possible so that it is not shared with 0thers.2~ examples of conditions in the neonate that require contact precautions include neonatal mucocutaneous herpes simplex virus infection, respiratory syncytial virus infection, varicella (also see airborne precautions), infection or colonization with a resistant organism such as mrsa or a multiple drug-resistant gram-negative bacillus, and congenital rubella syndrome. droplet precautions are intended to reduce the risk of transmission of infectious agents in large-particle droplets from an infected person. such transmission usually occurs when the infected person generates droplets during coughmg, sneezing, or talking, or during procedures such as suctioning. these relatively large droplets travel only short distances and do not remain suspended in the air, but can be deposited on the conjunctiva, nasal mucosa, andfor mouth of persons working within 3 feet of the infected patient.25 patients requiring droplet precautions should be placed in private rooms (see earlier discussion of isolation rooms in nurseries in the paragraph on contact precautions), and staff should wear masks when working within 3 feet of the patient.25 examples of conditions in the neonate that necessitate droplet precautions are pertussis and invasive n. meningitidis infection. airborne precautions are designed to reduce the risk of airborne transmission of infectious agents.25 because of their small size, airborne droplet nuclei and dust particles containing infectious agents or spores can be widely spread on air currents or through ventilation systems and inhaled by or deposited on susceptible hosts. special air-handling systems and ventilation are required to prevent transmission. patients requiring airborne precautions should be placed in private rooms in negative air-pressure ventilation with 6 to 12 air changes per hour. air should be externally exhausted or subjected to high-efficiency particulate air (hepa) filtration if it is recirculated. 222 examples of conditions in the neonate for which airborne precautions are required are varicella-zoster virus infections and measles. susceptible hcws should not enter the rooms of patients with these viral infections. if assignment cannot be avoided, susceptible staff members should wear masks to deliver care. if immunity has been documented, staff members need not wear masks.222 airborne precautions also are required for active pulmonary tuberculosis, and although neonates are rarely contagious, the cdc recommends isolating patients while they are being e~aluated.~'~ a more important consideration is the need to isolate the family of a suspected tuberculosis patient until an evaluation for pulmonary tuberculosis has been completed, because the source of infection frequently is a member of the child's before the 1990s, well-baby nurseries and many nicus were constructed as large, brightly lit open wards with rows of incubators surrounded by equipment. sinks could be provided in such rooms only around the periphery, limiting access to hand hygiene facilities for staff and families. in these nicus, parents' time with their infant was severely restricted, and the units were designed for the convenience and function of the hcw.225 more recently, perinatal care professionals have come to understand that neonates (and especially preterm infants) can benefit from a quiet, soothing atmosphere and protection from unnecessary light, noise, handling, uncomfortable positioning, and sleep disruptions?26 if infants are kept in a central nursery rather than roomingin with mothers, at least 30 square feet of floor space should family.223,z24 be provided per neonate, and bassinets should be at least 3 feet apart.216 teams designing units delivering higher levels of perinatal care, including nicus, should plan individual bed areas large enough for families to stay at the bedside for extended periods of time without interfering with the staffs ability to care for the child. if individual rooms cannot be provided, at least 150 square feet of floor space should be allowed for each neonate in an nicu, incubators or overhead warmers should be separated by at least 6 to 8 feet, and aisles should be at least 8 feet a scrub sink with foot, knee, or touchless (electronic sensor) controls should be provided at the entrance to every nursery and should be large and deep enough to control splashing. sinks in patient care areas should be provided at a minimum ratio of 1 sink for at least every 6 to 8 stations in the well-baby nursery and 1 sink for every 3 or 4 stations in higher-level nurseries, including the nicu.'i6 every bed position should be within 20 feet of a hand-washing sink and accessible for children and persons in wheelchairs.227 for nicus composed of individual rooms, a hand-washing sink should be located in each room near the door to facilitate hand hygiene on entering and leaving the room. environmental surfaces should be designed so that they are easy to clean and do not harbor microorganisms. sink taps and drains, for instance, have been implicated in outbreaks of infection.2283z29 installing sinks with seamless construction may minimize this risk by decreasing areas where water can pool and microorganisms proliferate. faucet aerators have been implicated in outbreaks of infection and should be avoided in the intensive care unit.230 although carpeting can reduce noise levels in a busy nicu, the cdc "guidelines for environmental infection control in health-care facilities" recommend against use of carpeting in areas where spills are likely, including intensive care units. the guidelines further recommend against upholstered furniture in nicus.~~' if, for reasons of noise reduction and developmentally appropriate care, porous surfaces such as carpeting and cloth upholstery are selected for the nicu, cleaning must be performed carefully. carpet should be vacuumed regularly with equipment fitted with hepa filters, and upholstered furniture should be removed from inpatient areas to be cleaned. attention also should be paid to air-handling systems. according to the perinatal guidelines, minimal standards for inpatient perinatal care areas include six air changes per hour, and a minimum of two changes should consist entirely of outside air. air delivered to the nicu should be filtered with at least 90% efficiency. in addition, nurseries should include at least one isolation room capable of providing negative pressure vented to the outside, observation windows with blinds for privacy, and the capability for remote m~n i t o r i n g .~~~'~~~ floors and other horizontal surfaces should be cleaned daily by trained personnel using environmental protection agency (epa)-registered hospital disinfectantddetergents. these products (including phenolics and other chemical surface disinfectants) must be prepared in accordance with manufacturers' recommendations and used carefully to avoid exposing neonates to these products. phenolics should not be used on surfaces that come in direct contact with neo-nates' skin.231 high-touch areas, such as counter tops, work surfaces, doorknobs, and light switches, may need to be cleaned more frequently because they can be heavily contaminated during the process of delivering care. hard, nonporous surfaces should be "wet dusted" rather than dry dusted, to avoid dispersing particulates into the air, and then disinfected using standard hospital disinfectant^.'^^ sinks should be scrubbed daily with a disinfectant detergent. walls, windows, and curtains should be cleaned regularly to prevent dust accumulation, but daily cleaning is not necessary unless they are visibly soiled. bassinets and incubators should be cleaned and disinfected between infants, but care must be taken to rinse cleaning products from surfaces with water before use. care units should not be cleaned with phenolics or other chemical germicides during an infant's stay. instead, infants who remain in the nursery for long periods of time should periodically be moved to freshly cleaned and disinfected units.23' patient care equipment must be cleaned, disinfected, and, when appropriate, sterilized between patients. sterilization (required for devices that enter the vascular system, tissue, or sterile body cavities) and higher levels of disinfection (required for equipment that comes in contact with mucous membranes or that has prolonged or intimate contact with the newborn's skin) must be performed under controlled conditions in the central processing department of the hospital. examples of patient care equipment that require these levels of processing are endotracheal tubes, resuscitation bags, and face masks.216,232,233 low-level disinfection is required for less critical equipment, such as stethoscopes or blood pressure cuffs, and usually can be performed at point of use (e.g., the bedside), although this type of equipment should be dedicated to individual patients whenever possible. requirements for linen handling and management for neonates do not vary appreciably from those for other hospitalized patients. although soiled linen can contain large numbers of organisms capable of causing infections, transmission to patients appears to be rare. studies suggesting linen as a source of infection often have failed to confirm it as the source of infection.234 at least one report, however, has implicated linen in the transmission of group a streptoco~ci.~~ investigation of this outbreak revealed that clothing worn by the neonates was being washed in the local hospital "mini laundry," rather than being processed under the usual laundry contract. investigation of the dryers revealed extensive contamination with the outbreak organism. this case illustrates the importance of having standard hospital laundry protocols and ensuring that appropriate water and dryer temperatures are maintained. when such protocols are followed, the mechanical actions of washing and rinsing, combined with hot water and/or the addition of chemicals such as chlorine bleach, and a final commercial dryer and/or ironing step significantly reduce bacterial few hospitals in the united states use cloth diapers, but regardless of type used, soiled diapers should be carefully bagged in plastic and removed from the unit every 8 hours.216 hcws caring for neonates have the potential both to transmit infections to infants and to acquire infections from vaccine should be considered for all hcws, including those born before 1957, who have no proof of immunity (receipt of 2 doses of live vaccine on or after first birthday, physician-diagnosed measles, or serologic evidence of immunity). hcws believed t o be susceptible can be vaccinated; adults born before 1957 can be considered immune. hcws, both male and female, who lack documentation of receipt of vaccine on or after first birthday or serologic evidence of immunity should be vaccinated; adults born before 1957 can be considered immune, except women of childbearing age. hcws without a reliable history of varicella or serologic evidence of varicella immunity should be vaccinated. patients. educating hcws about infection control principles is crucial to preventing such transmission. hospitals should provide education about infection control policies, procedures, and guidelines to staff in all job categories during new employee orientation and on a regular basis throughout employment. the content of this education should include hand hygiene, principles of infection control, the importance of individual responsibility for infection control, and the importance of collaborating with the infection control department in monitoring and investigating potentially harmful infectious exposures and outbreaks. transmission of infectious organisms between patients and hcws has been well documented. several studies have indicated that a high proportion of hcws acquire rsv (34% to 56%) when working with infected children, and these workers appear to be important in the spread of the illness within hospital^.^^'.^^^ although 82% of the infected hcws in one of these rsv studies were asymptomatic, staff should be aware of the importance of self-screening for communicable disease. they should be encouraged to report personal infectious illnesses to supervisors, who in turn should report them to occupational health services and infection control. in general, hcws with respiratory, cutaneous, mucocutaneous, or gastrointestinal infections should not deliver direct patient care to neonates.2i6 in addition, seronegative staff members exposed to illnesses, such as varicella and measles, should not work during the contagious portion of the incubation period. 232 staff members with hsv infection rarely have been implicated in transmission of the virus to infants and thus do not need to be routinely excluded from direct patient care. those with herpes labialis or cold sores should be instructed to cover the lesions and not to touch their lesions, and to comply with hand hygiene policies. persons with genital lesions also are unlikely t o transmit hsv so long as hand hygiene policies are followed. however, hcws who are unlikely or unable to comply with the infection control measures and those with herpetic whitlow should not deliver direct patient care to neonates until lesions have healed. 240 acquisition of cmv often is a concern of pregnant hcws because of the potential effect on the fetus. approximately 1% of newborn infants in most nurseries and a higher percentage of older children (up to 70% of children 1 to 3 years of age in child-care centers) excrete cmv without clinical manifestation^.'^^ the risk of acquiring cmv infection has not been shown to be higher for hcws than for the general for this reason, pregnant caregivers need not be excluded from the care of neonates suspected to be shedding cmv. they should be advised of the importance of standard precautions. hcws in well-baby nurseries and nicus should be as free from transmissible infectious diseases as possible?16 and ensuring that they are immune to vaccine-preventable diseases is an essential part of a personnel health program. the cdc recommends several immunizations for health care personnel (table 35-8) . staffing levels in a patient care setting also can affect patient outcomes. a number of studies suggest that as patient-tonurse ratios in intensive care units increase, so do nosocomial infections and mortality rate^.^^,'^^,'^ although optimal staffing ratios have not been established for nicus and will vary according to characteristics of individual units, one study demonstrated that the incidence of clustered s. aureus infections was 16 times higher after periods when the infantto-nurse ratio exceeded 7: 1. decreased compliance with hand hygiene during a period of understaffing frequently is cited as contributing to nosocomial infection rate increases." further study is necessary to determine best practice surrounding staffing levels in nicus. the first nicus in the late 1960s grouped infants together in large, brightly lit rooms with incubators placed in rows. parents were allowed very little time with their babies and even less physical contact. in the decades since, it has been recognized that "the parent is the most important caregiver and constant influence in an infant's life"225 and that hcws working in nicus should encourage parents to become involved in the nonmedical aspects of their child's care. principles of family-centered care also include liberal nicu visitation for relatives, siblings, and family friends and the involvement of parents in the development of nursery policies and programs that promote parenting skills.226 care must be taken, however, to minimize risk of infection for the neonate. mothers can transmit infections to neonates both during delivery and post partum, although separation of mother and newborn rarely is indicated. in the absence of certain specific infections, mothers, including those with postpartum fever not attributed to a specific infection, should be allowed to handle their infants if the following conditions are met: 0 they feel well enough. they wash their hands well under supervision. a clean gown is worn. contact of the neonate with contaminated dressings, a mother with a transmissible illness not requiring separation from her infant should be carefully educated about the mode of transmission and precautions necessary to protect her infant. personal protective equipment, such as cover gowns, gloves, and masks, and hand hygiene facilities should be readily available to her, and she should perform hand hygiene and don a long-sleeved cover gown before handling her infant. if wounds or abscesses are present, drainage should be contained within a dressing. if drainage cannot be completely contained, separation from the infant may be necessary. care should be taken to prevent the infant from coming in contact with soiled linens, clothing, dressings, or other potentially contaminated items. the mother with active genital hsv lesions need not be separated from her infant if the foregoing precautions are taken. those with herpes labialis should not kiss or nuzzle their infants until lesions have cleared; lesions should be covered and a surgical mask may be worn until the lesions are crusted and dry, and careful hand hygiene should be stressed. mothers with viral respiratory infections should be made aware that many of these illnesses are transmitted by contact with infected secretions as well as by droplet spread, that soiled tissues should be disposed of carefully, and that hand hygiene is critical to transmission prevention. masks can be worn to reduce the risk of droplet t r a n s m i s s i~n .~~~'~~~ as previously mentioned, although very few infections require separation of mother and infant, women with untreated active pulmonary tuberculosis should be separated from their infants until they no longer are contagious. mothers with group a streptococcal infections, especially when involving draining wounds, also should be isolated from their infants until they no longer are contagious. less certain is the necessity of separating mothers with peripartum varicella (onset of infection within 5 days before or 2 days after delivery) from their uninfected infants. the perinatal guidelines recommend that such infants remain with their mothers after receiving varicella-zoster immune globulin (vzig) but caution that infant and mother must be carefully managed in airborne and contact precautions245 to prevent transmission within the nursery. some experts recommend separating these mothers from their infants until all lesions are dried and crusted.246 numerous studies support the value of human milk for infants (see chapter 5). besides providing optimal nutritional content for infants, it has been shown to be associated with a lower incidence of infections and sepsis in the first year of linen, clothing, or pads is avoided.245 although contraindications to breast-feeding are few, mothers who have active untreated tuberculosis, human immunodeficiency virus (hiv) infection, breast abscesses (as opposed to simple mastitis that is being treated with antimicrobial therapy), or hsv lesions around the nipples should not breast-feed. mothers who are hepatitis b surface antigen positive may breast-feed, because ingestion of an infected mother's milk has not been shown to increase the risk of transmission to her child, but the infant must receive hepatitis b virus immune globulin (hbig) and vaccine immediately after birth. 248 because systemic disease may develop in preterm infants with low concentrations of transplacentally acquired antibodies to cmv following ingestion of milk of cmv-seropositive mothers, decisions regarding breast-feeding should consider the benefits of human milk versus the risk of cmv transmission. freezing breast milk has been shown to decrease viral titers but does not eliminate cmv; pasteurization of human milk can inactivate cmv. either method may be considered in attempts to decrease risk of transmission for breast-feeding nicu neonates. 249 neonates in the nicu frequently are incapable of breastfeeding because of maternal separation, unstable respiratory status, and immaturity of the sucking reflex. for these reasons, mothers of such infants must use a breast pump to couect milk for administration through a feeding tube. pumping, collection, and storage of breast milk create opportunities for contamination of the milk, and for cross-infection if equipment is shared between mothers. several studies have demonstrated contamination of breast pumps, contamination of expressed milk that had been frozen and thawed, and higher levels of stool colonization with aerobic bacteria in infants fed precollected breast milk. 16,250 *251 consensus is lacking on the safe level of microbiologic contamination of breast milk, and most expressed breast milk contains normal skin flora. although breast milk containing greater than 100 cfu/ml of gram-negative bacteria has been reported to cause feeding intolerance and to be associated with suspected sepsis, routine bacterial culturing of expressed breast milk is not recommended.249v250 instead, efforts to ensure safety of expressed milk should focus on optimal collection, storage, and administration techniques. cleaning and disinfection of breast pumps should be included in educational material provided to nursing mothers (table 35-9 ). in addition, mothers should be instructed to perform hand hygiene and cleanse nipples with cotton and plain water before expressing milk in sterile containers. 1923249 expressed breast milk can be refrigerated for up to 48 hours and can be safely frozen (-20" c f 2â°c [-4" f f 3.6" f]) for up to 6 months.192 it can be thawed quickly under warm running water (avoiding contamination with tap water) or gradually in a refrigerator. exposure to high temperatures, as may be experienced in a microwave, can destroy valuable components of the milk. thawed breast milk can be stored in the refrigerator for up to 24 hours before it must be discarded. to avoid proliferation of microorganisms, milk administered through a feeding tube by continuous infusion should hang no longer than 4 to 6 hours before replacement of the milk, container, and for mothers who choose not to breast-feed, commercial infant formula is available. most hospitals now use sterile, ready-to-feed formulas provided by the manufacturer in bottles, with sterile nipples to attach just before use. nipples each mother is supplied with a personal pumping kit. nursing staff instruct mothers in techniques of milk expression and appropriate procedures for cleaning breast pump parts: wipe all horizontal surfaces at the pumping station with hospital disinfectant before and after pumping. wash hands with soapy water before and after pumping. wash all parts of the breast pump kit that have been in contact with milk in hot water and dish detergent or in a dishwasher. expressed milk is collected in sterile, single-use plastic (polycarbonate or polypropylene) containers. breast milk containers are labeled with infant's name and the date and time of collection. administration containers (bottle or syringe) are similarly labeled when breast milk is transferred from collection containers. all hcws wear gloves when handling and administering breast milk. two persons check t h e labeled administration container against the infant's hospital identification band before administering breast milk (may be two hcws or one hcw and a family member). hcw. health care worker. from infection control policy, children's hospital and regional medical center, seattle, 2003 are best attached at the bedside just before feeding, and the unit should be used immediately and discarded within 4 hours after the bottle is ~ncapped.'~' specialty and less commonly used formulas may not be available as a ready-to-feed product, and breast milk supplements do not come in liquid form. after a recent report of a case of fatal enterobacter sakazakii meningitis in a neonate fed contaminated powdered infant formula:7 concerns have risen about the safety of these products. although powdered formulas are not sterile, preparation and storage practices can decrease the possibility of proliferation of microorganisms after preparation. the cdc, the food and drug administration, and the american dietetic association offered updated recommendations on the safe preparation and administration of commercial formula after the recall of the product linked to the e. sakazakii case. these recommendations instruct the care provider as follows: use alternatives (ready-to-feed or concentrated formulas) to powdered infant formula whenever possible. prepare formula using aseptic technique in a designated formula preparation room. refrigerate prepared formula so that a temperature of 2" to 3" c is reached by 4 hours after preparation, and discard any reconstituted formula stored longer than 24 hours. limit ambient-temperature hang time of continuously infused formula to no longer than 4 hours. use hygienic handling techniques at feeding time, and avoid open delivery systems. have written guidelines for managing a manufacturer's recall of contaminated formula.252 the fda also recommended that boiling water be used to prepare powdered formulas, but concerns about this recommendation include potential damage to formula components from the high temperature of the water, a lack of evidence that using this method would lull potential pathogens in the formula, and risk of injury to persons preparing the f0rmula.2~' the concept of co-bedding, or the bunlung of twin infants (or other multiples) in a single isolette or crib, is being explored in nicus for the potential benefits offered to the babies. co-bedding as a component of developmentally supportive care is based on the premise that extrauterine adaptation of twin neonates is enhanced by continued physical contact with the other twin.253 potential benefits need further study but may include increased bonding, decreased need for temperature support, and easier transition to home. it is certainly possible, however, for one of a set of multiples to be infected while the others are not, and for parents to be implicated as vectors in infection transmission. it also is possible for invasive devices and intravascular catheters to be dislodged by close contact with an active sibling. therefore, exclusion criteria for co-bedding infants should include clinical findings suggesting infection that could be transmitted to a sibling (e.g., draining wound) and the need for drains and central venous or arterial line^.*^^-'^^ the principles of family-centered care encourage liberal visitation policies, both in the well-baby nursery (or roomingin scenario) and in the nicu. parents, including fathers, should be allowed unlimited visitation to their newborns, and siblings also should be allowed liberal visitation. expanding the number of visitors to neonates may, however, increase the risk of disease exposure if education and screening for symptoms of infection are not implemented. written policies should be in place to guide sibling visits, and parents should be encouraged to share the responsibility of protecting their newborn from contagious illnesses. the perinatal guidelines regarding persons who visit newborns are listed in table 35-10. adult visitors to neonates, including parents, have been implicated in outbreaks of infections including p aeruginosa infection, pertussis, and salmonella i n f e c t i~n . '~'~~~~~~~~ ac cordingly, the principles for sibling visitation should be applied to adult visitors as well. they should be screened for symptoms of contagious illness, instructed to perform hand hygiene before entering the nicu and before and after touching the neonate, and should interact only with the family member they came to the hospital to visit. families of neonates who have lengthy nicu stays may come to know each other well and serve as sources of emotional support to one another. nevertheless, they should be educated about the potential of transmitting microorganisms and infections between families if standard precautions and physical separation are not maintained, even though they may be sharing an inpatient space. sibling visits should be encouraged for healthy and ill newborns. parents should be interviewed at a site outside the nursery to establish that the siblings are not ill before allowing them to visit. children with fever or other symptoms of an acute illness such as upper respiratory infection or gastroenteritis, or those recently visiting children should visit only their sibling. children should be prepared in advance for their visit. visitors should be adequately observed and monitored by hospital staff. children should carefully wash their hands before patient contact. throughout t h e visit, siblings should be supervised by parents or another responsible adult. exposed to a known communicable disease such as chickenpox, should not be allowed to visit. bathing the newborn is standard practice in nurseries, but very little standardization in frequency or cleansing product exists. if not performed carefully, bathing actually can be detrimental to the infant, resulting in hypothermia, increased crying with resulting increases in oxygen consumption, respiratory distress, and instability of vital signs.257 although the initial bath or cleansing should be delayed until the neonate's temperature has been stable for several hours, removing blood and drying the skin immediately after delivery may remove potentially infectious microorganisms such as hepatitis b virus, hsv, and hiv, minimizing risk to the neonate from maternal infection.249 when the newborn requires an intramuscular injection in the delivery room, infection sites should be cleansed with alcohol to prevent transmission of organisms that may be present in maternal blood and body for routine bathing in the first few weeks of life, plain warm water should be used. this is especially important for preterm infants, as well as full-term infants with barrier compromise such as abrasions or dermatitis. if a soap is necessary for heavily soiled areas, a mild ph-neutral product without additives should be used, and duration of soaping should be restricted to less than 5 minutes no more than three times per week. 257 few randomized studies comparing cord care regimens and infection rates have been performed, and consensus has not been reached on best practice regarding care of the umbilical cord stump. a review published in 2003 described care regimens used for more than 2 decades, including combinations of triple dye, chlorhexidine, 70% alcohol, bacitracin, hexachlorophene, povidone-iodine, and "dry care" (soap and water cleansing of soiled periumbilical skin) and found variable impact on colonization of the stump.258 the study authors suggested that dry cord care alone may be insufficient and that chlorhexidine seemed to be a favorable antiseptic choice for cord care because of its activity against gram-positive and gram-negative bacteria. they went on to stress, however, that large, well-designed studies were required before firm conclusions could be drawn. the current perinatal guidelines do not recommend a specific regimen but warn that use of alcohol alone is not an effective method of preventing umbilical cord colonization and ~mphalitis.'~~ the perinatal guidelines further recommend that diapers be folded away from and below the stump and that emollients not be applied to the although blindness resulting from neonatal conjunctivitis is rare in the united states, with a reported incidence of 1.6% or less, the rate among the 80 million infants born annually throughout the world is as high as 23%. 259 chlamydia trachornatis has been the most common etiologic agent in the united states, but other organisms such as neisseria gonorrhoeae, s. aureus, and e. coli also can cause ophthalmia neonatorum.260 use of 1% silver nitrate drops, at one time the agent of choice, is no longer recommended because of concerns about associated chemical irritation. agents thought to be equally efficacious and now recommended include 1% tetracycline and 0.5% erythromycin ophthalmic ointments, administered from sterile single-use tubes or vials. 156~245 povidone-iodine (2.5%) ophthalmic solution also can be used and in one study was shown to be more effective than silver nitrate or erythromycin in the prevention of ophthalmia neonatorum. bacterial resistance has not been seen with this agent, it causes less toxicity than either silver nitrate or erythromycin, and it is less expensivea definite consideration in developing countries.259 whatever the agent selected, it should reach all parts of the conjunctival sac, and the eyes should not be irrigated after application. ophthalmic agents will not necessarily prevent ocular or disseminated gonorrhea in infants born to mothers with active infection at time of delivery. these infants should be given parenteral antimicrobial therapy as well as ocular p r o p h y l a~i s . '~~,~~~ some experts also advise giving infants born to mothers with untreated genital chlamydia1 infections a course of oral erythromycin beginning on the second or third day of life.261 primary bloodstream infections (defined by the cdc nnis system as being due to a pathogen cultured from one or more blood specimens not related to an infection at another site) account for a large proportion of infections in nicu infants:' and most are related to the use of an intravascular catheter.36 peripheral intravenous catheters (pivs) are the most frequently used devices for the neonate for intravenous therapy of short duration. when longer access is necessary, nontunneled cvcs such as umbilical catheters and piccs most commonly are the most recent data available conduct surveillance in nlcus to determine catheter-related bloodstream infection rates, monitor trends, and identify infection control lapses. investigate events leading to unexpected life-threatening or fatal outcomes. select the catheter, insertion technique, and insertion site with the lowest risk for complications for the anticipated type and duration of intravenous therapy. use a cvc with the minimal number of ports essential f o r management of t h e patient. designate one port for hyperalimentation if a multilumen catheter is used. educate hcws who insert and maintain catheters, and assess their knowledge and competence periodically. use aseptic technique and maximal sterile barriers during insertion of cvcs (cap, mask, sterile gown, sterile gloves, and a large sterile barrier). do not routinely replace cvcs, piccs, or pulmonary artery catheters to prevent catheter-related infections. do not remove on the basis of fever alone. in pediatric patients, leave peripheral venous catheters in place until intravenous therapy is completed unless a complication (e.g., phlebitis, infiltration) occurs. remove intravascular catheters promptly when no longer essential. observe proper hand hygiene procedures either by washing with antiseptic-containing soap and water or use of waterless alcohol-based products before and after working with intravascular lines. disinfect skin with an appropriate antiseptic before catheter insertion and during dressing changes. a 2% chlorhexidine-based preparation is preferred. do not use topical antibiotic ointment or creams on insertion sites, except when using dialysis catheters. use either sterile gauze or sterile, transparent, semipermeable dressing to cover t h e catheter site. replace gauze dressings on short-term cvc sites every 2 days and transparent dressings at least weekly, except in pediatric patients, in whom the risk of dislodging the catheter outweighs the benefit of changing the dressing. change if damp, loosened, or visibly soiled. replace dressings on tunneled or implanted cvc sites no more than once per week until the insertion site has healed. chlorhexidine sponge dressings are contraindicated in neonates younger than 7 days or those born at a gestational age of less than 26 weeks. clean injection ports with 70% alcohol or an iodophor before accessing the system. use disposable transducer assemblies with peripheral arterial catheters and pressure monitoring devices. keep all components of such systems sterile, and do not administer dextrose-containing solutions or parenteral nutrition fluids through them. from nnis (august 2003) revealed that the mean umbilical catheter-and cvc-associated bloodstream infection rates for nicus ranged from 10.6 per 1000 catheter days for infants whose birth weight was less than 1000 g to 3.7 per 1000 catheter days in infants whose birth weight was 2500 g or more.'94 the cdc recommends implementing strategies to reduce the incidence of such infections that strike a balance between patient safety and cost-effectiveness. few large studies of risks related to intravascular devices have been performed in nicu patients. as a result, intravascular device recommendations for neonates are based on those developed for adults and older pediatric patients (table 35-1 1) . several differences in their management should be considered. although the cdc recommends, in certain circumstances, using antimicrobialor antiseptic-impregnated cvcs in adults whose catheters are expected to remain in place more than 5 days,36 these catheters are not available in sizes small enough for neonates. of more importance, studies to evaluate their safety in neonates, especially premature neonates of very low birth weight, have not been performed. in addition, although the cdc recommends changing the insertion site of pivs at least every 72 to 96 hours in adults, data suggest that leaving pivs in place in pediatric patients does not increase the risk of complications. 262 the 2002 cdc guidelines recommend that pivs be left in place in children until therapy is completed, unless complications occur. careful skin antisepsis before insertion of an intravascular catheter is critical to prevention of intravascular devicerelated bacteremia, although care in the selection of a product for use on neonatal skin is required. chlorhexidine preparations are recommended by the cdc because these products have been found to be superior to povidone-iodine in reducing the risk for peripheral catheter colonization in neonates. residues left on the skin by chlorhexidine prolong its half-life, providing improved protection for catheters in neonates that must be left in place for longer periods of timef5' umbilical veins and arteries are available for cvc insertion only in neonates and are typically used for several days; thereafter, the cvc is replaced with another, nontunneled cvc or picc if continued central venous access is required. the umbilicus provides a site that can be cannulated easily, allowing for collection of blood specimens and hernodynamic measurements, but after birth, the umbilicus quickly becomes heavily colonized with skin flora and other microorganisms. colonization and catheter-related bloodstream infection rates for umbilical vein and umbilical artery catheters are similar. colonization rates for umbilical artery catheters are estimated to be 40% to 55%; the estimated rate for umbilical artery catheter-related bloodstream infection is 5%?6 colonization rates are from 22% to 59% for umbilical vein catheters; rates for umbilical vein catheter-related bloodstream infections are 3% to 8?40.~~ a summary of the cdc recommendations for management of umbilical catheters36 is presented in table 35 -12. as mentioned earlier, nnis data indicate that nosocomial pneumonia is the second most common infection type in cleanse umbilical insertion site with an antiseptic before catheter insertion. avoid tincture of iodine; povidone-iodine can be used. add low doses of heparin to fluid infused through umbilical artery catheters. remove and do not replace umbilical catheters if signs of catheter-related bloodstream infection, vascular insufficiency, or remove umbilical catheters as soon as possible when no longer needed or if any sign of vascular insufficiency to the lower umbilical artery catheters should not be left in place for longer than 5 days. umbilical venous catheters should be removed as soon as possible when no longer needed but can be used for up to 14 nicu patients. risk factors for ventilator-associated pneumonia can be grouped as host-related (prematurity, low birth weight, sedation or use of paralytic agents), devicerelated (endotracheal intubation, mechanical ventilation, orogastric or nasogastric tube placement) and factors that increase bacterial colonization of the stomach or nasopharynx (broad-spectrum antimicrobial agents, antacids, or h, b l o c k e r~) . 5~,~"~~~ ventilator-associated pneumonia generally refers to bacterial pneumonia that develops in patients who are receiving mechanical ventilation. aspiration and direct inoculation of bacteria are the primary routes of entry into the lower respiratory tract; the source of these organisms may be the patient's endogenous flora or transmission from other patients, staff members, or the e n~i r o n m e n t .~~'~~~ few studies have been performed to assess the effectiveness of prevention strategies in pediatric patients. strategies to prevent ventilator-associated pneumonia in the nicu patient are therefore based primarily on studies performed in adults (table 35-13) . hand hygiene remains critical to the prevention of ventilator-associated pneumonia, and hcws should consistently apply the principles of standard precautions to the care of the ventilated patient, wearing gloves to handle respiratory secretions or objects contaminated by them, and changing gloves and performing hand hygiene between contacts with a contaminated body site and the respiratory tract or a respiratory tract device. because mechanical ventilation is a significant risk factor for the development of nosocomial infection or ventilatorassociated pneumonia, weaning from ventilation and removing endotracheal tubes as soon as indication for their use ceases are key infection control strategies. as an alternative to endotracheal intubation, noninvasive nasal continuous positive airway pressure (cpap) ventilation avoids some of the common risk factors for ventilator-associated pneumonia and has been used successfully for neonate^?^^"^^ respiratory care equipment that comes in contact with mucous membranes of ventilated patients or that is part of the ventilator circuit should be single use (discarded after one-time use with a single patient) or be subjected to sterilization or high-level disinfection between patients. wet heat pasteurization (processing at 76oc for 30 minutes) or chemical disinfectants can be used to achieve high-level disinfection of reusable respiratory equipment.263 ventilator circuits should be changed no more frequently than every 48 hours, and evidence suggests that extending the length of time between changes to 7 days does not increase the risk of ventilator-associated pneumonia.z70s271 circuits should be monitored for accumulation of condensate and drained periodically, with care taken to avoid allowing the condensate, a potential reservoir for pathogens, to drain toward the sterile fluids should be used for nebulization, and sterile water should be used to rinse reusable semicritical equipment and devices such as in-line medication nebulizers. 263 basic infection control measures, such as hand hygiene and wearing gloves during suctioning and respiratory manipulation, also can reduce the risk of nosocomial pneumonia. both open, single-use and closed, multiuse suction systems are available. if an open system is used, a sterile single-use catheter should be used each time the patient is suctioned. closed systems, which do not need to be changed daily and can be used for up to 7 have the advantage of lower costs and decreased environmental cross-contamination258 but have not been shown to decrease the incidence of nosocomial pneumonia when compared with open systems.273v274 although not well studied in pediatric patients, aspiration of oropharyngeal secretions is believed to contribute to development of ventilator-associated pneumonia in adults. 275 placing the mechanically ventilated patient in a semirecumbent position or elevating the head of the bed in an attempt to minimize aspiration is recommended unless medically contraindicated. also, placement of enteral feeding tubes should be verified before their to prevent regurgitation and potential aspiration of stomach contents by the sedated patient, overdistention of the stomach should be avoided by regular monitoring of the patient's intestinal motility, serial measurement of residual gastric volume or abdominal girth, reducing the use of narcotics and anticholinergic agents, and adjusting the rate and volume of enteral fee ding^.^^^,^^^ oral decontamination, with the intent of decreasing oropharyngeal colonization, has been studied in adults and seems to lower the incidence of ventilatorassociated pneumonia (although not duration of ventilation or mortality but further work is needed to determine whether this is an effective strategy in neonates. in addition, medications such as sucralfate, as opposed to histamine h, receptor antagonists and antacids, which raise gastric ph and can potentially result in increased bacterial colonization of the stomach, have been used to prevent development of stress ulcers and have been associated with lower incidence of ventilator-associated pneumonia in adults.277 two studies suggest, however, that this approach is of no benefit in pediatric patients, but the authors stress that additional studies with larger sample sizes are needed to confirm these nosocomial infections in a neonatal intensive care unit: incidence and risk factors. am 1 infect control bektas s, goetze b, speer cp. decreased adherence, chemotaxis and phagocytic activities of neutrophils from preterm neonates surgery, sepsis, and 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a vector of salmonella brandenburg nosocomial infection in a neonatal intensive care unit role of antimicrobial applications to the umbilical cord in neonates to prevent bacterial 47757-782,2000. colonization and infection: review of the evidence a controlled trial of povidone-iodine as prophylaxis against ophthalmia neonatorum ocular applications of povidoneiodine peripheral intravenous catheter complications in critically ill children: a prospective study guidelines for prevention of nosocomial pneumonia risk factors for nosocomial infections in critically ill newborns: a 5-year prospective cohort study nosocomial pneumonia hospital-acquired pneumonia: perspectives for the health care epidemiologist the prevention of ventilator-associated pneumonia non-invasive mandatory ventilation in extremely low birth weight and very low birth weight newborns with failed respiration ventilator-associated pneumonia with circuit changes every 2 days versus every week cost analysis and clinical impact of weekly ventilator circuit changes in patients in intensive care unit weekly versus daily changes of inline suction catheters: impact on rates of ventilator-associated pneumonia and associated costs incidence of colonization, nosocomial pneumonia, and mortality in critically ill patients using a trach care closed-suction system versus an open-suction system: prospective, randomized study prevention of ventilatorassociated pneumonia by oral decontamination: a prospective, randomized, double-blind, placebo-controlled study oropharyngeal decontamination decreases incidence of ventilator-associated pneumonia. a randomized, placebo-controlled, double-blind clinical trial stress ulcer prophylaxis in critically ill patients. resolving discordant meta-analyses occurrence of ventilator-associated pneumonia in mechanically ventilated pediatric intensive care patients during stress ulcer prophylaxis with sucralfate, ranitidine, and omeprazole ventilator-associated pneumonia and upper airway colonisation with gram negative bacilli: the role of stress ulcer prophylaxis in children rr-1):l-79 key: cord-001455-n7quwr4s authors: rapin, noreen; johns, kirk; martin, lauren; warnecke, lisa; turner, james m.; bollinger, trent k.; willis, craig k. r.; voyles, jamie; misra, vikram title: activation of innate immune-response genes in little brown bats (myotis lucifugus) infected with the fungus pseudogymnoascus destructans date: 2014-11-12 journal: plos one doi: 10.1371/journal.pone.0112285 sha: doc_id: 1455 cord_uid: n7quwr4s recently bats have been associated with the emergence of diseases, both as reservoirs for several new viral diseases in humans and other animals and, in the northern americas, as hosts for a devastating fungal disease that threatens to drive several bat species to regional extinction. however, despite these catastrophic events little information is available on bat defences or how they interact with their pathogens. even less is known about the response of bats to infection during torpor or long-term hibernation. using tissue samples collected at the termination of an experiment to explore the pathogenesis of white nose syndrome in little brown bats, we determined if hibernating bats infected with the fungus pseudogymnoascus destructans could respond to infection by activating genes responsible for innate immune and stress responses. lesions due to fungal infection and, in some cases, secondary bacterial infections, were restricted to the skin. however, we were unable to obtain sufficient amounts of rna from these sites. we therefore examined lungs for response at an epithelial surface not linked to the primary site of infection. we found that bats responded to infection with a significant increase in lungs of transcripts for cathelicidin (an anti-microbial peptide) as well as the immune modulators tumor necrosis factor alpha and interleukins 10 and 23. in conclusion, hibernating bats can respond to experimental p. destructans infection by activating expression of innate immune response genes. bats, members of the mammalian order chiroptera, have evolved a range of characteristics that allow them to adapt to changing environmental conditions. they are the only mammals capable of powered flight, most bat species undergo torpor to conserve energy and species that inhabit high northern latitudes hibernate for up to eight months with body temperatures below 10uc [1] . bats are extremely diverse, making up a fifth of all known mammalian species. they occupy a variety of niches across most of the world where they contribute in many ways to ecological balance [2] . bats have been implicated as reservoirs for human bacterial [3] [4] [5] and viral pathogens [6] . in recent years bats have also been associated with the emergence of several devastating viral diseases in humans and other animals [7] [8] [9] [10] [11] [12] [13] [14] [15] . circumstantial evidence even suggests that most human and domestic animal pathogens of at least four viral families -coronaviridae, paramyxoviridae, lyssaviridae and filoviridae may have arisen in bats [15] [16] [17] and bats are more likely than rodents to host zoonotic viruses [18] . interestingly, with the exception of rabies and other lyssaviruses, viruses do not appear to cause overt pathology in bats, suggesting the evolution of benign relationships between bats and their pathogens [14, 19] . despite the growing realization of the importance of bats in environmental and human health and disease, we know relatively little about how bats interact with their pathogens and commensal microbes. data on their immune responses to infection during torpor or long-term hibernation are particularly scarce because individuals of some species spend as long as 8 months a year in hibernation (norquay and willis, in press) and are therefore difficult to sample. the few studies on bats and other mammals suggest that the energy conserving benefits of torpor and hibernation may be enhanced by a state of immunosuppression [20] . the fungus pseudogymnoascus destructans (formerly known as geomyces destructans) is a cold-adapted saprophytic fungus that targets hibernating bats. it appears to be a recent introduction to the northern americas [21] and in recent years has been responsible for the death of an estimated 6 million bats and the potential regional extinction of some species [22, 23] . this disease is called white nose syndrome (wns) for the white fungal mycelia on the faces and wings of affected bats. although damage caused by the fungus is restricted to the superficial skin, infected bats clearly show signs of systemic physiological perturbation such as dehydration, hypovolemia and metabolic acidosis [24] . infected bats arouse from torpor more frequently than uninfected bats [25, 26] possibly leading to emaciation. recently, we investigated the pathogenesis of p. destructans [24, 25] . we used samples collected during the experiment to address the question: can hibernating bats respond to infection by activating genes responsible for innate immune and stress responses? while the site of primary site of fungal infection and occasional secondary bacterial infection was the skin of the wings [25] , most of this tissue was needed for histopathology and fungal detection and we were unable to obtain sufficient amounts of rna from these sites. to test the hypothesis that bats mount a systemic response to infection we examined lungs as an example of an epithelial surface which could have come in contact with aerosolized fungal spores despite being remote from the site of infection or may have responded to infection of peripheral areas [27, 28] . fifty four male m. lucifugus bats were collected from a wnsfree cave in manitoba, canada. protocols for collecting and transporting bats, infection with p. destructans, maintenance of bats in hibernation and sample collection have been described previously [24, 25] . briefly, bats in groups of 18 were either sham inoculated or inoculated with n. american or european isolates of p. destructans. bats were housed at 7uc and greater than 97% humidity with ad libitum water. all bats were equipped with data loggers to monitor skin temperatures. bats were euthanized during the experiment when humanely required or at the termination of the experiment 120 days after inoculation. immediately following euthanasia samples from segments of wing as well as various tissues were preserved in rnalater (life technologies, am7021) or in formalin. samples in rnalater were kept at 220uc until they were processed. we did not find p. destructans isolate-specific differences and treated the bats as either infected or sham-infected (control). during necropsy we collected representative samples for histopathology from all major organ systems. in addition, representative samples were taken from all areas of the wing and rolled on dental wax before placing in 10% neutral buffered formalin. tissues were processed routinely for histology, 5 mm sections cut and stained with periodic acid-schiff stain to highlight fungal hyphae. liver and other tissues were processed routinely and stained with hematoxylin and eosin. wings were subjectively scored on a scale of 0 to 5 with 5 being very severe with .50% of wing covered in fungal hyphae. bacterial score was from 0 to 5 with 5 indicating wide-spread and abundant bacteria being present in many areas within the dermis and underlying connective tissues. interstitial lung neutrophil assessment was similarly evaluated on a scale of 0 to 5, with 5 being very severe. tissues were homogenized in 2 ml sealed vials with a 5 mm steel bead, 0.1 g of 0.1 mm zirconium silica beads, 350 ml of rlt buffer (with b-mercaptoethanol) (rneasy plus kit, qiagen, 74136) using a retsch mm400 tissue homogenizer at 30 hz twice for 2 minutes each. rna from tissues was extracted using the procedure provided with the rneasy plus kit. cdna synthesis cdna synthesis was performed with 1 mg of rna per reaction using the quantitect reverse transcription kit (qiagen, 205313). cdna samples were either stored at 4uc if proceeding to use in qrt-pcr, or stored at 280uc until they were needed. to identify myotis genes for various cytokines and proteins involved in immune responses we scanned the m. lucifugus genome for regions with sequences similar to transcripts of proteins from human, mouse and rat. for genome sequences that were not annotated, m. lucifugus exons detected were compared with known exon-intron junctions in corresponding genes in human, mouse and rat to ensure that the sequences did indeed represent homologous genes. for mrnas where qrt-pcr primers had been described for other species, m. lucifugus primers were designed from the sequence of corresponding m. lucifugus genes. for mrnas where such information was not available we designed primers to optimize for annealing temperature and specificity, and to minimize dimer formation. in all cases the veracity of the pcr products was determined by electrophoresis and confirmed by sequencing. details of primers are in table 1 . stratagene's mx3005p pcr cycler was used in conjunction with quantifast sybr green pcr kit (qiagen, 204056). in addition to the primer sets mentioned, gapdh and cytochrome b were both used for every sample as normalizers. for each sample tested, 2 ml (1 mm, final concentration) of diluted forward primer, 2 ml (1 mm) of diluted reverse primer, 12.5 ml of sybr green master mix, and 8.5 ml of cdna was used. samples were tested in duplicate in each run and values used in the analysis were averages of the duplicates. the thermal profile used was as follows: 95uc for 5 minutes for initiation, then 40 cycles of 95uc for denaturing for 10 seconds, and 60uc for annealing and extension for 30 seconds with readings at the end of every cycle, then 1 cycle of 95uc for 1 minute, 55uc for 30 seconds, and 95uc for 30 seconds. the denaturation characteristics of the products were determined by incremental increase from 55uc to 95uc. a no template control (ntc) was used in every sample tested. reactions that generated primer dimers or products with spurious denaturation profiles were not considered. in all cases the identity of the pcr products was confirmed by determining their nucleotide sequences (macrogen). only results from reactions that yielded unambiguous results (products of the expected size and sequence, homogenous denaturation profiles, no products for no-template controls) were used for analysis. data from qrt-pcr and histopathological scores were analysed with spss statistics version 21. for qrt-pcr the relative levels of a transcript for each bat were calculated as cycle threshold (ct) normalized separately (dct) for levels of transcripts for two ''house-keeping'' genes -glycerol 6 phosphate dehydro-genase (gapdh) and cytochrome b. a lower ct (or dct) of one (1) indicates approximately a two-fold higher concentration of rna. the significance of differences of mean values of dct between infected and mock-infected (control) bats were determined using a student t-test. pearson's coefficients were calculated for the dct levels for cytokine transcripts for bats in each treatment class and lung interstitial neutrophil scores and mean bacterial and hyphae scores for 5 wing sections for each bat. methods were approved by the university of saskatchewan committees on animal care (protocol #20100120) and biosafety (permit #vmb03). recently we demonstrated that the european and north american strains of p. destructans were equally pathogenic for hibernating m. lucifugus [24, 25] . during these experiments histological sections from wings and other tissues were examined and graded for the presence of hyphae, edema, necrosis, bacteria (epidermal or invasive), neutrophil infiltration and inflammation. we also preserved samples which were then used in this study. we determined levels of transcripts for several immune and stress response genes (table 1) in lungs from infected and control bats. only those qrt-pcr reactions that yielded unambiguous results were considered. there were significant differences for transcripts for the interleukins (il) 10 and 23, the pro-inflammatory cytokine tumour necrosis factor alpha (tnfa), and the anti microbial and immunoregulatory peptide cathelicidin (figure 1 ). in figure 1a lower dct of one indicates approximately a two-fold higher concentration of rna. infected bats as a group had lower dct values (on an average 8 to 14 fold higher concentrations of rna, histograms in figure 1 ) for the four genes. while the group of infected hibernating bats had higher levels of transcripts for il10, il23, cathelicidin and tnfa than hibernating uninfected bats, the levels varied over a wide range with some infected bats having levels comparable to (or lower than) those of uninfected animals. since the level of fungal infection and pathology varied considerably among infected bats, we attempted to correlate transcript levels with scores for fungal infection, fungal hyphae and bacterial infection in wings and neutrophil accumumyotis homologues for innate response genes were identified by scanning the myotis genome for sequences that matched those of well-characterized reference organisms. where qrt-pcr primers for reference organisms d had been validated in the literature b,c , we used the corresponding myotis sequences as primers. myotisspecific substitutions in these primers are in bold a . for other genes (hsp70, tnfa, nos2, il-10, grp78) myotis-specific primers were designed from potential myotis exon sequences using parameters for optimal use in pcr reactions. in all cases the identity of pcr products were confirmed by sequencing. primers were used in qrt-pcr at a final concentration of 1 mm. doi:10.1371/journal.pone.0112285.t001 lation in lungs (table 2 ). il23 and cathlicidin levels correlated with all four parameters of disease and inflammation. il10 correlated with level of infection, and hyphae and bacterial scores in the wing, and tnfa correlated with level of infection and wing hyphae score. there was an inverse relationship between transcript levels and dct. the correlation coefficients are therefore negative. our results suggest that hibernating m. lucifugus are capable of inducing the expression of genes for cytokines in response to infection. the bats we examined were kept under conditions that closely mimicked temperature and humidity encountered in their natural hibernacula (i.e., 7uc and .97% relative humidity) [24, 25] . during hibernation m. lucifugus body temperature is maintained within 1-2uc of ambient temperature for weeks at a time with return to normothermic temperatures during periodic arousal [24, 25] . it is difficult to imagine mammalian gene expression at temperatures this low and it is possible that the genes were transcribed during the arousal periods. our previous study indicated that p. destructans-infected bats aroused more frequently than uninfected bats [25] , a pattern which has also been observed in the wild [26] and this may also have contributed to higher basal levels of gene expression in their cells. however, our observation that the levels for il 23, cathelicidin, il 10 and tnfa and not the other transcripts examined (dectin 1, defensin b1, hepcidin, il 17, cytchrome b, heat shock protein 70, nitrogen oxide synthetase) were higher in the lungs of infected bats, suggest that the response was a specific to infection. we observed mild to moderate levels of interstitial neutrophils in the lung of infected bats. on average, the lungs contained 8 to 20 times more transcripts for the anti-microbial peptide cathelicidin as well as for the immune modulators tnfa and il 10 and 23. the increase in levels of il 23, cathelicidin and il10 correlated with the lung interstitial scores ( table 2) . little is known about the role in bats of these bioactive proteins, information from other species indicates that they are intimately involved in the response to infection. for instance: cathelicidins belong to a family of antimicrobial peptides with an intrinsic ability to kill bacteria and fungi but they also function as chemoattractants for inflammatory cell recruitment and cytokine release. they are found in the lysosomes of neutrophils, macrophages and epithelial cells after activation or infection with a wide variety of pathogens [29] . humans, mice and dogs have a single gene for cathelicidin while several related genes have been discovered in pigs, horses, cattle and chickens. m. lucifugus has at least two genes with similarity to the human gene. our primers were designed to amplify transcripts for the gene most similar to that for human cathelicidin. interleukin 23 is a multifunctional pro-inflammatory cytokine involved in both innate and adaptive responses [30] . it is an important mediator of dectin-dependent response of the protective neutrophils to lung infections by aspergillus [31] . it is also important in the maturation of t helper cells. in addition to being protective il23 may also play a negative role by inducing chronic inflammation and exacerbating the effects of aspergillus and candida infections [32] . interleukin 10 is also known as cytokine synthesis inhibitory factor (csif) because of its role in modulating the inflammatory response. its effect is to suppress th1 responses and stimulate th2 responses leading to b-cell survival and proliferation and antibody synthesis. il10 is produced by a number of cell types including monocytes, and activated subsets of regulatory and helper t-cells b-cells. its synthesis is tightly regulated and is mediated by the nfkb and ap1 pathways after stimulation by commensal and pathogenic microorganisms [33] . if the role of il10 in wns is indeed to direct the immune system to a th2 response, our results are in contrast with those of moore et. al. [34] who found increased levels of il4 in the serum of wns-affected bats. work in other species suggests that il4 directs the immune system to a th1 response. tumour necrosis factor alpha (tnfa) is a monocyte derived cytotoxin implicated in tumour regression, septic shock and cachexia. tnfa binding to its receptor stimulates the activation of the transcription factor, nfkb, which activates gene expression required for several defensive responses [35] . the presence of neutrophilia in the lung interstitia of infected bats, despite a lack of obvious pathology or the presence of microorganisms, is puzzling. however, these observations may be explained by: a) injury in the skin can lead to complement-induced tnfa expression in the lungs followed by inflammation and neutrophil infiltration [27] . b) immune responses at mucosal surfaces are coordinated, with stimulation at one surface leading to responses at other mucosae [36] . the pulmonary response may have been due to stimulation of the upper respiratory tract by fungal spores. c) there have been suggestions that hibernating mammals show marked leukopenia [20] raising the possibility that circulating leukocytes are sequestered in some organ, possibly the lungs. the infected animals may have responded to inflammation in the wings with an increase in neutrophils, which then migrated to the lungs. d) lung neutrophils in infected bats, and the accompanying increase in the transcripts of the immune modulators may represent the end of the infectious process in the lungs and the removal of fungal spores and hyphae. this suggests that bats are capable of suppressing a lung or systemic infection by p. destructans and may explain why wns, the disease, occurs only after bat-to-bat contact rather than by infection through inhalation. while relatively little is known about the response of bats to infections, work with other species indicates that the host's response to pathogens is complex involving the interconnected innate, relatively non-specific mechanisms as well as the more specific adaptive systems of humoral and cellular immunity. the increased transcripts we detected in infected bats represent but a few components of this complex network. while our study shows that hibernating bats can respond to an infection, it does have some limitations and our results should be interpreted with caution. for instance, we have used increase in the stable levels of transcripts for various cytokine genes as a surrogate for increases in their respective proteins. however mrna and protein levels do not always correlate, largely because of complex regulatory processes that separate transcription of mrnas from their translation into protein [37] [38] [39] . in addition, only one of the transcripts, for cathelicidin, is directly linked to an anti-fungal response. the remainder, il 23, il 10 and tnfa may be indirectly related or may represent a non-specific response to the stress of infection. the importance of temporal heterothermy in bats bat ecology bats and bacterial pathogens: a review hemotropic mycoplasmas in little brown bats (myotis lucifugus) bats as reservoir hosts of human bacterial pathogen, bartonella mayotimonensis bats and viruses: friend or foe? bats: important reservoir hosts of emerging viruses isolation of nipah virus from malaysian island flying-foxes virology. what links bats to emerging infectious diseases? isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus fruit bats as reservoirs of ebola virus middle east respiratory syndrome coronavirus in bats, saudi arabia bat flight and zoonotic viruses isolation of genetically diverse marburg viruses from egyptian fruit bats bats host major mammalian paramyxoviruses evolutionary insights into the ecology of coronaviruses a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? antiviral immune responses of bats: a review hibernation: the immune system at rest? bat white-nose syndrome: an emerging fungal pathogen an emerging disease causes regional population collapse of a common north american bat species experimental infection of bats with geomyces destructans causes white-nose syndrome pathophysiology of white-nose syndrome in bats: a mechanistic model linking wing damage to mortality inoculation of bats with european geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome chemotactic mediator requirements in lung injury following skin burns in rats lung injury secondary to chemotactic factor-induced leukocyte activation innate immune defense system of the skin the il23/th17 pathway as a therapeutic target in chronic inflammatory diseases neutrophils produce interleukin 17a (il-17a) in a dectin-1-and il-23-dependent manner during invasive fungal infection th17 cells in the setting of aspergillus infection and pathology the regulation of il-10 production by immune cells hibernating little brown myotis (myotis lucifugus) show variable immunological responses to white-nose syndrome molecular basis of nf-kappab signaling role of inducible bronchus associated lymphoid tissue (ibalt) in respiratory immunity aebersold r (1999) correlation between protein and mrna abundance in yeast correlation of mrna and protein in complex biological samples insights into the regulation of protein abundance from proteomic and transcriptomic analyses regulation of cathelicidin antimicrobial peptide expression by an endoplasmic reticulum (er) stress signaling, vitamin d receptor-independent pathway dectin-1 expression at early period of aspergillus fumigatus infection in rat's corneal epithelium expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection the small molecule, genistein, increases hepcidin expression in human hepatocytes expression of interleukin-17 associated with disease progression and liver fibrosis with hepatitis b virus infection: il-17 in hbv infection the immunomodulatory effect of bone marrow stromal cells (bmscs) on interleukin (il)-23/il-17-mediated ischemic stroke in mice discovery of herpesviruses in bats key: cord-019977-kj0eaw6v authors: nan title: neonatal bacterial infection: a changing scene? date: 2005-04-14 journal: j infect doi: 10.1016/s0163-4453(82)91569-9 sha: doc_id: 19977 cord_uid: kj0eaw6v nan the foetus and newborn infant have a particular vulnerability to infection of all kinds, yet most escape this hazard. immune mechanisms are developing at each stage of existence which are appropriate to the likely challenges at such times; 1 the unusual challenge however may not be met, for the immune and inflammatory responses produced by healthy older children and adults cannot be mounted as effectively, particularly if the infant is born pre-term. 2 the risk therefore is ever present, and-at least where neonatal bacterial infection is concerned -its nature may be ever changing. the reasons for such change are sometimes obvious, sometimes obscure, and have to be sought in the maternal and infant hosts, in improved laboratory techniques, and in altering clinical practices which may impose change on host and infecting organism alike. records have been kept over a 5o-year period (i928-i978) at yale of the isolates recovered from the bloodstream of infants in the first weeks of lifef1-6 and accord well with those reported intermittently from other centres. in addition to showing changes in the infecting organisms, they also reveal the growing importance of maternally transmitted intrapartum (early) infections over later infections in recent years, many of the latter being environmentally acquired. in the first quarter of the 5o-year period, two-thirds of the isolates were gram-positive, the majority staphylococci (mostly staphylococcus aureus) and beta-haemolytic streptococci. the introduction of lancefield typing in the early i93os showed these to be mainly group a streptococci. over the next 20 years the position was to be gradually reversed and between i958 and i9655 two-thirds of yale's newborn bacterial isolates were gram-negative. this time period coincided with the entry of paediatricians to newborn nurseries on a much larger scale than hitherto, with an increase in the use of antimicrobial drugs, and with the introduction of apparatus such as incubators, resuscitation and suction units, the humidification parts of which often harboured gramnegative organisms, all capable of causing lethal disease in the infant. the last period (i966-i978) 6 has shown a decrease in gram-negative isolates, and a rise in gram-positive ones, the latter largely due to greatly increased isolation of group b streptococcus. during the half century under review, mortality from neonatal bacteraemia fell from 9o per cent in the period i928-i933, ~ to z6 per cent in i966-i978 ;6 and the proportion of isolates recorded as recovered in the first 48 hours of life (early infections) increased from io per cent to 57 per cent of the total respectively. we can only guess whether this last change was due to a growing awareness on the part of paediatricians of the possibility of maternally transmitted illness, or to a genuine increase of such infection. as the number of blood cultures drawn increased considerably over the years, the former may be more likely. on the other hand an increasing vogue for surgical induction of labour, and the use of pressure transducers for foetal monitoringfl if practised there may have introduced the organism into the amniotic fluid more frequently in recent years. a report from hammersmith hospital 8 suggests a further change may be under way in newborn nurseries, as the io6 editorial predominant isolate from cases of neonatal bacteraemia there since 1976 has been staphylococcus epidermidis. this organism has ousted the group b streptococcus from its prominent place in early infections at the hospital in previous yearsfl and has also displaced gram-negative bacilli as the most important cause of later infection. elsewhere in this issue (p. ii7) oto discusses some of the reasons-the increasing preoccupation of neonatal intensive care units with infants of very low birth weight and the increasing complexity of their care, involving much more intravenous therapy -which may be responsible for this change. twenty years ago the great majority of such infants would have died within 24 hours of birth, and it is likely that intrapartum infection, even as a contributory cause of their death, may have gone unsuspected and undiagnosed. today they live, and their immaturity makes them at risk throughout the many weeks of their hospital stay. oto also records the major infections found in such a unit over a six month period, and bacteraemia is far the commonest. much less often seen, but next in importance, is necrotising enterocolitis. this is a condition which is known to have occurred for over one hundred years, but which reached almost epidemic proportions in certain newborn intensive care units in the late 196os and i97os. 1° bacteria are responsible for the progression and complications of the illness, but whether they have a primary role, or merely invade ischaemic bowel wall which has been damaged by a variety of other factors, is still unsure. the condition has certainly accompanied genuine bowel pathogens (bacterial n and viral12), other cases often occur in clusters, tm may be curtailed by infection control measures, lz and clostridium spp. have occasionally been implicated. 14, 15 some believe it to be part of a spectrum of disease which includes pseudomembranous or antibiotic-associated colitis. 16 clostridium difficile and its toxin may be present in the stools of many well newborn babies, but this organism has not been implicated in the genesis of neonatal necrotising enterocolitis as it has been with pseudomembranous colitis. 17 meningitis is now fortunately rare (o.26/iooo live births in a geographically defined population, excluding that associated with neural tube defects18), but there is still controversy about treatment of the most commonly occurring gram-negative enteric infections, despite controlled trials. omphalitis, the bacteriology of which is described by brook in this issue (p. i27), is now very infrequently seen. many newborn nurseries use some form of antiseptic cord care; and a daily antibiotic spray containing polymyxin, bacitracin and neomycin, prevents bacterial colonisation, 19 and hence infection, at this site in the great majority of babies. improved laboratory techniques for the recognition of organisms such as clamydia trachomatis are leading to a gradually increasing awareness of the extent of its involvement in neonatal conjunctivitis and respiratory illness, s° bacterial infections can be permanently damaging to developing and rapidly growing organs; and constant surveillance of the patterns of infection is nowhere more important than in newborn care. preventive measures such as handwashing and disinfection of apparatus have to be carried out to a uniformly high standard in intensive care units. this is especially important if outbreaks of infection are to be prevented among the immature residents, for the inevitable overuse of antimicrobial therapy in such units leads to a disturbance of bacterial flora which may make the hosts even more unusually vulnerable. the development of the immune response. characterization of the response of the human infant and adult to immunization with salmonella vaccines host defences in the human neonate septicemia in the new-born. am`7 dis child septicemia of the newborn septicemia of the newborn a half century of neonatal sepsis at yale. 1928 to i978 nasal colonization of infants with group b streptococcus associated with intrauterine pressure transducers changing blood culture isolates in a referral neonatal intensive care unit early neonatal bacteraemia. comparison of group b streptococcal, other gram-positive and gram-negative infections neonatal necrotizing enterocolitis. monographs in neonatology. new york: grune and stratton gastroenteritis with necrotizing enterocolitis in premature babies association of coronavirus infection with neonatal necrotizing enterocolitis clustering of necrotizing enterocolitis. interruption by infection-control measures outbreak of necrotising enterocolitis caused by clostridium butyricum fulminant necrotising enterocolitis associated with clostridia pseudomembranous enterocolitis (antibiotic-related colitis) clostridium difficile and the aetiology of pseudomembranous colitis acute bacterial meningitis in childhood. incidence and mortality in a defined population an investigation into the aerobic and anaerobic bacterial flora of normal and ill~low birthweight newborn babies prospective study of chlamydial infection in neonates key: cord-003099-a0acr28o authors: koch, r. m.; diavatopoulos, d. a.; ferwerda, g.; pickkers, p.; de jonge, m. i.; kox, m. title: the endotoxin-induced pulmonary inflammatory response is enhanced during the acute phase of influenza infection date: 2018-07-05 journal: intensive care med exp doi: 10.1186/s40635-018-0182-5 sha: doc_id: 3099 cord_uid: a0acr28o background: influenza infections are often complicated by secondary infections, which are associated with high morbidity and mortality, suggesting that influenza profoundly influences the immune response towards a subsequent pathogenic challenge. however, data on the immunological interplay between influenza and secondary infections are equivocal, with some studies reporting influenza-induced augmentation of the immune response, whereas others demonstrate that influenza suppresses the immune response towards a subsequent challenge. these contrasting results may be due to the use of various types of live bacteria as secondary challenges, which impedes clear interpretation of causal relations, and to differences in timing of the secondary challenge relative to influenza infection. herein, we investigated whether influenza infection results in an enhanced or suppressed innate immune response upon a secondary challenge with bacterial lipopolysaccharide (lps) in either the acute or the recovery phase of infection. methods: male c57bl/6j mice were intranasally inoculated with 5 × 10(3) pfu influenza virus (ph1n1, strain a/netherlands/602/2009) or mock treated. after 4 (acute phase) or 10 (recovery phase) days, 5 mg/kg lps or saline was administered intravenously, and mice were sacrificed 90 min later. cytokine levels in plasma and lung tissue, and lung myeloperoxidase (mpo) content were determined. results: lps administration 4 days after influenza infection resulted in a synergistic increase in tnf-α, il-1β, and il-6 concentrations in lung tissue, but not in plasma. this effect was also observed 10 days after influenza infection, albeit to a lesser extent. lps-induced plasma levels of the anti-inflammatory cytokine il-10 were enhanced 4 days after influenza infection, whereas a trend towards increased pulmonary il-10 concentrations was found. lps-induced increases in pulmonary mpo content tended to be enhanced as well, but only at 4 days post-infection. conclusions: an lps challenge in the acute phase of influenza infection results in an enhanced pulmonary pro-inflammatory innate immune response. these data increase our insight on influenza-bacterial interplay. combing data of the present study with previous findings, it appears that this enhanced response is not beneficial in terms of protection against secondary infections, but rather damaging by increasing immunopathology. patients with influenza infection often suffer from severe secondary bacterial infections, which are associated with high morbidity and mortality rates [1, 2] . a striking example of this relationship was provided by bacteriological and histopathological analysis of infected lung tissue obtained from people who died of influenza during the 1918-1919 "spanish flu" pandemic, in whom bacterial pneumonia was found to be the predominant cause of death [1] . these data suggest that an influenza infection profoundly influences the immune response upon a secondary bacterial infection. several studies have evaluated immunological interactions between influenza and bacterial infections, including infections with gram-negative bacteria [3] . in vitro studies in which influenza-infected alveolar macrophages were subsequently stimulated with bacterial lipopolysaccharide (lps), a bacterial compound that induces a profound innate immune response, revealed increased levels of pro-inflammatory cytokines tumor necrosis factor (tnf) α, interleukin (il)-1β, and il-6 [4] [5] [6] [7] [8] , indicative of a priming effect on these cells by influenza. data from in vivo animal studies are ambiguous. similar to the in vitro data, some report enhanced responses. for instance, influenza infection in mice was shown to enhance the inflammatory response and neuropathogenicity resulting from lps administration on days 3 and 4 after influenza inoculation [9] . likewise, murine influenza infection resulted in increased levels of pro-inflammatory cytokines in both plasma and lungs, and enhanced pulmonary neutrophil influx upon pneumococcal infection 7 days later [10] . similar results were observed in mice 14 days after influenza infection [11] . however, two otherwise largely comparable studies demonstrated reduced pulmonary pro-inflammatory cytokine concentrations upon streptococcus pneumoniae and staphylococcus aureus infections in mice infected with influenza 7 days before, indicative of influenza-induced immunosuppression [12, 13] . these equivocal results may be due to differences in the severity or kinetics of the influenza infection or the use of different bacteria as secondary challenges, thereby targeting various complex multi-receptor signaling pathways. also, the use of live bacteria could have contributed to these ambiguous results. for instance, if influenza would induce immunosuppression and thereby facilitate outgrowth of bacteria upon a secondary live infectious challenge, the increased bacterial burden can eventually result in fulminant inflammation, which would wrongfully suggest influenza-induced augmentation of the immune response. in the present study, we investigated whether influenza infection results in an enhanced or suppressed innate immune response upon a secondary challenge with lps. furthermore, we assessed the kinetics of these influenza-induced effects by performing the lps challenges in either the acute or the recovery phase of influenza infection. all procedures described were in accordance with the requirements of the dutch experiments on animals act, the ec directive 86/609, and approved by the animal ethics committee of the radboud university nijmegen medical center (ru-dec 2013-029). forty-eight male c57bl/6j mice (charles river, sutzfield, germany) aged 10-12 weeks and weighing 23-29 g were used. mice were housed in individually ventilated cages, with five mice per cage at the central animal facility of the radboud university. at day 0, six groups of eight mice (total n = 48) were anesthetized by isoflurane and intranasally inoculated with a sublethal dose of influenza virus (ph1n1, strain a/ netherlands/602/2009, 5 × 10 3 pfu) or mock treated (nacl 0.9%) in a volume of 50 μl. following infection, all mice were monitored and weighed daily. the temperature was recorded with an infrared thermometer on the skin, and physical condition was scored using a scoring and weight sheet (weight, body temperature, ruffled coat, hunched back, reduced mobility, and moribund). at either day 4 (acute phase) or day 10 (recovery phase), mice were placed in a temperature-controlled chamber to receive lps (e coli, serotype 0111:b4, 5 mg/kg) or nacl 0.9% by intravenous injection in the tail vein. ninety minutes after lps or nacl administration, mice were deeply anesthetized with isoflurane and exsanguinated through orbital extraction, followed by cervical dislocation after which organs were collected. ethylenediaminetetraacetic acid (edta)-anticoagulated blood was centrifuged at 13000×g for 2 min at room temperature after which plasma was stored at − 80°c until analysis. subsequently, perfusion of the lungs was performed by intracardiac injection with phosphate-buffered saline (pbs), after which lung lobes were harvested and snap frozen in liquid nitrogen and stored at − 80°c until homogenization. lung tissue was placed in 1 ml lysis buffer containing pbs, 0.5% triton x-100, and a protease inhibitor cocktail (complete edta-free tablets, roche, woerden, the netherlands, 1 tablet per 50 ml lysis buffer). subsequently, lung lobes were homogenized at 50 hz, using a polytron homogenizer, and subjected to two rapid freeze-thaw cycles using liquid nitrogen. finally, homogenates were centrifuged (10 min, 14,000×g, 4°c), and the supernatant was stored at − 80°c until cytokine analysis. concentrations of tnf-α, il-1β, il-6, and il-10 in plasma and lung homogenates were measured using a luminex assay (milliplex, millipore, billerica, ma) according to the manufacturer's instructions. the lower detection limit of the assay was 32 pg/ml for all cytokines. plasma il-1β levels were below the detection limit in the majority of animals. lung homogenate cytokine concentrations were normalized to total protein content determined by bicinchoninic acid assay (bca protein assay; thermo fisher scientific). myeloperoxidase (mpo) content was measured in lung homogenates using an enzyme-linked immunosorbent assay (hycult biotech, uden, the netherlands) according to the manufacturer's instructions. concentrations were normalized to total protein content as described above. all data were normally distributed according to the shapiro-wilk test. the grubbs test (extreme studentized deviate method) was used to exclude significant outliers from analysis (maximum of one exclusion per dataset). to determine the number of animals required per group, we performed a power calculation based on a minimal detectable difference of 50% in lps-induced plasma tnf-α levels between influenza-infected and non-influenza-infected mice. mean ± sd (533 ± 163 pg/ml) tnf-α plasma levels were obtained from previous work from our group, in which male c57bl/6j mice were also injected intravenously with 5 mg/kg lps and sacrificed 90 min later [14] . using a two-sided α of 0.05 and a power of 80% (β of 0.2) in an unpaired t test design, six animals per group were required. to account for potential loss of animals due to influenza infection, eight animals per group were used. the effect size was based on previous work [9] , in which influenza infection modulated the plasma cytokine response to lps administration by at least 50%. comparisons were analyzed using unpaired student's t tests and repeated measures one-way analysis of variance (anova) as indicated in the figure legends. statistical analyses were performed in graphpad prism 5.03 for windows (graphpad software, san diego, ca). two-tailed p values < 0.05 were considered statistically significant. all influenza-inoculated mice showed clinical signs of infection, including weight loss, lethargy, and pyrexia. four influenza-infected mice were prematurely taken out of the experiment because of signs of severe infection. body weight decreased in all influenza-infected mice in the acute phase of infection, whereas it remained stable in mock-inoculated mice (fig. 1) . from day 7 onwards, body weight started to increase, marking the recovery phase of influenza infection (fig. 1) . influenza infection by itself did not result in increased plasma levels of any of the cytokines measured at both 4 and 10 days post-infection (fig. 2) . expectedly, lps fig. 1 body weight of influenza-or mock-inoculated mice. data are presented as mean with sem. dagger indicates the two time points at which mice in the respective groups were sacrificed administration led to profoundly increased plasma concentrations of tnf-α, il-6, and il-10. although tnf-α and il-6 plasma levels appeared to be somewhat higher in mice challenged with lps 4 days after influenza infection compared with mock-inoculated mice, this did not reach statistical significance (p = 0.12 and p = 0.86, respectively). plasma concentrations of the anti-inflammatory cytokine il-10 were however significantly enhanced in mice challenged with lps 4 days after influenza infection. no differences in any of the plasma cytokine levels were measured between influenza-infected and mock-inoculated mice at 10 days. in lung homogenates, influenza by itself caused mildly elevated levels of tnf-α, il-1β, il-6, and il-10 at 4 days post-infection and to a lesser extent at 10 days after infection (fig. 3) . similar to what was found in plasma, lps challenge also led to increased concentrations of all measured cytokines in lung tissue. a synergistic increase of all pro-inflammatory cytokines in the lungs was found in influenza-infected mice challenged with lps 4 days later and, to a lesser extent, in mice challenged with lps 10 days post-influenza infection. for il-10, the potentiating effect was additive rather fig. 2 plasma levels of tnf-α, il-6, and il-10 in mice that received influenza/mock followed by lps/nacl 4 or 10 days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < 0.05, **p < 0.01, ***p < 0.001 (calculated by unpaired student's t tests) fig. 3 levels of tnf-α, il-1β, il-6, and il-10 in lung homogenates of mice that received influenza/mock followed by lps/nacl 4 or 10 days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < 0.05, **p < 0.01, ***p < 0.001, # p = 0.05-0.10 (calculated by unpaired student's t tests) than synergistic, only observed at 4 days post-influenza infection, and reached a trend towards statistical significance. in accordance with pulmonary cytokine levels, influenza infection by itself led to increased mpo content in the lungs 4 days after infection and tended to result in increased mpo content 10 days post-infection (fig. 4) . again, lps administration also resulted in increased mpo levels in lung tissue, and there was a trend towards enhanced mpo content in influenza-infected mice challenged with lps 4 days after infection. in the present study, we demonstrate that a systemic lps challenge in the acute phase of influenza infection (4 days post-infection) results in an enhanced pulmonary, but not systemic pro-inflammatory cytokine response. this effect was synergistic rather than additive, indicating that influenza infection actually modulates the immune response to a subsequent challenge with lps. furthermore, this effect remained present, although less pronounced, in the recovery phase of influenza infection (10 days post-infection). the lps-induced increase in mpo content in lung homogenates, reflecting pulmonary neutrophil influx or sequestration, tended to be enhanced in the acute phase of influenza infection as well. these results suggest that influenza infection, especially in the acute phase, may cause a more pronounced pulmonary pro-inflammatory immune response upon a secondary bacterial infection. our results are in accordance with in vitro data reporting a cellular priming effect of influenza observed upon secondary stimulation with lps [4] [5] [6] [7] [8] , as well as with other murine in vivo studies that report increased inflammation and pulmonary neutrophil influx or sequestration upon a secondary bacterial infection or lps challenge in the acute phase of influenza infection [9, 10] . for example, a preceding influenza infection in mice gravely enhanced lung injury induced by a secondary infection with streptococcus pneumoniae 7 days later, resulting in a severe necrotic pneumonia accompanied by increased mortality [10] . also, fig. 4 mpo content in lung homogenates of mice that received mock/influenza followed by nacl/lps 4 or 10 days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < 0.05, **p < 0.01, ***p < 0.001, # p = 0.05-0.10 (calculated by unpaired student's t tests) the increased mpo content observed in our study is an important hallmark of acute respiratory distress syndrome (ards) [15, 16] , a severe complication of influenza infection caused by excessive pulmonary inflammation. these and our study reveal that the enhancing effect on the pro-inflammatory innate immune response is most evident in the lungs, probably because the influenza-induced damage and consequent inflammatory effects are most pronounced at this site. in this context, our data are in line with the recommendation to use corticosteroids in patients with severe influenza infections in the intensive care unit to counteract the pulmonary hyperinflammatory response causing ards. several underlying mechanisms may contribute to the observed effects. at the cellular level, studies have shown that influenza and certain bacterial pathogens, such as haemophilus influenzae and streptococcus pneumoniae, utilize similar immunological pathways and that the overlap in the inflammatory mediators produced thereby creates augmentation of the immune response during sequential infection, in turn causing immunopathology [17, 18] . furthermore, it has been hypothesized that influenza stimulates tnf-α gene transcription activators or may interfere with labile transcription repressor proteins and stabilizes tnf-α mrna by delaying its degradation [8] . alternatively, the increased lung mpo levels observed do not necessarily reflect pmn infiltration into the lungs, but may (also) result from pmns trapped in the vasculature, as circulating activated neutrophils become rigid and can be trapped within the small capillaries of the lung [19] . as such, increased entrapment of leukocytes in the pulmonary vasculature during influenza infection could also contribute to the enhanced inflammatory cytokine levels upon lps challenge. we can only speculate on this, because no histological data are available, which represents a limitation of this work. it may be argued that the enhanced pro-inflammatory immune response induced by influenza serves as a means to efficiently eliminate the primary pathogen and to enhance host defense towards a secondary infection. for instance, pro-inflammatory cytokines are induced in influenza-infected cells to limit viral replication and to initiate downstream immune responses [20] . however, this is not supported by previous work, where an increased bacterial burden was observed irrespective of an enhanced or suppressed response [11] [12] [13] . several explanations for this observation may be put forward. first, next to potentiating pro-inflammatory cytokine responses, the present study and work by others [11] have shown that influenza infection also potentiates production of the key anti-inflammatory cytokine il-10, which was demonstrated to be crucial in facilitating bacterial outgrowth upon secondary challenge with streptococcus pneumoniae [11] . second, influenza may on the one hand prime for production of innate cytokines produced by myeloid cells, but impair t cell-derived cytokines that are instrumental for the adaptive immune response. this was elegantly demonstrated by kudva et al., who showed that, in line with our results, infection with staphylococcus aureus 6 days after influenza resulted in increased pulmonary levels of innate cytokines such as il-6 and mcp-1, and increased neutrophil influx to the lungs, but decreased concentrations of t-cell-derived il-17 and il-22, which were demonstrated to play a pivotal role in fending off the staphylococcal infection [21] . whereas the enhancing effects of influenza on pro-inflammatory innate immune parameters were less pronounced at 10 days post-infection, a suppressed response was neither evident. this could be partly biased by the exclusion of two mice in both recovery groups due to severe influenza infection. however, it might also be argued that 10 days post-infection is too soon for these effects to manifest. for example, profound desensitization towards lps and flagellin, another toll-like receptor (tlr) ligand, was observed in alveolar macrophages obtained from mice up to 6 weeks after influenza infection [22] . furthermore, the direction of the response upon a secondary challenge is probably highly dependent on the pathogen or stimulus used, each using distinct intracellular signaling pathways. with regard to this, it is well-known that influenza virus particularly predisposes to aspergillus fumigatus, which is present in 25% of all influenza patients [23, 24] , causing infections such as invasive pulmonary aspergillosis that are associated with very high mortality rates. as different mechanisms may be important in host defense towards various pathogens, the specific response towards aspergillus fumigatus could be suppressed by a preceding influenza infection. the use of corticosteroids may be another important factor in the observed vulnerability towards particular secondary infections, as steroid use was shown to be independently associated with the presence of aspergillus fumigatus in sputum of cystic fibrosis patients [25] and with a substantially increased risk of community-acquired staphylococcus aureus bacteremia [26] . furthermore, a meta-analysis revealed that the use of corticosteroids was significantly associated with nosocomial infections [27] . to the best of our knowledge, these putative detrimental effects of corticosteroid treatment during influenza on secondary infections have yet to be studied systematically in animal models. in any case, it remains to be determined whether the overall effects of corticosteroid treatment are beneficial or not, as they may lead to increased susceptibility in a subset of influenza virus-infected patients but may also provide health benefits in another subset of influenza virus-infected patients. an lps challenge in the acute phase of influenza infection results in an enhanced pulmonary pro-inflammatory innate immune response. these data increases our insight concerning viral-bacterial interplay. combined with previous findings, it appears that this enhanced pro-inflammatory response does not lead to protection against secondary infections but rather causes immunopathology leading to damage, and thereby to organ failure. predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness secondary bacterial infections associated with influenza pandemics 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vascular inflammation new fronts emerge in the influenza cytokine storm influenza a inhibits th17-mediated host defense against bacterial pneumonia in mice sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory influenza infection invasive pulmonary aspergillosis is a frequent complication of critically ill h1n1 patients: a retrospective study influenzaassociated aspergillosis in critically ill patients inhaled corticosteroids and aspergillus fumigatus isolation in cystic fibrosis use of glucocorticoids and risk of community-acquired staphylococcus aureus bacteremia. a population-based case-control study mayo corticosteroids for the treatment of human infection with influenza virus: a systematic review and meta-analysis. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases the authors thank fred van opzeeland, elles simonetti, francine van der poll, ilona van de brink, and jelle gerretsen for their help with the mouse experiments and laboratory analyses. this work was supported by an efro (dutch: "europees fonds voor regionale ontwikkeling," english: "european regional development fund") grant (2011-013287). efro had no role in the design of the study; in the collection, analysis, and interpretation of data; and in writing the manuscript. data sharing is not applicable to this article as no reusable datasets were generated or analyzed during the current study.authors' contributions rk designed and conducted the study, analyzed and interpreted the data, and drafted the manuscript. dd and gf aided in the study design and conduct, interpreted the data, and critically revised the manuscript. pp and mdj supervised the study, interpreted the data, and critically revised the manuscript. mk designed and supervised the study, interpreted the data, and critically revised the manuscript. all authors read and approved the final manuscript. all procedures described were in accordance with the requirements of the dutch experiments on animals act, the ec directive 86/609, and approved by the animal ethics committee of the radboud university nijmegen medical center (ru-dec 2013-029). not applicable. the authors declare that they have no competing interests. key: cord-018907-c84t1bo5 authors: bin-hussain, ibrahim title: infections in the immunocompromised host date: 2012 journal: textbook of clinical pediatrics doi: 10.1007/978-3-642-02202-9_68 sha: doc_id: 18907 cord_uid: c84t1bo5 nan infections are considered a major cause of morbidity and mortality in immunocompromised children. the survival rate in this particular population has increased over the last 3 decades. this is mainly due to the advancement in medical technology leading to improvement in diagnosis capabilities as well as supportive care including antimicrobial therapy. immunodeficiency can be divided into primary and secondary immunodeficiency disorders. primary immunodeficiency disorders including combined t-cell and b-cell immunodeficiencies, antibody deficiency, disease of immune dysregulation, congenital defects of phagocyte number or function or both, defects in innate immunity, autoimmunity disorders, complement deficiencies, and cytokine defects. secondary immunodeficiency disorders include human immunodeficiency virus (hiv) and acquired immune deficiency syndrome (aids) -both of which lead to altered cellular immunity -dysgammaglobulinemia, defective phagocytic function or neutropenia. cancer leading to neutropenia, lymphopenia, humoral deficiencies and altered physical integrity especially with the use of chemotherapeutic agents leading to disruption barrier integrity with mucositis leading to easy access of microorganisms, solid organ transplant leading to deficiencies in cellular and phagocytic immunity, malnutrition which leads to impaired immunity, and complement activity. fever is the main manifestation and occasionally the only sign of infection in immunocompromised children. when approaching a patient with immunodeficiency in the context of infection, one needs to look at the net state of immunosuppression. the net state of immunosuppression can be evaluated by the host defense defects caused by the primary disease, dose and duration of the immunosuppressive therapy (the longer duration of immunosuppressive therapy, the higher risk of infection), presence of neutropenia, and anatomical and functional integrity because defect in the skin or mucosa can lead to easy access for the microorganisms, metabolic factors, and infection with immunomodulating viruses (hiv, hbv, hcv, cmv, ebv, and hhv-6). risk of infections can be classified as high, intermediate, and low. high risk includes hematologic malignancies, aids, hsct, splenectomized patient, and congenital immunodeficiency especially severe combined immune deficiency (scid). intermediate risk includes solid tumors, hiv/aids, and solid organ transplantation. low-risk patients include patients with corticosteroid therapy, local defects, and diabetes. the pathogens in immunocompromised patients can be predicted based on the immune defect. for example, if there is an anatomical disruption in the oral cavity it lead to infections caused by alpha hemolytic streptococci, anaerobes, candida species, and herpes simplex virus (hsv). patients with urinary catheters will be at risk for infection caused by gram negative bacteria including pseudomonas spp., enterococci, and possibly candida. if there is a skin defect including central venous catheter (cvc), the patient will be at risk of staphylococcus species (both coagulase-negative staphylococci and staphylococcus aureus, bacillus species, atypical mycobacterium, and gram-negative organism. if a defect in the phagocytic function, either quantitative or qualitative, predispose what to invasive diseases like invasive pneumonia caused by bacterial pathogens: gram-positive (staphylococci, streptococci, and nocardia species) and gram-negative bacilli (escherichia coli, klebsiella pneumoniae, p. aeruginosa), other enterobacteriaceae, and fungal pathogens like candida species and aspergillus species. patients with defective cell-mediated immunity are at risk of infections caused by intracellular pathogens (i.e., viral, fungi, mycobacterial, and intracellular bacteria). intracellular pathogens include legionella species, salmonella species, mycobacteria, and listeria species, histoplasma capsulatum, coccidioides immitis, cryptococcus neoformens, candida species, pneumocystis jiroveci, cytomegalovirus, varicella-zoster virus, epstein-barr virus, live viral vaccines (measles, mumps, rubella, and polio) and protozoal, toxoplasma gondii, strongyloides stercoralis, cryptosporidia, microsporidia, and isospora species. patients with immunoglobulin deficiencyo are at risk of sinupulmonary infection caused by s. pneumoniae, haemophilus influenzae, and cns infection from viral infections, especially enterovirus, leading to chronic meningoencephalitis as well as gastrointestinal infection due to giardiasis. patients with complement deficiency are at risk of diseases caused by s. pneumoniae, h. influenzae, and neisseria species. splenectomized patients are at risk of invasive diseases (e.g., sepsis, meningitis) caused by encapsulated organism including s. pneumoniae, h. influenzae, and neisseria meningitidis. in evaluating patients with immunodeficiency, one can predict the pathogen based on the primary immune defects, the organs involved, and the clinical presentation of the patient. for instance, staphylococcus aureus, burkholderia cepacia, serratia marcescens, pseudomonas and aspergillous infection should be considered for a chronic granulomatous diseased (cgd) patient with soft tissue infection, lymphadenitis, liver abscess, osteomyelitis, pneumonia, and sepsis. in centers dealing with immunocompromised patients, the microbiology laboratory as well as the radiology service need to be well equipped and trained in diagnosing these patients. patients with fever should be worked up with complete blood count with differential, renal, and hepatic profile, blood culture from central line (if present), and peripheral culture. chest x-rays are not done routinely unless the patients have respiratory symptoms. other investigations need to be guided by the presentation of the patient. patients with diarrhea should have stool checked for bacterial culture, ova and parasite, viral culture, rotavirus, and electron microscopy for viral studies, in addition to microspora, cryptosporidium, and isospora. in addition to chest x-ray, patients with respiratory symptoms required nasopharyngeal aspirate for rapid test for viruses and pcr multiplex -a newly developed laboratory procedure that can screen multiple viruses and other respiratory pathogens in the same setting. patients with skin lesions should have skin biopsy from the lesion, which will be sent for culture (bacterial, fungal, and mycobacterium) in addition to histopathology for gram-stain and special staining for fungal as well as acid fast stain (afb stain). there are several objectives in managing infections in immunocompromised patients. the first and foremost objective is to assure patients' survival and prevent infectious morbidity. decrease days of hospitalization and decrease exposure to multidrug resistance organism, decrease number of days of antibiotic use to minimize selection of resistance organism. modification of antimicrobial therapy in immunocompromised patients is the rule rather than the exception. timely modification of antibiotic therapy is very important to control breakthrough infection. there are several questions to be addressed to choose the effective antimicrobial therapy when evaluating patients. in addition to history and physical examination, it is important to determine which arm/arms of the immune systems that is/are affected? what the clinical syndrome/site of infection is? (to predict what are the likely pathogens), what clinical specimen(s) should be obtained (empiric/definitive therapy)? and which antimicrobial agents have predictable activity against pathogens? with these in mind, one can predict pathogen and choose the right antimicrobial agents. patients with wiskott-aldrich syndrome are at risk of bacterial pneumonia as well as sepsis with gram-positive organisms including mrsa. in this situation, medication should include agents active against gram-negative pathogen plus anti-staphylococcus agents, for example, cefotaxime or ceftriaxone plus naficillin; if mrsa or penicillin resistant s. pneumonia is suspected, one can use vancomycin. the pathogen in immunocompromised patients can be predicted by the system involved during the presentation. for example, the presentation and etiological agents in pneumonia in immunocompromised patients are different than immunocompetent persons. in evaluating pneumonia in immunocompromised patients, one needs to know that the pulmonary complication is present in up to 60% of immunocompromised patients and mortality is up to 80% of those who require mechanical ventilation. the initial evaluation needs rapid assessment of the vital signs including oxygen saturation, complete blood count with differential, renal profile, blood culture, and imaging of the lung either chest x-ray or ct scan. the organism can be predicted based on the primary immune defect. at certain point in the history, the defect in the immune system, the presence or absence of neutropenia, history of antimicrobial exposure, the presence of potential pulmonary pathogens in previous cultures, and the presence of indwelling catheters should be looked at. the pattern and distribution of radiological abnormalities can predict the pathogen and the time and the rate of progression and time to resolution of pulmonary abnormalities. for definitive diagnoses invasive procedures may be needed including bronchoalveolar lavage (bal), transbronchial biopsy, needless biopsy, thorascopic biopsy, and open lung biopsy. in obtaining the biopsy from this patient, it is very important to send it for histopathology for special staining, for viruses, bacteria, fungi, pneumocystis, mycobacterial pathogen, and also culture for viral, fungal, bacterial, and mycobacterium. other laboratory tests that will help in diagnosing pneumonia are nasal washings or swabs for direct fluorescent antibody, pcr for respiratory viruses and atypical pneumonia, culture and staining, cmv antigenemia or cmv viral load testing, aspergillus galactomannan assay, and 1,3 beta d glucan. the radiological finding in immunocompromised patient can be focal (lobar or segmental infiltrate), diffuse interstitial infiltrate or nodular (with or without cavitation). focal infiltrate can be due to gram-positive or gram-negative bacteria, legionella, mycobacteria, and fungal infection. also the noninfectious etiology includes infarction, radiation, and drug-related bronchiolitis obliterans organizing pneumonia (boop). diffuse interstitial infiltrate is caused by viral infection, pneumocystis jiroveci, less likely mycobacterium, disseminated fungal infection, atypical pneumonium including chlamydia, legionella, and mycoplasma. other noninfectious etiology causing diffuse interstitial infiltrate include edema, acute respiratory distress syndrome (ards), and drug-related radiation. for nodular infiltrate with or without cavitation the infectious etiology include aspergillus infection, and other mycoses, nocardia, bacteria either gram-positive or gram-negative, anaerobes, and mycobacterium tb, as well as noninfectious etiology including disease progression like metastasis and drug toxicity. the management of immunocompromised patients with pulmonary infiltrate will depend on the patient presentation. if the patient is acutely ill, it is very important to begin empiric therapy to cover the likely pathogen based on the presentation of the patient and the primary immune defect with simultaneously comprehensive evaluation. subsequently, therapy should be adjusted based on culture and clinical response. in providing empirical antibiotic therapy in patient with pulmonary infiltrate and defect in cell-mediated immunity one need to consider pneumocystis jiroveci, nocardia, legionella, mycoplasma, in addition to aerobic gram-positive cocci and gram-negative bacilli therefore it is advised to use trimethoprim-sulfamethoxazole, macrolides including erythromycin or clarithromycin and agent active against gram-positive and gram-negative; for example, thirdgeneration cephalosporin with or without aminoglycoside with anti-gram-positive either nafcillin or vancomycin based on the incidence of methicillin-resistant staphylococcus aureus (mrsa) and penicillin resistant streptococcus pneumoniae. the fever is defined in the context of febrile neutropenia as a single oral temperature of more than 38.3 c or more than 38.0 c for at least 1 h and is not related to the administration of pyrexial agents including blood, blood product, ivig, and pyrogenic drugs, especially ara c. neutropenia is defined as absolute neutrophil count (anc) less than 500/mm 3 or less than 1,000/mm 3 with predictive decline to less 500/mm 3 48 h. the most important risk factor is the presence of neutropenia as well as the degree and duration of neutropenia. the lower the neutrophil count, the higher the risk of infection. the longer the duration of neutropenia, the higher the risk of infection. usually, neutropenia is considered high risk if 7 days and low risk < 7 days. other risk factors include associated medical comorbidity, primary disease, and status (remission or relapse). low-risk patients are clinically defined by neutropenia as anticipated lasting less than 7 days, clinically stable, and having no medical comorbid conditions. about 50% of neutropenic patients who become febrile have established or occult infections and about 25% of patients with anc less than 100 cells/mm 3 have bacteremia. the risk varies depending on the underlying disease, for example, patients post allogenic bone marrow transplantation are at higher risk than autologous bone marrow transplantation while aml has the higher risk than all. the lowest risk is in patients with cyclic neutropenia. in evaluating a patient with fever and neutropenia, it is important to keep in mind that signs and symptoms can be muted or subtle. profoundly neutropenic patients can sometime have life-threatening infections and yet be afebrile especially if they presented with abdominal pain. careful and comprehensive physical examination is critical and should be repeated at least daily because these patients are dynamic and their condition can change rapidly. other important points in the history include the nature of chemotherapeutic agents, steroids, or other immunosuppressive agents because these can predict the degree of immunosuppression, the duration of neutropenia, and the severity of neutropenia. the history of antibiotic prophylaxis is also important because the antibiotic used as prophylaxis should be avoided in treating these patients. reviewing the recent documented infection with susceptibility can help in determining the empiric therapy. for example, if the patient has a previous infection with multidrug resistance pathogen, empiric therapy can be used to cover these pathogens. if the patient had recent surgical procedure, this means there is break of the skin and is at risk for certain pathogens including grampositive cocci (coagulase negative staphylococci and staphylococcus aureus). allergy history is an important factor in selecting empirical therapy as allergic medications need to be avoided. detailed and thorough physical examination is important with focus on certain sites that can be a portal of entry of pathogens including periodontium, pharynx, lower esophagus, lung, skin, perineum, bone marrow aspiration site, and catheter entry and exit sites. after history and thorough physical examinations, blood culture from central and peripheral lines should done in order to identify the source of infection. for example, if the blood culture is positive from the central culture but negative from peripheral culture, the likely source is the central line. if both are positive, time is needed to positively determine the source of infection. routine surveillance culture is not indicated as it is not cost effective and has low predictive value. other cultures should be guided by the sites of infection. for example, a patient with respiratory symptoms needs to have nasapharyngeal aspirate for viral study, pcr multiplex, and atypical pneumonia. patients with gastrointestinal symptoms, for example, with diarrhea, the stool needs to be sent for viral study, culture and sensitivity, ova and parasite. chest x-ray should not be done routinely in all patients with fever and neutropenia because it has low yield in patients without respiratory symptoms. it is only done in children who have respiratory symptoms. if negative, a chest ct scan to be considered to better evaluate patient not responding to therapy. most patients with fever and neutropenia have no identifiable site of infection and no positive culture results. bloodstream infection is documented in about 20% of patients with fever and neutropenia. disruption of the skin or soft tissue including vascular access or catheter insertion site can be a point of entry. in those centers, who are dealing with cancer patients, it is very important to monitor the infection rate and pathogen as well as the resistance pattern in the same center. the local data will help to select the appropriate empirical antimicrobial therapy (> table 68 .1). there is no ideal regimen because there are variables which include the risk status of the patient, microflora and their sensitivity patterns, toxicity indication, preference, and the cost. prompt initiation of broad-spectrum therapy when neutropenic patients became febrile is the key to successful management. in 1960 the mortality rate was up to 80% initially but with the introduction of empiric therapy against gram-negative organism the mortality rate now is close to 5%. there is no ideal regimen because this can be determined based on the isolate and its susceptibility in the same center as each center for example, one cannot extrapolate from different centers the likely pathogen, the same thing that a center can have a different pathogen and different susceptibility pattern in adult versus pediatric population with febrile neutropenia ( > table 68 .2). monotherapy and combination therapy has equal efficacy. the monotherapy needs to have antipseudomonal activities including antipseudomonal penicillin with or without beta-lactamase inhibitor, carbapenem, and third-or fourth-generation antipseudomonal cephalosporins. the combination therapy includes antipseudomonal betalactam with aminoglycoside. both monotherapy and combination therapy have equal efficacy but it is important to look at the local data to be able to predict the empiric therapy either combination therapy or monotherapy. it is worth stressing that vancomycin should not be used routinely for empiric therapy in febrile neutropenia and there is a special indication for vancomycin. the vancomycin indication includes hemodynamic instability or other evidence of severe sepsis, pneumonia documented radiographically, positive blood culture for gram-positive bacteria before final identification and susceptibility testing is available, clinically suspected catheter-related infections (e. g., chills or rigors with infusion through catheter and cellulitis around the catheter entry/exit site), skin or soft-tissue infection at any site, colonization with methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococcus, or penicillin-resistant streptococcus pneumoniae, and severe mucositis, if fluoroquinolone prophylaxis has been given and ceftazidime is employed as empirical therapy. if the patient started empirically on vancomycin the need for continuation of vancomycin should be re-assessed on daily basis. overuse of vancomycin in more than 90%, and selection for resistant organism and emergence of vancomycin resistance enterococci. the factors influencing antimicrobial selection include the types of bacterial isolates found in the institution, antibiotic susceptibility patterns, drug allergies, presence of organ dysfunction, chemotherapeutic regimen whether the patient was receiving prophylactic antibiotics, and condition of the patient at diagnosis, for example, presence of signs and symptoms at initial evaluation and presence of documented sites requiring additional therapy. the center-specific factors include the patterns of resistance, effect on microbial ecology, high presence of vancomycin resistance enterococci (vre), or extended spectrum beta-lactamase (esbl) producing organism. the patient-specific factors including recent antibiotic use such as current prophylaxis as drug allergy, and the underlying organ dysfunction. the signs and symptoms present at the initial evaluation determine. in the recent year more interest in the outpatient therapy for patient with fever and neutropenia. the advantages of ambulatory management of febrile patients with neutropenia especially those at low risk include lower cost particularly with oral outpatient therapy, fewer superinfections caused by multidrug-resistant nosocomial pathogens, improved quality of life for patient, greater . not recommended for routine use *other antimicrobials (aminoglycosides, fluoropuinolone, and/or vancomycin) may be added to initial regimen for complicated presentation or if resistance is suspected or proven infections in the immunocompromised host convenience for family or other caregivers, and more efficient utilization of valuable and expensive resources. the disadvantage includes the potential risk for developing serious complications such as septic shock at home, risk of noncompliance particularly with oral therapy, false sense of security or inadequate monitoring for response to therapy or toxicity, and the need to develop a team and infrastructure capable of treating substantial numbers of low-risk patients. there are several requirements for successful outpatient treatment programs for patients with febrile neutropenia which include institutional infrastructure and support, a dedicated and experienced team of healthcare providers, availability of institution-specific epidemiological data and susceptibility and resistance data, microbiologically appropriate treatment regimen, frequent follow-up monitoring of outpatient, adequate transportation and communication capabilities, and access to management team 24 h a day, 7 days a week. there are certain clinical events or manifestations that require modifying the initial antimicrobial therapy; for example, if a patient has breakthrough bacteremia and if gram-positive is isolated (add vancomycin especially if there is a risk of mrsa or pneumococcal resistance penicillin). if gram-negative organism is isolated consider resistant gram-negative and can change the regimen or broaden the coverage (carbapenems if the data in the center showed that the carbapenems has better sensitivity than cephalosporin or beta-lactam antibiotic). if the patient has catheter-associated soft tissue infection, vancomycin should be added. patients with severe oral mucositis or necrotizing gingivitis are at risk of anaerobic bacteria as well as viruses; add agent that is active against beta-lactamase-producing anaerobic bacteria including clindamycin, metronidazole, and acyclovir should be considered. if the patient has diffuse pneumonia, continue with the broad-spectrum anti-gramnegative coverage (add trimethoprim-sulfamethoxazole and macrolide to the therapy). increasing neutrophil count on patients who developed new infiltrates while on antibiotic can be related to the recovery of neutropenia. if the patient is stable observe if the neutrophil count is not rising, antifungal therapy should be considered as the patient is at risk for fungal infection. in addition to other evaluation aspergillus galactomannan and b-d glucan (fungitell) should be done with chest ct scan. depending on the ct scan findings bronchoalveolar lavage or lung biopsy should be considered. patient with prolonged fever and neutropenia needs to be observed if recovery of neutropenia is not imminent. antifungal therapy can include either regular amphotericin b, or lipid formulation of amphotericin b including liposomal amphotericin b (ambisome) or amphotericin b lipid complex (ablc), caspofungin or voriconazole depending of the availability of medications and epidemiology of the institution. infections in the immunocompromised low risk episodes of fever and neutropenia in pediatric oncology: is outpatient oral antibiotic therapy the new gold standard care? predicting events in children with fever and chemotherapy-induced neutropenia: the prospective multicenter spog 2003 fn study cefepime and death: reality to rescue clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious society of america etiology and clinical course of febrile neutropenia in children with cancer risk prediction in pediatric cancer patients with fever and neutopenia advances in the management of primary immunodeficiency immunocompromised children: conditions and infectious agents emprical oral antibiotic therapy for low risk febrile cancer patients with neutropenia meta-analysis of a possible signal of increased mortality associated with cefepime use febrile neutropenia: a critical review of the initial management fever and neutropenia in pediatric patients with cancer ) b lactam monotherapy versus b lactam-aminoglycoside combination therapy for sepsis in immunocompetent patients: systemic review and meta-analysis of randomized trials fever in immunocompromised patients bloodstream infections in hematology: risks and new challenges for prevention pediatric cancer patients in clinical trials of sepsis: factors that predispose to sepsis and stratify outcome key: cord-004586-i8tacj63 authors: nan title: empfehlung zur prävention nosokomialer infektionen bei neonatologischen intensivpflegepatienten mit einem geburtsgewicht unter 1500 g: mitteilung der kommission für krankenhaushygiene und infektionsprävention beim robert koch-institut date: 2007-10-05 journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: 10.1007/s00103-007-0337-0 sha: doc_id: 4586 cord_uid: i8tacj63 nan ein neugeborenes wird als solches bis zum 29. lebenstag bezeichnet. es handelt sich um ein frühgeborenes, wenn das gestationsalter (ga) bei geburt -gerechnet vom ersten tag der letzten normalen regelblutung -kleiner ist als 37 vollendete wochen (< 259 tage) 1 . je nach geburtsgewicht (gg) werden des weiteren unterschieden 1. < 2500g: untergewichtige neugeborene (lbw), 5-15 % der lebendgeborenen, 2. 1000-1499g: sehr untergewichtige neugeborene (vlbw), 0,8-1,5 % aller lebendgeborenen, 3. < 1000g: extrem untergewichtige neugeborene (elbw), 0,3-0,6 % aller lebendgeborenen. ausgehend von einem anteil an allen lebendgeborenen 2 von 8,5 % für die frühgeborenen sowie von 1,1 % für vlbw-und 0,45 % für elbw-neugeborene ergeben sich als mittelwert für die jahre 1991-2003 folgende kalkulationen für deutschland: die absolute zahl der lebendgeborenen in der jeweiligen gruppe pro jahr beträgt im median frühgeborene 65 513 kinder, frühgeborene unter 1500g geburtsgewicht 8478 kinder, vlbw frühgeborene (geburtsgewicht 1000-1499g) 5010 kinder, elbw frühgeborene (geburtsgewicht 500-999g) 3468 kinder. entsprechend verteilen sich die im rahmen des neo-kiss surveillanceprojektes dokumentierten patienten (n=7469) folgendermaßen: < 499g 2,5 %, 500-999g 40 %, 1000-1499g 57,5 % (http://www.nrzhygiene.de/surveillance/neo.htm). f bis zur verwendung ist der inkubator mind. 1 h bei laufendem motor zu belüften; siehe auch angaben der hersteller (kategorie ib). f nach der aufbereitung muss der inkubator in einem "reinen" und abgetrennten bereich (nicht auf dem stationsflur) vor kontamination geschützt werden (kategorie ib). f die patientenseitige desinfektion eines belegten inkubators ist nicht möglich, da eine schädigung des frühgeborenen durch exposition gegenüber marktüblichen flächendesinfektionsmitteln nicht auszuschließen ist (kategorie ib). f die reinigung der innenseite des belegten inkubators kann mit wasser von trinkwasserqualität erfolgen (siehe oben), wobei für jeden inkubator (patientenbezogen) ein frisches, keimarmes tuch verwendet werden muss (kategorie ib). f alle außen gelegenen handkontaktflächen am inkubator (inklusive steu-erungstastaturen) müssen arbeitstäglich wischdesinfiziert werden (kategorie ib). f die frage, in welchen abständen ein patient einen frisch aufbereiteten inkubator bekommen muss, wurde bisher nicht untersucht und ist daher ungelöst (kategorie iii). während in speziellen situationen, z. b. im verlauf einer sanierungsbehandlung bei mrsa-besiedlung, ein täglicher wechsel im rahmen der grundpflege erforderlich ist, spricht bei stabilen kindern, die zumindest einmal täglich mit frischer wäsche versorgt werden, nichts gegen einen wöchentlichen inkubatorwechsel. in einigen neonatologischen intensivbehandlungseinheiten ist es üblich, die abgepumpte muttermilch für sehr unreife frühgeborene in regelmäßigen intervallen mikrobiologisch zu untersuchen und je nach ergebnis freizugeben oder zu verwerfen. im rahmen von ausbruchsuntersuchungen wurden in der muttermilch e. [188, 191, 192, 193] (kategorie ii). die überbelegung einer station, die definitionsgemäß mit einem mangel an angemessen ausgebildeten schwestern, pflegern und ärzten einhergeht, korreliert mit einem erhöhten risiko nosokomialer infektionen [89, 90, 194, 195] . zahlreiche studien aus der neonatologie und aus anderen fachdisziplinen bestätigen dies übereinstimmend [196, 197, 198, 199, 200, 201, 202, 203, 204] . sie beweisen, dass bei gleich bleibendem personalbestand eine überbelegung das risiko nosokomialer infektionen erhöht bzw., dass eine bessere austattung mit fachschwestern-/pflegern das risiko von nosokomialen infektionen senkt [25] . jedoch kann auch eine quantitativ ausreichende personalausstattung nosokomiale infektionen nicht verhindern, wenn das vorhandene personal schlecht ausgebildet oder mit den arbeitsabläufen und den hygienestandards vor ort nicht ausreichend vertraut ist [196] . f es ist wissenschaftlich gesichert, dass eine nicht angemessene ausstattung der nips mit qualifiziertem und vor ort eingearbeitetem personal das risiko nosokomialer infektionen erhöht (kategorie ia). f die empfehlung der gesellschaft für neonatologie und pädiatrische intensivmedizin (zur personalausstattung) [205] ist diesbezüglich wegweisend (kategorie ib). die postnatale reinigung der haut bei frühgeborenen < 1500g erfolgt je nach allgemeinzustand und hautbeschaffenheit. hierzu benötigte pflegeutensilien und -mittel sind patientenbezogen einzusetzen (zur mikrobiologischen wasserqualität siehe oben), da sie zum ausgangspunkt nosokomialer ausbrüche werden können [206] . f in der besonders vulnerablen phase (erste lebenswoche) nach der geburt sollte bei extrem unreifen frühgeborenen (< 26. ssw) aufgrund der erhöhten permeabilität und verletzlichkeit der haut nur so viel wie unbedingt nötig manipuliert werden (z. b. antisepsis vor invasiven prozeduren, körperwaschung) (kategorie ib). entsprechende studien liegen für level 1 und 2 nips nicht vor (kategorie iii). vor jeder durchtrennung der haut (z. b. blutentnahmen, injektionen, punktionen) muss ein hautantiseptikum aufgetragen und die deklarierte einwirkzeit eingehalten werden (haut trocknen lassen). es empfiehlt sich die begrenzung auf präparate mit möglichst geringem resorptivem risiko [207, 208] . polyvidoniod 10 % (pvp-iod) hat den nachteil der systemischen jodresorption und ist daher bei extrem unreifen frühgeborenen primär kontraindiziert [207, 209, 210] (kategorie ib). chlorhexidin kann bei extrem unreifen frühgeborenen lokale unverträglichkeitsreaktionen auslösen, die einer zweitgradigen verbrühung ähneln [211, 212] . es weist wirkungslücken im gramnegativen bereich auf [213, 214] , was bei kontamination der lösung nosokomiale epidemien zur folge haben kann [215] . es hat zytotoxische und im tierversuch mutagene eigenschaften [207] . seit 1993 wurde über mehr als 30 durch chlorhexidin ausgelöste, zum teil lebensbedrohliche anaphylaktoide reaktionen berichtet, so dass die u.s.-amerikanische food and drug administration sich 1998 zu einem speziellen warnhinweis veranlasst sah [216] . f chlorhexidin wird aufgrund von wirkungslücken, ungünstiger beeinflussung der wundheilung, potenzieller mutagenität und der klinisch relevanten gefahr von überempfindlichkeitsreaktionen nicht zur hautdesinfektion empfohlen (kategorie ib). octenidinhydrochlorid (0,1 %) in kombination mit 2 % phenoxyethanol (octenispet®) ist ein farbloses, nicht mutagenes haut-und schleimhautantiseptikum mit breiterem wirkungsspektrum als chlorhexidin und guter lokaler verträglichkeit [217] . es gibt inzwischen eine reihe klinischer anwendungsbeobachtungen u. a. auch in der kinderurologie und bei extrem unreifen frühgeborenen [218] . die sicherheit des präparats in der anwendung bei extrem unreifen frühgeborenen kann jedoch nicht allein aus dem nachweis des abbauproduktes phenoxyessigsäure im urin geschlussfolgert werden. f aus toxikologischen gründen wird eine hautdesinfektion bei frühgeborenen mit octenidin 0,1 % ohne phenoxyethanol empfohlen (kategorie ii). der hersteller von octenisept® bietet klinikapotheken octenidin als grundsubstanz zur eigenen herstellung einer gebrauchsfertigen lösung (zielkonzentration 0,1 %) nach einer arzneirezeptur an. vor blasenkatheterisierung soll ein antiseptikum mit sterilem, satt getränkten tupfer aufgetragen und von der harnröhre nach dorsal abgewischt werden (einwirkzeit 1 min). octenisept® ist wegen der resorption des zusätzlich enthaltenen phenoxyethanols problematisch [218] . f zur schleimhautantiseptik bei frühgeborenen < 1500g ist octenidin 0,1 % ohne phenoxyethanol mittel der wahl (kategorie ib), (s. o.). eine evidenzbasierte empfehlung zur mundhöhlenantiseptik bei apparativer beatmung ist nicht möglich (kategorie iii). spezielle indikationen zur antiseptischen hautwaschung können bei frühgeborenen bestehen, die mit mrsa besiedelt sind oder unter der geburt/sectio äußerlich durch das blut der mutter mit he unmittelbar postnatal wird die nabelschnur nach anlegen einer sterilen kunststoffklemme durchtrennt und mit einer sterilen kompresse abgedeckt. dabei wird die kompresse unter der nabelklemme durchgezogen, um den kontakt des nabelschnurstumpfes mit der bauchhaut und mit urin zu verhindern. sofern der nabelstumpf nicht verunrei nigt ist, oder als zugang für intravaskuläre katheter genutzt wird, ist eine antiseptik nicht erforderlich (der nabelstumpf wird nicht als wunde angesehen, stellt jedoch eine potenzielle eintrittspforte für sekundärinfektionen dar). bei lokalen entzündungszeichen insbesondere bei einer rötung des nabelrings ist nach entnahme eines abstrichs zur erregerdiagnostik ein antiseptikum indiziert (siehe hautantiseptik). die verhinderung des hustenreflexes, die reizung/verletzung des trachealepithels, die leitschiene für die mikroaspiration von bakterien des mund-und rachenraumes und die kunststoffoberfläche, auf der sich ein mikrobieller biofilm bildet, machen den intratrachealen tubus zum wichtigsten risikofaktor der vap [220, 221] . intrinsische faktoren des beatmungspflichtigen frühgeborenen kommen hinzu, wie der oft kritische und katabole allgemeinzustand, eine zelluläre oder humorale immundefizienz sowie die an-tibiotische vorbehandlung. das beatmete frühgeborene ist einer vielzahl von faktoren in seiner belebten und unbelebten umgebung ausgesetzt, durch die eine infektion begünstigt wird [222] (. tabelle 2). die diagnostik der vap bei frühgeborenen ist schwierig, was bei der beurteilung entsprechender studien berücksichtigt werden muss [50, 223, 224] . risikofaktoren für eine vap bei frühgeborenen sind: . [246, 247] ist auch bei frühgeborenen möglich. ebenso ist eine intermittierende bauchlagerung zur besseren ventilation und sekretdrainage der basalen lungenabschnitte in der regel zu empfehlen. die erstversorgung geht in aller regel mit invasiven maßnahmen einher und findet oft räumlich angrenzend an den gynäkologischen operationssaal statt. vor dem hintergrund der besonderen infektionsgefährdung bei extrem unreifen frühgeborenen ergeben sich folgende empfehlungen: die derzeit verfügbaren beatmungsfilter zur passiven befeuchtung des systems [243, 249] sind für frühgeborene nicht geeignet, da sie für diese patienten nicht validiert sind und durch totraumerhöhung zu einer kritischen co 2 -retention führen könnten (kateorie iii). wegen der hochgradigen immundefizienz wird für fg < 1500g geburtsgewicht zusätzlich zur thermischen desinfektion in einem automaten eine definiert geschützte (geschlossene) lagerung von beatmungszubehör empfohlen (kategorie ii in einer prospektiven studie mit 66 vs. 67 beatmeten neugeborenen < 1250g gg [230] traten in beiden gruppen (geschlossene absaugung vs. offene konventionelle absaugung) je 5 vap-episoden auf. unabhängig von der methode des absaugens (offen oder geschlossen) muss die spülflüssigkeit steril sein. f wenn durch entsprechende maßnahmen flüssigkeits-und sekretrückstände im geschlossenen absaugsystem vermieden werden, ist das system als bestandteil des beatmungssystems zu betrachten und kann 7 tage am patienten verbleiben [251, 252] . studien zu dieser frage bei frühgeborenen fehlen (kategorie iii). f eine empfehlung zum infektionspräventiven einsatz und zum wechselintervall geschlossener absaugsysteme bei frühgeborenen kann nicht ausgesprochen werden (kategorie iii). f wenn der patient endotracheal mit multiresistenten krankheitserregern besiedelt ist, wird eine geschlossene absaugung zur verringerung der umgebungskontamination empfohlen (kategorie ib). es ist bisher unbekannt, ob spezielle maßnahmen der schleimhauthygiene, die sich in zahlreichen studien bei erwachsenen intensivpatienten als effektiv in der vap-prävention erwiesen haben [253] , die kolonisation der schleimhäute bei frühgeborenen günstig beeinflussen und die vap-inzidenz vermindern können. vor allem wurden die entsprechenden studien bei erwachsenen mit chlorhexidin oder oralen antibiotika durchgeführt, deren einsatz bei sehr unreifen frühgeborenen nicht empfohlen werden kann. f mit antibiotika behandelte, sehr unreife frühgeborene sollten eine lokale prophylaxe mit nystatin-oder amphotericin b (cave: hohe osmolarität von am-phomoronal®) erhalten [254] (kategorie ii). f eine prophylaktische antibakterielle chemotherapie bei beatmeten frühgeborenen wird nicht empfohlen [255] (kategorie ia). das gleiche gilt für eine prophylaktische substitution von immunglobulinen zur prävention der vap [256] (kategorie ia). f über die passive immunisierung mit palivizumab zur prävention der rsv-pneumonie bei hochrisikofrühgeborenen, die während der rsv-saison noch stationär behandelt werden, sollte im einzelfall entschieden werden [257, 258, 259, 260 , 261] (kategorie ib). zentrale venenkatheter werden bei frühgeborenen < 1500g in der regel als nabelkatheter oder als perkutane zentrale venenkatheter (peripherally-inserted central catheter, "silastik"-katheter, picc) angewendet [ f die indikation zum gebrauch eines picc muss täglich geprüft werden; er muss so früh wie möglich entfernt werden (kategorie ib). f piccs müssen nicht routinemäßig gewechselt werden (kategorie ib). [311, 312] . bei kindern im neugeborenenalter wurden bei 0,5 % lokalinfektionen beschrieben [313] . studien bei frühgeborenen < 1500g wurden bisher nicht publiziert. periphere arterienkatheter bei frühgeborenen werden gewöhnlich in die a. radialis oder die a. tibialis post. gelegt [314, 315] . eine bevorzugte insertionsstelle aus infektiologischen/hygienischen gründen konnte bisher in studien und insbesondere bei frühgeborenen < 1500g nicht gezeigt werden. obwohl keine daten zu den hygienischen maßnahmen während der insertion peripherer arterienkatheter vorliegen, sollten alle maßnahmen eingehalten werden, die bei der insertion peripherer venenverweilkanülen getroffen werden, und darüber hinaus sterile handschuhe getragen werden, da in der regel auch während der punktion die palpation des arterienpulses notwendig sein kann. f eine empfehlung zur wahl der insertionsstelle von peripheren arterienkathetern aus infektiologischer sicht kann nicht gegeben werden (kategorie iii hinsichtlich verband und verbandswechsel sollen periphere arterienkatheter wie periphere venenverweilkanülen behandelt werden. f eine punktionsnahe applikation von unsterilen pflasterstreifen ist zu vermeiden (kategorie ib). f es können sowohl transparente als auch gazeverbände verwendet werden (kategorie ib). f die verbände müssen täglich inspiziert werden (kategorie ib). f transparentverbände müssen nicht routinemäßig, sondern nur bei bedarf (verschmutzung, ablösung, durchfeuchtung, infektionsverdacht) gewechselt werden (kategorie ib). f gazeverbände müssen tgl. gewechselt werden (kategorie ib). f es ist eine hygienische händedesinfektion vor und nach verbandswechsel erforderlich (kategorie ib). f der verbandswechsel erfolgt mittels no-touch-technik oder mit sterilen handschuhen (kategorie ib). f die insertionsstelle wird mit steriler 0,9 % nacl-lösung und sterilem stieltupfer gereinigt (kategorie ib). da infektionen mit gram-negativen stäbchenbakterien bei verwendung von mehrwegdruckmesssystemen beschrieben wurden, sind einwegartikel, wie heute auch allgemein üblich, den mehrwegsystemen vorzuziehen [316, 317, 318, 319] . auch bei neonatologischen patienten wurden infektionen über das messsystem beschrieben, insbesondere mit candida parapsilosis aber auch gram-negativen bakterien [320, 321, 322] . als druckmesssysteme sollten geschlossene systeme ohne dreiwegehahn verwendet werden. die gesamten systeme sollten alle 96 stunden gewechselt werden [312, 323, 324] . die blutentnahme kann aus solchen systemen über eine durchstichmembran mit einer geeigneten sterilen nadel/spritze nach vorheriger alkoholischer wischdesinfektion der membran erfolgen [318] . grundsätzlich sollten die anzahl der manipulationen, insbesondere blutabnahmen so niedrig wie möglich gehalten werden [318, 325] . f es sollen einmalartikel als druckmesssysteme verwendet werden (kategorie ib). f es sollen geschlossene druckmesssysteme eingesetzt werden (kategorie ib). f die handhabung der nicht konnektierten druckmesssysteme muss im bereich der verbindungsstücke unter aseptischen kautelen erfolgen (kategorie ib). f druckmesssysteme müssen alle 96 stunden gewechselt werden (kategorie ib). f bei blutabnahmen über eine gummimembran mit einer sterilen nadel/ spritze muss vorher eine alkoholische wischdesinfektion der membran erfolgen (kategorie ib). wie bei peripheren venenverweilkanülen nimmt die kolonisationsrate bei längerer liegedauer zu [326, 327, 328, 329, 330] . zusammenfassend sind die infektionsraten auch bei längerer liegedauer niedrig, sodass keine empfehlung für einen routinemäßigen wechsel gegeben werden kann [312, 328] . wegen der begünstigung einer bakteriellen oder mykotischen besiedlung des systems sollten keine glukosehaltigen spüllösungen verwendet werden [318, 321, 322] . die zugabe von 0,25-1 u/ ml heparin führt zu einer verlängerten nutzungsdauer auch bei kindern und neonatalen patienten [331, 332, 333] . eine metaanalyse von randolph et al. kommt deshalb zu der allgemeinen empfehlung, der arteriellen spüllösung heparin zuzusetzen [334] . aus infektiologischer sicht existieren keine daten, die einen nutzen für die kontinuierliche heparingabe zur prophylaxe der katheter-assoziierten infektionen zeigen. f periphere arterienkatheter können in situ belassen werden, solange eine klinische indikation besteht bzw. keine komplikation aufgetreten ist (kategorie ib). f ein routinemäßiger wechsel peripherer arterieller katheter ist nicht notwendig (kategorie ib). f die indikation muss täglich neu geprüft werden (kategorie ib). f katheter sind bei sichtbarer entzündung an der eintrittsstelle sofort zu entfernen (kategorie ib). f glukosehaltige spüllösungen dürfen nicht verwendet werden (kategorie ib). f eine empfehlung zur verwendung von heparin kann aus infektiologischen gründen nicht gegeben werden (kategorie iii). speziell geschulte katheterteams und personalschulungen können die rate katheter-assoziierter infektionen signifikant reduzieren. studien, die dies für die anlage und pflege von nvk/nak untersucht haben, fehlen jedoch. der einfluss verschiedener kathetermaterialien wurde im hinblick auf infektions-präventive effekte für nabelkatheter nicht untersucht [341, 342] . katheter aus polyurethan oder silikon sollten bevorzugt verwendet werden, da in-vitro-daten eine schlechtere adhäsion von mikroorganismen an diesen materialien im vergleich zu polyvinylchlorid oder polyethylen zeigen [343] . mehrlumige nabelvenenkatheter sind im vergleich mit einlumigen kathetern nicht mit einer erhöhten infektionsrate assoziiert [344, 345, 346] . die position (hohe vs. tiefe position) des nabelarterienkatheters wurde hinsichtlich katheter-assoziierter infektionen nicht untersucht. hinsichtlich der entwicklung einer nec (s. u.) zeigte sich kein signifikanter unterschied [342] . f es können handelsübliche nvk/ nak aus silikon oder polyurethan verwendet werden (kategorie iii). f es können sowohl einlumige als auch mehrlumige katheter zum einsatz kommen (kategorie ib). f eine empfehlung für die hohe oder tiefe position beim nak kann nicht ausgesprochen werden (kategorie iii). es existieren keine studien, in denen der einfluss bestimmter hygienemaßnahmen bei der insertion auf die infektionsrate von nabelkathetern untersucht wurde. die anlage von nabelkathetern erfolgt in steriler technik unter maximalem barriereschutz. zur desinfektion des nabelstumpfes sollte wie bei den piccs und den peripheren venen-und arterienkathetern in der patientengruppe < 1500g gg octenidin 0,1 % verwendet werden. f katheter können im kreißsaal, op oder auf der station gelegt werden (kategorie ib). weitere empfehlungen sind: f hygienische händedesinfektion vor der anlage (kategorie ia), f haut-und nabelstumpfdesinfektion mit octenidin 0,1 % unter beachtung der einwirkzeit von mind. 1 min (kategorie ib), f anlage von sterilen einmalhandschuhen, sterilen schutzkitteln, mund-nasen-schutz und haube, großflächige abdeckung der punktionsstelle mit sterilem lochtuch (kategorie ia), f durchtrennung der nabelschnur und präparation der nabelgefäße mit sterilem instrumentarium (kategorie ib), f insertion unter aseptischen bedingungen (kategorie ia). die nabelgefäßkatheter können mit hilfe einer annaht am nabelstumpf fixiert werden. der nabelstumpf kann mit einem gazeverband verbunden werden. transparentverbände eignen sich hierfür nicht. nicht-transparente gazeverbände müssen täglich erneuert werden, um eine inspektion der eintrittsstelle durchzuführen. dem vorteil eines verbandes der nabelregion (z. b. schutz vor verunreinigungen aus der perianalregion) stehen die nachteile der schlechteren beurteilbarkeit der einführtiefe des nabelgefäßkatheters gegenüber. ob eine offene nabelpflege (ohne pflasterverband) zu einem höheren risiko für katheter-assoziierte infektionen führt, wurde nicht untersucht. die reinigung der insertionsstelle sollte mit sterilen tupfern und nacl 0,9 % und die lokale antisepsis mit octenidin 0,1 % erfolgen. daten über die anwendung von antimikrobiellen salben auf der insertionsstelle bei nabelgefäßkathetern existieren nicht. f eine aussage zur notwendigkeit eines pflasterverbandes bei liegendem nabelkatheter ist nicht möglich (kategorie iii). empfohlen wird eine f lokale antiseptik mit octenidin 0,1 % (kategorie ib), f und keine routineapplikation von antibakteriellen substanzen an der nabelöffnung bei liegenden nabelgefäßkathetern (kategorie iii). in einer kontrollierten studie konnte unter antibiotischer prophylaxe zwar eine reduktion der kolonisierung der nak, jedoch keine reduktion von klinischen infektionen beobachtet werden [335, 347] . gleiches gilt für nvk [348, 349] . f die prophylaktische gabe systemischer antibiotika zur prophylaxe nabelkatheter-assoziierter infektionen wird nicht empfohlen (kategorie ib). es gibt keine studien, welche den einfluss eines routinemäßigen wechsels bzw. eine entfernung von nabelkathetern nach einer definierten liegedauer untersucht haben. der systemwechsel erfolgt analog zu dem bei piccs (siehe dort). f ein routinemäßiger wechsel bzw. eine routinemäßige entfernung von nabelkathetern nach einem bestimmten zeitpunkt wird nicht empfohlen (kategorie iii). f nabelkatheter müssen bei zeichen einer omphalitis (eitrige sekretion, rötung der periumbilikalregion) sofort entfernt werden (kategorie ib). die durchgängigkeit von nak wird durch den kontinuierlichen zusatz von heparin zur infusionslösung ("arterienspülung") günstig beeinflusst und damit die liegedauer verlängert [350, 351] . ob der heparinzusatz zu einer geringeren infektionsrate führt, ist nicht untersucht. f intermittierende spülungen können, falls notwendig, mit steriler 0,9 % nacl-lösung erfolgen (kategorie ib). f eine empfehlung zum einsatz von heparin als kontinuierliche infusion zum erhalt und zur infektionsprävention bei nak kann nicht gegeben werden (kategorie iii). f siehe entsprechender abschnitt zu den arterienkathetern [384, 385, 386] , ciprofloxacin [387, 388, 389] oder linezolid [353, 390] . in bezug auf den gesamten verbrauch von glycopeptiden entfällt in pädiatrischen behandlungszentren ca. ein drittel auf die nips (27 % bei [391] ). bei der überwiegenden mehrzahl der so behandelten episoden (84 % bei [391] ) wird vancomycin empirisch ohne den nachweis eines methicillin-resistenten erregers in der blutkultur eingesetzt [392, 393] . aufgrund der hohen anwendungsrate breit wirksamer antibiotika bei früh-geborenen < 1500g geburtsgewicht ist der selektionsdruck für erreger mit speziellen resistenzen und multiresistenzen besonders hoch [172] . die anwendung bestimmter antibiotika korreliert mit der selektion von resistenten erregerspezies [394, 395, 396] . zum beispiel begünstigt der einsatz von cephalosporinen der gruppe iii die selektion von cephalosporin-resistenten enterobacter spp. [397] , von klebsiella spp. [398] und aufgrund der primären wirkungslücken auch die von enterokokken und clostridium difficile [399] . der empirische einsatz von vancomycin erhöht den selektionsdruck für glycopeptid-resistente erreger, wie vancomycin-oder teicoplanin-resistente koagulase-negative staphylokokken [369, 400, 401, 402] oder vancomycin-resistente enterococcus faecium [159, 163, 403, 404, 405] . bis zu 90 % der bei neonatologischen intensivpatienten isolierten koagulasenegativen staphylokokken (cons) sind meca gen-positiv und damit phänotypisch meist methicillin-resistent [406, 407] . der frühe empirische einsatz von vancomycin beruht somit auf der befürchtung, das akut erkrankte kind könnte ohne ein glycopeptid durch eine unwirksame therapie gefährdet werden. eine signifikant höhere letalität bei inadäquater initialer antimikrobieller therapie ist in dieser patientengruppe z. b. für candida-infektionen in einer multivariaten analyse beschrieben [408] . einige studien zur late-onset-sepsis deuten jedoch darauf hin, dass -im unterschied zu gram-negativen bakterien und systemischen candidainfektionen [409] -die letalität von cons-infektionen sehr gering ist (maximal 1,4 %-4 von 277 episoden bei karlowicz et al. 2000 [392] ). somit besteht vor allem beim empirischen einsatz von vancomycin in der neonatologie -wie in vielen anderen fachdisziplinen auch [395] -offensichtlich ein erhebliches einsparpotenzial, solange in einer abteilung nicht ein relevanter anteil der s.aureus methicillin-resistent ist [391] . der selektionsdruck der glycopeptide könnte durch den gezielten und restriktiven einsatz von vancomycin reduziert werden [392, 393, 403, 410, 411] . toxinbildende clostridium difficile werden vorübergehend von mehr als der hälfte aller mit antibiotika behandelten frühgeborenen ausgeschieden, sind in dieser altersgruppe jedoch als krankheitserreger von untergeordneter bedeutung [412, 413] . erhebliche probleme durch nosokomiale übertragung können daraus resultieren, dass ältere kinder mit neugeborenen auf der gleichen intensivstation betreut werden [414, 415] . neonatologische intensivpatienten mit kompliziertem verlauf und langem aufent halt können durch eine persistierende besiedlung der atemwege oder von dauerhaft eingesetzten hilfsmitteln (tubus, tracheostoma, magensonde, peg) zu einem bedeutsamen "reservoir" der nosokomialen ausbreitung multiresistenter infektionserreger werden [169, 170, 416, 417] . hier sind besonders gramnegative erreger wie acinetobacter spp. [387, 418, 419, 420] , enterobacter spp. [199, 397, 421, 422, 423, 424] , klebsiella spp. [197, 425, 426] im folgenden wird nur in stark komprimierter form auf eine begrenzte auswahl übertragbarer krankheitserreger eingegangen. zur weiteren vertiefung wird auf die zitierte literatur verwiesen. frühgeborene sind u. a. aufgrund der fehlenden kolonisationsresistenz und der häufigen anwendung breit wirksamer antibiotika besonders empfänglich für eine kolonisation mit nachfolgender infektion durch multiresistente nosokomiale infektionserreger. methicillin-resistente s.aureus (mrsa) [191, 192, 467, 468, 469] und vancomycin-resistente enterokokken (vre) [163, 405, 470, 471, 472, 473] weisen unter den gram-positiven erregern die größte bedeutung auf, da sie ein erhebliches epidemisches potential haben. zum teil haben aus der kolonisation hervorgehende epidemische infektionen auch dann, wenn das frühgeborene überlebt, schwerwiegende langzeitfolgen, wie etwa bei der meningoencephalitis mit ausgedehnten hirnabszessen verursacht durch serratia marcescens [431, 474] . zu gram-negativen bakterien finden sich zahlreiche berichte über ausbrüche durch multiresistente klebsiella spp. [475, 476, 477] , acinetobacter spp. [478, 479, 480, 481] , serratia spp. [185, 430, 432, 433, 482, 483, 484, 485, 486, 487, 488] oder zu solchen isolaten, die in vitro durch die produktion von betalaktamasen mit erweitertem wirkungsspektrum (esbl) eine resistenz gegen cephalosporine der gruppe iii aufweisen [398, 477, 489, 490] . vor allem für die gram-negativen erreger dieser auflistung [491, 492] , jedoch auch für vre und mrsa [11, 395, 493, 494, 495, 496] , ist die besiedlung des gastrointestinaltraktes von epidemiologischer bedeutung [497, 498, 499] . bei intubierten kindern sind oft auch die atemwege mit multiresistenten isolaten besiedelt [416, 428] . alle können über die hände des behandlungsteams (und der eltern) sowie durch kontaminierte gegenstände und pflegemittel von kind zu kind übertragen werden [ 500 501, 502] . acinetobacter baumanii nimmt eine sonderstellung unter den gram-negativen hospitalkeimen ein, weil diese spezies resistent ist gegen trocknungsschäden [479, 503, 504] zusätzlich zu den bakteriellen nosokomialen infektionserregern treten bei frühund neugeborenen virale erkrankungen auf, die sich aufgrund der fehlenden immunität auf seiten der patienten (z. b. fehlender nestschutz durch mütterliche an-tikörper) [539] [228] . zur prävention der influenza [549] ist v. a. die immunität aller kontaktpersonen des fg entscheidend. f das gesamte behandlungsteam einer nips und alle angehörigen von fg sollten (jährlich) gegen influenza aktiv immunisiert werden [550] bei herpes labialis muss von den eltern oder vom personal ein mund-nasen-schutz getragen werden (tröpfcheninfektion) und sehr sorgfältig auf die durchführung der hygienischen händedesinfektion vor jedem patientenkontakt (übertragung über kontaminierte hände) geachtet werden. die läsion sollte abgedeckt sein und nicht während der arbeit mit den händen berührt werden. da das cytomegalovirus (cmv) von infizierten frühgeborenen über monate im urin ausgeschieden wird, ist eine nosokomiale übertragung z. b. durch ungeschützten handkontakt mit "sauberen" windeln oder cmv in muttermilchproben und fehlende händedesinfektion möglich [552, 553, 554] . auch nicht-immunes personal (bis zu 50 % in abhängigkeit vom lebensalter und den lebensumständen) kann auf diesem wege cmv-infektionen bis hin zur akuten cmv-encephalitis erwerben [555, 556] . f in der pflege cmv-ausscheidender fg sind strikt befolgte standardhygienemaßnahmen zur vermeidung einer nosokomialen übertragung ausreichend (kategorie ib). f dem behandlungsteam sollte der eigene cmv-serostatus bekannt sein (kategorie ii). f augenärztliche vorsorgeuntersuchungen müssen mit desinfizierten händen und mit sterilisiertem oder mit einem umfassend viruzid wirksamen mittel desinfizierten instrumentarium erfolgen, weil sonst die gefahr einer übertragung von adenoviren bzw. einer epidemischen keratokonjunktivitis besteht [557, 558, 559, 560, 561] (kategorie ib) . das humane parvovirus b19 (erreger der ringelröteln) kann über tröpfcheninfektion (auch kontaminierte gegenstände) nosokomiale epidemien auslösen, wozu v. a. seine hohe tenazität und unempfindlichkeit gegenüber handelsüblichen desinfektionsmitteln beiträgt [562, 563, 564, 565] . der serostatus der mitarbeiter sollte diesen bekannt sein, insbesondere, wenn es sich um frauen mit kinderwunsch handelt (gefahr des hydrops fetalis bei infektion in der frühschwangerschaft) [566, 567, 568, 569, 570, 571] . eine exposition gegenüber masernvirus [572, 573, 574, 575, 576, 577, 578, 579] in einer nips erfordert die passive immunisierung aller nicht durch maternale antikörper geschützten frühgeborenen mit standardimmunglobulin und die überprüfung des serostatus (ggf. die auffrischimpfung) des behandlungsteams. unter den viralen erregern der gastroenteritis sind v. a. rotavirus [368, 580, 581, 582, 583, 584] insbesondere in ausbruchssituationen kann die zusätzliche rekrutierung von personal für reinigungs-und desinfektionsaufgaben erforderlich sein, da die oben aufgeführten kohortierungsstrategien das pflegepersonal bereits stark belasten [196] . schutzkittel sind immer bestandteile eines multibarrierekonzeptes und daher nur in wenigen studien als einzelne komponente der eindämmung untersucht (z. b. bei vre) [589, 590, 591] . wenn andere barrieremaßnahmen, wie die händedesinfektion oder die umgebungsdesinfektion oder ggf. der mund-nasen-schutz bei engem kontakt nicht 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amoxicillin prophylaxis for prevention of catheter-related infections in newborn infants with central venous silicone elastomer catheters neuraminidase inhibitors for preventing and treating influenza in children oral oseltamivir treatment of influenza in children vogelgrippe (geflügelpest) und mögliche influenzapandemie. stellungnahme zur verwendung von neuraminidasehemmern bei kindern und jugendlichen prospective controlled study of four infectioncontrol procedures to prevent nosocomial infection with respiratory syncytial virus. key: cord-022163-7klzsrpu authors: broder, christopher c.; wong, kum thong title: henipaviruses date: 2016-09-09 journal: neurotropic viral infections doi: 10.1007/978-3-319-33133-1_3 sha: doc_id: 22163 cord_uid: 7klzsrpu the first henipaviruses, hendra virus (hev), and nipah virus (niv) were pathogenic zoonoses that emerged in the mid to late 1990s causing serious disease outbreaks in livestock and humans. hev was recognized in australia 1994 in horses exhibiting respiratory disease along with a human case fatality, and then niv was identified during a large outbreak of human cases of encephalitis with high mortality in malaysia and singapore in 1998–1999 along with respiratory disease in pigs which served as amplifying hosts. the recently identified third henipavirus isolate, cedar virus (cedpv), is not pathogenic in animals susceptible to hev and niv disease. molecular detection of additional henipavirus species has been reported but no additional isolates of virus have been reported. central pathological features of both hev and niv infection in humans and several susceptible animal species is a severe systemic and often fatal neurologic and/or respiratory disease. in people, both viruses can also manifest relapsed encephalitis following recovery from an acute infection, particularly niv. the recognized natural reservoir hosts of hev, niv, and cedpv are pteropid bats, which do not show clinical illness when infected. with spillovers of hev continuing to occur in australia and niv in bangladesh and india, these henipaviruses continue to be important transboundary biological threats. niv in particular possesses several features that highlight a pandemic potential, such as its ability to infect humans directly from natural reservoirs or indirectly from other susceptible animals along with a capacity of limited human-to-human transmission. several henipavirus animal challenge models have been developed which has aided in understanding hev and niv pathogenesis as well as how they invade the central nervous system, and successful active and passive immunization strategies against hev and niv have been reported which target the viral envelope glycoproteins. the genus henipavirus in the family paramyxoviridae is presently represented by three known virus isolate species hendra virus (hev), nipah virus (niv) and cedpv (cedpv) and are enveloped, single-stranded negative-sense rna viruses (wang et al. 2013b ; marsh et al. 2012 ) . hev and niv are bat-borne disease-causing zoonoses while cedpv also resides in the same bat species as does hev in nature. studies have shown that cedpv is not pathogenic in animals susceptible to hev and niv disease, nor is it known to be zoonotic. to date, bats appear to be predominant natural reservoir hosts for henipaviruses (clayton et al. 2013 ) and recently, by nucleic acid based detection surveys, there has been a signifi cant species expansion of the henipavirus ranks including at least two full genome sequences , and also a report of one henipavirus from a rodent, but to date hev, niv, and cedpv are the only virus isolates that have been reported (wu et al. 2014 ; drexler et al. 2012 ) . central pathological features of both hev and niv infection in humans and several susceptible animal species is a severe systemic and often fatal neurologic and/or respiratory disease (abdullah and tan 2014 ; wong and ong 2011 ; playford et al. 2010 ) . of additional concern in people, both viruses, but particularly niv, can also manifest as relapsing encephalitis following recovery from an acute infection resulting from a recrudescence of virus replication in the central nervous system (cns) (wong and tan 2012 ; wong et al. 2009 ). spillovers of hev have continued to occur in australia since its identifi cation, as does niv in bangladesh and india, since its recognition in malaysia, which continue to make these henipaviruses an important transboundary biological threat . niv in particular possesses several features that highlight a pandemic potential, such as its ability to infect humans directly from natural reservoirs or indirectly from other susceptible animals along with a capacity of limited human-to-human transmission (luby 2013 ) . several henipavirus animal challenge models have been developed which has aided in understanding how hev and niv invade the central nervous system de wit et al. 2014 ) , and successful active and passive immunization strategies against henipaviruses have been reported which target the viral envelope glycoproteins broder 2012 ; broder et al. 2012 ). a new paramyxovirus was isolated and identifi ed in 1994 in an outbreak of fatal cases of respiratory disease in horses and humans in the brisbane suburb of hendra, australia, and was shown to be distantly related to measles virus and other morbilliviruses (murray et al. 1995a ) . thirteen horses and their trainer succumbed to the infection by this previously unknown virus, along with the non-fatal infection of seven other horses and a stable hand. in an unrelated and only retrospectively identifi ed spillover of this same virus near mackay in central queensland, ~1000 km north of brisbane, a farmer experienced a brief aseptic meningitic illness after caring for and assisting at the necropsies of two horses that were only later shown to have died from this virus infection (hooper et al. 1996 ; rogers et al. 1996 ) . thirteen months later this individual suffered severe fatal encephalitis resulting from that initial virus infection characterized by uncontrolled focal and generalized epileptic activity (o'sullivan et al. 1997 ) . the virus was provisionally termed equine morbillivirus but was later re-named hev where the initial recognized outbreak had occurred. to date, hev has since reemerged in eastern australia on 55 occasions with more than 97 horse deaths, 2 hev antibody positive euthanized dogs, and 4 of 7 human case fatalities anonymous 2012 anonymous , 2013a anonymous , b , 2014a . although hev infection was detected in two dogs in recent years, the extent of hev transmission from bats to dogs in australia is unknown, and all recognized hev spillovers and all cases of confi rmed human infections, the horse has served as an intermediate host between the virus-shedding bat reservoir and humans. the epidemiological features and potential mechanisms at play of hev emergence and continued spillovers have been examined (plowright et al. 2011 ) and reviewed elsewhere (field et al. 2007 . niv emerged just a few years later following the initial recognition of hev. a large outbreak of encephalitis among pig farmers in peninsular malaysia began in 1998 and continued into the next year (chua et al. 1999 ). this outbreak was initially attributed to japanese encephalitis virus because it occurred among people in close contact with pigs. however, several features distinguished this outbreak from japanese encephalitis such as patients were primarily adults not children, along with household clustering of cases being noted, and many of those affl icted had previously been vaccinated against japanese encephalitis (chua et al. 1999 ) . a syncytia-forming virus in vero e6 cell culture was obtained from the cerebrospinal fl uid (csf) of two patients which cross-reacted with antibodies against hev and several patients had igm antibodies in their csf that were reactive against hev (chua et al. 1999 ) . later molecular genetic studies confi rmed the close relationship of this new paramyxovirus, termed niv, to hev (chua et al. 2000a ) . there were at least 265 cases of human infection with 105 fatalities in malaysia along with an additional 11 cases and 1 fatality among abattoir workers in singapore (chua et al. 2000a ; paton et al. 1999 ) . the chronology of the events and the epidemiological features of this outbreak, including potential causes and the factors that exacerbated this outbreak, as well as the pathological observations made in both animals and humans have been critically reviewed and recently examined elsewhere (wong and tan 2012 ; wong and ong 2011 ; chua 2003 ; pulliam et al. 2012 ). niv has not reappeared in malaysia, however nearly annual outbreaks of niv infection have now been recognized since 2001, occurring primarily in bangladesh but also india. the most recent cases of human infections occurred in early 2015 with two fatalities (anonymous 2015 ) . the spillovers of niv in bangladesh and india have had lower numbers of human infections; however the fatality rates have been notably higher from 75 to 100 %. in addition, direct transmission of niv from bats to humans from the consumption of contaminated date palm sap along with signifi cant human-tohuman transmission has now been documented homaira et al. 2010a , b ; luby et al. 2009b ) . the epidemiological details of the spillovers of both hev and niv into people since their emergence and recognition have recently been reviewed and summarized in detail luby and broder 2014 ) . there have been ~613 human cases of niv infection with 315 fatalities (reviewed in luby et al. 2009b ; broder 2012 ; anonymous 2014c anonymous , 2015 . both hev and niv are highly pathogenic in a number of mammalian species and possess several characteristics that distinguish them from all other known paramyxoviruses and are classifi ed as biosafety level-4 (bsl-4) agents. finally, although not associated with a zoonotic event, the third recognized henipavirus species as a virus isolate was recently identifi ed . urine sample collecting for pcr and virus isolation experiments were being carried out as part of fi eld studies on hev genetic diversity and infection dynamics in fl yingfox populations in queensland, australia. from these studies a syncytia-inducing virus was identifi ed in pteropus bat kidney cell culture isolated from samples collected in september 2009 from a fl ying-fox colony in cedar grove, south east queensland . molecular analysis indicated that this virus was a new paramyxovirus most closely related to hev and niv and the virus was named cedpv after the location of the bat colony sampled. animal challenge studies with cedpv in guinea pigs and ferrets which are susceptible to infection and disease with hev and niv, revealed that while cedpv replication occurred and induced neutralizing antibodies, no clinical disease was apparent ). soon after the discovery and isolation of hev, a state-wide serologic survey of 2411 horses reported no evidence of infection and only horses involved in the initial brisbane outbreak were positive (ward et al. 1996 ) . this was followed by a large serological survey conducted across eastern queensland, australia in an effort to identify the potential natural host(s) of the virus, and 5264 sera samples across 46 species, mostly wildlife, were screened and no evidence of hev neutralizing antibody was found (young et al. 1996 ) . however, the additional screening of potential animal reservoirs that overlapped the two initial but distant hev spillover events led to the testing of the four fruit bat species (fl ying foxes) native to mainland australia, and here serological evidence was found in all four species of pteropus fruit bats (young et al. 1996 ) . hev was later isolated from the gray-headed fl ying fox ( pteropus poliocephalus ) and the black fl ying fox ( p. alecto ) (halpin et al. 2000 ) . following the fi rst appearance of niv in peninsular malaysia, a serological surveillance study on samples from 324 bats across 14 species revealed the presence of niv neutralizing antibodies in island fl ying-foxes ( p. hypomelanus ) and malayan fl ying foxes ( p. vampyrus ) . a follow-up study focusing on virus isolation by collecting pooled urine samples from island fl ying foxes, as well as partially eaten fruit, reported the isolation of niv . niv has since been isolated from the urine of p. lylei in cambodia (reynes et al. 2005 ) . serological assays as a means of detection of the presence of niv and/or hev in nature, from wildlife, domestic animals and human populations, is more readily achievable as compared to either virus isolation or nucleic acid detection (mcnabb et al. 2014 ) . a number of serological surveys have been carried out over the past several years to screen for the presence of henipaviruses in bats, domestic livestock and people. the preponderance of data indicates that the pteropus bat species appear to be the major natural reservoir hosts for henipaviruses yadav et al. 2012 ; wacharapluesadee et al. 2010 ; epstein et al. 2008 ; iehle et al. 2007 ) and all bat isolates of hev, niv and also cedpv have been derived from pteropus bats (halpin et al. 2000 ; chua et al. 2002 ; reynes et al. 2005 ; rahman et al. 2010 ; marsh et al. 2012 ) (see also chap. 26). further, as natural hosts, a lack of any observed overt disease in wild bats is also in agreement with a lack of elicited clinical signs in experimentally infected pteropid bats (middleton et al. 2007 ; williamson et al. 1998 williamson et al. , 2000 halpin et al. 2011 ) . pteropus bat species are distributed as far west as madagascar, through the indian subcontinent to southeastern asia and australia, and eastwards through oceania (clayton et al. 2013 ; breed et al. 2013 ; field et al. 2001 ) . however, there is evidence of henipaviruses in wide variety of other bat species in both megachiroptera and microchiroptera suborders (hayman et al. 2008 ; peel et al. 2012 peel et al. , 2013 hasebe et al. 2012 ; wacharapluesadee et al. 2005 ; li et al. 2008 ; drexler et al. 2009 drexler et al. , 2012 . most recently, a novel henipa-like virus, mojiang paramyxovirus (mojv), was identifi ed in rats ( rattus fl avipectus ) in china by nucleic acid analysis, with a genome length of 18,404 nt; however no virus isolate was obtained (wu et al. 2014 ) . also, serological and/or nucleic acid evidence of henipa-viruses in domestic livestock and in human populations have been reported providing evidence of sporadic henipavirus spillover events and also suggesting the existence of less pathogenic-related henipavirus. these fi ndings included henipavirus presence in domestic pigs in ghana, west africa; cattle, goats, and pigs in bangladesh; horse and humans in the philippines, and human populations in cameroon, africa (ching et al. 2015 ; pernet et al. 2014 ; chowdhury et al. 2014 ; hayman et al. 2011 ) . only the incident in the philippines was associated with a disease outbreak with evidence of horse-to-human and human-to-human transmission with niv as the likely cause (ching et al. 2015 ) . genomic sequence analysis revealed that hev isolates obtained from horses and a fatal human case in 1994 were essentially identical and both were highly similar to genomic sequences later obtained from p. poliocephalus and p. alecto 2 years after the initial outbreak (halpin et al. 2000 ; murray et al. 1995b ) . also, sequence analysis of fi ve hev isolates obtained from horses in australia; murwillumbah, in new south wales (2006) , and peachester (2007), clifton beach (2007), redlands (2008), and proserpine (2008) all in queensland, revealed identical genome lengths of 18,234 nt and sequence variation across the full genomes was <1 % (marsh et al. 2010 ) . similarly, in the initial malaysian outbreak of niv, both pig and human isolates were genetically similar to those obtained some years later from island fl yingfoxes ( p. hypomelanus ) (abubakar et al. 2004 ; chan et al. 2001 ; chua et al. 2002 ; harcourt et al. 2000 ) . however, a greater diversity among niv isolates is seen when comparisons are made between the malaysian isolates to the more recent niv isolates from other areas of southeast asia. the fi rst niv isolate from outside of malaysia came from bangladesh . characterization of the genome of niv-bangladesh revealed a length of 18,252 nt, 6 nt longer than the prototype niv-malaysian isolate, with a genome homology between them of 91.8 % . also, in that study, four niv-bangladesh isolates were examined showing a 99.1 % nt homology with interstrain nucleotide heterogeneity suggesting multiple spillovers of niv-bangladesh into people from varying bat sources. a third lineage of niv was isolated from lyle's fl ying fox ( p. lylei ) in cambodia and nucleocapsid (n) gene sequence analysis revealed this isolate to be more closely related to niv-malaysia than to niv-bangladesh (reynes et al. 2005 ; wacharapluesadee et al. 2010 ) whereas an analysis of nucleic acid sequences of niv derived from human sources from an outbreak in siliguri, india in 2001 revealed an isolate similar to niv-bangladesh (chadha et al. 2006 ) and a full niv genome amplifi ed from patient lung tissue from an outbreak in 2007 in west bengal, india showed 99.2 % nt with the niv-bangladesh isolate from 2004 (arankalle et al. 2011 ). more recently, partial genome sequence analysis of niv derived from an indian fl ying fox ( p. giganteus ) obtained from myanaguri, west bengal, india, revealed an n gene with 100.0 % homology with niv sequences from those prior outbreaks in india and with niv-bangladesh sequences, and a 96.0 % identity with niv isolates from cambodia and malaysia (yadav et al. 2012 ). in addition to the demonstration of at least three distinct virus isolate lineages of niv; malaysia, bangladesh and cambodia (wang et al. 2013b ), other nucleic acid based studies have signifi cantly expanded the genus henipavirus (drexler et al. 2012 ) . nineteen newly identifi ed virus species classifi ed into the genus henipavirus have been identifi ed, along with one full genome sequence , 18,530 nt, (gh-m74a) from a bat spleen ( eidolon helvum ) from ghana confi rmed classifi cation in the genus henipavirus (drexler et al. 2012 ) . cedpv is the third recognized species of henipavirus as a virus isolate . cedpv was isolated from pooled urine samples from a colony of predominantly p. alecto also with some p. poliocephalus . the cedpv genome is 18,162 nt and its organization was shown to be similar to that of hev and niv. also, some antigenic cross-reactivity of the cedpv n protein was noted with that of niv and hev; and cedpv was shown to utilized ephrin-b2 as entry receptor (discussed in the next section). henipavirus particles are enveloped and pleomorphic, with a size ranging from 40 to 1900 nm and can vary from spherical to fi lamentous forms when imaged by electron microscopy (hyatt et al. 2001 ; goldsmith et al. 2003 ; murray et al. 1995b ) . the viral envelope carries surface projections composed of the viral transmembraneanchored fusion (f) and attachment (g) glycoproteins (fig. 1 ). henipavirus genomes are unsegmented, single-stranded, negative-sense rna (wang et al. 2013b ). at the time of their discovery, the genomes of niv and hev were the largest amongst all members of the paramyxoviridae family, a factor considered in their classifi cation into their own genus, henipavirus (wang et al. 2000 ) . this increase in genome length is primarily attributable to additional nucleotides in 3′ untranslated regions of each transcription unit except the large/polymerase (l) gene (wang et al. 2000 harcourt et al. 2000 ) . as with all characterized members of the subfamily paramyxovirinae , the hev, niv and cedpv genomes and are divisible by six, conforming to the "rule of six" which relates to the way each n protein molecule interacts with every six nucleotides (lamb and parks 2013 ; wang et al. 2013b ). the rna genome in association with the n protein is also referred to as the ribonucleoprotein core that has a characteristic herringbone appearance by electron microscopy (wang et al. 2013b ) and is contained within a lipid bilayer (envelope) that is derived from the infected host cell during virus assembly and budding (fig. 1 ) . the relative gene order is conserved as compared to other paramyxoviruses, with the n gene being fi rst, followed by the p (phosphoprotein), m (matrix), f, g and l genes in a 3′ to 5′ order (fig. 1 ). gene transcription occurs in a gradient manner because of a failure of the rna polymerase to reinitiate transcription at downstream genes and those genes located towards the 3′ end are transcribed more abundantly than genes towards the 5′ (lamb and parks 2013 ) . the n, p, and l proteins form a complex that is responsible for replication of viral rna; polymerase activity resides within the l protein (lamb and parks 2013 ) . in addition to the full-length unedited p gene product, the henipavirus p gene (the largest among the paramyxoviruses) also encodes the v and w proteins which are produced through a transcriptional editing mechanism involving addition of nontemplated g nucleotides, as well as the c protein, which is encoded by an alternative start site within the p gene (lamb and parks 2013 ) . products of the p gene can antagonize both double-stranded (ds) rna signaling and interferon (ifn) signaling (reviewed in shaw 2009 ; basler 2012 ) . the v protein functions in anti-ifn induction or dsrna signaling, similar to that of other paramyxoviruses, by targeting the helicase encoded by the melanoma differentiationassociated gene 5 (mda5) . whereas the niv w protein could also inhibit dsrna signaling but does so by nuclear translocation, targeting interferon regulatory factor 3 (irf-3) and effectively blocking both dsrna signaling via mda5 and through the cell surface expressed toll-like receptor 3 (tlr-3) signaling pathway. henipaviruses also target the paracrine signal transduction pathway that is initiated by the binding of type i ifn to the two cell surface interferon alpha and beta receptors, ifnar1 and ifnar2 which assemble into a functional receptor complex leading to the activation of signal transducers and activators of transcription (stat) factors where they later direct the expression of genes possessing an interferon stimulated response element (isre) within the nuclease (reviewed in de weerd et al. 2007 ). the henipavirus v, w and p proteins block the type i ifn signaling pathway with the niv v and p proteins forming high-molecular weight complexes in the cytoplasm with stat1, and the niv w protein targeting stat1 within the nuclease (reviewed in detail (shaw 2009 ; basler 2012 ) ). in contrast, major difference between niv and hev with cedpv was noted in that the p gene lacks both rna editing and also the coding capacity for the v protein which may be a factor that limited its observed in vitro pathogenesis . the diverse ways that niv and hev can antagonize the host interferon responses are believed to be important factors that infl uence their pathogenic potential. the henipavirus m protein, which underlies the viral membrane ( fig. 1 ) , plays a key role in organization of viral proteins during the process of virion assembly and budding from the host cell, and the niv m protein possesses the ability to bud from expressing cells independent of any other viral proteins forming virus-like particles (ciancanelli and basler 2006 ; patch et al. 2007 ). sequence motifs with the m protein have been identifi ed that may act as traffi cking signals to facilitate the budding process (patch et al. 2008 ; ciancanelli and basler 2006 ; harrison et al. 2010 ). finally, the g and f envelope glycoproteins are located on the surface of the virion, appearing as spikes projecting from the envelope membrane of the viral particle ( fig. 1 ) and are essential for the binding and entry steps of the virus into permissive host cells (reviewed in bossart et al. 2013 ; steffen et al. 2012 ). the henipavirus g glycoprotein is a homo-tetramer and responsible for attachment of the virion to entry receptors on the host cell and the f glycoprotein is a homotrimer responsible for facilitating the fusion of the viral membrane with that of the host cell (reviewed in steffen et al. 2012 ) . additional details of the henipavirus envelope glycoproteins will be discussed below with regard to cellular tropism and as the targets of antiviral strategies. the exceptionally broad species tropism of henipaviruses, as represented by niv and hev, distinguishes them from all other known paramyxoviruses (wang et al. 2013b ). in addition to their principle natural hosts, pteropid bats, niv is known to have naturally infected pigs, horses, cats, dogs and humans, and experimental infections with disease in guinea pigs, cats, hamsters, ferrets, squirrel monkeys and african green monkeys have been demonstrated. in addition, niv can also productively infect chicken embryos with severe pathology (tanimura et al. 2006 ) . hev in nature appears less transmissible and naturally acquired infections have been observed only in bats, horses, dogs and humans; however, experimentally, hev can infect and cause disease in guinea pigs, cats, hamsters, ferrets, mice and african green monkeys (reviewed in geisbert et al. 2012 ) taken together, henipavirus infections seven orders (six mammalian and one avian). the henipavirus membrane anchored envelope glycoproteins (g and f) are the mediators of virus attachment and host cell infection and a major determinant of cellular tropism. the g glycoprotein is the henipavirus attachment glycoprotein and has neither hemagglutinating nor neuraminidase activities; activities associated with many other paramyxovirus attachment glycoproteins known as hemagglutinin-neuraminidase (hn) or the hemagglutinin (h) protein (wang et al. 2013b ; lamb and parks 2013 ) . the niv and hev g glycoprotein engage host cell membrane proteins as entry receptors and bind to ephrin-b2 and ephrin-b3 (negrete et al. 2005 (negrete et al. , 2006 bonaparte et al. 2005 ; bishop et al. 2007 ). the ephrin-b2 and -b3 molecules are members of a large family of cell surface expressed glycoprotein ligands that bind to eph receptors, the largest subgroup of receptor tyrosine kinases (drescher 2002 ; poliakov et al. 2004 ). the eph receptors and their ephrin ligands comprise an important group of bidirectional signaling molecules in a variety of cell-cell interactions including those of vascular endothelial cells and are modulators of cell remodeling events within the nervous, skeletal and vascular systems (pasquale 2010 ; lackmann and boyd 2008 ) . ephrin-b2 expression is prominent in arteries, arterioles and capillaries in multiple organs and tissues (gale et al. 2001 ) while ephrin-b3 is found predominantly in the nervous system and the vasculature (reviewed in poliakov et al. 2004 ; pasquale 2008 ) . the ephrin-b2 and -b3 molecules are highly sequence conserved across susceptible hosts including human, horse, pig, cat, dog, mouse and bat with amino acid identities of 95-96 % for ephrin-b2 and 95-98 % for ephrin-b3 . the identifi cation of ephrin-b2 as a major receptor for niv and hev has aided in the understanding and clarifi cation of both their broad species and tissue tropisms, as well as the resultant pathogenic processes that are seen in humans and animal hosts (reviewed in hooper et al. 2001 ; wong and ong 2011 ) . similar to most paramyxoviruses, the henipaviruses have two membraneanchored glycoproteins that are required for virus entry. the henipavirus attachment glycoprotein (g) is a type ii membrane protein with the amino (n)-terminus oriented towards the cytoplasm and the carboxy (c)-terminus extracellular . the g glycoprotein is comprised of a stem (or stalk) and a globular head domain which binds ephrin receptors. the native conformation of g is a tetramer, which is comprised of a dimer of dimers . the crystal structures of both niv and hev g globular head domains have been determined both alone and in complex with the ephrin-b2 and -b3 receptors, revealing the exact g-receptor interactions and identical receptor binding sites; with four binding pockets in g for the residues in the ephrin-b2 and -b3 g-h loop that are highly conserved (bowden et al. 2008a (bowden et al. , b , 2010 xu et al. 2008 xu et al. , 2012 . the second protein is the fusion (f) glycoprotein that facilitates the fusion of the viral and host cell membranes. f is a type i membrane glycoprotein with an extracellular n-terminus and is a class i viral fusion protein sharing several conserved features with other viral fusion glycoproteins . f is initially expressed as a precursor (f 0 ) which forms an oligomeric trimer that is cleaved into two disulfi de bond-linked subunits (f 1 and f 2 ) by the endosomal protease cathepsin l (pager and dutch 2005 ) . unique to the henipaviruses, the processing of f 0 into its biologically active form is a multi-step process requiring recycling of f 0 from the cell surface into an endosomal compartment, mediated by an endocytosis motif present in the cytoplasmic tail of f (meulendyke et al. 2005 ; vogt et al. 2005 ) . after cleavage, the homotrimer of disulfi de bond-linked f 1 and f 2 subunits is traffi cked back to the cell surface. the f glycoprotein contains two α-helical heptad repeat domains that are involved in the formation of a trimer-of-hairpins structure which facilitates membrane merger and peptides corresponding to either heptad repeat domains can inhibit the fusion activity of f when present during the fusion process (reviewed in bossart et al. 2013 ) . the henipavirus g and f glycoproteins work cooperatively to mediate membrane fusion and particle entry into the host cell. following virus attachment to a receptor-bearing host cell, the fusion-promoting activity of the g glycoprotein is initiated by engaging ephrin receptors and the g glycoprotein then facilitates the triggering of conformational changes in f, transitioning f conformation from a prefusion to post-fusion form driving the membrane fusion process between the virion and plasma membranes, resulting in delivery of the viral nucleocapsid into the cytoplasm (reviewed in aguilar and iorio 2012 ; lee and ataman 2011 ). in a related process, virus-infected cells expressing attachment and fusion glycoproteins on their surface can fuse with receptor-bearing cells leading to the formation of multinucleated giant cells (syncytia)-a hallmark of many paramyxovirus infections including the henipaviruses (wang et al. 2013b ) . the incubation period of human niv and hev infections ranges from a few days to about 3 weeks mahalingam et al. 2012 ) . to date, there have been only seven known cases of human hev infection, so much less is known about its clinical manifestations compared to niv infection. following an infl uenza-like illness (fever, myalgia, headaches, lethargy, vertigo, cough, pharyngitis, and cervical lympadenopathy), the majority developed severe disease and died; only two patients survived (mahalingam et al. 2012 ; selvey et al. 1995 ; playford et al. 2010 ). thus the mortality was about 60 %. three patients had an acute encephalitic syndrome characterized by drowsiness, confusion, ataxia, ptosis, dysarthria and seizures and died soon after. one patient had an acute pulmonary syndrome described as a pneumonitis with chest radiograph fi ndings of diffuse alveolar shadowing (selvey et al. 1995 ) . although clinical acute encephalitis was never suspected, apart from pulmonary pathology, this patient's brain at autopsy also showed features of acute encephalitis ). interestingly, abnormal chest radiographs were also described in two other clinical encephalitis cases. in one patient following relatively mild aseptic meningitis associated with headache, drowsiness, vomiting and neck stiffness, clinical features of probable meningoencephalitis , he presented 13 months later with full blown fatal encephalitis (o'sullivan et al. 1997 ) . in retrospect, this was the fi rst case of relapsing henipavirus encephalitis. the brain magnetic resonance (mr) scans available in three acute encephalitis patients showed multifocal hyperintensive lesions in the cerebrum and brainstem, and leptomeningeal enhancement . in the case of relapsing encephalitis, extensive, predominantly cortical hyperintense lesions were observed (mahalingam et al. 2012 ). based on a large cohort of 94 patients with niv infection from a single institution, the main features of acute infection was fever, headache, dizziness, and vomiting . a majority of patients had reduced consciousness levels and signs of brainstem dysfunction. other distinctive clinical signs included segmental myoclonus, arefl exia, hypotonia, hypertension, and tachycardia. the cerebrospinal fl uid obtained from lumbar puncture showed elevated leukocyte counts and protein levels. electroencephalogram abnormalities consisting of diffuse slow waves (continuous or intermittent) with or without focal sharp waves were observed, and in general correlated with disease severity. brain mr scans of acute niv infection were characterized by disseminated, multiple hyperintense lesions mainly in subcortical and deep white matter of the cerebrum with no associated edema or mass effect or correlation with severity of neurological signs. chest radiographs were reported to be abnormal in some patients paton et al. 1999 ) . the risk factors for severe disease and poor prognosis included abnormal doll's eye refl ex, tachycardia, and the presence of virus in the cerebrospinal fl uid (chua et al. 2000b ) , and diabetes mellitus (chong et al. 2001b ) . a small number, probably <10 %, of patients with acute niv infection developed a late-onset encephalitis (in symptomatic patients with no previous encephalitis or patients with asymptomatic seroconversion) or a relapsing encephalitis (in patients with previous encephalitis) a few weeks later. although potentially fatal, the mortality at about 18 % is considerably lower that acute encephalitis . the clinical features of late-onset encephalitis and relapsing encephalitis are similar to acute encephalitis. however, some features like fever, coma, brainstem signs, segmental myoclonus and meningism were less commonly observed, while seizures and focal cortical signs were more frequent. cerebrospinal fl uid pleocytosis was common but no virus could be isolated. the brain mr scans showed confl uent geographical abnormalities, especially in the cortical gray matter that is strikingly different from acute niv encephalitis . although most niv-infected human patients presented with acute encephalitis, some 25 % of patients also presented with respiratory signs, some cases also presented as a non-encephalitic or asymptomatic infection with seroconversion (chua 2003 ) . niv infection could also take a chronic and quiescent course with neurological disease occurring later (>10 weeks) following a non-encephalitic or asymptomatic infection. a recrudescence of neurological disease, also termed relapsing encephalitis, was also observed in some patients who had previously recovered from an acute encephalitic infection. here, there is a recrudescence of virus replication in the cns. most reported cases of relapsed encephalitis presented from a few months to approximately 2 years following the initial acute infection, however two cases of relapsed encephalitis were observed in 2003 4 years later (wong et al. 2001 ; chong and tan 2003 ; tan and wong 2003 ) and the longest reported case of niv encephalitic recrudescence is 11 years (abdullah et al. 2012 ) . this recrudescence of henipavirus encephalitis was fi rst noted in the second fatal human case of hev infection which presented with similar fi ndings (o'sullivan et al. 1997 ; wong et al. 2009 ). interestingly, evidence of recrudescence of niv infection in pteropus bats has also been reported (sohayati et al. 2011 ) as well as hev infection modeling in fl ying-fox populations (wang et al. 2013a ). there is no evidence of hev shedding in people who have recovered from infection (taylor et al. 2012 ) . persistent neurological defi cits have been observed in >15 % of niv infection survivors ). in addition, recent studies have also assessed the long-term neurologic and functional outcomes of >20 individuals surviving symptomatic niv infection in bangladesh (sejvar et al. 2007 ). in bangladesh, the outcomes among 22 of 45 serologically confi rmed cases of niv infection revealed neurological sequelae in survivors, and patients who initially had encephalitis could continue to exhibit neurological dysfunction for several years (sejvar et al. 2007 ). both persistent and delayed-onset neurological sequelae were noted, including a higher proportion of persistent behavioral disturbances including violent outbursts and increased irritability among pediatric patients (sejvar et al. 2007 ). viral persistence and/or recrudescence within the cns are suspected to be at play in these individuals . the mechanisms that allow niv and hev to escape immunological clearance for such an extended period and later result in disease are unknown, and this characteristic of niv and hev has important implications for therapeutics development. hev spillovers in australia have occurred annually since 2006 and to date there have been seven human cases of which four have been fatal (playford et al. 2010 ) . all human cases of hev infection was the result of exposure and transmission of the virus from infected horses to humans. the fi rst human case presented as an acute severe respiratory disease but no clinical evidence of acute encephalitis. at autopsy, the lungs showed macroscopic evidence of congestion, hemorrhage and edema (selvey et al. 1995 ) associated with focal necrotizing alveolitis and evidence of syncytia and multinucleated giant cell formation, and viral inclusions. focal vasculitis was also noted in some pulmonary vessels. viral antigens were localized by immunostaining to alveolar type ii pneumocytes , intra-alveolar macrophages and blood vessels ). although clinical encephalitis was apparently absent, the brain pathology clearly showed acute encephalitis characterized by mild meningitis, parenchymal and perivascular infl ammation . more importantly, there was evidence of neuronal viral inclusions, vasculitis and necrotic/vacuolar plaques. viral antigens/rna were demonstrated in blood vessels, neurons (fig. 2d ) , and ependyma. mild infl ammation could also be found in the lymph node and kidney where viral antigens were detected in glomeruli and renal tubules. panels ( a , b , d , f ) from wong and ong ( 2011 ) , panels ( c , e ) from wong et al. ( 2002 ) a second fatality occurred in an individual who fi rst experienced an aseptic meningitic illness associated with drowsiness caused by hev infection acquired after assisting at the necropsies of two horses that were only later shown to have died from hev infection. approximately 13 months later this individual suffered a recurrence of severe encephalitis characterized by uncontrolled focal and generalized epileptic activity. infl ammatory lesions were only found in the cns, not in other organs obtained at ). extensive lesions were found mainly in the meninges and cerebral cortex, but focal lesions were also found in the cerebellum, pons and spinal cord. there was intense infi ltration of the parenchyma and perivascular areas by macrophages, lymphocytes, and plasma cells together with severe neuronal loss, reactive glial, and vascular proliferation. although viral inclusions were not prominent, viral antigens/rna were detected in neurons, glial, and/or infl ammatory cells. interestingly, there was no evidence of vasculitis or endothelial syncytia in the cns, as well as absence of these and other features of infl ammation in all the non-cns organs examined. in the fi rst niv outbreak in malaysia and singapore, autopsies were conducted on >30 individuals which has afforded a better understanding of the pathology of niv in comparison to that of hev infection. these autopsies were mostly in individuals, including pig farm workers and farmers, who in one way or another had contact with sick pigs. the macroscopic features were generally non-specifi c. perhaps the most distinctive microscopic feature is the disseminated vasculitis found in most organs examined, particularly in the cns and lungs. the fully developed, typical vasculitic lesion comprised focal segmental infl ammation of the vascular wall, endothelial ulceration and thrombosis (fig. 2a ) . the rare endothelial multinucleated syncytia may occasionally be found in early vasculitis (fig. 2b ) . viral antigens and nucleocapsids can be demonstrated in blood vessels. extravascular necrotic lesions and infl ammation in many organs can also be seen. in the cns parenchyma, distinct necrotic plaques (fig. 2e ) arising from vasculitis-induced vascular obstruction, ischemia and infarction and/or neuronal infection were commonly found. neurons in or around necrotic plaques and other infl amed neuronal areas often showed the widespread presence of viral antigens (fig. 2c ) . glial cells were much more rarely involved. viral inclusions in neurons in the cns and other cells in non-cns tissues were also observed. apart from vasculitis, infl ammation, necrosis, and the rare multinucleated giant cells or syncytia involving extravascular tissue in the lung, spleen, lymph node, and kidney (fig. 2f ) , were reported hooper et al. 2001 ; wong 2010 ) . the combination of disseminated, vasculitis-induced thrombosis, vascular occlusion, and microinfarction, together with direct infection of parenchymal cells suggest a unique dual pathogenetic mechanism for tissue injury in acute niv infection. this appears to hold true for acute hev infection as well. certainly in the cns, extensive virus-associated vasculopathy, with or without neuroglial infection, as a signifi cant cause of tissue injury is probably unique. the pathological features in the few autopsy cases of niv relapsing or late-onset encephalitis and the single case of hev relapsing encephalitis were similar and confi ned mainly to the cns (wong and tan 2012 ; tan et al. 2002 ) . there was extensive and severe meningoencephalitis with parenchymal and perivascular infl ammation, severe neuronal loss and reactive gliosis. viral inclusions , antigens/ rna could be detected but vasculitis were absent (wong 2010 ) . indeed, vasculitis or other vasculopathies which were readily found in the acute infection, were absent in the cns and extra-cns organs. in addition to hev and niv infection of bats (middleton and weingartl 2012 ) , detailed reviews of the disease manifestations observed in natural and experimental infections of animals with hev and niv have recently been reported (dhondt and horvat 2013 ; geisbert et al. 2012 ; wong and ong 2011 ) . as mentioned previously, natural hev infections have almost exclusively been observed in horses, and only recently have two dogs been reported hev antibody positive. whereas in addition to pigs, naturally acquired niv infection was noted in dogs, cats and horses in the initial malaysian outbreak . serological studies of natural niv infection revealed that dogs in areas associated with farms in the malaysian outbreak were susceptible to infection ). however, diseased dogs were not prevalent with only two animals examined (one dead and one sick) wong and ong 2011 ) . in bangladesh, a few cases of human niv infection were associated with sick animal contact including cows (hsu et al. 2004 ) , pigs, and goats , and recently serological evidence of henipavirus infection in cattle, goats and pigs in bangladesh has been reported (chowdhury et al. 2014 ). the development of animal models of henipavirus infection and pathogenesis has been critical for understanding henipavirus pathogenesis and also needed for the evaluation of potential vaccines and therapeutics. several well-established animal models of hev and niv infection and pathogenesis have been developed and include the guinea pig (williamson et al. 2000 ; 2001 #3773; middleton et al. 2007 ), hamster (guillaume et al. 2009 ; wong et al. 2003 ) , cat middleton et al. 2002 ; williamson et al. 1998 ) , pig (li et al. 2010 ; weingartl et al. 2005 ; middleton et al. 2002 ) , ferret (pallister et al. 2011 ; bossart et al. 2009 ), african green monkey (agm) geisbert et al. 2010 ) , squirrel monkey (marianneau et al. 2010 ) and horse (marsh et al. 2011 ). among these models, the pathogenic processes of henipavirus infection in the hamster, ferret and agm best represent the pathogenesis observed in humans; whereas the most appropriate models for livestock are the pig and horse. the syrian golden hamster and niv challenge was the fi rst successful small animal model of henipavirus infection and pathogenesis . niv infection in the hamster produced severe lesions in the brain, with animals succumbing to infection 5-9 days after intraperitoneal infection, 24 h following the development of tremors and limb paralysis. hamsters inoculated intranasally survived ~5 days longer post-challenge, displaying progressive neurological signs and breathing difficulties. vascular pathology was widespread, involving the brain and lung, with endothelial cell infection. the vascular and parenchyma lesions were consistent with cns-mediated clinical signs . another study showed that higher doses of niv resulted in an acute respiratory distress syndrome (ards) while lower doses would yield the development of neurological signs and more widespread infection throughout the endothelium . hev infection of hamsters also produces both respiratory and brain pathology, with endothelial infection and vasculitis, and direct parenchymal cell infection in the cns (guillaume et al. 2009 ). similar to niv infection in hamsters, higher doses of hev resulted in ards and lower doses produced a more neuropathogenic syndrome ). niv infection of ferrets produces both a severe respiratory and neurological disease along with systemic vasculitis following oral-nasal challenge by 6-10 days postinfection (bossart et al. 2009 ; pallister et al. 2009 ). clinical signs in infected ferrets included severe depression, serous nasal discharge, cough and shortness of breath, and tremor and hind limb paresis. pathological fi ndings included vascular fi brinoid necrosis in multiple organs, necrotizing alveolitis, and syncytia of endothelium and alveolar epithelium. severe focal necrotizing alveolitis vasculitis and focal necrosis in a wide range of tissues was observed along with signifi cant levels of viral antigen in blood vessel walls. niv antigen was present within the brain along with infected neurons, and virus isolation from the brain and other organs was reported. hev challenged ferrets, also by the oral-nasal route, rapidly progressed with severe disease 6-9 days following infection with essentially identical fi ndings as seen in nivchallenged ferrets (pallister et al. 2011 ) . the henipavirus disease processes in the ferret accurately refl ects those reported in niv-infected humans and the ferret model has been used in the evaluation of vaccines and therapeutics against henipavirus infections. the fi rst successful nonhuman primate models for both niv and hev infection were developed using the african green monkey (agm) rockx et al. 2010 ) . both niv and hev will produce a uniformly lethal disease process following low dose virus challenge by intratracheal inoculation within 7-10 days post-infection. hev and niv spread rapidly to numerous organ systems within the fi rst 3-4 days following challenge. monkeys begin to develop a progressive and severe respiratory disease ~7 days post-infection rockx et al. 2010 ) . the lungs become enlarged and with high levels of virus replication, congestion, hemorrhage, and polymerized fi brin. widespread vasculitis with endothelial and smooth muscle cell syncytia with viral antigen, along with viral genome was detected in most organs and tissues along with associated pathology. monkeys infected with either niv or hev also exhibit neurological disease signs with the presence of meningeal hemorrhaging and edema, and vascular and parenchymal lesions in the brain including infection of neurons with in the brainstem particularly involved (fig. 3 ) rockx et al. 2010 ). rockx et al. ( 2010 ) the squirrel monkey was also found to be susceptible to experimental niv infection via intravenous and intranasal routes demonstrating fi ndings similar to agm and human infection (marianneau et al. 2010 ) . vasculopathy and parenchymal cell infection were found in the cns, lungs and other organs. niv infection of pigs revealed the respiratory system as a major site of virus replication and pathology, with viral antigen and syncytia formation present in the respiratory epithelium (tracheal, bronchial, bronchiolar, and alveolar) and small blood and lymphatic vessels (middleton et al. 2002 ; hooper et al. 2001 ; wong and ong 2011 ) . virus was also observed in the kidneys and in endothelial and smooth muscle cells of small blood vessels (middleton et al. 2002 ) . cns involvement was less common, with meningitis or meningoencephalitis observed as opposed to encephalitis (middleton et al. 2002 ) . niv infection of piglets generally resulted in a mild clinical disease with fever and respiratory signs and virus replication noted in the respiratory system, lymphoid tissues and the cns (weingartl et al. 2005 ) . recoverable virus was recorded in the respiratory, lymphatic and nervous systems, and virus shedding in nasal , pharyngeal, and ocular fl uids was reported. hev infection of pigs also presents as a primarily respiratory disease in both landrace piglets and older gottingen minipigs, with possible cns involvement observed in minipigs, and similar patterns of virus shedding (li et al. 2010 ) . overall, hev appeared to cause a more severe respiratory syndrome in pigs in comparison to niv. although hev and niv disease in pigs is often less severe in comparison to other animal models, the virus does replicate and disseminate to a variety of organs along with signifi cant levels of virus shedding. natural hev infection in horses is often associated with severe disease and experimental infections are essentially uniformly fatal (marsh et al. 2011 ) . naturally infected horses appear to have an incubation period of ~8-11 days and animals initially present as anorexic and depressed with general uneasiness and ataxia, with the development of fever and sweating. respiration becomes rapid, shallow and labored with pulmonary edema and congestion, along with nasal discharge 1-3 days following the onset of clinical signs. in severe cases the airways of horses are often fi lled with a blood-tinged frothy exudate. there was hemorrhage, thrombosis of capillaries, necrosis, and syncytial cells in the endothelium of pulmonary vessels noted. viral antigen was also observed within endothelial cells across a wide variety of organs, with recoverable virus from a number of internal organs as well as from saliva and urine. neurologic clinical signs can also present (rogers et al. 1996 ) . however, in experimentally infected horses, only meningitis (with vasculitis) was noted in all animals (marsh et al. 2011 ) and viral antigen was detected in the meninges of each case. one horse in this study also presented with vasculitis of blood vessels in the brain parenchyma, and hev antigen was also identifi ed within the cerebral blood vessels of this animal (fig. 4 ) (deborah middleton, personal communication) . also, an experimental control horse in middleton et al. ( 2014 ) also had vasculitis with hev antigen in blood vessels within the brain. however, to date hev antigen has not been reported to be present in the neurons of infected horses, but this may be a sampling artefact and/or an observation exacerbated by the fact that the horses are being euthanized and the hev infection is not reaching its full pathogenic expression under experimental conditions. however, the meningitis and infl ammation of cerebral blood vessels in the experimentally infected horses may be suffi cient explanation for the clinical signs of neurological disease in naturally acquired cases of hev infection (deborah middleton, personal communication) . experimental infection of horses with niv has not been performed but the brain and spinal cord of one naturally infected horse was examined and immunohistochemical staining of viral antigen observed revealing non-suppurative meningitis . an array of viruses across many families are known to exhibit neurotropism and there are two central routes of cns invasion; hematogenous spread or via infection of nerve cells (swanson and mcgavern 2015 ; koyuncu et al. 2013 ) . many viruses that cause viremia following the establishment of an initial infection have an opportunity to breach the blood-brain-barrier (bbb); a highly selectively permeable barrier that separates the cns from the peripheral blood circulation (ransohoff et al. 2003 ) . once in the blood, a number of viruses including some herpesviruses, paramyxoviruses, retroviruses, picornaviruses, fi loviruses, and fl aviviruses can directly infect vascular endothelial cells (koyuncu et al. 2013 ) which could allow passage of virus into the cns and/or promote infl ammation and breakdown of the bbb which may also facilitate virus access to the cns (obermeier et al. 2013 ) . alternatively, some viruses can infect myeloid and lymphoid cells and these infected cells can naturally traverse the bbb delivering virus into the cns by the "trojan horse" mechanism (mcgavern and kang 2011 ). a number of neurovirulent paramyxoviruses, particularly the morbilliviruses like measles virus and canine distemper virus, but also mumps virus and newcastle disease virus, can productively infect lymphocytes (joseph et al. 1975 ; krakowka et al. 1975 ; fleischer and kreth 1982 ; hao and lam 1987 ) (see also chap. 2). these infected lymphocytes serve as a cell-associated viremia which can then lead to the delivery of virus into the cns by transmigration through bbb (lossinsky and shivers 2004 ) . cns invasion by niv and hev is a key feature of their pathogenic features in humans and as discussed earlier several animal models have also demonstrated niv and hev cns disease. the widespread and disseminated endothelial infection and vasculitis in henipavirus encephalitis strongly suggest that bbb disruption is an important, if not the most important route, for viral entry into the cns. plaque-like, groups of infected neurons were frequently observed near to infected/vasculitic vessels suggesting centrifugal viral spread from focal bbb damage. however, although niv was shown not to infect human lymphocytes and only low levels of monocyte infection have been reported, human lymphocytes could bind niv and facilitate its transfer and infection to other susceptible cells (mathieu et al. 2011 ) . the traffi cking of such cell-associated infectious niv within a host disseminates the virus and also could potentially deliver niv into cns by leukocyte transmigration . in pigs, however, niv infection of cd6+ cd8+ t lymphocyte has been observed, along with monocytes and nk cells (stachowiak and weingartl 2012 ) . cd6 is a costimulatory molecule involved in lymphocyte activation and differentiation (gimferrer et al. 2004 ) which engages activated leukocyte cell adhesion molecule (alcam/cd166) which is known to promote leukocyte migration across the bbb (cayrol et al. 2008 ) . in this instance, it was suggested that nivinfected cd6+ t cells would elaborate a strong interaction alcam expressed on microvascular endothelial cells which could determine the observed tropism of niv for small blood vessels and also facilitate cns invasion by leukocyte migration. similar studies have not been reported with hev. alternatively, some neurotropic viruses can invade the cns via infection of peripheral nerves (swanson and mcgavern 2015 ) . for example, some neurotropic viruses begin the infection process in one cell type or tissue such as the oropharyngeal and intestinal mucosa in case of poliovirus (see also chap. 1) or in myocytes at the bite site in the case of rabies virus (see also chap. 4) and both later use peripheral motor neurons and retrograde transport to infect the cns (koyuncu et al. 2013 ). in the case of some herpesviruses, initial infection of sensory neurons is followed by retrograde transport and establishment of latency in the peripheral nervous system, and fortunately anterograde transport of herpesviruses to the cns is rare (koyuncu et al. 2013 ) (see also chap. 18). olfactory receptor neurons provide a unique opportunity for neurotropic pathogens to invade the cns because of the direct exposure of dendrites to the environment within the olfactory epithelium, and a few members of several virus families, including fl aviviruses, togaviruses, and bunyaviruses are known to invade the cns via an initial infection of olfactory receptor neurons within the olfactory epithelium and once infected virus can gain access to the cns by transported anterograde transport (mori et al. 2005 ; koyuncu et al. 2013 ) . certain paramyxoviruses have also been shown capable of neuroinvasion via anterograde transport following infection of olfactory neurons (rudd et al. 2006 ; ramirez-herrera et al. 1997 ) . niv infection in pigs is often asymptomatic as discussed above. when disease was noted in naturally infected pigs, neurological disease manifested as trembling, twitches, muscle spasms, and uncoordinated gait (mohd nor et al. 2000 ) . experimental niv infection challenge of landrace female piglets by the ocular and oronasal routes revealed that virus replication occurs in the oropharnyx and then spreads sequentially to the upper respiratory tract and submandibular lymph nodes, followed by replication in the lower respiratory tract, and additional lymphoid tissues, and niv was detected in the nervous system of both sick and apparently healthy animals; including cranial nerves, trigeminal ganglion, brain, and cerebrospinal fl uid. niv invaded the cns via cranial nerves, most importantly via the olfactory nerve, as early as 3 dpi, as well as by crossing the bbb (weingartl et al. 2005 ) . one report of hev infection of landrace and gottingen minipig breeds by oronasal or nasal inoculations produced clinical signs that were primarily respiratory with suggestive neurological involvement seen only in the gottingen minipig. an aged mouse model of intranasal challenge with hev revealed that animals could consistently develop encephalitic disease, and an anterograde route of neuroinvasion of the cns via olfactory nerves was proposed (dups et al. 2012 ) , however in a follow-up study using the same model with niv-bangladesh and niv-malaysia, animals did not exhibit cns disease (dups et al. 2014 ) . as was discussed earlier, in the hamster model for both niv and hev challenge, lower doses of virus allowed for a more neuropathogenic disease state. in an elegant spatial-temporal model of niv infection in the hamster by intranasal inoculation (10 5 tcid 50 ), individual niv-infected neurons were observed extending from the olfactory bulb by 4 dpi, demonstrating direct evidence for virus transport in the cns via olfactory neurons (munster et al. 2012 ) (fig. 5 ) . at 6 dpi, meningoencephalitis was observed, characterized by multifocal men-ingeal and perivascular lymphocytic infi ltration, and in the olfactory bulb neurons and axons of the olfactory nerve layer, glomerular layer and external plexiform layer of the olfactory bulb were positive by niv antigen staining. niv dissemination from the olfactory bulb to the olfactory tubercle region was noted by 6 dpi. from olfactory tubercle region, which is highly innervated to other brain regions including the hypothalamus, thalamus, amygdala, hippocampus and brain stem, spread of niv within the cns is readily possible. similarly, in oronasal challenge models of both niv and hev in the ferret (pallister et al. 2011 ; bossart et al. 2009 ), henipavirus genome and viral antigen were consistently detected in the olfactory lobe of brains along with many animals demonstrating neurological disease such as tremors and hind limb weakness or paralysis. finally, in the agm nonhuman model of niv and hev infection described earlier, consistent neurological disease was observed even though an intratracheal route of challenge is performed, with those animals surviving longer, or those challenged with lower doses of virus, showing more severe neurological disease with signs such as tremors, paralysis and convulsions geisbert et al. 2010 ) (geisbert and broder unpublished) . however, in human niv autopsy studies, involvement of the olfactory bulb has not been demonstrated so far . presently, there are no approved therapeutics for treating hev or niv infection in people, but there have been a few approaches tested in animal models (reviewed in broder 2012 ) . ribavirin is often a fi rst line treatment course for suspected viral infections of unknown etiology, having antiviral activity against many rna and some dna viruses (sidwell et al. 1972 ) and is an accepted treatment against several viruses including respiratory syncytial virus and arenaviral hemorrhagic fevers (reviewed in snell 2001 ) . during the initial niv outbreak in malaysia, some patients were treated with ribavirin and there was some evidence that this therapy may have been clinically benefi cial (chong et al. 2001a ; snell 2004 ) . of the recorded human hev cases , three individuals were treated with ribavirin, and of these, two succumbed to disease and one survived (playford et al. 2010 ) . chloroquine, an antimalarial drug, was shown to block the critical proteolytic processing needed for the maturation and function of the hev f glycoprotein discussed earlier (pager et al. 2004 ) and could block infection in cell culture (porotto et al. 2009 ). however, chloroquine and ribavirin treatment of a hev-infected individual had no clinical benefi t (reviewed in broder et al. 2013 ) . animal studies have also revealed no therapeutic benefi t of either chloroquine or ribavirin. two studies in hamsters and one study in monkeys showed that ribavirin treatment only delayed death after virus infection (freiberg et al. 2010 ; georges-courbot et al. 2006 ; rockx et al. 2010 ) , with hev challenge monkeys treated with ribavirin having marked increases of neurological symptoms. chloroquine treatment was also unable to prevent niv disease in ferrets (pallister et al. 2009 ). also, various forms of poly(i:c) are strong inducers of ifn-α and -β production, have been explored as antiviral therapies for over 40 years. polyic 12 u is very specifi c in triggering the toll-like receptor (tlr)3 pathway (reviewed in nicodemus and berek 2010 ). polyic 12 u was shown capable of blocking niv replication, and continuous administration of polyic 12 u for 10 days beginning at the time of challenge was shown to prevent lethal niv disease in fi ve of six hamsters (georges-courbot et al. 2006 ) , suggesting that use of tlr3 agonists such as polyic 12 u, perhaps in combination with other antiviral strategies, should be explored. but for hev and niv, the development of new therapeutics and vaccines has primarily focused on targeting the attachment and infection stages mediated by the viral f and g glycoproteins. as discussed earlier, peptides, typically 30-40 residues in length that are homologous to either of the heptad repeat domains of several paramyxovirus f glycoproteins, including the henipaviruses, can potently inhibit membrane fusion by blocking the formation of the trimer-of-hairpins structure (reviewed in bossart et al. 2013 ) . the fi rst henipavirus-specifi c peptide fusion inhibitor was a 36 amino acid heptad repeat-2 sequence (niv-fc2) (bossart et al. 2001 ) analogous to the approved hiv-1 specifi c therapeutic peptide enfuvirtide (fuzeon™) . other studies showed that a heptad repeat-2 peptide from human parainfl uenza virus type-3 (hpiv3) f blocked hev mediated fusion (porotto et al. 2006 ) and a sequence-optimized and cholesterol-tagged hpiv3-based heptad repeat-2 peptide appeared effective in the niv hamster (porotto et al. 2010 ). this cholesterol-tagged antiviral peptide could also penetrate the cns and exhibit some effective therapeutic activity against niv. additional in vivo effi cacy testing of peptide fusion inhibitors as henipavirus therapeutics merits further investigation. almost without exception all virus-neutralizing antibodies to enveloped viruses are directed against the viral envelope glycoproteins on the surface of the virion particle. initial passive immunization studies were conducted in the hamster nivchallenge model and showed that antibody immunotherapy against henipavirus infection by targeting the viral envelope glycoproteins was possible. protective passive immunotherapy using either niv g and f-specifi c polyclonal antiserums, or mouse monoclonal antibodies (mabs) specifi c for the henipavirus g or f glycoproteins has been shown (guillaume et al. 2004 (guillaume et al. , 2006 (guillaume et al. , 2009 . these studies demonstrated a major role of viral glycoprotein specifi c antibody in protection from henipavirus-mediated disease (reviewed in broder et al. 2012 ). using recombinant antibody technology, henipavirus-neutralizing human mabs reactive to the g glycoprotein were previously isolated (zhu et al. 2006 ) . one mab, m102, possessed strong cross-reactive neutralizing activity against hev and niv and was affi nity maturated (m102.4) and converted to an igg1 format and produced in a cho-k1 cell line ). the m102.4 mab epitope maps to the receptor binding site of g and engages g in a similar fashion as the ephrin receptors ). the m102.4 mab can neutralize niv-malaysia, hev-1994, hev-redlands and niv-bangladesh isolates (bossart et al. 2009 ). in a post-exposure niv-challenge experiment in the ferret model, a single dose of mab m102.4 administered by intravenous infusion 10 h after lethal challenge could prevent lethal infection (bossart et al. 2009 ). the therapeutic effi cacy of mab m102.4 has also been examined in monkeys against both niv and hev challenge with a study design refl ecting a potential real life scenario that would require a post-exposure treatment geisbert et al. 2014 ) . in one study, animals were challenged intratracheally with hev and later infused twice with m102.4 (~15 mg/kg) beginning at 10, 24, or 72 h post-infection followed by a second infusion ~48 h later. all subjects became infected following challenge, and all animals that received m102.4 survived whereas all control subjects succumbed to severe systemic disease by day 8. animals in a 72 h treatment group did exhibit neurological signs but all recovered by day 16, but there was no evidence of hev-specifi c pathology in any of the m102.4-treated animals, and no infectious hev could be recovered from any tissues from any m102.4-treated subjects. a follow-up study evaluated the effi cacy of m102.4 against niv disease in the agm model at several time points after virus exposure by intratracheal challenge, including at the onset of clinical illness ). here, subjects were infused twice with m102.4 (15 mg/kg) beginning at either 1, 3, or 5 days after virus challenge and again 2 days later. all subjects became infected after challenge and all subjects that received m102.4 therapy survived infection, whereas the untreated control subjects succumbed to disease between days 8 and 10 after infection. animals in the day 5 treatment group exhibited clinical signs of disease, but all recovered by day 16. together, these studies revealed that mab m102.4 could prevent widespread henipavirus dissemination in challenged subjects, and were the fi rst successful post-exposure in vivo therapies against hev and niv in nonhuman primates. a variety of active immunization strategies for henipavirus have been examined using recombinant virus platforms, protein subunit, virus-like particles and dna vaccines. several of these strategies have only been examined in terms of their ability to generate a henipavirus-specifi c neutralizing response (kong et al. 2012 ; kurup et al. 2015 ; wang et al. 2006 ; walpita et al. 2011 ) , whereas other studies examined immune response and effi cacy in animal challenge models. the fi rst report used the hamster model and the attenuated vaccinia virus strain nyvac, using recombinant viruses encoding either the niv f or g, both individually and in combination to immunize animals, and the study revealed that complete protection from nivmediated disease was achievable and that an immune response to the viral envelope glycoproteins can be important in protection (guillaume et al. 2004 ) . another poxvirus-based vaccine was examined as a potential livestock vaccine using recombinant canarypox virus in pigs (weingartl et al. 2006 ) . here, the niv f and g glycoprotein genes were used to generate recombinant canarypox viruses (alvac) vaccine vectors and used to immunize pigs. alvac vectors expressing f and g were tested alone and in combination, and piglets were challenged intranasally with niv. here, protection from niv-mediated disease was seen in all vaccinated pigs by either alvac vector alone or in combination and that vaccinated animals shed only low levels of nucleic acid detectable virus with no isolatable virus (weingartl et al. 2006 ) . more recently, several viral vector-based henipavirus vaccines have also been examined in animal challenge studies; these have included immunizations using the vesicular stomatitis virus based platform (vsv) expressing either the niv g or f glycoprotein in the hamster model (debuysscher et al. 2014 ; lo et al. 2014 ) and also vsv-based vaccines using niv f or g in the ferret model (mire et al. 2013 ) . all these studies demonstrated that a single dose of vaccine could induced strong neutralizing antibody responses and could afford protection from niv challenge, highlighting their potential usefulness as either a livestock vaccine or one suitable in an emergency use or outbreak scenario . vaccination and challenge experiments have also been examined using an adeno-associated virus platform with niv g showing protection against challenge in the hamster model and low level crossprotection (three of six animals) against a hev challenge (ploquin et al. 2013 ) , and also a recombinant measles virus vector with niv g which showed two of two agms were protected from niv challenge (yoneda et al. 2013 ) . a protein subunit vaccine strategy for henipaviruses has been extensively examined because of the inherent safety of such an approach. soluble, secreted, oligomeric forms of the g glycoprotein (sg) from both niv and hev were developed . the hev-sg glycoprotein is a secreted version of the molecule with a genetically deleted transmembrane and cytoplasmic tail that is produced in mammalian cell culture systems and is properly n-linked glycosylated (colgrave et al. 2011 ) . hev-sg retains many native characteristics including oligomerization and ability to bind ephrin receptors (bonaparte et al. 2005 ) , and it elicits potent cross-reactive neutralizing (hev and niv) antibody responses in a variety of animals including mice, rabbits, cats, ferrets, monkeys and horses. studies using the hev-sg subunit immunogen in the cat model demonstrated that it could elicit a completely protective immune response against a lethal subcutaneous niv challenge showing that a single vaccine (hev-sg) could be effective against both hev and niv. further studies in the cat model demonstrated that pre-challenge virus-neutralizing antibody titers as low as 1:32 were completely protective from a high-dose oronasal challenge of niv (50,000tcid 50 ) ). hev-sg immunization studies in the ferret model using either 100, 20 or 4 μg doses of hev-sg formulated in cpg and allhydrogel tm could all afford complete protection from a 5000 tcid 50 dose of hev (100 times the minimal lethal dose) with no disease or evidence of virus or viral genome in any tissues or body fl uids in the 100 and 20 μg vaccine groups; and only a low level of viral genome detected in the nasal washes from one of four animals in the 4 μg vaccine group. no infectious virus could be recovered from any vaccinated ferrets. the hev-sg subunit vaccine has also been evaluated in nonhuman primates (agms). in one study, doses of 10, 50, or 100 μg of hev-sg were mixed with allhydrogel ™ and cpg and vaccine was given to three subjects in each dosing group twice, 3 weeks apart, and subjects were challenged by intratracheal administration with a tenfold lethal dose of niv (1 × 10 5 tcid 50 ) 21 days later. complete protection was observed in all vaccinated subjects. some subjects had pre-challenge niv neutralizing titers as low as 1:28. no evidence of clinical disease, virus replication, or pathology was observed. a second study examined hev-sg vaccination and protection from hev challenge in agms, and also evaluated the hev-sg subunit (100 μg doses) in allhydrogel ™ and cpg as well as formulated with only allhydrogel ™ . subjects were vaccinated twice, 3 weeks apart, and were challenged intratracheally with a tenfold lethal dose of hev (∼5 × 10 5 plaqueforming units) 21 days after the boost vaccination. none of the eight vaccinated animals showed any evidence of clinical illness, virus replication, or pathology. the study also clearly demonstrated that hev-sg-allhydrogel ™ alone is capable of providing complete protection from a hev challenge providing crucial data for supporting preclinical development as a henipavirus vaccine for use in people. the simplicity and inherent safety of the hev-sg subunit vaccine approach together with the numerous successful vaccination and challenge studies that have been carried out in multiple animal models, the hev-sg subunit vaccine was chosen for the development of an equine vaccine to prevent infection in horses and also reduce the risk of hev transmission to people. hev-sg was licensed by zoetis, inc. (formerly pfi zer animal health) and developed as an equine vaccine for use in australia. horse hev-sg vaccination and hev challenge studies were conducted in australia the bsl-4 facilities of the australian animal health laboratories (aahl) in geelong, australia . here, hev-sg was formulated in a proprietary adjuvant (zoetis, inc.) and in two initial effi cacy studies in horses, either a 50 or 100 μg dose of the same sourced hev-sg which was used in all the animal challenge studies described earlier. two additional studies used 100 μg hev-sg produced from clarifi ed cho cell culture supernatant ( zoetis, inc. ) that was then gamma irradiated. immunizations were two 1-ml doses administered intramuscularly 3 weeks apart. horses in the effi cacy studies were exposed oronasally to 2 × 10 6 tcid 50 of hev. seven horses were challenged 28 days, and three horses were challenged 194 days, after the second vaccination. all vaccinated horses remained clinically healthy after challenge showing protection with hev neutralizing titers as low as 1:16 or 1:32 pre-challenge. at study completion, there was no gross or histologic evidence of hev infection in vaccinated horses; all tissues examined were negative for viral antigen by immunohistochemistry; and viral genome was not recovered from any tissue, including nasal turbinates, pharynx, and guttural pouch. in nine of ten vaccinated horses, viral rna was not detected in daily nasal, oral, or rectal swab specimens or from blood, urine, or feces samples collected before euthanasia, and no recoverable virus was present. only in one of three horses challenged at 6 months after vaccination, low viral gene copy numbers were detected in nasal swab samples collected on post-challenge days 2, 4 and 7, a fi nding consistent with self-limiting local replication, but no recoverable virus was present ). the horse vaccine against hev (equivac ® hev) is the fi rst commercially deployed vaccine developed against a bsl-4 agent and is the only licensed treatment for henipavirus infection. to date, more than 430,000 doses of equivac ® hev vaccine have been administered to horses (zoetis, inc.). hev and niv are the fi rst and only examples of zoonotic paramyxoviruses that can infect and cause lethal disease across a broad range of mammalian species including humans and there are currently no approved treatment modalities for people. because of the potential environmental accessibility of hev and niv and their highly pathogenic characteristics, the development of effective countermeasures against these biothreats has been a major research focus over the past decade. much of this research has focused on the virus binding and entry processes, including the processing, maturation and function of the envelope glycoproteins and the attachment to host cellular receptors and the membrane fusion process. these efforts have led to the development and testing of potential vaccine candidates and antiviral therapeutics. in 2010, the m102.4 mab producing cell line was provided to the queensland government, queensland health, australia to produce the m102.4 mab for emergency use on a compassionate basis in future cases of high-risk human hev exposure. queensland health authorities have completed in may, 2016, the fi rst phase 1 clinical safety trial of m102.4 in human subjects (queensland 2013 ). to date, 11 individuals exposed to either hev in australia (10 people) or niv in the united states (1 person) have been given high-dose m102.4 therapy under emergency use protocols, and all have remained well with no associated adverse events. in addition, the vaccine against hev (equivac ® hev) is vaccine for horses that is also expected to provide a substantial health benefi t to humans, and has fi t well within the spirit of a "one health" approach for the human and animal interface and also in respect to environmental health. studies on niv and hev have also provided important model systems to examine how pathogenic viruses interact with their natural reservoir hosts and also with animals susceptible to disease, providing insight into the dynamics of virus infection and maintenance in an animal reservoir; model systems to develop a variety of intervention strategies; details on how neurotropic viruses gain access to cns and cause disease; and will serve as tools to examine and evaluate potential therapies for virus-mediated cns disease. late-onset nipah virus encephalitis 11 years after the initial outbreak: a case report henipavirus encephalitis isolation and molecular identifi cation of nipah virus from pigs henipavirus membrane fusion and viral entry hendra virus, equine-australia (09): new south wales dog affected promed hendra virus, equine-australia: (08) queensland, new south wales. promed. international society for infectious diseases 2557020. www. promedmail.org anonymous (2014b) hendra virus, equine-australia (03): queensland. pro-med genomic characterization of nipah virus nipah and hendra 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nipah virus attachment glycoprotein: a template for antiviral and vaccine design dimeric architecture of the hendra virus attachment glycoprotein: evidence for a conserved mode of assembly the distribution of henipaviruses in southeast asia and australasia: is wallace's line a barrier to nipah virus? henipavirus outbreaks to antivirals: the current status of potential therapeutics immunization strategies against henipaviruses a treatment for and vaccine against the deadly hendra and nipah viruses activated leukocyte cell adhesion molecule promotes leukocyte traffi cking into the central nervous system nipah virus-associated encephalitis outbreak complete nucleotide sequences of nipah virus isolates from malaysia outbreak of henipavirus infection treatment of acute nipah encephalitis with ribavirin relapsed and late-onset nipah encephalitis, a report of three cases occupational exposure, age, diabetes mellitus and outcome of acute nipah encephalitis encephalitis serological evidence of henipavirus exposure in cattle, goats and pigs in bangladesh nipah virus outbreak in malaysia nipah virus: a recently emergent deadly paramyxovirus fatal encephalitis due to nipah virus among pig-farmers in malaysia isolation of nipah virus from malaysian island fl ying-foxes high mortality in nipah encephalitis is associated with presence of virus in cerebrospinal fl uid mutation of ymyl in the nipah virus matrix protein abrogates budding and alters subcellular localization henipaviruses: an updated review focusing on the pteropid reservoir and features of transmission site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of hendra virus type i interferon receptors: biochemistry and biological functions foodborne transmission of nipah virus in syrian hamsters single-dose live-attenuated nipah virus vaccines confer complete protection by eliciting antibodies directed against surface glycoproteins henipavirus infections: lessons from animal models eph family functions from an evolutionary perspective henipavirus rna in african bats bats host major mammalian paramyxoviruses subclinical infection without encephalitis in mice following intranasal exposure to nipah virus-malaysia and nipah virus-bangladesh a new model for hendra virus encephalitis in the mouse henipavirus infection in fruit bats (pteropus giganteus) ecological aspects of hendra virus the natural history of hendra and nipah viruses epidemiological perspectives on hendra virus infection in horses and fl ying foxes mumps virus replication in human lymphoid cell lines and in peripheral blood lymphocytes: preference for t cells combined chloroquine and ribavirin treatment does not prevent death in a hamster model of nipah and hendra virus infection ephrin-b2 selectively marks arterial vessels and neovascularization sites in the adult, with expression in both endothelial and smooth-muscle cells development of an acute and highly pathogenic nonhuman primate model of nipah virus infection animal challenge models of henipavirus infection and pathogenesis therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody poly(i)-poly(c12u) but not ribavirin prevents death in a hamster model of nipah virus infection relevance of cd6-mediated interactions in t cell activation and proliferation clinical features of nipah virus encephalitis among pig farmers in malaysia elucidation of nipah virus morphogenesis and replication using ultrastructural and molecular approaches nipah virus: vaccination and passive protection studies in a hamster model antibody prophylaxis and therapy against nipah virus infection in hamsters acute hendra virus infection: analysis of the pathogenesis and passive antibody protection in the hamster model pteropid bats are confi rmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus interaction between chicken lymphocytes and newcastle disease virus genetic characterization of nipah virus molecular characterization of nipah virus, a newly emergent paramyxovirus paramyxovirus assembly and budding: building particles that transmit infections serologic evidence of nipah virus infection in bats evidence of henipavirus infection in west african fruit bats antibodies to henipavirus or henipa-like viruses in domestic pigs in ghana, west africa nipah virus outbreak with person-to-person transmission in a district of bangladesh cluster of nipah virus infection comparative pathology of the diseases caused by hendra and nipah viruses the retrospective diagnosis of a second outbreak of equine morbillivirus infection nipah virus encephalitis reemergence ultrastructure of hendra virus and nipah virus within cultured cells and host animals henipavirus and tioman virus antibodies in pteropodid bats replication and persistence of measles virus in defi ned subpopulations of human leukocytes newcastle disease virus-vectored nipah encephalitis vaccines induce b and t cell responses in mice and long-lasting neutralizing antibodies in pigs virus infections in the nervous system effects of canine distemper virus infection on lymphoid function in vitro and in vivo rhabdovirus-based vaccine platforms against henipaviruses eph, a protein family coming of age: more confusion, insight, or complexity? fields virology modes of paramyxovirus fusion: a henipavirus perspective experimental inoculation study indicates swine as a potential host for hendra virus antibodies to nipah or nipah-like viruses in bats single-dose replication-defective vsv-based nipah virus vaccines provide protection from lethal challenge in syrian hamsters structural pathways for macromolecular and cellular transport across the blood-brain barrier during infl ammatory conditions the pandemic potential of nipah virus paramyxoviruses: henipaviruses epidemiology of henipavirus disease in humans transmission of human infection with nipah virus recurrent zoonotic transmission of nipah virus into humans hendra virus: an emerging paramyxovirus in australia experimental infection of squirrel monkeys with nipah virus cedar virus: a novel henipavirus isolated from australian bats experimental infection of horses with hendra virus/australia/horse/2008/redlands genome sequence conservation of hendra virus isolates during spillover to horses nipah virus uses leukocytes for effi cient dissemination within a host a recombinant subunit vaccine formulation protects against lethal nipah virus challenge in cats illuminating viral infections in the nervous system henipavirus microsphere immuno-assays for detection of antibodies against hendra virus endocytosis plays a critical role in proteolytic processing of the hendra virus fusion protein hendra virus vaccine, a one health approach to protecting horse, human, and environmental health experimental nipah virus infection in pteropid bats (pteropus poliocephalus) henipaviruses in their natural animal hosts experimental nipah virus infection in pigs and cats a recombinant hendra virus g glycoprotein subunit vaccine protects nonhuman primates against hendra virus challenge single injection recombinant vesicular stomatitis virus vaccines protect ferrets against lethal nipah virus disease nipah virus infection of pigs in peninsular malaysia olfactory transmission of neurotropic viruses feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine rapid nipah virus entry into the central nervous system of hamsters via the olfactory route a novel morbillivirus pneumonia of horses and its transmission to humans a morbillivirus that caused fatal disease in horses and humans ephrinb2 is the entry receptor for nipah virus, an emergent deadly paramyxovirus two key residues in ephrinb3 are critical for its use as an alternative receptor for nipah virus tlr3 agonists as immunotherapeutic agents fatal encephalitis due to novel paramyxovirus transmitted from horses development, maintenance and disruption of the blood-brain barrier cathepsin l is involved in proteolytic processing of the hendra virus fusion protein subcellular localization and calcium and ph requirements for proteolytic processing of the hendra virus fusion protein chloroquine administration does not prevent nipah virus infection and disease in ferrets a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge eph-ephrin bidirectional signaling in physiology and disease eph receptors and ephrins in cancer: bidirectional signalling and beyond quantitative analysis of nipah virus proteins released as virus-like particles reveals central role for the matrix protein the yplgvg sequence of the nipah virus matrix protein is required for budding outbreak of nipah-virus infection among abattoir workers in singapore henipavirus neutralising antibodies in an isolated island population of african fruit bats continent-wide panmixia of an african fruit bat facilitates transmission of potentially zoonotic viruses evidence for henipavirus spillover into human populations in africa human hendra virus encephalitis associated with equine outbreak protection against henipavirus infection by use of recombinant adenoassociated virus-vector vaccines urban habituation, ecological connectivity and epidemic dampening: the emergence of hendra virus from fl ying foxes (pteropus spp.) diverse roles of eph receptors and ephrins in the regulation of cell migration and tissue assembly inhibition of hendra virus fusion simulating henipavirus multicycle replication in a screening assay leads to identifi cation of a promising candidate for therapy inhibition of nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entry agricultural intensifi cation, priming for persistence and the emergence of nipah virus: a lethal bat-borne zoonosis world-fi rst hendra treatment one step closer date palm sap linked to nipah virus outbreak in bangladesh characterization of nipah virus from naturally infected pteropus vampyrus bats experimental infection of swine and cat central nervous systems by the pig paramyxovirus of the blue eye disease three or more routes for leukocyte migration into the central nervous system nipah virus in lyle's fl ying foxes a novel model of lethal hendra virus infection in african green monkeys and the effectiveness of ribavirin treatment clinical outcome of henipavirus infection in hamsters is determined by the route and dose of infection investigation of a second focus of equine morbillivirus infection in coastal queensland canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion mr imaging features of nipah encephalitis long-term neurological and functional outcome in nipah virus infection infection of humans and horses by a newly described morbillivirus nipah virus in the fruit bat pteropus vampyrus in sumatera, indonesia henipaviruses employ a multifaceted approach to evade the antiviral interferon response broad-spectrum antiviral activity of virazole: 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide ribavirin-current status of a broad spectrum antiviral agent ribavirin therapy for nipah virus infection evidence for nipah virus recrudescence and serological patterns of captive pteropus vampyrus nipah virus infects specifi c subsets of porcine peripheral blood mononuclear cells henipavirus mediated membrane fusion, virus entry and targeted therapeutics viral diseases of the central nervous system relapsed and late-onset nipah encephalitis nipah encephalitis outbreak in malaysia distribution of viral antigens and development of lesions in chicken embryos inoculated with nipah virus no evidence of prolonged hendra virus shedding by 2 patients endocytosis of the nipah virus glycoproteins a longitudinal study of the prevalence of nipah virus in pteropus lylei bats in thailand: evidence for seasonal preference in disease transmission vaccine potential of nipah virus-like particles recrudescent infection supports hendra virus persistence in australian flying-fox populations fields virology molecular biology of hendra and nipah viruses the exceptionally large genome of hendra virus: support for creation of a new genus within the family paramyxoviridae effi cacy of dna immunization with f and g protein genes of nipah virus negative fi ndings from serological studies of equine morbillivirus in the queensland horse population invasion of the central nervous system in a porcine host by nipah virus recombinant nipah virus vaccines protect pigs against challenge transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats experimental hendra virus infection in pregnant guinea-pigs and fruit bats (pteropus poliocephalus) a guinea-pig model of hendra virus encephalitis emerging epidemic viral encephalitides with a special focus on henipaviruses a golden hamster model for human acute nipah virus infection pathology of acute henipavirus infection in humans and animals human hendra virus infection causes acute and relapsing encephalitis nipah virus infection: pathology and pathogenesis of an emerging paramyxoviral zoonosis clinical and pathological manifestations of human henipavirus infection late presentation of nipah virus encephalitis and kinetics of the humoral immune response novel henipalike virus, mojiang paramyxovirus, in rats new insights into the hendra virus attachment and entry process from structures of the virus g glycoprotein and its complex with ephrin-b2 host cell recognition by the henipaviruses: crystal structures of the nipah g attachment glycoprotein and its complex with ephrin-b3 crystal structure of the hendra virus attachment g glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody detection of nipah virus rna in fruit bat (pteropus giganteus) from india nipah virus infection in bats (order chiroptera) in peninsular malaysia recombinant measles virus vaccine expressing the nipah virus glycoprotein protects against lethal nipah virus challenge serologic evidence for the presence in pteropus bats of a paramyxovirus related to equine morbillivirus exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody potent neutralization of hendra and nipah viruses by human monoclonal antibodies acknowledgments c.c.b. is supported nih grant ai054715-06. portions of fig. 1 were illustrated by andrew hickey. brain stem immunohistochemistry-stained images in fig. 3 were provided by thomas geisbert. brain parenchyma immunohistochemistry-stained images in fig. 4 were provided by debora middleton. key: cord-013837-x95r6bz8 authors: chai, qiyao; wang, lin; liu, cui hua; ge, baoxue title: new insights into the evasion of host innate immunity by mycobacterium tuberculosis date: 2020-07-29 journal: cell mol immunol doi: 10.1038/s41423-020-0502-z sha: doc_id: 13837 cord_uid: x95r6bz8 mycobacterium tuberculosis (mtb) is an extremely successful intracellular pathogen that causes tuberculosis (tb), which remains the leading infectious cause of human death. the early interactions between mtb and the host innate immune system largely determine the establishment of tb infection and disease development. upon infection, host cells detect mtb through a set of innate immune receptors and launch a range of cellular innate immune events. however, these innate defense mechanisms are extensively modulated by mtb to avoid host immune clearance. in this review, we describe the emerging role of cytosolic nucleic acid-sensing pathways at the host–mtb interface and summarize recently revealed mechanisms by which mtb circumvents host cellular innate immune strategies such as membrane trafficking and integrity, cell death and autophagy. in addition, we discuss the newly elucidated strategies by which mtb manipulates the host molecular regulatory machinery of innate immunity, including the intranuclear regulatory machinery, the ubiquitin system, and cellular intrinsic immune components. a better understanding of innate immune evasion mechanisms adopted by mtb will provide new insights into tb pathogenesis and contribute to the development of more effective tb vaccines and therapies. introduction tuberculosis (tb) remains a serious global public health threat, accounting for over 1.2 million deaths per year. 1 mycobacterium tuberculosis (mtb), the etiological agent of tb, is estimated to have infected 1.7 billion people worldwide. 1 despite the availability of anti-tb medications, cure rates are low (~56% globally) for continuously emerging drug-resistant tb cases, which necessitate the use of more complex and toxic regimens and even pose risks of transmitted resistance. 1,2 therefore, rational design of novel tb vaccines and therapeutics based on an in-depth understanding of the intimate interplay between mtb and host immunity is required. innate immunity plays a dominant role in protecting the host from early infection with mtb, as indicated by the majority of mtb-exposed individuals being able spontaneously control the infection despite a conspicuous delay of acquired immunity; 3 however, an intact adaptive immune system is insufficient to restrict mtb growth within a host deficient in innate immune responses. 4, 5 as first-line defensive patrols that quickly respond to mtb infection, innate immune cells perform the duty of immune surveillance via a range of pattern recognition receptors (prrs). activation of these immune receptors leads to a range of cellular events that contribute to host anti-mtb immunity, such as phagocytosis and apoptosis. 6 however, long-standing coevolution with the human host protects mtb from the effects these antibacterial mechanisms, leading to its persistent infection. furthermore, in recently emerging pathogenic strategies, mtb can directly target and modify various aspects of the molecular regulatory machinery of host innate immunity, such as the intranuclear regulatory machinery, the ubiquitin system and cellular intrinsic immune components, to evade host clearance. in this review, we summarize recently emerging aspects of innate immune evasion mechanisms adopted by mtb to benefit its own intracellular survival, including the role of cytosolic nucleic acidsensing pathways at the host-mtb interface; novel mechanisms adopted by mtb to circumvent host cellular innate immune events, such as membrane trafficking and integrity, cell death, and autophagy; and newly elucidated mtb strategies to manipulate the host molecular regulatory machinery of innate immunity. a better understanding of the intricate interplay between mtb and the host innate immune system may provide new insights into tb pathogenesis and contribute to the development of valid vaccines and therapies. emerging roles of cytosolic nucleic acid-sensing pathways in host-mtb interactions the core duty of the mammalian innate immune system to recognize infective pathogens is evolutionarily designed to rapidly sense and eliminate foreign threats. to prevent the successful establishment of mtb infection in the lungs, host immune cells, and various nonclassical immune cells in the airway are equipped with a set of cell-surface and intracellular prrs to recognize the invading mycobacteria, such as toll-like receptors, c-type lectin receptors, nod-like receptors (nlrs), complement receptors, and scavenger receptors (srs). these innate immune sensors play critical roles at the interface of host mucosal immunity and mtb pathogenesis and have been extensively reviewed elsewhere. [6] [7] [8] in this section, we focus on the recently emerging role of cytosolic nucleic acid-sensing pathways in host-mtb interactions (fig. 1 ). cytosolic dna-sensing pathways although the immunostimulatory effects of mycobacterial dna on mammalian hosts were receiving attention decades ago, 9 hostresponsive dna-dependent cytosolic surveillance pathways were not elucidated until recently. initially, mtb was thought to be able to translocate from phagosomes into the cytosol by virtue of its esat-6 secretion system-1 (esx-1) system during infection of host cells, 10, 11 and this process provides a potential opportunity for host cytosolic receptors to sense mycobacterial extracellular dna. in addition, the blood of patients with active tb is characterized by a distinct transcriptional signature related to type i interferon (ifn) signaling, 12 and this hallmark was proposed to be associated with the activation of the host cytosolic surveillance pathway, which can result in the robust production of type i ifns. 13 based on these observations, manzanillo et al. first tested the role of two putative cytosolic dna sensors, z-dna binding protein 1 (zbp1) and ifnactivable protein 204 (ifi204; the mouse ortholog of human ifi16), in host cytosolic surveillance of mtb and found that only ifi204 contributes to the type i ifn response to mtb infection via the stimulator of ifn genes (sting)/tank binding kinase 1/ifn regulatory factor 3 (irf3) axis in macrophages. 14 interestingly, the deletion of irf3 to subvert this signaling pathway in mice decreased the host expression of type i ifns and enhanced host resistance to long-term mtb infection. 14 these results indicate a negative regulation of type i ifns in host anti-mtb immunity and suggest a potential strategy by which mtb hijacks the cytosolic surveillance pathway to facilitate its own infection. cyclic gmp-amp synthase (cgas) is a recently characterized dna sensor. upon direct binding with cytosolic dna, cgas is activated to catalyze the production of cyclic gmp-amp (cgamp), leading to the activation of the downstream sensor sting. 15, 16 according to pioneering studies, cgas functions in the cytosol, where it cooperates with sting to activate both nuclear factor-κb (nf-κb) and irf3 signaling pathways to induce the transcription of type i ifns and various pro-inflammatory t helper type 1 (th1) cytokines with action against viral infections. [15] [16] [17] [18] nevertheless, our recent findings and those of others suggest that cgas can change its subcellular location and enter into the nucleus or reside on the plasma membrane, which is a possible strategy adopted by the host to distinguish self-and nonself dna through the exertion of distinct cgas-dependent functions. [19] [20] [21] the involvement of the cgas-mediated dna-sensing pathway in host anti-mtb immunity is indicated by the findings that cgas expression is upregulated and that cgas is colocalized with mycobacteria in human tb lesions, and its deficiency impairs the induction of type i ifn responses and autophagy in mtb-infected macrophages. [22] [23] [24] recent studies also suggest that the cgas/sting immunesensing pathway is necessary for host dendritic cell (dc) activation because it increases the expression of type i ifns upon mycobacterial infection. 25, 26 interestingly, despite confirmation of cgas/sting-dependent bacterial control in macrophages, cgas −/− and sting −/− mice show comparable lung bacterial burden and inflammation levels to those of wild-type control mice after mtb exposure, 22, 24, 25 suggesting that additional host dna sensors or other immune receptors may compensate for cgas/ sting-dependent antimycobacterial immune responses in vivo. apart from type i ifn stimulation, the detection of intracellular dna may also lead to inflammasome activation with the production of mature pro-inflammatory cytokines, including interleukin-1β (il−1β) and il-18, via the absence of melanoma 2 (aim2). 27, 28 in macrophages, aim2 responds to mtb genomic dna and results in increased caspase-1 cleavage and il-1β and il-18 release, a finding consistent with the observation that aim2deficient mice show an increased susceptibility to mtb infection with impaired pro-inflammatory responses. 29 similarly, infection with virulent mycobacterium bovis can also activate the aim2 inflammasome in macrophages. 30 notably, compared with nonvirulent mycobacteria containing a compromised esx-1 secretion system, such as mycobacterium smegmatis, mycobacterium fortuitum, mycobacterium kansasii, and attenuated mtb h37ra strains, virulent mtb h37rv has a significant inhibitory effect on aim2dependent innate cytokine responses. 31 this finding seemingly contradicts the accepted idea that esx-1 is essential for activating host cytosolic surveillance pathways. most likely, esx-1 is required for mtb to deliver a number of effectors into the host to remodel the intracellular environment to improve its chance for survival, despite its role in inducing immune recognition. in addition, it should be noted that individual effectors delivered by the mtb esx-1 secretion system may play independent immunoregulatory roles with different host targets, and thus, the mechanisms underlying esx-1-dependent stimulation or evasion of the host cytosolic surveillance pathway both function during host-mtb interactions. this notion is supported by the finding that blocking the secretion of esxa, a major substrate of esx-1, significantly reduced cgas/sting-mediated ifn production while leaving the inflammasome-mediated il-1β response virtually intact. 23 therefore, specifically targeting mycobacterial esx-1 products or host regulatory factors might enable the selective regulation of inflammasome and cgas/sting pathway activation and, hence, contribute to the recovery of the equilibrium between th1-type cytokine and type i ifn responses in tb patients to improve their anti-mtb immunity. cytosolic rna-sensing pathways the immunomodulatory activity of mycobacterial rna in mammalian hosts received attention as early as the 1960s and 1970s. 33 recently, it was reported that mtb-infected macrophages can deliver extracellular vesicles (exosomes) containing abundant mycobacterial rna to recipient cells, suggesting that mtb rna is probably released into host cells to trigger the rna-dependent cytosolic surveillance pathway. 34 the cytosolic rna-sensing pathway was initially identified as a key part of host immune surveillance against rna virus infection. in mammalian cells, retinoid acid-inducible gene i (rig-i)-like receptors (rlrs) are wellconserved cytosolic prrs that recognize cytosolic viral rnas and activate downstream immune pathways to promote the production of type i ifns and other pro-inflammatory cytokines. 35 rig-i and melanoma differentiation-associated protein 5 (mda5) are the best characterized rlrs, which preferentially recognize short polyphosphorylated double-stranded rna (dsrna) and long dsrna, respectively. 36 after sensing foreign rnas, rig-i, and mda5 transmit signals via a common adapter, mitochondrial antiviral signaling (mavs), which forms large prion-like polymers and recruits tumor necrosis factor (tnf) receptor-associated factors (trafs) to further activate nf-κb and irf3 immune signaling pathways. [37] [38] [39] recently, the rlr-mediated cytosolic surveillance pathway was also shown to participate in the host immune response to various bacterial pathogens, such as mtb, legionella pneumophila, helicobacter pylori, and listeria monocytogenes. [40] [41] [42] the involvement of the rlr-dependent rnasensing pathway during host-mtb interactions is implied by the increased expression of rig-i and mda5 mrnas in mtb-infected macrophages. 43 further investigation of recombinant mtb strains demonstrated that mtb seca2 and esx-1 secretion systems are critical for the delivery of mtb rna into the host cell cytosol, resulting in ifn-β production through the host rig-i/mavsmediated rna-sensing pathway. 44 the role of the mda5mediated rna-sensing pathway in detecting mtb infection was also confirmed by a recent study, which showed that deletion of mda5 impaired ifn-β production and bacterial control in human macrophages, results similar to those obtained by the deletion of rig-i or mavs. 45 nonetheless, rig-i, not mda5, appears to interact with the mtb-specific mrnas pola and ppe11 (ref. 44 ), suggesting that these rlrs probably play nonredundant roles in detecting different types of mycobacterial rnas. furthermore, mavsdeficient mice showed obviously increased resistance to mtb infection with attenuated bacterial growth in their lungs, 44 as was also observed in irf3-deficient mice, 14 supporting a potentially negative role of type i ifns in host anti-mtb immunity in vivo. in addition to the rig-i/mda5/mavs axis, protein kinase r (pkr) has been identified as another host sensor of cytosolic dsrna, which can interact with the natural rna derived from diverse viruses or bacteria, 45 leading to the activation of irf3, nf-κb, and other various innate immune signaling pathways. 46 according to an infection model based on the interaction of m. bovis bacillus calmette-guérin (bcg) and primary human blood monocytes, the mycobacteria-induced production of inflammatory cytokines is regulated by the phosphorylation and activation of pkr. 47 a recent study also demonstrated that mtb infection results in +increased expression of pkr and increased phosphorylation of its substrate, eukaryotic translation initiation factor 2 a, in human cells, and pkr deficiency leads to enhanced intracellular growth of mycobacteria. 48 however, the in vivo role of pkr in host immunity challenged by mtb infection remains unclear. although a research group has reported that mice lacking pkr show reduced mycobacterial burden with less severe pulmonary pathology than shown by wild-type mice, 49 they recently attributed this observation to different genetic backgrounds of the mice rather than to a direct role of pkr. 50 aside from the rig-i/mda5-and pkr-mediated cytosolic rnasensing pathways, intracellular nlr family members, including nlrp3 and nod2, can also recognize foreign dsrna and singlestranded rna (ssrna), respectively. 51, 52 mtb infection activates both of these nlrs in an esx-1-dependent manner to trigger various host downstream innate immune responses, such as nlrp3 inflammasome formation, autophagy initiation and nf-κb and irf3 pathway activation, which have been extensively reviewed elsewhere. 6, 52 however, it is still unclear whether nlrp3 and nod2, which respond to a range of pathogen-derived stimuli, 52, 53 can be activated by direct binding to mycobacterial extracellular rnas, although a recent study reported that dsrna from mtb cultures is able to induce caspase-1 activation in retinal pigment epithelium. 54 in summary, host cytosolic dna-and rna-sensing pathways are newly emerging innate immune recognition mechanisms of host-mtb interactions. growing evidence indicates that there is intimate cross talk among the components of different cytosolic nuclear acid-sensing pathways, 23, 44 and these immune surveillance pathways probably play nonredundant roles in host anti-mtb immunity. however, the in vivo data from animal infection models show that activation of cytosolic cgas-or rlr-mediated sensing pathways can induce a strong type i ifn response that appears to impair host resistance to mycobacterial infection, 14, 44 suggesting that mtb may exploit the host cytosolic surveillance pathways to facilitate its own growth. in contrast, activation of other cytosolic pathways during mtb infection, such as that mediated by aim2, nod2, and nlrp3, can promote the production of protective inflammatory cytokines. hence, further investigation may be focused on how to spatiotemporally and selectively regulate these cytosolic surveillance pathways to optimize host anti-mtb immunity. furthermore, a recent study demonstrated that drug treatment targeting cytosolic rna sensors benefited the host by controlling mycobacterial intracellular growth, 48 highlighting the potential value of targeting the cytosolic immune surveillance pathway for novel host-directed anti-tb therapy. new aspects of mtb-modulated cellular innate immune events the activation of host innate immune-sensing pathways by mtb infection leads to a range of subsequent cellular antimicrobial events, such as phagocytosis and apoptosis; however, these effects can be modulated by mtb to benefit its long-term intracellular survival. 6 in this section, we focus on recently emerging aspects of regulatory strategies adopted by mtb to interfere with host membrane trafficking and integrity, cell death, and autophagy processes (fig. 2 ). the leveraging of host membrane trafficking in infected cells is a key strategy for the notorious success of mtb as a highly adapted intracellular pathogen. upon infection, mtb is engulfed by host phagocytic cells such as macrophages, neutrophils, and dcs and internalized in a phagosome, the organelle responsible for routine clearance of pathogens. notably, while phagosomes in macrophages and neutrophils are generally designated to rapidly eliminate pathogen-associated cargo, dc phagosomes tend to moderately degrade their internalized substrates to preserve antigenic peptides for priming adaptive immune responses. 55 however, it has been well documented that mtb recruits the gtpase rab5, but not rab7, away from the phagosome to inhibit phagolysosome maturation. 56, 57 the prevention of the biogenesis of phagolysosomes plays a vital role in mtb infection, transmission, latency, and immune evasion. 56, 57 multiple routes and numerous effectors are employed by mtb for the suppression of phagosome maturation and acidification, which have been extensively summarized elsewhere. 6, 57 notably, the ability of mtb to manipulate host membrane trafficking may also contribute to the targeting of the host endosomal sorting pathway by human immunodeficiency virus during viral budding, thus favoring synergism of these two pathogens during coinfection. 58 recently, the spatiotemporal dynamics of mtb phagosomal morphology and composition have received growing attention. during maturation, phagosomes associate with early and late endosomes, as well as other intracellular organelles such as golgiderived vesicles, the endoplasmic reticulum (er) and mitochondria, 59 and these interactions are very dynamic and can promptly change both the phagosomal membrane and luminal components with the principal aim of restraining the growth of internalized pathogens. upon infection, mtb alternatively localizes to two morphologically different types of phagosomes, tight and spacious phagosomes, which are consistently observed in both tb patients and other animal hosts. [60] [61] [62] a recent study revealed that ifn-γ can facilitate endosomal interactions with mtb phagosomes via the regulation of the rab20-dependent vesicular trafficking pathway, which promotes membrane influx into tight phagosomes and shifts them into spacious and proteolytic compartments that restrict mtb growth. 63 however, mtb can avoid being directed to rab20-positive spacious phagosomes via its esx-1 system. another study has demonstrated that patient-derived mtb strains can produce large amounts of 1-tuberculosinyladenosine (1-tbad), which acts as a bacterial antacid and selectively accumulates in host cellular acidic compartments, resulting in phagosomal swelling and the obliteration of the lysosomal multilamellar structure. 64 the phagosomal components also appear to be fine-tuned by mycobacteria, given that the mtbspecific phagosome proteome shows distinct characteristics from that of latex bead-or other bacterial pathogen-containing phagosomes. 65 it is conceivable that mtb must remodel the intravacuolar microenvironment to establish a pathogen-friendly niche. for example, mtb can encode various effectors, such as ptpa, 1-tbad, and marp, to elude, neutralize or tolerate the acidic environment of phagosomes. 64, 66, 67 mycobacteria also avoid being trafficked with bactericidal molecules, such as lipocalin 2, an innate immune protein that disrupts bacterial iron acquisition, to their compartments while retaining access to transferrin for iron uptake through the rab11 + endocytic recycling pathway. 68 the change in mtb phagosomal content is also a hallmark of accumulated lipid droplets, which probably depends on rab7, according to a recent study. 69 although it was proposed that mtb can disrupt mitochondrial fatty acid oxidation to promote lipid body deposition in macrophages for utilization, 70 another study demonstrated that increased formation of lipid droplets in mtbinfected cells actually facilitates host biosynthesis of eicosanoids and restricts bacterial growth. 71 therefore, the multifaceted role of lipid bodies in mtb phagosomes requires further elucidation. membrane rupture, which depends on the mycobacterial esx-1 system, is another typical characteristic of mtb phagosomes. 10, 11, 72 this phenomenon has long been considered a pathogen-driven event utilized by mtb to escape from a bactericidal phagosome and enter the host cell cytosol, where it can obtain abundant nutrients. however, a recent study demonstrated that the inhibition of phagosomal maturation and acidification is a precondition for mtb phagosomal damage. 72 furthermore, as identified in other successful intracellular pathogens, such as l. pneumophila and brucella abortus, the establishment of a sheltered niche within a vacuolar compartment mimics a normal cellular organelle and enables the pathogen to avoid host immune surveillance and clearance. 73, 74 this finding one to wonder why a mycobacteria departs from a cozy niche to enter the cytosol where it must confront a series of cytosolic immune sensors? to date, no direct evidence indicates an obvious advantage of mycobacterial extra-phagosomal survival. one possible explanation is that the success of persistent mtb infection requires the esx-1 secretion system to damage the phagosomal membrane and deliver numerous secretory effectors into the cytosol to target and regulate cellular immune components. this assumption is supported by accumulating evidence that indicates an indispensable role for the esx-1 system in mtb pathogenesis, as it has been linked to host cytosolic surveillance evasion, 23,31 phagosome maturation arrest, 63,75 cell death reprogramming, 76,77 autophagy inhibition, 78,79 etc. alternatively, escape from phagosomes facilitates mtb esx-1-dependent plasma membrane damage, facilitating efficient mtb replication and spread to neighboring cells and, eventually, to new hosts. by using time-lapse microscopy at the single-cell level, ruptured host cell plasma membranes were observed at the contact points of mtb with the plasma membrane, which provides direct evidence for this assumption. 80 however, this evidence does not exclude the other possibility: the host may actively promote mtb phagosome rupture at the early stage of infection to eliminate the pathogen. as described above, host cytosolic immune sensors, [22] [23] [24] 29, 43, 44, 48, 51, 52 as well as other diverse defense molecules, 63, 81, 82 can recognize and target either damaged mtb phagosomes or cytosolic mycobacteria for immune clearance. accordingly, a recent work revealed that a host deficient in endosomal sorting complex required for transport, machinery thought to be important for repairing esx-1-dependent damage of mycobacteria-containing vacuoles, shows restricted intracellular bacterial growth. 83 in addition, several independent studies using different experimental methods consistently found that the majority of intracellular mycobacteria are not localized in the host cytosol until a very late stage of infection, 10, 11, 72 suggesting that mtb may prepensely escape from phagosomes for rapid replication and preparation for further transmission, which occurs only after the host cells are compromised by immune responses that are attenuated after prolonged interaction with the mycobacteria. aside from membrane changes related to phagosomelysosome fusion (and autophagosome formation, which is discussed below), recent studies have indicated that mtb is also involved in the modulation of other cellular membranes. for example, the translocation of the golgi apparatus and lysosome-derived vesicles to the plasma membrane is required for the repair of mycobacteria-induced disruptions of the macrophage plasma membrane, whereas virulent mtb strains are able to prevent this process and induce necrosis of infected cells. 76 in addition, mtb infection has also been associated with the induction of mitochondrial membrane permeability transition (mpt), which causes host cell necrosis. [84] [85] [86] interestingly, pathogenic mycobacteria may also coopt the host autophagic machinery to break through the plasma membrane and depart from their host cells through an f-actin-based vacuolar compartment termed an "ejectosome", which is proposed to be a nonlytic cell-to-cell bacterial transmission mechanism. 87, 88 furthermore, mtb can alter the protein composition of exosomes secreted by infected human macrophages. 89 these actions indicate that mtb is involved in the host exosomerelated vesicular trafficking pathway, but its significance for tb pathogenesis remains largely unexplored. in conclusion, the success of the intracellular lifestyle of mtb largely depends on the establishment of an easeful niche within a nonfusogenic phagosome. in fact, growing evidence suggests that the phagosome is more likely serving as a signaling platform than as clearance machinery, 90 and mtb is likely to promptly interact with the cellular membrane trafficking system to sense and change the host immune and metabolic conditions. these assumptions, as well as the potential interplay between mtb and other host cellular organelle membranes, warrant further indepth investigations. the development of central necrosis in granulomatous lesions, which induces lung cavity formation and promotes mtb transmission to another human host, is a hallmark characteristic of severe tb cases. 91 hence, mtb-induced host cell death during infection likely plays a crucial role in tb pathogenesis. initially, virulent mtb strains were thought to induce host cell apoptosis in an esx-1-dependent manner, as indicated by an in vitro infection model using immortalized murine macrophage cell lines. [92] [93] [94] however, several studies using human macrophage cell lines have indicated that virulent mtb leads to a lower apoptosis rate than attenuated strains [95] [96] [97] and even inhibits apoptosis by employing a wide variety of effector proteins (which are effectively summarized in ref. 98 ) to evade host downstream immune responses. most likely, the integrity of cell deathassociated molecular pathways in certain cell lines accounts for these discrepancies. further investigations suggested that virulent mtb strains can switch the induction of host cell apoptosis to necrosis via manipulation of eicosanoid metabolism pathways. 76, 77 in contrast to apoptosis, which is proposed to result in the containment of mycobacteria, 98 the propensity of mtb for inducing necrotic death likely benefits the release of bacteria into the permissive extracellular microenvironment they have modulated for better growth. 99 however, a recent study using time-lapse imaging suggested that mtb-induced necrosis predominantly benefits the growth of the bacteria within dead cells, as indicated by the observation of the accelerated intracellular replication of mtb after host macrophage death, which was much faster than it was in either live cells or in the extracellular milieu. 100 in addition, the phagocytosis of dead infected cells containing aggregated mycobacteria by bystander macrophages would cause further necrosis. 100 regardless of the debate on the benefit of necrosis on intra-or extracellular mycobacterial growth, these studies have established the currently accepted concept suggesting that mtb can reprogram host cell death and that it preferentially induces necrosis rather than apoptosis to facilitate its survival and dissemination. more recent studies have pointed out that mycobacteriainduced host cell necrosis is a programmed cell death process, termed "necroptosis", which is stimulated by host tnf via tnf receptor 1 (tnfr1) and is dependent on receptor-interacting serine-threonine kinases 1 (ripk1)/ripk3. 84, 85 mtb infection markedly increases mixed-lineage kinase domain-like protein (mlkl), the effector protein in the ripk1/ripk3-mediated necroptosis pathway, and other pronecroptotic molecules such as tnfr1 and zbp1 (ref. 84, 101 ). however, deletion of mlkl or inhibition of ripk1 in macrophages does not completely rescue mtb-infected cells from death, 84, 101 suggesting that, although the deficiency of mlkl or ripk1 can abrogate the canonical necroptosis pathway, upstream tnf/tnfr1-mediated signaling may stimulate the induction of other cell death pathways during mtb infection. alternatively, mtb may bypass the tnf/tnfr1/ripk1 cascade to cause necroptosis, a notion supported by a recent study showing that mtb can secrete a nicotinamide adenine dinucleotide (nad + ) glycohydrolase to induce host cell necroptosis independent of ripk1 and tnf. 102 furthermore, mlkl-deficient or ripk1-inhibited humanized mice exhibited bacterial burdens and lung histopathology indistinguishable from those of the control mice in response to mtb infection. 101 these results imply that, although tnf/tnfr1/ripk1-dependent necroptosis is activated by mtb, this type of cell death seems to play a restricted role in tb pathogenesis. hence, additional mechanisms underlying mtbinduced host cell death and their association with tb pathogenesis should be taken into account. in addition to those identifying necroptosis, a number of studies have identified multiple other types of programmed necrosis in mammalian host cells in response to mtb infection, such as inflammasome-mediated pyroptosis and neutrophil extracellular trap (net)-associated netosis, which have recently been extensively reviewed. 98 notably, it was reported that mtb inhibited macrophage inflammasome activation and pyroptosis via its secreted effectors zmp1 and rv3364c, thus limiting host pro-inflammatory immune responses. 103, 104 furthermore, netosis seemingly facilitates the interactions between neutrophils and other immune cells rather than killing mtb directly. 105, 106 more recently, amaral et al. found that mtb-induced macrophage necrosis was characterized by elevated levels of intracellular iron and mitochondrial superoxide. increased lipid peroxidation, and downregulated glutathione and glutathione peroxidase-4, findings that are in line with the hallmark characteristics of a typical and regulated necrosis process termed "ferroptosis". 107 using a mouse model of acute mtb infection, the same group confirmed the association between lung necrosis and mtb-induced ferroptosis, which indicated that ferroptosis probably contributes to tb pathology and allows mtb to thrive and spread. 107, 108 more importantly, treatment with the ferroptosis inhibitor ferrostatin-1 reduced the bacterial burdens and attenuated pulmonary necrosis in acutely mtb-infected mice, 107 suggesting that the targeting of the host ferroptotic pathway may be a potential strategy to control tb infection and reduce lung damage. in summary, diverse host cell death pathways are involved in mtb infection, acting either as host protective mechanisms or as bacterial survival strategies. notably, the preference for these different cell death modalities likely depends on both the mycobacterial strains and molecular integrity of cell death pathways in a certain host cell type. therefore, identification of and interference with mycobacterial effectors or potential host molecular switches that can control the death modes of infected cells might be a new approach to control tb infection and diminish mtb-caused tissue damage. exploitation of the autophagy process by mtb our knowledge of the physiological and immunological roles of autophagy has recently expanded greatly. 109 autophagy is a cellular mechanism evolutionarily conserved from yeast to mammals that involves the degradation of cellular materials such as damaged organelles, unwanted proteins or foreign pathogens by capturing them in a double-membrane structure termed the "phagophore", which can subsequently develop into a mature autophagosome and fuse with lysosomes. 109, 110 the protective role of autophagy in host defense against mtb was first investigated by gutierrez et al., who noted that a portion of mycobacteria are sequestered into autophagosome-like compartments during infection in macrophages and that exogenous stimulation to enhance autophagy restricted mtb intracellular survival. 111 a subsequent study confirmed this observation and revealed that while the th1 cytokine ifn-γ can induce host macrophage autophagy to control mtb infection, the th2 cytokines il-4 and il-13 abrogate such autophagy-mediated killing of intracellular mycobacteria. 112 furthermore, it has been reported that autophagy is involved in regulating other multiple anti-mtb mechanisms, such as the mycobactericidal capacity of the lysosomal soluble fraction, 113 the expression of srs on macrophages, 114 and mycobacterial antigen presentation. 115 taken together, these findings indicate an essential role of autophagy in both host innate and adaptive immunity in mtb infection. more recently, researchers noted that eukaryotic cells could allocate specific cellular materials to the autophagy pathway, which is considered a selective process. host selective autophagy of foreign pathogens is termed "xenophagy". 109 deletion of xenophagy-associated genes leads to significantly enhanced mycobacterial survival in macrophages and in mice, 22, 24, 81, [116] [117] [118] [119] [120] further supporting a protective role of autophagy in host anti-mtb immunity. during mtb infection, ubiquitin-ligating (e3) enzymemediated ubiquitin attachment to bacteria is a key step for host initiation of xenophagy, through which various autophagy receptors, such as p62 (sqstm1), nbr1, ndp52, and optineurin, are recruited and subsequently engage with autophagosomal membrane-associated protein lc3 to capture bacteria into autophagosomes. 81, [116] [117] [118] [119] [120] to date, only two e3 ubiquitin ligases, parkin and smurf1, have been found to control ubiquitin targeting of mtb for xenophagy initiation, which was realized through the mediation of k63-and k48-linked ubiquitination of mtb-associated substrates, respectively. 118, 119 in addition, a recent study demonstrated that human makorin ring finger protein 1 (mkrn1) is an mtb-specific e3 ubiquitin ligase that can mediate the ubiquitination of mtb in vitro in conjunction with ubiquitin-activating enzyme e1 (ube1) and ubiquitin conjugating enzyme e2 d3 (ube2d3), 121 although its intracellular role during mtb infection has not been illustrated. however, the protein substrates on mtbcontaining phagosomes or mycobacterial surfaces that can be ubiquitinated by these e3 ligases remain unidentified. parkin −/− mice fail to restrict mtb replication during acute infection, and smurf −/− mice display an attenuated capacity to control mtb infection during the chronic phase, 118, 119 suggesting that they have different roles in host anti-mtb immunity. apart from e3 ligase-mediated xenophagy, we recently identified an mtb surface protein, rv1468c, which can directly bind host cytosolic ubiquitin chains via a eukaryotic-like ubiquitin-associated (uba) domain to recruit autophagy components and trigger a xenophagic response. 81 therefore, both e3 ligase-dependent and e3 ligaseindependent mechanisms are involved in host ubiquitin targeting of intracellular mtb for xenophagy initiation. furthermore, it is notable that the host can also drive ubiquitin-independent xenophagy. in salmonella typhimurium-infected cells, host galectin-8 detects invading bacteria by binding glycans on damaged bacteria-containing vacuoles and further interacts with the autophagy receptor ndp52 to recruit lc3 and activate antibacterial autophagy. 122 given that galectins also participate in the cytosolic recognition of mtb-damaged phagosomes, 63, 82 ubiquitin-independent xenophagy may also occur during mtb infection. in addition, in view of growing eukaryotic-like effectors identified in mtb, 123, 124 it is not surprising that mtb might retain certain surface proteins that can be directly recognized by new insights into the evasion of host innate immunity by mycobacterium. . . q chai et al. autophagy receptors or lc3 family proteins via protein-protein interaction motifs to trigger host xenophagy. in response, mtb adopts multiple strategies to avoid autophagyrelated immune clearance during infection, and an effective mechanism involves directly or indirectly targeting autophagy machinery by delivering effector proteins into host cells. for example, mtb-secreted acid phosphatase (sapm) has been found to target host rab7 to prevent autophagosome-lysosome fusion. 123 another mtb effector, enhanced intracellular survival (eis), which is an n-acetyltransferase that has been reported to increase the acetylation level of histone h3 to upregulate il-10, results in autophagy suppression via the activation of the akt/ mtor/p70s6k pathway. 125 recently, a host noncanonical autophagy pathway, named lc3-associated phagocytosis (lap), was identified in the context of a fungal infection and involved in the recruitment of lc3 and other components of the canonical autophagy machinery on pathogen-containing phagosomes for lysosomal degradation. 126 notably, lap does not rely the preinitiation complex in ulk1 signaling, instead requiring rubicon and nadph oxidase 2 (nox2), molecules, which are not involved in the canonical autophagy pathway. 126 interestingly, mtb is insensitive to nadph oxidase and lap trafficking, and nox2deficient mice show few differences compared with the control mice in controlling mtb infection. 127 the mtb protein cpsa has been proven to cause autophagy resistance, 127 but its direct target in the host lap pathway remains unclear. interference with host micrornas (mirnas) is another efficient strategy by which mtb disturbs the host autophagy pathway, as shown by mirna often simultaneously targeting multiple interrelated genes, thereby leading to a potent cumulative effect on a certain molecular pathway. 128 mycobacteria can modulate the expression of diverse host mirnas, such as mir-33 and its passenger strands mir-33*, mir-125a, mir-17, mir-155, and mir144*, which results in autophagy inhibition through the direct repression of a wide range of key autophagy effectors. 70, [129] [130] [131] [132] in addition, we recently found that mtb infection induces the expression of mir-27a, the mirna that targets the er-located ca 2+ transporter cacna2d3 to inhibit the downstream calcium-associated xenophagy pathway in the host. 133 taken together, these findings support a prevailing view that autophagy is a host mechanism of intrinsic defense against intracellular bacteria, and under certain circumstances, mtb attempts to adopt it for its own benefit. several recent studies have raised questions about the exact role of autophagy in host-mtb interactions. on the one hand, growing studies support an autophagy-independent role of the autophagy machinery during infection. 134 for example, a study showed that mice lacking atg3, atg7, atg12, atg14, or atg16l1 in myeloid cells displayed few differences in bacterial loads compared with those of the control mice during acute mtb infection and argued that host atg5-dependent resistance to mtb predominantly depends on its regulatory functions in neutrophilrelated immunopathology rather its function in the autophagy pathway. 135 hence, the multifaceted protective role of autophagyrelated genes in host anti-mtb immunity should be taken into consideration and need to be further characterized. on the other hand, it has been shown that the mycobacterial esx-1 secretory system is required for activation of the host xenophagy pathway, 24, 117 which might support the supposition that mtb prevents autophagosome-lysosome fusion at the late stage of infection. 78, 79 furthermore, by monitoring autophagosome formation and subsequent degradation of autophagic cargo (a process termed autophagy flux) in infected cells, a research group found that virulent mtb strains selectively prevented autophagosomes from fusing with lysosomes, while the autophagosomes that did not contain mtb developed normally. 79, 136 these findings imply that mtb has probably adapted to persist in autophagosomal vacuoles by inhibiting their degradation, which means it creates a sheltered environment for prolonged intracellular survival. moreover, mtb appears to selectively prevent xenophagic flux rather than the entirety of autophagic flux in host cells, which would likely result in hyperinflammatory responses and cell death. 137 these hypotheses are supported by our finding that cytosolic mtb can induce autophagy recognition and activation via a highly conserved ubiquitin-binding associated (uba) domain on its surface to avoid excessive host inflammatory responses. 81 consistently, it has also been reported that, in a certain case, xenophagy can be beneficial for mtb replication. 63 notably, the host autophagy pathway has been proposed as a potential target for host-directed anti-tb therapy, 138 and based on these new concepts, a promising candidate of drugs or agents is expected to selectively target mtb-containing autophagosomal vacuoles rather than cause nonselective overall interference in host autophagic flux. in addition, these drugs should not only enhance autophagy activation but also overcome the mtbinduced blockade of autophagosome-lysosome fusion. novel mechanisms by which mtb targets innate immune regulatory machinery the increase in the number of studies has tremendously expanded our understanding of multifaceted molecular mechanisms by which mtb modulates the host immune regulatory network for its own advantage. in this section, we discuss the newly elucidated strategies adopted by mtb to manipulate the host regulatory machinery of cellular innate and intrinsic immune responses via direct host-pathogen molecular interactions (fig. 3) . mtb targeting of intranuclear immune regulatory machinery nucleus targeting has been emerging as a new aspect of the regulatory mechanism adopted by bacterial pathogens to manipulate host cell physiology and subvert immune defenses. in particular, an increasing number of bacterial effectors have been found to enter the infected cell nucleus to hijack host nuclear processes, and these nuclear attackers are named "nucleomodulins". 139 bacterial nucleomodulins may mimic eukaryotic transforming factors, transcription factors, chromatinregulatory factors or posttranslational modifiers, intervening in host gene transcription, chromatin reorganization, rna processing or dna replication and repair. 139 recent studies have identified several mycobacterial nucleomodulins that exert a range of intranuclear regulatory functions, which are described below. first, some mtb nucleomodulins function as histone-modifying enzymes to engage in epigenetic regulation of host immune responses. histone modification probably plays an essential role in the regulation of host anti-mtb immunity, since inhibition of histone deacetylases (hdacs) in human monocytes leads to attenuated host immune clearance of mtb. 140, 141 in addition, suppression of hdacs decreases matrix metalloproteinase-1 and -3 in mtb-infected macrophages, whose proteins drive tb lung immunopathology. 142 furthermore, histone methylation and acetylation are closely associated with bcg-induced host-trained immunity against mtb. 143, 144 pulmonary tb patients undergo obvious changes in histone modification in blood leukocytes; 145 similarly, individuals with clinical resistance to mtb infection (known as tb resisters) display an altered expression pattern of genes related to histone modification in blood monocytes. 140 to date, three mtb effectors that target and modify host histones have been identified: eis, rv1988, and rv3423.1. as previously described, mtb eis increases the acetylation level of histone h3 to regulate host autophagy activation during infection. 125 mtb rv1988 localizes to the host chromatin during infection, serving as a functional methyltransferase that dimethylates an arginine residue at h3r42 to repress a range of host genes involved in reactive oxygen species (ros) production, such as nox1, nox4, and noxa1 and nitric oxide synthase 2 (nos2). 146 although the significance of rv1988 on mtb pathogenesis has not been identified, the expression of rv1988 in nonvirulent m. smegmatis new insights into the evasion of host innate immunity by mycobacterium. . . q chai et al. markedly enhanced bacterial survival in infected mice. 146 mtb rv3423.1 was isolated from the chromatin of mtb-infected human macrophages where it displayed histone acetyltransferase activity and targeted host h3k9 and h3k14 (ref. 147 ). similarly, recombinant m. smegmatis rv3423.1 exhibited advanced intracellular survival in macrophages. 147 second, mtb nucleomodulin rv2966c was identified as a 5methylcytosine-specific dna methyltransferase that participates in the methylation of host genomic dna primarily at non-cpg cytosines upon infection. 148 however, the immunomodulatory role of rv2966c in host-mtb interactions has not been clarified. despite limited knowledge of the pathogenic contribution of mtbinduced host dna methylation changes, in mtb-infected macrophages, hypermethylation was predominantly observed on genes related to host immune responses, such as nlrp3 inflammasome activation and pro-inflammatory cytokine production. [149] [150] [151] these characteristics have been consistently observed in blood monocytes isolated from tb patients. 152 in addition, blood monocytes from bcg-vaccinated individuals also displayed a different dna methylation pattern and advanced capacity for mycobacterial control, indicating the involvement of dna methylation in hosttrained immunity against mtb. 153 third, some mtb protein effectors exhibit dual regulatory functions that not only target host cytosolic components but also mimic eukaryotic transcription factors involved in host intranuclear processes. for instance, the mtb secretory protein ppe2 was found to directly interact with the host cytosolic subunit of nadph oxidase, p67 phox , via an sh3-like domain to inhibit ros production and favor intracellular survival of mtb in macrophages. 154 intriguingly, ppe2 also contains a eukaryotic-like nuclear localization signal (nls), by which it can be translocated into the host nucleus via the classical importin α/β pathway. 155 thereafter, ppe2 binds to the nos2 promoter and limits host ros production. 155 in another example, early studies have demonstrated that mtb ptpa is delivered into the host cytosol, where it directly targets the vacuolar-h + -atpase machinery to inhibit phagosome acidification and the nf-κb pathway to suppress host inflammatory immune responses. 67, 156 moreover, we recently found that mtb ptpa can also enter the nucleus of infected cells, where it binds to and modulates the expression of diverse host genes, such as gadd45a, to affect cell proliferation and migration. 157 the host nucleus plays a central role in governing the all cellular activity, through which both genetic and epigenetic regulation of host immune responses to mtb are driven. 8 however, our understanding of the mechanistic and pathological implications of mtb-hijacked intranuclear processes in the host remains limited. for example, it remains unclear how mtb spatiotemporally regulates the intra-and extranuclear functions of these nucleustranslocated effectors. furthermore, the majority of the identified nucleomodulins do not contain a classic nls or nuclear export signal; what is the mechanism by which they shuttle between the mtb targets the host ubiquitin system the ubiquitin system refers to a network of proteins comprising enzymes that engage in ubiquitination and deubiquitination of cellular targets and ubiquitin receptors that decipher the ubiquitin code and translate it into cellular responses. 158 this elaborate system regulates a wide range of cellular immune responses and plays a vital role in host-pathogen interactions. 159 162 furthermore, it was recently reported that another mtb-secreted virulence factor, lpqn, directly interacts with the human e3 ubiquitin ligase cbl, which plays a regulatory role in cell-intrinsic responses to infection. 163 intriguingly, mycobacteria possess pupylation, the covalent modification of protein lysine residues with a ubiquitin-like protein called pup, but not ubiquitination as in eukaryotic cells. 164 our recent study noted that to efficiently interfere with host immunity, mtb not only simply inhibits ubiquitin ligase-mediated immunomodulatory functions but also subtly exploits the host ubiquitin system for its own advantage. we found that, by direct interaction with ubiquitin via a unique ubiquitin-interacting motif-like region, mtb ptpa is activated to dephosphorylate host p-jnk, p-p38, and p-vps33b, leading to suppression of innate immune responses. 156 similarly, we verified that another mtb ubiquitin-binding protein, rv1468c, resides on the bacterial surface, as mentioned above, and it directly recruited host cytosolic ubiquitin to trigger xenophagy to restrict host inflammatory responses. 81 more recently, we identified a mtb-secreted protein effector, rv0222, as a key suppressor of host nf-κb activation, showing that it undergoes k11-linked ubiquitination mediated by the host e3 ubiquitin ligase anaphase promoting complex (apc) subunit 2 (anapc2). 165 interestingly, rather than inducing the apcmediated canonical ubiquitin-proteasome degradation pathway, 166 k11-linked ubiquitination of rv0222 facilitates the interaction between src homology region 2 domain-containing phosphatase-1 and its adapter protein traf6, which blocks the k63-linked ubiquitination and activation of traf6, leading to inhibition of the nf-κb signaling pathway. 165 in conclusion, targeting the host ubiquitin system is a recently emerging aspect of the tactics mtb uses for immune evasion, which has received growing attention. curiously, growing evidence suggests that the ubiquitin system is often coopted by invading pathogens and then plays an altered regulatory role in host immune responses. future research will continuously expand our understanding of the ubiquitin system at the interface of host-mtb interactions, particularly the undefined roles of host-originated and mtbmimicking e3 ubiquitin ligases, deubiquitinases, and ubiquitin receptors. mtb targets intrinsic cellular immune components mtb has evolved to secrete a wide range of protein effectors via its sophisticated esx secretion systems to counter host immunity. 124, 167 in particular, growing numbers of mycobacterial effectors have been linked to direct protein-protein interactions with the host to target and modify key cellular intrinsic antibacterial machinery. for example, it has been found that mtb encodes eleven eukaryote-like serine-threonine protein kinases, including pkna to pknl (but not pknc), and two tyrosine phosphatases, ptpa and ptpb. 124 among these proteins, pkng is likely to selectively downregulate host pkc-α to inhibit the biogenesis of phagolysosomes. 168 ptpa dephosphorylates host p-vps33b, p-jnk, and p-p38 as described above, inhibiting phagosome acidification and the production of tnf and il-1β in macrophages 67, 156 ; ptpb decreases the phosphorylation of host p65, ikkα, erk1/2, and p38, suppressing macrophage apoptosis and the secretion of inflammatory cytokines. 169, 170 both ptpa and ptpb are indispensable for mtb intracellular survival. 156, 171 although the host substrates of these mtb eukaryotic-like kinases/phosphatases remain largely unknown, their essential roles in mtb virulence have been well documented. 124, 172 apart from phosphorylation-associated regulation of host factors, it was found that mtb eis can target and acetylate mitogen-activated protein kinase phosphatase-7 to prevent host jnk-dependent immune responses. 173 in addition, in our recent work, we revealed an mtb disulfide-bond-forming-like protein, mpt53, that can directly oxidize thiols on tak1 to facilitate tak1-mediated host hyperinflammatory immune responses. 174 in contrast to the abovementioned cellular factors that control pathogen infection indirectly through the activation of signaling cascades followed by innate immune responses, some other host proteins are constitutively expressed in certain cell types and directly act to restrict pathogen growth, and they are termed "restriction factors". 175 cellular restriction factors provide a frontline defense against invading microorganisms in a system known as host "intrinsic immunity"-a form of innate immunity initially elucidated in hosts as a mechanism to control viral infections. as discussed before, a range of antiviral immune mechanisms, such as cytosolic immune surveillance and the type i ifn response, are also involved in the host control of mtb infection, indicating that the host might adopt certain shared cellular immune machinery, which may include the similar restriction factors, upon infection by viruses and bacteria. for example, ifn-induced transmembrane (ifitm) family proteins are well-characterized host antiviral restriction factors critical for controlling the entry and intracellular replication of viral pathogens, 176 which has recently been associated with host anti-mtb defense mechanisms as well. specifically, iftm1, iftm2, and iftm3 are required for the host restriction of mtb intracellular growth in both human macrophages and lung alveolar cells, among which iftm3 was shown to colocalize with mtb phagosomes and contribute to phagosomal acidification. 177 another group of host intrinsic antiviral restriction factors, tripartite motif proteins (trims), have also been demonstrated to engage in the host control of mtb infection. 162, 178, 179 in turn, these host restriction factors may be targeted by mtb for immune evasion. as discussed above, mtb lpqn is able to interact with host cbl, which is a restriction factor that regulates the balance between cellular intrinsic antibacterial and antiviral responses. 163 similarly, mtb ptpa can directly bind host trim27 to antagonize its intrinsic immune functions. 162 furthermore, it was reported that trim14 is recruited to mtb phagosomes in macrophages to act as a negative regulator of host cytosolic dnasensing pathway-dependent mycobacterial restriction. 180 together, these findings suggest potential strategies utilized by mtb to avoid host intrinsic immunity. despite the compelling findings supporting an essential role for various cellular intrinsic protein factors in host anti-mtb immunity, the determinant molecules of host resistance to tb infection new insights into the evasion of host innate immunity by mycobacterium. . . q chai et al. remain largely unexplored. the application of recently developed research methods, such as genome-wide association analysis of human tb patients, may help to reveal the genetic etiology of tb and to identify key anti-mtb intrinsic immune components. 181 furthermore, there is still a limited understanding of the direct interactions between mtb-secreted proteins and host proteins, which play central roles in tb pathogenesis. thus, more studies based on valid screening systems, such as the affinity tag purification mass spectrometry system, the mycomart7 transposon system, and the crispr-cas9 screening system, 163, 182, 183 are warranted to further improve our understanding of the mtb-host network of molecular interactions. our understanding of the interplay between mtb and the host innate immune system has extensively expanded in recent years. as summarized in this review, upon mtb infection, various cellular antimicrobial components respond to the activation of host innate immune surveillance pathways, which might be modulated by mtb for its benefit. moreover, an increasing number of studies have revealed emerging mtb strategies to exploit the host molecular regulatory machinery of the innate immune system, including mtb-mediated disruption of the host intranuclear immune regulatory machinery, the ubiquitin system and intrinsic cellular immune components. thus, recent research on host-mtb interactions has changed the traditional view that the pathogen is incompatible, and in conflict with its host until one is overwhelmed. as a particularly successful intracellular pathogen, mtb has evolved much more moderate and nuanced strategies for immune modulation and evasion, with the principal aim of adapting to an intracellular niche for prolonged survival, rather than simply destroying the host. therefore, it is not surprising that some mycobacterial factors have an inhibitory effect on host cellular antibacterial mechanisms (e.g., interfering with protective th1-type cytokine production, vacuolar membrane trafficking, or autophagy activation), while others appear to play an opposite regulatory role. in fact, host immune responses are spatiotemporally regulated and dynamically changed throughout the course of tb. 184, 185 therefore, mtb probably tends to employ distinct effectors at different stages to bilaterally modulate the host immune machinery to establish a successful long-term infection. this concept is supported by compelling evidence indicating that, while an early protective th1-type response favors a host-controlled infection, the machinery is often suppressed or exploited by mtb, for example, to induce hyperinflammation at the late stage of infection, which causes lung cavitation and thus benefits bacterial transmission. 186 therefore, more in-depth studies are warranted to gain further insights into the regulatory mechanisms by which mtb establishes innate immune evasion, providing knowledge that may help in the identification of either hostor pathogen-directed anti-tb therapeutic targets and contribute to the design of more efficient vaccines. transmission of extensively drug-resistant tuberculosis in south africa t cells and adaptive immunity to mycobacterium tuberculosis in humans fatal mycobacterium tuberculosis infection despite adaptive immune response in the absence of myd88 tlr2-dependent mast cell activation contributes to the control of mycobacterium tuberculosis infection innate immunity in tuberculosis: host defense vs pathogen evasion recognition of mycobacterial lipids by immune receptors host defense mechanisms against mycobacterium tuberculosis antitumor activity of deoxyribonucleic acid fraction from mycobacterium bovis bcg. i. isolation, physicochemical characterization, and antitumor activity m. tuberculosis and m. leprae translocate from the phagolysosome to the cytosol in myeloid cells esx-1-mediated translocation to the cytosol controls virulence of mycobacteria an interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis innate immune dna sensing pathways: sting, aimii and the regulation of interferon production and inflammatory responses mycobacterium tuberculosis activates the dna-dependent cytosolic surveillance pathway within macrophages cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna cytosolic-dna-mediated, sting-dependent proinflammatory gene induction necessitates canonical nf-kappab activation through tbk1 phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf3 activation cgas is essential for cellular senescence nuclear cgas suppresses dna repair and promotes tumorigenesis phosphoinositide interactions position cgas at the plasma membrane to ensure efficient distinction between self-and viral dna cyclic gmp-amp synthase is an innate immune dna sensor for mycobacterium tuberculosis mycobacterium tuberculosis differentially activates cgasand inflammasome-dependent intracellular immune responses through esx-1 the cytosolic sensor cgas detects mycobacterium tuberculosis dna to induce type i interferons and activate autophagy the cgas/sting pathway is important for dendritic cell activation but is not essential to induce protective immunity against mycobacterium tuberculosis infection cgas/sting/tbk1/irf3 signaling pathway activates bmdcs maturation following mycobacterium bovis infection aim2 activates the inflammasome and cell death in response to cytoplasmic dna aim2 recognizes cytosolic dsdna and forms a caspase-1-activating inflammasome with asc critical role of aim2 in mycobacterium tuberculosis infection the aim2 inflammasome is involved in macrophage activation during infection with virulent mycobacterium bovis strain new insights into the evasion of host innate immunity by mycobacterium cutting edge: mycobacterium tuberculosis but not nonvirulent mycobacteria inhibits ifn-beta and aim2 inflammasome-dependent il-1beta production via its esx-1 secretion system deficiency of the aim2-asc signal uncovers the sting-driven overreactive response of type i ifn and reciprocal depression of protective ifn-γ immunity in mycobacterial infection stable extracellular rna fragments of mycobacterium tuberculosis induce early apoptosis in human monocytes via a caspase-8 dependent mechanism a. from mycobacterium tuberculosis-infected cells is functional in recipient macrophages rig-i-like receptor regulation in virus infection and immunity master sensors of pathogenic 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to remember a role for double-stranded rna-activated protein kinase pkr in mycobacterium-induced cytokine expression cytoplasmic rna sensor pathways and nitazoxanide broadly inhibit intracellular mycobacterium tuberculosis growth improved control of tuberculosis and activation of macrophages in mice lacking protein kinase r evidence for dispensability of protein kinase r in host control of tuberculosis human nlrp3 inflammasome senses multiple types of bacterial rnas nod1 and nod2: beyond peptidoglycan sensing molecular mechanisms regulating nlrp3 inflammasome activation nlrp3 inflammasome activation by mycobacterial esat-6 and dsrna in intraocular tuberculosis phagocytosis and antigen presentation in dendritic cells cell biology of mycobacterium tuberculosis phagosome several routes to the same destination: inhibition of phagosome-lysosome fusion by mycobacterium tuberculosis endosomal membrane traffic: convergence point targeted by mycobacterium tuberculosis and hiv quantitative 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the rab11 + recycling endocytic pathway and avoid lipocalin 2 trafficking to the lysosomal pathway rab7 controls lipid droplet-phagosome association during mycobacterial infection mycobacterium tuberculosis induces the mir-33 locus to reprogram autophagy and host lipid metabolism lipid droplet formation in mycobacterium tuberculosis infected macrophages requires ifngamma/hif-1alpha signaling and supports host defense cytosolic access of mycobacterium tuberculosis: critical impact of phagosomal acidification control and demonstration of occurrence in vivo formation of the legionella replicative compartment at the crossroads of retrograde trafficking a brucella type iv effector targets the cog tethering complex to remodel host secretory traffic and promote intracellular replication a unique mycobacterium esx-1 protein co-secretes with cfp-10/ esat-6 and is necessary for inhibiting phagosome maturation mycobacterium tuberculosis evades macrophage defenses by inhibiting plasma membrane repair eicosanoid pathways regulate adaptive immunity to mycobacterium tuberculosis esx-1 dependent impairment of autophagic flux by mycobacterium tuberculosis in human dendritic cells mycobacterium tuberculosis inhibits rab7 recruitment to selectively modulate autophagy flux in macrophages plasma membrane damage causes nlrp3 activation and pyroptosis during mycobacterium tuberculosis infection a mycobacterium tuberculosis surface protein recruits ubiquitin to trigger host xenophagy association of a macrophage galactoside-binding protein with mycobacterium-containing phagosomes the escrt and autophagy machineries cooperate to repair esx-1-dependent damage at the mycobacterium-containing vacuole but have opposite impact on containing the infection bcl-xl mediates ripk3-dependent necrosis in m. tuberculosisinfected macrophages tnf dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species corticosteroids inhibit mycobacterium 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thp-1 affects apoptosis differential responses by human macrophages to infection with mycobacterium tuberculosis and non-tuberculous mycobacteria cell death at the cross roads of hostpathogen interaction in mycobacterium tuberculosis infection host genotype-specific therapies can optimize the inflammatory response to mycobacterial infections intracellular growth of mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells necroptotic signaling is primed in mycobacterium tuberculosisinfected macrophages, but its pathophysiological consequence in disease is restricted nad + depletion triggers macrophage necroptosis, a cell death pathway exploited by mycobacterium tuberculosis mycobacterium tuberculosis prevents inflammasome activation inhibition of the plasma-membrane-associated serine protease cathepsin g by mycobacterium tuberculosis rv3364c suppresses caspase-1 and pyroptosis in macrophages mycobacterium tuberculosis-induced neutrophil extracellular traps activate human macrophages in vivo induction of neutrophil extracellular traps by mycobacterium tuberculosis in a guinea pig model a major role for ferroptosis in mycobacterium tuberculosisinduced cell death and tissue necrosis die another way: ferroptosis drives tuberculosis pathology molecular definitions of autophagy and related processes ubiquitin-dependent and independent signals in selective autophagy autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages t helper 2 cytokines inhibit autophagic control of intracellular mycobacterium tuberculosis lysosomal killing of mycobacterium mediated by ubiquitin-derived peptides is enhanced by autophagy autophagy regulates phagocytosis by modulating the expression of scavenger receptors autophagy enhances the efficacy of bcg vaccine by increasing peptide presentation in mouse dendritic cells delivery of cytosolic components by autophagic adaptor protein p62 endows autophagosomes with unique antimicrobial properties extracellular m. tuberculosis dna targets bacteria for autophagy by activating the host dna-sensing pathway the ubiquitin ligase parkin mediates resistance to intracellular pathogens the ubiquitin ligase smurf1 functions in selective autophagy of mycobacterium tuberculosis and anti-tuberculous host defense the selective autophagy receptors optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection in vitro ubiquitination of mycobacterium tuberculosis by e3 ubiquitin ligase, mkrn1 galectin-8 targets damaged vesicles for autophagy to defend cells against bacterial invasion mycobacterium tuberculosis: an adaptable pathogen associated with multiple human diseases virulence factors of the mycobacterium tuberculosis complex mycobacterium tuberculosis eis gene inhibits macrophage autophagy through up-regulation of il-10 by increasing the acetylation of histone h3 molecular characterization of lc3-associated phagocytosis reveals distinct roles for rubicon, nox2 and autophagy proteins mycobacterium tuberculosisis protected from nadph oxidase and lc3-associated phagocytosis by the lcp protein cpsa micrornas play an essential role in autophagy regulation in various disease phenotypes microrna-125a inhibits autophagy activation and antimicrobial responses during mycobacterial infection microrna 17-5p regulates autophagy in mycobacterium tuberculosis-infected macrophages by targeting mcl-1 and stat3 mycobacterium tuberculosis-induced mir-155 subverts autophagy by targeting atg3 in human dendritic cells mir144* inhibits antimicrobial responses against mycobacterium tuberculosis in human monocytes and macrophages by targeting the autophagy protein dram2 microrna-27a controls the intracellular survival of mycobacterium tuberculosis by regulating calcium-associated autophagy autophagy-independent functions of the autophagy machinery unique role for atg5 in neutrophil-mediated immunopathology during m. tuberculosis infection selective autophagy gets more selective: uncoupling of autophagy flux and xenophagy flux in mycobacterium tuberculosis-infected macrophages autophagy regulates inflammatory programmed cell death via turnover of rhim-domain proteins autophagy: a new strategy for hostdirected therapy of tuberculosis when bacteria target the nucleus: the emerging family of nucleomodulins transcriptional networks are associated with resistance to mycobacterium tuberculosis infection phenylbutyrate is bacteriostatic against mycobacterium tuberculosis and regulates the macrophage response to infection, synergistically with 25-hydroxy-vitamin d 3 epigenetic regulation of matrix metalloproteinase-1 and −3 expression in mycobacterium tuberculosis infection bacille calmette-guerin induces nod2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes bcg educates hematopoietic stem cells to generate protective innate immunity against tuberculosis histone h3k14 hypoacetylation and h3k27 hypermethylation along with hdac1 up-regulation and kdm6b down-regulation are associated new insights into the evasion of host innate immunity by mycobacterium with active pulmonary tuberculosis disease mycobacteria modulate host epigenetic machinery by rv1988 methylation of a non-tail arginine of histone h3 hypothetical protein rv3423.1 of mycobacterium tuberculosis is a histone acetyltransferase the interaction of mycobacterial protein rv2966c with host chromatin is mediated through non-cpg methylation and histone h3/h4 binding nlrp3 activation was regulated by dna methylation modification during mycobacterium tuberculosis infection genome-wide non-cpg methylation of the host genome during m. tuberculosis infection unraveling methylation changes of host macrophages in mycobacterium tuberculosis infection dna hyper-methylation during tuberculosis dampens host immune responsiveness anti-mycobacterial activity correlates with altered dna methylation pattern in immune cells from bcg-vaccinated subjects mycobacterium tuberculosis ppe2 protein interacts with p67 phox and inhibits reactive oxygen species production the ppe2 protein of mycobacterium tuberculosis translocates to host nucleus and inhibits nitric oxide production mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system the mycobacterial phosphatase ptpa regulates the expression of host genes and promotes cell proliferation snapshot: expanding the ubiquitin code ubiquitin in the immune system the ubiquitin system: a critical regulator of innate immunity and pathogen-host interactions mycobacterium tuberculosis alters the metalloprotease activity of the cop9 signalosome the ubiquitin ligase trim27 functions as a host restriction factor antagonized by mycobacterium tuberculosis ptpa during mycobacterial infection an mtb-human protein-protein interaction map identifies a switch between host antiviral and antibacterial responses the pupylation pathway and its role in mycobacteria host-mediated ubiquitination of a mycobacterial protein suppresses immunity building a regulatory network with short linear sequence motifs: lessons from the degrons of the anaphase-promoting complex esx secretion systems: mycobacterial evolution to counter host immunity downregulation of protein kinase c-α enhances intracellular survival of mycobacteria: role of pkng targeting mycobacterium protein tyrosine phosphatase b for antituberculosis agents mptpb promotes mycobacteria survival by inhibiting the expression of inflammatory mediators and cell apoptosis in macrophages disruption of mptpb impairs the ability of mycobacterium tuberculosis to survive in guinea pigs mycobacterium tuberculosis serine/threonine protein kinases mycobacterium tuberculosis eis protein initiates suppression of host immune responses by acetylation of dusp16/mkp-7 oxidization of tgfβ-activated kinase by mpt53 is required for immunity to mycobacterium tuberculosis intrinsic antiviral immunity the broad-spectrum antiviral functions of ifit and ifitm proteins a role for ifitm proteins in restriction of mycobacterium tuberculosis infection trim22 regulates macrophage autophagy and enhances mycobacterium tuberculosis clearance by targeting the nuclear factor-multiplicity kappab/beclin 1 pathway trims and galectins globally cooperate and trim16 and galectin-3 co-direct autophagy in endomembrane damage homeostasis trim14 is a key regulator of the type i interferon response during mycobacterium tuberculosis infection genome-wide association study identifies two risk loci for tuberculosis in han chinese comprehensive identification of conditionally essential genes in mycobacteria improved vectors and genome-wide libraries for crispr screening inflammatory signaling in human tuberculosis granulomas is spatially organized spatial and temporal localization of immune transcripts defines hallmarks and diversity in the tuberculosis granuloma ecology and evolution of mycobacterium tuberculosis competing interests: the authors declare no competing interests.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-008499-tl3i7uzd authors: robb, james a.; benirschke, kurt; barmeyer, robert title: intrauterine latent herpes simplex virus infection(): i. spontaneous abortion date: 2007-11-06 journal: hum pathol doi: 10.1016/s0046-8177(86)80561-5 sha: doc_id: 8499 cord_uid: tl3i7uzd herpes simplex virus (hsv, probably type 2) antigen was detected in nonpregnant and pregnant endometria, placentae, umbilical cords, and neonatal tissues (companion paper) by avidinbiotin complex immunohistochemical studies. hsv cytologic abnormalities were not detected in any of the 380 cases examined: included were specimens from therapeutic and spontaneous abortions (200 cases) and endometrial curettage (180 cases). the presence of inflammation was not correlated with hsv positivity. endometrial hsv positivity was significantly correlated with normal late secretory phase (40 per cent of specimens positive), abnormal secretory phase (67 per cent positive), and therapeutic (33 per cent positive) versus spontaneous (26 per cent positive) abortions. placental hsv positivity was significantly correlated with spontaneous (39 per cent positive) versus therapeutic (14 per cent positive) abortions and with blighted ova (67 per cent positive). no significant correlation was found between hsv positivity and a clinical history of oral or genital hsv infection in either the patient or the male partner. the data support the concept of a subclinical latent intrauterine endometrial hsv infection that is hormonally regulated and can produce transplacental infection of the embryo or fetus, with variable consequences. acute intrauterine herpes simplex virus (hsv) infection, although rare, t has been increasing in frequency. 2 our stndy was prompted by the following case of acute intrauterine hsv infection. a full-term infant was delivered alive with acute hsv infection of the skin and died a few days later of systemic infection. the presence of acute hsv infection in the skin and brain was confirmed by morphologic hsvspecific cytologic studies, culture, and transmission electron microscopy. no evidence of hsv infection was found in routine studies of the placenta and umbilical cord by the same techniques. transplacental infection, however, must have occurred, because the infection was not acquired from the birth canal during delivery. a newly developed and highly sensitive glucose oxidase-avidin-biotin immunohistochemical technique for formalin-fixed tissues 3 demonstrated hsv-specific antigen ill phagocytic cells of the placental subamnionic chorion, in hofbauer cells in chorionic villi, and in the subamnionic mesenchyme of the umbilical cord. no inflammation or hsv cytologic abnormalities were present in these areas. spontaneous and therapeutic abortion material, endometrial curettage tissue, full-term placentae and umbilical cords, and stillborn and liveborn neonatal tissues were studied by hsv-specific immunohistochemical methods to evaluate the prevalence and distribution of these hsv antigens in cases in which hsv infection was not suspected or detectable as hsv cytologic abnormalities by light microscopy, virus particles by transmission electron microscopy, or infectious hsv by direct cultnre. the presence and tissne distribution of the hsv antigens were then correlated with the finding for normal and pathologic conditions. the data concerning the fullterm neonates are presented in the companion paper. 4 the data concerning the detection of persistent intranterine hsv infection and its correlation with pathologic states in abortion and curettage material are presented below. all tissues were routine stn'gical (products of conception, endometrial curettage, placentae, and umbilical cords) or autopsy (neonates and infants) specimens fixed in 10 per cent buffered formalin or bouin's solution (placentae and cords only) and embedded in paraffin. the products of conception and endometrial tissues had been collected from 1981 to 1984 at the green hospital of scripps clinic. the placentae, umbilical cords, and neonatal or infant tissues had been collected from 1981 to 1984 at the university of california san diego medical center. some neonatal cases had been submitted from other hospitals for consultation. serial 4-i.tm sections were attached to glass slides with either 1 per cent elmer's glue in deionized water or chromic acidfpoly-d-iysine (50 per cent chromic acid in deionized water for 30 minntes at room temperature, followed by rinsing and 0.01 per cent poly-d-lysine [sigma, st. louis, missouri] in deionized water for 30 minutes at room temperature, followed by rinsing). the paraffin sections were not dried in an oven, but were left at room temperature for at least two hours before routine rentoval of paraffin and immunohistochemical staining. all of the following steps were done at room temperature in a humidified box. the tissue sections were treated with 0.25 per cent crude porcine trypsin (sigma, catalog no. t-8128) in phosphate-buffered saline (pbs, ph 7.4) for 60 minutes and rinsed with pbs by a jet-dribble technique. primary polyclonal rabbit anti-hsv type 1 and type 2 igg (dako, santa barbara, california; lots 059a and 1188, respectively) and nonimmune rabbit igg (cappell labs, west chester, pennsylvania) were incubated for 22 to 24 hours at 1:2,000 (hsv types 1 and 2) and at 10 txg/ml (nonimmune rabbit igg) in pbs+ (pbs with 0.2 per cent sodium azide [sigma] and 0.1 per cent bovine serum albumin [calbiochem, la jolla, california; type v]). the tissues were again rinsed with pbs, and biotinylated goat antirabbit igg (3.25 lag/ntl pbs + ; vector labs, burlingame, california), biotinylated horse antimouse igg (30 ixg/ml, vector), or biotinylated goat anti-guinea pig igg (7.5 ixg/ml, vector), depending on the species of primary antibody (see table 2), was added for 30 minutes. after pbs rinsing, a glucose oxidase-avidin-biotin complex (abc-go, vector) was added for 30 minutes at room temperature. the staining reaction was identical to the previously described glucose oxidaseavidin-biotin (gab) technique originally used in this study. 3 tetranitroblue tetrazolium (tnbt) formazan formation (vector) was used to detect sites of antibody binding. this "colorization reaction" was identical to that originally used in this study. 3 after a deionized water rinse, aqueous counterstaining was accomplished with 0.25 per cent metanil yellow in deionized water for 1 minute, deionized water rinse, and 0.1 per cent nuclear fast red for 5 minutes; a 0.45-~m filter (gelman acrodisc, gelman sciences, ann arbor, michigan) was used at the time of addition to remove microcrystals. aquamount (lerner labs, new haven, connecticut) was used for coverslipping. no loss of stain occurred during a four-year period. for statistical analyses a clinfo software program was used in the scripps clinic general clinical research center. all data were nonparametric, except for the patient age data, which were normally distributed and analyzed by the anderson-darling t-test. the nonparametric data were analyzed with the wilcoxon nonpaired rank sum test. the rabbit anti-hsv 2 antibody staining in placental tissues was cytoplasmic and occurred in individual cells in the subamnionic chorion ( fig. 1 ) and in single hofbauer cells in chorionic villi ( fig. 2 ). umbil-ical cord staining occurred in single cells in tile subamnionic mesenchyme ( fig. 3 ), perivascular mesenchyme ( fig. 4 ) and, rarely, vascular smooth muscle ( fig. 4 ). no nuclear, amnionic, trophoblastic, or endothelial staining was detected in placental tissues. apical and/or diffuse cytoplasmic staining occurred focally in the endometrial epithelium in both products of conception and nonpregnant endometrium, without specific nuclear staining ( fig. 5 ). single choriodecidual cells were positive, as in the placentae ( fig. 6 ). endothelial staining was rare (three of 380 cases) and was fotmd only in choriodecidual tissue, where it was both cytoplasmic and nuclear ( fig. 6 ). acute hsv endometrial epithelial infections, although rare, have been documented. 5,6 altered endometrial epithelial nuclei with features suggestive of hsv infection, but without viral particles by transmission electron microscopy, were found in 7 per cent of a group of 200 cases in which endometrium was associated with trophoblastic tissue. 7 endothelial cells can be infected by hsv, with subsequent altered function and death. 8,9 histologically occult cytomegalovirus (cmv) infection has been detected in human tissue by immunohistochemical methods i° and by in situ dna hybridization. ii either the detected antigens or their detecting antibodies were at a low concentration, because the screening anti-hsv 2 antibody titer difference between acute and chronic or latent infection was about 100-fold (acute--1:20,000 for two hours; chronic or latent--1:2,000 for 20 hours). the pattern of embryonic and neonatal staining is described in the companion paper. 4 other virus-infected tissues. table 1 demonstrates the specificity of the anti-hsv antibodies when tested against culture-proven acute hsv 1 infection in the skin ( fig. 7 ), brain, and esophagus, and against hsv 2 infection in the liver (acute) and adrenal gland (subacute, i.e., resolving intrauterine infection, several weeks after acute infection; fig. 7 ). anti-hsv 1' and hsv 2 antibodies strongly cross-react in tissues acutely infected with hsv 1 and hsv 2 (see table 1 for end-point titers). the antibodies were absolutely negative in other tissues with culture-proven and/or immunohistochemically proven infections with the other three herpetoviruses (herpes zoster virus, cmv, and epstein-barr virus) and other miscellaneous viruses (hepatitis a and b, papillomavirus, and echovirus). a placenta with culture-proven listeria infection was also negative. the virus used as antigen for antibody production was purified from culture medium containing fetal bovine serum. for that reason, the antibodies were tested against tissues containing both human and bovine alpha-fetoprotein and were found to be negative (table 1) . furthermore, adsorption of the anti-hsv 2 igg with fresh fetal bovine liver powder (acetone-extracted) did not decrease the specific staining in target tissues. cnlt n re/cytopat hology cytopathology cnltn re/c)'topathology cnhure/cytopathology * hsv 1 and ttsv 2 antibody tilers against tile tlsv 1 acutely infected skin were i:16,000 and 1:8,000, respectively; tile liters against the hsv 2 subacutely infected adrenal gland were 1:4,000 and 1:!6,000, respectively. other antiviral antibodies. table 2 lists the staining results for various hsv-specific and nonspecific antibodies with the various target tissues. all of the ucsd/dr rabbit antisera and the yale/gdh guinea pig antisera were coded samples at the time of tissue staining and evaluation. t h e following findings strongly support our hypothesis that the staining detected in the target and patient tissues with the dako rabbit anti-hsv 2 igg (lot 1188, our screening antibody) is specific for hsv and, probably, for type 2 infection in t h e products of conception, placentae, umbilical cords, and endometrial curettage specimens: 1. all preinfection, noninfected, and non-hsv sera were negative with all target tissues, with one exception. one of 11 ucsd/dr hsv 1 antisera produced 4 + staining of the vascular smooth muscle of the umbilical cord target ( fig. 4 ). the meaning of this finding is not known, although histologically occult cmv infection has been detected in human smooth muscle by immunohistochemical methods, l° 2. all hsv-infected animal sera were positive with at least one target tissue. there was wide individual variation, however, when the same target was used with different sera and different targets were used with the same sera. this phenomenon of variation in individual animal antibody expression to individual virus antigens is to be expected, both in humans 12 and in syngeneic mouse littermates, r3 as observed when cloned virus was used for "identical infection." one example o f the specificity o f the rabbit anti-hsv 2 antibody is shown in figure 8 . this gastric biopsy specimen from a patient with the acquired immunodeficiency syndrome shows cmvpositive cells (goat anti-cmv, polysciences, 1:200 with trypsin pretreatment), which are negative for hsv 2 antigen, while adjacent gastrothelial cells show latent hsv 2 infection. 3. all of the nih/mz mouse anti-hsv monoclonal antibodies were positive in at least three targets. these antisera stain cells infected acutely with both hsv 1 and hsv 2 in cell culture. 4. the nih/mz mouse monoclonal antibody icp4 recognizes an immediate/early (alpha) antigen in a nuclear location in both hsv 1 and 2 acute infections in cell culture. scattered anmionic nuclei in the placental target were unequivocally positive with this antibody ( fig. 9 ), an indication of latent infection in the amnion. intranuclear staining by icp4 was also detected in chorionic nuclei in one case ( fig. 9 ). negative control antibodies (negative in all above target tissues): rabbit--normal nonimmune igg, anti-fetuin, anti-fetal bovine serum, anti-callous keratin, anti-carcinoembryonic antigen, and auti-ebv. antibovine alpha-fetoprotein was positive only in phagocytic cells of sac, a subset of which were hsv-2-positive by dual labeling experiments. guinea pig--normal nonimmune igg, anti-insulin, and anti-varicella/zoster. mouse--mopc-21 and anticytokeratius 35bhi 1 and aei. goat--normal nonimmune igg and anti-cmv. abbrevtations: an, anmiotic nuclear staining; c, cytoplasmic staining; cc, chorionic cell cytoplasnfic staining; hisv, hyperimmunization with solubilized virions; macro, adrenal adventitial macrophages; n, nuclear staining; nd, not done due to scarcity of target tissue; nsb, nouspecific background staining; post, postinfection; pre, preinfection; pvm, perivascular mesenchymal cells; sac, subamnionic chorion cells; sam, subamnionic meseuchymal cells; vh, chorionic villias hofbauer cells; vsm, vascular smooth muscle cells; neut tit, neutralization titer. * lot of anti-hsv 2 igg used to screen all patient tissues. staining grade of 1 to 4: positive cell number and intensity of stain were compared with the staining observed in tissues stained by the lot 1188 rabbit anti-hsv 2 igg as follows: 4 +, 75-100%; 3 +, 50-75%; 2 +, 25-50%; i +, less than 25%. endothelial nuclei were positive in three of 380 abortion cases ( fig. 6 ); the rabbit anti-hsv 2 antibody stained specimens in all three cases, while staining with the mouse anti-icp4 antibody was observed in only two cases. weak intranuclear staining of amnionic cells in the placental target cells was observed with the mouse anti-icp5 antibody, which detects nuclear antigen in hsv 1 and 2 acute infection in cell culture. 5. the dako hsv 2 individual sera were from rabbits that had not had the extensive repeat immunizations undergone by tile rabbits in the pool of the screening antibody (lot 1 188). they were, therefore, of much lower titer. the "new" lot (no. 012c) of adsorption experimer~ts. two identical adsorption experiments were periormed four months apart with coded samples of noninfected frozen cells, or cells infected with cmv or hsv l or hsv 2. the thawed cell lysates were mixed with the screening hsv 1 and hsv 2 antibodies for 30 minutes at room temperature and centrifuged, and the supernatant fluid was tested on the target tissues. in the first experiment, the anti-hsv 2 antibody was easily identified by decreased staining, compared with that produced by the nonadsorbed antibody assayed at the same time in serial sections of target tissues. in the second experiment, no reduction in any of the adsorbed antibodies was detected. the reason for the discrepancy is not known, although adsorption experiments such as this can be difficult to replicate and interpret, because of the frozen, then thawed, nonfixed antigens and complement receptors. false-positive res'tdts. only two false-positive staining patterns were identified, both involving the endometrial epithelium. the first, and more frequent (20 of 380 cases), was a basal cytoplasmic granular staining pattern, without the usual diffuse and/ or apical staining ( fig. 10 ). this pattern occurred with several of the hsv-specific and nonspecific anti-bodies and was independent of the secondary antibody, abc complex, and colorizing solution. the mechanism of staining is not known. the second, and quite rare (two of 380 cases), false-positive pattern was confined to endometrial nuclei in a focal pattern ( fig. ] 0) . this intranuclear staining was independent of the primary and secondary antibodies, the enzymes attached to the abc complex (peroxidase, alkaline phosphatase, and glucose oxidase), and the various colorizing solutions. it was dependent only on the abc complex itself. the mechanism remains unknown, because neither biotin or avidin preincubations blocked the staining. no viral cytologic abnormalities were detected in any of the 200 specimens. material from spontaneous abortiops was more inflamed than that from therapeutic abortions (92 versus 66 per cent, p < 0.001), but there was no difference between the two groups with regard to acute versus chronic choriodeciduitis (p "-0.565). although total hsv positivity (endometrial epithelial positivity and/or placental subamnionic chorion positivity and/or chorionic villus positivity) was similar in the material from spontaneous and therapeutic abortions (48 and 41 per cent, p = 0.386), placental subamnionic chorion positivity and chorionic villus positivity were significantly more frequent in spontaneous abortion specimens (39 and 14 per cent) than in those from therapeutic abortions (13 and 0.0 per cent, p < 0.001 and p < 0.001, respectively), while endometrial epithelial positivity was more frequent in therapeutic than in spontaneous abortion material ( table 3 lists the statistical correlations between the endometrial phases, abnormalities, and inflammation for endometrial epithelial negativity and endometrial epithelial positivity for 180 cases. there was no significant difference between the ages of the two groups (hsv-negative, 41.5 years; hsv-positive, 39.8 years; p = 0.765) or for the volume of endometrial tissue removed, excluding blood clots and mucus (<3 mm 3 versus 3 to 5 mm 3 versus >5 mm 3, p = 0.797). the detection of endometrial epithelial positivity was significantly different in tile different phases of the cycle. normal proliferative (i0 per cent), hyperplastic (14 per cent, p = 0.697), and atrophic (6.7 per cent, p > 0.990) endometria were not statistically different. normal secretory (40 per cent) and abnormal secretory (67 per cent) endometria were significantly different from normal proliferative (p = 0.017 and <0.001), hyperplastic (p = 0.001 and <0.001), and atr-ophic (p = 0.014 and <0.001) endometria, as well as from each other (p = 0.040). furthermore, the cycle date in the cases of normal secretory endometrium was significantly earlier for the hsv-negative cases (day 20.6) than for the hsv-positive cases (day 22.6, p = 0.004), suggesting activation of hsv protein synthesis in the latter portion of the normal secretory phase. the percentage of hsv positivity in normal secretory phase endometriuln (40 per cent) was similar to that found in material from both therapeutic (41 per cent) and spontaneous (48 per cent) abortions. the presence of hsv positivity in chronic active endometritis depended on the phase and was similar to the degree of hsv positivity found in the corresponding noninflamed normal phases. * parameters: total inflammation, presence of acute or chronic choriodeciduitis (cd); normal, no acute or chronic cd; total i isv +, staining in one or more of the following patterns--chorionic villus llofbauer cells (vh), snbamnionic chnrion (sac), or endometrial epithelium (emet) (see results section anti figures i and 3); blighted ovmn, destruction of the embr)o as defined by lack of nucleated embryonic erythrocytes and capillaries in the chorionic villi; muhiparity, more than one pregnancy; sab and tab, spontaneous and therapeutic abortions, respectively; recurrent sab, more than one spontaneous abc)rtion; gestational age, the gestation:d age of the embryo at the time of the abortion; patient or partner cold sore, clinical history of at least one episode of ~wal hsv infection in the patient or partner at the time of pregnancy, respectively; patient or partner genital tlsv, clinical ifistory of genital hsv infection in the patient or partner, respectively. i the patient ages were normally distributed for all categories and were analyzed t))" the anderson-darling test, while all other parameters were nonparametric and were analyzed by the wilcoxon mmpaired rank sum test. the specificity of the screening rabbit anti-hsv antibody in detecting only hsv antigens underlies the concepts about latent intrauterine and neonatal hsv infection presented in this study. our contention that the antigens that we detected in endometrial, placental, and cordal tissues are specific for hsv, probably of type 2 origin, is strongly supported by the control data. the control studies included numerous non-hsv virtts-infected tissues, hsv-negative tissues, and bacterium-infected tissue. in addition, numerous hsv-positive and -negative antibodies derived from different animals and from different routes of infection with hsv viruses of both'type 1 and 2 were used as controls. confirmation of this hsv specificity will have to be accomplished by dna and/or rna extraction and/or in situ hybridization techniques, if the necessary technical sensitivity can be achieved, lt.ta.t5 mouse monoclonal antibodies lmve been useful in defining the hsv-specific nature of tile detected antigens, especially the amnionic intranuclear staining of ttte immediate/early antigen, ici'4. exact delineation of the hsv-specific proteins present in our tissues was difficult, for at least two reasons. first, tltere are 15 to 35 different virion proteins in the hsv virion, 16,t7 and the ntunber of available monoclonal antibodies to these proteins is limited. virus-specific nonvirion and celhtlar proteins also are present in hsv-infected cells; some of these proteins may have been detected with the dako antibody, which recognizes both hsv-specific virion and nonvirion antigens, ts the antibody response to hsv is polyclonal, and, in humans, at least 31 hsv 1 and 27 hsv 2 virus-specific proteins can be antigenic with great individual variation, t2 second, little is known about the fate or degradation sequence of the various hsv-spccific virion and nonvirion proteins in phagocytic cells (primarily macrophages) tltat organize (i.e., clean up) the cellular and viral debris after acute hsv infection. hsv proteins are degraded differentially in different cell lines after acute infections in cell culture. 19 therefore, redundant antigenic sites on the virus-specific proteins 9°-23 may be either exposed or lost during the degradation process in macroplmges or other phagocytic cells, such as some choriodecidual cells and the hofbauer cells of the chorionic villi. tiffs uncertainty may make identification of specific hsv protdins by imnumohistochemistry and/or isolation difficult in subacute or chronic infections in animal tissues. antigenic variants of the viral glycoproteins may also occur. 24 the long-term (weeks to months) fate of the numerous hsv virion and nonvirion protein antigenicities in acutely infected tissues has not been studied in animal models. our working hypothesis is that a latent endometrial epithelial infection is established by hsv type 2 intrauterine infection through one or more of the following routes: ascending cervical, transneural, maternal primary viremia during pregnancy, postnatal primary viremia in childhood or adulthood, and activated endometrial intrauterine embryonic or fetal infection with or without germ cell involvement. once this latent endometrial infection is established, sporadic transient focal acute infections may be activated, as in the oral and genital regions. this endometrial activation is maximal at the end of the secretory phase, when endometrial prostaglandin synthesis is maximal 25 and when local immunosuppression is occurring to protect the implanting embryo (l. olding, unpublished data) if fertilization has occurred. prostaglandins produce local and systemic immunosuppression, 26 and both immtmosuppression 27 and prostaglandins 8'29 activate latent hsv infection. the level of endometrial prostaglandin synthesis is also quite high during pregnancy, s° and genital hsv activation and asymptomatic shedding of hsv are increased during the gestational period. 31 furthermore, the presence of icp4, the immediate/early protein detected in our study, is necessary, but not sufficient, for hsv activation and replication. further events are required for virus production, s2,s3 possibly prostaglandin synthesis and/or local immunosuppression. the immune response to hsv infection in both tile pregnant and nonpregnant states is quite complex and involves antibody and complement, antibody and natural killer ceils, macrophages, and polymorphonuclear leukocytesj '~7 maternal antibody in the anmionic fluid may protect the embryo or fetus. 34 the cellular immtme response to hsv 2 infection is decreased during pregnancy. 35 if conception has not occurred, the acute activated infection is not detected clinically, because tile endometrium has no sensory pain nerves, and there is no transcervical discharge or spotting. if conception has occurred, however, the infection has the potential to spread to the placenta and amniochorionic sac. if the infection is not inhibited by maternal antibody in the amnionic fluid, 34 it may continue into the embryonic or fetal circulation and/or tissues, as evi-denced by antigen psotivity in the chorionic villi. if the embryo or fetus is infected, the probability of spontaneous abortion, stillbirth, or neonatal complications is high. if the infection is limited to ttle amniochorionic sac, however, tile chance of embryonic or fetal damage is greatly reduced (see companion paper4). spontaneous abortion and infants small for the gestational age are associated with nonspecific villitis, ~° a condition that we found to be associated with hsv antigen positivity. specimens from maternal placental floor infarction ~7'3s were hsv antigen-positive in five of the eight cases assayed. our companion paper 4 describes the neonatal complications associated with hsv antigen positivity. the observational and clinical data in support of this hypothesis are as follows. first, endometrial epithelial positivity is maximal in tile late secretory phase (day 22.4, 40 per cent positive) as compared with the proliferative phase (10 per cent, p = 0.017), endometrial hyperplasia (14 per cent, p = 0.001), and tile atrophic state (7 per cent, p = 0.014). maximal positivity occurred in abnormal secretory phase endometria (68 per cent, p = 0.040 versus normal secretory phase), probably a time of altered prostaglandin synthesis. 39 second, the mean gestational age increases progressively for endometrial epithelial positivity,.placental subamnionic chorion positivity, and chononic villus positivity (8.1 weeks, p = 0.006, versus 9.8 weeks, p = 0.006, versus 11 weeks, p = 0.028, respectively). the increasing gestational age for antigen positivity at these sites supports tile concept that the infection begins in the endometrium and progresses into the anmiochorion and, finally, into tile chorionic villi (i.e., the embryo or fetus). third, placental subamnionic chorion positivity is dependent on endometrial epithelial positivity (p = 0.004), and chorionic villus positivity is dependent on placental subamnionic chorion positivity (p = 0.001). this finding supports the concept that amniochorionic infection cannot occur without endometrial infection, and chorionic villus infection cannot occur without amniochorionic infection. the reason for which chorionic villus positivity is not statistically dependent on endometrial epithelial positivity could be that most epithelium-positive endometria may not produce placental subamnionic chorion-positive or chorionic villus-positive infections. another reason for the discordance in individual specimens between endometrial epithelial positivity, p.lacental subamnionic chorion positivity, and chorlonic villus positivity is that very little of the endometrium in the uterus accompanies the placental tissue at the time of abortion. furthermore, little of the removed endometrium may be submitted for examination, because of the difficulty in separating it from the placental and decidual tissue during gross examination. fourth, material from spontaneous abortions is more likely to be inflamed (92 per cent, p < 0.001) than is that from therapeutic abortions (66 per cent). both placental subamnionic chorion positivity and chorionic villus positivity show trends (p = 0.139 and 0.078, respectively) toward an association with inflammation, while endometrial epithelial positivity is significantly associated with noninflamed normal tissue (p = 0.022). some therapeutic abortions are associated with inflammation and blighted ova, because (hey are incipient spontaneous abortions that have not been recognized clinically. therapeutic abortions occur earlier than spontaneous abortions (at'8.5 versus 9.2 weeks of gestation, p = 0.038). fifth, placental subamnionic chorion positivity (39 versus 13 per cent, p < 0.001) and chorionic villus positivity (14 versus 0.0 per cent, p < 0.001) are more frequent in spontaneous than in therapeutic abortions, while the incidences of endometrial epithelial positivity are similar (26 versus 33 per cent, p = 0.230). sixth, blighted ova are associated more frequently with spontaneous than with therapeutic abortions (78 vs 27 per cent, p = 0.001), with acute (48 per cent, p = 0.043) and chronic (73 per cent, p = 0.007) inflammation more frequently than with no inflammation, with placental subamnionic chorion positivity more frequently than with subamnionic chorion negativity (67 versus 44 per cent, p = 0.008), and with chorionic villus positivity more frequently than with chorionic villus negativity (70 versus 49 per cent, p = 0.207, trend), but not with endometrial epithelial positivity or negativity (51 versus 51 per cent, p = 0.820). the data presented in the fourth, fifth, and sixth points support the concept that hsv infection of the amniochorion (placental subamnionic chorion positivity) and chorionic villi (chorionic villus positivity) can destroy the embryo (blighted ovum) and produce spontaneous abortion, as suggested previously. 4°-43 it is difficult to determine the percentage of spontaneous abortions caused by hsv infection. the identified causes of spontaneous abortion are chromosomal abnormalities (about 50 per cent), bacterial infection (about 10 per cent), cmv (rare), and chlamydial infection (unkown frequency); the cause is unknown in up to 40 per cent of cases)j since our data show an increase of at least 50 per cent in blighted ova when spontaneous abortion material has placental subamnionic chorion positivity (67 per cent/44 per cent = 152 per cent) and since hsv infection itself may produce chromosomal damage (double-stranded dna virus), a significant proportion of the 40 per cent of spontaneous abortions of unknown etiology may be caused by hsv infection. recurrent spontaneous abortions may be associated with endometrial epithelial positivity (30 versus 28 per cent, p = 0.143) and chorionic villus positivity (50 versus 23 per cent, p = 0.207), but the present data show only a trend, and the number of mothers with recurrent spontaneous abortions in this study was small (38 cases). recurrent spontaneous abortions are not associated with placental subamnionic chorion positivity (26 vs 22 per cent, p = 0.521). this finding might correlate with the amount of maternal anti-hsv antibody in the amnionic fluid, a4 that is, the infection is blocked at the amniochorionic level, without transmission into the embryo or fetus (i.e., chorionic villi). recurrent neonatal hsv infection has not been documented in successive pregnancies of the same mother. 43 two of the hsv-positive patients in this study who had spontaneous abortions subsequently had normal full-term infants with hsv antigen-positive placentae and cords, but with negative chorionic villi. in a guinea pig model of placental cmv infection, only 27 per cent of the fetuses that had cmv-positive placentae were cmv-positive by direct culture. 44 muhiparity is associated with chronic inflammation more than with the absence of inflammation (79 versus 58 per cent, p = 0.034), and shows a trend toward associations with endometrial epithelial positivity (75 versus 69 per cent, p = 0.179) and placental subamnionic chorion positivity (78 versus 65 per cent, p = 0.084). the greater the number of pregnancies, the greater is the potential for latent endometrial infection (endometrial epithelial positivity) to activate and produce acute infection with intrauterine infection (placental subamnionic chorion positivity). muhiparity was not associated with increased risk of embryonic infection (chorionic villus positivity, 75 versus 67 per cent, p = 0.635). the only significant association between oral and genital hsv infections in the patient and/or the father of the aborted embryo or fetus was between genital hsv infection in both individuals (50 per cent of patients with clinical histories of genital hsv infection had partners with clinical histories of genital hsv infection, and 6.7 per cent of patients with positive clinical histories had partners with negative clinical histories, p = 0.001). only 3.6 per cent of 83 hsv antigen-positive patients and 5.6 per cent of 107 hsv antigen-negative patients had clinical histories of genital herpes infection (p = 0.522). asymptomatic genital hsv 2 infection, however, is common, occurring in more than 50 per cent of nonpregnant women with primary infections, 45 25 per cent of nonpregnant women with recurrent infections, 46 and 13 to 33 per cent of women during pregnancy, a~,41,48 the optimal time to detect asymptomatic shedding from an activated endometrial infection in nonpregnant women would be the first day of menstrual flow, as maximal antigen detection occurs in the late secretory phase (day 22 to 23). the data reported in this study suggest that latent intrauterine hsv infection is etiologically important in spontaneous abortion. the accompanying paper describes the neonatal problems produced by infections of this type. 4 future studies should include the following investigations: determination of maternal anti-hsv antibody status; extraction of dna from, and in situ dna/rna hybridization of, aborted and curettage tissues to determine whether hsv-specific nucleic acid sequences are present; and direct culture of first-day menstrual flow material for infectious hsv. note added in proof. in collaboration with dr. david myerson, we have detected hsv-specific dna in amnionic and chorionic nuclei in serial sections from the specimen in figures 1 and 9. in situ hybridization using a biotinylated hsv dna probe as described in reference 1 1 was used. evaluation of endo'rnetrial tissues is underway. + sac* (48) 16.7 + tab (99) 13.1 + emet + (58) 39.7 + tot inflam ÷ (157) 25.6 + acute cd (98) 23.5 + tab (97) 8.5 ± sac ÷ (44)9.8 ± + chronic cd (45) 73.3 + sac* (48)66.7 + partner genital + (6) 50.0 + chronic cd (56) 78.6 + prolif (19) 10.5 + abn secretory (21) 66.7 + atrophic (15) 6.7 + hyperplastic (35) 14.3 + prolif (19) 10.5 + hyperplasia (35) 14 + chronic cd (58) 29.3 + tot inflam* (158) 28.5 + chronic cd (59) 30.5 + partner oral + (17) 17.6 + tot inflam'-(123) 8.1 + acute cd* (78) 7.7 + chronic cd ÷ (45) 8.9 + tab (65) 60.0 + tab (99) 41.4 + tab (99) 33.3 + chronic cd (58) 29.3 + partner oral* (18) 22.2 + partner genital ÷ (6) 33.3 partner oral/genital + (24) 25.0 4 chronic cd (59) 42.4 + acute cd (99) 27.0 + chronic cd (59) 30.5 + chronic cd (5-t) 9.2 ± 2 acute cd (92) 8.6 chronic cd (54) 9.2 ± 2.7 blighted ovum + (73) 9.4 herpes simplex neonatal herpes simplex virus infection in king county robbja: a new enzyme immunolectin tissue stain intrauterine latent herpes simplex virus infection. ii. latent neonatal infection herpetic inclusions in the endometrium herpesvirus hominis endometritis in a young woman wearing an intrauterine contraceptive device optically clear nuclei, an alteration of endometrial epithelium in the presence of trophoblast leukocytoclastic vasculitis assnciated with cutaneous infection by herpesvirus kefalides na: herpes simplex virus type 1 infection of endothelium reduces collagen and fibronectin synthesis cytomegalovirus antigen within human arterial smooth muscle cells widespread presence of histologically occult cytomegalovirus antibody response in humans to individual proteins of herpes simplex viruses pathogenic murine coronaviruses iii. biological and biochemical characterization of temperature-sensitive mutants of jhmv detection of herpes simplex virus in clinical specimens by dna hybridization localization of herpes sireplex virus in tile trigeminal and olfactory systems of tile mouse central nervous system during acute and latent infections by in situ hybridization herpesviruses and their replication dako labs rabbit anti-hsv 1 and 2 igg package insert. november 1975 (5 references listed in insert from the scandinavian literature monoclonal antibodies to herpes simplex virus type 1 proteins, including tile immediate-earl)' protein icp4 serological analysis of herpes simplex virus types 1 and 2 with monoclonal antibodies localization of a type-specific antigenic site on herpes simplex virus type 2 glycoprotein d extensive homology between the herpes simplex virus type 2 glycoprotein f gene and the herpes simplex virus type 1 glycoprotein c gene antibodies to a synthetic oligopeptide that react with herpes simplex virus type 1 and 2 glycoprotein c antigenic variants of herpes simplex virus selected with glycoprotein specific monoclonal antibodies best fa, et ah prostaglandins in endometrium and menstrual fluid from normal and dysmenorrhoeic subjects regulation of the immune response by prostaglandins, a review herpes simplex virus. in fields bn reactivation of herpes simplex virus infection by uhraviolet light and possible involvement of prostaglandins the effect of prostaglandins on the multiplication and cell-to-celt spread of herpes simplex virus type 2 in vitro prostaglandins and the regulation of uterine blood flow in pregnancy genital herpes in pregnancy: risk factors associated with recurrences and asymptomatic viral shedding expression of herpes simplex virus type 2 antigens in premalignant and malignant human vulvar cells cells that constitutively express the herpes simplex virus intmediate-early protein icp4 allow efficient activation of viral delayed-early genes in traits neutralization of herpes simplex virus by antibody in amniotic fluid humoral and cell-mediated responses to herpesvirus antigens during pregnancy--a longitudinal stud clinical and pathologic aspects of recurrent placental villitis recurrent maternal floor infarction: a preventable cause of fetal death maternal floor infarction. hum pathot the menstrual stimulant in puberty mfiternal herpes-simplex infection causing abortion: intrauterine hsv infection: spontaneous abortion (robb et ol.) histopathologic study of the placenta perinatal risk associated with maternal genital herpes simplex virus iqfection pathology of the female genital tract herpes simplex the placenta as a site of cytomegalovirus infection in guinea pigs neonatal herpes simplex virus infection of the central nervous system genital herpes simplex virus infections: clinical manifestations, course, and complications herpesvirus hominis type ii infections in asymptomatic pregnant women herpesvirns infections of pregnancy enzyme immunofihration technique for rapid diagnosis of herpes simplex virus eye infections in a rabbit model complement-requiring neutralizing antibody in guinea pigs with primary and recurrent genital herpes neonatal herpes simplex virus infection in guinea pigs key: cord-005007-pofm8b5x authors: higgins, p. g. title: interferons and viral infections date: 1984 journal: eur j clin microbiol doi: 10.1007/bf01977473 sha: doc_id: 5007 cord_uid: pofm8b5x nan interferons are among the most biologically active of known substances. they are proteins, the natural product of eukaryotic ceils exposed to specific inducers, the best known of which is a virus infection. interferons protect cells from the effect of virus replication by inhibiting the formation of new, infectious virus particles. however, this protection is, generally speaking, only conferred on cells of the same species as that in which the interferon itself was produced. although the antiviral effect was the first property of interferons to be recognised, subsequent studies have revealed that they have many other activities including the inhibition of cell growth and division, and modulation of the immune response, both humoral and cell mediated. of the three types of interferon, interferon a (leucocyte) and interferon fl (fibroblast) possess many similarities although they are antigenically distinct. interferon 3' (immune) differs from interferon a and fl in many of its physical, chemical and biological activities as well as its antigenicity. supplies of human interferons have been limited in the past because of the need for human ceils for their production, and early preparations suffered the disadvantage of containing no more than 1% actual interferon. in the last few years recombinant dna technology has permitted the production of human interferons by microorganisms, and improved techniques together with the use of monoclonal antibodies have resulted in preparations with a very high degree of purity. this applies not only to the three types of interferon but also to a number of the twenty or so subtypes of interferon a that are known to exist. since such genetically engineered, highly purified interferons have been available for only a short time, much of the reported work with interferons in viral infections has been performed with interferons produced by leucocytes of the buffy coat of human blood or by. human fibroblasts. however, trials with the new interferon preparations have confirmed that the activity observed with the earlier, crude products was the result of the interferon they contained and not caused by impurities. during influenza infections in humans the production of interferon parallels that of the clinical symptoms and virus excretion. by the time that antibodies are detected, interferon has virtually disappeared from the circulation (1). this suggests that interferons play an all important role in the limitation of, and recovery from, virus infections. interferons are effective against practically all viruses and this makes them antivirals of great clinical potential. they are obviously indicated as replacement therapy in those who, for one reason or another, produce little or no interferon. this has been shown to occur in some children who are abnormally susceptible to upper respiratory tract infections (2) and also in some instances of fulminating hepatitis, when giving exogenous interferon appears to activate the patient's own interferon production (3). this latter observation may be an example of priming, in which the induction of cells pre-treated with a small quantity of interferon produces greater quantities of interferon than does the stimulation of cells without prior exposure. as interferon's antiviral effect is mediated through the protection of non-infected cells and not by any direct action on the virus particle, interferon's prophylactic effect will probably be greater than its therapeutic action. intradermal interferon prevents the 'take' of subsequently inoculated vaccinia virus (4), and interferon given intranasally before and after virus challenge protects volunteers against infection with rhinovirus (5-8) or coronavirus (9) . systemic interferon has been shown to reduce the incidence of herpetic lesions and the excretion of herpes virus following section of the trigeminal nerve in the treatment of tic douloureux (10) . it also delays the excretion of cytomegalovirus (cmv) in the urine and reduces the incidence of cmv viraemia in renal transplant patients (11) . more prolonged prophylaxis can also reduce the clinical manifestations of cmv infections in such patients (12) . in established acute viral infections the results have been less encouraging. in cases of herpes simplex virus infection leading to dendritic ulceration of the cornea, interferon alone has little effect, but in combination with wiping debridement (13), thermocautery (14) or trifluorothymidine (15) it is more effective than any of the treatments alone. interferon does not influence the course of zoster in normal individuals, but in the immunosuppressed, where the appearance of endogenous interferon is delayed and the infection is more likely to become disseminated, interferon can reduce the incidence of serious complications and post-herpetic neuralgia. in addition it shortens the periods in the acute phase during which pain is experienced and new lesions appear (16) . similar but less conclusive results have been obtained following interferon treatment of varicella in children suffering from malignancies (17) . interferon therapy has also been tried in chronic infections, notably those with hepatitis b virus. quite dramatic reductions or elimination of the circulating markers of hepatitis b virus can be achieved with interferon (18) . however, this may require repeated courses, with or without synthetic antivirals (19) , and success is obtained in only a proportion of patients. furthermore, this phenomenon is known to occur spontaneously in some untreated cases. treatment of congenital rubella (20) and cytomegalovirus (21, 22) infections has not resulted in any consistent clinical improvement, although reduction in viral replication can be achieved. warts and the much rarer juvenile laryngeal papillomas result from infection with papiuomaviruses. warts respond to interferon applied locally (23, 24) , while systemic interferon halts the progress of laryngeal papilloma and prevents its recurrence after surgical removal (25) . it is by no means certain which of interferon's activities, antiviral, growth inhibition or immunomodulatory, is responsible for the effect observed in these cases. the results summarized here represent very modest success for a substance which has been heralded as the ideal antiviral and recognised for over 25 years. progress has been slow mainly because the limited supplies of interferon available until recently have restricted the number of trials. furthermore, the optimal dosage, either prophylactically or therapeutically, has had to be determined by trial and error. for example it is now known that there is a critical concentration of interferon and frequency of administration below which intranasal interferon fails to protect against experimental rhinovirus infection (26) and that the beneficial effect of interferon in zoster in patients with malignancies is directly related to the amount given (16, 27) . unfortunately it has now become apparent that even the highly purified interferon is, itself, toxic. more than one million units given systemically results in fever, headache and malaise and if medication is continued it induces anorexia and fatigue. such dosage also depresses the bone marrow and affects liver function. even when given topically to the nasal mucosa, for any period of time greater than a few days, mild inflammatory changes occur which prevent its use for long-term prophylaxis (7). however, much work needs to be done before interferon can be discarded as mainly of academic interest and of little clinical importance. virtually no trials have been performed with interferon 3' or many of the interferon a subtypes, and it is possible that one or more of these may have a greater antiviral to toxic ratio than those already studied. it is not known if synergism exists between different types and subtypes of interferon. this would seem worthwhile exploring in view of the fact that the greatest success in therapy so far has been achieved with interferon in combination with synthetic antivirals. p. g. higgins mrc common cold unit, coombe road, salisbury, wiltshire, england sp2 8bw. general consideration of the interferon system deficient production of leucocyte interferon (interferon-a) in vitro and in vivo in children with recurrent respiratory tract infection interferon system in acute virus hepatitis effect of human fibroblast interferon on vaccination in volunteers inhibition of respiratory virus infection by locally applied interferon purified interferon as protection against rhinovixus infection efficacy and tolerance of intranasally applied recombinant leucocyte a interferon in normal volunteers intranasal interferon '~2 for prevention of rhinovirus infection and illness intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers prevention of reactivated herpes simplex infection by human leukocyte interferon after operation on the trigeminal root controlled clinical trial of prophylactic human-leukocyte interferon in renal transplantation. effects on cytomegalovixus and herpes simplex virus infections effects of interferon-alpha on eytomegalovixus reactivation syndromes in renal transplant recipients topical therapy of ulcerative herpeetic keratitis with human interferon successful treatment of dendritic keratitis with human leucocyte interferon -a controlled clinical study graefes archly f'tir klinische und experimentelle opthalmologie 1976 combination therapy of dendritic keratitis with trifluorothymidine and interferon human leukocyte interferon for the treatment of herpes zoster in patients with cancer human leukocyte interferon for the treatment of varicella in children with cancer effect of human leukocyte interferon on hepatitis b virus infection in patients with chxonic active hepatitis antiviral treatment of chronic hepatitis b virus infection. 1. changes in viral markers with interferon combined with adenine arabinoside alpha interferon administration to infants with congential rubella effect of human exogenous leukocyte interferon in cytomegalovirus infections effect of leukocyte interferon on urinary excretion of cytomegalovirus by infants double blind clinical study with human leukocyte interferon in the therapy of condylornata acuminata effect of injections of small doses of human fibroblast interferon into genital warts: a pilot study interferon therapy in juvenile laryngeal papillomatosis an effective dosage regimen for prophylaxis against rhinovirus infection by intranasal administration of hulfn-~2. antiviral research short-course human leukocyteinterferon in treatment of herpes zoster in patients with cancer key: cord-011095-79ce5900 authors: meskill, sarah d.; o’bryant, shelease c. title: respiratory virus co-infection in acute respiratory infections in children date: 2020-01-24 journal: curr infect dis rep doi: 10.1007/s11908-020-0711-8 sha: doc_id: 11095 cord_uid: 79ce5900 purpose of review: this investigation aims to understand the role and burden of viral co-infections for acute respiratory illnesses in children. co-infection can be either viral-viral or viral-bacterial and with new technology there is more information on the role they play on the health of children. recent findings: with the proliferation of multiplex pcr for rapid diagnosis of multiple viruses as well as innovations on identification of bacterial infections, research has been attempting to discover how these co-infections affect each other and the host. studies are aiming to discern if the epidemiology of viruses seen at a population level is related to the interaction between different viruses on a host level. studies are also attempting to discover the burden of morbidity and mortality of these viral-viral co-infections on the pediatric population. it is also becoming important to understand the interplay of certain viruses with specific bacteria and understanding the impact of viral-bacterial co-infections. summary: rsv continues to contribute to a large burden of disease for pediatric patients with acute respiratory illnesses. however, recent literature suggests that viral-viral co-infections do not add to this burden and might, in some cases, be protective of severe disease. viral-bacterial co-infections, on the other hand, are most likely adding to the burden of morbidity in pediatric patients because of the synergistic way they can infect the nasopharyngeal space. future research needs to focus on confirming these conclusions as it could affect hospital cohorting, role of molecular testing, and therapeutic interventions. acute respiratory illnesses are the most common cause of under-5-year-old mortality worldwide [1] . in particular, pneumonia is responsible for approximately 1.4-1.8 million fatal cases in children under age five globally [2] . beyond the mortality burden, there is significant morbidity with symptomatic viral infections estimated at more than five episodes per year in children under age three [3] . while single viral infections are relatively straightforward, there is interest in understanding the role of dual respiratory infections in pediatric patients. dual infections could either be viral-viral or viral-bacterial as the human respiratory tract is a reservoir for many organisms. molecular diagnostics have increased the ability to diagnose causative agents in patients with respiratory illnesses. real-time polymerase chain reaction (rt-pcr) allows for the detection of multiple viruses at once and is found to be more reliable and expedient than viral culture [4] . furthermore, newer methods, such as multiplex pcr and next-generation sequencing are producing more accurate and quicker results towards identifying these organisms [5] . given this ability, studies have attempted to discern if the positive test for pathogen is actually causative or if it is asymptomatic shedding leading to a positive test result. one study evaluated adult visitors to a tourist attraction taking clinical history of symptoms as well as testing pcr for common respiratory illnesses [6] . of those participants who tested negative for respiratory viral infections, 81.9-99.6% reported to be asymptomatic depending on the definition applied. only 6.2% of patients tested positive for a respiratory virus. of these positive results, up to 48.1% were considered symptomatic. another study focused on respiratory viral infection positivity specifically in the pediatric population [7] . testing children biweekly whether symptomatic or not, 56% of symptomatic episodes had detection of a viral pathogen compared to 40% of asymptomatic episodes. the younger the patient, the less likely a pathogen positive episode was asymptomatic (p = 0.01); in addition, multiple pathogens were found in significantly less asymptomatic episodes (p = 0.02). further studies attempted to discern which viruses were more likely to cause symptoms. one study in children under age 5 years old found that respiratory syncytial virus (rsv), human metapneumovirus (hmpv), and parainfluenza viruses (piv) were more likely to be causative of disease [8] . another study still found high rates of asymptomatic shedding of rsv and piv with more than half of positive tests associated with an asymptomatic patient [9] . however, within these data, younger patients were associated with higher rates of positive test results correlating with a symptomatic event demonstrating the importance of age in clinical manifestation of viral infections. rsv is responsible for an extremely high burden of disease in children. infection with rsv is one of the leading causes of death in children under 1 year of age worldwide, second only to malaria [10] . in a surveillance of acute respiratory infections in children under age 5, rsv was responsible for 20% of annual hospitalizations, 18% of emergency department visits, and 15% of office visits [11] . in an evaluation of children under 18 years old admitted to the hospital with a diagnosis of pneumonia, the most common cause of infection, whether bacterial or viral, was rsv quickly followed by rhinovirus [12] . rhinovirus is the most common cause of respiratory viral illness during all seasons except winter when rsv is predominant [13] . rhinovirus is second only to rsv in causing bronchiolitis in hospitalized patients [14] . unfortunately, the true burden of rhinovirus is under-reported as many studies do not include this virus in there molecular diagnostic testing as the rates of asymptomatic shedding are quite high and it is difficult to differentiate shedding from actual cause of disease [13] . while some studies do have rsv and rhinovirus as the leading cause of pneumonia in children, another important common viral contributor is influenza [15] . influenza and its complications are the leading cause of morbidity and mortality [15] . despite antiviral medications and vaccines, it is estimated by the world health organization (who) that the annual influenza epidemics cause 3 to 5 million severe infections and 250,000-500,000 deaths each year in developed countries and 300,000 hospitalizations and 35,000 deaths in the usa [16, 17] . influenza has recently been recognized as having a higher burden of disease than previously thought because of poor recognition and diagnosis even with molecular testing [18] . although vaccination against influenza decreases the risk of infection, now with less vaccination we are witnessing less herd immunity and those more vulnerable are becoming severely ill [19] . in my practice, i've witnessed a 2-month-old infant-too young for the influenza vaccine and caregivers opted out of the influenza vaccination for themselves-develop respiratory failure requiring endotracheal intubation due to influenza a epiglottitis [20] . human metapneumovirus (hmpv) also causes acute respiratory infections globally. hmpv has been shown to cause high hospitalization rates of 1 per 1000 in children 5 years old or younger, with an estimated 20,000 hospitalizations annually which is similar to the rates seen with influenza [21] . outpatient visits were estimated at one million clinic visits and 263,000 emergency department visits annually in the same population [21] . admitted pediatric patients with hmpv were more likely than those without the infection to require supplemental oxygen and had longer intensive care stays [21] . at the host level, the outcome of dual infection is commonly viral interference, such as when one virus competitively inhibits the replication of another virus, but it can also enhance replication in some cases [22] . it is postulated that the sequence of infections, the time interval between viral exposure, and the route of infection affect the pathogenicity of the co-infection [22] . one example of this used mice models. when the mice cell models that were infected with rhinovirus were then infected with influenza a virus 2 days later, there was an attenuated response to the influenza infection with less severe manifestation of disease [23] . this demonstrates that the preceding infection altered the host response and the timing and order of infection of the host are crucial in outward disease manifestation. co-infections can also alter the epidemiology of viral infections. for example, rhinovirus is a rapidly replicating virus and can interfere with the replication of other viruses while piv is an extremely slow replicator and its replication can be interrupted by the presence of other viruses [22] . viral loads can be compared among patients with one or multiple infections. one study found that viral loads were consistently high regardless of co-infection status (such as rsv, influenza a, and hmpv) but others had viral load vary based on coinfection status (such as piv1 and adenovirus) [24] . of these viruses, influenza a is least likely to be identified in coinfected patients. in mathematical modeling, the idea of resource competition can explain viral loads as the fasterreplicating viruses overcome slower replicating viruses by leaving no resources [25] . in this model, influenza a replicates faster than rsv and therefore keeps the rsv viral load below detection level. the pathophysiology behind dual viral infections can explain some of the epidemiology of viral-viral co-infections seen at the population level. given the high rates of rsv and rhinovirus infections, it makes sense that they are the most commonly identified viruses in co-infected patients across multiple studies [14, [49] [50] [51] [52] [53] [54] [55] . however, one study did conclude that the odds of rhinovirus detection were lower when rsv was present and the odds of rhinovirus were significantly higher in those patients who received rsv immunoprophylaxis [56] . this indicates that despite the high prevalence of co-infection with these viruses, there is still viral interference occurring. there is also evidence of viral competition when it comes to rsv and influenza. in terms of viral competition, when rsv infection rates are high, influenza rates of infection are low and the converse is true [57] . in addition, when both viruses are circulating in a small population the rates of co-infection of rsv and influenza are over 6 times less than expected [58] . on review of influenza interactions with other viral pathogens, there is evidence of competitive interference with rsv, rhinovirus, other influenza strains, and hmpv virus by evaluating population incidence and co-infection detection [59] . multiple studies have attempted to evaluate the clinical importance of viral co-infections. it would seem intuitive to think that having more than one virus causing disease in a person would lead to more severe symptoms and sequelae. one study did support this conclusion. in it the authors looked at all pediatric patients who had a respiratory viral panel sent, they found an unadjusted increase in the risk of moderate-severe illness, non-invasive ventilation, ecmo, and death in coinfected patients however in the adjusted analysis only the risk moderate-severe disease continued to be increased [60] . some studies found certain clinical outcomes to be at increased risk but not a persistence of high risk in all outcomes. one study evaluated patients under 12 months in france and found that viral co-infections were 2.7 times more likely to be in the picu however once in the picu there was no difference in length of stay, duration of mechanical ventilation, duration of supplemental oxygen [52] . another study supported this finding by evaluating children in the picu retrospectively comparing one virus vs co-infected viral status and found that the co-infected patients had longer average length of stay in the picu and longer time of intubation [61] . however, there was no difference in rates of needing highflow nasal cannula, mechanical ventilation, or having cardiovascular dysfunction. it is possible that the prolonged intubation times could be because patients who were co-infected with viruses had higher odds of bacterial co-infection. there are some studies that also found increased risk of clinical outcomes but these actually were based on specific viral combinations. one study found that specifically influenza viral co-infection with another influenza strain significantly increased the risk of icu admission or death but this did not carry through to any other co-infected status [62] . another study did a multicenter evaluation of bronchiolitis over 3 years and found longer length of stay in those with rsv/rv coinfection but no proof that these patients were sicker (as in no difference in icu admission or support with cpap or mechanical ventilation) [14] . another study also found an increase in length of stay and oxygen use in those patients with specifically rsv/rv co-infected status [63] . there are a few studies that found no difference in clinical outcomes on co-infected patients. the study looking at tourists did not find any association between viral co-infections and the likelihood of being symptomatic or presenting with more severe symptoms [9] . another one looking at hospitalized pediatric patients found no increased risk of icu admissions in those with multiple viruses identified [64] . beyond no difference in clinical outcomes, quite a few found less severity in those patients with multiple viral infections. one study found age to be important in understanding the risk of severe disease. in this study, they evaluated children with bronchiolitis in the netherlands and found those patients with dual infection had no difference in severity under 3 months of age but significantly less severe disease in patients over 3 months [65] . another study in the netherlands looking at pediatric patients had shorter mean length of stay and no difference in oxygen supply needs or intensive care (icu) admission [53] . another found that viral co-infected patients were significantly less likely on the inpatient ward (or 0.55), icu stay (or 0.32), require oxygen supplementation (or 0.55), or have length of stay greater than 3 days (or 0.32) [24] . one study was actually able to see a linear response to length of stay with a shorter length of stay the more viruses detected when evaluating all pediatric patients with hospitalized respiratory infections [66] . the best evaluation of the literature on viral co-infections and clinical severity is going to be in systematic reviews. there are three looking at viral co-infections that warrant discussion. scotta et al. [67• ] did a large systematic review of respiratory viral coinfections with illness severity in children with over 17,000 patients in the combined evaluation. in this review, viral co-infections did not influence risks of all outcomes assessed: mean length of stay, length of supplemental oxygen, need for hospitalization, need for intensive care admission, mechanical ventilation, or death. they also looked at sub-analyses of specific viral combinations and did not see any influence on outcomes. lim et al. [68] did a systemic review for children under age 5 years with respiratory illness and found insufficient evidence to suggest a difference in any clinical outcome based on co-infected status. in a very small subset of patients, they found a suggestion that children without co-morbidities actually did worse when only a single virus was identified. goka et al. [55] did a systemic review of patients of all ages with respiratory illnesses and found that studies that recruited young children were more likely to report high rates of co-infection and that there were inconclusive results on risk of hospitalization or icu admission. to cause respiratory illnesses, bacterial pathogens first need to colonize the nasopharyngeal space [26] . organisms achieve this colonization via positive and/or negative associations: positive association exists through mutualism, symbiosis or helping to evade the host's immune system; negative association exists through ammensalism, predation, or the host immune system disproportionally affecting one organism over the other [26] . overall, there are multiple mechanisms, for viruses and bacteria, to aid in the success of invasion and colonization of the human body. 1) viral pre-disposition to bacterial adherence: alteration of the host's respiratory epithelium causes viruses to increase the susceptibility of bacterial colonization during a simultaneous infection and after full recovery of a viral illness [26, 27] . examples include: influenza and streptococcus pneumoniae and adenovirus and streptococcus pneumoniae [26, 28] . 2) disruption of the epithelium barrier: viruses can intracellularly disarrange cellular processes or destroy infected cells through metabolic exhaustion or lysis [26] . the destruction of cells leads to the denuding of the epithelial layer, exposing the basement membrane; therefore, causing introduction of bacterial organisms [29, 30] . examples of this mechanism are s. pneumoniae binding to fibronectin after the denudation of the epithelium layer, and staphylococcus aeurus and morexella catarrhalis binding to extracellular matrix protein after destruction to the epithelium [31] [32] [33] . loss of the epithelium integrity and promotion of bacterial translocation are also seen in rhinovirus-induced paracellular migration of haemophilus influenzae [34] . 3) upregulation of adhesion proteins: viral infected cells may decrease the innate immune response by altering the expression of antimicrobial peptides (defensins), which are secreted in the respiratory mucosa [35] . during viral infections, there are cascades of proinflammatory responses leading to the upregulation of adhesion proteins found on epithelial cells, which leads to the cellular invasion of pathogenic organisms [26] . for example, rsvand parainfluenza viruses upregulate intracellular and outer membranes proteins such as intracellular adhesion molecule 1 (icam-1), p5-homologous fimbriae (p5 fimbriae), carcinoembryonic adhesion molecule-1 (ceacam-1), and platelet-activating factor receptor (pafr) [36, 37] . with the expression of these proteins, several bacterial organisms, s. pneumoniae and h. influenzae, are able to adhere to these molecules leading to invasion of the host's cells [36, 37] . 4) production of viral factors: production of viral components such as neuramindase (na), a glycoprotein produced by influenza and parainfluenza, and protein-gexpressed on rsv cells-destroy the integrity of infected cells. this destruction exposes bacterial receptors and aids in bacterial co-infections [38] [39] [40] [41] . 5) dysfunction of immune system components: respiratory viruses may affect the immune system by impairing neutrophil function, decreasing oxidative burst, and enhancing neutrophil apoptosis, thus increasing the susceptibility to bacterial superinfection [42, 43] . also, viruses can predispose to bacteria superinfection by rendering natural killer (nk) cells recruitment and activation ineffective, which is seen with influenza and s. pneumoniae [44] . viruses also alter monocyte function, decrease production and activity of cytokines, and prevent appropriate immune response routing, leading to enhanced bacterial colonization and increasing risk for mortality [45] [46] [47] [48] . because of the interaction between viruses and bacteria, there are many known specific virus-bacteria relationships ( table 1) . for example, influenza interacts with both streptococcus pneumoniae and staphylococcus aureus. the most common bacteria found in viral, secondary bacterial infections is s. pneumoniae [15] . s. pneumoniae has over 97 distinct serotypes and is a common cause of acute otitis media (aom), pneumonia, sepsis, and bacterial meningitis [85] [86] [87] . one of the most common complications of influenza infection is aom from either s. pneumonia or s. aureus [15, 88] . since influenza is one of the common viral contributors to pneumonia, it also increases the rates of bacterial pneumonia from s. pneumoniae and s. aureus. this is because influenza with both of these bacteria has synergistic relationships using the mechanisms listed above [15, 26] . in addition, the bacteria increase influenza's infectivity by activating hemagglutinin on the membrane, thus neutralizing antibodies and allowing more virions to be replicated inside the host cell [15, 89] . table 1 reviews the most common viral-bacterial co-infections and their associated complications. there are also reports of preceding bacterial infections leading to increase viral susceptibilities [26] . for example, s. pneumoniae and hmpv have a unidirectional synergistic relationship where s. pneumoniae predisposes children less than 2 years old to hmpv infections [90] . similarly, h. influenza stimulates adhesion proteins on human epithelial cells, creating an entry point for rhinovirus [91] . rhinovirus also has a bi-directional relationship with s. aureus: rhinovirus aids in the bacterial adhesion and engulfment into the epithelium cell and s. aureus promotes the replication of rhinovirus [70] . rhinovirus can actually increase the nasal load of s. aureus by 39% over baseline [70] . the evaluation of viral-bacterial co-infection on disease severity is an advancing field however no study reports a decrease in severity with viral-bacterial co-infection. one study [92] evaluated children presenting with bronchiolitis using nasal swabs to identify a plethora of viruses in addition to s. pneumoniae, m. catarrhalis, and h. influenzae. in this study, rsv and s. pneumoniae co-detection was significantly associated with severe disease in the regression analysis. in a cohort study of children admitted with acute respiratory disease, bacterial superinfections were significantly associated with higher illness severity scores (or = 2.12), more severe respiratory distress (or = 4.4), required more respiratory support (or 3.4), and have longer hospital length of stay [93] . it is encouraging to note that the children who received the pneumococcal vaccine had lower illness severity, less respiratory distress, required less respiratory support, and had less admissions to the picu [93] . in a review of the clinical significance of viral-bacterial coinfections in pediatric patients [94•] , 16 studies were found to evaluate clinical severity in this co-infected group. only four of these studies did not observe increased clinical severity, such as more frequent and longer picu admissions or longer ventilation requirements. it is also important to place this discussion in a historical context and discuss the influenza a pandemic of 1918. this pandemic had a mortality rate of 40-50 million people worldwide [15] . it is now known that secondary bacterial pneumonia caused the majority of the deaths during this time [95] . this further underscores that the viral infection alone was not as detrimental as the viral-bacterial co-infection. parainfluenza s. pneumoniae aom, acute rhinosinuisitis [78] a invasive pneumococcal disease: pneumococcus is isolated from a sterile site, i.e., sepsis, meningitis [84] cap community acquired pneumonia, aom acute otitis media, copd chronic obstructive pulmonary disease, lrti lower respiratory tract infection there is still a lot to learn about pediatric respiratory illness co-infections, as the interplay is intricate and not fully understood. looking at viral-viral co-infections, it seems that ultimately rsv seems to be the major decider of severity of infection whether or not the child has one or multiple viruses identified [96] . this information could influence the role of molecular testing in routine hospitalized patients. viralbacterial co-infections, on the other hand, usually lead to more severe diseases. overall, being able to better understand coinfection relationships can aid in the development of therapeutic methods as well as potentially affect the role of molecular testing. conflict of interest all authors declare no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. papers of particular interest, published recently, have been highlighted as: • of importance •• of major importance global, regional, and national causes of under-5 mortality in 2000-15: an 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increased severity during respiratory syncytial virus infection in young children does viral co-infection influence the severity of acute respiratory infection in children? review of the literature on the role of viral-bacterial co-infections in pediatric respiratory illnesses predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness respiratory syncytial virus coinfections with rhinovirus and human bocavirus in hospitalized children publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-021424-kocwsyi7 authors: shannon, m. frances title: genomic approaches to the host response to pathogens date: 2009-01-30 journal: genomic and personalized medicine doi: 10.1016/b978-0-12-369420-1.00107-4 sha: doc_id: 21424 cord_uid: kocwsyi7 nan following a period of relative disinterest in infectious disease research due to the enormous impact of vaccines and antibiotics on the spread of and mortality from these diseases, there is now renewed and growing interest in this area of research. this has been driven by several recent worldwide developments including: (i) the rising incidence of diseases such as acquired immune defi ciency syndrome (aids) and antibiotic-resistant tuberculosis; (ii) antibiotic-resistant bacterial strains presenting a severe health threat in hospitals; (iii) the rapid spread of new pathogens such as severe acute respiratory syndrome (sars); and (iv) the threat of bioterrorism. indeed, nearly 25% of annual deaths worldwide are due to infectious disease ( morens et al., 2004 ) . thus, the need to develop new diagnostic methods, more effective vaccines and better therapeutic strategies is urgent. in order to effectively deal with infectious disease threats, it is important to understand both the pathogen and the response of the host, since the outcome of infection is determined by complex host-pathogen interactions. pathogens are initially detected by the surveillance cells of the innate immune system using cell surface receptors known as toll-like receptors (tlrs) (reviewed in ). these tlrs recognize specifi c components of the pathogen, for example, bacterial lipopolysaccharide (lps) or double-stranded (ds) rna from viruses. while many cell types express tlrs, cells of the innate immune system such as dendritic cells (dcs) and macrophages play particularly important roles in detecting and responding to pathogens. the response of these cells to a pathogen is determined by the specifi c pathogen component that interacts with the tlr and the specifi c tlr family member that is activated. widespread changes in gene expression are detected following tlr activation and the activated cells produce a plethora of cytokines and chemokines that then activate the adaptive arm of the immune system. the specifi c cytokines and chemokines produced by the tlr-activated cells tailor the response of the adaptive immune system to deal with the specifi c pathogen . thus, the initial host response to a pathogen through the tlrs determines the outcome of the infection. host response to infection can be a double-edged sword in that sometimes the response itself can create an adverse outcome for the host. in addition, the aberrant response of the host to self instead of foreign pathogens can create severe pathologies involving chronic infl ammatory and autoimmune diseases. the urgent need to better understand host-pathogen interactions has come at a time when genomics and related technologies are expanding rapidly. the availability of complete genomic sequences of an expanding number of pathogens, the human and mouse genome sequences and the advent of genome-wide genotyping and gene expression profi ling has opened up new avenues of investigation in the fi eld. the genotype of the pathogen plays a major role in the response of the host to infection with more virulent pathogenic strains often possessing the capability to interfere with the host immune response ( fitzgerald and musser, 2001 ; kato-maeda et al., 2001 ; schoolnik, 2002 ) . in addition, different individuals in a population can have very different responses to a genetically identical pathogen. while there are many complex reasons for this, it is clear that part of the differential response is governed by underlying genetic differences between individuals ( clementi and di gianantonio, 2006 ; zhang and zhang, 2006 ) . studies in mouse models of infection have clearly demonstrated that these genetic differences are complex and may involve more than one genetic locus for a given susceptibility or resistance trait (e.g., [delahaye et al., 2006 ] ). while there are some classic examples of genetic mutations affecting the response of the host to a pathogen (e.g., malaria and sickle cell mutations) there is much to be learned before the genetics of host susceptibility is fully understood. the advent of genome-wide genotyping using single nucleotide polymorphisms (snps) or microsatellite markers, leading to major advances in molecular epidemiology, will revolutionize our ability to determine the complexities of the genetic component of pathogen-host interactions ( weiss and terwilliger, 2000 ) . it is well known that the cells of the host immune system are activated upon detection of a pathogen by tlrs as described above. this activation process includes widespread changes in the gene expression profi le of the cells with hundreds of genes being either switched on or off in response to signals generated from the pathogen-detecting tlrs. the response of individual genes has been studied in minute detail for a handful of genes and while this has produced an understanding of some aspects of host response to infection it by no means gives us the total picture. understanding the molecular response of the host to infection has been greatly improved by using microarray-based technologies and these technologies are opening up new diagnostic possibilities as well as presenting new therapeutic options ( aderem and smith, 2004 ; bryant et al., 2004 ; feezor et al., 2005 ; hedeler et al., 2006 ; korth and katze, 2002 ; ng et al., 2006 ; ricciardi-castagnoli, 2005 ; ricciardi-castagnoli and granucci, 2002 ; smith and bolouri, 2005 ) . this chapter will focus on two aspects of the host response to pathogens where major advances are being made using genomics approaches and will describe the future impact of these approaches on the development of diagnostics and therapeutics for infectious disease. these are (i) defi ning the basis of genetic susceptibility to infection and (ii) the defi nition of the system-wide molecular response to a pathogen. it is now relatively easy to map genes associated with genetic diseases that show a mendelian pattern of inheritance. however, these diseases account for only a very small proportion of the human disease burden and many of the more common and fatal diseases have a complex etiology with many genetic and environmental contributions. while it is clear that most complex disease has a genetic component, defi ning that genetic component has been diffi cult to date since many complex diseases such as coronary heart disease, diabetes and others are polygenic with different genetic loci contributing in major or minor ways to disease susceptibility. in addition, in different populations or under different environmental conditions, distinct but overlapping sets of genetic loci are likely to contribute. the sequencing of the human genome and the genome-wide genetic and functional mapping that has followed have raised hopes of mapping the genetic component of complex disease and there are many large efforts around the world with this aim. studies in both animal models and human populations have shown that infectious disease and the response of the host to a specifi c infection also has a complex genetic component ( clementi and di gianantonio, 2006 ; lipoldova and demant, 2006 ; marquet et al., 1996 ; mira et al., 2004 ) . thus, inbred mouse models have been developed that clearly show a genetic component to susceptibility for specifi c pathogens and in some cases at least part of the underlying genetic reason has been defi ned ( beck et al., 2000 ; mak et al., 2001 ; rogner and avner, 2003 ) . mapping the genetic components of susceptibility to infection in human populations has been much more difficult due to the large natural variation in humans, the polygenic nature of this trait and the low penetrance of many of the susceptibility alleles. for infectious disease, this is complicated even more by the complex nature of the environmental infl uences particularly the fact that these diseases, unlike other complex diseases, are transmissible in populations. however, a combination of animal and human population studies, combined with the latest genomic technologies, is beginning to unravel the issues of genetic susceptibility to infection. the use of inbred and congenic strains of mice are well established systems for identifying susceptibility loci ( beck et al., 2000 ; rogner and avner, 2003 ) . in recent years genetic manipulation of specifi c loci by deletion or mutation has provided many mouse models for screening ( mak et al., 2001 ) . the use of ethylnitrosourea (enu) mutagenesis to randomly create point mutations in the mouse genome has opened up a new forward genetics approach to identifying susceptibility loci (papathanasiou and goodnow, 2005 ) . this chemical mutagen, when used at appropriate doses and at the correct stage of development, can introduce single point mutations into the mouse genome. by screening libraries of mutant mice for susceptibility to specifi c pathogens, it should be possible to identify genetic loci that dictate susceptibility or resistance to a range of pathogens on a large scale. it is relatively straightforward to identify a chromosomal region involved in susceptibility in these mouse strains by genotyping with microsatellite markers, but identifying the specifi c gene that is mutated is still very time-consuming. the speed with which this can be achieved depends on the presence of candidate genes within the chromosomal interval or the ability to resequence large amounts of dna. the latter is becoming achievable with the advent of new rapid sequencing technologies and is set to revolutionize forward genetic approaches to disease understanding ( bennett et al., 2005 ; serre and hudson, 2006 ) . the recent explosion in genetic information for the human genome including the complete genome sequence and detailed genetic and physical maps has increased our ability to fi nd variation in the human genome and correlate it with disease. family studies, especially twin studies, and population studies have clearly shown a genetic component to susceptibility to infectious disease ( frodsham and hill, 2004 ; lipoldova and demant, 2006; strunk and burgner, 2006 ) . susceptibility generally follows a complex pattern of inheritance and there are two main methods of mapping and identifying genetic loci involved in such complex heredity. these are either association studies or linkage studies. association studies involve screening populations for specifi c mutations in a candidate gene(s) in case-control studies or in family studies. this type of approach identifi ed the link between human immunodefi ciency virus (hiv) resistance and the chemokine receptor, ccr5 described below ( dean et al., 1996; samson et al., 1996 ) . genome-wide association studies although still quite expensive are now becoming feasible and being used to defi ne linkage between specifi c markers and susceptibility. these linkage studies depend on the availability of markers and the density of these markers is rapidly increasing t a b l e 1 0 7 . 1 list of well-described susceptibility loci for resistance or susceptibility to infectious disease. genes references with the large scale identifi cation of new snps across the human genome. the selection of which snps to use and the large numbers of samples needed to generate statistically significant associations for low penetrance alleles are still challenges. the haplotype map (hapmap) project is starting to identify haplotypes within different population groups and together with improvements in large scale genotyping technology and bioinformatics should be useful in studies of complex disease inheritance. table 107 .1 summarizes the best studied genetic susceptibility loci for response to different infectious agents in both mouse models and human studies. one of the classical examples of genetic susceptibility to infection is the role of the hemaglobinopathies in the outcome of malaria infection ( patrinos et al., 2005 ) . there are also certain chromosomal regions and families of genes that have attracted attention in terms of searching for susceptibility alleles or polymorphisms. because the tlr family of receptors plays a major role in recognizing pathogens, it was speculated that genetic variation in these receptors or their signaling pathways might be responsible for some susceptibility phenotypes (reviewed in [schroder and schumann, 2005 ] ). one of the best examples to date is the occurrence of a single polymorphism in the region of the human tlr4 gene encoding the extracellular domain of the receptor which confers reduced sensitivity to inhaled escherichia coli ( e. coli) lps ( arbour et al., 2000 ) . interestingly, when septic shock patients were compared with a control group, these lower-responding alleles were found only in the septic shock group and these individuals had a higher incidence of gramnegative bacterial infection ( feterowski et al., 2003 ) . such studies need further confi rmation since there are also a number of studies that failed to fi nd any linkage between tlr4 mutations and response to various infections ( schroder and schumann, 2005) . there is also enormous variation in the response of individuals to lps even in the absence of tlr4 mutations implying that variation may occur in other components of the tlr4 signaling system. an example of this is the link between irak4 mutations and increased susceptibility to infection with pyogenic bacteria ( picard et al., 2003 ) . variation in other tlr genes has also been associated with disease susceptibility. for example, a mutation in the extracellular domain of tlr2 is linked to susceptibility to leprosy ( alcais et al., 2005 ) and a mutation in tlr5 increases susceptibility to legionella . taken together these data support the idea that variation in the innate immune recognition of pathogens play an important part in governing susceptibility to an array of infectious diseases. however, caution needs to be exercised until larger population groups have been studied. the extensive polymorphism at the chromosomal regions encoding major histocompatibility complex (mhc) proteins is thought to have arisen through natural selection in response to selective pressure from infectious disease. although human leukocyte antigen (hla) association with resistance or susceptibility to infectious disease has been diffi cult to identify because of the complex array of antigenic epitopes involved, a number of studies have implicated this locus in genetic susceptibility to infectious disease ( ghodke et al., 2005 ; little and parham, 1999 ) . mhc molecules fall into two classes, class i that present foreign antigens to cd8 ϩ cytotoxic t cells and class ii that play a similar role for cd4 ϩ helper t cells. variation in specifi c class i genes has been shown to confer susceptibility to pulmonary tuberculosis and to hiv whereas mutations in other class i genes confer resistance to hiv and to severe malaria. class ii mutations that confer resistance to hepatitis b or hepatitis c have been identifi ed and susceptibility to typhoid fever and leprosy are also associated with specifi c class ii mutations. further molecular analysis of these and other associations may in the future have an impact on the development of new vaccines and immunotherapeutics. to date the most successful manner of identifying susceptibility genes in human populations has been the candidate gene approach. candidate genes have emerged from many sources including mouse genetic studies as well as biochemical and function dissection of the immune system. once a candidate gene is identifi ed, the chromosomal region spanning this gene in the human genome is then scanned for the occurrence of specifi c mutations or for functional polymorphisms in casecontrol studies across populations or in linkage studies in family groups. such studies have identifi ed a number of well described susceptibility loci for infection with various pathogens. one of the most heralded example was the identifi cation of a deletion in the chemokine receptor, ccr5, which was shown to confer resistance to hiv infection ( dean et al., 1996 ; samson et al., 1996 ) . biochemically, this can be explained by the fact that ccr5 is a co-receptor for hiv on the surface of t cells (dragic et al., 1996 ) . a mutation in another chemokine receptor, ccr2, has also been shown to confer hiv resistance in certain caucasian populations ( o'brien and moore, 2000 ) . some genes have been associated with susceptibility or resistance to multiple pathogens. for example, variation in the nramp1/slc11a1 gene is associated with susceptibility to leishmania and to specifi c intracellular bacteria such as tuberculosis ( barton et al., 1999 ; govoni et al., 1996 ; lipoldova and demant, 2006 ; sebastiani et al., 1998 ) . mutations in the tumor necrosis factor (tnf) locus, mainly gene promoter mutations, have been linked with malaria and leprosy susceptibility ( lipoldova and demant, 2006 ) . gene promoter or control region mutations have an impact on the level of protein produced from the gene rather than the function of the protein. this is an area of great interest but more diffi cult to study for several reasons, including the inability to identify control regions simply from sequence information and the complexity and fl exibility of transcriptional control. a recent review detailing the genes associated with leishmania susceptibility describes a number of genes that can affect disease outcome including the interferon-gamma receptor type 1 ( ifngr1), the interleukin-4 (il-4 ) gene and the nramp-1/slc11a1 gene ( lipoldova and demant, 2006) . these genes and others such as interleukin-12 ( il-12) and its receptor are also linked with salmonella and certain mycobacterial infections ( lipoldova and demant, 2006 ) . thus, it is likely that variation in many genes can contribute to disturbing the fi nely balanced tuning of the immune system and lead to an altered response to a pathogen. it is clear from such studies that the same genes may be involved in susceptibility to an array of pathogens indicating a core immune response critical for any pathogen. the identifi cation of susceptibility loci for infection with various pathogens will aid in developing new diagnostic screens based on the detection of genetic variants in these loci. it could be envisaged that a person's susceptibility or resistance to a pathogen could be defi ned by a simple genotyping screen either prior to exposure to any pathogen or upon presentation with an infection. it may also be possible to determine the likely outcome of the infection through a genotyping screen. the defi nition of susceptibility loci will also contribute to our ability to develop new vaccines and therapeutics. the use of microarray technology to generate expression profi ling data is becoming common place in biomedical research (reviewed in [ quackenbush, 2002 ; sherlock, 2000 ] ). this technology allows the documentation of mrna levels for thousands of genes from total rna prepared from cells or tissue samples. the data obtained can be compared from sample to sample allowing the changes between samples to be documented and quantifi ed. the "expression profi le " for any cell or tissue is simply the list of genes whose expression can be detected using microarrays. the differences in the expression profi le from one cell or tissue to the next or in cells treated in a specifi c manner is a surrogate measure of the cell/tissue phenotype and shows how that phenotype responds to its environment. expression profi ling is most useful when large datasets become available and when the data is combined with other data types and detailed bioinformatics studies. for example, using functional clustering of expression profi ling data can help identify pathways that are important for a particular process and co-expression clustering combined with other technologies can help defi ne regulatory networks within the cell. over the last 5-6 years, this technology has been applied to identifying the changes in gene expression that occur in response to infection by various pathogens ( aderem and smith, 2004 ; boyce et al., 2004 ; bryant et al., 2004 ; feezor et al., 2005 ; foti et al., 2006 ; jenner and young, 2005 ; korth and katze, 2002 ; korth et al., 2005 ; ricciardi-castagnoli and granucci, 2002 ) . to date there are more than 150 papers in the literature that describe gene expression changes that occur in response to infection with a plethora of pathogens and in many cell types (reviewed in [ jenner and young, 2005 ] ). many of these are in vitro studies, taking specifi c cell types and infecting them with specifi c agents including bacteria, viruses, parasites and yeasts. in addition, cellular responses to bacterial components have also been documented, helping to identify pathogen-specifi c responses as well as determining the pathogenic component responsible for the major gene expression effects. virulent or non-virulent strains of specifi c pathogens as well as mutant organisms have been used to determine the gene expression profi le associated with a negative or positive clinical outcome. few in vivo studies have been carried out and have shed light on the more complex responses seen in whole animals and helped to validate the in vitro data. several pioneering studies demonstrated that microarrays could be used to determine changes in the gene expression profi le of cells in response to virus or bacterial infection ( boldrick et al., 2002; gao et al., 2002 ; huang et al., 2001 ; nau et al., 2002 ) . these studies paved the way for the analysis of the host response to a wide variety of infectious agents. the most signifi cant of these studies compared the response of macrophages or dcs to a variety of infectious agents in a single study. in these studies a strong shared response to all infections, be they bacterial, viral or parasitic in nature, was identifi ed. not only was there commonality from one infectious agent to another but there was also some commonality across cell types. this expression signature has been interpreted as a general "alarm signal " for infection (reviewed in [ jenner and young, 2005 ] ). studies of infection with gram-positive and gram-negative bacteria also revealed a common expression signature in peripheral blood mononuclear cells. recently, the young lab has interrogated all of the publicly available expression profi ling data related to the host response to infection ( jenner and young, 2005 ) . the dataset includes 785 experiments in cells ranging from macrophages and dcs to cells of the adaptive immune response, endothelial and epithelial cells and spanning a wide range of infecting agents. this meta-analysis revealed that a "common host response " can be detected across all of these cell types and infectious agents and show that although cells such as macrophages and dcs specialize in detecting infection, other cells of the body can mount the same " alarm response " as described above ( figure 107.1 ) . not surprisingly this expression signature contains many genes associated with the immune system particularly those encoding infl ammatory cytokines, chemokines and their receptors. however, some more surprising patterns of expression were also detected. it has been long known that interferon-stimulated genes (isgs) are regulated by virus infection, but it has only recently been recognized that bacteria and other infecting agents can also illicit the interferon response. this was borne out in these meta-analyses where upregulation of an isg set is observed across a broad range of infecting agents and cell types. not only do these cells change the expression of secreted factors and their receptors during infection but the intracellular milieu is also modifi ed. once again, there is a common pattern of change observed in all of these studies. the upregulation of signaling and transcription pathways that both augment and attenuate the immune response are observed leading to the interpretation that both positive and negative feedback loops operate within the cell to heighten or dampen the immune response. temporal profi ling can reveal extra layers of complexity and in one study a pro-infl ammatory profi le followed by an anti-infl ammatory profi le was identifi ed in macrophages activated with lps ( wells et al., 2005 ) . meta-analysis of temporal studies of activation through different tlrs also revealed that the infl ammatory chemokines/cytokine signature was an early response while the isg response was later, presumably refl ecting the need for an indirect activation of the isgs through interferon production ( jenner and young, 2005 ) . changes in the expression level of genes involved in both activation and repression of apoptosis also fall under the "common signature " banner and this is interpreted as sending the cells into a state of high alert where apoptosis can be either initiated to eliminate infected cells or terminated if the infection resolves ( jenner and young, 2005 ) . although these in vitro studies have provided an overview of the response of isolated cell types to pathogenic infection, in vivo studies are needed to validate any of these results before application to clinical medicine. a number of animal models have been used to profi le the host response to infection with a variety of agents. these studies are complicated especially if whole tissue samples are used in that changes in gene expression can result not only from genuine changes within the cells of the tissue but also from the recruitment especially of immune cells into the infected or infl amed tissue. nevertheless, in vivo studies have, in some cases, shown good correlation with the expression profi les found from in vitro studies. for example, profi ling the brains of mice infected with virulent sindbis virus revealed that isgs as well as infl ammatory chemokines were upregulated ( johnston et al., 2001 ) and in terms of diagnosis or treatment the exact reason behind these changes in expression signature may be irrelevant. in vivo studies also have some advantages in that the gene expression profi les detected in infected tissues will often make more sense when combined with other physiological or cell biology data from studies of the infected host. thus an "infection signature " would not only describe the altered gene expression of the immune cells that are recruited to the sites of infection but also would include changes in the gene expression of the resident cells of the tissue and may provide a more robust profi le of the infection process for use in diagnostic applications. what can be applied to clinical medicine from these studies? the ability to detect an "infection signature " using focused microarrays could potentially be used as a diagnostic tool. there would be an immediate need to identify a core set of genes with suffi ciently robust changes in gene expression to form the basis of a diagnostic array. arraying technology would need to be priced for diagnostic use and the technology would have to be deemed suffi ciently robust to pass all the quality control requirements of a diagnostic laboratory. no doubt progress will be made toward these goals in the near future. in addition to the "common host response " described above, microarray studies have revealed that pathogen-specifi c responses also exist. this is not surprising since it has long been known that different pathogens induce distinct arms of the adaptive immune response. for example, distinct types of helper t cells are activated in response to bacterial and viral infection compared to parasite infection and dc products such as cytokines and chemokines control the differentiation of t helper cell into the correct response type ( mosmann et al., 2005 ) . thus, the fact that cells of the innate immune system display a pathogenspecifi c transcriptional response as well as a general alarm signal helps to dictate the subsequent immune response. different tlrs are involved in recognizing and responding to different pathogens. for example, tlr2 is responsible for activation in response to gram-positive bacteria while tlr4 responds to lps, a component of gram-negative bacteria (beutler and rietschel, 2003 ; . tlr3 responds to dsrna and thus dictates the viral immune response for many dsrna viruses ( beutler and rietschel, 2003 ; . studies using bacterial components such as lps and fl agellin as well as dsrna have revealed that each tlr induces a specifi c as well as general transcriptional response. the transcriptional response of macrophages and peripheral blood mononuclear cells is more robust in response to gram-negative (tlr4) compared with gram-positive (tlr2) bacteria and the isg response is considerably reduced for the gram-positive expression signature ( boldrick et al., 2002 ; nau et al., 2002 ) . these differences are further observed when bacterial components are used to activate cells. for example, lps from gram-negative bacteria, a specifi c ligand for tlr4, can activate the isg profi le but tlr2 ligands such as lta and mdp cannot ( jenner and young, 2005) . not only the activation of specifi c gene sets but also the strength of the specifi c response signature may be important for the immune detection of the type of pathogen involved. e. coli infection of dcs strongly upregulates the chemokines/cytokine infl ammatory cluster whereas infection with infl uenza or other single stranded (ss) rna viruses (through tlr7) has a weaker ability to regulate this cluster but a stronger ability to regulate the isg signature ( huang et al., 2001 ; lund et al., 2004 ) . these types of results raise the possibility that the diagnosis of the type of pathogen involved in an infection would be helped by the development of customized microarrays that could distinguish the gene expression profi les elicited by particular pathogens. additionally, arrays that also detect rnas produced by the pathogen may be even more signifi cant as a diagnostic tool. an infecting agent can either be cleared from the body by the immune system mounting an appropriate response or cause severe or terminal pathology. the outcome depends on a multiplicity of events ranging from the genotype of the host, that is, whether the host displays a resistant or susceptible phenotype, the genotype of the infectious agent, that is, virulent or non-virulent strains and many other less defi ned environmental factors. can genomic approaches be used to determine the outcome of infection? clearly, as discussed above, the technology is developing to defi ne susceptible and resistant host genotypes especially in animal models of infection but the ability to do this routinely in human populations is some way into the future. given the smaller genomes of pathogenic organisms, defi ning virulence genotypes is progressing at a faster rate ( chan, 2003 ; dorrell et al., 2005 ; fitzgerald and musser, 2001 ; kato-maeda et al., 2001 ; macfarlane et al., 2005 ; schoolnik, 2002 ; zhang and zhang, 2006 ) . expression profi ling studies have been used to investigate the differences in the host response to pathogenic and nonpathogenic strains of specifi c infectious agents. in one example, mice infected with a pathogenic strain of pneumonia virus upregulated the expected infl ammatory chemokines/cytokine profi le as well as the isg profi le but an attenuated strain of the same virus could not, although the virus replicated in the lungs of these mice to the same degree ( domachowske et al., 2001 ) . temporal profi ling of the infection process in animals will also help to defi ne the expression signatures associated with the ability of the host to clear specifi c pathogens. while considerable information and progress is being made with the approaches described above, there is now much interest in combining both a genetic screening approach with an expression profi ling approach in a new area of investigation being dubbed "genetical genomics " (reviewed in de koning et al., 2005 ; schadt et al., 2003 ] ). the rationale behind this combination is twofold: fi rst, expression profi ling data can be used to identify those genes in a chromosomal region previously identifi ed from genetic approaches, whose expression is altered between two different genotypes. these genes then immediately become strong candidates for a susceptibility gene at that genetic locus. in this way, mutations that affect the expression but not the function of a protein can also be identifi ed. second, expression profi ling combined with genotyping can identify functional pathways that can be affected by a single mutation. for example, if a mutation affects a transcription factor that controls expression of a group of functionally related genes then not only the transcription factor but the entire pathway will be identifi ed opening up better opportunities to design new therapeutics. in a prelude to these types of approaches, hume and colleagues ( wells et al., 2003 ) carried out expression profi ling studies on lps-stimulated macrophages from various mouse strains that show differential response to infection. these studies showed that while there was a common core response to tlr4 activation, each strain of mice showed a unique gene expression program implicating many different genetic loci in this variable response. mapping the genetic loci responsible for these different transcriptional responses will help to shed light on genetic loci that may play a role in human susceptibility to infection. these type of approaches move into the realms of " systems biology " which is the study of an organism, viewed as an integrated and interacting network of genes, proteins and biochemical reactions, focusing on all the components and the interactions among them, as part of one system. given the complexity of the immune response to infection, ultimately this type of approach may provide more answers. although genomic approaches to understanding the host response to infectious disease are still very much at the developmental stage, it is likely that such approaches will be applied to diagnosis in the near future. in fact there are already some examples of the application of these approaches. a number of studies have been described using oligonucleotide-based arrays for the detection of multiple pathogens in a single experiment (campbell and ghazal, 2004 ; dietel and sers, 2006 ; hervas, 2004; kostrzynska and bachand, 2006 ; pompe et al., 2005 ) . recent studies have investigated the use of expression profi ling of whole blood to determine human subjects at risk of recurrent tuberculosis ( mistry et al., 2007 ) and to identify high and low responders to lipopolysaccharide ( wurfel et al., 2005 ) . a study in macaques has used whole blood expression profi ling to examine molecular signatures of infl uenza infection ( baas et al., 2006 ) . there are many efforts currently to optimize expression profi ling for 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(2004) . toll-like receptors in the pathogenesis of human disease. nat immunol 5(10), 975-979. this review describes the role of toll-like receptors in detection of pathogens and summarizes their involvement in infectious disease susceptibility. http://www.genmapp.org/ gene map annotator and pathway profi ler is a computer application designed to visualize gene expression data on maps representing biological pathways and groupings of genes. http://www.ensembl.org/index.html ensembl is a joint project between embl-ebi and the sanger institute to develop a software system which produces and maintains automatic annotation on selected eukaryotic genomes. http://www.informatics.jax.org/ mgd includes information on mouse genetic markers, molecular clones (probes, primers and yacs), phenotypes, sequences, comparative mapping data, graphical displays of linkage, cytogenetic and physical maps, experimental mapping data, as well as strain distribution patterns for recombinant inbred strains (ris) and cross haplotypes. http://www.geneontology.org/ the gene ontology project provides a controlled vocabulary to describe gene and gene product attributes in any organism. http://www.ncbi.nlm.nih.gov/geo/ gene expression omnibus is a gene expression/molecular abundance repository supporting miame compliant microarray data submissions, and a curated, online resource for gene expression data browsing, query and retrieval. http://www.genome-www5.stanford.edu/ the stanford microarray database stores raw and normalized data from microarray experiments, and provides data retrieval, analysis and visualization. http://www.expression.microslu.washington.edu/expression/index. html public microarray data download site powered by expres-sion array manager. http://www.ncbi.nlm.nih.gov/projects/snp/ the national center for biotechnology information has established the single nucleotide polymorphism (dbsnp) database to serve as a central repository for both single base nucleotide substitutions and short deletion and insertion polymorphisms.recommended resources ■ 1323 key: cord-023143-fcno330z authors: nan title: molecular aspects of viral immunity date: 2004-02-19 journal: j cell biochem doi: 10.1002/jcb.240591009 sha: doc_id: 23143 cord_uid: fcno330z nan mechanisms of t-cell mediated clearance of viruses from the central nervous system are poorly understood, but likely to differ from those employed in the periphery because the cns lacks lymphatic drainage and constitutive expression of mhc class i antigen, and the unique structure of the cns vasculature imposes constraints on access by leukocytes and soluble immune mediators. to study the mechanism by which viruses are cleared from neurons in the central nervous system, we have developed a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (oblv60). grew preferentially in the olfactory bulbs of balbk mice. using in situ hybridization, we found viral rna localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. virus was cleared rapidly from the olfactory bulb between 5 and 11 days. athymic nude mice failed to eliminate the virus demonstrating a requirement for t lymphocytes. immunosuppression of normal mice with cyclophosphamide also prevented clearance. both cd4+ and cd8+ t-cell subsets were important as depletion of either of these subsets delayed viral clearance. gliosis and infiltrates of cd4+ and cd8+ cells were detected by immunohistochemistry at 6 days. the role of cytokines in clearance was investigated using an rnase protection assay for il-la, il-lp, il-2, il-3, il-4, il-5, il-6, tnfa, tnfp and ifny. in immunocompetent mice there was upregulation of rna for il-la, il-lp, il-6, tnfa and ifny at the time of clearance. nude mice had comparable increases in these cytokine messages with the exception of ifny. induction of mhc-i molecules on cells in infected brains was demonstrated by immunohistochemistry in normal and nude mice, suggesting that ifny may not be necessary for induction of mhc-i on neural cells in vivo. luca g. guidotti, kazuki ando, tetsuya ishikawa, lisa tsui and francis v. chisari. the scripps research institute, la jolla, ca 92037 although cytotoxic t lymphocytes (ctl) are known to clear viral infections by killing infected cells, recent studies suggest that they can also suppress the replication of certain viruses by noncytolytic mechanisms. we have examined this area by monitoring the immunopathological and antiviral consequences of antigen recognition by hepatitis b virus (hbv) specific ctl in hbv transgenic mice that express the viral gene products in their hepatocytes. we have shown that intravenously injected ctl rapidly trigger their target hepatocytes to undergo apoptosis, but that the direct cytopathic effect of the ctl is minimal in comparison with the cytopathic effects of the antigen-nonspecific intrahepatic inflammatoly response that they activate. in addition to killing the hepatocyte, the same ctl also downregulate hbv gene expression and completely abolish hbv replication in the hepatocytes that they don't destroy. this noncytolytic antiviral ctl effect is mediated by at least two distinct processes in these animals. first, the ctl cause a quantitative reduction in the steady state content of all hbv mrna species in the hepatocyte, and this is followed by disappearance of all of the corresponding viral proteins in the liver and serum. the ctl initiate this process by secreting ifny and tnfa when they are activated by antigen recognition. since the regulatory effect of the ctl can he prevented completely by prior administration of the corresponding antibodies. nuclear run-on experiments reveal that viral mrna transcription is unaffected despite the profound reduction in hbv mrna content in the liver, suggesting that the ctl-derived cytokines accelerate viral mrna degradation in the hepatocyte. a second noncytolytic antiviral pathway is also activated by the ctl. we have recently shown that hbv nucleocapsid particles, and the replicative hbv dna intermediates that they contain, disappear from the transgenic mouse liver following either ctl administration or partial hepatectomy. the latter of which triggers hepatocellular regeneration without any change in hepatocellular hbv mrna content. these results suggest that preformed hbv nucleocapsid particles may be actively degraded during hepatocyte turnover, and they raise the possibility that similar events might also occur in nondividing hepatocytes that are activated by noncytolytic signals delivered by the ctl. we propose that, in addition to their pathogenetic effect, the comhined effects of the ctl response at die hbv mrna. nucleocapsid and rcplicative dna levels may represent a curative antiviral stimulus during hbv infection. since the virus must contain molecular elements that iespond to these ctl-induced antiviral signals. inactivating mutations at these loci could be very efficiently selected by immune pressure, because a single mutation could abrogate the antiviral effect of a wide spectrum of t cell responses, irrespectrve of epitope specificity. identification of these viral response elements and the intracellular pathways that interact with them may lead to the development of new strategies for antiviral drug design. human fibroblasts infected with hsv are resistant to lysis by cd8+ cytotoxic t lymphocytes (ctl), yet human b cell lines can be efficiently lysed by these ctl. the effect on human fibroblasts is rapid (within 2 hr of infection of cells), occurring before synthesis of mhc class i is altered by virus infection. a recombinant hsv, f-usbmhc, which expresses mouse mhc class i proteins does not render human fibroblasts sensitive to lysis by mouse ctl. mhc class i molecules are retained in the er of hsv-infected fibroblasts i n a misfolded, unstable form and stability of the mhc complex can be restored by addition of exogenous peptides. using a panel of hsv mutants and ad expression vectors we demonstrated that the hsv ie protein icp47 was both necessary and s f i c i e n t to cause retention of class i and icp47 expression in fibroblasts caused the cells to resist lysis by cd8+ t lymphocytes. icp47 is a soluble, cytosolic protein and we have found no evidence of membrane association. therefore, it appears that icp47 inhibits cytosolic stages of the antigen presentation pathway so that antigenic peptides do not reach the er. to date, polyclonal and monoclonal antibodies directed to icp47 have not specifically precipitated any of the previously described components of the antigen presentation pathway and we have not found icp47 associated with tap transporter proteins or proteosomes i n these experiments. the effects of icp47 are being assessed in proteosome and tap transporter assays. gst-icp47 fusion proteins tightly bind a 8.5 kda cytosolic cellular protein which is found in a number of adherent human cell lines but not lymphocytes. the protein has been purified and sequencing is in progress. in addition, radiolabelled icp47 binds to a single cellular protein of =55 kda on ligand blots. these proteins are good candidates as cellular targets of icp47 and as novel components of the antigen presentation pathway. preliminary experiments support the hypothesis that icp47 is very effective i n blocking cd8+ t lymphocyte responses in vivo, perhaps explaining the predominance of cd4+ vs. cd8+ anti-hsv ctl i n vivo. we expect that icp47 may be very useful, not only to elucidate antigen presentation pathways, but also to prevent immune recognition of gene transfer vectors and a s a immunosuppressive agent. susceftibility to polyoma virus-induced tumors is conferred by an endogenous mmtv superantigen. aron e. lukacherl, yupo ma2, john p. carroll2, sara r. abromson-leeman2, joseph c. laning2, martin e. dorf2, and thomas l. benjamin2. idepartment of pathology, emory university school of medicine, atlanta, ga 30322, and 2department of pathology, harvard medical school, boston, ma 02115. susceptibility to tumors induced by mouse polyoma virus varies among inbred mouse strains. we have previously shown that polyoma tumor susceptibility is controlled by products of mhc as well as non-mhc genes. in crosses between mhc-nonidentical strains differing in tumor susceptibility, resistance correlates with dominantkodominant inheritance of the resistant h-2 haplotype. we have observed the opposite pattern of inheritance of susceptibility in crosses between mhc-identical strains. in crosses between the highly susceptible c3wbida mouse and the highly resistant but mhc-identical (h-2k) c57bwcd.i mouse, polyoma tumor susceptibility is conferred by a single autosomal dominant gene, which we have designated pyvs. pyj does not encode cell receptors for the virus, affect viral dissemination or anti-viral antibody responses, or affect intracellular events essential for productive infection or cell transformation by the virus. whole-body irradiation renders cs7bwcd.i mice fully susceptible to polyoma-induced tumors, indicating an immunological basis for this strain's resistance. we hypothesized that p y j encodes an mtv superantigen (sag) that confers susceptibility to c3wbida mice by deleting precursors of polyoma-specific t cells. we found that tumor susceptibility in (c3wbida x c57bwcd.i) x c57bwcdj backcross mice cosegregated with mtv-7. inheritance of mtv-7 showed perfect concordance with absence of peripheral vp6+ t cells. genotyping of backcrossmice using markers of simple sequence repeat polymorphisms flanking mtv-7 showed no evidence of recombination between pyvs and mtv-7. strongly biased usage of vp6 by (a) polyoma-specific cd8+ ctl from virus-infected c57bwcdj mice and by @) cd8+ t cells infiltrating a polyoma tumor in a virus-immune c57bwcd.i host provide further evidence that t cells bearing this mtv-7 sag-reactive vp domain are critical anti-polyoma tumor effector cells. these results indicate identity between p y j and mtv-7 sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's t cell repertoire. infection of mice with lymphocytic choriomeningitis virus (lcmv) causes a transient to longlasting immunosuppression dependent upon virus-isolate dose of virus and age, h-2, non h-2, level of cd4+ t cells, of cd8+ t cells and kinetics of neutralizing antibodies of the host. the immunohistological analysis suggests that cd8+ t cell dependent disappearence of marginal zone macrophages of follicular dendritic cells and of virus infected cells in general correlates with immunosuppression. the details of mechanisms responsible for these findings are now being analysed. a role of this cd8+ t cell dependent immunosuppression in the establishment of a lcmv carrier state in immunocompetent mice is suggested by the following experiments: the otherwise slow and low neutralizing antibody response agamst lcmv is accelerated and enhanced by cd8+ t cell depletion at the time of infection, suggesting virus-specific immunopathology being responsible at least partially. the elisa antibody response is not significantly altered under the same conditions but is abrogated if lcmv-specific t cell receptor transgenic mice are infected with high doses of lcmv, indicating, that suppression of the specific antibody response depends upon the relative kinetics of ctl versus antibody responses. whether exhaustion of specific ctl responses is enhanced by similar mechanisms remains to be tested. the role of interleukins of the relative distribution of virus in the mouse and in the various aspects of immunosuppression are now being studied. immunosuppression, caused by cd8+ t cell-dependent immunopathology, may also be operational in hiv infection in humans. such a pathogenesis of hiv-triggered aids could explain several aspects of the disease process not readily fitting the (unproven) conventional idea that hiv is causing immunodeficiency via direct viral pathogenicity. the cellular immunity against two dna tumor viruses (i.e. human adenovirus type 5 (ad 5) and human papillomavirus type 16 (hpv16)) was studied with respect to possible immune escape mechanisms and to the development of ctl epitope based peptide vaccines. after identifying an immunorelevant ctl epitope in the ad 5e1a protein to which ctl clones were directed that could eradicate ad 5e1 induced tumors in nude mice, an amino acid replacement study of this epitope revealed a point mutation that totally eliminated the possibility to recognize the mutant peptide by the ctl clones directed against the wild-type peptide sequence. new viral constructs were made that contained this point mutation and used to transform mouse embryo cells. however, these mutant tumor cells were still immunogenic and ctl clones specific for these mutant tumor cells were shown to react with a peptide derived from the ad 5e1b protein. these ad 5eib specific ctl clones, however, were as effective as the ad 5e1a specific ctl clones in the eradication of ad 5e1 induced tumors in nude animals, indicating that a choice can be made of immunorelevant epitopes to which an immunization strategy could be developed. in addition, we discovered that by supertransfection of ad 5e1 induced tumor cells with the activated ras oncogen the possibility of ad 5e1b specific ctl to recognize the ad 5e1 induced tumors was eliminated whereas the ad 5e1a specific ctl could still kill these tumor cells. this might indicate a new mechanism of tumors to escape ctl. in an hpv16 induced mouse tumor model an immunosubdominant ctl epitope was identified in the e7 protein that could, upon immunization with that peptide,protect mice against a subsequent challenge with hpv16 induced tumor cells. by changing the anchor residues in that peptide an even more immunoprotective peptide could be generated. combined, these data indicated a successful use of a ctl epitope based peptide vaccine in the prevention of hpv16 induced tumors in mice. subsequently this led us to identify relevant ctl epitopes of hpv16, that is highly associated with cervical carcinoma in humans, for the major hla-a alleles (i.e. hla-a *0101, a *0201, a"0301, a*1101 and a *2401). together these alleles cover a majority of all humans. ctl epitopes were identified through peptide-mhc binding assays followed by in v i m peptide immunizations with high affinity binding peptides to induce primary ctl responses and immunogenicity studies in hla-a transgenic mice. thereafter, memory ctl responses were measured in cervical cancer patients against selected peptides. combined, these data led us to develop a ctl epitope based peptide vaccine that could be of use in hpv16 induced cervical cancer patients. a clinical trial for this disease is scheduled to start in the fall of 1994. class ii presentation of an endogenously synthesized glycoprotein. carol s. re is^'.'.^, shirley m. bartido', miriam stein', and stephanie diment3.4, biology department', and center in contrast to class i presentation which is well characterized to use peptide fragements of proteins sythesized in the cytoplasm, exogenously administered experimental antigens enter the class ii mhc pathway through endocytosis. we have been studying the recognition of the glycoprotein of vesicular stomatitis virus (vsv) which can enter either the exogenous or endogenous pathways for presentation to cd4 + t cells. investigations of the intracellular sites involved, the proteolytic processes involved in the epitope generation, will be discussed. the glycoprotein studied in detail is a truncated form of the wt type 1 glycoprotein, termed poison tail (gpt) . expressed with a vaccinia virus vector, the gpt remains endo h sensitive and never becomes endo d sensitive, indicating that it is restricted to the endoplasmic reticulum. gpt is degraded in the er, and w e believe the degradation products include the immunogenic epitopes recognized by a panel of lad and i-ed t cell clones and hybridomas. lmmunofluorescence studies have confirmed the er localization. flow cytometric evaluations s h o w that the gpt never appears on the cell surface, in contrast to the wt g. the peptides generated are not secreted; using an innocent bystander assay, gpt-infected cells are incapable of sensitizing 5'cr-labeled uninfected apc. this contrasts with the rapid ability of supernatants from wt g-vaccinia virus-infected cells to sensitize apc for t cell recognition. investigations of the characteristics of the enzymes contributing to the degradation of the gpt have shown that a reducing environment is essential, as diamide treatment of cells prevents degradation. lysosomotropic drugs (eg. nh,ci and leupeptin) d o not alter the half-life of the protein, but do prevent presentation of the peptides; this is inconsistent with an autophagic component to the proteolysis. ph optima are physiological, as ph8 environment inhibits the enzyme activity. inhibitors of enzyme classes are consistent with a trypsin-like, and not cystein-, cathepsin b-, or chymotrypsin-like class. supported by nih grant al 18083 to csr. (emcv) and mengovirus are related members of the cardiovirus genus of picomaviruses. their rna genomes encode a large polyprotein which is cleaved proteolytically in co-and post-translational reactions to yield all mature viral proteins necessary to establish an infection. although originally thought to be exclusively murine in host range, both viruses actually infect a wide range of mammals. emcv has caused devastating epizootics in captive primates (eg: macaques, chimps and baboons), domestic pigs and exotic zoo collections (elephants, lions and tigers). death, following ingestion of virus-contaminated material, is rapid, and caused by extensive meningoencephalitis and virus-induced damage to the cns. myocarditic lesions are common in older animals. when administered intracerebrally, the ld,, for emcv strain r is about 1 pfu. we are studying the pathogenesis of emcv and mengo with engineered cdna plasmids containing infectious viral sequences. many plasmids contain h'uncated versions of the unusual 5' noncoding homopolymeric poly(c) tract that is a hallmark of these cardioviruses. short poly(c) mengoviruses grow very well in tissue culture but are 106-10'z fold less pathogenic to mice than the wild-type strains. animals receiving sublethal doses of short-tract mengo strains develop high titers of neutralizing antibodies, exhibit potent ctl responses and acquire lifelong protective immunity against challenge with wild-type virus. the genetic stability of the short-tract strains, even upon serial brain passage, mark them as safe, efficacious live vaccines. currently, we believe the poly(c) phenomenon is due to interference by the wild-type virus sequences (long poly(c) tract) with normal cellular cytokine induction mechanisms (ie: ifa and ifp) during the initial stages of animal infection. the targeted cells are probably macrophages, and their singular ability to correctly respond or not respond to poly(c) tract length during the first few hours of infection determines whether an inoculated animal will live (protectively vaccinated) or die. the short-tract viruses probably induce if in the macrophages, and are consequently killed then rapidly cleared from the host in related experiments we've found that attenuated mengo strains can easily carry large heterologous insertions within their genomes, and express these sequences into protein them during replication in animals. the resulting immune response (b cell and ctl) to the chimeras is directed towards the foreign sequences (epitopes) as well as towards the mengo proteins. a chimeric hiv vaccine, a rabies vaccine and an lcmv vaccine have been developed and tested. the lcmv chimera seems especially effective, as a single pfu of this engineered mengo strain, administered orally to a mouse, is sufficient for complete immunogenic protection against intracerebral challenge with wild-type lcmv virus. rsv is the most common cause of serious viral lower respiratory tract disease in infants and children. we have recently renewed our efforts to generate a safe and effective live attenuated rsv vaccine for topical administration that will overcome the deficiencies of previously studied live and non-living rsv vaccines. this vaccine will be a bivalent vaccine consisting of subgroup a and b live attenuated virus components. since the peak incidence of severe disease caused by rsv is in the 2-month old infant, an rsv vaccine will need to be effective when given to 1-month old infants. based on the success of live poliovirus vaccines given early in infancy, it is anticipated that the intranasally administered live virus vaccine will infect and induce a protective local and systemic immune response even in infants with passively acquired maternal antibodies. the main approach that we have taken in this effort to develop the live rsv vaccine is to introduce one or more ts mutations by chemical mutagenesis into a cold-passaged virus (cprsv) that had been partially attenuated by the acquisition of host-range mutations selected by passage in cells of a heterologous host species. we have developed a large set of cprsv subgroup a rs mutants (termed cprs mutants) that contain the host-range mutations selected during cold passage and two or more ts mutations introduced by chemical mutagenesis. these mutants have been evaluated in virro for their level of temperature sensitivity and in vivo in rodents, chimpanzees, and humans. a large set of rsv subgroup b cpts mutants has been similarly produced and evaluated. the immunogenicity and protective efficacy of three candidate live attenuated rsv vaccine strains that represent a specaum of attenuation were evaluated for protective efficacy in chimpanzees. prior to infection some of these animals were given rsv immune globulin by the iv route to simulate the condition of the very young infant who possesses passively-acquired maternal rsv antibodies. the three candidate vaccine strains were immunogenic and induced significant resistance to rsv challenge in both groups of chimpanzees. interestingly, the chimpanzees infused with rsv antibodies prior to immunization were primed more effectively for an unusually high serum neutralizing antibody response to infection with challenge virus than chimpanzees which did not receive such antibodies. this high booster response occurred despite marked reshiction of replication of the challenge virus. the evaluation of two candidate vaccines in seronegative human infants will also be described. rs virus is immunologically interesting for at least t w o reasons: 1) upper respiratory reinfection occurs despite previous exposure and demonstrable immunological memory: 2) humans or rodents previously immunised against virus infection can show enhanced disease during reinfection. others have shown that passive transfer of antiviral antibody either protects against virus infection or has no effect, and there is no evidence of antibody enhancement of disease in vivo. by contrast, t cell immunity appears closely associated with disease augmentation. we have focused on examining the immunological mechanisms of disease enhancement in mice. initial studies showed that transfer of cd8+ cytotoxic t lymphocytes (ctl) causes rapid virus clearance from the lungs of rs virusinfected mice, but also increased disease severity with alveolar haemorrhage and polymorphonuclear (pmn) cell recruitment to the lung. this disease (reminiscent of shock lung) could sometimes be fatal, whereas normal mice recover well from similar doses of rs virus. next, we compared the effects of cd4' and cd8+ t cells, using polyclonal t cells separated immunomagnetically from mixed lines grown in vitro with viral antigen. cd4' t cells were more pathogenic than cd8+ t cells in a dose-for-dose comparison, but that the type of pathology varied depending on the type of cell injected. while testing recombinant vaccinia viruses expressing single rs viral proteins for their ability to protect mice against infection, we observed that animals sensitised t o the major surface glycoprotein g (attachment protein) developed lung eosinophilia after challenge with rs virus intranasally. t cell lines from the spleens of mice sensitised with various recombinant vaccinia viruses were established. those form mice primed with the m 2 (22k) protein were predominantly cd8' ctl, and that produced few cytokines. those from mice primed with fusion protein (f) generated mixed t cell lines with both t h l cd4+ t cells, and ctl. mice primed to g protein gave rise to predominantly cd4' t cells producing th2 cytokines. ln vivo transfer of these cell lines into na'ive rsv infected mice reproduces the patterns of disease seen in mice sensitised in vivo with the respective antigens. the mouse model of rs virus disease therefore has excellent potential for illustrating mechanisms of lung immunopathology. the eye is a complex organ whose function is to transmit light images through different cell and tissue layers and liquid media to a neurosensory retina. elements as could occur when invading pathogens arrive and an inflammatory response with its swelling, plasma protein extravasation, leukocyte infiltration and tissue damage results. inflammatory responses when possible and rely on immune defenses which do not involve tissue distortion and damage. restricting tissue damaging responses is not always effective and the process is best developed in response to agents delivered to locations such as the anterior chamber. where an inflammatory response is initiated which may result in ocular impairment. such herpetic stromal keratitis (hsk) is a common cause of blindness in man. animal model studies indicate that hsk is a multi-step process initiated by virus in an avascular structure. hsk fails to occur in the absence of t cells or replicating virus. disappears several days before a visible inflammatory response becomes evident. evidence will be presented that the secondary agonists which drive the inflammatory response may not be viral antigen(s) per s e . multiple cell types are involved in hsk, with the respective role of functional sets of lymphocytes changing according to the clinical phase of the disease. in addition, nonspecific inflammatory cells such as neutrophils and nk cells also influence the severity of lesions. basically the reaction begins with t cells that produce type one cytokines, particularly ifn-y, dominating the scene, but during remission type 2 cytokines, notably il-10, appear as mechanistically involved. from the use of knockout mice for various immunological parameters, evidence will be presented that numerous mechanisms of pathogenesis may be at play during hsk. damage to corneal tissues in all systems appear to involve tnfo. a second ocular damaging event in which immunopathology is at least partially involved is herpetic retinal necrosis. evidence that this disease may involve the immunopathological role of cd4 t cells and protective effects by cd8' t cells will be presented, as will be suggestions by which the pathological events are mediated at the molecular level. thus it is in the eye's functional interest to limit acute viral infections and live vaccines often confer long-term immunity the nature of t and b cell memory is different. b cell memory is manifested not only by the presence of memory b cells but also by continuous antibody production in contrast, the effector phase of the t cell response i s shortlived and long-term t cell memory is due to the presence of 'quiescent' antigen specific memory t cells that are present at higher frequencies and are able to respond faster upon re-exposure to virus due to increased levels of adhesion molecules in this talk i w i l l present our results on. (i) the bone marrow as a major site of long-term antibody production after acute viral infection, (ii) the role of c d 4 ' t cells and b cells (immune complexes) in maintaining cd8+ t cell memory, (iii) the role offas antigen in regulating t cell responses, and (iv) the efficacy ofvarious antigen delivery systems in inducing long-term t cell memory sendai virus is a natural respiratory viral pathogen of mice. intranasal infection of mice with the virus provokes a virus specific antibody-forming-cell reaction that exhibits a distinct kinetic pattern in the lymph nodes that drain the respiratory tract, in the spleen, and in the bone marrow. the bone marrow afc population is extremely long-sustained, and supports an active humoral response that essentially persists for the lifetime of the infected animal. thus the conventional categories of "primary" and "secondary" response may not apply to the humoral response of mice naturally exposed to respiratory viruses. paradoxically, the population of b cells that reacts most rapidly to sendai virus infection does not itself secrete antibody, but can he demonstrated by the recovery of hyhridomas that secrete "polyspecific" antibodies. the activation of this polyspecific b cell population is, like the humoral response, extremely persistant. viral infection thus sets in train multiple b cell "memory" processes. variation in the rules of development and turnover of different b cell populations constrains the mechanisms that may operate to generate these different forms of memory. establishment and maintenance of t cell memory to respiratory viruses, peter c. doherty, sam hou, christine ewing, david topham, anthony mcmickle, james houston, and ralph tripp, department of immunology, st. jude children's research hospital, memphis, tn 38105. the analysis of the development and memory phases of the cd4+ "helper" n h ) and cd8+ cytotoxic t lymphocyte (ctl) responses to the respiratory pathogens, influenza virus and sendai virus (parainfluenza type 1) have been characterized by a combination of limiting dilution analysis (lda) for determining th and ctl precursor @) frequency and facs separation of lymphocytes with different activation phenotypes. the interpretation at this stage, largely based on the analysis of the ctl response, is that the development phase of t cell memory and the primary response are synonymous. virus-specific ctlp are produced in considerable excess of the numbers required to provide the effector ctl that terminate the primary infection, with only a fairly small proportion localizing to the target organ (the lung) that supports virus growth. even when many of the proliferating ctlp are killed by administration of a small dose (20 mgkg) of the dna-targeted drug cyclophosphamide (cy), there is no indication of immune exhaustion. the cd4+ th response has, at this stage, not been analyzed through the course of the primary infection. use of the lda approach to determine thp frequencies is inherently more difficult, as the "read-out'' is lymphokine production and there is considerable "bystander" activation in these primary responses to respiratory viruses. memory thp and ctlp are characterized initially by the expression of an "activated" phenotype: cd44-high, l-selectin-low, cd49d (vla-4) high. after some months, an increasing proportion of the memory t cells revert to the l-selectin-high cd49d-low form typical of naive ctlp. the change, which is never absolute, seems to occur first with cd49d and the rate varies for different viruses. current experiments are addressing the possibility that intercurrent infection, particularly with the mouse y-herpesvirus 68 which causes persistent infection of lymphoid tissue, may be inducing a switch back to the activated pattern, as a consequence of "bystander" effects, or "low affmity" stimulation via the clonotypic tcr in responding lymphoid tissue. the question of such cross-reactivity and/or exposure to "high lymphokine" environments for the long-term maintenance of memory is also being addressed. to study the factors which regulate the generation and persistence of specific t cell memory we have used model systems utilizing t cell receptor transgenic mice as a source of enriched naive cells which can be either cultured in vitro to generate effector populations or restimulated in adoptive hosts. in either case one can visualize the development of an expanded effector population. we have documented that the proliferation and il-2 production of the naive t cells depends on their activation by apc expressing high levels of co-stimulatory molecules. we find that b7.1 and icam-i as costimulators strongly synergize and that increased t cell receptor triggering can both increase the magnitude of the response and decrease its dependence on costimulation. when cytokines il-4 vs il-i2/ifny are present at the initiation of the response of either cd8 or cd4 cells they dictate that the effectors generated will be polarized either towards il-4 and il-5 secretion or il-2 and ifny secretion, respectively. the fate of the effector population generated and followed in vitro, also is tightly regulated by ag, cytokines and probably by costimulation. cd4 effector cells not re-exposed to ag, produce no cytokines and they die within 3-4 days. effectors restimulated with ag make massive amounts of cytokines, regardless of the presence of cytokines, at low densities of ag and with little dependence on costimulation. when there is little il-2 produced and no cytokines added, effectors die rapidly by apoptosis. however the combination of il-2 and tgfp block apoptosis and support expansion of the effector population which is greatly enhanced by periodic ag stimulation. some conditions favor the reversion of effector-like cells to a more resting memory phenotype and these are being further explored. we have also examined the development and maintenance of memory after transfer of effector cells to adoptive hosts. long-lived polarized memory populations are generated from the polarized effectors and these persist for prolonged period in the absence of apparent ag stimulation. this supports the idea that factors other than antigenic stimulation, present in situ can support the expansion and maintenance of memory cells. the rabies glycoprotein (g) is the only external protein of the virion and is therefore responsible for any interaction that rabies makes with the host cell during the first steps of the virus cycle. the g protein is also the target of neutralizing antibodies. there are around 450 trimers of g at the virion surface which constitute the spikes visible by electron microscopy. upon exposure to slightly acidic ph, the glycoprotein undergoes a conformational change which results in ion er and less regular spikes. strikingly and quite differently from influenza hemagglutinin, this conformational change is reversible: if the p d is risen back to 7.0, the s ikes re ain their neutral configuration (1). probably as a consequence, the viral infectivity is totally preserved after an exposure of 2 hours at p 8 6.4 an cf 37t, which induces the conformational change, followed by an incubation at neutral ph. since the conformational change is reversible, there is a ph-dependant equilibrium between the native and the low-ph conformation: the higher the ph, the more spikes are in their native configuration. two main antigenic sites and several minor sites have been identified on the native rabies glycoprotein (2). specific amino acids belonging to each of the two major antigenic sites are important or essential for viral virulence. for instance a lysine in position 198, which is part of antigenic site 11, is important, although not essential, for the viral virulence. similarly, the arginine 333, which belongs to antigenic site 111, is essential for pathogenicity while dispensable for multiplication in cell culture (reviewed in 3). viral strains mutated at arginine 333 have lost the capability to penetrate certain categories of neurons, suggesting that this mutation affected the recognition of specific receptors or subsequent interactions necessaly for the penetration of the virus at nerve terminals. therefore the two main antigenic sites are regions of the glycoprotein which also interact specifically with neurons in the animals. we have found that neutralization requires the fixation of at least one or two igg for every three spikes, irrelevant of the anti enic site recognized by the antibody (4). most neutralizing antibodies recognize conformational epitopes which are accessible on the native configuration of the protein. some epitopes remain accessible also on the acidic configuration while others are not. in addition, a minority of antibodies recognize epitopes which are only accessible on the acidic conformation. this is not unlikely in view that each spike has a certain probability to undergo a conformational change, even at neutral ph. in consequence the surface of the virus probably fluctuates and g epitopes which are not accessible on the native glycoprotein could be transiently exposed. conformational flexibility at neutral ph and physiological temperatures has also been observed for poliovirus (5). structural flexibility of external proteins could have important implications in virus-host interactions. katpus, norlhwestern university medical school, chicago, il 6061 1 theiler's murine encephalomyelitis viruses (tmev) are endemic enteric pathogens of wild and colony-reared mice. lntracerebral inoculation of susceptible mouse strains leads to a chronic, progressive inflammatory demyelinating disease of the central nervous system (cns) characterized clinically by an abnormal gait, progressive spastic hind limb paralysis and urinary incontinence, and histologically by parenchymal and perivascular mononuclear cell infiltration and demyelination of cns white matter tracts. demyelination is related to persistent cns viral infection. due to the similarity in clinical and histological presentation, tmev-induced demyelination is considered to be a highly relevant model of multiple sclerosis (ms). our current interests are in determining the phenotype, fine specificity, lymphokine profile and tcr usage of cns-infiltrating cells involved in the effector stages of tmev-induced demyelination. based on a variety of experimental evidence, it is clear that demyelination induced in sjuj mice by infection with the bean strain of tmev is a thl-mediated event: (a) disease induction is suppressed in t cell-deprived mice and by in vivo treatment with anti-i-a and anti-cd4 antibodies; (b) disease susceptibility correlates temporally with the development of tmev-specific, mhc-class il-restricted dth responses and with a predominance of anti-viral lgg2a antibody; (c) activated (le., ll-2rc) t cells infiltrating the cns are exclusively of the cd4+ phenotype, and (d) proinflammatory cytokines (ifnq and tnf-p) are predominantly produced in the cns. we have mapped the predominant thl epitope on the virion to amino acids 74-86 of the vp2 capsid protein. a thl line specific for vp274-86 exacerbates the onset of demyelination in recipient mice infected with a suboptimal dose of tmev. tmev-infected sjuj mice fail to exhibit peripheral dth and t cell proliferative responses to the major myelin proteins, mbp and plp, and pre-tolerization with neuroantigens has no affect on the incidence or severity of tmev-induced demyelinating disease, whereas neuroantigen-specific tolerance prevents the induction of relapsing experimental autoimmune encephalomyelitis (eae). in contrast, tolerance induced with intact tmev virions specifically anergizes virus-specific thl responses and results in a dramatic reduction of the incidence and severity of clinical disease and cns dernyelination in sjuj mice subsequently infected with tmev. these results have important implications for a possible viral trigger in ms as they indicate that chronic demyelination in tmev-infected mice is initiated in the absence of demonstrable neuroantigen-specific autoimmune responses and are consistent with a model wherein early myelin damage is mediated via primarily by mononuclear phagocytes recruited to the cns and activated by pro-inflammatory cytokines produced by tmev-specific thl cells. the concept that prions m novel pathogens which are different fium both viroids and viruses has received increasing support from many avenues of investigation over the past decade. enriching fractions from syrian hamster (sha) brain for scrap= prion infectivity led to the discovery of the prion protein 0. prion diseases of animals include scrapie and mad cow disease; those of humans present as inherited, sporadic and w o r n neurodegenemive disorders. the inhecited human pion diseases m genetically linked to mutatim in the prp gene that result in non-conswative amino acid substitutions. transgenic v g ) mice expressing both sha and m o w @lo) prp genes were used to demonstrate that the "specie9 bank?' for -pie prions resides in the primary structure of pip. this concept was strengthened by the results of studies with mice expressing chimeric mdsha transgenes &om which "artificial" prions have been synthesized. similar chimeric mdhuman (hu) rp transgenes were constructed which differ from m o w by 9 amino acids between residues 96 and 167. au of the tg(mhu2m) mice developed neurologic drsease -200 days after inmulation with brain homogenates from three patients who died of creutzfeldt-jakob disease (cjd). inoculation of tg(mhu2m) mice with cjd prims produced mhu2mprpsc, inoculation with mo prions produced moprw. ihe patterns of meluzmprpc and mom% accumulation in the brains of tg(mhu2m) mice wen differenl about 10% of tg(huprp) mice expressing huf" and non-tg mice developed neurologic diseane >500 days after inoculation with cn, prions. the different susce@uies of tg(hw) and tg(mhu2m) mice to human prions indiate that additional species specific factors such as chaperone proteins are involved in prion replicaton. diagnosis, prevention and treament of human @on diseases should be faciliated by tg(mhu2m) mice. in other sindies, tg mice were compared expressing wt and mutant moprp. overexpression of the wtmoprp-a aansgene -8-fold was not deleterious to themiw but it did shorten scrapie incubation times from -145 d to -45 d after inoculation with murine m p i e pnons. in contrast, overexpression at the same level of l morp-a transgene mutated at codon 101 (corresponding to codon 102 in hurp) pmdnced spontaneous, fatal neurcdegeneration between 150 and 300 d of age in two lines of tg(mohp-pio1l) mice designated 2866 and 2247. genetic crosses of tg(moprp-p101l)2866 mice with gene targeted mice lacking both rp alleles ( p m -p ) produced anhats with a highly synchronous onset of illness between 150 and 160 days of age. the t g~o p r p -p l o l l ) 2 8 6~~ mice had numerous prp plaques and widespread spongiform degeneration in contrast 10 the tg2866 and 2247 mice that exhibited spongifonn degeneration but only a few prp amyloid plaques. another line of mice designated tg2862 overexpress the mutant transgene -32-fold and develop fatal neurodegeneration behveen 200 and 400 d of age. tg2862 mice exhibited the most severe spongiform degeneration and had numerous, large pip amyloid plaques. while mutant moprpccploll) clearly produces neurodegeneration, wtmoprpc profoundly modifies both the age of onset of illness md the mumpathology for a given level of transgene expression. our tidigs and those from other smdies suggest that mutant and wtprp interact, phaps through achaperone-like protein as noted above in sndies of tg(mhu2m) mice, to modify the pathogenesis of the dominantly inhe&ed prion diseases. anton, heidi t. link, and jonathan w. yewdell, laboratory of viral diseases, niaid, bethesda, md 20892-0440. cd8' lymphocytes (tcd8+) play an important role in host immunity to viruses and other intracellular parasites. virus-specific tcdi+ recognize mhc class i molecules in association with peptides of 8 to 10 residues derived from viral proteins. this presentation will focus on how and where antigenic peptides are generated by cells. to begin to characterize the nature of proteases involved in the generation of antigenic peptides from cytosolic proteins, we used a panel of recombinant vaccinia viruses expressing different forms of influenza virus nucleoprotein (np). we found that the efficiency of generation of two np peptides is related to the metabolic stability of the source gene product. there has been considerable speculation that such short lived proteins are degraded by proteasomes in a ubiquitin-targeted process. our observations, however, call into question the importance of ubiquitin targeted-proteolysis in generating antigenic peptides from exogenously provided or endogenously synthesized viral proteins. we also examined the extent to which antigenic peptides can be generated in the endoplasmic reticulum (er). we found that antigenic peptides could be produced from short precursors (17 residues) hut not from a number of full length proteins (influenza virus hemagglutinin, np, ovalbumin) that are targeted to the er by a nh2-terminal signal sequence. peptides were generated much more efficiently from the cooh-terminus of the 17 residue precursor than from the nh2-terminus. these findings indicate that the er has a much more limited capacity than the cytosol to generate antigenic peptides, but that er proteases (particularly aminopeptidases) could perform the final proteolytic steps in the generation of class i binding peptides from precursors imported from the cytosol by tap, the mhc encoded peptide transporter. potential advantages of synthetic peptide or engineered recombinant vaccines are that they can be limited to contain only the specific antigenic determinants for desired responses without other determinants that elicit unwanted responses, and that the sequences of the determinants themselves can be modified to enhance potency or breadth of crossreactivity. however, they can have the disadvantage that any single determinant may be presented by only a limited selection of major histocompatibility complex (mhc) molecules of the species. to overcome the problem of mhc polymorphism, we have identified determinants presented by multiple mhc molecules, and have also located multideterminant regions of the hiv-1 envelope protein that contain overlapping determinants each presented by different class i1 mhc molecules, so that the whole multideterminant region is presented by multiple mhc molecules of both mouse and human. we have made use of "cluster peptides" spanning these multideterminant regions of the hiv-1 envelope to provide help for neutralizing antibody (ab) and cd8+ cytotoxic t lymphocyte (ctl) responses to peptides attached to these helper regions. these synthetic peptide vaccine constructs containing the p18 peptide from the v3 loop of hiv-1 iiib or mn, elicited both neutralizing ab and ctl in multiple strains of mice. the cluster peptides inducing helper t cells were essential for elicitation of ab and ctl to the p18 segment of both iiib and mn strains of hiv-1 in mice of several mhc haplotypes. several adjuvants were compared for their ability to elicit both ctl and ab simultaneously, without one response inhibiting the other. a single formulation in incomplete freund's adjuvant (ifa) could elicit all 3 responses, neutralizing ab, ctl, and th1 helper cells. the ctl specific for the mn strain p18 peptide crossreacted with strains sc, sf2,2321, and cdc4. the peptides in f a also elicit high titers of antibodies in rabbits. boosting was found to enhance ctl responses as well as ab responses. these constructs are being prepared for a human immunotherapy trial. these vaccine constructs are potent and also avoid sites on gp160 that are known to elicit enhancing antibodies or autoimmune responses that might conmbute to disease pathogenesis. however, we can potentially improve on these by tinkering with the internal structure of the individual epitopes. we have found that replacing a negatively charged glutamic acid residue with an uncharged amino acid in one of the helper determinants makes it 10 to 100-fold more potent in binding to the class i1 mhc molecule and in eliciting murine helper t cells that still recognize the natural hiv-1 sequence. thus, such a modified peptide should be more potent as a vaccine, while retaining the ability to elicit t cells that will respond to hiv proteins that of course do not have the altered sequence. we are currently mapping the critical residues for presentation of one of these peptides by human hla-a2, with the intent of developing modified peptides that will be more potent as components of a human vaccine. thus, by leaming how these peptides bind to mhc molecules and tcell receptors, we can design internally modified determinants to construct more potent or more crossreactive second generation vaccines. we are testing these vaccine approaches in a mouse model in which mice can be protected against tumor cells expressing hiv proteins as would an hivinfected cell. dna vaccines, comprised of non-replicating plasmids encoding viral proteins, are capable of generating protective immunity in animal models of several viral diseases. in preclinical models of influenza infection, reduced viral shedding was observed in dna-vaccinated ferrets after challenge with the human clinical virus strain, a/georgia/93. cross-strain protection was conferred by dna encoding the major internal proteins (nucleoprotein, np, and matrix, m1) and the surface protein haemagglutinin (ha) from the antigenically-distinct previous virus strains, a/beijing/89 and a/hawaii/91. this protective efficacy was greater than that seen by immunization with the widely-used clinical vaccine composed of killed a/beijing/89 virus. thus, compared to a killed virus vaccine, protection seen with the dna vacane against a drifted virus strain was greater. we previously demonstrated that immunization of mice with np dna generated mhc class i-restricted cytotoxic t lymphocytes. mice likewise were protected from death and morbidity following cross-strain challenge'. ha dna vaccines generated neutralizing antibodies in mice, ferrets and primates, and provided protection in m i d and ferret models of influenza. in animal models of other viral diseases, immune responses and protection against viral challenge have been seen after immunization with dna encoding viral proteins. dna encoding hiv gp120 generated ctl and neutralizing antibodies in monkeys. antigen-specific proliferative responses and, in mice, secretion of high levels of yifn relative to levels of il-4, months after immunization were also observed . immunization of rabbits with dna encoding l1, the major viral capsid protein of cotton tail rabbit papilloma virus (crpv), resulted in neutralizing antibodies and protected against the development of warts after inoculation with crpv. mice immunized with dna encoding the glycoprotein gd from herpes simplex virus type 2 (hsv-2), developed neutralizing antibodies and were protected from death when subsequently challenged with hsv-2. dna vaccines were protective in animal models of various viral diseases. neutralizing antibodies, helper t cells (thl) and cytotoxic t cells were generated. cross-strain protection due to cellular immunity was demonstrated. ' science, 1993 2593745-1749 , 2dna cell biol, 1993 the profile of a neurovirulent virus is determined by its mechanism of entry into the cns (neuroinvasion), the type of cns cell in which it replicates (neurotropism) and its ability to cause pathologic effects in the brain (neurovimlence). whereas neuroectodermal cells, especially neurons, are the target cells of most neurovirulent viruses, the main target cell in the brain for siv and other lentiviruses is the macrophage. infection in, expression of viral antigens by and products of siv replication exported from these cells result in inflammation and degenerative changes in the brain and concomitant loss of neurons. siv strains that are mainly t-cell tropic cause transient activation of t-cells and during this period, infected t-cells cross the blood brain barrier and localize in the brain causing persistent hut minimally productive infection and minimal neuro pathologic effects. viral proteins but not virions are produced continuously. by virtue of the tropism of the virus for cd4 t cells, many infected animals eventually become immunosuppressed and develop aids, but not classical ueurological disease. viruses which are macrophage tropic invade the brain presumably also in t lymphocytes and the viruses infect macrophages in the brain. however, productive virus replication is minimized by antiviral cd8 t cells which suppress (kill?) all virus producing cells throughout the body, including the cns. productive virus replication in brain macrophages and accompanying inflammatory changes develop only when cd8 cells fail i.e. after profound immunosuppression sets in. the neurological disease that results from productive virus replication in macrophages in the brain therefore depends on presence of an appropriate macrophage-tropic viral phenotype invading the neuropil and development of immunosuppression in the host. the neurological disease could therefore be defined as one of the aids syndromes. the adenovirus (ad) early transcription region (e3) codes for more than 7 polypeptides, four of which have already been shown to alter the immune response to ad infection. the amount of the class i major histocompatibility complex (mhc) on the plasma membrane can be reduced by the binding of the ad e3 gpl9k protein to the mhc heavy chain, which prevents transport of the complex out of the endoplasmic reticulum. this process interferes with presentation of viral peptides to cytotoxic t lymphocytes. cytolysis by tumor necrosis factor-o (tnf) is inhibited by 4 distinct viral polypeptides, 3 of which (the ad e3 14.7k or the complex of the 10.4k and 14.5k proteins) are coded in the e3 region. the e3 polypeptides are translated from a family of viral mrnas, that are synthesized from a single viral promoter and processed by alternative splicing. we have studied the functions of the e3 polypeptides in several murine models. the goals of these experiments were to determine the effects of the ad e3 polypeptides in acute and persistent viral infections as well as in a transplantation model designed to measure whether these viral immunoregulatory proteins would abrogate allogeneic graft rejection. in a vaccinia virus (v.v.) pneumonia model, in which the isolated ad e3 14.7k or ad e3 gpl9k genes were inserted into the v.v. pathogen, the ad anti tnf polypeptide increased viral virulence but the ad anti mhc had no effects. in addition to manipulating the ad e3 genes in viral constructs, several transgenic mouse lines containing the ad e3 genes have been constructed for these experiments. the e3 genomic dna behind the rat insulin promoter (rip) has been used to generate transgenic animals. islets from rip-e3 transgenic animals (h-2b'd) have been transplanted allogeneically to h-2d recipients and remained viable, secreting insulin until the end of the experiment at 94 days; in contrast, control nontransgenic islets of the same genotype were rejected by 21-28 days. the e3 genes behind the native e3 promoter have been inserted into mouse embryos to generate transgenic animals, and the expression of the transgene monitored in multiple organs. the e3 promoter of the transgene is responsive to stimulation by the ad e1a following infection with an e3 minus ad 7001 and can also be upregulated by administration of bacterial lipopolysaccharide. the effects of this transgene on ad pathogenesis are currently being studied. thus, these viral immunoregulatory genes have been shown to alter viral pathogenicity during acute infection and to downregulate the host immune response sufficiently to permit islet cell transplantation. these results on manipulating the ad e3 genes for the control of the host immune response also have implications for designing adenovirus vectors for gene therapy. "emerging" infections can be defined as infectious diseases that either have newly appeared in the population, or that are rapidly increasing their incidence or expanding their geographic range. recent viral examples include aids, ebola, and hantavirus pulmonary syndrome (fnst identified in a 1993 outbreak in the southwestern u.s.). emerging viral infections show a number of common features. most "new" viruses derive from existing viruses that move into new areas or acquire new hosts ("viral traffic"). many are zoonotic (originating from animal sources) (even pandemic influenza appears usually to be a reassortant originating in wildfowl). ecological or environmental changes (either natural, or, often, man-made) may precipitate emergence of new diseases by placing people in contact with a previously unfamiliar zoonotic reservoir or by increasing the density of a mtud host or vector of a pathogen, increasing the chances of human exposure. upon introduction into a human population from a zoonotic reservoir, the newly introduced virus may cause localized outbreaks of disease. some may show rapid variation and evolution upon introduction, and some evidence suggests a role for immune selection in this process. a few viruses (such as hiv) may succeed in establishing themselves and disseminating in the human population, becoming truly "human" infections. human activities can also play an important role in establishment and dissemination. migrations from rural areas to cities, now an accelerating worldwide phenomenon, or other displacements, can introduce remote viruses to a larger population; the virus may then spread along highways and (globally) by air travel. the development of an effective system of surveillance and rapid response is essential, but resources for this are presently inadequate. vaccine development, production, and deployment problems also need to be addressed. immunopathology may be a key feature of many of these infections, a number of which manifest as hemorrhagic fevers. many of the life threatening complications are due to increased vascular permeability. the resemblances to septic shock suggest that cytokines (such as tnf) are likely to be important in the pathogenesis of these infections. the response of cells, such as the macrophage, that induce or synthesize key cytokines, may be an important element, and the ability to infect these cells may be one common denominator. why some viruses elicit this response, while other closely related viruses do not, cannot yet be predicted from molecular data. better understanding of these aspects of the immune response should lead to additional therapeutic strategies. (supported by nih grant roi rr03121.) genetic approaches have been used to detect and characterize numerous previously unidentified hantaviruses. puumalarrospect hilvsin nombre-like viruses or virus variants are present throughout north and south america, europe and russia. several of the american viruses identified are associated with the newly recognized hantavirus pulmonary syndrome (hps), a severe respiratory illness with high mortality. the genetic relationships of these and previously characterized hantaviruses have been studied by phylogenetic analysis of the nucleotide sequence differences located in pcr bgments amplified from the g2 encoding region of the virus m segments. the relationships observed are consistent with a long-term association of viruses with their primary rodent reservoirs and suggestive of coevolution of host and virus. a sin nombre virus isolate is now available and its genetic characterization has been completed. various virus antigens have been expressed and are being used to probe the interaction of the virus with the host immune system. hantaviruses cause significant morbidity and mortality throughout the world. more than 200,000 cases of hemorrhagic fever with renal syndrome (hfrs) are reported annually in asia, europe and scandinavia. the etiologic agents of hfrs are hantaan, seoul and puumala viruses, with hantaan virus causing the most severe form of the disease. in 1993, a new hantavirus was discovered in the united states (initially termed four comers virus), and was identified as the etiologic agent of hantavirus pulmonary syndrome (hi's). vaccines for hantaviruses are not readily available, although a number of inactivated viral preparations have been made and tested in asia. recurrent problems with inactivated hantaviral vaccines have been lot to lot variability, the need for repeated immunizations, and their inability to elicit long-lasting neutralizing antibody responses in immunized volunteers. because of such limitations on traditional vaccine development for these viruses, as well as the viruses' hazardous nature and slow, low-titer replication in cell culture, we used a recombinant dna approach to develop a vaccine for i-ifrs. our vaccine is a recombinant vaccinia virus expressing the m segment of hantaan virus under control of the vaccinia virus 7.5 k promoter and the s segment under control of the 11 k promoter. the m segment, which encodes the g1 and g2 envelope proteins, was included because of our findings that: (1) immunization with vaccinia or baculovirus-expressed g1 and g2 induced a neutralizing and protective immune response in hamsters; and, (2) neutralizing antibodies to g1 or g2 could passively protect hamsters from challenge with virulent virus. the s segment, which encodes the nucleocapsid protein (n), was included because of our finding that hamsters immunized with baculovirus-expressed n also were protected from subsequent infection. although the protective immune response to n is probably cell-mediated, the importance of such a response is presently not well defined. assessment of our vaccine in preclinical studies, indicated that immunized hamsters developed neutralizing antibodies and were protected from displaying viral antigen in their lungs after challenge. in a phase i, dose escalation, clinical study, the vaccine induced neutralizing antibodies in individuals immunized subcutaneously with approximately lo7 pfu of the recombinant virus. in addition to humoral responses, immunized volunteers developed a cell-mediated immune response as indicated in lymphocyte proliferation assays. larger clinical studies, including alternate routes or booster immunizations, are planned. based on these studies, we anticipate that the vaccine will be efficacious for preventing hfrs caused by hantaan and the antigenically closely related seoul virus. we are studying the cross-protective properties of this vaccine with more distantly related hantaviruses such as puumala virus. although we expect this vaccine to be safe as well as effective, we also are investigating the use of more attenuated pox-viruses as vaccine vectors. infection of mice with lymphocytic choriomenigitis virus (lcmv) results in a profound expansion in the number of spleen cd8 t cells and in the induction of virus-specific ctl activity. thereafter, the cd8 t cell number declines, and the ctl activity diminishes, though the frequency of lcmvspecific precursor ctl per cd8 cell, as assessed by limiting dilution assays (lda), is remarkably stable throughout long-term immunity. the decline in t cell and total spleen leukocyte number at the late stages of acute infection is associated with high levels of apoptosis, as detected by the in situ nucleotidyl transferase assay. apoptosis occurred in both the t cell and b cell populations, with the b cells dying in clusters. this apoptosis was also seen in tfansgenic mice ectopically expressing bcl-2 in the t and b cells and in c57bl/6 ipr/@r mice, which have a mutation in the fas gene. t cells from the infected animal underwent apoptosis in vitro when stimulated through the tcr with anti-cd3, thereby explaining some of the immunosuppression seen during acute viral infections. memory cells persisted for over a year and could be found in blast-size cell populations. challenge of lcmv-immune mice with either pichinde virus, vaccinia virus, or murine cytomegalovirus led to the reactivation of the lcmv-specific ctl response. lda analyses showed unexpectedly that these heterologous viruses crossreacted with subpopulations of lcmv-specific memory t cells. this memory t cell response to virus from an earlier infection was associated with enhanced immunopathology and enhanced clearance of virus during a heterologous virus challenge. over the course of the acute infection, ctl specific for the second virus were preferentially expanded over the crossreactive ctl, and after the acute infection, when the t cell response had subsided, ctl memory to the first infection had decreased. there is therefore a network of memory t cells which contribute to and are modulated by infections with putatively unrelated viruses, and apoptosis plays a homeostatic role in the course of these t cell responses. immune responses to live attenuated retroviral vaccines, r. paul johnson*?, cara wilsont, kelledy mansons, michael wyands, bruce walker?, ronald c. desrosiers* *new england regional primate research center, southborough, ma 01772 thfectious disease unit, massachusetts general hospital, boston, ma 021 14 â§tsi/mason, worcester, ma immunization of rhesus macaques with live attenuated retroviruses deleted in nef can induce protective immunity against challenge with pathogenic siv. development of protective immunity in these vaccinated animals occurs only after several months of infection, with maximal protection observed after one year. the specific immune responses responsible for mediating protection have not been defined, and little is known about the cellular immune responses in animals vaccinated with these live attenuated retroviruses. we have analyzed cellular and humoral immune responses in rhesus macaques and chimpanzees infected with live attenuated retroviruses. siv-specific neutralizing antibodies were present in vaccinated animals, but did not clearly correlate with protection against challenge. ctl specific for envelope and gag were identified in vaccinated macaques studied 12 or more months after vaccination. quantitation of siv-specific ctl activity in one of these animals using limiting dilution analysis revealed a relatively high precursor frequency of cytotoxic t lymphocytes, up to moo0 for gag and 1/8500 for envelope. cd8+ lymphocytes obtained from vaccinated macaques were also able to suppress siv replication in autologous cd4+ cells. suppression mediated by unstimulated cd8 autologous cells was maximal when cells were in direct contact with siv-infected lymphocytes, but cd8+ cells activated by an anti-cd3-specific monoclonal antibody were able to release. a potent soluble inhibitor of siv replication. in contrast to the relatively vigorous ctl response present in vaccinated macaques, we were not able to detect consistent ctl activity in chimpanzees infected with a hiv-1 molecular clone (nl43) or attenuated viruses at periods up to one year after infection, despite the use of a variety of stimulation techniques. proliferative responses to hiv p24 and gp160 were observed in chimpanzees infected with n u 3 and attenuated variants. although the relative contribution of these immune responses to protective immunity is not known, the relative vigor of the cellular immune responses observed in vaccinated macaques suggest they may play a role in mediating resistance to challenge. obiectives: to analyze the magnitude and specificity of the ctl response to hiv-1, and to determine the tcr usage by clonal ctl responses in infected persons, including persons with documented infection of up to 15 years with cd4 cells > 500/mm3. methods: hiv-l-specific ctl activity was evaluated in pbmc as well as in pbmc stimulated in vitro with hn-1 infected autologous cd4 cells, using target cells infected with recombinant vaccinia viruses expressing hn-1 proteins. ctl epitopes recognized by these individuals were determined using cloned effector cells. quantitative cultures were performed by endpoint dilution, and viral quantitation was determined by qc-pcr tcr analysis was performed by pcr, using both family-specific primers and anchored pcr, followed by sequencing. sequence analysis of ctl epitopes in autologous viruses was determined by pcr amplification and sequencing. clonal frequency was analyzed in pbmc by oligonucleotide probe to the cdr3 region of the tcr. studies performed in long-term non-progressing persons indicate the presence of a vigorous and broadly directed ctl response. detailed epitope mapping in a person infected for 15 years, who by qc-pcr had <i67 viral rna molecules per ml and who tested negative by bdna assay, revealed ctl clones directed at six different epitopes, with detectable recognition of three of these epitopes using freshly isolated pbmc as effector cells. the ctl clones were broadly reactive against many hown hiv-1 variants, and both the p17 and p24 epitopes were cross reactive with siv. sequence analysis of autologous virus within the three dominant ctl epitopes revealed the lack of immune escape variants in pbmc, plasma, and virus obtained from coculhue. supernatant. analysis of tcr usage by clones to defined epitopes in persons at different disease stages revealed heterogeneity in tcr usage and heterogeneity in recognition of virus variants withii these epitopes. analysis of clonal frequency using an oligonucleotide probe to the cdr3 region of the tcr in an individual with a vigorous response to gp41 suggested that approximately 6% of circulating t cells were a single hiv-l-specific clone. conclusions: our results indicate that cytotoxic t lymphocytes are part of the immune response in persistent non-progressive hiv-1 infection, and that prolonged infection can occur without the development of viral immune escape variation within defined ctl epitopes. in addition, vigorous circulating ctl responses can occur in the setting of an e x m e l y low viral load. heterogeneity in the ability of clones from different individuals to recognize sequence variation within ctl epitopes may be important in the pathogenesis of infection. ctl to conserved epitopes or ctl which are broadly reactive with multiple viral mains may be important in conmbuting to maintenance of the asymptomatic state. factor-i (irf-1) gene is essential for the transcriptional activation of inducible nitric oxide synthase (nos) in murine macrophages. it also plays an important role in the activation of interferon and il-6 and il-7 receptor genes. to investigate the role of irf-1 related systems of defense against viral myocarditis, the transgenic mice were innoculated with l o 5 pfu of coxsackie virus 83 (cvb3). homozygous irf-1 (+) and heterozygous irf-1 (+/-) mice were randomized into groups to determine heart and spleen viral titres, cardiac pathology and mortality rates at early and late stages of the disease. the mortality was dramatic in the irf-1 -imice. the hearts of the homozygous animals also had increased mononuclear cell infiltration, necrosis and viral replication. we condude that irf-1 regulated gene products such as inos and ifn are essential for the early defense against cvb3-induced myocarditis by attenuating viral replication and inflammatory responses. the flu season is a yeariy problem, especially in the elderly population. annual vaccination lacks broad protection and antibody titers induced in the elderly are frequently lower than those acheived by younger vaccinees. using the mouse model, we are investigating the differences in immune response between old and young mice to influenza vaccine and at the same time testing the ability of a potent adjuvant to boost the response in the the elderly. our studies show defects in the immune response of the elderly. antibody titers after immunization are lower in the elderly for both seronegative and seropositive mice. cmi studies show that helper t-cell response (proliferation) to in-vitro antigen stimulation is also decreased in the elderly. facs analysis of wbcs show the elderly mice have lower leukocyte and lymphocyte populations, lower cd4 and b-cell populations and a higher proportion of macrophages and cd8 cells than young mice. it was also noted that the young mice had very consistent wbc profiles while the elderly profiles had greater variability. serum cytokine studies show lower levels of il2, il4 and il5, but higher levels of il6 and ifng in the elderly as compared to the young mice. the addition of mf59 to the immunization increased the antibody titers of both naive and seropositive young and old mice. the helper t-cell response of the young mice was unaffected, while the response of the elderly group was increased. cytokine profiles show an increase in il2, il4 and il5 levels for both young and old, while il6 and ifng levels went up for young animals but remained constant for the elderly mice. coxsackievirus 8 3 (cvb3), a member of the picornavirus family, is a common cause of acute or chronic human myocarditis. however, whether cell damage results primarily from direct virusmediated effects or from immune-mediated destruction of the heart tissue during the infection remains unclear. we used a cardiovirulent variant of cvb3 which is able to infect several strains of mice. the infection results in a disease which strongly implicates immunopathogenetic mechanisms in myofiber necrosis. to characterize the role of the immune system in this disease we used various transgenic knockout (ko) mice with different immune defects. cvb3 is able to infect normal c57bl/6 mice and immunocompromised (cd4 ko and 62m ko) mice and causes a lethal disease after i.p. injection in a viral dose dependent manner. we found elevated ld5os in immunocompromised mice, which also die slightly later than normal mice. this virus replicates in the hearts of all mice used with maximal titers 3-5 days pi.. large pathological changes were detectable only in heart tissue of cd4 ko mice, and were maximal at 1-2 weeks after infection. these results suggest the possibility that cd4' t cells may limit tissue damage by an as yet undefined mechanism; the roles of cd4+ and cd8' t cells in this disease are under investigation. research supported by a grant of the daad, germany. mice lacking p56lck are resistant to coxsackievirus b3 induced heart disease. tamara martino, karen aitken, joseph penninger, jan0 nikhilanandhan, tak mak, fayez dawood, wen-hu wen, lily wee, martin petric, and peter liu, the toronto and princess margaret hospitals, university of toronto, canada. coxsackievirus 83 (cvb3) causes severe heart disease in adult mice, and is a useful model for the human cardiac conditions of myocarditis and dilated cardiomyopathy. in this study, we have demonstrated that transgenic mice lacking the t-cell signalling molecule p56kk are resistant to cvb3 induced heart disease. the virus replicates in the heart, and is cleared from the myocardium at a rate comparable to "normal" littermates, but there is only minimal mortality, or damage to the cardiac tissue. however, in ick-deficient mice depleted of natural killer (nk) cells prior to viral infection, there is increased cvb3 replication, and some infiltration by mononuclear cells in the myocardium. to further elucidate the role of the immune system in cvb3induced heart disease, cytokine expression in the hearts of cvb3 infected normal, ick-deficient, and nk-cell depleted mice is under investigation. we conclude that t-cell activation is critical to produce substantive myofiber necrosis after cvb3 infection, that nk-cells play an fundamental role in protecting the mice from severe virus infection, and that ick transgenic mice serve as an important model for understandina the dathogenic mechanisms involved. we now show that cd4+ t cells from 62m-/-mice exhibit antigen specific release of serine esterase and interferon-gamma. further, as quantitated by flow cytometry, the early decrease in cd4+ t cells seen in normal mice 3 days after lcmv infection, previously presumed to be due to the activity of cd8+ ctl, still occurs in 62m-/-mice. however, 621114mice show an earlier rise in percentage and absolute numbers of cd4+ t cells by 7 days after infection. cd4+ t cells from am-/mice rise to above baseline levels with kinetics paralleling the early rise in cd8+ t cells in normal mice after lcmv infection. cd4+ ciz activity is lower in 62m-/mice and have a later peak than cd8+ ctl from normal mice. these differences may explain the lower mortality and longer time course of immunopathologic meningitis in 621114-mice. mice can assume some of the cytotoxic functions of the cd8+ cell population from normal mice, including immune-mediated pathology. further, cd4+ t cells from 62m-/-mice have the capacity to release serine esterase, and show kinetics similar to cd8+ t cells from normal mice. mice genetically engineered to be deficient in 62-microglobulin in conclusion, these data suggest that cd4+ t cells from am-/australia. molecule is an essential component of the t cell help required for an antibody response. the interaction between cd40 and its ligand, in the presence of b cell acting cytokines, such as interleukin 4 (il-4), is sufficient to trigger b cells to proliferate and differentiate and to induce immunoglobulin class switching. a recombinant vaccinia virus encoding both factors, cd40l and il-4, did not, however, stimulate an enhanced antibody response in mice infected with the construct. instead, the antibody levels were lower than those of mice infected with a control virus. the reduced antibody response reflected the diminished growth of cd4ol-expressing viruses, to the extent that immunocompromised mice were able to resolve these infections. the kinetics of virus clearance indicate that the anti-viral mechanism of cwol is rapidly activated and has generalized tissue distribution. the cwolmediated clearance of virus is independent of the anti-viral cytokines, tnf and ifn-y. the anti-viral activity of c w l may represent a surprising and potent effector mechanism of t cells activated during a virus infection. mary's medical school, norfolk place, london w2 lpg, u. k. and *nationallnstirute formedica1 research, london, nw7 1aa. respiratory syncytial (rs) virus is the most important respiratory pathogen of infants, causing the majority of cases of bronchiolitis, it can be life threatening in very young infants and those with underlying cardiopulmonary disease or immunodeficiencies. t cell immunodeficiency can lead to prolonged infection in children and t cell transfers clear virus in the murine disease model. mice can be readily infected with rs virus and have proved a valuable model of its pulmonary pathology and the t cell response to it. il-3 has a broad spectrum of activities on proliferation and terminal differentiation of early blast cells and committed progenitors of many lineages. less is known about the effects of il-3 on t cell development and proliferation and its effect on the host response to infection by common pathogens. the il-3 gene was disrupted in 129/sv embryonic stem (es) cells by homologous recombination after transfection with omega-type constructs flanked with a herpes simplex thymidine kinase gene. these cells were used to produce a mouse strain deficient of il-3. mice were infected intranasally with a 2 strain rs virus; pathological changes in the lung were monitored by cytology of bronchial lavage, and virus persistence by nested rt-pcr of lavage fluid and lung homogenate. no differences were found in pathology or virus persistence between knockouts and normal mice. il-3 deficiency is therefore compatible with apparently normal responses to viral infection. recombinant adenoviruses are an atuactive vehicle for gene therapy to lung in the treatment of cystic fibrosis (cf). first generation viruses deleted of ela and elb transduce genes into airway epithelial cells in vivo, however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. in this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive nansfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. our studies indicate that mhc class i resmcted cd8+ cytotoxic t lymphocytes (ctls) are activated in response to viral antigens leading to destruction of virus infected cells and loss of transgene expression. mhc class ii associated presentation of viral antigens activates cd4+t helper (th) cells of the -1 subset and, to a lesser extent, of the tm subset. ldv establishes a life long viremic infection in mice which is not controlled by anti-ldv immune responses. anti-ldv antibodies are rapidly generated but they neutralize ldv infectivity only poorly. here we show that ldv-specific cytotoxic t cells (ctls)are generated within 7 days pi. c t l s were detected by h-2 specific lysis of ldv-specific target cells. these target cells were generated by transfection of 3t3-nm cells with a retrovirus vector carrying the gene (ow 7) for the ldv nucleocapsid (n) protein. spleen cells from ldv-infected mice were incubated in vitro with ldv-infected macrophages or transfected 3t3-nih cells and cultured for 5 days in the presence of il-2. the ctls had largely disappeared in mice by 30 days pi. the following experiments were conducted to further explore the mechanism of immune escape. in situ hybridization was used to localize ldvinfected cells in tissues of infected mice. these were found in persistently infected mice only in the liver. testis, and lymphoidal tissues. in addition large amounts of virion or virion debris accumulated in the germinal centers of the spleen and lymph nodes beginning about 3 days p.i. the accumulation of large amounts of ldv antigen in the germinal centers may be causally related to the suppression of the ctl response as well as to the polyclonal activation of b cells associated with ldv infection. to determine whether immune selection plays a role in ldv immune escape, ldv was reisolated from nude and immunocompetent balb/c mice at various times pi. the ows for the nand the two envelope glycoproteins were r t k r amplified and sequenced. the role of cytokines in initiating the immune response to venezuelan equine encephalitis (vee) was investigated. mice were infected in the left rear foot pad with either cloned virulent vee (v3000) or an isogenic single-site attenuated vee mutant (v3032) (single amino acid change at e2 glycoprotein position 209 from glu-lys) and at 1,6,24,48,72, and 96 hours post-infection, the popliteal lymph nodes and spleen were harvested and examined for tnf-a, il-12, ifn-y, il-2, il-4, il-6, and il-10 gene expression using a quantitative rt-pcr. in both strains elevations of tnf-a, il-12, ifn-y, il-10, and il-6 were detected in the lymph node, with no changes in either il-2 or il-4; however, whereas cytokine elevations were detected by 6 hours after injection of v3000, elevations were delayed until 24 hours following infection with v3032. cytokine mrna elevations in the spleen were less substantial, but elevated levels of ifn-y, il-10, and il-12 were also observed. these findings suggest that vee infection induces an early highly specific cytokine pattern associated with simultaneous elevations in both ifn-y and il-10 and that a single amino acid mutation can induce temporal changes in this pattern without alterring either the actual cytokines induced or the degree of their elevation. we are currently performing intervention experiments to investigate the effect of intravenous anti-il-12 andor anti-ifn-alp antibody administration on this early in vivo cytokine response to examine in particular whether these cytokines may be required for the observed elevations in either ifn-y or il-10. to confirm the importance of the immune system in clearing rotavirus infection we infected rag 2 knockout mice with murine rotaviruses. both knockout p u p s and adults developed chronic infections. to determine if a t cell independent antibody could be mediating the clearance of rotavirus infection in nude mice we measured igg, igm and iga in faeces and serum of rotavirus infected mice. a low and transient igm response was found in serum but not faeces of infected nude mice but not in age matched naive nude mice. the protein specificity of these antibodies is being determined. to determine if a primary infection in n u d e mice would induce protection (memory) against a secondary infection, we challenged six week old mice that had been infected as pups with the murine ew strain of rotavirus with the murine cambridge strain of rotavirus. the previously infected mice, as well as naive nude mice, both shed very low but equal levels of rotavirus in the stools. interestingly adult naive heterozygous littermates shed more virus than the nudes. the factors responsible for this relative resistance of adult nude mice to rotavirus infection as well as the mechanism for viral clearence in nude mice will be discussed. symp. 1990, 121:149-160) . the great majority of the poxvirus encoded proteins actively mediating the evasion of host defense are secretory proteins termed "virokines". viruses produce proteins which can either compete with or antagonizz molecules critically involved in generating immune responses. the vaccinia virus complement control protein (vcp) was the first virokine to be reported that has structural similarity to humadmouse complement control proteins, with the greatest similarity to the human complement 4b binding protein (bc4-bp) and the first protein to have a postulated role in the viral evasion of host defense (kotwal and moss, nature 1988, 355:176-179) . bioactive vcp, purified from serumfree medium has been shown to be more potent than the hc4bbp ( kotwal, am. biotech. lab., 1994) and has also been shown to bind to two important complement components, c3 and c4, and block the complement cascade at multiple sites in vitro (kotwal et al., science 1990, 2502327-830; mckenzie, kotwal et al., j. inf. dis. 1992 , 1661245-1250 . the importance of this protein was demonstrated in vivo using recombinant virus lacking an intact dna coding for vcp (isaacs, kotwal and moss, pnas 1992, 89:628-672) and further underscored by the smallpox virus genomic sequence analysis, indicating the presence of a highly conserved homolog of vcp ( massung et al., nature 1993,366:748-751) . in order to determine the precise effects of vcp at the site of infection, a mouse model has been developed. measurement of the specific swelling response and microscopic examination of the histological changes in balb/c and dbn2 mice has revealed that vcp is capable of regulating inflammatory response in vivo (jayaraman and kotwal, 1993 mol. immunol. 30:20) . in order to ensure that the possiblity of the unrelated mouse strains sharing identical h-2 haplotypes but differing at numerous other loci was not overlooked, we performed similar experiments in congenic strains. well characterized c5deficient mice injected with poxviruses in comparison to normal mice, produce a significant inflammatory response, accompanied with severe ulceration that persists for several days. the data suggests that the host complement response is a critical factor in egulating poxvirus pathogenesis. previous studies have suggested that immune response was one of the major factors that contributed to the host resistantlsusceptible mechanism to sendai virus infection (brownstein, 1981) . various immune regulatory molecules were investigated in inbred susceptible 129/j (129) mice and resistant c57bl/6j (b6) mice which share the same mhc haplotype. when they were given 200 eid, of sendai virus intranasally, 129 mice showed histologically diffuse interstitial pneumonia compared to the local moderate inflammation in 86 mice. virus was eliminated from the infected lung 3 days earlier in b6 mice than in 129 mice. the cytokine levels and cytokine-producing cells were investigated in the acute pneumonic lung. also the recall cytokine production in the draining mediastinal lymph node (mln) were studied. the maximal numbers of il-2, ifn-y, il-10, and il-4 producing cells were comparable for each strains of mice, while more il-6 producing cells were present in the lung of 129 mice. however, the numbers of these cytokine producing cells peaked in 129 mice 3 days later than in b6 mice. analysis of soluble cytokines in bronchoalveolar lavage fluid showed that at least two-fold higher levels of ifn-y, il-10, and il-6 were present in 129 mice than in 86 mice. recall cytokine production in the draining mln showed only the presence of il-2, ifn-y, il-10, and il-6, while no il-4 was detected. generally, higher levels of these cytokines were detected throughout the whole infection process in 129 mice. therefore, the susceptibility of 129 mice to sendai virus is not due to insufficient cytokine production in these two anatomical sites, yet heavier and prolonged virus burden in susceptible mice may drive the more rigorous and protracted induction of immune regulatory molecules. cytokines play a decisive role in determining the type of immune response elicited by an antigen, both in driving th cell differentiation along thl or th2 pathway and, ultimately, the effector and regulatory activity of these subsets. we have studied the role of cytokines in virus infections by constructing recombinant virus vectors encoding cytokine genes. during replication in mice, these vectors produce the encoded factor which is secreted from the infected cells with profound immunological consequences. cytokines studied todate fall into two categories; either altering viral pathogenesis or selectively stimulating specific immune responses. the expression of il-2, ifn-y, tnf-a or il-7 in recombinant vaccinia virus (rvv) dramatically alters pathogenicity, such that immunodeficient mice resolve the normally lethal infection. on the other hand, expression of il-4 by rvv suppresses the induction of cytotoxic t cells (ctl) inhibiting the clearance of virus which leads to an increase in viral pathogenicity. the expression of il-4 by rvv was shown to inhibit the host il-2 response required for the development of ctl's. no production was also significantly suppressed by il-4. rvvs expressing il-5 or il-6, although not affecting pathogenicity, preferentially stimulates iga reactivity to coexpressed antigens. encoding cytokines in recombinant viruses has clearly demonstrated the important role of cytokines as anti-viral effector molecules and in regulating appropriate immune responses. in mice infected with lymphocytic choriomeningitis virus (lcmv), interleukin-i2 (il-12) doses of >i00 ngld induce profound immunotoxicities characterized as almost complete inhibition of virus-induced cd8+ t cell expansion and ctl activation, and up to 2 log increases in viral replication [orange, wolf, and biron, j. immunol. 152:1253, 19941 . serum tumor necrosis factor (tnf) is also observed under these conditions. the studies reported here further characterize the expression and function of tnf in this context. northern blot and in sifu hybridization analyses demonstrated that il-12 induced tnf-cx expression and that lcmv infection synergized with il-12 for this induction. administration of antibodies neutralizing tnf reversed the il-12-induced immunotoxicities in lcmv-infected mice and restored anti-viral defenses. the tnf-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and cd8+ t cells isolated from lcmv-infected mice were more sensitive to tnfmediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. additional physiological changes were observed in il-12-treated uninfected mice and were dramatically elevated in il-12-treated virus-infected mice, including: 1) decreases in body weights; 2) elevation of circulating glucocorticoid levels: and 3) decreases in thymic mass. these changes were also reversed by anti-tnf. the results delineate a unique tnf-mediated immunotoxicity and have significant implications concerning detrimental consequences of in vivo tnf andlor il-12 for protective anti-viral responses. lactate dehydrogenase-elevating virus (ldv), a naturally occurring virus, causes a persistent infection in mice and presents an ideal model for the study of immune modulation during acute and persistent virus infections. within a few days following infection with ldv there is a pronounced polyclonal activation of b cells followed by the suppression of primary b cell responses to t-dependent ag. we investigated the effect of acute and persistent ldv infection on the development of a memory b cell response to the model protein antigen, horse cytochrome c (cyt), by employing a modification of the splenic fragment assay. about a 50% decrease in the frequency of responding agspecific memory b cells was observed in balb/c mice infected with ldv, whether the mice were immunized with cyt at the time of ldv infection or three weeks later. this may be due in part to a defect in t cell help, since in cultures of normal memory b cells and t cells derived from ldv acutelyinfected mice the frequency of responding b cells was also decreased two-fold. in situ hybridization using a cdna probe specific for ldv revealed two patterns of ldv rna within the spleen. twenty-four hr p.i. ldv rna was located within the marginal zone, surrounding each follicle. this pattern is consistent with permissive macrophages. during persistence viral rna could no longer be detected in the marginal zone, but was located within the follicles. the absence of ldv-permissive cells within the follicular region suggests that the source of ldv rna is not due to ongoing viral replication. one possibility is that circulating virus is trapped by a specific cell population within the follicle. the effect of virus trapping within the spleen provides a mechanism by which ldv and other viruses can modulate immune cell function during persistent infections. ifn-y can be produced by activated nk cells. this cytokine enhances immune responses by augmenting macrophage antigen presentation. viral infection induces ifn-dp and nk cell activation. changes in splenic architecture, cell trafficking, and cytokine expression were examined during viral infections of c57bu6 mice. at times coinciding with ifn-dp production and nk cell activation, there was a redistribution of nucleated cells from red pulp to white pulp regions in spleens isolated from mice infected with either lymphocytic choriomeningitis virus (lcmv) or murine cytomegalovirus (mcmv). cell transfer experiments with dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-or pkh26-gl-labeled bone marrow cells isolated from normal mice demonstrated an infection-induced accumulation of non-t/non-b cell populations along recipient splenic marginal zones. flow cytometric analyses demonstrated that approximately 10% of the transferred bone marrow cells accumulated in spleens after 20 hrs and 30% of these expressed the nk cell marker, nkl.l+. in vivo antibody treatment procedures, to eliminate cell subsets in donor mice, demonstrated that the cells localizing at the marginal zone were derived from agmi+ and n k l . l + populations. a small subpopulation of marginal zone cells in infected mice were shown to be expressing high levels of ifn-7 mrna by in sifu hybridization. treatment with anti-agmi or anti-nk1 .i antibodies eliminated both endogenous nk cells and the ifn-y mrna positive cells. these data demonstrate that newly derived nk cells accumulate along marginal zones. the results also suggest that this trafficking pattern may act to enhance immune responses by facilitating delivery of cytokines to specialized antigen presenting cells. david segal, janet ruby, alistair ramsay and ian ramshaw. depamnent of cell biology, john curtin school of medical research, po box 334, canberra, act, 2601 australia cytokine expression has been shown to correlate with protective or ineffective immune responses in a number of disease models. recently there has been the suggestion that immunity to some retroviruses is associated with the production of ceaain patterns of cytokines. to explore this further we have have used rauscher murine leukemia virus (r-mulv) infection of c57bu6 (resistant) and balb/c (susceptible) mice to elucidate the role of cytokines immunity to retroviruses. initially the in viho proliferation of spleen and lymph node cells from infected mice was examined. in response to stimulation with immohilised anti-cd3 antibodies the proliferation of spleen hut not lymph node cells from infected mice. was found to he rapidly suppressed. suceptible balb/c mice exhibited a much greater suppression than resistant c57bu6 mice. the cause of this suppression is under investigation however, the immunosuppressive molecules nitric oxide and prostaglandins are not involved. in vitro cytokine production by spleen and lymph node cells from r-mulv infected mice was determined. in response to stimulation with immobilised anti-cd3 antibodies, spleen cells from infected balb/c mice produced diminishing amounts of ifn-7 and il-2. in contrast spleen cells from infected c57bu6 mice produced ifn-y and l-2 to levels that were only slightly less than uninfected controls. a-6 production by spleen cells from infected mice of both strains was at levels higher uninfected controls. anti-cd3 stimulated lymph node cells from infected mice produced elevated ifn-1 suggesting that suppressed cytokine production is spleen specific. expression of cytokine genes in vivo is currently being investigated using rt-pcr to detect cytokine mrna in the spleens of infected mice. we have previously shown that primary resting murine b lymphocytes are non-permissive for vesicular stomatitis virus (vsv), however, a productive infection can be induced when infected b cells are activated with anti-immunoglobulin (a-lg) plus il-4 or lipopolysaccharide (lps). we posit vsv in unactivated primary b cells provides a paradigm of persistently infected lymphocytes and activation dependent recall of an active infection. analysis of the behavior of virus in unstimulated b cells during long term culture and the requirements for subsequent induction of productive infection has been limited by the poor survival of primary cells in culture. we circumvented this limitation by using highly purified small b cells from mice transgenic for the bcl-2 proto-oncogene, expression markedly extends in vitro survival of unstimulated primary b cells. overexpression of bcl-2 does not alter b cell infection or induction of a productive infection by activators during acute infection. infection does not effect b cell survival in culture. unstimulated virus infected b cells produce primary viral mrnas but not viral proteins or infectious particles (pfu) during culture. persistently infected b cells stimulated with a-lg plus il-4 produced a fully productive vsv infection at all times analyzed, up to 3 weeks post infection. in contrast, vsv production in persistently infected b cells activated with lps markedly declined relative to acutely infected activated cells (50-1 00 fold by week 1 and 1,000 fold by week 2). cells were not completely refractory to lps activation as vsv protein was produced. the selective lps deficiency is unique to persistently infected cells as uninfected cultured b cells proliferate and differentiate to produce antibody upon lps activation. these data show that a persistent infection may selectively alter the host cell response to previously productive activators which may as a consequence interfere with immune regulation. rsv-g glycoprotein specific t cells preferentially secrete il-5 and predispose to pulmonary eosinophillia., anon srikiatkhachorn. and thomas j. braciale, the beirne b. carter center for immunology research and the departments of microbiology, pathology, and pediatrics, university of virginia health sciences center, charlottesville, va 22908 we studied the immune responses to two different glycoproteins of respiratory syncytial virus (rsv) in a murine model. balb/c mice were immunized with recombinant vaccinia virus expressing either rsv-fusion glycoprotein ( vac-f), attachment glycoprotein (vac-g) or 8-galactosidase (as a control). these mice were given rsv intranasally three weeks after priming and then sacrificed 5 or 14 days later. spleens and bronchial lymph nodes were harvested for in vitro culture and lungs were harvested for histologic studies . we found that bulk cultures obtained from both vac-f and vac-g immunized animals secreted both thl and th2 type cytokines when stimulated with rsv infected spleen cells . however, the levels of 11-5 and 1fn-y were higher in bulk cultures derived from vac-g primed animals while the levels of il-2 were higher in the bulk culture from vac-f primed animals. the il-4 and il-5 production was relatively short lived since spleen cells and bronchial lymph node cells obtaind form mice sacrificed 14 days after intranasal inoculation produced much lower levels of il-4 and 11-5 while the levels of il-2 and ifn-y production were comparable to bulk cultures obtained from mice at the peak of infection. there was little inflammatory response in the lungs obtained from mice immunized with the control vaccinia. in contrast , lungs from mice immunized with vac-f or vac-g showed significant infiltration of inflammatory cells. there was a striking infiltration of eosinophils in the lungs from mice primed with vac-g. these eosinophils could be detected aroud major bronchi and blood vessels, as well as, in some cases, in lung parenchyma. this study suggests that the immune responses to different viral glycoproteins may be distinct and may play important roles in viral pathogenesis. during infection of normal mice with lymphocytic choriomeningitis virus (lcmv), nk cell responses peak on day 3 and subside as cd8+ t cell responses are activated at day 7 post-infection. in contrast, 02m-/-mice, lacking cd8+ t cells, have dramatically elevated nk cell responses on day 7 postinfection. the 02m-/-response is evidenced by increased nk cell activity, as well as up to 5-fold increases in blast and total nki.i+cd3-cell numbers. nk cell responses in normal mice are cyclosporin a (csa)-resistant and interleukin (il)-2independent, whereas day 7 nk cell responses in 02m-/-mice are csa-sensitive and il-2-dependent. to investigate the role of additional cytokines in regulating cellular responses during acute viral infections, production and function of il-4 and transforming growth factor4 (tgf-0) were examined. induction of il-4 mrna, at late times post-infection of normal mice, was shown by in situ hybridization of t cell-enriched splenic leukocytes and polymerase chain reaction (pcr) amplification of cdna from rna. ellsas of media cor.aitioned with cells isolated on days 0, 3, 5, 7, 9, and 14 post-infection demonstrated delayed induction of il-4 protein as compared to ctl activation. tgf-0, evaluated in biological and elisa assays, was induced maximally at days 7 to 9 post-infection. the kinetics of tgf-0 production by cells from infected 82m-/mice was similar to that of normal mice. however, cells from 02m-/-mice produced il-4 at early but not at late times postinfection. together, these results suggest that either il-4 is a critical cytokine for shutting off nk cells during normal responses to viral infection, or that the 02m-l context modulates responsiveness of nk cell subsets to other late cytokines. studies are in progress to distinguish between these two possible mechanisms. the induction of fever in response to infection is an important host defense mechanism that enhances aspects of the immune response and restricts the replication of some microorganism. vaccinia virus, a member of the poxvirus family, is a complex cytoplasmic dna virus that encodes a variety of proteins that interfere with host immune functions, such as complement regulatory factors and soluble receptors for il-lp, tnf and ifny. here we show that expression of the vaccinia virus il-1p receptor (vil-lpr) in the w r strain prevents the febrile response and reduces the severity of infection in intranasally inoculated mice. fever was recorded on days 1-6 after infection of mice with a vil-lpr deletion mutant, but not in animals infected with wild type wr or a virus revertant. these studies were extended to other virus strains that were used as smallpox vaccines, and expression of the vil-lpr was consistently found to prevent the onset of fever. vaccinia virus induced a severe hypothermia after 6 days in infected mice that was independent on vil-lpr expression and correlated with virus replication in the brain, the organ that controls body temperature. these results represent the first example of a virus mechanism to inhibit the host febrile response and suggest a central role for soluble il-lp in the induction of fever in poxvirus infections. measles virus (mv) infection can depress cell-mediated immune responses for months following clinical disease. mv is known to infect the thymus during human illness and this may contribute to immune suppression. we have used the scid-hu m o w with co-implants of human fetal thymus and liver to determine the effect of virulent and avirulent strains of mv on the thymus. scid-hu mice were. infected by direct inoculation of the graft with 103 pfu of either a wild type strain of mv(chicago-1,chi-1) or an attenu-ated strain (moraten, mor) and sacrificed at intervals over 28 days. peak viral titers, as judged by plaque assay on vero cells, were reached by chi-1 on d4 (105.7 pfu/ third of implant), and moron d21 (103.2 pfu/ third of implant). hematoxylideosin stained sections of chi-1-infected thymuses showed marked distortion of the cortex and medulla by d4 with thymocyte poilolosis and decreased cellularity. by d14, these. implants were mostly devoid of normal thymocytes. mor-infected thymuses showed relatively preserved architecture and cellularity. suspensions of the cells from implants stained with mabs to cd3,cd4 and cd8 were analyzed by flow cytomehy. there were significant decreases in the cd4+cd8+ cell pop-ulation by d10 with complete loss of all such cells by d28 with chi-1, and only modest reductions with mor. immune fluorescence staining of sections with a mv mab to hemagluttinin(ha) and abs for either human cytokera-tins(ael/ae3) or cd15 co-localized mv predominantly to epithelial and monocytic cells. additionally, mv antigen was present diffusely by d4 in both cortex and medulla in chi-i infection whereas mor-infected implants had only patchy distribution by d21. only rare cells stained both with mv ha and cd2 or cd4. mv ha was not expressed over background on any cd4+ cells judged by facs. we conclude that mv replicates in the scid-hu thymic implant primarily in epithelial and monocytic cells, and that the attenuated virus reproduces more slowly and with less cellular disruption. little mv ha could be demonstrated in thymocytes, therefore the data suggest that significant infection of the thymic epithelial stroma disrupts the thymic microenvironment which normally supports and aids in selection of immature t cells. part of the long-term immune suppression seen in mv infection may be due to infection of the thymic epithelial stroma with subsequent loss of thymocytes. it is becoming increasingly evident that many poxviruses contain genes that enable the virus to evade the host's immune system. myxoma virus is a leporipoxvirus and is the causative agent of myxomatosis, a rapidly lethal disease in the european rabbit (oryctolagus cuniculus). one possible mechanism of immune evasion is virus-induced downregulation of cell-surface receptors important for an immune response. cell-surface levels of several receptors on a rabbit t cell lymphoma cell line (rl-5) were monitored by flow cytometry. following infection with myxoma virus, cellsurface levels of cd4 were found to drop dramatically. other cell surface antigens such as cd18, cd43, and cd45 were unaffected during infection with myxoma virus. further more, the downregulation of cd4 by myxoma virus could be inhibited by treating cells for an extended period of time with pma, suggesting that the downregulation was not simply a masking of the epitope via viral antigens. analysis of cd4 levels in the presence of cytosine arabinoside indicates that late gene expression is not necessary for the modulation. since the tyrosine specific protein kinase p56lck associates with the cytoplasmic domain of cd4 we have also examined the association of p56lck with cd4 as well as steady state levels of p56lck during viral infection. the modulation of surface cd4 has also been described in hiv infected t cells suggesting that the loss of cell-surface cd4 may be a common viral immune evasion tactic by lymphotrophic viruses. i n addition, stably-transfected cell l i n e s expressing e i t h e r u s 1 1 o r us2-6 gene products s i g n i f i c a n t l y reduced l e v e l s of mhc class i heavy chain. studies are i n progress t o f u r t h e r d e f i n e t h e mechanism by which t h e s e v i r a l gene products a l t e r immune recognition. cytotoxic t lymphocytes (ctl) may play a significant role in containing the spread of hiv in infected individuals. although hiv-infection is associated with immune suppression, a vigorous ctl response has been detected in infected adults. hiv can be transmitted from mother to child. one third of vertically infected children has a rapid evolution toward disease, with onset of aids before 18 months. the other two thirds remain asymptomatic for years. the bimodal course of disease evolution in hiv-infected children could be related to differences in the host immune control of viral replication. hiv-specific ctl response from fresh and in vitro activated pbmc of hiv-infected children was measured. the vast majority of infected chidren had detectable hiv-specific ctl, which where cds+cd8+. we previously showed that among children with a slow disease progression, fresh ctl were more frequent in the p2a(paucisymptomatic) group than in the pl(asymptomatic) and the p2b-f groups (symptomatic group). the cohort of children has now been followed during 4 years, and 46 children have been tested at least once. we found that ctl responses were less frequent in the children with a rapid disease progression than in the children with a slow disease progression at the same age. our data suggest that ctl response is an important factor in delaying disease evolution. we, as well as others. have proposed that sag function is critical to the ability of milk-borne m m n to infect mice. to determine whether this is the case, we created transgenic mice (hyb pro/cla) with a frameshift mutation int the sag gene. young hyb pro/cla mice (c 10 weeks of age) showed no deletion of their cognate vp14* t cells, unlike transgenic mice carrying a functional sag gene however, a slow, progressive loss was seen in the hyb prolcla mice as they aged, indicating that it was due to expression of wild type sag protein. thus, as the hyb pro/cla mice aged, there was production of virus that appeared to lose the cla mutation. the hyb pro/cla mice produced transgene rna in their lactating mammary gland and shed virus in their milk. their nontransgenic offspring of showed infection with transgene-encoded mmtv because they had the typical slow deletion of vp14+ t cells characteristic of c3h mmtv infection and because we detected transgene-derived m m n rna in their mammary glands. cloning and sequencing of the viral rna produced by the nontransgenic offspring of the hyb pro/cla mice showed that recombination between the mtv-1 endogenous viral rna and the transgene-encoded rna occurred, such that the frameshift introduced by the cia mutation was repaired. these results show that there is selection of infectious virus that contains a functional sag gene. thus, it appears that the only virus that is capable of being transmitted by the milk borne infection pathway is that which encodes a functional sag protein. hepatitis b virus (hbv) causes acute and chronic liver diseases and is closely associated with hepatocellular carcinoma. in order to understand the cellular immune response against hbv in chronic hbv infection, t cell proliferation, cytotoxicity and cytokine production were studied. we found that although the majority of asymptomatic hbsag carriers and patients of chronic hepatitis b (chb) had no proliferative response to hbsag, some individuals in both groups showed significant t cell proliferation against hbsag. in contrast, the proliferative t cell response to hbcag in asyrnpatomatic hbsag carriers was significantly stronger than that in patients of chb with acute exacerbation. in addition, the frequency of hbcag-reactive t cell precursors measured by limiting dilution assay was much higher in asymptomatic hbsag carriers than in patients of chb. therefore, t cell responses against hbsag and hbcag are regulated differently in chronic hbv infection. furthermore, we demonstrated hbsag-and hbcag-specific cytotoxic t lymphocyte (ctl) activity in asymptomatic hbsag carriers, using autologous hbsag-and hbcag-expressing lymphoblastoid cell lines (lcl) as target cells, respectively. the cloned ctl were able to produce ifn-y, tnf-a or gm-csf after stimulation. these findings demonstrate that t cell response to hbv is not completely suppressed in asymptomatic hbsag carriers. most of them have strong hbcag-specific response and some of them have hbsag-specific response. transcription and tax the human t-lymphotropic virus type i (htlv-i) promoter contains the structural features of a typical rna polymerase i1 (pol 11) template. the promoter contains a tata box 30 bp upstream of the transcription initiation site, binding sites for several pol i1 transcription factors, and long poly a+ rna is synthesized from the integrated htlv-i proviral dna in vivo. consistent with these characteristics, htlv-i transcription activity was reconstituted in v i m using tbp, tfiia, rtfiib, rtfiie, rtfiif, tfiih and pol 11. in hela whole cell extracts, however, the htlv-i ltr also contains an overlapping transcription unit (otu). htlv-i otu transcription is initiated at the same nucleotide site as the rna isolated from the htlv-i-infected cell line, mt-2, but was not inhibited by the presence of a-amanitin at concentrations which inhibited the adenovirus major late pol i1 promoter (6 pglml). htlv-i transcription was inhibited when higher concentrations of a-amanitin were used (60 pglml), in the range of a typical polymerase in (pol 111) promoter (va-i). purified tax, transactivates this promoter 5-to 10-fold in v i m . interestingly, basal and tax,-transactivated transcriptional activity of the htlv-i ltr could be reconstituted with the 0.5 m phosphocellulose fraction. these observations suggest that the htlv-i ltr contains overlapping tax,responsive promoters, a typical pol i1 promoter and a unique pol i11 promoter which requires a distinct set of transcription factors. tax, further in vifro transactivates a polymerase i1 template containing the 21 base pair repeats cloned upstream of the ovalbumin promoter and g-free cassette. tax,-transactivated transcription was concentration dependent and inhibited by low concentrations of a-amanitin. flaviviruses are arthropod-borne viruses whose route of infection is via the skin. they are mostly neurotropic and responsible for significant human morbidity and mortality. the classic cell-mediated immune response to a viral infection may be influenced by the ability of these viruses to modify expression of cell-surface molecules involved in the presentation of antigen to, and activation of, t cells. the skin langerhans cell is the prototypic nonlymphoid dendritic cell and as such is uniquely placed to participate in a response against epidermally-acquired viral infections. the migratory properties of these cells contribute to their role as initiators of t cell-mediated immune responses within the draining lymph node. we have previously shown infection of epidermal cells in vifro by the flavivirus west nile (wnv) results in an increase in mhc class i and i1 expression on the majority of epidermal cells and langerhans cells respectively. in this study a technique for infecting the epidermis with wnv in vivo was developed. tme-dependent increases in the surface expression of a number of antigens which are involved either directly or in a co-stimulatory capacity in initiating a cell-mediated immune response, were detected on both the majority of epidermal cells and the langerhans cell population using flow cytometry. these increases were detectable as early as 16 hours after infection. a significant decrease in the percentage of langerhans cells remaining in the epidermis was observed within 48 hours of infection. the phenotypic changes observed in vivo are analogous to those described following in vifro culture of langerhans cells. these results, together with the reduction in langerhans cell numbers, may represent the in situ maturation and concomitant migration of these. cells as a consequence of virus-induced cytokines within the skin microenvironment. which cause a wide variety of illnesses with high morbidity and mortality in humans throughout the world. their high genomic stability argues for a survival strategy related more to interaction with the vertebrate host immune response, than a dependence on viral genetic mutation. our previous work has shown that west nile virus (wnv) infection of many cell types directly induces functional increases in class i and 11 mhc expression. we report here that wnv infection of human embryonic fibroblasts (hef) results in the increased expression of cd54 by two distinct mechanisms. an early, direct cytokine-independent mechanism operates within 2 h of virus infection, while an indirect mechanism, regulated by type 1 interferon (ifn), operates within 24 h of virus infection. cd54 expression increased by 4-5 fold within 2h of wnv infection on hef, and by 6-7-fold within 24h. wnv-inactivated, conditioned supematants removed from infected hef cultures after 4 h incubation did not alter cd54 expression on unqimulated hef. whereas conditioned supernatants from 24 h-infected cultures increased cd54 expression by about 1.5-2-fold after incubation for 24 h, but not after 4 h, similar to cd54 induction by 200ulml of ifn-p. increased cd54 expression on hef by wnv was also cell-cycle dependent. cd54 increased only in quiescent, contact-inhibited infected hef in go phase. in contrast, induction of cd54 by types 1 and 2 ifn was not cell-cycle dependent. other viruses, including double-stranded dna viruses, vaccinia, and adenovirus 2 and 5, and the single, positive-stranded rna alphavirus, semiliki forest virus, did not induce cd54 expression on hef after 24 h. another alphavirus, ross river, was able to induce cd54 but only by the indirect mechanism of type 1 ifn-dependent release. poly i.c, also, increased cd54 expression to the same extent as ifn-p after 24 h, making it unlikely that the early increase was due to a nonspecific viral effect. the closely related flavivirus, kunjin, induced increased cd54 expression in a manner similar to wnv. the ability of flavivhses to induce increased cd54 expression directly within a few hours of infection may be an important virus-host survival strategy promoting cell-cell adhesion and hence possible further viral infectiodreplication. recognition of viral peptides presented on the cell surface in association with class i mhc molecules leads to lysis by cytotoxic t cells (ctl) and forms an important part of the immune response to hiv infection. hiv virus has a high mutation rate and variation in the region of the viral epitope may allow evasion of this immune response. variation could theoretically affect processing of the antigen, binding of the epitope to the hla molecule or recognition of the presented epitope on the cell surface. we have studied proviral sequence variation in gag and ctl responses in a number of hla b8 patients infected with hiv. amino acid substitutions, such as a lysine to arginine change at position 3 of the pi7 gag nonamer cckkkyklk, lead to loss of recognition of the peptide by ctl from the patient whose provirus contained this sequence. these variant peptides bind to hla 68 with comparable affinity to the index peptide suggesting that this loss of recognition is likely to be caused by changes in the interaction between the hla-peptide complex and the t cell receptor. other changes, such as lysine to arginine or glutamine at position 7, not only cause loss of recognition, but also lead to inhibition of lysis of targets bearing the index peptide. thus it appears that in addition to loss of recognition by cytotoxic t cells, naturally occurring epitope variants may act as "antagonists", as has been demonstrated in mhc class ii systems. antagonism may be an important mechanism allowing immune escape by the hiv virus. genes. subsequent complex formation between peptide, class i and p2microglobulin in the er results in stable cell surface expression of the trimeric mhc-1 molecule. in previous studies we showed that in hpv-16 positive cervical carcinomas there was a loss of mhc-1 protein expression, which correlated at the single cell level with loss of tap protein. in this study we investigated whether loss of tap and mhc-1 is mediated by an hpv-16 encoded protein. human keratinocytes were transfected withvarious hpv-16 constructs including pat16, the full length genome, pat16esx the full length genome with a premature stop codon in e5, puc.et16, the e6 and e7 oncogenes only, and pkve5, expressing e5 from mouse moloney ltr the different constructs were transfected into primary keratinocytes, cloned cells grown in medium supplemented with and without y-interferon ( y -a r ) for 48 hours. cells were harvested and total rna and protein harvested for northern and western blots respectively. western blots showed very low steady state levels of tap-1 and mhc-1 heavy chains in the cells with pat16 as well as those containing es alone, which was marginally increased by y-lfn. in contrast, primary keratinocytes, pat16esx and puc.et16 lines showed comparable tap-1 and mhc-1 protein levels, which increased a & y-ifn treatment. northem blots showed no differences in the amounts of tap-1 and mhc-1 mrna between the different cell lines. the data indicate that expression ofhf'v-16 e5 leads to post-transcriptional loss of mhc-1, presumably by interfering with tap. to map and characterize functional differences between e1a of ad5 and adl2, we previously constructed a series of hybrid ad5/12 e1a genes and used them with ad12 e1b to transform primary hooded lister rat kidney cells. at least two regions within the first exon of ad12 e1a were identified which influenced tumorigenicity. this study further examines the role of these regions in tumorigenicity by analyzing their affect on cell surface mhc class i expression and sensitivity to class i-restricted cd8+ as well as to non-class irestricted nks. the bcrfl open reading frame of epstein-barr virus exhibits remarkable sequence homology with the coding sequences of interleukin-10 from a variety of organisms. many of the numerous immunological properties ascribed to interleukin-10 are shared by the product of bcrfl and this has led to it being termed viral interleukin-10. in order to investigate the activity of viral interleukin-i0 (vil-10) and its interactions with the human interleukin-10 receptor we have expressed the protein in a bacterial and the eukaryotic cos-7 expression systems. the bacterially expressed vil-i0 was partially purified and used to set up two assays to measure i l l o activity: i)the increase in igm secretion from an ebv transformed b cell line -mt4.l and ii)the downregulation of class ii hla expression on the human monocytic cell line thp-1. a series of deletion mutants (both n-and c-terminal as well as an internal deletion to remove a putative heparin binding domain) were constructed to identify possible domains within the vil-10 protein that interact with the hil-10 receptor and confer its biological activity. a number of these mutants have been expressed in the cos-7 expression system and their structure and biological activity are currently being assessed. the identification of the domains within vil-10 that interact with the receptor or accessory proteins may aid in the understanding of the possible role of vil-i0 within the ebv life cycle and in the pathogenesis of the numerous diseases associated with the virus. generation. to further test the role of ctl in ad pathogenesis, viruses lacking the cll epitopes were tested when mutants that lack the immunodominate ctl epitope in eia where used, a second immun-ssive epitope in elb becorns the predominate target of clu. these findings arc important since human ad is currently being tested as a vector for gene therapy of cystic fibrosis. our data suggest that when consuucting ad vectors to be. used for gene therapy, one must retain either the 10.4k or 14.7k genes to decrease pathology and that meting the genes that encode the antigens that a n recognized by clu does not prevent the generation of ad specific clu. the interferons (ifns) a n ? a family of cytokines whose functions include the protection of cells against viral infection. type i ifns include the 15 ifna subtypes and ifnp that compete for binding to the same cell surface receptor, while type ii ifn (ifny) binds to a different receptor. the orthopoxviruses, of which vaccinia virus (vv) is the prototypic member, have developed a number of anti-ifn strategies. the vv e3l protein competitively binds dsrna and prevents the activation of ifninduced and dsrna-activated protein kinase (pkr), while the vv k3l protein shows sequence similarity to the eukaryotic initiation factor 2a (eif2a) that is phosphorylated and inactivated by pkr. the k3l protein competitively binds the kinase and blocks host eif2a phosphorylation and hence ifn-induced inhibition of host protein synthesis. onhopoxviruses also suppress cytokine action by expressing soluble cytokine receptors that bind and sequester the ligand; to date soluble receptors for interleukin-18, tumour necrosis factor and ifny have been described. supernatants from vv-infected cells were found to contain a soluble inhibitor of type i ifn that was conserved in most of the orthopoxviruses tested. the inhibitor was produced early in infection and did not inhibit ifny. the ifna/p inhibitor was mapped and the gene expressed from recombinant baculovirus. the inhibitor blocked the binding of 125i-ifna to u937 cells and binding of 125i-ifna to supernatants from baculovirus and vv-infected cells demonstrated that the inhibitor functioned as a soluble receptor for 1fnc1fp. direct binding of 1251-ifna to vv wr supernatants revealed that the soluble ifna/p receptor had a high affinity for type i ifn. deletion of the gene from the vv genome and ligand blotting of the soluble receptor demonstrated that ifn binding was encoded by a single protein. competitive binding curves using ifna from other species revealed that the poxvirus soluble ifndp receptor bound human and bovine ifn with high affinity but murine ifn with relatively low affmity. interestingly, the soluble ifncrip receptor is highly conserved in variola virus. given the importance of ifn in antiviral defense it is likely that the soluble ifndp receptor plays an important role in the virulence of the orthopoxviruses. endogenous processing of a viral glycoprotein for presentation t o cd4+ t cells has defined a previously under-investigated pathway in antigen processing and presentation. it may be important not only for pathogens, but also for self-proteins, and thus may be involved in self-tolerance. we have been characterizing the processing o f the er-restricted gpt glycoprotein of vesicular stomatitis virus (vsv) biochemically and enzymatically, by cellular localization using confocal immunofluorescence, cellular fractionation, and by t cell recognition assays. by flow cytometry, gpt is undetected on the plasma membrane; in contrast, the wild type protein (g) is readily found following infection of a20 cells with a vaccinia virus vector, leading t o endogenous synthesis. the gpt can be found exclusively in the er compartment using co-localization with markers for er (signal peptide binding protein, calnexin), and not in the golgi compartment (a-mannosidase 11, wheat germ agglutinin), endosome, lysosome, or surface plasma membrane. this is consistent with the characteristics o f the localization of the proteases which appear to be responsible for its degradation. work is in progress to localize the site of peptide binding to mhc heterodimers. supported by nih grant a118083 t o csr. presentation of an out-of-frame class i restricted epitope. t.n.j.bullock and l.c.eisenlohr, department of immunology, thomas jefferson university, philadelphia, pa 19107. antigen presentation by class i mhc molecules is thought to require the degradation of fully formed proteins in the cytosol. this degradative process supplies oligopeptide epitopes for transport into the endoplasmic reticulum (er) where they can interact with and stabilize class i molecules. stable class i molecules, associated with p2-microglobulin, can then proceed to the cell surface where they present the epitopes to t cell receptors. the generally accepted model for protein translation, the scanning hypothesis proposed by ko&, is thought to describe the traditional method of translation for the majority of proteins. we wished to test the hypothesis that any internal methionine that is in good translation initiation context can be a source of short peptides, which may then be processed into class i epitopes. nucleoprotein gene (np), the target of the ctl response of several inbred mouse strains. np contains three class i restricted epitopes at amino acids 50-57 (h2-kk), 147-155 (h2-kd) and 366-374 (h2-db). the frameshift was introduced 26 amino acids upstream of the h2-kd epitope. the mutated genes were then recombined with vaccinia virus and tested for presentation using ctl restricted to each of the epito s described above. we found that, whilst presentation of the h2-i@ epitope was unaffected by the frame shift, the epitope proximal to the frameshift (h2-kd) was no longer presented to appro riately restricted ctl. however, presentation of the distal h2-dg epitope was retained. therefore we have shown, using a viral protein and a viral expression system, that out-of-frame epitopes can be processed and presented to ctl. work is ongoing to c o n f m that internal methionines are capable of providing a platform for the initiation of translation for in-frame and out-of-frame epitopes. we have created a frameshift mutation in the influenza pr8 the fine specificity of t cell recognition of peptide analogues of the influenza nucleoprotein epitope np 383-391 srywairtr was studied using hla b27-restricted influenza-specific cytotoxic t cell (ctl) clones, of defined t cell receptor (tcr) usage, derived from unrelated individuals following natural infection. synthetic analogue peptides were synthesized containing single amino acid substitutions, and tested both for binding to hla b'2705 in vitro, and for presentation to ctl clones by hla 827positive targets. even conservative amino acid substitutions of the peptide residues p 4 , 7, and 8 profoundly influenced ctl recognition, without affecting binding to hla 8'2705. these amino acid side chains are thus probably directly contacted by the tcr. ctl clones which used the tcr v a l 4 gene segment (but not those using tcr va12) were also sensitive to p1 substitutions, suggesting that the tcr alpha chain of these clones lies over the n terminus of bound peptide, and that the "footprint" of certain tcrs can span all exposed residues of a peptide bound to mhc class 1. these results, taken together with previous structural and functional data, suggest that, for nonarner peptides bound to hla 827, p i , p4 and p8 are "flag" residues with tcr accessible side chains. the e3/19k protein of human adenovirus type 2 (ad2) is a resident transmembrane glycoprotein of the endoplasmic reticulum. its capacity to associate with class i histocompatibility (mhc) antigens abrogates cell surface expression and the antigen presentation function of mhc antigens. at present, it is unclear exactly which structure of the e3/19k protein mediates binding to mhc molecules. apart from a stretch of approximately 20 conserved amino acids in front of the transmembrane segment, e3/19k molecules from different adenovirus subgroups (b and c) share little homology. remarkably, the majority of cysteines is conserved. in this report, we examined the importance of cysteine residues (cys) for structure and function of the ad2 e3/19k protein. we show that e3/19k contains intramolecular disulfide bonds. by using sitedirected rnutagenesis, individual cysteines were substituted by serines and alanines, and mutant proteins were stably expressed in 293 cells. based on the differential binding of monoclonal antibody tw1.3 and cyanogen bromide cleavage experiments, a structural model of e3/19k is proposed, in which cys 11 and cys 28 as well as cys 22 and cys 83 are linked by disulfide bonds. both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human mhc antigens. this was demonstrated by three criteria: loss of e3/19k coprecipitation, lack of transport inhibition and normal cell surface expression of mhc molecules in cells expressing mutant e3/19k molecules. mutation of the three other cysteines at position 101, 109 and 122 had no effect. this indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the e3/19k molecule, namely, to bind and to inhibit transport of mhc antigens. previous studies have suggested that several abundant cmv proteins are major immunogenic targets in seropositive adults. we are interested in defining the major viral protein targets of a cd8' ctl response, in order to derive a vaccine strategy for individuals who are unable to mount immune responses which are lymphokinedependent because of immunosuppression. hla-typed and cmv-pgsitive normal volunteers who have hla-a alleles that represent -75% of the u.s. population are being tested to determine which of 5 abundant cmv proteins they recognize by a cd8' ctl response: p28, p65, p150, ie, and gb. t cell lines will be derived in order to unambiguously determine the hla restriction of the cd8' ctl response to each of these proteins. proteins which are recognized by the most hla diverse population will be further characterized in terms of mapping of class 1 epitopes through the use of t cell clones derived from the polyclonal cell lines by limiting dilution. the defined epitopes will form the basis of a vaccine strategy to augment the memory responses of seropositive volunteers against cmv. these epitopes will be used to boost the ctl precursor frequency of bone marrow transplant donors as a means to transfer cellular immunity to immunosuppressed hematologic transplant recipients. an alternative strategy is to immunize seropositive individuals with recombinant viral proteins as a means to boost immunologic memory. we are pursuing that strategy in a transgenic murine model of hla-a2.1 developed by dr. l. sherman (scripps institute, la jolla). we are vaccinating the transgenic mice with two well defined cmv proteins, p65 and gb together with either of two lipid-based adjuvants, commercially available d0tapm (bcehringer-mannheim) or mf5gth (chiron, emeryville, ca). our preliminary studies with hsv-2 gb demonstrate that both adjuvants are effective at eliciting murine class i restricted responses against the protein. current studies are evaluating the recognition properties of the adjuvant-cmv protein complexes by hwa2 as a restriction element in the transgenic model. the ctl response to sendai virus in c57by6 mice is directed almost exclusively to a single h-2kb-restricted epitope derived from the virus nucleoprotein, npj24-332 (sev-9). analysis of 18 independent t cell hybridomas generated from c57by6 mice following primary sendai virus infection has shown that a very diverse repertoire of tcr is selected in response to this epitope. crystallographic analysis of sev-9 bound to kb has shown that the side chaiis of peptide residues phpi, gl484, a d s , and alaps protrude towards the solvent and are potentially available for recognition by the tcr notably, residues gi484 and a d 5 protrude prominently from the peptide binding site due to their l o c a l i o n on a bulge in the center of sev-9. to determine the importance of each of these residues for t cell recognition, we analyzed hybridoma responses to sev-9 analogs substituted at each of these four positions. preliminary data showed there generally appeared to be dominant recognition of glyp4 and asnm. however, individual hybridomas exhibited distinct patterns of fine specificity for residues phep1 and alaps. thus, individual hybridomas were dependent on one, both, or neither of these residues for recognition of sev-9. these data are consistent with a critical role for the gi94 and a d 5 in governing tcr-sev-9eb recognition and suggest a structural basis for the diversity of the tcr repertoire selected by this @tope. previous results from this laboratoty demonstrated that the dominant influenza a epitope recognized by hla42.1 restricted ctl from hla-a2.1 uansgenic mice was the m1 peptide epitope that is immunodominant in human ctl responses. however, analysis of a large number of ctl lines revealed a subset of influenza a/pr/8/34-specific murine ctl that recognized an hla-a2.1 restricted epitope distinct from m1. using recombinant vaccinia viruses encoding werent influenza gene segments, the epitope recognized by these ctl was shown to be derived from the a/pr/8 nsl protein. because these ctl did not recognize targets infected with the a/alaska/6/77 saain of influenza, candidate peptide epitopes were synthesized based on sequences that included an hla-a2.1 specific binding motif and that differed between a/pw and nalaska all of these ctl recognized a nonamer and a decamer peptide which contained a common 8 amino acid sequence and two distinct sets of bmding mtif residues. however, the n0name.r peptide was able to sensitize ctl for half maximal lysis at 80-2500 fold lower doses than either the octamer or decamer. the homologous peptide derived from nalaska nsl contained conservative amino acid changes at positions 4 and 8 and was not recognized at any tested concentration, although it bound with higher &ity to hla-a2.1 than the peptide from a/pw8. the a/pr/8 nsl nonamer epitope was also recognized by human influenza a specific ctl derived from two individuals. these results substantiate the general utility of hla class i aansgenic mice for the identification of human cn epitopes for other pathogens. furthemore, the recombinant dhfr was functional in the induction of gb epitope-specific ctl response upon immunization of c57bv6 mice. these results indicate that an viral epitope expressed in a cellular protein can be. efficiently processed, presented and recognized by epitope-specific ctl, and suggest that the cellular proteins can be used to express ctl epitopes for induction of cd8+ immune responses. virus-specific cytotoxic t lymphocytes (ctl.) were generated a day later at this site. to determine which apc was capable of stimulating virusspecific ctl precursors in the mln, b, t and dendritic cells from the mln of influenza-infkcted mice were separated and examined for the presence of virus. the predominant cell type which contained infectious virus was the dendritic cell. b and t cells from the mln contained little, ifany, virus. the apc capacity ofthese populations was tested by their ability to stimulate vir~~-~pecific t cell hybridomas. only dendritic cells from the mln of influenza-infected mice were able to stimulate virusspecific t cell hybridomas, althwgh all apc populations from both naive and influenza-infected mice were effective stimulators after in y h pulsing with the appropriate intluenza peptide. potential apc populations were also separated from the lung. v i s was detected in bronchioalveolar macrophages and dendritic cells but not b or t cells. both macrophages and dendritic cells isolated from intlum-infected lungs could stimulate virus-specific t cell hybridomas. the ability of the mln and lung apc populations to stimulate naive cd8' t cells and generate virus-specific ctl is currently being examined. virus infected cells present only a very limited number of peptides intracellularly processed from a viral protein to ctl even when many peptides hearing the mhc class i-restricted binding motif are present in the protein. infection of h-2b mice w i t h lymphqtic choriomeningitis virus (lcmv) induces a cd8+ ctl response directed against three wellcharacterized epitopes presented by h-2db molecules: "396-404 (fqpq-ngqfi), gp33-43 (kavynfatcgi) and gp276-286 (sgven-pggycl). the h-2db motif is characterized by a sequence of 9 to 11 a.a. with two anchor residues: asn at position 5 and hydrophobic (met, ile, leu) at the c-terminus. the lcmv np and gp proteins contain thirly-one other peptides exhibiting the db motif. however, no ctl response against one (or more) of these peptides has been characterized. peptide binding to mhc is a critical step in antigen presentation. the aim of this study was therefore to analyze the binding properties of the potential db lcmv peptides. the 34 lcmv peptides and 11 known db-selective peptides were synthesized and their mhc binding affinities measured in two db-specific binding assays. most of the lcmv peptides (28/34) did not bind to db. the other 6 (including the 3 epitopes) and all the known db peptides showed good affinity. comparison of the sequences (good vs. non binders) allowed the identification of auxilliary anchors required for high binding affinity or of negative elements hampering mhc binding. in addition to the main anchors, the positive and negative factors at secondary residues play a crucial role in governing peptidemhc interactions. knowledge of such factors might he of importance for the prediction of mhcrestricted ctl epitopes. etienne joly, andrea gonzalez, carol clarkson, jonathan c. howard and geoffrey w. butcher. laboratory of immunogenetics, department of immunology, the babraham institute, cambs cb2 4at, uk. tap transporters from rats can be divided into two allelic groups, depending on their capacity to provide the rt1.aa molecule with an appropriate level of suitable peptidesl. recent results suggest that this might correlate with the rt1.aa molecule requiring arginine-ended peptides (powis et al., manuscript submitted), which the tapb allele of the transporter is unable to translocate across the er membrane efficiently2~3. rt1.a alleles are naturally linked with the tapa or the tapb allelic group4. we have set out to characterise various alleles for the rt1.a molecule, and find that, for the majority of tapaassociated rt1.a molecules, 3 acidic residues line the c/e pocket, dictating arginine as c-terminal anchor residue for the bound peptides. on the other hand, in tapb-associated rt1.a molecules, one acidic residue at the most is found in the c/e pocket, which certainly results in a different anchor residue for the bound peptides. the selective pressure of viral infections must have driven this coevolution which affects dramatically the array of peptides presented to cytotoxic t lymphocytes. cytotoxic t lymphocyte responses in hiv infection can be impaired due to variation in the epitope regions of viral proteins such as gag. we show here an analysis of variant epitope peptides in three gag epitopes presented by hla b8. seventeen variant peptides were examined for their binding to hla b8; all but one bind at concentrations comparable to known epitopes. all except two could be seen by ctl clones grown from hla b8 positive hiv-1 infected patients and were therefore immunogenic. however, in one haemophiliac patient studied in detail, there was a failure to respond to some of the peptides that represented virus present as provirus in his peripheral blood. in one case his ctl had previously responded to the peptide. thus there was a selective failure of the ctlresponse to variant epitopes. this impaired reaction to new variants and failure to maintain responses to some epitopes late in hiv infection could contribute to the loss of immune control of the infection. pira, anna ferraris, daniele saverino, peifang sun and annalisa kunkl; dept. immunology, san martino hosp. univ. of genoa, 16132 genoa, italy. th epitopes present on viral proteins can be recognized by specific th cells if appropriately expressed by antigen presenting cells (apc) as a result of uptake and processing. since viral epitopes are not simply present in the context of viral proteins, but also in the context of whole viral particles, it is important to determine the role of the molecular and/or structural context on antigen uptake-processing-presentation. therefore we have generated panels of cd4+ human t cell lines and clones specific for different hiv antigens (gp120, p66, p24), in order to test their ability to respond to the same epitopes present within synthetic peptides, recombinant proteins or inactivated virions (provided by g. lewis, dept. microbiology, univ. maryland, baltimore). we could identify t cell lines and clones that were able to discriminate the molecular and structural context of the epitops. certain t cells, in fact, responded to peptides and proteins, but not to viral particles, whereas other t cells were also able to proliferate when challanged in vitro with autologous apc and viral particles. the data suggest that in the human th cell repertoire specific for viral antigens t cells exist that can discriminate the molecularstructural context of th epitopes. it will be interesting to ascertain whether t cells specific for epitopes that can only be recognized when provided in the context of a soluble molecule, but not of a viral particle, have any relevance in viva protection, or are a simple by-product of the cellular immune response. eric g. pamer, merceditas s. villanueva, section of infectious diseases, yale university school of medicine, new haven, ct 06520 listeria monocytogenes is a gram positive bacterium that infects macrophages and secretes proteins into host cell cytosol. the murein hydrolase p60 is secreted by l. monocytogenes and is required for complete bacterial septation. in the infected macrophage secreted p60 is processed by the host cell into the nonamer peptide p60 217-225 and is presented to cytotoxic t lymphocytes by the h-2kd mhc class i molecule. we have used strains of l. monocytogenes that secrete different amounts of p60 to show that the rate of p60 217-225 production is proportional to the amount of antigen secreted into the host cell cytosol. p60 is degraded in the host cell cytosol with a half life of 90 minutes. the appearance of p60 217-225 is coupled to the degradation of newly synthesized p60. we have determined the rate of intracellular p60 secretion and by accounting for the rate of p60 degradation we estimate that approximately 35 p60 molecules are degraded to produce one p60 217-225 epitope. this ratio is maintained over a range of intracellular antigen concentrations. our findings provide an estimate of the efficiency of antigen processing and demonstrate the remarkable capacity of the mhc class i antigen processing pathway to accommodate new epitopes. we have isolated and characterized three cytotoxic t lymphocyte (ctl) clones from the peripheral blood of two acute seroconversion patients and one patient in the first trimester of pregnancy. these clones were cd8+ and class i hlarestricted by the b7 molecule. all three clones recognized lllb and rf but not mn strains of hiv-1. using vaccinia vectors expressing truncated versions of the hiv-1 envelope, the clones were found to recognize an epitope within amino acids 287-364, but not including 312-328 of gp120. further mapping of the epitope with synthetic 20-mer peptides overlapping by 10, or 25-mers overlapping by 8, was unsuccessful. the sequence of the region of gp120 recognized by these clones was compared to the predicted hla-87 peptide binding motif and a possible matching region was found. using shorter peptides corresponding to this potential epitope recognition site, the minimum epitope recognized by the clones was determined to be the 10 aa sequence rpnnntrksi spanning amino acids we have further pursued a strategy to define a minimal cytotoxic epitope for a vaccine against cmv infection using t cell clones derived from individuals who have the mhc 835 gene (kind gifts of drs. riddell and greenberg, fred hutchinson cancer research center and dr. robert siliciano, johns hopkins university medical center). we tested by chromium release assay (cra) the recognition of a series of 835 allelic variants of ebv-lcl. by 835 restricted and cmv or hiv-specific t cell clones. several conclusions quickly became apparent. the previously described 8'3501 peptide epitope from pp65 was not able to prime the autologous 835 ebv-lcl for killing by the pp65-specific ctl, whereas a recombinant vaccinia virus expressing whole pp65 could cause the same cell line to be recognized and killed in the same experiment. in addition, an hiv gp41-specific cd8' ctl which has a defined minimal cytotoxic epitope will only recognize and kill a subset of 835 ebv-lcl. the two t cell clones will not recognize each other's autologous ebv-lcl. the resolution of this interesting phenomena comes from sequence analysis of the hla class i b genes from both ebv-lcl. ebv-lcl which contain the b'3502 allele are recognized and killed by the pp65-specific t cell clone, and cell lines carrying 8'3501 alleles are recognized by the hiv gp41-t cell clone. we conclude that the reported cmv pp65 b"3501 restricted epitope is not correct, since the ctl in question will only recognize 6'3502 alleles in combination with the correct pp65 epitope. fragments with or without a signal sequence sensitize rma-s/kd to a similar limited extent. this data i s consistent with an inefficient movement of peptides from the cytoplasm into the er by a tap independent mechanism and does not reveal a processing competent compartment within the secretory pathway. peptide transport by the transporter associated with antigen processing (tap) was studied using a microsome system as previously reported by heemels et. al.. in this system, a radiolabeled synthetic peptide which can be n-link glycosylated is used as the indicator peptide for the transport studies. the transport efficiency of synthetic peptides corresponding to antigenic peptides restricted to the murine kd molecule was measured by inhibition of labeled peptide transported into the microsomes. the transport efficiency of three kd epitopes in the type a influenza virus "147-155, ha204-212 and ha210-219 was found to be similar. an 11 amino acid peptide corresponding to ha204-214 which contains the 204-212 epitope was transported at a similar efficiency as the 9 amino acid minimum epitope. however, when the peptide sequence is further extended by one amino acid to residue 215, this peptide is poorly transported. these results suggest that the flanking region of an epitope can dramatically influence the transport of the epitope. when the transport kinetics of tap was studied using the microsome system, the vmax for transporting the indicator peptide (a variant of np epitope that has the sequence tynrtrali) was found at 260.8 fmolelminute (+/-30.5). the km for this peptide was found to be 231.9nm(+/-31.8). bypassing a block in antigen processing for class i-restricted cytotoxic t cell recognition. amy j. yellen-shaw and laurence c. eisenlohr. thomas jeferson universitv. hiladelphia, pa., 19107. previous work from our laboratory showed that processing of an influenza nucleoprotein (np) epitope (amino acids 147-155) expressed endogenously from a recombinant vaccinia virus "minigene" is severely impaired when a flanking sequence (the dipeptide threonine-glycine) is appended to the cterminus of the construct (147-158/r-). the inhibition of processing is overcome by placing the unprocessed peptide in the context of the fulllength np molecule, demonstrating that regions of a protein outside the epitope itself critically affect the ability of the proteolytic machinery to fragment the protein appropriately. to determine the requirements for bypassing the block in antigen processing, we have constructed an array of "minigene"-expressing vaccinia recombinants in which the unprocessed epitope is extended by varying lengths toward either the c-terminus or the n-terminus of the np molecule. our results show that while an extension of the c-terminus by only one amino acid restores processability, a much longer extension of the n-terminus (75 < n < 100 amino acids) will also allow the substrate to be processed. it is therefore clear that a full-length, properly folded molecule is not required for liberation of the blocked epitope, and that probably more than one mechanism can contribute to enhancement of substrate proteolysis. we hypothesize that the c-terminal extension allows recruitment of an endopeptidase versus exopeptidase ("trimming") activity which is capable of cleaving the difficult bond. we considered the possibility that the n-terminal extension rescues processing by recruitment of the ubiquitin-dependent degradation system. to address this possibility we replaced all available ubiquitination sites (lysine residues) in one of the rescued constructs (50-158/r-) to see if the construct would still be processed and presented. the six available lysine residues were changed to arginine using pcr-based mutagenesis. the resulting construct (termed 6r) was recombined into vaccinia virus and tested for presentation to np-specific ctl. the 6r construct was presented at a level equivalent to that seen with the wild-type 50-158/rconstruct. this result provides clear evidence that entry into the ubiquitindependent degradation pathway is not responsible for rescue of presentation in this system and more importantly, that ubiquitination is not required for processing of all large substrates. chia-chi ku, li-jung chien ,and chwan-chuen king, institute of epidemiology, national taiwan university, taipei, taiwan, r.o.c. dengue virus (den) can cause dengue fever (df) and dengue hemorrhagic fever (dhf) i dengue shock syndrome (dss) and den-2 was the most common serotype found in dhf outbreaks globally. current hypotheses suggested that dhf may be associated either with antibodydependent enhancement (ade) or with viral virulence. den can replicate predominantly in monocytedmacrophages (mim), but whether peripheral blood lymhocytes (pbls) are the target cells of den still remain controversial. in order to compare whether various clinically derived den-2 will interact with mim and lymphocytes in different manners, we used two isolates --plo46 strain (obtained from a df patient during taiwan 1981 outbreaks) and 16681 strain (isolated from a dhf patient in thailand by cdc, usa) to infect primary mim and lymphocytes as well as several types of cell lines. primary lymphocyte culture was nonadherent cells obtained after 24 hr adherence of pbmcs, whereas the primary mim culture was collected by depletion of lymphocytes using anti-cd3icdi9 mab and complement prior to adherence procedure and the purity of mim culture was checked by cd14 surface marker staining. supernatants (sn) of virus were harvested at various time points post infection after with several or without treatments. our prelimanary data showed that dhf-associated den-2 strain had higher viral yield in certain age of mim and a promonocytic cell line (hl-cz) than taiwan df-associated den2 strain. in addition, this dhf-den2 strain was more likely to infect the promonocytic (hl-cz) than well differentiated monocytic (ctv-1) and lymphocytic (h9) cell lines and also had higher peak yields than den-i virus in hl-cz cells. interestingly, dhf-den2 strain replicated much more efficiently in primary lymphocytes no matter these cells were activated with pha or not, whereas taiwan df-den2 strain virus was hardly detectable in sn of both activated and non-activated lymphocyte cultures. therefore we conclude that (1) different strains of dengue virus could orchestrate quite differently with immune cells, (2) different stage of mim differentiation might be an important permissive determinants for dengue virus infection and replication, and (3) den virus strain virulence -a more important factor than lymphocyte activation status -seemed to determine whether this strain would infect human pbls. further studies should be focused on searching for detaied mechanisms of virus and immune cell interactions. (2) when viral yields were enhanced early than day5 post infection, it provided tremendous opportunity to attack the immune system and finally may lead to severe disease. hiv-1 using recombinant immunoglobulin molecules, marie-claire gauduin, graham p. allaway, paul j. maddon, carlos f. barbas, dennis r. burton, and richard a. university school of medicine, new york. ny 10016. primary isolates of hiv-1 have been shown to be less sensitive to neutralization by immune sera, monoclonal antibodies and cd4-based molecules than t cell line-adapted strains of hiv-i. we studied two immunoglobulin molecules for ability to neutralize primary isolates of hiv-i. lgg12 is an immunoglobulin molecule created from a combinatorial phage expression library and reacts with the cd4 binding site (cd4-bs) on gp120. cd4-lgg2 is a recombinant molecule in which the variable domains of both heavy and light chains of lgg2 were replaced with the first and second immunoglobulin-like domains of human cd4. both molecules have been previously shown to effectively neutralize hiv-i in vitro. ex vivo neutralizations were performed as follows: lgg12 and cd4-lgg2 were added at 25 pg/ml to wells containing serial dilutions of plasma from hiv-i-infected patients and phastimulated peripheral blood mononuclear cells from seronegative donors. p24 production was measured over 14 days of culture and an end-point titer of hiv-1 in the presence and absence of added antibody was determined. both igg12 and cd4-lgg2 were found to reduce the original hiv titer from seven plasma samples with high virus titer (>250 tcid50/ml) by up to 625-fold. this is in comparison to soluble cd4 which only reduced viral infectivity by 55-fold at the same concentration. in vitro binding and neutralization assays on isolates recovered from plasma confirm the potency and breadth of neutralization by these two molecules. these studies suggest that recombinant antibodies directed at the cd4-bs of hiv-1 gp120 are able to effectively neutralize primary isolates of hiv-1 and may be useful in dissecting the mechanisms of resistance to neutralization by other antibodies. dillner and p. heino, microbiology & tumor biology center, karolinska institute, stockholm, sweden hpv 16 the major cause of anogenital precancers in man. the search for neutralizing epitopes that could form the basis for a preventive vaccine has shown that the surface-exposed imunodominant epitopes of the capsid are strongly conformationdependent, which has precluded detailed epitope analysis. similarly, immunization with whole, denatured capsid proteins has only identified linear immunodominant epitopes positioned on the inside of the capsid. reasoning that linear surface-exposed epitopes should exist, but might be cryptic, a set of 66 overlapping synthetic peptides corresponding to the entire hpv16 capsid proteins was used to generate hyperimmune sera. several antisera against 3 different peptides were reactive with intact hpv16 capsids at titers up to 1:150.000. hiv-1 serum antibodies and mucosal iga. basil golding, john inman, paul beining, jody manischewitz, robert blackburn and hana golding. div. of hematology and viral products, cber, fda, and lab. of immunology, niaid, bethesda md 20892. previously, we showed that hiv-1 proteins conjugated to 8. abortus (ba) could generate anti-hiv-1 neutralizing antibodies in mice even after depletion of cd4* t cells. in this study a 14-mer peptide from the v3 loop of hiv-1 (mn) was synthesized 013) and coupled to ba and klh. balb/c mice were immunized twice i.p. with these conjugates at two week intervals. v3-klh induced mainly igg1, whereas v3-ba induced all igg isotypes but lgg2a predominated. fecal extracts from mice immunized with v3-ba were shown by elsa to contain iga antibodies. sera from these mice bound gp120, expressed on the surface of infected cells. sera from mice immunized with v3-ba inhibited syncytia formed between cd4' t cells and chronically infected [hiv-i (mn)] h9 cells. inhibition of syncytia, formed by other hiv-1 lab. strains correlated with the degree of their homology with the v3 region of hiv-i (mn). to mimic the efffect of hiv-1, mice were depleted of cd4' cells using anti-l3t4 at the time of primary or secondary immunization. following primary immunization, cd4+ t cell depletion abrogated v3-klh antibody responses, whereas responses to v3-ba were retained and sera from these mice were able to inhibit gp-120mediated syncytia. in secondary responses, cd4' t cell-depletion prevented boosting to v3-klh, but v3-ba increased anti43 and syncytia-inhibiting antibodies. these results suggest that: 1. 8. abortus, can provide carrier function for a peptide and induce both serum and mucosal antibody responses, and 2. that infection with hiv-1 with subsequent impairment of cd4' t cell function would not abrogate anti-hiv-1 antibody responses if 8. abortus is used as a carrier to stimulate memory responses. nucleotide sequence analysis of the vh genes revealed the usage of one particular vh germline element (vh61-1p) in all clones. this finding allowed the determination of somatically mutated positions in the vh regions. two vsv-ind neutralizing antibodies expressed vh and vl genes in complete germline configuration whereas the rest of the clones showed somatic mutations which obviously were antigen dependently selected for. however, binding affinities of mutated and unmutated antibodies were comparably high. in order to determine the influence of somatic point mutations on one single antibody we generated a monovalent single chain antibody (fv-ck) of a mutated clone and reversed it stepwise to germline configuration by means of site directed mutagenesis. surprisingly, already the germline configuration of fv-ck could neutralize vsv-ind, even though the binding affinty was lower than that of the mutated fv-ck. every single somatic point mutation tested improved the binding avidity although some mutations reduced affinity. thus, during the course of vsv-ind infection some antibodies are subjected to avidity maturation although this is not required for the generation of high affme, efficiently virus neutralizing antibodies, lisa hyland'", sam hou'.~, and peter c. doherty'. 'department of immunology, st. jude children's research hospital, memphis, tn 38 10 i, 2departments of immunology and microbiology, and 'pathology, university of otago,dunedin, new zealand. the b and t cell responses in c57bl/6j(b6) mice treated with the mab mel-14 to l-selectin have been analysed following i.n. infection with sendai virus. mel-14 treatment caused a 70-90% decrease in the lymphocyte recruitment to the mediastinal (h4ln) and cervical (cln) lymph nodes following infection with sendai virus. the cellularity of the spleen was unchanged. the clonal expansion of cd8+ ctl precursors in the mln was slightly delayed, but potent ctl effectors were present in the virusinfected lung by day 10 after infection and the overall magnitude of the response was not compromised. the prevalence of iga antibody forming cells (afcs) was greatly increased in both the mln and the cln of the mice given the mel-14 antibody. the igm response was prolonged and the igg response, particularly iggl, was delayed compared to controls. the altered pattern of the antibody response may reflect the limited availability in mel-14-treated mice of th cells secreting lymphokines which are involved in ig class switching, by blocking the entry of cd4+ th precursor cells into lymph nodes. facs sorting for l-selectin+, 8220+, and l-selectin-, b220+ cell populations from the mln and the cln of normal b6 mice 9 days post sendai virus infection, showed that the afcs were from the l-selectin-, b220+ cell population, a population which comprised 6-10% ofthe total cell population. we have distinguished targets of broadly neutralizing antibodies present in hiv-1 infected individuals by imunoselection in vitro and by the use of chimeric virus. one target of neutralizing antibodies, defined by an escape mutant with an ala to thr substitution at position 582 in gp41, is resistant to human monoclonal antibodies that map to a site closely congruent with that for cd4 binding. substitution of gly, ser, and val fail to confer resistance. a second, defined by an ala to val substitution at position 281, upstream from the v3 loop, does not involve the same site and does not involve v3. substitution of thr or ile also confers resistance. replacement of the v3 loop of hiv-l(mn) into a clone of hiv-l(iiib) allows the detection of two other broadly neutralizing targets. one recognizes the v3 peptide of mn but is affected by regions outside v3. the other appears to be conformational and outside v3, but its functional recognition is influenced by the v3 loop. all of these sites seem to depend on the overall conformation of the envelope protein rather than a single discrete linear epitope. antibodies against amino acids 579-613 of the hiv transmembrane (tm) glycoprotein have been shown to enhance hiv infection in vitro in the presence of complement. there has been no study demonstrating that enhancing antibodies to this region of hiv, despite increasing levels of infectious virus 10 to 100 fold in vitro, adversely affect disease pathogenesis. in two separate studies reported herein, it is shown that animals which have high levels of antibody against this region of siv, amino acids 603-622 of the envelope, fair poorly compared to animals with lower antibody levels against this region when subsequently challenged with siv. when actively immunized with a synthetic peptide from this region of siv, animals died earlier and failed to clear antigen at two weeks after infection compared to animals that received a control peptide (p<0.05). when animals were passively immunized with antibodies from a longterm survivor of siv infection, those animals that received higher levels of antibody against the tm peptide died within six months compared to longer intervals for those animals that had lower levels of antibody to this region. when taken together, these data suggest that antibody to the tm region of siv and hiv in general, and to this highly conserved peptide in particular, are detrimental to the host. therefore, immunization strategies that minimize the immune response against tm or treatment protocols that decrease antibody levels against tm may lead to prolonged survival following exposure to lentiviruses. we have developed a mouse model to examine the immune response to hpv 16 proteins when these proteins are presented to the immune system via the epithelial route. in this model animals are grafted with keratinocytes expressing hpv e6 a n d e7 genes using a transplantation procedure which permits epithelial reformation. animals so grafted when challenged intradermally with e7 either as protein or via a recombinant vaccinia virus exhibit a delayed type hypersensitivity response which is e7-specific and cd4+ t cell mediated. animals grafted with a sub optimal priming inoculum of cells develop immune non-responsiveness and have an abrogated dth response when challenged subsequently with a priming cell graft. in the present study w e have examined the antibody status in these animals. the e7 protein of hpv 16 was expressed in e. coli as a maltose binding fusion protein using the plasmid vector pmalc. after cleavage and affinity purification this protein was used in a n elisa assay to measure antibody levels in 4 groups of mice (1) those not challenged with e7 (2) mice not grafted but challenged with e7 protein in the ear (3) mice primed by grafting with 107 hpv e7 expressing cells and challenged with e7 protein (4) mice primed by grafting with 5 x 105 hpv 16 e7 cells on day 7, grafted again with lo7 hpv 16 e7 cells on day 14 and challenged with e7 protein in the ear. mice optimally grafted and challenged (group 3) exhibited high titres of igg antibodies, particularly elevated levels of iggza. mice sub-optimally grafted (group 4) exhibited igg antibody levels comparable to the control group (1). the possible mechanisms of this immune attenuation are discussed. the hepatitis c virus is a frequent cause of chronic liver disease. a proposed mechanism responsible for virus persistence is evasion of the host immune response through a high mutation rate of crucial regions of the viral genome. the portion of hcv genome coding for the amino-terminal part of the putative envelope protein (gp70) undergoes frequent mutation during the course of infection. we have cloned and sequenced the hypervariable region (hvri) of the virus isolated from an hcv asymptomatic patient at three time points during 18 months follow up. sequence analysis has allowed the identification of variants of this region and multiple antigenic peptides (map), corresponding to three hvrl variants, sequentially foundin the blood stream of the patient, have been synthesized. maps have been used as antigens for detection of specific antibodies in elisa. our results show that anti-hvri antibodies and their cognate viral sequence coexist in the blood stream but a viral sequence becomes undetectable when the specific antibodies reach maximum levels of reactivity. thus humoral immunity against the hvrl may play a role for virus clearence. the presence of anti-hvr1 antibodies was also investigated in 100 hepatitis c viremic individuals and 25 non-viremic patients. a high frequency of positive reaction (90%) against at least one of the three hvrl variants analysed in this study was detected in the viremic patients. finally, competition experiments show that antibodies crossreacting with more than one hvrl variant are produced by hcv infected individuals. this results suggest that complex cross-reactivity exist between hcv isolates for antibodies against the hvrl region as described for antibodies against the gp120 v3 loop of hiv. we propose as mechanism for viral escape in hcv chronic infections the one described as the "original antigenic sin", observed firstly in influenza, in togavirus, paramixovirus, enterovirus, and recently in hiv infection. using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge witb the murine rotavirus strain edim (ward et al., 1992) . to determine i f a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of 18 reassortants between the fully protective edim strain and a partially protective heterologous rotavirus strain (rrv-g). reassortants that contained genes for edim proteins responsible for protection were anticipated to provide complete protection; however, no edim proteins were found to be both necessary and sd3cient for full protection. instead, protection was found to be highly correlated with viral shedding (p = ,005) and with serum rotavirus iga titers stimulated by the different reassortants (p < ,001). this indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of edim proteins. this conclusion was supported by the finding that the titers of serum rotavirus i& but not igg, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against edim. if these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization. h a l l medical center, 55 individuals with adequate serum samples were identified as either rapidly progressing (rp) or slowly progressing (sp) by clinical and surrogate marker criteria. anti-v3 profiles were determined using synthetic proteins derived from the amino acid sequences of the v3 region of 5 laboratory strains of hiv-1 in standard capture elisa format. serum obtained from each patient at multiple different time points was screened against these peptides. the majority of individuals in both groups demonstrated broad recognition, with reactivity to peptides corresponding to the v3 regions of mn, sf2, ny5 and han/sc. less than 50% of individual in each group recognized the v3 peptide derived from iiib, @=ns, between groups). as the rp progressed to aids there was significant nonspecific narrowing of response, while the sp remained broadly reactivity (p< .001). in v i m neutralizing activity of the homologous laboratory isolates was determined with cytotoxicity, cytopathic effect and p24 ag inhibition assays. although most patient serum was capable of inhibiting p24 ag production in homologous lab strains while aids-free, there was no relationship with the ability to inhibit homologous virus effects on target cells and anti-v3 profiles. model, we show that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appear in the bone m w and constitute the major sou~ce of longterm antibody production. following infection of adult mice, wspecific antibody secreting cells (asc) peaked in the spleen at 8 days postinfection, but were at this time undetectable in the bone marmw. the infection was essentially cleared by 15 days and the asc numbers in the spleen rapidly declined while an increasing population of lcmv-specific asc appeared in the bone marrow. when compared to the peak response at 8 days post-infection, timepoints from 30 days to more than one year later demonstrated greater than a l@fold reduction in splenic asc. in contrast, jltvlv-specific plasma cells in the bone marrow remained at high numbers and correlated with the high levels of antiviral serum antibody. the prewnce of antiviral plasma cells in the bone marrow was not due to a persistent infection at this site, since virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and pcr assays. the igg subclass profile of antibody m e t i n g cells derived from bone manuw and spleen correlated with the igg subclass distribution of lcmv-specific antibody in the serum. upon rechallenge with w , the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. bone marrow asc populations and lcmv-specific antibody levels in the serum did not change during the early phase of the reinfection but both increased about 2-fold by 15 days post-challenge. after both primary and secondary viral infection, lcmv-specific plasma cells were maintained in the bone marrow showing that the bone marrow is a major site of long-term antibody production after acute viral infection. "memory" t cells, and associated with responsiveness to soluble and recall antigens. cd4+ lymphocytes staining bright, dim, or negative (equivalent to an isotype control) for cd29 were evaluated in 49 uninfected controls (group l), 84 hiv-1 positive patients with 220% cd4+ t cells (group 2), and 47 hiv-i-infected patients with ~2 0 % cd4+ t cells (group 3). most of these subjects also had 3-color staining for cd4\cd45ro\cd45ra. the appearance of positive cd29 and cd45ro on hivinfected and uninfected cells correlated well (r=.82 p<.ool). the percentage of cells staining cd4+\cd29+.(bright plus dim) was 43.3 (95%cl 37.3-49.4) in group 1, 28.9 (27.5-30.4 ) in group 2, and 10.2(8.6-11.9) in group 3. the respective values for these groups that were cd4+\cd2gbwm was 30.6 (26.9-34.3), 20.7 (1 9.3-22.2), and 7.4(6.3-8.6). values for cd4+\cd45ro+ were 33.7 (31.8-35.5), 21.8 (20.5-23.1), and 9.9 (8.5-11.3), respectively. in single factor discriminate function tests, the %cd4+\cd29+ cells best predicted subject group (87% correct), proving to be a better discriminator than %cd4+\cd29b'h' (77% ~orrect),cd4+\cd29~"" (5 l%), cd4+\cd45ro+ (75%) and cd4+\cd45ro+\cd45ra-(63%). overall, no advantage was seen to splitting the cd4+\cd29+ cells into bright and dim positive subsets in the subjects studied for the purpose of stratifying early vs. late hiv infection. likewise, splitting the cd4+\cd45ro+ compartment into cd45ra+ subsets did not improve the ability to distinguish between uninfected and early or late hiv-1 infected patients. the relationship between the virus-specific cytotoxic response in hiv infected patients and disease progression support the concept that a vaccine candidate should also induce a virus-specific ctl activity. immunization of uninfected adult volunteers by a hiv-gpl60 recombinant canarypox virus was carried out in a phase i trial.two injections of a recombinant canarypox expressing the hiv-l/mn gp160 were performed at month 0 and 1 and two boosts of recombinant gpl60mn/lai at month 3 and 6 in alum or incomplete freund adjuvant(1fa). hiv-envelope specific cytotoxic activities were detected from ctl lines derived from pbmc stimulated by specific stimulation with autologous hiv infected blasts. ctl lines were obtained from 18 out of 20 donors : seven out of eighteen (39%) were found to present envelope specific cytotoxic activity at months 2, 4, 7 or 12 post immunization ; this activity was characterized as a cd3+,cd8+, mhc class-i restricted cytotoxic activity, and for at least two volunteers, this activity was still present two years after the first canari-pox/env injection. because avian poxviruses are incapable of complete replication and undergo abortive replication in mammalian cells , this is a n example of the persistence of long term memory cd8+ cytotoxic t lymphocytes in the absence of the priming antigen, indicating that t-cell memory might be independent of continued antigenic exposure. the university of alabama at birmingham, al 35294. mhc class i restricted cd8' ctl activity plays an important role in the control of influenza virus infection as indicated in studies in mice and humans. cytokines such as il-2 and ifn-y regulate the generation of virus-specific ctl responses. we recently demonstrated a good correlation between the induction of influenza virus-specific ctl activity and the production of ifn-y by the cd8' t cells at the single cell level using an if?-specific elispot assay, secreted ifn-y by an elisa, and ifn-y specific mrna expression by rt-pcr. several recent studies have characterized cd4+ and cd8' t cells by their expression on the surface of distinct d45r isoforms. cd45ra is expressed on naive or virgin t cells, while cd45ro is expressed on memory t cells. in the present study, pbmc of healthy young adult subjects were stimulated with influenza a virus and then enriched for cd8+ t cells. the cd8' cells were stained for cd45ro' (pe) and cd45ra' (fitc) cells and sorted. ctl activity against virus-infected autologous target cells was determined in a 4 hour 'lcr release assay while ifn-y production and expression was assessed by elispot and quantitative rt-pcr, respectively. cds+/cd45ro+ (memory) cells exhibited significant mhc class i ctl while cds+/cd45ra+ cells exhibited no lytic activity. no activity was exhibited by freshly isolated or unstimulated cd8+/cd45ro+ t cells. similarly, cd8+/cd45rot t cells contained significantly higher numbers of ifn-y spot forming cells and higher quantity of ifn-y-specific mrna than cd8+/cd45rac cells. these data support our previous findings that ifn-y may serve as a useful surrogate marker for influenza virus-specific ctl activity in humans. in studying the kinetics of the cd8+ t cell response in lcmv infection we have observed a profound activation and proliferation of cd8+ t cells with a 10-40 fold increase in total number peaking at day 8-9 post infection. in c57bw6 mice, most of the viral antigen is cleared by day seven, and after day 9 the total cd8+ number per spleen drops about 10-fold. however, the relative specificity of the viral peptidespecific precursor ctl frequencies @ctwf) per cd8+ cell remains remarkably stable between day 7-8 of the acute infection and for many months thereafter. thus, the decline in the cd8' t cell number is not a function of the tcr specificities but is rather an across-the-board event. in contrast, we found that subsequent to the decline of the ctl response to a second heterologous virus infection such that the mouse was in a "resting, immune'' state, there often was a reduction in pctl/f to the first virus. for example, infections with w or mcmv substantially reduced the pctuf to lcmv or pv in all memory compartments, including spleen, lymph nodes, peritoneal exudate cells. reinfection with the original virus substantially elevated its pctuf and restored the pctuf that had been reduced by a heterologous viral infection. analyses of the progression of ctl responses during a heterologous virus challenge of a virus-immune mouse indicated a high frequency of crossreactive ctl appearing early during infection, but as the infection progressed there was a higher proportion of ctl specific only for the second virus. thus, we believe that when the across-the-board apoptosis of t cells occurs late in the infection, ctl specific for the first virus are diluted by those responding to the second virus. this may cause the reduction in memory to the first virus and may be one of the mechanisms contributing to the waning of secondary immune responses to certain viruses over time if there is no re-exposure to the original infectious agent. t-cells which arise after virus infection will aid our understanding of tcell memory and be useful in the design of vaccines which augment the memory response. to estimate the sendai virus specific precursor frequency in memory mice, cd4+ cells from c57bl6 female mice which had been infected with sendai virus intranasally (i.n.) more than two months earlier were subjected to limiting dilution analysis. responder cell populations were enriched for cd4+ cells either by magnetic bead depletion of non-cd4+ cells, or by facs after staining with anti-cd4 monoclonal antibody these enriched (>90% cd4+) responders were cultured with sendai virus-infected, irradiated, t-cell depleted splenic antigen presenting cells (apc). supernatants from these cultures were tested for activity on the cytokine-dependent ctll cell line. duplicate cultures of responders on uninfected apc were used to set the level of rejection (mean cpm + 3x std. dev.). using this type of analysis we were able to demonstrate a frequency of memory thp at 111600 cd4+ cells, compared to a frequency greater than 1/1ooooo in naive controls. the memory cd4+ cells were further characterized as cd45rb-low (1/472) , cd44-high (1/294), lselectin-low (1/364), and cd49d-high (vla-4-high) (v102). this is close agreement with other phenotyping studies on cd4+ memory cell specific for soluble antigens. t cells, ralph a. tripp, sam hou, anthony mcmickle, james houston and peter c. doherty, department of immunology, st. jude children's research hospital, memphis, tn 38105. the immune response of influenza a and sendai-virusspecific, memory cd8' cytotoxic t lymphocyte precursors (ctlp) have been analyzed in c57bu6 mice infected intranasally with unrelated or cross-reactive respiratory viruses. the numbers of influenza a-specific memory t cells increased in the regional lymph nodes (ln), spleen and bronchoalveolar lavage through the course of an irrelevant infection (influenza b). memory t cells showed evidence of enhanced steady-state activation. profiles of ctlp recruitment were analyzed in association with t cell proliferation and activation to determine whether signaling via the t cell receptor is necessary to induce "bystander" stimulation of the memory t cell pool. the extent of t cell proliferation was addressed by treating mice with low doses of cyclophosphamide (cy). "resting" sendai virus-specific memory t cells were unaffected by cy treatment, however upon challenge with influenza and treated 5 or 6 days later, the emergence of influenzaspecific ctlp was severely diminished. cell cycle analysis showed that cy eliminated the majority of cd8' t cells from the ln and spleen resulting in dna fragmentation of 12-18% ofthis lymphocyte subset. a decrease (though smaller) in the numbers of sendai virus-specific ctlp indicated that some of the cycling cells killed by cy were memory t cells, presumably activated in a "bystander" manner. the decrease in ctlp numbers for both influenza and sendai virus-specific ctlp was still apparent 9 days after cy treatment, long after the viral elimination. thus, immune responses to unrelated antigens may be a mechanism involved in maintaining the pool of memory t cells. experimentally vsv can result in an acute cns infection of mice. data from our in vitro experiments indicate that no has inhibitory effect on productive vsv infection. vsv infection at neuroblastoma nb41 a3 cells was significantly inhibited by loopm of a no donor s-nitro-n-acetylpencillamine (snap), while 1oopm of the control compound n-acetylpencillamine (nap) had no effect. when vsv infected nb41a3 cells were treated with 500pm of a constitutive no synthase (cnos) activator n-methyl-d-aspartate (nmda), a significant inhibition of vsv production was observed. inhibition by 500pm of nmda was reversed by 300pm of nos inhibitor n-methyl-l-arginine (l-nma). work is in progress to determine the effects of inducible nos (inos) in a glioma cell line c6 on vsv infection. levels of no and expressions of both cnos in neurons and inos in glial cells in the cns following vsv will be further investlgated. supported by nih grant a118083 to carol s. reiss. pediatrics, university of iowa, iowa city, ia. 52242 mouse hepatitis virus, strain jhm (mhv-jhm), is a neurotmpic coronavirus which causes acute encephalitis and acute and chronic demyelinating encephalomyelitis in susceptible rodents. 40.90% of suckling c57bu6 (kbdb) mice inoculated intranasally with mhv-jhm at 10 days and nursed by dams immunized against the virus develop a chronic demyelinating encephalomyelitis characterized clinically b hindlimb paralysis, at 3-8 weeks postinoculation. the chronic demyelinating encephalomyelitis nor the clinical symptoms. recently, it was shown that lymphocytes isolated from the central nervous system (cns) of c57bu6 mice both acutely and persistently infected with mhv-jhm display a cytotoxic t lymphocyte (ctl) response to the s protein of mhv-jhm. this response was further characterized by identifying the ctl epitopes that are recognized by a bulk population of ctls from the cns of mhv-jhm infected c57bv6 mice. three epitopes were identified using synthetic peptides and truncated forms of the s protein in primary ciz assays. the epitopes recognized were amino acids 510-518 (cslwngphl, db), 598-605 (rcqifani, kb), and 1143-1151 (nfcgngnhi, db). thus, the results indicate that cytotoxic t lymphocytes responsive to the s protein of mhv-jhm in c57bu6 mice recognize both kb and db-restricted cil epitopes. ctl lines and clones specific to these peptides and the entire s protein are being developed to test their biological significance in vivo with respect to the acute encephalitis and chronic demyelinating disease caused by mhv-jhm. a marked change in susceptibility to some neurotropic viruses during the first few postnatal weeks has long been recognised in rodents. infection of neonatal or suckling mice with the neurotropic alphavirus, semliii forest virus results in lethal encephalitis. infection of weaned animals is not lethal. earlier investigations focusing on changes in specific immunity have shown this not to be the explanation. infection of 3-4 week old mice with severe combined immunodeficiency does not result in acute rapidly fatal encephalitis. we have studied mortality, neuroanatomical distribution and spread of infection in mice of different ages and the effect of gold compounds on rendering infection of 3-4 week old mice lethal. neuroanatomical distribution of infection correlates with synaptogenesis. as this is completed in different systems within the first two weeks postnatal, systems no longer transmit virus and infection switches from disseminated to focal and restricted. complete productive replication and transmission of infection require smooth membrane synthesis which is present in neurones undergoing synaptogenesis, absent in mature neurones but inducible by administration of gold compounds. infection of neurones undergoing synaptogenesis is productive and virus is transmitted along neuralpathways, infection spreads rapidly around the brain, destroys cells and animlas die of a fulminant encephalitis. in mice infected after 14 days of age replication in mature neurones is restricted, nonproductive, cannot be transmitted, does not spread, is non-destructive and non-lethal. as a consequence, in the absence of immune responses virus can persist in isolated cns cells for life and can even be detected by reverse transcriptase pcr in immunocompetent mice months after infection. in the presence of an immune response, cd8+ t-cells recognise and destroy infected glial cells leading to dem yelination. a~k e r m a n n ,~ virology swine,' virology cattle,' and avian diseases3 research units, national animal disease center, usoa, agricultural research service, ames, ia 5001 0 a recombinant pseudorabies virus (prv) (lltbap) was constructed which contains a 3.0 kb deletion spanning the standard recombination junction of the unique long and internal repeat sequences replaced by e lacz expression cassette. this deletion interrupted the large latency transcript gene (llt) and truncated one copy of the diploid immediate early iel80 gene. replication and viral gene expression of lltbaz in madin-darby bovine kidney cells was similar to that of the parental virus and a virus rescued for the deleted sequences (lltbres). when inoculated intranasally in 4-week-old or 4-day-old pigs, lltba2 replicated efficiently at the site of inoculation yet caused markedly reduced fatality when compared to the parent or lltbres viruses. in particular, the lltba2-infected pigs did not exhibit neurological symptoms characteristic of prv infection. to further examine the pathogenesis of lltba2, 4-day-old pigs were infected intranasally with lltpa2 or lltbres and necropsied at various times postinfection. virus isolation from the nasal turbinate, tonsils, and trigeminal ganglia was comparable between the two viruses. although both viruses spread to the brain and induced an inflammatory response in cns tissues, virus isolation from brain tissues was reduced about 20-fold for lltpa2. abundant prv antigen was detected in the cerebrum and cerebellum of lltpresinfected pigs, but only a few antigen positive neurons were observed in the cerebrum of lltba2-infected pigs. while replication of lltbres in the brain progressed until death at 7 days post-infection, replication of lltpa2 in the brain ceased by 9 days post-infection and the pigs exhibited only mild clinical signs. since lltba2 is capable of spread to the cns, reduced neurovirulence of lltbaz is likely the result of its decreased ability to replicate in cns tissues. the cns is a target for hiv infection, and in individuals with aids this can lead to a devastatin dementia. only certain viral variants appear capable 07 invading the cns and infecting microglia and brain macrophages. in order to determine whether the virus entering the brain may be particularly pathogenic to the cns, we isolated microglia from the brains of siv-infected rhesus monkeys. transfer of these cells into naive animals indicated that productive siv infection could indeed be transferred. furthermore, cns infection occurred within a relatively short time span, and was associated with viral gene expression in the brain and pathology characteristic of hiv encephalitis. serial transfer of microglia into additional animals also resulted in successful transfer of infection, neuroinvasion, and neuropathology. behavioral analysis in a trained group of animals is ongoing. this result demonstrates that neuropathogenic virions partition into the cns during natural siv infection, likely driven by mutational events that occur during the course of infection. molecular characterization of the microglia-associated virus has revealed that a distinct pattern of sequence changes in the envelope gene occurs concomitantly with this in vivo selection. our approach will allow the dissection of functional neuropathogenic elements present in these viruses. in non-specific host defense mechanisms. ifn-y-induced nitric oxide (no) in murine macrophages was previously shown to inhibit the replication of poxviruses and herpes simplex virus type 1 (hsv-1) . we now demonstrate that murine macrophages activated as a consequence of vaccinia virus (vv) infection in viva express inducible nitric oxide synthase (ios). the vvelicited macrophages were resistant to infection with w and efficiently blocked the replication of w and hsv-1 in infected bystander cells of epithelial and fibroblast origin. this inhibition was arginine dependent, correlated with no production in cultures and was reversible by the nos inhibitor nqjmonomethyl-l-arginine. the mechanism of no mediated inhibition of virus replication was studied by treating vv-infected 293 cells with the noproducing compound, s-niuoso-n-afetyl-penicillamine. antibodies specific for temporally expressed viral proteins, a vv-specific dna probe and transmission electron microscopy were employed to show that no inhibited late gene protein synthesis, viral dna replication and virus particle formation, but not expression of the early proteins analyzed. further, we have also identified putative enzymatic targets of inactivation by no that results in inhibition vv replication. although antiviral ctl are important for virus elimination. they can only halt further virus spread, and cannot reduce the number of infectious particles already present. the beneficial effect of ctl-mediated lysis is apparent only if infected cells are lysed before assembly of progeny virus. if infectious virus was released from infected cells in solid tissues before the generation of neutralizing antibody or in sites where antibody did not readily penetrate, then recruitment of mononuclear phagocytes, which phagocytose and destroy infectious material and/or become non-productively infected, would definitely help control virus dissemination. in this context, inos induction in macrophages may be an important antiviral strategy. in addition, the inhibition of virus replication in infected contiguous cells by inos-expressing macrophages at infectious foci would prevent release of mature viral particles after lysis by nk cells and ctl. since viral early proteins are expressed in such infected cells, their recognition and subsequent lysis by ctls will not be hindered. cns persistence, tropism and genetic j. pedro s i s , anthony a. nash and john k. fazakerley, department of pathology, university of cambridge, cb2 iqp, uk theiler's murine enchephalomyelitis virus, a natural occuring enteric pathogen of mice, is a picomavirus belonging to the curdovirus genus. following intracerebral inoculation of 3-4 week old cba or balb/c mice, the bean strain causes a chronic persistent cns demyelinating infection in a proportion of the cba that survive acute infection. balb/c mice are resistant to chronic demyeliating disease. we have studied the tropism, persistence and genetic variability of bean, in cba and balb/c mice in the chronic phase of this disease. by in situ hybridisation and reverse transcription (rt) pcr and southern blot analysis, no viral rna could be detected in the cns of any balb/c mice later than day 60 post-infection. in contrast, in a large group of cba mice studied up until 393 days post-infwtion, viral rna could be detected by both techniques in 50% of mice until as late as 268 days post-infection. by employing a combination of, in situ hybridisation for viral genome followed by immunocytochemistry for cell phenotypic markers, bean rna was observed predominantly in oligodendrocytes and occasionally in astrocytes during persistent infection, in both brain and spinal cord. in the persistently infected mice, the striking total destruction of the pyramidal layer of the hippocampus, substantia nigra and anterior thalamic nuclei indicated that these were the mice that had had greatest dissemination of virus and highest virus titers during the preceeding acute phase of infection. direct pcr t h d cycle sequencing of uncloned rt-pcr products, revealed that during persistent infection, loops i and ii of the vpi capsid protein gene did not undergo any genetic variability. furthermore, no changes were detected in this region in sequenced pcr products amplified from the cns of mice with severe combined immunodeficiency in which no selective immunological pressure would have been operative. infection, thomas e. lane, michael j. buchmeier, dorota jakubowski, debbie d. watry, and howard s. fox, department of neuropharmacology, the scripps research institute, la jolla, ca 92037 our laboratory is interested in the effects of siv infection in the central nervous system of rhesus macaques. to enrich for neuroinvasive and neurovirulent viruses, microglia were isolated from infected monkeys and used t o infect new, uninfected monkeys. such microglia-mediated infection resulted in the production of neuropathological changes, including giant cells, macrophage infiltrates and microglial nodules in recipient animals within 4 months. microglial cells isolated from siv-infected monkeys produced virus in vitro as measured by reverse transcription (rt) and p27 production. treatment of microglia with recombinant human interferon alpha (rhulfn-a) resulted in a sharp decrease in viral activity (both rt and p27 production) suggesting that rhulfn-a is able t o modulate viral activity in infected microglia. we have analyzed slvenv sequences by pcr amplification directly from microglia dna preparations from monkeys. nucleotide sequence analysis results in an enrichment of unique sequences in the v1 region of the siv env gene. the majority (>95%) of nucleotide changes encoded amino acid changes, indicating that these envelope sequences evolved as a result of selection. moreover, sequential passage of sivassociated microglia resulted in an increase in potential n-linked glycosylation sites within the v1 region of the env gene when compared with the parental virus. these data suggest that sequential passage of microgliaassociated siv may select for neuroinvasive, neurovirulent variants. the adoptive transfer of ctl specific for an ld-restricted epitope within the nucleocapsid protein of the jhmv strain of mouse hepatitis virus both protect from acute infection and reduce virus replication in the mhc class 1 positive cells within the cns. the source of these ctl and the route of their delivery is critical in the outcome of this protection. for example, 10 fold less spleen cells activated in vitro with the pn peptide are required for protection via the direct i.c. route than the i.v. route. in addition, ctl clones are unable to protect via the i.v. route and are very efficient via the i.c. route. these data suggested the possibility that the cd4+ t cells within the polyclonal activated spleen cell population derived from in vitro culture on the pn peptide were facilitating access to the cns. to examine this question, polyclonal pn-specific t cells were either depleted of cd4+ t cells prior to transfer to infected recipients or untreated cells were transferred to recipients depleted of cd4+ t cells with monoclonal antibody gk1.5. both of these treatments eliminated the ability of the ctl to reduce virus replication within the cns, suggesting that cd4+ t cells in the peripheral compartment are required for the entry of ctl into the parenchyma of the cns during acute cns encephalomyelitis. division of retrovirology, walter reed army institute of research and henry m. jackson foundation, rockville, md 20850; department of retrovirology, armed forces research institute of medical sciences, bangkok, thailand background the hn-1 epidemic in thailand is largely due to two highly divergent subtypes of virus, b and e. dual infection with distinct hn-1 subtypes, which has not been reported previously, would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. merhoak: pcr typing and serologic typing were used to screen a panel of non-random convenience specimens from hiv-1 infected subjects in thailand. specimens that showed dual subtype reactivity in these assays were subjected to differential pmbe hybridization and nucleotide sequence analysis of multiple molecular clones to c o n f m the presence of dual infection. results. two individuals were shown to simultaneously harbor hiv-1 of env subtypes b and e (table) . additionally, both subtypes were identified in co-cultured pbmc from one individual. conclusions. these data provide the fmt evidence of dual hiv-i infection in humans and reinforce the need for polyvalent vaccines. infection by herpes simplex virus wsv) induces in man and in mice cytolytic t lymphocytes (ctl) which recognize the immediateearly protein icp27. because of its early expression during the hsv replication cycle, lcp27 represents a prime target for specific t cell responses susceptible of controlling virus replication. we have expressed in e. coli a remmbinant construct coding for a fusion protein consisting of a fragment of influenza virus non-structural protein4 (nsi) and the lcp27 sequence of hsv-2. the nsi-icp27 protein was purified by preparative eleclmplmresis and formulated in oil-in-water emulsions with monophosphoryl lipid a (mpl) and qszl adjuvants. balwc mice were immunized by two intrafootpad injections of formulations containing 5 pg of nsi-icp27. responder cells obtained from draining lymphnodes were re-stimulated in vitro with p815 cells lransfected with icp27 and then lesled for cytolytic activity on icp27-p815 and control p815. the induction of icp27 specific ctl by different formulations was observed and will be discussed. the induction of heterologous cytotoxic t lymphocytes (ctl) using cassettes of multiple conserved t cell epitopes derived from different proteins and/or virus strains is envisioned as a promising vaccine approach. to study the effects of antigen processing on peptide presentation from chimeric epitope precursors we are using a model system comprising two distinct viral epitopes which are immunodominant in the h-2d haplotype: a dd restricted epitope from the gp160 protein of hiv-1 and an ld restricted epitope from the murine hepatitis virus nucleocapsid protein (mhv n). the influence of proximity and flanking sequences of epitopes on antigen presentation was analyzed using vaccinia virus (vv) recombinants in which the epitopes were expressed as chimeras containing the individual epitopes in reverse order or separated by different spacer residues. whereas individually expressed epitopes were efficiently recognized by protein-specific ctl, recognition of peptides derived from tandem constructs varied significantly with closer epitope proximity and sequential order. following immunization with the recombinant viruses, the chimeras were all able to induce antiviral ctl specific for the native proteins. however, cn, frequency analysis indicated that the number of responder cells to the same epitope dramatically depends on its context within the chimera and correlates with antigen recognition in vitro. the profound effect of flanking regions on ctl induction suggests that the context of an epitope will require careful evaluation in the design of recombinant multivalent minigene vaccines to induce an optimal t cell mediated immune response. and robert e. johnston', departments of 'microbiology & immunology and %iochemistxy, univ. of north carolina, chapel hill, nc 27599 a hll-length cdna clone of venezuelan equine encephalitis virus w e ) has been altered to contain two strongly attenuating mutations and a second subgenomic rna promoter immediately downstream of the structural gene region. expression ofthe influenza ha protein from this second promoter in baby hamster kidney (bhk) cells was approximately 50?? of the level in influenza virus-infected cells, as measured by immunoprecipitation. fourweek-old cd-1 mice were inoculated subcutaneously with 2 x 10' p h of the ha vector, vector alone or diluent. expression of ha mrna was detected in the draining lymph node of ha vector-inoculated mice by in situ hybridization, consistent with the organ tropism of vee. mice were challenged three weeks after imnmization by intranasal administration of lo5 ed, of influenza h s . au 24 corn1 mice suffered severe disease and 50% died. only one of 12 ha vector-inoculated mice died, and another exhibited signs of disease for one day and recovered. the geometric mean elisa titer of anti-ha serum igg in the ha-vector inoculated mice was 246, while only three control mice had measurable serum reactivity, and that was at the lowest dilution tested, 150. in a parallel experiment, no influenza infectivity was detected in the lungs of 12 ha vector immunized mice at 4 days postchallenge. in contrast, 8/12 pbs-inoculated mice and 5/12 inoculated with vector alone were positive for influenza infectivity and had geometric mean titers of 3.04 and 1.93 x lo6 pwgm, respectively. this vector also has been used to express the h n w c a protein in a form recognized by patient sera and a specific antibody on western blots. these experiments demonstrate the feasibility of using vectors based on attenuated vee cdna clones for protective immunization against heterologous human and animal pathogens. dose/response curves have been used to compare different routes of immunization with plasmid dna encoding the h1 hemagglutinin glycoprotein of influenza virus. routes of inoculation included intramuscular, intradermal and gene gun delivery of dna. from 100 to 0.1 ug of dna was inoculated by intramuscular and intradermal routes. from 0.4 ug to 0.0004 ug of dna was inoculated by gene gun. each route was evaluated for single and boosted immunizations. antibody titers were followed over a 20 week period, following which animals were evaluated for protection against a lethal challenge. each of the routes raised both antibody and protective responses. gene gun-delivery of dna required 250 to 2,500 times less dna to raise responses than the intramuscular and intradermal inoculations. boosts did not have much of an effect on antibody titer or protection except at low dose inoculations (4 ng and lower for the gene gun). for each of the routes, antibody responses showed good persistence over the 20 weeks of the experiment. inoculation of mice with plasmid vectors carrying a microbial gene under the control of an appropriate promoter results in a full spectrum of immune responses to the vectorencoded antigen. using a murine rabies model a plasmid termed psgsrab.gp expressing the full-length rabies virus glycoprotein regulated by an sv40 promoter was shown to induce upon inmuscular inoculation a rabies virus specific t helper cell response. of the thl type, cytolytic t cells and virus neutralizing antibodies resulting in protection against a subsequent challenge with live rabies virus given either peripherally or directly into the cenaal nervous system. a response comparable in magnitude was also induce upon inoculation of a vector expressing a secreted form of the rabies virus glycoprotein. the immune response to the dna vaccine could be modulated by co-injection of the rabies virus glycoprotein-expressing vector with plasmids expressing mouse cytokines. inoculation of mice with the psg5rab.gp vector and a vector expressing granulocyte/macrophage colony stimulating factor (gm-csf) enhanced both the t helper and the b cell response to rabies virus thus improving vaccine efficacy. co-inoculation with vectors expressing interferon-g failed to improve the response. co-inoculation of the antigen-expressing vector with a plasmid encoding mouse i l 4 caused a reduction of both the t helper cell response and the b cell response to rabies vlns. hpvl6 e7 hpv associated cervical cancer cells express hpv16e7 protein and antibody to hpv16 e7 can be detected in the blood of cancer patients, yet the twnours are. not rejected. a mouse transgenic for the e7 protein of hpv16, and expressing e7 protein in the skin, has recently been described (1) and these mice develop spontaneous humoral immunity to e7 protein similar to patients with cervical cancer(2). to determine whether immunisation could induce immunity to e7 sufficient to allow tumour rejection, we firstly demonstrated that immunisation of h-zb mice with hpv16e7 protein with quil a as adjuvant could induce cytotoxic t cells able to kill hpv16 e7 expressing tumour cells in nrro. we then used similar immunisation with e7iquil a to induce e7 specific immunity in fvb (h-29) mice. h-2qskin @s expressing e7 were not rejected by e7 immunised h-zq mice, though immunisation induced antibody to e7, and similar grafts were rejected, as expected, across an doantigen mismatch in h-zb mice. we conclude either that hpvl6 e7 lack a tc epitope in the context of h-zq, or that expression ofe7 in the skin from the e7 transgenic mice is insufficient for recognition by primed effector cells, and further experiments will address this distinction. cervical carcinoma is strongly associated with infection by human papillomavirus (hpv) types 16 or 18, and continued expression of the e6 and e7 gene products. this provides an opportunity for an immunotherapeutic approach to the treatment of cervical carcinoma by activation of immune reponses directed against these virally encoded tumour specific antigens. we have constructed a recornbinant vaccinia virus expressing e6 and e7 from hpv16 and 18 with the aim of inducing e6 and e7 specific hla class i restricted cytotoxic t lymphocytes (ctl). the sequences have been inserted into the wyeth vaccine strain of vaccinia virus at a single locus in the form of two separate fused e6/e7 reading frames, each under the control of an early v a d n i a promoter, and each modified to inactivate the rb binding site. the virus has been characterised with respect to its ability to synthesise the expected hpv proteins, its genetic stability, and growth and virulence in a mouse model prior to use in human clinical trials. analysis of hpv16 â�¬7 specific ctl from c57bu6 mice immunised with this recombinant virus show the response to be equivalent to that generated by a control vaccinia recombinant expressing non-modified hpvi 6 e7 alone, with similar recognition of the defined immunodominant h-2db restricted epitope, e7 residues 49-57. ability of mice to resist influenza challenge, arthur friedman, douglas martinez, john j. donnelly and margaret a. liu, department of virus and cell biology research, merck research laboratories, west point, pa 19486 mice infected with the laboratory strains of a/pr/8/34 (hln1) or the mouse adapted a/hk/68 (h3n2) show complete protection against challenge with a different strain of influenza a. humans, however, undergo multiple influenza infections as previous infections appear to provide weak or short-lived protection against the continual antigenic change of strains. we have previously shown that immunization of naive mice with dna encoding the conserved internal antigen nucleoprotein (np) provides protection against both h1 and h3 strains of a/influenza. although such mice became infected they were resistant to weight loss and death this differed substantially from a/pr8 and a/hk recovered mice which were resistant to subsequent infection. to produce a more representative model of human infection, we infected the lungs of mice with currently circulating strains of human influenza. mice that had been given lung infections with a/beijing/92 were susceptible to subsequent infection with the a/hk/68 strain although they were resistant to weight loss and death. other strains such as a/beijing/89 or a/georgia/93 provided only marginal protection against weight loss and death against a/hk challenge. mice that were immunized with np dna had greater resistance to weight loss and death after a/hk/68 challenge than mice previously infected with a/bei/89 and a/ga/93, and were similar to mice that had been previously infected with a/bei/92. thus, infection with different virus strains provide various levels of cross strain protection and the level of protection provided by immunization with dna can exceed that induced by live influenza infection. the development of sendai virus-specific cytotoxic t lymphocyte (ctl) effectors and precursors (p) has been compared for mice that are homozygous (-/-) for a disruption of the h-21-ab class ii major histocompatibility complex (mhc) glycoprotein, and for normal (+i+) controls. the generation of cd8+ ctlp was not diminished in the (-/-) mice, although they failed to make virusspecific igg class antibodies. while the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (bal) of the infected lung was not modified and potent ctl effectors were present in bal populations recovered from both groups at day 10 after infection. there was little effect on virus clearance. as found previously with cd4-depleted h-2b mice, the absence of a concurrent class il-mhc-restricted response does not compromise the development of sendai virus -specific cd8+ t cell-mediated immunity. the importance of cytoxic t lymphocytes in defense against acute and chronic viral infections is gaining increasing recognition. our approach to investigating the structure-function relationship between immunogens and their in vivo ability to elicit cytotoxic t lymphocyte responses has been to formulate simple, well-defined structures that vary in their ability to introduce associated antigens directly into the cytoplasm of antigen presenting cells. we have introduced methods for the preparation of unique, lipid-matrix based immunogens, which are highly effective in mice and monkeys for stimulating strong cd8+ cytotoxic t cell responses, (ctl). antigens used have been proteins or peptides derived from influenza, parainfluenza, and hiv viruses, and whole formalin-fixed siv. ctl can be induced by parenteral as well as oral administration. comparing the physical and chemical nature of our formulations with those from other laboratories which have reported the use of subunit preparations to induce cd8+ ctl, leads us to propose that a minimal immunogenic formulation capable of eliciting cd8+, mhc class i restricted cytotoxic t lymphocytes includes: i) a peptide that represents a mhc class i epitope; ii) a component that enhances the aftinity of the immunogen for mhc class i positive antigen presenting cells ; iii) properties that can compromise the integrity of a lipid bilayer, facilitating delivery of the antigen directly into the cytoplasm for class i presentation. cd8+ responses to peptides, glycoproteins, and even whole fixed viruses, makes them attractive candidates for diseases where clearance of infected cells is important in protection and recovery. cani ne rabies is uncontrolled. rabies also is epizootifally active in several species in most areas of the wor!d. thm, vaccination of animals, both wild and domestic, as well as postexposure treatment of humans remains a global concern. unfortunately, in those countries in which,people most need postexposure prophylaxis, the best vaccines are expenswe and in limited supply, whereas available vaccines are of questionable immunogenic efficiency, are otten contaminated and may produce neurological complications. the goal of this study was to determine whether a rabies vaccine for global use is complete one round of replication have the potential to be used as vaccines. we have previously reported the abiliity of a ghdeleted herpes simplex virus type 1 (hsv-1) to protect mice and guinea-pigs from subsequent challenge with wild-type hsv. this virus, which we have called disc (disabled infectious single cycle) virus, can infect normal cells but the absence of gh in the progeny virus prevents further rounds of infection. as disc hsv clearly has potential as a vaccine, it is important to determine the durability of the immune response elicited by this virus. we have investigated the ability of disc hsv-1 to protect mice from a wild-type virus challenge six months post vaccination using the ear model of hsv infection. two immunisations on day 0 and day 21 resulted in a considerable reduction in virus titres in the challenged ears, and an almost complete absence of virus in the dorsal root ganglia. hsv-specific antibody titres as determined by neutralisation and ellsa were maintained for the six months period. it was possible to demonstrate an hsvspecific cytotoxic t-cell response in the disc hsv-1 vaccinated mice following challenge; this ctl activity was similar to that observed in mice vaccinated with wild-type virus and challenged after the same time period. animals vaccinated with inactivated virus or control mock-vaccinated mice showed a low level of ctl activity typical of a primary ctl response following challenge. these results indicate that an effective cell-mediated and humoral anti-hsv immune response can be maintained for at least six months following vaccination with disc hsv-1. viruses which lack an essential gene and thus can only the lmmunogenicity of two ctl @topes. influenza npl47-158 and plasmodium berghei cs protein 252-260 were studled in balblc mice. paptides were formulated as a) a iipopepudepeplkle conlugated to irlpalmltoyl-sgly~~l cysteine (pam3cyj) and dissolved in a 1% dmsolglycerol solution. b) micmparticles prepared with poly @.l ladide-coglywlide) using a solvent evaporation technique. the micropaltides were administered as a suspension in phosphate buffered saline or c) an emulsion prepared wilh egg lecithin and 10% soya oil in water. 1 wpg of peptide or controls (the welght equivalent of blanks) were administered to groups of 3 mice intra-peritoneally or subcutaneously at 1.10 and 20 days. 7 days following the last immunization splenocytes were cukured in vlro in the presence of appropriate pepwe or wntml m h rat con a supematant as a source of omwth fadors. ctl adivity was measured in a standard 4 hour chromium release assay and results expressed as % specific lysis. ctl could be elicited in vivo with all three formulations. at an ewedoctarget ratio of 1w:l the plasmodium berghei peptide encapsulated in micmpartides gave 47% iysls on peptide pulsed target calls. levels of lysis were similar for the peptide in emulslons. the iipopeplide p3ccs252-260 gave a level of lysis of 82% at an e:t ralioof1w1. these results demonstrate that peplides edminldered in a variety of formulations can induce a systemic ctl response in vivo. peplide vaccines using such formulations wuld be used to stimulate ctl responses as part of a prophyladic vaccines or as immunotherapeulics. attenuated the attenuated sabin strains of poliovirus have been used for many years to elicit protective immunity to poliovirus. oral vaccination with replicating polioviruses generates both mucosal and systemic immunity. therefore, use of recombinant polioviruses expressing heterologous antigens as vaccine delivery vectors should provide a system for generating protective immunity to those antigens. cdna copies of the poliovirus genome has been used to construct vectors containing a multiple cloning site for insertion of heterologous genes. a pilot enhanced-potency inactivated poliovirus vaccine (ejpv) with assumably improved immunogenicity containing win-treated type 3 poliovirus (shah sauketf) together with the regular type 1 and type 2 canponena was subjected to s*mdard safety and potency tesls in the labmatory and laken through wase i and i1 clinical aials. in balb/c mice, the lrypin-mcdit%d e-if' v cfryipv) was found to induce antibodies targeted ouaide the uypsin-sensitive bc-loop of capsid protein vp1. as previously shown for hypsin-mated type 3 poliovirus @vm alone. trypsin used to modify the type 3 component at the bulk phase was removed by the vaccine manufacturer (rivm) in the regular purification process. absence of uypsin in the final product was further confumed by immunizing mice and rabbits with 10-fold concentrated type 3 component of tryipv. assays for lrypin antibodies using eia and westem blot techniques were newve. in the clinical phase 1 aial six adult volunteers with existing immunity to poliovirus were given increasing doses of tryipv. already one tenth of the regular dose induced a booster effect in neuarlizing antibodies to both intact and mypsin-treated type 3 poliovirus. no unexpected sideeffects were recorded phase i1 trials comprised 50 adult volunteers with at least 5 years since the last dose of poliovirus vaccine and 50 children who were due to receive the third dose of the regular immunization schedule at about 2 years. in both groups. 25 individuals received tryipv and 25 were injected with the regular enhanced potency ipv (e-ipv). serum specimens drawn before injection and one month after were tested for neunalizing antibodies using standard microneuwlization assays (all mutypes) and the racina test (intact and uypsin-mated type 3 poliovirus). in all volunteers tryipv was at least as immunogenic t w the regular e-ipv according to all assays. no statistically significant differences in side effects were reported. a murine/influenza virus model has been used to evaluate the longevity of antibody and protective responses raised by gene gun delivery of a hemagglutinin-expressing dna. mice were immunized and boosted at one month with 0.4 ug of an h1 expressing plasmid dna (pcmvri1). antibody responses and protection against a lethal challenge were followed over the next year. antibody responses had good longevity exhibiting comparable titers at one year post boost as at 10 days post boost protection against the lethal challenge was complete at 10 days, 1 month and four months post boost, but only partial at one year. a transgenic mouse model for identifing htlv-1 t-cell epitopes: generation of hla-b*3501-restricted ctl directed against synthetic peptides and naturally processed viral antigens, christian schiinbach*, ai kariyone*, kiyoshi nokiharaa+, karl-heinz wiesmulle6 and masafumi takiguchil, departments of tumor biology* and immunology#, institute of medical science, university of tokyo, tokyo "tokyo university of agriculture and technology, tokyo +biotechnology instruments department, shimadzu corp., kyoto, japan $natural and medical science institute at the university of tubingen, reutlingen, germany the majority of human t-cell leukemia virus type-i (htlv-l), hla class i-resmcted t-cell epitopes have been identified by cloning htlv-1 patient-derived t cells. here we describe for the frst time a rapid method (reverse immunogenetics) for identifing t-cell epitopes, together with a transgenic mouse model as a guide for testing the cellular immune response to a mixture of the lipohexapeptide immunoadjuvant pamgcys-ser-(lys)4 and synthetic htlv-1 peptides which seem suitable for vaccine design. htlv-1 amino acid sequences were searched for eight to 14mer patterns carrying the anchor residues of the hla-b*3501 peptide motif at positions two and eight to fourteen. 65 candidate peptides were synthesized according to the matched sequence patterns. their hla-b*3501 affinity was quantitatively analyzed in an indirect immunofluorescence peptide binding assay using rma-s-b*3501 cells. the fourth group (controls) were inoculated with h3n2 (in) thereby providing heterotypic ctl immunity in the context of a natural infection without the confounding effects of humoral immunity against surface antigens. all four types of inoculations have been shown to protect normal (class i expressing) mice from a lethal challenge with influenza, presumably mediated by class i restricted cytotoxic t cells. the two groups inoculated via the intranasal route may gain additional protection by activating the mucosal immune system (iga). none of these types of inoculations has been evaluated in the context of class i1 restricted cytotoxic t cells, the only ctls found in class i deficient mice. for all four types of inoculations, mhc class i deficient mice lost significantly more weight than the class i expressing control groups (seven mice per group) indicating the importance of class i restricted t-cells in protection. within the class i expressing groups, there was no significant difference between the four types of inoculations; within the class i deficient groups the vac-np im immunized mice lost significantly more weight than the h3n2 group;the other two groups, vac-np in and genetically immunized groups had intermediary results. these data lend support for a protective role for mucosal immunity. results on both class i and class i1 ctl activity for the four types of inoculations will also be presented. we tested the pbmcs of patients participating in two vaccine therapy trials for their ability to recognize overlapping peptides of the gp120 la1 sequence. seventeen patients participating in a phase i gp160 protocol and 13 patients participating in a phase i gp120 protocol had their pbmcs isolated by ficoll separation of heparinized venous blood. the fresh pbmcs were plated, in triplicate, into 96 well plates containing peptides overlapping the la1 sequence of gp120, pulsed on day 7 with tritiated thymidine and harvested and counted on day 8. results: the percentage of patient's pbmcs from each trial with an lsi 2 5 to each peptide are depicted below. conclusions: the pbmcs of hiv-infected volunteers who have been multiply immunized with either gp160 or gp120 proliferate to multiple peptides within the gp120 molecule. reactivity from the end of c1 through early c2 (lai #i 12-21 1) is particularly prominent and contains previously undescribed th epitopes (asterisks). conspicuously missing is reactivity to the v3 loop peptide (la1 #300). although the percent reactivity to the entire gp120 molecule is similar between the immunization groups, there is differential recognition of some of the individual peptides, particularly peptides in early c3 (lai #319-348). the intracytoplasmic lifecycle of listeriu mmcytogenes (lm) enables it to be a convenient vaccine vehicle for the introduction of foreign proteins into the mhc class i pathway of antigen presentation. taking advantage of these properties, we have inserted the nucleoprotein (np) gene from lymphocytic choriorneningitis virus (lcmv) into the lm chromosome by site specific homologous recombination. infection of mice with recombinant lm expressing lcmv-np elicited a virus-specific ctl response. we were able to recover lcmv-np specific ctl precursers from recombinant lm vaccinated mice as shown by vigorous secondary ctl responses after in vitro stimulation. in contrast to mice immunized with wild type lm, mice vaccinated with np-recombinant lm were protected against challenge with immunosuppressive lcmv variants. protection was demonstrated by reduced viral titers or complete clearance of lcmv from serum and various organs including, spleen, liver, lung, kidney, and brain. the kinetics of the lcmv challenge indicate that mice vaccinated with recombinant lm were able to arrest viral growth early in the infection due to a strong ctl response and did not exhibit the immunopathology associated with infection of naive mice. since lm not only delivers antigens into the mhc class i pathway but also induces il-12 production, it has the potential to function simultaneously as a vehicle for expressing foreign antigens and as an adjuvant promoting cell mediated immunity. , latent membrane protein [lmp] 1 and 2a) in chromium release assays. we were fortunate in identifying one child from whom cryopresetved pbmc samples were available before. and during ebv seroconversion. ebv-specific ctl activity was demonstrated concurrent with initial detection of virus in the peripheral blood by ebv-dna pcr. in the absence of detectable serum antibody. ctl lines from all nine children recognized one or more ebv latent gene prcduct(s). all children demonstrated ctl responses against one or more ebna 3 proteins (3a, 3b, 3c). and ebna 3c was recognized most frequently. no ctl responses were detected against the ebv latent proteins ebna 1, 2, lp or lmp 1. the ebv-specific ctl lines expressed cd3/cd8 and mab blocking experiments demonstrated that the majority of target cell lysis was inhibited by antibody against mhc class-i but not antibody against mhc class-11. these results represent one of the first reports characterizing ebv-specific ctl responses in young children. the striking similarity between ebv-specific ctl responses described here in young children and those reported for adults suggests that the ebna 3 family of proteins and lmp 2a should be considered for inclusion in candidate ebv vaccines. evaluation of cellular immune responses to adenovirus vectors in the cotton rat. soonpin yei,' gary kikuchi,' ke tang' and bruce c. trapnell.' departments of virology' and immunology,2 genetic therapy, inc., gaithersburg, maryland 20878 replication deficient recombinant adenovirus (av) vectors are efficient gene delivery vehicles currently being developed for a variety of in vivo gene therapy strategies such a s for the fatal pulmonary component of cystic fibrosis. the cotton rat (sigmodon hispidus) is one of the most widely accepted animal models for studying these av vectors because wild type human adenovirus replicates in cotton rats and because of the histopathologic similarities of infected respiratory epithelial tissues from humans and cotton rats. despite this, methods for studying immunologic responses in the cotton rat have not been developed. importantly, recent studies in the cotton rat (gene tber. 1 :192-200; 1994) in our laboratory suggest that a dose-dependent specific immune response to av vectors can limit expression of the transgene. in this context, w e have established methods to evaluate cytotoxic lymphocyte (ctl) responses to av vectors in the cotton rat. to accomplish this, a ctl target cell line was established consisting of primary cotton rat lung fibroblasts (crlf). splenocytes from cotton rats exposed previously to an av vector were harvested, cultured in virro with irradiated, addb274nfected crlf. cultured (effector) splenocytes were then incubated with s'cr-labelled crlf (target) cells a t effectoctarget (e:t) ratios of 100, 50 and 10. in parallel, splenocytes from naive cotton rats served as negative controls. results demonstrated vector-specific ctl lysis of target cells significantly greater than controls: 80.3 f 1.3% vs 6.2* 0.5%. 49.6*1.6% v s 5.7*0.4%. and 22.8*3.5% vs4.8*0.5% (meanrts.e.m., n=3; p<o.ol, all comparisons) a t e:t ratios of 100, 50 and 10, respectively. thus, a specific ctl response does develop in cotton rats following in vivo av vector administration. this observation will be useful in guiding vector modifications in the rational development of improved vectors for clinical use. the identification of ctl epitopes is essential for subunit vaccine design against aids. a major portion of the anti-viral ctl responses after infection with htv in humans or siv in rhesus macaques is made up by anti-gag specificities. here, effector cell populations were established from hiv seropositive individuals by specific stimulation of pbl with pfa fixed autologous b-lcl infected with tw gag. expanded cultures were then tested for ctl activity against autologous b-lcl pulsed with overlapping 20-mer peptides derived from p24. two individuals (008 and 617) had ctl against p24.14 (a(mino) a(cids) 263-282, krwiilglnknrmysftsi), two others (038 and 157) recognized p24.17 (aa293-312, frdwdrfyktlraeqasqd). similarly we found two siv infected rhesus macaques reacting against the analogous aa sequences of siv; it. p26.14 (8653) and p26.17 (ivy). this led us to further fine map the human and rhesus ctl reactivities using sets of 9 aa overlapping 10-mers and to test for hiv-1isiv crossreactivity. both two p24.14 epitopes (possibly restricted by hla-a2) and the p26.14 epitope were non-overlapping, and all three epitopes were non-crossreactive. finemapping of the p24.17 epitopes is in progress. the sn p26.17 epitope was also non-crossreactive, despite only one aa difference (position 6, s+t) between the siv and hiv sequence. others have also mapped cl'l epitopes to the same regions of p24 in humans (johnson ji 147,1512 ,1991 , buseyne jv 67,694,1993 and to p26.17 in cynomolgus macaques (gotch jmprim 22.1 19,1993) . in conclusion, multiple non-cross reactive ctl epitopes are clustered within corresponding regions of hiv and siv gag. ctl hotspots may be important for inclusion into vaccines. forming virus) with cd-4 and cd-8 1-cells, b-cells and adherent cells, while an average of 5% of virus was associated with lps-stimulated 8-cells and adherent cells. in the second experiment the combined function of antigen presenting cells, 1-helper cells and 8-cells (humoral immune response) to a third party antigen (srbc) during the acute phase of enterovirus induced disease was increased at day 0, and decreased at days 2, 3 and 4 post-cvb3, infection relative to unlnlected animals. conclusions: these data indicate that cvb3, associates with and replicates in rnurine splenic immune cells, and that infection of susceptible mice with cvb3, modifies the immune response to a third party antigen during the acute stages of disease. an understanding of such cvb3,-i mmune cell interactions is criitical for gaining insight into the pathogenesis and persistence of enteroviral infection both within and outside of the immune system. understanding the pathogenic mechanisms of virus infections depends upon identification of viral gene products involved in host-virus interactions in vivo. many viral gene products which affect pathogenesis in vivo are nonessential for virus replication in vitro. therefore, we constructed insertion or deletion mutants in the hind i11 j and i regions of murine cytomegalovirus (mcmv) to assess the importance of these genes in in vivo replication of virus as well as interaction with immune regulatory and effector cells. three mutants replicated in nih 3t3 fibroblasts as efficiently as wild-type virus, thus confirming the nonessential nature of the genes. deletion of 2.8 kb in hind i11 j and of 7.7 kb in hind i11 j and i resulted in mutants with reduced replication in target organs in vivo. an insertion in hind i11 j had no significant effect on replication in mouse salivary gland, but curtailed growth of the virus in footpad fibroblasts and subsequent priming of mcmvspecific cpl precursors in the draining lymph node. in addition, the 7.7 kb deletion mutant was unique in its failure to replicate in a permissive, differentiated macrophage cell line. current studies aim to further define the role of this gene region in hcmv pathogenesis. that in vv5-4 progression o f v v life cycle is blocked at the the uncoating ii or replication stage. furthermore, host protein synthesis in v v 5 -4 cells is not shut-off after v v infection suggesting uncoatingll/replication is important for determining cell fate. since this mutant clone vv5-4 was isolated b y retroviral insertional mutagenesis. a cellular gene that was integrated by single retrovirus was isolate and codes for a 5kb transcript in northern blot analysis. a 2 kb cdna was isolated and codes f o r a novel polypeptide o f 800 amino acids that show no homology with known proteins. experiments are in progress to understand t h e role of this cellular gene in vv infection. in addition, a two-hybrid system will be employed to identify a n y viral gene product interacting with this cellular target u s i n g o u r laboratory's s t a n d a r d s t r a i n o f a/japan/305/57 in in uitro assays of viral infectivity of the mouse b-lymphoma a20-1.11. we have found that although infection sensitizes the a20 cells for recognition by both class i and class i1 restricted ctl.'s and leads t o high surface expression levels o f hemaglutinin (ha), the infected a20 cells grow through t h e infection, ha expression declines, and few of the infected cells die. in contrast, using a second s t r a b of a/japan/305/57 from a different passage history, we find that n o t only are the infected a20 cells sensitized for lysis by both class i and class ii restricted ctl's, exhibiting even higher levels o f ha surface expression, but in addition, most cells in the infected population die with characteristics of apoptosis. the two virus strabs appear to be highly related because in various assays we have found t h e m antigenically indistinguishable in both their t-and b-cell epitopes. using these two virus strains in a n in uiuo dose response assay, we have found that o u r laboratory's standard strain will kill all mice infected with a 10-2 the therapeutic and prophylactic antiviral efficacy of interleukin-12 (il-12) was studied using murine models of herpes simplex virus (hsv) and murine cytomegalovirus (mcmv) infection. therapeutic intraperitoneal administration of il-12 commenced 6 hours after mice were infected w i t h hsv, and was continued daily for a total of 5 days. il-12 therapy improved the survival rates of mice w i t h systemic hsv infection, compared to that of placebo treated infected mice. since neutrophilia and thrombocytopenia were common in dengue patients,we are investigating whether bm may be accompanied with abnormal hematopoiesis. using ficoll-hypaque separation, mononuclear cells prepared from the total bm cells. 1 .0x106we~ells/well of both adherent and nonadherent cell types were infected with two different den-2 virus strains : (1) thiland strain isolated from the dengue hemorrhagic fever(dhf1 patient and (2) taiwan strain isolated from the dengue fever(df) patient at moi=l and collected the suprnatants at various time points for assay. the preliminary data showed that : (a) adherent cells were infected with den2 and dhf virus strain replicated much more efficiently (2.3x104pfu/ml on day 3 p.i.1 than df strain; (b) nonadherent cells were 10 times less efficient to produce viral yields than adherent cells (dhf and2 df strains peaked at 6.3~10' pfu/ml and 1.5~10 pfu/ml on day 3 p.i., respectively); (c) addition of gm-csf to nonadherent cells increased virus infectivity and productivity. in conclusion, den viruses were able to infect bm cells and the more virulent virus strain isolated from dhf patients had better replication capability in human bone marrow cells. future studies on cytokines and immunologic evaluation are in progress. number of gene products whose function is to modify host responses. recent evidence suggests that a subclass of poxvirus-encoded host response modifiers interferes with host defense responses, and thus can be termed immune or inflammatory response modifiers (irms). several poxvirusencoded irms possess homology to mammalian proteins which are associated with host defenses, while other poxvirus irms have little sequence homology to immune-effector molecules or their receptors. the ultrecht strain of rabbitpox (rpv) virus induces a large reddish pock on the chorioallantoic membrane (cam) of the chicken embryo. rpv mutants lacking the spi-2/crmn38k gene trigger an inflammatory response by 36-48 hr after inoculation, similarly to that found for cowpox virus (brighton red strain) mutants. rpv mutant viruses with a deletion of the ps/hr gene, a gene which has recently been associated with virus host range and virion maturation, elicited an inflammatory response on the chicken embryo cam at 48-72 hr after virus inoculation. thus, another poxvirus-encoded gene product has been identified that has irm functional activity which would not have been predicted by sequence analyses. poxviruses have recently been found to possess a herbert w. virgin iv, center for immunology and division of infectious diseases, washington university school of medicine, st. louis, mo 63110 murine cytomegalovirus (mcmv) follows three stages of infection in the immunowmpetent host. following the initial acute infection, a persistent infection remains in which lytic virus resides in the acinar cells of the salivary glands. finally a third stage of infection exists which is characterized by a lack of detectable infectious virus. it is not known whether mcmv remains persistent at some low level during this thiid stage or establishes molecular latency. to test this, spleens, kidneys and salivary glands from mice 2-8 months post infection were evaluated by two sensitive assays. sensitivity was tested using virus co-cultured on murine embryo fibroblasts (mefs) for 14 days. by this method, 1 pfu mcmv was reproducibly detected in the presence of sonicated spleen, kidney and salivary glands diluted 1:lo. scid mice were used as a second detection assay for mcmv. using 46 scid mice and different doses of virus, the lds, of mcmv in scid mice was 2-3 pfu. mcmv infections were detected when 10-25 pfu were added to sonicated spleens, kidneys, and salivary glands prior to injection into scid mice. using both co-culture with mefs and injection of tissue into scid mice, we have tested a total of 34 spleens, 34 kidneys, and 37 salivary glands and have detected no persistent virus. while no preformed virus existed in these tissues, reactivation from explanted spleen, kidney and peritoneal exudate cells occurs in culture after 10-40 days. in addition, lytic mcmv was detected in scid mice 28 days after injection with 2(10)' kidney cells from latently infected mice. following identical transfers of latent spleen cells, mcmv was not detected in scid mice although explants from the same organs reactivated in vitro. using facs sorting, panning, magnetic bead separation, and reactivation assays, individual cell types have been analyzed for the presence of mcmv genome and the ability to reactivate virus. heat shock proteins (hsp) are expressed in all organisms in response to a variety of stresses, including virus infection. however, since viruses are not known to encode hsp genes, this represents expression of cellular hsps. we have found a dramatic induction of the 72kda hsp in the ovaries of w-infected mice, and using primary ovarian fibroblasts, demonstrated hsp72 mrna within virus-infected cells. the physiological role of hsps expressed during virus infection is unknown, although hsps act as molecular chaperones to facilitate protein folding and assembly in unstressed cells, and a number of bacterial hsps are essential for bacteriophage replication and morphogenesis. in order to determine whether hsp72 expression was beneficial to vv replication, we constructed a recombinant vv which encoded murine hsp72, but found that this virus grew to similar titres as control viruses, both in vitro and in vivo. in particular, kinetic studies of virus growth in an hsp72-negative cell line showed no difference from control viruses, and the presence of hsp72 did not influence the virulence of infection in immunocompromised nude mice. these results are interesting given that hsp70 proteins act as molecular chaperones and that hsp72 can be found in association with vv proteins. we suggest that this association may simply reflect the inherent affinity of hsp70 for non-native proteins, and the close physiological proximity of hsp and nascent viral proteins within the virus infected cell. our results indicate that the functional significance of these interactions are minimal with respect to the outcome of virus infection. furthermore, although the expression of hsw2 in vv-infected mice coincides with the peak anti-viral cellular immune response, hsp72 does not appear to be immunologically significant, as we cannot demonstrate any immunological responses to hsp72 during the course of vv infection. the effect of in vivo neutralisation of ifn-y on cytokine production during influenza infection was compared in cs7/bl6 and balb/c mice. similar cytokine profiles were obtained on in v i m restimulation of mln or spleen cells from virus-infected mice of each strain. at days 7 and 10 postinfection, mln or spleen cells from both strains of mice produced il-2, il-6, il-10 and ifn-y, but little or no il-4 or il-5. however, following in vivo treatment with anti-ifn-y antibody, production of 1l-4 and il-s was induced in mln and spleen cells of balb/c mice whereas those from cs7/bl6 mice showed little change in cytokine profile. an increase in il-6 and 1l-10 production was also observed on in virro restimulation of splenocytes from balb/c mice. however, significant amounts of 1l-2 and ifn-y were still secreted in these cultures. in v i m neutralisation of ifn-y in the restimulated cultures had little effect on the production of other cytokines, regardless of whether this cytokine had also been neutralised in vivo .despite the change in cytokine profile, anti-ifn-y treatment had no effect on the kinetics of viral clearance in balb/c mice. a similar situation was seen for c57/bl6 mice. congenic strains of mice are currently being used to determine whether the effect on cytokine profiles is associated with the mhc haplotype. wunner, and steven l. spitalnik, department of pathology and laboratory medicine, university of pennsylvania and the wistar institute, philadelphia, pa 19104 rabies virus glycoprotein (rgp) is the only glycoprotein on the viral surface, is the target of viral neutralizing antibodies, and is the viral protein that interacts with the host cell receptor. rgp of the era strain is a 505 amino acid type i membrane glycoprotein with 3 potential n-glycosylation sites at asn37, asn247, and asn319. due to rgps central role in the immunology and biology of rabies, it is important to determine its three-dimensional structure. towards this end we produced a recombinant form of rgp that may be more amenable to analysis by x-ray crystallography. to avoid the use of detergents, we constructed a soluble 434 amino acid form of rgp lacking a transmembrane domain (rgpt434). rgpt434 was secreted by transfected cells and was appropriately glycosylated, assembled, antigenic, and immunogenic. since n-glycosylation was required for rgpt434 secretion, we minimized the number of n-glycans by site-directed mutagenesis. interestingly, rgpt434 was secreted only when asn3l 9 was n-glycosylated. in contrast, full-length rgp was expressed at the cell surface a any of the three potential sites was n-glycosylated. soluble inhibitors of n-glycosylation were used to simplify the structures of the n-glycans on rgpt434. although inhibiting alpha-glucosidases with castanospermine blocked secretion, inhibiting any further processing with deoxymannojirimycin had no effect on secretion. finally, to simplify the purification process, a tail of 6 his residues was added to the cooh-terminus of rgpt434. this modification did not affect secretion, antigenicity, of assembly of the recombinant glycoprotein, but did allow purification using ni+2-agarose chromatography. in summary, we constructed a soluble form of rgp with a minimal number of homogeneous n-glycans and an "epitope" tag. this form of rgp is structurally similar to the extracellular domain of the full-length protein and may be amenable to analysis by x-ray crystallography. endogenous retroviral genes (ev) are well characterized in chickens. our laboratory has established chicken lines with single and multiple ev loci and, compared to ev negative birds, these lines exhibit a delayed and suppressed titer of neutralizing antibody in responses to avian leukosis virus (alv) challenge. to address the effects of ev genes on the cell mediated immune response we have developed an in-vitro assay to monitor the in-vivo induction of mhc restricted cytotoxic t cells (ctl) induced by alv. using this assay, we find the ctl response in birds expressing the ev-21 locus is reduced 85 to 95% compared to ev negative birds having nearly identical (eighth backcross) genetic backgrounds. other ev loci (1,6,10 and 11 ) also reduce the ctl response to alv in mhc matched chickens with different genetic backgrounds. ' national public health institute. helsinki. finland universities of tampere, qulu and 'helsinki, finland coxsackie b virus infections have been associated with clinical iddm in seveml previous studies but their initiating role in the slowly progressing beta-cell damage was for the fmt time directly implied in our recent prospective. nationwide dime study. siblings of the index iddm cases who progressed to clinical iddm experienced enterovim infections more frequently than nonprogressing siblings during prospective follow-up period. enterovim infections were initially demonstrated by detecting significant increases in serum class-specific cross-reactive enteroviral antibodies by using radioimmunoassay (ria) with the heavy-chain capture principle. causal association benveen these infections and the pathogenesis of iddm was suggested by temporal association of sercconvenions of viral antibodies with increases in islet cell antibody levels which are considered to be markers ok ficell damage. the possibility of the existmce of specific diabetogenic s & s of enternviruses was then considered. to identify the serotypzs responsible for the infections, successive sera from subjects exhibiting seroconvenion of autoantibodies coinciding with an increase in enteroviral antibodies were anlyzed by plaque n e u n a t i o n assay using a set of different enterovhes. the results indicate that several enternvirus serotypes may be associated with the progressive prediabetic p~ocess. not only different coxsackie b but also coxsackievirus a9 -infections were found to coincide with seroconvenions of islet-cell and insulin auwantibodies. it is known that an immunogenic region of gad 65. one of the major isletcell antigens, shows a high degree of homology with the 2c protein of cbv-4. studies on potential immunological cross-reactions between enteroviruses and two significant autoantigens, hsp60 and gad65, are in progress. this is carried out by using immunohistochemistry in infected cell lines and in frown sections of human pancreas with antibodies induced by synthetic peptides and natural and expressed viral proteins, the role of p53 alterations in human malignant pleural mesotheliomas (mpm) is unclear. immunostaining (ipox) of snap frozen mpm specimens revealed p53 expression in 31 of 52 (60%). p53 detection by ipox is usually associated with mutated p53. sscp for exons 5-9 revealed p53 alterations in 2 of 25 specimens, and loh at exon 4 was seen in only 2 of 12 evaluable specimens. thus, most of these specimens appeared to contain wild-type (wt) p53. we recently reported that sv40 induces mesotheliomas in rodents, and that sv40-like sequences are present and expressed in mpm. of note, sv40 large t antigen (tag) binds and inactivates wt p53 i n ! , abolishing its tumor suppressor function. we theorized that the immunohistochemical persistence of p53 may result from its physicdl binding with tag. tag was detected by ipox in 32 (62%) of these mpm specimens. expression of tag was significantly associated with coexpression of wt p53 (p2 = 0.008, fisher's exact text), and preliminary experiments suggest co-precipitation of p53 and tag. finally, we demonstrate that waf-1 is not detectable in mpm expressing wt p53 suggesting that p53 is inactive. we speculate that tag expression in mpm binds to and inactivates p53 contributing to the malignant phenotype. human papilloma virus (hpv) infection is involved in 90% of cervical carcinomas and possibly 95% of cervical intraepithelial neoplasia (cin). at present it is unclear whether patients infected with the transforming hpv types can mount efficient t cell responses. to address this issue we examined the ctl response of peripheral blood lymphocytes from patients attending a cervical neoplasia outpatient clinic. all were diagnosed hpv positive with various stages of c m and nine patients were selected for hla-a2 expression by facs analysis. pbmc were stimulated with caski, a cervical carcinoma cell line demonstrated to be hf' v type 16' and hla-a2'. culture media consisted of rpmi-10% human serum supplemented in three cases with 15 u/ml il-7, in another three cases with 3 u/ml il-2 and 15 u/ml il-7, and in the final three cases with 10 u/ml il-2 and 15 u/ml il-7. the ctl were screened for reactivity to caski and in addition the cervical carcinoma cell line c33a (hpv, hla-a2') transfected with either the hpv type 16 e6 or e7 genes. one patient's cells maintained with 10 uiml il-2 and 15 ulml il-7 showed hpv e7 protein specific cytotoxicity in a hla-a2 restricted fashion. these ctl lysed the caski stimulator cell line but not the nk sensitive target k-562. the ctl efficiently lysed a c33a-e7 clone but in contrast a vector only transfectant was not lysed. the lysis of c33a-e7 was blocked with a-cd3 and a-cd8 antibodies at 66% and 41% respectively. facs analysis determined that this ctl population was 92.8 % cd3'cds' and 7.3% cd3'cd4'. limited e6 recognition was also observed but has not been hlly characterized to our knowledge this report represents the first demonstration of hpv e7 responsive ctl isolated from the peripheral blood of hf' v infected patients. we have previously shown that proviral variants found in the peripheral blood mononuclear cells (pbmc) of hiv-1 infected individuals can interfere with recognition by autologous cytotoxic t-lymphocytes (ctl). abolition of recognition could be caused by interference with t h e processing of viral peptides, by a failure of the variant peptides to bind to mhc class-i molecules or by a failure of the mhc class-vpeptide complex to efficiently activate the tcr (t-cell receptor). these mechanisms could allow the virus to escape the immune surveillance by ctl. most studies so far have addressed the question of viral variation in t-cell epitopes by analysing proviral dna, extracted from the pbmc of hiv-1 infected individuals. however, the analysis of virion-associated rna offers several advantages. sequences found in viral rna are likely to be functional, a t least u p to the point of packaging into virus particles. furthermore, rna sequences represent virus which is currently actively produced. here, we present the sequence variation i n two hla-b8 restricted epitopes: p17-3 gag and amino acids 18-26 of the pol gene. both proviral dna from pbmc and viral rna sequences from plasma are discussed. the ability of viral variants to hind to mhc class-i molecules was assessed and the recognition of variant peptides by ctl was tested. variants were also tested for their ability to antagonize recognition of the index sequences. we show that viral variants with the ability to interfere with recognition by ctl are not only found in proviral dna but also in virion associated rna. objective: clinical course of hiv-1 infection may be influenced by an effective host immune response against hiv-1 and/or by viral properties. to investigate the cellular immune response to hn-1 we analyzed ctl activity against different hiv-1 proteins in long-term asymptomatic hiv-1 infection as well as during rapid progression to aids. methods: 10 longterm asymptomatic individuals with cd4 counts >500 celllpl after more than 8 years of infection were selected from the amsterdam cohort study on aids versus 10 subjects who progressed to aids < 5 years. ctl activity was measured on "cr labelled hla matched or autologous b-lcl, infected with rvv expressing hiv-1 ag. both bulk and limiting dilution ctl assays were performed longitudinally with pbmc after ag-specific stimulation. sequences of ctl epitopes were determined in homologous virus isolates resulrs: different kinetics of anti-gag ctl responses were observed in rapid progressors. in any case ctl responses disappeared during progression to aids. in long-term asymptomatic subjects persistent ctl responses were observed together with low viral load. conclusions: sustained, broad anti-hiv cellular immunity may correlate with maintenance of the asymptomatic state in long-term survival by controlling viral replication. enteroviruses are a large group of positive stranded rna viruses known to be responsible for a number of distinct disease entities. recombination is thought to be capable of generating new enterovirus strains that cause significant morbidity. for example, enterovirus 70 which was responsible for a pandemic of haemorrhagic conjunctivitis and poliomyelitis is thought to have originated by recombination between a coxsackie b like virus and another unidentified enterovirus. we are studying a group of echovirus 11 isolates from an outbreak of disease in southem india. sequence analysis within the 5' untranslated region reveals that these isolates fall into two groups that differ by -20% (equivalent diversity to that seen between between published sequences of poliovirus 1 and coxsackie a 9 virus). these two groups of viruses also differ in their cell tropism. isolates defined as group 1 by their 5'utr sequence grow equally well on ht29 cells (a human colon carcinoma cell line) and vero cells. isolates of group 2, with one exception, grow only on ht29 cells. analysis of the structural proteins of these isolates revealed differences in migration that correlated with their cellular tropism. thus, significant genotypic and biological diversity exists amongst these virus isolates. one virus isolate had the 5' untranslated region sequence of a group 1 virus but the protein profile and cellular tropism of a group 2 virus. the best explanation of these findings is that this anornolous isolate is a natural recombinant between the parented strains. both the ease with which viable recombinants are generated and the diversity present within this one enterovirus serotype increase the potential for the production of novel pathogenic enterovirus strains. dominant susceptibility to polyoma tumors in inbred wild mice, sharon r. nahill, yupo ma, john carroll and thomas l. benjamin, department of pathology, harvard medical school, boston, ma 021 15 polyoma virus (f' y) is a mouse dna tumor virus which, under appropriate conditions, causes tumors in a wide variety of cell types. generation of tumors is a function of both the viral and host genomes. lukacher et al. have recently described a dominant gene, pyv', carried by the c3-i mouse strain, which confers susceptibility to py-induced tumors mapping and immunological analyses indicate that py4 is the mouse mammary tumor virus 7 superantigen (mtv 7 sad gene, which deletes t cells required for py tumor immunosurveillance in h-2' mice. to determine the generality of endogenous superantigens as determinants of susceptibility and to reveal potentially novel mechanisms of susceptibility, we have looked for dominant susceptibility @ s ) gene(s) id newly established and genetically diverse inbred wild mouse strains, czech i1 and pedatteck (peru). both strains are susceptible to py as 100% of infected animals develop a full profile of tumors. crosses between cs7br, whose resistance is contributed by the major histocompatibility (mhc) locus, and susceptible peru or czech 11, yield f1 progeny which are fully susceptible, indicating a dominant inheritance pattern of susceptibility the incidence of tumor-bearing backcross animals [((peru x cs7br) x c57br) and ((czech i1 x cs7br) x c57br)i suggests that ds is due to at least one, but not more than two genes. amplification of genomic dna from the czech i1 and peru mice by pcr using primers specific for mtv 7 sag indicates that both strains are negative for proviral mtv 7 sag. furthermore, the mechanism ofds in these mice may be independent of all mtv sag as pcr using primers specific for the highly conserved region of mtv sag is unable to amplify mtv dna from peru or czech i1 genomic dna. these results indicate that, like the c3hibi, the pedatteck and czech i1 contain gene(s) which overide the resistance to py-induced tumors contributed by the mhc of the c57br parent and which may cause tumors via a novel, mtv sag-independent mechanism. we have initiated efforts to map the ds in peru and czech i1 mice using pcr and primer pairs flanking simple sequence length polymorphisms. fis-2 is a low leukemogenic, but relatively strong immunosuppressive variant of friend murine leukemia virus (f-mulv). this variant was originally isolated from t-helper cells of flc-infected adult nmrl mice. compared to f-mulv, fis-2 suppresses primary antibody response more efficiently in infected mice. some of the fts-2 infected adult nmri mice developed a disease resembling the acquired immunodeficiency syndrome induced by hiv. restriction mapping and nucleotide sequence analysis of fis-2 show a high degee of homology between this variant and the prototype f-mulv clone 57. in this study we have attempted to localize the genomic determinant of fis-2 which is responsible for induction of a strong suppression of primary antibody response. six chimeric viruses of fis-2 and f-mulv were constructed. the primary antibody response of the mice infected with these chimeric viruses were investigated. the results of these experiments will be presented. anti-fmdv antibodies, as measured in an elisa capture assay, were cross reactive. b) cellular: proliferative (cd4) t cell responses of peripheral blood mononuclear cells (pbmc) were low or undetectable during primary responses to vaccine or virus, and frequently low during secondary responses. for good t cell proliferation in vitro, multiple immunisation is required. this may reflect preferential stimulation of the th2 cd4 t cell subset. interestingly, when cd4 responses were observed, cd8 tcell responses were also detectable. 2 . recognition of individual viral proteins a) expression cloning: structural and non-structural protein pseudogenes were cloned from cdna by pcr. expressed in pgex-3xuc. and purified by sds-page. b) humoral: structural and non-structural proteins were recognised by infected animals. a good anamneetic antinon-structural response was only observed when the boosting serotype differed from the serotype stimulating the primary response. c) cellular: both structural and non-structural proteins were recognised and some were cross reactive. interestingly, vp1 was strain specific, and the polymerase (3d) was the most immunogenic and cross reactive. d) a construct comprising 3d and the immunodominant vp1 epitopes was prepared and tested. in common with other herpesviruses, the envelope glycoproteins of equine herpesvirus 1 (ehv-1; equine abortion virus) are major determinants of the infectious process and pathogenicity, and are inducers of humoral and cell-mediated immune responses. as such, they are candidates for components of subunit vaccines against ehv-1. to generate useful amounts of individual ehv-1 glycoproteins, we have constructed recombinant badoviruses capable of expressing glycoproteins c, d, h (gc, gd, gh ) in insect cells, and have evaluated the recombinant products as innnunogens in a murine model of ehv-1 infection. au three glycoproteins induced serum (elisa) antihodies to ehv-1, and ehv-1 gc and gd also induced neutralizing antibody responses. following intranad challenge with infectious ehv-1, protective immunity, as demonstrated by acelerated clearance of virus fiom respiratory tissues to below detectable levels, was evident in mice immunized with either recombinant gc or gd. in contrast, gh-immmkd mice did not develop detectable neutralizing antibody, and did not clear challenge virus more rapidly than controls. delayed type hypersensitivity and lymphoproliferation responses to ehv-1 antigen were observed for each of the ehv-1 glycoproteins, and in experiments with gdimmunized mice, a role for cell mediated immunity in protection was confirmed by adoptive transfer and t-cell depletion experiments. the data provide support for the potential of glycoproteins c and d as a subunit vaccine against ehv-1. molecular pathogenesis of ural infeetiom 52-331 enterovirus-immune cell interactions: implications in enterovirus-induced diseases we have also evaluated the effect of virus infection on the humoral immune response to cvb3, infection in adolescent c3h/hesnj mice. antigen presenting cell, 1-helper cell and 8-cell function were evaluated utllizlng a sheep red blood cell (srbc) plaque assay. mice were injected intrapentoneally (ip) with lo5 plaque forming units of cvb3, at day 0 and with lo7 srbc's at days 0, 2, 3 and 4 post-cvbb, infection. splenocytes were harvested 4 days post-srbc injection, mixed with target srbc's and guinea pig complement and incubated. plaques were then quantitated. results: cvb3, was associated with 12.9% to 17.4% of cd-8 positive t-cells and w a 11 % to 26% of adherent splenocytes. after mitogen (lps and con a) stimulation, b-cells and adherent cells were demonstrated to be permissive for viral replication. a 248% and 738% under non-stimulated conditions. an average of 1 % of virus is cell-associated (plaque north america. bruce anderson, teny yates, norah torrez-martinez, wanmin song, brian hjelle. university of new mexico, albuquerque, n.m.we recently identified a new species of hantavirus (hmv) associated with the harvest mouse reithrodontomys megalotis (hjelle b et al, j. viroj. 1994, in press ). an arizona woodrat (neotoma mexicana) was found to he infected with hmv, presumably through "spillover". hmv is most closely related to the four comers hantavirus (fcv) of deer mice (genus peromyscus). the nucleocapsid gene and protein of hmv differ from those of fcv by 24% and 15% of residues, and the 1896 nt s genome is shorter by 163 nt. we surveyed 174 reithrodontomys animals captured in the u.s. and mexico for hantavirus antibodies; 27 (15.6%) were positive. s segment cdnas were amplified and sequenced from seropositive animals captured in california (4), arizona (3), new mexico (l), and mexico (2). a monophyletic clade of hmv-like agents was identified at all sites, although an r. megalotis infected with an fcv-like virus was also identified in the state of zacatecas, mexico. nucleotide sequence distances among members of the hmv clade were up to 15.5%. but amino acid distances were less than 2%. hmv is enzootic in harvest mice throughout much of north america, and can also infect wood rats. htlv i-associated myelopathy/tropical spastic paraparesis (hamnsp) is a slowly pro ressive neurological disease characterized by perivascuaar mononuclear infiltrates in the cns. htlv i-specific cd8+ ctl are found in pbl and csf of infected patients with htlv i-associated neurological disease but not in htlv i seropositive individuals without neurological involvement. previous studies have shown that in hla-a2+ patients, htlv i-specific cd8+ ctl restricted by hla-a2 recognize a peptide derived from the htlv i tax protein (tax 11-19 llfgypvw). in the present study, we have analyzed the potential of these tax-specific ctl to recognize addtional peptides. our results demonstrate that a subpopulation of high affinity cd8' tax 11-19 specific ctl clones cross-react on a self peptide derived from the se uence of myelin-associated glycoprotein (mag 556-564 vl&sdfri) presented by hla-a2. these obsenatlons suggest that the demyelination process in hamltsp may be,due, in part, to virus-specific ctl recognition of a self myelin component that is independent of htlv i infection. development of pathology varies widely between different strains of mice after intracerebral inoculation with the so-called 'docile' isolate of lymphocytic choriomeningitis (lcm) virus. the c3hebfej and blo.br/sgsnj mouse strains have been of special interest because they display autoimmune hemolytic anaemia with varying degrees of apparent immunological involvement. in this study, we examined the role of cd4+ t helper cells in this autoimmune response by treating mice with the cw-specific gk1.5 monoclonal antibody. we also determined if polyclonal activation of b lymphocytes, induced either by lcm virus or by lactate dehydrogenase-elevating virus, another well known b cell activator, correlated with the development of anaemia in these mice. our results strengthened the central role of the immune system in the anaemia in c3h mice by showing that depletion of cd4+ cells largely, if not completely, abrogated this anti-erythrocyte autoimmune reaction. as reported by others, we found that the anaemia was more mild in b 1o.br mice than in c3h mice. however, we could not confirm the difference in the degree of b lymphocyte polyclonal activation between these mice. furthermore, lactate dehydrogenase-elevating virus had no apparent effect on erythrocytes, even though this virus also induced a sharp increase in plasma igg levels. one of the two class i mhc (h-pkd)-restricted immunogenic sites identified on the influenza strain aijapanl57 (h2n2) hemagglutinin (ha) encompasses two distinct partially overlapping epitopes, mapping to residues 204-212 and 210.219. when we investigated the magnitude of the ctl responses of balwc mice to the two overlapping epitopes, we found that while the nhrterminal nonamer epitope is immunodominant, eliciting vigorous ctl responses in njapanl57-immunized balb/c mice, the ctl responses to the cooh-terminal decamer epitope are weak and variable. the c-terminal epitope subdominance seems to be due to factors other than inefficient processing of the epitope in vivo because ctls generated by priming mice with recombinant sindbis viruses expressing only one of the ha 204-219 subsites displayed patterns of responsiveness similar to that of influenza virus primed ctls. limiting dilution ctl assays showed that the ctl precursor frequency (pctl) of the nterminal epitope is at least ten fold higher than the pctl of the cterminal epitope, implying that the low and variable pattern of cterminal specific responsiveness was due to the limited t cell precursors in the c-terminal specific ctl repertoire. this was further confirmed by the limited heterogeneity in the cross reactivity patterns displayed by the c-terminal specific ctl for an ig vh fragment and the ha 210-219 epitope of influenza strain a i m 5 7 in short term bulk cultures, and the facs analysis of tcr vg chain usage. taking these together with our previous observation that some jha 210-219 specific ctls can also crossrecognize an ig vh fragment. these studies had provided a strong evidence that ig gene products may influence t lymphocyte function and repertoire development. we have previously described the identification of homologous regions in the c-terminus of hiv-1 gp41 and in the n-terminus of hla class i1 beta chains. forty percent of patients infected with hiv-i virus were shown to have antibodies which bind to the homologous sequences, as well as to native hla class i1 molecules. affinity purified crossreactive antibodies (crab) were shown to have direct blocking effects on normal t cell responses to recall antigens, and could mediate adcc of hla class ii+ cell lines.in order to determine the contribution of such antibodies to disease progression, we obtained longitudinal plasma samples from patients in the macs study. in a first study, it was found that the presence of high titers crabs correlated with a more rapid disease progression (p = 0.027 by fisher two tail analysis)in a second, 7 year-longitudinal study of 12 progre.ssors and 12 stable patients we found: (1) the production of crab was seen in 70 -80% of rapid progresson, while the true stables produce only infrequent low-titers crab. (2) in rapid pmgressors, production of crab preceded by 2-3 years the marked drop in cd4 counts. (3) crab production did not correlate with the degree of hyperglobulinemia in these patients. (4) the presence of crab during the asymptomatic stage correlated with early loss of t-helper responses to recall antigens.we are currently establishing whether periodic measurements of crab in patients sera could be valuable in predicting a drop in cd4 counts and disease progression. the lymphokine ifn-y is i pleiotropic insnunomodulator and possesses intrinsic antiviral activity. we studied its significance in the development of antiviral immune responses using ifn-7 receptor deficient (ifn-yr-'.) mice. after inoculation with live attenuated pseudorabies virus (prv) the mutant mice showed no infectivity titers in various tissues and transient viral ag expression only in the spleen similar as in wild-type mice. however, the absence of the ifn-yr resulted in increased proliferative splenocyte responses. the prv-immune animals showed a normal ifn-1 and 11-2 production, without detectable 11-4, and with decreased 11-10 secretion in response to viral ag or con a. immunohistochemically, an increased ratio of ifny/i1-4 producing spleen cells was found. after immunization with either live attenuated or inactivated prv, ifn-yr"' mice produced significantly less antiviral antibody (ab), and more succumbed to challenge infection than the intact control animals. the reduction in ah titers in the mutant mice correlated with lower protection by their sera in transfer experiments. thes? findings are in line with the strong enhancing effect of exogenous ifn-y on rabies virusand prv-specific igg responses. our data demonstrate that a physiological ifn-y system is surprisingly not critical for the generation of antiviral th-i-type and the suppression of th-2-type cytokine responses. the lymphokine, however, is an important mediator in the generation of protective antiviral ab. key: cord-271130-6s79q1c1 authors: filoni, claudia; helfer-hungerbuehler, a. katrin; catão-dias, josé luiz; marques, mara cristina; torres, luciana neves; reinacher, manfred; hofmann-lehmann, regina title: putative progressive and abortive feline leukemia virus infection outcomes in captive jaguarundis (puma yagouaroundi) date: 2017-11-17 journal: virol j doi: 10.1186/s12985-017-0889-z sha: doc_id: 271130 cord_uid: 6s79q1c1 background: feline leukemia virus (felv) is an exogenous gammaretrovirus of domestic cats (felis catus) and some wild felids. the outcomes of felv infection in domestic cats vary according to host susceptibility, virus strain, and infectious challenge dose. jaguarundis (puma yagouaroundi) are small wild felids from south and central america. we previously reported on felv infections in jaguarundis. we hypothesized here that the outcomes of felv infection in p. yagouaroundi mimic those observed in domestic cats. the aim of this study was to investigate the population of jaguarundis at fundação parque zoológico de são paulo for natural felv infection and resulting outcomes. methods: we investigated the jaguarundis using serological and molecular methods and monitored them for felv-related diseases for 5 years. we retrieved relevant biological and clinical information for the entire population of 23 jaguarundis held at zoo. post-mortem findings from necropsies were recorded and histopathological and immunohistopathological analyses were performed. sequencing and phylogenetic analyses were performed for felv-positive samples. for sample prevalence, 95% confidence intervals (ci) were calculated. fisher’s exact test was used to compare frequencies between infected and uninfected animals. p-values <0.05 were considered significant. results: in total, we detected evidence of felv exposure in four out of 23 animals (17%; 95% ci 5–39%). no endogenous felv (enfelv) sequences were detected. an intestinal b-cell lymphoma in one jaguarundi was not associated with felv. two jaguarundis presented felv test results consistent with an abortive felv infection with seroconversion, and two other jaguarundis had results consistent with a progressive infection and potentially felv-associated clinical disorders and post-mortem changes. phylogenetic analysis of env revealed the presence of felv-a, a common origin of the virus in both animals (100% identity) and the closest similarity to felv-faids and felv-3281 (98.4% identity), originally isolated from cats in the usa. conclusions: we found evidence of progressive and abortive felv infection outcomes in jaguarundis, and domestic cats were probably the source of infection in these jaguarundis. jaguarundis (puma yagouaroundi) are small diurnal felids that have several unpatterned color morphsbrownish-black, gray and reddish-yellow furand are protected across most of their range. the species occurs at low densities and has a decreasing population in the wild, despite being widely distributed throughout south and central america and occupying a broad range of habitats [1] . mainly due to displacement from nature and, to a lesser extent, to captive breeding, jaguarundis are commonly found in zoos and similar captive settings in brazil [2] . the feline leukemia virus (felv) is an exogenous, oncogenic, immunosuppressive gammaretrovirus that can establish persistent infections in domestic cats (felis catus) [3] . the prevalence of felv ranges from 1% to 8% in healthy cats almost everywhere in the world and is usually higher when sick cats are included [4] . felv is naturally transmitted by oronasal exposure to viruscontaining secretions, mainly saliva, but also in feces and urine [5] [6] [7] . the effects of felv are cytoproliferative diseases including lymphomas and myeloproliferative disorders; degenerative illnesses, such as anemia and leukopenia; and immunosuppressive diseases associated with opportunistic infections [3, 8] . felv infections tend to be rare or absent in many nondomestic felid species [9] [10] [11] [12] [13] [14] , except for the european wildcat (felis silvestris silvestris), a species very closely related to the domestic cat, in which felv appears to be endemic [15] [16] [17] . however, documentation of felv is becoming more common in wild felid species less closely related to the genus felis, which highlights the omnipresent threat that felv represents to the conservation of wild felids worldwide. felv has been shown to represent a major threat to the survival of critically endangered populations of iberian lynxes (lynx pardinus) in europe [18, 19] and to florida panthers (puma concolor coryi) in north america [20, 21] . in brazil, antibodies against felv have been detected in two freeranging pumas (puma concolor) and two jaguarundis; felv dna was detected in the following captive felids: an ocelot (leopardus pardalis), an oncilla (leopardus tigrinus) and two jaguarundis [22] [23] [24] . for domestic cats, the detection of the structural viral protein felv p27 in serum or plasma is used as a marker of infection and, in most cases, as a parameter for viremia. the outcomes of felv infection vary according to infectious challenge dose, route of challenge, and possibly host susceptibility and virus strain. the classification of felv outcomes in the domestic cat has been refined using sensitive molecular assays that detect and quantify proviral felv dna and viral felv rna, in addition to traditional serological and virological methods [25] [26] [27] [28] [29] [30] . in this regard, the main host response categories of felv infection in domestic cats have been redefined as abortive, regressive, and progressive infection. in short, those cats that abort infection do not show any evidence of virus infection, except for seroconversion. cats with regressive infection overcome viremia after an undetectable or transient initial phase by means of efficient cellular and humoral immune responses. these cats seroconvert, permanently harbor low to moderate felv proviral loads integrated in mononuclear cells, i.e., lymphocytes, and may or may not clear their plasma viral rna loads [25, 27, 31] . cats with progressive infection are constantly viremic, have low or no antibodies to felv, show elevated felv proviral and viral loads in peripheral blood cells and plasma and may develop felv-associated disease [25, 26, 28, 29] . in previous surveillance work, we detected felv infection in two captive-born jaguarundis (#1 and #4) as well as previous exposure to felv in two other captive-born jaguarundis (#2 and #22) among a population of 23 jaguarundis held at fundação parque zoológico de são paulo (fpzsp), brazil [23] . jaguarundis #1 and #4 tested positive for felv p27 antigen by sandwich elisa. felv proviral dna was detected in blood from both animals by quantitative real-time polymerase chain reaction (qpcr). the sampling for this previous study occurred between 2003 and 2004 (table 1) . by analogy, we hypothesized here that felv infection in p. yagouaroundi mimics the main outcomes observed for the domestic cat. thus, the aim of this study was to perform additional serological and molecular tests and monitor the population of jaguarundis at fpzsp for felv infection and development of felv-related diseases for 5 years (2003) (2004) (2005) (2006) (2007) . we retrieved relevant biological and clinical information for the entire population of 23 jaguarundis held at fpzsp, from birth or admission of the animals into the zoo until 2007. most animals were mature adults (n = 16), five were immature and two were geriatric; kittens were absent. three jaguarundis were wild born, while 20 were captive born at fpzsp. six females (two of wild origin) and three zoo-born males were reproductively active. eleven animals from the population were full siblings, and 16 were half siblings. the females (n = 9) averaged 4.28 ± 0.57 kg in body weight, and the males (n = 14) averaged 5.08 ± 0.49 kg in body weight. detailed biological data such as age, sex, weight, origin (captive or wild born) and the parental history for the 23 jaguarundis are presented in table 2 . for hematological analysis, several laboratories were used over time, and registries of results from the animals were not completed and standardized. all jaguarundis notes: felv-infected (#1, #4) and felv seropositive (#2, #22) jaguarundis are identified in bold. a for the wild-born jaguarundis (#3, #10 and #18), the dates refer to entrance in the zoo as their dates of birth are unknown. b immature adults (n = 5) were defined as jaguarundis in the age range of 6 months ≤ age < 24 months for males and 6 months ≤ age < 36 months for females; mature adults (n = 16) were defined as those in the age range of 24 months ≤ age < 10 years (120 months) for males and 36 months ≤ age < 10 years for females; geriatric jaguarundis (n = 2) were those aged ≥10 years for both sexes. c for two captive-born jaguarundis (#8, #9), the identities of the father and the mother, respectively, were not available were vaccinated regularly against feline herpesvirus 1 (fhv-1), feline calicivirus (fcv), feline parvovirus (fpv) and rabies lyssavirus. we monitored the potential development of felv-related diseases through clinical data provided by the veterinarian staff at the zoo and registered at the zoo archives. serological and molecular felv tests were performed, in addition to those previously published [23] , using samples that had been previously obtained or additional samples collected throughout the observation period. felv tests previously performed included p27 felv antigen detection by a sandwich elisa and the commercial immunoassay snap™ combo felv antigen/fiv antibody test kit (idexx laboratories inc., westbrook, me, usa), detection of antibodies against the recombinant non-glycosylated form of the felv gp70 surface glycoprotein (p45) by indirect elisa, detection of antibodies against felv p12, p15, p27, p58, and gp70 antigens by western blot and detection of felv proviral dna by real-time pcr. we investigated the presence of antibodies to felv transmembrane protein p15e in serum samples from six jaguarundis (#1, #3, #4, #5, #14 and #17) using the previously described assay [32, 33] . six jaguarundis (#1, #2, #4, #5, #10 and #14) died between 2005 and 2008 and underwent necropsy at the zoo; for these jaguarundis, relevant clinical and post-mortem findings from necropsies were recorded, and several tissues were collected for analyses. fragments of tissues from these animals were fixed in 10% formalin, embedded in paraffin wax and sectioned. the sections were stained with hematoxylin-eosin (he) on glass slides for histopathological analyses. tissue specimens from animals #1 and #5 (bone marrow, mesenteric lymph node and spleen) were also stored at ≤ −70°c. a saliva specimen from animal #1 was collected at first sampling using a sterile cotton swab immersed in virus transport media, which consisted of phosphate-buffered saline (pbs) balanced salt solution supplemented with 0.5% bovine albumin (bsa), antimicrobial agents (200 u/ml penicillin g, 200 u/ml streptomycin, 25 μg/ml fungizone and 6 μg/ml gentamycin) and was kept at ≤ −70°c. no additional material could be obtained from animal #2. to clarify the histogenesis of an intra-abdominal mass of tissue that developed in jaguarundi #5, we performed immunohistologic assays by the streptavidin-biotinperoxidase complex technique with a commercial immunoperoxidase kit (lsab kit, dako, glostrup, denmark) according to the manufacturer's protocol. we used silanized microscope slides to produce histologic slides, which were processed by usual deparaffination techniques in xylol and hydrated in alcohols (absolute, 95%, 70%, and distilled water). a microwave antigen retrieval technique using edta buffer, ph 9.0, was applied [34] . the sections were incubated overnight at 4°c with primary antibodies against the t-cell marker cd3 (rabbit polyclonal antibodies a0452; dako; diluted 1 in 500), the b-cell marker cd79 (mouse monoclonal antibodies; clone hm57; m7051; dako; diluted 1 in 500), and vimentin (mouse monoclonal antibodies; m0725; dako; diluted 1 in 100) as well as antibodies against proliferating cell nuclear antigen antibodies (pcna) (mouse monoclonal antibodies; m0879; dako; diluted 1 in 800). for all reagents, negative controls were generated by substituting the primary antibody with a class-matched immunoglobulin. these procedures were conducted using the logistics of the department of pathology, school of veterinary medicine and animal sciences, university of são paulo (usp), são paulo, sp, brazil. we investigated the presence of felv antigens in several paraffin-embedded tissues from the deceased jaguarundis #1, #4, #5 and #14. this investigation was carried out using the methods and logistics of the institute of veterinary pathology, university of giessen, giessen, germany [35] . total nucleic acid (tna) was extracted from 100 μl of edta-anticoagulated whole blood or buffy coat or from 200 μl of edta-anticoagulated plasma using the magna pure lc total nucleic acid isolation kit (roche diagnostics, rotkreuz, switzerland). tna was eluted into 100 μl of elution buffer and stored at −70°c until pcr testing was performed. rna was extracted from the saliva, plasma samples collected from jaguarundi #1 and from serum samples from jaguarundis #1 and #4 using the qiamp® viral rna mini kit (qiagen). gdna and rna were extracted from tissues (bone marrow, mesenteric lymph node and spleen) from animals #1 and #5 upon necropsy, as previously described [36, 37] . during all extractions, negative controls consisting of 100 μl of pbs were concurrently prepared with each batch of samples to monitor for cross-contamination. felv viral rna loads and proviral loads were investigated using real-time rt-pcr and pcr and primers targeting the u3 region of exogenous felv, as previously described [30] . for tissue samples, the total copy numbers detected per reaction were normalized to the glyceraldehyde 3-phosphate dehydrogenase (gapdh) gene as described [38] . felv subgroups a, b and c were investigated in the bone marrow, mesenteric lymph node, and spleen of the jaguarundi #1 and in the serum and plasma of jaguarundi #4 by conventional pcr using the felv-a specific primers rb59 and rb17; the felv-b specific primers rb53 and rb17 and the felv-c specific primers rb58 and rb47 as described [39, 40] . serum from jaguarundi #5 was tested for the presence of antibodies against feline immunodeficiency virus (fiv) using a commercial immunoassay snap™ combo felv antigen/fiv antibody test kit (idexx laboratories) and by western blotting, as described [41] . fiv real-time and conventional rt-pcr were performed from whole blood from jaguarundi #5, as previously described [42, 43] . endogenous felv-like sequences (enfelv) pcr tna samples from the whole blood or buffy coat of all 23 jaguarundis were tested for the presence of endogenous felv (enfelv) by the three qpcr assays enfelv-u3-1, enfelv-u3-2, and enfelv-env, as previously described [44] . felv env sequences were analyzed for the felv-positive jaguarundis #1 and #4 from tna of previously collected buffy coats. for comparison, a second sequence obtained from bone marrow of animal #1 was analyzed 1.4 years later, at the time of euthanasia. for the analysis of the full-length felv env sequences, genomic dna from these samples was amplified using pcr with the primers 5847f (5′ acatatcgtcctcctgaccac 3′) and 8197r (5′ gaaggtcgaaccctggtcaact 3′) [43] , yielding an approximately 2´370 bp product. to ensure high-fidelity amplification, the pcr was performed using phusion polymerase and hf buffer (finnzyme, ipswich, uk). pcr products were sequenced by microsynth (balgach, switzerland) after purification using the genelute pcr clean-up kit (sigma, fluka gmbh, buchs, switzerland). the jaguarundi felv-a env sequence was submitted to genbank [kr349469]. phylogenetic analyses were conducted using mega version 6 [45] . the felv env sequences were aligned using clustal w [46] . bootstrap support (1000 replicates) was calculated by the neighbor-joining (nj) [47] and maximum parsimony (mp) [48] methods, and results >70% were considered significant [49] . the mp tree was obtained using the subtree-pruning-regrafting (spr) algorithm [48] with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). all positions containing gaps and missing data were eliminated from the dataset (complete deletion option). for sample prevalence, 95% confidence intervals (ci) were calculated. fisher's exact test was used to compare frequencies between infected and uninfected animals. pvalues <0.05 were considered significant. the population of jaguarundis kept at fpzsp from 2003 to 2007 consisted of 23 captive jaguarundis. the animals presented good general condition at sampling. twenty animals presented discrete to moderate oral disorders, including one or more of the following conditions: gingivitis, presence of tartar, periodontitis, tooth loss and/or fractured teeth. no oral disorders were registered in three immature adults, #19, #21 and #23. at least once during the experiment, 15 animals presented episodes of diarrhea, 11 jaguarundis demonstrated transitory weight loss and three suffered from dermatological disorders, such as alopecia; for all these clinical conditions, the underlying causes of the problems were unknown. despite regular deworming, intestinal parasites were detected in 18 animals during the study period. in hematological analysis, most animals (n = 16) showed some degree of leukopenia (marked: n = 1; mild: n = 8) and leukocytosis (marked: n = 2; mild: n = 5). jaguarundi #1, which was felv positive, and jaguarundi #2, which was felv seropositive, showed mild transient leukocytosis. several (n = 12) animals showed mildly increased packed cell volume (pcv) and two showed markedly increased pcv in at least one sampling. jaguarundis #1 and #2 showed mildly increased pcv, and jaguarundi #22, which was felv seropositive, showed a markedly increased pcv. the increased pcv could be due to dehydration as the animals had been fasted before immobilization. six jaguarundis (#1, #2, #4, #5, #10 and #14) in the investigated population died from 2005 to 2008. jaguarundi #1 was euthanized 1.4 years after being diagnosed with felv infection [23] . this jaguarundi was euthanized because the zoo could not keep it segregated under good ethological conditions, although the animal was in good clinical condition. jaguarundi #5 developed a large intra-abdominal mass of tissue about 1 year after testing negative for felv. after clinical and ultrasonographic examinations, the animal underwent laparotomy for excision of the mass and died 3 days after surgery due to clinical deterioration. animals #2, #4, #10 and #14 died naturally during the period of study. for the six deceased animals, clinical conditions and main post-mortem findings are presented in table 3 . antibodies against p15e antigen were not detected to a level that is considered positive for privately owned domestic cats and the results from all the jaguarundis tested (#1, #3, #4, #5, #14 and #17) were negative (table 1) . the excised mass of tissue from jaguarundi #5 weighed approximately 0.25 kg and affected all layers of the wall of the jejunum. in addition, several small foci were present across the mesentery. there was multifocal necrosis as well as intense reactional fibrosis and proliferation of round cells arranged in dense blocks limited by delicate conjunctive septa. the cells were neoplastic and malignant and presented anisokaryosis, anisocytosis, and a high mitotic index. the tumor was found to be an intestinal b-cell lymphoma, as the cells stained positive for a b-cell marker (cd79) but not a t-cell marker (cd3) by immunohistology. no felv rna or provirus was detected in the tissues collected upon necropsy, including tumoral tissues, bone marrow, mesenteric lymph nodes and spleen, and no felv antigens were detected in paraffin-embedded tissues by immunohistological analysis (table 1) . because b-cell lymphomas can also be associated with fiv infection, jaguarundi #5 was tested for fiv by serology and pcr. the animal tested negative for antibodies to fiv in the commercial assay. in the fiv western blot, one band against p24 was detectable. felv rna and proviral dna were detected in two out of the 23 animals, namely, jaguarundis #1 and # 4. from jaguarundi #1, a saliva sample was available; it was felv rna positive, indicating shedding of felv at the time of sampling. felv was also demonstrated in the plasma sample collected at a second sampling from jaguarundi #4 upon necropsy 1.4 years later, indicating persistent antigenemia in this animal. for jaguarundis #1 and # 4, respectively, the absolute copy numbers of proviral dna were 4.26 × 10 7 and 3.49 × 10 7 copies of dna/ml of buffy coat; the absolute copy numbers of viral rna were 2.63 × 10 8 and 2.97 × 10 9 copies of rna/ml of serum. the results from all felv tests are presented in tables 1 and 4 . furthermore, felv could be demonstrated in the tissue samples collected at necropsy from jaguarundi #1, and felv antigens were confirmed by immunohistological analysis of paraffin-embedded tissue samples from deceased jaguarundis #1 and #4. for both jaguarundis, #1 and #4, felv-a was the only felv subtype identified; no felv-b or felv-c was detected. no enfelv was detected in the blood or buffy coat samples from any of the 23 jaguarundis. (fig. 1) . when the env surface unit from the felv detected in jaguarundis was compared with those found in iberian lynxes [eu293175 to eu293194], a sequence identity of approximately 97% was obtained (data not shown). we detected evidence of felv exposure in four (#1, #2, #4 and #22) out of 23 jaguarundis in the fpzsp (17%; 95% ci 5-39%). the remaining 19 jaguarundis were negative for felv in all serological and molecular tests performed. the population of jaguarundis presented clinical disorders that were common at fpzsp, as in any other captive setting in brazil [2, 12] , and could be associated with a multitude of causes. although the felv-positive jaguarundis also presented some conditions that could be related to felv [8] , our data were insufficient to prove a causal association. two captive-born male jaguarundis, the geriatric #1 and the mature adult #4, presented serological and molecular felv test results similar to the progressive felv infection outcome in domestic cats [25] . jaguarundis #1 and #4 were both antigenemic. antigenemia is usually used as a measure for viremia in domestic cats and is consistently found in domestic cats with a progressive felv infection outcome [26, 28] . additionally, blood, buffy coat, serum and tissue samples from both jaguarundis were positive for felv proviral dna and viral rna with high proviral and viral loads, respectively (table 4 ). while the presence of proviral dna attests to the integration of provirus into blood cells, the detection of viral rna usually indicates replicating virus. once the infection outcome has been established, high proviral and felv rna loads are characteristically found in cats with progressive infection, while low loads are detected in cats with regressive infection that are provirus positive but not viremic at the time [25, 29, 50] . felv proviral and viral rna loads were measured in the two felv-positive jaguarundis #1 and #4. comparison of the felv tissue loads of jaguarundi #1 revealed loads similar to those reported earlier for domestic cats with progressive infection using identical methods. viral rna loads in serum from the two felv-positive jaguarundis were comparable to those described earlier in persistently antigenemic cats [30] . the viral rna load in the clinically healthy jaguarundi #1 was slightly lower than that in jaguarundi #4, which showed weight loss, vomiting, anorexia and diarrhea before death. this is also similar to previous findings from a study in cats, comparing viremic healthy and viremic ill cats [51] . moreover, consistent with findings in domestic cats with a progressive felv infection, no antibodies to felv antigens were detected in jaguarundis #1 and #4. felv rna and felv provirus were also detected in the saliva of jaguarundi #1. this finding is indicative of virus shedding; thus, jaguarundi #1 was a potential source of felv infection to other felids. jaguarundi #1 was euthanized in good clinical condition as a biosafety measure. however, upon necropsy, histopathologic evaluation revealed splenic lymphoid depletion and muscular atrophy of the hind limbs; these findings may be associated with immunosuppression and neurological impairment in this animal. jaguarundi #4 died presenting weight loss and gastrointestinal disorders including vomiting, anorexia and diarrhea. histopathology revealed splenic alterations and evidence of enteritis (table 3) . although these conditions are commonly associated with felv in domestic cats, it was not possible to ascertain whether they were caused by felv infection in this case. two captive-born jaguarundis, #2 and #22, presented test results similar to those reported for domestic cats with abortive felv infection and seroconversion as the only marker of felv exposure [28] . the female jaguarundi #2 had a history of direct contact with the two jaguarundis with progressive felv infection, #1 and #4: she was a sibling of jaguarundi #1 and had mated with the male #4. relationships at the protein level. the most parsimonious tree, with length = 637, is shown. the consistency index is (0.750000), the retention index is (0.747664), and the composite index is 0.620901 (0.560748) for all sites and parsimony-informative sites (in parentheses). there were a total of 605 positions in the final dataset. the percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. the tree is drawn to scale, with the length being relative to the number of changes over the entire sequence. the mp tree was obtained using the subtree-pruning-regrafting (spr) algorithm with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). all positions containing gaps and missing data were eliminated. analyses were conducted in mega 6 [44] [45] [46] [47] 63] this would explain how she was exposed to felv, either at the same time as her sibling #1 or during mating with #4. subsequently, jaguarundi #2 gave birth to jaguarundi #23, who did not show any signs of felv exposure (tables 1, 2, 4). this may well be the case, since the mother, #2, had developed an antibody response and probably had never shed felv (abortive infection). it is unknown for how long and at what virus challenge conditions jaguarundi #2 was exposed, as animals were naturally and not experimentally infected in the present study. overall, we speculate that jaguarundi #2 developed an abortive felv infection for similar (and possibly several) reasons that may drive felv abortive infections in some domestic cats. the parents of male jaguarundi #22 were the felv-negative male jaguarundi #5 and the female #7. thus, it is unknown at what time point jaguarundi #22 was exposed to felv. overall, we provide evidence of progressive and abortive felv infection in p. yagouaroundi. similar results have been found in florida panthers (puma concolor coryi), which belong to the felid lineage 6 (genus puma), the same phylogenetic lineage as the jaguarundis [52] . in florida panthers, infection outcomes resembled those of domestic cats with progressive, regressive and abortive infection (previously persistent, regressive, and latent infection) [20, 21] . we speculate that felv infection in jaguarundis is similarly unpredictable, and it is influenced for the same diverse aspects cited above for domestic cats. complimentary to the fact that felv causes various tumors in domestic cats [53] , the literature has reported a felv-associated multicentric t-cell lymphoma in a captive non-domestic cheetah (acinonyx jubatus) [54] and non-felv-associated t-and b-cell lymphomas in geriatric african lions (panthera leo) [55] . these data motivated the search for possible felv involvement in the appearance of the neoplastic intra-abdominal mass in jaguarundi #5. although jaguarundi #5 had not shown any evidence of felv infection intra vitam (table 1) , we further investigated whether felv antigens or proviruses were present in intestinal lymphoid cells and whether these cells produced felv viral rna locally without it entering the bloodstreamas would be expected in a case of sequestered felv infection [4] . however, no felv antigens were detected by immunohistochemistry and no felv provirus or viral rna were detected by molecular assays. infection with fiv, which is a lentivirus associated with lymphomagenesis in domestic cats [56] , was not detected by the commercial immunoassay, and only antibodies specific to fiv p24 capsid proteins were detected by western blot, which may be indicative of an early or very late fiv infection or may have resulted from unspecific cross-reactivity. the fiv rt-pcr assays performed from whole blood were negative; however, this may be due to low viral loads or lack of specificity of the assays due to sequence diversity of different fiv isolates. in conclusion, jaguarundi #5 had developed a non felv-associated intestinal b-cell lymphoma; involvement of fiv in the development of the neoplasia cannot completely be ruled out. notably, a spleen sample from jaguarundi #5 had been previously analyzed for the presence of the felv receptor fthtr1, which permits the virus to enter the cell. the fthtr1 complementary dna (cdna) from that animal had shown 99% nucleotide and amino acid identity to the fthtr1 cdna sequences of the domestic cat and other wild felid species, such as the lynx (lynx pardinus), african lion (p. leo bleyenberghi), asiatic lion (p. leo persica), and european wildcat (f. silvestris silvestris) [57] . in addition, fthtr1 was quantified by real-time pcr in a few tissues from two jaguarundis where tissue was available (the viremic jaguarundi #1 and jaguarundi #5, which developed a tumor, but was not felv-infected).these limited results showed no significant difference between the fthtr1 tissue loads in cat and jaguarundi tissue (results not shown). however, it also needs to be mentioned that the real-time pcr assay was designed for the domestic cat thtr1 and not the jaguarundi thtr1. two-point mutations can be found in the region of the assay: one-point mutation in the middle of the forward primer and one in the probe. thus, although similar fthtr1 expression levels were found in jaguarundi tissue compared with cat tissue, we cannot exclude differences in the efficiency of the real-time pcr assay. these findings support the perspective that the first phase of the felv virus cycleviral entrymight be similar among domestic cats and other wild felid species. we did not detect enfelv in the jaguarundis by qpcr. this is consistent with earlier data [58] in which enfelv was not detected in jaguarundis. accordingly, felv-b, which has greater pathogenicity than felv-a in domestic cats and arises by recombination of exogenous felv-a with enfelv sequences [59] , was not found in the two felv-positive jaguarundis #1 and #4. moreover, the highly virulent felv-c was not detected. in felvinfected iberian lynxes, enfelv sequences were also not detected, but their felv-a variants were shown to be highly virulent, suggesting that the mechanisms inducing disease in these wild felids might be distinct from those in domestic cats [19] . the env sequence obtained from the felv infections of jaguarundis #1 and #4 [kr349469] clustered with the highest identity with the felv-faids and felv-3281 strains, which represent members of a highly conserved group of horizontally transmitted, minimally pathogenic felv-a present in all naturally occurring infections in domestic cats. both felv strains were originally isolated from cats in the united states of america (usa) [60, 61] . interestingly, the surface unit of the env sequence of felv detected in the jaguarundis also showed 97% identity to those of the env gene of felv isolated from the critically endangered iberian lynxes [eu293175 to eu293194] in spain, whose small wild population suffered from an outbreak of felv with several deaths [18] . both pcr-positive jaguarundis, #1 and #4, were infected with a felv with an identical env sequence, indicating a common origin of the virus. it is unknown at what time point each of the jaguarundis became infected. the reproductive success achieved for jaguarundis in captivity is probably a result of the breeding program conceived for small felids at fpzsp [62] and a number of measures for improving the well-being of the animals, including the location of the jaguarundi enclosures in an isolated and forested area of the zoo away from the public. nonetheless, domestic cats commonly are seen invading the zoo, especially the more isolated areas, and they were probably the source of infection of felv for the jaguarundis. domestic cats are the main reservoir for felv worldwide and greatly outnumber felv-infected wild felids. given this, it is prudent to prevent the presence of domestic cats in nature reserves, zoos and other settings with captive wild felids and to reinforce the importance of control measures for felv in domestic cats, such as testing and vaccination. a severe felv outbreak occurred in a previously naïve population of florida panthers (puma concolor coryi) in north america from 2002 to 2005 in which five felv antigen-positive panthers died [20, 21] . during a six-month period in 2007, six provirus-positive antigenemic iberian lynxes (lynx pardinus) died in spain [18, 19] . in both situations, felv vaccination programs were initiated. we recommend safe recombinant subunits or inactivated felv vaccines for captive jaguarundis, especially considering that invading domestic cats, potential sources of infection, are a frequent problem faced by zoos. in addition, we recommend the inclusion of felv testing in breeding programs for jaguarundis. our findings support our hypothesis that felv infection in p. yagouaroundi mimics the outcomes observed for the domestic cat. in addition, we found phylogenetic evidence that domestic cats may have been the source of the felv-a infection of the jaguarundis. despite the minimal pathogenicity of felv-a for domestic cats, this virus may represent a threat to the jaguarundi population, and regular felv testing and vaccination are encouraged. herpailurus yagouaroundi. in: the iucn red list of threatened species tratado de animais selvagens: medicina veterinária feline leukemia virus infection and diseases clinical aspects of feline retroviruses: a review excretion of feline leukaemia virus by naturally infected pet cats shedding of feline leukemia virus rna in saliva is a consistent feature in viremic cats fecal shedding of infectious feline leukemia virus and its nucleic acids: a transmission potential diseases associated with spontaneous feline leukemia virus (felv) infection in cats a serosurvey of viral infections in lions seroprevalences to viral pathogens in free-ranging and captive cheetahs (acinonyx jubatus) on namibian farmland a serologic survey of wild felids from central west saudi arabia serosurvey for feline leukemia virus and lentiviruses in captive small neotropic felids in são paulo state, brazil prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging lions in east africa serological detection of viral infections in captive wild cats from costa rica viral infections in free-living populations of the european wildcat feline viruses in wildcats from scotland prevalence and pathogenicity of retroviruses in wildcats in france feline leukemia virus and other pathogens as important threats to the survival of the critically endangered iberian lynx (lynx pardinus) feline leukemia virus infection: a threat for the survival of the critically endangered iberian lynx (lynx pardinus) epizootiology and management of feline leukemia virus in the florida puma genetic characterization of feline leukemia virus from florida panthers survey of feline leukemia virus and feline coronaviruses in captive neotropical wild felids from southern brazil surveillance using serological and molecular methods for the detection of infectious agents in captive brazilian neotropic and exotic felids first evidence of feline herpesvirus, calicivirus, parvovirus, and ehrlichia exposure in brazilian free-ranging felids how molecular methods change our views of felv infection and vaccination vaccination against the feline leukaemia virus: outcome and response categories and long-term follow-up exposure of cats to low doses of felv: seroconversion as the sole parameter of infection re-examination of feline leukemia virus: host relationships using real-time pcr real-time pcr investigation of feline leukemia virus proviral and viral rna loads in leukocyte subsets quantitation of feline leukaemia virus viral and proviral loads by taqman real-time polymerase chain reaction cellular segregation of feline leukemia provirus and viral rna in leukocyte subsets of long-term experimentally infected cats detection of antibodies to the feline leukemia virus (felv) transmembrane protein p15e: an alternative approach for serological felv detection based on antibodies to p15e retroviral dna -the silent winner: blood transfusion containing latent feline leukemia provirus causes infection and disease in naïve recipient cats use of avidin-biotin-peroxidase complex (abc) in immunoperoxidase techniques: a comparison between abc and unlabeled antibody (pap) procedures expression of viral proteins in feline leukemia virus-associated enteritis gapdh pseudogenes and the quantification of feline genomic dna equivalents quantitative taqman real-time pcr assays for gene expression normalisation in feline tissues quantitative real-time pcr for the measurement of feline cytokine mrna recombinant feline leukemia virus genes detected in naturally occurring feline lymphosarcomas pathogenicity of a subgroup c feline leukemia virus (felv) is augmented when administered in association with certain felv recombinants specificity assessment of feline t-lymphotropic lentivirus serology quantification of proviral fiv dna using competitive pcr influence of preassay and sequence variations on viral load determination by a multiplex real-time reverse transcriptasepolymerase chain reaction for feline immunodeficiency virus association between endogenous feline leukemia virus loads and exogenous feline leukemia virus infection in domestic cats mega6: molecular evolutionary genetics analysis version 6.0 clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice the neighbor-joining method: a new method for reconstructing phylogenetic trees phylogenetic inference: maximum parsimony methods confidence limits on phylogenies: an approach using the bootstrap feline leukaemia provirus load during the course of experimental infection and in naturally infected cats long-term follow up of feline leukemia virus infection and characterization of viral rna loads using molecular methods in tissues of cats with different infection outcomes the late miocene radiation of modern felidae: a genetic assessment clinical aspects of feline immunodeficiency and feline leukemia virus infection multicentric t-cell lymphoma associated with feline leukemia virus infection in a captive namibian cheetah (acinonyx jubatus) malignant lymphoma in african lions (panthera leo) role of feline immunodeficiency virus in lymphomagenesis -going alone or colluding? quantification and molecular characterization of the feline leukemia virus a receptor genomically intact endogenous feline leukemia viruses of recent origin naturally occurring feline leukemia virus subgroup a and b infections in urban domestic cats experimental transmission and pathogenesis of immunodeficiency syndrome in cats strong sequence conservation among horizontally transmissible, minimally pathogenic feline leukemia viruses small felid breeding project at sao paulo zoo subtle mutational changes in the su protein of a natural feline leukemia virus subgroup a isolate alter disease spectrum vaccination of cats experimentally infected with feline immunodeficiency virus, using a recombinant feline leukemia virus vaccine recombinant felv vaccine: long-term protection and effect on course and outcome of fiv infection monoclonal antibodies to three epitopic regions of feline leukemia virus p27 and their use in enzyme-linked immunosorbent assay of p27 we are deeply indebted to all veterinarians and staff of fundação parque this work was supported by grants of coordination for the improvement of higher education personnel (capes) and the post-graduate pro-rector from usp in brazil and the international relations office from the university of zurich in switzerland. partial writing of this manuscript work was also supported by the são paulo research foundation (fapesp) (grant 2013/ 12253-0). the funding bodies had no specific role in the design of the study, collection, analysis, and interpretation of data. the datasets supporting the conclusions of this article are included within the article.authors' contributions cf, jlcd and rhl concepted and designed the study. cf and mcm handled, sampled, and retrieved data from jaguarundis at zoo. cf and khh conducted the serological and molecular tests. cf, jlcd, lnt and mr performed the histopathological and immunohistopathological analyses. khh conducted the sequencing and phylogenetic analyses. all of the authors drafted and approved this version of the manuscript. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-017012-yl0vanuh authors: herberg, jethro; pahari, amitava; walters, sam; levin, michael title: infectious diseases and the kidney date: 2009 journal: pediatric nephrology doi: 10.1007/978-3-540-76341-3_52 sha: doc_id: 17012 cord_uid: yl0vanuh the kidney is involved in a wide range of bacterial, viral, fungal, and parasitic diseases. in most systemic infections, renal involvement is a minor component of the illness, but in some, renal failure may be the presenting feature and the major problem in management. although individual infectious processes may have a predilection to involve the renal vasculature, glomeruli, interstitium, or collecting systems, a purely anatomic approach to the classification of infectious diseases affecting the kidney is rarely helpful because most infections may involve several different aspects of renal function. the kidney is involved in a wide range of bacterial, viral, fungal, and parasitic diseases. in most systemic infections, renal involvement is a minor component of the illness, but in some, renal failure may be the presenting feature and the major problem in management. although individual infectious processes may have a predilection to involve the renal vasculature, glomeruli, interstitium, or collecting systems, a purely anatomic approach to the classification of infectious diseases affecting the kidney is rarely helpful because most infections may involve several different aspects of renal function. in this chapter, a microbiologic classification of the organisms affecting the kidney is adopted. although they are important causes of renal dysfunction in infectious diseases, urinary tract infections and hemolytic uremic syndrome (hus) are not discussed in detail because they are considered separately in chapters 54 and 48 respectively. elucidation of the cause of renal involvement in a child with evidence of infection must be based on a careful consideration of the geographic distribution of infectious diseases in different countries. a history of foreign travel; exposure to animals, insects, or unusual foods or drinks; outdoor activities such as swimming or hiking; and contact with infectious diseases must be sought in every case. the clinical examination should include a careful assessment of skin and mucous membranes and a search for insect bites, lymphadenopathy, and involvement of other organs. a close collaboration with a pediatric infectious disease specialist and hospital microbiologist will aid the diagnosis and management of the underlying infection. a tantalizing clue to the pathogenesis of glomerular disease is the marked difference in the incidence of nephrosis and nephritis in developed and underdeveloped areas of the world. in several tropical countries, glomerulonephritis (gn) accounts for up to 4% of pediatric hospital admissions; the incidence in temperate climates is 10-to 100-fold less. this difference might be explained by a complex interaction of several different factors, including nutrition, racial and genetically determined differences in immune responses, and exposure to infectious diseases. a growing body of evidence, however, suggests that longterm exposure to infectious agents is a major factor in the increased prevalence of glomerular diseases in developing countries. renal involvement in infectious diseases may occur by a variety of mechanisms: direct microbial invasion of the renal tissues or collecting system may take place in conditions such as staphylococcal abscess of the kidney as a result of septicemic spread of the organism or as a consequence of ascending infection; damage to the kidney may be caused by the systemic release of endotoxin or other toxins and activation of the inflammatory cascade during septicemia or by a focus of infection distant from the kidney; ischemic damage may result from inadequate perfusion induced by septic shock; the kidney may be damaged by activation of the immunologic pathways or by immune complexes resulting from the infectious process. in many conditions, a combination of these mechanisms may be operative. in the assessment of renal complications occurring in infectious diseases, the possibility of druginduced nephrotoxicity caused by antimicrobial therapy should always be considered. the nephrotoxic effects of antibiotics and other antimicrobial agents are not addressed in this chapter but are covered in chapter 53. bacterial infections associated with renal disease and the likely mechanisms causing renal dysfunction are shown in > impaired renal function is a common occurrence in systemic sepsis (1) . depending on the severity of the infection and the organism responsible, the renal involvement may vary from insignificant proteinuria to acute renal failure requiring dialysis. the organisms causing acute renal failure as part of systemic sepsis vary with age and geographic location and also differ in normal and immunocompromised children. in the neonatal period, group b streptococci, coliforms, staphylococcus aureus, and listeria monocytogenes are the organisms usually . responsible. in older children, neisseria meningitidis, streptococcus pneumoniae, and s. aureus account for most of the infections. in people who are immunocompromised, a wide range of bacteria are seen, and, similarly, in tropical countries other pathogens, including haemophilus influenzae, salmonella species, and pseudomonas pseudomallei, must be considered. where h. influenzae type b vaccine has been introduced, however, the incidence of severe systemic infections due to this organism has shown a sharp fall. systemic sepsis usually presents with nonspecific features: fever, tachypnea, tachycardia, and evidence of skin and organ underperfusion. the pathophysiology of renal involvement in systemic sepsis is multifactorial (1, 2) . hypovolemia with diminished renal perfusion is the earliest event and is a consequence of the increased vascular permeability and loss of plasma from the intravascular space. hypovolemia commonly coexists with depressed myocardial function because of the myocardial depressant effects of endotoxin or other toxins. the renal vasoconstrictor response to diminished circulating volume and reduced cardiac output further reduces glomerular filtration, and oliguria is thus a consistent and early event in severe sepsis (1, 3) . a number of vasodilator pathways are activated in sepsis, including nitric oxide and the kinin pathways. this may lead to inappropriate dilatation of vascular beds. vasodilation of capillary beds leading to warm shock is common in adults with sepsis due to gram-negative organisms but is less commonly seen in children, in whom intense vasoconstriction is the usual response to sepsis. if renal underperfusion and vasoconstriction are persistent and severe, the reversible prerenal failure is followed by established renal failure with the characteristic features of vasomotor nephropathy or acute tubular necrosis. other mechanisms of renal damage in systemic sepsis include direct effects of endotoxin and other toxins on the kidney, and release of inflammatory mediators such as tumor necrosis factor (tnf) and other cytokines, arachidonic acid metabolites, and proteolytic enzymes (3) . nitric oxide (no) is postulated to play a key role in the pathophysiology of renal failure in sepsis. whether the renal effects of increased no are beneficial or harmful remains unclear. trials of selective no synthetase inhibition did not offer any advantages over saline resuscitation (4) . no in endotoxemia is possibly beneficial because it maintains renal blood flow and glomerular filtration. activation of coagulation is an important component of the pathophysiology of septic shock and may contribute to renal impairment. activation of multiple prothrombotic and antifibrinolytic pathways occurs, together with downregulation of antithrombotic mechanisms such as the protein c pathway. treatment with activated protein c has been shown to improve outcome of adult septic shock, but has not been confirmed to have benefit in pediatric sepsis, and may carry a risk of bleeding particularly in infants (5) . the renal findings early in septic shock are oliguria, with high urine/plasma urea and creatinine ratios, low urine sodium concentration, and a high urine/plasma osmolarity ratio. once established, renal failure supervenes, and the urine is of poor quality with low urine/ plasma urea and creatinine ratios, elevated urine sodium concentration, and low urine osmolarity. proteinuria is usually present, and the urine sediment may contain red cells and small numbers of white cells (1) . the management of acute renal failure in systemic sepsis depends on early diagnosis and administration of appropriate antibiotics to cover the expected pathogens. . in addition, management is directed at improving renal perfusion and oxygenation. volume replacement with crystalloid or colloid should be undertaken to optimize preload. central venous pressure or pulmonary wedge pressure monitoring is essential to guide volume replacement in children in severe shock (1, 2) . the use of low-dose (2-5 pg/kg/min) dopamine to reduce renal vasoconstriction together with administration of inotropic agents such as dobutamine or epinephrine to improve cardiac output may reverse prerenal failure. early elective ventilation should be undertaken in patients with severe shock. if oliguria persists despite volume replacement and inotropic therapy, dialysis should be instituted early, because septic and catabolic patients may rapidly develop hyperkalemia and severe electrolyte imbalance. in most children who develop acute renal failure as part of systemic sepsis or septic shock, the renal failure is of short duration, and recovery can be expected within a few days of achieving cardiovascular stability and eradication of the underlying infection. occasionally, renal cortical necrosis or infarction of the kidney may result in prolonged or permanent loss of renal function. meningococcus n. meningitidis continues to be a major cause of systemic sepsis and meningitis in both developed and underdeveloped parts of the world (6) . in developed countries, most cases are caused by group b and y strains, particularly after introduction of meningococcal c vaccination, whereas epidemics of meningococcus groups a, c and w135 continue to occur in many underdeveloped regions of the world (6, 7) . infants and young adults are most commonly affected, but cases in adolescents and young children are also common. there are two major presentations of meningococcal disease (6) : meningococcal meningitis presents with features indistinguishable from those of other forms of meningitis, including headache, stiff neck, and photophobia. lumbar puncture is required to identify the causative agent and distinguish this from other forms of meningitis. despite the acute nature of the illness, the prognosis is good, and most patients with the purely meningitic form of the illness recover without sequelae. meningococcemia with purpuric rash and shock is the second and more devastating form of the illness. affected patients present with nonspecific symptoms of fever, vomiting, abdominal pain, and muscle ache. the diagnosis is only obvious once the characteristic petechial or purpuric rash appears. patients with a rapidly progressive purpuric rash, hypotension, and evidence of skin and organ underperfusion have a poor prognosis, with a mortality of 10-30%. adverse prognostic features include hypotension, a low white cell count, absence of meningeal inflammation, thrombocytopenia, and disturbed coagulation indices (8) . renal failure was seldom reported in early series of patients with meningococcemia, perhaps because most patients died rapidly of uncontrolled septic shock. with advances in intensive care, however, more children are surviving the initial period of profound hemodynamic derangement, and renal failure is more often seen as a major management problem. approximately 10% of children with fulminant meningococcemia develop renal failure, which usually occurs 24-48 h after the onset of illness (9) . the pathophysiology of meningococcal septicemia involves the activation of cytokines and inflammatory cells by endotoxin (6, 7) . mortality is directly related to both the plasma endotoxin concentration and the intensity of the inflammatory response, as indicated by levels of tnf and other inflammatory markers (10) . patients with meningococcemia have a profound capillary leak leading to severe hypovolemia. loss of plasma proteins from the intravascular space is probably the major cause of shock (11) , but myocardial suppression secondary to il-6 production is also important (12) . intense vasoconstriction further impairs tissue and organ perfusion, and vasculitis with intravascular thrombosis and consumption of platelets and coagulation factors is also present (6) . oliguria is invariably present in children with meningococcemia during the initial phase of the disorder. this is prerenal in origin and may respond to volume replacement and inotropic support. if cardiac output cannot be improved and renal underperfusion persists, established renal failure supervenes. occasionally, cortical necrosis or infarction of the kidneys occurs. children with meningococcemia should be aggressively managed in a pediatric intensive care unit, with early administration of antibiotics (penicillin or a third-generation cephalosporin), volume replacement, hemodynamic monitoring, and the use of inotropic agents and vasodilators. if oliguria persists despite measures to improve cardiac output, elective ventilation and dialysis should be instituted early (6, 7) . because activation of coagulation pathways occurs, severe acquired protein-c deficiency may result and is usually associated with substantial mortality (13) . protein c is a natural anticoagulant which also has important antiinflammatory activity. despite evidence for impaired function of the activated protein c pathway in meningococcal diseases (14) , and adult trials suggesting benefit of activated protein c administration in septic shock (prowess trial) (15) , pediatric trials of activated protein c showed no clear benefit, and were associated with increased risk of intracranial bleeding in very young infants (5) . the role of apc therapy in pediatric sepsis remains unclear. most patients who survive the initial 24-48 h of the illness and regain hemodynamic stability will ultimately recover renal function even if dialysis is required for several weeks. the least common presentation of meningococcal sepsis is chronic meningococcemia. patients with this form of the illness present insidiously with a vasculitic rash, arthritis, and evidence of multiorgan involvement. the features may overlap those of henoch-schonlein purpura or subacute bacterial endocarditis (sbe), and the diagnosis must be considered in patients presenting with fever, arthritis, and vasculitic rash, often accompanied by proteinuria or hematuria. response to antibiotic treatment is good, but some patients may have persistent symptoms for many days resulting from an immunecomplex vasculitis. staphylococcal infections may affect the kidneys by direct focal invasion during staphylococcal septicemia, forming a renal abscess; by causing staphylococcal bacteremia; or by toxin-mediated mechanisms, as in the staphylococcal toxic shock syndrome. staphylococcal abscess. staphylococcal renal abscess presents with fever, loin pain and tenderness, and abnormal urine sediment, as do abscesses caused by other organisms (16) . the illness often follows either septicemia or pyelonephritis. the diagnosis is usually considered only when a patient with clinical pyelonephritis shows an inadequate response to antibiotic treatment. the diagnosis is confirmed by ultrasonography or computed tomographic scan, which shows swelling of the kidney and intrarenal collections of fluid. antibiotic therapy alone may result in cure, but if the patient remains unwell with evidence of persistent inflammation despite use of appropriate antibiotics, surgical intervention may be required. percutaneous drainage under ultrasonographic or computed tomographic scan guidance is often effective and may avoid the need for a more direct surgical approach (16, 17) . staphylococcal toxic shock syndrome. the staphylococcal toxic shock syndrome is a systemic illness characterized by fever, shock, erythematous rash, diarrhea, confusion, and renal failure. the disorder was first described by todd et al. in 1978 in a series of seven children (18) . during the 1980s, thousands of cases were reported in the united states. most cases were in menstruating women, in associated with tampon use. although most cases worldwide are seen in women and are associated with menstruation, children of both sexes and of all ages are affected (19) . the illness usually begins suddenly with high fever, diarrhea, and hypotension, together with a diffuse erythroderma (20) . mucous membrane involvement with hyperemia and ulceration of the lips and oral mucosa or vaginal mucosa, strawberry tongue, and conjunctival injection are usually seen. desquamation of the rash occurs in the convalescent phase of the illness. confusion is often present in the early stages of the illness and may progress to coma in severe cases. multiple organ failure with evidence of impaired renal function, elevated levels of hepatic transaminases, thrombocytopenia, elevated cpk and disseminated intravascular coagulation (dic) is often seen. according to cdc criteria, the diagnosis is made on the basis of the clinical features of fever, rash, hypotension, and subsequent desquamation along with deranged function of three or more of the following organ systems: gastrointestinal (gi), mucous membranes, renal, hepatic, hematologic, central nervous system, and muscle. other disorders causing a similar picture, such as rocky mountain spotted fever, leptospirosis, measles, and streptococcal infection, must be excluded. the staphylococcal toxic shock syndrome is now known to be due to infection or colonization with strains of s. aureus that produce one or more protein exotoxins (21) . most cases in adults are associated with toxic shock toxin i; in children, many of the isolates associated with the syndrome produce other enterotoxins (a to f). the staphylococcal enterotoxins appear to induce disease by acting as superantigens (22) , which activate t cells bearing specific v beta regions of the t-cell receptor; this causes proliferation and cytokine release (23) . the systemic illness and toxicity are believed to result largely from an intense inflammatory response induced by the toxin. the site of toxin production is often a trivial focus of infection or simple colonization, and bacteremia is rarely observed. renal failure in toxic shock syndrome is usually caused by shock and renal hypoperfusion. in the early stages of the illness, oliguria and renal impairment are usually prerenal and respond to treatment of shock and measures to improve perfusion. in severe cases and in patients in whom treatment is delayed, acute renal failure develops as a consequence of prolonged renal underperfusion, and dialysis may be required. in addition to underperfusion, direct effects of the toxin or inflammatory infectious diseases and the kidney mediators may also contribute to the renal damage. recovery of renal function usually occurs, but in severe cases with cortical necrosis or intense renal vasculitis, prolonged dialysis may be required. the management of staphylococcal toxic shock syndrome depends on early diagnosis and aggressive cardiovascular support with volume replacement, inotropic support, and, in severe cases, elective ventilation. if oliguria persists despite optimization of intravascular volume and administration of inotropic agents, dialysis should be commenced early (20) . anti-staphylococcal antibiotics should be started as soon as the diagnosis is suspected and the site of infection identified. initial empiric antimicrobial therapy should include an anti-staphylococcal antibiotic effective against betalactamase-resistant organisms and a protein synthesisinhibiting antibiotic such as clindamycin to stop further toxin production (24) . if there is a focus of infection such as a vaginal tampon, surgical wound, or infected sinuses, the site should be drained early to prevent continued toxin release into the circulation. the intravenous administration of immune globulins may be considered when infection is refractory to several hours of aggressive therapy, an undrainable focus is present, or persistent oliguria with pulmonary edema occurs (24) . with aggressive intensive care, most affected patients survive, and renal recovery is usual, even in patients who have had severe shock and multiorgan failure. relapses and recurrences of staphylococcal toxic shock syndrome occur in a proportion of affected patients because immune responses to the toxin are ineffective in some individuals. panton valentine leucocidin (pvl) producing staphylococcal infection: in recent years there have been increasing reports of severe staphylococcal disease, associated with shock and multiorgan failure, caused by strains of staphylococci producing the pvl toxin. panton-valentine leukocidin (pvl) is a phage-encoded toxin, which profoundly impairs the host response due to its toxic effect on leucocytes (see review (25) ). pvl producing strains are associated with tissue necrosis and increased propensity to cause abscesses in lung, bone, joint, and soft tissue infections. perinephric abscesses have been reported (26) . there are increasing numbers of children and adults admitted with fulminant sepsis, and shock due to pvl producing strains, and renal failure is a significant component of the multiorgan failure. in addition to intensive care support, antibiotic treatment of pvl strains should include antibiotics which reduce toxin production, such as clindamycin, linezolid or rifampicin, as well as vancomycin if the strain is resistant to methicillin. beta-lactam antibiotics should be avoided, as there is some data to suggest that pvl toxin production can increased by these antibiotics under some conditions (27) . immunoglobulin infusion may also be of benefit. aggressive surgical drainage of all collections requires close consultation with orthopedic and surgical teams. the group a streptococci (gas) are a major worldwide cause of renal disease, usually as poststreptococcal nephritis. however, in addition to this post-infection immunologically mediated disorder, in recent years there have been increasing reports of gas causing acute renal failure as part of an invasive infection with many features of the staphylococcal toxic shock syndrome (28) . acute poststreptococcal glomerulonephritis. acute poststreptococcal gn (apsgn) is a delayed complication of pharyngeal infection or impetigo with certain nephritogenic strains of gas. different strains can be serotyped according to the antigenic properties of the m protein found in the outer portion of the bacterial wall. apsgn after pharyngeal infection is most commonly associated with serotype m12. in contrast, in apsgn after impetigo, serotype m49 is most commonly identified (29) . on occasions, other serotypes and non-typeable strains have been described as causing gn. the pathology and pathogenesis of the disorder is discussed in detail in chapter 30. apsgn has a worldwide distribution. epidemiologic differences are observed between pharyngitis-associated and impetigo-associated streptococcal infections. pharyngitis-associated apsgn is most common during school age and has an unexplained male/female ratio of 2:1. it occurs more often in the cooler months, and familial occurrences are commonly described. the latent period is 1-2 weeks, in notable contrast to impetigo-associated cases, which have a latent period of 2-6 weeks. in many developing countries, children have chronic skin infections, and it may be difficult to establish the latent period with accuracy. impetigoassociated cases are more common in the warmer months, sex distribution is equal, and children tend to be younger. introduction of a nephritogenic strain into a family often results in the occurrence of several cases within that family, and in some cases, attack rates of up to 20% have been described (30) . the incidence is linked to poor socioeconomic conditions. renal involvement in apsgn can be mild, and in many patients, the disease may not be manifested clinically. studies of epidemics with nephritogenic strains of streptococci have shown that up to 50% of those infected had subclinical evidence of renal disease (30, 31) . in a typical case a sudden onset of facial or generalized edema occurs. hypertension is usually modest but is severe in 5% of cases, and occasionally may lead to encephalopathy or left ventricular failure. the urine is smoky or tea colored in 30-50% of cases. pallor, headache, backache, lethargy, malaise, anorexia, and weakness are all common nonspecific features. the urine volume is decreased. proteinuria is present (up to 100 mg/dl), and microscopy shows white cells, red cells, and granular and hyaline casts. urea, electrolyte, and creatinine levels are normal in subclinical cases but show features of acute renal failure in severe cases. it may be possible to culture gas from the skin or the throat in some patients. other evidence of infection with a gas can be obtained through the antistreptolysin-o titer (asot), which is increased in 60-80% of cases. early antibiotic treatment can reduce the proportion of cases with elevated asot to 30%. anti-deoxyribonuclease b and anti-hyaluronidase testing has been shown to be of more value than asot in confirming group a streptococcal infection in impetigo-associated cases. measurement of anti-m protein antibodies is of more value for epidemiologic purposes than for the diagnosis of individual cases (31) . decreased c3 and total hemolytic complement levels are found in 90% of cases during the first 2 weeks of illness and return to normal after 4-6 weeks. penicillin should be given to eradicate the gas organisms. erythromycin, clindamycin, or a first-generation cephalosporin can be given to patients allergic to penicillin. antibiotic treatment probably has no influence on the course of renal disease but will prevent the spread of a nephritogenic strain (32) . close contacts and family members who are culture-positive for gas should also be given penicillin, although antibiotic treatment is not always effective in eliminating secondary cases. recurrent episodes are rare, and immunity to the particular nephritogenic strain that caused the disease is probably lifelong. antibiotic prophylaxis is therefore unnecessary. most studies suggest that the prognosis for children with apsgn is good, with more than 90% making a complete recovery. however, 10% of cases may have a prolonged and more serious course with long-term chronic renal failure (33) . other streptococci. apsgn has also been described after outbreaks of group c streptococcus infection (34) . this has occurred after consumption of unpasteurized milk from cattle with mastitis. patients developed pharyngitis followed by apsgn. endostreptosin was found in the cytoplasm of these group c strains, and during the course of the illness, patients developed anti-endostreptosin antibodies. this antigen has been postulated to be the nephritogenic component of gas. in addition, strains of group g streptococci have been implicated in occasional cases of apsgn (35) . isolates possessed the type m12 protein antigen identical to the nephritogenic type m12 antigen of some group a streptococcal strains. streptococcal toxic shock syndrome and invasive group a streptococcal infection. since 1988, there have been several reports of an illness with many similarities to the staphylococcal toxic shock syndrome, occurring in both children (36) and adults, associated with invasive group a streptococcal disease (32, 37, 38) . patients with this syndrome present acutely with high fever, erythematous rash, mucous membrane involvement, hypotension, and multiorgan failure. unlike staphylococcal toxic shock syndrome, in which the focus of infection is usually trivial and bacteremia is seldom seen, the streptococcal toxic shock syndrome is usually associated with bacteremia or a serious focus of infection such as septic arthritis, myositis, or osteomyelitis (36, 38) . laboratory findings of anemia, neutrophil leukocytosis, thrombocytopenia, and dic are often present, together with impaired renal function, hepatic derangement, and acidosis. acute renal failure requiring dialysis occurs in a significant proportion of cases. it is not clear why there are increasing numbers of cases with invasive disease caused by gas, nor why there has been an emergence of streptococcal toxic shock syndrome, and indeed a similar syndrome caused by some pseudomonas and klebsiella strains. the most common antecedent of invasive gas disease is varicella infection, with the streptococcal infection developing after the initial vesicular phase of the disease is subsiding. strains causing toxic shock syndrome and invasive disease appear to differ from common isolates of gas in producing large amounts of pyrogenic toxins that may have superantigenlike activity. another important mechanism is the production by invasive gas of an il8 protease. il8 serves as a molecular bridge between receptors on neutrophils and the vascular endothelium. cleavage of this protein prevents neutrophil attachment to the endothelium, and results in uncontrolled spread of the bacteria through the tissues (39) . in severe cases necrotizing fasciitis occurs with extensive destruction of the subcutaneous tissues, and is often associated with multiorgan failure. the pathophysiology of streptococcal toxic shock syndrome and that of invasive disease is similar in that superantigen toxins that induce release of cytokines and other inflammatory mediators play a role in both conditions. however gas toxic shock is usually more severe, carries a higher mortality, and is more often associated with focal collections or necrotizing fasciitis. treatment of streptococcal toxic shock syndrome depends on the administration of appropriate antibiotics, aggressive circulatory support, and treatment of any multiorgan failure. surgical intervention to drain the infective focus in muscle, bone, joint, or body cavities is often required. antibiotic therapy with beta-lactams should be supplemented by treatment with a protein synthesisinhibiting antibiotic, such as clindamycin, and it is suggested that this limits new toxin production (40, 41) . a number of new therapies are in development. firstly, pooled intravenous immunoglobulins are now in widespread use in the treatment of toxic shock, particularly when caused by streptococcus (42, 43) . the role of steroids remains unclear, with their hemodynamic benefit set against the detrimental effects of hyperglycemia secondary to gluconeogenesis. (44) . the benefit of insulin therapy to control hyperglycemia is unclear. a recent study in adults found that intensive insulin therapy increased the risk of serious adverse events (45) . in contrast to adult patients, in children with severe sepsis, the use of activated protein c (drotrecogin) cannot be recommended, as in a multicenter trial, fatality was increased in the treatment group (5) . recovery of renal function occurs in patients who respond to treatment of shock and the eradication of the infection. leptospirosis is an acute generalized infectious disease caused by spirochetes of the genus leptospira (46) . it is primarily a disease of wild and domestic animals, and humans are infected only occasionally through contact with animals. most human cases occur in summer or autumn and are associated with exposure to leptospiracontaminated water or soil during recreational activities such as swimming or camping. in adolescents and adults, occupational exposure through farming or other contact with animals is the route of infection. the spirochete penetrates intact mucous membranes or abraded skin and disseminates to all parts of the body, including the cerebrospinal fluid (csf). although leptospires do not contain classic endotoxins, the pathophysiology of the disorder has many similarities to that of endotoxemia. in severe cases, jaundice occurs because of hepatocellular dysfunction and cholestasis. renal functional abnormalities may be profound and out of proportion to the histologic changes in the kidney (47) . renal involvement is predominantly a result of tubular damage, and spirochetes are commonly seen in the tubular lesions. the inflammatory changes in the kidney may result from either a direct toxic effect of the organism or immunecomplex nephritis. however, hypovolemia, hypotension, and reduced cardiac output caused by myocarditis may contribute to the development of renal failure. in severe cases, a hemorrhagic disorder caused by widespread vasculitis and capillary injury also occurs (47, 48) . the clinical manifestations of leptospirosis are variable. of affected patients, 90% have the milder anicteric form of the disorder, and only 5-10% have severe leptospirosis with jaundice. the illness may follow a biphasic course. after an incubation period of 7-12 days, a nonspecific flu-like illness lasting 4-7 days occurs, associated with septicemic spread of the spirochete. the fever then subsides, only to recur for the second, ''immune,'' phase of the illness. during this phase, the fever is low grade and there may be headache and delirium caused by meningeal involvement, as well as intense muscular aching. nausea and vomiting are common. examination usually reveals conjunctival suffusion, erythematous rash, lymphadenopathy, and meningism. the severe form of the disease (weil's disease) presents with fever, impaired renal and hepatic function, hemorrhage, vascular collapse, and altered consciousness. in one series the most common organs involved were the liver (71%) and kidney (63%). cardiovascular (31%), pulmonary (26%), neurologic (5%), and hematologic (21%) involvements were less common (49) . vasculitis, thrombocytopenia, and uremia are considered important factors in the pathogenesis of hemorrhagic disturbances and the main cause of death in severe leptospirosis (50) . urinalysis results are abnormal during the leptospiremic phase with proteinuria, hematuria, and casts. uremia usually appears in the second week, and acute renal failure may develop once cardiovascular collapse and dic are present (48) . the clinical features of leptospirosis overlap with those of several other acute infectious diseases, including rocky mountain spotted fever, toxic shock syndrome, and streptococcal sepsis. the diagnosis of leptospirosis should be considered in febrile patients with evidence of renal, hepatic, and mucous membrane changes and rash, particularly if a history of exposure to fresh water is found. diagnosis can be confirmed by isolation of the spirochetes from blood or csf in the first 10 days of the illness or from urine in the second week (48) . the organism may be seen in biopsy specimens of the kidney or skin or in the csf by dark-field microscopy or silver staining. serologic tests to detect leptospirosis are now sensitive and considerably aid the diagnosis. immunoglobulin m (igm) antibody may be detected as early as 6-10 days into the illness, and antibody titers rise progressively over the next 2-4 weeks. some patients remain seronegative, and negative serologic test results do not completely exclude the diagnosis. in one series levels of igm and igg anticardiolipin concentrations were significantly increased in leptospirosis patients with acute renal failure (50) . leptospirosis is treated with intravenous penicillin or other beta-lactam antibiotics. the severity of leptospirosis is reduced by antibiotic treatment, even if started late in the course of the illness (51) . supportive treatment with volume replacement to correct hypovolemia, administration of inotropes, and correction of coagulopathy is essential in severe cases. dialysis may be required in severe cases and may be needed for prolonged periods until recovery occurs. infection with s. pneumoniae is one of the most common infections in humans and causes a wide spectrum of disease, including pneumonia, otitis media, sinusitis, septicemia, and meningitis. despite the prevalence of the organism, significant renal involvement is relatively rare but is seen in two situations: pneumococcal septicemia in asplenic individuals or in those with other immune deficiencies presents with fulminant septic shock in which renal failure may occur as part of a multisystem derangement. the mortality from pneumococcal sepsis in asplenic patients is high, even with early antibiotic treatment and intensive support. the second nephrologi syndrome associated with s. pneumoniae is a rare form of hus. in 1955, gasser and colleagues described hus as a clinical entity in children, and they included two infants with pneumonia among the five patients they described (52) . hus associated with pneumococcal infection is induced by the enzyme neuraminidase released from s. pneumoniae (53, 54) . thomsen-friedenrich antigen (t antigen) is present on the surface of red blood cells, platelets, and glomerular capillary endothelia against which antibodies are present in normal serum. neuraminidase causes desialation of red blood cells, and possibly other blood cells and endothelium, by the removal of terminal neuraminic acid, which leads to unmasking of the t antigen. the resultant widespread agglutination of blood cells causes intravascular obstruction, hemolysis, thrombocytopenia, and renal failure. results of the direct coombs test are frequently positive, either from bound anti-t igm or from anti-t antibodies. the diagnosis of thomsen-friedenrich antibody-induced hus should be suspected in patients with acute renal failure, thrombocytopenia and hemolysis after an episode of pneumonia or bacteremia caused by s. pneumoniae. fragmented red blood cells will usually be present on blood film. association with s. pneumoniae is defined by culture of pneumococci from a normally sterile site within a week before or after onset of signs of hus. clues to a pneumococcal cause, in addition to culture results, include severe clinical disease, especially pneumonia, empyema, pleural effusion, or meningitis; hemolytic anemia without a reticulocyte response; positive results on a direct coombs test; and difficulties in abo crossmatching or a positive minor crossmatch incompatibility (55) . however, when renal disease is seen in the context of severe pneumococcal infection, it is important to maintain a broad diagnostic perspective, because the occurrence of acute tubular necrosis due to septic shock and dic is well described (56, 57) . therapy for this syndrome should be with supportive treatment and antibiotics (usually a third generation cephalosporin); dialysis may be required if renal failure occurs. because normal serum contains antibodies against the thomsen-friedenrich antigen, blood transfusion should be undertaken with washed red blood cells resuspended in albumin rather than plasma (53, 54) . exchange transfusion and plasmapheresis have been used in some patients, with the rationale that these procedures may improve outcome by eliminating circulating neuraminidase (53, 57, 58) . intravenous igg has been used in a patient and was shown to neutralize neuraminidase present in the patient's serum (59) . in comparison to patients with the more common diarrhea-associated hus, s. pneumoniae-induced hus patients have a more severe renal disease. they are more likely to require dialysis. their long-term outcome maybe affected by the severity of the invasive streptococcal disease itself, and a significant proportion of surviving patients (30-70%) develop end-stage renal failure (60, 61) . a recent review of uk cases found an eightfold increase in early mortality as compared to diarrhoea-induced hus (62) . gastrointestinal infections (escherichia coli, salmonella, campylobacter, yersinia, shigella, vibrio cholerae) the diarrheal diseases caused by escherichia coli, salmonella, shigella, campylobacter, vibrios, and yersinia remain important and common bacterial infections of humans. although improvements in hygiene and living conditions have reduced the incidence of bacterial gastroenteritis in developed countries, these infections remain common in underdeveloped areas of the world, and outbreaks and epidemics continue to occur in both developed and underdeveloped countries. renal involvement in the enteric infections may result from any of four possible mechanisms. regardless of the causative organism, diarrhea results in hypovolemia, abnormalities of plasma electrolyte composition, and renal underperfusion. if severe dehydration occurs and is persistent, oliguria from prerenal failure is followed by vasomotor nephropathy and established renal failure. e. coli, shigella, and salmonella (particularly salmonella typhi) may invade the bloodstream and induce septicemia or septic shock. acute renal failure is commonly seen in infants with e. coli sepsis but is also reported with klebsiella, salmonella, and shigella infections. its pathophysiology and treatment were discussed previously. enteric infections with e. coli, yersinia, campylobacter, and salmonella have been associated with several different forms of gn, including membranoproliferative gn (mpgn), interstitial nephritis, diffuse proliferative gn, and iga nephropathy (63) (64) (65) . in typhoid fever, gn ranging from mild asymptomatic proteinuria and hematuria to acute renal failure may occur (64, (66) (67) (68) . renal biopsy findings show focal proliferation of mesangial cells, hypertrophy of endothelial cells, and congested capillary lumina. immunofluorescent studies show igm, igg, and c3 deposition in the glomeruli, with salmonella antigens detected within the granular deposits in the mesangial areas. in the iga nephropathy after typhoid fever, salmonella vi antigens have been demonstrated within the glomeruli. yersinia infection has been reported as a precipitant of gn in several studies (65, 69) . transient proteinuria and hematuria are found in 24% of patients with acute yersinia infection, and elevated creatinine levels in 10%. renal biopsy reveals mild mesangial gn or iga nephropathy. yersinia antigens, immunoglobulin, and complement have been detected in the glomeruli. yersinia pseudotuberculosis is well recognized as one of the causes of acute tubulointerstitial nephritis causing acute renal failure, especially in children; patients have histories of drinking untreated water in endemic areas (70) (71) (72) . the illness begins with the sudden onset of high fever, skin rash, and gi symptoms. later in the course, periungual desquamation develops, mimicking kawasaki disease. elevated erythrocyte sedimentation rate, c-reactive protein level, and thrombocytosis are noticeable, and mild degrees of proteinuria, glycosuria, and sterile pyuria are common. acute renal failure, which typically develops 1-3 weeks after the onset of fever, follows a benign course with complete recovery. renal biopsy mainly reveals findings of acute tubulointerstitial nephritis. antibiotic therapy, although recommended, does not alter the clinical course, but reduces the fecal excretion of the organism (73, 74) . hus is characterized by three distinct clinical signs: acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. it was first described in 1955 and was associated with infection by shiga toxin-producing shigella dysenteriae. a major breakthrough in the search for the cause of hus occurred in the 1980s when karmali et al. reported that 11 of 15 children with diarrheaassociated hus had evidence of infection with a strain of e. coli that produced a toxin active on vero cells (75) . in diarrhea-associated hus in the united states and most of europe, e. coli 0157:h7 is the most important of these strains. e. coli 0157:h7 occurs naturally in the gi tract of cattle and other animals, and humans become infected through contaminated food products. most outbreaks have been associated with consumption of undercooked meat, but unpasteurized milk and cider, drinking water, and poorly chlorinated water for recreational use have also been implicated as vehicles for bacterial spread. hus is discussed in detail in chapter 67. the global epidemic of mycobacterium tuberculosis is growing. several factors have contributed to this increase, including the emergence of the human immunodeficiency virus (hiv) infection epidemic, large influxes of immigrants from countries in which tuberculosis (tb) is common, the emergence of multiple-drug-resistant m. tuberculosis, and breakdown of the health services for effective control of tb in various countries. it is generally estimated that, overall, one-third of the world's population is currently infected with the tb bacillus. there are more than 8 million cases of tb, which result in the death of approximately 2 million people each year. furthermore, 5-10% of people who are infected with the tb bacillus develop tb disease or become infectious at some time during their lives (76, 77) . after respiratory illness in children, mycobacteria are widely distributed to many organs of the body during the lymphohematogenous phase of childhood tb (78) . tubercle bacilli can be recovered from the urine in many cases of miliary tb. hematogenously-spread tuberculomata develop in the glomeruli, which results in caseating, sloughing lesions that discharge bacilli into the tubules. in most cases, the renal lesions are asymptomatic and manifest as mycobacteria in the urine or as sterile pyuria. tuberculomata in the cortex may calcify and cavitate or may rupture into the pelvis, discharging infective organisms into the tubules, urethra, and bladder. dysuria, loin pain, hematuria, and pyuria are the presenting features of this complication, but in many cases, the renal involvement is asymptomatic, even when radiologic and pathologic abnormalities are very extensive. continuing tuberculous bacilluria may cause cystitis with urinary frequency and, in late cases, a contracted bladder (79) . the intravenous urogram is abnormal in most cases. early findings are pyelonephritis with calyceal blunting and calyceal-interstitial reflux. later, papillary cavities may be seen, indicating papillary necrosis. ureteric strictures, focal calcification, hydronephrosis, and cavitation may also be seen. renal function is usually well preserved, and hypertension is uncommon. in some cases, either the infection itself or reactions to the chemotherapeutic agents may result in renal failure with evidence of an interstitial nephritis (79) (80) (81) . classic symptomatic renal tb is a late and uncommon complication in children, rarely occurring less than 4 or 5 years after the primary infection, and therefore is most commonly diagnosed after adolescence (78, 80) . adult studies have shown that 26-75% of renal tb coexists with active pulmonary tb and 6-10% of screened sputumpositive pulmonary tb patients have renal involvement. the diagnosis is established by isolation of mycobacteria from the urine or by the presence of the characteristic clinical and radiographic features in a child with current or previous tb. renal tb is treated with drug regimens similar to those used for other forms of tb, with isoniazid, rifampicin, pyrazinamide and ethambutol administered initially for 2 months, and isoniazid and rifampicin then continued for a further 7-10 months. late scarring and urinary obstruction may occur in cases with extensive renal involvement, and such patients should be followed by ultrasonography or intravenous urogram. mycobacteria, both m. tuberculosis and atypical mycobacteria, have also emerged as important causes of opportunistic infection in immunocompromised patients undergoing dialysis and in patients undergoing renal transplantation. the possibility of mycobacterial disease must be considered in patients with fever of unknown origin or unexplained disease in the lungs or other organs. results of the mantoux test are often negative, and diagnosis depends on maintaining a high index of suspicion and isolating the organism from the infected site. renal involvement has been well documented in both congenital and acquired syphilis, with an estimated occurrence of 0.3% in patients with secondary syphilis and up to 5% in those with congenital syphilis (82, 83) . the most common manifestation of renal disease in congenital syphilis is the nephrotic syndrome, with proteinuria, hypoalbuminemia, and edema. in some patients, hematuria, uremia, and hypertension may be seen. the renal disease is usually associated with other manifestations of congenital syphilis, including hepatosplenomegaly, rash, and mucous membrane findings. nephritis in congenital syphilis is usually associated with evidence of complement activation, with depressed levels of clq, c4, c3, and c5. histologic findings are a diffuse proliferative gn or a membranous nephropathy. the interstitium shows a cellular infiltrate of polymorphonuclear and mononuclear cells (84) . immunofluorescent microscopy reveals diffuse granular deposits of igg and c3 along the glomerular basement membrane (gbm). mesangial deposits may also contain igm. on electron microscopy, scattered subepithelial electron-dense deposits are seen, with fusion of epithelial cell foot processes (84) . good evidence exists that renal disease is due to an immunologically mediated reaction to treponemal antigens. antibodies reactive against treponemal antigens can be eluted from the glomerular deposits, and treponemal antigens are present in the immune deposits. treatment of both congenital and acquired syphilis with antibiotics results in rapid improvement in the renal manifestations (82, 84) . renal involvement is surprisingly rare in mycoplasma pneumoniae infection considering the prevalence of this organism and its propensity to trigger immunologically mediated diseases such as erythema multiforme, arthritis, and hemolysis. acute nephritis associated with mycoplasma infection may occur 10-40 days after the respiratory tract infection (85, 86) . renal histopathologic findings include type 1 mpgn, proliferative endocapillary gn, and minimal change disease (87) . antibiotic treatment of the infection does not appear to affect the renal disease, which is self-limited in most cases (85, 86) . since its recognition in 1976, legionnaires' disease, caused by legionella pneumophila, has emerged as an important infectious diseases and the kidney cause of pneumonia. the disease most commonly affects the elderly but has been reported in both normal and immunocompromised children (97, 98) . renal dysfunction occurs in a minority of patients (98) . patients who develop renal impairment present with oliguria and rising urea and creatinine levels. they are usually severely ill, with bilateral pulmonary infiltrates, fever, and leukocytosis. shock may be present, and the renal impairment has been associated with acute rhabdomyolysis with high levels of creatine phosphokinase and myoglobinuria. renal histologic examination usually shows a tubulointerstitial nephritis or acute tubular necrosis (98, 99) . the pathogenesis of the renal impairment is uncertain, but the organism has been detected within the kidney on electron microscopy and immunofluorescent studies, which suggests a direct toxic effect. myoglobinuria and decreased perfusion may also be contributing factors, however. mortality has been high in reported cases of legionnaires' disease complicated by renal failure. treatment is based on dialysis, intensive care, and antimicrobial therapy with erythromycin (98) . steroid therapy may be effective for tubulointerstitial nephritis (99) . the rickettsial diseases are caused by a family of microorganisms that have characteristics common to both bacteria and viruses and that cause acute febrile illnesses associated with widespread vasculitis. with the exception of q fever, all are associated with erythematous rashes. there are four groups of rickettsial diseases: 1. the typhus group includes louse-borne and murine typhus, spread by lice and fleas, respectively. 2. the spotted fever group includes rocky mountain spotted fever, tick typhus and related mediterranean spotted fever and rickettsial pox, which are spread by ticks and mites, with rodents as the natural reservoir. 3. scrub typhus, which is spread by mites. 4. q fever, which is spread by inhalation of infected particles from infected animals. rickettsial diseases have a worldwide distribution and vary widely in severity, from self-limited infections to fulminant and often fatal illnesses (88) . in view of the widespread vasculitis associated with these infections, subclinical renal involvement probably occurs in many of the rickettsial diseases. however, in rocky mountain spotted fever, tick typhus, and q fever, the renal involvement may be an important component of the illness. rocky mountain spotted fever is the most severe of the rickettsial diseases (89, 90) . the onset occurs 2-8 days after the bite of an infected tick. high fever develops initially, followed by the pathognomonic rash, which occurs between the second and sixth days of the illness. the rash initially consists of small erythematous macules, but later these become maculopapular and petechial, and in untreated patients, confluent hemorrhagic areas may be seen. the rash first appears at the periphery and spreads up the trunk. involvement of the palms and soles is a characteristic feature (88) . headache, restlessness, meningism, and confusion may occur together with other neurologic signs. cardiac involvement with congestive heart failure and arrhythmia are common. pulmonary involvement occurs in 10-40% of cases. infection is associated with an initial leucopenia, followed by neutrophil leukocytosis. thrombocytopenia occurs in most cases. histopathologically, the predominant lesions are in the vascular system (91) . rickettsiae multiply in the endothelial cells, which results in focal areas of endothelial cell proliferation, perivascular mononuclear cell infiltration, thrombosis, and leakage of red cells into the tissues. the renal lesions involve both blood vessels and interstitium, and acute tubular necrosis may occur. acute gn with immune-complex deposition has been reported (92) , but in most cases the pathology appears to be a direct consequence of the invading organism on the renal vasculature (90, 93) . renal dysfunction is an important complication of rocky mountain spotted fever. elevation of urea and creatinine levels occurs in a significant proportion of cases, and acidosis is common. prerenal renal failure caused by hypovolemia and impaired cardiac function may respond to volume replacement and inotropic support, but acute renal failure may subsequently occur, necessitating dialysis. rocky mountain spotted fever is diagnosed by the characteristic clinical picture, the exclusion of disorders with similar manifestations (e.g., measles, meningococcal disease, and leptospirosis), and detection of specific antibodies in convalescence. culture of rickettsia rickettsii, immunofluorescent staining, and polymerase chain reaction (pcr) testing of blood and skin biopsy specimens are available only in reference laboratories. antibiotics should be administered in suspected cases without awaiting confirmation of the diagnosis (93) . doxycycline is the drug of choice for children of any age. chloramphenicol is also effective (94) . intensive support of shock and multiorgan failure may be required in severe cases, and peritoneal dialysis or hemodialysis may be required until renal function returns. before the advent of specific therapy, mortality was 25%. today the overall mortality in the united states is still 5-7%. death predominantly occurs in cases in which the diagnosis is delayed. q fever is caused by coxiella burnetii and has a worldwide distribution, with the animal reservoir being cattle, sheep, and goats. human infection follows inhalation of infected particles from the environment. the clinical manifestations range from an acute self-limited febrile illness with atypical pneumonia to involvement of specific organs that causes endocarditis, hepatitis, osteomyelitis, and central nervous system disease (95) . proliferative gn may be associated with either q fever endocarditis, rhabdomyolysis or a chronic infection elsewhere in the body (96) . renal manifestations range from asymptomatic proteinuria and hematuria to acute renal failure, hypertension, and nephrotic syndrome. renal histologic findings are those of a diffuse proliferative gn, focal segmental gn, or mesangial gn. immunofluorescent studies reveal diffuse glomerular deposits of igm in the mesangium, together with c3 and fibrin. c. burnetii antigen has not been identified within the renal lesions. treatment of the underlying infection may result in remission of the renal disease, but prolonged treatment may be required for endocarditis. tetracycline has been used in conjunction with rifampicin, co-trimoxazole, or a fluoroquinolone. nephritis has been reported in association with the presence of a wide range of microorganisms that cause chronic or persistent infection (> table 52 -2) (63, 100). it is likely that any infectious agent that releases foreign antigens into the circulation, including those of very low virulence, can cause renal injury either by deposition of foreign antigens in the kidney or by the formation of immune complexes in the circulation, which are then deposited within the kidney. nephritis is most commonly seen in association with intravascular infections such as sbe or infected ventriculoatrial shunts, but it is also seen after focal extravascular infections; ear, nose, and throat infections; and abscesses. renal involvement is one of the diagnostic features of bacterial endocarditis. virtually all organisms that cause endocarditis also produce renal involvement ( > table 52 -2). although endocarditis caused by bacteria is the most common and is readily diagnosed by blood culture (100), unusual but important causes of culture-negative endocarditis include q fever (101) and legionella infection (102) . in the immunocompromised individual, opportunistic pathogens such as fungi and mycobacteria are important causes. the usual renal manifestations of sbe are asymptomatic proteinuria, hematuria, and pyuria. loin pain, hypertension, nephrotic syndrome, and renal failure may occur in more severe cases. the renal lesions occurring in endocarditis are variable, and focal embolic and immune-complex-mediated features may coexist (100, 103, 104) . embolic foci may be evident as areas of infarction, intracapillary thrombosis, or hemorrhage. more commonly, there is a focal necrotizing or diffuse proliferative gn. immunofluorescent studies show glomerular deposits of igg, igm, iga, and c3 along the gbm and within the mesangium. electron microscopy reveals typical electron-dense deposits along the gbm and within the mesangium (100, 103, 104) . early reports suggested that the renal lesions were caused by microemboli from infected vegetations depositing in the kidney, a hypothesis supported by the occasional presence of bacteria within the renal lesions. most subsequent evidence, however, indicates that immunologic mechanisms rather than emboli are involved in the pathogenesis in most cases: bacteria are rarely found within the kidney, and renal involvement occurs with lesions of the right side of the heart, which would not be likely to embolize to the kidney. immune complexes containing bacterial antigens are present in the circulation, and both bacterial antigens and bacteria-specific antibodies can be demonstrated within the immune deposits in the kidney. serum c3 level is usually low, and complement can be found within both the circulating and the deposited immune complexes. these features all support an immune-complex-mediated pathogenesis of the renal injury (100, 103, 104) . treatment of the endocarditis with antibiotics usually results in resolution of the gn and is associated with the disappearance of immune complexes from the circulation and return of c3 levels to normal. the prognosis of the renal lesions in sbe generally depends on the response of the underlying endocarditis to antibiotics or, in cases of antibiotic failure, to surgical removal of the infective vegetations (105) . in patients previously treated by shunting for hydrocephalus, there is a well-documented association of gn with infected ventriculoatrial shunts. this condition is another example of an immune-complex nephritis similar to that seen in endocarditis (106) . coagulase-negative staphylococci are the causative organisms in 75% of cases. the clinical and pathologic findings are similar to those in sbe. presenting features are proteinuria, hematuria, and pyuria, and they may progress to renal failure. immune complexes containing the bacterial antigens and complement are present in the serum, and c3 is depressed. histologic findings are those of a diffuse mesangiocapillary gn. immunofluorescent microscopy demonstrates deposits of immunoglobulin and c3 along the gbm, and bacterial antigen can be demonstrated in the renal lesions (107) . the prognosis for the renal lesion is good if the infection is treated early. this usually involves removal of the infected shunt and administration of appropriate antibiotics (106, 108) . the possible progression to end-stage renal disease requires frequent nephrologic monitoring of patients with ventriculoatrial shunts (106) . there are a few reports in the literature of a similar renal complication occurring in chronic infection of ventriculoperitoneal shunts. gn has been reported after chronic abscesses (63), osteomyelitis, otitis media, pneumonia, and other focal infections ( > table 52 -2). acute renal failure has been the presenting feature of focal infections in various sites, including the lung, pleura, abdominal cavity, sinuses, and pelvis. many different organisms have been responsible, including s. aureus, pseudomonas, e. coli, and proteus species. this is probably another example of immunecomplex gn. c3 level is decreased in approximately onethird of reported cases, and immunofluorescent studies reveal diffuse granular deposits of c3 in the glomeruli of all reported instances, with a variable presence of immunoglobulin. the renal lesion is that of mpgn and crescentic nephritis. the renal outcome is reported to be good with successful early treatment of the underlying infection. the role of viral infections in the causation of renal disease has been less well defined than that of bacterial infections. clearly defined associations of renal disease have been made with hepatitis b virus (hbv), hepatitis c virus (hcv), hiv, and hantaviruses, but the role of most other viruses in the pathogenesis of renal disease is not clearly defined. most viruses causing systemic infection may trigger immunologically mediated renal injury. with increasing application of molecular techniques, it may be that a significant proportion of gns currently considered to be idiopathic will ultimately be shown to be virus induced. in children with immunodeficiency states and those undergoing renal transplantation, viruses such as cytomegalovirus (cmv) and polyoma virus have been recognized to be associated with nephropathy. since the discovery of hepatitis b surface antigen (hbsag) in 1964, hepatitis virus has been shown to infect more than 5% of the world's population and is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma worldwide. some 350 million people have hbsag in the circulation (who figures). the infection is most common in africa and the orient, where it is acquired in childhood by vertical transmission from infected mothers or by horizontal transmission from other children or adults. in developed countries, transmission in adults occurs more often by blood product exposure, sexual contact, or intravenous drug use. the epidemiology of hbv infection in children is changing following the widespread use of effective vaccination at birth, in both developed and developing countries. hbv is a complex dna virus with an outer surface envelope (hbsag) and an inner nucleocapsid core containing the hepatitis b core antigen (hbcag), dna polymerase, protein kinase activity, and viral dna. incomplete spherical and filamentous viral particles consisting solely of hbsag are the major viral products in the circulation and may be present in concentrations of up to 1014 particles per ml of serum. hepatitis b e antigen (hbeag) can be released from hbcag by proteolytic treatment and may be found in the circulation either free or complexed to albumin or igg antibodies. the presence of hbeag correlates with the presence of complete viral particles and the infectivity of the individual (109) . infection with hbv may result in either a self-limited infectious hepatitis followed by clearance of the virus and complete recovery, or a chronic or persistent infection in which the immune response is ineffective in eliminating the virus. chronic hbv infection with continued presence of viral antigens in the circulation caused by an ineffective host immune response provides the best-documented example of immunologically mediated renal injury caused by persistent infection (110) . development of chronic hbv infection is positively associated with infection at a younger age, particularly in infancy, where the rate of chronic infection is up to 90%. in contrast, the likelihood of an acute, symptomatic illness increases with age, to a level where approximately 40% of infections are symptomatic in children above 15 years. in the early prodromal phase of hbv hepatitis, before the onset of jaundice, some patients develop fever, maculopapular or urticarial rash, and transient arthralgias or arthritis. occasionally, proteinuria, hematuria, or sterile pyuria are observed. the syndrome usually lasts 3-10 days and often resolves before the onset of jaundice (110, 111) . there have been no histologic studies of the renal changes during this prodromal period. since 1970, numerous reports have linked hbv infection with polyarteritis nodosa (pan). most of these cases have been in adults, but the disorder has also been reported in children (112, 113) , where it is estimated that approximately one third of pan cases are caused by hbv (114) . hbv pan appears to be uncommon in africa and the orient, where infection is usually acquired in childhood, and has declined in incidence following the introduction of hbv vaccination (115) . hbv pan presents weeks to months after a clinically mild hepatitis but may occasionally predate the hepatitis. after a prodromal illness, frank vasculitis affecting virtually any organ appears. abdominal pain, fever, mononeuritis multiplex, and pulmonary and renal involvement may occur. the renal involvement may appear as hypertension, hematuria, proteinuria, or renal failure (see chapter 61) . laboratory investigations reveal a florid acute-phase response, leukocytosis, and anemia. transaminase levels are usually elevated, and hbsag is present in the circulation. the pathology consists of focal inflammation of small and medium-sized arteries, with fibrinoid necrosis, leukocyte infiltration, and fibrin deposition. renal pathology may be limited to the medium-sized arteries or may coexist with gn (110, 116) . circulating immune complexes containing hbsag and anti-hbs antibodies are usually present in the circulation (110, 116) . c3, c4, and total hemolytic complement levels are depressed. hbsag, igg, and igm antibodies to hbv and c3 have been identified by immunofluorescence in the blood vessels (116) . a positive anca excludes hbv-pan (115) . although most evidence suggests that the pathogenesis involves an immunecomplex-mediated vasculitis, autoantibody or cell-mediated vascular injury may coexist. if the condition is untreated, the mortality is high (112) . most studies suggest that steroids or immunosuppressants help to suppress the vasculitis but potentially predispose to chronic infection . (110, 112) . successful treatment of hepatitis b-associated pan with nucleoside analogues such as lamivudine or newer anti-viral drugs, either alone or in combination with interferon-alpha and conventional immunosuppressive therapy, has been reported (117) (118) (119) . hbv is now the major cause of membranous gn (mgn) in children worldwide. the proportion of patients with mgn caused by hbv is directly related to the incidence of hbsag in the population, with 80-100% of all cases of mgn in some african and oriental countries being associated with hbv (110, 120) (see chapter 26) . hbv mgn usually presents in children aged 2-12. there is a striking male predominance; in the united states, 80% of patients are males (121) . the virus is usually acquired by vertical transmission from infected mothers or horizontally from infected family members. unlike adults with hbv mgn, children do not usually have a history of hepatitis or of active liver disease, but liver function test results are generally mildly abnormal. liver biopsy specimens may show minimal abnormalities, chronic persistent hepatitis, or (occasionally) more severe changes (121) . the renal manifestations are usually of proteinuria, nephrotic syndrome, microscopic hematuria, or (rarely) macroscopic hematuria. hypertension occurs in less than 25% of cases, and renal insufficiency is rare. hbsag and hbcag are usually present in the circulation, and hbe antigenemia is seen in a high proportion of cases. occasionally, hbsag may be found in the glomeruli but is absent from the circulation. c3 and c4 levels are often low, and circulating immune complexes are found in most cases. immunohistologic study reveals deposits of igg and c3 and (less commonly) igm and iga in subepithelial, subendothelial, or mesangial tissue. hbv particles may be seen on electron microscopy, and all the major hepatitis b antigens, including hbsag, hbcag, and hbeag, have been localized in the glomerular capillary wall on immunofluorescence. immunologic deposition of hbv and antibody in the glomerular capillary wall is clearly involved in the glomerular injury, but the underlying immunologic events are incompletely understood (110, 122) . passive trapping of circulating immune complexes may be involved, but the circulating immune complexes containing hbsag are usually larger than would be expected to penetrate the basement membrane. hbsag and hbcag are anionic and are therefore unlikely to penetrate the glomerular capillary wall. in contrast, hbeag forms smaller complexes with anti-hbe antibodies and may readily penetrate the gbm. this may explain the observation that hbeag in the circulation frequently correlates with the severity of the disease (110) . an alternative mechanism for immunemediated glomerular injury is the trapping of hbv antigens by antibody previously deposited in the kidney. anti-hbe antibodies are cationic and may readily localize in the glomerulus and subsequently bind circulating antigen and complement. the third possibility is that the depositions of hbv and antibodies are consequences of glomerular injury by cellular mechanisms or autoantibodies. little evidence supports this view at present (110) . a transgenic mouse model of hbv-associated nephropathy has been developed, in which hbsag and hbcag is expressed in liver and kidney, particularly tubular epithelial cells, without viral replication. in these mice, gene expression analysis revealed upregulation of acute-phase proteins, particularly c3, although measurable serum c3 levels were reduced. this supports the notion that local persistent expression of hbv viral proteins contributes to hbv-associated nephropathy (123) . hbv infection has been associated with a variety of other forms of gn in both adults and children. in one small series in children, mpgn was found to be equal in incidence to mgn in the spectrum of hbv-associated gns (124) . both mpgn and mesangial proliferative gn may be triggered by hbv. in several countries where hbv is common, the proportion of patients with these forms of nephritides who test positive for hbv greatly exceeds the incidence of positivity in the general population (122) . as with mgn and hbv-associated pan, circulating immune complexes and localization of hbv antigens in the glomeruli have been reported in both mpgn and mesangial proliferative gn, and it is likely that similar mechanisms are occurring (110, 125) . several other forms of gn have been associated with hbv, including iga nephropathy, focal glomerulosclerosis, crescentic nephritis, and systemic lupus erythematosus, but the evidence for these associations is less consistent than for the entities discussed earlier (125) . hbv is normally cleared as a result of cell-mediated responses in which cytotoxic t cells and natural killer cells eliminate infected hepatocytes. it is not surprising, therefore, that the administration of steroids and immunosuppressive agents either may have no effect on hbv disease or may increase the risk of progressive disease (126) . children with hbv mgn have a good prognosis, and two-thirds undergo spontaneous remission within 3 years of diagnosis. steroid therapy does not appear to provide any additional benefit (110, 120, 110) . antiviral therapy with pegylated interferon-alpha and lamivudine shows promise in facilitating clearance of hbv, and in some cases, elimination of the infection with antiviral therapy in both children and adults is associated with improvement or resolution of the coexisting renal disease. there is considerable effort being put into the development of newer anti-viral agents which avoid the common problems of resistance associated with lamivudine (127-129). hcv is an enveloped, single-stranded rna virus of approximately 9.4 kb in the flaviviridae family. there are six major hcv genotypes. hepatitis c is a common disease affecting approximately 400 million people worldwide. in the united states, 4.1 million persons are estimated to be anti-hcv positive, and 3.2 million may be chronically infected (130) . an estimated 240,000 children in the united states have antibody to hcv and 68,000-100,000 are chronically infected (131) . children become infected through receipt of contaminated blood products or through vertical transmission. the risk of vertical transmission increases with higher maternal viremia and maternal co-infection with hiv. acute hcv infection is rarely recognized in children outside of special circumstances such as a known exposure from an hcv-infected mother or after blood transfusion. most chronically infected children are asymptomatic and have normal or only mildly abnormal alanine aminotransferase levels. although the natural history of hcv infection during childhood seems benign in the majority of instances, the infection can take an aggressive course in a proportion of children, leading to cirrhosis and end-stage liver disease during childhood. the factors responsible for this more aggressive course are unidentified (131) . even in adults, the natural history of hcv infection has a variable course, but a significant proportion of patients will develop some degree of liver dysfunction, and 20-30% will eventually have end-stage liver disease as a result of cirrhosis. the risk of hepatocellular carcinoma is significant for those who have established cirrhosis. hepatitis c is currently the most common condition leading to liver transplantation in adults in the ''western world.'' gn has been described as an important complication of chronic infection with hcv in adults. the clinical presentation is usually of nephrotic syndrome or proteinuria, hypertension, or hematuria, with or without azotemia (132) . mpgn, with or without cryoglobulinemia, and mgn are most commonly described. isolated case reports of other, more unusual patterns of glomerular injury, including iga nephropathy, focal segmental glomerulosclerosis, crescentic gn, fibrillary gn, and thrombotic microangiopathy, have also been associated with hcv infection (132, 133) . glomerular deposition of hepatitis antigens and antibodies has been described and is believed to play a role in pathogenesis. cryoglobulinemia is a common accompaniment of gn that is associated with the depression of serum complement levels (132) . renal failure may develop in 40-100% of patients who have mpgn (132, 134) . the presence of virus-like particles as well as viral rna within the kidney sections of patients with hcv-associated glomerulopathies has been reported (135) . the diagnosis should be suspected if glomerular disease is associated with chronic hepatitis, particularly with the presence of cryoglobulins, but renal biopsy is necessary to establish a definitive diagnosis. hcv infection is relatively common in children with end-stage renal disease and is an important cause of liver disease in this population. acquisition of hcv infection continues to occur in dialysis patients because of nosocomial spread (136) . elevation of transaminase level is not a sensitive marker of infection in children and hcv enzymelinked immunosorbent assay or pcr testing should be used to increase sensitivity (137) . hcv-infected renal transplant recipients had higher mortality and hospitalization rates than other transplant recipients (138) , and hcv infection has been reported to be associated with de novo immune-mediated gn, especially type 1 mpgn, in renal allografts, resulting in accelerated loss of graft function (139, 140) . no large randomized, controlled trials of treatment of children with chronic hepatitis c have been performed, although one study (peds-c) is currently recruiting patients into a trial of pegylated interferon +/ã� ribavirin (141) . small heterogeneous studies of interferon monotherapy have reported sustained virologic response rates of 35-40% (131) . in adults, improvement of proteinuria and renal function often follows interferon-alpha treatment (132, 134) , but relapses are common after cessation of treatment. combination of interferon with ribavirin in infectious diseases and the kidney patients with chronic liver disease has been shown to increase the rate of sustained response in these patients (142) . as yet, however, there are few data regarding the use of combination therapy with interferon and ribavirin in children. moreover, interferon-alpha therapy is associated with acute or subacute renal failure in more than one-third of the patients with renal transplants (143) . hepatitis c may be complicated by systemic mixed cryoglobulinemic (mc) vasculitis, and in some cases by a polyarteritis nodosa (pan)-type non-cryoglobulinemic vasculitis (144) . treatment with interferon-a (ifn-a) and ribavirin mostly is associated with an improvement of vasculitic symptoms. in some cases, exacerbation and rarely new onset of vasculitis of the peripheral nervous system have been described after this treatment. in fulminant cases immunosuppressive therapy with steroids, and cyclophosphamide, or rituximab may be needed to control life threatening vasculitis prior to antiviral treatment (144) . cytomegalovirus cmv is one of the eight human herpes viruses. transmission of the virus requires exposure to infected body fluids such as breast milk, saliva, urine, or blood. individuals initially infected with cmv may be asymptomatic or display nonspecific flu-like symptoms. after the initial infection cmv, like all herpes viruses, establishes latency for life but will be periodically excreted by an asymptomatic host. cmv replicates within renal cells, and on biopsy samples from immunocompromised hosts, viral inclusions can be visualized by light microscopy in cells of the convoluted tubules and collecting ducts (145) . glomerular cells and shed renal tubular cells may have characteristic inclusions, but clinically evident renal disease is rare and is seen virtually only in immunocompromised or congenitally infected children (145, 146) . the clinical manifestations of cmv-induced renal disease in congenitally infected infants are variable and range from asymptomatic proteinuria to nephrotic syndrome and renal impairment. in congenital cmv infection, histologic changes of viral inclusions commonly occur in the tubules. in addition, proliferative gn has been reported, with evidence on electron microscopy of viral immune deposits in glomerular cells (146, 147) . in cmv-infected immunocompromised patients, immunecomplex gn has been documented with mesangial deposits of igg, iga, c3, and cmv antigens within glomeruli. eluted glomerular immunoglobulins have been shown to contain cmv antigens (148) . cmv is the most common viral infection after kidney transplantation. experience with pediatric kidney transplant recipients suggests a 67% incidence of cmv infection (149) . the direct and indirect effects of cmv infection result in significant morbidity and mortality among kidney transplant recipients. cmv-negative patients who receive a cmv-positive allograft are at risk for primary infection and graft dysfunction. patients who are cmv seropositive at the time of transplantation are also at risk of reactivation and superinfection. tubulointerstitial nephritis is a well-characterized pathologic feature of renal allograft cmv disease, which can be difficult to distinguish from injury caused by rejection. histologic evidence of endothelial cell injury and mononuclear cell infiltration in the glomeruli has been reported (148) . cmv glomerular vasculopathy in the absence of tubulointerstitial disease, causing renal allograft dysfunction, has also been reported (150) . beyond the acute allograft nephropathy associated with cmv viremia, cmv is known to cause chronic vascular injury. this may adversely affect the long-term outcome of the allograft and may be the explanation for the observed association with chronic allograft nephropathy (151) . newer techniques for rapidly diagnosing cmv infection are becoming widely available and include shell vial culture, pp65 antigenemia assay, pcr, and the hybridcapture rna-dna hybridization assay for qualitative detection of cmv pcr. quantitative plasma pcr testing (pcr viral load) is increasingly used for diagnosis and monitoring of cmv viremia in renal transplant recipients. antiviral agents that have been shown to be effective against cmv include ganciclovir, valganciclovir, foscarnet, and cidofovir. ganciclovir remains the drug of choice for treating established disease. intravenous ganciclovir therapy is preferred in children because of the erratic absorption of oral ganciclovir. major limitations of ganciclovir therapy are the induction of renal tubular dysfunction and bone marrow toxicity, principally neutropenia and thrombocytopenia. dosage adjustments are necessary for recipients with renal dysfunction. oral valganciclovir is now used for cmv prophylaxis post-transplant (152) . use of other antiviral agents such as foscarnet and cidofovir is limited because of nephrotoxicity and difficulty of administration. a number of reports have demonstrated the effectiveness of high-titer cmv immune globulin therapy in reducing severe cmv-associated disease when used in combination with ganciclovir (149, 153) . the association of varicella with nephritis has been known for more than 100 years since henoch reported on four children with nephritis that occurred after the appearance of varicella vesicles. varicella, however, is rarely associated with renal complications (154) . in fatal cases with disseminated varicella and in the immunocompromised individual, renal involvement is more common. cases in which varicella infection caused gn in renal transplant recipients have been reported (155) . histologic findings in fatal cases include congested hemorrhagic glomeruli, endothelial cell hyperplasia, and tubular necrosis. in mild and nonfatal cases and in non-immunocompromised individuals, varicella is occasionally associated with a variety of renal manifestations, ranging from mild nephritis to nephrotic syndrome and acute renal failure (156) . histologic findings include endocapillary cell proliferation, epithelial and endothelial cell hyperplasia, and inflammatory cell infiltration (154) . rapidly progressive nephritis has also been reported. immunohistochemical studies reveal glomerular deposition of igg, igm, iga, and c3. on electron microscopy, granular electron-dense deposits have been found in the paramesangial region, and varicella antigens may be deposited in the glomeruli. the features suggest an immune-complex nephritis. elevated circulating levels of igg and iga immune complexes and depressed c3 and c4 levels support this possibility (154) . fulminant disseminated varicella and varicella in immunocompromised patients should be treated with intravenous acyclovir. renal involvement is common during acute infectious mononucleosis, usually manifesting as an abnormal urine sediment, with hematuria in up to 60% of cases. hematuria, either microscopic or macroscopic, usually appears within the first week of the illness and lasts for a few weeks to a few months. proteinuria is usually absent or low grade. more severe renal involvement with proteinuria, nephrotic syndrome, or acute nephritis with renal failure is much less common. acute renal failure may be seen during the course of fulminant infectious mononucleosis with associated hepatic failure, thrombocytopenia, and encephalitis. it is usually caused by interstitial nephritis that is likely the result of immunopathologic injury precipitated by epstein-barr virus (ebv) infection. however, the identification of ebv dna in the kidney raises the possibility that direct infection might play a role (157) . the renal involvement must be distinguished from myoglobinuria caused by rhabdomyolysis, which may occur in infectious mononucleosis, and from bleeding into the renal tract as a result of thrombocytopenia. renal histologic findings in ebv nephritis are an interstitial nephritis with mononuclear cell infiltration and foci of tubular necrosis. glomeruli may show varying degrees of mesangial proliferation. on immunohistochemical study, ebv antigens are seen in glomerular and tubular deposits. the prognosis for complete recovery of renal function is good. treatment with corticosteroids may have a role in the management of ebv-induced acute renal failure and may shorten the duration of renal failure (158) . ebv-associated post-transplantation lymphoproliferative disease is a recognized complication in renal transplant recipients. latent infection of ebv in renal proximal tubular epithelial cells has recently been described as causing idiopathic chronic tubulointerstitial nephritis (159) . the herpes simplex virus (hsv) causes persistent infection characterized by asymptomatic latent periods interspersed with acute relapses. as with other chronic and persistent infections, immunologically mediated disorders triggered by hsv are well recognized, and it is perhaps surprising that hsv has rarely been linked to nephritis. acute nephritis and nephrotic syndrome have been associated with herpes simplex encephalitis. renal histology shows focal segmental gn with mesangial and segmental deposits of igm, c3, and hsv antigens. as with other herpes viruses, hsv has been suggested as a trigger for iga nephritis, mpgn, and membranous nephropathy. elevated levels of hsv antibodies have been reported in patients with a variety of forms of gn, but no conclusive evidence exists of an etiologic role for hsv (160) . adenovirus and enterovirus, are unrelated ubiquitous pathogens that infect large proportions of the population annually and yet are rarely associated with renal disease. the literature contains scattered reports of acute nephritis after infection with each of these viruses. adenovirus is a major cause of hemorrhagic cystitis and was implicated as the cause of hemorrhagic cystitis in 23-51% of children with this disorder (161) . boys are affected more often than girls, and hematuria persists for 3-5 days. microscopic hematuria, dysuria, and frequency may occur for longer periods. adenovirus types 11 and 21 are the usual strains isolated. picornaviruses, including enteroviruses, echovirus and coxsackieviruses, have been linked with acute nephritis and acute renal failure associated with rhabdomyolysis. coxsackie b virus can be isolated in urine. direct infection of kidney cells is supported by in vitro work demonstrating lytic infection of human podocyte and proximal tubular epithelial cell cultures, although different strains exhibit variable degrees of nephrotropism. renal damage in vivo may have both a direct lytic mechanism and an immune-complex basis (162) . in the newborn, enteroviruses cause fulminant disease with dic, shock, and liver failure, and acute renal failure may occur. renal involvement from measles virus is uncommon, although measles virus can be cultured from the kidney in fatal cases. an acute gn has been reported to follow measles with evidence of immune deposits containing measles virus antigen within the glomeruli. the nephritis is generally self-limiting (163) . mild renal involvement is common during the acute phase of mumps infection. one-third of children with mumps have abnormal urinalysis results, with microscopic hematuria or proteinuria. mumps virus may be isolated from urine during the first 5 days of the illness, at a time when urinalysis findings are abnormal. plasma creatinine concentrations usually remain normal, despite the abnormal urine sediment, but more severe cases in adults have been associated with evidence of acute nephritis with impaired renal function. renal biopsy specimens demonstrate an mpgn with deposition of iga, igm, c3, and mumps virus antigen in the glomeruli, which suggests an immune-complex-mediated process (164) . despite the increasing availability of interventions to limit vertical hiv transmission, an estimated 1,500 children renal involvement in hiv infection was first described in 1984 in adults (165) (166) (167) and in children (168) , and renal involvement occurs in 2-15% of hiv-infected children in the united states (169) (170) (171) . since the development of highly active antiretroviral therapy (haart), however, the incidence of end-stage renal disease in hiv infection in both adults and children in industrialized countries has declined, but it is predicted that the dramatic decline in aids-related deaths will lead to an ageing population of hiv-infected individuals who will be at risk of non-hiv related renal problems, such that the numbers of hiv-positive esrd patients will increase in the united states (172) . hiv infection is associated with a number of renal pathologies. hiv-associated nephropathy (hivan) is a syndrome of glomerular and tubular dysfunction, which can progress to end-stage renal failure. it is discussed more fully below. glomerular syndromes other than hivan include mgn that resembles lupus nephritis and immune-complex gn, with iga nephropathy and hcv-associated mpgn being the most common forms. there have also been several case reports of amyloid kidney (171, 173, 174) . the kidneys may be affected by various other mechanisms. opportunistic infections with organisms such as bk virus (bkv) that give rise to nephropathy and hemorrhagic cystitis have been reported in association with hiv infection (175) . systemic infections accompanied by hypotension can cause prerenal failure leading to acute tubular necrosis. acute tubular necrosis has also been reported in hiv patients after the use of nephrotoxic drugs such as pentamidine, foscarnet, cidofovir, amphotericin b, and aminoglycosides. intratubular obstruction with crystal precipitation can occur with the use of sulfonamides and intravenous acyclovir. indinavir is well recognized to cause nephropathy and renal calculi (176) . mpgn associated with mixed cryoglobulinemia and thrombotic microangiopathy/atypical-cal hus in association with hiv infections have been reported (177, 178) . hivan is characterized by both glomerular and tubular dysfunction, the pathogenesis of which is not entirely known. hivan is a clinico-pathologic entity that includes proteinuria, azotemia, focal segmental glomerulosclerosis or mesangial hyperplasia, and tubulointerstitial disease (171) . in adults in the united states, there is a markedly increased risk of nephropathy among african american persons with hiv infection. this appears to be true in children as well, but the data are sparse. the spectrum of hivan seems to be coincident with the degree of aids symptomatology. it is thought that hivan can present at any point in hiv infection, but most patients with hivan have cd4 counts of less than 200 ã� 10 6 /200 cells/ml, which suggests that it may be primarily a manifestation of late-stage disease (179) . although a spectrum of clinicopathologic entities including mesangial hyperplasia, focal segmental glomerulosclerosis, minimal change disease, and systemic lupus erythematosus nephritis has been described, the classic pathologic feature of hivan is the collapsing form of focal and segmental glomerulosclerosis (180) . in the affected glomeruli, visceral epithelial cells are hypertrophied and hyperplastic, and contain large cytoplasmic vacuoles and numerous protein resorption droplets. there is microcystic distortion of tubule segments, which contributes to increasing kidney size. podocyte hyperplasia can become so marked that it causes obliteration of much of the urinary space, forming ''pseudocrescents'' (173) . capillary walls are wrinkled and collapsed with obliteration of the capillary lumina. the interstitium is edematous with a variable degree of t-cell infiltration (181) . the bowman capsule can also be dilated and filled with a precipitate of plasma protein that represents the glomerular ultrafiltrate. one of the most distinctive features of hivan, however, is the presence of numerous tubuloreticular inclusions within the cytoplasm of glomerular and peritubular capillary endothelial cells (173) . immunofluorescence testing is positive for igm and c3 in capillary walls in a coarsely granular to amorphous pattern in a segmental distribution (180, 181) . the presence of the hiv genome in glomerular and tubular epithelium has been demonstrated using complementary dna probes and in situ hybridization. proviral dna has been detected by pcr in the glomeruli, tubules, and interstitium of micro dissected kidneys from patients who had pathologic evidence of hivan, but it has also been detected in the kidneys of hiv-positive patients with other glomerulopathies (182) . a combination of both proliferation and apoptosis of renal cells may cause the loss of nephron architecture. apoptosis has been demonstrated in cells in the glomerulus, tubules, and interstitium of biopsy specimens from hiv-positive patients with focal segmental glomerulosclerosis. in addition, the role of various cytokines and growth factors, specifically transforming growth factor beta (tgf-beta), in the development of sclerosis has been studied (183, 184) . transgenic murine models provide some of the strongest evidence for a direct role of hiv-1 in the induction of hivan. these mice do not produce infectious virus but express the hiv envelope and regulatory genes at levels sufficient to re-create the hivan that is seen in humans (183) . serial deletion experiments have concluded that the nef and vpr genes are necessary though not sufficient for hivan pathogenesis. additional factors such as genetic predisposition are thought to explain the fact that african americans have a far greater likelihood of developing hivan than other racial groups, and that hivan is more likely in patients with a family history of esrd. hivan can manifest as mild proteinuria, nephrotic syndrome, renal tubular acidosis, hematuria, and/or acute renal failure (168) (169) (170) (171) . nephrotic syndrome and chronic renal insufficiency are late manifestations of hivan. children with hivan are likely to develop transient electrolytic disorders, heavy proteinuria, and acute renal failure due to systemic infectious episodes or nephrotoxic drugs. early stages of hivan can be identified by the presence of proteinuria and ''urine microcysts'' along with renal sonograms showing enlarged echogenic kidneys. urinary renal tubular epithelial cells are frequently grouped together to form these microcysts, which were found in the urine of children with hivan who had renal tubular injury (171) . advanced stages of hivan typically present with nephrotic syndrome with edema, heavy proteinuria, hypoalbuminemia, and few red or white blood cells in urinary sediments. hypertension may be present, but usually blood pressure is within or below the normal range. hivan in adults follows a rapidly progressive course, with end-stage renal disease developing within 1-4 months, but in children this rapid progression does not necessarily occur. definitive diagnosis of hivan should be based on biopsy results, and biopsy should be performed if significant proteinuria is present, because in approximately 50% of hiv-infected patients with azotemia and/or proteinuria (>1 g/24 h) who undergo renal biopsy, the specimen will have histologic features consistent with other renal diseases (179) . when available, haart should be given to children with symptomatic hiv disease. specific treatment of hivan remains controversial. several studies have looked at the role of haart, angiotensin i-converting enzyme (ace) inhibitors, steroids, and even cyclosporin with somewhat encouraging results. however, as yet no randomized case-controlled trials have been undertaken. most of the studies have been small and retrospective, and many have included patients both with and without renal biopsy-proven hivan. cyclosporin has been used to treat hivan in children with remission of nephrotic infectious diseases and the kidney syndrome (169) . similar responses have been reported to treatment with corticosteroids in various studies (185) (186) (187) (188) . ace inhibitors have been used with encouraging results (189) . the general regimen used to treat patients with hiv, including haart, should be applied to children with hivan. the dosages of some medications must be adjusted to the patients glomerular filtration. there are reports of spontaneous regression of hivan with supportive management and treatment with haart, particularly with regimes containing protease inhibitors (190) (191) (192) (193) . it should be emphasized that the improvement reported with other modalities of treatment such as corticosteroids, cyclosporin, and ace inhibitors always occurs when these agents are given in conjunction with antiretroviral therapy. the kidneys of transgenic mice have been found to have elevated levels of tgf-beta messenger rna and protein (184) . furthermore, gene expression analysis on tubular epithelial cells from a patient with hivan found upregulation of several inflammatory mediator genes downstream of interleukin 6 and of the transcription factor nfkb (194) . several other therapeutic options have been suggested, aimed specifically at the presumed role of tgfbeta in the pathogenesis of hivan. treatment directed at its synthesis using gene therapy to block tgf-beta gene expression is being explored. therapy directed at decreasing the activity of tgf-beta using anti-tgf-beta antibodies or other inhibitory substances is also an area of investigation. in addition, blocking renal receptors for chemokines such as rantes (regulated upon derivation, normal t cell expressed and secreted), interleukin-8, and monocyte-chemoattractant protein-1 has been proposed as another possible treatment alternative (195) . in the haart era, the outlook for hiv patients with esrd has improved, but these patients fare worse than esrd patients without hiv (196) . most reports of hivinfected patients on hemodialysis have shown poor prognosis, with mean patient survival times ranging from 14-47 months. mortality is therefore still close to 50% within the first year of dialysis. in general, improved survival is associated with younger age at initiation of hemodialysis and with higher cd4 counts. access complications such as infection and thrombosis tend to occur at a higher rate in hiv-infected hemodialysis patients. cross infection with hiv in dialysis patients is very rare. no patient-topatient hiv transmission has yet been reported in a hemodialysis unit in the united states, although several such cases have occurred in south america (195, 197) . peritoneal dialysis is an alternative for hiv-infected patients. the incidence of peritonitis varies across studies, but some studies did report a higher incidence of pseudomonas and fungal peritonitis in the hiv-positive population (195) . infections with unusual organisms such as pasteurella multocida, trichosporon beigelii, and mycobacterium avium intracellulare complex have also been reported. several studies, however, have suggested that there is no significant difference between the hiv-infected and non-hiv-infected populations. of note is that virus capable of replication in vitro has been recovered from the peritoneal dialysis effluent, and it can be recoverable for up to 7 days in dialysis bags at room temperature and for up to 48 h in dry exchange tubing (195) . previously, long-term dialysis had been thought to be preferable to renal transplantation, primarily because of the concern that the immunosuppressive therapy required after transplantation could promote progression of hiv/ aids. a multicenter prospective study has been addressing these questions (198) . data so far indicate that the outcome for liver and kidney transplantation is not considerably different from patients without hiv, with good graft persistence, and a low rate of development of opportunistic infections in those with well-controlled hiv and relatively high cd4 counts (199) . the human polyoma viruses are members of the papovavirus family and have received increasing attention as pathogens in immunocompromised patients. they are nonenveloped viruses ranging in size from 45-55 nm, with a circular, double-stranded dna genome that replicates in the host nucleus. the best-known species in this genus are the bkv, the jc virus (jcv), and the simian virus sv40. bkv was first isolated from the urine of a 39-year-old man who developed ureteral stenosis 4 months after renal transplantation (200) . the name of the virus refers to the first patients initials, which is also true of jcv. bkv establishes infection in the kidney and the urinary tract, and its activation causes a number of disorders, including nephropathy and hemorrhagic cystitis. bkv-associated nephropathy has become an increasingly recognized cause of renal dysfunction in renal transplantation patients (201) (202) (203) (204) (205) . jcv establishes latency mainly in the kidney, and its reactivation can result in the development of progressive multifocal leukoencephalopathy. there are a few reports of nephropathy in association with jcv infection (see references in (206) ), but bkv poses a much bigger problem in this regard. recent studies have reported sv40 in the allografts of children who received renal transplants and in the urine, blood, and kidneys of adults with focal segmental glomerulosclerosis, which is a cause of end-stage renal disease and an indication for kidney transplantation (207) . seroprevalence rates as high as 60-80% have been reported among adults in the united states and europe. the peak incidence of primary infection (as measured by acquisition of antibody) occurs in children 2-5 years of age. bkv antibody may be detected in as many as 50% of children by 3 years of age, and in 60-100% of children by 9 or 10 years of age; antibodies wane thereafter. bkv infection may be particularly important in the pediatric transplantation population, in whom primary infection has a high probability of occurring while the children are immunosuppressed (208) . primary infection with bkv in healthy children is rarely associated with clinical manifestations. mild pyrexia, malaise, vomiting, respiratory illness, pericarditis, and transient hepatic dysfunction have been reported with primary infection. investigators hypothesize that after an initial round of viral replication at the site of entry, viremia follows with dissemination of the virus to distant sites at which latent infection is established. the most frequently recognized secondary sites of latent infection are renal and uroepithelial cells. secondary infection has been reported to cause tubulointerstitial nephritis and ureteral stenosis in renal transplantation patients. it may be that renal impairment in immunocompromised patients and in non renal solid organ transplant recipients is found to be frequently associated with bkv infection. the reported prevalence of bkv nephropathy in renal allografts is between 1 and 8% (201, 202, 205, 209, 210) . asymptomatic infection is characterized by viral shedding without any apparent clinical features. viruria, resulting from either primary or secondary infection, can persist from several weeks to years. tubulointerstitial nephritis associated with bkv in renal transplant recipients is accompanied by histopathologic changes, with or without functional impairment. ''infection'' and ''disease'' must be differentiated carefully. bkv infection (either primary or reactivated) can progress to bkv disease, but will not always do so (208) . furthermore, not all cases of bkv disease lead to renal impairment. however, infection can progress to transplant dysfunction and graft loss, although the diagnosis may be complicated by the coexistence of active allograft rejection. bkv nephritis is reported to have a bimodal distribution, with 50% of bkv-related interstitial nephritis cases occurring 4-8 weeks after transplantation and the remainder of patients developing disease months to years after transplantation (211) . allograft failure is due mainly to extensive viral replication in tubular epithelial cells leading to frank tubular necrosis (203) . although damage is potentially fully reversible early in the disease, persisting viral damage leads to irreversible interstitial fibrosis. tubular atrophy and allograft loss has been observed in 45% of affected patients (203, 212) . in most cases, bkv nephropathy in adult renal transplant recipients represents a secondary infection associated with rejection and its treatment. in children, however, primary bkv infection giving rise to allograft dysfunction may occur (208) . the definitive diagnosis of bkv nephropathy requires renal biopsy. histopathologic features include severe tubular injury with cellular enlargement, marked nuclear atypia, epithelial necrosis, denudation of tubular basement membranes, focal intratubular neutrophilic infiltration, and mononuclear interstitial infiltration, with or without concurrent tubulins. this constellation of histologic features, particularly severe tubulitis, is often misinterpreted as rejection, even by the experienced pathologist. the presence of well-demarcated basophilic or amphophilic intranuclear viral inclusions, primarily within the tubular and parietal epithelium of the bowman capsule, can help distinguish bkv disease from rejection (202, 203, 205) . additional tests such as immunohistochemistry, pcr analysis, or electron microscopy of biopsied tissue aimed at the identification of bkv may be required. a practical diagnostic approach for identifying bkv in renal transplant patients is summarized in > table 52-3. bkv infection may cause ureteral obstruction due to ureteral ulceration and stenosis at the ureteric anastomosis. bkv-associated ureteral stenosis has been reported in 3% of renal transplant patients and usually occurs between 50 and 300 days after transplantation. ulceration due to inflammation, proliferation of the transitional epithelial cells, and smooth muscle proliferation may lead to partial or total obstruction. high-level bkv replication is implicated in acute, late-onset, long-duration hemorrhagic cystitis after bone marrow transplantation (213) . there are two case reports in children of renal carcinomas arising in the transplanted kidney in association with bk virus nephropathy. it remains unclear whether infectious diseases and the kidney bk virus itself has oncogenic potential in the transplant setting, but this is possible given that the big t antigen (t-ag) expressed by polyomavirus family viruses has been shown to have the ability to disrupt chromosomal integrity (214, 215) . whether patients with asymptomatic viremia or viruria need specific therapeutic intervention is not certain. review of the literature suggests that careful reduction of immune suppression, combined with active surveillance for rejection, will result in clinical improvement. reduction in immunosuppression may precipitate episodes of acute cellular rejection, which need to be judiciously treated with corticosteroids. the outcome of bkv nephropathy is unpredictable, and stabilization of renal function may occur regardless of whether maintenance immunotherapy is altered or not (216) . some reports favor the use of cidofovir. cidofovir has important nephrotoxic side effects in the usual therapeutic dosage recommended for the treatment of cmv infection, and for bkv nephropathy a reduced dosage regime is generally used. the efficacy of cidofovir in reducing viremia has been demonstrated (see review in (210)). however, spontaneous clearance of viral infection after reduction of immunosuppression (without cidofovir) has also been reported. there are also case studies of the use of leflunamide. presence of bkv by pcr or decoy cells in urine signifies bkv replication. decoy cells are caused by infection of the urinary epithelial cells with human polyoma viruses. the nuclei are enlarged and nuclear chromatin is completely homogenized by viral cytopathic effect. positive pcr results for bkv viruria and presence of decoy cells have poor predictive value. specificity is increased if >10 cells/ cytospin along with presence of inflammatory cells. presence of antibody is usually indicative of previous infection; however, positive results for bkv dna pcr on serum signifies bk viremia. bkv pcr testing of plasma has proven to be a sensitive (100%) and specific (88%) means to identify bkv-associated nephropathy in adults. viral load has also been used to monitor infection and clearance. however, because primary infection occurs in childhood, it might not be applicable to the pediatric population. the definitive diagnosis of bkv nephropathy requires renal biopsy. histopathology might mimic rejection or drug toxicity. however, characteristic findings have been described. electron microscopy and immune staining are helpful in confirming the diagnosis. pcr assays of viral load in tubular cells have been reported to be a sensitive marker for diagnosis and monitoring. viral hemorrhagic fever involves at least 12 distinct rna viruses that share the propensity to cause severe disease with prominent hemorrhagic manifestations ( > table 52 -4) . the viral hemorrhagic fevers, widely distributed throughout both temperate and tropical regions of the world, are important causes of mortality and morbidity in many countries. most viral hemorrhagic fevers are zoonoses (with the possible exception of dengue virus), in which the virus is endemic in animals and human infection is acquired through the bite of an insect vector. aerosol and nosocomial transmissions from infected patients are important for lassa, junin, machupo, and congo-crimean hemorrhagic fevers, and marburg and ebola viruses (217) . viral hemorrhagic fevers have many clinical similarities but also important differences in their severity, major organs affected, prognosis, and response to treatment. in all viral hemorrhagic fevers, severe cases occur in only a minority of those affected; subclinical infection or nonspecific febrile illness occurs in the majority. fever, myalgia, headache, conjunctival suffusion, and erythematous rash occur in all the viral hemorrhagic fevers (218) . hemorrhagic manifestations range from petechiae and bleeding from venepuncture sites to severe hemorrhage into the gi tract, kidney, and other organs. a capillary leak syndrome, with evidence of hemoconcentration, pulmonary edema, oliguria, and ultimately shock, occurs in the most severely affected patients (218) . renal involvement occurs in all the viral hemorrhagic fevers, proteinuria is common, and prerenal failure is seen in all severe cases complicated by shock. however, in congo-crimean hemorrhagic fever and hemorrhagic fever with renal syndrome (hfrs), an interstitial nephritis, which may be hemorrhagic, is characteristic, and renal impairment is a major component of the illness. dengue is caused by a flavivirus that is endemic and epidemic in tropical america, africa, and asia, where the mosquito vector aedes aegypti is present (219). classic dengue is a self-limited nonfatal disease; dengue hemorrhagic fever and dengue shock syndrome, which occur in a minority of patients, have a high mortality if not aggressively treated with fluids. after an incubation period of 5-8 days, the illness begins with fever, headache, arthralgia, weakness, vomiting, and hyperesthesia. in uncomplicated dengue the fever usually lasts 5-7 days. shortly after onset a maculopapular rash appears, sparing the palms and the soles, and is occasionally followed by desquamation. fever may reappear at the onset of the rash. in dengue hemorrhagic fever and dengue shock syndrome, the typical febrile illness is complicated by hemorrhagic manifestations, ranging from a positive tourniquet test result or petechiae to purpura, epistaxis, and gi bleeding with thrombocytopenia and evidence of a consumptive coagulopathy. increased capillary permeability is suggested by hemoconcentration, edema, and pleural effusions (219) . in severe cases, hypotension and shock supervene, largely as a result of hypovolemia. renal manifestations include oliguria, proteinuria, hematuria, and rising urea and creatinine. acute renal failure occurs in patients with severe shock, primarily as a result of renal underperfusion. however, glomerular inflammatory changes may also occur. children with dengue hemorrhagic fever show hypertrophy of endothelial and mesangial cells, mononuclear cell infiltrate, thinning of basement membranes, and deposition of igg, igm, and c3. electron microscopy shows viral particles within glomerular mononuclear cells (220) . the diagnosis of dengue is made by isolation of the virus from blood or by serologic testing. there is no specific antiviral treatment, and management of patients with dengue shock syndrome or dengue hemorrhagic fever depends on aggressive circulatory support and volume replacement with colloid and crystalloid (221, 222) . with correction of hypovolemia, renal impairment is usually reversible, but dialysis may be required in patients with established acute renal failure. yellow fever is caused by a flavivirus, and is transmitted by mosquito bites, typically aedes species. it remains an important public health problem in africa and south america. renal manifestations are common and include albuminuria and oliguria. over the next few days after first manifestation of infection, shock, delirium, coma, and renal failure develop, and death occurs 7-10 days after onset of symptoms. laboratory findings include thrombocytopenia and evidence of hemoconcentration, rising urea and creatinine levels, hyponatremia, and deranged liver function test results. pathologic findings include necrosis of liver lobules, cloudy swelling and fatty degeneration of the proximal renal tubules, and, often, petechiae in other organs. the oliguria appears to be prerenal and is due to hypovolemia; later, acute tubular necrosis supervenes. at present, there is no effective antiviral agent for yellow fever. . congo-crimean hemorrhagic fever, first recognized in the soviet union, is now an important human disease in eastern europe, asia, and africa (223) . severely affected patients become stuporous or comatose 5-7 days into the illness, with evidence of hepatic and renal failure and shock. proteinuria and hematuria are often present. the disease is fatal in 15-50% of cases. the virus is sensitive to ribavirin, but in one small trial of i.v. ribavirin versus supportive treatment only, there was no significant improvement in outcome in the treatment group (224) . rift valley fever is found in many areas of sub-saharan africa. in humans, most infections follow mosquito bites or animal exposure. the infection may present as an uncomplicated febrile illness, with muscle aches and . (226) . clinical entities include korean hemorrhagic fever, nephropathia epidemica in scandinavia, and epidemic hemorrhagic fever in japan and china. in general, hfrs due to hantaan, porogia, and belgrade viruses is more severe and has higher mortality than that due to puumala virus (nephropathia epidemica) or seoul virus. hantaan is predominant in the far east, porogia and belgrade in the balkans, and puumala in western europe; seoul has a worldwide distribution (225) . the clinical features of the disease vary. the incubation period is 4-42 days. although hfrs occurs with the same clinical picture in children as in adults, both incidence rates and antibody prevalence rates are very low in children under 10 years of age. men of working age make up the bulk of clinical cases (226) . mild cases are indistinguishable from other febrile illnesses. in more severe cases, fever, headache, myalgia, abdominal pain, and dizziness are associated with the development of periorbital edema, proteinuria, and hematuria. there is often conjunctival injection, pharyngeal injection, petechiae, and epistaxis or gi bleeding. the most severely affected patients develop shock and renal failure. the disease usually passes through five phases: febrile, hypotensive, oliguric, diuretic, and convalescent. laboratory findings include anemia, lymphocytosis, thrombocytopenia, prolonged prothrombin and bleeding times, and elevated levels of fibrin degradation products. liver enzyme levels are elevated, and urea and creatinine levels are elevated during the oliguric phase. proteinuria and hematuria are consistent findings. the renal histopathologic findings are those of an interstitial nephritis with prominent hemorrhages in the renal medullary interstitium and renal cortex. acute tubular necrosis may also be seen. immunohistochemical analysis reveals deposition of igg and c3, and the gbm, mesangial, and subendothelial deposits may be seen on electron microscopy (227) . recovery from hantavirus-associated disease is generally complete, although chronic renal insufficiency is a rare sequela of hfrs. in mildly affected patients, the disease is self-limiting and spontaneous recovery occurs. however, in severe cases, with shock, bleeding, and renal failure, dialysis and intensive circulatory support may be required (228) . mortality rates vary depending on the strain of virus; rates are 5-15% for hemorrhagic fever and renal syndrome in china and significantly lower for the milder finnish form associated with the puumala virus strain. ribavirin is active against hantaan viruses in vitro, and clinical trials indicate that both mortality and morbidity can be reduced by treatment with this antiviral agent if it is administered early in the course of illness. dosages of 33 mg/kg followed by 16 mg/kg every 6 h for 4 days and then 8 mg/kg every 8 h for 3 days have been used (229) . lassa fever is a common infection in west africa, caused by an arenavirus, and usually manifests as a nonspecific febrile illness. in 10% of cases, a fulminant hemorrhagic disease occurs. in severe cases, proteinuria and hematuria are usually present, and renal failure may occur. ribavirin is effective in decreasing mortality. as in other hemorrhagic fevers, intensive hemodynamic support and correction of the hemostatic derangements are important components of therapy (230) . junin and machupo viruses, the agents of argentine and bolivian hemorrhagic fever, respectively, cause hemorrhagic fevers with prominent neurologic features and systemic and hemorrhagic features similar to those of lassa fever. oliguria, shock, and renal failure occur in the most severe cases. marburg and ebola viruses have been associated with outbreaks of nosocomially transmitted hemorrhagic fever. both viruses cause fulminant hemorrhagic fever. onset is with high fever, headache, sore throat, myalgia, and profound prostration. an erythematous rash on the trunk is followed by hemorrhagic conjunctivitis, bleeding, impaired renal function, shock, and respiratory failure. the mortality rate is high. renal histopathologic findings in fatal cases are of tubular necrosis, with fibrin deposition in the glomeruli. there is no specific treatment for these disorders. the important role played by a number of other recently characterized viruses is only now being recognized, as improved molecular diagnostic techniques allow identification of hitherto unrecognized viruses. two examples of recently described viruses are metapneumovirus (237) and bocavirus (238) . while both have significant prevalence, and may make an important contribution to the burden of childhood viral infection, as yet there are no reports indicative of significant renal pathology in association with these infections. influenza virus has been linked with nephritis and acute renal failure. an emerging infectious disease is avian flu, caused by highly pathogenic h5n1 strains which have hitherto been confined to an avian reservoir, and there have been several outbreaks of infection in humans, particularly in the first part of this decade. commonly, these patients develop a flu-like illness with prominent respiratory and gastrointestinal symptoms. renal failure may develop alongside multi-organ failure in the context of acute respiratory distress syndrome (231) . as yet, there is no clear correlation of degree of initial renal insufficiency, and outcome (232) . there is little data available on treatment, but based on the known resistance patterns of h5n1 strains, oseltamivir and zanamivir are the preferred agents to be used for treatment of infection with h5n1. severe acute respiratory syndrome (sars) is a newlyemerged infectious disease which was first seen in south china in 2002. it is caused by a sars coronavirus (sars cov). predominantly, it causes a viral pneumonia, with diffuse alveolar damage; it has considerable mortality (233) . renal effects are not generally significant in the pathophysiology of sars. however, sars cov has been found in kidney tissue at post-mortem (234) (235) . sars cov enters cells via angiotensin converting enzyme 2 (ace2) (236) , and it is thought that the invasion of kidney tissue reflects the virus' tropism for ace2, which is expressed on kidney cells. chronic exposure to infectious agents is a major factor in the increased prevalence of glomerular diseases in developing countries. malaria is the best-documented parasitic infection associated with glomerular disease, but other parasitic infections including schistosomiasis, filariasis, leishmaniasis, and possibly helminth infections may also induce nephritis or nephrosis. malaria is estimated to cause up to 500 million clinical cases of illness and more than 1 million deaths each year (239) . the association of quartan malaria and nephritis has been well known in both temperate and tropical zones since the end of the nineteenth century. epidemiologic studies provide the most conclusive evidence for a role of plasmodium malariae in glomerular disease (240, 241) . chronic renal disease was a major cause of morbidity and mortality in british guiana in the 1920s. the frequent occurrence of p. malariae in the blood of these patients led to detailed epidemiologic studies that implicated malaria as a cause of the nephrosis. after the eradication of malaria from british guiana, chronic renal disease ceased to be a major cause of death in that country (240) . the link between malaria and nephrotic syndrome was strengthened by studies in west africa in the 1950s and 1960s that demonstrated a high prevalence of nephrotic syndrome in the nigerian population (242) . the pattern of nephrotic syndrome differed from that in temperate climates, with an older peak age, extremely poor prognosis, and unusual histologic features. the incidence of p. malariae parasitemia in patients with the nephrotic syndrome in nigeria was vastly in excess of that occurring in the general population, whereas the incidence of plasmodium falciparum parasitemia was similar to that in the general population. the age distribution of nephrotic syndrome also closely paralleled that of p. malariae infection (242) . in some affected patients, circulating immune complexes and immunoglobulin, complement, and antigens were present in the glomeruli that were recognized by p. malariae-species antisera. there is now a view that the patterns of childhood renal disease described in the last century may no longer be representative of the current situation. the variable patterns of renal disease throughout africa may no longer reflect a dominant role for ''malarial glomerulopathy,'' and the relative causative role of tropical infections in nephropathy remains an unanswered question (243) . most patients have poorly selective proteinuria and are unresponsive to treatment with steroids or immunosuppressive agents. the characteristic lesions of p. malariae nephropathy are capillary wall thickening and segmental glomerular sclerosis, which lead to progressive glomerular changes and secondary tubular atrophy (242) . cellular proliferation is conspicuously absent. electron microscopy shows foot-process fusion, thickening of the basement membrane, and increase in subendothelial basement membrane-like material. immunofluorescent studies show granular deposits of immunoglobulin, complement, and p. malariae antigen in approximately one-third of patients. in addition to the histologic pattern, termed quartan malaria nephropathy, p. malariae infection is associated with a variety of other forms of histologic appearance, including proliferative gn and mgn (244) . although quartan malaria nephropathy has been clearly linked to p. malariae infection in nigeria, a number of studies from other regions in africa have not revealed the typical histopathologic findings described in the nigerian studies (245) . furthermore, quartan malaria nephropathy may be seen in children with no evidence of p. malariae infection or deposition of malaria antigens in the kidney. this, together with the fact that antimalarial treatment does not affect the progression of the disorder, raises the possibility that factors other than malaria might be involved in the initiation and perpetuation of the disorder. although there is undoubtedly a strong association between p. malariae infection and nephrotic syndrome on epidemiologic grounds, the direct causal link is not proven. most likely, a number of different infectious processes, including malaria, hepatitis b, schistosomiasis, and perhaps other parasitic infections that cause chronic or persistent infections and often occur concurrently in malaria areas, may all result in glomerular injury and a range of overlapping histopathologic features. the prognosis for the nephrotic syndrome in most african studies has been poor, regardless of whether the histologic findings were typical of quartan malaria nephropathy or whether p. malariae parasitemia was implicated. treatment with steroids and azathioprine is generally ineffective, and a significant proportion of patients progress to renal failure. p. falciparum appears to be much less likely to cause significant glomerular pathology. epidemiologic studies have failed to show a clear association between p. falciparum parasitemia and the nephrotic syndrome. whereas renal failure appears to be a common complication of severe malaria in adults, it seldom occurs in children. renal biopsy specimens from adult patients with acute p. falciparum infections who have proteinuria or hematuria show evidence of glomerular changes, including hypercellularity, thickening of basement membranes, and hyperplasia and hypertrophy of endothelial cells (246) . electron microscopy reveals electron-dense deposits in the subendothelial and paramesangial areas. deposits of igm, with or without igg, are localized mainly in the mesangial areas. p. falciparum antigens can be demonstrated in the mesangial areas and along the capillary wall, which suggests an immune-complex gn. the changes, generally mild and transient, are probably unrelated to the acute renal failure that may complicate severe p. falciparum infection (246) . heavily parasitized erythrocytes play a central role in the various pathologic factors (247) . renal failure occurring in severe p. falciparum malaria is usually associated with acidosis, volume depletion, acute intravascular hemolysis or heavy parasitic infection that leads to acute tubular necrosis. recent studies have confirmed an important role for volume depletion in children with severe falciparum malaria, who characteristically have evidence of tachycardia, tachypnoea, poor perfusion and in severe cases hypotension (248) . volume expansion with either colloid or crystalloid results in improvement in hemodynamic indices and reduction in acidosis (249) . there is growing evidence that volume expansion with albumin is associated with a better outcome than saline or synthetic colloids (250, 251) . treatment with antimalarials, correction of hypoglycemia and infectious diseases and the kidney electrolyte imbalance, and volume expansion reduces mortality to less than 5%. although renal failure is usually associated with infection by p. falciparum, acute renal failure has been described with plasmodium vivax infection and mixed infections (252) . the term blackwater fever refers to the combination of severe hemolysis, hemoglobinuria, and renal failure. it was more common at the start of the twentieth century in nonimmune individuals receiving intermittent quinine therapy for p. falciparum malaria. blackwater fever has become rare since 1950, when quinine was replaced by chloroquine. however, the disease reappeared in the 1990s, after the reuse of quinine because of the development of chloroquine-resistant organisms. since then, several cases have been described after therapy with halofantrine and mefloquine, two new molecules similar to quinine (amino-alcohol family) (253) . renal failure generally occurred in the context of severe hemolytic anemia, hemoglobinuria, and jaundice. the pathophysiology of the disorder is unclear; however, it appears that a double sensitization of the red blood cells to the p. falciparum and to the amino-alcohols is necessary to provoke the hemolysis. histopathologic findings include swelling and vacuolization of proximal tubules, necrosis and degeneration of more distal tubules, and hemoglobin deposition in the renal tubules. recent studies indicates a better outcome with earlier initiation of intensive care and dialysis combined with necessary changes in antimalarial medications. schistosomiasis affects 200 million people living in endemic areas of asia, africa, and south america (254) . the infection is usually acquired in childhood, but repeated infections occur throughout life. schistosoma japonicum is found only in the orient, whereas schistosoma haematobium occurs throughout africa, the middle east, and areas of southwest asia. schistosoma mansoni is widespread in africa, south america, and southwest asia. human infection begins when the cercarial forms invade through the skin, develop into schistosomula, and move to the lungs via the lymphatics or blood. they then migrate to the liver and mature in the intrahepatic portal venules, where male:female pairing takes place. the adult worm pairs then migrate to their final resting site -the venules of the mesenteric venous system of the large intestine (s. mansoni) or in the venules of the urinary tract (s. haematobium). the females release large numbers of eggs, which may remain embedded in the tissues, embolize to the liver or lungs, or pass into the feces or urine. clinical manifestations may occur at any stage of the infection. cercarial invasion may cause an intense itchy papular rash. katayama fever is an acute serum sicknesslike illness that occurs several weeks after infection, as eggs are being deposited in the tissues. deposition of the eggs in tissues results in inflammation of the intestines, fibrosis of the liver, and portal hypertension. with s. haematobium, chronic inflammation and fibrosis of the ureters and bladder may lead to obstructive uropathy (255) . renal manifestations of schistosomiasis occur most commonly in s. mansoni infection. schistosomal nephropathy usually presents with symptoms including granulomatous inflammation in the ureters and bladder, but glomerular disease (probably on an immune-complex basis) may also occur. renal disease usually occurs in older children or young adults with long-term infection, but serious disease may also occur in young children (255) . the early renal tract manifestations of schistosomiasis are suprapubic discomfort, frequency, dysuria, and terminal hematuria. in more severe cases, evidence of urinary obstruction appears. poor urinary stream, straining on micturition, a feeling of incomplete bladder emptying, and a constant urge to urinate may be severely disabling symptoms. the fibrosis and inflammation of ureters, urethra, and bladder may be followed by calcification and may result in hydroureter, hydronephrosis, and bladder neck obstruction. renal failure may ultimately develop, and there is a suspicion that squamous cell carcinoma of the bladder may be linked to the chronic infective and inflammatory process. secondary bacterial infection is common within the obstructed and inflamed urinary tract (254) . the hepatosplenic form of s. mansoni infection may be accompanied by a glomerulopathy in 12-15% of cases, manifested in the majority as nephrotic syndrome (256) . histopathologic findings include mesangioproliferative gn, focal segmental glomerulosclerosis, mesangiocapillary gn, mgn, and focal segmental hyalinosis (257) . immune complexes may be detected in the circulation of these patients, and glomerular granular deposition of igm, c3, and schistosomal antigens are seen on immunofluorescence. usually schistosoma-specific nephropathy is a progressive disease and is not influenced by antiparasitic or immunosuppressive therapy (258) , but isolated case reports of remission after treatment with praziquantel have been reported (259) . the diagnosis is confirmed by the detection of schistosoma eggs in feces, urine, or biopsy specimens. eggs are shed into the urine with a diurnal rhythm, and urine collected between 11 am and 1 pm is the most useful. urinary sediment obtained by centrifugation or filtration through a nuclepore membrane should be examined. in cases in which studies of urine and feces yield negative results in patients in whom the diagnosis is suspected, rectal biopsy specimens taken approximately 9 cm from the anus have a high diagnostic yield for both s. mansoni and s. haematobium infection. biopsy of liver or bladder may be required to establish the diagnosis. antibodies indicating previous infection can be detected using enzyme-linked immunosorbent assay or radioimmunoassay. the tests are sensitive but lack specificity and may not differentiate between past exposure and current infection. praziquantel is the drug of choice for treatment of schistosomiasis. a single oral dose of 40 mg/kg is effective in s.haematobium and s. mansoni infection and is usually well tolerated. the alternative drug for s. mansoni infection is oxamniquine. complete remission of urinary symptoms may occur in renal disease of short duration, but in late disease with extensive fibrosis, scarring, and calcification, obstructive uropathy and renal failure may persist after the infection has been eradicated. there are reports of a drastic decrease in the number of severe hepatosplenic forms of s. mansoni infection after mass treatment of the population in endemic areas with oxamniquine. this also reduced schistosomal nephropathy (256) . visceral leishmaniasis is a chronic protozoon infection characterized by fever, hepatosplenomegaly, anemia, leukopenia, and hyperglobulinemia. proteinuria and/or microscopic hematuria or pyuria have been reported in 50% of patients with visceral leishmaniasis (260) . acute renal failure in association with interstitial nephritis has also been reported (261) . renal histologic analysis in patients with visceral leishmaniasis reveals glomerular changes, with features of a mesangial proliferative gn or a focal proliferative gn, or a generalized interstitial nephritis with interstitial edema, mononuclear cell infiltration, and focal tubular degeneration. immunofluorescence reveals deposition of igg, igm, and c3 within the glomeruli, as well as electron-dense deposits in the basement membrane and mesangium on electron microscopy (260) . circulating immune complexes together with immunoglobulin and complement deposition in the glomeruli suggests an immune-complex cause. renal disease in leishmaniasis is usually mild and may resolve after treatment of the infection. renal dysfunction may be associated with treatment for visceral leishmaniasis with antimony compounds. proteinuria is more common in filarial hyperendemic regions of west africa than in nonfilarial areas. renal histologic analysis has shown a variety of different histopathologic appearances; the most common is diffuse mesangial proliferative gn with c3 deposition in the glomeruli (262) . renal biopsy specimens also demonstrate large numbers of eosinophils in the glomeruli, and microfilariae may be seen in the lumen of glomerular capillaries. filarial antigens have been detected within immune deposits within the glomeruli. echinococcus granulosus causes chronic cysts within a variety of organs. in addition, nephrotic syndrome in association with hydatid disease has been reported. membranous nephropathy, minimal change lesions, and mesangiocapillary gn have been described in association with hydatid disease (263, 264) . immunofluorescence reveals deposits of immunoglobulin, complement, and hydatid antigens within the glomeruli. remission of nephrotic syndrome has been reported with treatment by antiparasitic agents such as albendazole (263, 264) . few reports have been published of renal disease occurring in patients with trypanosomiasis. the trypanosomal antigens can induce gn in a variety of experimental animals (265) . nephrotic syndrome has occasionally been reported as a manifestation of congenital toxoplasmosis. dissemination of previously latent toxoplasma infection in patients undergoing treatment with immunosuppressive drugs has been increasingly recognized in recent years. reactivation of toxoplasmosis or progression of recently acquired primary infection should be considered in patients undergoing renal transplantation or immunotherapy for renal disease who develop unexplained inflammation of any organ. fungal infections of the kidneys and urinary tract occur most commonly as part of systemic fungal infections in patients with underlying immunodeficiency, as focal urinary tract infections in patients with obstructive lesions, or as a result of indwelling catheters. although candida infection is the most common fungal infection in both immunocompromised and non immunocompromised hosts, virtually all other fungal pathogens may invade the renal tract during severe immunocompromise. urinary infection with candida albicans is most commonly a component of systemic candidiasis in patients who are severely immunocompromised. systemic candidiasis is also seen in premature and term infants with perinatally acquired invasive candidiasis. presentation is usually with systemic sepsis, fever or hypothermia, hepatosplenomegaly, erythematous rash, and thrombocytopenia. systemic candidiasis may be seen on ophthalmologic investigation as microemboli in the retina. the first clue to the underlying diagnosis may be the presence of yeasts in the urine (266) . candida involvement of the urinary tract may affect all structures including the glomeruli, tubules, collecting system, ureters, and bladder. microabscesses may form within the renal parenchyma, and large balls of fungi may completely obstruct the urinary tract at any level. acute renal failure caused by systemic candidiasis or obstruction of the renal tracts with fungal hyphae is a wellrecognized complication of systemic candidal infection (266, 267) . indwelling catheters (which form a nidus for persistent infection) should be removed. successful treatment of non-obstructing bilateral renal fungal balls by fluconazole either alone or in combination with liposomal amphotericin b has been reported (268, 269) . in the presence of obstruction, however, percutaneous nephrostomy to relieve the obstruction with antegrade amphotericin b irrigation, coupled with systemic antifungal therapy, is the mainstay of treatment (267) . amphotericin b is the most effective antifungal agent, but it is not excreted in the urine. local irrigation via nephrostomy provides good results, however. for treatment of urinary tract candidiasis, it is usually combined with fluconazole or 5-flucytosine, both of which are excreted in high concentrations in the urine. treatment is required for weeks to months to ensure complete elimination of the fungus, and the ultimate outcome is largely dependent on whether there is a permanent defect in immunity. in 1983, levin et al. first described hemorrhagic shock and encephalopathy, which appeared to be distinct from previously recognized pediatric disorders (270) . other cases have subsequently been reported from several centers in the united kingdom, europe, israel, the united states, and australia, and the syndrome is now recognized as a new and relatively common severe childhood disorder (271) . hemorrhagic shock and encephalopathy usually affects infants in the first year of life, with a peak onset at 3-4 months of age. a prodromal illness with fever, irritability, diarrhea, or upper respiratory infection occurs 2-5 days before the onset in two-thirds of cases. affected infants develop profound shock, coma, convulsions, bleeding and evidence of dic, diarrhea, and oliguria. laboratory findings include acidosis, falling hemoglobin and platelet levels, elevated urea and creatinine levels, and elevated levels of hepatic transaminases. despite vigorous intensive care, the prognosis is poor, and most affected infants die or are left severely neurologically damaged (271, 272) . a small number of patients have been reported to survive without residual sequelae. the renal impairment appears to be largely prerenal in origin, and when aggressive volume replacement and treatment of the shock results in improved renal perfusion, rapid improvement in renal function is usually observed. in patients with profound shock unresponsive to initial resuscitation, vasomotor nephropathy supervenes and dialysis may be required. myoglobinuria in association with hemorrhagic shock and encephalopathy has been reported. following the description of the mucocutaneous lymph node syndrome by kawasaki in 1968, kawasaki disease has been recognized as a common and serious childhood illness with a worldwide distribution. although the etiology remains unknown, epidemiologic features clearly suggest an infective cause. the disease occurs in epidemics, and wavelike spread has been demonstrated during outbreaks in japan. deposition of amyloid within the kidney is an important complication of chronic and persistent infection. amyloidosis is most common in patients with chronic osteomyelitis and chronic pulmonary infections such as bronchiectasis and is seen occasionally in those with persistent infections such as leprosy or malaria (273) (274) (275) . paediatric emergencies, 2nd edn. black ja shock in the paediatric patient vasoactive hormones in the renal response to systemic sepsis renal effects of nitric oxide in endotoxemia drotrecogin alfa (activated) in children with severe sepsis: a multicentre phase iii randomised controlled trial treatment of meningococcal 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diagnosis pathogenesis and management of polyomavirus infection in transplant recipients bk virus infection in renal transplant recipients prospective study of polyomavirus type bk replication and nephropathy in renal-transplant recipients bk virus infection, replication, and diseases in pediatric kidney transplantation occurrence and significance of papovaviruses bk and jc in the urine polyoma-virus induced interstitial nephritis in two renal transplant recipients: case reports and review of the literature late-onset hemorrhagic cystitis associated with urinary excretion of polyoma-viruses after bone marrow transplantation collecting duct carcinoma arising in association with bk nephropathy posttransplantation in a pediatric patient. a case report with immunohistochemical and in situ hybridization study association of renal adenocarcinoma and bk virus nephropathy post-transplantation clinical course of polyoma virus nephropathy in 67 renal transplant patients epidemiology of 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outcome? pathogenesis of severe acute respiratory syndrome fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a newly discovered human pneumovirus isolated from young children with respiratory tract disease cloning of a human parvovirus by molecular screening of respiratory tract samples malaria in 2002 malaria and renal disease with special reference to british guiana. ii. the effect of malaria eradication on the incidence of renal disease in british guiana immunopathology of malaria the nephrotic syndrome and other diseases in children in western nigeria malaria-induced renal damage: facts and myths clinicopathological features of childhood nephrotic syndrome in northern nigeria 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shock and encephalopathy: reflections about a new devastating disorder that affects normal children haemorrhagic shock and encephalopathy: clinical, pathologic, and biochemical fea-mks use of protein-c concentrare, heparin, and haemodiafiltration in meningococcus-induced purpura fulminans quantitative viral load monitoring and cidofovir therapy for the management of bk virus associated nephropathy in children and adults antiviral therapy and prophylaxis for influenza in children infectious diseases and the kidney key: cord-016704-99v4brjf authors: nicholson, felicity title: infectious diseases: the role of the forensic physician date: 2005 journal: clinical forensic medicine doi: 10.1385/1-59259-913-3:235 sha: doc_id: 16704 cord_uid: 99v4brjf infections have plagued doctors for centuries, in both the diagnosis of the specific diseases and the identification and subsequent management of the causative agents. there is a constant need for information as new organisms emerge, existing ones develop resistance to current drugs or vaccines, and changes in epidemiology and prevalence occur. in the 21st century, obtaining this information has never been more important. population migration and the relatively low cost of flying means that unfamiliar infectious diseases may be brought into industrialized countries. an example of this was an outbreak of severe acute respiratory syndrome (sars), which was first recognized in 2003. despite modern technology and a huge input of money, it took months for the agent to be identified, a diagnostic test to be produced, and a strategy for disease reporting and isolation to be established. there is no doubt that other new and fascinating diseases will continue to emerge. infections have plagued doctors for centuries, in both the diagnosis of the specific diseases and the identification and subsequent management of the causative agents. there is a constant need for information as new organisms emerge, existing ones develop resistance to current drugs or vaccines, and changes in epidemiology and prevalence occur. in the 21st century, obtaining this information has never been more important. population migration and the relatively low cost of flying means that unfamiliar infectious diseases may be brought into industrialized countries. an example of this was an outbreak of severe acute respiratory syndrome (sars), which was first recognized in 2003. despite modern technology and a huge input of money, it took months for the agent to be identified, a diagnostic test to be produced, and a strategy for disease reporting and isolation to be established. there is no doubt that other new and fascinating diseases will continue to emerge. for the forensic physician, dealing with infections presents two main problems. the first problem is managing detainees or police personnel who have contracted a disease and may be infectious or unwell. the second problem is handling assault victims, including police officers, who have potentially been exposed to an infectious disease. the latter can be distressing for those involved, compounded, in part, from an inconsistency of management guidelines, if indeed they exist. with the advent of human rights legislation, increasing pressure is being placed on doctors regarding consent and confidentiality of the detainee. therefore, it is prudent to preempt such situations before the consultation begins by obtaining either written or verbal consent from the detainee to allow certain pieces of information to be disclosed. if the detainee does not agree, then the doctor must decide whether withholding relevant details will endanger the lives or health of those working within custody or others with whom they may have had close contact (whether or not deliberate). consent and confidentiality issues are discussed in detail in chapter 2. adopting a universal approach with all detainees will decrease the risk to staff of acquiring such diseases and will help to stop unnecessary overreaction and unjustified disclosure of sensitive information. for violent or sexual assault victims, a more open-minded approach is needed (see also chapter 3) . if the assailant is known, then it may be possible to make an informed assessment of the risk of certain diseases by ascertaining his or her lifestyle. however, if the assailant is unknown, then it is wise to assume the worst. this chapter highlights the most common infections encountered by the forensic physician. it dispels "urban myths" and provides a sensible approach for achieving effective management. the risk of exposure to infections, particularly blood-borne viruses (bbvs), can be minimized by adopting measures that are considered good practice in the united kingdom, the united states, and australia (1) (2) (3) . forensic physicians or other health care professionals should wash their hands before and after contact with each detainee or victim. police officers should be encouraged to wash their hands after exposure to body fluids or excreta. all staff should wear gloves when exposure to body fluids, mucous membranes, or nonintact skin is likely. gloves should also be worn when cleaning up body fluids or handling clinical waste, including contaminated laundry. single-use gloves should only be used and must conform to the requirements of european standard 455 or equivalent (1) (2) (3) . a synthetic alternative conforming to the same standards should also be available for those who are allergic to latex. all staff should cover any fresh wounds (<24 hours old), open skin lesions, or breaks in exposed skin with a waterproof dressing. gloves cannot prevent percutaneous injury but may reduce the chance of acquiring a bloodborne viral infection by limiting the volume of blood inoculated. gloves should only be worn when taking blood, providing this does not reduce manual dexterity and therefore increase the risk of accidental percutaneous injury. ideally, a designated person should be allocated to ensure that the clinical room is kept clean and that sharps containers and clinical waste bags are removed regularly. clinical waste must be disposed of in hazard bags and should never be overfilled. after use, the clinical waste should be doublebagged and sealed with hazard tape. the bags should be placed in a designated waste disposal (preferably outside the building) and removed by a professional company. when cells are contaminated with body fluids, a professional cleaning company should be called to attend as soon as possible. until such time, the cell should be deemed "out of action." there is a legal requirement in the united kingdom under the environmental protection act (1990) and the control of substances hazardous to health regulations 1994 to dispose of sharps in an approved container. in the united states, the division of health care quality promotion on the centers for disease control and prevention (cdc) web site provides similar guidance. in custody, where sharps containers are transported off site, they must be of an approved type. in the united kingdom, such a requirement is contained within the carriage of dangerous goods (classification, packaging and labelling) and use of transportable pressure receptacles regulations 1996. these measures help to minimize the risk of accidental injury. further precautions include wearing gloves when handling sharps and never bending, breaking, or resheathing needles before disposal. sharps bins should never be overfilled, left on the floor, or placed above the eye level of the smallest member of staff. any bedding that is visibly stained with body fluids should be handled with gloves. there are only three acceptable ways of dealing with contaminated bedding: the bbvs that present the most cross-infection hazard to staff or victims are those associated with persistent viral replication and viremia. these include hbv, hcv, hepatitis d virus (hdv), and hiv. in general, risks of transmission of bbvs arise from the possible exposure to blood or other body fluids. the degree of risk varies with the virus concerned and is discussed under the relevant sections. figure 1 illustrates the immediate management after a percutaneous injury, mucocutaneous exposure, or exposure through contamination of fresh cuts or breaks in the skin. hbv is endemic throughout the world, with populations showing a varying degree of prevalence. approximately two thousand million people have been infected with hbv, with more than 350 million having chronic infection. worldwide, hbv kills about 1 million people each year. with the development of a safe and effective vaccine in 1982, the world health organization (who) recommended that hbv vaccine should be incorporated into national immunization programs by 1995 in those countries with a chronic infection rate of 8% or higher, and into all countries by 1997. although 135 countries had achieved this goal by the end of 2001, the poorest countries-often the ones with the highest prevalence-have been unable to afford it. in particular these include china, the indian subcontinent, and sub-saharan africa. people in the early stages of infection or with chronic carrier status (defined by persistence of hepatitis b surface antigen [hbsag] beyond 6 mo) can transmit infection. in the united kindgom, the overall prevalence of chronic hbv is approx 0.2-0.3% (6, 7) . a detailed breakdown is shown in table 1 . the incubation period is approx 6 weeks to 6 months. as the name suggests, the virus primarily affects the liver. typical symptoms include malaise, anorexia, nausea, mild fever, and abdominal discomfort and may last from 2 days to 3 weeks before the insidious onset of jaundice. joint pain and skin rashes may also occur as a result of immune complex formation. infections in the newborn are usually asymptomatic. * in the united kingdom, written consent from the contact must be sent with the sample, countersigned by the health care practitioner and, preferably, an independent police officer. the majority of patients with acute hbv make a full recovery and develop immunity. after acute infection, approx 1 in 300 patients develop liver failure, which may result in death. chronic infection develops in approx 90% of neonates, approx 50% of children, and between 5 and 10% of adults. neonates and children are usually asymptomatic. adults may have only mild symptoms or may also be asymptomatic. approximately 15-25% of chronically infected individuals (depending on age of acquisition) will develop cirrhosis over a number of years. this may also result in liver failure or other serious complications, including hepatocellular carcinoma, though the latter is rare. the overall mortality rate of hbv is estimated at less than 5%. a person is deemed infectious if hbsag is detected in the blood. in the acute phase of the illness, this can be as long as 6 months. by definition, if hbsag persists after this time, then the person is deemed a carrier. carriers are usually infectious for life. the degree of infectivity depends on the stage of disease and the markers present table 2 . the major routes include parenteral (e.g., needlestick injuries, bites, unscreened blood transfusions, tattooing, acupuncture, and dental procedures where equipment is inadequately sterilized), mucous membrane exposure (including mouth, eyes, and genital mucous membranes), and contamination of broken skin (especially when <24 hours old). hbv is an occupational hazard for anyone who may come into contact with blood or bloodstained body fluids through the routes described. saliva alone may transmit hbv. the saliva of some people infected with hbv contains hbv-dna concentrations 1/1000-1/10,000 of that found in their serum (8) . this is especially relevant for penetrating bite wounds. infection after exposure to other body fluids (e.g., bile, urine, feces, and cerebrospinal fluid) has never been demonstrated unless the fluids are contaminated with blood. intravenous drug users who share needles or other equipment are also at risk. hbv can also be transmitted through unprotected sexual contact, whether homosexual or heterosexual. the risk is increased if blood is involved. sexual assault victims should be included in this category. evidence has shown that the virus may also be spread among members of a family through close household contact, such as through kissing and sharing toothbrushes, razors, bath towels, etc. (9) (10) (11) . this route of transmission probably applies to institutionalized patients, but there are no available data. studies of prisoners in western countries have shown a higher prevalence of antibodies to hbv and other bbvs than the general population (12) (13) (14) ; the most commonly reported risk factor is intravenous drug use. however, the real frequency of transmission of bbvs in british prisons is unknown owing to the difficulty in compiling reliable data. hbv can be transmitted vertically from mother to baby during the perinatal period. approximately 80% of babies born to mothers who have either acute or chronic hbv become infected, and most will develop chronic hbv. this has been limited by the administration of hbv vaccine to the neonate. in industrialized countries, all prenatal mothers are screened for hbv. vaccine is given to the neonate ideally within the first 12 hours of birth and at least two more doses are given at designated intervals. the who recommends this as a matter of course for all women in countries where prevalence is high. however, the practicalities of administering a vaccine that has to be stored at the correct temperature in places with limited access to medical care means that there is a significant failure of vaccine uptake and response. in industrialized countries, hbv vaccination is recommended for those who are deemed at risk of acquiring the disease. they include the following: 1. through occupational exposure. 2. homosexual/bisexual men. 3. intravenous drug users. 4. sexual partners of people with acute or chronic hbv. 5. family members of people with acute or chronic hbv. 6. newborn babies whose mothers are infected with hbv. if the mother is hbsag positive, then hepatitis b-specific immunoglobulin (hbig) should be given at the same time as the first dose of vaccine. 7. institutionalized patients and prisoners. ideally, hbv vaccine should be administered before exposure to the virus. the routine schedule consists of three doses of the vaccine given at 0, 1, and 6 months. antibody levels should be checked 8-12 weeks after the last dose. if titers are greater than10 miu/ml, then an adequate response has been achieved. in the united kingdom, this is considered to provide protection for 5-10 years. in the united states, if an initial adequate response has been achieved, then no further doses of vaccine are considered necessary. vaccine administration after exposure varies according to the timing of the incident, the degree of risk involved, and whether the individual has already been partly or fully vaccinated. an accelerated schedule when the third dose is given 2 months after the first dose with a booster 1 year later is used to prevent postnatal transmission. where risks are greatest, it may be necessary to use a rapid schedule. the doses are given at 0, 7, and 21-28 days after presentation, again with a booster dose at 6-12 months. this schedule is currently only licensed with engerix b. hbig may also be used either alone or in conjunction with vaccine. the exact dose given is age dependent but must be administered by deep intramuscular injection in a different site from the vaccine. in an adult, this is usually into the gluteus muscle. hbig is given in conjunction with the first dose of vaccine to individuals who are deemed at high risk of acquiring disease and the incident occurred within 72 hours of presentation. it is also used for neonates born to mothers who are hbeag-positive. between 5 and 10% of adults fail to respond to the routine schedule of vaccine. a further full course of vaccine should be tried before deeming the patients as "nonresponders." such individuals involved in a high-risk exposure should be given two doses of hbig administered 1 mo apart. ideally, the first dose should be given within 48 hours after exposure and no later than 2 weeks after exposure. other measures include minimizing the risk of exposure by adopting the safe working practices outlined in subheading 2. any potential exposures should be dealt with as soon as possible. in industrialized countries blood, blood products, and organs are routinely screened for hbv. intravenous drug users should be encouraged to be vaccinated and to avoid sharing needles or any other drug paraphernalia (see subheading 6.9.2.). for staff or victims in contact with disease, it is wise to have a procedure in place for immediate management and risk evaluation. an example is shown in fig. 1 . although forensic physicians are not expected to administer treatment, it is often helpful to inform persons concerned what to expect. tables 3 and 4 outline treatment protocols as used in the united kingdom. detainees with disease can usually be managed in custody. if the detainee is bleeding, then the cell should be deemed out of action after the detainee has left until it can be professionally cleaned. contaminated bedding should be dealt with as described in subheading 2.2. if the detainee has chronic hbv and is on an antiviral agent (e.g., lamivudine), then the treatment course should be continued, if possible. hcv is endemic in most parts of the world. approximately 3% (200 million) of the world's population is infected with hcv (15) . for many countries, no reliable prevalence data exist. seroprevalence studies conducted among blood donors have shown that the highest prevalence exists in egypt (17-26%). this has been ascribed to contaminated needles used in the treatment of schistosomiasis conducted between the 1950s and the 1980s (16) . intermediate prevalence (1-5%) exists in eastern europe, the mediterranean, the middle east, the indian subcontinent, and parts of africa and asia. in western europe, most of central america, australia, and limited regions in africa, including south africa, the prevalence is low (0.2-0.5%). previously, america was included in the low prevalence group, but a report published in 2003 (17) indicated that almost 4 million americans (i.e., 1.8% of the population) have antibody to hcv, representing either ongoing or previous infection. it also states that hcv accounts for approx 15% of acute viral hepatitis in america. the lowest prevalence (0.01-0.1%) has been found in the united kingdom and scandinavia. however, within any country, there are certain groups that have a higher chance of carrying hcv. these united kingdom figures are given in table 5 . after an incubation period of 6-8 weeks, the acute phase of the disease lasts approx 2-3 years. unlike hepatitis a (hav) or hbv, the patient is usually asymptomatic; therefore, the disease is often missed unless the individual has reported a specific exposure and is being monitored. other cases are found by chance, when raised liver enzymes are found on a routine blood test. a "silent phase" follows the acute phase when the virus lies dormant and the liver enzymes are usually normal. this period lasts approx 10-15 years. reactivation may then occur. subsequent viral replication damages the hepatocytes, and liver enzymes rise to moderate or high levels. eighty percent of individuals who are hcv antibody-positive are infectious, regardless of the levels of their liver enzymes. approximately 80% of people develop chronic infection, one-fifth of whom progress to cirrhosis. there is a much stronger association with hepatocellular carcinoma than with hbv. an estimated 1.25-2.5% of patients with hcv-related cirrhosis develop liver cancer (18) . less than 2% of chronic cases resolve spontaneously. approximately 75% of cases are parenteral (e.g., needle-stick, etc.) (19) . transmission through the sexual route is not common and only appears to be significant if there is repeated exposure with one or more people infected with hcv. mother-to-baby transmission is considered to be uncommon but has been reported (20) . theoretically, household spread is also possible through sharing contaminated toothbrushes or razors. because the disease is often silent, there is a need to raise awareness among the general population on how to avoid infection and to encourage high-risk groups to be tested. health care professionals should also be educated to avoid occupationally acquired infection. an example of good practice blood or blood-stained body fluids need to be involved for a risk to occur. saliva alone is not deemed to be a risk. the risk from a single needlestick incident is 1.8% (range 0-7%). contact through a contaminated cut is estimated at 1%. for penetrating bite injuries, there are no data, but it is only considered a risk if blood is involved. blood or blood-stained body fluids have to be involved in transmission through mucous membrane exposure. this may account for the lower-than-expected prevalence among the gay population. follow the immediate management flow chart, making sure all available information is obtained. inform the designated hospital and/or specialist as soon as possible. if the contact is known and is believed to be immunocompromised and he or she has consented to provide a blood sample, it is important to tell the specialist, because the antibody tests may be spuriously negative. in this instance, a different test should be used (polymerase chain reaction [pcr] , which detects viral rna). the staff member/victim will be asked to provide a baseline sample of blood with further samples at 4-6 weeks and again at 12 weeks. if tests are negative at 12 weeks but the risk was deemed high, then follow-up may continue for up to 24 weeks. if any of the follow-up samples is positive, then the original baseline sample will be tested to ascertain whether the infection was acquired through the particular exposure. it is important to emphasize the need for prompt initial attendance and continued monitoring, because treatment is now available. a combination of ribavirin (antiviral agent and interferon a-2b) (18) or the newer pegylated interferons (15) may be used. this treatment is most effective when it is started early in the course of infection. unless they are severely ill, detainees can be managed in custody. special precautions are only required if they are bleeding. custody staff should wear gloves if contact with blood is likely. contaminated bedding should be handled appropriately, and the cell cleaned professionally after use. this defective transmissible virus was discovered in 1977 and requires hbv for its own replication. it has a worldwide distribution in association with hbv, with approx 15 million people infected. the prevalence of hdv is higher in southern italy, the middle east, and parts of africa and south america, occurring in more than 20% of hbv carriers who are asymptomatic and more than 60% of those with chronic hbv-related liver disease. despite the high prevalence of hbv in china and south east asia, hdv in these countries is rare. hdv is associated with acute (coinfection) and chronic hepatitis (superinfection) and can exacerbate pre-existing liver damage caused by hbv. the routes of transmission and at-risk groups are the same as for hbv. staff/victims in contact with a putative exposure and detainees with disease should be managed as for hbv. interferon-î± (e.g., roferon) can be used to treat patients with chronic hbv and hdv (21) , although it would not be practical to continue this treatment in the custodial setting. hiv was first identified in 1983, 2 years after the first reports were made to the cdc in atlanta, ga, of an increased incidence of two unusual diseases (kaposi's sarcoma and pneumocystis carinii pneumonia) occurring among the gay population in san francisco. the scale of the virus gradually emerged over the years and by the end of 2002, there were an estimated 42 million people throughout the world living with hiv or acquired immunodeficiency syndrome (aids). more than 80% of the world's population lives in africa and india. a report by the joint united nations programme on hiv/aids and the who in 2002 stated that one in five adults in lesotho, malawi, mozambique, swaziland, zambia, and zimbabwe has hiv or aids. there is also expected to be a sharp rise in cases of hiv in china, papua new guinea, and other countries in asia and the pacific during the next few years. in the united kingdom, by the end of 2002, the cumulative data reported that there were 54,261 individuals with hiv, aids (including deaths from aids) reported, though this is likely to be an underestimate (22) . from these data, the group still considered at greatest risk of acquiring hiv in the united kingdom is homosexual/bisexual men, with 28,835 of the cumulative total falling into this category. among intravenous drug users, the overall estimated prevalence is 1%, but in london the figure is higher at 3.7% (6, 23) . in the 1980s, up to 90% of users in edinburgh and dundee were reported to be hiv positive, but the majority have now died. individuals arriving from africa or the indian subcontinent must also be deemed a risk group because 80% of the world's total cases occur in these areas. the predominant mode of transmission is through unprotected heterosexual intercourse. the incidence of mother-to-baby transmission has been estimated at 15% in europe and approx 45% in africa. the transmission rates among african women are believed to be much higher owing to a combination of more women with end-stage disease with a higher viral load and concomitant placental infection, which renders it more permeable to the virus (24, 25) . the use of antiretroviral therapy during pregnancy, together with the advice to avoid breastfeeding, has proven efficacious in reducing both vertical and horizontal transmission among hiv-positive women in the western world. for those in third-world countries, the reality is stark. access to treatment is limited, and there is no realistic substitute for breast milk, which provides a valuable source of antibodies to other life-threatening infections. patients receiving blood transfusions, organs, or blood products where screening is not routinely carried out must also be included. the incubation is estimated at 2 weeks to 6 months after exposure. this depends, to some extent, on the ability of current laboratory tests to detect hiv antibodies or viral antigen. the development of pcr for viral rna has improved sensitivity. during the acute phase of the infection, approx 50% experience a seroconversion "flu-like" illness. the individual is infectious at this time, because viral antigen (p24) is present in the blood. as antibodies start to form, the viral antigen disappears and the individual enters the latent phase. he or she is noninfectious and remains well for a variable period of time (7-15 years). development of aids marks the terminal phase of disease. viral antigen reemerges, and the individual is once again infectious. the onset of aids has been considerably delayed with the use of antiretroviral treatment. parenteral transmission included needlestick injuries, bites, unscreened blood transfusions, tattooing, acupuncture, and dental procedures where equipment is inadequately sterilized. risk of transmission is increased with deep penetrating injuries with hollow bore needles that are visibly bloodstained, especially when the device has previously been in the source patient's (contact) artery or vein. other routes include mucous membrane exposure (eyes, mouth, and genital mucous membranes) and contamination of broken skin. the higher the viral load in the contact, the greater the risk of transmission. this is more likely at the terminal stage of infection. hiv is transmitted mainly through blood or other body fluids that are visibly blood stained, with the exception of semen, vaginal fluid, and breast milk. saliva alone is most unlikely to transmit infection. therefore, people who have sustained penetrating bite injuries can be reassured that they are not at risk, providing the contact was not bleeding from the mouth at the time. the risk from a single percutaneous exposure from a hollow bore needle is low, and a single mucocutaneous exposure is even less likely to result in infection. the risk from sexual exposure varies, although it appears that there is a greater risk with receptive anal intercourse compared with receptive vaginal intercourse (26). high-risk fluids include blood, semen, vaginal fluid, and breast milk. there is little or no risk from saliva, urine, vomit, or feces unless they are visibly bloodstained. other fluids that constitute a theoretical risk include cerebrospinal, peritoneal, pleural, synovial, or pericardial fluid. management in custody of staff/victims in contact with disease includes following the immediate management flow chart (fig. 1 ) and contacting the designated hospital/specialist with details of the exposure. where possible, obtain a blood sample from the contact. regarding hbv and hcv blood samples in the united kingdom, they can only be taken with informed consent. there is no need for the forensic physician to go into details about the meaning of the test, but the contact should be encouraged to attend the genitourinary department (or similar) of the designated hospital to discuss the test results. should the contact refuse to provide a blood sample, then any information about his or her lifestyle, ethnic origin, state of health, etc., may be useful for the specialist to decide whether postexposure prophylaxis (pep) should be given to the victim. where only saliva is involved in a penetrating bite injury, there is every justification to reassure the victim that he or she is not at risk. if in doubt, then always refer. in the united kingdom, the current recommended regime for pep is combivir (300 mg of zidovudine twice daily plus 150 mg of lamivudine twice daily) and a protease inhibitor (1250 mg of nelfanivir twice daily) given for 4 weeks (27) . it is only given after a significant exposure to a high-risk fluid or any that is visibly bloodstained and the contact is known or is highly likely to be hiv positive. ideally, treatment should be started within an hour after exposure, although it will be considered for up to 2 weeks. it is usually given for 4 weeks, unless the contact is subsequently identified as hiv negative or the "victim" develops tolerance or toxicity occurs. weekly examinations of the "victim" should occur during treatment to improve adherence, monitor drug toxicity, and deal with other concerns. other useful information that may influence the decision whether to treat with the standard regimen or use alternative drugs includes interaction with other medications that the "victim" may be taking (e.g., phenytoin or antibiotics) or if the contact has been on antiretroviral therapy or if the "victim" is pregnant. during the second or third trimester, only combivir would be used, because there is limited experience with protease inhibitors. no data exist regarding the efficacy of pep beyond occupational exposure (27) . pep is not considered for exposure to low-or no-risk fluids through any route or where the source is unknown (e.g., a discarded needle). despite the appropriate use and timing of pep, there have been reports of failure (28, 29) . unless they are severely ill, detainees can be kept in custody. every effort should be made to continue any treatment they may be receiving. apply universal precautions when dealing with the detainee, and ensure that contaminated cells and/or bedding are managed appropriately. cases of this highly infectious disease occur throughout the year but are more frequent in winter and early spring. this seasonal endemicity is blurring with global warming. in the united kingdom, the highest prevalence occurs in the 4-to 10-years age group. ninety percent of the population over the age of 40 is immune (30) . a similar prevalence has been reported in other parts of western europe and the united states. in south east asia, varicella is mainly a disease of adulthood (31) . therefore, people born in these countries who have moved to the united kingdom are more likely to be susceptible to chicken pox. there is a strong correlation between a history of chicken pox and serological immunity (97-99%). most adults born and living in industrialized countries with an uncertain or negative history of chicken pox are also seropositive (70-90%). in march 1995, a live-attenuated vaccine was licensed for use in the united states and a policy for vaccinating children and susceptible health care personnel was introduced. in summer 2002, in the united kingdom, glaxosmithkline launched a live-attenuated vaccine called varilrix. in december 2003, the uk department of health, following advice from the joint committee on vaccination and immunisation recommended that the vaccine be given for nonimmune health care workers who are likely to have direct contact with individuals with chicken pox. any health care worker with no previous history of chicken pox should be screened for immunity, and if no antibodies are found, then they should receive two doses of vaccine 4-8 weeks apart. the vaccine is not currently recommended for children and should not be given during pregnancy. following an incubation period of 10-21 days (this may be shorter in the immunocompromised), there is usually a prodromal "flu-like" illness before the onset of the rash. this coryzal phase is more likely in adults. the lesions typically appear in crops, rapidly progressing from red papules through vesicles to open sores that crust over and separate by 10 days. the distribution of the rash is centripetal (i.e., more over the trunk and face than on the limbs). this is the converse of small pox. in adults, the disease is often more severe, with lesions involving the scalp and mucous membranes of the oropharynx. in children, the disease is often mild, unless they are immunocompromised, so they are unlikely to experience complications. in adults (defined as 15 yr or older), the picture is rather different (32) . secondary bacterial infection is common but rarely serious. there is an increased likelihood of permanent scarring. hemorrhagic chicken pox typically occurs on the second or third day of the rash. usually, this is limited to bleeding into the skin, but lifethreatening melena, epistaxis, or hematuria can occur. varicella pneumonia ranges from patchy lung consolidation to overt pneumonitis and occurs in 1 in 400 cases (33) . it can occur in previously healthy individuals (particularly adults), but the risk is increased in those who smoke. immunocompromised people are at the greatest risk of developing this complication. it runs a fulminating course and is the most common cause of varicella-associated death. fibrosis and permanent respiratory impairment may occur in those who survive. any suspicion of lung involvement is an indication for immediate treatment, and any detainee or staff member should be sent to hospital. involvement of the central nervous system includes several conditions, including meningitis, guillain-barre, and encephalitis. the latter is more common in the immunocompromised and can be fatal. this is taken as 3 days before the first lesions appear to the end of new vesicle formation and the last vesicle has crusted over. this typically is 5-7 days after onset but may last up to 14 days. the primary route is through direct contact with open lesions of chicken pox. however, it is also spread through aerosol or droplets from the respiratory tract. chicken pox may also be acquired through contact with open lesions of shingles (varicella zoster), but this is less likely because shingles is less infectious than chicken pox. nonimmune individuals are at risk of acquiring disease. approximately 10% of the adult population born in the united kingdom and less than 5% of adults in the united states fall into this category. therefore, it is more likely that if chicken pox is encountered in the custodial setting, it will involve people born outside the united kingdom (particularly south east asia) or individuals who are immunocompromised and have lost immunity. nonimmune pregnant women are at risk of developing complications. pneumonia can occur in up to 10% of pregnant women with chicken pox, and the severity is increased in later gestation (34) . they can also transmit infection to the unborn baby (35) . if infection is acquired in the first 20 weeks, there is a less than 3% chance of it leading to congenital varicella syndrome. infection in the last trimester can lead to neonatal varicella, unless more than 7 days elapse between onset of maternal rash and delivery when antibodies have time to cross the placenta leading to either mild or inapparent infection in the newborn. in this situation, varicella immunoglobulin (vzig) should be administered to the baby as soon as possible after birth (36). staff with chicken pox should stay off work until the end of the infective period (approx 7-14 days). those in contact with disease who are known to be nonimmune or who have no history of disease should contact the designated occupational health physician. detainees with the disease should not be kept in custody if at all possible (especially pregnant women). if this is unavoidable, then nonimmune or immunocompromised staff should avoid entering the cell or having close contact with the detainee. nonimmune, immunocompromised, or pregnant individuals exposed to chickenpox should seek expert medical advice regarding the administration of vzig. aciclovir (or similar antiviral agent) should be given as soon as possible to people who are immunocompromised with chicken pox. it should also be considered for anyone over 15 years old because they are more likely to develop complications. anyone suspected of severe complications should be sent straight to the hospital. after chicken pox, the virus lies dormant in the dorsal root or cranial nerve ganglia but may re-emerge and typically involves one dermatome (37) . the site of involvement depends on the sensory ganglion initially involved. shingles is more common in individuals over the age of 50 years, except in the immunocompromised, when attacks can occur at an earlier age. the latter are also more susceptible to secondary attacks and involvement of more than one dermatome. bilateral zoster is even rarer but is not associated with a higher mortality. in the united kingdom, there is an estimated incidence of 1.2-3.4 per 1000-person years (38). there may be a prodromal period of paraesthesia and burning or shooting pains in the involved segment. this is usually followed by the appearance of a band of vesicles. rarely, the vesicles fail to appear and only pain is experienced. this is known as zoster sine herpete. in individuals who are immuno-compromised, disease may be prolonged and dissemination may occur but is rarely fatal. shingles in pregnancy is usually mild. the fetus is only affected if viremia occurs before maternal antibody has had time to cross the placenta. the most common complication of shingles is postherpetic neuralgia, occurring in approx 10% of cases. it is defined as pain lasting more than 120 days from rash onset (39) . it is more frequent in people over 50 years and can lead to depression. it is rare in children, including those who are immunocompromised. infection of the brain includes encephalitis, involvement of motor neurones leading to ptosis, paralysis of the hand, facial palsy, or contralateral hemiparesis. involvement of the oculomotor division of the trigeminal ganglion can cause serious eye problems, including corneal scarring. shingles is far less infectious than chicken pox and is only considered to be infectious up to 3 days after lesions appear. shingles is only infectious after prolonged contact with lesions. unlike chickenpox, airborne transmission is not a risk. individuals who are immunocompromised may reactivate the dormant virus and develop shingles. people who have not had primary varicella are at risk of developing chickenpox after prolonged direct contact with shingles. despite popular belief, it is untrue that people who are immunocompetent who have had chicken pox develop shingles when in contact with either chicken pox or shingles. such occurrences are merely coincidental, unless immunity is lowered. staff with shingles should stay off work until the lesions are healed, unless they can be covered. staff who have had chickenpox are immune (including pregnant women) and are therefore not at risk. if they are nonimmune (usually accepted as those without a history of chicken pox), they should avoid prolonged contact with detainees with shingles. pregnant nonimmune women should avoid contact altogether. detainees with the disease may be kept in custody, and any exposed lesions should be covered. it is well documented that prompt treatment attenuates the severity of the disease, reduces the duration of viral shedding, hastens lesion healing, and reduces the severity and duration of pain. it also reduces the likelihood of developing postherpetic neuralgia (40) . prompt treatment with famciclovir (e.g., 500 mg three times a day for 7 days) should be initiated if the onset is 3 d ays or less. it should also be considered after this time if the detainee is over age 50 years. pregnant detainees with shingles can be reassured that there is minimal risk for both the mother and the unborn child. expert advice should be given before initiating treatment for the mother. this tiny parasitic mite (sarcoptes scabiei) has infested humans for more than 2500 years. experts estimate that in excess of 300 million cases occur worldwide each year. the female mite burrows into the skin, especially around the hands, feet, and male genitalia, in approx 2.5 min. eggs are laid and hatch into larvae that travel to the skin surface as newly developed mites. the mite causes intense itching, which is often worse at night and is aggravated by heat and moisture. the irritation spreads outside the original point of infection resulting from an allergic reaction to mite feces. this irritation may persist for approx 2 weeks after treatment but can be alleviated by antihistamines. crusted scabies is a far more severe form of the disease. large areas of the body may be involved. the crusts hide thousands of live mites and eggs, making them difficult to treat. this so-called norwegian scabies is more common in the elderly or the immunocompromised, especially those with hiv. after a primary exposure, it takes approx 2-6 weeks before the onset of itching. however, further exposures reduce the incubation time to approx 1-4 days. without treatment, the period of infectivity is assumed to be indefinite. with treatment, the person should be considered infectious until the mites and eggs are destroyed, usually 7-10 days. crusted scabies is highly infectious. because transmission is through direct skin-to-skin contact with an infected individual, gloves should be worn when dealing with individuals suspected of infestation. usually prolonged contact is needed, unless the person has crusted scabies, where transmission occurs more easily. the risk of transmission is much greater in households were repeated or prolonged contact is likely. because mites can survive in bedding or clothing for up to 24 hour, gloves should also be worn when handling these items. bedding should be treated using one of the methods in subheading 2.2. professional cleaning of the cell is only warranted in cases of crusted scabies. the preferred treatment for scabies is either permethrin cream (5%) or aqueous malathion (0.5%) (41) . either treatment has to be applied to the whole body and should be left on for at least 8 hours in the case of permethrin and 24 hours for malathion before washing off. lindane is no longer considered the treatment of choice, because there may be complications in pregnancy (42) . treatment in custody may not be practical but should be considered when the detainee is believed to have norwegian scabies. like scabies, head lice occur worldwide and are found in the hair close to the scalp. the eggs, or nits, cling to the hair and are difficult to remove, but they are not harmful. if you see nits, then you can be sure that lice are also present. the latter are best seen when the hair is wet. the lice bite the scalp and suck blood, causing intense irritation and itching. head lice can only be passed from direct hair-to-hair contact. it is only necessary to wear gloves when examining the head for whatever reason. the cell does not need to be cleaned after use, because the lice live on or near skin. bedding may be contaminated with shed skin, so should be handled with gloves and laundered or incinerated. the presence of live lice is an indication for treatment by either physical removal with a comb or the application of an insecticide. the latter may be more practical in custody. treatment using 0.5% aqueous malathion should be applied to dry hair and washed off after 12 hours. the hair should then be shampooed as normal. crabs or body lice are more commonly found in the pubic, axillary, chest, and leg hair. however, eyelashes and eyebrows may also be involved. they are associated with people who do not bath or change clothes regularly. the person usually complains of intense itching or irritation. the main route is from person to person by direct contact, but eggs can stick to fibers, so clothing and bedding should be handled with care (see subheading 6.5.3.). staff should always wear gloves if they are likely to come into contact with any hirsute body part. clothing or bedding should be handled with gloves and either laundered or incinerated. treatment of a detainee in custody is good in theory but probably impractical because the whole body has to be treated. fleas lay eggs on floors, carpets, and bedding. in the united kingdom, most flea bites come from cats or dogs. the eggs and larvae fleas can survive for months and are reactivated in response to animal or human activity. because animal fleas jump off humans after biting, most detainees with flea bites will not have fleas, unless they are human fleas. treatment is only necessary if fleas are seen. after use, the cell should be vacuumed and cleaned with a proprietary insecticide. any bedding should be removed wearing gloves, bagged, and either laundered or incinerated. bedbugs live and lay eggs on walls, floors, furniture, and bedding. if you look carefully, fecal tracks may be seen on hard surfaces. if they are present for long enough, they emit a distinct odor. bedbugs are rarely found on the person but may be brought in on clothing or other personal effects. bedbugs bite at night and can cause sleep disturbance. the detainee does not need to be treated, but the cell should deemed out of use until it can be vacuumed and professionally cleaned with an insecticide solution. any bedding or clothing should be handled with gloves and disposed of as appropriate. staphylococcus aureus is commonly carried on the skin or in the nose of healthy people. approximately 25-30% of the population is colonized with the bacteria but remain well (43) . from time to time, the bacteria cause minor skin infections that usually do not require antibiotic treatment. however, more serious problems can occur (e.g., infection of surgical wounds, drug injection sites, osteomyelitis, pneumonia, or septicemia). during the last 50 years, the bacteria have become increasingly resistant to penicillin-based antibiotics (44) , and in the last 20 years, they have become resistant to an increasing number of alternative antibiotics. these multiresistant bacteria are known as methicillinresistant s. aureus (mrsa). mrsa is prevalent worldwide. like nonresistant staphylococci, it may remain undetected as a reservoir in colonized individuals but can also produce clinical disease. it is more common in individuals who are elderly, debilitated, or immunocompromised or those with open wounds. clusters of skin infections with mrsa have been reported among injecting drug users (idus) since 1981 in america (45, 46) , and more recently, similar strains have been found in the united kingdom in idus in the community (47) . this may have particular relevance for the forensic physician when dealing with idus sores. people who are immunocompetent rarely get mrsa and should not be considered at risk. the bacteria are usually spread via the hands of staff after contact with colonized or infected detainees or devices, items (e.g., bedding, towels, and soiled dressings), or environmental surfaces that have been contaminated with mrsa-containing body fluids. with either known or suspected cases (consider all abscesses/ulcers of idus as infectious), standard precautions should be applied. staff should wear gloves when touching mucous membranes, nonintact skin, blood or other body fluids, or any items that could be contaminated. they should also be encouraged to their wash hands with an antimicrobial agent regardless of whether gloves have been worn. after use, gloves should be disposed of in a yellow hazard bag and not allowed to touch surfaces. masks and gowns should only be worn when conducting procedures that generate aerosols of blood or other body fluids. because this is an unlikely scenario in the custodial setting, masks and gowns should not be necessary. gloves should be worn when handling bedding or clothing, and all items should be disposed of appropriately. any open wounds should be covered as soon as possible. the cell should be cleaned professionally after use if there is any risk that it has been contaminated. during the last decade, there has been an increasing awareness of the bacterial flora colonizing injection sites that may potentially lead to life-threatening infection (48) . in 1997, a sudden increase in needle abscesses caused by a clonal strain of group a streptococcus was reported among hospitalized idus in berne, switzerland (49) . a recent uk study showed that the predominant isolate is s. aureus, with streptococcus species forming just under one-fifth (50% î²-hemolytic streptococci) (50) . there have also been reports of both nonsporing and sporing anerobes (e.g., bacteroides and clostridia species, including clostridia botulinum) (51, 52) . in particular, in 2000, laboratories in glasgow were reporting isolates of clostridium novyi among idus with serious unexplained illness. by june 12, 2000, a total of 42 cases (18 definite and 24 probable) had been reported. a definite case was defined as an idu with both severe local and systemic inflammatory reactions. a probable case was defined as an idu who presented to the hospital with an abscess or other significant inflammation at an injecting site and had either a severe inflammatory process at or around an injection site or a severe systemic reaction with multiorgan failure and a high white cell count (53) . in the united kingdom, the presence of c. botulinum in infected injection sites is a relatively new phenomenon. until the end of 1999, there were no cases reported to the public health leadership society. since then, the number has increased, with a total of 13 cases in the united kingdom and ireland being reported since the beginning of 2002. it is believed that these cases are associated with contaminated batches of heroin. simultaneous injection of cocaine increases the risk by encouraging anerobic conditions. anerobic flora in wounds may have serious consequences for the detainee, but the risk of transmission to staff is virtually nonexistent. staff should be reminded to wear gloves when coming into contact with detainees with infected skin sites exuding pus or serum and that any old dressings found in the cell should be disposed of into the yellow bag marked "clinical waste" in the medical room. likewise, any bedding should be bagged and laundered or incinerated after use. the cell should be deemed out of use and professionally cleaned after the detainee has gone. the health care professional managing the detainee should clean and dress open wounds as soon as possible to prevent the spread of infection. it may also be appropriate to start a course of antibiotics if there is abscess formation or signs of cellulites and/or the detainee is systemically unwell. however, infections can often be low grade because the skin, venous, and lymphatic systems have been damaged by repeated penetration of the skin. in these cases, signs include lymphedema, swollen lymph glands, and darkly pigmented skin over the area. fever may or may not be present, but septicemia is uncommon unless the individual is immunocompromised (e.g., hiv positive). co-amoxiclav is the preferred treatment of choice because it covers the majority of staphylococci, streptococci, and anerobes (the dose depends on the degree of infection). necrotizing fasciitis and septic thrombophlebitis are rare but life-threatening complications of intravenous drug use. any detainee suspected of either of these needs hospital treatment. advice about harm reduction should also be given. this includes encouraging drug users to smoke rather than inject or at least to advise them to avoid injecting into muscle or skin. although most idus are aware of the risk of sharing needles, they may not realize that sharing any drug paraphernalia could be hazardous. advice should be given to use the minimum amount of citric acid to dissolve the heroin because the acid can damage the tissue under the skin, allowing bacteria to flourish. drugs should be injected at different sites using fresh works for each injection. this is particularly important when "speedballing" because crack cocaine creates an anerobic environment. medical help should be requested if any injection site become painful and swollen or shows signs of pus collecting under the skin. because intravenous drug users are at increased risk of acquiring hbv and hav, they should be informed that vaccination against both diseases is advisable. another serious but relatively rare problem is the risk from broken needles in veins. embolization can take anywhere from hours to days or even longer if it is not removed. complications may include endocarditis, pericarditis, or pulmonary abscesses (54, 55) . idus should be advised to seek medical help as soon as possible, and should such a case present in custody, then send the detainee straight to the hospital. the forensic physician may encounter bites in the following four circumstances: a detailed forensic examination of bites is given in chapter 4. with any bite that has penetrated the skin, the goals of therapy are to minimize soft tissue deformity and to prevent or treat infection. in the united kingdom and the united states, dog bites represent approximately three-quarters of all bites presenting to accident and emergency departments (56) . a single dog bite can produce up to 220 psi of crush force in addition to the torsional forces as the dog shakes its head. this can result in massive tissue damage. human bites may cause classical bites or puncture wounds (e.g., impact of fists on teeth) resulting in crush injuries. an estimated 10-30% of dog bites and 9-50% of human bites lead to infection. compare this with an estimated 1-12% of nonbite wounds managed in accident and emergency departments. the risk of infection is increased with puncture wounds, hand injuries, full-thickness wounds, wounds requiring debridement, and those involving joints, tendons, ligaments or fractures. comorbid medical conditions, such as diabetes, asplenia, chronic edema of the area, liver dysfunction, the presence of a prosthetic valve or joint, and an immunocompromised state may also increase the risk of infection. infection may spread beyond the initial site, leading to septic arthritis, osteomyelitis, endocarditis, peritonitis, septicemia, and meningitis. inflammation of the tendons or synovial lining of joints may also occur. if enough force is used, bones may be fractured or the wounds may be permanently disfiguring. assessment regarding whether hospital treatment is necessary should be made as soon as possible. always refer if the wound is bleeding heavily or fails to stop when pressure is applied. penetrating bites involving arteries, nerves, muscles, tendons, the hands, or feet, resulting in a moderate to serious facial wound, or crush injuries, also require immediate referral. if management within custody is appropriate, ask about current tetanus vaccine status, hbv vaccination status, and known allergies to antibiotics. wounds that have breached the skin should be irrigated with 0.9% (isotonic) sodium chloride or ringer's lactate solution instead of antiseptics, because the latter may delay wound healing. a full forensic documentation of the bite should be made as detailed in chapter 4. note if there are clinical signs of infection, such as erythema, edema, cellulitis, purulent discharge, or regional lymphadenopathy. cover the wound with a sterile, nonadhesive dressing. wound closure is not generally recommended because data suggest that it may increase the risk of infection. this is particularly relevant for nonfacial wounds, deep puncture wounds, bites to the hand, clinically infected wounds, and wounds occurring more than 6-12 hours before presentation. head and neck wounds in cosmetically important areas may be closed if less than 12 hours old and not obviously infected. â�¢ dog bites-pasteurella canis, pasteurella multocida, s. aureus, other staphylococci, streptococcus species, eikenella corrodens, corynebacterium species, and anerobes, including bacteroides fragilis and clostridium tetani â�¢ human bites-streptococcus species, s. aureus, e. corrodens, and anerobes, including bacteroides (often penicillin resistant), peptostreptococci species, and c. tetani. tuberculosis (tb) and syphilis may also be transmitted. â�¢ dog bites-outside of the united kingdom, australia, and new zealand, rabies should be considered. in the united states, domestic dogs are mostly vaccinated against rabies (57) , and police dogs have to be vaccinated, so the most common source is from racoons, skunks, and bats. â�¢ human bites-hbv, hbc, hiv, and herpes simplex. antibiotics are not generally needed if the wound is more than 2 days old and there is no sign of infection or in superficial noninfected wounds evaluated early that can be left open to heal by secondary intention in compliant people with no significant comorbidity (58) . antibiotics should be considered with high-risk wounds that involve the hands, feet, face, tendons, ligaments, joints, or suspected fractures or for any penetrating bite injury in a person with diabetes, asplenia, or cirrhosis or who is immunosuppressed. coamoxiclav (amoxycillin and clavulanic acid) is the first-line treatment for mild-moderate dog or human bites resulting in infections managed in primary care. for adults, the recommended dose is 500/125 mg three times daily and for children the recommended does is 40 mg/kg three times daily (based on amoxycillin component). treatment should be continued for 10-14 days. it is also the first-line drug for prophylaxis when the same dose regimen should be prescribed for 5-7 days. if the individual is known or suspected to be allergic to penicillin, a tetracycline (e.g., doxycycline 100 mg twice daily) and metronidazole (500 mg three times daily) or an aminoglycoside (e.g., erythromycin) and metronidazole can be used. in the united kingdom, doxycycline use is restricted to those older than 12 years and in the united states to those older than 8 years old. specialist advice should be sought for pregnant women. anyone with severe infection or who is clinically unwell should be referred to the hospital. tetanus vaccine should be given if the primary course or last booster was more than 10 years ago. human tetanus immunoglobulin should be considered for tetanus-prone wounds (e.g., soil contamination, puncture wounds, or signs of devitalized tissue) or for wounds sustained more than 6 hours old. if the person has never been immunized or is unsure of his or her tetanus status, a full three-dose course, spaced at least 1 month apart, should be given. penetrating bite wounds that involve only saliva may present a risk of hbv if the perpetrator belongs to a high-risk group. for management, see subheadings 5.1.6. and 5.1.7. hcv and hiv are only a risk if blood is involved. the relevant management is dealt with in subheadings 5.2.5. and 5.4.6. respiratory tract infections are common, usually mild, and self-limiting, although they may require symptomatic treatment with paracetamol or a nonsteroidal antiinflammatory. these include the common cold (80% rhinoviruses and 20% coronaviruses), adenoviruses, influenza, parainfluenza, and, during the summer and early autumn, enteroviruses. special attention should be given to detainees with asthma or the who are immunocompromised, because infection in these people may be more serious particularly if the lower respiratory tract is involved. the following section includes respiratory pathogens of special note because they may pose a risk to both the detainee and/or staff who come into close contact. there are five serogroups of neisseria meningitidis: a, b, c, w135, and y. the prevalence of the different types varies from country to country. there is currently no available vaccine against type b, but three other vaccines (a+c, c, and acwy) are available. overall, 10% of the uk population carry n. meningitidis (25% in the 15-19 age group) (59) . in the united kingdom, most cases of meningitis are sporadic, with less than 5% occurring as clusters (outbreaks) amongst school children. between 1996 and 2000, 59% of cases were group b, 36% were group c, and w135 and a accounted for 5%. there is a seasonal variation, with a high level of cases in winter and a low level in the summer. the greatest risk group are the under 5 year olds, with a peak incidence under 1 year old. a secondary peak occurs in the 15-to 19-year-old age group. in sub-saharan africa, the disease is more prevalent in the dry season, but in many countries, there is background endemicity year-round. the most prevalent serogroup is a. routine vaccination against group c was introduced in the united kingdom november 1999 for everybody up to the age of 18 years old and to all firstyear university students. this has since been extended to include everyone under the age of 25 years old. as a result of the introduction of the vaccination program, there has been a 90% reduction of group c cases in those younger than under 18 years and an 82% reduction in those under 1 year old (60, 61) . an outbreak of serogroup w135 meningitis occurred among pilgrims on the hajj in 2000. cases were reported from many countries, including the united kingdom. in the united kingdom, there is now an official requirement to be vaccinated with the quadrivalent vaccine (acwy vax) before going on a pilgrimage (hajj or umra), but illegal immigrants who have not been vaccinated may enter the country (62). after an incubation period of 3-5 days (63,64) , disease onset may be either insidious with mild prodromal symptoms or florid. early symptoms and signs include malaise, fever, and vomiting. sever headache, neck stiffness, photophobia, drowsiness, and a rash may develop. the rash may be petechial or purpuric and characteristically does not blanche under pressure. meningitis in infants is more likely to be insidious in onset and lack the classical signs. in approx 15-20% of cases, septicemia is the predominant feature. even with prompt antibiotic treatment, the case fatality rate is 3-5% in meningitis and 15-20% in those with septicemia. (65). a person should be considered infectious until the bacteria are no longer present in nasal discharge. with treatment, this is usually approx 24 hour. the disease is spread through infected droplets or direct contact from carriers or those who are clinically ill. it requires prolonged and close contact, so it is a greater risk for people who share accommodation and utensils and kiss. it must also be remembered that unprotected mouth-to-mouth resuscitation can also transmit disease. it is not possible to tell if a detainee is a carrier. nevertheless, the risk of acquiring infection even from an infected and sick individual is low, unless the individual has carried out mouth-to-mouth resuscitation. any staff member who believes he or she has been placed at risk should report to the occupational health department (or equivalent) or the nearest emergency department at the earliest opportunity for vaccination. if the detainee has performed mouth-to-mouth resuscitation, prophylactic antibiotics should be given before receiving vaccination. rifampicin, ciprofloxacin, and ceftriaxone can be used; however, ciprofloxacin has numerous advantages (66) . only a single dose of 500 mg (adults and children older than 12 years) is needed and has fewer side effects and contraindications than rifampicin. ceftriaxone has to be given by injection and is therefore best avoided in the custodial setting. if the staff member is pregnant, advice should be sought from a consultant obstetrician, because ciprofloxacin is not recommended (67) . for anyone dealing regularly with illegal immigrants (especially from the middle east or sub-saharan africa) (e.g., immigration services, custody staff at designated stations, medical personnel, and interpreters), should consider being vaccinated with acwy vax. a single injection provides protection for 3 years. detainees suspected of disease should be sent directly to the hospital. human tb is caused by infection with mycobacterium tuberculosis, mycobacterium bovis, or mycobacterium africanum. it is a notifiable disease under legislation specific to individual countries; for example, in the united kingdom, this comes under the public health (control of disease) act of 1984. in 1993, the who declared tb to be a global emergency, with an estimated 7-8 million new cases and 3 million deaths occurring each year, the majority of which were in asia and africa. however, these statistics are likely to be an underestimate because they depend on the accuracy of reporting, and in poorer countries, the surveillance systems are often inadequate because of lack of funds. even in the united kingdom, there has been an inconsistency of reporting particularly where an individual has concomitant infection with hiv. some physicians found themselves caught in a dilemma of confidentiality until 1997, when the codes of practice were updated to encourage reporting with patient consent (68) . with the advent of rapid identification tests and treatment and the use of bacillus calmette-guã©rin (bcg) vaccination for prevention, tb declined during the first half of the 20th century in the united kingdom. however, since the early 1990s, numbers have slowly increased, with some 6800 cases reported in 2002 (69) . in 1998, 56% of reported cases were from people born outside the united kingdom and 3% were associated with hiv infection (70, 71) . london has been identified as an area with a significant problem. this has been attributed to its highly mobile population, the variety of ethnic groups, a high prevalence of hiv, and the emergence of drug-resistant strains (1.3% in 1998 ) (phls, unpublished data-mycobnet). a similar picture was initially found in the united states, when there was a reversal of a long-standing downward trend in 1985. however, between 1986 and 1992, the number of cases increased from 22,201 to 26,673 (72) . there were also serious outbreaks of multidrug-resistant tb (mdr-tb) in hospitals in new york city and miami (73) . factors pertinent to the overall upswing included the emergence of hiv, the increasing numbers of immigrants from countries with a high prevalence of tb, and perhaps more significantly, stopping categorical federal funding for control activities in 1972. the latter led to a failure of the public health infrastructure for tb control. since 1992, the trend has reversed as the cdc transferred most of its funds to tb surveillance and treatment program in states and large cities. from 1992 to 2001, the annual decline averaged by 7.3% (74) , but the following year this was reduced to 2%, indicating that there was no room for complacency. the who has been proactive and is redirecting funding to those countries most in need. in october 1998, a global partnership called stop tb was launched to coordinate every aspect of tb control, and by 2002, the partnership had more than 150 member states. a target was set to detect at least 70% of infectious cases by 2005. the acquisition of tb infection is not necessarily followed by disease because the infection may heal spontaneously. it may take weeks or months before disease becomes apparent, or infection may remain dormant for years before reactivation in later life especially if the person becomes debilitated or immunocompromised. contrary to popular belief, the majority of cases of tb in people who are immunocompetent pass unnoticed. of the reported cases, 75% involve the lung, whereas nonrespiratory (e.g., bone, heart, kidney, and brain) or dissemination (miliary tb) are more common in immigrant ethnic groups and individuals who are immunocompromised (75) . they are also more likely to develop resistant strains. in the general population, there is an estimated 10% lifetime risk of tb infection progressing to disease (76) . there has been an increase in the number of cases of tb associated with hiv owing to either new infection or reactivation. tb infection is more likely to progress to active tb in hiv-positive individuals, with a greater than50% lifetime risk (77) . tb can also lead to a worsening of hiv with an increase in viral load (78) . therefore, the need for early diagnosis is paramount, but it can be more difficult because pulmonary tb may present with nonspecific features (e.g., bilateral, unilateral, or lower lobe shadowing) (79). after an incubation period of 4-12 weeks, symptoms may develop (see table 6 ). the main route is airborne through infected droplets, but prolonged or close contact is needed. nonrespiratory disease is not considered a risk unless the mycobacterium is aerosolized under exceptional circumstances (e.g., during surgery) or there are open abscesses. a person is considered infectious as long as viable bacilli are found in induced sputum. untreated or incompletely treated people may be intermittently sputum positive for years. after 2 weeks of appropriate treatment, the individual is usually considered as noninfectious. this period is often extended for treatment of mdr-tb or for those with concomitant hiv. patient compliance also plays an important factor. the risk of infection is directly proportional to the degree of exposure. more severe disease occurs in individuals who are malnourished, immunocompromised (e.g., hiv), and substance misusers. people who are immunocompromised are at special risk of mdr-tb or mycobacterium avium intracellulare (mai). staff with disease should stay off work until the treatment course is complete and serial sputum samples no longer contain bacilli. staff in contact with disease who have been vaccinated with bcg are at low risk of acquiring disease but should minimize their time spent in the cell. those who have not received bcg or who are immunocompromised should avoid contact with the detainee wherever possible. detainees with mai do not pose a risk to a staff member, unless the latter is immunocompromised. any staff member who is pregnant, regardless of bcg status or type of tb, should avoid contact. anyone performing mouth-to-mouth resuscitation with a person with untreated or suspected pulmonary tb should be regarded as a household contact and should report to occupational health or their physician if no other route exists. they should also be educated regarding the symptoms of tb. anyone who is likely to come into repeated contact with individuals at risk of tb should receive bcg (if he or she has not already done so), regardless of age, even though there is evidence to suggest that bcg administered in adult life is less effective. this does not apply to individuals who are immunocompromised or pregnant women. in the latter case, vaccination should preferably be deferred until after delivery. detainees with disease (whether suspected or diagnosed) who have not been treated or treatment is incomplete should be kept in custody for the minimum time possible. individuals with tb who are immunocompromised are usually too ill to be detained; if they are, they should be considered at greater risk of transmitting disease to staff. any detainee with disease should be encouraged to cover his or her mouth and nose when coughing and sneezing. staff should wear gloves when in contact with the detainee and when handling clothing and bedding. any bedding should be bagged after use and laundered or incinerated. the cell should be deemed out of action until it has been ventilated and professionally decontaminated, although there is no hard evidence to support that there is a risk of transmission from this route (70). on march 14, 2003 , the who issued a global warning to health authorities about a new atypical pneumonia called sars. the earliest case was believed to have originated in the guandong province of china on november 16, 2002. the causative agent was identified as a new corona virus-sars-cov (80, 81) . by the end of june 2003, 8422 cases had been reported from 31 different countries, with a total of 916 deaths. approximately 92% of cases occurred in china (including hong kong, taiwan, and macao). the case fatality rate varied from less than 1% in people younger than 24 years, 6% in persons aged 25-44 years, 15% in those aged 44-64 years, and more than 50% in persons 65 years or older. on july 5, 2003, the who reported that the last human chain of transmission of sars had been broken and lifted the ban from all countries. however, it warned that everyone should remain vigilant, because a resurgence of sars is possible. their warning was well given because in december 2003, a new case of sars was detected in china. at the time of this writing, three more cases have been identified. knowledge about the epidemiology and ecology of sars-cov and the disease remains limited; however, the experience gained from the previous outbreak enabled the disease to be contained rapidly, which is reflected in the few cases reported since december 2003. there is still no specific treatment or preventative vaccine that has been developed. the incubation period is short, approx 3-6 days (maximum 10 days), and, despite the media frenzy surrounding the initial outbreak, sars is less infectious than influenza. the following clinical case definition of sars has been developed for public health purposes (82) . a person with a history of any combination of the following should be examined for sars: â�¢ fever (at least 38â°c); and â�¢ one of more symptoms of lower respiratory tract illness (cough, difficulty in breathing, or dyspnea); and â�¢ radiographic evidence of lung infiltrates consistent with pneumonia or respiratory distress syndrome or postmortem findings of these with no identifiable cause; and â�¢ no alternative diagnosis can fully explain the illness. laboratory tests have been developed that include detection of viral rna by pcr from nasopharyngeal secretions or stool samples, detection of antibodies by enzyme-linked immunosorbent assay or immunofluorescent antibody in the blood, and viral culture from clinical specimens. available information suggests that close contact via aerosol or infected droplets from an infected individual provide the highest risk of acquiring the disease. most cases occurred in hospital workers caring for an index case or his or her close family members. despite the re-emergence of sars, it is highly unlikely that a case will be encountered in the custodial setting in the near future. however, forensic physicians must remain alert for the sars symptoms and keep up-to-date with recent outbreaks. information can be obtained from the who on a daily basis from its web site. if sars is suspected, medical staff should wear gloves and a surgical mask when examining a suspected case; however, masks are not usually available in custody. anyone suspected of sars must be sent immediately to the hospital, and staff who have had prolonged close contact should be alerted as to the potential symptoms. the most consistent feature of diseases transmitted through the fecaloral route is diarrhea (see table 7 ). infective agents include bacteria, viruses, and protozoa. because the causes are numerous, it is beyond the remit of this chapter to cover them all. it is safest to treat all diarrhea as infectious, unless the detainee has a proven noninfectious cause (e.g., crohn's disease or ulcerative colitis). all staff should wear gloves when in contact with the detainee or when handling clothing and bedding, and contaminated articles should be laundered or incinerated. the cell should be professionally cleaned after use, paying particular attention to the toilet area. this viral hepatitis occurs worldwide, with variable prevalence. it is highest in countries where hygiene is poor and infection occurs year-round. in temperate climates, the peak incidence is in autumn and winter, but the trend is becoming less marked. all age groups are susceptible if they are nonimmune or have not been vaccinated. in developing countries, the disease occurs in early childhood, whereas the reverse is true in countries where the standard of living is higher. in the united kingdom, there has been a gradual decrease in the number of reported cases from 1990 to 2000 (83, 84) . this results from, in part, improved standards of living and the introduction of an effective vaccine. the highest incidence occurs in the 15-to 34-year-old age group. approximately 25% of people older than 40 years have natural immunity, leaving the remainder susceptible to infection (85) . small clusters occur from time to time, associated with a breakdown in hygiene. there is also an increasing incidence of hav in gay or bisexual men and their partners (86) . an unpublished study in london in 1996 showed a seroprevalence of 23% among gay men (young y et al., unpublished). the clinical picture ranges from asymptomatic infection through a spectrum to fulminant hepatitis. unlike hbv and hcv, hav does not persist or progress to chronic liver damage. infection in childhood is often mild or asymptomatic but in adults tends to be more severe. after an incubation period of 15-50 days (mean 28 days) symptomatic infection starts with the abrupt onset of jaundice anything from 2 days to 3 weeks after the anicteric phase. it lasts for approximately the same length of time and is often accompanied by a sudden onset of fever. hav infection can lead to hospital admission in all age groups but is more likely with increasing age as is the duration of stay. the overall mortality is less than 1%, but 15% of people will have a prolonged or relapsing illness within 6-9 months (cdc fact sheet). fulminant hepatitis occurs in less than 1% of people but is more likely to occur in individuals older than 65 years or in those with pre-existing liver disease. in patients who are hospitalized, case fatality ranges from 2% in 50-59 years olds to nearly 13% in those older than 70 years (84). the individual is most infectious in the 2 weeks before the onset of jaundice, when he or she is asymptomatic. this can make control of infection difficult because the disease is not recognized. the main route is fecal-oral through the ingestion of contaminated water and food. it can also be transmitted by personal contact, including homosexuals practicing anal intercourse and fellatio. there is a slight risk from blood transfusions if the donor is in the acute phase of infection. it should not be considered a risk from needlestick injuries unless clinical suspicion of hav is high. risk groups include homeless individuals, homosexuals, idus, travellers abroad who have not been vaccinated, patients with chronic liver disease and chronic infection with hbv and hcv, employees and residents in daycare centers and hostels, sewage workers, laboratory technicians, and those handling nonhuman primates. several large outbreaks have occurred among idus, some with an epidemiological link to prisons (87, 88) . transmission occurs during the viremic phase of the illness through sharing injecting equipment and via fecal-oral routes because of poor living conditions (89) . there have also been reports of hav being transmitted through drugs that have been carried in the rectum. a study in vancouver showed that 40% of idus had past infection of hav, and they also showed an increased prevalence among homosexual/bisuexual men (90). staff with disease should report to occupational health and stay off work until the end of the infective period. those in contact with disease (either through exposure at home or from an infected detainee) should receive prophylactic treatment as soon as possible (see subheading 8.3.7.). to minimize the risk of acquiring disease in custody, staff should wear gloves when dealing with the detainee and then wash their hands thoroughly. gloves should be disposed of only in the clinical waste bags. detainees with disease should be kept in custody for the minimum time possible. they should only be sent to the hospital if fulminant hepatitis is suspected. the cell should be quarantined after use and professionally cleaned. any bedding or clothing should be handled with gloves and laundered or incinerated according to local policy. detainees reporting contact with disease should be given prophylactic treatment as soon as possible (see subheading 8.3.7.). contacts of hav should receive hav vaccine (e.g., havrix monodose or avaxim) if they have not been previously immunized or had disease. human normal immunoglobulin (hnig), 500 mg, deep intramuscular in gluteal muscle should be used in the following circumstances: â�¢ has the detainee traveled to africa, south east asia, the indian subcontinent, central/south america, or the far east in the last 6-12 months? â�¢ ascertain whether he or she received any vaccinations before travel and, if so, which ones. â�¢ ask if he or she took malaria prophylaxis, what type, and whether he or she completed the course. â�¢ ask if he or she swam in any stagnant lakes during the trip. â�¢ if the answer to any of the above is yes, ask if he or she has experienced any of the following symptoms: a fever/hot or cold flushes/shivering. diarrhea â± abdominal cramps â± blood or slime in the stool. a rash. persistent headaches â± light sensitivity. nausea or vomiting. aching muscles/joints. a persistent cough (dry or productive) lasting at least 3 weeks. â�¢ take temperature. â�¢ check skin for signs of a rash and note nature and distribution. â�¢ check throat. â�¢ listen carefully to the lungs for signs of infection/consolidation. staff at higher risk of coming in to contact with hav should consider being vaccinated before exposure. two doses of vaccine given 6-12 months apart give at least 10 years of protection. there is no specific treatment for hav, except supportive measures and symptomatic treatment. although the chance of encountering a tropical disease in custo1dy is small, it is worth bearing in mind. it is not necessary for a forensic physician to be able to diagnose the 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chemoprophylactic agent for meningococcal diseaselow risk of anaphylactoid reactions joint formulary committee 2002-03. british national formulary notification of tuberculosis an updated code of practice for england and wales statutory notifications to the communicable disease surveillance centre. preliminary annual report on tuberculosis cases reported in england, wales, and the prevention and control of tuberculosis in the united kingdom: uk guidance on the prevention and control of transmission of 1. hiv-related tuberculosis 2. drug-resistant, including multiple drug-resistant, tuberculosis. department of health, scottish office control and prevention of tuberculosis in the united kingdom: code of practice epidemiology of tuberculosis in the united states nosocomial transmission of multi-drug resistant tuberculosis among hiv-infected persons-florida the continued threat of tuberculosis tuberculosis-a clinical handbook the white plague: down and out, or up and coming? a prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection influence of tuberculosis on human immunodeficiency virus (hiv-1): enhanced cytokine expression and elevated b 2 -microglobulin in hiv-1 associated tuberculosis the chest roenterogram in pulmonary tuberculosis patients seropositive for human immunodeficiency virus type 1 coronavirus as a possible cause of severe acute respiratory syndrome epidemiological determinants of spread of causal agents of severe acute respiratory syndrome in hong kong alert, verification and public health management of sars in post-outbreak period age-specific antibody prevalence to hepatitis a in england: implications for disease control phls advisory committee on vaccination and immunisation. guidelines for the control of hepatitis a infection control of a community hepatitis a outbreak using hepatitis a vaccine seroprevalence of and risk factors for hepatitis a infection among young homosexual and bisexual men outbreaks of hepatitis a among illicit drug users identifying target groups for a potential vaccination program during a hepatitis a community outbreak multiple modes of hepatitis a transmission among metamphetamine users past infection with hepatitis a among vancouver street youth, injection drug users and men who have sex with men implications for vaccination programmes key: cord-017184-1ewi3dka authors: nan title: primary immunodeficiencies date: 2008 journal: pediatric allergy, asthma and immunology doi: 10.1007/978-3-540-33395-1_22 sha: doc_id: 17184 cord_uid: 1ewi3dka primary immunodeficiencies (pids), once considered to be very rare, are now increasingly recognized because of growing knowledge in the immunological field and the availability of more sophisticated diagnostic techniques and therapeutic modalities [161]. however in a database of >120,000 inpatients of a general hospital for conditions suggestive of id 59 patients were tested, and an undiagnosed pid was found in 17 (29%) of the subjects tested [107]. the publication of the first case of agammaglobulinemia by bruton in 1952 [60] demonstrated that the pid diagnosis is first done in the laboratory. however, pids require specialized immunological centers for diagnosis and management [33]. a large body of epidemiological evidence supports the hypothesis of the existence of a close etiopathogenetic relation between pid and atopy [73]. in particular, an elevated frequency of asthma, food allergy (fa), atopic dermatitis and enteric pathologies can be found in various pids. in addition we will discuss another subject that is certainly of interest: the pseudo-immunodepressed child with recurrent respiratory infections (rris), an event that often requires medical intervention and that very often leads to the suspicion that it involves antibody deficiencies [149]. structural genes, and also perhaps on the lack of 2/4 c4 genes [506] . mbl deficiency is due to one of three point mutations in the gene for mbl, each of which reduces levels of the lectin by interfering with the protein oligomerization [351] . in children with this kind of deficiency, the level of mbl is 4.9 mg/l compared to the 143 mg/l in controls [487] . regardless of whether the children are homozygote (hz) or heterozygote (het) in relation to a given mutation, the defect appears to be more consistent in small babies aged 6-18 months [487] , who show an immaturity in providing immune response to capsular bacteria and in whom low levels of opsonin are incapable of compensating for this [506] . the risk of contracting infections is similar in hzs [175] and hets [486] , though it persists throughout life in hzs because of an abnormal allele, while it exhausts itself in the hets, where the frequency of abnormality is similar to that of the general population [175] . anomalies in immunoglobulins (ig) and in opsonization have been observed, respectively in 13%-20% of children suffering from frequent asthma and igg subclass deficiencies. children suffering from cystic fibrosis also present an elevated prevalence of immediate cutaneous reactions to aeroallergens, and although without primary defects of adoptive immunity, they are susceptible to severe rris; therefore it may be possible that they suffer from the mucosal antigenic exclusion [61] . unlike asthmatic children, in whom a relatively high concentration of ige for respiratory viruses was observed [172, 173, 457] , positive skin prick tests (spts) are more common for aspergillus fumigatus [61] . the hypothesis suggested by these observations is that atopy derives from an unbalanced immune response to foreign antigens, with a consequent lack of their early identification or the capacity to neutralize or eliminate them. this hypothesis is based on the evidence that id precedes the development of atopy: in the taylor et al studies, 22 newborn babies, the children and/or siblings of atopic patients, presented a significant reduction in serum concentrations of iga when aged 3 months: this was transient hypogammaglobulinemia (hgg) of infancy (thi). the association of very low iga levels with atopy has been proposed again in the classic prospective study on the association of viral respiratory infections (vri) and the onset of allergic manifestations, which proved serum iga levels at the lowest normal levels in the children studied [173] . this data has been confirmed within primary immunodeficiencies (pids), once considered to be very rare, are now increasingly recognized because of growing knowledge in the immunological field and the availability of more sophisticated diagnostic techniques and therapeutic modalities [161] . however in a database of >120,000 inpatients of a general hospital for conditions suggestive of id 59 patients were tested, and an undiagnosed pid was found in 17 (29%) of the subjects tested [107] . the publication of the first case of agammaglobulinemia by bruton in 1952 [60] demonstrated that the pid diagnosis is first done in the laboratory. however, pids require specialized immunological centers for diagnosis and management [33] . a large body of epidemiological evidence supports the hypothesis of the existence of a close etiopathogenetic relation between pid and atopy [73] . in particular, an elevated frequency of asthma, food allergy (fa), atopic dermatitis and enteric pathologies can be found in various pids. in addition we will discuss another subject that is certainly of interest: the pseudo-immunodepressed child with recurrent respiratory infections (rris), an event that often requires medical intervention and that very often leads to the suspicion that it involves antibody deficiencies [149] . in several pids (table 22 .1) [61, 65, 76, 101, 162, 351, 414, 480, 514, 543] , atopic symptoms are present: gastroenteric and rhinitis in selective iga deficiency (sigad), severe ad in wiskott-aldrich syndrome (was) and hyper-ige syndrome (higes), in which it spreads over the entire body, and to which other allergic manifestations can also be associated such as asthma, rhinitis and angioedema. various acquisitions indicate that pid is also an opsonization deficiency, observed in 5% of the normal population [469] . in this disease, microorganism phagocytosis by polymorphonuclear (pmn) leukocytes appears annulled, and the patient is subject to severe infections supported by capsular bacteria: the deficiency, described in association with severe and recurrent infantile infections [175, 485, 487] , depends on the lack of mannose-binding lectin (mbl) [487] , its primary immunodeficiencies a possible atopy dependence on iga underproduction rather than on ige hyperproduction ( fig. 4.1 ): in children with levels of iga at the minimum normal level, and followed from birth until the age of 18-23 months, a greater severity of atopic manifestations and an increased cumulative incidence of asthma, ad and otitis media with effusion (ome) were observed compared to controls. the close links between id and atopy are confirmed by symptoms similar to ad present in some forms of was (70%), higes (85%), xla (x-linked agammaglobulinemia) or autosomal recessive (ar), ataxia-telang1268 chapter 22 primary immunodeficiencies proposed that in these patients cd4-th2 levels are sufficient for modulating ige synthesis, but cd8 t-cell levels are inadequate for inhibiting ige synthesis, which results in increased ige synthesis. this hypothesis is supported by the observation that omenn syndrome, was and especially higes, with an immunological phenotype characterized by a quantitative and qualitative reduction of cd8 t cells, are accompanied by extremely high levels of serum ige [61, 162, 196] . lymphocytes in subjects with normal levels of ige are incapable of producing them, not even after stimulation with polyclonal activators such as pwm (pokeweed mitogen) or ebv (epstein-barr virus), while patients with high antibody levels spontaneously synthesize in culture sige (specific) levels between 200 and 2,000 pg/ml, also releasing factors capable of increasing ige secretion (ige-pf) [287] . supernatant derivatives from the t cells of patients with higes are in fact capable of inducing in vitro the pre-b cells to increase ige production; furthermore, when the t lymphocytes in these patients are isolated on the basis of receptors for the ige fc fragment, the remaining cells release ige-pf [287] . considering the suppressive activity of human lymphocytes with cd8 phenotype on sige, it has been observed that these lymphocytes are able to suppress sige synthesis in patients with high antibody levels; similarly cd8 + cells from a bone marrow transplant (bmt) can suppress ige production in the hla-compatible recipient [478] . the study of patients with id associated with hyper-ige has supplied useful information concerning ige system biology, although the immune defect essentially responsible for ige increased production and for severe atopic iectasia (ata), thymic alymphoplasia, scid (severe combined id) (48%) [44] and, occasionally, by digeorge syndrome (dgs), id with hyper-igm (higms) now cd154/cd40l deficiency, selective igm deficiency, biotin-dependent carboxylase deficiency, cgd (chronic granulomatous disease), primary neutropenia, and in netherton, nezelof, omenn and shwachman syndromes [434] . other forms, in addition to those discussed, are associated with gastrointestinal symptoms: diarrhea and malabsorption of xla and thi, diarrhea in was and dgs, food-related allergies (43%) in sigad and also an elevated frequency of asthma [36] .among secondary id, only aids is associated with ad (chap. 23). the association between a deficiency of t cells and high levels of ige, observed in patients with higes, nezelof syndrome, ata, was and other diseases, has been known for some time (table 22. 2) [62] . experimental studies on animals indicate that there may be an inverse correlation between serum ige levels and t-cell functions: this could be attributed to a t-lymphocyte deficiency in atopics, genetically determined, which makes them more vulnerable to the camp inhibiting activity, and consequently causing an imbalance between the two subclasses of t cells, which could lead to ige hyperproduction and atopy development; however, in no case is there evidence of a relationship between cd8 deficiency, ige levels and allergic symptoms. it has been 1269 serum ige concentrations 54 7 chronic granulomatous disease 10 6 m-17 y <1-3, 160 88 hyper -ige syndrome 11 3-31 y 3150-40,000 11,305 nezelof syndrome 3 8 m-3 y 5-7,000 55 non-x-linked agammaglobulinemia 15 6-35 y 1-10 3 other variable immunodeficiency normal infants and adults 106 2-55 y 2549 55 data from reference [62] . m months, y years, gm geometric mean. manifestations has not yet been identified. the most interesting syndromes from this point of view are the three syndromes analyzed above, characterized by common clinical indications such as early ad onset, increased susceptibility to all varieties of pathogens, as well as an exceptionally high ige serum level [162, 394] ( table 22. 2). several pids have autoimmune features, including chédiak-higashi syndrome, cgd, complement deficiencies c1q, c1r, c1s, c2, c4, griscelli syndrome, higms (cd154 deficiency), lad (leukocyte adhesion deficiency), hla class i deficiency, hla class ii deficiency, omenn syndrome, was, and xlp (17) , which will be dealt with subsequently. in 25 children with a mean age of 44 months, autoimmunity was chronic and severe requiring prolonged immunosuppression, however with no spontaneous remission of such manifestations [44] . definition pids ( fig. 22 .1) [413] consist of a heterogeneous spectrum of congenital, individual and combined anomalies of the immune system (humoral deficiencies, combined deficiency of b and t cells, the complement, phagocytes, neutrophils, etc.), as well as syndromes and diseases associated with id that are traditionally classified as pids. the updated classification (table 22 .1) has divided ª100 pids into six main groups, also including secondary id with infections (first among them all aids) that cause deficiency and immunosuppression [414] . the classifications of pids is based on characteristic clinical features and specific alterations in immune status. advances in molecular genetics now make it possible to complete the table according to the types of genetically altered molecules involved [63] . to complete this data, see table 22.1 and table 22 .3 [236, 246, 407, 453] showing the behavior of antibodies and circulating b and t cells. pids occur infrequently and are highly heterogeneous in nature, relatively few centers gain extensive experience in the diagnosis, so it is difficult to estimate the prevalence of these disorders from routinely collected health statistics [33] . studies in 13 countries on all continents have included 10,895 patients: tables 22.4 and data concerning incidence has increased considerably thanks to a greater availability of specific tests and more widespread knowledge in the medical profession related to these pids, including ata [94] . however, because 1271 primary immunodeficiencies [236, 453] ; other data from [246] (omenn syndrome) and [408] (jak3). ada adenosine deaminase, id immunodeficiencies, jak janus-family kinase, pnp purine nucleoside phosphorylase, ø decreased, øø markedly decreased, ≠ increased, -absent, + present, n normal. a progressive. b not functional. predominantly t-cell defects 3 the total may not correspond to the sum of the cases because it may include some pid with very low incidences. the figures should be divided into the years that were considered. a incidence × 10 6 live births; the thi figure includes probable cases. thi 14 60 11 selective igg subclass deficiency 10 39 autosomal hyper-igm syndrome 3 34 2 selective antibody deficiency with normal igs 4 20 10 cellular and antibody id syndromes associated with other major defects 20 ata 7 149 12 wasp syndrome 31 34 2 digeorge anomaly 1 18 6 hie 4 63 6 nijmegen anomaly 1 immunodeficiency associated with or secondary granulocyte dysfunctions 9 defects of phagocyte number and function cgd 14 85 3 cyclic neutropenia 1 11 1 kostmann's syndrome 4 14 schwachman syndrome 1 complement deficiency -undefined 1 total pid 166 1, 428 122 time period 15 years 20 years 11/1983-12/1999 latin america includes eight countries. xla x-linked agammaglobulinemia, cvid common variable immune deficiency, thi transient hypogammaglobulinemia of infancy, scid severe combined id, was wiskott-aldrich syndrome, ata ataxia-telangiectasia, cgd chronic granulomatous disease. to be reliable and can also be used for other recessive x-linked pids to identify cell lines with genetic defects, as is the case of was [537] . one must, however, properly consider the phenomenon of mutations, that can render useless the inactivation method, as has been proved in was, in xla and also in scid, in which the mutation is not in the maternal t cells but in the germlines [389] . the main clinical aspects of humoral and cellular pid are schematized in table 22 .6 [80] ; further in numerous pids there is a deficiency of chemotaxis (table 1 .65) as in cgd [416] . in antibody deficiencies, current treatment, while waiting for genetic treatment to become available, complicated in xla by several btk mutations, is based on the prophylactic administration of ivig, combined with quick antibiotic treatment during infectious episodes. the genes responsible for id linked to chromosome x have been recently mapped on the respective chromosome bands (fig. 22. 2): the bands on the short limb are designated "p" and those on the long limb "q" (table 22 .1). this was possible thanks to the refinement of dna recombinant technology (rdna), including dna probes (sequences of radio-marked dna) and restriction fragment length polymorphism (rflp). the closer the gene segregates to rflp, the lower the chance that they might be separated by recombination phenomena when meiosis occurs: the identification of deficient genes allows early diagnosis, even prenatal, and if necessary gene therapy or bmts [76] . furthermore, the observation that numerous pids are transmitted with an x-linked modality allows a relatively simple diagnosis of males with a positive family history (fh); if fh is negative (40%-50% of xla cases) or there are females presenting a clinical pattern of pid, or when sporadic cases are caused by a new mutation, carrier identification is based on the study of immunologically normal female carriers, with two populations of b precursors, using x-chromosome inactivation analysis. this test does not take into account the existence of possible gene mutations and the availability of already affected relatives, and it is also relatively simple and fast [537] . molecular studies follow the hypothesis that, at an early stage during embryogenesis, one of the two x chromosomes is randomly inactivated in the cells of all tissues of female embryos (persisting as barr's chromatin) [300] . therefore in normal conditions, one has a cell mosaic that actively expresses for 50% the paternal x chromosome and for the remaining 50% the maternal x chromosome (lionization) [300] (fig. 22 .3a) [355] . in female carriers of xla, the cell mosaic expresses 50% for an x chromosome with btk in an active form and the remaining 50% for an x with a mutated btk (bruton tyrosine kinase). this means that in the carrier mother it is inactivated in preference to the x chromosome carrier of the defective gene in b, which matures therefore in an unbalanced manner (not randomly), while in all other cells activation occurs randomly. it follows that in fixed carriers only the b lymphocytes that have the x carrier of a normal gene complete the differentiating route, while the precursors that express the x chromosome with a mutated btk do not mature into b cells, but remain blocked [100, 413] (fig. 22.3b) . in x-scid, the study of fixed carriers follows the corrected lyon hypothesis, because the cells with a normal active x develop into normal t lymphocytes; however, when t precursors with a mutant x reach the stage where the x is needed, they do not find it and consequently do not develop: thus female carriers have only one normal and active x, instead of the random mixture of cells with one of the two active x [389] . the inactivation test appears predominantly b-cell immunodeficiency inherited in an x-linked trait, only 50% of males have a fh positive for pid; female cases are also known, supporting an ar trait [100] . classically affected subjects present levels of igg at <100 mg/dl, with very low circulating iga, igm and b cells (table 22. 3), in which are found, in addition to bm, pre-b lymphocytes in an almost normal quantity [100] . xla is characterized by a blocking of b-cell differentiation that results in an arrest of the evolution of pre-b1a cells: low levels of cytoplasmic igm and high levels of surrogate light (l) chains (cd179b) into later-stage b cells [348] .the b-cell differentiation arrest in the majority of xla patients appears to be homogeneous, with approximately 80% of the pro b-cell compartment being negative for cytoplasmic igm expression [349] . the size and nature of the residual more mature b-cell population (leakiness) varied among patients, independent of the type of btk mutation. further, it appears that the pro b-cell compartment composition in bone marrow (bm) of some xla patients can be influenced by low levels of wild-type btk mrna [349] . on the contrary, t cells are normal both in function and in number, as is thymus architecture, including hassall's corpuscles and thymus-dependent areas of spleen and lymph nodes. b lymphocyte zones are typically depleted, with an absence of gcs, plasma cells, and cortical and medullar differentiation compared to normal (figs. 22.4 and 22.5) and an absence of adenoidal tissue (fig. 22.6 ). the intestinal lamina shows a similar deficiency [227] , even if both b and t cells use the same recombination (chap. 1). in the bm, increased pre-b lymphocytes without cd19 and cd21 can be observed. the pre-b cells are capable of transcribing and translating microgram intracytoplasmic (ic) h chains, but not the l chains [453] , thus pre-b only form microgram chains not associated with v h , while only 5% of normal cells produce incomplete chains [278] . experimental data currently indicate that the defect lies in the xla gene mutations that codify for btk [505, 513] . the xla gene is expressed by b cells during differentiation, but is not transcribed in the t cells, thereby explaining the b lymphocyte maturative block at the pre-b level [61] (fig. 2.6 ), immediately after their appearance in the bm [302] . xla, however, presents a genetic heterogeneity, explained by mutations in the 5 btk domain (ph, th, sh3, sh2, kinase), with a frequency proportional to the pertinent domain dimensions [514] . the mutation size was ascertained by finding 175 mutations in 236 patients ( fig. 22.7) . equally, genes codifying for marker proteins and receptors that are essential for b-cell maturation and development are also involved: in fact many of these proteins, including btk, hm chains and surface proteins are crucial for b-cell differentiation [395] . studying chil-dren of both sexes with xla, various mutations of hm germline have been identified, in addition to deletions affecting the d, j h and cm genes and other gene alterations capable of blocking h chain synthesis on b lymphocytes [548] . an equivalent molecular defect was observed in an infant girl with xla, with differentiation block preceding the ig gene rearrangements by early pre-b cells [316] . there are also the so-called leaky forms, with absent or few b cells and various antibody deficiencies [240] , which can be attributed to individual mutations of btk [267] , for example in the non-kinase domain, which permits the expression of normal btk levels [427] . however, btk mutations can be even more detrimental for b lymphocyte proliferation compared to the total kinase absence [370] . xla is clinically characterized from its onset in male babies, at 5-6 months of life (but also at the end of the 1st year), when the maternal igg passive protection ceases. it usually attracts attention due to delayed growth and mostly recurrent and severe bacterial infections dominate (sinusitis, otitis, bronchitis, [476] . although representing the phenotypic picture of humoral id, confirmed as a separate deficiency [459] , xla associated with ghd is mapped in the same region as the x chromosome of the isolated xla. the observation of hz deletion of one or more c h genes for the h chains of ig in 5%-10% of normal patients has led to the identification of various polygenic deletions concerning the genes of one or more isotypes and subclasses [282, 414] . some subjects are lacking in genes of all or some igg subclasses, associated with iga 1 and ige deficiency, with no clinical symptoms in 94% of cases [282, 414] . in italy these deletions have a frequency of 2.7% and the expected frequency in hzs is of 1:1,400 [385] . only some of the families that produce l chains and not the k chains are known. in one family the molecular bases of the deficiency were ascribable to two different punctiform mutations, one in each cκ allele that prevented the formation of -s-s bridges between the k and h chains. the k:l ratio in human ig is 2:1, and the relative alterations can be observed in numerous primary or secondary ids [414] . only one patient is known with an l chain deficiency, hgg and rri (upper and lower respiratory tract) [508] . [61, 79, 302] . rotavirus and echo viruses also cause severe meningoencephalitis in 5%-15% of patients [100] . phenotypic variability may occasionally be present, as in a family spanning three generations [332] . in 33 patients with a median age of 9.4 years the median age at the xla onset was 8 months and the median age of diagnosis was 4 years, with a median diagnosis delay of 33 months. the common infectious diseases were pneumonia, otitis, diarrhea, sinusitis, and arthritis. the most common chronic infections were seen in 75.8% of the patients: in the respiratory tract in 93.9%, in the gastrointestinal tract in 75.8%, in the central nervous system (cns) in 33.3%, and in the musculoskeletal system in 21.2% of patients [324] . bronchiectasis, malabsorption, arthritis, autoimmune and tumor-related diseases are the most common complications, as well as edema, contractures, etc. (fig. 22 .8). one must predict the onset of bronchiectasis and intervene quickly with specific physiotherapy, because forms that are initially localized later spread, causing respiratory failure in older children and adolescents. one-third of all cases start with mono-or rheumatoid arthritis (ra) caused by ureaplasma urealyticum with a sterile exudate, which usually regresses following treatment with ivig [355] .anti-polio vaccinations with live attenuated viruses should be forbidden, because they can cause very severe pneumonia [278] . about ten cases of xla associated with ghd are known. in addition to reduced growth, clinical symptoms are typical of xla, though it is not a variant, sporadic cases have been described, occasionally associated with sigad (10%-20%) and more often with ata (80%) and susceptibility to infections [227] or without rris [385] , differentiating patients with probable pid from those with low levels of igg 2 (table 22 .7) [387] , in whom it may represent delayed maturation [450] . selective deficiency of igg subclasses presents three different aspects: ∑ total lack of a subclass ∑ two sd levels below average ∑ inability to produce antibodies relative to the subclass in question, even when hematic concentrations are normal [465] the following selective deficiencies are present [283, 465] : ∑ isolated igg 1 : deficiency in only a few cases has been described, also because this subclass represents 60%-70% of all iggs (the others account for 25% [igg 2 ], 6% [igg 3 ] and 3% [igg 4 ]), its absence is very probably an indication of an evident hgg; these patients usually have a reduced level of total iggs and react normally to antigens with a polysaccharide capsule. ∑ other combined id (cid): for example igg 2 +igg 4 , igg 2 +igg 3 , igg 1 +igg 2 +igg 4 , igg 1 +igg 2 , igg 2 +igg 3 + igg 4 , some of which are associated with a deficiency of iga or its subclasses [153, 465] . the association of sigad and defects of igg subclasses is explainable in view of ig production ontogenesis by b cells: one starts with igm, moving on to igd, and then to igg ending up with iga passing by ige [296] ; therefore the deficiency could originate with an immunological defect involving the t lymphocyte regulating work or b cells secreting ig, with an effect on the final stages of their production. in rare cases, more or less extended deletions in chromosome 14 have been observed, in the region that codifies the h chains; in most cases the genome is instead intact, confirming a possible defect in b lymphocyte switching [367] . very rarely this deficiency depends on gene hz deletions [227] . this pid is probably the most common of all (tables 22.4, 22 .5), especially in nonselected populations of caucasian origin [367] . it is defined by the presence of a serum level of iga <5 mg/dl and the absence of siga in the total deficiency; in the partial sigad levels are <5 mg/dl but <2 sd compared to normal levels for age, with measurable siga [367] . in total deficiencies, igm and igg levels can be normal [414] (table 1. 15), but igg 2 and igg 4 levels are low [153] . in several cases, the partial deficiency is transient [383] , returning to normal levels of iga in 50% of cases by the age of 14 and in 80% by 18 ( fig. 22.9 ) [383] . the sigad is transmitted sporadically; however, cases of multifactorial and dominant ar, with a variable or incomplete expression [105, 536] transmitted within the same family have been reported. functional alterations reflect on the final maturing process of b lymphocytes, given that about 80% of b iga + lymphocytes show an igm+igd+iga+ membrane phenotype, a normal aspect only in newborn babies [103] . studies on chromosome 18 have not led to conclusive results, because deletions in children with sigad are associated with mental retardation, facial dysmorphisms, failure to thrive, etc. an association with hla haplotypes situated on chromosome 6 is instead more consistent, and common in patients with cvid. interesting indications for understanding the pathogenesis come from molecular genetic studies that have allowed the formulation of a hypothesis of multifactorial origin, given that the combinations of more widely involved hla haplotypes and extended haplotypes involve the class i-iii genes, to the extent that they are more often encountered in the general population [105] .among the class iii alleles the most studied is the gene that dictates the c4a, among class i and ii the most common are a1, a28, b8, b13, b40, cw6, dr1, dr3, dr7, dqw1, associated with haplotypes such as a1, b8, dr3; b13, dr7; a1, b14 or a28, b14. other haplotypes are extended, such as b8/sco1/dr3, bw65/sc2 [1, 2] /dr1, bw57/sc61/dr7 and b44f/fc31/dr7, the first of which is increased in patients with a combined iga, igg 2 and ige deficiency [170, 313, 536] . the association with dr3 gives sigad a risk factor of 13 (table 18. 3). one hla supertype is also found in deficiencies of 21-hydroxylase with a late onset, suggesting an important locus for iga differentiation close to class iii hla genes [99] . it has been thought that to induce sigad a non-hla gene or an environmental penetration factor might be necessary, due to the possible sigad discordant expression in hla-identical twins [536] . however, the analyzed sequence of involved alleles showed a significant sigad correlation with some alleles belonging to hla-dq locus, composed of a protective allele with aspartic acid and a susceptible one with valine or alanine in the b chain in position 57 [358] . we have observed that the presence of aspartic acid ensures protection in dm. furthermore, the immunological deregulation extends to the typical formation of auto-antibodies and characterized by the presence of igg anti-iga [61, 196] . in these subjects, there is a wide symptom range, mostly represented by rris, allergic and autoimmune diseases (aids), among which is diabetes. allergic diseases are twice as common in partial deficiencies, unlike aids [507] . from a clinical point of view, the frequency of chronic diarrhea and malabsorption, associated with celiac disorders and giardia lamblia infestation are not surprising, considering siga's prominent iga 1 , igg 2 , igg 4 and ige due to deletion of ig h chain constant region genes were associated with undue susceptibility to infection [384] (see "rri"). a few cases of selective igm deficiency are known, associated with rris and various other symptoms [414] . igm deficiency was detected in four children with rris. isolated igm defect was present in two children, and two more children had an associated igg 3 subclass deficiency [160] . cvid includes a heterogeneous group of unhealthy conditions that have in common hgg and rris; it has an incidence of between 1:50,000 and 1:200,000 [414] . the inheritance of two susceptibility genes within the hla on the short arm of chromosome 6: one located near the class ii region and the other near the junction between the class iii and class i regions is a serious risk for the development of cvid [441] . there are autosomal dominant or ar forms also linked to sex; sporadic cases are the most common [413] . the molecular bases are not totally clarified as yet: the pathogenetic mechanisms may depend on b lymphocyte (80% of patients) and t lymphocyte (20%) defects [227] . the b-cell intrinsic defect is attributable to an alteration of the differentiating line at different stages of maturation, resulting in a poor formation of antibodies, with hgg of variable degrees, while in patients with xla the circulating b lymphocytes are virtually absent. the iggs are <500 mg/dl (with a reduction in all the subclasses: a normal phenotype is observed in only 14% of patients) [367] . more often there is a hierarchical order in the shortage: igg 3 < igg 1 < igg 2 < igg 4 [528] . iga and igm antibodies are <5-50 mg/dl [102] , reflecting the potential cd154 underexpression, implying an activation deficiency [150] or a t-b cooperation defect [232] . a study of t lymphocyte subpopulations indicates various subgroups of patients: 60% have t cells with scarce il 2 , il 4 and il 5 levels, while 30% have a reduced cd4:cd8 ratio, with an increase in cd8 bearing the cd57 marker, which suppresses igg production, elaborates normal il 2 levels and increases ifn-g production [232] . it can also accompany a deficiency of interleukins (ils: il 10 , ifn-g), suggesting a defect in the signaling mechanisms based on the tcr/cd3 [191] . a t-lymphocyte deficiency is therefore difficult to evaluate [527] , also because this could be a vri effect [232] . cvid can also be observed following congenital rubella or ebv infections; it can also be induced by some drugs such as phenytoin [355] . in 2/9 cvid families, 5 subjects were identified with identical large mutations in the icos (inducible costimulator) gene, expressed on the surface role in the formation of a barrier against the penetration of polypeptidic macromolecules through the intestinal mucosa. sigad therefore facilitates the penetration of food antigens through the mucosa followed by the formation of specific antibodies. for example, 50% of patients present cics and precipitins to cm, 23% to bovine anti-serum and 13% to anti-serum of calf fetus [106] . symptoms affecting the respiratory tract are also caused by the absence of siga, as in 30/36 children aged 1-15 with increased susceptibility to rris [327] . patients balance the siga deficiency with the sigm, but in some cases the compensation is insufficient for exempting them from rris and asthma [227] . sigad should be diagnosed on the basis of both serum and secretory iga, because normal levels in adults are achieved at different times (table 1. 15) . some drugs such as phenytoin (an anticonvulsant) can determine sigad, sometimes persisting in time after the drug has been discontinued. the clinical symptoms in these cases are not different from those of patients with sigad [507] . there is no random treatment; these patients do not benefit from therapy with ivig, even when enriched in iga. there are no counter-indications for obligatory and optional vaccinations [507] . whole blood or plasma transfusions containing iga can sensitize patients or cause anaphylactic shock in those already sensitized [105] . life expectancy is excellent; however, the random discovery of sigad in asymptomatic children should not be underestimated. they should in fact undergo periodic clinical and laboratory controls so as to identify as early as possible any possible pertinent symptoms. at the same time, there is the need to ensure a good life quality with adequate prevention of rris in those patients whose respiratory tract is affected [383] . sadni translates into the inability to respond to certain antigens, especially if polysaccharide. while some individuals are normal, others contract sinopulmonary infections. the reduction of igg 2 levels is more of an associative relationship than a random one; igg 2 levels, on the other hand, do not predict antibody responses. subjects who do not respond to anti-hepatitis vaccination may fall into this category [414] . in one retrospective survey at a pediatric tertiary care center, sadni was the most frequent diagnosis, accounting for 23% of id diagnoses [236] . there are cases of patients in good health, without ige due to gene deletion [62] . in two siblings, deficiency of of activated t cells, which interacts with the icos ligand gene expressed on b cells. an additional 181 patients with sporadic cvid were examined, and no mutations were found. only 9 in 226 patients with cvid screened thus far (<4%) have been found to have icos mutations. one unexplained feature of cvid is that the onset of clinical symptoms does not occur until late childhood or adulthood [429] . pid is variable either in the clinical and immunological pattern, or in the onset period, more common during the school years or in adults, but also between the ages of 1 and 3 [102] . the acute bacterial recurrent and/or severe lower respiratory tract infections (lrti) are characteristic: sinusitis (60% of pediatric cases), otitis media (47%), bronchitis, pneumonia (87%) and/or digestive tract infections (diarrhea 57%) [213] . the prevalence of infections caused by mycetes has increased as well as cases of pneumonia caused by pneumocystis carinii, a signal for cell-mediated immunity (cmi) [152] . the gastroenteric tract is dominated by symptoms similar to those seen in celiac disease, with generalized malabsorption, steatorrhea, lactose intolerance, protein-losing enteropathy, inflammatory bowel disease (ibd), saccharidase deficiency and malabsorption of vitamin b 12 and folic acid, supported also in this case by intestinal infestation caused by giardia lamblia [527] . the tumor necrosis factor receptor family (tnfr) member taci (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) mediates isotype switching in b cells. in 4/19 unrelated subjects with cvid and 1/16 subjects with sigad there was a missense mutation in one allele of tnfrsf13b (encoding taci). none of these mutations were present in 50 healthy subjects. tnfrsf13b mutations cosegregated with the phenotype of cvid or sigad in family members of the 4 index subjects. b cells from subjects with taci mutations expressed taci but did not produce igg and iga in response to the taci ligand april (a proliferation-inducing ligand), probably reflecting impaired isotype switching [87] . other characteristics are hemopathies, hepatosplenomegaly, autoimmune hemolytic anemia (aiha) and x-linked lymphoproliferative disease (xlp), and cutaneous and internal organ granulomas (which differentiate it from xla), in particular ra, thrombocytopenia, and neutropenia [102] . offspring of cvid patients are at risk throughout their lives for cvid development and should be monitored with a high index of suspicion [441] . based on experimental evidence, it has been hypothesized that iga-and cvid-associated deficiencies may be the extreme opposites of one clinical spectrum: there is a block of b-cell differentiation, different only in the isotype involved. both defects often appear in different members of the same family groups and more or less the same alleles are present [313] . the most accredited hypothesis is that a number of extended haplotypes of the hla system are shared, to which gene duplications, deletions and polymorphisms codifying for some class ii and iii alleles correspond [22] . in fact, a number of common hla haplotypes, especially belonging to class iii, are observed in patients, and at least two haplotypes in 77% of cases [518] , such as hla-dqb1*0201, hla-dr3, c4b-sf, c4a, g11-15, bf-0.4, c2a, hsp-70-7.5, tnfa-5, hla-b8 and hla-a1, postulating therefore the existence of a common genetic basis [22] , with a susceptible gene (6p21.3) possibly the association marker [61] . for example in five members of a large family with one of the two pids, duplications of the c4 genes were associated with a selected group of hla class ii and iii genes [22] . the fact that four members without pid also had these haplotypes indicates that their presence alone is not sufficient for expressing pid, leaving room therefore for other factors [22] such as overlapping relations with celiac disease. the analysis of linked genes has confirmed a strong association with locus 4a, suggesting that an important role in both pid is played by the gene codifying c4a or an adjacent one [162] . see "x-linked hyper-igm (or hyper-igd) or cd154 deficiency (xhigms)" for further discussion. unlike transient hgg that occurs when the maternal iggs gradually disappear from circulation (table 1 .15), in the original study by taylor et al iga levels had fallen, becoming regular in newborn babies with thi after 1 year, corresponding to the nonatopic levels [493] . it consists therefore of a pathological delay in the normal antibody production maturing process. walker et al have calculated that the prevalence of thi is 23 × 10 6 in children, equal to the prevalence of symptomatic sigad (24 × 10 6 ) [525] . in all 15 children the igg and in 12/15 (80%) the iga were <5th percentile, 9/15 (60%) had igm levels <20th percentile resolved around the 22nd month; further confirmation consisted in the fact that the 12 children had symptoms either of ad or of fa or food intolerance [525] . from table 22 .4 the mean incidence is from 1 to 61 × 10 6 . during 7 years 30 children aged 6-46 months were diagnosed with thi and an incidence of 4.3/year [124] . in other studies the main defect was in the igg: in one it had normalized between 18 and 40 months [129] , in another trial 13/247 babies (5.3%) exhibited at the age of 10 months an absence of serum igg levels and of specific antibodies to viral agents, which in eight children were detected before the serum igg levels returned to normal, whereas in basis is an il 2 r deficiency [529] , more precisely of the g receptor mapped on chromosome xq13 [347] . the sole deficiency of il 2 r is not sufficient for producing an immunological phenotype as devastating as scid [529] . because the g chain of il 2 r (il 2 rg), a shared component of il 4 r, il 7 r, il 9 r, il 13 r, il 15 r, il 21 r and il 23 r [351] , gc mutations interfering with its link to the ils deprive the lymphoid progenitor cells of the crucial signals for normal lymphocyte intrathymic development [424] . mutations in any of the genes: il 2 rg, il 7 ra, jak3, artemis, rag1, rag2, cd38, ada, cd45 cause scid [68, 69, 211, 247, 272, 347, 350, 365, 391, 424, 443] . a total of 264 il 2 rγ gene mutations have been sequenced, of which 169 are unique [341] . each of these mutations has resulted in γc deficiency with varying degrees of id. the mutations are distributed throughout the eight exons of the gene, as well as in the regions necessary for proper transcription and translation. the penetrance of each of the above il 2 rg mutations is unknown. exons 5 and 7 have mutation hot spots. the types of mutations identified include missense, nonsense, insertions, deletions, splice mutations, and mutations that affect rna processing and translation [341]. among 93 mutations in 136 patients, the most numerous (67%) are punctiform mutations (fig. 22 .11) plus one missense related to amino acid residues [390] , with a lack of jak3 and gc interactions [424] . in an atypical form, the substitution with residual cysteine of the arginine at position 115 appears to be decisive for gc chain expression, probably a mutation reversion at the basis of the molecular defect, with a numeric and functional t-cell normalization [475] . the mutations, by inactivating the common g chain, render the t cells of boys with scid-x1 unresponsive to several ils. the result is a block in t-cell development and a severe deficiency of mature t cells. b cells, although pre-two children normal igg levels were detected even before the appearance of specific viral antibodies. igg levels usually normalize at between 15 and 36 months [78] , at the age of 2 years (fig. 22 .10) or before 36 months of age in 33/40 children; however, 7/40 still had low ig levels at 40-57 months of age [253] . at 27 was in 9/30 children ig levels were still <2 sd for age and in 5/9 various igg subclass deficiencies were detected [124] . a prospective study with an 8-year follow-up found that igg and iga deficiency is normalized by the age of 6, but in a minority of cases this may be a prodrome of sigad or another humoral deficiency [315] . a study with a 10-year follow-up of 35 children with igg deficiency as well as iga deficiency in 34% of the cases, observed multiform clinical symptoms. since thi can gradually normalize, some children have low antibody titers, and others low igg levels. however, both groups experienced significant infections [110] . in some cases, thi is asymptomatic; in others infections, especially of the respiratory tract, are present. the designation of thi may be a misnomer, and an alternative designation could be added to thi such as "with recovery" or "with development of other dysgammaglobulinemia" [315] . general characteristics of combined t-cell/b-cell immunodeficiency are summarized in table 22 .8 [453] . t -b + scid is a heterogeneous group with an incidence of between 1:50,000 and 1:75,000 livebirths [509] . xlinked fh is positive in 53% of cases [68] . the genetic sent in normal or even increased numbers than in other forms of scid, are dysfunctional [347] . b cells do not mature or produce antibodies due to a complete b-cell differentiation arrest at the pre-bcr checkpoint, showing the absence of complete vdj recombination [350] . other forms are also known with an attenuated phenotype and a partial t-cell function [162] . typical scid-x1 represents the most common form, with 45.5% of cases [68] (5.5% in tables 22.4, 22.5) . in the thymus, there is a severe hypocellularity, without lymphocytes and hassall's bodies where thymic epithelial cells predominate without grossly evident corticomedullary differentiation (fig. 22.12 ). severe lymphopenia is often associated with eosinophilia; nk cells are within the norm or rare. the majority of in-fants with scid-x1 lack both t and nk cells (t -b + nkphenotype) [68, 391] . cd3 t cells, if present, are of maternal origin, because the block, as also in scid ar, occurs at the level of cd4 -, cd8 -, cd44 -, cd3 + and cd1a + ; developing t cells and cd83 + thymic dc are reduced >50-fold when compared to age-and gendermatched control thymus [209] (fig. 2. 2, pre-t, tn), thus scid t -b + . the study of other subpopulations distinguishes the scid subtypes: t cells are reduced in all variants, the absolute cord blood (cb) number is 158-2,400 lymphocytes/mm 3 (tables 1.34, 1.35, so that any count below 4,000/mm 3 is lymphopenic). moreover, in ada deficiency (adenosine-deaminase) there is a maximum reduction in total lymphocytes, in scid-x1 and in jak3 defi-1284 chapter 22 primary immunodeficiencies alies) and against a common t and nk cell drop [68] . furthermore, in two cohorts [68, 474] the affected females had the same phenotype, indicating a possible complex molecular defect [68] . there is no response to delayed spts, and there is an absence of lymphocyte proliferative responses to mitogens and to a specific antigen such as tetanic toxoid [61, 189, 474] . the average age at diagnosis was 4.4 months in 31 children [21] , similar to the ages reported in >200 children with scid from all causes [68, 474] . the male:female ratio is 3:1 and diagnosis is often missed or occurs too late to save the lives of those infected infants who may manifest gvhd early on with morbilliform eruption in the first few days of life due to the transplacentally acquired maternal t cells, intractable diarrhea resulting in a severe malabsorption ( fig. 22 .13), severe interstitial pneumonia (fig. 22. 14), or giant cells caused by anti-measles vaccination or bcg (bacillus calmette guérin), with death caused by chickenpox or infections caused by pneumocystis carinii, herpes, adenovirus, cmv (cytomegalovirus), etc. [63] . the absence of tonsils is observed and also lymphoid tissue [508] and thymus [453] hypoplasia. these children must be transferred urgently to a specialized center and be placed in a sterile room to receive a bmt [474] . on rare occasions, il 2 rγ mutations have caused an atypical mild scid that presented beyond infancy [390] . up-regulation of bcl-2 by an il 2 r lacking il 2 rb tyrosine residues leads to increased cell survival after il deprivation; astonishingly, this survival signal does not occur when gc tyrosine residues are absent. thus, if ciency, the number of b cells is the highest and that of nk cells is the lowest (b + >t ->nk -); nk cells on the contrary reach their highest levels in the ar form [68] . in scid there can also be b alymphocytosis [474] . this divergent data is, however, characteristic of t -b + nkmolecular defects (gc, jak3 defects), b + t -nk -(ada deficiency), or t and b (possible recombination anomgc-dependent signals are revealed only in the absence of il 2 rb tyrosine, il 2 r engages at least two distinct signaling pathways to regulate apoptosis and ccl-2 expression [293] . in two clinical series, patients with mutations in il 2 rg represent 28%-45% of all scid cases [68, 474] . children with il 2 rg mutations have lymphopenia in 95% of cases, with total lymphocyte counts <2,000/mm 3 (normal levels, 4,000-13,500/mm 3 ), based on clinical case series [68, 474] . all patients have very low or absent t cells, and approximately 88% have low or absent nk cells [390] . ar mutated genes on autosomal chromosomes have been identified in ada deficiency, jak3 deficiency, and rag1 or rag 2 deficiency [65] . the existence of b-and t-lymphocyte lymphoid precursor differentiated defect is particular, in some cases a rag1 and rag2 mutation, the two genes that activate vdj recombination [443] was observed; however, this rag2 gene function has been questioned [411] since a rag defect is more present in t -b -scid and the omenn syndrome [491] . in babies suffering from scid, there is a marked reduction of t and b lymphocytes (table 22. 3) and all in vivo and in vitro responses are absent. onset and clinical and histological pattern is similar to that of x-scid. the jak3 gene mutation (tables 1.31-1.33) variant has a frequency of 5.9%-7.4% [68, 408] among babies affected by scid and in the absence of t (3±2%) and nk cells (1±1%) [407] . the molecular base is the mutation affecting the jak3, which prevents it from associating with the gc chain and from sending signals to the abovementioned ils [354] and to other marker proteins belonging to the jak-stat complex [301] . at the origin is a lack of t lymphocytes that transform into the scid phenotype [424] . these patients present b + t -nk -: the b (70±12%) with iga equal to 2±2% [407] , and those with x-scid present a defective differentiation, but are capable of producing elevated levels of ige in the absence of other isotypes [354] . this data indicates that gc and jak3 are essential for t-and nk-cell development [407] . the clinical characteristics are identical to those in x-scid, with the difference that the scid-jak3 phenotype is also observed in females (50%) [408] . furthermore, a jak3 deficiency could be an important cause of scid ar and should be considered in all patients with the b + t -nkphenotype, without an x-recessive heritage [68] . in a 6-month scid-x1 infant presenting with a history of recurrent infection and failure to thrive, a novel splice mutation, gc-dependent, was described, characterized by near-normal count of functionally deficient nk cells (b + t -nkcell phenotype). cell surface gc expression was undetectable on nk cells and in trace amounts in the minority of b cells. t cells were absent, igg and iga undetectable, and igm were within the normal range [183] . bmt is not a perfect therapy, because b-cell function developed in 3/9 children, and nk functions normalized in 2/9 children after bmt [408] . the family pedigree shows an inbred family with consanguinity across five generations. two brothers were diagnosed with scid. one, at the age of 4 months, presented with persistent oral thrush, oral ulcers, and failure to thrive. he had no palpable lymph nodes and no thymus shadow on a chest x-ray film. the second was diagnosed soon after birth and the third brother has always been healthy. three other male cousins died in infancy from severe infections consistent with scid; a 4 th cousin presented with oral candidiasis at the age of 2 weeks and failure to thrive. no thymic shadow was detected on chest x-ray film and peripheral blood lymphocytes showed persistent lymphopenia. he had no lymph nodes, failed to reject a skin allograft and did not show an increase in the blood igg and igm antibodies for dtp after three vaccinations. the three affected patients were hz for a caet transition at nucleotide 394 in exon 4, leading to a proline to serine substitution (p132s) in the extracellular domain of il 7 r. the cousins and their parents harbored both wild and mutant alleles. this partial deficiency is sufficient to block t-cell development and lead to a scid phenotype. the fh of severe pid with multiple affected male infants strongly suggested an x-linked inheritance. nevertheless, this family consanguinity is in favor of an ar inheritance [410] . defective il 7 r expression caused in three patients a t -b + nk + scid, indicating that the t-cell defect in scid-x1 resulted from inactivation of il 7 ra signaling. thus il 7 r-mediated signaling is required for t cells but not for nk ontogenes. mutations in the gene for the il 7 r chain on chromosome 5p13 were found in all three patients [391] . these infants resemble those with other types of scid with respect to their susceptibility to infection and the absence of functional t cells and b cells. however, they differ in that their circulating lymphocytes are primarily nk cells. rag1 and rag2 are required for the rearrangement of tcr and bcr genes [343] . half of the patients with t -b -scid had mutations in their rag1 or rag2 genes, thus highlighting the crucial role of these genes in normal v(d)j recombination machinery [443] . rag-thymocytes lack a functional pre-tcr and hence arrest at the cd44-/cd 25+ stage of differentation [491] : without rag1 and rag2, mature ig and tcr genes cannot be assembled, and lymphocyte development is arrested at very early stages [53] . abnormalities of the chest, scapula and iliac bones and short and stumpy limbs [219, 224] . x-ray abnormalities are documented in fig. 22 .17: the absent thymic shadow and a notable cupping and flaring of the ribs' ends (arrows) can be observed, while histological studies of the chondrocostal junctions document their total cellular disorganization (fig. 22.18 ). this deficiency is the object of a great deal of attention because it was the first to be treated using gene therapy [313] . the lack of ada is observed in 14.8% of patients with scid [68] . the ada enzyme catalyzes the conversion of adenosine and deoxyadenosine into inosine and deoxynosine; although ada is found in all cells (cd26 anchors ada to the lymphocyte cell surface (table 1. 2), the deficiency damages above all the immune system [219] . table 22 .9 [219] summarizes the biochemical foundations of this pid. more than 50 ada mutations are known, including >30 amino acid substitutions, deletions and punctiform mutations or anomalies of the gene itself, such as exon 1 deletion and exons 4, 5, 7 and 9-11 mutations with a total of 15, nine of these in patients with ada-scid and six in those with a partial deficiency [219] . an additional 29 mutant alleles have been found (28 missense and 1 single-codon deletion) [21] . adenosine and deoxyadenosine are also apparent suicide inactivators of the enzyme s-adenosylhomocysteine (sah) hydrolase, with consequent accumulation of sah, a powerful inhibitor of virtually all cellular methylation reactions [61] . the accumulation of metabolites, including camp, deoxy-atp and 2'-o-methyladenosine, has a toxic effect on the cells by blocking dna synthesis and dividing and resting t lymphocyte proliferation [61] (fig. 22.15 ). four different clinical phenotypes have been described for ada-deficient subjects (table 22 .10) [219] , which cover a broad spectrum of immunological aberrations, from the complete absence of b and t immunity, indicating scid (85%-90% of patients) (the thymus in fig. 22 .16) to forms with a delayed onset or partial deficiency (10%-15%) [219] . in children, the delay between onset of symptoms and diagnosis has been estimated to average 2 months [474] . if clinical symptoms indicate an early onset, in addition to typical scid symptoms, there are also x-ray pathognomonic skeletal abnormalities of the chondrodysplasia type, especially this very rare ar type of scid was observed for the first time in 1959 in identical twin male infants who exhibited a total lack of both lymphocytes and granulocytes in their peripheral blood and bone marrow. it has a frequency of 1% in cases of scid [68] . the children are symptomatic in 90% of cases within the first days after birth [46] and is usually fatal within the 3rd month of life without a bmt [224] . due to the common stem cell (sc) non-maturation [224] , it is characterized by total block in lymphoid and myeloid precursor differentiation, therefore not only by an extraordinary lymphopenia, but also by a marked cytopenia in all sections (table 22 .11) [474] , in the spleen, in the lymph nodes and in the gastroenteric tract, and a high frequency of severe successive infections [474] . the thymus is always much reduced in volume, no hassall's bodies are seen [224] . seven of the eight infants reported by who with this defect died between 3 and 119 days of age from overwhelming infections; the eighth underwent complete immunological reconstitution from a bmt [543] . an additional three of five children who required two hscts (hematopoietic sct stem-cell transplantation) and received intensive conditioning therapy before haploidentical hsct (matched for 3 of the 6 hla loci) are alive and well an activated phenotype and poor functional capacity [117] . studies involving hla typification and dna polymorphism show that t cells belong to the host, ruling out, therefore, the etiology of maternal cell engraftment [117] , unlike other types of scid [474] . the absence of circulating b is also characteristic [117] , equal to 3.8%-7.1% of normal levels [474] , reaching 0% [520] , high ige levels (526 ui/l), hypereosinophilia reaching 3,000¥10 9 cells/l (normal, 0-0.5 cells/l), and low ig levels at the beginning [246] then declining to the point of agammaglobulinemia [520] , comparable to that in reticular digenesis (table 22 .11) [474] . the marked b-cell depletion can also bear rag1 and rag2 gene missense mutations that decrease the efficiency of vdj recombination, which results in impaired but not absent rearrangement of both bcr and tcr. four missense mutations were detected in the rag-2 in 6/8 patients [491] . in 13/16 patients (81%) the mutations affected the rag1 gene, and in 3/16 (19%) the rag2 gene [515] . increased ige is linked to th2 primary infiltration, with spontaneous production of il 2 ifn-g, il 4 , il 5 and il 10 , which is down-regulated by ifn-g therapy [437] . clonal expansion of vb14 + cd3 + , cd4 -cd8secreting high il 5 levels and low il 4 and ifn-g levels [318] could indicate an analogy with the fas (cd95) defect. t lymphocytes show an activated phenotype and a spontaneous apoptosis associated with reduced expression of bcl-2 gene product, and a higher cell death of cd4 + cd45r0 + cells [59] . given that high cd30 levels in the lymph nodes, skin and serum of three children generated th2 lymphocytes [95] , a th2-mediated pathogenesis is possible: the cd30 are th2 markers (chap. 1). as in human scid, b and t cells are found in mice with scid, but with a final repertoire that is decidedly oligoclonal and lacks the heterogeneity characteristic of a normal immune system, so lymphopenic scid and omenn syndrome could be two aspects of the same disease with different clinical expressions, especially of time [224] . clinically, young babies soon after birth show a generalized exudative erythroderma and desquamation, often mistaken as ad, alopecia, widespread lymphadenopathy, hepatosplenomegaly, persistent and profuse diarrhea, failure to thrive with malnutrition ( fig. 22.19) , aiha, recurrent infections caused by common and opportunistic germs ( fig. 22 .20), and markedly elevated serum ige levels [136, 241, 474, 520] . this outline included four babies from the same family with the same symptoms until death occurred at 10-19 months, but who did not present hypereosinophilia and were diagnosed as dm [375] . differential diagnosis may be challenging since omenn syndrome and gvhd show dyskeratosis and basal vacuolation, but the first always shows acanthosis and usually parakeratosis. gvhd shows a flat epidermis and rarely parakeratosis. both can be distinguished after immunohistochemical staining for cd45 and cd68, which shows predominantly lymphocytes in the dermal infiltrate in omenn syndrome, and relatively more macrophages in gvhd [438] . with myeloid and t-and b-cell lymphoid reconstitution [46] ; another child is alive and well after 32 months [13] . b-scid, characterized by increased cell sensitivity to radiation secondary to mutations of the artemis gene, could carry a poorer prognosis because of defective repair of dna breaks [406] , occurring around the time of bmt, from the effects of chemotherapy, infections, and gvhd [206, 247, 332] . one group of patients with scid with an additional sensitivity to radiation was found to harbor large deletions or truncation mutations in the artemis gene mapped on chromosome 10p [65] , implying a role for artemis in dna double-strand break repair, which is mutated in human scid [350] . omenn syndrome is classified as a scid because newborn babies exhibit symptoms similar to a gvhd, due to 1a antigen expression and cd1a absence [241] , and because it can coexist in families with alymphocytosis [117] . this is an ar syndrome with an unknown pathogenesis, sharing characteristic clinical and immunological abnormalities with t + b -scid [515] . severe cutaneous lesions with hyperkeratosis, apoptotic malpighian necrosis and basal membrane destruction can be associated [241] . no lymphoid cells or hassall bodies are found in the thymus [241] . the immunological structure reveals histiocyte infiltration of the skin, bm and lymph nodes, with proliferation of t infiltrating the epidermis and the enteric mucosa, increased t cells with 1289 combined t-cell and b-cell deficiency igg (g/l) 7 2.28-6.16 igm (g/l) 0 neutrophils (cells/ml) 200 1,000-8,500 data from [474] . in a child with scid and circulating t cells within the norm, a gene transcription deficiency was ascertained [414] . a male infant of first cousin parentage presented at the age of 6 months with cmv pneumonia, persistent oral and esophageal candidiasis, adenovirus gastro-enteritis, and failure to thrive. he developed lymphadenopathy, hepatosplenomegaly, iron deficiency anemia with no evidence of hemolytic anemia, and chronic inflammation of his lungs and mandible. biopsies showed extensive lymphocytic infiltration of his lung, liver, gut, and bone. serum igg and igm were elevated, but iga was low. he had t-cell lymphocytopenia, with an abnormal cd4:cd8 ratio of 1:1. the t cells responded poorly to anti-cd3, phytohemagglutinin and other mitogens, and to il 2 . he was found to have a truncated mutation of the il 2 ra chain (cd25). he was given a successful allogeneic bmt after cytoreduction [452] . about 225 cases have been reported, [75, 290, 539] , 78% [75] or 100% [539] of which have the x-linked form of cd154 deficiency [75] . patients are generally male, but there can be a non-x-linked form [224] , in which 22% of patients are females [75] . an estimated minimal incidence was calculated of 1 in 1,030,000 live births. over half of 79 patients developed id symptoms and were diagnosed by 1 year of age, and over 90% by 4 years of age [539] .although carriers of xhigms are considered to be asymptomatic, an extreme lyonization of the normal x can lead to a mild expression of the xhigms which is similar to cvid [118] . it can be secondarily caused by environmental factors and also stem from congenital rubella [278] : this indicates its heterogeneity. mutations in the tnfrsf5 encoding cd154 in xhigm patients result in a lack of b-cell signaling by activated t cells [53] . however, 21 boys out of 56 failed to express cd154, and tnfrsf5 mutations were found in 20 of these boys, whereas no tnfrsf5 mutations were found in 16 boys with weak expression of cd154 [184] . as a result, xmigm b cells fail to undergo isotype switching and produce only igm due to a defect in the rna editing enzyme, activation-induced cytidine deaminase (aicda), an enzyme expressed only in b cells and required for the processes of class-switching and somatic hypermutation of ig genes [357] . the marked reduction of igg (<150 mg/dl), ige and iga is accompanied by a sharp increase in mature igm and circulating igd, but b cells do not express other ig [227] . interestingly, 25% of patients with confirmed xhigms who had tnfrsf5 mutations had low concentrations of igg, iga, and igm. most of the remaining patients with xhigms had the classic pattern of normal or raised igm with low concentrations of iga and igg [184] . the cd154 gene defect is usually expressed on the membrane by activated t lymphocytes, which therefore cannot bind b-cell cd40 [266, 346] . figure 22 .21 shows 75 cd154 localizations and mutation frequencies, in 39.5% of cases mistakenly. for example, a sense codon substitutes for a missense one, creates a premature stop signal: therefore specific pertinent mutations, such as g144e, tunistic infections. this disorder is related to mutations in the gene that encodes the nuclear factor kb (nf-kb), which is required for activation of the transcription factor nf-kb, or nemo (nf-kb essential modifier), also known as ikk (inhibitor of b kinase). the phenotype observed in x-higms-eda patients shows that the putative zinc-finger domain of nemo has a regulatory function and demonstrates the definite requirement of cd40-mediated nf-kb activation for b cell ig classswitching [233] . three other genes, expressed by b cells, have been associated with the higm phenotype giving place to higm 2-4. mutations of activation-induced cytidine deaminase (aicda) (higm2) and uracil glycosylase (ung) (higm4), both expressed by follicular b lymphocytes, lead to defective class switch recombination and somatic hypermutation. mutations of cd40, the cd154 receptor, cause a rare autosomal form with a clinical phenotype similar to cd154 deficiency (higm3). these rare pids may shed light on the complex events leading to the production of high-affinity, antigen-specific antibodies of different isotypes [146, 355] . early treatment with ivig associated with antibiotic prophylaxis have reduced the incidence of life-threatening infections and improved the growth of children with higms [290] . cycles of g-csf (granulocytecolony stimulating factor) in the presence of severe neutropenia are advised [290] . substitute therapies with soluble forms of recombinant or gene type cd154 [76] are being studied. a recent review of cd154-deficient patients showed that 75% develop liver disease and only 20% survive into the third decade of life [290] . bmt has a successful outcome in young children (65%); older patients with more ad-can interfere directly with the link site for cd40 ( fig. 22.22 ). consequently the signal which indicates that b cells should begin isotype switching, limited to the production of low-affinity igm, is missing [346] . without isotype switching, gc formation is minimal [508] (fig. 22 .23) and follicular dendritic cells (fdcs) are reduced in number, also having an abnormal phenotype [147] . as shown by figs. 1.31-1.33, the lack of cross-linking of cd40 by cd154 results in b-cell failure to up-regulate cd80 and cd86, important costimulatory molecules that interact with immunoregulatory molecules on t cells such as cd28 and ctla-4. two patients with normal levels of cd154 have also been described [359] . as in males with xla, infections start during the 12th month, those most often observed are otitis, pneumonia or sepsis cased by pyogenic bacteria, opportunistic infections, in particular caused by pneumocystis carinii [290] , and also ulcerative stomatitis, ra, neutropenia, aiha, lymphoproliferating complications and type b gastroenteric lymphomas with igm [224, 413] . the most prominent clinical infections were pneumonia (81% of patients), upper respiratory infections (urti) (49%-87%) including sinusitis (43%) and recurrent otitis (43%), lrti (82.1%) recurrent/protracted diarrhea (34%-55.3%), cns infections (12.5%-14%), sepsis (13%-14.3%), cellulitis (13%), hepatitis (9%-16.3%), and osteomyelitis (1%) [290, 539] . lymphoid tissues are normal or hyperplastic [75] . recently, a rare form of higms associated with hypohydrotic ectodermal dysplasia (eda) characterized by the absence or hypoplasia of hair, teeth, and sweat glands has been described. unlike patients with higms, these patients failed to have a history of oppor purine-nucleoside phosphorylase (pnp) deficiency, ar, for which 35 patients have been reported [53] , is characterized by the absence of an enzyme necessary for the catabolism of purines, which converts inosine, deoxynosine, guanosine and deoxyguanosine into hypoxanthine and guanine ( fig. 22 .15); the responsible gene has been mapped to chromosome 14q at position 13.1 [414] . this has also been observed in 33 patients with nezelof syndrome [304] . a variety of mutations have been found in the pnp gene in patients with pnp deficiency [432] . although ada and pnp are both purine salvage pathway enzymes, pnp deficiency does not lead to as severe an id as ada deficiency. patients have considerably reduced concentrations of serum and urinary uric acid. numbers of t cells fall progressively, more than that of b cells (table 22. 3), just like the proliferating responses to mitogens and antigens, especially because pnp deficiency causes an intracellular accumulation of deoxy-gtp (guanosine triphosphate) inhibiting ribonucleotide-reductase and t-and b-lymphocyte proliferation, so combined t and b defects are critical. pnpdeficient patients are as profoundly lymphopenic as those with ada deficiency, with absolute lymphocyte counts usually <500/mm 3 . ig levels and production of specific antibodies are all normal [19] . onset may be early, as for scid, but also delayed until the age of 3-5 years. the clinical pattern is dominated by recurrent bacterial, viral and fungal infections, with an abnormal susceptibility to opportunisic germs. two-thirds vanced liver disease may die because cryptosporidia infection that has progressed rapidly following pretransplantation cytotoxic conditioning therapy [252] . therefore, a patient with end-stage liver disease related to cd154 deficiency first received a liver graft, and as soon as liver-graft function was satisfactory, bmt was performed with a nonmyeloablative conditioning protocol of fludarabine and melphalan [208] . the screening for cd154 deficiency should include children with severe rri, and with dysgammaglobulinemia with a normal or increased igm level [197] . conventional allogeneic hsct from an hla-matched or a matched unrelated donor (mud) is curative and feasible, if performed before significant infections and organ damage occur [503] . an approach for high-risk patients including nonmyeloablative hsct was workable in a retrospective analysis of 38 european patients undergoing hsct for cd154 deficiency in eight european countries between 1993 and 2002. the donor sc source included 14 hla-identical siblings, 22 muds, and two phenotypically matched parental stem cells (scs) (12 tcd [t-cell depleted]). of these patients, 12 (32%) died from infection-related complications, with a positive result in 68.4% of patients [180] . carriers can be detected, and this is useful for making a prenatal diagnosis [407] . of patients suffer from neurological alterations, ranging from spastic symptoms and alterations, etc., to mental retardation and one-third from aids, the most common of which is aiha. the consequence of severe infections, generalized vaccination, severe chickenpox, lymphosarcoma and gvhd caused by blood transfusions in the first decade of life is death [304, 397] unless bmt is successful [25, 58, 67, 83, 98] . however, poor neurodevelopmental progression may result [25] or may not [98] . since the biochemical bases of pnp and ada deficiencies are similar, it is hoped that genetic treatment will also be effective in children with this pid [76] . this deficiency of hla molecule expression occurs in the more severe forms of pid if they are class ii: about 80 cases [140, 428] are known of this ar syndrome [140] , heterogeneous for the numerous complementation groups the patients are divided into [76] . hla class ii molecules are absent in all tissues [400] , to the extent that the cells of patients maintained in cultures for years preserve the negative phenotype [400] . there is a deficiency of class ii gene transactivator (ciita) codified by chromosome 15, the expression of which plays an important role in t-cell activation: its absence makes class ii gene expression impossible [473] . this function is shared with another protein mapped on chromosome 2, rfx5, with a binding site in the promoter region of genes codifying class ii chains [401] . two additional class ii-specific transcription factors are rfxap and rfxank [314] . these act on the class ii promoter region and are essential and also nonreplaceable, to the extent that alternative routes cannot compensate for their absence [311] . furthermore inactivation, or the deficiency of these factors, has a specific effect on the genes dictating hla class ii, the li chain and hla-dm, because there is no indication that other regulating systems may be involved [401] . the absence of hla class ii is associated with a cd4 lymphopenia. hla class i expression is normal in the patients tested and cd8 lymphocyte numbers are not reduced [428] . interestingly, in a twin study, despite the deficiency, there were antibody responses and class ii-dependent t cells; hence the authors envisage that this represented a hla class ii residual expression below the test sensitivity [540] . the clinical outline is dominated very early on, before the age of 6 months (range, 2 weeks to 12 months) [428] , by severe and recurrent gastroenteric and pulmonary infections, with a severe and prolonged course, associated with malabsorption and failure to thrive [77] . bacterial and viral infections, bronchopneumonia, hepatitis, cholangitis, viral meningoencephalitis and various autoimmune manifestations are common complications [140] . even though an hla class ii deficiency is clinical-ly less severe than scid, the result is uniformly fatal during the first or second decade of life [162] . the most evident immune defect consists in the complete lack of reactivity to exogenous antigens, which in vivo reflects an anergy to spts, as well as the complete lack of hla class ii expression and absence of cellular and antibody responses to antigen stimulation [140] , which are instead positive to mitogens (table 22 .8; fig. 22 .24) [508] . laboratory investigations show a normal b lymphocyte number, but children may be agammaglobulinemic [140] . the thymus and other lymphoid organs are remarkably hypoplastic, with a severe cd4 lymphocyte depletion, while cd8 and b-cell levels are normal. the syndrome involving a deficiency of hla antigens confirms an important hla biological role in the complex system of t-b cooperation [77, 140] . some studies suggest that there are more types of deficiency. when placed together in a culture, the b lymphocytes of these patients, previously transformed by ebv, the lymphocytes correct each other so as to allow hla class ii molecule expression. this has led to the identification of so-called complementary groups [251] . the specification that the gene is mapped on chromosome 19p13.3 can lead to an earlier prenatal diagnosis [166] . longterm survival seems to depend primarily on hla-identical and hla-haploidentical bmt performed in the first 2 years of life, before the acquisition of chronic virus carriage and sequelae of infections [257, 140] . a child recently received a transplant [428] with a novel protocol [208] : the cd4 count increased up to 300 cells/ml [428] . a direct correction of the genetic defect is based on the transduction of cells from patients with lentiviral vectors encoding ciita, rfxank, rfx5, or rfxap. the rfxank vector restored class ii expression in a t-cell line from one patient. the rfxap vector corrected primary cells from a second patient [314] . the study of the common association of hla class i molecule deficiency, already known as bare lymphocyte syndrome, has led to the identification of various patients with an isolated deficiency and of one patient with a deficiency associated with class ii, the most severe [85] . the deficiency is caused by tap-1-tap-2 mutation, accompanied by severe and chronic bacterial rris [115] . in two brothers with the ar hla class i defect, the onset of rris took place between the ages of 4 and 7; the poor expression of nk cells was so severe that it led to the development of bronchiectasis [125] . the immunological structure is characterized by few cd8: the deficient expression of hla class i molecules is diagnostic [115] (tables 22.1, 22.3). mutations with no symptoms referable to an id, therefore integrating a genetic heterogeneity. two other brothers and both parents were healthy [5] . while the ε chain deficiency produces modest clinical symptoms, the other two are severe also from an immunological point of view: in the γ chain deficiency resulting from the profound cd8 and cd45ra decrease caused by altered thymic activity that leaves the cd45ro unaffected [249] , and in those of the ζ chains due to the severe thymic atrophy [5] and thymocytes falling to 15% of normal levels, with limits at between 1% and 50% [249] . the cd3δ deficiency due to a heritable mutation of the cd3 gene that prevents the synthesis of the cd3 protein has been reported in 3 cases hz for the cd3 mutation. two cousins died at 2-3 months of age because of overwhelming infection. the thymus shadow is clearly visible on chest x-rays. the thymus becomes populated with developing thymocytes, with an arrest of differentiation at the cd4 -cd8stage of t-cell development. a girl (3rd patient) survives after a bmt [111] . this rare deficiency transmitted as an ar trait is caused by mutations of the zap-70 gene, a non-src family protein tyrosine kinase (ptc) important in t-cell signaling (tables 1.31-1.33). zap-70, known to be crucial for t cell activation, is a key player in tcr down-modulation and z degradation [136] . zap-70 has an essential role in the cd3g chain deficiency due to g or e gene mutations [19] determines a lack of cd8 and the absence of cd45ra [249] . in the first two cases described, one brother died at 31 months because of viral pneumonia after a clinical history indicating scid with severe aiha, while the other was asymptomatic at the age of 10 years, although with the same molecular defect [18] . the study of these brothers proved that, in spite of the absence of functioning g chains and 50% of the expressive levels of the cd3/tcr complex, the lymphocytes were normal. according to the authors, other chains may act in the place of missing ones; however, the correlated scarcity of cd8 may have negatively interfered with the mechanisms discriminating between self and non-self, while g chain deficiency could have modulated the onset of the deceased brother's severe autoimmune disease (aid) [18] . a cd3e deficiency was found in a 4-year-old child with mild rri symptoms and otitis media; the expression of the cd3/tcr complex was only 10%, but the stimulation with anti-cd3 induced a normal proliferating response. in fact, despite the ongoing mutation, a northern blot analysis showed production of a low amount of transcribed rna, corresponding to a small quantity of e normal chains, even though their dimensions were smaller than normal ones [471, 496] . the z chain deficiency found in the two brothers is similar to the deficient expression of cd3/tcr [5] . in the younger brother, the thymus, markedly reduced, showed no hassall bodies; the elder brother had similar chain [342] . in several babies (most of mennonite origin) with scid [20, 92, 139, 179] , the nonfunctional cd4 t cells (table 22. 3) were either normal or increased (cd3 + cd4 + , 75%). cd8 absence in the thymus and in circulation (cd3 + cd8 + , 0%-2%) [139, 179] suggests that the selective process is arrested during the transition from double-positive (dp) to mono-positive (mp) t cells [20, 140, 179] .arrested thymocytes had terminated rag gene expression and up-regulated tcr and bcl-2 expression, but failed to differentiate into mature cd4 or cd8 mp thymocytes, to be rescued from death by neglect or to sustain il 7 ra expression [294] . zap-70 deficiency results in an impairment of transendothelial migration that can be rescued by the transfection of zap-70 because cross-talk between the zap-70 signaling pathway and the chemokine receptor cxcr4 is required for t-cell migration [502] . although the thymic architecture is normal with presence of hassall bodies [179] and cd8 seem normal in the cortex, very few migrate to the medulla [20] . the near absence of cd8 + cells and an increased cd4:cd8 ratio dominate [27] . the few cd8 coexpress cd56 + , the nk-cell marker; b cells appear normal and functional, cd3 -cd19 + is at a level of 20%-40% [139, 179] , and serum ig values are normal [27] . the same phenotype was found in the brothers [92] ; other relatives were het [20, 139] . the absence of cd8 expression was shown to correlate with a missense mutation in both ig alleles of the cd8 a gene domain in a 25-year-old man and his sister, whereas high percentages of cd4 -cd8 -tcrab + t cells were found in the three siblings [114] . the proliferative responses in vitro to phorbol myristate acetate (pma) and ionomycin, pkc activators (protein kinase c), were normal, unlike pha (phytohemagglutinin), pwm, tetanic toxoid, anti-cd3, etc. [20, 139] . the positives operate below the tcr, while the negatives react directly with the cd3/tcr complex [92, 140, 179] , confirming the zap-70 deficiency [20] . the cd4 are present despite the deficiency because syk, the other member of the family, ensures a compensatory role in the infrathymic cd4 selection, although with a limited efficacy [179] . seven months after bmt, a child was clinically well and immunologically recovered [27] . studies in two siblings hz for a stop mutation in the tap-2 gene suggest that nk cells express still unknown inhibitory receptor(s) (the missing receptor, discussed in chap. 1) capable of down-regulating the nk cell cytotoxicity on binding to surface ligand(s) expressed by t cell blasts. functional analyses were consistent with the concept that this putative inhibitory receptor is expressed by virtually all tap-2/nk cells, whereas it is present only in rare nk cells from healthy persons. another prospect would be that tap-2/nk cells are actually missing this still unidentified triggering receptor involved in nk cell-mediated killing of pha blasts. since cells derived from patients displaying defective expression of either of the tap subunits are characterized by a strong reduction of mature hla class i molecules at the cell surface, a tap deficiency is connected with hla class i deficiency [517] . as discussed in chap. 1, nfat (nuclear factor of the activated t cells) is a transcription factor that forms a powerful transcriptional activating complex and, by linking with specific dna-regulating sites, plays a critical role in the synthesis of various t-cell ils which, due to the deficiency or excessive migratory mobility of nfat, although normal in number and in distribution, are incapable of activating and/or secreting the genes of il 2 , il 4 and ifn-g [86] . a 4-year-old girl with scid presented during infancy with severe recurrent infections and failure to thrive; her mrna was not produced for il 2-5 and ifn-g due to poor t-cell proliferation, although these were normal in number and in distribution, to initiate the transcription of the relative genes, regulated by nfat, with a binding site in the proximity in the 5¢ region. this severe clinical picture is accompanied by evident hgg [19, 86] . nk-cell deficiency is found in scid, cvid, reticular dysgenesis, chédiak-higashi syndrome, xlp, lad in tap-2 deficiency and in cfs (chronic fatigue syndrome), in particular cid such as scid, suggesting an association between nk-and t-cell deficiencies [478] . there is one known case of an adolescent with an isolated numerical and functional deficiency of nk cells and of precursors, recurrent neutropenia, severe and recurrent ebv, cmv, herpes simplex virus (hsv) infections and life-threatening chickenpox. another child, diagnosed at the age of 2.5 years with a cd8 deficiency, suffers from severe viral and bacterial infections although he has antibodies to various viruses [49] . the growing list of human genetic defects that impair nk-cell function has been recently joined by nemo-id [356] which occurs in a group of patients with antibody deficiency combined with exquisite susceptibility to infection with nontuberculous mycobacteria. infectious susceptibilities common to these disorders stress the important role for nk cells in host defense [59] . the natural history of three boys with nemo mutations outside of the 10th exon has been described. including these boys, there have been 22 families described as having nemo-id. the resulting estimated incidence of nemo-id is 1:250,000 live male births, making this disorder significantly less common [356] . cd45ra cells and a marked clinical improvement. these data indicate that the thymus is differentially required in the maintenance of the tcr repertoire complexity [380] . known also as idiopathic lymphocytopenia, primary cd4 t-cell deficiency is revealed by a profound and persistent reduction in circulating cd4 and with a cmi deficiency. it is documented in patients suffering from infections caused by opportunistic germs such as cryptococcus-induced meningitis and oral candidosis, also including ten children and a number of adolescents, for whom the following minimum levels of cd4 per age have been established: <1,000 cells/mm 3 from 0 to 23 months and <300/mm 3 from 2 to 12 years, or a total lymphocyte count of <20% on two separate occasions without being hiv-infected [463] . a family has been reported involving two brothers aged 13 and 18 with t counts between 150 and 200/mm 3 , recurrent respiratory, intestinal and cutaneous infections, and failure to thrive. the mother showed a low cd4:cd8 ratio [122] , while the entire family showed normal levels of ig and subclasses and hla molecules [128] . other symptoms included mental retardation, pansinusitis, bronchiectasis [168] , but no infections caused by opportunistic germs such as those reported by the who scientific group [414] . one case of primary cd7 deficiency is known of a child with scid without genetic transmission of the deficiency. t-cell proliferative responses to mitogens were defective and il 2 r expression was deficient on his t lymphocytes, and b cells did not differentiate into antibodysecreting cells when provided with the help of normal t cells [245] . the index patient for primary cd45 deficiency was the first child of consanguineous kurdish parents. she presented aged 2 months with a rash, pyrexia, hepatosplenomegaly, lymphadenopathy, pneumonitis, pancytopenia, and disseminated cmv infection. laboratory analysis showed absolute lymphopenia, low t cell numbers, with markedly low cd4 + and low cd8 + and normal b cell numbers. she responded well to anti-cmv treatment and at 8 months underwent a mud bmt. t-cell engraftment was demonstrated 3 weeks after bmt. despite continuous anti-cmv treatment, her undifferentiated scid human p56lck deficiency p56lck deficiency is an ar scid due to a defect of an src kinase critical for the generation of mature thymocytes in adult mice. p56lck is important in tcr signaling and phosphorylation of the itams of the cd3/tcr complex proteins. mutant mice lacking p56lck have pronounced thymic atrophy, a critical reduction in dp (cd4 + cd8 + ) thymocytes, no detectable mp thymocytes, and only a few peripheral t cells. both proliferation and development of a given defined cell subpopulation depend on meuse age. the absolute numbers and proliferation of dn and isp (immature single positive) thymocytes only proliferate during fetal and early postnatal life up to 14 days after birth, whereas the proliferation is significantly decreased beyond that age, thus lck may have differential roles in the proliferation and maintenance of dn, isp, and mp/dp thymocyte populations [151] . the first demonstration of a human scid patient with an abnormal expression of p56lck is an scid infant hospitalized at 2 months for dehydration, failure to thrive, and sepsis. the immune phenotype included hgg, selective cd4 lymphopenia, lack of cd28 expression on cd8 + t cells and poor t cell blastogenic responses to various mitogens and il 2 . p56lck protein expression was only minimal with an unusual mrna splicing pattern of the lck gene. the levels of p59fyn were normal and it is therefore possible that p59fyn played a role, albeit incomplete, in the development of his mature t cells. the child has since undergone an allogeneic bmt (at 32 months) from a matched unrelated donor (mud) [185] . unfortunately the boy died 2 months later due to cmv infection and gvhd (fd goldman, pers. comm., 8 nov. 2005 ). whn (winged-helix-nude) encodes for a transcription factor that is crucial for maturation of the thymus microenvironment [185] . nu/nu mice fail to develop a thymus and mature t cells due to a defect in the whn gene encoding a transcription factor necessary for terminal epithelial cell differentiation. a defective whn gene could lead to the disrupted early t cell development in the bm. t cell progenitors were associated with a lack of pta gene expression and a failure to give rise to mature t cells in adoptive euthymic hosts. wild-type hscs rapidly matured into functional t cell progenitors in the marrow of euthymic or thymectomized but not nu/nu hosts. therefore defects in bm prethymic t cell development can contribute to t cell deficiency in nu/nu mice [90] . in two sisters a severe scid caused by mutation of the whn gene was associated with complete alopecia. hla-identical bmt in one of the two girls resulted in a clear reconstitution of cd4 + and cd8 + cmv reactivated, and she died 55 days after bmt [74] . a 6-bp deletion in the gene encoding cd45 resulted in the loss of glutamic acid 339 and tyrosine 340 in the first fibronectin type iii module of the extracellular domain of cd45, identifying a region important for cd45 structural integrity and lack of surface cd45 expression. this was almost certainly responsible for the id in this girl [494] . a second child presented at 2 months of age with severe cid, showing similar t-cell defects. despite normal b-lymphocyte numbers, serum ig levels decreased with age [272] . introduction of a functional cd45 minigene was sufficient to overcome the main scid-associated defects and represents a potential route to a gene therapy for human cd45-deficient scid [516] . two male infants born to consanguineous parents had scid despite phenotypically normal blood lymphocytes. their t cells were unable to produce il 2 , ifn-g, il 4 and tnf-a [154] . another child with scid had defective transcription of il genes encoding il 2 -il 5 [86] . dna binding of activation protein 1 (ap-1), oct, creb, sp1, and nf-k b was normal, but the binding of nfat to its il 2 promoter response element [154] , or the ability of nuclear factors from the child's t lymphocytes to bind response elements present in the il 2 regulatory region [86] was barely detectable [86, 154] both before and after t-cell stimulation [154] . these results indicate that the nfat abnormality may underlie the multiple il deficiency in these boys. nezelof syndrome, also known as cellular id with ig, or combined with a predominant t-cell defect, or as a scid variant, clinically less severe compared to the previous ones, is characterized by a form of ad, concentrations of ige that may also be extremely elevated (table 22. 2), and normal or increased serum levels of other ig classes [62] . the cmi study emphasized the mature t-cell reduction or absence, various expressions of immature cells, with cutaneous anergy to spts and a reduced or absent in vitro lymphocyte response to mitogens. from infancy, patients present recurrent or chronic pulmonary infections, pondostatural retardation, oral and/or cutaneous candidosis, chronic diarrhea, recurrent cutaneous and urinary tract infections, gram-negative bacterial sepsis and a particularly severe form of chickenpox [394] . differential diagnosis must include pediatric aids, also marked by proportionably increased ig and a lack of antibody and t-cell function [79] . inherited through ar modalities, cd95 deficiency has been observed in 8 children, two of whom were brothers, with mutations of the fas gene, one hz and 7 het [166, 281] , as well as in 9 unrelated children [468] . these mutations most often arise as a result of mutations in the gene encoding the lymphocyte apoptosis receptor fas/apo-l/cd95. a novel mutation has been identified in the intracellular apoptosis signaling domain of fas in 11 members of a family, with several members monitored for up to 25 years [228] . thus, the deficiency is inherited in an autosomal dominant fashion but with a high degree of variability in clinical expression [228] , but also in an ar fashion [510] . the clinical picture is dominated by imposing hepatosplenomegaly with an early onset, even neonatal, accompanied by t-cell hyperproliferation, chronic and persistent lymphadenopathy, and failure to thrive [468] . an extensive lymphocyte infiltration of lymph nodes, spleen and liver is observed, with t cells reaching 35,000/ml [cd3 + , cd4 -cd8 -(dn) equal to 35-60 cells/ml compared to 0-3 in controls], as in omenn syndrome, also in the bloodstream, with possible oligoclonality of t cellularity. dn t cells expressed the a/b tcr [468] . immune dysregulation is associated with g and a hgg (hypergammaglobulinemia, auto-antibodies and aids, especially of the hematological type, such as aiha, and with a severe and recurrent thrombocytopenia [166, 281] . autoimmune features are discussed in chap. 18. an overlapping mechanism could belong to the etiopathogenesis of xlp and omenn syndrome. was has a prevalence of approximately 4 × 10 6 live births [402] . it is transmitted as a recessive hereditary trait linked to the chromosome x, localized in a pericentrometric position on the short limb of chromosome x (xp11.22-p11.3) [274] . it is therefore possible to identify the female carriers and to provide prenatal diagnosis [61] . the gene that codifies the was defective protein (wasp) has been isolated [458] and has 167 mutations distributed among all 12 exons of the entire gene, 110 of which are unique and 38 familiar, with two large deletions, one embracing exons 1-7 and one intron 8 [275, 444] (fig. 22.25 ). six novel mutations have been identified that involve nonsense mutations, or small deletions, all of which result in predicted truncation of wasp synthesis [57] . a new, recurrent mutation is v75m, due to a cpg island was found in a hz girl, who showed microthrombocytopenia and infections to the same degree as her hemizygous father and brother. the amount of was protein was about 10% in platelets and 15% in mononucleated white cells [388] . involved in ensuring the t lymphocyte functional polyvalence, also explaining why microvilli and platelet defects are absent [152] .wasp and several related proteins (the wasp family) are all involved in the organization of the actin cytoskeleton. to carry out vital functions, cells have to rearrange their actin cytoskeletons [467] . the characteristics peculiar to wasp as a meeting point for the marking pathways is illustrated in fig. 22 .26 [152] . the wasp function is absent in 135 cases of was, in ten with attenuated was and in 23 with xlt [372] . in normal subjects, it is found in the cyto-molecular biology has proven that wasp found only in blood cells binds the small gtpase cdc42h2 in the gtp but not in the gdp [23, 265, 490] . cdc42h2 plays a critical role in the assembly of actin filaments [490] and in t-cell polarization when they encounter a b-lymphocyte apc [481] . wasp activity is regulated by several proteins acting in concert to control wasp configuration. the wasp-interacting protein, when phosphorylated, releases wasp from its grip, allowing wasp to be activated by rho-family gtpases [433] . experimental data also indicate that cdc42, wasp and actin might be plasm but not in the nucleus of various cells such as platelets, t and b lymphocytes and monocytes [477] . the xlt gene is located on the same locus as the was and could therefore be a variant [444] ; the main immunological anomalies are summarized in table 22 .12 [287, 413] . children with was have significantly elevated levels of il 4 and ige (table 22. 2) and decreased levels of ifn-γ [217] . the pathogenetic mechanism unifying the symptom triad is not clear; the glycosylation defect has been proved, primarily concerning sialidation, therefore resulting in an instability on the membranes of platelets, neutrophils and lymphocytes expressing a glycoprotein sialopherin (cd43) [402] , localized on chromosome 16, which makes it an improbable candidate, even though cd54 is indeed the binding agent of cd43 and could therefore play a role in t-cell maturation, differentiation and activation, thereby acquiring marking capacities that are independent of tcr/cd3 [19] . however, the tcr-mediated signaling defect is characteristic of was [259] , in addition to the reduced expression of cd23 [458] , which can explain immune and hematological defects. in the lymph nodes, there is a shortage of lymphatic follicles and the thymus-dependent and -independent areas are depleted, moderately at the age of 4 years ( fig. 22.27 ) and to a greater extent at 8 years ( fig. 22.28 ). the predominant immunological outline is constituted by elevated iga and ige levels, low igm and all igg levels, as well as the absence of a response to polysaccharide antigens, which is why the children's serum lacks isohemagglutinin [278, 528] . in unweaned babies, the most striking finding is the cd4:cd8 ratio =5 [549] , compared to 2.65 in normal children aged 0.63-3.06 (tables 1.36-1.39). was usually starts at 13.7 months (range, 1-58) [132] with hemorrhagic manifestations, petechiae and prolonged bleeding from the umbilical scar or the circumcision site, observed in newborn babies [402] . the clinical triad is characterized by cutaneous lesions that are practically indistinguishable from rather severe ad (70%), congenital thrombocytopenia (100%), a marked susceptibility to rris (91%) [132] (figs. 22.29, 22.30) and gastroenteric symptoms such as hematemesis, melena and chronic diarrhea [132] . other complications may include neutropenia (25%), arthritis (29%), skin vasculitis (22%), cerebral vasculitis (7%), inflammatory bowel disease (9%), and renal disease (3%) [132] . a reduced thrombopoiesis (level <50,000/ml), with microthrombocytes and an accelerated turnover in boys must allow for a suspected diagnosis [413] . it has recently been proven that the classic presentation is more common in children aged 6.8 months than in those aged 7.2 months (60% compared to 25%), unlike platelet counts [549] . however, only 27% of 154 unselected children with persistent thrombocytopenia, positive fh, small platelets and defects associated with t and/or b lines had the classic triad and 20% only thrombocytopenia before diagnosis [484] . primary immunodeficiencies infections, appearing during the first months of life, are often marked by otitis media, pneumonia, meningitis and sepsis, caused by viruses (cmv and herpesvirus) and by bacteria (pneumococci or other capsular polysaccharide). these are followed by more common infections caused by opportunistic germs, pneumocystis carinii and mycetes such as candida albicans. differential diagnosis should also include a rare ar syndrome similar to was, also reported in female patients, characterized by ad, rris and thrombocytopenia with microthrombocytes [62] . when caring for these children one must monitor the platelet count, the immunological structure (ig, lymphocyte and subpopulation counts) and the potential onset of autoimmunity and tumors [224] .aiha may be found in 36% of children [132] . prophylactic treatment for infections is done with ivig; 500 mg/kg every 3 weeks) and sulfamethoxazole (25 mg/ kg/2 days) after diagnosis [132] . splenectomy may decrease the bleeding tendency [224] , but early relapse of thrombocytopenia after splenectomy is predictive of a poor prognosis [132] . on average death occurs around the age of 11 (8 in untreated children), but can occur between 0.5 and 4.5 [549] , with survival also >18. death is caused by massive hemorrhages (23%), tumors (26%) and severe infections (44%) [484] . the second largest group of patients with id given bmts since 1968 are those with was, with 78.8% of children aged <5 years [158] . fourteen out of 18 patients underwent phenoidentical (n=1) or haploidentical (n=13) hscts; the other four died before hsct could be undertaken [132] . boys who had received a mud hsct transplant <5 years had survival rates similar to those receiving hla-identical sibling transplants, but the success rate decreases dramatically at the age of 5-6 [158].wasassociated t-cell signaling defects can be improved upon retrovirally transduced hscts [258] . recently, correcting the t-cell defects has been proposed. the potential for correction of the t-cell defects has recently been demonstrated by transduction with an oncoretroviral vector encoding the wasp, which resulted in correction of the deficient proliferative response to tcr stimulation characteristic of was [483] . ata is a complex ar inherited syndrome, associated with neurological, immunological, endocrinological, hepatic and cutaneous abnormalities, characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, and increased susceptibility to rris [379] with an incidence estimated at 1:100,000-1,300,000 live births [61] . in italy the frequency on the general population, is of 1.3 × 10 6 , with an increase in hets from 1.7% to 3.43% [94] . it is characterized by a genetic heterogeneity, which is reflected in the division into four main groups of complementation, to which one must add the nijmegen and at-fresno variants, perhaps caused by other well-defined id syndromes the same gene, also localized on the long arm of chromosome 11q22.23 [176] . the 12-kb gene, called atm (at mutated) because of its mutations by defective splicing in all patients with ata, permits het identification [435] . a dna clone complementary to atm shows considerable affinity to factors responsible for signals involved in regulating the cell cycle and codifying a protein similar to phosphatidylinositol-3-kinase (pi 3k) [435] , involved in mitotic signal transduction, meiotic recombination, and cell cycle control. a result could be a recombination defect which interferes with b and t lymphocyte gene rearrangement, involving tcr and isotype switching, consequent to a damaged dna triplication and therefore accounting for ig deficiencies [176] . cells from these patients progress too rapidly from the g 1 phase, in which they receive ionizing radiations, to the s phase, then continuing irradiation, to the g 2 /m phase with further delay, evolving in apoptosis [279] . this hypothesis has received further credit after observing that the p53 gene expression does not increase in human cells exposed to radiations [250] . the p53 gene is part of the normal cell cycle and during the s phase provides time for the dna physiological repair after exposure to radiation that may also be cosmic [279] . the thymic tissue is either absent or degenerated with a fetal appearance (fig. 22.31) : some follicles, also with b cells, are visible at the age of 2 (fig. 22.32) , at 8 there is complete cellular depletion (fig. 22.33 ). immune deficiencies are humoral and cellular (cutaneous anergy and depressed proliferative responses) [70] . the karyogram shows that the lymphocytes have common rupture points at the chromosomal level with inversions and translocations involving precisely the tcr and ig genes [320] . most chromosomal translocations involve the genes encoding tcr on chromosome 7 and the ig h chains on chromosome 14: most breakpoints occur at the loci that encode ig and tcr for antigen (regions 7q35, 7p12, 14q32, 14q12) [264] , in areas typical for cod-ification of molecules of immunological importance (chap. 1). an important role is played by genes belonging to ig gene superfamily (igsf) (table 1.4) . possibly the progressive id of ata, like its apparently unlinked manifestations, is at least in part linked to the accumulation of clonal anomalies affecting the tcr and the igsf: this suggests the intervention of "illegitimate" recombinations damaging above all the t cells [162] . t-cell immunological deficiency is completed with lymphopenia, a decreased cd4:cd8 ratio due to the drop in cytotoxic cd8, and a rise of immature forms with tcrgd [80] . another consequence is the isotype deficiency: about 70% of patients present sigad; >50% are also affected by an igg 2 -igg 4 deficiency with igm becoming monoclonal, and 30% by serum igg deficiency [364, 379, 528] . fig. 22 .32,the degree of depletion of lymphocytes is extensive in both thymus-dependent and thymus-independent areas chromosome 11 into cells, the chromosomal aberrations induced by x-rays were suppressed [261] . the rare ar nijmegen breakage syndrome, so called because it was initially seen in two brothers of secondcousin parents living in that city, and at the moment observed in approximately 80 patients, has various characteristics of ata but without ataxia, telangiectasia, or high concentrations of afp. clinical characteristics are singular: short stature and microcephaly with prenatal onset, bird-like profile, prominent midface, a long nose, low-set ears, cutaneous depigmentation with caféau-lait spots, an almost normal intelligence, and also rris and bronchiectasis. humoral and cellular id includes reduction of antibodies and lymphoproliferative responses [70, 224] . during an 8-year period of observation, the id was found to be profound, highly variable, and with a tendency to progress over time in 40/50 children [193] . there is a high proclivity to expressing rearrangements of chromosomes 7 and 14 as in ata [70, 224] . dgs is usually sporadic, with known cases of positive fh [224] . it is caused by a defective development of the 3 rd and 4 th branchial pouches which takes place before the 12 th week of gestation, with consequent thymic hypoplasia or aplasia and parathyroid hypoplasia; the 5 th and 6 th pouches and branchial arches can also be affected [497] . the cause can be found in the neural crest cell incapacity to migrate and interact appropriately with endothermic cells of the brachial pouches and arches [295] . deletions (often microdeletions) at the pericentrometric region of chromosome 22q11-pter have been described in 80%-90% of cases [130] . a microdeletion 22q11.2 was recorded in 112 children aged 4-70 months, 54% of whom had developmental delays, mild hypotonia, as well as language and speech delays [181] . another 80 children had deficits in the areas of attention, story and visuospatial memory, arithmetic performance relative to other areas of achievement, psychosocial functioning [541] , and mental retardation in 73% of 44 children [11] , thus indicating the need for early intervention beginning in infancy [181] . overlapping alterations are present in the syndrome complex known as catch 22, which in turn includes the charge association. other cases of dgs can derive from microdeleted chromosome 10p (fetal-alcoholic syndrome, retinoic embryopathy, maternal diabetes) [414] . this variable phenotype is reliably referred to microdeletion 22q11.2; the greater it is the more complex is the associated phenotype [508] . another difference depends on the variable spectrum of t-cell abnormalities in individuals with dgs who might have normal t-cell numbers and function, low t-cell numbers but fairly normal t-cell proliferative function [32] or no t cells purkinje cells (pcs) and degenerated granular cells. that the number of basket cells, so called because they form with the axons bunches of fibrils distributed so as to form a nest in which the nucleus of each pc settles, is almost normal, proving that pcs are probably normal at birth and degenerate only later [176] . typical clinical manifestations are ataxia, telangiectasia of both auricular lobes and sclera, rris and an elevated incidence of neoplasia [489] . from a review of 331 patients [70] , the percentages of symptoms are as follows: progressive ataxia (100%), typically cerebellar, becomes evident when children start walking or a little later, affecting intentional movement and becoming complicated by dysarthria (100%) and involuntary choreic movements (92%), causing the majority to be unable to walk by about the age of 10-12 [276] . at a later stage it is possible to observe nystagmus (67%), strabismus, oculomotor apraxia (88%), reduction or absence of reflexes (77%), and dyslalia, increasingly amplified and, in some patients, also mental retardation [176] . telangiectasias develop between the ages of 1 and 6 on the bulbar conjunctiva (97%) (fig. 22.34) , on the flexor surfaces of the limbs and areas exposed to sun rays (17%). height and weight are <10th percentile (64%) and the appearance is progeric (63%). severe rris are common (70%), encouraged by antibody deficiencies. the pathogens involved can be bacterial or viral, often resulting in lung bronchiectasis, all starting after the onset of neurological manifestations [276, 489] . associated neoplasia (6%) is usually lymphoreticular, less common than adenocarcinoma, with an eightfold increased trend for all kinds of tumors [224] . in cultures the fibroblasts of these patients are three times as sensitive, compared to controls, to ionizing radiations and to radiomimetic chemical substances, but not to uv rays, unlike what is observed in the cells of subjects affected by xeroderma pigmentosum [70] . in addition, the persistence of elevated serum a-1-fetoprotein (afp) levels was observed in all patients. an interesting in vitro study has reported that by introducing a normal human 1303 other well-defined id syndromes fig. 22.34 . conjunctival telangiectasia in a girl with ata [305] . a second group is referred to as having partial dgs (dgsp) or transient forms (dgst), with mild symptoms. the designation "complete digeorge syndrome" (dgsc) is reserved for the third group of infants who have absence of thymic function in addition to other defects of the 3 rd and 4 th pharyngeal pouches, <1% of patients with dgs, although they can have high t-cell numbers that respond to mitogens [32, 305] . these patients have profound id, with its associated clinical findings [308] . dgst includes cases with a spontaneous quantitative and qualitative t lymphocyte recovery [162] . the thymus can also be ectopic: in dgsc the t zones are depleted, the cd4/cd8 markedly reduced both in number and in function with spt anergy, and b cells appear unaffected or increased [162] . in dgsp, the most common type, t-cell number and function are instead usually normal, as are the cd56/cd16 cells with a nk phenotype, or they may be moderately reduced [162] . the proliferative response to mitogens can be pathologically reduced [224] and the response to polysaccharide antigens may be absent [442] . from neonatal age, there are malformations of other structures that form during the first weeks of embryogenesis, presenting a suggestive but not pathognomonic picture (table 22 .13) [79] . diagnosis is usually suspected within the first 2 days after birth, due to the presence of hypocalcemic tetany caused by hypoparathyroidism and cardiac malformation. the facial dysmorphism is also characterized by a small mouth with thin lips described as fish-like [79, 192] (fig. 22.35) . the two rare cardiopathies indicated in table 22 .13 depend on neural crest nonintegration, as mentioned, which accounts for >50% of the alterations alone [295] . others can be observed affecting the right heart, such as fallot tetralogy, pulmonary athresia with an interventricular septum defect, and pulmonary infundibular stenosis [508] . babies surviving the neonatal period manifest from the very first months an increased susceptibility to infections, particularly those of the respiratory and digestive tract, viral and/or fungal, but also caused by pneumocystis carinii, which can be fatal in dgsc [162] . other findings include gastroesophageal reflux, speech delay, laryngomalacia, absent kidney, conductive or sensorineural deafness, 6th cranial nerve palsy, and hypothyroidism [535] . treatment with high doses of vitamin d and diets enriched with ca gluconate are needed immediately, also ensuring that calcemia remains at the lower limit of normal values so as to avoid snc and renal damage. subsequently the possible correction of cardiac malformations should be evaluated. id may be severe, but can regress spontaneously with reconstitution of cmi and t functions; compensating hyperplasia of the residual parathyroid tissue can make it possible to discontinue ca and vitamin d treatment [192] . dgs natural history is, however, complicated by mental retardation and the difficulties encountered in correcting cardiac malformations and in controlling hypoparathyroidism [224] . because of variability in the id severity, it is difficult to evaluate claimed benefits of bmt: in two cohorts of 8 [305] and 5 transplanted infants [306] , the survivors were 3 out of 13 (23.1%). recently, 5/6 and 7/12 infants underwent postnatal transplantation with cultured unrelated thymic tissue, with immunosuppression, with positive results [307] . del22q11.2 syndrome, characterized by a 3-mb deletion on chromosome 22q11.2 is the most frequent known chromosomal microdeletion syndrome, with an incidence of 1 in 4,000-5,000 livebirths. patients show 1304 chapter 22 primary immunodeficiencies clinical outlines not unlike gvhd [446] (fig. 22.36 ) [508] , suggesting a possible connection to a fas deficiency (cd95) or apoptosis syndrome. the sh2d1a gene was found altered in two families, thus indicating that xlp must be considered when more than one male patient with cvid is encountered in the same family, and sh2d1a must be analyzed in all male patients with cvid [330] . recently a critical revisitation of this experiment in nature has allowed the identification of links between id and allergy [62, 178, 196, 287] . the rare higes is associated with bacterial rris, chronic ad, coarse facial features and very elevated ige levels [60, 250] ( table 22. 2), up to 40,000 iu/ml [549] . linkage to a region on chromosome 4q has been demonstrated in several affected families; however, neither the fundamental host defect nor the defective gene has yet been identified [195] . fh is frequently positive for atopic disease, at times higes is combined with an unusual predisposition to staphylococcus aureus infections [62, 178, 196, 287] . in buckley's study, it was present in 36.4% of cases, of both sexes, indicating an autosomal dominant transmission with incomplete penetrance [62] . onset occurs in the pediatric age in 90% of cases [64] . clinical presentation is unusual: there are no complaints during the first months of life, toward the 3rd-4th month a severe form of chronic ad appears all over the body, which can be associated with other allergic manifestations, including asthma in 13.6% of cases [62] . skin biopsy specimens reveal spongiosis and perivascular dermatitis and/or folliculitis with a predominance of eosinophils [91] . there is an excessive predisposition to cutaneous and respiratory tract infections (deep and superficial abscesses, otitis, pneumonia, sepsis) ( fig. 22 .37), also encouraged by neutrophil chemotactic deficiency caused by defective cellular functions (table 1. 65), which, if present, is so pronounced that it becomes a characteristic, unlike ad where it is secondary [226] . the subcutaneous abscesses, described as cold, not covered by warm and reddened skin, are pathognomonic to higes but not essential to the diagnosis [144] . the abscess is filled with pus that always grows staphylococcus aureus; in some cases mucocutaneous candidosis and chronic herpetic keratitis are associated [178] . infections appear within the first 18 months [62] . the face shows coarse and dysmorphic features, midline facial defects such as a prominent nose and a high, arched palate, and disproportionate cheekbones and mandible; pondostatural growth notably retarded [64] , pneumatocele (fig. 22 .38) and osteoporosis caused by reduced bone density with a tendency to fracture [287] complete the picture. six consanguineous families have been reported with an ar form of higes, including 13 cardiac abnormalities, t-cell deficits, cleft palate facial anomalies, and hypocalcaemia. at least 30 genes have been mapped to the deleted region. recently, in 5/13 patients with del22q11.2 syndrome without 22q11 deletion mutations were found in t-box 1 that is a major genetic determinant of the del22q11.2 syndrome [545] . xlp is caused by a defect in the sh2d1a gene (table 22 .1), which binds to the cytoplasmic domains of cd150 slam (signaling lymphocyte activation molecule) and 2b4, and may regulate signals transmitted by these receptors in t and nk cells, respectively [345] . xlp has been reported in >270 males from >80 families [446, 460] , and in an other 27 males [381] , it is inherited with the x-linked model. it is set off in males aged 5-6 by an ebv infection that became manifest with a very polymorphous pattern, often with unusually severe or fatal infections mononucleosis caused by the immune system incapacity to respond to ebv, or evolving into a hgg with iga and igg deficiency and higms, or medullar aplasia and/or a burkitt type lymphoma [446] . the disease has been reported in 15 female subjects [381] . xlp polymorphism could be explained by the fact that the ebv receptor is expressed on differentiating b lymphocytes starting with the preceding isotypic conversion stage [500] . it has recently been verified that before ebv infection, males already suffer from dys-or pan-hgg, incapable of regulating the expression of ig and/or containing b or t lymphoproliferation. even after ebv infection, the immune system is unable to provide adequate th2 responses, and therefore releases cytotoxic alloreactive cd8 and th1-like t cell ils, causing extensive damage to the entire parenchyma, exemplified by fulminating hepatitis, cellular infiltrations and tissular necrosis. the lymphoid tissues with an altered structure are also affected by necrosis, with a high incidence of mostly nonlocalized lymphomas [381] . the thymus is also affected by thymocyte rarification, with 1305 other well-defined id syndromes fig. 22.36 . bone marrow biopsy specimen in a 3-year-old boy: numerous histiocytes in erythrophagocytosis affected children aged 15 months to 12.5 years, with ar-higes presenting with the classic immunological findings, including rri, eczema, elevated serum ige, hypereosinophilia, and severe recurrent fungal and viral infections [64] . notably, patients with ar-higes did not have skeletal or dental abnormalities and did not develop pneumatoceles, as seen in autosomal dominant-higes [404] . among the immunological characteristics (table 22. 14) [93, 287] , cutaneous anergy to several antigens such as candida and tetanic toxoid is characteristic, which is associated with the anomaly of proliferative responses by the t cells to antigens and mitogens, in contrast with the integrity of other functions tested in vitro [196] . t subpopulations appear to be normal [64] . however, the lymphocyte proliferation to anti-cd3/cd28 monoclonal antibodies can be impaired [226] . as noted, ifn-γ deficiency associated with a pathological th2 prevalence has a fundamental impact on ige hyper-production [178, 287] . in higes, some studies have confirmed ifn-g deficiency compared to controls [120, 368] , also due to an impaired response to il 12 [56] , while others have not [62, 512] ; however, compared to ad, normal levels of t producers of il 4 are characteristic [120] . considering the ifn-g/il 4 + correlation of ad, in higes no specific t-cell anomalies are noted, nor does the hyper-ige explain this pediatric abnormal susceptibility to infections: high ige levels are also seen in children with ad, who do not, however, have an unusual predisposition to abscess formation [226] . one typical characteristic is sige directed against microbial antigens: the anti-staphylococcal sige rise to 8.9% compared to normal levels of 0.2%-0.6%. another constant finding is the increase in 100% of cases of eosinophil concentrations, which make up 6%-12% of leukocytes [64] , reaching 30%-50% [178] . by expressing the cd40-cd154 duo, they stimulate the isotype b-cell switching to ige.we studied children affected by severe ad, chronic fa-induced diarrhea and asthma. the allergens responsible were cm and der p [73] . in case of higes caused by fa, atopic manifestations can clearly improve following an exclusion diet, reducing the frequency of infections and partially correcting the immune defect [420] , revealing how fa can induce several immunological anomalies. diagnosis is made on the basis of the data in table 22 .14; differential diagnosis with ad is schematized in table 22 .15 [287] . treatment with cromolyn is extremely effective, anti-staphylococcal antibiotic treatment [64, 226] and if necessary antifungal therapy provide good results [144] . griscelli disease, mapping to chromosome 15q21 [372] , is an ar syndrome caused by mutations in the myo5a (gs1), rab27a (gs2), or mlph (gs3) genes, all of which lead to a similar pigmentary dilution [50, 312] . the disease is also characterized by partial oculocutaneous albinism, predisposition to pyogenic infections and in most patients by abnormal regulation of the immune system, which results in a syndrome of macrophage hyperactivation, known as hemophagocytic lymophohistiocytosis [15] . mutations in the gtp-binding protein rab27a (gs2), which appears to be involved in an uncontrolled t lymphocyte and macrophage activation syndrome, leading to death in absence of bmt, occur in this syndrome [319] . a mutation was found in the myo5a gene (gs1) associated primarily with neurological impairment [319] . two identical twin boys aged 3 months were reported with persisting fever, mouth ulcers, hepatosplenomegaly, pancytopenia and failure to thrive [431] , as was an 8-month-old infant [397] . both infants had silvery-gray hair and pigment clumps on the hair shafts, and skin biopsy showed accumulation of melanocytes on melanosomes. their parents were first cousins and a sibling with similar manifestations had already died, as did the twins. a genetic study revealed a 5-bp deletion in the rab27a gene (510 del aagcc in exon 5) [431] . in a 4-year-old child with hemophagocytic syndrome, id, and secondary neurological disorders, typical melanosome accumulation was found in skin melanocytes and pigment clumps were observed in hair shafts. two heterozygous mutant alleles of the rab27a gene, a c-t transition (c352t) leading to q118stop and a g-c transversion on the exon 5 splicing donor site (g467+1c) were found [50] . the finding of gray strands of hair, gray eyebrows, and eyelids in childhood should alert pediatricians to considering griscelli syndrome since an early diagnosis is life-and health-saving [203] . the phagocyte system with the biochemical basis of cgd is analyzed within the framework of innate immunity. chronic granulomatous disease (cgd) has an overall prevalence of 1:500,000 to 1:10 6 , although this could be underrated (table 22 .4), considering that some subjects may have a very mild clinical phenotype that escapes diagnosis [501] . a us registry of birth rates found a prevalence of 1:200,000 to 1:250,000 live births for the period 1980-1989 [538] . the youngest patient was 27 days old [337] and in 12 children with cgd the mean age at the onset of infections was 5 months, with a median delay in diagnosis of 2.5 years [371] . otherwise the the clinical features of this rare ar disease include oculocutaneous albinism and susceptibility to especially s. aureus and b-hemolytic streptococcus [549] . approximately 85% of patients develop an accelerated phase of the disease, with deposition of lymphohistiocytes in the liver, spleen, lymph nodes and bm, resulting in hepatosplenomegaly, lymphadenopathy, bm infiltration hemophagocytosis, pancytopenia as well as fever, jaundice, prolonged bleeding, easy bruisability, neurological changes (nystagmus and neuropathy), mild mental retardation, and partial ocular and cutaneous albinism [285, 334, 526] . the cellular hallmarks of the disease include large lysosomal granules in leukocytes, giant melanosomes in melanocytes and affecting other cells of the body such as neural schwann cells, renal tubular cells, gastric mucosa, pneumocytes, hepatocytes, langerhans cells of the skin, and adrenal cells [229, 157] . the fundamental defect in this disorder was found to be caused by mutations in a gene mapped to chromosome 1q42-q43 [31] encoding a cytosolic protein on chromosome 1 named lysosomal-trafficking (lyst) regulator, encoding a 425-kd protein whose function remains unknown [285] . bmt is resolutive in these children [205] . normal protein expression (x91 + form), but with a total absence of oxidase due to incorrect binding [412] . ar-cgd is caused by a mutation in the genes encoding the remaining oxidases of 47 kd (p47 phox ) (phox, phagocytic oxidase) [538] (ncf-1), p22 phox of 22 kd (cyba), and p67 phox of 67 kd (ncf-2) [109, 501] . the ar-cgd forms (18.5%-22% of cases) [285, 458] are identified using the immunoblotting technique, depending on whether they affect the p22 phox , the p47 phox or the p67 phox [84, 97, 109, 501] , with greater prevalence in an american study [97] . patients with the x-cgd appear to have a more serious clinical phenotype than patients with the ar-cgd, based on the fact that they are diagnosed significantly earlier (mean, 3.01 years of age vs 7.81 years of age, respectively), have a significantly higher prevalence of infections and a higher mortality (21.2% vs 8.6%) [537] . mutations in any of the 6 structural molecules (table 22 .16) lead to cgd. mutation of rac2 (see lad), the predominant g protein in neutrophils, leads to defects in so production, as well as in chemotaxis [416] . activation of the nadph oxidase requires complex rearrangements between the protein median age at onset was 1.12 months, and the median age at diagnosis was 1.1 years [81] . the deficiency appears in two forms ( [335, 538] , with an h-chain deficiency, is divided into four x91 subtypes (table 22. 16 [84, 97, 109, 112, 185, 501] ), also identified on the basis of nbt results, depending on whether the x91 is absent (the most common form), reduced or present but inactive; subtype x91is divided into two variants: in one of them the nbt is slightly positive in 80%-100% of cells (6% of patients), in the other in 5%-10% (3% of patients) [84, 109, 412, 501] . more precisely, the four subtypes are caused by mutations in the four gp91 phox regions, many of which depend on cybb gene mutations, causing the x91 0 form, while 17 mutations depend on the nadphoxidase activity (x91form) and eight others lead to a 1309 phagocyte deficiency subunits, which are in part mediated by noncovalent binding between src-homology 3 domains (sh3 domains) and proline-rich motifs [447] . cgd is a hereditary disease (table 22 .16) characterized by severe recurrent pyogenic infections. this marked susceptibility is caused by the phagocytes' incapacity to kill in particular the catalase-positive bacteria, because of a genetic defect of the nadph-oxidase enzymatic system situated in the wall of the phagocytic vacuole. in cgd, phagocytosis occurs normally, but the nadph-oxidase is unable to markedly produce anion superoxide (o 2 •-), h 2 o 2 and other o 2 free radicals, thereby permitting the survival of microorganisms within the cells, where they are protected from the antibodies and from most antibiotics [501] . another consequence of the lack of o 2 radicals is the development with countless inflammatory episodes, which then result in typical granulomas [109] . the nitroblue tetrazolium (nbt) reduction test is based on the chemical characteristics: in fact, the phagocytes without o 2 •are unable to reduce the yellow nbt of products activated by pha aspecifically stimulated phagocyte o 2 , or specifically with corpuscle particles such as preopsonized yeasts (fig. 22.41) . the result was 0% in 14 children [81] . at a molecular level, the genes that codify the two subunits of flavocytochrome b588, gp91 phox and p47 phox have been cloned: respectively the cytochrome, h (b) and l (a) chains situated on the phagosome vacuole membrane, and also the cytosolic factors p40 phox , p22 phox and p67 phox , deriving from the nadph-oxidase activation, all proteins placed inside the cytoplasm and that belong to innate immunity. it has therefore been possible to identify molecular lesions at the cgd origin, with the exception of the p21 rac1 [16, 412, 533] . the clinical pattern is severe in the x91 0 form and variable in the other two x91 forms; onset occurs within the 1st year of life in 2/3 of cases, and in others within the 2nd year [159] , although it can appear also at the age of 16 [335] . purulent recurrent infections, with a granulomatous evolution, predominantly affect the epithelial surfaces normally colonized by bacteria, such as cutaneous, subcutaneous, mucous membranes, the respiratory tract and the intestine: cutaneous and mucosal infections, and lymphadenitis lead to suppuration and fistulation (fig. 22.42) , pneumonia or lung abscesses (fig. 22.43 ) are more frequently characterized by persistent fever and diarrhea [159] (table 22. 17) [109, 538] . pneumonia was the most prevalent infection in 369 patients (79%) (mostly by aspergillus), followed by suppurative adenitis (53%), subcutaneous abscess (42%) and liver abscess (27%); mostly by staphylococcus, osteomyelitis (25%) mostly by serratia, and sepsis (18%), and by salmonella [538] . in a long-term trial, pneumonitis was the most prevalent infection (91%) followed by lymphadenitis (83%), aphthous stomatitis (58%), liver abscesses (25%) and chronic lung disease the rates (%) consist of a first [109] and of a second number related to the european study [84] ; us data regarding ar cgd are in parentheses [97] . data from [84, 97, 109, 185, 501] . x x-linked, ar autosomal recessive, ad autosomal dominant inheritance, nd not done. a the superscript symbols indicate the level of immunoreactive proteins: 0 undetected,diminished, + normal protein levels. granulomas in the entire lung parenchyma, which are formed by mononucleates (fig. 22 .44a) with giant cells (fig. 22.44b) . the chronology of infection onset is summarized in table 22 .18 [335] : lymphadenitis is the earliest. osteomyelitis is usually a worrying complication: (58%) [371] . lymphadenitis, lung infections, enteral infections, and hepatic abscesses were the most frequent infections in a cohort of 48 children [335] . staphylococcal liver abscesses are almost pathognomonic of cgd [447, 538] . because the infections develop in areas drained by lymphatics, they tend to diffuse via the lymphohematogen route, thus causing arthritis and osteomyelitis and abscess formation, especially affecting the bones, which are the most severe manifestation, and hepatitis with common upsurge of hepatosplenomegaly. lung infections are almost the rule: those initially segmented and parallel tend to gradually spread over the entire lobe [109, 538] . histological examination shows widespread gastric outlet obstruction 10 15 urinary obstruction extensive bone destruction involves various segments, for example the vertebra, the metacarpus and the metatarsus, causing widespread damage, which is difficult to treat and is also irreversible [109, 501] . aspergillus, pulmonary, bone (fig. 22 .44c) or encephalic infections constitute a severe therapeutic problem and are threatening events, with a mortality rate of 26%, but with specific treatment the prognosis is good as far as recovery is concerned [335] . the treatment includes prophylaxis with trimethoprim-sulfamethoxazole (tmp/smx) (5 mg/day given in two divided doses), and ifn-g (50 mg/m 2 subcutaneously thrice weekly) in all patients with cgd, regardless of genotype [81, 416] . itraconazole therapy (5 and then 10 mg/kg/day) has an excellent tolerance in all cases and was effective in 29 of 32 children (90.6%) [336] . survival until the age of 21 and beyond is achieved by 20% of patients with cgd xl and 37% of those with cgd ar [339] . because the prognosis is uncertain, as observed, the only possibility for a definite resolution is with a bmt, from family donors who are x-cgd or x-cgd-identical [393] . bmt was successful in 27 children out of 31 (87.1%) (see table 22 .30), including a 4-year-old boy with x-cgd who underwent successful hla-identical peripheral blood sc transplantation during invasive pulmonary aspergillosis and osteomyelitis, which was unresponsive to antifungal treatment [48] . lad is due to mutations in the gene on chromosome 21 at position q22.3 encoding cd18 (table 22 .1). it is divided into five types: lad type i to lad type v [24, 71, 138, 367] . the three subunits of the cd11/cd18 complex are involved in pid (lad type i syndrome), ar, linked to the lack of a m b 2 equal to cd11b/cd18 (table 1.46) surface expression on all leukocyte populations caused by 20 different mutations in the cd18 encoding gene, often severe in infancy [145, 202] . children with a deficiency of these integrins have a defect above all in phagocyte action, suffer from severe infections from the neonatal period [202] due to absent b2-integrin activity, which impairs neutrophil ability to exit the circulation and travel to sites of infection. on the contrary, leukocyte movements are not prevented, indicating the normal involvement of cd54 and cd102 (table 1.4) . the clinical basis for defining this disease, described in over 200 cases [145] , dates back to a study at the soothill school in 1979 [215] . there are two forms of lad type i [71] : if the deficiency is full blown (no detectable cd18), the clinical symptoms (table 22. 19) [71, 138] are dominated by severe and recurrent infections with a negative prognosis in the first years of life unless corrected by an allogenic bmt, the only resolutive treatment [492] . if instead it is a partial deficiency with residual cd18 expression, the clinical outline is less severe and some patients, with appropriate treatment, can live to adult age [24] . lad type ii, ar (cd18 levels are 1%-10% of the normal levels), with a molecular base represented by an slex ligand (cd15s) is a defect common to cd62 e and p, which mediate neutrophil rolling. in the absence of a gdp-fucose transporter, the slex is not made. lad type 2 results from mutations in this transporter that takes fucose into the golgi apparatus for posttranslational fucosylation of newly synthesized proteins. this is the ligand for cd62e; without it, leukocytes cannot make initial attachment to vascular endothelium [7] . lad has been described in two children aged 3 and 5 with mental retardation, from different families, but both with parents who were blood relatives [145] . it has a lower mortality rate [138] . mice with a deficiency of both selectins show a lad-like syndrome, providing a useful model for studying these syndromes [171] . lad type iii shows defective tethering and adhesion and bleeding diathesis. this is a new syndrome where in vitro leukocytes showed normal rolling along endothelial cell cultures but defective tethering and tight adhesion. thus this is a defect in the capability of vascular integrins on circulating leukocytes to rearrange with their endothelial ligands at adhesive contacts and rapidly arrest on target vascular endothelium in response to endothelial-displayed chemoattractants. however, the expression levels of the major integrins on lymphocytes and neutrophils were largely conserved in the patient cells, ruling out a lad-i syndrome. patient leukocytes showed no lad-ii like fucosylation defect, since they expressed normal levels of the fucosylated marker cd15s, comprising the slex carbohydrate selectin ligand [7] . defects in both leukocyte and platelet functions that are biochemically and molecularly distinct from the adhesion disorders previously described suggest a mutation in an early myeloid pathway. the defect is associated with regulation of the gtpase activating protein rap1, as demonstrated by the intact rap1 expression and activation by phorbol esters, thus ruling out an lad defect in rap1 gtp loading [255] . lad type iv manifests defective cd62e expression or tethering. a girl developed pseudomonas omphalitis at 5 weeks of age, recurrent ear and urinary tract infections, and had clinical evidence of impaired pus formation reminiscent of a lad syndrome, but her neutrophils were functionally normal and expressed normal levels of cd18, cd62e, and slex. however, the patient showed an absence of cd62e from the endothelium, although e-selectin mrna was present. in contrast to patients with lad 1, she had mild chronic neutropenia but appropriate leukocyte increases in response to infections or gm-csf. a bm biopsy performed during a period of health showed normal cellularity for her age. her fh is remarkable only for a previous sibling who had died at 32 weeks of gestation of a staphylococcal infection of the fetus, amniotic fluid, and 1313 phagocyte deficiency placenta. she also has two half-sisters who are completely well. the fh is negative for recurrent infections in either parent or more distant relatives [121] . lad type v caused by rac2 deficiency. a 5-week-old boy born to unrelated parents had delayed uc separation, perirectal abscesses, poor wound healing, and absent pus at sites of infection in the setting of neutrophilia, suggesting a neutrophil defect. his neutrophils exhibited decreased chemotaxis, polarization, azurophilic granule secretion, as well as significantly reduced stimulated superoxide production but had normal expression and up-regulation of cd11b. rac2 constitutes more than 96% of the rac in neutrophils [9] . a 1-yearold boy who had multiple recurrent, life-threatening infections characterized by leukocytosis and notable for the absence of pus in the inflamed tissues was reported. the presence and density of cd11b, cd11c, and cd18 were normal. the expression of cd62p and cd62l were also normal. a bmt was curative. the boy shared a phenotype that closely mimicked that of a mouse mutant deficient in the rho gtpase, rac2 [534] . the disease was shown to be attributable to an ad mutation in the rho gtpase rac2 at an amino acid needed for proper interaction with other intracellular proteins. rac2 comprises >96% of the critically important g protein rac in neutrophils. each member of the family appears to control a distinct function of the actin cytoskeleton (chemotaxis and degranulation) and nadph oxidase (superoxide production) function [9] . a male child from the mother's first pregnancy was born at term from parents of arab ethnic origin who were first cousins. he had a severe genetic disorder associated with functional defects in multiple leukocyte integrins, reflected in recurrent infections, profound leukocytosis and a bleeding diathesis. platelet transfusions and antibiotic courses reduced the symptoms, which remained a significant clinical problem. at age 6 years, he died from disseminated fungal infection after a mismatched bmt. a younger brother presented with the same clinical and hematological phenotypes at birth and died at age 1 week from sepsis. g6pd converts g6p to 6-phosphogluconolactone, generating nadph and a h + ion from nadp + . nadph oxidase catalyzes the monovalent reduction of o 2 to o 2 •-, with the subsequent conversion to h 2 o 2 by superoxide dismutase [285] . in the form of a partial deficiency, known as the cause of hemolytic anemia or favism, the enzyme's residual activity (20%-25%) permits nor-mal bactericidal activity. the 400 g6pd variants have been classified by the level of residual enzyme activity and propensity for hemolysis and grouped into five classes: class i, severely deficient with chronic hemolytic anemia; class ii, severely deficient with occasional hemolytic anemia (<10% residual activity); class iii, moderately deficient (10%-60% residual activity); class iv, normal activity (60%-150%); class v, increased activity [310] . in a trial on 161 g6pd-deficient subjects originating from different parts of italy, a greater molecular heterogeneity than described by others was observed, especially in sardinia [310] . in a complete deficiency, sexually transmitted, whose gene is localized on the chromosome x at position p28 and characterized by several mutations and their variants, with a consequent deficiency of bactericidal activity, the neutrophils are unable to kill s. aureus, e. coli and serratia, and therefore there is an increased susceptibility to infections, rather like cgd [109] . the diagnostic work-up of children may reveal a child with recurrent infections who initially received the diagnosis of g6pd deficiency, subsequently shown to have the phenotype of x-linked cgd [3] . the disorder has a higher incidence in mediterranean countries and asia, in japan (10.6%) than in indonesia (4.3%), as ascertained with a novel screening kit [234] , and is low in newborns in tehran, iran (2.1%) [1] . within the framework of oxygen-dependent killing defects, hereditary myeloperoxidase deficiency (mpo) is the most common neutrophil biochemical defect and plays an important role in the host defense mechanism against microbial diseases. the neutrophil disorder characterized by the lack of mpo activity is speculated to be associated with a decreased level of immunity. mpo is unusually accompanied by a specific pathology. ar transmitted, it appears far more common than previously suspected (1:2,000 for the partial deficiency to 1:4,000 for the total deficiency). it is a disorder that is prevalently recorded in entirely healthy patients and therefore, in most cases, a random laboratory finding. in addition to three already-known mutations, the genetic characterization of an italian population showed the presence of six novel mutations: four missense mutations, a deletion of an adenine within exon 3 (c.325dela) and a mutation within the 3¢ splice site of intron 11 (c.2031-2a>c). the c.325dela deletion causes a shift in the reading frame with the occurrence of a premature stop codon within the pro-peptide. the activation of a cryptic 3¢ splice site located 109nt upstream of the authentic 3¢ splice site causes a shift in the reading frame that may lead to the generation of an abnormal mpo precursor lacking the enzymatic activity [303] . in a japanese patient with complete mpo deficiency, neutrophil function analysis revealed that mpo activity was alkaline phosphatase [414] . monocyte functional alterations in the second individual suggest that c/ebph plays a critical role in monocyte/macrophage development of humans and implicates abnormalities in monocytes/macrophages and neutrophils in the onset and development of the disorder [455] . severe congenital neutropenia (scn) and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. recently, mutations of the gene encoding neutrophil elastase 2 (ela2) have been indicated as the most common cause for scn as well as the cause for autosomal dominant cyclic neutropenia [225] . deficiency of ela2 leads to regularly fluctuating levels of neutrophils [112] . linkage analysis on 13 affected pedigrees have shown that cyclic neutropenia and sporadic cases of this disease are due to a mutation in the gene for ela2, located at 19p13.3 [112] . this enzyme is synthesized in neutrophil precursors early in the process of primary granule formation [225] . a mutation in the ela2 gene was detected in one of three apparently autosomal dominant kindreds with familial scn. no mutations were identified in the apparently ar families [12] . these results fit those showing that mutations were found in all five scn families [112] , but they suggest that not all cases of autosomal dominant scn caused by mutations in ela2 [12] . however, the high frequency of het mutations in the neutrophil elastase gene in sporadic scn confirms a previous report [112] . considering that four novel mutations and a low-frequency polymorphism were detected, nearly all cases of sporadic scn may result from de novo het mutations in ela2 [12] . in recurrent scn, an absolute neutrophil count of <200 cells/mm 3 (or <0.1¥10 9 /l) [12] oscillates with an approximate 21-day periodicity. circulating neutrophils vary between almost normal numbers and zero [225] . in about 30% of patients with cyclic neutropenia, however, the cycles range from 14 to 36 days [42] . in 26 children referred during a 22-year period pids were as follows: cyclic neutropenia (30.7%), shwachman-diamond syndrome (26.9%), kostmann syndrome (23%), and chédiak-higashi syndrome (19.2%). the mean absolute neutrophil count of children was 398.2±259.3 cells/mm (range, 74-1,152/mm) at the first visit. the children first experienced symptoms of infection suggesting neutropenia at a median age of 7.5 months (range 1 month to 10 years), also suffering from oral ulcer, otitis, pneumonia, diarrhea, cutaneous abscess, and oral candidiasis [398] . fever, stomatitis, and periodontitis and skin infections occur during periods when the neutrophil count is low. significantly diminished with slightly elevated superoxide production. mutational analysis of the patient revealed a glycine to serine substitution (g501s) in the exon 9 region [356] . because the granulocytes without mpo cannot kill candida, some subjects, presumably carriers of a more extensive mutation and in association with other diseases, present severe and recurrent candida infections. the mpo defect can be diagnosed via a cytochemical investigation or a quantitative count of enzyme levels [303] . neutrophil-specific granule deficiency is a rare autosomal dominant disorder characterized by recurrent pyogenic infections, defective neutrophil chemotaxis and bactericidal activity, and lack of neutrophil secondary granule proteins [284] . the markedly decreased level of mrna expression for the bactericidal/permeability-increasing (bpi) protein, the activation factor pu-1 and defensins in these patients suggests a role for ccaat/enhancer binding protein (c/ebpη) gene in earlier phases of the myeloid differentiation program [187] . c/ebpη is a member of the leucine zipper family of transcription factors, expressed primarily in myeloid cells [284] . recessive mutations in the c/ebpη gene were described in one patient; analyses of the c/ebpη locus indicated that the disorder could have resulted from hz recessive inheritance of the mutant allele from an ancestor shared by both parents [187] . loss of c/ebph function is the primary genetic defect in this disease [455] . in a second individual lacking functional c/ebph, analysis of peripheral blood leukocytes revealed aberrant expression of cd45, cd11b, cd14, cd15, and cd16 on the proband cells [455] . a male patient lacking neutrophilspecific granules died from complications of pneumonia at age 20 [284] . neutrophil-specific granules contain important microbicidal components (table 1.23) . among other deficiencies of oxygen-independent killing, this ar defect is characterized by severe recurrent bacterial deep-tissue skin infections without patients showing an increased susceptibility to a particular pathogen. they have defects in chemotaxis, disaggregation, and receptor up-regulation. deficiencies of the oxidoreduction and microorganism-killing mechanisms have also been described. the markedly decreased level of mrna expression for the bactericidal/ permeability-increasing (bpi) protein, the activation factor pu-1 and defensins in these patients suggests a role for c/ebph in earlier phases of the myeloid differentiation program [187] . the defect is identified through a blood test colored with a wright reactive in which polymorphonucleates do not present the specific granules that normally contain lactoferrin. from a morphological point of view, the nuclei appear bilobated and the nuclear membrane may show intro-and extroversions. it is also possible to identify the membrane's lack of cyclic neutropenia is an autosomal dominant disorder in which cyclic hematopoiesis causes intervals of neutropenia and susceptibility to opportunistic infection. in nine families whose children displayed typical blood patterns, pedigrees confirmed dominant inheritance without evidence of heterogeneity or decreased penetrance; three pedigrees suggested new mutations [369] . a wide spectrum of symptom severity, ranging from asymptomatic to life-threatening illness, was observed within the nine families. the phenotype changed with age. children displayed typical neutrophil cycles with symptoms of mucosal ulceration, lymphadenopathy, and infections [369] . patients are usually asymptomatic, but during the period of severe neutropenia, recurrent overwhelming infections, inflammation, and ulcers occur in about 10% of patients and can lead to significant chronic morbidity [298] . severe neutropenia was shown by 21 children, moderate by 4, and mild by 1: 16 of these children had leukopenia, 7 anemia, 2 thrombocytopenia, and 1 monocytosis. during follow-up, respiratory infections developed in 24, oral manifestations in 20 children. the most common infections, in descending order of frequency, were otitis media, abscesses, pneumonia, oral ulcers, acute diarrhea, cutaneous infections, oral candidiasis, and periodontits. sinusitis, cystitis, conjunctivitis, meningitis, and osteomyelitis were less frequently observed. hepatomegaly was also detected in 10 children and splenomegaly in one; 3 children died of recurrent infections. therefore, recurrent infections always deserve further evaluation for detecting such disorders [398] . abdominal pain must be assessed aggressively because of the high frequency of clostridium infections during the period of severe neutropenia [369] . during the course of scn, bm shows lack of maturation of granulocyte precursors beyond myelocytes, and there is myeloid hyperplasia during the remainder of the cycle. occasionally, there is a reduction in the severity of neutropenia and the accompanying infections over time [369] . a complete clearing of symptoms and a significant increase in quality of life is noteworthy in children [298] . however, while the disease is commonly described as benign, four children in three of the nine families died of clostridium or e. coli colitis, documenting the need for urgent evaluation of abdominal pain [369] . pediatric cyclic neutropenia is effectively treated with rhug-csf (recombinant human g-csf), usually at doses of 1-5 mg/kg/day (median dose, 2.5 mg/kg/day) [449] or twice weekly, or once a month. typically, children are noted in early infancy to have persistent scn with absolute neutrophil counts <0.2¥10 9 /l lasting for months or years [12] . in children aged 4 days to 19 months, the initial and lowest median absolute neutrophil counts were 0.29¥10 9 /l and 0.06¥10 9 /l, respectively [289] . usually, children suffer from long-term recurrent bacterial infections, and maturation arrest of myelopoiesis at the promyelocytemyelocyte stage of bm development [12] . the disease begins during the 1st year of life, and its infectious complications include cellulitis, perirectal abscess, peritonitis, stomatitis, and meningitis, commonly as a result of infections with s. aureus, e. coli and pseudomonas aeruginosa [42] . the numbers of circulating monocytes and eosinophils are often increased [42] . missing the most important cells in the defense against bacterial infections, the neutrophil granulocytes, children suffer from episodes of severe, often life-threatening bacterial infections [42] . they spend many days in hospital, requiring iv antibiotic treatment. recurrence of bacterial infections leads to irreversible tissue damage, for example in the lungs, requiring often disabling surgical interventions. a high incidence of significant bone mineral loss was seen in children with scn [545] . the presence of qualitative and quantitative abnormalities of primitive myeloid progenitor cells expressing g-csfr may play an important role in the impairment of granulopoiesis in these patients, thus nearly all patients have a response to pharmacological doses of rhug-csf: neutrophil counts rise, infection rates fall, and mortality is reduced [343] . since the introduction of rhug-csf, most children enjoy a normal life span and a greatly improved quality of life, although they still have problems with infections, especially chronic gingivitis and periodontitis [82] . it is more likely that the bone loss was caused by the pathophysiological features of the underlying disease, but it is possible that rhug-csf accelerates bone mineral loss [545] . prolonged administration of rhug-csf at a dose of 3 u/kg bw twice daily may be associated with increased bone resorption, mediated by osteoclast activation and leading to bone loss. in children, the resulting osteopenia can be successfully managed with antiresorptive bisphosphonate therapy with significant improvement in bone density [449] . a child maintained on long-term rhug-csf therapy developed acute myelogenous leukemia associated with a g-csfr mutation.after having undergone successful allogeneic bmt, both ela-2 mutation and g-csfr mutation became undetectable by pcr [237] . shwachman syndrome, a rare ar condition, characterized by pancreatic insufficiency, reduced mobility and neutrophil chemotaxis, cyclic neutropenia, thrombocytopenia, metaphyseal dysostosis, delayed growth and recurrent pyogenic infections, in two cases was associated with isolated gh deficiency [96, 268] . in addition to metaphyseal chondrodysplasia, neutropenia, and pan-poor to absent granuloma formation [242] . salmonella and certain viral infections [hsv, cmv, parainfluenza, and respiratory syncytial virus (rsv)] are also seen [126] . most patients bearing an ifn-gr1 deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome [6] . mortality in these children is high, and infections are severe and recurrent [242] , as in an 8-year-old girl before receiving a bmt [405] . a point mutation may be fatal: an individual, probably hz for the mutation, died from meningitis due to mycobacterium bovis [6] .a hz missense ifn-gr1 mutation was identified in two siblings who did not respond to low or intermediate concentrations, yet responded to high ifn-g concentrations, probably for a reduced affinity of ifn-gr1 for its ligand [243] . otherwise the mutation results in normal surface expression of ifn-gr1 that do not bind ifn-g [244] . a dominant deletion in the ifn-gr1 gene has been reported in a female patient hz for a 4-bp deletion in exon 5 of ifn-gr1 who developed postvaccinal disseminated bcg infection [417] . the ar form of partial ifn-gr1 deficiency was reported in 18 patients of 12 unrelated kindred with susceptibility to mycobacterial infection [403] . an 8-year-old girl with ifn-gr1 deficiency, also with recurrent mycobacterial infections and liver cirrhosis with portal hypertension, received red cell-depleted bmt from her hla-identical sister. the transplantation course was uneventful and 4 years later the child remains in excellent clinical condition and free of mycobacterial infections [405] . a complete ifn-gr2 deficiency was found in a child due to a hz dinucleotide deletion resulting in a premature stop codon in the protein extracellular domain. this gene defect emphasizes the critical role that ifn-g plays in host defense against mycobacteria [126] . a girl with bcg and salmonella enteritidis infection and a hz recessive deletion in the p40 subunit of il 12 leading to a complete il 12 p40 deficiency has been reported. a large hz deletion within the il 12 p40 subunit gene was found, precluding il 12 p70 (composed of p40 and p35 subunits) functional expression by activated dcs and phagocytes. the net result was a markedly impaired ifn-g production by lymphocytes. however, addition of recombinant exogenous il 12 p70 in the assay was able to restore normal ifn-g production in vitro [8] . the girl suffered from well-organized granulomas, possibly due to residual il 12 -independent ifn-g production [8] . another kindred [377] and two siblings and one unrelated patient [142] carried the same large deletion, also accompanied by disseminated infections. a 3-year-old female was repeatedly hospitalized since the age of 5 weeks for recurrent episodes of pneumococcal pneumonia with sepsis and other infections in the absence of creatic exocrine insufficiency, the findings in children are noted as variable extremity shortening, cup deformation of the ribs, metaphyseal widening and hypoplasia of the iliac bones, and increased echogenicity of the pancreas with no change in size [43] . recurrent infections begin during the 1st year of life and commonly involve the sinuses, lungs, bones, skin, and urinary tract [42] . neutropenia, either cyclic or intermittent, occurs in all patients, and 10%-25% of patients also have pancytopenia [464] . immune functions may be involved in this syndrome, including marked pan-hgg, especially of the iga, normal/increased cellular immunity, but depressed humoral and nk cell immunity [268] . in 13 patients diagnosed in infancy, a significant growth improvement and a decreasing frequency of infections were observed over time, in addition to improvement or normalization of exocrine pancreatic function [96] . a continuous spectrum from systemic bcg infection to local recurrent nontuberculous mycobacterial infection covered by the clinical features of affected children has recently helped to identify several genetic defects in the monocyte-macrophage-th1 t-cell pathway [509] . different types of mutations in four genes (ifn-gr1, ifn-gr2, il 12 p40, il 12 rb1) forming the ifn-γ/il 12 axis [123] have revealed both allelic and nonallelic heterogeneity and result in different disorders whose common pathogenic pathway is impaired ifn-g-mediated immunity [123, 403] . several children have been reported who presented a new kind of hereditary id with severe and/or recurrent infections caused by only one microorganism family, in opposition to other patients with classic pid. five new syndromes may encompass these children with a genetic predisposition to infectious diseases. if the ifn-g/il 12 axis is impaired, the host becomes highly susceptible to infection with organisms that replicate intracellularly (susceptibility to mycobacterial disease). stat-1 (signal transducer and activator of transcription-1) deficiency predisposes to viral disease, nemo and irak-4 (il 1 r-activating kinase-4) deficiencies predispose to infections caused by pyogenic bacteria [376] . this pid encompasses several defects: complete, partial, and ar ifn-gr1 deficiency, and complete, partial, and ad ifn-gr2 deficiency [122] . ifn-g and the cellular responses induced by it are essential for controlling mycobacterial infections. patients with ar mutations leading to complete loss of ifn-gr1 or ifn-gr2 expression have the most severe phenotypes, and they present early in life with disseminated severe infections, especially if they have received bcg vaccination, and have fever. she exhibited il 12 deficiency that was associated with an abnormality of the il 12 p40 gene. although present, ifn-g was reduced [211] . a genetic lack of il 12 rb1 surface expression predisposes to severe infections by pathogenic mycobacteria or salmonella and causes strongly decreased, but not completely abrogated ifn-g production [408] . the deficiency may be complete as well as partial [291] . several patients with these features have been reported [291] . three unrelated individuals with severe, idiopathic mycobacterial and salmonella infections were found to lack il 12 rβ1 chain expression. il 12 rβ1 sequence analysis revealed genetic mutations that resulted in premature stop codons in the extracellular domain [116] . a patient with severe infections as above and multiple adverse drug reactions had t cells unable to produce ifn-g or proliferate in response to il 12 , despite the expression of wild-type il 12 rβ1 and il 12 rβ2 [186] . defective il 12 r signaling leads to low t-cell and nk-cell ifn-g production [509] . il 12 rb1 and il 23 rb1 chains are associated in an ar deficiency with susceptibility to mycobacteria and salmonella infections [351] . the stat4 (2 forms, ad and ar [351] ) s721 mutant failed to restore ifn-g production in stat4-deficient il 12 rb2 transgenic cells [329] . stat1, -3, and -5 activation by il 12 was lost, an impairment specific for il 12 ; nor is activation of stat4 alone sufficient for il 12 -induced ifn-g production and proliferation [186] . two unrelated infants hz with respect to mutated stat1 suffered from mycobacterial disease, but unlike patients with ifn-gr deficiency both died of viral disease [133] the complement is an integral part of the humoral defense system against infections and also for promoting inflammatory process (figs. 1.63, 1.65 ). complement deficiency was found in 6/176 dutch patients (3.4%) over a 33-year period (0.1% × year) [156] . from the study of blood donors the prevalence may be of 0.03% in the general population [531] . congenital deficiencies have been described for most of the proteins it is composed of (tables 22.20 and 22 .1f [167, 169, 453, 531] , usually following the ar model. properdin deficiency is the only complement deficiency that is x-linked [531] . hets can be easily identified because their relevant component is present in the serum with a 50% concentration. the lack of one component at the hz level serologically involves the blockage of enzyme release below and the absence of hemolytic activity, while that of controlling proteins causes its uncontrolled activation, consuming the factor that is the object of control and, in various ways, also of successive components [137] . nonfunctional c1q variants have been observed, c1r and c1s deficiencies are often associated, probably because they are mapped on contiguous genes of chromosome 12 (c1q on 1) [167] . the b, c2 and c4 genes, situ-ated on the short limb of chromosome 6, constitute along with others the hla class iii (chap. 1). c6 and c7 are codified on chromosome 5p and have a similar structure; c8 shows a different structure, because the molecule consists in three a, b and g chains, united to form two subunits, a-g and b dictated by different genes [495] . alternative pathway deficiencies are extremely rare [328] . complement deficiencies are accompanied by an increased frequency of infectious pathologies [155] , although it is not rare to come across them in individuals who are apparently in good health, as in the case of c2 hereditary deficiency [422] . also frequent are teens and young adults with autoimmune manifestations (chap. 18). classic pathway deficiencies are often associated with sle-like diseases (systemic lupus erythematosus), id of the early components of complement (c1-c3) are associated with risks of infections caused by encapsulated bacteria such as streptococcus pneumoniae, haemophilus influenzae type b, as well as by meningococci [363] . the incidence of sle in patients with c1q, c4, or c2 deficiency is 90%, 75%, and 15%, respectively [378] . partial c4 deficiency is also associated with sle; 15% of patients with sle exhibit c4a deficiency [531] . several components are associated with development of membranoproliferative glomerulonephritis (table 22 .20 [167, 169, 453, 531] ). in alternative pathway id, the infections recognize pyogens as the most common etiological agents, while final common pathway id (c5-c9) or properdin (p) have been associated with recurrent or invasive infections by neisseria (n) gonorrhoeae or n. meningitidis, gramnegative bacteria, and asplenia, agammaglobulinemia [167, 169, 363, 531] . it is estimated that the frequency of meningitis in subjects with hz deficiency of the final c5-c9 pathway is 10%, 6,000-fold higher than in non-id individuals [363, 488] . some characteristics appear to associate the patients with complement deficiency and meningococcal disease: frequent recurrent episodes, an older age at the first onset, lower mortality compared to patients with a normal complement, and a prevalence of males [167] . over 50 patients with c1q, c1r and c1s deficiencies have been described, c1s deficiency only in two cases [239] . a selective and complete c1s deficiency in a 2-year-old girl with complex aids including sle-like syndrome, hashimoto's thyroiditis, and autoimmune hepatitis has been reported. exon-specific amplification of genomic dna by pcr followed by direct sequence analysis revealed a mz nonsense mutation in the c1s gene exon xii at codon 534. both parents were het for this mutation [127] . a deficiency in one of these proteins is sufficient to block the classic pathway activation; deficiency results as a consequence of non-synthesis, which in the unlike c2, hz c4 deficiency is very rare and is caused by the non-expression of all 4 alleles (2 of c4a and 2 of c4b, 2 maternal and 2 paternal alleles), which can occur due to punctiform mutations, gene deletions, or other gene alterations that prevent gene transcription [28] . the two 4a and 4b genes are polymorphous, just like c2, c3, c6, c4a and c4b and the b factor (bf); polymorphic variants of the other proteins are rare, with 12 different alleles for c4a and 23 for c4b identified at the moment. moreover, two loci c4a and c4b null alleles q0 (quantity 0), do not codify for any phenotype, although often present in the general population [167] . in a 4-year-old caucasian child who suffered from several bouts of pneumonia caused by respiratory viruses, eight episodes of acute otitis media, prolonged respiratory and urinary tract infections, molecular studies of the c4 gene region revealed hz deletion of hla class iii cyp21a-tnxa-rp2-c4b, generating total deficiency of case of c1q amounts to 60% of cases, while in the remaining 40% the molecules are malfunctioning but cross-reacting with the native molecule [328] . c1r and c1s deficiencies are usually combined, due to the contiguity of the two genes; typically in these patients c1r is absent and c1s levels are reduced (20%-40%) [422] . affected patients suffered from a sle-like syndrome and sporadically from an extended predisposition to infections [155] . associated symptoms in c1q deficiency are sle-like syndrome, rheumatic disease, and infection. several children suffered from meningitis, recurrent septicemia, recurrent otitis media, pneumonia, and stomatitis; two died from meningitis septicemia [241] . the incidence is based on the data from [167] ; the inheritance is always ar, with the exception of c1-inh deficiency (autosomal dominant) and factor p deficiency (autosomal recessive or x-linked). data from [167, 169, 453, 531] . gn glomerulonephritis, jra juvenile rheumatoid arthritis. c4b and the flanking 5¢ region up to c4a [230] . moreover, in 7/13 cases the c4a*q0 alleles were related to a c4a/cyp21p gene deletion within the hla-b8 c2c bfs c4aq0b1 dr3 haplotype. in 3/13 cases, the c4b*q0 allele was related to a c4b/cyp21p gene deletion within the hla-b18 c2c bff1 c4a3bq0 dr3 haplotype [212] . the c4 null allele incidence is so elevated that 60% of the population expresses all c4 genes, while 30% lacks 1-3 alleles [328] . the elevated number of q0 probably derives from the marked similitude of the two genes, which facilitates the unequal crossover, but this crossover in the hla can modulate the expression of three c4a alleles and one c4b or vice versa; the c4aq0 allele spreading amplifies the risk of contracting sle and juvenile ra (jra). hz c2 deficiency, the most common in the caucasian population, has an incidence that varies between 1:10,000 and 1:28,000, whereas the het carrier rate is 1.2% [456] . it is usually found in the a25, b18, dr2, bfs, c2q0, c4a, c4b2 haplotype context which, due to its considerable rarity, could assume a predictive value [238] . two different types of c2 deficiency are known: in type i the synthesis is deficient due to the protein nontranslation, in type ii there is a selective absence of secretion but not of synthesis, therefore c2 levels are 0.5%-2% of normal values [238] . hets have a nonfunctioning gene, the complement profile is characterized by serum c2 concentrations equal to 50% of normal values; about 50% are asymptomatic, while the other half exhibit frequent infections and quite a few suffer from sle and correlated syndromes [155] . het c2 deficiency was associated with a 28-bp deletion in the c2 gene (type i), mainly within the hla-a25 b18 c2q0 bfs c4a4b2 dr2 haplotype [212] . in a certain number of cases, a c2 deficiency is accompanied by a partial bf malfunctioning, genetically close to it. hzs can also have a deficient function of the alternative pathway [445] . possibly this deficiency, like other quite common ones, may not always be reported in the literature: the total number of cases therefore underestimates the real prevalence, as is also found in children with c7 deficiency [167] . c2 deficiency must be suspected in all patients presenting pneumococcal infections after the age of 2 years [239] . both c2 and c4 predispose to sle, but this is not the expression of a particular genetic association caused by the same gene localization, because c1q, r, s, deficiencies, which also cause sle, are situated as mentioned outside the hla system [422] . the molecular bases of this pid appear heterogeneous. the c3 gene exists in different allelic forms, some of which have reduced functionality. one must remember the c3 important role in immune responses, also as far as apc and b cells are concerned, as well as the defensive role played in innate immunity along with c4. since both pathways converge in the cleavage and activation of c3, there is no way that this defect can be corrected, and furthermore the opsonic power is greatly deficient, as is the c5 chemotaxis; therefore patients affected by hz deficiency mostly present clinical symptoms totally similar to a congenital hgg with severe recurrent infections and at times the symptoms of cic disease [239] . although id is severe, some patients apparently remain in good health and the syndrome may also in time become less severe, probably due to the higher number of immune experiences that allow a better effector function to antibody reactions mediated by the fc receptor [167] . c3 protein was defective in noninfected nigerian children with protein-energy malnutrition (pem), but rose significantly in the presence of bacterial infection, thus sharing the values found in healthy controls [137] . in its clinical expressions, c5 deficiency does not differ from the other deficiencies discussed here; the clinical consequences of absent c5a anaphylotoxin are unclear [328] . one-fourth of all patients are asymptomatic. in caucasians incidence is 1:60,000 [495] . deficiencies associated with c6-c7 are rare but reflect the close genetic proximity of their pertinent genes; in c6-c8 deficiencies, 63% of patients lacking one component experience at least one severe episode of neisseria infection and 5.5% one aid [239] . rare in europe, c7 deficiency is the second most common complement deficiency in the japanese (0.005%) [339] . in italian children it has a prevalence of 10% and has been identified also in healthy siblings [531] . in japan c7 deficiency is more associated with meningococcal meningitis than with neisseria infections [339] . in a highly inbred arab population, a c7 deficiency was associated with a mutation (g1135c) that is also prevalent among israeli jews of moroccan ancestry [34] . a total deficiency of this 150-kd protein, inherited as an ar trait, has been described in a young patient with a hemolytic-uremic syndrome and in one case in italy whose parents were first cousins [531] . among 21 relatives of the proband studied, encompassing 3 generations, ten had low factor h levels, including her two children, indicating a het factor h deficiency [157] . h deficiency results in uncontrolled breakdown of c3, and in depletion of bf, p and c5 [157] . c3 and c9 components are decreased in varying degrees, while c3 and c5 are found in plasma in traces and only as activated molecules [167] . inheritance of factor d deficiency is for the moment uncertain [488] (table 22 .20): a partial deficiency (6%-12% of normal concentrations) has been described in two mz twins, and a total deficiency in one male. this deficiency, serologically characterized by the non-functionality of the alternative pathway, is clinically accompanied by an increased susceptibility to neisseria infections [239] , leading us once more to emphasize the alternative pathway significance as a substantial means of defense for a broad spectrum of damaging actions caused by bacterial infections. properdin deficiency is the only one inherited as a characteristic linked to the chromosome x and only affects males [190] . at the moment, >50 cases have been described. the deficiency can be materialized by a total p absence, with levels reduced to 10%, with normal levels, however, showing an altered functional activity [239] . specific research has shown a remarkable reduction of c3a and b titers, which represent the c3-convertase proteins, with a consequent heightened consumption due to the alternative pathway spontaneous activation. males are affected by septic episodes caused by neisseria, sometime fulminating, with onset even occurring during the 1st year of life [488] . there is no evidence of increased susceptibility to cic diseases or infections caused by other organisms [155] , thus implying that a functioning alternative pathway is particularly important for a defense against infections [328] . correctly identifying children with complement abnormalities is important and worthwhile if any of the following factors are present: id (such as repeated or unusual infections with other organisms, fh, unusual course of the illness, etc.), repeated neisserial infections, infection with an unusual serogroup, fulminant disease in males (p deficiency), coexisting angioedema, autoimmune, or connective tissue disorders [220] . in the two forms of c8 deficiency (c8a + c8g and c8b mapped on chromosome 1), the subunit not involved is present in the serum, also with reduced levels, and accompanied by altered functionality of the one involved. c8 deficiency has different characteristics due to the diverse associations of the b and a-g chains; therefore caucasians with this deficiency lack the b chain (10%), while colored patients lack the a and g chains (90%) [495] . c9 deficiencies are poorly considered because they are often asymptomatic [339] ; however, in japan there is an incidence of 0.1% [339] and between 33% [155] and 25% of cases [339] present meningococcal meningitis. congenital c9 deficiencies are very common among the japanese (0.036%-0.1%) and represent 11.3% of all deficiencies (table 22 .20). there have been >150 cases of congenital c5-c9 deficiencies reported, distributed unevenly between the various ethnic groups: c5 and c6 deficiencies are prevalent in colored patients and the c7 deficiencies appear to be more common in caucasians [422] . an analysis of published studies shows that 14% of patients with sporadic meningococcal infections may have a c5-c9 deficiency [495] . clinical patterns are overlapping. the most common complement deficiency is c1-inh deficiency (c1 inhibitor), responsible for hereditary angioedema that causes symptoms in hets (chap. 8). at least 15 cases of factor i deficiency are known [328] , with autosomal co-dominant transmission because parents show normal complement levels at 50% of factor i [239] . as in an h deficiency, serologically one has the alternative pathway activation, due to c3b non-catabolization that continuously forms c3 conversion with severe c3 deficiency, the levels of which do not exceed 15% of normal values [155] . in hzs, in addition to subsequent pyogenic infections, as in c3 deficiency [422] , cutaneous rashes and urticaria caused by massive release of histamine and pro-inflammatory cellular products by anaphylotoxic fragments are reported [167] . the fact that a child during the initial period of life should experience a certain number of urtis or lrtis is within the norm: rris are mainly caused by immunological immaturity or inexperience, both transient. a typical symptom outline is difficult to define, and prevalence is also little known. in id children, a basic pathology is present instead, which encourages the recurrence of infections. although a distinction between infection and associated disease is important, childhood infections might have a key role in stimulating the maturation of the immune system, and the microbial burden in early life has been invoked as a protective factor against wheezing and asthma (chap. 4). preschool-age children have an especially high frequency of vris, with most having three to eight infections per year and 10%-15% have ≥12 vris per year. the rate in children aged 0-4 has been about 1 in 200 children compared with about 1 in 500 for children aged 5-10, and about 1 in 1,000 for those aged 11-17 [413] . rris may be defined as >six episodes of urti and/or >3 lrtis in the previous year, or based on age and ≥8 episodes per year if aged <3 or ≥6 episodes per year if aged ≥3. several risk factors can influence the onset and recurrence of infections [217] . it is clear that the younger the child is, the more he/she may fall ill: this is also related to serum ig levels (table 1 .15). the dogma of primary and secondary responses also may not apply to infections, at least those caused by rotavirus, which confers considerable protection only after various infectious events and in children aged 1 [511] . environmental factors in the absence of a basic pathology are important. it is also obvious that the more crowded the environment the child lives in, the more probable that infection becomes. in addition to the number of siblings, other factors such as socioeconomic status, age (preschool children), contact with outside persons, especially babysitter, early social contacts, exposure to passive smoke, indoor and outdoor pollution may be found to be related to the hygiene hypothesis (chap. 4). however, daycare attendance, which was considered to be an indicator of exposure to respiratory pathogens, and the presence of siblings, increased the risk of urti in preschool children aged 4-5 [273] , and in the 1st year of life for children with fha [88] . among children with fha, the protective effect of day care attendance in early life against the development of atopy only begins by 2 years, and against wheezing this may not be observed until after 4 years [89] . the particular ease of smoking parents and/or relatives who fall ill with influenza has been known for some time, to the same extent that the children who live with them are affected by rris (table 4 .24). the impact on rri incidence as it is related to children in kindergartens, has been reflected by a significantly increased morbidity observed in babies aged 3 months to 3 years who go to daycare, who show a number of more severe and longer lasting infections per year [523] , with an incidence of 6.5% in children who stay at home compared to 13.1% of those in kindergartens [524] and an increase of 49% of ome persistence (chap. 15). in children exposed to cigarette smoke, the risk increased 3-fold for lrti [221] or by 3.5-fold, equal to ≤3 episodes of respiratory infections each year [26] . studies on environmental pollution have identified the most damaging agents: conclusive data on fine particles in suspension and polluting derivatives is available, proving a significantly increased risk of infantile rris: table 4 .20 indicates that no 2 reduces immune defenses against rtis, provoking alterations of the epithelium and of the lymph node cells, with negative effects on mucociliary clearance and macrophages. the biological role played by no 2 in the domestic pollution derived from the home has been ascertained to be related to cooking and the smoke released by combustion [26] . using wood for heating leads to so 2 development, while radiators cause the air to dry, which in turn causes potentially infected particles to remain in suspension. pollutants are increasingly responsible for indoor pollution (chap. 4). although levels of micro-pollution are not easily ascertained, significant associations with acute rris and conditions such as polypnea and dyspnea have been reported, especially in <2 year-old children [507] . these children's capacity to evoke adequate responses is genetically controlled; however, it is commonly known that their parents or siblings have suffered similar illness as children. in subjects with physiological immaturity of the immune system, vris more easily cause infectious episodes, which are important factors to be considered only in the presence of recurrent or incompletely cleared conditions [165] . predisposing factors related to a basic pathology derive from perinatal factors, more common in premature babies, which can lead to respiratory tract alterations and consequently to bronchopulmonary dysplasia; anatomical anomalies; cystic fibrosis, which can become recurrent pneumonia; adenoiditis causing otitis and ome; congenital ciliary dyskinesia; humoral deficiency and pid characterized by recurrent sinopulmonary infections [101] (table 22 .21) [79] . viruses are the principal etiological agents, and over 159 a novel member of the coronavirus family has been characterized, which is associated with cases of sars (severe acute respiratory syndrome). phylogenetic analyses and sequence comparisons showed that sarscoronavirus is not closely related to any of the previously characterized coronaviruses [421] . as investigated by a 15-year study the overall prevalence is age-related, and different between children aged 0-4 ( fig. 22.46) [326] and those aged 5-19 ( fig. 22.47 ) [326] : rsv and rhinovirus have a different impact on the first group (58% vs 28%) and the influenza viruses on the second group (9%-48%). incidence in young children was 1.75-fold higher than in those aged 5-19 [326] . rsv causes bronchiolitis in breast-fed babies, with a higher frequency the younger the child is (tables 11. 24, 11.25) , and rhinitis in older siblings. even an infectious agent neglected for some time, such as ureaplasma urealyticum, causes a lung pathology in younger children while sparing those >3 years old [271] . the bacteria most commonly involved are: streptococcus pyogenes (pharynx and larynx); haemophilus in-1323 complement deficiency fluenzae (middle ear and larynx reaching the epiglottis); and streptococcus pneumoniae (middle ear). there are often secondary bacterial infections such as complications caused by vris, which certainly contribute to recurrent infections and/or the onset of chronicity [508] . in chap. 4 we reported several studies which concluded that early infections may protect from atopy development (hygiene hypothesis). we must distinguish pid outlines and pseudo-id in children with rris. summarizing the aforementioned, severe and recurrent lrti and sinusitis are the principal clinical manifestations in children affected by deficiencies prevalently involving humoral immunity (table 22 .1). these children fall ill during the first weeks of their lives and often contract infections caused by opportunistic agents, fungi, protozoa and viruses, and as months go by are also affected by malnutrition and failure to thrive. episodes affecting the airways, particularly common in cellular and combined id, tend to become longer or severe, especially if complicated by pneumonia [44] . chronic disorders such as sinusitis and bronchiectasis (sinobronchial syndrome) are not rare; interstitial pneumonia is in most cases caused by pneumocystis carinii and results in tachypnea and lung hyperinflation [409] . infections supported by the herpes simplex, ebv and cmv are also common: males with xlp exhibit a deficient response to ebv [446] . severe and recurrent sinopulmonary, was, higes and ata infections as well as severe rris in cgd, complement deficiencies and lad 1 to v must also be borne in mind. cases of recurrent pneumonia should be warning signals to rule in 4.8% pediatric cases of pids [392] . the second most important manifestation is chronic diarrhea: in some cases the infections are caused by rotavirus and enterovirus among which the echo: giardia lamblia, salmonella and campylobacter can also cause chronic enteric infection; malabsorption resistant to treatment can be ascribed to cryptosporidium [80] . rris are common in children. they reflect the immaturity of the immune system in its encounter with environmental antigens; this developmental delay during the first years of life fosters the development of rris. thus, rris are part of the growing-up process of any child [47] . the consequences of rris can be of a profound and sometimes protracted alteration of the different immune defense mechanisms, which place the child in an undefended position, similar to the condition observed in children with pids, compromising the phagocytes, lymphocytes, nk cells, antibody production, and ils at every occasion [419] . the responsible viruses for these infections have a development limited to surface mucous cells, spreading from cell to cell due to contiguity, while the viremic stage is absent or remains marginal. the incubation period is therefore brief, normally <3 days; consequently the immune response may not be capable of ensuring a protective function, or it only intervenes partially, clarifying the potentially unlimited number of infectious episodes [392] . from a pathogenetic point of view, the virus works by triggering the development of ige and allergic sensitization and/or damaging the immune structures [322, 519] . in the first case, it is known that experimental infection in mice with rsv is capable of significantly increasing the absorption of ovalbumin (ova) administered by aerosol, of igg, ige and anti-ova siga (fig. 4.26) and of increasing the synthesis of ige and specific igg to ragweed also administered by aerosol. the result is that the majority of infants become infected with rsv, although lrtis develop in only about 20% [382] . approximately 25%-50% of those subsequently experience recurrent acute asthma from vri [530] . the mechanism by which vris induce atopic sensitization in experimental models is identified with antigen penetration and sige synthesis [322] . studies show that viruses increase mucosal permeability, by modulating antigen uptake and altering antigen processing by the mucosa, which results in the ige-suppressor t-cell depression, while ifn-g-modulated histamine release further increases mucosal permeability [162] . it is probable that the immune deficiency is secondary to vris, because many viruses are capable of inducing transient [521] . the lack of nk cells, which are in the front line of defense against viral infections, and with which the altered production of il 2 and factors activating the phagocytes are associated, appears significant, but it is unclear whether the deficiency is primary or secondary to viral infections [419] . the basic question with potential therapeutic consequences therefore remains unanswered, and is probably destined to remain so until more sophisticated tests are available to clarify this issue, although the nk-cell reduction in these children corroborates the first hypothesis. recent data emphasizes the important defensive activity of cytotoxic cd8 t cells and by different ils, including il 10 , il 28 and il 29 active in antiviral defense: if the infected cells express on the surface the viral antigen processed in association with hla class i molecules, cytotoxic cd8 take care of killing the cell. normally the cd8 are activated by the virus itself or by soluble factors released by the infected cells and mediate the cellular lyses after recognizing hla class i antigens on the same cell. however, if the apc is a macrophage, it can be parasitized by the virus, with consequently reduced chemotaxis and microbicidal activity and in particular the capacity to cooperate with t cells [322] . considering that macrophages may have evolved specific mechanisms for directing t-cell development toward the th1, since cmi can solve some infections, it is clear that their dysfunctions appear in the insufficient nk cell activation and inadequate th1 development in response to infections. ifn-g, produced not as a direct consequence of infection, but probably by il 12 and/or il 15 , stimulates the nearby cells to block the nucleic acid transcription and therefore the viral replication, preventing the infection of those cells to which virus spreads due to contiguity. therefore the ifn-g species-specific antiviral activity takes place at the very first stages of the infection, preceding the antibodies [322] . the response to germs' capsular polysaccharides, particularly deficient until the age of 2, although encounters with these germs are abundant during this period of time, still remains to be evaluated [508] . however, selected children, although normal from an immunological point of view, may have a deficient antibody response even aged 4-8 [195] with a percentage of modifications of both humoral and cmi, therefore not only of antibody synthesis or phagocyte and neutrophil functions, but also of t lymphocytes and related ils [457] . it is also possible to hypothesize that persistent vris are capable of inducing th2 activation by antigens or super-antigens: th2 t cells with il 4 help induce virus-specific cd8 + to produce il 5 , which recruits eosinophils in the respiratory tract, thus reducing ifn-g secretion. thus there may be an increased interaction of ige mast cells in these subjects with immunoregulatory alterations, due to a marked lymphoproliferative response and the elaboration of other ils, which consequently amplifies ige production in the respiratory tract. the ige response is thought to lead to a greater production of bronchoconstrictor mediators by effector cells; viral infections themselves may induce these cells to release histamine. it is also known that humoral deficiency, especially of iga, very often opens the way to gram-positive germs causing viral and bacterial infections, thereby completing the circle [409] . interestingly, only 2/13 children have at least a significant production of antigen-specific salivary iga against klebsiella pneumoniae [418] . even if the alterations are transient and aspecific in children with rris, their occurrence and persistence for a number of months, for a year and even longer, leads to believe that the immunosuppressive mechanisms set off by the first episode occasion more severe, profound and lasting consequences for the immune functions than those occurring in their normal peers [36, 273] . having at least one physician-diagnosed lrti in the 1st year of life was significantly associated with recurrent wheezing (or, 2.0) and asthma (or, 2.5) [89] . at the base of this exclusive predisposition in children for contracting rris, there is a nk cell reduction [419] and immune deficiencies related to the global lymphocyte population, the cd4 and the cd4:cd8 ratio, unbalanced toward cd4 t cells, prevalent in children expressing coughing compared to those with bronchial hyperreactivity (bhr) [374] . other t-cell deficiencies in children with humoral anomalies include a considerable spontaneous production of il 2 and il 4 , both generated by th phenotypes, or alternatively by the th0, which express both ils [216] . it is similarly feasible that virus-specific cd8 deprived of cytolytic activity are converted into th2-like t cells when il 4 is present [216] . cmi in these children consists above all in the transient t-cell numeric and functional depression, coinciding with a deficiency of ils necessary for their activation, proliferation and differentiation. the virus toxic effect also acts directly on the t cells, inciting rough structural modifications including giant polynucleate cells, which jeopardize homing and recirculation capacities, to the point of immunosuppression, affecting the specific lymphocytes for that particular virus, thus favoring the attacking organism [552] . a condition of immunosuppression occurs also as a result of superantigen (sa) orchestration, which, stimulating a large num-nonreactive children of 4%-19% [143, 430] , rising to 13%-42% if with an iga and/or igg subclass deficiency [198, 216, 430] . while 50% of children have low antipneumococcus igg 2 , antibody responses are totally absent in 40% of dysimmunoglobulinemics with a virtual absence of iga and igg 2 [430] , confirming previous data [198] . iga and igg 2 -deficient children show a clinical pattern with elevated susceptibility to s. pneumoniae infections [430] . von waldeyer ring (nalt) is a significant constituent of immune response, and of resistance to nalt-dependent infections. among the consequences of chronic adenotonsillitis (table 15. 2) the statistically significant decrease in ig levels, including siga, must be evaluated eventually in relation to rri complications (chap. 15). igg subclass deficiencies can be present in children with chronic and/or severe asthma, associated or not with sigad, in children affected or not by asthma with severe rris or with chronic nonallergic respiratory clinical symptoms, in children with sigad, ata, was, cvid, scid and in healthy subjects [80] . the anti-virus antibodies generally belong to the igg 1 and igg 3 isotypes while the igg 3 protect from microbes with polysaccharide antigens, such as s. pneumoniae and group a and type b h. influenzae [153] . table 22 .7 shows normal values for igg subclasses in subjects aged 0-15. unlike total igg concentrations, igg 1 and igg 3 reach normal levels within the 1st year, igg 2 mature more slowly, ensuring an effective antibody response only after the 2nd year, and igg 4 develop even more slowly. various authors believe that the role played by igg subclasses is unique and vital in defending from infections: igg 2 deficiency is associated with an increased susceptibility to infections by bacteria expressing capsular polysaccharides, such as pneumococci, meningococci, h. influenzae, bordetella pertussis, etc., as well as other factors capable of setting off an inflammation [113, 465] . igg 4 deficiency instead seems associated with a marked predisposition for rris [333] . however, undetectable igg 4 subclass levels are a common finding in normal individuals and an accurate detection of very low levels of igg 4 is technically difficult to achieve [450, 465] . babies aged ≥1 month have levels of circulating lymphocytes secreting all four subclasses in a higher number than adults; therefore the capacity to produce antibodies exists well before a full humoral response is developed. subnormal igg subclass concentrations, especially of igg 2 , are also observed in healthy children who do not present an increased susceptibility to infections. low subclass levels do not necessarily indicate that the subject will experience immune disorders exclusively linked to these, nor do normal concentrations guarantee that the child will be spared complications. sub-jects have been observed both with normal igg 2 levels and with recurrent infections and with a subclass deficiency, which often do not form other types of antibodies [450] . igg subclass determination does not indicate what the humoral level restricted to that molecule is: this is a characteristic in children, unlike adults, even though research was carried out using the same methods in the two studies [465] . interesting hints come from studies involving children with rris: of children aged 2-10, with a confirmed diagnosis of susceptibility to infections, 67/567 (11.8%) had a igg subclass deficiency, almost all concerning igg 4 , with several associations [283] . in other children, the selective igg 4 deficiency was statistically significant compared to atopic controls without rri, associated not so much with a well-defined state of id as to a respiratory tract defense mechanism deficiency, in view of the fact that the relative prevalence of this subclass in secretions may indicate a role in the mucosal defense [333] . other children with typical symptoms of recurrent infections, lymphadenopathies, failure to thrive and hgg exhibited low igg 2 levels, confirming that normal levels of total igg do not exclude a subclass deficiency [454] . in a cohort of young babies, igg 4 deficiency was only present in 78/267 subjects (37%) [198] ; however, the absence of a control group makes the results incomparable. an igg subclass deficiency is therefore able to induce or worsen chronic respiratory symptoms in allergic and nonallergic children, especially if predisposed to developing these affections [113] , or in subjects with sigad [327] . with time these deficiencies, and eventually also those associated with iga, can normalize [29] . in conclusion, transient and persistent iga and/or igg 2 deficiencies have been reported in a small percentage of asymptomatic children [446] , but even if iga and igg subclasses are not always required as such for a normal immune response, their deficiency may predispose to rris [160] . as previously illustrated in chap. 11, the close links between atopy and rris are known, and this is confirmed by the observation that asthmatic children have a higher incidence of rris than their nonasthmatic siblings. it has been known that rris during the early periods of life can play a role in the development of bhr and atopy: in the classic study by frick et al [173] , in 11 out of 13 allergic children sensitization was propitiated by rris. with continued observation, the authors noted the presence of high ige levels, positive rast and histamine released by leukocytes after infections [172] . in a cohort of 73 asthmatic children aged 0.8-3.1, the 21 affected by 20th week of pregnancy is possible, so as to evaluate the immune phenotype in the fetal blood [491] . wccs (white-cell counts) in cb and differential counts can be used to detect the lymphopenia that is commonly present in infants with scid. however, subset analysis by flow cytometry is necessary to enumerate t, b and nk cells. subsequently, scid diagnosis will be suspected when overwhelming opportunistic infections occur [320] . depending on whether one suspects a humoral, cellular or innate immune deficiency, we begin with the algorithm in table 22 .23 [107] , positive if infants or children have ≥2 of these signs, then 478, 522] on laboratory tests can be consulted. however, children with variable levels of antibody id may end up with different diagnosis [269] . children with higms presented initially with a history of an increased susceptibility to infection including pneumocystis carinii pneumonia [539] in 43% of children [290] . in pids affecting phagocytes, because of the relatively narrow spectrum of disease-specific infections (such as aspergillosis in cgd), careful attention to the microbiology laboratory early in the course of evaluation of a patient suspected of having a pid is crucial to orient the work-up in the appropriate direction [416] . in a male newborn referred to hospital at 27 days of age for fever, hemodynamic failure and an inflammation syndrome caused by pulmonary infection, culture of tracheal, bronchoalveolar lavage samples and lung biopsy grew positive for a. fumigatus, enabling the diagnosis of cgd [337] . to investigate whether patients with undiagnosed id could be identified in diverse inpatient hospital populations, a scoring algorithm and computer screening method was updated [107] on the basis of icd-9 codes to survey the discharge diagnoses of all hospitalized patients over periods of time. thus 17 id patients were identified, eight of whom were children aged 2-10 (47%), two with neutropenia, two with igg deficiency, one with lad, one with dgs, etc. [108] . we also suggest including congenital phagocytic defects in the differential diagnosis of recurrent bacterial or fungal infections in a child [285] . a congenital complement deficiency should be suspected if levels of even one component are reduced [167] . early diagnosis is essential for choosing the necessary treatment [44] . the differential diagnosis of pid will emphasize the different characteristics schematized in table 22 .27, to which one must add objective rarity, while fh and child gender become important [10] . in subjects suffering from omenn syndrome, was, severe combined and cellular ids, the screening of clinical symptoms may be useful at birth and during the first few months after birth (table 22. 28) [61, 80, 399] . the localization of infections is multiple, the id child usually appears to be ill, and the peripheral lymph nodes and lymphatic rris had a higher incidence of fh positivity (p=0.015), increased ige (p=0.021), as well as a combined iga (p=0.038) and igg (p=0.018) deficiency [296] . ige hyperproduction could be the result, not only of the wellknown association between igg subclasses and ige and their coregulation of il 2 expression [296] , but also of a virus-caused unbalanced cd4:cd8 ratio [374] . these results link atopy to rris, confirming that the state of chronic inflammation and bhr induced by allergic sensitization is an ideal substratum for the adhesion and chronic evolution of bacterial and/or viral infections. there are no specific clinical outlines for rris. on the contrary, symptoms are extremely varied, with, as previously mentioned, infections caused by bacteria and viruses. urtis are common at age 4. during the last 12 months, 9.5% of the children experienced more than one bout of acute otitis media, 6.9% had more than one pharyngotonsillitis episode, 47.7% contracted >2 common colds, and 3.2% had rhinitis weekly or monthly [273] . there are children who, during the period of maximum exposure due to biological immaturity and immunological inexperience, suffer from one episode each month affecting different organ systems, as well as lymphadenopathies and failure to thrive. the capacity for inducing bhr in normal subjects and worsening the symptoms in those already ill are precisely caused by vri, also facilitating greater penetration of inhaled viral allergens [530] (table 11 .10); rris in turn predispose to sinusitis. lower ifn-g levels produced by 18 of 53 children at 6 months of age were even greater if the comparison was made between children with rris and those with no or maximally one rri during the follow-up period [382] . rhinovirus-induced infections (table 11 .11) take the appearance of common rhinitis, but stimulate mastocytes to release histamine, contributing to bhr development and the perspective of delayed reactions. a differentiating feature is the respiratory infections in the id child that may also result from opportunistic pathogens [80] (table 22 .21). respiratory infections should be under control. a screening of humoral immunity revealed low ig levels in 4.6%, low iga levels in 2.3%, and sigad in 1.3% of children [270] . during the last few years, increasingly sophisticated diagnostic techniques have permitted prenatal diagnosis in many cases (table 22. 22) [61, 166, 389, 406, 414, 491] : in forms supported by rag-1 and/or rag-2 mutations a diagnosis even at the 10th-12th or at the pharyngeal tissue are almost imperceptible [102, 196] . the seriously undernourished appearance should be noted, more often observed in children with scid [162] . rris can be observed in other cmi forms [162] : deficiencies of cd3 g and e chains [18, 249, 471] , zap-70 [92, 139] and hla class ii [77, 256] . finally, children with aids will seem to be in severe general condition and this disease is a paradigmatic example of how hiv can overturn the t lymphocyte immune defense with regards to opportunistic infections [79] . in some cases of pediatric aids there is hgg that is indistinguishable from pid and that belongs to the differential diagnosis of severe recurrent infections during the first few months after birth [79] . as far as tih is concerned, the confirmation of a normal presence of the b lymphocytes and low levels of intrinsically produced ig is resolutive, compared to agammaglobulinemia [61] . an articulate case history often identifies the familiarity of rris, usually with an absent basic pathology and the frequent predisposing environmental factors (table 22 .21), among which passive cigarette smoking stands out. maximum prevalence occurs during the first 2 years of life or during first contacts with school, the disease is limited in time, and there is usually a single location. in most cases the pseudo-immunodepressed child is clinically normal in all other respects [374] . in ten reported sars-infected children from hong kong, fever, cough, and runny nose were 1329 complement deficiency lateral pharyngeal x-ray to visualize adenoidal tissue cytokine production (il 2 , il 3 , il 4 , il 5 , ifn-g, il 12 ) chromosome fragility (ataxia-telangiectasia, bloom's syndrome, etc.) data from [101, 478, 522] . ada adenosine-deaminase, adcc antibody dependent cellmediated cytotoxicity, ctl cytotoxic t lymphocytes, ltt lymphocyte transformation test, pha phytohemagglutinin, pma phorbol myristate acetate, pnp purine nucleoside phosphorylase, ppd purified protein derivative. sars seems to have a less aggressive clinical course in younger children [223] . when in doubt, a broad spectrum of laboratory tests are available: cbc, proteinemia and protidogram, serum ig levels, or in secretions and igg subclasses (table 22 .7), immunoelectrophoresis (homogeneous components, k/l), dosage of isohemagglutinin, five other natural antibodies, the sweat test [10] , in strictly selected cases also a lymphocyte population and subpopulation count (tables 1.34-1.39), and x-ray of paranasal sinuses. analysis of the lymphocyte profile sometimes shows a number of deficiencies, statistically differentiated from those found in other children affected by an asthmatic pathology; however, none of the immunological deficiencies indicated (table 22 .29) are characteristic in pseudo-immunodepressed children. common diagnostic methods may not be capable of revealing a deficiency of igg subclasses or of selective igg 4 : the chance that there may be abnormal igg 2 or igg 4 levels is not excluded by the normality of igg serum concentrations [153] . furthermore, the distribu-tion of igg into four subclasses makes it difficult to identify these deficiencies simply by measuring total serum igg levels [113] ; only for the past few years have there been highly specific reagents for measuring individual subclass levels and methods such as radial immunodiffusion (rid) [198, 317] . the aaaai has recommended not relying on subclass levels [10] , especially igg 4 levels, which seem to be unmeasurable in 25% of the population [198] . rid, which has proven to be more sensitive than the elisa used by the cdc in atlanta, has shown that 25% of normal children have values below normal for at least one subclass [317] ; a similar deficiency was present in 58% of children with rris [198] . a recent study measuring the igg with both methods, has proved that the rid can show higher values of igg 1 and igg 2 in low serum levels of both ig [387] , data with an unquestionable negative effect in pediatrics. in conclusion, at the moment our knowledge suggests that we should also carefully interpret low levels of one or more subclasses, because on the one hand this might indicate a transient or paraphysiological condition, on [174] . igg levels in all patients also rose considerably compared to previous treatment: the average levels in different determinations was >700 mg [174] . finally, all children grew normally; the height achieved by each child is between the 3 rd and 50 th percentile, within the limits of theoretical values calculated on the height of their parents. substituting therapy with ivig has allowed patients to return to their normal activities, with a considerable improvement in quality of life. in all these years, we have never come across substantial unwelcome reactions or infectious complications [174] , as also found by other authors [177, 461] . ivig could also be effective for reducing the allergic symptoms discussed thus far: presuppositions are not lacking, such as the blocking of allergens and mastocyte fcr thanks to the modest quantities of igg 4 present in the preparations [196] . in addition, the increased understanding of the igg transplacental passage (chap. 2) can absolve the function of timing their transfusion in the case of mothers with antibody id, so that the fetal defenses can be complete and quantitatively adequate. in sigad common ivig preparations cannot be used, even if with a low content of iga, nor enriched, both because of the extremely short iga life-span, which would therefore suggest iga administration every 2-3 days, and because the infused iga do not reach the secretions [507] . should ivig be indicated for a deficiency associated with igg 2 , or should transfusions of blood derivatives become necessary, one must first investigate serum antibody anti-iga levels (igg and ige) and, should these be positive, avoid infusions or administer them in a hospital under strict medical supervision, or use washed red blood cells [507] . the same precaution must be taken for subjects with ata for whom ivig, if is appropriately administered, also ensure beneficial effects on quality of life, while there are no known therapies for contrasting neurological symptoms. in patients with humoral deficiency, alongside ivig, if appropriate, an antibiotic prophylaxis is suitable with monthly cycles, alternating amoxicillin, cephalosporin, co-trimoxazole, etc., bearing in mind family compliance. in cvid recurrent infections caused by giardia lamblia should be treated using furazolidone (8 mg/kg/day) or methronidazole (15 mg/kg/day) for 10 days, if necessary to be repeated. cvid treatment in specialized centers involves recombinant il 2 , il 10 and cimetidine [456] . some t-cell pids represent a severe clinical emergency, such as omenn syndrome, in which hypovolemic shock and reticular dysgenesis are immanent in the battle for survival. although precise figures are unavailable, thousands of patients worldwide with different forms of the other a modest deficiency can result in clear hgg [451] . subnormal igg 2 levels can indeed be associated with various manifestations of immune dysfunctions; it is therefore advisable in this case to proceed with specific investigations, measuring the response to polysaccharide antigens and studying the lymphocyte activity in vitro [451] . in children with normal serum levels, who are instead lacking in antibody responses to polysaccharide antigens [143, 430] , this is a conclusive investigation [470] (fig. 22.47) . a study of children aged >5, half atopic and half not, has confirmed this thesis, concluding that the answers were similar in both groups, therefore excluding a greater rri predisposition in the atopic children [344] . many patients with high ige levels do not present atopic manifestations: it is thought that an increased concentration is related to a reduced inhibiting activity of the thymus in ige synthesis [457] . recurrent sinopulmonary infections must make one also consider cystic fibrosis and immotile cilia syndrome [457] . children with malnutrition (chap. 21) suffer from numerous ids, prevalently concerning cmi; their vulnerability makes them succumb to severe bacterial me infections and urtis, often also risking death. obese subjects may also be affected by rris due to a possible adipose tissue hypovascularization or to a defect in the granulocyte microbicidal activity [457] . in the presence of antibody deficiency, antibiotic treatment is chosen as a preventive therapy in less severe cases, otherwise the preferable therapy consists in ivig (fig. 22.48 ). this treatment is restricted to a limited number of diseases, including some forms of id, secondary or cytopenic id, in which effectiveness has been proved in dbpc studies [135, 426, 439] , like other positive forms of intervention described, while it appears to be of no use in uncomplicated thi [426, 439] . two children aged 2.5 and 3 with higes and kawasaki disease were administered 400 mg/die of ivig for 5 days and one 2.5-year-old with higes received only one dose, with ige levels falling from 4,000-14,000 to 600-5,000 ui/ml on the 28th day. hence there was almost a normalization of ige production with symptom relapse after 6 months; similar results using a single dose were also obtained in two children with higes and severe ad [254] . the following data represent a number of clinical and immunological parameters in children suffering from humoral pid and ata, with igg levels <100 mg/dl. treatment with ivig, also at a higher dosage, was very well tolerated by patients: all children presented a clearverely affected by several factors [26] . either hla-identical marrow or t-cell-depleted (tcd) haploidentical parental marrow is the standard of care for scid ( fig. 22.51) . when histocompatible related donor bmt is unavailable, a bmt either with hla-identical unfractionated or tcd haploidentical parental marrow is the standard of care for scid [338] . all but one (95%) of 21 scid infants who received tcd identical or haploiden-genetically determined id have been given bmt in attempts to correct their underlying id [66] , including a recent series [14] . specific treatment for cellular pid consists in a bmt from a hla-compatible donor [10] . the ideal sc donor is normally a sibling who shares identical hla class i and class ii loci. without such a donor, these transplantations usually resulted in fatal gvhd. if death did not occur, event-free survival was se-1335 treatment tical bmt in the first 28 days of life are currently alive, with the period of survival ranging from 8 months to >19.2 years after transplantation. this compares favorably with a 74% survival rate of 96 infants receiving transplants at a median age of 190 days (range, 45-516 days) [338] .a girl with t -b -scid received a full matched bmt from her sister at age 2 weeks [491] . a worldwide survey conducted by buckley from 1994 through 1997, with subsequent additions of published cases from the literature, revealed that 239 of 302 (79%) patients with pid transplanted with hla-identical marrow during a period of 34 years were alive [65] . there are >375 patients worldwide who have survived scid as a result of successful transplantation of hla-identical or haploidentical bm [67] . most importantly, 34 of 35 infants (97%) undergoing transplantation in the first 3.5 months of life are currently alive [69, 338] , compared with a cut-off at 6 months (97% vs 86%, children younger vs those aged >6 months) receiving bmt (or 5.0 [14] ). we stress that neonates developed higher lymphocyte responses to phytohemagglutinin and higher numbers of cd3 + and cd45ra + t cells in the first 3 years of life than those receiving bmts late. t-cell antigens peaked earlier and with higher values in the neonatal bmts (181 days to 1 year) than in the late bmts (1-3 years) [338] . over the past 22 years 78% of all scid patients (110/142) receiving bmt at duke university medical center survive to varying ages up to ≥20 years after bmt. only 16 had an hla-identical donor. all others received rigorously tcd haploidentical bm from a parent, most often the mother. the soy lectin, srbc rosetting technique was used (r. buckley pers. comm. november 20th, 2004 and april 20th, 2005). an uncommon bmt to treat ar scid was undertaken in a 1-month-old girl. the donor was her hla-mismatched 6-year-old sister, who had previously received a bmt from her father [479] : presently, they are aged 5 and 13 and are affected by molloscum contagiosum infection [547] . bmt, both hla identical unfractionated and tcd hla aploidentical [63] .a recent trial found that because only 10%-15% of affected children have a familial hlaidentical donor (rid), the alternative therapeutic options are bmt from a mud or a haploidentical bmt or from hla-mismatched related donors (mmrds). only 40% of these children may find a matched donor; therefore, the remaining pid-affected children are candidates for a tcd haploidentical bmt [277] . mud hsct is successful in young children [132] , but the success rate decreases dramatically above the age of 5-6 years [158] (table 22 .30) [13, 15, 25, 27, 39, 46, 52, 55, 56, 58, 64, 67, 69, 80, 83, 85, 98, 111, 119, 131, 132, 163, 185, 188, 197, 205-208, 231, 233, 235, 237, 245, 252, 256, 257, 259, 260, 277, 280, 286, 288, 290, 305, 307, 334, 360, 366, 380, 405, 408, 428, 440, 448, 452, 456, 466, 474, 491, 496, 499, 503, 534] . bmt survived. compared with mmrd bmt, survival was significantly higher with rid or with mud (45/54 = 83.3%) [201] . when an hla-identical sibling as the donor is unavailable, a phenotypic hla-matched unrelated bmt is needed, also used in cd154 deficiency [290, 499] , with a clinical and immune outline normalization [246] and a variable effect on igg subclasses 1337 treatment [58] . c median (1.5-13 years after bmt). p, at the time of publication. [153] . possibly because of earlier diagnosis before untreatable opportunistic infections develop, the results have improved considerably during the last two decades [65] . bmts have been successful when applied within the first 7-24 days of life in 21 infants with scid, 20 (95%) of those still alive range from 8 months to 19 years, not justifying in utero transplants. a completely normal t-cell function was obtained within 82-118 days [338] and in an other 83 patients was still present after 10-17 years [373] . of the 58 children who received a mismatched parental bmt from 1980 to 1998, 43 (74%) remain alive with t-cell immune reconstitution, a median of 128.8 (range, 13-224. 3) months after bmt [362] . of 96 children out of 117 who received allogeneic bmt after the first 28 days of life, 71 (74%) are alive [69] . three out of five children who received a hsct are alive and well after 18-32 months [13] . breast feeding appeared correlated to an earlier reconstitution when the donor was the mother [338] . intra-amniotic gene transfer has been successfully carried out on a laboratory animal, registering in a dose-dependent manner the fetal gastroenteric and respiratory effects [222] . if confirmed in human beings, this method of treatment will certainly prove useful for prenatal correction of pid. bmt/hsct should be completed by conditioning regimens with busulfan and cyclophosphamide, less toxic than total lymphoid irradiation or a combination of nucleoside analogs and anti-lymphocyte antibody preparations [65] . for example, busulfan (16 mg/kg), melphalan (90 mg/m 2 ) and anti-thymocyte globulin (36 mg/kg) [83] . to enhance the engraftment rate in haploidentical bmt in pid, it was recently suggested to add donor peripheral scs after mobilization with g-csf (16 mg/kg for 5 days) and bm cells. with this procedure the cell load is increased, which allows intensification of the conditioning regimen for induction of faster engraftment [277] . in utero bmt suggested advantages include the sterile environment in utero, and immaturity of the fetal immune system enabling the prevention of clinical manifestations of the disease in the neonate, and the engraftment without the use of cytotoxic conditioning regimens: a child thus treated was well at age 11 months [164] . two series of six and four patients [386, 504] and two additional case reports [182, 532] of in utero transplants have been published, yet failure of b-cell engraftment and function may result in long-term dependence on ivig replacement [248] . better results than those published for in utero bmt for scid were implicit in 13 infants admitted and diagnosed at a median age of 3 days because of a fh of a previously affected infant. bmt was successful and all children are alive and well with follow-up to 11.5 years [248] or <30 days vs approximately 4 months [194] . however, it is suggested that in utero transplants may carry the risks associated with injecting the fetus and the inability to detect gvhd during gestation [65] . umbilical cb transplantation (ucbt) was done in two children affected by a zap-70 deficiency and an omenn-like syndrome. both are alive and well at 4.5 and 2.2 years after ucbt [148] . unrelated ucbt in eight children with severe t-cell id [260] and in three with was [259] resulted in consistent and stable t -, b -, and nk cell development [259, 260] . faster availability of ucbts is a meaningful advantage for patients requiring urgent transplantation: a median of 25 days more rapidly than did those receiving bone marrow [30] . in a large report [423] , 40 patients with scid, seven with was, and other unspecified pid received an unrelated ucbt. ucb was evaluated as a sc source for immune reconstitution in children with severe primary t-cell ids such as scid, reticular dysgenesis, thymic dysplasia, cid, dgs, and was when a matched sibling donor was unavailable, and has been used to date in more than 2,000 patients [194] . three infants who rejected a tcd-mismatched parental bmt without prior cytoreduction engrafted after infusion of ucbt [69] . a 4.5-year-old girl with hla class ii deficiency had a successful related ucbt for graft failure following tcd nonidentical bmt [52] . a girl with reticular dysgenesis failed to engraft following her first transplant, but fully engrafted after a second unrelated ucbt. five of six patients showed grade i gvhd, although one child experienced grade iv skin and gut gvhd. immunological ucbt resulted in consistent and stable t-cell, b-cell, and nk-cell development [260] . long-term event-free survival (≥27 months) with recovery of antigen-specific responses was reported following an unrelated ucbt in a child with omenn's syndrome [38] . gene therapy, which has revolutionized and could revolutionize even more pid treatment in the near future, is analyzed in table 22 .31 [76, 104] . the requisite for applying this form of genetic engineering treatment is that the responsible gene must be cloned; the most common techniques involve knock-out mice and inactivating a particle [104] , or employing retroviral vectors (table 22 .32) [263] , totally deprived of their genomic factors except for the normal copy of the gene to be inserted. this is indispensable for allowing the vector to reach the human nucleus, where it will integrate with the cellular genome [263] . in this case, pbls are collected through leukapheresis and cultivated in vitro with retroviral particles containing a normal gene rna copy: thus the healthy gene is introduced into the cell genome using a vector and the manipulated cells are reinfused, so as to restore normal immune functions. this system has been used to treat two children aged 2 months suffering from scid [508] and from ada deficiency by employing autologous pbls [51] , bm cells [54] , and cb cells [262] . three reports [4, 88, 204] of successful gene therapy in infants with x-linked scid [88, 204] and in t -b -scid [4] are a major step forward among repeated efforts to achieve better immune reconstitution in ada-scid with gene therapy than with bmt/sct [161] . immediately after the diagnosis had been made in two capacity of wild-type, replication-competent retroviruses to cause leukemia in immunologically immature neonatal mice [263] , and in humans (two children out of 11) [63, 88, 204] . retroviruses can cause insertional oncogenesis, a long-known potential complication of retroviral gene transfer attempts, because gene integration occurs at random in the genome, thus deregulating the expression of cellular oncogenes [263] . this complication has been thought to be unlikely with such vectors, because they are capable of inserting only once into the cell's chromosomes and cannot repeatedly reproduce and integrate. lentiviruses may be more effective than murine retroviruses for gene transfer into human hematopoietic scs and t lymphocytes [263] . in ada-scid, the safety and efficacy of hsc gene therapy combined with nonmyeloablative conditioning for the treatment of scid has allowed two children to live at home and clinically well, with normal growth and development [4] . on january 14, 2003, fda placed on "clinical hold" all active gene therapy trials using retroviral vectors to insert genes into blood scs after having learned that a second child treated in the french gene therapy trial developed a leukemia-like condition. gene therapy is on hold despite enormous promise for certain scid/ cid variants. survival. in scid, the european experience with unfractionated hla-identical and tcd or non-tcd haploidentical or mud bmts in patients with scid reported that between 1968 and 1999, a 3-year survival with sustained engraftment was significantly better after hla-identical than after mismatched transplantation (77% vs 54%).within the hla-identical group, survival after bmt from genotypically or phenotypically identical related or muds was 81%, 72%, and 63%, respectively [14] . in non-scid, 3-year survival after genotypically hla-matched, phenotypically hla-matched, mmrd, and mud bmt was 71%, 42%, 42%, and 59%, respectively [14] . in a retrospective analysis of bmts performed between 1977 and 1991 at 13 european centers in 149 children as young as 1 month with 11 different pids (excluding scid), the overall survival among 53 recipients of hla genetically identical bmt was 66%, 45.5% in 22 patients who received closely matched bmt, and 38% in 71 recipients of bmt with two or three mismatched hla antigens. a significant improvement in survival has been achieved in most pids (overall survival, 81.5% vs 51.7%, primarily because of a decrease in the frequency of infectious complications [163] . in the similar analysis performed in 193 children with scid at 18 european centers between 1982 and 1993, 116 out of 193 (60.1%) patients were alive with evidence of engraftment 6 months after bmt. however, 24 patients died >6 months post-bmt, mainly due to cgvhd and/ or viral infection. thus gvhd 6 months after bmt and b -scid vs b + scid were the main factors associated with a poor outcome [206] . the disease-free survival was significantly better for patients with b + scid (60.7%) than for those with b -scid (33.3%) [14] . in a children aged 8 and 11 months including a novel splice imitation in the common gc chain [183] , haploidentical cd34 + peripheral progenitor cells mobilized with gm-csf were isolated to a purity of more than 99%. these cells were infused with no prior chemoablation and no prophylaxis against gvhd. both children showed signs of t-cell reconstitution beginning 3 weeks after the cd34 + infusion and were weaned from continuous cures. they are in excellent health, without gvhd, 34 and 68 months after transplantation. one child does not need replacement ig. the other received a booster infusion of cd34 + scs from the original donor 1 year later to improve b-cell function and now receives ig every 3 months. both were followed for 10 months after gene transfer [88] . however, retroviral vectors have the 1339 treatment data from [4, 76, 104] . ciita class ii transactivator, cgd chronic granulomatous disease, xla x linked agammaglobulinemia; for other abbreviations see table 22 .1. a done with success, see text for details. b in utero transplant of maternal stem cells. trial on children with pid receiving bm from hla-nonidentical related donors or from hla-identical unrelated donors at 13 european centers between august 1990 and june 1993, 22 out of 28 children (76.6%) survived 22-58 months. bm was tcd by use of either erythrocyte rosetting or monoclonal antibodies to prevent gvhd [231] . additional survival rates were reported previously (table 22 .30). in a series of consecutive ud bmts 31/33 children with scid and non-scid pids who received a bmt with reduced-intensity conditioning (ric) regimen between 1998 and 2001 survived after a 3.3-year follow-up, as well as 10/19 children who received a bmt with myeloablative conditioning (mat) between 1994 and 1998 and survived after an 8.6-year follow-up. therefore a ric regimen results in improved survival and reduced bmt-related mortality compared with mat in hr children undergoing an ud bmt [396] . in 170 transplanted patients with was, the 5-year probability of survival differed according to donor type: 87% with hla-identical sibling donors, 52% with other related donors, and 71% with mud. significantly, boys who had received a mud transplant before 5 years of age had survival rates similar to those receiving hlaidentical sibling transplants [158] . however, the time required to develop immune function after haploidentical scts is quite different from that after unfractionated hla-identical bm. lymphocytes with mature t-cell phenotypes and functions fail to rise significantly until 3-4 months after bmt; normal t-cell function is reached between 4 and 7 months [338] . b-cell function develops much more slowly, averaging 2-2.5 years for normalization; many do not have b-cell function, despite normal t-cell function. [338] . ex vivo rigorous depletion of post-thymic t cells from donor marrow that cause gvhd is efficient and feasible, even in haploidentical settings [13] , presumably because of more effective infection-control measures and better transplantation strategy [514] . for non-scid, sct can provide a cure, and grafts from unrelated donors are almost as beneficial as those from genetically hla-identical relatives [45] . in most patients, deficient b-cell function persists after transplantation and requires lifelong ivig therapy [69, 207] , which is necessary to prevent bacterial and common viral infections [69, 514] . some patients also have persistent deficiencies of t-cell function after sct [206, 373] . in children with rris, depending on the nature of the infection, the pediatrician will prescribe the most appropriate symptomatic and/or antibiotic therapy. in the presence of persistent inflammation, or during the winter, when the risk of close acute recurrent episodes is higher, anti-inflammatory preparations will be prescribed via aerosol, chromones, ketotifen, b 2 -adrenergic and if necessary steroids for topical use, strictly depend-ing on the need. we suggest monitoring measures, such as keeping a clinical diary, in which each acute episode should be briefly noted, continuing registration until clinical symptoms have not regressed for at least 15 days and returning to keep notes in the diary each time there is a cough and/or nasal and/or bronchial inflammation, completing this with pef as well as some respiratory parameters right at the beginning and then every 6 months. it is obvious that if medical intervention is not resolutive a center specialized in infantile respiratory physiopathology should be contacted [47] . children with sars were treated with high-dose ribavirin, oral prednisolone, or iv methylprednisolone, with no shortterm adverse effects [223] . antibiotics must be used very carefully in these children because they can influence positively or negatively the innate, cellular or humoral immunity (chap. 18), interaction with ils and growth factors are not known, repeated use often causes phenomena involving allergy/ intolerance [470] , and most infection-prone children suffering from vris are given antibiotics unnecessarily. in italian children (54.6% of males) aged 6 months to 14 years (median, 4 years) with a history of rris, macrolide therapy of acute respiratory infections influenced the natural history of rris, probably because of their elective activity on atypical bacteria [508] . considering the emergence of antibiotic-resistant bacterial stock, as for example s. pneumoniae, immunotherapy has been proposed as a means of preventing rris by providing children with small doses of inactive bacterial antigens liable to trigger specific and protective immune responses (table 22 .33) [36] . for example, om-85 bv significantly reduces the urti rate, particularly in a dbpc study in 232 children aged 3-8 with a history of acute urtis [448] , is active in preventing rri episodes [36] with a meaningful reduction in the number of days of suffering acute urtis [448] . bacterial ribosomal and membrane proteoglycans of s. pneumoniae, which stimulate b cells with secretory responses, as well as memory cells, may be used for responding to future infections [36] . ribosomal immunotherapy appears to be not only well tolerated, but also ideally targeted to induce mucosal responses [37] . among the preparations reserved for specific use, a study of pidotimod in dbpc trials proved its effectiveness in a sample of 101 children with rris, also showing increased cd25, absent in placebo-treated children [72] . the use of immunostimulants should be limited to children with proven high susceptibility to acute urti, or overexposed children attending daycare facilities, or attending kindergarten or elementary school [508] . however, according to a meta-analysis, immunostimulants are an effective treatment for the prevention of acute urti in children [40] . furthermore the indiscriminate and purely empiric use of ivig must be discouraged in every child with rri, whereas in a prospective, dbpc study of ivig and co-trimoxazole, 106 of 130 children <8 years referred for recurrent bacterial rris became infection-free over a 4-month obser-if caused by pneumococci. other kinds of vaccines have provided disappointing results: fig. 22 .52 [340] indicates the immune bases of a specific immunization and the possibility of specific interventions. in children with pid, one should bear in mind all the aforementioned facts. until the past few years, there was a busy motion into the fundamental problems underlying a majority of these conditions. many have now been mapped to specific chromosomal locations, and an impressive number vation period [353] , in addition to having an extremely unfavorable cost-benefit ratio [426] immunization children with rris and deficient antibody responses to germs expressing a capsular polysaccharide can be successfully vaccinated, but avoiding the administration of live virus vaccines and integrating this if necessary with an igg replacement therapy [218] . furthermore, in view of the availability of conjugated vaccines, it will be possible to induce antipneumococcus-igg 2 , providing an effective treatment for children with rris, especially 1341 treatment of the fundamental biological errors have been identified. the pediatrician is entrusted with a more difficult job, that of identifying as early as possible the possible existence of pid, remembering the suggestions for case history in chap. 6, with the exception of clinical emergencies such as omenn syndrome and reticular dysgenesis. this specific research becomes a necessity thanks to the new diagnostic and therapeutic advances that have been conceived over the past few years: the earlier one acts, on the one hand with a prenatal diagnosis and on the other with a bmt or sct therapy, the greater the chance to increase life expectancy for these children, in addition to ensuring better quality of life. the discovery and cloning of the genes for these diseases have obvious implications for the potential of gene therapy. the rapidity of these advances suggests that there will soon be many more to come. one of the most common differential diagnoses will occur with a child affected by rris, for whom we believe the number of infections must be immediately clarified, although evaluated according to different numeric and epidemiologic factors, not associated with those which instead concern the severity and the site of the infection as well as the type of the pathogenic agent that characterize children with pid. however, antibiotics are banned by the supporters of the hygiene hypothesis (chap. 24). an update on the prevalence of glucose-6-phosphate dehydrogenase deficiency and neonatal jaundice in tehran neonates primary immunodeficiency in iran: first report of the national registry of pid in children and adults association of glucose-6-phosphate dehydrogenase deficiency and x-linked chronic granulomatous disease in a child with anemia and recurrent infections correction of ada-scid by stem cell gene therapy combined with nonmyeloablative conditioning familial defect in the surface expression of the t-cell receptor-cd3 complex a point mutation in a domain of gamma interferon receptor 1 provokes severe immunodeficiency a novel genetic leukocyte adhesion deficiency in subsecond triggering of integrin avidity by endothelial chemokines results in impaired leukocyte arrest on vascular endothelium under shear flow inherited interleukin 12 deficiency in a child with bacille calmette-guerin and salmonella enteritidis disseminated infection human neutrophil immunodeficiency syndrome is associated with an inhibitory rac2 mutation committee report: the diagnosis and management of immunodeficiency devenir intellectuel des enfants atteints d'une microdélétion 22q11.2 : suivi longitudinal monocentrique de 44 patients mutations in the ela2 gene encoding neutrophil elastase are present in most patients with sporadic severe congenital neutropenia but only in some patients with the familial form of the disease immune reconstitution without graft-versus-host disease after haemopoietic stem-cell transplantation: a phase 1/2 study long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies: report of the european experience 1968-99 successful treatment of griscelli syndrome with unrelated donor allogeneic hematopoietic stem cell transplantation genetic heterogeneity in patients with x-linked recessive chronic granulomatous disease autoimmunity in human primary immunodeficiency diseases brief report: primary immunodeficiency caused by mutations in the gene encoding the cd3g subunit of the t lymphocyte receptor human t-cell activation deficiencies defective t cell receptor signaling and cd8+ thymic selection in humans lacking zap-70 kinase adenosine deaminase deficiency: genotype-phenotype correlations based on expressed activity of 29 mutant alleles genetic and immunologic analysis of a family containing five patients with common variable immune deficiency or selective iga deficiency the two gtpases, cdc42 and rac, bind directly to a protein implicated in the immunodeficiency disorder wiskott-aldrich syndrome sindrome da deficiente adesione leucocitaria persistent developmental delay despite successful bone marrow transplantation for purine nucleoside phosphorylase deficiency objective passive-smoking indicators and respiratory morbidity in young children influence of severe combined immunodeficiency phenotype on the outcome of hla non-identical, t-cell-depleted bone marrow transplantation: a retrospective european survey from the european group for bone marrow transplantation and the european society for immunodeficiency reticular dysgenesis: hla non-identical bone marrow transplants in a series of 10 patients approach to recurrent respiratory infections successful treatment of invasive aspergillosis in chronic granulomatous disease by granulocyte transfusions followed by peripheral blood stem cell transplantation activation and function of natural killer cell responses during viral infections griscelli syndrome: characterization of a new mutation and rescue of t-cytotoxic activity by retroviral transfer of rab27a gene t lymphocyte-directed gene therapy for ada-scid: initial trial results after 4 years successful related umbilical cord blood transplantation for graft failure following t cell-depleted non-identical bone marrow transplantation in a child with major histocompatibility complex class ii deficiency primary immunodeficiency diseases gene therapy in peripheral blood lymphocytes and bone marrow for ada-immunodeficient patients bone marrow transplantation as treatment for x-linked immunodeficiency with hyper-igm defective interleukin-12/interferon-gamma pathway in patients with hyperimmunoglobulinemia e syndrome identification of six novel wasp gene mutations in patients suffering from wiskott-aldrich syndrome correction of purine nucleoside phosphorylase deficiency by transplantation of allogeneic bone marrow from a sibling in vitro cell death of activated lymphocytes in omenn's syndrome breakthroughs in the understanding and therapy of primary immunodeficiency primary immunodeficiency secondary to zap-70 deficiency genetic basis of human complement c4a deficiency: detection of a point mutation leading to nonexpression longterm follow-up of patients with igg subclass of iga deficiencies (letter) searching for unrelated donor hematopoietic stem cells: availability and speed of umbilical cord blood versus bone marrow genetic and physical mapping of the chediak-higashi syndrome on chromosome 1q42-43 prediction of persistent immunodeficiency in the digeorge anomaly the spectrum of primary immunodeficiency disorders in australia c7 complement deficiency in an israeli arab village les déficits immunitaires primitif en tunisie: étude de 152 cas current concepts of immune interventions in children with respiratory diseases. respiration 61 ribosomal immunotherapy for recurrent respiratory tract infections in children successful unrelated umbilical cord blood transplantation in a child with omenn's syndrome correction of fas (cd95)deficiency by haploidentical bone marrow transplantation compilation and meta-analysis of randomized placebo-controlled clinical trials on the prevention of respiratory tract infections in children using immunostimulants aspect clinique et épidémiologique des déficits immunitaires diagnosis and management of chronic neutropenia during childhood shwachman-diamond syndrome: clinical, radiological and sonographic aspects clinical consequences and treatment of primary immunodeficiency syndromes characterized by functional t and b lymphocyte anomalies (combined immune deficiency) disorders of the ige system molecular defect 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combined immune deficiency ("bare lymphocyte syndrome") severe combined immunodeficiency with selective t-cell cytokine genes taci is mutant in common variable immunodeficiency and iga deficiency day care attendance in the first year of life and illnesses of the upper and lower respiratory tract in children with a familial history of atopy day care attendance, respiratory tract illnesses, wheezing, asthma and total serum ige level in early childhood early defect methynic in bone marrow t cell progenitors in athymic nu/nu mice cutaneous manifestations of hyper-ige syndrome in infants and children zap-70 deficiency in an autosomal recessive form of severe combined immunodeficiency cytokine and chemokine dysregulation in hyper-ige syndrome ataxia-telangiectasia in italy: genetic analysis cd30 cell expression and abnormal soluble cd30 serum accumulation in omenn's syndrome. evidence for a th2-mediated condition shwachman's syndrome: pathomorphosis and long-term outcome genetic variants of chronic granulomatous disease: prevalence of deficiencies of two cytosolic components of the nadph-oxidase system successful hla-identical bone marrow transplantation in a patient with pnp deficiency using busulfan and fludarabine for conditioning the coexistence of iga deficiency and 21-hydroxylase deficiency marked by specific supra types cd40 ligand expression deficiency in a female carrier of the x-linked hyper-igm syndrome as a result of x chromosome lyonization haploidentical bone marrow transplants for two patients with reticular dysgenesis defective in vitro production of gamma interferon and tumor necrosis factor alpha by circulating t cells from patients with the hyperimmunoglobulin e syndrome loss of endothelial surface expression of e-selectin in a patient with recurrent infections genetic heterogeneity of mendelian susceptibility to mycobacterial infection inherited disorders of il-12-and ifngamma-mediated immunity: a molecular genetics update transient hypogammaglobullinemia of infancy and early infancy: outcome of 30 cases mutation in the signaltransducing chain of the interferon-gamma receptor and susceptibility to mycobacterial infection molecular basis of a selective c1s deficiency associated with early onset multiple autoimmune diseases familial congenital cd4 t cell deficiency: a new syndrome of immune deficiency transient hypogammaglobulinemia of infancy a genetic etiology for digeorge syndrome: consistent deletions and microdeletions of 22q11 immunologic reconstitution following bone marrow transplantation for x-linked hyper igm syndrome autoimmunity in wiskott-aldrich syndrome: risk factors, clinical features, and outcome in a single-center cohort of 55 patients impaired response to interferon-alpha/beta and lethal viral disease in human stat1 deficiency tcr/ cd3 down-modulation and zeta degradation are regulated by zap-70 manipulating the immune system with immune globulin omenn's syndrome serum c-reactive protein and c3 complement protein levels in severely malnourished nigerian children with and without bacterial infections carrier detection in typical and atypical x-linked agammaglobulinemia immunodeficiency disorders: general considerations. in: stiehm er (ed) immunologic disorders in infants and children, 4th edn childhood common variable immunodeficiency with autoimmune disease deficient iga synthesis viewed in the contest of normal development of iga b cells gene therapy for immune deficiency disorders immunologic disorders in infants and children, 5th edn milk precipitins, circulating immune complexes and iga deficiency identifying undiagnosed primary immunodeficiency diseases in minority subjects by using computer sorting of diagnosis codes identifying undiagnosed primary immunodeficiency diseases in minority subjects by using computer sorting of diagnosis codes disorders of granulopoiesis and granulocyte function the outcome of patients with hypogammaglobulinemia in infancy or early childhood effect of cd3d deficiency on maturation of a/b and g/d t-cell lineages in severe combined immunodeficiency mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia igg subclass specific antibody response in recurrent bronchitis familial cd8 deficiency due to a mutation in the cd8 alpha gene homozygous human tap peptide transporter mutation in hla class i deficiency severe mycobacterial and salmonella infections in interleukin-12 receptor-deficient patients restricted heterogeneity of t lymphocytes in combined immunodeficiency with hypereosinophilia (omenn's syndrome) leucocyte adhesion deficiency human severe combined immunodeficiency due to a defect of zap70, a t cell tyrosine kinase severe combined immunodeficiency with absence of peripheral blood cd8+ t cells due to zap-70 deficiency primary immunodeficiency diseases in cape town clinical and genetic heterogeneity of inherited autosomal recessive susceptibility to disseminated mycobacterium bovis 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binding of the nuclear factor of activated t cells in t lymphocytes of two male siblings infectious diseases associated with complement deficiencies assessment of complement deficiency in patients with meningococcal disease in the netherlands heterozygous and homozygous factor h deficiency states in a dutch family impact of donor type on outcome of bone marrow transplantation for wiskott-aldrich syndrome: collaborative study of the international bone marrow transplant registry and the national marrow donor program prognosis of chronic granulomatous disease evaluation of the relevance of humoral immunodeficiencies in a pediatric population combined immunodeficiencies immunodeficiencies of genetic origin bone marrow transplantation (bmt) in europe for primary immunodeficiencies other than severe combined immunodeficiency: a report from the european group for bmt and the european group for immunodeficiency treatment of x-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow clinical and laboratory assessment of immunity genetic markers on chromosome 19p and prenatal diagnosis of hla class ii-deficient combined immunodeficiency complement deficiencies hereditary cd4+ t lymphocytopenia l'exploration du système du complément en pratique clinique central mhc genes, iga deficiency and autoimmune disease susceptibility to infection and altered hematopoiesis in mice deficient in both p-and e-selectin effect of respiratory and other virus infections on ige immunoregulation development of allergy in children. i. association with virus infections treatment with gammaglobulin preparation for use in children with humoral immunodeficiency: clinical and immunological follow-up increased frequency of homozygosity of abnormal mannan-binding-protein alleles in patients with suspected immunodeficiency ataxia-telangiectasia: an interdisciplinary approach to pathogenesis genetic linkage of hyper-ige syndrome to chromosome 4 manifestations allergiques et déficits immunitaires héréditaires two children with severe recurrent infections and the x-linked hyper-igm syndrome role of immunoglobulin subclasses and specific antibody determinations in the evaluation of recurrent infection in children cure of x-linked lymphoproliferative disease (xlp) with allogeneic hematopoietic stem cell transplantation (hsct): report from the xlp registry brazilian report on primary immunodeficiencies in children: 166 cases studied over a follow-up time of 15 years bone marrow transplantation for severe combined immune deficiency an infant with severe leukocyte adhesion deficiency griscelli syndrome: a case report sustained correction of x-linked severe combined immunodeficiency by ex vivo gene therapy treatment of chediak-higashi syndrome by allogenic bone marrow transplantation: report of 10 cases long-term immune reconstitution and outcome after hla-nonidentical t-cell-depleted bone marrow transplantation for severe combined immunodeficiency: a european 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meningococcal disease child day-care, smoking by caregivers, and lower respiratory tract illness in the first three years of life intraamniotic administration of an adenoviral vector for gene transfer to fetal sheep and mouse tissues clinical presentations and outcome of severe acute respiratory syndrome in children disorders of the t-cell system mutations in ela2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis the hyperimmunoglobulin e syndrome immunoglobulin deficiency syndromes and therapy the clinical spectrum in a large kindred with autoimmune lymphoproliferative syndrome caused by a fas mutation that impairs lymphocyte apoptosis clinical, molecular, and cell biological aspects of chediak-higashi syndrome bone marrow transplantation from genetically hla-nonidentical donors in children with fatal inherited disorders excluding severe combined immunodeficiencies: use of two monoclonal antibodies to prevent graft rejection t-cell abnormalities in common variable immunodeficiency specific missense mutations in nemo result in hyper-igm syndrome with hypohydrotic ectodermal dysplasia rapid epidemiologic assessment of glucose-6-phosphate dehydrogenase deficiency in malaria-endemic areas in southeast asia using a novel diagnostic kit genetic and immunological assessment of a bone marrow transplantation in a patient with a primary immune defect: leukocyte adhesion deficiency distribution of primary immunodeficiency diseases diagnosed in a pediatric tertiary hospital spontaneous remission of granulocyte colony-stimulating factor-associated leukemia in a child with severe congenital neutropenia molecular heterogeneity of c2 deficiency disorders of the complement system x linked agammaglobulinemia with a "leaky" phenotype omenn's syndromepathologic arguments in favor of a graft versus host pathogenesis: a report of nine cases interferongamma-receptor deficiency in an infant with fatal bacille calmette-guerin infection partial interferon-gamma receptor 1 deficiency in a child with tuberculoid bacillus calmette-guerin infection and a sibling with clinical tuberculosis in: a novel form of ifn-gamma receptor 1 deficiency, cell surface receptors fail to bind ifn-gamma defective expression of t cell-associated glycoprotein in severe combined immunodeficiency clinical and immune recovery from omenn syndrome after bone marrow transplantation direct interaction of the wiskott-aldrich syndrome protein with the gtpase cdc42 defective expression of t-cell cd40 ligand causes x-linked immunodeficiency with hyper-igm atypical xlinked agammaglobulinemia (letter) shwachman-diamond syndrome associated with hypogammaglobulinemia and growth hormone deficiency x-linked agammaglobulinemia presenting as transient hypogammaglobulinaemia of infancy incidence of humoral immunodeficiency in children with recurrent infections ureaplasma urealyticum isolations from young children with respiratory problems mutations in the tyrosine phosphatase cd45 gene in a child with severe combined immunodeficiency disease upper respiratory morbidity in preschool children: a cross-sectional study localization of the gene for the wiskott-aldrich syndrome between two flanking markers timp and dxs255 on xp11 identification of mutation in the wiskott-aldrich syndrome gene and characterization of a polymorphic dinucleotide repeat at dxs6940 adjacent to the disease gene progressive peripheral neuron degeneration in ataxia-telangiectasia: an electrophysiological study in children haploidentical peripheral blood and marrow stem cell transplantation in nine cases of primary immunodeficiency. haematologica 85 x-linked immunodeficiencies involving the lymphoid system defect in radiation signal transduction in ataxia-telangiectasia successful hla nonidentical bone marrow transplantation in three patients with the leukocyte adhesion deficiency clinical, immunological, and pathological consequences of fasdeficient conditions mutations in genes required for t-cell development: il7r, cd45, il2rg, jak3, rag1, rag2,artemis, and ada and severe combined immunodeficiency neonatal bone marrow transplantation for severe combined immunodeficiency t lymphocyte receptor deficiencies a mammalian cell cycle checkpoint pathway utilizing p53 and gadd45 is defective in ataxia-telangiectasia three in vivo promoter phenotypes in mhc class ii deficient combined immunodeficiency bone marrow transplantation for cd40 ligand deficiency: a single centre experience transient hypogammaglobulinemia of infancy: clinical and immunologic features of 40 new cases high-dose intravenous g-globulin treatment for hyperimmunoglobulinemia e syndrome lad-iii, a leukocyte adhesion deficiency syndrome associated with defective rap1 activation and impaired stabilization of integrin bonds major histocompatibility complex class ii deficiency: clinical manifestations, immunologic features, and outcome bone marrow transplantation in major histocompatibility 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novel mutation with loss of function of the transcription factor ccaat/enhancer binding protein epsilon immunodeficiency diseases caused by defects in phagocytes elective bone marrow transplantation in a child with x-linked hyper-igm syndrome presenting with acute respiratory distress syndrome clinical and immunologic aspects of the hyperimmunoglobulin e syndrome bone marrow transplantation for chronic granulomatous disease: long-term follow-up and review of the literature severe chronic neutropenia in chinese children in hong kong clinical spectrum of x-linked hyper-igm syndrome severe mycobacterium bovis bcg infections in a large series of novel il-12 receptor beta1 deficient patients and evidence for the existence of partial il-12 receptor beta1 deficiency primary immunodeficiency diseases in singapore -the last 11 years anti-apoptotic signaling by the interleukin-2 receptor reveals a function for cytoplasmic tyrosine residues within the common gamma (gamma c) receptor subunit restricting zap70 expression to cd4+cd8+ thymocytes reveals a t cell receptor-dependent proofreading mechanism controlling the completion of positive selection cardiovascular anomalies in digeorge syndrome and importance of neural crest as a possible pathogenetic factor reduced levels of igg subclasses and iga in young children with asthma incidence of primary immunodeficiencies in a population of south moravia, czechoslovakia cyclic neutropenia: an unusual disorder of granulopoiesis effectively treated with recombinant granulocyte colony-stimulating factor primary immunodeficiency syndrome in italy: a report of the national register in children and adults x-chromosome inactivation and developmental patterns in mammals mutations of jak-3 gene in patients with autosomal severe combined immunodeficiency agammaglobulinemia and insights into b-cell differentiation genetic characterization of myeloperoxidase deficiency in italy purine nucleoside phosphorylase deficiency complete digeorge syndrome: persistence of profound immunodeficiency transplantation of thymus tissue in complete digeorge syndrome thymus transplantation in complete digeorge syndrome: immunologic and safety evaluations in 12 patients complete digeorge syndrome: development of rash, lymphadenopathy, and oligoclonal t cells in 5 cases postnatal thymus transplantation with immunosuppression as treatment for digeorge syndrome molecular heterogeneity of glucose-6-phosphate dehydrogenase (g6pd) variants in italy a gene encoding a novel rfx-associated transactivator is mutated in the majority of mhc class ii deficiency patients mutations in mlph, encoding a member of the rab effector family, cause the melanosome transport defects observed in leaden mice progress in primary immunodeficiency direct genetic correction as a new method for diagnosis and molecular characterization of mhc class ii deficiency transient hypogammaglobulinemia of infancy: need to reconsider name and definition a human non-xla 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cd40 and transduces the signal for cognate activation of b cells interleukin-2 receptor g chain mutation results in x-linked severe combined immunodeficiency in humans genetic defect in human x-linked agammaglobulinemia impedes a maturational evolution of pro-b cells into a later stage of pre-b cells in the b-cell differentiation pathway composition of precursor b-cell compartment in bone marrow from patients with x-linked agammaglobulinemia compared with healthy children radiosensitive scid patients with artemis gene mutations show a complete b-cell differentiation arrest at the pre-b-cell receptor checkpoint in bone marrow primary immunodeficiency diseases: an update primary immunodeficiency in colombian children a prospective, double-blind, placebo-controlled trial of i.v. immunoglobulin and trimethoprim-sulfamethoxazole in children with recurrent respiratory tract infections signaling via il-2 and il-4 in jak3-deficient severe combined immunodeficiency lymphocytes: jak3-dependent and independent pathways expansion of cd3 + , cd4 -, cd8 -t cell population expression high levels of il-5 in omenn's syndrome mutations in rab27a cause griscelli syndrome associated with haemophagocytic syndrome meyn ms 1993) high spontaneous intrachromosomal recombination rates in ataxia-telangiectasia registro español de inmunodeficiencias primarias (redip) interactions of viruses with the immune system microbial antigens and superantigens. clinical and immunological significance xlinked agammaglobulinemia: a survey of 33 iranian patients differential requirement for p56lck in fetal and adult thyreopoiesis acute respiratory illness in the community: frequency of illness and the agents involved susceptibility to infections in children with selective iga-and iga-igg subclass deficiency complement deficiency and disease stat4 serine phosphorylation is critical for il-12-induced ifn-gamma production but not for cell proliferation alterations of the x-linked lymphoproliferative disease gene sh2d1a in common variable immunodeficiency syndrome phenotypic variability: clinical presentation between the 6th year and the 60th year in a family with x-linked agammaglobulinemia (letter) artemis, a novel dna double-strand break repair/v(d)j recombination protein, is mutated in human severe combined immune deficiency deficiency of igg4 in children: association of isolated igg4 deficiency with recurrent respiratory tract infection chédiak-higashi syndrome: four cases from northern finland incidence, severity, and prevention of infections in chronic granulomatous disease long-term itraconazole prophylaxis against aspergillus infections in thirty-two patients with chronic granulomatous disease chronic septic granulomatosis revealed by neonatal pulmonary aspergillosis antibody deficiencies novel missense mutation found in a japanese patient with myeloperoxidase deficiency the aid enzyme induce class switch recombination in fibroblasts different amino acids at position 57 of the hla-dq b chain associated with susceptibility and resistance to iga deficiency immunodeficiency with hyperimmunoglobulinemia m in two female patients is not associated with abnormalities of cd40 or cd40 ligand expression bone marrow transplantation for t-b-severe combined immunodeficiency disease in athabascan-speaking native americans the presentation and natural history of immunodeficiency caused by nuclear factor kb essential modulator mutation hematopoietic cell transplantation for immunodeficiency disorders indications for the immunological evaluation of patients with meningitis igg2 deficiency in ataxia-telangiectasia a critical role for il-21 in regulating immunoglobulin production bone marrow transplantation in 26 patients with wiskott-aldrich syndrome from a single center nuovi aspetti diagnostici e patogenetici dei difetti primitivi dell'immunità umorale selective deficiency of interferon-gamma production in the hyper-ige syndrome: relationship to in vitro ige synthesis genetics, phenotype, and natural history of autosomal dominant cyclic hematopoiesis atypical x-linked agammaglobulinemia (letter) long-term followup and prognosis of chronic granulomatous disease in yugoslavia: is there a role for early bone marrow transplantation? griscelli disease maps to chromosome 15q21 and is associated with mutations in the myosin-va gene thymic function after hemapoietic stem cell transplantation for the treatment of severe combined immunodeficiency il bambino che si ammala spesso: profilo immunologico-clinico xlinked immune dysregulation, neonatal insulin dependent diabetes, and intractable diarrhoea new hereditary immunodeficiencies and genetic predisposition to infective diseases in children inherited interleukin-12 deficiency: il12b genotype and clinical phenotype of 13 patients from six kindreds links between complement abnormalities and systemic lupus erythematosus ataxia telangiectasia syndrome: clinical picture and immunological abnormalities human equivalent of the mouse nude/scid phenotype: longterm evaluation of immunologic reconstitution after bone marrow transplantation immunodeficiency-related lymphoproliferative disorders: prospective data from the united kingdom children's cancer study group registry lowered yields of virus-induced interferon production in leukocyte cultures and risk of recurrent respiratory infections in children clinical heterogeneity and reversibility of selective immunoglobulin a deficiency in 80 children two siblings with deficiency of iga1, igg2, igg4 and ige due to deletion of immunoglobulin heavy chain constant region genes extensive deletion of immunoglobulin heavy chain constant region genes in the absence of recurrent infections: when is igg subclass deficiency clinically relevant? il trapianto prenatale e postnatale di cellule staminali emopoietiche in bambini affetti da immunodeficienza primitiva methodologic problems in establishing normal values for igg subclass concentrations in a 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diseases bcl10 activates the nf-kappab pathway through ubiquitination of nemo virally induced immunosuppression the xlinked hyper-igm syndrome: clinical and immunologic features of 79 patients brief report: twin boys with major histocompatibility complex class ii deficiency but inducible immune responses neuropsychological profile of children and adolescents with the 22q11.2 microdeletion case definition for surveillance of severe acute respiratory syndrome sars primary immunodeficiency diseases role of tbx1 in human del22q11.2 syndrome high incidence of significant bone loss in patients with severe congenital neutropenia (kostmann's syndrome) key: cord-018659-rxzy6k3b authors: danziger-isakov, lara; munoz, flor m.; estabrook, michele title: posttransplant complications and comorbidities date: 2018-01-08 journal: solid organ transplantation in infants and children doi: 10.1007/978-3-319-07284-5_71 sha: doc_id: 18659 cord_uid: rxzy6k3b infectious complications cause significant acute morbidity and mortality after pediatric lung transplantation. with the lung graft in direct communication with the environment, it is susceptible to a variety of bacterial, fungal, and viral pathogens. appreciation for pretransplant risk factors in addition to perioperative and posttransplant exposures is necessary to anticipate, diagnose, and treat infections in this population. further, epidemiologic associations between infection and chronic allograft dysfunction have been reported and suggest consequences of infectious events may have substantial impact. infectious complications cause significant acute morbidity and mortality after pediatric lung transplantation. with the lung graft in direct communication with the environment, it is susceptible to a variety of bacterial, fungal, and viral pathogens. appreciation for pretransplant risk factors in addition to perioperative and posttransplant exposures is necessary to anticipate, diagnose, and treat infections in this population. further, epidemiologic associations between infection and chronic allograft dysfunction have been reported and suggest consequences of infectious events may have substantial impact. bacteria · cytomegalovirus · infectious complication · nontubercular mycobacteria · pediatric lung transplantation · respiratory virus bacteria account for about 50% of infections post lung transplant with pneumonia being most frequent. other sites include nosocomial central lineassociated bacteremia, urinary tract, and surgical site infections. the greatest risk is within the first year after transplantation, particularly in the first 3-6 months. donor infection and recipient airway colonization are also risk factors (speich and van der bij 2001; aguilar-guisado et al. 2007; parada et al. 2010; burguete et al. 2013; yun et al. 2015) . one of the largest studies of primarily adults found that 75% of infections occurred within the first year posttransplant and 42% occurred within the first 3 months. the majority, 48%, was bacterial (parada et al. 2010; burguete et al. 2013) . another study showed similar results but found that bacteremia, both primary and catheter associated, was the most common infection in the first month after transplant with pneumonia becoming most frequent after 2 months. multidrug-resistant bacteria including methicillin-resistant staphylococci, vancomycin-resistant enterococci, and carbapenem-resistant or extended-spectrum betalactamase producing gram-negative bacilli were involved in 66% of infections. bacterial infections were significantly more common in those colonized with multidrug-resistant gram-negative bacilli than those who were not (yun et al. 2015) . a pediatric study of 42 children and 49 lung transplants found that half of the infections were bacterial with 42% occurring within 3 months after transplant and 80% in the first year. the lung was the most common site (72%) and pseudomonas aeruginosa was the most common organism. bacterial infections were felt to contribute to pulmonary dysfunction (bronchiolitis obliterans) but were not the primary cause of death (metras et al. 1999) . recent data from the registry of the international society for heart and lung transplantation reported that non-cytomegalovirus infection was the cause of death in 24% of lung transplant recipients in the first year after transplant (benden et al. 2013) . one of the largest studies of pneumonia in 236 lung transplant recipients (aguilar-guisado et al. 2007) found that the most common etiology was bacterial in 83%. gram-negative bacilli accounted for 60% with pseudomonas aeruginosa being the most frequent isolate in 25%, followed by acinetobacter baumannii in 14%. staphylococcus aureus was the etiology in 14%. the probability of 1-year survival was significantly higher in those recipients who did not have an episode of pneumonia (aguilar-guisado et al. 2007 ). late-onset communityacquired pneumonia with streptococcus pneumoniae also occurs (de bruyn et al. 2004 ). survival after lung transplantation is limited by the high incidence of chronic lung allograft dysfunction (clad) that has two forms: bronchiolitis obliterans syndrome (bos) and restrictive allograft syndrome (ras). the role of infection in the development of clad has recently been reviewed (martin-gandul et al. 2015; gregson 2016) . while acute infection with community-acquired respiratory viruses is recognized as a risk, pseudomonas aeruginosa and staphylococcus aureus are increasingly recognized as well. one study of lung transplant recipients with cystic fibrosis found that loss of colonization with pseudomonas was protective against the development of bos (gottlieb et al. 2009 ). two further studies found that infections due to gram-positive bacteria, primarily staphylococcus aureus, increased the hazard risk for bos (gupta et al. 2009; valentine et al. 2009 ). the underlying allograft inflammatory state in the setting of infection also appears to be important in determining the development of clad (gregson 2016). cystic fibrosis (cf) is a common, underlying diagnosis in children who undergo lung transplantation. the registry of the international society for heart and lung transplantation reported that half of children <10 years of age and 70% of children aged 11 through 17 years had cf (benden et al. 2013) . cf-specific bacterial pathogens including multidrug-resistant (mdr) or pan-resistant bacteria persist in the paranasal sinuses and upper airways and can be a cause of posttransplant pneumonia. pseudomonas aeruginosa is most common, but other organisms include stenotrophomonas maltophilia, achromobacter xylosoxidans, and burkholderia cepacia complex (shoham and shah 2013) . in lung transplant recipients, there is increasing resistance in gram-negative bacilli including extended-spectrum beta-lactamase, ampc betalactamase, and carbapenemase (shoham and shah 2013; van duin and van delden 2013) . pseudomonas aeruginosa infection occurs in up to 80% of patients with cf and bronchiectatic lung disease, and pretransplant colonization is a significant risk factor for infection after transplant. mdr p. aeruginosa has a prevalence rate from 10% to 45% in patients with cf (shoham and shah 2013) . survival posttransplant in patients colonized with pan-resistant p. aeruginosa before transplant was similar to those with sensitive organisms at 1 year (88% vs. 96%) but lower at 3 years (63 vs. 91%) (hadjiliadis et al. 2007; shoham and shah 2013) . however, the 2006 update of the international guidelines for the selection of lung transplant candidates stated that colonization with multidrug or pan-resistant p. aeruginosa was not a contraindication because it has not been shown to significantly affect shortterm survival (orens et al. 2006) . a recent study in lung transplant recipients with cf reported that infection with pan-resistant achromobacter xylosoxidans and stenothrophomonas maltophila also did not reduce survival after lung transplantation (lobo et al. 2015) . burkholderia cepacia complex (bcc) is comprised of several different species that colonize the respiratory tract in 15-22% of patients with cf. most infections are caused by b. cenocepacia (genomovar iii) and b. multivorans (genomovar ii). pretransplant colonization with b. cenocepacia has been associated with increased posttransplant mortality (relative risk 8.4) with one study reporting 1-year survival of 29% compared to 92% in those uninfected and is considered by many centers as a contraindication to transplant (shoham and shah 2013) . recipients colonized with b. multivorans did not have decreased survival while b. gladioli had an increased mortality risk but not as high as b. cenocepacia. bcc has an 80% prevalence of multidrug resistance (shoham and shah 2013) . methicillin-resistant staphylococcus aureus (mrsa) has been increasingly recognized as a significant bacterial pathogen post lung transplantation. a study of lung transplant recipients found that 18% had s. aureus infection in the first 90 days with 62% being methicillin sensitive (mssa) and 35% being mrsa. the site of infection was pneumonia 48%, tracheobronchitis 26%, and bacteremia 12%. colonization before transplant with mrsa was a risk factor for mrsa infection posttransplant but was not associated with increased mortality at 30 and 90 days post onset of infection (shields et al. 2012) . a second study had a calculated incidence rate of mrsa of 76 cases per 1000 transplant years with the median onset of 3 months posttransplant. the most common site was the lower respiratory tract and 31% of mrsa infections were associated with bacteremia. the direct mortality after mrsa infection was 17.6% (manuel et al. 2009 ). nontuberculous mycobacteria (ntm) are ubiquitous bacteria found in environmental sources including water, soil, plants, and animals. exposure is felt to be from the environment but more recently patient-to-patient transmission has been proposed for m. abscessus complex (bryant et al. 2013) . pretransplant infection is confined primarily to the lungs, with abnormal parenchyma such as cystic fibrosis or bronchiectasis being a risk factor. posttransplant infection can involve asymptomatic colonization, invasive lung disease, skin and soft tissue infection, and central lineassociated bacteremia (griffith et al. 2007; keating and daly 2013; smibert et al. 2016) . ntm isolation from respiratory cultures in lung transplant candidates is common particularly in those with cystic fibrosis. one study (chalermskulrat et al. 2006 ) reported a 20% colonization rate with 45% of isolates being m. avium complex (mac) and 41% m. abscessus. isolation after transplant is also common from 13 to 22% with mac accounting for about 70%. invasive disease after transplant is much less common, however, occurring in fewer than 5% of lung transplant recipients (chalermskulrat et al. 2006; chernenko et al. 2006; huang et al. 2011; knoll et al. 2012) . pretransplant colonization has been associated with an increased risk of posttransplant ntm infection as well as invasive disease but only for m. abscessus (chalermskulrat et al. 2006) . while ntm isolation and disease particularly with m. abscessus is associated with increased complications post lung transplant, it has not been associated with increased mortality and is not considered an absolute contraindication to transplantation (chalermskulrat et al. 2006; knoll et al. 2012; qvist et al. 2013) . case reports of two adolescents with cystic fibrosis and pretransplant m. abscessus infection showed that when antibiotic therapy led to afb stain negativity at the time of transplant, the outcome was favorable even in the face of positive cultures (zaidi et al. 2009 ). diagnosis of ntm disease is based on criteria published by the american thoracic society/ infectious diseases society of america that include clinical and microbiological criteria (griffith et al. 2007) . compatible symptoms and radiological changes consistent with ntm infection with other etiologies excluded must be accompanied by: positive culture from at least two sputum samples, positive culture from one bronchial lavage or wash, or lung biopsy with consistent pathology and positive culture. treatment depends on accurate identification and susceptibility testing of the organism. guidelines are available and consultation with an infectious disease expert is recommended (griffith et al. 2007; keating and daly 2013) . obtaining cultures of respiratory, blood, urine, and wound samples with accurate identification and determination of drug sensitivity is critical in the treatment of bacterial infection post lung transplant. consultation with a transplant infectious diseases physician and pharmacist is recommended when designing antibiotic therapy for multi-and pan drug-resistant organisms to maximize effectiveness and minimize toxicity. removal of sources of infection such as central venous lines and drainage of focal fluid collections should be undertaken when feasible. there are no well-conducted studies that have addressed the optimal choice of agent, duration, and efficacy of antibiotic prophylaxis for lung transplantation. in the absence of positive cultures from the donor or recipient, prophylactic regimens of 48-72 h and no longer than 7 days are recommended (bratzler et al. 2013 ). in recipients with cf, broad-spectrum antibiotics are administered at the time of transplant and are selected to cover the pretransplant bacterial pathogens and associated resistance patterns (hirche et al. 2014) . most centers treat recipients with a history of p. aeruginosa infection with two-drug antipseudomonal therapy for 2-3 weeks postoperatively to reduce the risk of pneumonia and colonization of the allograft (shoham and shah 2013). large multicenter prospective studies of adult sot recipients reported that the most common fungal organisms in lung recipients were aspergillus (63%), candida (23%), and other molds (10%) while zygomycosis, cryptococcosis, and endemic fungi were uncommon (neofytos et al. 2010 ). more recent data suggest an emergence of non-aspergillus mold infections (neofytos et al. 2013; chong et al. 2015) . pediatric studies have reported an incidence of pulmonary fungal infection from 10.5 to 20%, with aspergillus being the most common organism in two studies (danziger-isakov et al. 2008; liu et al. 2009b) . a single center study of 55 lung transplant recipients (liu et al. 2009b) found a higher incidence of posttransplant fungal colonization (60%) compared to adult patients (30-40%). however, posttransplant colonization was not associated with invasive pulmonary disease, and pulmonary fungal infections were not associated with chronic allograft rejection or death (liu et al. 2009b) . a larger retrospective multicenter study with patients transplanted in 1988-2006 found tacrolimusbased immunosuppression, cytomegalovirus sero-mismatch, age over 15 years, and prior episode of rejection greater than a2 were risks for pulmonary fungal infection, but the study did not investigate colonization as a risk factor (danziger-isakov et al. 2008) . additionally, pulmonary fungal infection was independently associated with decreased 12-month survival. mortality for proven and probable infection was 38 and 11%, respectively, similar to what has been reported for adults (danziger-isakov et al. 2008) . bronchial airway anastomotic complications occurred in 14% of 214 pediatric lung transplant recipients in a single center cohort, and this complication was associated with prior episodes of posttransplant fungal infection (choong et al. 2006) . these studies indicate that fungal infection in pediatrics significantly impact posttransplant morbidity and potentially mortality. while candida species isolated from respiratory secretions may represent normal commensal flora, invasive infections due to candida species have been reported in pediatric lung transplant recipients. in addition to bronchial anastomotic infection, pleural infection, pulmonary fungal infections, and bloodstream infections appear in the pediatric literature (danziger-isakov et al. 2005; danziger-isakov et al. 2008; liu et al. 2009b ). non-albicans species including c. krusei, c. glabrata, c. parapsilosis, and c. dubliniensis can all cause disease but may have differing antibiotic susceptibilities. identification to the species level is important to facilitate optimum treatment especially as non-albicans species have been associated with increased mortality (andes et al. 2016). aspergillus species cause posttransplant infections including tracheobronchitis with anastomosis infection, invasive pulmonary disease, and disseminated disease (hosseini-moghaddam and husain 2010). risk factors for invasive disease include ischemia at the anastomosis site, single lung transplant, hypogammaglobulinemia, placement of bronchial stent, cmv infection, and colonization (robertson et al. 2009; hosseini-moghaddam and husain 2010; chong et al. 2015) . as with candida species, speciation of aspergillus is important. while a. fumigatus causes the majority of disease, other species including a. niger, a. terreus, a. flavus, and a. ustus appear to be increasing in prevalence especially with the use of inhaled amphotericin as prophylaxis (hosseini-moghaddam and husain 2010; peghin et al. 2016) . prompt diagnosis of invasive aspergillus infection is imperative to improve outcome; however, newer diagnostics have not been specifically evaluated in pediatric lung transplantation. in pediatric cancer patients, the sensitivity and specificity of galactomannan (gm), beta-dglucan, and pcr-based assays were highly variable (lehrnbecher et al. 2016) . in adult lung transplant recipients, the serum gm assay has excellent specificity but poor sensitivity while bronchoalveolar lavage gm appears more promising for diagnosis with a sensitivity of 88-100% and specificity of 89-90% depending on the cutoff used for diagnosing invasive pulmonary aspergillosis (husain et al. 2004; pasqualotto et al. 2010; luong et al. 2011 ). further, a pan-aspergillus real-time pcr assay also performed well with a sensitivity and specificity of 100% and 93%, respectively (luong et al. 2011). as newer diagnostics emerge, their utility in pediatric lung transplantation will need assessment. histoplasmosis, blastomycosis, and coccidioidomycosis are endemic fungi with restricted geographical distribution. they are found in the environment as molds and the route of infection is inhalation of spores. posttransplant disease with these organisms is rare in adults and has not been reported in the pediatric lung transplant literature to date (neofytos et al. 2010; assi et al. 2013 ). treatment of invasive fungal infection in pediatric lung transplant recipients should include input from an infectious diseases specialist particularly regarding drug choice and dosage. several national and international guidelines present treatment recommendations for invasive fungal infections (pappas et al. 2016; patterson et al. 2016) . new antifungal agents have emerged in the past decade including second-generation azole medications and echinocandins (lewis 2011). while these agents are improving outcomes related to fungal infections, clinicians must pay careful attention to therapeutic drug monitoring, interactions with immunosuppressive agents (both calcineurin inhibitors and mtors), and medication side effects to reduce potential complications. despite the significant morbidity and mortality associated with fungal infections following lung transplantation, there are not established guidelines for prophylaxis. in pediatrics, the impact of prophylaxis to prevent colonization and progression to infection is uncertain. several small, nonrandomized clinical trials in adult recipients have demonstrated efficacy ranging from 80 to 100% (hosseini-moghaddam and husain 2010; brizendine et al. 2011). three main approaches have been used in lung transplant recipients: universal prophylaxis, targeted prophylaxis, and pre-emptive therapy. universal prophylaxis is given to all recipients immediately post transplantation while targeted prophylaxis is given to patients with known risk factors (neoh et al. 2011) . further, response to positive cultures on routine posttransplant bronchoscopy may prompt initiation of pre-emptive therapy, but the optimal response to positive bal cultures is unclear (avery 2011). while inhaled amphotericin has recently been linked to a decrease in posttransplant aspergillus, amphotericin-resistant strains have emerged indicating that intervention is not benign (peghin et al. 2016) . a recent world-wide survey of antifungal prophylaxis (neoh et al. 2011 ) showed a highly variable approach with the majority (58%) using universal prophylaxis that primarily focused on preventing aspergillus infections. a survey of centers performing pediatric lung transplantation (50% exclusively pediatric) revealed an equally variable approach. universal prophylaxis is provided in 28% of centers, while 48% use targeted prophylaxis primarily to patients with cystic fibrosis or pretransplant fungal colonization (mead et al. 2014) . the focus of prophylaxis includes both aspergillus and candida species with most centers reporting the use of either voriconazole or inhaled amphotericin. additionally, the duration of prophylaxis is widely distributed from 3 to 6 months to more than 12 months. the optimal approach for fungal prophylaxis in pediatric lung transplant recipients is undefined and there are sparse data for this population to guide recommendations. the introduction of preventative antiviral regimens has improved the natural history of cytomegalovirus (cmv) after adult lung transplantation (patel and paya 1997; zamora et al. 2004; chmiel et al. 2008) ; however, cmv remains associated with increased morbidity and mortality after transplantation (husni et al. 1998; monforte et al. 2001; ruttmann et al. 2006; chmiel et al. 2008) . to improve the clarity of cmv reporting in the literature, specific definitions have been suggested and updated with diagnostic evolution (humar and michaels 2006; husain et al. 2011; ljungman et al. 2017) . cmv infection refers to the presence of active replicating virus by any method without associated symptoms. cmv syndrome includes the presence of virus plus one or more associated symptoms including fever, fatigue/malaise, leukopenia, atypical lymphocytes, thrombocytopenia, or transaminitis. those with evidence of tissue invasion are defined as end-organ cmv disease. newer definitions take into account the availability of quantitative cmv pcr testing, but a specific viral load in bal to determine cmv pneumonitis has not yet been established (ljungman et al. 2017 ). cytomegalovirus (cmv) occurs in approximately 30% of pediatric lung transplant recipients (danziger-isakov et al. 2003b; danziger-isakov et al. 2009 ) and is associated with decreased survival in this population (metras et al. 1999; danziger-isakov et al. 2003b; danziger-isakov et al. 2009 ). the largest multicenter study from the international pediatric lung transplant collaborative identified cmv donor seropositivity, a2 rejection, and transplant in the earliest era of transplantation (1985) (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) as increased risks for cmv. progression from cmv infection to disease occurred in 22% (danziger-isakov et al. 2009 ). interestingly, cmv developed in 7% of cmv dà/rà recipients and induction therapy increased the risk for cmv in this group (danziger-isakov et al. 2009 ). the optimal preventative regimen against cmv remains uncertain in pediatric lung transplant recipients. controversies include choices around the use of universal prophylaxis, risk-based prophylaxis, or pre-emptive therapy and duration of prevention strategy (danziger-isakov et al. 2003a) . as the merits and potential disadvantages in the limited population of pediatric transplant recipients are difficult to discern, extrapolation from the adult lung transplant population directs current practice (kotton et al. 2013) . data from adult lung transplantation indicates that prolonged prophylaxis (9-12 months) with valganciclovir has both short-and long-term benefits preventing cmv events and decreasing risk for bronchiolitis obliterans syndrome (finlen et al. 2011; mitsani et al. 2010; palmer et al. 2010 ). pre-emptive therapy is not currently recommended for high-risk cmv d+/rà lung transplant recipients (kotton et al. 2013; . antiviral complications including nephrotoxicity, bone marrow suppression, gastrointestinal manifestations, and the development of viral resistance mutations must be considered when developing prevention strategies (mitsani et al. 2010; danziger-isakov and mark baillie 2009) . a study in pediatric transplantation showed safety and efficacy of an oral valganciclovir dosing algorithm, but no pediatric lung transplant recipients were enrolled (vaudry et al. 2009 ). pediatric studies have assessed long-term intravenous ganciclovir and the adjunctive use of cmv hyperimmune globulin (cmvig) (spivey et al. 2007; ranganathan et al. 2009 ). in a study of nine pediatric lung transplant recipients, 12 weeks of intravenous ganciclovir was feasible, safe, and effective prevention, although cases of catheter-related bloodstream infections did occur when the catheters remained in place beyond the 12-week ganciclovir administration period (spivey et al. 2007 ). cmvig administration as part of a prevention regimen was associated with a threefold decrease in cmv infection but did not impact the incidence of cmv disease or other posttransplant morbidities and mortality in a multinational retrospective study (ranganathan et al. 2009 ). each institution should assure that a consistent prevention strategy and adequate monitoring are in place (kotton et al. 2013 ). cmv monitoring is an integral part of any prevention strategy and potentially allows identification of cmv infection prior to the development of cmv symptoms or end-organ disease. viral culture or a pp65 antigenemia assay has been replaced by more sensitive polymerase chain reaction (pcr) (weinberg et al. 2000) . as interassay and intercenter variability has been reported for pcr testing even in controlled research settings; utilization of a consistent assay is crucial so that results can be compared for an individual subject over time (pang et al. 2009; rychert et al. 2014 ). based on multicenter retrospective evaluation of pediatric lung transplant recipients (danziger-isakov et al. 2009 ), the highest risk period for cmv infection occurs during the first 6 weeks after discontinuation of prophylaxis; thus, appropriate monitoring should occur during this high-risk period. additionally, evaluation for cmv should occur with new onset symptoms suspicious for cmv infection including fever, fatigue, and lymphadenopathy even in cmv dà/rà patients. increased frequency of cmv surveillance is suggested during periods of increased immunosuppression, but not limited to cytolytic therapy for refractory rejection, plasmapheresis, or prolonged lymphopenia (kotton et al. 2013) . additional monitoring for cmv-specific immunity continues to develop (westall et al. 2008; kumar et al. 2009; snyder et al. 2011; manuel et al. 2013b ) and may be employed in the future to personalize cmv prevention strategies. treatment of cmv infection and disease relies primarily on antiviral therapy and, if possible, decreasing immunosuppression. a multicenter randomized clinical trial of predominantly adult kidney transplant recipients reported noninferiority of oral valganciclovir compared to intravenous ganciclovir for nonlife-threatening cmv disease; however, no pediatric patients were enrolled (asberg et al. 2007 ). current recommendations from the transplant society consensus statement include the use of intravenous ganciclovir for pediatric-aged patients as first-line therapy with acknowledgement that some experts would use oral valganciclovir for cmv infection (kotton et al. 2013) . oral ganciclovir, acyclovir, famciclovir, or valacyclovir should not be used to treat cmv. adjunctive therapy with immunoglobulins for severe pneumonitis (either intravenous immunoglobulin or cmvig) can be considered (kotton et al. 2013) . resistant cmv is rarely reported in pediatric transplant recipients (martin et al. 2010; kim et al. 2012 ), but concern may prompt consideration for alternative antiviral therapy including high-dose ganciclovir, foscarnet, and cidofovir (kotton et al. 2013) . newer antiviral agents are under investigation as options for either prevention or treatment including brincidofovir, letermovir, and maribavir. emerging data on the adoptive transfer of cmv-specific t-cells and the use of small-molecule drugs such as sirolimus, leflunomide, and artesunate may alter the future of treatment, but currently no data related to these interventions exist for pediatric lung transplant recipients. human herpes virus 6 and 7 epidemiology and risk human herpes virus (hhv) 6 and 7 are ubiquitous, common viruses that cause mild infections in young children so frequently that by 5 years of age, practically all children have been infected. there are two types of hhv-6, and although the epidemiology of hhv-6a is not clearly defined, hhv-6b is the most common cause of pediatric infections. young infants, especially those under 2 years of age, are at risk for community-acquired or nosocomial hhv-6 infection after solid organ transplantation, while infection may also be acquired through the allograft or as a reactivation of a prior infection in older children. overall, symptomatic or significant infection with hhv-6 after lung transplantation is uncommon, and the overall incidence in solid organ transplant recipients has been reported to be less than 1% . hhv-7 infection seems to be common, but its clinical manifestations are less well characterized. the most typical disease manifestation of hhv-6 infection is roseola infantum (also known as exanthem subitum or sixth disease), a classic febrile illness in young children where the resolution of a high fever of short (3-5 days) duration is followed by the appearance of a characteristic erythematous rash. while young children may present with roseola after lung transplantation, the most likely clinical manifestation in these patients may be a nonspecific febrile illness that may or may not be associated with an erythematous diffuse rash, hepatitis, pneumonia, encephalitis, and leukopenia. hhv-7 coinfection with hhv-6 is reported frequently, and hhv-7 infection alone appears to be asymptomatic or associated with milder clinical manifestations. diagnosis hhv-6/7 infection is confirmed by detection of the virus in otherwise sterile samples (blood, csf, tissue) by nucleic acid identification (pcr) or consistent histopathologic changes. quantification of viral load might be helpful to assess the progression of viremia. however, there is no known clinically relevant viral load threshold to predict progression or severity of disease. immunohistochemical staining is available in some laboratories and might be helpful to determine the presence of infection in specific organs. however, hhv-6/7 latency in human cells may result in the identification of these viruses in samples without correlation with infection. the first step in the treatment of suspected or confirmed hhv-6/7 infection in immunocompromised solid organ transplant patients is decreasing immunosuppression to allow the host's immune system to control the virus. there are no specific antivirals recommended for treatment of hhv-6/7. however, antiviral activity has been described with ganciclovir and its oral form valganciclovir, foscarnet, and cidofovir. consultation with an infectious diseases expert for the antiviral management of these infections is recommended. there are no vaccines available for the prevention of hhv-6/7 infections. suppression may be observed with antivirals used after transplantation for cmv, such as ganciclovir and valganciclovir; however, specific antiviral prophylaxis for hhv-6/7 is not recommended. hand hygiene is the most effective method to prevent transmission. hhv-8, known as the cause of kaposi's sarcoma, is an oncogenic virus associated with a variety of malignancies (primary effusion lymphoma and castleman disease) and other disease syndromes such as febrile illness, bone marrow suppression, hemophagocytosis, and multiorgan failure in highly immunocompromised patients, including transplant recipients . however, the incidence of hhv-8 infection and disease in children is very rare in the united states. residence in hhv-8 endemic areas is a risk factor, as is receipt of an organ from a donor coming from an endemic area. hhv-8 serology is not routinely obtained in solid organ recipients or donors. as with other human herpesviruses, latency can be established. decreasing immunosuppression is recommended, while treatment of associated malignant disease may also include surgical debulking, cytotoxic chemotherapies, and antivirals (for which the efficacy has not been established). adenoviruses commonly cause self-limited respiratory and gastrointestinal infections in immunocompetent children, but infections can be severe in lung and other solid organ transplant recipients. adenovirus infections are more common in pediatric than in adult transplant recipients. primary adenovirus infections may be acquired after transplantation in young children, while reactivation of latent infection or infection from the transplanted organ may occur in older children and adolescents (florescu et al. 2013) . lung transplant patients are at particularly high risk for complicated respiratory tract infection though inhalation of infected aerosol particles or direct contact transmission from infected individuals. gastrointestinal infection may occur via fecal-oral transmission. most infections occur in the first few months after transplantation, or during periods of enhanced immunosuppression. nosocomial and community exposures may be the source of infection. clinical manifestations of adenovirus depend on the organ affected. adenovirus infection can result in severe respiratory disease in lung transplant recipients, including rapidly progressive, necrotizing and potentially fatal pneumonia, as well as development of chronic lung disease with fibrosis and bronchiectasis (liu et al. 2010) . adenovirus may also cause gastroenteritis, hepatitis, meningoencephalitis, and disseminated disease with multiorgan involvement (florescu et al. 2013) . asymptomatic infection, defined as the identification of adenovirus in clinical specimens by nucleic acid detection (pcr) or culture, has been reported more commonly in adults. in children, persistent and rising viremia should be considered a concerning sign of end organ infection and risk for disseminated disease. graft failure may result from acute adenovirus infection after lung transplantation (doan et al. 2007 ). the diagnosis of adenovirus infection requires the presence of consistent clinical symptoms and the identification of adenovirus by viral culture, molecular methods, direct antigen detection, or characteristic histopathology. most adenovirus serotypes (with the exception of adenovirus 40 and 41 which cause gastroenteritis) can be isolated in cell culture; however, diagnosis by pcr is more commonly used and available. the sensitivity of rapid antigen detection tests is variable and not reliable in immunocompromised hosts. while adenovirus can be identified in respiratory secretions, stool, and urine, these are places where prolonged shedding after infection may occur. therefore, the diagnosis of acute infection is more reliable when viral identification is associated with consistent clinical symptoms, or when adenovirus is found in otherwise sterile specimens such as blood and cerebrospinal fluid or in tissues. rising viremia and detection of virus in two or more sites is considered consistent with invasive adenovirus disease. a viral load cutoff or threshold does not exist to predict the progression of disease or its outcome. however, higher and/or persistent viral loads are concerning for progressive or disseminated disease and typically indicate the need to intervene. decreasing the level of immunosuppression to allow for the host's immune response to handle the virus is the most important treatment strategy to manage adenovirus infections in young solid organ transplant patients. in certain cases, antiviral treatment may be useful, if instituted with the guidance and follow-up of a pediatric infectious diseases specialist. while there are no approved adenovirus-specific antivirals, some agents such as cidofovir have activity against adenovirus and have been used empirically for treatment. however, use of this agent is limited by its propensity to cause nephrotoxicity and bone marrow suppression. close follow up and monitoring for these side effects is recommended. the standard dose of cidofovir in children is 5 mg/kg once weekly. however, more frequent, lower dosage (1 mg/kg three times per week) and pre-and post-dose hydration have been used in an attempt to reduce the risk of renal toxicity (doan et al. 2007) . treatment is usually continued until resolution of viremia and/or symptoms, with close monitoring for side effects. other antivirals have been evaluated for treatment of adenovirus, including a lipid conjugate of cidofovir (cmx001, chimerix inc.), which is administered orally and has a lower risk for nephrotoxicity; however, its use remains experimental at this time. lung transplant patients with severe infection may have hypogammaglobulinemia, and in these cases, administration of intravenous immunoglobulin (ivig) for replacement has been used, although its benefit has not been proven (mawhorter and yamani 2008 ). an effective novel treatment strategy has been developed with the use of antigen-specific cytotoxic t lymphocytes (ctl) directed against adenovirus in hematopoietic stem cell transplant recipients; ctls have not been evaluated in lung or other pediatric solid organ transplant recipients (leen et al. 2009 ). prolonged shedding after resolution of the acute infection may occur; therefore, strict hand hygiene and disease prevention strategies need to be implemented. there are no licensed vaccines for the prevention of adenoviruses. epidemiology and risk pediatric solid organ transplant recipients and particularly lung transplant recipients are at increased risk of medical complications and mortality when acquiring common respiratory viral infections (manuel et al. 2013a) . common respiratory viruses that circulate with well-described seasonality in the united states include influenza virus, respiratory syncytial virus (rsv), human metapneumovirus, human rhinovirus, parainfluenza viruses, coronaviruses, and other respiratory viruses that are being more frequently described, such as bocaviruses. lung transplant recipients are at risk for community and nosocomial exposures during the typical time of circulation of these viruses. infection with respiratory viruses may also increase the risk for secondary bacterial pneumonia and other bacterial or fungal infections, particularly in the first few months after transplant (liu et al. 2009a) . after an acute lower respiratory virus infection, the risk for graft rejection or chronic allograft dysfunction may increase as shown in adult lung transplant recipients; however, this is controversial and has not been shown in pediatric lung transplant recipients to date (liu et al. 2010; liu et al. 2009a; vu et al. 2011) . although upper respiratory infections may present similarly in solid organ transplant recipients as in immunocompetent hosts, progression to lower respiratory tract disease manifestations with tachypnea, cough, abnormal breath sounds, hypoxemia, and respiratory failure is a concern in lung transplant recipients. clinical deterioration due to respiratory viruses is more frequently reported in the period of highest immune suppression shortly after transplant. prompt diagnosis with viral detection using nucleic acid amplification methods (pcr) is recommended in immunocompromised hosts. viral cultures could be obtained but are not as useful given that results are delayed in comparison with pcr. pcr platforms that test for multiple viruses at the same time are most helpful in lung transplant recipients. rapid antigen detection tests are no longer recommended for influenza due to their variable sensitivity; but they could still be useful for the diagnosis of rsv. respiratory secretions including nasal wash or swabs, naropharyngeal aspirates, and tracheal or broncheoalveolar lavage can be used. these viruses do not tend to be associated with viremia. supportive measures must be instituted promptly in lung transplant recipients with progression to lower respiratory tract disease. the need for invasive mechanical ventilation or other higher level of supporting measures such as extracorporeal membrane oxygenation (ecmo) is not uncommon in patients with severe disease. specific antiviral treatment is available for influenza a and b infection. immediate initiation of neuraminidase inhibitor (oseltamivir and zanamivir) therapy in lung transplant patients with fever and/or other respiratory symptoms during the period of influenza circulation may decrease the risk of complications and death associated with influenza. although influenza antivirals are usually preferred within 48 h of the onset of clinical symptoms, lung and other solid organ transplant patients have improved outcomes even with later treatment initiation (kumar et al. 2010 ). in some cases, a more prolonged duration of antiviral therapy has been used given these patient's immune-suppressed status and prolonged viral shedding. intravenous peramivir (also a neuraminidase inhibitor) is now licensed for adults, with clinical studies underway in children and adolescents. intravenous administration might be preferred in patients who have inadequate enteral absorption and who are severely ill with influenza. ribavirin, an aerosolized antiviral with in vitro activity against rsv, parainfluenza, human metapneumovirus, and other viruses, is fda approved but not routinely recommended for treatment of these infections due to lack of definitive efficacy. however, ribavirin has been used early in the course of rsv and other respiratory virus infections, as well as in more severe cases of respiratory disease, in lung transplant patients due to its antiviral effects. no randomized controlled trials have been performed although data from adult lung transplantation has indicated a potential response to aerosolized, intravenous, and oral ribavirin (glanville et al. 2005; pelaez et al. 2009; li et al. 2012 ). an inhaled small-interfering rna that targets rna (aln-rsv-001) has also been investigated as a therapy for rsv in adult lung transplantation showing potential reduction in bronchiolitis obliterans syndrome after rsv infection (zamora et al. 2011; gottlieb et al. 2016) . utilization of an rsv antibody preparation (monoclonal antibody) along with antiviral treatment in severe cases has been reported to reduce rsv-associated mortality in some cases (chavez-bueno et al. 2007 ). similar to the management of other viral infections, decreasing immune suppression is advisable when respiratory viral infections are identified. influenza immunization prior to and/or after transplantation for the recipient and all close contacts and family members is recommended to prevent infection and severe disease. inactivated influenza vaccine should be administered ideally prior to the start of the season, to ensure optimal protection. however, after transplant, and in some patients prior to transplant depending on their underlying diagnosis or need for chronic steroid or other medication use, the immune responses to vaccination might be suboptimal in lung and other solid organ transplant recipients. therefore, vaccination of close contacts and avoidance of contact with sick individuals become important measures for prevention of infection (avery et al. 2013) . prophylactic antivirals may also help decrease the risk of infection and complications in exposed unvaccinated or unprotected transplant recipients. there are no other vaccines available for the prevention of respiratory infection in most pediatric lung transplant recipients. however, palivizumab, a monoclonal antibody against rsv, can be used during the rsv season in young children less than 2 years of age who are lung transplant recipients, immunosuppressed, or who have underlying chronic lung or hemodynamically unstable heart disease (american academy of pediatrics committee on infectious diseases and american academy of pediatrics bronchiolitis guidelines committee 2014). all lung and solid organ transplant patients with suspected or known respiratory viral infections need to be isolated from other patients using standard contact and droplet precautions. posttransplant, infections remain a significant factor causing both morbidity and mortality in pediatric lung transplant recipients. pathogens are diverse including bacteria, fungi, and viruses with timing of events dependent on time from transplant. all events can have both immediate and long-term consequences in this at-risk population. prevention, identification, and early intervention for infectious events can improve outcomes after pediatric lung transplantation. lung infections in pediatric lung transplantation: experience in 49 cases cytomegalovirus disease among donor-positive/recipient-negative lung transplant recipients in the era of valganciclovir prophylaxis nebulized amphotericin b prophylaxis for aspergillus infection in lung transplantation: study of risk factors epidemiology and outcome of invasive fungal infections in solid organ transplant recipients epidemiology, risk factors, and outcomes of clostridium difficile infection in kidney transplant recipients antifungal prophylaxis in lung transplantation-a world-wide survey international guidelines for the selection of lung transplant candidates: 2006 update -a consensus report from the pulmonary scientific council of the international society for heart and lung lransplantation extended valganciclovir prophylaxis to prevent cytomegalovirus after lung transplantation: a randomized, controlled trial interlaboratory comparison of cytomegalovirus viral load assays clinical practice guideline for the management of candidiasis: 2016 update by the infectious diseases society of america early and late infections in lung transplantation patients diagnosis of invasive aspergillosis in lung transplant recipients by detection of galactomannan in the bronchoalveolar lavage fluid infections in solid-organ transplant recipients practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the infectious diseases society of america 10 years of prophylaxis with nebulized liposomal amphotericin b and the changing epidemiology of aspergillus spp. infection in lung transplantation efficacy of oral ribavirin in lung transplant patients with respiratory syncytial virus lower respiratory tract infection nontuberculous mycobacterial disease is not a contraindication to lung transplantation in patients with cystic fibrosis: a retrospective analysis in a danish patient population cytomegalovirus immunoglobulin decreases the risk of cytomegalovirus infection but not disease after pediatric lung transplantation human herpesviruses 6, 7 and 8 in solid organ transplant recipients cytomegalovirus in solid organ transplantation hypogammaglobulinemia: incidence, risk factors, and outcomes following pediatric lung transplantation combined cmv prophylaxis improves outcome and reduces the risk for bronchiolitis obliterans syndrome (bos) after lung transplantation multicenter comparison of laboratory performance in cytomegalovirus and epstein-barr virus viral load testing using international standards staphylococcus aureus infections in the early period after lung transplantation: epidemiology, risk factors, and outcomes impact of multidrug-resistant organisms on patients considered for lung transplantation mycobacterium abscessus complexa particular challenge in the setting of lung transplantation polyfunctional cytomegalovirusspecific immunity in lung transplant recipients receiving valganciclovir prophylaxis epidemiology and management of infections after lung transplantation safety and efficacy of prolonged cytomegalovirus prophylaxis with intravenous ganciclovir in pediatric and young adult lung transplant recipients effect of etiology and timing of respiratory tract infections on development of bronchiolitis obliterans syndrome multidrug-resistant gram-negative bacteria infections in solid organ transplantation valganciclovir dosing according to body surface area and renal function in pediatric solid organ transplant recipients (2013) (6) key: cord-017393-kx8kmdej authors: herbers, alexandra; de pauw, ben e. title: acute myelogenous leukemia and febrile neutropenia date: 2009-08-31 journal: managing infections in patients with hematological malignancies doi: 10.1007/978-1-59745-415-5_5 sha: doc_id: 17393 cord_uid: kx8kmdej aggressive chemotherapy has a deleterious effect on all components of the defense system of the human body. the resulting neutropenia as well as injury to the pulmonary and gastrointestinal mucosa allow pathogenic micro-organisms easy access to the body. the symptoms of an incipient infection are usually subtle and limited to unexplained fever due to the absence of granulocytes. this is the reason why prompt administration of antimicrobial agents while waiting for the results of the blood cultures, the so-called empirical approach, became an undisputed standard of care. gram-negative pathogens remain the principal concern because their virulence accounts for serious morbidity and a high early mortality rate. three basic intravenous antibiotic regimens have evolved: initial therapy with a single antipseudomonal β-lactam, the so-called monotherapy; a combination of two drugs: a β-lactam with an aminoglycoside, a second β-lactam or a quinolone; and, thirdly, a glycopeptide in addition to β-lactam monotherapy or combination. as there is no single consistently superior empirical regimen, one should consider the local antibiotic susceptibility of bacterial isolates in the selection of the initial antibiotic regimen. not all febrile neutropenic patients carry the same risk as those with fever only generally respond rapidly, whereas those with a clinically or microbiologically documented infection show a much slower reaction and less favorable response rate. once an empirical antibiotic therapy has been started, the patient must be monitored continuously for nonresponse, emergence of secondary infections, adverse effects, and the development of drug-resistant organisms. the averageduration of fever in serious infections in eventually successfully treated neutropenic patients is 4–5 days. adaptations of an antibiotic regimen in a patient who is clearly not responding is relatively straightforward when a micro-organism has been isolated; the results of the cultures, supplemented by susceptibility testing, will assist in selecting the proper antibiotics. the management of febrile patients with pulmonary infiltrates is complex. bronchoscopy and a high resolution computer-assisted tomographic scan represent the cornerstones of all diagnostic procedures, supplemented by serological tests for relevant viral pathogens and for aspergillosis. fungi have been found to be responsible for two thirds of all superinfections that may surface during broad-spectrum antibiotic treatment of neutropenic patients. antibiotic treatment is usually continued for a minimum of 7 days or until culture results indicate that the causative organism has been eradicated and the patient is free of major signs and symptoms. if a persistently neutropenic patient has no complaints and displays no evidence of infection, early watchful cessation of antibiotic therapy or a change to the oral regimen should be considered. only 50 years ago, dealing with a patient with a disseminated malignant disease was relatively simple. there were no curative options and information on the inevitable dismal prognosis was not shared with the patient or his family. the mid sixties of the twentieth century witnessed the first successes of chemotherapeutic agents. this encouraged investigators to explore this route further, thereby escalating the dosage of the cytostatic drugs in the expectation of better results. it became rapidly clear that the destructive effects of cytotoxic compounds were not limited to malignant cells. infection has emerged as a prominent complication of chemotherapy, which was particularly worrisome in the sixties, a decade without powerful broad-spectrum antimicrobial agents. since a possible cure of the cancer was seen as the primary goal, complications of rigid cytotoxic regimens were taken for granted and when they occurred, treatment was more or less improvised. this situation remained unchanged until bodey [1] pointed out that patients in remission of their underlying disease could die suddenly of an overwhelming infection during cytotoxic therapy-induced neutropenia. neutropenia was and remains defined as an absolute neutrophil count of less than 0.5 × 10 9 /l (500/mm 3 ) or a count less than 1.0 × 10 9 /l (1,000/mm 3 ) expected to fall below 0.5 × 10 9 /l (500/mm 3 ) [1] . he even showed a positive correlation between the severity and duration of neutropenia and the risk of acquiring a life-threatening bacterial infection. this risk appeared even more pronounced in individuals who were treated for an acute leukemia or lymphoma as these disorders interfered directly with vital components of the immune system. next to gram-negative bacilli, staphylococcus aureus earned a notably bad reputation [2] . a few years later, schimpff and co-workers demonstrated convincingly that early administration of antimicrobial agents covering the above suspected pathogens while waiting for the results of the blood cultures saved lives. his so-called empirical approach became an undisputed standard of care [3] . however, better options to manage infections encouraged hematologists to intensify their antileukemic regimens further in an attempt to improve the remission rates in previously refractory cases. these intensifications, in turn, inspired more thorough clinical research into potentially more effective antimicrobial regimens, which was facilitated by the booming development of new antimicrobial agents such as broad-spectrum synthetic penicillins, third and fourth generation cephalosporins, fluoroquinolones, and carbepenems in conjunction with a keen eagerness of the respective pharmaceutical companies to put their compounds to test in large clinical trials that were usually conducted by cooperative trial groups [4, 5] . a cycle of several subsequent rounds of broader-spectrum antibiotics and further intensification of chemotherapeutic regimens has eventually lessened the mortal risk of neutropenia to only one of many problems in today's clinical practice. modern anti-leukemic therapy is inherently associated with ulceration of the pulmonary and gastrointestinal mucosa thereby allowing micro-organisms originating from the damaged mucosal tracts easy access to the body [6, 7] . these pathogens may be part of the original indigenous flora but are commonly acquired during hospitalization [8] . in the 1970s, it was considered logical to prevent invasion of the body by indigenous flora by prophylactic administration of anti-infective agents. since such prophylactic agents were mainly targeted against the gram-negative enterobacteriaceae, a shift from gram-negative to gram-positive micro-organisms, including coagulase-negative staphylococci, viridans streptococci, and enterococci, as the primary cause of fever in neutropenic patients was seen [9] [10] [11] [12] [13] [14] [15] . in the meantime, therapeutic regimens in the treatment of hematological malignancies have become so complex that use of surgically implanted venous access devices became universal in spite of the risk of catheter-associated infections and thrombosis [16, 17] . an epidemiological survey among hospitalized patients treated for hematological malignancies between 1995 and 2001 in the united states showed that approximately 70% (64% in 1995 and 76% in 2001) of all microbiologically confirmed febrile episodes were due to gram-positive bacteria and 18% (22% in 1995 and 14% in 2001) due to gram-negative bacilli [18] . this change in pathogens was facilitated by increased use of central venous catheters and other medical devices. introduction of immunomodulatory monoclonal antibodies into the therapeutic arena has extended the treatment-related immunodeficiency to t-cell functions and innate immunity. this, in turn, has brought viral and fungal infections, including pneumocystis jeroveci, into play, particularly when impaired cellular immunity coincided with prolonged, severe neutropenia [19, 20] . the modern chemotherapeutic regimens designed to treat acute lymphoblastic leukemia incorporate high doses of corticosteroids. as a result, patients treated with such regimens are at increased risk of infections typically related to an impaired cellular immunity. in addition, allogeneic bone marrow transplantations have become a fully accepted treatment modality for many hematological malignancies. nowadays, infections still account for substantial morbidity and mortality among patients who undergo myeloablative therapy for a hematological malignancy. in spite of all changes in the spectrum of infectious agents, gram-negative pathogens remain the principal concern because their virulence accounts for serious morbidity and high early mortality rate [21, 22] . administration of potentially curative chemotherapy is the starting point in treating acute leukemias. giving cytotoxic drugs is relatively straightforward since internationally accepted antitumor protocols have defined the optimal dosages. once the chemotherapy has been administered, the hematologist must wait patiently for the desired outcome a few weeks later. however, while the scientist in the hematologist has completed this first task, the general clinician in him or her has to step forward to monitor the patient, as the natural host defense system gradually disintegrates. close surveillance of the patients with attention to the emergence of infectious complications is mandatory. management of infections during this time of danger must be individualized because fixed protocols and algorithms are of limited usefulness given the complexity of infectious diseases management [23, 24] . it is here that the science and art of medicine meet; listening to the patient's complaints and meticulous physical examinations constitute the crucial factors for timely therapeutic interventions and eventual success. this applies to both patients who are treated with intensive chemotherapeutic regimens and to recipients of a stem cell transplant. during this period of neutropenia, appropriate coordination of information coming from different sources is important, since, next to the patient, family members, nurses, microbiologists, pulmonologists, radiologists, and pathologists can assist in the timely discovery of an emerging complication. different cancer centers approach these tasks in different ways but it occurs to us that the hematologist who is responsible for treating the underlying hematological disease must also act as the captain of the ship. this coordinating role obliges him or her to have at least some basic knowledge of likely infection problems and, perhaps even more importantly, to have fine communication skills to keep all parties on board as well as incorrect on the same course. since the symptoms of an incipient infection are usually rather subtle due to the absence of granulocytes, teamwork is crucial to ensure that antibiotics are administered at the first signs or symptoms of infection [25] . in most cases, fever defined as a single oral temperature of more than 38,3°c (101°f) or a temperature of more than 38,0°c (100,5°f) for more than 1 h, will serve as a trigger for action. at the onset of fever, attempts to identify the cause of fever deserve absolute priority (see table 5 -1), immediately followed by institution of appropriate broad-spectrum antibiotic therapy preferably within one hour of fever [22] . fever in a neutropenic patient is a warning sign that should be taken very seriously because self-limiting infection is virtually • consider determination of crp, galactomannan antigen, and viral serology nonexistent in neutropenic patients irrespective of whether they have been treated for acute leukemia or lymphoma or received a stem cell transplantation. in anticipation of the results of the diagnostic evaluation, fever denotes infection until proven otherwise. absence of phagocytic cells in combination with a damaged skin and mucosal surfaces allows micro-organisms residing at a superficial site of infection easy access to the bloodstream. under these circumstances, a relatively small inoculum, that easily can escape detection when limited volumes of blood are sampled for culturing, can cause a serious septic syndrome [3] . therefore, withholding antibiotics while waiting for a blood culture to become positive is a bad idea, even though fever can be of noninfectious origin [26] . a sudden onset of fever accompanied by chills, tachycardia with or without a drop in blood pressure, and tachypnea is associated with a higher rate of positive blood cultures. shock at the onset of fever is an ominous clinical sign but neither clinical manifestations nor the pattern of fever during neutropenia can serve as an indicator of a particular causative agent, not even when the most notorious pathogens such as pseudomonas aeruginosa or staphylococcus aureus are involved [19, 27] . a substantial minority of patients with true infections will have an insidious onset of fever. although more frequently related to noninfectious causes than acute fever, a slow rise of temperature does not exclude an infectious origin, although gram-negative rods, viridans streptococci, and staphylococcus aureus are less prevalent amongst these patients. acute fever following transfusion is often related to the presence of irregular blood group antigens or to cytotoxic antibodies acquired during previous transfusions or a pregnancy [28] . of note, relative bradycardia in patients who did not receive antiarrhythmic medication suggests either a viral or noninfectious origin of the fever. a possible relation between fever and frequently used drugs such as allopurinol, antibiotics, bleomycin, and cytarabine or with the underlying disease process itself should always be kept in mind [29, 30] . a dysfunctional immune system is presumed to be responsible for the high rate of drug allergy in patients with active acute leukemia; the allergy may abate when complete remission is achieved [29] . this phenomenon is well known in patients with infectious mononucleosis or acquired immunodeficiency syndrome. until recently, coagulase-negative staphylococcal bacteremia was thought to be entirely related to the use of central venous catheters but recent work points at mucosal sites as important portals of entry [31] [32] [33] . the clinical spectrum of catheter-related infections ranges from asymptomatic bacteremia as a manifestation of intraluminal colonization or a process confined to the site of insertion to marked inflammation of the tunnel tract and septicemia with metastatic emboli in the skin and other organs. suspicion of a tunnel or exit line infection should arise when the catheter tract becomes painful, red, or swollen or when signs of inflammation are visible at the exit site. malfunction of the catheter, illustrated by problems drawing blood through the line, is a common first warning of a possible lumen infection [16, 17] . in the selection of the initial antibiotic regimen, one should consider the type, frequency of occurrence, and antibiotic susceptibility of bacterial isolates recovered from other patients at the same hospital. in addition, the use of certain antibiotics may be limited by special circumstances, such as drug allergy, liver function disturbances, or renal insufficiency. despite numerous clinical studies, since the 1970s, no single empirical antibiotic regimen has been shown to be superior for initial treatment of patients who become febrile during a neutropenic episode after therapy with chemotherapy drugs for hematological malignancies (see table 5 -2) [4, 9, [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] . however, there is world-wide consensus that any initial antibiotic regimen should include drugs with reliable activity against escherichia coli, pseudomonas aeruginosa, klebsiella species, other enterobacteriaceae, and staphylococcus aureus [22] . three basic intravenous antibiotic regimens have evolved: initial therapy with a single b-lactam, the so-called monotherapy; a combination of two drugs, a b-lactam with an aminoglycoside, a second b-lactam or a quinolone but without a glycopeptide; and, thirdly, a glycopeptide in addition to b-lactam monotherapy or combination. numerous extensive studies have shown that traditional combinations, consisting of an antipseudomonal b-lactam and an aminoglycoside, are not more effective than monotherapy in the empiric treatment of uncomplicated episodes of fever in neutropenic patients. a third or fourth generation cephalosporin, a carbapenem, as well as piperacillin-tazobactam, have been found to be effective single agents in the majority of cases [43, 45] . it appears appropriate to reserve two-drug regimens for complicated cases or if antimicrobial resistance is a potential problem. the major disadvantages of an aminoglycoside are nephrotoxicity and ototoxicity, and the necessity to monitor serum levels [46] [47] [48] . combination of drugs such as amphotericin b, cyclosporine, and cisplatinum with an aminoglycoside is best avoided because of their additive renal toxicity, whereas high sodium content may limit the simultaneous use of two b-lactam antibiotics in elderly patients. an extensive study by european organization for research and treatment of cancer -national cancer institute of canada [11] showed unambiguously that vancomycin can be withheld and not administered empirically for persistent fever despite appropriate initial monotherapy or combination antibiotic treatment until the results of the cultures indicate the need for vancomycin. vancomycin must be included in an initial empiric regimen for patients known to be colonized with penicillin-and cephalosporin-resistant pneumococci, viridans streptococci, or methicillin-resistant staphylococcus aureus or in situations where b-lactam resistance is likely such as a catheter-associated cellulitis where coagulase-negative staphylococci predominate . the choice to implement a particular antibiotic regimen is, at least partly, based on the results of clinical trials as reported in the literature. yet, the results of such trials should be interpreted with great caution. definitions for response as well as inclusion and exclusion criteria for clinical study protocols are usually very rigid and quite different from common clinical practice [23, 24] . conduct of clinical trials in febrile neutropenic patients was a booming business in the mid-seventies and eighties when many new broad-spectrum antibiotics became available. the data derived from these trials expanded our knowledge of the possible infectious complications tremendously. for instance, analyses of these studies revealed that only half of the patients who develop fever during neutropenia will have a clinically or microbiologically documented infection, the majority being pulmonary infiltrates and bacteremias (see table 5 -3) [49, 50] . furthermore, it was obvious that neutropenic patients without a documented infection generally defervesced within a few days, whereas those with a clinically or microbiologically documented infection showed a much slower and less frequent fever defervescence rate [19, 27, 51] . this very consistent observation suggests that it might be prudent to select different antibiotic regimens for patients with different symptoms. although there is no statistically valid evidence to support a more individually tailored approach, it appears reasonable to assume that patients might benefit from timely administration of the antibiotics with the highest intrinsic potency against a given micro-organism. a known or suspected focus of infection, if present at the time of initial fever, could help in the selection of additional case-specific anti-infective agents because the location of an infection is, at least to a certain extent, predictive of specific infective pathogens (see table 5 -4) [52] . likewise, results of surveillance cultures and knowledge of clinically documented infection fever accompanied by a clinical infection, but pathogens cannot be identified, e.g., cellulitis, pneumonia microbiologically documented infection fever accompanied by a localized infection and microbiologically plausible evidence, or fever without a localized infection, but infectious agents can be demonstrated in a (blood) culture the common complications associated with particular antileukemic regimens may offer valuable input to individualizing appropriate initial treatment of a neutropenic patient with fever. a damaged integument probably plays a major etiologic role in virtually all infections that occur following aggressive cytoreductive therapy for a hematological malignancy but its involvement is most obvious in infections of the skin and gastrointestinal tract. the use of high-dose cytarabine in conjunction with the occurrence of diarrhea were found to be independent risk factors for streptococcal infections among 513 patients evaluated during first episodes of neutropenic fever [53] . it has been recognized that bacteremias due to oral streptococcus mitis and streptococcus oralis may result in serious complications such as sepsis or adult respiratory distress syndrome, which carry high mortality [54] [55] [56] . similarly, bacteremias due to staphylococcus aureus, pseudomonas aeruginosa, and clostridium species as well as candidemias are more frequently encountered in patients with acute leukemia who suffer from neutropenic enterocolitis or typhlitis, the most serious disturbance of the delicate balance between mucosal damage and microbial flora in the setting of prolonged exposure to antibiotics after intermediate or high-dose cytarabine chemotherapy. the signs and symptoms of chemotherapy-induced enterocolitis or typhlitis vary considerably from patient to patient and include nausea, vomiting, abdominal cramps, and severe abdominal pain with virtually no formed bowel movements but accompanied by profuse, watery diarrhea. many patients are in such pain that they only gain relief from narcotic analgesics which, in turn, induce constipation by reduction of bowel movements. this may create a very alarming situation as the clinical picture in severe cases resembles that of gut perforation, acute pancreatitis, or even toxic megacolon. because there is a high mortality rate for surgical interventions in neutropenic and thrombocytopenic patients with acute leukemia, it is essential for physicians to be aware of the existence of neutropenic enterocolitis/typhlitis with the accompanying symptoms. ultrasonography or ct, showing pathological thickening of the bowel walls, may be useful to establish the diagnosis of typhlitis. patients treated for acute myeloid leukemia with a bowel wall thickness of more than 10 mm had a significantly higher mortality rate than did those with a bowel thickness of less than 10 mm [57] . disproportional bacterial overgrowth in the gastrointestinal tracts of neutropenic patients with damaged mucosa can serve as a source of bacteremia for the endogenous gastrointestinal flora as well as for otherwise exclusively enteric pathogens such as clostridium septicum [58, 59] and bacteroides fragilis. in contrast, salmonella species are rarely found in the stool or blood of granulocytopenic patients; these organisms are obviously not major players in this field. this is also true for pathogens like campylobacter and shigella species. therefore, an adequate antibiotic regimen for patients with abdominal symptoms should cover gramnegative rods but due consideration should be given to the use of compounds with activity against anaerobes. next to glycopeptides and carbapenems, metronidazole is an attractive adjunct to a standard monotherapy/combination regimen under these circumstances. pseudomembranous colitis caused by clostridium difficile [60] [61] [62] [63] [64] constitutes a related but distinct entity that can be severe and even fatal. the stool should be tested immediately for clostridium difficile toxin if the diagnosis is suspected. enteric clostridia infections necessitate oral antibiotic therapy with either vancomycin or metronidazole. relapses are frequent and may follow cancer chemotherapy or courses with antibiotics such as clindamycin. relapse is harder to document because toxin may persist in the stool of successfully treated patients. diagnostic problems account for underestimating enteric viruses as causative agents in gastrointestinal infections. although a compromised cell-mediated immunity is known to predispose for parasitic and protozoan infections, their incidence is surprisingly low in patients who are treated for a hematological malignancy [65, 66] . folliculitis and cellulitis are the most common manifestations of infectious processes in the skin. sometimes it is difficult to differentiate infectious lesions from drug-induced toxic skin eruptions. infection-associated erythema and swelling are usually mild but, if left untreated, infiltration and abscess formation will involve extensive areas of the skin with necrosis and gangrene. since the lesions associated with the various organisms are rather alike, a simple needle aspiration or biopsy should be performed to establish an accurate diagnosis as early as possible in the course of the disease. causative micro-organisms include streptococci, staphylococci, and, less commonly, gram-negative bacilli and fungi [67] [68] [69] [70] . localized infections of the skin, particularly in the face, are usually caused by gram-positive bacteria that arise more frequently in carriers of organisms like staphylococcus aureus. none of the standard empiric regimens is the optimal choice for treating skin infections caused by the prevalent but usually indolent nons. aureus gram-positive cocci that are often methicillin-resistant, but the morbidity from these infections should not be underestimated either. pseudomonas aeruginosa acquired in a hot jacuzzi may cause a folliculitis that occasionally progresses to a destructive ecthyma gangrenosum [71] . this characteristic entity should be distinguished from similar lesions caused by other rare pathogens, such as actinomyces, stenotrophomonas maltophilia [68] and fungi [69, 70] as well as from pyoderma gangrenosum, a noninfectious cutaneous process in patients with a myeloid malignancy [72, 73] . sweet's syndrome, a dense, tender infiltration by neutrophils of the dermis on the head, neck, and upper extremities is associated with a leukocytosis [74] . varicella zoster is the leading dermatologic complication in patients with impaired cell-mediated immunity [75] [76] [77] . if skin or mucous membrane lesions due to herpes simplex or varicella-zoster viruses are present, even if they are not the cause of fever, treatment with valacyclovir or another suitable antiviral is indicated with the intention to speed healing of lesions that could become potential portals of entry for bacteria and fungi. the results of several prospective studies do not indicate a general need for a glycopeptide as part of the front-line therapeutic regimen unless one has a particular reason to suspect the presence of methicillin-resistant staphylococcus aureus or penicillin-resistant viridans streptococci on the basis of local patterns of resistance or surveillance cultures. nevertheless, most physicians intuitively prefer an up-front glycopeptide-containing regimen to cover catheter-related infections as these are frequently due to coagulase-negative staphylococci, although early glycopeptide treatment does not contribute to improved survival from these usually indolent infections. hence, when coagulase-negative staphylococci are involved, a few days of watchful waiting for a possible clinical response and the results of the cultures will have no detrimental impact. most catheter-associated infections will respond to antibiotic therapy without the removal of the catheter. rotation of antibiotics through each lumen of multilumen catheters to avoid microbial sequestration in one of the lines and the use of antibiotic-containing heparin lock solutions to supplement systemic therapy have been proposed by some investigators but such practices remain controversial. pulling the catheter is most likely to be required for the cure if a concurrent venous thrombosis is found, the tunnel tract appears involved, or if the infection, regardless of the etiology, is recurrent, or if after several days of therapy an eventual response to antibiotics appears doubtful [78] . gingivostomatitis and periodontal lesions occur frequently in patients with acute leukemia [79] . oral mucositis is characterized by pain, edema, erythema, superficial lesions, pseudomembranous formation in conjunction with excessive mucous production, reduced saliva secretion, and bleeding. a wide array of pathogens can be found and include herpes simplex, gram-negative bacilli, streptococci, anaerobes, and candida species [80] . with the introduction of aggressive chemotherapeutic regimens, hitherto unusual pathogens such as stomatococcus and aerococcus are increasingly seen in patients with mucositis. mixed and polymicrobial infections are more or less standard [80, 81] . given the range of prevalent pathogens, there is little need to deviate from one of the standard regimens, although, on theoretical grounds one might prefer to select a carbapenem, fourth generation cephalosporin, or extendedspectrum penicillin given their superior intrinsic activity against viridans streptococci and pneumococci. the course of herpes simplex stomatitis is usually prolonged in patients treated for leukemia or lymphoma, and relapses are common [82] . herpes simplex lesions are most commonly white painful plaques with or without serpiginous borders on the gums, tongue, buccal mucosa, or oropharynx and may be difficult to discriminate from oropharyngeal candidiasis and, indeed, co-infections do occur. swallowing can be so painful that saliva is expectorated and intake of food and fluids drastically reduced. it is not uncommon for oropharyngeal herpes simplex and candida infections to extend to the esophagus. although neither herpes nor candidiasis belong to the category of diseases that requires an empiric approach, it is generally accepted that early treatment with valacyclovir and fluconazole, respectively, is important to prevent extension into the esophagus and further dissemination, particularly among bone marrow transplant recipients. when the paranasal sinuses are involved in the infectious process, moulds have to be considered as possible causes. direct inspection of the nasal turbinates and a computer-assisted tomographic scan of the sinuses can be helpful to establish or reject the diagnosis. management of pulmonary infiltrates that are responsible for 70% of all fatal infections in febrile neutropenic patients is complex [83] [84] [85] . the importance of classic clinical complaints of cough, pain, and dyspnea should not be neglected but bronchoscopy and radiological examination of the chest by a computerassisted tomographic scan represent the cornerstones of all diagnostic procedures. typically, chest radiographs performed early in the evolution of infection in patients with profound granulocytopenia fail to show infiltrates. it may take more than 3 days for the infection to generate enough necrosis with hemorrhage and edema to produce a visible infiltrate. the critical decision faced by the clinician at the bedside of patients with pulmonary infiltrates is whether to undertake invasive procedures such as bronchoscopy with or without bronchoalveolar lavage, transbronchial biopsy, transthoracic aspiration, thoracoscopy-guided biopsy, or open lung biopsy. the exact role of these diagnostic procedures in the optimal management of patients is still controversial because the yield depends on the collaboration and skills of various specialists. moreover, concurrent thrombocytopenia precludes simple invasive diagnostic procedures such as transbronchial biopsies in many patients. the radiologic pattern of a possible infiltrate is often suggestive of its cause. a diffuse opacity, usually of both lungs, is seldom of bacterial or fungal origin. although viruses and pneumocystis jeroveci typically cause diffuse, bilateral pulmonary infiltrations, it should be kept in mind that a similar picture of pneumonitis can be seen secondary to radiation, fluid overload, cytotoxic drugs such as methotrexate, cytarabine and bleomycin, and in pulmonary hemorrhage. pneumocystis jeroveci pneumonia is manifested in patients with deficient cellular immunity as fever, progressive hypoxemia with dry cough, and dyspnea, typically beginning after discontinuation of corticosteroid therapy given for other reasons [85] . high-dose trimethoprim-sulfamethoxazole with adjuvant corticosteroids for hypoxemic patients (po 2 < 70 mmhg) has become the preferred therapy for these infections [86] . alternatives include intravenous pentamidine, oral dapsone in combination with trimethoprim, or oral atovaquone suspension alone. antiviral drugs are indicated only if there is clinical or laboratory evidence of viral disease. with the exception of a cytomegalovirus-related pneumonitis in allogeneic bone marrow transplant recipients with graft-versus-host disease, there appears to be no need for empiric coverage of respiratory viruses, such as respiratory syncytial virus, influenza [87, 88] , and adenoviruses. ganciclovir, valganciclovir, and foscarnet have established activity in the treatment of cytomegalovirus infection and their timely use might be lifesaving. mycoplasma pneumoniae with or without cold agglutinins is remarkably infrequent in patients treated for leukemia. in more acutely ill patients, the possibility of acute lung injury following transfusion of a cellular blood product or respiratory distress syndrome related to streptococcal sepsis should be considered. patients with an infection by streptococcus mitis, which has been linked with severe mucositis and high-dose cytarabine are at particular risk [53, 54] . the incidence of acute respiratory distress syndrome in such cases is more than 20% and mortality is substantial. the pathophysiology of adult respiratory distress syndrome following streptococcal bacteremia in a neutropenic patient is poorly understood. probably several factors are involved, such as deleterious effects of sepsis superimposed on preexisting tissue damage. even patients who had received appropriate antimicrobials at the onset of fever were reported to experience shock and death [54] [55] [56] . therefore, in addition to antibiotics, corticosteroids should be considered in the management of patients affected by ards and streptococcal bacteremia. bacterial infections of the lung, accompanied by bacteremia in about 50% of cases, usually create infiltrates on a computer-assisted tomographic scan that are confined to one or more lobes. pneumonias caused by pseudomonas aeruginosa and staphylococcus aureus do have a bad reputation but enterobacteriaceae [89, 90] , haemophilus influenzae and streptococcus species are hardly less dangerous. given the uniformly poor outcomes of pulmonary infections in clinical trials, the empiric use of a combination of antibiotics is recommended with the addition of vancomycin in centers that face resistance of s. pneumoniae to penicillin and macrolides. outbreaks of legionella pneumophila, an infection characterized by patchy interstitial or nodular pulmonary infiltrates and sometimes accompanied by headache or gastrointestinal symptoms, have been observed among compromised patients in units with contaminated water systems [91] . therefore, if a case of legionellosis is encountered, other patients with similar symptoms on the same ward should be treated with a macrolide or a fluoroquinolone from the start of antimicrobial therapy. a nodular pattern of pulmonary infiltrates should lead the physician to consider the possibility of atypical pneumonia or, more commonly, a pulmonary fungal infection. in the latter case, diagnostic procedures rather than immediate institution of antifungal drugs should be given priority. especially in patients with concomitant impairment of the cell-mediated immunity, pulmonary aspergillosis has to be distinguished from tuberculosis. infections with mycobacterium tuberculosis in patients with impaired cell-mediated immunity are manifested as either localized pulmonary disease or devastating miliary tuberculosis. nontuberculous mycobacteria are still rather rare in patients with acute leukemia, but the introduction of purine analogues such as cladribine and fludarabine, which cause severe and prolonged depression of cellular immunity, may change this picture in the near future [92] . urinary tract infections are astonishingly uncommon in patients who are treated for leukemia or lymphoma and, since gram-negative bacteria are the predominant urinary tract pathogens, the choice for a single broad-spectrum b-lactam is fully justified. malignant otitis externa is a very serious infectious complication that can emerge after administration of aggressive chemotherapy for a hematological malignancy. at the outset, the patient will complain of a painful, discharging ear, and physical examination will reveal a reddened edematous ear canal. local maceration and humid conditions favor the growth of pseudomonas aeruginosa which, indeed, can be isolated frequently from swabs taken from superficial lesions of the external canal. untreated, the infection will penetrate into underlying soft tissues, threatening the retromandibular and parotid area. likewise, spread to the middle ear, the mastoid air cells, and adjacent temporal bone is possible. once osteomyelitis becomes established, extension to the base of the skull with invasion of the cranial nerves and local thrombosis poses a direct danger to the patient's life. a computer-assisted tomographic scan may be helpful to identify tissue damage in the early phase. prolonged antibiotic therapy with ceftazidime, ciprofloxacin, or other antipseudomonal antibiotics in combination with surgical debridement constitutes the treatment of choice [93] . occasionally, a similar clinical picture can be the result of an infection by staphylococcus aureus or aspergillus fumigatus. in such cases, surgery should be combined with an antistaphylococcal penicillin or vancomycin, or with voriconazole, respectively. an insidious onset of fever accompanied by headache and confusion might be indicative of meningitis when causation by leukemia or lymphoma has been excluded by cytologic examination of the cerebrospinal fluid. in cases of infection, the cerebrospinal fluid is usually clear with moderate protein elevation. the prevalent pathogens are listeria monocytogenes, cryptococcus neoformans, and toxoplasma gondii [65] . recovery of one of listeria monocytogenes [94] and cryptococcus neoformans from blood cultures should, provided that no intracranial hypertension is detected, always prompt a lumbar puncture even in the absence of neurological symptoms. considering their low incidence and the relatively reliable diagnostic possibilities, there is no need to cover for these infections with a specific empiric regimen. outpatient management of infections in patients with hematological malignancies is discussed in more depth in chap. 6. when potent oral broad-spectrum antibiotics became available in the late eighties, many clinicians felt tempted to use these drugs in the treatment of febrile neutropenic patients. several groups around the world assessed the options and limitations of this seemingly revolutionary approach [95] [96] [97] systematically. these analyses showed that it is possible to define risk factors that can be used to classify patients into low or high-risk categories. in fact, these studies offered nothing more than identification of objective parameters that corroborate the gut's feeling of the experienced clinician. since the time of bodey [1] , it was already obvious that patients with absolute neutrophil count between 0,1 and 0.5 × 10 9 /l (100-500/ml) carry a minor risk compared to those with a granulocyte count of less than 0.1 × 10 9 /l (100/ml). but now other risk factors have been identified. patients with concurrent mucosal damage or impaired cellular immunity, as well as those with clinically documented infections or unstable vital signs, are at high risk and deserve increased vigilance. patients with these additional risks cannot be considered candidates for antibiotic treatment on an out-patient basis. the vast majority of patients with acute leukemia are considered high-risk patients and should continue to receive intravenous broad-spectrum antibiotics in the hospital or similar setting. the remaining low-risk patients, namely those with unexplained fever who are clinically stable, may be safely treated with oral antibiotics provided that they have been seen at a qualified medical center promptly after the onset of fever [95, 96] . the possible use of antibiotic prophylaxis does not pre-empt the need for a thorough check-up but limits the choice of drugs that can be used for treatment. patients with increasing granulocyte counts are considered to be better candidates for outpatient therapy than are patients without an indication of bone marrow recovery. among the oral regimens that have been evaluated are ofloxacin, ciprofloxacin, and ciprofloxacin plus amoxicillin-clavulanate. it is crucial to make sure that the patient is informed about the risk of unremitting fever during a neutropenic episode and that he or she fully understands the importance of seeking immediate medical advice in case any unexpected incident occurs. vigilant observation at home by a relative or professional health care worker and prompt access to appropriate medical care must be available 24 h per day, 7 days a week [98] [99] [100] [101] . as an alternative to initial outpatient therapy, early discharge with continued outpatient therapy for selected patients may be considered after a brief admission during which intravenous therapy is initiated, fulminant infection is excluded, and appropriate culture specimens are taken [102, 103] . two studies have demonstrated that children who lack signs of sepsis and severe mucositis, who are afebrile for >48 h, who have neutrophil counts of >100 cells/mm 3 (>0.5 × 10 9 /l), and who are at low risk for complications may have their intravenous antibiotic treatment safely stopped to be substituted by oral cefixime [104, 105] . after starting empiric antibacterial treatment, fevers will persist or return in about one third of patients. the average duration of fever in serious infections, in eventually successfully treated neutropenic patients is 4-5 days (table 5 -2, fig. 5-1) [4, 9, 13, 14, [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] . although fever can be inconvenient for the patient, it is important to realize that it is part of the body's defense system [106] . indeed, some retrospective studies have suggested that fever is associated with improved survival and shortened disease. uncontrolled studies have reported an association of increased mortality with the absence of fever in polymicrobial or gram-negative sepsis and in elderly patients with community acquired pneumonia [107, 108] . so, when the body temperature remains above normal during 3 or 4 days on apparently effective broad-spectrum antibiotics, this should not be considered a complete waste of time, particularly not if the time is used for an appropriate diagnostic work-up. it should be kept in mind that empiric administration of antibiotics is only meant as an immediate cover for rapidly fatal bacteria such as gram-negative rods and staphylococcus aureus, thereby, so to say, buying time for consideration of the next therapeutic interventions and for waiting for the results of the diagnostic procedures. when the results of the cultures become available and the infection has had time to blossom clinically, there is a more solid basis for decisions on necessary adjustments of an antibiotic regimen. unfortunately, all large, randomized clinical trials on empiric antibiotic therapy in the febrile neutropenic patients during the past 30 years have been pharmaceutical company-driven for purposes of attaining governmental agency approval [23, 24] . as a consequence, the design of these studies focused primarily on the efficacy of a particular drug in comparison with another drug or combination of drugs. according to the protocols for these trials, only patients who survived the febrile episode without a change in the allocated regimen could be labeled as successes, whereas any change in therapy, independent of the trigger, was denoted a failure, even if the patient survived unscathed and the infection was eradicated. therefore, modification of the test regimens was discouraged, which constitutes a rather artificial situation, as clinicians are inclined to adjust an antibiotic regimen for no other reason than a subjective feeling of unease with the original choice of antibiotics. changes often reflect impatience, nervousness, and lack of confidence on the part of the clinician concerned over the still febrile neutropenic patient rather than any deficiency in the original antibiotic regimen used. when restrictions surrounding a clinical trial do not apply, juggling antibiotics against an undulating line on a temperature chart is a well-known frequent occurrence on a ward full of patients suffering with hematological malignancies. indeed, in daily practice, many modifications are not based on objective criteria and are made outside office hours, i.e., by less experienced physicians on call [103, 109] . however, it is generally recognized that exposure to many different antibiotics as a result of haphazard changes of regimens enhances the risk of drug-related adverse events and seldom improves the outcome of the patient under treatment. moreover, such a policy of endless therapeutic trials of antibiotic changes might wrongly decrease the perceived need for further diagnostic procedures in poorly responding patients. since there is evidence from clinical trials on what to do after the empiric phase, some experts have been promoting the so-called algorithms of planned progressive antibiotic therapy to treat neutropenic patients with fever. a planned progressive strategy involves adjustment of therapy every 2-3 days, until the patient becomes febrile or until all the potential causes of infection are covered by the best available microbial agents, irrespective of the development of additional symptoms. it is clear that algorithms featuring planned progressive therapy are destined to lead to overtreatment with unnecessary expenses and drug exposures [108] . it appears more intellectually attractive not to rely on fixed algorithms but to weigh several different, patient-specific parameters, including fever and clinical response, as a guide for modification of an empiric regimen. it goes without saying that spending time at the bedside is crucial for those who feel attracted to the role of attending physician because careful observation often provides early clinical clues for a rational adaptation of the original empirical antibiotic regimen. the need for individualization is not only dictated by variations in the signs and symptoms of the patient that accompany persisting fever but also by differences in skills and expertise amongst attending specialists in various centers. for example, centers with excellent and interested departments of medical microbiology and pathology will rely more heavily on their findings than do centers with poorly functioning departments, whereas units with an active radiology service may benefit from the locally available know-how in this particular field. once an empiric antibiotic therapy has been started, the patient must be monitored continuously for nonresponse, emergence of secondary infections, adverse effects, and the development of drug-resistant organisms. this implies that the start of antibacterial agents cannot be seen as an impetus to stop diagnostic procedures. daily blood cultures are certainly justified as long as patients remain febrile and when a new temperature peak occurs because breakthrough bacteremia or fungemia may develop. close monitoring of sites that are prone to infection should start before the onset of fever and has to be continued after empirical antibacterial therapy has commenced. subtle changes must bring diagnostic tools into play to confirm or exclude the presence of an infectious focus. regular ct-scans of the chest, preferably in combination with serological monitoring for aspergillosis antigen, have an established value in patients who are at increased risk of fungal infections [92] . as a rule, approximately 65% of patients without a focus of infection, which includes 30% overall with positive blood cultures, will show some clinical improvement after 3 days of broad-spectrum empiric coverage in spite of persisting fever. in most cases, defervescence will follow rapidly. elements that should be incorporated in clinical decision making include the course of fever and clinical condition with special attention to the vital signs, evolving symptoms of infection in relation to the granulocyte count, c-reactive protein levels, antigen monitoring, and risk for relapses of latent viral infections determined by pretreatment antiviral titers. the results of all cultures taken at the onset of fever have to be assessed and it is recommended to analyze surveillance cultures, if any, to identify possibly colonizing resistant organisms. without clinical deterioration or proof of an infection caused by a micro-organism resistant to the initial antibiotic regimen, persisting fever after 72-96 h of empiric therapy in and of itself is an unsatisfactory basis for changing the original empirical antibacterial regimen. it is better to alter the regimen only when there are objective reasons to do so: deterioration of vital signs, isolation of a resistant pathogen without clinical improvement, persistence of a pathogen, antibiotic-related adverse events, occurrence of a new focus of infection or progression of an existing focus in the absence of granulocyte recovery, unexplained fever persisting for more than 5 days, new fever, a new pathogen or recognition of a local outbreak with a resistant organism (tables 5-5 and 5-6). in most patients, antimicrobial therapy can be adjusted objectively on the basis of clinical or microbiologic findings but such an individually tailored approach requires careful daily assessment of all possible parameters collaborating with consulting specialists, including microbiologists, pulmonologists, and radiologists. in contrast to the moment of the onset of fever, there is ample time for deliberation and contemplation in a situation where the patient's fever persists for 3 or more days while on antibiotics because the origin of fever is obviously not a rapidly fatal microorganism that needs immediate treatment. fever that persists for more than 3 days suggests that the patient has a nonbacterial infection, a resistant bacterial infection, a second infection, or a drug fever [22, 110] . despite extensive cultures, only around 30% of all febrile patients will be shown to have microbiologically defined infections. in 30% of patients, organ involvement is already apparent with the initial fever and an additional 10% will show clinically defined infection within the next 72 h (see fig. 5 table 5-7) . patients belonging to each of these three categories may have either a microbiologically documented infection, a clinically documented infection, or an explained fever. all these factors that are partly subjective and partly objective can be exploited to steer the modification of an empiric regimen when there is a perceived need to do so. ultimately, only 15-20% of patients with a persisting unexplained fever should require a continued empirical rather than a clinical or microbiologically directed approach after 72 h of broad-spectrum antibacterial therapy. whichever modification is planned, it cannot be overemphasized that maintenance of appropriate antigram-negative cover is mandatory as long as a patient is febrile and neutropenic. when the patient is improving or stable, there appears to be no imminent need to adjust an antibiotic regimen. depending on the micro-organism isolated, a change to an oral regimen could be considered with caution. when a gramnegative isolate is identified, broad-spectrum antibiotic coverage should be maintained in full dose. whereas the clinical relevance of a blood culture positive for gram-negative bacilli is never a matter of controversy, the implication of recovery of particular gram-positive cocci is less clear. single blood cultures positive for s. aureus, s. pneumoniae, or enterococcus faecalis in neutropenic patients should be regarded as significant and indicative of the need for further treatment. viridans group streptococci, with an average mortality of 15-20%, are perhaps the most feared among the bacteremias today [54] [55] [56] . although viridans streptococci are common blood contaminants in the general population, positive blood cultures in patients with oral mucositis should not be disregarded, certainly not when s. mitis or related streptococci are isolated [53, 54] . isolation of rare micro-organisms should prompt evaluation of the appropriateness of the starting antibiotic regimen, especially when the patient is not responding optimally. on the other hand, isolation of in vitro resistant organisms such as coagulase-negative staphylococci and, more rarely, stenotrophomonas maltophilia, from the blood of a clinically, evidently improving patient, pose an interesting challenge. many would be inclined to modify the initial regimen but in many cases other bacteria that were not recovered on the culture plate may have been the culprits in the current fever. a blood culture that yields candida species or another fungus should be taken very seriously and dictates immediate institution of antifungal therapy [111] [112] [113] . the availability of the candins has extended the therapeutic options [114] [115] [116] . adaptations of an antibiotic regimen in a patient who is clearly not responding is relatively straightforward when a micro-organism has been isolated; the results of the cultures, supplemented by susceptibility testing, will assist in selecting the proper antibiotics. all clinical trials so far have demonstrated consistently that patients diagnosed with a clinically documented infection respond much slower and remain febrile for a longer time than those without a focus of infection [19, 27, 51] . moreover, due to problematic penetration into avascular sites, infections associated with abscesses or prosthetic devices usually respond poorly to antimicrobial therapy. attending physicians should, therefore, be more hesitant to change antibiotics in patients who are not deteriorating. on the other hand, there are indications that early addition of specific agents might be useful for more rapid control of clinically documented infections. for instance, considering the probable involvement of anaerobes, switching to a carbapenem, if not given initially, or addition of metronidazole to a standard anti-gram-negative regimen, appears a logical choice when fever is accompanied by abdominal symptoms. in cases with a clinically documented site who do not improve or stabilize, coverage of micro-organisms known to prevail at the involved site of infection (see table 5 -4) appears appropriate. clinically documented infections that emerge later during the course of febrile neutropenia carry a dismal prognosis and are presumed to be related to the occurrence of resistant microorganisms, including invasive fungi, in combination with persisting immunodeficiency often as a result of a refractory underlying disease. if the patient with an unexplained fever clinically improves or remains stable after 72 h of empirical treatment and re-evaluation by physical examination and diagnostic tests yields no new information, and no isolate was found, the initial antibiotic regimen can be continued or can be switched to an oral compound. the latter option is more reasonable clinically if neutropenia is expected to resolve within the ensuing days. if vancomycin is a component of the initial antimicrobial regimen, withdrawal of the drug should be considered if the results of the cultures do not support its use. deteriorating cases without any microbiological or clinical sign of infection pose a dilemma. unexplained fever accompanied by deterioration can imply that the patient has a nonbacterial infection or a noninfectious cause of fever, but foremost, a resistant bacterial infection or the emergence of a second infection should be taken into account [19, 110] . an initial response rate of about 35% may be expected in patients with shock, compared with 70% in patients without shock, which suggests the possible presence of an undetected toxin-producing pathogen in the former. addition to the original empirical antibacterial regimen is mandatory in critically ill patients, independent of the level of fever. escalation might include filling theoretical gaps in antibiotic spectrum and enhanced monitoring for any changes in the patient's condition. under these circumstances, the selection of agents should be guided by knowledge of locally prevalent virulent pathogens and actual susceptibility patterns, which implies the necessity of close cooperation with the local microbiology laboratory. addition of vancomycin appears reasonable in view of the fact that the spectrum of antibacterial drugs in traditional empiric regimens usually does not cover coagulase-negative staphylococci, methicillin-resistant staphylococcus aureus, enterococci, and some strains of penicillin-resistant s. pneumoniae and viridans streptococci. on the other hand, liberal use of vancomycin has confronted the medical community with vancomycin-resistant enterococci and staphylococci, which has led to increasing use of new agents like quinupristindalfopristin and linezolid in the treatment of febrile neutropenic patients. when the starting regimen consists of a single, broad-spectrum b-lactam, addition of an aminoglycoside is an attractive option to provide a better coverage when infections by resistant gram-negative rods are suspected. however, it has to be emphasized that development of resistance during therapy is extremely rare and that aggressive gram-negative organisms typically cause the infection to deteriorate rapidly to a stage beyond cure within a few days after first fever in most cases. hence, if the local resistance pattern or a particular concern in an individual patient prompts the use an aminoglycoside for resistant gram-negative bacteria, then aminoglycosides should be prescribed from the start in optimal doses with monitoring of the peak and trough serum levels. clinical deterioration in a persistently neutropenic patient with unexplained fever is an important but rather rare event in daily practice and applies to only a quarter of the overall 10% of cases that deteriorate while on broadspectrum antibacterial treatment. moreover, it is noteworthy that the success rate of empiric modifications is less than 20%, whereas more than 50% of cases will respond to specifically customized modifications [41] . invasive fungal infections are encountered in up to 40% of autopsies in patients with hematological malignancies. fungi have been found to be responsible for two thirds of all superinfections, which surface during broad-spectrum antibiotic treatment of neutropenic patients. more than 20 years ago, when diagnostic capabilities were virtually nonexistent and the choice of effective antifungal agents limited, two prospective, randomized trials laid the scientific foundation for the addition of systemically active antifungals even though neither study was adequately powered to reach a statistically valid conclusion [111, 112] . this strategy appeared to reduce the incidence of invasive fungal infections in patients without any further sign of a clinically documented infection. solid statistical evidence to support the validity of this empiric approach was never obtained subsequently in further placebo-controlled trials because empirical antifungal treatment had become widely accepted as the standard of care. this so-called empiric antifungal therapy has remained popular as it seemed to make life easy for clinicians. the lack of reliable diagnostic tools combined with very poor outcomes of invasive fungal infections that were not timely treated contributed greatly to this popularity [117] [118] [119] . however, in most cases in 2007, antifungals prescribed empirically for fever alone are unnecessary because invasive fungal infection is present in a minority of cases. a better understanding of the pathophysiology of invasive fungal disease in combination with use of better diagnostics allows for a more individualized approach [117, 120] . an optimal diagnostic work-up in conjunction with careful clinical observation will likely render routine empiric antifungal therapy superfluous in most cases because appropriate application of presently available diagnostic tools enables timely pre-emptive institution of appropriate antifungal therapy by experienced clinicians [121] [122] [123] . the most common initial presentation of invasive aspergillosis is unremitting fever despite broadspectrum antibacterial treatment, accompanied eventually in most patients by pulmonary infiltrates or sinusitis. clinicians should suspect the diagnosis in a patient with pleuritic pain, hemoptysis, or a localized pleural rub. the halo sign (a dense central nodule with surrounding less dense infiltrate) on a computer-assisted tomographic scan of the chest, though not pathognomonic, is highly suggestive of an early phase of pulmonary aspergillosis or other mould pneumonia in immunosuppressed patients [124] [125] [126] . even when gramnegative pathogens, including pseudomonas aeruginosa and enterobacter cloacae, are isolated from the sputum or blood of such patients, aspergillosis should be the leading consideration when nodular chest ct findings are present. if no infiltrate is found in a high-risk patient with persisting fever, the investigation should be repeated within a few days, preferably supported by bronchoalveolar lavage if indicated and additional assays such as screening for the presence of galactomannan in the blood [121] . even in patients with aspergillosis who are responding adequately to antifungals, the computerassisted tomographic chest scan will usually show some enhancement of the lesion when the neutrophils return with eventual development of cavitation within the infiltrate, the so-called air-crescent sign [124] [125] [126] . this finding is suggestive of aspergillosis, although mucormycosis and other moulds may cause an identical picture. whether the increased incidence of non aspergillus mould is due to more extensive use of the new azoles like voriconazole or to the use of more intensive immunosuppressive treatment schemes remains to be seen [127, 128] . isolation of an aspergillus species from sputum or bronchoalveolar lavage specimens connotes either invasive infection or bronchial colonization, the latter conferring high risk for invasive aspergillosis. when voriconazole or posaconazole have been used as prophylaxis, it is sensible to select an antifungal compound with a different mode of action when therapy becomes mandatory [129, 130] . surgery is indicated for patients in whom lesions near the pulmonary hilus pose a direct threat of invasion of a major vessel with the risk of fatal hemorrhage or for debridement of dead tissue after a period of antifungal therapy [126] . low risk patients who test negative for aspergillus in all diagnostic procedures do not need to be started on intravenous antifungals. treatment should be stopped for those patients started on antifungals pending diagnostic test results. a more conservative wait-and-see approach can be implemented successfully once clinicians learn to accept that negative diagnostic results constitute sufficient evidence that there is no fungal infection in many persistently febrile neutropenic patients [121, 123] . fluconazole given as prophylaxis has virtually eliminated infections with candida albicans. however, candida species or other fungi are still occasionally identified as causes of disseminated infections in humans, albeit with a shift from candida albicans to nonalbicans species [131, 132] . a candidemic patient typically presents with an irregular fever sometimes accompanied by polymyalgia and polyathralgia. in about 10% of cases, characteristic pinkishpurple, nontender subcutaneous nodules may arise anywhere on the body. biopsy specimens should be cultured and histologically screened at multiple levels in an attempt to establish a final diagnosis. candida ophthalmitis is seldom seen in leukemic patients since the distinctive retinal exudates are the result of an inflammatory response that involves granulocytes. upon the return of the neutrophils or tapering of corticosteroids, complaints of abdominal discomfort and elevation of alkaline phosphatase levels with or without hepatosplenomegaly may emerge. at this stage, an abdominal ultrasound or computer-assisted tomographic scan will display rather distinctive multiple abscesses in the liver and/or spleen, known as "bull's-eyes" [133, 134] . mortality from an invasive yeast infection may be as high as 40%, particularly when the start of antifungal therapy has been delayed. trichosporonosis and fusariosis can produce a clinical syndrome identical to candidemia [135] [136] [137] . up to now, empirical antimicrobial therapy has been the backbone of improving survival of febrile neutropenia in leukemic patients. hematopoietic growth factors have been studied as adjunctive therapy for febrile neutropenic patients in several randomized, controlled trials. g-csf (filgrastim) and granulocytemacrophage colony-stimulating factor (sargramostim) when used as part of the treatment of febrile neutropenic patients were shown to consistently shorten the duration of neutropenia defined as a neutrophil count below 0.5 × 10 9 /l (500/ml). however, the duration of absolute neutropenia, i.e., count of less than 0.1 × 10 9 /l (100/ml), was not influenced, which might help to explain why neither a decrease in infection-related mortality rates nor a significant effect on morbidity, including duration of fever and use of anti-infectives, were observed [138, 139] . therefore, the use of growth factors should be restricted to complicated cases for which there appears to be no rational alternative therapeutic option [140] [141] [142] . this concept also applies to the use of granulocyte transfusions. transfusion of high numbers of granulocytes harvested after administration of g-csf, with or without dexamethasone, to a donor is done by some clinicians without there being any unequivocal evidence of its efficacy. patients with prolonged profound neutropenia and an uncontrolled clinically documented infection, such as severe cellulitis or sinusitis, appear to be the primary candidates for treatment with granulocyte transfusions, whereas administration of a colony-stimulating factor (g-csf) should be preferred when a return of the neutrophils is imminent. significant toxicities in granulocyte-transfusion recipients include transmission of cytomegalovirus, alloimmunization associated with fever, graft-versus-host reactions if granulocytes are not irradiated, progressive platelet refractoriness, and, possibly, respiratory insufficiency associated with concomitant administration of amphotericin b. new approaches with agents designed to protect the mucosa, like recombinant human interleukin 11 and keratinocyte growth factor palifermin, show promising results in terms of reducing severity of mucositis and occurrence of fever and bacteremia in neutropenic patients [143] [144] [145] . it is widely believed that antibiotic treatment should be continued for a minimum of 7 days or until culture results indicate that the causative organism has been eradicated, infection at all sites has resolved, and the patient is free of major signs and symptoms. ideally, the neutrophil count should be >500 mm 3 (0.5 × 10 9 /l) before treatment is stopped [146] . when no infection has been identified after 3 days of treatment and the patient has become afebrile for 48 h in association with a neutrophil count that has exceeded 500 cells/mm 3 (0.5 × 10 9 /l), antibiotic therapy may be stopped. in addition, if a persistently neutropenic patient has no complaints and displays no clinical, radiological, or laboratory evidence of infection, cessation of antibiotic therapy or a change to oral antimicrobials should be considered after 4 days without symptoms. if antibiotics are discontinued while the patient is still neutropenic, the patients must be monitored closely and intravenous antibiotics restarted immediately with recurrence of fever or any other evidence of bacterial infection, since the initial infection may have only been suppressed, not eradicated. one should consider continuous administration of antibiotics throughout the neutropenic period in patients who have profound neutropenia, mucous membrane lesions of the gastrointestinal tract, or any other identified risk factor. some experts suggest, in patients in whom hematological recovery cannot be anticipated, a change from the therapeutic regimen to a prophylactic scheme after 2 weeks of therapy with intravenous antimicrobials. when the suspicion of a noninfectious cause of the fever is high, interruption of antibiotic therapy after ~4 days seems warranted in clinically well patients without any evidence of infection apart from persisting fever. under these conditions, meticulous monitoring has to be maintained to guarantee the patients timely protection against subsequent infections that are likely to occur. the decision to start antifungals may appear complex but is not as difficult as the decision to discontinue. if a systemic fungal infection has been identified, the course of antifungal therapy will be determined by the causative agent and the extent of the disease. in patients with pulmonary infiltrates or other suspicious lesions, it is essential to see a clinical and, preferably, a radiological response before one ponders cessation of antifungal therapy. however, if no fungal infection is found, it is not clear how long antifungal drugs should be administered [147] . for clinically well patients with prolonged neutropenia, it is suggested that antifungal agents can be stopped after 2 weeks of treatment, provided that no conspicuous lesions can be found by clinical evaluation or by computer-assisted tomographic scanning of the chest and the abdominal organs. in the patient who appears ill or is at high risk, continuation of antifungal therapy throughout the neutropenic episode is recommended. conversely, when neutropenic fever subsides, the patient is clinically well and computer-assisted tomographic scan of the abdomen and chest reveals no suspicious lesions; antifungals may be discontinued, particularly when the criterion for commencing antifungal therapy had been simply fever unresponsive to antibiotics. this approach also applies when the presumptive diagnosis becomes questionable during the course of granulocytopenia. when a patient diagnosed with and treated for a proven or probable invasive fungal disease requires further chemotherapy or bone marrow transplantation, protection against the offending pathogen has to be provided, even if the patient responded completely to initial antifungal therapy. the risk of relapse of invasive fungal disease is so high that secondary prophylaxis is warranted, requiring that a full dose of the most effective antifungal is administered [148, 149] . after introduction of routine ct scanning it became apparent that solitary lesions caused by invasive fungal disease are rare and this observation reduced the enthusiasm for surgical interventions. however, if the number of lesions is limited or a difficult-to-treat pathogen, such as a zygomycosis, has been found, surgical excision has to be considered, especially when the lesions are located close to a large vessel [150] . modern chemotherapy offers hope of a cure to many cancer patients, but it confronts the medical community with new challenges continuously. infection remains an inevitable side-effect of the myeloablative therapy for acute leukemia and is the principal cause of morbidity and mortality amongst these patients. optimal care can be delivered only by those who pay scrupulous attention to the patient's clinical condition and are aware of the evolving therapeutic and diagnostic modalities. it cannot be denied that time remains an important factor in the management of infectious complications but we must try to distinguish more accurately between patients truly in need of immediate therapy and those who are not. fixed treatment algorithms are only acceptable if they allow individual interpretation and reasonable deviations. maintaining guidelines that dictate second line treatment of a population in which more than half of the patients do not have true infection is not justifiable in view of potential adverse events and the economical burden. the demand for an alternative strategy, built on clinical skills, modern and more accurate laboratory tests and imaging techniques, has become apparent and a broad application of this principle may change the approach to antimicrobial treatment in neutropenic patients completely. overuse of antimicrobial agents, both antibacterial and antifungal, has become all too common in the belief that broader coverage will benefit 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key: cord-016426-aw3wirmb authors: wohrley, julie d.; bartlett, allison h. title: the role of the environment and colonization in healthcare-associated infections date: 2018-07-16 journal: healthcare-associated infections in children doi: 10.1007/978-3-319-98122-2_2 sha: doc_id: 16426 cord_uid: aw3wirmb healthcare-associated infections (hais) can be caused by endogenous host microbial flora or by exogenous microbes, including those found in the hospital environment. efforts to decrease endogenous pathogens via decolonization and skin antisepsis may decrease the risk of infection in some settings. controlling the spread of potential pathogens from the environment requires meticulous attention to cleaning and disinfection practices. in addition to selection of the appropriate cleaning agent, use of tools that assess the adequacy of cleaning and addition of no-touch cleaning technology may decrease environmental contamination. hand hygiene is also a critical component of preventing transmission of pathogens from the environment to patients via healthcare worker hands. resulting from invasive devices or surgical procedures may be associated with a shift from colonization to infection. a complex interaction occurs throughout the body between commensal organisms and the barriers they colonize, with research on the skin and gastrointestinal tract microbiome shedding light on these interactions. the skin is a protective barrier with large numbers of colonizing bacteria. the skin microbiome is inhabited by bacteria, fungi, viruses, archaea, and mites; however most research has focused on bacteria. the presence of various microbes may influence disease as evidenced by the shift from staphylococcus epidermidis cultured from healthy skin in young children [3] to propionibacterium acnes in teenagers with acne [4, 5] . children with atopic dermatitis have a propensity to infection with staphylococcus aureus [6] . loss of skin integrity, as with wounds, burns, inflammation, or invasive devices, allows pathogens to enter. the balance between host and flora is also important in the gut and is influenced by antibiotic usage, diarrheal diseases, and critical illness [7] [8] [9] . the gut provides both an essential immune response in maintaining health with normal flora stimulating proliferation of epithelial cells in small and large intestines, participating in development of competent gut-associated immune responses, as well as providing a physical barrier function against pathogen invasion through colonization resistance [10] [11] [12] . inflammatory bowel disease is just one example of altered gut flora associated with a disease state. the secretory antibody system is important in the defense against mucosal infections. specific secretory immunoglobulin a (iga), transported through secretory epithelia to the mucosal surface, inhibits pathogen colonization through microorganism entrapment in mucus and promotion of clearance of entrapped microbes via peristalsis or mucociliary movement [13] . iga also plays a role in mucosal protection of the gut by binding to a mucous layer that separates commensal bacteria from the apical surface of intestinal epithelial cells [14] . patients admitted to the hospital bring with them their "normal" flora which may be very different in a previously healthy child than in a technology-dependent child who resides in a long-term care facility. colonization of children with organisms specific to their individual clinical conditions, such as pseudomonas aeruginosa in a tracheostomy-and ventilator-dependent child, or multidrug-resistant enterobacteriaceae in the gi tract of a neurologically impaired adolescent with neurogenic bladder and a history of frequent urinary tract infections, may lead to infection with these pathogens. these resident organisms may be transferred to the environment where they can be acquired directly by other patients or transmitted indirectly by hcw hands. high-risk populations for acquisition of multidrug-resistant organisms include those who are critically ill, who are immunocompromised, or who have been hospitalized for long periods of time, either in acute care or long-term care settings. additional risk factors include prolonged use of antibiotics and contact with colonized patients or the colonized/contaminated hands of hcw. colonizing organisms may produce invasive infection whether or not colonization is acquired in the hospital or the community. colonization with methicillinresistant staphylococcus aureus (mrsa) is a risk factor for subsequent invasive mrsa infection [15] . a study of relatedness between colonizing strains of s. aureus and those associated with invasive disease in adults found that more than 80% of s. aureus blood isolates were identical to those colonizing the patient's anterior nares [16] . genotypes of s. aureus strains from surgical site infections were also noted to be identical to colonizing strains in more than 80% of surgical patients [17] . the hospital environment represents a reservoir of organisms such as mrsa, vancomycin-resistant enterococcus (vre), and multidrug-resistant gram-negative pathogens as well as clostridium difficile. notably, these same drug-resistant organisms found on surfaces in acute care hospital settings can be found in outpatient settings [18] [19] [20] [21] . transmission between patients and the environment may occur directly from contaminated fomites, indirectly from fomites on hcw hands, or indirectly from patient to patient on hcw hands. c. difficile may be spread in this indirect fashion. for example, this organism may be first identified in the stool of hospitalized patients and then later found to be contaminating the hospital room and its contents, with spread to hcw hands and the hospital environment. hospital surfaces may serve as both a reservoir and a vehicle of transmission for pathogens. specific pathogens such as mrsa [22] , pseudomonas aeruginosa [23] , acinetobacter [24] , and c. difficile can contaminate hospital surfaces because of their ability to survive in the environment. the amount of hospital surface contamination varies depending on body site of infection/colonization, patient type, and cleaning practices. vre is commonly associated with environmental contamination, especially in the presence of diarrhea [25] . in a study of icu patients, the rates of environmental contamination were higher for patients with more than one body site positive for vre [26] . c. difficile is often identified from rooms of colonized and infected patients, proving difficult to eradicate due to resilience of the spores [27] . the frequency of positive environmental cultures for c. difficile is high; in one study 29% (11 of 38) of environmental cultures in rooms occupied by asymptomatic patients had positive cultures for c. difficile, and 49% (44 of 90) of cultures in rooms occupied by patients had c. difficile-associated diarrhea (p = 0.014) [25, 28] . over 80% of the environmental isolates characterized in this study had an immunoblot type identical to that of the patient [28] . during an outbreak investigation in an adult long-term care facility, c. difficile skin isolates from asymptomatic patients and from environmental surfaces matched the source patient's isolate in 13/15 (87%) and 11/19 (58%) cases, respectively [29] . c. difficile has also been recovered from physician and nurse work areas [27, 30] . given the potential for hospital surfaces to be contaminated with pathogens, it stands to reason that the hands, and even the gloves, of hcws can become contaminated as well. contamination of hands of hcws occurs after direct patient care or contact with contaminated surfaces [31] [32] [33] . positive environmental cultures were found to be a risk factor for development of hand/glove contamination [34] . not surprisingly, the level of hand contamination has been shown to correlate with level of environmental contamination [35] . prior room occupants infected with healthcare-associated (hca) pathogens may provide a source of exposure to other patients [36, 37] . admission to a room in which the prior occupant was infected or colonized with mrsa, vre, acinetobacter, or c. difficile is a risk factor for subsequent colonization or infection with these organisms [38] [39] [40] [41] . special terminal cleaning (after the patient has been discharged) of rooms previously occupied by patients with c. difficile infection, including the use of hydrogen peroxide vapor, has been implemented to reduce rates of subsequent infection [42] . these procedures have led to reduced rates of infection in patients subsequently admitted to a room where a prior room occupant was infected or colonized with c. difficile [43, 44] . since colonization may lead to infection, two basic strategies -horizontal and vertical -are employed to reduce hais. horizontal strategies seek to broadly reduce the burden of common healthcare-associated pathogens including s. aureus, enterococcus, gram-negative bacteria, and candida through interventions such as hand hygiene and environmental cleaning. vertical strategies target specific pathogens known to cause hais and utilize active surveillance testing as well as directed approaches to decrease colonization and prevent transmission and subsequent infection [45, 46] . general horizontal prevention strategies are approached elsewhere in this text and include hand hygiene, contact precautions, isolation, and ppe use (see chap. 1, principles of infection control). strategies applied to patients known or at risk for pathogen colonization when viewed from a vertical approach fall into three broad categories: active surveillance testing (ast), pathogen-specific isolation, and decolonization. screening (active surveillance testing, or ast) involves detection of colonized patients using culture or molecular methods and typically focuses on high-risk pathogens, including s. aureus (mrsa), enterococcus (vre), and c. difficile, that are transmitted from person-to-person from colonized or infected patients [45, 47, 48] . some of the principles, strategies, challenges, and controversies of ast will be discussed below. optimal samples for ast vary by pathogen. specimens for mrsa testing are most frequently obtained from nares; however s. aureus also colonizes the skin, perineum, pharynx [49] [50] [51] [52] , gi tract [49] , vagina [53] , and axillae [49, 50] , and additional sites of screening may be indicated depending on the clinical scenario and potential consequences of infection. specimens may undergo culture-based or molecular methods for detection of s. aureus/mrsa. vre colonization is based on samples from stool, rectal, and perirectal swabs, using both molecular methods and culture-based methods [54] . rectal or stool samples are also used for detection of multidrug-resistant gram-negative organisms such as extended-spectrum β-lactamase-producing enterobacteriaceae, as well as carbapenemase-producing enterobacteriaceae. pseudomonas and acinetobacter may be detected in multiple sites, depending on clinical situation, including the rectum, skin, nares, pharynx, wounds, urine, and trachea if the patient is mechanically ventilated. the use of ast without additional interventions to reduce risk for transmission has not proven effective. universal screening effort for pathogens has been most widely studied for mrsa and is considered to be controversial due to questions regarding its effectiveness in controlling spread, as well as cost [45] . screening alone has not shown to be effective in reducing colonization and infection for mrsa [55, 56] . studies have failed to show benefit for a combination of ast and isolation in reducing vre infection or colonization; however, outbreaks of vre have been successfully controlled in hospital settings with use of active surveillance, contact precautions, patient isolation, and cohorting [57] . similarly, active surveillance is most useful following outbreaks of mrsa [58] . a cluster randomized trial in intensive care units found that universal gown and glove use did not reduce overall acquisition of multidrug-resistant organisms (mdro); there was, however, a small reduction in mrsa transmission noted as a secondary outcome [59] . another prospective study of icu patients failed to show a difference in mrsa transmission [60] , with additional concerns for the psychosocial effect that isolation places on patients [61] . in observational studies, single room isolation was shown to reduce mrsa acquisition and infection among hospitalized patients [62, 63] . current recommendations for mrsa colonized and infected patients include isolation in single rooms or cohorting [64, 65] . however, experts have called for a review of the current recommendations for contact precautions and isolation for mrsa colonization in view of the above stated concerns [66] . the addition of targeted decolonization strategies to ast and isolation for control of spread of healthcare-associated pathogens has been most extensively studied to prevent mrsa spread in the hospital setting. patients who are nasally colonized with s. aureus are more than twice as likely as non-colonized patients to develop s. aureus infection [1, 67, 68] . carriage may be classified as persistent, intermittent, or noncarriage [69] . persistent colonization is associated with an increased risk of infection compared with intermittent or non-carriers [70] . carriers with high bacterial loads have a higher risk of infection and may be more likely to transmit the bacteria to their environment [70, 71] . greater quantities of s. aureus are found in the nares of persistent s. aureus carriers compared with intermittent carriers [72, 73] . much research exists regarding the efficacy of active surveillance cultures combined with decolonization to decrease s. aureus transmission and infection in adults, with growing literature in neonatal icus [74] [75] [76] . intranasal antibiotics (mupirocin), with or without antibacterial skin washes (chlorhexidine), have been used in order to decrease the bacterial burden and prevent transmission and infection. shortterm nasal mupirocin has been demonstrated to be effective in eradicating mrsa nasal carriage, with up to 90% success after 1 week of treatment, and 30-60% efficacy for longer duration of follow-up, depending on patient profile and body sites colonized [77, 78] . nasal mupirocin use in high-risk settings has been demonstrated to be effective in eradicating s. aureus nasal colonization and reducing the number of infections in icu, hemodialysis, surgical, and long-term care settings [79] [80] [81] [82] . in a study of nearly two million adult admissions, a significant reduction in the rate of mrsa transmission and infection was noted after introduction of an infection control bundle, which included decolonization of mrsa carriers and isolation [83, 84] as well as a hand hygiene program [84] . however, a crossover study of universal screening on surgical wards combined with targeted decolonization and contact precautions was unable to demonstrate reduction in mrsa infections despite high compliance with screening [85] . nasal mupirocin decolonization of nicu infants with mrsa colonization in two units with high prevalence (>25%) of mrsa colonization decreased the rate of mrsa infections [86] . however, a retrospective study in the usa failed to demonstrate benefit when nasal mupirocin was used for 5 days in colonized neonates in a unit with a baseline prevalence of around 2% [87] . this study showed that some nicu infants develop infection prior to detection of colonization and infants who remain in the nicu can become recolonized over time [87] . taken together, these data suggest that decolonization measures may be most beneficial when the baseline rate of colonization is high. additional nicu studies have found a high correlation between colonizing strains and infecting strains and confirmed high rates (42%) of infections occurring before colonization is detected suggesting universal, rather than targeted, decolonization should be used to control the spread of mrsa [74] . current recommendations suggest that decolonization may be considered in highrisk neonates during an mrsa outbreak or in cases of endemic mrsa when other measures are failing [88] . a recent society for hospital epidemiology association (shea) survey regarding practices for mrsa identification and eradication in nicus noted that most (86%) performed surveillance screening (ast) for mrsa in neonates with variability in timing of samples, sites sampled, isolation protocols, and decolonization strategies employed [89] . recommendations for mssa are less clear. invasive mssa infections occur 2.5 times more frequently than invasive mrsa infections in neonates, leading to significant morbidity and mortality [90] . targeted screening followed by mssa decolonization in a single nicu reduced incidence rates of mssa-positive clinical cultures and mssa infections by more than 50% [91] . mupirocin resistance among s. aureus isolates has been demonstrated in multiple studies, especially associated with prolonged use. high-level resistance has been associated with decolonization failure, and low-level resistance may be associated with early recolonization [71, 72] . therefore, the long-term use of mupirocin is questioned, and alternatives to mupirocin for decolonization in those with mupirocin-resistant strains of mrsa are needed. however, in a long-term study examining use of mupirocin prophylaxis in the nicu over a 7-year period, the rate of s. aureus (mssa and mrsa) infections decreased from 1.88 to 0.33 per 1000 patient days without any mupirocin-resistant isolates identified [92] . this finding is consistent with previous reports of low prevalence of resistance among s. aureus isolates from mupirocin-treated neonates [93] . the use of decolonization may be most effective for patients at risk of infection for short periods of time such as surgical patients, whose risk of infection may be less once the surgical site is closed, as well as icu patients, whose risk may lower once they are discharged from the icu [94, 95] . this is of import given that patients are recolonized within weeks or months following decolonization, and thus the effect is often shortlived [95, 96] . mupirocin decolonization has been used specifically to reduce the risk of surgical site infections (ssis) associated with gram-positive organisms. in a metaanalysis of 17 rcts or quasi-experimental studies including adult cardiac and orthopedic surgery patients, mupirocin decolonization was found to be significantly protective against gram-positive ssis, specifically s. aureus ssis [46, 81, 97] . preoperative s. aureus decolonization is not routinely recommended for most pediatric patients undergoing surgery, however the impact of preoperative colonization on risk of ssi in children has been examined in many small studies. risk of ssi was not elevated in s. aureus-colonized children undergoing cardiac surgery [98] . studies in adult cardiac surgery patients, however, suggest a benefit to mupirocinbased decolonization in prevention of ssi [99] ; this topic as it pertains to cardiac surgery is discussed further in chap. 11. chlorhexidine (chg) is a widely used broad-spectrum topical antimicrobial agent [100] . the centers for disease control and prevention recommend its use as a skin cleanser prior to insertion of central venous catheters (cvc) in both children and adults but do not recommend its use in infants less than 2 months of age due to lack of safety and efficacy data in this population [101] . in spite of these cautions, a national survey of neonatology training program directors indicates that most nicus use chlorhexidine for cvc site prep and maintenance but restrict use based on gestational age, chronological age, and birth weight [102] . risks to premature infants relate to the increased potential for chemical burns and contact dermatitis in the setting of underdeveloped skin [100] and the possibility of systemic absorption of chg, although no adverse events have been reported despite demonstrable blood chg levels [100, [103] [104] [105] . chlorhexidine bathing has been suggested as another adjunct to decrease colonization and has been studied in adults and children, including neonates. an adult randomized controlled trial demonstrated that daily chlorhexidine bathing did not reduce hai including central line-associated bloodstream infection (clabsi), catheter-associated urinary tract infection (cauti), or ventilator-associated pneumonia (vap) [106] . a number of other studies (including clinical trials) in adults, however, have shown positive benefits of chlorhexidine-containing products when used as part of a bundle approach for hai prevention [107] [108] [109] . in the pediatric scrub trial, daily chlorhexidine bathing was compared with standard bathing practices to evaluate effect on incidence of bacteremia in critically ill children [110] . there was a non-statistically significant reduction in bacteremia in the chg group in the intention-to-treat analysis and a 36% decrease in bacteremia in the per protocol arm [110] . the use of universal decolonization raises concerns about the possibility of chlorhexidine resistance. a study from texas children's hospital found that nearly half of nosocomial s. aureus carried one or both genes associated with chlorhexidine tolerance (qaca/qacb and smr), noting that smr-positive isolates were more often resistant to methicillin, ciprofloxacin, or clindamycin as well [111] . mupirocin resistance was also noted in 2.8% of the isolates in this study [111] . vertical and horizontal approaches to infection prevention have been compared in two studies: huang et al. compared three approaches to mrsa prevention among 74 adult icu patients in the reduce-mrsa study [112] . vertical approaches consisted of ast with and without targeted decolonization of mrsa carriers with chg bathing and intranasal mupirocin compared with a horizontal approach involving universal decolonization of all icu patients regardless of mrsa status. universal decolonization was found to be associated with the largest reduction in all-cause bsi (44%) and mrsa clinical culture rates (37%) [112] . another group showed that improved hand hygiene in addition to universal chg bathing reduced overall infection rate and specific rates of candida cauti and acinetobacter vap [113] . additionally, when there is high adherence to chg bathing and hand hygiene, there is no additional benefit to ast and isolation to reduce mdro acquisition rates [114] . selective digestive decontamination (sdd) and selective oral decontamination (sod) are additional methods of universal decolonization employed in an effort to reduce colonization with gram-negative organisms, particularly in critically ill patients. both methods use a polymyxin, an aminoglycoside, and an antifungal, applied to the oropharynx as a paste or gel (sod) or in a liquid form administered per nasogastric or orogastric tube (sdd), paired with systemic antimicrobials, usually an intravenous third-generation cephalosporin. these two strategies have been studied in more than 50 rcts and have been examined in 12 metaanalyses with demonstrated efficacy in reduction of colonization, morbidity, and mortality in adult icu patients [115] [116] [117] . widespread acceptance has been limited by concern over selecting for resistant organisms in universal applications, although long-term follow-up in units employing these strategies have not demonstrated an increase in resistant organisms [118] . microbiome studies of adults undergoing sdd compared to healthy adults revealed dramatic shifts in the gastrointestinal microbiome of sdd recipients (as would be expected) as well as an increase in the relative abundance of organisms expressing antimicrobial resistance genes [119] . the pediatric experience with these strategies is limited. in a single meta-analysis of 4 rcts including 335 children, ventilator-associated pneumonia rates were 69% lower in in those children receiving sdd [120] . the use in neonatal populations has not been studied. the evidence for perioperative antimicrobial prophylaxis is well established, and the use of antimicrobials prior to incision reduces rates of ssi [121] by reducing the concentration of potential pathogens within or near the surgical incision. the basic tenets of antimicrobial use to prevent ssi include use of prophylaxis for all elective operations requiring entry into a hollow viscus, involving insertion of intravascular or orthopedic prosthetic devices or implants, or operations in which occurrence of ssi would pose catastrophic risk to the patient (e.g., sternotomy). the choice of antimicrobial is based upon a need for bactericidal activity against the expected pathogens for specific surgical procedures as well as agents which are known to be safe and cost-effective. the goal is to provide bactericidal concentrations in tissues and serum at the time of incision and to be continued throughout the entire operation until the wound is closed. re-dosing of the antimicrobial agent may be required should the procedure last several hours or if there is significant blood loss. an important risk factor contributing to ssi risk is the number of organisms which gain entry into the wound intraoperatively. the greater the burden, the greater the risk of infection. when appropriate antimicrobial prophylaxis has been administered, a bacterial burden of 10 5 is required to cause ssi; however if a foreign body is present, the threshold to cause infection may be significantly reduced. virulence of the organism also contributes to ssi risk. pre-and perioperative antiseptics are utilized in order to decrease organism burden and thereby reduce the risk of ssi. preoperative bathing with agents such as chlorhexidine has been shown to decrease the amount of endogenous flora on the skin but has not been shown to reduce rates of ssi in pooled analyses of adult surgical patients [122] . in certain very high-risk populations, however, such as cardiac and orthopedic surgery patients, preoperative chlorhexidine bathing has been associated with reduced rates of ssi (especially those due to s. aureus or mrsa) [123] [124] [125] . it is likely that the benefits of chlorhexidine bathing are influenced by the type of surgical procedure (i.e., high-risk vs. low-risk) as well as the baseline rate of ssi at a given institution. in spite of these controversies, the use of chlorhexidine body wash prior to surgery is routine. there are several options for preoperative skin antisepsis with either chlorhexidine-alcohol or povidone-iodine as the active agent. the authors of a cochrane review conclude that other characteristics of skin prep agents such as potential side effects and cost should be taken into consideration as well until there are definitive data showing clinical superiority of one agent over another [126] . new cdc guidelines for prevention of ssi recommend an alcohol-based skin antiseptic, such as either chlorhexidine-alcohol or iodophor-alcohol products [127] [128] [129] . surgical site infections generally arise from endogenous sources such as bacteria present on skin surfaces or in a viscus, with greatest risk occurring while the wound is open. in addition to skin surface, bacteria may be found in skin appendages, including sebaceous glands, hair follicles, and sweat glands [130] . when infections related to exogenous sources occur, they may be sporadic or related to an outbreak. exogenous sources include contamination of the operating room environment, surgical instruments, equipment, or colonized or infected personnel [131, 132] . common nosocomial pathogens can persist for months on surfaces, contributing to transmission risk in the absence of regular and thorough cleaning and disinfection [133] . these pathogens importantly include gram-positive (enterococcus, including vre; s. aureus, including mrsa; and streptococcus pyogenes) as well as gramnegative (acinetobacter spp., e. coli, klebsiella spp., p. aeruginosa, serratia marcescens, or shigella) organisms. spore-forming bacteria, such as clostridium difficile, can survive for months as can fungi and yeast. viruses from the respiratory tract, such as coronavirus, influenza, coxsackie, and rhinovirus, survive a relatively short period of days, whereas viruses from the gastrointestinal tract, such as norovirus or rotavirus, may persist for up to 2 months [133] . surfaces in rooms of patients infected or colonized with pathogens may (and frequently do) become contaminated. mrsa, vre, acinetobacter spp., norovirus, and c. difficile have been detected on environmental surfaces in rooms of infected or colonized patients, can colonize healthcare workers' hands, and can then be transmitted to others [134] . contact with the environment is as likely as contact with the affected patient to result in contamination of hcw hands [32] . the presence of environmental contamination is a risk factor for hcw hand/glove contamination [33] . admission to a room previously occupied by a patient colonized or infected with mrsa, vre, acinetobacter spp., or c. difficile has been shown to be a risk factor for subsequent development of colonization or infection by these pathogens [38, 40, 41] . multiple studies have demonstrated that a lack of thorough cleaning [135, 136] contributes to persistence of environmental contamination. assessing adequacy of cleaning can be performed using various methods including observation for visible soiling, culture-based colony counts, fluorescent dye, and atp detection. for example, fluorescent dye can be applied as a dot to surfaces where it dries clear. if a surface was inadequately wiped, the area fluoresces when exposed to black light. atp bioluminescence systems measure atp, a marker for presence of residual organic material (e.g., human secretions or excretions and food). atp, however, does not indicate presence of viable pathogens, and its absence does not rule out the presence of contamination, and as such, use of fluorescent dye correlates more closely with colony counts than does atp bioluminescence [137] . focused efforts to eradicate pathogens can improve cleaning efficacy, which may involve specialized teams [138] or through use of improved monitoring of cleaning practices with markers such as atp and fluorescent dye [137, 139] . feedback to environmental services (evs) staff following use of enhanced methods has also been demonstrated to improve the frequency of achieving adequate cleaning [140, 141] . in a study of 36 acute care hospitals, only 48% (9910/20,646) of environmental surfaces were cleaned at baseline. after educational and procedural interventions combined with provision of objective performance feedback to evs staff, 77% (7287/9464) of surfaces were cleaned (p < 0.001) [141] . in addition to ensuring each surface is cleaned, it is important to select the correct cleaning product as microorganisms vary in their resistance to disinfectants. for example, disinfection of a room potentially contaminated by c. difficile requires use of hypochlorite-based solutions [142] rather than phenols or quaternary ammonium compounds generally used for general hospital-based cleaning. in spite of enhanced cleaning methods aimed at improving cleaning thoroughness and monitoring of cleaning practices, many surfaces remain inadequately cleaned. for this reason, no-touch room disinfection units that decontaminate environmental surfaces and objects utilizing either ultraviolet (uv) light or hydrogen peroxide (hp) vapor have been developed [143, 144] . these technologies are considered an adjunct to standard cleaning and disinfection since surfaces must be physically clean and the room must be emptied of people prior to use. uv irradiation with certain wavelengths breaks the molecular bond in dna, thereby destroying the organism. this has been shown to be effective against mrsa, vre, and acinetobacter baumannii, in experimentally contaminated rooms [145] . systems utilizing hp vapor have been found to be effective in eradicating pathogens such as mrsa, mycobacterium tuberculosis, serratia, and c. difficile spores from rooms and equipment [146] . both of these methods have been found to be effective at reducing hais [146] . their advantages include ability to substantially reduce c. difficile spores [147] as well as achieve substantial reductions in vegetative bacteria. failure to adequately clean and sterilize equipment may lead to transmission via contaminated equipment [148] . the level of disinfection or sterilization considered acceptable depends on the intended use of the object and 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as the source of legionella pneumophila in incubators of a neonatology unit first outbreak of nosocomial legionella infection in term neonates caused by a cold mist ultrasonic humidifier key: cord-018808-h2zb87oa authors: tantawichien, terapong; thisayakorn, usa title: dengue date: 2018-03-20 journal: neglected tropical diseases south asia doi: 10.1007/978-3-319-68493-2_10 sha: doc_id: 18808 cord_uid: h2zb87oa dengue is one of the most important mosquito-borne viral infections caused by single-stranded rna virus that are transmitted by the aedes aegypti and aedes albopictus mosquito species. dengue is endemic in over 140 countries in asia, the usa, the eastern mediterranean, and africa. the world health organization (who) estimated that there are more than 2.5 billion people—mainly occurs in children living in tropical and subtropical countries—at risk of dengue infection with one or more dengue viruses. there are estimated nearly 100 million symptomatic dengue infections occurring worldwide annually, nearly 75% in asia and the western pacific region [1]. during the past decades, the outbreaks of dengue infection have been reported throughout the world with increased severity. ecologic and demographic changes are considered to be the contributing factors to the emergence of dengue infection in the past decades. dengue has expanded into new countries and into urban settings associated with increased distribution of a. aegypti, population growth, urbanization, development of slums, migration of population, movement of dengue virus by infected travelers, trade development, and improved diagnostic capabilities in medical practice [2, 3]. increased transmission of dengue virus in tropical urban areas has been created by substandard housing and crowding as well as deterioration in water, sewer, and waste management systems, all of which are intimately associated with unplanned urbanization [4–7]. so it is likely that dengue will expand its geographic reach and become an increasing burden on health resources in affected areas during the next decade. an effective vector-control management is the only means to reduce dengue infection in endemic areas. because vector control has achieved only limited success so far in reducing the transmission of dengue, the usage of effective dengue vaccine in target population along with the preventive measures already used such as raising public awareness may be the means to effectively control of this disease in endemic area [8]. dengue is one of the most important mosquito-borne viral infections caused by single-stranded rna virus that are transmitted by the aedes aegypti and aedes albopictus mosquito species. dengue is endemic in over 140 countries in asia, the usa, the eastern mediterranean, and africa. the world health organization (who) estimated that there are more than 2.5 billion people-mainly occurs in children living in tropical and subtropical countries-at risk of dengue infection with one or more dengue viruses. there are estimated nearly 100 million symptomatic dengue infections occurring worldwide annually, nearly 75% in asia and the western pacific region [1] . during the past decades, the outbreaks of dengue infection have been reported throughout the world with increased severity. ecologic and demographic changes are considered to be the contributing factors to the emergence of dengue infection in the past decades. dengue has expanded into new countries and into urban settings associated with increased distribution of a. aegypti, population growth, urbanization, development of slums, migration of population, movement of dengue virus by infected travelers, trade development, and improved diagnostic capabilities in medical practice [2, 3] . increased transmission of dengue virus in tropical urban areas has been created by substandard housing and crowding as well as deterioration in water, sewer, and waste management systems, all of which are intimately associated with unplanned urbanization [4] [5] [6] [7] . so it is likely that dengue will expand its geographic reach and become an increasing burden on health resources in affected areas during the next decade. an effective vector-control management is the only means to reduce dengue infection in endemic areas. because vector control has achieved only limited success so far in reducing the transmission of dengue, the usage of effective dengue vaccine in target population along with the preventive measures already used such as raising public awareness may be the means to effectively control of this disease in endemic area [8] . hyperendemic dengue with a general increase in the number of dengue cases over time is a major public health problem in many countries in south and southeast asia where a. aegypti and a. albopictus are widespread in both urban and rural areas, where multiple virus serotypes are circulating, and where dengue is a leading cause of hospitalization and death in children. the extent of hyperendemicity and time between serotype introductions are key determinants in a population's serotypespecific immunity and, consequently, in the age distribution of clinically apparent dengue infection. in the past, the age distribution of indigenous dengue cases in south and southeast asia is different from that of the americas, where these syndromes occur in all age groups included elderly. recently several countries in asia have reported an epidemic shift of dengue from mainly affecting children to affecting adolescents and young adults with increased severity [9] [10] [11] [12] . in thailand, the affected adults aged over 15 years old are reported to comprise 30-40% of dengue virus infected cases [13] . there are some differences in clinical presentations, laboratory findings, and severe complications between children and adult with dengue [14] [15] [16] . increasing incidence of dengue among travelers (1.0-6.7%) has been recognized as a potential hazard to tourists as various reports in international travelers returning from endemic areas [17] [18] [19] [20] . recent reports revealed that adult dengue appears to occur more frequently than malaria among travelers returning from asia, so the healthcare providers in western countries are more likely to be confronted with travel-acquired dengue infections [21] [22] [23] [24] . in returning travelers with fever, clinical manifestations of dengue infection are comparable with observations in endemic area where dengue may go unnoticed. we emphasize the need for continued dengue surveillance in non-endemic countries with careful evaluation and follow-up of febrile travelers after visiting countries where are dengue-endemic areas [25, 26] . travelers should be encouraged to protect themselves from mosquito bites, in order to avoid infections and onward transmission of dengue in new areas where a. aegypti is established. dengue virus, single-stranded rna virus member of genus flavivirus in the family flaviviridae, is the etiologic agent of dengue infection. there are four serotypes of dengue virus (den-1, den-2, den-3, and den-4) that are transmitted by the aedes aegypti and a. albopictus mosquito species. peak transmission occurs in rainfall season and high temperature in hyperendemic and endemic areas. although dengue virus is transmitted by mosquitoes, unusual dengue transmissions through needlestick, receipt of infected blood component, tissue or organ transplantation, and transplacental infection have been reported [27] [28] [29] [30] [31] [32] [33] [34] . after an incubation period of 4-8 days, infection by any dengue virus can produce a wide spectrum of illnesses ranging from asymptomatic or subclinical infection to undifferentiated fever, dengue fever (df), and severe forms of the disease associated with plasma leakage (dengue hemorrhage fever: dhf), dengue shock syndrome (dss), severe bleeding, encephalopathy, and multi-organ failure [35] . dhf is characterized by rapid onset of capillary leakage accompanied by thrombocytopenia, hemoconcentration, vascular collapse, abdominal pain, and hemorrhagic manifestations [36] . despite the clinical classification of df and dhf as distinct entities, they are likely to be a continuum of the same disease process with divergent outcomes with regard to the perturbation of vascular integrity. in case of dengue infection, asymptomatic cases are more frequent than the symptomatic cases with the variable ratio of asymptomatic to symptomatic dengue infections of 0.9:1 to 18:1, dependent on the geographical areas, the epidemiological contexts, and individual immunological attributes [37, 38] . however, patients with asymptomatic infection may act as reservoir for dengue virus to transmit to mosquitoes and subsequently to humans and should be considered in estimation of disease burden. recovery from dengue infection with one serotype confers lifelong homologous immunity to that particular serotype but short-term protection against other serotypes, so secondary infection can occur with other dengue serotypes. previous epidemiologic data reveals that secondary heterotypic dengue virus infection is a risk factor to develop severe dhf/dss, mediated most likely by antibody-dependent enhancement (ade) of infection. pre-existing homotypic antibodies bind to heterotypic dengue virions (virusantibody complexes) and enable fcγ receptor-mediated uptake by target fcγ receptor-bearing cells (e.g., monocyte/macrophage) resulting in increased viral replication and viremia [38] . changing of inflammatory cytokine production (such as tnf1, interleukin-1, interleukin-2, interleukin-6, interleukin-12, macrophage migration inhibitory factor, hmgb1, mcp-1) produced by t-lymphocytes, monocytes/macrophages, and endothelial cell is observed in dengue patients who have increased vascular permeability, thrombocytopenia, and activation of coagulation and fibrinolysis [39, 40] . in addition, secreted ns1 protein, anti-ns1 antibodies, and increased complement activation (c3a, c5a) might be involved in increased production of inflammatory cytokines, triggering local and systemic effects implicated in intravascular coagulopathy and virus-induced vascular leakage. thrombocytopenia caused by bone marrow suppression, shortened platelet survival, and increased platelet consumption due to platelet adhesion occurs during the dengue infection and reaches nadir during the day of defervescence (toxic stage) [40, 41] . although secondary dengue infection remains the strongest known risk factor for dhf/dss, viral genetics, serotype sequence, host factors, and time interval between primary and secondary infections can modulate severity of illness [40, [42] [43] [44] [45] . dengue infection should be suspected if patients in dengue-epidemic or dengueendemic area have a fever of 10 days or less with myalgia, headache, flushing, anorexia, nausea or vomiting, arthralgia, bone pain, periorbital pain with no obvious respiratory tract symptoms or signs, and no organ-specific symptoms of other infectious diseases. the clinical spectrum of dengue infection ranges from mild illness (undifferentiated fever, non-severe df) to the life-threatening severe forms of the disease with plasma leakage (dhf/dss), severe bleeding, or multi-organ failure, which may be fatal. dengue fever in its classical form is nonfatal febrile illness with about 5-7 days associated with sudden onset, anorexia, myalgia, headache, and occasional rash. dhf is characterized by high continuous fever of 2-7 days and rapid onset of capillary leakage accompanied by thrombocytopenia, hemoconcentration, vascular collapse, abdominal pain, and hemorrhagic manifestations. shock (dss) occurs as a consequence of severe plasma volume loss into serous spaces (e.g., pleural space or peritoneum cavity) or severe internal hemorrhage. during the acute febrile phase, usually lasting 3-8 days, the clinical symptoms resemble those of df and severe dengue (dhf), including fever, nausea or vomiting, headache, rash, and myalgia; nonetheless, abdominal pain and severe or widespread bleeding are less frequent in df. minor hemorrhagic manifestations such as petechiae, epistaxis, gingival bleeding, and menorrhagia do sometimes occur in patient with dengue, although df is rarely associated with severe hemorrhage leading to shock. age-related differences in dengue severity are poorly understood; however, there are some differences in clinical courses between children and adults. plasma leakage (dhf) and dss appear to be more frequent in children than adults, possibly reflecting age-dependent differences in intrinsic vascular permeability, but some reports suggest that bleeding manifestations, especially severe internal hemorrhage and hepatic dysfunction, are both more common in adults and older age groups [14, 16, 41, 43, 44, [46] [47] [48] [49] [50] . the symptoms generally last for 3-7 days before the fever subsides and symptoms remit. during convalescent stage, the patients with dengue infection, even in dss, may have rapidly increasing appetite, convalescent rash on lower extremities (a confluent rash with characteristic, scattered, round areas on pale skin), and sinus bradycardia. most patients with dengue infection recover spontaneously, and abnormal hemostasis normalizes during the convalescent stage or within 1-2 weeks after defervescence. the emergence of severe bleeding, fulminant hepatic failure, and encephalopathy in df and dhf have been the causes of an apparent increase in the complications of dengue in the adolescent, adult, and elderly [47] [48] [49] [51] [52] [53] [54] . high mortality rate has previously been reported in elderly patients with dengue infection because of medical comorbidity and waning of host immunity [55] [56] [57] [58] [59] . the prognosis of dengue infection depends on early diagnosis, recognition of plasma leakage, and treatment with immediate replacement of fluid and intensive supportive care. the classification of severity has a high potential for being of physician's practice as to where and how intensively the patient should be observed and treated with intravenous fluids, blood, or plasma transfusion and medicines. who released a new classification in 2009, which is dengue with or without warning signs and severe dengue because who 1997 classification (df, dhf, dss) was poorly related to disease severity, difficult to use in clinical setting, and unhelpful in triage in outbreaks [35, 36, 60, 61] . these warning signs (persistent or severe vomiting, abdominal pain or tenderness, liver enlargement, drowsy or alteration of consciousness, fluid accumulation with respiratory distress, epitaxis, gum bleeding, gastrointestinal bleeding, retinal hemorrhage, oliguria, and hemoconcentration with severe thrombocytopenia) are used to alert the clinicians to monitor dengue infection progress. physicians should be aware of these warning signs in patients with dengue infections before they develop severe dengue [62] [63] [64] . severe dengue is defined by one or more of the following: plasma leakage (dhf) that may lead to shock (dss), severe bleeding, and severe organ impairment such as hepatic failure, acute renal failure, and encephalopathy) as fig. 1 [14, 35, [39] [40] [41] [42] [43] [44] [45] [46] [47] . if untreated, mortality can be as high as 20%, whereas appropriate case management and intravenous rehydration can reduce mortality to less than 1%. virus factors (serotypes, structural and nonstructural proteins of dengue virus, and viral load) and host factors (age, gender differences, genetic, nutritional status, immune reaction, and coexisting medical conditions) might be involved in the severity of dengue infection. typically dhf resembles df in many clinical respects, but it is characterized by high continuous fever of 2-7 days, hemorrhagic diathesis, hepatomegaly, and circulatory disturbance (dss). the critical stage associated with plasma leakage (20% increase in hematocrit over baseline) and marked thrombocytopenia (<100 â 10 9 /l) associated with bleeding frequently occur at the end of febrile phase of illness [36] . right side pleural fluid detected by chest roentgenogram or free fluid in the peritoneal cavity and thickening of gall bladder wall detected by ultrasonography has been interpreted as evidence of plasma leakage, which is usually only clinically detectable after intravenous fluid therapy unless plasma leakage is significant [65] [66] [67] . the right side or bilateral pleural effusion is generally not prominent but becomes increasingly more after excessive intravenous fluid administration. in mild dhf cases, the changes in blood pressure and pulse may be minimal and transient. patients recover shortly after treatment. in more severe [35] dhf cases, a rapid, weak pulse, narrowing of the pulse pressure to less than 20 mm hg, or an unobtainable blood pressure establishes dss [36] . clinical indicators of impending dss include severe abdominal pain, change from fever to hypothermia, restlessness, sweating, prostration, and tender hepatomegaly. if plasma loss continues and becomes excessive, the patient's situation can progress rapidly into profound shock. prolonged shock often complicates metabolic acidosis, severe gastrointestinal bleeding, and disseminated intravascular coagulopathy (dic). dss was an independent risk factor (odds ratio 220) for development of acute renal failure in adult patients with dhf [68] . cardiac involvement was observed in few patients ranging from abnormality of electrocardiogram, mild elevation of cardiac biomarkers to myocarditis and/or pericarditis and death [69] . acute respiratory failure is a rare complication but has a high mortality rate [70] . although children are more likely to develop hypovolemic shock than adults in dhf characterized by increased microvascular permeability, a high mortality rate is seen in the adults and elderly with dengue infection [42, 49, 51, 55, 57, 62] . high fatality rate of dengue in adults was significantly associated with pre-existing comorbid medical illnesses such as cardiac diseases and renal diseases [49, [56] [57] [58] [59] 62] . because the altered vascular permeability is short-lived and spontaneously converts to normal level, the period of clinically significant plasma leakage usually lasts 24-48 h after defervescence. diuresis ensues as plasma leakage terminates. convalescent rash, transient hypertension, and sinus bradycardia were described during convalescence in patients with dhf/dss. hemorrhage contributes to morbidity and mortality, especially during the severe thrombocytopenia, usually occurring in 5 to 8 days after onset of illness [41] . the pathogenesis of abnormal bleeding in dengue is multifactorial and encompasses severe thrombocytopenia, platelet dysfunction, blood coagulation defects, and vasculopathy. there are typical coagulopathies of increased aptt and low fibrinogen levels in most patients, but severe thrombocytopenia and platelet dysfunction are probably the major cause of clinical bleeding. variable degree of hemorrhage may occur at any sites, most commonly petechiae, epistaxis, gingival bleeding, or menorrhagia, and usually occurs on days 5-8 of the illness. bleeding from the nose, gums, and upper gastrointestinal tract are not uncommon in patients with dengue infection. vaginal bleeding (menorrhagia) is a common site of bleeding (24.6% in adults with dengue infection), and hormonal therapy such as premarin and primolut n is suggested for patients exhibiting excessive vaginal bleeding [48] . of the dengue patients with plasma leakage (dhf), severity of bleeding varied markedly with spontaneous petechiae, hematemesis, melena, menorrhagia, and epistaxis. risk factors of severe bleeding are platelets 20,000/mm 3 ( 20 â 10 9 /l), high aspartate aminotransferase (ast) or alanine aminotransferase (alt) level, prolonged prothrombin time (pt), severe plasma leakage (dss), dic, or fulminant hepatic failure [71] . massive hematemesis may occur in adults with df or dhf caused by pre-existing peptic ulcer or hemorrhagic gastritis, and it may be not associated with profound shock in adults as previously reported in children. in the few reports of endoscopic findings for dengue adults with upper gastrointestinal bleeding, hemorrhagic gastritis was the most common finding (40.9-58.5%), followed by gastric ulcer, and duodenal ulcer [53] . however, the role of endoscopic therapy in upper gastrointestinal bleeding of dengue patients is still unknown [72] . life-threatening internal hemorrhage such as subcapsular splenic bleeding and ruptures is rare but can happen spontaneously or as a result of trauma, which may be unnoticed. splenectomy is still the treatment of choice for splenic rupture, but numerous recent reports have documented favorable outcomes with conservative treatment [54, 73] . early diagnosis, intensive supportive care, and replacement therapy are needed to avoid a fatal outcome in dengue patients who have severe hemorrhage. there are pregnant women with df or severe dengue reported in asia, highlighting the concept that young women in hyperendemic and endemic area remain susceptible to dengue infection [74] [75] [76] . the obstetricians must be aware that dengue infection of pregnant women may occur and some history or laboratory results consistent with dengue infection must be identified. dengue during pregnancy is also particularly important in pregnant travelers from non-endemic countries to countries where dengue is endemic [77] . uterine hemorrhage resulting in spontaneous abortion and severe postpartum bleeding has also been reported in pregnant women [76] . surgical procedures such as cesarean section performed on patients with dengue infection may unmask dengue-induced hemostatic defects, resulting in unexpected hemorrhage in postoperative period that is difficult to control [78] . it also has been reported that dengue infection was vertically transmitted to the fetus and led to a full-blown illness in the neonate similar to that seen in children and adults [74] . although the effects of dengue infection on pregnant women and their fetuses or newborns are unclear, recent studies have demonstrated that this infection did not cause any infant abnormalities but may have been responsible for fetus deaths and morbidity in pregnant women [79] [80] [81] . hepatomegaly and increased levels of aspartate aminotransferase (ast) and alanine aminotransferase (alt) were more commonly found in patients with dengue infection especially dhf [82] [83] [84] [85] [86] [87] . so dengue infection should be included in the differential diagnosis of acute viral hepatitis in asia. unlike conventional viral hepatitis, patients with dengue have a level of ast that are greater than that of alt which may be due to excessive release of ast from damaged myocytes during dengue infections, and these levels of liver enzymes increase to a maximal 7-9 days after onset of illness, then decrease to normal levels within 2 weeks [48, 49, [82] [83] [84] . potential mechanisms of liver injury involve a variety of potential insults including direct effects of infected virus serotypes or an adverse consequence of dysregulated host immune responses on liver cells; compromised circulation and/or hypoxia caused by hypotension or localized vascular leakage inside the liver capsule; hepatotoxic effects of drugs such as acetaminophen or traditional herbal remedies, coinfection with other viruses such as virus hepatitis a, b, and c; and pre-existing underlying diseases (e.g., hemoglobinopathies, alcoholic liver diseases) [88] . attention must therefore be given to the use of hepatotoxic drugs such as acetaminophen, antibiotics, and antiemetic drugs, all of which have the potential to aggravate liver damage in patient with dengue. previous reports have found evidence that acetaminophen overdose may play an important role in causing acute liver failure in dengue patients [89, 90] . it is likely that relatively more adult dengue patients have more liver impairment than children. pre-existing liver diseases such as chronic infection with virus hepatitis b or c, alcoholic liver disease, and cirrhosis may aggravate the liver impairment of dengue. abnormal liver enzyme levels have been associated with severity and a poor outcome in patients with vascular leakage and abnormal bleeding [86, 87] . increased levels of bilirubin and alkaline phosphatase were observed in a few patients. severe liver impairment occurred in late stage of disease may complicate the outcome of dengue infection by causing acute hepatic failure and contributing directly to severe bleeding, as well as potentiating the severity of dic [89, 90] . severe jaundice and high mortality are observed in dengue patients with fulminant hepatic failure. the management of fulminant hepatic failure in dengue is primarily intensive supportive care; however, therapy with n-acetylcysteine (nac) or providing artificial liver support was previously described [87] . the unusual manifestations of dengue infection have been recognized including severe internal hemorrhage, fulminant hepatic failure, encephalopathy, cardiomyopathy, cardiac arrhythmia, adult respiratory distress syndrome (ards), rhabdomyolysis, pancreatitis, appendicitis, coinfection with other viruses or tropical infectious diseases, and neurological complications (e.g., altered consciousness, seizures, paresis, and coma resulting from encephalitis and encephalopathy) [91] [92] [93] [94] [95] [96] [97] [98] . the neurological manifestations secondary to dengue infection including encephalopathy, encephalitis, myelitis, neuro-ophthalmic complications, polyradiculopathy, neuropathy, and neuromuscular complications were ascribed in 0.5-21% of hospitalized patients [92, 94, 95, 99] . possible causes of encephalopathy in patient with dengue include hypotension, cerebral edema, focal hemorrhage, hyponatremia, fulminant hepatic failure, and the direct invasion of dengue virus in the central nervous system [100, 101] . acute renal failure is an accompanying presentation in dss or dengue-associated fulminant hepatic failure. previous studies revealed that 5.5% of the patients with dhf/dss also had dual infection (e.g., urinary tract infection, diarrhea, or bacteremia) [48, 102] . dual infection should be suspected in patients who have atypical manifestations, for example, fever for more than 10 days, mucus diarrhea, jaundice, persistent abdominal pain, recurrent fever, wbc > 10,000/mm 3 (>10 â 10 9 /l) with neutrophilia, or the presence of the band form of neutrophil and acute renal failure [103] . the patient with severe dengue infection may have secondary bacterial sepsis, e.g., bacteremia, and uti after hospitalization. failure in making a diagnosis of concurrent infection in patients with dengue may lead to otherwise preventable mortality [104] . attempts to differentiate dengue infection clinically from other acute febrile illnesses are unlikely to be successful; although, the diagnosis is aided if laboratory examination indicates leukopenia, thrombocytopenia, or mildly elevated ast levels. early definite diagnosis of dengue infection can help clinicians in initiation of early supportive care, adequate fluid administration, and identification of patients with severe dengue who should be closely monitored for signs of plasma leakage, bleeding, and organ damage. this information might promote early supportive therapies, prevent the use of potentially harmful drugs, encourage assessment of complications, ensure the adequate use of treatment guidelines, and lead to the effective control of dengue outbreaks. the tourniquet test considered by the who has been used as a clue for probable dengue infection for a long time [35] . unfortunately, the sensitivity and specificity of tourniquet test were not excellent, ranging between 34 and 56% and 68 and 94%, respectively, and a negative test does not exclude the disease [105] [106] [107] . laboratory diagnosis of dengue infection is established either directly by isolation or detection of viral components in serum or tissue or indirectly by detection of virus-specific antibodies in serum [108] . the sensitivity of each approach is influenced by the duration and severity of the patient's illness. within the first 2-3 days of illness, only reverse transcription polymerase chain reaction (rt-pcr) or dengue virus ns1 ag assay can reliably confirm the diagnosis of dengue. determination of dengue virus by rt-pcr in serum, tissues, saliva, or urine is definitely the most satisfactory test that might detect dengue viruses up to the 7th day after the onset of the symptoms, especially in severe cases [109] [110] [111] . a high circulating level of dengue virus ns1 was demonstrated in the early stage of dengue infection by different elisas in the plasma and/or sera of dengue patients [112] . until now, elisa used to detect acute phase (igm), and convalescent phase (igg) antibodies have been considered the most useful test for dengue diagnosis due to its high sensitivity and ease of use. because dengue antibodies are better detected around the 5th day after the onset of the illness, antibody detections are unfeasible for rapid diagnosis. there are several commercial kits of rapid tests of igm and igg detection, but the sensitivity, specificity, and accuracy vary among these tests [113, 114] . various combination tests for elevated levels of ns1 and dengue igm/igg in serum have yielded sensitivities between 75.5 and 92.9% with specificities ranging from 75.0 to 100% and are a pragmatic diagnostic approach in a patient in whom dengue infection is suspected. it should be stressed that in dengue-endemic areas, while early accurate laboratory tests are not widely available, dengue infection should be considered in every children and adults presenting with an acute undifferentiated febrile illness. no specific therapeutic agent to treat dengue infection is available, so treatment remains supportive care with particular emphasis on careful fluid administration. the early recognition of warning signs, plasma leakage, abnormal bleeding, signs of circulatory collapse, and other serious complications would reduce morbidity/ mortality rates in patients with dengue infection. dengue patient without warning signs may be treated at home with oral hydration and antipyretics with instructions to follow up at out-patient care and return to the hospital immediately if bleeding or warning signs suggestive of severe dengue develop. oral rehydration is indicated to replace losses from vomiting and high fever. acetaminophen, salicylates, nonsteroidal anti-inflammatory drugs (nsaids), and traditional medicines are commonly prescribed to febrile patients that these medications may cause severe bleeding or hepatic injury in dengue patients. development of any warning signs indicates the need for close observation and hospitalization with appropriate use of intravenous fluids in patients with inadequate oral intake or a rapidly increasing hematocrit [35] . monitoring all these patients for the development of warning signs of severity as recommended by who may impose a great burden on healthcare services in limited-resource countries. however, the patients with dengue infection should be hospitalized immediately if any of the followings are observed: severe nausea/ vomiting, restlessness or lethargy, severe hemorrhage (e.g., hematemesis or hematochezia), narrowing of pulse pressure ( 20 mmhg) or hypotension, sudden rise in hematocrit or continuous elevated hematocrit despite the administration of fluid, a platelet count of 20,000/mm 3 ( 20 â 10 9 /l), ast or alt >500 u/ml, oliguria or acute renal failure, liver failure, heart failure, severe hypoxemia, pregnancy, and no opportunity to be followed up in an out-patient setting [115, 136] . attentive clinical monitoring of patients with severe dengue or suspected dhf/dss and intensively supportive treatment are lifesaving and have reduced fatality rates. the critical activities for hospitalized dengue patients are monitoring of abnormal bleeding, circulation, and vascular leakage by serial clinical assessments of hypovolemia/shock and rising of hematocrit to trigger intravenous fluid or blood component transfusion. close monitoring measurements (e.g., vital signs, urine output, and serial hematocrit levels) to assess the severity of plasma leakage and promptly effective intravenous fluid resuscitation with crystalloid to counteract massive plasma leakage are required in critical stage of dhf to reduce morbidity and mortality. patients with dhf need to be monitored closely for signs of shock until at least 24-48 h after defervescence. for patients suffering from plasma leakage with shock (dss), the mainstay of therapy is early and effective replacement of plasma loss. the who recommends immediate volume replacement with ringer's lactate, or physiologically normal saline solution, followed by plasma expander such as fresh frozen plasma or colloid solutions (albumin and dextran) in the event that shock persists [116] . therapeutic responses to colloid and crystalloid solutions from two randomized controlled studies revealed that ringer's lactate performed the least well and that the more severely ill patients identified by a narrow pulse pressure would benefit more from initial resuscitation with colloid solution than with crystalloid solution [117] [118] [119] . in order to assess adequate volume replacement, the rate of intravenous fluid should be adjusted throughout the period of plasma leakage by frequent assessments of vital signs, hematocrit, and urine output and be kept to the minimum required to maintain cardiovascular stability until permeability reverts to a normal level. in cases with persistent shock despite a declining hematocrit after fluid resuscitation with crystalloid or colloid solutions, internal or concealed bleeding should be suspected. transfusion therapy with packed red cell, concentrated platelets, and fresh frozen plasma to correct the bleeding tendency, anemia, coagulopathy, and hypovolemia is still the mainstay of treatment of severe bleeding in dengue patients. the prophylactic blood or platelet transfusions for severe thrombocytopenia may be harmful and should be avoided in uncomplicated cases [120] . the invasive procedures should be minimized to avoid hemorrhagic complications. metabolic acidosis and hyponatremia occur more commonly in dss, so sodium bicarbonate infusion should be considered along with early adequate fluid replacement. medical comorbidities in adult and elderly patients, for example, coronary artery disease, peptic ulcer, hypertension, diabetes mellitus, cirrhosis, or chronic kidney disease, which should be considered for proper management, may contribute to the severity of dengue infection [55, 97, [121] [122] [123] . there is no evidence to support the use of chloroquine, corticosteroid, interferon, immune globulin, desmopressin, or carbazochrome sodium sulfonate (ac-17) for severe dengue infection [124] [125] [126] [127] . with intensive support through the critical period of illness, spontaneous resolution of vasculopathy and circulatory failure usually can be expected within 2-3 days with complete recovery afterward. in the recovery period, the patients usually have more appetite, bradycardia, and convalescent rash and may have fatigue or mood disturbance for several weeks. to reduce burden of dengue, the who has set out specific objectives in global dengue control strategy: estimation of the true burden of dengue by 2015 and a reduction of dengue morbidity and mortality by 2020 by at least 25 and 50%, respectively (using 2010 as the baseline) [128] . it seems clear that implementations of effectively sustainable vector control and effective dengue vaccines are keys to success for this disease control. the who has recommended integrated vector management (ivm), an evidence-based approach which encourages optimal use of resources by examining local in-country evidence. challenges of vector management remain in the development and deployment of vector-control strategies that effectively minimize dengue replication and transmission dengue prevention currently relies on public health and community-based a. aegypti control programs with chemical or biological methods to remove and destroy mosquito-breeding sites [129] . because this ivm approach has not succeeded in most of the asian countries especially dengue-endemic regions, the new tools are needed to prevent and control dengue, including the development of a safe and efficacious dengue vaccine. the potential dengue vaccine is a vaccine consisting of a tetravalent combination of attenuated dengue strains, which simultaneously induce protective and durable immune responses against all four dengue serotypes. recent studies in asia and latin america show that recombinant live-attenuated tetravalent dengue vaccine (cyd-tdv) was safe and moderately efficacious when given three injections at months 0, 6, and 12 to children and adolescents [130, 131] . overall vaccine efficacy of cyd-tdv was estimated to be 60% against virologically confirmed dengue (vcd) infection, with high levels of protection offered against hospitalization (80%) in subjects aged 2-16 years. however, variations of vaccine efficacy against vcd were observed in endemic settings, dengue serotype, and individual's pre-existing dengue antibody [130, 131] . recent pooled analyses of the first 2-3 years of long-term follow-up provided further supportive evidence of efficacy against hospitalized dengue in children 9 years of age or older [132] . this vaccine has recently obtained licensure for use in children 9 years of age or older (9-45 years old) in mexico, the philippines, brazil, thailand, singapore, and several other endemic countries. in view of the high disease burden in endemic countries, this vaccine, despite moderate overall efficacy, could have a substantial effect on public health [133] [134] [135] . the implementation of an efficacious dengue vaccine will shift the burden of disease; the age-related differences in clinical manifestations and prognoses described here indicate the importance of comparing a wide range of ages in future clinical studies of dengue. increase in the number of dengue cases is a major public health problem in many countries in south and southeast asia where a. aegypti and a. albopictus are widespread in both urban and rural areas. several countries in asia have reported an epidemic shift of dengue from mainly affecting children to affecting adolescents and young adults with increased severity. the clinical spectrum of dengue ranges from mild illness (undifferentiated fever and df) to the life-threatening infection (dhf/dss, severe bleeding, and multi-organ failure), which may be fatal. the early recognition of warning signs, plasma leakage, abnormal bleeding, circulatory collapse, and other serious complications would reduce mortality rates in patients with dengue infection. the implementations of effectively integrated vector management and efficacious dengue vaccines are keys to success for disease control in hyperendemic/endemic areas. conflicts of interest both authors have no conflicts of interest to declare. the global distribution and burden of dengue global spread and persistence of dengue the impact of the demographic transition on dengue in thailand: insights from a statistical analysis and mathematical modeling dengue and dengue hemorrhagic fever in the americas dengue hemorrhagic fever epidemiology in thailand: description and forecasting of epidemics an information value based analysis of physical and climatic factors affecting dengue fever and dengue haemorrhagic fever incidence developing 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10.1007/s00068-008-8074-0 sha: doc_id: 5902 cord_uid: 5zuij5i3 necrotizing fasciitis (nf) describes a life threatening soft tissue infection characterized by a rapid spreading infection of the subcutaneous tissue and in particular the fascia. various synonyms for this type of infection are used, often due to the difficult diagnosis. necrotizing fasciits of the extremities is found after simple skin lacerations and often in rural, farming or garden setting environments. many of the infections are found in immunologically healthy people, but persons revealing a compromised wound healing are endangered additionally, e.g., diabetes. in the majority of the microbiological analyses, streptococci alone or a mixture with mainly anaerobic bacteria may be detected. the management of infected extremities requires a rapid diagnosis, dedicated aggressive surgical management as soon as possible, and a wide debridement extending the border of the infected fascia. timely surgical revisions within the first day or days together with antibiotic treatment are the only measures to stop the infection. depending on the status of the patient a hyperbaric oxygenation treatment seems to be useful in order to limit the infection. in fulminated cases early amputations, maximal intensive care treatment of the septic patient are required, where all means are warranted to save the patients life. as a consequence, early clinical diagnoses with thorough surgical debridement of the infected liquid necrotic fascia as well as correct antibiotic treatment are needed. secondary plastic reconstruction of the soft tissue defects will generally be required. necrotizing fasciitis (nf) is an infection accompanied by spreading crepitating edema and blister formation. it was first described by hippocrates in the fifth century as erysipelas [1] . in the eighteenth century a detailed description of nf was provided by british naval physicians and in the early nineteenth century, it was named as gangrenous ulcer, putrid ulcer, malignant ulcer, phagedenic ulcer, phagedena, and phagedena gangrenosa and hospital gangrene [2] . in 1871, during the civil war, j. jones, an american surgeon, mentioned more than 2,000 cases very possible nf. afterwards pfanner described in 1818 in the german medical literature the clinical picture of necrotizing erysipelas and found streptococci related to the disease [3, 4] . in 1924, meleney used the name hemolytic streptococcal gangrene [5] . since then, various names have been given to nf (table 1) . finally, in 1952, wilson used the term necrotizing fasciitis (nf). the anogenital manifestation of nf was first described by fournier in 1883 and is since then called fournier's gangrene [6, 7] . the early onset of this type of soft tissue infection is a discrepancy between pronounced pain and clinical appearance. the aggressive progression induces finally skin and fascia necrosis, coagulopathia and cellulites. five diagnostic criteria can be defined ( table 2 , [8] ). the missing isolation of clostridium perfringens does not necessarily exclude gas gangrene nor prove nf. in patients suffering from nf, more often diabetes, hypertonic blood pressure, arterial occlusive disease, and obesity are found as well as higher percentage of alcohol and drug abuse, immunosuppressant therapy and hivinfections. even a varicella zoster virus infection in combination with the use of nsaids is discussed as risk factor [9] . it is more likely to appear in elderly people, but children also can be affected. in the literature various reasons have been related to the primary focus and include minor skin lesions, bites of insects and wounds after surgical procedures [10] . initial microbiological tests discover in a high amount streptococci (app. 30%), a mixture of different bacteria and less common pseudomonas aeruginosa (app. 5%, table 3 ). due to an increased amount of invasive streptococcal infections in the us during the 1980s, a ''streptococcal septic shock syndrome (stss)'' was defined. this is characterized by a systemic sepsis with multiple organ failure, especially the kidney. today, nf caused by streptococci group a is classified as a subgroup of this infection. also myositis caused by streptococcimostly due to direct inoculation/contaminationshould be differentiated. in such cases rapid lyses of the involved muscle, with edema formation, focal development of a compartment syndrome with consecutive necrosis is observed [11] [12] [13] [14] [15] [16] . therefore necrotizing soft tissue infections (nsti's) characterize various diseases. clinically they can be separated in superficial infections involving cutis/subcutis and deep infections affecting fascia and muscle (nf, myositis). these deep infections are further classified as type i (polymicrobial) and type ii (monomicrobial). bacterial factors play an important role in nsti. in the cases of invasive streptococcal infections, surface proteins (m1, m3) increase adhesion and prevent phagocytosis and exotoxines (a, b, c, streptococci super-antigen) induce the release of cytokines and could bind to t-cell receptors causing further release of tnf-a, il-1 and il-6 ending in a stss [17, 18] . although m-types 1 and 3 are common other types have been isolated in invasive infections; however, a stable genetic change was observed in m-type 1 group a streptococci in the 1980s, resulting in its ability to produce nicotinamide adenine dinucleotide glycohydrolase (nadase) and might be one factor in severe invasive infections [19] . the rapid tissue destruction is a result of toxin-induced vascular occlusion. as the infection progresses more toxins are produced and tissue destroyed. this microvascular occlusion contributes to shock and organ dysfunction. in the case of nf early diagnosis is critical with respect to the survival of the patient. this is primarily a clinical diagnosis with no typical changes in lab diagnostics. serum creatinine phosphokinase (cpk) might be useful in detecting deeper soft-tissue infections. even a mild leukocytosis could be combined with an increasing percentage of immature neutrophils and should be a reason to be suspicious. renal impairment precedes hypotension, as well as hypoalbuminemia and hypocalcemia are early signs. especially in the extremities the bacteria are inoculated mostly through a minor skin lesion. the infection is in the early stage characterized by un-proportional local pain due to fascia necrosis. after this, skin changes are visible with edema and erythema ( figure 1 ). the typical pattern of skin necrosis with or without blisters is found later; however, the necrosis generally spreads rapidly in the proximal direction. the necrosis of the fascia has already much further spread, compared to the changes of the skin ( figure 2 ). crepitating skin can be recognized in about 50% of the patients and is suspicious of a poly-bacterial infection. the typical clinical signs are listed in table 4 . since the time to surgery needs to be diminished no demanding diagnostics like histology or bacterial isolation is possible. to support the diagnosis of nf ultrasound can be used demonstrating fluid between the muscle and subcutaneous tissue because of fascia necrosis. sometimes x-rays can reveal gas formation which could be easily palpated. ct or mri could be of some use, but with little impact on the final decision to proceed to the operative treatment. mri has some impact due to soft tissue and multiplanar imaging and might be helpful in case the source of infection lies deep inside the body. during surgery an excision biopsy could be done, but the typical discoloration of the fascia (yellow to green) and the possibility to manually dissect the fascia (like chewing gum) will assure the diagnosis. in contrast to myositis caused by streptococci the muscles appear normal and not as necrotic with a discoloration, brown to grey, comparable to loam. stss is more often found in association with pharyngitis, or small lesions of the skin or mucosa (scratches, insect bites). this syndrome appears in normally healthy people of all ages, in children more often seen following chickenpox infection [16] . isolation of streptococci group a is typical -also in normally sterile body compartments -and of course the signs of a systemic shock. however, 50% of the stss cases are accompanied by nf. the typical serious soft tissue infections are listed in table 5 . the relevance of a surgical management could be proved by kaiser and cerra. a reduced surgical treatment was followed by a significant increase of death rate [20] . there is no space for incision and drainage or limited evacuation of the abscess. the only surgical option is the radical surgical excision of the infected subcutaneous tissue in particular including the grey pale fascia. in these cases, the fascia is grey, has lost all its strength and can more or less be pealed off. it is essential to resect the infected fascia and to debride into healthy tissue. furthermore, it is essential to follow up by surgical re-interventions after a short time -even again on the same day in fulminant cases or at least the subsequent day. in general this regime is followed a few days until the spreading in the proximal direction has been stopped. an amputation in the extremities is not the primary treatment, but in cases where the whole tissue is necrotic and most muscles involved, this might be the only option to stop further spreading and systemic sepsis with multiple organ failure. these amputations have to be performed as open amputations, again requiring second look operation and secondary closure. after primary intensive care and control of the infection and sepsis (mostly after 1 week) reconstructive procedures are initialized reaching from secondary wound closure and skin grafting to flap coverage saving viable tissue and restore function. histology histology of the excised tissue reveals infiltration of fascia by polymorphic nuclear cells, with peri-vascular focus. sometimes bacteria are detectable. later, a co-liquation necrosis of the fascia is visible, involving subcutaneous tissue and skin. the tissues cannot be differentiated anymore and muscle tissue is involved as well. demanding a radical debridement, therefore the resulting wound areas are extensive in most cases and therefore the increased fluid turnover already justifies an intense care treatment. an adjusted antibiotic regime is mandatory. in undefined cases gram-negative, gram positive and anaerobic bacteria must be addressed. mono-therapy includes imipenem-cilastatin, meropenem, ertapenem, piperacillin/tazobactam and tigecycline. a combination-therapy adds vancomycin, linezolid or daptomycin to a carbapenem or b-lactam/ b-lactamase inhibitor combination, if methicilin-resistant staphylococci are possible. another combination therapy includes penicillin, clindamycin and fluoroquinolone or aminogycoside to cover gram-negative bacteria. in a case of streptococcal infection clindamycin should be included into the medication, since it has been shown to inhibit the toxin production (m-protein and exotoxin) in severe cases [21] . especially streptolycin o (slo) induces changes to the leukocytes. it is specific to phenoloxidase important in the mechanism of host defense and much reduced in nf-cases due to streptococci infection. this significant immunosuppressive effect is accompanied by the effect that phenoloxidase catalyses the transformation of tyrosine to dehydroxy-phenylalanine necessary to produce catecholamine, one reason a patient with nf might need catecholamine substitution. additionally, some immunotherapies (e.g., immunoglobulins) are also suggested by some authors [13] . the mechanism is believed to be related to the neutralization of superantigen activity and reduction of tnf-a and il-6. intensive care therapy various efforts have been made to categorize patients with respect to the risk of mortality. negative parameters are age above 50 years, wbc > 40,000 cells/ mm 3 , hematocrit > 50%, hr > 100, temperature < 37°c and creatinine > 15 mg/dl [22] . if the patient develops a septic shock or stss an acute respiratory distress syndrome (ards) is also very likely (app. 50%) and needs mostly intubation and mechanical ventilation to achieve adequate oxygen supply. every patient with signs of sepsis or impaired immune response should have intensive care treatment, since organ failure is very common in the time course of nf. the patients are at an extremely high risk to run into systemic sepsis with a poor prognosis. good oxygenation, cardiac output and control of homeostasis are the primary goals in treating a systemic sepsis and septic shock according to the current guidelines [23] . these guidelines have to be included stepwise in the treatment of sepsis due to necrotizing fasciitis. additional treatment options to enhance systemic toxin and mediator reduction have been discussed, such as continuous hemofiltration [24] . due to the variation and limited number of patients in single centers, this approach has been only applied in isolated cases [25] . besides the basic treatment including intensive care medicine and surgical debridement numerous adjuvant therapies have been recommended with respect to the systemic management of these infections as well as possibilities for local wound treatment. hyperbaric oxygenation (hbo) necrotizing infections are considered to be one of the primary indications for hbo as well as decompression disease, gas embolism, co-and smoke intoxication, anaerobic infections (clostridia infections) and radionecrosis [26] . hbo-therapy is able to increase blood oxygen content by 25% and thereby tissue oxygenation tenfold [27] . other effects related to this treatment are vasoconstriction, reduced leukocyte sequestration, lipid peroxidation, free radical scavenging and reduction of tissue edema resulting in an increased tissue perfusion/microcirculation [28] [29] [30] . another important effect, thought to be helpful in treating nf by improving host defense, is the activation of leukocytes. also reparative processes might be stimulated due to fibroblast migration, proliferation and collagen synthesis [22, 31, 32] . these effects might be very helpful to support healing/granulation tissue formation of these mostly difficult wounds [33] . various treatment regimes are recommended to be followed-up; however the most intense is that in accordance with crush injury with three treatments within the first 48 hours (2-2.5 ata, 1-2 h o 2breathing), followed by two treatments the next 48 h and finally 48 h once a day. since the use of hbotreatment is connected with high medical and technical expenditure, especially if the patient is critically ill due to sepsis and needs breathing support, estimation is necessary, also the literature is controversial about the effects on morbidity and mortality rates [12, 34, 35] . however, this therapy needs to be considered focusing an increasing network and therapeutic standard in hbo-treatment. another possibility to increase oxygen in the body is an intravenous application (regelsberger's intravenous oxygen therapy). however the effect needs to be proved for nf, also with respect to an increase of granulocytes [36] . therefore the major focus in nf should be related to intensive care management and the vital surgical therapy with no delay. after primary surgical treatment in most cases a topical wound treatment is used. various substances according to the isolated bacteria can be chosen. mostly the following antiseptic substances are recommended: polyhexamid, povidon-iodine, silver sulfadiazine, actate mafenid. also wine vinegar and citric acid have been applied, especially to modify the wound environment and lower the ph in cases of pseudomonas infections. vacuum sealing is a widely used approach to condition destroyed soft tissue in order to allow granulation and a safe secondary reconstruction [37] . it is extremely useful in cleaned wounds of the extremities and the abdomen and reduces surgical interventions to intervals between 2 and 5 days. in acute stages of necrotizing fasciitis, however, vacuum sealing is not indicated during the early states of purulent infections and infected tissue necrosis. but subsequently, when infected and destroyed soft tissues have been removed, vacuum sealing seems to be very useful for conditioning the defects and occasionally allowing limited tissue closure using skin graft; if not, a complex reconstruction is required [38] . the clinical cases with nf and primary focus on the extremities are listed in tables 6 and 7 . most of these have had minor injuries with rapid spreading of the infection. one exemplary case is described to illustrate the significance of early operative treatment and excision of the devastated tissue. a 52-year-old gentleman was visiting a garden market. by lifting various flowers, he experienced a minor scratch, while handling various flowers, on the ulna side at the middle phalanx of the left ring finger. this happened on a thursday afternoon and since there was no obvious skin lesion he did not care about it. during the next night and day he experienced increasing swelling and pain of the whole hand -not only the finger. on saturday morning he consulted a local surgeon, who admitted him immediately to the hospital. the patient had already clear signs of local infection (figure 3 ), systemic sepsis with reduced blood pressure, tachycardia and fever up to 40°c. immediately, he was brought to the surgery room where a primary excision of the possibly involved tissue was done, up to the elbow. however, the patient did not recover very well afterwards in the intensive care unit. finally he was brought back to surgery -on the same day -and a radical debridement was performed, including the amputation of the ring finger and excision of all obvious involved fascias (figure 4 ). after this, four more wound care procedures were done in the operating theatre. finally after 6 days the wounds could be closed with a pedicle groin flap and skin graft. three more reconstructive procedures were followed including flap division, and two more correction with a final skin graft ( figures 5 and 6 ). during the treatment period at the intensive care unit also hbo-therapy was followed daily starting at day 1 for 5 days. conclusion necrotizing fasciitis (nf) is a life threatening soft tissue infection, characterized by foudroyant spreading necrosis of the involved fascias. since a high variety of bacteria can be isolated the two different types of nf can be differentiated: type 1 characterized by a poly-bacterial infection of aerobic and anaerobic bacteria and type 2 with streptococci group a as source of infection [39] . the type 2 infections are less common, but are more often found in the case of the involvement of extremities. every fascia in the body can be destroyed by this disease. early diagnosis is critical to the survival of the patient and must rely on the clinical picture. when there is doubt, there should be no delay in performing surgery with radical debridement. the suggested treatment strategy with adequate early surgical and intensive care medicine could help reduce the up to 70% lethality rate, as stated in some publications, to less than 10% [40] [41] [42] . infections of the extremities are less likely lethal, whereas an intra-abdominal occurrence leads mostly to the death of the patient. higher age, diabetes, arterial occlusive disease, immunosuppressant status and the onset of nf due to iatrogenic infections is linked to a much worse prognosis. nf is an infection still observed very seldom; however, some data indicate an increase of this type of infection in the last decade, therefore clinicians should be aware and alert in cases of a significant soft tissue infection to rule out nf. hippokrates: 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lower extremity fractures with soft tissue defects bacteriology of necrotizing fasciitis improved results from a standardized approach in treating patients with necrotizing fasciitis necrotising fasciitis due to group a streptococci in western norway: incidence and clinical features necrotizing fasciitis -a preventable disaster key: cord-003387-82573enr authors: nam, gyu-hwi; mishra, anshuman; gim, jeong-an; lee, hee-eun; jo, ara; yoon, dahye; kim, ahran; kim, woo-jin; ahn, kung; kim, do-hyung; kim, suhkmann; cha, hee-jae; choi, yung hyun; park, chan-il; kim, heui-soo title: gene expression profiles alteration after infection of virus, bacteria, and parasite in the olive flounder (paralichthys olivaceus) date: 2018-12-24 journal: sci rep doi: 10.1038/s41598-018-36342-y sha: doc_id: 3387 cord_uid: 82573enr olive flounder (paralichthys olivaceus) is one of economically valuable fish species in the east asia. in comparison with its economic importance, available genomic information of the olive flounder is very limited. the mass mortality caused by variety of pathogens (virus, bacteria and parasites) is main problem in aquaculture industry, including in olive flounder culture. in this study, we carried out transcriptome analysis using the olive flounder gill tissues after infection of three types of pathogens (virus; viral hemorrhagic septicemia virus, bacteria; streptococcus parauberis, and parasite; miamiensis avidus), respectively. as a result, we identified total 12,415 differentially expressed genes (deg) from viral infection, 1,754 from bacterial infection, and 795 from parasite infection, respectively. to investigate the effects of pathogenic infection on immune response, we analyzed gene ontology (go) enrichment analysis with degs and sorted immune-related go terms per three pathogen groups. especially, we verified various go terms, and genes in these terms showed down-regulated expression pattern. in addition, we identified 67 common genes (10 up-regulated and 57 down-regulated) present in three pathogen infection groups. our goals are to provide plenty of genomic knowledge about olive flounder transcripts for further research and report genes, which were changed in their expression after specific pathogen infection. viruses, viral hemorrhagic septicemia virus (vhsv) is affiliated to novirhabdovirus genus, which is a member of the rhabdoviridae family 4 . the six gene were contained in the vhsv genome of about 11 k bases and each of them coded nucleoprotein (n), phosphoprotein (p), matrix protein (m), glycoprotein (g), nonstructural viral protein (nv), and rna polymerase (l) in the following order 3'-n-p-m-g-nv-l-5' 4 . infection of vhsv results in contagious viral hemorrhagic septicemia (vhs) in diverse fish species regardless of their inhabitation; seawater or freshwater 5 . in east asia, a lot of infection cases into olive flounder have been reported steadily, since vhsv was detected in middle of 1990s [6] [7] [8] [9] . a variety of scuticociliates have been reported as cause of scuticociliatosis in marine species including turbot, guppy, and southern bluefin tuna [10] [11] [12] . in olive flounder, disease has been reported to be causing from various scuticociliates; uronema marinum, pseudocohnilembus persalinus, philasterides dicentrarchi, miamiensis avidus [13] [14] [15] [16] . interestingly, judging from infection experiments using various scuticociliates plus identification outcome of 8 isolates acquired from olive flounders with symptom of ulcers and haemorrhages, miamiensis avidus was suggested as the major aetiologic agent of scuticociliatosis because of high pathogenicity and mortality rate compared with other scuticociliates 14, 17 . infection of bacteria could sustain serious damage to fish. streptococcosis is known to be caused by a variety of streptococcic species; streptococcus parauberis, streptococcus iniae, streptococcus difficilis, lactococcus garvieae, lactococcus piscium, vagococcus salmoninarum, and carnobacterium piscicola, and has become major nuisance in olive flounder farms [18] [19] [20] [21] . in particular, streptococcus iniae, lactococcus garvieae, and streptococcus parauberis have been introduced to be related with streptococcosis in olive flounder [19] [20] [21] [22] [23] . the main issue of aquaculture industry is to reduce economic loss by preventing mortality of fish from various pathogens. a large number of immunologic studies have been proceeded about various immune-related gens against pathogen infection 3, [24] [25] [26] [27] . a huge quantity of genomic information from next generation sequencing (ngs) technique has been gradually increasing for the last few years, indicating that researchers could approach more comprehensive understanding view about genome of organisms than when they research a single gene level. with development of wide-sized analysis methods, it is not difficult to figure out change of gene expression level after any chemical treatment or environmental change. recently, studies to identify large-scale genes were conducted in the olive flounder genome for researches about vaccine, gonadal development, and sex determination [28] [29] [30] . in particular, characterizing of immune-related genes was reported in olive flounder spleen tissue 31 . a lot of studies reported earlier were focused on gene expression analysis of single pathogen and specifically defined the expression pattern of limited genes [32] [33] [34] [35] [36] . further, infection by two or more pathogens were reported in the olive flounder genome 37, 38 . in order to solve these problem, we need plentiful genomic information to respond rapidly to multiple infection of pathogens. however, researches, which were comprehensively analysed about change of gene expression pattern by different type of pathogens, have not been reported in the olive flounder genome, so far. in this research, we identified differentially expressed genes (degs) by transcriptome analysis and conducted gene ontology (go) analysis with genes identified. then, we tried to find important genes which showed consistently meaningful expression change in the results of three infection experiments. as a result, we determined 10 up-regulated genes and 57 down-regulated genes in common after infection of three pathogens. we aimed to provide essential genome information which is related with pathogen infection and explore the various consequences related to differential infections and find out the common strategies against specific candidates involved in disease progression in natural habitat of aquaculture. statistical summary of transcriptome analysis. to profile gene expression after infection of three pathogens (vhsv, streptococcus parauberis, and miamiensis avidus), transcriptome analysis was conducted using gill tissues of olive flounders, respectively. we prepared twelve olive flounders (three un-infected individuals as control, three virus-infected, three bacteria-infected, and three parasite-infected individuals) to raise confidence. to gain the sufficient number of transcripts, twelve independent rna samples acquired from normal and pathogen-infected olive flounder gill tissues were employed for construction of cdna library. then, these cdna libraries were sequenced using illumina hiseq2500, generating the numbers of approximately 78.4 million, 65.2 million, and 45.7 million raw reads from three control samples, 44.0 million, 56.9 million, and 62.0 million raw reads from bacteria-infected samples, 41.2 million, 62.4 million, and 40.0 million raw reads from virus-infected samples, 39.6 million, 41.6 million, and 53.1 million raw reads from parasite-infected samples, respectively (table 1) . after trimming of low-quality reads and adaptor sequences, the number of clean reads acquired from control samples were average 62.3 million reads from control samples, average 53.5 million reads from bacteria-infected samples, average 48.0 million reads from virus-infected samples, and average 44.3 million reads from parasite-infected samples, respectively. then, we checked gene coverage whether the reads that we acquired are sufficient for quantitative gene expression analysis ( supplementary fig. 1 ). the clean reads were assembled into 120,880 transcript sequences acquired from transcriptome analysis, we identified total 40,100 genes involving novel 19,560 genes from transcript sequences using interproscan database and non-redundant protein database in the ncbi ( table 2 ). to figure out the effects of external pathogen for gene expression, we sorted out genes which showed expressional change after pathogens infection having p-value of <0.05 when compared with control sample. as shown in table 3 and fig. 1 , the largest numbers of gene expression change were shown in vhsv infection group; total 12,415 degs were identified from transcriptome analysis. we showed information of degs derived from viral infection in supplementary to explore the functional enrichment of these degs, we performed go enrichment analysis using david tool 39 table 2 ). prediction following viral infection, we identified the degs with p-value of <0.05 after infection with bacteria (streptococcus parauberis) and parasite (miamiensis avidus) ( table 3 and table 2 ). miamiensis avidus affected gene expression pattern in olive flounder genome and selected genes which showed expression change (supplementary table 2 ). we identified 795 degs caused by infection of miamiensis avidus; description samples u-i 1 u-i 2 u-i 3 b-i 1 b-i 2 b-i 3 v-i 1 v-i 2 v-i 3 p-i 1 p-i 2 p-i 3 number distributional pattern of total degs acquired from transcriptome analysis. after comparison of gene expression level among twelve transcriptome analysis, we identified degs after pathogen infection. then, we focused on selection of genes showing expression change pattern after three types of pathogen infection in common. as a result, we summarized 10 up-regulated genes and 57 down-regulated genes, respectively (fig. 2) . we analyzed these 67 degs to identify their gene symbol correctly using their sequences in non-redundant protein database of the ncbi database, and 37 degs were annotated (table 4) . we showed the rest of 30 unannotated degs in supplementary table 3 . with development of sequencing technique, numerous genomic researches have been reported to understand infection results by virus 28, 31, 40 , bactria 26, 41, 42 , and parasite 43 in the olive flounder genome. a fundamental way to overcome disease outbreak from external pathogens is to approach from their genome level. it is essential to expand quantity of genomic information in pursuance of biological research about any target. investigation of overall gene expression change after pathogen infection would provide clues of cause of biological damage. gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed. phylogenetic diversity of pathogens (virus, bacteria and parasite) is also responsible for differential expression of genes in diseases. each individual pathogen causes disease in a different way, which makes it challenging to understand the basic biology of infection. in this study, we understood the relation between three types of pathogen infection and differential gene expression in the olive flounder genome through transcriptome analysis, respectively. the diverse pathogens used in this study, carry specific antigenic variations, which refers to the specific mechanism by which an infectious agent infect the fish and progress the disease. transcriptome analysis help us to understand the progression of disease in fish through pathogen infection based on diversity of pathogen (virus, bacteria and parasite). this study shows differentially expressed genes were up-and down-regulated at different extend in fish tissue. interestingly, virus and bacteria have more down-regulated genes while parasite have more up-regulated genes. this data signifies the fact that fish immune system interacts with bacteria and virus with the same strategy, while with parasite different due to difference in mode of infections between them. for efficient prevention against pathogen, it is important to understand which genes were activated/repressed after pathogen infection because their expression change means variation of metabolic system in body. in this study, we identified total number of 12,415 in vhsv infection group, 1,754 in streptococcus parauberis infection group, and 795 degs from miamiensis avidus infection group, respectively (table 3 ). given the difference in the number of degs among three pathogen groups, these results seemed that virus had the most impact on gene expression mechanism in the olive flounder genome among three pathogens. interestingly, 11,051 degs (89% of all degs) showed down-regulated pattern after viral infection. this phenomenon that global gene expression was decreased by viral infection must cause pathogenic disease by affecting immune-related gene expression level, finally leads to death. this view was supported by our findings (supplementary table 2 ), which showed expression decrease pattern of all genes in the immune-related go terms. especially, go terms in viral infection group showed that all genes tend to be down-regulated after pathogen infection, indicating loss of resistance against pathogens by down-regulating the expression of immune-related genes. like this situation, functional information of genes acquired from go enrichment could help researcher to figure out critical biological pathway against any external factor. the immune mechanism in fishes is composed of a set of cellular and humoral system and divided into innate (inherit), and adaptive (acquired) substances. the understanding of fish immune system structure and function is essential for the development of new technologies and products to improve productivity. the transcriptome analysis bring exposure to basic difference in expression profile of all pathogens in the host. these differences were due to nature of parasite, their mode of infection, antigenic variations and many other factors. additionally, along with all above differences, disease progressed in host due to external surface variation of pathogens (viral, parasite and bacterial) and their appropriate recognition by host immune systems for making the basis to initiate microbial clearance 44, 45 . disease research requires the knowledge of important key factors like method of avoiding host immune surveillance, antigenic variations, subversion of immune responses through phagocyte and inhibition of cytokines and chemokines in common with pathogen infections (viral, bacterial and parasite) 44, 45 . on the other hand, the disease progression was different in accordance to type of pathogens. in case of viral infection, understanding of complement inhibition and blockade of cellular immunity is the most important, while parasite and bacterial infections required knowledge and research of innate pathway and acquired immunity 44, 45 . our analysis indicates the complexity and difference of expression profile could be due to all the above reasons. in addition, important basis of fish vaccine is depending on innate and adaptive immunity 46 . there were many vaccine types which depend upon antigens, live microorganisms or specific dna segment of pathogens or polyvalent vaccines. all above vaccines required complete knowledge of pathogenicity and deep research of efficacy 47 . our study clearly indicates about various immune and antigenic genes which can be chosen for pathways analysis and use for therapeutic agents or as some vaccine candidates (supplementary table 2) . despite of their different infection pathway, we wondered common degs which were affected from infection. as shown in fig. 2 , total 67 degs (10 up-regulation and 57 down-regulation) were identified in common in three pathogens, and 1 of 10 up-regulated and 36 of 57 degs were annotated by genomic database (table 4 ; , 408 genes (bacteria), and 561 genes (parasites). these genes were specific for each pathogen so can be used as candidate genes for vaccination or therapeutic agents. in case of the down-regulation of 10168 gene (virus), 177 gene (bacteria) and 60 gene (parasites) in infection of fish, these genes were specific for specific pathogens so can be used as a diagnosis marker for specific pathogens. c-x9-c motif containing 4, col1a1; collagen type i alpha 1 chain, col1a2; collagen type i alpha 2 chain, slc14a2; solute carrier family 14 member 2) which showed highest expression change (down regulation) with fold change (log 2 ) of ' <â��2.0' on at least two infection groups. in this study, we investigated all the above candidate genes and found their role in disease progression. we have listed these genes and their role in below headings. anpep, called as gene aminopeptidase n (apn), is metallopeptidase that exerts strong influence on various immune response mechanisms. for example, apn has been known to cause decomposition of cytokines and peptides used by neurons [48] [49] [50] , and acts as receptor for viruses 51, 52 . in addition, relation between expression level of apn and stimulated t-cell was reported 53 . recently, it was suggested that apn controlled the balance of innate immune and adaptive immune by regulating tlr4 signal transduction pathway in myeloid cells 54 . bglap, also known as osteocalcin, is a noncollagenous protein, mainly found in bone, which needs vitamin k for its synthesis. this protein was thought to play a role in calcium ion homeostasis and used as biological marker for bone formation 55 . in addition, it concerns in endocrine regulation, especially in digestive system, by stimulating release of insulin hormone from î²-cell of the pancreas and adiponectin hormone from fat cells, respectively 56 . as well as these function, it has been reported to take a role in promotion of energy availability and sexual maturation of male by stimulation of testosterone biosynthesis 57, 58 . cmc4, called as mtcp1, has been mainly reported to be related in various diseases. it was reported that mtcp1 gene affected t-cell homeostasis prior to process of leukemogenesis in transgenic mice 59 . although the function of this gene has been entirely discovered, regulation error of mtcp1 gene affected on cell survival and cell growth 60 . besides, this gene was known to be related in the pathogenesis of a subset of t-cell lymphoproliferative diseases 61, 62 . col1a1 and col1a2 encodes the pro-î±1 chain and the pro-î±2 chain protein, respectively. the type i collagen, which is comprised by two pro-î±1 chains and one pro-î±2 chain, plays a role in reinforcement and support in most of all connective tissues such as bone, cartilage, skin, and tendon, and offers those tissues rigidity and elasticity 63, 64 . this protein was reported to stimulate expression of pro-inflammatory cytokines and professional phagocytes in teleost fish gilthead seabream 65, 66 . in addition, it has been reported that receptor-mediated interaction which is formed in between cells and collagen molecule might affect in wound healing, inflammatory, and immune response by activating various factors such as cytokines, growth factor, and matrix metalloprotease 63, [66] [67] [68] [69] [70] . slc14a2, also is known urea transporter 2 (hut2), is important gene involved in urea transport and play role in physiology. in mammals, two types of urea transporter (slc14a1 and slc14a2) has been reported 71 and were regulated by vasopressin hormone 72 . the kidney uses urea to maintain the appropriate concentration and volume of blood. without control of these proteins, organism would result in extreme damage in urinary system. besides, a previous study has reported that genetic variation including nucleotide change is known to significantly influence blood pressure (bp) and metabolism syndrome 73, 74 . as shown in supplementary table 2 , immune-related degs were revealed as results of three pathogens infection. infection of pathogens caused activation of immune system to respond to invasion of harmful external elements, indicated that change of expression level of immune-related genes. the down-regulation of gene could sequentially influence on expression of various molecules positioned in down-streams in metabolic pathway. in this view, cd13, which is one of down-regulated genes by infection of three pathogens on common, was reported to inactivate interleukin 8 49 . representative function of this cytokine is to induce migration of neutrophils and granulocytes toward infection site. in addition, absence of cd13 considerably improves cross-presentation of soluble antigen via regulation of receptor-mediated uptake 75 . thus, decrease of cd13 expression consequentially might activate immune response in the olive flounder. mtcp1 gene induced malignant t-cell transformation 59 and was related in the leukemogenic process of mature t-cell proliferation 61 . this gene was thought to maintain balance of immune response by t-cell. the innate immune system mediates the initial inflammatory response by pathogen infection or injury. for rapid response against external pathogens, infected cells secrete various cytokines to induce effector cells and complements. the type i collagen, which is comprised by proteins coded from col1a1 and col1a2, was involved in the expression of pro-inflammatory cytokines in the innate immune system. however, two genes (col1a1 and col1a2) showed decreasing expression pattern after infection in our study. given sampling period (7 days from infection) of olive flounders for this study, it might be explained that the adaptive immune response was activated in the olive flounder genome. it is hard to understand comprehensively about immune system of fish genome. however, our results were expected to contribute for further study by extend of genomic knowledge in the olive flounder. in conclusion, this study is helpful in understanding infection of the diversified pathogens (antigenic variation) and their role in disease progression in the olive flounder. the differentially expressed genes identified from transcriptome analysis using three types of pathogens could be useful to study the basic diagnosis and therapeutic mechanisms, and offer opportunities for designing the appropriate vaccines or drug targets for pathogen specific candidate genes. because of lack of genomic information or using one external infection factor, previous studies have been limited to understand global expression pattern of whole genes in the olive flounder genome. we hope that this research would contribute to achieve great outcome in various biological field. ethical statement. all experiments with the olive flounders in this study were carried out in accordance with the guidelines and regulation approved by ethical committee of pukyong national university. preparation of olive flounder gill tissues. gill tissues from twelve olive flounder (bw = ~50 g, n = 3/ group) including healthy and infected fish with each pathogen were used for this study. briefly, healthy fish (non-challenged), sampled fish at 7 days post challenge (dpc) with s. parauberis at 5.06 ã� 10 3 cfu/fish in 1/3 seawater of 21 â°c, sampled fish at 7 dpc with vhsv at 106 pfu/fish in 1/3 seawater of 19 construction of cdna libraries for transcriptome analysis. building of transcriptome libraries were conducted by illumina's truseq rna protocol, and 1-2 î¼g of total rna were used in each samples. ampure xp beads (beckman coulter) and ambion fragmentation reagents kit (ambion, austin, tx) were used for extraction of poly(a)+ rna and their fragment, respectively. as the following steps, cdna synthesis, end-repair, a-base addition, and ligation of the illumina indexed adapters were carried out according to illumina's protocol. the size-selected 250-300 bp cdna fragments were loaded on a 3% nusieve 3:1 (lonza) agarose gel for libraries. the cdna fragments were recovered using qiaex ii gel extraction reagents (qiagen), and amplified using phusion dna polymerase (new england biolabs) for 14 pcr cycles. the amplified libraries were purified by ampure xp beads, their concentration and product sizes were assessed on an agilent 2100 bioanalyzer. sequencing of paired-end libraries were conducted with the illumina hiseq2500, (2 ã� 100 nucleotide read length). transcriptome analysis and differential gene expression. transcriptome analysis were carried out with the rnaseq tuxedo protocol. mapping of sequences were conducted against the olive flounder draft genome (submitted at present) using tophat v2.0.9 with default options for paired-end sequences. transcripts expression were estimated using the cufflinks program v2.1.1. total sequencing reads were subjected to preprocessing as follows: adapter trimming was performed using cutadapt with default parameters, and quality trimming (q30) was performed using fastqc with default parameters. processed reads were mapped to the olive flounder draft genome (submitted at present) using tophat and cufflink with default parameters 76 . the differential analysis was performed using cuffdiff 76 using default parameters. further, the fpkm values from cuffdiff were normalized and quantitated using r package tag count comparison (tcc) 77 to determine statistical significance (e.g., p values) and differential expression (e.g., fold changes.). through these statistics analysis, we sorted degs having p < 0.05 and showed them as results. gene ontology analysis. deg set for go analysis was acquired from transcriptome analysis. degs were annotated from interproscan database and non-redundant protein database in the ncbi. david and uniprot tool were used for 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asian population cd13 regulates dendritic cell cross-presentation and t cell responses by inhibiting receptor-mediated antigen uptake differential gene and transcript expression analysis of rna-seq experiments with tophat and cufflinks tcc: an r package for comparing tag count data with robust normalization strategies this research was a part of the project titled "omics based on fishery disease control technology development and industrialization (20150242), " funded by the ministry of oceans and fisheries, korea. g.n. wrote the main manuscript text, a.m. and j.g. supported arrangement of contents in manuscript, h.l. and a.j. supported to complete fig. 2 , d.y. and a.k. prepared fish tissue samples, w.k. provided olive flounder genome reference sequence, k.a. provided bioinformatic advice for this study, d.k., s.k., h.c., y.c. and c.p. reviewed the manuscript, h.k. supervised entire flow of the manuscript and reviewed contents. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36342-y.competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-001397-nrq4ncdf authors: mlera, luwanika; melik, wessam; bloom, marshall e. title: the role of viral persistence in flavivirus biology date: 2014-05-12 journal: pathogens and disease doi: 10.1111/2049-632x.12178 sha: doc_id: 1397 cord_uid: nrq4ncdf in nature, vector-borne flaviviruses are persistently cycled between either the tick or mosquito vector and small mammals such as rodents, skunks, and swine. these viruses account for considerable human morbidity and mortality worldwide. increasing and substantial evidence of viral persistence in humans, which includes the isolation of rna by rt-pcr and infectious virus by culture, continues to be reported. viral persistence can also be established in vitro in various human, animal, arachnid and insect cell lines in culture. although some research has focused on the potential roles of defective virus particles, evasion of the immune response through the manipulation of autophagy and/or apoptosis, the precise mechanism of flavivirus persistence is still not well understood. we propose additional research for further understanding of how viral persistence is established in different systems. avenues for additional studies include determining if the multifunctional flavivirus protein ns5 has a role in viral persistence, the development of relevant animal models of viral persistence as well as investigating the host responses that allow vector borne flavivirus replication without detrimental effects on infected cells. such studies might shed more light on the viral-host relationships, and could be used to unravel the mechanisms for establishment of persistence. defining mechanisms of viral persistence will be critical for understanding vector-borne flavivirus infections. these viral infections account for considerable human morbidity and mortality worldwide. furthermore, incidence is increasing and infections are being appreciated in previously nonendemic geographic locations. prominent vector-borne flaviviruses (vbfvs) associated with significant human infections include both tick-borne and mosquito-borne agents. the tick-borne flaviviruses (tbfvs) are exemplified by the tick-borne encephalitis virus (tbev) sero-complex group, omsk hemorrhagic fever virus, kyasanur forest disease virus, alkhurma virus, powassan virus (powv), and deer tick virus (dtv), the latter two occurring in the united states (holbrook et al., 2005; brackney et al., 2008b; ebel, 2010) . the mosquito-borne flaviviruses (mbfvs) are perhaps better known and include yellow fever virus (yfv), west nile virus (wnv), japanese encephalitis virus (jev), and dengue virus (denv) serotypes 1-4. infections with all of these viruses can lead to severe disease, prolonged debilitating neurological sequelae, hemorrhagic fever, and/ or death in some cases (solomon et al., 1998; glass et al., 2002; haglund & gunther, 2003; madden, 2003; van gerpen, 2003; carson et al., 2006) . viral persistence is a hallmark of the ecology of vbfvs. both tbfvs and mbfvs are cycled between arthropod and vertebrate hosts (figs 1 and 2) , and in many cases, they are maintained without deleterious effects on the hosts. in nature, tbfvs, such as powv and tbev, alternately infect small vertebrates such as rodents, hares, some carnivores, and a range of hard-bodied (ixodid) ticks, although recent evidence suggests that the soft-bodied ornithodoros (argasid) ticks can also support tbfv (rajagopalan et al., 1969; charrel et al., 2007) . similarly, mbfvs, such as wnv and jev, primarily alternate in nature between small mammals, birds, and mosquitoes ( fig. 1 ). in addition, there is evidence that mbfv and tbfv persistence also occurs in humans, and persistence in cell culture is well documented (poidinger et al., 1991; lancaster et al., 1998; bugrysheva et al., 2001; farfan-ale et al., 2009; murray et al., 2010) . although the hosts for the vbfvs are highly varied, the genomic and structural organization of the viruses themselves is remarkably similar. all flaviviruses are spherical, enveloped particles that contain a genome of (+) ssrna measuring approximately 11 kb. the genome also functions as mrna and encodes a single polyprotein that is cleaved into 10 proteins (table 1) . reasonable progress has been made toward understanding how these viruses replicate (chambers et al., 1990; mandl, 2005; miorin et al., 2008; tuplin et al., 2011; pierson & diamond, 2012; brinton, 2013; pierson & kielian, 2013) . we present a simplified overview of the general aspects of a vbfv replication cycle in fig. 3 . the individual proteins are three structural proteins: capsid (c), precursor membrane/memfig. 1 flavivirus maintenance and transmission cycle in ticks and vertebrate hosts. ticks are crucial for viral persistence as they remain infected once they acquire viral infection. infected ticks are capable of transmitting tbfvs to other ticks when they feed in close proximity on the same animal, as well as the different stages of the tick life cycle. tbfvs persist also in a cycle between small mammals (e.g. rodents) and the ticks that feed on them. large mammals and humans tend to be incidental, dead-end hosts. fig. 2 a representation of a mosquito-borne flavivirus amplification and transmission cycle. wnv is cycled between the mosquito and avian hosts that play an amplification and maintenance role. swine are important amplifying hosts for jev, and mosquitoes that acquire blood meals on infected pigs can become infected and transmit the virus. similar to tbfvs, mbfv infection in humans and large animals, such as horses, is accidental. the flavivirus proteins, untranslated regions (utrs), and their known functions the 5 0 utr contains conserved rna stem loops (sl), cis-elements located upstream and downstream of the aug region (uar and dar, respectively), and complementary sequence (cs) sponsoring cyclization of the genome by interacting with the 3 0 utr to form a 'panhandle' (alvarez et al., 2005; villordo & gamarnik, 2009; friebe & harris, 2010; friebe et al., 2011) . the 'panhandle' structure is essential for certain steps in the flavivirus replication cycle such as replication/translation and viral assembly (gritsun & gould, 2007b ). sl1-2 or (sl-a and sl-b) is located within the 5 0 utr, whereas the sl3-4 is extended and situated in the n-terminal of the open reading frame orf (alvarez et al., 2005; lodeiro et al., 2009; villordo & gamarnik, 2009; friebe & harris, 2010; friebe et al., 2011) . the sl1 structure is essential for viral replication and acts as a promoter which is targeted initially by ns5 and then delivered to the 3 0 end via cyclization (filomatori et al., 2006; zhang et al., 2008; lodeiro et al., 2009) although there is low nucleotide conservation between flaviviruses and different cs homology (hahn et al., 1987) , the 5 0 utrs of tbfvs share the same genomic organization as the mbfvs (kofler et al., 2006) structural proteins capsid (c) 11 kda, 114 aa cytosol/nucleus the capsid protein is a dimeric alpha-helical (jones et al., 2003) protein and assembles into an icosahedral structure, measuring 30 nm in diameter, which initiates encapsidation of the associated genomic rna in virus-induced membrane invaginations of the er (welsch et al., 2009 ). the c protein is also a pro-apoptotic factor in various cell lines (yang et al., 2002 (yang et al., , 2008 oh et al., 2006; netsawang et al., 2010; morchang et al., 2011) . c of jev was reported to limit viral neurovirulence (mori et al., 2005) the c-encoding nucleotide sequence of tbev contains conserved rna structures that function as replication enhancer elements (tuplin et al., 2011) † precursor membrane (prm) 469-972, 26 kda, 165 aa er lumen during virion assembly, the prm forms a heterodimer with the e protein and acts as a chaperone for correct e protein folding (kuhn et al., 2002; lorenz et al., 2002; zhang et al., 2003) . in the final step of virion maturation in the trans-golgi network prior, to viral release, the precursor is cleaved by furin and only the c-terminal region (m) is retained in the viral membrane (heinz et al., 1994; elshuber & mandl, 2005; lindenbach et al., 2007) . prm protein of jev also limits viral neurovirulence (kim et al., 2008 ) † (bressanelli et al., 2004) . the glycoprotein forms 'head to tail' homodimers that completely cover the surface of the mature icosahedral virions. it is also responsible for receptor binding, which leads to virus internalization by clathrin-mediated endocytosis (lindenbach et al., 2007) . the e protein contains three b-sheets domains (di-diii): dii includes the membrane fusion peptide (beasley & barrett, 2002; bressanelli et al., 2004; sukupolvi-petty et al., 2007) ; diii is associated with receptor binding and is also a target for several neutralizing monoclonal antibodies. crystal structures of the e protein from several flaviviruses all show similar secondary structure and domain organization (modis et al., 2003 (modis et al., , 2005 zhang et al., 2004; kanai et al., 2006; nybakken et al., 2006) . the e protein is important in determining neuroinvasiveness and neurovirulence for several mbfvs (cecilia & gould, 1991; hiramatsu et al., 1996; ni & barrett, 1996; sanchez & ruiz, 1996; chambers et al., 1998; beasley et al., 2004; lee et al., 2004 lee et al., , 2006 bordignon et al., 2007) . for wnv, jev, and denv, mutations in the e protein (located in the lateral surface of diii and the base of dii) affect fusion and receptor binding with target cells (lee et al., 2004) the e protein was first described in 1995 and later has been extensively defined and crystalized for many other flaviviruses (rey et al., 1995) . tbfv e protein is important for determining neurovirulence and neuroinvasiveness (holzmann et al., 1990; mcminn, 1997; beasley et al., 2005; mandl, 2005; engel et al., 2010) nonstructural proteins ns1 2461-3516/46-55 kda/325 aa er lumen/cytosol/ secreted low-resolution structural studies found the ns1 structure to be an open barrel configuration with d3 symmetry measuring 10 nm by 7.5 nm and with a central cavity approximately 4.5 nm in diameter (gutsche et al., 2011; edeling et al., 2014) . all flavivirus ns1 genes share a conserved degree of homology. ns1 is found at different cellular locations such as vesicular compartments associated with the cell membrane or on the cell surface and is also secreted as an extracellular † westaway et al., 1997; lindenbach et al., 2007) . each subunit forms a homodimer located in the er lumen, and it has been shown to colocalize with viral dsrna. secreted and cell surface associated ns1 is immunogenic and induces an antibody response that can serve as an important diagnosis marker (falgout et al., 1990; avirutnan et al., 2006; sun et al., 2007) . ns1 is also implicated in immune evasion functions as it activates human complement and induces host vascular leakage by specific inhibition of the classical and lectin pathways of complement activation. this occurs through a direct interaction with complement components c4 and c1s (avirutnan et al., 2010) . denv, wnv, and yfv ns1 are shown to limit c4b deposition and c3 convertase activity by enhancing cleavage of c4 through the recruitment of the complement-specific protease c1s (avirutnan et al., 2010) . ns2a (blitvich et al., 1995 (blitvich et al., , 1999 (assenberg et al., 2009 ). ns3 is a highly conserved and multifunctional protein: the protein has protease activity in one domain, rna-stimulated ntpase activity and rna triphosphate activity (rtpase) in the other domain (chambers et al., 1991; valle & falgout, 1998; lescar et al., 2008) . ns3 protein is an essential member of the rc and is activated in association with ns5 to bind the genomic rna 3 0 sl prior to replication (chen et al., 1997; mackenzie et al., 1998; lindenbach et al., 2007) . beside its essential role in the viral replication cycle, ns3 can also induce apoptosis (shafee & abubakar, 2003; ramanathan et al., 2006; yang et al., 2009; yiang et al., 2013) . ns3 is involved in the inhibition of the transcriptional factor ap1 signaling pathway, which also directly affects apoptosis (lin et al., 2006) . studies on neurovirulence associated with denv-1 ns3 have shown that mutations in ns3 (leu480ser) enhance the ability of replication in cns causing leptomeningitis and encephalitis in mice (bordignon et al., 2007) . this mutation was also shown to induce cell death in denv-1-infected cells (duarte dos santos et al., 2000) . the ns3 protein can be targeted by antiviral drugs such as ivermectin, ribavirin as inhibitors of the atpase activity of helicases (mastrangelo et al., 2012) the (lindenbach et al., 2007) . its interaction with ns1 is essential for viral rna replication as ns4a acts as a docking site (bartenschlager et al., 1995; miller et al., 2007) . ns4a interacts with the polypyrimidine tract-binding protein (ptb), with the latter being implicated in translation, transcription, and viral processes for various viruses (bieleski et al., 2004; florez et al., 2005; shi & lai, 2005) including the role in regulation of hcv replication (tischendorf et al., 2004; domitrovich et al., 2005; aizaki et al., 2006; chang & luo, 2006) . ns4a-ptb plays an indirect role in stabilization of the viral genome dsrna intermediates by binding to host cell ptb protein in a similar manner as stabilizing wnv rna synthesis by the elongation factor 1 alpha (davis et al., 2007; jiang et al., 2009) . ns4a together with ns4b and ns3 is also responsible for inducing the rearrangement of the host er membrane leading to the formation of virus-induced membranous spherules and vesicles encasing the dsrna and rc; this is thought to minimize exposure of the replicating rna to innate immune sensors such as retinoic acid-inducible gene-i, rig-i roosendaal et al., 2006; miller et al., 2007) . the mature ns4a can also induce the pi3k-dependent autophagy signaling, which in turn lead to protection from camptothecin-and staurosporine-induced cell death (mclean et al., 2011) . the role of ns4a in the up-regulation of the autophagy and er rearrangement makes the protein a potential target for antiviral drug development † 2k 6841-6909, 2 kda, 23 aa complete cleavage of the vbfv polyprotein generates an er-spanning 2k peptide located between ns4a and ns4b (lin et al., 1993; roosendaal et al., 2006; miller et al., 2007) . the 2k peptide functions as a signal peptide for ns4b and might play a role in enhancing viral replication by conferring resistance to lycorine, a suppressor of flavivirus rna synthesis (zou et al., 2009 ) † miller et al., 2006) . ns4b appears also to be involved in er rearrangement and modification during viral replication (westaway et al., 1997) . the ns4b protein of wnv, jev, and denv inhibits type i interferon (ifn-a/b) response by blocking the phosphorylation of stat1 (muñoz-jord an et al., 2003 , 2005 lin et al., 2004; liu et al., 2005; evans & seeger, 2007) . the same is true for kunv and wnv strain ny99, which blocks the activation of stat1/ stat2 and its translocation to the nucleus (liu et al., 2005) . these events disrupt the host immune responses by blocking the ifnalpha/beta/gamma pathways † ns5 7666-10 374, 100 kda, 900 aa cytosol/er/nucleus ns5 is the largest and most conserved among the vbfv proteins. ns5 primarily functions as the rna-dependent rna polymerase (rdrp) through its c-terminal domain (lindenbach et al., 2007) . the ns5 has been crystalized and contains an n-terminal domain with s-adenosyl methionine methyltransferase activity (mtase), and possibly guanylyltransferase (gtase) for rna capping (egloff et al., 2002; malet et al., 2007; yap et al., 2007) . ns5 is also involved in activating other viral proteins, such as ns3 ntpase and rtpase activity in a dose-dependent manner. denv ns5 contains both nuclear localization signal (nls) and nuclear export signal (nes): these facilitate its importation into the nucleus by both importin b1 and the importin a/b complex, and an additional function of the signals could be enhancement of virus replication and hyperphosphorylation of ns5 (johansson et al., 2001; brooks et al., 2002) . for wnv, ns5 is exported from the nucleus in a chromosome region maintenance 1 (crm1)-dependent manner (pryor et al., 2007; rawlinson et al., 2009 ). ns5 is involved in different cellular pathways and has a crucial role in the escape from, or blocking, the interferonalpha/beta (ifn-alpha/beta) signaling pathway. denv ns5 inhibits host tyk2 and stat2 phosphorylation and prevents the activation of jak-stat signaling pathway (mazzon et al., 2009) in addition to the viral rdrp functions of ns5 described for the mbfvs, the tbfv ns5 was the first to be implicated in disrupting innate immune signaling. to suppress critical host responses, lgtv ns5 interacts with ifnar2 and ifngr2, two ifn receptor subunits, and antagonizes ifndependent responses by suppressing jak-stat signal transduction (best et al., 2005; park et al., 2007) . cellular responses targeted at restricting viral replication occur via lysosomal degradation of ns5, ns2b, and ns3. the ns5 of lgtv and tbev interacts with trim79-alpha, an ifn-inducible protein, and induces the degradation (taylor et al., 2011) . the cellular antiviral state is also compromised by tbev ns5 interaction with the scaffold protein scribble (werme et al., 2008) . this complex blocks the phosphorylation of stat1 and disrupts the jak-stat signaling pathway the extreme 3 0 terminal region contains secondary structures and rna elements important for cyclization of the genome and virus viability. the mbfv 3 0 utr can be divided into three regions (markoff, 2003; liu et al., 2009 ): a variable region directly upstream of the stop codon; a second region that is relatively conserved in nucleotide sequences containing hairpin motifs; and a highly conserved core element contains stable sl structures and cyclization motif (villordo & gamarnik, 2009) the 3 0 utr of tbfvs exhibits significant heterogeneity in length and sequence even among closely related strains. the tbfv 3 0 utr can also be divided into a variable region part and an extremely conserved core element (gritsun et al., 1997) , where the length variability of the variable region has been suggested to be related to the number of laboratory passages (mandl et al., 1998; mandl, 2005) . a hypothesis interprets the biological importance of the variable region that influences the replication/translation dynamics in mammalian and tick cells (elvang et al., 2011) . the conserved core element contains highly essential sequential and structural motifs defined as sl1-5 for viral maintenance (mandl et al., 1998; pletnev, 2001) . the pentanucleotide (gagag) motif exposed on the sl4 is strictly conserved among all the flavivirus genomes . other important elements present in the conserved region include the homopurine box, a homopyrimidine box, and cyclization sequences (mandl, 2005; gritsun & gould, 2007a, b) *nucleotide numbers are related to the strain neudoerfl of tbev. † shared function is presumed for both mosquito-and tick-borne flaviviruses. in many cases, no specific studies have been carried out in tbfv. brane (prm/m), and envelope (e), and seven nonstructural proteins: ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5 (chambers et al., 1990; ryan et al., 1998; bollati et al., 2010) . the defined functions of these proteins are presented in table 1 . however, the precise function of some of the proteins remains to be elucidated. furthermore, it should be noted that the bulk of the studies have been carried out in mbfv systems, and it is possible that the functions may not be identical in tbfv. recognizing that the vbfvs cycle between vertebrate and arthropod hosts and that both viral and host factors are likely to be involved, it is highly probable that viral persistence is exceedingly complex. the purpose of this review is to evaluate the current literature on flavivirus persistence as well as to suggest ideas for additional research into this interesting and important area. humans are inadvertent targets of vbfv infection (figs 1 and 2), and infection is often associated with debilitating, acute neurological syndromes or hemorrhagic fever. however, there are several lines of evidence suggesting flavivirus persistence in humans (murray et al., 2010; gibney et al., 2011; baty et al., 2012) . the establishment of flavivirus persistence in humans seems to be mainly associated with encephalitic flaviviruses (diamond, 2003) . however, yfv and denv persistence has been described in eukaryotic cells in culture and in successive generations of aedes mosquitoes (lodge et al., 1987; takasaki et al., 2001; farfan-ale et al., 2009) . yfv and denv commonly cause a hemorrhagic illness, jaundice, and dengue shock syndrome, and encephalitis is atypical and extremely rare , and delivered to endosomes, where a ph-dependent fusion of the particles with the endosome membrane occurs (c). subsequent to uncoating, the single-stranded, positive-sense rna genome (d) migrates to the endoplasmic reticulum (er) and is translated (e) as a polyprotein traversing the er membrane several times (f). the polyprotein is cleaved into the viral proteins by viral and cellular proteases, although prm and e remain covalently attached. through the agency of several viral nonstructural proteins and cellular proteins, there is a proliferation of er-derived membranes and the formation of spherules that maintain a pore-like connection to the cytoplasm (g); the viral genome is replicated within these spherules by the viral proteins comprising the replication complex. by an as yet uncharacterized mechanism, progeny genomes are delivered to adjacent er membranes where the capsid protein mediates assembly and inclusion of prm-e into immature virions (h). the immature virions transit the golgi membrane system (i), and as a mild ph change occurs, the cellular enzyme furin cleaves the prm-e linkage (j), allowing the virus particle to assume its final mature stage prior to release from the cell (k). defined roles for the individual viral proteins are enumerated in table 1 . (tomori, 2004; gulati & maheshwari, 2007; varatharaj, 2010) , but persistence cannot be excluded. human infection with the encephalitic viruses almost always occurs following a bite from infected ticks or mosquitoes (figs 1 and 2) . generally, illness tends to be biphasic. the first phase is characterized by a flu-like illness with symptoms such as headache, arthralgia, and malaise. the second phase may present with neurological symptoms, such as mild meningitis to severe encephalitis. survivors of encephalitis may develop persistent neurological sequelae, suggesting neurological tissue damage and/ or viral persistence. neurological tissue damage, such as loss of neurons, has been reported for wnv infection (guarner et al., 2004) . long-term morbidity following tbev infection is known to occur frequently (haglund et al., 1996; haglund & gunther, 2003) . a study conducted in sweden reported chronic postencephalitic syndrome in 36% of patients who had been infected with tbev (haglund et al., 1996) . in up to 60% of patients who develop encephalitis due to wnv infection, neurological and/or neuropsychological symptoms continue to be reported for up to 7 years following the infection (murray et al., 2010; sadek et al., 2010) . the most definitive evidence for viral persistence would be the isolation of viable virus or the demonstration of viral antigens or rna long after the acute illness. several reports describe the isolation of infectious vbfvs from persistently infected humans. for example, the siberian tbev strain za was isolated from an individual who had harbored the virus for 10 years (gritsun et al., 2003) . he later died following 2 years of a progressive form of tbev encephalitis (gritsun et al., 2003) . in addition, isolation of jev from the cerebrospinal fluid was possible for more than 3 weeks following infection in 19% of individuals that had developed encephalitis (ravi et al., 1993) . jev persistence was also demonstrated in peripheral mononuclear cells in infected children in northern india (sharma et al., 1991) . in these children, virus could be isolated by culture 8-9 months after acute infection (sharma et al., 1991) . furthermore, wnv has been transmitted to patients who had received blood transfusions or organ transplants from asymptomatic donors (pealer et al., 2003; montgomery et al., 2006; cdc, 2009) , suggesting viral persistence in the donors as well as stored blood for transfusion. long-term presence of viral nucleic acid is another indicator for persistent infection; however, study results are inconsistent. viral rna can be readily sought using molecular biology techniques, such as rt-pcr and transcription-mediated amplification (tma) (s anchez-seco et al., 2005; muñoz-jord an et al., 2009; patel et al., 2013) . using tma, wnv rna was detectable in the blood of donors for up to 104 days following the index donation prince et al., 2008) . using rt-pcr, wnv rna could also be detected in urine of persistently infected individuals for up to 6.5 years following the acute phase of infection (murray et al., 2010) , although virus could not be isolated from the rna-positive urine samples. in contrast, wnv rna could not be detected after 6 years in urine in another study reported a year later (gibney et al., 2011) . baty and colleagues also failed to detect wnv rna by rt-pcr, but were able to detect viral rna using tma (baty et al., 2012) . similarly for jev, viral rna could not be detected by rt-pcr in the individuals who had anti-jev igm antibodies (zhao et al., 2013) . in the study by gibney et al. (2011) , wnv persistence could not be excluded. even though the results in these studies are somewhat variable, it seems reasonable to conclude that the presence of viral rna can also be taken as an indicator of persistent infection. viral serology may be an additional surrogate measure for vbfv persistence. in general, infection or vaccination leads to a sterilizing immunity, but long-term persistence of anti-vbfv igm antibodies has often been assumed to indicate continued exposure to viral antigens or virus particles (ravi et al., 1993; stiasny et al., 2012) . an exception to this is, of course, denv where antiviral antibody plays a key role in pathogenesis (martina et al., 2009; pierson, 2010) . igm antibodies induced by flaviviruses, such as tbev and wnv, are known to persist in serum and csf for 12 months or more (kapoora et al., 2004; stiasny et al., 2012) . indeed, igm antibodies against tbev persisted for up to 32 months (stiasny et al., 2012) . furthermore, anti-wnv igm antibodies persist in previously exposed blood donors for up 16 months prince et al., 2008) . however, igm antibody persistence does not necessarily correlate with infectious virus persistence. for example, persistent igm antibodies against tbev can be detected in some cases following vaccination without active viral infection (rendi-wagner et al., 2004; stiasny et al., 2012) . for wnv infection, igm antibodies against the nonstructural protein ns5 cannot be used to distinguish between recent/active infection from past infection (prince et al., 2008) . thus, serology may be a less useful marker for viral persistence. a major consideration in vbfv in humans would be identification of sites of viral persistence. as flavivirus persistence seems to be mainly associated with the neurotropic and encephalitogenic viruses, the central nervous system may well be the preferred site for viral persistence. furthermore, this may account for why patients who recover from encephalitis often have prolonged neurological symptoms. however, other studies by murray and colleagues postulated kidneys as a preferred site for the establishment of persistent flavivirus infections following the detection of wnv rna in urine (murray et al., 2010) . certainly, additional work is required to define the true incidence, the biology, and the pathogenic potential in humans of persistent vbfv infections. arthropod hosts play crucial roles in the biology of both tbfv and mbfv and contribute to persistent infection. in nature, ticks are the important arachnid reservoirs of tbfvs. the persistence of the tbfvs in arachnids is well established, and the role of ticks as long-term reservoirs and vectors for the viruses is clear. estimates suggest that ticks transmit about 25% of the known flaviviruses. tbfvs are capable of infecting about 14 tick species, but the ixodid ticks, ixodes scapularis and ixodes ricinus, account for nearly all transmission of tbev to humans. ixodes cookei may also harbor powv and dtv, while the soft-bodied onithodoros tick transmits alkhurma virus (main et al., 1979; charrel et al., 2007) . to transmit virus, these ticks need only 15 min of attachment to the host (crowder et al., 2013) . the virus is present in the tick salivary glands, and saliva constituents enhance infectivity (nuttall & labuda, 2003; girard et al., 2004) . a recent publication revealed that a selected subset of salivary gland genes is expressed when infected ticks feed on na€ ıve mice (mcnally et al., 2012) . ticks acquire the virus while feeding on infected rodents, typically by a process called 'cofeeding' (fig. 1 ; labuda et al., 1993a, b) , and the virus persists throughout several life stages. ticks at the larvae, nymph, and adult life stages can become infected by feeding on infected animals (horizontal transmission) or can transmit virus vertically across instars (transstadial transmission) and through the eggs (transovarial transmission; fig. 1a ; labuda et al., 1993a labuda et al., , 1996 nuttall & labuda, 2003) . indeed, results of a carefully controlled study in our laboratory show that transstadial transmission seems to be very high (mitzel et al., 2007) . the blood meal remains in the midgut for long periods of time, allowing infection of the epithelial cells lining the midgut with subsequent translocation into the hemocoel (nuttall & labuda, 2003) . infection of the midgut cells may be facilitated by the heterophagous nature of ticks (i.e. blood meal digestion is principally an intracellular process). in the open circulatory system, tissues and organs are bathed in hemolymph, which acts as a medium for transporting nutrients, hormones, and immune effector molecules. therefore, the hemolymph likely serves as a viral dissemination medium. tbe virus was found in the esophagus and subesophageal ganglion in dermacentor marginatus larvae and in columnar epidermal cells of dermacentor reticulatus nymphs (nuttall & labuda, 2003) . in d. reticulatus nymphs, tbe virus was demonstrated in epidermal cells and in vacuoles in the region of golgi complexes of salivary gland cells (nosek et al., 1984) . the precise mechanisms by which tbfvs traverse various tissues in the tick and reach the salivary glands remain to be fully elucidated. prevalence surveys indicate that 0.5-5% of ixodid ticks carry the virus in europe, but prevalence of up to 40% has been reported in russia (ustinova et al., 1997) . in north-central usa, up to 4.9% of i. scapularis ticks are infected by powassan or dtv (brackney et al., 2008b; dupuis et al., 2013) . interestingly, in a study carried out in chicago, i. scapularis was found to infest 2.8% of wild birds, which figure prominently in the mbfv cycle ( fig. 1b; hamer et al., 2011) . however, identification of tbfv was not attempted in this study. in brief, tbfv persistence in ticks is well established, and the essential role of ticks in the biology of these viruses is not in doubt. as is the case for tbfvs, the role of mosquitoes in the maintenance of the mbfv cycle is also well characterized. mosquitoes are thought to account for transmitting approximately 50% of more than 70 known flaviviruses (gould, 2001) . diverse mosquito species can be infected by mbfvs. however, the two major mosquito vectors are aedes aegypti (e.g. transmission of yfv and denv) and culex species (e.g. transmission of wnv and jev). adult female mosquitoes become infected when they obtain blood meals from flavivirus-infected animals, and virus replication in the mosquito has been well described (girard et al., 2005; mcgee et al., 2010; colpitts et al., 2012) . girard et al. (2004) used immunohistochemistry to demonstrate that wnv infects epithelial cells of the culex pipiens midgut and that viral antigen can be detected as early as day 2 postinfection. viral antigen staining becomes more intense in the cells of the midgut over time until day 14 to 21 following infection (girard et al., 2004) . using denv-2, virus titer in the midgut peaks to about 9 9 10 3 pfu ml à1 by 10 days postinfection, but declines to about 7.4 9 10 2 pfu ml à1 by day 12 (s anchezvargas et al., 2009 ). dissemination of virus into various tissues occurs at various time points, and the amount of antigen also varies. similar to tbfvs in ticks, the hemolymph serves as a vehicle for viral dissemination. however, virus also spreads in a cell-cell fashion to the muscle of the posterior midgut from 6 to 27 days postinfection (girard et al., 2004) . studies with denv-2 showed that infected mosquitoes mount an rna interference (rnai)-mediated antiviral response, but impairing the vector rnai resulted in increased viral replication (s anchezvargas et al., 2009) . this mechanism could be associated with observations of flavivirus-related rna that persists as dna in mosquitoes (crochu et al., 2004) . as mosquitoes are such efficient vectors for the transmission of the mbfvs, these reports suggest that the viruses have evolved a mechanism of evading the host response to persist in the vector. mosquitoes also acquire and deliver virus horizontally during blood meals and are competent to transmit vertically to progeny by transovarial passage (rosen et al., 1983) . wnv can persist overwinter in mosquitoes that hibernate in cold months. cool temperatures also facilitate persistence of flaviviruses in adult female mosquitoes. for example, st. louis encephalitis virus (slev) survived for more than 100 days of winter in culex quinquefasciatus (kramer & ebel, 2003) . similarly, jev and wnv have been transmitted by mosquitoes that carried the viruses at cold temperatures when exposed to temperature increases equal to ambient levels (kramer & ebel, 2003) . however, at low temperature, mosquitoes are less likely to acquire new infections (colpitts et al., 2012) . at 18°c, low wnv dissemination to the legs of the c. pipiens was observed, and rapid viral dissemination occurred at higher temperatures (colpitts et al., 2012) . furthermore, higher temperatures increase vector population growth rate and the rate of viral evolution in the mosquito (girard et al., 2005) . the prevalence and maintenance of wnv across landscapes is mediated by environmental factors, such as local effects of agriculture on vector and host communities (crowder et al., 2013) . results of investigations carried out in the states of oregon and washington showed that the prevalence of wnv in both c. pipiens and culex tarsalis was similar at 14.5% or 13.5%, respectively (crowder et al., 2013) . however, a study conducted in stratford, connecticut, showed that the c. pipiens was the most dominant mosquito captured in this wnv focus area (anderson et al., 2004) . in the captured mosquitoes, more than 85% of wnv isolations were from the same species, whereas culex salinarius accounted for between 5% and 12% (anderson et al., 2004) . in a similar study conducted in mexico, c. quinquefasciatus was more common at 48.3% and mbfv rna was detected in 70% of the pools (farfan-ale et al., 2009) . it is evident that mosquitoes are important as viral hosts and vectors. furthermore, it can be concluded that the mbfvs have evolved mechanisms of evading the host innate responses to persist in the mbfvs. the principal vertebrate hosts for vbfvs are small mammals, marsupials, and birds (fig. 1) , but the viruses can also infect reptiles (mackenzie et al., 2002 (mackenzie et al., , 2004 steinman et al., 2003; jacobson et al., 2005; root et al., 2005; marschang, 2011) . in most instances, larger animals, such as cervids, goats, and sheep, are incidental hosts. the 1999 outbreak of wnv in humans in new york is thought to be associated with wnv infection in birds (strausbaugh et al., 2001) . the mosquito-borne wesselsbron virus could also be cycled between mosquitoes and birds, but not much is known about the vertebrate host(s). thus, in nature, numerous species are susceptible to vbfvs and might serve as reservoirs or secondary amplifying hosts. in this section, we will survey the literature and the three potential markers for viral persistence: isolation of virus, identification of viral rna or protein, and viral serology. small mammals, particularly rodents, are the principal vertebrate hosts and reservoirs for tbfvs (mansfield et al., 2009; dobler et al., 2012) . in europe, the yellow-necked mouse (apodemus flavicollis) and bank vole (myodes glareolus) are implicated as the most common hosts, and they develop sufficient viremia to infect ticks that feed on them (weidmann et al., 2011; dobler et al., 2012; knap et al., 2012) . in various wild rodent species captured in brandenburg, germany, an average tbev infection rate of 15% was reported (achazi et al., 2011) . in the captured rodents, tbev rna was detected by rt-pcr in the brains and spleens. in north america, deer mice (peromyscus), squirrels, and the striped skunk (mephitis mephitis) are important reservoirs of powv and dtv (main et al., 1979; telford et al., 1997) . based on serological studies, a 6.2% dtv prevalence in the red-backed voles (myodes rutilus) was found in siberia and alaska (deardorff et al., 2013) . dtv in new mexico was serologically prevalent in peromyscus truei and peromyscus maniculatus at 22.2% and 6.0%, respectively (deardorff et al., 2013) . therefore, it seems likely that persistent infection of these small mammals occurs. deer may stabilize and maintain tbfvs at levels that are important for transmission (pugliese et al., 2007; carpi et al., 2009; mcgee et al., 2010; dobler et al., 2012) . while deer are important sources of blood meals for ticks, they develop low virus titer and are therefore not competent in transmission of tbfvs. however, in a broader sense, deer are important in viral persistence because they help sustain tick populations (cagnacci et al., 2012) . the situation for domesticated animals is also less clear than for rodents. dogs, horses, and monkeys can be infected with tbfvs, but case reports in veterinary practice are relatively infrequent (jaenson et al., 2012) . large domesticated animals, such as goats, sheep, and cattle, become viremic for a short while and develop antibodies following infection with tbev, but do not show any specific clinical signs of illness (mansfield et al., 2009; klaus et al., 2012) . however, transmission of tbev via milk from these domesticated animals (fig. 1) has been documented, suggesting that virus may persist following the acute viremia in at least some instances (vereta et al., 1991; caini et al., 2012; hudopisk et al., 2013) . birds and primates are considered to be the primary reservoirs of mbfvs. other mammals are generally accidental hosts, but this may not always be the case, and when viremic, these domestic animals are able to infect mosquitoes. for jev, the major amplifying hosts are birds and pigs, which attain high levels of viremia. they provide a source of infection for the mosquito species that subsequently transmit jev to humans (hukkanen et al., 2006; van den hurk et al., 2009) . jev control has been achieved through vaccination of pigs and humans in korea, japan, and taiwan (igarashi, 2002) while horses are considered dead-end hosts of jev infection due to a very low level of viremia. in geographic regions where pig populations are low, swine may not be important for zoonotic transmission of jev. herons and egrets are important jev-amplifying hosts and source of infection for mosquito species that transmit jev to humans (nemeth et al., 2009) . wild-caught pigeons were shown to have antibodies that persist for up to 15 months. it is not clear whether the antibody persistence is associated with viral persistence. although various bird species do play a prominent role in jev infection, evidence of birds as a source of persistent infection is less certain. in some instances, birds can acquire jev viremia and then fail to develop or lose neutralizing antibodies. birds are also excellent amplifying hosts for wnv, and migratory birds can move the viruses to different areas because they become viremic for several days. near 100% mortality is associated with natural or experimental wnv infection in birds, such as american crows (corvus brachyrhynchos), blue jays (cyanocitta cristata), and greater sage-grouse (centrocercus urophasianus; steele et al., 2000; komar et al., 2003; clark et al., 2006) , but some birds are able to carry the virus for a longer time before they develop antibodies or succumb to disease (mckenzie & goulet, 2010) . for instance, a combined 37% of house sparrows that were either naturally or experimentally infected with wnv tested positive for wnv rna by rt-pcr (wheeler et al., 2012) . in the same study, wheeler et al. (2012) showed that 97% and 100% of wnv-infected house sparrows and finches, respectively, seroconverted following exposure to wnv. birds that develop neutralizing anti-wnv antibodies are protected from reinfection (nemeth et al., 2009 ), suggesting total virus clearance in seroconverted birds, and antibodies against wnv have been found in wild birds, suggesting exposure to wnv. early demonstration of wnv persistence was described in blue-gray pigeons, from which virus was isolated up to 100 days postinfection, and viral antigen could be detected in liver tissue for up to 180 days (ruiz et al., 2010) . american robins (tardus migratorius) are known to be competent reservoirs of wnv and slev (kilpatrick et al., 2006) . wnv persistence was also demonstrated by the detection of viral rna in the presence of antibody for up to 36 weeks in spleens of naturally infected house sparrows and finches (wheeler et al., 2012) . taken together, it seems that there is decent evidence for persistent infection by wnv in some bird species. however, many details about the biology of persistence remain to be studied. antibodies to various flaviviruses, such as slev, powv, jev, and wnv, have been identified in chelonians, snakes, and crocodiles in different geographic locations (steinman et al., 2003; marschang, 2011) , and it is known that alligators and crocodiles can be infected with wnv. however, there are no reports suggesting that reptiles host persistent mbfv infection. in summary, it is apparent that various animals and birds can sponsor persistent vbfv infection. nevertheless, the precise role that these persistent infections play in larger scheme of viral maintenance merits additional study. there are several animal models of persistent mosquito-borne flavivirus infection, including wnv and slev (charlier et al., 2004; kimura et al., 2010) . as shown in table 2 , mice and hamsters have been the main study models. in a nonhuman primate study, wnv was isolated from central nervous system tissues more than 5 months post-intra-cerebral inoculation , suggesting that these species might also be relevant system. persistent wnv has also been studied experimentally in the golden hamster. the clinical outcomes of wnv infection in hamsters vary and depend on animal age and immune competence, viral dose, and route of infection. wnv infection can lead to asymptomatic illness, encephalitis, severe paralysis, and acute death (xiao et al., 2001; morrey et al., 2004; tesh et al., 2005) . adult golden hamsters infected with wnv develop chronic infection, characterized by shedding of virus in urine for up to 8 months (xiao et al., 2001; ding et al., 2005; tonry et al., 2005) . the chronically infected animals exhibit antigens in tubular epithelial cells, interstitial cells, and macrophages of distal renal tubules . interestingly, na€ ıve hamsters inoculated intraperitoneally with wnv-containing urine from persistently infected hamster do not develop clinical disease, but the hamsters become viremic and develop antibody responses (ding et al., 2005) . this suggests possible viral genetic changes, which may facilitate persistent infection, although other explanations cannot be totally excluded. hamsters have also been used as experimental models to study the persistence of slev and are among the natural vertebrate hosts of the banzi flavivirus, a member of the yfv group of flaviviruses (grard et al., 2010) . golden hamsters infected with slev did not develop clinical signs of illness, but they developed viruria and antibodies against slev at 28 days postinfection (siirin et al., 2007) . (appler et al., 2010; pierson & diamond, 2012) : wnv rna persisted in a pantropic manner in 12% of infected mice for up to 6 months. infectious virus could be isolated in 12% of mice for up to 4 months. c3h mice survival rate was lower and 22% when compared to the survival rate of b6 mice, which was 78% macaque rhesus (pogodina et al., 1981) : virus persisted in asymptomatic animals for 5½ months and could be isolated from cerebellum, cerebral subcortical ganglia, lymph nodes, and kidneys golden hamster (mesocricetus auratus; tesh et al., 2005; tonry et al., 2005) : chronic renal infection with persistent shedding of virus for up to 8 months. virus could be recovered by culture, and genotypic and phenotypic changes were identified house sparrow (passer domesticus; nemeth et al., 2009) : infectious virus persisted in tissues for up to 43 days, but was not detectable in sera after 6 days. wnv rna persisted in tissues for up to 65 days slev golden hamster (siirin et al., 2007) : infected animals remained asymptomatic, but virus could be cocultivated in various organs for up to 185 days jev swiss albino mice (mathur et al., 1986a, b) : persistence was demonstrated by reactivation in 41% of congenitally infected pups. in adult mice, viral persistence was shown to last longer (16 weeks) in pregnant mice compared with 4 weeks in nonpregnant mice tick tbev macaque rhesus (pogodina, 1983; pogodina et al., 1984 pogodina et al., , 1981 : monkeys recovered from encephalitis and virus persisted for at least 738 days. in asymptomatic animals, virus persisted for 302 days liv* immunosuppressed guinea pigs (zlotnik et al., 1971) : liv was lethal in young animals, but older animals acquire a nonapparent infection with viral replication in the brain and spleen powv deer mouse (peromyscus leucopus; telford et al., 1997) : not a well-characterized model, but adult mice appear to survive infection *louping ill virus infects sheep. hamsters that become persistently infected with slev shed virus in their urine for up to 185 days postinfection. the shedding of virus in the urine of infected hamsters also suggests that the kidneys could be an organ preferred for viral persistence. c57bl/6j mice maintained a persistent wnv infection in the face of robust neutralizing antibody levels for more than 6 months (appler et al., 2010; stewart et al., 2011) . in this study, infectious virus was recovered in the skin, and viral rna was identified in the skin, as well as the spinal cord and brain (appler et al., 2010) . in contrast, wnv persistence was uncommon in kidneys and rare in the heart (appler et al., 2010) . however, in another study in which c57bl/6j mice were infected with wnv derived from the urine of persistently infected hamsters, the kidney was found to be the preferred organ for viral persistence (saxena et al., 2013) . the hamster-derived wnv was also found to be highly attenuated in both neuroinvasiveness and neurovirulence in infected mice (saxena et al., 2013) . these results also suggest that the virus acquires phenotypic changes to be able to persist. while the inbred laboratory mouse models are patently useful for understanding some aspects of flavivirus persistence, very few natural rodent hosts have been established as experimental animal models. evidence of exposure to wnv has been reported in rodent species, such as rats (rattus), bank voles (m. glareolus), and deer mice (peromyscus; molnar et al., 1976; root et al., 2005; docherty et al., 2006; gomez et al., 2008) . however, infectious virus was only identified in the bank vole, whereas antibodies were detected in all of the species (molnar et al., 1976; root et al., 2005) . although persistence in mammals obviously plays a significant role in tbfv infections, little attention has been devoted to experimental studies of this aspect of tbfv biology. while disease or therapy models have been established for tbfvs (kreil et al., 1997; holbrook et al., 2005; hayasaka et al., 2010; palus et al., 2013) , none of these specifically investigated viral persistence. experiments to determine the susceptibility of several wild and domesticated mammals to powv showed that viremia lasted for 0-3 days in the goat, pig, skunk, red fox, and gray fox (yiang et al., 2013) . in another study, adult deer mice (peromoyscus leucopus) survived challenge with powv without apparent illness, but evidence of viral persistence was not sought (telford et al., 1997) . development of suitable experimental models for persistent tbfv infection would clearly provide useful information about this aspect of these important pathogens. the previous sections have looked at flavivirus persistence in humans, animals, and arthropod vectors, as well as some relevant animal models. in this section, we will survey information related to the initiation and maintenance of persistence. when mammalian cell cultures are acutely infected with tbfv, a legion of general cellular defense and antiviral systems are triggered, as are specific factors designed to limit or restrict virus reproduction. some of these include type i interferon (ifn-a/ifn-b), type iii interferon (ifn-k), mitochondrial activated signaling, and the induction of inflammatory factors, such as interleukins (tam & messner, 1999; madden, 2003; van gerpen, 2003) . for example, the ifn-induced tripartite motif protein, trim79a, has been shown to restrict tbev replication by degrading ns5 (taylor et al., 2011) . the unimpeded deployment of these antiviral factors and systems would lead to cell death. cell death is thought to be mediated primarily through apoptosis (r u zek et al., 2009) , and programmed cell death induced by various tbfvs has been described in neurons, epithelial cells, hepatocytes, kupffer cells, and neuroblastoma cells (ramanathan et al., 2006) . impeding or evading the antiviral response is one characteristic of vbfvs, which plays an important role in viral persistence. during flavivirus infection, ifn production is induced within hours, but viral rna replication complexes are enclosed in vesicles, which may offer protection from recognition by pathogen recognition receptors, thus delaying ifn production (welsch et al., 2009; gillespie et al., 2010; overby et al., 2010; offerdahl et al., 2012; ye et al., 2013) . in addition, some vbfvs can directly affect ifn secretion by inhibiting ifn gene transcription, suppressing ifn signaling or impairing the functions of interferon-stimulated genes (best et al., 2005; robertson et al., 2009; ye et al., 2013) . the humoral and cell-mediated immune responses are also prone to inhibition by vbfvs. studies with wnv in c57bl/6 mice suggest that wnv-specific antibodies correlate with decreased spread to the cns (diamond, 2003) . antibody escape mutants could also evade the t cell recognition (diamond, 2003) , but a precise role of these in viral persistence is yet to be defined. the e protein is thought to be responsible for provoking the cytopathic effect (isaeva et al., 1998; prikhod'ko et al., 2001) . however, the ns3 protease and ns2b-ns3 protease precursor (table 1) are also known to induce apoptosis by binding to caspase 8 (shafee & abubakar, 2003; ramanathan et al., 2006; safronetz et al., 2013) . in addition, ns2a has also been implicated in causing ifn-independent cytopathic effect (chang et al., 1999; melian et al., 2013) . observations in our laboratory (l. mlera, d. offerdahl & m.e. bloom, unpublished results) and those of others (lancaster et al., 1998) indicate that tbfv infection in mammalian cell culture is characterized by an acute phase, which kills most infected cells; however, a small number of cells survive the initial cytolytic phase, and the culture is repopulated with cells almost all of which are infected. clearly, vbfv induces an acutely cytopathic infection in mammalian cells, and thus, the development of a persistent infection implies that cell death factors must be evaded or modulated, although the precise mechanisms are still obscure. the implication of specific viral proteins, domains, or sequences that combat cytolytic cell death has been rather limited, but any viral determinants that limit cell death in the face of acute infection might also enhance the initiation of persistent infection. for example, viral ns4a (table 1) was shown to induce phosphatidylinositol 3-kinase (pi3k)-dependent autophagy and thereby leading to protection from cell death (mclean et al., 2011) . the manipulation of apoptosis by jev, which activates pi3k in infected cells, was also reported (lee et al., 2005) . jev triggers apoptosis during the late stages of infection, and the activation of pi3k is thought to provide protection from early cell death. limited replication implies a low level of viral protein expression and may favor viral persistence. over the years, significant attention has been focused on defective interfering (di) virus particles, which limit replication of the wild-type virus (schmaljohn & blair, 1977; debnath et al., 1991; blitvich et al., 1999) . the di particles represent truncated genomes that can be replicated and encapsidated and compete with wild-type viral particles when they infect cells. in vesicular stomatitis virus (vsv), di particles are thought to modulate virulence (cave et al., 1985) . however, the role of vbfv di particles in persistence is not completely certain. for tbev, the c protein is reported to tolerate internal deletions ranging from 4 to 21 amino acids, and the deletions seem to favor attenuation and immunogenicity (kofler et al., 2002) . di particles of vbfvs, such as murray valley encephalitis (mve), tbev sofjin strain, and wnv, are known to generate truncated ns1 proteins, following infection at high multiplicity of infection (debnath et al., 1991; poidinger et al., 1991; chen et al., 1996; bugrysheva et al., 2001) . persistent infection of vero cells with mve was associated with a truncated ns1, whereas this form of ns1 was not noted in the acute infection (brinton, 1982; lancaster et al., 1998) . the truncated ns1 in mve virus was a result of the presence of di rna, which contained a large internal deletion (lancaster et al., 1998) . in this case, mve di particles reduced the wild-type mve titer by 75-95% (poidinger et al., 1991) . however, a causal role for the mve di particles in maintaining persistent infection was not conclusively demonstrated. studies of the far eastern sofjin strain of the tbev complex identified a 39-kda truncated ns1 in both acutely and persistently infected human kidney rh cells (bugrysheva et al., 2001) . for wnv, naturally occurring di particles interfere with transmission in mosquitoes and minimally impact pathogenesis in mice (pesko et al., 2012) . furthermore, truncated denv rna species, suggesting the presence of di particles, have been identified in acute human infections (li et al., 2011) , but have not been described in other acute vbfv infections in humans. similarly, banzi virus di particles seem to have been generated in resistant c3h/rv mice (smith, 1981) . although di particles and the truncated ns1 may be frequently observed in persistent infections, it is not at all clear that they are independently sufficient for the establishment and maintenance of a persistent infection in vitro or in vivo. stable expression of truncated ns1 failed to render persistent infection with jev, suggesting that truncated ns1 is a consequence rather than a cause for viral persistence (liao et al., 1998) . the role of di particles merits further investigation, but the role of other aspects of virus biology should also be scrutinized. restricted expression of the envelope (e) protein may also favor development of persistence. in kn73 cells that were persistently infected with jev, the e protein was found to be expressed at markedly low levels compared with acutely infected cells (feng et al., 2002) . in these studies, the expression of ns3 was found to be unchanged in the acute and persistent phases of jev infection (feng et al., 2002) . as the e protein is important for pathogenesis and immunity (cdc, 2009), low-level expression could result in immune tolerance and contribute toward viral persistence. in addition to low expression levels, mutations in the e protein of jev, yfv, and wnv might also play a role in vbfv persistence (ding et al., 2005; farfan-ale et al., 2009) . a 138e?k mutation in the e protein of jev and wnv was shown to inhibit cell-cell spread of the virus and to contribute toward the development of a small-plaque phenotype (carson et al., 2006) . while this mutation leads to viral attenuation (carson et al., 2006) and is key in the attenuation of jev sa14-14-2 vaccine (monath et al., 2002) , further elucidation of mutations that may play a role in persistence is required. observations that hamsters and mice infected with mbfvs obtained from urine of other mbfv-infected animals do not suffer severe disease and become persistently infected indicate that the virus is attenuated (rosen et al., 1983; ding et al., 2005) . a number of amino acid-changing mutations in c, e, ns1, ns2a, ns2b, and ns5 were reportedly associated with persistence of wnv in serially passaged hamsters (s anchezvargas et al., 2009) . these mutations are thought to have attenuated the virus (s anchezvargas et al., 2009) . however, the same mutations have not been reported by other groups, suggesting that the mutations may not be specific to development or maintenance of viral persistence. mechanisms not directly involving viral proteins may also play a significant role in persistence. for instance, jev delays the exposure of dsrna to innate sensors and inhibits phosphorylation of irf3 via noncoding viral short flaviviral rna (espada-murao & morita, 2011; chang et al., 2013) . similarly, wnv delays recognition by pathogen recognition receptors by activating irf3 in a rig-i-dependent manner without antagonizing the host ifn response (fredericksen & gale, 2006) . the mechanism of evasion is currently not clear, but membrane-bound vesicles that enclose the rig-i-activating dsrna of tbev have been described (overby et al., 2010) . as the delayed recognition of viral pathogen-associated molecular patterns is only temporary, these manipulations are probably just among the suite of mechanisms deployed for the establishment of viral persistence. the specific viral genes and how they manipulate apoptosis at a cellular level needs further examination. specific host genes that may contribute toward the development of flavivirus persistence in the vertebrate host have not been defined, but there are some suggestions. for instance, the overexpression of the proto-oncogene bcl-2 was reported to prevent apoptosis and promote persistence in jev-infected bhk and cho cells (liao et al., 1998) , suggesting that the control of apoptosis is likely to be implicated. in addition, some inbred laboratory mice strains can carry the 2 0 -5 0 -oligoadenylate synthetase gene, flv r , which confers flavivirus resistance (urosevic et al., 1997) . the animals are productively infected with flaviviruses, but produce low virus titer (brinton & perelygin, 2003; barkhash et al., 2010) . mice that are susceptible to flavivirus infection carry the flv s allele. the flv r -like allele has also been characterized in wild mice and could be a partial explanation of flavivirus persistence in rodents in nature (urosevic et al., 1997) . additional vertebrate genes, apart from the flv, could play a role in varied susceptibilities of different mouse strains to flavivirus infection. this was suggested from recent observations that tbev-infected balbc mice were moderately resistant, sts mice are highly resistant, whereas the balbc/sts recombinant mice were highly sensitive to infection (palus et al., 2013) . immunosuppression may also be a potentiating factor for the establishment of flavivirus persistence in animal hosts. for example, wnv persistence was demonstrated by the detection of wnv rna and immunohistochemistry in brain tissue of an immunosuppressed 57-year-old man 4 months after the initial diagnosis (penn et al., 2006) . the follicular b cell lymphoma, from which he had suffered, may have facilitated, or aggravated, the persistence of wnv. in mice, transient immunosuppression with cyclophosphamide leads to wnv recrudescence (appler et al., 2010) , an observation suggesting that some aspects of the immune system operate to restrict wnv replication during persistence. however, the situation is complicated because wnv is able to persist for up to 16 months in the face of a robust humoral immune response in c57bl/6 mice (appler et al., 2010) . furthermore, wnv persistence was reported in the brains of cd8 + t cell-deficient rodents (shrestha & diamond, 2004) , but the cd8 + t cell deficiency did not affect the antibody response in this mouse model. knowledge about specific immune system components that could facilitate or control viral persistence remains to be characterized. infection by vbfvs of arthropod cells has not received the same degree of study; however, acute infection is not accompanied by the same cytopathic response observed in mammalian cells. in addition, very little is known about how or in what tissues tbfvs persist in the ticks, but the viruses likely evolved mechanisms of modulating or evading the tick immune system over the millenia (robertson et al., 2009) . persistent infection of the i. scapularis cell line ise-6 (munderloh et al., 1994) was readily established and was noncytopathic (offerdahl et al., 2012) . detailed studies of persistent tbfv infection in arthropod cell lines using contemporary techniques and methods are certain to yield useful and interesting information. the mechanism(s) of how mbfvs persist in mosquitoes also remains a suitable topic for investigation. slev persists in the midgut of c. pipiens for hours before infecting the midgut epithelium (whitfield et al., 1973; brackney et al., 2008a) , but this is a short time. interestingly, approximately two-thirds of flavivirus-related sequences were reportedly detected as integrated dsdna form in laboratory-bred and wild aedes mosquitoes (crochu et al., 2004) , although detection of a complete flavivirus genome was not reported. furthermore, the finding of partial flavivirus-like sequences in dna form is not clear. clearly, research into the biology of flavivirus persistence in mosquitoes and ticks has been limited and is worthy of extensive additional research. in summary, it should be apparent that both viral and host factors play a role in the initiation and maintenance of persistent infection at both the cellular and organismal levels. in addition, successful persistence in nature almost certainly depends upon ecological and environmental forces, but these latter factors are wholly beyond the scope of this limited review. multiple rna and dna viruses are known to establish persistence in culture as well as in humans and animals. consideration of several may provide insight, if not direct parallels, useful in the study of biology of persistent vbfv infections. the hepacivirus genus, containing hepatitis c virus (hcv) species, belongs to the flaviviridae family, together with the flavivirus genus under which vbfvs are classified. despite the virus-specific cytotoxic t lymphocytes and antibodies, persistent hcv infections, which are established with high efficiency, are known to occur in humans and animals, such as chimpanzees (main et al., 1979; caini et al., 2012; mcnally et al., 2012) . hcv persistence is associated with various strategies, such as the high genetic variability that facilitates passive immune evasion. in vivo, hcv fails to activate cd4 + t cells, leading to exhaustion of cd8 + t cells. at cellular level, hcv can block interferon induction by blocking rig-i and mitochondrial antiviral signaling (mavs) using its ns3-ns4a protease, which cleaves the ifn promoter-stimulator 1 (gould, 2001; muñoz-jord an et al., 2005; baril et al., 2009; hudopisk et al., 2013; perera-lecoin et al., 2013) . hantavirus infections are another interesting example of viral persistence. hantaviruses are segmented, rna viruses that cause lifelong infections in their reservoir rodent hosts, despite high levels of neutralizing antibodies (botten et al., 2000; meyer & schmaljohn, 2000; easterbrook & klein, 2008) . pathogen recognition receptors, such as rig-i and tlr7, are not elevated in the lungs of infected rats, suggesting that evasion of viral recognition may contribute toward the establishment of a persistent infection. perhaps, the reason for noninduction of rig-i is the fact that hantaviruses do not produce detectable amounts of dsrna (wang et al., 2011) . ifns, such as ifn-b, ifn-k, mxa, and pro-inflammatory chemokines, cytokines, and transcription factor genes are elevated midway in the infection followed by a down-regulation that favors the expression of tgf-b (schountz et al., 2012) . a continued up-regulation of the cytokines could be detrimental to the cell, and the virus would fail to persist. results of a recently established animal model also show that host-adapted snv achieves prolonged and disseminated infection, with no disease in hamsters (safronetz et al., 2013) . therefore, hantaviruses may have evolved mechanisms of manipulating target genes, which establishes persistence when induced. one of the best-studied models of persistent rna virus infection is the rodent-borne arenavirus, lymphocytic choriomeningitis virus (lcmv). in lcmv clone 13 (cl13), a glutamine at position 1079 in the glycoprotein (lysine in the arm 53b strain of lcmv) is important for persistence, but its precise function is unknown (salvato et al., 1991; moskophidis et al., 1995) . the mechanisms of lcmv persistence are linked to the down-regulation of mhc and costimulatory molecules, inflammatory cytokines, as well as virus-induced production of immunosuppressive cytokines (ng et al., 2011) . the inability of cd134-deficient mice to control lcmv infection over time was reported to be a result of cd4 and cd8 responses (boettler et al., 2012) . in addition, ox40 also has a role in the establishment of persistent lmcv infection (boettler et al., 2012) . a recent report showed that ifn blockade of type i ifn signaling results in a cd4 t cell-dependent clearance of lcmv cl13 (teijaro et al., 2013) . this is an interesting observation considering that ifn induction is part of the antiviral response and that its induction can lead to apoptosis. however, the precise mechanism of persistence is not completely clear (easterbrook & klein, 2008) , but it also demonstrates 'clever' viral manipulation of the host system to establish persistence. coxsackievirus infections are another system from which lessons might be drawn. coxsackievirus b3 has been implicated toward the development of certain chronic muscle diseases, such as chronic inflammatory myopathy (lodge et al., 1987; tam & messner, 1999) . coxsackievirus persistence in cell culture can take two forms: (1) an incurable steady state characterized by nonlytic virus infection and (2) an antiviral-curable carrier culture system (brinton, 2013; pierson & kielian, 2013) . while mechanisms of coxsackievirus persistence are not completely understood, down-regulation of the coxsackievirus receptor (car) has been suggested to play a role in viral persistence (varatharaj, 2010) . down-regulation of car in coxsackievirus-infected hl-1 cells occurs rapidly from 60% following three passages to 90% at passage 8 (varatharaj, 2010) . the importance of car down-regulation is emphasized by the fact that in vitro car knockout results in reduced viral replication as well as virus-induced cell lysis (tomori, 2004; gulati & maheshwari, 2007) . these selected examples simply highlight the diversity of possible mechanisms that the vbfv might harness in initiating and maintaining persistence and provide concepts that might be useful to investigate. in the preceding sections, we surveyed a substantial literature with relevance to various aspects of flavivirus persistence. elucidation of how flaviviruses persist in humans could help toward the development of therapeutic interventions that could alleviate morbidity and budgetary burdens associated with neurological sequelae. despite the current knowledge, a relative dearth of knowledge still exists, and additional research is merited on these significant human pathogens. for instance, the definition of specific viral proteins and cellular factors and their interactions in the establishment and maintenance of persistent infection is very limited. as noted, for flaviviruses to persist in infected cells in culture or in vivo, specific host defenses need to be evaded or controlled. the role of ns5 as an interferon antagonist (especially in tbfvs) has been established (best et al., 2005) , but its ifn antagonism in the context of vbfv persistence has not been fully explored. this is also true for mbfv ns4b, which impairs ifn-a/b induction via jak/stat signaling (muñoz-jord an et al., 2005) . furthermore, mutations in ns2a of kunjin virus result in increased ifn levels, suggesting an ifn antagonistic role of ns2a (liu et al., 2004) . intriguing is the fact that vbfvs antagonize ifn even in the cells that will eventually die in the acute phase of infection. as ifn antagonism seems to be important for hcv, interrogating the role of these vbfv proteins to ascertain their role in the establishment of viral persistence will be critical. the specific mammalian host immune responses that are evaded or controlled also need to be identified precisely. although overexpression of bcl-2 in bhk and cho cells resulted in the inhibition of apoptosis and jev persistence, a direct viral effect on bcl-2 was not determined (liao et al., 1998) . it would be useful to understand which, and how, vbfv proteins interact with the bcl-2 pathway. indeed, other host pathways could be involved and need to be elucidated further. the exact correlates of persistent flavivirus infection in the arachnid or insect vector host also need to be determined. the biology of ticks and that of mammals is completely different. for ixodid tick vectors, identifying genetic or biological targets that could play a role in viral persistence is imperative. these vector mechanisms might find application, when known, in efforts to control natural flavivirus persistence cycles in ticks. the mechanism(s) could involve viral genetic changes, such as specific mutations that may render the virus less detectable in infected cells. an important question for transmission and viral evolution dynamics is why arthropod vectors do not appear to be killed by viral infection. the down-regulation of the car, as a coevolutionary development that favors persistence, is intriguing (fechner et al., 2007; pinkert et al., 2011) . for flaviviruses, various receptors have been identified as possible virus-entry mechanisms (smit et al., 2011; perera-lecoin et al., 2013) . investigations into whether there is a down-regulation of flavivirus receptors, as in coxsackievirus persistence, could be useful in defining flavivirus persistence mechanism (s). finally, the establishment of relevant animal models of vbfv persistence will also be crucial for understanding the dynamics of viral persistence and host responses. animals that serve as the natural host reservoirs will be key in developing these models. some models have been established, but may have failed to answer ecologically important question. thus, future work will combine studies encompassing the biology of vbfvs, molecular cell biology, animal models, and eventually virus-host ecology. rodents as sentinels for the prevalence of tick-borne encephalitis virus polypyrimidine-tract-binding protein is a component of the hcv rna replication complex and necessary 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shedding of west nile virus in urine of experimentally infected hamsters replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the flavivirus genus molecular characterization of virus-specific rna produced in the brains of flavivirus-susceptible and -resistant mice after challenge with murray valley encephalitis virus clinical and epidemiological features of tick-borne encephalitis in perm region ecology and geographical expansion of japanese encephalitis virus neurologic sequelae of west nile virus infection encephalitis in the clinical spectrum of dengue infection the transmission of the tick-borne encephalitis virus via cow's milk genome cyclization as strategy for flavivirus rna replication old world hantaviruses do not produce detectable amounts of dsrna in infected cells and the 5 0 termini of their genomic rnas are monophosphorylated relation of genetic phylogeny and geographical distance of tick-borne encephalitis virus in central europe composition and three-dimensional architecture of the dengue virus replication and assembly sites tick-borne encephalitis virus ns5 associates with membrane protein scribble and impairs interferon-stimulated jak-stat signalling ultrastructure of kunjin virus-infected cells: colocalization of ns1 and ns3 with double-stranded rna, and of ns2b with ns3, in virus-induced membrane structures kunjin rna replication and applications of kunjin replicons detection of persistent west nile virus rna in experimentally and naturally infected avian hosts st louis encephalitis virus: an ultrastructural study of infection in a mosquito vector west nile virus infection in the golden hamster (mesocricetus auratus): a model for west nile encephalitis induction of inflammation by west nile virus capsid through the caspase-9 apoptotic pathway west nile virus capsid protein induces p53-mediated apoptosis via the sequestration of hdm2 to the nucleolus japanese encephalitis virus ns2b-ns3 protease induces caspase 3 activation and mitochondria-mediated apoptosis in human medulloblastoma cells crystal structure of the dengue virus rna-dependent rna polymerase catalytic domain at 185-angstrom resolution immune evasion strategies of flaviviruses the ns3 protease and helicase domains of japanese encephalitis virus trigger cell death via caspase dependent and independent pathways structures of immature flavivirus particles west nile virus genome cyclization and rna replication require two pairs of long-distance rna interactions japanese encephalitis virus rna not detected in urine the persistence of louping ill virus in immunosuppressed guinea-pigs a single-amino acid substitution in west nile virus 2k peptide between ns4a and ns4b confers resistance to lycorine, a flavivirus inhibitor this work was supported by the intramural research program, national institute of allergy and infectious diseases, national institutes of health (nih). we wish to acknowledge the able assistance of anita mora and heather murphy for graphic support. danielle offerdahl and jennifer lam provided assistance in manuscript preparation. key: cord-004247-lagv3tp7 authors: hooft van huijsduijnen, rob; kojima, somei; carter, dee; okabe, hisafumi; sato, akihide; akahata, wataru; wells, timothy n. c.; katsuno, kei title: reassessing therapeutic antibodies for neglected and tropical diseases date: 2020-01-30 journal: plos negl trop dis doi: 10.1371/journal.pntd.0007860 sha: doc_id: 4247 cord_uid: lagv3tp7 in the past two decades there has been a significant expansion in the number of new therapeutic monoclonal antibodies (mabs) that are approved by regulators. the discovery of these new medicines has been driven primarily by new approaches in inflammatory diseases and oncology, especially in immuno-oncology. other recent successes have included new antibodies for use in viral diseases, including hiv. the perception of very high costs associated with mabs has led to the assumption that they play no role in prophylaxis for diseases of poverty. however, improvements in antibody-expression yields and manufacturing processes indicate this is a cost-effective option for providing protection from many types of infection that should be revisited. recent technology developments also indicate that several months of protection could be achieved with a single dose. moreover, new methods in b cell sorting now enable the systematic identification of high-quality antibodies from humanized mice, or patients. this review discusses the potential for passive immunization against schistosomiasis, fungal infections, dengue, and other neglected diseases. the last three decades have seen a dramatic rise in the use of monoclonal antibodies (mabs) as therapeutics. by 2017, a total of 78 antibodies had been approved by the us food and drug administration (fda) or the european medicines agency (ema) [1] , with a further 11 approved by the fda in 2018 [2, 3]. beyond this, over 570 antibodies are in clinical development [4] . the global mab market reached us$100 billion annually in 2017 [5], underscoring the considerable economic importance of these medicines. the success of mabs starts with the general applicability of the technology used to make them. antibodies can be developed that have not only high affinity for their targets but also high selectivity, meaning they are less likely to have unwanted side effects and unexpected safety problems. mabs are particularly good at targeting cell-surface proteins and circulating protein factors; this is in contrast to small molecules, in which cell surface protein-protein a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in infectious disease, the biggest success story has been the use of antibodies to provide prophylaxis against infection by respiratory syncytial virus (rsv). rsv is responsible for over 30 million episodes of new lower respiratory tract infections, particularly targeting children 5 years and younger, resulting in an estimated 48,000 to 74,500 deaths globally (2015 estimates) [14] . palivizumab, dosed at 15 mg/kg, is given as a monthly intramuscular injection throughout the rsv season, which is suitable for small babies but would not be appealing for adults. a follow-on antibody-medi8897, now in phase ii clinical studies [15]-is 100 times more potent than palivizumab in vitro. it is being developed as a single 50 mg injection to cover the typical 5-month season. taken together, these cases illustrate, that after initial proof of concept, it is possible to find second-generation antibodies with more simple routes of delivery and sufficient potency to support relatively infrequent dosing in the field. a major objection to the use of mabs in infectious diseases as a therapeutic class is their high price, which is presumed to be a consequence of a high cost of goods. manufacturing costs are a particularly important attribute for any medication that is developed for diseases of poverty in low-and middle-income countries (lmics). however, production efficiency of mabs has increased dramatically over recent decades, and cell-culture expression levels around 4 g/l or even higher are common. a published analysis from a decade ago already noted that the costs of mab production were dropping from us$300/g to us$20/g when being produced at the 10-tonne-per-year scale, potentially using large, 100,000 l reactors [16] . a more recent analysis has similar estimates [17] , which-depending on process and volume-range from us$20/g to us$80/g [18] . for a public health application against an infectious disease in an lmic, an intramuscular or subcutaneous delivery would be preferable (rather than the intravenous route). the intramuscular route effectively limits the dose; an injection volume of 1.0 ml is probably close to the maximum acceptable in small children, and antibodies are typically only soluble to around 100 mg/ml, which gives a limitation on the total dose of around 100 mg, corresponding to a range of 5 to 20 mg/kg [19] . taken together, the cost of goods of an injection based on current numbers would be in the range of us$1 to us$8. benchmarking such costs is difficult, but as a comparison, the recently launched malaria vaccine provides less than 50% protection for 3 injections at around us$5 each. the programmatic cost of protecting a child from malaria for a year in the sahel using existing low-cost oral medicines has been estimated at us$3.40 by unitaid [20] . an injectable therapeutic that could protect a child for a season for this price provides a cost-effective option. the other important factor to manage is the reduction of the frequency of administration, to make the prophylaxis useful in resource-poor settings. mabs used in therapy are generally iggs (immunoglobulin gs), which have plasma half-lives of 20 to 25 days in humans [21, 22] . such antibodies can be used to provide several months of protection, as in the case of the once-per-season anti-rsv antibody medi8897 (nirsevimab) discussed earlier or the anti-calcitonin gene-related peptide (anti-cgrp) fremanezumab, recently approved for prevention of migraines with a dose of 675 mg every 3 months. an alternative to simply increasing the dose would be increasing the half-life of the circulating mab. several strategies have been proposed here [23] : mutations can be made in the igg fc (fragment crystallizable) region, which interacts with the receptors responsible for the uptake of antibodies. such mutations, as for medi8897, can significantly extend an mab's plasma half-life. mutations derived from the neonatal fc receptor (fcrn) increase the strength of the interaction between the fc region and the fc receptors under the acidic ph conditions in lysosomes, and as a result, the receptor-fc complex is recycled back to the cell surface, escaping degradation [24, 25] . this approach was followed for bevacizumab and cetuximab, by mutating met 428 to leu and asn 434 to ser. discussion about even longer periods of protection has been stimulated by recent results from virally mediated expression of antibodies targeting hiv [26] . these technologies hold out the promise of sustained serum titers of antibodies for more than a year from a single injection, although the technological development is more complicated than for the direct injection of antibodies. one final objection to the goal of administering monoclonals is that vaccination would instead provide lifelong coverage. however, for some diseases, the development of long-lasting potent vaccines still remains elusive. many pathogens will not raise an adequate immune response, due to immunosuppression. finally, antibodies give immediate protection, important during rapidly evolving epidemics or pandemics and for individuals being deployed into zones of high transmission. antibodies would thus form part of the overall armamentarium, along with drugs, bed nets and insecticides, and vaccines. first and foremost for the development of mabs is the question of suitable antigen targets. viruses are the simplest pathogens, and their genomes allow limited means to evade host immunity; therefore, passive and active vaccinations have been most successful in this area. therapeutic antibodies have been developed and marketed for respiratory syncytial, varicella zoster, vaccinia, and hepatitis b viruses (hbvs) [27, 28] . historically, immune responses against viruses were considered purely cellular and those against other types of pathogens mostly humoral (antibody based) and less effective. however, nowadays the interplay between both systems is better understood, for instance, with therapeutic opportunities against viruses such as rabies that rely on passive immunization. mabs that target the initial stages of an infection may provide prophylaxis, and the repertoire of potential antigens for such approaches is greatly helped by the increased availability of genomic and transcriptional information. passive immunization is especially effective in infections, such as those with the rabies virus, which avoids apoptosis of infected neurons while killing protective t cells so that carriers do not mount an immune response themselves [29] . a recent review [30] lists 21 mabs (and a nanobody, a single-chain type of antibody produced by camelids) that are currently in clinical trials in the united states. in addition, polyclonal antibodies have been approved in the european union or the us for cytomegalovirus, hepatitis viruses a and c, and postexposure prophylaxis against measles, rabies, rubella, and tetanus [31]. these approaches appear most suitable for virus infections and for protection against the consequences of snake venom but might also be applied to other types of disease. there are fewer marketed antibodies that target bacteria, and these target toxins, namely anthrax and clostridium cytotoxin [27] . this paucity may be due to a lack of discovery efforts, a consequence of the historic availability of cheap and effective antibiotics. however, with the worrying rise of antibiotic resistance, the tide is turning [32] . immunization against protist parasites and fungi is generally ineffective. one problem is that organisms such as trypanosoma brucei (which causes sleeping sickness) and plasmodium (malarial parasites) encode dozens or even hundreds of different surface antigens that are sequentially exposed at their most abundant stage, outpacing the host's immune system. for some pathogens, clinical trials in infected patients are not possible or desirable. in the case of inhalational anthrax, raxibacumab has been approved using the fda "animal rule" [33]. this provides a route forward in disease areas in which trials with infected human patients are not possible or not ethical. recently, the case for discovering mabs for neglected tropical and other infectious diseases was reviewed [34] . this work took a broad, category-wide approach (viruses, bacteria, parasites) and also included venoms. snake bites, and the challenge of envenoming, represent a serious health problem, resulting in 81,000 to 138,000 deaths yearly [35] , and were recently included in the world health organization (who) list of neglected tropical diseases. the discovery of mabs in this area logically follows the long-standing therapeutic successes with passive immunization [36] , and the costs and feasibility of developing mabs were recently reviewed [34, 37] [38], including specific clinical development and regulatory challenges [39] . in this review, we selected diseases prioritized by the global health innovative technology (ghit) fund, an international collaboration between the public and private sectors, supporting collaborations between japanese and non-japanese entities to advance global health research and development [40, 41] . the present vaccine discovery statuses for these diseases are listed in table 1 . human schistosomiasis is caused by infection, mainly with one of 4 species of the blood fluke belonging to schistosomatoidea. it is estimated that there are over 200 million cases annually in africa, with two-thirds being schistosoma haematobium and the remainder s. mansoni. in southeast asia, other species such as s. japonicum and s. mekongi cause smaller numbers of infections [64] . the mortality associated with schistosomal infections is difficult to calculate. comorbidities linked to anemia are common, and infection with s. haematobium can lead to female genital schistosomiasis, which results in increased susceptibility to hiv infection [65] . the life cycle of the parasite is complex, involving both a snail and a human host. in the infective stage, cercariae migrate and penetrate the human host skin to become schistosomula, which then move with the venous circulation, initially to the lungs. after further maturation, the parasite migrates via the heart to the portal circulation and the intestines; from there, eggs are excreted with the feces or, passing through the bladder wall, in urine. prophylaxis could therefore either be causal-and prevent entry and survival of the cercariae-or suppressive, by killing the juvenile and adult worms. clearly, as with the malarial parasites, the more stages of the parasite life cycle that can be inhibited, the more effective the protection will be. the molecular targets best addressed by antibodies are still not clear. some life cycle differences can be characterized: schistosomula recovered from the lungs are elongated and more resistant to antibody-dependent, cell-mediated cytotoxicity than newly transformed schistosomula. praziquantel, the standard treatment for schistosomiasis, acts only against adult worms and has no effects on the juveniles, thus repeat treatment is required to eliminate all the worms in a patient. an ideal treatment would therefore kill both adult and juvenile worms at a submicromolar concentration. this is effectively the target candidate profile for a small molecule inhibitor [66] . no target-product profile (tpp) for prophylaxis against schistosomiasis has been published, but it is possible to start to build one, with the recent characterization of praziquantel for such use in murine models and in pediatrics. clinically, this treatment reduces infection by 70% to 80% using doses of 40 to 60 mg/kg [67] . in a murine model with infection by s. mansoni cercariae, the maximum dose of 400 mg/kg killed 96% of all adult worms [68]. however, this murine dose produced a peak exposure of 6 ng/ml, some 10-fold higher than the exposure observed in children at the recommended regimen of 40 to 60 mg/kg [69, 70] . it seems pragmatic to project that an antibody that can reduce the worm burden by 80% in the mouse model, at a dose that is clinically achievable in humans, could be acceptable. the maximally practicable injection of an antibody in children is 2 to 5 mg/kg, and given the relatively consistent allometry between humans and mice for mabs, this corresponds to a dose of 80 to 200 μg in mice [19] . target identification for schistosomiasis is not straightforward. unlike malaria, the vaccine field offers no clues as to useful antibody targets. two vaccine candidates are currently being tested in humans. the first is based on the fatty acid-binding protein (fabp) sm-14 (schistosoma mansoni-14), which is being tested in 2 small, open-label pilot studies in senegal (nct03041766 and nct03799510). a larger 300-participant trial in uganda, nct03910972, is being planned for a vaccine based on sm-tsp-2 (schistosoma mansoni-tetraspanin-2) but is not expected to report before 2023. clues about other possible ways forward, in terms of antigen selection, come from a variety of areas.first, schistosoma are helminths (or flatworms), and the initial immune response is a characteristically strong t helper 2 (th2) cytokine reaction, which is not seen in viral or bacterial infections and is less pronounced in protozoan infections. the cytokines that are released include interleukin (il)-5, which drives the production of eosinophils to attack the parasite, and il-4 and il-13, which drive the production of immunoglobulin e (ige). epidemiological studies in endemic areas suggest that an age-dependent immunity may develop against infection, or against reinfection after treatment [71-73]. there is a good correlation between this protection and the development of ige antibodies, resulting from th2 responses [73, 74] . a hybridoma that produces a monoclonal ige antibody to s. japonicum, sj18ε.1, was identified [57]. this mab was protective in an in vitro, antigen-dependent, cellular cytotoxicity assay with rat macrophages or eosinophils and also in vivo during the early phase of infection second, beyond the cell-surface proteins, schistosomes also express a large number of glycans as part of their glycoprotein and glycolipid repertoire, and an antibody response against those glycans is mounted by the infected host [80] . in 2 cases, the specific antibodies produced could be identified using b cell cloning [81] or after infection of mice genetically modified to express human antibody repertoires [82] . interestingly, there has been no systematic analysis of the antibodies produced that target the various stages of the parasite life cycle, although early work on s. mekongi suggests schistosomula and adult worm extracts would induce a better response [83] . in addition to stimulating a th2 response, schistosome infection can suppress t-cell activation [84-86]; s. mansoni uses 2 distinct mechanisms to suppress t-cell activation, resulting in the selective up-regulation of pd-l1 on the surface of splenic f4/80 + macrophages. the presence of fatigued or anergized t cells opens up the possibility for cotherapy of low doses of mabs against programmed cell death protein-1 (pd-1) or pd-l1 with antibodies that reverse the anergy. the treatment of invasive fungal infections (ifis) remains a major challenge worldwide. there are few broad-spectrum antifungal drugs; the most effective have substantial toxicity concerns, and well-tolerated drugs used prophylactically frequently induce resistance. even with the best current treatment, the risk for mortality due to an ifi can be higher than 40%. for low-income countries, this figure is substantially worse, as many invasive infections are uniformly fatal without treatment, and estimates of global mortality involving fungal infections are as high as 1.5 million annually [87-89]. in addition, fungal infections are the cause of significant comorbidity and mortality in hiv patients. modeling studies suggest that optimal therapy could save the lives of 1.6 million hiv patients over a 5-year period [90]. the discussion here will focus on mabs developed for the following significant fungal pathogens: cryptococcus, pneumocystis and paracoccidioides, and candida. in addition to antibodies that directly target and inhibit the fungal pathogen, mabs can be directed to checkpoints that control the host immune response. this approach may be particularly useful against fungal pathogens for which sustained infection is characterized by a shift from a protective th1 or th17 response to a noninflammatory th2 response. the clinical use of anti-il17 in rheumatoid arthritis clearly exacerbates fungal infection [91], although it remains to be seen whether the reverse-stimulating this pathway-has a clinically useful effect. cryptococcus neoformans predominantly causes opportunistic infection in patients with hiv/aids and is responsible for a large burden of aids-related disease and death in sub-saharan africa [92] . although the rate of infection has decreased in recent years through greatly improved access to antiretroviral therapy (art), mortality in infected patients has not declined, demonstrating the failure of antifungal development to keep pace with improved antiviral treatment. cryptococcosis first manifests as a pulmonary disease but spreads hematogenously to the cerebrospinal fluid and brain to cause meningitis and meningoencephalitis, adding the complexity of crossing the blood-brain barrier to drug development. the ability of mabs to protect against a lethal cryptococcal infection in mice was first demonstrated over three decades ago [93], with subsequent work demonstrating the efficacy of various mabs that target the polysaccharide capsule, an essential virulence factor and the primary host-pathogen interface of cryptococcus infection [94] [95] [96] . among these, mab 18b7 was evaluated in a phase i clinical trial [50] and was found to produce a modest reduction in circulating cryptococcal antigen. further development has been hampered, however, by difficulties in securing funding for a disease for which financial returns are likely to be low-a common problem for neglected infectious diseases. subsequent studies have examined mabs that target cryptococcal melanin [97] , β-glucan [98] , or glucosylceramide ( [99] [100] [101] ; see [102] for additional in vivo and in vitro examples). host cd40 has also been targeted to stimulate the immune response [103] . in addition to highlighting the potential of mabs as therapeutics, these studies have demonstrated the diversity of inhibitory actions that mabs can perform on cryptococcal cells, which can include opsonization and increased phagocytosis, inhibition of fungal growth, capsular polysaccharide release and biofilm formation, antibody-mediated target cleavage, and augmentation of the host response [104] [105] [106] [107] . pneumocystis, like cryptococcus, is an important opportunistic pathogen in hiv/aids and other immunosuppressed patient populations, with estimates of up to 500,000 cases per year [87]. however, unlike cryptococcus, which is acquired from the environment, pneumocystis spp. are commensals, with different species occurring in the lungs of many mammals. the capacity for endogenous infection and for acquisition from asymptomatic carriers makes pneumocystis an attractive target for immunoprophylaxis. to this end, a variety of programs are aimed at developing a pneumocystis vaccine [108, 109] , and passive immunization studies have been initiated using mabs raised to pneumocystis epitopes. intranasal administration of 4f11, an mab that recognizes the pneumocystis kexin-like protein kex1, was able to prevent transmission of pneumocystis pneumonia from infected to susceptible cohoused mice, demonstrating the feasibility of this approach [110] . emerging evidence of the importance of b cellmediated immunity to pneumocystis infection strongly supports further research in this area [111] . paracoccidioides brasiliensis, while less globally prevalent than cryptococcus or pneumocystis, is the most important cause of ifis in latin america. mabs have been raised against the major p. brasiliensis antigens glycoprotein 43 (gp43) and gp70. in an infected mouse model, anti-gp43 activity, mediated by mab e3-enhanced phagocytosis of p. brasiliensis cells, increased interferon-γ production and led to a reduction in the fungal burden [112] , while anti-gp70 mabs significantly reduced fungal colony-forming units and almost completely abolished granuloma formation in the lungs [113] . antibodies raised against mouse cd25 + regulatory t (t reg ) cells, which control immunity and excessive inflammation, depleted cd25 + cells, resulting in less severe tissue inflammation, with reduced mortality in susceptible mice [112] . again, this demonstrates the potential for mabs to exert control of fungal infection, either directly by incapacitating the fungal cells or indirectly via modulation of the host response. candida albicans is the most commonly isolated fungal pathogen globally and is associated with significant morbidity and mortality, particularly in patients with hiv or tuberculosis (tb) infection [114] . a recent paper demonstrated the cloning of antibody genes from b cell cultures derived from patients infected with c. albicans [115] . these antibodies were capable of stimulating opsonophagocytic macrophage activity and provided protection in a murine model of disseminated candidiasis. in summary, mabs offer tremendous potential to augment the antifungal arsenal: animal models have provided promising results, there is a wide range of potential targets, they have the capacity to both inhibit the pathogen and augment the host response, and it may be possible to target diverse fungal pathogens with an appropriate mab cocktail or by targeting panfungal antigens. use of mabs as adjuvants to existing antifungal drugs also shows promise [116] . however, it should be noted that not all mabs are inhibitory; indeed, some may worsen infection, species and strain specificity can be an issue, and the mechanistic basis of inhibition by different mabs can vary dramatically [117] . to date, only 2 mab treatments have advanced to clinical trials: the previously mentioned 18b7 in cryptococcus, and mycograb, a heat shock protein 90 (hsp90)-specific antibody fragment, which showed promise for treating candida infections but failed to make it to market due to production difficulties and unresolved safety issues. antifungal immunomodulation is a complex area, and the field is still emerging. these preliminary studies highlight the potential for exciting new advances in mab research and application, both for understanding fungal immunity and for manipulating it to tackle lifethreatening fungal infections. dengue fever is a mosquito-borne viral infection found in tropical and subtropical regions around the world. the dengue virus (denv) is transmitted by female mosquitoes, mainly of the species aedes aegypti and, to a lesser extent, a. albopictus. there are 4 distinct serotypes-denv-1 to denv-4-of the virus, and all serotypes are presently circulating in endemic areas. denv infects cells of the human immune system and other cell types, leading to symptoms that include high fever, severe headache, severe pain behind the eyes, joint pain, muscle and bone pain, rash, and mild bleeding. in severe cases, plasma leaks out of the circulatory system, which can be fatal. the global incidence of dengue has grown dramatically in recent decades. one recent study estimated that approximately 390 million people are infected, of which 96 million manifest clinically each year [118] . who estimates that, globally, 500,000 people with severe dengue require hospitalization each year and that 2.5% of these infections are lethal. antibody-dependent enhancement (ade) is problematic in dengue infection. the presence of subneutralizing levels of flavivirus cross-reactive serum antibodies (acting against one member of the virus family) may result in an increase in infectivity via ade of another virus member or serotype, which is observed particularly after secondary dengue infection [119, 120] . despite decades of effort, there is no effective treatment against dengue. currently, dengvaxia is approved by the fda and is the only licensed dengue vaccine in the world. this is also a live attenuated tetravalent dengue vaccine developed by sanofi pasteur [121] that has been approved in several countries. however, interim results from long-term safety follow-up studies demonstrated an increased risk for hospitalization of vaccine-sensitized individuals [122] , suggesting that ade-related concerns are relevant. it has been reported that non-neutralizing levels of anti-denv antibody can enhance viral entry into host cells by forming a denvantibody complex [123, 124] . there is concern that an incomplete antibody against denvs may cause ade-mediated severe dengue disease. hence, there is a need for a safe and highly efficacious dengue therapy or vaccine that provides immunity against all 4 serotypes simultaneously. mab therapy is an alternative to vaccines and other therapies against dengue. many mabs against dengue from mice and humans have been characterized, and the use of mabs has also been explored as a therapeutic option. antibody sign-3c, identified by the singapore immunology network, neutralized all 4 dengue serotypes and decreased viremia of all serotypes in mice when given 2 days after infection [43] . a humanized mab visterra 513 (vis513), a panserotype anti-denv developed by visterra [44, 45] , which binds e protein domain iii (ediii) and neutralizes all 4 serotypes of denv, also showed useful antiviral utility. vis513 (25 mg/kg or 50 mg/kg) was administered at 5 days post infection in nonhuman primates, and no infectious virus could be detected by either plaque assay or virus isolation after treatment, a finding that was, however, not mirrored by reverse transcription pcr (rt-pcr) findings. a human challenge model is now available for clinical research in dengue [125] . in this model, the efficacy and safety profile of therapeutic antibodies can be evaluated rapidly in small-scale clinical settings, prior to traditional large-scale field studies with naturally infected patients. to develop mabs for dengue therapy, it is important to consider an approach that prevents or reduces ade, and several studies that address this link have been undertaken. a neutralizing human mab, d23-1b3b9, that targets the fusion loop in domain ii showed strong neutralizing activity against all 4 denv serotypes [46] . however, at subneutralizing concentrations, it also elicited ade activity in vitro [47] . to reduce the ade, injampa and coworkers [48] modified the d23-1b3b9 antibody fc domain at position n297q. the modified antibody kept the same cross-neutralizing activity to all 4 serotypes as those of wild-type antibody but lacked ade activity against all 4 serotypes at subneutralizing concentrations. in another neutralizing mab, sign-3c, 2 leucine to alanine mutations were engineered in the fc part, abrogating binding to fc gamma receptors [43] . this mutant fc version (sign-3c-lala) protected mice, while ade was completely abrogated. similarly, mab11/mutated fragment crystallizable region (mutfc) (an mab that is unable to bind to cells with fcγ receptors [fcγr]) and potentiate ade have been used as a prophylactic therapy [49] . passive immunization with this mab (at 25 mg/kg) reduced viral load and disease progression in nonhuman primates. here again, therapeutic antibodies can also be used for prophylaxis, affording immediate and reliable protection. tb tb is a good example of the gap between a pathogen's prevalence and burden. mycobacterium tuberculosis spreads easily among human populations; presently, about one-third of all humans are infected, and new infections occur in 1% of the population each year. among these billions of carriers, there are, however, "only" 10 million active tb infections, with 1.3 million deaths in 2016. thus, the vast majority of carriers keep the pathogen in check. tb exerts 2 levels of immune evasion: one in which it is maintained in a latent state and one in which it breaks free and causes active disease [126] . several studies have tested antibody therapy in tb, with varying success (reviewed in [127] ). even if an mab treatment would not be curative, shortening the standard treatment of patients infected with multidrug-resistant (mdr) and extensively drug-resistant (xdr) strains would represent a major advance. in addition, "checkpoint blockade during chronic tb infection requires further consideration" [128] . the role of protective antibodies in malaria was demonstrated over 40 years ago with the finding that the passive transfer of sera from mice with radiation-attenuated sporozoites delayed the development of infection in other mice [129] . the tpps for malaria are well described [130, 131] . these include a profile for seasonal malaria chemoprevention, a treatment successfully launched in sub-saharan africa in the last 5 years, consisting of a full treatment course of 3 days of amodiaquine and 1 dose of sulfadoxine/pyrimethamine. it is given monthly to children during the rainy season. antibody therapeutics for malaria could (1) prevent the entry (initial infection) of the parasite, (2) block entry of the sporozoite into the liver cells, (3) block entry of the merozoites into the erythrocytes, or (4) block the uptake of the gametocytes into the mosquito (breaking the transmission cycle). one difficulty in targeting the merozoites in symptomatic malaria is that the extracellular phase of the pathogen is relatively short-lived and is only a small part of the life cycle. additionally, the number of merozoites invading erythrocytes is very large (up to 10 12 ), compared with the number of sporozoites invading the liver stages (dozens). as such, the most interesting place to intervene with an mab is at the initial infection of the liver. currently there are 3 mabs published with potent activity against the circumsporozoite protein, csp, and these can reduce the parasitaemia in sporozoite-infected frg (triple mutant fah/raγ2/il2rγ) mice that carry a human liver implant [132] . these mabs include mab317, cloned from b cells obtained from a patient in the recent rts,s vaccine trial [133] , cis43 (circumsporozoite protein 43 [56]), and a set that included mgu12 [134] , cloned from patients vaccinated with irradiated sporozoites. antibodies that block the invasion process of the red blood cells by merozoites represent another possible approach. in studies of the merozoite protein rh5 (reticulocytebinding protein homologue 5) as a potential vaccine, some antibodies were described that could block the cycle of erythrocyte infection [135] . the specific merozoite epitopes are now being characterized, and this could form the basis of a second-generation antibody [136, 137] . a general observation with plasmodium infections is that the human host is unable to mount a sterilizing immune response. one theory is that the parasite can control the t-cell response and induce a state of fatigue or anergy similar to that seen in tumor-invading lymphocytes in immuno-oncology. several studies in mice have demonstrated that blocking pd-l1 or ctla-4 (cytotoxic t-lymphocyte-associated protein 4) improves clearance in mice infected with plasmodium bergheii [138] , and from this and similar experiments, a strong argument can be made that reversal of this fatigue by mild checkpoint inhibitor blockade may be a way of facilitating the host response [11]. hiv mab therapies have also been proposed for hiv. the case for hiv was recently reviewed [139, 140] . two such mabs are in phase iii trials: pro 140 and cenicriviroc. pro 140 targets ccr5 (cysteine-cysteine chemokine receptor type 5) and recently entered phase iib/iii trials for weekly subcutaneous dosing for monotherapy maintenance [141] . cenicriviroc is a dual ccr2 and ccr5 antagonist investigated for a number of indications, including hiv infection [142] . as noted earlier, ibalizumab was recently approved as a second-line treatment for hiv treatment [13]. for hbv infection, the hypothesis is that high circulating hbsag levels prevent a proper immune response. a novel mab, e6f6, is being evaluated for reducing hbsag levels in patients [52] . in addition, the hbv s protein is being targeted for the discovery of therapeutic mabs [143] . in the case of visceral leishmaniasis (vl), il-10 and glucocorticoid-induced tnf-receptorrelated protein have been considered as mab targets [144] , but there has been no systematic analysis of antigens or reported cloning of b cells from infected patients. although mabs have made a massive impact in controlling autoimmune disease, inflammation, and cancer, the relative impact in the world of infectious disease has largely been confined to viral diseases. the use of mabs to protect against rsv infections and the profile of secondgeneration antibodies shows that it is possible to obtain mabs that are sufficiently potent to provide long-term protection with a single intramuscular or subcutaneous injection. with the development of new technologies for cloning antibodies from b cells or plasma cells taken from patients infected with bacteria, viruses, fungi, or even protozoal pathogens, it is possible to quickly obtain fully human antibody collections with potential activity against pathogens in vitro and in vivo. technologies for expressing antibodies are now at the stage in which it is not uncommon to see extremely high levels of expression in cell culture, and taken together with the progress in reducing costs of production, the cost of goods for an antibody injection is starting to enter the range of us$1 to us$10, reaching the edge of what is affordable for infectious diseases of neglected populations. studies of mutations in the fc region confirm that mab half-lives can be extended, and the goal of a single injection to cover an entire season for those infections with seasonality is now a possibility. new technologies with viral delivery offer the promise that a single injection could give protection for even longer periods. for many infectious diseases, we are now seeing the buildup of a portfolio of potential antibodies. in cases in which little progress has been made, a systematic attempt is needed to identify the antibodies resulting from successful control of an infection in patients. beyond these basic antibodies, the availability of new monoclonals with anti-infective activity in vivo would open up the door to even more creative options: bispecific antibodies could be used in key immune cells and could effectively support the natural response to infection. studies in animal models of chronic malaria infection led to the observation that this results in a reduction in the impact of cytotoxic t cells and modulates the t reg capacity, leading to an "exhausted" or ineffective t-cell response [145] . clinical trials are already underway that use immune checkpoint blockers for chronic hiv and hbv infection [11] . this has important implications for any infectious disease in which a single infection does not drive a sterilizing immune response. for such diseases, which include malaria, one question for the longer term is whether immune checkpoint inhibition, or interfering with interferon-α signaling, could be used [128] . two decades ago, one of the biggest challenges of working in anti-infective drug discovery was the need to have new medicines with activities against the widest range of pathogens. in more recent times, the tide has changed, and clinical diagnosis now is such that medicines with a high degree of selectivity and specificity are often preferred-as long as they show good clinical activity. this shift to highly specific medicines favors the use of mabs. given the overall rise in interest for new treatments in infectious diseases caused by concerns about antimicrobial resistance, there is a real opportunity now to progress the newly emerging families of mabs to target infectious diseases of neglected populations. because of outdated preconceptions about this class of therapeutics, few research funds are being allocated to their discovery, resulting in an egg-and-chicken problem (the absence of conspicuous success driving additional efforts). one of the goals of the analysis that we present here is to promote making additional funds available to pursue the initial discovery of mabs for neglected diseases. monoclonal antibodies approved by the ema and fda for therapeutic use protective murine monoclonal antibodies to cryptococcus neoformans antibody-mediated protection in mice with lethal intracerebral cryptococcus neoformans infection monoclonal antibodies to cryptococcus neoformans capsular polysaccharide modify the course of intravenous infection in mice passive immunization with melanin-binding monoclonal antibodies prolongs survival of mice with lethal cryptococcus neoformans infection protection by anti-betaglucan antibodies is associated with restricted beta-1,3 glucan binding specificity and inhibition of fungal growth and adherence human antibodies against a purified glucosylceramide from cryptococcus neoformans inhibit cell budding and fungal growth monoclonal antibody to fungal glucosylceramide protects mice against lethal cryptococcus neoformans infection fungal glucosylceramides: from structural components to biologically active targets of new antimicrobials immunotherapy of cryptococcus infections. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases immunomodulation with cd40 stimulation and interleukin-2 protects mice from disseminated cryptococcosis a monoclonal antibody to cryptococcus neoformans glucuronoxylomannan manifests hydrolytic activity for both peptides and polysaccharides antibody to cryptococcus neoformans glucuronoxylomannan inhibits the release of capsular antigen immunoglobulins in defense, pathogenesis, and therapy of fungal diseases the yin and yang of current antifungal therapeutic strategies: how can we harness our natural defenses? frontiers in pharmacology immunization with pneumocystis cross-reactive antigen 1 (pca1) protects mice against pneumocystis pneumonia and generates antibody to pneumocystis jirovecii vaccine-induced immunogenicity and protection against pneumocystis pneumonia in a nonhuman primate model of hiv and pneumocystis coinfection passive intranasal monoclonal antibody prophylaxis against murine pneumocystis carinii pneumonia an emerging role of b cell immunity in susceptibility to pneumocystis pneumonia. american journal of respiratory cell and molecular biology the monoclonal antibody against the major diagnostic antigen of paracoccidioides brasiliensis mediates immune protection in infected balb/c mice challenged intratracheally with the fungus characterization of gp70 and anti-gp70 monoclonal antibodies in paracoccidioides brasiliensis pathogenesis candida albicans, a major human fungal pathogen single human b cell-derived monoclonal anti-candida antibodies enhance phagocytosis and protect against disseminated candidiasis immune modulators as adjuncts for the prevention and treatment of invasive fungal infections immunotherapy of fungal infections the global distribution and burden of dengue antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications development of the sanofi pasteur tetravalent dengue vaccine: one more step forward the impact of the newly licensed dengue vaccine in endemic countries antibody-enhanced dengue virus infection in primate leukocytes long-term safety assessment of live attenuated tetravalent dengue vaccines: deliberations from a who technical consultation experimental dengue virus challenge of human subjects previously vaccinated with live attenuated tetravalent dengue vaccines mycobacterium tuberculosis: immune evasion, latency and reactivation the role of antibodies in the defense against tuberculosis. tuberculosis-current issues in diagnosis and malaria drives t cells to exhaustion. frontiers in microbiology protective immunity produced by the injection of xirradiated sporozoites of plasmodium berghei hit and lead criteria in drug discovery for infectious diseases of the developing world new developments in anti-malarial target candidate and product profiles assessing drug efficacy against plasmodium falciparum liver stages in vivo structural basis for antibody recognition of the nanp repeats in plasmodium falciparum circumsporozoite protein a public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein human vaccination against rh5 induces neutralizing antimalarial antibodies that inhibit rh5 invasion complex interactions kilchip v1.0: a novel plasmodium falciparum merozoite protein microarray to facilitate malaria vaccine candidate prioritization. frontiers in immunology human antibodies that slow erythrocyte invasion potentiate malaria-neutralizing antibodies programmed death-1 ligand 2-mediated regulation of the pd-l1 to pd-1 axis is essential for establishing cd4(+) t cell immunity antibody-mediated therapy against hiv/aids: where are we standing now? broadly neutralizing anti-hiv-1 monoclonal antibodies in the clinic the return of pro 140, a ccr5-directed mab apobec3g/3a expression in human immunodeficiency virus type 1-infected individuals following initiation of antiretroviral therapy containing cenicriviroc or efavirenz a human monoclonal antibody against hepatitis b surface antigen with potent neutralizing activity combined immune therapy for the treatment of visceral leishmaniasis immune checkpoints and their inhibition in cancer and infectious diseases we thank dr. kiyoshi kita, dean and professor at the school of tropical medicine and global health, nagasaki university; dr. yoshimasa tanaka, assistant professor at the center for therapeutic innovation, nagasaki university; and dr. simon croft, professor at the london school of hygiene and tropical medicine, all of whom provided insight and expertise that greatly assisted the research. key: cord-009101-376snefs authors: strodtbeck, frances title: viral infections of the newborn date: 2015-12-16 journal: j obstet gynecol neonatal nurs doi: 10.1111/j.1552-6909.1995.tb02548.x sha: doc_id: 9101 cord_uid: 376snefs viral infections of the newborn result in significant morbidity and mortality each year. the fetus and newborn are particularly wlnerable to viral infection. the range of expression may vary from no clinical disease to devastating illness and infection occurring before, during, or after birth. nursing management is determined by the specific viral infection, the severity of the illness, and the unique conditions of the newborn and his/her family. promising new therapies are on the horizon that may lessen the severity of viral disease. until such time, the major thrusts of management of neonatal viral disease are prevention of infection and supportive care for the acutely ill newborn. ical infection can vary from recovery to persistent infection with or without sequelae to death (overall, 1992; smith, 1993) . this article reviews the unique pathogenesis of newborn viral infection and the response of the neonatal immune system to viral attack; current information on the clinical features of specific viral diseases is summarized. the diagnosis, management, and nursing care of the newborn with a viral disease are addressed. the first goal and ultimate management of neonatal viral disease is prevention of the infection iral infections of the newborn are a serious con-v cern; such infections result in significant morbidity and mortality each year. the incidence of viral infections in the newborn is estimated at 6-8% of all live births (smith, 1993) . the fetus and newborn are particularly vulnerable to viral infection for numerous reasons, including a developing immune system that is inadequate for preventing infection and containing the spread of viruses, lack of immunologic experience with viruses, and the presence of rapidly growing cells and tissues (strodtbeck, 1986) . viral infection in the newborn can vary from no clinical disease to devastating illness. infection of the newborn with a virus produces a variety of clinical presentations, ranging from absence of symptoms to infection before (congenital), during (natal), or after birth (postnatal) (overall, 1992) . because of the affinity of viruses for rapidly growing cells, newborn infection results in multiple outcomes that are determined by the specific virus and the gestational age at the onset of infection. congenital viral infections may result in miscarriage or stillbirth, congenital defects of various organ systems, clinical infection, or asymptomatic infection. natal and postnatal infections can result in asymptomatic infection or clinical infection. outcomes of clinviruses are unique microorganisms in that they are obligatory intracellular parasites that must gain access to the inside of a host cell for replication. physical contact is necessary for this process, and many factors influence the attraction between the host cell and the invading virus. the structure of an individual virus and the presence of certain enzymes determine the mechanism by which the virus replicates inside the host cell. viral replication follows five basic steps. proteins using host cell components and metabolism; 4. assembly of new virions in the cytoplasm; and 5 . release of the new virions into the intracellular environment. release of the virus can occur quickly or slowly and frequently results in destruction of the host cell (strodtbeck, 1986; voyles, 1993) . or lysis. latency occurs when the provirus reproduces itself along with the host cell such that all daughter cells contain the viral genetic material. these cells can remain latent or be triggered into one of the other two outcomes at a future time. a classic example of latency is the herpes virus, which can remain dormant until a trigger such as stress activates the virus and a characteristic herpetic skin lesion is produced. in the controlled growth outcome, the host cell is not destroyed but becomes committed to the production of new virions, which are gradually released into the surrounding interstitial space. the last outcome, lysis, involves destruction of the host cell upon release of the newly formed virions. this process is referred to as the cytopathic effect and often results in patterned tissue damage that can be used in the clinical laboratory for identification of the virus (strodtbeck, 1986; voyles, 1993) . from this discussion, it should be clear why viral infections can result a such a wide array of presentations. because of the intracellular, parasitic nature of viruses and the resulting dependence on host cell metabolic machinery for proliferation, the use of antiviral drug therapy is limited. many of the drugs developed for viral disease also have an effect on rapidly growing cells, which limits their use in the newborn. thus, the newborn is not only vulnerable to infection, but also is disadvantaged for viable treatment options. the newborn infected with a virus is capable of mounting an immune system response. the response depends upon the gestational age of the newborn and is not as efficient or effective as the response of an adult. clinical viral infection in newborns usually results in a more rapid progression to full-blown disease and earlier onset of symptomatic organ involvement than would be seen in adults with the same infection (overall, 1992; smith, 1993; strodtbeck, 1986) . entry of the virus into the newborn triggers activation of the developing immune system. local antibodies such as iga are stimulated. because of their inability to contain the virus, a primary focus of infection is established. the virus replicates itself and releases new virions into the interstitial space, where they are carried away via the lymphatics or the bloodstream to secondary foci of infection. if tissue damage occurs at this point, the inflammatory and cell-mediated responses are activated. escaping virions in the bloodstream stimulate the production of circulating igm and igg antibodies. igm tends to be produced earlier if the virus is new to the immune system, whereas igg tends to be produced first if the virus is a repeat challenger. the rise in fetal igm during intrauterine infection allows fetal or cord blood levels to be used in the diagnosis of congenital viral infection. igm production peaks early in the infection and rapidly declines. igg peaks gradually and functions longer than igm. both of these immunoglobulins attempt to neutralize the invading virus by binding with the virus to activate complement. this mechanism is designed to prevent virus absorption to host cells (overall, 1992; strodtbeck, 1986) . once the virus gains entry to the host cell, it becomes the responsibility of the cell-mediated system and the inflammatory response to contain the infection. infected host cells develop new virus-induced, antigenic determinants on their cell membranes that alert these defenses. the new antigens trigger t lymphocytes to respond along with macrophages and other components of the immune system (overall, 1992; strodtbeck, 1986) . activation of the immune response at this point may result in the destruction of some host cells, rather than the virus, by the immune system. in the developing fetus, this can worsen the severity of birth defects associated with specific viral diseases, such as rubella or cytomegalovirus (cmv). for example, neurons coated with cmv may be destroyed by lymphocytes which, when added to the number of neurons undergoing viral lysis, increases the risk for microcephaly. antigen-antibody complexes also are formed that liberate chemotactic factors and cytokines. histamine and kinin release results in vasodilation, heat production, and increased capillary permeability. this results in increased temperature (fever) and stagnant anoxia locally, which decreases absorption of the virus because most viruses are susceptible to increased temperature and acidic environments (strodtbeck, 1986; voyles, 1993) . a side effect of this process is a less-than-ideal environment for the growth of healthy tissue. the final consideration in the newborn's response to viral infection is the production of interferon. interferons are highly active proteins produced by virus-infected cells that act upon neighboring cells to prevent the spread of the viral infection. they also are produced by cells of the immune system. interferons disrupt viral infectivity in a variety of ways that are still being studied. newborn interferon production appears to be decreased via one pathway and normal via the other (strodtbeck, 1986) . despite the deficiencies of the newborn immune system to viral attack, only a few of the hundreds of viruses known to cause human disease actually result in infection of the fetus or newborn (overall, 1992) . viral infection of the newborn usually is classified as congenital, acquired, or nosocomial (hospital-acquired) in origin. congenital viral infections occur when an infected mother transmits the virus across the placenta to the fetus. theoretically, all viruses are capable of causing congenital infections, but most do not (overall, 1992) . this may be attributable in part to maternal and placental immune defenses that protect the fetus or to the nature o f the maternal illness. most viral infections in pregnancy are mild respiratory or gastrointestinal infections that do not pose a serious threat to the health of the fetus (overall, 1992) . the viruses implicated in congenital infection are cytomegalovirus, herpes simplex virus, rubella (german measles), and varicella (chickenpox) (overall, 1992; smith, 1993) . there is debate within the medical community about a congenital viral syndrome caused by the human immunodeficiency virus (hiv). acquired viral infections occur after exposure of the newborn t o maternal vaginal tract flora and breast milk. genital secretions may be contaminated with cmv, enteroviruses, hepatitis b virus, and herpes simplex virus. contaminated breast milk has been implicated in newborn infection with hiv, herpes simplex, and other viruses (overall, 1992) . nosocomial viral infections may be acquired in the newborn nursery or the intensive care nursery (guerina 8r goldmann, 1993; strodtbeck, 1986) . current trends of short length of stay and rooming in minimize the risk of hospital-acquired infection in the healthy infant assigned to the newborn nursery. high-risk or premature infants transferred to the intensive care nursery are at risk for nosocomial viral infections from members of their family, health care providers, other infants, treatment therapies (such as blood transfusions), or contaminated objects (fomites) in the environment. specific features about the most common viral infections of concern to the newborn are summarized in table 1 . pertinent information on the incidence, epidemiology, and characteristics of the virus; transmission risk; and effect(s) on the newborn are identified. diagnosis and clinical manifestations of the infection, along with treatment, prognosis, and prevention strategies, are discussed (arvin 8r maldonado, 1994; cherry, 1994; gershon, 1994; mueller 8r pizzo, 1994; torok, 1994; whitley & arvin, 1994) . diagnosis of viral infection in the newborn usually is prompted by a strong suspicion based on physical characteristics of the newborn, history of exposure or maternal illness, or failure of sepsis testing to yield positive results. careful review of the maternal history for evidence of viral illness and serum antibody determinations can assist in making the diagnosis; however, definitive diagnosis is based on neonatal studies (overall, 1992; smith, 1993) . accurate diagnosis often is difficult. because many infected infants have no symptoms or the clinical manifestations are nonspecific, laboratory diagnostic tests must be used to determine the virus responsible for the illness. laboratory diagnosis may involve direct isolation of the virus in culture; detection of viral antigen or antibodies by immunologic, genetic, or electron microscopic study; or histopathologic methods (guerina & goldmann, 1993; overall, 1992; smith, 1993) . the best method of diagnosis varies, depending upon the specific characteristics of the virus. recovery of virus from clinical specimens usually is more difficult than recovery of other microorganisms (strodtbeck, 1986) . a negative result may mean an unsuccessful recovery of virus present. clinical judgment is important for determining the likelihood for additional management and treatment of the suspected viral illness. the most common management strategy for newborn viral infection is supportive care. the availability of specific chemotherapies such as antiviral drugs is limited. promising new therapies are being developed, and several are undergoing evaluation in clinical trials with select populations (filippell & rearick, 1993; kinney & eiden, 1994; overall, 1992; stagno, 1994) . such therapies include ganciclovir for cmv disease, hyperimmune intravenous globulins for cmv and respiratory syncytial virus (rsv) infections, and new vaccinations for rotavirus. most of these therapies, if approved for use in newborns, will most likely lessen the severity of the disease and subsequent sequelae, rather than cure the infection. other strategies include improvements in nutrition for the most susceptible newborns, interferon administration to enhance viral-specific immune defenses, and lymphokine enhancement of the neonatal immune system. the first goal and ultimate management of neonatal viral disease is prevention of the infection. diligent hand washing; improved screening of visitors and health care providers who come into contact with susceptible newborns; increased efforts to immunize susceptible populations, such as childbearing women and children, to known viral pathogens, such as rubella and measles; and immunization of health care providers who work with immunocompromised patients are strategies that can be used to achieve this goal. the american academy of pediatrics (1994) recommendation that all neonatal intensive care unit (nicu) staff members receive annual influenza immunizations to prevent the spread of infection to infants with chronic lung disease is routinely ignored by most units across the country (eisenfeld et al., 1994) . nursing care of the newborn with a viral illness is determined by the specific viral infection, the severity of the illness, and the unique conditions of the newborn and his/her family. newborns with a known viral infection who have no symptoms require no special nursing care while in the hospital. newborns with viruses such as rubella and cmv who have no symptoms should be followed up closely for the development of late onset secherry, 1994; cooper, preblud, 8r alford, rearick gershon, 1994; giacoia, 1994; kinney bz eiden, azt: zidovudine; cns: central nervous system; ddc: dideoxycytidine; ddi: dideoxyinosine; dic: disseminated intravascular coagulation; nec: necrotizing enterocolitis; mueller l(r pizzo, 1994; overall, 1992; smith, 1993; stagno, 1994; torok, 1994; whitley 8r arvin, nicu: neonatal intensive care unit. egies are important for protecting the ill newborn from other infections and preventing nosocomial spread of the usually is prompted by a strong suspicion based on physical characteristics of the newborn, history of exposure or maternal illness, or failure of sepsis testing to yield positive results. quelae (cooper, preblud, tk alford, 1994; overall, 1992) . family members should be provided with available educational materials. brochures, such as those on cmvfrom the children's biomedical research institute in st. paul, minnesota, can be helpful for some families (children's hospital, 1989 ). the first action of nursing care of the newborn with symptoms often is to raise the index of suspicion for viral disease. the presence of physical findings, such as characteristic rashes or vesicles, microcephaly, intrauterine growth retardation or small size for gestational age, should alert the nurse to the possibility of congenital infection. obtaining a detailed, accurate maternal history, including immunizations received, signs/symptoms of possible viral illness during the pregnancy, and exposure to possible sources of viral infection, is important. collecting correct specimens for diagnostic tests and cultures is as important as prompt processing by the clinical laboratory (strodtbeck, 1986) . nursing care of the newborn with severe viral disease is complex and varies according to the clinical presentation of the neonate. presentation of specific management plans for each viral infection is beyond the scope of this article. general nursing care may involve ventilator therapy, pharmacologic support of cardiovascular status, correction of acidosis and shock states, correction of coagulopathies, and treatment of severe neurologic disorders, such as meningitis and seizures. skin care and nutrition are other areas of concern to nursing because many infants may have rashes or be at risk for secondary infections. care of the family is important and can be complicated by maternal or paternal feelings of guilt regarding congenital infection or infection acquired at the time of delivery. the need for special care such as isolation also can present problems and increase the family's stress. meticulous attention to hand washing, aseptic technique, and the use of personal protective equipment are additional important aspects of nursing care. these last stratwithin recent years, there has been a developing awareness on the national level of the role of viruses as nosocomial pathogens (giacoia, 1994; guerina & goldmann, 1993; strodtbeck, 1986) . although viral infections are uncommon in the newborn nursery, the potential for outbreaks remains a constant concern, especially during peak seasons of viral infections within the community. infected visitors and health care workers may unknowingly expose healthy newborns to respiratory viruses such as rsv, influenza, and adenovirus; enteric viruses such as rotavirus and enteroviruses; herpes simplex virus; and varicella virus (overall, 1992) . early discharge results in newborns being discharged before the end of the minimum incubation period for most viruses. these infants often have symptoms develop at home. routine telephone follow-up of discharged newborns may alert the nursery staff to the presence of a problem. the nicu is especially susceptible to nosocomial viral infections for a variety of reasons, including extensive use of invasive technology for the care of sick infants, a fragile and immunocompromised patient population, and large numbers of health care workers and visitors who come in contact with the patients (guerina & goldmann, 1993; strodtbeck, 1986) . newborns with underlying cardiac and pulmonary dysfunction are particularly at risk for the development of nosocomial viral infections, especially rsv and cmv (guerina & goldmann, 1993; smith, 1993) . many viruses have been implicated as etiologic agents of nosocomial infections in the nicu. the most commonly identified viruses are the respiratory viruses (rhinovirus, adenovirus, rsv, parainfluenza, and influenza virus); the enteric viruses (rotavirus and coronavirus); the enteroviruses; and the herpes viruses (cmv and herpes simplex). nosocomial viral infections are of concern because they prolong hospital stays and increase health care costs, increase neonatal morbidity and mortality, and are difficult to recognize and diagnose. new evidence suggests that infection with cmv or influenza virus may complicate matters by predisposing the critically ill newborn to bacterial superinfection because of viral-induced changes in polymorphonuclear leukocyte function (abramson & wheeler, 1994) . reports of nosocomial infection range from case studies to unit outbreaks (singh-naz, brown, & ganeshananthan, 1993; strodtbeck, 5 8 6 ; watson et al., 1993) . risk factors linked to nosocomial viral infections include intubation and assisted ventilation; transfusion with cmv-positive donor blood; ingestion of contaminated breast milk; direct and indirect contact among patients, visitors, and hospital personnel; contact with contaminated objects in the environment; and inadequate hand washing (guerina & goldmann, 1993; strodtbeck, 1986) . nursing care of the newborn with severe viral disease is complex and varies according to the clinical presentation of the neonate. each year significant neonatal morbidity and mortality occur as a result of viral infections. the presence of a developing immune system inexperienced in response to viruses in a rapidly growing host make the newborn especially vulnerable to viral disease. the effects of viral infection are varied and range from no symptoms to mild or severe disease. although the newborn immune system is capable of mounting a defense to the virus, the response often is inadequate for preventing infection. the outcome of viral infection depends upon the specific virus, the gestational age of the newborn at the onset of infection, and the severity of the infection. in addition to limited specific antiviral drug therapy, many of the available drugs are not recommended for use in newborns. several new therapies are being tested and show promise; eventually they may lessen the severity of disseminated disease and minimize the long-term sequelae. until such time, the major thrusts of management of neonatal viral disease are prevention of the infection and supportive care for the acutely ill newborn. virus-induced neutro-phi1 dysfunction: role in the pathogenesis of bacterial infections the 1994 red book other viral infections of the fetus and newborn enteroviruses infectious diseases of the fetus and newborn infant rubella respiratorysyncytial virus chickenpox, measles and mumps uncommon pathogens in newborn infants neonatal nosocomial infections: prevention and management current therapy in pediatric infectious disease enteric infectious disease in neonates: epidemiology, pathogenesis, and a practical approach to evaluation and therapy acquired immunodeficiency syndrome in the infant viral infections of the fetus and neonate nosocomial adenovirus infection: molecular epidemiology of an outbreak congenital viral and protozoan infections cytomegalovirus the epidemiology of nosocomial viral infections in infants who are long term residents of the neonatal intensive care unit human parvovirus bl9 the biology of viruses vertical transmission of hepatitis a resulting in an outbreak in a neonatal intensive care unit herpes simplexvirus infection key: cord-021453-vf8xbaug authors: dysko, robert c.; nemzek, jean a.; levin, stephen i.; demarco, george j.; moalli, maria r. title: biology and diseases of dogs date: 2007-09-02 journal: laboratory animal medicine doi: 10.1016/b978-012263951-7/50014-4 sha: doc_id: 21453 cord_uid: vf8xbaug nan status as a cooperative companion animal of reasonable size. dogs were used in the mid-1600s by william harvey to study cardiac movement, by marcello malpighi to understand basic lung anatomy and function, and by sir christopher wren to demonstrate the feasibility of intravenous delivery of medications (gay, 1984) . the use of dogs continued as biomedical research advanced, and they were featured in many noteworthy studies, including those by pavlov to observe and document the conditioned reflex response and by banting and best to identify the role of insulin in diabetes mellitus. for a comprehensive but concise review of the use of the dog as a research subject, the readers are directed to the manuscript by gay (1984) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger-breed dogs for use in surgical research studies. some specific breeds with congenital or spontaneous disorders are also maintained by research institutions (see specific examples below). random-source dogs used in research are most frequently mongrels or larger-breed dogs (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for beagle for the years 1998-1999, approximately 40% of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. the next most common areas of research using beagles were dental and periodontal disease and surgery (12% of publications), orthopedic surgery and skeletal physiology (7%), and radiation oncology (4%). other research areas that utilized beagles included canine infectious disease, surgery, imaging, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established, and the organs of larger-breed dogs are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies is maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of blood, most notably the neutrophil population. these dogs are used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., 1995) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., 1994) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease), and the development of spontaneous diabetes mellitus and hypothyroidism in a variety of dogs has also been studied for comparisons with the human conditions. although historically the dog has been a common laboratory animal, the use of dogs in research has been waning over the past few years. according to the u.s. department of agriculture (1998) , the number of dogs used in research has declined from a high use of 211,104 in 1979 211,104 in to only 75,429 in 1997 . this decrease was caused by a variety of factors, including (but not limited to) increased cost, decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purpose-bred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against canine distemper virus, parvovirus, adenovirus type 2, parainfluenza virus, leptospira serovars canicola and icterohemorrhagiae, and bordetella bronchiseptica. rabies virus vaccination may also be included. purpose-bred dogs are also usually treated prophylactically for helminths and ectoparasites, intestinal coccidia, and bacterial ear infections (r. scipioni and j. ball, personal communication, 1999) . random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting), retired racing dogs, or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. randomsource dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations. options for procurement of dogs for biomedical research typically include purchase from a u.s. department of agriculturedesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title 9, chapter 1 (1-1-92 edition), subchapter a, animal welfare, 1.1, definitions, and 2.1, requirements and application. briefly, class a licensees are breeders who raise all animals on their premises from a closed colony (suppliers of purpose-bred dogs are typically class a dealers). class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and then resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and recordkeeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. regulations regarding the sale of pound dogs to research facilities or class b dealers vary from state to state and include some bans on this practice. the best resource for identification of possible vendors is the "buyer's guide" issue of the periodical lab animal. typically the last issue of each year, the "buyer's guide" lists sources for both purpose-bred and random-source dogs and denotes such features as pathogen-free status, documentation of health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within that issue of the journal. welfare act (7 cfr 2.17, 2.51, and 371.2[g] ) are described in 9 cfr chapter 1 (1-1-92 edition), subchapter a, animal welfare. regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats, of part 3 (standards) of subchapter a. particular attention should be paid to section 3.6c (primary enclosures--additional requirements for dogs), because the space required for housing dogs is calculated using the length of the dog rather than the body weight (which is used for other species and also for dogs, according to national research council (nrc) guidelines). section 3.8 (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute of laboratory animal research (ilar) has written the "guide for the care and use of laboratory animals" (seventh edition, 1996) . the "guide" is the primary document used by institutional animal research programs to develop and design their programs, as well as by the association for assessment and accreditation of laboratory animal care international (aaalac international) and other animal care evaluation groups to facilitate site visits and inspections. the ilar committee on dogs has also written "dogs: laboratory animal management " (1994) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. growth data for beagles from a purpose-bred dog breeding facility are provided in table i . table ii features hematology data from beagles from the same commercial facility. table iii lists serum and urine chemical data for beagles. normal physiologic data for dogs (no breed specified) are provided in table iv . the information presented in the tables represents a range of normal values that can vary, depending on the analytical method and equipment used as well as the age, breed, gender, and reproductive status of the animal. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal good nutrition and a sound, balanced diet are essential to the health, performance, and well-being of the animal. the basic nutrient requirements for dogs have been compiled by the nrc and represent the average amounts of nutrients that a group of animals should consume over time to maintain growth and prevent deficiencies (national research council, 1985) . the reader is referred to these guidelines for useful reference points for management of an animal's diet during various physiologic states (e.g., gestation, lactation, maturational age). most commercially available balanced dog diets are "closedformula" diets, in which the labeled specific minimum requirements for protein and fat, and the maximum values for ash and fiber, are met. these diets do not necessarily provide the identical composition of ingredients from batch to batch. ingredient composition varies, depending on the cost relationships of the various ingredients as the manufacturer attempts to achieve the label requirements at the lowest ingredient cost. an "openformula" (or "fixed-formula") diet provides more precise dietary control. in these diets the ingredients are specified, and the percentage of each ingredient is kept constant from batch to batch. "semipurified" diets provide for the strictest control of ingredients and are formulated from the purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are generally safe for consumption up to 6 months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf life, but the best strategy is to use each lot based on the date of manufacture in order to prevent food from expiring and to ensure that only fresh diets are fed. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the basal metabolic rate, or basal energy requirement (ber), refers to the amount of energy expended following sleep, 12-18 hours after food consumption, and during thermoneutral conditions (kleiber, 1975; lewis et al., 1987) . the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment, which in the dog is approximately twice the ber (lewis et al., 1987) . for dogs weighing greater than 2 kg, the mer may be calculated using this simplified linear equation: mer (metabolizable kcal/day) = 2(30 weightkg + 70) (national research council, 1985; lewis et al., 1987) . the quantity of a correctly balanced diet to be fed to each dog can then be determined by dividing the mer by the energy density of the diet. fat provides three major dietary functions, including absorption of fat-soluble vitamins (a, d, e, and k), enhancement of palatability, and provision of essential (unsaturated) fatty acids. dietary fat is an excellent, highly digestible energy source, providing 2.25 times more energy on a per weight basis than either soluble carbohydrates or proteins (lewis et al., 1987) . however, fats are not needed for this purpose when adequate carbohydrate and protein are present. consumption of fat in excess of an animal's ability to metabolize it results in steatorrhea and has been related to the development of acute pancreatitis, whereas lack of dietary fat may lead to a fatty acid/energy deficiency. fatty acid deficiency is associated with poor growth, poor physical performance, reduced reproductive performance, and weight loss. dogs are considered to be "easy keepers," because they do not have as many absolute nutritional requirements as their domestic counterpart, the cat. however, they do possess a unique requirement for certain polyunsaturated fatty acids, a deficiency of which may predispose them to decreased growth rates and dermatologic abnormalities, such as "hot spots." dogs require linoleic (f2-6) acid, an essential fatty acid (national research council, 1985) , and more recently it has been demonstrated that the f2-3 fatty acids may play a role in maintaining healthy skin (logas and kunkle, 1994) . supplementation with a balanced essential fatty acid product (e.g., derm caps) may alleviate allergy-related dermatoses such as flea-bite dermatitis and pyoderma (logas and kunkle, 1994; miller, 1989) . essential fatty acid deficiency can occur in dogs receiving low-fat dry dog food that has been stored too long, particularly under warm, humid conditions (lewis et al., 1987) . there are 22 a-amino acids, 10 of which cannot be synthesized in sufficient quantity to meet a dog's normal metabolic demands for growth and maintenance. hence, as their name implies, these essential amino acids are required by all dogs and must be provided in the diet. the essential amino acids and the minimal requirements for growth are listed elsewhere (lewis et al., 1987) . chronic excessive protein intake may be detrimental to the kidney by contributing to accelerated renal aging and subsequent glomerulosclerosis (lewis et al., 1987) . conversely, inadequate protein intake results in retardation of growth and adata graciously provided by r. scipioni and j. ball of marshall farms usa, inc., north rose, new york. beagles tested for period 2/28/99-9/01/99. b s.d., standard deviation; wbc, white blood cells; rbc, red blood cells; hgb, hemoglobin; hct, hematocrit; mcv, mean corpuscular volume; mch, mean corpuscular hemoglobin; mchc, mean corpuscular hemoglobin concentration; rdw, red cell distribution width; hdw, hemoglobin distribution width; plt, platelets; mpv, mean platelet volume; neut, neutrophils; lymp, lymphocytes; mono, monocytes; eos, eosinophils; baso, basophils; luc, large unstained cells; li, lobularity index; mpxi, mean peroxidase activity index reduction in production and/or performance. protein deficiency, a potential consequence of decreased food intake, results in decreased energy intake. as a compensatory mechanism for a lack of fat or carbohydrate, body protein catabolism ensues in order to meet energy demands, thus exacerbating the negative protein balance and contributing to the clinical signs of edema/ascites, unkempt appearance, lethargy, and weight loss. thus, caloric needs must be met before protein needs (lewis et al., 1987) , an important concept to bear in mind in the event of research experiments that may predispose to anorexia. in general, providing a good quality commercial diet that supplies the required amount of amino acids and caloric requirements of the animal, while avoiding excess protein, will ensure nutritional stability and promote longevity. appropriate mineral balance in the diet is very important. the best approach in the laboratory setting is to feed a commercial diet that has been formulated with the proper amount and balance of minerals for normal growth. the recommended amount of dietary minerals and the major causes and clinical signs of deficiencies are published elsewhere (lewis, 1987) . determining the specific mineral involved in an imbalance can be a diagnostic challenge, because the clinical signs for several excesses/ 2.0 (basal) 12.6 (anestrus) 2.0 c <0.5 c 20-100 c 4-73 r (continues) deficiencies are similar and nonspecific. a definitive diagnosis is often made only after the diet has undergone analysis of the mineral components. once the imbalance has been identified, the safest resolution to the problem is to discard the entire lot of misformulated diet. attempting to correct the imbalance through oral supplementation is likely to be more harmful than beneficial, and it risks intensifying the problem by creating additional mineral imbalances. vitamins function as enzymes that regulate a wide variety of physiologic processes. they are divided into two groups based on their solubility. the fat-soluble vitamins include a, d, e, and k, whereas the rest are water-soluble. a list of the vitamins, their requirements, and clinical signs associated with deficiencies and toxicities is published elsewhere (lewis et al., 1987) . cases of dietary deficiency are rarely encountered in the research setting, because laboratory dog chows are fortified with vitamins. additional vitamin supplementation may occasionally be required during prolonged clinical illnesses, such as polyuria or diarrhea, which predispose to loss of water-soluble vitamins (b complex and c) (lewis et al., 1987) . however, as with minerals, routine supplementation of vitamins may induce inadvertent toxicity and exacerbation of an imbalance. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. this section is largely based on information assimilated from texts such as "miller's anatomy of the dog" (evans and christensen, 1993) , "veterinary reproduction and obstetrics" (arthur et al., 1989) , and an issue of veterinary clinics of north america: small animal practice devoted to pediatrics of puppies and kittens (hoskins, 1999) . the ovaries of the bitch are attached to the dorsolateral walls of the abdominal cavity caudal to the kidneys by the broad ligaments and are not palpable abdominally. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the birchard and sherding (1994) . ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. catheterization of the cervix is usually not possible in the normal bitch at any stage of the reproductive cycle, except during or immediately following parturition. thus, semen is deposited at the external cervical os during natural or artificial insemination. the vagina is a long musculomembranous canal that extends from the uterus to the vulva. when the vagina is examined, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva so as to avoid the deep ventral clitoral fossa. examination should proceed at an angle of approximately 60 ~ until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the bitch has a monoestrous cycle, with clinical estrus occurring predominantly in january or february and again in july or august (although it can occur at any time of year). the estrous cycle consists of four stages: proestrus, estrus, diestrus, and anestrus. the average duration of proestrus is 9 days. during this stage the vulva is enlarged, turgid, and firm, and a sanguinous vaginal discharge is present. endocrinologically, proestrus is the follicular stage of the cycle, and estrogen levels peak at this time. estrus generally lasts 9 days, and the vulva is softer and smaller than in proestrus. a vaginal discharge persists during estrus and may remain serosanguinous or become straw-colored. the endocrine feature of estrus is the luteinizing hormone (lh) surge, followed by ovulation within 24-72 hours. diestrus begins approximately 9 days after the onset of standing heat. the end of this stage is 60 days later, which would be coincident with whelping if the bitch had become pregnant. serum progesterone levels peak during diestrus. the duration of anestrus is approximately 4 months. anestrus is the stage of reproductive quiescence, characterized by an absence of ovarian activity and serum progesterone levels of less than 1 ng/ml. components of the canine spermatic cord include the ductus deferens, the testicular artery and vein, the lymphatics and nerves, and the cremaster muscle. the cremaster muscle and pampiniform plexus aid in thermoregulation of the testicles, which are maintained at 2~176 lower than basal body temperature. sweat glands in the scrotum assist in lowering the scrotal temperature through evaporation. the penis is a continuation of the muscular pelvic urethra and is attached to the ischiatic arch by two fibrous crura. it is composed of fibrous tissue and three cavernous sinuses: corpus cavernosum, corpus spongiosum penis, and corpus spongiosum glandis. the accessory sex glands of the dog consist of only a well-encapsulated prostate gland that surrounds the pelvic urethra, and ampullary glands at the termination of the vas deferens in the urethra. the dog does not have seminal vesicles or bulbourethral glands. the onset of puberty ranges from 5 to 12 months of age and is affected by breed, season, and nutritional and disease status. testicular growth is rapid at this time, and the seminiferous tubules begin to differentiate. the sertoli cells form the bloodtestis barrier, the tubules become hollow, and spermatogenesis commences. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial, or leydig's, cells. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, while inhibin and estrogen play a role in a feedback loop on the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in 45 days, with subsequent maturation of sperm occurring in the epididymis for approximately 15 days. thus, the entire process from initiation of spermatogonial mitosis to delivery of mature sperm to the ejaculate is 60 days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality and number of sperm produced. problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities that suppress sexual behavior. animals with poor hindlimb conformation or with trauma to the back or hindlimbs may be unable to properly mount the female. there is a positive correlation with the size of the testicles as measured by scrotal circumference and the number of sperm produced. finally, parameters used to assess the quality of sperm include motility, morphology, volume, and concentration. an ejaculate (5 ml) that contains approximately 500 million progressively motile sperm without significant morphological abnormalities (such as a kinked tail) is a good indicator of normal male fertility. in general, erection, which involves muscular contractions and increased arterial blood flow to the penis, is controlled by the parasympathetic nervous system, whereas ejaculation is under sympathetic control. on mounting, the initial thrusting and ejaculation of semen last about 1 minute. the bulbus glandis becomes enlarged, which lodges the penis in the female reproductive tract. the male then dismounts and brings one hindleg over the female, and the two continue to be joined "rear to rear," a position classically termed "the tie." ejaculation of the accessory gland fluid continues for 5-30 minutes. the continued expulsion of prostatic fluid during the "tie" may serve to propel the semen from the vagina through the cervix into the uterus. fertilization occurs in the oviduct and may occur as late as 8 days after coitus, because of the long life span of sperm in the dog. however, once ovulated, oocytes generally remain viable for only 12-24 hours. therefore, the bitch should be bred prior to ovulation to ensure the presence of sperm for fertilization of live oocytes. cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation, nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear-cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear "anuclear" and are classified as "cornified" or "anuclear squames." cornification occurs approximately 2 days prior to the estrogen peak and 4 days prior to standing heat. the percentage of cornified cells (of the total number of epithelial cells) decreases gradually to zero after the onset of diestrus. the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified 6 days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear-cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, it is not a substitute for observation of behavioral estrus, which is the best criterion to use in breeding management. during proestrus the male is attracted to the bitch and will investigate her hindquarters, but she will not accept breeding. the behavioral hallmark of estrus is standing receptivity toward the male. during this stage the bitch will exhibit "flagging," or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate, and the number of pups whelped per egg ovulated, it is recommended to breed the bitch on days 1, 3, and 5 of the standing heat. fertilization is completed in the mid-to distal oviduct. implantation is evident by areas of local endometrial edema 17-18 days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary and deciduate, indicating that the placental villi are arranged in a belt and that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is 59-63 days. luteal progesterone is responsible for maintaining pregnancy, and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than 1 ng/ml in late proestrus to a peak of 30-60 ng/ml during gestation, then declines to 4-5 ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility. pregnancy detection can be performed by abdominal palpation of the uterus 28 days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings in the early gravid uterus. they are approximately 2 inches in length at 28-30 days, the time at which pregnancy is most easily and accurately diagnosed. by day 35 the uterus begins to enlarge diffusely, so that the vesicles (and, therefore, pregnancy) are difficult to identify by palpation. fetal skeletons become calcified and are radiographically evident by day 42. bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. real-time ultrasound can be utilized for pregnancy detection of vesicles as early as 25-28 days. an abrupt drop in body temperature to less than 100~ indicates impending parturition within 18-24 hours. the process of parturition has been divided into three stages, stage 1 of labor lasts 6-12 hours and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include hard panting and increased pulse and respiration rates. fetal expulsion occurs during stage 2, which lasts approximately 3-6 hours. as the fetus engages the cervix, the neuroendocrine system induces the release of oxytocin; this is referred to as the ferguson reflex. oxytocin strengthens the uterine contractions and may elicit voluntary abdominal contractions as well. the bitch is usually recumbent during stage 2 but is able to inhibit this stage if labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between delivery of each pup is irregular, but the average time lapse is less than 1 hour between pups until parturition is complete. veterinary assistance is necessary if the bitch remains in stage 2 for more than 5 hours without delivering the first pup, or for more than 2 hours before delivering subsequent pups. the placentas are expelled during stage 3 of labor, immediately following delivery of a pup, or up to 15 minutes thereafter. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats, and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged as they have the potential to coat the umbilical cord, which may predispose to ascending infections. heat lamps may be placed 24 hours prior to parturition and remain until all neonates dem-onstrate vigorous and successful suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. thus, monitoring of parturition is important, but human intervention should be minimal in order to prevent stress-induced cannibalism. weak or debilitated puppies may be cannibalized by the bitch before the research staff recognizes the need for veterinary attention. the postpartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. in these cases, 5-20 units of oxytocin may be administered intramuscularly. uterine involution occurs during anestrus within 4-5 weeks of parturition. during this time a greenish to red-brown vaginal discharge, or lochia, may be noted. although lochia is normal, the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the sixth postpartum week, with complete repair by 3 months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately 12 days, and ears are patent at approximately 12-20 days. solid food can be introduced between 4.5 and 6 weeks of age, and puppies can be weaned at 6-8 weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as strictures, or when the bitch refuses to stand for breeding. semen for ai is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the bitch's scent and manually stimulated. after collection of the first two fractions, a sufficient amount of the third fraction, which consists predominantly of prostatic fluid, is collected to bring the total semen volume to 4-6 ml. the semen is then drawn into a sterile 10 or 12 ml syringe attached to a sterile disposable insemination pipette. the bitch is inseminated either standing or with raised hindquarters. a gloved index finger is inserted into the dorsal commissure of the vulva and directed craniodorsally until it is over the ischial arch. the tip of the insemination pipette is introduced and guided by the gloved finger toward the external cervical os. the semen is injected, and 2-3 ml of air are then flushed through the syringe and pipette. the pipette is withdrawn, and the gloved finger is used to feather the ceiling of the vagina until contractions of the vaginal musculature are palpable. the bitch's hindquarters are subsequently elevated to promote pooling of semen around the external cervical os. as with natural breeding, ai should be performed on days 1, 3, and 5 of standing heat, or on the days of maximal vaginal cornification. the bitch should be palpated for pregnancy approximately 4 weeks after the first insemination. false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. however, in the event of extreme discomfort due to mammary gland enlargement, bitches may be treated with mibolerone (cheque drops) at an oral dose of 16 ~tg/kg q24 hr for 3-5 days (brown, 1984) . reproductive performance in the bitch is optimal prior to 4 years of age. although normal cycle lengths are reported to occur up to the ages of 5-7 years, the interestrous interval tends to increase by 4 years of age. cycling does not completely cease; however, after 8 years of age, bitches demonstrate significant decreases in conception rate and number of live pups whelped. by 8-9 years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia are extremely common. beagles have been a popular animal model because of their docile nature. they are easily handled and for the most part respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiogram (ecg) recordings, oral gavage, and venipuncture. dogs are sexually mature by 6-9 months of age, but they are not socially mature until 18-36 months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from 3-8 weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks 5 and 12, puppies are most capable of learning how to interact with people. by 10-12 weeks of age dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. the extent to which breed affects behavior has been the subject of popular speculation but is difficult to prove. in general, breed-specific patterns do tend to emerge. for example, it appears that beagle pups are very motivated by food reward (overall, 1997 ). this is not surprising, because the breed was selected to work with its nose, and this may be a useful attribute for laboratory investigations that are predicated on food restriction. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the third chapter of "clinical behavioral medicine for small animals" (overall, 1997). by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of the disorders that can affect this species. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting. especially noted in this chapter are infectious diseases associated with the use of random-source dogs that have unknown vaccination history and have had intensive contact with other similar animals at pounds and/or shelters, or conditions seen frequently in the beagle, the most common breed used in biomedical research. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks, such as the "current veterinary therapy" series (j. d. bonagura and r. w. kirk, eds.), "veterinary internal medicine" (s. j. ettinger and e. c. feldman, eds.), and "infectious diseases of the dog and cat" (c. e. greene, ed.) . full citations of some chapters from these texts are listed in the references (w. b. saunders co. of philadelphia publishes all three texts.) canine infectious tracheobronchitis (kennel cough complex) etiology. infectious tracheobronchitis (itb) is a highly contagious illness of the canine respiratory tract that usually manifests as an acute but self-limiting disease. several organisms have been incriminated as causative for this condition: bordetella bronchiseptica; canine parainfluenza virus (cpiv); canine adenovirus types 1 and 2 (cav-1, cav-2); canine herpesvirus; canine reovirus types 1, 2, and 3; and mycoplasms and ureaplasms. clinical signs. clinical infectious tracheobronchitis can be subdivided into mild or severe forms. the mild form is the more common presentation and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough is easily elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise the dog is typically asymptomatic, with normal body temperature, attitude, and appetite. mild tracheobronchitis usually lasts 7-14 days, even if left untreated. the severe form of tracheobronchitis generally results from mixed infections complicated by poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and may be the determinant of severity (sherding, 1994) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. these cases require more aggressive treatment and may be fatal. bordetella bronchiseptica is considered to be the respiratory tract of infected animals (bemis, 1992) . this bacterium is very easily spread by aerosol and direct contact, and fomite transmission is also possible (bemis, 1992) . transmission is favored by confined housing of multiple animals. in experimental studies, b. bronchiseptica transmission to susceptible individuals was 100% (thompson et al., 1976; mccandlish et al., 1978) . the incubation period is 3-10 days. cpiv and cav-2 are also spread by aerosols. of these two viruses, cav-2 is the most persistent, lasting for up to several months in the environment, whereas cpiv is fairly labile (hoskins, 2000a) . both viruses can be destroyed by quaternary ammonium disenfectants. pathogenesis. the most common clinical isolates are cpiv and bordetella bronchiseptica. however, b. bronchiseptica may be a commensal organism, and it is often recovered from asymptomatic animals. in cases of clinical infection, b. bronchiseptica attaches to the cilia on the mucosal surface of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv or cav-2 alone are usually subclinical; coinfections with b. bronchiseptica or other microbes may result in clinical itb (keil and fenwick, 1998; wagener et al., 1994) . the characteristic lesion from cpiv or cav-2 infection is necrotizing tracheobronchiolitis (dungworth, 1985) . pathogenic infection of the upper airways typically results in inflammation and ciliary dysfunction. diagnosis and differential diagnosis. diagnosis of infectious tracheobronchitis is often based on clinical signs. isolation of bordetella bronchiseptica or mycoplasma by nasal swabs allows only a presumptive diagnosis. viral isolation or paired serology can be done but is often impractical and expensive. if cough persists for more than 14 days, other disease conditions should be considered. canine distemper virus infection, pneumonia, heartworm disease, tracheal collapse, and mycotic infections are differential diagnoses for dogs with similar signs. bronchial compression as a result of left atrial enlargement, hilar lymphadenopathy, or neoplasia may also elicit a nonproductive cough (johnson, 2000) and should be considered as a differential for itb. prevention. prevention is best achieved by avoiding exposure to infected animals, but this is oftentimes not practical. dogs should be vaccinated prior to, or at the time of, admission to the animal research facility. intranasal vaccine combinations for bordetella bronchiseptica and cpiv are preferred. intranasal vaccines protect against both infection and disease, can be given to dogs as young as 2 weeks of age, and can produce immunity within 4 days. control. sanitation and ventilation are critical for control. the animal care staff must practice proper hygiene to prevent fomite transmission. symptomatic animals should be isolated, and animal-to-animal contact avoided. kennels should be disinfected with agents such as bleach, chlorhexidine (nolvasan) or quaternary ammonium chloride (roccal-d). proper ventilation and humidity are important in controlling spread of these infectious agents; 15-20 air changes per hour at 50% relative humidity are recommended (sherding, 1994) . no specific treatment is available for viral infections. bordetella bronchiseptica is typically sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur, and it should be continued for 14 days. use of empirical antibiotic treatment in mild cases may hasten the resolution of clinical signs. for severe or unresponsive infection, treatment should be based on bacterial culture sensitivity patterns; nebulized gentamicin may be helpful. cough suppressants (e.g., dextromethorphan) should be avoided if the cough is bringing up mucus (productive); however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction and minimizing discomfort. tis results in altered respiratory tract histology and impaired mucociliary clearance, infected animals should not be used for pulmonary studies. animals with clinical disease would also be poor surgical candidates. etiology. [3-hemolytic lancefield's group c streptococcus (streptococcus zooepidemicus) is a gram-positive non-spore-forming coccus and an etiologic agent for pneumonia and septicemia in dogs. clinical signs. clinical signs vary based on the organ system affected. pneumonic disease is typically associated with coughing, weakness, fever, dyspnea, and hematemesis. peracute death without clinical signs has been reported in a previously healthy research dog (bergdall et al., 1996) , and conjunctivitis can also be caused by this organism (murphy et al., 1978) . epizootiology and transmission. lancefield's group c streptococci have been isolated as commensal flora in the upper respiratory tract and the vagina of clinically normal dogs (olson et al., 1973) . epizootics have been reported in both racing greyhounds and research colonies (sundberg et al., 1981; garnett et al., 1982) . in these epizootics, and in the reported case of peracute death (bergdall et al., 1996) , recent transportation (within 7 days) was associated with the disease. as such, lancefield's group c streptococcus may be an opportunistic pathogen in dogs. pathologic findings. in the peracute case reported (bergdall et al., 1996) , hemorrhage from the mouth and nose and within the pleural cavity was the most striking lesion. ecchymotic and petechial hemorrhages were seen on other organ surfaces. the lungs were heavy and wet, and blood oozed from cut surfaces. "bull's-eye" lesions were observed on the pleural surface of affected lung lobes, similar to ischemic lesions seen with fungal infections (fig. 2) . histologically, the lungs were characterized by areas of hemorrhage surrounding foci of degenerative neutrophils, blood, and necrotic debris. gram-positive cocci were seen in both the lung and the tonsils. pathogenesis. the pathogenesis for disease caused by lancefield's group c streptococcus is unclear. strain variation with respect to virulence and host immune factors is probably significant. diagnosis and differential diagnosis. definitive diagnosis is made based on bacterial culture and identification. any cause of pneumonia and/or peracute death in dogs needs to be considered as a differential diagnosis. bacterial pneumonias or septicemias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and bordetella bronchiseptica. nonbacterial causes include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control. too little is known about the pathogenesis of lancefield's group c streptococcus to make any recommendations about prevention and control. treatment. antibiotic therapy should be provided, based on culture and sensitivity. intravenous fluids are indicated for febrile or systemically ill patients. for dyspneic patients, oxygen therapy and strict activity restriction are required. research complications. clearly, dogs with severe hemorrhagic pneumonia or septicemia are not appropriate for any research study. the association between epizootics of this disease and transportation shipment supports the philosophy of providing acclimation periods to animals upon arrival at research facilities to evaluate health status and enable the animals to normalize physiologically. etiology. serovars of the spirochete leptospira interrogans sensu lato cause canine leptospirosis. disease in dogs is primarily due to serovars canicola, icterohemorrhagiae, grippotyphosa, pomona, and bratislava. clinical signs. leptospirosis may present as either an acute or a chronic problem. clinical signs are nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, and vomiting. animals may be febrile and may be reluctant to move, because of muscle or renal pain or meningitis. icterus, congested mucous membranes, or signs referable to disseminated intravascular coagulation (petechial/ecchymotic hemorrhages, melena, epistaxis, or hematemesis) are also possible. animals with peracute leptospirosis are characterized by septicemia, shock, vascular collapse, andrapid death. uveitis, abortions, and stillbirths have also been associated with leptospirosis. epizootiology and transmission. vaccination and reduced exposure to reservoir hosts have markedly decreased the prevalence of leptospirosis over the past 30 years. wild animals, cattle, and rodents are reservoirs for leptospira. the epidemiology of the disease is not static, and recent changes have been observed. serovars pomona, grippotyphosa, and bratislava are becoming more common causes of canine disease, with canicola and icterohemorrhagiae becoming less common. this may be due to vaccination practices and increased movement of wildlife reservoirs (raccoons, skunks, and opossums) into urban/suburban areas. rats have been implicated as important in the transmission of serovars canicola and icterohemorrhagiae (rentko et al., 1992; brown et al., 1996; kalin et al., 1999) . transmission occurs primarily by environmental contact, and not directly from animal to animal. infected hosts shed leptospires in urine, thereby contaminating the environment; naive animals are infected when the organisms contact mucous membranes or abraded skin. recovered animals may shed organisms in their urine for months to years. the organisms are actually labile in the environment; moisture, moderate temperatures, and alkaline soil favor survival and subsequent transmission. close contact, bites, ingestion of infected meat, and transplacental and venereal transmission are also possible. leptospirosis is a zoonotic disease. pathologic findings. the kidneys consistently have gross and microscopic lesions. in the acute phase of the infection, kidneys are swollen and have subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs and liver may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and focal areas of necrosis (searcy, 1995) . in chronic stages of leptospirosis the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly. several days postinfection the renal tubular epithelium (and, to a variable extent, the liver) is colonized. the hematogenous phase lasts 4-14 days. acute renal failure or progressive renal failure leading to oliguria or anuria may occur. the most common clinical syndrome is chronic or subclinical infections after recovery from the acute phase (greene, 2000) . the nephritis may or may not be accompanied by hepatitis, uveitis, and meningitis. icterus, if it develops, is most common in the acute phase. the combination of azotemia and icterus should alert the clinician to the possibility of leptospirosis. disseminated intravascular coagulation is often a secondary complication. the severity and course of leptospirosis depend on the causative serovar and the age and immune status of the patient. diagnosis and differential diagnosis. zinc toxicity in dogs most closely mimics the clinical syndrome of leptospirosis. other causes of acute and chronic renal failure, icterus, and acute hepatic failure must also be considered. paired serology is the most reliable means of definitive diagnosis; however, seroconversion may not occur until after the first week of infection. prevention and control. vaccination for leptospirosis is standard veterinary practice. bivalent inactivated bacterins for serovars of l. interrogans canicola and serovars of l. interrogans icterohemorrhagiae are commercially available. however, immunization does not prevent development of the carrier state or protect against other serovars. for outdoor-housed dogs, an effective program to prevent contact with wildlife reservoirs is important. control requires identification and either treatment or elimination of carrier animals. treatment. penicillins are the drugs of choice for treating leptospiremia, and prompt use reduces fatal complications. aggressive fluid therapy and supportive care may also be needed. elimination of renal colonization and the carrier state can be accomplished with dihydrostreptomycin or doxycycline administration. should not be used in research studies because of the effects of the disease on renal and hepatic function. etiology. campylobacteriosis in dogs is caused by campylobacter jejuni, a thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rod. clinical signs. most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than 6 months of age (greene, 2000; burnens et al., 1992) . in cases of clinical illness, small volumes of mucoid or watery diarrhea, with or without frank blood, are most commonly noted. these signs are usually mild, may be intermittent, and typically last 5-21 days. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea. epizootiology and transmission. the role of c. jejuni as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, 1994) . clinical signs of disease most often occur in dogs less than 6 months of age, although any age may be affected. stress or immunosuppression may make animals more susceptible to clinical disease. pound and shelter populations have the highest rates of fecal excretion of c. jejuni (sherding and johnson, 1994) . transmission is via the fecal-oral route, mostly through fecally contaminated food or water. unpasteurized milk, poultry, and meat are other sources of infection. campylobacter jejuni can be zoonotic; children and immunocompromised individuals are at the greatest risk. pathologic findings. the actual lesions observed depend upon the mechanism of the enteropathy (van kruiningen, 1995) . enterotoxin production results in dilated fluid-filled bowel loops, with little or no histopathologic alteration. in cytotoxin-mediated disease, hyperemia and a friable, hemorrhagic mucosal surface are noted. on histopathology the mucosal surface is irregular and ulcerated, and a lymphocytic-plasmacytic ileitis or colitis may be seen. when translocation occurs, the lamina propria becomes edematous and congested, with focal accumulation of granulocytes in the crypts and lamina propria. focal areas of epithelial hyperplasia and decreased numbers of goblet cells are also noted. with warthin-starry silver staining, c. jejuni may be seen between enterocytes but only rarely inside them. pathogenesis. clinical disease may be produced by several different mechanisms after the campylobacter has populated the intestinal tract (van kruiningen, 1995) . after colonization of the enterocyte surface, c. jejuni can produce an enterotoxin that causes a secretory diarrhea. campylobacterjejuni can also cause an erosive enterocolitis by invasion of the ileal and colonic epithelium along with production of a cytotoxic agent; this may be the mechanism that causes hematochezia. in addition, c. jejuni can produce illness by translocation, i.e., multiplication in the lamina propria and transportation to regional lymph nodes by macrophages. this causes mesenteric lymphadenitis. diagnosis and differential diagnosis. fresh feces (per rectum) are best for ensuring an adequate diagnostic sample. presumptive diagnosis may be made by demonstration of highly motile curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of 42~ any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis, including canine parvovirus, coronavirus, distemper virus, giardia, and salmonella infections; helminth infestations; and hemorrhagic gastroenteritis. clinical signs. based on experimental infections in dogs, three phases to the disease have been described: acute, subclinical, and chronic. clinical signs observed vary with the phase of the disease, and the acute and subclinical phases are often missed or misdiagnosed (c. g. couto, personal communication, 1993; waddle and littman, 1988; woody and mcdonald, 1985) . a history of tick exposure may be noted prior to onset of signs. in the acute phase, clinical signs range from mild to severe and may last 1-2 weeks. they include inappetance, lethargy, fever, generalized lymphadenopathy, hepatosplenomegaly, exercise intolerance or dyspnea, petechial or ecchymotic hemorrhages, and peripheral edema. central nervous system (cns) signs may also be present such as hyperaesthesia, myoclonus, and cranial nerve deficits. clinical laboratory abnormalities noted during the acute phase include thrombocytopenia, anemia, neutropenia or neutrophilia, and bicytopenia or pancytopenia. hyperplastic bone marrow, mild hyperglobulinemia, and elevated hepatic enzymes may be noted during this phase (kuehn and gaunt, 1985) . clinical signs are generally absent during the subclinical phase. mild thrombocytopenia, anemia, or leukopenia may be seen. the chronic phase develops 1-4 months after the initial infection, and signs may be subclinical to severe. an extremely varied clinical picture can emerge during this time and can mimic several other clinical syndromes. the following constellation of clinical signs may be observed: chronic lethargy, weight loss, inappetance or anorexia, fever, generalized lymphadenopathy, hepatosplenomegaly, petechial or ecchymotic hemorrhages, epistaxis, hematuria, melena, pallor, anterior or posterior uveitis, chorioretinitis, peripheral edema, ataxia, upper and lower motor neuron deficits, altered mentation, cranial nerve deficits, and seizures. persistent thrombocytopenia is the most consistent laboratory abnormality noted for all three stages. many other hematologic abnormalites may be found, such as regenerative or nonregenerative anemia (more frequently the latter), positive coombs' test, bicytopenia or pancytopenia, and splenic plasmacytosis or lymphocytosis. on bone marrow evaluation, plasmacytosis along with hypoplasia of erythroid, myeloid, and/or megakaryocyte lines may be seen. hyperglobulinemia as a result of polyclonal or occasionally monoclonal gammopathy has been noted in 50-100% of e. canis seropositive or infected dogs (kuehn and gaunt, 1985; breitschwerdt et al., 1987; shimon et al., 1996) . proteinuria and/or hypoalbuminemia have also been seen. epizootiology and transmission. ehrlichia canis is an obligate intracellular parasite that infects mononuclear cells. the definitive hosts are arthropods; domestic and wild canids are parasitized secondarily. the primary vector and reservoir is the brown dog tick, rhipicephalus sanguineus. ehrlichia canis is found worldwide and follows the distribution of the vector. infection in dogs is most prevalent in tropical and subtropical areas (greene, 1991) . in the united states, cases are concentrated in the southeastern and southwestern states but have been reported in almost every state (breitschwerdt, 2000) . transmission is primarily by tick bites, but it can also occur via blood transfusions from dogs infected for as long as 5 years. ticks become infected by feeding on an infected dog that is in the first 10-15 days of an acute infection (lewis et al., 1977) , and ticks can shed the organisms for up to 5 months. within the tick population, e. canis is transmitted transstadially (within developmental stages) but not transovarially (from female to offspring) (groves et al., 1977) . pathogenesis. in experimental infections, the incubation period prior to the onset of the acute phase is 7-21 days. during the acute phase, which can last from 2-4 weeks, the bacteria replicate within circulating and tissue monocytes, resulting in lymphoreticular hyperplasia in affected tissues. infected monocytes then spread hematogenously to other organs in the body, in particular the lungs, kidney, and meninges. infected cells adhere to the vascular endothelium and induce vasculitis, which is the primary mechanism whereby the organism causes disease. the thrombocytopenia during the acute phase is due to both sequestration and destruction, and the development of anemia is a result of red blood cell destruction and suppression of erythrocyte production. the subclinical phase of the disease occurs 6-9 weeks after initial infection. during this stage, dogs that can mount an effective immune response clear the infection. those that cannot mount such a response progress to the chronic stage. infection does not confer protective immunity in dogs that recover. german shepherds and doberman pinschers seem to be more severely affected than other breeds. pathologic findings. gross lesions are varied and change, depending on the phase of the disease. the most common findings are petechial and ecchymotic hemorrhages and edema of dependent tissues (woody and hoskins, 2000) . the most common histologic abnormality noted is lymphocytic-plasmacytic inflammation of numerous organs. mononuclear phagocytic system hyperplasia, extramedullary hematopoiesis, and splenic erythrophagocytosis may also be seen. diagnosis and differential diagnosis. the most sensitive, specific, and commonly employed method for diagnosing e. canis infections is the indirect fluorescent antibody (ifa) test. antibodies can be detected as early as 7 days postinfection, although some dogs may not seroconvert until 28 days postinfection (buhles et al., 1974) . cross-reaction may occur between e. canis, e. chaffeensis, and e. ewingii. titers greater than 1:10 are considered positive and indicative of infection and may persist for up to 1 year. effective treatment typically produces seronegative results in 6-9 months. in some cases, asymptomatic dogs may remain seropositive for years after treatment or may be seropositive with a persistent hematologic abnormality (bartsch and greene, 1996) . the exact mechanism for this finding has not been elucidated. ehrlichia canis morulae can be demonstrated in circulating monocytes of giemsa-stained blood smears. however, this method is labor-intensive and has low sensitivity, as morulae are present transiently and in low numbers. using buffy coat smears from capillary blood may increase the diagnostic yield. polymerase chain reaction (pcr) assays are also available to identify e. canis. differential diagnoses include immune-mediated hemolytic anemia/thrombocytopenia, multiple myeloma, chronic lymphocytic leukemia, and lymphoma. prevention. preventing laboratory animals from contacting ticks is the primary means to avoid monocytic ehrlichiosis in research dogs. avoid exercising dogs in areas infested with ticks. use topical acaricides to prevent tick infestations. keep kennel areas tick-free. dogs used as blood donors and dogs from unproven sources should be tested for e. canis. treatment. doxycycline is the drug of choice for treating monocytic ehrlichiosis. oral doses of either 2.5-5 mg/kg q12 hr or 10 mg/kg q24 hr for 21 days are very effective at eliminating the organism. tetracycline, chloramphenicol, and enrofloxacin are also effective antibiotics; however, chloramphenicol should not be used in animals with cytopenias. in chronic cases, antibiotic treatment should be extended for an additional 1-2 weeks. research complications. the most significant research complication is the thrombocytopenia that persists for all stages of the disease. additionally, there is probable alteration in immune function and increased susceptibility to infectious agents. for these reasons, dogs positive for antibodies to e. canis should not be used in research. etiology. this disease, caused by ehrlichia platys, was first described as cyclic thrombocytopenia by harvey et al. in 1978 . clinical signs. in most cases, infection with e. platys results in subclinical disease. a generalized lymphadenopathy may be noted. epizootiology and transmission. the vector for e. platys is assumed to be a tick; however, this mode of transmission has not been established. experimental studies by simpson et al. ( 1991) failed to demonstrate rhipicephalus sanguineus as a vector for e. platys. coinfection with e. canis has been reported, which suggests a common vector for both organisms (french and harvey, 1983; kordick et al., 1999) . dogs have been experimentally infected by inoculation with infected blood or infected platelets from other dogs (harvey et al., 1978; gaunt et al., 1990) . the geographic distribution of thrombocytic ehrlichiosis is assumed to follow that of other ehrlichia organisms. the highest concentration of cases seems to be in southeastern states, but isolated cases have been reported as far north as michigan and as far west as oklahoma (wilson, 1992; mathew et al, 1997) . the prevalence of seropositive dogs can be high in some parts of the country. a study by bradfield et al. (1996) reported that 74% of the dogs entering a research institute's quarantine facility from sources in eastern north carolina were seropositive for e. platys. hoskins et al. (1988) reported a 54.2% seropositive prevalence in healthy dogs from kennels in louisiana. pathologic findings. gross and histopathologic findings during experimental e. platys infection in dogs have been described by baker et al. (1987) . generalized lymphadenopathy was the only gross lesion noted. follicular hyperplasia and plasmacytosis were the predominate findings in lymphoreticular tissues. all dogs also had extramedullary hematopoiesis, erythrophagocytosis, and crescent-shaped hemorrhages in the spleen. multifocal kupffer's cell hyperplasia was noted in the liver, and mild multifocal lymphocytic-plasmacytic interstitial inflammation was seen in the kidneys. pathogenesis. the pathogenesis of e. platys in dogs has primarily been determined through experimental infection (harvey et al., 1978) . after inoculation the organism directly infects platelets. thrombocytopenia occurs by day 10-14 and fluctu-ates, along with parasitemia, at 10 to 14 day intervals. in some cases the rebound may be within the normal range for thrombocyte counts. the nadir can be lower than 20,000 platelets/~d. concurrent with low platelet counts is the development of megakaryocytic hyperplasia in the bone marrow. interestingly, despite extremely low platelet counts, spontaneous bleeding has not been reported in cases of e. platys infection. the mechanism responsible for the cyclic nature of the infection has not been elucidated. diagnosis and differential diagnosis. ehrlichia platys infection may be diagnosed on stained blood smears by visualization of the organisms within platelets. however, this method is very unreliable due to the cyclic nature of the parasitemia and the low numbers of infected thrombocytes. available ifa assays are much more sensitive and specific, and there is reportedly no serologic cross-reaction with other ehrlichia species. dogs usually develop detectable titers 2-3 weeks postinfection. pcr assays for e. platys have now been developed as well (chang and pan, 1996; mathew et al., 1997) . differential diagnoses for thrombocytic ehrlichiosis include e. canis infection, immunemediated thrombocytopenia, and disseminated intravascular coagulation (dic). platys is the same as described for e. canis, above. research complications. ehrlichia platys infection may increase the risk of bleeding during surgical or traumatic procedures. coinfection with e. platys may potentiate the pathogenicity of other infectious agents, in particular e. canis (breitschwerdt, 2000) . etiology. lyme disease is caused by borrelia burgdorferi sensu lato, a microaerophilic spirochete that is primarily an extracellular pathogen. clinical signs. clinical signs may be highly variable; lameness due to polyarthritis has been reported as the most common sign. the onset of lameness may be acute or chronic, shift from limb to limb, and be accompanied by swelling and joint pain. synovial fluid analysis from affected joints is consistent with a diagnosis of suppurative arthritis. other clinical signs include fever, anorexia, lethargy, lymphadenopathy, and weight loss. over the course of the disease, signs may wax and wane over a period of weeks to months. dogs rarely develop erythema chronicum migrans (the characteristic rash seen in infected people) and do not exhibit the severe arthritis and neurologic sequelae seen in human beings (greene, 1991; manley, 1994) . hematologic and biochemical profiles are generally unremarkable. lyme disease is thought to be the most common arthropod-borne disease of human beings (and possibly of dogs) in the united states. it affects humans and dogs worldwide. the geographic distribution of canine borreliosis is assumed to follow that of the human disease and is related to the range of the arthropod vectors. three major endemic foci that have been identified in the united states account for 94% of reported human cases (appel and jacobson, 1995). the distribution of these cases is as follows: northeast/mid-atlantic focus, 85%; midwestern focus (michigan, wisconsin, minnesota, iowa, illinois, and missouri), 10%; and california and oregon, 4%. for the most part, dogs in the remainder of the country are not at risk for contracting lyme disease. borrelia burgdorferi is transmitted exclusively by ixodes ticks. other arthropod hosts may carry the organism but have not as yet been implicated in the transmission of disease. ixodes scapularis, a three-host tick with a 2 to 3 year life cycle, is the prototypical vector for north america. the spirochetes are spread by tick bites from both nymphs and adults. ticks become infected by feeding on an infected mammal and by transstadial transmission (transovarial passage is rare). in endemic areas, 50-80% of adult ticks may be infected (appel and jacobson, 1995) . the primary reservoir for the organism is the whitefooted deer mouse, peromyscus ieucopus, which can carry spirochetes for its life span without becoming ill. evidence also indicates that the eastern chipmunk, tamias striatus, is an important reservoir (slajchert et al., 1997) , and birds may also be a significant reservoir. deer, however, serve only as hosts for the tick vectors and not as a reservoir for the spirochete. pathogenesis. the pathogenesis of lyme disease is poorly understood, primarily because of a lack of good animal models and the chronic nature of the disease. infection can be induced experimentally by the bite of a single infected tick. clinical signs develop 60-90 days postinfection. some evidence points to the host's inflammatory response to the organism as etiologic for disease (pershing et al, 1994; greene, 1991) . seroconversion in dogs occurs 4-6 weeks after infection with b. burgdorferi. antibody titers may remain extremely elevated for at least 18 months. igm titers also remain elevated for several months and are indicative of neither acute nor active infection (appel and jacobson, 1995) . because antibiotic treatment may not eliminate the organism, persistent infections in dogs (treated for 30 days with antibiotics) can be reactivated by steroid treatment up to 420 days postinfection (straubinger et al., 1998) . diagnosis and differential diagnosis. appel and jacobson (1995) recommend that three of the following four criteria be met to establish a diagnosis of lyme disease in dogs: (1) history of exposure to ixodes ticks in an endemic area, (2) characteristic clinical signs, (3) positive serology, and (4) rapid resolution of clinical signs with antibiotic therapy. ifa or elisa tests for borrelia antibodies are the assays of choice. it should be re-membered, however, that a positive titer in an endemic area indicates exposure and not necessarily disease and that vaccinated dogs will also have a positive titer. responses to vaccine versus infection may be distinguished by western blot. culture or identification of the organism provides a definitive diagnosis but is very difficult to perform. differential diagnoses include immune-mediated polyarthritis and septic arthritis from other etiologic agents. prevention and control. prevention and control are the same as for the other tick-borne diseases (see discussion of monocytic ehrlichiosis, section iii,a,l,e above). a vaccine against b. burgdorferi is available but should not be necessary in a research setting. treatment. doxycycline is the drug of choice for treating lyme borelliosis. a typical dosing regimen is 10 mg/kg q12 hr for 3-4 weeks. amoxicillin, tetracycline, and the quinolones are also effective. of significant note is that antibiotic treatment results in resolution of clinical signs but may not result in elimination of the organism. (fox and lee, 1997) . "helicobacter heilmannii" and h. bizzozeronii are thought be the same species, with the latter being the updated nomenclature. this species, as well as h. rappini and h. canis, is considered to be zoonotic (fox and lee, 1997) . clinical infections may present with vomiting, diarrhea, fever, and anorexia, pica, or polyphagia. epizootiology and transmission. the epizootiology and transmission of helicobacter spp. in the dog remains to be elucidated. the prevalence of canine helicobacter infections in colony or shelter situations has been reported to range from 82% to almost 100% (fox, 1995; hermanns et al., 1995) . both oral-oral and fecal-oral routes for transmission have been suggested. pathologic findings. no gross lesions are noted; the primary lesion is that of histologic gastritis. this is typically characterized by reduced mucus content of the surface epithelium; vacu-olation, swelling, karyolysis, and karyorrhexis of parietal cells; and multifocal infiltrates of plasma cells and neutrophils into the subepithelium, primarily around blood vessels and between the gastric pits (hermanns et al., 1995) . focal areas of lymphocytic inflammation and lymphoid follicles may also be seen. pathogenesis. some helicobacter spp. colonize the gastric epithelium exclusively and other species colonize lower parts of the gastrointestinal tract. helicobacter felis and "h. heilmannii" infections have been linked to gastric lesions in laboratoryraised beagles (fox and lee, 1997) . the mechanism by which these organisms cause disease may be related to the host's inflammatory response to colonization and the helicobacter's ability to produce urease. urease splits urea into ammonia and bicarbonate; ammonia is toxic for the epithelial cells, and bicarbonate may help the organism survive the acidic environment (marshall et al., 1990; shimoyama and crabtree, 1998 ). diagnosis and differential diagnosis. any of the numerous causes of acute or chronic vomiting and diarrhea in the dog (including canine distemper, viral or bacterial gastroenteritis, and ingested toxicants) should be considered as differential diagnoses. definitive diagnosis for dogs requires either endoscopic or surgical biopsy. confirmation of infection with helicobacter spp. requires demonstration of the organism in biopsy samples by histopathology, culture, or recognition by pcr. a positive urease test on a biopsy sample may give a presumptive diagnosis, but only for those species that produce urease. the use of warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. prevention and control. until more is known about the epizootiology and transmission of helicobacter spp. in the dog, specific recommendations cannot be made about prevention and control in this species. treatment. combination therapy has proven to be the most effective method for treating helicobacter spp. infections in dogs. combination therapy of amoxicillin (10 mg/kg q12 hr), metronidazole (30 mg/kg q24 hr), and sucralfate (0.25-0.5 mg/kg q8 hr) for 21 days has been suggested for dogs (hall and simpson, 2000) . replacing the sucralfate with famotidine (0.5 mg/kg q24 hr), omeprazole (0.3 mg/kg q24 hr), or bismuth subsalicylate (0.2 ml/kg q4-6 hr) may also be effective (marks, 1997; jenkins and bassett, 1997; denovo and magne, 1995) . the benefits of antimicrobial therapy in dogs still need to be established by controlled therapeutic studies. research complications. helicobacter spp. infections could result in altered gastrointestinal responses to drugs and toxic or carcinogenic compounds. therefore, dogs used in gastric physiology or oral pharmacology studies should be free from helicobacteriosis. clinical signs. clinical signs of canine parvovirus usually appear 5 days after inoculation by the fecal-oral route and are characterized by anorexia, fever, depression, and vomiting. profuse, intractable diarrhea ensues, which may become hemorrhagic. approximately 85% of affected dogs develop severe leukopenia, with a total granulocyte/lymphocyte count ranging from 500-2000 wbc/~d or less. repeated hemograms may provide prognostic value, because rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. parvovirus can infect dogs of any age, but puppies between 6 and 20 weeks of age appear to be particularly susceptible. puppies less than 6 weeks of age are generally protected from infection by passive maternal antibody. adult dogs probably incur mild or inapparent infections that result in seroconversion. pathogenesis. canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes an acute, highly contagious enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis. parvovirus can be detected in fecal samples with a commercially available elisa from cite. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. other lesions include myeloid degeneration and widespread lymphoid depletion. parvovirus can also be demonstrated in frozen sections by fluorescent antibody techniques. differential diagnoses should include other viral enteritides, salmonellosis, and small intestinal obstruction. prevention and control. prevention of transmission begins with isolation of affected animals and quarantine for 1 week after full recovery. disinfection of potentially infected kennel and diagnostic areas with diluted bleach (1:30) or commercially prepared disinfectant (such as kennesol, available from alphatech, lexington, massachusetts) is essential for elimination of the virus. six-week-old puppies should be vaccinated every 2-4 weeks with a commercially available modified live vaccine until 16-18 weeks of age. young rottweilers and doberman pinschers appear to be predisposed to parvoviral enteritis and should be vaccinated every 3 weeks (5 times) from 6-18 weeks of age. treatment. treatment is largely supportive and is aimed primarily at restoring fluid and electrolyte balance. research complications. infection with parvovirus obviously precludes the use of a particular dog in an experimental protocol. given the potential for significant discomfort of the affected animal, and the cost of therapy, humane euthanasia is usually the option chosen in a research setting. canine coronavirus infection is usually inapparent or causes minimal illness. this epitheliotropic virus preferentially invades the enterocytes of the villous tips, resulting in destruction, atrophy, and fusion and subsequent diarrhea of varying severity. subclinical infections are most common, but abrupt gastrointestinal upset accompanied by soft to watery, yelloworange feces is possible. definitive diagnosis by virus isolation or paired sera is usually not made, because supportive therapy generally results in rapid resolution of the diarrhea. inactivated coronavirus is present in commercially available combination vaccines, which are administered immunoprophylactically at 6 -8, 10 -12, and 12-14 weeks of age and then annually thereafter. the role of these vaccines in protection from coronaviral infection is unknown, because the virus typically causes inapparent or mild illness (hoskins, 1998) . etiology. canine distemper virus (cdv) belongs to the family paramyxoviridae, within the genus morbillivirus, which includes human measles virus and rinderpest virus of ruminants. although there is only one serotype of cdv, there is a wide difference in strain virulence and tissue tropism. some strains produce mild clinical signs that are similar to tracheobronchitis, whereas other strains cause generalized infections of the gastrointestinal tract, integument, and central nervous system, resulting in enteritis, digital hyperkeratosis, and encephalitis, respectively. other factors contributing to the severity and progression of clinical signs include environmental conditions, immune status, and age of the host. a transient subclinical fever and leukopenia occur 4-7 days after exposure, with a subsequent fever spike 7-14 days later, accompanied by conjunctivitis and rhinitis. other clinical signs associated with acute distemper include coughing, diarrhea, vomiting, anorexia, dehydration, and weight loss. secondary bacterial infections can cause progression to mucopurulent oculonasal discharge and pneumonia. an immune-mediated pustular dermatitis may develop on the abdomen; this is usually a favorable prognostic sign (greene and appel, 1998) , because dogs that develop skin lesions often recover. neurologic complications of distemper infection may occur weeks to months after recovery from an acute infection. dogs that develop late-onset disease are usually immunocompetent hosts, suggesting that the virus may have escaped complete elimination by the immune system, possibly because of protective effects by the blood-brain barrier. classic neurologic signs that may occur in acute or chronic cdv infection include ataxia, incoordination, vocalization, "chewing gum" seizures, and myoclonus with or without paresis of the affected limb. canine distemper is the most common cause of seizures in dogs less than 6 months of age. dogs with extensive neurologic involvement often have residual clinical deficits, including flexor spasm and olfactory dysfunction. cdv has also been associated with two forms of chronic encephalitis in mature dogs: multifocal encephalitis and "old dog encephalitis." epizootiology and transmission. the virus is highly prevalent and contagious to dogs and other carnivores, especially at the age of 3-6 months, coincident with the waning of maternal antibody. transmission is primarily by aerosolization of infective droplets from body secretions of infected animals. pathologic findings. the predominant histopathologic lesion in neurologic forms of distemper is demyelination, which may .. be accompanied by gliosis, necrosis, edema, and macrophage infiltration. acidophilic cytoplasmic inclusions can be found in epithelial cells of mucous membranes, reticulum cells, leukocytes, glia, and neurons, while intranuclear inclusions are often present in lining or glandular epithelium and ganglion cells. diagnosis and differential diagnosis. diagnosis of cdv is based on history of exposure and clinical signs. young dogs who have not received routine immunoprophylaxis (or similarly, mature dogs with a questionable vaccination history) and present with rhinitis, mucopurulent oculonasal discharge, plus or minus hyperkeratosis of the footpads and neurologic signs, are highly likely to have cdv. ophthalmologic examination may reveal chorioretinitis with acute disease or retinal atrophy in chronic cases. definitive diagnosis of acute infection can be made by fluorescent antibody testing of intact epithelial cells from conjunctival and mucous membranes. attenuated strains of cdv, found in modified live vaccines, are not disseminated from lymphoid tissue to epithelial cells and thus are not detected by the fluorescent antibody. serologic testing is usually not useful, because dogs frequently fail to mount a measurable immunologic response. because of the variety of clinical signs, there are many differential diagnoses for canine distemper. an important differential diagnosis for respiratory illness is infectious tracheobronchitis (kennel cough). bacterial, viral, and protozoal causes of gastroenteritis must be considered for cases presenting with vomiting and diarrhea, and rabies, pseudorabies, bacterial meningitis, and poisonings are differential diagnoses for dogs with central nervous system disorder. prevention and treatment. a series of three immunizations from 6 to 14 weeks of age, followed by yearly boosters, is a recommended preventative. treatment is largely supportive, but because of the profound immunologic effects and significant morbidity of cdv, humane euthanasia is usually undertaken in the research setting. etiology. canine herpesvirus (chv) infection causes a generalized hemorrhagic disease with a high mortality rate in newborn puppies less than 2 weeks of age. in adult dogs, chv causes a persistent, latent infection of the reproductive tract with recrudescence and shedding during periods of physiologic stress. clinical signs. clinically affected puppies do not suckle, cry persistently, become depressed and weak, and fail to thrive. petechial hemorrhages of the mucous membranes and erythema of sparsely haired regions such as the caudal abdomen and inguinal area are evident. older puppies, aged 3-5 weeks, develop less severe clinical signs and are likely to survive with neurologic sequelae such as ataxia and blindness resulting from reactivation of latent infection. infection in adult dogs may result in stillbirths, abortions, and infertility. lesions in adult bitches include raised vesicular foci in the vaginal mucosa, accompanied by mild vaginitis. adult males have preputial discharge due to vesicular lesions at the base of the penis and on the preputial mucosa. passage of puppies through the birth canal or venereally in adult dogs. puppies can also be horizontally infected by littermates. entire primiparous litters may be lost, with subsequent litters protected by colostral antibody. pathologic findings. pathologic findings include multifocal ecchymotic hemorrhages of the kidneys, liver, lungs, and gastrointestinal tract. basophilic intranuclear inclusions in necrotic areas of parenchymal organs are characteristic findings. diagnosis and differential diagnosis. diagnosis of canine herpesvirus infection in adult dogs is based on a history of reproductive infertility and the presence of genital vesicular lesions. differential diagnoses for stillbirths, abortions, and infertility include canine brucellosis, canine distemper virus and parvovirus infections, and pyometra. the diagnosis in infected puppies is usually made based on clinical history and characteristic lesions (multifocal systemic hemorrhages) (carmichael and greene, 1998) . differential diagnoses for the disease in neonates would include canine ehrlichiosis and causes of disseminated intravascular coagulation, including bacterial endotoxemia. there is no effective curative treatment. supportive therapy is unrewarding, and death usually ensues within 48 hours in in-fected neonates. in general, adult bitches that have multiple abortions, stillbirths, or persistent infertility should be culled from the breeding colony. examination of these animals may reveal raised vesicular lesions on the vaginal mucosa. adult male dogs that have vesicular lesions on the base of the penis and preputial mucosa should be similarly culled. adult dogs would obviously interfere with production operations, and affected animals should be culled based on the criteria noted above in the discussion of prevention and treatment. because of the severity of clinical illness in puppies, such animals should be humanely euthanatized. etiology. rabies virus is a member of the rhabdovirus family and is essentially contagious to all species of warm-blooded animals. clinical signs. clinical progression of neurologic disease occurs in three stages. the first, or prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, or furious, stage animals are easily excited or hyperreactive to external stimuli and will readily snap at inanimate objects. the third, or paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death usually occurs within 2-7 days of the onset of clinical signs, due to respiratory failure. epizootiology and transmission. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact of infected saliva from a rabid to a naive animal (or human), usually via bite wounds. pathogenesis. the incubation period for rabies is generally 3-8 weeks from the time of exposure to the onset of clinical signs but can range from 1 week to 1 year. bites of the head and neck typically result in shorter incubation periods because of the proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to the central nervous system and eventually to neurons within the brain, resuiting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis. diagnosis of rabies is based on clinical signs; differential diagnoses include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. definitive diagnosis is based on fluorescent antibody demonstration of the virus in negri bodies of hippocampal cells. prevention and treatment. puppies should be vaccinated at 4-6 months of age, "boostered" in 1 year, then vaccinated annually or triennially, depending on state and local laws and which vaccine product is used. treatment of rabies is not recommended, because of the risk of human exposure. research complications. in a research setting, dogs are often not vaccinated for rabies, because of the low incidence of exposure to wild-animal reservoirs. a healthy, purpose-bred dog that bites a human in a research facility should be quarantined for 10 days and observed for signs of rabies. this quarantine interval is based on the knowledge that dogs do not shed rabies in the saliva for more than a few days before the onset of neurologic disease. a random-source dog with an unknown vaccination history that bites a human should be immediately euthanized. the brain should be examined for rabies virus to determine if the dog was infected, and if the test is positive, postexposure immunization should be initiated for the human patient. a rabies vaccine licensed for use in humans is available, and immunoprophylaxis is recommended for animal care and research personnel who may have high work-related risks of exposure. a. protozoa i. giardiasis etiology. giardiasis is a small-intestinal disease of the dog caused by giardia duodenalis (lamblia), a binucleate flagellate protozoan. clinical signs. most giardia infections are subclinical. when dogs are clinically affected, diarrhea is the most prominent sign. the diarrhea is a result of intestinal malabsorption and is often characterized as voluminous, light-colored, foul-smelling, and soft to watery. weight loss has also been associated with clinical infection. clinical illness is more often seen in young animals. epizootiology and transmission. giardia has a direct life cycle. dogs (and people) typically become infected when they consume water (or food) contaminated with giardia cysts. the ph change from the stomach (acid) to duodenum (neutral) causes excystation. trophozoites migrate to the distal duodenum and proximal jejunum and attach to the villus surface. eventually the trophozoites encyst and pass in the feces to perpetuate the life cycle. pathologic findings. giardiasis is rarely fatal. on histopathology of duodenal or jejunal specimens, giardia trophozoites can be seen attached to enterocytes. mucosal inflammation and ulceration, and villous atrophy, have been observed. pathogenesis. the exact pathogenesis of giardia-induced illness is unknown. it is thought that tissue invasion, although occasionally observed, is unimportant for pathogenesis. it is suspected that illness is caused by physical obstruction of enteric absorption, enterotoxicity, competition for nutrients, excess mucus production, and/or secondary bacterial overgrowth. diagnosis and differential diagnosis. definitive diagnosis requires observation of the organism in fecal or intestinal samples. direct fecal smears are considered best for observing trophozoites, and zinc sulfate flotation is preferred for detection of cysts. commercial elisa kits and direct immunofluorescent tests are available to detect fecal giardia antigens, but the diagnostic specificity and/or sensitivity of these tests may not be sufficient to warrant substitution for the less expensive direct fecal examination or zinc sulfate preparation (barr, 1998) . differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. prevention. high-quality water sources will eliminate the possibility of infection developing within an animal research facility. use of dogs with a known husbandry and medical background will minimize the chances of giardiasis developing in a research colony. control. once giardiasis has been diagnosed in a canine population, segregation of infected animals will help to reduce further infection (provided other dogs were not preinfected at the same source location as the signal case). disinfection with quaternary ammonium compounds, bleach, or steam is usually successful in eradication of giardia cysts. treatment. the most common treatment for giardiasis is metronidazole (flagyl) at 25-30 mg/kg per os twice per day for 5-10 days. quinacrine hydrochloride (atabrine) at 9 mg/kg per os once per day for 6 days, furazolidone (furoxone) at 4 mg/kg per os twice per day for 7-10 days, and the anthelmintics albendazole and fenbendazole have been proposed for use against metronidazole-resistant strains of giardia. a1bendazole is recommended at 25 mg/kg per os q12 hr for 2 days, and fenbendazole at 50 mg/kg per os q24 hr for 3 days. fenbendazole was thought to be safer for both puppies and pregnant females (nonteratogenic) (barr, 1998) . research complications. typical asymptomatic infections probably have no consequence on research protocols, with the exception of intestinal physiology or immunology studies. clinical diarrhea would clearly need to be treated before a dog could be used as a research subject. ii. coccidiosis etiology. intestinal coccidia that have been associated with enteropathy in dogs include cystoisospora canis, c. ohioensis, c. burrowsi, and c. neorivolta. clinical signs. dogs are typically asymptomatic when infected with intestinal coccidia, and oocysts are an incidental finding on fecal flotation or direct smear. dogs that are clinically infected usually develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. epizootiology and transmission. cystoisospora oocysts are typically spread by fecal-oral transmission, usually by ingestion of fecal-contaminated food or other objects in the environment. an indirect form of transmission is also possible, whereby the dog consumes a rodent or other animal that is serving as a transport host. once inside the small intestine, the cyst releases sporozoites that infect enteric epithelium. several generations of asexual reproduction can occur in the enterocyte before sexual reproduction produces gamonts. the gamonts fuse to become a zygote, which encysts, ruptures the enterocyte, and passes in the feces. once in the environment the cyst sporulates and is now an infective stage for ingestion by another host. pathologic findings. dogs with coccidiosis may have hyperemia or fluid retention at affected intestinal segments. the mucosa may appear normal, raised, or ulcerated. histologically, there may be necrosis of enterocytes, hyperemia, and submucosal inflammation. the oocysts are usually readily apparent within the epithelial cells (van kruiningen, 1995) . pathogenesis. intestinal coccidia are opportunistic organisms; they do not typically cause illness unless other predisposing factors are present. such factors include immunodeficiency, malnutrition, and/or concurrent disease. overcrowding and unsanitary conditions can also promote clinical coccidiosis by providing a high population of infective oocysts to stressed animals. diagnosis and differential diagnosis. diagnosis is somewhat difficult, as coccidian oocysts (of both cystoisospora and non-cystoisospora spp.) can be seen on fecal examinations of clinically healthy dogs, as well as animals with diarrhea. other causes for diarrhea (e.g., parvovirus, roundworms, giardia spp., campylobacter jejuni, and inflammatory bowel disease) should be excluded before a coccidial etiology is implicated. prevention. clinical coccidiosis can be readily prevented by adhering to proper sanitation guidelines, reducing any over-crowding, and providing as stress-free an environment as possible. treatment. treatment for the presence of coccidial oocysts may often not be necessary, because cystoisospora infections are typically self-limiting and clinically insignificant. treatment may, however, help to limit the number of oocysts shed in a kennel housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine (25-30 mg/lb per os for 10 days), trimethoprim sulfa (15 mg/lb per os for 10 days), or quinacrine (5 mg/lb per os for 5 days). amprolium, which is not labeled for dogs, can also be used as a coccidiostat. it can be given in gelatin capsules for 7-12 days at a daily dose of 100 mg for small-breed pups and 200 mg for larger breeds. research complications. as with any enteric disease, the presence of clinical coccidiosis can cause aberrations in gastrointestinal physiological parameters. dogs used in intestinal pharmacokinetic studies should be confirmed to be free of cystoisospora infections. b. nematodes i. ascarids etiology. the most common ascarid of dogs is toxocara canis. toxascaris leonina can also infect both dogs and cats. clinical signs. ascarid infestations are most commonly subclinical. however, large worm burdens can cause diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization. puppies may have a classical "potbellied" appearance and dull hair coat. heavy infestations can cause intussusception and/or intestinal obstruction, in which case the young dogs may be found dead. visceral larval migrans caused by toxocara canis can cause pneumonia. epizootiology and transmission. toxocara canis typically infects puppies. in fact, a unique characteristic of t. canis is its ability to infect prenatal puppies by transplacental migration, and neonatal puppies by transmammary migration. ingestion of infective eggs that have been shed in the feces is another common route of transmission, and infection by ingestion of a transport or intermediate host is also possible. pathologic findings. puppies that die from ascarid infestations typically have large worm populations in the lumen of the small intestine. such populations can cause intestinal obstruction and may also result in intussusception or intestinal perforation. puppies that experience lung migrations of large larval worm populations can have severe pulmonary parenchymal damage and develop fatal pneumonia. pathogenesis. the infective stage of t. canis is the third-stage larva (l3). infections initiated by ingestion of infective eggs have three possibilities for larval migration: liver-lung migration (which leads to intestinal infection), somatic tissue migration, and intestinal wall migration. older dogs that become infected typically have an age-related resistance to liver-lung migration and instead experience the other two migratory patterns. these larval migrations are often asymptomatic, and progression of the l3 larvae is arrested in the tissues. it is these larvae that become reactivated in a pregnant bitch, thus establishing the transplacental and transmammary routes of transmission. if the source of infection is transplacental, puppies may be born with l3 larvae in their lungs, because larval migration is already in progress (sherding, 1989 ). diagnosis and differential diagnosis. the characteristic large (70-85 ~tm in diameter) and relatively round ascarid eggs can be readily diagnosed by standard fecal flotation methods. prevention and control. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, 2000) . treatment. most anthelmintics are effective for treatment of ascariasis. pyrantel pamoate (nemex) and fenbendazole (panacur) are commonly used. treatment should be started early in puppies (2, 4, 6, and 8 weeks) because of the possibility of prenatal or neonatal infection. pyrantel pamoate, dosed at 5 mg/kg per os, is safe for puppies and is also effective in treatment of hookworms (see section iii,a,3,b,ii). in breeding colonies in which ascarid infestation is a known problem, treatment of the pregnant and nursing bitch may be advantageous. extended fenbendazole therapy (50 mg/kg per os twice per day for 14 days or once per day from day 40 of gestation through day 14 of lactation) has been shown to be experimentally safe and effective in decreasing ascarid burdens in puppies. research complications. puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. ii. hookworms etiology. the most common and most pathogenic hookworm of dogs is ancylostoma caninum. other, less pathogenic canine hookworms found in north america are a. braziliense, which can be found in the american tropics and southern united states, and uncinaria stenocephala, which is distributed in the northern united states and canada. clinical signs. only a. caninum infestation typically results in clinical illness, because of the amount of blood that it con-sumes. puppies with a. caninum infestations are typically pale and weak (from anemia), with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. epizootiology and transmission. infective larvae (l3) are typically ingested by puppies and develop directly in the intestinal tract. ingestion can be from the bitch's milk (transmammary migration occurs with a. caninum), from food or objects contaminated with infective larvae, or from ingestion of a paratenic host. transplacental migration does occur with a. caninum, but to a much lesser extent than is seen with toxocara canis. larvae can also penetrate intact skin, migrate to the lung via somatic or circulatory routes, and be coughed and swallowed to reach the intestine. the prepatent period is 3 weeks. pathologic findings. infected puppies often have severe anemia and eosinophilia. the anemia can be from acute blood loss or can also be an iron-deficiency anemia caused by chronic blood loss coupled with limited iron reserves. on gross necropsy, the small-intestinal tract contains worms admixed with intestinal contents containing fresh or digested blood (fig. 3a) . ulcerative enteritis caused by hookworm attachment is evident on histopathologic examination, and worms with mouthparts embedded in the mucosa can be identified in some sections (fig. 3b) . pathogenesis. the severe pathogenicity of a. caninum is a direct result of its voracious consumption of blood and body fluids. each adult hookworm can consume 0.01-0.2 ml of blood; thus an extensive infection could deplete a puppy of 20 ml of blood per day, which is approximately 15% of the blood volume of a 2.0 kg animal. in contrast, a. braziliense and u. stenocephala consume 0.001 and 0.0003 ml per worm, respectively. diagnosis and differential diagnosis. diagnosis of ancylostomiasis is made by identification of eggs or larvae from fecal samples by either flotation or direct smear. parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in the diagnosis of a young dog with anemia. prevention and control. purchase of purpose-bred animals will limit the exposure to hookworm larvae, and effective sanitation programs will easily eradicate the infective larvae. unlike ascarid eggs, hookworm eggs are readily killed by drying, sunlight, or cold; however, they do survive readily in warm, moist environments. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, 2000) . treatment. pyrantel pamoate (nemex) is the anthelmintic of choice because it is safest in young ill animals and is also effective against ascarids and other enteric helminths. because of the possibility of transplacental or milk-borne infection, puppies should be treated every 2 weeks from weeks 2-8. a follow-up treatment at 11 weeks is recommended to kill any larvae that have migrated and matured since the initial therapy. severely ill puppies may require supportive fluid therapy and possibly whole blood transfusions and iron supplementation. research complications. anemic puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. iii. strongyloides etiology. strongyloides stercoralis is a small strongyle that can cause hemorrhagic enteritis in puppies. it is found in warm, humid climates such as the southeastern united states. fects dogs and other animals by third-stage larval penetration of the skin or mucous membranes. larvae migrate via the circulatory system to the lung and then are coughed and swallowed to initiate the intestinal parasitism. the eggs of s. stercoralis hatch within the gut lumen, and so it is the first-stage larvae that pass in the feces and need to be identified by diagnostic examination. once passed, the larvae can either develop into the infectious third-stage larvae or mature into free-living, nonparasitic adults. diagnosis and differential diagnosis. the baermann procedure is usually performed on fresh feces in order to detect the motile first-stage larva (280-310 ~tm x 30-80 ~tm). the larvae must be distinguished from larva of filaroides hirthi and hatched ancylostoma caninum. treatment. the usual treatment for s. stercoralis is fenbendazole (panacur) at 50 mg/kg per day for 5 days. iv. whipworms etiology. trichuris vulpis, the canine whipworm, can cause acute or chronic large-intestinal diarrhea. the adult whipworm typically resides in the cecum or ascending colon. clinical signs. most whipworm infections are subclinical. in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss are also seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. severe dehydration with electrolyte imbalance has occurred occasionally as an acute crisis episode. life cycle. adult worms residing in the canine large intestine intermittently release eggs that pass in the feces. the eggs are very hardy and can persist for years. in optimal conditions, the eggs develop into an infective embryo within 10 days. after ingestion by a dog, the larvae hatch in the small intestine, burrow into the small-intestinal mucosa, and then reemerge several days later to travel and burrow into the cecal and colonic mucosa. the prepatent period is typically 2-3 months long. pathologic findings. dogs do not typically die from whipworm infestations. lesions seen as incidental findings feature adult worms embedded into the colonic and cecal mucosae, causing local granulomatous inflammatory reactions and mucosal hyperplasia. pathogenesis. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to the clinical development of diarrhea. factors that influence the possible.development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. diagnosis and differential diagnosis. whipworm infestation is diagnosed by the presence of characteristic trichurid eggs on fecal flotation. these eggs are barrel-shaped, with thick walls and bipolar plugs. because of the intermittent release of eggs by the adult female worms, negative fecal flotation does not exclude the possibility of clinical whipworm infection. adult worms can be seen on colonoscopy (jergens and willard, 2000) . differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. prevention and control. trichuris eggs are resistant to disinfection, making control difficult. dessication or incineration is the only completely effective means to eradicate whipworm eggs from the environment. treatment. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for 3 months (jergens and willard, 2000) . treatment is also suggested in cases wherein whipworm infestation is suspected but not confirmed by multiple fecal flotation. rapid response to treatment would be indicative of a correct diagnosis; lack of response should prompt further diagnostic efforts. research complications. whipworm infestation has not been documented to interfere with research protocols, although one would anticpate that aberrations in local enteric immune function and absorptive functions of the large intestine could result from trichuriasis. etiology. heartworm disease of dogs is caused by the filarial worm, dirofilaria immitis. adult heartworms reside in the pulmonary artery; severe infestations can result in the presence of worms in the right ventricle and atrium. microfilariae, the immature worms produced by the adults, circulate in the bloodstream until a mosquito (intermediate host) ingests them. clinical signs. most heartworm infestations are asymptomatic. the most common clinical signs observed are coughing and dyspnea. clinical signs of exercise intolerance and rightsided heart failure can be seen in severe infestations. epizootiology and transmission. successful heartworm transmission requires the presence of mosquitoes. for this reason, random-source dogs or dogs housed in outdoor kennels are much more likely to have heartworm infestations than indoor, purpose-bred dogs. mosquitoes become infested with heartworm microfilariae when they take a blood meal from the dog. the microfilaria progress through several larval stages within the mosquito, eventually terminating at the third stage. this stage is then returned to the canine bloodstream during feeding. this stage matures within the dog's circulatory system, and the adults reside in the pulmonary artery. male and female heartworms will then sexually reproduce to create more microfilariae and propagate the parasitic life cycle. in the united states, transmission of heartworm by mosquitoes occurs over a 6month or shorter period, except for the southeastern and gulf coast states. here, climatic conditions enable longer survival of the mosquitoes (possibly year-round), thus resulting in the highest prevalence of heartworm infestation (knight, 2000) . pathologic findings. on necropsy, the small, slender worms can be seen in the pulmonary artery, right ventricle, and/or right atrium (fig. 4a ). there may be no histologic abnormalities associated with a minor worm burden, although typically the arterial endothelium in these areas is hyperplastic (fig. 4b) . endothelial cell hyperplasia, vascular smooth muscle hyperplasia, inflammation, and thrombosis of the pulmonary arteries and arterioles characterize more significant infestations. severe infestations can lead to right-sided heart failure and its pathologic sequelae of ascites, pleural effusion, hepatomegaly, and right heart and pulmonary artery enlargement. verminous pulmonary embolism can result from treatment of dogs with anthelmintics when a worm burden is present. immune responses to circulating microfilariae can cause pathologic lesions, most commonly glomerulonephritis. pathogenesis. the physical presence of the worms in the pulmonary artery is partially responsible for clinical signs observed in severe cases. however, the host immunologic response to this infestation, coupled with secretion by the heart-worms of physiomodulative factors, contributes significantly to the complications seen with this disease. endothelial cell proliferation, damage, and sloughing stimulates periarteritis and proliferation of the vascular media of pulmonary arteries and arterioles. these changes lead to thrombosis of these vessels and the arterial truncation that can be seen radiographically in severe infestations. the heartworms also release circulating factors that affect vascular tone and can promote bronchoconstriction (dillon, 2000) . these factors are discussed in more detail below, under "research complications." diagnosis and differential diagnosis. for dogs used in biomedical research, diagnosis of asymptomatic heartworm disease is important, especially if the dogs are used in cardiovascular, pulmonary, or long-term studies. a diagnosis of dirofilariasis is typically made by detection of adult heartworm antigens in a blood sample. use of adult heartworm antigen tests has virtually eliminated the historical status of "occult" heartworm disease, which was caused by infestation of adult worms without corresponding microfilarial circulation. commercial test kits that assay for the presence of adult heartworm antigens, and designed for use by veterinary practitioners, are readily available. false-negative results can occur during the prepatent period after initial infection (first 6-7 months), and when the adult worm burden is light or predominantly male. infections consisting of more than three mature female worms are usually detected by antigenic serology (knight, 2000) . a significant feature of these tests for circulating antigen is that they have a very high specificity (low rate of false-positive resuits). if a dog were negative on initial testing because of prepatency or small worm burden, it will more than likely be detected on a follow-up test 7 months later. examination for circulating microfilariae could be used to confirm an antigenic diagnosis of dirofilariasis or to establish that microfilarial production had occurred. microfilarial detection can be done by microscopic examination of the buffy coat of a microhematocrit tube or by concentration techniques, such as the modified knott test and filter tests. tests that examine for microfilariae have the inherent problem of false positives caused by microfilariae of dipetalonema reconditum, a nonpathogenic filarial worm. other serologic diagnostic tests that were more common historically, and that may still be useful, include detection of antibodies to either adult heartworm antigens or microfilarial antigens. these same techniques can be used to diagnose clinical heartworm disease. additional diagnostic tests that can augment a diagnosis of clinical heartworm disease include thoracic radiography (pulmonary artery and right-heart enlargement), electrocardiography (right-heart enlargement), and hematology (eosinophilia). differential diagnoses for symptomatic heartworm disease (coughing, dyspnea, and exercise intolerance) include canine distemper, canine infectious tracheobronchitis (complicated), streptococcal or other bacterial pneumonia, nocardiosis, and congestive heart failure. prevention and control. for dogs used in biomedical research, prevention is primarily via insect control and housing of the dogs in a controlled, indoor environment. purpose-bred dogs reared in such an environment are usually free from dirofilariasis. however, any dog (random-source or purposebred) exposed to mosquitoes could become inoculated with infective larvae and, if untreated, could develop adult heartworm disease. there are many commercial anthelmintic preparations used to prevent heartworm infestation by killing the larval stages in the canine bloodstream before they become adult worms (e.g., ivermectin, milbemycin, and diethylcarbamazine). these could be used in a research setting in which heartwormnegative dogs are housed outdoors and thus could potentially be infected through mosquito bites. if a research facility is conditioning random-source dogs for long-term use, the presence of circulating adult heartworm antigen should disqualify an animal from the conditioning program. treatment. treatment for eradication of heartworms (adults, juveniles, and microfilaria) is a long process that can pose a significant risk to the patient with regard to both drug side effects (hoskins, 1989) and immunologic reactions to dead worms lodged in the pulmonary vasculature. for this reason, medical treatment of heartworm disease is not usually attempted in research dogs. in a rare instance when such treatment was in the best interest of a long-term canine experiment, thiacetarsamide (caparsolate) and ivermectin (ivomec) were used to eradicate adults and microfilariae, respectively (authors' personal experience). alternative choices include melarsomine (immiticide) as an adulticide and milbemycin (interceptor), levamisole (levasol), or fenthion (spotton) as microfilaricidal agents. dosing regimens for these agents are detailed in dillon (2000) . research complications. the physiomodulative properties of heartworm infection have been studied. such studies have looked at factors released by adult heartworms, as well as changes in the function of host tissues in response to the worm presence. probably the most consistent finding is that endothelial cell-dependent relaxation of pulmonary arterial smooth muscle is depressed in heartworm-infected dogs as compared with control dogs, indicative of alterations in local endothelial cell behavior (maksimowich et al., 1997; matsukura et al., 1997; mupanomunda et al., 1997) . the extension of this effect on peripheral arteries (in vivo and in vitro) has been supported in some studies (kaiser et al., 1992) but refuted in others (tithof et al., 1994) . it is thought that the endothelium is perturbed by a factor released from the adult dirofilaria, possibly a cyclooxygenase product such as prostaglandin d2 (kaiser et al., 1990 (kaiser et al., , 1992 . these products have also been demonstrated to cause constriction in in vitro rat tracheal ring preparations (collins et al., 1994) , suggesting that bronchoconstriction could be an aspect of the pathogenesis of the infestation. platelet reactivity was also been found to be enhanced in dogs naturally infected with dirofilaria, when compared with uninfected controis (boudreaux and dillon, 1991) . based on these data, dogs that are positive for adult heartworm antigen should be considered inappropriate for use as research subjects and, if used, should be restricted to nonsurvival preparations that do not require physiological measurements. etiology. several species of cestodes (tapeworms) parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. clinical signs. most cestode infestations are subclinical. severe infestations with dipylidium can be associated with diarrhea, weight loss, and poor growth. epizootiology and transmission. the cestode life cycle requires an intermediate host. for dipylidium caninum, the intermediate hosts are fleas and lice. thus this species of tapeworm can be readily transmitted by ingestion of arthropods that are canine parasites in and of themselves. taenia pisiformis requires small ruminants, rabbits, or rodents for intermediate hosts, so spread is less likely, especially in a research setting. echinococcus granulosus uses not only sheep as an intermediate host but also human beings, and thus the zoonotic potential of this cestode must be considered. pathologic findings. adult cestodes in the small intestine are usually an incidental finding at necropsy. diagnosis and differential diagnosis. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. dipylidium egg packets are large (100 x 150 bm) and contain 1-63 eggs per packet (hall and simpson, 2000) . prevention and control. the most significant means to limit cestode infestation is to control the population of fleas and/or lice infesting the colony. see the sections on these ectoparasites for effective means to treat infested dogs and kennels. treatment. praziquantel at 5-12.5 mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against dipylidium caninum (hall and simpson, 2000) . clinical signs. most lung fluke infestations are inapparent, but coughing can develop in cases that prompt a strong inflammatory response. pneumothorax has been a sequela of cyst rupture, in which case dyspnea with reduced lung sounds would be the typical presentation. epizootiology and transmission. the lung fluke life cycle requires two intermediate hosts: a snail and then a crayfish. dogs become infested after eating crayfish, which essentially limits this disease to random-source dogs. on ingestion, the immature flukes (metacercariae) migrate to the lungs and encyst in the pulmonary parenchyma. eggs produced by adult flukes are passed into the bronchioles, coughed up, swallowed, and passed in the feces to complete the life cycle. pathologic findings. grossly, the trematode cysts containing adult flukes can be seen in the lung parenchyma. areas of eosinophilic inflammation surround the cysts, and eosinophilic granulomas can also be seen encircling released eggs. pleural hemorrhages may also be caused by the migrating metacercariae (lopez, 1995) . pathogenesis. clinical illness is usually a result of a severe eosinophilic inflammatory response, pneumothorax caused by cyst rupture, or secondary bacterial pneumonia. diagnosis and differential diagnosis. definitive diagnosis of paragonimus infestation requires identification of the characteristic ovoid eggs (80-115 ~tm long) with a single operculum in either the feces or a transtracheal wash. identification from fecal samples requires sedimentation techniques. other causes of coughing in dogs (e.g., infectious tracheobronchitis, dirofilariasis, congestive heart failure) need to be considered. radiographically, the appearance of (multi)focal densities within the air-filled lung field needs to be differentiated from pulmonary neoplasia (primary or metastatic) or systemic fungal pneumonias. prevention. use of purpose-bred dogs virtually eliminates the chance of pulmonary trematodiasis in a research animal. treatment. praziquantel (at 25 mg/kg q8 hr x 3 days) or fenbendazole (25-50 mg/kg q12 hr x 10-14 days) are recommended for treatment of canine paragonimus infestation (hawkins, 2000) . effectiveness is monitored by fecal sedimentation tests for eggs and resolution of radiographic lesions (which may never resolve entirely). early diagnosis of pulmonary trematodiasis should warrant discontinuation of a dog from a long-term study because of the possibility of more serious clinical sequelae, such as pneumothorax. research complications. experimental studies involving the immune system, especially eosinophilic or local pulmonary responses, would be significantly affected by even minor infestations. clinical illness would complicate almost any research project and makes dogs poor anesthetic risks. radiographic lesions may confound diagnostic evaluation for pulmonary metastasis of tumors. e. mites i. demodicosis etiology. canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles. it is considered to be normal fauna of dog skin, but certain conditions (i.e., immunosuppression) cause development of clinical illness. clinical signs. demodex canis infestation is typically asymptomatic. clinical demodicosis presents with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and the face and around the ears (demanuelle, 2000a). secondary bacterial pyoderma is a common complication. epizootiology and transmission. demodex canis mites pass to nursing pups from the dam. they live their entire lives on one dog and are not considered contagious to other dogs or humans. certain breeds are predisposed to the generalized form of demodex dermatitis (see "pathogenesis," below). beagles are among the predisposed breeds, as are german shepherds, doberman pinschers, old english sheepdogs, collies, boxers, and shorthair brachycephalic breeds (muller et al., 1983) . pathologic findings. histologically, demodex infections are characterized by perifolliculitis and folliculitis with mites and keratin debris visible in the hair follicles. cases with generalized demodicosis (see "pathogenesis," below) may have a minimal cellular response with no eosinophils, indicative of severe immunosuppression . pathogenesis. when clinical demodicosis develops, it is classified into "localized" or "generalized" (e.g., more than one foot affected, or five or more small areas, or one large body area). localized demodicosis is typically seen in juvenile dogs (< 18 months) and usually resolves without treatment as natural immunological control develops. generalized demodicosis can develop in juvenile or adult populations. juvenile-onset generalized demodicosis occurs in dogs with a genetic predisposition, thought to be an inherited t-lymphocyte dysfunction. adult-onset generalized demodicosis is usually indicative of an underlying endocrine (hyperadrenocorticism, diabetes mellitus, hypothyroidism) or neoplastic disorder or can develop as a result of immunosuppressive therapy (such as corticosteroid administration). diagnosis and differential diagnosis. demodex is readily identified from deep skin scrapings of lesioned areas (campbell, 2000; noli, 2000) . demodex canis has a characteristic "cigar shape," with short, stubby legs on a body 100-400 ~tm long. differential diagnoses for local demodicosis include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma; remember, however, that bacterial pyoderma is a common secondary complication of the generalized form of this parasitism. prevention and control. dogs with generalized demodicosis should not be maintained in a breeding colony. treatment: ivermectin (ivomec) at 200-600 ~tg/kg and oral milbemycin (interceptor) at 1-2 mg/kg/day have been found to be effective treatments. these parasiticides are probably the most practical to use in a research setting, although they are not labeled for treatment of demodex canis. amitraz (mitaban) dips (250 ppm every 14 days) can be used for more problematic cases. treatment duration can be extensive and must be accompanied by repeated skin scrapings. research complications. dogs with generalized demodicosis should not be used in research studies, because this disease is indicative of another underlying disorder (endocrine or immunological). dogs that receive immunosuppressive agents or paradigms could develop generalized demodicosis as an unexpected consequence of the experimentation. ii. sarcoptic mange etiology. canine sarcoptic mange is caused by sarcoptes scabiei var. canis. clinical signs. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas such as the ear pinnae, elbows, and ventral thorax and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. epizootiology and transmission. sarcoptes mites live their entire lives in the stratum corneum of the host animal; however, they can survive for 1-3 weeks away from the host, and it is this ability that enables them to spread from dog to dog. sarcoptes scabiei var. canis can also infect cats and humans. pathologic findings. histologic examination can be unrewarding because mites are rarely seen on tissue sections, and the associated dermatitis is nondiagnostic: perivascular and interstitial dermatitis with hyperkeratosis, with or without eosinophilic infiltration. suggestive histopathologic lesions are epidermal "nibbles," small foci of edema, exocytosis, degeneration, and necrosis . pathogenesis. lesions and illness are a result of the female mites burrowing through the epidermal layers to deposit eggs, and the larvae migrating back to the surface. the typical locations of mange lesions are a result of the mite's preference for relatively hairless areas. diagnosis and differential diagnosis. sarcoptic mange can be difficult to diagnose because multiple skin scrapings can yield negative results with this parasitic disorder. hopefully, adult mites, mite eggs, or mite feces can be observed on superficial skin scrapings. even if scrapings are negative, however, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige in either the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, 2000 ). an important differential diagnosis is flea allergy dermatitis; in contrast, mange is nonseasonal and contagious. prevention and control. use of purpose-bred dogs limits the possibility of having research animals with sarcoptic mange. for random-source dogs, an ectoparasite control program should be in place to limit possible infestations. many institutions use ivermectin as a means to control both endoparasites and ectoparasites. treatment. unless treatment would interfere with research objectives, all dogs with sarcoptic mange (no matter how minor the lesions) and their kennel mates should be treated because of the contagious nature of the disease and its zoonotic potential. in research colonies, the usual means of treatment is either ivermectin (ivomec) at 200-400 ~tg/kg q14 days or milbemycin (interceptor) at 3 oral doses of 2 mg/kg q7 days . neither of these agents is approved for treatment of sarcoptic mange, but they are considered to be effective. acaricidal dips (e.g., lime sulfur, organophosphates, amitraz) can also be used. research complications. the local skin inflammation and systemic immune response to sarcoptic mange probably make infected dogs poor subjects for dermatologic and immunologic studies. f lice and ticks i. lice etiology. dogs can be infested by one species of sucking louse (linognathus setosus) and two species of biting lice (trichodectes canis and heterodoxus spiniger). clinical signs. mild cases of pediculosis may be asymptomatic or may cause pruritic areas of dry skin. more severe infestations can cause significant pruritus and produce alopecia, papules, and crusts. these lesions lead to excoriation and secondary bacterial dermatitis. severe linognathus infestations could cause anemia, because this species feeds on blood. epizootiology and transmission. louse infestations are uncommon in both pet animal practice and the research setting. they would most likely be seen in random-source dogs that were obtained from a pound or shelter. transmission is usually by direct contact, for lice spend their entire lives on the host species. lice are host-specific and not zoonotic. pathogenesis. the biting lice usually cause more local irritation than the sucking louse and therefore are more apt to induce clinical dermatologic signs. trichodectes canis can serve as vector for the canine tapeworm dipylidium caninum. the most severe complication of infestations by the sucking louse is the potential anemia. diagnosis and differential diagnosis. pediculosis is diagnosed by direct observation of the lice or nits (eggs) on the dog's skin. cellophane tape can be used to pick up surface debris from skin lesions, which may include nits or immobilized lice (muller et al., 1983) . differential diagnoses include dermal acariasis, flea allergy dermatitis, and seborrhea. prevention. use of high-quality conditioned dogs for research should prevent pediculosis from ever being seen within a research facility. random-source dogs should be shampooed or treated prophylactically with topical insecticide before being permitted to enter the research colony. treatment. most commercially available insecticide shampoos and dips readily treat louse infestations. treatment should be repeated in 10-14 days, because any nits that were not killed would have hatched by that time (muller et al., 1983) . there is probably minimal interference with research, unless severe linognathus infestations cause anemia. ii. ticks etiology. ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. except for the brown dog tick (rhipicephalus sanguineus), ticks have a wide host range and are not especially host-specific; so any number of tick genera and species can be found on dogs. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, and ixodes. the primary significance of tick infestation is the tick's ability to be a vector for many other infectious diseases, including rocky mountain spotted fever (caused by rickettsia rickettsii), lyme disease (borrelia burgdorferi), and the canine forms of ehrlichiosis (ehrlichia canis and e. platys), babesiosis (babesia canis), haemobartonellosis (haemobartonella canis), and hepatozoonosis (hepatozoon canis). clinical signs. as an entity unto itself, tick infestation causes minimal clinical signs. most infestations are subclinical, although some dogs may lick and bite at the site, aggravating the local lesion. some dogs can develop a hypersensitivity reaction after several tick bites; these dogs develop a more granulomatous response at the location of the bite (merchant and taboada, 1991) . some species of ticks (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that can cause an ascending flaccid paralysis (malik and farrow, 1991) . the paralysis develops within 5-9 days of tick attachment and can result from a single tick. this paralysis is fatal once the respiratory musculature is affected. epizootiology and transmission. in dogs used for biomedical research, tick infestation may occasionally be seen in randomsource dogs, because these dogs are more likely to have been in tick habitats than purpose-bred dogs. ticks commonly reside in wooded areas until they contact a suitable host for a blood meal. the brown dog tick may reside within kennels (attics, bedding, wall insulation) (garris, 1991) . pathologic findings. under most circumstances, tick infestation will be an incidental finding on necropsy (unless tick paralysis was the cause of death). pathogenesis. tick-bite paralysis is caused by the presence of a salivary neurotoxin released by female ticks of certain genera (e.g., dermacentor) while consuming a blood meal (malik and farrow, 1991) . interestingly, dogs seem to be most affected by this condition, whereas cats appear to be resistant. the primary dysfunction appears to be at the neuromuscular junction, as stimulation of the motor nerves fails to elicit a response, but direct stimulation of the muscle tissue results in contractions. tick bites can also transmit pathogen microorganisms to the dog, because ticks serve as vectors for several infectious diseases, including lyme borreliosis, ehrlichiosis, babesiosis, and rocky mountain spotted fever. diagnosis and differential diagnosis. for uncomplicated tick bites and tick-bite paralysis, definitive diagnosis is made by identification of the offending arachnid (and improvement of paralysis after removal). differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, 1991) . prevention. purpose-bred dogs should be free from all ectoparasites, but ticks can occasionally be seen on randomsource animals. research dogs should not be exercised in outdoor areas infested with ticks, and kennels must be cleaned properly and regularly so as to remain free of ticks and other parasites. treatment. removal of the offending tick is the primary treatment for both local inflammation as well as tick-bite paralysis. dogs with tick-bite paralysis usually show improvement within 24 hr, with complete recovery within 72 hr (malik and farrow, 1991) to remove an attached tick from a dog, forceps should be used to grasp the tick as close to the dog's skin as possible. the tick should not be grabbed by the body, as this may cause the parasite to either rupture or inject its body contents into the dog. the tick should be pulled away from the dog with steady pressure. many of the diseases transmitted by ticks are zoonotic so precautions, such as wearing gloves, should be taken. use of topical acaricide/insecticides on newly arrived random-source dogs should help to limit infestations. probably have minimal impact on research variables. the significant concern for tick infestation is the possible development of tick-bite paralysis or of any one of a number of systemic diseases spread by ticks (see sections iii,a,l,e-g). g. other i. flea infestation etiology. fleas are laterally flattened wingless insects that feed on animal blood. the most common flea to infest dogs is ctenocephalides felis, the cat flea. other fleas that can affect dogs are ctenocephalides canis, pulex irritans, and echidnophaga gallinacea. the fleas are speciated by the shape of their head and by the presence or absence of ctenidae (spiny combs on or behind the head) (campbell, 2000) . clinical signs. flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop the more severe "flea allergy dermatitis," which features papules and crusting. acute moist dermatitis ("hot spots") can also be seen in these cases, and secondary pyoderma or seborrhea can develop. lesions from flea allergy dermatitis generally appear in the dorsal lumbosacral region, as well as the flanks, thighs, and abdomen (muller et al., 1983) . the lesions are typically worse in the summer and autumn months and are progressively more severe as the dog ages. epizootiology and transmission. fleas are readily transmitted between animals and even between host species. they move readily between the host and the environment, making transmission easy and control difficult. because fleas require host blood for food, they can survive off of a host for only 1-2 months (muller et al., 1983) . pathologic findings. biopsy samples are usually nondiagnostic in cases of flea allergy dermatitis. lesions are typically characterized by perivascular eosinophilic inflammation and may feature pustules and folliculitis if secondary pyoderma develops (muller et al., 1983) . pathogenesis. fleas are parasites that require animal blood for their meals. when they bite host animals, they inject some saliva into the host's skin. if the host develops an allergic response to the flea saliva, it will develop the more pruritic flea allergy dermatitis. fleas can also transmit or serve as vectors for other pathogens (e.g., dipylidium tapeworms). flea allergy dermatitis are definitively diagnosed by observing the fleas on the host's skin. given that this may be difficult because of the mobility of the flea and the majority of the time it spends off of the host, diagnosis is often based on clinical signs, history, and lesion distribution. sometimes the presence of flea excrement ("flea dirt") on the dog's skin can support a presumptive diagnosis (demanuelle, 2000b) . circulating eosinophilia is seen in some dogs with flea allergy dermatitis. differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs (e.g., food, drug, or contact hypersensitivity). prevention. most dogs obtained from high-quality purposebred facilities should be free from flea infestations. dogs received from pounds, shelters, or licensed dealers would be more likely to be affected by fleas (or any ectoparasitism). thorough knowledge of prevention, control, and treatment measures at these facilities should be obtained, and dogs from sources where proper prevention and/or therapy are not practiced should be evaluated and/or empirically treated upon arrival at the facility. control. thorough cleaning of the dog's housing environment should remove the risk of perpetuating or transmitting flea infestation in the colony. treatment. treatment for fleas needs to address treatment of both the dog and the environment. many insecticide formulations such as shampoos, sprays, dips, powders, and oral systemics can be used for initial treatment of the individual dog. the active ingredients include pyrethrins, pyrethroids, carbamates, and organophosphates. flea control in the kennel may need to include outdoor areas in warm climates. typically combinations of adult insecticides and juvenile growth regulators are used for environmental treatment. directed sprays are the most effective means of treating housing areas, because flea "bombs" or foggers do not penetrate adequately into tight areas where fleas might hide (demanuelle, 2000b) . in addition to insecticide therapy, dogs with flea allergy dermatitis may also require anti-inflammatory medication to relieve clinical signs. oral prednisone or prednisolone at 0.5 mg/kg q12 hr for 5-7 days has been proposed as a starting therapy (muller et al., 1983) . the use of hyposensitization with flea-bite antigens is controversial and not practical for the research setting. research complications. mild flea infestation probably has minimal impact on most research protocols, and treatment measures may in fact be more detrimental to the experimental objective than the actual ectoparasitism. in a research setting, the residual effects of insecticides may preclude their use in experimental animals. such treatments should be used judiciously to ensure that experimental results are not more seriously affected by the therapy rather than the infestation. dogs with flea-allergy dermatitis are more severely affected by the flea infestation and should be treated apigropriately; however, systemic corticosteroids may also interfere with experimental objectives, especially in studies involving functions of the immune system. the ability of fleas to transmit other parasitic diseases must also be considered. etiology. dermatophytoses ("ringworm") are fungal skin infections, which in dogs in the united states are usually caused by either microsporum canis, m. gypseum, or trichophyton mentagrophytes (muller et al., 1983) . clinical signs. uncomplicated superficial dermatophytoses are characterized by circumscribed circular areas of alopecia, usually with minimal to no inflammation. these skin lesions are usually seen around the face, neck, and forelimbs but can be found anywhere on the body. secondary bacterial infections can develop; these lesions are called kerions and are selflimiting, for the fungus cannot survive in inflamed skin (muller et al., 1983) . ep&ootiology and transmission. the fungi that cause skin infections are very contagious and readily transmissible between dogs and other species (including human beings), but they can also be obtained from the soil. pathologic findings. on close inspection of skin samples, broken hair shafts (and not complete hair loss) would be seen with uncomplicated dermatophytosis. histologically, fungal elements can be seen within the stratum corneum or in and around the hair and hair follicles (muller et al., 1983) . stains that facilitate visualization of fungal elements include periodic acid-schiff (pas) or gomori methenamine-silver. the pattern of inflammation in the affected foci is very variable and can feature folliculitis, perivascular dermatitis, hyperkeratosis, and/or vesicular dermatitis. pathogenesis. the dermatophytes typically infect the hair shaft itself, the hair follicle, and possibly the skin around the affected hair. the hair follicle is not destroyed (unless by secondary bacterial infection), but the hair itself becomes brittle and breaks. this causes short stubbly hair to be seen within the lesion. as the lesion progresses, the hairs in the center recover from the infection, thus leading to the classic "ringworm" appearance of the alopecic areas. it is postulated that the inflammatory process produces an environment that is unfavorable for dermatophyte survival, whereas the periphery of the lesion still enables continued fungal growth (muller et al., 1983) . diagnosis and differential diagnosis. diagnosis of dermal fungal infection is typically made by scraping the affected area to obtain hair and superficial epidermal cells. these scrapings are then digested with potassium hydroxide to facilitate observation of fungal elements. fungal elements can also be seen on skin biopsy samples. for speciation of a fungus, skin scrapings can also be inoculated onto agars that promote fungal growth, such as sabouraud's medium or dermatophyte test medium (dtm). incubation should be at 30~ with 30% humidity for 14-30 days. lesions caused by m. canis may fluoresce when inspected using a wood's (253.7 nm ultraviolet) light. unfortunately, some strains of m. canis do not fluoresce, and neither does m. gypseum or t. mentagrophytes. differential diagnoses for dermatomycosis include seborrhea, localized demodecosis, folliculitis, histiocytoma, and acral lick dermatitis (muller et al., 1983) . prevention. purpose-bred dogs are typically free of infectious dermatophytes, but ringworm may be diagnosed on randomsource animals. control. in cases of dermatophytosis, isolation of the affected animal(s) is prudent, because the fungi are easily spread to other dogs, as well as to people. treatment, if acceptable, should be started immediately. treatment. topical antifungal therapy is most commonly used. shampoos, rinses, and creams containing miconazole, ketoconazole, enilconazole, or chlorhexidine are commercially available to treat ringworm (stannard et al., 2000) . severe cases may require systemic therapy with griseofulvin, ketoconazole, itraconazole, or fluconazole. however, these systemic antifungal agents may have considerable side effects (such as vomiting and teratogenicity with griseofulvin). many of the newer agents are also expensive and not labeled for use in dogs. impact on most research applications for dogs. unfortunately, the zoonotic implications of dermatophytoses force the issue of aggressive treatment, and many antifungal agents may not be compatible with biomedical research studies. systemic fungal infections disseminate to multiple organ systems from a single mode of entry (usually through the respiratory tract). dogs are susceptible to several fungi that characteristically cause systemic mycosis, including blastomyces dermatitidis, histoplasma capsulatum, coccidioides immitis, and cryptococcus neoformans var. neoformans. these diseases are not typically seen in the research setting, because of the low overall incidence and noncontagious nature of these disorders and because of the use of purpose-bred animals. these conditions could, however, present in the rare random-source dog that was subclinical at its point of origin, especially if the animal becomes immunosuppressed (either naturally or by virtue of experimental manipulation). typical clinical signs include weight loss, fever, lymphadenopathy, and cough and dyspnea (if the lungs are affected). the reader is advised to read veterinary medical text chapters (e.g., taboada, 2000) for more complete information on these disorders and their possible treatments. although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, 1994) , deficiency in thyroid hormone can significantly affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology. the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. both of these causes result in a gradual loss of functional thyroid tissue (kemppainen and clark, 1994) . lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, 1962; beierwaltes and nishiyama, 1968; manning 1979) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, 1989; kemppainen and clark, 1994) . clinical signs. because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including alopecia, hyperpigmentation, seborrhea, and pyoderma (peterson and ferguson, 1989; panciera, 1994) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, 1989; panciera, 1994) . normocytic normochromic nonregenerative anemia and increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, 1989; panciera 1994) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, 1989) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, 1989) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bischel et al., 1988; panciera, 1994) , and so the relationship between hypothyroidism and these problems has not been completely defined (panciera, 1994) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, 1989) . hypothyroidism can also cause bradycardia as a result of decreased myocardial conductivity. abnormalities that may be detected by ecg include a decrease in p and r wave amplitude (peterson and ferguson, 1989) and inverted t waves (panciera, 1994) . these electrocardiographic abnormalities are caused by lowered activity of atpases and calcium channel function. several reports have suggested that hypothyroidism is associated with von willebrand's disease and bleeding abnormalities. however, the relationship is probably one of shared breed predilection and not a true correlation. it has been demonstrated that dogs with hypothyroidism are not deficient in von willebrand's factor when compared with other dogs. in addition, the replacement of thyroid hormone in dogs did not increase the levels of vwf:ag in naturally occurring (panciera and johnson, 1994) or experimentally induced (panciera and johnson, 1996) hypothyroidism. epizootiology. the prevalence of hypothyroidism in the general canine population has been reported to be less than 1% (panciera, 1994) . the disorder occurs most often in large-breed dogs but has been reported in several other breeds as well as mongrels. doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism when compared with other breeds (panciera, 1994; peterson and ferguson, 1989; scarlett, 1994) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, 1979; tucker, 1962; beierwaltes and nishiyama, 1968) . in general, the problem is usually recognized in middle-aged animals, and some reports state that there is a higher incidence of hypothyroidism in spayed female dogs (panciera, 1994; peterson and ferguson, 1989 ). diagnosis and differential diagnosis. because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. the tests currently available and in popular use will be discussed further. however, a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, 1989; ferguson, 1994) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t 4 (thyroxine) and free t 4 (peterson and ferguson, 1989; ferguson, 1994) . t 4 serves primarily as a precursor for t 3 in the body and is heavily proteinbound. free t4 represents the unbound fraction that is available to the tissues (peterson and ferguson, 1989) . using the measurement of serum total t 4 and free t4, hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial is in order (peterson and ferguson, 1989) . however, it must be noted that nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, 1989; ferguson, 1994) . therefore, low values do not always indicate that hypothyroidism is present, and animals should not be treated solely on the basis of serum hormone levels if clinical signs are absent. if the clinical signs are equivocal or if only total t 4 or free t 4 is decreased, further diagnostic testing is warranted (peterson and ferguson, 1989) . although t 3 is the most biologically active form of thyroid hormone in the body, the measurement of serum t 3 levels is an unreliable indicator of hypothy-roidism (peterson and ferguson, 1989; ferguson, 1994) . like t4, serum t 3 can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t 3 may be preferentially released, and conversion of t 4 to t 3 may be enhanced in the hypothyroid dog (peterson and ferguson, 1989; ferguson, 1994) . t 3 was within normal limits in 15% of the hypothyroid dogs in one study (panciera, 1994) . autoantibodies can be responsible for false elevations in the concentrations of t 3 and t 4 found in these respective assays. it has been recommended that free t4, measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t 3 and t 4. autoantibodies have been found in less than 1% of the samples submitted to one laboratory (kemppainen and behrend, 2000) . other means of diagnosing hypothyroidism have been described. in humans, endogenous tsh (thyroid-stimulating hormone) levels provide reliable information on thyroid status, and an assay for endogenous tsh is now available in dogs. however, tsh levels can be normal in some dogs with hypothyroidism, and high tsh levels have been noted in normal dogs. therefore, it is recommended that tsh levels be considered along with other information (clinical signs, t4) prior to diagnosis and treatment (kemppainen and behrend, 2000) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, 1989; ferguson, 1994) . another drawback of tsh testing is that the test must be postponed for 4 weeks if thyroid supplementation has been given (peterson and ferguson, 1989) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is 0.045 u of tsh per pound of body weight (up to a maximum of 5 u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and 6 hours after. a normal response to the administration of tsh should create an increase of t 4 levels at least 2 ktg/dl above the baseline levels or an absolute level that exceeds 3 ~tg/dl (peterson and ferguson, 1989; wheeler et al., 1985) . treatment. the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is 0.02 mg/kg once a day or 0.05 mg/m 2 (body surface area)/day for very small or very large dogs. if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in 6-8 weeks, and blood samples should be drawn 4-8 hours after the morning pill. a clinical response is usually seen in 6-8 weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, 1994) . ecg abnormalities also return to normal (peterson and ferguson, 1989) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, 1994) . weight gain and eventual obesity are also frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must be aware of the development of obesity and the potential effect that it can have on research. etiology. obesity is defined as a body weight 20-25% over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy. excessive caloric intake resuits from overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, 1992) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household, because access to food is more restricted and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and thus the limitation to exercise reduces energy expenditure and predisposes dogs to weight gain. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy expenditure are followed (butterwick and hawthorne,. 1998 ). as in humans, genetics plays an important role in the development of obesity in dogs. it has been established that certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, 1986) . in addition to genetics, several metabolic or hormonal changes are associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese when compared with intact females (macewen, 1992) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, 1986) . in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, 1992) . epizootiology. ewen, 1992) . obesity affects up to 40% of pet dogs (mac-diagnosis and differential diagnosis. the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict total body fat (wilkinson and mcewan, 1991) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see section iii,b,l,a), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment. restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to 60% of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to 50% produces no adverse health effects. however, t 3 levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of 1-2% of body weight per week (laflamme et al., 1997) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to rebound and gain weight after restrictions are relaxed. there has been agreat deal of attention in humans as to the correct diet to be fed to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, 1992) . there has been much concern about the addition of fiber to the diet in both humans and animals as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorierestricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick and thorne, 1994; butterwick and thorne, 1997) . it is important to control weight gain in research animals, because of the association of obesity and several metabolic changes. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, 1992) , this relationship is not consistently apparent (edney and smith, 1986) . obesity in dogs over 10 years of age appears to be related to an increase in cardiovascular problems (edney and smith, 1986) , and obesity has been linked to hypertension. joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, 1992; kealy et al., 1997) . in addition, diabetes mellitus has been linked to obesity, and obesity induces hyperinsulinism in several experimental models (macewen, 1992) . in the laboratory setting, the majority of traumatic wounds will be small in size. in facilities with good husbandry practices and a diligent staff, traumatic wounds will generally be observed quickly and attended to promptly. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or. when the basic principles of wound management are not followed. to aid in the description of wounds and in decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a "golden period." it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the capability of the host's defense systems (swaim, 1980; waldron and trevor, 1993) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as either clean, clean-contaminated, contaminated, or dirty (see table v ). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds, which are seen infrequently in the laboratory setting, require more aggressive therapy. dirty wounds can occur as postsurgical infections or complications of initial wound therapy. when one is in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of the wound's classification. when first recognized, the wound should be covered' with a sterile dressing until definitive treatment is rendered. bleeding should be controlled with direct waldron and trevor (1993) . pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, 1980) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, 1980) . when the treatment of a wound begins, anesthesia or analgesia may be necessary, and the choice of anesthetic regimen will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed at this time. then a water-soluble lubricant gel may be applied directly to the wound. a wide margin of hair should then be clipped from around the wound, using a #40 blade. after the clipping, a surgical scrub is performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., 1990a,b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. two solutions, 0.05% chlorhexidine diacetate in water (lozier et al., 1992) and 1% povidone-iodine in saline, are most often recommended for wound lavage (waldron and trevor, 1993) . the chlorhexidine solution may offer the advantage of greater bactericidal activity but does not significantly alter wound healing when compared with povidone-iodine (sanchez et al., 1988) . actually, the type of solution chosen may not be as important to wound care as the volume and pressure at which the solution is delivered. it has been suggested that 8 psi is required to obtain adequate tissue irrigation, and this may be achieved by using a 35 ml syringe and an 18-or 19gauge needle (waldron and trevor, 1993) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, 1993) . if one is unsure about tissue viability in areas that are devoid of extra skin, the tissue may be left (swaim, 1980; waldron and trevor, 1993) , and nonviable areas will demarcate within 2-3 days (waldron and trevor, 1993) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, 1980) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure of the wound at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be place d. subcutaneous closure should be performed with absorbable suture such as polydioxanone (pds), polyglactin 910 (vicryl), or polyglycolic acid (dexon). it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon (3-0 or 4-0). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. initially, the wound can be covered by gauze sponges soaked in saline or chlorhexidine to create a wet-to-dry bandage. when the sponges are later pulled from the wound, dried exudates will also be removed. when the wound appears clean, the layer in contact with the wound may be changed to a nonadherent dressing such as vaseline-impregnated gauze (swaim, 1980) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within 3-5 days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after 5 days, this is considered secondary closure (waldron and trevor, 1993) . secondintention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, 1980) . it is important to note that second-intention healing will take longer than surgical repair of a wound, and in the case of large wounds it will be more expensive because of the cost of bandaging materials. several factors must be weighed concerning the use of antibiotics in traumatic wounds, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated, and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. in skin wounds, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. cephalosporins, amoxicillin-clavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, 1993) . etiology. pressure sores (decubital ulcers) can be a problem in long-term studies that require extended periods of recumbency. decubital ulcers usually develop due to continuous pressure from a hard surface contacting a bony prominence such as the elbow, the tuber ischii, tarsus, or carpus. the compression of the soft tissues between the hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, 1990) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores. these factors include poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting casts or bandages (swaim and angarano, 1990) . clinical signs. at first, the skin at the developing site will appear red and irritated. over time, constant trauma can result in full-thickness skin wounds and can progress to necrosis of underlying structures such as bone. the severity of the sores may be graded from i to iv, according to the depth of the wound and the tissues involved, from superficial skin irritation to bone necrosis. epizootiology. the problem usually occurs in large-breed dogs, but any type of dog can be affected. prevention and control. minimizing or eliminating those factors that can predispose to decubital ulcers is important to both the prevention and the control of this condition. if the dogs are going to experience long periods of recumbency, adequate bedding or padding must be provided. skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain good flesh and adequate healing is also important (swaim and angarano, 1990) . treatment. the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive therapy must be performed. the affected area should be radiographed to assess bone involvement, and the wound should be cultured. all of the damaged tissue should be debrided, and wound management guidelines should be followed (see section iii,c,1). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, 1990) . with extensive lesions, reconstruction with skin flaps may be necessary. bandaging should be performed on all full-thickness wounds; however, it is important to remember that ill-fitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded, because this will increase the pressure over the wound. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound. this will displace the forces acting on the wound over a larger area and over healthier tissue. then the doughnut is incorporated into the bandage. if a cast has been applied to the area for treatment or for research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, 1990 ). bandages should be removed at least once or twice a day to allow wound care. after wound care has been initiated the causative factors for the pressure sore must be addressed (see "prevention and control," above). recumbent animals should be moved frequently to prevent continuous compression on the wound. if the dog tends to favor a position that aggravates the problem, splinting the body part to reduce contact with hard surfaces may be necessary. etiology. acral lick granuloma is a psychodermatosis, a skin lesion caused by self-trauma. in a few cases, self-trauma begins because of identifiable neurologic or orthopedic causes (tarvin and prata, 1988) . however, the majority of the cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, 1990) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., 1988) . the laboratory setting is an environment that could promote this abnormal behavior and lead to acral lick granuloma. epizootiology. the lesions associated with acral lick granuloma are seen most often in large-breed dogs, but any type of dog can be affected (walton, 1986) . clinical signs. at first, lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, 1990 ). the predilection for the limbs may be due to accessibility or possibly may be caused by a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated, and the wound has a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis. acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast-cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of these problems can be ruled out by the history of the animal. when in doubt, a biopsy should be taken. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, 1986) . prevention and control. behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. the environment of a dog with this problem can be enriched with exercise and the introduction of toys. in addition, the relief of boredom or anxiety can be attempted through the use of drugs such as phenobarbital, megestrol acetate, and progestins. these drugs may produce side effects, however (swaim and angarano, 1990) , and may interfere with experimental results. treatment. several treatments have been reported for acral lick granuloma, and none of them have been proven to be successful in ah cases. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, 1990; walton, 1986) . opioid antagonists have been used in an attempt to treat acral lick granuloma by blocking endogenous opioids. in one study, either naltrexone (1 mg/kg sq) or nalmefene (1-4 mg/kg sq) successfully reduced the excessive licking behavior in 7 of 11 dogs; however, lesions returned after the drug was discontinued (dodman et al., 1988) . the use of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide (3 ml of banamine [schering] mixed with 8ml of synotic [diamond laboratories]) applied topically twice daily has also been shown to be effective (walton, 1986) . the prognosis for acral lick granuloma should be considered guarded, because the lesions often recur or new lesions develop when treatment is discontinued. etiology. hygromas are fluid-filled sacs that develop as a result of repeated trauma over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., 1974) . epizootiology. elbow hygromas are most frequently reported in large and giant breeds of dogs around 6-18 months of age (johnston, 1975; bellah, 1993) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment for research dogs predisposes them to hygromas, because these animals spend a large amount of time on hard surfaces such as cage bottoms or cement runs. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs. a dog with an elbow hygroma presents with a unilateral or bilateral, painless, fluctuant swelling over the point of the elbow. the animals are not usually lame. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma is secondarily infected, the animal may exhibit pain and fever (johnston, 1975; bellah, 1993) . pathology. the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is yellow or red and is a serous transudate. this fluid is less viscous than joint fluid, and elbow hygromas do not communicate with the joint (johnston, 1975) . treatment. the treatment of elbow hygromas should be conservative whenever possible, and surgical options should be reserved for complicated or refractory cases. conservative management of the elbow hygroma is aimed at relieving pressure at the point of the elbow by providing a padded cage surface and/or bandaging the elbow in a manner similar to that used to treat pressure sores (see section iii,c,2). more aggressive therapy, including needle drainage and the injection of corticosteroid into the hygroma, has been described but is not recommended, because infection is a serious complication of this treatment (johnston, 1975) . likewise, simple surgical excision of elbow hygromas can be associated with complications such as wound dehiscence and ulceration (johnston, 1975) . a technique that has been used successfully involves placement of multiple penrose drains. the drains are kept in place for 2-3 weeks, and the limb remains bandaged for 4 weeks with this technique (bellah, 1993) . another technique has been described that involves the removal of a crescent-shaped piece of the skin and capsule. the remaining dead space is closed with mattress sutures over stents, and then the wound is closed in a routine fashion. the stents are removed in 5-7 days, and the wound is bandaged until suture removal in 10-14 days (newton et al., 1974) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology. in the research environment, corneal ulcers are most often associated with either direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these cases would be rare in the laboratory setting. clinical signs. the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may not appear abnormal; however, in cases of deeper ulceration, the cornea may appear roughened or may have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers may be made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies or abnormal eyelids or cilia. treatment. the treatment of corneal ulcers will depend on the depth and size of the affected area. deep ulcers may require debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given 3 times a day for 2-3 days usually provides adequate treatment. ointments are preferred over drops, because use of the former requires less frequent. simple corneal ulcers are restained with fluorescein after 3 days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. indwelling intravascular catheters, including intracaths and vascular access ports, often play a vital role in research protocols. the catheters are most often placed in a central vein or artery where they may be used for repeated blood sampling, administration of anesthetics and experimental compounds, or measurement of hemodynamic parameters. although catheters vary in composition, number of ports, and port placement, the basic principles of their implantation and maintenance are similar. it is important that the laboratory animal veterinarian be familiar with these principles and the potential complications of catheter use. when appropriately maintained, indwelling catheters may remain functional for months without serious complication. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. this is due largely to the fact that many of the problems may be incidental findings or related to a particular research protocol. one study (hysell and abrams, 1967 ) examined the lesions found at necropsy in animals with chronic indwelling catheters (exact vascular locations not specified). the lesions found were categorized as traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. the traumatic cardiac lesions consisted primarily of masses of fibrin and inflammatory cells on the heart valves. the visceral infarcts were noted in the spleen, kidney (fig. 5) , and brain and resulted from fibrin embolization from either the valvular lesions or the catheter tip. fatal hemorrhages were most often found in animals with experimentally induced hypertension. these animals developed clinical signs of sepsis and later ruptured a major vessel associated with mycotic infection and aneurysm. etiology. the leading complication associated with the use of indwelling vascular catheters is infection, either systemic or local at the point of entry through the skin. septicemia may develop from bacterial colonization of either the tract around the catheter or the catheter lumen. clinical signs. the signs and treatment of systemic infection are covered in section iii,d,3. problems with the skin defect associated with the catheter port vary from mild skin irritation to obvious infection. the signs may include redness and swelling of the skin around the external port, discharge from the skin wound, or even abscess formation. prevention. because indwelling catheters play an important role in many research protocols, it is highly desirable to prevent catheter complications that may result in loss of the device. the catheter should be made of nonthrombogenic material. in addition, it is recommended that catheters be as simple as possible. a catheter with extra ports or multiple lumens requires additional management and supplies more routes for infection. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that a long extension of tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, 1984) . the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. the placement of the catheter should be verified by radiography. catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. after catheter placement, the animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide, as described below (see "treatment"). throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. treatment. the treatment of catheter infections almost invariably involves removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, 1984; darif and rush, 1983) . superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. systemic antibiotic therapy should be initiated for a 10-day period. the choice of drug will ultimately be based on previous experience and culture results. aerobic and anaerobic cultures of blood and locally infected sites should be performed (ringler and peter, 1984) . localized abscesses or sinus tracts may be managed by establishing drainage and flushing with chlorhexidine. again, the catheter should be removed. if retention of a catheter is important, the catheter lumen may be disinfected by filling with chlorine dioxide solution. it has been shown that there are no adverse effects from the use of chlorine dioxide in catheters (dennis et al., 1989) . the solution is removed after 15 min and replaced with heparinized saline. all of the extension lines and fluids used in the catheter should be discarded. the blood cultures should be repeated 3 days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., 1993) . when placed and managed correctly, these ports may remain in place for months without complications. the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., 1993 , kwei et al., 1995 . these infections lead to removal of the catheters despite treatment with local lavage and systemic antibiotics. there have also been reports of catheters dislodging from the intestinal tract and resulting in peritonitis. this complication has largely been eliminated with the improved security afforded by a synthetic cuff added to the end of the catheter (meunier et al., 1993) . the chapter authors have also seen migration of the catheter end within the lumen of the intestine (caused by peristaltic motion to egest the catheter), extensive intra-abdominal adhesions, and intestinal torsion (figs. 6a,b) as complications of intestinal access ports. the procedures for placement and maintenance of the catheters are similar to those outlined previously for indwelling vascular catheters. it is important that the catheters be firmly secured to the intestine to prevent migration or dislodgment. an omental patch placed over the site of entry may help form a firm adhesion. in addition, it is important to place the proper length of catheter within the peritoneal cavity; excess catheter length can promote adhesion formation, whereas insufficient catheter length to account for visceral organ motion can result in detachment. the placement and patency of the catheters can be verified periodically by contrast radiography using iodinated contrast material or by fecal occult blood testing after a small amount of blood has been injected through the catheter (meunier et al., 1993) . etiology. sepsis is defined as the systemic response to infection. most often, sepsis is a result of infection with gramnegative bacteria; however, sepsis may also be associated with gram-positive bacteria and fungi. in laboratory animals, sepsis is seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs. the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course of sepsis, dogs will present with signs of a hyperdynamic response, including an increased heart rate, increased respiratory rate, red mucous membranes, and a normal to increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals will show the classic signs of septic shock, including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, 1993) . pathogenesis. the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such ~ as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis. in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of bands than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., 1997) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several systems should be evaluated for infection, including urinary tract, reproductive tract, abdominal cavity, respiratory tract, teeth, and heart valves (kirby, 1995) . treatment. the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is associated with the source of infection, the implant should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or third-generation cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no "magic bullet" for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology. in research animals, aspiration into the lungs may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. clinical signs. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on a history consistent with aspiration and the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. pathogenesis. aspiration of gastric contents or other compounds can create lung injury of variable severity, depending upon the ph, osmolality, and volume of the substance. the compounds aspirated can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after 24-48 hr. treatment. the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. in cases in which a small amount of a relatively innocuous substance (e.g., barium) has been aspirated, treatment may not be necessary. when severe inflammation is present, systemic fluid therapy should be instituted. support of the cardiovascular system should be performed judiciously; fluid overload could lead to an increase in pulmonary edema. the use of colloids is controversial because of the increase in vascular permeability that occurs in the lungs. oxygen therapy is also controversial, because it may increase lung injury if administered at high concentrations for long periods of time (nader-djahal et al., 1997) . several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. in humans, antibiotics are reserved for use in cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be treated with antibiotics immediately when the aspirated material is either not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, 2000) . the presence of pneumonia should be verified by tracheal wash and cultures. etiology. in laboratory animals, accidental burns usually result from thermal injury (heating pads, water bottles) or harsh chemicals (strong alkalis, acids, disinfectants). the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs. the clinical signs vary with the type and degree of burn injury. initially, the injury may not be noticed. the first signs may be oozing from the skin and matting of the overlying hair. within a couple of days, progressive hair and skin loss may be observed (johnston, 1993) . the wounds may vary in severity from very superficial (involving only the epidermis) to those in which the epidermis and dermis are completely destroyed. superficial wounds appear as red, inflamed skin similar to sunburn in humans. the pain associated with these injuries usually subsides in 2-3 days, and the wound reepithelializes without complications in 3-5 days. deeper burns develop a thick covering, or eschar, composed of the coagulated proteins and desiccated tissue fluid. the wound heals by granulation under the eschar, which eventually sloughs or is removed to allow further healing by contraction and reepithelialization. within 2-3 days of injury, the burn wound will be colonized by grampositive bacteria that rapidly cover the entire wound. several days later, gram-negative organisms can appear in the burn wound (johnston, 1993) . at this point, signs of wound infection and sepsis may occur (see section iii,d,3). treatment. appropriate and timely treatment of a burn wound will reduce the extent of the injury. thermal injuries should be immediately cooled to reduce edema and pain (demling and lalonde, 1989) . chemical burns should be thoroughly lavaged for 60 min after wounding. the damaged tissues may be unable to mount appropriate responses to changes in temperature; therefore, the lavage should be performed with warm water to prevent hypothermia. after the initial treatment, all burn wounds should be gently cleansed 2-3 times a day (demling and lalonde, 1989) . burns involving the epidermis and part of the dermis can be extremely painful, and analgesia should be addressed throughout the treatment period. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, topical wound dressings are recommended in the early stages of treatment. a thin film of a water-soluble broad-spectrum antibiotic ointment should be applied to the wound surface after each cleaning. silver sulfadiazine has a broad spectrum, penetrates eschar well, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it is associated with pain upon application (demling and lalonde, 1989) . when signs of wound or systemic infection are present, systemic antibiotics should be employed, and their ultimate selection should be based on culture and sensitivity results. after the topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds. when the eschar over a burn wound has formed and become fully defined, a small or moderately sized wound may be completely resected. prevention. obviously, prevention of burn wounds is preferable to a long course of treatment. care should be taken to prevent direct exposure to harsh chemicals. tables, floors, and other surfaces should be rinsed thoroughly after chemical use, prior to allowing any animal contact. electric heating pads should be avoided, and only heated water blankets or circulating warm-air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. etiology. research and/or anesthetic protocols may require the intravenous injection of various solutions. when these substances have a ph or osmolarity significantly different from that of the surrounding tissues, the accidental perivascular extravasation of the solutions may result in tissue damage. several drugs have been shown to cause problems when injected perivascularly, including pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin (swaim and angarano, 1990; waldron and trevor, 1993) . clinical signs. the immediate signs of perivascular injection are swelling at the injection site and withdrawal of the limb or other signs of discomfort. later, the area may appear red, swollen, and painful as inflammation progresses. often there will be eventual necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a 1 to 4 month period. this is because the drug is released over time from the dying cells (swaim and angarano, 1990) . prevention. because the degree of injury and extensive treatment associated with perivascular extravasation of a drug can be detrimental to research protocols and can cause severe discomfort to the dog, prevention of these injuries is preferred. prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. if a potentially caustic compound is to be used in a fractious subject, sedation of the dog is warranted if this will not interfere with the research protocol. whenever possible, insertion of an indwelling catheter is extremely important. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. prior to use, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. treatment. the treatment of perivascular injections will depend on the amount and type of substance injected. in most cases, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions (swaim and angarano, 1990 ). the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, 1989) , and local infiltration of hyaluronidase accompanied by warm compresses has been suggested for use in cases of vinblastine injection (waldron and trevor, 1993) . despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, 1990) . in all cases, the condition can be painful, and analgesia should be addressed. etiology. hepatic encephalopathy is the result of the derangements in metabolism associated with abnormal liver function. this condition may be seen in young dogs with congenital portosystemic shunting of blood flow. however, in the research setting, encephalopathy occurs more often in canine models of hepatic disease that lead to liver failure. a well-developed knowledge of the pathophysiology of liver disease is necessary for the initial treatment and long-term management of hepatic encephalopathy. pathogenesis. when the liver function is severely impaired because of either portosystemic shunting of blood flow or loss of metabolically active hepatic tissue, the result is an accumulation of ammonia, toxic amines, aromatic amino acids, and short-chain fatty acids (hardy, 1989; center, 1998) . these compounds have several toxic effects that result in a decrease in cerebral energy metabolism and a decrease in excitatory neurotransmitter synthesis. concurrently, there is an increase in the concentration of false neurotransmitters and the inhibitory substance 7-aminobutyric acid (gaba). clinical signs. the signs of hepatic encephalopathy include lethargy, depression, muscle tremors, and convulsions. diagnosis and differential diagnosis. a presumptive diagnosis of hepatic encephalopathy may be based on the appearance of clinical signs following experimental manipulation of the liver. additional diagnostic tests to verify the loss of liver function can be performed to confirm the diagnosis. serum glucose and protein levels may be low if hepatic function is severely impaired. a low serum urea nitrogen level suggests that the normal hepatic metabolism of ammonia into urea has been impaired. elevated levels of serum bile acids and blood ammonia also verify the loss of liver function (hardy, 1989) . measurement of serum hepatic leakage enzymes are nondiagnostic, because they can be low, high, or normal. treatment. because of the severity of hepatic encephalopathy, treatment may be initiated based on a presumptive diagnosis. during initial treatment, supportive care with fluids and electrolytes should be instituted, based on the results of serum chemistry and blood gas analysis. the majority of animals with hepatic dysfunction will be hypokalemic, alkalotic, and hypernatremic; therefore, either 0.45% sodium chloride or 0.45% sodium chloride with 2.5% dextrose, supplemented with potassium chloride, is recommended (hardy, 1989) . the type of drug to be used for seizure control is controversial. the short halflife of diazepam makes it an attractive choice compared with barbiturates, which have prolonged metabolism when hepatic function is impaired (maddison, 1995) . however, endogenous benzodiazepines mediate some of the cns signs seen with hepatic encephalopathy. therefore, the use of diazepam has been discouraged in favor of phenobarbital (johnson, 2000) . the drug selected for seizure control should be titrated carefully, given the altered liver metabolism. most importantly, the treatment of dogs with hepatic encephalopathy must be aimed at reducing the levels of toxic metabolites in the bloodstream. because protein metabolism is a major source of ammonia, all oral food intake should cease until the signs of hepatic encephalopathy have abated. because gastrointestinal bleeding may occur in individuals with liver failure and this is also a source of protein, the use of h2 blockers such as cimetidine or ranitidine is suggested (swalec, 1993) . in addition, lactulose retention enemas should be performed (10-15 ml/lb of a 30% solution in water, retained for 20-30 min) (hardy, 1989) . lactulose is an indigestible semisynthetic sugar that is metabolized in the gut to lactic and other acids. the decrease in colonic ph reduces ammonia levels in the bloodstream by converting intestinal ammonia into less diffusible ammonium ions. lactulose will also cause an osmotic diarrhea. antibiotics such as neomycin (10 mg/lb, 3-4 times/ day) or metronidazole (9 mg/lb, 3 times/day) should also be used to reduce the intestinal load of urease-producing bacteria responsible for splitting urea into ammonia (hardy, 1989) . when the signs of hepatic encephalopathy have resolved, the dog may be fed a low-protein diet. diets suitable for dogs with renal insufficiency are recommended initially. this type of diet is not suitable for long-term use, however, because it appears that individuals with some types of hepatic disease actually have increased protein requirements. these requirements may be met by slowly increasing protein in the diet as long as signs of hepatic encephalopathy do not recur. to maintain the appropriate balance of aromatic and branched-chain amino acids, the diet should be based on vegetable and dairy protein instead of meat or fish protein (center, 1998) . in addition, the antibiotics suggested above should be continued to reduce the effects of increasing dietary protein levels. the prevalence of cancer in the general canine population has increased over the years (dorn, 1976) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, 1982) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. (1994) found death rates similar to the death rate of the at-large dog population (bronson, 1982) . approximately 22% of the male beagles died of cancer. the majority of the tumors were lymphomas (32%) and sarcomas (29%), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer (26% of the population studied), three-quarters had either mammary cancer (40%), lymphomas (18%), or sarcomas (15%). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, 1975; benjamin et al., 1996) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients but typically require local anesthesia. an instrument such as a tru-cut needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a 1 mm x 1 to 1.5 cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least 1 cm around the tumor, and 3 cm if mast cell tumors are suspected (morrison et al., 1993) . incisional biopsies are performed when large soft-tissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology. lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide 2,4-dichlorophenoxyacetic acid (2,4-d) as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, 1991) . clinical signs. multicentric and alimentary lymphomas account for most cases of canine lymphoma. in multicentric lymphoma, animals usually present with enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. alimentary lymphoma is associated with vomiting and diarrhea, in addition to previous clinical signs. less commonly, dogs develop mediastinal, cutaneous, and extranodal lymphomas. dogs with mediastinal lymphoma often present with respiratory signs secondary to pleural effusion. hypercalcemia is most frequently associated with this form of lymphoma and may result in weakness. cutaneous lymphoma varies in presentation from solitary to generalized and may mimic any of a number of other skin disorders. the tumors may occur as nodules, plaques, ulcers, or dermatitis. approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement. epizootiology. the incidence of lymphoma is highest in dogs 5-11 years old, accounting for 80% of cases. although the neoplasm generally affects dogs older than 1 year, cases in puppies as young as 4 months have been reported (dorn et al., 1967) . pathologic findings. enlarged neoplastic lymph nodes vary in diameter from 1 to 9 cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. pathogenesis. all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is 4-6 weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, 1991) . hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. diagnosis and differential diagnosis. differential diagnoses for multicentric lymphoma include systemic mycosis; salmonpoisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections are frequently needed. treatment. therapy for lymphoma typically consists of one or a combination of several chemotherapeutic agents. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. macewen and young (1991) provide a thorough discussion of therapeutic options for the treatment of lymphomas in the dog. research complications. given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with signifcant clinical illness. etiology. the fibrosarcoma group of tumors encompasses not only malignant tumors of fibroblasts but also a number of indistinguishable tumors, all of which are capable of collagen production (pulley and stannard, 1990) . frequently classified in this group are undifferentiated leiomyosarcomas, liposarcomas, malignant melanomas, and malignant schwannomas. clinical signs. although these neoplasms can arise throughout the body, they are most commonly found in the skin, subcutaneous tissues, and oral cavity. fibrosarcomas are extremely variable in size and can grow to be quite large. in general, they are irregular and nodular, poorly demarcated, and nonencapsulated, and they frequently invade deeper tissues. epizootiology. most fibrosarcomas develop in adult and aged animals but can affect dogs as young as 6 months or less. pathogenesis. fibrosarcomas exhibit rapid, invasive growth, recurring frequently after excision. metastasis occurs in only one-fourth of cases, usually by the bloodstream to the lungs. less frequently, spread to local lymph nodes is observed. diagnosis and differential diagnosis. differential diagnoses for fibrosarcomas vary with the location of the tumor. histopathologic exam should be used to distinguish these tumors from round cell tumors (mast cell tumors, histiocytomas, transmissible venereal tumors), papillomas, and other neoplasms. treatment. treatment of any soft-tissue sarcoma would begin with wide surgical excision. if the tissue margins indicate incomplete resection, radiotherapy could be used. for any highgrade tumors, adjuvant chemotherapy would be recommended (see macewen and withrow, 1991a , for a complete discussion). research complications. because fibrosarcomas are locally invasive and often recur, dogs with these neoplasms should not be considered good subjects for long-term studies. etiology. neoplasms of lipocytes and lipoblasts are welldifferentiated tumors referred to as lipomas. clinical signs. these growths can be found as single or multiple round, ovoid, or discoid masses in the subcutaneous tissues of the lateral and ventral thorax, abdomen, and upper limbs. generally they are well circumscribed, encapsulated, and soft on palpation. further, the skin is freely movable over the tumor. epizootiology. lipomas occur principally in aged animals (average 8 years), and the incidence increases with age (pulley and stannard, 1990) . the tumors are most commonly seen in overweight female dogs, but no breed predisposition is observed. pathologic findings. histologically, lipomas are indistinguishable from normal adipose tissue except when a fibrous capsule is present. pathogenesis: lipomas are typically slow-growing and do not recur after complete surgical excision. diagnosis and differential diagnosis. lipomas are not frequently confused with other tumors but can sometimes be difficult to distinguish from normal adipose tissue. generally, the distinction can be made from the clinical history. treatment. treatment for lipomas is not usually necessary unless the mass is causing problems with normal ambulation. in such cases, surgical excision is usually curative. research complications. lipomas usually do not complicate research studies unless they are interfering with other systemic functions or ambulation. etiology. histiocytomas are benign skin growths that arise from the monocyte-macrophage cells in the skin. some debate exists as to whether this growth is actually a neoplasm or a focal inflammatory lesion (pulley and stannard, 1990) . clinical signs. the most frequent sites for histiocytomas are the head (especially the pinna) and the skin of the distal forelegs and feet. the masses are usually domelike or buttonlike (often referred to as "button tumors") and usually measure 1-2 cm in diameter. epizootiology. histiocytomas are the most common tumors of young dogs, mostly occurring in dogs less than 2 years of age. pathologic findings. histologically, these tumors contain round to ovoid cells with pale cytoplasm and large nuclei. the cells infiltrate the dermis and subcutis, displacing collagen fibers and skin adnexa. despite being benign lesions, histiocytomas characteristically have a high mitotic index. pathogenesis. this tumor typically exhibits rapid growth (1-4 weeks) but does not spread. most histiocytomas will spontaneously regress in less than 3 months. diagnosis and differential diagnosis. histiocytomas must be distinguished from potentially metastatic mast cell tumors. this is accomplished by staining with toluidine blue, which would stain the cytoplasmic granules of mast cells red or purple. treatment. although most histiocytomas will spontaneously resolve, conservative surgery or cryosurgery will provide an expeditious resolution. research complications. histiocytomas should not interfere with most studies. etiology. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog (bostock, 1986) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin. clinical signs. well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, 1 to 10 cm nodules in the skin. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema. mast cell tumors can be found on any portion of the dog's skin but frequently affect the hindquarters, especially the thigh and in-guinal and scrotal areas. mast cell tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. epizootiology. these tumors tend to affect middle-aged dogs but have been observed in dogs ranging from 4 months to 18 years (pulley and stannard, 1990 ). pathologic findings. because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. (1986) has become widely accepted. in this system, grade i has the best prognosis, and grade iii the worst prognosis. grade i tumors are well differentiated, with round to ovoid uniform cells. the nuclei are regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have large, irregular nuclei with multiple prominent nucleoli. the cytoplasmic granules are few, but mitotic figures are much more frequent. in addition to skin lesions, mast cell tumors have been associated with gastric ulcers. these lesions are most likely secondary to tumor production of histamine. histamine stimulates the h2 receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (>75%) of dogs with mast cell tumors (howard et al., 1969) . the ulcers can be found in the fundus, pylorus, and/or proximal duodenum. although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis. mast cell tumors can be distinguished histologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue, which metachromatically stains the cytoplasmic granules of the mast cells red or purple. treatment. initial treatment for mast cell tumors is generally wide surgical excision (1 to 3 cm margins). even with wide surgical margins, approximately 50% of mast cell tumors may recur. if the site is not amenable to wide surgical excision, debulking surgery and radiation therapy may be used. other alternatives include amputation (if on a limb) or radiation therapy alone. as an adjunct to surgery, grier et al. (1990 grier et al. ( , 1995 found that deionized water injected into surgical margins reduced tumor recurrence by hypo-osmotically lysing any mast cells left behind. this technique has recently been refuted by jaffe et al. (2000) . for systemic mastocytosis, and nonresectable or incompletely excised mast cell tumors, chemotherapy can be used. treatment options would include oral prednisolone, intralesional triamcinalone, and the combination of cyclophosphamide, vincristine, and prednisolone (graham and o'keefe, 1994) . research complications. because of the possibility of systemic histamine release and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed on a regular basis (monthly). grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every 3-6 months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology. hemangiosarcomas are malignant tumors that originate from endothelial cells. clinical signs. these tumors may arise in the subcutis but are more commonly found in the spleen and the right atrium. clinical signs are associated with the site of involvement. vascular collapse is frequently observed secondary to rupture and hemorrhage from splenic masses. heart failure can be observed secondary to tumor burden or hemopericardium. when found in the skin, hemangiosarcomas are poorly circumscribed, reddish black masses that range in size from 1 to 10 cm in diameter. the most common cutaneous sites are the ventral abdomen, the prepuce, and the scrotum. epizootiology: hemangiosarcomas occur most frequently in 8-to 13-year-old dogs. the german shepherd dog is most commonly affected. pathologic findings. grossly, splenic hemangiosarcomas resemble nodular hyperplasia or hematomas (fig. 7) . the masses are spherical and reddish black and can range in size up to 15-20 cm in diameter. on cut section the masses may appear reddish gray or black and have cavernous areas of clotted blood. when the masses are found in the heart, the endocardium may be covered by a thrombus, giving the 2 to 5 cm tumors a reddish gray or yellow appearance. histologically, hemangiosarcomas are composed of immature endothelial cells that form vascular channels or clefts. these spaces may be filled with blood or thrombi. the neoplastic cells are elongated with round to ovoid, hyperchromatic nuclei and frequent mitotic figures. pathogenesis. hemangiosarcomas can be found in one or many sites. in cases where multiple sites are involved, it may be impossible to identify the primary tumor. this neoplasia is highly malignant and spreads easily. metastasis occurs most frequently to the lungs but can be found in any tissue. diagnosis and differential diagnosis. splenic hemangiosarcoma may resemble nodular hyperplasia or some manifestations of lymphoma. when the heart is affected, other causes of heart failure must be ruled out. echocardiography is a valuable tool for identifying the primary lesion. histopathology should be used to differentiate dermal hemangiosarcoma from hemangiomas and other well-vascularized tumors. treatment. surgery is generally the first choice of treatment for hemangiosarcoma. dermal tumors are treated with radical resection, splenic tumors by total splenectomy, and heart tumors by debulking and pericardiectomy. because of the high likelihood of metastasis, adjunct chemotherapy should always be considered. research complications. dogs with dermal hemangiosarcoma may be cured after complete resection with margins, but monitoring should be done regularly for recurrence. the other forms of hemangiosarcoma have a much poorer long-term prognosis, and treatment is typically unwarranted in the research setting. etiology. also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the transmissible venereal tumor is transmitted to the genitals by coitus (nielsen and kennedy, 1990) . the origin of this tumor is still unknown but has been described as a tumor of lymphocytes, histiocytes, and reticuloendothelial cells. although this tumor has been reported in most parts of the world, it is most prevalent in temperate climates (macewen, 1991) . clinical signs. the tumors are usually cauliflower-like masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to 10 cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis (fig. 8) . less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission. transmissible venereal tumors are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, 1990) . extragenital lesions are believed to be a result of trauma prior to exposure to the tumor. pathogenesis. tumor growth is rapid after implantation but later slows. metastasis is rare (<5% of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. diagnosis and differential diagnosis. transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. cytology may be of benefit in making a definitive diagnosis, so impression smears should be made prior to processing for histopathology. prevention. thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control. removing affected individuals from a breeding program should stop further spread through the colony. treatment. surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine (0.5-0.7 mg/m 2) iv once weekly for 4 -6 treatments will induce remission and cure in greater than 90% of the cases (macewen, 1991). research complications. experimental implantation of transmissible venereal tumors has been shown to elicit formation of tumor-specific igg (cohen, 1972) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology. dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs. single nodules are found in approximately 75% of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger and invasive and coalesce with adjacent tissues. epizootiology. mammary tumors are uncommon in dogs under 5 years of age with the incidence rising sharply after that. median age at diagnosis is 10-11 years. mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, 1977) . extensive discussions of classification, staging, and histopathologic correlations can be found in macewen and withrow (199 lb) and moulton (1990) . pathogenesis. mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in 60-70% of tumors. futher, schneider et al. (1969) showed that the risk of developing mammary tumors increased greatly after the first and second estrus cycles. dogs spayed prior to the first estrus had a risk of 0.8%, whereas dogs spayed after the first and second estrus had risks of 8% and 26%, respectively. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs. diagnosis and differential diagnosis. both benign and malignant mammary tumors must be distinguished from mammary hyperplasia and mastitis. prevention. mammary tumors can effectively be prevented by spaying bitches prior to the first estrus. this is commonly done in the general pet population at 6 months of age. recently, the topic of spaying sexually immature dogs (8-16 weeks of age) has received much attention for the control of the pet population. kustritz (1999) reviewed the techniques for anesthesia and surgery, as well as possible pros and cons of spaying at this young age. treatment. surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. lumpectomy or nodulectomy should be elected in the case of small discrete masses, while mammectomy and regional or total mastectomies are reserved for more aggressive tumors. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. inguinal lymph nodes should be removed any time the fourth and fifth glands are excised (macewen and withrow, 199 lb). research complications. because 50% of mammary tumors are benign, treatment may be rewarding, allowing dogs to con-tinue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. etiology. beagles are among the breeds with the highest prevalence of thyroid carcinomas. benjamin et al. (1996) reported a correlation between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in the beagle. clinical signs. thyroid carcinomas generally present as palpable cervical masses. affected animals may experience dysphagia, dyspnea, and vocalization changes. precaval syndrome resulting in facial edema is also observed in some cases. epizootiology and transmission. the mean age of dogs presented with thyroid carcinomas is 9 years, with equal distribution of cases between the sexes. pathologic findings. grossly, thyroid carcinomas are multinodular masses, frequently with large areas of hemorrhage and necrosis. they tend to be poorly encapsulated and invade local structures such as the trachea, esophagus, larynx, nerves, and vessels. the masses are unilateral twice as often as bilateral (capen, 1990) . histologically, thyroid carcinomasare divided into follicular, papillary, and compact cellular (solid) types (see capen, 1990 , for complete discussion). pathogenesis. thyroid carcinomas tend to grow rapidly and invade local structures. early metastasis is common and occurs to the lungs by invasion of branches of the thyroid vein. diagnosis and differential diagnosis. nonpainful cervical swellings such as seen with thyroid tumors are also consistent with abscesses, granulomas, salivary mucoceles, and lymphomas. often a preliminary diagnosis can be made by fineneedle aspirate. treatment. surgery is the treatment of choice for thyroid carcinomas that have not metastasized. when the tumor is freely movable, surgery may be curative. surgical excision may be difficult for tumors that adhere to local structures, requiring excision of the jugular vein, carotid artery, and associated nerves. when bilateral tumors are observed, preservation of the parathyroid glands may not be possible. in such cases, treatment for hypoparathyroidism will be necessary. both chemotherapy and radiation therapy have been suggested for extensive bilateral tumors and/or after incomplete excision, but no controlled trials have been performed (ogilvie, 1991) . in the research setting, treatment of this tumor may not be rewarding. only freely movable tumors can be practically treated without seriously affecting research efforts. euthanasia is warranted in the more advanced cases when clinical illness is apparent. beagles are subject to many of the inherited and/or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, 2000b) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm1 gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). in addition, there are defects that affect so many breeds that the author simply lists "many breeds" for the breeds affected by those disorders. thus these defects could also affect beagles and include pectus excavatum, polydactyly, radial and ulnar dysplasia, hypoadrenocorticism, entropion, lens coloboma, factor viii deficiency (von willebrand's disease), renal agenesis or ectopia, and developmental defects of the reproductive and lower urinary tracts. at a commercial breeder of purpose-bred beagles, the most common birth defects were umbilical hernia (1.82% of births) and open fontanelle (1.44% of births) (r. scipioni and j. ball, personal communication, 1999) . other defects observed include cleft palate and cleft lip, cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than 1.0% incidence. etiology. cataract is an opacification of the lens or the lens capsule. it is the pathologic response of the lens to illness or injury, because the lens has no blood supply. cataracts can be caused by metabolic, inflammatory, infectious, or toxic causes and can be congenital, juvenile, or degenerative. nuclear sclerosis is an apparent opacification of the lens caused by the compression of older lens fibers in the center of the lens (nucleus) as a consequence of the production of new fibers. because the nucleus increases in size as the animal ages, the sclerosis is more apparent in older animals and may be mistaken as a senile cataract. the ability to see the fundus during ophthalmoscopy persists with nuclear sclerosis but is obstructed by a true cataract. clinical signs. the first clinical sign is typically the ability to visualize the opaque lens through the pupil of the dog's eye. dogs have an impressive ability to tolerate bilateral lens opacity (especially when development is gradual), and often visual impairment is detected late in the development of the condition (helper, 1989) . moderate vision loss may cause the dog to be hesitant in moving in new surroundings or unable to locate movable objects (such as a toy). rapid cataract development can result in a sudden vision loss, such as can occur with diabetic cataracts. epizootiology and transmission. certain dog breeds can be predisposed to the development of juvenile or senile cataracts or to metabolic disorders that result in cataract development, such as diabetes mellitus. dogs in studies for diabetes mellitus should be observed regularly for cataract development. toxicological studies may also induce formation of cataracts. pathogenesis. lens fibers respond to all biological or chemical insults by necrosis and liquefaction (render and carlton, 1995) , because they have no blood supply with which to recruit an inflammatory and repair process. disruption of these fibers by any means, therefore, leads to opacification. the exact processes by which the varieties of congenital and juvenile cataracts are produced have not been determined. in diabetic cataracts, the excess glucose is metabolized to sorbitol and fructose. as these alcohols and sugars accumulate in the lenticular cells, they produce an osmotic imbalance, which brings fluid into the cells, causing swelling and degeneration of lens fibers and resultant opacity (capen, 1995) . diagnosis and differential diagnosis. the ability to visualize the retina and fundus during ophthalmoscopy differentiates true cataracts from nuclear sclerosis. dogs with cataracts should be evaluated for possible causes, especially diabetes mellitus. diabetes mellitus will typically affect middle-aged dogs and feature rapid cataract formation, whereas juvenile and senile cataracts are slow to develop and affect younger and older dogs, respectively. progressive retinal atrophy can also cause secondary cataract formation; pupillary light response is maintained with primary cataracts (even if the lens is completely opaque), whereas this reflex is obtunded by retinopathy. prevention. most forms of cataracts cannot be prevented, for their exact etiologic pathogenesis is unknown. diabetic cataracts, however, can be prevented by proper regulation of blood glucose concentrations with insulin therapy and proper diet. treatment. because dogs do not need to focus visual images as accurately as human beings, proper lens clarity and function are not necessary for an adequate quality of life. many dogs adjust quite well to the visual impairment caused by persistent cataracts. lens removal can be performed for dogs seriously affected by cataracts, but this would not be anticipated for dogs in the research setting. information on surgical lens extraction procedures can be found in helper (1989) or other veterinary ophthalmology textbooks. research complications. research complications would be minimal with cataracts, unless the dogs were intended for use in ophthalmologic or visual acuity-based studies. etiology. hip dysplasia is a degenerative disease of the coxofemoral joint. a specific etiology is unknown, but the development of hip dysplasia has a strong genetic component (pedersen et al., 2000) , modified by age, weight, size, gender, conformation, rate of growth, muscle mass, and nutrition (smith et al., 1995) . clinical signs. the initial clinical abnormality caused by hip dysplasia is laxity of the coxofemoral joint. this may present as a gait abnormality without any indication of lameness or stiffness. eventually, affected dogs will have periods of lameness and, in protracted cases, will be rendered immobile by severe pain. epizootiology and transmission. hip dysplasia has been seen in most dog breeds, but it typically affects larger breeds of dogs. in the research setting, it is primarily a condition of randomsource large-breed dogs used for surgical research. diagnosis and differential diagnosis. hip dysplasia is classically diagnosed by radiography of the pelvis and hip joints. radiographic abnormalities consistent with hip dysplasia include shallow acetabula with remodeling of the acetabular rim, flattening of the femoral head, subchondral bone sclerosis (caused by erosion of articular cartilage and exposure of underlying bone), and osteophyte production around the joint (pedersen et al., 2000) . hip dysplasia needs to be differentiated from other musculoskeletal or neurological conditions that can cause unusual gaits and/or lameness. this may be somewhat difficult, because clinical signs of hip dysplasia may develop before radiographic abnormalities. radiographic calculation of the distraction index (di) to measure joint laxity has proven to be a good means to predict future hip dysplasia before other radiographic changes are evident (smith et al., 1995) . prevention. because of the genetic component, dogs with hip dysplasia should not be used in breeding colonies. dogs should be provided a good plane of nutrition but not be allowed to become overweight. dogs that were limit-fed at 75% of the food amount eaten by ad libitum-fed dogs had lower body weights and decreased severity of radiographic lesions of hip dysplasia (kealy et al, 1997) . treatment. in the stages when clinical signs are episodic, cage rest and analgesics for several days can be used to treat the symptoms. more advanced cases may require continuous analgesia. sectioning of the pectineus muscle or tendon may provide some pain relief but does not affect the progression of the disease (pedersen et al., 2000) . surgical treatments for hip dysplasia include femoral head ostectomy and total hip replacement. neither surgical treatment is likely in a research setting. research complications. long-term studies using large-breed dogs may be affected by the eventual development of hip dysplasia. in studies where hip dysplasia would be a serious complication or confounding variable (e.g., orthopedic research), dogs should be radiographed upon arrival to assess possibility of early coxofemoral joint degeneration and suitability for use in the study. etiology. benign prostatic hyperplasia (bph) is an agerelated condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs. bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum as it passes through the pelvic canal. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs for bph. epizootiology and transmission. bph typically affects older dogs (>4 years), although it has been seen as early as 2 years of age. pathologic findings. in its early stages, canine b ph is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis. bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to 5a-dihydrotestosterone (kustritz and klausner, 2000) . diagnosis and differential diagnosis. bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis characterized by hemorrhage, at most. differential diagnoses include squamous metaplasia of the prostate, paraprostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve when castration is used for treatment of prostatic enlargement. prevention. castration is the primary means for prevention of benign prostatic hyperplasia. treatment. the first and foremost treatment for b ph is castration. in pure cases of b ph, castration results in involution of the prostate gland detectable by rectal palpation within 7-10 days. for most dogs in research studies this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases in which the dog is a valuable breeding male (e.g., genetic diseases), and semen collection is necessary. if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. newer drugs marketed for human males have also shown promise in treating canine bph. finasteride (proscar) is a 5a-reductase inhibitor that limits metabolism of testosterone to 5a-dihydrotestosterone. treatment at daily doses of 1-5 mg/kg has been effective in causing prostatic atrophy without affecting testicular spermatogenesis (kustritz and klausner, 2000) . dogs given 1.0 mg/kg were proven to still be fertile. there are also indications that lower doses may be effective in relieving b ph. androgen receptor antagonists (flutamide and hydroxyflutamide) have also been studied in the dog and found to be effective for treatment of bph while maintaining libido and fertility (kustritz and klausner, 2000) . unfortunately, both the 5areductase inhibitors and the androgen receptor antagonists are not presently labeled for use in male dogs in the united states. research complications. bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. it is presently unknown whether the use of the newer antihyperplastic agents systemically alters physiologic parameters outside of the prostate itself. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. etiology. juvenile polyarteritis syndrome (jps) is a painful disorder seen in young beagles (occasionally reported in other breeds). the lesion consistent with the syndrome is systemic necrotizing vasculitis. the cause of the vasculitis has not been established, but it appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs. clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to extend the neck ventrally. most dogs seem to be in pain when touched, especially in the neck region. the syndrome typically has a course of remissions and relapses characterized by 3-7 days of illness and 2-4 weeks of remission (scott-moncrieff et al., 1992) . there may be a component of this condition that is subclinical, given that a vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission. jps typically affects young beagles (6-40 months), with no sex predilection. pathologic findings. on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., 1995) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to medium-sized arteries are seen. these lesions are most noticeable in the three locations where gross lesions are observed, but they may be seen in other visceral locations. the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries (fig. 9a) . the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., 1995) . fibrinous thrombosis of the affected arteries is also seen (fig. 9b) . a subclinical vasculitis has also been diagnosed in beagles postmortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis. the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclical nature and respond to treatment with corticosteroids, and the affected dogs have elevated a2-globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis of some affected dogs has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resuited in 1/7 affected pups (scott-moncrieff et al., 1992) . diagnosis and differential diagnosis. differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). are known at this time. no prevention and control measures brane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs. the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit (fig. 10) . excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. treatment. clinical signs can be abated by administration of corticosteroids. prednisone administered orally at 1.1 mg/kg, q12 hr, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of 0.25-0.5 mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. pathologic findings. typically the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis. prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, 1989) . research complications. because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology. "cherry eye" is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating mem-prevention. hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment. corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, 1989 ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications. in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland, or its removal, might compromise ophthalmologic studies. etiology. interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes (fig. 11 ). the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym "sterile pyogranuloma complex"). clinical signs. dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission. interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, 1993) . pathologic findings. histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis. initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis. bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection, and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment. if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at 1 mg/kg ql2h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require fig. 11 . interdigital cyst between the third and fourth digits of the forelimb of a research beagle. surgical removal. excision includes removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, 1993) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weightbearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, 1993) . research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. post-therapy antibody titers in dogs with ehrlichiosis: follow-up study on 68 patients treated primarily with tetracycline and/or doxycycline dog thyroiditis: occurrence and similarity to hashimoto's struma surgical management of specific skin disorders bordetella and mycoplasma infections in dogs and cats associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles diagnostic exercise: peracute death in a research dog saunders manual of small animal practice neurologic manifestations associated with hypothyroidism in four dogs neoplasms of the skin and subcutaneous tissues in dogs and cats neoplasia of the skin and mammary glands of dogs and cats platelet function, antithrombin-iii activity, and fibrinogen concentration in heartworm-infected and heartworm-negative dogs treated with thiacetarsamide pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis ehrlichia platys infection in dogs the rickettsioses monoclonal gammopathy associated with naturally occurring canine ehrlichiosis variation in age at death of dogs of different sexes and breeds leptospira interrogans serovar grippotyphosa infection in dogs efficacy and dose titration study of mibolerone for treatment of pseudopregnancy in the bitch tropical canine pancytopenia: clinical, hematologic, and serologic response of dogs to ehrlichia canis infection, tetracycline therapy, and challenge inoculation comparison of campylobacter carriage rates in diarrheic and healthy pet animals. zentralbl advances in dietary management of obesity in dogs and cats effect of level and source of dietary fiber on food intake in the dog effect of amount and type of dietary fiber on food intake in energy-restricted dogs external parasites: identification and control tumors of the endocrine glands thomson's special veterinary pathology infectious diseases of the dog and cat nutritional support for dogs and cats with hepatobiliary disease specific amplification of ehrlichia platys dna from blood specimens by two step pcr detection of humoral antibody to the transmissible venereal tumor of the dog dirofilaria immitis: heartworm products contract rat trachea in vitro dogs: laboratory animal management management of septicemia in rhesus monkeys with chronic indwelling catheters client information series: canine demodicosis client information series: fleas and flea allergy dermatitis management of the burn wound chlorine dioxide sterilization of implanted right atrial catheters in rabbits current concepts in the management of helicobacter associated gastritis dirofilariasis in dogs and cats use of narcotic antagonists to modify stereotypic self-licking, self-chewing, and scratching behavior in dogs epidemiology of canine and feline tumors epizootiologic characteristics of canine and feline leukemia and lymphoma study of obesity in dogs visiting veterinary practices in the united kingdom miller's anatomy of the dog update on diagnosis of canine hypothyroidism helicobacter-associated gastric disease in ferrets, dogs, and cats the role of helicobacter species in newly recognized gastrointestinal tract disease of animals serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test hemorrhagic streptococcal pneumonia in newly procured research dogs control of ticks platelet aggregation studies in dogs with acute ehrlichia platys infection health benefits of animal research: the dog as a research subject soft tissue sarcomas and mast cell tumors textbook of veterinary internal medicine infectious diseases of the dog and cat canine lyme borreliosis mast cell tumor destruction by deionized water mast cell tumour destruction in dogs by hypotonic solution transmission of ehrlichia canis to dogs by ticks (rhipicephalus sanguineus) textbook of veterinary internal medicine diseases of the liver and their treatment cyclic thrombocytopenia induced by a rickettsia-like agent in dogs shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs textbook of veterinary internal medicine canine thyroid neoplasms: epidemiologic features magrane's canine ophthalmology helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats thiacetarsamide and its adverse effects infectious diseases of the dog and cat pediatrics: puppies and kittens canine viral diseases textbook of veterinary internal medicine antibodies to ehrlichia canis, ehrlichia platys, and spotted fever group rickettsia in louisiana dogs mastocytoma and gastroduodenal ulceration complications in the use of indwelling vascular catheters in laboratory animals deionised water as an adjunct to surgery for the treatment of canine cutaneous mast cell tumours helicobacter infection textbook of veterinary internal medicine textbook of veterinary internal medicine hygroma of the elbow in dogs thermal injuries dirofilaria immitis: do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta? depression of endotheliumdependent relaxation by filarial parasite products three cases of canine leptospirosis in quebec cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs role of bordetella bronchiseptica in infectious tracheobronchitis in dogs kirk's current veterinary therapy 12: small animal practice the fire of life kirk's current veterinary therapy 13: small animal practice coinfection with multiple tick-borne pathogens in a walker hound kennel in north carolina tarsal joint contracture in dogs with golden retriever muscular dystrophy clinical and hematological findings in canine ehrlichiosis early spay-neuter in the dog and cat textbook of veterinary internal medicine chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs the brown dog tick rhipicephalus sanguineus and the dog as experimental hosts of ehrlicha canis the clinical chemistry of laboratory animals double-blinded crossover study with marine-oil supplementation containing high-dose eicosapentaenoic acid for the treatment of canine pruritic skin disease thomson's special veterinary pathology effects of four preparations of .05% chlorhexidine diacetate on wound healing in dogs transmissible venereal tumors kirk's current veterinary therapy 11: small animal practice soft tissue sarcomas tumors of the mammary gland canine lymphoma and lymphoid leukemias kirk's current veterinary therapy 12: small animal practice effect of heartworm infection on in vitro contractile responses of canine pulmonary artery and vein tick paralysis in north america and australia saunders manual of small animal practice thyroid gland and arterial lesions of beagles with familial hypothyroidism and hypedipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think urea protects helicobacter (campylobacter) pylori from the bactericidal effect of acid characterization of a new isolate of ehrlichia platys using electron microscopy and polymerase chain reaction decreased pulmonary arterial endothelium-dependent relaxation in heartworm-infected dogs with pulmonary hypertension vaccination against canine bordetellosis: protection from contact challenge dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs clinical trial of dvm derm caps in the treatment of allergic diseases in dogs: a nonblinded study diagnosis of neoplasia tumors of the mammary gland dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior survey of conjunctival flora in dogs with clinical signs of external eye disease hyperoxia exacerbates microvascular injury following acid aspiration nutrient requirements of dogs surgical closure of elbow hygroma in the dog tumors of the genital system practical laboratory methods for the diagnosis of dermatologic diseases walker's mammals of the world tumors of the endocrine system beta hemolytic streptococcus isolated from the canine vagina comparison of three skin preparation techniques in the dog comparison of three skin preparation techniques in the dog. part 2: clinical trial in 100 dogs clinical behavioral medicine for small animals hypothyroidism in dogs: 66 cases (1987-1992) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in 83 dogs joint diseases of dogs and cats target imbalance: disparity of borrelia burgdorferi genetic material in synovial fluid from lyme arthritis patients textbook of veterinary internal medicine tumors of the skin and soft tissue thomson's special veterinary pathology canine leptospirosis: a retrospective study of 17 cases dogs and cats as laboratory animals effects of chlorhexidine diacetate and povidoneiodine on wound healing in dogs epidemiology of thyroid diseases of dogs and cats factors influencing canine mammary cancer development and postsurgical survival muller and kirk's small animal dermatology systemic necrotizing vasculitis in nine young beagles thomson's special veterinary pathology textbook of veterinary internal medicine canine infectious tracheobronchitis (kennel cough complex) saunders manual of small animal practice serum protein alterations in canine erhlichiosis bacterial factors and immune pathogenesis in helicobacter pylori evaluation of rhipicephalus sanguineus as a potential biologic vector of ehrlichia platys role of the eastern chipmunk (tamias striatus) in the epizootiology of lyme borreliosis in northwestern illinois evaluation of risk factors for degenerative joint disease associated with hip dysplasia in dogs pathologic features of naturally occurring juvenile polyarteritis in beagle dogs textbook of veterinary internal medicine clinical manifestations, pathogenesis, and effect of antibiotic treatment on lyme borreliosis in dogs streptococcus zooepidemicus as the cause of septicemia in racing greyhounds trauma to the skin and subcutaneous tissues of dogs and cats chronic problem wounds of dog limbs portosystemic shunts textbook of veterinary internal medicine lumbosacral stenosis in dogs experimental respiratory disease in dogs due to bordetella bronchiseptica dirofilaria immitis: depression of endothelium-dependent relaxation of canine femoral artery seen in vivo does not persist in vitro thyroiditis in a group of laboratory dogs: a study of 167 beagles of agriculture, animal and plant health inspection service thomson's special veterinary pathology a retrospective study of 27 cases of naturally occurring canine ehrlichiosis role of canine parainfluenza virus and bortedella bronchiseptica in kennel cough management of superficial skin wounds serum concentrations of thyroxine and 3,5,3'-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs ehrlichia platys in a michigan dog ehrlichial diseases of dogs canine ehrlichiosis. miss albert, r. e., benjamin, s. a., and shukla, r. (1994). life span and cancer mortality in the beagle dog and human. key: cord-023925-qrr7jcwe authors: verhoef, jan; van kessel, kok; snippe, harm title: a8 immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites date: 2011-07-12 journal: principles of immunopharmacology doi: 10.1007/978-3-0346-0136-8_8 sha: doc_id: 23925 cord_uid: qrr7jcwe in the middle of the 19th century, it became clear that micro-organisms could cause disease. effective treatment, however, was not possible at that time; prevention and spread of infectious diseases depended solely on proper hygienic means. at the beginning of the 20th century, passive and active vaccination procedures were developed against a number of these pathogenic micro-organisms to prevent the diseases in question (rabies, diphtheria, tetanus, etc.). thanks to the discovery of antimicrobial chemicals (by paul ehrlich) and antibiotics (by sir alexander fleming), the threat of infectious diseases seemed to be minimised. large scale vaccination programmes against childhood diseases (diphtheria, whooping cough and polio), started in the early 1950s, raised hopes of finally being able to eradicate these diseases from the planet. in the middle of the 19th century, it became clear that micro-organisms could cause disease. effective treatment, however, was not possible at that time; prevention and spread of infectious diseases depended solely on proper hygienic means. at the beginning of the 20th century, passive and active vaccination procedures were developed against a number of these pathogenic micro-organisms to prevent the diseases in question (rabies, diphtheria, tetanus, etc.) . thanks to the discovery of antimicrobial chemicals (by paul ehrlich) and antibiotics (by sir alexander fleming), the threat of infectious diseases seemed to be minimised. large scale vaccination programmes against childhood diseases (diphtheria, whooping cough and polio), started in the early 1950s, raised hopes of finally being able to eradicate these diseases from the planet. this approach was successful for smallpox (1980) . however, new infectious diseases have emerged [e.g., legionella, human immunodeficiency virus (hiv), helicobacter, sars, etc.] and new vaccines and antibiotics are needed. furthermore, due to intensive medical treatment with antibiotics and immunosuppressive drugs, hospital infections are a growing problem. bacteria hitherto deemed harmless are causing opportunistic infections in immunocompromised patients. the pathogens have developed resistance to many antibiotics and sometimes no effective antibiotics are available to treat these patients. to make the story even more serious, man is surrounded and populated by a large number of different non-pathogenic micro-organisms. in the normal, healthy situation, there is a balance between the offensive capabilities of micro-organisms and the defences of the human body. the body's defences are based on vital non-specific and specific immunological defence mechanisms. an infection means that the micro-organism has succeeded in penetrating those lines of defence, signalling a partial or complete breakdown of the body's defence system. the body's first line of defence comprises the intact cell layers of skin and mucous membrane, which form a physical barrier. the skin's low ph level and bactericidal fatty acids enhance the protection provided by this physical barrier. the defences in the respiratory tract and the gastrointestinal tract are mucus, the 'ciliary elevator' of the epithelium, and the motility of the small intestine. the presence of normal microbial flora (colonisation resistance) in the intestine also plays a role in protection against colonisation by external bacteria. the most important humoral natural resistance factors are complement, antimicrobial peptides, lysozyme, interferon, and a number of cytokines (see chapters a5 and a6). antimicrobial peptides are widely expressed as part of the professional phagocyte antimicrobial arsenal and rapidly induced at epithelial surfaces. they are found in mammals, invertebrates, and plants. in general, they are small, amphipathic molecules, contain positive charge and can be structurally divided into several categories. the mode of action goes beyond their antimicrobial capacities and they elicit a complex array of responses in different cell types. the most extensively studied mammalian gene families are the cathelicidins and defensins. in general the antimicrobial peptides disrupt lipid membranes and thereby induce microbial killing. micro-organisms have developed several countermeasures against antimicrobial peptides but the many structurally different peptide classes still provide protection against infection. lysozyme, which is found in almost all body fluids, degrades sections of the cell wall of gram-positive and -in combination with complement -gramnegative bacteria. this causes the otherwise sturdy cell wall to leak and the bacterium to burst. interferons are glycoproteins and may inhibit the replication of viruses. within several hours after the onset of a virus infection, interferons are produced in the infected cell and help protect the neighbouring unaffected cells against infection. this protection is brief, but high concentrations of interferons are produced at a time when the primary immunological response is relatively ineffective. cytokines, such as interleukin-2 (il-2), granulocytemacrophage colony-stimulating factor (gm-csf), and tumour necrosis factor-α (tnf-α), stimulate non-specifically the proliferation, maturation, and function of the cells involved in defence (see chapter a6). innate immune cells recognise microbes by tolllike receptors (tlr) (see section pathogenesis of shock), giving rise to the above production of cytokines in the early phase of the response. 128 immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites micro-organisms that succeed in penetrating the first line of defence are ingested, killed, and degraded by phagocytic cells [polymorphonuclear leukocytes (pmn) or neutrophils, monocytes, and macrophages], which are attracted to a microbial infection through chemotaxis. the ingestion by phagocytic cells of the micro-organism is enhanced by serum proteins (opsonins), such as antibodies and the c3b component of complement, which are recognised by specific receptors on the phagocytes. after ingestion, the particle is surrounded by the membrane of the phagocyte, forming a vacuole known as a phagosome. the phagosome then fuses with some of the countless granules in the phagocyte, thus allowing the lysosomal microbicidal agents and enzymes to do their work. the formation of toxic oxygen radicals greatly contributes to the killing and elimination of the ingested micro-organism ( fig. 1 ) (see chapter a7). a special role in cellular natural resistance is reserved for the natural killer cells (nk cells), which display considerable cytotoxic activity against virusinfected cells. this nk activity is stimulated by interferons and, at a very early stage in the infection, serves to reinforce the non-specific defence mechanism. in the specific immune response, elements of the natural defence mechanism are directed against a specific enemy. depending on the micro-organism, either the cellular defence mechanism (tuberculosis) or the humoral antibody-dependent defence mechanism (influenza) is of primary importance. in many cases, a joint cellular and humoral response is needed to provide an effective immune defence (typhus). both t lymphocytes and macrophages play a role in cellular defence. during the first contact with an antigen, macrophages process the antigen and present its protein fragments (t cell epitopes) to t cells, which then proliferate and remain present for years in the body as memory cells. when a second encounter occurs, t cells produce lymphokines, which activate the macrophages. these activated macrophages grow larger, produce more and better degrading enzymes, and are now able to eliminate micro-organisms, which otherwise would have survived intracellularly (tuberculosis, typhoid fever). macrophages from non-immune animals are not able to eliminate these micro-organisms. specific resistance schematic representation of the progressive steps of phagocytic endocytosis. five different classes of antibodies can be distinguished in man, namely, igg, iga, igm, igd, and ige. they differ from one another in size, charge, amino acid composition, and glycosylation (see chapters a3, c2). in principle, the structure of the antibodies is the same, i.e. two heavy and two light chains: it is the variable part of these chains that recognises the micro-organism. the biological function (see below) is determined by the constant part (fc) of the heavy chain. with the exception of igd, all these antibodies are important in antimicrobial activity. • iga, which is found in all external secretions, reacts with the surface of micro-organisms, preventing them from adhering to sensitive cells and mucous membranes. • igg neutralises microbial toxins. • igg, igm, and c3b serve as opsonins, which promote phagocytosis. • igg, igm, and to a lesser extent iga activate the complement system after binding to the micro-organism. activation products c3a and c5a ensure that the phagocytes are attracted to the inflammatory response. • igg and igm, in combination with complement and lysozyme, have a lytic effect on gram-negative bacteria and enveloped viruses. • igg and igm inhibit the mobility of micro-organisms by attaching specifically to the flagellum. thereby the chance of phagocytosis increases and the chance of spreading of disease decreases. • igg, together with the killer or k cells, can eliminate infected host cells which carry viral or other foreign antigens on their surface. • ige is of importance in parasite infections. at the site of the infection, mast cells, bearing specific ige, release large quantities of vasoactive amines, which cause the contraction of smooth muscle tissue and increase the permeability of the blood vessels. in the intestine, this results in worms being detached and eliminated. several non-invasive bacteria, i.e. those that do not invade the body, cause disease through the production of exotoxins (tetanus, diphtheria, cholera). the immune system neutralises the toxin with the aid of antibodies (igg, igm). if the individual has not been inoculated, the toxin will act on certain cells in the body directly through a receptor. this bond is very strong (i.e. has a high affinity), and is difficult to break by the administration of antibodies. in practice, if there are clinical symptoms of the disease, then large doses of antitoxins must be administered. if one is trying to prevent the development of the disease, then the presence of small quantities of specific antibodies (igg) is sufficient. the adherence of bacteria to cells is effectively blocked by iga. oral vaccination against cholera, for example, is aimed at obtaining sufficient specific iga in the intestine, so that no colonisation of this bacterium can take place, and the cholera toxin can no longer adhere to its receptor. in general, defence against invasive bacteria is provided by antibodies (igg, igm) that are directed against bacterial surface antigens. in many cases, these bacteria have a capsule, which interferes with effective phagocytosis. antibodies against the antigens of these capsules neutralise the interference, with subsequent elimination of the bacteria by phagocytes. antibodies (igm, igg, iga) in combined action with complement kill bacteria by producing holes in the cell wall of the bacterium. although intracellular bacteria (tuberculosis, leprosy, listeriosis, brucellosis, legionellosis, and salmonellosis) are ingested by macrophages, they are able to survive and multiply. in these cases, cellular immunity alone provides the defence, since antibodies are not effective. only activated macrophages are capable of killing and degrading these bacteria. antibodies neutralise viruses directly and/or indirectly by destroying infected cells that carry the virus antigen on their surface. the mechanisms of this defence resemble those of humoral defence against bacterial surfaces. the antibody-dependent cellular cytotoxicity reaction is specific for defence against viruses. cells that carry an antigen encoded by the virus on their surface are attacked by cytotoxic k cells, bearing antibodies that fit the antigen on the target cell (k cells have fc receptors for igg). (fig. 2) not only humoral, but also cellular immunity plays an important role in virus infections. people with a genetic t cell deficiency are highly susceptible to virus infections. in cellular defence, it is primarily the virus-infected cells that are attacked and eliminated. cytotoxic t cells recognise mhc class i-presented t cell epitopes on the surface of virusinfected cells and kill them. the fungi responsible for human diseases can be divided into two major groups on the basis of their growth forms or on the type of infection they cause. pathogens exist as branched filamentous forms or as yeasts, although some show both growth forms. the filamentous types (trichophyton) form a 'mycelium'. in asexual reproduction, the fungus is dispersed by means of spores; the spores are a common cause of infection after inhalation. in yeast-like types (cryptococcus), the characteristic form is the single cell, which reproduces by division or budding. dimorphic types (histoplasma) form a mycelium outside, but occur as yeast cells inside the body. candida shows the reverse condition and forms a mycelium within the body. in superficial mycoses, the fungus grows on the body surface, for example skin, hair, and nails (epidermophyton, trichophyton), the disease is mild, and the pathogen is spread by direct contact. in deep mycoses (aspergillus, candida, cryptococcus, histoplasma), internal organs are involved and the disease can be life-threatening and is often the result of opportunistic growth in individuals with impaired immunocompetence. many of the fungi that cause disease are freeliving organisms and are acquired by inhalation or by entry through wounds. some exist as part of the normal body flora (candida) and are innocuous unless the body's defences are compromised in some way. the filamentous forms grow extracellularly, while yeasts can survive and multiply within phagocytic cells. neutrophils kill yeasts by means of both intra-and extracellular factors. some yeasts (cryptococcus neoformans) form a thick polysac-charide capsule to prevent phagocytic uptake. in addition, many cell-wall components of yeasts cause suppression of cell-mediated immune responses. the role of humoral and cellular immunity in controlling infections caused by fungi is not yet well defined, but cellular immunity is the cornerstone of host defence against (some) fungal infections. as a consequence, hiv infection, which affects the cellular arm of the immune system, results in previously uncommon infections such as those caused by c. neoformans. the immunological defence systems against parasites are considerably more complex than those against bacteria and viruses. this is due to various factors. in the first place, each parasite has its own life cycle, consisting of various stages with specific antigen compositions. moreover, parasites are able to avoid the host defence system (mimicry), to combat it (immunosuppression), or to mislead it (antigenic variation). both humoral and cellular immunity are important for the defence against parasites growing intercellularly, as we have seen in the case of bacteria and viruses. antibody concentrations (igm, igg, ige) are often elevated. ige also plays a special role in the removal of parasites (especially worm infections) from the intestine (see above). defence against bacteria, viruses, fungi, and parasites sepsis is a systemic inflammatory response to presumed or known infection. the resulting inflammatory response becomes over amplified, leading to multiple organ failure and death. pathogen-associated molecular patterns (pamp) in bacteria, viruses, parasites, and fungi initiate the host response by triggering families of pattern recognition receptors (prr). in gram-negative (fig. 3) bacterial infections, the interaction between bacterial endotoxin or lipopolysaccharide (lps; a major structural component of the cell wall) and various host-cell systems has been implicated in the pathogenesis of septic shock. in particular, the release of tnf-α and interleukin-1 (il-1) after the activation of host cells by endotoxin induces haemodynamic shock. biochemical and genetic evidence has identified tlr4 as the receptor that mediates cellular activation in response to lps. this family of tlr proteins (fig. 4) , which resemble the antimicrobial toll proteins of drosophila (fruit fly), has been identified in humans and mice. tlr4 was identified as the missing link in lps-induced cell signal transduction and responsiveness that is associated with md-2 and cd14. it is known that c3h/hej mice are hyporesponsive to the biological effects of lps. this proved to be the result of tlr4 deficiency. the tlr family members are coupled to a signalling adapter protein (myd88) and form differential dimers that may explain the discrete responses to tlr ligands such as lipoproteins, heat shock proteins, unmethylated cpg dna, viral dsrna and bacterial flagellin. intracellular signalling involves several kinases depending on 132 immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites genotypical characteristics: chromosomal dna fragment analysis, nucleic acid sequence analysis, probes phenotypical characteristics: morphology, biotyping, serotyping, antibiotic resistance analytical characteristics: cell-wall analysis, lipid and protein analysis, enzyme typing (catalase) gram staining positive or negative aerobic, anaerobic: fermentation of different sugars naming and classification of viruses according to: structure: size, morphology (naked, enveloped), nucleic acid (rna, dna) molecular aspects: mode of replication, assembly and budding disease: encephalitis, hepatitis means of transmission: droplets, water, blood, insects host range: animal, plant, bacteria classification of fungi according to: structure: macroscopic morphology of hyphae (mycelium); microscopic morphology of hyphae, conidophores and conidia (spores); and shape and size cell features: nucleus, cytosol, plasmalemma (cell membrane which contains cholesterol), physiology, staining properties sexual characteristics: sexual and /or asexual reproduction, extended dikaryotic phase, basidium formation genotypical characteristics: chromosomal dna fragment analysis, nucleic acid sequence analysis, probes schematic illustration of the cell envelope of a gram-negative and a gram-positive bacterium. schematic illustration of cell activation through toll-like receptors (tlrs). the tlr involved and includes the map kinase and nf-κb pathways leading to a cellular response. pamp for tlr2 include a variety of agonists derived from gram-positive organisms such as peptidoglycan and lipoteichoic acid. therefore, it seems that tlr2 and tlr4 are activated primarily by different pamp to initiate the host response to gram-positive and gram-negative bacterial infection, respectively. this is further illustrated by the fact that tlr2 (but not tlr4) knockout mice are highly susceptible to gram-positive bacteria, like staphylococcus aureus, whereas tlr4 (but not tlr2) knockout mice are highly susceptible to gram-negative bacteria such as salmonella typhimurium. several lines of evidence support the current hypothesis that the monocyte/macrophage is the principal cellular mediator of endotoxicity. lpshyporesponsive tlr4-deficient c3h/hej mice are made responsive by transfer of macrophages of a closely related lps-sensitive strain. when the host is challenged with lps, soluble factors are produced by macrophages that mediate fever and an acute-phase response. these factors include the proinflammatory cytokines, il-1, il-6, il-8, and tnf-α. together, tnf-α and il-1 stimulate endothelial cells to produce and express proteins on their membrane that have adhesive properties for leukocytes, promoting the margination and passage of neutrophils from blood vessels through the endothelial layer, leading to neutrophil influx into the tissue. adhesion molecules that mediate the binding of neutrophils appear on the endothelium after an inflammatory stimulus, followed by molecules that are specific for adhesion of monocytes or lymphocytes, which may be why neutrophils enter before mononuclear cells. molecules that are currently known to be involved in leukocyte-endothelium interactions belong to three structural groups: the immunoglobulin gene superfamily, the integrin family, and the selectin family. concomitant with cytokine release, lps induces the activation of neutrophils, macrophages, and many other cells, resulting in the release of toxic oxygen radicals, which lead to tissue damage. at the same time, membrane-associated phospholipases are activated and products of the arachidonic acid cascade are released through the cyclooxygenase and/or lipoxygenase pathways (see chapter a7). platelet-activating factor (paf) is also generated, partly in response to the same signals. all these products contribute to a generalised inflammatory state with influx of neutrophils, capillary-leak syndrome, disturbances in blood coagulation, and myocardial suppression. endotoxin and tnf-α also trigger multiple abnormalities in coagulation and fibrinolysis, leading to microvascular clotting and diffuse intravascular coagulation. they also induce endothelial cells to produce plasminogen activator and il-6, which is an important modulator of the production of acutephase proteins by the liver. interestingly, despite having important structural differences, tnf-α and il-1 have multiple overlapping and few distinct biological activities, act synergistically, and mimic the whole spectrum of toxicity caused by lps (see chapter a5). il-8 is an important chemoattractant and activator of neutrophils and is crucial in the early stages of inflammation. infusion of endotoxin in healthy humans leads to an early and transient increase in plasma levels of tnf-α (detectable after 30 minutes, peaking after 90-120 minutes, and undetectable after 4-6 hours), which coincides with the development of clinical symptoms and pathophysiological responses encountered in gram-negative septicaemia. tnf-α, il-1, il-6, and il-8 levels are also increased in patients with sepsis syndrome, with high levels of these cytokines being correlated with severity of disease. all these observations support the concept that endotoxin largely acts by initiating an inflammatory response through the activation of monocytes/macrophages and the subsequent release of cytokines. it also activates the complement system (leading to the generation of c5a, which induces aggregation of neutrophils and pulmonary vasoconstriction) and factor xii of the intrinsic coagulation pathway (hageman factor). finally, it induces the release of endorphins, which are also involved in the complex interactions of the inflammatory response in endotoxic septic shock. gram-positive bacteria are frequently and increasingly cultured from blood obtained from patients in shock. unlike the pathophysiology of shock caused by gram-negative bacteria, not much is known about the sequence of events that controls the signalling of monocytes and macrophages that leads to the release of cytokines. cell-wall components, such as peptidoglycan and teichoic acid, are clearly important in the activation of these cells. exotoxins, however, may also play a role in the pathogenesis of gram-positive bacterial shock. recently, another protein family was identified that also participates by sensoring microbial components derived from bacterial peptidoglycan. the nod (nucleotide-binding oligomerisation domain) proteins nod1 and nod2 have that have been implicated in intracellular recognition of the core structure, γ-d-glutamyl-meso-diaminopimelic acid, present in peptidoglycan. a number of circulating inflammatory mediators have been investigated as marker tools to facilitate the early recognition of sepsis. these include il-1, il-6, tnf, pro-calcitonin, and triggering receptor on myeloid cells (trem-1). trem-1 is expressed on leukocytes and trem family members have been implicated in mounting the inflammatory response. pro-calcitonin (pct) and il-6 have proved to be the most prominent biomarkers of early sepsis. pct is the pro-hormone of the hormone calcitonin, and can be produced by several cell types and many organs in response to pro-inflammatory stimuli, in particular by bacterial products. more recently high mobility group box-1 (hmgb-1) has been implicated as a lethal mediator of systemic inflammation. hmgb-1 is a nuclear and cytosolic protein widely studied as a transcription and growth factor that is released into the extracellular environment. it has a weak proinflammatory activity by itself and it may work in concert with other pro-inflammatory cytokines. this molecule may also be useful as a biomarker in the stratification of sepsis. susceptibility to sepsis can be influenced by factors that include ethnicity, gender, age, genetic defects and environmental factors. single-nucleotide polymorphisms (involving single base-pair alterations) have been described in genes controlling the host response to infection such as alterations in tnf receptors, il-1 receptors, coagulation factors and tlr. it is now clear that sepsis is a complex, dynamic syndrome with great heterogeneity, and not a distinct disease. therefore, neutralisation of a single key mediator as a cure for all patients with sepsis is erroneous. the hiv is a retrovirus that infects cells bearing the cd4 antigen, such as t helper cells (th), macrophages, and dendritic cells. the cd4 molecule, together with other receptor molecules, like chemokine receptor ccr5, acts as a binding site for the gp120 envelope glycoprotein of the virus. in an attempt to respond to hiv antigens and concomitant secondary microbial infections, these cells are activated, thus inducing the replication of hiv in the infected cd4 t cells, which are finally destroyed. in contrast, hiv-1 infection of macrophages is self-sustained and results in an inexorable growth of chronic active inflammatory processes in many tissue compartments including the central nervous system. infected cells bear the fusion protein gp41 and may therefore fuse with other infected cells. this helps the virus to spread and accounts for the multinucleated cells seen in lymph nodes and brain. as a result of the decreased numbers of cd4 + th cells and defects in antigen presentation, depressed immune responses in these patients are observed. during the progression of the disease, opportunistic infections by otherwise harmless micro-organisms can occur. these include can dida albicans oesophagitis, mucocutaneous herpes simplex, toxoplasma in the central nervous system, and pneumonia caused by toxoplasma and pneumo cystis carinii; kaposi's sarcoma also occurs frequently in these patients. this has been linked to the presence of a previously unknown type of herpes virus (hhv-8). this immune deficiency syndrome is called 'acquired immune deficiency syndrome' (aids). it has been suggested that infected monocytes/macrophages carry the hiv virus into the brain where it replicates in microglia and infiltrating macrophages. as a consequence, many aids patients develop cognitive and motor brain impairments. however, the picture is complicated by the various persistent infections already present in these patients, which give rise to their own pathology in the brain. these include toxoplasma gondii, cryptococcus neoformans and jc virus. so far, no cure for hiv infection has been achieved. the main effort in the prevention of hiv infection lies 135 human immunodeficiency virus infection in mass public education programmes. treatment of infected individuals is possible but expensive. highly active antiretroviral therapy (haart) reduces morbidity and mortality among patients infected with hiv (see fig. 5 ). success is limited by the emergence of drug-resistance viruses that can be transmitted to newly infected individuals. resistance, drug toxicity, and poor patient adherence lead to treatment failure and necessitate continuous development of alternative treatment strategies that intervene with the hiv replication cycle: i.e. on the level of virus entry, critical viral enzymes [reverse transcriptase (rt), integrase (in) and proteases (pr)] or viral nucleocapsid (nc) protein. at this moment a triple therapy is being prescribed in western countries (two rt inhibitors and one pr inhibitor, fig. 4) , each of which interfere with specific steps in the process of hiv replication. one major problem that has arisen is the increasing resistance to these drugs. agents with novel mechanisms of action provide options for patients with drug-resistant virus. blocking of the chemokine receptor ccr5, a co-receptor on cd4 cells for hiv, is an alternative treatment for persons infected with the r5 hiv type. this notion is supported by a recent finding that a homozygous defect in this chemokine receptor accounts for resistance of multiple-exposed individuals to hiv-1 infection. currently a commercially available drug is being used that specifically binds to ccr5 on the surface of the cd4 cell and selectively blocks hiv-1 binding. pasteur and koch triggered the stormy development of vaccines (anthrax, rabies, cholera) at the end of the 19th century. while pasteur remained faithful to the principle of attenuated micro-organisms in preparing his vaccines, koch employed killed germs (cholera) as a vaccine. since diphtheria and tetanus cause disease by means of toxins, the next logical step in the development of vaccines was the use of detoxified toxins to induce protection against these diseases [diphtheria (von behring) and tetanus (kitasato)]. von behring and kitasato were the first to demonstrate that the source of the protective activity induced by vaccines was present in blood serum. von behring was also the first to prove that protective immunity could be passed on via serum. the development of new vaccines had its ups (yellow fever) and downs (tuberculosis). with the arrival 136 immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites the effect of single and triple therapy on viral load and cd4 cells over time in hiv-infected individuals. the immune system in health and disease pathogenesis, treatment, and prevention of pneumococcal pneumonia harmful molecular mechanisms in sepsis chemokine coreceptor signaling in hiv-1 infection and pathogenesis 25 years after hiv discovery: prospects for cure and vaccine monoclonal antibody-based therapies for microbial diseases antibiotic-resistant bugs in the 21st century -a clinical super-challenge co-evolutionary aspects of human colonisation and infection by staphylococcus aureus outbreaks of infectious diseases centers for disease control and prevention of antibiotics, all work on new bacterial vaccines was suspended or severely curtailed, although some researchers continued to work on viral vaccines, such as rubella, measles, polio, and mumps.since it has proved difficult to consistently develop new antibiotics to combat antibiotic-resistant bacteria, interest in vaccines has gradually increased over the last 15 years (see chapter c1). today, thanks to new insights into the immune system and modern molecular biological and chemical techniques of analysis and synthesis, it is possible to produce well-defined vaccines. these contain only those determinants of the pathogenic micro-organism that induce protection (epitopes). these epitopes are usually short peptide or oligosaccharide chains, which can be produced synthetically or by means of recombinant dna techniques. the immunogenicity of these products can be enhanced by coupling them to a carrier (tetanus toxoid, liposomes) and/or by adding an adjuvant (a substance that strengthens the immune response non-specifically). the recombinant dna technique can also be used to obtain attenuated strains of micro-organisms, which are fully immunogenic and thus provide protection, but which are no longer virulent. one example of this is the development of a new cholera vaccine based on a bacterium that has all the characteristics of a virulent strain, except the toxin. the bacterium has retained all its adherence factors, which allow it to adhere to the intestinal mucosa; the length of time it spends in the intestine is sufficient to stimulate the local immune system. the newest trend in vaccinology is immunisation by introducing plasmid dna into the host. success has been attained by this method for hepatitis b vaccination.not only are new vaccines being developed, but it is also possible to heighten natural resistance for longer or shorter periods. various interleukins (il-2, gm-csf) and interferons are being studied in order to use them to combat infectious diseases (see chapter c7). passive antibody therapy with polyclonal or monoclonal antibodies (mouse or human igg with single specificity) for infectious diseases is experiencing a renewed interest. targeting soluble factors (like neutralisation of bacterial toxins and viruses) or common structures (like bacterial adhesins or viral entry factors) in high-risk patient groups may be ben-eficial alone or may enhance the therapeutic efficacy of other drugs. targets for clinical development of monoclonal antibodies include multi-resistant staphylococci and enterococci, bacillus anthracis toxin, hiv, hepatitis c virus (hcv), and respiratory syncytial virus (rsv). as outlined above for a number of bacteria and viruses, effective vaccines have been developed and applied worldwide. the eradication of smallpox (variola major) virus in the 1970s was a milestone for the world health organisation. the next goal of the who is to eradicate poliovirus in the coming years. major problems to be dealt with are the distribution of these vaccines, the costs involved, the registration and the compliance of the vaccinees and molecular techniques to trace the final bug. meanwhile new unexpected microbiological threats become in focus. hospital infections caused by multi-resistant micro-organism due to the abundance use of antibiotics and exchange of genetic material between micro-organisms impose major problems on patients and healthcare workers. new antibiotics and/or vaccines should be developed and new strategies employed to contain these infections. due to crowding and the high mobility of the world population, old and new pathogens, e.g. influenza and sars, threaten our society. the recent influenza a h1n1 ("mexican") flu outbreak in 2009 demonstrated how rapidly a new strain of flu can emerge and spread around the world. the sudden outbreak of this novel flu virus has tested the world's public health preparedness. in the netherlands, q fever emerged with large epidemic outbreaks. q fever is a zoonotic disease, that is passed from infected (farm) animals (cattle, sheep, and goats are the primary carriers) to humans. q fever is a disease caused by the intracellular bacterium coxiella burnetii. organisms are excreted in milk, urine, and faeces of infected animals. most importantly, during birthing the organisms are shed in high numbers within the amniotic fluids and the placenta. c. burnetii is resistant to heat, drying, and many common disinfectants, allowing it 137 infections in the new millennium 138 immune response in human pathology: infections caused by bacteria, viruses, fungi, and parasites to survive for a long time in the environment. people can become infected by inhalation of the bacteria, but the risk of infection is low. only about one-half of all people infected with c. burnetii show signs of clinical illness (e.g. flu-like illness, pneumonia, and hepatitis). q fever can be treated with antibiotics, and most people will recover fully. as a control measurement pregnant goats on dairy farms had to be killed and a mandatory vaccination campaign was started.on top of this, terrorists might intentionally use micro-organisms (smallpox, anthrax, plague etc.), or bacterial toxins (botulism) to cause death and disease in humans or animals in a civilian setting. the recognition that an event was caused by a biological weapon presents a severe challenge to be prepared for such an attack, especially for medical care providers, and public health officials. strategies to combat bioterrorism have to be worked out but with the experience of 100 years of combating micro-organisms with hygiene measures, vaccination, antibiotic and anti-viral treatment, there must be a way out. key: cord-023724-5at0rhqk authors: cann, alan j. title: infection date: 2015-07-24 journal: principles of molecular virology doi: 10.1016/b978-0-12-801946-7.00006-7 sha: doc_id: 23724 cord_uid: 5at0rhqk virus infection of higher organisms is the cumulative result of all the processes of replication and gene expression described in the previous chapters. together, these determine the overall course of each infection. infections range in complexity and duration from a very brief, superficial interaction between the virus and its host to infections that may span the entire life of the host organism, from before birth to its eventual death. a common misconception is that virus infection inevitably results in disease. in reality, the reverse is true—only a small minority of virus infections gives rise to any disease symptoms. this chapter provides an overview of the numerous patterns of virus infection and forms an introduction to the discussion of virus pathogenesis in chapter 7. unlike previous and subsequent chapters, this chapter deals primarily with the interaction of viruses with intact organisms rather than with the molecular biologist’s usual concern about the interaction between a virus and the cell. i explain how the immune responses to viruses enables the body to resist infection, and how viruses respond to this pressure. i describe and understand how virus infections are prevented and treated. life on earth depends on the primary productivity of plants-the production of organic molecules from inorganic molecules such as co 2 -(with some an additional contribution from some bacteria). from the smallest single-celled alga in the ocean to the largest forest giant tree (and everything in between, such as broccoli), they are vitally important. photosynthetic algae in the oceans play a major role in controlling the atmosphere and the climate, and interaction with viruses is one of the major mechanisms which in turn control the algae. all higher animals depend on the primary productivity of plants for their food. so plants are a big deal, and anything which affects plant growth is of great importance. in purely economic terms, viruses are only of importance if it is likely that they will affect crops during their commercial lifetime, a likelihood that varies greatly between very short extremes in horticultural production and very long extremes in forestry. some estimates have put total worldwide cost of plant virus infections as high as us$ 6 3 10 10 per year. by which plant viruses are transmitted between hosts is therefore of great importance. there are a number of routes by which plant viruses may be transmitted: i seeds: these may transmit virus infection either by external contamination of the seed with virus particles or by infection of the living tissues of the embryo. transmission by this route leads to early outbreaks of disease in new crops which are usually initially focal in distribution but may subsequently be transmitted to the remainder of the crop by other mechanisms. i vegetative propagation/grafting: these techniques are inexpensive and easy methods of plant propagation but provide the ideal opportunity for viruses to spread to new plants. i vectors: many different groups of living organisms can act as vectors and spread viruses from one plant to another: i bacteria (e.g., agrobacterium tumefaciens-the ti plasmid of this organism has been used experimentally to transmit virus genomes between plants) i fungi i nematodes i arthropods: insects (e.g., aphids, leafhoppers, planthoppers, beetles, thrips) i arachnids (e.g., mites) i mechanical: mechanical transmission of viruses is the most widely used method for experimental infection of plants and is usually achieved by rubbing virus-containing preparations into the leaves, which in most plant species are particularly susceptible to infection. however, this is also an important natural method of transmission. virus particles may contaminate soil for long periods and be transmitted to the leaves of new host plants as wind-blown dust or as rain-splashed mud. some of the most original experimental biology currently being done involves plant science. biologists can do experiments with plants that they can only dream of being able to perform with animals. and yet the idea persists among many that botany is boring. much of the most exciting plant science involves plant viruses, either as experimental tools or in terms of finding ways to prevent infection. and as this section describes, the biology of plant viruses has some striking differences from that of animal viruses. so if you think botany is boring, you probably need to find out more about plant viruses. the problems plant viruses face in initiating infections of host cells have already been described (chapter 4), as has the fact that no known plant virus employs a specific cellular receptor of the types that animal and bacterial viruses use to attach to cells. transmission of plant viruses by insects is of particular agricultural importance. huge areas of monoculture and the inappropriate use of pesticides that kill natural predators can result in massive population booms of pest insects such as aphids. plant viruses rely on a mechanical breach of the integrity of a cell wall to directly introduce a virus particle into a cell. this is achieved either by the vector associated with transmission of the virus or simply by mechanical damage to cells. transfer by insect vectors is a particularly efficient means of virus transmission. in some instances, viruses are transmitted mechanically from one plant to the next by the vector and the insect is only a means of distribution, through flying or being carried on the wind for long distances (sometimes hundreds of miles). insects that bite or suck plant tissues are the ideal means of transmitting viruses to new hosts-a process known as nonpropagative transmission. however, in other cases (e.g., many plant rhabdoviruses), the virus may also infect and multiply in the tissues of the insect (propagative transmission) as well as those of host plants. in these cases, the vector serves as a means not only of distributing the virus but also of amplifying the infection. initially, most plant viruses multiply at the site of infection, giving rise to localized symptoms such as necrotic spots on the leaves. the virus may subsequently be distributed to all parts of the plant either by direct cell-to-cell spread or by the vascular system, resulting in a systemic infection involving the whole plant. however, the problem these viruses face in reinfection and recruitment of new cells is the same as the one they faced initially-how to cross the barrier of the plant cell wall. plant cell walls necessarily contain channels called "plasmodesmata" which allow plant cells to communicate with each other and to pass metabolites between them. however, these channels are too small to allow the passage of virus particles or genomic nucleic acids. many (if not most) plant viruses have evolved specialized movement proteins that modify the plasmodesmata. one of the best known examples of this is the 30-k protein of tobacco mosaic virus (tmv). this protein is expressed from a subgenomic mrna (figure 3 .12), and its function is to modify plasmodesmata causing genomic rna coated with 30-k protein to be transported from the infected cell to neighboring cells (figure 6 .1). other viruses, such as cowpea mosaic virus (cpmv; comoviridae) have a similar strategy but employ a different molecular mechanism. in cpmv, the 58-/48-k proteins form tubular structures allowing the passage of intact virus particles to pass from one cell to another (figure 6 .1). typically, virus infections of plants might result in effects such as growth retardation, distortion, mosaic patterning on the leaves, yellowing, wilting, etc. these macroscopic symptoms result from: i necrosis of cells, caused by direct damage due to virus replication, i hypoplasia-localized retarded growth frequently leading to mosaicism (the appearance of thinner, yellow areas on the leaves), i hyperplasia-excessive cell division or the growth of abnormally large cells, resulting in the production of swollen or distorted areas of the plant. plants might be seen as sitting targets for virus infection-unlike animals, they cannot run away. however, plants exhibit a sophisticated range of responses to virus infections designed to minimize harmful effects. plants fight virus infections in a number of ways. first, they need to detect the infection, which they do by means of sensing virus signature molecules (so-called pathogen-associated molecular patterns or pamps, e.g., particular proteins) via dedicated receptors. when this happens, the production of resistance proteins that activate highly specific resistance mechanisms is triggered. in response, plant viruses attempt to evade these defense mechanisms by altering protein structures where possible and by producing proteins which bind to and hide small rnas which would trigger rna silencing. infection results in a "hypersensitive response," manifested as: i the synthesis of a range of new proteins, the pathogenesis-related ("pr") proteins, i an increase in the production of cell wall phenolic substances, i the release of active oxygen species, i the production of phytoalexins, i the accumulation of salicylic acid-amazingly, plants can even warn each other that viruses are coming by airborne signaling with volatile compounds such as methyl salicylate. the hypersensitive response involves synthesis of a wide range of different molecules. some of these pr proteins are proteases, which presumably destroy virus proteins, limiting the spread of the infection. there is some similarity here between the design of this response and the production of interferons (ifns) by animals. systemic resistance to virus infection is a naturally occurring phenomenon in some strains of plant. this is clearly a highly desirable characteristic that is prized by plant breeders, who try to spread this attribute to economically valuable crop strains. there are probably many different mechanisms involved in systemic resistance, but in general terms there is a tendency of these processes to increase local necrosis when substances such as proteases and peroxidases are produced by the plant to destroy the virus and to prevent its spread and subsequent systemic infection. an example of this is the tobacco n gene, which encodes a cytoplasmic protein with a nucleotide-binding site which interferes with the tmv replicase. when present in plants, this gene causes tmv to produce a localized, necrotic infection rather than the systemic mosaic symptoms normally seen. there are many different mechanisms involved in systemic resistance, but in general terms there is a tendency toward increased local necrosis as substances such as proteases and peroxidases are produced by the plant to destroy the virus and to prevent its spread and subsequent systemic disease. virus-resistant plants have been created by the production of transgenic plants expressing recombinant virus proteins or nucleic acids which interfere with virus replication without producing the pathogenic consequences of infection, for example: i virus coat proteins, which have a variety of complex effects, including inhibition of virus uncoating and interference of expression of the virus at the level of rna ("gene silencing" by "untranslatable" rnas), i intact or partial virus replicases which interfere with genome replication, i antisense rnas, i defective virus genomes, i satellite sequences (see chapter 8), i catalytic rna sequences (ribozymes), i modified movement proteins. this is a very promising technology that offers the possibility of substantial increases in agricultural production without the use of expensive, toxic, and ecologically damaging chemicals (fertilizers, herbicides, or pesticides). in some countries, notably in europe, public resistance to genetically engineered plants has so far prevented the widespread adoption of new varieties produced by genetic manipulation without considering the environmental cost of not utilizing these new approaches to plant breeding. the most significant response to virus infection in vertebrates is activation of both the cellular and humoral parts of the immune system. a complete description of all the events involved in the immune response to the presence of foreign antigens is beyond the scope of this book, so you should refer to the books mentioned in the further reading at the end of this chapter to ensure that you are familiar with all the immune mechanisms (and jargon!) described below. a brief summary of some of the more important aspects is worth considering however, beginning with the humoral immune response, which results in the production of antibodies. the major impact of the humoral immune response is the eventual clearance of virus from the body. serum neutralization stops the spread of virus to uninfected cells and allows other defense mechanisms to mop up the infection. figure 6 .2 shows a very simplified version of the mammalian humoral response to infection. virus infection induces at least three classes of antibody: immunoglobulin g (igg), igm, and iga. igm is a large, multivalent molecule that is most effective at cross-linking large targets (e.g., bacterial cell walls or flagella) but is probably less important in combating virus infections. in contrast, the production of iga is very important for initial protection from virus infection. secretory iga is produced at mucosal surfaces and results in "mucosal immunity," an important factor in preventing infection from occurring. induction of mucosal immunity depends to a large extent on the way in which antigens are presented to and recognized by the immune system. similar antigens incorporated into different vaccine delivery systems (see "prevention and therapy of virus infection") can lead to very different results in this respect, and mucosal immunity is such an important factor that similar vaccines may vary considerably in their efficacy. igg is probably the most important class of antibody for direct neutralization of virus particles in serum and other body fluids (into which it diffuses). direct virus neutralization by antibodies results from a number of mechanisms, including conformational changes in the virus capsid caused by antibody binding, or blocking of the function of the virus target molecule (e.g., receptor binding) by steric hindrance. a secondary consequence of antibody binding is phagocytosis of antibody-coated ("opsonized") target molecules by mononuclear cells or polymorphonuclear leukocytes. this results from the presence of the fc receptor on the surface of these cells, but as has already been noted in chapter 4, in some cases opsonization of virus by the binding of nonneutralizing antibodies can result in enhanced virus uptake. this has been shown to occur with rabies virus, and in the case of human immunodeficiency virus (hiv) may promote uptake of the virus by macrophages. nonphagocytic cells can also destroy antibody-coated viruses via an intracellular pathway involving the trim21 protein. antibody binding also leads to the activation of the complement cascade, which assists in the neutralization of virus particles. structural alteration of virus particles by complement binding can sometimes be visualized directly by electron microscopy. complement is particularly important early in virus infection when limited amounts of low-affinity antibody are made-complement enhances the action of these early responses to infection. despite all the above mechanisms, in overall terms cell-mediated immunity is probably more important than humoral immunity in the control of virus infections. this is demonstrated by the following observations: i congenital defects in cell-mediated immunity tend to result in predisposition to virus (and parasitic) infections, rather than to bacterial infections. i the functional defect in acquired immune deficiency syndrome (aids) is a reduction in the ratio of t-helper (cd4 ), which may have been present before the onset of aids but were previously suppressed by the intact immune system. cell-mediated immunity depends on three main effects ( figure 6 .3). these all act via molecular mechanisms that will be explained later in this chapter (see "viruses and apoptosis," below): i nonspecific cell killing (mediated by "natural killer" [nk] cells), i specific cell killing (mediated by cytotoxic t-lymphocytes [ctls]), i antibody-dependent cellular cytotoxicity (adcc). nk cells carry out cell lysis independently of conventional immunological specificity, that is, they do not depend on clonal antigen recognition for their action. they are not major histocompatibility complex (mhc) restricted. in other works, nk cells are able to recognize virus-infected cells without being presented with a specific antigen by a macromolecular complex consisting of mhc antigens plus the t-cell receptor/cd3 complex. the advantage of this is that nk cells have broad specificity (many antigens rather than a single epitope) and are also active without the requirement for sensitizing antibodies. they are therefore the first line of defense against virus infection. nk cells are most active in the early stages of infection (i.e., in the first few days), and their activity is stimulated by ifn-α/β. nk cells are not directly induced by virus infection-they exist even in immunologically naive individuals and are "revealed" in the presence of ifn-α/β. they are thus part of the "innate" rather than the "adaptive" immune response. their function is complementary to and is later taken over by ctls which are part of the "adaptive" immune response. not all of the targets for nk cells on the surface of infected cells are known, but they are inhibited by mhc class i antigens (which are present on all nucleated cells), allowing recognition of "self" (i.e., uninfected cells) and preventing total destruction of the body. it is well known that some virus infections disturb normal cellular mhc-i expression and this is one mechanism by which nk cells recognize virus-infected cells. nk cell cytotoxicity is activated by ifn-α/β, directly linking nk cell activity to virus infection. unlike nk cells which may be either cd4 1 or cd8 1 , ctls are usually of cd8 1 (suppressor) phenotype, that is, they express cd8 molecules on their surface. ctls are the major cell-mediated immune response to virus infections and are mhc restricted-clones of cells recognize a specific antigen only when presented by mhc-i antigen on the target cell to the t-cell receptor/ cd3 complex on the surface of the ctl. (mhc-i antigens are expressed on all nucleated cells in the body; mhc class ii antigens are expressed only on the surface of the antigen-presenting cells of the immune system-t-cells, b-cells, and macrophages.) ctl activity requires "help" (i.e., cytokine production) from t-helper cells. the ctls themselves recognize foreign antigens through the t-cell receptor/cd3 complex, which "docks" with antigen presented by mhc-i on the surface of the target cell ( figure 6 .4). the mechanism of cell killing by ctl is similar to that of nk cells (explained below). the induction of a ctl response also results in the release of many different cytokines from t-helper cells, some of which result in clonal proliferation of antigen-specific ctl and others that have direct antiviral effects-for example, ifns. the kinetics of the ctl response (peaking at about 7 days after infection) is somewhat slower than the nk response (e.g., 3à7 days cf. 0.5à3 days)-so nk cells and ctls are complementary systems. the induction of a ctl response is dependent on recognition of specific t-cell epitopes by the immune system. these are distinct from the b-cell epitopes recognized by the humoral arm of the immune system. t-cell epitopes are more highly conserved (less variable) than b-cell epitopes, which are more able to mutate quickly to escape immune pressure. these are important considerations in the design of antiviral vaccines. the specificity of cell killing by ctls is not absolute. although they are better "behaved" than nk cells, diffusion of perforin and local cytokine production frequently results in inflammation and bystander cell damage. this is a contributory cause of the pathology of many virus diseases (see chapter 7), but the less attractive alternative is to allow virus replication to proceed unchecked. adcc is less well understood than either of the two mechanisms mentioned above. adcc can be carried out by nk cells or by ctls. the mechanism of cell killing is the same as that described in the next section, although complement may also be involved in adcc. the distinguishing feature of adcc is that this mechanism is dependent on the recognition of antigen on the surface of the target cell by means of antibody on the surface of the effector cell. the antibody involved is usually igg, which is bound to fc receptors on the surface of the t-cell. adcc therefore requires a preexisting antibody response and hence does not occur early during primary virus infections-it is part of the adaptive immune response. the overall contribution of adcc to the control of virus infections is not clear, although it is now believed that it plays a significant part in their control. apoptosis, or "programmed cell death," is a critical mechanism in tissue remodeling during development and in cell killing by the immune system. there are two ways in which a cell can die: necrosis or apoptosis. i necrosis is the normal response of cells to injury caused by toxins or environmental stress. necrosis is marked by nonspecific changes such as disruption of the plasma membrane and nuclear envelope, rupture of membrane-bounded organelles such as mitochondria and lysosomes, cell swelling, random fragmentation of dna/rna, influx of calcium ions into the cell, and loss of membrane electrical potential. the release of cellular components from the dying cell causes a localized inflammatory response by the cells of the immune system. this frequently leads to damage to adjacent cells/tissue-"bystander" cell damage. i apoptosis is, in contrast, a tightly regulated process that relies on complex molecular cascades for its control. it is marked by cell shrinkage, condensation, and clumping of chromatin, a regular pattern of dna fragmentation, and "bubbling off" of cellular contents into small membrane-bounded vesicles ("blebbing") which are subsequently phagocytosed by macrophages, preventing inflammation. when triggered by the appropriate signals, immune effector cells such as ctls and nk cells release previously manufactured lytic granules stored in their cytoplasm. these act on the target cell and induce apoptosis by two mechanisms: i release of cytotoxins such as: (1) perforin (aka cytolysin), a peptide related to complement component c9 which, on release, polymerizes to form polyperforin, which forms transmembrane channels, resulting in permeability of the target cell membrane; and (2) serine proteases related to trypsin. these two effectors act collaboratively, the membrane pores allowing the entry of granzymes into the target cell. the membrane channels also allow the release of intracellular calcium from the target cell, which also acts to trigger apoptotic pathways. i in addition, ctls (but not nk cells) express fas ligand on their surface which binds to fas on the surface of the target cell, triggering apoptosis. binding of fas ligand on the effector cell to fas (cd95) on the target cell results in activation of cellular proteases known as "caspases," which in turn trigger a cascade of events leading to apoptosis. the process of induction and repression of apoptosis during virus infection has received much attention during the last few years. it is now recognized that this is an important innate response to virus infection. the regulation of apoptosis is a complex issue that cannot be described fully here (see further reading and figure 6 .5 for a summary), but virus infections disturb normal cellular biochemistry and frequently trigger an apoptotic response, for example: the pathways controlling apoptosis are very complex. this diagram represents only a simple summary of some of the mechanisms of major significance in virus infections. i pkr activation: the ifn effector pkr (rna-activated protein kinase may be activated by some viruses (e.g., hiv, reovirus). i p53 activation: viruses that interact with p53 (chapter 7) may cause either growth arrest or apoptosis (e.g., adenoviruses, sv40, papillomaviruses). i transcriptional disregulation: viruses that encode transcriptional regulatory proteins may trigger an apoptotic response (e.g., htlv tax). i foreign protein expression: overexpression of virus proteins at late stages of the replication cycle can also cause apoptosis by a variety of mechanisms. in response to this cellular alarm system, many if not most viruses have evolved mechanisms to counteract this effect and repress apoptosis: i bcl-2 homologues: a number of viruses encode bcl-2 (a negative regulator of apoptosis) homologues (e.g., adenovirus e1b-19k, human herpesvirus 8 [hhv-8] ksbcl-2). i caspase inhibition: caspases are a family of cysteine proteases that are important inducers of apoptosis. inhibiting these enzymes is an effective way of preventing apoptosis (e.g., baculovirus p35, serpins, viaps-"inhibitors of apoptosis"). i fas/tnf inhibition: viruses have evolved several mechanisms to block the effects of fas/tnf, including blocking signaling through the plasma membrane (e.g., adenovirus e3), tumor necrosis factor receptor (tnfr) mimics (e.g., poxvirus crma), mimics of death signaling factors (vflips), and interactions with signaling factors such as fas-associated death domain (fadd) and tnfr-associated death domain (tradd) (e.g., hhv-4 [ebv] lmp-1). i p53 inhibition: a number of viruses that interact with p53 have evolved proteins to counteract possible triggering of apoptosis (e.g., adenovirus e1b-55k and e4, sv40 t-antigen, papillomavirus e6). i miscellaneous: many other apoptosis-avoidance mechanisms have been described in a wide variety of viruses. without such inhibitory mechanisms, most viruses would simply not be able to replicate due to the death of the host cell before the replication cycle was complete. however, there is evidence that at least some viruses use apoptosis to their benefit. positive-sense rna viruses such as poliovirus, hepatitis a virus, and sindbis virus with lytic replication cycles appear to be able to regulate apoptosis, initially repressing it to allow replication to take place, then inducing it to allow the release of virus particles from the cell. by the 1950s, interference (i.e., the blocking of a virus infection by a competing virus) was a well-known phenomenon in virology. in some cases, the mechanism responsible is quite simple. for example, avian retroviruses are grouped into nine interference groups (a through i), based on their ability to infect various strains of chickens, pheasants, partridges, quail, etc., or cell lines derived from these species. in this case, the inability of particular viruses to infect the cells of some strains is due to the expression of the envelope glycoprotein of an endogenous provirus present in the cells which sequesters the cellular receptor needed by the exogenous virus for infection. in other cases, the mechanism of virus interference is less clear. in 1957, alick issacs and jean lindenmann were studying this phenomenon and performed the following experiment. pieces of chick chorioallantoic membrane were exposed to ultraviolet (uv)-inactivated (noninfectious) influenza virus in tissue culture. the "conditioned" medium from these experiments (which did not contain infectious virus) was found to inhibit the infection of fresh pieces of chick chorioallantoic membrane by (infectious) influenza virus in separate cultures ( figure 6 .6). their conclusion was that a soluble factor, which they called "interferon," was produced by cells as a result of virus infection and that this factor could prevent the infection of other cells. as a result of this provocative observation, ifn became the great hope for virology and was thought to be directly equivalent to the use of antibiotics to treat bacterial infections. the true situation has turned out to be far more complex than was first thought. ifns do have antiviral properties, but by and large their effects are exerted indirectly via their major function as cellular regulatory proteins. ifns are immensely potent; less than 50 molecules per cell show evidence of antiviral activity. hence, following isaacs and lindenmann's initial discovery, many fairly fruitless years were spent trying to purify minute amounts of naturally produced ifn. a b figure 6.6 discovery of ifns. in 1957, alick issacs and jean lindenmann discovered ifns by performing the following experiment. (a) pieces of chick chorioallantoic membrane were exposed to uv-inactivated (noninfectious) influenza virus in tissue culture. (b) the "conditioned" medium from these experiments (which did not contain infectious virus) was found to inhibit the infection of fresh pieces of chick chorioallantoic membrane by (infectious) influenza virus in separate cultures. they called inhibitory substance in the condition medium "interferon." this situation changed with the development of molecular biology and the cloning and expression of ifn genes, which has led to rapid advances in our understanding over the last 15 years. there are a number of different types of ifns: i ifn-α: there are at least 15 molecular species of ifn-α, all of which are closely related; some species differ by only one amino acid. they are synthesized predominantly by lymphocytes. the mature proteins contain 143 amino acids, with a minimum homology of 77% between the different types. all the genes encoding ifn-α are located on human chromosome 9, and gene duplication is thought to be responsible for this proliferation of genes. i ifn-β: the single gene for ifn-β is also located on human chromosome 9. the mature protein contains 145 amino acids and, unlike ifn-α, is glycosylated, with approximately 30% homology to other ifns. it is synthesized predominantly by fibroblasts. i other ifns: the single gene for ifn-γ is located on human chromosome 12. the mature protein contains 146 amino acids, is glycosylated, and has very low sequence homology to other ifns. it is synthesized predominantly by lymphocytes. other ifns, such as ifn-γ, -δ, -k, -τ, etc., play a variety of roles in cellular regulation but are not directly involved in controlling virus infection. because there are clear biological differences between the two main types of ifn, ifn-α and -β are known as type i ifn, and ifn-γ as type ii ifn. induction of ifn synthesis results from upregulation of transcription from the ifn gene promoters. there are three main mechanisms involved: i virus infection: this mechanism is thought to act by the inhibition of cellular protein synthesis that occurs during many virus infections, resulting in a reduction in the concentration of intracellular repressor proteins and hence in increased ifn gene transcription. in general, rna viruses are potent inducers of ifn while dna viruses are relatively poor inducers; however, there are exceptions to this rule (e.g., poxviruses are very potent inducers). the molecular events in the induction of ifn synthesis by virus infection are not clear. in some cases (e.g., influenza virus), uv-inactivated virus is a potent inducer; therefore, virus replication is not necessarily required. induction by viruses might involve perturbation of the normal cellular environment and/or production of small amounts of double-stranded rna. i double-stranded (ds) rna: all naturally occurring double-stranded rnas (e.g., reovirus genomes) are potent inducers of ifn, as are synthetic molecules (e.g., poly i:c); therefore, this process is independent of nucleotide sequence. single-stranded rna and doublestranded dna are not inducers. this mechanism of induction is thought to depend on the secondary structure of the rna rather than any particular nucleotide sequence. i metabolic inhibitors: compounds that inhibit transcription (e.g., actinomycin d) or translation (e.g., cycloheximide) result in induction of ifn. tumor promoters such as tetradecanoyl phorbol acetate or dimethyl sulfoxide are also inducers. their mechanism of action remains unknown but they almost certainly act at the level of transcription. the effects of ifns are exerted via specific receptors that are ubiquitous on nearly all cell types (therefore, nearly all cells are potentially ifn responsive). there are distinct receptors for type i and type ii ifn, each of which consists of two polypeptide chains. binding of ifn to the type i receptor activates a specific cytoplasmic tyrosine kinase (janus kinase, or jak1), which phosphorylates another cellular protein, signal transducer and activator of transcription 2 (stat2). this is transported to the nucleus and turns on transcriptional activation of ifn-responsive genes (including ifn, resulting in amplification of the original signal). binding of ifn to the type ii receptor activates a different cytoplasmic tyrosine kinase (jak2), which phosphorylates the cellular protein stat1, leading to transcriptional activation of a different set of genes. the main action of ifns is on cellular regulatory activities and is rather complex. ifn affects both cellular proliferation and immunomodulation. these effects result from the induction of transcription of a wide variety of cellular genes, including other cytokines. the net result is complex regulation of the ability of a cell to proliferate, differentiate, and communicate. this cellregulatory activity itself has indirect effects on virus replication. type i ifn is the major antiviral mechanism-other ifns act as potent cellular regulators, which may have indirect antiviral effects in some circumstances. the effect of ifns on virus infections in vivo is extremely important. animals experimentally infected with viruses and injected with anti-ifn antibodies experience much more severe infections than control animals infected with the same virus. this is because ifns protect cells from damage and death. however, they do not appear to play a major role in the clearance of virus infections-the other parts of the immune response are necessary for this. ifn is a "firebreak" that inhibits virus replication in its earliest stages by several mechanisms. two of these are understood in some detail, but a number of others (in some cases specific to certain viruses) are less well understood. ifns induce transcription of a cellular gene for the enzyme 2 0 ,5 0 -oligo a synthetase ( figure 6 .7). there are at least four molecular species of 2 0 ,5 0 -oligo a, induced by different forms of ifn. this compound activates an rna-digesting enzyme, rnase l, which digests virus genomic rnas, virus and cellular mrnas, and cellular ribosomal rnas. the end result of this mechanism is a reduction in protein synthesis (due to the degradation of mrnas and rrnas)-therefore the cell is protected from virus damage. the second method relies on the activation of a 68-kda protein called pkr (figure 6 .8). pkr phosphorylates a cellular factor, eif2α, which is required by ribosomes for the initiation of translation. the net result of this mechanism is also the inhibition of protein synthesis and this reinforces the 2 0 ,5 0 -oligo a mechanism. a third, well-established mechanism depends on the m x gene, a single-copy gene located on human chromosome 21, the transcription of which is induced by type i ifn. the product of this gene inhibits the primary transcription of influenza virus but not of other viruses. its method of action is unknown. in addition to these three mechanisms, there met-trna met .elf2.gtp (initiation of translation) gdp + "gef" the modified nucleic acid 2 0 ,5 0 -oligo a is involved in one of the major mechanism by which ifns counteract virus infections. are many additional recorded effects of ifns. they inhibit the penetration and uncoating of sv40 and some other viruses, possibly by altering the composition or structure of the cell membrane; they inhibit the primary transcription of many virus genomes (e.g., sv40, hsv) and also cell transformation by retroviruses. none of the molecular mechanisms by which these effects are mediated has been fully explained. ifns are a powerful weapon against virus infection, but they act as a blunderbuss rather than a "magic bullet." the severe side effects (fever, nausea, malaise) that result from the powerful cell-regulatory action of ifns means that they will never be widely used for the treatment of trivial virus infections-they are not the cure for the common cold. however, as the cell-regulatory potential of ifns is becoming better understood, they are finding increasing use as a treatment for certain cancers (e.g., the use of ifn-α in the treatment of hairy cell leukemia). current therapeutic uses of ifns are summarized in table 6 .1. the longterm prospects for their use as antiviral compounds are less certain, except for possibly in life-threatening infections where there is no alternative therapy (e.g., chronic viral hepatitis). in total, the many innate and adaptive components of the immune system present a powerful barrier to virus replication. simply by virtue of their continued existence, it is obvious that viruses have, over millennia, evolved effective "counter-surveillance" mechanisms in this molecular arms race. as described above, ctls can only respond to foreign antigens presented by mhc-i complexes on the target cell. a number of viruses interfere with mhc-i expression or function to disrupt this process and evade the ctl response. such mechanisms include downregulation of mhc-i expression by adenoviruses and interference with the antigen processing required to form an mhc-iàantigen complex by herpesviruses. the mhc-ii antigens are essential in the adaptive immune response in order to stimulate the development of antigen-responsive clones of effector cells. again, herpesviruses and papillomaviruses interfere with the processing and surface expression of mhc-iiàantigen complexes, inhibiting the ctl response. the poxvirus molluscum contagiosum encodes a homologue of mhc-i that is expressed on the surface of infected cells but is unable to bind an antigenic peptide, thus avoiding killing by nk cells that would be triggered by the absence of mhc-i on the cell surface. similar proteins are made by other viruses, such as hhv-5 (cmv), and herpesviruses in general appear to have a number of sophisticated mechanisms to avoid nk cell killing. see viruses and apoptosis earlier in this chapter. cytokines are secreted polypeptides that coordinate important aspects of the immune response, including inflammation, cellular activation, proliferation, differentiation, and chemotaxis. some viruses are able to inhibit the expression of certain chemokines directly. alternatively, herpesviruses and poxviruses encode "viroceptors"-virus homologues of host cytokine receptors that compete with cellular receptors for cytokine binding but fail to give transmembrane signals. high-affinity binding molecules may also neutralize cytokines directly, and molecules known as "virokines" block cytokine receptors again without activating the intracellular signaling cascade. ifns are cytokines which act as an effective means of curbing the worst effects of virus infections. part of their wide-ranging efficacy results from their generalized, nonspecific effects (e.g., the inhibition of protein synthesis in although direct humoral immunity is less significant than cell-mediated immunity, the antiviral action of adcc and complement make this a worthwhile target to inhibit. the most frequent means of subverting the humoral response is by high-frequency genetic variation of the b-cell epitopes on antigens to which antibodies bind. this is only possible for viruses that are genetically variable (e.g., influenza virus and hiv). herpesviruses use alternative strategies such as encoding viral fc receptors to prevent fc-dependent immune activation. poxviruses, herpesviruses, and some retroviruses encode mimics of normal regulators of complement activation proteins (e.g., secreted proteins that block c3 convertase assembly and accelerate its decay). poxviruses can also inhibit c9 polymerization, preventing membrane permeabilization. viruses do not set out to kill their hosts. virus pathogenesis is an abnormal situation of no value to the virus-the vast majority of virus infections are asymptomatic. however, for pathogenic viruses, a number of critical stages in replication determine the nature of the disease they produce. for all viruses, pathogenic or nonpathogenic, the first factor that influences the course of infection is the mechanism and site of entry into the body (figure 6 .9): i the skin: mammalian skin is a highly effective barrier against viruses. the outer layer (epidermis) consists of dead cells and therefore does not support virus replication. very few viruses infect directly by this route unless there is prior injury such as minor trauma or puncture of the barrier, such as insect or animal bites or subcutaneous injections. some viruses that do use this route include hsv and papillomaviruses, although these viruses probably still require some form of disruption of the skin such as small abrasions or eczema. i mucosal membranes: the mucosal membranes of the eye and genitourinary (gu) tract are much more favorable routes of access for viruses to the tissues of the body. this is reflected by the number of viruses that can be sexually transmitted; virus infections of the eye are also quite common (table 6 .2). i alimentary canal: viruses may infect the alimentary canal via the mouth, oropharynx, gut, or rectum, although viruses that infect the gut via the oral route must survive passage through the stomach, an extremely hostile environment with a very low ph and high concentrations of digestive enzymes. nevertheless, the gut is a highly valued prize for viruses-the intestinal epithelium is constantly replicating and a good deal of lymphoid tissue is associated with the gut which provides many opportunities for virus replication. moreover, the constant intake of food and fluids provides ample opportunity for viruses to infect these tissues (table 6 .3). to counteract this problem, the gut has many specific (e.g., secretory antibodies) and nonspecific (e.g., stomach acids and bile salts) defense mechanisms. i respiratory tract: the respiratory tract is probably the most frequent site of virus infection. as with the gut, it is constantly in contact with external virus particles which are taken in during respiration. as a result, the respiratory tract also has defenses aimed at virus infection-filtering of particulate matter in the sinuses and the presence of cells and antibodies of the immune system in the lower regions. viruses that infect the respiratory tract usually come directly from the respiratory tract of others, as aerosol spread is very efficient: "coughs and sneezes spread diseases" (table 6 .4). the natural environment is a considerable barrier to virus infection. most viruses are relatively sensitive to heat, drying, uv light (sunlight), etc., although a few types are quite resistant to these factors. this is particularly important for viruses that are spread via contaminated water or foodstuffsnot only must they be able to survive in the environment until they are ingested by another host, but, as most are spread by the fecalàoral route, they must also be able to pass through the stomach to infect the gut before being shed in the feces. one way of overcoming environmental stress is to take advantage of a secondary vector for transmission between the primary hosts ( figure 6 .10). as with plant viruses, the virus may or may not replicate while in the vector. viruses without a secondary vector must rely on continued host-to-host transmission and have evolved various strategies to do this (table 6 .5): i horizontal transmission: the direct host-to-host transmission of viruses. this strategy relies on a high rate of infection to maintain the virus population. i vertical transmission: the transmission of the virus from one generation of hosts to the next. this may occur by infection of the fetus before, during, or shortly after birth (e.g., during breastfeeding). more rarely, it may involve direct transfer of the virus via the germ line itself (e.g., retroviruses). in contrast to horizontal transmission, this strategy relies on long-term persistence of the virus in the host rather than rapid propagation and dissemination of the virus. having gained entry to a potential host, the virus must initiate an infection by entering a susceptible cell (primary replication). this initial interaction frequently determines whether the infection will remain localized at the site of entry or spread to become a systemic infection (table 6 .6). in some cases, virus spread is controlled by infection of polarized epithelial cells and the preferential release of virus from either the apical (e.g., influenza virus-a localized infection in the upper respiratory tract) or basolateral (e.g., rhabdoviruses-a systemic infection) surface of the cells (figure 6 .11). following primary replication at the site of infection, the next stage may be spread throughout the host. in addition to direct cellàcell contact, there are two main mechanisms for spread throughout the host: i via the bloodstream: viruses may get into the bloodstream by direct inoculation-for example, by arthropod vectors, blood transfusion, or intravenous drug abuse (sharing of nonsterilized needles). the virus may travel free in the plasma (e.g., togaviruses, enteroviruses) or in association with red cells (orbiviruses), platelets (hsv), lymphocytes (ebv, cmv), or monocytes (lentiviruses). primary viremia usually precedes and is necessary for the spread of virus to other parts of the body via the bloodstream and is followed by a more generalized, higher titer secondary viremia as the virus reaches the other target tissues or replicates directly in blood cells. i via the nervous system: as above, spread of virus to the nervous system is usually preceded by primary viremia. in some cases, spread occurs directly by contact with neurones at the primary site of infection; in other cases, it occurs via the bloodstream. once in peripheral nerves, the virus can spread to the central nervous system (cns) by axonal transport along neurones. the classic example of this is hsv (see "latent infection," below). viruses can cross synaptic junctions as these frequently contain virus receptors, allowing the virus to jump from one cell to another. the spread of the virus to various parts of the body is controlled to a large extent by its cell or tissue tropism. tissue tropism is controlled partly by the route of infection but largely by the interaction of a virus-attachment protein (vap) with a specific receptor molecule on the surface of a cell (as discussed in chapter 4) and has considerable effect on pathogenesis. at this stage, following significant virus replication and the production of virus antigens, the host immune response comes into play. this has already been discussed earlier and obviously has a major impact on the outcome of an infection. to a large extent, the efficiency of the immune response determines the amount of secondary replication that occurs and, hence, the spread to other parts of the body. if a virus can be prevented from reaching tissues where secondary replication can occur, generally no disease results, although there are some exceptions to this. the immune response also plays a large part in determining the amount of cell and tissue damage that occurs as a result of virus replication. as described above, the production of ifns is a major factor in preventing virus-induced tissue damage. the immune system is not the only factor that controls cell death, the amount of which varies considerably for different viruses. viruses may replicate widely throughout the body without any disease symptoms if they do not cause significant cell damage or death. retroviruses do not generally cause cell death, being released from the cell by budding rather than by cell lysis, and cause persistent infections, even being passed vertically to the offspring if they infect the germ line. all vertebrate genomes, including humans, are littered with retrovirus genomes that have been with us for millions of years (chapter 3). at present, these ancient virus genomes are not known to cause any disease in humans, although there are examples of tumors caused by them in rodents. conversely, picornaviruses cause lysis and death of the cells in which they replicate, leading to fever and increased mucus secretion, in the case of rhinoviruses, and paralysis or death (usually due to respiratory failure due to damage to the cns resulting, in part, from virus replication in these cells) in the case of poliovirus. the eventual outcome of any virus infection depends on a balance between two processes. clearance is mediated by the immune system (as discussed previously); however, the virus is a moving target that responds rapidly to pressure from the immune system by altering its antigenic composition (whenever possible). the classic example of this phenomenon is influenza virus, which displays two genetic mechanisms that allow the virus to alter its antigenic constitution: i antigenic drift: this involves the gradual accumulation of minor mutations (e.g., nucleotide substitutions) in the virus genome which result in subtly altered coding potential and therefore altered antigenicity, leading to decreased recognition by the immune system. this process occurs in all viruses all the time but at greatly different rates; for example, it is much more frequent in rna viruses than in dna viruses. in response, the immune system constantly adapts by recognition of and response to novel antigenic structures-but it is always one step behind. in most cases, however, the immune system is eventually able to overwhelm the virus, resulting in clearance. i antigenic shift: in this process, a sudden and dramatic change in the antigenicity of a virus occurs owing to reassortment of the segmented virus genome with another genome of a different antigenic type (see chapter 3). this results initially in the failure of the immune system to recognize a new antigenic type, giving the virus the upper hand ( figure 6 .12). the occurrence of past antigenic shifts in influenza virus populations is recorded by pandemics (worldwide epidemics; figure 6 .13). these events are marked by the sudden introduction of a new antigenic type of hemagglutinin and/or neuraminidase into the circulating virus, overcoming previous immunity in the human population. previous hemagglutinin/neuraminidase types become resurgent when a sufficiently high proportion of the people who have "immunological memory" of that type have died, thus overcoming the effect of "herd immunity." the other side of the relationship that determines the eventual outcome of a virus infection is the ability of the virus to persist in the host. long-term persistence of viruses results from two main mechanisms. the first is the regulation of lytic potential. the strategy followed here is to achieve the continued survival of a critical number of virus-infected cells (i.e., sufficient to continue the infection without killing the host organism). for viruses that do not usually kill the cells in which they replicate, this is not usually a problem; hence, these viruses tend naturally to cause persistent infections (e.g., retroviruses). for viruses that undergo lytic infection (e.g., herpesviruses), it this chart shows the history of influenza pandemics throughout the twentieth century. the first pandemic of the twenty-first century occurred in 2009 and was caused by an h1n1 type virus, although this was not as damaging as earlier pandemics. is necessary to develop mechanisms that restrict virus gene expression and, consequently, cell damage. the second aspect of persistence is the evasion of immune surveillance, discussed above. patterns of virus infection can be divided into a number of different types. abortive infection occurs when a virus infects a cell (or host) but cannot complete the full replication cycle, so this is a nonproductive infection. the outcome of such infections is not necessarily insignificant, for example, sv40 infection of nonpermissive rodent cells sometimes results in transformation of the cells (see chapter 7). this pattern is familiar for many common virus infections (e.g., "colds"). in these relatively brief infections, the virus is usually eliminated completely by the immune system. typically, in acute infections, much virus replication occurs before the onset of any symptoms (e.g., fever), which are the result not only of virus replication but also of the activation of the immune system; therefore, acute infections present a serious problem for the epidemiologist and are the pattern most frequently associated with epidemics (e.g., influenza, measles). these are the converse of acute infections (i.e., prolonged and stubborn). to cause this type of infection, the virus must persist in the host for a significant period. to the clinician, there is no clear distinction among chronic, persistent, and latent infections, and the terms are often used interchangeably. they are listed separately here because, to virologists, there are significant differences in the events that occur during these infections. these infections result from a delicate balance between the virus and the host organism, in which ongoing virus replication occurs but the virus adjusts its replication and pathogenicity to avoid killing the host. in chronic infections, the virus is usually eventually cleared by the host (unless the infection proves fatal), but in persistent infections the virus may continue to be present and to replicate in the host for its entire lifetime. the best-studied example of such a system is lymphocytic choriomeningitis virus (lcmv; an arenavirus) infection in mice (figure 6 .14). mice can be experimentally infected with this virus either at a peripheral site (e.g., a footpad or the tail) or by direct inoculation into the brain. adult mice infected in the latter way are killed by the virus, but among those infected by a peripheral route there are two possible outcomes to the infection: some mice die but others survive, having cleared the virus from the body completely. it is not clear what factors determine the survival or death of lcmv-infected mice, but other evidence shows that the outcome is related to the immune response to the virus. in immunosuppressed adult mice infected via the cns route, a persistent infection is established in which the virus is not cleared (due to the nonfunctional immune system), but, remarkably, these mice are not killed by the virus. if, however, syngeneic lcmv-specific t-lymphocytes (i.e., of the same mhc type) are injected into these persistently infected mice, the animals develop the full pathogenic symptoms of lcmv infection and die. when newborn mice, whose immune systems are immature, are infected via the cns route, they also develop a persistent infection, but, in this case, if they are subsequently injected with syngeneic lcmv-specific t-lymphocytes, they clear the virus and survive the infection. the mechanisms that control these events are not completely understood, but evidently there is a delicate balance between the virus and the host animal and the immune response to the virus is partly responsible for the pathology of the disease and the death of the animals. not infrequently, persistent infections may result from the production of defective-interfering (d.i.) particles (see chapter 3). such particles contain a partial deletion of the virus genome and are replication defective, but they are maintained and may even tend to accumulate during infections because they can replicate in the presence of replication-competent helper virus. the production of d.i. particles is a common consequence of virus infection of animals, particularly by rna viruses, but also occurs with dna viruses and plant viruses and can be mimicked in vitro by continuous high-titer passage of virus. although not able to replicate themselves independently, d.i. particles are not necessarily genetically inert and may alter the course of an infection by recombination with the genome of a replication-competent virus. the presence of d.i. particles can profoundly influence the course and the outcome of a virus infection. in some cases, they appear to moderate pathogenesis, whereas in others they potentiate it, making the symptoms of the disease much more severe. moreover, as d.i. particles effectively cause restricted gene expression (because they are genetically deleted), they may also result in a persistent infection by a virus that normally causes an acute infection and is rapidly cleared from the body. in a latent state, the virus is able to downregulate its gene expression and enter an inactive state with strictly limited gene expression and without ongoing virus replication. latent virus infections typically persist for the entire life of the host. an example of such an infection in humans is hsv. infection of sensory nerves serving the mucosa results in localized primary replication. subsequently, the virus travels via axon transport mechanisms further into the nervous system. there, it hides in dorsal root ganglia, such as the trigeminal ganglion, establishing a truly latent infection. the nervous system is an immunologically privileged site and is not patrolled by the immune system in the same way as the rest of the body, but the major factor in latency is the ability of the virus to restrict its gene expression. this eliminates the possibility of recognition of infected cells by the immune system. restricted gene expression is achieved by tight regulation of α-gene expression, which is an essential control point in herpesvirus replication (chapter 5). in the latent state, hsv makes an 8.3-kb rna transcript called the latent rna or latency-associated transcript (lat). the lat is broken down into even smaller strands called micrornas (mirnas), and these block the production of proteins which reactivate the virus. drugs which block production of these mirnas could in theory "wake up" all the dormant viruses, making them vulnerable to the immune system and to antiviral therapy, and this raises the eventual possibility of a cure for herpes infections. expression of the lat promotes neuronal survival after hsv infection by inhibiting apoptosis. this anti-apoptosis function could promote reactivation by: i providing more latently infected neurons for future reactivations, i protecting neurons in which reactivation occurs, i protecting previously uninfected neurons during a reactivation. when reactivated by some provocative stimulus, hsv travels down the sensory nerves to cause peripheral manifestations such as cold sores or genital ulcers. it is not altogether clear what constitutes a provocative stimulus, but there are many possible alternatives, including psychological and physical factors. periodic reactivation establishes the pattern of infection, with sporadic, sometimes very painful reappearance of disease symptoms for the rest of the host's life. even worse than this, immunosuppression later in life can cause the latent infection to flare up (which indicates that the immune system normally has a role in helping to suppress these latent infections), resulting in a very severe, systemic, and sometimes life-threatening infection. in a manner somewhat similar to herpesviruses, infection by retroviruses may result in a latent infection. integration of the provirus into the host genome certainly results in the persistence of the virus for the lifetime of the host organism and may lead to an episodic pattern of disease. in some ways, acquired immunodeficiency syndrome (aids), which results from hiv infection, shows aspects of this pattern of infection. the pathogenesis of aids is discussed in detail in chapter 7. there are two aspects of the response to the threat of virus diseases: first, prevention of infection, and second, treatment of the disease. the former strategy relies on two approaches: public and personal hygiene, which perhaps plays the major role in preventing virus infection (e.g., provision of clean drinking water and disposal of sewage; good medical practice such as the sterilization of surgical instruments) and vaccination, which makes use of the immune system to combat virus infections. most of the damage to cells during virus infections occurs very early, often before the clinical symptoms of disease appear. this makes the treatment of virus infection very difficult; therefore, in addition to being less expensive, prevention of virus infection is undoubtedly better than cure. to design effective vaccines, it is important to understand both the immune response to virus infection and the stages of virus replication that are appropriate targets for immune intervention. to be effective, vaccines must stimulate as many of the body's defense mechanisms as possible. in practice, this usually means trying to mimic the disease without causing pathogenesis-for example, the use of live attenuated viruses as vaccines such as nasally administered influenza vaccines and orally administered poliovirus vaccines. to be effective, it is not necessary to get 100% uptake of vaccine. "herd immunity" results from the break in transmission of a virus that occurs when a sufficiently high proportion of a population has been vaccinated. this strategy is most effective where there is no alternative host for the virus (e.g., measles) and in practice is the situation that usually occurs as it is impossible to achieve 100% coverage with any vaccine. however, this is a risky business; if protection of the population falls below a critical level, epidemics can easily occur. synthetic vaccines are short, chemically synthesized peptides. the major disadvantage with these molecules is that they are not usually very effective immunogens and are very costly to produce. however, because they can be made to order for any desired sequence, they have great theoretical potential, but none are currently in clinical use. recombinant vaccines are produced by genetic engineering. such vaccines have been already produced and are better than synthetic vaccines because they tend to give rise to a more effective immune response. some practical success has already been achieved with this type of vaccine. for example, vaccination against hepatitis b virus (hbv) used to rely on the use of australian antigen (hbsag) obtained from the serum of chronic hbv carriers. this was a very risky practice indeed (because hbv carriers are often also infected with hiv). a completely safe recombinant hbv vaccine produced in yeast is now used. dna vaccines are the newest type of vaccine and consist of only a dna molecule encoding the antigen(s) of interest and, possibly, costimulatory molecules such as cytokines. the concept behind these vaccines is that the dna component will be expressed in vivo, creating small amounts of antigenic protein that serve to prime the immune response so that a protective response can be rapidly generated when the real antigen is encountered. in theory, these vaccines could be manufactured quickly and should efficiently induce both humoral and cellmediated immunity. initial clinical studies have indicated that there is still some way to go until this experimental technology becomes a practical proposition. subunit vaccines consist of only some components of the virus, sufficient to induce a protective immune response but not enough to allow any danger of infection. in general terms, they are completely safe, except for very rare cases in which adverse immune reactions may occur. unfortunately, they also tend to be the least effective and most expensive type of vaccine. the major technical problems associated with subunit vaccines are their relatively poor antigenicity and the need for new delivery systems, such as improved carriers and adjuvants. virus vectors are recombinant virus genomes genetically manipulated to express protective antigens from (unrelated) pathogenic viruses. the idea here is to utilize the genome of a well-understood, attenuated virus to express and present antigens to the immune system. many different viruses offer possibilities for this type of approach. one of the most highly developed systems so far is based on the vv genome. this virus has been used to vaccinate millions of people worldwide in the campaign to eradicate smallpox and is generally a safe and effective vehicle for antigen delivery. such vaccines are difficult to produce. no human example is clearly successful yet, although many different trials are currently underway, but vvàrabies recombinants have been used to eradicate rabies in european fox populations. vv-based vaccines have advantages and disadvantages for use in humans-a high percentage of the human population has already been vaccinated during the smallpox eradication campaign, and this lifelong protection may result in poor response to recombinant vaccines. although generally safe, vv is dangerous in immunocompromised hosts, thus it cannot be used in hiv-infected individuals. a possible solution to these problems may be to use avipoxvirus vectors (e.g., fowlpox or canarypox) as "suicide vectors" that can only establish abortive infections of mammalian cells and offer the following advantages: i expression of high levels of foreign proteins, i no danger of pathogenesis (abortive infection), i no natural immunity in humans (avian virus). inactivated vaccines are produced by exposing the virus to a denaturing agent under precisely controlled conditions. the objective is to cause loss of virus infectivity without loss of antigenicity. obviously, this involves a delicate balance. however, inactivated vaccines have certain advantages, such as generally being effective immunogens (if properly inactivated), being relatively stable, and carrying little or no risk of vaccine-associated virus infection (if properly inactivated, but accidents can and do occur). the disadvantage of these vaccines is that it is not possible to produce inactivated vaccines for all viruses, as denaturation of virus proteins may lead to loss of antigenicity (e.g., measles virus). although relatively effective, "killed" vaccines are sometimes not as effective at preventing infection as "live" virus vaccines, often because they fail to stimulate protective mucosal and cell-mediated immunity to the same extent. a more recent concern is that these vaccines contain virus nucleic acids, which may themselves be a source of infection, either of their own accord (e.g., (1)sense rna virus genomes) or after recombination with other viruses. virus vaccines do not have to be based on virion structural proteins. the effectiveness of attenuated vaccines relies on the fact that a complete spectrum of virus proteins, including nonstructural proteins, is expressed and gives rise to cell-mediated immune responses. live attenuated virus vaccines are viruses with reduced pathogenicity used to stimulate an immune response without causing disease. the vaccine strain may be a naturally occurring virus (e.g., the use of cowpox virus by edward jenner to vaccinate against smallpox) or artificially attenuated in vitro (e.g., the oral poliomyelitis vaccines produced by albert sabin). the advantage of attenuated vaccines is that they are good immunogens and induce long-lived, appropriate immunity. set against this advantage are their many disadvantages. they are often biochemically and genetically unstable and may either lose infectivity (becoming worthless) or revert to virulence unexpectedly. despite intensive study, it is not possible to produce an attenuated vaccine to order, and there appears to be no general mechanism by which different viruses can be reliably and safely attenuated. contamination of the vaccine stock with other, possibly pathogenic viruses is also possible-this was the way in which sv40 was first discovered in oral poliovirus vaccine in 1960. inappropriate use of live virus vaccines, for example, in immunocompromised hosts or during pregnancy may lead to vaccineassociated disease, whereas the same vaccine given to a healthy individual may be perfectly safe. despite these difficulties, vaccination against virus infection has been one of the great triumphs of medicine during the twentieth century. most of the success stories result from the use of live attenuated vaccines-for example, the use of vv against smallpox. on may 8, 1980, the world health organization (who) officially declared smallpox to be completely eradicated, the first virus disease to be eliminated from the world. the who aims to eradicate a number of other virus diseases such as poliomyelitis and measles, but targets for completion of these programs have undergone much slippage due to the formidable difficulties involved in a worldwide undertaking of this nature. although prevention of infection by prophylactic vaccination is much the preferred option, postexposure therapeutic vaccines can be of great value in modifying the course of some virus infections. examples of this include rabies virus, where the course of infection may be very long and there is time for postexposure vaccination to generate an effective immune response and prevent the virus from carrying out the secondary replication in the cns that is responsible for the pathogenesis of rabies. other potential examples can be found in virus-associated tumors, such as hpv-induced cervical carcinoma. most existing virus vaccines are directed against viruses which are relatively antigenically invariant, for example, measles, mumps, and rubella viruses, where this is only one unchanging serotype of the virus. viruses whose antigenicity alters continuously are a major problem in terms of vaccine production, and the classic example of this is influenza virus (see earlier). in response to this problem, new technologies such as reverse genetics could be used to improve and to shorten the lengthy process of preparing vaccines. rna virus genomes can be easily manipulated as dna clones to contain nucleotide sequences which match currently circulating strains of the virus. infectious virus particles are rescued from the dna clones by introducing these into cells. seed viruses for distribution to vaccine manufacturers can be produced in as little as 1à2 weeks, a much shorter time than the months this process takes in conventional vaccine manufacture. using the same technology, universal influenza vaccines containing crucial virus antigens expressed as fusion proteins with other antigenic molecules could feasibly be produced, making the requirement for constant production of new influenza vaccines obsolete. although this has not yet been achieved, advances toward these goals are being made. the explosion of molecular techniques described in earlier chapters is now being used to inform vaccine design (as well as the design of antiviral drugs) rather than simply relying on trial-and-error approaches. however, developing safe and effective vaccines remains one of the greatest challenges facing virology. rna interference (rnai) is a posttranscriptional gene silencing process that occurs in organisms from yeast to humans. in mammals, small rnas include small interfering rnas (sirnas) and mirnas. sirnas, with perfect base complementarity to their targets, activate rnai-mediated cleavage of the target mrnas, while mirnas generally induce rna decay and/or translation inhibition of target genes (figure 6 .15). mammals, including humans, encode hundreds or thousands of mirnas. some viruses with eukaryotic hosts also encode mirnas. herpesviruses in particular encode multiple mirnas; most other nuclear dna viruses encode one or two mirnas. rna viruses and cytoplasmic dna viruses appear to lack any mirnas. virus mirnas may serve two major functions. several have been shown to inhibit the expression of cellular factors that play a role in cellular innate or adaptive antiviral immune responses, so reducing the effectiveness of the immune response. alternatively, virus mirnas may downregulate the expression of virus proteins, including key immediate-early or early regulatory proteins. in hsv, mirnas are expressed at high levels during latency, but not during productive replication, so their action is thought to stabilize latency. recently there have been controversial claims that mirna can exert antiviral activity in mice, at least in some circumstances. antiviral sirna activity is only seen in stem cells and in newborn mice and many scientists think sirna is not a major part of the innate immune system in adult animals. there is evidence that sirna may be turned on in responses to virus infection, but rather than acting directly against the virus, it may be used to regulate the ifn response. that still leaves the fact that in mammals mirna is a powerful regulator of gene expression, including virus genes. many viruses use mirna to control their own gene expression and that of their host cells. on infection of a host cell, viruses encounter a range of mirna species, many of which have been shown to restrict virus gene expression. thus they have had to evolve a range of mechanisms to evade mirna restriction is the same way that they have evolved other mechanisms to mitigate the impact of innate immunity. these include: i blocking mirna function i avoiding 3 0 utr targets complementary to cellular mirnas i evolving very short 3 0 utrs i evolving structured 3 0 utrs rnai expression can be induced by dsrna, and this approach has been used to investigate gene function in a variety of organisms including plants and insects. however, this method cannot be applied to mammalian cells as dsrnas longer than 30 nucleotides induces the ifn response (see earlier), which results in the degradation of mrnas and causes a global inhibition of translation. to circumvent this problem, chemically synthesized sirnas or plasmid-vectors manipulated to produce short hairpin rna molecules can be used to investigate gene function in mammals. in the future it may be feasible to treat virus diseases by shutting off gene expression by directing the degradation of specific mrnas, and many clinical trials are currently underway. although rna interference has been used widely in cultured cells to inhibit virus replication and to probe biological pathways, considerable problems must be overcome before it becomes a useful therapy, including the development of suitable delivery and targeting systems and solving the issue of stability in vivo. the natural world is a soup of bacteriophages. so how do bacteria survive against this constant onslaught? with their own form of adaptive immunity. crisprs (clustered regularly interspaced short palindromic repeats) are short, direct repeats of dna base sequences. each crispr contains a series of bases followed by the same series in reverse (a palindrome) and then by 30 or so base pairs known as "spacer" dna. the spacers are short segments of virus or plasmid dna. crisprs are found in the genomes of approximately 50% of bacteria and 90% of archaea. crispr loci are typically located on the bacterial chromosome, although some are found on plasmids. bacteria may contain more than one crispr locus-up to 18 in some cases. crisprs function as a sort of prokaryotic immune system, conferring resistance to exogenous genetic elements such as plasmids and bacteriophages. intriguingly, the crispr system provides a form of acquired immunity, allowing the cell to remember and respond to sequences it has encountered before. how do crisprs work? crisprs are often adjacent to cas (crispr-associated) genes. the cas genes encode a large and heterogeneous family of proteins including nucleases, helicases, polymerases, and polynucleotide-binding proteins, forming the crispr/cas system. (note: cas 5 genes, cas 5 the proteins encoded by these genes.) the interesting bits are the unique spacer elements (derived from exogenous sequences such as viruses and plasmids) rather than the repeats themselves. the spacer elements originate from exogenous dna the bacterium (or its ancestors) has previous encountered-they are typically pieces of phage or plasmid dna. this allows the cell to recognize these sequences via base homology if they enter the cell again, for example, if the bacterium is infected with a bacteriophage whose genome contains this sequence: 1. cas-encoded nucleases cleave invading dna into short pieces. 2. other cas proteins allow a fragment of the foreign dna to be incorporated as a novel repeat-spacer unit at the leader end of the crispr site. 3. the crispr array is then transcribed to form a pre-crispr rna (crrna) transcript. 4. the pre-crrna is cleaved within the repeat sequence by cas proteins to generate small crispr rnas, crrnas. 5. the crrnas to work in a similar way to rnai in eukaryotic cells, although there are important differences in the machinery by which this happens. crisprs are an important way in which bacteria are able to survive constant attack by bacteriophages in the environment, but phages have been around for a very long time too, so they must have found ways of counteracting the crispr system. eukaryotic viruses may express inhibitors such as dsrnabinding proteins that interfere with the rna silencing machinery, whereas bacteriophages acquire mutations or recombine the sequence corresponding to the crispr spacer to avoid recognition in an analogous way to how viruses of eukaryotes acquire mutations in b-cell and t-cell epitopes in proteins to evade the mammalian immune system. so who (apart from bacteria) cares about crisprs? altering the spacer via genetic manipulation can provide novel phage resistance, whereas spacer deletion results in loss of phage resistance. although crisprs originate in bacteria, they also work in eukaryotic cells if introduced by genetic engineering. this provides a convenient way of targeting genes in cells, including human cells. recent work suggests that crisprs might also be involved in control of bacterial gene expression as well as in immunity. we will undoubtedly see much more widespread use of crisprs in biotechnology over the next few years. phage therapy, the use of bacteriophages to treat or prevent disease, stretches back a century to the earliest days of the discovery of phages. long before the discovery of antibiotics, the thought that viruses which lyse bacteria could be used to treat diseases was highly attractive. yet this idea has never become a widespread practical reality. devotees of phage therapy defend their cherished belief with almost religious fervor, but there are serious obstacles to be overcome, such as the narrow host range of most phages (a few strains of bacteria, not even an entire species) and the speed at which bacteria develop resistance to infection. as the spectrum of clinically useful antibiotics dwindles, phage therapy increases in attractiveness, but is unlikely ever to replace the antibiotic golden era of disease treatment we are now leaving behind. another aspect of "virotherapy" is the growing interest in oncolytic virusesviruses engineered to kill only cancer cells. the usefulness of many different types of virus has been investigated, including adenoviruses, herpesviruses, reoviruses, and poxviruses. although safety is a concern even in patients with terminal illnesses, this is one area of medical research where optimism is considerable. many clinical trials are underway at it seems certain that this approach to cancer treatment will eventually become more common, possibly as an adjunct to other forms of therapy such as surgery, drugs, and radiotherapy. viruses have also developed as gene delivery systems for the treatment of inherited and acquired diseases. gene therapy offers: i delivery of large biomolecules to cells, i the possibility of targeting delivery to a specific cell type, i high potency of action due to replication of the vector, i potential to treat certain diseases (such as head and neck cancers and brain tumors) that respond poorly to other therapies or may be inoperable. the very first retroviral and adenoviral vectors were characterized in the early 1980s. the first human trial to treat children with immunodeficiency resulting from a lack of the enzyme adenosine deaminase began in 1990 and showed encouraging although not completely successful results. like most of the initial attempts, this trial used recombinant retrovirus genomes as vectors. in 1995, the first successful gene therapy for motor neurons and skin cells was reported, while the first phase three (widespread) gene therapy trial was begun in 1997. in 1999, the first successful treatment of a patient with severe combined immunodeficiency disease (scid) was reported, but, sadly, the first death due to a virus vector also occurred, and in 2002 the occurrence of leukemias due to oncogenic insertion of a retroviral vector was seen in some scid patients undergoing treatment. several different viruses are being tested as potential vectors ( integrate into cellular dna at high frequency to establish a stable latent state; not associated with any known disease; vectors can be constructed that will not express any viral gene products. only b5 kb of dna can be packaged into the parvovirus capsid, and some virus sequences must be retained for packaging; integration into host-cell dna may potentially have damaging consequences. herpesviruses relatively easy to manipulate in vitro; grows to high titers; long-term persistence in neuronal cells without integration. (long-term) pathogenic consequences? integrate into cell genome, giving long-lasting (lifelong?) expression of recombinant gene. difficult to grow to high titer and purify for direct administration (patient cells must be cultured in vitro); cannot infect nondividing cells-most somatic cells (except lentiviruses?); insertional mutagenesis/activation of cellular oncogenes. can express high levels of foreign proteins. avipoxvirus vectors (e.g., fowlpox or canarypox) are "suicide vectors" that undergo abortive replication in mammalian cells so there is no danger of pathogenesis and no natural immunity in humans. a high proportion of the human population has already been vaccinated-lifelong protection may result in poor response to recombinant vaccines (?). dangerous in immunocompromised hosts. the alternative to vaccination is to attempt to treat virus infections using drugs that block virus replication (table 6 .8). historically, the discovery of antiviral drugs was largely down to luck. spurred on by successes in the treatment of bacterial infections with antibiotics, drug companies launched huge blind-screening programs to identify chemical compounds with antiviral activity, with relatively little success. the key to the success of any antiviral drug lies in its specificity. almost any stage of virus replication can be a target for a drug, but the drug must be more toxic to the virus than the host. this is measured by the chemotherapeutic index, given by: dose of drug that inhibits virus replication dose of drug that is toxic to host the smaller the value of the chemotherapeutic index, the better. in practice, a difference of several orders of magnitude between the two toxicity values is usually required to produce a safe and clinically useful drug. modern technology, including molecular biology and computer-aided design of chemical compounds, allows the deliberate design of drugs, but it is necessary to "know your enemy"-to understand the key steps in virus replication that might be inhibited. any of the stages of virus replication can be a target for antiviral intervention. the only requirements are: i the process targeted must be essential for replication. i the drug is active against the virus but has "acceptable toxicity" to the host organism. what degree of toxicity is "acceptable" clearly varies considerably-for example, between a cure for the common cold, which might be sold over the counter and taken by millions of people, and a drug used to treat fatal virus infections such as aids. the attachment phase of replication can be inhibited in two ways, by agents that mimic the vap and bind to the cellular receptor or by agents that mimic the receptor and bind to the vap. synthetic peptides are the most logical class of compound to use for this purpose. while this is a promising line of research, there are considerable problems with the clinical use of these substances, primarily the high cost of synthetic peptides and the poor pharmacokinetic properties of many of these synthetic molecules. it is difficult to target specifically the penetration/uncoating stages of virus replication as relatively little is known about them. uncoating in particular is largely mediated by cellular enzymes and is therefore a poor target for intervention, although, like penetration, it is often influenced by one or more virus proteins. amantadine and rimantadine are two drugs that are active against influenza a viruses. the action of these closely related agents is to block cellular membrane ion channels. the target for both drugs is the influenza matrix protein (m 2 ), but resistance to the drug may also map to the hemagglutinin gene. this biphasic action results from the inability of drug-treated cells to lower the ph of the endosomal compartment (a function normally controlled by the m 2 gene product), which is essential to induce conformational changes in the ha protein to permit membrane fusion (see chapter 4). many viruses have evolved their own specific enzymes to replicate virus nucleic acids preferentially at the expense of cellular molecules. there is often sufficient specificity in virus polymerases to provide a target for an antiviral agent, and this method has produced the majority of the specific antiviral drugs currently in use. the majority of these drugs function as polymerase substrates (i.e., nucleoside/nucleotide) analogues, and their toxicity varies considerably, from some that are well tolerated (e.g., acyclovir) to others that are quite toxic (e.g., azidothymidine or azt). there is a problem with the pharmacokinetics of these nucleoside analogues in that their typical serum half-life is 1 to 4 hours. nucleoside analogues are in fact pro-drugs, as they must be phosphorylated before becoming effective-which is key to their selectivity: i acyclovir is phosphorylated by hsv thymidine kinase 200 times more efficiently than by cellular enzymes. i ganciclovir is 10 times more effective against cmv than acyclovir but must be phosphorylated by a kinase encoded by cmv gene ul97 before it becomes pharmaceutically active. i other nucleoside analogues derived from these drugs and active against herpesviruses have been developed (e.g., valciclovir and famciclovir). these compounds have improved pharmacokinetic properties, such as better oral bioavailability and longer half-lives. in addition to these there are a number of nonnucleoside analogues that inhibit virus polymerases; for example, foscarnet is an analogue of pyrophosphate that interferes with the binding of incoming nucleotide triphosphates by virus dna polymerases. ribavirin is a compound with a very wide spectrum of activity against many different viruses, especially against many (à)sense rna viruses. this drug acts as an rna mutagen, causing a 10-fold increase in mutagenesis of rna virus genomes and a 99% loss in virus infectivity after a single round of virus infection in the presence of ribavirin. ribavirin is thus quite unlike the other nucleoside analogues described above, and its use might become much more widespread in the future if it were not for the frequency of adverse effects associated with this drug. virus gene expression is less amenable to chemical intervention than genome replication, because viruses are much more dependent on the cellular machinery for transcription, mrna splicing, cytoplasmic export, and translation than for replication. to date, no clinically useful drugs that discriminate between virus and cellular gene expression have been developed. as with penetration and uncoating, for the majority of viruses the processes of assembly, maturation, and release are poorly understood and therefore have not yet become targets for antiviral intervention, with the exception of the antiinfluenza drugs oseltamivir and zanamivir, which are inhibitors of influenza virus neuraminidase. neuraminidase is involved in the release of virus particles budding from infected cells, and these drugs are believed to reduce the spread of virus to other cells. the most striking aspect of antiviral chemotherapy is how few clinically useful drugs are available. as if this were not bad enough, there is also the problem of drug resistance to consider. in practice, the speed and frequency with which resistance arises when drugs are used to treat virus infections varies considerably and depends largely on the biology of the virus involved rather than on the chemistry of the compound. to illustrate this, two extreme cases are described here. acyclovir, used to treat hsv infections, is easily the most widely used antiviral drug. this is particularly true in the case of genital herpes, which causes painful recurrent ulcers on the genitals. it is estimated that 40 to 60 million people suffer from this condition in the united states. fortunately, resistance to acyclovir arises infrequently. this is partly due to the high fidelity with which the dna genome of hsv is copied (chapter 3). mechanisms that give rise to acyclovir resistance include: i hsv pol gene mutants that do not incorporate acyclovir i hsv thymidine kinase (tk) mutants in which tk activity is absent (tk 2 ) or reduced or shows altered substrate specificity strangely, it is possible to find mutations that give rise to each of these phenotypes with a frequency of 1 3 10 23 to 1 3 10 24 in clinical hsv isolates. the discrepancy between this and the very low frequency with which resistance is recorded clinically is probably explained by the observation that most pol/tk mutants appear to be attenuated (e.g., tk 2 mutants of hsv do not reactivate from the latent state). conversely, azt treatment of hiv infection is much less effective. in untreated hiv-infected individuals, azt produces a rise in the numbers of cd4 1 cells within 2à6 weeks. however, this beneficial effect is transient; after 20 weeks, cd4 1 t-cell counts generally revert to baseline. this is due partly to the development of azt resistance in treated hiv populations and to the toxicity of azt on hematopoiesis, as the chemotherapeutic index of azt is much worse than that of acyclovir. azt resistance is initiated by the acquisition of a mutation in the hiv reverse transcriptase (rt) gene at codon 215. in conjunction with two to three additional mutations in the rt gene, a fully azt-resistant phenotype develops. after 20 weeks of treatment, 40à50% of azt-treated patients develop at least one of these mutations. this high frequency is due to the error-prone nature of reverse transcription (chapter 3). because of the large number of replicating hiv genomes in infected patients (chapter 7), many mistakes occur continuously. it has been shown that the mutations that confer resistance already exist in untreated virus populations. thus, treatment with azt does not cause but merely selects these resistant viruses from the total pool. with other anti-rt drugs, such as didanosine (ddi), a resistant phenotype can result from a single base pair change, but ddi has an even lower therapeutic index than azt, and relatively low levels of resistance can potentially render this drug useless. however, some combinations of resistant mutations may make it difficult for hiv to replicate, and resistance to one rt inhibitor may counteract resistance to another. the current strategy for therapy of hiv infection is known as haart (highly active antiretroviral therapy) and employs combinations of different drugs such as a protease inhibitor plus two nucleoside rt inhibitors. molecular mechanisms of resistance and drug interactions are both important to consider when designing combination regimes: i combinations such as azt 1 ddi or azt 1 3tc have antagonistic patterns of resistance and are effective. i combinations such as ddc 1 3tc that show cross-reactive resistance should be avoided. certain protease inhibitors affect liver function and can favorably affect the pharmacokinetics of rt inhibitors taken in combination. other potential benefits of combination antiviral therapy include lower toxicity profiles and the use of drugs that may have different tissue distributions or cell tropisms. combination therapy may also prevent or delay the development of drug resistance. combinations of drugs that can be employed include not only small synthetic molecules but also "biological response modifiers" such as interleukins and ifns. virus infection is a complex, multistage interaction between the virus and the host organism. the course and eventual outcome of any infection are the result of a balance between host and virus processes. host factors involved include exposure to different routes of virus transmission and the control of virus replication by the immune response. virus processes include the initial infection of the host, spread throughout the host, and regulation of gene expression to evade the immune response. medical intervention against virus infections includes the use of vaccines to stimulate the immune response and drugs to inhibit virus replication. molecular biology is stimulating the production of a new generation of antiviral drugs and vaccines. further reading rna-based viral immunity initiated by the dicer family of host immune receptors viral subversion of apoptotic enzymes: escape from death row understanding hiv-1 latency provides clues for the eradication of long-term reservoirs implications of high rna virus mutation rates: lethal mutagenesis and the antiviral drug ribavirin five questions about viruses and micrornas how do viruses avoid inhibition by endogenous cellular micrornas? clinical applications of dna vaccines: current progress modulation of natural killer cell activity by viruses pros and cons of phage therapy microrna in the immune system, microrna as an immune system crispr interference: rna-directed adaptive immunity in bacteria and archaea new viruses for cancer therapy: meeting clinical needs how do plant viruses induce disease? interactions and interference with host components interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures antiviral drugs for viruses other than human immunodeficiency virus plant virus ecology the prospects and challenges of universal vaccines for influenza viral subversion of the immune system viral tricks to grid-lock the type i interferon system oncolytic viruses as anticancer vaccines. front. oncol. 4, 188 key: cord-020010-q58x6xb0 authors: nan title: 19th icar abstracts: date: 2006-03-13 journal: antiviral res doi: 10.1016/j.antiviral.2006.02.001 sha: doc_id: 20010 cord_uid: q58x6xb0 nan the society was organized in 1987 as a non-profit scientific organization for the purpose of advancing and disseminating knowledge in all areas of antiviral research. to achieve this objective, the society organizes an annual meeting. the society is now in its 20 th year of existence, and has about 600 members representing 30 countries. for membership application forms or further information, please contact dr. amy patick, secretary, isar; pfizer global r&d, department of virology, 10777 science center drive, san diego, ca 92121; phone +1 858 622 3117; fax +1 858 678 8182; e-mail amy.patick@pfizer.com. membership application forms will also be available at the conference registration desk, or from our website www.isar-icar.com. enzymes of the pol gene of hiv have been identified as important viral targets for the discovery anti-hiv therapeutic agents. while the viral targets, hiv reverse transcriptase and hiv protease, have been successfully investigated for the development of clinically useful therapeutic agents, research efforts on drug discovery on the third enzyme of the pol gene, hiv integrase, have not resulted in a single fda-approved drug. nevertheless, as integrase is essential for hiv replication, it remains an attractive target for the discovery of new anti-hiv agents. in this presentation, we report the discovery of a conceptually new beta-diketo acid, constructed on a nucleobase scaffold, that is a potent inhibitor of both the 3 -processing and strand transfer steps of recombinant hiv integrase. this inhibitor and the positive control compound, azt, were tested in a pbmc cellbased, microtiter anti-hiv assay against the clinical isolate, hiv-1teki (nsi phenotype) and hiv-1nl4-3 (si phenotype), and in a magi-x4 assay against hiv-1nl4-3 with hela-cd4-ltr-beta-gal cells. our integrase inhibitor was found to have highly potent in vitro anti-hiv activity and efficacy. the discovery of this remarkably active molecule, representative of a unique set of diketo acids bearing nucleobase scaffolds, has uncovered a new chapter in the chemistry and biology of integrase inhibitors and their potential therapeutic applications. berta bosch 1 , imma clotet-codina 1 , julia blanco 1 , eduard pauls 1 , gemma coma 1 , samandhy cedeño 1 , francesc mitjans 2 , anuska llano 1 , margarita bofill 1 , bonaventura clotet 1 , jaume piulats 2 , jose este 1 1 retrovirology laboratory, fundacio irsicaixa, badalona, spain; 2 laboratorio de bioinvestigación, merck farma y química, barcelona, spain macrophages are key cells for hiv infection and spreading inside the organism. macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (m-csf). in the monocyte to macrophage differentiation process with m-csf, av-integrins are upregulated concomitantly to the capacity of hiv to generate a productive virus infection. in the present study we show that an anti-av antibody, 17e6, inhibited hiv-1 infection of primary macrophages. the effect of 17e6 on r5 or x4 hiv-1 replication in acutely infected macrophages was dose-dependent, with a 50% effective concentration (ec50) of 17 ± 2 g/ml in the absence of cytotoxicity. similarly, a monoclonal antibody targeting the avb6 integrin (14d9.f8) also inhibited hiv-1 infection in this cell type. 17e6 reduced the detection of hiv-1 bal proviral dna in acutely infected macrophages but was completely ineffective against hiv-1 bal production in chronically infected macrophages, suggesting that 17e6 inhibited hiv infection at an early stage of the virus cycle. finally, a small molecular weight antagonist of the avb6 integrin reduced hiv replication at subtoxic concentrations. therefore, our results suggest that av-containing integrins could play a role in hiv replication in macrophages and indicate that small molecular weight compounds may be developed to interfere with hiv replication in macrophages through the interaction with av integrins. andrew vaillant 1 , hong lu 2 , shuwen liu 2 , carol lackman-smith 3 , roger ptak 3 , jean-marc juteau 1 , shibo jiang 2 1 replicor inc., laval, que., canada; 2 f. lindsay kimball research institute, new york blood center, new york, ny, usa; 3 southern research institute, frederick, md, usa the sequence independent antiviral activity of phosphorothioate oligonucleotides in inhibiting hiv-1 by blocking interactions between the v3 loop and cd4 has been previously described. this activity was attributed to their polyanionic activity. here we show that ps-ons (and their fully 2 -o-methylated derivatives) are also potent inhibitors of hiv-1-mediated membrane fusion and hiv-1 replication in a sequence-independent, sizedependent (optimal size ∼50-60 bases) and phosphorothioation dependent manner (independent of stabilization). ps-ons interact with the heptad repeat regions of gp41 and the hiv-1 fusion inhibitory activity of ps-ons is closely correlated with their ability to bind to these heptad repeats and block gp41 six-helix bundle formation, a critical step during the process of hiv-1 fusion with the target cell. the requirement for ps-on interaction was also found to be dependent on phosphorothioation, suggesting that the v3 loop/ps-on interaction may also have a hydrophobic component. the increased hydrophobicity of longer (≥40 base) ps-ons may contribute to their inhibitory activity against hiv-1 fusion and entry because these longer ps-ons have a greater hydrophobicity and are more potent in blocking the hydrophobic interactions involved in the gp41 sixhelix bundle formation than shorter ps-ons (<30 bases). this novel antiviral mechanism of action of long ps-ons has important implications for therapy against infection by hiv-1 and other enveloped viruses with type i fusion proteins. chris meier 1 , soenke jessel 1 , bastian reichardt 1 , olaf ludek 1 , jan balzarini 2 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katholieke universiteit leuven, leuven, belgium carbocyclic nucleoside analogues like abacavir showed very interesting antiviral properties. therefore, we are interested in a convenient stereoselective access to this class of compounds as potential antiviral agents. by using a new convergent synthetic strategy, starting from a chiral cyclopentenol, enantiomerically pure carbocyclic thymidine (carba-dt) was obtained as a key intermediate for further variations at the 3 -position. this pathway allows an entry to d-and l-configurated nucleoside analogues. however, using this approach a mixture of side products avoids the formation of the product in very high yields. however, we will present that the side products can be recovered by a stereoselective hydroboration leading to one intermediate only that can be use as well for the synthesis of carbocyclic nucleosides. the condensation of the carbocyclic moiety and different pyrimidine and purine nucleobase was achieved by a mitsunobu reaction. various analogues have been prepared via this strategy, e.g. d-and l-carba-bvdu, nucleoside analogues known to be antivirally active against hsv-1. additionally, carbocyclic ␣nucleosides and carbocyclic iso-nucleosides are accessible by this reaction sequence. all new nucleoside analogues were tested for their antiviral activity. particularly carba-dt was found to be a potent anti-hiv active derivative showing no toxicity. however, it can not be excluded that a non-activity of a compound is related to a missing phosphorylation to the monophosphate. in order to prove that, all nucleosides were converted into their cyclosal-phosphate trimesters and transferred into the nucleotides. detailed chemistry, enzymatic and antiviral activity data will be presented. in some cases the nucleotide releasing system showed improved antiviral activity as compared to the parent nucleoside. michela pollicita 1 , candace pert 2 , maria-teresa polianova 2 , alessandro ranazzi 1 , michael ruff 2 , carlo-federico perno 1 , stefano aquaro 1 1 university of rome tor vergata, italy; 2 georgetown university, washington, dc, usa monocytes/macrophages (m/m) are a strategic reservoir of hiv-1 commonly infected by ccr5-using (r5) strains of hiv-1. ccr5 is an attractive target for inhibition of ccr5 mediated hiv-1 entry. thus, ccr5 antagonists are expected to be a power-ful new class of receptor-based therapeutic agents against hiv-1 infection. d-ala-peptide t-amide (dapta) is an octapeptide derived from the gp120 v2 region of hiv-1, able to bind ccr5. dapta acts as selective viral entry inhibitor, displacing the binding of gp120 with ccr5. dapta anti-hiv-1 activity was evaluated in m/m infected with two different hiv-1 r5 strains, bal and 81a, in presence of several doses of the compound. dapta inhibited hiv-1 replication in m/m (>80% compared to control), measured by the p24 gag ag released in the cell culture supernatants, at concentration of 10-3 nm. pcr analysis of integrated hiv-1 proviral dna on cultured m/m proved that dapta is able to block hiv entry and so, to prevent hiv infection in m/m. moreover, the capability of different hiv-1 r5 strains produced and released by infected m/m in affecting neuronal homeostasis was assessed in a neuroblastoma cell line, sk-n-sh, expressing ccr5. in sk-n-sh were evaluated cell morphology, propidium iodide binding and fluorescenceactivated cell sorting (facs) analysis. dapta, at concentration of 10-3 and 10-4 nm, strongly inhibited apoptosis in sk-n-sh of 58 and 56%, respectively, compared to control. unexpectedly, tak-779 (a nonpeptidic ccr5 antagonist with potent anti-hiv-1 activity) inhibited apoptosis only of 30% compared to control. our results suggest that the development of new anti-hiv-1 compounds, such as dapta, could be important in synergistic combination with other antiretroviral treatments in prevent both central nervous system hiv-infection and the consequent neural damage. the mechanisms of dapta inhibition may include both suppression of hiv-1 r5 strains in the brain as direct inhibition of hiv-1 replication in m/m and gp120 related damage by ccr5 binding. pradimicin a (prm-a) is an antifungal non-peptidic benzonaphtacenequinone antibiotic that specifically inhibits human immunodeficiency virus (hiv) in cell culture. it markedly suppresses a variety of different hiv-1 clades in pbmcs, in primary macrophages and several hiv-2 and siv strains in laboratory cell lines (range of 50% effective concentrations: 0.69-11 g/ml; 50% cytostatic concentration: >50 g/ml). prm-a also inhibits syncytium formation between persistently hiv-1-infected hut-78 cells and uninfected sup t1 cells. prm-a behaves as an artificial lectin that selectively binds mannose-containing glycans. consequently, biacore experiments revealed that it binds to gp120 of hiv-1/mn in the presence of ca 2+ . prm-a is endowed with a high genetic barrier with regard to drug resistance development against hiv-1. a variety of multiple mutations at n-glycosylation sites in hiv-1 gp120 are required before the virus looses marked sensitivity to the drug. there is no clustered pattern of hiv-1 gp120 glycan deletions that occur under prm-a drug pressure. the resistance spectrum and mode of action is unique among any of the existing anti-hiv drugs and warrant further (pre)clinical investigations. acknowledgement: this research was supported by the flemish "fonds voor wetenschappelijk onderzoek," the centers of excellence of the k.u. leuven (no. ef/05/15), and the european commission (empro). jan muench 1 , ludger ständker 2 , knut adermann 2 , axel schulz 2 , michael schindler 2 , raghavan chinnadurai 2 , wolf-georg forssmann 2 , frank kirchhoff 2 1 department of virology university of ulm, albert-einstein allee 11, 89081 ulm, germany; 2 ipf pharmaceuticals gmbh, feodor-lynen-strasse 31, 30625 hannover, germany a variety of components in human blood might influence hiv-1 replication in infected individuals. peptide libraries derived from hemofiltrate (hf), an aqueous blood solution, contain essentially all circulating blood peptides with a molecular mass below 30 kda, including chemokines, defensins, and cytokines. to identify the most potent natural occurring factors inhibiting hiv-1 replication, we screened a hf-derived peptide library for antiviral activities. the most active fraction contained a 20-residue peptide corresponding to a c-terminal fragment of ␣1-antitrypsin (␣1-at), a highly abundant serine proteinase inhibitor. further analysis of the corresponding chemically synthesized peptide, termed virus inhibitory peptide (virip), demonstrated that it inhibits infection by all hiv-1 variants tested, independently of their subtype and coreceptor usage. notably, virip also blocked multi-resistant hiv-1 variants and primary isolates. virip specifically inhibited hiv-1 env function, and did not affect infection by virions containing hiv-2, siv, mlv, hcv, ebola or vsv env proteins. the antiviral activity proved to be highly specific for the 20-residue virip sequence since structurally closely related peptides were inactive. we found that virip inhibits hemolysis of erythrocytes induced by the hiv-1 gp41 fusion-peptide (fp). nmr spectroscopy confirmed that virip interacts directly with synthetic gp41 fp. our observations are evidence that a naturally occurring human substance inhibits hiv-1 infection by a new mode of action, i.e. binding of the highly conserved fp. furthermore, we performed a structure-activity-relation study with more than 350 virip analogs and found that specific amino acid changes enhanced the antiviral potency of virip by up to two orders of magnitude. experiments in cell culture and animal models further demonstrated that virip exerted no cytotoxic effects. thus, virip derivatives might become a new class of hiv-1 entry inhibitors. stefano aquaro 1 , valentina svicher 1 , roberta d'arrigo 2 , ubaldo visco-comandini 2 , andrea antinori 2 , mario santoro 1 , giovanni di perri 3 , sergio lo caputo 4 , pasquale narciso 2 , carlo-federico perno 1,2 1 university of rome tor vergata, italy; 2 inmi l. spallanzani, italy; 3 university of turin, italy; 4 sm annunziata hospital, florence, italy to investigate gp41-variability and correlation with viroimmunological parameters in 54 hiv-infected patients (pts) receiving t20 added as a single active drug to a failing regimen. two hundred and ten hiv-gp41 sequences and clinical follow-up from 54 t20-treated patients were analyzed from baseline up to 48 weeks (weeks) of treatment. the association of mutations with viremia (vl)/cd4 count (c/ul) was assessed by mann-whitney test. the addition of t20 to the failing antiretroviral regimen induced at 4 weeks a significant vl decrease from 5.1 log (stable in the last 12 weeks prior t20) to 4.3 log (p = .0002) and a significant cd4 increase from 48 c/ul (decreasing in the last 12 weeks prior t20) to 106 c/ul (p = .008). while vl rebounded to 4.7-5 log at 12-48 weeks, respectively, cd4 increased to 129 c/ul at 48 weeks. t20 resistance mutations, absent at bl, occurred shortly after treatment and usually alone. v38a was the most common sign of t20 failure (27.8% of pts). the viroimmunological outcome of t20-treated pts varied according to gp41-mutations. v38a/e (38.5% of pts) was associated with a cd4 increase from bl (48 c/ul) of 4.5-fold (142 c/ul) at 24 weeks and 6.0-fold (196 c/ul) at 36 weeks (p = .004 and .02 compared without v38a/e, respectively). no significant correlation with vl was observed (from 4.9 log at bl to 4.4-4.8 at 24-36 weeks). by contrast, q40h + l45m (11.1% of pts) was associated with cd4 loss from 71 c/ul at bl to 26 c/ul at 36 weeks (p = .02), without significant changes in vl (from 5 log at bl to 5 log at 36 weeks). mutation n126k (observed in 6 pts, but never found at bl) abrogates the 4th gp41-glycosylation site and correlated with 1.7-fold cd4 increase at 24 weeks. conformational changes induced by v38a/e in the highly conserved giv motif of gp41-hr1, are tightly related with a loss of hiv-induced damage of immune system. this facilitates cd4-recovery through mechanisms that can be virus-(loss of fusion efficiency) and immune-mediated (exposure of new epitopes) not applicable to protease/rt-inhibitors, and thus important for innovative therapeutic strategies. the spread of highly pathogenic h5n1 influenza viruses in humans in asia, with high mortality rates among infected individuals is a major public health concern. in the absence of a vaccine antigenically matching the pandemic virus, antiviral drugs can play an important role. in the present study we reported the antiviral activity of neuraminidase inhibitor oseltamivir against lethal h5n1 influenza virus infection in ferrets, an appropriate animal model that closely resembles clinical signs of human influenza. inoculation of young adult ferrets with a viral dose as low as 10 2 eid 50 of a/vietnam/1203/04 (h5n1) influenza virus caused high fever (39.8-41.8 • c), weight loss (25.4% of initial), anorexia, extreme lethargy and death of animals on days 7-10 post-virus inoculation (p.i.). oral administration of oseltamivir at a dose of 5 mg/kg/day for 5 days twice daily initiated 4 h p.i. inhibited the febrile response, reduced weight changes (14.6% of initial) and, most importantly, completely protected ferrets from lethal h5n1 infection. in the treatment groups, virus replication in the upper respiratory tract of ferrets was prevented, whereas untreated animals shed virus at titers of 2.8-6.5 log 10 eid 50 /ml on days 3, 5 and 7 p.i. systemic spread of the h5n1 virus was observed in untreated ferrets: virus was detected in multiple internal organs, including the brain. treatment with oseltamivir resulted in complete inhibition of virus replication in the lungs and small intestine on day 5 p.i. in the brains of treated animals virus was detected in one of the two animals tested with >100-fold reduction of titer. sequence analysis showed no amino acid substitutions at conserved residues in na or ha1 subunit in viruses isolated from ferret's internal organs after treatment. these results suggest that oseltamivir earlier treatment can prevent h5n1 mortality in ferrets, however, further studies investigating optimal doses and treatment durations required to achieve protection against infection with highly pathogenic influenza viruses are much needed. natalia ilyushina, erich hoffmann, rachelle salomon, robert webster, elena govorkova st. jude children's research hospital, memphis, tn 38105, usa in the present study we tested in the mouse model the hypothesis that combination chemotherapy with drugs targeting dif-ferent virus proteins may lead to more potent and beneficial effects. we applied plasmid-based reverse genetics technique to generate two recombinant a/vietnam/1203/04-like (h5n1) viruses. one virus possessed asparagine at position 31 of the m2 protein that was found in the naturally circulating virus (rgvn-1203) and confers resistance to amantadine. the other recombinant virus possessed serine at that position and was sensitive to amantadine (rgvn-1203sens) . balb/c mice were administered oseltamivir (1 or 10 mg/kg/day) and amantadine (1.5 or 15 mg/kg/day) twice daily for 5 days by oral gavage; the first doses were given 24 h before inoculation with 10 mld50 of h5n1 virus. combination treatment with 10 mg/kg/day oseltamivir and 15 mg/kg/day amantadine was given on the same schedule. single-drug oseltamivir produced a dose-dependent antiviral effect against both recombinant h5n1 viruses (p < 0.01). treatment with oseltamivir at dosage of 10 mg/kg/day significantly inhibited virus replication in the lungs, brain, spleen, and blood of mice at days 3, 6, and 9 after inoculation (p < 0.05), but resulted in low survival rate (20%). single-drug amantadine showed dose-dependent effect only against rgvn-1203sens strain. notably, risk of death for mice that received 15 mg/kg/day of amantadine or 10 mg/kg/day of oseltamivir was similar (p < 0.05). in contrast, prophylactic treatment of mice with combinations of oseltamivir and amantadine completely inhibited virus replication in the animals infected with rgvn-1203sens (p < 0.05) compared to singledrug usage and protected 90% of animals. importantly combination chemotherapy completely protected h5n1 virus spread to the brain of the mice: virus was not detected in brain of treated animals on days 3, 6, and 9 after inoculation and neurological symptoms were not observed. our results suggest that combination chemotherapy provides an advantage over single-agent treatment. this strategy could be an option for the control of influenza virus infection, and combinations with other novel drugs should be explored. françois jean 1 , reid asbury 2 , meera raj 1 , david lawrence 3 , martin petric 3 1 the university of british columbia, vancouver, bc, canada v6t 1z3; 2 ge healthcare bio-sciences, piscataway, nj 08855, usa; 3 british columbia center for disease control, vancouver, bc, canada v5z 4r4 in late 2002, severe acute respiratory syndrome (sars) became the first new severe and easily transmissible human disease to emerge in the 21st century. although it abated after six months, sars serves as a modern paradigm for human emerging infections with 772 deaths reported from 29 countries. the causative agent was found to be a new sars-associated coronavirus (sars-cov) . while the sequence of sars-cov genome was first reported by the bc genome sciences center, the full set of viral and cellular proteins that compose the sars-cov virion remains unknown. to approach this problem, we have utilized two-dimensional gel electrophoresis and liquid chromatography-tandem ms (lc-ms/ms) to identify the viral and cellular proteins in purified sars-cov virions obtained from human infected cells [huh7: human liver] and primate (veroe6: monkey kidney) infected cells. interestingly, analysis of the proteins from purified sars-cov preparations has revealed that the enveloped virions contain not only the predicted viral structural proteins (e.g. spike glycoprotein, nucleocapsid protein, and membrane glycoprotein) but also an important number of differentially incorporated host cellular proteins into or onto the newly formed viruses. we have unambiguously identified over 50 host cellular proteins in sars-cov virions by lc-ms/ms. these proteins include members of the annexin superfamily, cytoskeletal proteins, chaperones, vesicular transport proteins, uracyl-dna glycosylases, and aldehyde oxidoreductases. this study provides the first comprehensive and comparative analysis of the viral and cellular proteins that compose infectious particles of sars-cov obtained from human and primate infected cells. the functions of these newly identified hostspecific proteins are currently being investigated using rna interfering systems; their contributions to structure, viral productive replication, and pathogenicity will be discussed. acknowledgement: supported by an early career ubc operating grant (f. jean) and cihr (m. petric) . dale barnard 1 , craig day 1 , robert montgomery 1 , kevin bailey 1 , matt heiner 1 , larry lauridsen 1 , robert sidwell 1 , kurt berg 2 1 institute for antiviral research, utah state university, logan, ut, usa; 2 panum inst., immi, the ifn-lab, copenhagen, denmark severe acute respiratory syndrome (sars) is a life-threatening respiratory illness caused by sars-cov. there are no approved therapies for sars. some drugs inhibit sars-cov replication in vitro including human interferons and selected antiinflammatory agents (chihrin and loufty, 2005. 3, 251-262) . interferons are very promising because of their potent in vitro inhibition of sars-cov. although anti-inflammatory agents are not very active in vitro, it is thought that they might be efficacious in reducing any deleterious inflammatory response associated with virus infections such as sars infections in humans. for example, troxerutin, a flavenoid with anti-inflammatory properties, is in clinical trials for treating rhinovirus (rv) infections, ameliorating rv-induced inflammation (turner et al., 2004. apmis 112, 605-611) . therefore, troxerutin was tested for inhibition of sars-cov replication in the lungs of infected mice using a mix of four hydroxyethylrutosides that included troxuretin. in addition, mouse interferon-alpha, used as a model compound for human interferon-alpha, was evaluated for inhi-bition of virus lung titers. both mouse interferon-alpha administered i.p. daily beginning 12 h pre virus exposure at doses of 100,000 and 10,000 iu and the hydroxyethylrutoside mix (100 and 10 mg/kg) administered i.p using the same schedule reduced virus replication in the lungs of mice to below detectable limits. when a hydroxyethylrutoside mix was given to mice in the drinking water at 1.32 mg/ml (likely equivalent to an i.p. dose of 100 mg/kg, assuming that the mice drank freely), virus lung replication was also completely inhibited. all treatments appeared to be well tolerated, since all groups of mice gained weight. we also report on the efficacy of various combinations of two doses of these drugs administered i.p., using the same dosing regimen as described. these data support the supposition that interferon might be a useful therapy for treating human sars infections and that hydroxyethylrutosides should be investigated further as a potential therapy. acknowledgement: supported by contract no. n01-ai-15435 from the virology branch, niaid, nih. treatment options for human respiratory syncytial virus (rsv) are limited. an effective vaccine is not yet available. neutralizing polyclonal antibody (respigam tm , medimmune) and a humanized monoclonal antibody (synagis tm , medimmune), are licensed for prophylactic use. ribavirin is the only approved antiviral against rsv, but its efficacy is controversial and its use is limited to treatment of high-risk patients. there is a clear need for new anti-rsv therapeutics with improved efficacy and ease-of-use. many early efforts to identify anti-rsv compounds focused on blocking the process of fusion. we have developed a cell-based screening platform to identify antivirals that inhibit rsv transcription and replication. the assay does not require infection with wild-type virus. it is based on an rsv subgenomic replication system in baby hamster kidney (bhk-21) cells that express the essential viral replication proteins (n, p, l and m2-1). the readout is expression of the reporter gene lacz from a subgenomic rna. screening of the apath small molecule library yielded 596 compounds (hit rate = 0.75%) with ec50 values ≤10 m and with selectivity index (si) values ≥10. seventy-two compounds demonstrated antiviral activity against wild-type rsv (strain a2) in a cytopathic effects inhibition assay (ec50 < 10 m; si > 10). these anti-rsv compounds represent nine different chemical classes. two compounds, a 4-aminoquinoline (ec50 = 0.25 m) and a thienopyrimidine (ec50 = 0.82 m), were shown to have desirable pharmacokinetic profiles and were chosen for efficacy testing in the cotton-tail rat model of infection. sar studies to identify the pharmacophore of the compounds have been initiated. preliminary studies to characterize the mechanism-ofaction in virological assays will be discussed. acknowledgement: supported by nih r44 ai047552-02. we have previously reported bicyclic furano pyrimidine nucleoside analogues (bcnas) as exquisitely potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for p-alkylphenyl substituted analogues such as lead compound cf1743(cf-1743) (1) . these compounds have entered preclinical development with fermavir pharmaceuticals. we now report the first chromatography-free synthesis of these agents, their scale up to multi-gramme amounts, and their pre-clinical characterisation. in addition, we were keen to address potential solubility and bioavailability issues of these highly lipophilic agents by the synthesis of more polar analogues in two categories; side-chain ethers (2) as new analogues in their own right, and 5 -phosphates (3) as potential more soluble prodrugs. we report data on both of these new families at this meeting. finally, we note the application of our phosphoramidate pro-tide approach to this family, with a series of bcna protides (4) designed as intracellular phosphate delivery forms to bypass the essential vzv thymidine kinase-mediated first phosphorylation step. graciela andrei 1 , joos van den oord 2 , pierre fiten 1 , ghislain opdenakker 1 , erik de clercq 1 , robert snoeck 1 1 rega institute for medical research, katholieke universiteit leuven, leuven, belgium; 2 pathology department, u.z. leuven, leuven, belgium varicella (chickenpox) , the primary infection caused by vzv, is characterized by viremia and skin lesions. reactivation of the latent virus results in skin lesions characteristic of herpes zoster (shingles). as keratinocytes are one of the main target cells for productive infection in vivo for vzv, human epithelial cells represent a relevant model for the study of vzv pathogenesis and evaluation of antiviral compounds. organotypic epithelial raft cultures permit full differentiation of keratinocytes via culturing of the cells on collagen matrix at the air-liquid interface. we have previously shown that the susceptibility of cultures to infection with vzv depends on the stage of differentiation of the rafts. we have now quantified the activity of reference anti-vzv compounds by measuring viral dna load by realtime pcr. quantitative pcr for vzv dna was performed by using specific primers and a mgb-probe for the orf29 gene (single-stranded dna binding protein) by the taqman method. two series of raft cultures were infected with the wild-type oka strain after 4 days of differentiation and treated with serial dilutions of the test compounds. at 12 days post-differentiation one series of the cultures was processed for histology and the other one for viral dna quantification. acyclovir (acv), penciclovir (pcv) and brivudin (bvdu) at 4 and 0.4 g/ml, foscarnet (pfa) at 40 g/ml and cidofovir (cdv) at 4, 0.4 and 0.04 g/ml inhibited viral dna content by more than 95%. these results were in agreement with histological examination of the rafts, no cytopathic effect being observed at these concentrations. as expected, only cdv and pfa inhibited the replication of the thymidine-kinase deficient (tk-) 07-1 strain. a correlation between the degree of protection as determined by histological examination and viral quantification could also be demonstrated for cdv and pfa against the tk-07-1 strain. since no animal model is available for the in vivo evaluation of antiviral agents against vzv, the organotypic cultures may be considered as a valuable ex vivo model to evaluate the efficacy of new anti-vzv antivirals. jae-seon hwang 1 , oliver kregler 1 , john c. drach 2,3 , leroy b. townsend 3 , elke bogner 1 1 institut für klinische und molekulare virologie, erlangen, germany; 2 department of biologic and materials sciences, school of dentistry; 3 interdepartmental graduate program in medicinal chemistry, college of pharmacy, university of michigan, ann arbor, mi, usa dna packaging is the key step in viral maturation and involves binding and cleavage of viral dna containing specific dnapackaging motifs. this process is mediated by a group of specific enzymes called terminases. we have previously demonstrated that the hcmv terminase is composed of two subunits, the large one encoding pul56 and the small pul89, where each protein has a different function. while the large subunit mediates sequence specific dna binding and atp hydrolysis, pul89 is only required for duplex nicking. inhibitors targeting pul56 and/or pul89 are attractive alternatives as hcmv antivirals since mammalian cell dna replication does not involve cleavage of concatameric dna. we now have screened several members of the benzimidazole ribonucleoside class of replication inhibitors in order to determine if a compound has the capacity to block the atpase activity of the large terminase subunit pul56. analysis by bioluminometric atpase activity assays identified bdcrb and one more compound [2-bromo-4,5,6-trichloro-1-(2,3,5-tri-o-acetyl-␤-dribofuranosyl)benzimidazole (btcrb)] with inhibitory effects. although only btcrb and bdcrb were inhibitors of the atpase activity, two other compounds, dbdcrb and cl4rb, inhibited virus replication in a plaque-reduction assay, thus indicating that those have a different mode of action. in addition, by electron microscopy of thin sections we observed that in the presence of btcrb only b-capsids and dense bodies were formed. furthermore, spherical capsids accumulated in the perinuclear cisternae indicating a block in nuclear egress thereby providing additional evidence that closely-related benzimidazole d-ribonucleosides may have differences in their antiviral modes of action. human cytomegalovirus (hcmv) is the cause of significant morbidity and mortality in a variety of immunocompromised patients. currently available anti-hcmv drugs interfere with dna replication; however, these drugs are highly toxic, pre-cluding their long-term use in humans. interrupting hcmv viral entry is largely unexplored as an antiviral drug development strategy and is potentially an ideal and tractable goal. hcmv is believed to rely upon formation of ␣-helical coiled coils in the viral glycoproteins gb and gh to promote virus-host membrane fusion; peptides encompassing heptad repeat sequences in these two proteins inhibit viral infection. we have explored nonnatural oligomeric molecules ("foldamers") that are designed to mimic elements of the putative ␣-helical segment of gb. this effort has led to the discovery of oligomers of ␤-amino acids ("␤-peptides") that block hcmv infection. the ␤-peptide scaffold offers several advantages for the design of protein-protein interaction inhibitors, as ␤-peptides are amenable to modular synthesis, resist proteolytic degradation, and can display large and tailored molecular surfaces. the most potent ␤-peptide inhibitor blocks hcmv infection with a micromolar ic 50 in a cell-based assay. these compounds show specificity for hcmv relative to closely related viruses. mechanistic studies suggest that these inhibitors interfere with membrane fusion between hcmv particles and host cells. current efforts are focused on understanding in greater detail the origin of the observed biological activity, exploring other foldamer scaffolds as bases for inhibitor design, and developing specific fusion inhibitors for other herpesviruses. previous reports have indicated that herpes simplex virus (hsv) activates nuclear factor-kappab (nf-kb) during productive infections. nonsteroidal anti-inflammatory drugs (nsaids) have significant inhibitory effects on nf-kb. therefore, two nsaids, indomethacin and aspirin, were assayed for antiherpetic effects and utilized as tools to further study the role of nf-kb in hsv-1 infection. we report that indomethacin and aspirin inhibited hsv-1 replication at non-cytotoxic doses. in vero cells, 500 um indomethacin and 20 mm aspirin reduced hsv-1 titers 99.999 and 99.5%, respectively. electromobility shift assays revealed that hsv-1 activation of nf-kb is inhibited by the nsaids at doses that coincide with reduction of hsv-1 titers. to investigate a pathway for nf-kb inactivation, protein levels of ikb-alpha, a cytoplasmic nf-kb inhibitor, were examined. ikb-alpha protein was present in uninfected samples, but decreased over time in all hsv samples, regardless of chemical treatment, suggesting localization of nf-kb to the nucleus. immunohistochemistry studies verified that p65, a component of the dimeric nf-kb complex, translocated to the nucleus of hsv-1 infected cells in the presence or absence of the nsaids. finally, direct effects on viral gene activity were assayed by real-time rt-pcr analysis. indomethacin and aspirin reduced mrna for icp4, an essential hsv immediate-early gene, 2.9and 2.5-fold, respectively, resulting in significant decreases of icp4 protein. but transcriptional analysis revealed that synthesis of mrna for thymidine kinase, an hsv early gene, was unaffected by chemical treatment. however, mrna for glycoprotein c, an hsv late gene was undetectable in indomethacin and aspirin treated samples. cumulatively, these data indicate that: (i) indomethacin and aspirin block hsv-1 replication and (ii) the in vitro anti-herpetic effects of nsaids may reside in their ability to block nf-kb activity within the nucleus, impairing activation of essentials hsv genes. increasing species-specificity constraints preclude study of human cytomegalovirus (hcmv) in animals, necessitating the use of rodent cmvs to model human disease. however, the susceptibility of animal cmvs to clinically useful antivirals is unpredictable. for example, the guinea pig cmv (gpcmv), a uniquely valuable virus for modeling congenital cmv infection, is highly resistant to ganciclovir (gcv) at medically relevant doses. we used a molecular virological approach to test the hypothesis that gcv susceptibility could be conferred on gpcmv by insertion of the human ul97 phosphotransferase gene into the gpcmv genome. the gpcmv genome, cloned as a bacterial artificial chromosome in e. coli, was modified by site-specific recombination, using a shuttle plasmid targeting the gp97 locus, and carrying the ul97 gene from hcmv strain towne. the resultant chimeric virus was replication competent, and was found to contain the hcmv ul97 by southern-blot and sequence analyses. northern-blot revealed that a hcmv ul97-specific transcript was expressed with late gene kinetics. western-blot, using a hcmv ul97-specific polyclonal antibody, detected protein in virus-infected cells. the chimeric virus was gcv-susceptible, compared to wild-type gpcmv, with an ic 50 of 15 m. chimeric virus also exhibited increased sensitivity to maribavir (mbv), exhibiting a 3-log reduction (compared to wild-type virus) in the presence of 50 m mbv, and an ic 50 of 5 m. to study the in vivo pathogenesis of chimeric virus, cyclophosphamide-immunocompromised strain two guinea pigs were challenged intraperitoneally, resulting in evidence of disseminated infection, and mortality. ganciclovir treatment (25 mg/kg/day) resulted in reduced weight loss, and mortality, compared to placebo. these studies confirm the key role of ul97 in cmv antiviral therapy, and demonstrate that a 'humanized' gpcmv can be generated with altered antiviral susceptibilities. genital herpes infections are a global health problem and impact hiv/aids epidemic. strategies to prevent transmission include treatment of infected subjects to suppress shedding and prophylaxis with vaginally-applied microbicides. we examined the in vitro and vivo activity of rep 9, a fully degenerate 40 mer phosphorothioated oligonucleotide against hsv-2 infection of human cervical cells and in a vaginal murine model. rep 9 has broad-spectrum anti-herpetic activity with potent in vitro activity against hsv-1, hsv-2, hcmv, vzv, ebv, and hsv-6 (vaillant et al., submitted for publication). at a concentration of 100 m, rep 9 inhibited 6-logs of hsv-2 infection if present during the entire experiment. synchronized infectivity assays demonstrate that, unlike sulfonated polyanions in clinical trials, which primarily block hsv attachment, rep 9 acts at multiple steps and inhibits binding, entry and post-entry gene expression. in our in vivo studies, mice were treated once intravaginally with rep 9 or pbs control at various times prior to vaginal challenge with a lethal dose of hsv-2 strain 186 (10 log 4 pfu). rep 9 prophylaxis provided protection to mice from hsv-2 infection and disease. protection was significant when challenged 30 min after treatment (p < 0.01). additionally, treatment with an analog of rep 9, which cannot activate tlr-9 mediated immune stimulation, was at least as active as rep 9, suggesting that direct antiviral activity and not stimulation of innate immunity is the mechanism of action in vivo. utilizing this analog, protection was significant when challenged 30 min after treatment (p < 0.001) with a trend toward protection when administered 60 min prior to challenge (p = 0.07). in summary, treatment with the rep 9 analog which has superior resistance to low ph and nuclease degradation was more effective than rep 9, in some experiments protecting 100% of mice from viral infection and disease. the testing of this ph resistant rep 9 analog in a gel formulation is currently underway. acknowledgement: supported by contract no1-ai-15438 from the virology branch, niaid, nih. a phosphorodiamidate morpholino oligomer (pmo) designed to hybridize to a highly conserved region including the aug translation start site of hcv, called avi-4065, has been evaluated for efficacy, toxicity, and pharmacokinetic properties. avi-4065 inhibits translation initiated at the aug start site with ec50 of 308 nm (2.1 ug/ml) and shows positive cooperativity. this pmo retains most of the activity in the presence of point mutations in the hcv genome. huh-7 cells were incubated with normal human serum (nhs) or hcv infected human serum (is) and hcv replication observed by rt-pcr. avi-4065 produced robust inhibition of hcv in a dose and sequence-specific manner. studies conducted in vivo with avi-4065 in the hcv infected trimera mouse (xtl) show reduction in viral titer which is dose dependent with approximately 90% of mice with undetectable viral titer and the remaining mice show 1 log reduction in viral titer with 0.1 mg/mouse/day for 7 consecutive days. the fractional bioavailability of avi-4065 from a sq dose is approximately 1. the apparent elimination half life in rat, nonhuman primate and humans was 2.3, 4.5 and 11.4 h, respectively. the volume of distribution ranged from 0.6 to 1.0 l/kg and the cmax is linearly related to the dose in mg/m 2 . a phase i study in healthy volunteers in which 14 daily sq doses of 50 and 100 mg has been completed. no serious adverse events have been observed. treatment of infected patients is currently planned. inhibition of hcv polyprotein synthesis is anticipated to contain therapeutic benefis of both protease inhibitors and polymerase inhibition. hcv infection can progress to fibrosis, reduced liver function, hepatocellular carcinoma, and death. currently, the standard treatment for hcv infection involves treatment with pegylated interferon in combination with the nucleoside analogue ribavirin. this treatment regimen effects a cure in approximately 40-60% of the genotype-1 (gt-1) population; therefore a significant unmet clinical need exists in hcv therapy. virus-encoded polymerases have proven to be excellent molecular targets for chemotherapeutic intervention in numerous viral mediated diseases. in the case of hiv, hbv and herpes virus infections, deoxy-nucleoside analogues, which act as chain terminating agents, have been shown to have invaluable clinical utility. by analogy, appropriate ribonucleoside analogues might be expected to inhibit the essential rna polymerase (ns5b) encoded by hcv. here we describe the preparation of nucleoside analogues as inhibitors of the hcv polymerase. in our design of nucleoside analogs as potential anti-hcv agents, we chose to investigate the effect of 4 -substituted ribonucleoside derivatives. we reasoned that after incorporation of a ribonucleoside containing a 4 -substituent, a disruption in elongation of the growing rna could be effected through either steric hindrance or via a conformational change of the carbohydrate moiety. our investigations on several such analogues will be presented. of particular interest is 4 -azido-cytidine, which shows good activity in the genotype 1b sub-genomic replicon (ic50 = 1.28 m) with no measurable cytotoxic or cytostatic behavior. in addition, we have shown that the triphosphate of 4 -azido-cytidine is a potent and highly selective inhibitor of ns5b (ic50 = 0.32 m). joanna e. boerner, sue ma, choilai tiongyip, michael p. cooreman, teresa compton, kai lin novartis institutes for biomedical research, 100 technology square, cambridge, ma 02139, usa current drug discovery efforts for hepatitis c virus (hcv) focus on developing specific inhibitors of two viral enzymes, ns5b polymerase and ns3-4a protease. however, resistant viral mutants are likely to emerge during therapy, compromising the effectiveness of these inhibitors. an alternative and complementary strategy is to target host factors that are also essential for viral replication. cyclophilins, a family of peptidyl-prolyl isomerases and the cellular targets of cyclosporin a (csa), present such an opportunity. it was reported recently that cyclophilin b bound to hcv ns5b polymerase and stimulated its rnabinding activity, and that these functions were blocked in the presence of csa (watashi k. et al., molecular cell 2005) . nim811, a csa derivative, is a more suitable candidate for hcv therapy because it binds to cyclophilins with higher affinity than csa while lacking the immunosuppressive activity associated with csa. using the hcv replicon system we demonstrated that nim811 exhibited potent anti-hcv activities in vitro. moreover, the combination of nim811 with a specific non-nucleoside inhibitor of hcv polymerase led to synergistic antiviral effects with no significant increase of cytotoxicity. resistant clones against both inhibitors were obtained in vitro, however, it was much more difficult to generate resistance against nim811 than the polymerase inhibitor. also, there was no cross-resistance between the two inhibitors. finally, addition of nim811 to the hcv polymerase inhibitor drastically reduced the emergence of resistance compared to polymerase inhibitor alone. taken together, nim811, with a novel mechanism of action and a favorable pharmacokinetics and safety profile, represents a promising clinical candidate for treating hepatitis c and provides a rationale for specific combination therapy. the nucleoside analog r1479 was identified as a specific inhibitor of hcv replication in subgenomic hcv replicon cells. r1479-tp is a competitive inhibitor of cmp incorporation by hcv polymerase ns5b. in a transient replicon system r1479 inhibited hcv rna replication driven by genotype 1b polymerase with similar potency as compared to that driven by genotype 1a polymerase. r1479-tp inhibited native hcv replicase and recombinant ns5b from genotype 1a and 1b with similar potency. in contrast, r1479-tp did not inhibit human dna polymerases alpha, beta or gamma, including reverse transcriptase activities of dna polymerases beta and gamma, which were highly sensitive to inhibition by azt-tp and 3tc-tp. no significant inhibition was observed with human rna polymerases i, ii and iii derived from hela cells. in addition, the functionally related native influenza virus rna dependent rna polymerase (rdrp) activity in vitro was not inhibited by r1479-tp at concentrations up to 1 mm, suggesting high selectivity for the hcv rdrp. thus, r1479 was identified as a potent and highly selective inhibitor of hcv polymerase mediated rna synthesis. guangxiang luo, zhaohui cai, chen zhang, kyung-soo chang, jieyun jiang microbiology, immunology, and molecular genetics, university of kentucky college of medicine, lexington, ky, usa the study of hepatitis c virus (hcv) replication and the search for specific antiviral agents against hcv infection have been hampered by the lack of an efficient stable cell culture system of hcv infection and propagation. we have successfully constructed stable human hepatoma cell lines that contain a chromosomally integrated-genotype 2a hcv cdna and constitutively produce and secrete high titers of infectious virus into the culture media. transcriptional expression of the full-length hcv rna genome is under the control of a cellular pol ii polymerase promoter at the 5 end and a hepatitis delta virus ribozyme at the 3 end. the resulting hcv rna was expressed and replicated efficiently, as shown by the presence of high levels of hcv proteins as well as hcv rna in the stable huh7 cell lines. hcv secreted from the stable cell lines was infectious, as determined by antibody neutralization, blockage of putative hcv receptors, and inhibition of hcv replication by interferon. our findings demonstrate the establishment of a stable cell culture system of infectious hcv production and propagation, which allows the study of the entire hcv infectious cycle. the stable hcv-secreting cell lines are now being pursued to develop high throughput screens for effective hcv inhibitors. additionally, we established a novel and powerful hcv replication system in the mouse hepatocyte and mouse embryo fibroblasts (mef). hcv rna was found to replicate efficiently in both pkr +/+ and pkr −/− mef cells, demonstrating that hcv rna replication in mef cells is a powerful system to study host-virus interaction by using diverse gene-knockout animals. interestingly, hcv rna replicates more efficiently in the pkr −/− cell than in the pkr +/+ cell, suggesting a role of pkr in the control of hcv rna replication. however, ifn inhibited hcv rna replication in the pkr −/− cell with an efficacy similar to that in the pkr +/+ cell, suggesting a pkr-independent antiviral mechanism. clearly both pkr-dependent and pkrindependent antiviral mechanisms are important for the control of hcv replication and the mediation of the ifn-induced anti-hcv response. our studies set a stage for the development of transgenic mouse models of hcv replication and open up new avenues to study hcv and host interactions in mefs derived from diverse gene-knockout animals. andrea cuconati 1 , haitao guo 2 , gael westby 1 , anand mehta 2 , timothy block 1,2 1 institute for hepatitis and virus research; 2 drexel institute for biotechnology and virology research, doylestown, pa, usa the high levels of hepatitis b surface antigen (hbsag)-bearing non-infectious particles in the serum of infected individuals is thought to play a role in suppressing hepatitis b virus (hbv)specific immune response by titering out hbv-specific antibodies and lymphocytes. current hbv therapeutics do not directly reduce this viral antigenemia. our group has focused on the enhancement of the immune response through the inhibition of viral antigen secretion in the infected hepatocytes, with the therapeutic goal being the use of hbv vaccination for the treatment of acute and chronic infection. the high-throughput screening of a small molecule library of 80,288 drug-like compounds was undertaken to discover novel inhibitors of hbsag secretion. using the stably hbv-transfected, human hepatoma cell line hepg2.2.15, we developed an hts-compatible elisa protocol for the detection of hbsag secreted in the culture media. the screen resulted in 1758 initially positive hits, a hit rate of 2.2%. subsequent retesting for activity and toxicity by mtt assay has narrowed the number of confirmed, non-toxic hits to 77, currently categorized in twelve chemical series. we have previously reported on a trio of related pyrazolo-pyridines with ec50 measurements below 5.0 m and cc50 measurements >50.0 m. nascent structure-activity relationship (sar) suggests that a central moiety of the molecules is essential to activity, with an aromatic side group contributing to potency. among recently confirmed inhibitors, two currently under investigation include: (1) an isobutyl-acetamide with an ec50 of 87.0 nanomolar, and a cc50 of >50 m, and (2) a carbothiamide with an ec50 of 1.6 micromolar and a cc50 of >50 m. measurement of secreted hbv l and m antigens and cellular markers indicated that the pyrazolo-pyridines are not specific inhibitors of viral antigens, while the isobutyl-acetamide and the carbothiamide are indeed specific. measurement of intracellular viral dna indicated that none of these molecules are inhibitors of replication. we will be reporting on our studies of the potency, specificity, and potential mechanisms of action of these novel anti-hbv compounds. background: entecavir (etv) is a potent competitive inhibitor of hepatitis b virus (hbv) polymerase with activity versus all three enzymatic functions including priming, minus, and plus strand dna synthesis. virologic rebound due to etv resistance (etvr) has only been observed in lamivudine resistant (lvdr) hbv (m204v/i ± l180m), and requires at least one additional change in the reverse transcriptase domain (rt) at residues t184, s202, or m250. these substitutions surround the dntp binding site or primer grip of rt. the objectives of this work were to further characterize etvr and its mechanism(s) using cell culture, in vitro enzyme, and molecular modeling studies. methods: hbv cell culture assays used transfected hepg2 cells and quantitation of released, immunocaptured hbv nucleocapsids. gradient-purified intracellular nucleocapsids were used for in vitro rt assays. a 3d homology model based on the hiv-1 rt structure was used to model resistance changes in hbv. results: reduced etv susceptibility of etvr hbv was observed both in culture and enzymatically in vitro. kinetic studies showed various etvr substitutions in lvdr hbv selectively reduced etv-triphosphate (etv-tp) binding (k i ) to rt without markedly changing the affinity for dgtp (k m ) or inhibition by ddgtp. etvr rts also displayed reduced enzymatic activity (k cat ) relative to wildtype and etvr hbv appeared growth impaired. modeling studies suggested a novel etv-tp binding pocket in hbv rt that became constrained with etvr changes. m250 changes in the primer grip region of rt were unique in that resistance was primarily seen during synthesis of minus strand dna. etvr changes in the absence of lvdr substitutions had greatly reduced impacts on etv susceptibility, confirming models suggesting etvr is imparted through lvdr changes. summary: etv provides a high genetic barrier to resistance, requiring additional changes at residues t184, s202 or m250 along with pre-existing lvdr substitutions m204v/i ± l180m. kinetic parameters and molecular modeling indicated that etvr substitutions selectively affected etv-tp binding and reduced the replication capacity of hbv. a nonhuman primate (nhp) model of classical, lesional smallpox has been used to test the efficacy of intravenous (iv) cidofovir treatment. cynomolgus macaques were infected with a high dose (10 8 pfu iv) of variola to produce an artificial primary viremia, and then treated with cidofovir at 0, 24, or 48 h postinfection (pi). later treatment times were not evaluated. treatment at 24 or 48 h pi halted increases in peak blood viral genome titers measured by quantitative taqman-mgb real-time pcr, which were more than 10-fold less in cdv-treated animals compared to placebo. historically, the number of pox lesions provided the best correlation with human smallpox clinical severity, and cdv treatment in our model significantly reduced maximum pox lesion counts by >90%; the number and size of skin lesions, and in untreated animals contributed significantly to the total viral burden with lesions containing 10 9 -10 10 genomes/g. to better understand the role of viral burden and disease progression in major organ systems, a serial sample study was undertaken. in untreated animals at 24 h pi, viral replication in spleen exceeded 10 9 genomes/g while liver and bone marrow yielded 10 8 genomes/ml. in comparison, titers in other tissues ranged between 10 5 and 10 6 genomes/g and blood yielded 10 4 genomes/ml at 24 h, suggesting that the liver, spleen, and marrow may be initial sites of replication. levels of virus in the bone marrow reached a peak of approximately 10 10 genomes/g at day 5, then decreased to quantities consistent with those in blood. viral load in the blood increased with time, peaking around days 7-9 at 10 8 genomes/ml. virus was also detected in intestine, skeletal muscle, and late in infection, testes. the ability to successfully treat with cdv 24 h pi despite early extensive organ infection in the accelerated nhp variola model suggests that this treatment could be effective in reducing viremia and mortality after onset of symptoms in human smallpox, which demonstrates a more protracted disease course. work involving variola virus conducted in who-sanctioned cdc, atlanta bsl-4 laboratory. earl kern 1 , kathy keith 2 , robert jordan 2 , dennis hruby 2 , debra quenelle 2 1 department of pediatrics, university of alabama school of medicine, birmingham, al, usa; 2 siga technologies, inc., corvallis, or, usa although cidofovir (cdv) has been approved as an investigational new drug for emergency treatment of smallpox, its lack of oral activity and dose limiting toxicity dictates a need for continued development of better therapeutic agents for this potential bioterror disease. it has been reported previously that st-246, a low-molecular weight compound, inhibits replication of all the orthopoxviruses in vitro and protects mice infected with vaccinia or ectomelia virus. in the present study, we have utilized cowpox virus (cv) and vaccinia virus (vv) infections in vitro and in vivo to evaluate the efficacy of st-246 for treatment of orthopoxvirus infections. in plaque reduction assays in human foreskin fibroblast cells, both cv and vv were inhibited by about 0.1-0.5 um of st-246. for in vivo studies, st-246 was administered once daily by oral gavage to mice using 100 mg/kg for 5, 7, 10, or 12 days beginning 4 or 24 h after intranasal inoculation with vv or cv. st-246 was highly effective (p < 0.001) in preventing mortality due to vv or cv even when treatment was delayed up to 24 h post-infection. a dosing duration of 5 days was adequate for vv infected mice, but duration of 7 days or longer was required for efficacy in cv infected mice. when st-246 was given once daily for 14 days at 100, 30, or 10 mg/kg daily at 24, 48, or 72 h post-cv inoculation, mortality was significantly altered at all dosage levels and time points. to determine the effect of treatment on virus replication in target tissues, mice were inoculated with cv or vv and treated once daily with 50 mg/kg of st-246. on various days post-infection tissues were harvested and assayed for virus. in cv or vv-infected mice, st-246 treatment successfully reduced virus titers from 3 to 5 logs 10 in liver, spleen, and kidney. little effect was noted in lung tissue. these results indicate that st-246 has significant activity against vv and cv infections in vitro and in vivo and may be a potential chemotherapeutic agent for treatment of human orthopoxvirus infections. cidofovir (hpmpc) is a broad-spectrum anti-viral agent that is used (vistide ® ) to treat aids-related cmv retinitis. currently, cidofovir is of particular interest as a potential therapy for orthopox virus infections, including smallpox. an important limitation of cidofovir and analogous nucleotide drugs in a therapeutic role is their low oral bioavailability and poor transport into cells. in principle, bioavailability of a drug can be improved by structural modification targeting transporters expressed in human intestine. to be effective, the transported prodrug must be cleaved by endogenous enzymes to its parent compound. we will present synthetic studies of novel cidofovir and cyclic cidofovir (chpmpc) prodrugs incorporating amino acids or small peptides, comparing different drug-amino acid linkage strategies. the compounds were evaluated for transporter-mediated uptake and cellular and plasma hydrolysis. the results will be compared with similar studies carried out on a series of peptidomimetic conjugates of foscarnet, the trisodium salt of phosphonoformic acid (pfa), an anti-viral agent that also has very low oral bioavailability and poor cell penetration. the question addressed in this study is if wnv-reactive antibody can improve disease signs in a hamster model after the virus is demonstrated to be in the brain. the hypothesis is based on the high activity of a humanized monoclonal antibody, he16, in a mouse model when administered later in infection (oliphant et al., 2005. nat. med. 11, 522) . in this study, virus was demonstrated to be in the brains of hamsters at 5 days post-viral injection (dpi) by cell culture assay, quantitative rt-pcr, and immunohistochemical staining of wnv in neurons. eighty percent of hamsters treated i.p. 5 dpi with 100 mg/kg of humanized monoclonal antibody, he16, survived wnv disease, whereas, 37% of placebo-treated hamsters survived ( *** p < 0.001). if administered at 2 dpi, 100% survived. we tested the hypothesis that he16 is effective if delivered directly into the brain instead of by peripheral administration. the antibody was delivered into the brain 5 dpi using convectionenhanced delivery through a cannula implanted into the brain. the he16 was detected in the cns, but none was detected in the kidney. the survival of he16-treated hamsters was 88% as compared to 22% of placebo-treated animals ( *** p < 0.001). for additional proof, the majority of hamsters having wnv in their cerebrospinal fluid, a marker for cns infection, were protected with he16 administered i.p. at 5 dpi. this humanized monoclonal antibody, therefore, is a possible treatment for the post-exposure, wnv-infected humans that develop signs of neuroinvasive disease. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih, and grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. hemorrhagic fever viruses are of serious worldwide health concern as well as potential biological weapons. lassa fever virus in particular annually infects several hundred thousand individuals in west africa, and the export of this pathogen outside of this region, either intentionally or unintentionally, presents a serious risk to the developed countries of the world. the cdc and niaid have identified lassa fever virus as a category a priority pathogen, indicating the highest degree of threat to public health. no arenavirus-specific antiviral drugs are currently approved for use in humans. the purpose of siga's biodefense program is to develop safe and effective drugs for preventing and treating diseases caused by category a viruses. to that end, a large and diverse library of small molecule compounds was screened using a viral pseudotype assay to identify inhibitors that target the essential lassa surface glycoprotein (gp) and thus block viral entry into the host cell. twenty-six compounds were identified as quality hits, as defined by potency, selectivity, and chemical tractability. antiviral activity against authentic lassa fever virus was assessed in cell culture through a collaboration with colleagues at usamriid. a number of these potent antiviral compounds and their related analogs have exhibited informative chemical structure-biological activity relationships (sar). two potential lead compound series have emerged from these studies, each with 50% effective concentrations (ec50s) of less than 100 nm against lassa fever virus and with ec50s of less than 2 nm against lassa gp-pseudotyped virus. characterization of the in vivo properties of these compounds is underway. the in vitro antiviral potency and selectivity, animal pharmacokinetics, and the development process will be presented. these inhibitors represent an important step toward the development of a small molecule antiviral drug for lassa fever virus. sven enterlein 1 , pramila walpita 1 , allison groseth 2 , heinz feldmann 2 , ramon flick 1 1 university of texas medical branch, department of pathology, galveston, tx, usa; 2 national microbiology laboratory, public health agency of canada, winnipeg, man., canada nipah (niv) virus (family paramyxoviridae) is a recently emerged human and animal pathogen that can cause severe encephalitis with fatality rates of up to 70%. since no treatment or vaccination is available, and cross-species spread was observed, the virus has been classified as biosafety level 4 (bsl-4) agent. to avoid bsl-4 containment for the study of cis-acting signals as target for antiviral strategies, we used an optimized plasmid-driven t7 minigenome rescue system (without the need for recombinant vaccinia virus mva-t7) as well as an newly established rna polymerase i-based approach. minigenome rescue is based on transfection of the minigenome niv-cat and the plasmids encoding for the three nucleocapsid proteins n (nucleoprotein), l (polymerase), and p (phosphoprotein) and measured by enzymatic cat assays. we used the established plasmid-based minigenome rescue systems to screen for potential antiviral compounds. in a first step we tried to determine the optimal strategy for the delivery of small hairpin (sh) interfering rna molecules. for this we compared three shrna delivery systems against another bsl4 agent-reston ebolavirus (family filoviridae); (i) plasmid-mediated pol i and (ii) pol iii-driven shrnas, and (iii) exogenously (t7) produced shrna, for their ability to induce gene silencing. interestingly, beside the in vitro-generated or pol iii-driven shrnas, pol i transcripts showed very efficient inhibition of minigenome rescue. however, the most efficient delivery method was transfection of in vitro transcribed shrnas. we will present the results of this comparison and, based on the most efficient approach, also first results of shrnas targeted either to niv n, p, and l genes or to the leader/trailer noncoding regions to interfere with minigenome replication. conformative data with live virus experiments under bsl4 conditions will be included. filoviruses, which include ebola virus and marburg virus, are among the most notorious human pathogens because they cause sporadic outbreaks of severe hemorrhagic fever. unfortunately, very few therapeutic agents are available to treat infections with these viruses. antiviral screening methods that determine the effect of compounds on viral replication involve working with infectious virus, which is obviously not practical for these biosafety level 4 (bsl-4) agents. we developed an antiviral screening method based on a cell-based, infection-independent, ebola subgenomic replication system in which the expression of an easily measurable enzyme is dependent on the rna replication and transcription factors of ebola virus. using this system we screened a synthetic compound library for antiviral activity against ebola virus and have identified a number of inhibitors. we also used it to identify a peptide inhibitor directed against vp30. anti-ebola virus activity for many of the inhibitors was confirmed in a viral replication assay using a gfp-expressing zaire '76 strain of ebola virus. fifty-two small molecule inhibitors from at least six classes of compounds had ec50 values in the low micromolar range and good selectivity. several of these compounds have promising chemical, biological, and pharmacological profiles to pursue as potential anti-filovirus drugs. we are currently preparing to test these compounds in a mouse model of ebola virus. we have also begun a lead optimization program to improve antiviral potency and selectivity of aryl sulfonamide and 4-aminoquinoline compounds. acknowledgement: supported by nih r44 ai052917-02 and r01 ai066502-01. human papillomavirus (hpv) has been a difficult virus to target by traditional antiviral methods due to its small size, its small number of obvious therapeutic targets, and its resistance to propagation in vitro. nevertheless, antiviral compounds that reduce hpv dna load have the potential to prevent carcino-genic progression in infected patients. to that end, we developed an approach that dramatically reduces the hpv episomal dna load of keratinocytes in vitro by targeting viral dna sequences. pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv31 ori. all fluorescent compounds rapidly localized to the nucleus of cultured keratinocytes following addition to the culture media. the compounds were then tested for their ability to alter keratinocyte hpv31 episomal dna content. two of the 19 compounds caused a dose-dependent reduction in hpv31 episomes as measured by taqman tm realtime pcr. while control and vehicle-treated cells maintained ∼1000 copies of hpv31 per cell, compounds 2-ta and 4-ta both reduced hpv31 dna levels to below 50 copies per cell after 48 h incubation with 10 m compound. an alternative taqman tm amplicon within the hpv31 e7 gene produced identical results. a multiplexed taqman tm real-time pcr reaction that followed the ratio of hpv31 dna to the human apoe gene also demonstrated dramatic loss of hpv31 dna copies, further confirming our initial observations. finally, cells were treated with polyamides for 48 h, polyamide-containing media was removed, and episome levels were followed for 8 days. at day 6, 4 days after removal of polyamide and 3 days after sub-culturing of the cells, viral episome levels remained approximately 60% lower than control samples. by day 8, 6 days after removal of polyamide, viral dna levels were beginning to recover but still remained significantly lower than control samples. together our results demonstrate that targeting the hpv origin of replication with dna-binding compounds dramatically reduces episomal dna levels. small interfering rnas (sirnas) are potent tools for gene down-regulation but are minimally stable in cells. to improve the efficacy of sirna, we replaced non-bridging oxygens in the phosphodiester linkages of natural rnas with bh3 groups. the resulting boranophosphates have unique properties, including enhanced nuclease resistance, altered hydrogen bonding of the phosphate, different interactions with metal ions, and increased thermal stability of rna:rna and rna:dna duplexes. anti-egfp sirnas containing boranophosphate modifications were prepared by in vitro transcription with t7 rna polymerase from ribonucleoside 5 -(alpha-p-borano)triphosphates, as well as normal and phosphorothioate sirnas. after confirming the presence of the borane modifications with maldi-ms, several properties of borano-modified sirnas were investigated: (1) the double stranded rna with borane modifications maintained the a-form conformation characteristics according to the circular dichroism (cd) spectra; (2) the borane groups in the sirnas increased the thermal stability, with an enhancement of t m by 0.5-0.8 • c per modification; and (3) sirnas with borano-modifications were shown to be at least 10-fold more resistant to rnase a digestion than normal ones. when these modified sirnas were used to down-regulate egfp expression in hela cell cultures, it was found that: (1) borano-modified sirnas were consistently more effective than sirnas containing the corresponding phosphorothioate modifications; (2) borano-sirnas were more effective than normal sirnas provided that the center of the antisense strand was not heavily modified; (3) borano-sirnas were more potent than normal or phosphorothioate sirnas at lower concentrations; and (4) finally, the silencing activity of boranophosphate singlestranded sirna (ss-rna) was comparable to that of unmodified ds-sirna. the borano ss-rna had excellent maximum silencing activity and was highly effective at low concentrations, and silencing activity was durable up to one week after transfection. results with anti-hpv sirnas will be discussed. boranophosphate modification is a potential new class of anti-viral therapeutic agents. this report describes the antiviral structure activity relationships that led to the discovery of phosphonomethoxy-2 -fluoro-2 ,3dideoxydidehydroadenosine (fd4ap, gs9148), a novel ntrti, with an excellent resistance profile toward hiv-1 variants containing major n(t)rti resistance mutations. methods: phosphonomethoxy analogs on purine and pyrimidine dideoxydidehydro (d4) and dideoxy (dd) ribose scaffolds were prepared. antiviral activity was measured against wildtype and n(t)rti-resistant recombinant viruses using cytopathic assay in mt-2 cells. mitochondrial toxicity was assessed in hepg2 cells by measuring mitochondrial dna content. results: the d4 scaffolds displayed superior antiviral activity compared to the dd scaffold and adenine was superior to other nucleobases. phosphonomethoxy-2 ,3dideoxydidehydroadenosine (d4ap) inhibited hiv-1 replication with a mean ec50 of 2.1 m and an 0.8-, 2.9-, and 2.9-fold change in potency against viruses containing m184v, k65r, and 6 thymidine analog mutations (tams), respectively. further exploration of d4ap was limited by its mitochondrial toxicity, which was then addressed in 2 ways: (i) preparation of l-d4ap or (ii) 2 fluorine substitution. l-d4ap exhibited an ec50 of 5.9 m but had substantially reduced potency (14-fold) toward m184v mutant viruses. fd4ap exhibited an ec50 of 12.3 m, with 0.8-, 1.2-, and 3.5-fold change in potency against viruses containing m184v, k65r, and 6 tams, respectively. no cytotoxic effects were measured up to 1 mm in mt-2 cells and no effects on mitochondrial dna were detected up to 300 m in hepg2 cells for both fd4ap and l-d4ap. conclusion: fd4ap is a novel phosphonate ntrti with antiretroviral activity toward wild-type and resistant mutant hiv-1 strains. compared to d4ap, the 2 -fluorine atom significantly improved the in vitro toxicity profile while retaining the favorable resistance profile. in subsequent studies, the monoamidate prodrug strategy was applied to fd4ap to achieve optimal in vivo pharmacokinetic properties. entry inhibitors, and ccr-5 antagonists in particular, have become one of the most actively pursued treatments for hiv within the pharmaceutical industry. recently, multiple groups have disclosed piperidine-based ccr-5 antagonists that -to the medicinal chemist's eye -might appear to share a common three-point pharmacophore comprised of a tertiary amine, a phenyl ring, and a carboxamide or sulfonamide group. in several of these cases, these pharmacophoric elements are tethered together by a flexible, aliphatic chain. we sought to improve the potency of and introduce structural novelty into this class of compounds by rigidifying this tether. herein, we describe stereoselective syntheses and sar of a series of ccr-5 antagonists wherein the tether has been replaced with four stereochemical isomers of a rigidified cyclopropyl scaffold. the regulation of hiv transcription is a complex, multistage process that requires the concerted action of viral and cellular proteins. we discovered the n-aminoimidazoles (naims) as a unique class of hiv inhibitors targeted at the viral transcription level. a prototype naim, nr-818, prevents the reactivation of dormant virus by inhibiting both the hiv-1 p24 and viral mrna production from latently hiv-1-infected cell lines upon stimulation with tnf-␣, pma, or tsa. extensive research revealed that nr-818 was unable to inhibit the nf-b activation pathway or chromatin remodeling at the viral promoter, both known to be crucial for viral transcriptional activation. focusing on the viral transcription process, chromatin immunoprecipitation (chip) experiments revealed that nr-818 was able to inhibit the ser5 phosphorylation of the c-terminal domain (ctd) of rna polymerase ii. this step is mediated by the cdk9 subunit of p-tefb, which is recruited to the viral promoter by the hiv-1 tat protein. since we did not find an inhibition at the level of cdk9 activity or tat-mediated transcription in tat-expressing cell lines transiently transfected with a ltr-gfp construct, we infer that nr-818 must interfere with the transcription process by a unique mode of action. evidence points towards a kinase, not belonging to the cdk family, to be the target of the naims, resulting in an antiviral action at the level of retroviral transcription. clara e. cases-gonzález 1 , sandra franco 2 , miguel a. martínez 2 , luis menéndez-arias 1 1 centro de biología molecular "severo ochoa", csic-uam, madrid, spain; 2 fundació irsicaixa, hosp. university germans trias i pujol, badalona, spain a ser-ser insertion at codons 69-70 together with substitutions t69s and t215y in the reverse-transcriptase (rt)-coding region of hiv-1 are known to confer resistance to zidovudine (azt) and stavudine (d4t). phenotypic resistance correlates with increased atp-dependent phosphorolytic activity on inhibitor-terminated primers. we have previously shown that an rt derived from a clinical isolate (ss rt) that contained the insertion and 10 additional mutations related to drug resistance (including t215y) showed >10-fold increased unblocking activity on azt-and d4t-terminated primers, when compared with an rt containing the insertion together with mutations t69s and t215y, in an otherwise wild-type bh10 sequence. these results suggested that other mutations associated with the complex t69sss/t215y in clinically relevant rts contributed to increase atp-mediated excision activity and conferred high-level resistance to azt and d4t in phenotypic assays. to identify residues increasing the excision activity, we obtained recombinant enzymes bearing ss rt residues 1-135 and wild-type bh10 rt residues 136-560 (l1 rt), or residues 1-135 of the bh10 rt and 136-560 of the ss rt (l3 rt), as well as an l1 rt variant with the substitution t215y (l2 rt) and an l3 rt derivative with t69sss (l4 rt). additional rts containing mutations m41l, a62v, or k70r together with the combination t69sss/t215y in the bh10 background were also obtained. atp-mediated excision activities on azt-and d4tterminated primers were determined and the effects of mutations were tested in phenotypic assays using recombinant hiv-1. the l2 rt containing mutations t69sss/t215y and additional changes in the n-terminal region showed the highest atp-dependent phosphorolytic activity on blocked primers, giving values similar to those reported for the ss rt. results were consistent with phenotypic data. in contrast, l1, l3, and l4 rts displayed low-level activity. further experiments revealed that three amino acid changes at the n-terminal region of the polymerase (m41l, a62v and k70r) were responsible for the increased excision activity shown by rts bearing mutations t69sss and t215y. from a series of phenyl-substituted thiazolobenzimidazoles, several compounds were identified as selective inhibitors of coxsackie b virus replication in vero cells. a structure-activity relationship was established, from which the 6-trifluoromethyl substituted analogs emerged as the most potent congeners. the compounds were active against all six coxsackie b strains tested. the in vitro antiviral activity of one of the most selective compounds, i.e. chi-033, was assessed by (i) mts-based cytopathic effect assays, (ii) virus yield reduction assays, (iii) real-time quantitative pcr (rt-qpcr) and (iv) by monitoring viral antigen expression. in all assays a clear concentration-response effect was obtained. the 50% effective concentration (ec50) was 0.30 ± 0.18 g/ml, while the cc50 (50% cytotoxic concentration) of chi-033 for vero cells was more than 100 g/ml, thus resulting in a selectivity index of >500. detailed single cycle time-of-drug-addition studies (in which viral replication was monitored by means of rt-qpcr) revealed that the compound interacts with viral replication at a time that coincides with the onset of intracellular viral rna synthesis. chi-033resistant virus is being generated by culturing the virus in the presence of increasing drug concentrations. drug-resistant virus will be genotyped, which should allow us to identify the (putatively viral) molecular target of this class of compounds. retroviruses 42 hiromichi tanaka 1 , kazuhiro haraguchi 1 , hiroki kumamoto 1 , takao nitanda 2 , masanori baba 2 , ginger e. dutschman 3 , yung-chi cheng 3 1 school of pharmaceutical sciences, showa university, tokyo, japan; 2 center for chronic viral diseases, kagoshima university, kagoshima, japan; 3 school of medicine, yale university, new haven, ct, usa our recent research program on the development of synthetic methods for 4 -carbon-substituted nucleosides has led to a new strategy, ring opening of 4 ,5 -epoxy-nucleosides with organoaluminum and organosilicon reagents. this enabled us to introduce alkyl, alkenyl, and alkynyl groups to the 4 -position. as a result of this study, 4 -ethynylstavudine (4 -ed4t) was found to be more anti-hiv active than the parent compound stavudine (d4t). this compound (4 -ed4t) has several additional appeals as a promising anti-hiv agent: much less toxic to various cells and also to mitochondrial dna synthesis, better substrate for human thymidine kinase than d4t, very much resistant to catabolism by thymidine phosphorylase, its activity enhances in the presence of a major mutation k103n known for nnrti-resistant hiv. in this conference, we present the synthesis and sar studies of 4 -ed4t analogues modified mainly in the sugar portion. negatively charged polymers (np) possess a broad immunoadjuvant and antiviral activity topically useful for vaccine, drug, and microbicide development. but their efficiency is limited over a reversibility of electrostatic kind of interference with virusspecific nano-objects. to overcome this limitation the purposemade intra-molecular modifications of np were studied among non-toxic maleic acid co-polymers (npsa), dextran and chitin derivatives (npps) within varied alicyclic modifiers application. the configurationally flexible alkyls (i), as non-alicyclic control, are ineffective synergist for np antiviral potency. monocycles (ii) are moderate active too. on the contrary the hardconformation frame-structured spheroids (iii-vi) exhibit ability (at optimal macromolecular parameters) to be super-effective synergists for strength and diapason of np antiviral action. unlike small molecular iii/iv-containing prototypes (amantadin, rimantadin, deitiforin, etc.), narrowly-effective inhibitors mainly of influenza a viruses, the np-coupled modifications become effective also against many other viruses, including the drugs resistant strains [antivir. res. 46 (1), 44]. in focus of the anti-hiv potency the ivs provide a 10-100-fold elevation of np activity. the more available and less toxic iii species are similarly active, but iii* (with spatial-optimally contactable double bond due to the exo-configuration) turns out the best synergist 20-500-fold amplifying the anti-hiv-1 selectivity up to is∼10000. augmentation of the frame cycles from iii-iv toward v-vi results in no essential enhancement of antiviral activity, but stimulates toxicity. the recently involved in the investigation vii, cholesterol-like systems, as tools for novel raft-targeted strategy, demonstrate capacity for at least 10-fold amplification of anti-hiv-1 potency our earlier studies showed that esterification of cidofovir (hpmpc) with alkoxyalkanols increased antiviral activity by more that two logs and promoted oral bioavailability. to evaluate this approach with purine based nucleoside phosphonates, we synthesized several alkoxyalkyl esters of acyclic purine phosphonates such as 2,6,-diamino-(9-[2-phosphonomethoxyethyl]purine (pme-dap) and 2-amino-6-cyclopropylamino-(9-[2phosphonomethoxyethyl]-purine (pme-cpr-dap) these purine phosphonates have been reported to be active against a wide range of viruses such as human immunodeficiency virus (hiv-1), other retroviruses, herpesviruses, poxviruses and hepatitis b virus. for this study several alkoxyalkyl analogs of acyclic 2,6diaminopurine nucleoside phosphonates were synthesized and evaluated against hiv-1. the alkoxyalkyl esters were more inhibitory than the unmodified compounds in p24 reduction assays in mt-2 cells infected with hiv-1. for example, hexadecyloxypropyl (hdp) and oleyloxyethyl (ole) esters of pme-cpr-dap were >3 logs more active than unmodified pme-cpr-dap. in spite of increased cytotoxicity in mt-2 cells, the selectivity indexes are more than 10-fold higher then for unmodified compound. in conclusion, esterification of pme-dap and pme-cpr-dap with hexadecyloxypropyl-or oleyloxyethyl-residues greatly increased their antiviral activity and selectivity against hiv-1 in vitro. victor kuz'min 1 , eugene muratov 1 , anatoly artemenko 1 , ludmila koroleva 2 , vladimir silnikov 2 , v. lozitsky 3 , a. fedchuk 3 1 a.v. bogatsky physical-chemical institute, odessa, ukraine; 2 institute of chemical biology and fundamental medicine, novosibirsk, russian federation; 3 ukrainian mechnikov research anti-plague institute, odessa, ukraine "chemical" ribonucleases hold promise as tools for studying the structures of rnas and rna-protein complexes, as reactive groups in conjugates intended for cleavage of particular rnas, as therapeuticals inactivating virus genome rnas or certain mrnas, and as a promising antiviral agents. drug design and development of new medicines directed against hiv are permanently actual tasks. the usage of modern quantitative structure-activity relationship (qsar) methods could allow us to solve these problems more effectively. the objective of the present work is qsar analysis of antiviral activity of various tetrapeptides-artifical ribonucleases and consequent molecular design of new antiviral agents. qsar approach based on simplex representation of molecular structure (sirms) has been used for the solution of the formulated problem. usage of sirms allows us to develop the molecular design of the new effective antiviral agents. thorough researches of relationship between antiviral activity (hiv-1, % of rna p-o bond cleavage) and a structure of artifical ribonucleases have been carried out. statistic characteristics for pls (partial least squares model) are quite satisfactory (r 2 = 0.836, q 2 = 0.788). on the base of these models the molecular fragments with positive or negative influence on the explored property have been determined. thus, for example, guanidine and triethylenediamine fragments promote antiviral action. it gives a possibility to realize based on elucidated rules molecular design of compounds with the high level of antiviral activity. the results of prognosis are verifying by the experimental investigations. thus, quite adequate simplex qsar model "anti-hiv activity-artifical ribonucleases structure" was obtained and used for drug design. the cyclotriazadisulfonamide (cada) compound specifically down-modulates the cd4 receptor expression on the surface of lymphocytes and monocytes/macrophages, the primary receptors utilized by hiv for infection of its target cells. cada thus inhibits the entry of hiv and hhv-7 (vermeire et al., 2002. virology 302, 342-353) . cada chemotherapy may not be susceptible to the production of drug resistant strains of viruses, as its mechanism of action is completely different from those of any other anti-hiv drugs currently in clinical use. the cd4 down-modulating and antiviral potencies of more than 25 cada analogs have been described (vermeire et al., 2003. mol. pharmacol. 63, 203-210) . structural modifications of cada were made to increase potency, reduce cytotoxicity, and improve physical properties. several head group analogs were synthesized with polar groups and good leaving groups ( fig. 1) . the anti-hiv and cd4 down modulation activities of these compounds are being studied. some of these head groups may regenerate the double bond of cada by elimination reactions, potentially producing water-soluble pro-drugs. isocada (sa05), an isomer of cada, was synthesized by cyclization of 1,5,7-triazabicyclo-[4.4.0]dec-5-ene (tbd) (fig. 1 ). this structural modification may reveal a relationship between the symmetry of the molecule and its biological activity. two new fluorine-containing analogs were also synthesized by modifying the toluenesulfonamide side arms (fig. 1) . the anti-hiv and cd4 down modulation activities of these new cada analogs are summarized. the center for drug discovery, university of georgia, athens, ga 30602, usa drug discovery targeted at the elusive viral enzyme, hiv integrase, has not resulted in a single fda-approved drug. in this presentation we describe our molecular modeling studies with conceptually novel inhibitors of hiv integrase that also possess potent in vitro anti-hiv activity. docking was performed on the catalytic core of integrase represented by chain c of pdb structure code 1bl3. building of molecules and primary modeling was done with sybyl 7.1 on a silicon graphics onyx3 (r14000) workstation. the program gold 3.0 (genetic optimization for ligand docking) was used extensively in evaluating the docking poses of these compounds with the active site of hiv integrase and to give information on key residues involved in the recognition and binding of these ligands. the gold function consists of three basic components: protein-ligand h-bonding energy, protein-ligand van der waals energy, and ligand internal energy. post-processing gold output was done with the program silver 1.1, a utility program supplied with gold for evaluating hydrogen-bonding interactions, metal coordination and van der waals factors. for comparison purposes, additional docking was performed using other docking protocols, notably the sybyl module flexx. data obtained from these and related studies including binding poses, binding affinities, functional and conformational considerations, and gold function scores will be presented and explained. the center for drug discovery, university of georgia, athens, ga 30602, usa hiv integrase is essential for hiv replication and is an attractive target for drug discovery against aids. however, research efforts on drug discovery pertaining to hiv integrase have not resulted in a single fda-approved drug for which mechanism of action is inhibition of hiv integrase. recently, we have been exploring a novel class of diketo acids that are constructed on nucleobase scaffolds and that have a specific arrangement of the functional and hydrophobic group on the scaffold. these compounds are inhibitors both key steps of hiv integrase. one lead compound from this group has also been found to have remarkable in vitro anti-hiv activity. however, the syntheses of the inhibitors are quite challenging. this presentation will describe the synthetic methodologies specifically developed in our laboratory for the preparation of some representative examples of these integrase inhibitors. purification approaches to produce highly purified compounds for biological studies will be explained. structural, functional and conformational data obtained from extensive spectroscopic studies will be discussed. representative anti-hiv integrase data and in vitro anti-hiv screening results will be presented. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz-51, that inhibits both hcv and hiv in vitro with ec 50 values ranging in micromolar concentrations or less, with little or low toxicity to the host cells. in this part i of the presentation on this subject, we report our preliminary findings on the mechanism of anti-hiv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues. in view of the fact that a number of hiv patients also suffer from hcv as a major coinfection, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. marina burshtein 1 , alexander serbin 2 , alissa bukrinskaya 1 d.i. ivanovsky institute of virology, moscow, russia; 2 health research and development found, moscow, russia introduction: amantadine is a well-known effective antiinfluenza drug. it was modified to enhance its antiviral activity by chemically linkage with the water-soluble polyanionic matrix via different spacer groups. the other group of used compounds was norbornene derivatives, as norbornene is an adamantane analogue on anti-influenza activity. methods: the absence of cytotoxic effect was shown by mtt test for estimating cytotoxic dose (ctd50). the antiviral effect of the compounds was analyzed in lymphoblastoid mt-4 cells and in hela cd4+/b-galactosidase cells ("magi" cells). the effect of the compounds was registered by immunoblotting of cell lysates and by measuring of b-galactosidase activity. results: the strong inhibition of hiv-1 replication was observed when the compounds were added with the virus and was expressed even when the compounds added with the virus were removed 1 h after infection. the anti hiv-1 effect of the compounds was gradually decreased if they were added 1 and 2 h after infection, no inhibition was observed when the compounds were added 4 h after infection. the compounds did not impair the virion structure. adamantane and norbornene derivatives were shown also to inhibit azt resistant viral strains. conclusion: adamantane and norbornene were shown to be active hiv inhibitors with the high selectivity index. the compounds are promising candidates for further investigation including preclinical studies. less is known about the effect of their intracellular half-lives on the maintenance of antiviral activity. to investigate this question, we developed a novel in vitro antiviral persistence assay. measurement of the antiviral persistence of tenofovir (tfv) and abacavir (cbv) was coupled to measurement of the half-lives of their tfv-dp and cbv-tp anabolites. methods: mt-2 cells or stimulated primary cd4+ t-cells were incubated with graded concentrations of tfv or cbv for 14 h (h); then extracellular drug was removed by washing. cells were further incubated without drug for 0-24 h and then infected with hiv-1 (iiib or bal). p24 was quantified on day 2; inhibition of hiv-1 replication due to intracellular drug persistence (pc50) was determined relative to a standard ec50. decay of intracellular dp/tps in cd4+ t-cells was measured using lc/ms/ms. results: in mt-2 cells, the pc50 value for tfv 12 h after drug removal remained unchanged relative to the ec50 (<3-fold shift) whereas the pc50 for cbv shifted >65-fold, indicating less persistence of cbv. in cd4+ t-cells, the pc50 value for tfv also showed a minimal shift relative to the ec50 (2.4-fold) 24 h after drug removal. cbv showed a much larger relative shift (>243-fold). quantification by lc/ms/ms of intracellular tfv-dp and cbv-tp in cd4+ t-cells in vitro demonstrated that tfv-dp had the longest intracellular half-life of the two drugs (tfv-dp, 21 h versus cbv-tp, 5 h). conclusions: a novel antiviral persistence assay was developed to study the relationship between intracellular nrti halflives and antiviral activity. in both mt-2 cells and primary activated cd4+ t-cells, tfv had the longest persistence of antiviral activity. in cd4+ t-cells, tfv-dp also had the longest half-life of the two nrtis. cbv-tp had a much shorter half-life than tfv-dp and showed less antiviral persistence. although both drugs are approved for qd dosing, the half-life of intracellular tfv-dp maintains antiviral suppression in vitro over a timeframe most consistent with qd dosing. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa isis 5320 is a phosphorothioate oligonucleotide with a molecular structure of t2g4t2. the g-quartet possessing molecule has been shown to be a potent inhibitor of hiv attachment and cell-cell fusion and acts by specifically interacting at the v3 loop of gp120. mapping studies with monoclonal antibodies targeting epitopes in and around the v3 loop have been used to define the binding site of isis 5320. in vitro, isis 5320 inhibits all laboratory and clinical strains of hiv-1 and hiv-2 tested, including representative subtype viruses, drug resistant viruses (including mdr viruses) and viruses that utilize the cxcr4 and ccr5 chemokine receptors. serial passage of virus in the presence of increasing concentrations of the oligonucleotide did not result in the selection of drug resistant virus strains and combination assays resulted in additive to synergistic interactions with other approved hiv inhibitors. the antiviral and toxicity profiles of isis 5320 resulted in the performance of human clinical trials for the therapeutic use of the oligonucleotide to treat hiv infection. the antiviral properties and mechanism of action of isis 5320 suggest that it may be an excellent anti-hiv topical microbicide. isis 5320 was found to be highly active in a cervical explant model of hiv infection with highly significant inhibition of ccr5-tropic strains of virus. activity was also observed in cell-free and cell-associated virus transmission assays, as well as in cd4-dependent and cd4-independent acute infection inhibition assays. in microbicidal specific combination assays, significant efficacy has been observed with isis 5320 used in combination with other microbicidal compounds. the results of these studies suggest that isis 5320 may represent a new and novel anti-hiv topical microbicide. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa though a variety of compounds are being developed as anti-hiv topical microbicides, such as polyanionic molecules, surfactants, natural products, peptides, proteins, heterocycles, and virucidal agents, clinical efficacy studies that demonstrate the ability of these agents to impact virus transmission are still in progress. it has been estimated that a microbicide that is only 60% effective would have the capacity to prevent millions of new infections each year. thus, one of the challenges in hiv drug development is the discovery of compounds that will inhibit the sexual transmission of infectious organisms between sexual partners. the rapid mutability of hiv and the known presence of drug resistant viruses in wild type virus populations suggests that microbicide development will suffer from the same problems that exist for all hiv therapies, namely the selection of resistant virus strains that will bypass the microbicide barrier and infect target cells in the vaginal or rectal environment even in the presence of the microbicide. thus, it is likely that haart-like combination drug therapies will become the most effective means of inhibiting the sexual transmission of hiv. we have evaluated a wide variety of anti-hiv and anti-sti compounds in vitro alone and in combination with one another and have demonstrated that certain patterns of inhibition (additivity, synergy, antagonism) occur between the various classes of compounds. recently, we have compared the combination anti-hiv activity of microbicide compounds in fresh human pbmcs infected with clinical isolates of hiv to the combination activity of the same test agents in cem-ss-based cultures. in general, these two assay systems yield similar combination assay results. to provide a rationale for the combination use of the compounds in a microbicide setting, the same combination of compounds was evaluated in a microbicide-like virus transmission assay. these combination results suggest that higher levels of synergy between virus attachment and reverse transcriptase inhibitors might be expected in the microbicide environment compared to levels predicted for the systemic therapeutic environment. the results of the combination assays with various microbicides will be presented. during the onset of the hiv disease, hiv rna is continually produced in the face of treatment with haart in circulating reservoirs and rt inhibitors are almost ineffective in the postintegration events. among the classes of anti hiv-1 drugs, protease inhibitors (pi) are the unique to inhibit the hiv-1 production in chronically infected macrophages. in the progression of hiv infection, the role of the monocytes-derived macrophages (m/m) is further confirmed as they represent chronologically the first cytotype where the viral replication restarts as a consequence of failure or interruption of antiviral therapy. aim of the work was to evaluate the rebound of hiv-1 production when pi have been removed in hiv-1 chronically infected m/m and, moreover, to verify the effect of this removal on virus maturation, infectivity and ability to trigger apoptosis in uninfected peripheral blood lymphocytes (pbl). a rebound of p24 gag protein was measured starting from 12 h after drug removal yet virus infectivity remained 1 log lower than control up to 1 week. inhibition of hiv-1 replication was still 48% and 18% upon amprenavir 20 and 4 m, respectively. these data were confirmed by western blotting and electronic microscopy showing production and release of immature viral particles. moreover, pi (amprenavir and indinavir) treatment dramatically reduced apoptosis of pbl co-cultured with chronically infected m/m and kept cd4/cd8 ratio above the levels of untreated controls until the 5th day of co-culture. taken altogether, these findings suggest a wide clinical importance for amprenavir and indinavir for their relevant long-lasting antiviral effect in persistently-infected reservoirs of hiv even in case of drug interruption and/or when hiv infection can restart in districts where drugs find not sufficient concentration. moreover, these results strengthen the evidence for an unique positive utilize of pi against ongoing and productive hiv infection. weili jin, salvatore santino, michael wang gilead sciences, foster city, ca, usa background: effective inhibition of hiv reverse transcriptase (rt) currently represents a crucial objective of antiretroviral therapy. capravirine is a second-generation non-nucleoside rt inhibitor (nnrti) that is capable of blocking the replication of certain nnrti-resistant strains of hiv and was recently in clinical development. in this study, we report on the in vitro selection and characterization of viral resistance to capravirine. methods: viral resistance selection experiments were performed in mt-2 cells with the hiv iiib isolate and increasing concentrations of capravirine. viruses were analyzed genotypically by population sequencing and by single genome sequencing (sgs). recombinant viruses with nnrti mutations were generated from proviral dna clones. phenotypic analyses were performed in mt-2 cells. results: capravirine resistance selections were initiated at 1 nm (ec 50 of 1.5 nm for capravirine). following nine passages in the presence of increasing concentrations of capravirine, the l100i mutation emerged in rt and additional passaging led to v179d and f227c mutations at higher concentrations (100-300 nm). further increases in capravirine concentrations led to the emergence of a l100i + v179d + f227c triple mutant, which confers >1000-fold resistance to capravirine. sgs of mixed viral populations from different passages showed that l100i, v179d and f227c were present on the same genome, with l100i as the primary mutation, and f227c and v179d were acquired sequentially at later passages. through sgs analysis, a l100i + k166r + v179d + f227c quadruple mutation on the same genome was also observed at higher capravirine concentrations (>1000 nm). recombinant viruses carrying these mutations were produced to assess their susceptibilities to capravirine. conclusions: after extensive in vitro passaging of hivinfected cells in the presence of capravirine, neither k103n nor y181c mutations in rt were observed. instead, the l100i mutation was initially acquired, followed by mutations f227c and v179d. addition of the k166r mutation to the triple mutant genome, l100i + v179d + f227c, appears to further enhance hiv resistance to capravirine. oluwafemi olawuyi 1 , adeyemi falegan 2 1 medical microbiology, university college hospital, ibadan, nigeria; 2 dentistry, university college hospital, ibadan, nigeria issue: the percentage of aids/hiv is increasing every year in the third worlds, and this is reinforced by the factor that majority of youth in third worlds do not know his/her hiv status. description: a self developed validated and reliable questionnaire [r = 0.87] was used to collect the data and percentage was used to analyze the data. the population of the study was made up of youth [female and male] in higher institutions, working places, market places and community streets in nigeria, 50,000-sample size, selected through simple random sampling technique. the mean age is 25.5 years old. relative risk [rr] calculated is 3.1, i.e. rr >1, indicating that the factor is the risk factor, and the confidential interval [ci] for rr at 95% significant level is 2.61 < 3.1 < 3.90 from the formula, ci lower limit < rr < ci upper limit. lessons learned: seventy percent of the sample population did know his/her hiv status and had had sexual intercourse in the past before, out which 20% had the unprotected intercourse once or more, 25% had protected sex while 25% were not sure of using protection means. while, 20% have knowledge about own hiv status and had had sexual intercourse before. ten percent have no knowledge about own hiv status and had no sexual intercourse before. conclusion: aids/hiv still remains a killer disease in the third world. however, the lack of knowledge of individual's hiv status remains the only highest risk factor for the spread of the disease in the third worlds. yuichiro habu 2,3 , jacob barnor 1,6 , norio yamamoto 4 , kahoko hashimoto 1,2 , naoko miyano-kurosaki 1,2 , koichi ishikawa 5 , naoki yamamoto 5 , david ofori-adjei 6 , hiroshi takaku 1,2,7 1 department of life and environmental sciences, chiba institute of technology, chiba, japan; 2 high technology research center, chiba institute of technology, chiba, japan; 3 japan foundation of aids prevention; 4 department of molecular virology, bio-response, tokyo medical and dental university, tokyo, japan; 5 aids research center, national institute of infectious disease, tokyo, japan; 6 department of virology, noguchi memorial institute for medical research accra-ghana, accra, ghana; 7 bach tech corp. rna interference (rnai) is a potentially strong gene interference tool, which had been successfully used to silence many pathogenic viruses including hiv. however, many recent reports have shown that, in long-term assay cultures involving rna viruses such as hiv, escape mutants breakthrough the silencing effect. in the light of this conundrum, it had been proposed that, vector designed to target multiple genes in a synergistic manner, may address the problem. hence, we designed a chimeric rna expression vector which express vif shrna and decoy tar rna by combining vif shrna and decoy tar rna with linker to which dicer was able to recognize for cleavage, as a second generation rnai expression vector system. the synergistic effect of these molecules enhanced the inhibition of hiv-1 replication in a long-term transduced pbmcs, h9, and jurkat cell culture assays (9 weeks) and prevented virus breakthrough associated with sirna-mediated escape variants. notably, hiv-1 replication was similarly suppressed in the control cells expressing only vif shrna for about 3 weeks, but an increase in virus replication was observed afterwards. hiv viral rna extracted and sequenced at this point indicated escape mutants in the cells expressing the vif target in hiv. we confirmed substitution of bases in the vif shrna target sequence. on the other hand, the incidence of mutation was not observed in a sequence of viral rna from the culture expressing the vif shrna-decoy tar rna at the fourth week. interestingly, virus production was inhibited for a long-term by an effect of decoy tar rna, through the rna-protein interaction. combining shrna with decoy tar rna as second-generation anti-hiv shrna may provide practical basis for applying sirna-based gene therapy to the treatment of hiv/aids. introduction: this is a designed efficient gene therapy against aids/hiv. the novelty of this aids vaccine design/concept is seen in the fact that the 'pol' gene encoding for nonstructural proteins (polyproteins that generate three enzymes: reverse transcriptase, integrase and protease) is cloned in a suitable retroviral vector and adult stem cells are transfected by this and reinfused into the circulation to effectively counter hiv replication and antigenic variation. method: the mrna are isolated from adult stem cells and transcribed into cdna with reverse transcriptase. the cdna are then cloned in a suitable retroviral vector (vacinia) carrying 'pol' gene that confers resistance to a strong reverse transcriptase inhibitor drug. the adult stem cells are transfected by the recombinant mixture, and reinfused into the circulation of hiv infected person. result: the transfected stem cells are reinfused to provide renewable source of more and better empowered normal blood cell types that would disrupt and half hiv replication in the circulation. there would be efficient induction of both humeral and cellular mediated immunity with prolonged expression of antigens and protective immunologic memory generation against hiv antigenic variation. conclusions: this aids vaccine design would lead to both efficient prophylactic and therapeutic therapy against aids in that it would effectively take care of the problematic factor of hiv antigenic variation which has long been the main obstacle to potent aids vaccine development. because of the real risk of interspecies transmission and/or reassortment between avian, swine and human influenza a strains, drug susceptibility monitoring of circulating avian and porzine virus strains appears to be warranted for effective application of antiviral drugs like amantadine. this study was designed to gain insight into amantadine susceptibility of 6 avian and 12 porcine influenza a viruses isolated in germany between 1981 and 2002. virus strains were isolated in embryonated chicken eggs and passaged one time in mdck cells. plaque reduction assays were applied to examine virus susceptibility to amantadine. genotyping was used to confirm drug resistance. in the result of these antiviral studies, only 3 of the 12 porzine isolates but all 6 avian isolates were shown to be amantadine-susceptible. interestingly, the three amantadinesensitive porzine strains were isolated between 1981 and 1987. all porzine influenza a viruses isolated later on were drugresistant and contained the aa substitutions g16e, s31n, and r77q in the matrix protein 2 (m2). additionally, l27a was detected in two h1n1 strains. s31n and/or l27a are well known amino acid substitutions in m2 that confer amantadine resistance. the role of the pig as an intermediate host of avian and human influenza a viruses, the possible involvement of genetic reassortment, and the high incidence of naturally amantadineresistant porcine influenza a viruses suggest a real risk of emergence of amantadine resistant human viruses. therefore, further studies are ongoing now to evaluate the circulation of the resistant phenotype in pigs, birds and human. recently much attention has been devoted to searching for effective chemotherapeutic agents and vaccines for eradication of this notorious disease. at present only chemotherapy is available to combat avian flu, for instance, tamiflu, approved for the treatment by the us-fda. development of a simple, novel molecule with potential antiviral activity against is essential to treat avian flu viral infection. isatin (2,3-dioxoindole), is a versatile lead molecule for designing of potential antiviral agents and its derivatives were reported to possess broad spectrum antiviral activity. methisazone (nmethylisatin-3-thiosemicarbazone) was first clinically approved for treatment of pox viral infections, and its derivatives were documented to have anti-influenza activity. based upon this evidence, the present work was initiated to determine the antiviral activity of novel isatin derivatives against avian flu (h5n1) in mdck cells. antiviral activity was studied by virus yield assay (ec90), and cytotoxicity by neutral red uptake assay by uninfected mdck cells. all five compounds of a series inhibited the replication of avian flu (h5n1) virus replication in mdck cells and compounds spiii-5h and spiii-5cl were most active (ec90 5.5 g/ml, cc50 >100 g/ml and si > 18). details of these studies and results of treatment of influenza-infected mice are discussed. acknowledgement: supported in part by contract noi-ai-15433 and noi-ai-30048 from virology branch, niaid, nih]. arginine-rich peptide conjugated phophorodiamidate morpholino oligomers (arp-pmo) are nuclease resistant antisense compounds that hybridize to target rna in a sequence-specific manner resulting in disrupted rna function. eight arp-pmo were designed to base-pair with various regions of a/pr/8/34 (h1n1) rna and were then evaluated by hemagglutination and plaque assays for their ability to inhibit fluav production in vero cell culture. arp-pmo targeting the aug translation start site of the np or pb1 segment mrnas, or the 3 -terminus of their respective vrnas, were highly effective, reducing influenza virus titer by 1-3 orders of magnitude in a dose-dependent and sequence-specific manner over a period of 2 days. two of the p-pmo, targeting the pb1 translation start site region (pb1-aug) and the 3 terminus of np vrna (np v3 ), were evaluated by endpoint dilution (tcid50) or elisa assays against another h1n1 strain (a/wsn/33), as well as a/memphis/8/88 (h3n2) and a/thailand/1(kan-1)/04 (h5n1). the pb1-aug arp-pmo generated over 85% specific reduction of virus level, regardless of viral subtype or methodology, at concentrations in the range of 10-20 m. the np v3 p-pmo yielded similar results, with the exception of considerably lower efficacy against the h3n2 strain, with which it has two base mispairings. studies are planned to further evaluate of at least two arp-pmos in animal models for h1n1 and h5n1 fluva subtypes. macroheterocyclic compounds containing crown fragments and nitrogen atoms show large-scale biological activity. we synthesized series of aza-crown ethers and their derivatives. we also studied anti-influenza and antiherpetic action of some of them. anti-hsv action of studied compounds was tested using cyto-morphological method. hep-2 cells were infected with hsv-1 strain us in dose 5 ifu/cell. the cells were incubated in eagle's medium that contained compounds in a dose of 10 −4 m in experimental samples, or without them in control samples. then cells were fixed with 96% ethanol and stained with 0.01% acridine orange solution. the amount of infected cells with dna-containing virus inclusion bodies was counted by fluorescent microscopy. anti-hsv activity of compounds was calculated as the difference between of the percentage of infected cells in treated cell cultures to the percentage of infected cells in untreated cell cultures. anti-influenza activity was studied on the model of replication of a/hong kong/1/68 (h3n2) strain in tissue culture of chorio-allantoic membranes of chicken embryos. compounds were used in a dose of 10 −3 m during the study of their anti-influenza action. diaza-18crown-6 and two of its derivatives have showmen neither anti-hsv nor anti-influenza activity. diaza-18crown-6 derivatives that contain 2-oxyethyl-or ethoxycarbonyl-fragments decreased amount of cells infected by hsv-1 by 13 and 29%, respectively. both of these compounds inhibited replication of influenza virus on 1.7 log 10 tid 50 aza-15crown-5 did not show antiviral activity, but both its derivatives proved to be active inhibitors of hsv and influenza virus reproduction. aza-15crown-5 derivatives that contain 2-amino-3-phenyl-propanoyl-or 5-benzyloxy-3-oxapentyl-fragments decreased amount of cells infected by hsv-1 with virus-specific intranuclear inclusions by 41 and 47%, respectively. first compound inhibited replication of influenza virus on 1.5 log 10 tid 50 and the second one decreased virus amount on 2.0 log 10 tid 50 . the results of this study show that aza-crown ethers are the perspective class of compounds for search of new antiviral agents. acknowledgement: this work was partially supported by stcu (grant # 3147) . robert w. sidwell 1 , kevin w. bailey 1 , min-hui wong 1 , donald f. smee 1 , dale l. barnard 1 , shanta bantia 2 1 institute for antiviral research, utah state university, logan, ut, usa; 2 biocryst pharmaceuticals, inc., birmingham, al, usa the cyclopentane neuraminidase inhibitor, peramivir (bcx-1812, rwj-270201) has striking inhibitory effects on a spectrum of influenza viruses in vitro, and has also demonstrated significant effects against influenza a (h1n1, h3n2) and b virus infections when administered orally to mice and ferrets. unfortunately, clinical trials with the drug administered orally were not successful, probably due to low blood levels obtained after oral administration. significant plasma drug levels of peramivir persist up to 6 h after intramuscular (i.m.) injection; more importantly, however, is the observation that peramivir remains tightly bound to influenza virus n9 neuraminidase for over 24 h, suggesting single i.m. or intravenous (i.v.) therapy with the drug may be highly effective against an influenza infection. experiments now in press have indicated that single i.m. peramivir therapy administered up to 48 h after virus exposure was protective to mice infected with influenza a (h1n1) virus. in the present study, peramivir was administered i.m. or i.v. in a single injection 1 h pre-virus exposure in separate experiments to mice infected with an influenza a (h5n1) virus; efficacy was compared to similar dosages of oseltamivir and oseltamivir carboxylate run in parallel. dosages of 20 and 10 mg/kg of peramivir administered by either route significantly prevented deaths, lessened arterial oxygen (sao 2 ) decline, inhibited development of lung consolidation, and inhibited lung virus titers. the lung assays were performed at varying times after virus exposure. oseltamivir and oseltamivir carboxylate, which do not have the same neuraminidase binding abilities seen with peramivir, were less efficacious in these experiments. delaying the single i.v. therapy up to 72 h after virus exposure also significantly inhibited the virus infection. peramivir appeared to be well tolerated in toxicity control animals run concomitantly with these studies. these data indicate parenterally administered peramivir may hold promise as a therapy for clinical influenza a (h5n1) virus infections. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. hiroshi saitoh 1 , naoko miyano-kurosaki 1,2 , hiroshi takaku 1,2 1 department of life and environmental sciences, faculty of engineering, chiba institute of technology, chiba, japan; 2 department of life and environmental sciences, faculty of engineering and high technology research center, chiba institute of technology, chiba, japan background: influenza virus causes widespread infection in the human respiratory tract, but existing vaccines and drug therapy are of limited value. recently, small interfering rnas (sirnas) are a powerful tool for sequence-specific, post-transcriptional gene silencing and have a potential therapeutic and prophylactic application against cancer, as well as infectious diseases. here we show that short interfering rnas (sirnas) specific for conserved regions of the viral genome can potently inhibit influenza virus production in cell lines. the influenza virus np gene is a potential target for rnai technology. on the other hand, the baculovirus (acmnpv) can infect a variety of mammalian cells, facilitating its use as a virus vector for gene delivery in viral entry into cells. in this study, we describe the inhibition of influenza virus production by baculovirus-mediated shrna expression vectors. methods: the psv2neo-u6 plasmid vectors and pvl1393based baculovirus vectors were used in this study. the influenza virus a and b np genes were made into the target and the shrna expression plasmid vectors were constructed under the control of the human u6 pol iii promoter. the shrna expression plasmids or shrna expression baculovirus vectors introduced into mdck cells, and 24 h later the cells were infected with either a/pr8 or b/ibaraki virus at a moi of 0.01. at 72 h postinfection, culture supernatants were harvested and assayed to determine the virus titer by plaque assay. conclusion: the findings reveal that newly synthesized np proteins are required for influenza virus replication and provide a basis for the development of shrnas expression plasmids as prophylaxis and therapy for influenza infection in humans. julia serkedjieva, ekaterina krumova, tsvetanka stefanova, nadja nikolova, maria angelova institute of microbiology, bulgarian academy of sciences, sofia, bulgaria a semi-standardized polyphenol-rich extract (pre), obtained from geranium sanguineum l., inhibited the reproduction of influenza viruses types a and b in vitro and in ovo and protected mice from mortality in the experimental influenza virus infection (serkedjieva and manolova, 1992) . the selective in vitro virus-inhibitory activity of pre was fairly modest and this was in contrast with the significant protection in vivo. thus, the therapeutic effect of pre needed explanation. it was presumed that it might be attributed to a combination of more than one biological activities known for natural polyphenols. we have demonstrated previously that pre manifested strong antioxidant and radical-scavenging activities in model systems (sokmen et al., 2004) . the current study was undertaken to investigate the effect of the plant extract on the levels of the antioxidant enzymes superoxide dismutase (sod), catalase (kt) and peroxidase (po) in mice lungs during influenza virus infection as well as the effect of pre on the production of reactive oxygen species (ros) and reactive nitrogen intermediates (rni) by alveolar macrophages in influenza virus infected mice. mice were challenged intranasally (i.n.) with 5-10 ld 50 of a/aichi/2/68 (h3n2) influenza virus. pre was administered by i.n. instillation 3 h before infection in the dose of 10 mg/kg. it was established that influenza infection induced an increase in sod, kt and po production and on days 6 and 9 after infection their levels reached 140-160% of placebo control. the application of pre brought enzymes values to control levels. influenza infection caused also a significant increase of h 2 o 2 , o 2 •− and no production by alveolar macrophages; the generation of ros and rni peaked on day 9. pre-treatment before viral challenge reduced this excessive production. in conclusion, the obtained results outlined the antioxidant and radical scavenging properties of the plant extract; pre beneficially modulated the oxidative stress response in influenza virus-induced pneumonia. this alternative mechanism of action might contribute to the overall protective effect in the lethal murine experimental influenza infection. the antiviral activity of s11, a natural herb extract, ji-sun kwon 1 , hyun-jeong lee 1 , chi-ung moon 2 , jong-hwan kwak 3 , youn-jeong lee 4 , chang-seon song 1 1 avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; 2 hanyang university, seoul, korea; 3 sungkyunkwan university, seoul, korea; 4 national veterinary research and quarantine services, seoul, korea the antiviral activity of s11, one of the traditional korean medical herb extract, against influenza virus was investigated. the 50% effective concentration (ec50) using plaque reduction assay was 31.25 ug/ml and the mean 50% cytotoxic concentration (cc50) using wst-1 assay in the mdck cells was 334 ug/ml. oral gavage treatment of the s11 to balb/c mice infected with a/pr/8/34 (h1n1) influenza virus showed the therapeutic effects as delaying clinical signs, significant inhibition of death and reduction of lung virus titers. to identify the lead molecules, the s11 was subjected to further fractionation, purification, and isolation of active compounds. the antiviral activity of these natural herb compounds will be discussed. these results suggest that the s11 is a possible candidate for the development of new antiviral medicine for influenza therapy. hiroshi takaku 1,2,4 , takayuki abe 3 , hitoshi takahashi 1 , naoko miyano-kurosaki 1,2 1 department life environ. sci., chiba inst. tech., chiba, japan; 2 high tech. res. center, chiba inst. tech., chiba, japan; 3 res. inst. microbial dis., osaka university, osaka, japan; 4 bach tech corp background: the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) has long been used as a biopesticide and as a tool for an efficient recombinant protein production in insect cells. in this study, we examined the immunization of a recombinant baculovirus expressing the influenza virus hemagglutinin (ha) against lethal influenza infection in mice. protection was observed in mice immunized intranasally with not only the recombinant baculovirus but also a wild-type baculovirus. baculovirus was also shown to induce secretion of inflammatory cytokines, such as tnf-␣ and il-6, in murine raw 264.7 macrophage cell line. results: a varied route of immunization with a recombinant baculovirus expressing the influenza virus hemagglutinin protein of a/pr/8/34 (h1n1) virus against lethal influenza infection was examined in mice. the recombinant baculovirus encoding the hemagglutinin gene under the control of chicken ␤ actin promoter was inoculated twice, 2 weeks apart, at a dose of 1.1 × 10 8 pfu per mouse by intramuscular, intradermal, intraperitoneal, and intranasal routes. mice intramuscularly and intraperitoneally immunized with the recombinant exhibited higher level of production of serum anti ha antibody than those immunized via the other routes, but protection was only achieved by the intranasal immunization. surprisingly, mice immunized with a wild-type baculovirus with intranasal route were also protected from the lethal influenza virus challenge. sufficient protection in mice was achieved by the intranasal immunizations with 10 8 pfu of either the recombinant or wild-type baculovirus, as evaluated by the reduction of virus titer, production of inflammatory cytokines, and pulmonary consolidations in the lung. these results indicate that infection with a baculovirus induces a strong innate immune response and protection of mice from lethal influenza virus infection. conclusion: baculovirus (cpg motifs) induces a strong innate immune response and protection of mice from lethal influenza virus a and b infection. andrew vaillant 1 , annie lebel 2 , nathalie goyette 2 , guy boivin 2 , jean-marc juteau 1 , phil wyde 3 1 replicor inc., laval, que., canada; 2 chuq-chul and laval university, st. foy, que., canada; 3 baylor college of medicine, university of texas, houston, tx, usa potent antiviral activity of phosphorothioate oligonucleotides (ps-ons) was observed against influenza viral infections. antiviral activity was sequence-independent, size dependent (optimally active ps-ons were ≥40 bases in length) and dependent on the presence of the phosphorothioate modification (hydrophobicity). binding studies showed that rep 9 (a 40 mer degenerate ps-on) interacts with both neuraminidase and hemagglutinin although the sialidase activity of neuraminidase was not affected, suggesting that the structural interactions of these proteins required for influenza activity are the target for this compound. the requirement for hydrophobicity further suggests that the alpha helical regions of hemagglutinin are one of the regions of interaction. the antiviral activity of rep 9 was conserved in many influenza a and b strains suggesting potential therapeutic activity against avian flu and other newly emerging influenza strains. rep 9 aerosol has excellent characteristics for lung deposition and aerosol treatment with rep 9 was well tolerated and highly effective against infections with influenza a both in prophylaxis and 24 h after infection. these results demonstrate the therapeutic potential of aerosolized ps-ons against influenza infection. acknowledgement: supported by nih contract no1-ai-15437. irina v. alymova 1 , y. sudhakara babu 2 , allen portner 1 1 virology division, department of infectious diseases, st. jude children's research hospital, memphis, tn 38105, usa; 2 biocryst pharmaceutical, inc., birmingham, al 35244, usa bcx 2798 is a novel selective inhibitor of human parainfluenza virus infections, which design was based on the threedimensional structure of the hemagglutinin-neuraminidase (hn) protein of newcastle disease virus. compound exhibited striking activity against parainfluenza viruses in vitro and in vivo, and was efficacious in prophylaxis of lethal synergism between parainfluenza virus and streptococcus pneumoniae in a mouse model. present study was conducted to determine if bcx 2798's resistant variants of the recombinant sendai virus whose hn gene was replaced with that of human parainfluenza virus type 1 (rsev(hpiv-1 hn) could be selected in tissue culture and animals. for this purpose virus was serially passaged in llc-mk 2 cells at moi 0.1 in the presence of increasing (from 100 to 3200 m) concentrations of compound; infected 129×1/svj mice were treated with 10 mg/kg/day of bcx 2798 twice for five days. treatment started 4 h before infection. individual clones of viruses were analyzed for the presence of mutations. one mutation, e527k, on the globular head region of the hn protein was selected in tissue culture after the fifth and eleventh passages of rsev (hpiv-1 hn) . several mutations in hn gene of rsev (hpiv-1 hn) were selected in an animal model after the second passage of virus from mice treated with bcx 2798. two mutations, n23s and p29q, were located in the cytoplasmic domain of hn protein; mutations n173s and t553a were found on the globular head region of the glycoprotein. only nonconserved amino-acid residues of hn protein were involved in substitutions. all isolated mutant viruses were stable after the five passages in llc-mk 2 cells without drug; did not develop other substitutions in the presence of drug and displayed no resistance to bcx 2798 both in vitro and in vivo. infectivity of all mutants was not altered to compare with the wild type of rsev (hpiv-1 hn) virus. taking together our results indicate that prophylaxis/treatment of human parainfluenza virus infections with bcx 2798 may not lead to appearance of clinically significant variant of viruses. kie-hoon jung 1 , michelle mendenhall 1 , lawrence m. blatt 2 , robert w. sidwell 1 , brian b. gowen 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa hantavirus pulmonary syndrome (hps) is an acute human respiratory disease with remarkably high case fatality rates (30-50%) for which the etiological agents are members of the bunyaviridae family, genus hantavirus. maporal (map) virus is a recently identified hantavirus isolated in western venezuela, which is most similar phylogenetically to hantaviruses known to cause hps in southern regions of south america. despite the lack of evidence that map can productively infect humans and cause hps, infection of hamsters closely resembles disease manifestations associated with human hps. hantaviruses, in general, are known to produce little to no cytopathic effect (cpe) in cultured cell lines. unexpectedly, we found that map produces remarkable cpe in several vero cell lines facilitating the evaluation of known antiviral agents, ribavirin and interferon alfacon-1. both drugs were highly effective at reducing cpe, as determined by visual examination and neutral red dye uptake, associated with map infection. since much of the observed cpe may be due to apoptosis of uninfected bystander cells, we also developed a quantitative (q)rt-pcr assay to detect copies of map genomic sequence to more directly assess the inhibition of viral replication. data obtained using the qrt-pcr-based assay were consistent with the visual cpe reduction and neutral red-uptake cytotoxicity findings. the development of in vitro antiviral testing methods for map are essential to the evolution of the in vivo hamster disease model of hps. the latter is of utmost importance considering the current need for effective antivirals for the treatment of hps and the lack of a suitable model that does not require biosafety level 4 containment facilities. acknowledgement: supported by contract no1-ai-30048 from the virology branch, national institute of allergy and infectious diseases, national institutes of health. nucleoside analogues are widely used in antiviral and anticancer chemotherapy. for this class of drugs, intracellular conversion of the nucleoside analogue into the corresponding 5mono-, 5 -di-, and 5 -triphosphate after target cell penetration is a prerequisite for biological activity. because of the structural differences from natural nucleosides, this conversion is often inefficient and, as a consequence, therapeutic efficacy is sometimes limited. the free phosphates, or nucleotides, have limited utility in therapy on account of their poor membrane permeability and chemical stability. one approach to improve the therapeutic potential of nucleoside analogues is the delivery of the corresponding nucleotide entities via neutral, lipophilic prodrugs, or protides. the nucleoside aryl phosphoramidate approach, developed by mcguigan and co-workers (2004) has been successfully applied to a number of different nucleosides (azt, d4t, dda, d4a). the general structure of aryl phosphoramidates encompasses two masking groups, an amino acid ester and an aryl moiety bonded to the phosphate group. in order to apply this protide technology to nucleosides with the potential for anti-hepatitis c virus (hcv) activity, we have undertaken studies designed to probe the effect of varying the natural and unnatural amino acid esters and the aryl groups used as masking groups in the target phosphoramidates. these compounds have been synthesised and evaluated using genotipe 1b sub-genomic hcv replicon. we have prepared a variety of arylphosphoramidate derivatives from a range of 4 -substituted nucleosides, including 4azido-cytidine, 4 -azido-uridine, and 2 ,3 -protected variants. with certain nucleoside phophoramidates, we have observed dramatic enhancement (>1000-fold) of replicon activity relative to the parent nucleoside. the synthesis, biological activity and sar of these compounds will be presented. reference mcguigan, d. cahard, balzarini, j., 2004. mini-review. med. chem. 4, 371-382 . we have identified a series of novel anthranilic acid derivatives that are potent, reversible inhibitors of hepatitis c virus (hcv) ns5b polymerase, an essential enzyme for viral replication. the micromolar ns5b polymerase inhibitors belong to the n-phenoxyacetylanthranilic acid chemotype. x-ray crystallography determined that the inhibitors bound to ns5b between the thumb and palm regions adjacent to the active site. guided by crystallography, subsequent modifications to the hydrogen bonding and lipophilic regions of the inhibitors resulted in greatly improved activity against ns5b. further sar studies revealed a second, more potent sub-series where the phenoxy group was replaced by an anilino group. analogs in both subseries showed antiviral activity in a cell-based replicon model of hcv. andrea brancale 1 , dimitrios vlachakis 1 , maria chiara barbera 1 , romano silvestri 2 , colin berry 3 , johan neyts 4 1 cardiff university, the welsh school of pharmacy, cardiff cf10 3 xf, uk; 2 universita' degli studi "la sapienza", dipartimento di studi farmaceutici, 00185 roma, italy; 3 cardiff university, cardiff school of biosciences, cardiff cf10 3us, uk; 4 rega institute for medical research, k.u. leuven, b 300 leuven, belgium hepatitis c is a viral infection that affects 170 million people worldwide, including 4 million in the united states and 8 million in europe. the virus establishes a chronic infection in 55-85% of cases and 20% of affected individuals develop cirrhosis. at the moment there is neither a vaccine nor an effective antiviral therapy available and efforts to identify a specific anti-hcv inhibitor have dramatically intensified in the last few years. many research groups have focused their interest on the enzymes involved in the viral replication and, among these enzyme, the viral helicase/ntpase has proven to be a suitable target for developing novel anti-hcv compounds. compound 1 is a potent inhibitor of the hcv helicase and, although its mode of action is still uncertain, it has been proposed that it acts as competitive inhibitor of rna binding. starting from this hypothesis, we have prepared a series of novel compounds based on the structure of 1 where the benzimidazole moiety has been replaced by different chemical groups, including the negatively charged carboxylate moiety, which should mimic the phosphate backbone of the nucleic acid. the synthesis, the enzyme inhibition and the biological evaluation in replicon of these novel compounds will be presented and analyzed. dale r. cameron migenix inc., 3650 wesbrook mall, vancouver, bc, canada v6s 2l2 hepatitis c virus (hcv), a leading cause of liver disease, continues to be an attractive target for new drug development. among the more favourable approaches to developing new hcv drugs is to target the rna-dependant rna (rdr) polymerase (ns5b), which has been shown to be an essential enzyme for replication. there are several published non-nucleoside inhibitors of this polymerase (some in clinical development) and several published allosteric binding pockets on the protein they target. to be successful, traditional lead identification can be timeintensive, costly and have large infrastructure requirements. increasingly, a push towards effective computational-based screening has led to the development of virtual screening tools. such tools allow investigation of large quantities of compounds in silico for particular properties without the need for compound synthesis or high throughput screening. moreover, these techniques require only a modest infrastructure investment and are very efficient. we employed the openeye set of screening tools (omega, rocs and eon) in concert with publicly available hcv inhibitor information, and commercial databases to identify novel leads. the inhibitor coordinates from a protein-inhibitor complex crystal structure were utilized as the target. available compound databases (asinex and chembridge) were utilized as the testset of compounds. filtered compound conformers were generated using omega and compared with the template using rocs with post-analysis by eon. visual analysis to maximize particular desirable binding features while minimizing protein-inhibitor steric clash allowed the list of potential hits to be further narrowed. multiple classes of compound were identified from the above procedure and after sourcing a subset of the actual compounds or close analogs, they were tested for enzymatic inhibition activity and further characterized. iteration of the process resulted in the identification of a lead compound class containing multiple active compounds, one with reasonable replicon activity. in conclusion, readily available structural and database information and virtual screening tools can be successfully utilized to identify novel inhibitors of hcv rdr polymerase which, in turn, can serve as novel leads for developing new therapies for treating hcv. synthesis, antiviral activity, and cytotoxicity of some novel quinazolin-4(3h)-one derivatives a series of novel 6-bromo/6,8-dibromo-4-(4-oxo-2-phenyl-4h-quinazolin-3y-l)-benzenesulphonamides were synthesized by condensation of 2-substituted benzo[1,3]oxazine-4-ones and sulphonamide. their chemical structures were assigned by means of spectral analysis (ft-ir, pmr, ms). synthesized compounds were screened for in vitro antiviral activity against human pathogenic viruses (hiv, hcv, hsv, vv). 6-bromo-4-(4-oxo-2-phenyl-4h-quinazolin-3y-l)benzenesulphonamide (sps-ii) and 4-(4-oxo-2-phenyl-4hquinazolin-3y-l)-benzenesulphonamide (sps-i) inhibits the replication of hiv-1 in acutely infected mt-4 cells at a concentration of approximately 10 g/ml, while not being toxic to the host cell at a concentration of 60 or >125 g/ml (selectivity index: 8 and >12), respectively. in huh 5-2 cells sps-i inhibited hcv rna synthesis at ec50 of 8 g/ml, while at cc50 for cell growth 32 g/ml. sps-ii inhibited the virus-induced cytopathicity in human embryonic lung (hel) cell infection with hsv-1, hsv-2 or vaccinia (vv) at a concentration of 50 g/ml, while not being toxic to the cells up to a concentration of 400 g/ml. further molecular modification in this series of compounds may help in optimising their antiviral activity. wengang yang, yongnian sun, avinash phadke, milind deshpande, mingjun huang achillion pharmaceuticals, new haven, ct 06511, usa hcv nonstructural protein ns5b is the catalytic subunit of the replication complexes, possessing a motif characteristic of rna-dependent rna polymerases. biochemical assays using recombinant ns5b have been used to investigate ns5b nonnucleoside inhibitors. however, the inhibitory effect of compounds often varies with the forms of recombinant ns5b and the concentrations of the template and/or primer used in the assays. in addition, it does not always correlate to that obtained with replicon-containing cells. these observations have cast concerns about the validity of these cell-free assays. in the report, we explored replication complexes, isolated as crude membrane fractions from replicon-containing cells, for their competency to synthesize viral rna in vitro as well as their responsiveness to ns5b inhibitors. after optimizing the experimental conditions, two species of nascent viral rna, one double-stranded and the other single-stranded, were readily detected. the addition of ns5b nucleotide inhibitor blocked synthesis of both species. the presence of nonnucleoside inhibitors, however, inhibited mostly single-stranded rna (ssrna) synthesis. in addition, the replication complexes isolated from the cells containing a replicon that carried a resistant mutation in ns5b to the nonnucleoside inhibitor were able to synthesize the same amount of ssrna in vitro regardless of the presence or absence of the inhibitor, demonstrating that the phenomenon is due to the specific inhibitory effect of the compound on ns5b. combining with kinetic studies that ssrna synthesis was inhibited only when the nonnucleoside inhibitor was present during the pulse period, we conclude that ssrna synthesis catalyzed by the replication complexes in vitro is likely derived from the de novo initiation. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz-51, that inhibits both hcv and hiv in vitro with ec 50 values ranging in micromolar concentrations or less, and little or low toxicity to the host cells. in this part ii of the presentation on this subject, we report our preliminary results on mechanistic studies of anti-hcv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues in the series. in light of the fact that hcv is a major co-infection in patients infected with hiv, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. nigel bourne, ronald veselenak, richard pyles, minkyung yi, stanley lemon the university of texas medical branch, galveston, tx, usa more than 170 million people worldwide are estimated to be infected with hepatitis c virus (hcv). in the majority of these people a chronic infection is established which can result in serious long-term liver damage including progressive fibrosis, cirrhosis and hepatocellular carcinoma. in fact, hcv is believed to cause more than 100,000 cases of liver cancer annually worldwide and accounts for at least 40% of liver transplants in the us. current treatment options are limited and there is a high treatment failure rate. thus, there is a real need for new treatment options. amantadine has been evaluated as a treatment for chronic hcv infection in a number of clinical studies both as a monotherapy and in combination with other therapeutics. however, the results of these trials have been contradictory and at this time the clinical potential of amantadine as a therapy for chronic hcv infection remains unclear. recent studies have shown that the small hydrophobic hcv p7 protein forms an amantadine sensitive ion channel providing a possible basis for antiviral activity. we examined the ability of amantadine to reduce hcv replication in both subgenomic and full-length hcv replicons of genotypes 1a strain h77c and genotype 1b strain n. in these studies amantadine failed to reduce viral rna replication in any of the replicons tested. further, in infectious virus assays using hcv genotype 2a strain jhf-1 10 um amantadine failed to reduce viral rna levels under any of the conditions tested. however, in these infectious virus studies, when the viral inoculum was treated with amantadine prior to infection of cell monolayers, or when the amantadine was added to cells 1 h after virus adsorption there was a significant reduction in the number of infectious viral foci observed after 72 h incubation (p < 0.05 and <0.01, respectively). these results suggest that even in the absence of a direct impact on rna replication amantadine has antiviral activity. we are currently evaluating amantadine for activity in infectious hcv genotype 1a assays to further define its antiviral spectrum of activity. studies to more fully define its mechanism of action in the virus life cycle are also underway. dominique dugourd, raymond siu, jeremy fenn migenix inc., vancouver, bc, canada celgosivir is an alpha glucosidase inhibitor that is being developed for the treatment of hepatitis c virus (hcv) infections in humans. the purpose of this study was to evaluate the in vitro antiviral activity of celgosivir and its primary active metabolite, castanospermine, when combined with current approved therapies (ribavirin, interferon ␣-2b, or both) in a surrogate model of hcv (bovine viral diarrhea virus (bvdv)). compounds alone or in combination were tested against bvdv in infected madin-darby bovine kidney (mdbk) cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). the celgosivirinterferon ␣2b combination was significantly more synergistic than the celgosivir-ribavirin combination (∼3-fold), or the ribavirin-interferon ␣2b combination (∼2-fold). similarly, the castanospermine-interferon ␣2b double combination was more synergistic than the castanospermine-ribavirin combination (∼5-fold), or the ribavirin-interferon ␣2b combination (∼3.3-fold). the combinations of celgosivir-interferon ␣2b or castanospermine-interferon ␣2b led to significant decreases in the ec50s of celgosivir (up to >20-fold) and castanospermine (up to >50-fold). the effective ec50s of celgosivir or castanospermine were further reduced by the addition of ribavirin. the cytotoxicity of the double and triple combinations was additive or less than additive, indicating that combinations of celgosivir or castanospermine with ribavirin and/or interferon ␣2b were generally less toxic than expected. these results indicate that the combination of celgosivir with interferon ␣2b or with interferon ␣2b and ribavirin may be effective in the treatment of hcv. pegylated interferon ␣ plus ribavirin is the current standard of care for the treatment of chronic hepatitis c virus (hcv) infections. this regimen results in sustained virologic response in only about 50% of patients and is associated with significant treatment-associated toxicities. a number of approaches are being used to identify novel therapeutic combinations with better tolerability and/or efficacy. inhibitors of endoplasmic reticulum (er) ␣-glucosidase have been shown to inhibit viral replication and secretion and may have utility as part of new multi-drug treatment cocktails. the ␣-glucosidase inhibitor celgosivir is currently being evaluated in combination with pegylated interferon ␣ and ribavirin in humans. the purpose of this study was to evaluate the antiviral effects of combinations of celgosivir and castanospermine, the primary active metabolite of celgosivir, with other antiviral agents having diverse mechanisms of action. the effect of the combination of celgosivir or castanospermine with the nucleoside analogue nm-107, amantadine, and another iminosugar, n-butyl-deoxynojirimycin (nb-dnj) was determined in a cytopathic assay using the hcv surrogate virus bovine viral diarrhea virus in madin darby bovine kidney cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). volumes of synergy indicated that the castanospermine and nb-dnj combination was additive, while the celgosivir and nb-dnj combination was synergistic at high nb-dnj concentrations (>100 m). celgosivir and castanospermine were synergistic with both amantadine and nm-107, with volumes of synergy between 60 and 150 m%. isobologram analysis confirmed these synergistic interactions. these results indicate that celgosivir could be considered in combination regimens containing drugs that directly target viral replication like nm-107. mechanism(s) of synergy are under investigation. department of biotechnology, yonsei university, seoul 120-749, korea hepatitis c virus (hcv) is an enveloped virus with positivestranded rna genome of approximately 9.6 kilobases and a major cause of non-a and non-b hepatitis, leading to liver cirrhosis and hepatocellular carcinoma. combination of interferon-␣ (ifn-␣) and ribavirin is the current standard therapy for the treatment of hcv infection, but there is no specific antiviral therapy available. the hcv viral genome encodes a single polyprotein of approximately 3010 amino acids, which is proteolytically processed by a combination of host and viral proteases into at least 10 distinct structural and nonstructural proteins. the structural proteins include c, e1, e2, and p7 and the nonstructural (ns) proteins include ns2, ns3, ns4a, ns4b, ns5a, and ns5b. as new hcv specific therapies, small-molecule inhibitors against hcv enzymes including ns5b protein, the viral rna-dependent rna polymerase (rdrp), and ns3 protease are in clinical tests. however, rapid emerging of drugresistant mutants has been hampering their practical clinical applications. recently, we have shown that phosphorylation of hcv rna polymerase by protein kinase c-like 2 (prk2) regulates virus rna replication. hcv rna replication was inhibited when prk2 expression level was down-regulated by using a prk2-specific sirna. in this study, we investigated the anti-hcv effect of prk2 inhibitors in an hcv subgenomic replicon system. treatment of the replicon cells with prk2 inhibitors suppressing the endogenous prk2 activity inhibited the phosphorylation of hcv rna polymerase and resulted in suppression of hcv rna replication in a dose-dependent manner. furthermore, the prk2 inhibitor in combination with ifn-␣ more effectively inhibited hcv rna replication than ifn-␣ alone. because the prk2 inhibitor did not show cytotoxicity in the cell-based drug inhibition studies and cellular proteins rarely get mutated, prk2 can serve as a cellular target for therapeutic intervention of hcv replication. specific inactivation of prk2 activity will provide an opportunity to interfere with hcv rna replication. haitao guo 1 , tianlun zhou 2 , ju-tao guo 1 , andrea cuconati 3 , anand mehta 1 , timothy block 1 1 drexel university college of medicine, doylestown, pa, usa; 2 nucleonic inc., irvine, ca, usa; 3 hepatitis b foundation, doylestown, pa, usa more than 400 million people worldwide are chronically infected with hepatitis b virus (hbv). the major complication of chronic hepatitis b is the development of primary hepatocellular carcinoma (hcc), which causes an estimated 500,000 deaths annually. currently clinical treatments (␣-interferon and nucleoside analogs) of chronic hepatitis b rarely cure the virus infection. this is due, at least in part, to their failure to eliminate viral covalently closed circular (ccc) dna from the nuclei of infected hepatocytes. hbv cccdna is essential to the virus life cycle by serving as the template for the transcription of the pregenomic rna and of the subviral rna species. its elimination during chronic infection is considered critical to long-term therapy. however, cccdna has not previously been targeted in high throughput screens of small molecule libraries. to screen compound libraries for antiviral drugs targeting cccdna, we set out to develop a cell-based assay suitable for high throughput screening. since cccdna is time-consuming to assay, it was desirable to use a viral gene product that could serve as a reporter for intracellular cccdna level. we predicted that the secretion of hbv e antigen (hbeag) by hepad38 cells, a hepg2-derived tetracycline inducible hbv expression cell line, would be cccdna-dependent. this is because a large portion of pre-core mrna leader sequence in the 5 terminus of integrated viral genome was deleted, preventing hbeag expression from transgene, but could be restored from the 3 terminal redundancy of pre-genomic rna during viral dna replication and subsequent cccdna formation. our experimental results showed that following induction, hepad38 produced and accumulated cccdna, which became detectable between 7 and 8 days. hbeag synthesis and secretion into culture fluid were dependent upon and proportional to the level of cccdna detected. therefore, the secretion of hbeag by hepad38 cells could potentially serve as a convenient reporter for the high throughput screening of novel antiviral drugs targeting hbv cccdna. kathy aldern, james beadle, karl hostetler university of california, san diego and the veterans medical research foundation, san diego, ca, usa (s)-hpmpa is a broad spectrum antiviral active against orthopoxviruses, hbv, cmv, hsv, and other herpes group viruses. we have shown that hdp-(s)-hpmpa has greatly enhanced antiviral activity against these viruses. in addition, while hpmpa itself is nearly inactive against hiv, we showed that hdp-(s)-hpmpa exhibited an ec50 >3 logs less than unmodified hpmpa in mt-2 cells by p24 reduction assay. to evaluate the metabolic basis for the increased antiviral activity, we studied and compared the cellular uptake of radiolabeled cdv, (s)-hpmpa and their hdp-esters and conversion to hpmpa-diphosphate (hpmpapp) and cdv-diphosphate (cdvpp) in mrc-5 human lung fibroblasts using hplc partisil sax ion exchange chromatography. cellular uptake of hdp-cdv and hdp-(s)-hpmpa was similar. however, when cells were exposed to the respective drugs for 6, 24 and 48 h, hpmpapp appeared much earlier than cdvpp and reached levels several fold greater than observed with hdp-cdv. drug wash out experiments were carried out in mrc-5 cells exposed to radiolabeled hdp-cdv and hdp-(s)-hpmpa. after 24 h, the culture medium was removed and replaced with complete medium without drug and the levels of hpmpapp and cdvpp were measured by hplc every 2 days for 0-10 days. levels of the diphosphates declined slowly with a t 1/2 of 5-10 days. in conclusion, hdp-(s)-hpmpa is converted to its diphosphate more rapidly than hdp-cdv and reaches higher intracellular levels. this may explain, in part, its greater antiviral activity. the antiviral activity and oral bioavailability of cidofovir (cdv) is enhanced when the phosphonate is esterified with various straight chain alkoxyalkyl groups. the length of this chain is an important determinant of antiviral activity and selectivity. however, in some cases, rapid metabolism to an inactive short chain metabolite was observed. to enhance the metabolic stability of these esters, we synthesized cidofovir alkoxyalkyl esters bearing methyl groups on the penultimate carbon of the alkyl chain. enzymatic stability of 15-me-hdp-cdv (1) was tested in liver s9 fractions from various species. in mouse and human liver s9 fractions, compound 1 was completely stable for 90 min while 15-20% of the straight chain hdp-cdv was metabolized. the branched alkoxyalkyl esters were then evaluated in cells infected with vaccinia, cowpox and ectromelia viruses. the branched methyl analogs were substantially more active than cdv and equal to or slightly more active than the straight chain analogs. compound 1 retained full activity compared to hdp-cdv and compound 2 showed greater activity against orthopoxviruses compared to its unbranched analog. we believe that the structural modification of the alkyl chain slows the formation of inactive metabolites, possibly by interfering with oxidation and may result in better pharmacokinetics and more potent antiviral activity against orthopoxvirus infection in vivo. cidofovir ([1-(s)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, hpmpc) is a broad spectrum antiviral agent clinically used for treatment of aids-related cmv retinitis. cidofovir has limited oral bioavailability (<5%), attributed to ionization of its phosphonic diacid moiety under physiological conditions. we have shown that masking of this group by conjugation of the cyclic form of the drug (chpmpc) via a ser side chain p-o ester linkage with x-ser dipeptides, where x = a hydrophobic amino acid, can result in prodrugs 1 that afford significantly improved biological availability of parent drug in an animal model. here we describe the total synthesis of novel cyclic cidofovir prodrugs 2ab of l-val and l-phe using an alternative conjugation strategy, viz. via an ethylene glycol link utilizing p-o and c-o ester bonds. the preparation of the hpmpc synthon from r-glycidol used our modification of the literature procedure (brodfuehrer et al., 1994) , involving reaction of tritylated (r)-glycidol directly with unprotected cytosine to achieve regiospecific opening of the epoxide ring, followed by reaction with benzoic acid anhydride to obtain the desired n-benzoyl intermediate needed to continue the synthesis. pybop was used as condensing agent in a convenient, one-pot conversion of hpmpc to chpmpc and subsequent esterfication of the latter by the ethylene glycol-modified amino acids. the prodrugs 2 were converted to drug by cellular (caco-2, hff) and tissue (liver and intestinal) homogenates, but did not show enhanced oral bioavailability when evaluated in a rat model, suggesting that such compounds may be useful for understanding the effectiveness of 1 in drug delivery. cidofovir (cdv) is a broad-spectrum anti-viral agent that is used to treat aids-related cytomegalovirus (cmv) retinitis and other cmv infections. cdv has good in vitro activity against orthopox viruses, including smallpox; however, its use is limited because of the drug's low oral bioavailability and poor transport into cells. in order to improve its oral bioavailability, our group has synthesized a series of dipeptide and amino acid prodrugs of the cyclic analog of cidofovir (ccdv). in the current project, we examined the cytotoxicity and antiviral activity of the prodrugs, showing that the compounds are not cytotoxic and have diverse activity against hcmv and orthopox viruses (vaccinia and cow pox) with 50% inhibitory concentrations ranging from 0.1 to 0.5 and 10 m and greater, respectively for the two virus types. in vitro and in situ perfusion studies established that the permeability of the prodrugs is enhanced more than 30-fold and that the transport is mediated, at least in part, by the intestinal dipeptide transporter. we also have found that the bioavailability of the prodrugs is dependent upon the prodrug structure and that we can achieve up to an eight-fold increase in bioavailability over the parent compound in vivo. drug stability experiments showed that in gastrointestinal and liver homogenates, the ccdv prodrugs are enzymatically hydrolyzed to the parent compound. it is clear from this work that the biologically benign dipeptide moiety, strategically linked to the drug to mask its anionic properties, significantly enhances intestinal transport of ccdv, creating the possibility of an orally bioavailable form of ccdv with low toxicity. acknowledgement: supported by funds from tsrl inc, the university of michigan, and nih grants r43ai056864 and u01ai061457. lawrence trost 1 , bernhard lampert 1 , lloyd frick 2 , merrick almond 1 , george painter 1 1 chimerix, inc., durham, nc, usa; 2 dmpk advisor, chimerix, inc., durham, nc, usa foscarnet, a pyrophosphate analog approved for the treatment of cmv retinitis and acyclovir-resistant herpes infections in immunocompromised patients, is active against highly drug resistant strains of hiv-1. however, the clinical utility of foscarnet is limited because it requires controlled intravenous infusion and is associated with high risks of renal impairment and seizure caused by alterations in plasma minerals and electrolytes. lipid conjugation has been shown to increase the in vitro activity, improve oral bioavailability, and reduce the toxicity of several antiviral drugs requiring intravenous administration because of poor bioavailability. in the case of foscarnet, conjugation to methylbatyl alcohol (cmx012) decreases the apparent ec 50 value against hiv-1 by up to 40-fold. cmx012 was esterified to produce cmx052 in order to increase solubility and to protect against decarboxylation of the foscarnet moiety during passage through the stomach. here we present the results of a preliminary toxicology and toxicokinetic study of the methylbatyl alcohol conjugate of foscarnet methyl ester (cmx052). rats were given oral doses of 10, 30 and 100 mg/kg cmx052 daily for 7 days. there were no clinical signs of toxicity. body weight and food consumption were comparable to controls and serum biochemistry, hematology, coagulation parameters and urinalysis were normal. there were no gross findings at necropsy, no effects on organ weights and no findings by histopathological examination of a wide range of tissues. importantly, there were no changes in serum biochemistry parameters or histopathological examination that were indicative of the renal impairment or serum electrolyte changes that are associated with foscarnet. oral dosing resulted in significant plasma exposure to cmx012 (c max > 4 g/ml), the biologically active deesterified form of cmx052. in conclusion, cmx052 is absorbed after oral administration, converted to cmx012, and has a good preliminary toxicity profile. these results support the development of cmx052 for the treatment of drug resistant hiv infection. zhiqian wu 1 , julie breitenbach 2 , ulrika erickson 1 , john hilfinger 3 , john drach 2 , gordon amidon 1 1 department of pharmaceutical sciences, college of pharmacy, the university of michigan, ann arbor, mi, usa; 2 school of dentistry, the university of michigan, ann arbor, mi, usa; 3 tsrl, inc., ann arbor, mi, usa vaccinia virus is a surrogate model system for study of pox virology and development of antiviral therapeutics. the potent anti vaccinia virus activity and various shortcomings of vidarabine make it a good candidate for improvement by utilizing prodrug strategy. vidarabine is a polar nucleoside drug with low membrane permeability and rapid degradation by adonesine deaminase. 5 -monoester prodrugs of vidarabine with various amino acids promoieties (l-valine, l-isoleucine, l-phenylalanine. laspartic acid, l-proline) are synthesized and evaluated for their stability, permeability and activity against vaccinia virus. prodrugs exhibit different hydrolysis rate in caco-2 cell homogenate (t1/2: 2-40 min). 5 -l-isoleucyl and 5 -l-valyl monoester prodrugs exhibit comparable bio-conversion rate and hpept1mediated uptake as well as caco-2 permeability with valacyclovir, a commercially marketed oral amino acid ester prodrug. both prodrugs have potent activity against vaccinia virus and are resistant to ada1. preliminary animal study shows 5 -lisoleucyl vidarabine results in >10-fold increase in circulating vidarabine level. the results suggest that it may be possible to use amino acids prodrug strategy to improve vidarabine as anti vaccinia virus agent. vidarabine [9-␤-d-arabinofuranosyl)adenine or ara-a) was originally investigated as an anti-tumor agent and was later found to be active against herpes simplex virus (hsv) types 1 and 2. it was the first fda-approved drug for treatment of systemic hsv infections. although replaced by acyclovir and analogs for most applications, vidarabine remains an alternative therapy for acyclovir-resistant hsv and varicella-zoster virus infections. despite its proven efficacy, vidarabine suffers some limitations including: (i) metabolism by adenosine deaminase (ada) to its inactive hypoxanthine homolog (ara-h); (ii) low lipophilicity and membrane permeability and (iii) poor aqueous solubility, thus limiting options for parenteral and peroral delivery. our recent interest in vidarabine was triggered by our discovery that it was ∼5-fold more active against vaccinia (vv) and cow pox (cpv) viruses than was cidofovir in plaque reduction assays. its activity was enhanced about 10-fold by combination with 1 m 2 -deoxycoformycin (pentostatin, a potent inhibitor of ada) thereby providing significant superiority to cidofovir. from these results and our earlier studies on 5 -substituted vidarabine analogs (lipper et al., 1978. mol. pharmacol. 14, 366-369), we determined that minimizing metabolism of vidarabine by synthesizing 5 -amino acid substituted prodrugs gave a significantly more potent anti-pox virus agent. we found that amino acid ester prodrugs of vidarabine are active against vv at non-cytotoxic concentrations. further, using cell homogenates, purified enzyme and intact cell systems, we showed that the prodrugs are resistant to inactivation by ada. the prodrugs also had enhanced transport potential, most likely targeting the intestinal dipeptide transporter. finally, oral delivery of the prodrug to the small intestine resulted in a 10-fold increase in vidarabine plasma levels when compared to unsubstituted vidarabine. these properties make the prodrugs of vidarabine good candidates as orally bioavailable anti-pox virus agents that are stable in the presence of ada. acknowledgement: supported by funds from tsrl inc. and the university of michigan. ulf goerbig 1 , anne baum 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katolieke universiteit leuven, leuven, belgium the cyclosal pronucleotide system has been designed for an intracellular delivery of therapeutically active nucleoside monophosphates. as part of recent work on the cyclosal approach, the interaction of cyclosal nucleotides with cholinesterases has been investigated. it is known that organo-phosphates may act as irreversible inhibitors of cholinesterases (suicide mechanism). in the case of cyclosal nucleotides, cholinesterase inhibition could lead to unwanted side effects in a possible therapeutic application. there are two types of cholinesterase found in humans, the highly specific, physiologically important acetylcholinesterase (ache) and the much more unspecific butyrylcholinesterase (bche) of unknown physiological importance. fortunately, no inhibition of ache was observed for a variety of different cyclosal nucleotides. in contrast, bche inhibition was found in some cases. the anti-hiv-active 3,5-bis-tertbutyl-6-fluoro-cyclosal-d4t monophosphate is the first cyclosal derivative combining three desired properties: successful intracellular delivery of nucleotides, sufficient hydrolytic stability and strongly reduced inhibitor activity towards bche. because of the promising properties of this compound, we combined this mask developed for d4t with the antiviral active nucleoside analogues like d4a, dda, azt and acyclovir. in this contribution we present the synthesis, hydrolysis stability, inhibition behaviour towards bche and anti-hiv data of these new compounds. henning jessen 1 , wolfgang fendrich 1 , tilmann schulz 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany, 2 rega institute for medicinal research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides are used for the delivery of antivirally active nucleotides into cells via a ph triggered selective hydrolysis. to distinguish between intra-and extra-cellular environment enzyme-cleavable side chains were introduced in the aromatic moiety of the pronucleotide to enrich the compound inside cells. this behavior will further be described as "lock-in"effect. many other different cyclosal pronucleotides have been designed, all showing different hydrolysis properties and antiviral data, both originating from the nature of the cyclosal-moiety as well as of the nucleoside. to examine these differences, analytical tools of high accuracy and sensitivity were needed, being structurally as close as possible to the lead compounds. these requirements are met by intrinsically fluorescent nucleosides coupled to different cyclosal masking groups. for analysis of the purine-type nucleosides iso-da with high intrinsic fluorescence properties was chosen and converted into iso-a, iso-dda and iso-d4a. for the pyrimidine-type series the fluorescent nucleoside m 5 k was synthesized as well as the dideoxy-compound dm 5 k. these nucleosides were transformed into different cyclosalpronucleotides and tested for their suitability for fluorescence analysis. in fact, an improvement of sensitivity by a factor of 5000 compared to uv-detection was found for some of the compounds (pmol detection). for all compounds fluorescence and absorbance spectra were recorded to determine the absorption and emission maxima. the new compounds lacked activity against hiv-1 and hiv-2 strains. however, the compounds showed low cytotoxicity, which is important for their usability as fluorescent probes in cells. due to the analytical sensitivity, a simple model uptake study could be carried out, employing an u-tube with two aqueous phases, which were separated by an unpolar organic solvent simulating a diffusion barrier. the properties of the aqueous phases were varied and an enzyme-driven enrichment of a "lock-in"-modified intrinsically fluorescent cyclosal-pronucleotide passing the diffusion barrier could be simulated. nicolas gisch 1 , jan balzarini 2 , chris meier 1 1 university of hamburg, institute of organic chemistry, hamburg, germany; 2 rega institute for medical research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides efficiently deliver therapeutically active nucleoside monophosphates in human cells. "lock-in"-cyclosal-pronucleotides -the so-called second generation of cyclosal-compounds -have been designed to trap the compound by intracellular cleavage of esterase-cleavable moiety. one disadvantage of the "lock-in"-compounds is their high chemical stability, which leads to a delayed drug delivery. therefore, conceptually different, enzymatically activated cyclosalpronucleotides have been developed. in this concept lipophilic donor substituents attached to the aromatic ring are converted into a polar acceptor substituent by intracellular enzymatic cleavage. as a consequence the liberated acceptor group leads to a strong decrease in hydrolysis stability and a rapid formation of a charged intermediate is the result. from the phosphodiester intermediate the nucleotide is released subsequently. the concept, synthesis, characterization and in vitro antiviral evaluation of the third generation of cyclosal-pronucleotides will be presented. tomas cihlar 1 , richard mackman 1 , adrian ray 1 , dean boojamra 1 , lijun zhang 1 , deborah grant 1 , hon hui 1 , jennifer vela 1 , neil parkin 2 , yolanda lie 2 , kirsten white 1 , michael miller 1 , gerry rhodes 1 , manoj desai 1 1 gilead sciences, foster city, ca, usa; 2 monogram biosciences, so. san francisco, ca, usa background: n(t)rtis are currently used as a backbone of antiretroviral combination therapy. however, their long-term benefit can be limited by adverse effects, resistance development, drug-drug interactions, and sub-optimal efficacy in treatment-experienced patients. therefore, we searched for novel nucleotide analogs with improved pharmacological profiles. methods: phosphonomethoxy-2 -fluoro-2 ,3 -dideoxydidehydroadenosine (gs9148) was selected from a broad range of nucleoside phosphonate analogs. phosphoramidate prodrug technology previously explored with tenofovir was applied to gs9148, resulting in the identification of gs9131 (ethylalaninyl phenyl ester of gs9148). results: gs9131 exhibits potent anti-hiv-1 activity in primary lymphocytes and t-cell lines (ec 50 < 150 nm). low cytotoxicity (cc 50 > 100 m) was observed in multiple cell types including renal cells. diphosphate metabolite of gs9148 was shown to act as an obligatory dna chain terminator and a competitive inhibitor of hiv-1 reverse transcriptase (rt) (k i = 0.8 m). unlike ddi, d4t, or d4fc, neither gs9148 nor its prodrugs inhibited mitochondrial dna replication in hepg2 cells. in a phenosense assay, gs9148 retained its full activity against hiv-1 variants with k65r, m184v or l74v mutations in rt. viruses with ≥4 thymidine analog mutations showed ≤2fold reduced susceptibility to gs9148, a shift that was smaller than that of any other tested nrti. following an oral dose of 3 mg/kg gs9131 in dogs, the bioavailability of prodrug exceeded 20%, resulting in high intracellular levels (9.0 ± 2.3 m) and prolonged retention (t 1/2 > 24 h) of gs9148 diphosphate in blood lymphocytes. conclusions: both gs9148 and its prodrug gs9131 exhibit favorable in vitro pharmacological profiles including less resistance due to rt mutations than approved nrtis. gs9131 possesses good in vivo pharmacokinetic properties and thus represents an attractive development candidate with potential for clinical efficacy in both treatment-naive and nrti-experienced patients. martin mcdermott, gabriel birkus, ruth wang, holly macarthur, xiaohong liu, nilima kutty, tomas cihlar, craig gibbs, swami swaminathan, arnold fridland, william lee gilead sciences, inc., foster city, ca, usa gs-7340 and gs-9131 are alkylalaninyl phenyl ester prodrugs of tenofovir (tfv; 9-[(2-phosphonomethoxy)propyl]adenine) and a novel nucleotide analog fd4ap (phosphonomethoxy-2 -fluoro-2 ,3 -dideoxydidehydroadenosine), respectively. both gs-7340 and gs-9131 exhibit potent in vitro anti-hiv-1 activity, favorable resistance profile, and low cytotoxicity. compared to tenofovir disoproxil (viread), both prodrugs are significantly more stable in plasma and deliver >10-fold greater levels of active diphosphate metabolites into pbmcs in vitro and in vivo. the initial step in the intracellular activation of gs-7340 and gs-9131 is the hydrolysis of the alanine carboxyester by an unknown hydrolytic enzyme. the isolation and identification of this enzyme from human pbmcs is reported here. results: a major enzyme capable of cleaving gs-7340 and gs-9131 was purified from human pbmcs and was separable from esterases able to cleave alpha napthyl acetate (ana). the increase in specific activity of prodrug hydrolase achieved was 3500-fold. sds-page analysis showed the presence of a prominent protein band at 29 kda, which was identified by ingel tryptic digestion and ms/ms sequencing of the resultant peptides as lysosomal carboxypeptidase a (cathepsin a, ec 3.4.16.5, cata). the biochemical properties of purified prodrug hydrolase matched those of cata. recombinant cata and the isolated prodrug hydrolase displayed nearly identical susceptibility to hydrolase inhibitors and substrate preference against a panel of tenofovir phosphoramidate prodrugs. incubation of both enzymes with [ 14 c]gs-7340 and [ 3 h]difluorophosphonate resulted in the labeling of an identical 29 kda protein (catalytic subunit). both labeled bands reacted with polyclonal antibodies specific for human cathepsin a. finally, following incubation with gs-7340, approximately 6-9-fold lower intracellular concentrations of tfv metabolites were detected in fibroblasts from patients expressing non-functional cat a (cat a-cells) compared to normal control fibroblasts (cat a+ cells). center for drug delivery and nanomedicine, university of nebraska medical center, omaha, ne, usa nucleoside 5 -triphosphate (ntp) is the biologically active form of many antiviral nucleoside analogs capable of efficiently blocking the production of viral nucleic acids in infected cells. we describe novel microparticulate formulations for encapsulation of ntp, drug delivery and antiviral therapy of respiratory infections. polymer networks (poloxagels) consisted of crosslinked poloxamers and cationic polymer molecules were designed, synthesized and characterized by loading with ntp and interaction with cells. poloxagel-ntp formulations could be obtained by simple mixing of the aqueous solution of ntp with the aqueous dispersion of poloxagel and subsequent lyophilization. drug loading was equal up to 30% by weight. in this form, phosphates groups of ntp are complexed with amino groups of polycationic backbone of poloxagels, and the formulations could be stored at room temperature for many months without degradation of ntp. the particle size of aqueous poloxagel-ntp dispersions was low, with a hydrodynamic diameter of 0.1-0.2 m. the rate of passive drug release in physiological solution was from 33 to 50% of loaded drug during the 24-h period. these formulations were effectively consumed by many types of cells. significant amounts of drug and poloxagels were detected in the cellular interior after only 1-2 h of incubation. in the presence of cellular membranes drug release from poloxagel-ntp formulations was dramatically increased. we attribute this effect to the triggered release of the bound ntp as a result of competitive interaction of polycationic backbone of poloxagels with phospholipids of cellular membranes. mucoadhesive properties of poloxamers may additionally enhance binding of poloxagels with airways/lung epithelium. 5 -triphosphate of 1-␤-d-ribofuranosyl-1h-1,2,4-triazole 3-carboxamide (ribavirin) was synthesized using phosphorylation with a tris(imidazolyl) phosphate in a convenient one-pot approach. formulations of different poloxagels with the ribavirin 5 -triphosphate were prepared and characterized as prospective antiviral formulations for prophylactic and therapeutic treatments of respiratory infections including influenza a virus. aerosolic route of application of these antiviral formulations and associated problems are discussed. edwin gong 1 , ebrima gibbs 2 , joel oger 2 1 department of pharmacology and therapeutics, the university of british columbia, vancouver, bc, canada; 2 neuroimmunology lab, ubc hospital, department of medicine, the university of british columbia, vancouver, bc, canada ifn-alpha and ifn-beta are currently employed in the treatment of many viral diseases, especially chronic hepatitis. ifn-beta is also employed for the treatment of multiple sclerosis (ms), a chronic and often debilitating disease of the central nervous system. however, as with other protein therapeutics, long-term ifn therapy can lead to the development of binding and neutralizing antibodies to ifns and thus lead to deceased clinical effect of ifns. in order to measure the bioavailability of ifns and the level of neutralizing antibody, we have developed a realtime rt-pcr (taqman) assay by quantitating the expression of mxa (an ifn-induced protein) mrna. the nucleotide sequences of mxa deposited in the genebank were aligned, and a pair of primers and the hybridization probe were designed based on the conserved regions. a house keeping gene, gapdh, was used as a calibrator for relative quantitation. the rna standards were generated by in vitro transcription from cloned mxa gene in a plasmid vector. the reaction parameters were optimized. the assay was validated using pbmcs of ms patients that were treated with ifn-beta. for evaluation of the ifn bioavailability, the total rna was extracted from pbmcs and quantitatively detected by one-step rt-pcr for both mxa and gapdh. the results calculated by the 2 (− ct) method showed that the difference (signal-to-noise ratio) between samples with neutralizing antibodies and samples from untreated ms patients or healthy donors were approximately 60-70-folds. this indicates that our assay is a reliable method for determination of ifn bioavailability. v. lozitsky 1 , i. kravchenko 2 , v. larionov 2 1 research anti-plague institute, odessa, ukraine; 2 national university, odessa, ukraine transdermal delivery (td) of drugs is a novel method for treatment of diseases. td is carried out by the help of transdermal therapeutic systems (tts), which are multilayer plasters that contain active ingredients. td have a number of advantages, such as: (1) prolongation of the drug's action; (2) drug's concentration is maintained in therapeutic range; (3) there is no trauma to patient's skin while using td; (4) removing tts from the skin immediately stops drug's entering to the organism; (5) first-pass effect in the liver is reduced; and (6) many highly active drugs are irritating the gastro-intestinal tract if administered orally, others have a short half-life time-these drugs do not have downsides mentioned above if used as tts. in our previous research we elaborated tts containing rimantadine (ttscr) and studied its efficacy during experimental influenza in mice. we had established that transdermal delivery of rimantadine is more effective than oral administration. the aim of this work was to increase the efficacy of ttscr. to solve this task we studied the influence of some permeability enhancers on anti-influenza efficacy of ttscr during experimental infection. applied tts had adhesion hydrogel matrix (polyvinyl alcohol and 1.2-propylenglycol). they consisted of a base and a plastificator, which improves the administration of active substances through the skin and does not induce irritation. ttscr (1 mg/mouse) were applied on shaven backs of experimental animals. tts for other groups additionally contained one of such permeability enhancers as: 10 mg/mouse of dmso or 10 mg/mouse of octanol or 1 mg/mouse of papain. tts were applied on shaven backs of mice and stayed there from 1 day before infection to 10th day after challenge. mice of all groups were infected intranasally with influenza virus a/pr/8/34 (h1n1), which is highly pathogenic for them. challenge was carried out using four animals for each virus dilution within the range of 10 −1 to 10 −7 . deaths of animals were recorded for 14 days. the results showed that proteolytic enzyme papain increased the anti-influenza efficacy of ttscr on 1 log 10 tid 50 . dmso and octanol did not demonstrate such activity. mikhail dobrikov 1 , serguei vinogradov 2 , barbara ramsay shaw 1 1 department of chemistry, duke university, durham, nc, usa; 2 center of drug delivery and nanomedicine, and college of pharmacy, university of nebraska, omaha, ne, usa nucleoside reverse transcriptase inhibitors (nrti) are widely used in the antiviral chemotherapy. most nrtis require stepwise phosphorylation to the respective nucleoside triphosphates, which inhibit the viral dna synthesis. however, the emergence of hiv-1 reverse transcriptase-dependent drug resistance limits the effectiveness of treatment by nrtis. the ␣-p-borano-nucleotide analogues show several unique physico-chemical and biological properties: (i) enzymatic studies indicate that the rp-isomer of ␣-p-borano-2 ,3 -ddndps is a better substrate for cellular ndp kinase than the parent ddndp; (ii) neither isomer of the ␣-p-borano-ddndps is a substrate for mammalian pyruvate kinase and shows very poor inhibitory properties to this enzyme; (iii) the rp-(␣-p-borano)-ddntp isomers are better inhibitors of drug-and multidrug-resistant viral reverse transcriptases and are poor substrates for dnadependent dna polymerises; and (iv) after incorporation into viral dna the borano-ddnmp residues are more resistant to atp-dependent removal from viral dna than parent ddntps. to by-pass inefficient phosphorylation of the nrtis, several prodrugs of ␣-p-borano-nucleotide analogues have been previously synthesized. a more efficient delivery system for ␣-p-boranonucleotide analogues based on nanosized cationic polymeric gel (nanogel) is proposed. selective inhibition of drug-and multi-drug resistant viral rts, poorer inhibition of intracellular kinases and dna polymerases by the ␣-p-borano-nucleotide analogues, and their specific delivery into infected cells in the complex with nanogel particles suggest a new approach to the design of more powerful antiviral drugs. acknowledgement: this work was supported by the nih grant r01 al52061 to b.r.s. we have developed a high-throughput, cell-based assay to address the critical need for antiviral drugs for the treatment of influenza. in consideration of the demand to screen high volumes of compounds, we targeted the development of a 384 microtiter plate format for the assay. in this assay, the inhibition of the influenza-induced cytopathic effect (cpe) in mdck cells was assessed using the celltiter-glo luminescent cell viability assay by promega. this reagent measures the amount of atp present in cells, which is directly proportional to the number of metabolically active cells. validation studies were executed to establish optimal cell density, viral concentration, dmso tolerance for compound dilution, incubation time for virus-induced cpe and effective control drug concentration. additional parameters, such as assay variability, reagent and read stability, edge effects, and ic50 stability were also investigated during validation. we are currently initiating use of the assay to screen chemical libraries and will report our findings from library screens in addition to the aforementioned validation. we believe the approach will also provide a mechanism for discovery of new antiviral leads for influenza as well as avian flu. fundacio irsicaixa, hospital universitari germans trias i pujol, 08916 badalona, spain we have developed bacteriophage lambda based genetic screen that can be used to isolate and characterize site-specific proteases. this genetic screen system is based on the bacteriophage lambda ci-cro regulatory circuit, in which the encoded repressor ci is specifically cleaved to initiate the lysogenic-to-lytic switch. we have adapted this simple, safe and rapid genetic screening system to predict the activities and phenotypes of human immunodeficiency virus type 1 (hiv-1) proteases in the course of viral infection and antiretroviral therapy. a specific target for the hiv-1 protease, p17-p24, was inserted into the lambda phage ci repressor. the target specificity of the ci-hiv repressor was evaluated by coexpression of this repressor with an hiv-1 protease construct. upon infection of escherichia coli cells expressing the two constructs encoding the ci-hiv-1 repressor and hiv-1 protease, lambda phage replicated up to 1000-fold more efficiently than in cells that did not express the hiv-1 protease. this assay responds appropriately to well-known hiv-1 proteases inhibitors and can be used to search for new proteases inhibitors. the high level of specificity of this system, in which modest differences in catalytic efficiency can be quantified, should be also useful for the characterization of different mutant viral proteases. we further demonstrated the broad applicability of this protease assay using other viral proteases and their cognate cleavage sites, including hepatitis c virus (hcv) ns3 protease and severe acute respiratory syndrome (sars) coronavirus (cov) (scov) 3c-like protease. compared with other protease assay methods, this assay has the following advantages: safe, highly sensitive, highly specific, easy quantification, and rapid generation of different protease cleavage substrates using molecular cloning and expression. this system may be useful for the development of a screening method to identify viral protease inhibitors and should be also useful to characterize cellular, viral, or other infectious agent proteases with different activities and specificities. karen m. watson, todd b. parsley, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa a virus transmission and rapid resistance selection assay has been developed in order to quickly evaluate the biological properties of anti-infective test compounds and rationally prioritize them for further development. the transmission assay specifically evaluates the ability of test agents to suppress and clear virus replication from cultures during the serial passage of virus in the presence of fixed concentrations of the test compounds alone or in combination. the growth and expansion of virus in the infected cultures has been shown to occur through the replication of originally infected cells in the absence of virus spread, through direct virus to cell infection, and through cell to cell transmission. in order to sterilize a culture a test compound must possess the ability to specifically and potently interfere with virus replication by each of these three methods and must be able to inhibit the replication of resistant viruses which pre-exist in the viral inoculum and which rapidly grow in the presence of the fixed concentrations of the test agent. twelve pyrimidinediones being evaluated for potential use as both anti-hiv therapeutic agents and topical microbicides were evaluated for their ability to inhibit virus transmission and to define their ability to rapidly select for resistant virus strains. these compounds were evaluated in parallel with known anti-hiv agents that inhibit virus entry (t20) and reverse transcription (sustiva, uc781 and azt). the results of the transmission assays suggest that significant biological differences exist between antiviral compounds and even between highly related congeners of the same class of pyrimidinediones, suggesting that the transmission and rapid resistant selection assay measures important antiviral properties of anti-hiv agents. biological studies that evaluate the mechanisms of virus growth in the presence of high concentrations of test compounds will be described. laure deflubé 1 , kerstin angner 1 , anna overby 1 , david stein 2 , patrick iversen 2 , ramon flick 1 1 utmb, department of pathology, galveston, tx, usa; 2 avi biopharma, inc., corvallis, or, usa in the family bunyaviridae, several members on the genus phlebovirus have been reported to cause disease in humans or livestock. among these, rift valley fever (rvf) virus is an important human/animal pathogen. its widespread geographic distribution and its ability to produce severe human disease makes this virus a worldwide public health concern. the phosphorodiamidate morpholino-oligomers (pmo) are a class of dna-like antisense agents typically synthesized to a length of about 20 subunits and contain purine or pyrimidine bases attached to a backbone composed of six member morpholine rings joined by phosphorodiamidate intersubunit linkages. they have been shown to be effective antiviral compounds for different virus families, e.g. coronaviridae and flaviviridae. pmo bind to rna preventing translation of the viral rnas. we used our recently developed plasmid-based minigenome rescue systems for uukuniemi (phlebovirus model virus) and rvf viruses to screen antiviral compounds based on the morpholino antisense oligonucleotide approach. for this the antiviral compounds were appraised on the basis of reporter gene activity (fig. 1b) . the inhibitory effects of the same compounds were also tested by measuring reduction in virus titer (fig. 1a) , by monitoring changes in viral antigen production using an indirect immunofluorescence procedure and facs analysis, and analysis of genome transcription/replication by rt-pcr. indeed several pmos could be identified with interfering effect at a low ic50 on bunyaviral minigenome rescue as well as virus proliferation. based on these results, we plan to confirm antiviral activity of the most promising compounds in suitable animal models. we have shown that the fractal approach to the problem of viruscell interaction gives the unique possibility to process the data through the sequence of the direct and inverse fourier transforms. the studies were carried on the herpes simplex virus us-1 interacting with the hep-2 sensitive cell culture. the object was imaged as system of bright peaks formed as a result of laser diffraction on the structural elements of the virus-cell system. the whole virus-cell interaction information is inserted into computer in a fastest parallel way. the laser intensity peaks, which form the speckle image of the system under consideration, could be transformed into the hierarchical system of the circles (or squares) according to the choice of the researcher, but conserving the same d value, which depends only on the true intermolecular interaction potential. this potential, being characteristic for every stage of virus-cell interaction, is responsible for the given structure of the dynamic virus-cell system and the unique, but the typical form of the fractal cluster corresponding both to the system itself and its image as well, was processed by computer techniques. the hierarchical fractal design of the virus-cell system, proposed here for the first time, gives the universality, needed for the quantitative description of any possible combination of the virus and corresponding sensitive cell. it should be noted, as well, that the fractal microscope use for viruscell dynamic system imaging have all the properties, required from all other experimental tools of monitoring, including the reliability, reproducibility and preciseness. this device could be used in drug design biological test stages with the scope of time and efforts economy during the compounds libraries screening. the fractal microscope combined with the qsar drug design technique makes the antiherpetic drug design more competitive as compared to the regular approaches. acknowledgement: the authors are indebted to the partial support of the stcu grant #3147. we have investigated experimentally the fractal properties of diffraction images obtained by laser irradiation of virus-cell system. it was shown that the diffraction process is mathematically equivalent to the direct fourier transform of the said system's components modeled with simple geometrical figures (e.g. circles). each viral family could be coded and described quantitatively with the average size of the free viral particle and the type of its symmetry. we propose here to use the inverse fourier transform of the virus-cell system in order to get the real enlarged image of the viruses attacking the sensitive cell as well as the cell's structural transformation caused by the sequent stages of virus reproduction process. the set of bright and dark spots, which forms the virus-cell system's diffraction image, could be coded into set of numbers (matrix form of correlation vector-function) using the quantification procedure. the correlation function was used as presented in polar coordinates because the system has the axial symmetry (laser beam taken as main physical axis). the full information included into the image peaks' diameters and color index is transformed using inverse fourier technique into set of intersecting bright and dark circles. the full in vitro dynamics of the structural changes of the virus-cell system are described by the changes of circles' diameters and the area of their intersection. it was shown, also, that the magnification of the fractal microscope could achieve 10,000× to 100,000×, depending on the laser power used. proposed fractal microscope could be applied as well in vivo experiments until the required magnification will not make us to use projection laser with the output exceeding 25 mw. we have shown that the fractal microscope based on the inverse fourier transform could be applied successfully in pharmaceutical antiviral drug design, laboratory and clinical trials of new antiviral preparations, especially effectively in hierarchic qsar research. acknowledgement: authors are grateful to the support under the stcu grant #3147. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for palkylphenyl substituted analogues . these compounds however are highly lipophilic and poorly soluble in water. we then reported a series of p-alkyloxyphenyl compounds containing a phenolic ether aiming to enhance water solubility whilst retaining antiviral activity (mcguigan et al., 2002) . we will now report the synthesis, characterisation and antiviral evaluation of a novel series of p-alkyloxyphenyls where there is at least one methylene spacer between the phenyl and ether group to potentially boost the pharmacokinetic profile. the alkyl chain length was fixed to retain a high clogp value, a parameter that has previously been shown to correlate with high antiviral potency . the target structures were prepared by the pd-catalysed coupling of a series of para-substituted alkoxyphenyl acetylenes with 5-iodo-2 -deoxyuridine, to give intermediate 5-alkynyl nucleosides, which were subsequently cyclised in the presence of cui to give the desired bicyclic systems. the antiviral activity, cytotoxicity, and solubility of these compounds are to be reported. we have previously reported on some novel nucleoside analogues containing and unusual furano bicyclic pyrimidine base and long side chain , which were discovered to be both potent exquisitely and selective towards the varicella zoster virus. following this discovery, three main sites for modification were identified and explored: (1) the side chain; (2) the bicyclic base; and (3) the sugar moiety. modification to the side chain by insertion of a phenyl ring, led to the most potent anti-vzv nucleoside to date (ec50 1 nm) . the investigation into modifications at the three sites stated above has continued and we herein report further adjustments to these analogues. replacement of the furo oxygen with sulfur on the parent nucleosides bearing an alkyl side chain has been reported to retain antiviral activity. however, those bearing a phenyl alkyl side chain are here shown to give a slight reduction in anti-vzv activity. modifications to the phenyl ring of the side chain have included halogen substitutions, and the fluorine in particular has produced some intriguing results in that, while the ortho and meta substitutions show some anti-vzv activity, the para analogue is completely devoid of antiviral activity. we now report further studies which include the di and tri substituted phenyl analogues. finally, we have also investigated sugar modification that has included substitutions of the 3 hydroxyl group. previous modifications which have replacements of the hydroxyl groups, resulted in loss of activity against vzv . we now present some new 3 substituted analogues which have provided interesting biological results. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) with subnanomolar activity for palkylphenyl substituted analogues srinivasan et al., 2001) . the sar is now further explored via the substitution of phenyl derivatives with electron withdrawing and electron donating groups. we now report the synthesis, characterisation, and biological evaluation of a novel series of mono substituted phenyl derivatives in order to probe the structure activity relationships in this region. the target compounds were synthesised under sonogashira conditions where a series of substituted phenyl acetylenes were coupled with 5-iodo-2 -deoxyuridine, to give intermediate 5alkynyl nucleosides that were subsequently cyclised in the presence of cui to give the desired bicyclic systems. diseases caused by herpes simplex virus (hsv) are widely distributed. prophylaxis and treatment of these infections are important health care tasks that require also the search, design and development of new antiherpetic drugs overcome drug resistance and toxic side effects of existing drugs. drug selection simply based on results of empirical screening is not very effective. computer-based technologies may help to optimize the structure of antiviral compounds as well as to design and develop new drugs. the objective of the present work is the quantitative structureactivity relationship (qsar) analysis of antiviral activity of various n,n -(bis-5-nitropyrimidyl)dispirotripiperazines in connection with consequent drug design. the well-established simplex representation of molecular structure (sirms) qsar approach has been used to fulfill this objective. it allows the molecular design of new effective antiviral drugs. thorough investigation of the relationship between: (a) cytotoxic (hela cells and gmk cells, cc 50 , g/ml); (b) antiherpetic activity (hsv-1 strain kupka, ic 50 , g/ml); and (c) selectivity index (ratio of cc 50 to ic 50 ) and the structure of 48 n,n -bis-5-nitropyrimidyl derivatives of dispirotripiperazine have been conducted. statistic characteristics for pls (partial least squares) models are quite satisfactory (r 2 = 0.816-0.991, q 2 = 0.637-0.868). the results are confirmed by experimental data. based on the obtained models, molecular fragments that promote and interfere with antiviral activity were defined. additionally, these models provide the possibility to predict molecular fragments that will enhance antiherpetic activity and to design new well tolerated highly virus-specific drugs. in summary, the developed simplex approach is an effective instrument for prediction and design of novel effective antiherpetic agents. several representatives of a series of 5-arylethynyl-2deoxyuridines (1a) bearing bulky aryl groups were recently shown to possess unexpected activity towards hsv-1. unlike common anti-hsv drugs, these compounds retain activity towards kinase-deficient acyclovir-resistant strains. therefore, an unusual mechanism of antiviral action is assumed. in order to investigate the mechanism and to discover more potent analogues we synthesized several novel 2 -deoxy (1a) and 2 -arabino (1b) uridine derivatives possessing different 5-arylethynyl substituents. dinucleosides 2 containing two uridine moieties coupled to a single polycyclic aromatic hydrocarbon (e.g. pyrene) represent another type of structural variation of nucleosides 1a. these compounds as well as some of 1a and 1b possess bright fluorescence that can be used in biological evaluations. cytomegalovirus (cmv) is a wide spread opportunistic pathogen which belongs to the beta subfamily of the herpesviridae. primary infection is generally asymptomatic resulting in life long latency. however, morbidity and mortality rates post-transplantation are greatly increased following reactivation or recrudescence of cmv. ganciclovir (gcv) and cidofovir (cdv) have both been successful in suppressing cmv viral replication in immunocompromised patients. although sustained use of these drugs has resulted in the emergence of multi-drug resistant strains of virus. in this study we used plaque reduction assays to determine the antiviral efficacy of two ribonucleotide reductase inhibitors, didox (dx; 3,4dihydroxybenzohydroxamic acid) and trimidox (tx; 3,4,5trihydroxybenzamidoxime) in inhibiting both wild type and drug-resistant strains of murine cmv (smith strain). the results presented here demonstrate that both dx and tx inhibit viral plaque formation in a dose dependent manner in both wild type and the resistant strain. a 43-and 39-fold increase in drug dose was required for cdv and gcv respectfully to inhibit plaque formation by 50% in the resistant strain (cdv wt: 0.03 m, r: 1.28 m/gcv wt: 3.17 m, r: 122.85 m). this compared to only a moderate increase in drug dose required for dx and tx to achieve 50% inhibition in the resistant strain (dx wt: 20.71 m, r: 28.88 m/tx wt: 7.12 m, r: 11.59 m), corresponding to a 1.4-and 1.6-fold increase respectfully. further work is currently underway to determine the possible mechanism of antiviral actions and toxicity profiles of these novel virostatics. in patients with human immunodeficiency virus (hiv) infection, coinfection with herpesviruses continues to be a problem for patients receiving antiviral hiv therapy. since treatment can be affected by the large number of drugs required for multiple infections, it would be useful to have antivirals that are active against both hiv and the herpesviruses. we reported previously that alkoxyalkyl ester prodrugs of cidofovir (cdv) are several logs more active against herpesvirus replication than unmodified cdv. to determine if this strategy would be effective for other acyclic nucleoside phosphonates which are active against hiv infections, hexadecyloxypropyl (hdp) esters were synthesized from 1-(phosphonomethoxyethyl)-cytosine (pme-c), 1-(phosphonomethoxyethyl)-5-bromo-cytosine (pme-5brc), 1-(phosphonomethoxyethyl)-5-fluoro-cytosine (pme-5fc), 9-(phosphonomethoxyethyl)-2,4-diaminopurine (pme-dap) and 9-(phosphonomethoxyethyl)-2-amino-4cyclopropylaminopurine (pme-cprdap) and assayed for activity against herpesvirus replication. overall, the hdp esters were more active than the unmodified acyclic nucleoside phosphonates, indicating that this is a useful strategy for increasing the antiviral activity of acyclic nucleoside phosphonates. one of the most active compounds was hdp-pme-cprdap which had ec 50 values of 0.2, 0.3, and 0.03 m in hff cells infected with hsv-1, hsv-2 or hcmv, representing a 12-26-fold increase in efficacy over the parent pme-cprdap. another promising compound was hdp-pme-dap, which had ec 50 values of 0.7, 0.2, and 0.6 um in hff cells infected with hsv-1, hsv-2, and hcmv, representing a 16-43-fold increase over the parent pme-dap. the results presented here indicate that modified acyclic nucleotides with antiviral activity against hiv also inhibit the replication of some of the herpesviruses. further evaluation of their activity against other herpesviruses that are a problem in hiv-infected patients, such as human herpesviruses type 6 and 8, is warranted and may provide new therapeutic options for patients with coinfections. julie m. breitenbach 1 , katherine z. borysko 1 , jiri zemlicka 2 , john c. drach 1 1 biologic & materials sciences, school of dentistry, university of michigan, ann arbor, mi, usa; 2 karmanos cancer institute, wayne state university school of medicine, detroit, mi, usa we previously described first (qiu et al., 1998. j. med. chem.) and second generation (zhou et al., 2004. j. med. chem.) methylenecyclopropane purines that have potent and selective activity against hcmv. strains selected separately for resistance to first-generation analogs (synadenol, synguanol) were 10-20-fold resistant to several first-generation purine analogs. similar resistance was observed to the second-generation guanine analog cyclopropavir [ic 50 's in plaque assays = 0.35 and 21 m, respectively for wild-type (wt) and synguanol-resistant (1982r) virus]. likewise a ul97 deletion mutant (prichard et al., 1996. j. virol.) was resistant to both first and second-generation compounds (ic 50 's = 2.1 and 0.25 m in wt; 100 and 15 m in ul97 del , respectively for synguanol and cyclopropavir). ul97 from the hcmv strain selected for resistance to the synadenol was sequenced and two mutations were identified: m460i and c603y. because hcmv with either m460i or the related c607y mutation alone was sensitive to synadenol and synguanol (baldanti et al., 2002 . antiviral res.), we hypothesize that two mutations are required for resistance to first-and second-generation analogs. this hypothesis was tested by construction of three strains of hcmv from hcmv ad169 bac with one, the other, or both mutations in ul97. as expected, the two strains with the single mutations were 3-to 6-fold resistant to ganciclovir but had little resistance to the first generation compounds synadenol and synguanol (0.5-to 1-fold). both strains were somewhat more resistant to the second-generation compound cyclopropavir (6to 8-fold) but less so than observed in the 1982r virus with two mutations. study of the resistance of the constructed virus with two mutations is underway. we conclude that a functional ul97 is required for activity against hcmv and that is likely that two mutations in ul97 are required for significant resistance. acknowledgement: supported by grants p01-ai46390 and r44-ai054135 from nih and funds from the university of michigan. svitlana zagorodnya 1 , nadiya nesterova 1 , inna alexeeva 2 , larisa palchikovskaya 2 , galina baranova 1 , alexander kobko 2 , anna golovan 1 1 zabolotny institute of microbiology and virology of nas of ukraine, kiev, ukraine; 2 institute of molecular biology and genetics of nas of ukraine, kiev, ukraine search of new effective preparations capable to inhibit herpesviruses reproduction is stipulated by their certain resistance to different groups of chemical preparations. new triazine bearing tricyclic bases and their n-glycosidic derivatives structures are widely used as potential antiviral agents. the objective of the present investigation was to study the activity triazine bearing tricyclic bases nos. 1 and 2, as well as n-glycosidic derivatives no. 3 against epstein-barr virus-lymphotropic and oncogenic virus from herpesviridae family. as a model of ebv-infection in vitro we used the line of lymphoblastoid b-cells raji, which infected by ebv. an inhibition of reproduction of ebv in a cell culture by no 1, no 2, and no 3 was determined by reduction of a number of genome-equivalents of ebv dna on a cell, which were revealed by quantitative pcr with use of primers and reagents "amply-senc-100 r" (russia). the first stage of investigation of substances was the analysis of their cytotoxicity for cell line raji. they have been studied in concentrations of 1000, 500, 250, 125, 64, 32, 16, 4 , 1, 0.5 and 0.1 g/ml. the concentrations that inhibited the quantity of live cells on 50% (id50) were equal to substances no. 1-750 g/ml, no. 2-625 g/ml and no. 3-125. the minimal inhibiting concentration (mic) of nos. 1, 2, and 3 was equal to 1 g/ml, because the amount of genome-equivalents of dna ebv on a cell was reduced with 28.0 up to 14. hence, the index of selectivity (is) was equal to 750 and 625 for triazine bearing tricyclic bases nos. 1 and 2, for n-glycosidic derivatives-125. in addition these compounds were also tested in transcription and replication model systems in vitro. our results indicate that bases and their n-glycoside derivatives effect rna and dna synthesis in different manner. r. sgarbanti 1 , l. nencioni 1 , g. macrì 2 , c. nucci 2 , u. benatti 3 , m. magnani 4 , e. garaci 5 , a.t. palamara 1 1 department public health sciences, university rome "la sapienza," rome, italy; 2 department biopathology, physiopathological optics, university rome "tor vergata," rome, italy; 3 department exp. medicine biochemistry section, university genova, genoa, italy; 4 inst. biochemistry, university urbino, urbino, italy; 5 department exp. med. biochem. sciences, university rome "tor vergata," rome, italy several studies have demonstrated that different viruses induce an imbalance in the intracellular redox state through a depletion of glutathione (gsh), the main intracellular antioxidant. the imbalance in the intracellular redox state represents a key event in the development of viral infection. indeed, our previous data showed that treatment with gsh prevents a decrease in intracellular gsh and inhibits replication of different rna and dna viruses in vitro and in vivo. our recent data demonstrated that a butanoyl derivative of gsh (gsh-c4), with increased hydrophobic properties, inhibited in vitro parainfluenza-1 and hsv-1 replication more efficiently than gsh. for this reason we evaluated the effectiveness of topical gsh-c4 administration in hsv-1-induced keratitis in rabbits. for infection, the corneal epithelium, previously scratched, was inoculated with 2 × 10 5 pfu/ml of hsv-1. gsh-c4, dissolved in a saline solution (150 mm, 100 ul/eye), was administered as eyedrops four times daily for ten days. a saline solution was used for the control group. the clinical evaluation of conjunctival and corneal involvement, performed by using 0.5% fluorescein sodium eyedrops and a slit lamp fitted with a cobalt blue filter, demonstrated that gsh-c4 treatment was effective in reducing the severity and progression of keratitis and conjunctivitis. moreover, in gsh-c4 treated animals, conjunctival hsv-1 titre, assayed by tcid50 on day 4 post-infection, was significantly reduced as compared to that of control animals (mean = 1.4 × 10 3 units/ml versus 1.1 × 10 5 units/ml, n = 10 for group). accordingly, similar results were obtained by measuring virus titre from the corneas of gsh-c4-treated animals versus placebo animals (mean = 3.5 × 10 3 units/ml versus 8.5 × 10 5 units/ml, n = 5 per group). such results highlight the antiviral activity of gsh-c4 in vivo and suggest that topical gsh-c4 treatment could be considered as complementary therapy of hsv-1-induced keratitis. debra quenelle 1 , deborah collins 1 , latisha pettway 1 , caroll hartline 1 , james beadle 2 , w. wan 2 , karl hostetler 2 , earl kern 1 1 university of alabama school of medicine, birmingham, al, usa; 2 department of medicine, university of california, san diego and veterans medical research foundation, san diego, ca, usa cytomegalovirus (cmv) can cause a wide variety of clinical manifestations in immunocompromised hosts or transplant recipients. we have utilized severe combined immunodeficient (scid) mice implanted with human fetal tissue and subsequently infected with hcmv or balb/c mice infected with mcmv to evaluate new antiviral therapies against cmv infection. in the current studies we used these two models to determine the efficacy of (s)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine ((s)-hpmpa), hexadecyloxypropyl-(s)-hpmpa (hdp-(s)-hpmpa), or octadecyloxyethyl-(s)-hpmpa (ode-(s)-hpmpa). in the hcmv model, human fetal thymus and liver (thy/liv) tissues were implanted under the kidney capsule of mice and inoculated 16-20 weeks later with 4700 pfu of hcmv. tissue samples were obtained at various time points for quantitation of hcmv titers by plaque assay. in general, replication of the toledo strain of hcmv in the implant tissue increased through 21-28 days and then gradually decreased to undetectable levels by 8 weeks post-infection. to determine efficacy of these compounds, oral treatment with vehicle or 10 mg/kg of (s)-hpmpa, hdp-(s)-hpmpa or ode-(s)-hpmpa was initiated 24 h after infection and continued for 28 days. cidofovir (cdv) at 20 mg/kg was administered i.p. daily as a positive control. results indicated that (s)-hpmpa, hdp-(s)-hpmpa and ode-(s)-hpmpa were highly effective in significantly reducing replication when compared to the vehicle control. in mcmv infected mice, hdp-(s)-hpmpa was highly effective in preventing mortality when administered orally at 30 or 10 mg/kg beginning 24 h post-viral inoculation and 30 mg/kg when treatment was delayed until 48 h postviral inoculation. these data indicate that these compounds were highly efficacious in two animal models of cmv infection and should be evaluated for use in hcmv infections in humans. cytomegalovirus (cmv) is a ubiquitous ␤-herpesvirus that asymptomatically infects immunocompetent individuals but leads to serious illness in immunocompromised individuals, such as transplant recipients, neonates and aids patients. thus, the need for well-tolerated and potent antiviral compounds with activity against cmv is well recognized. in our current studies, we have evaluated the in vivo activity of rep 9, a fully degenerate 40 mer phosphorothioated oligonucleotide against murine cytomegalovirus infection (mcmv) in mice. rep 9 has potent in vitro activity against hsv-1, hsv-2, hcmv, vzv, ebv, and hsv-6 (vaillant et al., submitted for publication). in our initial studies, infected mice were treated with rep 9 and compared to saline-treated infected control mice. compound was administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting at 2 days prior to infection. mice were infected with 5 × 10 5 pfu mcmv on day 0, at 3 h post-treatment. sera were collected at −22 h, at 36 hpi, and at 3 dpi for elisa analysis of ifn␥ production. spleens and livers were collected at 3 dpi for determination of virus titers. at 3 dpi, virus titers in the spleens and livers were significantly reduced by rep 9 treatment as compared to control mice. splenomegaly was observed in infected mice treated with rep 9 but not in saline treated, infected mice or in rep 9 treated, uninfected mice. ifn␥ levels in mice treated with rep 9 peaked at 36 hpi compared to 72 hpi for saline-treated control mice. these data suggests that immune stimulation might contribute to the antiviral activity of ps-ons, perhaps through ifn␥ levels. a second study comparing the in vivo activity of rep 9 with two oligonucleotide analogs that do not activate tlr-9 mediated immune stimulation suggests that direct antiviral activity of rep 9 and the analogs was the predominant therapeutic mechanism in vivo. moreover, one rep 9 analog exhibited even greater antiviral activity than rep 9 while causing no splenomegaly. additional experiments are underway to provide insights into the mechanism of action against mcmv infection. acknowledgement: supported by contract no1-ai-15438 from the virology branch, niaid, nih. kathy keith 1 , joseph maddry 2 , namita bansal 2 , kochurani jacob 2 , secrist john 2 , earl kern 1 1 department of pediatrics, university of alabama school of medicine, birmingham, al, usa; 2 southern research institute, birmingham, al, usa a series of novel antiviral agents was prepared based on lead compounds related to acyclic nucleoside phosphonates. these agents consist of a purine nucleus bearing a pendant phosphonic acid group. the design strategy was two-fold: (1) following the approach of the hostetler group, to mask or partially mask the anionic phosphonate as a lipophilic ester and/or as an amino acid phosphonamidate prodrug that could enhance cellular uptake and be cleaved intracellularly; and (2) to investigate new substituents at the purine 2-and 6-positions. for proof of concept, a phosphonomethoxyethyl adenine (pmea) scaffold was employed. over 100 analogs of pmea substituted at the purine 2-or at the adenine n-6 site have been synthesized and evaluated for activity against orthopoxvirus infections. many n-6 substituents other than the previously recognized n-cyclopropyl have shown antiviral activity, and these structure-activity relationships are being investigated. an exciting finding has been that introduction of several novel moieties at the purine 2-position particularly the hydrazino, hydroxylamino, or the cyclopropylamino groups resulted in several compounds with excellent in vitro activity. for example, octadecyloxyethyl (ode) 2-amino-n(6)-cyclopropyl pmea had ec 50 values of 0.03-0.07 m and ode 2-hydroxylamino-n(6)-cyclopropyl pmea had ec 50 values of 0.3-1.6 m against cowpox and vaccinia viruses, respectively, using a plaque reduction assay in hff cells. under these conditions the parent molecule, pmea, was completely inactive. these two compounds had cc 50 values of 30-70 m giving selective indices of 200-1000. these studies indicate that several modifications in the pmea scaffold can result in good antiviral activity against orthopoxvirus infections in vitro and the most active compounds are currently being scaled up for evaluation in animal models. isatin (2,3-dioxoindole), a versatile lead molecule for designing of potential bioactive agents, and its derivatives have been reported to possess inhibitory activity against a variety of pathogenic viruses. methisazone(n-methylisatin-3thiosemicarbazone) was one of the first synthetic antiviral agents used clinically for the treatment of orthopox virus infections. the presence of the thiosemicarbazone, however, can result in immunosuppresion and we have attempted to replace the thiosemicarbazone with a sulphonamide in order to modify the antiviral activity. the present work was performed to evaluate the antiviral activity and cytotoxicity of some novel 4-[(1,2dihydro-2-oxo-3h-indol-3-ylidene)amino]-n-(4,6-dimethyl-2pyrimidiny)-benzene sulphonamides against pox viruses such as vaccinia and cowpox virus in human fibroblast cells and the activity was compared with cidofovir(cdv). among the compounds tested,4-[(5-methyl-1,2-dihydro-2-oxo-3h-indol-3-ylidene)amino]-n-(4,6-dimethyl-2-pyrimidiny)-benzene sulphonamide(spiii-5me), was the most active compound with an ec50 value of 18 mol, compared with cdv, which had an ec50 of 20 mol against vaccinia virus. all the compounds were non-toxic (>300 lm)using a neutral red uptake assay. substitution of a halogen atom in 5th position of isatin was found to abolish the antiviral activity. this compound should be evaluated in orthopox infections in animal to determine its potential for development as an effective agents for treatment of these infections. acknowledgement: supported in part by contract no1-ai-30049 from virology branch, niaid, nih, usa. evgeny belanov 1 , svetlana kotovskaya 2 , nikolay bormotov 1 , sergey balakhnin 1 , olga serova 1 , nataliya perova 2 , zina baskakova 2 , galina dzhumbaeva 2 , valerii charushin 3 , oleg chupakhin 2 1 state research center of virology and biotechnology "vector", koltsovo, novosibirsk reg., russia; 2 ural state technical university, yekaterinburg, russia; 3 institute of organic syntheses, yekaterinburg, russia during this study, we synthesized a series of 1,2,4-benzotriazine ( fig. 1) derivatives in order to evaluate the structural features required for anti-orthopoxviruses activity. these derivatives were tested for cytotoxicity and activity against the vaccinia, cowpox, mousepox, monkeypox, and in some experiments with variola viruses in vero and mk-2 cells. the results from studies of structure-activity relationship revealed that only compounds containing phenyl group at c-3 and the alkoxy and fluoro substitutes in the benzene ring of benzotriazines showed anti-orthopoxviruses activity. the antiviral activity was reduced or lost after substitution with other substitutes. thus, we find a new class of heterocyclic compounds with antiviral activity. acknowledgment: this research was funded by istc project #1989. yali chen, guang yang, kady honeychurch, dennis hruby, robert jordan siga technologies, inc., 4575 sw research way-suite 230, corvallis, or 97333, usa we have recently discovered a highly specific and potent antiorthopoxvirus compound (st-246) via high throughput screening (yang et al., 2005. j. virol. 79, 13139-13149) . marker rescue of st-246 resistant variants localized compound resistance to the f13l gene that encodes a major orthopoxvirus envelope protein (p37), which is required for extracellular viral particle formation. p37 participates in wrapping of intracellular mature virus (imv) in membranes derived from the trans golgi or late endosomal compartment to produce intracellular enveloped virus (iev) that are transported to the cell surface to form extracellular virus particles. to gain insight into the mechanism of action of st-246, we examined the effects of st-246 on the production of the extracellular viral particles in bsc40 cells infected with recombinant vaccinia virus containing a gfp-tagged p37 protein. in the presence of st-246, iev particle formation was dramatically reduced, plaque formation was almost completely inhibited, and imv particles appeared to be retained in intracellular vesicles as revealed by electron microscopy. furthermore, st-246 prevented the intracellular localization of p37 to the late endosome compartment as measured by confocal microscopy. in contrast, st-246 did not affect localization of p37 expressed from a st-246 resistant virus variant. more intriguingly, the compound did not affect the intracellular localization of p37 in transfected cells. these results suggest that st-246 inhibits an unknown virusspecific activity that requires f13l. this work underscores the exquisite specificity of st-246 and supports continued development of st-246 as a potential anti-orthopoxvirus drug. guang yang, chris harver, dennis hruby, robert jordan siga technologies, inc. 4575 sw research way, suite 230 corvallis, or 97333, usa st-246 is a potent, orally bioavailable anti-orthopoxvirus compound that is active in vitro and in vivo. the frequency of naturally occurring st-246 resistant variants was measured by fluctuation analysis and found to be 3.58 × 10 −6 . marker rescue of drug resistant variants localized changes associated with reduced compound susceptibility to the vaccinia virus f13l gene. the spectrum of mutations that confer st-246 resistance was determined using an error-prone pcr procedure to increase the frequency of compound resistance by 100-fold relative to the frequency of naturally occurring resistance. using this procedure, random point mutations were introduced into the f13l coding sequence by error-prone pcr and the mutated f13l alleles were transferred into wild-type virus genome by marker rescue. sequence analysis of the input error-prone pcr products prior to marker rescue identified numerous nucleotide changes in the f13l coding sequence, some of which created nonsense mutations. virus recombinants were selected that formed plaques in the presence of drug selection. this powerful selection procedure enriched for viruses that produced functional, st-246 resistant, f13l proteins. sequence analysis of the compound resistant f13l alleles identified numerous silent mutations scattered throughout the f13l coding sequence and 27 point mutations leading to amino acid changes that clustered around amino acid positions 258-302 within the f13l gene. seven of these mutations resulted in single amino acid changes and could be correlated with reduced compound susceptibility. taken together, these results suggest that: (1) mutations in at least 7 positions within f13l can confer resistance to st-246 and (2) st-246 resistant mutations cluster to a 58 amino acid domain in a region of the protein of unknown function. several 5-substituted pyrimidine analogs were identified as having antiviral activity against cowpox virus (cv) and vaccinia virus (vv) in primary human foreskin fibroblast cells. molecules containing benzopyran, cyanovinyl, and pyrazolone moieties at this position exhibited significant antiviral activity against both these viruses. three compounds in this series had ec 50 values below 10 m in a plaque reduction assay against both cv and vv. the antiviral activity of these compounds was also determined against herpes simplex virus (hsv) in a plaque assay. two compounds with cyanovinyl derivatives at the 5 position had ec 50 values below 15 m against both hsv-1 and hsv-2, whereas other substituents at this position exhibited weaker activity against one or both of these viruses. analogs containing the benzopyran substituents were the most effective against varicella zoster virus (vzv) and yielded ec 50 values below 10 m in a plaque reduction assay. none of the compounds were active against human cytomegalovirus. interestingly, all of the compounds were much less effective in a thymidine kinase (tk) negative strain of cv suggesting that the activation by this enzyme was important in their mechanism of action. tk deficient strains of hsv were also comparatively resistant to some of the compounds. the tk dependence of these compounds in cv and hsv taken together with the lack of activity against cytomegalovirus replication suggests that activation by a viral tk is important in their mechanism of action. these results indicate that pyrimidine analogs with large substituents at the 5 position are substrates for the distinct tk homologs encoded by the herpesviruses and orthopoxviruses and suggest that they may be effective against infections with these viruses. synthesis and testing of additional analogs is warranted and should help identify the most potent analogs for in vivo testing. department of pediatrics, university of alabama school of medicine, birmingham, al 35233, usa n-methanocarbathymidine ((n)-mct) is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. this compound inhibits the replication of herpes simplex virus (hsv) with ec 50 values below 1 g/ml, and vaccinia virus (vv) and cowpox virus (cv) with ec 50 values of 0.55 and 1.5 g/ml, respectively. assays using a thymidine kinase (tk) negative strain of cv yielded ec 50 values 14-fold greater than a tk positive isolate. similarly, a tk negative strain of hsv-1 was 90-fold less sensitive to the drug than wild-type strains. thus, the antiviral activity of this molecule is dependent on the type i tk in hsv and the type ii tk expressed by vv and cv viruses, suggesting that it is a substrate for these divergent forms of the enzyme. the drug is also a good inhibitor of viral dna synthesis in both viruses and is consistent with inhibition of the viral dna polymerase once it is activated by the viral tk homologs. it is also possible that the phosphorylated forms of the drug may inhibit other enzymes such as thymidylate synthetase and inhibit viral dna synthesis indirectly. the interesting tk dependence of this molecule explains the rather unusual spectrum of activity that includes orthopoxviruses, alphaherpesviruses, epstein-barr virus (ebv), and human herpesvirus 8 (hhv-8), since these viruses all express molecules with tk activity that could phosphorylate and thus activate the drug. conversely, n-mct is ineffective against the betaherpesviruses because they do not encode tk homologs. the compound is also highly effective in reducing the mortality of mice infected with cv, vv, and hsv when treatment is initiated 24 h after infection and at doses as low as 17 mg/kg. these results indicate that (n)-mct is active in vitro and in vivo and its mechanism of action suggests that the molecule may be an effective and selective therapeutic for orthopoxvirus and certain herpesvirus infections and that it warrants further development. we have previously reported the isolation and characterization of drug-resistant mutants obtained following repeated passages of the vaccinia virus (vv, lederle strain) in the presence of increasing concentrations of cidofovir (cdv). cdvr mutants encoded two mutations (a314t and a684v) not related to genetic polymorphism. we have now introduced these mutations in the pathogenic strain w western reserve and characterized the drug-susceptibility profile of the recombinant viruses and their pathogenicity in mice. both the a314t and the a684v recombinant viruses proved to be resistant to cdv and related compounds, such as cyclic cdv and -propoxy]pyrimidine}. the virus bearing both substitutions proved to be more resistant to cdv than the single mutants. interestingly, the a314t and the a684v mutants differed in their sensitivity to phosphonoacetic acid (paa); the a314t and the a684v mutants being, respectively, hypersensitive and resistant to paa. in contrast, the double mutant showed no change in sensitivity to paa as compared to the wild-type strain. unlike the a684v mutant that showed only a two to three-fold decrease in susceptibility towards the 3-hydroxy-2-phosphonomethoxypropyl (hpmp) purine derivatives, the a314t mutant showed cross-resistance to the hpmp purine derivatives. it should be noted that in the process of selection of cdv-resistant mutants in the presence of increasing concentrations of the compound, the a314t mutation appeared before the a684v substitution, and the latter mutation only occurred in conjunction with a314t. when tested for virulence in a lethal intranasal infection model in mice, all cdvr recombinant viruses proved to be attenuated, suggesting that cdvr mutations are associated with reduced pathogenicity. furthermore, we found that cdv at a dose of 50 mg/kg/day for 5 days was still able to protect mice (in terms of body weight loss) against an intranasal challenge with the a314t + a684v recombinant virus. evaluating the use of cpg dna as an antiviral therapy amanda phelps, linda eastaugh, amanda gates, david ulaeto, arthur krieg 1 defence science and technology laboratories (dstl), salisbury, wiltshire, uk; 6 coley pharmaceuticals ltd., ottawa, ont., canada at present there are no licensed antivirals against orthopoxvirus infections such as variola or vaccinia virus (vacv). although a stockpile of smallpox vaccine exists and has utility as a post-exposure treatment to infection, it is a live viral vaccine and as such cannot be administered to those with contraindications. bacterial dna contains unmethylated motifs that, together with their flanking regions, can stimulate an innate immune response. synthetic cpg dna mimics the immunostimulatory activity of bacterial dna and is recognised by intracellular toll-like receptor 9. there are four classes of cpg dna all of which have different properties, eliciting distinct initial immune responses. previous studies using an established lethal respiratory model of poxvirus infection demonstrated that a class b cpg dna (7909) could provide protection from lethality against vacv in balb/c mice when administered up to 7 days prior to challenge. in order to evaluate efficacy balb/c mice were challenged intra-nasally with vacv and treated with doses of 7909 ranging between 15 and 100 ug/mouse. treatment was administered intra-nasally under light anaesthesia either on the day of challenge, 1, 2, 3, or 4 days post-challenge. efficacy was determined by percentage body weight loss post-challenge. the optimum survival rate observed was 60% when treated with 30 ug 1 day post-challenge (70 mld50 challenge). a survival rate of 80% was observed when treated with 50 ug 2 days post-challenge (40mld50 challenge). the delay of treatment to either 3 or 4 days post-challenge was ineffective, indicating that the window of opportunity for delivery of 7909 is within 2 days. multiple doses of 7909 were used to attempt to extend this window of opportunity, delivering 7909 twice within a 3-day period. interestingly, this had a considerable detrimental effect, actually accelerating the onset of disease and ultimately death. further work is required to optimise the use of cpg dna as a potential antiviral therapy, and there is evidence to suggest that they may have immense utility as part of a co-administration therapy with other antiviral compounds, an area of work currently under investigation. © crown copyright dstl 2005. department of virology, hebrew university, hadassah medical school, jerusalem, israel the pathogenicity and immunogenicity of the lister (elstree) strain of vaccinia virus, used for vaccination against smallpox, was studied in the mouse model. the virus did not reach the brain when inoculated intranasally, but when injected intracranially at a dose of 5 × 10 5 plaque forming units (pfu), was lethal for 50% of the mice. lower doses of virus caused the mice to initially loose some weight but they completely recovered thereafter. a significant level of protection against a lethal dose of the wr strain was achieved in mice following immunization with the lister strain, while higher doses and repeated vaccination procedure, were required with modified vaccinia virus ankara (mva). we found that the lister vaccine strain applied in israel is comprised of heterogeneous virus population. we isolated and plaque-purified three virus variants differing in their plaque size in bs-c-1 cell cultures. they were named: l-large plaque, m-medium plaque and s-small plaque variants. these isolates could be neutralized by rabbit antibodies prepared against the western reserve strain of vaccinia virus and their one-step growth curves in bs-c-1 cells were quite similar. however, they differ in their pathogenicity to mice following intranasal inoculation of 10 7 pfu, or an intracranial injection of 5 × 10 4 pfu; the s variant being more virulent than the other two variants and resembles the pathogenicity of the lister strain. activity was also determined against a thymidine kinase (tk) deficient vaccinia virus in mouse and monkey cells. the potency of (n)-mct was similar to that seen with wild-type virus, suggesting that a cellular enzyme may be more important than viral tk to phosphorylate the compound. mice were intranasally infected with cowpox and vaccinia viruses followed 24 h later by intraperitoneal treatment with (n)-mct (2x/day for 7 days) or cidofovir (1x/day for 2 days). (n)-mct treatment at 100 and 30 mg/kg/day resulted in 90 and 20% survival from cowpox virus infection, respectively, compared to 0% survival (placebo). statistically significant reductions in lung virus titers on day 5 occurred in 10, 30, and 100 mg/kg/day treated mice. these doses did not spare mice from lethal vaccinia virus challenge, however, but the 30 and 100 mg/kg/day treatments significantly reduced day 5 virus titers and lung weights, and the 100 mg/kg/day treatment reduced lung consolidation. cidofovir (100 mg/kg/day) protected all animals from death in both models. the evaluation of (n)-mct may be limited to mice based upon its greatly reduced efficacy in the cells of higher animals. acknowledgement: supported in part by contract no-1-ai-15435 from the virology branch, niaid, nih. chelsea byrd 1 , elena sbrana 2 , shu-yuan xiao 2 , marina siirin 2 , robert tesh 2 , dennis hruby 1 , robert jordan 1 1 siga technologies, inc., corvallis, or, usa; 2 university of texas medical branch, galveston, tx, usa st-246 is a potent small molecule inhibitor of orthopoxvirus replication that has been shown to protect mice from lethal challenge with vaccinia and ectromelia viruses. here we report the results of preliminary trials that show efficacy of st-246 against severe monkeypox virus infection in the ground squirrel model. ground squirrels infected with less than 1 pfu of monkeypox virus develop a fulminant disease resembling human hemorrhagic smallpox: the most severe and lethal form of the disease. oral administration of st-246 at 100 mg/kg once per day for 14 days protected ground squirrels from a lethal challenge with 100 and 1000 pfu of monkeypox virus. compound treated animals showed no weight loss or evidence of disease, and blood chemistry values were similar to uninfected animals. in contrast, placebo-treated animals showed elevated liver enzyme (alt and ast) levels and all animals died by day 12 post-infection. when treatment with 48, 72, and 96 h, 100% protection was observed in the 24, 48, and 72 h groups, and 66% protection in the 96 h group. severe pathologic changes were observed in the organs of the animals receiving placebo, especially in the lungs, liver, and spleen. in contrast, the organs of the animals receiving st-246 at 0, 24, 48, and 72 h postinfection appeared grossly and microscopically normal. thus, st-246 appears to be a promising candidate for continued development as a therapeutic agent for severe orthopoxvirus infection. inge vliegen 1 , guang yang 2 , dennis hruby 2 , erik de clercq 1 , robert jordan 2 , johan neyts 1 1 rega institute for medical research, k.u. leuven, belgium; 2 siga technologies, inc. corvallis, or, usa st-246 is a potent inhibitor of the replication of various orthopoxviruses. resistance of cowpoxvirus to st-246 maps to a mutation in v061, which is homologous to vaccinia virus f13l (yang et al., 2005. j. virol.) . the latter encodes the envelope protein p37 required for production of extracellular virus. deleting f13l resulted in a virus ( f13l-vac) that is replicationcompetent in cell culture but that produces smaller plaques than the wild-type wr-vac. whereas intravenous (i.v.) inoculation of nmri mice with 2 × 10 5 pfu of wr-vac resulted in 59 ± 11 pox tail lesions per mouse, the same inoculum of f13l-vac caused no lesions (p < 0.001). athymic nude (nu/nu) or scid mice inoculated iv with 2 × 10 5 pfu f13l-vac did not develop tail lesions. the mean day of death in nu/nu mice inoculated with f13l-vac was 22 ± 1 days as compared to 9 ± 1 days for wr-vac-infected mice (p < 0.001); scid mice survived the infection. we next studied whether f13l-vac is able to protect mice against a subsequent infection with wr-vac. to mimic the human vaccination protocol, nmri mice were infected intracutaneously (i.c.) by means of scarification at the lumbosacral area with 5 × 10 5 pfu f13l-vac or placebo. none of the infected mice developed lesions at the inoculation site. one week later, animals were infected ic with 5 × 10 5 pfu of wr-vac. all placebo animals, but none of the f13l-vac animals developed poxvirus lesions. in a second set of experiments, mice were again inoculated ic with placebo or f13l-vac and were infected one week later with 2 × 10 4 pfu of wr-vac by the iv route. placebo animals developed an average of 14 ± 4 pox tail lesions; no lesions developed in the f13l-vac animals (p < 0.001). in a third set of experiments, nmri mice were inoculated iv with either 2 × 10 4 pfu of f13l-vac or placebo, and none of the mice developed lesions. one week later, animals were inoculated iv with 2 × 10 4 pfu wr-vac. the placebo group developed an average of 11 ± 8 lesions as compared to 1.2 ± 0.7 lesions in the f13l-vac mice (p < 0.05). f13l-vac may thus be considered as a severely attenuated virus that may have potential for use as a smallpox vaccine. ji yuan 1 , travis lim 1 , shuan coughlin 1 , dexin qiu 1 , zhen liu 1 , dave stein 2 , decheng yang 1 1 the james hogg icapture centre for cardiovascular and pulmonary research, university of british columbia, vancouver, bc, canada; 2 avi biopharma, inc., corvallis, or, usa background: coxsackievirus b3 (cvb3) is the most common cause of viral myocarditis, but existing drug therapy is of limited value. antisense oligonucleotides (asons) designed to pair with viral rna could inhibit viral replication. however, the effectiveness of traditional asons is limited due to poor cellular uptake and degradation by nucleases. phosphorodiamidate morpholino oligomers (pmos) contain backbone modifications, which make pmos more resistant to nucleases. in addition, an arginine rich peptide (p007) is conjugated to the 5 end of the oligomer to improve its delivery into cells. these features make p007-conjugated pmos (p-pmos) promising candidates for the inhibition of cvb3 infection. methods: total 8 p-pmos targeting distinct regions of viral genome and one scrambled sequence were designed and chemically synthesized. fitc labeled p-pmos were used to observe their distribution of by confocal microscopy. viral protein vp1, viral titre, and cell viability were measured by western-blot, plaque assay, and mts assay, respectively. results: p-pmos showed increased cellular uptake compared to non-conjugated pmos. among the 8 p-pmos, p-pmo-6, targeting the internal ribosomal entry site in the 5 utr, showed the most potent anti-cvb3 ability in a dose-dependent manner. both infected hela and cardiomyocytes hl-1 cells treated with p-pmo-6 showed drastically reduced vp1 production and 3.0 log decreases in viral titres as compared to the controls. cell viability assay revealed that 83 and 89% of treated hela and hl-1 cells were still alive as compared to 11 and 10% of control-treated cells and 47% antiviral activity still existed after 5 days treatment. in addition, cells treated post-infection showed similar inhibition of viral replication. furthermore, the specificity of the p-pmos was demonstrated by their inability to inhibit rsv infection in hela cells. we have showed that p-pmos can effectively inhibit viral replication in vitro, providing a new possibility for antiviral intervention. picornaviruses are responsible for various human viral diseases including common cold, encephalitis, meningitis, myocarditis, etc. up to now, there is no specific antiviral therapy to treat or prevent such viral disease. the usage of modern computer technologies may help to solve this problem more effectively. the objective of the present study was the quantitative structure-activity relationship (qsar) analysis of antiviral activity of a set of [(biphenyloxy)propyl]isoxazole derivatives that inhibit cvb3 replication in hela cells. based on results from qsar, the structure of new potential antiviral agents should be predicted by using consequent molecular design. the qsar approach applied is based on simplex representation of molecular structure (sirms). the relationship between: (a) antiviral activity against the pleconaril-sensitive clinical cvb3 isolate 97-927 (ic 50 , g/ml); (b) cytotoxicity in hela cells (cc 50 , g/ml); and (c) selectivity index (si = ratio of cc 50 to ic 50 ), and structure of 21 [(biphenyloxy)propyl]isoxazole derivatives has been studied systematically. quite adequate qsar models (r 2 = 0.831-0.931, q 2 = 0.674-0.896) have been obtained using pls (partial least squares) method for all parameters studied. the models are in close correlation with experimental data. structural fragments with positive or negative influence on cytotoxicity as well as antiviral activity have been determined on the base of these models. for example, qsar analysis of antiviral activity of [(biphenyloxy)propyl]isoxazole derivatives revealed that the presence of m-nitrophenyl or p-trifluorophenyl fragment has distinctly negative influence on antiviral action. compounds with strong antiviral activity have to contain an oxadiazole fragment. moreover, our data allow the virtual screening and molecular design of new well-tolerated compounds with strong anti-cvb3 activity. ivanka nikolova 1 , roumena petkova 2 , boris atanassov 3 , stoyan chakarov 2 , angel s. galabov 1 1 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; 2 scientific technological service, ltd., sofia, bulgaria; 3 institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria analysis of the rna sequence of the disoxaril-resistant mutants of the coxsackievirus b1 was carried out. the wild-type disoxaril-sensitive strain (connecticut 5) and two disoxarilresistant mutants (one of them produced in fl cells and the other one isolated from brains of newborn mice) infected with coxsackievirus b1 and treated with disoxaril and a disoxarildependent mutant strain obtained from the resistant strain by 9 passages in cell culture were included in the present study. a rt-pcr assay with primer sets selected from a region of the coxsackievirus b1 genome coding for the capsid protein vp1 was carried out. a parallel comparative analysis of the sequences of resulting fragments from the disoxaril mutants studied and the genbank sequence of origin of the vp1 gene of coxsackievirus b1 was performed with the blast alignment tool. distinct alterations in the vp1 locus of the disoxaril-resistant and the disoxaril-dependent mutants compared to the sequence of origin from the genbank (namely, a deletion of uug at ntt. 2749-2751 and an insertion of uuu at nt.2769) were observed. high-degree similarity (97%) between the resistant mutant produced in cell cultures and the dependent strain was observed, while the similarity to the wild strain was only 91%. the resistant mutant obtained in mice was found to be very similar to the strain, developed in cell cultures. a putative 3-d model of the spatial folding of the target protein in disoxaril mutants is proposed. ralitsa vassileva-pencheva, angel s. galabov in previous study of ours we presented a new approach to combined application of antivirals-consecutive administration of the partners. this schedule could be considered especially suitable for treatment of enteroviral infections, in which the development of resistance is very rapid due to the extremely high viral mutation rate. this approach aims to restrict the resistance development in experiments in vivo, using antivirals with proved high efficiency in experiments in cell cultures. the screening of various double, triple, and quadruple combinations that we carried out showed that two of the triple combinations, namely disoxaril (win compound)/oxoglaucin (a new antiviral drug, developed in our laboratory)/ptu-23 (a classic enteroviral inhibitor) and disoxaril/oxoglaucin/guanidinehydrochloride (a classic enteroviral inhibitor) manifest significant effect of protection in newborn mice with neurotropic coxsackievirus b1 infection. in the current study the role of the chronology of arrangement of the antivirals included in the combinations was investigated. in the experiments carried out with the triple combination disoxaril/oxoglaucin/guanidine-hydrochloride, it was found that the optimal treatment course should start with disoxaril. the treatment course is quite successful when disoxaril is followed by guanidine-hydrochloride. the effect of the triple combination starting with oxoglaucine, followed by guanidine-hydrochloride was moderate. the course starting with guanidine-hydrochloride proved to be ineffective. furthermore, we studied the virus sensitivity to the inhibitorspartners (ic50 values) and some other phenotypic characteristics of the brain isolates, e.g. the size of the plaques and the pathogenicity for mice. recently our contribution to the development of new antipicornavirus agents has led to the discovery of 5methylthio-3-aryl-isoxazole-4-carbonitrile derivatives whose in vitro anti-coxsackievirus b1 activity were dependent on the nature of the substituents on the para position of the phenyl ring. particularly, compounds 5-methylthio-3-[4-(3-phenyl-1-propoxy)phenyl]isoxazol-4-carbonitrile (on-7) and 5-methylthio-3-[4-(4-phenoxy-1-buthoxy)phenyl]isoxazol-4-carbonitrile (on-10) exhibited an interesting antiviral activity with high selectivity indexes. in the present study, we investigated on the mechanism of action of these compounds. studies on time of addition experiments suggested that these compounds exert a different interference with an early step of the viral replicative cycle. in fact, compound on-7 was effective when added within 1 h after the end of the adsorption period and no reduction was observed if it was added during the adsorption period. whereas a reduction of virus titer was observed for on-10 when was added during the adsorption period, while no reduction was observed if the compound was added after this period (time 0). the influence of the compounds on virus adsorption step, studied by the infective center assays, indicated that on-10 primarily interferes with coxsackie b1 cellular attachment. at a concentration 100 times the id 50 , inhibition of adsorption of coxsackievirus by on-10 was complete, while similar concentration of on-7 had no effect. our experiments on neutralization of viral infectivity and on thermal stabilization demonstrated that the compounds were able to directly inactivate coxsackievirus, and the infectious titer was restored to the original value after extraction of the compound with chloroform. however, the compounds did not protect the viral infectivity against heat inactivation at the different concentrations used. the blood-brain barrier (bbb) fulfills a vital protective function by limiting entry of potential pathogens, toxins, and inflammatory cells into the central nervous system (cns). disruption of the bbb is a common component of many cns diseases, including viral diseases such as that caused by west nile virus (wnv). transforming growth factor-␤1 (tgf-␤1) has previously been shown to improve the function of an in vitro model of the bbb. we evaluated the role of the bbb in wnv infection in mice by determining the ability of intraperitoneally (i.p.) administered sodium fluorescein to move from the circulating blood to the central nervous system. to demonstrate bbb permeability a mean and normal range of permeability values was determined in 30 non-infected c57/bl6 mice. in subsequent experiments, any animal expressing a permeability value greater than 2 sd above the mean was considered abnormally high. we determined that elevations in bbb permeability can be detected in mice 8 days after subcutaneous inoculation with wnv. wnv inoculated animals were treated with doses of 3000, 1000, or 300 ng/kg/day of tgf-␤1 or with drug vehicle once daily via the i.p. route on 7 and 8 days post-virus inoculation (dpi), and then assayed for bbb permeability on 8 dpi. sixty-two percent (8/13) of placebo-treated animals had abnormally high permeability values, while animals treated with 3000 and 1000 ng/kg/day of tgf-␤1 had 29% (2/7) and 57% (4/7) of animals with abnormally high permeability values, respectively. in contrast, none of the animals treated with 300 ng/kg of tgf-␤1 (0/8) expressed abnormally high permeability values, which was significantly lower (p < 0.01) than placebo-treated animals. these results suggest that tgf-␤1 may improve the function of the blood-brain barrier in wnv infected mice. acknowledgement: supported by grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. and contract no1-ai-15435 from the virology branch, niaid, nih. people infected with west nile virus (wnv) usually see their physicians after showing symptoms suggestive of neurological infection. wnv infects the central nervous system (cns) of rodents 3-5 days after s.c. viral challenge. yet, most published animal studies begin therapeutic treatments before or soon after viral challenge. the question addressed in this study is if neuroprotective agents can be efficacious when administered early before brain infection or later after the virus is demonstrated to be in the brain. the drugs evaluated in wnvinfected rodents were nmda and ampa receptor antagonists, modulators of nitric oxide synthase (nos) and nitric oxide production, and riluzole for reducing glutamate excitotoxicity. serial doses of diethyldithiocarbamate (ddtc) and n(g)monomethyl-l-arginine (l-nmma), an inducer or inhibitor of nos, respectively, administered i.p. daily for 10 days beginning 4 h before viral challenge slightly improved survival of mice, but the difference was not statistically significant. tolerated doses of two nmda-receptor antagonists, flupertine (7 mg/kg) and mk-801 (1 mg/kg), and one ampa-receptor antagonist, gyki 52466 (10 mg/kg), were administered twice daily (b.i.d.) on 4 though 9 days post-virus inoculation (dpi). gyki 52466 slightly improved mouse survival and weight gain, but the difference was not statistically significant. talampanel, an ampa-receptor antagonist and a derivative of gyki 52466, slightly improved hamster survival (p ≤ 0.05) when treatment began on 5 dpi, but repeated experiments using different doses and slightly different protocols gave mixed results. riluzole, the only drug shown to improved survival of amyotrophic lateral sclerosis (als), presumably by reducing glutamate excitotoxicity, was not effective against wnv disease when administered b.i.d. beginning 5 dpi. overall, neuroprotective agents did not consistently improve wnv disease, although slight improvements in animal survival might be relevant to improvement of neurological sequelae in wnv-patients. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. hamster and mouse models for west nile virus (wnv) disease were used in this study to identify infected cells of the central nervous system (cns) early in the course of infection. this information may be relevant to therapeutic strategies since most wnv-infected people visit their physicians after showing symptoms suggestive of neurological infection. we subcutaneously infected adult mice and hamsters using 105.3 tissue culture infectious doses of wnv. tissues of infected and control animals from 3 to 11 days post-viral injection (dpi) were fixed by cardiac perfusion using 4% paraformaldehyde. we localized wnv, neuronal and astroglial markers in the paraffin embedded tissue sections by immunofluorescence. the images were captured using the confocal microscope (bio-rad, mrc 1020). we observed the presence of wnv antigen in cns tissues of mice and hamsters as early as 3 and 5 dpi, respectively. a strong wnv-specific immunofluorescence staining was observed in the cytoplasm of neurons from the spinal cord, cerebellum, cerebral cortex, and midbrain of these rodents. the wnv-specific staining co-localized with neuron-specific markers; however, astroglial markers were not co-localized with wnv antigen in brain sections. the lack of tropism by wnv for astrocytes was also confirmed in primary murine astrocyte cultures. interestingly, infected neurons in the midbrain of 7-day infected hamsters co-localized with calbindin, which is a calcium-binding protein and mostly expressed in the interneurons of the cns. therapies were evaluated in hamsters or mice at a time-point when wnv-stained neurons were identified in the cns. acknowledgement: supported by grant 1-u54 ai06357-01 from the rocky mountain regional centers of excellence, nih. laboratory of virology, department of biological chemistry, school of sciences, university of buenos aires, argentina dengue virus (denv) is an arthropod-borne flavivirus that has re-emerged in recent years as an increasingly important public health threat with nearly 50 million infections occurring each year. at present neither specific antiviral therapy nor vaccine exists for the treatment and prevention of denv infections. carrageenans are sulfated galactans that can be extracted from red seaweeds and comprise diverse structures with a wide range of biological properties useful in biomedicine. in a previous study we have demonstrated the antiviral activity of commercialand -carrageenans against denv type 2 and 3 in vero (monkey kidney cells) and hepg2 (human hepatoma cells), showing inhibitory concentration 50% (ic 50 ) values in the range 0.14-1.1 g/ml and selectivity indexes (cc 50 /ic 50 ) in the range 1000-10000. in the present work we studied the mode of action of these polysulfates against denv-2 in vero and hepg2 cells, first analyzing the influence of time of addition of compounds on anti-denv activity by an infectious centre assay. the highest inhibitory effect was observed when the compounds were added during adsorption or at 1 h p.i., being ineffective at later times. then, the effect of compounds on virus adsorption and internalization was studied separately by a virus yield inhibition assay. significant antiviral efficacy was attained if compounds were present either only during denv-2 adsorption or internalization. the possible effect of carrageenans on viral protein synthesis, the subsequent stage of the virus cycle occurring during the first hour of infection, was analyzed by pulse-labeling with ( 35 s)-methionine. no alterations in denv protein synthesis in carrageenan-treated cells were observed. when cells were transfected with purified denv-2 rna in the presence of -carrageenan no inhibition in fluorescent cell focus formation and virus production was detected. besides, no significant direct virucidal effect on denv-2 was shown by the compounds. these results indicate that both denv adsorption and internalization seem to be the main target for these compounds, lacking effect on the steps that occur once the viral genome is inside the cell during in vitro infection of human and monkey cells. multiple members of the flavivirus genus of the family flaviviridae cause lethal hemorrhagic fever or encephalitis. the public health significance of the hemorrhagic fever and encephalitis caused by such flaviviruses is enormous and global and there is a tremendous need for antivirals. imino sugar glucosidase inhibitors have been shown to have selective antiviral activity against viruses such as bovine viral diarrhea virus (bvdv) and west nile virus (wnv) that have common requirements for their glycoprotein processing during virus production. we are developing imino sugar deoxynojirycin (dnj) derivatives through chemical synthesis of compounds with various alkyl side chains and antiviral testing against bvdv and wnv as well as in wnv subgenomic replicon assays. briefly, using a single step growth (virus yield reduction) assay for bvdv and wnv, a series of dnj derivatives containing various conformational locking side chains were shown to have antiviral activity. pre-liminary structure-activity relationships (sar) were obtained for further modification of the alkyl side chain and improvement of these dnj derivatives. the activity and mechanisms of action of these compounds will be presented. several flaviviruses cause life-threatening diseases in man. currently, there is no therapy available for these infections. in recent years, several highly selective inhibitors of the replication of hepatitis c virus (hcv) were designed. most small molecule inhibitors of hcv that are in preclinical or clinical development are either protease or polymerase inhibitors. most of these compounds are highly selective for hcv and are unlikely to exhibit activity against flaviviruses. nucleoside polymerase inhibitors, however, may have the potential to inhibit the replication of flaviviruses as well. we evaluated in vitro whether the active component of the anti-hcv compound valopicitabine, i.e. 2 -c-methylcytidine inhibits the replication of flaviviruses in cell culture. the compound was found to exhibit specific antiviral activity against yellow fever virus 17d (ec 50 = 3.1 g/ml in cpe reduction assays and >98% reduction at 5 g/ml as assessed by qpcr) and dengue fever virus type 2 (ec 50 = 16.5 g/ml in cpe reduction assays). the compound also efficiently inhibited west nile virus replication (>98% at 5 g/ml as assessed by qpcr and >98% by plaque reduction neutralization test at 50 g/ml). in the absence of any drugs for the treatment of flavivirus infections, it may be envisaged that nucleoside polymerase inhibitors, when marketed for the treatment of hcv infections, could be used off-label for the treatment of lifethreatening flavivirus infections. even if such drug would not be able to completely inhibit flavivirus replication, a partial reduction of the viremia during the acute phase of the infection may be sufficient to prevent the development of a fulminate disease and thus protect against virus-induced mortality. yuri klimochkin 1 , andrew shiryaev 1 , igor moiseev 1 , v sabynin 2 , larisa rustamova 2 , alexandr petkevich 2 1 state technical university, samara, russia; 2 institute of epidemiology and microbiology, minsk, belaruss arenaviruses are one of the most dangerous tools of bioterrorism in the view of pathogenicity and epidemiological threat. for the purpose of searching new remedies for treatment are-naviruses infections the synthesis of new derivatives of cage compounds has been carried out. the prepared compounds are bridgehead derivatives of cage compounds bearing different functional groups such as hydroxy, acylamino, alkoxycarbonylamino, alkylthiocarbonylamino groups as well as iminoalkyl adamantane derivatives, some adamantylated heterocycles and compounds containing two adamantane moieties in a molecule. the antiviral activity of the cage compounds has been studied in respect to arenaviruses lassa (sierra-leone strain) and pichinde (an-3739 strain) on the vero cells culture. different level of antiviral activity was shown by 15 compounds. the most active compounds are monosubstituted adamantane derivatives having sufur and nitrogen-containing substituent in the bridgehead position. the data on the activity confirm the availability of searching inhibitors of arenaviruses reproduction in the cage compounds series. brian gowen 1 , donald smee 1 , min-hui wong 1 , anne pace 1 , kie-hoon jung 1 , kevin bailey 1 , lawrence blatt 2 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa several arenaviruses endemic to the south american (junin, machupo, and guanarito) and african (lassa) continents are known to cause frequently fatal hemorrhagic fever. with the exception of ribavirin, which has demonstrated efficacy in cases of lassa fever, there are no other effective therapeutics for the treatment of arenaviral hemorrhagic fever. the outcome of treatment is ultimately dependent upon early diagnosis and the tolerability of ribavirin by patients at the high doses required for effective antiviral activity. we have recently demonstrated that consensus interferon-alpha (ifn-alfacon 1) can protect hamsters from lethal pichinde virus (pcv) infection (gowen et al., 2005 . antimicrob. agents chemother.), which serves as a model for acute arenaviral disease in humans. here we demonstrate highly effective therapy through the combined use of ribavirin and ifn alfacon-1 for the treatment of pcv infection in hamsters. ribavirin was given orally, twice per day for 7 days, and ifn alfacon-1 was administered intraperitoneally, once per day for 10 days. treatments were initiated 1-5 days post-infection with various dose combinations, many which were less than optimal when the drugs were given independently. combining suboptimal doses of ribavirin (5-10 mg/kg/day) with ifn alfacon-1 (5-10 mg/kg/day), we were able to show increased protection from mortality, reduced viral burden and liver disease, and greatly extended survival times as compared to treatments where drugs were administered alone. our data indicate that synergistic activity resulted from combination therapy and that this activity may slow down the progression of disease and decrease fatality rates seen with severe arenaviral infections. further, combination therapy reduces the effective dosage of ribavirin, which would serve to limit its toxicity. acknowledgement: supported by contract no1-ai-15435 from the virology branch, national institute of allergy and infectious diseases, national institutes of health. slobodan paessler 1 , laure deflubé 1 , andrew vaillant 2 , jean-marc juteau 2 , ramon flick 1 1 department of pathology, university of texas medical branch, galveston, texas, usa; 2 replicor inc., laval, que., canada rift valley fever virus (rvfv; genus phlebovirus, family bunyaviridae) is an arbovirus transmitted by many species of mosquitoes. this virus is a major public health concern in sub-saharan africa and egypt, which spread to yemen and saudi arabia. in this area, rvfv is responsible for dramatic epidemics/epizootics underlining the need for efficient antiviral/prophylactic measures. rep 9 is a 40 mer phosphorothioate oligonucleotide, which has previously been shown to have broad-spectrum activity in several viruses (vaillant et al., submitted for publication) . we used a vaccine strain (mp12) as well as the wild-type rvfv (zh501), to test the ability of rep 9 to inhibit bunyavirus proliferation. in vitro data showed reduction of virus titer for both strains using rep 9 at nanomolar concentrations. moreover, the absence of the phosphorothioate modification in a stabilized rep 9 analog resulted in a loss of antiviral activity, suggesting that as in other viruses, the increased hydrophobicity of rep 9 is essential for its antiviral activity. based on the inhibitory activity observed in vitro, we started with in vivo efficacy studies by utilizing a validated mouse model used in our laboratory. more animal experiments are ongoing to confirm the in vitro results and to evaluate the antiviral effect of the rep 9. adriana garozzo 1 , rossella timpanaro 1 , aldo stivala 1 , gianna tempera 1 , christian c.c. cutrì 2 , angelo castro 1 1 department of microbiological sciences, university of catania, via androne 8, 95124 catania, italy; 2 department of pharmaceutical sciences, university of catania, viale a. doria 6, 95125 catania, italy our previous studies described the synthesis and the antiviral activity of 3,4,5-trisubstituted isothiazole derivatives that were found to be particularly effective against picornaviruses. compound 3-methylthio-5-phenyl-4-isothiazolecarbonitrile (is-2) exhibited an interesting anti-poliovirus activity with high selectivity index. in the present study, we investigated on the mechanism of action of this compound. studies on the time of is-2 addition to poliovirus type 1 infected cells suggested that the compound may inhibit some early processes of viral replication. in order to determine its mechanism of action, we evaluated the rate of attachment and internalization of purified [ 3 h]uridine-labeled poliovirus to hep-2 cells in the presence or absence of is-2. no effect on poliovirus adsorption and internalization to host cells was detected. we also investigated the influence of the compound on virus uncoating using labeled poliovirus and measuring the radioactivity of oligoribonucleotides formed from viral rna susceptible to ribonuclease. these experiments demonstrated that poliovirus uncoating is influenced by is-2 action. justin julander 1 , aaron olsen 1 , john morrey 1 , lawrence blatt 2 , kristiina shafer 1 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa alpha togaviruses are medically important arboviruses, with clinical cases occurring each year in north, south, and central america. the recent increase in the threat of the use of these viruses as bio-terrorism agents has led to increased efforts to develop therapeutic agents for treatment of these viruses. venezuelan (vee) and western equine encephalitis (wee) viruses have been listed as category b priority pathogens by the national institute of allergy and infectious disease (niaid). the goal of these studies was to characterize animal models for vee and wee for use in evaluation of antiviral therapies. c3h/hen mice were infected through the intranasal (i.n.) route with a vaccine strain of vee, tc-83. virus was detected in the brain 2 days post-viral injection (dpi). brain titers increased to a peak titer of 10 9.5 50% cell culture infectious doses per gram tissue (ccid 50 /g) on 4 dpi, maintained a titer of 10 9 ccid 50 /g through 7 dpi, and dropped slightly to 10 8.6 ccid 50 /g by 8 dpi. virus was also detected in spleen, liver, and kidney. treatment of vee-infected mice with interferon alpha b/d, a human consensus interferon, resulted in 100% survival, whereas all placebotreated animals died by 9 dpi. syrian golden hamsters were infected with 10 3 ccid 50 wee through intraperitoneal (i.p.) injection. morbidity, including hind limb paralysis, tremors, nasal bleeding, and hunching, and some mortality were seen as soon as 4 dpi. the majority of deaths occurred on 5 dpi. virus was detected in all organs assayed (brain, liver, and spleen) with peak titers occurring 4 dpi. interferon alfacon 1 (ifn alfacon), a human consensus interferon, active in hamsters, was effective in significantly reducing mortality (p < 0.001 as compared to placebo). there was a trend for reduction of brain titers in ifn alfacon-treated animals (mean titer 10 4.8 ccid 50 /g) as compared with placebo (mean titer 10 9.5 ccid 50 /g), although this difference was not statistically significant. these models will be useful in screening potential antiviral agents for efficacy against vee and wee. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. justin julander 1 , kristiina shafer 1 , john morrey 1 , lawrence blatt 2 , robert sidwell 1 1 institute for antiviral research, utah state university, logan, ut, usa; 2 intermune, brisbane, ca, usa yellow fever virus (yfv) has caused significant morbidity and mortality for centuries. primates were the only animal models for visceral yfv. recently, hamsters were found to have morbidity and mortality when injected with a hamster-adapted jimenez strain of yfv (tesh et al., j. infect. dis. 183, 1431 -1436 . the objective of this study was to characterize this model of yfv viscerotropic disease for the study of effects of antiviral compounds and to test compounds with known efficacy for use as a positive control. animals were challenged with a 10 −4 dilution (a dilution previously shown to cause high mortality) of a liver homogenate made from livers taken 3 days post-viral injection (dpi) from hamsters challenged with the jimenez strain. there was 66% mortality in animals challenged with the virus up to 9 dpi. virus titers in the liver peaked 6 dpi as determined by qrt-pcr. a significant increase in serum levels of alt (6 dpi), alkaline phosphotase (6 dpi) and bilirubin (6 dpi), and a significant decrease in amylase (8 dpi), albumin (6 dpi), and glucose (6 dpi) were observed. hepatic icterus was observed in hamsters that exhibited disease signs at the time of necropsy. hamsters were treated with ribavirin or interferon (ifn) alfacon 1, a consensus interferon. ribavirin and ifn alfacon 1 both significantly (p < 0.001) reduced mortality as compared with placebo-treatment. there was also significant reduction in weight loss with ribavirin (p < 0.05) and ifn alfacon 1 (p < 0.01) treatment as compared with placebo. disease signs, such as lethargy and lying prostrate, were also reduced with treatment of ribavirin and ifn alfacon 1. viral liver titers from treated animals were not significantly different from titers in placebotreated animals. the hamster model of yfv disease will serve as a suitable model for the evaluation of antiviral compounds for efficacy against the virus. acknowledgement: supported by contract no1-ai-15435 from the virology branch, niaid, nih. polyomaviruses are small dna tumor viruses that depend on the host cellular dna polymerase for their replication. three polyomaviruses have been associated with tumor formation in humans: jc virus (jcv), bk virus (bkv) and simian vacuolating virus (sv40). in addition, some of them have been associated with viral diseases. jcv can cause progressive multifocal leukoencephalopathy in immunosuppressed patients, while bkv is considered to be the causative agent of polyomavirusassociated nephropathy, which leads to kidney transplant failure. sv40 has not been associated with a well-defined disease, but viral dna sequences and protein expression have been detected mostly in central nervous system (cns) tumors which strengthens the evidence for the association of this virus with human cancer. the activity of various acyclic nucleoside phosphonates (anps) such as cidofovir and adefovir against murine polyomavirus and primate sv40 in vitro has already been demonstrated (andrei et al., 1998. antimicrob. agents chemother. 41, 587-593) . here, the activity of a new class of anp's, namely 6-[2-(phosphonomethoxy)alkoxy]-2,4diaminopyrimidines, against polyomaviruses was assessed. confluent uc1-b cells were infected with either of the four murine polyomavirus strains mn/rde toronto, pta, 2pta2 or lid-1, while bsc-1 cells were infected with either the primate sv40 strain a2895, the sv40 pml-1 strain ek or the sv40 pml-2 strain dar. after removal of the residual virus, serial dilutions of the test compounds were added. the viral cytopathic effect was recorded microscopically after 4-6 days (murine polyoma virus) or 5-7 days (sv40). hpmpo-dapy (2,4-diamino-6-(r)-[3-hydroxy-2-(phosphonomethoxy)propoxy]pyrimidine) and pmeo-dapy (2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine) were less active/selective than cidofovir and adefovir against the three sv40 strains tested. hpmpo-dapy and pmeo-dapy proved to be equally active as cidofovir and adefovir against the murine polyomaviruses. naresh sunkara 1 , sylvester mosley 1 , brian bakke 1 , joshua sadler 1 , katherine seley(radtke) 1 , sunny zhou 2 1 university of maryland-baltimore county, baltimore, md, usa; 2 washington state university, pullman, wa, usa inhibition of biologically significant enzymes critical to nucleotide metabolism and viral replication is a well-established chemotherapeutic approach to the treatment of many diseases. transcriptional 5 -capping of viral mrna has been implicated as an "elongation checkpoint" critical to the replication cycle of many viruses. this capping process is accomplished by various methyltransferases, therefore disruption of methylation becomes an attractive target for therapy. this can be accomplished in several ways; in particular, by direct inhibition of methyltransferases (metase) and/or indirect inhibition of s-adenosyl-l-homocysteine hydrolase (sahase), both established cellular targets for antiviral, antiparasitic and anticancer agents. modified nucleosides, in particular carbocyclic nucleosides, have exhibited potent inhibitory activity against both sahase and metase. inspection of the recent literature has revealed a close correlation between sahase inhibition and potent biological activity against negative stranded (−)-rna viruses (i.e. arenaviridae, paramyxoviridae, rhabdoviridae), double stranded (ą)-rna viruses (reoviridae), poxviridae, as well as hiv-1, thus supporting the importance of sahase as a viable chemotherapeutic target. herein we report the design, synthesis, and preliminary biological activity of a new class of structurally novel carbocyclic nucleosides. phosphorylation of ␣-p-borano substituted nucleoside diphosphates charlotta wennefors, mikhail dobrikov, barbara ramsay shaw chemistry department, duke university, durham, nc 27708-0346, usa most nucleoside antiviral agents require stepwise phosphorylation to their respective triphosphates in order to be activated in the cell. ␣-p-borano substituted nucleoside triphosphates are of interest because they have proven to be good substrates for hiv-1 reverse transcriptase (rt) and may therefore be useful antiviral agents. studies in our laboratory have indicated that the ␣-p-borano substitution of 2 3 -dideoxycytidine triphosphate (ddctp) resulted in a 28-fold increase in efficiency of incorporation by mmlv rt compared to non-substituted ddctp. however, the potency of these ␣-p-borano substituted nucleoside analogs as anti-viral drugs highly depends on their ability to be activated to nucleoside triphosphate (ntp). the phosphorylation of nucleoside analog diphosphates to their respective triphosphates has remained largely unexplored. here, the roles of several phosphorylating enzymes are examined. in our laboratory, nucleoside diphosphate kinase, creatine kinase, and pyruvate kinase are being evaluated for their specificity towards nucleoside analog diphosphates. the effects of nucleobase, ribose, ␣-phosphate substitution and stereochemistry of the boranophosphate group are of interest. the binding affinities of the substrates for creatine kinase (ck) and pyruvate kinase (pk) were determined using a fluorescence-quenching assay, which allowed us to investigate the substrate affinity in the pre-steady state. rabbit muscle ck and pk were titrated with a wide range of ndps and ntps by monitoring a decrease in enzyme intrinsic fluorescence. the affinities of these substrates were determined to establish a structure-activity relationship for ck and pk and to evaluate the effect of a substrate ␣-p-borano modification. ck showed stereospecificity towards the sp isomer of adp␣b whereas pk showed stereospecificity towards the rp isomer of adp␣b. negative cooperativity was observed for all studied substrates. steady-state experiments are also being performed directly following the product formation using uv-visible spectroscopy and high performance liquid chromatography (hplc). these kinases were investigated because they may serve as a means for activation of antiviral ␣-p-borano substituted ndps. traditional antiviral targets encoded by the small human papillomavirus (hpv) genome are lacking. for this reason, we chose to target dna sequences within the hpv genome in an effort to identify compounds that would block viral dna replication in cells. we chose compounds known as polyamides, which are related to distamycin and other natural products, as our dna binding agents. unlike many literature studies where polyamides were designed to block formation of the transcription complex for a particular gene, we chose to target sequences within the origin of replication (ori). thus, pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv31 ori. the principles used to design these compounds will be described. we used "traditional" hairpin polyamides and some more unusual structures related to very recent literature reports. from the focused library that we prepared, two highly active molecules were identified. the rest of the molecules had minimal or zero activity. no cellular toxicity was observed, either in this project or in a related program where polyamides were used to affect cox-2 transcription (and subsequent expression) in rheumatoid synovial fibroblasts. of particular interest is the difference between the active molecules and two closely related compounds that were inactive: the active species bind and recognize two more hpv dna base pairs than do the related but inactive structures. this presentation will provide detailed chemistry background and structural information to complement our cell work that is also being presented at the meeting. discovery of the chemokine receptor ccr5 as a co-receptor for hiv-1 infections revealed a novel approach to hiv-1 treatments and preventions. ccr5, a member from the family of 7tm g-protein coupled receptors, thus became an attractive target pursued in the pharmaceutical industry. with the recent successful developments of several small molecules in clinic, these ccr5 antagonists hold great promise to be the next generation of anti-hiv medicines. this poster will describe our efforts at the n-terminal piperidine ring of template a to improve pharmacological properties of derived molecules. according to current models, proteolytic processing of hiv-1 gag precursor occurs within the virions which detach from infected cells. meanwhile, the viral protease is activated much earlier, and gag p55 cleavage initiates in infected cells. we followed the fate of matrix protein cleaved in infected cells (cma) in comparison with ma cleaved in the virions (vma) and showed that both forms differ in their localization in the infected cells and in the virions, both forms are involved into virus pathogenesis and represent the targets for antiviral compounds. mt-4 cells were labeled with [3h]-leucine or myristic acid, and 2 h after labeling protease inhibitor was added to separate the cleavage of cma from vma. cma was found in the nuclear and membrane fractions of infected cells while cca resided in cytoplasm. 18-20 h after labeling cma was found in the virions localizing in the cores. vma was located under lipoprotein envelope of the virions. new membranotropic antiviral compounds based on adamantane-and norbornene-related derivatives not toxic for the host cells were added to mt-4 cells before infection or 1-2 h later and at concentration 2-6 ug/ml blocked reverse transcription, the transport of cma into the nuclei, and the production of infectious virus. the compounds inhibiting very early step of virus life cycle are optimal candidates for microbicides. to enhance their antiviral activity, we plan to associate polyanionic matrix with ma imitating peptides and cholesterol-like fragments. kurt vermeire 1 , thomas bell 2 , sreenivasa anugu 2 , noah duffy 2 , roger le grand 3 , erik de clercq 1 , dominique schols 1 1 rega institute for medical research, katholieke universiteit leuven, leuven, belgium; 2 department of chemistry, university of nevada, reno, usa; 3 service de neurovirologie, fontenay-aux-roses, france the cyclotriazadisulfonamide (cada) compounds were shown to be potent inhibitors of hiv replication in human t-cell lines, pha-stimulated pbmcs, and monocytes/macrophages (ec 50 : 0.3-3.2 m). the prototype compound, cada, had consistent activity against laboratory adapted and primary clinical isolates of hiv-1, irrespective of chemokine receptor preference (r5, x4, r5/x4). cada acted synergistically when evaluated in combination with various other hiv drugs, such as reverse transcriptase (rt), protease, and virus entry inhibitors. flow cytometric analysis revealed a significant decrease in the cell surface and intracellular expression of the cd4 receptor in human cells after cada-treatment. moreover, the anti-hiv activity of cada correlated with its ability to down-modulate the cd4 receptor in human t-cells. here, we report the consistent antiviral activity of cada against: (i) drug-resistant viruses (i.e. viruses resistant to rt inhibitors, protease inhibitors, and enfuvirtide); (ii) different hiv-1 subtypes (a, b, c, d, a/e, f, h, o); and (iii) various hiv-2 strains examined. in addition, cada potently inhibited sivmac251 infection of pbmcs isolated from macaques (ec 50 : 1.6 m). comparable results were obtained in human cells infected with sivmac251. flow cytometric analysis also demonstrated a significant and dose-dependent down-regulation of the cd4 receptor expression at the cell surface of simian pbmcs after treatment with cada. the combination of cada with cellulose acetate 1,2benzenedicarboxylate (cap), an enteric coating polymer for capsules and tablets, resulted in a synergistic antiviral activity. in summary, our data indicate that cada may qualify as a potential anti-hiv microbicide drug candidate for the prevention of the sexual transmission of hiv. the preparation of gel formulations of cada (as single drug and in combination with cap) for vaginal administration in non-human primates is currently under investigation. department of micro & immuno, suny upstate medical university, syracuse, ny, usa varicella zoster virus (vzv, human herpesvirus 3) infection causes chicken pox, latency is established in neurons, and reactivation leads to shingles. acyclovir and its derivatives are the treatment of choice for both manifestations of vzv. new therapeutics are needed because acyclovir-resistant strains exist, and treatment must begin within 48 h. we have studied the anti-vzv properties of roscovitine, a cyclin dependent kinase (cdk) inhibitor. here, we tested more compounds that block the cell cycle and determined that vzv is acutely sensitive to them. their effects on vzv replication were tested in human foreskin fibroblasts (hffs) because these primary cultures should have a normal cell cycle (unlike tumor cell lines). the cytotoxicity of the drugs was determined by neutral red dye uptake assays. hffs were inoculated with a low moi (0.01) of vzvinfected cells, which remains entirely cell-associated, and then treated with drugs or diluent for 48 h. vzv spread and replication were measured by infectious focus assay and quantitative real time pcr. all of the drugs tested (acyclovir [acv], phosphonoacetic acid [paa], aphidicolin, aloisine a, purvalanol a, roscovitine, r-roscovitine, s-roscovitine, indole-3-carbanol [i3c], l-mimosine, dichloro-␤-d-ribofurano-sylbenzimidazole [drb]) had some anti-vzv activity. the selective indices of aphidicolin (833), purvalanol a (570), and i3c (1000) were greater than the positive controls acv (250) and paa (60). aphidicolin inhibits mammalian dna polymerase and is in clinical use for cancer; purvalanol a, a 2,6,9-trisubstituted purine, primarily inhibits cdk1; and i3c is derived from cruciferous vegetables and inhibits cell proliferation. the concentrations of these compounds that inhibited vzv replication were much less than those needed to cause cell cycle arrest, suggesting that vzv depends on the enzyme activities targeted by these compounds and not on cell proliferation per se. these three drugs will be studied next in skin organ culture and in the scid-hu mouse model of vzv pathogenesis. the results presented here demonstrate that targeting cell functions can inhibit vzv replication and help us better understand virus-host interactions. the viruses could be identified as supra-biopolymeric nanoscale complexes, parasitic intervention in cells of which occurs on an inter-polymeric reactions level. so the antiviral safety can not be fully provided without adequate nano-responsible antivirals (nav). here we discuss a strategy and methodology for the multi-functional nav development by rational macromolecular sar-cooperation of: (1) polyelectrolyte-specific interferon induction and immunomodulation; (2) electrostatic-selective prevention of viruses absorption on plasma membranes; (3) membrane-targeted blocking of post-absorption steps (fusion); (4) macromolecular prevention of structure-specific interactions of viral and cellular receptors; as well as (5) polymericassociated disruption of the latest stage of viral replication (virions assembly and maturation). a cooperation of the (1) and (2) functions was explored by synthesis and sar-optimization of succinate and carbohydrate polymeric derivatives modified with controllable combinations of anionic (a1/a2) groups. the immune-mediated protectors against tick-born, rabies, and other viral infections in vivo, and hiv-1 absorption inhibitors in vitro, were developed. this pre-nav generation was used as a macromolecular platform to step-by-step targeted design and synthesis toward high effective multi-functional nav where virusresponsible nano-selectivity was achieved by rational intra-or inter-molecular cooperation of virus-specific membranotropic vectors (bi), raft-targeted anchors (ci), and peptide-kind mimickers of virus usable receptors of human cells (pi), particularly ccr5/cxcr4. as a result, the novel nano-sensitive systems possessed unique wide multi-synergistic antiviral potency on a high level selectivity up to si = 20,000 (against hiv-1 strains) were created and purposed for advancement of antiviral vaccines, drugs, and microbicides. marina kukhanova 1 , alexander ivanov 1,2 , georgii galegov 3 , valeria andronova 3 , maxim jasko 1 1 engelhardt institute of molecular biology, russian academy of sciences, moscow, russia; 2 centre for medical studies, university of oslo, moscow, russia; 3 ivanovsky institute of virology, russian academy of medical sciences, moscow, russia novel acyclic purine phosphonate derivatives bearing a double bond conjugated with the nucleic base, namely, (z)-and (e)-9-[3-(phosphonomethoxy)prop-1-en-1-yl]purines, were synthesized, and their efficacies against hiv-1 and hsv-1 were evaluated in cell cultures. the activity of (z)isomer was higher against hiv than that of the reference 9-[2-(phosphonomethoxy)ethyl]adenine (adefovir) and comparable in respect to the activity of adefovir against hsv. the (e)-isomer showed low antiviral activity against both viruses. the compounds were less toxic towards cell cultures if compared to adefovir. the diphosphates (z)-and (e)-9-[3-(phosphonomethoxy)prop-1-en-1-yl]purines were evaluated as substrates towards hiv-1 reverse transcriptase and hsv dna polymerase. (z)-isomer was shown to be a more efficient substrate for both enzymes than the (e)-isomer. human dna polymerase alpha could incorporate neither of the diphosphates into the 3 -end of the growing dna chain. available to this virus. jev genome is an approximately 11-kb single-stranded positive-sense rna that has a cap structure at its 5 terminus but lacks a poly(a) tail at its 3 -terminus. the coding region of the genome is flanked by 5 -and 3 -untranslated region (utr). the 3 -utrs on both plus-and minus-strand jev genome contain important cis-acting elements required for the replication of the viral rna genome. peptide nucleic acid (pna) is a synthetic oligonucleotide, in which the phosphodiester backbone of dna/rna is replaced with a polyamine-(2-aminoethyl) glycine skeleton. pna offers a potentially powerful approach for recognition of rna and silencing of gene expression. in this study, we investigated the antiviral effect of the pnas targeted to the 3 -utr region of jev genome. to evaluate the pnamediated inhibitory effect on rna synthesis in vitro, the rnadependent rna polymerase (rdrp) of jev, ns5 protein, which plays a major role in replication of the viral genomic rna, was expressed in escherichia coli and purified to near homogeneity by sequential column chromatographies. the recombinant jev ns5 protein exhibited a primer-dependent rdrp activity in vitro on a homopolymeric template, poly(a). in addition, it was able to accept both plus-and minus-strand 3 -utrs as templates for rna synthesis in the absence of an exogenous primer. it could utilize the 3 -end 83-nt of jev genome as a minimal template. in vitro rdrp assays using this functional recombinant jev rdrp in the presence of the pnas targeted to the jev 3 -utr 83-nt showed a dose-dependent rna synthesis inhibition. delivery of the inhibitory pnas to the jev-infected cells suppressed jev replication, as determined by western-blot analyses and plaque assays. our results showed a sequence specific inhibition of jev replication by antisense pnas, suggesting the possible application of pna as a novel anti-jev agent. julia serkedjieva 1 , reneta toshkova 2 , milena nikolova 3 , reneta tsvetkova 3 , stefka antonova 4 , ivana roeva 1 , munnever sokmen 5 , bektas tepe 5 , medine gulluce 6 , fikrettin sahin 6 , atalay sokmen 5 1 institute of microbiology; 2 institute of experimental pathology and parasitology; 3 institute of botany, bulgarian academy of sciences; 4 faculty of biology, department of microbiology, sofia university, sofia, bulgaria; 5 faculty of art and science, department of biology, cumhuriyet university, sivas, turkey; 6 faculty of art and science, department of biology, atatürk university, erzurum, turkey natural products can be an important source of new pharmaceuticals. research on antivirals of natural origin is mainly focused on plants, since, among other reasons, they can be selected on the basis of their ethnobotanical use. plant extracts and natural plant products exhibit also a variety of biological activities with pharmacophoric utility. the population of the balkan peninsula, like people from all continents, has long applied poultices and imbibed infusions of hundreds of indigenous plants. the present report summarizes the antiviral screening study of 134 plant products, obtained from 67 bulgarian and turkish medicinal plants. they were tested for inhibitory effect on the reproduction of selected influenza virus (flu) strains in mdck cells and herpes simplex virus (hsv) strains in mdbk cells. the reduction of virus-induced cpe and infectious virus yields were used as measures of viral inhibition. fifteen samples (11.2%) inhibited flu reproduction, and twelve samples (7.5%) were active against hsv. the anti-flu activity was confirmed in vivo for all tested samples. the most effective products were tested further for their antiproteolytic, antioxidant and immunogenic capacities and for potential antibacterial and antifungal effects. the following correlations among the variety of biological and pharmacological activities of the plant products were observed: the anti-flu effect was associated with anti-hsv effect and vice-versa in 55.5%; the antiviral effect was connected with antioxidant activity in 100%; the anti-flu effect was associated with immunogenic properties in 100%; there was found no correlation between the antiviral effect and the antiproteolytic capacity, the anti-viral properties and bacterial or fungal inhibition, the anti-viral activity and the polyphenol contents. our previous investigations have revealed antiviral activity of some proteolysis inhibitors such as e-aminocaproic acid (e-aca) and para-aminomethylbenzoic acid (pamba) in vitro, in vivo and in clinic. construction of qsar computer-assisted hierarchical system for the effective anti-herpetic (anti-hsv) and anti-influenza (anti-flu) agents' selection as well as the elaboration of new methods of their synthesis are permanently the object of keen interest of our team. the objective of this study was to investigate the efficacy 2,6-di-substituted pyridines and their analogs combined with the fragments of proteolysis inhibitors in the framework of the qsar approach. molecules of new compounds consisted of "nucleus" (py or ar) and two symmetrical fragments: e-aca-carbonyl or pambacarbonyl taken from the inhibitors' molecules. anti-flu activity in dose 10-3 m was studied in vitro on the model of a/hong kong/1/68 (h3n2) reproduction in tissue cultures of chorioallantoic membranes of 12 days old chick embryos. anti-hsv activity in doses 10-4 m was studied on models of reproduction of hsv-1 on cell culture hep-2. compounds with py-nucleus, contained pamba-carbonyl or e-aca-carbonyl fragments, demonstrated sufficient anti-hsv activity (39.4 and 61% of reduction of intra-nucleus virus-specific inclusions on infected cells account accordingly). 3,5-dihydrazine-carbonyl-2,6-dimethylpyridine showed high anti-hsv (52%) activity. the efficacy of the designed antiherpetic compounds obtained with the combined efforts of qsar computer-assisted design, properties prediction, synthesis, and biological testing as well as the correction introduced after the iteration circle passsage have proven to be the efficient modern way of drug design. acknowledgement: this research was supported in part by stcu grant # 3147 and all the authors are indebted to stcu foundation courtesy. lubomira nikolaeva-glomb 1 , angelina trifonova 1 , stephan filipov 2 , angel s. galabov 1* 1 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; 2 institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria a series of aporphinoid alkaloids isolated from glaucinum flavum l. or obtained synthetically, were tested in vitro for antiviral activity against viruses belonging to picorna-, orthomyxo-, paramyxo-and herpesviruses. one of them, oxoglaucine, manifested a well-pronounced inhibitory effect on poliovirus 1 replication in fl cells measured by the semi-quantitative agardiffusion plaque-inhibition test. in virucidal activity testing the compound did not show direct virucidal effect on the extracellular virus. oxoglaucine's 50% inhibitory concentration for poliovirus 1 (mahoney) was found to be 0.188 g/ml in the cpeinhibition test and 0.041 g/ml in the classical plaque-inhibition test. similar values were obtained for the vaccinal poliovirus type 1 strain, lsc-2ab. the antiviral effect of oxoglaucine on the replication of viruses belonging to another enterovirus species was tested, i.e. coxsackie and echoviruses (hev-b). cva-9, the six coxsackie b viruses and 6 echoviruses were tested for their sensitivity against the antiviral effect of oxoglaucine by the endpoint dilution method in the multi-cycle cpe inhibition set-up in fl cells. oxoglaucine revealed a marked inhibitory effect on all tested enteroviruses. the concentrations that reduced virus titer by 1 lg ranged from 0.01 to 1.0 g/ml. selectivity index was greater than 100 and even greater than 1000 for some of the viruses tested. time-of-addition study by the one-step virus growth cycle set-up showed strong virus inhibition during the early periods of virus replication. milka mileva 1 , angel s. galabov 2 1 department of medical physics and biophysics, medical university, sofia, bulgaria; 2 institute of microbiology, bulgarian academy of sciences, sofia, bulgaria in this study an investigation and comparison of the effects of plant flavonoid polyphenols quercetin and its sugar-containing homologue (rutinoside) rutin on the "oxidative stress" in liver, isolated from influenza virus a/aichi/2/68 (h3n2) (2.0 of ld50) inoculated mice, is carried out. it was found that experimental influenza virus infection is accompanied with graduated oxidative disturbances in the liver of mice, despite the absence of virus and inflammation in this tissue. it was found that experimental influenza virus infection is accompanied with a significant increase of lipid peroxidation products, a decrease of natural antioxidants (vitamin e, glutathione) and cyp, an inhibition of cytochrome c-reductase and liver monooxygenases (analgin-ndemethylase and amidopyrine-n-demethylase) as compared to control (non-infected) animals. the preliminary (5 days) supplementation of mice with rutin, quercetin or its combination, and their subsequent virus inoculation influence significantly all analyzed parameters as compared to controls. the protective effect of rutin against influenza virus-induced lipid peroxidation and activities of cyp and liver monooxygenases in liver was better expressed than the effect of quercetin may be due to containing of rutinoside part or difference of its metabolism. hyun-jeong lee 1 , ji-sun kwon 1 , chi-ung moon 2 , jong-hwan kwak 3 , youn-jeong lee 4 , chang-seon song 1 1 avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; 2 hanyang university, seoul, korea; 3 sungkyunkwan university, seoul, korea; 4 national veterinary research and quarantine services, seoul, korea one of the traditional korean medical herb extract named s22 was investigated to determine the anti-influenza virus activity in vitro and in vivo. the s22 showed potent antiviral activities against a/pr/8/34 (h1n1) influenza virus with the 50% effective concentration (ec50) values of 62.5 g/ml and the 50% cytotoxic concentration (cc50) values of 673.86 g/ml in mdck cells. treatment with the s22 appeared capable of significantly ameliorating the influenza virus infection in mice by oral gavage treatment. the s22 treated mice showed significantly higher survival rate and lower pathogenic index as well as lung virus titers than untreated control mice. further, the s22 was extracted with chcl3, etoac and n-buoh for isolation of active compounds. the anti-influenza effects of these active compounds will be discussed. the antiviral effect of aqeous and ethanol extracts of ocimum gratissimum (og), terminalia catappa (tc), gynostemma pentaphyllum (gp), newbouldia laevis (nl), aspilia africana (aa) and phyllantus amarus (pa) leaves was examined by cultivation of virus and extracts in embryonated chicken eggs. extracts were inoculated immediately after virus (zero time) or 2 h after virus inoculation. virus replication was compared with those of controls by haemagglutination assay. at zero time, aqeous extracts of og, tc, pa, and gp inhibited virus growth by 50, 61, 72, and 94%, respectively whereas those of nl and aa did not. ethanol extracts of og, tc, pa and gp at same time inhibited by 25, 100, 72, and 100%, respectively whereas nl and aa did not. at two h after virus inoculation aqeous extracts of og, tc, pa and gp inhibited virus growth by 92, 6, 67, and 100%, respectively whereas nl and aa had no effect. ethanol extracts of tc, pa and gp inhibited the virus by 94, 78, and 94%, respectively whereas those of og, nl, and aa did not. the herbs were studied because they were being used by some herbalists in the treatment of human infectious diseases. the 20th international conference on antiviral research will be held in the palm springs, california area. the conference will begin on sunday, april 29, 2007 and will end on thursday afternoon, may 3, 2007. all scientific sessions will be held at the westin mission hills resort, rancho mirage, ca. the purpose of the international conference on antiviral research is to provide an interdisciplinary forum at which investigators involved in basic, applied, and clinical research worldwide can meet to review recent developments in all areas of antiviral research. specific topics to be covered in the program include synthesis and chemistry, biochemistry and mechanism of action, molecular biology and drug targeting, in vitro evaluation, animal models, pharmacokinetics, toxicology, and clinical trials. within these areas of interest, there will be invited overview speakers, oral presentations, and poster presentations. the famous el paseo shopping district of palm desert and downtown palm springs offer not only a large variety of galleries, boutiques and shops too numerous to mention, but there are restaurants for virtually every palate. whether your tastes run to burgers, sushi, pizza, escargot, steak, mexican or continental you will find it here with a california flourish in every price range. we hope you will take advantage of this opportunity to combine an important learning experience with a magnificent travel experience and join us in palm springs, california for the 20 th international conference on antiviral research. isar conference committee future conferences acknowledgement: the study was supported by rfbs 05-04-49500 and russian ministry of sciences (lot 11). key: cord-014965-efmozngq authors: nan title: infectious diseases other than cmv (1st section) date: 2001-06-11 journal: bone marrow transplant doi: 10.1038/sj.bmt.1702942 sha: doc_id: 14965 cord_uid: efmozngq nan performed after culturing cd34+ cells for 3 days, we observed the same effect as described above. however, when cd34+ cells were cultured for 7 days prior to infection, a comparable suppression of cell expansion could be found for both hhv-6 variants a and b. our results suggest, that hhv-6a may influence the expansion of more immature progenitor cells, whereas hhv-6b displays a suppressive effect only on more mature, differentiated cells in vitro. w.h. krü ger, n. krö ger, m. abromeit, h. renges, f. tö gel, j. schrum, p. schafhausen, r. erttmann, h. kabisch, a.r. zander (hamburg, d) patients with a positive history of systemic fungal infection undergoing allogeneic sct are highly endangered by reactivation of their infection. for such patients some attempts of antimycotic prophylaxis with low dose conventional amphotericin-b (am-b) have been reported. however, the major side effect of conventional am-b is nephrotoxicity. here, we report data of patients grafted under the protection of liposomal (lip) am-b. 37 patients (w/m: 14/23) with a median age of 37 (2-65) years underwent allogeneic mrd (n=19), mud (n=14), syngeneic (n=2) or autologous sct (n=2) for treatment of al (n=24), cml (n=7), mds (n=4), saa (n=1), and granulomatosis (n=1). all patients had a history of fungal pneumonia with evidence for aspergillus spp. as infectious agent in 17 cases. three patients had suffered from aspergillus sinusitis (n=2) and aspergillosis of the bone, respectively. fungal infection was culture-documented in 8 cases. x-ray examination or clinical parameters led to the diagnosis in 29 cases. therapy with lip-amb was started either primarily (n=18) or after a short course of conventional am-b (n=15) or itraconazole (n=4). lip-amb was initiated on day +3 (-10-69) for a median duration of 19 days (2-63). the start dose of 2,7 (0,6-5) mg/kg was increased to 2,9 (1,0-8,2) mg/kg in some patients. lip-amb was excellently tolerated, fever and chills occurred in two patients. creatinine showed a slight increase to 137% (85%-667%) of the base line probably not related to lip-amb. after a median follow-up of 28 (8-78) days 22 (60%) patients were discharged without evidence for fungal infection. 3/8 (38%) of patients with history of culture-documented mycosis died from relapse of c. krusei septicaemia (n=1), from cns bleeding probably related to relapsed aspergillosis and from multi-organ failure without culture-proven mycosis. 5 patients have died from aspergillosis (n=2) or candidosis. one of these patients had to be grafted during active aspergillosis of the lung for disease-specific reasons. the remaining seven patients have died from multi-organ failure (n=4), gvhd and septicaemia (n=1) and pneumonia (n=2) related to bacterial infection in two cases. patients with a history of preceding mycotic infection are at high risk to acquire potentially fatal infections under sct. however, our data clearly show that only 7/37 (19%) of our patients developed culture-positive fungal infection during severe immunosuppression. we conclude that a history of preceding fungal infection is no contraindication for stem cell transplantation. between 1/96 and 9/98, 17 out of 270 pts manifested proven or possible txo after allogeneic sct. during this period 4/17 patients died from txo as primary cause of death. since 10/98 we started a prospective randomized monocentric study comparing tmp/smx with pym/dps to prevent reactivation in patients at risk. risk was defined as a positive serology for txo in recipients or donors. additionally, immunosuppression with a daily dose of more than 50 mg prednisolone was requested for inclusion. randomization was executed when hematopoiesis appeared stable at the end of hospitalization. arm a received tmp/smx 2 x 160 / 800 mg twice a week (a). arm b was treated with pym 50 mg once a week and dps 50 mg once a day (b). folinic acid was given 2 x 30 mg twice a week to all pts. 111/199 pts were at risk, 67/111 were randomized and evaluable. patients with drug related side effects discontinued prophylaxis and switched to the alternative arm after resolution. during the study, 1 pt developed lethal cerebral txo prior to randomization. the principal reason for exclusion was cytopenia (18/44 pts), immunosuppression with pdn ͻ50mg/d (n=25), and non signed informed consent (n=1). prophylaxis associated side effects were equally distributed in the two study arms (fisher exact test p=0,2). main reason for discontinuation was cytopenia. interestingly, the covered interval by prophylaxis was significantly longer for arm a: 160 versus 67 days, respectively (t-test: p=0.001). at a first glance, arm a seems to be better tolerated in view of the longer covered period of prophylaxis. however, longer follow up and further analysis will show whether this is due to side effects itself or to the practice of the physician to take arm b side effects more serious than those of arm a. to evaluate the influence of the helicobacter pylori (h.p.) infection on gastrointestinal complications after high-dose chemotherapy and stem cell transplantation (stx) we tested 114 patients (54 female, 60 male) by the 13c-urea breath test prior to initiation of conditioning therapy. at the initial testing 92 patients (solid tumors: 24pts., hematological malignancies: 68 pts.) showed a negative result and 22 patients (solid tumors: 9 pts., hematological malignancies: 13 pts.) a positive result without any gastrointestinal complaints. compared to the overall incidence of h.p. infection of 19% in these patients, the subpopulation of breast cancer-patients had a higher infection rate (41%). these 114 patients with a median age of 46 years (17-60) underwent an autologous (n=95) or allogeneic transplantation (n=19). beside gut decontamination they all received prophylaxis with intravenous ranitidin (150mg/d) which was replaced by a proton pump inhibitor (ppi) at a dose of 40mg/d i.v. if clinical symptoms of gastritis appeared. no main differences between both groups according to the occurrence of nausea/ vomitus, enteritis and mucositis could be seen. however, the incidence of severe mucositis whoiii/iv as well as enteritis who iii/iv was higher in h.p. positive patients (41% and 23%) in comparison with h.p. negative patients (29% and 10%). especially in breast cancer patients (n=22) mucositis whoiii/iv was more pronounced in the h.p. positive group (55% vs. 15% respectively). furthermore, the occurrence of clinical signs of gastritis could not be related to the helicobacter infection: 36% of the h.p. positive vs. 45% of the h.p. negative patients felt epigastrical pain. 68% vs.71% needed treatment with a proton pump inhibitor. in conclusion helicobacter pylori infection seems not to influence the incidence of gastrointestinal complications but possibly the severity of mucositis and enteritis. low-dose amphotericin b lipid complex (ablc) is safe and effective as empiric anti-fungal therapy in immunocompromised patients with hematologic malignancies r. powles, b. sirohi, j. mehta, s. kulkarni, k. murphy, b. cheung, a. conway, r. saso, a. riggs, s. singhal, d. cunningham, j. treleaven (sutton, uk) ablc (abelcet, the liposome company) is a ribbon-shaped liposomal formulation of amphotericin b consisting of dimyristoylphosphatidylcholine and dimyristoyl-phosphatidylglycerol in a 7:3 molar ratio which is especially concentrated in pulmonary tissue. it is known to be safe and effective in presumed and confirmed fungal infections in immunocompromised patients at the dose of 5 mg/kg. there are limited data on its use at lower doses. we explored low-dose ablc in immunocompromised patients with hematologic malignancies who had fever of unknown origin which had failed to respond to combination antimicrobials, and were presumed to have fungal sepsis. 26 immunocompromised patients (15-70 y, median 44; 17 leukemia, 7 myeloma, 2 lymphoma) received 32 courses of ablc at the median daily dose of 2 mg/kg rounded off to the nearest vial size (range, 1.3-2.7 mg/kg) after autologous (n=7) or allogeneic (n=8) stem cell transplantation or chemotherapy (n=17). the median neutrophil count at start of ablc therapy was 0.1 x 109/l (range, 0-8.6). 14 courses of ablc had been preceded by conventional amphotericin b and 11 by fluconazole without response. the median days of therapy with ablc was 6 (range, 1-35). courses of 4 doses (n=23) were considered evaluable for efficacy and all courses were evaluable for toxicity. 18 courses resulted in complete response, 3 in partial response, and 2 in failure (overall response rate 91%). the 2 failures were switched after 5 and 6 days of ablc to 3 and 2.5 mg/kg ambisome for 10 and 6 days respectively, grew candida and aspergillus from the sputum respectively, and died without responding. the change in serum creatinine from the beginning to the end of therapy was -56 to +54 micromol/l (median -1). 23 treatment courses had been premedicated with chlorpheniramine/hydrocortisone for the first few days. infusion-related toxicities comprised rigors (n=9), pyrexia (n=5), hypertension (n=1), and hypotension (n=1); at least one of these toxicities was seen in 9 (28%). these data suggest that low-dose ablc is very effective empiric anti-fungal therapy in immunocompromised patients with hematologic malignancies. viral pneumonia is an important cause of morbidity and mortality in patients undergoing allogeneic stem cell transplantation. a previous study from the usa has found viral pathogens such as respiratory syncytial virus (rsv), influenza a and b (flu a & b), parainfluenza (paraflu) and adenovirus emerging as significant causes of upper and lower respiratory tract infections (clin inf dis 1996; 22:778-82) . a retrospective analysis of the incidence of community respiratory viral infections in our population was carried out for the 72 allogeneic bm and pbsc transplants performed on 70 patients between january 1997 and december 1999. rsv, flu a & b, paraflu, adenovirus and picornavirus were detected using direct immunofluorescence testing of nasopharyngeal aspirate or bronchoalveolar lavage fluid as clinically indicated. the overall incidence of proven community respiratory viral infection was 26% in the first year post allograft (19 of 72 transplants). these consisted of 9 cases of rsv (48%), 4 of flu a (21%), 1 of flu b (5%), 3 of paraflu iii (16%), 1 of adenovirus (5%) and 1 of picornavirus (5%). these results are comparable to previous us studies. six of these 19 patients (32%) had upper respiratory signs only, 3 received specific anti-viral therapy with nebulised ribavirin for rsv (1), paraflu iii (1) and flu a (1). the other 13 patients (68%) had lower respiratory tract signs on examination and 7 had chest x-ray changes. of this group, 9 were treated with nebulised ribavirin for rsv (7), paraflu iii (1) and flu a (1) and 4 went on to receive iv ribavirin for rsv, one received zanamivir and amantidine for flu a and 3 had no specific antiviral therapy for flu a (1), paraflu iii (1) and picornavirus (1). overall of the 19 proven infections, 12 patients made a full recovery, 2 died of rsv pneumonitis, two patients with paraflu iii died of superimposed infection, one bacterial and one fungal; neither received specific antiviral therapy and three died of unrelated causes. to conclude, prompt treatment of community respiratory viral infections may reduce morbidity and mortality in adult allogeneic bmt and pbsct patients but respiratory viral infection was still the primary cause of death in 3% of our patients and probably contributed significantly in a further 3%. a.j. ullmann, k. weise, p. brandt, c. huber (mainz, d) objective: patients post stem cell transplantation are at very high risk for reactivating varicella-zoster virus [vzv] . herpes zoster infection is frequent and has the potential to disseminate. atypical generalized zoster is associated with an increased mortality rate. methods: retrospective analysis of 1282 blood samples originally collected from 53 patients for cmv-monitoring was performed. period of analysis was approximately one year with a minimum of 5 months. thirty-seven of the patients received an allogeneic transplantation and 16 underwent a cd34-selected autologous transplantation. pcr-primers were selected from the orf63 of the vzv-genome. confirmation of the positive results was performed by southern blot analysis. the patients' histories were available from their medical record and/or telephone interview. results: none of the patients with negative blood pcr-results developed vzv-disease. seven of the 12 allogeneic transplanted patients with at least one positive pcr-result had a vzv-disease. three of the positive patients developed a disseminated disease. four of the 5 patients with a positive result and no vzv-disease received at that time point polyvalent immunoglobulins. none of the five received antiviral medications. all (n=2) of the autologous transplanted patients with a positive pcr-result developed a vzv-disease. conclusion: this retrospective analysis reveals that the pcr testing of the blood for vzv has a high sensitivity and suggests that a vzv-viremia occurs even during localized disease. a prospective study to evaluate the possibility to predict a disseminated vzv-disease is warranted. antimicrobial prophylaxis in ebmt centers: a report from the ebmt infectious diseases working party h. akan, c. cordonnier for the infectious diseases working party of the ebmt (ankara, tr; creteil, f) in order to know about the anti-infectious prophylactic policies used in the ebmt centers, the idwp run a mail-in survey regarding antibacterial, antifungal, antiviral, and immunoglobulin (ivig) prophylaxis. the survey consisted of questions on indications and modalities (drugs, doses, duration) of prophylaxis in autologous and allogeneic sct. seventy-four centers from 18 countries responded. 1. allogeneic transplant centers 67/71 centers give antibacterial prophylaxis during neutropenia, mostly by quinolones (58%) and 8/67 centers (12%) continue prophylaxis after the neutropenic phase, mostly until discharge. 69/73 (94%) centers give antifungals, at least during neutropenia, with fluconazole (48%), oral ampho b (20.4%) or itraconazole (11.1%). three centers give prophylaxis until discharge. prolonging prophylaxis in case of gvhd is a common approach. the main selection criteria for antifungal prophylaxis are secondary prophylaxis (60%) and unrelated transplants (20%). 77/83 (93%) centers give antiviral prophylaxis during a mean duration of 90 days. 10 centers use ganciclovir and 5 use valaciclovir instead of aciclovir for selected pts (unrelated sct, cmv positivity). 25/73 (34%) centers give ivig to all allogeneic pts and half of them give 0.2-0.5 g/kg/w. 20/73 (27%) centers restrict ivig prophylaxis to selected pts (unrelated transplants, hypogammaglobulinemia, cmv positivity). 2. autologous transplant centers 33/37 (89%) centers give prophylactic antibacterials, mainly quinolones (67%) during neutropenia, and 6 centers continue prophylaxis after the first month. 38/42 (90%) centers give prophylactic antifungals mainly fluconazole (62%), itraconazole (14%) and oral ampho b (12%). 45/53 (85%) centers give antiviral prophylaxis mainly aciclovir (82%) during a mean duration of 60 days. 9/48 (18%) centers give ivig to all pts and 11/48 (23%) only to selected pts. our survey indicate that although antimicrobial prophylaxis is common in sct centers, there are great discrepancies in the policies used in the ebmt centers, with different regimens, selection criteria, and durations of prophylaxis. these findings indicate the need for guidelines. this survey will serve as a basis for future discussions and proposals of recommendations from the idwp. radiologically guided fine needle lung biopsies in the evaluation of focal pulmonary lesions in allogeneic stem cell transplant recipients e. jantunen, a. piilonen, l. volin, p. ruutu, t. parkkali, p. koukila-kahkola, t. ruutu (kuopio, helsinki, fin) lung problems are common in allogeneic stem cell transplant (sct) recipients. we have retrospectively evaluated feasibility and usefulness of radiologically guided fine needle lung biopsies(fnlb) in the evaluation of focal pulmonary lesions in this patient population. during a 10-year period, altogether 30 fnlbs in 21 patients were performed (1-3 biopsies/patient) guided by either ultrasound (n=17) or computed tomography (n=13). the median time from sct to the first fnlb was 131 days (20-343 d). in addition to complications of fnlb, also biopsy findings in relation to the final diagnosis were assessed. prophylactic platelet transfusions were given in 19 procedures (66 %). complications of fnlb included clinically insignificant pneumothorax in four procedures (13 %) and hemoptysis in one case. the first fnlb was suggestive of invasive pulmonary aspergillosis (ipa) in six patients (29 %). additional clinically useful findings of fnlb included pseudomonas aeruginosa (two patients) and nocardia (one patient). the final diagnosis was ipa in 14 patients, immunological lung problems in three patients and other in four patients. fnlb is feasible in allogeneic sct recipients with a low complication rate. the diagnostic yield is relatively high especially in suspicion of ipa. however, re-biopsy or other diagnostic methods are often needed in patients with non-confirmatory findings on fnlb. among chronic carriers of hepatitis b virus receiving cht for nhl, a reactivation of virus replication has been often observed and this may give rise to hepatitis, hepatic failure and death, and may prevent from performing further therapy including trasplantation procedures. herein we present our experience in four patients with nhl where a hepatitis flare-up was observed after 2 (in three patients) and 6 (in one patient) cycles of standard-dose chemotherapy. the patients were affected by mantle cell, follicle centre grade iii, peripheral t-cell unspecified and diffuse large b-cell lymhoma respectively, were male aged 39, 47, 52 and 62 years respectively, hcv and hiv negative. in all patients, pretreatment hbv serology was as follow: hbsag positive, hbeag negative, total anti-c ab positive, igm anti-c negative, anti-e ab positive and anti-s ab negative. three of them were treated with f-machop regimen, the oldest was treated with the chop regimen. hbv-dna, was negative before starting chemotherapy and became positive in all four patients. after spontaneous recovery they were treated with lamivudine 100 mg once daily and this allowed resuming and completing the chemotherapy program without another reactivation of hepatitis b. in two patients high-dose chemotherapy and autologous stem cell tranplantation was also performed under lamivudine as a part of our program for high-risk nhl. antiviral treatment was stopped 4 to 6 months after the last chemotherapy given. during the follow-up period they were monitored with twice-monthly blood counts, transaminases'levels and hbv-dna: all these parameters remained normal/negative all throughout the period. currently, patient b.s. is in cr 19 months from diagnosis and 13 months from the end of chemotherapy; patient d.f.a. is in cr 22 months from diagnosis and 12 months from the end of chemotherapy, patient v.e is in cr 34 months from diagnosis and 26 months from trasplantation; patient c.g. is in cr 23 months from diagnosis and 6 months from transplantation. this data suggests a possible role of lamivudine in preventing hepatitis b reactivation during administration of chemotherapy and asct to chronic carriers of hepatitis b virus. background: severe acute graft-versus-host-disease (agvhd) of the gut is still a major complication after allogeneic stem-cell transplantation (sct) as response rates to treatment (tx) of intestinal gvhd (igvhd) are lower than those observed for gvhd of the skin. since tnf-alpha (tnfa) is one of the key cytokines in agvhd, murine monoclonal antibodies (mab) against tnfa were used in gvhd-prophylaxis and also in the therapy of severe igvhd. infliximab, a chimeric human and mouse mab against tnfa has been introduced recently as a new tx option for pts with progressive crohn's disease. this antibody showed also promising results in the tx of steroid-resistant (sr) agvhd achieving response rates up to 80% in pts with igvhd. in previous studies of our own, the combination of okt3 and a murine mab against tnfa (mak 195) showed potent synergistic effects without major side effects in the tx of pts with sr igvhd. thus it seemed reasonable to combine okt3 and infliximab in the tx of severe sr agvhd of the gut. results: we report the results of a pilot study using the combination of okt3 and infliximab as second or third-line-tx in 3 pts who developed sr agvhd grade iii to iv with severe involvement of the gut after allogeneic sct. infliximab was given in a dose of 5 mg/kg weekly for two weeks (i.e. three doses) combined with okt3 5mg daily for seven days. all three pts responded well showing marked improvement of diarrhea and abdominal pain. but several days to weeks after the tx all three pts died due to severe infectious complications. one patient developed histologically proven aspergillosis of the liver, in another patient invasive aspergillosis of the gut was demonstrated at autopsy and finally the third patient developed hepatic dysfunction and pneumonia which clinically presented as fungal pneumonia and of which he died despite of antibacterial and antifungal tx. conclusions: t cell-depletion and blocking of tnfa is an effective tx of refractory intestinal agvhd but provokes life-threatening fungal infections. inhibiting the inflammatory t cell response by t cell depletion and blocking the effector functions of neutrophils and macrophages by tnfa-suppression gives way to breakthrough-infections of aspergilli as could be dramatically demonstrated in our small series of three pts. we therefore conclude that pts who are really at need to receive both, t cell depleting and tnfa blocking antibodies should receive antifungal tx with substances effective against aspergilli not only in prophylactic but in full therapeutic doses. objective: toxoplasmosis is a rare but serious infectious complication after allogeneic bmt. by pcr, parasitemia can be detected in the peripheral blood before symptomatic disease develops. the risk factors for reactivation of toxoplasmosis, however, are unknown. methods: we prospectively studied 28 consecutive patients seropositive for t. gondii with pcr at least fortnightly for the presence of t. gondii dna in the peripheral blood. the following potential risk factors were evaluated: gender, age, type of preparative regimen (myeloablative or non-myeloablative), presence or absence of gvhd before parasitemia, receiving methylprednisolone before parasitemia, underlying disease (cml versus other malignancies), cmv reactivation, amount of specific igg against t. gondii before bmt, and donor (family donor versus matched unrelated donor). results: 8 of the 28 patients (29%) showed t. gondii dna in the peripheral blood at a mean of 102 days after bmt (95% ci, 30 to 173 days). of those 8 patients, four had a persistent parasitemia which could be treated successfully in two patients. the other two patients died of fulminant toxoplasmosis despite treatment. 4 of the 8 patients demonstrated only a transient parasitemia on one or two occasions. 20 patients without parasitemia were compared to the parasitemic patients. there was no statistically significant difference between the two groups with respect to age, gender, length of follow-up, amount of specific igg against t. gondii before bmt, preparative regimen (myeloablative versus non-myeloablative), underlying disease (cml versus other malignancies), presence of gvhd, occurrence of cmv reactivation, or donor (family donor versus mud). only the use of methylprednisolone was observed more frequently in patients with t. gondii parasitemia (7 of 8 patients) versus the control group (10 of 20 patients); this, however, just missed statistical significance (p = 0.07 by the fisher exact test). conclusions: in this series of patients, no risk factor for t. gondii parasitemia could be detected. until a population at risk can be identified in larger series, regularly monitoring allogeneic bmt patients seropositive for t. gondii may be justified for the presence of t. gondii dna, especially if the patients receive steroid medication. abcd consists of amphotericin and sodium cholesteryl sulfate in a 1:1 molar ratio. between 7/99 and 7/00, 58 immunocompromised/neutropenic patients (2-71 y, median 48) with hematologic malignancies (31 acute leukemia, 16 myeloma, 4 lymphoma, 7 other) received 65 courses of abcd at daily dose of 1 mg/kg for presumed (n=55) and 3-4 mg/kg for proven/strongly suspected (n=9; 3 microbiologic, 6 radiologic) fungal infections. abcd administration followed allogeneic (n=14), autologous (n=17), or syngeneic (n=2) stem cell transplantation, or chemotherapy (n=32). the major indications for the use of abcd were renal dysfunction or potassium requirements ͼ3 mmol/kg/d on conventional amphotericin. in view of known problems with infusion-related toxicity of abcd, each dose was administered over 4-6 h after premedication comprising 1 g paracetamol, 10 mg chlorpheniramine, 25-50 mg pethidine, 100 mg hydrocortisone and 20 mg nefopam in various combinations. courses comprising 4 doses were evaluable for efficacy, and all courses were evaluable for toxicity. the median neutrophil count at initiation of treatment was 0 and the median baseline creatinine was 141 micromol/l (18-460). 46 courses in 43 patients (4-26 doses, median 9) were evaluable for efficacy. 9 courses in proven infections resulted in 2 complete and 4 partial responses, and 3 failures (67% response). 37 courses in presumed infections resulted in 23 complete and 4 partial responses, and 10 failures (73% response). the overall response rate was 72%. the change in serum creatinine from the beginning to the end of therapy was -145 to +331micromol/l (median -27). despite premedication, significant infusion-related toxicity was seen: 30 (46%) rigors, 26 (40%) pyrexia, 6 (9%) bronchospasm, 6 (9%) rash, and 4 (6%) anaphylaxis. at least 1 of these toxicities was seen in 37 (57%). tachyphylaxis developed to infusion-related reactions with subsequent doses. 12 (18%) patients experienced hepatotoxicity which could not be definitely attributed to abcd. the underlying intervention (type of transplant or chemotherapy) did not affect efficacy or toxicity. we conclude that despite its efficacy, the high incidence of infusion-related side effects seen in abcd-treated patients despite extensive premedication is likely to be a limiting factor in the usefulness of this drug. the frequent use of corticosteroids mandated by abcd may be detrimental in patients who are already immunocompromised and/or neutropenic. human herpes virus 6 (hhv-6) is ubiquitous through the human population, with more than 80% of adults being seropositive. the incidence of hhv-6 reactivation and its impact on morbidity in allografted recipients is poorly known. we have conducted in the last six months a prospective pcr screening of hhv-6 in 2o consecutive patients allografted in our institution (6 cml, 1 cll, 1 hd, 4 aml, 7 all, 1 mds) with 14 hla-identical (10 related and 4 unrelated) and 6 haplo-identical donors. acyclovir was used in all as prophylaxis. pcr screening for hhv-6 was performed twice a week on peripheral blood (pb) and on bone marrow or organs biopsies (liver, gut or skin) when available. a nested pcr using primers recognizing type a and b hhv-6 early antigen gene was used. out of the 20 patients, five became pcr-positive, four of which displayed clinical symptoms. p1 (matched unrelated t-cell depleted transplant) engrafted normally, with grade ii skin gvhd after the second dli. hhv-6 was initially detected in a liver biopsy (day 23) performed for hepatitis associated with viral encephalopathy. he was first successfully treated with foscavir but reactivated 7 months later with the same clinical pattern. p2 (t-cell depleted haploi-identical transplant) engrafted normally without agvhd. he was successfully treated with foscavir after detection of hhv-6 in pb at month 1 associated with encephalopathy confirmed by mri. at month3, pcr became positive again in pb and marrow without clinical symptoms and it was decided not to treat. p3 (matched related transplant) engrafted normally, and developed limited cgvhd after early cyclosporin withdrawal for lack of molecular response. he presented with mild hepatitis at month 4. a liver biopsy was performed and revealed positive for hhv-6, with marrow and pb screening remaining negative throughout the follow-up. foscavir treatment led to resolution of hepatitis. p4 (matched related transplant), engrafted normally without gvhd. he then became pcr positive for both cmv and hhv-6 in pb and marrow and was treated with ganciclovir with negativation of both pcr tests. we conclude that hhv-6 reactivation may be an underestimated factor of morbidity in allogeneic transplant and should therefore monitored carefully. however, pcr screening in pb does not appear as a sensitive marker as in cmv reactivation, some patients showing positivity only in the involved organ tissue and the role of pb pcr screening remains to be better defined. j. olson, j. adler-moore (pomona, usa) systemic candidiasis is difficult to completely eradicate using conventional treatment regimens, especially in continuously suppressed animals. it was hypothesized that with the unique pharmacokinetics of ambi (i.e., sustained bioavailability in tissues and reduced toxicity at high doses), an intermittent, high dose treatment regimen could be designed that would be effective in clearing the infection from the kidneys of both ic and is candida albicans infected mice. by monitoring the kidneys for clearance at various times during treatment, it could also be determined if the immune status of the animals effected the length of treatment needed. groups of ic or is mice (cyclophosphamide 75 mg/kg ip, 2x/wk throughout study)(n=20/group), were challenged iv with 5.9 log cfu (ic) or 4.8 log cfu (is). beginning d2 post-challenge, ic or is mice received iv, either 20 mg/kg ambi or 5% dextrose, 3x/wk. after 1, 3 or 5 weeks of treatment, the amount of candida in the kidney tissue was assessed 48h post-treatment. an additional five ic and is mice received no further treatment after wk 5. at the end of wk 9, their kidneys were examined for the presence of fungi. both is and ic mice had a dose dependent, significant (pͻ0.05) reduction in median introduction viral respiratory infections are thought to complicate treatment of hematological malignancies frequently, but current techniques do not always yield a causative agent. therefore, we evaluated the additional value of pcr in the detection of viral infections in haematologic cancer patients with pulmonary abnormalities on x-ray, of whom broncho-alveolair lavage (bal) samples had been stored. methods between october 1997 and june 2000, we collected bal sample from 43 patients (m/f: 28/15; median age:46). in 17 of these patients nose/troat (nt) swabs were collected within 1 week as well. after initial examination of these samples by virus culture, they were stored at -70 degrees c. pcr was done on both bal and nt samples for the following respiratory viruses: parainfluenza virus 1,2, and 3, respiratory syncytial virus (rsv), rhinovirus, influenzavirus a and b, enterovirus and coronavirus. we compared the results of pcr detection with those of virus culture and paired serology specimens (n=29). results twenty-eight patients had been treated with stem cell transplantation (sct) for their underlying diseases (allo-sct: 24, auto-sct: 4), and 15 patients had been treated with cytotoxic agents for leukaemia (n=11) or nhl (n=4). on presentation with respiratory symptoms 17 patients were neutropenic and 27 patients were on immunosuppressive medication. virus cultures of bal showed 9 respiratory viruses in 8 patients, that were also detected by pcr. in addition, pcr was able to detect 8 respiratory viruses in another 7 patients. interestingly, when from a patient both bal and nt samples were available (n=7) pcr on nt swabs yielded the same results as pcr on bal samples. serological examination only detected a rsv infection in 4 patients, and had no additional value over pcr. two of 17 viral infections were acquired nosocomially. four of 15 (27%) of patients treated with cytotoxic therapy and 11 of 28 (40%) sct recipients developed respiratory diseases related to respiratory viruses. six patients had a respiratory illness related to both respiratory virus and other cause (bacterial and fungal pneumonia: n=4, bronchiolitis: n=2). conclusion pcr is more sensitive compared to direct culture and serology for the detection of respiratory viruses in patients with haematologic malignancies. examination of nt swabs by pcr appears as sensitive as on bal samples. introduction : the diagnosis of invasive aspergillosis (ia) in neutropenic patients affected by hematological neoplasms is cumbersome, due to the difficulty of obtaining adeguate bioptic and cultural specimens, very scarce results of radiological detection of early lesions, inadeguate results of serologic tests. rt-pcr detection of dna is still under study, and detection of galactomannan by elisa is sensible, but not particularly specific. patients and methods : we performed 263 seriated elisa assays twice a week in 37 auto/allobmt and neutropenic patients at risk for invasive mycoses (neutropenia, gvhd, high-dose chemotherapy, immunosuppressive treatment): 6 allobmt (3 anll, 2 mm, 1 bc cml), 16 autobmt (8 mm,6 nhl,2 hd), 8 anll, 2 saa, 2 bc/ap cml, 1 cll, 1 hd,1 nhl. antifungal prophylaxis included fluconazole, itraconazole or i.v. low-dose amphotericin b (am-b). the results of elisa assay was considered positive in a single patient, if at least two consecutive positive tests were obtained. all febrile patients entered a program of radiological survey by weekly chest ct scan. results : 241 assays out of 263 were negative. there were 19 positive results for 9 affected (positive) patients, developing ia as follows : 2 proven cases, and 7 probable according the revised eortc mycoses study group criteria; in all the patients positivity of elisa assay was sustained and durable, and decreased with the efficacy of antifungal treatment. at the onset of elisa assay positivity the chest rx was negative; while the chest ct scan detected only subtle pulmonary changes, suggestive of fungal lesions, and thereafter became diagnostic of ia. the remaining 28 patients (including 2 with borderline single positive assay and one with only a single positive test) never developed ia. sensitivity and specificity of elisa assay (considering proven and probable ia diagnoses) were 100%. at onset of elisa assay positivity the patients were given 1-1.5 mg/kg/day am-b, eventually changed to 3-5 mg/kg/day liposomal am-b. in 2 anll patients, refractory to liposomal am-b, voriconazole determined improvement of pulmonary cavitations in 1 and regression in the other patient, enabling him to undergo allobmt. mortality was high, reaching 50% in allobmt and 12.5% in autobmt recipients. conclusion : we suggest that combination of routinary, twice a week, elisa assay and weekly chest ct scan could enable an early diagnosis of pulmonary ia, for targeted antifungal treatment. respiratory virus infections in adult t-cell depleted allograft recipients: risk factors and response to anti-viral therapy s. chakrabarti, k. collingham, k. holder, c. fegan, t. gentle, d. milligan (birmingham, uk) we prospectively evaluated respiratory virus infections in 40 allograft recipients conditioned with conventional myeloablative (n=23) or non-myeloablative treatment and t-cell depleted with campath antibodies. throat samples were obtained weekly for 180 days. upper and lower respiratory symptoms were evaluated by naso-pharyngeal aspirates and bal. parainfluenza (piv), rsv, influenza (if)b and adenovirus infections were treated with ribavirin (oral, inhaled or iv) in a dose escalation regimen. ifa infections were treated with amantadine or zanamivir if unresponsive. 16 episodes of respiratory virus infection were detected in 9 patients (22%). all infections were associated with upper respiratory illness and 5 had lower respiratory involvement. piv3 (piv3; 5, piv1; 1), and rsv(5) were the commonest isolates. there were four episodes of influenza (ifa 3; ifb 1) and 2 episodes of rhinovirus and a single episode of adenovirus. six patients had infection with multiple viruses and the median number of respiratory virus isolated was 2 (range 1-4). the median time to the onset of the infection was 95 days (range 0 to 700). the duration of treatment ranged from 7 to 46 days (median 23 days). two episodes of rhinovirus and one of ifb were self-limited and were not treated with antivirals. the adenovirus infection responded to donor lymphocytes. two patients each with piv and rsv required oral or iv ribavirin at 60 mg/kg/day. the 3 other episodes of piv and 2 episodes of rsv responded to inhaled ribavirin. ifa infection responded to amantadine in one patient and another patient needed zanamivir. there was one death (11%) related to respiratory virus infection (rsv and ifa). on analysis of the risk factors, the dose of campath antibody was the only significant factor (7 infections in 18 patients receiving 100mg campath versus 2/22 in patients receiving a lower dose p=0.03, or 4.5 (1.1-26). on comparing the immunological recovery, the median cd4 cell count at 3-6 months was lower in the infected group (72±45/mm3), compared to the non-infected group (186±72/mm3); p=0.003. in conclusion, multiple respiratory virus infections are frequent after t-cell depleted transplants, the dose of t-cell antibody being a significant risk factor. infection correlated with a poor cd4 recovery at 3-6 months post-transplant. although mortality was low, prolonged antiviral therapy added to the morbidity and cost. screening for aspergillus spp. by a pcr of whole blood samples from patients with haematological disorders c. lass-flö rl, e. gunsilius, d. nachbaur, h. einsele, m. dierich (austria, germany) we performed a screening for aspergillus spp. by polymerase chain reaction of whole blood samples in patients with haematological disorders. in a two-year study, 121 patients admitted to the university hospital of innsbruck for cancer chemotherapy without clinical signs of fungal infection were prospectively screened for aspergillus spp. in 28 of 121 (23%) patients aspergillus dnaemia was detected. of these patients 16 (57%) were positive only once for aspergillus dna but positivity was never associated with invasive aspergillosis. pcr positive episodes were short and resolved without antifungal treatment. five patients (18%) had intermittent pcr positive results. seven (25%) patients presented at least two consecutive positive pcr results, one of these patients developed invasive aspergillosis and another two were highly suspected of having aspergillosis. based on the criteria of eortc case definitions sensitivity and specificity of serial pcr monitoring were 75% and 96%. positive pcr results became negative shortly after commencement of antifungal treatment but the changes did not correlate with clinical responsiveness to treatment in three patients. our results indicate the potential usefullness of pcr for screening for aspergillus spp. in patients at risk yet without antifungal treatment. r. herbrecht, a. thiebaut, a. vekhoff, f. isnard-grivaud, p. moreau, g. michel, s. dupuis, f. bastides, a. datry, o. lortholary (strasbourg, lyon, paris, nantes, marseille, tours, bobigy, f) a pharmaco-epidemiology study to evaluate current therapeutic practice in treating invasive fungal infections (ifi) was carried out in 27 haematology departments between november 1998 and october 1999. two hundred fifty two ifi were recorded, leading to an incidence of 0.92%. therapeutic practice was analysed in the 5 last patients treated in each center for a total of 122 cases. the average age of patients was 41 years (range 1 week -78 years). one hundred and nine (89%) had a haematological malignancy. eighty one (66%) had aspergillosis, 35 (29%) candidiasis and 6 (5%) another ifi. according to the criteria of the eortc/msg, aspergillosis was retrospectively graded as "proven" in 17% of the cases, as "probable" in 62% and as "possible" in 21%. factors associated with ifi were antibiotherapy (93%), chemotherapy (74%), presence of a central veinous catheter (82%), neutropenia ͻ 1000 anc/µl (78% of which 42 % were severe ͻ 100 anc/µl), haematopoetic stem cells transplant (29% with two thirds of allografts), corticosteroid therapy (51%), immunosuppressive therapy (22%), radiotherapy (11.5%), prior ifi (9%) and diabetes (8%). eighty eight percent of patients received nephrotoxic agents and 62% prophylactic antifungal therapy. fifty six percent of the aspergillosis and 70% of the candidiasis were treated with more than 2 lines of antifungal treatment. first line treatments were amphotericin b (44%), azoles (16%), amphotericin b mixed in intralipid (16%), liposomal amphotericin b (11.5%), lipid complex of amphotericin b (5%) with a cure rate of 42%, 43%, 25%, 44% and 15% respectively. in haematopoietic stem cells transplant recipients, antifungal first line treatments were 31%, 22%, 8%, 22% and 6% respectively. the overall mortality rate was 52 % and was higher in aspergillosis (56%) than in candidiasis (45%). death was attributed to the ifi in 67% of the aspergillosis and in 28% of the candidiasis. in transplant patients, cure rate was 41 % and an overall mortality was 58 %. in conclusion, this first pharmaco-epidemiological study evaluates the incidence of ifi in haematology departments in france and reflects the variety of treatments used as first line antifungal therapy. the overall incidence of adenovirus infection following bone marrow transplant (bmt) has been reported in two comprehensive studies at 5% and 21%, with rates of disease of 1% and 6.5%, respectively. the clinical manifestations of adenovirus infection in bmt recipients range from fever with gastroenteritis or cystitis to disseminated disease with multi-organ involvement and high mortality. although there are several case-reports which support either the use of ribavirin with or without immunoglobulins, or ganciclovir, no conclusive evidence of benefit or clear treatment recommendations has been defined. the antiviral drug cidofovir ((s)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine, hpmpc) is an acyclic nucleoside phosphonate with broadspectrum activity in vitro against several dna viruses, including adenovirus. we report the use of intravenous (iv) cidofovir for the treatment of adenovirus infection from june 1999 to october 2000 in five haematology patients: four children and one adult (age range: 3 years -29 years). in four patients, the infection occurred post-allogeneic transplantation (3 hla-identical related; 1 haplotype mismatched) between days +22 and days +123; one patient had adenovirus infection before transplantation. in three patients, adenovirus was isolated only from faeces, but two patients had disseminated infection with isolates from multiple sites (oropharynx, urine, faeces, and conjunctiva). all patients received iv cidofovir at a dose of 5 mg/kg once a week (and thereafter every fortnight) for at least 2 weeks or until negative for adenovirus by viral culture. four patients cleared adenovirus within one month after commencing treatment and one patient died because of a fungal infection while still on cidofovir, and no follow-up sample was available. cidofovir was well tolerated, and no severe side effects were observed. conclusion: cidofovir has been used for the treatment of adenovirus infection in pre-/post-bmt patients. based on these preliminary results further randomised/multicentre studies are warranted to establish the efficacy of cidofovir in the treatment of adenovirus infection. prevalence of hepatitis c after bone marrow transplantation for solid tumours in children included in a randomised study from 1985 to 1987 comparing prophylactic versus therapeutic transfusion of platelet concentrates there was still no recoverable fungus in the is mice, and only one ic mouse had 1.31 log cfu/g. in conclusion, both is and ic mice responded similarly to 3x/wk treatment with 20 mg/kg ambi. there was a significant as the risk of post-transfusion hepatitis c was high during this period, we tested in 1999, post-bmt sera of 66 children (30 in ppt and 36 in tpt group) to determine whether either of these two strategies was associated with a higher risk of hepatitis c. children were screened for anti-hcv using a 3rd generation enzym linked immunosorbent assay (elisa)(elysis, ortho diagnostic) and we investigated the presence of viral rna by pcr (test cobas amplicor hcv v2.0, roche diagnostic). the median interval between the last blood products transfusion and the serum sample was 3 months (range: 1-18). pcr analysis of rna was positive in 28 children (42%): 12/30 in group ppt versus 16/36 in group tpt (p=ns). the mean total number of pc units transfused during the trial was not significantly different between the ppt and tpt groups: 65.5+/-82.2 versus 46.2+/-83.2 respectively. this mean total number of transfused pc units in relation to the weight and the total duration of neutropenia was 1.8 in ppt group versus 1.3 units in tpt group (p=0.07). anti-hcv antibodies were detected by the elisa assay in 12/28 pcr-positive serum rna samples (elisa sensitivity=0.43). all the 38 pcr-negative serum rna samples were negative by the elisa assay (elisa specificity=1). the interval between the last blood product transfusion and the serum sample one of 18 pre-bmt serum rna samples was pcr-positive and 17/18 were pcr negative. in conclusion, the prevalence of hepatitis c after bmt in children transfused with pooled random pc between 1985 and 1987 was 47% in our study. the elisa test used has a poor sensitivity in this population and pcr analysis of rna must be preferred to test hepatitis c after bmt one other patient treated with immunosuppressive therapy for saa has developed ebv-lpd at the same period. diagnosis of ebv-lpd was confirmed by pcr and several immunological studies at the time of first clinical symptoms in three. cause of death in one was not clear until autopsy. diagnosis of ebv-lpd was confirmed on autopsy in all three patients who died 6, 7, and 43 days after first clinical symptoms. lpd as a cause of death still cannot be excluded in one other patient. first signs of lpd appeared 64, 64 and 67 days after last dose of atg (16mg/kg) in transplant patients, 30 days after last dose of atg (30mg/kg) in patient treated with atg+csa for relapsing saa. signs of ebv-lpd included fever, pancytopenia, lymphadenopathy, splenomegaly, lymphocytosis, elevated igg. serological studies done prior exposure to atg were positive for latent ebv infection in all of them. all affected patients got same batch of rabbit-atg as a gvhd prophylaxis pre-transplant in three, as an is therapy in one. thirteen other transplanted patients got the same dose and batch of r-atg, which was followed with other unusual and otherwise less frequent complications (encephalitis in two, severe and relapsing bkv hemorrhagic cystitis in one, autoimmune cytopenia in two and unexplained anemia in two). transplant related mortality 180 days after transplant in our cohort of 16 patients who got the same batch of r-atg was extremely high (50%) compare to 24% trm over last two years in fully comparable cohort of 25 patients receiving r-atg (84% alternative donors) transplanted at the same center gonzá lez-fraile, m.c. del cañ izo 166 women with a median age of 46 years (21-60) diagnosed of breast cancer were treated with autologous peripheral stem-cell support using two different conditioning regimens: in 147 cases stamp-v regimen and, in 19 patients stamp-i. the median number of mnc infused was 5.6 x 108/kg and a median of 2.06 x 106/kg cd 34+ cells. granulocyte colony-stimulating factors were used in all patients febrile episodes were classified: 1) microbiologically confirmed with bacteriemia in 33 cases (21%), (gram-positive infections in 22 cases (66.6%), and only 11 (33.4%) gram-negative, 2) microbiologically confirmed infections without bacteriemia in 42 cases (26.2%), with a similar proportion of gram-positive and gram-negative agents, 3) clinically documented infections in 46 cases (29.4%), 10 of which were pneumoniae, 4) fever of unknown origin in 35 (23%), 56% responded to first-line antimicrobial therapy aim:to present a fungal pcr assay for detection and identification of fungal pathogens in blood and bronchoalveolar lavage (=bal) number of patients analysed were six, (2 males and 4 females, ages ranged between 4 months and 40 years). all had received allogeneic stem cell transplantation because of all (n=2),scid (n=1), cml (n=1), nhl (n=1) and aml (n=1) ultrasound confirmed with typical hepato-splenic candida lesions. blood cultures were negative.one patient was positive for c.parapsilosis in pcr (we get our samples after first blood culture was positive). nine blood cultures were positive for c.parapsilosis. one patient was positive in bal for c.albicans in pcr. culture of bal confirmed c.albicans. one patient was positive for fungi in pcr (detected by electrophoresis only ,negative for candida and aspergillus spp. in elisa) 3 days after blood culture revealed phichia etchellsii. conclusion: fungal pcr performed in blood and bal for detection and identification of fungal pathogens is a rapid and sensitive method ebv-infection/disease in allogeneic stem cell transplantation and cidofovir (cdf) treatment this study was a retrospective survey among ebv-infection/disease and the efficacy of cdf. 36 cdf applications were evaluated. 5 of 32 cdf-treated pts tested concommitantely positive for cmv, 6 for hhv6 and 1 for vzv reactivation/infection. 1 pt received previous antiviral therapy with ganciclovir and foscarnet, 2 pts received cidofovir combined with foscarnet and ganciclovir. 29 pts. received cdf as first line therapy. the dosage of cdf was 1-5 mg/kg/week. the duration was from 1 application to 7 times. all patients received probenecid and prehydratation. 4 pts suffered from presumed side effects of cdf (vomiting and nausea) key: cord-003915-kje8lvgl authors: pigeyre, laetitia; schatz, malvina; ravallec, marc; gasmi, leila; nègre, nicolas; clouet, cécile; seveno, martial; el koulali, khadija; decourcelle, mathilde; guerardel, yann; cot, didier; dupressoir, thierry; gosselin-grenet, anne-sophie; ogliastro, mylène title: interaction of a densovirus with glycans of the peritrophic matrix mediates oral infection of the lepidopteran pest spodoptera frugiperda date: 2019-09-17 journal: viruses doi: 10.3390/v11090870 sha: doc_id: 3915 cord_uid: kje8lvgl the success of oral infection by viruses depends on their capacity to overcome the gut epithelial barrier of their host to crossing over apical, mucous extracellular matrices. as orally transmitted viruses, densoviruses, are also challenged by the complexity of the insect gut barriers, more specifically by the chitinous peritrophic matrix, that lines and protects the midgut epithelium; how capsids stick to and cross these barriers to reach their final cell destination where replication goes has been poorly studied in insects. here, we analyzed the early interaction of the junonia coenia densovirus (jcdv) with the midgut barriers of caterpillars from the pest spodoptera frugiperda. using combination of imaging, biochemical, proteomic and transcriptomic analyses, we examined in vitro, ex vivo and in vivo the early interaction of the capsids with the peritrophic matrix and the consequence of early oral infection on the overall gut function. we show that the jcdv particle rapidly adheres to the peritrophic matrix through interaction with different glycans including chitin and glycoproteins, and that these interactions are necessary for oral infection. proteomic analyses of jcdv binding proteins of the peritrophic matrix revealed mucins and non-mucins proteins including enzymes already known to act as receptors for several insect pathogens. in addition, we show that jcdv early infection results in an arrest of n-acetylglucosamine secretion and a disruption in the integrity of the peritrophic matrix, which may help viral particles to pass through. finally, jcdv early infection induces changes in midgut genes expression favoring an increased metabolism including an increased translational activity. these dysregulations probably participate to the overall dysfunction of the gut barrier in the early steps of viral pathogenesis. a better understanding of early steps of densovirus infection process is crucial to build biocontrol strategies against major insect pests. the transmission of parvoviruses predominantly occurs by horizontal routes through inhalation or oral exposure, making interaction with mucosal epithelia a crucial part of their pathogenesis (for review [1] ). the oral route represents a major challenge for viruses as they need to overcome a diversity of barriers to invade their host. indeed, most animal epithelia are covered in their apical surface by a carbohydrate-rich meshwork of various complexity and thickness, the glycocalyx, which can be coated by an additional layer of secreted mucus [2] . these structures constitute successive protective surfaces where viruses aggregate and either access to attachment factors and receptors at the surface of the epithelial cells or are eliminated by luminal or cilia movements [3] . this dual fate depends on the virus affinity for glycans, which must allow to escape the trap of the mucus. the diversity of glycans present on the epithelial surfaces vary between and within species and therefore constitute an important component of the innate immunity and of the species barrier [4] . members of the parvoviridae family are non-enveloped viruses that have a simple capsid with t = 1 icosaedral symmetry protecting a 4-6 kb linear, single stranded (ss) dna genome [5] . they can cause diseases of various severity in a wide range of animals. parvovirus interest in human and animal health or in biomedicine as vectors for gene transfer, has long focused research to understand their cell tropism and entry mechanisms. a number of cellular attachment factors and receptors have been characterized, mostly for vertebrates' parvoviruses. they highlight the importance of glycans in capsid recognition and binding specificity [6] [7] [8] . however, how capsids interact with epithelia remains poorly known, mainly due to difficulties to reconstitute in cellular models the complexity of animal epithelial systems [9, 10] . insect parvoviruses, named densoviruses, can be highly pathogenic, a feature that can both represent threats for insect mass rearing or opportunities for biocontrol against harmful insects as alternative to chemicals. developing methods against infections or tools for biocontrol requires a deep understanding of the mechanisms driving host range and pathogenesis. like their vertebrate counterparts, densoviruses are mainly transmitted orally, gut recognition and binding constitute the primary step of their pathogenesis. the mechanisms determining densovirus specificity are poorly known. depending on species, densovirus replication can be either restricted to or exclude the gut, viral particles being then transported across the epithelium by transcytosis to reach internal organs where replication goes [11] [12] [13] [14] . only one cellular receptor has been so far characterized for a "gut restricted" densovirus. it is a mucin of the gut of the silkworm bombyx mori, whose inactivation makes silkworms resistant to infection with the bombyx mori densovirus type 1 (bmdv) [15] . we have previously reported that gut transcytosis of the junonia coenia densovirus (jcdv) involves a gut specific receptor-dependent mechanism in caterpillars [12] . however, the mechanisms used by viral particles to overcome the successive intestinal barriers of different structures and composition remain elusive. in insects, the intestinal tract is covered by a chitinous acellular layer, which has specific features due to the dual embryonic origin of the gut. indeed, anterior and posterior extremities of the gut are ectodermal and the acellular layer is covered by an impermeable cuticle. the midgut section is endodermal and has no cuticle but is covered in most insects by a semi-permeable membrane, named the peritrophic matrix (pm) (for review [16] ). the pm forms a highly organized lattice of chitin fibrils associated with glycoproteins, mainly peritrophins that have a chitin-binding domain [17, 18] . the midgut is thus the portal of entry for most pathogens and their interaction with the pm a critical step of their pathogenesis. the pm forms pores whose size varies between insect species (e.g., 7-36 nm in lepidoptera and up to 150 nm in coleoptera) and developmental stages [19, 20] . large entomopathogenic viruses have developed specific mechanisms to pass through extracellular matrices including virus-encoded enzymes and specific proteins that are associated with the viral particles of baculo-and entomopoxviruses [21] [22] [23] [24] . how densoviruses cope with the physical barriers that constitute the gut and in particular the pm is so far unknow. due to their small size, it was initially thought they could diffuse passively across the pores of the matrix, but measures of the size of pores, the complexity of the pm and the nature of the interactions between components make this hypothesis unlikely [16, 20] . we previously reported that following oral infection, viral particles of jcdv, a type species ofthe ambidensovirus genus, aggregate on the pm of spodoptera frugiperda caterpillars as a first step of the infection process [12] . such rapid virus concentration on a carbohydrate-rich surface suggested a lectin-like activities of the capsids. although there is no sequence similarity of the unique protein making the surface of the jcdv with lectin domains, its structure displays similarities with cellular carbohydrate binding proteins including lectins, which suggests that capsids could indeed recognize and bind carbohydrates [25] . herein, we used a combination of approaches, including microscopy, biochemistry, proteomics and transcriptomics, to decipher the interaction of jcdv with pm major components (i.e., chitin, glycans and proteins). we found that capsids affinity for the pm might result from multiple interactions with different glycans including chitin and glycosylated proteins. in addition, we showed that jcdv early infection results in (i) an arrest of n-acetylglucosamine (glcnac) secretion by epithelial cells associated with a disorganization of the pm structure mimicking the effect of chitin-binding plant lectin; (ii) substantial changes in the expression of gut genes, which may also contribute to an early gut dysfunction and participate to viral pathogenesis. caterpillars were reared under controlled conditions (25 ± 1 • c, 65 to 70% relative humidity [rh], 16-h light, 8-h dark photoperiod) on a wheat germ-based artificial diet. jcdv was amplified by oral infection. at death, larvae were crushed and virus extraction was processed by clarification and filtration on 0.22 µm to constitute a semi-purified virus stock (jcdv). to obtain a purified viral stock (p_jcdv), the semi-purified virus stock was loaded on optiprep tm (sigma-aldrich, lyon, france) density gradient and dialyzed against phosphate-buffered saline (pbs) 1x as previously described in [13] . viral concentrations were estimated by quantitative pcr (qpcr) as described in [12] and expressed as viral equivalent genomes ([veg]). virus titers were determined by the tissue culture assay method (50% tissue culture infective dose (tcid50) in the permissive ld652 cells as previously described [26] . calcofluor white m2r (calcofluor; f3543), n-acetyl-d-glucosamine (glcnac; a4106), n-acetyl-d-galactosamine (galnac; a2795), d-(+)-fucose (fucose; f8150), d-(+)-mannose (mannose; m6020), mucin from porcine stomach (porcine mucin; m2378) and wga-fitc conjugate (l4895) were all purchased from sigma-aldrich (lyon, france). for in vivo bioassays, third-instar (l3) s. frugiperda caterpillars were individually infected by feeding with jcdv (10 9 veg/caterpillar) or by intrahemocelic injection (10 9 veg/caterpillar). each treatment was applied to a cohort of 24 caterpillars and three independent experiments were performed. calcofluor (0.5% to 3%) was concomitantly administrated with the virus to l3 or l6 caterpillars (as described in the figures). for competition assays, jcdv was incubated with glycans (glcnac, galnac, fucose or mannose at 5 µm and/or 5 mm; for one hour before feeding or injection. in all experiments, control larvae were fed or injected with pbs. caterpillars mortality was recorded each day during 10 days and results were presented as survival rates per day. the time to death was assessed by comparing the survival curves using the kaplan meier method (graphpad prism software, version 7). the significance between groups were analyzed using log-rank (mantel-cox) tests and gehan-breslow-wilcoxon tests. rabbit erythrocytes cells were diluted at 3% (v/v) with 150 mm nacl and treated with 0.5 mg/ml of trypsin (sigma) for 30 min at 37 • c. after 3 washes with 150 mm nacl, 25 µl serially two-fold dilutions of viral inocula (jcdv or p_jcdv; started from 10 11 veg/µl) were mixed with 25 µl of erythrocytes in 96 well microtiter plates for 30 min at 37 • c. positive controls of hemagglutination was performed using 25 µl of wga (1 mg/ml). fifty µl of erythrocytes were subsequently added to each well for 30 min at 37 • c. hemagglutination was read under a light microscope. for calcofluor experiments, pms were isolated from sixth-instar (l6) s. frugiperda caterpillars, previously anesthetized on ice, then opened and washed with phosphate buffered saline (pbs) to remove the food bolus. pms isolated from caterpillars treated with calcofluor were fixed 1 h with 4% paraformaldehyde (pfa), then dehydrated with increasing concentrations of ethanol (50% to 100%) and in 1:1 (100% ethanol and hexamethyldisilazane [hmds]) for 10 min and finally in 100% hmds for 20 min. after overnight evaporation, samples were ultimately coated with platinium and observed with a scanning electron microscope (sem) hitachi s-4800, with an acceleration voltage of 2 kv and a working distance of 5 mm. for ex vivo infection experiments, pms were isolated as described above and incubated for 30 min at room temperature (rt) with jcdv (10 9 veg/µl; 500µl), or with jcdv pre-incubated for 1 h with glycans (glcnac, galnac, fucose or mannose; 5 µm to 5 mm) before infection. the pms were then washed with pbs and fixed with 4% pfa as above. immunolabelling of jcdv particles was performed using a specific rabbit anti-capsid antibody (1:1,000; eurogentec) incubated for 2 h at rt and an anti-rabbit secondary antibody (alexa fluor ® 568; 1:500; invitrogen, carlsbad, ca, usa) incubated for 45 min at rt. labeled pms were then mounted in dako (sigma) on coverslips for observation. for epifluorescence measurements, we worked on a zeiss axioimager z2, equipped with suitable filters for the dyes, using a 40×/1.3 plan-apochromat oil ph3 and a 63×/1.4 plan-apochromat oil ph3 objectives. for structured illumination images, the zeiss apotome-slider was introduced into the field-stop plane of the microscope to improve resolution of images. we used the zen software to operate the microscope and took at least 10 images (10 fields per pm) for each treatment. all images were taken with a cmos orca flash 4.0 b&w camera. images were processed with fiji software. the intensity of fluorescence (arbitrary unit) were measured on epifluorescence images. statistical analyses were performed using the non-parametric kruskal-wallis test (graphpad prism software, version 7, graphpad software, san diego, ca, usa). each experiment was repeated at least three times, and each independent experiment gave similar results. l3 s. frugiperda caterpillars were individually infected by feeding as in section 2.3. twenty four hours later, caterpillars were anesthetized on ice and sacrificed. in parallel, caterpillars were kept until death to insure their infection status. sacrificed caterpillars were fixed in 4% pfa + 2.5% glutaraldehyde for 1h at 4 • c and dehydrated by incubations in increasing concentrations of ethanol (50% to 100%) before progressive embedding in 100% unicryl resin (bb international) as described in [12] . semi-thin sections (1 µm) were cut in the central part of the caterpillars corresponding to the midgut. after 45 min of incubation with wga-fitc (1:300), to label glcnac and the pm, or with phalloidin-fitc (1:300; sigma), to label actin cytoskeleton, and 10 min with dapi (1 µg/ml; invitrogen) to label nuclei, semi-thin sections were mounted and observed with a zeiss axioimager z2 microscope using the structured illumination module zeiss apotome-slider as in 4.5. images were taken with a cmos orca flash 4.0 b&w camera using fixed parameters for all treatments and processed with fiji software. ten µl of chitin beads (new england biolab) were washed three times with pbs and added in 200 µl of virus suspension (10 9 veg/µl in pbs). after 1 h of incubation with gentle rotation, beads were pulled-down by centrifugation (500× g for 5 min à 4 • c), washed five times with pbs, then resuspended in laemmli buffer 2 × (4% sds, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 m tris hcl, ph 7) and heated at 95 • c for 5 min before western blot analysis. two µl (5% of the pull down and 1% of the initial input) was loaded on 4-15% polyacrylamide tris-hcl gel (mini-protean ® tgx™ precast gels, biorad, hercules, ca, usa) and separated by sds-page for 1 h at 150 v. next, samples were transferred to pvdf membrane (immobilon-p, merck) for 2 h at 200 ma. subsequently, the membrane was saturated with 5% of milk in pbs/tween 0.1% (pbst), then incubated 2 h at rt with the primary anti-capsid antibody (1:1,000; see above). after washes in pbst, the membrane was incubated 1 h at rt with an anti-rabbit secondary antibody hrp-conjugated (1:3000; biorad, hercules, ca, usa). proteins were revealed by enhanced chemiluminescence (millipore, burlington, ma, usa) using a chemidoc imager (biorad). peritrophins were extracted from pools of five pms from sixth instar s. frugiperda in 200 µl of laemmli buffer 2x, then boiled and heated at 95 • c for 5 min [27] . after centrifugation at 8000× g for 10 min 4 • c, the supernatant was collected and 1/15 was loaded on 4-15% polyacrylamide tris-hcl gel and separated by sds-page as above. thirty µg of porcine mucins were also loaded on the same gel. gel was stained with page blue (thermo fisher scientific, waltham, ma, usa) to analyze total proteins or with periodic acid-schiff (pas) as described in [28] to analyze glycosylated proteins. proteins were also transferred on nitrocellulose membranes (biorad) for 3 h at 70 v for viral protein overlay binding assay [29] . briefly, the membrane was saturated with 3% bsa in pbst for 2 h at rt, then incubated ovn at 4 • c with jcdv (10 9 veg diluted in pbst containing 1% bsa). after washes in pbst, the membrane was incubated with the rabbit anti-capsid antibody (1:1000; see above), then the anti-rabbit secondary antibody hrp-conjugated, and proteins were revealed by enhanced chemiluminescence as above. protein bands revealed by vopba were cut in sds-page gel stained with page blue (3 replicates, 10 bands) and destained with three washes in 50% acetonitrile and 50 mm triethylammonium bicarbonate (teabc). after protein reduction (with 10 mm dithiothreitol in 50 mm teabc at 60 • c for 30 min in the dark) and alkylation (55 mm iodoacetamide teabc at room temperature for 30 min) proteins were digested in-gel using trypsin (0.5 µg/band, gold, promega, madison, wi, usa) as previously described [30] . digested products were dehydrated in a vacuum centrifuge and resuspended with 0.05% trifluoroacetic acid (tfa) and 2% acn. the generated peptides were analyzed online by nano-flow hplc-nanoelectrospray ionization using a q-exactive plus mass spectrometer (thermo fisher scientific, waltham, ma, usa) coupled to an ultimate 3000 rslc (thermo fisher scientific). desalting and pre-concentration of samples were performed on-line on a pepmap ® pre-column (0.3 mm × 10 mm, dionex, sunnyvale, ca, usa). the capillary reverse-phase column (0.075 mm × 150 mm, acclaim pepmap 100 ® c18, thermo fisher scientific) fitted with an uncoated silica picotip emitter (new objective, woburn, ma, usa) was first equilibrated in solvent a (0.1% formic acid) and a multistep linear gradient of acetonitrile consisting of 0-25% of solvent b (0.1% formic acid in 80% acetonitrile) for 30 min, 25-40% for 5 min and 90% for 8 min, at 300 nl/min was used to elute peptides from. spectra were acquired with the instrument operating in the information-dependent acquisition mode throughout the hplc gradient. survey scans were acquired in the orbitrap system with resolution set at a value of 70,000. up to twelve of the most intense ions per cycle were fragmented and analyzed using a resolution of 17,500. peptide fragmentation was performed using nitrogen gas on the most abundant and at least doubly charged ions detected in the initial ms scan and an active exclusion time of 45 s. for all full scan measurements with the orbitrap detector a lock-mass ion from ambient air (m/z 445.120024) was used as an internal calibrant as described [31] . analysis was performed using the maxquant software (version 1.5.5.1) [32] . all ms/ms spectra were searched using andromeda against a decoy database consisting of a combination of s. frugiperda databases [33] and 236 classical contaminants, containing forward and reverse entities. the following settings were applied: spectra were searched with a mass tolerance of 7 ppm (ms) and 0.5 th (ms/ms). enzyme specificity was set to trypsin. up to two missed cleavages were allowed and only peptides with at least seven amino acids in length were considered. carbamidomethylation was set as fixed cystein modification and oxidation was set as variable methionine modification for searches. fdr was set at 0.01 for peptides and proteins. sequences which found homology were annotated according to the gene ontology (go) terms and classified using blast2go software (https://www.blast2go.com/; [34] ). the enrichment in go terms compared to the s. frugiperda reference (predicted proteins from the ogs2.2 genome; gouin et al., 2017) was analyzed with the same software (fdr set at 0.01). fourth instar s. frugiperda caterpillars were orally infected or not with jcdv (10 12 veg per caterpillar; twenty caterpillars per condition). at 1-day p.i. or 3 days p.i., caterpillars were anesthetized on ice and dissected. the midguts were washed in pbs to eliminate the food bolus and the pms. trachea, malpighi tubes and visceral muscles were removed and the epithelia were incubated with 2.5% trypsin for 30 min to dissociate the tissues. after washes, gut cells were lysed in 800 µl of trizol ® reagent (invitrogen) for total rna extraction according to the manufacturer's instructions. total rna amount and purity were checked by using spectrophotometrer nanodrop nd-1000 (thermo scientific) and the integrity of total rna was analyzed by capillary electrophoresis (2100 bioanalyzer instrument, agilent, santa clara, ca, usa). we used digital gene expression (dge) method that generates short sequences (tags) specific for mrna [35] [36] [37] . four dge libraries were constructed from midgut total rna extracted from s. frugiperda caterpillars infected (or not) for 1 and 3 days. sequence tag preparation was done with illumina's digital gene expression tag profiling kit according to the manufacturer's protocol (version 2.1b) as described in [36] . for fourth libraries, 10 µg of total rna were incubated with oligo-dt beads. first-and second-strand cdna syntheses were performed using superscript ii reverse transcription kit according to the manufacturer's instructions (invitrogen). the cdnas were cleaved using the nlaiii anchoring enzyme. subsequently, digested cdnas were ligated with the gex adapter 1 containing a restriction site for mmei. the second digestion with mmei was performed, which cuts 17 bp downstream of the catg site. at this point, the fragments detach from the beads. the gex adapter 2 was ligated to the 3 end of the tag. a pcr amplification with 15 cycles using phusion polymerase (finnzymes) was performed with primers complementary to the adapter sequences to enrich the samples for the desired fragments. the resulting fragments of 85 bp were purified by excision from a 6% polyacrylamide tbe gel. the dna was eluted from the gel using spin-x cellulose acetate filter (0.45 µm), precipitated, resuspended in 10 mm tris-hcl (ph 8.5) and quantified using nanodrop 1000 spectrophotometer. cluster generation was performed after applying 4 pm of each sample to the individual lanes of the illumina 1g flowcell. after hybridization of the sequencing primer to the single-stranded products, 18 cycles of base incorporation were carried out on the 1g analyzer according to the manufacturer's instructions. image analysis and base calling were performed using the illumina pipeline, where sequence tags were obtained after purity filtering. we could assign 63% of the tags (supplementary materials tables s2-s4) out of which 54% correspond to multiple matches and were discarded from functional analysis with go. functional annotation was performed using biotag software (skuld-tech, grabels, france). the statistical value of dge data comparisons, as a function of tag counts, was calculated by assuming that each tag has an equal chance of being detected. differential expression of the tag counts of the infected vs. mock conditions was performed to obtain a list of up-and down-regulated tags for each condition. tags for which differential expression was ≥ 5 fold change were assigned using the reference databases for s. frugiperda [33, 38] . sequences with homology were annotated according to the go terms and classified using blast2go software (https://www.blast2go.com/; [34] ) and represented at level 2 of biological process and molecular function. the enrichment in go terms compared to the s. frugiperda reference (predicted transcripts from the ogs2.2 genome) was analyzed with the same software (fdr set at 0.01). the junonia coenia densovirus (former jcdnv) corresponds to the complete genome of the oxford isolate (genbank accession number kc883978.1). early studies have estimated that the average pore size of the pm is around 8 nm in s. frugiperda caterpillars, which might exclude 25 nm densovirus particles [39] . to assess the pm barrier function against densovirus infection, we disrupted its structure by feeding third instar (l3) larvae with sub lethal doses (0.5%) of the chitin-binding agent calcofluor white prior jcdv infection [40] [41] [42] [43] [44] . we then measured larval mortality rates daily. results showed that jcdv infected larvae pre-treated with calcofluor displayed a significant shorter median time to death (lt50) compared to untreated infected larvae (7 vs. 9 days p.i. for control larvae; p < 0.01) ( figure 1a and supplementary materials figure s1 ), supporting that the pm can limit jcdv infection. noteworthy, an increased larval mortality was also observed at late time post-treatment with calcofluor in mock-infected caterpillars compared to untreated controls (18% at 10 days p.i.), confirming a detrimental inhibition of chitin assembly on larval development [41] . larvae (n = 24) were infected individually by feeding with jcdv (10 9 veg/caterpillar) concomitantly with 0.5% (5 µg) of calcofluor or pbs as a control. caterpillar mortality was recorded once a day during 10 days and results were presented as survival rates per day. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance. p values of less than 0.05 were considered significant (**, p < 0.01). pbs refers to control (pbs-treated and non-infected) caterpillars; calcofluor refers to calcofluor-treated and non-infected caterpillars; jcdv refers to jcdv-infected caterpillars and jcdv+calcofluor to calcofluor-treated and jcdv-infected caterpillars. (b) calcofluor disrupts the pm integrity. sem images of pm ultrastructure isolated from l6 caterpillars fed with pbs (0%), 0.5% (5 µg) or 3% (30 µg) of calcofluor. endoperitrophic face is shown (bars, 300 nm). we analyzed the effect of calcofluor on the pm integrity by scanning electron microscopy (sem). because the pm of l3 larvae has a gel-like structure that cannot be manipulated, we took l6 larvae for this experiment as the pm is thick and solid at this stage and can be easily dissected. l6 caterpillars were fed with up to 3% calcofluor (not lethal at 24 h post treatment) and pms were isolated 24 h post-treatment and prepared for sem analysis. as shown by figure 1b , pms from control larvae displayed a highly organized structure, similar to pms from caterpillars treated with 0.5% calcofluor. by contrast, pms from caterpillar fed with 3% calcofluor had a clear disrupted structure with enlarged pores, confirming that calcofluor binding to chitin fibrils compromised the integrity of the matrix. the rapid recognition of the pm by jcdv capsids suggests that their affinity for glycans is important for the oral infection process. to test this hypothesis, we first assayed the capsid ability to agglutinate erythrocytes, a feature displayed by vertebrate parvoviruses [45, 46] . we performed a typical hemagglutination assay, adding serial dilutions of the virus inoculum to rabbit erythrocytes ( figure 2 ). the first dilutions (1:2 and 1:4) of jcdv triggered a strong hemolysis of erythrocytes suggesting a toxic effect of the viral inoculum, ie capsids or some host-derived component associated with the inoculum. it is worthy to note that we use semi-purified inoculum as it mimics naturally occurring infections. jcdv was therefore further purified on a density gradient (p_jcdv) and similarly assayed for hemagglutination. a clear hemagglutination was obtained with p_jcdv, supporting that toxicity is likely due to a host-derived component that can be eliminated during the purification process. hemagglutination with p_jcdv was obtained up to the third dilution (hemagglutination titer of 1:8), which indicates a rather weak interaction of the capsids with glycans at the surface of (mammalian) erythrocytes. to better understand capsid affinity for glycans, we performed a competition bioassay using monomeric glycans as jcdv-binding competitors. jcdv binding was revealed with an immunofluorescence staining on the pms using a specific anti-capsid antibody. we quantified this fluorescence as a proxy of binding and competition. we first performed competition ex vivo on isolated pms incubated with jcdv in the presence of four monosaccharides commonly found in insects [47] , ie n-acetyl-d-glucosamine (glcnac), which is the monomeric unit of chitin, n-acetyl-d-galactosamine (galnac), d-fucose and d-mannose (figure 3 ). we first verified with a dot blot assay that capsids interaction with monosaccharides were not interfering with antibody recognition, which validated the competition bioassay (supplementary materials figure s2 ). as shown in figure 3 , jcdv binding resulted in an intense fluorescence signal on the pms (left panel), which was similarly competed away by the four monosacharides and within a similar concentration range (0.5 mm to 5 mm). we noted that fluorescence quantification did not result in a strictly linear dose-dependent effect. to further test the role of glycans in jcdv pathogenesis, we carried out these competition bioassays in vivo (figure 4 and supplementary materials figure s3 ). we mixed jcdv with each monosaccharide prior infection, fed caterpillars with these inocula and calculated mortality rates daily as in figure 1 . results showed that oral infection with 5 mm (but not 5 µm) of each monosaccharide significantly delayed the median time to death (lt50) of caterpillars (7 vs. 6 days; p < 0.05 for 5 mm) ( figure 4a and supplementary materials figure s3a ,b), further supporting that pm recognition is the first step of jcdv oral infection. to confirm these results and reveal jcdv binding and competition for binding the pm in vivo, we carried out midgut semi-thin sections and immunofluorescence as above. caterpillars were infected with jcdv mixed or not with 5 mm glcnac and sacrificed at 2 h p.i. for midgut isolation and preparation. as shown in figure 4b , we observed a red fluorescence signal in untreated infected caterpillars that typically lines the pm. in addition, labelling was also observed in the lumen, likely revealing jcdv interaction with food bolus and/or microbial components. both signals were strongly and specifically decreased following competition with glcnac ( figure 4b) , showing that different glcnac-containing glycans in the gut lumen can recognize the capsids. control caterpillars were fed with pbs. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance. p values of less than 0.05 were considered significant (ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001). (b) immunolabeling of midgut semithin transversal sections 2 h after ingestion of jcdv alone or jcdv (10 9 veg/caterpillar) incubated for 1 h with 5 mm of glcnac before oral infection. control caterpillars were fed with pbs. the pm is shown by an arrowhead. phalloidin-fitc is in green, jcdv is in red, and nuclei are labeled with dapi (blue). bars, 30 µm. lum, midgut lumen; hemol, hemolymphatic compartment. (c) survival curves of caterpillars (n = 24) infected by injection of jcdv alone (10 9 veg/caterpillar) or of jcdv incubated for 1 h with 5 mm of each glycan (glcnac, galnac, fucose or mannose) before infection. control caterpillars were injected with pbs. three independent experiments were performed, each independent experiment gave similar results, one is represented here. the log-rank (mantel-cox) and the gehan-breslow-wilcoxon tests were used to determine statistical significance, p > 0.05 were considered non-significant (ns). pbs refers to control (pbs-treated and non-infected) caterpillars; 'jcdv' to jcdv-infected caterpillars; 'jcdv + glcnac', 'jcdv + galnac', 'jc + fucose' and 'jc + mannose' refer to caterpillars infected with jcdv incubated with glcnac, galnac, fucose or mannose, respectively, before infection. last, we studied whether such "stickiness" was specifically required by the densovirus to cross the gut, i.e., for oral infection. jcdv infection of target cells (eg epidermis, trachea, hemocytes) proceeds by a receptor-dependent mechanism different from intestinal cells [12] . these cells express glycan structures of various complexity that are attached to the cell surface or secreted and forming extracellular matrices, whose glycans might be similarly targeted by jcdv for attachment. we performed competition bioassays in vivo, bypassing the midgut by injecting caterpillars with jcdv mixed or not with 5 mm of each monosaccharide. interestingly, the median time to death was similar for all conditions ( figure 4c and supplementary materials figure s3c ; p > 0.05), showing that none of the monosaccharides competed with jcdv infection proceeding by the systemic route. altogether these results show that jcdv capsid is a carbohydrate-binding protein and this feature is required for oral infection to target the pm of caterpillars. we next wanted to determine which component of the pm, i.e., chitin and/or glycosylated proteins were involved in capsid interactions, using biochemical assays. we first tested capsid physical interaction with chitin using a pull-down assay with chitin beads. jcdv from purified or semi-purified inocula were incubated with chitin beads, pulled-down by centrifugation and subsequently revealed by western blot using a specific jcdv anti-capsid antibody. figure 5a shows jcdv pull-down by chitin beads and we did not observed difference between inocula (purified vs. semi-purified), which confirmed that capsids can interact directly with chitin. second, we tested virus interaction with pm proteins using a viral overlay binding assay (vobpa). total proteins were extracted from isolated pms, separated with sds-page and either stained with page blue and periodic acid schiff (pas) in order to visualize total and glycosylated proteins respectively, or blotted onto nitrocellulose membranes for vopba. we included porcine mucins as a control of highly (o-)glycosylated proteins. at the first glance, vopba revealed that jcdv binds to most if not all the pm proteins labelled by page blue and pas combined, although with different intensities ( figure 5b ). interestingly, no binding was observed with the porcine mucins which might support some specificity for insect glycans. more specifically, a set of jcdv-interacting proteins was identified at high molecular weights (>250 kda) including proteins with a pattern similar to porcine mucins. these proteins were labelled with page blue and pas or only with pas suggesting that they are mostly and probably highly glycosylated ( figure 5b ). interestingly, proteins at 180 kda displayed a higher intensity as they are in a relative lower amount (according to page blue), which suggests higher affinity for jcdv. proteins interacting with jcdv were detected at 150-200 kda and 25-60 kda, and corresponding to proteins with low or no glycosylation (according to pas staining). thirty µg of porcine mucins were also loaded in the gel as a control of highly o-glycosylated proteins. proteins were then stained with page blue or periodic acid schiff (pas, pink) to visualize total or glycosylated proteins, respectively, and transferred to nitrocellulose membranes for probing with jcdv and anti-jcdv capsid antibody. proteins interacting with jcdv capsids were finally revealed by enhanced chemiluminescence (black arrowheads on the vopba jcdv membrane); the corresponding positions of these bands were reported on the page blue and pas gels and indicated as well by black arrowheads on the right of these gels. in total, 10 bands representing jcdv interacting proteins are reproducibly obtained with vopba. noteworthy, each band probably include several proteins and/or isoform/glycoform of the same proteins. proteins corresponding to these 10 bands were next analyzed by lc-ms/ms mass spectrometry. we only considered 155 proteins that were shared between 3 replicates ( figure 6a) , out of which 138 were annotated in the reference genome of s. frugiperda [33] . these proteins are pm structural proteins (i.e., peritrophins including intestinal mucins) and pm-associated proteins (enzymes, i.e., serine proteases and aminopeptidases n (apn) (supplementary materials table s1 ). gene ontology (go) annotation confirmed the enrichment in proteolytic activities (particularly serine-type endopeptidases) and chitin synthesis, which are consistent with the pm composition and the gut function ( figure 6b ). interestingly among the set of proteins >150 kda, we identified intestinal mucins, an atp binding cassette a type 5 (abca5) transporter and aminopeptidases n (supplementary materials table s1); the latter being known receptors for a number of viruses and for the cry toxins from bacillus thuringiensis [49] [50] [51] [52] . [33] . (b) go terms enrichment for the 138 common annotated pm proteins interacting with jcdv (in green), compared to the reference in grey (predicted proteins from ogs2.2 s. frugiperda genome) (fdr set at 0.01). specific enrichment in jcdv interacting proteins is considered when the green bars exceed the greys (controls). the 138 common pm proteins were assignated to the go terms using blast2go software. these results show that jcdv capsids can recognize and bind to the different components of the pm including chitin and several highly glycosylated proteins, both structural components of the pm (mucin, peritrophins) or associated proteins (enzymes). jcdv recognition and binding to glycans of the pm concentrates viral particles close to the epithelial surface, which raised questions about the mechanism involved to cross over and reach the midgut receptor(s). we hypothesized that capsids aggregation on the matrix can result in its disorganization, in a way similar to chitin-binding wheat germ agglutinin (wga) lectin or calcofluor [53] . to test this hypothesis, we used fluorescent wga-labelling (wga-fitc) to label chitin and thus examine chitin fibrils formation and pm organization. third-instar caterpillars were fed with jcdv and then sacrificed at 1 day p.i. to dissect and prepare midguts for semi-thin sections and wga labelling. as shown in figure 7 , the labelling of the pm (green) lined the apical surface of the epithelium in non-infected larvae (pbs condition, figure 7 , upper panel). in addition, we observed a specific labelling at the apex of columnar cells, probably corresponding to microvillar secretion of glcnac from these cells. by contrast, the labelling lining the epithelium appeared discontinuous following infection (figure 7, lower panel) , displaying a disorganized pattern reminiscent of the pm structure observed for caterpillars fed with the wga lectin [53] . moreover, intracellular labelling was no longer observed in the sections from infected caterpillars suggesting an arrest of glcnac secretion from the cells following early infection, i.e., before we can detect virus replication in subepithelial tissues [13] . these results thus support the hypothesis that jcdv binding on the pm and transcytosis is associated with a loss in its integrity, which might reveal gut dysfunction. to determine the midgut response following jcdv break in, we analyzed the transcriptomic response. we used digital gene expression (dge) based on the serial analysis of gene expression approach [35] . this method involves the sequencing and quantification of end tagged short cdna fragments (i.e., tags), which enables quantitative differential gene-expression analysis. we built four cdna libraries from midguts of mock-and infected larvae (supplementary materials tables s2 and s3) . tag sequences were mapped to the genome and transcriptome of s. frugiperda ( [33, 38] ) and to the jcdv genome. none of the tags were assigned to viral transcripts, which is consistent with jcdv pathogenesis excluding replication in midgut cells [13] . pie charts represented go assignment corresponding to unique transcripts at 1 and 3 days p.i. that displayed a differential expression at least 5-fold up-or down-regulated (supplementary materials figure s4a,b) . interestingly, the distribution of go terms was roughly similar at 1 and 3 days p.i., suggesting that the overall intestinal response to jcdv oral infection was poorly affected by virus replication going on in subepithelial tissues (supplementary materials figure s4a ,b). we observed only go terms enrichment for the over-represented transcripts at 1-day p.i., more specifically in functions involved in metabolic processes including translation (i.e., regulation of biological processes, response to stimuli and signaling) at 1 day p.i., that might indicate that jcdv intrusion induces a rapid metabolic response in the gut (figure 8 ). interestingly, these changes did not change significantly at 3 days p.i. suggesting that the gut response is rapidly initiated by jcdv transcytosis and was not affected by the viral replication that takes place in underlying tissues. we did not observe any significant activation of genes involved in "inflammation" nor in the canonical gut immune response. however, we observed an increased expression in cytochrome p450 and catalase genes that might indicate a response of the cells to the ongoing infection. table s4 ), we only observed few changes including a trehalose transporter and an intestinal mucin, both down-regulated from day 1 p.i., and a chitinase up-regulated at day 3 p.i.. except for an aminopeptidase n, no gene corresponding to the jcdv interacting proteins identified by vopba displayed a transcriptional change. we did not observe any significant activation of genes involved in "inflammation" nor in the canonical gut immune response. however, we observed an increased expression in cytochrome p450 and catalase genes that might indicate a response of the cells to the ongoing infection. altogether these results show that jcdv infection induces rapid changes in the gut, particularly in translation and metabolism, within 24 h p.i.. both increased molecular activities might favor viral invasion by supporting the increased energetic demand associated with virus replication in target tissues. interestingly, the canonical midgut immune system did not detect jcdv break in and transport across the epithelium. how densoviruses cope with the forest of glycans that constitute extracellular matrices and decorates insect cell surfaces has been so far a neglected step of their early pathogenesis. results presented here show that jcdv capsids display carbohydrate-binding properties that insure recognition of the peritrophic matrix and determines caterpillars oral infection. we found that capsids can bind to the different components of the pm and their agglutination on the pm surface is associated with the disruption of its organization. furthermore, we showed that this primary step of infection of caterpillars results in a series of physiological changes in the midgut including an arrest of chitin synthesis by epithelial cells. the pm is an obligatory binding platform for capsids to avoid elimination and get closer to the epithelial cell surface where receptor recognition can occur. however, strong attachment to glycans composing the pm would trap capsids there and thus impair their physical connection with the receptor(s). therefore, a first hypothesis is that the "stickiness" of the capsids is balanced to bind and unbind glycans. we used competitions assays with monosaccharides to test this "bind and release" hypothesis and our results showed that indeed, capsids have an affinity for glycans, although the concentration range of the monosaccharides (mm) we tested likely indicates their poor affinity for the capsids. as these monosaccharides could compete capsids away from the pm further suggests that glycan-capsids interaction are probably of low affinity. the issue for bound viral particles is then to move across the pm. our experiments show that capsid binding results in a structural disorganization of the pm similar to effects induced by chitin-binding lectin wga and calcofluor. such capsid-induced disruption of the pm thus favors a second hypothesis, involving a "saturate and pass through" mechanism, where bound capsids are not released but open a way for viral particles to cross over. such "cooperative" mechanism of the capsids to overcome the pm is supported by the fact that pm disruption is enhanced by virus concentration and decreased as caterpillars age. such developmental resistance of s. frugiperda has been also reported following baculovirus infections and a high synergism with calcofluor was obtained at late instars (e.g., > 60-fold at 4th instar vs. 3 to 6-fold at 2nd and 3rd instars) [54] .the structure and the composition of the pm can vary as caterpillars grow and feed, or between populations, which might impact virus-pm interactions and consequently insect susceptibility [16, 17, 55] . whether pm disruption results from mechanical stresses on chitin fibers similar to calcofluor and probably wga lectins or from an enzymatic activity of the capsids (i.e., of vp4) remains to be analyzed more thoroughly. understanding the early interaction of jcdv with the glycans of the pm within species, i.e., along the larval development is of importance to develop biocontrol strategies against insect pests. whether or not the pm could contribute to the species barrier against densovirus infection is unknown. a better understanding of the structure and the glycan composition of the pm in s. frugiperda together with comparative studies in different lepidopteran species are essential to go further on the role of the pm in densovirus infection. structure-fonction studies of the capsid of parvoviruses infecting vertebrates, in particular for species in the genera protoparvovirus and dependovirus, have highlighted the importance of glycans recognition on tissue tropism, pathogenicity, and host range adaption [6, 45, [56] [57] [58] [59] . regarding densoviruses, information and capsid structure-function studies is poor [60] . we performed preliminary assays with a glycan array from the consortium for functional glycomics (http: //www.functionalglycomics.org/fg/, as gosselin-grenet, unpublished data). although this array represents mammalian glycans, the specific recognition of jcdv capsids by paucimannose, which are particularly abundant in insects, suggests some specificity in the interaction of the capsid with glycans. an "insect array" would be of interest to explore glycan ligands affinity and specificity for densovirus capsids. morevover, insects are particularly "handly" animal models for structure-function assays in vivo. we showed that jcdv early infection triggers an arrest of glcnac secretion, which might considerably weaken the pm and explain the disorganized pattern observed with chitin-labelling at 1 day p.i. interestingly, these changes are observed before we could detect jcdv transcription/replication in primary targeted cells [13] , suggesting that these effects are induced as a consequence of the transport of the viral particles across the epithelium. it has been reported that specific drugs that disorganize microtubules induce an arrest of chitin synthesis [41, 61] . we speculate that jcdv transcytosis might induce some stress on the cytoskeletal network, similar to microtubules disorganizing drugs. it is worthy to note that chitin synthesis arrest was not associated with a drop in the expression of chitin synthase genes. however, we cannot exclude to have missed enzymes due to some lack in the annotation of the genome of s. frugiperda [33] . last, our results showed that jcdv capsids can also interact with components of the luminal compartment including food and bacteria. the competition of the capsid with monosaccharides for binding the pm, suggests that food components could interfere with infection. indeed, plants contain compounds that can interfere with pm synthesis, which has been shown to consequently affect baculovirus infection [55] (chen et al., 2018) . regarding bacteria, it has been shown recently that the pm controls commensal bacteria, and conversely that its synthesis and integrity can be microbiota-dependent, i.e., the gut microbiota inducing the expression of components of the peritrophic matrix [62, 63] . so, it is plausible that food and microbiota can modify the outcome of the densovirus infection, either by directly competing for binding the pm, or indirectly by modulating the composition of the pm. the binding of densovirus capsids to a wide array of glycans questioned about the role of this "stickiness" in the whole infection cycle including transmission. groundbreaking articles have shown the role of microbiota polysaccharides including glcnac, on the infectivity and thermostability of picornaviruses [64] [65] [66] , whose capsid share structural similarity with parvoviruses [25] . it is tempting to speculate that densovirus "stickiness" can similarly impact their transmission, which might participate to their success if only among arthropods that occupy extremely diversified ecosystems [67] . such consideration could also apply to parvoviruses going through faecal-oral route and environmental contamination [58, 68] . more generally, stickiness is a major issue for most viruses to and mathematical models have been applied to influenza. they predict that a maximum stickiness favors a maximum fitness [69] . however, trade-off probably exists in biological systems with an optimal "stickiness" that must be found to infect and leave a host for transmission. densoviruses can be highly pathogenic for insect pests and vectors, which have long stimulated their interest as biocontrol agents or genes vectors [70] . they are considered today with a renewed interest as solutions to control harmful insects are lacking, which encourages efforts to understand their pathogenesis and their specificity. altogether our results suggest that pm glycans are crucial interacting components of the early jcdv pathogenesis. exploring their diversity and their complexity in insects can also provide important cues on the extend of the mechanisms that determine densovirus specificity. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/11/9/870/s1, figure s1 . calcofluor increases s. frugiperda caterpillars susceptibility to densovirus oral infection. figure s2 . virus interaction with monosaccharides did not interfere with anti-capsid antibody recognition. figure s3 . replicates of survival curves of caterpillars infected by jcdv. figure s4 . jcdv oral infection induces midgut gene expression modulation. table s1 . annotation of pm proteins interacting with jcdv in vopba. table s2 . characteristics of dge libraries generated from caterpillars orally infected with jcdv. table s3 . characteristics of annotated tags and transcripts. table s4 . annotation of regulated intestinal transcripts following oral jcdv infection. the authors declare no conflict of interest. the mucosal barrier at a glance extracellular matrix in plants and animals: hooks and locks for viruses ruvoën-clouet, n. host-pathogen co-evolution and glycan interactions twenty-five years of structural parvovirology an essential receptor for adeno-associated virus infection binding site on the transferrin receptor for the parvovirus capsid and effects of altered affinity on cell uptake and infection aav transcytosis through barrier epithelia and endothelium secreted and transmembrane mucins inhibit gene transfer with aav4 more efficiently than aav5 detailed investigation of the sequential pathological changes in silkworm larvae infected with bombyx densovirus type 1 densovirus crosses the insect midgut by transcytosis and disturbs the 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evidence for a gap between polymerization and microfibril formation chitin synthesis inhibitors: old molecules and new developments calcofluor disrupts the midgut defense system in insects turnover, and functions of chitin in insects optical brightener m2r destroys the peritrophic membrane of spodoptera exigua (lepidoptera: noctuidae) larvae functional implications of the structure of the murine parvovirus, minute virus of mice analysis of the cell and erythrocyte binding activities of the dimple and canyon regions of the canine parvovirus capsid diversity and functions of protein glycosylation in insects role of the phosphatidylinositol-3-kinase/akt/target of rapamycin pathway during ambidensovirus infection of insect cells further characterization of aminopeptidase-n as a receptor for coronaviruses porcine aminopeptidase n is a functional receptor for the pedv coronavirus in vitro evidence supports membrane alanyl aminopeptidase n as a receptor for a plant virus in the pea aphid vector feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i effect of wheat germ agglutinin on formation and structure of the peritrophic membrane in european corn borer (ostrinia nubilalis) larvae effect of optical brighteners on the insecticidal activity of a nucleopolyhedrovirus in three instars of spodoptera frugiperda the effect of diet on midgut and resulting changes in infectiousness of acmnpv baculovirus in the cabbage looper protoparvovirus cell entry host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species four amino acids of an insect densovirus capsid determine midgut tropism and virulence chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases microbiota-induced peritrophic matrix regulates midgut homeostasis and prevents systemic infection of malaria vector mosquitoes pgrp-ld mediates a. stephensi vector competency by regulating homeostasis of microbiota-induced peritrophic matrix synthesis intestinal microbiota promote enteric virus replication and systemic pathogenesis interactions between enteric bacteria and eukaryotic viruses impact the outcome of infection bacteria and bacterial envelope components enhance mammalian reovirus thermostability discovery of parvovirus-related sequences in an unexpected broad range of animals transmission ecology of canine parvovirus in a multi-host, multi-pathogen system how sticky should a virus be? the impact of virus binding and release on transmission fitness using influenza as an example viral delivery of dsrna for control of insect agricultural pests and vectors of human disease: prospects and challenges we are grateful to c. gibard, r. bousquet and g. clabot for their great help with insect rearing and the piq quarantine plateform from vectopole sud. we especially acknowledge e. jublanc and c. cazevieille for their skillful technical assistance and the imaging facility mri, member of the national france-bioimaging infrastructure supported by the french national research agency (anr-10-inbs-04, pia «investments for the future»). we warmly thank m. agbandje-mckenna from the cfg consortium (university of florida) for the glycan array and p. clair from the qphd platform (montpellier genomix). m. younes, and d. piquemal are also acknowledged for their support in the sage analysis. special thanks to p.a. lafon for his help for statistical analyses. l. pigeyre is a doctoral fellow of the french ministry for higher education and research/ephe. key: cord-009380-5uptbat3 authors: evermann, james f.; eriks, inge s. title: diagnostic medicine: the challenge of differentiating infection from disease and making sense for the veterinary clinician date: 2007-09-28 journal: adv vet med doi: 10.1016/s0065-3519(99)80006-8 sha: doc_id: 9380 cord_uid: 5uptbat3 nan diagnostic medicine has taken on a new, broader meaning in the 1990s and reflects an expansion of clinical investigation from the diagnosis of disease to include detection of infection (evermann, 1998) . this leads to an entirely new perspective on how veterinary clinicians and diagnosticians view laboratory tests and how test results are interpreted. one must consider not only the specificity and sensitivity of the test, but the predictive value of the test, which relates directly to the clinical utility of the result (jacobson, 1997) . the definitive diagnosis of infectious diseases relies on a combination of clinical symptoms, history, and laboratory analyses of ante-mortem and/or postmortem specimens (evermann, 1998) . disease diagnosis has customarily used diagnostic assays for early recognition of disease and rapid implementation of therapy in an individual animal basis, and when appropriate use of corrective management (segregation, culling, vaccination, etc.) on a population basis. the detection of infection during preclinical stages has become more important as one considers the consequence of long-term infections that have prolonged incubation periods and inapparent transmission to susceptible animals in the population. this includes lifethreatening diseases, such as feline infectious peritonitis (fip), rickettsial and ehrlichial diseases and canine herpesvirus (chv) infections. of equal, if not more so, importance for the early detection of infection has been the increased recognition of zoonotic infections, such as rabies virus, salmonella typhimurium dt104, and escherichia coli o157:h7 (evans and davies, 1996; slutsker et al., 1997; smith, 1996) . together with the necessity to detect infections earlier during the preclinical stages, there has been a remarkable expansion in the availability of assays that can detect infectious microorganisms in low quantity. this increased sensitivity has been primarily through the detection of nucleic acid sequences after amplification by polymerase chain reaction (pcr, relman and persing, 1997) . pcr can allow not only early detection of infection, but rapid speciation of organisms as well as strain typing for epidemiologic analyses (fredricks and relman, 1996; mcdade and anderson, 1996) . the assessment of preclinical infections allows the veterinarian the opportunity to determine the relative risk of disease occurring, and to take preventive steps to reduce or eliminate the risks depending on the consequences of the disease in the animal and/or human (if zoonotic) population. this chapter focuses on one main issue, and that is differentiating the detection of infection from diagnosis of disease. in the course of differentiating infection from disease three questions will be addressed: (1) how early do we want to detect infection? (2) what are the consequences of the results? (3) where are we heading with veterinary diagnostics? historically, the primary aim of the diagnostic laboratory was to assist the veterinarian in the diagnosis of disease. this is presented in fig. 1 . this type of approach initially ignored the origin of the causative microorganisms and focused on the accurate diagnosis of the disease agent. an example of this type of approach was the testing of cats that were clinically ill for feline leukemia virus (felv). if tested positive, they were segregated or euthanized. further examples include fip, chv, johne's disease (mycobacterium paratuberculosis), and the mucosal disease form of bovine viral diarrhea (bvd) virus. expanded use of diagnostic results by the veterinarian and client allowed for some corrective management steps to be taken. these included the use of vaccination when available or segregation and culling to reduce the source of the infection in the population. based on this latter principle, the reduction of the source of the infection, a different approach has been taken. one may consider this an epidemiologic view of the disease process (susser and susser, 1996a) . with a combination of more sensitive diagnostic assays, the veterinarian's concern to know the state of the preclinical infection, economic incentives to minimize disease by effectively controlling the infection, and concern over potential zoonotic diseases, laboratory diagnosis has taken on a different strategy. this is presented in fig. 2 . the primary emphasis in this scheme is to view animals preclinical and determine the disease risk and/or zoonotic potential of the infection. this has been the approach for some retroviral infections (evermann and jackson, 1997; knowles, 1997) and bacterial infections with public health concerns, such as e. coli o157:h7 and salmonella spp. infections (evans and davies, 1996; firstenberg-eden and sullivan, 1997; mcdonough and simpson, 1996) . the ultimate goal of the assessment of preclinical testing is to initiate a preventive management type of control. this type of approach places more emphasis on early testing and management of infected animals rather than on diseased animals. with the shift in emphasis to preclinical testing, the knowledge of the ecology of the infectious microorganism has become very important in our overall understanding of how successful the control program may be (susser and susser, 1996b) . the control of infections with a low degree of transmissibility and narrow host range, such as caprine arthritis-encephalitis (cae) virus, is much more realistic than the control of diseases with a wide host range, such as chlamydia and salmonella spp., or those agents spread by arthropod vectors, such as arboviruses and rickettsia (gregory and schaffner, 1997; hewinson et al., 1997; knowles, 1997; raoult and roux, 1997; saluzzo and dodet, 1997) . the ecology of infection provides the veterinarian with vital information with which to make decisions. the ecology of six different agents is listed in table i . the ecology of infection is divided into infection rate, attack rate (progress to become clinical), and mortality rate. it can be seen that the infection rate usually exceeds the attack rate and mortality rate in the majority of cases. exceptions to this generalization are the mucosal disease form of bvd that occur in cattle that are tolerant to bvd and persistently infected (pi), and rabies infections in mammals (innocent et al., 1997; smith, 1996) . another way to view the ecology of an infection is demonstrated in fig. 3 . the schematic allows the veterinarian to readily use a graphic approach with clients to explain the differences between infection and disease. rabies virus is used as an example of a microorganism with a low infection rate, but high mortality. (this figure would be different if one were to diagram the ecology of rabies in bats, the natural reservoir for rabies in the united states.) the cae virus is used as an example of an infection in goats with a high infection rate, but lower attack rates, and much lower fatality rates (fig. 4) . with retroviruses, such as cae virus, bovine leukosis virus (blv), and equine infectious anemia (eia), the ecology can also be subdivided into progressor (progress onto clinical signs and/or fatality) or nonprogressor (remains clinically normal, but infected and potentially contagious). with persistent bacterial infections, such as salmonella and many members of the rickettsiaciae, the ecology can be subdivided into clinical disease leading to mortality or clinical disease leading to a chronic carrier state. this chronic carrier state can then be further subdivided into inapparent infections with constant shredding and inapparent infections with intermittent shedding. with potentially zoonotic diseases, such as salmonella, rickettsioses, and psittacosis (chlamydia psittici), the ability to shed or transmit the organism into the environment or vectors becomes particularly relevant (evans and davies, 1996; gregory and schaffner, 1997; raoult and roux, 1997) . early detection of infection is now feasible with a number of microorganisms affecting animals. the detection may take the form of specifically identifying the nucleic acid of the infectious agent, such as bovine herpesvirus-1 in semen samples, blv provirus is selected blood cells populations, and foodborne bacteria in dairy products (batt, 1997; masri et al., 1996; xie et al., 1997) . although this form of early microbial detection is preclinical at this time, with further research it may be determined that these "subclinical infections" are actually causing alterations in cell structure and function leading to endocrine imbalances and decreased productivity. this form of disease has been referred to a "lesionless pathology," and will be the subject of further research (de la torre and oldstone, 1996) . early detection of infection may take a "back door" approach by analyzing the host animal's genetic predisposition to infection and disease. this interesting approach has already been used in order to control the prion disease, scrapie (o'rourke et al., 1997) . sheep with a unique chromosome are highly susceptible to progress onto scrapie, an irreversible disease. animals that are bred for genetic resistance to infection and/or disease will be major factors in disease control in the future (gavora, 1996; malo and skamene, 1994) . the utilization of genetic testing is essential for some infections, such as the retroviruses, which serve to activate cellular oncogenes and promote disease. identifying these cellular oncogenes would be a major step in controlling retroviral-induced diseases (wiedemann et al., 1991) . it will be essential to clearly define what the diagnostic assay is detecting so that the veterinarian may utilize the information appropriately. figure 5a graphically presents the use of thresholds to differentiate subclinical infection from clinical manifestations of the disease. figure 5b shows five potential diagnostic assays, each with varying levels of sensitivity. it would be critical to understand the differences between a test with high sensitivity, which detects subclinical infection and a test with lesser sensitivity, but more accurately diagnoses disease. this question becomes more difficult the more one employs preclinical testing in preventive medicine programs (clementi et al., 1995; jacobson and romatowski, 1996; smith, 1995) . the predictive value of a positive result may be high when an animal is clinical, such as a cat with fip. however, with early testing the problems of detecting cross-reacting viruses (feline enteric coronaviruses) increases, as does the question of whether the preclinical result accurately identifies an animal that is just infected or will progress onto disease (evermann et al., 1995; foley et al., 1997) . in infections such as eia, the consequences of infection are just as severe as the horse that has clinical signs of eia. this is because the infection is regulated by the u.s. department of agriculture and all seropositive horses and mules are required to be reported, regardless of their health status. assays for early detection of eia infection have been reported to detect viral rna in plasma samples as early as 48 hours after infection (langemeier et al., 1996) . similarly in bovine tuberculosis, caused bymycobacterium bovis, the consequences of a positive test result can be economically devastating due to stringent government regulations. this becomes particularly problematic because many tests currently available may cross-react with other mycobacterial species (essey and koller, 1994; o'reilly and daborn, 1995) . to determine what consequences the test results will have on the animal and the owner it is important to ask five key questions (table ii) . is the infection and/or disease of economic concern, such as eia or m. bovis; is the infection and/or disease of zoonotic concern, such as e. coli 0157:h7; where is the microbial agent when not causing disease, such as with rabies reservoirs in bat populations; what are the contributing factors to the infection and/or disease process, such as pregnancy for chv and other herpesviruses; and what factors can animal owners/veterinarians/public health personnel control to minimize or eliminate the risk of infection and/or the disease process? table iii lists some of the consequences of the infection and/or disease process. these range from no sale, as with a goat that is cae seropositive, to euthanasia if a horse or mule is tested eia seropositive. v. where are we heading with veterinary diagnostics? veterinary diagnostics, like their human counterparts, are already directing efforts toward more sensitive assays, which are capable of detecting infections very early (within hours of initial infection); subclinical infections that are the result of persistent infections acquired during gestation and masked by immune tolerance; latent infections due to herpesviruses and retroviruses; and infections that pose a public health risk (barrett et al., 1997; burr, 1996; clarke, 1997; de la torre and oldstone, 1996; rodriquez, 1997) . the evolution of diseases and the emergence of newly recognized pathogens have placed considerable pressure on new diagnostic technologies. the newer assays will assist in tracking the emerging infections, as well as linking causal association with disease to a firm cause and effect of the disease (bryan et al., 1994; holtzman et al., 1997; hoet and haufroid, 1997; lipstich et al., 1996; mcdade and anderson, 1996; poland et al., 1996) . 1. is the infection and/or disease of economic concern? 2. is the infection and/or disease of zoonotic concern? 3. where is the microbial agent when not causing disease (microbial ecology)? 4. what are the contributing factors to the infection and/or disease process? 5. what factors can animal owners/veterinarians/public health personnel control to minimize or eliminate the risk of infection/disease process ? table iii immune function (immune competence), (2) assays to monitor genetic resistance/susceptibility, (3) assays to monitor infections, (4) assays to diagnose disease and monitor response to treatment, and (5) assays to track emerging infections. as infectious agents continue to evolve, disease expression will change, resulting in the necessity to develop new diagnostic assays (susser and susser, 1996b; wilson, 1994) . nucleic acid amplification technologies: application to disease diagnosis molecular diagnostics for dairy-borne pathogens emerging infectious diseases in the united states: improved surveillance, a requisite for prevention detection of canine herpesvirus 1 in a wide range of tissues using polymerase chain reaction paratuberculosis and molecular biology quantitative molecular methods in virology anatomy of viral persistence: mechanisms of persistence and associated disease status of bovine tuberculosis in north america case control study of multiple-resistant salmonella typhimurium dt104 infections in cattle in great britain laboratory diagnosis of viral and rickettsial infections laboratory diagnosis of viral and rickettsial infections laboratory diagnostic tests for retroviral infections in dairy and beef cattle feline infectious peritonitis ez coli rapid detection system: a rapid method for the detection of escherichia coli o157 in meat and other foods risk factors for feline infectious peritonitis among cats in multiple-cat environments with endemic feline enteric coronavirus sequence-based identification of microbial pathogens: a reconsideration of koch's postulates resistance of livestock to viruses: mechanisms and strategies for genetic engineering detection of chlamydia psittici dna in avian clinical samples by polymerase chain reaction biological monitoring: state of the art predictive genetic testing: from basic research to clinical practice the use of a mass-action model to validate the output from a stochastic simulation model of bovine viral diarrhea virus spread in a closed dairy herd. prey principles of validation of diagnostic assays for infectious diseases assessing the validity of serodiagnostic test results. semin laboratory diagnostic tests for retrovirus infections of small ruminants detection of equine infectious anemia viral rna in plasma samples from recently infected and long-term inapparent carrier animals by pcr the evolution of virulence in pathogens with vertical and horizontal transmission genetic control of host resistance to infection rapid detection of bovine herpesvirus 1 in the semen of infected bulls by a nested polymerase chain reaction assay molecular epidemiology: applications of nucleic acid amplification and sequence analysis diagnosing emerging bacterial infections: salmonellosis, campylobacteriosis, clostridial toxicosis, and helicobacteriosis. semin the epidemiology of mycobacterium bovis infection in animals and man: a review prp genotypes and experimental scrapie in orally inoculated suffolk sheep in the united states two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus rickettsioses as paradigms of new or emerging infectious diseases genotypic methods for microbiol identification detection of animal pathogens by using the polymerase chain reaction (pcr) factors in the emergence of arbivirus diseases escherichia coli o157:h7 diarrhea in the united states: clinical and epidemiologic features new aspects of rabies with emphasis on epidemiology, diagnosis and prevention of the disease in the united states evaluation of diagnostic tests choosing a future for epidemiology: i. eras and paradigms choosing a future for epidemiology: ii. from black box to chinese boxes and eco-epidemiology chromosome rearrangement, oncogene activation and other clinical events in cancer. their use in molecular diagnostics disease in evolution: introduction detection of proviral dna of bovine leukemia virus in cattle by a combination of in situ hybridization and the polymerase chain reaction 9 no sale 9 public health risk 9 early cull 9 regulatory quarantine 9 shedding of microbial agent to susceptible animals in the population 9 segregation of animal 9 euthanasia of animal(s)the future of veterinary diagnostics is now. there are at least five directions to be pursued (table iv) , none of which is new, but continuing to evolve as the needs mandate the detection of infection earlier and the diagnosis of disease at a manageable stage (wilson, 1994) . these five directions are the development of (1) assays to monitor 1. assays to monitor immune function (immune competence) 9 foal check 9 calf failure of passive transfer 9 llama/alpaca immunoglobulin status 9 cmi response 2. assays to monitor genetic resistance/genetic susceptibility 9 cellular receptors 9 cellular oncogenes 9 cellular prion proteins 3. assays to monitor infections 9 in the environment 9 in asymptomatic vectors (potential transmissibility) 9 in asymptomatic carriers (potential shedders) 4. assays to diagnose disease 9 prognosis 9 monitor response to treatment via cytokines (il-2, il-4, etc.) 5. assays to track emerging infections 9 culture invitro 9 conserved pcr 9 disease potential 9 develop new detection assays 9 develop new vaccines key: cord-007367-e31zhty6 authors: tassier, troy; polgreen, philip; segre, alberto title: network position and health care worker infections date: 2015-09-07 journal: j econ interact coord doi: 10.1007/s11403-015-0166-4 sha: doc_id: 7367 cord_uid: e31zhty6 we use a newly collected data set coupled with an agent-based model to study the spread of infectious disease in hospitals. we estimate the average and marginal infections created by various worker groups in a hospital as a function of their network position in order to identify groups most crucial in a hospital-based epidemic. surprisingly, we find that many groups with primary patient care responsibilities play a small role in spreading an infectious disease within our hospital data set. we also demonstrate that the effect of different network positions can be as important as the effect of different transmission rates for some categories of workers. vaccines are a primary way to stop or slow the spread of many infectious diseases, perhaps most notably, influenza. the lack of appropriate vaccination levels is a major health problem. for instance, influenza is a major cause of morbidity and mortality throughout the world despite the availability of a highly effective and inexpensive vaccine. in the us alone, influenza causes an estimated 36,000 deaths and 120,000 hospitalizations annually yet only around 1/3 of healthcare workers are vaccinated each year (thompson et al. 2003) . efficient provision of vaccinations poses a difficult problem in that the positive externality associated with a vaccination is the product of the probability of infection, the cost of the infection, and the marginal infections generated by an agent if infected (all of which may vary across agents). there is great concern over the spread of infectious diseases in hospitals, but little knowledge is available to identify healthcare workers who are most likely to acquire and transmit infectious diseases in hospitals. the problem is especially difficult because the transmission of many infectious diseases is not observable. for instance if someone in your household acquires influenza, you likely do not know which of the potentially hundreds of people you come in contact with each day that may have caused the infection. further if a vaccine is available for an infectious disease and the vaccine is in short supply or is expensive, it is imperative to know which individuals should have the highest priority in vaccine campaigns. in this paper we use a newly collected data set on hospital worker contacts in order to identify hospital worker groups that have the potential to create the largest number of infections based on their location in a hospital contact network. to achieve this goal, we have collected person-to-person contact information on 140 individuals belonging to one of 15 types of healthcare workers at the university of iowa hospitals and clinics (uihc). the data contain information on the contacts between healthcare workers and patients and between healthcare workers and other healthcare workers at the hospital. with this information we develop a network model describing the spread of an infectious disease in a hospital. we estimate, using an agent-based model, the effect of network position of different hospital worker groups on the spread of infectious diseases in a hospital. through this model we are able to identify the hospital worker groups that create the largest externality if removed from the network (perhaps through a vaccination or a quarantine). we argue that methods such as those used in this paper can help hospitals, health care professionals, and epidemiologists to design efficient programs for healthcare worker vaccinations. of importance, we note that we only study the externality in terms of network position within the hospital. in this paper we do not consider other potential heterogeneity among agents such as differences in transmission rates across workers, or differences in behavior outside the hospital that may play a role. 1 while these effects are also important, the large differences across classes of workers shown below are worthy of independent study. this is the first paper to use specific micro-level contact data within a hospital that can be used to help guide policy makers and public health officials in the problem of efficiently allocating vaccines within hospitals. the data used in this paper is unique and detailed in comparison to other studies. the data consists of shadow data (where a research assistant follows a specific, randomly chosen hospital worker for an entire shift) for the 15 major groups of healthcare workers at the uihc, a 700-bed major medical center. this results in over 600 h of direct hospital worker observations and the notation of over 6500 specific worker to worker or worker to patient contacts throughout the hospital. to the best of our knowledge, the data that we have collected comprises the most detailed micro-level healthcare worker contact data set in existence. as a comparison, ueno and masuda (2009) collect data on contacts between doctors, nurses, and patients. their data is based on two calendar days from a small, 129 room, community hospital in tokyo. they model contacts between nurses, physicians and patients. their data does not consider contacts with and between other healthcare worker groups (other than nurses and doctors). based on our data at the uihc, these assumptions would ignore over 60 % of hospital staff, including most of the groups we identify as most crucial to the spread of an infection disease. we begin by discussing the background and motivation for our study and then move to develop a simple model of infectious disease transmission. in the model, we initially assume homogeneous contacts as in traditional epidemiological models. we then discuss a similar model with heterogeneous contacts and discuss the difficulties of achieving efficient vaccination levels. following the theoretical discussion, we use our newly collected data on healthcare worker and patient contacts to model the spread of an infectious disease in a hospital setting. the model allows us to identify the healthcare worker groups that would be expected to play the largest role in the spread of infectious diseases, in terms of network position, in this hospital setting. traditionally, epidemiology research has focused on well-mixed (randomly mixed) populations where agent contacts are homogeneous. in these models, every agent in a population may "bump into" any other agent with equal probability, much as a gas molecule may bump into any other gas molecule with an equal probability over a fixed time period. only recently have epidemiologists and other researchers begun to study the heterogeneous contact structures between people over which infectious diseases spread (early studies include comins et al. 1992; grenfell and harwood 1997; wallinga et al. 1999) . we focus this study on healthcare workers and a particular class of infectious disease, of which influenza is an example. healthcare workers are at especially high risk of contracting influenza. one study of healthcare workers with a low rate of influenza vaccination demonstrated that 23 % of healthcare workers had evidence of influenza infection during a single influenza season (elder et al. 1996) . two features of influenza make its spread difficult to control in hospitals. first, people with influenza are infectious 1-4 days before the onset of symptoms. thus, they can spread the virus when they are still feeling well and are unaware of their own infectious state. second, only about 50 % of infected persons develop classic influenza symptoms (cdc 2002 (cdc , 2003 consequently, restricting healthcare workers with influenza-like symptoms from the workplace will not completely prevent transmission of influenza because healthcare workers with atypical symptoms could continue working and spreading the virus. furthermore, studies show that healthcare workers are more likely than other workers to return to work early or to keep working when they have influenza-related symptoms (weingarten et al. 1989) . because of the ease with which influenza may be contracted and spread by healthcare workers, the centers for disease control and prevention (cdc) have, for the past two decades, recommended influenza vaccination for all healthcare workers. yet, in the us, only 36 % of workers who have direct patient contact are immunized against influenza annually (smith and bresee 2006) . outside of concerns about traditional influenza, there are additional reasons to study the spread of infectious diseases in hospitals. first, healthcare-associated infections affect about 2 million patients in us hospitals each year (jarvis 1996). second, there is a growing fear that hospitals could become a breeding ground for new strains of influenza such as the recent h1n1 influenza outbreak, the potential emergence of person-to-person transmission of avian flu, or other "new viruses." much as sars spread widely in hospitals to begin the sars epidemic in toronto (chowell et al. 2004) , person-to-person transmission of avian flu may start in hospitals as well, and, if a more lethal version of h1n1 were to develop, hospitals again could be a breeding ground for new infections. this last point is of particular salience. with the recent h1n1 outbreak and the subsequent work to develop a vaccine, controversies arose concerning which individuals to vaccinate first. healthcare workers were high on the list. but, as we show below, not all healthcare workers are equal in terms of their importance in spreading infectious diseases. thus, one primary focus of this paper is identifying the individual hospital workers who are most important to vaccinate should a similar outbreak occur again. there is a growing literature in economics on the vaccination choices of individuals and of the externalities associated with vaccinations. but scant attention is paid to the network effects determined by heterogeneous contacts that we focus on in this paper. for example, francis (2004) solves for the optimal tax/subsidy policy for influenza in an sir model with a constant contact rate and random mixing among the population. geoffard and philipson (1997) examine how the individual incentives for vaccination decrease as disease incidence decreases and thereby argue that relying exclusively on private markets is unlikely to lead to disease eradication. boulier et al. (2007) , the most similar paper to ours, investigate the magnitude of the externality associated with a vaccination as a function of the number of vaccinations in the population, the transmission rate of the disease, and the efficacy of the vaccination. they find nonmontonic relationships between each of these items and the magnitude of the vaccine externality. more specifically, the externality and the number of infections prevented per vaccination initially increases before eventually decreasing. however, like francis, they do not consider the case of heterogenous contact number or heterogenous sorting among the population. finally, much of the recent literature on the economics of infectious disease is summarized in philipson et al. (2000) . we begin by describing a simple model where agents in a population have contacts with each other with a uniform probability. this is the traditional random mixing model in epidemiology pioneered by kermack and mckenrick (1927) . the important results in this paper describe exceptions to this homogeneous contact assumption, but we use the simplified model to develop intuition before describing a richer model with heterogeneous contacts. in this simple model, we assume that all agents are homogeneous in that all agents have the same number of contacts with other agents and that these contacts are randomly drawn with uniform probability from the population at large. suppose that agents are assigned to one of three states: susceptible (s), infected (i), or recovered (r). a susceptible agent can transition to being infected with probability α if she is in contact with an infected agent. once infected, an agent transitions to the recovered state according to a parameter κ. once recovered, the agent is immune to the possibility of future infection. this is a classic susceptible-infected-recovered (sir) model for infectious diseases such as influenza (kermack and mckenrick 1927) . the description and parameters yield the following differential equations describing the flows of agents among the various states, assuming a constant population of size n and contact rate of γ . each equation describes the rate of growth for one of the three populations in the sir model. equation 1 describes how susceptible agents contact γ other agents in the population, of which i t /n are infected, and how, of these contacts with infected agents, a percentage α cause the susceptible agent to transition to being infected. equation 2 describes the previously mentioned flows from susceptible into infected and that each infected agent moves to being recovered at rate κ. finally, eq. 3 describes the flows from infected to recovered. we can write these equations in terms of population shares by dividing each of the above equations by the population size n and using lower case letters to denote these population shares, s t = s t /n , i t = i t /n , and r t = r t /n , yielding the following population share equations: the number of infected agents will increase in the population if the flows into the infected state from the susceptible state exceed the flows out of the infected state into the recovered state, di t dt > 0. this condition is equivalent to αγ s t > κ or s t > κ αγ . if this inequality holds then we say that the population is above the epidemic threshold. note that we cannot remain above the epidemic threshold forever without an introduction of new susceptible agents since ds t dt < 0: eventually the population will run out of susceptible agents to infect unless the susceptible population is replenished at a sufficient rate. one goal of healthcare policy is to attempt to place a population below the epidemic threshold so that the number of infectious agents in a population does not grow subject to some cost constraint. a population is most vulnerable to being above the epidemic threshold when the infectious disease first enters a population because s t ≈ 1. this implies that each infected agent infects approximately αγ κ new agents in the population. this fraction is sometimes referred to as the initial reproduction number in the population and is commonly denoted as r 0 ≡ αγ κ . without new individuals entering a population in the susceptible state this reproduction number can only decline as the infectious disease spreads. a successful vaccination moves an agent from state s to state r without incurring the costs of infection. if we reduce the initial population of susceptible individuals, s 0 , by enough we can push the population below the epidemic threshold. specifically, if s 0 < κ αγ then the infectious disease dies out of its own accord without further action. thus an epidemic is prevented whenever s 0 < κ αγ which occurs when (1 − κ αγ )n agents are successfully vaccinated. vaccinating enough agents to produce this effect is called herd immunity (smith 1970) ; once enough people are vaccinated, the entire population (herd) is effectively protected without everyone being vaccinated. the question then becomes, given a cost of vaccination, c(v), what is the efficient level of vaccinations to provide in a population and how do we obtain this efficient level? we begin to approach this question by introducing standard value function notation. in this initial model, once an agent enters state r she remains there forever. thus the value of being in state r is simply the lifetime discounted utility received in state r. we also introduce the possibility of having heterogenous contacts at this stage by indexing agent j's contacts (γ j ) and other terms that we allow to vary across agents. where u j ( ) = utility of agent j from the specified state and β is the discount rate. if an agent is in state i, she will remain in state i for 1/κ periods, on average, until recovered and then enter state r. if an agent is in state s, she receives the same utility as she would if she was recovered, unless she becomes infected. the value to an agent of being in state s is the value of being in state r less the product of the probability that the agent becomes infected and the cost of the infected period. where π(γ j , α, i) is the probability of becoming infected over the course of the epidemic as a function of the contacts of the agent and the transmission rate of the infectious disease. the cost of being infected is the difference in utility between states s and i during the time spent in state i , with the value functions specified we can now specify the vaccination problem for the individual and the social planner. to simplify the vaccination choice of individuals we assume that an agent can only be vaccinated at time period 0. at this time the agent will choose to be vaccinated if the value of being in the recovered state less the cost of the vaccination is greater than the value of being in the susceptible state. thus the agent will choose to be vaccinated if which implies the agent will choose to be vaccinated if c(v) < π(γ j , α, i)c j . the social planner's vaccination problem is more difficult than the individual vaccination problem. essentially, the social planner's problem is to vaccinate agent j if the cost of the vaccination is less than the expected dis-utility of the increase in infections created by agent j if agent j is infected weighted by the probability that agent j is infected. we define the term "marginal infections" of agent j to be the additional infections that occur if j is infected that would not occur if j was not infected. note that this is not simply the number of infections that agent j creates. as an example, agent k maybe infected by agent j. but, if k would have been infected by another agent had she not been infected by j then this would not be a "marginal infection" of agent j. k will be infected regardless of the vaccination choice of agent j. along these lines of thinking, measuring marginal infections is a difficult problem for epidemiologists for at least three reasons: 1. as mentioned earlier, many infectious disease transmissions are not observable. thus it is not easily known how many infections a given agent causes. 2. even if transmissions are observable, the marginal infections created by agent j are not simply the number of other agents that j infects. this is because some agents that j infects may get infected by agents other than j even if j does not infect them herself. further one needs to know how many additional agents are infected by those that j infects and any additional infections created by these agents and so forth. thus one needs to know information on the dynamics of the entire epidemic to measure the true marginal infections of a given agent. 3. the marginal value of vaccinating an agent depends on the behavior and vaccination choices of other agents. it eventually must be decreasing in the number of other vaccinations that are performed. in the extreme, if there are enough vaccinations in the population to produce herd immunity the marginal value of vaccinating an additional agent only involves the probability that the agent is infected from outside the agent population. in effect, the only value is preventing a single agent from infection because she will not, on average, infect anyone else. because of these difficulties we use a simulation approach to help us measure the average and marginal effects of individuals belonging to different worker groups in our hospital contact data. with simulations one can monitor the various infections that occur and also perform controlled experiments to sort out the effects of various groups on potential hospital epidemics. define m j (γ j , α, κ, i, v) as the true marginal infections created by agent j if infected, where v is the number of agents vaccinated in the population. for the majority of the paper we will suppress the notation that does not differ across agents and simply refer to marginal infections as m j (γ j ) since the primary focus of the paper will be on the effect of heterogeneous contacts on the spread of infectious diseases. as shown in boulier et al. marginal infections may be increasing in v for sufficiently small v. 2 but, marginal infections must eventually decrease in v; at the extreme, marginal infections are 0 for any level of v above the point at which herd immunity is reached. thus marginal infections may be increasing or decreasing in the number of vaccinations depending on the specifics of disease transmission, contact patterns, and the number of vaccinations performed. we can now state the social planner's vaccination problem. vaccinate agent j if: note that the individual and social planner's vaccination problems differ by the term π(γ j , α, i)m j (γ j )c j (i). this is the positive externality created by a vaccination when a vaccinated agent j protects other agents which he contacts from acquiring the disease from him. the key terms to investigate in this externality are the probability agent j gets infected, and the marginal infections created by agent j if infected. note that if these marginal infections, m j (γ j ), go to 0, the social planner's problem and the individual problem converge, and the externality is removed. similarly the externality is removed if enough vaccinations are performed to reach herd immunity, again because m j (γ j ) = 0 when herd immunity is achieved because the epidemic never occurs. the main focus of this paper concerns the network position of hospital workers. as such, we assume throughout the paper that the cost of an infection is equal across all agents, c j (i) = c k (i) for all j and k. further, without loss of generality, we can normalize c j (i) = 1. thus the externality above is the product of the probability of infection and the marginal infections produced by the agent if infected. one question then emerges: how are π(γ j ) and m(γ j ) related? at a simple level, if contacts only vary in degree, that is if the only difference in contacts between two agents is the number of contacts and not other, qualitative, aspects of the contacts, then you would expect π(γ j ) and m(γ j ) to be highly correlated. if an agent has a high likelihood of being infected because he has many contacts then he also has many contacts to pass on the infection. suppose that each agent pair is connected with a fixed probability. then, by chance, some agents will have a higher than average number of contacts and some a lower than average number of contacts. in this case, any agent who has a large number of contacts will also generate a large number of secondary infections since there is a lack of structure within the network population. thus any agent with a high probability of infection, π(γ j ) will also be expected to generate a high level of marginal infections m(γ j ). example one is fairly direct. however, various relationships are possible as we show below. in this case each agent in a population is directly connected to every other agent in the population. if this is the case, then any agent that becomes infected is directly tied to all other agents and can infect anyone in the population. thus once someone is infected each agent has a high probability of becoming infected (either from the original agent or from secondary infections). but, since the first agent contacts everyone in the population, and many agents will be infected from him, the other agents in the population may have a low m(γ j ). thus it is possible to have a high probability of infection π(γ j ) and low marginal infections generated m(γ j ) from the same agent. imagine that there are three groups of agents in a population. two of these groups, call them a and b, are separate fully connected graphs containing equal numbers of agents, who do not have any connections to the other group. in other words an agent a ∈ a is connected to every agent a ∈ a but no agent a ∈ a is connected to any agent b ∈ b. suppose that group b is formed in a similar manner. the third group is composed of one agent, j. agent j has only two contacts: one to agent x ∈ a and one to agent y ∈ b. in this example it may be unlikely that agent j gets infected, especially if there is a low transmission rate, because he only has two contacts in the populations. but, if agent j is infected, he may be integral to spreading the disease to the second fully-connected group. suppose an agent in a becomes infected and subsequently infects agent x (or any of the other agents in a) as well as several other agents in a. agents in group b are safe from infection as long as agent j is not infected. but, if j becomes infected, then it is possible that a large fraction of agents in b may become infected as well. thus agent j may have a low probability of being infected, π(γ j ), but create a large number of marginal infections, m(γ j ), if he does become infected. note that each of these three examples offer different implications for public policy approaches to encouraging vaccinations. in the first example, each agent has a probability of being infected that is in line with the number of marginal infections generated. in the other two examples, the infection rate and the number of marginal infections generated may not have a simple relationship with each other. this is an important observation if one considers using subsidies or other means to encourage increased vaccination rates. in the remaining portion of the paper we examine a data set on contacts of and between healthcare workers and patients in a large hospital on the university of iowa campus. we discuss the data and use agent-based models to identify the healthcare workers whose position in the hospital contact network has the potential to create large numbers of infections in the hospital. observational data on contacts between healthcare workers and patients was collected during the winter and early spring of 2006-2007 (the 2006-2007 "flu season") at the university of iowa hospitals and clinics (uihc). the uihc is a 700-bed comprehensive academic medical center and regional referral center in iowa city. data were collected by randomly selecting uihc employees from each of 15 job classifications (specified below) and then using research assistants to "shadow" the 140 selected employees. the research assistants manually recorded every human contact of the subjects (within approximately three feet) over a work shift. note that these observed contacts include anyone contacted within the hospital, not just with the other workers in the shadow sample. this resulted in a total of 6654 recorded contacts over 640 h of observation. additionally, the ra recorded the worker or patient group category for each observed contact (patient or category of healthcare worker) in our data set, and the location in the hospital where the contact occurred. 3 the job categories and number of observed subjects in the data set are as follows: floor nurse (8), food service (11), housekeeper (8), intensive care nurse (8), nurse assistant (10), pharmacist (8), phlebotomist (10), physical/occupational therapist (9), resident/fellow/ medical student (8), respiratory therapist (11), social worker (8), staff physician (11), transporter (7), unit clerk (9), and x-ray technician (14). the data for each group contain approximately 40 h of shadowing. the data were summarized into tallies of contacts over 30-min intervals and then aggregated into contacts per 8 h shift by the authors. table 1 lists the average number of non-repeated contacts 4 per 8 h that occur between the various worker (and patient) categories. note that we were not allowed to choose patients as subjects in our shadow data directly because of privacy concerns. we were only able to observe patient contacts as a result of shadowing other members of the hospital. thus they do not appear as a row in the table. we use this contact data to model the spread of an infectious disease across the uihc hospital. with this data we create a probabilistic contact network for the hospital worker groups. the network is constructed to match the distribution of worker groups at the university of iowa hospitals and clinics. this totals 5232 employees. the distribution of workers across the categories is given in table 2 . we create a contact network among these agents. in the model, each worker in a given group connects to other workers according to the rates observed in our shadowed subjects given in table 1 . as an example, all floor nurses in the model create 11 contacts to other randomly selected floor nurses on average, 0 contacts to food service workers, etc. we assume that the contacts are symmetric in our model, that is, a contact from a given floor nurse to a given housekeeper is also a contact from the housekeeper to the floor nurse. there are at least two reasons for this assumption. first, if our subject is in close enough proximity to pass on the influenza virus to a second agent, the second agent is also within close enough proximity to pass on the influenza virus to our subject. thus the ability to acquire or to pass on virus is a symmetric relationship. second, the reader may note in the table that the matrix of observed contacts is not symmetric because of randomness in the observation of subjects. for instance one notices that the subject floor nurses were not observed to contact food service workers, but a small number of food service worker to floor nurse contacts were observed. thus by assuming that all contacts that occur in the matrix are undirected, we create a symmetric contact matrix where the total number of contacts from a member of group x to group y (and from group y to group x) is one-half the sum of the observed average contacts from group x to group y and from group y to group x. we create the contacts in a uniform random manner within groups. let ρ i j be the ratio of the average contacts between a member of group i and j (taken from table 1 ) to the total number of group j employees (taken from table 2 ). we then take each pair of employees across each group and create a contact with probability ρ i j . specifically, let agent a be a member of group i and agent b be a member of group j. then the probability that a and b are connected is average number of contacts observed between members of groups i and j and n j is the number of employees belonging to group j. 5 before moving to the computational model, we mention two limitations of our data set. human contact networks frequently have a small number of individuals with a much larger than average number of contacts, perhaps differing by orders of magnitude. these individuals are often called hubs. these hubs have the potential to significantly influence disease transmission because they are highly likely to be infected, and if so, to pass on infections to a large number of individuals. because our sample includes approximately 2.5 % of the hospital worker population, we may be missing hubs in our sample if they exist in this setting. however, we note two things in relation to this: first, the relatively homogeneous workday responsibilities of workers within categories, likely limit the variation of contacts within a category. for instance two physicians or two floor nurses are likely constrained to see a relatively similar number of patients each day. this is unlike many other social network data sets, (such as general friendship or online networks) where there are not these work responsibility constraints on individual contacts. thus the possibility of hubs with orders of magnitude differences in numbers of contacts is more limited in our data context. second, our data set is more comprehensive than any other within hospital contact data set in existence in terms of the worker categories included. recall from earlier in the paper that the ueno and masuda data set only includes physicians, nurses, and patients in a hospital much smaller than is studied here. our results below suggest that many of the most important groups in the hospital are not included in their study. thus, at a minimum, our study highlights the importance of funding for studies that aim to collect even more comprehensive data sets that include individuals with nonpatient care responsibilities and more comprehensively cover a larger share of hospital workers. in addition, we note that we consider all contacts in our data set to be equivalent, or "equally weighted." there may be some concern that not all contacts are created equal in our context. for instance, a contact between a physician and a patient that occurs during a physical examination, may be more likely to spread an infectious disease than other contacts in our data set. we attempt to control for this possibility later in the paper when we consider heterogeneous transmission rates and repeated contacts. as mentioned above, transmissions of infectious disease are not usually observable. thus studying infectious disease transmission using an agent-based model can be a useful tool. in the remainder of the paper we model the spread of an infectious disease across the simulated hospital contact networks described above. once created, we use the contact network in a model of the spread of an infectious disease in the hospital as follows: agents can be in one of three states, susceptible to being infected (s), infected and able to infect others (i), or recovered (and therefore immune) (r). we assume that each infected agent recovers after 5 periods which is in-line with the infectious period for influenza. once recovered the agent enters state r and is therefore immune to further transitions to the infected state. initially, all agents in the model are in state s. agents may be vaccinated against infection. vaccinations occur only in the initial period of the model. once vaccinated, an agent moves immediately from state s to state r and is thus immune for the remainder of the model. in the initial period of the model, each agent in state s (all agents that have not been vaccinated) is subject to infection with probability α 0 = 0.005. these are the agents of our model that seed the potential epidemic. once these initial infections occur we assume that each contact in our network occurs once in each subsequent period of the model. if a contact occurs between a state i and a state s agent, the state s agent transitions to state i with probability α, which we vary across experiments. we continue the model until no agents remain in state i. in each period of the model we calculate the fraction of agents in each worker category in each state (s, i, or r). for each of the results reported below we run 100 replications of each parameter set or computational experiment reported. the results reported are averages over these replications. in addition to the results reported below, we have studied a wide range of parameters for our model and find the results reported below to be robust to changes in all of the parameters within reasonable bounds. 6 the purpose of the model is to estimate m(γ j ) and the externality generated for the network of contacts in the uihc shadow data and, in turn, to identify the classes of workers most important to vaccinate. this is a two step process. first we perform a series of base-line models as described above with none of the healthcare workers and patients vaccinated. from this baseline, we observe the rate of infection for each class of agents in the hospital population (15 worker groups and patients for a total of 16 groups). we denote the infection rate of group k in the base model as a function of the transmission rate α as π k b (α) and the overall infection rate in the entire population as a function of α as π 0 b (α). second, we want to calculate the average and marginal infections generated by each group. to do so, we change the vaccination rate for each group, one group at a time, and re-run the model. as an example, we run the model with all floor nurses vaccinated and no other vaccinations and observe π 0 1 (α). then we run the model with all housekeepers vaccinated and no one else and observe π 0 2 (α) and so on for each group. we then compare the change in the average number of infections between the models, δ(b, k) = (π 0 b (α)−π 0 k (α))n , which is the difference between the overall infection rate in the base model with no vaccinations and the overall infection rate in the model with all of group k vaccinated, multiplied by the total population size n . now, using the notation described above, the change in the number of infections δ(b, k) is equal to the change in number of people vaccinated, n k , multiplied by the probability that each of these agents becomes infected if not vaccinated, multiplied by the number of additional infections each infected agent would generate. simplifying, if we assume each agent infects the same number of individuals, we can write the average number of secondary infections generated per infected person in group k, as a k and write: one can then find an estimate of the average secondary infections per infected person as:â effectively, this process removes each group from the hospital, one at a time. we then can observe the effect of each individual worker group on the size of the modeled epidemic. instead of vaccinating all agents of a group at once, we can vaccinate a fraction of a group. as we change this fraction at intervals n k we can view the effect of increases in vaccination rates for each group (one at a time). we then have an estimate of the marginal infections prevented per vaccination as: we now proceed in two steps. first we investigate the effect of each group in total on the epidemic process by vaccinating an entire group one at a time and calculatinĝ a k for each group. we then choose a sample of interesting groups and study the details of the epidemic process as we vary the number of vaccinations performed in each of these groups, at specified intervals between 0 and 100 %. interestingly, as we vary the percent of each group vaccinated, we will see that there are different outcomes across these different groups in terms of marginal infections generated, probability of infection and the overall effect of a vaccination (in terms of reducing the number of infections). we begin by varying the transmission rate, α, over the range [0.0004, 0.006] and observing the base infection rate π 0 b (α). the results are displayed in fig. 1 . as one can see in the figure, a sufficiently large transmission rate is needed to generate an epidemic of reasonable size. further, as expected, the number of infections generated monotonically increases as a function of the transmission rate, α. our primary interest is in intermediate ranges of epidemic outbreaks. if the transmission rate is too high then almost everyone in a population needs to be vaccinated in order to reach herd immunity. and, if the transmission rate is too low, then there is not a large need to worry about vaccine priority. thus, we concentrate on two intermediate levels of the transmission rate α = 0.0030 and α = 0.0035. with no vaccinations, these levels yield an epidemic where between one-third and one-half of the population is infected over the course of the epidemic. we now find the average effect of vaccinations across the hospital worker groups using the procedure described above for α = 0.0030 and α = 0.0035. we present results for the average "secondary infections" generated per infected person,â k , and the product of average infections and probability of infection, which yields the "decrease in infections per vaccination," δ(b,k) n k in tables 3 and 4 . from the decrease in infections per vaccination we have an indication of how much the vaccination of an individual group member is contributing to preventing the spread of an epidemic. the results of this experiment suggest where efforts should be directed in the event of an influenza vaccine shortage or in the event of the development of new disease for which a vaccine may be developed (e.g., avian flu, swine flu, etc.) but is initially in short supply until mass quantities may be made available. note that some of the groups have vaccinations that prevent less than one infection per vaccination. this occurs because these groups have a low probability of infection and sufficiently low number of average infections that each member generates if infected. groups with large decreases in infections per vaccination are the ones to prioritize in times of a vaccine shortage, assuming equal costs of infection. in these experiments we see three clear groups that stand above the others in terms of the effect of vaccinations. for the parameters of the experiments, each vaccination of a unit clerk, social worker, and phlebotomist, results in a decrease of 3.1 infections or more on average for α = 0.0030 and of 2.2 or more for α = 0.0035. 7 in addition vaccinating unit clerks is extremely effective; each unit clerk vaccination results in a decrease of over 7 infections for α = 0.0030 and over 6 infections for α = 0.0035. somewhat surprisingly, some of the groups that are seen as central to the functioning of a hospital play a very small or moderate role in spreading an infectious disease. vaccinating staff physicians results in a lower than average decrease in infections. we revisit this result in our discussion of transmission rates later in the paper. also of note, as one would expect, as the transmission rate increases, the probability of infection increases. but, this has the effect of making individual vaccinations less beneficial on average. note thatâ k is smaller for each group for a higher transmission rate. this has the effect of lowering the variance of average infections. for the α = 0.0030 case above the standard deviation is 1.79, and for the α = 0.0035 case, the standard deviation is 1.41. as the transmission rate increases, a larger fraction of individuals are infected throughout the population. thus there are more opportunities for each individual to be infected if she has not already been infected. vaccinating a given person in the population will only prevent one of these multiple channels for infection. so, as the infection rate increases, the effectiveness of a vaccination becomes more uniform across the groups. this has direct policy applications. an infectious disease that is highly contagious could best be met with a uniform vaccination strategy since each individual in the population will create a similar level of infections on average. but an infectious disease with a low level of contagiousness could most effectively be met with a targeted vaccination campaign (bansal and pourbohloul 2007) . we next look at the marginal effect of a vaccination as the number of vaccinations increase. we present results in figs. 2, 3, 4 , 5, 6 and 7 for five interesting worker group categories for the same two transmission rates discussed above. unit clerks, social workers and phlebotomists are chosen because of their large number of secondary infections generated. we also choose floor nurses and staff physicians because of interest in the effect of worker groups with primary patient care responsibilities. in fig. 2 we plot the marginal infections prevented per vaccination as a function of the number of vaccinations performed for the five groups. recall that the number of marginal infections is the additional number of infections that an agent generates if the agent becomes infected. in fig. 3 we plot the probability of infection for the five groups. and, in fig. 4 we plot the product of marginal infections and probability of infection which yields the total number of infections prevented per vaccination. these figures all consider a transmission rate of 0.0030. figures 5, 6 , and 7 plot the same relationships for a transmission rate of 0.0035. we begin with marginal infections. recall that marginal infections may be increasing or decreasing in the number of vaccinations performed for small numbers of vaccinations (boulier et al. 2007 ). (here the number of vaccinations performed is small relative to the entire population as we are only vaccinating some members of one of the 15 groups. so, even if we vaccinate an entire group, this is a small number relative to the entire population.) here, we see two interesting outcomes. first, we see that marginal infections for both unit clerks and floor nurses increase as more vaccinations are performed. for these two groups, each additional vaccination prevents a larger and larger number of infections. this is particularly extreme for a transmission rate of 0.0030 and the case of unit clerks where a small number of vaccinations results the product of the two previously discussed statistics yields the number of infections prevented per vaccination. again, unit clerks display a unique relationship in that the number of infections prevented per vaccination dramatically increases in the number of vaccinations performed. the other four groups result in much flatter plots. thus again there is little difference between the marginal and the average for these groups. there are two interesting points to be made from these results. the first is that the effect of vaccinating unit clerks in our data is most important both from a marginal and an average perspective regardless of how many vaccinations have been performed. particularly, the marginal effect of vaccinating a unit clerk increases at a greater rate than the probability of infection for a non-vaccinated unit clerk falls. thus, it is always more beneficial to vaccinate one more unit clerk as opposed to a worker from another group (assuming transmission rates are equal). the second is that there is little difference between the average and the marginal effect of a vaccination for the other groups considered here. this second point can be interpreted as good news from a policy making perspective in the sense that the optimal allocation of vaccinations does not switch as more of a group is vaccinated. in other words it is not the case that group a is the optimal group to target up to some vaccination percentage, after which group b should be targeted. switching such as that would indicate a much more complicated solution to the optimal vaccine allocation problem. here, because there is little difference between the marginal and the average effect of a vaccination for most groups, one can pragmatically target the groups with the largest average effect of a vaccination. we now move to discuss the important features of the contact network that creates the externality. as we will see below, it is not just the number of contacts that an agent has but also which specific agents and groups the agent contacts, as well as who the agent's contacts connect to in turn. we begin by looking at some basic statistics of the contacts in our data in table 5 . for each group, the table displays the total number of contacts, the percentage of total contacts that are with members of an agent's own group, and the number of patient contacts. total contacts and contacts with patients could be directly correlated with the likelihood of being infected and with passing on infections. the percentage of contacts within one's own group can indicate how varied one's network is and how widespread one's connections are. for instance having few contacts within one's own group provides the possibility of introducing an infection to other groups within the hospital. in addition the table also contains a common network characteristic measure, betweenness centrality, which we discuss below. on a network, the geodesic distance, g a,b , between nodes a and b is the length of the shortest path between the two nodes that traverses connections on the network measured as the number of connections traversed (or "hops" required) to reach node b from node a. as one measure of the centrality of a node on a graph one can calculate the average distance to all other nodes on the graph. thus if a graph g is composed of n nodes, average distance for node a is calculated as: a short average distance indicates that a node is close to other nodes on average an thus may be likely to be infected and to pass on infections. thus it is sometimes considered a measure of the centrality or importance of a node in a network. betweenness is another measure of the centrality of a node in a network. let c i jk be the proportion of all geodesics linking node j and node k which pass through node i. let c i be the sum of all c i jk for i = j = k. letc i be the maximum possible value for c i . (normalized) betweenness for node i, b i is then: betweenness for node i is therefore a measure of the proportion of shortest paths between nodes that go through node i. 8 in the the table below we report group level values for betweenness centrality. the values are created in the following manner. first we create a network using the same methods as described above. second, we then calculate the betweenness centrality measurement for each agent in the simulated network. third, we calculate the average value for each hospital worker group and report the result in the table below. this network variable is likely to be an indicator of importance for disease transmission in the network. if a hospital worker group has a high average betweenness value, then the nodes in this group are potentially important in passing infections on to other nodes as it plays a crucial role in location along many of the shortest paths between nodes in a network. as such, this measure should be closely related to the marginal infections that a group generates. what is most interesting in table 5 is the lack of a clear relationship between any of the variables in the first three columns and our previously-listed most important groups (highlighted in bold). the only measure that consistently aligns well is the betweenness measure. for a moment concentrate on the values in the first three columns. each of these three plausibly important characteristics fail to display a meaningful relationship with the average or marginal infections generated. if we concentrate on the top three most important groups, some have relatively large numbers of contacts (unit clerks), although not the largest, while others have contacts significantly below the average (phlebotomists). some have large numbers of patient contacts (phlebotomists) while others have some of the smallest number of patient contacts (unit clerks and social workers). one interesting thing that appears in the table is that phlebotomists have almost all of their contacts with patients and other phlebotomists. thus the network position of phlebotomists, is in some sense, very similar to that of patients. overall, what these observations imply is that there is not likely to be a simple relationship (or a small set of simple relationships) indicating which individuals are most important to vaccinate by looking at easily observed worker interaction patterns. instead the relationship depends on the intricate and complex web of relationships that make up the entire contact network of the hospital. this is what is captured in the betweenness centrality measure. betweenness measures the percentage of shortest paths in the entire network on which an agent is located. if we remove agents with high betweenness measures (by vaccinations) from the disease propagation network, we disrupt the flow of an epidemic. for concreteness, the correlation between the betweeness measure and the average infections generated is about 0.84 for both α = 0.0030 and α = 0.0035. as mentioned above, the primary focus of this paper concerns the effect of network position on the marginal infections generated within a hospital. here, we consider two robustness checks to the results presented above. first, we discuss a comparison of the magnitude of the above described network effects and the effect of transmission rates on marginal infections for an interesting example group, staff physicians. second, we do an additional set of experiments using an additional data set that includes observed repeated contacts. we perform these robustness checks for two main reasons: first, because of the job responsibilities of different worker groups, the different worker groups may have different transmission rates, durations of contacts, or frequency of contacts creating another source of heterogeneity in infections. as a few examples, a staff physician may be more likely to transmit an infection over the course of a patient exam that includes a series of physical contacts, compared to a nurse who has a brief arm's-length conversation with a hospital transporter. a floor nurse may have multiple interactions with the same patient during the course of the day. 9 we begin this analysis by varying the transmission rate of staff physicians. (recall that staff physicians created a lower than average number of infections in our earlier model.) again, this is primarily to account for the fact that physicians may have longer duration contacts with patients and the contacts may more frequently involve physical touch. we now assign a special transmission rate to staff physicians that we denote as α p . this will be the transmission rate for any contact between a physician and another agent where one of the agents is infected. we use α = 0.0030 for all other contacts in the population and vary α p from α p = 0.0030 and α p = 0.0200. as we do this we again measure average infections as described earlier and show the resulting average infections for unit clerks and staff physicians in fig. 8 . before we present the results we note that there are alternative ways to model this scenario. for instance, we could re-weight our contact matrix. if we knew, for instance, that physician to patient contacts lasted twice as long as other pairs of contacts in the hospital worker population, we could re-weight each physician to patient contact by a factor of two. but note that, on average, this is equivalent to increasing the transmission probability by a factor of two because the expected number of new infections is the number of susceptible to infected contacts in a period multiplied by the transmission rate. a doubling of the number of contacts is equivalent to a doubling of the transmission rate. 10 recall from our results above that when the transmission rates are equal the average secondary infections created by unit clerks are slightly greater than 8 and slightly greater than 1.5 for staff physicians. as we increase the transmission rate for staff physicians we see several things: first, as you would expect, the average infections for staff physicians increases. but the change is not large. for α p = 0.0200 the average secondary infections created by staff physicians is 2.86, slightly less than a two-fold increase and still well below the level of average infections noted for unit clerks when the transmission rates are equal. for unit clerks, as α p increases, the average infections of unit clerks drops rapidly. this occurs for at least two reasons: one is that unit clerks become less important relative to staff physicians as α p increases. another is that, as α p increases, the overall infection rates increase, and as we reported earlier, this causes average infections to become more uniform across groups. overall, the level of average infections between these two groups does not become similar until the α p increases to about 0.0150, a five-fold increase in the staff physician transmission rate. and, the average infections of staff physicians does not become greater than that of unit clerks until α p = 0.0200. most interesting about these results is the magnitude of the network effects relative to the magnitude of the transmission rates. in the case of staff physicians and unit clerks it takes a 5-6 times increase in the transmission rate of staff physicians to "make up" the difference in network position. this suggests that the network effect differences in average infections are very important in understanding overall transmission patterns. as a second robustness check we use an additional cut of our data that includes observed repeated contacts. in some instances members of the hospital population were observed to have come in contact more than once during the observation period. we use this data as one way to include the weighting of contacts mentioned above into the analysis. table 6 displays the observed contacts that were observed and had occurred previously in our data. as you see in the table many of these contacts involved repeated interactions with patients (often by members of the nursing staff). we re-perform the analysis above with these additional contacts added into the data. the only additional change is that we modify the transmission rate to α = 0.0020 so that the total number of infections in the population remains nearly constant compared to the non-repeat contact data. (recall from earlier in the paper that a larger epidemic smooths out differences in the population groups and makes the average and marginal values more similar across groups. thus we control for epidemic size by varying the transmission rate.) we present the results in table 7 . you will notice in the table that most of the groups that previously had the largest effect still do. unit clerks are still the most important group to vaccinate, but the difference in magnitude between unit clerks and other important groups is less than in the the previous model. further, as one would expect, groups that have more repeated contacts, such as all types of nurses, become more important. the important point though, is that the relative ranking of most of the groups changes very little. of course this is partially due to there being relatively few repeated contacts in the data set. taking these two robustness checks in combination, they demonstrate two important things. first, network structure is at least as important as transmission rate in determining the course of an epidemic in our data set. second, while the data we have collected is not perfect in terms of comprehensiveness, the relative ranking of group importance appears relatively robust to alternative measures of network structure. a a minimum, taken in combination, these results suggest a need for a greater emphasis on network based data collection in order to better understand both micro level and macro level epidemiology. we now make a small shift in focus. generally, a hospital's primary goal is to restore or improve patient health. thus prioritizing healthcare worker vaccinations so as to best protect patients may be a legitimate goal of a hospital. in other words hospital administrators may care about protecting patients from infection as much, or more, than they do about protecting the entire hospital population from infection. of course these may be closely related goals. in addition, in a large scale epidemic, it may be of great importance to have a healthy staff of physicians to treat patients. thus protecting physicians may be another important goal in vaccine priority within a hospital. in tables 8 and 9 we display the same relationships as displayed in our initial results section above but this time only with regard to patient infections generated and staff physician infections generated, not infections in the entire hospital. in this analysis we see very similar results to the overall population results. beginning with patients, the four groups (unit clerk, social worker, physical and occupational therapist, and phlebotomist) that played the most important role in transmitting to the hospital population as a whole also play an important role in their effect on patients specifically. however, we see some difference in the two groups of models. first, groups that have more direct patient contacts increase in importance. for instance, phlebotomists replace unit clerks as the most important group. second new groups emerge as important for transmitting to patients. for instance, hospital transporters are among the top four groups in transmitting to patients but are significantly below the average in terms of transmissions to the general population. with this in mind it seems that giving vaccination priority to health care workers with direct patient contacts is more important for protecting patients than it is for protecting the general population, as one would expect. but still, some of the groups with the largest impact on infecting patients have few direct patient contacts (unit clerks and social workers, for example). for staff physicians we see similar results. again, the four most important groups remain important, but other groups such as floor nurses increase in importance when considering staff physicians specifically. to summarize the results of this section, the same groups that create infections in the general population also create infections in the patient and staff physician population. but, groups that have direct patient or staff physician contacts have increased importance. still, one should not ignore other groups central to the network of the hospital that have only few direct patient or staff physician contacts (e.g., social workers and unit clerks). we utilize a newly collected data set on contacts of health care workers at a large university hospital to estimate network effects for infectious disease transmission. interestingly the most important groups to vaccinate tend to have heterogeneous contacts throughout the hospital. groups such as social workers and unit clerks are very important to vaccinate even though they have been given low priority in past vaccine campaigns because of their relatively limited number of patient contacts. for instance, the cdc recommended in their interim influenza vaccination recommendations in 2004-2005 that influenza vaccine priority be given to "health-care workers involved in direct patient care" and further stated, "persons who are not included in one of the priority groups described above should be informed about the urgent vaccine supply situation and asked to forego or defer vaccination." this mismatch of scientific results and past policy decisions suggests that future research in this area is warranted especially when one considers the public health dangers associated with the emergence of avian flu, a more lethal version of swine flu, or recent dangers such as sars. with that stated, we want to be careful to recognize that one reason to vaccinate primary care providers is to assure individuals are available to care for the sick. this important incentive is outside the scope of our model. the results of this paper lead to important public policy considerations. specifically, hospital workers with a low probability of infection may be likely to ignore recommendations for vaccination even if they are central to the spread of an infectious disease. one way to increase the overall vaccination level is with a subsidy program. but, as the results in this paper show, not all hospital workers are equal in terms of the positive externality generated by a vaccination. because of the heterogeneous contacts throughout the hospital, some workers are more important to the spread of an infectious disease than others. thus if hospitals and other public health organizations want to efficiently distribute vaccines they need to target specific worker groups, perhaps by allocating subsidies, on the basis of discrepancies in probability of infection and marginal infections generated. this paper is the first to use specific micro-level contact data within a hospital to guide policy makers and public health officials in this endeavor. to be clear, these results are not meant to be specifically calibrated to measure the exact effect of vaccinations in these groups. instead our hope is that the orderings of the hospital worker groups (which are robust across the parameters that we have explored) indicate where public health officials can effectively intervene in order to prevent widespread epidemics within hospitals. and these experiments reveal interesting and surprising groupings. prior to this study, as quoted above, it had been argued that groups like unit clerks be excluded from influenza vaccine campaigns, in times of vaccine shortages, because of their minimal patient contacts. the results of this study suggest that decisions such as these need to be more fully explored. a comparative analysis of influenza vaccination programs article 23 cdc (2002) epidemiology and prevention of vaccine-preventable diseases, 7th edn. public health foundation prevention and control of influenza: recommendations of the advisory committee on immunization practice (acip) model parameters and outbreak control for sars the spatial dynamics of host parasitoid systems incidence and recall of influenza in a cohort of glasgow healthcare workers during the 1993-94 epidemic: results of serum testing and questionnaire optimal tax/subsidy combinations for flu season disease eradication: private versus public vaccination meta)population dynamics of infectious diseases selected aspects of the socioeconomic impact of nosocomial infections: morbidity mortality, cost, and prevention a contibution to the mathematical theory of epidemics epidemiology economic, diseases infectious prospects of the control of disease prevention and control of influenza: recommendations of the advisory committee on immunization practices, morbidity and mortality weekly report 55 mortality associated with influenza and respiratory syncytial virus in the united states controlling nosocomial infection based on the structure of hospital social networks perspective: human contact patter ns and the spread of airborne infectious diseases barriers to influenza vaccination acceptance. a survey of physicians and nurses key: cord-021770-zn7na974 authors: slifka, mark k.; amanna, ian j. title: passive immunization date: 2017-07-17 journal: plotkin's vaccines doi: 10.1016/b978-0-323-35761-6.00008-0 sha: doc_id: 21770 cord_uid: zn7na974 nan passive immunization, passive immunity, and passive immunotherapy all refer to the transfer of antibodies to an unprotected individual for the prevention or treatment of disease. the first formal demonstration of passive immunization for successfully treating diphtheria and tetanus dates back to animal studies published in deutsche medizinische wochenschrift (german medical journal) in 1890. 1 the technique was quickly adapted to clinical use and as early as the mid-1890s, diphtheria-specific antitoxin was used successfully in the hospital setting to reduce mortality during diphtheria outbreaks. [2] [3] [4] indeed, in 1901 emil von behring was awarded the first nobel prize for physiology or medicine for the discovery of this important medical intervention. 5 the significance of this clinical advance cannot be overstated; behring estimated that 45,000 lives were saved each year using diphtheria-specific passive immunotherapy in germany alone. 6 in the 1890s, the mortality rate of hospitalized cases ranged from 47% to 60%, 7 and the work of emil von behring and his colleague, shibasaburo kitasato, provided the only hope for diphtheria patients in the preantibiotic era. according to behring, the discovery of passive immunization would not have occurred if it were not for his earlier work that focused on characterizing the protective mechanisms of active immunization against diphtheria 5, 8 and through the work of his collaborator, kitasato, on the mechanisms of vaccine-mediated immunity against tetanus. 1 when guinea pigs were infected with corynebacterium diphtheriae, the animals routinely died of the disease. however, when behring vaccinated animals and they mounted neutralizing antibodies to diphtheria toxin, he found that they were protected from a normally lethal dose of c. diphtheriae. to determine if protection was now an intrinsic property of the immune host that could be transferred to a susceptible host, he injected naïve guinea pigs with diphtheria toxin and then successfully treated them with immune serum from vaccinated animals. likewise, injection of clostridium tetani or purified tetanus toxin was typically lethal, but through a method developed by paul ehrlich, 5 animals could eventually become immune to high doses of tetanus toxin by sequentially inoculating them with lower, nonlethal doses of tetanus toxin. kitasato used this approach to demonstrate that the blood of vaccinated, tetanusimmune rabbits could be transferred to naïve mice and fully protect them from a normally lethal dose of virulent c. tetani or from filtered c. tetani culture supernatant containing tetanus toxin. 1 behring and kitasato may have said it best in the final sentence of their landmark 1890 study, "the result of our experiments remind us forcibly of these words: blut ist ein ganz besonderer saft [blood is a very unusual fluid]." 1 technology has advanced substantially in the more than 125 years since behring and kitasato's first formal demonstration of protective passive immunotherapy. 1 in those early days, it was infeasible to use human immune serum to treat diphtheria, so the first large-scale production of polyclonal diphtheria-immune serum was prepared by vaccinating dairy cows. 5 to this day, commercial antisera used to treat a broad range of toxins are still produced in animals (table 8 .1). passive immunotherapy with animal-derived antibody preparations should only be used under close medical supervision 9 or the resulting host immune response to the foreign 8 immunoglobulins and serum proteins may trigger serum sickness, urticaria, and/or anaphylaxis following administration. fortunately, the advent of several innovative technologies that reduce the need for animal-derived antibodies have forged new paths in terms of safety, feasibility, and the protective efficacy afforded by passive immunization. following the discovery of monoclonal antibody technology, 10, 11 further refinements have been made, including use of various display techniques (e.g., phage display, yeast display) to screen large antibody libraries. 12 other technological advances include the development of chimeric monoclonal antibodies in which the murine antibody is "humanized" by genetically replacing the heavy chain region of the molecule with the human immunoglobulin counterpart and the use of transgenic mice in which the endogenous murine immunoglobulin genes have been replaced by human immunoglobulin genes. 12 this latter approach has the advantage that hybridomas from immunized transgenic mice produce fully human monoclonal antibodies without requiring further genetic modifications. recently, development of epstein-barr virus (ebv)-transformed human memory b cells for the production of monoclonal antibodies has led to yet another surge in the production of new human monoclonal antibodies with rare antigenic specificities to uncommon pathogens and these can be produced directly from immune human subjects. 12, 13 before the era of antibiotics, antibodybased therapy was the only option available for combating many bacterial diseases. even today, there are only a handful of antiviral drugs available and no therapeutic options exist for most viral diseases. however, new antibody-based therapies are continuing to be developed with the potential to provide protection against a broad array of bacterial and viral pathogens. in this chapter, we describe the role of passive immunity in the protection of the naïve host, discuss the parameters involved with successful immunotherapy, and provide examples of protective efficacy in animal models as well as in human clinical studies. age who were born to mothers who received pertussis vaccination during pregnancy, 30,31 thus lending further support to the current recommendations for the vaccination of pregnant mothers against b. pertussis. 32 the age limit of younger than 2 months was chosen as this is the age at which primary pediatric vaccination is recommended and analysis beyond this age might be confounded by the protective effects of direct vaccination of the child. nevertheless, the protection afforded by maternally derived igg against respiratory infections involving viral (e.g., influenza) or bacterial (e.g., b. pertussis) pathogens together demonstrate the broad impact that maternal vaccination and the subsequently increased transfer of maternal antibodies can have on the health of young infants. before vaccines and antibiotics revolutionized modern medicine, antibody-based therapies represented the only effective medical treatment for many life-threatening diseases including diphtheria, scarlet fever, bacterial meningitis, and bacterial pneumonia. 33,34 today, most commercial forms of antibodybased immunotherapy for infectious disease still rely on polyclonal antibodies of human or animal origin, with the notable exceptions of the monoclonal antibodies, palivizumab and raxibacumab (see table 8 .1). the main advantage of using polyclonal antibodies for passive immunotherapy is that this approach will include antibodies to multiple epitope specificities that may work in an additive or synergistic manner with the potential contribution of multiple immunoglobulin isotypes and subclasses that have different biological functions (table 8. 2). 35 on the other hand, there are several potential challenges to using polyclonal antibodies for immunotherapy including low antigen-specific activity, supply limitations (especially for rare diseases), variability between manufacturing lots, and safety as well as quality control issues that are often associated with the use of human blood products. in ungulates. these differences also indicate that care should be taken when choosing an appropriate animal model for studying the role of maternal antibodies against infectious disease as the mechanisms may be more species-specific than typically realized. in a comprehensive study involving the analysis of antibodies to 16 viruses using samples from 58,500 patients, the relationship between maternal immunity and infant immunity is clear (fig. 8.1 ). 15 the prevalence of antibodies to each viral antigen among infants less than 1 month old is remarkably similar to those observed in the 20-to 40-year old adults who represented the main age group of the mothers. for instance, immunity to common childhood diseases such as measles and mumps was comparable between newborns and their mothers. immunity to less-common viral pathogens, such as influenza b, was relatively low among infants and adults in the cohorts examined in 1971-1972, but higher among those sampled in 1973-1974,1975-1976, and 1977-1978 , coinciding with an influenza b epidemic that had occurred in 1974. 15 this shows that the prevalence of maternal antibodies is dynamic and that recent outbreaks involving a specific pathogen will result in a higher frequency of pathogenimmune mothers and a concomitant increase in the number of infants who are likewise bestowed at least transient immunity to that particular microbe. as expected, maternal antibodies wane rapidly during the first 6 months of life and then exposure to pathogens over the following months and years results in an accumulation of different antibody specificities as children reach adulthood (see fig. 8 .1). the overall protective efficacy of maternal antibodies is perhaps most pronounced among children with genetic immunodeficiencies such as severe combined immunodeficiency (scid), resulting in the lack of functional t and b cells or agammaglobulinemia, in which patients lack functional b cells while still having the ability to mount pathogen-specific t-cell responses. the clinical presentation of scid is not apparent at birth but relatively uniform diagnosis occurs at a mean of 6.59 months of age, 16 which is also about the age that maternal antibodies have reached their lowest levels 15 (see fig. 8 .1). likewise, agammaglobulinemic patients also begin to present with symptoms of immunodeficiency around this same age. 17 maternal antibodies represent an immunological "doubleedged sword" in the sense that they are known to interfere with live attenuated virus vaccines such as the mmr (measles, mumps, rubella) [18] [19] [20] and rotavirus vaccines, 21, 22 whereas direct immunization of mothers in the third trimester of pregnancy can significantly increase protection of infants against common respiratory viruses such as influenza. [23] [24] [25] indeed, maternal vaccination may result in a 45% to 91% reduction in influenzarelated hospitalizations among infants younger than 6 months of age. [23] [24] [25] likewise, the importance of maternal vaccination against bordetella pertussis (i.e., whooping cough) was recognized as early as the 1930s to 1940s with studies showing higher antibacterial antibody responses and potential protection from exposure to whooping cough among infants born to vaccinated mothers. [26] [27] [28] [29] recent studies verify these earlier results, demonstrating a 90% to 91% vaccine efficacy against whooping cough among infants younger than 2 months of nonlymphoid tissues and to penetrate mucosal sites of infection is likely to explain why it is often considered the best immunoglobulin isotype for routine passive immunization and has shown clinical benefit ranging from reduced clinical symptoms to nearly complete protection from lethal infection in a number of infectious disease models (table 8 .3). over the last century, it has been well established that high specific antibody titers and early timing of antibody transfer in relation to disease onset are the two most important parameters involved with determining the protective efficacy of passive immunization ( fig. 8.2 ). in one account of the early days of clinical diphtheria-specific immunotherapy developed by behring and ehrlich, 5 initial failures in patients after treatment with weak or unstandardized diphtheria-immune serum brought ehrlich to describe three points that he believed were important for successful immunotherapy: (a) treatment has to be initiated at the onset of disease; (b) the more the disease has progressed, the higher the serum quantities necessary for cure; and (c) depending on the severity of the case, certain minimal doses can be specified. later studies confirmed these results: if diphtheria immunotherapy was initiated on the first day of disease, there was 0% mortality (n = 183). 49 however, if therapy was delayed to 2, 3, or 4 days after disease onset, then the accompanying diphtheria case-fatality rate subsequently increased to 1.6% (n = 905), 4.4% (n = 632), and 6.9% (n = 436), respectively. 49 these results are similar to those observed during antibiotic-based therapy of bacterial sepsis. in an ideal setting, it is recommended that antibiotics be administered within 1 hour of diagnosis of severe sepsis or septic shock as these drugs provide clinical benefit only if administered early in the course of disease and are generally ineffective during late-stage disease. 50 the importance of high-dose immunotherapy given at the earliest sign of disease is not unique to bacterial anti-toxin therapy. the same rules apply to preventing or treating viral infections as well. during a measles epidemic in 1931-1932, 72% of exposed individuals (n = 32) who received no passive immunization contracted measles. if convalescent serum was administered within 10 days of exposure, then the attack rate was reduced to 16% (n = 219) whereas if therapy was not initiated until 12 to 16 days postexposure, approximately 80% of contrast, monoclonal antibodies are, by definition, limited to a single epitope specificity but they have several advantages over polyclonal antibodies since they can be manufactured in vitro at large scale, with inherently high specificity and lot consistency (table 8 .2). for example, the combination of 0.7 mg of two tetanus-specific human monoclonal antibodies has the same neutralizing capacity observed with administration of 100 to 170 mg of polyclonal tetanus immunoglobulin. 36 likewise, administration of 0.023 mg of a vaccinia virus-specific monoclonal antibody provides the same level of protection afforded by 5 mgs of vaccinia immunoglobulin (vig). 37 although neutralization escape mutants are a valid concern when using monoclonal antibody therapy, 38,39 this has not yet been a major problem during clinical use of palivizumab for respiratory syncytial virus (rsv). initially, sequencing of 371 rsv isolates demonstrated that there were no mutations in the neutralizing epitope of the f protein. 40 subsequent studies identified rsv escape mutants in approximately 5% of 146 breakthrough cases, indicating that selective pressure for escape mutations is still relatively uncommon under current conditions of use. 41 this suggests that monoclonal antibodies can remain effective when used clinically in the long-term, as long as they are specific for a stable epitope for that particular pathogen. the functional characteristics of the immunoglobulins used for passive immunization is an important consideration in determining protective efficacy in vivo. 35 for example, serum igg molecules equilibrate into extravascular space whereas igm is largely confined to intravascular space. 14 igm molecules also have a short half-life (5 days 14 ) and are typically of low affinity, which is why igm is not an optimal choice for passive immunotherapy. serum iga is monomeric and, although it also equilibrates into extravascular space, 14 it has only a 6-day half-life 14 and does not appear to contribute significantly to functional iga in the lungs of mice. 42,43 human igg on the other hand, has an average half-life of approximately 21 days (except igg 3 , which has a 7-day half-life), 14, 44 is typically of high affinity, and transudation across mucosal barriers can protect against pathogens that invade through mucosal routes. interestingly, serum igg (and serum iga) responses elicited in response to vaccination against neisseria meningitidis correlate strongly with the levels of antibacterial antibodies present in the saliva at 1 month and 1 year after vaccination, 45 indicating that circulating serum antibodies may be an important contributor to the antibodies released in mucosal secretions. indeed, after intravenous administration of an hiv-specific monoclonal antibody into rhesus macaques, serum antibody titers of 690 to 725 µg/ml resulted in mucosal antibody titers of 17 to 30 µg/ml in vaginal fluids and provide complete protection against intravaginal challenge with shiv (chimeric simian immunodeficiency virus expressing hiv envelope). 46 influenza virus is another mucosal pathogen with strict tropism to the respiratory tract, but influenza-specific serum antibody titers correlate with protection in humans. 47 in mice, the relative roles of influenza-specific polymeric iga and igg were compared in terms of antiviral protection in the upper respiratory tract versus the lung after influenza challenge. 42 when polymeric iga was transferred 4 hours prior to influenza infection, this prevented pathology in the upper respiratory tract but was not effective in the lung, whereas transfer of igg prevented pathology in the lung, but required higher doses to protect against infection of the upper respiratory tract. the authors concluded that different antibody isotypes may function preferentially at different anatomical sites in vivo. these results are in contrast to experimental influenza infection in humans in which inactivated influenza vaccinederived igg is believed to be a major contributor to protection of the nasal compartment. 48 overall, the ability of igg to enter efficacy of passive immunity decreases with disease progression. full protection from symptomatic disease is best achieved through prophylactic administration of antibody therapy prior to exposure or infection. however, antibody therapy may also be highly effective at early points postexposure, prior to the onset of disease symptoms. passive immunity is generally less effective when administered after the onset of symptomatic disease, and typically shows little to no clinical benefit once severe late-stage disease has occurred. to 16 iu/ml, the postexposure incidence of measles increased from 17% to 57% despite either lot being administered within 5 days of exposure. 53 likewise, the timing of passive immunotherapy is also important for enteric (e.g., polio) and respiratory pathogens (e.g., rsv). an outbreak in 1934 involving 2992 polio patients showed that if convalescent serum was administered within 0 to 2 days of meningitis, then paralysis was reported in 5.4% of patients (n = 2367). if treatment was delayed until 3 to 6 days after meningeal disease onset, 15.5% reported paralysis (n = 536), and if treatment was delayed for more than 6 days, then paralysis was noted in 30.3% of polio patients (n = 89). 54 for rsv, polyclonal rsv-immunoglobulin reduced the incidence of rsv-associated hospitalization by contacts subsequently contracted measles (n = 5). 51 in a study published in 1945 involving 1024 cases of measles exposure, 36% of the individuals who received immunotherapy within 0 to 2 days of exposure contracted measles compared to 48% for those whose treatment was delayed to 6 to 8 days postexposure. 52 the dose used in these studies was also critical: 67% of patients who received 0.01 ml/kg of gammaglobulin contracted measles whereas only 16% of patients who received 0.06 ml/kg of gammaglobulin contracted the disease. the titer of virus-specific antibodies will often differ between lots of polyclonal immunoglobulin preparations (see table 8 .2). in another study, when the measles-specific titer of gammaglobulin from different lots decreased from 33 iu/ml for animal studies, prophylaxis is defined as antibody administration prior to experimental infection and treatment is defined as antibody administration after infection. for clinical studies, prophylaxis is defined as antibody administration prior to disease onset and treatment is defined as antibody administration after disease onset. b evidence provided through maternal immunization studies. c anecdotal results are defined as small studies that indicate passive immunization may provide clinical benefit but are too limited in scope to be conclusive. d not available, although two studies 433,434 demonstrated prophylaxis by direct mixing of mumps virus and antibody prior to inoculation. confirmed lassa fever who received immune serum within 10 days of hospitalization survived (4 of 4; 100%). however, if treatment was not initiated until more than 10 days after hospitalization, then only 1 of 4 (25%) patients survived, similar to the untreated group in which only 1 of 5 (20%) patients with virologically confirmed lassa fever survived. although passive immunity against toxins and systemic infections such as measles 51,53,80-84 and smallpox [64] [65] [66] is well established, the impact of this approach for the prevention or amelioration of disease caused by respiratory and enteric pathogens may not be as well recognized. however, several studies support the role of passive immunity against mucosal pathogens (see table 8 . 3) , including examples such as influenza (respiratory virus), haemophilus influenzae (respiratory bacterium), rotavirus (enteric virus), and escherichia coli (enteric bacterium). influenza is a significant cause of morbidity and mortality throughout the world, including both seasonal transmission and pandemic outbreaks. [85] [86] [87] [88] the clinical correlation between homotypic influenza immunity and vaccine-associated protection was recognized early in the development of the influenza vaccine. [89] [90] [91] early animal studies confirmed this result, with passive transfer of antibodies (both systemic and mucosal delivery) able to protect naïve animals against subsequent challenge, or provide therapeutic benefit when administered postexposure. [92] [93] [94] more recent animal studies with defined monoclonal antibodies continue to support and extend these earlier results. 95,96 passive immunization against influenza in humans has also been successful. in a comprehensive retrospective metaanalysis of eight passive immunization studies performed during the spanish influenza outbreak (1918) (1919) (1920) (1921) (1922) (1923) (1924) (1925) , a significant 21% decrease in mortality (95% confidence interval [ci], 15-27%; p < .001) was observed. 97 subset analysis of studies that recorded early (treatment initiated within 4 days of pneumonia complications) versus late intervention (>4 days) showed a significant advantage for early treatment, with mortality decreasing from 59% (49 of 83) to 19% (28 of 148) with earlier intervention, consistent with general considerations for effective passive immunity against infectious diseases (see fig. 8 .2). in a recent double-blinded, randomized controlled study during the 2009 influenza pandemic, the use of hyperimmune intravenous immunoglobulin (ivig) (from recovered convalescent donors) was compared to normal ivig in the treatment of severe infection in 34 subjects. 98 the hyperimmune treated group (n = 17) demonstrated more rapid viral clearance than the control group (n = 17), with a greater than 90% drop in viral loads by day 5 posttreatment. in those patients receiving immunoglobulin within 5 days of symptom onset (n = 22), all 12 who received hyperimmune ivig survived (12 of 12), whereas only 60% of patients receiving normal ivig survived (6/10, p = .02). h. influenzae type b (hib) is an extracellular gram-negative bacterium that initially infects the host via the respiratory tract and represents another important human pathogen that can be controlled through passive immunization. several early reports described the use of concentrated rabbit immune serum as a successful adjunct therapy to sulfonamide treatment for patients suffering from hib meningitis. 99-101 indeed, a full course of serum therapy (in addition to antibiotics) was able to reduce mortality to 14% (3 of 19) when compared to 78% mortality rate (7 of 9) in those patients only receiving sulfonamides. 101 a more recent study established the prophylactic 41% among children with a history of prematurity or bronchopulmonary dysplasia. 55 prophylactic administration of a neutralizing monoclonal antibody, palivizumab, was shown to significantly improve clinical outcome by reducing rsvassociated hospitalizations of children with congenital heart disease by 45%. 56 among premature infants or those with bronchopulmonary dysplasia, rsv-associated hospitalizations were reduced by 55%. 57 a third palivizumab study confirmed these results by showing a 70% reduction in hospitalizations among premature infants and infants with chronic lung disease. 58 another monoclonal antibody, motavizumab, 59 demonstrated a further 26% relative reduction in rsv hospitalizations compared with patients receiving palivizumabbased prophylaxis. 60 in contrast, once rsv infection has been established, the use of palivizumab, 61 motavizumab, 59 or rsvimmunoglobulin 62 shows no clinical benefit, although rsvimmunoglobulin may provide limited protection in the most severe cases. 62 passive immunotherapy can be highly successful for severe, even life-threatening human diseases such as smallpox, or hemorrhagic fever caused by arenaviruses including junin or lassa fever virus (see table 8 .3). successful intervention, however, typically requires initiating treatment before or very shortly after symptom onset. when convalescent serum from smallpox survivors was administered to smallpox patients during the late stages of confluent or hemorrhagic smallpox, there was no clinical benefit observed in comparison to untreated controls (80% vs. 72% mortality, respectively). 63 when vaccinia-immune gammaglobulin (vig) was administered to smallpox contacts prior to disease onset in addition to postexposure vaccination (i.e., standard of care), the number of smallpox cases was reduced by 70% compared to contacts who received postexposure smallpox vaccination alone. 64 likewise, administration of vacciniaimmune serum of animal origin along with postexposure vaccination resulted in 0 of 13 cases (0%) of smallpox among close contacts compared to 13 of 29 cases (45%) among controls who received smallpox vaccination alone. 65 during a smallpox outbreak in 1941, 3 of 10 patients (30%) died while undergoing standard clinical care. 66 to determine if addition of passive immunotherapy would reduce mortality after smallpox diagnosis, 250 cases of smallpox were treated with convalescent serum or blood, with no smallpoxassociated deaths reported (0 of 250). approximately 75 patients were described as having severe or hemorrhagic smallpox at the time of treatment and yet all survived. this appears to be the result of using convalescent serum obtained at the peak of the humoral immune response shortly after recovery from smallpox and the use of an optimized dosing schedule with higher doses administered to patients with the more severe disease manifestations. 66 argentine hemorrhagic fever is caused by infection with the junin virus and untreated cases result in 15% to 40% mortality. [67] [68] [69] convalescent serum is protective in animal models of junin infection [70] [71] [72] and when administered within 8 days of symptom onset, the mortality rate among human cases drops to 1% to 3%. 68, 69 likewise, in 35% to 50% of hospitalized cases, lassa fever virus causes severe disease including diffuse capillary leakage and hemorrhagic diathesis. 73 prophylactic administration of immune serum protects guinea pigs 74, 75 and nonhuman primates 76,77 from subsequent lethal challenge, indicating that antibodies play a clear role in protection against this virulent viral pathogen. in one small clinical study, if passive immunotherapy was administered within 0 to 5 days after admission to the hospital, 4 of 4 (100%) patients survived whereas if immunotherapy was initiated 7 to 9 days after hospitalization, 0 of 3 (0%) patients survived. 78 in another study, 79 patients with virologically with any new scientific advance, there is controversy. in 1890, when behring demonstrated that immune serum therapy could protect against diphtheria, it went against the current dogma at that time in which the cellular theory of phagocytosis was believed to be the primary mechanism of host protection. 5 there were also skeptics who, as early as 1896, discussed why antibody immunotherapy would not work. 114 however, the science not only prevailed but today a number of passive immunotherapy products are in clinical use (see table 8 .1) and an ever-increasing number of human diseases benefit from the use of this technology (see table 8 .3). some believe that antibody plays a more important role in protection against cytopathic viruses and extracellular bacteria, but that t cells must be required for protection against infection by noncytopathic viruses and other intracellular pathogens. 115 although this is partially refuted by the protective efficacy of maternal antibodies and ivig therapy in scid patients who do not have functioning t cells, it is important to bear in mind that antibody-mediated protection by passive immunotherapy in immunocompetent individuals does not function in isolation, but instead works best in conjunction with other immune defenses, including host t cells, b cells, natural killer (nk) cells, etc. although the role of antibody-mediated protection against intracellular bacteria and chronic viral infections was thought to be relatively minor, there are examples in each of these instances in which passive immunity provides substantial clinical benefit. as noted previously, prior to the advent of antibiotics, passive immunotherapy was the only option for clinical treatment of most bacterial infections including francisella tularensis, a facultative intracellular bacterium that causes tularemia, a severe disease associated with up to 30% mortality in untreated cases. 116,117 when streptomycin became available, a comparative study in 1946 was performed with 542 tularemia patients who received only symptomatic treatment, 832 who received immune equine serum, 60 who received hyperimmune equine serum, and 9 who received streptomycin. 118 the untreated tularemia cases required an average of 3.78 months to recover and only three modes of therapy showed substantial improvement-treatment with immune serum within 9 days of disease onset (2.41 months until recovery), treatment with hyperimmune serum (2.15 months until recovery), and treatment with streptomycin (2.40 months until recovery). two clinical cases were extensively described, with the following summary: "the clinical responses to each agent [i.e., immune serum, and streptomycin] were similar, prompt amelioration of the symptoms of intoxication-headache, mental dullness or lethargy, sense of prostration and severe malaise; reduction of fever and of the sizes of the buboes, acceleration in the healing of ulcers and in the resolution of pulmonary exudates." in other words, passive immunotherapy appeared in many ways to mimic antibiotic therapy in terms of protective efficacy. however, it was noted that treatment with equine serum caused serum sickness in 51% of the patients and had a more variable outcome than the antibiotic approach, leading to the recommendation that streptomycin would be the agent of choice for future treatment of this disease. 118 with the recent development of polyclonal and monoclonal antibodies that show protective efficacy against tularemia in animal models, [119] [120] [121] it may be possible to incorporate both passive immunotherapy and antibiotic treatment into clinical practice not only for tularemia, but for other bacterial diseases, especially in cases in which antibiotic resistance is becoming more widespread. 122, 123 mycobacterium tuberculosis is another intracellular bacterium that, despite the availability of antibiotics, remains one use of human immunoglobulin in at-risk populations. 102 santosham and colleagues administered hyperimmunoglobulin (n = 353), or saline placebo (n = 350) to infants at 2, 6, and 10 months of age and examined the rates of invasive hib. for the first 90 days following the passive immunization protocol, none of the treated infants experienced invasive hib (0% incidence), compared to 7 of 350 placebo-treated children (2.0% incidence, p = .007). 102 rotavirus represents an enteric viral pathogen wherein protective passive immunotherapy has been demonstrated. [103] [104] [105] [106] [107] [108] in one example of postexposure treatment in infants, oral administration of hyperimmune antibody (in addition to standard supportive care) was able to efficiently reduce rotavirus shedding compared to placebo controls; treated patients (n = 26) exhibited no evidence of viral shedding by day 8 posttreatment as compared to 25% of controls (n = 26). 105 in a separate study, prophylactic passive immunity using orally administered bovine colostrum from immunized animals was tested in a blinded and randomized trial among infant children (3-15 months old) admitted to a hospital, typically for respiratory conditions. 103 following admission, infants were given a 10-day course of the bovine colostrum or placebo. infants who received placebo contracted symptomatic rotavirus at a rate of 14% (9 of 65) whereas no symptomatic rotavirus disease was observed in the colostrum-treated infants (0 of 55; p < .001). analysis of rotavirus vaccine failures also indicates that maternally derived antibodies play a role in passive immunity to rotavirus infection. in a study involving 177 vaccinated infants, a strong inverse correlation was observed between maternally derived rotavirus antibodies and the ability of infants to seroconvert following vaccination with a live rotavirus vaccine. 21 this is an important demonstration not only of passive immunity to an enteric pathogen, but also has broader implications on the timing of vaccine administration, especially in developing countries where preexisting immunity is relatively high, and rotavirus vaccine immunogenicity appears impaired. 109 e. coli is a significant enteric pathogen wherein prophylaxis through passive immunity has been demonstrated in several clinical studies. 110-113 tacket and colleagues were able to passively protect human subjects against experimentally induced e. coli diarrhea with specific bovine antibody. 110 using heatinactivated or glutaraldehyde-inactivated e. coli for vaccination, pregnant cows were hyperimmunized with a large number of enterotoxigenic o serogroups. milk collected during the first 10 days of lactation was purified, concentrated, lyophilized, and formulated for oral administration. as a control, a similar preparation was made using rotavirus as the immunizing antigen. subjects received daily treatment (3 times daily) for 7 days, with e. coli challenge administered 3 days into the treatment regimen. of the 10 subjects who received the e. coli antibody prophylaxis, all remained diseasefree following challenge, compared with clinical diarrhea in 9 of 10 placebo subjects (p < .0001). using a closely related clinical protocol, otto and colleagues also demonstrated good efficacy with hyperimmune bovine colostrum tablets. 111 in the first study conducted in this trial, 11 of 15 (73%) of placebo subjects contracted diarrhea following challenge, but this was reduced to only 1 of 15 (7%) in treated subjects (p = .0005). in a second study investigating the impact of omitting buffer to the oral prophylaxis, the authors also examined dose sparing. in these studies, the standard dose still conferred significant protection with 3 of 15 (20%) treated subjects contracting diarrhea, compared with 12 of 14 (86%) of controls. interestingly, if the dose was reduced by one-half then disease incidence increased to 5 of 14 subjects (36%), indicating a key role played by treatment dose in achieving successful passive immunotherapy. developed at similar rates among all three groups (p = .97). however, because administration of immunoglobulin is typically only performed during the first 4 months after transplantation and antiviral antibody half-life is estimated to be approximately 25 days, 134 it is not surprising that the protective effects of passive immunotherapy were only maintained through the first year. nevertheless, the inadvertent discovery of the protective role of antibodies in preventing ebv-induced non-hodgkin lymphoma represents a potential breakthrough in clinical management of this vulnerable patient population. despite decades of research aimed at finding a vaccine or a cure for hiv infection, this virus remains a scourge of global proportions. early attempts at passive immunotherapy using first-generation hiv-specific monoclonal antibodies were not highly effective [135] [136] [137] and this approach was not further pursued until a new generation of highly potent and broadly neutralizing antibodies were identified. 138, 139 in particular, a recent phase i clinical trial 140 involving a single administration of a broadly neutralizing antibody, 3bnc117, has renewed interest in the study of passive immunotherapy for hiv prevention and therapeutic intervention. 3bnc117 is an anti-cd4 binding site antibody that neutralizes 195 of 237 hiv strains comprising six different clades and was tested in a doseescalation study among hiv-positive patients with different levels of viremia. at a dose of 10 or 30 mg/kg, patient viral load was reduced by up to 2.5 log 10 (average decline: 1.48 log 10 ) in 10 of 11 individuals. the subject that did not respond to antibody treatment at 10 mg/kg was infected with a resistant strain of hiv. although the effect of antibody therapy on viremia was mainly transient after a single administration, the viral load remained lower than their preexisting set point in 3 of 10 patients at 56 days and one subject exhibited viremia levels that remained near the limits of detection throughout the 56-day study. it is currently unclear if hiv viremia in these patients will eventually rebound to their original levels. similar results were observed during antibody-based therapy of shiv-infected rhesus macaques in which most animals showed a rebound in viral replication after the transferred monoclonal antibodies declined to undetectable levels but a subset of animals maintained virological control in the absence of further infusions. 141 combinations of antiretroviral drugs are currently the standard of care for treatment of hiv infection and it is unlikely that one dose of a single monoclonal antibody will be sufficient to have a long-term clinical benefit among a broad patient base. however, there is growing optimism that combining a cocktail of potent, broadly neutralizing monoclonal antibodies with antiretroviral drugs and/ or agents that activate latent virus reservoirs could theoretically provide long-term reduction in viral load and reduce the rates of transmission. with substantial advances in monoclonal antibody technologies and an increasing appreciation for the role of antibodies in the control of infectious disease, the development of sophisticated new passive immunotherapies is likely to continue at an accelerated pace. antibiotic resistance among clinically relevant bacteria including multidrug-resistant (mdr) and xdr m. tuberculosis, methicillin-resistant staphylococcus aureus (mrsa), and dominant strains of antibiotic-resistant salmonella typhi and other gram-negative bacterial species is a growing concern. 122, 125, [142] [143] [144] this, coupled with the knowledge that fewer new antibiotics are moving through the drug pipeline, 122,123 may further motivate research into the development of antibody-based therapies to overcome these challenges to clinical intervention against microbial disease. one drawback to passive immunization is that antibody half-life in vivo of the most common human diseases and it is estimated to infect up to one-third of the world's population. 124 the development of strains of extensively drug-resistant (xdr) tuberculosis (tb), 125 some of which are resistant to all current antibiotic therapies, 126, 127 is also a growing concern, especially as there are few antibiotic drugs in the pipeline. 122, 123 there is considerable debate over the role of antibodies in controlling tb, with many believing that antibody plays little or no role in protective immunity (reviewed in references 124 and 128). in a comprehensive historical review by glatman-freedman and casadevall, 128 the clinical benefit of antibody-mediated immunotherapy, albeit quite variable, provides evidence to suggest that antibody plays a role in protection against tb. in studies reported by paquin in 1895, a group of patients with pulmonary tb confirmed by the presence of bacterium in their sputum showed clinical benefit. after 2 months of passive immunotherapy, 82% of patients showed reduced cough, reduction in bacterial load in sputum, clearance of pulmonary infiltrates, reduction in hemoptysis, improved appetite, and weight gain. 128, 129 at 6 months after initiating treatment, all the treated patients were alive and more than half were discharged from the hospital. in contrast, more than 30 untreated tb patients from another ward in the hospital had died within 4 months of starting the study. experimental proof of antibody-mediated protection against tb was also published in 1897 by fisch. 128, 130 after lethal tb challenge of guinea pigs, administration of immune serum was performed on days 4, 7, and 10, with further doses administered every other day for 4 weeks and once a week after that. fisch reported that 16 of 18 treated animals were alive after 2.5 months (89% survival). if treatment was delayed until day 14 postchallenge, then 2 of 3 (66%) animals survived but showed signs of illness. if no antibody treatment was performed, then 0 of 3 (0%) of the animals survived past day 28. the same approach was used to treat 50 patients with pulmonary tb. 131 all of the 19 patients treated at the earliest stages of disease improved rapidly after passive immunotherapy and were tuberculin negative at the end of the study. of the 11 patients treated at the "incipient" stage of disease, 36% no longer had bacilli in their sputum and were considered cured and 64% showed substantial improvement in disease symptoms. the 20 patients with advanced tb showed only modest or no improvement after therapy and it was concluded that immune serum was only beneficial in early but not advanced cases of disease. 131 ebv is a common human pathogen that causes a chronic infection and is a leading cause of posttransplant non-hodgkin lymphoma resulting from the uncontrolled proliferation of ebv-infected b lymphocytes in patients undergoing immunosuppressive therapies. 132 in a large retrospective study involving 44,828 kidney transplant patients, the effect of prophylactic treatment for cytomegalovirus (cmv) on posttransplant incidence of non-hodgkin lymphomas was examined. 133 the standardized incidence ratio (sir) for non-hodgkin lymphoma was expressed as the number of lymphoma cases per 100,000 persons and calculated after normalizing for age, sex, and geographical origin. the 30,255 patients who did not receive cmv prophylaxis had a sir = 26.4, which remained unchanged (sir = 24.2, p = .62) among the 12,470 patients who received antiviral drugs (acyclovir or ganciclovir). in striking contrast, the 2103 patients who received anti-cmv immunotherapy showed a complete absence of lymphomas during the first year after transplantation (sir = 0, p = .016 vs. antiviral treatment). the most common anti-cmv immunoglobulin products were shown to contain antibodies against ebv and it is believed that this is the mechanism of action for the protection afforded during the first year posttransplantation. 133 in the subsequent 5 years of follow-up, new cases of lymphoma sufficient for protection or therapeutic intervention of acute or remittent disease, active immunization through improved vaccine design may still be needed to train the host immune system to maintain long-term levels of protective immunity. importantly, examples of successful passive immunization approaches may provide a useful framework for developing new and improved vaccines that elicit the most protective antibody responses. references for this chapter are available at expertconsult.com. often provides only 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role of serum antibody in immunity to chlamydial genital infection human monoclonal antibodies directed against toxins a and b prevent clostridium difficile-induced mortality in hamsters mechanisms of protection against clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab treatment with monoclonal antibodies against clostridium difficile toxins chicken egg yolk antibodies against f18ab fimbriae of escherichia coli inhibit shedding of f18 positive e. coli by experimentally infected pigs prevention and therapy of experimental escherichia coli infection with monoclonal antibody stx2-specific human monoclonal antibodies protect mice against lethal infection with escherichia coli expressing stx2 variants field trial of an infant formula containing anti-rotavirus and anti-escherichia coli milk antibodies from hyperimmunized cows treatment of enterotoxigenic and enteropathogenic escherichia coli-induced diarrhoea in children with bovine immunoglobulin milk concentrate from hyperimmunized cows: a double-blind, placebo-controlled, clinical trial a summary of certain aspects of the disease including methods for early diagnosis and the results of serum treatment in 600 patients efficacy of human hyperimmune globulin in prevention of haemophilus influenzae type b disease in infant rats monoclonal anti-lps inner core antibodies protect against experimental hematogenous haemophilus influenzae type b meningitis protection from infection with haemophilus influenzae type b by monoclonal antibody to the capsule serum treatment of influenzal meningitis a mycobacterial lipoarabinomannan specific monoclonal antibody and its f(ab′) fragment prolong survival of mice infected with mycobacterium tuberculosis therapeutic efficacy of high-dose intravenous immunoglobulin in mycobacterium tuberculosis infection in mice passive serum therapy with polyclonal antibodies against mycobacterium tuberculosis protects against post-chemotherapy relapse of tuberculosis infection in scid mice passive immunization with neisseria meningitidis pora specific immune sera reduces nasopharyngeal colonization of group b meningococcus in an infant rat nasal challenge model protective efficacy of monoclonal antibodies to class 1 and class 3 outer membrane proteins of neisseria meningitidis b:15:p1.16 in infant rat infection model: new prospects for vaccine development experimental meningococcal infection in the mouse experimental cerebro-spinal meningitis and its serum treatment protection against infection with neisseria meningitidis group b serotype 2b by passive immunization with serotype-specific monoclonal antibody the results of the serum treatment in thirteen hundred cases of epidemic meningitis a novel anti-pcrv antibody providing enhanced protection against pseudomonas aeruginosa in multiple animal infection models a multifunctional bispecific antibody protects against pseudomonas aeruginosa in vitro and in vivo properties of a fully human igg1 monoclonal antibody that combats multidrug resistant pseudomonas aeruginosa pseudomonas hyperimmune globulin passive immunotherapy for pulmonary exacerbations in cystic fibrosis intravenous immune globulin treatment of pulmonary exacerbations in cystic fibrosis safety and pharmacokinetics of an anti-pcrv pegylated monoclonal antibody fragment in mechanically ventilated patients colonized with pseudomonas aeruginosa: a randomized,double-blind, placebo-controlled trial immunoprotection by monoclonal antibodies to the porins and lipopolysaccharide of salmonella typhimurium a new antigen of b. typhosus: its relation to virulence and to active and passive immunisation oral passive immunization against experimental salmonellosis in mice using chicken egg yolk antibodies specific for salmonella enteritidis and s. typhimurium clinical trials with a new antitiyphoid serum multi-serotype outer membrane vesicles of shigellae confer passive protection to the neonatal mice against shigellosis passive immunity with multiserotype heat-killed shigellae in neonatal mice protection against keratoconjunctivitis shigellosa induced by immunization with outer membrane proteins of shigella spp efficacy of bovine milk immunoglobulin concentrate in preventing illness after shigella flexneri challenge hyperimmune bovine colostrum in the treatment of shigellosis in children: a double-blind, randomized, controlled trial protein a-neutralizing monoclonal antibody protects neonatal mice against staphylococcus aureus a human monoclonal antibody targeting the conserved staphylococcal antigen isaa protects mice against staphylococcus aureus bacteremia functional antibodies targeting isaa of staphylococcus aureus augment host immune response and open new perspectives for antibacterial therapy a blinded, randomized, multicenter study of an intravenous staphylococcus aureus immune globulin antistaphylococcal immunoglobulins to prevent staphylococcal infection in very low birth weight infants multicenter study to assess safety and efficacy of inh-a21, a donor-selected human staphylococcal immunoglobulin, for prevention of nosocomial infections in very low birth weight infants phase ii, randomized, multicenter, double-blind, placebo-controlled trial of a polyclonal anti-staphylococcus aureus capsular polysaccharide immune globulin in treatment of staphylococcus aureus bacteremia. antimicrob agents chemother phase ii, randomized, double-blind, multicenter study comparing the safety and pharmacokinetics of tefibazumab to placebo for treatment of staphylococcus aureus bacteremia human monoclonal antibodies to group b streptococcus. reactivity and in vivo protection against multiple serotypes monoclonal antibodies in the therapy of experimental neonatal group b streptococcal disease monoclonal antibodies targeting different cell wall antigens of group b streptococcus 95.e6 section 1 general aspects of vaccination mediate protection in both fc-dependent and independent manner immunological fingerprinting of group b streptococci: from circulating human antibodies to protective antigens maternal antibody at delivery protects neonates from early onset group b streptococcal disease correlation of maternal antibody deficiency with susceptibility to neonatal group b streptococcal infection pro tection against pneumococcal pneumonia in mice by monoclonal antibodies to pneumolysin human antibodies to phtd, pcpa, and ply reduce adherence to human lung epithelial cells and murine nasopharyngeal colonization by streptococcus pneumoniae preclinical evaluation of the pht proteins as potential crossprotective pneumococcal vaccine antigens effective combination therapy for invasive pneumococcal pneumonia with ampicillin and intravenous immunoglobulins in a mouse model studies on experimental pneumonia: vii. treatment of experimental pneumococcus type i pneumonia in monkeys with type i antipneumococcus serum the treatment of lobar pneumonia with concentrated anti-pneumococcus serum bacterial polysaccharide immune globulin for prophylaxis of acute otitis media in high-risk children the serum treatment of lobar pneumonia defense from the group a streptococcus by active and passive vaccination with the streptococcal hemoprotein receptor active and passive immunizations with the streptococcal esterase sse protect mice against subcutaneous infection with group a streptococci mechanism of protection induced by group a streptococcus vaccine candidate j8-dt: contribution of b and t-cells towards protection scarlet fever prophylaxis: use of blood serum from persons who have recovered from scarlet fever the prevention of scarlet fever intravenous immunoglobulin g therapy in streptococcal toxic shock syndrome: a european randomized, double-blind, placebo-controlled trial clinical efficacy of polyspecific intravenous immunoglobulin therapy in patients with streptococcal toxic shock syndrome: a comparative observational study intravenous immunoglobulin therapy for streptococcal toxic shock syndrome-a comparative observational study. the canadian streptococcal study group antitoxin versus no antitoxin in scarlet fever the antitoxin treatment of erysipelas passive immunity in experimental cholera passive oral immunization by egg yolk immunoglobulin (igy) to vibrio cholerae effectively prevents cholera experimental cholera in infant mice: protective effects of antibody further investigation of a new anti-cholera serum treatment of cholera with a new anti-cholera serum: preliminary note human anti-plague monoclonal antibodies protect mice from yersinia pestis in a bubonic plague model synergistic protection of mice against plague with monoclonal antibodies specific for the f1 and v antigens of yersinia pestis la peste bubonique (duexiéme note) treatment of plague: promising alternatives to antibiotics protection of mice from fatal bubonic and pneumonic plague by passive immunization with monoclonal antibodies against the f1 protein of yersinia pestis the scid/beige mouse as a model to investigate protection against yersinia pestis human immune response to a plague vaccine comprising recombinant f1 and v antigens administration of antibody to the lung protects mice against pneumonic plague passive immunity to yersiniae mediated by anti-recombinant v antigen and protein a-v antigen fusion peptide roles of v antigen in promoting virulence and immunity in yersiniae les épidémies de peste en extrême-orient. xiiie congrès international de médecine sur la peste bubonique (sérothérapie) prophylaxis and therapy for chikungunya virus infection chikungunya viruses that escape monoclonal antibody therapy are clinically attenuated, stable, and not purified in mosquitoes case reports of neuro-chikungunya in southern thailand antigenic sites of coxsackie a9 virus inducing neutralizing monoclonal antibodies protective in mice a murine model of coxsackievirus a16 infection for anti-viral evaluation effect of intravenous immunoglobulin for neonates with severe enteroviral infections with emphasis on the timing of administration vaccination for the prevention of maternal and fetal infection with guinea pig cytomegalovirus rat cytomegalovirus vaccine prevents accelerated chronic rejection in cmvnaive recipients of infected donor allograft hearts complete protection of mice against lethal murine cytomegalovirus challenge by immunization with dna vaccines encoding envelope glycoprotein complex iii antigens gh, gl and go passive immunization during pregnancy for congenital cytomegalovirus infection prophylaxis of primary cytomegalovirus disease in renal transplant recipients. a trial of ganciclovir vs immunoglobulin cmv-hyperimmune globulin for preventing cytomegalovirus infection and disease in solid organ transplant recipients: a meta-analysis the development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1 epitope determinants of a chimpanzee dengue virus type 4 (denv-4)-neutralizing antibody and protection against denv-4 challenge in mice and rhesus monkeys by passively transferred humanized antibody development of a humanized antibody with high therapeutic potential against dengue virus type 2 ebola gp-specific monoclonal antibodies protect mice and guinea pigs from lethal ebola virus infection successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies therapeutic intervention of ebola virus infection in rhesus macaques with the mb-003 monoclonal antibody cocktail treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee preventive effect of igg from ebv-seropositive donors on the development of human lympho-proliferative disease in scid mice a mouse monoclonal antibody against epstein-barr virus envelope glycoprotein 350 prevents infection both in vitro and in vivo active and passive vaccination against hantavirus pulmonary syndrome with andes virus m genome segment-based dna vaccine dna vaccine-derived human igg produced in transchromosomal bovines protect in lethal models of hantavirus pulmonary syndrome a non-randomized multicentre trial of human immune plasma for treatment of hantavirus cardiopulmonary syndrome by andv the use of human immune serum globulin (gamma globulin) in infectious (epidemic) hepatitis in the mediterranean theater of operations i. studies on prophylaxis in two epidemics of infectious hepatitis prevention of infectious hepatitis with gamma globulin the prevention and attenuation of infectious hepatitis by gamma globulin gamma globulin in the prevention of infectious hepatitis; studies on the use of small doses in family outbreaks preclinical evaluation of two human anti-hepatitis b virus (hbv) monoclonal antibodies in the hbv-trimera mouse model and in hbv chronic carrier chimpanzees hepatitis b immune globulin as a prophylactic measure for spouses exposed to acute type b hepatitis liver transplantation in hbsag-positive hbv-dna-negative cirrhotics: immunoprophylaxis and long-term outcome liver transplantation in hbs antigen (hbsag) carriers. prevention of hepatitis b virus (hbv) recurrence by passive immunization human monoclonal antibody hcv1 effectively prevents and treats hcv infection in chimpanzees sexual transmission of the hepatitis c virus and efficacy of prophylaxis with intramuscular immune serum globulin. a randomized controlled trial human monoclonal antibody mbl-hcv1 delays hcv viral rebound following liver transplantation: a randomized controlled study immunotherapeutic potential of neutralizing antibodies targeting conserved regions of the hcv envelope glycoprotein e2 clinical evaluation (phase i) of a human monoclonal antibody against hepatitis c virus: safety and antiviral activity successful passive and active immunization of cynomolgus monkeys against hepatitis e role of immune serum globulins in pregnant women during an epidemic of hepatitis e passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in pathogenicity analysis of the role of antibody-dependent cellular cytotoxic antibody activity in murine neonatal herpes simplex virus infection with antibodies to synthetic peptides of glycoprotein d and monoclonal antibodies to glycoprotein b effect of antibody alone and combined with acyclovir on neonatal herpes simplex virus infection in guinea pigs l or more. effect on viral, opportunistic, and bacterial infections. the national institute of child health and human development intravenous immunoglobulin clinical trial study group intravenous immunoglobulins suppress the recurrences of genital herpes simplex virus: a clinical and immunological study antibody and antiretroviral preexposure prophylaxis prevent cervicovaginal hiv-1 infection in a transgenic mouse model antibody-based protection against hiv infection by vectored immunoprophylaxis hiv therapy by a combination of broadly neutralizing antibodies in humanized mice hiv-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice passive immunotherapy in aids: a double-blind randomized study based on transfusions of plasma rich in anti-human immunodeficiency virus 1 antibodies vs. transfusions of seronegative plasma delay of hiv-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies safety and efficacy of hiv hyperimmune globulin for prevention of motherto-child hiv transmission in hiv-1-infected pregnant women and their infants in kampala passive immunotherapy in the treatment of advanced human immunodeficiency virus infection a murine genital-challenge model is a sensitive measure of protective antibodies against human papillomavirus infection in vivo mechanisms of vaccine-induced protection against hpv infection the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory metaanalysis protection of mice against japanese encephalitis virus by passive administration with monoclonal antibodies passive protection of mice, goats, and monkeys against japanese encephalitis with monoclonal antibodies protection of monkeys against machupo virus by the passive administration of bolivian haemorrhagic fever immunoglobulin (human origin) protection against measles virus encephalitis by monoclonal antibodies binding to a cystine loop domain of the h protein mimicked by peptides which are not recognized by maternal antibodies correlation between epitopes on hemagglutinin of measles virus and biological activities: passive protection by monoclonal antibodies is related to their hemagglutination inhibiting activity smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus a protective therapy for mumps mumps; use of convalescent serum in the treatment and prophylaxis of orchitis serum prophylaxis of epidemic parotitis use of human blood in protection against mumps intravenous immunoglobulin therapy for pure red cell aplasia related to human parvovirus b19 infection: a retrospective study of 10 patients and review of the literature guidelines on the use of intravenous immune globulin for hematologic conditions the relation of the meninges and choroid plexus to poliomyelitic infection passive immunity in poliomyelitis. iv. protection of rhesus monkeys against cerebral challenge experimental poliomyelitis in monkeys seventh note: active immunization and passive serum protection evaluation of red cross gamma globulin as a prophylactic agent for poliomyelitis. iv. final report of results based on clinical diagnoses evaluation of red cross gamma globulin as a prophylactic agent for poliomyelitis. 5. reanalysis of results based on laboratory-confirmed cases resume of the results of therapy with convalescent serum in poliomyelitis intramuscular use of convalescent serum in treatment of poliomyelitis preparalytic poliomyelitis: observations in one hundred and six cases in which convalescent serum was used recherches sur la vaccination antirabique evaluation of human rabies immune globulin and homologous and heterologous antibody antiserum in the prophylaxis of rabies comparison of an anti-rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin use of hyperimmune anti-rabies serum concentrates in experimental rabies laboratory data supporting the clinical trial of anti-rabies serum in persons bitten by a rabid wolf management of rabies in humans development of a humanized monoclonal antibody (medi-493) with potent in vitro and in vivo activity against respiratory syncytial virus quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats studies of passive immunotherapy for infections of respiratory syncytial virus in the respiratory tract of a primate model isolation of a second recombinant human respiratory syncytial virus monoclonal antibody fragment (fab rsvf2-5) that exhibits therapeutic efficacy in vivo respiratory syncytial virus-enriched globulin for the prevention of acute otitis media in high risk children active and passive immunization against rift valley fever virus infection in syrian hamsters topological mapping of antigenic sites on the rift valley fever virus envelope glycoproteins using monoclonal antibodies passive protection against rotavirus-induced diarrhea by monoclonal antibodies to surface proteins vp3 and vp7 serum igg mediates mucosal immunity against rotavirus infection a gastrointestinal rotavirus infection mouse model for immune modulation studies rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection prophylaxis of german measles with immune serum globulin prevention of rubella by gamma globulin during an epidemic in barrow, alaska, in 1964 trial of high-titre human rubella immunoglobulin rubella epidemic, 1964: effect on 6,000 pregnancies passive immunization against rubella: studies on the effectiveness of rubella-immunoglobulin after intranasal infection with rubella vaccination virus human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (sars) coronavirus s protein neutralizes the virus in a rhesus macaque sars model therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and viral burden in golden syrian hamsters passive immunization of newborn rhesus macaques prevents oral simian immunodeficiency virus infection passive immune globulin therapy in the siv/macaque model: early intervention can alter disease profile passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies fc receptor but not complement binding is important in antibody protection against hiv protection of macaques against pathogenic simian/human immunodeficiency virus 89.6pd by passive transfer of neutralizing antibodies determination of a statistically valid neutralization titer in plasma that confers protection against simian-human immunodeficiency virus challenge following passive transfer of high-titered neutralizing antibodies highly potent hiv-specific antibody neutralization in vitro translates into effective protection against mucosal shiv challenge in vivo antibody-mediated immunotherapy of macaques chronically infected with shiv suppresses viraemia passive transfer of modest titers of potent and broadly neutralizing anti-hiv monoclonal antibodies block shiv infection in macaques neutralizing antibody directed against the hiv-1 envelope glycoprotein can completely block hiv-1/siv chimeric virus infections of macaque monkeys pre-and postexposure protection by passive immunoglobulin but no enhancement of infection with a flavivirus in a mouse model passive immunization of mice with monoclonal antibodies raised against tickborne encephalitis virus efficiency of use of immunoglobulin preparations for the postexposure prevention of tickborne encephalitis in russia (a review of semi delayed humoral immunity in a patient with severe tick-borne encephalitis after complete active vaccination combination therapy of vaccinia virus infection with human anti-h3 and anti-b5 monoclonal antibodies in a small animal model disparity between levels of in vitro neutralization of vaccinia virus by antibody to the a27 protein and protection of mice against intranasal challenge protection of rabbits and immunodeficient mice against lethal poxvirus infections by human monoclonal antibodies postexposure prevention of progressive vaccinia in scid mice treated with vaccinia immune globulin prophylactic effect of antivaccinia gamma-globulin against post-vaccinal encephalitis experience of anti-vaccinia immunoglobulin in the united kingdom complications of smallpox vaccination. effects of vaccinia immune globulin therapy prevention of varicella by zoster immune globulin the use of serum gamma globulin antibodies to control chicken pox in a convalescent hospital for children congenital varicella syndrome: the evidence for secondary prevention with varicella-zoster immune globulin a humanized murine monoclonal antibody protects mice either before or after challenge with virulent venezuelan equine encephalomyelitis virus antibody prophylaxis and therapy against west nile virus infection in wild-type and immunodeficient mice efficacy of killed virus vaccine, live attenuated chimeric virus vaccine, and passive immunization for prevention of west nile virus encephalitis in hamster model using high titer west nile intravenous immunoglobulin from selected israeli donors for treatment of west nile virus infection development of a humanized monoclonal antibody with therapeutic potential against west nile virus possible benefit of intravenous immunoglobulin therapy in a lung transplant recipient with west nile virus encephalitis treatment of west nile virus encephalitis with intravenous immunoglobulin lethal 17d yellow fever encephalitis in mice. i. passive protection by monoclonal antibodies to the envelope proteins of 17d yellow fever and dengue 2 viruses the duration of passive immunity in yellow fever persistence of yellow fever immunity on the use of immune serum at various intervals after the inoculation of yellow fever virus into rhesus monkeys notes on laboratory infections with yellow fever observations on laboratory and hospital infections with yellow fever in england immunoprotection against systemic candidiasis in mice rats clearing a vaginal infection by candida albicans acquire 95.e10 section 1 general aspects of vaccination specific, antibody-mediated resistance to vaginal reinfection a vaccine and monoclonal antibodies that enhance mouse resistance to candida albicans vaginal infection vaccine and monoclonal antibody that enhance mouse resistance to candidiasis immunoglobulin g3 blocking antibodies to the fungal pathogen cryptococcus neoformans human immunoglobulin g2 (igg2) and igg4, but not igg1 or igg3, protect mice against cryptococcus neoformans infection more is not necessarily better: prozone-like effects in passive immunization with igg phase i evaluation of the safety and pharmacokinetics of murine-derived anticryptococcal antibody 18b7 in subjects with treated cryptococcal meningitis enteral human serum immunoglobulin treatment of cryptosporidiosis in mice with severe combined immunodeficiency efficacy of monoclonal antibodies against defined antigens for passive immunotherapy of chronic gastrointestinal cryptosporidiosis prophylactic effect of bovine anti-cryptosporidium hyperimmune colostrum immunoglobulin in healthy volunteers challenged with cryptosporidium parvum demonstration of passive immunity in experimental monkey malaria model for in vivo assessment of humoral protection against malaria sporozoite challenge by passive transfer of monoclonal antibodies and immune serum the importance of human fcgammari in mediating protection to malaria gamma-globulin and acquired immunity to human malaria antibodies that protect humans against plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes monoclonal antibodies to toxoplasma cell membrane surface antigens protect mice from toxoplasmosis generation of a neutralizing human monoclonal antibody fab fragment to surface antigen 1 of toxoplasma gondii tachyzoites protection against experimental toxoplasmosis by adoptive immunotherapy impact of trough igg on pneumonia incidence in primary immunodeficiency: a meta-analysis of clinical studies high-vs low-dose immunoglobulin therapy in the long-term treatment of x-linked agammaglobulinemia history of immunoglobulin replacement use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology effect of intravenous gammaglobulin therapy in igg2 deficient and igg2 sufficient children with recurrent infections and poor response to immunization with hemophilus influenzae type b capsular polysaccharide antigen intravenous immune globulin for the prevention of bacterial infections in children with symptomatic human immunodeficiency virus infection. the national institute of child health and human developments intravenous immunoglobulin study group polyclonal intravenous immunoglobulin for the treatment of severe sepsis and septic shock in critically ill adults: a systematic review and metaanalysis supplemental immune globulins in sepsis: a critical appraisal prevention of infection in multiple trauma patients by high-dose intravenous immunoglobulins the role of intravenous immunoglobulin for the prevention and treatment of neonatal sepsis immunity in mumps: i. experiments with monkeys (macacus mulatta). the development of complement-fixing antibody following infection and experiments on immunization by means of inactivated virus and convalescent human serum an experimental study of parotitis key: cord-016882-c9ts2g7w authors: ribeiro, edna; leitão, céu; cristovam, elisabete; dias, ana title: viruses present indoors and analyses approaches date: 2017-06-12 journal: exposure to microbiological agents in indoor and occupational environments doi: 10.1007/978-3-319-61688-9_7 sha: doc_id: 16882 cord_uid: c9ts2g7w through human history viruses have shown enormous epidemiological and pandemic potential as the occurrence and spread of viruses in pandemic dimensions poses a threat to the health and lives of seven billion people worldwide. scientific evidence has associated harmful health effects to indoor air hazards recognizing the existence of a vital concern in public health sector. thus the assessment of human exposure to biological aerosols and droplets indoor became an imperative requirement of investigation. environmental bioburden assessment of viruses relies in both culture-dependent approaches that comprise classical methodologies, still prominent and vital in the field of modern biotechnology, and culture-independent approaches based on nucleic acid amplification techniques, which are considered the gold standard in clinical virology. the main factor influencing indoor microbiology is the human being and their activities. indoor environments to be considered are those regularly occupied by humans: residences, offices, schools, industrial buildings, health care facilities, farming activities and other settings occupied all the time, or in which occupant density is high. it’s well known that approximately 60% of total human respiratory and gastrointestinal infections are acquired indoor, since viruses have a rapid spread in the community and can be transmitted easily, especially in crowded and poorly ventilated environments, causing high morbidity and decline in quality of life and productivity. studies have shown that respiratory syncytial virus, rhinovirus, metapneumovirus, influenza and parainfluenza virus, and human enterovirus infections may be associated with virus-induced asthma, leading to diseases such as pneumonia. gastroenteritis infectious (about 30±40% of cases) is attributable to viruses. rotavirus, astrovirus, norwalk-like viruses and other caliciviruses are responsible for 48% of all reported outbreaks of infectious intestinal disease. safe working conditions are essential for healthy living, that’s why the programmes conceived as a result of strategic and preventive policy maintenance, in refrigeration and ventilation systems, are the determining factor for the control of biological pollutants. moreover, the development of highly sensitive and specific detection and identification methodologies with capacity to be used in diverse applications, such as diagnosis, public health risk assessment, research and for the implementation of preventive measures and protocols are imperative. indoor air pollution is a major global public health threat requiring increased hard work, linking research to policy-making. the evidence of the effects of physical and chemical pollutants on human health, present in the external and internal environment has no superior aggression than the existing bioaerosols. health effects from indoor air pollutants may be experienced soon after exposure or, possibly, years later, according to the nature of air contaminants, being classified, in an abbreviated approach, as physical, chemical or biological (nazaroff, 2016) . several studies have related hostile health effects to indoor air hazards and adequate assessment of human exposure to biological aerosols has been recognized as an imperative requirement and a very important concern in the area of public health (douwes et al., 2003) . scientific evidence has showed that the air within buildings can be more seriously polluted than the outdoor air, even in developed and industrialized countries, and it is well known that indoor environments occupied by humans, contain abundant material of microbial origin, consequently, the risks to human health may be greater due to exposure to air pollution indoors (fisk et al., 2007) . on the other hand, investigation indicates that people spend approximately 90% of their time indoors, with mechanical heating, cooling and ventilation systems, influencing irrefutably the quality of life (klepeis et al., 2001; nazaroff, 2016) . complex syndromes arise associated with indoor air quality, such as sick building syndrome, building-related disease leading to loss of productivity and absence at work. these syndromes are associated with increased incidence and prevalence of asthma and other chronic diseases worldwide. exposure to bioaerosol material can cause or can contribute to several relevant diseases. human occupancy and activities are major factors influencing indoor microbiology. humans are important primary sources of certain bacteria and viruses. the recent development of quantitative polymerase chain reaction (pcr) and other rna/dna-based measurement technologies has permitted studies that measure pathogenic material in indoor air (lax et al., 2014; nazaroff, 2016) . droplet transmission is not to be confused with airborne transmission. aerosols are suspensions in air (or in a gas) of solid or liquid particles, small enough to remain airborne for a prolonged period of time because of their low settling velocity. droplets do not remain suspended in the air. airborne transmission depends on viruses from evaporated droplets or dust particles that can remain suspended in the air for long periods. droplet transmission occurs when viruses travel on relatively large respiratory droplets (>10 μm) that people sneeze, cough, or exhale during conversation or breathing (primary aerosolization) (la rosa et al., 2013) . respiratory droplets initially all move forward with the exhaled air jet; very large droplets leave the jet quickly and fall on the ground and small droplets completely desiccate within the jet (tellier, 2009) . the transport and the settling of a bioaerosol are affected by its physical properties and the environmental parameters that it encounters. size, density and shape of droplets or particles, air currents, relative humidity and temperature, determine the capacity of generation of airborne bioaerosols from liquid suspensions, undergo desiccation, whereas those generated as dusts or powders partially rehydrate. the presence of moulds indicates a problem with water penetration or high humidity. bioaerosols can be transmitted at long distances. small particle aerosols, as shown during endotracheal intubation, are transmitted to persons in the immediate area near the patient. viruses' inductors of severe acute respiratory syndrome (sars), influenza and norovirus are transmitted from patients primarily by contact and/or droplet routes, while airborne transmission occurs over a limited distance (srikanth et al., 2008) . indoor-outdoor air exchange (mechanical ventilation), penetration (air filter), deposition, sources and aerosol resuspension, are extremely relevant for spread contamination. once aerosolized, aerosols viruses' particles may travel significant distances through buildings before being captured and retained by hvac filters, or they pass through as well, because most hvac filters are not 100% efficient in capturing particles. the rapidity with which airborne viruses are inactivated during transport or after filter capture is uncertain and merits additional study. analysis of ventilation filters certainly could play a role in the epidemiology of infectious diseases caused by pathogens released into the environment, suiting all the different situations of confinement (goyal et al., 2011) . the main source of indoor viruses is the human being. viruses are spread by air currents after resuspension of material scattered by aerosols droplets or saliva. we can say that viral infections are probably the most common acquired diseases indoor that affect man, knowing approximately a thousand types of different viruses involved. it is estimated about 60% of total human respiratory and gastrointestinal infections, with a rapid spread in the community, being a cause of high morbidity and decline in quality of life and productivity, since viruses can be easily transmitted, especially in crowded and poorly ventilated environments. it is well-known that viruses are shed in large numbers, with transmission routes extraordinary diverse, including direct contact with infected persons, faecal-oral transmission (through contaminated food and water), droplet and airborne transmission, and can survive for long periods on surfaces or fomites, emphasizing the possible role of surfaces in the transmission of viruses (barker et al., 2001; la rosa et al., 2013) . for instance, faeces can contain up to 10 12 viruses particles per gram and vomit up to 10 7 per millilitre, so the potential transfer contamination from hands to surfaces is frighteningly considerable. the most important source of potentially pathogenic viral aerosol is other humans and other means, as the flushing of a toilet that can aerosolize significant concentrations of airborne viruses. viruses' survival on fomites is influenced by temperature, humidity, ph and exposure to ultraviolet light. particle size, depth of penetration and the minimum dosage of the agent capable of causing disease are implicated in the infectivity. in addition, it is also important to be conscious of the risk groups, the most susceptible to contracting infection when exposed to microorganisms, conditioned by factors such as a weakened immune system, the children, the elderly, the pregnant women, the chronically ill, especially those suffering from respiratory or cardiovascular disease. chronic obstructive pulmonary disease (copd) and acute exacerbations are frequent complications, thought to be caused by interactions between host factors, bacteria, viruses and changes in air quality producing increased inflammation in the lower airway with a long-lasting adverse influence on health status (celli and macnee, 2004; celli and barnes, 2007) . approximately of 50% of acute exacerbations of copd are associated with symptoms of viral infections of the respiratory tract by rhinovirus, respiratory syncytial virus and influenza. studies have shown that respiratory syncytial virus (rsv), human rhinovirus (hrv), human metapneumovirus (hmpv), influenza and parainfluenza virus (hpiv), and human enterovirus infections may be associated with virus-induced asthma, leading to diseases such as pneumonia or death (tsukagoshi et al., 2013) . respiratory and enteric viruses are opportunistic pathogens transmitted mainly via other routes are able to spread via droplet nuclei or dust in certain circumstances (la rosa et al., 2013) . numerous studies identify the factors that are involved in the transmission of infection by aerosol indoor with the correlation between the pulmonary mechanism, the different human activities and the critical concentration of particles expelled. a virus (h3n2) had a higher transmissibility and uncontrollable potential than the a (h1n1) and b viruses (chen et al., 2009; chen and liao, 2010) . a major cause of morbidity and mortality, leading to diseases such as bronchiolitis, asthma and pneumonia (tsukagoshi et al., 2013; paba et al., 2014) . despite all the progress of the last decades in the prevention and care of health, respiratory infections represent one of the major causes of disease in humans. this group remains the leading cause of outpatient consultation as well as antibiotic prescription and work absence. the chronic infections, causing disability, have the most important impact on quality of life. the main effect of the inadequate quality of indoor air is in respiratory system. most studies focus on the influenza virus, but in general, we can say that a total concentration of viruses, in a variety of environments such as classrooms, health institutions, restaurants, offices and others corresponds to an average 4.7 ± 2.5 × 10 5 particles per cubic meter, without significant differences between the different indoor environments. also, is estimated that the viral particles inhaled daily indoor correspond approximately to 5 × 10 6 . respiratory viruses can be transported over considerable distances by air currents and be inhaled, penetrating deep into the respiratory system (prussin ii et al., 2015) . droplet transmission occurs when viruses travel on relatively large respiratory droplets (>10 μm) that people sneeze, cough, or exhale during conversation or breathing, primary aerosolization. a single cough can release hundreds of droplets, up to 40,000, at speeds of up to 50-200 miles per hour, each droplet containing millions of viral particles. aerosol droplets travel only short distances (1-2 m) before settlings on surfaces, where viruses can remain infectious for hours or days. secondary aerosolization can occur when air displacements disperse the viruses back into the air from contaminated surfaces (la rosa et al., 2013) . transmission occurs through air droplets, aerosol and fomites that may come into contact with nasal and conjunctival epithelium. the etiological viral agents involved include: influenza virus types a and b, parainfluenza viruses types 1, 2, 3, and 4, respiratory syncytial virus, adenovirus, and rhinoviruses/enteroviruses (paba et al., 2014) . the acute respiratory infections are the result of active multiplication of microbiological agents in the respiratory system when favourable conditions of the host exist. viral or bacterial ethology, predisposing factors related to anatomical factors, immune changes, colonization of the naso and oropharynx, and the spread of these infections is favoured by the continuity of the epithelia of the respiratory system and a continuum between upper and lower airways. the upper and lower respiratory infections are in most cases (60%) of viral origin. adenovirus (type 4), the first virus to be isolated from indoor aerosol, was identified in 1966 in aerosol samples from the quarters of military recruits infected with acute respiratory disease (artenstein and miller, 1966) . since then, human infections due to viral aerosol (or contact with contaminated surfaces) have been studied in various environments, including office building, hospitals, restaurants, transport systems and schools. in the last few years, other respiratory viruses have been discovered and linked to the upper and lower respiratory tract infections: human metapneumovirus, sars coronavirus, hku1 coronavirus, nl63 coronavirus, mers coronavirus and bocavirus. in 2007, two novel human polyomaviruses named kipyv and wupyv were discovered in the respiratory secretions of patients with acute respiratory symptoms. the outbreak of the influenza a virus (h1n1) infection in 2009 has reminded us again of the importance of monitoring and controlling airborne microorganisms in public facilities (lee et al., 2012) . unlike conventional viral cell cultures, with the introduction of the real-time pcr assay, the diagnosis of respiratory infections improved greatly. it is possible to search for up to 21 different respiratory pathogens including viruses and bacteria. in addition, it is possible to detect co-infections that may have implications on disease severity or therapeutic strategies (paba et al., 2014) . for bioaerosol particles, maybe the most important exposure pathway is inhalation followed by deposition in the respiratory tract. the probability of deposition varies with particle size, with lung morphology, and with breathing characteristics. airborne exposure indoors makes a meaningful contribution to the occurrence or spread of disease. rhinovirus (rv) is a small rna virus belonging to the picornaviridae family. more than 100 immunologically distinct serotypes have been identified and new serotypes are continuously emerging. this virus is responsible for more than 50% of cases of common cold, being the virus with the highest morbidity among respiratory diseases. in children causes bronchitis. although the method of transmission of rvs is in doubt, they are thought to be mainly transmitted via large droplets, but indirect contact with contaminated fomites has also shown to transmit infection or by aerosols. it's known that rv need only 10 s of contact (hand-to-hand) for infecting another person. the recurrence of viral infections is predominantly associated with an immune response to a serotype that does not provide immunity to reinfection with another serotype for the same virus (ballow, 2008) . rhinoviruses can survive on environmental surfaces for several hours. infectious viruses have been recovered from naturally contaminated objects in the surroundings of persons with rv colds. aerosols are generated by coughing, talking, sneezing and even simply breathing (huynh et al., 2008) . rhinovirus outbreaks in health care facilities, capable of determining severe infections and also death have been documented (macintyre et al., 2012) . rvs have also been detected in transport vehicles (la rosa et al., 2013) . even in the common cold, rhinoviruses and coronaviruses predominate (barker et al., 2001; heikkinen et al., 2003) , but the role of rhinoviruses is the most prominent, in particular are responsible for outbreaks of the common cold in the general community such as schools, day care centres and hospitals (barker et al., 2001; heikkinen et al., 2003; la rosa et al., 2013) . rhinoviruses and coronaviruses also cause a greater disease burden in elderly people living at home, compared with influenza or syncytial viruses. the infections by rhinovirus may be triggering factors of exacerbations of asthma, probably because they induce inflammation in airways that are already damaged and sensitized. rhinoviruses were detected by rt-pcr among infants (von mutius, 2004) , in the acute exacerbation of asthma when infection was prolonged (tsukagoshi et al., 2013) . some authors describe that bronchial epithelial patient cells with asthma are more effectively infected with rv than normal epithelia. it was shown that viral are upregulated in the epithelial cells of patients with allergies (canonica et al., 1995; tantilipikorn and auewarakul, 2011) . respiratory syncytial virus (rsv) is a single-stranded rna virus belonging to paramixoviridae family. rsv infections occur all over the world and outbreaks are common in the cold season in temperate climates and in the rainy season in tropical climates. is one of the most common viruses which can cause a type of potentially lethal pneumonia in older people and is a major cause of respiratory illness in young children producing bronchitis and pneumonia worldwide, affecting about 90% of children by the age of 2 years (24), inducting a preparative stage in the development of asthma (tsukagoshi et al., 2013) . school-aged children often carry rsv to their homes and extent infection to younger brothers as well as when admitted to hospital, tend to shed the virus abundantly and for prolonged periods permitting sufficient opportunity for spread in adults. sometimes this virus produces a flu-like syndrome indistinguishable from influenza (barker et al., 2001) . rsv is highly contagious and transmission can occur when infectious material comes into contact with mucous membranes of the eyes, mouth or nose, and possibly through the inhalation of droplets generated by a sneeze or cough. also result from contact with contaminated environmental surfaces, the commonest mode of transmission in school classrooms and day care centres. transmission with fomites predominates over droplet contact can be transmitted by the airborne route. rsv rna is detected in air samples from the hospital rooms of infected patients at large distances from the patient's bedside. particles containing rsv rna were detected in airborne throughout a health care facility, particles small enough to remain in the air for an extended period and to be inhaled deeply into the respiratory tract (6). evidence shows that direct and indirect contact is a key factor in transmission hands, touching surfaces contaminated with fresh secretions from rsv-infected infants (barker et al., 2001) . influenza virus (iv) is one of the most common and highly contagious infectious diseases and can occur in people of any age. influenza or flu produces an acute infection of the respiratory system with high transmissibility and global distribution. is a rna virus belonging to the orthomixoviridae family which is subdivided into three distinct antigenic serotypes: a, b and c, causing moderate to severe acute febrile illness, resulting in variable degrees of systemic symptoms, ranging from mild fatigue to respiratory failure and death. strains a and b have greatest potential epidemic, causing more versions of the flu. the flu vaccine only protects against viruses a and b. influenza c viruses are antigenically stable, cause subclinical disease and do not cause epidemics. the influenza a and b have multiple subtypes that still suffer mutations, emerging new strains with consequent increased risk of epidemics or pandemics. the continuing threat of pandemics by the human influenza virus suggests an urgent and constant surveillance. despite the vaccination continues to be considered the intervention of containment of infection, the viruses a (h1n1), a (h3n2) and influenza b persist with a global distribution and with such a power of infectivity that influenza activity management has been undertaken by the centres for diseases control. influenza a (h3n2) has a uncontrolled and potential infectivity demonstrating a much greater transmissibility than a (h1n1) and b viruses (chen and liao, 2010) . influenza affects all age groups, but it is the elderly and persons with underlying health problems who are at particular risk from complications of influenza and are more likely to require hospitalization. both influenza virus a and b revealed to survive on hard surfaces such as stainless steel and plastic for 24 ± 48 h and on absorbent surfaces such as cloth, paper and tissues for up to 12 h (barker et al., 2001) . asymptomatic patients shed virus and can transmit the disease, thus creating a reservoir for the virus. influenza virus is transmitted by droplets, through the coughing and sneezing of infected persons, but it can also be transmitted by airborne droplet nuclei as well as by contact, either through direct skin-to-skin contact or through indirect contact with contaminated environments (la rosa et al., 2013) . influenza viruses have been detected in different indoor environments, homes, schools, offices and others public buildings. places such as hospitals, where the presence of a susceptible population is often combined with a high population density, may harbour high concentrations of pathogens and therefore pose a considerable risk for the transmission of the virus, with potentially fatal consequences for hospitalized patients. schools are known to have an important role in influenza transmission in the community since children have a higher influenza attack rate than adults (children get the flu twice as often as adults) (zhao et al., 2007) . on buildings ventilation systems influenza a and b were detected (along with other groups of viruses), meaning that contamination exists in the surrounding environment. several reviews consider three modes of transmission of influenza, not mutually exclusive, by large droplets, self-inoculation of the nasal mucosa by contaminated hands and the aerosol transmission, the mode of the greatest impact for infection, since it requires specialized personal protective equipment. influenza virus rna was directly detected in aerosol particles generated by normal breathing in patients with influenza and collected through an orinasal facemask; particles of 5 mm or less have a significant penetration into the respiratory tract all the way to the alveolar region. increasing evidences point towards a role for aerosol transmission in the spread of influenza, at least over short distance where exposure to both aerosol and large droplets occurs. in most settings where there is adequate ventilation, long-range transmission does not appear to occur so frequently (tellier, 2009) . the relative importance of the aerosol transmission route for influenza remains controversial. to determine the potential for influenza to spread via the aerosol route, the authors 6x measured the size distribution of airborne influenza a virus. collected size-segregated aerosol samples during the 2009-2010 flu season in a health centre, a day-care facility and on board of aeroplanes. filter extracts were analysed using quantitative reverse transcriptase polymerase chain reaction. half of the 16 samples were positive, and their total virus concentrations ranged from 5800 to 37,000 genome copies m23. on average, 64% of the viral genome copies were associated with fine particles smaller than 2.5 mm, which can remain suspended for hours. modelling of virus concentrations indoors suggested source strength of 1.6 + 1.2 × 10 5 genome copies m23 air h21 and a deposition flux onto surfaces of 13 + 7 genome copies m22 h21 by brownian motion. over 1 h, the inhalation dose was estimated to be 30 +18 median tissue culture infectious dose (tcid50), adequate to induce infection. these results provide quantitative support for the idea that the aerosol route can be an important mode of influenza transmission (yang et al., 2011) . parainfluenza viruses (pivs) are a further major group of respiratory pathogens. they cause severe colds, croup, bronchitis and pneumonia in children and adults and in infants the virus can cause life-threatening disease. infection is probably spread by aerosols in addition to direct contact with contaminated surfaces. the persistence of piv on hospital surfaces contaminated with patients' secretions was noted as a potential source of transmission (barker et al., 2001) . goals for epidemiological surveillance of influenza include supervise the currently circulating virus subtypes and offer quick response to spread of new subtypes; follow the tendency of the morbidity and mortality to plan strategies to reduce the burden of the disease in public health; define strategies to reduce the occurrence of deaths; monitor the severity standard of the disease by detecting any virulence changes. severe acute respiratory syndrome (sars) is a respiratory illness caused by a type of coronavirus from the coronaviridae family, which can cause mild to moderate upper respiratory illness, such as the common cold or develop into potentially severe pneumonia. this virus is known as sars-cov. the sars epidemic broke out in 2002-2003 in southern china, and spread to other regions of asia and also to europe and north america. it caused more than 8000 infections worldwide with an approximately 10% fatality rate, along with enormous economic losses. one or a few trivial mutations at the receptor-binding surface of a virus may lead to dramatic epidemic outcomes by facilitating cross-species infections and human-to-human transmission of the virus (li, 2013). sars is a condition associated with substantial morbidity and mortality with patterns suggesting droplet or contact transmission (poutanen et al., 2003) . the earliest symptom is a sudden onset of high fever. some patients may also have chills and headaches followed by pneumonia; others showed respiratory distress (severe breathing difficulty) and sometimes death (la rosa et al., 2013) . the most common mode of transmission is contamination by warm air indoor, by water droplets generated by coughs or sneezes, but may be transmitted through the airborne route as well. transmission in an aircraft from an infected person to passengers located seven rows of seats ahead had been described. aerosol generated by the building's sewage systems is also responsible. many health care workers were infected after endotracheal intubation and bronchoscopy procedures which often involve aerosolization. these observations indicate the possible role of more remote modes of transmission, including airborne spread by small droplet nuclei, and emphasize the need for adequate respiratory protection in addition to strict contact and droplet precautions when managing sars patients. contaminated fomites or hospital surfaces might contribute to spread. it is known that these viruses may live on hands, tissues and other surfaces for up to 6 h and up to 3 h after droplets have dried. airborne spread of the virus appears to explain the happened large community outbreaks of sars (ignatius et al., 2004; hui and chan, 2010; la rosa et al., 2013) . a novel coronavirus, mers-cov (ncov, hcov-emc/2012), originating from the middle-east, has been discovered. incoming data reveal that the virus is highly virulent to humans. the members of this group (c) are likely to persist in the environment for a longer period of time and possess the highest oral-fecal components but relatively low respiratory transmission components. oral-urine and saliva transmission are also highly possible (goh et al., 2013) . human adenovirus (adv) is a non-enveloped, icosahedral virus of the genus mastadenovirus, family adenoviridae. there are more than 60 types classified into seven strains from a to g, defined via biological and molecular characteristics. clinical manifestations are highly heterogeneous, ranging from upper and lower respiratory tract infections to gastroenteritis, pneumonia, urinary tract infection, conjunctivitis, hepatitis, myocarditis and encephalitis. the burden of disease manifests as pneumonia, bronchiolitis, otitis media, conjunctivitis, and tonsillitis. the adenoviral detection rates indicate the potential contamination of the environment, with adverse effects on public health. adenoviruses can cause severe or lifethreatening illness, particularly in immunocompromised patients, children and the elderly. some types are capable of establishing persistent asymptomatic infections in tonsils, adenoids, and intestines of infected hosts, and shedding can occur for months or years (lessa et al., 2009; osuolale and okoh, 2015) . adenoviruses can occur anytime throughout the year but adenoviral respiratory infections are most common in the late winter, spring, and early summer. modes of transmission are also diverse, primarily spread by the respiratory route, person-to-person contact, fomites, and occasionally by airborne aerosols. since advs are able to infect a wide range of tissues, they can be excreted in large numbers in different body fluids during the acute illness, including oral secretions and faeces. the spread by the fecal-oral route happens through the ingestion of contaminated food or water. small doses of adv in aerosols resulted in infection accompanied by febrile acute respiratory disease, sometimes with pneumonia. humidity affects the viability and dispersal of advs in aerosol. these viruses tend to survive best at high relative humidities. aerosols seem to be very resistant to uv air disinfection. adenovirus outbreaks have been documented in different indoor environments, including health care facilities, schools, military hospitals and barracks, throughout the year (la rosa et al., 2013) . in developed countries, it is estimated that 30% ± 40% of infectious gastroenteritis cases are attributable to viruses. rotavirus, astrovirus, norwalk-like viruses, also known as small round structured viruses and other caliciviruses are responsible for 48% of all reported outbreaks of infectious intestinal disease. rotaviruses belong to the reoviridae family; they are segmented bicatenary rna viruses, which explain their genetic variability. is the most common cause of severe diarrhea among children, resulting in the death of over 500,000 children annually worldwide. rotaviral gastroenteritis is a serious public health problem in both developed and developing countries. the disease occurs most often in the winter, with annual epidemics occurring from december to june. the highest rates of illness occur among infants and young children. a large proportion of hospital admissions due to gastroenteritis in children less than 5 years old are caused by rotavirus. some adults acquired rotavirus infections a few days after their children's illnesses, suggesting that the children rather than the parents brought infection into the home, though disease tends to be mild. immunity after infection is incomplete, but repeated infections tend to be less severe than the original infection. rotavirus is shed in large numbers from an infected person by animate and non-porous inanimate surfaces. excretion of the virus can persist for up to 57 days after diarrhea has stopped in symptomatic patients, contributing to an increase of the number of environmental surfaces contaminated with rotavirus. it has been suggested that low humidity and people spending more time indoors contribute to the spread of rotavirus infections. handwashing is a very important means of preventing the spread of rotavirus (barker et al., 2001; anderson et al., 2004; bernstein, 2009) . norwalk-like viruses (nlvs), rna virus belonging to caliciviridae family, also known as small round structured viruses or caliciviruses, are an important cause of gastroenteritis outbreaks and are spread frequently through contaminated food or water. projectile vomiting associated with nlvs is probably a major source of cross-infection because it is estimated that 3×107 particles are distributed as an aerosol into the environment during a vomiting attack. carpets can also serve as reservoirs of infection. aerosols produced by vomiting can be inhaled or can contaminate hands or work surfaces, with the potential for subsequent transfer to foods or direct handto-mouth transfer. the importance of airborne transmission was demonstrated in a recent outbreak in a restaurant where no food source was detected but an analysis of the attack rate showed an inverse correlation with the distance from a person who had vomited. it was found that the risk of gastroenteritis amongst workers and customers who shared toilet facilities was twice of those who had a private bathroom (green et al., 2000; barker et al., 2001) . enteroviruses (evs) are members of the picornaviridae family, a large and diverse group of small rna viruses present worldwide. in humans, evs target a variety of different organs causing gastrointestinal, respiratory and myocardial and central nervous system diseases. in temperate climates, enteroviral infection occurs primarily in the summer and early fall. although the majority of infections are asymptomatic or result in a self-limited illness, fatalities do occur, especially in neonates or individuals with b-cell immunodeficiency. enterovirus outbreaks in neonatal units and school nurseries, reflects the susceptibility of infants to evs infection, leading to extensive discussion on control measures and interventions. faecal-oral transmission is the major mode of transmission. other important routes of transmission are person-to-person contact and the inhalation of airborne viruses in respiratory droplets. infectious coxsackievirus, a member of the ev genus, in large droplets and droplet nuclei generated by coughs and sneezes as well as in the air of rooms contaminated by such discharges, transmit this viral infection by the airborne route. aerosol transmission is suspected of having contributed significantly to the ev epidemic which infected up to 300,000 children and caused 78 deaths in taiwan in 1998 (chang et al., 2004; la rosa et al., 2013) . noroviruses (novs), emerging as the leading cause of epidemic gastroenteritis, are rna viruses belonging to the family caliciviridae, currently subdivided into five genogroups. novs are responsible for nearly half of all gastroenteritis cases and for more than 90% of non-bacterial infection epidemics worldwide. the illness can be severe and sometimes fatal, especially among vulnerable populationsyoung children, the elderly and the immunocompromisedand is a common cause of hospitalization. encephalopathy, disseminated intravascular coagulation, convulsions, necrotizing enterocolitis, post-infectious irritable bowel syndrome, and infantile seizures highly contagious with a low infectious dose, occurs repeatedly. faecal-oral spread is the primary transmission mode and the foodborne and waterborne transmission. airborne transmission of nov is also a cause of acute viral gastroenteritis. sources of contaminated aerosol are diverse. droplets being inhaled can be deposited in the upper respiratory tract, and subsequently be swallowed along with respiratory mucus. aerosol droplets produced during vomiting could settle onto indoor surfaces that might then be transferred to hands of exposed individuals through physical contact, or deposited on the floor from which they can be resuspended by human movement and turbulence. aerosol droplets can also be generated from toilet flushing. transmission via fomites is documented. the viruses were identified in indoor environments such as hospitals, schools, kindergartens, restaurants, care facilities, hotels and concert halls as well as airplanes, buses and cruise ships (morillo and timenetsky mdo, 2011; la rosa et al., 2013) . human adenoviruses (adv) are classified into 47 serotypes and six subgenera (a-f) with different tropisms are associated with outbreaks of gastroenteritis in schools, paediatric hospital and nursing homes. they may be second to rotavirus as a cause of gastroenteritis in young children, especially newborns, mainly caused by serotypes adv 40 and adv 41 of subgenus f. the clinical characteristics include watery diarrhea accompanied by vomiting, low grade fever and mild dehydration. institutionalized persons, immunocompromised persons and transplant recipients seem to be among the most severely affected, with mortality rates as high as 60%. respiratory symptoms are infrequent but some studies have suggested that adenovirus infections may be involved with chronic airway obstruction, pulmonary dysplasia, myocarditis and dilated cardiomyopathy, mononucleosis-like syndromes, sudden infant perinatal death and, perhaps most intriguingly, the development of obesity. although these associations may or may not be causal, understanding adenovirus transmission seems to be the key to their further study. adenoviruses are the most uv-resistant viruses, and their detection is now a key indicator of water quality (uhnoo et al., 1990; zlateva et al., 2005; gray, 2006) . indoor air quality proves to be of great importance in hospitals due to the spread of air microorganisms maximizing nosocomial infections. reports about infections correlated with the presence of viral aerosols in indoor air remain scarce. during and after illness, viruses are shed in large numbers in body secretions, including blood, faeces, urine, saliva, and nasal fluid (la rosa et al., 2013) . the microbial load in hospital indoor air is highline nuanced by the number of occupants, their activity and the ventilation. occupants are a potential source of microorganisms as they shed the microorganisms from the skin squamous and the respiratory tract. ventilation causes dilution thus reducing the microbial load. sinks, wash-basins and drains, nebulisers, humidiphiers, and cooling towers are the potential sources which colonize on the moist surfaces. sweeping of floors, changing of bed linens and entry into the hospital buildings through ventilation ducts also can be the sources of airborne microorganisms. since exposure levels are high, this may be an issue for immunocompromised patients (srikanth et al., 2008) . during the 2009-2010 flu seasons, size-resolved particle samples were collected on filters in a day care center and a health center. influenza a virus was identified in 50% of the samples with concentrations ranging from 5800 to 37,000 genomes per cubic meter and a substantial proportion of the detected viruses was associated with fine particles (<2.5 μm) that can remain airborne for extended periods and that can also penetrate and deposit deeply in the respiratory tract when inhaled (barker et al., 2001; nazaroff, 2016) . establishing how viruses are transmitted under different circumstances, and whether transmission requires close contact, is of great importance as such information will affect the choice of infection control measures in health-care settings (la rosa et al., 2013) . in a hospital paediatric unit when there was an increase in the number of children suffering from rotavirus gastroenteritis, on the surfaces in direct contact with children (thermometers, play mats and toys) rotavirus was detected in 63% of samples compared with 36% for surfaces without direct contact (telephones, door handles and washbasins). rotavirus was also found in hand washings of 19% of attendants of patients with non-rotavirus diarrhoea, indicating that they may have come into contact with other attendants and patients in adjacent beds. this highlights the potential for contaminated hands to spread the infection (soule et al., 1999) . norwalk-like virus gastroenteritis in an elderly care unit spread rapidly within and between wards, affecting both patients and staff. although infectious aerosols were probably the main route of dissemination of infection within a particular cohort of guests, contact with contaminated fomites was the most likely factor responsible for maintaining the outbreak by forming the link between successive cohorts (barker et al., 2001) . farming, one of the oldest professions of mankind, is by far the one that employs the largest number of individuals worldwide. although outdoor country work is supposedly healthy, farmers are at risk of respiratory diseases because of their work environment. it is very well established that chronic and acute respiratory diseases have been associated with work in confinement operations, like underfloor manure pits. respirable dust and asphyxiating gases such as hydrogen sulphide, carbon dioxide, methane, and ammonia in high concentrations were measured on these area stations (reeve et al., 2013; manbeck et al., 2016) . but bioaerosols are recognized as a serious threat on these environments. mice are prevalent on farms. faeces of deer mice can be contaminated with the hantavirus, which can cause a devastating infection in humans, an influenzalike syndrome that often leads to respiratory failure. farmers should wear protective respiratory equipment when cleaning building areas where mouse droppings are present. farms are also the usual sources of influenza outbreaks. influenza viruses infect pigs or poultry that can be transferred from animals to humans even without a mutation. this has so far been the case of the influenza a virus subtype h5n1 that, to date, only infected farmers in close contact with birds (cormier, 2007) . in animal slaughterhouses handling out cattle and sheep is hypothesized that these environments would contain significant amounts of bioaerosol due to the mechanical processes used to kill and process animals, a high degree of splashing and fluid handling, and also a high relative humidity of the environment. workers on these conditions and meatworkers having exposure to a number of significant zoonotic diseases including leptospirosis, parapoxvirus, human papillomavirus subtypes hpv2, hpv4 and hpv7 are known to be at occupational health risk. these workers are also affected by a higher-rate of malignancies of the lung compared to the general population (hall et al., 2013) . farmers and consumers of fresh farm products from farms irrigated with river water may be at risk of infection from adenoviruses. the findings highlight the lurking dangers of using contaminated surface water and the need for routine monitoring of such waters for protection of public health (sibanda and okoh, 2012) . workers in concentrated animal feeding operations are at risk of adverse respiratory outcomes from exposures to indoor contaminants. is indispensable an optimal management of indoor air quality, preventing the transmission of infectious respiratory disease to workers and animals (kim et al., 2005) . considering that viruses are obligate intracellular parasites, the use of culturedependent methods is achieved with the use of suitable hosts, such as whole animals or cultured cells. currently the most commonly used methods for virus cultures are inoculation of viruses into embryonated eggs and tissue cultures. virus started to be propagated in whole animals or embryonated chicken eggs before the use of cell culture methods. virus cultivation in embryonated eggs is intrinsically dependent on the utilized egg, which must be sterile and the shell should be intact and healthy. the inoculation of the samples is made by injection into the fluid of the egg through a hole drilled in the shell. viral growth and multiplication is revealed by embryo death, cell damage or through the formation of typical pocks or lesions on the membranes. the selection of the sites of viral inoculation in embryonated eggs is dependent of the studied virus, as each virus has different preferential location for growth and replication. chorioallantoic membrane (cam): virus growth and replication is indicated by visible lesions (pocks) derived, under optimal conditions, from a single virion and a grey white area in transparent cam. allantoic cavity: usually utilized for growth and replication of virus for vaccine production, provide a rich yield of influenza, some paramyxoviruses and avian viruses isolation. amniotic cavity: virus growth and replication of virus can be analyzed by haemagglutination assay. yolk sac: frequently utilized for growth, multiplication and isolation of mammalian viruses. currently, embryonated egg inoculation is conventionally considered the "gold standard" method for isolation and propagation of virus such as the influenza virus (jianqiang zhang, 2014) . mammalian cell culture technology has become a prominent and fundamental field in modern biotechnology, especially in the area of human health and has replaced embryonated eggs as the preferred methodology for virus growth and replication. cellular cultures rely on techniques such as media changes, passaging, and transfection under aseptic conditions to avoid contaminations (e.g. bacteria, yeast, among others). presently, numerous valuable cell monolayers are commercially available, and are regularly utilized in clinical laboratories for the diagnosis of virus infections. some of the most utilized cell are hela derivative (hep2), rhesus monkey kidney cells (rhmk), human lung fibroblasts (mrc-5), human lung carcinoma cells (a549), among others. these cell lines are selected for their ability to support the replication of a wide variety of clinically relevant viruses due to their ability to express cell type-specific factors that contribute to pathology during viral infection. for instance a549 cell line is considered representative of the alveolar type ii pneumocytes of the human lung as it exhibits features of an atii epithelial cell phenotype. applying such methodology enables the isolation of diverse viruses, such as adenovirus, cmv, rsv, influenza a & b, parainfluenza viruses types 1 to 3, vzv, as well as the ebola virus, severe acute respiratory coronavirus (sars-cov), and human metapneumovirus (hmpv). the recent adaptation of cellular cultures to shell vials with subsequent direct or indirect immunofluorescence technique has dramatically decreased the time of diagnostics for clinical samples from weeks to less than 48 h by staining for early antigens of viral infections. currently, this methodology is the most sensitive, non-molecular viral detection method, utilized for the identification of viruses such as respiratory viruses, enterovirus and adenovirus among others. although embryonated chicken egg inoculation is still considered the "gold standard" method for influenza virus isolation and propagation, several primary cells as well as continuous cell lines have also been developed for influenza virus isolation and replication (jianqiang zhang, 2014). the development of nucleic acid amplification techniques has endorsed the advance of molecular tools for virus identification using low specimen quantity with higher sensitivity and specificity, at the same time dramatically decreasing the time for identification. currently these methodologies are accepted as the gold standard for clinical virology and have been utilized for the identification of different viruses in environmental samples from diverse contexts. the fundamental principles of nucleic acid amplification techniques are based on the thermostable polymerase-based target nucleic acid amplification which results in the production of millions of copies of the targeted sequence. these amplification products are then analyzed through diverse techniques. the advances in molecular analysis techniques lead to the development of the real-time pcr where the target amplification and detection steps, using fluorescent dyes, occur concomitantly. this methodology uses copulated software that monitors the thermal cycler data at every cycle and produces a quantitative amplification plot for each reaction. thus, rt-pcr allows the performance of viral load assays to quantitatively assess the amount of virus in a sample. furthermore, a modification of polymerase chain reaction, nested polymerase chain reaction (nested pcr), has been developed in order to decrease non-specific binding in products. this methodology encompasses two sets of primers, used in two successive pcr runs, whereas the second set intended to amplify a secondary target within the first run product. currently, nucleic acid amplification techniques are considered the gold standard for rapid and accurate detection of viruses, compared to methodologies such as shell vial cell cultures. multiplex pcr assays were developed based on single target nucleic acid amplification methodologies with the aim to quantify multiple nucleic acid targets using specific probes to diverse viral targets in a single pcr reaction, allowing the assessment of viral co-infections. currently, for respiratory viruses three fdaapproved platforms for multiplex pcr assays are available. multiplex pcr combined with liquid-phase bead-based array technology. multiplex rt-pcr followed by electrochemical detection of hybridized capture probes on gold-plated electrodes. multiplex-pcr preceded by nested rt-pcr with detection trough melt curve analysis. currently, dna sequencing is considered one of the most valuable, accurate and consistent methodologies for microorganisms identification and is applied in contexts were quick and precise identifications are required. this method is used to determine the exact sequence of a certain stretch of dna. several new sequencing technologies have been developed (next generation sequencing) with the aim to provide fast/efficient techniques for analysis of microorganisms bioburden. genotyping assays utilize a combination of pcr and nucleic acid sequencing to identify viral genotypes. genotyping methodology allows for the identification of genetic variants and can be performed via genotyping chips or arrays, depending on the variants of interest and resources available. viral genotyping is mostly utilized to provide relevant clinical data to predict therapeutic responses to antiviral drugs and/or epidemiologic comparison. currently, it is a consensus among researchers that methodologies based on nucleic acid amplification techniques offer advantages compared with traditional methods such as inoculation on embryonated eggs and cell culture, due to higher sensitivity, specificity and fast results, which will be further discussed. a comparison between the different viral identification methods is presented in table 7 .1. the efficacy of sampling methodologies for viruses in environmental samples is still a matter of intense debate. a meta-analysis study performed with the aim to assess the efficacy of virus concentration methods associated to the molecular detection of adenovirus demonstrated that for detection in environmental samples qpcr or nested-pcr should be prioritized over pcr; in water samples (e.g. rivers or lakes) ultracentrifugation should be associated with nested-pcr and that microfiltration membrane, ultrafiltration, and qpcr must be associated for assessment of treated and untreated sewage samples (silva and melo, 2010) . it is currently acknowledged that viral contamination of recreational water presents a high risk of infection and is considered a significant public health hazard. published studies, summarized in table 7 .2, have addressed the assessment of virus bioburden in water environmental samples. the most commonly found viruses in aquatics environments are enteric viruses, such as enterovirus, rotavirus and norovirus, adenovirus and hepatitis virus a and c. begier and coworkers have correlated swimming in polluted seawater with enterovirus infection (begier et al., 2008) . for enteroviruses, the most common mode of transmission is the fecal-oral route, although aerosol transmission has also been reported. in a similar manner, drinking water contaminated with virus, such as norovirus, also presents a risk for human health, highlighting the importance of good hygiene practices with respect to water storage, shin and sobsey suggested that water chlorination could inactivate enteric viruses (shin & sobsey, 2008) . sample concentration is a critical step in viral diagnosis, since the number of viral particles in water is generally very low, which often results in false results if samples are tested directly using pcr (katayama et al., 2002) . therefore, some authors choose an adsorption-elution method, followed by ultrafiltration, to concentrate the viruses, before nested-pcr or quantitative real time pcr. in severally contaminated environments such as hospital wastewater treatment plants, prado and coworkers utilized pcr/rt-pcr, quantitative real-time pcr (qpcr) and genome sequencing, after sample concentrations techniques, to assess the presence of viruses associated with human pathologies such as acute gastroenteritis and (prado et al., 2011) . in intermediate contaminated environments, such as urban and bathing waters the assessment of microbial hazards with potential public health risk associated with viral contamination also preferentially utilizes nucleic acid amplification methodologies, after sample concentration. some authors chose to utilize the combination of culture dependent and independent methodologies for virus identification from environmental samples. in a study performed by roberto a. rodríguez and coworkers that aimed to assess the effects of sewer overflows to the viral contamination of receiving waters, concentrated samples were assayed for total culturable viruses using the plc/prf/5 cell line with associated confirmation by pcr/rt-pcr (rodríguez et al., 2012) . viruses can be transmitted directly from individual to individual via sneezing, coughing and touching, or indirectly via the environment. the prevalence of pathogenic viruses in healthcare settings potentially transmitted by airborne, droplet and contact represents a significant threat for both workers' and patients' health. thus, most of the presently performed studies regarding virus contamination of surfaces have focused on clinical settings, as demonstrated in table 7 .3. the data resultant from these studies is of foremost importance since it can allow the identification of critical locations and direct the selection of infection control measures for health-care settings to decrease viral associated nosocomial infections. protocols regarding transmission based precautions in both hospital personnel and patients are continuously updated at the international level (la rosa et al., 2013) . et al., 2014) in aerosol samples trough filtration method combined with real-time quantitative polymerase chain reaction (qpcr) technique (tseng et al., 2010) . in hospital settings, torque teno virus has been detected in samples collected with an impactor sampler with posterior culture in tryptone soy agar (tsa) and identification trough nested rt-pcr (carducci et al., 2011) or trough burkard c90m cyclone sampler and qrt-pcr (d'arcy et al., 2014) . nevertheless, culture-dependent methods and nucleic acid amplification techniques can also be used simultaneously. as an example, a study performed by goyal and coworkers utilized embryonated eggs and tissue cultures of vero, mdck, and rk-13 cell lines combined with nucleic acid extraction, pcr, rt-pcr and nucleic acid sequencing to assess contamination of respiratory viruses (vsr, influenza a and b, parainfluenza 1, 2 and 3, rinovirus, enterovirus, coronavirus, filoviruses, adenovirus and orthopoxvirus) and viruses with bioterrorism potential in ventilation filters from two large public buildings (goyal et al., 2011) . the assessment of viral contamination and/or infections play a key role in reducing global viral disease associated burden. it is currently acknowledged that the advances and development of molecular assays such as pcr, qrt-pcr, genotyping and multiplex assays endorsed important improvements in viral assessments regarding sensitivity, specificity, speed, simplicity and cost-effectiveness. nevertheless, the developments of new approaches that can provide alternatives to evade the limitations of nucleic acid amplification techniques are of foremost importance. in this context, currently nanobiosensors represent a new promising tool for virus detection. this methodology, still at research stage, encompasses the technology of viral disease biosensors with nanoparticles and nanomaterials, focused on the development of miniaturized biosensors with high sensitivity, specificity, and stability. this state of the art technology aims to deliver an alternative tool for effective and rapid viral disease diagnosis with no requirement of highly trained personnel or heightened laboratory facilities (kizek et al., 2015) . during the last two decades, there has been increasing concern within the scientific community over the effects of indoor air quality on human health. everything looked definitive when vaccines were produced, diseases had the end announced, but the truth is that infections insist on persisting in our common environment although the better knowledge. changes in building design formulated to improve energy efficiency have meant that modern homes and offices are frequently more airtight than older structures. indoor pollutants can emanate from a range of sources and we know much less about the health risks from indoor air pollution than we do about those attributable to the contamination of outdoor air (jones, 1999) . the indoor environments to be considered are those ordinarily and commonly occupied by humans: residences, offices, schools, industrial buildings, hospitals and other settings occupied a high proportion of the time, or in which occupant density is high (hanski et al., 2012) . biological hazards to man arise from exposure to high concentrations forms of bio-aerosols and three major groups of diseases associated with bio-aerosol exposure are infectious diseases, respiratory diseases and cancer. current knowledge is unclear regarding risk to cancer whether these excess risks occur from exposures to biological agents or are due to various chemicals used in industries (srikanth et al., 2008) . acclimated environments have an artificially multitude of chemical compounds (toxic, carcinogenic, radioactive) and biological (pathogenic) issued by a variety of sources, depending on the physical conditions (air humidity, air temperature, inadequate ventilation) of the environment. the air recirculation phenomenon is responsible for the increase of pathogenic microorganisms in the order of 1000 to 100,000 times in relation to the external air (lee et al., 2006) . incorrect cleaning filters and ducts of air conditioning provide the development of microbial particles including viruses that may lead the occupants of air-conditioned environments contracting respiratory infections or allergic diseases. viruses can persist in sufficient number to act as sources of infection for several hours, weeks or even months. the level of information and awareness of agricultural health and safety risks, disease, and injury prevention among the dairy farmers is low. training on health and safety in agriculture field is urgently needed. safe working conditions are essential for healthy living. the lack of a preventive policy maintenance programmes in refrigeration and ventilation systems is the determining factor for the occurrence of biological pollutants. take corrective actions preventing the spread of pathogens by the airborne route requires the use of special air handling and ventilation systems, such as airborne infection isolation rooms to contain and then safely remove the infectious agents. in addition, respiratory protection with validated and certified equipment is recommended (fisk et al., 2007) . environments have been studied more extensively than other issues due to their greater clinical significance. however, more work is still needed to provide a clearer picture regarding the rates of viral diseases transmission, airborne transmission in particular, in closed environments, and potential ways for reducing the levels of indoor viral pollution and transmission must be investigated. studies including homes, non-industrial workplaces and public buildings, are scarce (prussin ii et al., 2015) . ventilation filters of two large public buildings were sampled to determine the presence of human respiratory viruses by polymerase chain reaction and reversetranscription polymerase chain reaction. nine of the 64 filters tested were positive for influenza a virus, two filters were positive for influenza b virus, and one filter was positive for parainfluenza virus 1. filters are installed in hvac systems of buildings to protect ventilation equipment and maintain healthy indoor air quality. these filters process enormous volumes of air. building hvac filters may be used as a method of detection for airborne viruses. they may yield valuable information on the epidemiology and aerobiology of viruses in air that can report to the development of methods to prevent airborne transmission of viruses (goyal et al., 2011) . regrettably, several investigations have revealed that many hvac installations have a lot of operational and maintenance problems. numerous practical recommendations for design and operation of hvac systems are needed. following the recommendations will result in less pollution and increased indoor environmental quality (hanssen, 2004) . basic strategies of source control should not to make indoor air sterile but keep indoor environments dry, maintain good hygienic conditions in ventilation systems, apply effective filtration on mechanical supply ventilation, and use masks in the event of respiratory illness. the importance of hands in the transmission of viruses is well recognized and many of the studies relate specifically to handwashing. the number of asthma patients in most industrial countries has greatly increased, resulting in a morbidity rate of around 10−15% of the population. may be aerosol transmission is responsible for the most severe cases of disease involving viral infection of the lower respiratory tract (tellier, 2009; tsukagoshi et al., 2013) . rotavirus infection in adults air sampling for respiratory disease agents in army recruits approach to the patient with recurrent infections spread and prevention of some common viral infections in community facilities and domestic homes an outbreak of concurrent echovirus 30 and coxsackievirus a1 infections associated with sea swimming among a group of travelers to mexico rotavirus overview icam-1 on epithelial cells in allergic subjects: a hallmark of allergic inflammation environmental survey to assess viral contamination of air and surfaces in hospital settings standards for the diagnosis and treatment of patients with copd: a summary of the ats/ers position paper exacerbations of chronic obstructive pulmonary disease transmission and clinical features of enterovirus 71 infections in household contacts in taiwan viral kinetics and exhaled droplet size affect indoor transmission dynamics of influenza infection probabilistic indoor transmission modeling for influenza (sub) type viruses respiratory health and farming: an essay environmental viral contamination in a pediatric hospital outpatient waiting area: implications for infection control bioaerosol health effects and exposure assessment: progress and prospects meta-analyses of the associations of respiratory health effects with dampness and mold in homes surveillance of human viral contamination and physicochemical profiles in a surface water lagoon environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit contamination of the hospital environment with gastroenteric viruses: comparison of two pediatric wards over a winter season group a rotavirus detection on environmental surfaces in a hospital intensive care unit prediction of intrinsic disorder in mers-cov/hcov-emc supports a high oral-fecal transmission detection of viruses in used ventilation filters from two large public buildings adenovirus transmission -worthy of our attention taxonomy of the caliciviruses metagenomic detection of viruses in aerosol samples from workers in animal slaughterhouses environmental biodiversity, human microbiota, and allergy are interrelated hvac -the importance of clean intake section and dry air filter in cold climate the common cold severe acute respiratory syndrome and coronavirus a new method for sampling and detection of exhaled respiratory virus aerosols effect of cleaning and disinfection of toys on infectious diseases and micro-organisms in daycare nurseries evidence of airborne transmission of the severe acute respiratory syndrome virus isolation of swine influenza virus in cell cultures and embryonated chicken eggs indoor air quality and health development of a virus concentration method and its application to detection of enterovirus and norwalk virus from coastal seawater temporal and spatial distributions of aerial contaminants in an enclosed pig building in winter nanoscale virus biosensors: state of the art the national human activity pattern survey (nhaps): a resource for assessing exposure to environmental pollutants viral infections acquired indoors through airborne, droplet or contact transmission longitudinal analysis of microbial interaction between humans and the indoor environment relationship between indoor and outdoor bioaerosols collected with a button inhalable aerosol sampler in urban homes health care transmission of a newly emergent adenovirus serotype in health care person nel at a military hospital in texas receptor recognition and cross-species infections of sars coronavirus viral contamination source in clinical microbiology laboratory respiratory viruses transmission from children to adults within a household online design aid for evaluating manure pit ventilation systems to reduce entry risk norovirus: an overview indoor bioaerosol dynamics incidence of human adenoviruses and hepatitis a virus in the final effluent of selected wastewater treatment plants in eastern cape province, south africa screening of respiratory pathogens by respiratory multi well system (mws) r-gene™ assay in hospitalized patients identification of severe acute respiratory syndrome in canada quantification and molecular characterization of enteric viruses detected in effluents from two hospital wastewater treatment plants total virus and bacteria concentrations in indoor and outdoor wintertime factors affecting contaminant distribution in a swine farrowing room the impact of combined sewage overflows on the viral contamination of receiving waters inactivation of norovirus by chlorine disinfection of water assessment of the incidence of enteric adenovirus species and serotypes in surface waters in the eastern cape province of south africa: tyume river as a case study artículo original avaliação de métodos de concentração e detecção molecular de adenovírus em águas não tratadas -uma metanálise monitoring rotavirus environmental contamination in a paediatric unit using polymerase chain reaction bio-aerosols in indoor environment: composition, health effects and analysis airway allergy and viral infection aerosol transmission of influenza a virus: a review of new studies detection of airborne viruses in a pediatrics department measured using real-time qpcr coupled to an air-sampling filter method molecular epidemiology of respiratory viruses in virus-induced asthma enteric adenoviruses influences in allergy: epidemiology and the environment limiting spread concentrations and size distributions of airborn e influenza a viruses measured indoors at a health centre, a day-care centre and on aeroplanes characteristics of bacterial and viral contamination of urban waters: a case study in outbreaks of influenza and influenza-like illness in schools in england and wales surveillance of viral contamination of invasive medical instruments in dentistry chromatography paper strip sampling of enteric adenoviruses type 40 and 41 positive stool specimens key: cord-007796-zggk0x2q authors: lindemans, caroline a.; kimpen, jan l. l. title: the immune response to viral lower respiratory tract infection date: 2005 journal: hot topics in infection and immunity in children ii doi: 10.1007/0-387-25342-4_4 sha: doc_id: 7796 cord_uid: zggk0x2q viruses are responsible for the majority of respiratory infections in childhood,causing considerable morbidity and mortality. it is estimated that in the united states approximately $ 652 million per year is spent on medical costs for respiratory syncytial virus (rsv) related disease alone (paramore et al., 2004). viruses cause a variety of respiratory diseases in children from the common cold to life-threatening pneumonia and bronchiolitis. the host reacts to a viral infection with a combination of innate and adaptive immune mechanisms, usually resulting in the clearance of the virus and clinical recovery. however, there is an accumulating evidence for a number of viral infections that the host immune response actually enhances disease in the course of clearing virus from the infected organs. interestingly, the effectiveness of the immune response seems to be dependent on the age and probably genetic background of the child. this has important implications for treatment as well as vaccine development. viruses are responsible for the majority of respiratory infections in childhood, causing considerable morbidity and mortality. it is estimated that in the united states approximately $ 652 million per year is spent on medical costs for respiratory syncytial virus (rsv) related disease alone (paramore et al., 2004) . viruses cause a variety of respiratory diseases in children from the common cold to life-threatening pneumonia and bronchiolitis. the host reacts to a viral infection with a combination of innate and adaptive immune mechanisms, usually resulting in the clearance of the virus and clinical recovery. however, there is an accumulating evidence for a number of viral infections that the host immune response actually enhances disease in the course of clearing virus from the infected organs. interestingly, the effectiveness of the immune response seems to be dependent on the age and probably genetic background of the child. this has important implications for treatment as well as vaccine development. viral infections play an important role in both childhood and adult asthma. they might be instrumental in the inception of asthma and are associated with the majority of exacerbations in asthmatic individuals (johnston et al., 1995; bont et al., 2000) . in respect to the role of viruses in the pathogenesis of acute and chronic airway disease in children, it is of utmost importance that we gain a proper understanding of the underlying mechanisms involved in order to design effective therapeutic and preventive strategies. although viral respiratory tract infections are considered to be mainly pediatric diseases, there is an increasing acknowledgement of their pathogenic potential in the immunocompromised host of all age groups and in the elderly. causative agent is not identified. when an upper respiratory tract infection in an infant progresses to lower respiratory tract disease, bronchiolitis and pneumonia are most common. both disease entities are hard to differentiate and no clinically relevant differences with regard to outcome have been identified (van woensel et al., 2003) . rsv belongs to the paramyxoviridae family and the genus of pneumovirus. it is an enveloped unsegmented single-stranded rna-virus of which two subtypes are known (a and b) . a clear relationship between subtype and disease severity has not been established (kneyber et al., 1996) . since rsv infection does not lead to complete immunity, reinfection is common. immaturity of the immune system during initial infection seems to be the main cause of incomplete memory-response although an as yet undefined mechanism of partial immune evasion by rsv cannot be ruled out . recently, it was suggested that rsv could cause persistent infection or latency (dakhama et al., 1997; schwarze et al., 2004) , although the significance of this is not clear. rsv affects 70% of infants in the first year of life, and by the age of two nearly all children have been infected. (figure 4 .1) it is likely that a specific balance and timeframe of changes in air temperature and humidity are responsible for the well-defined yearly winter outbreaks of rsv (stensballe et al., 2003) . in most infants as well as in older children and adults, rsv is the cause of upper respiratory tract infection with mild symptoms. however, in the very young, the infection spreads to the lower respiratory tract in approximately 40% of cases. one to three percent of infants develop bronchiolitis or pneumonia requiring hospitalization, with a considerable number requiring mechanical ventilatory support. apart from the obvious respiratory symptoms, very young children frequently present with atypical symptoms such as impaired feeding, vomiting, lethargy, and apnea (kneyber et al., 1998) . several risk factors for more severe disease have been identified, including age less than 6 weeks, prematurity, pre-existent cardiorespiratory disease and immunological impairment (bont and kimpen, 2002) . respiratory symptoms are directly related to airway pathology. necrosis of the airway epithelium is a key phenomenon resulting in sloughing of the epithelial cells. together with a dramatic influx of inflammatory cells into the airways and increased mucus production, this leads to the formation of copious secretions that block the small airways. mucosal edema and bronchospasm through irritation of subepithelial nerve endings further compromise airway diameter. although antibiotics are prescribed for up to 50% of children with lower respiratory tract infections, proof of bacterial superinfection is only found in a minority of patients and the role of this event in the pathogenesis of severe disease remains controversial (purcell and fergie, 2002; bloomfield et al., 2004) . on the other hand, it has been demonstrated that rsv infection of airway epithelial cells in vitro enhances adherence of s. pneumoniae (hament et al., 2004) . the influenza virus belongs to the orthomyxoviridae family and is an enveloped segmented single-stranded rna (ssrna) virus (table 4 .1). the structural proteins of the virion are encoded by separate gene segments and include three viral rna polymerases, nucleoprotein, matrix, and the hemagglutinin (ha) and neuraminidase (na) surface glycoproteins. on the basis of their nucleocapsid and matrix protein antigens, the influenza viruses are divided into three distinct immunological types (a, b, and c). although all three influenza viruses cause respiratory disease in humans, only a and b are known to cause epidemics. induction of a memory-response results in long-lasting immunity and it is the antigenic variation that is responsible for frequent reinfection with the virus. the most antigenic variation is seen in the virus that infects both animals and humans, influenza a. fourteen subtypes of hemagglutinin (h1-h14) and nine types of neuraminidase (n1-n9) are circulating in nature. the segmentation of the genome makes exchange of genetic material between subtypes possible, resulting in the structural changes observed in influenza, which has caused pandemics in the past. when two different subtypes of influenza a virus infect the same cell, major changes (antigenic shifts) can occur through rearrangement of genetic segments from both infecting viruses. minor changes (antigenic drifts) in na and/or ha proteins occur through accumulation of point mutations, and provide a mechanism for the virus to escape protective antibodies and cause respiratory symptoms every year. influenza virus epidemics are difficult to separate in time from rsv epidemics, and the diseases caused by both viruses can also be difficult to differentiate (zambon et al., 2001) . (figure 4 .1) compared to other viruses, morbidity caused by influenza is high in all age groups. children with influenza infection of the respiratory tract are more likely to present with fever. infants aged less than 6 months and older children with an impaired immune system, or other serious health problems, have a higher risk of hospitalization and mortality. subclinical infections with influenza in children are common, suggesting children can be an important reservoir and source of transmission. yearly updated vaccines are available and effective, and recently it has been proposed to extend the current recommendations to children less than 2 years of age, children with recurrent acute otitis media or respiratory tract infections, and healthy children attending day-care centers or elementary schools (principi and esposito, 2004 ). adenoviruses, belonging to the adenoviridae family and the genus mastadenovirus, are a group of dna-viruses of which at least 47 serotypes are known. for lower respiratory disease, subtypes 1, 2, 3, 4, 5, 7, and 21 are most important. it is an icosahedral capsid virus with extruding fiber proteins, which are required for viral entry to epithelial cells (howitt et al., 2003) . although human adenoviruses are ubiquitous, and cause primary infection in the first year of life, there is geographical variation in the distribution of serotypes and in the association of serotypes with different age groups. in europe, adenovirus is the cause of infection in approximately 5% of hospitalized patients with viral lower respiratory tract disease. however, in some south american and asian countries, adenovirus is the second most prevalent pathogen for acute lower respiratory tract infection in children after rsv (carballal et al., 2002) . although adenovirus infections in general occur the whole year round, respiratory adenovirus infections are most common during late winter, spring, and early summer. adenovirus type 7, acquired by inhalation, has been associated with more severe lower respiratory tract disease (larranaga et al., 2000) . subtype-specific immunity occurs. however, some types are capable of establishing persistent asymptomatic infections in tonsils, adenoids, and intestines of infected hosts, and shedding can occur for months or years. children under 6-years old. though it is the causative agent of similar disease entities, hospitalizations occur four times less frequently than for rsv infections (hall, 2001) . two subtypes are clinically important respiratory pathogens in children, piv-1 and piv-3. piv-1 is the main cause of croup in 2-6-year olds, while piv-3 is responsible for parainfluenza bronchiolitis in children under 6-months old. piv-4 only causes mild upper respiratory tract infections. because of acute narrowing of the subglottic region of the larynx, moderate to severe croup may require emergency management with systemic or inhaled corticosteroids, which are effective in improving stridor in a few hours (cetinkaya et al., 2004) . the hallmark cytopathic effect of acute infection with piv-3 is comparable to that of rsv with extensive cell fusion resulting in syncytium formation. for fusion to occur, two piv glycoproteins are required, including the hemagglutinin-neuraminidase (hn) glycoprotein interacting with host cell sialic acid receptor and the viral fusion (f) glycoprotein. rhinovirus infections account for the largest number of respiratory tract infections in children. however, rhinovirus infections produce mild symptoms compared to rsv, piv, and influenzavirus. most of the symptoms caused by rhinovirus are confined to the upper respiratory tract. although present in the community the whole year round, rhinovirus infections peak at the onset of fall, which is probably related to schools starting after summer break. rhinoviruses can cause severe lower respiratory tract infection (guittet et al., 2003; papadopoulos, 2004) and in immunocompromised patients, life-threatening pneumonia. rhinovirus is a positive-stranded rna-virus belonging to the picornavirus family and over 100 serotypes exist, making it difficult to develop an effective vaccine. rhinovirus subtypes have been divided into a major and minor group with respect to the receptor used for cell entry (table 4 .1). major group rhinoviruses use epithelial intracellular adhesion molecule l (icam-1) for cell entry, while minor group viruses bind to the low-density lipoprotein (ldl) receptor. during rhinovirus infection, a predominant granulocyte and monocyte recruitments are observed. while specific antibody production occurs, it is probably not required for viral clearance, although neutralizing antibodies can provide some temporary protection against rhinovirus reinfection (van kempen et al., 1999) . icam-1 blocking antibodies have also been utilized and have been shown to decrease inflammation in vitro. there are, however, indications that rhinovirus can adapt to this with changes in receptor usage (reischl et al., 2001 ). in 2001, a new respiratory virus was identified in the netherlands causing infections similar to rsv in children (van den hoogen et al., 2001) . the reported incidence rate of human metapneumovirus (hmpv) infection in children with acute respiratory symptoms varies between 4% and 16%, of which three-quarters occur in children less than 1-year old (williams et al., 2004) . by the age of 5 years, approximately 70% of children have developed antibodies to hmpv. hmpv very much resembles rsv in its clinical spectrum, varying from coryza to bronchiolitis and pneumonia. however, hmpv is less likely to cause pneumonia than rsv and influenza virus. children with hmpv infection present less frequently with atypical symptoms such as vomiting, and on physical examination, rales and wheezing are found less often. co-infection with rsv and hmpv does occur and has been suggested to result in more severe disease (greensill et al., 2003) . there is some evidence that secondary hmpv infection occurs frequently in childhood, probably accompanied only by mild symptoms (ebihara et al., 2004) . several investigators have found chemokine profiles during acute infection to be different in children with hmpv infections compared to those with rsv infections, with higher interleukin-8 (il-8) and lower rantes concentrations in hmpv patients. however in another study, inflammatory cytokine (il-8, tnf-␣, il-1␤) levels in respiratory secretions were 6-fold lower than in children infected with rsv (jartti et al., 2002; laham et al., 2004) . tthe physiological relevance of these observations remains unclear. the outbreak of severe acute respiratory distress syndrome (sars), which started in late 2002 in east asia, and spread throughout the world during that winter, was found to affect mainly health-care workers and close contacts of diseased individuals. sars, which was proved to be caused by a new coronavirus, induces an atypical pneumonia with fever, dry cough, and shortness of breath. many adults also suffer from myalgia, dizziness, chills, and rigors. it was concluded from postmortem examinations that sars-pathology is primarily caused by immunological damage to the lungs. the interstitial space of the lungs was mainly filled with mononuclear infiltrates and there was diffuse hemorrhage on the lung surface. sars coronavirus (sars-cov) spreads mainly via the respiratory route, the epithelial cell being its primary target cell. as for other coronaviruses, the spike proteins, s1 for cell entry and s2 for fusion, also seem to be important for sars entry of host cells, despite the fact that sars is only 20-30% homologous to other coronaviruses. very recently, angiotensine converting enzyme (ace2) was identified as the host cell receptor for sars-cov (li et al., 2003) and surface expression of ace2 on alveolar epithelial cells was demonstrated (hamming et al., 2004) . transmission of sars-cov occurs by droplets and most cases have occurred through close contact exposure. however, recently evidence of airborne transmission has emerged (yu et al., 2004) . although many individuals were infected in the initial 2 weeks of the epidemic and the disease spread rapidly over several countries, the numbers of infected children stayed relatively low in all regions (ͻ5%). furthermore, children tend to develop less severe disease. after an incubation period of 5-10 days, similar to adults, infected children developed symptoms of a mild upper respiratory tract infection, clinically indistinguishable from other common colds. none of the 100 pediatric sars cases in hong kong turned out to be fatal and only one adolescent required mechanical ventilation (leung et al., 2003) . adolescents are more likely to develop severe disease, as observed in adult sars patients (leung et al., 2004) . a sore throat and a high initial and peak peripheral blood neutrophil count were found to be independent risk factors for severe disease in children with a laboratory-confirmed sars infection. furthermore, children seemed to spread the disease less easily to others and there have appeared no reports in the literature demonstrating transmission from children to other individuals. many children with laboratory-confirmed sars do not meet the who criteria for diagnosis of sars. as children seem to have a much milder clinical course, the term "sars" may not represent the disease in children very well. the role of the innate immune system in viral lower respiratory tract infection has not been studied intensively until recently. studies have focused on the adaptive immunity with the goal of developing a vaccine, for example, for rsv. understanding the mechanisms underlying primary and recurrent viral infection has attracted increased attention. the immunological response against viral invasion of the lower respiratory tract comprises both adaptive and innate immune response mechanisms with both beneficial as well as detrimental characteristics. the innate response occurs in the early phase of the infection and increasing evidence suggests that these early events determine disease course and possibly even long-term outcome (garofalo and haeberle, 2000; tasker et al., 2000) . for the rest of the discussion on immunological phenomena, focus will be on rsv as a prototype. epithelial cells are key regulators of the innate immune response against viral infections (garofalo and haeberle, 2000) , producing a number of inflammatory mediators in response to rsv infection. these include cytokines (interleukin-6, -1, tumor necrosis factor (tnf)-␣), several chemokines (il-8, macrophage inflammatory protein (mip)-1␣, monocyte chemotactic protein (mcp-1), rantes), type-l interferon (ifn-␣/␤), and growth factors (gm-csf, g-csf). epithelial-derived levels of chemokines correlate with disease severity (bont et al., 1999; smyth et al., 2002) . surfactant proteins produced by epithelial cells (sp-a and sp-d) may also play a role as opsonins for viruses and bacteria. thus, epithelial cells provide a potential mechanism for serum-independent phagocytosis. many of these mediators are induced both at the level of secretion and transcription. interestingly, some mediators (e.g., il-8 and rantes) are also upregulated by inactive forms of the virus (harrison et al., 1999) . rsv uptake by immune and non-immune cells is a receptor-mediated process. experiments with blocking antibodies against g-protein revealed inhibition of binding of rsv to epithelial cells. the fractalkine receptor, also known as the cx3cr1 chemokine receptor, is involved in g-protein-mediated uptake by epithelial cells . other receptors may very well be involved in uptake by dendritic cells, macrophages, and other cells of the innate immune system (harris and werling, 2003) , and toll-like receptors (tlrs), especially tlr-4, are being investigated as possible candidates for mediating viral uptake (haeberle et al., 2002a, b; monick et al., 2003) . several groups have demonstrated activation of the transcription factor nf-kb in rsv-infected epithelial cells (tian et al., 2002) . many of the exhibited effects observed in epithelial cells can be explained by activation of nf-kb. several cytokines associated with rsv infection have nf-kb binding sites in their promoter or enhancer regions (bitko et al., 1997) . epithelial nf-kb activation has also been observed in other viral infections, including parainfluenza, influenza a, and rhinovirus (pahl and baeuerle, 1995; kim et al., 2000; bose et al., 2003) . nf-kb could be an exciting target for therapy development and experiments in which balb/c mice were treated with perflubron have confirmed this concept. perflubron has already been shown to be effective in clinical trials of patients with respiratory distress syndrome because of its physical characteristics. besides the beneficial physical effect in improvement of gas exchange and of lung compliance, this agent was found to have anti-inflammatory effects. rsv-infected balb/c mice treated with perflubron intranasally showed a reduction in cellular inflammatory infiltrates and decreased chemokine expression in the lung tissue. both the anti-inflammatory effects were directly linked to interference of perflubron with nf-kb-mediated transcription (haeberle et al., 2002a, b) . the chemokines produced by epithelial cells attract t-cells, neutrophils, monocytes, and possibly eosinophils to the respiratory tract. besides induction of secreted products, epithelial cells upregulate expression of adhesion molecules for neutrophils on their surface, allowing neutrophils to adhere firmly to infected cells (wang and forsyth, 2000) . furthermore, neutrophils are the dominant cell type found in bronchoalveolar lavage (bal) fluid of rsv patients (everard et al., 1994 . however, their role in fighting viral infection is not as well established as in bacterial infections. pathological studies of lungs of rsv-infected calves have shown a major influx of neutrophils in the infected airway mucosa, observed earlier than any other cell type involved. furthermore, neutrophils are the dominant cell type found in bronchoalveolar lavage (bal) fluid of rsv patients (everard et al., 1994 " several chemokines and cytokines involved in neutrophil activation have been associated with rsv lower respiratory tract infections. recently, local neutrophil il-9 production has been linked to rsv bronchiolitis (mcnamara et al., 2004) . as shown by wang et al., a major increase in epithelial damage occurs, when rsvinfected epithelial cells are co-cultured with neutrophils (wang and forsyth, 2000) . this is suggestive of a detrimental role for neutrophil-induced immunopathology in lower respiratory tract infections. rantes and mip-1␣ are produced by the epithelium in response to rsv infection and these chemoattractants recruit eosinophils to the inflammatory site. the analogy between clinical features of virus-induced wheezing illnesses and asthma has made eosinophils an attractive subject for studies aimed at improving understanding of rsv pathogenesis. however, mainly because of their absence in bal of rsv patients, their involvement remains controversial. however, eosinophil-derived cationic protein (ecp) has been linked to bronchiolitis and postbronchiolitic wheezing pifferi et al., 2001; dimova-yaneva et al., 2004) . in vitro, eosinophils have also been shown to be susceptible to rsv. eosinophil priming, superoxide production, and degranulation were induced by incubation with rsv kimpen et al., 1992; olszewska-pazdrak et al., 1998; tachibana et al., 2002) . rosenberg and domachowske (2001) have suggested a beneficial role for eosinophils in rsv bronchiolitis. they identified antiviral properties for the eosinophil based on ribonuclease activity of eosinophil-derived neurotoxin (edn) and ecp. this enzymatic activity leads to destruction of extracellular ssrna virions and delayed replication both in vitro and in vivo. recently, there has also been great interest in the involvement of macrophages and dendritic cells in rsv pathogenesis. these cells were already appreciated for their role in antigen presentation, at which dendritic cells are by far superior. macrophages express phagocyte activity, which may be of importance in clearance of infected epithelial cellular debris. fascinating new players in host defense against viruses are pattern recognition receptors. toll-like receptor-4 (tlr-4) and cd14, both present in a complex on these cells, have been found to interact with rsv and receptor-binding results in triggering of the innate immune system. tlr-4 has been shown to activate nf-kb in macrophages of rsv-infected mice (haeberle et al., 2002a, b) and tlr-4-deficient mice have impaired nk-cell and cd14ϩ cell trafficking and delayed viral clearance . furthermore, intracellular pattern recognition receptors tlr-3 and -7, may be involved in recognizing doubleand single-stranded rna (dsrna/ssrna), respectively (akira and hemmi, 2003; lund et al., 2004) . dsrna is produced during replication of rna-viruses and is a potent inducer of ifn-␣/␤. all human cells can produce ifn-␣/␤ in response to viral infection, while only t-cells and nk-cells produce ifn-␥. dsrna also activates dsrna-dependent protein kinase r (pkr) and nf-kb via distinct pathways. transcription of pkr is under control of ifn-␣/␤. pkr controls enzymes directly involved in protein synthesis, thereby inhibiting cellular and viral protein translation. ifn-␣/␤-deficient mice as well as pkrϫ/ϫ mice are extremely sensitive to influenza infection (balachandran et al., 2000) . several viruses, including rsv, have evolved mechanisms to escape the interferon system, which will be discussed below. respiratory epithelial cells are the principal host cells for viral pathogens in lower respiratory tract disease. the degree of replication and the mechanism of spread along the epithelial layer depend on the virus family characteristics. through the fusion (f) protein, rsv is capable of syncytium formation, which allows it to replicate and spread relatively undetected by the immune system for a relatively long period. the virus itself is directly responsible for cytopathology and viral envelope proteins are expressed on the surface of infected epithelial cells. dendritic cells, lining the basal membrane of the respiratory epithelium encounter rsv, pick up viral antigens and migrate to mediastinal lymph nodes where viral antigen is presented to naïve cd4ϩ t-cells. antigen presentation and co-stimulatory molecule expression lead to maturation to the t-helper phenotype. this then induces b-cell proliferation with the production of specific antibodies as well as proliferation of virus-specific cytotoxic cd8-cells. cellular responses are responsible for controlling and terminating acute infection with rsv. in primary infections, the adaptive cellular immune response develops within 10 days. these cd8ϩ cells can recognize and eliminate virus-infected epithelial cells resulting in perforin-mediated cytotoxity. epithelial cells are nonprofessional antigen presenting cells (apc) expressing mhc class l on the surface (garofalo et al., 1996) . when infected, epithelial cells present viral antigen in association with mhc class l molecules. mhc class l restricted antigen presentation to cd8ϩ cells, among other factors, may determine the strength of the cytotoxic response. in cd8-deficient mice, there is delayed viral clearance; however, these mice also exhibit decreased disease severity (graham et al., 1991) . therefore, it is conceivable that cd8ϩ t-cells are crucial in viral clearance while a surplus of cytotoxicity may result in pulmonary injury. in humans, a cytotoxic t-cell response is elicited against all viral proteins, except the g-(attachment)-protein, which is required for cell entry (bangham et al., 1986; hacking and hull, 2002) . it is suggested that a defective response against g-protein is directly associated with enhanced disease. however, g-protein can induce a cd4ϩ response in mice, which is associated with th2-cytokine production and eosinophilia both during primary and secondary infection (openshaw, 1995) . the immune response to the f-protein is dominated by ifn-␥ production and subsequent polarization toward a th1-type cellular response, and therefore it has been postulated that responses to the other viral proteins can modulate the strong th2response to g-protein (graham et al., 2000) . a stronger th1-response seems to induce a more rapid viral clearance and milder disease (bont et al., 1999; legg et al., 2003) . besides activated t-cells, nkcells also produce considerable amounts of ifn-␥ (hussell and openshaw, 1998) . ifn-␥ has important antiviral effects and provides a link between adaptive and innate immune system. it can induce expression of tnf-related apoptosis inducing ligand (trail) on immune cells, which has the potential to trigger apoptosis of virus-infected cells (sedger et al., 1999) . in vitro findings suggest that rsv-infected cells in vivo are susceptible to killing by immune cells through the trail pathway (kotelkin et al., 2003) . nk-cells are also thought to play a role in activating cd8ϩ cells, further modulating the degree of cytotoxicity (hussell and openshaw, 1998) . in summary, in rsv lower respiratory tract infections, cytotoxic cd8ϩ t-cells are involved in viral clearance while the humoral response is required for the protection against reinfection. however, as has been discussed before, memory is incomplete and repeated infections with rsv are common. both igm and igg as well as secretory iga against rsv are formed in infants, and a more vigorous antibody response seems to be protective against rsv infections (meurman et al., 1984; welliver et al., 1989 ). rsv infections are most severe in the youngest age group, which is the least mature in terms of immunity to infections. relative deficiencies in both innate and antigen-specific immunity in infancy have been characterized. these include delayed trafficking of immune cells, less-efficient antigen presentation by dendritic cells, and impaired production of ifn-␥ by t-cells in response to antigen presentation (bont and kimpen, 2002) . the fetus derives maternal igg-antibodies via the placenta fairly late in gestation. this partly explains why prematurity is an important risk factor for severe disease caused by rsv, as well as the physiological characteristics of the small airways. antibody titers produced by infants are relatively low compared to older children. trials with humanized monoclonal antibodies against rsv-f-protein have shown a 50% reduction in rsv lower respiratory tract-related hospitalizations in this highrisk group for severe disease (impact study group, 1998). the cytokine milieu at the time of infection is another factor possibly contributing to the occurrence of severe rsv bronchiolitis especially in the youngest age group. at birth, there is skewing toward a th2-phenotype and rsv bronchiolitis was long thought to be a th2-type disease. this role of th2-skewing is an attractive concept, because it provides some explanation for the association between rsv bronchiolitis and the development of asthma. asthma and allergy have long been acknowledged to beth2-mediated conditions. however, convincing evidence that primary rsv infections are mediated by th2 cytokines is lacking. dendritic cells are thought to have an important function in skewing the th1/th2-ratio. viruses may be important in maturation of dendritic cells, which can then drive differentiation of naive t-cells into either a th2-or a th1-phenotype. the role of regulatory t-cells that suppress both th1 and th2 differentiation has not been studied in rsv bronchiolitis so far. gene polymorphism studies have been undertaken to identify a genetic background to explain the individual susceptibility to rsv lower respiratory tract infection. several polymorphisms situated in genes relevant for the adaptive and innate immunity have been found to correlate with occurrence of rsv infection. polymorphisms of interleukin-4, il-4r, and its receptor, have been associated with rsv bronchiolitis, which is consistent with the th2-hypothesis (choi et al., 2002; hoebee et al., 2003) . very recently, a polymorphism of the gene coding for interleukin-10 (il-10) was found as well (hoebee et al., 2004) . this is particularly interesting since il-10 is a cytokine produced by t-regulatory cells and monocytes, thought to be primarily involved in development of allergy. gene polymorphisms involved in innate immunity include surfactant proteins spa and d (lahti et al., 2002) , the chemokine il-8 (hull et al., 2001) , tlr-4 (tal et al., 2004) , and the chemokine receptor for rantes and mip-1␣, ccr5 (hull et al., 2003) . immunocompromised patients have a higher risk of developing severe disease from viral respiratory tract infections. in particular, the presence of defects in cellular immunity result in an increased duration of viral shedding and enhanced risk of developing severe disease. most cellular immunodeficiencies are iatrogenic in nature. an important cause is intensive immunosuppressive treatment. the number of pediatric patients undergoing organ or stem-cell transplantation is increasing and high doses of chemotherapeutic and immunosuppressive agents are often used in the pre-and posttransplant regimens. immunosuppressive drugs are used in cancer treatment regimens and for a number of inflammatory conditions. community acquired respiratory viruses such as rsv, rhinovirus, adenovirus, influenza a, influenza b, and the parainfluenza group are frequent causes of respiratory disease in these patients (soldatou and davies, 2003) . adenovirus infections have a particularly high risk of adverse outcome, mortality rates are high, and no effective treatment exists. the presence of lower respiratory tract infection and infection in the pre-engraftment phase of hsct is believed to have a particularly poor prognosis (khushalani et al., 2001) . the risk of severe disease is higher during allogenic hsct than autologous hsct. besides causing increased morbidity and mortality, respiratory tract infections are associated with a greater risk of delayed engraftment (abdallah et al., 2003) . in solid organ transplant patients, respiratory virus infections are also associated with a higher incidence of rejection (wendt, 1997) . prolonged shedding of respiratory viruses for weeks or months has been documented in hiv-infected adults and children. this has important implications for infection control in medical facilities. in addition, respiratory viral infection may result in increased hiv replication and, theoretically, hiv disease progression (king, 1997) . in hiv-infected children, rsv infections are less limited by season (madhi et al., 2000) . however, generally, the course of rsv infections in hiv patients is not more severe, unless there is profound lymphopenia or pre-existing lung disease (soldatou and davies, 2003) . other viruses may also cause respiratory complications in the immunocompromised patients. in particular herpesviruses, such as cytomegalovirus (cmv) and varicella zoster virus, can cause severe pneumonia. with a cmv-negative donor and a cmv-positive recipient, there is an especially high risk of reactivation which may lead to severe disease. this reactivation also occurs with epstein barr virus (ebv), human herpes virus (hhv)-6, -7, and -8, although these are much less frequent causative agents of pneumonia. the innate immune defense to viral respiratory tract infections consists of the mucosal layer, type 1 interferons, activated phagocytes, and nk-cells. the impact of primary defects in the innate immune defense has not been well documented. phagocyte defects are primarily related to a higher incidence of bacterial infections. one indication that impaired phagocyte function also leads to increased severity of respiratory viral infection can be derived from a report of severe abnormalities on lung-ct-scans of rsv patients with phagocyte defects (uzel et al., 2000) . interferon-gamma receptor deficiency, which may have implications for both the adaptive and innate immune system, has also been associated with increased susceptibility to viral respiratory pathogens (dorman et al., 1999) . chronic lung disease also increases susceptibility to respiratory viruses (meert et al., 1989; griffin et al., 2002) . premature patients with bronchopulmonary dysplasia are candidates for rsv-immunoprophylaxis because of their increased risk of developing severe lower respiratory tract infections. in children with cystic fibrosis (cf), 39% are already hospitalized with respiratory virus infection in their first year of life. furthermore, there is a correlation between viral infections in infancy and disease progression. infants with cf suffering from a respiratory virus infection are at significant risk for lower respiratory tract disease, hospitalization, and deterioration in lung function that persists months after the acute illness (hiatt et al., 1999) . cf infants were found to be four times more likely to develop an lrti compared with controls. it has been shown that cfderived airway epithelial cells allow a higher degree of piv replication and have an increased production of pro-inflammatory cytokines (zheng et al., 2003) . cfderived epithelial cells are also unable to express no-synthase 2, which results in a decrease production of nitric oxide (no), which has antiviral capacity, reducing effects on replication. furthermore, in cf-cells there is no viral induction of 2ј5јoligoadenylate synthetase (oas), an enzyme that is normally induced by dsrna and ifn-␥. oas is involved in inhibition of cellular protein synthesis, thereby inhibiting viral replication. respiratory viruses can be isolated from the secretions of approximately 75% of children and of more than half of adults during asthma exacerbations (johnston et al., 1995; lemanske, 2003) . recently, copd exacerbations have also been attributed to viral infection by rhinovirus, rsv, and piv (seemungal and wedzicha, 2003) . the underlying mechanisms for this are, however, still a matter of debate. from experimental rhinovirus (rv) infections in humans, it has been shown that rv infection causes increased bronchoconstriction in atopic non-asthmatic and asthmatic individuals, while symptoms in normal individuals are relatively mild. this implies that induction of a wheezing episode requires both rv infection and a preexisting tendency to develop allergic or asthmatic disease (message and johnston, 2001) . rv-specific t-cell responses can be activated by either serotype-specific or shared viral epitopes. cross-reactivity between rv-subtypes could result in vigorous t-cell responses and may amplify allergic inflammation. other proposed mechanisms linking viral infections to asthma exacerbation are epithelial dysregulation, airway remodeling, the immune response to virus, and alterations of neural responses (message and johnston, 2001; gern, 2002) . upregulation of icam-1-expression, which is the entry receptor for major group rhinoviruses, has been found in susceptible individuals. this may be one mechanism predisposing atopic individuals to rv-induced exacerbations. rhinovirus can induce a number of inflammatory mediators (kinins, arachidonic acid) and cytokines (e.g., il-1, il-6, ifn-␣/␤, gm-csf, tnf-␣) that can further enhance inflammation. th1 cytokines seem to have a general antiviral effect while a predominant th2-cytokine response leads to enhanced disease, failure to clear the virus, and amplification of allergic inflammation (message and johnston, 2001) . eosinophil numbers were found to be increased in bronchial biopsies from both healthy and asthmatic human volunteers after experimental rhinovirus infection. this cell type is associated with allergic inflammation in the lung. in allergic rhinitis patients, the increased level of eosinophils in bal even persisted for 6 weeks. these data suggest a potential role for eosinophils in virus-induced asthma, which can be either pathogenic or protective. virus-induced exacerbations of asthma tend to be resistant to treatment with corticosteroids and may require a different therapeutic approach. in vitro, blocking icam-1 has been tried with positive results, which may be of particular relevance to rhinovirus infections. the possibility of other immunomodulating drugs is being investigated and may be of significant benefit to future asthma treatment. a causal relationship between viral respiratory tract infections and asthma exacerbations is generally acknowledged. however, the suggestion that respiratory virus infection is a causal determinant in the development of asthma is highly controversial. according to the hygiene hypothesis, viral infection would be expected to have an inhibitory effect on the development of asthma (an allergy), and this is supported by a study from matricardi et al. (1997) , showing an inverse relation between hepatitis a seropositivity and atopy among soldiers. the hygiene hypothesis is based on the theory that the immune system is directed toward a more th1-skewed immune response with each viral infection. however, this hypothesis is not supported by the observation that rsv infections, severe enough to cause bronchiolitis, are significantly associated with a higher incidence of asthma up to the age of 7-11 years (stein et al., 1999; sigurs et al., 2000) . these data convincingly show a link between rsv bronchiolitis and recurrent wheezing in childhood. in a recent study, wheezing following rsv lower respiratory tract infection was found to develop independent of atopy . it may, however, be true that the transmission route, the organs involved, and exposure to microbial products may be important in determining the final effect of a virus infection on the development of asthma and allergy (gern and busse, 2002) . the link between rsv infection and atopy is even less clear than the one with recurrent wheezing and asthma, at least in humans. animal studies have yielded conflicting results. one group found that rsv infection in mice enhances subsequent allergic inflammation (schwarze et al., 1997) , while others reported a decrease in allergic sensitization and bhr after rsv infection (peebles et al., 2001) . no proof exists that severe rsv infections are associated with atopy that persists into adulthood (peebles, 2004) . a key question is whether the association with the development of asthma is merely an expression of increased susceptibility to both asthma and rsv-induced lower respiratory tract infections or whether true causality is involved. the prevailing theory on this subject involves maturation effects of the th1/th2-balance. the system shifts from a th2-polarization in fetal life, which is an optimal environment for the placenta, to a more balanced th1/th2-phenotype in adulthood. most viruses are known to induce a th1 cytokine response (ifn-␥). this theory states that when infections occur early in infancy, there is a reduced ability to react with an appropriate antiviral th1-response. low ifn-␥ production may result in spread of the virus to the lower respiratory tract. this is in agreement with findings that in children with severe rsv lower respiratory tract infections, lower amounts of ifn-␥ are produced . the dynamics of this shift toward a more balanced th1/th2 immune response may differ between individuals. both environmental factors and genetic make-up may contribute to a slower maturation of th1 competence in some individuals. respiratory virus infections in infancy and an atopic sensitization to aero-allergens, both of which are related to th2-skewed responses and intermittent wheeze, may than synergistically result in persistent wheeze (holt and sly, 2002) . it is likely that more links between atopic sensitization and respiratory infections exist, while preventive rsv-ivig treatment of children results in a decreased sensitization to aeroallergens as well. rsv-prophylaxis may therefore have a long-term benefit in the development of persistent wheeze (piedimonte and simoes, 2002; wenzel et al., 2002) . another theory linking viral infections in childhood to the development of asthma involves the pathologic effects of viral lower respiratory tract infections on airway physiology. wall thickening with consequent increased resistance may predispose the airway to more infections and thus influence bronchial hyperreactivity (bardin et al., 1992) . however, it may also be true that small airways predispose to both asthma and airway symptoms from viral infections. remodeling of the submucosal neural networks by rsv, as observed by piedimonte et al., is also proposed to result in increased responsiveness to airway irritants (piedimonte, 2002) . walter et al. (2002) have proposed that paramyxoviral infection has the ability not only to induce acute hyperresponsiveness, but also to result in long-lasting changes in airway behavior. from mouse-studies with a piv (sev), it has been concluded that viruses cause long-term effects in epithelial cells, associated with airway reactivity and goblet cell hyperplasia. long-term effects are induced by the virus in the acute phase, and later on, the presence of virus is no longer required for the persistence of symptoms. it is speculated that primary paramyxoviral infection within the proper genetic background may result in chronic dysfunction of epithelial cell behavior. their results have indicated that different mechanisms are responsible for the induction of the acute and the chronic response (walter et al., 2002) . several theories on the induction of asthma have thus been proposed. the current view is that virus infection modulates the development of an asthmatic phenotype in a susceptible host. the relationship is, therefore, not purely causal but certainly requires an intrinsic vulnerability. the effectiveness of respiratory virus infections in the host depends partly on the ability to evade the immune system (figure 4 .2). while several viral evasion mechanisms have evolved, not all have been intensively studied in respiratory viruses. viral entry into host cells is one of the first obstacles viruses have to overcome. since the cell membrane is in principle impermeable to macromolecules, viruses 56 caroline a. lindemans and jan l. l. kimpen must first have an effective method to attach to the cell membrane. some viruses bind putative cell surface receptors that do not simply play a role in viral attachment, but also allow viral entry by inducing endocytosis. for some viral pathogens, such as rhinovirus, these receptors have been identified (icam-1 and ldl-r), while for others, such as rsv, the receptor that is used for cellular entry has not been unequivocally defined. the cx3cr1-chemokine receptor, also known as fractalkine-receptor, may be involved and tlrs have also been proposed to play a role. many enveloped viruses have glycosylated proteins, which not only bind to cellular receptors, but also have additional functions as membrane fusion factors, or receptor-destroying enzymatic activity. membrane fusion factors such as the rsv fprotein also allow cell-to-cell transmission of virus, which keeps it relatively hidden from the cellular immune system (smith and helenius, 2004) . escaping the "interferon signaling system" is one of the common mechanisms most viruses have acquired. as mentioned earlier in this chapter, both ifn-␣/␤ and ifn-␥ have potent antiviral properties. both types of interferon regulate transcription of a variety of target genes through activation of interferon inducible transcription factors. ifn-stimulated genes encode a variety of cellular enzymes, including pkr and 2ј5 ј-oligoadenylate synthetase, both involved in inhibition of viral protein synthesis. furthermore, interferons induce cellular apoptosis and upregulate mhc1 expression, targeting cells for cd8ϩ t-cell-mediated cytotoxicity. additionally, ifn-␥ activates the adaptive cellular immune system. rsv infection leads to an increase in ifn-␣/␤, but does not induce ifn-␥ from mononuclear and nk-cells as efficiently. despite the fact that interferons are known for their antiviral properties, intranasal administration of either lfn-␣/␤ or ifn-␥ in the airway does not lead to reduction of symptoms of viral respiratory infections (ramaswamy et al., 2004) . this is suggestive of a viral mechanism to evade the host's interferon response. the paramyxoviridae family (figure 4 .3), responsible for a large part of children's respiratory infections, consists of two subfamilies based on the structural differences in the gene encoding for the polymerase complex (p-protein). the paramyxovirinae subfamily members are able to block interferon-mediated promoter activity. these paramyxovirinae, to which parainfluenza-, measles-, and mumps virus belong to, have a p-gene that encodes for additional proteins besides the p-protein, the v-proteins. it is these v-proteins that have been found to be responsible for evading the interferon signaling pathways in this group of viruses. ifnmediated transcription is predominantly mediated by signal transducers and activators of transcription (stat). v-proteins have the ability to block interferon-mediated signaling by targeting stats for proteosomal degradation (horvath, 2004) . in contrast, rsv, belonging to the pneumovirinae subfamily, fails to inhibit ifn-induced promoter activity. the pneumovirinae consist of only one genus, the pneumovirus, which also includes hmpv. in this subfamily, p-genes only encode for the p-protein and therefore rsv cannot block interferon-mediated signaling (young et al., 2000) . however, recently it was demonstrated that, although rsv does not inhibit interferon-induced promoter activity, rsv replication is still resistant to ifn treatment of infected cells. apparently, an alternative mechanism to circumvent the interferon antiviral response exists. this has been attributed to additional proteins, characteristic of these pneumoviruses (spann et al., 2004) . these are nonstructural proteins (ns1 and ns2) that have no homologs in the paramyxovirinae. however, the underlying molecular pathway has not yet been elucidated. rsv infection of epithelial has been shown to lead to an upregulation of trail-receptor expression on these cells (kotelkin et al., 2003) . apoptosis of infected cells is an effective way to eliminate intracellular pathogens without damage to the surrounding tissue (figure 4.4) . however, several respiratory viruses have developed mechanisms to inhibit apoptosis. it has been demonstrated that rsv is able to effectively inhibit apoptosis of epithelial cells in vitro, in accordance with the limited pathology induced by rsv in epithelial cells during the first few days of the infection (thomas et al., 2002) . eventually, necrosis is observed when mature viral particles are released from the cells, after 2-3 days. furthermore, experiments with both adult and cord blood monocytes have shown a prolonged longevity of cells, when cultured in the presence of rsv (krilov et al., 2000) . of all respiratory viruses, viral evasion techniques of adenoviruses have been studied most intensively. it appears that approximately a third of the adenovirus genome is devoted to counteracting innate and adaptive immune defenses (burgert et al., 2002) . adenoviruses encode the protein e1a that blocks interferon-induced gene transcription. through the va-rna protein that blocks activation of pkr, they also interfere with the antiviral enzymes that are synthesized under interferon control. additionally, adenoviruses have developed several mechanisms to inhibit both constitutive and death receptor induced apoptosis (figure 4.4) . the e1b/55k protein inhibits p53-mediated apoptosis, e3/19k interacts with pro-apoptotic proteins bax and bak, whereas several e3 proteins are involved in removing fas and trail (death) receptors from the cell surface by promoting their degradation in lysosomes (wold et al., 1999) . finally, adenoviruses interfere with recognition of infected cells by cytotoxic lymphocytes. the adenovirus e3/19k protein inhibits transport of mhc1 molecules to the cell surface, resulting in decreased viral antigen presentation to cd8ϩ cells. future studies may unravel further mechanisms of immune evasion that may also be important in viruses involved in lower respiratory tract disease. this knowledge extrinsic and intrinsic signaling pathways of apoptosis. some viruses such as adenovirus interfere with cellular apoptosis. cellular apoptosis can normally occur via activation of the extrinsic (death receptor) pathway or the intrinsic pathway. both lead to activation of effector caspases 3 and 7, which will inevitably result 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respiratory syncytial virus immune globulin human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic paramyxoviridae use distinct virusspecific mechanisms to circumvent the interferon response evidence of airborne transmission of the severe acute respiratory syndrome virus contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study impaired innate host defense causes susceptibility to respiratory virus infections in cystic fibrosis is likely to be crucial to the improvement of immunotherapies for prevention and treatment of viral respiratory tract infections. key: cord-016990-ot1wi3xi authors: zaki, sherif r.; paddock, christopher d. title: viral infections of the lung date: 2008 journal: dail and hammar&#x02019;s pulmonary pathology doi: 10.1007/978-0-387-68792-6_11 sha: doc_id: 16990 cord_uid: ot1wi3xi the lungs are among the most vulnerable to microbial assault of all organs in the body. from a contemporary vantage, lower respiratory tract infections are the greatest cause of infection-related mortality in the united states, and rank seventh among all causes of deaths in the united states.2,3 from a global and historic perspective, the scope and scale of lower respiratory tract infection is greater than any other infectious syndrome, and viral pneumonias have proven to be some of the most lethal and dramatic of human diseases. the 1918–1919 influenza pandemic, perhaps the most devastating infectious disease pandemic in recorded history, resulted in an estimated 40 million deaths worldwide, including 700,000 deaths in the u.s.4 the global outbreak of severe acute respiratory syndrome (sars) during 2003, although considerably smaller in scale, resulted in 8098 cases and 774 deaths5 and is a dramatic contemporary example of the ability of viral pneumonias to rapidly disseminate and cause severe disease in human populations. the lungs are among the most vulnerable to microbial assault of all organs in the body. from a contemporary vantage, lower respiratory tract infections are the greatest cause of infection-related mortality in the united states, and rank seventh among all causes of deaths in the united states. 2 ,3 from a global and historic perspective, the scope and scale of lower respiratory tract infection is greater than any other infectious syndrome, and viral pneumonias have proven to be some of the most lethal and dramatic of human diseases. the 1918-1919 influenza pandemic, perhaps the most devastating infectious disease pandemic in recorded history, resulted in an estimated 40 million deaths worldwide, including 700,000 deaths in the us. 4 the global outbreak of severe acute respiratory syndrome (sars) during 2003, although considerably smaller in scale, resulted in 8098 cases and 774 deaths s and is a dramatic contemporary example of the ability of viral pneumonias to rapidly disseminate and cause severe disease in human populations. although viruses are commonly identified causes of pneumonia of infants and young children, they are relatively infrequently recognized as agents of communityacquired pneumonia in adults. 6 in several large series that investigated a microbiologic cause of community-acquired pneumonia, viral etiologies were identified in only 9% to 18% of cases (table 11 .1).7-12 however, it is likely that viral pneumonias are underrecognized and underdiagnosed for various reasons. although some agents may cause distinct cytopathology or inclusions (e.g., adenoviruses, herpesviruses, and paramyxoviruses), many important pathogens (e.g., influenza viruses) do not, and none of these agents are resolved specifically in tissue by routine histologic stains. viruses require live cells for cultivation, and are generally more difficult than bacteria to isolate from clinical samples. for some viral pneumonias, 426 the pathogen appears to initiate a cascade of destructive host responses that continue or progress even in the absence of the specific infectious agent, and in these patients the etiologic agent may be absent from host tissues at the time of autopsy.13 thirty to sixty percent of community-acquired pneumonias are etiologically undetermined, 14 and it is entirely possible that viruses directly cause more episodes of pneumonia than currently appreciated. because viral infections of the lower respiratory tract often precede bacterial pneumonias, viruses may indirectly exert considerable influence on the cumulative morbidity and mortality of infectious pneumonias. 15 , 16 the mechanisms by which viruses may facilitate bacterial invasion of the respiratory tract are complex and varied. certain viruses cause the death of ciliated respiratory epithelium and thereby disrupt normal ciliary activity. viruses may also inhibit the phagocytic or bactericidal activities of neutrophils, t lymphocytes, and alveolar macrophages, and predispose the host to secondary infections. certain gram-positive and gram-negative bacteria adhere to and colonize virus-infected epithelium more readily than to noninfected cells by various hypothetical mechanisms, including alteration and induction of receptors at the host-cell surface and changes in the extracellular environmenty-19 finally, viral infections of the lung may exacerbate noninfectious pulmonary conditions (e.g., asthma and chronic obstructive pulmonary disease) and indirectly contribute to aggregate morbidity and mortality associated with these conditions. 20 although influenza viruses remain the most frequently identified cause of viral pneumonia in adults (table 11 .1), the diversity of agents identified as causes of viral pneumonias has expanded considerably. several newly recognized viral pneumonias have been identified since 1992 that are among the most feared and lethal of all emerging infections, including those caused by hantaviruses, nipah virus, and sars corona virus (co v). !3,21-23 certain causes "rubella (2), rhinovirus (1) , mixed viral infections (3) . source: adapted from greenberg. 6 of viral pneumonia, particularly those that occur in vulnerable patient cohorts, have diminished during this same interval. by example, the u.s. incidence of varicella pneumonia has dropped by two thirds since universal childhood vaccination for varicella was implemented in 1995, 24, 25 and advances in the clinical management of transplant recipients have reduced the incidence of cytomegalovirus (cmv) pneumonia?6 also occurring during the last decade has been the development and use of powerful molecular techniques that have unveiled the identity of historic pathogens (e.g., the hini "spanish" influenza a virus),27.28 and have facilitated the rapid characterization of emerging agents (e.g., sars-co v). 13 it should be noted that the disease manifestations of several of these agents (e.g., human parainfluenza virus [hpiv] , respiratory syncytial virus [rsv] , and influenza) are often confined to the upper airway and are not invariaorb rsv hpiv adeno cmv varicella other' 10 0 3 0 0 3 27 1 4 3 0 0 0 7 2 0 0 1 0 19 5 5 0 0 1 7 8 13 15 0 0 0 37 5 11 5 0 0 3 21 (11) 32 (16) 30 (15) 1 (0.5) 1 (0.5) 6 (3) ably associated with pneumonia. with some of these pathogens (e.g., influenza viruses, rsv, hantaviruses, and hpiv viruses) respiratory disease is the primary manifestation. for other agents, such as measles, nipah virus, and herpesviruses, typically the lungs are involved as part of a multisystem syndrome. the diagnosis of viral pneumonia, suspected by patient history and clinical manifestations, also can be supported histopathologically, and the general pattern of histopathologic lesions may suggest a specific diagnosis. many viruses can be identified in lung by examining the tissue response and cytopathic changes. some of these viruses cause recognizable tissue reaction patterns including necrotizing tracheobronchitis, bronchiolitis, and interstitial pneumonia. a summary of the key diagnostic histopathologic and ultrastructural features for the most common viral pathogens that cause a majority of pulmonary infections is provided in tables 11.2 and 11.3. yes (cytoplasmic; ill-defined) yes (nuclear and cytoplasmic) pulmonary tissue reaction necrotizing bronchiolitis; smudge cells; dad severe edema, early dad interstitial pneumonia; dad; occasional multinucleation interstitial pneumonia; dad; cytomegaly dad; necrosis and rare multinucleation dad; necrosis and rare multinucleation dad; necrotizing bronchiolitis interstitial pneumonia with multi nucleation dad dad; interstitial pneumonia with occasional multinucleation necrotizing bronchiolitis, interstitial pneumonia with occasional multinucleation recently recognized; human pathology not well described interstitial pneumonia with multinucleation; dad only certain viruses can cause cytopathic changes that are sufficiently distinct to enable the pathologist to recognize a specific diagnosis on routine histologic examination of lung specimens. with the availability of special figure 11 .1. negative stain electron microscopic images of different viruses that can cause pulmonary infections. a. adenovirus. adenoviruses are protein-shelled icosahedral-shaped nonenveloped viruses that measure approximately 70 to 90nm in diameter. two of the viruses are stain penetrated revealing the dna-containing nucleoprotein. b. sin nombre virus. sin nombre virus, the causative agent of hantavirus pulmonary syndrome, belongs to the genus hantavirus in the family bunyaviridae. the envelopes of hantaviruses are checkerboard in appearance, and particles measure 90 to 150nm in diameter. c. herpes simplex virus. the stain has penetrated the envelope of several of these herpesvirus particles, delineating the icosahedral-shaped nucleocapsids, which measure 90 to 100nm in diameter. d. cytomegalovirus. another herpesvirus; one virus particle (left) is intact while two nearby particles are stainpenetrated and show the viral nucleocapsids. the nucleocapsid of the upper right particle shows a central core that harbors the dna of the virus. e. influenza virus. influenzaviruses belong to the family orthomyxoviridae; viral particles are pleomorphic s.r. zaki and cd. paddock diagnostic techniques, such as immunohistochemistry (ihc) and in-situ hybridization (ish), many viruses can be detected in formalin-fixed, paraffin-embedded tissue samples even if specific viral inclusions cannot be found in histologic examination of tissue sections. among the techniques, ihc utilizing specific antibodies can be routinely performed on formalin-fixed tissue and can enhance the pathologist's accuracy in identifying organisms in tissue specimens. in addition to histologic pattern recognition, ihc, and ish in tissue, several other diagnostic tests are available to aid the pathologist. cell culture techniques, serology, polymerase chain reaction (pcr), and electron microscopy (em) all play vital roles in the diagnosis of these infections. while histologic techniques can be an excellent means of demonstrating organisms, tissue culture isolation remains essential for definitive identification of the virus. when a viral pneumonia is suspected, samples of lung tissues should be evaluated by cell culture, which has the advantage of being a nonbiased method for screening purposes that does not rely on the availability of specific antibodies or probes. electron microscopy offers the same utility as a broad scope diagnostic tool and has been especially critical in outbreaks of unknown etiology. it played a critical role during the hendra and nipah virus outbreaks in 1994 and 1999, respectively, and more recently, in the early recognition of a novel coronavirus associated with sars in 2003 and in the diagnosis of emerging transplant-associated infections. 13.22.29-33 the advantage of this approach is that viral particles may be demonstrated by negative stain or thin section em, either directly in clinical material or after amplification in cell culture. like culture, em is not limited by narrow specificity of reagents or by prior clinical bias ( fig. 11 .1). and can be filamentous or spherical in shape. the evenly spaced spikes cover the entire virus surface and contain both the hemagglutinin and neuraminidase surface glycoproteins. f. human metapneumovirus. these paramyxoviruses are heterogeneous in size and shape, and range in size from 150nm to 1 j.lm in diameter. g. parainfluenza virus. another paramyxovirus, the viral nucleocapsid, with its typical herringbone appearance, can be seen both within the stain-penetrated particle as well as partially extruded from the virion. an important feature that can help distinguish between paramyxovirinae (parainfluenza and measles viruses) and pneumovirinae (human metapneumovirus and respiratory syncytial virus) is the diameter of the nucleocapsids, which measure 18nm and 14nm, respectively. h. severe acute respiratory syndrome (sars) coronavirus. these 80-to 100-nm particles are named for the characteristic crown-like fringe on the surface. scale bars, 100nm. (a,b,d,f,h: courtesy of c. humphrey; c,g: courtesy of e. palmer; e: courtesy of ea. murphy, all at centers for disease control and prevention, atlanta, ga.) adenoviruses were first cultured and identified during the early 1950s by investigators searching for etiologic agents of acute respiratory infections. the initial adenovirus isolate was made serendipitously from adenoid tissues obtained from children during efforts to establish a primary human adenoid cell line. 34 a related virus was identified the following year by investigators studying respiratory disease in military recruits?5 these agents were subsequently named adenoviruses after the original source of tissue from which the prototype strain was identified. 36 adenoviruses are non enveloped viruses with a single, linear, double-stranded dna genome that is contained within an icosahedral capsid that measures 70 to 90nm in diameter (fig. ilia) . the capsid is comprised of seven known polypeptides, including the hexon capsomere, which contains group-specific antigenic determinants. 37 adenoviruses are a ubiquitous and diverse group of viruses found naturally in the upper respiratory tracts and gastrointestinal systems of humans, other mammals, and birds. most adenoviruses infect mucosal epithelium, although some pathogens of animals are trophic for endothelial cells, and endothelial infection has been identified in some immunocompromised humans. 38 adenoviruses are represented by at least 51 serotypes on the basis of resistance to neutralization by antisera to other known adenovirus serotypes, and comprise six subgroups or subgenera (a through f) that are distinguished by differential hemagglutination with erythrocytes from various animal species. 37 ,39ao more than 50% of the known adenovirus serotypes are associated with human diseases of the upper and lower respiratory tract, conjunctiva, urinary tract, intestine, and occasionally heart, liver, and central nervous system. the others are rarely encountered and mayor may not act as pathogens in recognizable disease. 37 it is estimated that approximately 5% to 10% of all pneumonias in infants and young children are caused by adenoviruses. 41 ,42 most pediatric cases of adenovirus pneumonia occur between 6 months and 5 years of age, and serotypes 3, 7, and 21 (all members of the b subgenus), are the most common causes of pneumonia in this patient cohort. 43 -45 serotypes 3 and 7 are particularly pathogenic adenoviruses that can cause disseminated and often fatal disease in previously healthy children. 46 in adults, pneumonia is generally associated with serotypes 3,4, and 7.47 periodic epidemics of adenovirus pneumonia in young adults have been identified, particularly among military recruits. 48 ,49 in a manner similar to other pathogens, adenoviruses take advantage of impaired or destroyed immune systems to establish persistent and disseminated infections in immunocompromised hosts. in this patient cohort, the s.r. zaki and cd. paddock case fatality rate of adenoviral pneumonia approaches 60%, compared with an approximately 15% mortality in immunocompetent patients. 37 immunocompromised patients are also susceptible to a broader range of different adenovirus serotypes. by example, the commonly recognized serotypes in normal children account for only about 50% of the adenovirus serotypes reported for children with congenital immune deficiencies. 37 .4 6 because some adenoviruses establish latency in lymphoid tissues and the kidneys of their host, it is believed that many, possibly most, cases of clinical disease caused by adenoviruses in immunocompromised patients are reactivated infections. 37 the lungs of patients with adenovirus pneumonia are typically heavy and edematous, and the bronchi are generally filled with mucoid, fibrinous, or purulent exudates. the histopathologic findings ( fig. 11 .2) include necrotizing bronchitis and bronchiolitis with extensive denudation ofthe surface epithelium,particularly in medium-sized (1 to 2mm in diameter) intrapulmonary bronchi ( fig. 11 .2a). affected airways may be occluded by homogeneous eosinophilic material, mixed inflammatory cells, detached epithelium, and cellular debris. the lamina propria of bronchi and bronchioles is typically congested and infiltrated by predominantly mononuclear inflammatory cell infiltrates. bronchial serous and mucous glands are also often involved and show necrosis and mixed infiltrates. 5o as the infection progresses, there is involvement of the pulmonary parenchyma, forming bronchocentric necrosis with hemorrhage, neutrophilic and mononuclear cell infiltrates, and karyorrhexis. these findings generally occur against a background of exudative diffuse alveolar damage, with filling of the air space by macrophages, fibrin, and detached pneumocytes, and hyaline membrane formation. 51 patients with fatal pneumonia may develop disseminated intravascular coagulopathy and demonstrate fibrin thrombi in vessels of the lungs, kidney, heart, adrenals, and central nervous system (see fig. 4 .20 in chapter 4). 48 adenoviruses form intranuclear inclusions in respiratory epithelial cells of the trachea, bronchi, and bronchioles, in the acinar cells of bronchial glands, and in alveolar pneumocytes, and are generally most abundant at the viable edges of necrotic foci. by using the hematoxylin and eosin (h&e) stain, early inclusions appear as small, dense, amphophilic structures surrounded by a cleared zone and peripherally marginated chromatin, similar to herpetic inclusions. as the cellular infection progresses, the inclusion becomes larger (as large as 14/-lm in some cells) and more basophilic, and the margins of the nuclear membrane become blurred to form the characteristic "smudge cell" (fig. 11 .2b,c).5o,51 tracheal aspirates of patients with adenovirus pneumonia may show distinctive features on cytologic preparations that include cells with fine strands of chromatin that radiate from a central inclusion to the marginated chromatin at the nuclear membrane ("rosette cells"), and cells with foamy, "honeycomb" nuclei, as well as typical smudge cells (see fig. 7 .45 in chapter 7). 52 various methods can be used to diagnose adenovirus infections that include antigen detection, cell culture, electron microscopy, molecular assays, and serology. direct detection techniques that identify the common group-reactive hexon antigen in tissues or body fluids include fluorescence antibody assays and enzyme immunoassays.53 immunohistochemistry staining methods have been used successfully to detect adenovirus-infected cells in formalin-fixed, paraffinembedded tissues using various commercially available, adenovirus group-specific antibodies ( fig.11 .2e,f).38,51 electron microscopy of adenovirus-infected tissues reveals a paracrystalline array of virions ( fig. 11 .2d ). 54, 55 most adenoviruses can be isolated in cell culture from bronchial washings, tracheal aspirates, or lung biopsy specimens during the early stage of the illness and grow well in various cell lines, including human embryonic kidney, hela, and hep-2 cells. 47 cell cultures infected with adenoviruses exhibit a relatively characteristic cytopathologic effect, described as a "cluster of grapes," within 3 to 5 days after inoculation. serotyping of the isolate is accomplished by using hemagglutination inhibition and neutralization tests with hyperimmune type-specific animal antisera. 56 molecular assays, particularly gene amplification using pcr, and ish methods, have been developed to detect adenovirus nucleic acid in respiratory secretions and in formalin-fixed, paraffin-embedded tissues. 51 ,57,58 broad-range, sensitive assays that can detect any adenovirus amplify common genomic sequences (e.g., the hexon gene region). other more specific assays detect specific adenovirus types with unique genomic sequences. 59 serologic assays include tests for groupspecific antibodies (e,g., complement fixation and enzyme immunoassays), or type-specific antibodies (e.g., neutralization and hemagglutination-inhibition assays). pitfalls associated with serologic testing for adenoviruses include occasional rises in heterotypic antibodies when typespecific assays are used, and relatively low sensitivity demonstrated by complement fixation assays. 59 human hantaviral diseases are caused by a group of closely related, trisegmented, negative-sense rna viruses of the genus hantavirus, of the family bunyaviridae. 60 -62 members of the genus hantavirus have similar morphologic features. 63 ,64 virus particles are 70 to 130nm in diameter and generally appear spherical to ovoid, although pleomorphic forms may be seen. a lipid envelope containing glycoprotein spikes surrounds a core consisting of the genome and its associated proteins (nucleocapsids) s.r. zaki and cd. paddock arranged in delicate tangles of filaments showing occasional granulation. the presence of characteristic inclusion bodies in thin section electron microscopy and a unique grid-like pattern on negative-stain electron microscopy differentiate these viruses from other members of the family bunyaviridae ( fig. 11.1b) . 65, 66 the severity and disease type largely depends on the viral serotype. two categories of hantavirus-associated illnesses are described: hemorrhagic fever with renal syndrome (hfrs) for disease in which the kidneys are primarily involved, and hantavirus pulmonary syndrome (hps) for disease in which the lungs are primarily involved. 67 -69 the isolation of the first recognized hantavirus (hantaan virus, named for the river in south korea), and its subsequent identification as the causative agent of hfrs was reported in 1978. 70 in 1993, the deaths of several previously healthy individuals due to a rapidly progressive respiratory disease in the southwestern united states were etiologically linked to a previously unrecognized hantavirus. clinically, the disease differs from hfrs in its pronounced pulmonary involvement and higher mortality rates and is known as hps. 23.69,71.72 hantavirus-associated diseases primarily affect blood vessels and result in different degrees of generalized capillary dilatation and edema. 73 in contrast to severe hfrs where abundant protein-rich, gelatinous retroperitoneal edema fluid is found, all hps patients have large bilateral pleural effusions and heavy edematous lungs. 7 in fatal far eastern hfrs, a distinctive triad of hemorrhagic necrosis can be seen in the renal medullary junctional zone, cardiac right atrium, and anterior pituitary. 75, 78 however, in patients with hps, hemorrhages are rare, and ischemic necrotic lesions, except those attributed to shock, are not seen. 72, 74 histologically, morphologic changes of the endothelium are uncommon but, when seen, consist of prominent and swollen endothelial cells. vascular thrombi and endothelial cell necrosis are rare. in hfrs, the most severe and characteristic microscopic lesions involve the kidney; however, an interstitial pneumonitis can also be seen in some fatal cases. in contrast, the microscopic changes of north and south american hps are principally seen in the lung and spleen. 72 ,74 the lungs show a mild to moderate interstitial pneumonitis characterized by variable degrees of edema and an interstitial mononuclear cell infiltrate comprised of a mixture of small and enlarged mononuclear cells with the appearance of immunoblasts ( fig.11 .3a). focal hyaline membranes composed of condensed proteinaceous intraalveolar edema fluid, fibrin, and variable numbers of inflammatory cells are observed ( fig. 11 .3b). typically, neutrophils are scanty and the alveolar pneumocytes are intact with no evidence of cellular debris, nuclear fragmentation, or hyperplasia. in fatal cases, with a prolonged survival interval, tissues show features more characteristic of the exudative and proliferative stages of diffuse alveolar damage. lung biopsies taken from patients who survive their illness appear similar with proliferated reparative type ii pneumocytes, severe edematous and fibroblastic thickening of the alveolar septa, and severe air-space disorganization with distorted lung architecture (see chapter 4) . other characteristic microscopic findings in hps cases include variable numbers of immunoblasts within the splenic red pulp and periarteriolar white pulp, lymph nodal paracortical zones, hepatic portal triads, and peripheral blood ( fig. 11.3d,e) . similarly, in severe hfrs cases, large mononuclear cells can be present in the spleen, lymph nodes, blood, and hepatic portal triads. 75 . 76 electron microscopic studies of hps lung tissue demonstrate infection of endothelial cells and macrophages. 69 . 72 the virus or virus-like particles observed are infrequent and extremely difficult to identify in autopsy tissues because of the considerable degree of viral pleomorphism and the postmortem deterioration of tissues. however, typical hantaviral inclusions are seen more frequently and their identity can be confirmed by immunolabeling ( fig. 11 .3f,g). similar inclusions are observed in epithelial cells in hfrs and are considered to be ultrastructural markers of hantavirus-infected cells. 63 .79.8o using immunohistochemistry, viral antigens are found primarily within capillary endothelium throughout various tissues in both hps and hfrs. in hps, marked accumulations of hantaviral antigens are in the pulmonary microvasculature and in splenic and lymph nodal follicular dendritic cells (fig. 11 .3c).72 despite the extensive endothelial cell accumulations of hantaviral antigens, there is little ultrastructural evidence of cytopathic effect. hantavirus pulmonary syndrome should be suspected in cases of adult respiratory distress syndrome (ards) without a known precipitating cause among previously healthy individuals. the level of suspicion should be particularly high when patients have a known exposure to rodents in areas where peromyscus maniculatus or other reservoirs of hantavirus are found. physicians need to differentiate hps from other common acute respiratory diseases, such as pneumococcal pneumonia, influenza virus, and unexplained ards. the diagnosis of hps, suspected by patient history and clinical manifestations, can also be supported histopathologically. although there is no single pathognomonic lesion that would permit certain histopathologic diagnosis of hps, the overall constellation of histopathologic hematologic findings suggests the diagnosis.72.7 4 diseases that need to be distinguished pathologically from hps include a relatively large number of different viral, rickettsial, and bacterial infections, as well as various noninfectious disease processes. virus-specific diagnosis and confirmation can be achieved through serology, pcr for hantavirus rna, or ihc for hantaviral antigens. 2 1,72 serologic testing can detect hantavirus-specific immunoglobulin m or rising s.r. zaki and cd. paddock titers of immunoglobulin g in patient sera and is considered the method of choice for laboratory confirmation of hps. immunofluorescent assays and enzyme-linked immunosorbent assays (elisas), which demonstrate the presence of specific antihantaviral antibodies, are currently used as rapid diagnostic tests and provide results within a few hours. recently, synthetic hantaviral nucleocapsid proteins have been used to improve the sensitivity and specificity of serologic assays. these proteins are more available than inactivated hantaviral antigens. 81 . 82 polymerase chain reaction detects viral rna in blood and tissues and is extremely useful for diagnostic and epidemiologic purposes. hantaviral rna can also be detected in formalin-fixed, paraffin-embedded archival tissue by reverse-transcriptase (rt)-pcr.83 immunohistochemistry testing of formalin-fixed tissues can be used to detect hantavirus antigens, and is a sensitive method to confirm hantaviral infections. 72 it has a unique role in the diagnosis of fatal hps cases when serum samples and frozen tissues are unavailable but formalinfixed autopsy tissues are obtainable. 84 • 85 severe acute respiratory syndrome the causative agent of sars is an enveloped, positivestranded rna virus that is a member of the genus coronavirus, of the family coronaviridae. corona viruses have the largest genomes of all rna viruses and replicate by a unique mechanism that results in a high frequency of recombination. maturation of sars coronavirus (sars-co v) is similar to features previously described for other coronaviruses. 8 6-9 0virions form by alignment of the helical nucleocapsids along the membranes of the endoplasmic reticulum or golgi complex and acquire an envelope by budding into the cisternae. the cellular vesicles become filled with virions and progress to the cell surface for release of the virus particles; large numbers of particles remain adherent to the plasma membrane at the cell surface. severe acute respiratory syndrome was recognized during a global outbreak of severe pneumonia that began in late 2002 in guangdong province, china, and gained prominence in early 2003 as cases were identified in more than two dozen countries in asia, europe, north america, and south america. the disease causes an influenza-like illness with fever, cough, dyspnea, and headache, and in severe cases it can cause death in humans. person-toperson transmission, combined with international travel of infected persons, accelerated the worldwide spread of the illness. 5 • 13 . 91 several reports have described diffuse alveolar damage with various levels of progression and severity as the main histopathologic findings in sars patients ( fig. 11 .4a,b)y·92-98 lungs typically show changes described for the proliferative phase of diffuse alveolar damage, with hyaline-membrane formation, desquamation of epithelial cells, fibrin deposit in the alveolar space, and hyperplasia of type 2 pneumocytes. increased mononuclear infiltrate in the interstitium can be seen in some cases. other findings identified in some patients included focal intra alveolar hemorrhage, necrotic inflammatory debris in small airways, and organizing pneumonia. in addition, multinucleated syncytial cells may be seen in the intraalveolar spaces of some patients who died 14 days or more after onset of illness ( fig. 1l .4c). infection with some coronaviruses, including sars-co v, is known to induce cell fusion in culture producing syncytial cells similar to those sometimes observed in lungs of patients who die from sars. these cells contain abundant vacuolated cytoplasm with cleaved and convoluted nuclei, but without obvious intranuclear or intracytoplasmic viral inclusions. the ish and ihc studies of tissues from sars patients have identified corona virus infection of upper airway bronchiolar epithelium ( fig. 11 .4d_f).92,99-101 infected ciliated columnar epithelial cells can be seen focally in lining epithelium of trachea and larger bronchi ( fig. 11 .4e). many of these infected cells slough from the epithelium and can be observed by using ish within the bronchial lumen. abundant viral antigens can also be found distributed focally in parenchyma of lungs of some patients and are seen predominantly in the cytoplasm of pneumocytes, in occasional macrophages, and in association with intra alveolar necrotic debris and fibrin (fig. ll.4d). double-stain studies indicate that most sars-co v-infected cells are type 2 pneumocytes. double-stain studies also detected viral nucleic acids with a distribution similar to that seen in ihc studies, mainly in pneumocytes and in some macrophages.looelectron microscopic examination of lung tissues selected from areas with abundant ihc staining shows numerous coronavirus particles and nucleocapsid inclusions. virions are seen in cytoplasmic vesicles and along the cell membranes of pneumocytes, in phagosomes of macrophages, and associated with fibrin in alveolar spaces (fig.ll.4g-i). because corona virus particles may be confused morphologically with other non viral cellular components, definitive ultrastructural identification can be achieved by using immunogold labeling electron microscopy. the primary histopathologic lesions seen in the lungs of patients who die from sars are somewhat nonspecific and can also be seen in acute lung injury cases caused by infectious agents, trauma, drugs, or toxic chemicals. 102 multinucleated syncytial cells similar to those seen in some sars patients can also be found in a number of virus infections, including measles, parainfluenza viruses, rsv, and nipah virus infections. 102 -104 in an early study of four human sars patients,13 viral antigens were not detected in the lung by ihe. the most likely explanation is that all patients in the study had a clinical course aver-s.r. zaki and cd. paddock aging more than 2 weeks. for many virus infections, viral antigens and nucleic acids are cleared within 2 weeks of disease onset by the host immune response. it is also possible that the pulmonary damage associated with sars is not caused directly by the virus, but represents a secondary effect of cytokines or other factors induced by the virus infection. similarly, in influenza virus infections, viral antigens are seen predominantly in respiratory epithelial cells of large airways and are only rarely identified in pulmonary parenchyma despite concomitant and occasionally severe interstitial pneumonitis. !os in recent reports by shieh et al. 100 and chong et al.,92 the temporal relationship between the duration of illness and clearance of sars-co v in human lung tissue was examined. viral antigens and nucleic acids were detected only in pulmonary tissues of patients who died early in the disease. the development of specific ihc, ish, and immunoelectron microscopy (iem) assays to identify sars-co v in formalin-fixed, paraffin-embedded samples has facilitated the assessment of the cellular tropism of sars-co v infection in human lung tissues. localization of sars-co v in the lung occurs mainly in the cytoplasm of pneumocytes, primarily type 2, and occasionally in alveolar macrophages ( fig. ll.4f ). type 2 pneumocytes are known to secrete pulmonary surfactant, resulting in reduced surface tension and preservation of the integrity of the alveolar space. these cells also play an important role in tissue restitution following lung damage. moreover, there is mounting evidence to support their contribution to the development of acute inflammatory lung injury following exposure to biological or chemical agents. additional studies are needed to further define the role of type 2 pneumocytes and alveolar macrophages in sars-co v infection. cynomolgus macaques inoculated with sars-cov develop pathologic findings of pneumonia and have been proposed as an animal model. 106 haagmans et a1. 107 showed extensive sars-co v antigen expression in experimentally infected macaques 4 days after infection. the antigens were mainly in alveolar lining epithelial cells with morphologic characteristics of type 1 pneumocytes, indicating type 1 pneumocytes are the primary target for sars-co v infection early in the disease. type 1 pneumocytes normally represent 90% of the alveolar epithelial cell volume and are easily damaged during pulmonary infections or other types of injury. in a more recent study on nonhuman primates,108 evidence of infection of type 1 pneumocytes in addition to some type 2 pneumocytes and macrophages was found. small animal models, such as rodents, would be very useful for evaluating vaccines, immunotherapies, and antiviral drugs, and we have recently identified the mouse as an animal model for this purpose. 10 9 in those studies, microscopic examination of trachea, bronchus, lung, thymus, and heart on day 2 after infection revealed mild and focal peri bronchiolar mononuclear inflammatory infiltrates with no significant histopathologic change in other organs. viral antigens and nucleic acids were focally distributed in bronchiolar epithelial cells, and virions were found in these same areas by ultrastructural analysis. data suggest that sars-co v replicates in mice to a titer sufficient to evaluate vaccines and antiviral agents. the mouse and other small animal models l1o might also be used to test the ability of the virus to replicate and cause disease and facilitate identification of host-immune mechanisms that contribute to the resolution of sars-co v infection. cytomegaloviruses (cmv) comprise a distinct and ancient group of herpesviruses that are widely distributed in nature, share similar growth characteristics in cell culture, and cause cellular enlargement and form distinctive inclusions in infected cells. these cytopathic changes, identified by early pathologists in the salivary glands of children dying from various unrelated diseases,111-113 led to the early designation of cytomegalic inclusion disease many years before the causative agent was isolated in the mid-1950s. the name cytomegalovirus was proposed in 1960 to reflect the cytopathic changes caused by these viruses. 114 cytomegaloviruses are highly host-specific, and various mammalian hosts, including nonhuman primates, rodents, and domesticated animals, are infected with their own distinct cmy. in this context, human cmv is stringently species-specific and, with rare exception, only infects cells of human origin. ll5 several cell types are permissive for cmv replication, including alveolar pneumocytes, vascular endothelium, fibroblasts, monocytes, dendritic cells, and exocrine and endocrine glandular epithelial cells.ll6 cytomegalovirus is a ~-herpesvirus with the largest genome (230 kilobase pair [kbp]) of all the herpesviruses known to infect humans. the double-stranded linear dna genome is contained within a 90 to 100nm icosahedral capsid and is surrounded by an amorphous material known as the tegument. these components are enclosed in a lipid bilayer envelope that is derived from the host cell nuclear or golgi membranes and contains several vir ally encoded glycoproteins necessary for infection of other cells. mature enveloped virions range from 150 to 200nm, making cmv one of the largest viruses that infect humans (fig. i1.1d ).ll7 the structure of cmv is typical of other human herpesviruses, but demonstrates some subtle ultrastructural differences from other viruses in this group including greater pleomorphism of the lipid envelope and dense body inclusions in the cytoplasm of infected cells.ll8 437 cytomegalovirus is a ubiquitous human pathogen, and in north america infects approximately 50% to 90% of the population.ll7 most of these infections are inapparent, although some cases of primary infection in otherwise healthy individuals result in a self-limited mononucleosis syndrome similar to that caused by epstein-barr virus; it is estimated that 20% to 50% of cases of heterophile-negative mononucleosis, and 8% of all cases of mononucleosis, are caused by cmy' 119 pulmonary involvement in cmv mononucleosis is infrequent and occurs in approximately 6% of these casesyo congenitally acquired cmv infection has various deleterious effects on the fetus, including mental retardation, neurologic abnormalities, sensorineural hearing loss, and retinitis, and in one series pulmonary involvement occurred in 64 % of symptomatic infants. 121 like all herpesviruses, cmv remains with its host for life after primary infection and establishes latency in various cell types, including vascular endothelial cells, monocytes and macrophages, neutrophils, and renal and pulmonary epithelial cells. 122 activation of viral replication occurs in persons with severely compromised immunity. patients with advanced hiv disease and recipients of hematopoietic stem cell or lung transplants are particularly at risk of developing cmv pneumonia. before the use of cmv screening and effective antiviral prophylaxis regimens, 10% to 30% of all patients undergoing allogeneic bone marrow transplantation for leukemia, and 15% to 55% of solid organ transplants, developed cmv pneumonia with case fatality rates of greater than 80% in some series. 26 ,123,124 the relatively high frequency of cmv pneumonia in lung transplant recipients may be a correlate of animal model data that indicate the lungs are a major site of latent cmv infection. 125 before the use of ganciclovir as therapy for cmv disease in aids patients, the case fatality rate for cmv pneumonia in this patient cohort was 75% when cmv was the only pathogen identified. mixed infections with cmv and another pathogen had an even worse prognosis, and the case fatality rate for patients with pulmonary disease caused by cmv and pneumocystis jiroveci (formerly carinii) was 92%. 126 cytomegalovirus pneumonia can show various histopathologic patterns (fig. 11 .5). extensive intra alveolar hemorrhage with scattered cytomegalic cells and relatively few inflammatory cell infiltrates may occur ( fig. 11 .5a).127 in a similar manner, extensive involvement of the alveolar epithelium with minimal inflammation or overt evidence of parenchymal injury has also been described. 128 other patterns include multifocal lesions with mixed inflammatory cell infiltrates, hemorrhage, necrosis, and cytomegalic cells, or a diffuse, predominantly mononuclear cell infiltrate, interstitial pneumonitis with intraalveolar edema and fibrin deposition, and diffusely distributed cytomegalic cells (fig. 11 .5e).129-131 the cytomegalic changes of cmv-infected cells are evident by standard h&e staining and are virtually pathognomonic of active cmv infection. the cells are enlarged (25 to 40!j,m) and contain amphophilic to deeply basophilic intranuclear and intracytoplasmic inclusions ( fig. 11 .5b,e). the single intranuclear inclusion is composed of viral nucleoprotein and assembled caps ids, and is a large (up to 20 !j,m), round to ovoid body with a smoothly contoured border that is generally surrounded by a clear halo that gives the inclusion a distinctive owl'seye appearance. the host cell nucleolus is often retained in the inclusion. 131 cytoplasmic inclusions are small (1 to 3!j,m), granular bodies that appear after the intranuclear inclusion is well developed and are not uniformly present in all cmv-infected cells (fig. 11 .5b). these inclusions represent a mixture of virions and various cellular organelles, and increase in size and number as the infection progresses. 132 unlike the intranuclear inclusion, the cytoplasmic inclusions stain with periodic acid-schiff stain and are deeply argyrophilic with gomori's methenamine silver stain.!33 cytomegalovirus pneumonia is defined by the presence of signs or symptoms of pulmonary disease combined with the detection of cmv in bronchoalveolar lavage (bal) fluid or lung tissue samples. in this context, detection methods that support this definition include virus isolation, histopathologic observation of cytomegalic cells, ish, or ihc stains (fig. 11 .5f). detection by pcr alone is considered too sensitive for the diagnosis of cmv pneumonia and is insufficient for this purpose. 134 cytomegalovirus is most often cultured in human diploid fibroblasts such as human embryonic lung and human foreskin fibroblasts. it grows slowly in conventional cell culture, and the cytopathic effect is generally not detected until the second week or longer after inoculation. for this reason, a shell vial method using centrifugation to enhance infectivity has become the standard isolation technique and can usually yield diagnostic results within 48 hours. 135 for centuries, the term herpes, derived from the greek erpein (to crawl), was used in medicine to describe any spreading cutaneous lesion. by the end of the 19th century, investigators surmised that the herpetic lesions of the lips and genitalia were manifestations of a single infectious agent, and recognized that it was a disease distinct from herpes zoster. 136.137 as with all human herpesviruses, hsv is a large, enveloped, double-stranded dna-containing virion with an icoshedral nucleocapsid approximately 100 to 1l0nm in diameter, composed of 162 cap somers. the nucleocapsid is surrounded by an amorphous, sometimes asymmetric material (the tegument) that is surrounded by a thin, trilaminar envelope that contains numerous glycoprotein spikes (fig. 11.1c ). the assembly of hsv begins in the nucleus of its host cell and the virus acquires its envelope as the capsid buds through the inner lamella of the nuclear membrane.120.l38 two serologic types are recognized and each is most frequently associated with particular disease syndromes; however, either serotype may cause any of the aggregate clinical syndromes. herpes simplex virus-1 causes gingivostomatitis, pharyngitis, esophagitis, keratoconjunctivitis, and encephalitis, and is the serotype most commonly associated with adult hsv pneumonia. herpes simplex virus-2 typically infects genital sites such as the penis, urethra, vulva, vagina, and cervix, and is the serotype associated with approximately 80% of disseminated disease and pulmonary infections in newborn infants. 136 all herpesviruses have the ability to persist in an inactive state for varying periods of time and then recur spontaneously following undefined stimuli associated with physical or emotional stress, trauma to nerve roots or ganglia, fever, immunosuppression, or exposure to ultraviolet radiation. 138 during the primary infection, hsv replicates at the portal of entry (typically oral or genital mucosae), and infects sensory nerve endings. the virus is transported centripetally along peripheral sensory nerves to central axons and finally to nerve cell bodies in the trigeminal, sacral, and vagal ganglia, where it replicates briefly before becoming latent.139-141 antiviral drugs have no effect on latent infection with hsv. following cues that initiate viral reactivation, hsv replicates in sensory ganglia and is transported centrifugally along sensory nerves to epithelial cells on mucosal surfaces. 138 reactivation of hsv from the trigeminal ganglion is associated with asymptomatic excretion of virus in saliva, and with the development of herpetic ulcers on the vermillion border of the lip, oral mucosa, or external facial skin. 142 newborn infants, severely immunosuppressed or burned patients, and patients with severe trauma are at greatest risk of developing hsv pneumonia.143-146 lower respiratory tract disease in neonates is most commonly associated with disseminated herpetic infections. disseminated hsv infection in the newborn was first described in 1935 as "hepatoadrenal necrosis"147 because of the prominent and frequent necroses that occur in the livers and adrenal glands of affected neonates. 148 most cases of neonatal disease represent primary hsv infections and are acquired during parturition from hsvinfected mothers. the incidence of neonatal hsv infection is approximately 1 in 3200 deliveries, and disseminated disease develops in approximately 25% of infected neonates. 149 in disseminated infections, signs and symptoms appear a mean of 5 days after birth (range, 0 to 12 days), and approximately 40% to 50% of these patients develop pneumonia. in the pre antiviral era, 85 % of neonates with disseminated disease died from the infection. with early diagnosis and high-dose acyclovir therapy, mortality has been reduced to approximately 30%?6,138,149 the disease can be exceedingly difficult to diagnose in a timely manner as only 10% of mothers of affected infants have clinically apparent hsv infection at the time of delivery.15o neonates that survive severe disseminated disease may develop hepatic and adrenal calcifications evident on abdominal radiographs. 151 in adults, infection of the respiratory tract with hsv may be associated with disseminated herpetic infection, but is more commonly identified as an isolated disease manifestation resulting from reactivation of latent herpetic infections in the oropharynx. herpetic tracheobronchitis is an ulcerative process characterized by large areas of denuded mucosal epithelium and fibrinopurulent exudate containing necrotic cells with densely eosinophilic cytoplasm. despite extensive tissue damage, cells with intranuclear inclusions may be sparse, and, when identified, are found most often at the margins of the ulcerated epithelium or occasionally in the mucous glands subjacent to ulcerated surfaces. ls2 aspiration of viruscontaining secretions into the lower respiratory tract is believed to be the most frequent cause of pulmonary infection with hsv; however, oral lesions may be absent in patients with herpetic laryngotracheobronchitis and bronchopneumonia. 153 disease can be also associated with airway trauma caused by tracheal intubation or from hematogenous dissemination of hsv. 144 ,1s2,1s4 chest radiographs of hsv pneumonia generally show ill-defined nodular or reticular densities of various sizes scattered in both lung fields. during the early stages of disease, these nodules measure 2 to 5 mm and are best seen in the periphery of the lungs. as the disease progresses, these lesions coalesce and enlarge to form more extensive infiltrates. 137 herpetic infections of the airways and lung are characteristically difficult to diagnose clinically and hsv pneumonia was not described as a distinct clinical entity until 1949.155 several studies attest to the relative infrequency with which this diagnosis is considered in patients with respiratory disease. for example, none of the 15 cases of hsv disease of the middle and lower respiratory tract identified in a review of autopsies at brooke army medical center during 1965 to 1968 were suspected prior to autopsy. 143 in a 1982 review of 20 culture-confirmed cases of hsv pneumonia, none of the patients had been diagnosed prior to death and all 16 had oral herpetic lesions at the time of death.144 recent investigations also suggest that lower respiratory tract disease caused by hsv may be more common than currently appreciated. in a study from sweden, hsv was cultured from proa diagnosis of tracheobronchitis and pneumonia is best established histologically (fig. 11.6 ). because lower respiratory tract hsv infections are often focused in the tracheobronchial tree, open lung biopsy may be less sensitive than bronchoscopy.154 herpetic lesions show extensive necrosis and karyorrhectic debris and are associated with hemorrhage and a sparse-to-moderate neutrophilic infiltrate (fig. 11.6a,b) . intranuclear inclusions are best appreciated in cells at the leading edge of necrotic foci ( fig. 11 .6b,c). inclusions appear either as homogeneous, amphophilic, and glassy (cowdry type b inclusions), or as eosinophilic with a halo separating the inclusion from the nuclear membrane (i.e., cow dry type a inclusions). 158 cowdry type b inclusions contain actively replicating virus. type a inclusions, considered noninfectious and devoid of viral nucleic acid or protein, represent the nuclear "scar" of hsv infection. 136 ,141 other changes associated with hsv, including multi nucleation and nuclear molding, and ballooning degeneration of the cytoplasm, are more frequently associated with squamous epithelium and are seldom encountered in the lung. because of the high frequency of hepatic and adrenal involvement with disseminated hsv infection in young children ( fig. 11 .6f,g), liver biopsy has been suggested as a diagnostic technique in this patient cohort. 148 commercially available antibodies exist for ihc detection of hsv in tissues (fig. 11.6d ).ls9 virus isolation remains an important diagnostic method; however, because hsv can be isolated from oropharyngeal secretions and occasionally from the lower respiratory tract of patients who lack overt pulmonary disease, virologic cultures must be interpreted in the context of complementary clinical, radiographic, and histopathologic findings as much as possible. cell culture systems susceptible to hsv include vero cells and foreskin fibroblasts. cytopathic effects generally develop within 24 to 48 hours after cultures are inoculated with infectious specimens. suitable specimens include scrapings made from mucocutaneous lesions, tracheobronchial aspirates, or bal specimens. in infants with evidence of hepatitis, it may also be useful to obtain duodenal aspirates for hsv isolation. 138 polymerase chain reaction methods that amplify hsv dna from clinical specimens, including tissue and blood, can be particularly useful by specifically distinguishing between hsv-l or hsv-2 infections.138,149 varicella-zoster virus (vzv), also known as human herpesvirus 3 (hhv-3) , is a human a-herpesvirus most closely related to hsv. it has a linear, double-stranded dna genome with approximately 125 kbp that encodes more than 70 proteins. the icosahedral nucleocapsid is indistinguishable in appearance from other herpes viruses. the nucleocapsid and the tegument are surrounded by a lipoprotein envelope derived from the host cytoplasmic membranes. the enveloped viral particle is pleomorphic to spherical in shape and 180 to 200nm in diameter. 16o the primary infection is initiated by inoculation of respiratory mucosa by the virus through infectious aerosols or by direct contact of skin lesions of patients with varicella or herpes zoster. after a primary viremia in the reticuloendothelial system, and secondary viremia in circulating mononuclear cells, the virus is disseminated to the skin, where it initiates a vesicular rash, and back to mucosal sites in the lungs. the release of infectious virus into respiratory droplets is a pathogenic characteristic that distinguishes vzv from other human herpesviruses. the attack rate for previously uninfected household contacts exposed to varicella is approximately 90%. for less sustained exposure, it is estimated to be approximately 10% to 30%. 160 during primary infection with vzv, viral replication in keratinocytes (fig. 11.7 a) and vascular and lymphatic endothelial cells of the superficial dermis produces the generalized, pruritic, vesicular rash of varicella commonly known as chickenpox. varicella-zoster virus also establishes latent infection within satellite cells and neurons of the trigeminal and dorsal root ganglia and can reactivate under various conditions to cause herpes zoster, a painful vesicular rash commonly referred to as shingles. 161 the origin of the term chickenpox is speculative, but believed to derive from gican, an old english term for itching. the term shingles originates from the medieval latin cinguls, or girdle, and alludes to the partial encircling of the trunk by the rash of herpes zoster. 137 ,160 varicella-zoster virus is ubiquitous in human populations around the world, and humans are the only known host. during the prevaccine era in the u.s., approximately 4 million cases, 4000 to 9000 hospitalizations, and 50 to 140 deaths were reported annually.25.162 the risk of severe illness during primary or recurrent vzv infection appears to depend more on host factors rather than a particular viral strain. chickenpox is considered a relatively benign infection in children, but adult patients are approximately 25 times more likely than children to develop pneumonia. the greatest risk of severe disease and pneumonia occurs in those patients with chronic lung disease, immunesuppressing conditions, neonates, and pregnant women. 163 varicella-zoster virus-related deaths have declined sharply in the u.s. since universal childhood vaccination was implemented in 1995. 24 ,25 s.r. zaki and cd. paddock varicella pneumonia was first described in the medical literature in 1942 164 and is the most frequently reported complication of chickenpox in adult patients. 24 ,165 pneumonia occurs in approximately 10% to 15% of adults infected with vzv. 166 ,167 the occurrence of pneumonia during herpes zoster is rare, and limited primarily to profoundly immunosuppressed patients, particularly bone marrow transplant recipients. m varicella-zoster virus pneumonia generally develops within 2 to 7 days following the onset of rash and may be characterized by fever, cough, tachypnea, chest pain, and hemoptysis. 26 .168 hypoxemia is common and may be severe. radiographically, the lungs show multiple, scattered, defined, nodular densities. untreated adult varicella pneumonia is fatal in approximately 10% of cases, but mortality is as high as 25% to 40% in certain high-risk cohorts, including pregnant women, transplant recipients, and neonates. 137 ,lfj9-171 massive pulmonary hemorrhage is a frequent terminal event. gross examination reveals lungs that are generally two to three times heavier than normal, firm, and plumcolored. there are often multiple necrotic and hemorrhagic lesions on the visceral pleura that resemble the pox lesions of skin. 169 ,172,173 pox may also be seen on the parietal pleura, although pleural effusions are uncommon and rarely prominent. 163 ,173 the trachea and bronchi are generally edematous and erythematous with occasional vesicles on the mucosal surfaces, and there may be lobular consolidation of the lungs. microscopically, pulmonary involvement consists primarily of interstitial pneumonitis and diffuse miliary foci of necrosis and hemorrhage in the pulmonary parenchyma that involve alveolar walls, blood vessels, and bronchioles ( fig. 11.7b,d) . 172 other findings may include intraalveolar collections of edema, fibrin, or hemorrhage, diffuse alveolar damage, and septal edema. 1m ,168.173 virally infected cells with intranuclear inclusions may be identified in respiratory epithelial cells, pneumocytes, interstitial fibroblasts, or capillary endothelium ( fig. 11 .7e,f).167 eosinophilic intranuclear inclusions and multinucleated syncytial cells may be difficult to locate but are best identified at the edges of necrotic foci (fig. 11.7c ). in cases of disseminated disease, similar necrotizing hemorrhagic lesions and occasional viral cytopathic changes in epithelial cells or fibroblasts may be observed in other tissues and organs, including esophagus, pancreas, liver, renal pelves, ureters, urinary bladder, spleen, bone marrow, thymus, lymph nodes, adrenal glands, and brain. 172,174 in those patients who recover from severe vzv pneumonia, some necrotic parenchymal foci may mineralize, and can be identified by chest radiograph years later as miliary, 1 to 5 mm, nodular opacities. microscopically, the lesions are characterized as discrete collections of dense fibrous connective tissue that surround multiple, small, calcified bodies. the periphery may include a cellular zone of fibroblasts and occasional giant cells.175 a radiographic survey of 16,894 persons identified pulmonary calcifications in eight (1.7%) of 463 patients who had chickenpox during adulthood compared with only eight (0.05%) of the remaining 16,431 who did not report having varicella as an adult. 176 a positive history of varicella predicts immunity in >95% of persons. 160 because pulmonary symptoms most often occur several days following the onset of the characteristic rash of varicella, a pathologic diagnosis is seldom required for a real-time diagnosis of vzv pneumonia. however, hematopoietic cell transplant recipients may present with signs of visceral dissemination and pneumonia 1 to 4 days before the localized cutaneous eruption of herpes zoster appears, and lower respiratory tract disease has been described in the absence of skin lesions, particularly in neonates and bone marrow transplant recipients. 26 ,137,177 commercially available antigen detection kits can be used for rapid diagnosis of cutaneous vzv infection. epithelial cells are scraped from the base of a newly formed vesicle, applied to a slide, and stained by using fluorescein-conjugated vzv monoclonal antibodies to detect specific viral proteins in the specimen. in a similar manner, the tzanck test uses wright-giemsa stain to demonstrate multinucleated giant cells in these specimens; however, this test does not differentiate between hsv and vzv, and false-negative results are common. commercially available antibodies are also available for ihc detection of vzv in tissue specimens ( fig. 11 .7e,f); however, relatively few laboratories are able to provide well-validated assays. some commercial laboratories offer pcr amplification to detect viral nucleic acid in clinical specimens. isolation of the virus in cell culture remains the reference standard for the diagnosis of vzv. in human melanoma cells, an excellent substrate for vzv isolation, the average time for visible cytopathic effect from the virus is 3 to 5 days.178 infectious vzv is usually recoverable from the clear fluid of cutaneous vesicles of varicella for approximately 3 days after the appearance of these lesions and for approximately 1 week from herpes zoster lesions. the lungs are the most common organ from which vzv is isolated at autopsy, but isolates have also been obtained from heart, liver, pancreas, gastrointestinal tract, brain, and eyes. 160 influenza is derived from the term influentia, meaning epidemic in the italian form of latin, originally used because epidemics were thought to result from astrologic or other occult influences. influenza is a highly contagious, acute respiratory illness with a spectrum of clinical illness ranging from asymptomatic or mild disease with rhinitis or pharyngitis to primary viral pneumonia with s.r. zaki and cd. paddock fatal outcome. influenza may also be associated with a broad range of other disorders affecting the heart, brain, kidneys, and muscle. influenzaviruses belong to the orthomyxoviridae family, which consists of four genera that include the two important influenza viruses types a and b associated with significant human disease. 179 ,180 influenza a viruses are further classified into subtypes based on the antigenicity of their hemagglutinin (ha) and neuraminidase (na) surface glycoproteins. only one type of ha and one type of na are recognized for influenza b. influenza a occurs in both pandemic and interpandemic forms. fortunately, pandemics, defined as worldwide outbreaks of severe disease, occur infrequently. interpandemic influenza, although less extensive in its impact, occurs virtually every year. the epidemiologic pattern of influenza in humans is related to two types of antigenic variation of its envelope glycoproteins, namely antigenic drift and antigenic shift. during antigenic drift, new strains related to those circulating in previous epidemics evolve by accumulation of point mutations in the surface glycoproteins. this enables the virus to evade the immune system leading to repeated outbreaks during interpandemic years. antigenic shift occurs with the emergence of a "new" potentially pandemic, influenza a virus that possesses a novel ha alone or in combination with a novel na. there are 16 recognized ha subtypes and nine na subtypes of influenza a virus. viruses from all ha and na subtypes have been recovered from aquatic birds, but only three ha subtypes (hi, h2, and h3) and two na subtypes (nl and n2) have established stable lineages in the human population since 1918. since 1997, widespread avian infection with influenza a (h5n1) and associated clusters of human disease have aroused concern about the threat of a pandemic, and attention has been appropriately focused on control measures to deal with such an event. all influenzaviruses have a segmented, negative-sense rna core surrounded by a lipid envelope. influenzavirus particles are pleomorphic. among isolates that have undergone a limited number of passages in cell culture or eggs, more filamentous than spherical particles are seen. spherical morphology becomes dominant when the virus is extensively passaged in the laboratory. a 10-to 12-nm layer of ha (rod-shaped) and na (mushroomshaped) spikes project radially on the surface of the influenza a and b viruses. hemagglutinin facilitates entry of virus into host cells through its attachment to sialic-acid receptors. because neutralizing antibodies are directed against this antigen, it is a critical component of current influenza vaccines. neuraminidase, the second major antigenic determinant, catalyzes the cleavage of glycosidic linkages to sialic acid and the release of progeny virions from infected cells. accordingly, it has become an important target for drug inhibitors such as oseltamivir and zanamivir. the m2 surface component and channel of influenza a (not present in influenza b virus) regulates the internal ph of the virus and is blocked by the antiviral drug amantadine. influenzaviruses are spread person-to-person primarily through the coughing and sneezing of infected persons. the typical incubation period for influenza is 1 to 4 days, with an average of 2 days. adults can be infectious from the day before symptoms begin through approximately 5 days after illness onset. children can be infectious for ~1o days, and young children can shed virus for several days before their illness onset. severely immunocompromised persons can shed virus for weeks or months. uncomplicated influenza illness is characterized by the abrupt onset of constitutional and respiratory signs and symptoms (e.g., fever, myalgia, headache, malaise, nonproductive cough, sore throat, and rhinitis). among children, otitis media,nausea,and vomiting are also commonly reported with influenza illness. respiratory illness caused by influenza is difficult to distinguish from illnesses caused by other respiratory pathogens on the basis of symptoms alone. influenza typically resolves after 3 to 7 days in most patients, although cough and malaise can persist for >2 weeks. among certain persons, influenza can exacerbate underlying medical conditions (e.g., pulmonary or cardiac disease), lead to secondary bacterial pneumonia or primary influenza viral pneumonia, or occur as part of a co-infection with other viral or bacterial pathogens. young children with influenza infection can have initial symptoms that mimic bacterial sepsis. more than 20% of children hospitalized with influenza can have febrile seizures. influenza has also been associated with encephalopathy, transverse myelitis, reye syndrome, myositis, myocarditis, and pericarditis. the risks for complications, hospitalizations, and deaths from influenza are higher among persons aged ~65 years, young children, and persons of any age with certain underlying health conditions than among healthy older children and younger adults. influenza-related deaths can result from pneumonia or from exacerbations of cardiopulmonary conditions and other chronic diseases. the histopathologic features of nonfatal and fatal influenza have been well described and include necrotizing bronchitis, thrombosis, interstitial inflammation, hemorrhage, hyaline membrane formation, and intra alveolar edema (figs. 11.8a,b, 11 .9a,c, and 11.10a). 105, [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] [191] the pathology is more prominent in larger bronchi, and inflammation may vary in intensity in individual patients, viral inclusions cannot be identified by light microscopy (fig, 11 .8d), secondary bacterial infections with organisms such as streptococcus pneumoniae (group a streptococcus [gas]), staphylococcus aureus, and haemophilus influenzae may occur as a complication in about 50% to 75% of fatal cases and make it difficult to recognize the pathologic changes associated with the primary viral infec-445 tion ,190,192,193 the histopathologic features in other organs may include myocarditis, cerebral edema, rhabdomyolysis, and hemophagocytosis (figs, 11.8h and 11.9e,f), immunohistochemistry and ish assays demonstrate that viral antigens and nucleic acids are usually sparse and are primarily seen in the bronchioepithelial cells of larger bronchioles (figs. 11.8c,e,f and 11.9d) . 105.189,190 antigens are more readily identified in patients who die within 3 to 4 days of onset of illness. recent studies suggest that unlike human influenza viruses, avian virus h5n1 preferentially infects cells in the lower respiratory tract of humans, resulting in extensive damage of the lungs with minimal pathology in the upper respiratory tract (fig. 11.10a,c) . this may help explain why the h5n1 avian influenza virus is so lethal to humans but so difficult to spread from person to person. these studies show that the avian virus preferentially binds to the a-2,3-galactose receptors, which are found only in and around the alveoli. this is in contrast to the human influenzaviruses that preferentially bind to the a-2,6-receptors, which are found throughout the respiratory tract from the nose to the lungs. 194.195 in birds and other animals, viral antigens can be detected in the lung as well as a variety of extrapulmonary tissues (fig. 11.10b) . the diagnosis of influenza, suspected by history and clinical manifestations, can also be supported histopathologically. however, because of the absence of any characteristic viral inclusions and because the overall pathologic features of influenza may resemble other viral, rickettsial, and certain bacterial infections, an unequivocal diagnosis can be made only by laboratory tests such as viral culture, direct fluorescent antibody and rapid antigen assays, serology, and ihc. 190 ,196,197 measles measles (rubeola) is an infectious, acute febrile viral illness characterized by upper respiratory tract symptoms, fever, and a maculopapular rash. the causative agent, a member of the genus morbillivirus, of the family paramyxoviridae,198,199 is an enveloped virus that contains a negative sense, single-stranded rna genome of 16,000 nucleotides. other human pathogens in this family include parainfluenza, mumps, and respiratory syncytial viruses. measles virions are pleomorphic, generally spherical, enveloped particles from 120 to 250 nm in diameter. the virus is morphologically indistinguishable from other members of the paramyxoviridae family when viewed by negative contrast electron microscopy. a lipid envelope surrounds a helical nucleocapsid composed of rna and protein. two transmembrane glycoproteins, hemagglutinin (h) and fusion (f), are present in the envelope and appear as surface projections. these proteins mediate viral attachment and figure 11.8. influenza a. a. alveolar damage in a patient with fatal influenza a showing prominent congestion with intraalveolar macrophages and fibrin deposits. b. extensive ulceration of respiratory epithelium in a large airway of a patient with fatal influenza a. the lamina propria shows florid vascular congestion and focal hemorrhage, and predominantly mononuclear inflammatory cell infiltrates. c-e. immunohistochemical staining (c,e) of influenza virus a hemagglutinin antigens in the cytoplasm of residual respiratory epithelial cells of a large airway. this same focus of extensively infected respiratory epithelium (d) demonstrates that, unlike many other viral respiratory pathogens, influenza viruses do not elicit specific cytopathic effects in infected cells. f. in-situ hybridization assay demon-• fusion with respiratory epithelium. they are also believed to playa role in virus maturation through their interaction with the matrix (m) protein, which, in turn, is thought to interact with the nucleocapsid structure. 2oo measles is a highly communicable disease of worldwide distribution. before the introduction of measles vaccines, epidemics occurred about every 2 to 5 years when the percentage of nonimmune members of a population reached critical levels. recently, epidemics have occurred in cycles of about 10 years. in small and isolated communities, measles circulation may cease altogether unless it is reintroduced. if introduced in non immune populations, the disease tends to be more severe and may involve more than 90% of the population because of the highly infectious nature of the virus. although still a significant problem in underdeveloped countries, measles infection became uncommon in the us. after the development and widespread use of an effective measles vaccine. however, a recrudescence of measles infection occurred in several large us. urban centers in recent years, associated with reduced use of the vaccine among children and young adults. during the peak of this activity (i.e., between 1989 and 1991), greater than 50,000 measles cases and approximately 150 measlesassociated deaths were reported. 20l measles virus is highly contagious, spread by aerosols and droplets from respiratory secretions of acute cases.202-204 less frequently, contaminated fomites are involved in transmission. a person with acute measles is infective from just before onset of symptoms to defervescence of fever. in developed countries, likely settings for exposure to measles virus are infectious disease clinics, pediatric emergency rooms, and physicians' offices. 20s children are usually infected by 6 years of age, resulting in lifelong immunity, and almost all adults are immune. clinical infection in children younger than 9 months of age is generally uncommon because of passive protection afforded the infant by the transfer of maternal antibodies. however, with the resurgence of measles in the us. came the realization that most women of childbearing age 447 strating active replication of influenza a in respiratory epithelium in a large airway. g. electron micrograph of influenza a particles (arrows) attached to the cilia of a rodent tracheal epithelial cell. viral ribonucleoproteins are evident in the central aspect of the particles. the hemagglutinin and neuraminidase surface glycoproteins make up the peripheral spike layer. (courtesy of f.a. murphy.) h. rhabdomyolysis in a patient with fatal influenza a. bar, 100nm. a,b,d,h, h&e; c,e, immunoalkaline phosphatase stain, naphthol fast-red, and hematoxylin counterstain; f, digoxigenin-iabeled probe followed by immunoalkaline phosphatase staining, naphthol fast-red. and hematoxylin counterstain; g, uranyl acetate, lead citrate stain. acquired immunity to measles through vaccination and not through natural infection. lower levels of maternal antibodies were found to be transferred from an immunized mother to her infant; thus, a substantial number of measles cases occurred in children younger than 1 year of age in the us. in recent years. after an incubation period of about 1 to 2 weeks, the prodromal phase of measles begins with fever, rhinorrhea, cough, and conjunctivitis. koplik's spots, which are small, irregular red spots with a bluish-white speck in the center, appear on the buccal mucosa in 50% to 90% of cases shortly before rash onset. an erythematous maculopapular rash begins on the face 3 to 4 days after prodromal symptoms and usually spreads to the trunk and extremities. the symptoms gradually resolve, with the rash lasting for approximately 6 days, fading in the same order as it appeared. although recovery is rapid and complete in most cases, complications can arise as a result of continued and progressive virus replication, bacterial or viral superinfections, or abnormal host immune response. 204.206.207 the most common complications are secondary bacterial pneumonia and otitis media. 206 other complications include febrile convulsions, encephalitis, liver function abnormalities, chronic diarrhea, and sinusitis. several pulmonary and central nervous system (cns) syndromes that are often fatal have been described. death occurs in about 1 of every 1000 measles cases; however, the risk of death and other complications is substantially increased in infants, adults, malnourished and immunocompromised individuals, persons with underlying illnesses,and nonimmunized populations in underdeveloped countries.208-212 the first step in measles infection is attachment of the virus to cd46 cell surface receptors on the respiratory epithelium.213 adhesion and fusion of the virus to the respiratory epithelium is mediated by both the hand f viral glycoproteins. 214 this stage is followed by local replication in respiratory mucosa and draining lymph nodes. a primary viremia follows, with dissemination two types of multinucleated giant cells have been described in patient tissues during measles infection. 215 ,216 the reticuloendothelial giant cell (warthin-finkeldey) appears first during the incubation period and is seen in 449 the nucleus of a pneumocyte in the lung of a patient with fatal avian influenza. unlike other influenza viruses that cause disease in humans, h5n1 preferentially infects alveolar epithelial cells, and causes relatively minimal pathology in the upper airway. a, h&e; b,c, immunoalkaline phosphatase stain, naphthol fast-red, and hematoxylin counterstain. different lymphoid tissues throughout the body. the second type is the epithelial giant cell, which has been observed in the epithelium of essentially every major organ. the onset of the rash temporally coincides with the appearance of detectable serum antibody to measles virus. interestingly, virus replication and giant cell formation cease with the appearance of rash. t-cell immunity is essential in the process of viral clearance from lymphoid tissue and respiratory tract. while children with congenital agammaglobulinemia respond normally to measles virus infection, patients with cell-mediated immunodeficiency develop severe disease that presents as giant-cell pneumonia or encephalopathy in the absence of an exanthem. [208] [209] [210] [211] 217, 218 immunity to the f surface glycoprotein is necessary to prevent the spread of measles infection. 219 an atypical measles syndrome characterized by pulmonary consolidation with pleural effusions and hilar adenopathy has been reported in children exposed to wild-type measles virus who had previously received the killed-measles virus vaccine.220-222 recipients of the formalin-inactivated vaccine have a good antibody response to the h protein, but antibodies to the functional region of the f protein and to the nucleoprotein are weak or absent. it has been suggested that the lack of a functional f antibody response may playa role in virus spread. 223 the pathologic features of measles have been well described and several references containing detailed morphologic descriptions are recommended. 13 1,209.224-230 the typical morbilliform skin lesions, koplik's spots, and measles lymphadenitis are seldom seen by the surgical pathologist since the clinical diagnosis is usually apparent. histopathologic changes in the skin include mild congestion, edema, and a predominantly mononuclear infiltrate surrounding small vessels of the dermis, as well as other nonspecific features. occasional diagnostic multinucleated epithelial giant cells with eosinophilic cytoplasmic and nuclear inclusions are observed. 228 .231 pathognomonic reticuloendothelial multinucleated giant cells can be observed in appendix specimens from patients mistakenly operated on for acute appendicitis before the emergence of diagnostic koplik's spots and rash. these cells, which have been reported in various lymphoreticular tissues throughout the body, are typically large and contain from a few to occasionally up to 100 nuclei. these cells do not usually contain viral inclusions. the lymphoid tissues are typically hyperplastic, and the architecture is partially or totally obliterated by diffuse proliferation of immunoblasts. [232] [233] [234] a focal or generalized interstitial pneumonitis, similar to that seen in many other viral infections, is seen in the lungs of measles patients. histopathologic features seen include various degrees of peribronchial and interstitial mononuclear cell infiltrates, squamous metaplasia of bronchial endothelium, proliferation of type ii pneumocyte alveolar lining cells, and intraalveolar edema with or without mononuclear cell exudates and hyaline membranes. secondary changes, such as bacterial or viral superinfection, or organizational changes may alter the original pathology. the hallmark of the disease is the formation of multinucleated epithelial giant cells (fig. ll.lla,b) . these cells, which are often numerous, are formed by fusion of bronchiolar or alveolar lining epithelial cells (fig.ll.l1a) . in contrast to the reticuloendothelial giant cells, these cells generally contain characteristic nuclear and cytoplasmic inclusions. the intranuclear inclusions are homogeneous, eosinophilic, and surrounded by a slight indistinct halo (fig. 11.11c,d) . the cytoplasmic inclusions are deeply eosinophilic, vary in size, and some form large masses with a "melted tallow" appearance ( fig. 11.11d ). these giant cells may undergo degenerative changes with progressive loss of cytoplasm, increasing basophilia, and shrinkage of nuclei. the presence of measles virus in these giant cells may be demonstrated by immunofluorescent,235.236 ihc,21o.237 and ish techniques (fig.ll.lle,f) . these giant cells can also be seen in extra pulmonary tissues (fig. 11.11 g,h) . the diagnosis of typical cases of measles can usually be made on the basis of clinical signs and symptoms. other causes of a similar rash, but without other features of measles, include rubella, dengue virus, enteroviruses, and drug reactions, especially to ampicillin. the typical case of measles giant-cell pneumonia generally presents little diagnostic difficulty for the surgical pathologist. the presence of giant cells with both intranuclear and intracytoplasmic inclusions in a setting of interstitial pneumonitis is highly specific for measles infection. however, multinucleated giant cells are not seen in all cases of measles pneumonia and their absence should not exclude the j diagnosis. other viral and rickettsial agents may also cause a similar interstitial pneumonitis, but without the typical giant cells, and should be differentiated. 229 as previously noted, the histopathologic features in measles pneumonia can be somewhat variable,226 and secondary bacterial and viral infections may modify the histology, further complicating the pathologic diagnosis ( fig. 11.5d ).207.229 other viral pathogens, such as respiratory syncytial virus,238 parainfluenza,239.240 vzv,241 and a recently discovered hendra virus,242 as well as granulomatous diseases of the lung, may give rise to pneumonia with giant cells and should also be considered in the differential diagnosis. however, these clinical entities can be distinguished by history, histopathologic features, and laboratory tests. immunohistochemistry237.243,244 or ish 243 ,244 tests demonstrate viral antigens or nucleic acids in the majority of cases. laboratory confirmation is useful to avoid possible confusion with other rash-causing illnesses. diagnostic laboratory procedures consist of direct detection of either the virus or the viral antigens, usually by indirect immunofluorescence or by serologic methods using hemagglutination inhibition, neutralization, or enzyme immunoassay. specimens for serologic testing consist of acute-and convalescent-phase serum pairs. antibody appears within 1 to 2 days after onset of rash, and titers peak approximately 2 weeks later. alternatively, the presence of specific immunoglobulin m (igm) antibody can be used to diagnose recent infection. 245 human parainfluenza viruses (hpivs) are second only to rsv as a cause of lower respiratory tract disease in young children. human parainfluenza viruses are negative-sense, nonsegmented, single-stranded, enveloped rna viruses that possess fusion and hemagglutinin-neuraminidase glycoprotein "spikes" on their surface (fig. 11 .ig). the four serotypes of hpiv belong in the family paramyxoviridae, subfamily paramyxovirinae, and genera respirovirus (hpiv-l and -3) and rubulavirus (hpiv-2 and -4). the virions are variable in shape and size, ranging from 150 to 300nm. 246 human parainfluenza viruses are spread from respiratory secretions through close contact with infected persons or contact with contaminated surfaces or objects. infection can occur when infectious material contacts mucous membranes of the eyes, mouth, or nose, and possibly through the inhalation of droplets generated by a sneeze or cough. human parainfluenza viruses are unstable in the environment (surviving a few hours on environmental surfaces), and are readily inactivated with soap and water. they are ubiquitous, and infect most s,r. zaki and cd. paddock people during childhood. the highest rates of serious hpiv illnesses occur among young children. serologic surveys have shown that 90% to 100% of children aged 5 years and older have antibodies to hpiv-3, and about 75% have antibodies to hpiv-l and -2. the different hpiv serotypes differ in their seasonality, with hpiv-l causing biennial outbreaks of croup in the fall and hpiv-2 causing annual or biennial fall outbreaks. human parainfluenza virus-3 peak activity occurs during the spring and early summer months each year, but the virus can be isolated throughout the year. similar to rsv, hpivs can cause repeated infections throughout life, usually manifested by an upper respiratory tract illness (e.g., cold and sore throat). human parainfluenza viruses can also cause serious lower respiratory tract disease with repeat infection (e.g., pneumonia, bronchitis, and bronchiolitis), especially among the elderly, and among patients with compromised immune systems. each of the four hpivs has different clinical and epidemiologic features. the most distinctive clinical feature of hpiv-l and hpiv-2 is croup (i.e., laryngotracheobronchit is ); hpiv-l is the leading cause of croup in children, whereas hpiv-2 is less frequently detected. both hpiv-l and -2 can cause other upper and lower respiratory tract illnesses. human parainfluenza virus-3 is more often associated with bronchiolitis and pneumonia. human parainfluenza virus-4 is infrequently detected, possibly because it is less likely to cause severe disease. the incubation period for hpivs is generally from 1 to 7 days. 247 most hpiv infections cause a mild, self-limited illness; however, hpiv-3 infections are an important cause of bronchiolitis, croup, and pneumonia that may be lifethreatening in infants and newborns. 247 human parainfluenza virus infections are also increasingly being recognized as an important cause of severe morbidity and mortality in immunocompromised patients. 20 ,248--252 the mortality of bone marrow transplant patients with hpiv-3 infection has been reported to be as high as 60%. 249, 253, 254 in patients with severe hpiv infection, multinucleated giant cells derived from the respiratory epithelium may be seen in association with an interstitial pneumonitis and organizing changes (fig. 11.12a,b ) . 239,240.249,255-260 these giant cells, which may contain intracytoplasmic eosinophilic inclusions, (fig. 11.12c) , have also been reported in extrapulmonary tissues such as kidney, bladder, and pancreas. 258 other viral causes of giant cell pneumonia, including measks, rsv, vzv, and hsv, should be considered in the histopathologic differential and laboratory testing, including ihe, can be useful in making this differentiation possible ( fig. 11.12d) . diagnosis of infection with hpiv scan also be made by virus isolation, direct detection of viral antigens py enzyme-linked immunoassay (eia) or immunofluorescent assay (ifa) in clinical specimens, detection of viral rna by rt-pcr, demonstration of a rise in respiratory syncytial virus (rsv) is the most common cause of bronchiolitis and pneumonia among infants and children under 1 year of age. the causative agent is a negative-sense, nonsegmented, single-stranded, enveloped rna virus. the virion is variable in shape and size and ranges from 120 to 300 nm. respiratory syncytial virus is a member of the family paramyxoviridae, subfamily pneumovirinae, in the genus pneumovirus, and can be 453 ing poorly defined cytoplasmic eosinophilic inclusions. d. parainfluenza virus antigens in giant cells localized by using immunohistochemistry. a-c, h&e; d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. further distinguished genetically and antigenically into two subgroups, a and b. the subgroup a strains are usually associated with more severe infections. two surface glycoproteins, g and f, are present in the envelope and mediate attachment and fusion with respiratory epithelium. the f protein also mediates coalescence of neighboring cells to form the characteristic multinucleated syncytial giant cells for which the virus name is derived. 265 respiratory syncytial virus is spread from respiratory secretions through close contact with infected persons or contact with contaminated surfaces or objects?66 infection can occur when infectious material contacts mucous membranes of the eyes, mouth, or nose, and possibly through the inhalation of droplets generated by a sneeze or cough. respiratory syncytial virus is unstable in the environment, surviving a few hours on environmental surfaces, and is readily inactivated with soap and water. in temperate climates, rsv infections usually occur during annual community outbreaks, often lasting several months, during the late fall, winter, or early spring months. the timing and severity of outbreaks in a community vary from year to year. respiratory syncytial virus spreads efficiently among children during the annual outbreaks, and most children will have serologic evidence of rsv infection by 2 years of age. illness begins most frequently with fever, runny nose, cough, and sometimes wheezing. during their first rsv infection, between 25% and 40% of infants and young children have signs or symptoms of bronchiolitis or pneumonia, and 0.5% to 2% require hospitalization. most children recover from illness in 8 to 15 days. the majority of children hospitalized for rsv infection are under 6 months of age. respiratory syncytial virus also causes repeated infections throughout life, usually associated with moderate-to-severe cold-like symptoms; however, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems. the major histopathologic changes described in fatal rsv infections include necrotizing bronchiolitis and interstitial pneumonia (fig. 11.13a,b) .238,267-272 bronchiallumina and airways are usually filled with necrotic debris and inflammatory cells. these findings may be accompanied by various degrees of diffuse alveolar damage (fig. 11.13e ), organizational changes, and secondary bacterial superinfection. giant cell pneumonia is a feature seen in some cases ( fig. 11.13c,d) . the multinucleated giant cells contain irregular, intracytoplasmic, eosinophilic inclusions surrounded by a clear halo. these inclusions are extremely difficult to identify with any degree of certainty and are only seen in about half the cases (fig. 11.13d) . other viral causes of giant cell pneumonia should be considered in the histopathologic differential and laboratory testing, including ihc,271,273,274 can be useful in making this differentiation possible ( fig. 11.13b,f) . diagnosis of rsv infection can also be made by virus isolation, direct detection of viral antigens in clinical specimens by eia or ifa, detection of viral rna by rt-pcr, demonstration of a rise in rsvspecific serum antibodies, or a combination of these approaches (see also fig. 7.43, chapter 7) .264,275-281 in 2001, van den hoogen et a1. 282 described the identification of this new viral agent from clinical specimens obtained from patients with respiratory illness, which 455 they designated human metapneumovirus (hmpv). it is a negative-sense, nonsegmented, single-stranded, enveloped rna virus. the virion is variable in shape and size, ranging from 150 to 300nm (fig. 11.1f ). it has been categorized in the family paramyxoviridae, subfamily pneumovirinae, genus metapneumovirus, based on genomic sequence and gene constellation. human metapneumovirus can be further distinguished genetically and antigenically into two subgroups, a and b. similar to rsv, hmpv infection is ubiquitous and occurs during infancy and early childhood, with annual epidemic peaks occurring in the winter and spring months in temperate regions. seroprevalence studies reveal that 25% of all children aged 6 to 12 months have antibodies to hmpv; by age 5 years, 100% of patients have evidence of past infection. like rsv, hmpv has been associated with a wide spectrum of respiratory illnesses.283-287 the patient may be asymptomatic, or symptoms may range from mild upper respiratory tract illness to severe bronchiolitis and pneumonia. although rsv, hpiv-l, and hpiv-3 have been definitively linked to cases of lower respiratory tract disease in infants and young children, the relative contribution of hmpv remains undetermined. like rsv and the hpivs, studies suggest that hmpv may also contribute to respiratory disease in elderly adults and the immunocompromised. 288-29o histopathologic descriptions of features of hmpv infections are few and have not been well described. [291] [292] [293] [294] this is partly related to interpreting the clinical significance of virus detection in context with the ubiquitous nature of the virus. virus detection in such cases is usually made by culture isolation of the virus from upper airways or by pcr assays performed on nasopharyngeal aspirates or bal washings. in nonhuman primates viral antigens are observed in ciliated epithelial cells, type 1 pneumocytes, and alveolar macrophages. this distribution is associated with mild, multifocal, erosive, and inflammatory changes in airways, and an increased number of foamy macrophages in alveoli. 291 the bal specimens collected from patients within a few days of a positive hmpv assay show degenerative changes and cytoplasmic inclusions within epithelial cells, multinucleated giant cells, and histiocytes. the intracytoplasmic inclusions are ill-defined, eosinophilic structures that measure 3 to 4 ~m. 293 lung biopsy or autopsy tissue obtained and examined later in the disease show chronic airway inflammation, intraalveolar foamy and hemosiderin-laden macrophages, and acute and organizing lung injury including areas of diffuse alveolar damage with hyaline membrane formation and foci of a bronchiolitis obliterans/organizing pneumonia like reaction ( fig. 11 .14). in such cases, typical multinucleated giant cells or viral inclusion cannot be identified. 292 -294 in-situ hybridization studies on limited number of human cases human metapneumovirus is difficult to identify with commonly used viral diagnostic procedures. the virus replicates slowly in primary and tertiary monkey kidney cell lines, and the cytopathic effect can be difficult to discern. commercial monoclonal antibody reagents to hmpv are not widely available. most hmpv studies have been conducted using rt-pcr assays or by demonstration of a rise in hmpv-specific serum antibodies. two novel paramyxoviruses, hendra and nipah, have been recently identified in australia and malaysia; these viruses have been associated with acute febrile encephalitis and respiratory tract disease. both infections are zoonotic. hendra was first identified in 1994 when patients who came in close contact with sick horses developed an influenza-like illness. two patients died with pneumonitis and multiorgan failure. 30 the closely related nipah virus was identified during an outbreak in malaysia and singapore during 1998-1999 that included more than 250 patients. patients presented with a severe acute encephalitic syndrome, but some also had significant pulmonary manifestations. 22 ,295-302 most of the patients had a history of contact with pigs, most of them being pig farmers. in bangladesh in 2001 and 2003, outbreaks of nipah encephalitis occurred. 303 ,304 similar to the malaysian outbreak, the most prominent symptoms were fever, headache, vomiting, and an altered level of consciousness. respiratory illness was much more common in the bangladesh cases, however, with 64 % having cough and dyspnea. the reason for increased involvement of the respiratory tract in this outbreak is not known. epidemiologic and laboratory investigations identified fruit bats of the pteropus genus as asymptomatic carriers of hendra and nipah viruses and possible animal reservoirs. [305] [306] [307] [308] [309] hendra and nipah viruses belong to the recently designated genus henipavirus within the family paramyxoviridae, subfamily paramyxovirinae. both viruses are nonsegmented, negative-stranded rna viruses composed of helical nucleocapsids enclosed within an envelope to form roughly spherical, pleomorphic virus particles.3io-312 the structure of their genome is consistent with the other members of the subfamily. 30 histopathologic findings in fatal cases of hendra and nipah infections are similar with varying degrees of cns and respiratory tract involvement. 104 ,313-315 findings include a systemic vasculitis with extensive thrombosis, endothelial cell damage, necrosis, and syncytial giant cell formation in affected vessels ( fig. 11.15a ,b,f). plaques with various degrees of necrosis, in association with inclusion-bearing neurons, can be found in both the gray and white matter of the cns (fig. 11.15e ). multinucleated giant cells with intranuclear inclusions can occasionally be seen in lung, spleen, lymph nodes, and kidneys ( fig. 11 .15c,g). in the lung, vasculitis and fibrinoid necrosis can be seen in majority of cases. fibrinoid necrosis often involves several adjacent alveoli and is frequently associated with small vessel vasculitis. the multinucleated giant cells with intranuclear inclusions are usually noted in alveolar spaces adjacent to necrotic areas. histopathologic changes of bronchiolar epithelium are uncommon; rarely, the large bronchi may show transmural inflammation and ulceration. widespread presence of nipah virus antigens can be seen by ihc in endothelial and smooth muscle cells of blood vessels as well as in various parenchymal cells (fig. 11.15d ). the diagnosis of nipah virus infection, suspected by patient history and clinical manifestations, can be supported by characteristic histopathologic findings. from a diagnostic standpoint, perhaps the most unique histopathologic finding is the presence of syncytial and parenchymal multinucleated endothelial cells. this feature 457 occurs in only approximately one fourth of the cases and cannot be used as a sensitive criterion for the diagnosis of henipah virus infections; furthermore, these cells can also be seen in measles virus, rsv, hpiv, herpesviruses, and other infections. unequivocal diagnosis can be made only by laboratory tests such as ihc, cell culture isolation, pcr, or serology.l04,304,316-319 the parvoviruses are small (18 to 26nm) naked viruses that possess a single-stranded dna genome and require actively dividing cells to complete the viral replication cycle. in 2005, allander et a1. 320 identified a new parvovirus (genus bocavirus) associated with lower respiratory tract infections in children; however, there is no information as yet that describes specific pulmonary pathology attributed to this newly identified agent. on the other hand, human parvovirus b19, a member of the genus erythrovirus, has long been known to cause human disease and has been well studied. 321 ,322 the most commonly recognized manifestation of b19 infections is erythema infectiosum, and approximately one third of the cases of maternal parvovirus infections result in intrauterine parvovirus b19 infections. this places the fetus at increased risk for severe anemia, hydrops, and death. hydrops fetalis is the most commonly recognized complication of intrauterine parvovirus infection, accounting for 4% to 18% of all cases of nonimmune hydrops. cases tend to be clustered during community outbreaks of erythema infectiosum. 321 . 3 22 pathophysiologic effects and histopathologic findings are a result of the tropism of b19 parvovirus for erythroid precursor cells. villi from placentas from patients with bl9-associated nonimmune hydrops are edematous, and fetal capillaries show numerous nucleated erythroid precursors, some containing parvovirus inclusions. the infected cells with eosinophilic "ground glass" intranuclear inclusions and ring-like margination of nuclear chromatin are easily recognized and in the context of a hydropic fetus are pathognomonic of b19 infection. the liver is the major site of blood production in the fetus and the principal organ affected by intrauterine b19 infection. inclusion-bearing nucleated erythrocytes can also be frequently identified in the lung, making histopathologic examination of this organ a worthwhile endeavor for confirming the diagnosis of intrauterine bi9 infection ( fig. il.i6a,b) .323-332 however, these cells may be infrequent and irregularly distributed, requiring examination of multiple sections and use of ihc to confirm the diagnosis (fig.l1.16c,d) . the combination of fever and hemorrhage can be caused by different viruses, rickettsiae, bacteria, protozoa, and fungi. however, the term viral hemorrhagic fever (vhf) is usually reserved for systemic infections characterized by fever and hemorrhage caused by a special group of viruses transmitted to humans by arthropods and rodents. the vhfs are febrile illnesses characterized by abnormal vascular regulation and vascular damage and are caused by small, lipid-enveloped rna viruses. this syndrome can be caused by viruses belonging to four different families that differ in their genomic structure, replication strategy, and morphologic features (table 11 .4). arenaviruses, bunyaviruses, and filoviruses are negative-stranded, 459 in the nucleus and cytoplasm of erythroid precursors by using immunohistochemistry (ihe) (same patient as in a,b). a,b, h&e; c,d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. whereas fiaviviruses are positive-stranded rna viruses. hemorrhagic fever viruses are distributed worldwide, and the diseases they cause are traditionally named according to the location where they were first described. the oldest and best known is yellow fever virus; others include lassa fever, lymphocytic choriomeningitis, ebola, and dengue viruses. viral hemorrhagic fevers share many common pathologic features, although the overall changes vary among the different diseases. the similar pathologic and immunopathologic findings in cases of vhf suggest that microvascular involvement and instability is an important common pathogenic pathway leading to shock and bleeding in many instances. infection of the mononuclear phagocytic system and endothelium are thought to play c s.r. zaki and cd. paddock 11 .17. ebola virus hemorrhagic fever. a. pulmonary congestion and lack of inflammation. b. numerous filamentous ebola virus inclusions are seen within hepatocytes in association with hepatocellular necrosis. c. ebola virus-infected intra alveolar macrophages as seen by colorimetric in-situ hybridization using digoxigenin-labeled probes. d. viral anti-gens are seen in endothelial cells and other interstitial cells in this lung section. a,b, h&e; c, digoxigenin-labeled probes followed by immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain; d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. figure 11.18 . yellow fever. a. pulmonary congestion in fatal case of yellow fever associated with vaccination. b. yellow fever antigens in the pulmonary interstitium of the same patient. in natural infections, yellow fever antigens are usually not seen outside the liver in fatal cases. in contrast, in vaccine-associated cases viral antigens can be found in a variety of extrapulmonary a critical role in the pathogenesis of vhfs through the secretion of physiologically active substances, including cytokines and other inflammatory mediators (figs. 11.17c,d, 11.18b, 11.19b,c, and 11.20b). at autopsy common findings include widespread petechial hemorrhages and ecchymoses involving skin, mucous membranes, and internal organs. however, in many hf patients manifestations of bleeding may be minimal or absent. effusions, occasionally hemorrhagic, are also frequently seen. widespread, focal, and sometimes massive necrosis can be commonly observed in all organ systems and is often ischemic in nature. necrosis is usually most prominent in the liver and lymphoid tissues. the most con-461 organs, including heart, lung, and spleen. c. extensive midzonal hepatic necrosis characteristic of yellow fever. d. abundant antigens of yellow fever virus are observed in midzonal area of hepatic lobule in this immunohistochemical preparation. a,c, h&e; b,d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. sistent microscopic feature is found in the liver and consists of multifocal hepatocellular necrosis with cytoplasmic eosinophilia, councilman bodies, nuclear pyknosis, and cytolysis (figs. 11.17b and 11.18c). inflammatory cell infiltrates and necrotic areas are usually mild and, when present, consist of neutrophils and mononuclear cells. commonly observed histopathologic changes in the lung include various degrees of hemorrhage, intra alveolar edema, interstitial pneumonitis, and diffuse alveolar damage (figs. 11.17 a, 11.18a, 11.19a, 11.20a, and 11.21a). several references containing detailed pathologic descriptions in human cases are recommended. 29.72.333-35o .' a. pulmonary hemorrhage and diffuse alveolar damage in a patient who was infected through organ transplantation. note that, unlike this case, which occurred due to immunosuppression, lcmv infections are rarely fatal and usually resolve with no specific treatment. b. abundant lcmv antigens in areas of the diagnosis of vhf should be suspected in patients with appropriate clinical manifestations returning from an endemic area, particularly if there is travel to rural areas during seasonal or epidemic disease activity. the diagnosis suspected by history and clinical manifestations can also be supported histopathologically, and the overall pattern of histopathologic lesions may suggest a specific diagnosis. however, because of similar pathologic features seen in vhf and a 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west nile virus in the united states acknowledgments. the authors gratefully acknowledge the invaluable assistance of gillian genrich, of the cdc, for her comments and help with organization of the references, cynthia goldsmith for her comments, and mitesh patel, of the cdc, for his help with preparation and assembly of the images. key: cord-008716-38sqkh9m authors: schmidt, alexander c; couch, robert b; galasso, george j; hayden, frederick g; mills, john; murphy, brian r; chanock, robert m title: current research on respiratory viral infections: third international symposium date: 2001-06-01 journal: antiviral res doi: 10.1016/s0166-3542(01)00136-x sha: doc_id: 8716 cord_uid: 38sqkh9m nan the third international symposium on respiratory viral infections was convened by the macrae group (new york, ny) in st. lucia, windward islands, on 1 -3 december 2000. for the third time, this symposium provided a forum for virologists, vaccinologists, clinicians, pharmacologists and public health specialists to discuss recent advances in respiratory virus research in an interdisciplinary fashion (kaiser et al., 1999; munoz et al., 2000) . the spectrum of discussion ranged from basic virology and pathogenesis to vaccinology, immunology, and management strategies for respiratory viral infections. epidemiology of respiratory viral disease and possible preparations for the next influenza pandemic were also an important part of the agenda. until 1953, influenza viruses were the only known filterable human respiratory tract pathogens. in 1931, shope recovered an influenza a h1n1 virus from swine, which probably was the first human influenza virus isolated. two years later smith, andrews and laidlaw recovered the first influenza isolate from humans, and soon after, efforts to develop an inactivated influenza a vaccine began. influenza b and c were isolated in 1940 and 1947 by francis and taylor, respectively. in 1953 in the laboratory of infectious diseases (lid) at the national institutes of health, and hilleman, then working at the walter reed army medical center, recovered the first human adenoviruses and established their importance in acute febrile respiratory tract disease (rowe et al., 1953; hilleman, 1954) . in the following years, robert chanock discovered most of the remaining respiratory viruses that are considered important lower respiratory tract pathogens today. the first of them, a croup-associated myxovirus, was discovered during an outbreak of croup in cincinatti in 1954, and it was later designated human parainfluenza virus type 2 (piv2). in 1956, morris and colleagues recovered the chimpanzee coryza agent (cca) during an outbreak of a cold-like illness in a chimpanzee colony, and a year later chanock and colleagues recovered two similar isolates from an infant with bronchopneumonia and from another infant with laryngotracheobronchitis, and characterized the human virus now known as respiratory syncytial virus (rsv) (chanock et al., 1957) . the discovery in 1958 of piv1 and piv3, the single most common cause of croup and the second most common cause of serious viral pediatric lower respiratory tract disease, respectively, broadened our understanding of the etiology of acute lower respiratory tract disease (chanock et al., 1958) . the discovery of piv4 in 1960 followed in short order. in 1961, a double-blind prospective study evaluating the use of tetracycline in the treatment of cold agglutinin-positive atypical pneumonia led to the identification of the etiologic agent of this disease. the agent, originally recovered by eaton from patients with this form of pneumonia, was known to be filterable and thought to be a virus but its role as an etiologic agent was heavily disputed. a large double-blind study by chanock, kingston and mufson, in which antibodies to the eaton 'virus' were used to define a subset of patients with pneumonia, showed that tetracycline therapy decreased duration of the disease, thereby excluding a virus etiology. subsequently, the eaton agent was shown by chanock, hayflick and barile to be a mycoplasma that grew in cell-free medium; later it was named mycoplasma pneumoniae (chanock et al., 1962) . serologic analyses and studies in adult volunteers confirmed the etiologic role of this organism in cold agglutinin-positive atypical pneumonia. renewed efforts in vaccine development against respiratory viruses began in the 1960s with the observation that infants and young children, after having recovered from respiratory tract infection with adenoviruses, shed virus from their gastrointestinal tract for an extended period of time without experiencing gastrointestinal symptoms. this led to the hypothesis that one could potentially use the gastrointestinal tract to vaccinate against respiratory tract disease caused by these viruses. wild-type adenovirus type 4 and 7 administered orally in enteric-coated capsules was found to protect military recruits against respiratory tract disease caused by these viruses. gastrointestinal symptoms were not observed, and although virus was shed from the intestine, it did not infect close contacts (couch et al., 1963) . the development of vaccines against respiratory viruses suffered a major setback in 1966 when formalin-inactivated rsv vaccine not only failed to protect infants against rsv infection but instead potentiated rsv disease upon subsequent rsv infection (kim et al., 1969) . the inactivated vaccine did not induce a potent neutralizing antibody response but it stimulated an exaggerated cd4 + t cell response without stimulating cytotoxic cd8 + t cells. this unanticipated failure of a non-living vaccine reoriented the research agenda of the laboratory of infectious diseases towards the development of live-attenuated virus vaccines. a cold-passaged rsv strain (cp52) was selected in 1966 as the first candidate live-attenuated rsv vaccine strain (friedewald et al., 1968) . this candidate vaccine was safe and immunogenic in adults and older children but was insufficiently attenuated in seronegative infants (kim et al., 1971) . since then, the search for a live rsv vaccine strain has been a central focus of the lid. developing a live rsv vaccine candidate that is attenuated yet immunogenic in seronegative infants has proved to be a formidable task. since incidence and morbidity of rsv are highest in the second and third month of life, a vaccine candidate has to be safe for administration to neonates, able to stimulate an immature immune system, and able to overcome the immunosuppressive and antiviral effects of passively acquired maternal rsv antibodies. initial vaccine candidates were derived in the late 1960s and early 1970s by passage of virus at low temperature (cold passage) or by chemical mutagenesis. several different lineages of mutants, such as temperature sensitive mutants generated by 5-fluorouracil (5fu) mutagenesis, were evaluated in infants and young children but were insufficiently attenuated or genetically unstable. the cold-passaged (cp) mutant that was subsequently further attenuated by the acquisition of two missense mutations that conferred temperature sensitivity (ts) to yield cpts rsv 248/404, has provided the most promising vaccine candidate tested thus far. this candidate vaccine virus was infectious, safe and immunogenic in 1-month-old seronegative infants, conferred protection against challenge with a second dose of vaccine virus 6 weeks later, and caused only mild upper respiratory tract symptoms (wright et al., 2000) . recent development of a method for rescue of infectious rsv from cdna by collins enhanced our ability to develop rsv vaccine candidates rapidly (collins et al., 1995) . site-directed mutagenesis can now be used for the first time to construct viruses with one or more additional attenuating mutations. using recombinant cdna technology, viable rsv mutants with deletion of the ns1, ns2, sh or m2-2 gene have been constructed as vaccine candidates that bear genetically stable attenuating mutations. for these reasons, it is likely that a live-attenuated vaccine that exhibits an acceptable balance between attenuation and immunogenicity can be developed within the next several years. this vaccine virus might possess one or more gene deletion mutations together with or without the earlier characterized cold-passaged (cp) and temperaturesensitive (ts) mutations. the first piv vaccine candidates were also prepared by formalin-inactivation. similar to the experience with formalin-inactivated rsv, these vaccines did not protect against piv disease. in the early 1980s, belshe attenuated a piv3 isolate by 45 passages at 20°c (cp45) (belshe and hissom, 1982) . in clinical studies, the piv3cp45 vaccine candidate has proved to be safe, genetically stable and immunogenic in seronegative in-fants (karron et al., 1995) . this vaccine candidate is currently being tested in phase ii clinical trials. a recombinant version of piv3cp45 has been rescued from cdna, and the genetic basis of its attenuation (att), temperature-sensitivity (ts) and cold-adaption (ca) phenotypes has been determined (skiadopoulos et al., 1999) . influenza a and b virus vaccine development followed this same path of serial passage at 20°c (cold-adaptation) to generate mutants with att, ts and ca phenotypes (maassab, 1969; maassab and bryant, 1999) . in contrast to piv and rsv, live attenuated influenza a vaccine strains were virus reassortants that were generated by mating the attenuated donor virus with an epidemic wild type virus so that the reassortant virus vaccine was a chimera that contained the attenuating genes of the donor virus, while the ha and na genes were derived from the current epidemic virus (murphy et al., 1980) . this strategy, developed by john maassab, of using the cold-adapted mutant virus a/aa/6/60 as the donor of the six attenuating internal and non-structural genes for construction of reassortant vaccine strains was validated by the large series of consecutive reassortants that have proven to be attenuated and immunogenic. analysis of the genetic basis of attenuation showed that the influenza a pb1 and pb2 genes each consistently specified the ts phenotype, and pa specified the ca phenotype. however, all three of these genes of the viral polymerase complex contribute to the attenuation of the trivalent influenza a (h1n1 and h3n2) and b vaccine viruses (murphy, 1993; maassab and bryant, 1999) . the safety, protective efficacy and phenotypic stability were confirmed in large phase iii trials, and licensure is expected in the near future (belshe et al., 1998) . evidence for the prophylactic effect of serum rsv neutralizing antibodies was demonstrated in the 1980s (prince et al., 1985b) . passive transfer of homologous rsv convalescent serum to cotton rats protected them against rsv replication in the lungs following subsequent intranasal challenge with wild type virus. a serum rsv neutralizing titer of 1:300 in recipient cotton rats conferred almost complete protection. this amount of neutralizing antibody necessary for protection was later confirmed in clinical trials and today forms the basis for passive rsv prophylaxis in high-risk infants (groothuis et al., 1993) . an increased incidence and severity of rsv disease is seen in preterm infants with or without chronic lung disease (cld), in children with congenital heart disease (chd), and in immunosuppressed children and adults. respiratory disease in general is a common cause for re-hospitalization of preterm infants. cunningham and colleagues compared a cohort of preterm infants (mean gestational age at birth 289 2 weeks) to a cohort of term infants and found a 10-fold increase in readmission for respiratory disease in preterm infants without cld, and a 18-fold increase for preterm infants with cld (2.5, 25 and 45%, respectively) (cunningham et al., 1991) . cld patients in a home oxygen program were at even higher risk (53% hospitalization, 13% icu admission) (groothuis et al., 1988) . children with pulmonary disorders such as cystic fibrosis, lung malformation or recurrent aspiration pneumonitis, when admitted for rsv disease, are as likely to require icu treatment (13-50%) and mechanical ventilation (up to 33%) as children with cld (35 and 28%, respectively) (arnold et al., 1999) . earlier studies of high-risk infants admitted for rsv disease yielded similar results, with icu treatment necessary in 13-34% and mechanical ventilation necessary in 12-16% (meert et al., 1990; navas et al., 1992) . apart from preterm infants with or without cld, children with congenital heart disease (chd) are a second group of high-risk patients, particularly when they suffer from pulmonary hypertension. in a prospective study of 699 children hospitalized in five consecutive rsv seasons (1976) (1977) (1978) (1979) (1980) , 63% of 27 patients with rsv disease and chd (compared with 14% of patients without chd) required icu treatment and 37% died (macdonald et al., 1982) . in a prospective study of 214 children with chd, the incidence of rsvrelated hospitalization during one rsv season was 15% for children younger than 4 years and 24% for infants younger than 6 months of age (simoes et al., 1998) . immunocompromised children are a very diverse population and not all of them are at equal risk for severe rsv infection. children receiving corticosteroid therapy have a much lower risk for rsv-related hospitalization and death than children receiving chemotherapy for malignancies or children with primary immunodeficiencies. rsvrelated mortality was 15% for chemotherapy-recipients and 40% for children with primary immunodeficiencies compared with 0% for steroid-treated children (hall et al., 1986) . rsv grew to very high titer in children receiving chemotherapy, and more than half the patients shed rsv for 3 weeks or longer. for immunocompromised adults, the picture is not much different from that described for children. infections are often acquired nosocomially, virus shedding is prolonged, and the incidence of pneumonia and death is high. rsv is the most important viral respiratory pathogen in these patients, followed by picornaviruses, influenza and parainfluenza viruses: 60% of confirmed rsv infections in leukemia and bone marrow transplant patients resulted in pneumonia, and the fatality rate was greater than 30% . whimbey and colleagues reported rsv case fatality rates for bone marrow transplant recipients as high as 31% when therapy was administered early and adequately (ribavirin and ivig), and 100%, when therapy was initiated late or inadequately . hiv-infected children in an urban setting in south africa also have an increased burden of viral lower respiratory tract illness (lri) although respiratory viruses are less frequently isolated from nasopharyngeal aspirates of hiv-infected children than from children without hiv infection. the relative risk for severe lri caused by rsv was twice as high in hiv-infected than in uninfected children two years of age or younger (madhi et al., 2000) . little information is available regarding genetic and environmental factors in susceptibility to rsv infections. most rsv epidemiologic studies are conducted in affluent countries and temperate climates although rsv is thought to be the leading cause of severe viral acute respiratory infections (ari) in infants around the globe (weber et al., 1998) . a 3-year prospective surveillance study was conducted in the yukon-kuskokwim (yk) delta of south-western alaska to determine the rate and severity of rsv infections requiring hospitalization for infants in this yupik eskimo population (karron et al., 1999) . the annual rate of rsv hospitalization for yk delta infants less than 1 year of age was unusually high, i.e. 53-249/1000. one in 125 children born in the yk delta, compared with between one in 550 to one in 11 000 infants in affluent countries (sims et al., 1976; martin et al., 1978; glezen et al., 1981) , required ventilatory support for rsv disease. rsv infection was the single most frequent cause of hospitalization of yk delta infants. as in temperate climates, rsv epidemics in the yk delta occur annually from november through june, with peak hospitalizations for rsv disease occurring between november and february. within sub-regions of the yk delta, epidemics were as brief as 1 month, probably because there is very limited traffic between villages in the winter months. most of the infants admitted to hospital were less than 1 year of age and had no medical risk factors for rsv disease. surprisingly, 9% of the admitted infants in the yk delta and 8% of infants in a comparison group admitted to johns hopkins hospital (jhh) were less than one month old. of children with severe disease, 34% in the yk delta and 24% at jhh were less than 2 months old. disease severity in non-high risk children did not differ between children admitted to jhh or yk delta regional hospital, suggesting that differences in hospitalization practices could not account for the high rates of hospitalization for rsv in yk delta infants. in the yk delta, 19% of those admitted for rsv disease were readmitted within a single rsv season. severity of rsv disease, age at first illness and receipt of ribavirin were all associated with readmission. in infants less than 6 months of age, a low neutralizing antibody titer in cord blood samples was strongly associated with severe disease. a questionnaire-based case control study that was matched for age and sub-region in the yk delta detected three risk factors influencing rsv hospital admission. medical risk factors (prematurity, chronic lung disease, congenital heart disease) increased the risk of admission 6.25-fold. more than eight people living in one household doubled the risk of hospitalization while breastfeeding had a protective effect. smoking, food-pre chewing and economic status were not significantly associated with rsv hospitalization. this study may be useful in the continued analysis of the impact of rsv in developing countries. as in the yk delta, rsv is the leading cause of viral lower respiratory tract disease in most developing countries, but lack of access to diagnostic reagents and hospital facilities has made it difficult to quantify the impact of rsv. also, the rate of severe rsv disease in term neonates was much higher than in earlier studies, both in the yk delta population and the comparison group in baltimore. these findings should be confirmed in other populations because they have important implications for rsv vaccine development. 2.4. respiratory syncytial 6irus (rsv) and human rhino6irus (hrv) infections in children with aids and lower respiratory tract illness (lrti) acute respiratory infections (ari) cause a great burden of disease in developing countries (de arruda et al., 1991) . although a growing number of children from developing nations are hiv infected, there is little knowledge about the frequency and severity of viral ari in hiv infected children. earlier studies of viral pathogens in immunocompromised adults indicated that cmv, herpes simplex, influenza, parainfluenza, rhinovirus, adenovirus, enterovirus, and rsv cause lower respiratory infection (connolly et al., 1994) . a recent study assessed the frequency of rsv and rhinoviruses (hrv) in hospitalized children with or without aids, who presented with lower respiratory tract infection (lrti). about 73 episodes of lrti in children with aids and 73 in children without hiv infection, matched by age and sample collection month, were studied in a rural area of southern brazil. the frequency of rsv infection was highest in the fall and winter, between february and july, whereas hrv was detected throughout the year. rsv was found in 8/73 (11%) and 9/73 (12%) of lrti episodes in aids and non-hiv infected children, respectively. hrv was found in 16/73 (22%) and 12/73 (16%) of the episodes in children with aids and hiv-uninfected children, respectively. no difference was detected in frequencies of hrv and rsv infections between the two groups. hrv infections, however, tended to be more frequently associated with pneumonia in children with aids (7/16, 44%) than in the control group (1/12, 8%) (p= 0.09). other clinical presentations of lrti were observed with equal frequency. these findings did not confirm hrv as a causative agent of pneumonia in children with aids, but suggest that further studies of lrti are desirable and that interventions for hrv could be considered for immunocompromised children with lrti. the human adenoviruses (ads) are a large family of over 50 serotypes, as well as numerous variants and intermediates. they are divided into six subgroups (a-f) that exhibit different tissue tropism. the clinical manifestations of adenoviral disease are protean. the most common are respiratory syndromes in both children and adults. it is estimated that ads cause 5-15% of all respiratory disease in children, including pharyngitis, tonsillitis and pertussis-like syndrome. in children and adults, ads are also associated with lower respiratory tract infections such as bronchitis and pneumonia. subgroup d ads are associated with both epidemic and sporadic ocular infections including conjunctivitis and keratoconjunctivitis. due to their stability in the environment, these viruses are highly transmissible, particularly in nosocomial settings. in adults, ads cause largescale epidemics of acute respiratory disease (ard) in closed populations of military recruits, dormitory residents and long-term care facility occupants, which are primarily associated with ad serotypes 4 and 7a, and to a lesser extent with serotypes 2, 3, 11, 21 and 35. while replication in the gastrointestinal tract is a feature of most ad infections, only ad40 and ad41 (subgroup f) are associated with gastroenteritis in infants and young children. ad infections in immunocompromised subjects are increasing as their numbers increase, with severe consequences. clinical manifestations of ad infection in immunocompromised patients include pneumonia, hepatitis, encephalitis, and systemic and disseminated disease, with case fatality rates from 18 to 60%, depending on the nature of the immunodeficiency. nearly all ad serotypes have been associated with these infections, but the higher numbered subgroup d serotypes that are usually not associated with clinical disease in the immunocompetent host have been especially common. a number of 'new' disease associations with ad infection have recently been reported, probably due to improved highly sensitive molecular diagnostic techniques that also increase the probability of laboratory contamination. detection of viral genome in the absence of positive viral culture has been described in cases of myocarditis and pericarditis in children and adults (martin et al., 1994; bowles et al., 1999; pauschinger et al., 1999) , sudden infant death (shimizu et al., 1995) , toxic shock-like syndrome (price, 2000) and 'unexplained death' (perkins et al., 1996) . isolation of ads from patients with central nervous system manifestations of fatal acute flaccid paralysis (cardosa et al., 1999) and encephalitis with cerebral edema (chatterjee et al., 2000) has also been reported. it is, perhaps, not surprising that the clinical manifestations of ad infection are evolving because the viruses themselves are in the process of continuous evolution. the ad mutational repertoire includes homologous recombination, illegitimate recombination, and single base mutation (sbm). homologous recombination occurs in conserved regions of the genome between closely related viruses within the same subgroup, and it requires regions of homology in the two parent strands. it is the primary mechanism responsible for intermediate ads, which are mosaic viruses with shared hexon characteristics, or with the hexon characteristics of one type and fiber of another. illegitimate recombination requires only short regions of homology of one to three nucleotides (short direct repeats), and is thought to be the result of polymerase stuttering or slippage. it causes deletions, insertions and duplications of short regions of dna. in ads it occurs in noncoding regions and hypervariable regions (hvrs) of hexon capsid proteins that tolerate structural variation. the hvrs of the hexon contain the viral neutralization epitopes, so that mutations in these regions result in deletion, formation or alteration of these epitopes, leading to antigenic shift. it is the primary mechanism by which new serotypes arise, particularly among the fastest growing group, the subgroup d ads. single base mutations accumulate gradually across the viral genome but can occur at a 30-fold higher rate in the hvrs, where they cause incremental antigenic drift and the creation of variant strains. ad evolution is compounded by all three mechanisms and perhaps others that have yet to be defined. as a result, serological identification of subgroup b2 and d ads has become extremely difficult. it seems reasonable that the time has come to consider a sequence-based ad classification system, similar to that in use for papillomaviruses and enteroviruses. adenovirus has re-emerged as a leading cause of febrile respiratory disease among military recruits. large and frequent epidemics were common at trainee camps before 1971 but were eliminated with the introduction of the live enteric type 4 and 7 vaccines. in 1996, the sole vaccine manufacturer discontinued production of these live enteric coated vaccines because of contractual issues. while limited vaccine stores were still available in 1997 and 1998, vaccine stores were completely depleted by early 1999. to monitor the effect of discontinued vaccination, weekly surveillance for febrile respiratory infections (fri), defined as oral temperature \100.5°f with respiratory disease symptoms, was conducted from october 1996-june 1998 at four military training camps. during this interval, 1814 (53%) of 3413 throat cultures yielded adenovirus. during the winter of 1997-1998, adenovirus infections caused more than 90% of fri at each of the four camps. ad 4, 7, 3, and 21 accounted for 57, 25, 9, and 7% of the isolates, respectively. three training camps experienced a high prevalence of adenovirus type 4 and the fourth camp experienced a type 7 outbreak. among symptomatic trainees, those who did not receive vaccine were 28 times more likely to be infected by ad 4 or 7 than vaccinated subjects (gray et al., 2000) . surveillance was extended to eight sites in june 1998 and virus isolation was attempted for adenovirus, influenza a and b, rsv, and parainfluenza 1-3. large ad 4 epidemics were observed in six training camps throughout the us, while rsv and influenza a and b viruses were isolated less frequently. the impact of adenovirus epidemics on basic training can hardly be overestimated. recruit camps were forced to convert barracks into special infirmaries to care for the ill, and hospitals were forced to halt elective surgeries. at one camp, the number of trainees that had to repeat their basic training because of extended illness increased 20-fold. this 'recycling' has an extremely negative effect on the morale of trainees and it impacts on the military's readiness. as many as 1800 preventable adenoviral trainee medical encounters occurred during the winter months of 1999 and in 2000; two military trainees died with molecular evidence of acute adenoviral infection, one with encephalomyelitis and another with acute respiratory distress syndrome (ards). vaccination against ad 4 and 7 has proven to be extremely safe and effective, and to prevent an enormous burden of disease in military trainees (howell et al., 1998) . it is urgent that a new manufacturer for adenoviral vaccines be identified, and vaccine production must resume as soon as possible. in recent years, much progress has been made in understanding virus-induced modulations of the host immune response to viral infections. for ad, more than 20 viral gene products are known to participate in the modulation of immune responses, and many of these gene products are expressed from genes clustered in the early region 3 (e3) (wold et al., 1999; horwitz, 2001) . the overall effect of ad e3 gene products on immune responses in vivo can be appreciated from the results of three studies in mice that investigated transplant rejection and development of autoimmune disease. in the first study, the expression of the complete ad e3 cassette in pancreatic islet cells as transgenes under the control of the rat insulin promoter (rip) enabled allogeneic islet donor cells containing the h-2 bxd class i mhc to be accepted long-term by h-2 d recipient mice (efrat et al., 1995) , indicating that ad e3 gene products could potentially be used as a powerful tool in the control of transplant rejection. the second study used the lymphocytic choriomeningitis virus (lcmv) model of autoimmune diabetes mellitus, in which the lcmv proteins np or gp are expressed on the surface of islet cells, and diabetes is induced by infection with lcmv that induces cd8+ (gp) or cd8 + and cd4 + (np) t-cell mediated immune responses. in this model, the co-expression of rip-e3 with lcmv-np or gp completely prevented the onset of diabetes after lcmv infection (von herrath et al., 1997) . similar protective effects of ad e3 transgenes were seen in a third study that used the non-obese diabetic mice (nod) model of diabetes mellitus, and the underlying mechanisms are currently being investigated (efrat et al., 2001) . thus, as the understanding of the mechanism of action of the ad e3 immunoregulatory genes are being pursued in various systems, they are being utilized to control selected immune reactions that might be involved in the genesis of autoimmune diabetes. some of the better-characterized gene products of the e3 region are (in order of increasing distance from the e3 promoter) gp19, 11.6, 10.4, 14.5 and 14.7k. only the functions of gp19 and 14.7k shall be discussed here; the other ad e3 gene products have been reviewed elsewhere (horwitz, 2001) . in vitro, gp19k reduces the expression of class i major histocompatibility complex (mhc) molecules by retaining the mhc heavy chain in the endoplasmic reticulum or retrieving it back from the golgi, and also by inhibiting peptide processing (bennett et al., 1999) . in the cotton rat model of adenovirus pneumonia, gp19k deletion mutants replicate like wild-type virus but they induce a much stronger inflammatory response (ginsberg et al., 1989) , whereas in c57bl mice an increase in pulmonary pathology is not seen (sparer et al., 1996) . the ad e3 14.7k protein inhibits tnfa-induced cell death by a process that does not involve down-regulation of the tnfa-receptor. in cotton rats (ginsberg et al., 1989) and c57bl mice (sparer et al., 1996) deletions of ad e3 14.7k modify the pulmonary inflammatory response, i.e. an increase of polymorphonuclear leukocytes in cotton rats and more pronounced alveolar infiltration in mice. in order to determine how the ad 14.7k protein prevents cell death, the cell proteins that interact with this viral protein were determined. using a yeast two-hybrid system, four 14.7k-interacting proteins (fips) were identified. three of them have been characterized and have been shown to participate in quite diverse cellular pathways (li et al., 1997 (li et al., , 1998 (li et al., , 1999b . fip-1 (also known as rag-a, a ras-related small gtpase) can bind to tctel, a component of the microtubule motor protein dynein, forming 14.7k-fip-1-tctel complexes (lukashok et al., 2000) , and 14.7k has been postulated to affect microtubule dependent macromolecular transport or even modulate the transport of virus. however, because 14.7k is not a structural protein of adenoviruses and must be made de novo from early viral transcripts, it is unlikely to play a role during viral entry, even though the process is known to be microtubule dependent. the role of 14.7k during viral exit from cells has not been studied. fip-1 also binds to a second gtpase (gip-2) that localizes in the centrosome and in addition to potential effects during mitosis may be involved in transporting macromolecules between the nucleus and the cytoplasm. fip-2 binds to abnormal huntingtin, and more specifically to the expanded polyglutamine tract that appears to be associated with cell death of neurons in huntington's disease (faber et al., 1998) . whether or not ad e3-14.7k and/or fip-2 can prevent huntingtin-induced cell death is currently being investigated. over-expression of fip-3, which is also called nf-kb essential modulator (nemo) or inhibitor of kappa kinase gamma (ikkg) causes morphologic changes and eventually apoptosis in a variety of cell lines. the amount of apoptosis induced by fip-3 can be reduced by 70% when ad e3-14.7k is present. apart from 14.7k, fip-3 seems to interact with a number of key molecules in the tnf receptor and nf-kb signaling pathways such as the receptor interacting protein (rip), the inhibitor of kappa b kinase beta (ikkb) and the nf-kb inducing kinase (nik) (li et al., 1999b) . these few examples of the effects ad e3 gene products have on the pathobiology of diseases as different as autoimmune diabetes, transplant rejection and huntington's disease indicate how much remains to be learned from studying adenovirus-host interactions. the rsv (strain a2) genome is a single stranded negative-sense rna of 15 222 nucleotides that is transcribed into 11 major subgenomic mrnas. three of the eleven encoded proteins are transmembrane proteins. the g protein mediates attachment to cell surface receptors, the f protein mediates virus-cell and cellcell fusion, and the function of the sh protein is unknown. other structural proteins are the m protein, which plays a role in virion assembly, the n, p and l proteins that make up the viral polymerase, and the m2 orf1 protein that functions as a transcription anti-termination factor. the other proteins include two non-structural species, ns1 and ns2, and the m2-2 protein encoded by the second orf of the m2 mrna. using a reverse genetics system to rescue infectious rsv from cdna, five of the 11 genes of rsv can be ablated individually and in some cases in combination without rendering the virus non-viable jin et al., 2000b) . these five non-essential genes are ns1, ns2, sh, g, and m2-2. since all of these genes confer a selective advantage to rsv in vitro and/or in vivo, they can be described as virulence factors -the deletion of which will lead to attenuation of the virus. deletion of the small hydrophobic (sh) transmembrane protein yields a recombinant rsv called rsva2dsh, which replicates in vitro as well as wild type (wt) rsv and induces plaques in hep-2 cells that are larger than wt rsv plaques. there is no reduction in synthesis of rna or protein associated with the deletion of sh. in chimpanzees, however, the virus is slightly attenuated (whitehead et al., 1999a) . deletion of the ns1 or ns2 gene results in a substantial reduction in replicative efficacy in vitro, and this reduction is more pronounced in hep-2 cells than in vero cells (which lack interferon a and b genes), suggesting that these two genes act as antagonists to type 1 interferon effects. deletion of ns1 and ns2 from bovine rsv provided direct evidence that ns1 and ns2 cooperatively antagonize a/b interferon-induced antiviral responses (schlender et al. 2000) . in chimpanzees, the level of replication of both rsva2dns1 and rsva2dns2 is reduced greater than 10 000-fold in the lower respiratory tract. in the upper respiratory tract, the dns1 virus is more attenuated than the dns2 virus (teng et al., 2000) . deletion of the m2 orf2 not only identifies a markedly attenuated rsv mutant but reveals an important role for this orf in the replicative cycle of rsv (bermingham and collins, 1999) . during infection with wildtype rsv, transcription appears to shut off at approximately 12-16 h post infection while rna replication increases concurrently. in contrast, this apparent switch from transcription to rna replication was not observed for the rsvdm2-2 virus, implying that m2-2 is a regulatory protein involved in the shift. instead, transcription continued to increase while rna replication remained low compared to wild-type rsv. overall, gene expression was increased 7-18 fold. the synthesis of the g and f proteins also was increased and resulted in increased syncytium formation. replication of the rsvdm2-2 virus in vitro was attenuated, probably due to reduced rna replication (bermingham and collins, 1999; jin et al., 2000a) . in chimpanzees, comparison of the four rsv gene deletion mutants mentioned above with wt rsv and the incompletely attenuated rsv cpts-248/ 404 vaccine candidate results in a hierarchy of increasingly more attenuated viruseswt rsv b dsh b dns2 b 248/404 b dns1 b dm2-2. the final rsv gene deletion mutant, rsva2dg, was not evaluated in chimpanzees because the absence of this major protective antigen would not be desirable in a vaccine virus. rsva2dg replicates as efficiently as wt rsv in vero cells, showing that g is not essential for efficient virus replication. however, the rsvdg virus is highly attenuated in balb/c mice, indicating the importance of the rsv g protein in vivo. it is evident from the above ranking that rsv reverse genetics is able to generate mutants exhibiting gradations in their level in attenuation ). this menu of viruses with different levels of attenuation is crucial in identifying an rsv vaccine that exhibits the desired balance between attenuation and immunogenicity in seronegative infants. since clinical data indicate that rsv 248/404 is just slightly under-attenuated in the 1-month-old target population, the dns1 mutant could be exactly what is needed. in order for rsv assembly to be an efficient process, viral structural proteins must be brought together in a coordinated fashion (peeples, 1991; lenard, 1996) . compared with other paramyxoviruses rsv exhibits several unique features. the g, m2-1 and m2-2 proteins are found only in the pneumo6irus genus of the paramyxo6iridae, and the role these proteins play in rsv assembly is much less well understood than the role of the f, hn and m proteins of other paramyxo6iridae (collins et al., 1996) . comparison of multiple human, bovine and ovine rsv strains shows that the cytoplasmic domains of the f and g proteins are well conserved amongst human rsv strains and subtypes, and conserved to some degree between the three species. in analogy to other paramyxoviruses, it is likely that the cytoplasmic domain of f and g interact in the process of virion assembly with cellular proteins involved in protein trafficking and in the polarized budding process, as well as with other viral proteins. the rsv g and m proteins co-localize in the golgi apparatus, not only in rsv-infected cells but also in cells transfected with only the g and m proteins, indicating that other viral proteins are not needed for this interaction (peroulis et al., 2001) . the g-m interaction is seen with fulllength g protein but not with a secreted form of g that lacks the conserved cytoplasmic domain and transmembrane domains. systematic deletion and substitution mutagenesis of the cytoplasmic domain of g has identified a sequence-specific, six amino acid motif that directly interacts with m (peroulis et al., 2001) . during rsv infection m protein can initially be detected in the nucleus, but later in the infectious cycle it is found in inclusions within the cytoplasm (ghildyal et al., unpublished) . the n and p proteins of rsv were earlier shown to be necessary and sufficient for these inclusions to form, and the rsv m2-1 and l proteins were also shown to be present in these inclusions (garcia et al., 1993) . these same investigators were unable to identify m protein in the inclusions (garcia et al., 1993) . using confocal immunofluorescent microscopy of infected and cotransfected cells the m protein was shown to be present in these inclusions . m protein does not localize to the inclusions unless m2-1 is also present . the m and m2-1 proteins not only co-localize by confocal microscopy but also interact in a protein overlay assay (ghildyal et al., unpublished) . taken together, these data suggest that, as with other single stranded negative-sense viruses (peeples, 1991; lenard, 1996) , the rsv m protein seems to play a crucial role in rsv assembly; bringing the nucleocapsid together with the envelope proteins by binding to the cytoplasmic domains of g and f and to the nucleocapsid proteins n and p together with or via m2-1. the interaction between m, m2-1 and the nucleocapsid proteins might be more complex than outlined here, and might involve additional cellular or viral proteins. to better understand rsv assembly, the domains in the m protein that interact with g, f and m2-1 will have to be defined. while influenza viruses readily develop resistance to older antivirals such as amantadine or rimantadine, resistance to neuraminidase inhibitors occurs much less frequently. nonetheless, resistant influenza a virus mutants can be isolated from patients treated with oseltamivir (treanor et al., 2000) . one of the resistant mutants carries an arginine to lysine mutation at position 292 (r292k) of the neuraminidase protein, causing a reduction in substrate binding and enzymatic activity, as well as resistance to oseltamivir (mckimm-breschkin, 2000) . the infectivity of influenza a viruses carrying the r292k mutation was earlier found to be markedly reduced in mice and ferrets. transmission of an influenza a h3n2 clinical isolate with a r292k mutation was studied in ferrets in comparison to transmission of the parent wt h3n2 virus that was isolated from the same patient. donor ferrets (four per group) were inoculated intranasally with the r292k mutant or wt virus and housed with three naïve contact ferrets per donor ferret. the four donors inoculated with wt virus were infected and transmitted virus to each of the 12 contacts. wild type virus replicated to between 10 4 and 10 5 pfu/ml nasal aspirate. only two of four donor ferrets inoculated with mutant virus became infected, and the level of replication of mutant virus was reduced 10-100-fold compared with that of wt virus. however, both infected ferrets transmitted virus to contacts. one of them transmitted the r292k virus to one of three contacts only, with virus detected at very low titer on 1 day only. the other donor transmitted virus to all three contacts, and the transmitted virus replicated to titers greater than 10 4 pfu/ml in the contact ferrets. sequence analysis showed that the donor virus was a mixed population of mutant and wt virus and that only wt virus was recovered from contacts. these data confirm the reduced infectivity of oseltamivir resistant r292k mutants. effective transmission of the r292k mutant virus in ferrets was not observed, suggesting that the transmission of oseltamivir-resistant virus from human to human will be unlikely, even during widespread use of na inhibitors in the treatment of influenza. respiratory virus infections in immunocompromised patients are characterized by persistence of viral infection, prolonged shedding of virus, a high rate of nosocomial acquisition, and a high frequency of pneumonia and death. similarly, respiratory virus infections in the elderly are responsible for a substantial amount of morbidity and mortality. detection of respiratory virus infections in these high-risk patients is important for several reasons. it enables the initiation of specific isolation procedures, initiation of specific antiviral therapy, cessation of unnecessary therapy, tests and procedures, and it can aid in identifying and preventing potential outbreaks. respiratory virus infections can be diagnosed using serology and culture techniques, as well as newer methods such as antigen-detection by enzyme-linked immunoassay (eia) or immunofluorescence (ifa), enzymatic detection by chemical reactions, or amplification of parts of the viral genome (pcr). serology is not useful in the acute phase of most illnesses and is of limited usefulness in immunocompromised patients, the elderly or those receiving blood products. recovery of virus in cell culture is still seen as the gold standard by many but the importance of obtaining a good specimen to ensure that the culture is not falsely negative is often overlooked. a good clinical specimen is the most critical factor in ensuring a correct diagnosis regardless of the diagnostic method used, although it is perhaps most important for culture. use of nasal wash to obtain a specimen for virus isolation is well-tolerated in cooperative adults and, compared to nasal swabs or throat swabs, increases the sensitivity of cell culture for virus detection. other factors critical to laboratory success are the use of appropriate transport media, temperature of transport/incubation and time until processing (atmar and englund, 1997) . this is particularly important in the case of rsv infections in immunocompromised or elderly patients, who often have a relatively low viral titer such as 10 2 -10 3 pfu/ml, whereas children often have a titer greater than 10 6 pfu/ml of nasal wash or bronchoalveolar lavage fluid (englund et al., 1996) . for rsv, parainfluenza and influenza virus there are a number of commercially available rapid detection kits. multiple simultaneous rt-pcr for detection of rsv, influenza a and b, and piv1, 2 and 3 (hexaplex ® ) was tested in 763 pediatric samples and yielded 100% sensitivity and 98% specificity as compared with culture (fan et al., 1998) , with only 8 h processing time. in a separate study, pcr for respiratory viruses in adult patients with hematologic malignancies was found to be as sensitive as culture (van kraaj and van elden, 2000) . for influenza, several antigen detection kits ((directigen, fluoia and quick-vue) and one neuraminidase (zstatflu) assay are available to detect virus, with sensitivities ranging from 50 to 96% and specificities ranging from 52 to 99%. rapid diagnosis of influenza in pediatric patients leads to a decrease in the frequency and duration of antibiotic use and an increase in the frequency of antiviral therapy (noyola and demmler, 2000) . rapid diagnosis kits for the detection of rsv in children range in sensitivity from 61 to 87% and in specificity from 93 to 97%, with some test kits performing better than others (dominguez et al., 1993) . in immunocompromised adults, however, the sensitivity of antigen detection kits from nasal wash or throat swab sample was only 15%, while endotracheal or bronchoalveolar sampling increased sensitivity to 70 or 80%, respectively, (englund et al., 1996) . several studies in recent years have highlighted the importance of upper respiratory tract infections in the exacerbation of asthma in children. freymuth and colleagues reported human rhinovirus (hrv) (46.9%) and rsv (21.2%) as the most frequent pathogens detected in patients with exacerbation of asthma, and noted that pcr increased detection rates 5.8-and 1.6-fold in hrv and rsv infections, respectively, over that of conventional assays (freymuth et al., 1999) . in a study of wheezing children between 2 months and 16 years of age, respiratory viruses were detected in 82%, with rsv in infants (detected in 68% of subjects) and hrv in older children (71%) as the predominant pathogens. both were strongly associated with wheezing (rakes et al., 1999) . in a community-based longitudinal study, respiratory viruses were detected in 80% of episodes of acute illness with reduced peak expiratory flow, 80% of episodes of wheezing, and in 85% of episodes of upper respiratory symptoms, cough, wheezing, and a fall in peak expiratory flow (johnston et al., 1995) . in these settings, rt-pcr provides a fast and sensitive method to detect rna viruses. real time quantitative pcr assays can be similar to conventional pcr in specificity and speed. however, real-time pcr can also quantify viral load (quantity of virus in respiratory secretions) during asthma exacerbations, and it is sometimes more sensitive than conventional pcr. real time taq-man quantitative pcr allows estimation of the input viral genome copy number by including a fluorescence reporter on one end and a quenching molecule on the other. the reporter does not fluoresce until the quencher has been cleaved off by the exonuclease activity of taq polymerase, permitting an estimate of the quantity of pcr product. fluorescence is being measured continuously every seven seconds, and quantification of the target is based on the number of pcr cycles it takes to produce detectable fluorescence. a retrospective study was conducted in 117 asthmatic children 9-11 years old to compare viral loads in quiescent and exacerbation periods of asthma using the taqman technology. nasal aspirates had been collected earlier and records of peak flow measurement and clinical scoring were available. a quiescent period was defined as absence of clinical symptoms for two weeks or longer. real time quantitative pcr detected respiratory viruses (mostly rhinoviruses) in 97% of the children with an asthma exacerbation and in 42% of children in quiescence. viral load was higher in children with exacerbation of asthma than in those with quiescent asthma and higher viral loads also correlated with more severe clinical disease. although the study showed that real time quantitative pcr is more sensitive than nonnested conventional pcr and also allowed esti-mation of viral load, there is the caveat that the study had a retrospective design. modalities of immunity to acute viral respiratory infections are both specific and nonspecific, humoral and cell mediated. fever, interferon (ifn), tumor necrosis factor (tnf), natural killer cells and activated macrophages are non-specific modalities stimulated by infection that are capable of mediating antiviral effects. lung collectins such as sp-a, have also been implicated in the control of viral respiratory infections (ghildyal et al., 1999) . specific modalities are antibody in serum and secretions, lymphocyte proliferation responses with cytokine release, and cytotoxic lymphocytes (ctls). all modalities participate, to some degree, in containing an infection and promoting recovery either via inactivation of free virus or elimination of infected cells. thus, there is considerable redundancy in the mechanisms controlling the virus infection and promoting recovery. on the other hand, protection against infection is primarily conveyed by specific antibody. sufficient data is available to conclude that serum igg neutralizing antibody is the primary mediator of resistance to influenza virus infection, presumably because infection is initiated in the lower respiratory tract and igg antibody derived from serum is the dominant antibody isotype at that site (couch and kasel, 1983) . in contrast, iga antibody is the primary mediator of resistance to rhinovirus and coronavirus infection because evidence indicates these infections are initiated in the nasopharynx where iga is the dominant antibody isotype (cate et al., 1966) . since primary infections with influenza virus, rsv, or piv induce disease in both the upper and lower respiratory tract, both igg and iga antibodies are correlates of immunity to infection and disease (crowe, 1999) . although adenoviruses also cause upper and lower respiratory disease, only serum antibody has been shown to correlate with immunity. reinfection with homologous rsv, piv, rhinovirus and coronavirus can occur but is generally confined to the upper respiratory tract, presumably because igg antibody in protective quantities is more durable in serum (and lower respiratory secretions) than is iga antibody in nasopharyngeal secretions. for rsv, the risk of infection declined from 74% for naïve subjects to 56 or 33% after one or two infections, respectively; the risk of lower respiratory disease in these groups declined from 18 to 15 to 3%, respectively, (glezen et al., 1986) . for a rhinovirus, resistance to reinfection of the nasopharynx correlated with the titer of iga antibody in secretions (cate et al., 1966) . rodent models of viral respiratory disease provided much of the early data on immune modalities conferring protection (crowe, 1999) . both cd4 + and cd8 + t cells can effect clearance of influenza a virus, rsv and piv in mice in the absence of the other cell type (lightman et al., 1987) . while cd8+ cytotoxic lymphocytes (ctl) mediate immunity through lysis of infected cells and expression of antiviral cytokines, cd4+ t cells exhibit limited direct antiviral activity but play a role in activating b cells and in inducing antiviral cytokine expression (epstein et al., 1998) . there is a general consensus that ctls contribute significantly to the resolution of primary infections with influenza, rsv and piv in rodents. while the correlation between antibody response and protection in humans was established decades ago, cellular immune responses were studied much later, and understanding of the development of these responses in infants and immunosenescence of them in the elderly is still incomplete. whether or not ctls in humans convey immunity to respiratory virus infection in the lower respiratory tract and whether they promote clearance of virus in the upper respiratory tract of humans remains to be elucidated. however, for rsv disease in humans, a correlation has been observed between the presence of rsvspecific ctls in year 1 and absence of severe rsv disease in year 2 (mbawuike, in press). morever, the level of influenza-specific ctls correlated inversely with the quantity of virus in nasal secretions after challenge of humans, and the ability of ctls to function at this site was demonstrated by a reduction in influenza virus titer in nasal turbinates of mice which were adoptively immunized with purified cd8 + ctls before intranasal challenge (mcmichael et al., 1983 mbawuike, personal communication). knowledge regarding viral-bacterial interactions in the respiratory tract goes back at least to the 1918 influenza pandemic, and interactions have been described for later influenza pandemics as well. epidemics and pandemics of influenza have been followed regularly by an increase in the incidence of bacterial pneumonia (schwarzmann et al., 1971; cartwright et al., 1991) . associations between rsv and haemophilus influenzae, bordetella pertussis, neisseria meningitidis and staphylococcus aureus infections have also been described (patel et al., 1992; jiang et al., 1999) . interactions between viral and bacterial diseases are fairly complex and the underlying mechanisms are only beginning to be elucidated. it is known that up-regulation of tnfa and il-1 increases adherence and uptake of pneumococci (cundell et al., 1995) , and that several cellular receptors for bacterial adherence are up-regulated by viral infections. s. pneumoniae and h. influenzae interact with paf receptors (swords et al., 2000) , and n. meningitidis interacts with cd14 and cd18 (raza et al., 1999) . not all interactions are regulated at the level of cell surface receptors. in the mouse influenza model, for instance, severe damage and desquamation of the respiratory epithelium enables access of s. pneumonia to the basal membrane and thereby increases the risk for invasive disease (plotkowski et al., 1986) . otitis media was traditionally thought to be a purely bacterial infection, with s. pneumoniae, h. influenzae and moraxella catarrhalis as the main pathogens. more recent studies, however, indicate that the majority of acute otitis media (aom) cases are a result of mixed bacterial and viral infection. heikkinen and colleagues reported an increase of rsv, parainfluenza virus, influenza or adenovirus infection in children with otitis media (heikkinen et al., 1999) . all respiratory viral infections of the nasopharynx are thought to predispose to aom but some viruses, e.g. rsv, are frequently found in the middle ear during aom. the mechanism underlying this respiratory tract infection-aom sequence involves eustachian tube obstruction, leading to negative middle ear pressure and inspissation of bacteria into the middle ear (giebink et al., 1980) . children with rsv, adenovirus or influenza virus infections have a 30% risk of developing aom within 2 weeks of the onset of the respiratory tract infection (henderson et al., 1982) , and coinfection with bacteria and viruses also adversely influences the outcome of aom. if aom does not respond to antibiotic therapy within 48 h, it is more likely to involve a viral infection (arola et al., 1990) . the effects of viral co-infection complicate the evaluation of clinical efficacy of anti-bacterial drugs in the treatment of aom (marchant et al., 1992) . with bacterial-viral co-infection in aom, there is also delayed clearance of bacteria from the middle ear during anti-bacterial therapy compared with disease attributable to bacterial infection alone (chonmaitree et al., 1990) . it is unclear whether the decrease in efficacy of antibiotics is due to impaired host responses (poor neutrophil function) or due to poor penetration of antibiotics into the middle ear. in a recent clinical trial heptavalent pneumococcal vaccine had 90% efficacy for prevention of bacteremia and 57% efficacy for prevention of aom caused by serotypes included in the vaccine. however, the frequency of aom caused by pneumococci not included in the vaccine increased (replacement phenomenon), so that the overall effect of the vaccine was reduced (eskola et al., 2001) . in contrast to the pneumococcal vaccine, viral vaccines seem very effective in preventing aom. belshe and colleagues reported considerable efficacy for an attenuated live influenza vaccine in preventing aom, and rsv prophylaxis with rsv antibodies was also associated with a marked decrease in otitis media (belshe et al., 1998; group, 1998a ). the human adenoviruses consists of over 51 known serotypes which have been divided into six subgroups (a-f) with distinctly different organ tropism. although other factors may influence infectivity and replication of these viruses, high affinity attachment of virions to host cell receptors represents a key determinant of tissue tropism. examples of this distinctly different organ tropism are a predominance of subgroup a adenoviruses (ad) such as ad31 in pneumonia in patients with primary immunodeficiencies; the preference of subgroup b viruses such as ad11, 34 and 35 for the urinary tract in patients with kidney transplants; and the predominance of subgroup c viruses in hepatitis in liver transplant patients. receptor binding is mediated by the ad fiber protein, a homotrimeric molecule composed of an amino terminal region that anchors the fiber to the penton base capsid protein, an elongated central shaft domain (van raaij et al., 1999) , and the carboxy-terminal receptor binding knob. a high resolution structure of the ad12 fiber knob bound to its receptor, the coxsackie-adenovirus receptor (car), has recently been obtained by x-ray diffraction (bewley et al., 1999) , and amino acid residues directly involved in receptor binding were defined for several adenoviruses through mutagenesis studies (roelvink et al., 1999) . adenoviruses differ remarkably in the isoelectric point of the fiber protein knob domain, e.g. ph 8.9 for ad8 versus ph 4.5 for ad35, suggesting that these fiber proteins cannot use the same receptor. besides car, heparin sulfate proteoglycans (dechecchi et al., 2000) and sialic acid (arnberg et al., 2000) mediate adenovirus attachment. since car is only expressed on the basolateral but not the luminal surface of epithelium, car can probably not be used to target adenoviral vectors in the therapy of cystic fibrosis. the important role that charge plays in virus-cell interactions can be deduced from the differential effect that removal of sialic acid from the cell surface by neuraminidase treatment has on adenovirus attachment. while ad19p attachment is not affected by neuraminidase treatment, ad5 attachment is increased and ad37 attachment is decreased. the difference in ph optima may well be a determinant of tissue tropism. the major pathogens in adenoviral eye infections, ad8, ad19 and ad37, belonging to subgroup d, are very similar in their knob charge. chimerization of fiber proteins, whether evolved in nature or generated by mutagenesis, can cause a significant change in adenovirus pathogenesis. in 1984, a new ad7 genotype (ad7h) appeared in argentina, uruguay and chile, where it caused severe respiratory tract infections in children. analysis of the ad7h fiber protein revealed that it was a chimera containing ad7 and ad3 sequences. in 1996 this new genotype and ad7d2 also emerged in japan, where ad7 had been absent for 30 years, and caused outbreaks of respiratory disease. the fiber protein, however, is not the only factor that determines adenovirus tropism. virus uptake is thought to also be mediated by an interaction between the penton base protein with integrin avß 3 or avß 5. adenoviruses also differ in their ability to induce inflammatory responses. while ad 7 is a potent inducer of interleukin 8 (il-8), a hallmark cytokine of viral pneumonia, ad5 is not; this might explain why ad7 but not ad5 causes significant respiratory disease. in summary, it can be concluded that neither subgroup classification alone nor fiber protein knob charge alone determine adenovirus tissue tropism. key amino acids in the knob, as well as certain motifs of the penton base protein and possibly other adenovirus proteins interact in virus attachment and internalization. avian influenza viruses of the h5n1 subtype were found to be transmitted directly from poultry to humans in 1997 in hong kong. these viruses were highly pathogenic in chickens and also caused severe clinical symptoms in humans, leading to the death of six of 18 infected individuals. in order to obtain a better understanding of the pathogenesis, tropism and kinetics of replication of these viruses in primates, cynomolgus monkeys (macaca fascicularis) were infected with the highly pathogenic h5n1 a/hong kong/156/ 97 isolate that was obtained from the index case. four monkeys were inoculated with 2.5× 10 4 tcid 50 in a 5 ml inoculum that was administered intratracheally, orally (tonsils) and onto the conjunctiva. two of the monkeys were euthanized on day 4 and the remaining two on day 7 post infection. the two monkeys that were euthanized on day 7, post infection developed a respiratory distress syndrome with high respiratory rate and fever on day 5. by day 7, one of the monkeys was lethargic and severely ill, with central cyanosis. the other monkey was also ill and developed fever. the virus replicated to a titer greater than 10 6 tcid 50 per g lung tissue on day 4 but could not be isolated on day 7. macroscopic lung pathology was dominated by peribronchial consolidation and necrotic lesions. histopathologic examination of the tissues collected 4 and 7 days post infection revealed extensive pathologic changes in the respiratory tract characteristic of a viral necrotizing interstitial pneumonia. although rt-pcr for h5n1 influenza virus was positive not only in the respiratory tract but in spleen, heart and also the cerebrum and cerebellum of one monkey, virus could only be demonstrated by immunoperoxidase staining in, and isolation from, the respiratory tract. the respiratory tract seems to be the major and probably the only target for the h5n1 influenza virus. although cynomologous monkeys have been used earlier as a model for h3n2 influenza disease (rimmelzwaan et al., 1997) , this is the first time a primate model for h5n1 viruses has been described. influenza h5n1 clinical symptoms observed in this study correlate well with what was seen in human disease caused by these highly virulent viruses, and was more severe than what is seen with h3n2 viruses. therefore, cynomologous monkeys may provide a useful model for studying influenza h5n1 pathogenesis and for developing h5n1 vaccines. 4.5. sb-242235, an orally acti6e, selecti6e p38 mitogen acti6ated protein (map) kinase inhibitor impro6es pulmonary functions in a murine influenza pneumonia model influenza infections are responsible for significant morbidity, especially in high-risk groups with underlying cardiopulmonary disease and in the elderly. the pathology results from a vigorous inflammatory response in the respiratory tract and damage to respiratory epithelial cells. in cells exposed to inflammatory cytokines, p38 mitogen activated kinase (map) activation leads to the upregulation of cytokines such as il-6, il-8 and tnfa (ono and han, 2000) . a recent study examined the effect of sb-242235, a highly selective orally bioavailable inhibitor of p38 map kinase, on pulmonary function in mice infected with influenza a virus. mice were infected intranasally with influenza a/pr8/34 and treated with sb-242235 at different time points post infection. initiation of treatment on day 0, 3 or 5 post-infection resulted in a 57% (p b0.01), 32% (pb 0.01) or 10% improvement in pulmonary capacity, respectively, compared with placebotreated control animals. no effect on virus clearance, survival, or antiviral immunity was observed. the efficacy of sb-242235 in reducing pulmonary resistance and increasing blood oxygenation was similar to that of the neuraminidase inhibitor oseltamivir, the steroid dexamethasone, and the cox-2 inhibitor nimesulide. sb-242235 was superior to non-specific nsaids indomethacin, naproxen, and ibuprofen. in mice and ferrets, sb-242235 reduced airway neutrophilia, and treatment was well-tolerated without any adverse effects. these data suggest that p38 map kinase is involved in influenza-induced cell signaling and that inhibition of this enzyme might reduce the severity of pulmonary disease. fortunately, the number of viruses that cause respiratory disease severe enough to require hospitalization is limited. in children younger than five years, rsv (subgroup a and b viruses), the parainfluenza viruses (piv1, -2 and -3), influenza a and b, and adenovirus types 1, 2, 3 and 5 are the major respiratory pathogens. influenza a and b remain, due to antigenic drift and shift, important agents in all age groups with very severe disease most commonly occurring in the elderly. rsv is the single most important cause of severe respiratory disease in infancy and childhood but it also causes significant morbidity in the elderly and in immunocompromised individuals. kim, chanock, brandt and parrott were the first to quantify the contribution of these viruses to the severe respiratory tract disease leading to hospitalization of infants and young children. rsv, piv3, piv1, piv2, adenoviruses and influenza b caused 23, 12, 6, 3, 6, and 1%, respectively, of respiratory disease leading to hospitalization, while influenza a was responsible for 3% between 1957 and 1968 (h2n2 era) and 7% between 1968 and 1976 (h3n2 era) (brandt et al., 1972; parrott et al., 1973; kim et al., 1979; murphy et al., 1988) . what are the general principles underlying vaccine development for these viruses? first, the protective antigens of the virus and mediators of immunity to reinfection are largely known. it is generally accepted that neutralizing antibodies to surface glycoproteins (g and f of rsv, hn and f of piv, and ha and na of influenza) or to capsid proteins (hexon and fiber proteins) of adenoviruses are the major mediators of resistance to reinfection. second, serum and mucosal antibodies make independent contributions to immunity against reinfection. whereas serum antibody is needed to mediate immunity in the lower respiratory tract, mucosal antibody is needed to protect the upper respiratory tract (with the exception of adenovirus, where serum antibody alone can prevent uri). third, live virus vaccines are generally more immunogenic than nonliving virus vaccines in immunologically naïve subjects, but identifying a live vaccine virus that is both safe and sufficiently immunogenic can prove to be a difficult task. fourth, different age groups might need different vaccines. infants younger than four months of age make less antibody than older children due to immunosuppression by maternal antibodies and immaturity of the immune system, and only potent immunogens, e.g. live virus vaccines, will be successful in this population (murphy et al., 1986) . on the other hand, a live virus vaccine that is sufficiently attenuated for use in infants might be over-attenuated in the elderly (gonzalez et al., 2000) . fifth, viruses differ in their ability to cause disease in the presence of maternal serum antibody. while mucosally delivered rsv, piv3 and influenza are infectious in the presence of maternal antibody, piv1, piv2 and adenovirus seem to be restricted in their replication in young infants by maternal antibodies. these five principles need to be considered when determining the optimal vaccination strategy (systemic versus mucosal, vectored antigen versus live attenuated virus) and the target age for vaccination. which vaccines are available? against influenza, the vaccine currently in use in the us is mostly a non-living, subvirion vaccine made from egg-grown purified virus disrupted with detergents. for practical purposes, the only relevant protein in the vaccine is the influenza hemagglutinin. the strains to be included in the vaccine for annual vaccination are selected by the public health service based on the antigenic profile of the currently circulating strains. subivirion or subunit vaccines plus adjuvant are also being developed (minutello et al., 1999; gluck et al., 2000) . live attenuated virus vaccines based on cold-adaptation of influenza a strain a/aa/6/60-h2n2 and influenza b strain b/aa/1/66 have been evaluated in phase iii clinical trials and should soon become available as a mucosal vaccine administered by nasal spray (maassab and bryant, 1999) . to date, an rsv vaccine has not been licensed. two non-living virus vaccines against rsv are being developed. one is a purified f protein subunit vaccine and the other consists of a g protein-specific peptide conjugated to the albumenbinding site of the streptococcus g protein (cano et al., 2000) . the purified f subunit vaccine is being evaluated for use in seropositive populations such as the elderly or high-risk older children (gonzalez et al., 2000) . used by itself, the f subunit vaccine has the disadvantage of not inducing a potent mucosal antibody response. also, this antigen induces a serum antibody response that is dominated by non-neutralizing antibodies (high ratio of titer of f protein-specific binding antibody to titer of neutralizing antibody). subgroup a and b live rsv vaccine candidates are being developed . one candidate vaccine bearing multiple attenuating mutations has been evaluated in 1-2 months old infants and was found to be attenuated and immunogenic but retained mild reactogenicity for the upper respiratory tract (wright et al., 2000) . it is likely that recombinant cdna technology will be used to develop or improve live attenuated rsv vaccines in the near future (whitehead et al., 1999a) . for example, a subgroup b rsv vaccine candidate was developed by substituting the g and f glycoprotein genes of rsv b for the corresponding genes in an attenuated recombinant subgroup a virus (whitehead et al., 1999b) , thereby rapidly generating a live attenuated subgroup b rsv vaccine candidate. to date, a piv vaccine has not been licensed. subunit and vectored vaccine candidates against piv3 have been developed and tested in animal models, but there are no reports of ongoing clinical trials. two live-attenuated piv3 vaccine candidates, a bovine piv3 (bpiv3) and a cold-adapted piv3 (cp45), have been evaluated in clinical trials, and both viruses were found to be safe and immunogenic in seronegative infants (karron et al., 1995 (karron et al., , 1996 . recombinant versions of these two viruses are also being evaluated as vaccines against piv3 and as vectors for the expression of foreign viral glycoproteins such as rsv g or measles ha (karron et al., 1995; durbin et al., 2000; schmidt et al., 2000) . the recombinant piv3cp45 vaccine candidate is currently being tested in phase ii trials in seronegative infants. for adenoviruses, a live virus vaccine against serotypes 4 and 7 has been used successfully in military recruits from 1971 to 1998. the vaccine made use of site-specific attenuation, i.e. a wt virus capable of inducing respiratory disease if administered to the respiratory tract was administered orally in an enteric coated capsule. the vaccine virus replicated only in the gastrointestinal tract and did not spread to the respiratory tract. the oral vaccine was found to be com-pletely attenuated and highly efficacious in inducing serum antibodies and protecting the upper and lower respiratory tract (couch et al., 1963; howell et al., 1998) . a similar tetravalent vaccine against serotypes 1, 2, 3 and 5 should be evaluated for use in young pediatric patients since these four viruses are responsible for over 80% of the adenovirus-related respiratory tract disease in this population. in virology, reverse genetics refers to the ability to rescue infectious virus from cdna. reverse genetic systems have been developed for all major viral respiratory pathogens, i.e. influenza a virus, paramyxoviruses, adenoviruses and most recently coronaviruses. this short review will only address the impact of reverse genetics on vaccine development for selected members of paramyxoviridae. for rsv, the generation of attenuated vaccine viruses started with conventional biological methods. first, wild-type rsv was passaged extensively at low temperature, and the resulting cold-passaged (cp)rsv was demonstrated to be attenuated in chimpanzees, as well as in seropositive adults and young children (friedewald et al., 1968 ). the cprsv virus was further modified by two rounds of chemical mutagenesis and biological selection for temperature-sensitivity (crowe et al., 1994a) . a panel of resulting mutant viruses was evaluated in mice and chimpanzees and ranked according to increasing attenuation. several promising candidate vaccines then entered clinical trials. rsv cpts248/404, a cold-passaged virus with two additional attenuating point mutations, is the most promising vaccine candidate tested so far, but it still causes transient nasal stuffiness in infants (wright et al., 2000) . the development of a reverse genetics system for rsv provided a method for expediting development of further attenuated viruses. as a first step, the biologically derived mutant viruses were sequenced completely and their mutations were identified and evaluated by introduction, individually or in combination, into wt recombinant (r)rsv. the direct identification of attenuating mutations made it possible, for example, to improve the above mentioned cpts248/404 mutation by incorporating one or more additional attenuating mutations from other vaccine candidates. furthermore, in many cases, the amino acid substitutions can be engineered to involve two nucleotide substitutions relative to wt, which should confer increased genetic stability. similarly, a reverse genetics system for piv3 was established and used to analyze all 15 mutations in the biologically derived cold passaged piv3cp45 (skiadopoulos et al., 1999) . however, the capabilities of reverse genetics go far beyond the analysis of existing biological mutants. a whole new set of methods for generating attenuated paramyxoviruses has been developed as described above. gene knock-out mutations, i.e. the deletion of non-essential genes, such as the rsv sh, ns1, ns2, m2-2 or g gene, generated a number of vaccine candidates with different levels of attenuation. as another example, in some cases an attenuating point mutation was found to involve a residue conserved, for example, between the l proteins of rsv and piv3 or between the c proteins of sendai virus and piv3. transfer of the attenuating mutation between the heterologous paramyxoviruses resulted in transfer of the attenuation phenotype. host range restriction could be employed as a basis of attenuation by creating antigenic chimeric viruses that use an animal virus as a platform for the expression of human rsv or piv glycoprotein protective antigen genes schmidt et al., 2000) . in this manner, an hpiv3 vaccine candidate was generated by replacing the bpiv3 f and hn glycoprotein genes in rbpiv3 with their hpiv3 counterparts (schmidt et al., 2000) . thus, the menu of available attenuating mutations for rsv and hpiv3 include numerous attenuating point mutations (which in some cases specify temperature sensitivity and in other cases do not), gene deletions, and host range restriction elements. the attenuating mutations or elements from each menu can be combined as desired to develop appropriately attenuated vaccine candidates. reverse genetics also expedites vaccine development because attenuated platforms can be used to make vaccines against additional viruses. for ex-ample, attenuated derivatives of rsv a2 (subgroup a) can be used to generate rsv subgroup b vaccine candidates by replacing the a2 f and g glycoprotein genes with their counterparts from the b1 strain of subgroup b (whitehead et al., 1999b) . as another example, a live-attenuated vaccine candidate was developed for hpiv1 by replacing the f and hn coding regions of hpiv3 with their hpiv1 counterparts. the resulting virus thus bears the antigenic determinants of hpiv1 in a wt hpiv3 backbone, which can then be attenuated by the introduction of mutations from the hpiv3 attenuation menu. reverse genetics can potentially help us to make vaccines that are satisfactorily attenuated but more immunogenic than wt virus. protective antigen genes can be moved closer to the genomic promoter and codon usage can be optimized for translation in mammalian hosts. cytokines and/or chemokines can be co-expressed from additional genes (bukreyev et al., 1999) , and reactogenicity can potentially be reduced, for example by ablating the secreted form of rsv g, which might be a decoy for antibody and might also perturb the t helper lymphocyte response. vaccine specificity can be broadened by adding additional genes to existing vaccine viruses, e.g. the measles hemagglutinin (ha) gene can be expressed from an additional orf in rhpiv3, generating a vaccine candidate that protects against both hpiv3 and measles (durbin et al., 2000) . this use of recombinant piv3 as vaccine and as a vector has several advantages over existing vector systems. it reduces the number of viruses to be administrated to infants and enables intranasal administration and thereby mitigates neutralization and immune suppression by maternal antibodies. the eradication of measles could be facilitated by a vaccine that does not include an infectious measles virus, which might cause prolonged infection in immunocompromised hosts. in summary, vaccines can be optimized through reverse genetics in a number of ways, such as the creation of novel combinations of mutations, the fine-tuning of the attenuation phenotype, the provision of a common attenuated platforms, and the generation of multivalent vaccines. 5.3. predicti6e 6alue of animal models in rsv 6accine de6elopment rsv was initially isolated from chimpanzees that developed a common cold-like disease in 1956. since then chimpanzees and other animal models for rsv disease have been studied extensively. principally, these higher primates have been used for three different purposes: the development of live virus vaccines, the recovery of rsv antibodies, and lastly to evaluate formalininactivated and subunit vaccines, and to study enhanced disease following vaccination with formalin-inactivated vaccine. a number of animal models are used to study rsv disease. among the apes, chimpanzees have been used most extensively for several reasons. they are permissive for rsv, their core body temperature is similar to that of humans, and symptomatic scoring of uri is possible (rhinorrhea score). the permissiveness of chimpanzees makes it possible to perform quantitative virologic studies in the upper and lower respiratory tract over the time course of an acute rsv infection. also, the restriction of rsv replication in the respiratory tract of chimpanzees correlates well with attenuation in seronegative human infants. in addition, human immunologic reagents can be used in chimpanzee studies. however, only young, carefully raised chimpanzees are rsv seronegative. other primates used include old world monkeys such as african greens, rhesus, cynomologous or bonnet monkeys and new world monkeys such as marmosets, tamarins and owl monkeys. although all these monkeys are semi-permissive for rsv, their core body temperature is higher than that of humans so that the level of attenuation of temperature-sensitive rsv mutants might be overestimated. although primate studies provide essential data for vaccine development, their use can be difficult. transport and care of primates, as well as sample collection are difficult and potentially dangerous, and they require well-trained personnel. primate research centers must provide an environment similar to that of a child-care facility and, therefore, maintenance costs are very high. lambs and calves are also used to study rsv infection, mostly because ovine and bovine rsv causes significant disease in their natural host. also, these viruses are important economically. cotton rats, mice and other rodents are used as small animal models. mice have the advantage of having a body temperature similar to that of humans and immunological reagents are readily available. also, gene knockout mice, transgenic mice and various inbred strains are available to study pathogenesis. the mouse model is not without difficulties, however. mice acquire a large portion of their maternal antibodies by suckling, and, therefore, they are not an optimal model for study of immunosuppression by maternal antibodies. peak rsv titers vary 100-fold among different mouse strains, and subgroup b rsv is poorly infectious and immunogenic in mice. immune mechanisms may also vary among different mice strains. rodent models can be used to study quantitative virology, immunology and airway pathophysiology (a model of wheezing), and weight loss can be used as a surrogate marker for rsv disease. whichever animal model one chooses to study respiratory syncytial virus, it is essential to clearly define whether the ultimate goal of the study is to prepare for clinical vaccine trials or to understand basic mechanisms of disease. several cold-passaged (cp) live attenuated rsv candidate vaccines have been evaluated in clinical trials. most of these candidate vaccines were derived by further attenuating the original cprsv through chemical mutagenesis and selection of ts mutants. the first two vaccines evaluated in the cpts lineage -cpts-248/955 and cpts-530/1009, were either insufficiently attenuated or were transmitted amongst seronegative children (karron et al., 1996) . a study of a more attenuated vaccine candidate, cpts-248/404, has recently been completed (wright et al., 2000) . this virus initially was evaluated in chimpanzees where it replicated to a peak titer of only 10 1.3 pfu/ml in the upper respiratory tract. due to this restriction in the respiratory tract, it was subsequently evaluated in clinical studies. cpts-248/404 did not replicate in adults or older children. in seronegative children 6-24 months of age, however, cpts-248/404 replicated to a peak titer of 10 4 pfu/ml in the upper respiratory tract and induced an antibody response against rsv f and g glycoproteins. the virus was well tolerated in this age group, and the frequency of upper or lower respiratory tract disease, otitis media or fever was not different from that of the placebo group. based on these data cpts-248/404 was selected to be the first rsv vaccine to be administered to 1-2-month-old infants, which represent the primary target group for vaccination. in this youngest age group, 17 of 24 vaccine recipients developed a clinical syndrome characterized by nasal congestion that occurred most typically between days 8 and 12 and lasted for approximately 24 h. since young infants are obligate nose breathers, this interfered with feeding and caused fussiness and difficulty in falling asleep. most vaccine recipients shed approximately 10 3 pfu of rsv per ml nasal wash. age of the infant or level of maternal antibodies against rsv did nor affect virus shedding, indicating that virus replication in the nasopharynx was independent of these variables. in these young infants, neutralizing antibody responses and igg elisa responses to rsv f or g were rarely detected, most likely because these responses were masked by the presence of maternally derived antibody. these young infants did, however, develop serum and mucosal iga responses preferentially to the rsv g glycoprotein and detection of serum iga correlated with protection from re-infection with a second dose of vaccine. although cpts-248/404 is not an acceptable vaccine in one to two month-old infants, several lessons were learned from the study of this vaccine candidate. first, seronegative infants are a much more susceptible host to rsv than chimpanzees. this means that for further attenuated vaccine candidates, the chimpanzee model of rsv infection will not be very useful. second, only viruses that do not replicate in adults and older children are attenuated enough for vaccination of infants. and third, reverse genetics is needed to further attenuate the existing biological rsv vaccine candidates. two of these recombinant rsv vaccine candidates are currently being evaluated in clinical trials. cpts-248/404dsh was generated by deleting the sh gene from the recombinant cpts-248/404 virus, and cpts-248/404/1030dsh was engineered to contain an additional (1030) mutation in the polymerase protein. preliminary data suggest that both recombinant viruses are well tolerated in 6-24-month-old children and are not associated with lower respiratory tract illness. whereas cpts-248/404dsh replicates as well as its parent biological virus without the sh gene deletion, the cpts-248/404/1030dsh mutant appears to be much more restricted in its replication in the nasopharynx. the more restricted cpts-248/ 404dsh induced both neutralizing and elisa igg antibodies in this age group. however, since this virus replicates, as well as cpts-248/404, it is likely not to be suitable for vaccination of one to two month old infants. cpts-248/404/1030?sh is currently being evaluated in one to two month old infants. in summary, although cpts-248/404 is close to an ideal vaccine candidate, further modifications of the virus through reverse genetics, such as addition of the 1030 mutation, are likely to be needed to generate an rsv vaccine that is immunogenic yet attenuated enough to be given to young infants. antibody preparations that neutralize free virus have been used as passive immunoprophylaxis to prevent a number of viral diseases, including hepatitis a and b, varicella and respiratory syncytial virus (rsv) disease. the efficacy of antibody preparations in preventing rsv disease was established first in cotton rat and chimpanzee models, and later in extended clinical trials. therapy of rsv infections with rsv antibodies, however, have so far failed to prove efficacious (malley et al., 1998; van woensel and kimpen, 2000) . the most important mechanism of action of antibody preparations is probably neutralization of free virus. this can occur via aggregation of free virus by bivalent or multivalent antibody, via receptor blockade, via antibody-complement lysis, or via fusion inhibition. receptor blockade is thought to result from steric inhibition of receptor binding rather than binding of antibody directly to the receptor-binding site itself. even if receptor binding does occur, virus infectivity can be neutralized through fusion inhibition. most rsv neutralizing antibodies are thought to use this mechanism of action. although complete antibody is most effective in neutralizing free virus, antibody binding fragments (fabs) alone can also neutralize. the minimal unit necessary to ablate infectivity is probably a peptide corresponding to one loop of the complementarity-determining region 3 (cdr3). although neutralizing and non-neutralizing antibodies against infectious virus are often distinguished, it is not clear whether there really are antibodies that bind glycoproteins in virions without neutralizing the virion (sakurai et al., 1999) . many so-called non-neutralizing antibodies bind purified (conformationally relaxed) rsv f glycoprotein but not the (conformationally correct) f protein on infected cells. different immunoglobulin subclasses neutralize virus with varying efficacy, and mouse and human igg subclasses also bind complement with varying efficacy. the replication of sendai virus (murine piv1) and influenza a virus can be inhibited intracellularly by iga and similar effects of iga on rsv replication may occur. a last but essential determinant of neutralizing activity is defined by the amount of antibody available to neutralize virus. to prevent rsv disease in the lower respiratory tract, a serum neutralizing antibody titer of approximately 1:300 is required (groothuis et al., 1993; top et al., 2000) . upper respiratory tract infections, however, can only be prevented with serum antibody titers as high as 1:5000-1:15 000. such titers can only be achieved in experimental settings in small rodent models. there is no single monoclonal antibody directed against rsv g protein that neutralizes completely on its own but cooperative neutralization occurs when multiple mabs are used. effective treatment for rsv illness is very limited. corticosteroids, bronchodilators, ribavirin, and, more recently, rsv mabs have been used but none of these therapeutic interventions are accepted as reproducibly efficacious. it seems that neither antivirals nor anti-inflammatory agents alone improve the outcome of rsv disease. a single dose of topically administered human fabs has been shown to clear free virus from the respiratory tract of rodents, but their effect is shortlived since infected cells release newly synthesized infectious virus within a day or two (crowe et al. 1994b ). whereas peak virus titers in humans usually occur around day 4, most patients with clinical rsv disease likely present no earlier than day 7. at this point, virus load is already in decline and it may be too late for antivirals, and antiinflammatory drugs such as map kinase inhibitors may be more effective in reducing cell injury. are there immunological consequences of passive immunoprophylaxis? in chimpanzees therapy with rsv antibodies suppresses the primary antibody response to rsv. this effect is mostly antigen-specific but may also have a non-specific aspect mediated by the fc portion of the antibody. the secondary antibody response to rsv in chimpanzees is enhanced by prior antibody therapy. however, this effect was not observed in mice or in clinical studies. t cell responses do not seem to be as affected by antibody therapy as humoral responses, and t cells might fill in for absent primary antibody responses. whether this is a desirable effect or not, remains uncertain. without a safe and effective rsv vaccine available, monthly infusion of rsv-igiv (respigam ® ) was the first effective measure to prevent rsv-induced lower respiratory tract infection (lri) and hospitalization in infants. the evaluation of rsv-ivig in cotton rats correctly predicted the serum concentrations necessary to protect against rsv-induced lri (prince et al., 1985a) . protective concentrations in children were achieved by monthly infusions of 750 mg/kg of rsv-ivig. in order to increase the potency of a rsv antibody preparation, and in order to be able to replace intravenous with intramuscular administration, a humanized monoclonal anti-rsv fusion protein antibody was developed. mab1129, a mouse monoclonal antibody developed in the laboratory of infectious diseases at nih against the rsv f protein antigenic site a, one of two antigenic sites that are conserved amongst different rsv strains, was selected for humanization based on in vitro and in vivo studies, and the complementarity determining regions (cdr) were transferred from the mouse antibody to a human igg. this humanized igg should have pharmacokinetics similar to human igg and permit repeated administration at monthly intervals. the chosen mab, medi-493, had no crossreactivity with adult or neonatal tissue, broadly neutralized rsv subgroup a and subgroup b isolates at concentrations of 20 ng/ml, and proved to be 50-100 times more active on a weight basis than rsv-ivig in the cotton rat model (johnson et al., 1997) . adult volunteers tolerated doses of the humanized antibody medi-493 from 1 to 30 mg/kg well, and only some volunteers developed a low-titer, transient anti-idiotypic antibody response. medi-493 had a half-life of 17 days, as is expected for igg, and serum concentration after iv and im administration were comparable except for the initial bioavailability. medi-493 was safe and well tolerated in phase i/ii studies in high-risk children, and had no specific immunogenicity in and of itself. monthly dosage of 15 mg/kg maintained serum concentrations greater than 40 mg/ml, and a single intravenous dose of 15 mg/kg reduced rsv titers in tracheal secretions of intubated children with rsv infection (malley et al., 1998) . the impact-rsv study that led to fda approval of medi-493, also called palivizumab, was a 2:1 randomized, double-blinded, placebo-controlled phase iii multicenter study conducted at 139 sites in the us, canada and the uk. study subjects received five doses of medi-493 or placebo at intervals of 30 days and were followed for a total of 150 days. the primary endpoint of this study, overall rsv-related hospitalization, was significantly reduced (pb 0.001) from 10.6% for placebo recipients (n= 500) to 4.9% for medi-493 recipients (n=1002). for premature infants with chronic lung disease, the incidence of rsv hospitalization was reduced from 12.8 to 7.9% (p= 0.038), and for premature infants without chronic lung disease, it was reduced from 8.1 to 1.8% (pb 0.001). total rsv hospitalization days, total days with increased oxygen requirement, total days with severe lri, rate of icu admission and total days in icu were secondary endpoints that occurred less frequently in the medi-493 treated group (p b 0.05). thus, administration of 15 mg/kg medi-493 by im injection was found to be safe, well-tolerated, and to lead to a 55% reduction of rsv hospitalization in high-risk children (group, 1998b) . this conclusion was confirmed in an outcome survey conducted in nine centers in 1998 and 1999, in which 1839 high-risk children received 15 mg/kg palivizumab. although direct comparison to the impact-rsv study is not possible, rsv hospitalization rates were low and similar to those observed in the impact-rsv study: 2.1% (vs. 1.8% in the impact-rsv study) of premature infants without cld and 4.0% (vs. 7.98%) of children with cld were hospitalized in the 1998-1999 rsv season (sorrentino and powers, 2000). an alternative strategy to protect neonates and young infants against severe rsv disease is vaccination of pregnant women. transfer of high concentrations of maternal rsv-specific igg to the fetus is expected to protect the infant against severe disease (glezen et al., 1981) . the safety and immunogenicity of respiratory syncytial virus purified fusion protein-2 (pfp-2 wyeth lederle vaccine and pediatrics, pearl river, ny) were recently evaluated for use in pregnancy. thirtyfive pregnant women were randomized at a ratio 2:1 to receive rsv pfp-2 or saline placebo at 30-34 weeks of gestation and were followed until the time of delivery. infants were followed during their first year of life and their first rsv season. rsv pfp-2 was safe and well tolerated by pregnant women, and there were no systemic reactions or serious adverse events associated with vaccine administration. all 35 infants were born healthy, and there were no differences in the frequencies and outcomes of neonatal events between the groups. during the first rsv season, there was no increase in the frequency or severity of respiratory tract illnesses in infants of vaccine recipients. there is ample material from which to draw lessons relevant to needed preparations for pandemic influenza. but, to date, the response, and specifically preparations for dealing with a serious pandemic of influenza remain more in the realm of academic reflection than meaningful action. although much time has been devoted to the development of a national response plan, we do not seem to be much better equipped to deal with a new pandemic of influenza than we were in the spring of 1957, when the h2n2 strain of influenza a virus emerged. at that time, we faced a burgeoning epidemic, whose virulence and propensity for spread were as yet unknown. a program of surveillance and field epidemiology to better define the epidemic had to be developed and, at the same time, preparations were needed for distribution and use of a new influenza vaccine, if it arrived in time. it did not. it appears today that not much progress has been made over the past 30 years, and still no real sense of urgency in dealing with the essentials of the problem can be felt. although much has been written about the 1918 epidemic, it is still perceived by most as an interesting but questionably relevant tale of death and disease during an earlier, pre-antibiotic era of medicine. many doubt that an epidemic of this severity would be possible today. but, here are a few of the facts. first, it is important to recognize that, for the us, it dwarfed all other outbreaks of the 20th century. at that time, more than 20 million died worldwide and in the united states, there were more than 500 000 registered deaths. from various studies, it is thought that, overall, perhaps 40% of the population became ill, of whom about 2% died. medical services were overwhelmed but, other than supportive care, there was little that curative medicine could offer. the deaths were so numerous that burials were greatly delayed because of the lack of morticians and grave diggers. pictures from the time provide at least a pale illumination of that catastrophic period. the effect of the disease was anything but uniform. surveys revealed morbidity rates ranging from 15% to more than 60% in different parts of the country. some remote and rural areas escaped the disease entirely but in some areas, it was remarkably lethal. western samoa, then a new zealand protectorate, registered 8500 deaths in a population of 38 000. this represented 22% of the entire population. curiously, american samoa, only 50 miles away but with a quarantine in effect, was one of the few political entities to escape the epidemic entirely. what was surprising and unique about the 1918 epidemic was that more than half of all deaths were in persons between 15 and 45 years of age -an age bracket in which death is a relatively uncommon phenomenon. pregnant women and those with cardiac problems were at highest risk but most, who died, were otherwise in good health. a substantial number in this age group died of a fulminant, rapidly progressive pneumonia marked by severe cyanosis with death occurring within a matter of 1-3 days, in brief, what seemed to be almost certainly, a primary influenza pneumonia that would have benefited little from antibiotics, had they been available. today, a better outcome might be foreseen with better ventilatory support in intensive care units; with the administration of antiviral agents; and with the administration of antibiotics for the treatment of secondary pulmonary infections. but under epidemic circumstances, the bulk of cases in a new pandemic would occur over a period of only 3-6 weeks and only a fraction of patients could be accommodated in suitable hospital settings, let alone appropriate intensive care units. what quantity of antiviral drugs is available for emergency use? where do we obtain the added quantities of the standard antibiotics that today, are produced on a just-in-time basis -when, during a period when clinical facilities are not stressed, we are regularly experiencing antibiotic shortages on a regular, rotating basis. a cogent question is whether under epidemic circumstances, our modern health care system could provide a standard of clinical care that would be better than it was in 1918. but there is still another dimension to the problem. do we appreciate that a 'pandemic', so characteristic of the emergence of a new influenza strain, means exactly that -a worldwide epidemic. many countries -developed and developing, have made no preparations and will inevitably turn to those with resources to help them in dealing with a catastrophe -a catastrophe that poses an international security threat. the rapid production and administration of large volumes of vaccine effective against the emergent new strain has been a basic building block of the strategy for dealing with pandemic influenza and, understandably so, given the limitations of curative medicine and the capacity of clinical services. the first real test of this strategy came during the 1957 epidemic. the first notice of a major outbreak occurred in mid april; specimens were received in the us on 13 may; and field testing of new lots of the vaccine began in july. not a bad record. it was foreseen that 60 million doses would be required. with good fortune in adapting the virus to grow at reasonably high titer on egg membrane and provided that sufficient fertilized eggs could be obtained, it was expected that a production target of 1 february could be met. even this was a problematical date given that the seasonal peak of epidemic influenza usually occurs between the end of december and the end of february. still, it was believed that substantial numbers would be able to be protected. however, 1957 was not a typical year, and such, one must note, was the case when the 1918 epidemic strain first appeared. widespread epidemics began occurring in mid to late september, two months before they were anticipated. more than half of all counties reported epidemics by mid-october and by the end of october, the peak incidence had past, long before any substantial quantity of vaccine was available. given these experiences, could we expect another new strain to behave differently today? over the past 30 years, extensive studies have been conducted in the search for a satisfactory live attenuated vaccine and for various approaches in production which would permit new antigenic variants to be produced rapidly and in quantity in tissue cell culture. however, today we are still producing influenza vaccine in the allantoic cavity of hens' eggs, as we were in 1957; procuring adequate supplies of fertile eggs in a timely manner remains a serious problem; and difficulties in adapting new strains to eggs remain. were we able to solve the vaccine production problem for our own country, this would not be the end to the practical quandary of dealing with the pandemic. few countries have influenza vaccine production capability and the international implications of the us having all or much of the vaccine supply are profound. following the 1976 swine influenza outbreak at fort dix, new jersey, the us had embarked on a program to rapidly produce an appropriate vaccine. vaccine production capability in europe and other countries was so marginal that a world health organization committee could only recommend that the fort dix situation be carefully monitored -a strategy of wait and hope. but as those at the who meeting commented in corridor conversations, they would not wish to face the ethical and political dilemma the united states would face were there a pandemic and the us was the only nation with a vaccine. in recent meetings with national and local hospital authorities, current capabilities of the medical system in us to deal with sudden surges in demand such as might follow release of a biological weapon were explored. such a release would result in an epidemic that would stress the system not unlike the way it would be stressed by a pandemic of severe influenza. from these meetings, it was evident that the elasticity of the nation's bed supply has been significantly reduced as drives for financial efficiency and the increasing use of out-patient procedures have sharply reduced the numbers of beds in all hospitals. meanwhile, managed care-driven market pressures and federal government reimbursement reductions have driven large numbers of hospitals into operating deficits. many of the municipal hospitals, once a primary source of care for the less prosperous and uninsured have been privatized. meanwhile, the hospitals are experiencing severe labor shortages, especially for nursing and technical personnel. few have either the resources or motivation to prepare to respond to the challenges posed by mass casualties due to any cause. reserves of antibiotics, as noted earlier, are marginal to nil; the public health infrastructure needed to deal with epidemic disease is grossly understaffed, underpaid and under trained; mechanisms for the development and implementation of community-wide plans are largely unexplored. it is not unreasonable to suggest that we are today less well-prepared to deal with an epidemic of influenza than we were 30 years ago in most parameters that one can identify. what might a new strain of influenza mean in terms of numbers of cases and deaths. estimates of past pandemics suggest that perhaps 40% of the population were affected in a first wave. with rising proportions of the population in urban areas, the greater and more rapid mixing of populations through travel, a figure of not less than 60% would seem more reasonable. thus, in a city of 3.0 million, one might expect 1.8 million cases of influenza. it is doubtful that either antibiotics or antiviral agents would be of much help given the number of cases and the dearth of reserve supplies. the number of deaths would vary greatly depending on the strain. in hong kong, a recent new strain, h5n1, resulted in death among six of 18 persons infected. it did not spread readily, however. it is estimated that the 1918 strain killed 2% of those who became ill, while the 1957 case-fatality rate was about one-tenth as large or 0.2%. thus, in a city of 3 million persons, one might anticipate between 3600 and 36 000 deaths over a period of 3-6 weeks. medical care would consist largely of supportive therapy given the numbers of those ill. public health measures, likewise, would consist of little more than reassurance given the likelihood that vaccine supplies would not be available and that stocks of antiviral drugs would be too small to be of significance. many would argue for the closing of schools, churches and other places of public gathering but experiences suggests that such actions produce little benefit. the wearing of masks was once a favored intervention but this, too, has been discredited. in brief, without vaccine, there would be little that could be done of practical benefit except to reassure the community that the epidemic would someday pass and to accept the criticism of the public and political leadership for failure to make reasonable preparations. at least four lessons can be derived from past experience. first, the threat of pandemic influenza caused by an especially virulent strain is a continuing threat that has to be taken seriously. second, adequate supplies of an effective vaccine, available in a timely manner, are absolutely critical to a preventive effort. solving this problem should command top priority for research and development funding. third, special plans, programs and funding are needed within the health care system to permit development of an adequate community-wide response to the occurrence of mass casualties whatever the cause. lastly, additional research in influenza is needed to better understand its pathogenesis and epidemiology with the expectation that better preventative measures might eventuate. pandemics are the most dramatic presentation of influenza a virus and they cause considerable excess mortality as a result of pneumonia and exacerbation of cardiopulmonary or other chronic diseases. the epidemiologic success of influenza a is in large part due to antigenic variation that takes place in the two surface glycoproteins of the virus, i.e. its hemagglutinin (ha) and neuraminidase (na) proteins. to date, 15 ha and 9 na subtypes have been defined and are used to classify influenza a viruses. antigenic variation occurs either gradually through accumulation of point mutations (antigenic drift) or more abruptly though introduction of a new ha gene into virus circulating in the human population, be it through reassortment of animal and human viruses or through a change in host-specificity of an animal virus (antigenic shift). a 'pandemic virus' can be defined as a virus with a new ha with or without a novel na gene, acquired through antigenic shift, that spreads readily from person-to-person in a population that is highly susceptible to infection. the 20th century saw three pandemics caused by new ha subtypes. influenza a h1n1 caused the 'spanish flu' in 1918, subtype h2n2 ('asian flu') was the causative agent of the 1957 pandemic and the h3n2 subtype ('hong kong flu') caused a pandemic in 1968. the excess mortality for the 1918, 1957 and 1968 pandemics in the us alone can be estimated to have been 500 000, 70 000, and 34 000 deaths, respectively. if one applies mathematical modeling to estimate the effects of a future pandemic, the us alone can expect between 89 000 and 207 000 excess deaths in the next pandemic (meltzer et al., 1999) . what are the viruses with pandemic potential? in 1997, 18 individuals in hong kong were infected with h5n1 influenza, an avian subtype earlier not known to infect humans (subbarao et al., 1998) . of the 18 patients 1-60 years of age, six died. molecular analysis established that all eight genes of the h5n1 virus were of avian origin and that reassortment with human viruses had not occurred (subbarao et al., 1998) . the reported 18 infections were most likely acquired from poultry; human-to-human spread, however, was a rare event. sequence analysis of the hong kong h5n1 virus suggests that it is a reassortant made up from two or three different avian parent viruses: ha from a goose h5n1 virus (xu et al., 1999) , na from a teal h6n1 virus (hoffmann et al., 2000) , and internal genes from a quail h9n2 (guan et al., 1999) or a teal h6n1 virus (hoffmann et al., 2000) . although this virus did not spread readily from person to person, deep concern was caused by the fact that this was the first known avian influenza a virus that caused disease in humans. until this outbreak it was thought that the receptor specificity of avian ha proteins limited their infectivity to avian species. this notion, however, needs to be revised. in march 1999, h9n2 influenza a viruses were isolated from two children with febrile upper respiratory tract illness (peiris et al., 1999) . this virus, again, was an avian virus that was able to infect humans without passage through an intermediate host (lin et al., 2000) . what are our options for vaccination against potentially pandemic viruses? in the case of the h5n1 viruses, there are four options for generating an effective influenza vaccine. a conventional inactivated vaccine can theoretically be generated by reassortment of internal genes from influenza a/pr/8/34 with h5n1 glycoprotein genes. there are, however, problems with incompatibility between certain gene segments. secondly, a cold adapted live attenuated vaccine approach could be taken. the influenza a/ann arbor/6/60 coldadapted strain has been used to generate an h5n1 vaccine in which the ha cleavage site was modified (li et al., 1999a) . this virus could also be used to produce an inactivated vaccine. thirdly, a surrogate virus that is nonpathogenic but antigenically related (takada et al., 1999) could be sought. one of the closest antigenically related viruses found to date was of the h5n3 subtype, but this virus was not highly immunogenic in humans. fourthly, purified protein could be used but again limited immunogenicity may be a problem. the development of vaccines against potential pandemic viruses poses a number of challenges. to begin with, most of the work has to be conducted in biosafety level 3+ laboratories. there is a large array of avian viruses that could potentially become pandemic viruses and those with a genotype that confers transmissibility have to be identified. h5n1, h9n2 and h6n1 viruses that were the genetic precursors of the h5n1 and h9n2 viruses that caused human infections in hong kong (guan et al., 1999; xu et al., 1999; hoffmann et al., 2000) and that continue to circulate among birds should certainly be given priority in vaccine development. an adequate strategy for vaccine development has to be selected, and safety testing must be expedited in mice, ferrets, chickens, and eventually humans. use of recently described plasmid based reverse genetics systems may lessen the technical challenges faced in generating vaccine candidates. assays to evaluate immunity have to be optimized (hemagglutination inhibition assays do not perform well with avian ha proteins, yet neutralization assays are time consuming and technically more difficult), and serologic and molecular diagnostic reagents must be made available. collaboration between veterinary and public health authorities must be maintained and new technologies such as plasmid based reverse genetics systems must be used. last but not least, alternative substrates for vaccine production and new adjuvants are urgently needed. although influenza epidemics occur every winter, we have only had three pandemics this past century -1918, 1957 and 1968 . while annual influenza epidemics generally cause an excess mortality of 20 000-40 000 people in the us, it is only the pandemics that remain in memory. of the three pandemics that occurred in the twentieth century, the 1918 pandemic was the most devastating and claimed the most victims. although there is wide spread public interest in the 1918 pandemic, the event is mostly remembered as an interesting event in the distant past. whether due to denial or not, few feel that an influenza pandemic is a current threat. most journalists see their role not only as educators and advocators but also as entertainers. the interest of the readership has to be captured, and it can only be maintained if their curiosity is satisfied. while the aids pandemic and its effect on people's lives found a wide audience through most of the early 1990s, the late 1990s were characterized by fatigue and a reduced interest in the pandemic (although it was still on the increase). as a result, editors were much less willing to include related stories. reportage on influenza pandemics suffers a similar fate: the disease itself is old, and the last pandemic occurred too long ago for most to remember. the mere warning of a coming pandemic is almost perceived as an unfulfilled promise if the event does not occur shortly after the article is published. breast cancer, in contrast, is one of a few examples, where interest in a disease process can be maintained over an extended period of time. the risk of suffering from breast cancer is felt much more acutely, it seems, and there is a sense that proactive behavior will lead to an improved outcome. whether the mass media will take on a role in changing the public's attitude toward influenza as a threat is very much in doubt as long as journalists themselves perceive the next pandemic as an event as anonymous and inevitable as an earthquake. rsv infection is initiated by g glycoprotein attachment to cell surface receptors and followed by virus-cell fusion that is mediated by the f protein. the cleavage-activated rsv f1 protein is thought to interact with the target cell membrane through its n-terminal fusion peptide, which is released from a shielded position within the f homotrimer through a major conformational change. insertion of the f1 n-terminus into the cell membrane destabilizes the cell membrane and induces lipid mixing that is followed by mixing of contents, thereby enabling the viral nucleocapsid to enter the cytoplasm. the rsv f protein was recently found to interact with a small gtpase called rhoa through a domain that lies just carboxyterminal to the f1 fusion peptide (pastey et al., 1999) . rhoa is a member of the ras superfamily that is expressed intracellularly in all cell types. it induces bundling of actin filaments into stress fibers, focal adhesion plaque formation, cell-to-cell adhesion and organization of integral membrane proteins; it has additional roles in cell morphology and motility as well as cell cycle transition from g1 to s. a series of overlapping rhoa peptides from the interaction domain were evaluated in rsv plaque reduction neutralization assays. rhoa peptide 77-95 has an ic 50 of about 1 mg/ml against an inoculum of 100 pfu. neutralizing activity is also seen against parainfluenza virus type 3 (piv3), but not against a variety of other paramyxo, orthmyxo, corona and filoviruses (pastey et al., 2000) . intranasal administration of 500 mg of peptide 77-95 prior to intranasal infection of mice with rsv reduces rsv replication more than 100-fold and also diminishes weight-loss. studies with recombinant rsv expressing green fluorescent protein indicated that rhoa peptide 77-95 inhibited rsv replication at a very early stage of infection. subsequently, it was shown that fusion of cells transfected with rsv f, g and sh was inhibited by this peptide, suggesting that it exerted an effect on rsv replication at the level of membrane fusion or entry. since rhoa is an intracellular molecule, one would have to hypothesize that the interaction of the n-terminal heptad repeat and fusion peptide with the target membrane causes enough membrane disruption to allow an interaction between rhoa and rsv f. alternatively, rsv f may not interact with rhoa during the entry process, and the rhoa-derived peptides may simply disrupt the f structure or function to render the virus noninfectious. the question of whether rhoa and rsv f interact during a natural infection is not yet resolved. rhoa can be found in the cytoplasm bound to gdp and a guanine nucleotide dissociation inhibitor, and, upon gtp exchange and isoprenylation, rhoa associates with the cell membrane. if the proposed model of rhoa-rsv f interaction at the membrane is correct, then inhibition of isoprenylation should inhibit rsv infection. indeed, inhibition of isoprenylation through hmg coa reductase inhibitors, such as lovastatin, inhibits rsv infection of hep-2 cells at an ic 50 of 3 mm and reduces peak rsv titers in lungs of mice greater than 100-fold with oral gavage of 1 mg per day (gower, t.l., graham, b.s., submitted). the antiviral effect of lovastatin is also seen with piv3 in vitro. although it has not been formally proven that rsv f interacts with rhoa in the infected cell membrane, rhoa signaling activity is triggered in rsv-infected cells (gower, t.l. et al., submitted) . while rhoa signaling is not required for rsv replication, syncytium formation is diminished when rhoa signaling activity is inhibited. in addition, rhoa signaling may play a role in other aspects of rsv pathogenesis. rhoa kinase inhibitors reduce the transcription of il-6 and il-8 mrna in rsv infected cells (cytokines abundant in the nasal secretions of rsv-infected patients), and obviate airway hyperresponsiveness, one of the cardinal symptoms of rsv disease (hashimoto, k. et al., submitted). the balb/c mouse model was used to explore whether mycophenolic acid (mpa), an inhibitor of de novo purine synthesis in t and b lymphocytes, could improve the outcome of rsv disease, which was monitored by clinical symptoms such as ruffling of fur, increased respiratory rate and weight loss. oral administration of 100 mg/kg mmf (the prodrug of mpa) daily from day 1 to 6 post-infection reduced weight loss on day 7 post-infection from 23% in untreated animals to 8% in mpa treated mice (p b 0.001). a reduction of weight loss (7.8%) was also observed when initiation of treatment was delayed until day 5 post-infection. virus titers in lungs of mmf treated mice were similar to those of untreated controls but histological changes were reduced. on day 7 post-infection, ifng levels were elevated 2.5-fold in the treatment group while il4, il5 and il10 levels were unchanged, indicating a shift toward a t helper cell type 1 response that is thought to correlate with improved rsv disease outcome. these data suggest that inflammatory responses contribute to rsv disease in mice and that an immunomodulatory approach to the treatment of human rs virus disease is worth further consideration. using a cell-based assay to identify compounds which are able to inhibit fusion of hela cells infected with rsv, more than 300 analogues of a lead compound were synthesized and one compound (termed r170591) was selected for further evaluation. the in vitro 50% inhibitory concentration (ic 50 ) of this benzimidazole derivative (mw 395) was150 pm, and thus its potency exceeds that of ribavirin almost 100 000-fold (ic 50 =10 mm). r170591 exhibits in vitro antiviral activity against human rsv (subgroup a and b) and bovine rsv but not against pneumovirus of mice or other paramyxoviruses. rsv-induced cytopathic effect was reduced by r170591 at 0.1 nm, and concentration of 10 nm reduced rsv titers 1000-fold in multi-cycle growth curves. time of addition studies indicated that both virus-cell fusion and cell-cell fusion were inhibited by this compound. selection of resistant viruses in vitro yielded two mutants with single point mutations in the f-protein: one upstream of the second heptad repeat motif and another within it (s398l and d486n). rsv titers, determined by quantitative rt-pcr, were reduced 10-fold in bronchoalveolar lavage fluid and in lung tissue of cotton rats treated once by inhalation with r170591 prior to rsv infection. rfi-641, a compound that was derived by chemical optimization of the earlier described antiviral cl-387626, is a small molecule antiviral drug that selectively inhibits rsv (wyde et al., 1998) . the molecule is water-soluble and not orally bioavailable, but it proves to be efficacious when administered intranasally or by inhalation. the in vitro ic 50 varies between 3 and 180 ng/ml for laboratory strains or clinical isolates of rsv subgroup a and b. viral specificity and the large therapeutic window of rfi-641 (\100 fold) indicate that the antiviral activity of the compound is not due to adverse effects on normal cells. addition of rfi-641 to cell culture prior to adsorption reduces rsv yield 1000-fold at 48 h post infection (wyde et al., 1998) . temperature shift experiments suggest that the rsv f protein is the target for rfi-641, and this observation is confirmed by the inhibitory effect that rfi-641 has on rsv b1 cp-52/2b5, a viable rsv mutant in which both the g and sh open reading frames are deleted. if rfi-641 is added to cell culture 5 h post infection, it inhibits syncytium formation indicating that fusion inhibition occurs both in the early and late phase of the infectious cycle. rfi-641-resistant viruses can be selected, albeit much less easily than amantadine resistant variants. resistance to rfi-641 is acquired by point mutations solely in the f protein, mostly upstream of the second heptad repeat motif, but not by mutations in the g or sh protein. when administered prophylactically by the intranasal route, rfi-641 inhibits rsv replication in vivo, with mice and cotton rats exhibiting a 10-1000-fold reduction in rsv replication on day 5 post infection. in african green monkeys, rfi-641 reduces peak rsv titers not only when administered prophylactically but also when therapy is initiated at 24 h post infection and continued for a total of 9 days. a nebulized form of rfi-641 has been shown to be active in monkeys. the preclinical profile of this drug supports its development for treatment and prophylaxis of rsv disease in pediatric, adult and geriatric populations. phase 1 clinical trials confirmed that rfi-641 is a potent antiviral, and that the safety profile of this drug is encouraging. 7.5. de6elopment of picona6irus 3c protease inhibitors ag7088 is a potent, irreversible inhibitor of human rhinovirus (hrv) 3c protease, the enzyme that is responsible for the cleavage of the viral polyprotein into its functional protein subunits (matthews et al., 1999) . ag7088 was discovered by protein structure based rational drug design, and the compound exhibited activity against a large set of different hrvs. the 50% effective concentration (ec 50 ) ranges between 3 and 81 mm. other piconaviruses such as coxsackievirus a21 or b3, enterovirus 70, and echovirus 11 are also sensitive to the compound. in a placebo controlled challenge study in which adult volunteers were infected with hrv39 and treated with ag7088 (8 mg per dose intranasally, 5 times per day), ag7088 reduced mean viral titers, as well as mucus weight, respiratory symptom score and total symptom score significantly. in this study viral titers were determined by culture and also by quantitative pcr (taqman) to exclude ex-vivo effects of ag7088 on the reduction of virus titers. a phase ii clinical trial with 868 subjects was conducted to determine the efficacy of ag7088 in naturally acquired piconavirus infections. patients selected for the study had to present within 36 h of onset of symptoms and had to suffer from at least two mild or one moderate cold symptom. a five step symptom score was used to record severity of rhinorrhea, cough, sneezing, sore throat, chills, headache, and malaise. the treatment group was stratified into two or four daily doses of 8 mg ag7088 per dose intranasally, and the mean respiratory symptom score for days 1-5 was chosen as the primary endpoint. only 29% of all enrolled patients were infected by picor-naviruses. no significant difference in respiratory or total mean symptom score for days 1-5 was detected. however, the drug was well tolerated and safe. the lack of efficacy in this particular study might have been due to a lower than expected frequency of picornavirus infections. in a retrospective analysis, stratification for start of therapy within 24 h detected a trend to fewer respiratory symptoms and fewer total symptoms. there was a trend to earlier onset of relief, but the difference between treatment and placebo group again did not reach significance. 7.6. pleconaril (p) treatment reduces the incidence of acute otitis media (om) in children with 6iral respiratory illness: a pilot study pleconaril, an orally bioavailable picornavirus 3c protease inhibitor, has in vitro antiviral activity against 93% of all rhinoviruses. to evaluate the efficacy of pleconaril in preventing acute otitis media (aom) in an outpatient population, a double-blind, placebo-controlled pilot study was conducted in children with viral respiratory illness. eighty-seven children with a median age of 3 years and a history of 2-3 episodes of aom were randomized to pleconaril treatment (5 mg/kg or 2.5 mg/kg) or placebo thrice daily for 7 days following presentation with upper respiratory symptoms. picornavirus rna was detected by rt-pcr in nasal samples in 51% of patients at baseline. the overall incidence of physician-diagnosed aom during the 14 day follow-up were 18.8% (9/48), 12.5% (3/24), and 0% (0/15) in the placebo, low dose and high dose groups, respectively. of the 87 children developing aom, 86 tested positive for picornavirus rna. pleconaril treatment reduced the frequency of nasal symptoms by 29% in the high dose (5.0 mg/kg, p= 0.006) and 9% in the low dose (2.5 mg/kg, p= 0.393) group. it reduced frequency of systemic symptoms by 40% in the 5.0 mg/kg group (p= 0.020) and 26% in the 2.5 mg/kg group (p= 0.096). pleconaril was well tolerated, and adverse events did not differ from the control group. these results encourage further expanded trials of pleconaril in children with viral respiratory infections. 7.7. oral oseltami6ir reduces influenza-related complications in all age groups influenza illness is associated with the development of secondary complications, often requiring antibiotic treatment or even hospitalization. insurance data indicate that up to 64% of influenza related hospital admissions occur in 15-64 year old individuals, often without recognized underlying disorders. influenza complications affect all age groups but the type of complication differs among them. while acute otitis media has been reported in over 20% of children with influenza, lower respiratory tract infections (lrti) such as bronchitis and less often pneumonia, are the most common complication in adults. in recent years six phase iii clinical trials were completed; two of them in pediatric populations, three in healthy adults and one in the elderly. the neuraminidase inhibitor oseltamivir (o) is already approved for the treatment of influenza in adults, and it was recently approved for treatment in children aged 1 year and older by the fda. in six phase iii trials, most of the patients were enrolled within 36 h of onset of symptoms, and oseltamivir was administered at 2 mg/kg twice daily for children and 75 mg twice daily for adults. subjects with febrile ( \37.8°c) influenza were randomized to a 5 day regimen of oseltamivir or placebo, and an important endpoint of all studies was the effect of oral oseltamivir (o) on the incidence of influenza-related complications requiring antibiotics. one thousand and twenty nine children between the age of 1 and 12 (mean age 6.4 years) were enrolled in the pediatric trials. in the adult and elderly populations, 953 patients with a mean age of 35 years and 736 elderly patients with a mean age of 73 years were enrolled. in the pediatric, adult and elderly trials 61, 64 and 65%, respectively, tested positive for influenza a or b by culture or fourfold increase in hai titers. physician-diagnosed secondary complications (bronchitis, pneumonia, lrti, sinusitis or otitis media) requiring antibiotics were assessed in patients with confirmed influenza infection. oseltamivir reduced the rate of complications compared with placebo by 40% in children (p=65/235, o =36/217; p =0.005), by 51% in adults (p =23/309, o = 11/301; p = 0.05), and by 28% in the elderly (p= 49/254, o= 31/223; p= 0.14). in the pediatric population, aom was the most common complication, followed by bronchitis, pneumonia and sinusitis. the frequency of aom was reduced from 17% in the placebo group to 10% in the oseltamivir group, a significant 40% reduction. in adults and the elderly, bronchitis and sinusitis were the most common complications of influenza. the frequency of bronchitis in the elderly was reduced from 15 to 11%. thus, oral oseltamivir reduced the incidence of secondary complications and associated antibiotic use in all age groups. when all age groups were combined, antibiotic use for any indication was also significantly reduced. twelve patients in the placebo group versus four patients in the oseltamivir group required hospitalization for probable or possible influenza-related complications, suggesting a possible effect on hospitalization. 7.8. neuraminidase inhibitors: clinical update 7.8.1. oseltami6ir oseltamivir was recently approved in the us for prophylaxis of influenza in adults and adolescents over 12 years of age after three successful phase three trials. one was conducted in healthy adults, one in a family setting with an infectious index case, and another evaluated seasonal prophylaxis in the elderly. the efficacy of oseltamivir in all three trials ranged between 70 and 90%. an application for the approval of oseltamivir for treatment of influenza in children over one year of age was recently approved by the fda. in otherwise healthy children of 1-12 years, treatment with oseltamivir reduced the median time to freedom from illness by 1.5 days (p= b 0.0001) and also reduced the relative risk of otitis media (aom) by 40%. in 6 -12 year old asthmatic children, oseltamivir treatment, started within 24 h of first symptoms, decreased the median illness duration by 1.7 days (p =0.07), and airway function was significantly improved (1 s forced expiratory volume on day 5 improved by 10.8% compared with baseline values among oseltamivir recipients and 4.7% among placebo recipients (p= 0.0147)). the incidence of development of drug resistant virus under therapy was evaluated in more than 1000 subjects. none of 542 subjects sampled on day 3 of treatment harbored resistant virus. on day 5, however, 4/451 subjects (1%) shed virus with a resistant phenotype. in children, the frequency of viral resistance was 4% (10/247). all of these subjects recovered normally despite this incidental finding. so far, resistant mutants have been less infectious than wild type virus; no evidence of transmission has been detected in an experimental infection model in ferrets. the impact study evaluated the benefit of early intervention in reducing the total time with symptoms starting from the time of first onset of fever. this reflects the total burden of illness from a patient's point of view. a total of 958 patients, 13-70 years of age were enrolled and treated within 48 h of the first onset of symptoms. treatment was initiated within 12 h in 25% of those recruited. using an accelerated time to failure model, earlier intervention was shown to strongly correlate with shorter duration of illness. the total duration of illness could be halved if treatment was started within 12 h of the onset of symptoms compared with intervention at 48 h. in phase iii trials, zanamivir reduced the duration of illness in adults over 12 years of age and in children between 5 and 12 years of age by 1.5 days (95% ci= 1-2.5 days). in high risk patients (525 subjects, 65% with asthma and 35% with chronic obstructive pulmonary disease) duration of illness amongst all influenza positive subjects was reduced by 1.5 days. duration of illness was reduced in both vaccinated (1.25 day) and nonvaccinated (2 day) subjects. during therapy, pulmonary function improved slightly between days 1 and 5. zanamivir was well tolerated, and there were no differences in adverse effects between treatment and placebo groups. with 9000 subjects studied in clinical trials, resistant mutants were not isolated by sampling on days 3 and 5. efficacy for treatment of influenza b was assessed in 300 subjects and found to be similar to that of influenza a. a prophylaxis study in a family setting showed 79% protective efficacy. finally, in a nursing home study, zanamivir exhibited a 60% in-creased efficacy in preventing influenza disease compared with rimantadine. rwj-270201, an orally available once daily neuraminidase inhibitor that is active against influenza a and b in vitro and in animals, was evaluated in phase ii trials of experimentally infected healthy adults. therapy was started approximately 24 h post infection with influenza a/texas h1n1 or influenza b/yamagata. viral load over time was the primary endpoint, and nasal wash titers were determined every 12 h on day 1-3 and daily thereafter. in the influenza a study, 90 subjects were enrolled into four treatment (100 to 400 mg per day) and one placebo arm. high dose treatment (400 mg once daily or 200 mg twice daily) reduced viral load by 68-73%, whereas 100 or 200 mg per day led to a reduction by 34%. shedding was reduced by 1-2 days in the high dose groups, and the incidence of fever declined from 25% in placebo controls to less than 5% in all treatment groups combined. to evaluate efficacy against influenza b, 56 subjects were enrolled into two treatments (800 or 400 mg per day) and one placebo group. a 61% reduction of viral load was shown for the high dose treatment (800 mg per day). no evidence for the emergence of resistant virus was detected, and oral rwj 270201 was well tolerated without excess gastrointestinal side effects. world-wide, acute respiratory infections (ari) are responsible for more than 4 million deaths per year in children under five years of age. viral respiratory infections contribute significantly to this burden of disease, not only in children but also in adults. although there is a myriad of viral respiratory pathogens, the number of virus families that cause significant burden of disease is limited. in children, the paramyxoviridae are the most important family of viruses, with respiratory syncytial virus (rsv) and the parainfluenza viruses (piv) causing most disease, followed by adeno-and influenza viruses. in adults, influenza a virus with its constantly changing antigenic composition surpasses other viral pathogens, but the impact of rsv and rhinoviruses are being increasingly appreciated. the epidemiology of respiratory virus infections very much depends on host and environmental factors. while adenovirus infections are a major concern in military training camps and in immunocompromised patients, they are not as important in the general adult population. the immunocompromised are not only at increased risk for viral respiratory disease but diagnosis and management are much more difficult in this group. newer diagnostic methods based on antigen detection or viral genome amplification make rapid diagnosis possible and thereby affect clinical management of viral respiratory disease. vaccines are still the most powerful tool in preventing viral respiratory disease but for many important pathogens there is still no vaccine available. the inactivated influenza vaccine, with its antigenic composition changing annually, is still the mainstay of influenza prevention. coldadapted live attenuated influenza virus vaccines are on the horizon but are not yet approved by the fda. adenovirus vaccines have been used successfully in the military for many years, and the recent resurgence of large outbreaks of respiratory disease after discontinuation of vaccination emphasizes the importance of maintaining a preventive strategy. live virus vaccine candidates against rsv and piv, derived biologically or by reverse genetics, are currently in clinical trials, and there is hope that safe and efficient vaccines will become available within this decade. new insight into virus-host and virus-bacteria interactions in viral respiratory illness may help us understand the complexity of these disease entities and allows a re-evaluation of management options. diseases earlier thought of as purely bacterial, such as otitis media, might be more effectively prevented by viral vaccines than by bacterial vaccines. populations at high risk for complications resulting from respiratory viral infections are now better defined and a more targeted prophylaxis is possible, be it passive prophylaxis against rsv disease with monoclonal antibody preparations or active prophylaxis with influenza-or adenovirus vaccines. in the management of influenza virus disease, much has changed for the better. neuraminidase inhibitors (ni) are now established as an effective intervention to decrease the severity and to shorten the duration of illness when therapy is initiated within 2 days of the onset of symptoms. zanamivir and oseltamivir are now licensed in the us and elsewhere for adults and adolescents twelve years of age and older, and a license for use in children over one year of age has recently been obtained for oseltamivir. both drugs are safe and well tolerated, and development of resistance occurs much less frequently than with older antivirals such as amantadine or rimantadine. rhinovirus infections, the cause of most colds, may now be amenable to treatment with the experimental 3c protease inhibitors pleconaril (given orally) and ag7088 (given intranasally), and initial studies indicate that pleconaril treatment of colds in children might reduce their risk of developing otitis media. antivirals effective against rsv and piv are still in an early phase of development, but inhibitors of the viral fusion protein look promising. despite all progress, severe deficiencies remain in the preventative efforts. our level of preparedness for an influenza pandemic does not seem much different from that 40 years ago. streamlining of hospital infrastructure and just-in-time production of antibiotics and antivirals limit our ability to respond effectively should a pandemic strike. although potential pandemic influenza viruses have been defined and novel techniques such as reverse genetics are being employed in vaccine development, the implementation of pandemic preparedness is still a daunting task. 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receptors for non-pilate neisseria meningitidis on hep-2 cells induction of protective immunity against influenza virus in a macaque model: comparison of conventional and iscom vaccines identification of a conserved receptorbinding site on the fiber proteins of car-recognizing adenoviridae isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines bovine respiratory syncytial virus nonstructural proteins ns1 and ns2 cooperatively antagonize a/b interferon-induced antiviral response bovine parainfluenza virus type 3 (bpiv3) fusion and hemagglutinin-neuraminidase glycoproteins make an important contribution to the restricted replication of bpiv3 in primates bacterial pneumonia during the hong kong influenza epidemic of 1968 -1969 molecular identification of viruses in sudden infant death 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strain against a potential human pandemic recombinant respiratory syncytial virus that does not express the ns1 or m2-2 protein is highly attenuated and immunogenic in chimpanzees efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial therapy for respiratory tract infections caused by respiratory syncytial virus expression of adenoviral e3 transgenes in beta cells prevents autoimmune diabetes respiratory syncytial virus infection in tropical and developing countries community respiratory virus infections in immunocompromised patients with cancer recombinant respiratory syncytial virus (rsv) bearing a deletion of either the ns2 or sh gene is attenuated in chimpanzees replacement of the f and g proteins of respiratory syncytial virus (rsv) subgroup a with those of subgroup b generates chimeric live attenuated rsv subgroup b vaccine candidates immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy cl387626 exhibits marked and unusual antiviral activity against respiratory syncytial virus in tissue culture and in cotton rats genetic characterization of the pathogenic influenza a/goose/guangdong/ 1/96 (h5n1) virus: similarity of its hemagglutinin gene to those of h5n1 viruses from the 1997 outbreaks in hong kong key: cord-011438-imbpgsub authors: zhang, yun; xu, zhichao; cao, yongchang title: host–virus interaction: how host cells defend against influenza a virus infection date: 2020-03-29 journal: viruses doi: 10.3390/v12040376 sha: doc_id: 11438 cord_uid: imbpgsub influenza a viruses (iavs) are highly contagious pathogens infecting human and numerous animals. the viruses cause millions of infection cases and thousands of deaths every year, thus making iavs a continual threat to global health. upon iav infection, host innate immune system is triggered and activated to restrict virus replication and clear pathogens. subsequently, host adaptive immunity is involved in specific virus clearance. on the other hand, to achieve a successful infection, iavs also apply multiple strategies to avoid be detected and eliminated by the host immunity. in the current review, we present a general description on recent work regarding different host cells and molecules facilitating antiviral defenses against iav infection and how iavs antagonize host immune responses. influenza a virus (iav) can infect a wide range of warm-blooded animals, including birds, pigs, horses, and humans. in humans, the viruses cause respiratory disease and be transmitted by inhalation of virus-containing dust particles or aerosols [1] . severe iav infection can cause lung inflammation and acute respiratory distress syndrome (ards), which may lead to mortality. thus, causing many influenza epidemics and pandemics, iav has been a threat to public health for decades [2] . the virus is an enveloped, segmented, negative-strand rna virus, belonging to the orthomyxoviriae family. the eight viral gene segments encode as many as 18 proteins. besides polymerase basic 1 (pb1), pb1-n40, pb1-f2, pb2, polymerase acid (pa), hemagglutinin (ha), nucleoprotein (np), neuraminidase (na), matrix 1 (m1), matrix 2 (m2), nonstructural protein 1 (ns1) and ns2 (also known as nuclear export protein, nep), new viral proteins were recently uncovered, such as pb2-s1 [3] , pa-x (product of ribosomal frameshifting) [4] , pa-related proteins pa-n155 and pa-n182 [5] , m42 [6] , and ns3 [7] . ha, na, and m2 proteins constitute surface of the iav virion, where ha is the most abundant surface protein. according to the genetic and antigenic diversity of the ha and na proteins, iavs were divided into 18 ha and 11 na subtypes. h17n10 and h18n11 subtypes were recently identified in bats [8, 9] . ha is a type i glycosylated protein, which is responsible for virus entry to host cell. functional ha protein is a homotrimer structurally composed of a stem region and a globular head region in each monomer. the head region bearing n-acetylneuraminic acid (sialic acid, sa) binding pocket is critical for receptor attachment, and contains most antigenic determinants. the stem region undergoing conformational changes is responsible for low ph-triggered membrane fusion [10] , and plays an important role in cross protection against heterosubtypic iav infection [11] . n38 glycan at this region iavs can infect a broad spectrum of host species, including both wild and domestic birds, as well as many mammalian species. the virus is capable of interspecies transmission to new species. however, no interspecies transmission of the bat iavs has been reported so far [54] . furthermore, the high frequency of mutations and recombination increases the risk of iav adaptation in humans. besides three pandemic subtypes (h1n1, h2n2, and h3n2), other subtypes, including h5n1, h5n6, h7n7, h7n9, n9n2, and h10n8 could cross the species barrier and cause human infections [55] [56] [57] [58] . several effect factors are essential in iav host switch events, including the receptor-binding properties of ha [16] , as well as cellular receptors [59] [60] [61] [62] . long and his colleagues summarized the role of host factors in iavs adaption to humans, and the review is recommended here for further reading [63] . noteworthy is the fact that most phylogenetically diverse iavs with different origins could successfully replicate in swine [64] . since pigs have both sa α-2,6 and sa α-2,3 galactose receptors [65] , they can serve as a suitable mixing reservoir for both human and avian iavs, thus raising global concern on periodic zoonotic infections. take the emergence of influenza a (h1n1) pdm09 (ph1n1) and influenza a (h3n2) a/canada/1158/2006 strains for instance, both strains are swine-origin iavs and were the consequence of adaption and reassortment of several swine lineages [66, 67] . furthermore, some genes of these strains originated from avian iavs [68] . with the development of gene sequencing technology, machine learning (ml) facilitated with large genomic datasets are used in prediction about sequence changes in newly invaded viruses from other animal hosts, take the "batch-learning self-organizing map (blsom)" method for instance [69] . ml is also applied in characterization of distinct host tropism protein signatures [70] , and prediction of amino acid changes for interspecies transmission [71] . these studies provided measures in identification of potential high-risk strains. in addition, the nucleotides and dinucleotide compositions of viruses play important roles in prediction of viral host species [72] . combining gene sequencing technology viruses 2020, 12, 376 4 of 23 and ml methods, researchers applied large iav genomic datasets to analyze species selection bias of iav mono-/dinucleotide composition and predict human-adaptive swine or avian iavs [73, 74] . the application of multi-disciplinary subjects would provide useful information for prediction of pandemic influenza. in general, the life cycle of the iav is generally divided into four steps: virus entry into the host cell, transcription and replication of the viral genome, assembly, and virus budding. though alveolar epithelial cell is the primary target cell for iavs, different iav subtypes have different patterns of viral attachment (pva). for human iavs, alveolar type ii epithelial cells, as well as immune cells such as alveolar macrophages and dendritic cells, are major target cells for an established infection [75, 76] . two seasonal iavs and pandemic h1n1 virus, preferred to attach to ciliated epithelial cells and goblet cells in the upper respiratory tract (urt), and avian iavs, take h5n1 for instance, attached seldom to these cells [77] . in the lower respiratory tract (lrt), human iav h1n1 and h3n2 attached to more cell types than avian iav h5n1, a highly pathogenic avian iav (hpaiv) strain. however, h5n1 could bind to type ii pneumocytes [78] . considering the fact that metabolism in the type ii pneumocytes is quite active, infection of hpaivs is more likely to cause severe pneumonia [78] . other research on low pathogenic avian iavs (lpaivs), which generally do not cause severe pneumonia, showed that these viruses usually attach to human submucosal gland cells, thus can be cleared by the mucus [79] . iav infection starts from recognition of sa by ha protein, though in vitro research claimed that these n-linked glycans were not essential for virus entry [80] . the cleavage of ha precursor protein ha0 into ha1 (containing receptor binding domain) and ha2 (containing fusion peptide) in low ph environment during ha transport is critical for virion internalization [81] . some research showed that type ii transmembrane serine protease such as transmembrane protease serine 2 (tmprss2), human airway trypsin-like protease (hat), transmembrane protease serine 4 (tmprss4), homo sapiens serine protease desc1 and homo sapiens transmembrane protease, serine 13 (mspl) can cleave human and avian iav ha proteins at an arginine residue [82] . in addition, for avian iavs, ha0 of hpaivs can be cleaved by subtilisin-like protease, while that of lpaivs is cleaved by trypsin-like proteases [83] or thrombin [84] . therefore, in avian iavs, the cleavage sites are considered to be the major determinants for virus virulence [85] , and rna folding in the cleavage region could be an important factor for virulence determination [86, 87] . proteins in the vrnp complex contain different nuclear localization signals (nlss), thus helping the vrnp complex to enter the host cell nucleus via active transport, take the crm1-dependent pathway for instance [88] . the acidic environment of the endosome also activates m2 ion channel, hence acidifies the viral core, resulting in entrance of vrnp complex into the host cell [34] . replication of viral genome does not require a primer but a full-length complementary rna (crna), which is essential for the newly formed vrnp complex. the viral rna polymerases first bind to the 3 end and the 5 end of the segmented viral rna and crna, respectively, then start replication with the help of the 5 cap of host pre-mrnas via a pb1-pb2-mediated "cap snatching" mechanism [27, 89] . the conserved segment-specific nucleotides at the 3 and 5 ends of the viral genome could modulate genome expression and replication during infection [90] . in addition, dephosphorylation at a specific position of the h1n1 ns1 protein results in attenuated virus replication [91] . mature viral mrnas are transported to the cytoplasm by a "daisy-chain" complex and translated subsequently [88, 92] . new synthesis of ha occurs on the rough endoplasmic reticulum (er). glycosylation and palmitoylation of the protein are completed later in the golgi [93, 94] . after synthesis and maturation of na and m2 proteins, the trans-golgi network (tgn), together with coat protein i (copi) complex and gtpase rab proteins, transport the newly synthesized ha, na, and m2 proteins to the apical plasma membrane (pm). these proteins then assemble with viral genomic segments. the virions are finally closed and m1 and m2 proteins mediate virion budding from the apical side of the viruses 2020, 12, 376 5 of 23 cells [28, [95] [96] [97] [98] . na protein cleavages the sa residues, which allows the virions to be released from the plasma membrane [99] . since iav has a relatively small genome, host machinery is required in order to accomplish the viral life cycle. to uncover host dependency factors that are necessary for iav replication, numerous large-scale rna interference (rnai) screens and genome-wide crispr/cas 9 screen were performed [29, [100] [101] [102] [103] . for instance, son dna binding protein was important for iav virion trafficking in an early infection stage and cdc-like kinase 1 facilitated aiv replication [29] . usp47 facilitated viral entry, whereas tnfsf12 (april) and tnfsf12-tnfsf13 (twepril) helped with viral replication [101] . using genome-wide crispr/cas9 screen, several genes of sialic acid biosynthesis and related glycosylation pathways were involved with h5n1 infection [102] , and wdr7, ccdc115, and tmem199 were essential for viral entry and regulation of v-type atpase assembly [103] . furthermore, single-cell transcriptome sequencing (rna-seq) was applied to explore host-virus interactions, revealing a correlation between defective viral genomes and virus-induced host transcriptional programs [104] . these data provide valuable information for developing host-targeted therapeutics. host immune system functions immediately after detection of the virus. host mucosal immune system (mis), induced after virus invasion, serves as the first line to prevent iav from adhering to the susceptible cells. in the urt, mucosal response is induced in the naso-associated lymphoid tissues (nalt), while in the lrt, it occurs in bronchus-associated lymphoid tissues (balt). host innate immunity, including phagocytic cells, interferons (ifns), proinflammatory cytokines, etc., applies multiple mechanisms in defending iav infection [105] . host adaptive immunity, mediated by b lymphocytes and t lymphocytes, together with other immune mechanisms, reacts specifically to neutralize and eliminate the virus. on the other hand, to establish a successful infection, iavs also employ a plethora of strategies to avoid being detected or being cleared by the host immunity. notable strategies include regulation of ifn signaling [106] , inhibition of cytokine expressions [107, 108] , modulation of apoptosis [109] [110] [111] , interference of autophagy [112] , and effects on antibody production [113] . the iav-host immunity interaction was summarized by several reviews [114, 115] . upon detection of infection, innate effector cells, including natural killer (nk) cells, neutrophils, and dendritic cells (dcs), etc., are recruited to the infected sites. nk cells are large granular lymphocytes, making up 10% of the resident lymphocytes in the lung. after recruitment from the blood, nk cells interact with dcs and macrophages to secret various cytokines and restrict infection via lysis of the iav-infected cells. the lysis process is mediated by interaction between nk receptors p46 (most nkp46) and iav ha protein expressed by the infected cell [116, 117] . interestingly, liver nk cells other than lung nk cells possessed a memory phenotype to protect mice against subsequent iav infection, though the lung nk cells are important in control of primary iav infection [118] . however, nk cells are also shown to exacerbate iav pathology, since depletion of nk cells led to increased resistance to high dose h1n1 infection in mice [119, 120] . the contribution of nk cells to anti-iav defense in mouse models was later shown to be strain and dose dependent. in addition, the host genetic background also played an important role [121] . neutrophils are key innate immune cells recruited to infection sites by cellular migration through vascular endothelium. they function in clearance of pathogens via phagocytosis, producing extracellular traps, and degranulation [122] . in addition, they also regulate adaptive immunity via guiding influenza specific cd8 + t cells to the infection sites [123] . the function of dendritic cells (dcs) is to monitor invading pathogens. after iav infection, the conventional dcs migrate from lung to lymph nodes through interaction between ccr7 and its ligand, and present antigens to t cells [124, 125] . one study based on a mouse model showed that, during iav infection, immature and mature dcs were specialized in iav ha processing, since both types of dcs could present one epitope of h1n1 ha (ha amino acids 107-119), whereas another epitope (ha amino acids 302-313) could only be processed by mature dcs [126] . the complex role of dcs in initiation of robust immunity against iav infection is reviewed by waithman and mintern [127] . t cells and b cells are critical components in adaptive immunity against iav infection. cd8 + t cells differentiate into cytotoxic t lymphocytes (ctls) and defend iav infection via producing cytokines and effector molecules, and cytotoxic effects (i.e., lysis) of infected cells mediated by mhc class i. cd4 + t cells target iav-infected epithelial cells through binding with mhc class ii molecules and contribute to b cell activation thus consequently promote antibody production. the activation of t cells and b cells in iav infection will be exposited in section 3.5. the reaction of innate immunity is nonspecific. it is triggered by recognition of pathogen associated molecular patterns (pamps) via host pathogen recognition receptors (prrs). toll-like receptors (tlrs), retinoic acid-inducible gene-i proteins (rig-i), and nod-like receptors are common prrs, the activation of which leads to activation of innate immune signaling and further production of cytokines as well as other antiviral molecules. toll-like receptors are responsible for sensing pathogens at cell membranes, endosomes, and lysosome [128] . tlr3 and tlr7 are shown to be involved in iav detection at endosomes [105] . tlr3 recognizes double stranded rna (dsrna) which may be released by cellular stress and cell death [105] and unidentified rna structures in phagocytosed cells infected with iavs [129] . in macrophages and dendritic cells, tlr3 interacted with tir-domain-containing adapter, then activated the serine-threonine kinase iκkε (ikkε) and tank binding kinase 1 (tbk1) to phosphorylate interferon regulatory factor 3 (irf3), the process of which further led to expression of ifn-β [130] . in addition, an over-reacting tlr3 activation promoted iav pathogenesis, which could be reduced by a single-stranded oligonucleotide (sson) functioning as a tlr3 inhibitor, resulting in restrained viral loads both in vitro and in vivo [131] . tlr7 recognizes single stranded rna (ssrna). in plasmacytoid dendritic cells (pdcs), after activation of tlr7 during iav infection, irf7 or nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb) were activated via myeloid differentiation factor 88 (myd88) to induce type i ifns [132] . in avian macrophages, activation of tlr7 produced pro-inflammatory molecules such as interleukin (il)-1β [133] . in addition, in mouse models, tlr7 played an important role in activation of nk cells [134] . it was also shown to be involved in development of adaptive immunity to prevent iav infection [135, 136] . rig-i recognizes ssrnas and transcriptional products of iavs, which triggers activation of the caspase activation and recruitment domains (cards) via dephosphorylation or ubiquitination by e3 ligases, resulting in activation of transcription factors including irfs and nf-κb [137] . otub1 played an essential role in regulation of rig-i [138] . in addition, melanoma differentiation-associated gene 5 (mda5) was also involved in sensing transcriptional products of iavs in the cytoplasm [139] . for nod-like receptor family, pyrin domain containing 3 (nlrp3) and nlr apoptosis inhibitory protein 5 were activated after iav infection [140] . iav m2 ion channel and pb1-f2 were involved in activation of nlrp3 inflammasome and stimulate il-1β secretion subsequently [141, 142] . the role of the nlrp3 inflammasome in regulation of anti-iav responses is discussed in detail by sarvestani and his colleagues [143] . delayed oseltamivir and sirolimus combined treatment could suppress nlrp3 inflammasome mediated secretion of il-1β and il-18, resulting in attenuation of h1n1-induced lung injury [144] . after detecting viral components, transcription factors including nf-κb and irfs are activated, leading to transcription of ifns and pro-inflammatory cytokines. ifns bind to receptors, resulting in upregulation of multiple interferon-stimulated genes (isgs) [145] . it is well known that type i ifns viruses 2020, 12, 376 7 of 23 (ifn-α and ifn-β) and type iii ifns (ifn-λ 1-4) play critical roles in antiviral responses. mice failed to restrict non-pathogenic iav when both type i and type iii ifn receptors were knocked out [146] . the expressed ifns consequentially bind to different receptors. type i ifns interact with ifn-α/β receptors (ifnar), whereas type iii ifns interact with ifn-λ receptors (ifnlr). janus kinase-signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [147, 148] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [149] . furthermore, ifn-λs served an important role in programming dcs to direct effective t cell immunity against iav infection [150] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [151, 152] . cholesterol 25-hydroxylase (ch25h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [153] . guanylate-binding protein 3 (gbp3) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [154] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim14 could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [155] . trim22 degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [156] . trim 25 regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [157] . trim32 recognized iav pb1 protein and reduced its polymerase activity [158] . trim41 targeted np for ubiquitination and degradation in vitro [159] . for further reading on other isgs, several reviews regarding ifn responses during iav infection are recommended here [160, 161] . a general description of activation of innate immunity and ifn signaling pathway after iav infection is illustrated in figure 1 . signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [147, 148] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [149] . furthermore, ifnλs served an important role in programming dcs to direct effective t cell immunity against iav infection [150] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [151, 152] . cholesterol 25-hydroxylase (ch25h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [153] . guanylate-binding protein 3 (gbp3) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [154] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim14 could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [155] . trim22 degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [156] . trim 25 regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [157] . trim32 recognized iav pb1 protein and reduced its polymerase activity [158] . trim in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [162] . the follow-up work showed that poly (adp-ribose) polymerase 1 (parp1) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [163] . ns1 is the most important ifns antagonist protein via mechanisms including inhibition of the trim25-mediated rig-i ubiquitination, suppression of protein kinase r (pkr), in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [162] . the follow-up work showed that poly (adp-ribose) polymerase 1 (parp1) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [163] . ns1 is the most important ifns antagonist protein via mechanisms including inhibition of the trim25-mediated rig-i ubiquitination, suppression of protein kinase r (pkr), phosphorylation of iκb kinases (ikk) α and β in the nf-κb pathway, interruption of the phosphorylation of stat1, stat2, and stat3 [39, 115] , and degradation of otub1 [138] . phosphorylation of ns1 is crucial for its function of antagonizing ifn-β expression, since dephosphorylation at position 73 and 83 of the protein induced a high level of ifn-β [91] . non-structural protein pb1-f2, identified from a+1 open reading frame (orf) of pb1 gene segment [164] , is multifunctional in deregulation of type i interferon [165, 166] . it counteracted rlr-mediated activation of ifn pathway not only by targeting mitochondrial mavs [115, 165, 167] , but also by binding to the dead-box helicase ddx3 to induce proteasome-dependent degradation [166] . furthermore, pb1-f2 interacted with mitochondrial tu translation elongation factor (tufm) to mediate formation of autophagosome, thus inducing complete mitophagy, which is critical for mavs degradation [167] . novel pa-x protein could also modulate innate immune responses. a review regarding the function of ns1 and pa-x proteins in antagonizing host innate immunity is recommended here [114] . though autophagy is essential for cellular metabolism and homeostasis, it also plays important roles in innate immune responses against pathogen infection. for cellular homeostasis, the mtor pathway is one of the most conserved autophagic pathways. the mtor complex 1 (mtorc1) negatively regulates the ulk1 kinase activity, thus affecting the autophagy induction [168] . c-jun n-terminal protein kinase 1 (jnk1) disrupts the bcl-2/beclin-1 complex through phosphorylation, thus regulating the autophagy induction [169, 170] . jnk1 is also reported to upregulate beclin-1 expression through phosphorylation of transcription factor c-jun in vitro [171] . in contrast to the autophagic pathways for cellular metabolism and homeostasis, less is known about autophagosome formation after iav infection [172] . to restrict infection of multiple viruses including iavs, trim23 is essential to mediate autophagy via its ring e3 ligase and adp-ribosylation factor (arf) gtpase activity [173] . beclin-1 and tufm-regulated autophagy also inhibited iav replication [112] . in hela cells and a549 cells, iav infection activated jnk1 to induce autophagosome formation and tgf-β-activated kinase 1 might contribute to the process [174, 175] . furthermore, autophagy was involved in maintaining memory b cells to counteract iav infection [176] . iav also utilizes autophagy to complete its life cycle. ns1 protein is proposed to suppress jnk1-mediated autophagy induction [174] . m2 could also block autophagosome maturation and mediate microtubule-associated protein 1 light chain 3 (lc3)-bound membrane redistribution, thus allowing filamentous budding of iav [177] [178] [179] . circ-gatad2a (gata zinc finger domain containing 2a), induced by iav infection, could inhibit autophagy and promote iav replication [180] . for a comprehensive reading on iav-induced apoptosis, a review is recommended here [181] . upon detection of iavs, dcs trigger production of ifns and cytokines, which in turns assist maturation of the dcs into antigen presenting cells (apcs), and initiate t cell immune responses. through the activation of ag-bearing dcs, naïve cd4 + t cells differentiate into th1, th2, th7, regulatory t cells (treg cells), follicular helper t cells, and killer cells. th1 and follicular helper t cells are the most abundant cd4 + t helper cells. they can secret antiviral cytokines, regulate cd8 + t cell differentiation, promote b cell activation, and maintain immunological memory [182, 183] . th17 cells induced pulmonary pathogenesis and could decrease mortality of iav-infected mice [184, 185] . in addition, γδ t cells, expanding in the late stage of iav infection with a t cell receptor (tcr)-independent viruses 2020, 12, 376 9 of 23 manner, could efficient eliminate iav-infected airway epithelial cells, resulting in lower viral titers [186] . new surrogate markers cd49d and cd11a were used to explore the kinetics of iav-specific cd4 t cells responses, revealing endogenous cd4 t cell response to primary iav infection is predominantly composed of t-bet+ cells [187] . cd8 + t cells are major components for virus clearance in adaptive immunity. after activated by dcs, cd8 + t cells undergo rapid expansion, differentiation, and migration to the infected sites. in general, to establish effective primary cytotoxic t lymphocyte (ctl) responses, cd4 + t cells play an essential role, with a mouse model as an exception [188] . ctls produce cytotoxic granules containing perforin and granzymes (gra and grb) to induce apoptosis and interrupt iav replication [189] . in addition, ctls produce cytokines, such as tnf, fasl, and trail, which recruit death receptors to induce apoptosis [190] . in addition, il16 deficiency enhanced the th1 and ctl responses upon iav infection [191] . furthermore, as cd8 + cells could last for two years in murine models, iav-specific memory ctls reacted specific to epitopes in conserved iav proteins [192] . in the nasal epithelia, they could prevent the spread of the virus from the urt to the lung [193] . to establish memory cd8 + t cells, autophagy plays an important role [194] , while the function of cd4 + t cells in memory ctl responses is "context-dependent". a recent study showed that cd4+ t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [195] . grant and her colleagues summarized and discussed the importance of cd8 + t cell immunity against iavs [192] , and this review is recommended for further reading. with the help of cd40 ligand (cd40l), cd4 + cells contribute to b cell activation [183] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [196] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blocking iav transmission [197] . in addition, iav-specific antibody-dependent cell-mediated cytotoxicity (cdcc) also plays a role in cross-protection against iav infection. a general description of adaptive immunity against primary iav infection is illustrated in figure 2 . antigenic shift and drift, resulting in reassorted and mutated ha and/or na, are responsible for aiv escaping from host immunity [50] [51] [52] . furthermore, additional glycosylation on h5 ha could also induce virus escape from neutralizing antibodies [198] . apoptosis represents programmed single cell death that occurs in cell physiological remodeling, cell proliferation, or immune response to invading pathogens [199] . besides prototypical changes, cells undergoing apoptosis can be detected through dna and biochemical assays, take the tunel and in situ end-labeling (isel) techniques for instance. two primary pathways are involved in activation of apoptosis: the intrinsic or mitochondrial pathway, and the extrinsic or death receptor pathway. the intrinsic pathway is also known as "the mitochondrial pathway", which operates in response to various intracellular stress. several factors such as nitric oxide (no), cytochrome c, and second mitochondria-derived activator of caspases (smac) can activate this pathway, and the key player of this pathway is proteins in the bcl-2 family, which are activated by stress signals and then release apoptotic factors via destabilizing the mitochondrial membrane [200, 201] , resulting in release of mitochondrial cytochrome c. cytochrome c then binds to apoptosis protease activating factor-1 (apaf-1) and forms a complex with pro-caspase 9 (then cleaved into caspase 9), the function of which is to cleave its effector pro-caspase 3 [202] . in addition, smac, localizing in the cytosol, could initiate activation of caspase 9 via blocking the activity of iap [203] . the extrinsic pathway is regulated by extracellular ligands acting on transmembrane "death receptors": the first apoptosis signal (fas) receptor-fas ligand (fasr/fasl) and the tnf-αtnf receptor 1 (tnfα/tnfr1) [199] . in the fasr/fasl model, fas ligand binds to its receptor fasr [204] , forming the death-inducing signaling complex (disc) with pro-caspase 8, resulting in activation of caspase 8 and downstream activation of other caspases (caspase-3, caspase-6, and caspase-7) [199] . in the tnfα/tnfr1 pathway, tnfr1-associated death domain protein (tradd) is activated after binding of tnfα to tnfr1, leading to recruitment of fadd and receptor interacting protein (rip) [205] . fadd then associates with pro-caspase 8 to form the disc, resulting in activation of caspase 8 and apoptosis. could prevent the spread of the virus from the urt to the lung [193] . to establish memory cd8 + t cells, autophagy plays an important role [194] , while the function of cd4 + t cells in memory ctl responses is "context-dependent". a recent study showed that cd4+ t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [195] . grant and her colleagues summarized and discussed the importance of cd8 + t cell immunity against iavs [192] , and this review is recommended for further reading. with the help of cd40 ligand (cd40l), cd4 + cells contribute to b cell activation [183] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [196] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blockin during iav infection, viruses modulate host apoptotic responses in a time-dependent manner [206] . for instance, in order to earn enough time for replication and virion formation, iav inhibited apoptosis via upregulating the anti-apoptotic phophoinositide-3-kinase-protein kinase b (pi3k-akt) pathway at the beginning of infection. however, in the later phase of infection, the virus suppressed this pathway to upregulate the pro-apoptotic p53 pathway, thus allowing successful release of virions [207] . several viral proteins are involved in regulation of host apoptosis. np protein induces host apoptosis to favor viral replication through interaction with ring finger 43 (rnf43) [208] , apoptotic inhibitor 5 (api5) [209] , or clusterin [111] . pb1-f2 also induced apoptosis and promoted viral replication through dysregulating mitochondrial potential [206] . furthermore, m1 promoted apoptosis by binding to heat shock protein 70, thus activating caspase and the subsequent apoptosis [210] . in addition, ns1 expression was reported to induce apoptosis in mdck and hela cells [211] . however, mutant iav lacking the ns1 gene could induce apoptosis in cultured cells [212] . the function of ns1 in inhibiting apoptosis may be explained by its ability to inhibit type i ifn [213, 214] . these data demonstrate sophisticated mechanisms of iav in regulating host apoptosis. furthermore, the role of these viral proteins in apoptosis suggests that these proteins may present suitable targets for anti-iav therapies. a comprehensive review on influenza a virus-induced apoptosis discussed by ampomah and lim is recommended here [181] . in addition, recent in vitro research found that apoptosis was induced at early iav infection stage, while later the cell death pathway was shifted to pyroptosis. the switch process was promoted by the type i ifn-mediated jak-stat signaling pathway through expression of the bcl-xl gene [215] . during iav infection, multiple immune systems coordinate together to protect the host. accordingly, viruses antagonize the immune system through multiple measures to establish a successful infection. considering the high frequencies in genome mutations and recombination, vaccination is the most effective way to defend against the viruses via inducing cross-protective antibodies and/or enhancing immune responses. several studies in vaccine development have tried to enhance host immune responses. for instance, vaccine candidate containing ha targeted to chemokine receptor (porcine mip1α) was shown to enhance t cell responses, resulting in a strong and cross-reactive cellular immunity in vaccinated pigs [216] . another example is an attempt to intranasally administer a polyanhydride nano vaccine (iav-nanovax), which could promote robust lung-resident germinal center (gc) b cells with lung-localized iav-specific antibody responses as well as lung-resident memory cd4 + and cd8 + t cell responses [217] . for anti-iav drugs, currently, na inhibitors (relenza tm and tamiflu tm ) are applied clinically as anti-influenza drugs [218] . these drugs inhibit the activity of na by preventing viral budding [21] . in addition, cap-dependent endonuclease inhibitor (baloxavir marboxil) targeting pa is also applied against influenza a and b virus infection [219] . our progressing understanding of the iav life cycle of the virus and iav-host interaction could contribute to anti-influenza drug design. since the recognition of ha protein to sa linked glycoproteins is the first step in iav infection, effective blocking of the interaction between viral ha and sa receptor serves as a favorable target in drug design [220, 221] . favipiravir, a nucleotide analogue that selectively inhibits the rna-dependent rna polymerase, is licensed in japan to be applied against emerging influenza viruses resistant to other antivirals [222, 223] . oleanolic acid (oa), a kind of pentacyclic triterpene natural product, and its analogues, as well as its derivatives, were shown to bind to ha, thus blocking the attachment of iavs to mdck cells [224] [225] [226] . pvf-tet is a peptide-based ha inhibitor, which was shown to sequester ha into amphisome (fusion of late endosome with autophagosome) and protected mice from the lethal iav infection [227] . new effective drugs targeting the polymerase would be a promising strategy against iav infection, since they would directly reduce or eliminate viral replication. numerous sites, including the cap-binding site [228] , the endonuclease [229, 230] , and pa-pb1 inter-subunit interface [231] can serve as potential targeting sites for new drug design. coumarin compounds, including eleutheroside b 1 , isofraxidin, fraxin, esculetin, fraxetin, and scoparone, were investigated for their antiviral and anti-inflammatory activities against influenza virus in vitro [31] . other candidates, such as naproxen, a non-steroidal anti-inflammatory drug, was shown to target np protein at residues f209 and y148, thus antagonizes the crm1-mediated nuclear export of np. it is suggested to have a broad-spectrum anti-influenza activity [232] . verdinexor (kpt-335), a novel orally bioavailable drug, blocks crm1-mediated nuclear export of np and repress nf-κb activation, thus reducing cytokine production and eliminating virus-associated immunopathology [233] . for further reading on candidate anti-iv therapeutics, a review summarized by davidson is recommended here [234] . with the increasing knowledge obtained through massive investigations on host immunity against iav infection, promoting host immune responses not limited to antibody enhancement would have good prospects not only for vaccine design, but also for development of novel antiviral agents. author contributions: manuscript preparation, y.z.; revision, z.x.; supervision, y.c.; funding acquisition, y.z. all authors read and approved the final version of the manuscript. funding: this study was supported by the "zhujiang talent program" overseas youth talent introduction program (post-doctoral program) and doctoral initiative project of natural science foundation of guangdong province (18zxxt49). the authors declare that they have no financial and personal relationships with other people or organizations that can influence the work. there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in this review. they do not have any commercial or associative interest that represents conflicts of interest in connection with the work submitted. influenza virus aerosols in the air and their infectiousness influenza: the once and future pandemic identification of a novel viral protein expressed from the pb2 segment of influenza a virus an overlapping protein-coding region in influenza a virus segment 3 modulates the host response identification of novel influenza a virus proteins 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viruses treating influenza infection, from now and into the future key: cord-009577-29u7pdpk authors: gonzalez‐scarano, f.; tyler, kenneth l. title: molecular pathogenesis of neurotropic viral infections date: 2004-10-08 journal: ann neurol doi: 10.1002/ana.410220502 sha: doc_id: 9577 cord_uid: 29u7pdpk classical virologists defined a number of viruses that affect the nervous system and identified tissue tropism, extraneural replication, and viremia as important parameters that determine whether viral infections will affect the central nervous system. molecular techniques are expanding this knowledge by permitting us to relate specific genes and gene products to two defined phenotypes: neuroinvasion and neurovirulence. two converging situations make this knowledge particularly useful: (1) the development of antiviral drugs and subunit vaccines, which mandate that pathogenesis be related to specific regions of the viral genome; and (2) the expanding problem of central nervous system infections in immunodeficient states. the recent revolution in the biological sciences has had a tremendous impact on virology, which benefited early from the technological advances in molecular biology. viruses are now used to study gene expression and receptor-ligand interactions and to express cloned genes in eukaryotic systems. it has nevertheless remained easier to study viral infections in vitro rather than viral-host interactions in whole organisms. recently a number of significant insights into the molecular and genetic basis for the pathogenesis of viral infections of the nervous system have been reported by different groups. in this review we will highlight some of the major findings in the field. viral pathogenesis is the process by which a virus causes disease in a susceptible host. pathogenesis can be analyzed in terms of a series of stages (see 132) and 154) for reviews). to cause systemic illness, a virus must first enter the host animal, undergo primary replication at a site near its portal of entry, and then ultimately spread to distant target tissues, such as the central nervous system (cns). by definition, all animal viruses are intracellular pathogens, and the process of replication must commence with entry into a susceptible cell. an infecting animal virus faces two main blocks to penetration of the cns or any other specific target organ: (1) a variety of barriers prevent the free access of viruses to target cells, and (2) even when these barriers are ineffective, only certain cell types will support the internalization and replication of a particular virus. it is useful to think of the capacity of a virus to establish a lethal infection within the cns as the property of neumirulence and the ability to penetrate the cns after inoculation and growth at a peripheral site as the property of neuroinvasiveness. experimentally, it is easy to bypass the barriers to cns infection (by intracerebral inoculation, for example), and therefore each of these properties can be tested for independently. a major goal of much of the current work in virology has been the correlation of viral properties such as neurovirulence and neuroinvasiveness with specific viral genes or proteins or, when the systems have been more advanced, with specific regions of these genes andor proteins. each of the potential entry routes into the organismskin, mucosal membranes, gastrointestinal (gi) tractis utilized by viruses capable of eventually infecting the cns. the specific entry point will be determined by the biology and physicochemistry of the virus as well as by the need for vectors like mosquitoes, ticks (arboviruses), or mammals (rabies). for a number of neurotropic viruses, the physical barrier provided by the skin is breached by the bite of an animal or arthropod vector, or through the use of contaminated needles or other foreign bodies. many other neurotropic viruses enter the host via natural portals such as the respiratory and gi tracts. entry via these routes may be direct (via contaminated saliva, for example) or through aerosols. the molecular mechanisms that influence the capacity of viruses to become aerosolized and their subsequent stability are unknown. most nonenveloped viruses (poliovirus, reovirus) appear to lose infectivity in conditions of low (< 50%,) relative humidity. for example, aerosolized poliovirus shows a lo3-fold drop in infectivity when kept for 1 hour at 15 % relative humidity (when compared with 72% humidity) {58]. conversely, the infectivity of aerosols of most enveloped viruses is not notably altered by changes in the relative humidity, although in general enveloped viruses are more sensitive to environmental inactivation. the g i tract presents formidable chemical barriers to the entry of viruses. the mechanisms by which agents such as those present in the gi tract (e.g., acids, bile salts, proteolytic enzymes) affect viral structure, and hence determine the ability of a virus to survive transit through the local environments present at entry sites such as the gi tract, are gradually becoming understood. in the case of nonenveloped viruses, the outer capsid proteins appear to be the major determinants of viral stability to a wide variety of physicochemical agents. for example, rhinoviruses are rapidly inactivated at acidic p h levels, whereas other picornaviruses, such as polio, are not. one practical consequence of this is that rhinoviruses do not survive transit through the stomach and hence rarely produce disease outside the limited confines of the respiratory tract. exposure of rhinoviruses to an acidic p h results in the loss of the viral capsid protein v p 4 . this in turn produces a "hole" in the virus particle through which the viral nucleic acid leaks out, leaving a noninfectious "empty" viral capsid. interestingly, the same type of structural alteration can occur in polioviruses exposed to certain adverse physical conditions, which again emphasizes the role of the viral capsid in determining virion stability [45, 731. another example of the role of outer capsid proteins in determining viral stability can be seen in the reoviruses. these viruses, like the enteroviruses, are neurotropic viruses whose natural portal of entry is via the gi tract. the three serotypes of reovirus show profound variations in their in vitro sensitivity to a number of physicochemical agents, including temperature, ph, alcohol, and detergent solutions. studies using reassortant viruses containing genes derived from "sensitive" and "resistant" serotypes of virus (see fig. 2 for an analogous experiment) allowed specific genes to be identified and associated with particular phenotypes. sensitivity to alkahne p h and guanidine mapped to the viral s 1 gene, sensitivity to high temperature and the detergent sodium dodecyl sulfate mapped to the s4 gene, and sensitivity to phenol and ethanol mapped to the m2 gene. in each case these genes encode proteins that are components of the outer cap-sid of the virus, indiccating again the importance of these proteins in determining susceptibility to environmental conditions [17., 181. just as they differ with respect to sensitivity to p h changes, viruses vary in their response to proteolytic enzymes. some viruses are extremely sensitive to trypsin-foot and mouth disease virus demonstrates a 103 drop in infectious titer upon treatment with it-and others, like influenza, need the effects of a trypsin-like protease to become infectious e23, 411. the human rotaviruses, which are enteric pathogens, show dramatic enhancement of infectivity in the presence of trypsin, which cleaves the v p 3 outer capsid protein 1691. reoviruses also differ in their sensitivity to proteolytic digestion with enzymes such as chymotrypsin [4, 343. genetic studies have shown that the viral m 2 gene, which encodes an outer capsid polypeptide (mlc), is responsible for determining sensitivity or resistance to proteolysiij by chymotrypsin [66] . the effects of proteolytic enzymes on the infectivity of ortho-and paramyxoviruses have been investigated extensively. in the case of influenza virus, a trypsin-like protease present in host cells cleaves the hemagglutinin protein into two smaller peptides, ha1 and ha2, held together by a tlisulfide bond. the cleavage and subsequent removal (of an arginine residue exposes the hydrophobic n h 2 terminal of ha2, which is essential for the fusion function of this virus (see below) [23}. if influenza virus is grown in cells that lack this peptidase activity, h a cleavage does not occur, and, though the virus still binds to cellular receptors, it is not infectious [20, 41, 67, 681. enveloped viruses-those that incorporate a lipid bilayer on their out'er surface-are particularly sensitive to inactivation b y bile salts, which dissociate the lipoprotein components of the viral envelope. this may explain why enveloped neurotropic viruses only rarely enter the h0:jt via the gi tract (the coronaviruses, which include the neurotropic mouse hepatitis virus, are a known exception to this rule). the neurotropic viruses that commonly use the gi portal of entry are all nonenvel.oped and therefore insensitive to the action of bile salts. many viruses, such as those that infect the gi and upper respiratory tracts, attack target cells that are near the point of entry. for those agents, infection of contiguous cells satisfies the requirement of their life cycle. except in special circumstances 131, viruses that infect the cns rnwt reach it after entering the body at a distant site. in general, this means primary replication must occur in a target cell outside the cns, and then the virus must reach the cns by either the bloodstream (hematogenous spread) or via nerves (neural spread). it is (1) cns target cell, (2) the efficiency of spread after the primary infection of these target cells, and (3) the success of the host's immune response that determine whether a virus will be neuroinvasive. incidentally, under most circumstances this combination of factors reduces the incidence of successful cns infections, making these a small proportion of the total number of infections caused by viruses, even those that are traditionally considered neurotropic. most neurotropic viruses reach their target tissue through the bloodstream. arboviruses, for example, are inoculated directly into the subcutaneous tissue or bloodstream, then replicate in extraneural tissues such as muscle or regional lymph nodes 148, 52). figure 1 is a schematic diagram of the spread of an arbovirus from the blood to the nervous system. following primary replication, the virus is released into the bloodstream, where it is transported in the plasma. for a viremia to serve as a source of dissemination of virus to target organs, it must be of adequate magnitude and duration. studies with a variety of arboviruses have shown that the degree and persistence of viremia is directly related to the ability to penetrate the cns c30, 52). the viruses of the california encephalitis group, which are transmitted by mosquito species endemic to the midwestern united states and to upper new york state, are responsible for the majority of the cases of arboviral encephalitis in this country. their rna genome is segmented (a quality they share with the influenza viruses, arenaviruses, and rotaviruses and reoviruses), and different "pieces" can be recombined artificially in the laboratory. by taking genomic segments from neuroinvasive and noninvasive strains, one can ask the question: which genome segment is responsible for the ability to penetrate the cns? figure 2 summarizes such an experiment, in which viruses representing neuroinvasive and noninvasive strains were used to coinfect cells. "reassortant" viruses, containing genomic elements from two different strains, were thus generated and then tested for their ability to penetrate the murine cns. using this approach, it has been determined that the gene coding for the envelope glycoproteins cosegregates with the phenotypic property of neuroinvasiveness, although the genes coding for the other structural proteins of the virus can modulate this effect 13 1). the same line of reasoning can be extended by using viral mutants that have changes in a single protein product. one frequently used approach, primarily with rna viruses, is the selection of antigenic variants with monoclonal antibodies c26, 39, 71, 72) . in this approach, a virus mutant present in the initial stock at low concentration is amplified by neutralizing the wildtype virus with a monoclonal antibody (fig 3) . the resultant virus variant contains a mutation in the protein against which the neutralizing antibody is directed, which in most instances is the protein responsible for attachment to cellular receptors. that mutation can usually be mapped to one or two amino acids which, being altered, prevent binding of the mutant virus by the monoclonal antibody. monoclonal antibody variants have been used to map the antigenic sites of the influenza hemagglutinin 122, 76, 771 and have been used successfully to define important regions of the cellular binding proteins of rabies virus, reovirus, coronaviruses, and the california serogroup-all cns pathogens. antigenic variants of the california serogroup obtained with a single monoclonal antibody have altered neuroinvasiveness; if injected directly into the cns their pathogenesis is not altered. thus, the property of neuroinvasiveness can be specifically associated with a single protein in this system 1261. by sequencing the genome of the variant virus and comparing this sequence with that of the wild-type parent, it may be possible to show that the property of neuroinvasiveness is associated with specific regions within the glycoprotein molecule, as has been done in other systems f34). besides arboviruses, other agents, like poliovirus, also use the bloodstream as their primary route to the nervous system. in polio the organ of primary repli-cation is the gut, yet it is release into the bloodstream that determines whether the characteristic myelitis will occur. in addition to plasma viremia, some viruses, including human irnrnunodeficiency virus (hiv), are transported in the bloodstream in association with lymphocytes or macrophages f2, 371. following hemsttogenous spread, neuroinvasive agents penetrate the cns through the choroid plexus or through the endothelial cells. the lack of endothelial or choroid plexus tissue culture systems has made it difficult to analyze .in detail the molecular mechanisms of penetration of these tissues. classic investigations by pasteur and colleagues on rabies virus, and b,y goodpasture and colleagues on herpesviruses clearly established that neurotropic viruses could spread to the cns via nerves [32] . more recent investigations with a number of viruses including rabies, polio, and herpes have provided abundant confirmation and more detailed insights into the process of neural spread 112, 48, 50, 51, 751 . however, until recently it wm not possible to identify the role of specific viral genes in influencing the capacity of viruses to spread through nerves. in the case of rabies virus, after an initial period of replication in skeletal muscle, the virus concentrates at the neuromuscular junction in close proximity to neuromuscular and neurotendinous spindles. the virus then enters nerve terminals in proximity to these sites and is transported to the dorsal root ganglia and spinal cord [so, 511. rabies virus mutants with alterations in the virion glycoprotein (selected with monoclonal antibodies, as described for california encephalitis virus) appear to have altered capacity to enter certain types of nerve fibers 113-161. these findings suggest that the rabies glycoprotein may play an important role in viral entry into and transport within cells. reovirus type 3 has also been shown to spread from peripheral tissue tc) the cns via nerve cells. furthermore, the microtubule-associated system of fast axonal transport has been implicated in this spread, because experimental infection of the cns following footpad inoculation of mice is inhibited by low concentrations of colchicine. selective inhibitors of slow axonal transport had no effect on spread to the cns {751. like the california encephalitis viruses, reoviruses have a segmented genome (double-stranded rna in this case). experiments similar to the one outlined in figure 2 can be used to generate reassortant viruses. unlike type 3 reovirus, type 1 reoviruses spread to the cns via a hematogenous route. tyler and collaborators exploited this fact to test reassortants of type 1 and type 3 viruses and again ask the question: which gene segmeiit accounts for the different patterns of spread in these two strains? these reassortant viism, because they confer resistance to proteolysis, dehydration, and p h changes. in most instances, this has been determined through a combination of genetic methods, usually involving either reassortant viruses or selected antigenic variants. in the next section we discuss the role of these proteins in determining tropism at the cellular level. ruses, containing gene segments from both type 1 and type 3 viruses, have clearly shown that the property of spread can be associated with the gene that codes for the sigma 1 polypeptide-an outer protein that functions as the viral hemagglutinin and cell attachment protein. additional insights into the molecular basis for neural spread have recently come from the study of herpes simplex virus recombinants. for these experiments, two strains (hsv-1, 17; hsv-2, 186), which differ in their ability to spread to the mouse cns after corneal inoculation, were utilized to generate recombinant viruses [55] . the region of the hsv-1 genome which codes for a number of proteins including the gb glycoprotein, a nucleocapsid protein (p40), and a dna-binding protein (icp-8), as well as the dna polymerase, was found to be important in the spread of viruses from cornea to cns. in summary, the available evidence all points to the conclusion that, in several different viral systems, the envelope proteins (in the case of enveloped viruses) or the capsid proteins (in the nonenveloped agents) are the major determinants of spread to the nervous system. the outside proteins naturally play a very important part in other aspects of penetration into the organthe process of entry of viruses into typical mammalian cells is outlined in figure 4 . experimentally and biologically it can be divided into two main steps: (1) binding to the plasma membrane, and ( 2 ) penetration and uncoating. many of the concepts regarding virus binding to plasma membranes have developed from the pioneering work of brown and goldstein on the low-density lipoprotein receptor {zs}. in this model, macromolecules destined for the cytoplasm first interact with cellular receptors. the interaction with these receptors is quite specific and accounts for the restriction of unwanted macromolecules from the cells. viruses bind to the plasma membrane of susceptible target cells through specific receptors which may be proteins (hiv), lipids (vesicular stomatitis virus), or contain sialic acid (reovirus, influenza) [21, 641. in some instances, neurotropic viruses have been thought to bind to pharmacological receptors. this has been best demonstrated for reovirus type 3, which in some tissues appears to bind to the beta-adrenergic receptor [9, lo] , but it has also been suggested for rabies virus, which may use the acetylcholine receptor as its entry point [43, 5 11 . to a large extent, the specificity of this virus-receptor interaction can dictate the pathogenesis of a particular agent. a topical example is the virus that causes the acquired immunodeficiency syndrome (aids), now termed hiv c21. this virus binds to the t4 molecule, a membrane protein of unknown function which defines a particular subclass of lymphocytes many viruses can infect cells if their nucleic acid genome is introduced directly into them-in the absence of any viral polypeptides. introduction of infectious dna clones has been used to bypass cellular receptors in infections with hiv. in such an experiment a variety of cell types can be infected, not necessarily only those containing the appropriate membrane receptor [ 17. another well-known receptor-virus interaction involves the influenza hemagglutinin, which binds to receptors containing sialic acid. unlike the hiv system, where knowlege of the interaction between the receptor and the virus is still at a descriptive stage, the relationship of the influenza hemagglutinin to its receptor has been characterized to the single amino acid level, because the molecular structure of the influenza hemagglutinin has been determined through x-ray crystallography c76, 77) . the hemagglutinin molecule is present as a trimer on the virion surfaces. this trimer contains a "pocket" that appears to function as the site of attachment of the receptors present on susceptible cells. viruses selected from different hosts (avian and equine, for example), which have naturally different enzymatic activities and slightly different sugar residues on their sialic acid molecules, have variations in the amino acids surrounding this pocket [6, 641. thus, even though the target cells are presumably the same, small changes in the receptor-binding site of influenza virus may account for species specificity. different technology has been used to identify the receptor-binding site of rhinoviruses. the large number of rhinovirus serotypes has made production of a general vaccine impractical. the majority of serotypes use a single specific receptor present on the surface of infectable cells. this receptor was initially identified through the use of a polyclonal antibody, then by monoclonal antibodies ell]. the virus itself has subsequently been crystallized c651, and a large cleft or "canyon" present on each icosahedral surface is thought to be the receptor-binding site, by virtue of its relationship to antibodies capable of neutralizing the virus. drugs are now being designed to block this cleft. although rhinoviruses are not neurotropic, they form part of the same family (picornaviridae) as poliovirus, and many of the features of the viral topology are being extended to that group 1471 as well as to footand-mouth disease c71 and theiler's virus, other members of the picornaviridae. in summary, a necessary condition for the entry of viruses into all cells, and nerve cells in particular, is the availability of specific receptor molecules on the sur-face of such cells. some receptors are undoubtedly present in all cns cells, and viruses that utilize such may cause a generalized infection, or panencephalitis. in the cns, the pattern of illness a virus produces is determined in large part by the specific regions of the brain that are infected and by the population of cells in the affected regions that are injured or destroyed. some viruses, like jc papovavirus, which is responsible for progressive multifocal leukoencephalopathy, infect astrocytes an'd oligodendroglia while largely sparing neurons, whereas herpes simplex infects all cell populations. striking differences also occur in the topographical distribution of viral injury to the cns. rabies virus causes pathology in the cerebellum, hippocampus, and limbic areas, whereas polio affects the motor nuclei in the cortex, brainstem, and anterior horns of the spinal cord c32, 501. many of these specific topographic ;associations have been postulated to be the result of specific receptor-binding interactions. after a virus has bound the appropriate receptors, the process of internalization can proceed in one of two ways: (1) viruses can fuse with the plasma membrane, discharging their contents directly into the cytosol, or (2) viruses may be internalized through the endocytotic pathway. fusion at the plasma membrane has been observed for viruses like herpes simplex and coronaviruses, both of which are capable of forming giant syncytia during routine infection. presumably there are no special requirements for internalization under these circumsi:ances, and if the appropriate receptors are present the virus will appear within the cell. other viruses are not capable of fusing directly with the cell membrane, and for those the endocytotic pathway (outlined in figure 4 ) is the method of internalization. this pathway, which again resembles the mechanism of interrlalization of low-density lipoproteins [2 51, involves ithe collection of receptor-ligand complexes at regions of the membrane characterized by electron-dense material at their cytoplasmic side. the viruses are then transported within endocytotic vesicles, which have been determined to have a mildly acidic ph 142, 461. for many enveloped virusesthose viruses that inc:orporate a lipid bilayer into their structure-it has been shown that exposure to such a low p h leads to alterations in the conformation of the envelope proteins c681. again using the influenza hemagglutinin as a model, such a change probably brings to the surface stretches of hydrophobic amino acids normally buriecl within the viral protein. the hydrophobic residues interact with the membrane of the endosome, fusing the viral envelope to it and releasing the contents of the virus into the cytoplasm, where the process of replication can then begin. in influenza and related viruses, a host protease is responsible for the cleavage step that prepares this portion of the hemagglutin for this exposure (from a precursor protein). this proteolysis is essential for replication and accounts for some of the host specificity of the viruses, as noted above. in fact, epidemics of influenza in avian rpecies have been ascribed to the ease of proteolysis in different strains. for nonenveloped viruses (poliovirus, rhinovirus), there is also evidence that a ph-dependent step may be important in infection, but the process has not been observed clearly, as it has been in the enveloped agents. the degree of acidity that can elicit the changes in the viral proteins necessary for fl5ion to occur varies for different viruses. because of the inherent difficulties of such measurements, the endosomal p h has not been determined for a large number of cell lines (and for none of the cns cell lines). however, one can easily theorize that the relationship between the viral requirements and the acidity of these cellular organelles must influence the potential for infection of a particular cell type. in fact, the ability to fuse has been correlated with the encephalitogenic potential of bunyaviruses and mumps virus strains 126, 78) . once the contents of the viral envelope (genome and proteins necessary for replication) have been discharged into the cytosol, the process of viral replication can begin. it is likely that for most nononcogenic rna viruses the specificity of the virus-target interaction is determined prior to this step. an efficiently replicating virus, like vesicular stomatitis virus, for example, is probably capable of initiating replication of its genome in any cell line that it can penetrate. however, not all proteins or genome segments may be reproducible in any given system, and for lymphocytic choriomeningitis virus it appears that restriction at the segment level may be responsible for the outcome of infection {621. for many dna viruses like herpes simplex and varicella-zoster, persistence is clearly associated with the arrest of replication. such mechanisms are highly specific for different families. recently the role of regulatory signals in the enhancement of replication has become a prominent area of investigation. signals within the viral genome that stimulate gene transcription or translation appear to play an important role in determining viral host range and tissue tropism in certain systems 1191. these signals, variously termed enhancers, promoters, and transactivators depending on their position and orientation, are capable of stimulating the transcription of genes. some of these enhancers determine tissue tropism (for example, in polyoma virus), whereas others are active in all tissues (simian virus {svi-40, herpes simplex) {38, 40, 61, 701. transcriptional activators are genes whose products can activate the transcription of other genes. the viruses associated with aids have prominent transactivating activity, and they may also have regulatory activities at posttranscriptional events. in any case this kind of "tat" activity can greatly amplify the production of viral gene products and may be responsible for increased cytopathological changes. in some instances it may be responsible for host specificity. tat activity had previously been shown to be an important feature of sv-40 1357, an experimentally important virus that is related to jc virus, the etiological agent for progressive multifocal leukoencephalopathy. this kind of activity may be important in the selectivity that this virus shows for glial cells and could play a role in a relationship between hiv and jc. in several experimental models, the relative contribution of jc virus regulatory sequences in conferring tissue specificity has been studied (8, 361. it has been argued that the jc virus promoter may contribute substantially to neurovirulence. outcome of infection the final determinant of pathogenesis is the outcome of infection of the host cell. four major pathways exist: (1) death of the cell; ( 2 ) alteration of its growth pattern and change into a cancerous phenotype (transformation); (3) persistence of infection without obvious cellular change; and (4) persistence of infection with alteration of specialized cellular functions (luxury functions). neurotropic viruses can be responsible for any of these final results. transformation is a highly intricate subject and will not be discussed further. interested readers should refer to general reviews (27, 441. cytopathic effect significant enough to stop cellular metabolic activity is obviously the most common outcome of viral infection in all acute and some subacute viral infections. for the most part, morphological evidence has been utilized as the primary way of determining cell death. at a molecular level, mechanisms of viral cytopathology include the inhibition of production of host cell dna, rna, or protein. for some rna viruses it is known that interference with the transfer of a methylguanosine "cap"-m'gpppn-to host messenger rna (mrna) occurs in order to direct the host machinery to translate viral proteins preferentially. for influenza viruses and bunyaviruses (california encephalitis), the stolen cap is essential for transcription of the viral rna to proceed efficiently [57, 59}. except for these and a few other examples, the specific virally mediated mechanism for inhibition of cellular metabolism is unknown. much has been written about the role of the host immune response in mediating cell death. the immune response is frequently capable of harming the host instead of protecting it from viral infection as has been clearly demonstrated for a variety of neurotropic agents, including rabies, lymphocytic choriomeningitis virus, coronaviruses, and others (for review, see 153)). there has been a recent surge of interest in the role of viruses in inducing cell membrane molecules involved in the recognition of foreign antigens. these h-2 antigens are generally expressed at a low level in neural cells, but they increase in the presence of certain viral infections and interferons 1741. it has been postulated that the presence of these antigens on neural cells may make them a better target for cellular immunity directed at viral components and thus increase the possibility of virally triggered immunopathology. although this work is clearly just beginning, the availability of nucleic acid probes for the mrna's coding for these antigens should make it possible to study potential immunopathological mechanisms at a molecular level. similarly, the wealth of information now available about the t-cell receptor molecules {27) should allow exploration of the genetic correlation with immunopathological diseases. because of the complexity of the work involving virally induced immunopathology, we will not discuss it further here. particularly when dealing with clinical specimens, it can be very useful in terms of analyzing the state of the latent viral dna in experimental infections. although considerable effort has been spent on this question, to date there is no clear indication whether herpes simplex virus is integrated into host dna or is present as an "episome" in latently infected ganglia [51. work by fraser and co-workers indicates that the dna is present in a form distinct from that of the isolated virions 147, and personal communication). similar techniques can also be used to demonstrate that varicella-zoster virus is present latently in human dorsal root ganglia, and probably reactivated to cause zoster dermatitis 1241. this particular herpesvirus has not been cultured in explants of such ganglia. when the histological features so allow, the particular cell type that is latently infected may be identifiable-in varicella-zoster virus the genome is thought to be harbored exclusively by neurons of the dorsal root ganglia. because of the lack of a suitable experimental animal, the state of the varicella-zoster virus latent genome is even less known than that of herpes simplex virus. persistent infection with alteration of lux-ury functions. oldstone and co-workers have recently introduced the concept that some persistent viral infections may lead to the alteration of cellular function without any morphological change. in young mice infected with lymphocytic choriomeningitis virus, changes of growth and glucose metabolism are associated with persistent infection of the pituitary 156). such effects are subjelct to significant variation, depending on the age and strain of the mice as well as the viral strain. however, when present, this persistent infection of the mouse cns is associated with the absent expression of the viral glycoproteins. the synthesis of these glycoproteins may be decreased, allowing the virus to escape detection by the cellular immune mechanisms. summary a variety of factors affect the ability of viruses to infect cells within the cns. neurotropic viruses must penetrate the external barriers that protect the organism, replicate in an appropriate peripheral site, and then spread through either the bloodstream or through nerves to the cns itself. genetic analysis in several systems has pointed to lrhe external proteins as the main determinants of neuroinvasiveness and neurovirulence. however, other factors, including host enzymes, activating factors, and the replicative machinery of the virus itself, must come into play. ultimately, the balance between the viral machinery and the host immune system will determine the outcome of any infection. supported by tida ns007 17 (to f.g-s.) and physician-scientist award a100610 (to k. l. t.) and by the william p. anderson foundation. f.g.4. is a harry weaver neuroscience scholar of the national multiple sclerosis society, and k. l. t. is an alfred p. sloan research fellow. we thank neal nathanson for his helpful comments. presented in part at a conference sponsored by the american society for neurologic investigation, chicago, il, october 1, 1985. production of acquired immunodeficiency syndrome associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone isolation of 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influenza hemagglutinin and their involvement in antigenic variation structure of the hemagglutinin membrane glycoprotein of influenza virus at 3a resolution. nature 289366-373 virulence and persistence of three prototype strains of mumps virus in newborn hamsters key: cord-024651-578c9ut5 authors: nan title: 2020 cis annual meeting: immune deficiency & dysregulation north american conference date: 2020-05-11 journal: j clin immunol doi: 10.1007/s10875-020-00764-z sha: doc_id: 24651 cord_uid: 578c9ut5 nan abstract/case report text oral lichen planus (olp) is a t-cell mediated chronic inflammatory tissue reaction in which presentation can range from asymptomatic plaques to painful, erosive, bullous, or ulcerative lesions. here, we present a 15 year-old female with a novel ctla-4 variant, multiple autoimmune conditions, and unusual tongue lesions. our patient was healthy until 9 years of age when she developed hashimoto's thyroiditis. at 11, she developed psoriasis. at 13, she was diagnosed with alopecia totalis and epstein-barr virus (ebv) with resultant and persistent anemia, thrombocytopenia, lymphopenia and neutropenia. she had chronic abdominal pain and diarrhea since age 13. esophagogastroduodenoscopy revealed lymphocytic esophagitis and active duodenal inflammation with increased intraepithelial lymphocytes. colonoscopy revealed mildly active chronic colitis with eosinophils. whole exome sequencing revealed a heterozygous c.239dela (p.q80rfs*2) pathogenic mutation in exon 2 of ctla-4. family history is remarkable: father (splenomegaly and psoriasis) and brother (autoimmune hemolytic anemia) have ctla4 haploinsufficiency with the same mutation. abatacept was initiated with re-growth of hair, improvement in cytopenias, improvement in psoriasis, and some reduction of gastrointestinal symptoms. since her abdominal pain persisted repeat endoscopies after six months of abatacept revealed persistent active lymphocytic esophagitis with some improvement in inflammatory injury in her duodenum and colon. physical exam revealed glossitis with a gel-like coating and ulceration on her tongue, xerosis along her face and scalp without other abnormalities ( figure) . she denied recent dental procedures, appliances, or tongue biting. her wbc ranged from 3-4 x10^9 cells/l and hemoglobin 9.4-12.7 g/dl. absolute lymphocyte count ranged from 1.0-1.7 x10^9 cells/l. immunologic evaluation revealed low iga and pan-low lymphocyte subsets (table) . ebv pcr ranged from 430-1,700 copies/ml. tongue scraping revealed candida dubliniensis and she responded to 5 days of fluconazole. two months later, she developed painful white patches along her tongue and subsequent 4 kilogram weight loss recalcitrant to viscous lidocaine, antacids, and 14 days of fluconazole. incisional tongue biopsy revealed ulceration with underlying granulation tissue with lymphocyte and plasma cell infiltration consistent with olp ( figure) . periodic acid-schiff diastase stain and grocott stain were negative. aerobic culture was normal. no fungus was isolated within 14 days. epstein-barr encoding region in situ hybridization was negative. two weeks of topical dexamethasone lead to temporary improvement. her tongue lesions waxed and waned over the following months. due to persistent psoriasis, methotrexate was initiated without worsening in her tongue lesion. to our knowledge, this is the first case of olp reported in a patient with ctla-4 haploinsufficiency. ctla-4 haploinsufficiency may present with variable clinical phenotypes including increased risk of ebv viremia and malignancies. therefore, after ebv and malignancy are ruled out, olp may be a prudent diagnosis to consider in a ctla4 insufficient patient with unusual oral lesions. mutations of bcl6b appear to be associated with lymphoproliferation and autoimmunity as well as susceptibility to herpes virus infections. additional research focusing on characterization of dna binding sites of bcl6b as well as the downstream expression of associated target genes is needed. these data combined with longitudinal analysis of additional patients with confirmed bcl6b mutations, will help clarify determinants of bcl6b pathogenesis and highlight potential therapeutic strategies. consanguineous marriages in tribal cultures, such as that in the united arab emirates significantly increase the prevalence of autosomal recessive disorders. premarital genetic screening and counseling, thus, are expected to reduce the frequency of these diseases. in this pilot study, diagnostic exome sequencing was used in the premarital screening program to identify recessive pathologic variants preventable by premarital counseling. a total of 487 pathologic or likely pathologic variants were identified in 176 studied emiratis (88 couples), averaging 2.8 variants per person. four percent of the persons had negative diagnostic exome sequencing; the remaining had one to eight variants per person. of the 351 distinct variants, 162 (46%) were novel. twenty (23%) couples had pathologic or likely pathologic variants of inborn errors of immunity (iei). two couples (10%) had iei pathologic or likely pathologic heterozygous variants in both partners imposing risk for autosomal recessive disease in the offspring. other eighteen couples (90%) had pathologic/ likely pathologic heterozygous variants present in only one person of the couple. total of sixteen (4.5%) iei variant identified and eight (50%) were novel. fourteen known phenotypic iei diseases were recognized (table. 1 ). these preliminary results support a need for nationwide premarital genetic screening, and primary immunodeficiency registry to identify common and novel pathogenic variants with high heritability rate. these results will aid adopting a preand post-connectional reproductive carrier counseling to reduce autosomal recessive diseases. also, it will assist the diagnosis of these complex diseases in our community. table 1 pathologic or likely pathologic variants of primary immunodeficiency (pid). abstract/case report text background: chronic granulomatous disease (cgd) is a primary immunodeficiency disorder caused by defects in the phagocytic nadph oxidase complex, leading to increased susceptibility to infection and inflammatory or autoimmune disease. up to 50% of patients have gastrointestinal (gi) involvement and meet diagnostic criteria for inflammatory bowel disease (cgd-ibd). objectives: we analyzed cgd patients from the united states immunodeficiency network (usidnet) registry to determine whether ibd may change the presentation, treatment, and outcomes of cgd patients, as compared to those without ibd. methods: a retrospective evaluation of cgd cases from the usidnet registry was completed. cgd-ibd was defined as the presence of any major physician-reported inflammatory, non-infectious gi tract disease manifestation, including crohn disease, ulcerative colitis, ibd endoscopy findings, gi fistulas, gi strictures, gi obstruction, and proctitis. demographic information, genotypes, symptoms and conditions, infections, antimicrobial therapies, immunomodulator use, and allogeneic hematopoietic stem cell transplantation (hsct) data were analyzed. results: 194 patients with a diagnosis of cgd were identified. 96 met criteria for ibd; 98 were categorized in the non-ibd group. crohn disease and colitis were the most common gi disease manifestations in the cgd-ibd group (n=79), followed by gi fistulas (n=22). cgd-ibd patients had an increased average frequency of infections (10.6 events/patient) compared to the cgdnon-ibd group (5.1 events/patient). in both groups, lower respiratory tract infections were the most common infection type and aspergillus was the most common organism. enteric organism infections were more common in ibd patients. temporal data regarding the timing of infections were not available. immunomodulators, including biologics and interferon-gamma, were used at a significantly higher rate in ibd patients compared to non-abstract/case report text down syndrome (ds) is characterized by the occurrence of three copies of human chromosome 21 (hsa21). these patients often develop chronic mucocutaneous candidiasis (cmc) and autoimmune thyroiditis, mimicking patients with heterozygous gain-of-function (gof) stat1 mutations, which enhance cellular responses to the three types of interferon (ifn). hsa21 contains a cluster of four interferon receptor (ifn-r) genes: ifnar1, ifnar2, ifngr2 and il10rb. a gene dosage effect at these four loci may contribute to the infectious and autoimmune manifestations observed in individuals with ds. we report high levels of ifn-αr1, ifn-αr2 and ifn-γr2 expression on the surface of monocytes and ebv-transformed-b (ebv-b) cells from ds patients. levels of ifn-ɣr1, encoded by a gene on chromosome 6, were similar in the immune cells of ds patients and healthy controls. total and phosphorylated stat1 (stat1 and pstat1) levels were constitutively high in unstimulated and ifn-α-and ifn-γ-stimulated monocytes from ds patients, although less so than those in patients with gof stat1 mutations. following stimulation with ifn-α or -ɣ, but not with il-6 or il-21, pstat1 and ifn-ɣ activation factor (gaf) dna binding activities were significantly higher in the ebv-b cells of ds patients than in controls, this response resembling the dysregulated responses observed in patients with stat1 gof mutations. plasma type i ifns concentrations were high in about 12% of the ds patients tested. a genome-wide transcriptomic analysis involving principle component analysis and a comparison of interferon modules was performed on circulating monocytes. it showed that ifn-stimulated genes (isgs) were expressed more strongly in ds than in controls. ds monocytes have intermediate levels of ifn-α-and ifn-γ-induced isgs relative to monocytes from healthy controls and from patients with gof stat1 mutations. by contrast to patients with gof stat1 mutations, circulating th17 counts were normal and the proportion of terminally differentiated cd8+ t cells was high in ds patients. the constitutive upregulation of type i and type ii ifn-r, at least in monocytes of ds patients, may therefore contribute to the autoimmune diseases observed in these individuals. scid screens (positive screen defined as trec values less than 250 units/ul per statewide criteria). results forty nine neonates were identified with low trec values. 46 (94%) of these infants had repeat trec screening, 14 (29%) of which were found to have a positive second trec screen. lymphocyte subsets were evaluated in 23 of these infants and 5 of which were noted to have lymphopenia (defined as absolute lymphocyte count less than 2500). 2 infants were noted to have low cd4 levels (defined as < 300 cells/ul) and 6 were noted to have low cd8 levels (defined as less than 500 cells/ul). of note, 50% of the infants with cd4 and cd8 lymphopenia had normal repeat trec levels. 14 of the infants were noted to have low b cell levels (defined as < 610 cells/ul). 12 infants had quantitative immunoglobulin levels and of these two were noted to have igg levels less than 100. of note one infant was diagnosed with partial digeorge syndrome via microarray. in our study population, no infants were diagnosed with scid. discussion our study shows that testing for trec levels on newborn screen may be beneficial in identifying not only scid, but also other immunologic conditions. infants in our study had evidence for both cell mediated and humoral immunodeficiency which necessitated further workup and follow up from allergy and immunology specialists. it may be beneficial to develop further programs to track infants identified with abnormal trec levels on newborn screens to determine if they develop signs of immunodeficiency syndromes later in life. abstract/case report text the patient was transferred to our adult clinical immunology transition clinic for low igg and iga, elevated igm and b cell lymphopenia, treated with subcutaneous gamma globulin. his medical history was relevant for recurrent respiratory infections since two years-old, failure to thrive and developmental delay. he also developed chronic auto-immune hemolytic anemia (aiha) at ten years old, accompanied by prominent lymphoid hyperplasia. our initial evaluation at the age of twenty years old showed massive polyadenopathy and splenomegaly. work-up confirmed flair-up of aiha. at that point, the diagnosis of apds was raised. he also had mild intellectual impairment and dysmorphic features such as a mild degree of ocular depression, deep-set eyes, vaguely triangular face, small chin, but had normal stature. a customized panel for usual genes involved in classic hyperigm syndromes, apds, noonan and kabuki syndromes came back negative. at 23 years old, an urgent coloscopy was performed because of acute abdominal pain and showed diffuse ileal lymphoid hyperplasia. biopsies confirmed reactional lymphoid hyperplasia without infection nor malignancy. a second 232 genes ngs panel associated with pid identified a heterozygote mutation in tap1; expression of hla class 1 was normal on flow cytometry. the patient was then started on sirolimus for an "apds-like syndrome" despite the lack of genetic confirmation. six months after introduction of mtor inhibitor, his abdominal pain had completely disappeared. tep scan showed complete resolution of axillar, retroperitoneal and inguinal lymph nodes and significant regression of splenomegaly. a third large non-biased 6700+ genes ngs panel revealed a 30 pb de novo deletion that included the splice site of pik3r1 exon 11 typically involved in apds2: c.1392_1425+4del p.(asp464glufs*5) , which was missed by the first two panels. indeed, oligonucleotide-selective sequencing technology used for the previous panels was associated to mapping errors of short reads and difficult detection of large deletions. interestingly, the patient also presented some but not all dysmorphic features of short syndrome which is related to pik3r1 haploinsufficiency. in this new era of genetic testing, this case is a reminder that we need to be aware of the pitfalls of genetic tests and that clinical judgment is still our best diagnostic tool. abstract/case report text intro: patients with chronic granulomatous disease (cgd) are theorized to have a lower risk of malignancy related to their lack of free radical formation. there are relatively few reports of malignancy described in patients with cgd. we report three cases of malignancies in the large cohort cgd population at the national institutes of health followed between 1972-2018 to add to the seven cases described in the literature. case 1: a 48-year-old man with x-linked chronic granulomatous disease with a history of severe inflammatory bowel disease, who presented with progressive left-sided chest pain in 2017, decreased appetite and weight loss. transthoracic lung biopsy showed atypical cells and a pet/ct showed abnormally dense mesentery and widespread hypermetabolic abnormalities. a mesenteric biopsy showed metastatic pancreatic adenocarcinoma. palliative care was initiated. patient expired four months after diagnosis. case 2: a 24-year-old man with x-linked chronic granulomatous disease and severe inflammatory bowel disease requiring total proctocolectomy and who had been remotely treated with infliximab, presented in 2015 with right upper quadrant pain. abdominal ultrasound and mri of the liver showed multiple liver lesions. biopsy of these lesions revealed hepatocellular carcinoma. patient underwent two courses of radiolabeled itrium spherules. however, his disease progressed and he expired approximately five months after diagnosis. c a s e 3 : a n 1 8 -y e a r-o l d m a n w i t h x -l i n k e d c h r o n i c granulomatous disease and inflammatory bowel disease who presented in 2007 with fevers, abdominal pain and pancytopenia. during the course of his hospitalization, he developed sepsis which led to his demise. on autopsy, an incidental finding of papillary thyroid carcinoma was made. discussion: these three patients all had poorly controlled inflammatory bowel disease. additionally, patients with cgd are typically exposed to higher doses of radiation, leading one to expect higher rates of radiation induced malignancies. however, there are still relatively few case reports of cancer in the cgd population. tissue biopsy is necessary for diagnosis. due to end organ damage secondary to the underlying disease in the first two cases, treatment options were limited. managing infections during chemotherapy can be complex due to drug interactions with chemotherapeutic agents. abstract/case report text myeloperoxidase (mpo) deficiency is the most common inherited defect of phagocytes that impairs microbial killing since the toxicity of the respiratory burst is dampened without myeloperoxidase release from the azurophilic granules. a significant portion of these patients remain asymptomatic, however there is a clinically variable phenotype that can present if they do become symptomatic. fungal infections with candida strains appear to be the most frequently reported. we present an adulthood case of recurrent invasive candidal disease due autosomal recessive myeloperoxidase deficiency from a pathogenic missense variant in the mpo gene (c.1705c>t (p.arg569trp)). a 23-year-old caucasian male was in his normal state of health without any major illnesses until 18 years of age when he was diagnosed with candida osteomyelitis of the heel, followed by cryptococcal meningitis the following year which ultimately required a ventriculoperitoneal shunt. in 2019, he had a prolonged hospitalization after presenting with lethargy, headache and vomiting that culminated in seizure activity and prompted an emergency room visit. imaging at the time showed ventriculomegaly, and fluid from the shunt revealed yeast, but no bacteria. he was started on broad spectrum antifungal therapy and admitted for further management. cerebral spinal fluid and blood cultures confirmed invasive candida albicans meningitis. during this hospitalization, he also developed sepsis secondary to serratia marcescens. because of the pathogens that were being isolated, our service was consulted. of note, our patient does not have diabetes mellitus. a neutrophil oxidative burst assay showed an absent respiratory burst compared to control. a primary immunodeficiency panel to identify genetic variants was also sent to invitae. variants in cyba, cybb, ncf2, and ncf4 were not identified, making chronic granulomatous disease less likely. peroxidase staining was negative on neutrophils and normal on eosinophils, suggesting a diagnosis of mpo deficiency. this led to mpo gene sequencing for deletion and duplication analysis. a homozygous pathogenic variant consistent with a molecular diagnosis of a mpo related condition was identified. immunoblotting of patient-derived immune cells demonstrated an absence of mature enzyme. although not typically indicated, given the severity of his presentation, our patient remains on fluconazole for long term prophylaxis. his younger brother also had a history of invasive disease with candidaosteomyelitis and meningitis. a neutrophils oxidative burst assay showed similar results in his brother and similar results with peroxidase staining, also suggesting a diagnosis of mpo deficiency. confirmatory genetic testing has not been performed yet. their father, who reported severe skin infections with candida, had peroxidase stains performed on neutrophils and eosinophils which were both normal. we have presented a patient without a significant history of diabetes mellitus who developed invasive disease from candida and serratia and was ultimately diagnosed with myeloperoxidase deficiency. abstract/case report text introduction: juvenile xanthogranuloma (jxg) is an often benign, histiocytic proliferative disorder of the mononuclear phagocytic system. patients typically present with localized cutaneous lesions. systemic disease, especially central nervous system involvement, rarely occurs but has significant morbidity and mortality risk. no standard evaluation nor therapy regimen exists for systemic jxg and little is known about the genomic alterations underlying its pathology. case report: a full-term male infant presented at 4 months of age with post-prandial abdominal pain, fevers, altered mental status and weight loss. abdominal ultrasound and ct identified renal masses. a chest ct was obtained showing a paraspinal mass with possible neural foramina extension. mri brain and total spine was consistent with diffuse leptomeningeal disease involving the left frontal convexity, brainstem, cerebellum, and multiple cranial nerves. abnormal enhancement was also present along the entire surface of the spinal cord extending into the cauda equina with additional enlargement of the cervical/upper thoracic cord with intramedullary enhancing masses and a right paraspinal mass. renal biopsy yielded a pathologic diagnosis of disseminated jxg. integrative clinical sequencing of the mass identified a somatic driving alk rearrangement (kif5b-alk in-frame fusion). tumor and matched germline dna sequencing did not detect any alterations in the ras/mapk pathway. bone marrow biopsy was negative for disease with cerebrospinal fluid analysis showing numerous monocytes and macrophages consistent with jxg. the patient was started on therapy consisting of systemic dexamethasone, intrathecal methotrexate/hydrocortisone and systemic intravenous cytarabine. his first cycle was complicated by pseudomonas aeruginosa bacteremia and gangrenous cellulitis of the perianal region, treated with systemic/topical antibiotics and topical gm-csf. following completion of the initial cycle of therapy, the patient was noted to have declining neurologic status, including seizure-like activity. repeat mr imaging revealed worsening cns disease with new subdural fluid collection and progression of leptomeningeal enhancement and intramedullary cervical lesion. in light of disease progression, the decision was made to continue dexamethasone treatment, but add adjunct intrathecal cytarabine, and transition to targeted alk inhibition via daily oral ceritinib, given its predicted cns penetrance followed by ceritinib in combination with systemic intravenous clofarabine. significant clinical and radiographic improvement was noted with the new targeted treatment regimen. ceritinib therapy was tolerated well overall after a 25% dosing reduction made for initial grade 2 gastrointestinal toxicity and grade 4 hypertriglyceridemia (non-life threatening but level > 1000 mg/dl). following continued treatment with daily ceritinib and completion of 12 cycles of clofarabine therapy, our patient experienced complete disease remission. he continues to do well on daily ceritinib monotherapy with plan to complete an additional year of therapy. conclusion: our report highlights the potential benefit of real-time integrative clinical sequencing in the management of systemic histiocytic lesions, specifically non-langerhans cell conditions. it has the potential to identify novel somatic genetic alterations, other than the typical lchassociated braf mutations of the mapk pathway, that may be therapeutically targetable. treatment with 2nd generation alk-inhibition in our pediatric disseminated jxg patient was a novel, biologicallyrationale management approach with minimal toxicity and potentially contributed to his complete remission. abstract/case report text background: c3 glomerulonephropathy (c3gn) is a progressive kidney disease with the predominant pathological feature of c3 deposits around the glomerular capillaries. c3gn patients suffer from dysregulated activation of the alternative pathway as the result of autoantibodies or congenital genetic defects that stabilize cleavage of c3. despite therapy involving immunosuppression and complement-pathway inhibition, the prognosis for c3gn is poor. we report a patient with autoantibody-mediated, refractory c3gn who demonstrated no improvement on rituximab but achieved sustained remission on bortezomib. follow up studies after one year demonstrated clearance of the culprit autoantibody, normalization of c3 levels, and improved pathologic appearance of the kidneys. this case supports the idea that c3gn is frequently driven by pathogenic autoantibodies that may not clear with rituximab alone. plasma cell directed therapy has the potential to clear these autoantibodies and halt the progression of disease. case presentation: we report the case of a hispanic male with chronic renal dysfunction initially diagnosed with membranoproliferative glomerulonephritis on renal biopsy at 12 years of age. despite cellcept and prednisone, over the next 4 years, he had worsening proteinuria and an increase in protein-to-creatinine ratio. renal biopsy suggested c3gn, and lab studies revealed a factor-h autoantibody and c3 level below the assay limit of detection. after initiating eculizumab, the proteinuria temporarily improved; however, the proteinuria eventually worsened, and he was referred to immunology. we hypothesized that the factor h-binding autoantibody was the cause of dysregulated c3 cleavage and disease progression, and blocking the terminal complement pathway with eculizumab would not halt upstream c3-mediated kidney injury. at the age of 20, rituximab and plasmapheresis were administered to clear the factor-h autoantibody. three months after rituximab administration, the factor-h autoantibody level decreased to the normal range, but he continued to have significant proteinuria with low serum albumin and undetectable c3 level. we concluded that the relevant autoantibody was not solely produced by differentiating memory b cells, so we decided to target the plasma cell compartment. bortezomib was started at the age of 21, and eculizumab was continued given his initial response to treatment. after adding bortezomib, factor h autoantibody levels dropped below prior levels and serum c3 level normalized. renal biopsy at the age of 22 showed evidence of imp r o v i n g c 3 d e p o s i t i o n a n d l e s s p r o m i n e n t g l o m e r u l a r hypercellularity, with stable mesangial hypercellularity, interstitial fibrosis, tubular atrophy, and sclerotic glomeruli. although his proteinuria did not worsen, it remained persistent, suggesting that earlier introduction of bortezomib could have prevented disease advancement. there has been no further progression of kidney failure. conclusions: the majority of c3gn patients harbor autoantibodies to components of the alternative pathway of complement. this case provides evidence that at least some of these autoantibodies are indeed the cause of complement dysregulation, and thus are prime targets for therapy. b cell targeting therapies may be inadequate to decrease autoantibody levels for some patients. early initiation of bortezomib, or other plasmacell directed therapy, may effectively induce complement normalization and disease remission in these cases. inhaled corticosteroid and a long-acting bronchodilator. he has no family history of immunodeficiencies or congenital disorders. computed tomography(ct) chest showed bronchiectasis. his complement studies and isohemagglutinin titers were also normal. his serum immunoglobulin(ig) and lymphocytes on presentation are shown in table 1 . he had a poor response to polysaccharide pneumococcal vaccination. he was diagnosed with combined igg2/igg4 subclass/iga deficiency and was started on immunoglobulin replacement therapy and prophylactic rotating antibiotic therapy. thereafter, his clinical course markedly improved with a reduction in the frequency of rti's as well as the number of bronchiectasis exacerbations. there was high suspicion for an underlying genetic disorder based on his constellation of neurodevelopment disorders and immunodeficiency. cytogenetic evaluation with array comparative genomic hybridization(cgh) analysis showed duplication of xq25 and xq28 consistent with mds. discussion: mds is caused by duplications involving the mecp2 gene locus of the x chromosome at xq28. it has a 100% penetration rate in males whereas females act as carriers and are usually unaffected. rarely, cases of de novo mutations causing mds have been reported. chromosome microarray analysis is currently the best initial clinical test when mecp2 duplication syndrome is suspected. management needs a multidisciplinary approach involving geneticists, neurologists, ophthalmologists, physical medicine and rehabilitation specialists, psychologists, gastroenterologists, and allergy and immunology specialists. prophylactic treatment with ivig and antibiotics has been the standard of care for immunodeficiency in these patients. prognosis is guarded and most male patients die in the mid to late 20's because of severe rti's secondary to immunodeficiency. conclusions: this case confirms the association of mds with combined iga and igg subclass deficiencies. clinicians should consider pursuing genetic evaluation for mds in patients with neurodevelopmental disorders and immunodeficiency because the diagnosis of the syndrome can change the overall approach to management and expectations in prognosis. abstract/case report text introduction: c3 nephritic factor is an autoantibody that binds to the alternative pathway c3 convertase (c3bbb). this results in unchecked overactivation of the alternative complement pathway, which can lead to renal disease, partial lipodystrophy, retina disease, and frequent infections. in this case, we present a patient with partial lipodystrophy and low c3, subsequently found to have c3 nephritic factor. case description: a 5 year old female presented with a 19 month history of low c3 levels. she was diagnosed 21 months ago with poststreptococcal glomerulonephritis (psgn) after presenting with hematuria and elevated aso titers. she had c3 levels drawn 1-2 months after time of diagnosis and c3 level was low at 27 (normal range 81-157), which was consistent with psgn. it was rechecked 3 months after time of diagnosis and was still low. she was referred to rheumatology at this time and was found to have a positive ana titer 1:160. tests for lupus and anti-phospholipid syndrome were negative. c3 normalized to the low-normal range at 6 months after time of diagnosis to 81. her pediatrician checked to make sure it remained normal around 17 months after initial diagnosis and c3 was low again at 22. c4 was normal at 26. she was referred to immunology for further evaluation. during this time she was asymptomatic with no fevers, infections, hematuria, rashes, joint pain, or joint swelling. she has no history of hospitalizations other than the first for psgn. mother denied family history of autoimmune disorders. physical exam: physical exam was notable for abnormal subcutaneous facial fat with normal fat distribution in the rest of her body. the rest of the exam was unremarkable with normal cardiac, pulmonary, abdominal, and skin exam. testing: c3 level was rechecked and low at 26. c3 nephritic factor was elevated at 1.07 (normal range 0.00-0.26). alternate pathway complement (ah50) was confirmed twice and was undetectable, < 10 (normal level greater or equal to 46). total hemolytic complement (ch50) was low at 22 (normal level 42-95). other complement levels were checked and c1q, c2, c4, c5, c6, c7, c8, and c9 complement were within normal range. discussion: the overactivation of the alternative complement pathway by c3 nephritic factor can result in various clinical manifestations, such as c3 glomerulopathy and acquired partial lipodystrophy in predominantly the face and the upper torso. the exact mechanism of how c3 nephritic factor is related to facial and upper body lipodystrophy is not known. one proposed mechanism is that adipocytes in the face and upper body produce more factor d, which is a complement protein utilized by c3 nephritic factor. overactivation of the alternative complement pathway on the adipocyte then leads to formation of the membrane attack complex, resulting in adipocyte lysis. eye disease, such as retinitis pigmentosa and macular degeneration can develop. c3 nephritic factor can also lead to more frequent infections and renal disease. patients need to be closely monitored. if patients develop c3 glomerulopathy, they may need to be considered for immunomodulatory therapy, such as steroids and other immunosuppressants. 4 project manager/ucsf benioff children's hospital 5 senior clinical research associate/ucsf benioff children's hospital 6 associate professor/department of clinical pharmacy, ucsf 7 staff research assistant iv/ucsf benioff children's hospital 8 senior supervisor/ucsf benioff children's hospital 9 laboratory specialist/ucsf benioff children's hospital 10 research specialist/ucsf benioff children's hospital 11 assistant professor/ucsf benioff children's hospital 12 clinical professor/ucsf benioff children's hospital 13 assistant professor/ucsd rady children's hospital 14 staff pediatrician/tuba city indian health service 15 staff pediatrician/phoenix children's hospital 16 associate professor/seattle children's hospital 17 chief, genetic immunotherapy section/niaid, nih 18 professor/university of minnesota 19 professor/ucsf benioff children's hospital abstract/case report text background: artemis-deficient scid (art-scid) represents 3% of all scid, but occurs in 1/2000 births in navajo and apache native americans. artemis protein, encoded by dclre1c, is essential for repairing dna double-stranded breaks, including those generated during v(d)j recombination of antigen receptor genes as t and b cells develop. artemisdeficiency causes not only t-b-nk+ scid, but also increased sensitivity to alkylating drugs and radiation. art-scid is the most difficult scid to treat with allogeneic hematopoietic cell transplantation (hct) due to high rates of rejection and gvhd, incomplete immune reconstitution, and toxicity following intensive conditioning regimens. as an alternative, we developed a self-inactivating lentiviral vector containing the human artemis promoter and dclre1c cdna (aproart). we are evaluating its toxicity and efficacy in a phase i/ii gene transfer trial in art-scid patients. methods: newly diagnosed infants with art-scid and older patients with insufficient immunity despite prior allogeneic hct were eligible if organ function was acceptable. infants needed to have no matched sibling donor and be at least 2 months old at conditioning. cd34+ cells were isolated from bone marrow or cytokine-mobilized peripheral blood, cultured with cytokines, transduced x2 with aproart, and cryopreserved. patients received 2 daily doses of busulfan, targeted for a cumulative exposure (cauc) of 20mg*hr/l, with infusion of thawed cells on the following day. results: we treated 5 newly diagnosed infants (art001-3&007-8) with median age 2.6m (range 2.3-3.7) and 3 previously-treated patients (art004-6) (5.5y, 12.7y and 20.9y), with a median follow-up of 9.6m (range 1.6-17.2). the mean (sd) bu cauc was 19.4±1.0 mg*hr/l. patients received a median of 6.5x106 aproart-transduced cd34+ cells/kg (range 3.9-12.4). the average vector copy number (vcn) and transduction efficiency in the marrow grafts exceeded those in the pbsc grafts: 2.1±1.0 copies/cell vs 0.83±0.1 (p=0.02) and 75±9% vs 59±4.6% (p=0.03), respectively. there were no serious busulfan side effects. all patients had transduced peripheral blood leukocytes by 4w and 7 of 8 developed gene marking in t, b, nk and myeloid cells by 8w (fig. 1) . gene-corrected cd3, cd4, cd4/ 45ra/ccr7, cd8 and cd19 cells appeared in 7 of 8 patients (fig. 2) , with art005 having t, nk and myeloid marking without b cells at 6m post infusion. normalization of lymphocyte proliferation to pha occurred in the 3 evaluable (>12w) infants (fig. 3) , all 3 now outpatients off isolation. two infants and 1 previously treated child developed autoimmune hemolytic anemia (aiha), with 2 requiring immunosuppressive therapy. infections included rhinovirus at presentation in art001 that resolved with t cell reconstitution. after discharge art001 acquired and recovered from norovirus and art002 acquired and recovered from cmv and rotavirus. analyses of insertion sites and t cell receptor diversity are pending. conclusion: infusion of aproart-transduced autologous cd34 cells into art-scid patients pretreated with very low exposure busulfan resulted in multilineage engraftment of transduced cells with evidence for t and b cell immune development. aiha, the only complication to date, occurred early and appears to resolve following restoration of t cell immunity. these encouraging results suggest potential effectiveness of ex vivo gene therapy for art-scid. (26) submission id#798344 mailan nguyen, md 1 , susan canny, md, phd 2 , andrea ramirez, md 1 , ivan chinn, md 3 abstract/case report text background: systemic lupus erythematosus is a heterogeneous disorder of the immune system. systematic genetic evaluation of patients with childhood-onset sle (csle) has begun to identify phenotypic clusters of csle patients with classic sle-causing genetic variants, as well as revealed unexpected genetic mimics of lupus. we report 3 patients diagnosed with csle with similar typical and atypical lupus features, who were subsequently found to carry pathogenic nras variants that are the cause of ras-associated autoimmune leukoproliferative disorder (rald). cases: all 3 patients (2 females, 1 male) presented at < 3 years of age (average age 21.7 months, range 17-27 months) with antinuclear antibodies, anti-double-stranded dna antibodies, autoimm u n e h e m o l y t i c a n e m i a , s e v e r e t h r o m b o c y t o p e n i a , antiphospholipid antibodies, hypocomplementemia and nephritis. additionally, the patients all displayed fevers, organomegaly, lymphadenopathy and hypergammaglobulinemia. two out of 3 patients had a malar rash, leukopenia, lymphopenia, anti-smith antibodies, serositis or arthritis. no patient had oral or nasal ulcers or photosensitivity. despite the fevers, lymphoproliferation and systemic autoimmunity, the patients did not display overwhelming immune dysregulation (peak ferritin 128-623 ng/ml). interestingly, the patients were found to have monocytosis (19-45%), as has previously been reported in rald. double negative t cells were within normal range in the 2 patients in which this was tested. all 3 patients required aggressive immune modulation for control of their disease manifestations. two of the 3 developed severe infections, specifically pneumococcal sepsis, during therapy. current follow-up covers an average of 9.2 years (range 0.6 to 17 years). the patients responded to corticosteroids and were given sequential trials of various steroid-sparing therapies. in general, they appeared to benefit from both b cell depletion and t cell-directed modalities (cyclosporine, rapamycin), which are not first line therapy in csle. unfortunately, 1 patient developed a fatal pulmonary infection while on treatment; her underlying disease was felt to be quiescent. due to the early-onset of disease, each patient was selected for genetic evaluation (2 by exome sequencing, 1 by gene panel). this lead to the discovery of pathogenic nras variants (c.38g>a, p.g13d) in all patients, assumed to be somatic, although this was confirmed in only 1 case. conclusion: ras-associated autoimmune leukoproliferative disorder can present indistinguishable from csle with positive autoantibodies, immune cytopenias, arthritis, nephritis and hypocomplementemia. clinicians should consider evaluating for rald in csle patients who present at an early age ( < 3 years) with predominant features of lymphoproliferation and hematologic abnormalities, particularly monocytosis. t cell-directed therapy with cyclosporine or rapamycin should be considered for rald. (1) human ctla4 loss-offunction causes dysregulation of foxp3+ regulatory t (treg) cells, hyperactivation of effector t cells, and lymphocytic infiltration of target organs. patients also exhibit progressive loss of circulating b cells, associated with an increase of predominantly autoreactive cd21(lo) b cells and accumulation of b cells in non-lymphoid organs. inherited human ctla4 loss-of-function demonstrates a critical quantitative role for ctla4 in governing t and b lymphocyte homeostasis. (2) this case highlights the importance of next generation sequencing (ngs) in diagnosing and managing complex presentations with multi-system involvement. case presentation: patient was diagnosed with diffuse large b cell lymphoma at age 22 and treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (r-chop) in 9/2012. he underwent autologous stem cell transplant with preparative carmustine, etoposide, cytarabine, and melphalan (beam) in 10/2012. he subsequently developed recurrent giant condyloma acuminata following transplantation requiring 9 surgical resections, refractory immune thrombocytopenic purpura (itp) requiring aggressive systemic steroids and high dose ivig at least yearly, experiencing hypogammaglobulinemia, recurrent sinopulmonary infections, disseminated herpes zoster, and kaposi sarcoma. in 6/2014, he underwent ct/pet, revealing extensive hypermetabolic lymphadenopathy and splenomegaly. bone marrow biopsies were negative for lymphoma, although showed a slightly hypocellular marrow (40-60% cellularity), 2% blasts, and eosinophilia without peripheral eosinophilia. repeated evaluations for hiv, syphilis, histoplasma, cmv, hhv6, hhv8, bartonella, coxciella, brucella, htlv, toxoplasma were negative. htlv1 and 2 antibodies had been negative prior to transplantation. tonsillectomy 12/2015 due to progressive enlargement showed reactive follicular hyperplasia with focal acute tonsillitis without granulomas or viral inclusions. repeated lymphocyte enumeration and proliferation studies were normal. a repeat pet scan 3/2016 revealed persistent diffuse lymphadenopathy involving the neck, chest, abdomen, and pelvis. left lung biopsy revealed non-caseating granulomas without lymphoma. stains for ebv were negative. repeat pet scan 11/2016 indicated disease progression prompting a left axillary excisional lymph node biopsy, revealing ebv lymphadenitis with large, reactive follicles with interspersed inflammation and loosely formed granulomas and cd20 positive b cells within the follicles. ebv blood pcr was negative. afb and fungal stains were negative on all biopsies. in 1/2017, his igg was 615 (762-1488), iga 33 (70-390), and igm 58 (38-328) with only 1 out of 23 protective serotypes to pneumococcus post-vaccination at 1.3 or greater. in 3/2018, igg was 380 (762-1488). custom ngs panel showed a heterozygous missense variant in ctla4 c.534c>g (p.s178r) located in the transmembrane domain. this variant of uncertain significance is suspicious and strongly suggests the diagnosis of ctla4 -related autoimmune lymphoproliferative syndrome . patient was referred to the national institute of health, where he received a bone marrow transplant. conclusion: primary immunodeficiency diseases comprise a group of highly heterogeneous immune system diseases and around 300 forms of pid have been described. ngs has recently become an increasingly used approach for gene identification and molecular diagnosis of human diseases guiding treatment to patients who may otherwise have poor outcomes. (3) (28) submission id#799299 erik newman, md 1 , cullen dutmer, md 2 1 allergy and immunology fellow/university of colorado and children's hospital colorado 2 assistant professor of pediatrics/section of allergy & immunology, children's hospital colorado, university of colorado school of medicine, aurora, co, usa abstract/case report text introduction: inherited defects of the complement system are rare disorders that can result in unique susceptibility to infections with select bacteria. patients with a deficiency of a complement protein early in the complement pathway (affecting c1qrs, c2, c3, factor h, or factor i) have increased susceptibility to infection with encapsulated bacteria, most notably streptococcus pneumoniae and neisseria species. in contrast, patients with a deficiency of a complement protein at the terminal end of the complement pathway (affecting c5, c6, c7, c8 alpha/beta/gamma, or c9) almost universally present with severe, recurrent, or disseminated neisseria species infections. most genes encoding complement proteins are found on autosomes, in which specific complement deficiencies result from biallelic mutations. although particular complement deficiencies occur at higher frequencies in certain populations, the prevalence of specific complement deficiencies is unknown in many parts of the world, especially in underdeveloped regions, including sub-saharan africa. herein, we describe a young congolese boy with an atypical presentation of c8 alpha deficiency. case description: a 17-month-old congolese boy with consanguineous parents (first cousins) presented with recurrent infections. prior to an evaluation of his immune system, he was hospitalized five times. his infections included episodes of acute otitis media, bacterial pneumonia, and viral pneumonitis. a bronchoscopy revealed diffusely edematous airways and growth of candida albicans, moraxella catarrhalis, and streptococcus pneumoniae in bronchoalveolar lavage cultures. concurrently, his respiratory pcr panel was positive for adenovirus. his initial immune evaluation included assessments of his serum immunoglobulin levels, vaccine titers (tetanus, diphtheria, haemophilus influenzae, and streptococcus pneumoniae), neutrophil oxidative burst, lymphocyte subsets, and ch50, in which only his ch50 was abnormal (16 u/ml). his ch50 remained low on repeat assessment (16 u/ml), at which time an ah50 was pursued and also returned with a low result (1% of normal). a complement system genetic panel identified a homozygous intronic variant in c8a (c.856-12g>a). functional confirmation of the variant revealed that the patient had a significantly decreased c8 level (18 mcg/ml) and absent c8 function. discussion: we present a case of a young congolese boy with c8 alpha deficiency and a clinical presentation atypical for defects in terminal complement proteins. our patient presented primarily with recurrent respiratory infections, including streptococcus pneumoniae pneumonia, but without a preceding history of meningococcal disease. while it is well established that patients presenting with terminal complement pathway defects have an increased susceptibility to meningococcal disease, it is less clear if they have increased susceptibility to pneumococcal infections. occurring between exons 5 and 6, the homozygous intronic variant in c8a identified in our patient is predicted to result in abnormal splicing. while the allele frequency of this mutation is relatively high in the african population (0.017), functional confirmation of the variant demonstrated a decreased c8 level and absent c8 function that support the pathogenesis of the mutation. the discrepancy between the allele frequency and reported disease cases could be explained in part by varying clinical manifestations seen in c8 deficiency or underrecognized disease in sub-saharan africa. abstract/case report text rationale: scid is a syndrome characterized by profound t, b, and (in some cases) nk cell defects that is universally fatal unless immune reconstitution is achieved. a total of 177 scid infants have been given allogeneic bone marrow transplantation at duke university medical center without pre-transplantation chemotherapy or post-transplantation graft-versus-host disease (gvhd); 90% received t cell-depleted haploidentical parental marrow and 47(26%) are known to be deceased. post-transplantation follow-up ranged from 22 months to 37 years. the aim of this cross-sectional study is to characterize the clinical status of a large cohort of survivors treated at a single medical center. methods: clinical status was assessed by detailed questionnaires delivered by mail or electronically. adult (≥ 18 years old) and pediatric questionnaires were based on patients' age. patients were also contacted by telephone and evaluated at clinic visits. molecular type of scid, demographics, type, date and age at transplant were obtained from a clinical database. results: fifty questionnaires were completed to date from survivors ranging in age from 5 to 37 years. twenty-nine/50 were adults ≥18 years at the time of the questionnaire. genetic defects were known for all 50 patients-xlinked scid was the cause in about half ( figure 1 ). twenty-three of 50 patients were on immunoglobulin replacement. thirty of 50 reported having received immunizations, and about half of those received live vaccines. fifteen of 50 reported they were taking no regular medications; 11 reported taking prophylactic antibiotics. >we found substantial scholastic achievement, with 17/29 adult patients reporting college attendance. two had post graduate education including doctorate level degrees. occupations included physician, nurse, factory worker, musician, teacher, and engineer. one patient had 3 children. twenty-seven /29 adult patients shared their height and weight and 78% (21/27) had a healthy bmi (bmi 18.5-24.9), while 15% (4/27) were overweight, and 7% (2/27) were underweight (< 18.5) . in pediatric patients, the average age and sex-adjusted bmi was at the 47th percentile and only 3 had a bmi that was < 5th percentile. thirty-four/50 patients reported seeing an immunologist regularly. in the adult group, 38% reported no longer seeing an immunologist. the health conditions reported were similar to those common in the general population, and included rashes, warts and mouth ulcers. most reported these were transient, self-resolving issues. thirteen of 50 (26%) reported having adhd, higher than nih reported rates which estimate adhd in 8.1% of adults and 11% of children). ten of 50 (20%) reported having anxiety, similar to the nih reported prevalence of 19.1% in the general population. 34/50 (~68%) reported having no active concerns about their health. conclusions: overall, our findings are consistent with those in the last update done by railey et al, j. peds. 155: [834] [835] [836] [837] [838] [839] [840] 2009 in this population. patients are doing well with most problems similar to those common in the general population. most have a healthy bmi. adhd had a higher prevalence than in the general population. more than 1/3 of scid patients are not seeing an immunologist regularly, and a majority do not have any active concerns. . genetic causes of scid in the 50 patients whose questionnaire data are presented. x-linked scid was the most common, followed by ada and il-7r deficient scid clinical fellow/national institute of allergy and infectious diseases (niaid/nih) 2 research nurse/nih-nhgri 3 professor of pediatrics and allergy and immunology/ann & robert h. lurie children's hospital of chicago 4 chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih abstract/case report text rationale: myopathy has been occasionally documented in patients with primary immunodeficiency (pid). however, data on frequency and patient characteristics associated with myopathy are lacking. we performed a descriptive analysis of patients with primary immunodeficiency (pid) in the usidnet having myopathy as a feature of their primary disease. methods: the usidnet registry was queried for the spectrum of myopathic disorders in pid patients that had been entered into the registry as of november 26, 2019. results: a total of 63 pid patients with myopathy were identified, 36 of which (57.1%) were female. median age at onset of symptoms related to pid was 4 years (range 0.1-72 years, iqr 0.5-25 years). median age of diagnosis of pid was 8.3 years (range 0.4-77 years, iqr 3-32.5 years). age at onset of myopathic disorders was not known. twenty-eight (44.4%) patients had a diagnosis of common variable immunodeficiency (cvid), 6 (9.5%) had agammaglobulinemia, 3 patients each (4.8%) were diagnosed with severe combined immunodeficiency, hypogammaglobulinemia, or 'hlh and pigmentary disorders', while 2 patients (3.2%)were reported in each category of combined immunodeficiency (cid), autoimmune lymphoproliferative syndrome (alps), and autoinflammatory disease. thirty-five patients (55.6%) had a causative gene variant identified attributable to pid. the most common variant identified was btk (6 patients) followed by aire, lyst, cybb (3 patients each) and pi3kcd (2 patients). eighteen individual patients had other variants identified ( figure 1 ). 15 patients had cellulitis or skin/ subcutaneous tissue infection, 7 patients had a 'skin or subcutaneous tissue abscess', 2 had pyoderma gangrenosum, and 1 patient with eczema herpeticum. within this cohort of 63 patients, the most common myopathy listed was myositis (18) followed by 'muscle weakness' (11), dermatomyositis (8) , myalgia/s (6) , myalgia/myositis (4), myopathy (4), polymyositis (2) , steroid-induced myopathy (2) . no patient had an infectious myositis or muscle abscess listed. eighteen patients (28.6%) had a myopathic disorder at the time of diagnosis of their pid. thirtyone patients (49.2%) received prednisone, 11 (17.5%) received hydrocortisone and 2 (3.2%) received dexamethasone. two patients had a diagnosis of adrenal insufficiency. nine patients (14.3%) underwent hematopoietic stem cell transplantation. nine patients (14.3%) died; median age of death 16 years (range 0.4-40.6 years, iqr 3.6-21.1 years). ). one patient with chronic granulomatous disease had myopathy due to duchenne muscular dystrophy, which was listed as a cause of death. no other myopathic disorders were listed as a cause of death for the other patients. conclusion: myopathy and inflammatory myopathic disorders occur at relatively high frequency in pid, and may be present even at the onset of clinical symptoms. the underlying etiology can be speculated to be multifactorial. further subgroup analysis is warranted to elucidate possible variant-specific or treatment-associated characteristics of myopathy in pid. laboratory studies at 4 years revealed normal igg/iga/igm but markedly elevated serum ige (42,550 ku/l), anemia (hb 8.7 g/dl), thrombocytopenia (78 x109/l) and lymphopenia (520 cells/l), with low t and b cell counts, very low proportion of naïve t cells, skewed repertoire of cd8+ t cells, undetectable trec levels, and impaired t cell proliferation to mitogens and antigens. there was an elevated percentage of circulating plasmablasts (7.4%) and of dysreactive cd21low cd38low b cells (53.8%). at the age of 5, hsct with reduced intensity conditioning was performed from her phenotypically hla-matched father, with improvement of t and b cell count and function. whole exome sequencing (wes) identified a homozygous missense variant in the mannosidase alpha class 2b member 2 (man2b2) gene (p.asp38asn), that segregates with disease in the pedigree. the man2b2 asp38 residue is evolutionary conserved. the p.asp38asn allele has a minor allele frequency of 0.0002687 in gnomad, with no homozygotes. the cadd score for this variant is 28.300, significantly higher than the mutation significance cutoff score (3.313). man2b2 is involved in the lysosomal degradation of glycoproteins and demannosylation of free n-glycans. in particular, man2b2 cleaves man2glcnac1 to generate man1glcnac1. serum n-glycan profiling revealed elevated man5/man6 and man5/ man9 in the patient. n-linked and free glycan profiling by mass spectrometry (ms) showed accumulation of man2glcnac2, man2glcnac1 and man3glcnac1glycans in patient fibroblasts as compared to control cells, consistent with defective lysosomal glycoprotein degradation. lentiviral transduction of wild-type man2b2 into patient fibroblasts led to normalization of the n-linked glycan profile, with reduction of man2glcnac2 from 8.4 to 0.9 times control levels, and of man2glcnac1 from 26.9 to 1.5 control levels, indicating rescue of the impaired deglycosylation ( figure 1a ). western-blotting demonstrated defective n-glycosylation of lamp2 and icam1 proteins in patient fibroblasts ( figure 1b) , which were corrected upon lentiviral transduction of wild-type man2b2 ( figure 1c ). overall, our results indicate that loss of man2b2 enzymatic activity leads to dysregulation of deglycosylation and abnormal mannosylation of glycans. in conclusion, we have demonstrated that man2b2 deficiency accounts for a novel autosomal recessive cdg with prominent features of immune deficiency and immune dysregulation. abstract/case report text heme oxygenase-1 (hmox1) is a rate-limiting enzyme that catalyzes the degradation of heme to carbon monoxide, ferrous iron, and biliverdin, which becomes bilirubin. these byproducts are implicated in inflammation, cell homeostasis, and antioxidant defense(1). hmox1-deficiency is an extremely rare autosomal recessive disorder with a complex presentation of a wide spectrum of symptoms, including hemolytic anemia and hyperinflammation, requiring genetic testing for confirmed diagnosis (2) . we report the fifth known case of hmox1-deficiency (3) (4) (5) , a boy who presented at 4 years of age with aspects of the characteristic phenotype, but also had early onset asplenia, interstitial lung disease, and previously undocumented immune deficiency. patient's presentation was notable for hyperinflammatory exacerbations triggered by viral and bacterial infections as well as vaccinations. episodic flares occurred every few months lasting weeks to months with fevers of 102-104f, hypoxia, leukocytosis above 40,000/mm3, hemolytic anemia with negative coombs, thrombocytosis exceeding 1 million/ mm3, transaminitis, hemoglobinuria, hyperferritinemia to 4,000ng/ml, and elevated ldh to 28,000iu/l. immune evaluation revealed normal immunoglobulin levels and adequate vaccine titers to both protein and carbohydrate antigens. although class switched populations were normal, b-cell phenotyping showed absent immature and transitional b-cells, low mature memory, and reduced cd27+ memory b-cells at 6% (normal >8%). mitogen stimulation with phytohemagglutinin and anti-cd3 were decreased (24.7% of control and 21.5% of control, respectively). t-cell phenotyping demonstrated cd4 population heavily skewed to immaturity with 65% of cells with naïve phenotype cd45ra+cd27+ccr7+. there were few effector-memory t cells and the cd8 population was skewed towards immaturity with >65% of the cells naïve. liver biopsy was performed secondary to hepatomegaly yielding mild to moderate sinusoidal fibrosis. bone marrow biopsy revealed a normocellular marrow with 3% blasts, increased megakaryocytes, and extensive hemophagocytosis. natural killer cell function was very low, while soluble il-2ra level was normal. further workup for hemoglobinopathies, metabolic defects, congenital disorders of glycosylation, lysosomal storage disorders, wilson's disease, autoimmune hepatitis, inherited and autoimmune hypercoagulability disorders, connective tissue disorders, myositis, and myopathies were all unremarkable. imaging demonstrated asplenia and howell-jolly bodies were present. hemophagocytic lymphohistiocytosis (hlh) genetic testing showed no variants. he was suspected to have systemic juvenile idiopathic arthritis (sojia) with episodes of macrophage activation syndrome. the frequency of his autoinflammatory flares increased such that he was corticosteroid dependent by age 9, having failed methotrexate, azathioprine, and anakinra. he was started on tocilizumab with laboratory improvements, but his lung disease progressed and became oxygen dependent. lung biopsy confirmed nonspecific interstitial pneumonitis (nsip) with cholesterol granulomas also seen in sojia. ultimately, chronic lung disease led to his death at age 10. whole exome sequencing yielded a paternal frame shift hmox1 and maternal splice donor hmox1 resulting in absence of protein. bone marrow transplantation (bmt) in hmox1 deficient mice have rectified phagocytotic defects and thereby their autoinflammatory phenotype, but no human reports for bmt treatment of hmox1-deficiency has been described. here we describe a phenotype expansion for hmox1deficiency to include not only asplenia and hepatomegaly, but also interstitial lung disease with cholesterol granulomas and inflammatory flares. abstract/case report text introduction: mutations in the gene encoding signal transducer and activator of transcription 3 (stat3) cause autosomal dominant hyperimmunoglobulin e syndrome (ad-hies) characterized by recurrent skin and sinopulmonary infections, atopic dermatitis, and elevated serum immunoglobulin e (ige) levels. treatment is largely aimed at controlling symptoms and preventing infections with no standard of care. there is a paucity of literature describing the utilization of biologic therapies in the ad-hies patient population. we present 3 patients from one family with ad-hies successfully treated with monoclonal antibody therapies targeted at il-5, il-4 and il-13. case descriptions: patient 1: 13-year-old female with stat3 lof c.1003c>t (p.arg335trp) with a history of atopic dermatitis and asthma requiring 2-5 steroid courses per year with frequent school absences. she developed a severe pruritic rash covering her upper body 18 months ago that failed to respond to antihistamines and topical antibiotics prescribed by her primary care provider. given her poorly controlled asthma and our concern for a follicular type morphologic variant of atopic dermatitis, dupilumab was initiated. her scoring atopic dermatitis (scorad) prior to initiation of biologic therapy was 53.8 and improved to 4.5 following 12 doses (24 weeks) of dupilumab with clear dramatic improvement in her skin and quality of life ( figure 1 ). she also reports decreased asthma severity with no steroid courses, reduced albuterol usage, and significant decline in school absences since initiation of dupilumab. patient 2: 17-year-old female with stat3 lof c.1003c>t (p.arg335trp) who is the sister of patient 1. she has a history of severe asthma requiring frequent emergency department visits, hospitalizations, and 4-5 steroid courses per year despite therapy with high-dose fluticasone-salmeterol. spirometry prior to april 2017 demonstrated an obstructive pattern with an fev1 ranging from 51-64%. she was initiated on mepolizumab in april 2017. subsequent spirometry demonstrates an fev1 average of 115% with a range of 96-126%. she had one hospitalization in early 2018 but otherwise no hospitalizations for asthma since initiation of biologic therapy. patient 3: 15-year-old female with stat3 lof c.1003c>t (p.arg335trp) who is the paternal first cousin of patients 1&2. she has a history of severe atopic dermatitis with associated pruritus and picking behaviors, poorly controlled despite daily triamcinolone application. she previously failed ultraviolet therapy and crisaborole. she also has a history of severe asthma requiring 2-5 steroid courses per year despite high-dose fluticasone-salmeterol. dupilumab was started in may 2019. scorad prior to initiation monoclonal antibody therapy was 80.3 and declined to 21.2 following 13 weeks (7 doses) of dupilumab therapy with marked improvement in skin appearance and pruritus ( figure 2 ). discussion: we present three cases of ad-hies caused by stat3 loss-of-function mutations treated successfully with monoclonal antibody therapies targeted at il-5 or il-4 and il-13. to the best of our knowledge, there is no published data describing the use of these biologic agents in the treatment of ad-hies. future studies are needed to clarify the role of these cytokines in the pathogenesis of ad-hies and to elucidate clinical indications for biologic therapy in this patient population. informed consent was obtained from all individual participants included in the study. abstract/case report text ctla-4 is a potent inhibitor of t cell proliferation that competes with costimulatory receptor cd28 for its ligands cd80 and cd86 expressed on antigen presenting cells. heterozygous loss-offunction mutations in ctla-4 have been identified in patients with lymphocytic infiltration of multiple nonlymphoid organs (lo et al). the patient is a 2-year-old jordanian male born at term to nonconsanguineous parents, hospitalized at 1 mo for lll pneumonia, and at 2 mo and at 4 mo he was evaluated in the ed and diagnosed with non rsv-bronchiolitis with lll infiltrate thought to be secondary to atelectasis. at 2 yo he developed lll pneumonia and respiratory failure requiring picu admission. he was treated with ceftriaxone. after discharge, he had 6 weeks of intermittent fever, progressive fatigue, productive cough, and ftt. he received courses of cefdinir, clindamycin, and tmp-smx without improvement. chest ct revealed left lung consolidation, lll bronchiectasis, lul tree in bud opacities, and hilar lymphadenopathy. bronchoscopy with bal revealed no bacterial growth and no acid-fast bacilli. 16srrna ngs was positive for h. influenzae. lung biopsy demonstrated acute and chronic bronchiolitis with bronchiolitis obliterans and intraluminal polyps, with lymphocytic infiltration involving the bronchi and bronchioles. the lung parenchyma showed airspace filling with foamy macrophages and chronic interstitial inflammation. acid fast and fungal stains were negative. he was treated with systemic steroids for bronchiolitis obliterans with noted improvement. the severity of lung disease at such an early age prompted an immune evaluation. sweat test, anca, anti-pr3 and hiv were negative. total immunoglobulins were normal for age, and titers to s. pneumoniae, diphtheria and tetanus were protective. lymphocyte enumeration revealed elevated t and nk cell numbers for age. lymphocyte proliferation to pha, pwm, candida and tetanus were normal. dihydrorhodamine assay was normal. b cell phenotyping was normal. there was normal expression of cd69, hla-dr and cd25 on activated t cells; of note the patient was on systemic steroids when tested. invitae 207 gene pidd panel revealed a variant in ctla4: c.326g>a (p.gly109glu) that has been shown to be pathogenic in one patient (schawb et al). flow cytometry showed normal frequency of t follicular helper cells and t regulatory cells compared with controls, however ctla-4 expression by t regulatory cells was lower than control. due to the severe and progressive nature of the patient's lung disease, therapy with 3x weekly azithromycin and abatacept 50mg sq weekly was initiated. we report a case of ctla-4 haploinsufficiency presenting with recurrent pneumonia and bronchiolitis obliterans in a 2-year-old child. based on patient registry data, our patient appears to be the youngest child diagnosed with ctla-4 haploinsufficiency reported in the literature to date (schawb et al). notably, our patient l a c k s o t h e r f e a t u r e s c o m m o n l y d e s c r i b e d i n c t l a -4 haploinsufficiency, including autoimmune cytopenias, gastrointestinal disease, lymphoproliferation, and hypogammaglobulinemia. this case illustrates the importance of consideration of this diagnosis in young children with severe lung disease without other evidence of immune dysregulation. our hope is that prompt recognition and early treatment administration will prevent disease progression and further decrease in pulmonary function. splenectomy, he had an episode of pneumococcal meningitis at age 35, and sepsis of unknown origin at age 55. over the past several years he has developed chronic tinea corporis, onychomycosis, and otitis externa infections despite numerous antimicrobial regimens. at age 47, the patient developed urinary retention, walking, and balance difficulties. he was found to have diffuse white matter changes on mri, elevated wbc, and positive oligoclonal bands. initially, he was diagnosed as progressive ms treated with steroids with partial improvement. csf microbiology studies including afb stains, bacterial, fungal, mycobacterial cultures, cryptococcal antigen, vdrl, t. pallidum particle agglutination (tppa), as well as, pcr for cmv, ebv, vzv, enterovirus, hsv 1-2, jc virus and t. pallidum were all negative. peripheral blood studies included mycobacterial blood culture, pcr for cmv, ebv, hhv-6, in addition to serology for cryptococcal antigen, and coccidioides species, all of which were negative. additional neurological complications include granulomatous uveitis and oscillopsia, which he developed around age 47. immune evaluation performed at age 51 revealed low igg2 and igm, and the patient was started on 30 grams of monthly ivig. cbc with differential was notable for normal monocyte count and thrombocytopenia, mild neutropenia (table1). immunophenotyping revealed absent b cells and nk cells, while the cd8 t cells were elevated. cd4 t cells were normal (table 1) . at age 55, whole-exome sequencing identified a heterozygous missense mutation in gata2 c.1186c>t, p.(arg396trp). after the diagnosis of gata2 haploinsufficiency, he was found to have myelodysplastic syndrome with multilineage dysplasia (mds-mld) on bone marrow biopsy. he is currently awaiting bone marrow transplant discussion: we present a 55-year-old male with cytopenias, splenomegaly, leukoencephalomyelopathy, granulomatous uveitis, and recurrent fungal infections found to have a pathogenic heterozygous missense mutation in gata 2. leukoencephalomyelopathy in gata 2 haploinsufficiency has been associated with jc virus and ebv infection. our patient did not have any evidence of a chronic csf infection. to our knowledge, myelopathies have not been reported with gata2 c.1186c>t, p.(arg396trp). this case highlights the variable nature of presentation in gata 2 haploinsufficiency, and the need for clinical awareness of this entity in order to facilitate early diagnosis and appropriate therapy. immunoglobulins* white blood cells 4.3 x10*9 /l (n: 4-11) magnetic resonance imaging (mri) of the brain and spine showed numerous enhancing parenchymal nodules (figure one). brain or spinal biopsy was requested, but not recommended by our neurosurgery service. lumbar puncture evaluation was performed. cerebral spinal fluid showed no bacterial or fungal elements. both quantiferon gold for tuberculosis and three consecutive sputum cultures for acid fast bacilli were negative. he continued his sirolimus and the intravenous immunoglobulin replacement was increased to two grams/kg. the patient was cleared from respiratory isolation and discharged after two weeks in our facility with mild improvement in his neurologic status. within five days he was sent to a nationally renowned hospital. at this facility, extensive evaluation for his neurologic deficits were performed including culture and pcr for bacteria, virus, mycobacteria from csf bone marrow, lymph node, blood, and induced sputum. these were noncontributory. he was given high dose corticosteroids for two days. one of our facility's sputum cultures was reported with acid fast bacilli. but sputum mycobacterium tuberculosis pcr was negative. repeat mri scans of the brain and spine showed improvement (figure 2), so no brain biopsy was performed. he was sent back to our facility with the recommendation to start a targeted pi3kinase inhibitor on compassionate grounds as he was not eligible for the clinical trial because his weight was less than 45 kg. but pretreatment abdominal ct revealed multiple low-density lesions scattered throughout the liver (figure 3) not previously seen on prior noncontrast ct four months prior. discussion: although this gain in function mutation of the pi3kδ signaling pathway disorder has been well characterized, this is a rare report of a patient with pasli immunodeficiency with central nervous system and later liver lesions pet imaging showed subcarinal, mediastinal, retroperitoneal lymphadenopathy, splenic enlargement to 23cm and bilateral lung nodules ( figure 1 ). excisional biopsies of left axillary and left lower lobe of lung were performed and showed low-grade b-cell lymphoma (mucosal) and underlying lymphoproliferative disease. invitae alps and cvid panels (37 genes) revealed a heterozygous variant of unknown significance in exon 3 of fas (c.323a>g(p.asp108gly) unlike most fas mutations causing alps, this mutation is in the extracellular region rather than the death domain2. the c.323a>g variant has been reported in a single patient with alps phenotype, affecting fas protein function by inhibiting binding to fas ligand (fas-l), reducing fas-l induced apoptosis2. this fas c.323a>g mutation was found in alps affected brother and was absent in the unaffected father. the patient's mother passed away prior to testing. since diagnosis, the patient's malt lymphoma has been treated with rituximab weekly for the first month and then monthly. he continues to receive monthly ivig for hypogammaglobulinemia. after two years, ct demonstrates a significant decrease in pulmonary nodules and splenomegaly ( figure 2 ). the patient's forced vital capacity (fvc) improved from 3.78l (67% predicted) to 4.39l (79% predicted). conclusion: we report the first case of malt lymphoma seen in a patient with alps. it is unknown whether the unique fas c.323a>g mutation in the non-death domain contributes to malt lymphoma progression. we propose malt lymphoma is a malignant transformation of chronic inflammation that has the potential to occur in patients with alps. in the future, improved knowledge of mechanistic pathways of inflammation in lymphoma development and progression is important in the optimal management of alps. abstract/case report text background wiskott-aldrich syndrome (was) is a rare x-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, infections, autoimmunity and increased risk of hematological malignancies. gene therapy (gt) using autologous cd34+ cells is an emerging alternative treatment with possible advantages over standard allogeneic hematopoietic stem cell transplant. we report the outcomes of a phase i/ii clinical trial in which 5 was patients underwent gt using a self-inactivating lentiviral (sin-lv) vector expressing the human was cdna under the control of a 1.6kb fragment of the human was promoter. subjects and methods: five patients with severe was (clinical score 3-5) were enrolled (table 1) . cd34+ cells were transduced ex-vivo and reinfused after conditioning with busulfan and fludarabine. two subjects (p4, p5) had autoimmunity pre-gt, manifested as skin vasculitis and autoimmune cytopenias. results: all subjects were alive at median follow-up of 5.1 (range 2.8-6.3) years. multi-lineage vector gene marking was sustained over time. all had clinical improvement of eczema, infections and bleeding diathesis. was protein (wasp) expression was increased over baseline but remained below normal levels. proliferation of t cells in response to anti-cd3 improved post-gt. humoral immune deficiency improved, with normalization of igm, and independence from ig replacement and vaccine responses in those tested. platelet levels increased to >50 x 103 cells/ul in only the two subjects with a vcn ≥2 in transduced stem cells. podosome formation in monocyte-derived dendritic cells was near absent pre-gt and improved in all subjects post-gt, but only reached healthy control levels in the 2 subjects with highest vcn. in contrast to other trials using this sin-lv, two patients (p4 and p5) had flares of autoimmunity post-gt, offering the opportunity to study the poorly understood mechanistic features of immune dysregulation in this disease. selfreactive vh4-34-expressing b cells and cd21lo b cells remained elevated in most patients. however, despite wasp expression in foxp3+ tregs, those with autoimmunity had poor numerical recovery of t cells and tregs at the time of clinical symptoms ( fig 1a) . in addition, il-10 producing regulatory b cells (bregs) were highly deficient pre-gt, recovered in subjects who did not experience autoimmunity, but failed to recover in p4 and p5 ( fig 1b) . moreover, transitional b cells, which are enriched in bregs and are potent inducers of treg populations, also recovered poorly in those two subjects ( fig 1c) . there have been neither severe gt-related adverse events nor abnormal clonal expansion in transgene-marked cells to date. conclusion in summary, our data confirm and extend the safety and efficacy of gt in correcting disease manifestations associated with was, with the longest overall follow-up reported so far in studies using sin-lv. in addition, our findings suggest that higher vcn is needed in order to correct myeloid compartments such as platelets and monocytes. finally, we report the novel finding of the restoration of bregs and suggest that recovery of this compartment, along with tregs, is protective against development of autoimmunity post-gt. overall, these data suggest a mechanism for breakdown of immune tolerance in was with important therapeutic implications and prognostic value. this is an 8-year-old hispanic female who initially presented with failure to thrive, recurrent fevers and intermittent cough with episodes of perioral cyanosis. symptoms started at age 6 months and were attributed to recurrent viral and bacterial infections. at 14 months old, she was hospitalized with fever and hypoxemia (o2 saturations 70%). cxr showed prominent interstitial lung markings and she was diagnosed with pneumonia. ct scan confirmed cxr findings and ruled out anatomical anomaly. she was lost to follow up for 2 years, and re-presented with worsening respiratory status. a repeat ct scan demonstrated worsening interstitial thickening. immune workup, including quantitative immunoglobulins, ch50, lymphocyte subsets and vaccine response titers (pneumococcal and tetanus), was unremarkable, except for elevated igg levels. genetic testing for surfactant dysfunction mutations was negative. thoracoscopic lung biopsy revealed interstitial fibrosis, pas-positive granular alveolar proteinosis, type ii cell hyperplasia, and lymphoid follicles. at age 6, she was admitted for a pericardial effusion. rheumatology was consulted for evaluation frequent fevers and persistently elevated inflammatory markers, with concern that the pericarditis was autoinflammatory. she had an elevated ana (>1:2560 homogeneous pattern), il-6 (378.88 pg/ml), and igg (2020 mg/dl) at that time. she had an atypical anca pattern with positive myeloperoxidase antibodies. anti dsdna, smith and scl70 were negative. she was treated with steroids and hydroxychloroquine with some improvement in her oxygen requirement. one year later she had an additional episode of pericarditis, treated with colchicine. a few months later, she was admitted with newonset gross hematuria and elevated serum creatinine (to 2 mg/ dl). kidney biopsy showed anca vasculitis with glomerulonephritis (75% crescents, no scarring or fibrosis). she provisionally received a diagnosis of microscopic polyangiitis, with lung and kidney involvement. she did not have peripheral vasculopathy. she was started on cyclophosphamide, rituximab, and iv steroid pulses. cyclophosphamide was discontinued due to recurrent episodes of posterior reversible encephalopathy syndrome (pres) after infusion. her igg level decreased as she developed nephrotic range proteinuria. a primary immunodeficiency genetic panel was sent to evaluate for monogenic immune dysregulation syndromes and revealed a tmem173 gene mutation (c.463g>a) which has previously been reported in 4 other subjects with savi (stingassociated vasculopathy of infancy syndrome). sting is a cytosolic dna sensor that leads to type i interferon production upon stimulation. this gain-of-function mutation was confirmed by measuring interferon signature gene expression at the nih (fig 1) , and her diagnosis was revised accordingly. the patient was started on a jak-inhibitor (tofacitinib) to block interferon signaling. unfortunately, the patient is now deceased, due to overwhelming infection and multi-organ system failure. conclusion: genetic testing can be crucial in aiding the diagnosis of complex patients with immune dysregulation and can provide an opportunity for targeted therapy, which should be employed as soon as able to stop disease progression. abstract/case report text background: activated phosphoinositide 3-kinase δ syndrome (apds-1) was first described in 2013 as a monogenetic immune dysregulation syndrome with a variable phenotype. increased sinopulmonary and herpesvirus infections are well described, but fungal infections such as candidiasis have been rare. to date, disseminated histoplasmosis has not been described. history: a 38 yo caucasian male who was previously diagnosed with common variable immunodeficiency (cvid) in late childhood due to recurrent sinopulmonary infections presented with recurrent fever, pancytopenia, severe splenomegaly, and lymphadenopathy. urine histoplasmosis antigen and beta-d-glucan were elevated. a bone marrow biopsy demonstrated granulomatous inflammation. transbronchial biopsy of a subcarinal lymph node was consistent with granulomatous disease. this led to a diagnosis of disseminated histoplasmosis. he was treated with amphotericin b and then 11 months of itraconazole, with improvement of his symptoms. he was admitted to the hospital about 7 years later when he presented with fatigue, fever, chills, dark urine, and scleral icterus. he was found to have an acute worsening of chronic anemia with a hemoglobin of 4.7 g/dl. due to elevated ldh, presence of schistocytes on peripheral smear, and undetectable haptoglobin, he was diagnosed with autoimmune hemolytic anemia, despite a negative direct coombs. a bone marrow biopsy specimen was hypercellular with marked erythroid predominance, with normal flow cytometry and no blasts identified. infectious workup was negative. ct chest during the workup revealed new right hilar and mediastinal lymphadenopathy, in addition to calcified right hilar and subcarinal lymph nodes, bronchiectasis, and stable hepatosplenomegaly. transbronchial biopsy of lymph nodes showed benign lymph nodes with calcified necrotizing granulomata and presence of non-viable fungal species, presumably "old" histoplasmosis. family history: family history was significant for mom dying at 29 years-old from undefined cns infection. immune labs: · panlymphocytopenia: absolute lymphocyte count of 380/ul, cd3+ t cells 283/ul, cd4+ 174/ul, cd8+ 99/ul, cd19+ 65/ul, cd16+cd56+ 25/ul. cd4+/cd8+ ratio 1.8 · decreased class-switched memory b cells and plasmablasts · elevated t central memory cells and activated (hla-dr+) cd4+ and cd8+ t cells · hemoglobin 10.3 g/dl, platelets 77,000/ul, anc ranging from 360/ul to 1610/ul · iga 107 mg/dl, igm 273 mg/dl; reportedly had low igg prior to initiating ivig in childhood · ebv pcr and cmv pcr negative genetics: · pik3cd (c.3061g>a), consistent with diagnosis of autosomal dominant apds-1. discussion: gain-of-function variants leading to increased pi3kδ activity have been shown to cause both b and t cell dysfunction, leading to impaired immunologic responses to bacterial and viral infections. recurrent sinopulmonary infections and herpesvirus infections are commonly seen and while mucocutaneous candidiasis has been reported in cohorts of patients with pik3cd, other fungal infections are not common. severe disseminated histoplasmosis infections have been described in primary immunodeficiencies characterized by signaling defects in the il-12/ifn-γ pathway, stat3 deficiency, cd40l deficiency, gata2 deficiency and in stat1 gain-of-function mutations. to our knowledge, disseminated histoplasmosis has not been previously reported in patients with pik3cd immunodeficiency. abstract/case report text the reported case represents the first case of nbas disease detected by newborn screening program for primary immunodeficiency, based on krec assay. the patient came to our attention due to the complete absence of krecs and normal trecs on dbs (dried blood spot) while hospitalized for low weight at birth (1,520 g), intolerance for enteral feeding, hepatosplenomegaly, slightly elevated liver transaminase, head and face eczematous dermatitis. during the 1st month, he also presented klebsiella pneumoniae urinary tract infection and methicillin-resistant staphylococcus aureus sepsis. peculiar phenotypic features including triangular face, proptosis, flat philtrum, mild retrognatia, hirsutism, loose and slightly wrinkled skin, and apparent reduction of subcutaneous fat were noticed at birth. complete blood count showed lymphocytopenia, marked hypereosinophilia. serum immunoglobulin g (igg) were markedly decreased, iga and igm were undetectable. extended immune-phenotyping showed complete absence of cd19+ cells, low count of cd8+ lymphocytes, and reduced natural killer (nk) levels. at 9month of age a colonoscopy was carried out for persistent diarrhea and reduced tolerance to enteral feeding. the histological examination of mucosal intestinal biopsies showed signs compatible with autoimmune enteropathy. for this reason immunosuppressive therapy with rapamycin was started without consistent clinical amelioration. many cvc-sepsis occurred in the last months, associated with persistent gastrointestinal symptoms and severe growth restriction. despite the absence of experience data in literature for nbas syndrome, we retain that hsct represents the only resolutive therapy for him. abstract/case report text case: a 31-year-old female with asthma and allergies presented to immunology clinic with a history of chronic fatigue and sinusitis. fatigue occurred daily every 2-3 weeks and was described as not feeling rested even after 12 hours of sleep. chronic sinusitis required 4-5 prolonged antibiotic courses per year. nasal cultures grew methicillin-sensitive and -resistant staphylococcus aureus and haemophilus influenzae type b (hib). three separate sinus surgeries over the prior few years reduced her sinus symptoms. other infectious history was significant for recurrent urinary tract infections with e. coli and klebsiella, recurrent otitis media as a child, and a diagnosis of transient hypogammaglobulinemia of infancy that resolved at 4 years of age. review of systems revealed axillary lymphadenopathy for 2-3 days twice per year not related to infection. she had longstanding eczema that responded to topical tacrolimus, multiple environmental allergies, and recently diagnosed asthma that improved with inhaled budesonide/formoterol. as a teenager she received allergy immunotherapy for a few years but stopped due to frequent adverse reactions. family history revealed that father died from cancer. physical exam was unremarkable. laboratory evaluation demonstrated normal igg 874 mg/dl, igm 133 mg/dl, iga 166 mg/dl, elevated ige 457 mg/dl, normal t and nk cell enumeration, mildly low total b cells (180 cells/mcl, 5.6%), and normal b cell subsets (cd19+igm+cd27-67%, cd19+igm+cd27+ 17%, cd19+igm-cd27+ 13%). tetanus antibody titer was protective, but hib antibody titer was undetectable at < 0.15 mcg/ml with marginal response after vaccination (0.53 mcg/ml). pneumococcal serotype specific igg levels (mayo) were mostly undetectable with 3 of 23 serotypes protective at baseline and only 4 protective post vaccination with pneumovax 23. cd3/ cd28 blastogenesis was poor. due to poor antibody response and continued sinus infections she was started on igg replacement. her fatigue and sinus symptoms improved moderately but she continued to require antibiotics and sinus ct scans continued to demonstrate significant disease. a focused exome sequencing panel was pursued and a novel heterozygous card11 variant was found (c.215g>t, p.r72l). discussion: the card11/bcl10/malt1 (cbm) complex is a critical signaling adapter that facilitates several downstream immune responses predominately through nf-kb. mutations in several different domains of card11 result in a clinical entity collectively referred to as card11-associated atopy with dominant interference of nf-kb signaling (cadins). cadins is associated with a broad range of clinical manifestations but most have marked atopy with infections, poor t cell proliferation, and varying levels of poor antibody response. both our variant (p.r72l) and a previously reported pathogenic variant in the same amino acid (p.r72g) involve a change from a charged arginine to a non-polar amino acid in the critical bcl10/ card11 binding interface. iκbα degradation was not present in b cells from our patient ( figure 1 ) confirming the functional defect in nf-kb signaling. thus, we present a novel variant that fits cadins both clinically and genetically. clinicians should be aware of cadins when patients present with recurrent infections in the setting of significant allergic disease. i b degradation assay. stimulation: 200 μl of whole blood was stimulated in a 5 ml facs tube with 50 ng/ml phorbol 12-myristate 13-acetate (pma; sigma, cat# p1585) at 37°c for 5, 10, or 20 min, at which point 4 ml of pre-warmed 1x lyse/fix buffer (bd, cat# 558049) was added. cells were fixed for 10 min at 37°c, centrifuged and washed twice with facs buffer (pbs supplemented with 2% fbs and 1 mm edta). staining and permeabilization: fc receptors were blocked for 5 min at rt (human trustain fcx; biolegend), followed by a 20 min stain on ice with anti-cd4 af647 (clone rpa-t4; biolegend), and anti-cd19 bv421 (clone hib19; biolegend). cells were washed with facs buffer and permeabilized for 30 min on ice with 1 ml phosflow perm buffer ii (bd biosciences, cat# 558052) that had been precooled to -20°c. after permeabilization, two ml facs buffer was added and the samples were centrifuged. after three additional washes, the cells were stained with anti-iкbα pe (clone 25/ikba/mad-3; bd biosciences) for 30 min at rt. samples were washed three times and data were collected on a cytek dxp10 flow cytometer. data were analyzed with flowjo software. abstract/case report text introduction: wiskott-aldrich syndrome (was) is a rare, but well-defined x-linked disorder. loss-of-function mutations in the was gene result in classic was and x-linked thrombocytopenia (xlt), while gainof-function mutations lead to x-linked neutropenia (xln). classic was phenotypic features include recurrent infections, microthrombocytopenia and eczema along with increased susceptibility to autoimmune disorders and malignancy. most males with classic was are diagnosed in early childhood and early death can result from its various clinical manifestations. case: we present a 32-year-old male who was referred to immunology for hypogammaglobulinemia. as an infant he had moderate eczema, and at the age of two was diagnosed with immune thrombocytopenia (itp) with baseline platelets of 50-60 x 10^9/l. infectious history was notable for one episode of pneumosepsis and recurrent otitis media, influenza, and herpes labialis infections. around the age of 22, he was diagnosed with common variable immunodeficiency (cvid) based on the finding of low immunoglobulins. he developed diffuse large b cell lymphoma at age 26, and was treated with cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab (chop-r). at age 32 he developed abdominal pain with bloody stools. investigations confirmed an endoscopic and pathologic diagnosis of ulcerative colitis. due to the severity of his disease, he has required maintenance therapy with vedolizumab. he was again noted to have hypogammaglobulinemia at which point he was referred to immunology at our centre. there was no significant family history of immunodeficiency, malignancy or autoimmunity. blood work was notable for normal white blood cell and lymphocyte counts, platelets of 60 x 10^9 g/l, low igg at 4.82 g/l (7.0-16.0 g/l) with normal iga, igm and ige. lymphocyte subsets including t, b and nk cells were within the normal range. genetic testing was performed and he was found to have a known pathogenic mutation in the was gene (c.1453g>a, p.asp485asn) which has been previously reported in association with was and xlt. he has since been placed on immunoglobulin replacement and has been referred for consideration of hematopoietic stem cell transplantation. discussion: was is a rare syndrome that can have a similar phenotype to other immunodeficiency disorders including cvid, omenn syndrome and ipex (immune dysregulation, polyendocrinopathy, x-linked). individuals with cvid present with hypogammaglobulinemia and recurrent infections, and these individuals also have an increased susceptibility to autoimmune disorders, gastrointestinal disease and malignancies, especially lymphoma. although eczema is a common disorder, its presence in addition to features of early onset thrombocytopenia, immunodeficiency, autoimmunity and/or malignancy in male patients should heighten the suspicion for was. it is important to make the diagnosis of was as hematopoietic cell transplantation and gene therapy are potentially curative treatment options. abstract/case report text secondary immune deficiencies (sid) are caused by varied mechanisms and are common in patients with hematological malignancies such as chronic lymphocytic leukemia (cll) and multiple myeloma (mm). in this setting, both the disease and its treatment (such as b cell ablation therapy) contribute to the development of secondary antibody deficiency. infections remain a major cause of morbidity and mortality in cll and mm patients. this underscores the need for early recognition and stratification of risks in order to guide appropriate treatment, including immunoglobulin replacement therapy (igrt). new guidelines for the use of human normal immune globulins in sid patients were implemented by the european medicines agency (ema) in 2019. despite these new guidelines, significant variations remain across european countries in the assessment and approaches aiming to achieve reduction in infection burden, including different strategies for initiation, dosing and discontinuation of igrt. the same is true for north america where igrt is widely used off-label to prevent infections in patients with sid due to hematological disease or other reasons. in order to address this variability, a task force comprising both immunologists and hemato-oncologists drafted 20 statements aiming to test for consensus. statements were related to six major areas: definition of infections, measuring igg levels, initiating igrt, igrt dosing, scig usage and discontinuing igrt. this was followed by an international delphi consensus exercise in three rounds which aimed to develop recommendations on how to diagnose, treat and follow-up patients with antibody deficiency associated with hematological malignancies. the first delphi round consisted in testing the 20 statements with a panel of sid specialists and subsequently their comments were used by the task force to refine the statements. in the second delphi round, the refined statements were presented via phone interviews to the same panel to assess their level of agreement with each statement (ranging from 1 "i totally disagree" to 6 "i totally agree"). consensus was considered to be reached per statement if 70% of the experts agreed with each statement overall. the cut-off for overall agreement was 4 "i somewhat agree". if the expert chose level 4 or less the reasons underpinning his/her choice were discussed. consensus was achieved for all statements on level 4 ("i somewhat agree"). only 5 statements did not achieve consensus on level 5 "i mostly agree". in delphi round 3, panelists who had not "mostly agreed" with these five statements were given the opportunity to reconsider their assessment based on the feedback from other panelists, which was shared with them. the panelists then chose to maintain or refine their assessment. analysis of the full results on the six key areas identified by the task force will be presented at the conference to offer recommendations and help guide the management of sid in patients with hematological malignancies. abstract/case report text introduction: mast cells (mcs) are hematopoietic-derived immune cells, whose precursors migrate within tissues reaching maturation and differentiation. masitinib, a selective tyrosine kinase inhibitor, is efficient in controlling the survival, differentiation, and degranulation of mcs. aim: to optimize mast cell-differentiation from human bone marrow (bm) hematopoietic stem cells, and to find best cell culture conditions for proliferation, differentiation, and maintenance of mcs, which is important when studying particularly mcs' response to cytotoxic compounds. material-methods: to produce mcs in vitro, the first method (m1) we used was a modified semi-solid culture method (1). briefly; human bm mononuclear cells (mncs) were obtained with ficoll gradient from bm sample of a patient with idiopathic thrombocytopenic purpura. colonyforming unit (cfu)-mast was developed from mncs in methylcellulose medium supplemented with scf (200ng/ml) + il-6 (50ng/ml), and il-3 (1ng/ml; only first week). 5-6 weeks later mast cell colonies were transferred into suspension cultures, in which mcs matured and multiplied up to 7-8 weeks and were used in experiments till 10th week of culture. on the other hand, in our second method (m2); mncs were separated by ficoll, seeded in 6 well-plates with imdm containing fbs 2%, pen/ strep, and a little amount of methylcellulose, and incubated at 37 o c, 5%co 2 . cultures were then supplemented with imdm (fbs 1%) + scf (100ng/ml) + il-6 (50ng/ml) on day 4; and imdm (fbs 2%) + scf (100ng/ml) + il-6 (50ng/ml) + il-3 (1ng/ml) on day 9. beginning on day 18 till the end, imdm (fbs 2%) + scf (100ng/ml) + il-6 (50ng/ml) were added to cultures. for both methods, morphological assessment of colonies/cells were evaluated under an inverted microscope (figure 1 and 2). verification of mcs was performed by immunoflorescence staining for anti-tryptase andchymase antibodies, and by toluidine blue staining. macrophages were verified by anti-cd-68 immunoflorescence staining. mcs were exposed to masitinib or dmso for the evaluation of dose-related effects of masitinib, and cytotoxicity was evaluated by mtt assay. results: in m2, culture conditions were easier to handle compared to m1. in m2, high amounts of mcs in immature and pre-mature forms were appeared as early as 15-18 days, and peak levels of proliferation rate was around 2-4 weeks of culture, which was about 3 weeks earlier than m1. culture could be maintained till 10 weeks in both methods. although mcs are non-adherent cells, in liquid method adherent bm cells such as fibroblasts, endothelial cells and mesenchymal stem cells have adhered to the plate and grown up, providing an attachment site for mcs and serving as a natural bm nest, mimicking in-vivo environment, for mcs to grow and proliferate ( figure 2 ). attachment of mcs has provided medium exchange available without changing culture dishes. when mcs were exposed to masitinib (0.5, 1, and 2 μm/μl), approximate survival rates were 75%, 72%, 69%, respectively. discussion: in our liquid medium method, the adherent bm cells not only provided a natural nest supporting mc development and differentiation, they also served as an attachment site for mcs. as the cells slightly adhered, when trypsinized shortly, they easily detached and used for experiments. and we also report for the first time that adding a little amount of methylcellulose to the liquid medium provides ease of aggregation of cfus, and easy development of mcs. we suggest that our liquid culture may be superior to semi-solid method, that it is faster and easier to handle. in 2 studies subjects crossed over to subcutaneous (sc) igiv-c 10%, and in the third study crossover was to immune globulin sc (human), 20% caprylate/chromatography purified (igsc 20%). a total of 95 pi patients from these 3 studies were included in the poppk analysis and 1841 serum igg concentrations were included in the final pk analysis. the pk of igg following iv and sc administration was adequately described by a two-compartment model with first-order elimination from the central compartment. administration of igiv was modeled as an infusion directly into the central compartment. absorption of exogenous igg from the depot site of sc infusions into the central compartment was modeled as a first-order process with an absorption rate constant (ka). the full model was constructed by incorporation (forward selection process) of covariates of interest into the model. after completion of the covariate model development, the final model showed that igg pk was not influenced by (a) the igsc formulation used in the different studies (10% vs. 20%), (b) gender, and (c) age (pediatric vs. adult). body weight was identified as a significant covariate having an effect on clearance and volume of distribution. based on the final pk results, serum clearance of igg for the reference population was estimated to be 0.150 l/day. the volume of distribution of the central and peripheral compartments accounted for 3.06 l and 1.93 l, respectively. the intercompartmental clearance was 0.474 l/day, and the absorption constant from the depot (ka) was 0.246 day-1. the absolute bioavailability of igg after sc administration was calculated as 70.5%. the developed method was used to evaluate alternative dosing intervals following sc administration. the equivalent of a weekly igsc maintenance dose administered 1, 2, 3, 5, or 7 times per w e e k , o r b i w e e k l y p r o d u c e d o v e r l a p p i n g s t e a d y -s t a t e concentration-time profiles and similar area under the concentration versus time curve (auc), maximum concentration (cmax), and minimum concentration (cmin) values. the results of the evaluation and simulations for igg exposure following a switch from igiv-c 10% dosing (every 3-or 4-weeks) to sc dosing further suggest that a range of dose-adjustment factors (daf), from 1:1 to 1:1.37 would be sufficient to provide clinically effective trough igg concentrations throughout the course of treatment at various treatment frequencies. current us product labeling for igsc 20% specifies a daf of 1:1.37 for transitioning immune globulin dosing from iv to sc, and specifies igsc 20% dosing frequencies of weekly or more frequently (2-7 times per week). in this poppk analysis all sc dosing regimens evaluated theoretically would provide viable alternative administration options for maintaining adequate immunoprotection in pi patients with dosing flexibility over a range of regimens. however, in 50% or more, no causative gene can be found going down to undefined inflammatory syndromes (uis). anti-il1 drugs (ail1d) revolutionized some il-1 mediated diseases, such as traps, caps, hyper-igd/mkd and fmf. nevertheless, treatment response among disorders are not the same as well as no specific study was designed for uis. papa et al, 2019, recently suggested the use of anakinra for the treatment of uis, especially those refractory/intolerant to colchicine with severe or very symptomatic phenotype. this paper aims to retrospectively report for the first time the experience with canakinumab in monogenic and multifactorial disorders in a single, private center in brazil. patient and methods: patients's records that received canakinumab from january 2016 to december 2019 at clinica croce, ima-brazil, were revised. demographic and clinical data were extracted and descriptively described. all statistical analysis are presented as: average (minimal; maximum; standard deviation). results: a total of 23 patients with autoinflammatory diseases were enrolled and 65% (n=15) are female. of them, 65% (n=15) patients had a monogenic disease: 26% (n=6) caps, 21% (n=5 fmf), 4% (n=1) mkd, 4% (n=1) homozygous nlcr4 and 4% (n=1) pami syndrome. multifactorial disorders were 39% (n=9) patients : 8% (n=2) recurrent idiopatic pericarditis, 8% (n=2) schnitizler syndrome and 21% (n=5) uis. the average age of the first symptoms was 12,51 years (0;69;19,78) and the average age of diagnosis was 24,43 years (0;72;21,76) while the aveage of diagnosis delay was 11,59 years (1;59;15,16). all patients had used, prior to anti-il1, corticosteroids with 100% prevalence of cushing syndrome and 69% (n=16) tried at least one steroid sparing agent without clinical success due to: intolerance or non-effective disease control or side effects. in the fmf group (n=5) 100% tried colchicine prior to canakinumab and this drug was not effective to 60% because of amyloidosis status and in 40% colchicine-induced hepatitis was observed. canakinumab was effective for disease control in 75% (n=23) considering: control of clinical manifestations, amyloidosis reversion and normalization of acute reactants markers. the only side effect observed during the follow up were acute flu-like symptoms and psicomotor agitation (33,34% , n = 8). the average time of follow up is of 12,51 months (1;37;12,46). canakinumab could be discontinued in just one patient with uis. conclusions: this is the first report of canakinumab use for autoinflammatory disorders in brazil. canakinumab is an effective and safe drug for monogenic and multifactorial disorders control. no serious adverse effect could be observed in the 3 years maximum follow up of this drug. neither, no specific infectious disease more prevalent in south america, such as yellow fever, dengue, zika or chikungunya was observed. abstract/case report text a 21-year-old caucasian male with autosomal recessive hyper igm syndrome type 2 (higm2) due to aicda mutation, diagnosed at age 1, presented with a newly developed mediastinal mass. he receives routine ivig, pulmonary function tests (pft's) and chest x-rays. at age 16, patient was noted to have cervical and inguinal lymphadenopathy. ct scan indicated left mediastinal, hilar and pleural lymphadenopathy with soft tissue infiltration around the descending thoracic aorta and esophagus. biopsy indicated no evidence of a lymphoma or infection. 5 years after initial workup, routine pft's showed a declining diffusion capacity by 50%. patient complained of intermittent chest pain but displayed no clinical symptoms of cough, dyspnea, dysphagia or reflux. ct scan which revealed an extensive illdefined soft tissue mass extending from the thoracic outlet to the level of the esophageal hiatus that encased vascular structures resulting in narrowing and occlusion of left upper lobe pulmonary artery and left lower lobe pulmonary arteries respectively. imaging demonstrated homogenous ventilation to bilateral lungs and decreased perfusion in the left lung compared to the right lung. infectious workup was negative for atypical infections. biopsy revealed miced cellular infiltrate with no predominenant cell type or evidence of malignancy, consistent with previous lymph node biopsy 5 years prior. cd20 and cd3 stains revealed aggregates and scattered b-cells and t-cells, respectively. removal of mass was proposed but due to the ambiguous borders and location, surgical excision was not possible. patient was given 4 doses of rituximab (75g), 3 doses 10mg/kg pulse steroids 18 hours apart, and daily sirolimus (level was adjusted based on sirolimus level). follow-up ct scan indicated significant interval improvement with 50-60% reduction of the soft tissue mass. blood flow in the left lower lobe pulmonary artery has still not returned. this may be due to collaterals and may be a separate problem from compression due to the mass. cytotoxic t-lymphocyte antigen 4 (ctla-4) is an inhibitory immune regulator critical for governing t and b cell homeostasis. heterozygous ctla4 mutations can cause a syndrome of immune dysregulation with a variable clinical phenotype including hypogammaglobulinemia , autoimmune cytopenia and endocrinopathies, lymphoproliferation, predisposition to malignancy, tissue specific lymphocytic infiltration of brain, lung and gi tract as well as colitis. methods: we retrospectively reviewed medical records of all patients with ctla4 haploinsufficiency evaluated at the nih between 2014-2019. a pathologic variant in ctla4 was confirmed in all patients. we analyzed frequency of campylobacter species detected in the stool samples by pcr based biofilm rapid array as well as reflex bacterial stool and blood cultures when available. results: forty-six patients aged 8-75 years were evaluated at the nih between 2014 and 2019. six of 46 patients (13%) had at least one episode of campylobacter species associated acute or worsening diarrhea, with one patient also having campylobacter bacteremia. all patients with positive campylobacter species in stool samples had clinical histories and/or endoscopic biopsy findings consistent with enteropathy or colitis predating the incidence of campylobacter infection. two of the six patients (33%) had recurrent or chronic campylobacter infection, while four of the six patients (67%) had multiple gastrointestinal pathogens detected by stool pathogen screening at various times. conclusions: campylobacter species infection of the gastrointestinal tract seem to occur at an increased incidence in our ctla4 haploinsufficient cohort. to the best of our knowledge, this is the initial report for the association between ctla4 haploinsufficiency and campylobacter species infection of the gastrointestinal tract. although ctla-4 is a critical immune checkpoint involved in mucosal immune homeostasis and gut microbiota-immune system cross talk, the underlying mechanism predisposing to campylobacter infection in ctla-4 deficient patients remains to be explored. our study suggests screening of stool for campylobacter species in patients with ctla4 haploinsufficiency associated enteropathy. (62) submission id#806771 abstract/case report text ikaros transcription factor and ikaros family members are critical for development of lymphocytes and other blood cell lineages. full length ikaros (isoform 1) contains six c2h2 zinc fingers (zf), four nterminal dna binding zf and two c-terminal dimerization zf. somatic ikaros mutations and deletions have been associated with increased predisposition to b-acute lymphoblastic leukemia (all) as well with poor disease prognosis. recently, germline ikaros mutations affecting the n-terminal dna binding domain and acting in a haploinsufficiency or dominant negative manner were reported to be associated with common variable immunodeficiency (cvid) and combined immunodeficiency (cid), respectively. herein we describe a novel set of germline heterozygous ikaros allelic variants affecting the c-terminal dimerization domains in four unrelated families. clinical manifestations include hematopoietic cytopenias presenting as evans syndrome, and hematologic malignancies including t-cell all and burkitt lymphoma; other manifestations observed were b-cell lymphopenia and hypogammaglobinemia, but recurrent or severe infections were not prevalent or characteristic. we demonstrate that mutants affecting dimerization abolish ikaros homodimerization as well as heterodimerization with ikaros family members aiolos and helios. these variants also affect dna binding at dimerization sites and pericentromeric targeting. opposed to previous allelic variants reported, dimerization changes alter post-translational sumoylation and gene transcription regulation. our data show that mutations affecting ikaros dimerization are mainly associated with cytopenias and/or malignancies, have a different mechanism of action than previously reported variants, present with incomplete clinical penetrance, and contribute to the growing spectrum of genotype-phenotype ikaros associated diseases. introduction: there has been much discussion regarding the return of secondary findings in genetic sequencing research. opinions differ on whether researchers should return secondary findings to participants at all and if so, what the best method is to do so. we have opted to systematically identify and return pertinent secondary findings to participants in our cohort of patients with immune-mediated diseases that undergo exome sequencing. additionally, exome sequencing may determine multiple or other genetic diagnoses in addition to the primary diagnosis, which we call "incidental findings." here, we discuss the secondary and incidental findings discovered in our cohort thus far. methods: individuals in our protocol underwent consent for exome sequencing, including a discussion of the possibility of secondary findings. exome sequencing data was analyzed, and variant pathogenicity was scored using the acmg criteria (richards et al); variants determined to be likely pathogenic, pathogenic, or otherwise clinically important were confirmed via clia-certified sanger sequencing. confirmed variants were returned to participants. we then queried internal databases for cases involving secondary and incidental findings. results: as of november 2019, exome sequencing, interpretation and reporting had been completed for 629 participants. we detected a total of 18 secondary findings in 17 (2.7%) participants, including variants in apob, brca1(2), brca2 (5), dsp, fbn1, kcnh2, ldlr, mybpc3 (2), ryr1, pkp2 (2), and vhl. additionally, we detected possible dual/multiple genetic diagnoses in 18 (2.9%) participants, some of which explained an unusual clinical presentation or symptom. these included individuals with variants in multiple immune-related genes, including one individual with variants in gata2 and tnfrsf1a, and those with variants in genes related to multiple organ systems, including an individual with variants in ifngr1 and sco2. discussion: exome sequencing in this cohort detects not only important secondary findings, but also discovers a significant portion of individuals with multiple genetic diagnoses. notably, exome sequencing may provide further context or explanation for unusual phenotypic presentation and help determine specific symptom etiology even when a primary genetic etiology is already known. additionally, these secondary and incidental finds may be important to consider when delineating risks and symptoms of novel or recently-discovered conditions. abstract/case report text background: immune dysregulation and lymphoproliferative disorders including alps like disease, hlh ebv driven lymphoproliferative disease leading to rare lymphomas require a multidisciplinary approach utilizing expertise in immunology and hematology/oncology to care for these patients as we learn the molecular etiology of their underlying disorders. at texas children's hospital, the immunology lymphoproliferative evaluation and diagnostic (ilead) clinic was created to provide a comprehensive clinical and research approach to caring for patients with these rare disorders. in an effort to streamline care and access, we recently on-boarded an advanced practice provider (app) . methods: a chart review was conducted 6 months before and after onboarding the app for ilead patient visits. we reviewed the following patient care and access parameters to determine increase in efficient and effective patient care as well as improved access to the clinic. these parameters included: referral process, time of referral placement to appointment, number of patient visits, wait time in clinic, lab interpretation and reporting time for disseminating results to families, and collaboration process with other specialties. results: within 2 months of the app starting our average wait from placing the referral to first appointment fell by an average of 45%. in addition, we created an algorithm to prioritize patients with immediate need to be seen. by streamlining the referral process and patient priority, we developed a "pre-clinic" conference process by which all patients are reviewed and preliminary plans are made prior to the patient's arrival. this has translated into our ability to increase the number of patients seen in clinic from 4 to 6 and decreased the wait time in clinic by approximately 30 minutes. since the app started, no patient has been in clinic for more than 60 minutes. this has also led to an increase in rvu generation. in terms of efficiency in patient care, all labs are now ordered while in the room with the patient by the app and physician providers. in turn, all labs are resulted directly to the app who reviews labs, collaborates with physicians for care and reports to families in a timely fashion within 1-2 weeks of labs being resulted compared to greater than 1 month previously. to improve collaborator communication and post visit plans, a post-visit clinic summary was created. this has been effective in reducing the time to other specialty referrals, follow up visits and effective care for ongoing clinical needs. conclusions: the addition of an app in our ilead multidisciplinary clinic which provides specialized care for patients with immune dysregulation and lymphoproliferative disorders effectively increases work productivity of providers and enhances patient care by increasing access to care, decreasing wait time in clinic and time of reporting of results and future plans. the app with knowledge and expertise in immunology and immune dysregulation is a cost effective way to enhance provider and patient support. with the overwhelmingly positive results, future plans include expanding our multidisciplinary clinic to other services that care for patients with suspected immune deficiency. abstract/case report text introduction: pediatric lymphoproliferative disorders represent a clinically and genetically heterogeneous group of conditions. misdiagnosis and delayed diagnosis can contribute to substantial morbidity and mortality. identification of molecular etiologies and underlying disease mechanisms may facilitate timely interventions and guide targeted or curative therapies. methods: the study was performed through retrospective chart reviews in accordance with all local ethics and irb committees. the study was designed to investigate a cohort of pediatric patients who met criteria for non-malignant lymphoproliferative disorders from texas children's hospital and collaborating centers for underlying genetic etiologies. results: a total of 51 affected individuals from 47 families met criteria. distribution between male and females was nearly equivalent: males (n = 26) and females (n = 25). approximately half of the cohort was hispanic (n = 25). overall kaplan meier survival was 67% (n = 39). whole exome sequencing was performed in all subjects and available family members. likely disease-causing genetic defects were identified in 29 of 47 families (62%). within these 29 families, 20 (69%) carried variants in genes in international union of immunological societies established primary immunodeficiency diseases. potential novel genetic causes of immune deficiency or immune dysregulation were also discovered. mechanistically, all of the implicated genes had roles in modulating lymphocyte activity; initial activation, cytoskeletal organization, or apoptosis of lymphocytes; or regulation of inflammation. all subjects less than one year of age had an identified gene in one of the three mechanistic categories with the dominant mechanistic genetic category being defective control of lymphocyte signaling (57%). in addition, 72% of patients between 1 and 8 years of age were found to have a potential genetic diagnosis underlying the lpd, with a more equal distribution of mechanistic categories compared to patients greater than 15 years of age where only 33% have a genetic cause. other important disease manifestations identified were ebv-associated disease in 21 subjects (41%) and 15 subjects (24%) met hlh-2004 criteria. conclusion: primary immunodeficiency diseases and other genetic abnormalities of the immune system underlie a significant percentage of pediatric lymphoproliferative disorder cases. greater than 72% of patients less than 8 years of age have a genetic etiology underlying the lymphoproliferative disorder. many of these gene defects can be treated with targeted therapies or hematopoietic stem cell transplantation. genetic testing therefore plays an essential role in the diagnosis and management of children with these conditions. abstract/case report text a 21-year-old gentleman with a history of immune dysregulation polyendocrinopathy enteropathy x-linked (ipex) syndrome with known pathogenic variant in foxp3 presented to our emergency department with two witnessed episodes of tonic-clonic seizures earlier that day. he has had a longstanding history of recurrent infections and autoimmune conditions since birth, and was being treated with monthly ivig infusions and sirolimus while awaiting bone marrow transplantation. his symptoms on admission included foaming at the mouth, generalized shaking, bladder incontinence, and tongue biting that lasted about five minutes. family reported recent sores inside his mouth and lips, but denied any recent fevers, neck pain, headaches, chest pain, abdominal pain, nausea, vomiting, and sick contacts. he lives on a farm with livestock and reportedly had recent tick exposure. his last ivig infusion was two weeks prior to admission, at which time he also received inactivated flu vaccine. in the ed, a third seizure was witnessed by multiple medical providers. he subsequently received lorazepam and lacosamide with interval improvement. he underwent diagnostic lumbar puncture, as well as extensive evaluation for infections. he was started on empiric antibacterial and antiviral meningitis coverage. analysis of the csf showed a lymphocytic pleocytosis; bacterial cultures and hsv 1/2 pcr were negative, as was a 14-pathogen meningitis/ encephalitis panel performed by pcr. eeg was negative for seizure-like activity, and brain mri showed mild atrophy without sclerosis in the left hippocampus. subsequently, anti-infectious therapy was stopped, and patient was discharged with outpatient followup scheduled for suspected non-infectious aseptic meningitis that was potentially triggered by flu vaccination versus ivig. on day four post-discharge, however, pcr for ehrlichia chaffeensis in the serum returned positive, and he was started on oral doxycycline. ehrlichiosis is a rare tick-borne illness that may cause various nonspecific symptoms including fever, headaches, myalgias, and generalized malaise. most prevalent in the mid-atlantic regions of the united states, tick-borne ehrlichia spreads through the mononuclear phagocytic system and can infiltrate many organs including the kidney, liver, lungs, and heart. csf penetration can cause sometimes fatal meningoencephalitis. aseptic meningitis due to ehrlichiosis has been described in recent literature. cases in hiv patients and transplant patients on chronic immunosuppressive therapy have been severe, resulting in organ dysfunction in many instances and death in a few. however, this case marks the first documented ehrlichia infection in a patient with primary immunodeficiency. this patient's presentation of aseptic meningitis and clear exposure history fits the clinical picture. his relatively benign course could be due to preserved t effector function not seen in persons with hiv or transplant patients with significant immunosuppression. patients with ipex usually present with autoimmunity and allergies, but are also prone to significant infections. it is important to perform a comprehensive workup, including testing for atypical infections, in patients with immune dysregulation syndromes who present with symptoms of unclear etiology. special attention should be paid to patients who live in areas with known endemic exposure risks. empiric antibiotic therapy may need to be considered early to prevent delays in treatment. abstract/case report text introduction autosomal recessive hypomorphic mutations in pgm3 have been described to result most commonly in either hyper-ige or severe combined immunodeficiency (scid) clinical phenotypes in humans, with one report of an individual with combined immunodeficiency without atopy. herein, we describe a series of individuals newly diagnosed with pgm3 deficiency functionally confirmed using lectin-based flow cytometric analysis of peripheral blood mononuclear cells, that broadens the associated clinical phenotypes to confirm cid without atopy and childhood evans syndrome. in addition, we present 3 new disease-causing pgm3 variants, and functionally confirm the pathogenicity of a fourth (p.i350t). classical hies phenotype cases 1.1 and 1.2 identify 2 sisters of spanish descent with a classical hyper-ige phenotype. the younger sibling demonstrated severe atopic dermatitis, mild-moderate asthma, multiple food allergies, one episode of itp, and adhd. the older sibling demonstrated atopic dermatitis, skin infections, and c. albicans otomastoiditis. the siblings were found to have the damaging compound heterozygous variants p.t492i and p.q506x in pgm3. case 2 is a 15 year-old guatemalan boy with prominent atopy including asthma, allergic rhinitis, food allergy, elevated ige, atopic dermatitis, as well as oral hsv who was found to be homozygous for the damaging pgm3 variant p.i350t. cid phenotype with a paucity of atopy case 3 is a 10-year-old turkish girl who is the daughter of a consanguineous union. she presented with infantile nephrotic syndrome at 6 months of age, and subsequently developed leukopenia, neutropenia, and low igg. complications include bronchiectasis, sinusitis, pseudomonas urinary tract infection, and inflammatory skin lesions without atopy. she was found to be homozygous for the damaging pgm3 variant p.r69h evans syndrome case 4 is a 5-year-old girl from guatemala. she developed multilineage autoimmune cytopenias including immune thrombocytopenic purpura (itp), autoimmune hemolytic anemia (aiha) and autoimmune neutropenia (ain) at the age of 2 years, refractory to multiple treatments and finally responding to mycophenylate mofetil. she has a history of mild eczema but is without other atopy and suffered from multiple invasive bacterial infections. an additional patient, case 5, was diagnosed with coombs positive aiha and itp at age 3 years refractory to multiple treatments and finally responsive to cyclosporine. cytopenias recurred 1 year later, resulting in hypoxic brain injury. he died of infectious complications at the age of 7 years. both patients were found to be homozygous for the damaging pgm3 variant p.i350t. discussion this is the first report of pgm3 deficient individuals presenting with evans syndrome as a primary presentation without additional pathology. while disease-associated mutations appear to cluster around the 4 key conserved domains of the protein, no clear genotype-phenotype correlation is readily observed. in addition to autoimmune cytopenias, pgm3 deficient individuals have also been reported with splenomegaly, lymphoma, and ebv viremia. thus, in particular for children with lymphoproliferative disease, pgm3 deficiency should also be considered in the differential diagnosis. abstract/case report text introduction: meningitis is a life-threatening manifestation of cryptococcus neoformans (c. neoformans). it occurs in increased frequency in those with impaired cell-mediated immunity, especially those with hiv/aids. infection with c. neoformans has been seen in previously healthy individuals diagnosed with idiopathic cd4 lymphopenia (icl). icl is defined by an absolute cd4+ count of less than 300 cells/m3 on multiple occasions, usually 2 to 3 months apart, without other apparent cause such as hiv infection, immunodeficiency, or immunosuppressive medications. case description: our patient is a previously healthy 50-yearold female with cryptococcus meningitis and fungemia. her course was complicated by elevated intracranial pressure requiring extraventricular drain. she was treated with amphotericin and flucytosine for 1 month. notably, the patient was also found to have moraxella catarrhalis (m. catarrhalis) bacteremia without identifiable source. she denied history of environmental risk factors, was not up to date on cancer screening, and recently returned from a trip to italy. initial evaluation revealed lymphopenia (422 cells/ul), low cd3+ (283 cells/ul), cd4+ cells (42 cells/ul), and cd16/56+ (31 cells/ul), but normal cd8+ (234 cells/ul) and cd19+ cells (121 cells/ul). hiv, ana, leukemia/ lymphoma flow cytometry panel was negative. she also had a normal lymphocyte proliferative responses to pha (66.7%), normal cd45ra:ro, and protective tetanus titers (2.31 iu/ml), but only 1/23 protective pneumococcal serotypes. initial immunoglobulins demonstrated slightly low igg (616 mg/dl). laboratory studies 2 months after presentation demonstrated improved lymphopenia (800 cells/ul) continued low cd4+ cells (86 cells/ul), but normalized igg levels (645 mg/dl). followup labs also demonstrated decreased cd19+ b cells (44 cells/ ul) and insufficient response to polysaccharide vaccine (9/23 pneumococcal serotypes). three months after discharge, she is continued on daily fluconazole without recurrence of infections although she still has diplopia and headache. discussion: in a review of cryptococcosis in 53 patients with icl, 7 of them had cryptococcal infection in both the cns and blood. of these 7 patients, 1 was cured, 2 improved, 3 relapsed and then improved, and 1 died. three of these patients were treated with amphotericin and flucytosine. five of these patients had underlying disease and 3 had notable infections with vzv, tb, or hpv, however other infections such as m. catarrhalis were not mentioned. m. catarrhalis bacteremia has been described in children with underlying immune dysfunction and respiratory infection as well as secondary to pneumonia with m. catarrhalis. in 24 cases of m. catarrhalis bacteremia in adults, most had underlying malignancy and/or neutropenia, predisposing respiratory factors, or source for infection. conclusion: this report of c. neoformans meningitis and m. catarrhalis bacteremia in the setting of icl is unusual in that to our knowledge, m. catarrhalis bacteremia has not been reported in icl. cases like this also raise the question as to whether some laboratory abnormalities are secondary to infection, treatment, or underlying disease. it is important to report these cases with icl in order to group disease phenotypes, as continued monitoring and data collection of these cases may lead to discovery of new disease processes. abstract/case report text cytokines play critical roles in regulating the development, survival, differentiation and effector function of immune cells. cytokines exert their function by binding specific receptors on the surface of immune cells and typically activating intracellular jak/stat signaling pathways, resulting in induction of specific transcription factors and regulated expression of target genes. in order to differentiate into an appropriate effector fate, lymphocytes need to integrate multiple signals often provided concomitantly by numerous cytokines that activate shared transcription factors. how these signals are balanced and regulated to yield the optimal class of immune response remains to be completely determined. inborn errors of immunity, or primary immunodeficiencies (pids), result from germline mutations in defined genes, leading to loss-of expression, loss-of function, or gain-of function of the encoded protein. pids are characterised by defects in immune cell development, or their differentiation into effector cells during immune responses, thereby rendering patients not only highly susceptible to infectious diseases, but also autoimmunity, autoinflammation, allergy and cancer. pids are thus an unprecedented model to link defined monogenic defects to immune dysregulation in clinical settings. indeed, pids have unequivocally revealed non-redundant roles of single genes, molecules, signaling pathways and lymphocyte subsets in host defense and immune regulation, and formed the basis of better therapies for immunopathologies. our indepth analysis of inborn errors of immunity of cytokine signalling pathways have identified fundamental requirements for generating long-lived humoral immune responses in humans. here, i will present data relating to our recent studies of how inactivating mutations in il21r, il6r, znf341, stat1, stat3, and stat5, disrupt or dysregulate the generation and function of human memory b cells and tfh cells, thereby precipitating humoral immunity, as well as allergic disease and autoimmunity. abstract/case report text introduction: flow cytometry is a powerful diagnostic tool for detecting hematologic malignancies in a variety of patient specimens including body fluids and lymph node aspirates. cytopathologists are frequently confronted with lymphocyterich effusions, and the definite decision of whether the lymphocytosis is of a purely reactive nature or a presentation of an indolent lymphoma may be an extremely difficult based on microscopy alone. moreover, small proportions of malignant cells that may be missed out by routine morphology can be detected by flow cytometry. objective: the purpose of this study was to evaluate the usefulness of multiparametric flow cytometry immunophenotyping (fci) to confirm the presence of leukemia or lymphoma cells in body fluids and fna specimens. methods: body fluids and fna specimens simultaneously obtained for fci, cytologic analysis and real time pcr from 30 patients were submitted to our flow cytometry laboratory from january 2017 to september 2019. the samples studied were 16 body fluids ( 11 pleural fluids and 5 ascitic fluids) and 14 fna samples (13 enlarged lymph nodes and 1 lung mass).four color fci method was performed and the following fluorescent monoclonal antibodies were used: cd45, cd19, cd5, cd20, cd22, cd23, cd79b, fmc7, kappa and lambda light chains, cd200, cd123, cd10, cd11c, cd2, cd1a, cd3, cd5, cd7, cd4, cd8, tdt, cd52, cd25, cd30, cd40, cd56, cd95, bcl2, cd34. fci analysis was performed on a beckman coulter cytomics fc500 flow cytometer using software cxp to analyze data. the cases were diagnosed as leukemia or lymphoma as per tuberculosis (1 case). ascitic fluid (n=5) samples showed positivity for angioimmunoblastic t-cell lymphoma (4 cases) and dlbcl (1 case). fna of lymph nodes (n=13) were positive fort-lymphoblastic lymphoma (2cases), angioimmunoblastic tcell lymphoma (2 cases), dlbcl (3 cases), hodgkin lymphoma (1case), nodular lymphocyte predominant hodgkin lymphoma (1 case), peripheral t-cell lymphoma (nos) (1 case), splenic b-cell marginal zone lymphoma(1 case), tuberculosis (2 cases). one fna of lung mass were tumor of neural cell origin. both immunophenotype and cytomorphology positive for malignancy were in 19/30(63.33%) cases.cytomorphology was negative/ suspicious in 11/30(36.67%) cases, of which both cytomorphology a n d i m m u n o p h e n o t y p e n e g a t i v e w e r e 3 ( 1 0 % ) a n d cytomorphology negative but immunophenotype positive cases were 8(26.67%). mtb dna was detected in pleural fluid in 1 case and fna sample in 2 cases. conclusion: multiparametric flow cytometry by using comprehensive panel of monoclonal antibodies is a useful diagnostic test to evaluate body fluids or fna as it can demonstrate small malignant populations that may be missed out by routine cytomorphology. clinical laboratory geneticist/department of genetics, university of groningen, groningen, the netherlands abstract/case report text the phenotypes of primary antibody deficient (pad) patients range from milder (e.g. specific antibody deficiency) to severe (e.g. x-linked agammaglobulinemia) deficiency of the immune system. pad patients form a clinically, immunologically as well as genetically heterogeneous group. often, the genetic background has not been elucidated; it probably is not monogenetic in a large subgroup of patients. pad patients suffer most frequently from recurrent bacterial infections of the respiratory or gastrointestinal tract due to immune deficiency, but may also have varying degrees of autoimmune and lymphoproliferative comorbidities due to immune dysregulation. unfortunately, a standardized description of pad phenotypes is lacking rendering robust definitions of pad-subtype diagnoses, including cvid, difficult. this impairs the formation of homogeneous cohorts that can form the starting point for future clinical and genetic research. the pad subgroup of the human phenotype ontology (hpo) immune mediated disorders consortium supported by ern rita and esid is addressing the gaps in standardized phenotypic description of pads. using the hpo dataset, literature mining, and esid, iuis and omim classifications, we aimed to reevaluate and complete the pad-related hpo terms to allow efficient data exchange and matching of phenotypically similar pad patients. as a principle, it was decided to avoid the ongoing variance in pad-subtype definitions and to build the pad-related hpo tree based as much as possible on unambiguously interpretable items. 'hypogammaglobulinemia' was deleted as hpo term, and replaced by separate hpo terms such as 'decreased total igg in blood', subdivided in 'transient' vs. 'chronic', and '(near) absent' vs. 'partially decreased' (the same for igg1, igg2, igg3, igg4, iga and igm). 'decreased specific antibody level in blood' was specified further into 'decreased natural antibody level to blood group antigens in blood', subdivided in '(near) complete' vs. 'partial' absence (the same for protein, polysaccharide and protein-conjugated polysaccharide vaccination). relevant hpo terms related to infection and to specific organ manifestations like bronchiectasis, autoimmunity and lymphoproliferation were re-evaluated and completed, and will be linked to pad diseases in the hpo online system by the pad subgroup experts. once finalized, existing pad cohorts will be classified according to the new hpo pad-related terms, and studied by clustering technologies (example of two patients shown in figure 1 ; white = absent, color = present). acceptance and widespread use of this pad-related hpo tree for standardized phenotyping will be essential to empower future multicenter clinical research and related genetic discoveries as well as support clinicians in diagnosing pad through the linkage of hpo terms to pad disease entities. senior investigator oral immunity & infection section/nih/nidcr abstract/case report text leukocyte adhesion deficiency type 1 (lad1) is an autosomal recessive disorder characterized by the inability of granulocytes to emigrate from the bloodstream to sites of inflammation. lad1 is caused by mutations in the itgb2 gene (21q22.3), encoding the beta-2-integrin, cd18, which is essential for firm adhesion of leukocytes to the endothelium. in lad1 survival is compromised, morbidity from inflammatory lesions is high, and treatment is poor. the moderate form of lad1 is often managed with antibiotics for prophylaxis and during acute infections. after infancy severe gingivitis and chronic periodontitis are universal. periodontal findings affect primary and permanent teeth, causing intense oral mucosal (gingival) inflammation and destruction of tooth supporting bone, which are hallmarks of the disease periodontitis. blocking the il-23/il 17 cytokines, which are up regulated in lad1 gingiva, can reduce bacterial load and resolve inflammatory gingivitis. ustekinumab binds to the shared p40 subunit of human il-12 and il-23, cytokines that modulate lymphocyte function, including t helper (th) 1 cells and th17 subsets, thereby blocking them. objective: explore the effect of ustekinumab on lad1 inflammatory disease. method: prospective study using ustekinumab for oral inflammation. patients receive five doses over 1 year, 45-or 90 mg depending of weight. results: (two patients have enrolled, p1 is >1 year post treatment, p2 is still on study) patient characteristics · patient 1: age at diagnosis, 9yrs.(itgb2 mutation c.2070delt (null)); cd18(%pmn control):32.6%; cd11a(%pmn);6.6%. at the initiation of the protocol (17yrs old) wcc:7.41k/ul; crp: 6.10 · patient 2: age at diagnosis, 4yrs.(itgb2 mutation c.850a>g,p.g284s c.809c>t,p.a270v) cd18(%pmn control): 13%; cd11a(%pmn): 3.9%. at the initiation of the protocol (17yrs old) wcc:7.41k/ul; crp: 6.10 response: patient 1: oral ulcers before treatment: episodes every two months. during ustekinumab therapy: · oral ulcers: 1 episode in a year · reduction in bleeding on probing: 57.5% · gingival index reduction: 95% patient 2: oral ulcers before treatment : monthly. during ustekinumab therapy: · oral ulcers: none in first 4 months · reduction in bleeding on probing : 68.3% · gingival index reduction: 37.5% safety: no significant adverse events were documented during the therapy p1 had a previous skin lesion that flared leading to iv antibiotics. p2 had a previous sebaceous cyst drain spontaneously. discussion: two patients showed improvement in chronic periodontitis and a substantial decrease in oral ulcers while on ustekinumab. no clear safety signals were seen. durability of these findings is still unknown. ustekinumab in lad1deficiency appears to be safe and potentially effective. post doctoral fellow/servicio de inmunología, inst. multidisciplinario de investigación en patologías pediátricas (imipp), hospital de niños ricardo gutiérrez. 2 chief resident/servicio de inmunología-hospital de niños "dr. r.gutierrez" 3 immunologist/centro de inmunología clínica "dra. liliana bezrodnik y equipo"-servicio de inmunología htal. de niños "dr. r.gutierrez" 4 immunologist/centro de inmunología clínica "dra. liliana bezrodnik y equipo" 5 immunologist/servicio de inmunología-hospital de niños "dr. r.gutierrez" abstract/case report text introduction: primary immunodeficiencies with dysregulation associate defects in the immune homeostasis leading to inappropriate immune response (lack or excess) that causes autoimmunity, allergy and/or inflammation. impairment of different subsets of t and b compartments may be associated with these pids. aim: 1) describe t and b memory compartment of 18 pid patients (pts) with dysregulation: 1 cd25 deficiency, 3 stat1 gof, 1 stat5b deficiency, 4 ctla4 variant, 2 pi3kcd variant and 6 cvid-like (with no molecular defect) and compare them with a group of healthy donors (hd). 2) associate ctfh profile with b cell compartment impairment. results: 1) pts showed a significant decrease of naïve cd4+ t cells (cd45ra+cd27+) (52,2% vs 17,6%) (p < 0.0001) with expanded central memory t cells (cd45ra-cd27+) (32.8% vs 60.8%) (p < 0.0001); cd4+ t cells had higher levels of activation markers (cd4+hla-dr+) (6,1%vs 27,4%) (p < 0,0001). pts showed a significant increase of circulating follicular t cells (ctfh) (cd45ra-cxcr5+) compared with hd (mean 30,5% vs 11,6%) (p < 0,0001) with pd-1 overexpression (p < 0.0001). stat1 gof, ctla4, pi3kcd and cvid-like pts showed a skew towards ctfh1 (cxcr3+). regulatory t cells (cd4+cd25++ foxp3+) were absent in cd25 and stat5b deficiency and decreased in the other pts. within cd8+ cells, although effector memory (cd45ra-cd27-) (p < 0.002), temra (cd45ra+ cd27-) (p < 0.009) and hla-dr+ cd8+ (p < 0.002) subsets showed a significant increase compared with hd, the behaviour was variable between different mutations. regarding b cell compartment, pts with stat1gof, pi3kcd and cvid-like showed a severe impairment of switched-memory b cells (sw-mbl) (cd27+igd-igm-); the stat5b deficient patient had increased frequencies of this subset, while ctla4 pts had a variable b defect. 2) lower sw-mbl values were significantly associated with lower values of ctfh17 cells (p < 0.007) (r=0.6975). cd21low b cells were exclusively high in cvid-like pts, and transitional b cells were increase in pi3kcd and almost all cvid-like pts. discussion: in summary, patients with dysregulatory syndromes associate a defect of t and b homeostasis (survival, activation and differentiation). specific mutations can differentially affect the quantity and/or the quality of ctfh. there is a strict association between the differentiations of tfh with th17 profile with the generation of sw-mbl. these alterations may play a role in the pathophysiology of primary immunodeficiencies with b lymphocyte functional impairment. immune monitoring of lymphocyte subsets of patient with dysregulation may approach to the diagnosis of specific monogenic mutations. objective: the purpose of this study is to increase awareness and improve diagnosis of primary immune deficiency (pid) in the heterogenous group of patients with autoimmune cytopenia (aic) by identifying clinical characteristics and laboratory biomarkers that distinguish those with underlying pid, disease activity and guide mechanism-based targeted therapy. methods: patients with aic (autoimmune hemolytic anemia (aiha), immune thrombocytopenia (itp), or autoimmune neutropenia (ain)) were referred to our immune dysregulation team and prospectively enrolled during 2016-2019. detailed immune phenotyping (igg, iga, igm, lymphocyte subsets, vaccine titers, lymphocyte proliferation to mitogens/ antigens), serum lipopolysaccharide (selps) and autoantibodies were measured and/or collected by chart review and genetic testing for pid was pursued. results: from 2016 to 2019, 93 patients were enrolled; two subjects were removed due to parental request or lack of aic diagnosis. of the 91 remaining patients, 43 (46%) were classified as "aic-pid" based on genetic testing and/or immune phenotyping; 41 (45%) were classified as aic-only, and 7 (9%) were asymptomatic family controls. the patients were predominantly children (ages 1-82 years, average age 17.3 years); 47% (44/93) were male. among patients who have had genetic testing to date (n=66)(72%), pathogenic genetic mutations were confirmed in 23/66 (35%) of patients. mutations include fas/fasl (n=8, including 3 family members without aic), ctla4 (n=4), 22q11 (n=4), and one patient each with nfkb1, was, pole-1, pi3k, casp10, card11, and cgd; the remainder of aic-pid patients were classified as combined immune deficiency or common variable immune deficiency based on immune phenotyping. lymphocyte subsets (cd4+t, cd8+t, cd19+b, cd56+ nk) and immune globulins (igg, iga, igm) tended to be lower in aic-pid patients vs aic-only (p < 0.05). evans syndrome was more common in aic-pid patients (13/43, 30%) compared to aic-only (4/41, 10%). lps was elevated in the serum of aic patients compared to healthy controls (mean 719 vs 87 pg/ml, p < 0.001). excluding partial digeorge syndrome patients (average lps 222pg/ml), selps levels were significantly higher in genetically-defined untreated pid patients (average 1463 pg/ml) vs. other pid (average 444 pg/ml)(p=0.02) or patients with aic alone (average 667 pg/ml)(p=0.03). studies are ongoing on specific subsets that are linked to immune dysregulation (switched memory b cells, t-regulatory cells, double negative t cells, t follicular helper cells) and the use of soluble il-2 as a biomarker of disease activity. conclusions: a high fraction of aic patient were identified with underlying pid in our study. basic immune evaluation with immunoglobulin levels and lymphocyte subsets expedited diagnosis of pid. genetic evaluation distinguished a group of patients with aic-pid and highly elevated lps level, reflecting high bacterial load, which may distinguish them from the rest the aic cohort. the source of bacterial lps can be multifactorial and is yet to be determined. our studies continue focusing on biomarkers that can be applied to the heterogenous group of patients with aic. this will allow early detection and timely initiation of targeted therapies. investigator/dermatology branch, niams, nih 6 head, dermatology consult service/dermatology branch, niams, nih 7 chief, fungal pathogenesis section/laboratory of clinical immunology and microbiology, nih abstract/case report text introduction/background: autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced) is a monogenic autoimmune disease resulting from biallelic mutations in the aire gene. although typically characterized by the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism and adrenal insufficiency, we recently reported that the clinical spectrum of the syndrome is far broader and that incorporation of an adjunct triad of apeced rash, intestinal dysfunction, and enamel hypoplasia in the classic triad could lead to earlier diagnosis (ferre et al., jci insight, 2016). among the adjunct triad manifestations, apeced rash occurs in 66% of american apeced patients by age 3, most often developing in the first year of life. objectives: to report and describe the clinical features of apeced rash as the first manifestation in a 10-month old patient with apeced. methods: following enrollment in a niaid irb-approved protocol (11-i-0187) the patient was evaluated with history and physical examination, aire sequencing, measurement of interferon-autoantibodies, and skin biopsy with immunohistochemical analyses. results: a 10-month-old girl with a family history of genetically confirmed apeced in her 13-year old sister developed discrete circular, maculopapular erythematous lesions on her torso that spread to the face, arms, and legs while sparing the palms and soles. the rash was partially blanching, non-painful and non-pruritic and was preceded by low-grade fever (38□c) without other accompanying symptoms. she had not received medications or vaccinations prior to the rash onset. the lesions increased in size with associated central clearing and resolved 2.5 months after onset. the rash recurred with similar appearance 10 times over 14 months with each recurrence lasting between 10 days and 2.5 months. as with the first rash episode, recurrences were often preceded by fever (38-39□c) without accompanying symptoms or inciting factors. neither topical nor oral antihistamines improved the rash. aire sequencing identified the same compound heterozygous mutations (c.967_979del13 and c.769c>t) that the sister has. high titers of interferon-□ autoantibodies were measured in serum. skin biopsy revealed superficial perivascular chronic inflammation and intraepidermal lymphocytes composed predominantly of mixed cd4 and cd8 t lymphocytes with few perivascular b lymphocytes. no eosinophils or vasculitis was observed. myeloperoxidase immunostaining revealed extensive karyorrhexis. laboratory studies revealed normal white count and esr, negative anti-ige receptor antibody, and positive anti-ige antibody. at 22 months, she developed oral candidiasis as second manifestation of apeced, thus reaching a diagnostic dyad when applying our proposed expanded diagnostic criteria. she has not developed hypoparathyroidism or adrenal insufficiency; thus, she has not yet reached a classic diagnostic dyad. systematic screening for these endocrinopathies will be needed to avoid lifethreatening complications of acute endocrine failure. conclusions: we report the clinical and histologic features of apeced rash manifesting as the first disease component of apeced in a 10month old girl. apeced should be considered in the differential diagnosis of recurrent erythematous maculopapular urticaria-like eruptions characterized by mixed lymphocytic and neutrophilic infiltration unresponsive to antihistamines. our case illustrates the clinical utility of incorporating the expanded diagnostic criteria of apeced rash, enamel hypoplasia and intestinal dysfunction into the classic diagnostic triad, which can lead to earlier apeced diagnosis. who presented with prolonged severe neutropenia despite g-csf and seven hospitalizations for febrile neutropenia in the span of ten months. prior to his neutropenia, patient was on monthly ivig, with igg trough 700-900 in the past year. he was evaluated for bmt in 2015 but declined. patient was first found to be neutropenic in aug 2017 when he was admitted with pseudomonas thigh abscess, hsv stomatitis and rhinovirus infection. he was treated with broad-spectrum antibiotics with improvement in neutropenia. the following month, he was hospitalized again with neutropenic fever, left axilla pseudomonas abscess and rhinovirus infection. he underwent bone marrow biopsy revealing left shifted myeloids with decreased maturing forms and t cell predominant lymphoid aggregates, suggestive of autoimmune neutropenia vs. hyper igm syndrome associated with neutropenia. anti-neutrophils antibodies were negative. he was then admitted the following month (10/2017) with febrile neutropenia with cxr concerning for viral pneumonitis vs. atypical pneumonia. he was started on g-csf therapy with significant initial response in anc. however, this response was short-lived as he was again admitted in 12/2017 with febrile neutropenia and upper respiratory rhinovirus infection. he was continued on daily g-csf. due to persistently normal anc for approximately three weeks, he was weaned off g-csf in 1/2018. in 3/2018 and 4/2018, he had two more hospitalizations for febrile neutropenia. g-csf was restarted with dose uptitrated to 8mcg/kg during his hospitalization in april. he was found to be thrombocytopenic with splenomegaly on abdominal ultrasound. anti-platelet antibodies and repeat anti neutrophil antibodies were not detected. he was discharged with close follow up with immunology and hematology. due to his age, he was transitioned to penn allergy/immunology in 4/2018. there was close communication between chop allergy/immunology, chop hematology and penn allergy/immunology during this transition period. patient was admitted to hup in 5/2018 with febrile neutropenia (despite higher dose of g-csf), rhinovirus infection, pseudomonas sinusitis and ct chest findings suggestive of possible fungal pneumonia. due to persistent neutropenia refractory to g-csf treatment, hematology was consulted and repeat bone marrow biopsy showed hypercellular bone marrow with markedly left shifted granulocytic hyperplasia, compatible with g-csf therapy. flow cytometry showed no evidence of plasma cell neoplasm. dose of ivig was adjusted and increased based on his weight. per hematology, he also received an additional high dose ivig 1g/kg x 2 days for presumed immune mediated neutropenia with immediate increase in anc. despite anc of 0 for 8days,anadditional1g/kgofivigimprovedhisancto>1000within12hours of his first dose. thrombocytopenia also improved to normal range. since then, patient has been on monthly 500-600mg/kg ivig with no recurrence in neutropenia. this patient's prolonged persistent neutropenia with immediate response to high dose ivig is suggestive of autoimmune neutropenia, which should be taken into consideration in hyper igm patients with persistent neutropenia. abstract/case report text background: children with digeorge anomaly (dga) represent a heterogenous group, often classified as either partial dga (pdga) or complete dga (cdga) based upon the degree of thymic hypoplasia. this paucity of t-cell parameters and function has serious implications for infection risk, autoimmunity, and malignancy. however, there are limited studies stratifying children with dga by these subgroups, especially in regard to immune function and subsequent infection risk. study design: single-center, retrospective cohort analysis evaluating the relationship between pdga and cdga to infection risk with particular focus on infection-related hospitalization, pathogenic organism identification, and antimicrobial resistance profiles. the source population includes all pediatric patients < 18 years of age diagnosed with either pdga or cdga while receiving care at duke university from january 1, 2014 to june 30, 2019. the final analysis sample included 145 patients. methods: to evaluate the differences in immune function between dga subgroups, we will report the proportion of low ( < 10th percentile for age) t cell immune biomarkers for both subgroups and compare populations using a chi-squared test. to compare per year incidence of infectionrelated hospitalization for dga subgroups, a poisson model with number of hospitalizations per patient as the outcome, an offset equal to the time at risk for hospitalization, and either pdga or cdga diagnosis as the exposure will be used. models will be bivariate. we will report an incidence rate ratio (irr) and 95% confidence interval (95% ci). to evaluate the impact of cellular and humoral immune function on infection-related hospitalization, we will use poisson models where the outcome is the number of hospitalizations per patient, an offset equal to the time at risk for hospitalization, and low immune biomarker as the exposure. all models will be bivariate. we will report an irr and 95% ci. infection type and resistance profiles will be completely descriptive. results: as expected, children with cdga had a significantly higher probability of a low ( < 10th percentile for age) values for total t cells (cd3+), helper t cells (cd3+cd4+), cytotoxic t cells (cd3+cd8+), and naïve helper t-cells (cd4+cd45ra+cd62l+) as well as a significantly lower probability of low pan memory t-cells (cd3+cd45ro+) compared to children with pdga. no differences were detected in the percentage of low natural killer (nk) cells (cd15+cd56+) or b cells (cd19+) between subgroups. cdga patients had a significantly higher incidence of hospitalization per year (1.52 (0.61, 3.80)) compared to pdga patients (0.20 (0.12, 0.32)). the irr is 7.62 (2.71, 21.3). across both subgroups, the incidence of hospitalization was higher in dga patients who had low helper and naïve t-cells. there is ongoing analysis into hospital-related infection and resistance profiles. notable frequencies include bacteremia ( > 5%), invasive viral disease ( > 1%), and opportunistic infections ( > 1%). conclusions: children who had cdga were 662% more likely to have an infection requiring hospitalization than children who had pdga, emphasizing the need for thymus transplant for cdga. further analysis of infection type and patient outcomes is critical to enhancing management of this unique patient population. abstract/case report text antibody cross-reactivity among flavivirus has been documented. in recent times zika virus has been emerging in pockets of the mosquito-infested regions, while southwestern saudi arabia is known for arthropod-borne viral diseases and we do not know the incidence or even presence of zika virus in this region. it is restricted to predict the igm and igg antibody detection ranges owing to limited data and colossal cross-reactivity among the zika and other flaviviruses. we tested sera from 217 pregnant women irrespective of their clinical presentation for zika and dengue igm, igg respectively. the zika positive samples were further confirmed by plaque reduction neutralization tests (prnt). from our results, 3.6% (8) cases were positive for zika igm against 1.8 % (4) positivity to igg. when these samples were assessed for dengue igm and igg, we observed 1.3% (3) seropositivity for igm and igg respectively. there was no single sample positive for both igm and igg of zika or dengue. however, we observed one sample positive for both zika and dengue igm. upon mapping the overlapping serotiters, there was no significant correlation observed between the dengue igm and igg. whereas zika igg positive sample showed high serotiter for dengue igg indicating the contribution of cross-reactivity for observed zika positivity. screening for the incidence of zika, therefore, becomes particularly hard in a population that has the presence of pre-exposure of dengue and this cross-reactivity makes it hard to determine the zika incubation and antibody prevalence confounded with other flaviviruses. abstract/case report text introduction humoral pid diagnostic protocol includes the analysis of the immune response to different protein and polysaccharide antigens (ags) (1) . although the analysis of the immune response against the polysaccharides vaccine from pneumococca has been the standard method, the use of s. typhim vi vaccine has appeared as a good alternative (2) . in this report we show the results obtained with the use of s.typhim vi in 27 adults patients attending the pid outpatient clinic. material and methods patients with humoral-suspected pids were challenged with typhoid polysaccharide vaccine (typhim vi®; sanofi-pasteur). serum was obtained on basal and after 4 weeks of vaccination. specific igg levels against s.typhim were measured using "vacczyme tm human anti-salmonella typhi vi igg enzyme immunoassay kit" (binding-site). results a total of 90 adult patients attended the pid clinics during nov 2018-nov 2019. from those, 27 patients were fully evaluated using a humoral-suspected pid algorithm that includes the s.typhi vaccination. in total 13 male and 14 female patients completed the protocol and were analyzed. twenty patients were considered as responders (ratio pre/ post >3x) whereas 7 patients were non-responders. discussion the main advantage of assessing polysaccharide immune response using s.tyhpim is the usual lack of specific igg at the moment of the initial evaluation. in this serie, just one patient has a high basal level (#2-vaccinated in the past). thirteen patients had basal levels below the detection limit of the test (7,4u/ml) and 13 patients between 7,4 and 23,35u/ml, that has been described as a cut otf level for nonimmunised individuals (personal experience,3,4). regarding the polysaccharide immune response as a tool to distinguish pid vs non-pid patients, the results showed a good correlation between those non-responders with more clinical relevant pid diagnostics. seven non-responders patients were subsequently diagnosed with a primary (* on table i ) and/or secondary id (** on table i ). despite this, there were 16 patients that we could have classified as strong responders (ratio >3x, absolute specific igg postvaccination level >100u/ml) and 4 patients considered as weak responders (ratio post/pre >3x, absolute specific igg postvaccination level < 100u/ml). strong responders were considered non-pid after including other clinical investigations and laboratory tests (cell subpopulation study, pcp response) whereas weak responders group consisted in some "minor" forms of pid, like isolated igm or ig subclasses deficits. more patients are needed to confirm this functional classification of pid patients regarding their s.typhi immune response. abstract/case report text activated pi3kδ syndrome (apds) is a primary immunodeficiency characterized by recurrent respiratory infections, as well as increased risk of chronic viremia with herpes family viruses, benign lymphadenopathy and b cell lymphoma. it is caused by heterogeneous germline gain-of-function mutations which ultimately lead to the hyperactivation of the phosphoinositide-3-kinase δ (pik3δ). pik3δ exists as a heterodimer composed of a catalytic and a regulatory subunit. it interacts with b cell receptors, t cell receptors, costimulatory and cytokine receptors, and is a key player in a signaling pathway involved in cell growth, proliferation and survival. apds1 is caused by mutations in the pik3cd gene, affecting its protein product p110 δ (catalytic subunit). apds2 is caused by mutations in the pik3r1 affecting p85a (regulatory subunit). short syndrome is a rare multisystem disorder characterized by short stature, hypertextensible joints, ocular depression, reiger anomaly and tooth eruption delay. the primary causes of short syndrome are heterozygous loss-of-function mutations in the pik3r1 gene. the combination of apds2 and short syndrome is very rare, with only few cases described in the literature. in this report we present a teenager with a pathogenic variant in the pik3r1 gene, and phenotypic characteristics of both apds2 and short syndrome. our patient is a 17-year-old female with a history of growth delay and short stature, delay tooth eruption, recurrent sinopulmonary infections and hypogammaglobulinemia. evaluation performed at a prior institution for recurrent infections revealed low igg levels. she did not initiate therapy at that time and was lost to follow up for several years. at the time of our initial evaluation she reported continued recurrent episodes of upper respiratory infections and sinus infections requiring antibiotic treatment that often did not clear the infections. her physical exam was relevant for short stature (1%ile, z=-2.33), low weight for age ( < 1%ile, z=-2.69) and hyperextensibility. her facial features were significant for prominent forehead and triangular face. given concern for immune deficiency, a complete immune evaluation was obtained. her workup revealed low igg levels, with igm and iga within normal limits. she did not have protective titers to s. pneumoniae, h. influenza or diphtheria and tetanus. after administration of vaccine boosters, she was able to generate a response to all vaccines except for tetanus. she had remarkably low absolute b cells (34 cells/ul) and percentage (1 %), and low cd4:cd8 ratio (0.84). she was started on amoxicillin prophylaxis and monthly ivig replacement therapy. invitae immunodeficiency panel genetic testing was sent and revealed a pathogenic loss of function variant in an intronic splice site in the gene pik3r1 (c.1425+1g>c). after initiating treatment with ivig, her sinus infections significantly improved and she has not had any further episodes. igg levels have remained within normal limits with monthly ivig therapy. this pathogenic variant had been previously associated with apds2; however, it had not been associated with short syndrome. the mechanisms that link both conditions is yet to be identified. this case report emphasizes the importance of screening for comorbidities associated with short syndrome in apds2 patients, and vice versa. finding the genetic diagnosis for patients with suspicion of primary immunodeficiency (pid) is becoming increasingly important in the management of primary immunodeficiency and estimating the risk for family members. we constantly increase the diagnostic yield for pids by improving the sequencing technology, updating the panels with new genes discovered related to pid, and finding diagnoses from difficult to sequence regions and regions with high homology. here we report our experiences with nearly 1700 patients suspected with pid. moreover, we provide a case example, how we increase the diagnostic yield by developing unique techniques for specific genes which cannot be reliably analyzed by ngs alone. diagnostic yield including all immunology related panels was 14.9% (253/1698). the majority of the tested individuals were males (1343/1698, 79.1%) and the most common age of testing was between 10 to 20 years (468/1698, 27.6%). the highest diagnostic yield 20.6% (75/364) is in children from ages 0 to 5 years, whereas in patients over 60 years of age the diagnosis was found for only 6.5% (4/62) of the patients. in two patient cases, our cnv detection algorithm indicated a homozygous deletion in the index patient samples potentially covering the whole ncf1 gene. additional bioinformatic analysis targeting specifically two coding positions that differ between the ncf1 gene and the two pseudogenes showed that all reads in those positions originated from the pseudogenes. homozygous deletion in the ncf1 gene was further confirmed by sanger sequencing two regions in ncf1 with primers that specifically bind to either ncf1 or the pseudogenes. while clean ncf1 sequences from both regions were obtained for a control sample, no ncf1-specific amplification product was obtained for the index patient samples. pseudogenes were amplified and sequenced successfully in both index patient samples and positive control samples. loss-of-function of ncf1 is a well-established mechanism leading to cgd and by overcoming the difficulties regarding ncf1 deletion detection by ngs, we can improve diagnostic rate in individuals affected with cgd. gata2 deficiency can lead to a broad spectrum of clinical and hematological phenotypes; in some cases, nk cell deficiency is the primary manifestation, resulting in a greatly increased susceptibility to viral infections and malignancy. gata2-deficient patients, particularly those who suffer from severe viral infections, have reduced frequencies of peripheral blood nk cells and loss of function in the existing nk cells. specific loss of the less mature (cd56^bright) nk cell subset is a hallmark of the immune phenotype in gata2 deficiency, suggesting that generation or survival of nk cell precursors is impaired. given the remarkable spectrum of clinical phenotypes in gata2-deficient patients and the poorly understood biology underlying their nk cell defect, we sought to characterize circulating nk cells on a single-cell level. we performed single-cell (sc)rnaseq of lineage-depleted innate lymphocytes from a patient with gata2 deficiency. as expected from flow cytometric phenotyping of peripheral blood cells from this and other patients, scrnaseq revealed decreased representation of canonical cd56^bright cells. within the cd56^dim population, we identified two nk cell populations that were seemingly unique to the gata2 deficient patient relative to a healthy donor. pathway analysis defined the first of these populations (population 1) by the expression of genes associated with cellular response to stress, extracellular stimulus and inflammation, as well as programmed cell death and regulation of proliferation and apoptosis. the second population (population 2) was defined by genes associated with nk cell chemotaxis, cytokine responses and interferon signaling. to extend our findings, we performed scrnaseq of 6 additional healthy donors and analyzed an additional gata2-deficient individual who was clinically asymptomatic (yang et al. 2019). of note, we detected population 2 in seemingly healthy cmvnegative individuals, suggesting it was not uniquely a result of gata2 deficiency but associated with an inflammatory response and not related to adaptive nk cells generated in response to cmv infection. population 1, on the other hand, only appeared in our symptomatic gata2-deficient patient. we additionally performed bulk gene expression analyses from an unrelated gata2-deficient patient that confirmed the altered expression of genes associated with both novel cell populations. current efforts are focused on better defining the functional response of nk cells in these patients and confirming the identification of our novel populations by mass cytometry (cytof). together, our data define the heterogeneity and complexity of nk cells in gata2 deficient and healthy individuals. perforations have been reported with tocilizumab, a monoclonal antibody of the interleukin 6 (il-6) receptor, suggesting that il-6 signaling plays a role in intestinal wall integrity. as il-6 signals through stat3, we sought to investigate the potential association between lof stat3 and intestinal perforations, as well as the incidence and outcome in our patient cohort. methods: we performed a retrospective chart review of patients with lof stat3 (n=158) followed at our institution, looking for those with non-malignancy associated spontaneous gastrointestinal perforations. the demographic information, stat3 mutation, comorbidities at the time of perforation, clinical presentation, management, and clinical outcomes were compiled results: ten lof stat3 patients were identified as having documented intestinal perforations, an approximate rate of 6%. one perforation was the initial presentation of diffuse large b cell lymphoma (dlbcl) of the duodenum and liver, and was excluded from the rest of the analysis. the other nine perforations occurred between 4 to 60 years old (mean:26), and 55% were female. stat3 mutations were localized to the dna binding domain (n=4) and the sh2 domain (n=5). two of the perforations occurred while inpatient for lung infection. another occurred while recovering from pneumonia at home. two perforations were associated with the initial diagnosis of diverticulitis (at age 26 and 60). one perforation occurred in the terminal ileum, one in the cecum, one in the transverse colon, and six in the sigmoid. five patients underwent primary closure of the bowel. four patients required a temporary ostomy, with subsequent successful ostomy reversal. only one patient has since died of pulmonary hemorrhage, the other patients are alive with a mean of 6 years post perforation follow-up, with no recurrence of perforation. one patient with prior sigmoid resection required ileal resection 6 post perforation due to as massive intestinal bleed. conclusion: spontaneous gastrointestinal perforations occurred in our lof stat3 cohort at a rate of approximately 6%. one case was associated with malignant infiltration of the gastrointestinal tract and two cases were associated with diverticulitis both known risk factors for perforation. although the pathogenesis of the perforations in lof stat3 remains unclear, the connective tissue phenotype likely contributes as well as the association with diminished il-6 signaling, as has been demonstrated with the perforations and tocilizumab. abstract/case report text background granulomatous and lymphocytic interstitial lung disease (glild) is a life-threatening complication that occurs in patients with common variable immunodeficiency (cvid) and monogenic cvid-like disorders, but the optimal treatment is unknown. objective to determine if the use rituximab and azathioprine (rtx-aza) or rituximab and mycophenolate mofetil (rtx-mmf) would improve the radiographic abnormalities as determined by high-resolution computed tomography (hrct) of the chest and/or pulmonary function tests (pfts) in patients with cvid and glild. methods this is a retrospective study of patients seen from july 2006 to december 2018 with cvid and glild who completed immunosuppressive therapy (rtx (375mg/m2) for 4 weeks, repeated at 6-month intervals for 3 or 4 total courses, and aza (0.5-2.0 mg/kg/day) or mmf (250mg-1000mg-bid) for 18 months). complete pfts and hrct scans were performed prior to therapy, at the conclusion of therapy, and periodically thereafter. hrct scans were blinded, randomized, and scored independently (in pairs) by two radiologists. all patients underwent whole exome sequencing (wes). number (percentage) and median (interquartile range) were reported for categorical and continuous variables, respectively. differences between pre-and post-treatment and between relapse and post-relapse hrct scores and pft parameters were analyzed with wilcoxon signed ranks test. kaplan-meier survival curves were also done. unadjusted one-sided p-values < 0.05 were considered statistically significant. results the glild cohort (n=39) had a 2:1 female predominance, and age at glild diagnosis was 36 (25-43) years (table 1) . autoimmunity was present in the majority of patients, with thrombocytopenia (28 (72%)) the most common manifestation. enteropathy (3 (8%)), inflammatory bowel disease (1 (3%)), and nodular regenerative hyperplasia of the liver (6 (15%)) were also present. splenomegaly (31 (79%)) was present in the majority, but polyarthritis (0 (0%)) was notably absent. twenty (51%) patients had been previously treated with systemic steroids. hrct scores substantially improved between pre-and posttreatment for rtx-mmf (p=0.0005) and rtx-aza (p < 0.0001, figure 1 ). fev1 (p=0.019), fvc (p=0.0011), and tlc (p=0.0071) also improved, but dlco (p=0.060) was unchanged (figures 2 and 3 ). excluding two (5%) patients who died 2.3 and 2 years after therapy of respiratory failure (1 (3%)) and septicemia (1 (3%)) respectively, 9/37 (24%) patients relapsed 3.2 (2.8-3.5) years following therapy with an estimated 60% relapse rate after 5 years ( figure 4 ). as of december 2018, 6 of 9 patients that relapsed showed improvement in hrct scores (p=0.031), and the remaining 3 patients are still undergoing retreatment ( figure 5 ). four (10%) pneumonias occurred during immunosuppressive therapy, all with severe restrictive lung disease . eight (21%) patients had a damaging mutation in a gene known to predispose (tnfrsf13b, n=3 (8%)) or cause a cvid-like primary immunodeficiency (ctla4: 2 (5%); kmtd2: 2 (5%); birc4: 1 (3%)). immunosuppressive treatment improved the hrct scores regardless of the absence (p < 0.0001) or presence of a damaging mutation (p=0.0039) ( figure 6 ). conclusion combination chemotherapy appeared to be effective in improving the radiographic abnormalities and pulmonary function of patients with cvid and glild. a majority of patients had sustained remissions, regardless of the presence or absence of a monogenic disorder. iqr=interquartile range, cvid=common variable immunodeficiency, glild=granulomatous and lymphocytic interstitial lung disease, vats=video-assisted thoracoscopic surgery, tbx=transbronchial biopsy, ms=mediastinoscopy excluding b cell malignancy, pft=pulmonary function test, h/o=history of, dz=disease, nrh=nodular regenerative hyperplasia, ibd=inflammatory bowel disease table 1 . baseline patient characteristics since its first description the number of cases has increased progressively [1] . although described as a predominant antibody deficiency [1] , various complex phenotypes have been associated with mutations in this gene [2] . case description. female patient with no remarkable history until 2 years old, when she suffers from a persistent fever associated with purulent abscesses in venipuncture areas and hyperleukocytosis with neutrophilia, so she was treated for about 8 months in 3 hospitals in the city of barranquilla before she was referred to our institution. the patient's clinical picture consisted of persistent fever unresponsive to broad-spectrum antibiotic treatments, skin abscesses, left subphrenic abscess and toes osteomyelitis. the microbiological studies documented a bacteremia by acinetobacter baumannii and isolation in bone marrow of candida parapsilosis. during her care stay in barranquilla, she was approached as a chronic granulomatous disease versus job's syndrome, she received two doses of immunoglobulin with partial control of symptoms. due to the recurrence o f fever, abs ces ses and hyperleukocytosis, they decided to refer to our institution for further studies. upon admission to our institution, the patient presented nutritional compromise, with spontaneous resolution of fever but persistence of high acute phase reactants, with significant improvement of leukocytosis. all the cutaneous lesions she presented were debrided at the site of remission. immunoglobulin levels, lymphocyte populations and dyhidrorhodamine test were normal. she remained with no weight gain, constipation, abdominal distension and hepatic involvement with elevated liver enzymes and prolonged coagulation times. new bone compromise was documented. inflammatory bowel disease, neoplastic or chronic infectious disease involvement was ruled out. during the stay in our institution no microbiological isolation was documented. skin, colon and bone tissue biopsies were performed and extra-institutionally performed liver biopsy were examined, showing as a single common finding leukocytoclastic vasculitis in all tissues. given the heterogeneous nature of the condition, the diagnostic possibility of an immune dysregulation disorder was considered and a therapeutic trial with nsaids and prednisolone at 1mg/kg/day was started, as well as genetic studies by exome sequencing. the exome results documented a novel mutation in the pik3cd gene [c.1895t> a (p.phe632tyr)] as probably pathogenic. the patient has presented a clinical improvement and a significant decrease in inflammation markers. at the moment, we are waiting for the performance of functional tests to define the definitive therapy for this patient. conclusions. this case description highlights the diagnostic difficulties that face in developing countries, where the nonavailability of functional testing has implications on the diagnosis opportunity and establishment of optimal therapeutic for patients with complex diseases such as primary immune regulatory disorders abstract/case report text background: autoinflammatory syndromes, a wide family of diseases, defined as attacks of inflammation that are unprovoked (or triggered by a minor event) and are primarily related to dysregulation of the innate immune system. periodic/recurrent fever syndromes were the former name of these diseases. however, only in two conditions: cyclic neutropenia (cn) and periodic fever, aphthous stomatitis, pharyngitis, and adenitis (pfapa) are febrile episodes truly periodic. for pfapa, although diagnostic criteria differ and there is no consensus research definition, patients are usually not difficult to recognize based on clinical course and presentation1. high index of suspicion and understanding the parental experiences and descriptions of febrile episodes is imperative in facilitating early recognition and timely diagnosis. aims: to standardize and summarized the clinical presentation of pfapa, based on parental descriptions and providers observation of febrile episodes. methods: utilizing a query for the icd-10 diagnosis code m04.8( + ) we identified a cohort of children diagnosed and managed for periodic fever, excluding those with monogenetic mutation (e.g. blau, majeed) and those with chronic illness. we reviewed the charts for documented parental report and provider observation of febrile episodes. standardized signs and symptoms were recorded for each patient [ table 1 ]. results: a cohort of 75 children, 35 boys (47%) and 40 girls (53%) with documented, cyclic episodes of fever >101, was identified. the average age at diagnosis was 3.77 +/-2.34 years. classic symptoms were reported or observed in 85% of patients (64). more than half (68%, 51 patients) had documentations of other symptoms, usually reported by parents to occur sporadically during some fever episodes. decreased oral intake and general "ill appearance" was reported by parents in 91% of patients. when reporting the time intervals, parents usually reported similar length for each episode, typically between 3-5 days, and regular interludes, typically between 3-4 weeks. the findings are summarized in table 2. discussion: the findings presented here are in concordance with previously published data describing pfapa as a syndrome affecting young, generally healthy children with identical episodes of fever lasting for a few days, recur with regularity. our data support the approach that parental observation is fundamental in identifying the unique pattern of illnesses. engaging the parents with directed interview is crucial to establish this clinical diagnosis in a timely fashion, prevent misdiagnoses of future febrile episodes as presumptive infections, and avert unnecessary antibiotics courses. this cohort adds the observation that up to 15% of patients who display this identifiable pattern of illnesses, do not present with aphthous stomatitis, pharyngitis, or adenitis. these classic symptoms although common, are not the rule. the cyclic chronicity of febrile episodes associated with general ill appearance (but not lethargy), and decreased po, in an otherwise healthy child is the clinical gold-standard of this condition. abstract/case report text heterozygous mutations in nfkb1 are frequently identified among immunodeficient patients with highly variable clinical symptoms. in a world-wide collaborative effort, we characterized the clinical and cellular phenotype and the management of 251 of these patients harboring 106 distinct nfkb1 variants. nfkb1 encodes the transcription factor precursor p105 which is processed to p50 (canonical pathway). known pathogenic variants cause p50 haploinsufficiency (due to protein decay) or p105-skipping (with expression of p50-like forms). most variants however are single amino acid changes with yet unknown effects. all sequence changes were assessed in silico for their probability of pathogenicity including 32 variants which were additionally tested in vitro. these analyses include the sub-cellular protein localization (microscopy), protein expression, stability and processing (western blotting), transcription factor activity (reporter assay) and dna-binding ( figure 1 ); the associated unadjusted odds ratio (or) was not significant, suggesting igrt had restored cases to a similar baseline infection risk as the controls. in a multivariate conditional logistic regression model adjusting for the significantly higher occurrence of risk factors in cases compared with controls in the preindex period, cases were associated with a 31% lower adjusted odds of major/severe infections in the post-index period versus controls (or=0.69; p=0.04). conclusions: patients who required treatment with igrt (privigen®/ hizentra®) had previously experienced more major/severe bacterial infections than those not needing treatment with igrt. however, igrt was associated with a reduction in the risk-adjusted odds of major/severe bacterial infections compared with non-igrt treated sid patients with haematological malignancies. abstract/case report text introduction: real-world data are lacking as far as identifying patients with secondary immunodeficiency (sid)/hypogammaglobulinemia who may benefit most from interventions to protect them from potentially fatal infections. this study aimed to identify risk factors for major/severe infections in patients with sid with underlying haematological malignancies. methods: a retrospective database analysis was conducted using the iqvia real-world data adjudicated claims -us database (study period: january 2010-september 2018). inclusion criteria were adults newly diagnosed with sid (first diagnosis termed the index date), with ≥12months continuous health plan enrolment pre-index (baseline period) and a minimum of 3 months' data post-index (mean: 615 days), with chronic lymphocytic leukaemia, multiple myeloma and/or non-hodgkin's lymphoma and without claims for any ig therapy in the 12-month baseline period. patient characteristics in the 12-month baseline period were assessed. over the post-index period, antibiotic/antiviral use and frequency of infections were assessed. the frequency of major/severe infections was determined using diagnosis codes for bacterial, viral, fungal, parasitic, other or unspecified causal pathogen infections. major/severe infections were defined as those requiring inpatient hospitalisation with an infection diagnosis code and/or use of intravenous (iv) antibiotics or iv antivirals in an outpatient setting. a multivariate cox proportional hazards (ph) model evaluated baseline patient characteristics associated with risk of major/severe infections post-index. results: a total of 4,066 patients met the inclusion criteria. the mean age of patients was 57 years and 56.0% were male. in the 12-month baseline period: 75.6% of patients received cancer treatments and 87.0% of patients received antibiotics (27.1% iv antibiotics). a total of 79.5% of patients experienced any infection, 65.6% experienced ≥2 infections and 30.4% experienced major/ severe infections. the mean number of infections over the baseline 12 months was 9.5 for any infection (at the unique diagnosis code level) and 0.7 for major/severe infections (unique hospitalisations with any infection diagnosis code and/or unique days with an outpatient iv antibiotic or iv antiviral). in the post-sid diagnosis period, 46.4% of patients had major/severe infections; of the major/ severe infections, 34.0% were identified as bacterial, 11.6% were viral, 6.3% were fungal, while 48.2% did not have a causal pathogen specified. a total of 21.3% of patients experienced one severe/ major infection and 10.5% experienced ≥4 severe/major infections ( figure 1 ). the mean annualised number of major/severe infections post-index was 1.5. receiver operating characteristic (roc) curve analysis to optimise sensitivity versus false positives in identifying those at risk of major/severe infections post-index identified a cutoff point of three bacterial infections in the baseline pre-index period as a potential optimal trigger to consider treatment to avoid major/severe infections post-index ( figure 2 ). the multivariate cox ph analysis suggested that hospitalisations, infections (≥3), or antibiotic use in the 12-months pre-index (prior to sid diagnosis) were predictive of major/severe infections post-index (post-sid diagnosis) (all p < 0.0001). conclusion: infections are common in patients with haematological malignancies and sid. key baseline predictors for major/severe infections in patients with an sid diagnosis were a history of infections, hospitalisations or antibiotic use. unfortunately, ms/ms detection is limited by the extremely low (e.g., pmol/l) protein concentrations in blood cells. peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-srm) is a robust method for quantification of low abundance proteins in complex matrices, including dried blood spots (dbs). in a study of 37 patients, immuno-srm reliably identified wiskott-aldrich syndrome (was) and x-linked agammaglobulinemia (xla) patients using direct quantification of proteins responsible for disease (front. immunol., 2018). we further expanded our approach for x-linked chronic granulomatous disease (x-cgd), ada and dock8 deficiency. marker proteins representing platelets, nk cells, and t-cells have also been analyzed to provide additional information about disease processes. these results demonstrate the utilization of immuno-srm as a sensitive platform for multiplexed signature peptide quantification and its potential for pidd newborn screening and clinical diagnosis from dbs. methods: candidate peptides were selected based on ms/ms sensitivity and uniqueness in the proteome. anti-peptide monoclonal antibodies (mabs) were then generated for peptide enrichment from dbs. blood from normal controls, xla, was, xl-cgd, dock8 and ada deficiency patients was collected after consent on filter paper, dried, and stored at -20°c. proteins were extracted from dbs, digested with trypsin, and enriched using mabs bound to magnetic beads. the enriched peptides were then eluted and analyzed with a waters xevo tq-xs. results: a multiplexed immuno-srm panel has been generated for screening eight signature peptides representing five pidd-specific and three cell-type specific proteins from dbs. limits of detection and quantification were femtomoles of peptide, the assay showed a broad linear range, and intra-assay and inter-assay coefficients of variation were < 20%. in samples from 13 xla, 8 was, 3 xl-cgd, 1 dock8 and 1 ada deficiency patients, signature peptides are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. also included in the multiplex panel are cell specific markers for platelets (cd42), t-cells (cd3ɛ), and nk cells (cd56). diagnostic cutoffs for each peptide concentration have been established. in was patients, cd42 levels were significantly reduced consistent with characteristic thrombocytopenia. immuno-srm also has the ability demonstrate the effects of pidd treatment. a was patient analyzed before and after bone marrow transplant showed normalized was protein and cd42 after treatment. two ada deficiency patients showed normal levels of ada enzyme after rbc transfusion. finally, a high-throughput (ht) immuno-srm method screens pidd-specific peptides in a 2.5-minute runtime meeting high volume nbs workflow requirements. this ht method returned identical results to the standard immuno-srm pidd panel. conclusions: the data herein demonstrate the feasibility of using immuno-srm as a broad clinical diagnostic for identifying and studying pidd patients from easily collected and shipped dbs. significantly, ht immuno-srm workflows represent a promising potential option for nbs of pidds and other congenital disorders. 7 chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih abstract/case report text we have previously used the artificial thymic organoid (ato) system, based on the 3d aggregation and culture of a delta-like canonical notch ligand 4-expressing stromal cell line (ms5-dll4) with cd34+ cells, to study t cell differentiation from cd34+ cells obtained from patients carrying defects that are intrinsic to hematopoietic cells (rag1-2, ak2, il2rg) or that affect thymus development (digeorge syndrome). we now report results of in vitro t cell differentiation of cd34+ cells obtained from patients with either haploinsufficiency or dominant negative (dn) mutations of the ikzf1 gene. ikzf1 is an essential transcription factor expressed throughout hematopoiesis and involved in both lymphocyte and myeloid differentiation. heterozygous germline mutations in ikzf1 give rise to distinct clinical phenotypes, depending on the nature of the mutation. in particular patients with ikzf1 haploinsufficiency present with common variable immunodeficiency (cvid) associated with b cell immune deficiency, b-all susceptibility, and autoimmune manifestations. no clinical t cell defects are evident among these patients, except for elevated naive and central memory cd3+cd8+ t cells. in contrast, patients carrying dn ikzf1 mutations present with combined immunodeficiency (cid) characterized by the presence of an increased proportion of naïve t cells, associated with defective generation of memory t cells, impaired t cell activation, signaling and proliferation, reduced t-helper (th) polarization, and susceptibility to pneumocystis pneumonia. different mouse models of ikzf1 mutations have been developed, however their phenotype does not fully match what reported in patients, and in some models indicates a more severe defect in t cell development. to address these controversies and to gain novel insights into the effects of distinct ikzf1 mutations on human t cell development, we used the ato system to analyze progression of t cell development from cd34+ cells obtained from one patient with ikzf1 haploinsufficiency and one patient with dn ikzf1 mutation. both patients showed a similar early block in t-cell differentiation a t p re -t c el l s ta g e. ho w ev e r, th e p a ti e nt wi t h i kz f1 haploinsufficiency showed a more pronounced leakiness, with a residual production of cd3+tcrab+ cells, which could account for the milder t-cell phenotype presented in this type of patients. interestingly, the dn patient presented an increased accumulation of cd4-cd8b-cd8aa+ cells. these results show an unexpected role for ikzf1 in humans in early stages of t-cell differentiation and indicate ikzf1 as a necessary factor for the induction of cd8b expression in t cells. abstract/case report text background: pediatric acute liver failure without an identifiable cause (indeterminate palf/ipalf) is associated with increased rates of liver transplant and mortality. aplastic anemia (aa) may develop weeks after the diagnosis. the immunologic mechanisms that contribute to disease pathogenesis have not been clearly elucidated. we report detailed immunophenotyping of a patient with ipalf/aa. case: a previously-healthy 7-year-old male was admitted for acute hepatitis presenting with jaundice and hepatosplenomegaly. evaluation for infectious, toxic, metabolic, autoimmune, and rheumatologic disorders was negative. he was pan-lymphopenic (cd45+alc 445 cells/μl) with an inverted cd4:cd8 ratio of 0.2 on admission. liver biopsy showed severe portal, interface, and lobular inflammation characterized by activated sinusoidal macrophages and perforinexpressing cd8+t-cells. compared to a healthy control, the percentage and number of peripheral blood cd8+t-cells expressing perforin (20%v.94%, 85 v.153 cells/μl) and granzyme-a/b (15%v.90%, 66 v.147 cells/μl) was also increased, while percentages of perforin+ (96%) and granzyme-a/b+(98%) nk cells were normal. bone marrow (bm) showed 75% cellularity with rare hemophagocytosis. serum cytokine analysis demonstrated il-18 4052 pg/ml, il-18-binding-protein 23003 pg/ml, cxcl9 4436 pg/ml, and sil-2rα 7979 u/ml, consistent with smoldering hemophagocytic lymphohistiocytosis (hlh), but he did not meet hlh diagnostic criteria. genetic sequencing did not identify pathogenic variants in 207 genes associated with primary immunodeficiencies. he was diagnosed with ipalf and treated with three doses of anakinra and two weeks of ruxolitinib, followed by prednisone 1-2 mg/kg/day and intravenous immunoglobulin 1g/kg/month. immunophenotyping performed after two months of therapy showed persistent inversion of the cd4:cd8 ratio with small expansions of cd3+cd4-cd8-cells and tcr··+t-cells. in the cd4+t-cell subset, there was a substantial paucity of naïve cells, with effector memory t cells (tem) being more abundant than central memory t cells (tcm). in the cd8+t-cell subset, the majority of cd45ro+cells were tem with no detectable tcm, and 49% of all cd3+t-cells were cd8+temra. in both cd4+ and cd8+t-cell subsets, activated (hla-dr+) and senescent (cd57+) subpopulations were increased, and the majority of cells expressed the exhaustion marker pd-1. the hepatic inflammatory infiltrate similarly reflected repetitive antigenic stimulation, with expansion of cd103+cd8+t-cells. quantitative immunoglobulins and total memory b-cells and plasmablasts (cd19+cd27+) were normal for age. however, there were no circulating iga-memory b-cells and a reduced number of iggswitched memory b-cells (table 1) . given the severity of his phenotype and bm hypocellularity (5%), allogeneic hct was performed using a matched-related-donor (10/ 10) with conditioning of flu+cy+alemtuzumab. at d+30, he shows improved liver function but persistent pancytopenia, with transfusion-dependence for platelets. discussion: to our knowledge, this is the first description of detailed immunophenotyping in blood from a patient with ipalf/ aa. other studies have identified distinguishing hepatic infiltrates and cytokine/chemokine profiles that suggest excessive activation of cytotoxic t-lymphocytes and macrophages contribute to disease pathogenesis (alonso et al, 2017). our preliminary data supports this hypothesis and expands the spectrum of immune dysregulation in the t and b cell compartments, proposing a primary immune etiology. immune dysregulation may be concordant with hyperinflammation and cytokine storm, the latter offering potential therapeutic targets. early diagnosis and treatment of immune dysregulation may prevent development of aa. background: x-linked agammaglobulinemia (xla) is one of the first inborn errors of immunity identified, with thousands of patients described to date. infections originally dominated the clinical phenotype, but early diagnosis and immunoglobulin replacement allowed for long term survival as well as recognition of late-onset complications. nodular regenerative hyperplasia (nrh) of the liver is a silent cause of non-cirrhotic portal hypertension. nrh underlying pathophysiology remains blurry and the disease has no specific treatment. nrh has been increasingly reported in primary immunodeficiency but data in xla are very limited. objectives: to assess and characterize nrh in patients with xla. methods: we retrospectively reviewed the medical records of all xla patients referred to the nih between 1994 and 2019. hepatology evaluation and liver biopsies were performed when clinically indicated. patients were stratified into nrh+ or nrhgroups, according to their nrh biopsy status (patients with no liver biopsies were classified as unknown). laboratory values are presented as medians. fisher's exact test and mann-whitney test were used to compare categorical and continuous variables, respectively. results: twenty-one xla patient records were reviewed, with a median age at start of follow-up (f/u) of 17y and a median duration of f/u of 10 years. eight patients underwent at least one liver biopsy of whom 6 (29% of nih xla cohort) were nrh+. the median age at nrh diagnosis was 20y (17-31). among patients who had liver biopsies, alanine aminotransferase (alt) levels were mildly elevated in all, while alkaline phosphatase (alp) levels were only increased in nrh+ patients (p=0.04). both nrh+ and nrhgroups had similar aspartate aminotransferase (ast) levels at baseline but higher values were observed at the end of f/u in the nrh+ group (85 vs. 32 u/l, p=0.04). persistently low platelet count ( < 100k/μl for more than 6 months), mildly to highly elevated hepatic venous pressure gradient (hvpg) and either hepatomegaly and/ or splenomegaly were present in all nrh+ patients. in opposition, neither persistently low platelet counts, nor hepato-or splenomegaly were present in the two nrh-patients evaluated. hvpg was normal in the only nrh-patient tested. all-cause mortality was higher among nrh+ patients (5/6, 83%) than in the rest of the cohort (1/15, 7% among nrh-and unknown patients, p=0.002). conclusions: based on our retrospective analysis, nrh appears as an underreported, frequent and severe late-onset complication in xla, which is highly associated with increased mortality. persistent thrombocytopenia, elevated alp, elevated hvpg, hepato-and/or splenomegaly were common in liver biopsyproven xla/nrh+ patients and distinguish them from xla/ nrh-patients. based on nrh prevalence, severity, lack of specific treatment and poor outcome in xla, immune-reconstitution (rather than igg replacement and infectious prophylaxis) should be considered early in this population in order to prevent fatal long term complications. abstract/case report text introduction: barth syndrome (bths) is an x-linked recessive disorder caused by a mutation in the tafazzin (taz) gene resulting in an inborn error of cardiolipin phospholipid metabolism (an important mitochondrial inner membrane lipid). it is commonly characterized by intermittent neutropenia and cardiac and skeletal myopathies. we present a case of bths with associated lymphopenia and hypogammaglobulinemia, which has not been previously described in the literature. case report: a two-month old male, born full term with normal newborn screening, was first admitted for rsv bronchiolitis. at this time, patient underwent an echocardiogram given his older brother with hydrops had died hours after birth and on autopsy was found to have dilated cardiomyopathy (dcm). patient was similarly noted to have dcm and thus had whole exome sequencing done that showed a hemizygous mutation in the taz gene (c.639g>a). this novel variant resulted in early termination of the protein (p.trp213ter) with concern for loss of function. in regard to patient's first year of life, he had frequent uri symptoms, 6 episodes of acute otitis media requiring tympanostomy tubes, but no documented pneumonias or other serious bacterial infections. patient also had gross developmental delay, particularly motor, and feeding difficulties with persistent failure to thrive requiring g tube placement. his absolute neutrophil count ranged from 900-6800 cells/mm3 in the first year. at age 13 months, patient was found to be in acute decompensated heart failure with concern for myocarditis (ck 15,313 u/l, troponin i 3.006 ng/ml) as well as acute hypoxic respiratory failure with respiratory cultures growing pseudomonas. he was incidentally found to have an igg level of 188 mg/dl (normal for age 345-1213) and treated empirically with ivig. when seen by immunology, further workup showed persistent b cell lymphopenia (absolute cd19 of 167-377/mm3). he also had a low initial nk cell count (78-84/mm3, later normal) with normal cd8 and cd4 t cell counts. tetanus and hib titers could not be assessed as he had recently received ivig. his igg trended up to 931 mg/dl a few days after initial ivig and then subsequently dropped to 632 mg/ dl, with a level of 241 mg/dl two weeks following initial dose. workup for gastrointestinal or renal losses of immunoglobulin were negative. he also shortly after developed enterobacter bacteremia. his igg levels at this time continue to remain around 270 mg/dl. he subsequently required a heart transplant at age 14 months for his dcm. after transplant, he continued to improve from a cardiac standpoint, but his lymphopenia persisted and each time he was weaned off ivig, his hypogammaglobulinemia persisted at 275-372 mg/dl thus requiring additional ivig replacement over the course of the next 8 months. the remainder of immunoglobulins were normal initially, but the igm slowly dropped over time to 31-33.8 mg/dl. patient was started on weekly subcutaneous immunoglobulin replacement at 22 months, doing well clinically at age 24-month follow up. conclusion: here we present a patient with bths, with a novel variant, who had b-cell lymphopenia as part of his presentation with persistent hypogammaglobulinemia requiring ivig replacement. year fellow/ucla 2 associate professor/division of allergy, immunology, and rheumatology, university of california los angeles 3 chief of pediatric allergy and immunology/harbor-ucla 4 chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih 5 biologist/laboratory of clinical immunology and microbiology, division of intramural research, national institute of allergy and infectious diseases, national institutes of health 6 project scientist/ucla abstract/case report text a 28-year-old female presented for combined immunodeficiency. at 2 years of age she was diagnosed with rag1 hypomorphism and started on ivig. as a child, she was hospitalized for pneumonias and cryptococcal meningitis. she suffered sinusitis, hepatitis, tooth abscess, cmv and herpes stomatitis. later, she experienced recurrent cutaneous abscesses, utis, vaginal yeast infections, and hidradenitis. she twice hospitalized recently for pneumonias and diagnosed with mycobacterium abscessus on bronchoscopy. she suffers onychomycosis, osteomyelitis and oral and esophageal candidiasis with odynophagia. on exam, she had white plaques on tongue and buccal mucosa. she had hyperpigmented plaques on forehead and cheeks and thickened nails. immune evaluation was significant for lymphopenia with alc 770 and thrombocytopenia with platelets 102k. b cells were nearly absent (2 absolute count) and nk cells were low at 19 absolute count. ige was absent, igm 23 mg/dl, iga 72 mg/dl and igg 872mg/dl (on replacement). her total cd3+ count was 791, cd4+ t cells were low at 17%, but cd8+ cells normal at 78%. the cd4+ t cells were mostly memory phenotype, which probably reflects lymphopenia-induced proliferation of a small number of clones. her cd8+ t cells also had an elevated amount of memory cells for age, but still had presence of naive cd8+ t cells. as expected with perpetual lymphopeniainduced proliferation, there was evidence of terminal memory (temra) in the cd8+ lineage. proliferative responses of t cells were modest. cd4+ t cells did respond to pokeweed, but less to pha and cona. there were no antigen specific responses. trecs were normal. esr was mildly elevated at 31. of note, her liver enzymes were elevated with alkaline phosphatase 556 and ast 116, presumably secondary to prolonged fluconazole use. w es r e v e a l e d a k n o w n p a t h o g e n i c v ar i a n t i n s tat 1 (nm_007315.3: c.537c>g (p.n179k)) as well as a heterozygous variant in rag1 p.m355t. the stat1 mutation is de novo and was previously published as a gain of function mutation. however, when we performed validation studies to evaluate cd4+ cells with stimulation to ifna, the patient had decreased pstat1 as compared with control. va7.2 analysis was performed to evaluate rag1 defect and showed 12% of t cells with va7.2 expression confirming that the rag1 defect is not clinically significant. she developed severe thrombocytopenia refractory to platelet transfusions and ivig. she was started on ruxolitinib which improved platelet counts. however, she presented with shortness of breath, persistent tachycardia and was found to have cmv carditis and hepatitis significant for echocardiogram with ef 23%. cmv pcr is improving with last check 815 iu/ml after 1 month of therapy with ganciclovir. we now are looking for evidence of socs1 to explain the decreased stat1 phosphorylation. genetic testing is critical when evaluating a patient with immunodeficiency. our patient demonstrates that genetic mutations cannot be taken at face value and should be evaluated and validated fully to optimize patient care. fellow/university at buffalo / oishei children's hospital abstract/case report text opportunistic infections (oi) are commonly seen in patients undergoing hematopoietic cell transplantation (hct). different strategies for antimicrobial prophylaxis are often employed in the transplant setting to reduce the likelihood of encountering infection. the predisposing risks for infections include the expected neutropenia and lymphopenia following conditioning, prolonged defects in cell-mediated and humoral immunity during the engraftment period, and iatrogenic immunosuppression by medications for graft versus host disease (gvhd). we report the case of a 4-year-old male with acute lymphoblastic leukemia, which relapsed to chronic myelogenous leukemic blast crisis, and failed a subsequent allogeneic hct with central nervous system relapse. he was subjected to a second allogeneic hct. his immediate post-second transplant course was complicated with skin and gut gvhd, and infection and/or reactivation of coronavirus, respiratory adenovirus, epstein-barr virus, and human herpesvirus 6. while the herpesviral infections were controlled with antivirals and rituximab, adenovirus c1 infection proceeded to involve the gastrointestinal tract, and proved persistent over several months despite use of cidofovir. the patient's gvhd and transplant-associated thrombotic microangiopathy necessitated use of further immunosuppressants, including the complement protein c5-binding eculizumab (an inhibitor of formation of the terminal c5b-9 complex), ruxolitinib (a janus kinase [jak] 1/2 inhibitor) and low-dose interleukin-2. h i s c l i n i c a l c o u r s e w a s f u r t h e r c o m p l i c a t e d b y stenotrophomonas maltophilia gut colonization and subsequent bacteremia, as well as multiple gram-positive bacteremia courses. at around day +190, there was a life-threatening pericarditis with pericardial effusion and respiratory distress, associated with pneumocystis and stenotrophomonas being isolated from bronchoalveolar lavage. this occurred despite the patient being on pentamidine prophylaxis. the patient eventually recovered on trimethoprim/sulfamethoxazole therapy. we discuss the various risk factors potentially contributing to each oi in this illustrative case. in particular, complement and jak inhibitor therapy are fairly new drugs approved for other indications, whose off-label use in transplant patients is increasing. both have recently been associated with certain oi in the literature, as they are in this patient. abstract/case report text background: autoimmune lymphoproliferative syndrome (alps) is characterized by chronic nonmalignant lymphadenopathy, splenomegaly, hepatomegaly, cytopenias, and other autoimmune manifestations. typically, the biomarker profile of patients with alps includes elevated tcr αβ+ dnt cells, serum igg, serum b12, serum il10 and soluble fas ligand (sfasl). hdl cholesterol can also be significantly low. alps is caused by lymphocyte accumulation due to defects in the fas-mediated apoptosis signaling pathway. these defects cause resistance to physiological apoptosis in lymphocyte populations that results in chronic lymphoproliferation. the molecular defect underlying most alps etiologies is attributed to heterozygous germline or somatic (limited to dnt cell subpopulation) pathogenic single nucleotide variants (snv) in fas. we describe copy number variants (cnvs) at the fas locus underlying alps in 3 unrelated families. methods: through the centralized sequencing initiative at at the national institute of allergy and infectious diseases (niaid), patients undergo genomic workup to identify molecular defects contributing to clinical phenotypes of immune system disorders. all patients receive exome sequencing and a subset of patients also receive array-cgh analysis. patients and results: we performed exome sequencing on 132 patients with a clinical diagnosis of alps. for 30 patients with no molecular defect through exome, we performed cnv analysis. in this cohort, we identified three patients with a copy number variant involving the fas locus. all patients presented with splenomegaly and lymphadenopathy in childhood with ages of onset ranging from 8 months to 9 years old. all patients experienced anemia, autoimmune neutropenia, and thrombocytopenia. they had biomarker evidence showing elevated serum b12 levels, sfasl levels, and elevated αβ+dnt cell populations. they were found to have very low hdl cholesterol in early childhood ranging from 5-8mg/dl (38-55 mg/dl). all patients had negative family histories for lymphoproliferative disorders and immunodeficiency. these patients had clinical presentations and biomarker profiles similar to alps patients with germline and somatic fas variants. patient 1: we detected a~1.03 mb copy number loss encompassing all of fas. parental studies were not performed. patient 2: we detected a~1.004 mb copy number loss encompassing all of fas. parental studies showed this to be maternally inherited. in addition, prior karyotype testing of the bone marrow showed the same deletion. patient 3: we detected a~0.044 mb copy number loss encompassing exons 7-9 of fas. parental studies were not performed. these results are consistent with the pathogenic nature of copy number variant losses involving fas. the mechanism of disease in these patients is consistent with haploinsufficiency. in family 2, the mother harboring the fas deletion is unaffected. this is consistent with prior observation of reduced penetrance within a family in alps. conclusion: these three cases harbored causative deletions in fas in the presence of biomarkers indicative of alps and negative results for germline and somatic genetic variant testing. these patients demonstrate that copy number variant analysis should be pursued if there is robust clinical and biomarker evidence of alps as it can lead to a molecular diagnosis and appropriate treatment when exome or next generation panel based fas sequencing is inconclusive. abstract/case report text rationale: the thymus is essential for the development of tcells. patients with thymoma have decreased aire expression and have an abnormal thymic microenvironment where the negative selection of t-cells is compromised, resulting in a broad spectrum of autoimmune-mediated diseases. besides myasthenia gravis, which is found in 15 to 20% of patients with thymoma, other autoimmune diseases have been reported including erythroblastopenia, systemic lupus erythematosus, inflammatory myopathies, thyroid disorders and good's syndrome. recent studies have described additional autoimmune conditions such as pneumonitis in thymoma patients. we identified a patient who developed chronic cough post-thymectomy and was found to have lymphocytic pneumonitis with associated autoantibodies against lung antigen kcnrg and lung immunopathology consistent with apeced pneumonitis, which implies a common pathogenic mechanism between these conditions. methods: we describe a patient with thymoma who developed autoimmune pneumonitis associated with kcnrg autoantibodies and a characteristic pattern of immunopathology recently described in patients with monogenic disorder caused by primary aire deficiency (apeced) and secondary aire deficiencies (thymoma, rag deficiency). results: patient is a 32-year-old male with no significant past medical history who was in good state of health until age 20 when he was diagnosed with and received treatment for guttate psoriasis (resolved with uv therapy) and alopecia areata. at age 30, he developed severe abdominal pain and weight loss. he had an abdominal ct performed that showed chronic pancreatitis and thymoma. one month later, the patient underwent thymectomy and subsequently, underwent ercp and pancreas biopsy, revealing atrophic pancreatitis with negative staining for lgg and lgg4. at that time, he was started on pancreatic enzymes with improvement of abdominal symptoms. following thymectomy, he developed persistent dry cough and recurrent symptoms of sinusitis which did not respond to several courses of oral antibiotics to treat his positive culture for pseudomonas. he had a negative work up for vocal cord dysfunction and cystic fibrosis, and negative autoantibodies against ifn-gamma, il-17a, and gm-csf. for work up of chronic cough, the patient underwent ct imaging of the chest which revealed diffuse peri-bronchial thickening, mucus plugging, and tree-in-bud nodularity through most of his lungs. he underwent bronchoscopy with bal which revealed n o r m a l b r o n c h i a l m u c o s a a n d a i r w a y n e u t r o p h i l i a . endobronchial biopsies showed basement membrane thickening and dramatic lymphocyte infiltration in intraepithelial and submucosal areas. his bal cultures revealed mycobacterium intracellulare/chimaera. patient was also tested for autoantibodies against lung-specific bactericidal/permeabilityincreasing fold-containing b1 (bpifb1) and the potassium channel regulator kcnrg that have been associated with the development of pneumonitis in patient with apeced, thymoma and rag deficiency, and was found to have kcnrg-targeted autoantibodies. conclusions: thymoma is a disease associated with secondary aire deficiency. this case illustrates common clinical, radiographic, histological, and autoantibody features in thymomaassociated and apeced-associated pneumonitis, indicating that disorders with primary and secondary aire deficiencies may have common pathogenetic mechanisms. bpifb1 and kcnrg should be included in the autoantibody profile testing of patients with thymoma and lung disease. immune suppression and antimycobacterial antibiotic treatment are planned. abstract/case report text introduction/background: activated phosphoinositide 3-kinase δ (pi3kδ) syndrome (apds) is a primary immunodeficiency caused by a gain-of-function mutation in the pik3cd gene that encodes the p110δ catalytic subunit of pi3kδ. it is characterized by recurrent respiratory tract infections, lymphoproliferation, nodular mucosal lymphoid hyperplasia, enteropathy, ebv and/or cmv infection, reduced t cell function and high levels of igm. there is not evidence of this disease in peruvian patients. methods: a case series of two pediatric patients with apds. results: the first patient is a girl of non-consanguineous parents. family history shows four maternal uncles died at pediatric ages with unknown diagnosis. at the age of 2, she presented lymphadenopathy and fever being treated as cat scratch disease without improvement of symptoms. 8 months later, she was hospitalized due to anemia, mild hepatosplenomegaly, ascites and chronic diarrhea and diagnosed with gastrointestinal tuberculosis (tb). a hepatic biopsy only showed reactive hepatitis. however, the patient did not improve her symptoms despite anti tb treatment. 2 years later, she was hospitalized for lymphadenopathy, pancytopenia, chronic diarrhea, ascites and severe hepatosplenomegaly. cmv igg was positive and lymph node biopsy revealed paracortical and follicular lymphoid hyperplasia due to ebv infection without neoplastic proliferation. low cd4+ t and cd19+ b cells and high igg levels were found (table 1) . at this time, it was suggested the diagnosis of apds which was confirmed by next generation sequencing (ngs) identifying a heterozygous mutation in the pik3cd gene (c.3061g>a, p.glu1021lys). she was treated with sirolimus and ivig for 2 years. the symptoms persisted despite treatment and died at the age of 6. the second patient is a 6-year-old girl also of non-consanguineous parents. family history includes eczema (father) and colorectal cancer (mother). she has had recurrent respiratory infections, chronic diarrhea and poor weight gain since 4 months old receiving symptomatic treatment only. at the age of 3, she was hospitalized for persistent pneumonia ( p s e u d o m o n a s p o s i t i v e ) , l y m p h a d e n o p a t h y a n d m i l d hepatosplenomegaly. a ct scan showed bilateral bronchiectasis and the sweat chloride test was negative. based on this, a diagnosis of cystic fibrosis was made and treatment was started. however, a genetic study only showed heterozygous mutations in the cftr gene (g551d and g542x). 1 year later, she presented a neck-located skin abscess. at the age of 5, she was hospitalized for complicated pneumonia, diarrhea, lymphadenopathy, ascites and severe hepatosplenomegaly. multiple polyps in the duodenum and colon with lymphoid hyperplasia were detected, ebv igm and igg were positive and a lymph node biopsy showed paracortical hyperplasia without neoplastic proliferation. cd8+ t cells and igm levels were increased (table 1) . a diagnosis of apds was suspected and ivig was started. ngs showed the same mutation as the first patient (c.3061g>a, p.glu1021lys). conclusion: apds should be considered in patients with recurrent respiratory tract infections, lymphoproliferation, enteropathy and abnormal immunologic function without another explanation. ngs is a useful tool to identify these cases in low-income countries. acknowledgments: we thank drs. raif geha and janet chou, division of immunology, boston children's hospital, harvard medical school for the genetic diagnosis. background: granulomatous-lymphocytic interstitial lung disease (glild) is an increasingly recognized pulmonary complication associated with common variable immunodeficiency (cvid) but the natural history and long term prognosis remains poorly defined. imaging findings with computed tomography (ct) are heterogeneous and visual features do not consistently predict a patient's progression to fibrotic lung disease. computer-aided lung informatics for pathology evaluation and rating (caliper) provides an objective analysis of lung parenchymal texture and quantifies the extent of normal lung, along with abnormal features such as honeycombing, reticular/consolidative and groundglass opacity. this may be useful in cvid patients to monitor changes in character or extent of disease and may facilitate early intervention before the disease becomes more aggressive or advanced. case description: our patient is a 46-year-old non-smoking female with cvid who has been followed for her cvid and associated interstitial lung disease. for more than twenty years, she has had varying abnormalities found on chest ct and these appear consistent with glild. specifically, she has had variable regions of mixed consolidation, ill-defined nodularity and septal thickening. the changing morphology and distribution made assessment of overall severity and extent of fibrosis versus parenchymal infiltration inconsistent. for clinical decision support we used caliper to analyze the current ct (2019) and compared caliper results for previous ct data. caliper provided a comprehensive analysis of the extent and characteristics of parenchymal features, and objectively determined normal and abnormal regions, some of which were not visually apparent. the caliper color overlay was able to highlight subtle regions of ground-glass opacity in areas that visually were regarded as uninvolved lung and quantify the extent of the reticular densities/ consolidation over time. caliper does not differentiate reticulation from consolidation, does not detect nodularity or septal thickening, and ct imaging cannot distinguish inflammation from fibrosis. however, caliper has the power to quantitatively assess overall disease extent and demonstrate subtle abnormalities that would otherwise have been dismissed as normal, given relative sparing compared to other regions. caliper may also provide evidence for disease progression or therapeutic response that is not otherwise radiographically apparent. conclusion: caliper assessment may be a useful tool as an adjunct for a patient with glild to help quantify the extent and character of lung parenchymal involvement. this information may serve as an important guide for clinicians in the assessment of successful management and early intervention to prevent irreversible fibrosis. patients had a trial of fingolimod without any beneficial changes in immune status. both patients receive pneumocystis jirovecii pneumonia prophylaxis with sulfamethoxazole-trimethoprim. conclusions: these results indicate that s1pl deficiency due to sgpl1 mutations is a syndromic primary immunodeficiency leading to profound lymphopenia and hypogammaglobulinemia. our data emphasize the importance of sphingolipid metabolism for an efficient immune response and the need for more studies to delineate the exact mechanisms on how this happens in humans. after 1d at 4°c there was a median -9% (range -44% to +3%; p=0.017) change in activity. after 2d -11% (range -48% to +8; p=0.006), 3d -13% (range -55% to +4%; p < 0.001), 4d -16% (-60% to +6%; p < 0.001) and after 7d -21% (-68% to +2% p < 0.001). a 4°c stability of 3d was determined from the median percentage reduction; total allowable error adjusted stability data indicated a 4°c stability of 5d. samples stored at -20°c following repeat freeze thawing saw a freeze/thaw cycle dependent decrease in ch50 activity. after 1 freeze/thaw cycle there was a median -9% (range -22% to +1%; p=0.018) change, 2 cycles -11% (range -26% to +2%; p=0.003), 3 cycles -13% (range -38% to +2%; p < 0.001), 4 cycles -23% (range -69% to -6%; p < 0.001) and after 5 cycles -25% (range -68% to -7%; p < 0.001). allowable error adjusted stability data indicated a maximum of 3 freeze/thaw cycles. conclusion: sample storage and handling can have a significant impact on functional complement assessments. room temperature storage should be avoided unless samples will be analysed on the day of collection, 4°c storage is tolerable providing that assessment is within 3d; freezing samples at -20°c with limited freeze/thaw analysis would be optimal. however, further investigations into longer-term storage at -20°c and -80°c would be beneficial. conclusions: gi disease is common in cvid affecting 63% of patients in our cohort. gi+ cvid patients have a higher frequency of autoimmune manifestations than those without gi complaints. the odds of itp, hypothyroidism, and evans syndrome all showed significantly increased odds in the gi+ group. the results of our study may have implications for both gastroenterologist and immunologist. recurrent infections especially those of the sinopulmonary tract are often the trigger for cvid evaluation. autoimmune and gi symptoms however may be the initial presentations of cvid and overlooked until other more recognizable manifestations evolve. the combination of gi issues and autoimmunity especially thrombocytopenia, evans syndrome, and hypothyroidism should include cvid in the gi differential. for the immunologist, a cbc is standard in the work-up of cvid and may reveal autoimmune cytopenia. evaluation for autoimmune disease and in particular hypothyroidism is not. given our findings an initial immune work up specifically for thyroid disease may be indicated. is the transcriptional factor for many cytokines such as il-17, responsible for t cell and neutrophil defense again fungal infection. stat3 mutation leads to defect of neutrophil proliferation and chemotaxis to inflammatory site as well as production of antimicrobial peptides by respiratory epithelial cells. the poor tissue repair in the cavitary lesions and bacterial superinfection in patient's lung created a culture dish for fungal growth and dissemination. traveling to the endemic area and patient's noncompliance to antifungal prophylactic treatment further increased the risk of histoplasmosis infection. pediatric immunologist/john hunter children's hospital abstract/case report text we present the case of a 20 month old boy, the first child to his nonconsanguineous parents of european descent. he first presented at 9 months of age with a cellulitis of his right fourth finger culture positive for staphylococcus aureus which responded to a prolonged course of flucloxacillin. at 12 months he presented with norovirus positive gastroenteritis leading to a brief admission and slow resolution. the first of two severe episodes of oral stomatitis and respiratory distress occurred at 13 months of age. hsv1 was isolated from the oral lesions and blood culture during that admission was positive for kingella kingae. no cardiac or bone involvement was identified. a more severe episode of oral stomatitis occurred two months later (age 16 months) swab positive for an enterovirus (not typed). due to airway compromise and rapid deterioration he was admitted to the pediatric intensive care unit. again, kingella kingae was cultured from blood cultures with no obvious focal systemic source. the only notable clinical finding was rapid deterioration and, in retrospect, the absence of any significant recorded fever ( < 38oc). crp elevation was observed (max. 188 mg/l) and neutropenia was found with each of the more severe infectious presentations but recovered in the interval. baseline immunological investigations were normal (lymphocyte subsets, naïve t cell populations, lymphocyte proliferation, serum immunoglobulins and vaccine responses). serial measurement of circulating neutrophils did not identify a cyclical pattern and they were morphologically normal. a panel of genes relevant to primary immunodeficiency (invitae©) revealed a homozygous mutation in irak4 ((c.877c>t (p.gln293*)) which leads to a premature stop codon. this is a known pathogenic mutation leading to disease and is most prevalent in the european population (allele frequency (gnomad) = 0.0005). prophylaxis with sulfamethoxazole / trimethoprim and amoxicillin was commenced along with monthly ivig. he has been well since diagnosis with no further severe infectious presentations. functional testing is underway to assess in vitro host viral defence in our patient and potential novel mechanisms relevant to this rare innate immunodeficiency. case studies will be presented on the five cases of fmp that were diagnosed and treated in 2019. potential exposures were identified in four out of five cases: gardening exposure in one case and vaping exposure in three cases. all five were male, age range 14-45. four were gp91 deficient, and one was p47-phox deficient. historically, the vast majority of cases of fmp could be traced to a significant gardening exposure such as lawn mowing or spreading mulch. this was the first year that we saw patients with no identifiable gardening exposure in the setting of significant vaping exposure. with vaping at epidemic levels, especially among teenagers and young adults, it is important to consider that a vaping history is potentially a risk factor for fmp and counseling regarding the potential risks of vaping should be included in infection risk modification for all patients with cgd. abstract/case report text background: granulocyte-macrophage colony-stimulating factor (gm-csf) plays a critical role in macrophage and dendritic cell maturation and host defense against fungus. autoantibodies to gm-csf are associated with susceptibility to cryptococcus and nocardia infections as well as pulmonary alveolar proteinosis (pap) in otherwise healthy individuals. we report a case of a 23-year-old previously healthy female who presented with cryptococcal meningitis and was found to have autoantibodies against gm-csf. case presentation: 6 weeks prior to admission, our previously healthy 23-yearold taiwanese female developed a headache associated with tinnitus and visual changes. the headache worsened over the next few weeks and she developed photophobia, phonophobia, and severe nausea/vomiting. at presentation, her exam was notable for papilledema, bilateral cn vi palsy and right foot & left hand paresthesia. mri brain showed ring-enhancing lesions in the anterior frontal lobe, caudate head, and the inferior globus pallidus. she underwent a diagnostic and therapeutic lp. opening pressure was elevated at 60 and csf studies were notable for low glucose, elevated protein, pleiocytosis (94% lymphocytes) and positive cryptococcal antigen. csf culture grew cryptococcus gattii. ct chest revealed a right upper lobe and a left lower lobe nodule. workup: cbc with diff was unremarkable. hiv was negative. lymphocyte subsets were unremarkable with only mildly decreased nk cells, normal immunoglobulin panel including ige, protective titers to tetanus, diphtheria, and ppsv23. targeted genetic sequencing did not identify any known mutations in primary immunodeficiency. notably, anti-gmcsf autoantibodies were detected by elisa and were able to neutralize gm-csf phosphorylation of stat5 detected by flow cytometry. autoantibodies to ifn-γ were not detected. management: patient was initiated on a 6-week course of liposomal amphotericin b and flucytosine. her csf cultures were cleared of cryptococcus after 20 days of treatment, but her hospital course was complicated by persistently symptomatic intracranial hypertension, worsening pleiocytosis, and elevated cytokine levels in the csf, all of which were consistent with post-infectious inflammatory syndrome (piirs). she received therapeutic lps 2-7x/week until subsequent ventriculoperitoneal shunt placement. concurrently, methylprednisolone was administered for 7 days with a gradual prednisone taper. these interventions led to improvements in her symptoms, including diplopia, and reduction in opening pressures and inflammatory markers in the csf. lifelong fluconazole prophylaxis was recommended. from a pulmonary standpoint, she remained asymptomatic without signs of pap and has had normal pulmonary function tests (normal dlco) and stable chest imaging. conclusion: in otherwise healthy hiv-negative patients presenting with extrapulmonary cryptococcus or nocardia infections, autoantibodies to gm-csf should be suspected and testing for functional autoantibodies to gm-csf (and ifn-γ) should be sent, as genetic testing will not pick up this disease entity. genetic testing should be considered to rule out gata2 deficiency and x-linked cd40l deficiency. idiopathic cd4 lymphopenia can be ruled out with lymphocyte enumeration. immediate treatment of cryptococcosis is not necessarily different from patients without gm-csf autoantibodies. long-term prophylaxis (fluconazole if presenting with cryptococcus; trimethoprim-sulfamethoxazole if with nocardia) is likely warranted in addition to monitoring for the development of pap. recognizing piirs in patients with cryptococcal meningitis and management with corticosteroids are critical steps. abstract/case report text background: non-infectious complications cause most morbidity and mortality in common variable immunodeficiency (cvid). cvid with complications (cvidc) is defined by elevated t helper 1 (th1) responses attributed to increased circulating microbial products resulting from mucosal iga deficiency. however, complications do not uniformly occur in those with iga deficiency. objective: we tested whether cvidc occurs preferentially in those with hyper-responsiveness to microbial stimuli, manifested by elevated nf-κb-driven cytokines and resultant th1 responses in cvid patients with increased circulating microbial products. methods: we applied unbiased high-throughput seromics and mass cytometry, cellular and molecular biology approaches, and clinical record review in a 78 subject cvid cohort. results: cvidc was defined by increased nf-κb-driven cytokines that promote th1 immunity in blood in association with elevated soluble cd14, a marker of circulating microbial products, and elevated tnf production by peripheral blood mononuclear cells stimulated with lipopolysaccharide. this cytokine upsurge was associated with mutation of full-length nfkb1 p105 gene product (1375delt) but not mutations that also involved the nfkb1 p50 product involved in transactivation. cytokine elevation corresponded with increased cd14+cd16-monocytes expressing higher cd86 and hla-dr and more central and effector memory cd4+ t cells, t cell chemoattractants, and t cellpredominant tissue pathology. those with granulomatous or neutrophilpredominant, rather than t cell, pathology had the highest tnf. tnf antagonism improved neutrophilic gastritis in cvid with nfkb1 1375delt after t cell targeted therapy failed. conclusion: nf-κb dysfunction underlies th1 immunopathology and tnf-associated innate inflammation in cvidc. both forms of nf-κb immune dysregulation may divergently shape cvid immunopathology. staff clinician/laboratory of clinical immunology and microbiology, immunopathogenesis section, national institute of allergy and immunology, national institutes of health, abstract/case report text introduction: patients with autoantibodies to ifn-γ develop severe and progressive infections with intracellular pathogens, despite aggressive antimicrobial treatment. we describe the use of daratumumab (anti-cd38, targeting plasma cells) in a patient with autoantibodies to ifn-γ and progressive disseminated mycobacterium avium infection. she had progressive disease despite treatment with multi-drug antimycobacterials rituximab, and bortezomib. methods: clinical symptoms, total cd19/cd20, anti-ifn-γ autoantibody titers, and specific imaging were obtained before and after treatment with daratumumab. anti-ifn-γ autoantibody titers were determined by serial 10-fold dilutions of plasma and measuring anti-ifn-γ autoantibody levels by a particle-based technique as previously described. results: a 31-year-old filipino woman had progressive disseminated m. avium with extensive bone and soft tissue involvement (calvarium, ribs, bilateral arm soft tissue, paraspinal muscles, bilateral glutei, left inferior pubic ramus, bilateral iliac bones, sacrum, and bilateral humeri) and a tracheo-esophageal fistula. she received bedaquiline, azithromycin, ethambutol, tedizolid, moxifloxacin, clofazimine and meropenem as well as rituximab 1g once monthly for 5 months. despite these she had progression of clinical and radiographic disease. bortezomib 1.3 mg/m2 twice weekly for 8 weeks was added, but discontinued for ast and alt elevations. rituximab was continued to maintain cd20 numbers undetectable but clinical and radiographic disease progressed. while on rituximab, total igg level and anti-ifn-γ autoantibody levels decreased from 1521mg/dl to 1069mg/dl and 3058 to 2504, respectively. while on bortezomib, total igg levels remained stable (1031mg/dl to 1051mg/dl) and anti-ifn-γ autoantibody levels fell slightly (2504 to 1275). after starting daratumumab, there was clinical and radiographic improvement, with reduced pain and disappearance of multiple soft tissue lesions. igg levels decreased from 1100mg/dl to 434mg/dl and anti-ifn-γ autoantibody levels decreased from 1275 to 157. adverse effects of daratumumab were urticaria, pruritus and shortness of breath after the first infusion and aseptic meningitis after the 5th infusion. conclusions: daratumumab resulted in clinical and radiographic improvement of disseminated m. avium in a patient with rituximab and bortezimib-refractory autoantibodies to ifn-γ. daratumumab is another potentially effective therapeutic agent for anti-ifn-γ autoantibodies. abstract/case report text next-generation sequencing (ngs) is now routinely used as a clinical diagnostic tool. however, regions of high sequence homology continue to be a major challenge for short-read technologies. regions within ikbkg, ncf1, sbds, c4a, c4b, coro1a, fcgr3a, fcgr3b, pms2, slfn11, slfn13, stat5b, unc93b1, and ups18 are not available by standard ngs. we discuss strategies for analysis of these special regions. we have developed a strategy for supplementing our disease targeted panels which are performed using capture chemistry and a standard reference file. the supplemental method uses gene specific long range amplicon and a special gene specific reference file for alignment. the genes of interest are separated from their homologous counterparts using specific long range amplification primers. multiple amplicons may be pooled together and prepared for sequencing on an illumina miseq instrument using truseq nano dna library prep. bioinformatic analysis proceeds with a custom reference file in which non-specific regions of homology have been removed. this allows reads to be uniquely mapped despite significant homology; a requirement for variant calling. we prepared specific amplicon for several homologous gene targets including the ikbkg gene and the ikbkg pseudogene (ikbkgp1). both amplicons were sequenced in separate reactions and were compared with the standard capture method. variants which are not called in the standard-capture method due to poor mapping scores (non-uniquely mapped reads) are called in the amplicon method. in the capture method, the variants are visualized in the bam as a mixture of gene and pseudogene, while gene and pseudogene variants are clearly separated and identified in the amplicon method. due to high variability in alignment, many homologous regions do not provide reliable copy number variant (cnv) results and must be removed from cnv analysis. however in some situations, we are able to creatively leverage cnv analysis to identify alleles that mis-align to the pseudogene. the pathogenic ncf1 gt deletion in exon 2 appears to resemble a copy number deletion event when present as reads from one allele mis-align to the ncf1b and ncf1c pseudogenes. complement genes, c4a and c4b, share alignment due to their high homology with each other. cnv analysis in normal samples represents four alleles rather than two alleles. cnv events may have a weak signal with no indication of which gene is affected. variant frequencies from the capture and supplemental pcr analysis can be used in tandem with cnv analysis to detect events and may indicate which gene is affected. we plan to include these strategies in our new inborn errors of immunodeficiency gene panel (ieigp) which will enable us to provide a more comprehensive analysis than is currently available. the im diagnosed were inflammatory bowel disease-like (n = 5, with perianal fistula in 2/5), mouth ulcers (n = 1), discoid lupus (n = 1), autoimmune dermatitis (n = 1) and eczema (n = 1), chronic lung disease (n = 2) and granulomas (pulmonary n = 2; ocular n = 1; bladder n = 1; oropharynx n = 1). three patients presented more than one site of inflammatory disease. all patients were treated with systemic or topical immunosuppressive or immunomodulatory therapy, most of them corticosteroids. five patients underwent hematopoietic stem cell transplantation (hsct), median age at hsct was 13 years (4 -17), and two died 1 month after hsct. conclusions: although infections are more frequent and have a major impact on patient morbidity and mortality, im are increasingly prevalent in patients with cgd. awareness regarding this possible comorbidity is of major importance, since earlier diagnosis and adequate treatment may be crucial for patients survival and quality of life. there is a gap in clinical knowledge regarding associations between specific pid and different rheumatological diseases. in this study, we are reporting the incidence of various rheumatological conditions reported in a large pid population using the usidnet (united states immunodeficiency network) registry. methods: we used the retrospective usidnet registry to conduct the analysis. we included all primary immunodeficiency patients with physician diagnosed rheumatological diseases. results: the total number of pid patients in our query was 5058. 278 (5.49%) patients had a diagnosis of rheumatological disease. this cohort included 172 (61.8%) female and 106 (38.2%) male patients. rheumatologic complications were highest in the interferonopathies (66.6%), complement deficiencies (14.2%) and autoimmune lymphoproliferative syndrome (alps) (13.7%). additionally, disease patterns were noted to be different in each pid. dermatomyositis was found to be the most common rheumatologic condition in patients with x-linked agammaglobulinemia (xla) with a rate of 1.65%, which was remarkably higher than the reported prevalence in the united states (0.005%). alps patients had a higher (6.85%) numbers of sjogren syndrome diagnoses as compared to the general population (0.01-0.09%). systemic lupus erythematosus was increased in patients with mucocutaneous candidiasis (7.41%) as compared to the general population (0.001%) and other pids. rheumatoid arthritis (ra) was reported in patients with specific antibody deficiency (3.66%), common variable immunodeficiency (cvid) (2.93%) and alps (2.74%). wiskott-aldrich syndrome patients had the highest numbers of cases diagnosed with vasculitis (6.50%). 0.29% of patients with severe combined immunodeficiency (scid) had reported rheumatologic disease. juvenile rheumatoid arthritis (jia) and systemic sclerosis were reported in 0.19% of patients with digeorge syndrome. conclusions: this study reports that higher numbers of rheumatologic diseases are diagnosed in pids compared to the general population. the incidence of different rheumatological disease was variable based on the pid diagnosis. early diagnosis of these diseases is crucial, given the high risk of irreversible complications. limitations of our study include possible selection bias as majority of cases were enrolled from tertiary care centers. abstract/case report text background disorders of immune dysregulation are associated with autoimmune features. this feature could potentially have an impact on the outcome post hematopoietic stem cell transplantation (hsct). hsct, although curative, can be challenging with the underlying immune dysregulation resulting in significant morbidity and mortality. we present the journey through hsct for these children and the factors affecting the outcome. we analysed the data on children up to the age of 18 years diagnosed to have a disorder of immune dysregulation through gene mutation analysis and who underwent hsct at our centre from 2015 to 2019. results 1. xiap mutation a 10-year-old boy underwent a haploidentical hsct from his father using fludarabine, treosulfan, and 2 gray radiotherapy with post-transplant cyclophosphamide. after initial complete chimerism and cytomegalovirus reactivation responsive to valganciclovir, he developed progressive diarrhoea almost 15 months post-hsct. a rectal biopsy confirmed cmv reactivation and features of inflammation. he has since been treated for the same and is on follow up for inflammatory bowel disease. his chimerism had dropped to 67% and has remained stable. the second child is a 1-year-old girl who underwent tcr alpha/beta depleted haplo sct and is 12 months post-hsct, with no features of gvhd or infections, and is doing well with complete chimerism. 2. il10r deficiency three boys aged eight months, one year, and two years of age, diagnosed to have il10r deficiency underwent hsct. all three children needed nasogastric tube feeding, parenteral nutrition, and vigilant monitoring for electrolyte disturbances. in the first two children, we had performed tcr alpha/beta depleted pbsc transplants from their haplo matched fathers. the 1-year-old engrafted by d+17 and is doing well two years post hsct with complete chimerism, no gvhd, and infections. his autoimmunity, including recurrent skin scarring, has resolved entirely. the 8-month-old, however, had primary graft failure and succumbed to his illness. the 2-year-old boy underwent matched unrelated donor hsct and engrafted by d+14 with completed chimerism documented on three occasions. he, however, had secondary graft failure around d+60, and he succumbed to the illness. 3. lrba deficiency an 18-month-old girl with lrba deficiency had presented at four months of age with excessive sweating, hepatosplenomegaly, and recurrent chest infections. she was started on monthly intravenous immunoglobulin replacement and abatacept. she received myeloablative conditioning with thiotepa, treosulfan and fludarabine and underwent a matched sibling donor hsct. she engrafted by d+17 and has been well ten months post hsct with complete chimerism, no gvhd, and infections. conclusion disorders of immune dysregulation are a heterogeneous group with a varied spectrum of immune dysfunction. myeloablative conditioning is essential, and there is a high risk of cytokine release syndrome and the need for supportive care. the autoimmune features need to be followed for progression in organs other than the hematopoietic system and may require interventions. as long-term data evolves, more precise definitions for patient and donor selection will enable improving outcomes. we performed a retrospective observational analysis of case records of children up to 18 years of age, diagnosed to have variants of scid, and underwent hsct at our centre from 2002 to 2019. results 1. zap 70 deficiency a 6-month-old girl presented with oral thrush and submandibular cellulitis from one week of life with failure to thrive. she underwent a tcr alpha/beta depleted haploidentical hsct. conditioning included treosulfan/thiotepa/fludarabine/anti-thymocyte globulin. she engrafted by d+15; now three years post-hsct with complete donor chimerism without gvhd or infections. 2. orai-1 mutation a 15-month-old girl presented with failure to thrive, generalized hypotonia, oral thrush, and recurrent respiratory infections. she underwent haplo-sct with post-transplant cyclophosphamide with pbsc from her haplo-matched father. conditioning included fludarabine/treosulfan. she had cytokine release syndrome grade 4, which responded to tocilizumab. she had hypertension throughout the peri-engraftment period and had an episode of pres with seizures. her symptoms abated with neutrophil engraftment by d+ 17. the post-transplant period was complicated by grade 2 skin gvhd and cytomegalovirus reactivation. she has remained disease-free with complete chimerism three years post-hsct. her hypotonia is steadily improving with physiotherapy. 3. cernunnos-xlf deficiency a 27-year-old male presented with recurrent infections from 14 years of age, aplastic anemia diagnosed at 20 years of age, subsequent transformation to acute myeloid leukemia at 27 years of age. he had developed multiple fusarium abscesses during the neutropenic period post-chemotherapy for aml. he was referred for a matched sibling sister hsct when in remission. conditioning included fludarabine/treosulfan. he engrafted by d+15 with complete chimerism. he developed progressively worsening skin, gut, and liver toxicity secondary to chemotherapy and succumbed to the illness two months post-hsct. 4. ikzf mutation an 18-month-old girl presented with failure to thrive, massive splenomegaly, persistent pneumonia, anemia, and thrombocytopenia. she underwent a matched sibling donor pbsc transplant after myeloablative conditioning with thiotepa/treosulfan/fludarabine. she engrafted by d+20, following which all her symptoms abated. she had secondary graft failure two months post-hsct and succumbed to her illness. 5. mhc class ii deficiency (bare lymphocyte syndrome) three children, aged 18 months, two years, and four years underwent matched sibling donor hsct. myeloablative conditioning with thiotepa/treosulfan/fludarabine resulted in engraftment. the first child died of invasive intestinal aspergillosis 30 days post-hsct. the other two children are well 11 months post-hsct with complete chimerism without gvhd or infections. the two-year-old girl received one cycle of pre-transplant immunosuppression with fludarabine/dexamethasone to prevent graft rejection pre-hsct as she was referred for a second transplant. conclusion children with scid have traditionally been transplanted using reduced intensity (ric) conditioning with immunomodulation. scid variants require myeloablative conditioning with a vigilant follow up for the detection of graft rejection. radiation sensitive scid associated with dna breakage repair defects require ric and close monitoring for gvhd. advances in hsct, including supportive care and haplo-sct, have provided a ray of hope for these hitherto rare conditions. j clin immunol abstract/case report text immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) (omim #304790) is a monogenic autoimmune disorder that occurs due to loss of function variation in foxp3 causing dysfunctional t regulatory cells. although immunosuppression is a mainstay of treatment for autoimmunity, ipex treatment is frequently limited by insufficient response to therapy or side effects of immune suppression. we present a 28 year old male with ipex whose prior immunosuppressive treatment was complicated by inefficacy and medication side effects, requiring a new approach to treat his colitis and erosive dermatitis. he initially presented with infantile diabetes and subsequently developed dermatitis, squamous cell carcinomas, alopecia totalis, and colitis. his clinical diagnosis of ipex was confirmed by foxp3 sequencing, demonstrating known pathogenic variant c.1150g>a (p.ala384thr). this variant has been described in ipex affected individuals in multiple publications (ref 1). his variant affects at the frkhead domain of foxp3 and has been associated with others with severe psoriasiform dermatisis and alopecia universalis (ref 2). his prior immunosuppressive therapies included at different times combinations of corticosteroids, tacrolimus, sirolimus, azathioprine, infliximab, adalimumab, rituximab, dupilumab, and oral mesalamine. the relative efficacy of these agents based on experiences in a cohort of ipex patients was reviewed in 2018 (ref 3), with the exception of duplimab, which was not listed in that review. for our patient, management of his widespread autoimmunity has been limited by toxicity or lack of efficacy of medications. notably, his dermatitis had no improvement with duplimab, consistent his low total ige and lack of allergic manifestations. at age 27, after initiation of treatment with sirolimus, he had spontaneous colonic perforation requiring descending colectomy. after stabilization of his colonic perforation, his multi-disciplinary team of allergy-immunology, gastroenterology, and dermatology initiated tofacitinib. tofacitinib is small molecule inhibitor of janus kinase (jak) signaling pathways that mediate cytokine driven autoimmune activation. it is fda approved to treat rheumatoid arthritis, psoriatic arthritis and ulcerative colitis. the decision to use this jak inhibitor was due to its fda approved use for ulcerative colitis, to target our patient's colitis and his other autoimmune manifestations, specifically his dermatitis. its off label for primary immune dyregulatory disorders including candle, stat1-gain of function and stat3-gain of function disorders has been published (ref 4), but thus far its use to treat autoimmunity due to ipex has not been published. he experienced leukopenia while on 15 mg of tofacitinib, which resolved after lowering his dose. currently, he has had improvement in his colitis and dermatitis, and partial improvement in alopecia. he has been on tofacitinib 10mg daily for 11 months, with only prednisone 10mg daily as additional immune suppression. as the number and types of selective immune modulators increases, there is continued need to share the experiences of treating physicians of which therapies have been successfully able to decrease disease manifestations with tolerable side effect profiles. we present a 28 year old male with ipex syndrome with severe dermatitis and colitis complicated by colonic perforation despite standard immunosuppressive therapy, who is safely and effectively being treated with tofacitinib. is not frequently associated with autoimmunity, likely due to impaired il-6 and il-17 pathways. however, in our relatively large cohort of lof stat3 patients, we have noted an increased incidence of systemic lupus erythematosus (sle) diagnoses and sle-like symptoms. herein, we characterized the clinical and laboratory features of the patients in our cohort with sle and sle-like disease, with the aim to better understand the pathogenesis by evaluating ifn stimulated genes and neutrophil net formation. methods a retrospective chart review was performed of patients with lof stat3 to identify those with sle and sle-like presentations, and included clinical features, laboratories including inflammatory markers, auto-antibodies, and complement levels. rt-pcr was performed for interferon stimulated genes (isgs) from neutrophils and pbmcs of lof stat3 patients with and without sle, and healthy controls. neutrophil net formation was assessed for lof stat3 patients with and without sle, and healthy controls. results out of a cohort of 158 patients, five patients (ages 12-39) were identified who carried the diagnosis of sle, and 4 with slelike disease (ages 15-34). for those with sle, age of presentation was 8-21 years, 4 of 5 were female. clinical features included nephritis (4), alopecia (2), autoimmune cytopenias (2), arthritis (3), discoid rash (2), and raynaud (1). all had positive auto-antibodies, and 4 of 5 had low c3 and/or c4. for those with sle-like disease, age of presentation was 12-24, and 2 of 4 were female. clinical features included alopecia (1), autoimmune cytopenias (1), raynaud(1), and nephritis (2). all had positive autoantibodies, and 1 of 4 had low complements. lof stat3 patients with and without clinical features of sle had increased expression of isgs from both pbmcs and neutrophils. increased spontaneous net formation was observed for lof stat3 patients both with and without sle symptoms. discussion although autoimmunity is not a common finding in lof stat3, we have identified sle or sle-like disease in about 6% of our cohort, with a high incidence of kidney disease, including one patient who required kidney transplant. the interferon signature and net formation were unexpectedly high in both the patients with and without the sle features. ongoing studies include whole exome sequencing for possible second mutations or modifiers, the role of ige in the kidney disease, and further autoantibody detection. the increased ifn signature raises the question about jak-stat modulation for therapy. 8 chief, genetic immunotherapy section/niaid, nih 9 abstract/case report text chronic granulomatous disease (cgd), a rare immunodeficiency with decreased reactive oxygen species (ros) production, increased susceptibility to infection, and increased mortality is caused by mutations in any one of 5 distinct phagocyte oxidase (phox) components of the nadph oxidase, nox2. in the past, identification of the specific protein defect was primarily determined by immunoblotting using specific antibodies to the phox proteins. recently, however, we have shown using fluorescenceactivated cell sorting (facs) analysis of neutrophils in whole blood permeabilized and stained with specific anti-p47phox antibody that p47phox protein expression was absent in p47phox cgd patients and significantly reduced in p47phox cgd carriers [kuhns et al. 2019. blood adv. 3(2):136-147]. these findings demonstrated that determination of phox protein expression by facs analysis provide an alternative to immunoblotting and can aid in the identification of p47phox cgd patients and carriers. we now have extended these studies to patients and carriers with p67phox cgd. facs analysis of p67phox expression in permeabilized neutrophils demonstrated that p67phox expression was absent in four patients with different mutations in ncf2 [two patients homozygous for c. e12(+1) g>a, one patient homozygous for c.287_289 del aag, p.glu96 del; and one patient compound heterozygous for the mutations, e3(+1) g>a and c.55_63 del aagaaggac]. moreover, the expression of p67phox in nine p67phox cgd carriers was significantly reduced >50% compared to expression in neutrophils from healthy volunteers. another cytosolic phox protein, p40phox, has been shown to associate with p67phox in a 1:1 molar ratio [tsunawaki et al. 1994 . biochem biophys res comm. 199(3): 1378-1387]. the expression of p40phox was reduced in both carriers and patients with mutations in ncf2. despite reduced expression of p67phox and p40phox, neutrophils isolated from carriers of p67phox cgd exhibited normal dihydrorhodamine (dhr) oxidation after stimulation with phorbol ester and fell within the normal range for ros production (measured by luminol-enhanced chemiluminescence) after stimulation with either fmlf, opsonized zymosan, or phorbol ester with one notable exception. included in this cohort of p67phox carriers was a p47phox cgd patient (homozygous for a gt deletion at the start of exon 2 in ncf1) who also carried a heterozygous damaging mutation in ncf2 [c.1256 a>t; p. asn419ile]. normal ros production in the presence of reduced p67phox and p40phox expression suggest that these proteins are not rate-limiting components for maximum nox2 activity in neutrophils. finally, determination of the expression of specific phox components by facs analysis of permeabilized neutrophils from whole blood provides a rapid and alternative approach to immunoblotting to determine the specific protein defect in cgd, and, importantly, one that could be easily established in most clinical labs. funded by nci contract no. 75n910d00024. the original clinical observation that defined patients with hyper-ige syndrome (hies) was the presentation of cold abscesses ("job's syndrome"), which indicated a deficient inflammatory response. mutations in the stat3 gene have now been identified in most classic autosomal dominant hies patients, but we do not fully understand how these mutations cause the clinical presentation. since the discovery of stat3 mutations, research on hies focused largely on the adaptive arm of the immune system and suggested that the innate immune defects could be secondary. for example, the discovery that there is a th17 cell and il-17 cytokine deficiency in hies provided a possible explanation to the neutrophil chemotaxis defects in hies, as il-17 is one of the chemokines critical for neutrophil recruitment in vivo. the goal of this study was to investigate myeloid cells from hies patients. first we used c5a, fmlp, il-8, cxcl1, and cxcl2 to study neutrophil chemotaxis in vitro. responses to c5a, fmlp, and il-8 were equally robust in hies compared to healthy controls, demonstrating that neutrophils from patients are capable of efficient directed migration in vitro. neutrophils from all hies patients responded to cxcl1 and cxcl2 significantly below that of the healthy controls. cxcl1 and cxcl2 are cxcr2-specific chemokines. these results indicated a neutrophil intrinsic cxcr2-specific defect. we also found that patient-derived cells express comparable levels of cxcr2 on the cell surface, suggesting a cxcr2 chemokine receptor signaling defect. after identifying a neutrophil defect in hies, we wanted to get a broader view of myeloid cells in hies in addition to identifying the cxcr2specific defect. stat3 is a transcriptional regulator, therefore we performed transcriptional profiling of hies and healthy control-derived neutrophils and monocytes. as it was shown before, the expression of stat3 was not different between patients and controls, since hies is usually caused by the decrease in stat3 activity not by decrease in expression. we found, however, an increase in stat1 and stat2 expression as well as significant changes in the expression of genes regulated by interferons. increased expressions of stat1/2 in both neutrophils and monocytes likely provide and explanation for the increase in interferon regulated genes. multiple genes were identified as potential regulators of cxcr2 signaling. the balance between the stat3 and stat1 signaling has long known to be a regulator of immune cell activation, especially in t cells, but less studied in myeloid cells. stat3 and stat1/2 signaling pathways crossregulate each other in healthy cells. we propose that in hies the decreased stat3 signaling leads to not only changes in expression of effector (e.g. inflammatory) genes, but also decreases expression of genes in the regulatory (negative) feed-back loop, which are required for decreasing stat1/2 activity. therefore, the immune cell defects caused by decreased stat3 activity are compounded by the increase in stat1/2 activity. increase in stat1/2 signaling can cause pathologies in the absence of stat3 defects, as well as further decrease stat3 signaling, thus contributing to hies. interfering with stat1/2 signaling in hies may represent a therapeutic opportunity. abstract/case report text rationale: t-cell receptor excision circles (trecs) testing on newborn screening (nbs) has been vital for identifying patients with severe combined immunodeficiency (scid). we aimed to determine whether one or more abnormal trecs result on a nbs might predict higher mortality rates despite the absence of an identifiable underlying etiology. methods: newborns with a positive trecs nbs result without the diagnosis of scid or 22q11.2 deletion syndrome born from october 2011 to december 2014 were included (n=467). newborns were divided into three groups: group 1 infants had a subsequent normal repeat screen (n=375); group 2 infants did not undergo repeat screening as the majority expired before a repeat screen could be conducted (n=36); group 3 infants had a normal initial screen but subsequent abnormal screen (n=56). cases were matched 3:1 to controls on gestational age, birth weight, nicu status, race, birth quarter, and birth year. nbs records were linked to birth and death certificate records. demographic characteristics were compared and mortality rates were calculated between the groups. results: the mortality rate of group 1 was 2.4%, group 2 was 91.7% and group 3 was 46.4%. when compared with matched controls, there was no difference in the mortality rate of group 1 when compared to the control group. there was a significant difference in the mortality rate between cases and controls in both group 2 (p < 0.001, 95% ci 0.711, 0.950) and group 3 (p < 0.001, 95% ci 0.256, 0.551). the apgar scores in group 1 infants were comparable to their matched controls. infants in group 2 (p = 0.01) and group 3 (p = 0.003) had significantly lower apgar scores than the controls. the majority of the infants in all three groups were less than 37 weeks gestation, however, group 2 had a higher percentage of infants born very premature (less than 32 weeks). there was no significant difference in maternal age, maternal education, prenatal care status, cigarette use, or maternal steroid use between the cases and controls in all three groups. conclusions: infants with an initial abnormal screen who had a subsequent normal repeat screen did not have an increased rate of mortality compared to their matched controls (group 1). however, group 2 infants (with unresolved repeat screen) and group 3 infants (with a first abnormal value on a repeat screen) did have increased mortality rates when compared to their controls. overall, an abnormal trecs level on nbs without a confirmed negative repeat screen, was associated with higher mortality in our study population. further studies will be needed to determine if the trecs assay can serve as a predictor for mortality in newborns with an abnormal screen. abstract/case report text introduction: primary immunodeficiency refers to a heterogeneous group of diseases characterized by altered function or composition of the immune system, and are grouped into adaptive or innate system defect. immunoglobulin g subclass immunodeficiencies (iggscs) are classified as a b-cell-related adaptive system disorder and are therefore associated with recurrent sinopulmonary infections with encapsulated bacteria, presenting with pneumonia, recurrent bronchitis, rhinosinusitis, and herpes zoster. its primary mechanisms are still unclear, although the cause for this deficiency might be related to gene deletions, transcription errors, or be an effect of allotype. igg4 immunodeficiency reaffirms its association with the patient's clinical condition and is often associated with igg2 deficiency. objective: to evaluate the prevalence of igg4 immunodeficiency in ferraroni's clinic, classify it by gender, age, igg4 dosage and other subclasses, correlate it with igg2 immunodeficiency and the clinical presentations presented by the patients under analysis. method records of 24 patients with igg4 immunodeficiency whose clinical pictures were followed throughout 15 years were evaluated, patients aged from 4 to 85 years. all tests were done at the same laboratory and all patients have consented to be part of this study, which has been approved by the ethics committee. results twenty-four patients with igg4 deficiency, 79,16% (n=19) were women and 20,83% (n=5) were male, with average of 47 and 28 years, respectively. the average of igg4 was 7.76 mg/dl, and that of igg2 was 299 mg/dl. of the patients evaluated, 62.5% had upper airway infections (sinusitis, rhinitis, otitis and tonsillitis), 25% herpes simplex, 33.3% asthma. less prevalent cases were reported as 4.1% of patients had bronchiectasis, 12.5% candidiasis and 4.1% herpes zoster. 41.67% presented the association of igg4 and igg2 deficiency. discussion: the role of specific igg4 deficiency in the infectious setting is still unknown, but it usually occurs in association with other isotypic deficiencies and sinopulmonary infections. furthermore, the igg4 subclass is relevant on the study of environmental antigens -suggesting its involvement with allergic disordersand has been described in association with other diseases, such as chronic mucocutaneous candidiasis, ataxia-telangiectasia and allergic colitis. igg2 deficiency is related to increased susceptibility to bacterial infections. studies show a correlation between igg2 and igg4 immunodeficiency that generally imply clinical features characterized by recurrent infections by encapsulated bacteria. the data obtained through the analysis of patients' charts corroborated this information, since it was evident that most of the patients had really similar clinical conditions. conclusion: igg4 deficiency has a direct correlation with higher prevalence of upper airway infections, such as rhinitis, sinusitis and pneumonia, and with an increased incidence of allergic disorders, here presented by our cohort. additionally, research suggests that hies may cause impaired cd8+ t cell function. we hypothesized that a low percentage of both th17 and th1 cells would be predictive of hies and would differentiate hies from atopic disorders. to evaluate this hypothesis, we examined the percentage of th17, th1, and ifng+cd8+ t cells, laboratory parameters, and genetic diagnoses from a large cohort of patients to determine which parameters distinguish patients with stat3 loss-of-function variants. methods: we conducted a retrospective, multi-institutional chart review of over 200 patients who received a th17 assay at the medical college of wisconsin clinical immunology research laboratory. the th17 assay is performed by activating pbmcs with pma/ionomycin/brefeldin a and staining for cd4, cd8, ifng and il-17a. the following parameters were included in the chart review: the percentage of th17, th1, and cd8+ifng+ cells, immunoglobulin levels, atopy scores, infectious history, and genetic diagnoses. results: using logistic regression, we demonstrated that the percentage of th17, cd8+ifng+, and th1 cells were positively correlated with age, and percentage of cd8+ifng+ cells was higher in females than males. we found that the percentage of th17 and th1 cells were decreased in both atopic disease and hies, with hies having the lowest values. interestingly, one subject with a stat3 gain-of-function (gof) variant had an elevated percentage of th17 and th1. in addition, we determined that ige levels were inversely correlated with the percentage of th17, cd8+ifng+, and th1 cells, while iga and igm were positively correlated with the percentage of th17 cells. several different monogenic defects characterized by increased fungal infections exhibited a low percentage of th17 including tatton-brown-rahman syndrome and cornelia de lang syndrome. conclusions: we confirmed that the percentage of th17 cells is low in both hies and atopy in a large cohort of subjects, and that the percentage of th1 cells may be helpful in distinguishing hies from atopic disease. however, since the percentage of th17, cd8+ifng+, and th1 cells correlate with age, caution should be used when testing young children. the inverse correlation between ige levels with th1 and th17 responses suggests that similar pathway(s) may drive both hies and atopy. additionally, the decreased th1 responses in stat3 lof and increased th1 responses in stat3 gof hies raise questions about the role of stat3 in regulating ifng levels. we also identified patients with different genetic disorders with fungal infections in which the th17 percentages were low, suggesting that the th17 test may be useful in evaluating individuals with unusual fungal infections. abstract/case report text background:primary and secondary autoimmuune neutropenia (pan/san) are well described entities. several autoimmune neutropenias do not fit the criteria of either pan or san showing peculiar characteristics mainly for older age at onset and/or for duration of the disease; moreover they are not associated , at least at the beginning, with autoimmune markers/diseases aim of the study: to describe a cohort of subjects affected with autoimmune neutropenia, defined as "atypical" (aan), registered in the italian neutropenia registry (inr) and to compare these data with those from subjects diagnosed with pan still in the inr. patient and methods: subjects with neutropenia and positivity of indirect antibodies against neutrophils (registered in the inr from 2013 to 2019) lasting for more than 3 years, or diagnosed after 5 years of age ( up to 18 y), without any associated autoimmune, signs/markers were considered eligible for the present study. results: data from 248 patients were collected: 79/128 subjects (32%) were defined as aan and 169/248 (68%) as pan. among 79 aan affected patients 61%, were "long lasting" aan, while 39% were defined as "late onset" aan .the degree of neutropenia in aan group was mild in 20 %, moderate 44 % and severe in 36 % of the subjects . leukopenia at onset was a common hall mark seen in 46% of aan patients (median values 3700/mm³ ; range 2750-5140/mm³ ) especially in the "late onset" aan if compared with pan and " long lasting" one ( p=0.003). as for clinical features, almost half of the aan cohort suffered from recurrent or "significative infections", while severe episodes (namely sepsis, meningitis , osteomyelitis , pneumonia, deep abscess or flemmon) were shown in 28% being more frequent, but non significantly higher than those reported in the pan group (6% )( p=ns) interestingly, recurrent apthae were significantly more seen in the "late onset" aan group if compared with the "long lasting" aan (p=0.006). during follow up, markers and/or symptoms of autoimmunity appeared in 31% of the aan cohort, being another element of peculiarity in respect to pan (p < 0.0001). as for immunological pattern in aan, immunoglobulin values were lower than the references for age in 12%,while were above them in 17% of the cohort . lymphocytes subsets evaluation showed decreased value of cd3+cd19+ cells in 40% of cases, followed by depletion of cd3-cd16+cd56+ subtype in 33%, cd3+cd4+ in 24 % of cases and cd3+ cd8+ in 17% . preliminary study on b memory and t-reg cells values, showed a quantitative deficiency respectively of in 59% and 27% of the studied subjects. mutation analysis performed by ngs in 20% of the subjects identified pathogenic variants of : taci (2), tinf 2 (1) and lrba (1) .comparison between pan and aan is detailed in table 1 . conclusions atypical neutropenia in childhood is a disorder which show many difference with pan; indeed appears an epiphenomenon of a complex immunological disturbances rather than a disease itself. occasionally mutations of genes of immunodeficiency/disimmunity can be demonstrated abstract/case report text background: medications treating ra typically include systemic corticosteroids used to treat inflammation flares, and disease modifying therapies (dmards). traditional dmards include methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine. recently, biologic/immuneresponse modifiers have come to the forefront for overall therapeutic benefit, however, an unfortunate side-effect may be the risk of increased immunosuppression. this study seeks to determine the occurrence rates of immune deficiencies among patients initiating ra therapies. methods: using the pharmetrics plus commercial claims database from 2012-16, ra patients (icd-9 714 and -10 codes: m05, m06) over the age of 18 were indexed on their first use of a new biologic therapy. all patients were required to have enrollment six-month pre and one-year post index. cohorts of patients were grouped by medication: methotrexate, adalimumab, etanercept, and rituximab. ra patients receiving adalimumab, etanercept, and rituximab were allowed concomitant use of methotrexate, but could not use any other biologic medications in the post period. a minimal adherence of 40% was required of all biologic treated ra patients. an additional cohort of ra patients untreated with biologic therapies was indexed on their first ra diagnosis within the time window and used as a control. ra patients with comorbid conditions who would also require biologic treatment were excluded including crohn's disease and ulcerative colitis. between group comparisons were made with the no treatment group as the referent. to account for differences in age, gender, and elixhauser comorbidity conditions patients in each cohort were matched 1:1 to the rituximab group. results: 52,013 ra patients met inclusion criteria: 6,608 in the methotrexate group, 2,826 receiving etanercept, 2,808 receiving adalimumab, and 467 receiving rituximab. a total of 12,709 in the treated groups and 39,304 in the no biologic treatment group. demographic information including age and gender were significantly different but numerically similar between the groups, with rituximab group having the highest proportion of female patients but limited dispersion with the lowest proportion being in the etanercept group. healthcare utilization metrics highlighted a significantly higher average number of office visits (21.18, sd: 13.48 vs no treatment 15.61, sd: 13.29, p < 0.01) and a higher proportion of rituximab patients being hospitalized (14.35% vs no treatment 10.37%, p < 0.01). the diagnosis of immune deficiency was highest among the rituximab group with 7.92% followed by methotrexate 2.91%, adalimumab 2.88%, etanercept 2.80%, and no treatment 2.80%. after matching, similar rates were seen for healthcare utilization to the pre-match results. the post-match odds of being diagnosed with immune deficiency were significantly greater for the rituximab group (or 3.76, ci: 1.61-8.85) than the no treatment group. conclusions: the purpose of dmards is to modulate the immune system and decrease autoimmunity in ra. however, this treatment may lead to significant immunosuppression. this study suggests that treatment with certain biologic/immune-response modifier therapies may be associated with higher rates of healthcare utilization. in particular, the increased post-treatment diagnostic coding of immune deficiency demonstrates the heightened awareness among healthcare providers of the chronic immunosuppressive potential of rituximab. evaluation of potential secondary immunodeficiency pre-and post-dmard use should be incorporated into routine practice. abstract/case report text introduction: autoimmune lymphoproliferative syndrome (alps) is a rare inherited disorder of lymphocyte homeostasis due to a fasmediated apoptosis and characterized by non-infectious and nonmalignant lymphoproliferation, autoimmunity, and secondary malignancies (national institute of health criteria). in spite of recent progress, one third of alps patients still remain gene orphan and they have been previously categorized as alps-u. in some cases, patients fitting alps diagnostic criteria have been shown to carry mutation on genes involved in other immune-dysregulation syndromes. aims: the aim of this study is to compare the clinical and immunological features, and the outcome of a cohort of alps patients with mutations on the typical causing genes (fas, fasl, fadd and casp 10)-here defined as alps-g -vs the ones without a molecular diagnosis or carrying mutations on other genes (both defined as alps-u). patients and methods the demographic, clinical, biochemical, genetic informations and details about treatment are derived from the alps italian network. search of mutations was performed with sanger pcr and/or next generation sequencing techniques (extended to immunodeficiency genes panel). results: 68 alps patients were registered in our data base; the genetic analysis was performed in 42 subjects (62%): 14/42 pts (33%) were alps-g and the remaining 28 (66%) alps-u. six-teen out of 28 (57%) alps-u patients resulted to carry mutations on other genes (lrba, stat3+cecr, ctla4, baffr, taci, nmlrc4, ikbkg, gaucher), and the remaining 12 (43%) were negative. the alps-u subjects showed a more complex phenotype compared to the alps-g group, which was characterized by multi-organ involvement (p=0.003) and positivity of autoimmune markers (p=0.002). (table 1 ). cytopenia affecting one or more haematopoietic lineages was present in both groups (69% and 82%) with no significant difference, apart from lymphocytopenia that was more frequent in alps-u group (p=0.03) ( table 1) . as for lymphocyte subets and immunoglobulin dosage no differences were shown within the two groups. vitamin b12 and il-10 were more frequently raised in alps-g group (p=0.01, p=0.001) (table 1). four out of 42 (9%) patients did not require any treatment. first-line treatment (steroid or intravenous immunoglobulins) controlled the disease only in 4/38 (10%) cases. the response rate to second line therapy -micofenolate mofetile (mmf) or rapamycin-was 100% and 40% in alps-g and alsp-u group, respectively. moreover, target therapies or drug combinations were more commonly applied in alps-u subjects (p=0.02) ( table 1) . conclusions: our study showed that alps-u subjects, despite the alps phenotype, represent distinct clinical entities and that genes associated with other immune-dysregulation syndromes are frequently represented in this group (16/ 28, 57%). the identification of such disorders is crucial for the management of second-line treatment and/or the administration of target therapies abstract/case report text background: we have shown previously that allergic reactivity to ovalbumin (ova) could be regulated in mice following perturbation of immune networks using combinations of an immune ig along with antiidiotypic ig. we have explored features of this regulation including: its persistence after cessation of administration of combined igs; the ability of heterologous igs to produce immunoregulation; a role for treg induction in regulation; and the ability to attenuate responses in mice presensitized to an allergic stimulus. methods: balb/c mice were sensitized to ova. mice also received 5 weekly injections of immune ig or anti-idiotype ig (at separate sites) from either homologous (mouse) or heterologous (human) sources. in the latter case pooled ivig (given im, hence hereafter imig) was used as a source of anti-idiotype ig, and human anti-tet as immune ig. injections of the ig were given from the time of ova sensitization (to attenuate development of immunity), or after pre-sensitization of mice (to attenuate existing allergic responses). all mice were assayed for development of ova-specific serum ige and igg, as well as the production of ova-induced il-2, il-4, il-13, il-31 and il-33 in splenocytes cultured for 72hrs. in studies examining possible mechanism(s) responsible for inhibition of immunity mice received, in addition to the ig treatments described, infusion of depleting anti-cd4, and/or anti-cd8 antibodies, or a mab to tnfsfr25, known to expand tregs implicated in regulation of allo immunity. results: combinations of both heterologous and homologous immune igs and anti-idiotype igs attenuated ova allergic responses in both naïve and pre-sensitized mice. this attenuation persisted in mice greater than 14 weeks after cessation of treatment with the igs used. finally, depletion of either cd4 or cd8 cells ameliorated the suppressive effect seen, while the combination of anti-cd4 and anti-cd8 essentially abolished suppression. suppression was further enhanced by anti-tnfsfr25 mab. conclusions: we conclude that the combine ig treatment protocols used produced a long-lasting suppression of allergic immunity, even in pre-sensitized animals. the effects seem to depend upon induction and expansion of tregs and represents a novel approach to treatment of allergic disease in humans and other animals. abstract/case report text background wiskott-aldrich syndrome protein (wasp) is found in the cytoplasm of hematopoietic cells but can transit to t lymphocyte nuclei at distinct developmental timepoints. wasp deficiency is a rare, x-linked combined immunodeficiency disease. affected patients display qualitative but not quantitative t cell defects. we report two immune deficient subjects with nearly identical exon 11 frameshift mutations in was, the gene encoding wasp. one subject lacked circulating t cells, the other possessed several distinct cd8 t cell populations each expressing quantitatively different amounts of wasp. objective to determine how similar was mutations can cause scid in one person and generate b and t cells with heterogenous wasp expression in another. methods to identify somatic was mutations, we deeply sequenced was exons, introns, promoters and 5' untranslated regions at 10,000 read depth in genomic dna from various b and t cell populations of each subject and their unaffected relatives. we confirmed genomic variants were transcribed and translated by sequencing was transcripts and analyzing wasp in primary cell lysates, both fractionated and not. to model our subjects' diseases we transfected primary cells and cell lines with mutant was transcripts and then measured viability and nuclear localization via confocal microscopy. results deep sequencing of genomic dna revealed all of subject one's cells carried the same germline exon 11 frameshift was mutation. the mutation was incorporated into subject one's was transcripts and translated into a truncated form of wasp, which was relegated primarily to the cell nucleus. subject two possessed three distinct cd8 t cell subsets that each carried either the germline exon 11 frameshift was mutation or a variety of somatic mutations that circumvented frameshift wasp expression. evasion strategies included exon 11 skipping, adoption of a cryptic exon 11 splice site and reversion to wild type amino acid sequence. subject two incorporated somatic mutations into was transcripts which encoded either stable near full-length proteins or unstable non full-length ones. subject one's sister and subject two's mother, who both carried the germline exon 11 frameshift mutation, produced only wild type transcripts and proteins. conclusion we report two patients with was mutations encoding truncated wasp. if expressed, truncated wasp localized to the cell nucleus, and this was associated with t cell developmental arrest and severe combined immune deficiency. if, through a variety of epigenetic and somatic strategies, t cells could avoid expression of truncated wasp, they would survive but display phenotypical abnormalities and functional defects. patients with pidds show a higher susceptibility to hematopoietic malignancies, in particular to non-hodgkin lymphomas (nhl) that, generally, account for approximately 6-7% of paediatric cancers and their incidence increases with age. recently new gene defects responsible for pidds with lymphoproliferation as a key clinical sign have been identified. our goal is to investigate possible immune-mediated mechanisms underlying malignant lymphoproliferation in children who did not show other typical symptoms of pidds. we retrospectively selected and reviewed the clinical history of nine patients with nhl (6 burkitt lymphoma, 2 large b cell lymphoma and 1 lymphoblastic tcell lymphoma). immunophenotyping and exome analysis of known pidds genes were performed after lymphoma remission. six out of nine patients showed a mild hypogammaglobulinemia at time of presentation, not noticed before. moreover, one patient had history of recurrent respiratory infections, one of hematologic autoimmunity and two of nine were ebv-positive at diagnosis. preliminary results show an aberrant b cell phenotype in four patients; exome analysis reveals a novel heterozygous genetic variation in ikzf1 gene in one patient with burkitt lymphoma and autoimmune cytopenia was identified. concerning the remaining patients, further studies are ongoing. a detailed review of clinical history of paediatric patients affected from nhl as well as an impaired immunophenotyping can be important indicators of immune-mediated disorder underlying lymphoproliferation and helpful signs of possible pidds that should promptly be investigated by genetic analysis. this will allow an appropriate diagnosis and disease management. abstract/case report text introduction: caspase activation and recruitment domain 11 (card11) encodes a scaffold protein that links antigen receptor activation to intracellular signaling. dominant heterozygous loss of function (lof) mutations in card11 cause a syndrome of severe atopic dermatitis, elevated ige, and allergic disease. atopic dermatitis can be difficult to control leading to substantial morbidity. dupilumab is a humanized monoclonal antibody that blocks il-4 and il-13 signaling approved for treatment of refractory atopic dermatitis. we present a case of a 10-year-old female with card11 deficiency successfully treated with dupilumab. case: a 10-year-old puerto rican female with history of recurrent sinopulmonary infections with 5 episodes of pneumonia, moderate persistent asthma, food allergies, recurrent skin boils, and severe atopic dermatitis was referred for further management and evaluation for autosomal dominant hyper-ige syndrome (ad-hies). her atopic dermatitis was refractory to conventional therapy with topical corticosteroids, twicedaily emollient use, and bleach baths; it was also refractory to immunosuppression with mycophenolate mofetil and cyclosporine. on exam the patient exhibited coarse facial features and a high palate. she had eczematous lesions on the face, trunk, and extremities (scorad 84). laboratory evaluation showed: eosinophilia (1400 cells/ul), elevated ige (>2000 ku/l), low igm (20mg/dl), and elevated iga (568 mg/dl). lymphocyte subsets and mitogen response were normal but antigeninduced proliferation was abnormal. autosomal dominant hyper ige score was 42 indicating a high likelihood of ad-hies. no mutations in stat3 were identified and th17 cell expression was elevated. dedicator of cytokinesis 8 (dock8) deficiency was also considered but dock8 protein expression was normal. further genetic testing revealed an 18 base pair deletion in card11 (c.518_535del) predicted to be pathogenic. the combination of the patient's phenotype and large deletion was consistent with card11 deficiency. despite continued immunosuppression with cyclosporine and aggressive skin care, the patient's atopic dermatitis was still severe and poorly controlled. off label (patient < 12 years) treatment with subcutaneous dupilumab 300mg every 2 weeks was initiated. at last follow-up, 4 months after dupilumab start, the patient had substantial improvement in dermatitis with clear skin on the face, trunk, and extremities (scorad 40). cyclosporine was discontinued and topical medications were applied less frequently. discussion: hypomorphic heterozygous dominant negative loss of function mutations in card11 have recently been associated with severe atopic dermatitis and allergic disease. treatment of atopic dermatitis in card11 deficiency remains challenging, but dupilumab appears to be an effective alternative to refractory disease. longer follow-up and a larger cohort of card11-lof patients treated with dupilumab are necessary to understand the long-term efficacy and safety for use of dupilumab in these patients. 6 abstract/case report text purpose: the micromilieu within premalignant respiratory papillomas supports persistent hpv6/11 infection and disease recurrence in recurrent respiratory papillomatosis (rrp). these patients show polarized (th2-/treg) adaptive immunity in papillomas and blood, enriched immature langerhans cell (ilc) numbers, and overexpressed cox2/pge2 in the upper airway. to better understand the adaptive and innate dysregulation in rrp, we studied blood-derived monocytes, ilcs, and tissue-derived ilcs from rrp patients and controls. experimental design: monocyte subpopulations were isolated, differentiated into ilcs, activated, and then assessed by flow cytometry. monocytes were induced to differentiate into ilcs with/ without added pge2, and then activated by il-36γ, pge2, pge2+il36γ, or lps. ilc cd83 expression was identified by flow cytometry. monocyte-derived ilcs, papilloma, foreskin, and abdomen skin ilcs, were also analyzed by qpcr for select chemokine/cytokine mrna expression after isolation, 24 hrs later in culture, and again after poly(i:c) or tnfα stimulation. results: the three monocyte sub-populations differed between patients and controls, and patients' monocytes generated fewer ilcs. classical monocytes generated most, but not all ilcs. pge2 levels were higher in rrp plasma, and added pge2 reduced control, but not patients' monocyte-ilc differentiation. pge2 had no effect on ilc maturation identified by cd83 expression. papilloma-derived ilcs expressed low ccl-1, and high ccl-20 mrna and were unresponsive to poly(i:c) or tnfα. tissuespecific cytokine/chemokine responses between ilcs from papillomas, foreskin and abdominal skin differed. only papilloma ilcs expressed il-36γ after isolation, and they up-regulated ccl1 mrna 24 hrs later without further stimulation. conclusions: monocyte/ilc innate immunity is impaired in rrp, in part due to increased pge2 exposure. the immunosuppressive papilloma micromilieu likely alters ilc responses that skew, hpv6/11-specific th2/treg adaptive immunity in rrp. abstract/case report text introduction: familial mediterranean fever is a hereditary auto inflammatory disorder that typically manifests with recurrent fevers, abdominal pain and in some patients there is an associated with amyloidosis leading to eventual renal failure. while there are several common mutations in the mefv gene that when homozygous give these classic symptoms, patients with atypical mutations or heterozygous mutations often have a different clinical course. we present identical twin siblings with compound heterozygous mefv mutations but differing clinical phenotypes. case description: the index patient is a 3 year old girl, conceived via ivf, who began having fevers at age 2.5. her fevers occurred every 4 weeks for 4 months before she was referred to immunology for evaluation. her parents describe her as happy and otherwise not ill appearing during these episodes. genetic testing for familial mediterranean fever revealed compound heterozygous e148q and p369s mutations in the mefv genes. initiation of colchicine therapy in the affected sibling has resulted in a complete resolution of her symptoms. a trial off colchicine resulted in return of cyclic fevers. her identical twin sister was also tested, and carries the same mutation, but is still asymptomatic. this created great concern amongst their parents who had genetic testing prior to undergoing ivf that revealed no parental mutations in mefv. in consultation with genetics the mother was tested again through the same laboratory that had performed testing on the children. this revealed an identical mutation in mom who is also asymptomatic. conclusions: although classic homozygous mefv mutations have resulted in well described fever syndromes, there is considerably less data on heterozygous and compound heterozygous mefv mutations. in these two identical siblings only one patient has a classic manifestation of familial mediterranean fever. while it is possible that the other twin will develop similar symptoms later on in life, it is also possible that another factor is necessary to trigger symptoms in this unusual genetic presentation of fmf. in addition this case highlights the importance of understanding the testing method used by the laboratory performing the genetic testing. while the mother was initially reported as negative the laboratory that performed her testing only tested for the most common mefv mutations. more complete testing, that included the entire gene sequence, revealed that she did contain an mefv mutation in e148q, which although more rare is thought to be pathologic when combined when combined with a second mutation. abstract/case report text there are several lines of evidence that link the pi3k/akt/mtor signaling pathway to primary immunodeficiencies. hyperactivation of the pi3k/akt/mtor/s6k signaling pathway in immune cells can be the consequence of dominant gain-of-function mutations in the genes encoding for pi3kδ that cause the activated pi3kδ syndrome (apds). patients with these mutations may develop immunodeficiency and immune dysregulation as well as neurodevelopmental delay and growth retardation. in addition, mutations of genes within the pi3k-akt-mtor pathway were also known to cause megalencephaly and segmental cortical dysplasia. mutations in akt3, a member of the akt family of proteins and a downstream effector of pi3k-mediated signaling, was shown to be associated with autosomal dominant megalencephalyassociated syndromes. here we describe a 4 year old girl, born to consanguineous healthy parents, who presented with megalencephaly, developmental delay, hypotonia, cervical lymphadenopathy and hepatosplenomegaly. the patient had recurrent hospital and icu admissions for idiopathic thrombocytopenia (treated with ivig), recurrent laryngitis, recurrent peritonsillar abscess, preorbital cellulitis, conjunctivitis with purulent discharge, otitis media, pneumonia with pleural effusion (required drainage), metapneumovirus pneumonia with respiratory failure, recurrent skin cellulitis, and abscesses that grew mrsa (required drainage). in addition, the patient is known to have asthma and allergic rhinitis. mri of the brain showed megalencephaly, ventriculomegally, thin and dysplastic corpus callosum, a normal cerebellum, and myelination appropriate for age. immunoglobulin levels, lymphocyte subsets and the oxidative burst test were all within normal limits. cmvand ebv were not detected. bacterial cultures grew mrsa (skin), strept. pneumoniae, h. influenzae, and e. coli (urine). extensive metabolic workup was done, which was inconclusive (metabolic/mitochondrial diseases). whole exome sequencing identified an akt3 variant c.958g>a; p.(asp320asn) in exon 10. the variant was identified in the patient but not in the parents and it was confirmed by sanger sequencing. further molecular testing concluded that the variant is caused by a de novo mutation during early development. although pathogenic variants in akt3 gene were shown to be associated with megaloencephaly-associated syndromes, no associations with immune deficiency have be reported. functional studies will be pursued to confirm the link between the clinical phenotype and the identified variant in the akt3 gene. complex is a critical signalling adaptor that regulates lymphocyte activation, proliferation, survival, and metabolism. primary immunodeficiencies affecting each component (termed 'cbm-opathies') result in broad clinical manifestations ranging from combined immunodeficiency (cid) to atopic disease or lymphoproliferation. we present the laboratory and clinical findings of two canadian first nations patients found to be homozygous for the same novel card11 mutation (c.2509c>t; p.r837*) causing complete card11 deficiency. results: we recently identified an 8-month-old boy who presented with a severe case of entero/rhinovirus bronchiolitis with interstitial lung disease and a 17-year-old boy with a history of severe pulmonary infections with bronchiectasis (including pjp), chronic sinusitis, candidiasis, invasive bacteremia, and severe ileo-colitis and oral ulceration requiring total colectomy. testing of both patients demonstrated absent tregs, elevated naïve b cells with absent memory b cells, and panhypogammaglobulinemia. next generation sequencing revealed that both patients were homozygous for the same novel variant of card11 (c.2509c>t; p.r837*), which rendered card11 protein undetectable by immunoblot. card11 deficiency was confirmed by stimulating patient b cells with phorbol 12-myristate 13 acetate (pma) and ionomycin and immunoblotting for signalling proteins in both the nf-κb (ikkα/β, iκbα, p65) and mapk (mek1/2, mkk4, jnk1/2, erk1/2) pathways as well as cleavage substrates of the malt1 paracaspase (relb, cyld, bcl10, hoil1). nf-κb and jnk activation were completely absent and m a lt p a r a c a p a s e a c t i v i t y w a s l o s t . f u r t h e r m o r e , c oimmunoprecipitation experiments revealed that card11 was required for optimal malt1 association with bcl10 in response to stimulation. to define the impact of card11 deficiency on the b cell transcriptome, rna-seq experiments were performed. this revealed an inability to upregulate critical genes involved in immunity and tolerance (e.g. cd40lg, ctla4, il2, il10), decreased enrichment in cytokine pathways (e.g. ifn-α, il-6, tgf-β), and decreased enrichment in malt1-dependent genes. furthermore, rna-seq confirmed the developmental block observed in patient b cells and suggested that b cells were halted at the centroblast to centrocyte transition. both patients ultimately underwent hematopoietic stem cell transplantation (hsct), which restored lymphocyte signalling and activation as measured by nf-κb, jnk, and malt1 paracaspase substrate cleavage. conclusions: we have presented the most comprehensive clinical and molecular characterization of human card11 deficiency to date. these two cases highlight the crucial role of card11 in regulating b cell development, function, and humoral responses, as confirmed by signalling and transcriptomic analyses. furthermore, hsct is potentially helpful for these patients as assays performed on post-transplant cells demonstrated restored signalling and activation. abstract/case report text introduction: primary immune deficiencies (pid) can have a significant impact on the quality of life of patients and their families. as more patients with pid are surviving to adulthood, the need to monitor them closely and ensure they are transitioned appropriately is even more crucial. we compared the perspectives of pediatric and adult immunologists toward the transition of patients with pid at our institution. methods: pediatric allergy/immunology providers at lurie children's hospital and adult allergy/immunology physicians at northwestern university both in chicago, il completed respective surveys anonymously (www.surveymonkey.com). questions were derived from the validated 'attitude' and 'quartt' instruments for transition. respondents were asked to rate their level of agreement on a 5-point likert scale, ranging from strongly disagree to strongly agree. results: overall, 9 pediatric and 11 adult providers participated (response rate 71.4%). of total respondents, 55% thought the transition process should be initiated at age 18-20. about 36% of the adult immunologists selected 21 and older, whereas pediatric providers would begin earlier; 33.3% of pediatric providers note they would initiate transition at age 15-17. both pediatric and adult immunologists agreed that patients should be transferred when the provider felt they were ready (80%) and when they were in stable condition (70%). both adult and pediatric immunologists selected transfer of complete medical file as a preferred communication method for transition. other strategies preferred by adult providers were a referral letter with brief summary of medical history (90.9%) and staff meeting with pediatric and adult immunologists (63.6%), whereas pediatric providers would prefer a joint outpatient dedicated transition clinic (77.8%). pediatric and adult immunologists, patient, parent, and transition liaison were considered the most important active participants in the transition process. the most prominent barriers to a formal transition were unavailability of a transition coordinator or nurse specialists (100%) or of all disciplines of the interdisciplinary team (95%), and limited time (95%). on the other hand, limited demand (too few patients) was strongly rejected as a barrier. all participants agreed during transition patients should be educated about medications and their side effects, their condition and related potential future complications, and symptoms that require seeking health care. over 95% of participants also agreed that education about how to set up ivig and further insurance needs were important. all participants agreed that the transition process should include assistance on how to promote the patients' independence and self-management skills, medication management/adherence, and understanding of immunoglobulin replacement and side effects. other important transition components included knowing how frequently lab draws are required for monitoring (95%) and having a written individualized transition plan (70%). pediatric providers also thought having an email or telephone help line would be beneficial. conclusion: this study adds to the growing body of literature examining attitudes of immunologists toward transition, and it highlights important transition components and barriers. further work is ongoing to determine the transition needs identified by patients and parents and define markers for successful transfer in order to build a transition policy at our institution specific to immunodeficiency patients. abstract/case report text introduction: bronchiectasis (bq) is an abnormal and irreversible dilatation of bronchi secondary to repeated cycles of airway infection and inflammation. predominantly antibody deficiency is the main group of primary immunodeficiencies (pid) in adults and had been reported up 10% of subjects with non-cystic fibrosis bronchiectasis (ncfb). hypergammaglobulinemia (igg level higher than 1,600 mg/dl) had been observed in 9.2 % of ncfb cases (retrospective data). diagnostic delay and inappropriate management of patients with predominantly antibody deficiency can lead to irreversible lung damage or even death from serious infections. the effect of hypergammaglobulinemia on ncfb is unknown. here we present the frequency of immunoglobulin abnormalities (pad and hyperigg) in adults with ncfb in cali, colombia. methods: we present preliminary data of a descriptive prospective study that will include 260 patients with ncfb. women and men >14 and < 65 years old will be included. all volunteers will be evaluated by a clinical immunologist, complete blood count and serum igg, iga, igm and ige levels will be determined. according with clinical suspicious, igg subclasses, anti-pneumococcal igg response and b cell subpopulations will be performed. the project will be executed in 24 months. written informed consent has been obtained for all subjects included. this project count with irb approvals at universidad del valle and hospital universitario del valle. results: a total of 103 ncfb cases have been included in the study. the mean age was 46.7 years (14-65 years) with a female:male ratio 64:39. moderate-severe dyspnea was observed in 17/103 cases (medical research council -mrcdyspnea scale 4 to 5). recurrent pneumonia was found in 33/103 cases (32%). the main etiologies of bronchiectasis were: post-infection 30/103 (29%); idiopathic 13/103 (12%); autoimmunity 12/103 (12%); primary immunodeficiency 11/103 (10%) asthma 11/103 (10%); copd 4/103 (3.8%); primary ciliary diskinesia 3/103 (3%); reflux 3/103 (3%) and others. primary immunodeficiencies, 10.6% of ncfb cases, were classified as: predominantly antibody deficiency (10cases) including cvid 5 cases, igm deficiency 2 cases, igg subclasses deficiency 1 case, selective iga deficiency 1 case and hypogamaglobulinemia 1 case. combined immunodeficiency (dock8 deficiency) 1 case. interestingly igg hypergammaglobulinemia was observed in 29/103 cases (28.1%) suggesting humoral immune response deregulation. conclusion to the best of our knowledge this is the first prospective study evaluating the etiology of non-cystic fibrosis bronchiectasis (ncfb) in colombia. hypergammaglobulinemia and predominantly antibody deficiencies affect 39% of adults with ncfb in colombia. our study reinforced the necessity to evaluate humoral immune response in patients with bronchiectasis. conflict of interest: authors disclosure any potential financial conflict of interest related to this abstract. acknowledgements: this investigator-initiated research was supported with a grant from baxalta us inc, a member of the takeda group of companies bt16-35583/iir-col-bxlt-001923. abstract/case report text background: early-onset inflammatory bowel disease (eoibd) is defined as ibd diagnosis in children less than 10 years of age. the occurrence of autoimmune disease in children (where it is relatively rare, compared to adults) may be caused by a highrisk predisposition gene (monogenic disorders). mayo clinic children's center has a unique care model, where a patient who is referred for eoibd meets with a team of physicians, including gastroenterology, immunology, genetics, and nutrition. we describe our experience of our eoibd clinic from an immunologic perspective. methods: we conducted a retrospective cohort study through emr chart review of pediatric patients who were referred to our eoibd program (2011 -2019). first diagnosis of ibd under the age of 10 was the inclusion criteria. we assessed the presentation, clinical correlates, and immunologic evaluation. approval was obtained from mayo's institutional review board. data abstraction and analysis was done using the software jmp. results: 51 pediatric patients met the inclusion criteria, with 31(61%) males and 20(39%) females. the median age of ibd diagnosis was 5 years (3-7 years range). median values and the distribution of variables used in the nutritional and immune evaluation were assessed (fig 1) . nutritional assessment was remarkable for low to low normal hemoglobin and ferritin levels. vitamin d and albumin levels were overall within the normal range. growth parameters indicated that the median bmi percentile was 57 (28-77). with immune and genetic screening, one patient was found to have x linked chronic granulomatous disease (cgd). immune evaluation of other patients was overall within normal limits. fecal calprotectin served a reliable non-invasive biomarker for inflammation with the median being 220.5 (62.7-499.3). 41 of these patients underwent gi pathogen panel testing of which 17 (41%) tested negative, four (10%) tested positive for c. diff, and two (5%) others to shiga toxin-producing e. coli. it was also noted during the chart review that most patients had poor disease control despite undergoing treatment with various anti-inflammatory and immunosuppressive drugs. the patient diagnosed with cgd underwent bone marrow transplantation. a higher proportion of patients referred to our program in recent years underwent a more comprehensive multispecialty evaluation. conclusion: awareness of monogenic causes of inflammatory disorders in children has increased in recent years. it is also important to rule out intestinal infections that can act as ibd mimic. identifying monogenic disorders and other ibd mimics helps with targeted therapy and symptom improvement in these patients who have a difficult-to-treat disease. the group of children with eoibd, regardless of whether there is an inborn error of immunity, suffers from very high morbidity and a high burden of disease. comprehensive immune-nutrition assessment of eoibd patients paves way for further in-depth immunogenic assessments and allows for global management. abstract/case report text background: chronic granulomatous disease (cgd) is a primary immunodeficiency (pid) affecting the nadph oxidase system in phagocytes resulting in increased susceptibility to catalase-positive organisms. holland presented the first report of dual impact of cgd and hiv in a patient with disseminated nocardiosis. because their cgd patient admitted to history of iv drug use, he was frequently screened for hiv. case: a 19-year-old african american male with known cgd tested positive for hiv1 by western blot in the ed in 2009 when he presented with complaints of intermittent fever and cervical lymphadenopathy. his cgd was diagnosed by nbt blood testing at 4 years of age. he had frequent skin infections and fever prior to diagnosis. clinically, he did so well that his cgd diagnosis was questioned by his immunologists. however, cgd was confirmed by 2 additional abnormal nbt tests and, ultimately, dhr flow cytometry testing. during his second infectious disease consultation for hiv, at age 19, he disclosed that he was bisexual. previously, the patient was screened for hiv1 and hiv2 antibodies in 2003 due to anal fissure. he was screened again in 2007 for marked cervical and supraclavicular lymphadenopathy. his cd4+ t cell absolute count was noted to be low (293/mm3) in 2006 at age 17. until 2009, his prior hiv screenings were negative. during his cgd treatment course as an adult, he was known to be variably adherent with administration of interferon gamma due to adverse effects, particularly pain at the site of injection and malaise. at the time of his positive hiv western blot in 2009, his cd4+ t cell count was 237/ mm3. after starting hiv antiretroviral treatment, his viral load became undetectable. at age 23, he had burkholderia cepacia pyelonephritis resulting in left nephrectomy. sepsis from b. cepacia was fatal (positive blood cultures without known primary source) in 2019 at age 29. his recent viral load was still undetectable and cd4+ count was 240/mm3. summary: our case reveals the complexities of treating a patient with both primary and acquired immune deficiencies. it illustrates the importance of taking a thorough social and sexual history starting in adolescence, including those patients with pid. patients with pid should be followed closely by a primary care physician, in addition to an allergist-immunologist and infectious diseases specialist, to ensure age appropriate medical and developmental screening. the recognition of pids is improving due to better screening, awareness, and treatment. currently, the rate of hiv infection is highest among young homosexual african american males. it remains important to understand the epidemiology of primary and acquired immunodeficiencies to best identify those at highest risk. (155) submission id#812008 phosphatase and tensin homolog (pten) hamartoma tumor syndrome identified by newborn tcell receptor excision circle screening for severe combined immunodeficiency δ) subunits that are critical for cellular signaling. heterozygous gain-offunction (gof) mutations in pik3cd (encoding p110δ) result in activated pi3k δ syndrome 1 (apds1), while heterozygous loss-of-function (lof) mutations in pik3r1 (encoding p85α) result in activated pi3k δ syndrome 2 (apds2). given its role as a negative regulator of the pi3k signaling pathway, heterozygous lof mutations in pten (encoding phosphatase and tensin homolog, pten) result in a clinical phenotype that approximates that of apds1/apds2 and is therefore referred to as activated pi3k δ syndrome-like (apds-l). however, sequelae of heterozygous pten lof mutations extend beyond the immune system and include a group of disorders collectively known as pten hamartoma tumor syndrome (phts). although severe t cell lymphopenia at birth would be unexpected in apds1, apds2, or apds-l, below normal t cell receptor excision circle (trec) counts have been reported in apds1, but only in individuals outside of the neonatal period. herein, we describe an infant girl with a low trec count at birth who was found to have phts. case description: a 1-day-old girl, born at a gestational age of 39 weeks, was found to have a low trec count of 22/microliter (normal => 40). a second trec count obtained at 2 weeks of age resulted as 16/microliter. arguing against a diagnosis of severe combined immunodeficiency (scid), flow cytometric analyses performed at 3 weeks of age revealed only a modestly diminished cd3+ t cell count (1599/microliter; 1240 cd4+ and 277 cd8+) with a normal percentage of naïve and memory cd4+ t cells (78% and 22%, respectively). by 7 months of age, her cd3+ t cell count dropped to 828/microliter, which was accompanied by a significantly decreased percentage of naïve cd4+ t cells (56%). sequencing and deletion/duplication analysis was pursued via a commercially available 207-gene panel aimed at genetically defined primary immunodeficiency (pid), in which no clearly pathogenic mutations were identified. over the following months, the patient was noted to have macrocephaly, tall stature (99th percentile), axial hypotonia, and gross motor delays. sequencing and deletion/duplication analysis was then pursued via a commercially available 29-gene panel aimed at genetically defined macrocephaly and overgrowth syndromes, in which a hemizygous pathogenic mutation in pten (c.512a>g, p.gln171arg) was identified. subsequent flow cytometric analyses demonstrated findings characteristic of apds-l, including expanded transitional and cd21lo b cells, decreased isotype switched memory b cells, increased effector memory t cells, a lowered threshold for intracellular calcium mobilization upon b cell receptor engagement, and increased basal akt (protein kinase b) and s6 (ribosomal protein s6) signaling. discussion: we report the first case of phts identified by newborn trec screening for scid. as pten is not included in most commercially available, scid-or pid-tailored gene panels, phts would be missed by conventional genetic testing. therefore, analysis for variants in pten should be considered in neonates with low trec counts, macrocephaly, developmental delay, and other suggestive sequelae. abstract/case report text primary (or familial) hemophagocytic lymphohistiocytosis (hlh) is a rare, life-threatening hyper-inflammatory syndrome affecting mainly young children. it is caused by mutations in genes involved in the granule-dependent cytotoxic pathway, inducing extreme inflammation and massive tissue infiltration by activated t cells and macrophages. standard chemotherapy-based treatment regimens are toxic and induce remission in only 80% of patients. to this day, hsct is the only available curative treatment, but the inability to efficiently control the inflammation in many patients prior to transplantation often leads to graft failure, with transplant-related mortality around 25%. thus, the development of new, more potent and less toxic anti-inflammatory regimens would be a major advancement in the treatment of hlh. here, we hypothesize that combination therapies targeting several jak-dependent cytokines will be more effective than monotherapy to reduce the life-threatening symptoms induced by this pathology. using a perforin-deficient (pko) mouse model, we first tested the effects of blocking antibodies against ifnγ, the dominant cytokine secreted during hlh, in combination with antibodies targeting other highly elevated cytokines, such as il-6 and il-18, on the manifestations of the disease. we found that anti-il-6r and anti-il-18 antibodies, when used in combination with anti-ifnγ antibodies, did not significantly improve the symptoms of hlh compare to anti-ifnγ antibodies alone. further, we found that targeting the jak-stat signaling pathway with ruxolitinib, a specific inhibitor of jak1 and jak2, molecules downstream of ifnγ and il-6, but not il-18 signaling, was as beneficial as anti-ifnγ monotherapy. next, we tested the efficacy of ruxolitinib in combination with anti-il-18 antibody, as this later cytokine is not jak-dependent and was shown to drive macrophage activation syndrome in other contexts. unfortunately, this combination did not result in better symptom resolution than the use of ruxolitinib only. in contrast, combination therapy using ruxolitinib and anti-ifnγ antibodies showed a striking synergistic effect on the resolution of most disease manifestations, to such an extent that our pko mice presented a clinical phenotype indistinguishable than that of a c57bl6 control mice. our findings demonstrate that jak-dependent cytokines are the main cytokines driving the progression of hlh in pko mice. collectively, our results suggest that anti-ifnγ antibodies and ruxolitinib, although effective independently, should be used in combination to more efficiently suppress hlh progression. these results are particularly relevant since the emapalumab, an anti-ifnγ monoclonal antibody was recently approved by the fda for the treatment of hlh while ruxolitinib will soon be in clinical trials for this indication. this project was supported by funds from the fondation de cancérologie charles bruneau and the canadian institutes of health research (mop-130469). abstract/case report text introduction: transcription factor 3 (tcf3), also known as transcription factor e2-alpha (e2a), is a helix-loop-helix transcription factor which plays a critical role in lymphopoiesis. tcf3 is required for b and t lymphocyte development. defects in tcf3 have been associated with agammaglobulinemia 8, autosomal dominant, characterized by low levels of immunoglobulin and early onset recurrent bacterial infections. deletion or diminished activity of tcf3 may also play a role in lymphoid malignancies. runs of homozygosity (roh) are contiguous stretches of homozygous genotypes at consecutive polymorphic dna marker positions. roh are important reservoirs of homozygous deleterious variation. the homozygosity heterogeneous hmm (h3m2) algorithm was specifically developed for analyzing whole exome sequencing (wes) data. the branch point sequence (bps) is an essential splicing signal located 15-55 bases upstream of splice acceptor sites. while bps variants are rare, they may result in aberrant pre-mrna splicing and genetic disorders. these variants may be overlooked by standard wes analysis methods because they are intronic and the mammalian bps is a degenerate motif. objective: describe the method used to identify a bps variant in consanguineous brothers with immunodeficiency, including early onset recurrent infections and b-all, hypogammaglobulinemia, t and nk lymphocytosis, low b cells, and low naïve t cells. methods: wes was performed for all family members. data were analyzed using standard read mapping, variant calling and annotation methods. roh were analyzed using the h3m2 algorithm (magi et al. 2014). roh from the siblings was intersected (bedtools) and the output was submitted to the genomic oligoarray and snp array evaluation tool (v3.0). variants were confirmed by sanger sequencing. results: no candidate disease variants were detected in the coding regions, 5' or 3' splice sites, or utrs for both brothers; however, analysis of intersected roh revealed a homozygous tcf3 intronic variant within a putative bps (tcf3 c.1451-18a>t). sanger sequencing of the mutant cdna revealed activation of a cryptic splice site. heritability for routine childhood vaccines has been shown to range from 38-89%. the genetic component of vaccine response suggests we should be able to predict vaccine response in infants with biomarkers. methods: multi-center study of 93 infants born vaginally at full term and followed through 12 months of life. cord blood was collected at birth & peripheral blood was collected at 6 and 12 months of life. six-month collection was 2 weeks post administration of routine vaccinations, while 12-month collection was immediately prior to receiving the 12-month booster vaccines. b cell subsets were analyzed with flow cytometry. vaccine titers and cytokines were measured via multi-plex elisa. study was irb approved. results: our data confirmed the immaturity of the newborn humoral immune system with a lower overall b-cell abundance, a predominance of naïve b cells, an inability to class-switch and produce igg or iga, and a th2 bias. maturation was observed over the first year with increasing overall b-cell abundance, frequency of memory b-cells producing iga, igg, and igm, and frequency of plasmablasts. scd14 levels also increased throughout the first year due to microbial translocation reflecting establishment of the microbiome. conversely, cord blood contained high levels of baff, april, scd40l, il-4, and il-21, with levels decreasing thereafter. all infants displayed evidence of humoral immune system activation after getting 6-month vaccines. total plasmablast levels peaked 2 weeks after receipt of 6month immunizations. a decrease in total plasmablasts was evident between 6 and 12 months, although levels remained above those at birth, corresponding with the need for 12-month booster vaccinations to maintain long-lasting immunity. il-21 and ifn-gamma had a significant positive correlation with memory b cells and plasmablasts at subsequent time points suggesting that these cytokines play a role in b cell differentiation and vaccine response. baff and april cytokines were elevated at birth, consistent with germinal center formation and underwent a compensatory decrease thereafter. april & scd163 levels in cord blood significantly correlated with higher tetanus titers at 12 months suggesting that vaccine response may be predicted by cytokine biomarkers at birth. response to vaccines was also dynamic with il-2 levels being significantly correlated with tetanus titers at 2 weeks after receipt of 6 month immunizations. conversely, scd40l levels did not correspond to b cell development consistent with a known b cell hyporesponsiveness to cd40l in infants. conclusion: humoral immune development is both predictable and dynamic. biomarkers in the cord blood, produced by the infant, are predictors of b cell development and vaccine response in infancy. background: adult-onset immunodeficiency with anti-ifnɣ autoantibodies is a newly described immunodeficiency syndrome characterized by disseminated nontuberculous mycobacterial and other opportunistic infections in previously healthy middle-aged individuals typically from southeast asia. it is caused by the presence of autoantibodies directed against the cytokine ifnɣ, which is required for intracellular pathogen killing by macrophages as well as phosphorylation of the transcription factor stat1, which is involved in cell survival gene expression. successful treatment of the immunodeficiency has been described in prior case reports with immunomodulatory therapies, including rituximab. however, there are no standard recommendations for dosing or timing of these agents, or recommendations for longterm monitoring of disease activity. case presentation: a 49-year-old laotian woman with a history of type 2 diabetes and possible prior hepatitis c infection presented to the immunology clinic for evaluation of immunodeficiency. in the two years prior to presentation, the patient was diagnosed with mycobacterium avium infection involving the parotid gland and lymph nodes of the neck, mycobacterium avium complex bacteremia, histoplasma capsulatum involving the lymph nodes of the neck, and leukocytoclastic vasculitis of the lower extremities. as a child and young adult, she had no severe or recurrent illnesses and did not suffer from any chronic disease. preliminary immunologic testing demonstrated normal t, b, and nk cell subsets, elevated immunoglobulin g, a, and m levels, and protective titers to tetanus, diphtheria, and 23/23 pneumococcal serotypes. measurement of anti-ifnɣ autoantibodies was positive, which led to the diagnosis of adult-onset immunodeficiency with anti-ifnɣ autoantibodies. the patient was treated with four doses of monthly rituximab with resolution of the anti-ifnɣ autoantibodies, restoration of normal stat1 phosphorylation, and depletion of cd19-positive b cells. after a 4-year period of being lost to follow-up, during which she continued to receive rituximab every 6 months at the direction of a local provider, the patient re-presented to the immunology clinic to re-establish care. at that time, the patient had no evidence of anti-ifnɣ autoantibodies based on titers and normal stat1 phosphorylation. cd19-positive b cells remained depleted. the patient also confirmed subjective clinical improvement and denied any interim infectious complications. conclusion: this case provides an example of successful treatment of a patient with adult-onset immunodeficiency with anti-ifnɣ autoantibodies with rituximab. it also highlights the utility of ifnɣ functional testing with stat1 phosphorylation, which may be used to monitor disease activity and to make decisions about ongoing immunomodulatory treatment. it is necessary to monitor additional immunological markers to refine the diagnosis of a secondary post-lt immunodeficiency to take preventive measures before infections occur. we sequentially measured t lymphocytes and antibodymediated immunity in a 67-year-old male receiving a lung transplant for idiopathic pulmonary fibrosis to determine which immune indicators could improve the identification of a secondary immunodeficiency. methods: t and b cell numbers, igm, igg, iga, ige and igg subclasses and 13 specific antibodies to s. pneumonia capsular polysaccharides were assessed over a 5 months-long post lt period. the clinical progress, infections and pulmonary function were monitored prior to transplantation and at regular intervals thereafter. results: the patient had progressive idiopathic pulmonary fibrosis starting with an episode of pulmonary hypersensitivity 4 years earlier. he developed increasing respiratory failure progressing to complete 02 dependency requiring a lt in june 2019. he was treated with prednisone, 20mg/ day continuously for 3 months, then decreased to 15 mg/day. other immunosupressants included mycophenolic acid and tacrolimus and on/ off antibiotics that eventually led to severe tendinitis at 4-5 months post lt. at 4½-month post lt he developed an early onset bronchiolitis obliterans syndrome (bos) that was controlled by increasing the prednisone dose. sequential immunologic evaluation showed his igg dropping from 1,400 mg/dl to 800 after two weeks and then remaining stable at that level for the rest of the observation period. igm and iga had minor variations and ige remained very low. igg2 fell from 700-800mg/ml pre-lt to 180-200 in 2 weeks and remained stable at that level thereafter. antibodies against s. pneumoniae polysaccharides started high, between 5-15 ·g/ml for all 13 serotypes and fell rapidly in the first 2 weeks post lt, then continued a steady decline with > 50% serotypes falling < 1.3·g/ml at 5 months. twelve of 13 pneumococcal serotype antibodies increased above 1.3·g/ ml after igg replacement at 5 months. cd4 t lymphocytes decreased from ±1,000 cells/ul to 500-600 at 1 month, remaining at that number after that. cd4/th17 cells increased from 4-32 cells/ul to 178 at 5 months when prednisone was tapered down and then decreased to 12 after increasing the prednisone dose again. this decrease coincided with a reduction in bos manifestations. conclusions: immune monitoring revealed an independent decrease of immunoglobulins with a stronger decrease in igg2 and specific pneumococcal antibodies. the role of th17 cell increase in developing bos needs further investigation. stat3 is a frequent target of cancer therapies due to its role in certain malignancies for cell proliferation and metastasis. with lof stat3, decreased incidence of some cancers may be expected, however increased rates of lymphoma are described. we sought to describe the incidence and spectrum of malignancy in our relatively large lof stat3 cohort. methods: we performed a retrospective analysis of 158 lof stat3 patients evaluated at the nih clinical center to determine the type of malignancies diagnosed, treatments received, and outcomes following therapy. results: a total of 9 patients with 10 malignancies were identified (cancer incidence 6%). six patients (4%) were diagnosed with non-hodgkin lymphoma (nhl); 5 with diffuse large b-cell lymphoma (dlbcl) and 1 with burkitt lymphoma (bl) with age at diagnosis ranging from 4 years to 66 years with median age of 31 years. pathology staining for ebv was available in four patients; all of whom were negative by eber. all 5 dlbcl patients received da-epoch-r for 3-6 cycles, and all achieved complete remission. five of 6 patients with lymphoma are alive and disease-free. one patient died of heart failure 16 years post chemotherapy without disease relapse. two patients were diagnosed with papillary thyroid carcinoma at ages 26 and 27, one of whom was subsequently diagnosed with nhl. two other patients were diagnosed with basal cell carcinoma of the skin at ages 42 and 48; both of whom had prior voriconazole exposure. conclusion: malignancy, most commonly nhl, occurs in patients with lof stat3 mutations. nhl should be considered in patients with progressive lymphadenopathy, and thyroid carcinoma should be considered in patients with thyroid nodules. patients treated with voriconazole are at an increased risk of skin cancer and require careful skin monitoring. as survival increases, it will be important to monitor the incidence of malignancies diagnosed, as it is possible that decreased stat3 signaling may prove to be protective of some cancers, such as colon and breast carcinoma, in which increased stat3 signaling is implicated in pathogenesis. abstract/case report text introduction: recurrent pneumonia is defined as 2 or more episodes of pneumonia in one year or more than 3 pneumonias throughout life (with radiological resolution between episodes). in retrospective studies, up to 30% of adult subjects with recurrent pneumonia coursed with primary immunodeficiencies (pid). prospective studies evaluating the etiology of recurrent pneumonia are scarce. diagnostic delay and inappropriate management of patients with pid (predominantly antibody deficiency for example) could lead to irreversible lung damage or even death from serious infections. here we present the frequency of primary immunodeficiencies in adults with recurrent pneumonia in cali, colombia. methods: we present preliminary data of a descriptive prospective study that will include 100 patients with recurrent pneumonia. women and men >14 and < 65 years old will be included. all volunteers will be evaluated by a clinical immunologist, complete blood count and serum igg, iga, igm and ige levels will be determined. according with clinical suspicious, igg subclasses, anti-pneumococcal igg response and b cell subpopulations will be performed. the project will be executed in 24 months. written informed consent has been obtained for all subjects included. this project count with irb approvals at universidad del valle and hospital universitario del valle. results: a total of 52 recurrent pneumonia cases have been included in the study. the mean age was 38.8 years (14-65 years) with a female:male ratio 28:24. moderate-severe dyspnea was observed in 4/52 cases (medical research council -mrcdyspnea scale 4 to 5). non cystic fibrosis bronchiectasis was found in 33/52 cases (63%). the main etiologies of recurrent pneumonia were: primary immunodeficiency 17/52 (32%); asthma 4/52 (7.6%); autoimmunity 3/52 (5.7%); primary ciliary diskinesia 3/52 (5.7%) and others. hypergammaglobulinemia represented 14/52 (27%) of cases. primary immunodeficiencies, 32% of recurrent pneumonia cases, were classified as: predominantly antibody deficiency (15cases) including cvid 7 cases, igm deficiency 3 cases, selective iga deficiency 2 cases, igg subclasses deficiency 1 case, hypogamaglobulinemia 1 case and agammaglobulinemia 1 case. combined immunodeficiency: dock8 deficiency 1 case and ataxia telangiectasia 1 case. conclusion: to the best of our knowledge this is the first prospective study evaluating the etiology of recurrent pneumonia in colombia. predominantly antibody deficiencies and igg hypergammaglobulinemia affect 60% of adults with recurrent pneumonia in colombia. this study allows us diagnosed more than 10 new cases of adult onset pid. immunological evaluation is critical in the assessment of patients with recurrent pneumonia. conflict of interest: authors disclosure any potential financial conflict of interest related to this abstract. acknowledgements: this investigator-initiated research was supported with a grant from baxalta us inc, a member of the takeda group of companies bt16-35583/iir-col-bxlt-001923. abstract/case report text introduction: lysinuric protein intolerance (lpi) is an autosomal recessive metabolic disorder due to pathogenic mutations in slc7a7. it is distinguished by decreased plasma concentrations and increased urinary excretion of lysine, arginine and ornithine and can present with multiorgan involvement and a spectrum of immune deficiency. we present a five-year-old female with lpi, early-onset juvenile systemic lupus erythematous (sle), hemophagocytic lymphohistiocytosis (hlh), and granulomatous skin lesions that were positive for vaccine-strain rubella. methods: retrospective chart review was conducted. laboratory investigations included lymphocyte immunophenotyping by flow cytometry, lymphocyte proliferation to mitogen, quantitative serum immunoglobulins, vaccine titers, autoantibodies, metabolic studies, and genetic evaluation by next generation and whole exome sequencing. results: a five-years-old female of mixed native american and african american race presented at 3 years of age with severe failure to thrive, history of recurrent fevers, joint swelling, recurrent skin lesions, severe anemia and neutropenia, and hypergammaglobulinemia. upon further evaluation, she demonstrated hyperferritinemia and ana, rnp, smith, and ss-a autoantibodies and was diagnosed with early-onset juvenile sle. laboratory immune evaluation revealed age-appropriate lymphocyte subpopulations and lymphocyte proliferative responses to mitogens and antigens, markedly elevated igg, iga, igm with no associated monoclonality, and protective tetanus and pneumococcal titers. given her severe clinical manifestations at an early age, concern for immunodeficiency prompted further genetic evaluation with next generation dclre1c sequencing, which was negative, and whole exome sequencing, which revealed two heterozygous mutations in slc7a7, consistent with lpi. laboratory metabolic evaluation was also consistent with a diagnosis of lpi. she continued to experience recurrent cutaneous lesions on her upper and lower extremities. biopsy findings were consistent with a granulomatous lesion and subsequently identified by the cdc to have vaccine-strain rubella infection. due to recurrent pneumonia and concern for pulmonary alveolar proteinosis (pap), pulmonology was consulted and eventually confirmed pap, and she has required home oxygen supplementation. given her history of recurrent infections and vaccine-strain rubella infection, supplemental ivig was initiated. her sle has been fairly refractory to medical management, including systemic corticosteroids, mycophenolate, rituximab, and cyclosporine. she recently developed hlh at 5 years of age and is currently maintained on canakinumab, mycophenolate, and systemic corticosteroids, yet continues to have sle and pap that has been difficult to control. conclusion: the range of clinical and immunologic findings in lysinuric protein intolerance has varied widely in the literature. our patient presented with early-onset juvenile sle and did not develop hlh and pap until 2 years after initial presentation. despite relatively normal cellular immunity by laboratory evaluation, our patient was identified to have chronic infection with vaccine-strain rubella virus, indicating severe t cell dysfunction, and poses challenges for future immunomodulatory treatment. introduction: x-linked immunodeficiency with magnesium defect, ebv infection, and neoplasia (xmen) disease is caused by lossof-function (lof) mutations in the magnesium transporter 1 (magt1) gene. it is a rare x-linked combined immunodeficiency and selective congenital disorder of glycosylation. clinical manifestations include chronic ebv viremia, recurrent bacterial and viral infections, lymphadenopathy, splenomegaly, autoimmunity, liver and central nervous system (cns) abnormalities. magt1 deficiency was first noted to result in chronic ebv infection and an increased susceptibility to ebv+ lymphomas. we recently recognized merkel cell carcinoma at a very young age in two xmen patients, leading to our review of the malignancies in this cohort. methods: we reviewed the records of 25 male patients (22 seen at the nih) with confirmed hemizygous lof mutations in magt1 for diagnosis of malignancy, therapy, and outcome. results: we identified malignancy in 11 patients of 25 with magt1 deficiency (44%). four patients had hodgkin's lymphoma (hl) (ages 15-29 years), three had non-hodgkins lymphoma (nhl) (ages 7-57 years), one had kaposi sarcoma (5years) , one patient developed eber-negative liposarcoma (age 27 years) after receiving chemo and radiotherapy for severe lymphoproliferative disease (lpd) at age 13 years and two had merkel cell carcinoma at exceedingly young ages (14 and 22 years). all patients had chronic ebv viremia. they all received treatment according to established protocols. currently, all except for three patients are alive and in remission, including one post-hsct. overall malignancy survival of 73%. it is important to note that three patients who did not have malignancy had ebv lpd so severe that it warranted treatment with a malignancy protocol, with one mistaken as having lymphoma. conclusion: xmen immune deficiency, an x-linked glycosylation disorder, is a multisystem disease associated with increased susceptibility to malignancies. initially, ebv driven lymphoproliferation and lymphoma was described with xmen; however, with increasing diagnoses, more malignancies are being recognized. all the recognized malignancies are associated, at least in part, with dnaviruses, including ebv, hhv-8, and merkel cell virus. understanding the clinical phenotype and pathogenesis of this disease will improve monitoring and early diagnosis of malignancies for patients with magt1 deficiency. abstract/case report text background: primary immune deficiencies (pid) constitute a heterogeneous group of over 400 individually rare congenital diseases that involve genes coding for proteins of the immune system, and which result in increased susceptibility to infection, inflammation, autoimmunity, allergy and cancer. the complexity of the diagnostic task, and the intrinsic biases and limitations of the human mind, can be aided by computational tools. among the available machine learning approaches, decision tree algorithms select the best node to split based on entropy and information gain; random forests build dozens or thousands of decision trees randomly to improve accuracy and reduce overfitting. aim: to implement a machine learning-assisted clinical decision support system for the diagnosis of pid. methods: with a local database of patients with suspected iei, we built a decision tree using c4.5 dtc, and a random forest on python 2.7 (jupyter notebook, scikit, mathplotlib, pandas, numpy). the database was obtained by conducting an electronic search on medsys of patients with the term "immunodeficiency" in their electronic medical records, and then hand-picking cases in which a pid had been confirmed or ruled out. it consisted of 234 patients, of which 185 had been diagnosed with iei. we first split the dataset randomly into training (60%) and testing (40%) sets. the decision tree was tasked with classifying correctly pid or not. after running the algorithm in the training set, we evaluated in the testing set through cross-validation. results: accuracy was greater than 80% for the dataset (pid/not). 0.819 for the dtc with 15 levels. the attribute with the lowest gini coefficient was low iga (0.044). accuracy for the random forest classifier was 0.808 with 25 trees. feature importance was highest for lung infection (0.054), high igg (0.043), low iga (0.041), skin infection (0.046), no isolate (0.057), and allergy (0.043); it was lowest for consanguinity, high igm, central nervous system infection, parasites and no infections. during the random generation of trees, accuracy reached up to 87%. discussion: we built two classification models. decision trees lend themselves more easily to learning and deriving rules of thumb from their sequences. random forests are more robust and better suited for categoric (as opposed to binary) classification. we next want to develop a chatbot, currently under construction, that will ask relevant questions in optimal sequence, and extract undiagnosed patients with suspected iei, based on statistical "red flags". we also have preliminary results of this process applied to a usidnet database with over 3,000 patients, and are also working on multinomial logistic regression and naïve bayesian classifiers for this and other databases. abstract/case report text objectives: acute viral respiratory infections (avri) are associated with significant healthcare resource use and cost. the use of intravenous immunoglobulin (ivig) may be an effective treatment for immunosuppressed patients and reduce overall healthcare resource utilization. the goal of this study was to assess hospital resource utilization associated with ivig use among patients hospitalized for avri. methods: using data from the 2011-17 premier hospital database, we identified patients hospitalized with a diagnosis of avri [respiratory syncytial virus (rsv), parainfluenza virus, rhinovirus, or metapneumovirus], and who had an immune deficiency (chemotherapy treatment, transplant, primary immunodeficiency disorder (pidd), specific antibody deficiency, other immunodeficiency, or disorders of the immune or lymphatic systems). patients receiving ivig within the first 48 hours were compared to patients who did not receive ivig at all. due to the nature of the need to better understand the treatment effect associated with ivig, we used an inverse probability weight-based regression model. since there were substantially more controls than cases, we randomly drew 5,000 controls. a logistic regression model was developed to adjust for factors associated with the probability of ivig use within 48 hours of admission. this propensity score was then used to weigh subsequent models to assess length of stay (total and icu) using negative binomial models and logistic regression for inpatient death. results: a sample of 1,927 immunocompromised inpatients were identified, 65 receiving ivig within the first 48 hours of admission and 1,862 who did not receive ivig. the ivig group was older (mean age 54 vs 35, p < 0.001), had more antiviral use (40% vs 22%, p < 0.001), and had less cancer (40% vs 75%, p < 0.001). after adjustment for immunity type (transplant, cancer), rsv, pidd, age, prednisone, antiviral use, ribavirin use, urban hospital setting, teaching status, intubation and lung disease, patients with ivig use had 3.24 less days of hospitalization (p=0.027) and 1.83 less days in the icu (p=0.003) than non ivig users. conclusions: this data analysis suggests that hospital length of stay and icu length of stay were significantly shorter for immunocompromised patients hospitalized for acute viral respiratory infections who were administered ivig within the first 48 hours of admission, as compared to patients who did not receive ivig. it is possible that ivig use may have an impact on hospital resource utilization and costs. future prospective studies would help further assess the role of ivig in patients hospitalized with acute viral respiratory infections. associate professor/yale university abstract/case report text background: cd40 ligand deficiency is an x-linked combined immunodeficiency associated with opportunistic infections and increased risk of malignancies. expansion of memory cd8+ t-cells with senescent features is known to be associated with chronic immune stimulation including aging, chronic infection and malignancy. cd8+ t-cell characteristics of cd40l deficient (cd40ld) patients in relation to their clinical history have not been described. objective: we studied correlation between cd8+ t-cell senescence with clinical histories of cd40ld patients. methods: we analyzed the frequency and phenotypic characteristics of peripheral cd8+ t-cell subsets in four cd40ld patients (5, 28, 33 and 34 years old (yo)) and healthy controls (hcs). t cell excision circle (trec) counts and telomere lengths of the patients and hcs were measured using quantitative pcr. in-depth analysis of cd8+ t-cells of the 5 yo patient and hcs was done using high-dimensional cytometer time of flight analysis (cytof). results: three patients (5, 28 and 34 yo) with histories of recurrent infections and poor compliance with immunoglobulin therapy (ivig) showed an increased frequency of effector memory cd8+ t-cells with the senescent phenotype compared to age matched hcs. whereas 33 yo patient with excellent ivig compliance starting at infancy did not show any senescence phenotypes of the cd8+ t-cells. the telomere length and trec count of each patient correlated with the degree of cd8+ t-cell senescence and their current ages, respectively. in-depth analysis showed similar expression patterns of molecules related to senescence and cytotoxicity in cd8+ t-cells including cd57, t-bet, eomes, granzyme b and perforin in the 5 yo patient and mid-elderly hcs. conclusion: our findings suggest that prompt diagnosis and compliance with ivig starting at the infancy may prevent early onset cd8+ t-cell senescence in cd40l deficiency. abstract/case report text introduction: immunoglobulin g4-related disease (igg4-rd) is an immune-mediated fibroinflammatory condition that affects multiple organs. when igg4-rd is found in the ocular adnexa, the term "igg4related ophthalmic disease (igg4-rod)" is used. objective: our case describes a patient with igg4-rod without systemic involvement. case: mr. x is a 46-year-old male with a pmh of cml (on imatinib) and allergic rhinitis who presented to clinic with orbital swelling for twenty years. his swelling had always been responsive to steroids, but would return once steroids were tapered. patient was diagnosed with biopsy proven cml in 2011 and is currently taking imatinib. because his peri-orbital edema persisted, a right lacrimal gland biopsy was done which showed "marked lymphocytic infiltrate of soft tissue with lymphoid follicles, many plasma cells, and eosinophils. no atypical histiocytes." flow cytometry was negative for malignancy. results: crp 4.12 mg/l. esr 14 mm/hr. igg4 elevated at 655 mg/dl. ct chest from 2013 and ct chest, abdomen, pelvis from 2019 were without fibrotic changes. assessment: when diagnosing igg4-rd, we categorize diagnosis into three levels (possible, probable, or definite) by three criteria (clinical manifestation, elevated serum igg4, and histopathology). this is detailed as follows: clinical exam showing organ specific swelling or masses, elevated serum igg4 (>135 mg/dl), and histopathology with either lymphocyte and plasmacyte infiltration and fibrosis or infiltration of igg4+ plasma cells (ratio of igg4+/igg+ cells ≧40 % and ≧10 igg4+ plasma cells per high power field). not all these components are required for diagnosis, but meeting histopathologic criteria makes diagnosis more probable. our patient's disease was localized to his eye, and patients with igg4-rod have unique diagnostic criteria. these criteria are similar to the criteria for igg4-rd, but emphasize enlargement of the ocular adnexa, less frequent fibrosis, and ≧50 igg4+ plasma cells per high power field. our patient's histopathology revealed a lymphoplasmacytic infiltrate, but lacked storiform fibrosis or obliterative phlebitis. his serum igg4 level was 655 mg/dl, and his biopsy was positive for an igg4+/igg+ ratio of 50% and more than 100 igg4+ plasma cells per high power field. based on this, he meets criteria for igg4-rod. conclusion: igg4-rod is a rare condition that is usually associated with systemic organ involvement. our case is unique, as no systemic disease has been detected. we also suspect our patient has been living with igg4-rod for several years, as his orbital swelling began in high school. it is important to note that he has been on imatinib, a tyrosine kinase inhibitor, for treatment of his cml. imatinib inhibits c-abl and platelet-derived growth factor receptor, tyrosine kinases involved in profibrotic pathways. patient's lack of fibrosis could also be due to his longstanding use of this drug. it is also possible that he has a rare form of igg4-rod without systemic involvement. a limited number of such cases have been reported, but no consensus has been made on why disease course was localized. our patient was started on rituximab, and his serum igg4 decreased to 295 mg/dl after the first cycle. we hope his disease achieves remission. informed consent: informed consent was obtained from all individual participants included in the study. abstract/case report text platelet abnormalities with eosinophilia and immune-mediated inflammatory disease (plteid) is a recently discovered combined immunodeficiency with inflammatory and allergic manifestations with few cases reported. we describe a female patient with compound heterozygous mutation in arpc1b gene with suggestive clinical findings of plteid. a 2-year-old girl presented with chronic diarrhea since neonatal period, with bloody stools and failure to thrive. she also presented atopic dermatitis, recurrent cutaneous and mucosal ulcers, recurrent respiratory infections (4 episodes of otitis media, 2 pneumonias) and many episodes of mucocutaneous candidiasis. family history revealed a sibling deceased in the second month of life, who presented a similar clinical picture and a paternal uncle and second degree cousin that died in the first year of life. there is no history of consanguinity. laboratory evaluation revealed peripheral eosinophilia (1000/mm3), normal platelet numbers with low platelet volume (8,3 fl -reference value 9,4-12,4 fl), normal igm levels with elevated igg (1114 mg/dl-rv 453-916 mg/dl), iga (517 mg/dl -rv 20-100 mg/dl) and ige (1141 iu/ml -rv a) associated with plteid. t h e i n f a n t r e c e i v e s a n t i m i c r o b i a l p r o p h y l a x i s w i t h sulfamethoxazole-trimethoprim and fluconazole, intravenous immunoglobulin replacement and was referred to hematopoietic stem cell transplantation (hsct). this case was the first one described in brazil and highlights the importance of seeking for a genetic diagnosis in patients with complex clinical phenotypes. precise diagnosis can impact on treatment approach. live vaccines are generally contraindicated in patients with combined immunodeficiency (cid). however, in less severe cid, such as partial dgs, those vaccines can be considered depending on the immunologic status of the patient. there are recommendations regarding to measles, mumps, rubella (mmr) and varicella vaccines, but yellow fever vaccine (yfv) is generally contraindicated in this population. considering the severity of the yellow fever disease and the absence of specific treatment, the use of this vaccine is an important topic for debate in cases of patients from endemic areas. objective: this study aimed to describe the use of yfv and other live attenuated vaccines in patients with dgs, associating it with their immunological profiles and the presence of adverse effects. methods: retrospective study of medical records of patients with dgs confirmed by mlpa or fish, followed in a pediatric reference center for primary immunodeficiencies between 2009 and 2019. collected data included: demographic characteristics, medical history, history of immunization with live vaccines, postvaccination adverse reactions and immunological profile, including immunoglobulins levels, serologic vaccination responses, lymphocyte immunophenotyping, lymphocyte proliferation responses to mitogens and prophylactic treatments (antibiotic or immunoglobulins). results: thirty-five patients with confirmed dgs and median age of 12 years (2-21y) were included (22m:13f). thirty-three children (94%) received mmr vaccine: nine presented t lymphopenia. two of the 9 patients had cd4 < 300, one of them with normal mitogenic proliferation response and the other was not tested. three of the 33 patients had low immunoglobulins levels (2/33 low igg, 2/33 low igm and 1/33 low iga), and one of them received intravenous immunoglobulin (ivig). twentynine of 33 had normal serologic vaccination responses. adverse effect was only reported by one patient, who had one episode of fever after the administration of all vaccines. yellow fever vaccine was administrated to 14 children (40%): 2 had t cell l y m p h o p e n i a ( b u t c d 4 > 5 0 0 ) , a n d a n o t h e r p a t i e n t h a d hypogammaglobulinemia and received ivig and prophylactic antibiotics. twelve of 14 showed adequate serologic responses to mmr and hepatitis b. only 1 patient reported mild reaction (tremors) two days after the yfv administration. the same patient had normal t cells, immunoglobulins and vaccine responses. twenty patients (57%) received bacillus calmette-guerin vaccine (bcg), 15 (42%) received oral polio, 9 (25%) rotavirus and 8 (22%) received varicella vaccine. no severe adverse events were documented in any patient that received live vaccines, and no patient developed measles, mumps, rubella or yellow fever diseases as a consequence of administration of the vaccine. conclusions: in this cohort of pediatric patients with dgs, yfvand other live vaccines were well tolerated, and no severe adverse events were reported, suggesting that widespread contraindication of yfv may endanger unvaccinated patients with less severe phenotype living in endemic areas. immunological evaluation and individualized decisions are always recommended, and further studies are needed to assess the safety of the yfv in this pediatric population. abstract/case report text introduction: lad-i is a rare inherited disorder of leukocyte (primarily neutrophil) adhesion to endothelial cell surfaces, migration, and chemotaxis resulting from itgb2 gene mutations encoding for the β2-integrin component, cd18. severe lad-i (i.e., cd18 expression on < 2% of neutrophils) is characterized by recurrent serious infections, impaired wound healing, and childhood mortality. although allogeneic hematopoietic stem cell transplant (allohsct) is potentially curative, its utilization and efficacy are limited by hla-matched donor availability and risk of graftversus-host disease (gvhd). rp-l201-0318 (clinical trials.gov # nct03812263) is a phase 1/2 open-label clinical trial evaluating the safety and efficacy of autologous cd34+ cells transduced with a lentiviral vector (lv) carrying the itgb2 gene encoding for cd18 (chim-cd18-wpre) in severe lad-i. methods: pediatric patients ≥ 3 months old with severe lad-i (demonstrated by cd18 expression on < 2% neutrophils and at least one prior significant bacterial or fungal infection) are eligible. peripheral blood (pb) hematopoietic stem cells are collected via apheresis after mobilization with granulocyte-colony stimulating factor (g-csf) and plerixafor. cd34+ hspcs are selected, transduced with chim-cd18-wpre lv, and cryopreserved. myeloablative conditioning with busulfan (therapeutic drug monitoring (tdm) dosing with adjustments to enable target area under the curve (auc)) is administered over 4 days, followed by infusion of the thawed investigational drug product (rp-l201). patients are followed for safety assessments including replication competent lentivirus (rcl) and insertion site analysis (isa), and for efficacysurvival to age 2 (24 months) and at least 1-year post-infusion without allohsct, increase in neutrophil cd18 expression, pb vector copy number (vcn), decrease in infections and/or hospitalizations, and resolution of skin or periodontal abnormalities. results: an initial lad-i patient (age 9 years) with recurrent severe infections and documented itgb2 mutations has been treated as of november 2019. baseline cd18, cd11a, and cd11b expression were < 1%. mobilization and apheresis procedures were performed successfully and busulfan conditioning was administered at the target auc. investigational product was comprised of 4.2x10e6 cd34+ cells/kg with vcn of 3.8 copies/cell (liquid culture), and was infused without complications. no serious treatment-emergent adverse events were reported. neutrophil engraftment (3 consecutive days of anc ≥ 500) was observed 18 days post-infusion. pb pmn cd18 expression 3 months posttreatment was 44.9% with comparable cd11a and cd11b expression levels; pb cd15 (myeloid) vcn at 2.5 months was 1.5. safety and efficacy data 6 months post-treatment will be available at the time of presentation, in addition to preliminary data regarding a potential additional patient. conclusion: preliminary evidence demonstrates that rp-l201 enables itgb2 genetic correction with robust cd18/cd11 neutrophil expression in this frequently fatal primary immunodeficiency. abstract/case report text introduction: the complement system plays an integral role in the innate immune system and links innate and adaptive immunity. complement deficiencies, hereditary or acquired, are rare. acquired deficiencies are more prevalent, occurring in nephrotic syndrome, reduced hepatic synthesis or transiently in sepsis/viremia. they are also seen in the presence of autoantibodies known to cause depletion of complement factors, such as c3 nephritic factor (c3nef). c3 deficiency is associated with infection susceptibility, particularly to encapsulated bacteria, and immune complex disease. case description: a 47 year old male was evaluated for recurrent infection. in childhood, he had recurrent sinusitis, otitis media requiring tympanostomy tube placement and persistent pharyngitis despite tonsillectomy. as a teenager, he developed glomerulonephritis, progressing to end stage renal disease and requiring transplant at age 22. the kidney allograft failed 4 years later, with biopsy demonstrating recurrent glomerulonephritis. the patient was transitioned to peritoneal dialysis and later hemodialysis, due to recurrent pd-related infections. his adult course was complicated by recurrent methicillin sensitive staphylococcal aureus (mssa) catheter and soft tissue infections (cellulitis and abscess), sinusitis, sepsis (streptococcal, mssa and tularemia), multifocal pneumonia and a left below knee amputation for osteomyelitis that required revision surgery. patient reported other autoimmune phenomena including a presumptive diagnosis of vasculitis and possible lupus-like syndrome. the constellation of recurrent infections and autoimmune features was most concerning for an early complement deficiency. prior work up was notable for low c3, ch50 and ah50 with normal c4, factor h and factor i. extensive laboratory work up revealed normal c1q, c4 level and function, serum immunoglobulins, vaccine titers, factor b and factor d levels. atypical hus (ahus) panel revealed a heterozygous silent variant in exon 17 of cfh and a heterozygous polymorphism within an intron in mcp/cd46, seen with increased prevalence in the patient population with ahus. wes was notable for a variant of uncertain significance in the vcl gene only. c3 level and function were markedly decreased, alongside low ch50 and ah50. both sc5b-9 level and c3 nephritic factor were elevated. a diagnosis of acquired c3 deficiency due to c3nef was made and patient was started on bactrim prophylaxis. he has remained free of serious infection since starting antibiotic prophylaxis. discussion: c3nef stabilizes the alternative pathway c3 convertase, c3bbb, increasing its half-life and blocking dissociation. this leads to unregulated consumption of c3 with subsequent deficiency. c3nef has been associated with c3 glomerulopathy, infection and partial lipodystrophy. however, there is marked heterogeneity in clinical phenotypes with reported asymptomatic individuals. our patient's glomerulonephritis likely represents c3 glomerulopathy. case reports and series of successful treatment of c3 glomerulopathy with rituximab and eculizumab have not commented on immune outcomes beyond the kidney. other potential therapeutic strategies include plasma cell depletion with either bortezomib or daratumumab. further study is needed to evaluate these therapies influence on both reversal of c3 depletion and overall impact on immune function in the setting of c3nef. abstract/case report text introduction: patients with heterozygous signal transducer and activator of transcription 1 (stat1) gain of function (gof) pathogenic variants exhibit an array of phenotypes including susceptibility to viral, bacterial, fungal and mycobacterial infections, autoimmunity, and cancer predisposition. progressive disseminated histoplasmosis (pdh) is well-described to affect infants. however, no reports have evaluated underlying monogenic immune dysregulation in previously healthy infants presenting with pdh. we report an infant who presented with pdh and associated hemophagocytic lymphohistiocytosis (hlh) leading to the diagnosis of a heterozygous stat1 gof mutation. case report: a previously healthy 9-month old male presented with persistent fever, pancytopenia, transaminitis, elevated ferritin, hepatosplenomegaly and coagulopathy. his clinical and laboratory evaluations were concerning for hlh syndrome. he had no prior history of immune hyperactivation or atypical infections. secondary causes of hlh were investigated, and patient was diagnosed with pdh based on marked histoplasma antigenemia. targeted genetic testing did not reveal a genetic etiology of familial hlh. he was successfully treated with a pulse and taper of dexamethasone as well as liposomal amphotericin b with transition to itraconazole. immunologic evaluation at the time of initial presentation demonstrated increased mean channel fluorescence for both perforin and granzyme noted in his nk cells. his nk function was decreased; however, he had a normal cd107a degranulation assay. his b-cell panel demonstrated low non-switched memory b-cells, low switched memory b-cells and low total memory b-cells. given his extreme immune activation with histoplasmosis, abnormal immunologic testing, and persistent lymphopenia despite resolution of his infection, a primary immunodeficiency next generation sequencing panel was sent. the results demonstrated a pathogenic variant in stat1 (c.800c>t; p.ala267val). this single nucleotide variant has been previously shown to be pathogenic (clinvar). abstract/case report text introduction: cytotoxic t lymphocyte antigen-4 (ctla-4) is known to have an important role as a negative regulator of immune responses, participating in the control of regulatory t cells and effector t cells. in mice its absence is associated with fatal autoimmunity and several ctla-4 mutations, leading to low or absent ctla-4 expression, have been shown in humans to be associated with a phenotype that includes hypogammaglobulinemia (with recurrent respiratory infections) and several manifestations of autoimmunity (enteropathy, granulomatous lymphocytic interstitial lung disease, organ infiltration, splenomegaly, autoimmune cytopenias, lymphadenopathy, amongst others), in an autosomal dominant mode of transmission. one of the published mutations, c.c257t, that results in an alanine to valine substitution (p.a86v), with a highly conserved alanine at that position, had a cadd score of 24 and was associated with the phenotype above, and was shown to be associated with a low expression of ctla-4 on regulatory t cells and with low ctla-4 function (reduction of ctla-4-mediated transendocytosis). methods: after irb approval, we searched for ctla-4 mutations present in the biome biobank· biorepository, containing whole exome sequencing data on 30845 patients, with data obtained using illumina· v4 hiseq 2500 sequencing platform. sifting through all the ctla4 mutations in the data, we identified four patients with the c.c257t mutation described above. extensive chart review of the four patients was performed. results: four patients were found with the ctla-4 c.c257t mutation. none of them had any of the described phenotypical characteristics of ctla-4 deficiency. patient 1 is a 68-year-old male with history of coronary artery disease, atrial fibrillation, stroke, hypertension, brain aneurysm, chronic kidney disease, gout and depression. patient 2 is a 29-year-old female with history of morbid obesity. patient 3 is a 51-year-old female with history of hypertension, obesity, pre-diabetes, dyslipidemia and iron deficiency anemia. patient 4 is a 70-year-old female with history of peripheral artery disease, hypertension, dyslipidemia, chronic kidney disease and lung cancer. conclusion: prior literature has attempted to characterize the clinical penetrance of ctla-4 mutations, suggesting it to be around 67%, with that number applying to 45 different mutations in 133 ctla-4 mutation carriers. we screened a large biorepository of more than 30 thousand patients for ctla-4 patients and identified four patients that carry one of the best described ctla-4 mutations, previously validated from a functional standpoint and associated with a severe phenotype. none of the four patients demonstrated any of the previously described phenotypical characteristics, and all four have ages above the median age of onset of 11 years. with the increasing use and broad population application of genetic studies, it is crucial to define the value of identifying presumed pathogenic variants in the absence of the adequate phenotype, with all the prognostic, therapeutic and ethical considerations it may imply. prior case reports of pil patients with b-cell malignancies have discussed treatment regimens with chemotherapy, radiation, and/or surgery, but neither the use nor the outcomes of allogeneic hematopoietic stem cell transplantation (hsct) in the management of recurring b-cell malignancies have been readily reported. case description: a 21-year-old man with pil and an accompanying history of lymphopenia, hypoproteinemia, hypoalbuminemia, and hypogammaglobulinemia was diagnosed with diffuse large b-cell lymphoma (dlbcl) of the liver following a preceding history of burkitt lymphoma of the ileum at 6 years of age and dlbcl of the liver at 16 years of age, in which each malignancy was genetically distinct. in addition, the patient had a history of benign nodular adenomatoid hyperplasia of the thyroid at 17 years of age that required a hemi-thyroidectomy. treatment considerations for the patient included chimeric antigen receptor t-cell therapy, autologous hsct, and allogeneic hsct, in which allogeneic hsct was ultimately pursued. prior to hsct, the patient was lymphopenic (670 cells/microliter) with significant t-cell lymphopenia (290 cells/microliter) and an increased proportion of memory t-cells (67% of his cd4+ t cells were cd45ro+), as well as hypogammaglobulinemic (igg 296 mg/dl; iga 47 mg/dl; igm 62 mg/dl). immediately following treatment of his dlbcl with rituximab, ifosfamide, carboplatin, and etoposide, the patient underwent a matched-related sibling donor hsct with a preparative regimen of busulfan, thiotepa, and fludarabine. now 18 months status-post hsct, the patient has maintained full-donor chimerism and has no evidence of graft-versus-host disease or malignancy. as expected, hsct has not corrected abnormalities in certain parameters associated with his pil, as he continues to display significant hypoproteinemia, hypoalbuminemia, and hypogammaglobulinemia, but he has an improved lymphocyte count (1,530 cells/microliter). discussion: there is no definitive or curative treatment for pil; furthermore, the genetic etiology of pil remains unknown. supportive regimens to help mitigate or offset manifestations of pil exist, such as adherence to a low-fat diet with medium-chain triglyceride supplementation, but there are no therapies available to prevent or reduce the risk of developing bcell malignancies in this patient population. although previous case reports have detailed successful treatment of b-cell malignancies in pil patients with chemotherapy, radiation, and/or surgery, there are no published consensus guidelines regarding management of b-cell malignancies in the setting of pil, especially if recurrent in nature. for non-pil patients with chemotherapy-refractory disease, or recurrent disease following autologous hsct, allogeneic hsct is a potentially curative option. herein, we describe a pil patient with a history of multiple b-cell malignancies who underwent a successful allogeneic hsct, indicating that allogeneic hsct may be an effective treatment option for similarly affected patients. abstract/case report text introduction diarrhea in young infants is common and generally self-limited. in persistent cases, the differential diagnosis is broad and includes infections, food protein-induced allergic proctocolitis, congenital diarrheas and enteropathies. in addition to monogenic inflammatory bowel diseases, many cellular, humoral, and combined immunodeficiencies should be considered, including but not limited to cvid, ipex and ipex-like phenotypes, lad, dyskeratosis congenita, intestinal lymphangiectasia, omenn syndrome, cartilage hair hypoplasia, cgd, il-10 axis defects, aid deficiency and wiskott-aldrich syndrome. case presentation a full term infant born after an uncomplicated pregnancy to nonconsanguineous honduran parents presented with non-bloody, non-bilious vomiting and dehydration at 21 days of life. the infant later developed frequent loose stools, some of which were bloody, and failure to thrive. his family and prior medical history, including newborn screen, were normal. an extensive workup was initiated which showed: -persistent and severe anemia with a hemoglobin nadir of 3.5 mg/dl -hypoalbuminemia requiring multiple infusions -elevated alpha-1-antitrypsin and calprotectin level in stool -profound hypogammaglobulinemia with normal iga, igm and ige for age -normal gross and histologic findings on esophagogastroduodenoscopies and colonoscopies besides a gastric ulcer thought not be the cause of his anemia -normal abdominal imaging including ultrasound, ct angiography and mri -no source of bleeding on meckel scan or exploratory laparotomy -normal dhr assay, g6pd level and positive myeloperoxidase stain -immunophenotyping showing t cell lymphocytosis affecting cd4+ more than cd8+ compartment, with normal lymphocyte proliferation to mitogens -normal sweat chloride level genetic testing was initiated with a targeted immunodeficiency panel which showed variants of unknown significance in adar, dock8, lyst, ptprc and tbx1 genes, none of which adequately explained his presentation. whole exome sequencing showed that he was a compound heterozygote in the dgat1 gene. a pathogenic variant c.751+2t>c (ivs8 + 2t>c) was inherited from the father and a likely pathogenic variant c.1073g>c (p.r358p) was inherited from the mother. patient was diagnosed with dgat1 deficiency, an inborn error of lipid metabolism resulting in protein-losing enteropathy (ple). under gastroenterology's guidance, a low fat diet was initiated, resulting in rapid improvement in stool consistency, weight gain, albumin level and stool alpha-1-antitrypsin level. he remains on subcutaneous immunoglobulin replacement therapy for ongoing hypogammaglobulinemia. conclusion: protein-losing enteropathies commonly present with intractable diarrhea and significant laboratory derangements due to malabsorption including hypogammaglobulinemia. as a result of these findings and since many of the etiologies are immunologic in origin, immunologists are an integral part of the evaluation of such cases. in cases where immune system interrogation reveal normal results, genetic testing is crucial in guiding the diagnosis. in our case, whole exome sequencing not only provided the diagnosis but also characterized a variant that was previously of unknown significance as likely pathogenic. dietary management provided rapid improvement in growth and nutritional status. ongoing monitoring will reveal if this management also assists in igg level maintenance and hematologic abnormalities or if even more stringent control of dietary fat will be required abstract/case report text background: foxp3 gene mutations are associated with immune dysregulation polyendocrinopathy x-linked (ipex) syndrome, a rare xlinked monogenic disease of immune dysregulation and autoimmunity. the classic presentation consists of severe enteropathy, dermatitis, and endocrinopathies (commonly early onset insulin dependent diabetes mellitus). clinical presentation and severity can be variable even in family members with the identical variant. we present a patient with ipex symptomatology and a hemizygous variant in the polyadenylation (polya) signal of foxp3 that is classified as a variant of uncertain significance (vus). this specific variant was reported in a single case study in which the patient improved after hematopoietic stem cell transplantation (hsct). case presentation: a 2 month-old ex 34-week gestation boy was admitted with lethargy, hypovolemia, electrolyte disturbances, and acute kidney injury. he developed persistent diarrhea and vomiting after receiving rotavirus vaccine. his family history is significant for early deaths of three maternal unclesone stillborn, one death at 6 months and another at 2 years from unknown gastrointestinal problems. he demonstrated peripheral eosinophilia (to 4.26 k/ul), elevated ige, and anemia requiring multiple transfusions. he developed severe enteropathy with hypoproteinemia requiring total parenteral nutrition, continual albumin infusions and maintenance of npo. he had generalized edema, respiratory distress requiring high flow nasal cannula, and repeatedly spiked fevers with negative infectious evaluation. acute kidney injury improved but renal ultrasound showed persistent nephrocalcinosis. endoscopy yielded biopsies demonstrating duodenitis with severe villous atrophy, scanty isolated intraepithelial eosinophils and neutrophils, a few crypts with mucin, reactive epithelial changes and increased lamina propria eosinophils. colon biopsies showed mucosa with focally increased lamina propria eosinophils with scanty neutrophils and surface epithelium without cryptitis. esophagitis with reactive epithelial changes, spongiosis, and many intraepithelial eosinophils was also present. the patient's lymphocytes showed unremarkable proliferation to pha and pwm. cd4+ cd25+ t cells demonstrated intracellular foxp3 expression by flow cytometry. a commercially-available immunodeficiency targeted panel revealed that he was hemizygous for a vus in foxp3 (exon 12, c.*878a>g non coding). this variant is also referred to as an aauaaa>aauaag or aataaa>aataag change in the polya site. he was also heterozygous for vus at these additional loci: cd79a c.341c>t abstract/case report text introduction: implantation of allogeneic cultured thymus, partially depleted ex vivo of t cells, can result in naïve t cell development in patients with complete digeorge syndrome (dgs). in a few patients, early and transient skin rash, often characterized as "atypical dgs" or late autoimmune manifestations have been reported following implantation. here we describe a patient with complete dgs who developed immune reconstitution inflammatory syndrome (iris) or atypical dgs following thymus implantation. case description: a female patient was diagnosed at birth with complete dgs due to absent t cell receptor excision circles (trec), hypoplastic thymus, profound hypocalcemia with hypoparathyroidism and cardiac defects. the patient also had microretrognathia, oral motor dysfunction, sialorrhea, recurrent aspirations and reflux requiring a gastro-jejunum feeding tube, low-set ears with right ear microotia, semicircular canals atresia, alopecia and mal-rotated kidneys. prior to thymus implantation, the patient was thriving, had no skin rash, no eosinophilia and no t cells. detailed genetic analyses, did not reveal a cause for her syndrome. at 9 months of age pulmonary aspergillosis was diagnosed presumptively. at 10 months of age the patient received an allogeneic t-cell depleted thymus implant from a male donor, without prior conditioning or post-implantation immune suppressive medications. the procedure was uneventful and the patient returned home after 7 days. results: four months after implantation, a pruritic maculopapular rash appeared on the head and trunk that spread to the extremities including the palms and soles. there was no lymphadenopathy or splenomegaly. an infectious etiology could not be found. eosinophilia and an increase in liver enzymes were noted. there was an increase of cd4+ and cd8+ t cells with predominantly memory phenotype, which had been undetectable 1 month earlier. analysis of t cell diversity showed a restricted repertoire with expansion of two v-beta families. there was no evidence of donor cells to suggest graft versus host disease. skin biopsy showed minimal superficial perivascular inflammatory infiltrate composed mainly of cd163+ histiocytes and rare cd3+ t cells. the patient was treated with prednisone and cyclosporine. a liver biopsy was performed 3 weeks after initiation of treatment that showed moderate and diffuse peri-portal ductular reaction but no duct associated lymphocytic infiltrate or significant duct epithelial injury or ductopenia. the skin rash rapidly resolved with desquamation, while the liver enzyme abnormalities persisted for two more months. cyclosporine and prednisone were weaned over 2 months. t cell numbers, their response to stimulation and diversity have since normalized, as well as trec and naïve t cell production. the patient is producing appropriate antibodies to protein and polysaccharide vaccines. sixteen months after implantation the patient developed grave's disease with markedly elevated free-t4, undetectable tsh and elevated antibodies to the thyroid receptor, which rapidly normalized with ongoing methimazole treatment. the patient is currently 30 months after the implantation and is free of infections, thriving and developing appropriately. conclusions: this patient developed atypical dgs or iris, often associated with autologous and allogeneic hematopoietic stem cell transplants, organ transplants or effective treatment of hiv, after successful thymus implantation for complete dgs. abstract/case report text congenital disorders of glycosylation are a rare group of genetic disorders due to defects in protein glycosylation. phosphoglucomutase 3 (pgm3) is an enzyme necessary for the synthesis of uridine diphosphate n-acetylglucosamine, an important precursor for protein glycosylation. patients with autosomal recessive pgm3 deficiency have a multisystemic disorder characterized by a neurologic impairment and clinical features classically observed in autosomal dominant hyper-ige syndrome due to stat3 mutations; including recurrent pneumonias, skin abscesses, elevated levels of ige, and abnormalities in connective tissues and bones. we hypothesized that gp130, a highly glycosylated protein and coreceptor of the cytokine il-6, would be weakly expressed on pgm3deficient cells, due to impaired glycosylation. we studied 6 pgm3-deficient patients from 3 kindreds and showed that il-6-driven stat3 phosphorylation was impaired in their pbmcs and ebv-transformed b cells. accordingly, the induction of socs3 target gene was significantly decreased. in contrast, the patients had normal stat3 phosphorylation and socs3 induction downstream of il-10, a cytokine whose signaling is independent of gp130. flow cytometry and immunoblotting showed significantly lower gp130 expression in peripheral t-cells and ebv-transformed b cells from pgm3-deficient patients compared to healthy donors. we did also show that in vitro inhibition of n-glycosylation, using tunicamycin in ebv-transformed b cell line from healthy donor, alters gp130-mediated signaling. collectively, our findings demonstrate that defective glycosylation in pgm3-deficient patients results in reduced expression of gp130 and consequently, impaired gp130 dependent stat3 phosphorylation and defective il-6 signaling. this may account for the overlapping clinical features shared by pgm3 and stat3 deficient patients. abstract/case report text introduction: there are no known effective therapeutic modalities for patients hospitalized with moderate to severe acute viral respiratory infections, and treatment is primarily supportive. intravenous immunoglobulin (ivig) has been reported in limited cases to be used in this setting, especially in immunocompromised patients. the primary objective of this retrospective study is to compare clinical and economic outcomes among immunocompromised patients hospitalized with viral respiratory infections who received ivig to those who did not receive ivig at a large academic center hospital. methods: we performed a double-center, retrospective cohort study of all immunocompromised patients who were hospitalized for acute documented respiratory viral infections between 2011 and 2016. we divided patients into two groups: those who received ivig therapy for respiratory infections, and those who did not receive ivig therapy. data on age, gender, immune status, viral type, immunosuppression type, respiratory support, microbiological data, length of hospital stay (los), icu los, as well as death and readmission rates were extracted from medical records. in order to adjust for severity bias typically present in observational data such as these, we employed inverse probability weighting (ipw) using all collected baseline covariates. outcomes (death, length of stay in hospital and icu, readmission) were examined using a series of logistic and poisson regression models adjusting for baseline covariates and employing ipw. results: a total of 282 individual hospital admissions were analyzed; 99 patients received ivig and 183 did not receive ivig. there were no significant differences between the two groups in terms of mean age, gender . average age was 40.3, 50% were female, 74.5% were transplant patients of which 26.6% had lung transplant, 26.6% had liver transplant, 23.1% had bone marrow transplants (bmt), 8.5% had kidney transplant, 7.8% had heart transplant and 4.3% had both solid organ and bmt. 32.3% of patients had a hematologic malignancy, and 2.5% had a primary immunodeficiency. the most common isolated respiratory virus w a s r h i n o v i r u s ( 5 1 . 4 % ) , f o l l o w e d b y r s v ( 2 5 . 9 % ) , parainfluenza (11.4%) and metapneumovirus (10.6%). overall, the use of ivig as associated with a significantly shorter icu length-of-stay, with an (or=-2.46, p=0.001), and a higher hospital readmission rate. in the sub-analysis of patients who received ivig within the first 48 hours of hospitalization (n=39), ivig use was associated with a significantly shorter icu los (or=-6.01, p=0.0), significantly shorter overall hospital los (or=-4.341, p=0.008), and no significant change in readmission rate. conclusions: to our knowledge, this is the first retrospective cohort analysis evaluating the effect of ivig in immunocompromised patients hospitalized with respiratory viral infections. the results suggest that immunocompromised patients receiving ivig may have a shorter hospital and icu los, especially if ivig is provided within the first 48 hours of admission. this may result in reduced healthcare costs. this study is limited by its retrospective nature, and the potential bias that patients treated with ivig are sicker to start with. future prospective studies are suggested to further evaluate these findings. (1, 2) . the most common precipitant in children is medication, followed by infection (2, 3) . although a clear association between mycoplasma pneumoniae and sjs has been established, there is a scarcity of literature exploring the role of this infection in recurrent sjs in children (2) (3) (4) . case presentation: a 15-year-old female with prior history of sjs was admitted for mucosal and skin lesions in the setting of community acquired pneumonia. her past medical history included sjs with eye involvement, secondary to mycoplasma pneumoniae (ig m positive), occurring five years prior to this admission. she also had frequent episodes of acute otitis media and sinusitis in early childhood. family history was negative for immunodeficiency. her clinical presentation included respiratory symptoms and fever for 8 days treated with ceftriaxone, followed by cefdinir and levofloxacin. her fever improved the day prior to admission, but she developed conjunctival injection, ocular pain, and ulcerative lesions in her mouth and nares. on physical examination, she had low grade fever with mucosal lesions including conjunctival erythema with serous discharge, painful blisters and denudated skin in lips, perioral area, nares, tongue and oropharynx. initial testing included negative blood hsv pcr, blood culture, rapid antigen testing for group a streptococcus and influenza a/b, and elevated crp in 4.4mg/dl and esr 55mm/hr. right lower lobe p n e u m o n i a w a s c o n f i r m e d w i t h a c h e s t r a d i o g r a p h . nasopharyngeal pcr and serum igm were positive for mycoplasma pneumoniae. she had a mildly elevated anticardiolipin igm (17 mpl), a mildly decreased c3 (75 mg/dl) and a negative ana. she was diagnosed with recurrent sjs secondary to mycoplasma pneumonia infection. she completed treatment with levofloxacin for mycoplasma pneumonia, and received cyclosporine and high-dose methylprednisolone. she had bilateral amniotic membrane transplantation to prevent corneal ulceration . she was discharged after clinical improvement, and recurrent oral lesions were noted at followup. immunological work up as an outpatient revealed normal serum immunoglobulins, normal lymphocyte subsets and low pneumococcal titers with adequate response post-vaccination. sjs secondary to mycoplasma pneumonia infection has predominance of mucosal involvement over rash, which was observed in our patient (1, 2) . some case series reported a recurrence of sjs up to 18% within a 7-year follow up. almost half of patients with recurrent sjs developed multiple sequelae (2, 3) . early diagnosis of sjs, especially in those with prior history of sjs, helps to provide appropriate supportive care, monitoring of complications and treatment of possible superinfections (5, 6) . conclusions: there is limited information in the literature regarding the role of mycoplasma pneumoniae associated recurrent sjs in children. it is possible that these episodes are triggered by and/or immune predisposition. there is ongoing discussion regarding whether these clinical presentation should be labeled sjs secondary to mycoplasma pneumonia infection or, depending of the skin involvement, m. pneumonia-associated mucositis (mpam) and m. pneumonia-induced rash and mucositis (mirm) (5,6,7). mycoplasma should be treated appropriately in patients with recurrent sjs. abstract/case report text background: growing access to genetic testing has facilitated the genetic evaluation of primary immunodeficiencies but has also greatly increased the number of variants of uncertain significance (vus) encountered in clinical practice. interpreting the significance of vus requires multiple lines of evidence. w e d e s c r i b e a n e u t r o p e n i c i n d e x p a t i e n t w i t h hypogammaglobulinemia, unusual hpv susceptibility, and dual heterozygous pathogenic loss-of-function nfkb1 and heterozygous missense cxcr4 vus. family analysis showed the nfkb1 variant was inherited from his mother, while the novel cxcr4 variant was present in his father and sister. all four patients presented with recurrent infections, warts, and hypogammaglobulinemia. (figure 1 ) the nf-κb1 gene encodes p50/p105 transcription factor of the canonical nf-κb pathway, the most common autosomal dominant monogenic cause of common variable immunodeficiency (cvid). cxcr4 is a g-protein-coupled chemokine receptor with cxcl12 as cognate ligand. autosomal dominant pathogenic gain-offunction cxcr4 variants lead to impaired receptor downregulation and retention of neutrophils and other leukocytes in the bone marrow defining whim (warts, hypogammaglobinemia, infections, and myelokathexis) syndrome. all cxcr4 pathogenic variants truncate the carboxyl-tail of the cxcr4 receptor, a region responsible for receptor internalization, with the exception of one missense non-truncating variant p.e343k. case series: the proband (p1) is a 19-year-old male with history of recurrent bacterial respiratory tract infections, warts, moderate neutropenia, thrombocytopenia and hypogammaglobulinemia requiring immunoglobulin replacement therapy (igrt). bone marrow biopsy didn't show myelokathexis. next-generation panel sequencing identified a novel heterozygous missense cxcr4 (c.1022c>a, p.s341y) vus. the serine residue is highly conserved up to zebrafish. this variant was present in heterozygous form in two cases in gnomad database (277,984 alleles). additional whole-exome sequencing revealed a heterozygous pathogenic nfkb1 variant (c.980dup, pa328sfs*12) located in the nterminal rel homology domain, consistent with nfkb1 loss-offunction. both, the patient's sister (p2) and their father (p3), carry the heterozygous cxcr4 vus but not the pathogenic nfkb1 variant, and have history of warts, hypogammaglobinemia, and recurrent infections. the hpv susceptibility is particularly striking in p3 manifesting by genital warts and hpv-positive oropharyngeal cancer. bone marrow evaluation didn't identify myelokathexis in p2 (p3 is pending). the mother of the index case (p4) has cvid requiring igrt and immunomodulation. she shares the nfkb1 variant with p1 but is negative for the cxcr4 vus. extensive t and b cell phenotyping revealed low class-switched memory b cell count (0-7 counts/ul) in all subjects, and loss of transitional and mature naïve b cells in p1 and p4 with nfkb1 variant. proband b cells showed the highest tendency for apoptosis (35-55%) within the family. we describe members of a family with similar presentation (infections, hypogammaglobinemia, warts), however variable combination of nfkb1 and cxcr4 variants, where either genetic defect or their combination could explain the clinical phenotype. biochemical consequence of our novel cxcr4 variant is pending. as the proband showed the most severe immune phenotype and neutropenia, we hypothesize that cxcr4 has a synergistic effect on nkfb1 loss-of-function. the contribution of cxcr4 vus of the clinical phenotype of the two other family members is yet to be determined. background: the yield of diagnosis by exome sequencing for some primary immunodeficiencies (pid) has been less than the typical diagnostic rate for clinical exome analysis (~30-35%). the relatively low diagnostic rates for certain subtypes of the pids may be attributed to variable expressivity and/or an incomplete understanding of the genetic basis, among others. additionally the extent of multiple diagnoses and phenotypyic expansion in pid is not well explored. cohorts with highresolution clinical and genetic data are instrumental for exploring these questions. we evaluated the use of human phenotype ontology (hpo)annotated datasets to systematically address the prevalence of these issues using a cohort of 1000 individuals with pid who participated in research exome sequencing at the niaid. results: we generated a phenotype dataset of 1000 individuals with pids by annotating the clinical features of these subjects obtained from electronic health records (ehr) with hpo terms. exome sequencing of these 1000 individuals identified 313 probands with a pathogenic or likely pathogenic (p/lp) variant in a gene associated with the respective clinical presentation. we identified 118 probands where the same gene harbored a p/lp variant in at least three unrelated individuals. we used the clinical and genetic data of 118 individuals in the following areas: 1) we identified p/lp variants in aire, pik3cd, nlrp3, fas, ctla4, gata2, cybb, stat1 and tnfrsf13b in at least ten patients that explained their clinical presentations. this dataset allowed us to characterize variable expressivity of diseases associated with these genes by capturing the variability in the observed hpo terms among probands with p/lp variants in the same gene. dimensional reduction of clinical features of probands allowed us to cluster patients sharing similar phenotypic profiles. we found clinical presentation of individuals with monoallelic p/ lp variants in aire were relatively less variable and clustered more compactly compared to that of individuals with gata2 variants. 2) the extent of multiple diagnoses in pid is not well explored. the benchmark cohort we developed allowed us to identify candidates for multiple diagnosis or phenotype expansion by comparing the phenotype profile of each patient expressed in hpo terms to the hpo terms typically observed for a given pid. for example, we identified gain-of-function pathogenic variant in pik3cd in a patient that explained the clinical features of the pid observed in this patient. however, the patient also displayed developmental delay, congenital hemiplegia, cerebral palsy and absent speech. these features are not known to be associated with pik3cd variants, making this individual a candidate for >1 genetic diagnoses. conclusions: we developed a benchmark dataset where clinical features of patients were described using hpo terms. this dataset allowed us to quantify variable expressivity for certain pid subtypes and to systematically identify potential candidates for multiple diagnosis or phenotypic expansion. abstract/case report text warts, hypogammaglobulinemia, recurrent infections and myelokathexis syndrome is a rare combined immunodeficiency due to autosomal dominant gain-of-function mutations of cxcr4 chemokine receptor. the late diagnosis of whim syndrome in two ukrainian adolescents highlights the diagnostic challenges in this disease. patient 1, 12 year-old girl, had recurrent pneumonia since the first year of age; overall she had 7 episodes of pneumonia. she has suffered from chronic bronchitis for last several years. she had recurrent otitis media and chronic pyelonephritis. neutropenia was revealed when she was 3 year old. during episodes of bacterial infections she occasionally had normal value of neutrophils. the girl does not receive any treatment. patient 2, 14 year-old boy, had three episodes of pneumonia when he was 2, 7 and 14 year old. others symptoms include recurrent herpetic infection, warts on the hands. since 2 years of age he has haven persistent low neutrophil counts. the child was followed by hematologist and since 5 years of age he has received g-csf (5 mg/kg) twice a month. both children have leukopenia 750 -1200 cells/mm3, neutropenia -100 -300 cells/mm3, lymphopenia 560-830 cells/mm3, low number of bcells -30-50 cells/mm3. hypogammaglobulinemia was not prominent in both children, they have slightly decreased level of igg (7,1 g/l), normal level of igm (1,16 -1,44 g/l), patient 1 has low level of iga 0,24 g/l. patient 1 does not have protective level of antibodies to diphtheria and tetanus anatoxin, and anti-hbs antibodies were absent despite complete immunization. bone marrow aspirate revealed hypercellular marrow with granulocytic hyperplasia which was characterized by hypersegmented nuclei and cytoplasmic vacuolization of neutrophils. on molecular analysis of cxcr4, heterozygous mutation c.1000c>t (p.arg334*), known as r334x mutation, was detected in both patients, confirming the diagnosis of whim syndrome. replacement therapy with intravenous immunoglobulin was started in both children together with antibacterial prophylaxis and g-scf. vaccination with 4-valent vaccine against hpv infection was recommended for both patients. whim syndrome is very rare immunodeficiency but may be underdiagnosed. the awareness about rare forms primary immunodeficiency is very important in clinical practice for early diagnosis and treatment. methods: clinical providers recruited from nicer institutions electively completed web-based survey questions related to provider characteristics as well as initial diagnostic evaluation of itp, aiha, ain and es via securequestionpro® software. likert scales ranging from 1 ("rarely" < 20%), 2 ("sometimes" 21 to 40%), 3 ("half the time" 41% to 60%), 4 ("frequently" 61 to 80%), and 5 ("almost always" 81 to 100%) were used to ascertain frequency of evaluation for each diagnostic study. statistical analysis and plotting was done using rv3.6.1. plots were created using packages ggplot2, v3.2.0 and ggiraphextra v0.2.9. mean likert scale scores were calculated for each study for each suspected disease and plotted on radar charts. results: the survey was completed by 93 providers, including hematology/oncology (48.6%), rheumatology (16.4%), allergy/ immunology (17.1%) and other sub-specialties (17.9%). a slight majority of physicians (51%) were fellows or within 5 years of graduation; physician extenders and clinical pharmacists were also respondents. the majority (62.4%) of respondents indicated that ≤ 50 new immune-mediated cytopenia patients were seen at their institution annually. the vast majority of respondents (90.4%) reported evaluating ≤ 25 new es patients per year at their institution with 60% evaluating ≤ 10 cases annually. collated data from all respondents showed that in all disease states, the primary evaluation was focused on peripheral destruction mechanisms; the majority of patients are only "sometimes" or "rarely" evaluated for bone marrow failure syndromes, connective tissue disease, immunodeficiency and non-malignant lymphoproliferative disorders, but when done were more likely in es ( figure 1 ). evaluations were biased by sub-specialty with higher degrees of connective tissue focus by rheumatology and immunodeficiencies by allergy/immunology (table 1) . genetic sequencing was "frequently" or "almost always" sent in 4.5% of itp, 7.0% of aiha, 6.2% of ain and 32.2% of es p a t i e n t s . p e r s o n a l o r f a m i l y h i s t o r y o f a u t o i m m u n e / hyperinflammatory disease, malignancy or cytopenias most strongly influenced the decision to send genetic testing. lack of insurance coverage/negative financial impact on the patient and concerns about the inability to resolve variants of uncertain significance were the biggest barriers for obtaining genetic testing. conclusions: current practices in the evaluation of immunecytopenias are heterogeneous by sub-specialty and globally limited in scope with few patients being evaluated for underlying etiologies. in particular, despite a known high frequency of pathogenic variants in es, less than a third of patients are undergoing sequencing, highlighting a need to reduce barriers to genetic testing. development of a consensus guideline with multi-disciplinary engagement to harmonize an optimal evaluation for patients with immune-mediated cytopenias is needed. interferon regulatory factor-2 (irf2) binding protein-2 (irf2bp2) was originally identified as a transcriptional co-repressor of irf2(1). mutated irf2bp2 was identified in a 3-member family with recurrent sinopulmonary infections, progressive hypogammaglobulinemia, and poor response to protein vaccines (2) . we have now identified 10 additional families (18 subjects) with irf2bp2 mutations. clinical histories show an expanded phenotype with 15/21 having chronic gastrointestinal disease; 7 with gastrointestinal manifestations as the initial clinical complaint. five had granulomata in liver(x2), spleen, lung(x2) and gastrointestinal tract. five out of six tested had poor pneumococcal vaccine responses and four patients reported viral infections including varicella zoster(x2), influenza a and sapovirus. irf2bp2 is a 589 amino acid protein containing a highly conserved cterminal protein-protein interaction ring domain (rd). constraint metrics from gnomad indicate mild tolerance to missense changes and intolerance to loss-of-function alleles. we identified 3 categories of mutations: rd mutation or deletion (n=9 patients), null alleles (n=3) and non-rd missense changes (n=9). functional studies assessing the ability to affect nfatdriven luciferase expression were performed. rd mutations (4/5) had more profound loss-of-repression than wild-type, while missense changes had lesser, but still measurable effects. further, mutation categories and functional studies correlated with clinical phenotypes. of 9 patients with rd mutations, 8/9 had infections as presenting symptoms, 6/6 tested had hypogammaglobulinemia and 7/9 were diagnosed with cvid. one patient with a missense rd mutation had only an infectious phenotype (pulmonary mycobacterium avium) with slight decrease in immunoglobulins; in functional studies this mutation had the least effect of the rd mutations. haploinsufficient patients reported respiratory infections (3/3), recurrent urinary tract infections (2/3), gastrointestinal disease (2/3) and hypogammaglobulinemia (3/3). in contrast, 8/9 patients with non-rd missense changes presented with gastrointestinal complaints while only 2 patients had infections (recurrent bronchitis, shingles). gi disease prevalence is consistent with high levels of irf2bp2 expression in the colonic crypt cells (human protein atlas). to confirm this, immunohistochemical staining of colon biopsies from two patients was performed, identifying epithelial and glandular cells of the colon. irf2bp2 is involved in multiple processes, including the negative regulation of nfat signaling(3), tcr signaling(4), inflammatory macrophages (5) , and pd-l1 transcription (6) . interaction with the glucocorticoid receptor affecting anti-inflammatory and metabolic transcription (7) has also been reported. these observations highlight the irf2bp2 response to type-i interferons (irf2) and tcr stimulation (nfat), regulation of inflammatory macrophages and co-regulation of glucocorticoid receptor mediated signaling. the expanding role of irf2bp2 in multiple biologic systems correlates with the broad clinical presentation we observed in our patients. further studies utilizing irf2bp2 mutation knock-in mice will help characterize the gastrointestinal, lung and immune pathology seen in our cohort. abstract/case report text common variable immunodeficiency (cvid) is a disorder of antibody deficiency arising from over 20 genetic lesions. the clinical presentation of patients with cvid varies from recurrent, severe infections to autoimmunity. the immune dysregulation in cvid is especially difficult to treat and the lifespans of patients suffering from autoimmunity are much shorter than those without such complications. unfortunately, we have no way to identify which patients fall into which categories, or even know how many sub-categories of cvid there are. therefore, the field requires a method to classify patients into categories to precisely recognize and aggressively treat the more severe phenotypes. we address this goal by integrating analyses of patient exomes with analyses of cellular signaling. by analyzing stimulation assays with phospho-protein mass cytometry and high-dimensional data analytics, we aimed to elucidate signaling and phenotyping deficiencies in patients with cvid. importantly, our panel identifies all circulating immune cell subsets in whole blood. in eosinophils, we found amplified responses of pp38, pstat3, and cleaved caspase-3 in response to tlr1/2 stimulation. we found additional amplified responses of pstat3 and pstat5 in cd16lo monocytes. this finding suggests a previously unidentified role for eosinophils and cd16lo monocytes to contribute to the pathophysiology of cvid. we found abormal numbers of memory b cell counts, total switched b cell counts, and igm+, cd38+ b cell (plasmablasts) counts between cvid patients and healthy controls. cd21 expression on b cells was significantly reduced in cvid patients as well. these b cell results mirror findings from prior, seminal studies on cvid. notably, we have found higher pd-1 expression in the effector cd8 t cells of patients. integrating phenotype data, genetic analysis, and mass cytometry data will provide a deeper understanding of each patient's phenotype and how the are clustered. we also expect that a better understanding of alterations in the exomes and functions of the circulating immune cells of cvid patients will lead to new therapeutic approaches. abstract/case report text objectives: primary atopic disorders are monogenic disorders leading to profoundly dysregulated allergic responses. studying patients with these disorders has been instrumental in expanding our understanding of the pathogenesis of allergic inflammation with therapeutic implications for common polygenic versions of allergic disease. clinical findings: we have identified a now 8-year old boy who presented with severe eczema, extremely high blood eosinophil counts (5.8x109 cells/l, normal range: 0-0.85x109 cells/l) after birth and very high serum ige levels (2645υg/l, normal range: 0-500ug/l) since birth. known allergic disorders and parasitic infections are ruled out. given the extreme phenotype, whole exome sequencing was performed on the trio of patient and parents, and the patient was found to have a homozygous mutation in the evolutionarily conserved fibronectin iii domain of the osmr gene (c.1307t>a, p.v436d) (figure 1 ). osmr encodes oncostatin m receptor-beta, a component of both the osm type ii receptor and the il31 receptor, and is important for keratinocyte cell proliferation, differentiation, apoptosis and inflammation. mutations in osmr have been reported in association with familial primary localized cutaneous amyloidosis, however this condition was ruled out in this patient through skin biopsy which showed no amyloid deposits. methods and results: we modelled the c.1307t>a osmr mutation in hek293 cells and observed a loss of expression of the osmr receptor on the cell surface (with normal intracellular protein levels). this observation was mirrored in primary fibroblasts obtained from the patient. signal transduction through phosphorylation of stat1 and stat5 and gene expression (il6 and ccl2 measured via qpcr) was absent after stimulation with osm in patient fibroblasts. these signaling defects were rescued using a lenti-viral transduction approach to introduce the wild-type (wt) osmr gene. whole transcriptome analysis using rna sequencing confirmed that osm mediated jak-stat signalling pathways were deficient in the patient fibroblasts and were rescued after lenti-viral transduction of wt osmr. rna sequencing analysis also suggested significantly enhanced expression of genes in the nf-κb signalling pathway (e.g.: il18 and cxcl1) and decreased expression of genes in the tgf-β signalling pathway (e.g.: smad6 and smad7) in patient fibroblasts at baseline. this was also rescued upon lentiviral transduction. conclusion and future directions: our findings shed light into the disease mechanism of a novel primary atopic disorder, caused by a homozygous missense mutation in osmr. abstract/case report text 32-year-old caucasian female presented to immunology clinic with hypereosinophilia, eosinophilic esophagitis, peptic ulcer disease, severe gi bleeds, and chronic hepatitis. healthy throughout childhood, with minimal infectious history. in adolescence developed chronic severe myalgias and nsaid overuse, to which the peptic ulcer disease and bleeding were attributed. parents healthy and non-consanguineous. son with severe bleeding episodes and small stature. on exam she weighed 82lb, bmi 16. sclerae anicteric. tongue deeply furrowed. cervical nodes palpable. heart and lung exam normal. no hepatosplenomegaly. no clubbing of the digits or edema. skin was clear. wbc 11,600/ul, eosinophils 4730/ul, hemoglobin 10g/dl, normal platelet count. however, platelet aggregation testing abnormal. bone marrow normocellular, and flow cytometric and molecular analysis did not show hematolymphoid malignancy, primary hypereosinophilic syndrome, or systemic mastocytosis. lymph node biopsies did not show lymphoma or aberrant t cell populations. noted to have chronically elevated creatine phosphokinase, ranging from 706-3164u/l over two years at our institution. deltoid muscle biopsy showed non-specific myelopathic changes. an adult dystrophy immunostaining panel was normal. ultrastructure examination showed no abnormal storage material. a genetic panel for metabolic myopathies failed to reveal a cause. total igg, iga and igm normal. ige elevated at 934ku/l, and igg subclasses showed igg4 elevated at 354mg/dl. flow cytometry showed normal t, b and natural killer cell numbers. normal proportions of naïve, mature and activated t cells. vaccine response assessment was normal. evaluation for autoimmune/rheumatologic diseases was negative. liver biopsy demonstrated findings consistent with primary or secondary sclerosing cholangitis (without increased igg4 staining). given her inflammatory phenotype, additional genetic analysis was sent, assessing for primary immunologic disorders. this identified heterozygous variants of uncertain significance in ctla4 (c.553t>a; ps185t), zap70 (c.981c>g; p.d327e), and stim1 (c.752t>c; p.l251s). analysis of the ctla4 variant in vitro revealed that it was expressed normally. foxp3 expressing regulatory t cells were present in normal proportions in vivo and appeared phenotypically normal. this variant was found in her unaffected father. the zap70 variant is present in population databases (rs201605654, exac 0.07%), and was felt unlikely to be clinically relevant. the stim1 l251s variant, although not shown previously in human patients, has been previously shown in vitro to be a gain of function mutation [1] [2] [3] . furthermore, familial analysis revealed that this was a de novo mutation arising in the patient, and present in her son. humans with other gain of function mutations in stim1 and the orai1 channel it activates have overlapping syndromes including storkmorken syndrome, tubular aggregate myopathy and york platelet syndrome, characterized by chronic myopathy and platelet aggregation defects [4] . the stim1 l251s mutation is predicted to cause constitutive stim1 activation and calcium influx and likely provides an explanation for the patient's chronic myopathy and abnormal platelet aggregation. neither eosinophilic disease, nor cholangitis, have been described previously in stim1 gain of function-related diseases. it is unclear whether these issues are related to this novel stim1 mutation, or to other genetic or environmental influences. treatment of diseases caused by overactive crac channels is challenging as no pharmacologic inhibitors are yet clinically available. nomid/cinca syndrome is one of the periodic syndromes associated with cryopyridines. it is a defect in the innate immune system causing excessive activation of the inflammasome, with consequent il-1 secretion and neutrophil recruitment. clinically, damage occurs to organs such as the skin (neutrophilic urticaria), central nervous system (meningitis and deafness) and joint (arthritis). levy et al. (2015) evaluated a large series of 136 patients and median onset age was 0.8 years, while the median age at diagnosis was 15 years, although the symptoms initiate in the first days of life. treatment includes corticosteroids, which act by nonspecifically blocking all inflammatory cytokines, or by blocking il-1 specifically. if early diagnosis and treatment of the disease is not made, natural evolution leads to motor and adaptive disability and death in 20% of cases already in adolescence due to infection, neurological complications or secondary amyloidosis. we report a 10-month-old male child from nonconsanguineous parents who presented shortly after birth, multiple scaling and erythematous lesions throughout the body, evolving with following symptoms: abdominal abscess, hepatitis, meningitis and pioarthritis. laboratory tests showed elevation of inflammatory tests (esr, crp, amyloid protein a) and leukocytosis. the diagnosis was suspected at the nursery where the patient remained hospitalized for 51 days. a personalized multigene panel was requested. it was identified the variant p.gly309val, heterozygous for nlrp3 gene, not described in the literature, confirming the diagnosis of cinca/nomid syndrome. after discharge, it was introduced prednisolone (1,5mg/kg/day) and antiinterleukin-1 (il-1). after the second dose, skin lesions and joint edema regressed, weight gain, and neuropsychomotor development improved. this case reports a very early diagnosis of nomid/cinca syndrome. it warns neonatologists and pediatricians about the need of precocious recognition of the syndrome, probably improving the prognosis of the patient. professor/university center health abc abstract/case report text background: leprosy affects more than 208,000 people worldwide. brazil represents the 3rd. country in the world in leprosy frequency and maranhão state is an hyperendemic region. the city of imperatriz (ma) stands out as a reference center in the care of these patients. according to few reports, lectin pathway of complement system may play a role in susceptibility to leprosy. mannose binding lectin (mbl) and ficolins (fcns) recognize patterns of sugars and acetylated residues (pamp), respectively, in a wide variety of pathogens, including m. leprae. high levels of ficolins and mbl may act unfavorably promoting the spread of m. leprae. the present study evaluated the role of ficolin 3 and mbl in m.leprae patients and contacts. methods: a cross-sectional case-control analytical study was carried out, evaluating clinical and epidemiological data and serum levels of mbl and fcn3 (elisa) from july 2018 to april 2019. the study was approved by ethics committee and informed consent forms were signed before sample collection. data analysis was performed using the spss 22.0 for windows statistics program. results: we evaluated 169 serum samples (90 patients and 79 healthy family contacts), 54.4% were female, 32% under 15 years old, 78% african-brazilian, 65% of the families had more than 3 contacts at home. clinical data showed multibacillary forms in 73.3%; dimorphic (51%) and virchowian clinical forms (22.2%), up to 03 affected nerves in 26 (64.4%) and more than 5 lesions in 38 (42.2%). it was observed that 32 (35.6%) had a reaction, being type 1 (75%) more predominant. disability grade 2 was found in 16 patients (17.8%). in children under 15 years, 62.8% were multibacillary, 48.5% dimorphic and 20% undetermined; 11 (31.4%) also had reactions, 90% type 1 reaction and degree of disability 2 in 11.4% of children with the disease. the evaluation of serum fcn3 and mbl levels for the patients (n = 90) and contacts (n = 79) were 363.36ng/ml and 365.81ng/ml, (p = 0.76), and 3035.91ng/ml (p) and 2744.66ng/ml (c) (p = 0.29), respectively. there were lower values of fcn3 in patients with type 1 reaction (sudden and intense inflammatory processes) versus no reaction (337.23 ng/ml vs 372.86 ng/ml) (p = 0.03) and in patients with disability grade 2 (severe sequelae) versus disability grade 1 (319.74 ng/ml vs 343.25 ng/ml) (p = 0.003). higher fcn3 values was observed in patients with no disability (383.82 ng/ml) (p = 002). mbl concentrations were higher for patients above 15 years in comparison with patients below that age (3482.69 ng/ml vs 2626.04 ng/ml)(p = 0.02)) and correlated with the occurrence of a multibacillary clinical form. conclusions: mbl and fcn3 levels were not different in the patients and contacts of m. leprae, nevertheless the presence of severe forms with sequelae (reaction type 1 and disability grade 2) were associated with lower levels of fcn3. in addition, it is possible that lower mbl levels could influence the higher frequency of multibacillary disease below 15 years old. abstract/case report text introduction: hyper ige syndrome (hies) is a primary immunodeficiency characterized by elevated ige levels. symptoms can range from severe eczema, recurrent skin infections or pneumonias, and typical dysmorphic facies. there have been wide non-immunologic presentations in patients with hies, including retained primary teeth, scoliosis, craniosynostosis, arterial aneurysms and joint hyperextensibility. an association between hies and autoimmune hemolytic anemia (aiha) has further been described in the literature. however, there have been no reported cases of hies in association with iron deficiency anemia and concurrent pica. we present a unique case of a patient with a history of eczema, recurrent skin infections and pica found to have hies and iron deficiency anemia. case presentation: a 5-year-old boy with a history of allergic rhinitis presented to the allergy & immunology clinic for evaluation of chronic eczema and recurrent skin infections. the patient had a history of multiple hospitalizations requiring intravenous antibiotics for cellulitis and superinfected eczema since he was an infant. symptoms were refractory to the use of multiple skin barrier ointments and oral antihistamines. his mother further noted that for the past two months prior to initial evaluation, he developed a fixation with eating crayons, baby powder and chewing on drywall. physical exam was notable for a dysmorphic face, broad based nose, pale nasal mucosa with ample clear discharge, high-arched palate and lower incisor supernumerary teeth. his skin was characterized by generalized dryness, lichenification and scaly desquamation with boils on extensor surfaces of knees and elbows. initial screening for hies via t-helper 17 functional assay was consistent with decreased expression of il-17. genetic testing revealed stat3 s614g missense pathogenic variant consistent with hies. cbc was also notable for decreased hemoglobin at 9.9 g/l and mcv of 69 fl. patient was diagnosed with concurrent hies and pica in the setting of iron deficiency anemia. iron supplementation was started and patient's pica improved. discussion and conclusion: our patient with hies had a peculiar initial presentation with the classic signs and symptoms of hies and pica. the diagnosis of hies can often be delayed due to the wide range of clinical presentations. to our knowledge, the association of hies with iron deficiency anemia and pica has been underreported in literature. screening for anemia should be considered when evaluating patients with hies in order to rule out comorbid iron deficiency anemia which can be easily treated with iron supplementation. abstract/case report text introduction: common variable immunodeficiency is a primary immunodeficiency with variable and diverse phenotypic presentations. the two main phenotypes include a group which primarily exhibits recurrent infections and a group with or without infections and primarily inflammatory and autoimmune complications. the latter, may lead to a delay in diagnosis and is associated with poorer outcomes and higher morbidity and mortality. (1) another group of patients present with t-cell defects, lung disease, autoimmunity, and infections and may be diagnosed as having cvid but instead can have mutations in lrba or pi3 kinase. this subset of patients has been referred to as "cvid-like" in the literature. (2) case presentation: patient is an 11 year old female who initially presented to an outside facility due to 2 days of fatigue, fever, and abdominal pain. upon presentation, she was found to have massive splenomegaly, hepatomegaly, and an abnormal chest x-ray showing mediastinal lymphadenopathy and pleural effusion. laboratory results demonstrated pancytopenia, hypogammaglobulinemia, and low b cells, t cells, and nk cells via flow cytometry. she was transferred to our institution for further work up. she did not have any prior history of recurrent infections, asthma/lung disease, or autoimmune conditions. initial ct of the chest was consistent with granulomatous lymphocytic interstitial lung disease. patient was diagnosed with common variable immunodeficiency with granulomatous lymphocytic interstitial lung disease and was treated initially with high dose ivig, corticosteroid taper, rituximab, and imuran. she had interval worsening of pft and lung disease as shown by ct scan. genetic panel for cvid and related conditions revealed 2 variants of unknown significance. one heterozygous mutation in blnk gene (c.616g>a) and one heterozygous mutation in lrba gene (c.3914g>a). she was started on infliximab with plans to repeat ct scan in 6 months. discussion: mutations in both blnk and lrba have been associated with primary immunodeficiency. mutations in blnk, which is located on chromosome 10, have been associated with autosomal recessive agammaglobulinemia. homozygous or compound heterozygous mutations in lrba on chromosome 4, can lead to lrba deficiency which encompasses a wide range of clinical presentations including hypogammaglobulinemia, autoimmune disease, inflammatory bowel disease, antibody deficiency, organomegaly, and recurrent infections. (3) without genetic testing, the clinical presentation can be difficult to distinguish from common variable immunodeficiency. the patient presented has clinical features that can be seen with mutations in both blnk and lrba, however she is heterozygous for both mutations. further analysis, including measurement of lrba protein expression, is needed to further define her underlying immunodeficiency so appropriate treatment can be administered. abstract/case report text a 5 month-old, previously healthy, unvaccinated male presented with one week of diarrhea and cough and was admitted for dehydration and hypoxemia. his mother and sister both had a history of incontinenti pigmenti (ip). on physical exam, he was alert, afebrile, with tachypnea and subcostal retractions. enterovirus/rhinovirus and parainfluenza 3 were detected, but he became progressively hypoxemic and eventually required intubation and high-frequency oscillatory ventilation. chest x-ray showed multifocal bilateral airspace opacities. empiric treatment for pjp with trimethoprim/ sulfamethoxazole and glucocorticoids was started. tracheal aspirate pcr confirmed p. jiroveci. hiv rna pcr was negative. ivig was started due to suspicion for primary immunodeficiency. although his respiratory status gradually improved, he subsequently developed multiple skin lesions. skin biopsy grew mycobacterium szulgai. m. szulgai osteomyelitis of the right fibula and the left nasal bone was also detected, indicating hematogenous spread of the infection. he was started on four-drug anti-mycobacterial therapy and interferon-gamma (actimmune) at doses ranging from 50 μg/m^2 three times weekly to 100 μg/m^2 qod. immune work-up revealed t-cell lymphopenia [cd3+/cd4+ 202/μl (1400-5,100/μl) and cd3+/cd8+ 222/μl (600-2,200/μl)] with an abnormally increased proportion of memory cd4 t-cells compared to naïve cells for age. b-cell numbers were normal, and nk cells were decreased [cd56+cd16+/cd3-17/μl (100-1000/μl)]. nk cell lytic function by k562 lysis was normal, whereas cd107a degranulation was decreased. the serum igm level was normal [93 mg/dl (31-103 mg/dl) whereas iga [124 mg/dl (8-83 mg/dl)] and igg [1020 mg/dl (165-781 mg/dl)] were elevated. mononuclear cell cytokine response to ligands for tlr2-tlr1, tlr2-tlr6, tlr3, tlr4, and tlr7-tlr8 was normal. dna sequencing revealed a novel nonsense mutation in exon 5 of the ikbkg (p.gln201ter (q201x) (cag>tag): c.601 c>t, confirming the diagnosis of nemo deficiency, which was suspected based on the infectious disease presentation and the maternal history of ip. the diagnosis was further supported by signs of ectodermal dysplasia of teeth that appeared starting at 10 months of age. he underwent hsct using bone marrow from a 10/10 matched unrelated donor after conditioning with atg, busulfan, fludarabine and rituximab. actimmune therapy was continued until 10 days prior to transplant. for gvhd prophylaxis, he received tacrolimus and low-dose methotrexate. he achieved full donor chimerism post-transplant and has had no significant gvhd. interesting features of this case include the prominence of ip in mother and sister, which is usually due to female heterozygosity for an ikbkg null allele. such null alleles when inherited by the male fetus are embryonic lethal. our patient's nonsense mutation would be expected to result in severely impaired ikbkg protein expression and function. however, the fact that he had was born at term and initially was healthy coupled with his preservation of normal tlr function suggests that his ikbkg allele is likely to be a hypomorphic mutation. studies are in progress using ebv-transformed b-cell lines from the patient to evaluate ikbkg expression and function. also of interest, our patient was able to tolerate relatively high doses of interferon-gamma therapy without inflammatory side effects or an adverse impact on engraftment or gvhd. abstract/case report text background: primary atopic disorders are caused by genetic mutations that skew the immune system towards severe allergic disease. germline gain-of-function (gof) mutations in jak1 are a newly described monogenic cause of severe atopy, with affected patients demonstrating profound eosinophilia and allergic inflammation. our initial report of this novel condition identified a dramatic clinical response to the combined jak1/2 inhibitor ruxolitinib. we aimed to determine the long-term clinical response to ruxolitinib in patients carrying a germline jak1 gof mutation, and to characterize the effect of enhanced jak1 signaling on t lymphocyte effector functions and hematopoiesis. methods: clinical outcomes were evaluated in two pediatric patients carrying the c.1901 c>a (p.a634d) gof mutation in jak1 after 3.5 years of ruxolitinib treatment. t cell phenotyping was performed using extracellular surface marker and intracellular cytokine staining by flow cytometry, and by gene expression signature profiling of rna sequencing data. to evaluate the effect of enhanced jak1 activity on myelopoeisis, we reprogrammed jak1 gof patient-derived peripheral blood mononuclear cells into induced pluripotent stem cells (ipsc) and performed directed myeloid differentiation. rna sequencing was performed on rna collected during ipsc myeloid differentiation and from whole blood of affected patients before and after ruxolitinib treatment. results: long-term use of ruxolitinib was associated with improved growth, reduced eosinophilia, and control of allergic inflammation without significant infectious complications, however, anemia represented a dose-limiting adverse effect. t cell immunophenotypic analysis revealed severe t helper (th) cell skewing towards a th2 phenotype preruxolitinib treatment, in keeping with the allergic clinical manifestations. analysis of myeloid differentiation revealed an increased myeloid to erythroid ratio in colonies derived from jak1 gof ipscs compared to controls. rna sequencing analysis of jak1 gof human whole blood and ipscs compared to controls revealed upregulation of cytokine and cytokine receptor genes implicated in allergic inflammation and early eosinophil precursor commitment, including csf-1 and the interleukin-33 receptor. reactome pathway analysis of genes upregulated in both jak1 gof ipsc and whole blood compared to controls showed enrichment of several pathways including interferon alpha/beta, interleukin-4/-13 and interleukin-33 signaling. conclusions: this work demonstrates a critical role for jak1 in atopic immune dysregulation, specifically driving a th2 phenotype and eosinophilia. combined jak1/2 inhibition can reverse much of the allergic inflammation, with dramatic clinical effects. this has important implications for our understanding of the pathogenesis and potential therapeutic targets for early life allergic immune dysregulation. had severe combined immunodeficiency (scid) and/or severe disease in association with their combined immunodeficiency (cid) necessitating haematopoietic stem cell transplantation (hsct). we present clinical and laboratory features of 4 new zealand patients from the same family with a novel heterozygous missense variant in rac2 [c.62t>g, p.ile21ser (i21s)]. the index patient (p1 -age 8 y, m) has a history of infectious gastroenteritis, staphylococcal aureus conjunctivitis, recurrent otitis media and recurrent herpes simplex virus (hsv)-1 cutaneous infections. his 2 siblings (p2age 9 y, m; p3 -age 6 y, f) and his mother (p4age 42 y, f) all have a history of recurrent viral (hsv-1) and bacterial (staphylococcal aureus, streptococcal pyogenes) cutaneous infections and/or recurrent sinopulmonary infections that respond to empiric antimicrobial therapy. their neutrophils all had enhanced superoxide production in response to stimulation by fmlp and pma as compared to healthy controls'. these findings suggest that rac2 i21s is an activating mutation causing notable abnormalities in neutrophil morphology and nadph oxidase activation similar to other recently reported mutations. this novel mutation expands the phenotypic spectrum of rac2 activating mutations. clinical management of affected patients needs to be tailored to their phenotype and disease severity. background and aims: heterozygous mutations in cytotoxic tlymphocyte antigen-4 (ctla4) are associated with recurrent infections, lymphoproliferation, autoimmunity and lymphocytic infiltration of target organs. disease penetrance can be highly variable even among related family members carrying the same ctla4 mutation. our evaluation of a subset of the ctla4 patient cohort followed at the national institutes of health (nih) revealed that 50% of ctla4 mutation carriers have gastrointestinal (gi) manifestations which include diarrhea and diffuse lymphocytic enteropathy. our aim was to determine whether the intestinal microbiome, metagenome and metabolome could distinguish patients with ctla4 haploinsufficiency (ctla4-h) based on disease severity, and the presence or absence of gi manifestations. methods: clinical metadata and fecal samples were collected from healthy individuals (n=16) and patients with ctla4-h (n=32). patients with ctla4-h were classified as having minimal (n=7, only endocrine and/or dermatological manifestations) or systemic disease (n=25, hematological and multi-organ involvement). they were further classified based on whether they had a history of enteropathy (n=18) or active gi disease ( < 2 bowel movements per day and/or blood or mucus in stool) at time of sampling (n=8). metabolomic profiling (using a panel of 150 metabolites) and 16s rrna gene sequencing (v4 region) was performed on fecal samples (total samples: 62; number of reads/sample: 18,341 to 226,027; median: 74,857). a subset of 20 samples were subjected to shotgun metagenomic sequencing based on findings from the 16s rrna gene sequencing analysis. results: all patients with ctla4-h and a history of enteropathy or active gi disease also had systemic disease. fecal samples from patients with a history of enteropathy had a distinct microbial community structure (fig. 1 ) which was significantly less diverse (fig. 2 ) compared to healthy individuals and patients with minimal vs. systemic ctla4-h. patients with a history of enteropathy had significantly higher relative abundance of 5 bacterial taxa including shigella-escherichia (fig. 3) . shotgun metagenomic sequencing confirmed that samples from patients with a history of enteropathy were dominated by subsets of 12 identified escherichia coli strains, all of which share 30 genes coding for specific types of virulence factors such as curli fibers (facilitate uptake into host cells), flagellar proteins (increase motility) and enterobactins (increase bacterial iron transport). meanwhile, samples from patients without active gi disease at the time of collection were enriched for several taxa including bacteroides nordii and akkermansia muciniphila compared to patients with ctla4-h and active gi disease (fig. 4) . metabolomic analyses showed that asparagine, 3-hydroxybutyrate, cytosine and cystine were enriched in samples with abundant e. coli, whereas samples without e. coli were enriched in metabolites involved in pyrimidine (holm p=0.0005), purine (holm p=0.004), and alanine/aspartate/glutamate metabolism (holm p=0.01) (fig. 5) . conclusions: fecal samples from ctla4-h patients with a history of enteropathy were heavily colonized with e. coli strains that are associated with a specific metabolomic profile and that share virulence factor genes that may facilitate host invasion. these data suggest that the microbiome and metabolome can distinguish patients with ctla4-h and gi disease, and support the potential use of antibiotics or even antimetabolites to treat ctla4-hrelated enteropathy. the dna polymerase delta (pol δ) complex is essential for leading and lagging dna strand synthesis. its catalytic subunit (pold1), carries both polymerase and exonuclease activities and plays a crucial role in dna replication and repair. heterozygous pold1 mutations have been associated with inherited colorectal cancer and mandibular hypoplasia, deafness, progeroid features and lipodystrophy (mdpl) syndrome. more recently a biallelic loss of function mutation in pold1 (p.r1060c) that impairs the stability of the pol δ complex, has been reported in 3 related subjects with recurrent infections, deafness and combined immunodeficiency (cid) with t-cell lymphopenia, cd8+ t cell oligoclonality but preserved b cell proliferation. we report here a second family in which a novel biallelic missense mutation in pold1 gene was associated with cid. the proband is a 9-year-old boy born to consanguineous pakistani parents. since infancy he suffered from failure to thrive and recurrent infections, including 5 episodes of pneumonias, multiple otitis media, sinusitis, recurrent cellulitis at the g tube site, bk viruria and shingles. live and dead vaccines were well tolerated. at 2 years of age sensorineural hearing loss together with profound leukopenia (anc 260 cell/μl, alc 680 cells/μl) and hypogammaglobulinemia (420 mg/ dl) were identified. intermittent ivig replacement and antimicrobial prophylaxis were initiated. immunophenotyping at 9 years of age showed severe t cell lymphopenia (122 cd3+ cells/μl, 68 cd4+ cells/μl, 39 cd8+ cells/μl, figure 5 . volcano plot of metabolites present in fecal samples enriched with e. coli vs. samples without e. coli. c e l l s / μ l , 1 3 c d 4 + c d 2 5 h i f o x p 3 + c e l l s / μ l ) , a n d hypogammaglobulinemia (igm 23 mg/dl, igg 276 mg/dl, iga < 10). physical exam was remarkable for multiple acquired nevi in the groin area, teeth abnormalities and global developmental delay. whole exome sequencing analysis revealed a homozygous pold1 missense variant (nm_001256849 c.3175c>g, p.q1059e) absent in public databases (cadd score of 24). parents were heterozygous. tcr-vβ family expression was normal in both cd4+ and cd8+ t cells, but the proportion of t cells expressing vα 7.2 (encoded by the distal trav1-2 gene) was markedly reduced (less than 1%), consistent with impaired vdj recombination at the tra locus and/or with defective thymocyte survival. constitutive expression of γh2ax was observed in t and nk cells after 1 h and 24h of culture in unirradiated conditions. at 1 h post-irradiation (2 gy), reduced levels of p-atm were detected in t and nk cells, and lack of atm, smc1 and h2ax phosphorylation was observed in a subset of b cells, suggesting inability of these cells to mount an effective dna repair response. bone marrow examination showed normal trilineage hematopoiesis but decreased proportion of cd10-cd20+ mature b cells and increased proportion of pre-b cells. conclusion: we report the second mutation associated with autosomal recessive pold1 deficiency. our findings broaden the understanding of the mechanisms underlying the immune defect in this disease to include b cell maturation arrest in the bone marrow and a dna repair defect that may support the generation of a restricted tcr repertoire in the thymus and increased malignancy risk. abstract/case report text following allogeneic hematopoietic cell transplantation (hct) for scid, the development of a diverse t cell repertoire is essential for optimal immune recovery. high-throughput sequencing (hts) of the trb repertoire is the best tool for the evaluation of clonotype dynamics during immune reconstitution as compared to cdr3 spectratyping and staining of vβ families. we investigated whether longitudinal hts analysis of trb would accurately assess development of tcr repertoire diversity over time and reflect the quality of t cell reconstitution following hct for scid. we wanted to study the effect of conditioning regimen, scid genotype, donor type on tcr diversity post hct. we hypothesized that repertoire diversity may represent an early biomarker to predict long-term immune reconstitution vs. need for a second intervention. we assessed if the trb repertoire post-hct carried a molecular signature of selfreactivity. methods: the composition and diversity of trb repertoire of 27 scid infants, pre-hct and at 100 d, 6 and 12 mo and yearly posttreatment(s) was studied by hts. median time of follow-up was 48 mo. subjects were part of a prospective study of scid by the primary immunodeficiency treatment consortium. equal amounts of total rna extracted from peripheral blood was used as template to semi-quantitatively amplify trb rearrangements. the vdj statistics file (past program) was used to calculate a shannon entropy (h) index of repertoire diversity and simpson (1-d) index of repertoire clonality. results: trb sequence analysis of 27 scid patients showed poor diversity at baseline, followed by improvement to normal complexity (h index >8.0) after hct. similar kinetics of development of trb diversity were seen in patients with il2rg, jak3, and il7r defects (n=16) as in those with rag and artemis defects (n=14). in the latter group, however, hct with no conditioning or immune suppression only was associated with persistently lower diversity than hct with conditioning (p < 0.01), a difference not found in the il2rg/jak3/il7r group (fig.2) . hct from a matched donor (6/13 conditioned) correlated with higher diversity than hct from a mismatched donor (5/15 conditioned) (p=0.01). having > 500 cd4+ t cells/ul at 6 mo post-hct correlated with higher trb diversity at 24 and 36 mo post-hct (p < 0.01). the trb repertoire 100 d post-hct was enriched for the presence of central cysteines at the apex of the cdr3 (p < 0.001), a biomarker of self-reactivity ( fig.1 ). an h-index of 4.7 or lower at 100 d after hct predicted need for second intervention (hct or gt) (fig.3) . conclusions: analysis of trb diversity allows for detailed assessment of development of a diverse t cell repertoire following cellular therapies for scid and confirms the need for patienttailored treatment strategies based on scid genotype. t-cell repertoire 100 d post-hct is characterized by a molecular signature that may contribute to the increased rate of autoimmunity early post-transplant. furthermore analysis of trb diversity at 100 d post-hct may identify patients at risk for failure of sustained immune reconstitution, thus prompting a second intervention without delay. abstract/case report text background atopic dermatitis is a chronic, multifactorial, relapsing inflammatory skin condition which is one of the main known health problem worldwide. atopic dermatitis lesions are frequently colonized by staphylococcus aureus and staphylococcus epidermidis. their susceptibility to form biofilms, ability to form adhesive skin colonies which lead to extremely resistant to antibiotics and immune responses. formation of skin biofilm resulted in complex bacterial communities that have unique effects on human keratinocytes, mouse fibroblasts and host immunity. aims: the aims of this study to confirm the specificity of s. aureus or its secreted factors in induction of pro-inflammatory cytokines il-33, tslp and toxicity on human keratinocytes and mouse fibroblast. the second aim to study the inhibitory effect of co-culture of s. epidermidis with s. aureus in term of production of pro-inflammatory cytokines and toxicity. method and materials: human epidermal keratinocytes and mouse embryonic fibroblasts cell lines from 3t3 were used as a control strain to examine production of inflammatory response (il-33 and tslp) and cell death induced by s. aureus in the presence and absence of s. epidermidis. tslp and il-33 were detected by elisa and the apoptosis of s. aureus and s. epidermidis on these cells was evaluated by flow cytometry. result: recent findings propose the important role of skin biofilms in the pathogenesis of atopic dermatitis. s. aureus have been found to induce secretion of pro-inflammatory cytokines and cause apoptosis of human keratinocytes and mouse fibroblasts. presence of s. epidermidis as skin biofilm found to protects the human keratinocytes and mouse fibroblasts from induction of proinflammatory cytokines and cytotoxicity. conclusions and future work: s. aureus are essential in production of inflammatory response and cell death of mouse fibroblasts and human keratinocytes. future work will be carried out to identify the soluble factors that responsible in induction of pro-inflammatory cytokines. in addition, more studies are needed to be able to understand the mechanism by how s. epidermidis reduce the induction and cytotoxicity caused by s. aureus. j clin immunol in adult patients, in whom arbitrarily defined diagnostic criteria for antibody deficiency syndromes are not fulfilled, is subject to interpretation and decision differences reported by immunologists world-wide. in this study, we explored whether training in one particular program would decrease the variability in diagnostic and treatment approaches seen in the responses to two nationwide questionnaires in the uk and the usa. methods: a 10-minute online survey originally administered to a cross-sectional sample of 203 us allergists/immunologists (usa/i) in january, 2019, was also answered by 43 a/i subspecialists who had trained in the last 25 years at the louisiana state university health science center allergy immunology training program in new orleans (laa/i). respondents were asked questions on patient assessment, antibiotic use, initial igrt, and immune response assessment in decisionmaking to prescribe igrt. usa/i participants were recruited from the dynata physician professional panel. laa/i participants were recruited by the louisiana primary immunodeficiency network (lapin). results: overall, laa/i had consensus responses to the various practice questions close to 90% of the time, but outliers were always present, as was also observed in the usa/i. there was a higher frequency in the reported care of patients as described in the questionnaire by laa/i. over 98% of laa/i assessed vaccine responses prior to commencing igg replacement vs only 90% of usa/i p < 0.5. all la a/i used the pneumococcal vaccine for assessment purposes while few used tetanus and hemophilus influenza, and none used meningitis or salmonella vaccines. these vaccines were still used by some of usa/i. a high level of concordance was observed among all respondents in that only few regarded pneumococcal antibody testing as the definitive test to commence igrt. high resolution chest ct scan was used more often by laa/i before starting igrt. assessment of effectiveness of igrt was decided after only 3 months by more usa/i, vs laa/i, who tended to wait 6 months to decide to continue or discontinue igrt. conclusions: all a/i responders saw a significant number of patients who do not conform to strict diagnostic criteria for antibody deficiency syndromes. there is diversity in the approach of usa allergists/ immunologists in determining the indication for igrt for non-classical antibody deficient patients. laa/i responses made it obvious that post graduate influences always play a role in shaping the way a/i practice evolves after graduation. drawing on clinician experiences through questionnaires offers a valid contribution to developing consent approaches to improve patients' clinical conditions. diagnostic criteria and treatment guidelines would benefit from practice-based realistic recommendations based on a/i experience. abstract/case report text background: autoimmune lymphoproliferative syndrome (alps) is a rare genetic disorder secondary to a defective fas-mediated apoptotic pathway of mature lymphocytes. it is characterized by chronic nonmalignant lymphoproliferation in the form of lymphadenopathy and/or splenomegaly, autoimmune manifestations such as cytopenias, increased risk of lymphoma, and expansion of tcrαβ+ cd4-/cd8-(dnt)t-cells. germline or somatic pathogenic variants in fas, fasl, and casp10 are well described genetic defects associated with alps. the definitive diagnosis for alps, based on the revised 2009 nih diagnostic criteria, include both required criteria (chronic non-malignant, non-infectious lymphadenopathy, splenomegaly, or both and elevated tcrαβ+ dnt t-cells) and one of the primary accessory criteria (defective lymphocyte apoptosis or mutation in the genes mentioned above). patients who do not meet the current diagnostic criteria are considered for alps-related disorders. case presentation: we report a 2-year-old male who presented with recurrent infections, splenomegaly and chronic lymphadenopathy since 8 month of age. due to its chronicity he was evaluated by multiple specialists for malignant and infectious causes. hematological workup including bone marrow biopsy was unremarkable except for an elevated ldh level. infectious workup identified a past cmv infection. clinical course is pertinent for chronic splenomegaly which was identified incidentally at 1.5 years of age during an evaluation for intussusception. family history is pertinent for a father with recurrent infections, paternal grandmother with thrombocytopenia of unknown cause requiring platelet transfusions, and paternal cousin with neutropenia. there is no family history of lymphomas. history of chronic lymphoproliferation and recurrent infections prompted an evaluation for lymphoproliferative disorder. full immune workup was notable for elevated plasma il-10 and il-18, normal immunoglobulin levels, lymphocytes subsets, vitamin b12 level, soluble fasl, and relative frequency (%) but borderline increased absolute count of tcrαβ+ (dn) t-cells. in addition, he was noted to have presence of anti-platelet antibodies, poor lymphocyte proliferation to antigens, and low pneumococcal antibody titers. genetic testing with a 207 pid gene panel identified a likely pathogenic heterozygous variant in prf1 c.487del (p.his163thrfs*96), a heterozygous variants of uncertain significance in casp10 c.683c>t (p.pro228leu) and stim1 c.304a>g (p.thr102ala). the casp10 variant is present in 63 alleles in gnomad (282k total allele count) and reported deleterious by sift. discussion: unlike the typical alps presentation, characterized by dominantly lymphoproliferation and autoimmunity, our patient's clinical phenotype is striking for recurrent infections, abnormal t-cell function, and poor antibody response. our patient does not the meet diagnostic criteria for alps due to normal relative frequency of dn t-cells. however, presence of elevated of il-10, il-18, platelet autoantibodies raise concern for alps-related disorder. in addition, family history of recurrent infections and cytopenias raises concern for familial autoimmunity and alpslike phenotype. although casp10 is associated with autosomal dominant and autosomal recessive alps, the role of this vus is yet to be determined. conclusion: we continue to investigate the pathogenicity of our novel casp10 vus. further studies include pedigree analysis, fas apoptosis assay and apoptosis pathway testing to assess for the etiology of this alps-related disorder. (word count 487, max 500) abstract/case report text rationale: ocrelizumab is a recombinant anti-cd20 monoclonal antibody, which binds to a different, but overlapping cd20 epitope than rituximab. there have been increasing reports evaluating hypogammaglobulinemia and morbidity and mortality in patients receiving rituximab, but there is a paucity of data on hypogammaglobulinemia in patients treated with ocrelizumab. methods: we performed a retrospective review of patients who received ocrelizumab in our healthcare system. we evaluated the demographics, indication for ocrelizumab, frequency of immunologic evaluation, and h y p o g a m m a g l o b u l i n e m i a p r e -a n d p o s t -o c r e l i z u m a b . hypogammaglobulinemia was stratified as mild (igg < 600mg/ dl or less than lab reference range), moderate (igg < 400mg/ dl) or severe (igg < 200mg/dl). results: we identified 185 patients who received ocrelizumab for multiple sclerosis (average number of ocrelizumab cycles = 4; range 1-9 cycles). there were 120 (65%) female patients, with a mean age of 49 years old (range 23-74; standard deviation ±13 t-cells. tnfα, ifnγ, and il-10 were not statistically different between cgd patients and healthy controls. tnfα, ifny, il-10, and il-17a expression in patients with cgd who had active colitis or history of colitis were increased as compared to cgd patients without a history of colitis but did not reach statistical significance. in two patients, il-17a expression that was elevated pre-hct normalized post-hct. discussion: the mechanism for increased susceptibility to inflammatory disorders in patients with cgd has not been well elucidated. our results agree with previous studies demonstrating increased il-6 and il-17a production from cd4+ t-cells in patients with cgd indicating a proinflammatory state in these patients at baseline. also, there appears to be an increase in tnfα, ifny, il-10, and il-17a expression from cd4+ t-cells that correlates with presence of inflammatory disease vs. those without inflammatory disease indicating that these cytokine perturbations may be able to serve as biomarkers of disease activity. a larger sample size with prospective collection will be analyzed in the future. abstract/case report text background: glucose-6-phosphatase catalytic subunit 3 (g6pc3) deficiency, is characterized by severe congenital neutropenia, recurrent bacterial infections, mild intermittent thrombocytopenia and a high incidence of congenital cardiac and uro-genital defects. we report the case of a 28-yo male with chronic neutropenia and thrombocytopenia, who was found to have homozygous pathogenic variants in g6pc3 (c.210del, p.phe71serfs*46). unique to this case is the patient's long history of misdiagnosis of evans syndrome (chronic autoimmune neutropenia with thrombocytopenia). case presentation: a 28-yo male with reported diagnosis of chronic autoimmune neutropenia and thrombocytopenia since age 7 was referred to our a b clinic with concern for an underlying immune dysregulation syndrome. he had a history of oral ulcers, gingivitis, recurrent bacterial infections (otitis media, pneumonia, skin abscess) concomitant with severe neutropenia ( < 500 cells/ul), for which he had received treatment with systemic steroids and g-csf since he was 9 years old. he also had history of asthma and short stature, thought to be secondary to his chronic systemic steroid use. we present the first chilean patient with stat1 gof immunedysregulation . moreover, to our knowledge this is the first stat1 gof patient presenting with lymphomatoid granulomatosis. this is a severe pulmonary disease in which primary immunodeficiencies including stat1 gof should be considered in the differential. in this case rituximab successfully resolved pulmonary nodules and respiratory symptoms. there was persistence of mild hepatosplenomegaly but otherwise clinical stability and monitored expectantly until the age of 12. at that time he presented with fever, left knee and ankle arthritis. he underwent arthrocentesis of the left knee and left ankle, both aspirates were sterile, with notable leukocytosis with heavy neutrophilic predominance. an extensive rheumatologic and infectious workup was non-diagnostic. both sil-2r and il-6 were elevated, 5,026 units/ml ( < 1105) and 44pg/ml ( < 6), respectively. he was treated with systemic corticosteroids, ultimately arthritis resolved after 2 months. at age 13 he presented for the first time with periorbital pain and conjunctival injection of the left eye that persisted after minor trauma. he was found to have nongranulomatous uveitis, which responded ultimately to systemic corticosteroid. he then presented at age 14 with fever and right knee and great toe arthritis. again he underwent arthrocentesis which revealed aseptic arthritis, and at that time was started on anakinra (anti il-1β) and prednisone. there was clinical improvement over several weeks followed by return of right knee arthritis, coupled with onset of symptomatic uveitis of the left eye. despite systemic corticosteroids and anakinra and il-1 blockade, the patient was again admitted shortly thereafter to the hospital with arthritis, fevers, rash and abdominal pain. there was concern for evolving hlh and the patient was ultimately transferred to cincinnati children's for further evaluation and treatment. pertinent inflammatory biomarkers at that time included sil-2r of 3,394 units/ml and il-18 level of 9,624 pg/ml. in addition to anakinra and systemic corticosteroids, the patient was started on tadekinig alfa (recombinant human il-18 binding protein) as part of a prospective study. hlh flare ultimately resolved without use of antineoplastic agents, and the patient was discharged home. soluble il-2r levels since normalized, and il-18 levels decreased to less than 1000 pg/ml. the patient has been doing well on anakinra and tadekinig alfa, though continues to experience mild to moderate right knee effusion. this case suggests il-18 inhibition may be an effective therapeutic approach for patients with xiap deficiency. in the absence of neurological involvement and infectious trigger, ruxolitinib was initiated at a dose of 50 mg/m 2 /day, in combination with dexamethasone (10 mg/m 2 /day). this treatment led to rapid normalization of the neutropenia (48 hours), complete resolution of the splenomegaly (10 days) and disappearance of hlh biological markers (triglycerides levels in 1 week, activated hla-dr+ cd8 t cells in 2 weeks, fibrinogen levels in 1 month), without the need for etoposide or serotherapy. dexamethasone was weaned every two-weeks and stopped after 8 weeks. ruxolitinib was well-tolerated with no side effects. while in complete remission of her hlh, the patient then received alemtuzumab (0.5 mg/kg total dose) and a fludarabine-based myeloablative conditioning regimen. ruxolitinib was weaned over one week, and a 9/10 unrelated transplant was performed with success. the immediate posttransplant period was complicated by a veno-occlusive disease that responded rapidly to defibrotide and a corticosteroidresistant skin and ocular graft-vs-host disease (gvhd) despite a prophylaxis with ciclosporine and mycophenolate mofetil. gvhd was controlled by the reintroduction of ruxolitinib. at 3 months post-hsct, her chimerism is 100% donor. to our knowledge, this case is the first description of a patient with primary hlh successfully treated in first intent by a combination of dexamethasone and ruxolitinib prior to hsct. our observation suggests that this targeted and less-toxic treatment regimen, that does not include etoposide nor high-dose alemtuzumab, is effective, well-tolerated and could be used in first intent to treat primary hlh. abstract/case report text presentation a 15-year-old girl, presented in ambulatory consultation, with a 3-year history of recurrent fever, influenza-like symptoms (sore throat, malaise), associated with self-limited painful genital ulcers (just within the period of fever). the first episode was characterized for an fournier's infection, requiring in-hospital treatment, multiple surgical procedures, antibiotics and hyperbaric oxygen therapy. after that catastrophic debut, she was diagnosed approximately 6 episodes per year pharyngitis (with fever, malaise and sore throat) treated with corticoids, antibiotics and topic medication. the last year, noticed that every episode of fever (total of 4) were associated with one or several genital lesions. the patient had no relevant medical history, she didn't receive long-term medication, she received all immunizations, she was sexually inactive, and hadn't apply any topic medication or product on the vulva, there was no trauma history, psychological medical history or sexual abuse. episodic gynecologic examination showed her labia minor several lesions, fibrinous, soft ulcerations on their inner aspect, these lesions had a symmetrical appearance, known as kissing lesions; no vulvar swelling, vaginal discharge or lymphangitis were noticed. there were no other skin or mucous membrane lesions (figure 1 ). investigations viral (hiv, hbv, hcv, ebv, cmv) and treponemal (tpha-vdrl) serologies were negatives. erythrocyte sedimentation rate (esr) and pcr analysis within ulcers episodes were positive. specific antibodies (cardiolipin, anti ro/ss-a, anti la/ss-b, anti ccp, anti ena, anti gliadin, anti tpo anti tpo) serologies were negative. otherwise, important elevation immunoglobulin d was observed (9,3 mg/dl, twice the normal value): mild elevations of immunoglobulin m and immunoglobulin a were observed. serum subtypes of immunoglobulin g and immunoglobulin e were normal. leukocytosis with monocytes elevation and an increase of lymphocytes b were present. (table 1 ). discussion lipschütz ulcers are uncommon and an often unknown entity for physicians, but it is important to recognize and include it in the differential diagnosis of vulvar ulcerations. this condition is characterised by self-limited painful ulcerations of the vulva or lower vagina in adolescent or young women, non-sexually transmitted, and usually preceded by influenza or mononucleosis-like symptoms. hyperimmunoglobulin d syndrome (hids) is characterized for unremitting fever lasting four to seven days and the presence of palpable tender lymphadenopathy, splenomegaly, arthralgia/arthritis, abdominal pain, and mucocutaneous manifestations. laboratory findings suggestive of hids include elevated age-specific serum immunoglobulin d (igd) and/or immunoglobulin a (iga) levels, elevation of acute phase reactants, and urinary excretion of mevalonic acid during, but not between, attacks. the diagnosis is established if an elevated age-specific level of igd is detected. iga levels are typically measured at the same time but are not required for diagnosis. elevated serum igd is not specific for hids and can occur in patients with certain neoplastic, infectious, heritable, and idiopathic disorders. in the present case report, the patient was treated with colchicine, with favorable evolution and free from new events. levels of ig d, platelets and monocytes remain high. we describe a young female patient presenting recurrent lipschütz ulcers, fever and elevation of serum immunoglobulin d, suggesting that hids could be associated with genitalia ulcers. (2), and transmission in vivo in extremely low birth weight infants was considered (3) . based on these considerations, the risk/benefit was considered favorable for restarting pasteurized donor breastmilk feeds. given his small size and young age, we had significant concerns about using ganciclovir prophylaxis and opted to hold this and monitor weekly cmv pcr. the child has been titrated up on these feeds, is gaining weight appropriately, and has had weekly cmv pcrs which are negative x5 since restarting donor breastmilk. t cells, last checked at 7 weeks of age (37 weeks gestational age) remain essentially absent. given the lower risk of nec in premature infants with breastmilk-based enteral feeds, a broader, multi-institutional study is warranted to best examine the safety of pasteurized donor breastmilk in infants with scid and complete digeorge syndrome. transfected with a reporter plasmid (for luciferase), wt or mutant-stat1 plasmids. nk cell cytotoxicity was measured by cr 51 release assay. we used multiparametric immune profiling to dissect the effect of stat1-gof mutations on nk cell developmental phenotype. results: similar to our previous studies, we observed higher levels of stat1 phosphorylation after two hours of stimulation from the dbd mutation compared to the ccd mutations. the stat1 activity assay confirmed gain of function observed by flow cytometry, but this activity was higher in k388q mutant and d292e mutant (ccd-closer to dbd) than v266i mutant. all patients demonstrated low nk cell lytic unit compared to healthy donors. interestingly, we observed a correlation between low lytic unit and lower numbers of cd56 dim perforin + cd16 + nk cells; much lower in patient with k388q mutation. stat1-gof patients showed a significant decrease in total nk cell numbers and impaired nk cell maturation was characterized by low expression of cd57, and higher levels of immature nk cell markers (cd117, nkg2a, cd158b). conclusions: these data suggest that impairment of nk cell function is affected by the location of the stat1 mutation and continues to be the case in novel mutations identified. the identification the genotype/ phenotype correlation in the spectrum of the nk cell defect in stat1 gain-of-function mutants may help to better understand the molecular basis for stat1 activation and/or function to predict clinical manifestations of disease and ultimately treatment regimens. mutation specific analysis after an amniocentesis showed that one twin was a carrier (l152m) and the other was a compound heterozygote (r156c and l152m). after birth, twin a had a mildly low erythrocyte ada level (12.8 nmol/h/mg; normal range 63+41) with normal metabolites and a normal immunophenotype, similar to both parents. twin b showed a normal absolute lymphocyte count and mitogen proliferation, normal t lymphocyte subsets, mildly low b and nk cells with 85% naïve t cells and a normal trec assay. erythrocyte ada levels were absent in peripheral blood, with mildly elevated metabolites [daxp=0.041 μmol/ml rbc (normal < 0.002) and %axp=0.9 (normal < 0.2)]. weekly recombinant ada enzyme replacement therapy (ert) was started at 1 week of life with subsequent normalization of the metabolites by week 14. absolute lymphocyte, t cell subsets were normal at birth but continued to rise slightly above normal range after starting ert. b and nk cell counts were mildly low at birth but normalized by week 3. genetic testing confirmed the prenatal genotypes in the twin girls. the patient is now 11 months old and doing well with no history of infections. her twin was not an hla match and family is currently awaiting gene therapy approval. discussion: ada deficient patients show substantial clinical and metabolic heterogeneity that tends to correlate with the genotype but phenotypic discordance occurs even within the same genotype. we describe an infant with prenatally diagnosed compound heterozygous mutations in the ada gene (grade-i: r156c and grade-ii: l152m). ada alleles are graded from 0-iv with increasing ada expression and decreasing severity respectively. there are reports of children with grade i/iii allele combinations with delayed, late and partial phenotypes. two siblings have been reported with l152m allele (grade iii) in combination with a different grade i allele (r235q), presenting with combined immunodeficiency at 1 and 13 months. the specific allele combination from our patient has not been previously reported, however, we expected that the grade-i allele likely would be more deleterious than the grade-iii allele. in our case, predicting a future phenotype remains a challenge, creating a dilemma regarding management strategies. however, with only mild metabolite elevations in our patient after birth, we may speculate whether the prenatal diagnosis with early ert precluded the development of a full immunophenotype and it remains to be seen whether nonimmune sequeli may be prevented. conclusion: children with compound heterozygous mutations in the ada gene can pose diagnostic and therapeutic challenges, especially due to the associated metabolic and clinical phenotypic variability. early recognition and treatment may potentially alter long-term morbidity and mortality. (ipex)-like phenotype. immunodeficiency is often combined with impairment of the humoral and cellular compartments. hematopoietic cell transplant (hct) can resolve disease-related manifestations in stat1-gof, but overall survival is poor and there is a high rate of secondary graft loss in transplanted patients. jakinibs are a class of medications that block cytokine-induced jak/stat activation. ruxolitinib preferentially inhibits jak1 and jak2 and has been used as precision-directed therapy for treatment of stat1-gof related manifestations with success in stabilizing and in some cases reversing organ-specific manifestations. the utility and safety of jakinibs for long term treatment of stat1-gof and in the prevention of disease-related manifestations is not known. as such, hct is often pursued for patients once disease-related manifestations are controlled with jakinibs. we present a patient with stat1-gof mutation with gradual secondary graft loss following hct 10 years ago, that has had continued disease progression despite chronic ruxolitinib treatment. case presentation this is a 14 years-old male diagnosed with a de novo heterozygous stat1 mutation (c.983a>g/a) at age 9, 5 years following hct for ipex-like disease. he has been treated with ruxolitinib for the last three years. this patient initially presented at 5 months of age with wasting enteropathy, failure to thrive, early-onset type 1 diabetes and hypothyroidism. he had frequent upper respiratory infections during childhood including mycobacterium fortuitum mediastinal lymphadenitis. at 4 years he underwent 10/10 matched, unrelated bone marrow transplant following reduced-intensity conditioning. mixed donor chimerism was present in the first 100 days following hct, and he continued to have a slow progressive decline of donor chimerism with full graft loss (0% whole blood donor chimerism) by age 13. at age 11, enteropathy returned leading to cachexia and tpn dependence. concurrently, he had recurrent upper respiratory tract infections, lymphopenia, and hypogammaglobulinemia. imaging showed bronchiectasis and lung function was consistent with obstructive lung disease (fev1:1.88 l fvc:1.96 l dlco:13.9 ml/min/mmhg). initiation of ruxolitinib at age 11 resolved his enteropathy with discontinuation of tpn and >25-pound weight gain. enteropathy has not returned. pulmonary clearance measures have also been employed. dlco initially improved (dlco: 19.11 ml/min/mmhg) but obstructive lung pattern continued (fev1: 1.91 l fvc: 2.08 l). after initial improvement, dlco began to decline. over the last 2 years and despite treatment with ruxolitinib, lung function has deteriorated with worsened fev1 (1.64 l), fvc (2.03l), and dlco (17.91 ml/min/mmhg). with this progressive decline, the family is now pursuing second hct. discussion jakinibs apply precision-directed therapy for immune dysregulatory features of stat1-gof. their use leads to substantial disease control and clinical improvement but does not prevent disease progression. jakinibs should be used as a bridge to definitive therapy with hct in patients with stat1-gof mutation. a recent large registry study showed a higher risk of infections (hr 1.42, 95% c.i 1.33-1.52) in children with thymectomy as compared to surgery controls, in addition to demonstrating differences in the risk of cancers, autoimmunity and atopy. limited small studies have described some risk factors for altered immune consequences; however, specific predictors of infections among children with congenital heart disease (chd) undergoing thymectomies have not been systematically assessed. among children with chd and thymectomy, we sought to characterize children with and without reported infections within 4 years postthymectomy and identify predictors of bacterial and viral infections. methods: using a retrospective chart review (institutional irb approved) from 7/2/2013 and 3/31/2017, we identified children with chd that underwent thymectomy and excluded any known conditions associated with immunodeficiency and those with less than 6-month follow-up post-thymectomy. first absolute lymphocyte count (alc) after thymectomy was stratified using a cutoff at 50% of the lower limit of age-adjusted normal values (alc value < 50% vs alc value >50% of the lower limit of age-adjusted normal levels). we sought to assess predictors of reported bacterial (positive blood, cerebrospinal fluid, respiratory cultures and chest-x-ray confirmed pneumonia) and viral infections (positive viral pcr tests) within 4 years postthymectomy. results: we identified 128 children with chd who had thymectomies, of which, 65% (84/128) were male. the median age at thymectomy was 6 months (interquartile range 2 months-2.2 years); 51% (66/128) underwent a complete thymectomy; and 3% (4/128) developed a chylothorax within 1 week post-thymectomy. a substantial proportion of children had an alc below 50% of the lower limit of age-adjusted normal levels after thymectomy (2% [3/127] pre-thymectomy vs 65% [82/127] postthymectomy). among children with chd post-thymectomy, 51% (65/ 128) and 45% (57/128) reported bacterial and viral infections within 4 years, respectively. children with post-thymectomy alc values below 50% of the lower limit of age-adjusted normal levels had higher odds of reported bacterial (or 3.44, 95% c.i 1.37-8.64, p=0.008) and viral (or 5.86, 95% c.i 2.02-16.96, p=0.001) infections post-thymectomy as compared to those with an alc greater than 50% of the lower limit of ageadjusted normal levels (multivariate logistic regression). there was no association with the type of thymectomy (partial vs complete), age at thymectomy, weight at thymectomy, sex or prematurity. conclusions: among children with congenital heart disease with no known immunodeficiency undergoing thymectomy, alc below 50% of age-adjusted normal levels post-thymectomy may be associated with higher odds of bacterial and viral infections. a retrospective study design with a small sample size poses several limitations; however, this study suggests that post-thymectomy absolute lymphocyte values may be a potentially useful marker to identify higher risk patients in this population. radiological assessments esp. in the ct chest is commonly performed, but has associated radiation exposure and pulmonary function testing, at times, maybe insensitive to small changes in lung pathophysiology. many pids may have overlapping features with short telomere syndromes (sts) a, which are accelerated aging syndromes affecting hematopoietic, pulmonary, hepatobiliary and/or immunological systems, unified by a high cell turnover in these organs. clinical assessment of ageappropriate telomere length (tl) is performed using flow cytometry & fluorescence in-situ hybridization (flowfish). methods: we retrospectively analyzed telomere lengths in lymphocytes and granulocytes using the flow cytometry and fish method .flowfish testing was done at reference laboratories in johns hopkins university (jhu, usa).approval was obtained from mayo's institutional review board. data abstraction and analysis was done using the software jmp. results: 24 patients were included in our analysis with 13 females (54%) and 11 males (45%).the median lymphocyte count of our cohort was 0.98 (0.51-1.78).the telomere length was strongly associated with the presence of lung disease (p=0.02*) and the presence of interstitial lung disease closely paralleled the changes in telomere length (delta-as compared to age adjusted normal percentiles lengths). shorter lymphocytic telomere length was associated with more severe reduction on total lung capacity (tlc; p=0.006*). conclusion: shorter lymphocytic telomere length served as a reliable biomarker for interstitial lung disease in pid patients. this may open up newer avenues for assessment of aging pathways in pid and may offer the option of using senolytic therapies in pids. mutations in the il-2 receptor common gamma chain gene (il2rg) result in x-linked severe combined immunodeficiency (scid). the common gamma chain is shared by il-2, il-4, il-7, il-9, il-15 and il-21 receptors. x-linked scid typically presents with low or absent t and nk cells and normal or elevated numbers of b cells. we report a case of x-linked scid with elevated b and nk cell numbers (t-b+nk+). the male patient had an abnormal newborn screen for scid in north carolina. lymphocyte enumeration performed at 12 days of life showed 8 cd3+ cells/mm3, 370 b cells/mm3, and 997 nk cells/mm3. he had no naïve t cells. repeat lymphocyte enumeration two weeks later showed that the cd3+ count had increased to 389/mm3. only 1.5% (6 cells/mm3) were cd45ra+ naïve t cells. he continued to have elevated b cell and nk cell numbers. chimerism studies revealed the presence of 6% female cells in mitogen-stimulated pbmc by fluorescence in situ hybridization, indicating the presence of transplacentally transferred maternal cells. lymphocyte proliferation responses to pha and cona mitogen stimulation were very low (less than 10% of normal). immunoglobulin levels were igg 921mg/dl, igm 18mg/dl, and undetectable iga and ige. genetic studies revealed a missense mutation in il2rg, c.467c>t, resulting in an amino acid substitution (p.ala156val) in the extracellular domain. family testing showed that the patient's mother was a carrier for this variant. the father and the two healthy older brothers did not have this variant. of note, the family history was significant for lateral maternal male early deaths. at 7 weeks of age, the patient received an unfractionated bone marrow transplant from his hla-identical brother without conditioning or gvhd prophylaxis. at the time of this report's submission, he is 4 weeks post-transplantation and has had successful engraftment (whole blood-cd3+ fraction was composed of >95% donor cells). he also now has normal t cell proliferation in response to mitogens and normal levels of all immunoglobulins. genetic defects that cause primary immunodeficiency can have variable phenotypic presentations. the patient's phenotype was atypical in that he had elevated nk cell numbers. to further evaluate these cells, we checked for stat4 phosphorylation following il-12 stimulation of abstract/case report text background: stat3 gain-of-function (gof) mutations cause a multisystem disease of early onset autoimmunity and lymphoproliferation, severe post-natal growth restriction, and recurrent and/or invasive infections. treatment of the autoimmune and auto-inflammatory features of stat3 gof patients relies heavily on immunosuppression and is often challenging. the full scope of phenotypes, treatments and outcomes may be broader when analyzing a substantially larger cohort than those already reported. methods: we gathered and analyzed data on 144 patients from 56 centers world-wide with confirmed gof mutations in stat3. retrospective chart reviews were performed in accordance with all local ethics and irb committees to determine clinical manifestations, immunophenotype, treatment regimens, success of treatment methods, and overall survival. funcitonal transcriptional activity was assessed by luciferase reporter assay on each individual mutation. results: fifty-nine individual mutations were identified and all conferred gof by a validated luciferase assay. there were 5 mutations in the nterminal domain, 17 in the coiled-coil domain, 28 in the dna binding domain, 6 in the sh2 domain, and 3 in the transactivation domain with the overwhelming majority being missense mutations. median age at presentation was approximately 5 years; 56% of subjects are male and 44% are female. immunodysregulatory features presented in all patients. autoimmune cytopenias were the most common occurring in 72% of subjects (n=105), followed by lymphoproliferation in 69% (n=100) with increased frequencies of double negative (cd4-cd8-)t cells being found in 71% of of patients tested, enteropathy in 53% (n=76), endocrinopathy in 35% (n=50), interstitial lung disease in 45% (n=65), dermatitis in 41% (n=42), and inflammatory brain disease in 6.25% (n=9). growth failure was present in 54% (n=77) with half of those patients having concurrent enteropathy. infections were reported in 60% of the cohort to include recurrent and/or invasive viral, bacterial, opportunistic, fungal, and mycobacterial infections. prominent abnormalities of immunophenotyping included t cell (54%) and b cell (36%) lymphopenia with reduced t cell proliferation in response to mitogens or antigens in 30% of those evaluated patients. fifty-nine percent of the patients hypogammaglobulinemia while 40% exhibited poor specific antibody responses to recall antigens. overall survival was 86% at data collection.treatment of stat3 gof patients often included multiple agents: ivig , chronic and pulse steroids, mtor inhibitors, calcineurin inhibitors, rituximab, mycophenolate mofetil, alemtuzumab, tocilizumab, and jakinibs. those started on jak inhibition showed improvement in clinical symptoms and, to date, there are 20 stat3 gof patients on targeted jak inhibition. thus far, 18 patients have undergone bone marrow transplant with a 61% survival rate. discussion: stat3 gof mutations were first reported in 2014 to cause a heterogeneous syndrome of autoimmunity and lymphoproliferation with immunodeficiency and infection susceptibility. earlier treatment with targeted therapy such as jak inhibitors has led to reduced disease morbidity. we report the largest cohort of stat3 gof patients collected through a multi-national collaboration of the longitudinal data and natural history of stat3 gof disease. understanding the heterogeneity of presentation and key features that will lead to proper diagnosis and early treatment in an effort to prevent long term disease associated sequelae. we present the case of 5 month old male with a novel heterozygous mutation in tcf3 and two previously unreported phenotypes: 1) absent circulating cd19+ b cells yet preserved immunoglobulin synthesis and vaccine responses and 2) significant thrombocytopenia that improved with immunosuppression. there is also a striking family history of two half-sisters who died during early infancy with similar clinical and lab findings and the same genetic change. the infant boy was born at term with respiratory failure and generalized rash. at birth he had thrombocytopenia (24k/ul) and lymphopenia (465/ ul). initial absolute cd3+ t cell count was low (442/ul), yet he had normal thymic output and proliferative responses to mitogens ruling out scid. cd19+ b cells were < 5/ul and bone marrow biopsy revealed decreased hematagones, yet he had a normal igm level (23.1 mg/dl) elevated iga (56 mg/dl), and elevated ige (27 iu/ml). igg levels were initially obscured by maternal igg and ivig; in turn he made positive titers to diphtheria and tetanus vaccination. the infant has maintained his own igg production. rapid genome sequencing revealed a heterozygous predicted deleterious vous in tcf3, in the second transactivation domain (c.1138 c>t, p.pro380ser). the same change in tcf3 was identified in the deceased half-sisters as well as the father: all 3 infants had different mothers, suggesting autosomal dominant inheritance. the sisters had similarly severe thrombocytopenia and absent circulating b cells; their causes of death were not completely understood. the 33 year-old father has normal platelet levels, very low cd19+ b cells (35/ul), elevated igg (1,580 mg/dl) and ige (1,030 iu/ml), and normal levels of iga and igm. the father also has an elevated number of cd3+ t cells (3,455 /ul) with an increased percentage of t cells expressing hla-dr (46%). in the months after birth, the infant boy continued to require frequent platelet transfusions. despite the persistent t lymphopenia, there was evidence of increased t cell activation with elevated levels of soluble il-2r (2510 pg/ml) and increased percentage of cells expressing hla-dr (30%) cd95 (98%), cd25 (86%), cd71 (55%), and cd69 (14%). a 10-day trial of prednisone was associated with an increase in his platelet count to >100k/ul. he was switched to rapamycin as a steroid-sparing agent, and his platelet count has remained > 200k/ul for several weeks without transfusions. interestingly, his b cell counts also improved after the steroid trial (50/ul) and his absolute lymphocyte count is normalizing on rapamycin. a potential mechanism could be rapamycin decreasing t cellmediated destruction of platelets or b cells. reassessments of t cell activation markers and b cell phenotyping while on rapamycin will be done in the future. in contrast to multiple published cases of tcf3 mutations associated with complete agammaglobulinemia and absent b cells, we present a case of an infant with absent b cells yet preserved humoral function as well as severe thrombocytopenia responsive to rapamycin. in collaboration with colleagues at nih, studies are underway to understand whether/how the unique change in tcf3 is related to either phenotype described above. abstract/case report text background: childhood-onset, chronic, multi-system inflammatory diseases are increasingly being characterized as monogenic inborn errors of immunity. arpc1b deficiency is a recently described, rare combined immunodeficiency characterized by recurrent/severe infections, a variety of autoimmune manifestations and platelet defects. we describe a case of arpc1b deficiency identified in an adult patient with recurrent ulcers/ bechet-like disease, non-malignant lymphoproliferation and intermittent microthrombocytopenia. patient case: at 1 year of age, our female patient was diagnosed with behcet disease based on a history of bloody stools at 3 months, oral ulcers at 9 months and vulvar lesions at 1 year. she underwent rheumatology evaluations for inflammatory arthritis, episcleritis, eczema, vasculitic ulcerating nodules of the trunk, perineum and extremities, and verrucae forming flat plaques similar to epidermodysplasia verruciformis without a unifying diagnosis. other infections include otitis media, sinusitis, pseudomonas ecthyma gangrenosum, cervical lymphadenitis, and pneumonia. at 28 years old, the patient was referred to our immuno-hematology comprehensive program clinic with a concern for malignancy versus a primary immune regulatory disorder (pird). she had a 6-month history of drenching night sweats, urticarial plaques, edema in her extremities and diffuse cervical, axillary and inguinal lymphadenopathy. past complete blood counts showed intermittent mild microthrombocytopenia. lymph node biopsies were negative for a neoplastic process but identified plasmacytosis, including focally increased iga-kappa+ plasma cells. expert review of the lymph node biopsy, and further evaluation excluded multicentric castleman disease. consideration was also given to autoimmunune lymphoproliferative syndrome (alps)-like disorders; however, her alps flow cytometry panel was nondiagnostic. her basic immune evaluation showed severe t cell lymphopenia (cd3+ 229 cells/ cm, cd4+ 222 cells/cm, cd8+ 52 cells/cm) with adequate b and nk cells, normal lymphocyte proliferation to pha and pwm, and dysgammaglobulinemia with igg 1579 g/dl, iga 638 g/dl, igm 50 g/ dl and ige 592 g/dl. due to concern of an underlying pird, a primary immunodeficiency panel was sent for 207 gene analysis with negative results. however, trio clinical exome sequencing identified biallelic variants in the gene arpc1b. one allele has a truncating, nonsense pathogenic variant in exon 8 denoted as c.898g>t, p.glu300ter. the other allele has a likely pathogenic variant in intron 4 denoted as c.393-2a>g, resulting in disruption of the canonical splice acceptor for exon 5. this is predicted to cause exon skipping, with an in-frame deletion of amino acids coded by exon 5. conclusion: this case highlights the value for evaluation for pirds in patients presenting with behcet-like disease, particularly in the context of other autoimmune manifestations and/or microthrombocytopenia. it also underscores that patients with arpc1b deficiency may present with chronic non-malignant lymphoproliferation. moreover, this patient emphasizes the value of exhaustive genetic testing for complex immunologic phenotypes. abstract/case report text lipoyltransferase 1 gene defect is associated with severe mitochondrial dysfunction disrupting lipoic acid biogenesis. clinical manifestations associated with early seizures, hypotonia, cardiomyopathy and pulmonary hypertension and encephalopathy. early neonatal death due to sepsis and cardiovascular collapse is commonly seen. the patient is a 36 week preemie male with congenital heart disease who developed severe intractable lactic acidosis on day of life 1 with increased excretion on organic acids of 2-methyl-2,3dihydroxybutyric acid. a mitochondrial disorder , echs1 or hibch deficiency was suspected. at 3 mo of age the patient was admitted for apneic spells and respiratory compromise. he was found to have elevated crp associated with rhinovirus infection and gram-negative bacteremia. due to the history of failure to thrive and sepsis, immunology was consulted. immunologic work up indicated normal b, t and nk cells with normal dhr, but showed agammaglobulinemia. the patient was started on ivig and whole exome sequencing was done. molecular analysis showed compound heterozygote mutations in the lipt1 gene: c.293g>a (p.arg98gln) and c.635t>g (pval212gly). subsequent biochemical analysis also showed biochemical abnormalities consistent with lipt1 defect. lipoyltransferase 1 is an enzyme involved in activation of a number of enzymes requiring lipoic acid. it is involved in lipoic acid synthesis. lipoic acid is required for the activity of pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched-chain alpha-ketoacid dehydrogenase. the literature indicates that most patients with lipt1 defect have a severe, often fatal course. the patient is now almost 3 years old and has stable clinical course without any major infections. he certainly has significant hypotonia and developmental delay. in conclusion, we are presenting the first case of lipt1 gene mutations associated with agammaglobulinemia who responded well to ig supplementation therapy. our immunologic findings in this case highlights the importance of immunodeficiency work up in challenging cases. as we see more cases lipt1 gene mutations, we will better understand the clinical spectrum. abstract/case report text a now 8-year-old male was initially evaluated for concerns regarding food allergy, eczema, food protein-induced enterocolitis syndrome, and failure to thrive. he had reactions of varying severity to multiple foods. these usually involved immediate urticaria or prolonged vomiting, diarrhea, and abdominal pain. ige and skin prick testing was performed to suspected foods and was positive to milk, egg, pork, wheat, peanut, pecan, coconut and corn. these foods had historically caused reproducible immediate symptoms. testing was negative to other suspected foods. he developed an oral aversion and extremely restricted diet. symptoms of abdominal pain, hematochezia, rashes, arthralgias, headaches, fatigue, dyspnea, and palpitations increased. urticaria and severe abdominal pain with vomiting and diarrhea continued intermittently without identifiable triggers on a restricted diet. laboratory markers demonstrated elevated inflammatory markers, anemia, iron deficiency, vitamin b12 deficiency, and vitamin c deficiency (scurvy). gastroenterology work up did not identify any pathology. gastrointestinal symptoms did not respond to treatment with multiple gerd medications or oral steroids. baseline tryptase was elevated. low histamine diet was initiated and repeat tryptase remained elevated. fractionated tryptase revealed normal mature (beta) tryptase with elevated total tryptase, negative genetics for c-kit mutation, normal urine prostaglandins. family members had tryptase levels drawn. one parent and sibling had elevated tryptase levels, while the other parent's tryptase was normal. hereditary alpha tryptasemia syndrome is defined by elevated blood tryptase levels and symptoms involving multiple organ symptoms. patients with elevated tryptase levels without symptoms are defined as having hereditary alpha tryptase trait. there is significant variability regarding which patients are symptomatic. organ symptoms that may be involved include skin, gastrointestinal, neurologic, connective tissue, cardiac, neuropsychiatric. severe allergic reactions such as anaphylaxis can occur. increased blood levels of the protein tryptase are caused by extra copies of the alpha tryptase gene (tpsab1). treatment is usually directed at specific symptoms, antihistamines, and mast cell stabilizers. research continues into additional treatment options. this patient was started on cromolyn and long-acting antihistamine. his gastrointestinal symptoms and rash/urticaria improved, and he began tolerating a small, but increased, variety of foods. the majority of his constitutional symptoms of fatigue, arthralgias, weakness resolved as he began gaining weight, and hemoglobin, vitamin c and b12 normalized. his sibling was evaluated and noted to have food allergy, asthma, abdominal pain, gerd, and eczema. she was also started on cromolyn and antihistamines which improved her gastrointestinal symptoms. parent with elevated tryptase was recommended to be evaluated further with allergist. this is an example of a patient with elevated tryptase and multiple organ system involvement. some of his signs and symptoms responded to mast cell stabilizing and antihistamine medications. patients with history of recurrent episodes of allergic reactions to foods and multiple constitutional symptoms would benefit from baseline tryptase levels. family members should also be tested if the patient has elevated tryptase. multiple studies have been published looking at the rates of scid in the united states. the estimated rate of scid prior to screening was 1 per 100 000 live births. post screening implementation, on average rates of scid were found to be closer to 1 in 60 000 live births. results : development of a t cell receptor excision circles (trec) scid screen in alberta involved the screening of 4000 anonymous term neonates using quantitative pcr for trecs. the cycle threshold for the control gene, rnasep, was set at 30.5 as 95% of our population had a cycle threshold < 30.5 (90% ci [30.4,30.6]). from those bloods spots with adequate dna, a final trec cut off of 40 was chosen, as it would give an accuracy of 99.6%, and fairly low false positive rate of 0.4% (95% ci [0.002, 0.006]). since starting a population based screen for scid in june of 2019, we have identified 4 cases of scid and 13 cases of low trec not caused by scid. to date we have detected one case of reticular dysgenesis, 2 cases of ada scid and one case of x-linked scid. other causes of lymphopenia in the neonatal period detected with abnormal trecs include one syndrome associated with variably affected cellular immunity (charge) and 12 cases of secondary lymphopenia including four cases of prematurity, three cases of diaphragmatic hernia or gastroschisis, four patients with underlying cardiac disease, and one patient with severe hydrops. discussion : canada has multiple unique populations with increased risk of scid. the estimated rate of scid in canada prior to implementation of a population based screen was 1.4 per 100 000 live births. the rate within canada's first nations, métis and inuit populations is 4.4 per 100 000 live births. prior to scid screening, alberta had 13 cases of scid identified between 2005-2014 with an estimated rate of 1 per 60 000 live births. to date, our screen in alberta has identified 4 cases of scid with a rate of 1 per 7000 live births which is significantly higher than previously estimated. given that early diagnosis and definitive management through bone marrow transplant or gene therapy has been shown to reduce mortality this screen will help reduce morbidity and mortality in this vulnerable population. abstract/case report text introduction: human herpesvirus 6 (hhv-6) has the ability to integrate its genome into host telomeres. if this integration occurs in gametes, then the virus can be genetically transmitted and offspring will carry a copy of chromosomally integrated hhv-6 (cihhv-6) in each somatic cell. this can lead to false attribution of infectious and non-infectious presentations of hhv-6, and make the diagnosis of active hhv-6 infection difficult. we present the case of a patient with meningoencephalitis attributed to hhv-6 and persistently elevated blood levels of hhv-6 by pcr concerning for primary immunodeficiency who was discovered to have cihhv-6. case description: a 7-year-old female who carried a past diagnosis of hhv-6 meningoencephalitis was seen in immunology clinic for follow up of persistently elevated levels of hhv-6 dna in her blood by pcr. she was born at 35 weeks and as an infant had failure to thrive (ftt), anemia and a varicella like rash after varicella immunization. at the age of 3, she received flumist vaccine and developed a fever the following day. over the next few days, she developed lethargy, altered mental status, headache, photophobia, seizure, papular rash and oral ulcers. she was admitted to the hospital and csf studies were consistent with viral meningoencephalitis (270 wbc, 30l, 63m, 100 rbc) although hsv, cmv, ebv and enterovirus were negative. she was treated for presumed hsv encephalitis with 21 days of iv acyclovir and 5 days of high dose steroids. one week after discharge, she again developed papular rash on feet, headache, oral ulcers and lethargy. she was admitted and csf studies this time showed only 3 wbc but positive hhv-6. blood and skin swab were also positive for hhv-6. immunology was consulted while admitted and work up for primary immunodeficiency was initiated. her work up was normal including responses to vaccine titers, complement studies, moderate in t cells, monocytes, neutrophils, lung and muscle, and low in skin, liver, heart and kidney tissues. analysis of each individual gene expression level by nanostring in comparison with healthy controls demonstrated significantly higher levels of the following irgs: ddx60, epsti1, gbp1, ifi6, isg15, ly6e, oas1, oas2, oas3, rsad2, rtp4 and socs1. whole blood rna-seq was performed in 3 patients and pathway analysis with the differentially upregulated genes demonstrated an enrichment of intraluminal vesicle formation and negative regulation of apoptotic signaling pathways. stimulation of pbmcs with the tlr ligands poly i:c, odn, and lps induced a 200fold increase in ifi27 and a 30-fold increase in ifna1 and ifnb1 transcription compared to baseline. one patient had constitutive upregulation of stat1 and stat6 in monocytes and of stat1 and stat3 in t cells. conclusion: we describe a novel immunedysregulatory disease caused by de novo truncating variants in samd9l that presents similar to candle with neutrophilic pannicultis and points to an important role of samd9l on regulation of adaptive and innate immune responses. acknowledgements: this work was supported by the nih irp of niaid abstract/case report text introduction: ataxia telangiectasia (at) is caused by a defect in the atm gene which is responsible for repair of damaged dna. it is a rare, devastating neurodegenerative disease that results in ataxia and telangiectasias, particularly of the sclera and skin. those with at are at increased risk for immunodeficiency and cancer. the immunodeficiency is variable and may result in deficiency in humoral and cellular immunity in some patients. here we present a patient with ataxia telangiectasia and hypogammaglobulinemia on immunoglobulin therapy who developed recurrent urinary tract infections (uti) and sepsis. case presentation: the patient is a 23-year-old female with ataxia telangiectasia and hypogammaglobulinemia who presented with three episodes of uti, one of which resulted in prolonged hospitalization due to sepsis and acute kidney injury (aki). she presented with 4 days of flank and back pain and was hospitalized for 3 days for e coli uti. she improved on iv antibiotics and was discharged home to complete treatment with oral ciprofloxacin. due to persistent emesis, she was readmitted 2 weeks later with urosepsis and aki with a creatinine of 2.06 mg/dl, over 5 times her baseline creatinine. after additional antibiotics and iv fluids, she improved clinically, renal function normalized and she was discharged home. renal ultrasound was unremarkable with no anatomical abnormalities. she was relatively healthy prior to this with only one episode of bacterial pneumonia in 2006. she receives weekly subcutaneous immunoglobulin therapy dosed at 120 mg/kg with normal igg levels (1023, 931, 1078 mg/ dl). at baseline, she had high igm (550 mg/dl) and low iga ( < 4 mg/ dl) levels, as well as decreased t cells but normal nk and b cells (489 cells/ul cd3+, 329 cells/ul cd4+, 107 cells/ul cd8+, 211 cells/ul cd16+cd56+, and 357 cells/ul cd19+). she has hyperglycemia (on metformin), hypertriglyceridemia (on atorvastatin) and hypertension (on losartan). she is thin and wheelchair bound with bilateral telangiectasias to the sclera, neck, and chest. she has occasional eye bleeding and epistaxis, presumably from her telangiectasias. she has good hygiene and good adherence to medications. she voids voluntarily, has no indwelling urinary catheter and is not sexually active. discussion: patients with ataxia telangiectasia may have frequent viral and bacterial infections, most frequently upper and lower respiratory tract infections, as well as wart and skin infections. based on our review, this is the first reported case of ataxia telangiectasia with hypogammaglobulinemia on immunoglobulin therapy with recurrent utis complicated by urosepsis and aki. despite adequate igg levels on immunoglobulin therapy, our patient continued with recurrent utis. it is uncertain whether her non-ambulatory status, hyperglycemia or related immunodeficiency are the causes for her increased susceptibility to utis. the literature reports patients with at and bladder wall telangiectasias can result in significant hematuria, and perhaps this may be a source of entry for bacteria and consequent development of uti. this suggests that patients with ataxia telangiectasia and recurrent utis may benefit from renal ultrasound and possible cystoscopy to better visualize telangiectasias. we recommend consideration of workup for recurrent utis in patients with ataxia telangiectasia. abstract/case report text objectives: patients with partial rag deficiency frequently present with humoral autoimmunity suggesting breach in tolerance mechanisms and subsequent expansion of autoreactive b cell clones. here we aim to trace polyreactive b cells and their descendants at b cell developmental stages through our in-house bioinformatic pipeline, immchaintracer (ict). methods: the b cell receptor (bcr) was expressed as monoclonal antibodies from single sorted mature naive b cells (n=30-50 per donor) from patients with hypomorphic rag deficiency and healthy donors. xthe recombinant monoclonal antibodies were screened for polyreactivity (dsdna, insulin, lps and ifnα) by elisa. in parallel, igh repertoires were deep sequenced from sorted mature naïve, activated naïve and memory b-cell compartments. our in-house assembled bioinformatic pipeline called immchaintracer (ict) was applied to track down the descendants of cloned autoreactive igh sequences in repertoires of subsets above. results: igh sequences (n=125 including 45 polyreactive, 80 nonpolyreactive clones) from mature naive b cell from six patients with partial rag deficiency and 3 healthy donors (n=91 including 7 polyreactive, 84 non-polyreactive) were analyzed with our novel inhouse bioinformatic approach to track lineage fate in repertoire at specific developmental stages. interestingly, 28.8% of the patients' sequences and their descendants were identified in their mature naive, activated naive or memory b cell repertoires, while none of the analyzed healthy donor clones were found at later subsets. furthermore, genealogical analyses of related clones revealed lineage expansion and progressive positive antigen selection of the autoreactive clones in the patients. conclusions our findings demonstrate that peripheral tolerance checkpoint is broken in hypomorphic rag patients. our novel method enables tracing the fate of autoreactive naive b cells in the effector repertoires. we have shown that impaired b cell tolerance allows the expansion and combination of sirolimus and rituximab therapy controlled his autoantibody production which was an important goal for his autoimmune condition. we present a new treatment approach for anti-nmda receptor encephalitis. rituximab was tried on these cases before but the combination of sirolimus and rituximab therapy was never given before. we now recommended that on refractory cases of anti-nmda receptor encephalitis, combination of rituximab and sirolimus therapy can be tried. (247) submission id#812639 mosaic variants in immune function genes identified through exome sequencing stable with no interim infections and is doing well on thyroid hormone replacement. the current plan is for the patient to undergo a stem cell transplant for the arpc1b deficiency as he is at high risk for recurrent infections and severe disease. although this gene mutation is rare, review of the current literature describes patients with this condition that have undergone stem cell transplant and have done well. at this time, this seems to be the best option for management, and it may potentially be curative. abstract/case report text introduction: common variable immunodeficiency (cvid) is a disorder characterized by impaired immunoglobulin production and frequent or recurrent infections, but also associated with an increased risk for developing malignancies such as lymphomas. although intravenous and subcutaneous immunoglobin g replacement has been successful in reducing the number of bacterial infections and prolonging survival, it fails to address other complications that arise from this disorder. we report a case of a patient with cvid who developed mycosis fungoides (mf). mf is a rare form of cutaneous t-cell lymphoma, occurring in about 1 in 100,000 to 350,000 individuals, lack of treatment could potentially be fatal. case presentation: a 44-year-old caucasian woman with a history of ulcerative colitis, allergic rhino-conjunctivitis and tonsillectomy was referred to the immunology clinic for evaluation of low serum immunoglobulins. there was no family history of infections or immune deficiencies, but paternal grandfather had colon cancer and maternal grandmother had lung cancer. the patient reported frequent episodes of bronchitis, and sinus infections. immunizations were up to date for her age. medications included azathioprine, cetirizine, fluticasone nasal spray, hyoscyamine, montelukast, lactobacillus, omeprazole, and olopatadine ophthalmic solution. no history of frequent use of systemic steroids. initial serum immunoglobulins revealed normal ige and igm but low iga (72 mg/dl, normal range 81-463 mg/dl) and low igg (522 mg/dl, normal range 694-1618 mg/dl). cbc with differential, lymphocyte subsets, c3 and c4 levels were normal. while she had adequate protective titers against haemophilus influenza type b, diphtheria and tetanus, titers against pneumococcus were < 50% protective and she failed to mount an adequate response to pneumococcal polysaccharide vaccine. given her diagnosis of cvid, she started scig (500 mg/kg every 2 weeks). a year later, she developed a bilateral nonpruritic rash in the abdomen and upper trunk. initial skin biopsy suggested a drug reaction. a subsequent biopsy revealed a superficial perivascular lymphoid infiltrate with focal epidermotropism and positive t cell receptor gamma gene rearrangement, consistent with mf. testing for cell t receptor beta gene rearrangement was negative. while mf treatment consisted of triamcinolone 0.1% ointment, treatment for ulcerative colitis transitioned from azathioprine to vedolizumab. conclusions: cutaneous t cell lymphomas, although uncommon, can be seen in cvid. there are several reasons for the increased risk of lymphoma in cvid. the role of chronic infections and the development of lymphoma as of yet, is not clear. skin reactions to scig products in the areas of infusion are relatively common and resolve promptly. high index of suspicion is crucial in obtaining tissue sample to confirm or rule out malignancy therefore avoiding delaying proper treatment. abstract/case report text patients with lipopolysaccharide responsive beige-like anchor protein (lrba) deficiency present with a plethora of immune related defects including a defective humoral response characterized by low numbers of switched memory b cells and plasma cells, as well as an impaired production of antibodies, leading to recurrent infections. however, the molecular mechanisms behind the defective b cell response remain unknown. to gain better insights into the possible roles of lrba in b cell physiology, we screened for lrba-interacting proteins using computational predictions. twenty-seven proteins involved in vesicle trafficking and autophagy were identified as potential lrba-interacting partners. to validate those potential lrba interactions, we performed coimmunoprecipitations and proximity ligation assays (pla), finding that endogenous lrba interacts with the phosphoinositide 3kinase regulatory subunit 4 (pik3r4) in b cells. pik3r4 (aka vps15) is the regulatory subunit of vps34, the catalytic subunit of the pi3k-iii complex, which acts as a positive regulator of autophagy by producing phosphatidyl inositol-3 phosphate (pi(3)p). autophagy is a catabolic mechanism essential for cell survival and plasma cell differentiation. in fact, we observed that reduced lrba impaired the production of pi(3)p upon autophagy induction. in addition, we observed in both lrba-deficient hela and b cells reduced mobility, abnormal accumulation and increased size of autophaghosomes, accompanied by an atypical lysosomal positioning. these abnormalities are due to a blockade of the autophagosome-lysosome fusion, as detected by reduced lc3-ii lipidation upon autophagy induction in the presence of lysosome inhibitors. interestingly, lrba-deficient hela and b cells exhibited enhanced activity of mammalian target of rapamycin complex 1 (mtorc1) signaling, a key suppressor of autophagy whose activation possibly contributes to defective autophagy. taken together, b lymphocytes lacking lrba can form autophagosomes but they fail to fuse with lysosomes. thus, we propose a role of lrba at late stages of autophagy through the binding to pik3r4. abstract/case report text apds caused by gain-of-function mutations (gof) in the genes (pik3cd and pik3r1), encoding for the p110δ and p85 subunits of phosphoinositide 3-kinase δ (pi3kδ), results in hyperactivation of the pi3k/akt/mtor/s6k pathway and lead to immune dysregulation, lymphoproliferation and immunodeficiency. apds manifests with respiratory tract infections, bronchiectasis, susceptibility to herpes group viruses, autoimmunity, cytopenia, lymphoproliferation and lymphoma. gastrointestinal system manifestations include enteropathy, colitis, and liver disease. eosinophilic esophagitis (eoe) or eosinophilic gastrointestinal disease (egd) have been under diagnosed in reported apds cohorts. objectives: to review the incidence, demographics and relevant clinical data for eosinophilic gastrointestinal disease in a single center apds cohort. methods: review of clinical and laboratory findings from 70 apds patients followed at the nih clinical center, from 2005 to 2019. results: 12 patients were either historically diagnosed or actively studied at our center for egd. incidence of all egd is 17 % in our cohort and all patients had mutations in pik3cd, none in pik3r1. most patients also had multiple gi manifestations. conclusion: immunopathology and genetic predisposition leading to eoe is complex. eosinophilic gi disease including eoe appears to represent significant gi pathology in apds. this implies that activation of pi3k pathway may be directly involved in the etiology of eoe. abstract/case report text introduction: dominant negative mutations in stat3 (lof stat3; job's syndrome) cause a primary immune deficiency characterized by eczema, recurrent skin and lung infections, and connective tissues and skeletal abnormalities. over the last several years, vascular abnormalities causing tortuous and aneurysmal middle-sized arteries have increasingly been recognized. our institution has been imaging prospectively the coronary and cerebral arteries since 1999 -2019 for brain imaging and from 2004-2019 for heart imaging. the purpose of this review is to provide an update on the extent of clinical manifestations noted in hies (continued) pain improved but rapidly worsened with tapering of steroids. the pet/ ct at that time, showed extensive hypermetabolic areas in lymph nodes, femurs, left acetabulum, left pubic ramus, right ischium, sacrum, both iliac bones, eight rib, t4 and t8, both clavicles, both humeral, manubrium, and extension into musculature. initial biopsies were culture negative, 16s was positive for mac. she was referred to the national institutes of health where she was diagnosed with autoantibodies to interferon-γ . prior to referral, she initially was treated with azithromycin, ethambutol (need to discontinue due adverse effects), amikacin, rifampicin, linezolid, with not no evidence of clinical response. she underwent debridement of epidural anterior abscess to t8. surgery involved t8-9 laminectomy, and curettage on several bones. she presented unable to walk secondary to pain and neuropathies. initial laboratory: crp:90mg/l; wc 13.51; hgb: 8.2g/dl; ana :4.6(strongly positive) cd20: 373 ul her first course of rituximab consisted of 7 doses of 1gm at d0, 14, 42 and monthly thereafter for a total of 7 doses with clinical and radiographic improvement. she was maintained on optimal antibiotics therapymeropenem, rifampin, azithromycin, moxifloxacin, and clofazimine. two years after she completed rituximab, she presented to her home hospital with increased left hip pain and biopsy grew mac. retreatment with rituximab failed to show clinical improvement. her medical regimen was augmented with tedizolid and bedaquiline, however no iv antibiotics were added. rituximab was reinitiated, after the progression of symptoms despite treatment with rituximab; bortezomib, was trialed using the schedule based on the multiple myeloma literature. she completed 5 full cycles (two at the nih, three at home). she subsequently has had clinical improvement and is working again and has not had progressive neurologic decline. the titers of antibodies to interferon-· did not follow the clinical improvement. however, we plan to keep her cd20+ cells zero and continue the bortezomib given her clinical and radiologic improvement. abstract/case report text for patients with primary immunodeficiencies (pid), finding a genetically defined diagnosis can be critical for prognosis, treatment, and counseling. however, for many patients, determining a genetic etiology remains elusive despite routine gene panel and exome sequencing because of an inability to resolve variants of uncertain significance (vus). crispr-based genome editing could be used to address this need by introducing patient-derived vus into primary human immune cells for further study; however, existing techniques are limited by poor efficiency. we recently developed novel non-viral techniques for large gene editing in primary human t cells and hematopoietic stem cells (hscs). we achieve up to 8fold greater efficiency than existing tools using crispr cas9 ribonucleoprotein nanoparticles that are non-covalently linked to homology directed repair (hdr) template dna. whereas mutation analysis has previously been limited to expensive and time-consuming animal models and transformed cell lines, we now have the ability to rapidly recreate any mutation in the native gene locus in otherwise healthy primary human cells. we demonstrate this ability by using our technique to knock-in well characterized loss-of-function mutations in jak3 and il2rg and gain-of-function mutations in jak3 that are known to cause severe combined immunodeficiency and tumor growth, respectively. we show that these recreated mutations have the expected effects on t cell proliferation and intracellular signal transduction in the setting of il-2 stimulation. we then use our technique to investigate a prototype case of an adult patient with the unusual combination of common variable immunodeficiency, inflammatory arthritis and uveitis, and neutrophilic urticaria. genetic testing in this patient had previously revealed heterozygous coding vus's in four genes previously associated with a pid disease, including jak3, but none of the specific variants have been previously reported. it is thus not clear which mutation (if not more than one) causes this patient's dysregulated cell activation, which limits targeted treatment options with kinase inhibitors or future gene or cell therapy. by knocking our patient's jak3 variant into primary human t cells and comparing these cells to those carrying wild-type, known loss-of-function, and known gain-of-function mutations, we are able to rapidly characterize the functional impact of our patient's variant and isolate its effect on his complex phenotype. this in vitro genetic engineering approach thus allows patient-specific vus to be modeled directly in primary human immune cells with a rapid turnaround time that is relevant for clinical applications, including molecular diagnosis and screening of pharmacologic or gene therapies. further, similar strategies could be leveraged as a potential basis for future gene correction therapy. abstract/case report text definition : "at variants" comprise a heterogeneous group characterized by the later onset of clinical symptoms, a slower progression, a prolonged lifespan compared to most patients with at and decreased levels of chromosomal instability and cellular radiosensitivity. in these patients, telangiectasia and / or immunodeficiency may be absent, while neurological features are present.(1). material and methods :a 4 years old girl, born out of a non-consanguineous parents with clinical picture consisting in progressive alteration of the march (15 months), associated to exotropia, no weight gain or height, alteration of balance while she is sitting, she walks by herself. she has 1 acute bronchiolitis. results and discussion: brain magnetic resonance without alterations. abdominal ultrasonography and cpk normal. ophthalmologist assessment found exotropia, he did not find telangiectasia. motor and sensitive neuro-conduction were reported normal. alpha fetoprotein 102 ng / ml increased. karyotype xx, non-structural alterations. normal auditory-visual evoked potentials. whole exome sequencing (wes): identified the small homozygous pathogenically deletion ¬c.5496 + 2_5496 + 5del taag; p.? have a spelling effect in the splicing. it is not present in the population database or not as a known variant in function mutation of smad4 has been shown to increase smad phosphorylation in the nucleus in fibroblasts. further research is needed to examine the role of this mutation in t and b lymphocytes, given the interesting immunological phenotype of this patient. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. outbreak of mycoplasma pneumoniae-associated stevens-johnson syndrome characterization of children. with recurrent episodes of stevens johnson syndrome recurrent stevens-johnson syndrome secondary to mycoplasma pneumoniae infection recurrence and outcomes of stevens-johnson syndrome and toxic epidermal necrolysis in children syndrome after mycoplasma pneumoniae infection a review of causes of steven-johnson syndrome and toxic epidermal necrolysis in children submission id#812369 expanded phenotype and genotype-phenotype correlations bsc(hons) msc bmbs mrcp 6 dominant activating rac2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects an autosomal dominant scid form due to a gain of function mutation in the rac2 gene national institute of allergy and infectious diseases (niaid) professor biochemistry/molecular pharmacolog/nyu langone health canada 5 head of the ircm bioinformatics core facility/montreal clinical research institute (ircm) associate investigator/national human genome research institute (nhgri) division of intramural research (dir), national institute of allergy and infectious diseases (niaid) translational and functional genomics branch/chief and senior investigator of the translational and functionanational human genome research institute (nhgri) division of intramural research (dir), national institute of allergy and infectious diseases (niaid) formula versus donor breast milk for feeding preterm or low birth weight infants cytomegalovirus (cmv) inacɵvaɵon in breast milk: reassessment of pasteurizaɵon and freeze-thawing prevenɵon of cytomegalovirus transmission via breast milk in extremely low birth weight infants submission id#812554 the costa rican registry for primary immunodeficiencies department chief/department of pediatric immunology and rheumatology, national children´s hospital "dr. carlos sáenz herrera national children´s hospital "dr. carlos sáenz herrera" cid 13% (n=38), cid with syndromic features 54% (n=161, at 132), antibody deficiencies 13% (n=38, xla 15) ada deficiency, 1 cd40l-, 2 flh-4 (syntaxin 11 deficiency), 1 ipex and 8 osteopetrosis. nineteen patients developed malignancies, mainly non-hodgkin lymphomas among at patients conclusions: during the study period, 298 pid were registered, mainly at and osteopetrosis cases. scid represented 12% and xla 11% of patients allergy and immunology/ department of pediatric immuunology and texas children's hospital director immunogenetics program/department of immunology, allergy and rheumatology at baylor college of medicine 8 associate professor, director food allergy program/department of pediatric immuunology shearer center for human immunobiology 10 associate professor/department of pediatric immuunology, allergy, and retrovirology granuloma faciale: successful treatment of nine cases with a combination of cryotherapy and intralesional corticosteroid injection intralesional steroid injection: a novel method to treat the symptoms of idiopathic granulomatous mastitis long-term effectiveness of intralesional triamcinolone acetonide therapy in orofacial granulomatosis: an observational cohort study sarcoidosis in the periocular scar as the first finding of systemic sarcoidosis: clinical-radiological characteristics a case of scar sarcoidosis of the eyelid submission id#812563 null mutations: a gene dosage effect shubham goel, phd 1 , hye sun kuehn clinical centre, nih, 2 staff scientist/immunology service certified molecular geneticist/national institute of allergy and infectious diseases, national institutes of health genetic counselor/national institute of allergy and infectious diseases, national institutes of health division of intramural research; chief, immunopathogenesis section/laboratory of consultant/university hospital freiburg center for pediatrics and adolescent medicine cci -center for chronic immunodeficiency 16 medical director professor/division of blood and marrow transplantation and cellular therapies new south wales, australia. 23 professor of medicine and pediatrics/icahn school of medicine at mount sinai 24 senior investigator/laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health 25 functional nk assay, t/b/nk panel, tlr assay phd 11 staff scientist/translational autoinflammatory disease section, lcim, niaid, nih, bethesda, md 2 research fellow/translational autoinflammatory disease section post-baccalaureate fellow/translational autoinflammatory disease section pediatric rheumatologist/department of pediatric infectious diseases and immunology shupyk national medical academy for postgraduate education alder hey children's nhs foundation trust hospital, liverpool, uk 13 pediatric rheumatologist/department of pediatrics dra. liliana bezrodnik y equipo dra. liliana bezrodnik y equipo"-servicio de inmunología htal. de niños hospital de niños ricardo gutiérrez 19 pathology department/children's hospital of philadelphia, philadelphia, pa 20 post-doctoral fellow/translational autoinflammatory disease section results: we identified 6 patients with 4 de novo frameshift variants in samd9l (c.2626dela, p.i876lfs*15 reduced toxicity allogeneic hct with a busulfan, fludarabine regimen: a promising approach for non-cgd primary immune deficiencies requiring myeloablation? phd 2 , blachy davila saldana college of medicine 2 assistant professor/division of bone marrow transplant, immunedysregulation and immuno-hematology program, aflac cancer and blood disorders center 6 instructor of pediatrics/division of bone marrow transplantation and immune deficiency, cincinnati children's division of bone marrow transplantation and immune deficiency, cincinnati children's national institutes of health 2 certified molecular geneticist/national institute of allergy and infectious diseases genetic counselor/national institute of allergy and infectious diseases, national institutes of health operations manager and genetic counselor/national institute of allergy and infectious diseases collaborative bioinformatics resource/national institute of allergy and infectious diseases genome in a bottle analysis team/genome in a bottle consortium division of intramural research (dir), national institute of allergy and infectious diseases (niaid) medical genomics and metabolic genetics branch/national human genome research institute, national institutes of health table 1 clinical and demographic characteristics, severity measures, and previous treatments for the pid cohort geographic region, n (%) north central 66,736 (17.5) 406 (15.6) 3,022 (19.7) northeast 69,027 (18.1) 302 (11.6) 2,179 (14.2) south 9) 601 (23.1) 4,497 (29.3) high-potency oral antibiotics 130,153 (34.1) 967 (37.1) 4,720 (30.8) systemic high-dose corticosteroids copd, chronic obstructive pulmonary disease cd4 / cd8: 2.73 , lymphocytes b 161 , natural killer 314 cell/mm3. conclusions: although the atm activity corresponding to this new splicing mutation is unknown, it is presumed that it has some residual function, since splicing mutations is associated with better neurological prognosis have been reported (pidd) . at the immunodeficiencies research unit we discuss and pursue the molecular and genetic diagnoses for patients with suspected pid from all around the country. starting this year, we are processing and analyzing our own patients' wes results at the unit. evaluating the diagnostic yield of wes, as a measure of effectiveness or quality control, may result in process optimization and perhaps allow for better patient selection and resource allocation. objective: to describe and characterize our patients with suspected pidd whose dna samples were sent out for whole-exome sequencing; to analyze and compare our wes diagnostic yield after the first 3 batches of patients; to identify patient attributes that may predict a positive diagnostic wes result. methods: genomic dna was obtained from whole-blood samples of patients with suspected pidd from hospitals in mexico city, monterrey and puebla. wes was performed using a ng sequencer (illumina hiseq) in new jersey (admera health, llc), with 90% coverage and a 50x depth of the idt xgen library, human genome version 38 (december 2013). two fastq files for each patient sample were transferred back to our unit, where the bioinformatic workflow was completed. we used galaxy in the cloud for quality control, mapping & alignment, and detection of variants; variant effect predictor to process, map, annotate and filter variants; and igv (broad institute) and genome browser (ucsc) for visualization. we defined diagnostic yield as the proportion of patients with a genetic diagnosis after analysis of their wes results. we performed multivariate logistic regression, tree partitioning algorithm and linear abstract/case report text a 15-year-old male with x-linked chronic granulomatous disease (cgd) complicated by severe perianal disease and proctocolitis presented with two weeks of open draining lesions on the thighs, bilateral inguinal regions, and gluteal cleft. the wounds became excruciating and prevented normal ambulation. the patient was admitted for iv antimicrobial therapy, local wound care and systemic steroids. wound cultures, throughout his course, yielded growth of klebsiella pneumonia, candida parapsilosis, malassezia globose, escherichia coli, enterococcus faecalis and staphylococcus epidermidis allowing for directed antibiotic and antifungal therapies. despite improvement, the wounds persisted after several weeks of treatment. faced with recalcitrant cutaneous lesions despite aggressive systemic and topical therapies, we looked to alternative options. noting that other granulomatous diseases show response with intralesional corticosteroid therapy, we considered this for our patient (1, 2) . for example, patients with idiopathic granulomatous cheilitis had a complete response after three monthly injections of intralesional corticosteroids (3) . sarcoidosis patients also improve with intra-granuloma corticosteroid injection (4, 5) . our patient received 20 mg triamcinolone acetonide injections two separate occasions, administered in multiple open lesions at eight-week intervals. the cutaneous lesion improvement was gradual and complete resolution of the first open wound was noted fifty-two days from initial steroid injection. to our knowledge, intralesional glucocorticoid therapy has not previously been used to treat cutaneous disease in cgd patients. we are reporting the first cgd patient with successful lesion resolution following steroid injection as part of therapy. as such, we believe this case is significant and suggests that direct lesion injection with glucocorticoids can add to treatment options for cgd patients with recalcitrant cutaneous disease. serum igg, alone or in combination with iga and/or igm, were reduced in all patients. t-follicular helper cells were reduced only in patients carrying biallelic mutations (a.ii.1, a.ii.2 and a.ii.3) but not in any patients with monoallelic tcf3 null mutation. t cell enumeration and function by means of proliferation was normal in all mutation+ individuals. no mutated (truncated) protein expression was detected from patients with either biallelic or monoallelic tcf3 null mutations. however, wildtype tcf3 protein was detectable in about half amount in heterozygous patients. cdna data showed either 0/100 or 50/50 wt/mutated transcripts ratios in homozygous or heterozygous individuals, respectively, suggesting mutated proteins instability; and all together, protein haploinsufficiency for the heterozygous cases. ex-vivo, cd40l and il21-induced plasmablast differentiation was found to be reduced in 4/5 patients tested (1 biallelic patient a.ii.2 and 3 monoallelic patients a.i.2, b.i.1 and c.i.1). moreover, decreased igg, iga and igm production in vitro correlated with reduced plasmablast cell differentiation.in conclusion, all individuals carrying either mono-or biallelic null mutations have immunological penetrance of the b cell defect. however, while clinical penetrance was complete in patients with biallelic mutation, it was partial for those with monoallelic tcf3 null mutation suggesting a gene dosage effect for clinical penetrance. in addition, our study emphasizes that tcf3 is relevant to the plasmablast differentiation process as well as for ig production. further studies are being conducted to evaluate the individual roles of e12 and e47 on the immune and clinical features. background: heterozygous gain-of-function (gof) mutations in the stat1 gene result in a hyperphosphorylated state where patients develop recurrent or persistent chronic mucocutaneous candidiasis (cmc), other cutaneous mycosis, bacterial infections, disseminated dimorphic fungal infections and autoimmune disease. furthermore, the nk defect we characterized illustrated an immature cd56 dim nk cell subset with decreased expression of cd16, perforin, cd57, and impaired cytotoxic capacity associated with increased susceptibility to viral infections observed in these patients. methods: in this study, we evaluated 2 patients with novel stat1 mutations (d292e mutation, located in coiled-coil domain and k388q mutation, located in dna-binding domain). a third patient with the previously reported v266i mutation (ccd) was also recruited for this study. in vitro, pbmcs from these patients were stimulated with ifn-α for 30, 60, and 120 minutes and levels of phospho-stat1 on cd56 dim nk cell subset were measured by flow cytometry. the stat1 activity (firefly and renilla luciferase activities) was evaluated in u3a-stat1 deficient cells abstract/case report text background: t cell lymphopenia associated with genetic syndromes can be identified with low t cell receptor excision circle (trecs) up on the newborn screen for severe combined immune deficiency (scid). jacobsen syndrome (js), 11q terminal deletion, is a rare genetic disorder seen in 1/100,000 births characterized by facial dysmorphisms, platelet abnormalities, neurologic complications, immune system abnormalities including t and b cell defects. we report an infant with js found to have low trecs on nbs and review the immune phenotypes of our cohort of 7 patients with js. methods: a retrospective chart review of all patients with js seen by the allergy immunology service at a large tertiary referral center from 12/1/ 14-12/1/19 was performed in accordance with irb standards. result: the index patient had two newborn screens 24 hours after birth and two weeks later in accordance with texas state law. the first nbs resulted normal trecs while the second nbs had low trecs. a third nbs was done per protocol and again showed low trecs. subsequently, lymphocyte subsets at 2 months of age showed severe t-cell lymphopenia: cd3 575 cells/ dl (61%), cd4 351 cells/dl (21%), cd8 208 cells/dl, and low recent thymic emigrants (cd4+cd45ra+ccr7+cd31+) 42 cells/dl (12%) with normal lymphocyte mitogen proliferation. a chromosomal microarray (cma) revealed a 11q deletion known to cause js.over the five year study period we evaluated seven patients with js referred to our center. the majority of patients (85%) presented to clinic with history of recurrent infections including recurrent pneumonia, sinusitis, otitis media, skin abscesses and warts. t-cell lymphopenia was found in 3 of 7 (43%), 2/7 (29%) had abnormal lymphocyte proliferation (mitogens and antigens) and 2 met criteria for pjp prophylaxis. in addition, 4/ 7 (57%) had antibody deficiency requiring igg replacement therapy. of the 7 cases reviewed, only 2 patients were born during the period of time that texas was performing the nbs. conclusion: jacobsen syndrome can present with a spectrum of immune defects most notably t cell lymphopenia and antibody deficiency. these patients can present at birth with low trecs. this cohort analysis highlights the importance of considering chromosomal genetic syndromes with features of primary immunodeficiency in evaluating patients with low trecs. further evaluation of larger cohorts gathered from neurology or genetics clinics at multiple centers would be helpful for future study in identifying those who need close immunology care. abstract/case report text introduction: recognized as sentinels of the immune system, mast cells (mcs) and dendritic cells (dcs) are both derived from hematopoietic/ progenitor stem cells in bone marrow (bm). the crosstalk and direct contact between these cells have been well documented and play an important role in modulating immune response. we showed that presence/absence of il-3 directs cell fate; whether progenitor cells will be differentiated into mcs or dcs. we also report an easy method in which in vitro generation of dcs is possible without external induction of gm-csf or il-4. material-methods: to produce mcs and dcs in vitro, briefly bm samples were collected from patients with idiopathic thrombocytopenic purpura., bm mononuclear cells (mncs) were seperated by ficoll gradient, and seeded in plates with imdm medium containing fbs 2%, pen/ strep, and a little amount of methocult, and incubated in 37oc, 5% co2 (day 0). the treatments on the following days were as follows: day 4, imdm (fbs 1%) + scf (100ng/ml) + il-6 (50ng/ml); day 9, imdm (fbs 2%) + scf (100ng/ml) + il-6 (50ng/ml) + il-3 (1ng/ml). on day 18, 3 groups were formed: group i: imdm (fbs 2%) + scf (100ng/ml) + il-6 (50ng/ml) + il-3 (30ng/ml), group ii: imdm (fbs 2%) + scf (100ng/ml) + il-6 (50ng/ml), group iii: dmem + fbs 10%. the cultures were evaluated every 2 days under an inverted microscope. verification of mcs was performed by toluidin blue and tryptaseimmunoflorescence staining. macrophages were verified by cd-68 immunoflorescence staining. dendritic cells of different stages of abstract/case report text introduction a reduced toxicity busulfan, fludarabine regimen with alemtuzumab or anti-thymocyte globulin (gungor et al.) was efficacious in patients undergoing allogeneic hct for cgd. we report our experience with a similar approach for patients with non-cgd primary immune deficiencies needing a reduced toxicity myeloablative approach. methods we retrospectively reviewed records of consecutive patients who underwent allogeneic hct for primary immune deficiencies with a preparative regimen containing busulfan, fludarabine and alemtuzumab or anti-thymocyte globulin(atg), at three transplant centers between 2015-2018. busulfan was given either every 6 hours over 4 days with target auc of 875 to 1025 μmol/min (based on q6 hr. dosing) or twice daily over 4 days with target auc of 1800 to 2000 μmol/min (based on q12 hr. dosing) or once daily over 4 days with a target auc of 3600-4400 μmol/min (based on q24 hr. dosing). fludarabine 150 mg/m2 to 180 mg/ m2 was given divided over 4-6 days. serotherapy included alemtuzumab 0.3 -1.0 mg/kg or atg 7.5 mg/kg given divided over 3 days. gvhd prophylaxis consisted of cyclosporine and mycophenolate mofetil. results forty patients (was=12, hlh=10, cd40l deficiency=7, ipex/veoibd=4, scn=2, ifngr1 def./cid/x-scid/msn1/ lad=1) received busulfan, fludarabine and alemtuzumab or atg for allogeneic hct (first hct in 31 patients and second hct in the 9 patients with hlh). median age was 2.0 years (range, 0.25 years -19.8 years). patients received a graft from an hla-matched related (n=11), unrelated (n=27), or single allele mismatched related or unrelated donor (n=2). all except one patient engrafted at a median of 13 days (range,11-34 days). one patient developed veno-occlusive disease and two patients developed diffuse alveolar hemorrhage. notably, it was the second transplant for all 3 patients. eight patients (20%) developed grade 2-3 acute gvhd and 2 patients (5%) developed chronic gvhd. one patient developed primary graft failure and two patients secondary graft failure. nineteen patients (48%) maintained full donor (>95%) chimerism following allogeneic hct. twenty patients (50%) developed mixed chimerism, predominantly in the t-cell lineage, but t-cell donor chimerism progressively increased post-hct. at 1-year post-transplant, 15 of 20 patients (75%) with mixed chimerism had donor myeloid chimerism >90% and t-cell chimerism >75%. two patients underwent a second transplant for graft failure. there were 6 deaths in the cohort. overall survival was 85%(34 of 40) and event free survival was 80%(32 of 40) at 1 year. conclusion our experience suggests that a reduced toxicity busulfan, fludarabine regimen with alemtuzumab or atg as serotherapy offers a promising approach with low toxicity, durable myeloid engraftment, low incidence of grade 2-4 gvhd and excellent survival and can be considered for a variety of primary immune deficiencies where myeloablative hct is desired. abstract/case report text this is an 18-year-old patient who has the diagnoses of anti-nmda receptor encephalitis with associated catatonia. the patient has history of a pineal gland germinoma diagnosed in august 2018 after 8 months of double vision. however, after several months, the patient developed difficulty in sleeping, anxiety and nightmares. the patient presented to emergency department in february 2019 with personality changes. he was diagnosed with nmda encephalitis based on the clinical findings as well as presence of elevated serum and csf anti-nmda antibodies. the patient was initially treated with high dose systemic steroids with poor response. due to the worsening of his clinical condition, he was started on plasmapheresis, but had poor response to this therapy as well. these treatments are known standard treatment options for this condition. during his hospital stay, different therapies were discussed. cyclophosphamide was one of the treatment options, but because of the side effect profile and severe toxicity, we recommended different treatment modality. we started the patient on rituximab as well as sirolimus therapy to suppress both t and b-cell responses. after receiving two doses of rituximab in addition to daily sirolimus, the patient showed improvement of his symptoms. and declining nmda receptor antibody titers. this treatment plan was chosen for autoantibody mediated encephalomyelitis due to the fact that rituximab has inhibitory effect on naive b cells, but not on the proliferation of memory b cells and sirolimus has profoundly inhibiting role on memory b cells as well as a t-cell responses. a mosaic gene variant is one which is present in some, but not all, cells within an individual. they are increasingly associated with a number of diseases, including a number of primary immunodeficiency disease (pid). here we systematically analyzed mosaic variants in known or putative immunodeficiency genes from exome sequencing data from 998 individuals. lofreq was used to identify mosaic variants with a variant allele fraction (vaf) of 0.05-0.30 (0.50 is the vaf for a non-mosaic, heterozygous variant). we removed variants from extreme read-depth (> 2 standard deviations), unmappable, repeat-rich, and duplicated genomic regions. the average number of detected variants per mb was 3.2 and the total number of genomic locations with variants was 65,446. mosaic variants were underrepresented in exonic regions, suggesting that coding variants may be deleterious. more mosaic variants were detected in saliva exomes compared to peripheral blood exomes (p-value < 1 x 10-5), suggesting tissue-specific mosaic variants in buccal epithelial cells and white blood cells of saliva. to understand the clinical relevance of these findings, the variants were further filtered to include only nonsynonymous variants found in fewer than 1% of samples, leaving a total of 6,808 variant locations. of the remaining variant locations, 40 had a pathogenic assertion in the human gene mutation database, and 140 were in international union of immunological societies pid genes. further variant interpretations and clinical correlations are underway. these data suggest that mosaic variants in pid genes are common, vary by location of collection, and may have clinical diagnostic relevance. abstract/case report text introduction/background: the arpc1b (actin related protein 2/3 complex subunit 1b) gene is a protein coding gene prominently expressed in blood cells and is necessary for the assembly and maintenance of the human actin-related protein 2/3 complex (arp2/3). actin polymerization plays a central role in many immune functions including proliferation and differentiation of immune cells, migration, intercellular and intracellular signaling and activation of both innate and adaptive immune responses. defects in the actin cytoskeleton affect hematopoietic cells in the bone marrow and the immune response giving rise to a distinct primary immune deficiency, which is phenotypically similar to wiskott-aldrich syndrome (was). arpc1b deficiency clinically presents as a severe multisystem disease which includes platelet abnormalities, recurrent infections, failure to thrive, inflammatory changes in the intestine, eczema, cutaneous vasculitis, eosinophilia, and elevated inflammatory markers. arpc1b deficiency is rare and has only recently been described in the literature. here we present a clinical case of a patient found to have a pathogenic arpc1b mutation via whole exome sequencing. case report: here we describe a 15 month old somalian boy who presented to the immunology team at 9 months of age with hematemesis, hematochezia, melena, failure to thrive, atopic dermatitis, hypothyroidism, autoimmune thrombocytopenia and recurrent infections concerning for a primary immune deficiency. family history was notable for parental consanguinity and an older sibling with a similar presentation of hemorrhagic gastroenteritis who died in kenya around 3 months of age due to complications of his symptoms. the initial primary immunodeficiency evaluation revealed normal inflammatory makers, normal igg and protective vaccine (prevnar and tetanus) response. iga and ige were elevated at 364 mg/dl and 99.6 ku/l respectively. flow cytometry was remarkable for t cell lymphopenia (cd3 1541, cd4 1163, and cd8 363) with reduced naïve cd4 and cd8 t cells. b and nk cell count were normal. alps panel and was protein expression was unremarkable. whole exome sequencing was performed and revealed homozygotic mutation of arpc1b c.392t>c, ivs4+2t>c which was predicted to be a pathological variant. subsequently, dhr flow cytometry with fmlp showed significant increase of dhp fluorescent (mfi 9.7 when compared to control mfi 3.7) consistent with findings from other arpc1b deficiency patients. at the time of the most recent clinic visit, the patient has remained abstract/case report text introduction: activation of the nod-like receptor family cardcontaining 4 protein (nlrc4) leads to the formation of an inflammasome. the inflammasome, a large cytosolic multiprotein complex of innate immunity, promotes proteolytic cleavage, maturation, and secretion of pro-inflammatory cytokines, including il-1 and il-18. a gain-of-function mutation (gof) in the gene encoding nlrc4 or nlrc4-inflammasomopathy is characterized by hyperinflammation with persistent elevated il-18, infantile enterocolitis, and early-onset macrophage activation syndrome (mas). objectives: to describe phenotypic variation among three siblings with a novel nlrc4 gof variant and to expand our current understanding of the clinical manifestations of the disease methods: clinical and laboratory features were studied in three siblings of a hispanic family with a novel nlrc4 gof variant. results: a novel variant, c.1475g>a (p.arg492gln) on the nlrc4 gene, was identified in a 3-year-old male with recurrent febrile episodes since one year of age. his laboratory findings showed highly elevated esr, crp, il-18, and fecal calprotectin. his endoscopic finding was unremarkable. the recurrent fever partially responded to canakinumab. a 10-year-old sister with ileocolonic crohn's disease for two years was found with the same nlrc4 variant and highly elevated il-18. crohn's disease was well controlled after adding infliximab infusion to methotrexate therapy. a15-year-old sister, who has been asymptomatic and healthy, was tested with the same positive nlrc4 variant and highly elevated il-18. the nlrc4 variant is inherited from their father, who currently has a diagnosis of psoriasis vulgaris. the il-18 levels of the three siblings show in the figure 1 patients with flh-4 develop the classic hlh phenotype early in life, with periods of remission. the pathogenesis is associated with a defect of perforin-dependent cytotoxicity. t-ctl and nk cells fail to remove abnormal cells, consequently, an uncontrolled proliferation and activation of cd8+ t cells and macrophages develops and generates an inflammatory cytokine storm and a solid organs infiltration. mortality of fhl-4 is very high without treatment; allogeneic hsct is the only curative therapy. we report the outcome of a child with fhl-4, four years after his treatment with an allogeneic hsct using an hla haploidentical donor. case report: a boy born in december 2007, previously healthy, the only child of parents without consanguinity. he has a normal family and perinatal history. when he is 7 years old, he begins suffering from with fever, arthralgia, hepatosplenomegaly and pancytopenia. he meets the diagnostic criteria for hemophagocytic lymphohistiocytosis (hlh) and received treatment with iv methylprednisolone and entered in remission of its hlh. six months later, he restarts his clinical picture of hlh and j clin immunol receives treatment with the hlh-2004 protocol, which leads to remission. ayear later he presents a new episode of hlh that responds to ivig, cyclosporin a and dexamethasone. the possibility of primary hlh is then suggested. when he is 8 years old, the molecular diagnosis is made with the identification of a mutation in the stx11 gene, the case was classified as fhl-4 due to syntaxin 11 deficiency. in october 2015, a hsct was performed. at the age of 12, his clinical status is evaluated and laboratory studies show that his immunodeficiency due to syntaxin 11 deficiency was cured. his 42-year-old mother, hla haploidentical was used as a donor and a protocol developed for the treatment of osteopetrosis was applied. the hsct protocol did not use a tcell depleted graft. hstc conditioning was done with melphalan days -11 and -3, fludarabine days -7 to -2, anti-thymocyte globulin days -7 and -6, and cyclophosphamide day -2. (figure) . clinical and demographic characteristics, including the use of a novel claims-based weighted algorithm (risk vital sign; rvs) and those initiating ivig and scig, were described. stratified analysis based on pid diagnosis codes was performed. probability of receiving available ig treatments based on baseline characteristics was evaluated by logistic regression and propensity score methods. results: selected clinical and demographic characteristics, severity measures, and previous treatments between the overall pid population (n=382,131), pid patients initiating scig (n=2,604), and pid patients initiating ivig (n=15,327) are presented in the table. patient characteristics and previous treatments tended to be stable, although hypertension, obesity, and corticosteroid use increased during the study period. new ig users tended to be older and female, with increased depression, dyslipidemia, and hypertension than all pid patients. new scig users had more diagnoses of respiratory (e.g., asthma, copd) and inflammatory (e.g., arthritis, fibromyalgia, inflammatory bowel disease) comorbidities and less cancer than all pid patients. new scig users compared with new ivig users had increased asthma and copd, fibromyalgia, and inflammatory bowel disease and decreased cancer and peripheral vascular disease. previous corticosteroid use was higher in ig users than all pid patients. among scig users, prior pid treatments of iv antibiotics, and oral high potency antibiotics were similar to all pid patients. ivig users had higher iv antibiotic and antifungal use. rvs was initially developed to identify patients likely to have undiagnosed pid. this analysis applied rvs to patients diagnosed with pid to assess severity. rvs based on 1-year history in the overall pid cohort was predominantly low, with only 18.0% of patients scoring in the medium and high ranges. rvs was increased in incident ig users, with (37.4% medium/high for scig; 46.4% medium/high for ivig). in other markers of severity, scig users had more sinusitis and ivig had more pneumonia than all pid. ig users had fewer abscesses, cellulitis, and otitis media than the full pid cohort. conclusions: this exploratory analysis showed a trend toward increased hypertension, inflammatory and respiratory comorbidity, higher rvs, and previous corticosteroid treatment in patients initiating on ig compared with all pid patients. results could be confounded based on pid diagnosis codes used and warrants further research. author disclosures: mp, cas, and zh are employees and stockholders of the takeda group of companies. jo is a consultant to takeda. jbl is an employee of rti health solutions, an organization funded by takeda to conduct this research. mer was an employee of rti health solutions at the time this research was conducted.presenting author: colin anderson-smits submission topic: immunoglobulin replacement therapy patients in a larger cohort group and to determine if there are patterns of disease not previously reported. methods: we performed a retrospective chart review of patients with lof stat3 (n=81) followed at the national institutes of health. we specifically looked at tortuosity, aneurysms, and dilation of both coronary and cerebral arteries. epidemiologic information, stat3 mutation, co-morbidities, and laboratory information were reviewed along with imaging studies, specifically brain mri, brain mra, heart mri, and coronary ct. results: most recently, patients with hies are found to have vascular abnormalities including tortuosity, dilatation, narrowing, and aneurysms of middle sized, cerebral, and coronary arteries. in an effort to determine the extent of vascular involvement in addition to miscellaneous organ involvement, we are reviewing a cohort of 81 patients with hies who were evaluated at the nih. of these 81 patients, 53 are women and 28 are men. of these 81 patients, two have passed away due to vascular events leading to their deaths. there are four patients under the age of 20, 43 patients between the ages of 21 and 40, 33 patients between the ages of 41 and 60, and three patients above the age of 60 (age range 15-68, mean age 35.6).of the 81 patients, five of these patients were found to have abnormal brain mri/mra at an approximate rate of 6.2%. two of these patients were found to have at least one cranial aneurysm, two of these patients were found to have a level of narrowing or stenosis, and one patient was found to have dilatation.in terms of coronary abnormalities, 36 of the 81 (44.4%) patients were noted to have at least one coronary abnormality including dilatation, aneurysm, or tortuosity on heart mri or coronary ct. eight patients (9.8%) were found to have dilatation of which four patients were female and 4 patients were male. of the 81 patients, ten patients (12.3%) were found to have at least one aneurysm. there were 30 patients (37%) that were found to have at least mild tortuosity. conclusion: vascular abnormalities in our lof stat3 patients occurred at an exceedingly high rate-cerebral and coronary artery, 6.2% and 44.4% respectively. due to this, patient's with lof stat3 should be considered for screening with brain and heart imaging. currently, there are no guidelines which outline the appropriate timeline for screening in these patients however following these patients over time will allow us to determine the most appropriate interval for imaging follow up. abstract/case report text 63 year old woman with personal history of severe and recurrent upper and lower respiratory infections, chronic pulmonary disease with bilateral bronchiectasis and several micro nodules, chronic diarrhea without diagnosis (colonoscopy with mild colitis, without cmv. no bacterias no parasits were found in stools), mild osteopenia, focal lesion in right hepatic lobe, atopic dermatitis and anemia. she was followed up in other center and in 1996 she was diagnosed with common variable immunodeficiency (cvid) and started treatment with intravenous immunoglobulin (ivig), but with low adherence to it. she did not have referred history of lymphoproliferation nor significant viral infections. she had a daughter with spherocytosis who required esplenectomy and also had bronchiectasis and cvid diagnosis, she deceased at 28 years old due to pulmonary infection. one 30 years old son has anemia. her other daughter and son are healthy. objective: to describe unique management of recurrent viral respiratory infections in a 22qds patient. case: 5-year-old male with a history of 22qds complicated by truncus arteriosus, vsd, hypoparathyroidism, asthma, and autism was followed for a history of "frequent pneumonias." he had daily rhinitis and a history of frequent otitis media which resolved after tympanostomy tube placement at age 3y. however, over the next two years, he had 11 admissions with various respiratory viral infections resulting in respiratory distress and prolonged oxygen need. viruses detected during these separate admissions varied and included parainfluenza 3, metapneumovirus, rsv, b and coronavirus nl63, coronavirus hku1, and rhino/enterovirus (5 times total). he was previously on a prophylactic course of antibiotics which made no difference in his symptoms. laboratory evaluation showed protective adaptive immunity with normal immunoglobulin numbers for age (igg 589 mg/dl), along with normal b and t cell numbers. he had protective pneumococcal titers and mounted a normal mitogen response. while his nk cell numbers were normal, his nk t cells were low. his tlr functioning also appeared normal. in order to decrease his overall illness burden and to keep him out of the hospital, ivig infusions (~500mg/kg) were initiated monthly. shortly after initiation of treatment, his nasal purulence and drainage resolved and his family noted that he became more active and playful. treatment was continued for 18 months, during which time he had only one episode of influenza infection needing inpatient management. he has been off of ivig for 12 months without recurrence of his viral infections. conclusion: in this patient, severe recurrent viral respiratory infections despite apparently normal adaptive and cellular immunity presents a unique management dilemma. this was not an issue of recurrent bacterial infections as prophylaxis did not make a difference in the frequency of his infections. his nk t cells were low, which could have contributed to his frequent viral infections as nkt cells are known to play a role in viral immunity. the successful use of ivig treatment in his case points to a different use for ivig, namely for the anti-respiratory virus antibodies which are presumably contained within the formulation.given this finding, it may be prudent to consider ivig in management of 22qds patients, even with normal immune evaluation, in order to decrease risks of complications associated with severe and recurrent viral respiratory infections. abstract/case report text introduction: smad 4 is a critical downstream signaling molecule for transforming growth factor-β (tgf-β) and bone morphogenic protein (nmp1). initially, smad4, also known as dpc4 deleted in pancreatic cancer locus 4, was described as a tumor suppressor gene, and somatic deletion of the smad4 is seen in 90% of pancreatic carcinomas. subsequently, a germline point mutation in the smad4 gene (p. i500v) was reported to cause myhre syndrome (mim#139210). myhre syndrome is an autosomal dominant disease characterized by cognitive impairment, hearing loss, and musculoskeletal anomalies. the immunological phenotype of these patients has not been previously described, despite the critical role of tgf-β in regulating t cell response and the prevention of excessive inflammation. case report: a 9-year-old boy with myhre syndrome was referred to immunology clinic for evaluation of recurrent ear infections. he developed acute otitis media infections as an infant and had tympanostomy tubes placed at one year of life. he also had a recurrent sinus infections. at two years of age, he was diagnosed with autism and sensory neuronal hearing loss. brain mri showed a mildly hypoplastic pituitary gland, and a thickened corpus callosum with decreased myelination. given these findings, whole-genome sequencing was performed, which revealed a heterozygous de novo mutation in smad4 (p.i800v / c.1498 a>g) consistent with the diagnosis of myhre syndrome. through age 5, he was in the 5th percentile for height until he was started on growth hormone, which he responded to robustly. he is now he is in the 50th percentile for height. he had adenoidectomy due to sleep-disordered breathing at the age of 7 years. he is maintained on montelukast and inhaled corticosteroids for treatment of rhinitis and mild persistent asthma. he is on atenolol for the treatment of primary hypertension. on physical exam, he has facial dysmorphisms, thickened skin, and contraction of the fingers consistent with myhre syndrome. immunologic elevation showed significant hypogammaglobulinemia (igg 311 mg/dl), low iga (11 mg/dl) and normal igm 50 mg/dl. igg subclasses showed low igg1 and igg2 at 227 mg/dl and 58 mg/dl respectively. although he was fully vaccinated, his tetanus antibody was low at 0.14 iu/ml. however, this improved after repeat vaccination to 4.38 iu/ml. total t and b lymphocyte counts were normal; however, his memory cd4 and cd8 t cells were low for age at 2.26% and 0.5%, respectively. additionally, his switched memory b cell count was low at 1.9%. conclusion: smad4 gain of function (myhre syndrome) can lead to impaired memory t and b cell formation with significant hypogammaglobulinemia and low iga. although the patient was able to respond to protein vaccination (tetanus), it is not clear if he will be able to maintain a long-term response. in a previous study, a similar gain of key: cord-010162-hfo35gsq authors: saikku, pekka title: atypical respiratory pathogens date: 2014-12-29 journal: clin microbiol infect doi: 10.1111/j.1469-0691.1997.tb00464.x sha: doc_id: 10162 cord_uid: hfo35gsq the main atypical pathogens in respiratory tract infections are classified on the basis of their ability to cause atypical pneumonia. this is not a well-defined clinical entity, and it is evident that atypical pathogens can sometimes cause ‘typical’ pneumonias and vice versa. this emphasizes the need for microbiological diagnosis, since it affects the selection of proper treatment, in which β-lactam antibiotics and aminoglycosides are not effective. moreover, mixed infections caused by atypical and typical pathogens together are common. at this moment rapid and sensitive diagnostic methods are lacking. besides numerous viruses, the main bacterial pathogens causing atypical pneumonias are mycoplasma pneumoniae, two chlamydial species, chlamydia pneumoniae and c. psittaci, one rickettsia, coxiella burnetti, and several legionella species. the majority of these pathogens cause upper respiratory tract infections more often than overt pneumonias. an atypical agent, chlamydia pneumoniae, has also been associated with chronic inflammatory conditions in the cardiovascular system. the most recently discovered pathogen in atypical pneumonias is a hantavirus causing hantavirus pulmonary syndrome. 'atypical pathogens' in community-acquired respiratory tract infections are purely microbiological entities: apart from pneumonias, where the pneumococcus is the leading causative agent, the overwhelming majority of respiratory infections are caused by 'atypical pathogens' (table 1 ). the list of atypical pathogens is long, and the more microbial diagnostic methods are used, the more pathogens are found. a recent example comprises the hantaviruses recently found in hantavirus pulmonary syndrome [l] . although pneumonia and pneumonic symptoms had earlier been described in hantavirus infections, as had pulmonary edema in rickettsioses [2] , no one expected a hemorrhagic fever virus to be the causative agent in severe pneumonias. the use of advanced microbial diagnostic methods established a new disease syndrome. the history of atypical pathogens starts with psittacosis, caused by chlamydia psittaci [3], influenza viruses [4] and q fever, caused by rickettsia, coxiella burnetti [5] . influenza virus continues to be a great scourge of all mankind, and a possible appearance of a new killer strain is in every autumn a greater menace than the feared ebola virus. the other two agents also continue to be important respiratory tract pathogens, but being zoonoses, are limited in their occurrence mostly to cases with animal contacts. luckily, 'atypical pneumonias' are usually not clinically severe; rather, the converse is the case. some clinical features of atypical pneumonias are presented in table 2 . in the patient history, family cases and the epidemiologic situation can aid in the diagnosis, as well as contact with a sick parrot when there is suspicion of psittacosis. the onset can be delayed and insidious. sputum production can be nlinimal and polymorphonuclear leukocytes are not present. in the chest x-ray, no lobar infiltrates, but more diffuse alterations 'typical of atypical pneumonia', are seen. leukocytosis can be absent in cases without massive pulmonary destruction, the erythrocyte sedimentation level is usually elevated, but c-reactive protein does not reach the levels found especially in severe pneumococcal pneumonias. however, none of these symptoms and signs readily differentiates an 'atypical' disease from a 'typical' one. a u the values overlap, and even a lobar infiltrate in the x-ray can be caused by an atypical pathogen [7] . without proper microbial diagnosis, the first clue to the etiology can too often be only the lack of response to the standard antimicrobial treatment used. in the following sections the main bacterial agents causing atypical respiratory tract infections are discussed, with a special emphasis on the latest bacterial addition, chlamydia pneumoniae. mycoplasmal pneumonias concentrate in the younger age groups, and this is illustrated in figure 1 , comparing the prevalence of antibodies against m . pneumoniae and chlamydia pneumoniae in the finnish population. the clinical description of mycoplasmal infections is classical but there are some open questions. one is the lack of reliable diagnostic methods, since conventional serologic methods, cold-agglutinin and complementfixation tests, are neither sensitive nor specific, although the former is easy to perform. a second question is, how effective is the antibiotic treatment in mycoplasmal pneumonias? mycoplasmal diseases in general are a neglected area, and we should not rely on serology only in the diagnosis of mycoplasmal pneumonias [8] . the epidemic of a curious disease named afterwards as 'legionellosis' in pittsburgh in 1976 brought a special bacterial genus to our attention [9] . although its first member had been discovered over thirty years earlier [lo], only then was it discovered to be the causative agent of several serious epidemics of respiratory infections. relatively new also is its association with environmental constructions and air-conditioning techbeen associated with clinical diseases, especially with severe pneumonias, but the importance of these environmental bacteria varies considerably between different regions and settings. in some places they are causing a considerable proportion of all pneumonias [i21 and are listed among the three major causes. in other areas, e.g. finland, they are, despite an intensive search, rarities, usually imported by tourists. respiratory tract infections caused by chlamydia psittaci are directly dependent on exposure to birds carrying the pathogen. 'therefore, cases are seen in connection with curkcy and duck farming (chickens seem not to be iziportant), pigeon breeding, and sick pet birds. casual contacts with synanthropic birds are common, and ruling out a bird contact is much more difficult than finding one. there has been a debate over whether chlamydia pneumoniae is a more common agent than chlamydia psittaci. even in the microimmunofluorescence (mif) test it is sometimes difficult to differentiate these two chlamydial species from each other [13] , and it demands expertise [14] . fortunately, the treatment is the same in both chlamydial pneumonias. according to seroepidemiologic surveys, chlamydia pneumoniae infections are 20-50 times more common than chlamydia psitfari infections [15,16]. there is a possibility that the severity of chlamydia psittaci infections could be due to the sensitization of the patient to this species by an earlier, mild chlamydia pneumoniae pulmonary infection. the most recent addition to the long list of important atypical bacterial pathogens is chlamydia przeumoniae. like the first legionella strains, the first strains of this new chlaniydial agent were isolated [17] years before an epidemic in northern finland brought them to our attention [i 81. in the beginning, chlamydia ptzeumoniae seemed to be an agent causing beiiign and mild disease, but later it was shown to cause-the typical feature of all chlamydia-silent, slowly creeping infections, gradually leading to severe tissue damage 1191. the pathogenesis of chlamydia przeumoniae infections has been studied using mouse models 120,211. when chlamydia pneumoniae is given intranasally, acute infection with polymorphonuclear leukocytes in the lungs is seen only after massive challenge doses. otherwise a silent pneumonitis with a clear histologic picture of mononuclear inflammation around bronchioles and vessels develop without overt illness. repeated inocula-tions aggravate this inflammation temporarily, but the presence of the agent is difficult to demonstrate by isolation [22] . the demonstration of nucleic acids by polymerase chain reaction (pcr) gives, however, a positive finding and cortisone treatment, after alleviating inflammation, makes isolation of the agent possible [23] . chlamydia pneumoniae is also demonstrable after intranasal challenge in the blood circulation, alveolar and peritoneal macrophages, and the liver and spleen, pointing to a disseminated infection 1241. upper respiratory tract carriage has been described [25, 26] , and there is a possibility that carriers do not develop antibody responses or, if they do, only after a prolonged period. pneumonias due to chlamydia pneumoniae haw been described in infants [27] , and from japan there is a report of an epidemic in daycare centres [28] . however, in industrialized countries antibodies usually start to appear when children enter school [29, 301 . in a recent finnish study on childhood pneumonias, the youngest patient with a chlamydia pneumoniae infection was 7 years old, and the majority of patients were over 10 years of age (korppi et al, unpublished data). other respiratory syndromes associated with chlamydia pneumoniae are rhinitis, sinusitis, pharyngitis, otitis, and bronchitis. a recent review of chlamydia peumoniae infections in children concentrated on respiratory tract infections [31] . however, during a chlamydia pneumoniae epidemic in northern finland, only a third of the children presenting a seroconversion were hospitalized because of respiratory tract symptoms (uhari et al, unpublished data). the disease picture in children can thus be quite variable and demands further study. primary infection in young adults leads to pneumonia in about 10% of cases [32] . this is usually a mild disease, but can be prolonged with a long convalescence. in young age groups, reinfections seem not to lead to pneumonias (321. however, reinfection pneumonias, especially in elderly patients with underlying diseases, can be very severe 1331. one possibility is that when the resistance to infection has decreased enough to allow the agent to invade the lungs, or the invading strain is different enough to be able to colonize the lungs, partial immunity can lead to hypersensitivity reactions typical of all chlamydia1 infections. the role that chlamydia pneumoniae plays in other acute respiratory tract infections is still under study. in adults, it has been associated with 0.5-7'% of pharyngitis 134,351 cases (the last figure during an epidemic), and 5-100/;, of acute bronchitis cases. sinusitis and otitis in adults have also been reported, but wider studies are so far lacking. mixed infections are coninion in chlamydia p i mrnoniae infections. in the studies on finnish children, half or even the majority of patients have had a concomitant infection caused by another pathogen; virus, mycoplasma or bacterium. similarly, during an epidemic in north finland, nearly half of the chlamydia pneumoniae pneumonias were combined with invasive pneumococcal infections [36] . chlamydia pneumoniae has a ciliostatic effect [37] , which in these cases may help pneumococci carried in the upper respiratory tract to invade deeper layers. these double infections were more severe than usual and the response to antibiotics effective against the pneumococcus only was poor [38] . one should not be satisfied when one pathogen is diagnosed, but always keep in mind the possibility of mixed infection. serology has traditionally been used to diagnose infections caused by atypical pathogens. antigens and complete kits for antibody assays are commercially available from several sources. the most serious disadvantage is the need to demonstrate a seroconversion in the majority of the cases. this delays the diagnosis and is then of no aid to the clinician treating the acutely ill patient. attempts have been made to overcome this delay by the demonstration of igm antibodies in the acute phase or by using 'diagnostic' high titers in the first serum sample. the pitfalls are the lack of igm in reinfections and the uncertainty of the diagnostic value of high titers in acute diseases caused by several atypical agents. serology, even though inadequate, has remained the main diagnostic tool in mycoplasmal and chlamydia1 pneumonias as well as in q fever. culture of the atypical pathogens demands special media not widely used in microbiology laboratories or, in the case of obligatory intracellular pathogens, cultured living cells in specialized units. moreover, coxiella burnetti and chlamydia psittaci present dangers to laboratory workers handling the agent, and even chlamydia pneumoniae has caused laboratory-acquired pneumonias [39] . in legionellosis, however, culture is a standard diagnostic procedure [40] . it should also be attempted in chlamydia pneumoniae infections in order to obtain information on disease associations and strain variability of this newly recognized pathogen. antigen detection in respiratory tract infections has been utilized mainly in viral infections, with good success. its use in the case of atypical bacterial pathogens has not been as rewarding. moreover, techniques based on immunofluorescence demand experience and patience from the reader, since numbers of pathogens are often limited and their reliable identification is difficult. lack of commercially available reagents and luts is a problem in the diagnosis of uncommon atypical pathogens. however, in legionellosis, detection of antigen in urine seems to be a reliable diagnostic method [11, 41] . in pneumonias, the presence ofbacterial components in the circulation, alone or in immune complexes, has been used successfully in the diagnosis of pneumococcal pneumonias [42] , but has remained unstudied in the case of atypical pathogens. these types of complex are commonly, seen, however, in chronic chlamydia pneumoniae infections, which lessens their diagnostic value in acute infections, especially in elderly males with arteriosclerotic lesions [43] . nucleic acid (na) detections seems to be the diagnostic method of the future for atypical respiratory pathogens. the commercial kit for m . pneumoniae direct na detection is no longer available, but diagnostic companies are developing kits based on na amplification for legionella spp., m . pneumoniae and chlamydia pneumoniae. these, whether based on the polymerase or ligase chain reaction, would provide a sensitive and specific diagnosis for these pathogens in 24 h. this would finally give a firm basis for a rationally targeted therapy. table 3 shows the recommended treatment for infections caused by atypical pathogens. the therapeutic response of bacterial atypical pathogens to p-lactam antibiotics and aminoglycosides is lacking or marginal. the drugs used are tetracyclines, macrolides, and azalides. time will tell how much the advent of newer quinolones will alter these recommendations. prolonged treatment of 2-4 weeks has been used in legionellosis and in chlamydia pneumoniae pneumonias. the possibility of mixed infections should always be kept in mind. appropriate therapy can be efficient in preventing long-term sequelae caused by some of these atypical pathogens. q fever endocarditis is a feared complication, which often leads to valvular operations or drug therapy for the rest of the patient's life 1441. recently discovered chlamydia pneumoniae seems to be associated with complications of unexpected severity and ubiquity. signs of chronic chlamydia pneumoniae infection have been found in both childhood [45] and adult-onset [16] asthma, chronic bronchitis [47] , and sarcoidosis [48] . most surprising is its association with arteriosclerosis, the leading cause of death in industrialized countries. markers of chronic chlamydia yneumoniae infections are found in acute myocardial infarction [49, 50] , and they have repeatedly been shown to be a risk factor for cardiac events [51, 52] ; and, finally, the pathogen has been demonstrated in atherosclerotic lesions 153,541. animal experiments have been positive [55, 56] , as have preliminary intervention trials [57, 58] . it may be possible that appropriate treatment of chlavnydin pneumoniae respiratory infection in the acute phase prevents infection from progressing to a chronic state, which can be much more difficult to cure. this would demand acute diagnosis and targeted therapy for atypical pneumonias. hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized hsease lower respiratory tract involvement in rocky mountain spotted fever a virus obtained from influenza patents studies on the etiology of primary atypical pneumonia: a filterable agent transmissible to cotton rat, hamsters, and chick embryo comparative radiographic features of community acquired legionnaires' disease, pneumococcal pneumoniae, mycoplasma pneumonia, and psittacosis detection of mycoplarma pneurtioniae in clinical samples from pediatric patients by polymerase chain reaction legionnaires' disease: description of an epidenlic of pneumonia new and emerging etiologies for community-acquired pneumonia with implications for therapy: a prospective multicentre study of 359 cases specificity of the niicronnmunofluorescence assay for the serodiagnosis of chlaniydia pneurnoniae infections utility of complement fixation and microinimunofluorescence assays for detecting serologic responses in patients with clinically diagnosed psittacosis a comparison of the seroepidemiology of chlamydia1 infection in pigeon fanciers and farmers in the uk acute lower respiratory trxt infection associated with chlamydia pneumoniae in germany a new chlartiydiii prithici strain, twar, isolated in acute respiratory tract infections an epidemic of mild pneumonia due to an unusual strain of chlamydia psittaci the epidemiology and significance of chlamydia pneumoniac experimental infection of chlamydia pnrumoriiac in mice a mouce model of chlamydia pneumonia strain twak pneumonitis experimental chlaniydia prieirmoniae infection in mice: effect of reinfection and passive protection by immune serum saikku 't? keactivation of clilanzydia pueumoniae infection in mice by corticone treatment syrtemic dissemination of chlamydia pneumoniae following intranasal inoculation in mice asymptomatic respiratory tract infection with chlamydia pnerrnzoriiae twar prevalence of asymptomatic nasopharyngeal carriage of chlamydia pnenmoniae in subjectively healthy adults: assessment by polymerase chain reaction-enzyme immunoassay and culture chlamydia pneumoniae (twar) in neonates an outbreak of chlamydia pneumoniae infection in households and schools seroepidemiology of chlamydia pneumoniae twar infection in seattle families, 1966-1979 a new respiratory tract pathogen: chlamydia pnenmoniae strain twar chlamydia pneumoniae (twar) infections in children epidemics of pneumonia caused by twar, a new chlamydia organism, in military trainees in finland chlamydia pneumoniae as a new source of infectious outbreaks in nursing homes chlamydia pnenmoniae, strain twar, mycoplasma pneumoniae and viral infections in acute respiratory disease in a university student health clinic population pharyngitis in adults: the presence and coexistence of viruses and bacterial organisms shemer-avni y. lieberman d. chlamydia pnrumoniaeinduced ciliostasis in ciliated bronchial epithelial cells clinical picture of cominunity-acquired chlamydia pncumoniae requiring hospital treatment: a comparison between chlamydia1 and pneumocozcal pneumonia cell rnediated immunity to chlanzydia prirunzoniaf measured as lymphocyte blast transformation in uifw diagnosis of legionnaires' disease; an update of laboratory methods with new emphasic on isolation by culture frequency and diagnosis of legionella pneumonia: a 3 year prospective study with emphasis on application of urinary antigen detection demonstration of pneumolysin antibodies in circulating immune complexes-a new chagnostic method for pneumococcal pneumonia presence of chlamydia pnenmoniae specific antiboches in circulating immune complexes in coronary heart disease the association of chlamydia pneumoniae infection and reactive airway disease in children association of chlamydia pneumoniae (strain twar) infection with wheezing, asthmatic bronchitis, and adult-onset asthma chlamydia pneumoniae infection in acute exacerbations of copd antibodies to twar-a novel type of chlamydia-in sarcoidosis sarcoidosis and other granulomatous disorders serologic evidence of an association of a novel chlamydia, twar, with chronic coronary heart disease and acute myocarchal infarction circulating immune complexes containing chlamydia lipopolysaccharide in acute myocardial infarction chronic chlamydia pneumoniae infection as a risk factor for coronary heart disease in the helsinki heart study chlamydia pnenmoniae strain twar antibody and angiographically demonstrated coronary artery chsease. arteriosclerosis thrombosis 53. shor a, kuo cc, patton dl. detection of chlamydia pneumoniae in coronary arterial fatty streaks and atheromatous plaques demonstration of chlamydia pneumoniae in atherosclerotic lesions of coronary arteries chlamydia pneumoniae multiplies in human endothelial cells in vitro chlamydia pneumoiziae infection induces inflammatory changes in the aorta of rabbits elevated chlamydia pneumoniae antibodies, cardiovascular events, and azithromycin in male survivors of myocardial infarction roxis study group. randomised trial of roxithromycin in non-q-wave coronary syndromes: roxis pilot study key: cord-018017-c8myq6bi authors: iversen, patrick l. title: the threat from viruses date: 2018-09-30 journal: molecular basis of resilience doi: 10.1007/978-3-319-98164-2_3 sha: doc_id: 18017 cord_uid: c8myq6bi infectious disease represent the most significant threat to human health. significant geologic cataclysmic events have caused the extinction of countless species, but these “wrath of god” events predate the emergence of homo sapiens. pandemic infections have accompanied the rise of human civilization frequently re-occurring leaving a lasting imprint on human history punctuated by profound loss of life. emerging infections become endemic and are here to stay marking their presence with an annual death toll. each decade brings a new onslaught of emerging infectious agents. we are surprised again and again but are never prepared. the long-term consequences often remain unrecognized and are always inconvenient including cancer, cardiovascular disease and immune associated diseases that threaten our health. reliance on clusters of clinical symptoms in the face of diverse and non-descriptive viral infection symptoms is a foolhardy form of crisis management. viral success is based on rapid replication resulting in large numbers. single-stranded rna viruses with their high replication error rate represent a paradigm for resilience. contemplating recent career paths, i reviewed a broad range of scientific questions. most prominent was, why study an area of science that has no significant impact? in fact, why study anything that is not the most highly impactful area? this question demands a definition, what constitutes high impact? i decided to create a definition that impact and threat to life are related. further, threat to human life is the highest impact and it is likely that things threatening human life may also threaten all life. what presents the greatest threat to human life today? history should provide critical insights to answer this question. a comprehensive look would seek causes of mass extinctions over the past 3.5 billion years of life on earth. this perspective has been a trending subject in science with focus on five mass extinctions. there are inherent biases in focusing on mass extinctions such as these events fail to appreciate small living things such as single celled organisms or bacteria. another bias is in order to appreciate life in the past, the life form must occasionally produce a fossil when it dies. those concerns aside-geologic events have threatened the survival of living things (table 3 .1). the actual events causing these extinctions are frequently debated, for example, the asteroid impact 66 million years ago off the coast of mexico was accompanied by a massive tsunami that was responsible for mass extinctions in montana, the impact may have also triggered an enormous volcano in india resulting in ash and lava flow responsible for regional species extinctions, and the collective dust resulted in prolonged climate change which was probably responsible for even more species extinctions. the lesson remains the same, volcanic eruptions; climate change and asteroid impact threaten life in a highly significant way and are responsible for extensive selection pressure. unfortunately, these events are unpredictable and so enormous little can be done about them. a mass extinction may not represent a legitimate threat or selection pressure because large numbers of species are completely eradicated leaving few survivors to drive evolution. finally, outside of removing dinosaurs so that mammals could thrive, what impact did these events have on human life; humans were not yet on the earth when these events took place. refining the search to dramatic changes in human populations moves the period up to the last 100,000 years. mass migrations took place about every 23,000 years, which is in line with the precession of the earth leading to a possible linkage between earth's precession and human migration. this may have been due to regional climate change like ice ages in the northern hemisphere. however, human population changes on this timescale are not easily documented so estimates of climate and human population dynamics will be limited. time to refine the search. consider the exponential growth, outbreak, of tent caterpillars, malacosoma disstria, in montana reported by david quammen in his book spillover (quammen 2012) . the extensive caterpillar population consumed all of the leaves from the local elm and cottonwood trees in the summer of 1993. the caterpillar activity produced a crackle sound, "like a distant brushfire." the city attacked these insects with a broad arsenal of modern countermeasures but to no effect. however, a nuclear polyhedrosis virus (npv), uses the caterpillar host density like a critical mass in a nuclear weapon to explode destroying the caterpillars in an epic battle. the caterpillar population retreated to undetectable levels in a single season as a result of npv. human populations changed over the past 20,000 years with particular emphasis on the most recent 7000 years. a human population curve over this segment of time shows an exponential growth period interrupted in the middle ages by the plague. hypothesis: the plague and other pandemic infectious disease events appear to be the greatest threat to human life. infectious disease kills 15 million people each year, 26 percent of the total 57 million annual deaths in the global population (fauci and morens 2012) . a brief review of pandemic infections over the past 3000 years illuminates several events that reduced human populations. the plague of athens 430 bce killed 25 percent of the human population (table 3 .2). both bacterial and viral pandemic yersinia pestis is a bacterium causing plague. fleas can be infected with y pestis which transmit the bacterium to rodents, the primary hosts. changes in the environment may lead to the movement of rats into populated areas where humans become infected. homer points to such an infection in the iliad in his description of the trojan war in 1190 bce. plague has returned several times since the trojan war imposing enormous loss of human life (table 3 .2). the most recent plague epidemic killed over ten million people in india in the early 20th century. y pestis is still out there ready for favorable conditions to pounce on human populations but outcomes are likely to be less dramatic due to understanding of sanitation practices, quarantine, and availability of antibiotics. if infections are the greatest threat to human life, they should be critical drivers of evolution? clearly infections pose selection pressure on the human populations. origins of evolutionary thought did not include infection as darwin established key evolution concepts on the galapagos islands. these islands are isolated and an unlikely place for the spread of infections. the concepts speciation point to geographical separation of populations so infections would most likely be restricted to isolated populations. in many cases the survival selection pressure is not identified or ascribed to insufficient sources of food. unfortunately, common single-stranded rna viruses are so unstable that there are limited data for a viral fossil record. pandemic infections remain a threat to human survival in the presence of the information revolution, daily medical breakthroughs, and global travel. the human retrovirus hiv currently a global infection that infects up to 25% of the population in southern and eastern africa with a projected death toll of up to 100 million by 2025. measles killed 200 million people in the last 150 years and the development of an effective vaccine in 1963 reduced concerns for this infection but there were 777,000 deaths in the year 2000. vaccination programs are frequently disrupted due to complacence resulting from vaccine success, conflicts that shift healthcare focus, and social crisis such as the recent ebola outbreak in west africa. smallpox is also an ancient infection causing fever, skin lesions, and at times death. king ramses v of egypt is thought to have died from smallpox around 1200 bce. introduced into mexico in 1520, smallpox killed 3.5 million aztec indians or about half of the population in a period of 2 years and then proceeded to decimate the population of south america. variola is a highly infectious virus killing 300-500 million people during the 20th century inspiring the eradication campaign in 1967. variola was eradicated by december of 1979 (de cock 2001 , a rare triumph of public health. the who deserves acknowledgment for this unprecedented accomplishment and proof of concept that human suffering is not inevita-ble. however, variola is a dna virus with limited rate of mutation and a narrow host range so that animal reservoirs do not exist. the eradication of other viral infections will be more challenging. the world continues to confront a broad array of microbial threats. progress and preparedness make our engagement a likely success for those microbes that resurface and infections for which we have experience. medical and epidemiological uncertainties surround emerging infectious disease, those that challenge us with their novelty. pandemics dominate the infectious disease "fear factor" but each pandemic began as a much more frequent occurrence, an epidemic. most but not all epidemics come from emerging infectious agents, the most significant problem facing life on earth today. the concept of emerging is a human centric term as most of these infections are endemic in an animal host that serves as a viral reservoir. a 2005 report from the university of edinburgh identified 1407 human pathogens and 177 are emerging or re-emerging of which 75% are zoonotic, that is jump from an animal host to human. numerous emerging infections caused by viral agents have imposed high impact on human survival (table 3 .3). all the viral agents in table 3 .3 have genomes based on single-strands of rna except hbv which should focus scientific attention on rna. there are numerous questions that strike investigators as they ponder a collection of viral agents like those in table 3 .3. the viral polymerase errors in replicating single-stranded rna genomes are not corrected so the species are constantly changing. the apparent success of these viruses is that as they move from reservoir hosts to humans and as humans become immune to the initial infection, the population of diverse genomes offers multiple chances to adapt by finding a "fit" genome version which can propagate until the next transition requiring adaption. acquired immunodeficiency syndrome (aids) is caused by human immunodeficiency virus 1 (hiv-1), a retrovirus. these viruses have a single-stranded rna genome that is converted into dna, a paradigm shift in the flow of genetic information from dna to rna. the 36,000,000 human deaths caused by hiv-1 (table 3.3) is accompanied by a spectrum of clinical signs; eg. fever, diarrhea, peripheral neuropathy, pelvic inflammatory disease, cervical cancer, cytomegalovirus retinitis, kaposi's sarcoma, lymphoma, mycobacterium avium infection, recurrent pneumonia, and wasting syndrome. hiv-1 genome diversity includes base substitution, insertion, deletion, recombination, and gain or loss of glycosylation sites which all arises from the limited fidelity of the viral reverse transcriptase. these mutations are found in clusters or hypervariable regions indicating the fit virus is selected for from vast numbers of less-fit genome sequences. hiv-1 emphasizes key observations: (1) eid is a significant contemporary concern, (2) we are not prepared for the novel characteristics introduced by eid, (3) clinical signs can be diverse and often mimic symptoms of other diseases, and (4) the replication mechanisms are often error prone resulting in an array of fit viruses. dengue is a flavivirus with a positive sense single-stranded rna (+ssrna) genome carried by mosquitos to man. dengue has been a tropical disease for hundreds of years, typically a disease of young children but in 1953 an emerging severity was recognized in manila (table 3 .3), hemorrhagic fever (dhf) and dengue shock syndrome (dss). more than 2 billion people are at risk of dengue infection but only a small fraction of those infected will develop dhf or dhs. infected people develop antibodies that can lead to antibody-dependent enhancement (ade) in subsequent infections and more severe dhf or dss outcomes. it appears ade events occur in people that produce immunoglobins (iggs) with enhanced affinity to the activating fc receptor due to the igg1 subclass and lack of a fucose glycan modification of the igg (wang et al. 2017) aedes aegypti is the primary vector transmitting dengue but a new vector, aedes albopictus, now carries dengue to the southern united states. emergence of dengue in the southern united states is likely due to used tires imported from japan which provided a place for the asian tiger mosquito to live. outbreaks of dengue fever have become more numerous and more severe over the past three decades. viral "fitness" is constrained by the requirements imposed by the natural host so that it is a low probability event for a virus to move from the natural host to a human. while an insect frequently plays the role of vector carrying a virus from an animal reservoir to humans, several zoonotic viruses are transmitted by placing humans near rodents. several arenaviruses, minus sense single-stranded rna viruses, jump from their rodent natural hosts directly to humans through contact with rodent urine or saliva. notable arenaviruses are named for the hemorrhagic fever (hf) region of their zoonosis; bolivian hf is caused by machupo (macv), argentine hf is caused by junin (junv) and venezuelan hf is caused by guanarito (gotv). these south american outbreaks are caused by new world arenaviruses in contrast to lassa hf (lasv), an african or old world arenavirus. lasv is an endemic disease of west africa (table 3 .3) and can be confused with dhf or ebola infections. viruses that are transmitted by arthropod vectors are called arboviruses. in 1930, only yellow fever of six known arboviruses caused disease in humans. the discovery of arbovirus caused human disease expanded beginning in the late 1930s with western and eastern equine encephalomyelitis (weev, eeev) and st. louis encephalitis. both chickungunya and zika (table 3 .4) are arboviruses that have emerged to become global infections in the twenty-first century. zika not only became infamous for causing microcephaly in newborns of infected mothers and guillain-barre syndrome but is now sexually transmitted between humans. i worked on a therapeutic for the treatment of ebola infections from 2007 to 2013. the genome sequence recovered from each outbreak from 1976 to 2013 has been different. studies were conducted at usamriid in their bsl4 facility by expert ebola investigators. rhesus monkeys (macaca mulatta) were injected with 1000 plaque forming units (pfu) of zaire ebolavirus (zebov) kikwit into their thigh muscle. all of the untreated control monkeys died between days 8 and 10 following viral injection. we measured viral burden by quantitative polymerase chain reaction (qrt pcr) a measure of genome copies per milliliter (copies/ml) of plasma and plaque formation a measure of infectious viruses per ml (pfu/ml) plasma. we found an average of 1.7x10 8 genome copies per ml on day 8 post infection and 1.2x10 6 pfu/ml on day 8. nearly 100 genomes present for each successful virus in a nonhuvwas not observed, the dominant population of viral genome sequence did not change from one individual to another or from one time to another within the same individual. the existence of defective viral genomes may offer potential for rapid change but in a stable host, change was not rapid. when one considers the magnitude of differences in conditions as a virus jumps from a reservoir host to a human, rapid adaptability is a great advantage. a virus that kills all the hosts is not a successful virus. perhaps defective viral particles facilitate host immune responses giving them a chance to catch up to the rapidly proliferating virus. the ebola outbreak of 2014 illuminated the collateral damage that accompanies eid outbreaks. first, the first responders are local physicians and care givers are killed leaving a population lacking doctors and nurses. even physicians trained to exercise appropriate caution when interacting with patients such as dr. sheik humarr khan died of the ebola infection (bausch et al. 2014) . second, women are the main caregivers in their families and 75 percent of ebola deaths in the 2014 outbreak were women. this leaves families lacking traditional structure. third, the outbreak disrupts production, labor markets and trade resulting in scarcity and inflation of food prices. food security and nutrition are diminished which preferentially affects poor people as they spend 50 to 70% of their income on food. finally, public health measures are discontinued. before the 2014 outbreak, 97 percent of children in west africa were receiving routine vaccinations but that figure fell below 27 percent. the west africa loss of measles vaccinations was followed by measles out coronaviruses are unique in their large single-stranded rna genome (20,000 bases) and are often associated with mild disease, bronchitis and gastroenteritis. a 2003 outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in china rapidly spread to 8098 cases in 37 countries (table 3 .4). retrospective analysis of this outbreak linked zoonosis to a culinary trend in which people sought out exotic animals to eat. these nontraditional food animals appeared in street markets in urban areas bringing people close to the reservoir host triggering the outbreak. the civet cat was identified as the source of the first human cases but this is not the natural host. the natural host is the fruit bat, which infects a variety of small mammals including the civet cat. a point of intervention has been removal of the civet cat from markets but the natural bat host remains in the environment maintaining the virus for the next outbreak. a second coronavirus outbreak in 2014 of middle east respiratory syndrome coronavirus (mers cov) rapidly involved 23 countries including africa, western europe, and southeast asia. the number of cases is relatively low but the case fatality ratio is of great concern (table 3.4). the camel is reservoir host in this case but it may not be the natural host since camels often get sick as well. transition from animal-to-human transmission to efficient human-to-human transmission was observed quickly in the case of both sars-cov and mers-cov. this rapid adaptability of a highly infectious virus group should raise concern over endemic coronaviruses in companion animals since these viruses can clearly become pathogenic, can jump from animal to human and then become transmitted from human-to-human. an ongoing outbreak of yellow fever in africa (table 3 .4) is a striking reminder that eid can re-emerge into virtually naïve populations. some people recover from the acute symptoms but liver damage causes yellow skin, the reason for the name for the disease. the liver damage leads to bleeding and kidney damage and occasionally death. yellow fever is a flavivirus that originated in africa but was distributed to the world on barges and sailing ships to tropical ports. the virus arrived in the americas on slave ships. this virus interrupted the building of the panama canal which attracted the attention of walter reed, the military physician responsible for demonstrating transmission by mosquitos. an effective vaccine is used worldwide with immunity lasting over 10 years. the limitation of this vaccine like most is that populations need to be vaccinated regularly which in a world constantly changing due to political and economic drivers leads to vaccination gaps. cancer is one of the leading causes of human death but estimates are that 20 percent of all human cancers are directly caused by viruses (table 3 .5) (morales-sanchez and fuentes-panana 2014). the first tumor virus was identified by peyton rous in 1911. he took cell lysates from a chicken sarcoma which he passed through a filter known to hold back bacteria and then injected the filtered lysate into chickens. a tumor developed at the injection site. the virus is now known as rous sarcoma virus (rsv), a single-stranded rna virus. rous won a nobel prize in physiology or medicine for the discovery in 1966. rsv is a retrovirus that captured a tyrosine kinase, src gene, that triggers uncontrolled growth in infected host cells. the avian leukemia virus (alv) was discovered in denmark early in the twentieth century capable of causing disease in blood forming tissues of chickens. a strain of alv, avian myeloblastosis virus (amv) was described in 1952 that provided a convenient source of tissue for biochemical studies. during the 1930s a strain of inbred mice was found to develop leukemia between 6 and 18 months of (burkitt's lymphoma 2014) . after that he saw a second case at the jinja district hospital on the shores of lake victoria this became more than a curiosity. a review of hospital case notes confirmed the prevalence of these tumors of the jaw and they were accompanied by swellings in kidneys, ovaries, adrenal glands and liver. he assembled data from 38 patients and histological examination led to description of "lymphoma syndrome" or the "african lymphoma." burkitt received a grant in 1961 for ₤250 to visit 57 hospitals in eight african countries; uganda, kenya, tanzania, malawi, mozambique, zimbabwe, zambia, and republic of south africa to investigate african lymphoma. the tumor was found everywhere at the equator in areas where year-round temperature was above 60 °c but not in areas over 5000 feet in elevation. burkitt further determined the tumors only occurred where the annual rainfall was above 20 inches and not seen in the dry savannah of nigeria. burkitt contacted doctors in papua new guinea to discover these tumors were the most common childhood tumor in that country but only in wet coastal regions and not in the dry highland areas. burkitt's working hypothesis was that an insect transmitted virus infection was responsible for the lymphoma syndrome. he then observed adults with the lymphoma but only in individuals that moved into the "african lymphoma belt." these tumors were then observed in the united states and europe at a rate of 1-3 per million or about 100x less frequently than in africa. biopsy samples did produce viruses but none that came from insects so the working hypothesis was abandoned. a new hypothesis emerged between 1963 and 1966 in which p falciparum (a parasite causing malaria) was the causative agent for the lymphoma. it is believed that severe malaria does play a role in the development of burkitt's lymphoma. at the advice of peter clifford, burkitt administered methotrexate to treat patients which produced encouraging results. in 1961 burkitt gave a lecture at middlesex hospital, "the commonest children's cancer in tropical africa: a hitherto unrecognized syndrome." anthony epstein attended the lecture which changed his life and provides a punctuation mark in the history of science. epstein introduced himself after the lecture and the two sat down for tea. burkitt agreed to send tumor specimens to epstein and epstein received a grant from the us national institutes of health in 1963 for $45,000 which allowed him to employ two research assistants. yvonne barr was one of the research assistants that was able to propagate a virus after 26 tries with tumor specimens. the other research assistant was bert achong, an electron microscopist, who was first to identify the virus as a member of the herpes virus family. the virus bears the name epstein barr virus or ebv and is a causative agent of burkitt's lymphoma. an interesting historical note involves werner and gertrude henle at the children's hospital of philadelphia. they created antibodies to ebv infected cells and their survey of blood samples from american adults revealed over 90% to be ebv positive in 1966. they also showed that normal human lymphocytes could be made immortal by infecting them with ebv in 1967. we now know ebv silently infects children in the western world but in those infected a bit later in life become susceptible to infectious mononucleosis (the kissing disease) as adolescents. in 1972, a collaboration between george klein and the manolovs led to a discovery of a chromosome change that was specific to burkitt's lymphoma. this chromosome change was a translocation of 8:14 and rarely 8:2 or 8:22 (zech et al. 1976 ). the break points in chromosomes 2, 14, and 22 contained immunoglobin genes that become active in b lymphocytes as they produce antibodies during infection. a nobel prize in 1989 to michael bishop and harold varmus provided the significance to the break point in chromosome 8, bringing active immunoglobin genes near the oncogene c-myc. in 1970, a second cancer common in north and east africa as well as southern china was found to be ebv positive, carcinoma of the post-nasal space (nasal pharyngeal carcinoma or npc). the tumor is observed in epithelial cells not b-cells and is not geographically linked to endemic malaria regions. in this case, it appears that aerosol exposure to environmental carcinogens (possibly in preserved fish) may introduce mutations in nasal epithelial cells. the npc may be the result of silent ebv infection, carcinogen induced cellular mutations, and people carrying hla a*0207 and b*46 antigens. in 1975, several reports describing "fatal infectious mononucleosis" appeared. david purtilo described x-linked proliferative syndrome (xlp) based on an international registry of boys with fatal ebv infections (purtilo et al. 1977) . using genetic marker analysis from the international registry they narrowed xlp to a three million base pair segment on the x-chromosome. in 1998, a 384-base pair segment encoding the xlp protein of 128 amino acids was found to be lost or damaged in al xlp patients (nichols et al. 1998) . curiously, the xlp protein loss leads to a defect in nk and t lymphocytes which are necessary for recognition of ebv infected b lymphocytes. these individuals cannot make antibodies to ebv because their b cells require help from t lymphocytes. the development of potent immune suppressing drugs like cyclosporine a was linked to lymphoma after organ transplantation. two girls with acute lymphoblastic leukemia received bone marrow transplants from their hla-match brothers. the grafts were successful but they developed lymphoblastic leukemia from the transplant (male cells). these lymphomas do not present chromosomal translocation like burkitt lymphomas revealing a new path from ebv to lymphoma. the immune suppression upsets the ebv host homeostasis crippling t-cells that are required for keeping ebv under control. another situation where the virus can replicate without control is in hiv/aids patients and aids-lymphoma is yet another ebv induced casualty. ebv provides key insights into tumors associated with viral infections. first, the same virus is linked to multiple tumors in different populations including burkitt's lymphoma, hodgkin lymphoma, and nasopharyngeal carcinoma. second, tumorigenesis involves more than one mechanistic pathway but interest has focused on the latent membrane protein-1 (lmp1), ebv nuclear antigen 1 (ebna1), and bamh1-a reading frame-1 (barf1) genes. third, dissecting the association between infection with ebv and cancer has unfolded over a period of 30 years and led to a greater understanding of cancer, the immune system, and methods for immortalizing cells to study specific cell clonal populations. ebv has been a boon to scientific research while imposing a challenge to human survival. approximately 2 billion people have been infected with hbv and 360 million suffer from chronic infection. acute hbv infections are usually self-limiting associated with 5.2 million cases a year. chronic hbv infections lead to complications in 15-40 percent of cases resulting in 0.5 to 1.2 million deaths each year. hb v causes 60-80 percent of the world hcc cases which represents 5 percent of all cancers in the world. the hbx gene is considered key to oncogenesis. vaccination programs are effective in reducing mortality in infants and have reduced emerging prevalence of the disease. dietary exposure to aflatoxin enhances hbvrelated hcc (sun et al. 1999 ) as well as co-infection with hcv and excessive alcohol consumption. hepatitis b virus (hbv) was named due to differences in transmission: hepatitis a type virus follows fecal-oral transmission while b type virus is parenteral. the genome is circular dna that is not fully double-stranded with the end of the fulllength strand linked to dna polymerase. the genome is 3020-3320 nucleotides long (full length strand) and 1700-2800 nucleotides for the short strand. four genes are encoded including: (1) c the core protein (hbcag) synthesized by uorf aug to make pre-core protein, (2) p is the dna polymerase, (3) s is the surface antigen (hbsag) which has three aug start sites that divide the gene into pre-s1, pre-s2, and s, and (4) x is a gene that is not fully understood but may be a transcriptional transactivator. non-coding rna include hbv prealpha, hbv prebeta, and hbv rna encapsidation signal epsilon. there are 10 known genotypes which differ by 8 percent in sequence with distinct geographical distributions and are labeled a to j and at least 24 subtypes. type f is the most divergent form and found in central and south america, predominantly brazil. human hbv has a narrow host range infecting humans and higher primates, eg. chimpanzees. in the 1970s a woodchuck hepatitis virus (whv) was discovered which will not infect rodents. a ground squirrel form was identified (gshv) that is a distant relative of the marmot and woodchuck. indeed, dhbv infects ducks but not all species of duck, grey herons are infected with hhbv, the ross' goose with rghbv, the snow goose with sghbv, white storks with sthbv, and cranes with chbv. there are no hepadnaviruses in arthropods or insects. human t-cell lymphotropic virus (htlv-1) htlv-1 is a single-stranded rna retrovirus, defined by their use of reverse transcriptase, a polymerase, that makes a dna copy of the rna 7 kb viral genome. the dna viral genome is integrated into the host genome where it is referred to as a provirus and is replicated along with the host genome during cell division. only 1 percent of infected individuals will develop leukemia and this is observed 20 to 30 years after asymptomatic infection. the htlv-1 tax protein is likely to initiate cell transformation through interactions with transcription activators and cell cycle regulators. hepatitis c virus (hcv) hcv has infected approximately 3 percent of the world's population (~210 million) but screening of the blood supply has reduced prevalence. hcv is a flavivirus composed of a 9.4 kb single-stranded, positivesense rna. hcv is characterized by a single serotype but at least 6 major genotypes. genotype 1b is the most common genotype seen in the united states and taiwan. hcv becomes a chronic infection by evading host immune defenses through a combination of: (1) high replication rate (10 12 virions/day) and (2) lack of error proofreading by the viral polymerase leading to mutations in response to immune pressure. the genetic variability of hcv has limited efforts to design an effective vaccine. the polyomaviruses are small (3.4 kbp), double-stranded dna viruses. early studies with simian virus 40 (sv40) led to identification of the large tumor antigen (large t; lt). lt is also found in bk and jc viruses which are more suspect human tumor viruses and merkel cell polyomavirus (mcv) which is now well established as a human tumor virus (feng et al. 2008) . the n-terminus of lt contains an lxcxe motif that interacts with the retinoblastoma protein rb while the c-terminus contains an atpase/dna helicase domain that can inactivate p53. the ls-p53 complex activates insulin-like growth factor i (igf-i) which alone is capable of cell transformation. a recent report of morbidity and mortality reveals heart disease and cardiovascular events are the number one killer with neoplasia and infections following close behind. however, infections frequently cause neoplasia and cardiovascular disease leading to death. if we combine cardiovascular events and neoplasia caused by infection, then infectious disease is the most significant threat to human life and qualifies as the area of greatest impact. the picornavirus family are small (pico-), single-stranded, positive sense rna genome (7.4 kb) viruses that synthesize a single polyprotein that is cut into a small collection of functional proteins by virally encoded (2a and 3c) and cellular proteases. poliovirus is a picornavirus that has served as the prototype for the viral family. the enteroviruses are a group of picornaviruses that have been associated with cardiac disease. coxsackievirus b (cvb), coxackievirus a (cva), and echovirus infections lead to cardiac signs 3.2% of the time. about one-third of patients with acute cardiac disease (inflammation of the heart) are antibody positive for enteroviruses. while acute myocarditis is often self-limiting, chronic cardiac disease often leads to dilated cardiomyopathy (dcm) which is present with no heart inflammation. dcm is associated with heart failure which can be lethal (table 3 .6). these chronic infections lead to 50 percent of all cardiac transplants worldwide. the enteroviral 2a protease can also degrade dystrophin in the heart leading to cardiac necrosis, reduced ejection fraction, and then to dilated cardiomyopathy. transmission of these viruses is from contaminated food and water. the human herpesviruses are large double-stranded dna genome viruses. the human herpesvirus-5 (hhv5) or cytomegalovirus (cmv) has a 235 kbp genome encoding over two hundred genes. one problem in finding associations with cmv infections is that it is not an emerging disease, 50 to 99% of the population has been infected making comparisons to a control group challenging. most infections are observed in children and in newborns serious clinical findings can be observed. hence, most adults carry latent infections that are reactivated when individuals become immune suppressed following solid organ transplantation, malignant hematological disease, and aids. reactivation in immune suppressed individuals is associated with increased mortality. the association with cardiovascular disease has been demonstrated in two recent studies. in one, cmv infections were detected in 14.5 percent of coronary artery samples from bypass operations compared to 4 percent in of patients who needed cardiac surgery for reasons other than atherosclerosis hebar et al. 2015) . in the other, cmv reactivation (viremia) was detected in 16.5% of immunocompetent patients admitted for major heart surgery (roa et al. 2015) . the incidence of coronary artery disease is the major contributor to death from heart disease and if 14 to 16.5% of this disease is associated with cmv infections, this infection is a significant human health hazard. recognizing an emerging infectious disease involves well established strategies of surveillance; (1) identify unusual clusters of disease, (2) evaluate the spread of an outbreak, (3) estimate the magnitude of the problem, and (4) if possible identify the atheroscleotic plaque infectious agent. the strategy has proven valuable for known infectious and noninfectious diseases but has limited capacity to detect emerging infectious diseases. however, deviation from the traditional approach to surveillance is not likely to gain support. al smith, a veterinary virologist, approached me after a seminar i presented in 1997 in the college of veterinary medicine at oregon state university. i had just joined avi biopharma as the head of their research and development program. al was a veterinarian and professor and had devoted his career to the caliciviridae family of viruses dating back to his time in the naval research station in california. he had isolated the nonhuman vesivirus group of caliciviruses not only from suffering sea lions in the channel islands, but from reptiles in the san diego zoo and whales held in captivity. al and his capable technician, doug skilling successfully propagated the virus isolates in cell culture, an accomplishment not shared by other laboratories at the time. al developed nucleic acid probes and induced antibodies to these viral isolates. over decades of research he assembled an extensive collection of vesiviruses which were held in redundant −70° freezers. his singular vesivirus focus gave the appearance of a zealot but his ability to cultivate viral isolates, his extensive collection of isolates, and his one of a kind detection reagents made him a one of a kind virologist. al was well beyond retirement age and was an "old school" virologist ready at a moment's notice to take his bag into the real world and collect swabs from ailing animals. he maintained careful records of the condition of his patients, their location, and the setting of the animal in the community. every field trip led to work in the lab propagating virus from his swabs. al wanted to know if i would be interested in finding an antiviral agent for these viruses. my prime directive at avi biopharma was to explore the capabilities of our proprietary antisense technology and i had yet to investigate targeting a virus. the caliciviruses have positive sense single-stranded rna genomes which express three genes from a single polyprotein. we identified an active agent following an investigation with small collection of candidates (stein et al. 2001) . the vesivirus group of caliciviruses are considered animal only and are not believed to infect humans but al began exploring human samples with his collection of detection reagents. after several years making incremental progress, we found an association between human blood samples seropositive for vesivirus and markers of liver disease (smith et al. 2006) . we felt this evidence of an emerging infectious disease in humans and its potential to cause liver disease would be welcomed by the medical community. al and i made a trip to washington dc at our own expense to relate the findings in person. we meet with virologists at the national institutes of health but were met with judicious skepticism. harvey alter had been instrumental in the discovery of hepatitis c virus (hcv) and felt all viral liver disease is already accounted for by hepatitis viruses. hence, no interest in our findings. we met with the american red cross blood banking group in shady grove maryland and were met with concern. blood supplies are scrutinized by the nucleic acid test (nat) to eliminate viral contamination and any further elimination of blood samples would threaten an already limited inventory. our findings simply add complication to the blood supply finding an emerging infectious disease business. they asked for more compelling and more extensive data to add robustness to our claims. the only problem is that they were not interested in providing financial support for the recommended studies. the conundrum is all too common, you need more data to convince granting agencies to support the work but there is no support to add to the limited data. life on the cutting edge is frequently discouraging. our strategy took two paths: one to seek commercial support and the other to create proof of concept data for an antiviral solution. the most logical commercial solution was to meet with michael houghton at chiron. he was the driving scientific force in finding hcv and chiron might like adding to their reputation by finding another previously unrecognized hepatitis virus. indeed, dr. houghton invited us to bay area to discuss the project. he reviewed our data and the quality of al's detection reagents. chiron provided modest support and their in-house laboratory help to confirm our observations, a glimmer of hope. al created a company, calicitech, so that he could accept support and license his reagent patents from the university. testing an antiviral agent in humans would be expensive but in the absence of natural history of the infection would make human proof of concept impossible. however, al had been contacted by a cat rescue facility in atlanta, mommy cat, describing an outbreak of feline calicivirus (fecv) in their cattery. these cats had all been vaccinated with an approved fecv vaccine which failed to protect these cats. a veterinarian can decide to use alternative medicines if there are no alternatives and the owner oc the cats can sign the equivalent of informed consent. we provided our fecv specific antiviral to mommy cat along with a detailed protocol for treatment. the infected kittens we treated survived while those not treated died providing encouraging data. we then discovered a similar outbreak in the humane society facility in eugene oregon, our neighbors. again, we reached an agreement to treat infected kittens and were successful ). this was the first time we treated an infection based on symptoms in an "outbread" population of cats. with over 100 kitten patients and remarkable survival differences between treated and untreated, we had our proof of concept. there is no disease cluster to link to human vesivirus. norovirus is a calicivirus and is known to infect humans, in fact it is well known on cruise ships, day care centers, and extended care retirement facilities. our efforts to have human vesivirus recognized as an emerging infectious disease have failed. chiron was not impressed with our antiviral and no longer were interested in evaluating the detection reagents. if we are lucky, human vesivirus infections will remain mild clinical oddities. al smith has an interesting way of responding, "that virus is out there infecting humans. it won't go away." the existence of non-pathogenic viral infections led to the emergence of the study of the immune system, vaccination, gene therapy, and concern for future pandemics. we carry evidence of ancient retroviral infections in our genome from integration events that became vertically transmitted making up as much as 8 percent of the human genome (meyer et al. 2017) . these genomic fossils are called endogenous retroviral genomes (erv). most erv sequences accumulate sufficient mutations over evolutionary time that horizontal transmission is unlikely. these viral genome segments are trapped but can still be transcribed and encode some viral proteins. the significance of these integrated genomes is a current topic of investigation. adeno-associated virus (aav) is a single stranded dna virus that infects humans but are not known to cause disease. the lack of pathology has led to their use as viral vectors for human gene therapy. aav can infect dividing and quiescent cells and will persist extra chromosomally without integrating into the genome of the host cell. aav is a member of the parvoviridae family in the genus dependoparvovirus. is an arenavirus closely related to lassa virus and shares reservoir hosts. mv is naturally attenuated and nonpathogenic in humans. infection of humans with mv protects against lethal challenge with lv (wulff et al. 1977) . flaviviridae) named after g. barker, a surgeon, first identified in 1995. hgv infects one sixth of the world's population but does not cause human disease. a metaanalysis of 15 publications investigating gbv-c infections in hiv-positive individuals indicate coinfection with gbv-c slows the progression of hiv disease in individuals that have been seropositive for 2 years or more (zhang et al. 2006a) . two of the studies investigated gbv-c 5 years after documented hiv seroconversion estimate the hazard ratio of 0.36 (95% ci). is a retrovirus first identified in 1971 from a lymphoblastoid cell culture from a kenyan patient with nasopharyngeal carcinoma. hfv is homologous to primate foamy viruses and is most closely related to the chimpanzee foamy virus (sfvcpz). early studies raised alarm for association with autoimmune diseases but more extensive studies with more precise diagnostic reagents fail to find a disease associated with hfv. hfv is a rare human infection and concerns parallel sfv infections in humans. simian foamy virus (sfv; spumavirus-retroviridae) is a retrovirus infecting most primates born in captivity and people making contact with infected primates can become infected. human infections frequently occur in males probably requiring a bite from infected nonhuman primates but are harmless. the infected cells often fuse to form syncytia of giant foamy cells, which gives the virus its name. the error rate in sfv genomes is exceptionally low, 1.7 x 10 −8 substitutions per site per year, compared to hiv 10 −3 substitutions per site per year. since so-called crossspecies, infections have only been observed for a little over a decade the long term consequences are not known. these infections are watch and wait for everything from a zoonotic epidemic to identified disease clusters. perhaps this is exactly the sort of infection that will emerge as a significant human concern in the future. torque teno virus (ttv; alphatorquevirus) is a single-stranded, positive sense dna genome virus about 3.8 kb in size in the anelloviridae family. nearly 100 percent of even healthy individuals are infected in some countries. the virus was discovered in 1997 as the "transfusion transmitted virus (ttv)" in a japanese patient. it is often found in patients with liver disease but does not cause hepatitis on its own. closely related torque teno mini virus (ttmv) were isolated in 2007 and found to have smaller genomes of 2.8-2.9 kb. ttmv infections are also common but do not cause any described human disease. human adenovirus type 5 (rad5) is used to create an ebolavirus (ebov) vaccine encoding zaire ebolavirus glycoprotein failed to protect animals immune to ad5. a replication defective chimpanzee-derived adenovirus (chad3-ebo-z) provided protection against lethal ebov challenge in macaques but protection wane over several months. they boosted with a modified vaccinia ankara (mva-bn-filo) that led to durable protection (stanley et al. 2014 ). this vaccine progressed through phase i, single-blind, randomized human trials in mali between 2014 and 2015. a single dose of the chad3-evov-z is efficient as a prime vaccine strategy followed by mva-bn-filo as a boost was well tolerated in humans tapia et al. 2016) . is a 5229 base double-stranded dna virus infecting less than 5 percent of the human population. wu, named after washington university, is found as a co-infection in various respiratory infections but wu does not cause disease on its own. wu is closely related to ki virus that also is not known to cause clinical disease. however, related polyomaviruses that are clinically relevant include bk virus associated with nephropathy, jc virus associated with progressive multifocal leukoencephalopathy, sv40 virus associated with mesothelioma, and merkel cell polyomavirus associated with cancer. vaccinia virus (orthopoxvirus) is a large double stranded dna virus closely related to smallpox. edward jenner, the father of immunology, found the milkmaids exposed to cowpox (vaccinia) were immune to smallpox in 1798. this was the first vaccine (named after vaccinia) leading to the modern vaccine that has allowed for the eradication of smallpox. viral sequences are constantly mutating with no purpose other than seeking a survival/infectivity benefit. this means viruses with no current pathology represent a pre-mutation reservoir for the next catastrophic human pandemic. the popularity of rnaseq is likely to expand our catalog of nonpathogenic viral infections. however, the management of such information is in question. technology used to counter viral infections has resulted in over 90 approved drugs for the treatment of nine different human viral infections in just 50 years (de clercq and li 2016) . several different antiviral drug groupings have been reported, but the following arise from review of the mechanisms of action of antiviral drugs assembled in table 3 .7: (1) inhibition of viral attachment and entry, (2) inhibition of viral uncoating, (3) viral polymerase inhibitors, nucleotide analogues (ntrti) and non-nucleotide reverse transcriptase inhibitors (nnrti) and dna polymerase inhibitors, nucleic acid synthesis inhibitors and nucleotide pool size agents, (4) latency reversal agents, (5) integrase inhibitors, (6) protease inhibitors for both hiv and hcv, (7) neuraminidase inhibitors, (8) immune response modifiers, and (9) antisense inhibitors. administration of hyperimmune sera from immunized animals or human donors was the first effective treatment for infectious diseases. the practice has limitations but is still used to treat bacterial toxins and viral infections caused by cmv, rsv, hav, hbv, rabv, vzv and mev (keller and stiehm 2000) . the development of human or humanized monoclonal antibodies (humabs) has created a feasible way to rapidly generate novel antiviral therapeutics. humabs have advantages over serum therapy in that they are chemically defined reagents with minimal variability, greater activity per mass of protein, and they have no immunological consequences related to serum sickness. several mabs have been approved for treatment of infectious diseases including viral and bacterial pathogens (table 3 .8). the antiviral mab discovery field is exploding with activity particularly for hiv and hcv infections. a humanized mab targeting lymphocyte ccr5 receptors called pro140 has demonstrated potent and prolonged anti-hiv-1 activity and a large margin of safety (jacobson et al. 2010a) . administration of pro 140 by the subcutaneous route offers patients a way to self-administer the mab but importantly the mab is transported in the lymphatics providing enhanced access to binding to the cellular target (jacobson et al. 2010b) . the next generation of antiviral therapeutics are likely to be dominated by mabs. perhaps the only way to clear a viral infection involves a host immune response (table 3 .9). the innate immune response is particularly effective centered on a type 1-interferon pathway. unfortunately, many viruses carry mechanisms to evade host innate responses and innate immune effectors do not have the capacity for memory. the adaptive immune response can mitigate infection with antibodies, generally to surface antigens, which prevent the spread of the virus and t-lymphocytes, which can clear virus-infected cells. the first strategy will be to use an existing drug designed for another virus offlabel. this seems likely for antiviral drugs like cidofovir, foscarnet, and ganciclovir particularly for double-stranded dna viruses like ebv, hpv, and hhv6. ribaviran has been used for a number of single-stranded rna viruses including polio, junin, and lassa fever. secondary strategies will require investment of time and effort beginning with vaccine development and creation of monoclonal antibodies. platform technology rna-based therapeutics offer rational design for an expansive number of new antiviral strategies. this advantage is superimposed on the theoretical advantages of selectivity, specificity and affinity provided by watson-crick base pairing. rna-based therapeutics are expected to provide a substantially more narrow range of pharmacokinetic properties and toxicities thus are easier to compare to each other and ultimately combine into multi-agent cocktails. however, the mechanism of action may vary from rnase h or risc mediated degradation of the targeted rna or steric inhibition of rna function. the objectives of our antiviral program have been to exploit the broad understanding of rna-based therapeutics for antiviral activity with a common chemical type, the phosphorodiamidate morpholino oliogmer (pmo) and their enhanced derivatives. in this way the mechanism of action is common to all agents, which is steric blockade of rna function. studies reported by zamecnik and stevenson introduced the first approach to identification of an antisense antiviral agent stephenson and zamecnik 1978) . they used a 13-mer targeted to rous sarcoma virus. since the rous sarcoma virus pioneering efforts, antiviral rna based therapeutics have involved multiple oligomer chemistries with a variety of different mechanisms of action. chemical approaches to oligomers directed to hiv have been plentiful. a nonionic methylphosphonate oligonucleotide targeted to the splice acceptor site of hiv tat inhibited splicing of viral rna inhibiting syncytia formation and p24 synthesis at 3um concentration. poor aqueous solubility limited the utility of the methylphosphonate chemistry. phosphoramidate chemistry was investigated for inhibition of the splice-donor and splice-acceptor of hiv tat agrawal et al. 1988 ) and were more potent but these agents were cytotoxic and poorly water soluble. phosphorothioate chemistry targeting hiv-rev (matsukura et al. 1987 ) and hiv-tat were shown to be effective in inhibiting hiv replication, were not cytotoxic and were very soluble. further, the hiv-rev phosphorothioate oligodeoxynucleotide was stable in vivo with an acceptable pharmacokinetic profile (iversen et al. 1994) . a 25-mer phoshorothioate called gem91 targeting the initiation site of hiv-gag was evaluated in clinical trials (agrawal 1998 ) but the trials were discontinued. i focused on phosphorodiamidate morpholino oligomer chemistry which is both stable and net-neutral in charge at physiological ph. single stranded rna viruses with positive sense (ssrna(+)) these viruses are the most simple in terms of genome size, number of potential translated viral proteins, their genomes are all linear and they enter the cell ready for translation. the design of steric blocking rna-based therapeutics involves preventing translation, disrupting rna secondary structure and masking recognition sites for rna dependent rna polymerase (rdrp). the targeting of either the 5′-terminus and the first orf-aug are active. further, efficacy in animal challenge studies is observed with high fidelity when the most effective agent identified in vitro is employed. the family astroviridae with six different human astroviruses (huastv) responsible for 2-17% of all gastroenteritis. we found the 5′-terminus 1 to be the most effective site to target. this was also the optimal site for caliciviridae including vesivirus (vev; martin-alonso et al. 2005; stein et al. 2001), norovirus (nov; bok et al. 2008) , and feline calicivirus (fcv; smith et al. 2008) ; the flaviviridae including dengue (den; kinney et al. 2005; holden et al. 2006) , and west nile virus (wnv; deas et al. 2007; zhang et al. 2008) , arteriviridae including agriculturally important equine arterivirus (eav; van den born et al. 2005) , and porcine respiratory and reproductive virus (prrsv; zhang et al. 2006a, b) ; and the togaviridae exemplified by venezuelan equine encephalitis virus (veev; paessler et al. 2008 ). the family coronaviridae revealed a new active target site in the transcription regulatory sequence (5'-cgaac-3′) in both mouse hepatitis virus (mhv; neuman et al. 2004 ) and the severe acute respiratory syndrome virus (sars; neuman et al. 2005) . the picornaviridae active target site involved a highly conserved sequence in the internal ribosomal entry site (ires) in polio virus (pv; stone et al. 2008) , foot and mouth disease virus (fmdv; vagnozzi et al. 2007) , and the coxsackievirus (cvb3; yuan et al. 2006) . single stranded rna viruses negative sense (ssrna(−)) these viruses are generally more complex with respect to genome size, number of potential translated viral proteins, and multiple genome segments. the genome must be replicated prior to translation of viral proteins. the design of steric blocking rna-based therapeutics is similar to the positive sense rna genomes in that targets involve preventing translation and masking recognition sites for rna dependent rna polymerase (rdrp). we investigated 11 different targets in measles virus (mev), a member of the paramyxoviridae, finding the translation initiation start site of n the optimal target (sleeman et al. 2009 ). studies with the human respiratory syncytial virus (hrsv) found the translation site for l to be most active (lai et al. 2008) . the orthomyxoviridae studies investigated influenza a virus probing each of the 8 viral segments finding translation of pb1 active as well as the 5'terminal of vnp for h3n2 (ge et al. 2006 ) and h3n8 (lupfer et al. 2008 ) but a combination of targets was required in animal studies with a high pathogenic viral h7n7 isolate (gabriel et al. 2008 ). more extensive influenza a studies revealed a new target, the m-segment splice donor site. this target was evaluated in phase i clinical trials. the antisense platform technology has limitations in targeting viral sequences. inhibiting virally encoded proteins or blocking viral replication by interfering with the polymerase does not always work. the arenaviridae family proved difficult. we experienced some success with junin and lymphochoriomeningitis virus (lcmv) in cell culture observing 4 log 10 reductions in viral titer with a pmo targeting the highly conserved viral genome terminus. however, we failed to provide survival benefit to guinea pigs challenged with either junin or lassa virus. we then tried the mouse challenged with lcmv and failed again but we observed hemorrhagic disease in the mouse, a severe consequence of infection that mimics the worst aspects of arenaviral infection. we decided to try targeting host genes to mitigate disease in the mouse and found targeting il-17 provided a survival benefit (schnell et al. 2012) . the success in the face of failure puts targeting host genes at the center of attention for dealing with emerging infectious diseases. the antisense platform technology represents an excellent approach to rapid drug discovery for emerging infectious disease. rapid discovery demonstrated repeatedly but of course, the cost of advanced development is daunting. the application is best for immediate treatment of index cases, close contacts, and healthcare workers. we have an 800-pound gorilla in the room and everyone in the room considers it someone else's problem. the current course is to ignore the majestic creature until it starts tearing limbs from people and then the consensus is to kill the gorilla. agencies like the world health organization (who), the centers for disease control (cdc) and the national institutes of health (nih) can assemble highly skilled personnel and can confer with some of the greatest minds in the world. unfortunately, they are all aware of the potential problem that an emerging pandemic is likely to take us by surprise. significant speculation that a single-stranded rna virus will emerge killing tens of million people, costing hundreds of billions of dollars, and changing the course of human history. there is a high probability that the virus will be influenza a (h5n1 or h7n9) that will jump from a reservoir population of birds and establish human-to-human transmission. the pandemic will be a global event by the time an effective vaccine is available. neuraminidase (na) inhibitors as therapeutic and prophylactic agents in the setting of pandemic influenza a (flua) were called in to doubt in the past decade (michiels et al. 2013; jefferson et al. 2014) . indeed, 100% of isolates in the 2008/2009 a/h1n1 pandemic were found to be resistant to adamantanes, and resistance to oseltamivir (tamiflu; osl) was observed in virus recovered from individuals taking osl therapeutically or prophylactically (dharan et al. 2008; cdc mmwr 2009) , while effect on duration of shedding was not impacted. a recent outbreak of an influenza a (h7n9) virus caused 137 cases and 45 deaths in china revealed a novel na mutation r292k resulting in high level resistance to osl (wang et al. 2014) . therapeutic options for treatment of individuals with complicated influenza a are severely limited, perhaps no options. pandemic strains such as a/h1n1pdm09 carried significant morbidity and mortality, particularly in those who had not experienced h1-strain influenza in their lifetime. we are also witnessing more rapidly emerging highly pathogenic avian influenza strains that are resulting in human infection; some such as a/h5n1 and a/h7n9 appear to becoming more efficient in person-to-person transmission, and reports suggest osl resistance develops during treatment (de jong et al. 2005; lam et al. 2015) . we are not ready for an outbreak of avian flu or any other emerging single-stranded rna virus. why not? an estimate for the time and cost to develop a new drug is 10 years and $1 billion. the commercial use of a drug for an emerging infection is hard to estimate since by definition when you start development the infection has not emerged. most of us would be unlikely to use our retirement savings to invest in a drug development project with no reliable way to expect a return on our investment. it is a poor business model. when you consider there may be hundreds of emerging infectious diseases each times 10 years and $1 billion each the task is daunting. on which disease should we focus? consider the ebola drug avi-7537. the ebola therapeutic project began in 2004 following a laboratory accident at usamriid. we identified three compounds each with activity and when combined we observed unprecedented efficacy in a lethal challenge primate animal model (warfield et al. 2006 ). hundreds of experiments over the next 3 years optimized these agents (swenson et al. 2009 ). we were able to obtain research grants from the transformational medical technologies (tmt) division of the defense threat reduction agency (dtra) within the department of defense (dod) and we completed proof of efficacy studies (warren et al. 2010) . this led to submission of an investigational new drug application (ind) to the food and drug agency (fda) and phase i safety and tolerability studies were conducted i healthy human volunteers (heald et al. 2014) . after 11 years of continuous effort, we completed key aspects related to the fda approval process under the "animal rule" and we streamlined our treatment to a single agent, avi-7537 (warren et al. 2015) . however, shortly before the ebola outbreak in western africa, the us government "budget sequester" cancelled our project and the most advanced therapeutic on the planet was not deployed to treat those infected during the outbreak. as the outbreak continued, we found no viral resistance of avi-7537 unlike the monoclonal antibody therapy in use (khiabanian et al. 2014) . avi-7537 sits on a shelf, a political casualty and an unfavorable business model. the global virosphere may contain up to 10 31 virus/virus-like particles (suttle 2005) , the greatest reservoir of genetic diversity. the earth's atmosphere transports viruses all over the planet. viruses are found in soil at 1.5 x 10 8 to 6.4 x 10 8 particles per gram of dry soil (kimura et al. 2008) . the surface oceans carry approximately ten million viral particles in each milliliter of seawater, most of which are bacteriophages (phage). the small viral particles are easily carried into the upper environmental viral reservoirs atmosphere by up drafting winds. bacteria are deposited from the atmosphere at a rate of 0.3 to 8 x 10 7 per meter each day and viral deposition rates are 9-461 times greater (reche et al. 2018) . these phages influence bacterial lifecycles and play a role in natural energy and nutrient cycles fundamental to life on earth. the dynamics of phage-bacterial evolution drive changes in photosynthesis, phosphate, and nitrogen balance (breitbart 2012) . human accidental release of radioactive waste (discussed in chap. 2) and disposal of chemicals including potent antiviral and antibacterial compounds (discussed in chap. 7) may alter the eco-evolutionary dynamics producing unanticipated environmental consequences. the objective of this chapter has been to provide convincing evidence that infectious disease is the most significant threat to human health. the focus has been on viral infections because they rely on host ribosomes to produce their proteins, recent emerging infections have been from single-stranded rna genome viruses, and replication of rna viruses is error prone. pandemic infections have accompanied the rise of human civilization frequently re-occurring leaving a lasting imprint on human history punctuated by profound loss of life. emerging infections become endemic with an annual death toll. each decade brings a new onslaught of emerging infectious agents. we are surprised again and again but have never prepared for these inevitable catastrophies. the long-term consequences often remain unrecognized and are always inconvenient such as cancer, cardiovascular disease and immune associated diseases that threaten our health. reliance on clusters of clinical symptoms in the face of diverse and non-descriptive viral infection symptoms is a foolhardy form of crisis management. infectious disease will certainly continue to pose the most significant threat to human health in the age to cell phones, artificial intelligence, and global commerce. rapid replication of viral genomes combined with low fidelity polymerases provide the foundation for an unending source of new emerging infectious agents. these traits also make viral genomes sensitive to environmental contaminants in a way that may expand probabilities for zoonosis. infectious disease as part of our environment is not appreciated. the study of infectious disease is not a part of the curricula of students in environmental science/management. textbooks in in environmental studies do not include chapters in infectious disease. the integration of research at superfund sites focused on chemical contamination with infection and zoonosis would result in valuable insights into threat analysis. viruses with rna genomes lack sequence proofreading quality control during replication. the cumulative mutations in their genomes limits the genome size to under 30,000 bases. essentially, a larger genome would evolve out of existence, so called catastrophe evolution. the limited genome size makes these viruses exceptionally resilient to a changing environment. the virus must economize by combining functions. this means evolution and resilience are the same thing in the rna genome viruses. a unique insight is that in human evolution is restricted to the dna genome and resilience limited to rna, as it is in the rna genome viruses. antisense oligonucleotide-based therapy for hiv-1 infection from laboratory to clinical trials oligodeoxynucleotide phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus a tribute to sheik humarr khan and all the healthcare workers in west africa who have sacrificed in the fight against ebola virus disease: mae we hush inhibition of norovirus replication by morpholino oligomers targeting the 5'-end of the genome marine viruses: truth or dare cancer virus oseltamivir-resistant 2009 pandemic influenza a (h1n1) virus infection in two summer campers receiving prophylaxis-north carolina approved antiviral drugs of the past 50 years the eradicatio of smallpox: edward jenner and the first and only eradication of a human infectious disease oseltamivir resistance during treatment of influenza a (h5n1) infection in vitro resistance and in vivo efficacy of antisense oligomer against west nile virus outbreak of antiviral drug-resistant influenza a in long-term care facility the perpetual challenge of infectious disease clonal integration of a polyomavirus in human merkel cell carcinoma morpholino oligomers targeting the pb1 and np genes enhance survival of mice infected with highly pathogenic influenza a h7n7 virus inhibition of multiple subtypes of influenza a virus in cell cultures with morpholino oligomers safety and pharmacokinetic profiles of phosphorodiamidate morpholino oligomers with activity against ebola virus and marburg virus: results of two single ascending dose studies cytomegalovirus infection and atherosclerosis in candidate of coronary artery bypass graft inhibition of dengue virus translation and rna synthesis by a morpholino oligomer targeted to the terminal 3′ stem-loop structure pharmacokinetics of an antisense phosphorothioate oligodeoxynucleotide against rev from human immunodeficiency virus type 1 in the adult male rat following single injections and continuous infusion phase 2a study of the ccr5 monoclonal antibody pro 140 administered intravenously to hiv-infected adults anti-hiv-1 activity of weekly or biweekly treatment with subcutaneous pro 140, a ccr5 monoclonal antibody oseltamivir for influenza in adults and children: systemic review of clinical study reports and summary of regulatory comments passive immunity in prevention and treatment of infectious diseases viral diversity and clonal evolution from unphased genomic data ecology of viruses in soils: past, present and future perspectives inhibition of dengue virus serotypes 1 to 4 in cell culture with morpholino oligomers inhibition of respiratory syncitial virus infections in cell cultures and in mice with morpholino oligomers dissemination, divergence and establishment of h7n9 influenza viruses in china health impact of globalization: towards global governance inhibition of influenza a h3n8 virus infections in mice by morpholino oligomers isolation and characterization of a new vesivirus from rabbits phosphorothioate analogs of oligodeoxyribonucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus endogenous retroviruses: with us and against us the value of neuraminidase inhibitors for the prevention and treatment of seasonal influenza: a systematic review of systematic reviews antisense morpholino oligomers directed against the 5'-end of the genome inhibit coronavirus proliferation and growth inhibition, escape and attenuation of sars coronavirus treated with antisense morpholino oligomers inactivating mutations in an sh2 domain-encoding gene in x-linked lymphoproliferative syndrome inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oliogmers immunological disorders and malignancies in five young brothers spillover animal infections and the next human pandemic deposition rates of viruses and bacteria above the atmospheric boundary layer a prospective monitoring study of cytomegalovirus infection in nonimmunosuppressed critical heart surgery patients inhibition of acquired immunodeficiency syndrome virus by oligonucleotide methylphosphonates lymphocytic choriomeningitis virus infection in fvb mouse produces hemorrhagic disease ancient athenian plague proves to be typhoid inhibition of measles virus infection in cell cultures by peptide-conjugated morpholino oligomers vesivirus viremia and seroprevalence in humans virus specific antiviral therapy for controlling severe and fatal outbreaks of feline calicivirus infection chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge inhibition of vesivirus infetions in mammalian tissue culture with antisense morpholino oligomers inhibition of rous sarcoma viral rna translation by a specific oligodeoxynucleotide inhibition of multiple species of picornavirus using a morpholino oligomer targeting highly conserved ires sequence increased risk of hepatocellular carcinoma in male hepatiis b surface antigen carriers with chronic hepatitis who have detectable aflatoxin metabolite m1 viruses in the sea chemical modifications to phosphorodiamidate morpholino oligomer antisense molecules targeting vp24 modify their efficacy against ebola virus infection use of chad3-ebo-z ebola virus vaccine in malian adults with mva-bn-filo: a phase i, single-blind, randomized trial, a phase 1b, open label and double blind, doseescallation trial, and a nested, randomized, double-blind, placebo-controlled trial inhibition of foot-and-mouth disease virus in cell cultures with antisense morpholino oligomers antiviral activity of morpholino oligomers designed to block various aspects of equine arteritis virus amplification in cell culture pcr for detection of oseltamivir resistance mutation in influenza a(h7n9) virus igg antibodies to dengue enhanced for fcγriiia binding determine disease severity gene-specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers advanced antisense therapies for postexposure protection against lethal filovirus infections single component avi-7537 antisense compound provides greater protection than double component avi-6002 against lethal ebola virus infection in rhesus monkeys isolation of an arenavirus closely related to lassa virus from mastomys natalensis in south-east inhibition of coxsackievirus b3 in cell cultures and in mice by peptide-conjugated morpholino oligomers targeting the internal ribosomal entry site inhibition of rous sarcoma virus replication and transformation by a specific oligodeoxynucleotide characteristic chromosomal abnormalities in biopsies and lymphoid-cell lines from patients with burkitt and non-burkitt lymphomas effect of early and late gb virus c viremia on survival of hiv-infected individuals: a meta-analysis suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers west nile virus genome cyclization and rna replication require two pairs of long-distance rna interactions key: cord-006523-zxn4oqly authors: lodha, rakesh; chandra, uma; natchu, mouli; nanda, mrinal; kabra, s. k. title: nosocomial infections in pediatric intensive care units date: 2001 journal: indian j pediatr doi: 10.1007/bf02722358 sha: doc_id: 6523 cord_uid: zxn4oqly nosocomial infections are a significant problem in pediatric intensive care units. while indian estimates are not available, western picus report incidence of 6–8%. the common nosocomial infections in picu are bloodstream infections (20–30% of all infections), lower respiratory tract infections (20–35%), and urinary tract infections (15–20%); there may be some differences in their incidence in different picus. the risk of nosocomial infections depends on the host characteristics, the number of interventions, invasive procedures, asepsis of techniques, the duration of stay in the picu and inappropriate use of antimicrobials. most often the child had endogenous flora, which may be altered because of hospitalization, are responsible for the infections. the common pathogens involved arestaphylococcus aureus, coagulase negativestaphylococci, e. coli pseudomonas aeruginosa, klebsiella, enterococci, andcandida. nosocomial pneumonias predominantly occur in mechanically ventilated children. there is no consensus on the optimal approach for their diagnosis. bloodstream infections are usually attributable to the use of central venous lines; use of tpn and use of femoral site for insertion increase the risk. urinary tract infections occur mostly after catheterization and can lead to secondary bacteremia. the diagnostic criteria have been discussed in the review. with proper preventive strategies, the nosocomial infection rates can be reduced by up to 50%; handwashing, judicious use of interventions, and proper asepsis during procedures remain the most important practices. nosocomial or hospital acquired infections are all infections that occur during a patient's hospitalization and not present or incubating at admission. also any infection that appears to have been acquired in hospital but does not manifest until after discharge is judged to be a nosocornial infection. therefore, all infections diagnosed 48 hours after admission till 72 hours after discharge should be considered as nosocomial. 1, 2 nosocomial infections are a significant problem in pediatric intensive care units. the national nosocomial infection surveillance system (nnis) in the united states reports a rate of 14.1 nosocomial infections per 1000 patient days for pediatric icus. 3 the overall nosocomial infection rates in the picus in the west are reported to be 6-8%. 3.5 these rates are lower than those seen in adult intensive care units. 3, 6 the infections in pediatric intensive care units (picu) are more widespread than in general pediatric wards. the infection rates depend on the type of picu: multidisciplinary, medical, surgical, or cardiac. there are no data on the incidence of nosocomial infections in indian picus. it is possible that infection rates here are higher than those reported in the west. according to nnis, primary bloodstream infections are the commonest nosocomial infections (28%) in picus followed by the lower respiratory tract infections (21%) and urinary tract infection (uti) (15%). 3 in another study, lower respiratory tract infections were the commonest reprint requests : dr. rakesh lodha, assistant professor, department of pediatrics, all india institute of medical sciences, ansari nagar, new delhi-110029. e-mail : skkabra@hotmail.com (35%), followed by bloodstream infections (21%) and uti (16%). 4 these variations may arise from the differences in the proportion in the children ventilated in the picus. in adults, uti is the commonest nosocomial infection followed by surgical infection and lower respiratory tract infections. 6 staphylococcous aureus, coagulase negative staphylococci, e. coli, pseudomonas aeruginosa, klebseilla, enterococci and candida are the significant pathogens in pediatric services in the various sites examined. 3, 4 the risk of nosocomial infections in a picu is the direct consequence of the severity of illness, 4 the level of invasive monitoring, the indiscriminate use of antimicrobials and the nature of diagnostic procedures. the nnis data emphasizes that the duration of stay in an icu is an important determinant of nosocomial infection; therefore, the length of stay should be one of the denominators in calculation of nosocomial infection rates2 in addition, it highlight the importance of invasive devices (endotracheal tubes, intravascular catheters, urinary catheters) in development of nosocomial infections. 3 in order to control for device usage and duration the infection rates can be represented as ratios per thousand device days. according to the latest report, ventilator associated pneurnonia (vap) occurrence is 5.7 episodes per thousand ventilator days, uti 5.2 per thousand catheter days, and bloodstream infection is 8 per thousand central line days. 3 in the same study, it was observed that 91% of all nosocomial bloodstream infections occurred in children with central venous lines, 95% of nosocomial pneumonias occurred in those on mechanical ventilation and 77% of utis in children with urinary catheters. 3 these figures highlight the important role of various devices in nosocomial infections. nosocomial infections may be caused by organisms that originate from exogenous sources in hospital or from endogenous sources such as child's own flora. because of alteration of child's flora associated with the illness and hospitalization, such distinction may not be easy. a recent study has shown that the mean number of days after admission to picu to abnormal colonization were as follows: gastric aspirate, 2 days; tracheal aspirate, 5 days; urine 10 days; and stool, 4 days. the altered flora mainly included gram-negative bacilli and staphylococcus aureus, which were often antibiotic resistant/ the hospital environment contributes minimally to acquisition and spread of most endemic nosocomial infections directly. even if environmental surfaces are contaminated with microbes, they can be spread to patients by hand contact only ~. occasionally, environment may be responsible for life threatening nosocomial infections such as aspergillus in immunocompromised individuals. it is estimated that the overall mortality attributed to nosocomial infections is about 10%. 9 the mortality is associated with nosocomial infections is multifactorial; some of these factors are the type of patient, the number of altered organs, and the microbes responsible for the infection. blood stream infections caused by klebsiella pneumoniae or the fungi have mortality range of 18-20%2 ~ the nosocomial infections increase the duration of stay in hospital and also the cost of therapy. we restrict further discussion to nosocomial pneumonias, bloodstream infection and urinary tract infections. pneumonias constitute a common but potentially life threatening complication of hospitalization. the mortality rates associated with nosocomial pneumonia ranges from 20-50%22,~3 other studies have reported lower mortality rates. 14 while a few pneumonias represent hematogenous seeding of the lungs from a distant suppurative focus; in most patients, subclinical aspiration of oropharyngeal secretions containing bacteria that have colonized the upper airway of the patient is responsible for nosocomial pneu-monia~ this flora includes both gram-positive and gramnegative bacteria, which the patient acquires within 1-4 days after hospitalization. the risk factors for upper airway colonization includ(, acidosis, hypotension, endotracheal intubation, and broad-spectrum antimicrobial ther-apy25 only a few patients will develop nosocomial pneumonia in absence of colonization of the upper airways. 13 most nosocomial pneumonias are caused by gram-negative organisms. tm bacteria of enterobacteriaceae family eg. e. coli, klebsiella are usually isolated from the hypophar-ynx and rectum before they are isolated from the trachea. 12 however, non-enterobacteriaceae e.g.p, aeruginosa, acinetobacter are rarely demonstrated prior to their isolation from the trachea. 12 this suggests that colonization with enterobacteriaceae occurs from patient's endogenous flora and that non-enterobacteriaceae bacteria have environrnental origin. therefore, the hands of the health care workers and components of the respiratory therapy equipment may be important factors in the transmission of bacteria. the most important risk factor for nosocomial pneumonia in children is endotracheal intubation. nosocomial pneumonias are nearly four times more common in intubated patients than in non-intubated patients2 tm the risk may be greater with tracheostomy tubes. the filtration system of upper airways and mucociliary system of the large airways is bypassed during intubation, predisposing the patient to colonization with potential pathogens. children with asymptomatic or symptomatic aspiration are at risk22 children with tracheoesophageal fistula, pharyngeal aspiration, gastroesophageal reflux, pulmonary disease, malnutrition or immunodeflciency are at greater risk. the etiologic microorganisms for nosocomial pneumonia vary from hospital to hospital. the pediatrician must be familiar with the common microbes and the antimicrobial susceptibility of these microbes at his/her hospital. gram-negative bacilli are the most important cause; 3,~5 staphylococci are the most important gram-positive organisms. viruses such as respiratory syncytial virus are also important pathogens responsible for nosocomial pneumonia in picus. 3 however, some authors have suggested that viruses are less frequent as nosocomial pathogens in picus than in other areas of pediatric hospitals. 16 the optimal approach for diagnosing nosocomial pneumonia remains elusive. clinical features, chest radiographs and culture of respiratory secretions have been used to establish the diagnosis. however, this approach is likely to overestimate the diagnosis. fever and leukocytosis are also non-specific. cough and sputum production are infrequently diagnostic of pneumonia in intubated children. purulent tracheal secretions may be due to tracheitis, tracheobronchitis or pneumonia; differentiation can be difficult. conditions such as atelectasis, pulmonary edema, pulmonary hemorrhage and acute respiratory distress syndrome (ards) may be confused with pneumonia2 6 therefore, clinical suspicion has to be strong for diagnosing nosocomial pneumonia. a change in the child's status such as desaturation, increased requirement for supplemental oxygen, increase in ventilator settings or fever that are not explained by other events can be helpful in the diagnosis. in view of these problems and controversies centers for disease control and prevention have provided clinicians with guidelines for diagnosis of nosocomial lrti. 1,2 once nosocomial pneumonia is suspected attempts should be made to identify the etiologic agent. qualitative cultures obtained from endotracheal tubes do not predict the causative agent of lower respiratory tract infections. quantitative cultures appear to be superior to qualitative cultures. tm blood cultures are usually negative. transtracheal aspiration in non-intubated patients, percutaneous thin needle lung aspirations, bronchoalveolar lavage (bronchoscopic or blind), and protected bronchoscopic samples of the lower airways have all been suggested as methods for diagnosis, where contamination of lower respiratory secretions with upper airway flora is prevent-ed29~~ in the absence of gold standard criteria for the diagnosis of ventilator-associated pneumonia, quantitative cultures and microscopic examination of the lower respiratory tract secretions are the diagnostic tests of choice. this can provide accurate diagnosis and identification of causative organism. these can also help in diagnosis of ventilator-associated pneumonia in children with ards. 21 the colonization of upper airways by pathogenic microbes and thereby, the risk of nosocomial pneumonia can be reduced by several measures. effective hand washing by the health care personnel can reduce the risk of nosocomial pneumonias. 22 various chemicals such as chlorhexidine or rubs containing alcohol may be used. the reduction in the number of microorganisms on hands is related to the volume and number of times they are used. 23 in addition, the hospital workers should comply with hospital infection control policies. the gastrointestinal tract is an important source for endogenous upper airway colonization. use of antacids and h2 blockers raise gastric ph and facilitate gastric microbial colonization. when indicated, instead of h2 blockers and antacids, sucralfate may be used for prophylaxis against gastric bleeding as the gastric ph remains low with its use. 24 the use of selective decontamination of the gut using antimicrobial such as tobramycin, gentamycin, polymyxin and nystatin is controversial and is not recommended. 25 contaminated respiratory therapy equipments have been implicated in nosocomial pneumonias. 25 resuscitation bags, ventilator tubings, nebulizers should be disinfected. only sterile fluids should be nebulized or used in humidifiers. personnel taking care of intubated children should wash their hands before and after delivering care. the ventilator circuit tubings should be changed no more often than every 48 hours? 7,25 care should be taken to prevent contamination during suctioning; endotracheal suctioning should be performed as needed to remove secretions. 26 positioning of patients with head end elevation does reduce the risk of aspiration and nosocomial pneumonia. 27 bacterial tracheitis usually is secondary to a primary viral upper respiratory tract infection, most often parainfluenza virus. 17 the data on nosocomial bacterial tracheitis is scant. 14 most nosocomial upper respiratory tract infections are viral and appear 2 weeks after admission. 28 bacteria are implicated in sinusitis and otitis media. sinusitis may occur in a significant number of patients who have undergone nasotracheal intubation. 29 nasogastric tubes also predispose to sinusitis. the diagnosis usually has to be confirmed by radiology. while intravascular catheters provide lifesaving therapy, they also provide a route for microorganisms to bypa~ normal host defenses and can cause serious infections. primary bacteremia is defined as a bloodstream infection occurring in a patient with no evidence of localized infection while secondary bacteremia is defined as a bloodstream infection with evidence of infection at another site that is the source of the bloodstream infection. more than 90% of all nosocomial bloodstream infections are in children with central venous lines? infections may occur due to microbes from the skin moving along the catheter surface where catheter enters the skin or due to microbes gaining access to the catheter through the catheter hub and moving down the endoluminal surface of the catheter to the bloodstream. ~1 the former mechanism is more often seen in short-term catheters and the latter in long-term catheters. formation of biofilms over the implanted devices also may have a role in occurrences of the infections. the most common organisms in bloodstream infections are coagulase negative staphylococcus, staphylococcus aureus and enterococci. 3 among the gramnegative bacteria enterobacter, pseudomonas aeruginosa, klebsiella pneumoniae, and e. coli are the most important organisms. 3 in immunocompromised children and those who have received a variety of broad spectrum antibacterials, fungal infections are more frequent. the most important risk factor for catheter related bloodstream infections is the type of catheter used. central venous catheters account for about 2% of all catheters inserted and more than 90% of all catheterrelated bloodstream infections. 3 the risk is probably greater with multilumen central venous catheters. 32 sub optimal care of central venous catheters especially when used for total parenteral nutrition (tpn) is another risk factor. lipid infusions have specifically been associated with risk of catheter-related bloodstream infections due to coagulase negative staphylococcus? 3 the risk of infection increases linearly with the duration of catheterization. the site of insertion is an important determinant of the risk of infection; rates for femoral catheters are higher than that for jugular or subclavian catheters. 3a peripherally inserted central catheters (picc lines) may have lower infection rates. inadvertent contamination during insertion may be an important risk factor for infection. this is highlighted by an inverse relation between the total number of catheters inserted by the physician inserting the catheter and the risk of significant catheter colonization. 3s improper management of catheter after insertion significantly increases the risk of infection. transparent dressings may be associated with a higher rate of blood stream infections. 36 local catheter-related infections usually manifests with local inflammation erythema, tenderness and/or purulent discharge from the catheter tract. the presentation of bloodstream infections may be either acute or insidious. there may be signs of sudden deterioration, elevated body temperature, chills and tachycardia or there may be intermittent fever and failure to improve from their basic illness. whenever catheter related bacteremia is suspected, blood cultures should be obtained from peripheral blood as well as all catheter sites to correlate and differentiate colonization from infection. catheter related bloodstream infection is defined as the isolation of same organism from blood cultures that is shown to be significantly colonizing the catheter of a patient with clinical features of bloodstream infection in the absence of any other local infection caused by the same organism that could give rise to bloodstream infection. 1,2 the major factors associated with development of catheter-related nosocomial infections are : (i) the sterility of the technique of insertion and maintenance of the catheter throughout its life, (ii) type of solution being administered through the intravenous line, (iii) number of "break ins" into the catheters system and intravenous tubing, (iv) the presence of infection elsewhere in the body. 37 largely, the origin of the organisms causing the uti can be traced either to the patient's own large bowel, perineum or to the hands of care providers. contamination of the urinary tract by microbes occurs either through the catheter lumen or by ascending up along the catheter during or after insertion. 46 extraluminal contamination is the commonest; it can occur by direct inoculation when the catheter is inserted or later, by the ascent of microbes from the perineum by capillary action in the thin mucus film contiguous to the external catheter surface. 47 intraluminal contamination occurs by reflux of microorganisms gaining access to the catheter lumen because of failure of closed drainage or contamination of urine in the collection bag. pyelonephritis from bloodstream infections are rare but may occur in newborns and infants. the most important risk factor for nosocomial uti is catheterization. the timing, purpose and duration of catheterization are also important. malnutrition, renal dysfunction, urological procedures and stenting, open drainage, diarrhea, periurethral skin contamination also play a role in nosocomial utis. the criteria for the diagnosis of an uti would depend on the catheterization status as follows. some uncertainty still exists in the diagnosis in catheterized children. the catheter, by simple irritation to the bladder, may cause pyuria; the drainage provided by the catheter may mask the clinical symptoms, and in some patients, the underlying disease process may cause fever or render the patient incapable of indicating the symptoms. the following are the guidelines for diagnosis of uti 9 clean catch mid stream urine growing >10 5 bacteria/ml of urine. ~ 9 any growth in a suprapubic sample (gold standard for confirmation of diagnosis). 9 bacterial counts of >102/ml in samples aspirated with a needle from the catheter in those patients with indwelling catheters. 49 a multitude of practices have been recommended and used to prevent the occurrence of utis. controlled trials are yet to prove the efficacy of some of these. the catheterizations should be kept to a minimum. the need for catheterization must be strictly evaluated and catheterization must be replaced by a closed condom drainage whenever possible, s~ suprapubic catheterization may be linked to a lesser risk of a cauti and may be more comfortable to some patients, s~ when thought to have fulfilled their need the catheters must be immediately removed, sl strict asepsis should be maintained during insertion of the catheter using sterile gloves, drapes, and local antiseptics. closed drainage must be strictly maintained and this has been shown to bring down the rates of infection to less than 25% for up to 2 weeks of catheterization, s2 the closed drainage must be maintained with the collection tubing and bag below the level of the patient's bladder and the tubing must always be above the level of the bag. other positions, by allowing backflow of urine, increase the risk of infection by two-fold. 53 when in place the closed drainage system must be handled and manipulated as infrequently as possible. antibiotic prophylaxis does reduce the frequency of infections but is not universally recommended as it selects multidrug resistant strains when the infection occurs. antiinfective lubricants, sealed catheter tubing junctions, antireflux valves, bladder or bag irrigation with antiinfective solution, antibiotic, silver hydrogel impregnated catheter materials are the newer techniques being studied for their role in reducing the risk of nosocomial utis. 46 for reducing the incidence of nosocomial infection, each picu should have an infection control program. there should be a written description of the goals, objectives, and structure of program. a team of health professionals should ensure implementation of the policies and compliance on the part of the picu team. well-directed infection control activities can reduce the nosocomial infection rates by up to 50%. u the importance of hand washing and hand disinfection is well understood. the appropriate hand washing technique includes wetting the hands, taking soap, rubbing hands to produce a lather, and performing wash movements that include rubbing palm to palm, right palm over left dorsum and vice versa, palm to palm with fingers interlaced, backs of fingers to opposing palm with fingers interlocked, rotational rubbing of right thumb clasped in left palm and vice versa, rotational rubbing with clasped fingers of fight hand in palm of left hand and with changed roles. 5s the whole procedure should not take less than 30 seconds. after washing, hands should be dried with disposable paper or cloth towel. it has been noticed that health personnel practice hand washing in only 25-50% of the opportunities? 6 in order to improve compliance, various hygienic hand rubs can be used. rubbing of 3-5 ml of a fast acting antiseptic preparation on to both hands can be an effective substitute to hand washing. the various preparations available include n-propanol, isopropanol, ethanol, and chlorhexidine diacetate. 55 in addition to the specific measures mentioned earlier for prevention of specific nosocomial infections, proper sterilization/disinfection of various medical items is mandatory. aseptic precautions should be followed strictly whenever any invasive procedure is being carried out. a well-nourished child is less likely to acquire nosocomial infection than a malnourished one. even in picu, nutrition should be given due attention. enteral nutrition appears to be better than parenteral nutrition. enteral nutrition may have a favorable impact on gastrointestinal immunologic function and infectious morbidity. 57 there has been considerable interest in the role of immune-enhancing enteral diets (containing glutamine, arginine, mrna, omega-3 fatty acids from fish oil) in reduction of nosocomial infections and mortality in icus . 58, 59 appropriate and rational prescription of antibiotics is essential to prevent emergence of resistant strains, s6 there should be constant surveillance and periodic review of the antibiotic policies and prescriptions. with careful microbiologic monitoring, cycling of antibiotics for empiric therapy can help in reducing the emergence of drug resistance. 56 adequate and well trained staff-both nursing staff and physicians-are essential for infection control. various studies have demonstrated the adverse effects of understaffing on nosocomial infection rates. 6~ education of the staff about various infection control practices and procedure-specific guidelines has an important role in the reduction of incidence of nosocomial infections. the education program should be on a continuing basis with periodic evaluation of the knowledge and practices. surveillance of nosocomial infections is an essential element of any infection control program. the most important goal of surveillance is to reduce the risk of acq~g nosocomial infections. this provides data useful for identifying infected patients, determining the site of infections and identifying the factors that contribute to nosocomial infections. use of uniform definitions is critical for proper collection of data and inter-hospital comparisons. standard cdc definitions that include laboratory and clinical criteria may be used. 1,2 the major purpose of surveillance is to establish baseline infection rates and identifying outbreaks. this data can be used effectively to convince health care workers to accept recommended preventive practices. control measures can be evaluated objectively if the surveillance is good. cdc definitions for nosocomial infections surveillance of nosocomial infections the national nosocomial infection surveillance system 9 nosocomial infections in pediatric intensive care units in the united states risk factors for nosocomial infection in critically ill children: a prospective cohort study infections in a pediatric intensive care unit nosocomial infections in medical icus in the united states nosocomial colonization and infection in a pediatric respiratory intensive care unit environmental services nosocomial infections in a pediatric intensive care unit klebsiella pneumoniae bacteremia in children consequences of candidemia for pediatric patients nosocomial infection in children risk factors for pneumonia and fatality in patients receiving continuous mechanical ventilation nosocomial pneumonia and tracheitis in a pediatric intensive care unit: a prospective study sources of gram negative bacilli colonizing the trachea of intubated patients epidemiologic study of 4684 hospital acquired infections in pediatric patients nosocomial bacterial infections of the central nervous system, upper and lower respiratory tracts, and skin in pediatric patients specificity of endotracheal aspiration, protected specimen brush, and bronchoalveolar lavage in mechanically ventilated patients diagnostic efficiency of endotracheal aspirates with quantitative bacterial cultures in intubated patients with suspected pneumonia: comparison with protected specimen brush blind protected specimen brush and bronchoalveolar lavage in ventilated children ventilator-associated pneumonia or not? contemporary diagnosis the prevention of ventilator associated pneumonia a causal link between handwashing and risk of infection? examination of evidence nosocomial pneumonia in intubated patients given sucralfate as compared with antacids or histamine type 2 blockers: the role of gastric colonization guidelines for prevention of nosocomial pneumonia new strategies for preventing nosocomial pneumonia: which common interventions leave patients at increased risk? supine body position as a risk factor for nosocomial pneumonia in mechanically ventilated patients: a randomized trial epidemiology of nosocomial infections in pediatric patients incidence of sinusitis in patients with nasotracheal intubation aprospective study of the mechanisms of infection associated with hemodialysis catheters risk factors for central venous catheter related infections in surgical and intensive care units infection rate for single lumen vs triple lumen subclavian catheters potentiation of staphylococcus epidermidis catheter-related bacteremia by lipid infusions prospective multicenter study of vascular catheter-related complications and risk factors for positive patients prospective study of catheter replacement and other risk factors of infection of hyperalimentation catheters a prospective, randomized study comparing transparent and dry gauze dressings for central venous catheters nosocomial infections in the pediatric intensive care unit prevention of central venous catheter related infections by using maximal sterile precautions during insertion new technologies to prevent intravascular catheter-related bloodstream infections bacteriologic surveillance of indwelling urinary catheters in pediatric intensive care unit patients urinary tract etiology of bloodstream infections in hospitalized patients catheter-associated urinary tract infections in surgical patients: a controlled study on the excess morbidity and costs urinary tract infection: economic considerations mortality associated with nosocomial urinary tract infection hospitalacquired urinary tract infections in pediatric patient: a prospective study engineering out the risk for infection with urinary catheters a prospective study of the pathogenesis of catheter-associated with urinary tract infection asymptomatic infections of the urinary tract bacteriuria in the catheterized patient urethral catheters, condom catheters, and nosocomial urinary tract infections early catheter removal decreases incidence of urinary tract infections in renal transplant receipients prevention of catheter-induced urinary tract infections by sterile closed drainage risk factors for catheterassociated urinary tract infection: a prospective study showing the minimal effects of catheter care violations on the risk of cauti (abstract) the efficacy of infection surveillance and control programs in preventing nosocomial infections in us hospitals hand washing and hand disinfection controlling antimicrobial resistance in hospitals: infection control and use of antibiotics enteral nutrition in the critically ill patient: a critical review of the evidence an immune-enhancing enteral diet reduces mortality rate and episodes of bacteremia in septic intensive care patients randomised trial of glutamine-enriched enteral nutrition on infectious morbidity in patients with multiple trauma the role of understaffing in central venous catheter-associated bloodstream infections patient density, nurse-to-patient ratio and nosocomial infection risk in a pediatric cardiac intensive care unit at least one of the following: a. new onset of purulent sputum or change in character of sputum b. organisms cultured from blood c. isolation of an etiologic agent from a specimen obtained by transtracheal aspirate, bronchial brushing, or biopsy. criterion 2 : patient has a chest radiographic examination that shows new or progressive infiltrate, consolidation, cavitation, or pleural effusion criterion 4and at least one of the following: a. new onset of purulent sputum or change in character of sputum b. organisms cultured from blood c. isolation of an etiologic agent from a specimen obtained by transtracheal aspirate, bronchial brushing, or biopsy. d. isolation of virus or detection of viral antigen in respiratory secretions e. histopathologic evidence of pneumonia.: patient < 1 year of age has at least two of the following signs or symptoms: apnea, bradycardia, wheezing, rhonchi, or cough and at least one of the following : a. increased production of respiratory secretions b. new onset of purulent sputum or change in character of sputum c. organisms cultured from blood d. isolation of an etiologic agent from a specimen obtained by transtracheal aspirate, bronchial brushing, or biopsy. e. isolation of virus or detection of viral antigen in respiratory secretions f. histopathologic evidence of pneumonia : patient < 1 year has a chest radiographic examination that shows new or progressive infiltrate, consolidation, cavitation, or pleural effusion at least one of the following : a. increased production of respiratory secretions b. new onset of purulent sputum or change in character of sputum c. organisms cultured from blood d. isolation of an etiologic agent from a specimen obtained by transtracheal aspirate, bronchial brushing, or biopsy. isolation of virus or detection of viral antigen in respiratory secretions histopathologic evidence of pneumonia. *expectorated sputum cultures are not useful in diagnosis of pneumonia *findings from serial chest x-rays may be more useful than a single x-ray. a symptomatic urinary tract infection must meet at least one of the following criteria: criterion i : patient has at least one of the following sign or symptoms with no other recognized cause : fever (>38 ~ c), urgency, frequency, dysuria or suprapubic tenderness and patients has a positive urine culture that is >10 s microorganisms per cm 3 of urine with no more than two species of microorganisms. patients has at least two of the following signs or symptoms with no other recognized cause: fever (>38 ~ c), urgency, frequency, dysuria, or suprapubic tenderness and at least one of the following : a. pyuria (urine specimen with >10 wbc/mm 3 or >3 wbc/high power field of unspun urine) b. organism seen on gram stain of unspun urine c. at least two urine culture with repeated isolation of the same uropathogen (gram-negative bacteria or s.saprophyticus) with >102 colonies ml in non voided specimens d. < 10 s colonies/ml of a single uropathogen (gram negative bacteria or s. saprophyticus) in a patient being treated with an effective antimicrobial agent for a urinary tract infection e. physician diagnosis of a urinary tract infection f. physician institutes appropriate therapy for a urinary tract infection patient < 1 year of age has at least one of the following signs or symptoms with no other recognized cause: fever(>38 ~ c), hypothermia (<37~ apnea, bradycardia, dysuria, lethargy, or vomiting and patient has a positive urine culture, that is >10 s microorganisms per cm 3 of urine with no more than two species of microorganisms. patient < 1 year of age has at least one of the following signs or symptoms with no other recognized cause: fever (>38 ~ c), hypothermia (<37~ apnea, bradycardia, dysuria, lethargy, or vomiting and at least one of the following: a. pyuria (urine specimen with > 10 wbc/mm 3 or > 3 wbc/high power field of unspun urine) b. organisms seen on gram stain of unspun urine c. atleast two urine culture with repeated isolation of the same uropathogen (gram-negative bacteria or s. saprophyticus) with > 102 colonies/ml in nonvoided specimens d. < 105 colonies ml of a single uropathogen (gram negative bacteria or s. saprophyticus) in a patient being treated with an effective antimicrobial agent for a urinary tract infection e. physician diagnosis of a urinary tract infection f. physician institutes appropriate therapy for a urinary tract infection key: cord-017331-ru7mvfc0 authors: samanta, indranil; bandyopadhyay, samiran title: infectious diseases date: 2017-02-25 journal: pet bird diseases and care doi: 10.1007/978-981-10-3674-3_2 sha: doc_id: 17331 cord_uid: ru7mvfc0 the chapter describes bacerial, viral, parasitic and fungal infections commonly detected in pet birds. the chapter includes history, etiology, susceptible hosts, transmission, pathogenesis, clinical symptoms, lesion, diagnosis, zoonosis, treatment and control strategy of tuberculosis, salmonellosis, chlamydiosis, campylobacteriosis, lyme disease, other bacterial infection, newcastle disease, avian influenza infection, west nile virus infection, usutu virus infection, avian borna virus infection, beak and feather disease, other viral infection, toxoplasmosis, giardiasis, cryptosporidiosis, other parasitic infection, cryptococcosis, aspergillosis, other fungal infections. indranil samanta and samiran bandyopadhyay 2.1 bacterial diseases 2.1.1 tuberculosis 2.1.1.1 history tuberculosis (mycobacteriosis) is an ancient or 'heritage' disease which was observed even before the neolithic age and in egyptian mummies. in 1882, robert koch first isolated mycobacterium, stained with alkaline methylene blue and vesuvin and established its etiologic relationship with tuberculosis. koch described that the same human or bovine type mycobacterium may cause avian tuberculosis. strauss and gamaleia (1891) and maffucci (1892) cited in cobbett (1917) illustrated that the etiological mycobacterium of avian tuberculosis was different from human or bovine type. hinshaw (1933) and ackermann et al. (1974) reported occurrence of tuberculosis in amazon parrot (amazona farinosa). coyle et al. (1992) proposed a new mycobacterial species (mycobacterium genavense) isolated from human patients with aids. simultaneously in 1993, mycobacterium genavense was reported from pet birds in europe (hoop et al. 1993 ). mycobacterium is gram positive, straight or slightly curved rod with occasional coccobacillary, club and branched form. in the tissues, it measures 1-4 µm in length and 0.2-0.3 µm in width. it occurs singly, in pair or in bundle. it is difficult to demonstrate their gram positive nature due to high lipid content of the cell wall. the stains are relatively impermeable to the bacterial cell. they can be easily stained by ziehl-neelsen (zn) or acid fast staining technique. the genus mycobacterium (actinomycetes family) contains more than 100 species. some are pathogenic for man and animals which grow slowly in artificial media in laboratory (slow growers) than the fast growers (m. smegmatis). previously, slow growing mycobacterium was sub divided into three types based on their host specificity i.e. human type, bovine type and avian type. recent classification reveals that slow growing mycobacterium is composed of several species. the most common species are mycobacterium tuberculosis complex and m. avium-intracellulare complex (mac) . mycobacterium tuberculosis complex is comprised of m. tuberculosis, m. bovis, m. caprae, m. microrti, m. africanum and m. canettii. mac is composed of two major species-m. avium and m. intracellulare. m. avium is subdivided into four subspecies (ssp.) i.e. ssp. avium (maa), ssp. paratuberculosis (map), ssp. silvaticum and recently added ssp. hominissuis. mac is considered as 'atypical mycobacteria' and members of this group are highly resistant against environmental changes such as high and low temperatures, dryness, extreme ph and common disinfectants. there are total 28 serotypes of mac and the serotypes 1-6, 8-11 and 21 belonged to m. avium ssp. avium. mycobacterium genavense forms a deep branch of mycobacterial phylogentic tree with other members such as m. interjectum and m. simiae. this group is characterized by slow growth albeit contains the signature molecule of fast growers (short helix 18). m. genavense can be distinguished from other slow growers by their fastidious growth and preference for liquid medium. m. genavense was first reported from human aids patients with disseminated infections (hirschel et al. 1990 ). among psittacine birds, grey-cheeked parakeets (brotogeris pyrrophterus), amazons (amazona spp.), budgerigars (melopsittacus undulatus) and pinous parrots (pionus spp.) are the most commonly affected species with tuberculosis. other psittacines such as green-winged macaws (ara chloroptera), cockatoos (cacatua spp.), conures (aratinga auricapillus, cyanoliseus patagonus) and red-crowned parakeet (cyanoramphus novaezelandieae) are also detected to be infected with m. genavense and m. tuberculosis. m. avium subsp. avium is reported to cause infection in cockatiels (nymphicus hollandicus). the non-cultivable mycobacterium is detected in blue and yellow macaw (ara ararauna) and grey-cheeked parakeet (brotogeris pyrrhopterus). the common parrots (african grey parrot, senegal parrot) are not considered as natural host of m. tuberculosis although infection in african grey parrot is reported which was transmitted from human. m. bovis can produce natural infection in parrots and the budgerigars are experimentally infected with m. bovis producing clinical syndrome within 70 days. among non-psittacine group of birds, canary (serinus canaria), gouldian finch (chloebia gouldiae) and zebra finch (poephila guttata castanotis) are commonly infected with m. genavense. synergistic infection of m. genavense and avian polyoma virus was detected in european goldfinch (carduelis carduelis). occasionally canaries are also infected with m. tuberculosis. avian tuberculosis is a disease of adult birds although occasionally detected in young (<1 year old) canaries. mac is transmitted in birds chiefly by ingestion, inhalation and rarely through arthropods. m. avium ssp. avium is excreated through faeces of infected birds and survive in soil (up to 4 years), sawdust (8 months at 37°c) and water for a long period. bird to bird transmission in aviaries may occur through infected faeces or rarely by cannibalism. occasionally skin abrasion acts as a route of mycobacterial infection in pet birds. ingestion is considered as a potential route of transmission of m. genavense infection in pet birds. the environment specially drinking water is an identified source of m. genavense infection. lung involvement in pet birds suggests inhalation as an additional route of transmission. birds may act as reservoir of m. genavense. bird to bird transmission of m. genavense is rare although the possibility could not be excluded entirely. immunosuppression plays a role in transmission of m. genavense in human but whether the same condition facilitates the transmission in pet birds is obscure. transmission of m. tuberculosis in pet birds (green-winged macaws, blue-fronted amazon) from human is observed due to close contact with owners suffering from tuberculosis and feeding the birds with pre-chewed food. m. avium subsp. avium mac enters the host chiefly through ingestion route of transmission and become present in the intestine. the waxy cell wall of the bacteria protects it from gastric acids and enzymes. several pathogen associated molecular patterns (pamps) are expressed by virulent mycobacterium which can recruit 'microbicidal' macrophages through toll like receptor (tlr) mediated signaling. during m. tuberculosis infection in human, these pamps in the bacterial surface are masked with a lipid, namely phthiocerol dimycoceroserate (pdim). the pamps are not recognized by the host immune system and the bacteria can avoid the reactive nitrogen species (rns) generated by 'microbicidal' macrophages. mac (m. avium subsp. avium) does not contain pdim in their surface but use a different strategy (still unexplored) to resist rns. mac is benefited with these rns as commensal present in the gut are sensitive to it. commensal mediated competitive inhibition is thus excluded and probably mac enters m-cells like host adopted salmonella to invade the underlying blood monocytes. the m-cells are specialized cells of the follicle associated epithelium and the region is relatively free from commensal mediated competitive inhibition. the invasion of monocytes is followed by bacterimia and subsequent haematogenous spread to liver, spleen and other organs. mac enters the macrophages (histiocytes) of periarteriolar lymphoid sheath (pals) zone in spleen within 10 days post infection in birds. mycobacterium has several virulence factors which promote their survival within macrophages using different strategies such as acid resistance, avoidance of acidification etc. mac (m. avium subsp. avium) specifically restricts vacuole maturation and prevents the fusion of phagosome and lysosome for their survival within macrophages. haematogenous spread of the organism leads to infection of bone marrow, lungs, air sacs, gonads and rarely, kidney and pancreas. the organs become enlarged due to accumulation of macrophages within organ parenchyma. granuloma formation is an attempt of host tissue to localize the infection, although mycobacterium exploits it for their multiplication and further dissemination. the growth of mycobacterium occurs in the macrophages present in a granuloma. the infected macrophages undergo apoptosis and leave the encased bacteria which are engulfed by newly recruited macrophages. this process of apoptosis and re-phagocytosis within a granuloma is regulated by a mycobacterial secreation system (esx-1/esat6) detected in m. tuberculosis but absent in m. avium subsp. avium. typical tuberculous granulomas in different organs are not frequent in m. avium subsp. avium infection, although observed in lungs and periocular region of parrots. granulomas in different organs (liver, kidney, intestine, muscle and subcutaneous tissues) are observed in red-crowned parakeet (cyanoramphus novaezelandiae) and green-winged macaw (ara chloroptera) infected with m. tuberculosis. m. genevense infection in pet birds mostly occurs through the oral route like mac. there is every possibility that they follow the same pattern of pathogenesis although still unexplored. m. genevense causes non-tuberculoid form of mycobacteriosis in pet birds albeit occurrence of granulomas are observed in glottis of amazon parrot (amazona ochracephala), aorta of cockatiel (nymphicus hollandicus), small intestine of canary-winged parakeet (brotogeris versicolurus) and brain of spectacled amazon parrot (amazona albifrons). incubation period of mycobacteriosis in pet birds is 6 months to 4 years. clinical syndrome in psittacine birds varies widely. in acute form, sudden death without any symptom is common. in chronic form, constant loss of weight along with diarrhoea ( fig. 2.1 ), frequent micturition with excessively large quantity and low specific gravity of urine, depression, laboured breathing, distension of abdomen and poor feathering primarily suggests about mycobacteriosis. the condition fails to respond to common antibiotics. cutaneous masses are sometimes observed in skin and conjunctiva. inflammation of feather follicles (folliculitis) is occasionally observed which includes perifollicular swelling, erythema, pruritus and pain, restlessness, shivering and feather damaging behaviour. the liver and spleen are enlarged, mottled and whitish. miliary abscess in liver are observed in budgerigars experimentally inoculated with m. avium subsp. avium. the intestine becomes tubular, thickened and tan coloured. typical tuberculous granulomas in different organs are not frequent in m. avium subsp. avium infection, albeit observed in lungs and periocular region of parrots, pericardium of gang gang cockatoo (callocephalon fimbriatum) and cervical esophagus of blue-fronted parrots (amazona aestiva) occluding the lumen. involvement of lung is rare in pet birds affected with mycobacteriosis. some post mortem findings in canary (serinus canarius), eurasian goldfinch (carduelis carduelis) and the red siskin (spinus cucullatus) reported the occurrence of lung lesions. in a rare case of m. genevense infection in an amazon parrot (amazona albifrons), perivascular cuffs of macrophages in the grey and white matter of the brain and spinal cord, gliosis and mild vacuolation of white matter were observed. clinical specimens blood/sera, cloacal swabs, tracheal swabs, biopsies from organs can be collected as ante-mortem samples for diagnosis of mycobacteriosis in the laboratory. post-mortem samples include vital organs such as liver, spleen, intestine, heart, lung and bone marrow. all the specimens should be immediately sent to the laboratory following the standard regulations for sending biohazardous substances. if there is delay in sending, refrigeration of the samples should be done to prevent the growth of contaminants. addition of 0.5% boric acid may preserve the samples for 1 week. (a) clinical signs and history of direct contact with owner and other birds suffering from tuberculosis give a tentative diagnosis (b) haematological parameters: following haematological changes can be correlated with tuberculosis in pet birds although these changes are non-specific and are observed in other inflammatory and chronic infections also. • moderate to marked increases in white blood cell numbers (heterophilia, monocytosis, lymphocytosis) • decreased packed cell volume (pcv) (except during early stage of infection) • increased enzyme concentration (alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase) (c) demonstration of acid-fast organisms: demonstration of the organisms can be done in the smears prepared from ante-mortem samples by acid-fast (or zn) staining. mycobacterium appears as red coloured slender rods singly or in bundle ( fig. 2. 2). fluorescent acid fast stain may be used for better detection. in post mortem specimens, cytoplasm of infected cells is laden with acid-fast organisms. (d) gross and histopathology: enlarged liver, thickened intestinal loop, increased opacity in endosteal bone in the humerus, tibia, ulna, femur in advanced cases is suggestive for tuberculosis. presence of visible granuloma is not a constant feature although may be detected in lungs. in decomposed carcasses bone marrow is the best specimen. histopathogical findings such as presence of acid-fast bacilli in inflammatory cells can also tentatively diagnose mycobacteriosis. (e) isolation of organism: this is considered as gold standard method for confirmatory diagnosis of mycobacterium. to isolate the organism, the tissue sample must be processed in proper way. tissue samples are homogenized in pestle and mortar after keeping in the solution of hypochlorite (1:1000) for 4-16 h. it is decontaminated by the addition of acid (5% oxalic acid), alkali (2-4% sodium hydroxide) or detergent (0.375-0.75% hexadecylpyridiniumchloride, hpc). the acid or alkali mixture is neutralized, centrifuged and the sediment is used for culture. m. avium subsp. avium can be isolated in dorset egg medium, lowenstein-jensen (lj), herrold's and middlebrook's (7h10,7h11) medium containing pyruvate. glycerol and 'mycobactin' is also added as growth enhancer. mycobactin extracted from the environmental mycobacteria, acts as siderophore helping in acquisition of iron. mycobactin is produced by all cultivable mycobacteria except m. avium subsp. avium and m. avium subsp. paratuberculosis. incubation period is 8 week at 40°c. even it can grow at 42-43°c. it produces smooth or rough type of colonies. in liquid culture a radiometric method using 14 c labelled substrate can be used for rapid detection (bactec system). m. genavense isolation is difficult although can be done on special media with prolonged incubation for 2-9 months. conventionally, after decontamination of the samples with 2(n) naoh, the samples can be inoculated in herrold's egg yolk medium with and without mycobactin j and sula's liquid medium and incubated at 37°c. the growth is periodically checked in every two weeks. the successful isolation of m. genavense from blue headed parrot (pionus menstruus) was observed in herrold's egg yolk medium without mycobactin j after 270 days of incubation. in specialized laboratories, newly developed liquid culture systems [manual mycobacteria growth indicator tube (m-mgit), bactec system) are used for confirmatory isolation of m. genavense. subsp. avium in birds. however elisa produces variable results in different species of birds. immunological or serological tests for detection of mycobacteriosis in pet birds are not routinely followed. (g) molecular biology: polymerase chain reaction (pcr) can detect mycobacterium from fresh samples, faeces and paraffin-blocked tissues. the species-specific pcr targets is901 and hsp65 genes for detection of m. avium subsp. avium and m. genavense, respectively. differentiation of both the species can be done by sequencing of the 16srrna gene. a nested polymerase chain reaction (pcr) from the consensus sequences of the hsp65 gene, followed by analysis with restriction enzymes can also differentiate m. avium and m. genavense. real-time taqman pcr assay is developed to detect hsp65 gene of m. genavense and mac subsp. other recent technologies such as genotype assay and dna microarrays can be used for diagnosis of avian tuberculosis. for detection of genetic diversity among the strains of m. avium subsp. avium, mycobacterial interspersed repetitive units-variable-number tandem-repeat markers (miru-vntr) typing can be successfully used. 2.1.1.9 zoonosis m. avium subsp. avium is considered as a potential zoonotic risk in the immunocompromised persons albeit majority of human infection is caused by another member of m. avium group (m. avium subsp. hominissuis). m. genavense is associated with gastrointestinal or pulmonary mycobacteriosis in immunosuppressed patients associated with aids. other species of mycobacteria associated with pet bird causes opportunistic and sporadic infections in human. the cases of reverse zoonosis (anthroponosis) are also reported where m. tuberculosis was transmitted from the infected owners to their pet birds (green-winged macaws, blue-fronted amazon). successful treatment of pet birds is reported with various combinations of anti-tuberculous drugs at highest tolerable dose for a prolonged period (9 months or more). single anti-tuberculous drug is not preferred due to possibility of resistance development. emergence of multidrug-resistant tuberculosis (mdr-tb; resistant to isoniazid and rifampicin) and extremely drug-resistant tuberculosis (xdr-tb; in addition to being multidrug-resistant the bacteria are resistant to fluoroquinolone and 1 of 3 antibiotics such as capreomycin, kanamycin and amikacin) is a global problem now a days. in most of the cases, dose is fixed on the basis of human paediatric studies because pharmacokinetic properties of anti-tuberculous drugs in pet birds are still unknown. the anti-tuberculous drugs and antibiotics used against m. avium infections in pet birds are isoniazid, rifampin, rifabutin, ethambutol, clofazimine, ciprofloxacin, enrofloxacin, streptomycin and amikacin. successful therapy of m. genavense infections with combinations of moxifloxacin, clarythromycin, ethambutol and amikacin in humans has been reported but there is no specific drug recommended for m. genavense infection. treatment with clarithromycin, rifampin, and ethambutolm against m. marinum infection in a blue-fronted amazon parrot was not successful. experimentally, different combinations of anti-tuberculous drugs and antibiotics such as isoniazid (30 mg/kg) + ethambutol (30 mg/kg) + rifampin (45 mg/kg) for 12-18 months, clofazimine (6 mg/kg) + ethambutol (30 mg/kg) + rifampin (45 mg/kg) for 9-18 months, ciprofloxacin (80 mg/kg) + ethambutol (30 mg/kg) + rifampin (45 mg/kg) for 9-12 months was used successfully against confirmed cases of tuberculosis in different pet birds (grey-cheeked parakeet, double yellow-headed amazon, lilac-crowned amazon). although combination of azithromycin (43 mg/kg), rifampin (45 mg/kg), and ethambutol (30 mg/kg) administered orally once daily for 180 days in ring-neck doves (streptopelia risoria) naturally infected with m. avium subsp. avium failed to eradicate the infection. further, treatment with combination of clarithromycin (61 mg/kg bw), moxifloxacin (25 mg/kg bw) and ethambutol (60 mg/kg bw) administered in budgerigars experimentally infected with m. avium subsp. avium by crop gavage every 12 h for 18 weeks significantly improved the situation but failed to recover completely. combination of minocycline (10 mg/kg p.o. b.i.d.) and clarithromycin (10 mg/kg p.o. s.i.d.) significantly reduced oral plaques in blue penguins (eudyptula minor) naturally infected with m. intracellulare. due to zoonotic potential specially for children and elderly persons and immunocompromised patients, prolonged treatment and poor success rate, difficulty of drug administration to avian patients maintaining proper doses, natural and acquired antimicrobial resistance, poor owner compliance and moreover, lack of a proper treatment schedule, the debate exists regarding advice of treating pet birds against tuberculosis. nevertheless, euthanasia is the preferred measure in the prevention of tuberculosis in pet birds in relation to human health. for prevention of further transmission, removal of all organic matter and debris from cages, washing the cages and surroundings properly with disinfectants and maintenance of biosecurity measures are required. chlorohexidine and quaternary ammonium compounds can act as mycobacteriostatic disinfectants. 2.1.2 salmonellosis 2.1.2.1 history d.e. salmon (1885) first isolated salmonella from the infected pigs. it was considered as a cause of 'hog cholera' until the discovery of the etiological virus. the nomenclature of the bacteria ('salmonella') was done in memory of salmon. in 1889, klein (united kingdom) first isolated salmonella gallinarum from chickens with 'fowl typhoid'. loeffler first described salmonella typhimurium from a natural outbreak of typhoid like infection in mice. among the pet birds, salmonellosis was first described in ducks (vandervort 1954; keymer 1958) , and parakeets (kaye et al. 1961; madewell and mcchesney 1975) . the genus salmonella is classified under the family enterobacteriaceae that belongs to the order enterobacteriales. there are two major species under the genus salmonella i.e. s. enterica (>2440 serovars) and s. bongori (20 serovars). a third proposed species is s. subterranea, yet to be recognized as a true species. salmonella enterica is considered as type species of the genus at present. s. enterica has 6 subspecies (ssp.): salamae, arizonae, diarizonae, houtenae, indica, enterica. most of the pathogenic salmonella are designated as 'serovar' under the s. enterica ssp. enterica. virulent serovars are: typhi, typhimurium, dublin, choleraesuis, pullorum, gallinarum, abortusovis. salmonella typhimurium (var. copenhagen) is the most frequently isolated serovar from different outbreaks in psittacine birds. in passerine birds (perching/song birds), s. typhimurium phage types dt40, dt41, dt56, dt160 are adapted. it acts as either a primary pathogen or it causes sub-clinical infection in young and immunocompromised birds. if the density of flock is high and the quantity of available feed is low, most of the birds become debilitated and are susceptible to s. typhimurium infection. other sub-species and serovars of salmonella isolated from pet birds include s. houtenae, s. arizonae, s. rissen, s. enteritidis, s. pullorum, s. gallinarum, s. newport, s. panama, s. rublislaw, s. aberdeen, s. thompson and s. wasenaar. among them, s. gallinarum can infect canaries, ring dove, pheasants, peacocks and peafowl. a novel salmonella serovar (s. pajala) was isolated from peregrine falcon (falco peregrinus) nestlings. clinical outbreaks of salmonellosis are frequently detected in passerine and psittacine birds. among passerine birds, finches (fringillidae) and sparrows (passeridae) seem to be particularly susceptible to salmonella spp. infection. salmonellosis is reported from canary (serinus canaria), eurasian siskin (carduelis spinus), zebra finch (taeniopygia guttata), bengalese finch (lonchura striata domestica) and picoplat (sporophila intermedia). fatal outbreaks with high mortality were reported in psittacine birds such as lories and lorikeets (trichoglossus, lorius, eos spp.), budgerigars (melopsittacus undulatus), parakeets (psephotus spp., psittacula spp.), and sulphur crested cockatoo (cacatua galerita galerita). the birds with caecum [e.g. macaw (ara sp.), amazon parrot (amazona sp.)] can asymptomatically carry salmonella spp. like poultry. both free-ranging and captive blue-fronted amazon parrots (amazona aestiva) are detected to carry salmonella spp. although, lilac-crowned amazon parrots (amazona finschi schlater) was found died due to s. enteritidis infection. moreover, s. typhimurium is detected as a primary pathogen causing death of blue and gold macaws (ara ararauna). occasionally, raptors or hunter birds (e.g. falcon, red kite), game birds [e.g. red-legged partridge (alectoris rufa)], free-ranging sparrow (passer domesticus), gull (laridae), wild birds such as temminck's seedeater (sporophila falcirostris), chestnut-capped blackbird (chrysomus ruficapillus), brown-headed cowbirds (molothrus ater), white-throated sparrows (zonotrichia albicollis) can also harbour salmonella spp. infected pet birds, rodents, reptiles, wild birds, contaminated water, feed and eggs act as source of salmonella spp. when the pet birds are gathered in an exhibition the healthy birds come in direct contact to the infected birds. the rodents, reptiles and wild birds having access to the open-air aviary can contaminate the place. the bacteria can survive for extensive periods on wood and dust and can live for 28 months in avian faeces. the contaminated places become a constant source of infection. in a pet shop, iguana (iguana iguana) was identified as a source of salmonella spp. infection in a cockatoo. ingestion of contaminated feed and drinking water is the major route of transmission in pet birds. sometimes, the infection is also transmitted by owners or attendants through their contaminated hands, feet and clothes. trans-ovarian transmission is common in poultry, although, is not frequently observed in pet birds. salmonella typhimurium following oral route of transmission, salmonella is deposited in the intestine, where they invade enterocytes. the bacteria can invade the epithelial cells throughout the intestine, although, caeca and ileocaecal junction are the preferred site. in the intestine, low ph, peristalsis, intestinal mucus, lysozyme in secreations and moreover normal microbial flora try to destabilize the bacterial colonization. normal microflora in adults prevents the salmonella colonization by occupying their receptors, known as 'competitive exclusion'. young are more susceptible as their intestinal microfloral range is not fully developed. bacterial fimbriae, lipopolysaccharide (lps), pathogenicity island (sp-1-t3ss associated proteins) help in adherence with the enterocytes. the interaction between the t3ss proteins (sipa, sipc) with the actin cytoskeleton of the enterocytes causes cytoskeletal rearrangements to generate an uneven surface (membrane ruffle). the organisms are trapped within the ruffled membrane and are internalized by the enterocytes. within the enterocytes, the bacteria reside in a membrane bound vacuole i.e. salmonella containing vacuoles (scv). as the scv matures, it migrates from the luminal border of the enterocyte to the basal membrane. in the basal membrane, the scv enter the macrophages associated with peyer's patches in the sub mucosal space. the scv never fuses with lysosome within macrophages and thus avoid phago-lysosome fusion which is necessary to kill the bacteria. additional factors such as sp-2-t3ss (sipc protein), sp-3 associated proteins also help in intracellular survival. the formation of salmonella induced fibrils (sif) help in bacterial replication in an unknown way. it is evident that major portion of the infection is cleared by the macrophages, only certain part can survive leading to chronic infection or carrier state with persistent faecal shedding. sometimes, invasion of salmonella takes place beyond the intestine which causes bacterimia, survival and replication of the bacteria in reticulo-endothelial cells of liver and spleen. in passerine birds (canary, finch and starling), esophagus and crop are the preferred site for bacterial colonization after bacterimia. acute and chronic form of salmonellosis is detected in pet birds. in acute form, huge mortality without any prior cardinal signs was observed in a flock of canaries. a s. typhimurium infected macaw (ara ararauna) showed depression, anorexia, delay in the emptying of crops (ingluvies), laboured breathing and diarrhoea for 3-4 days before death. greenish-yellow diarrhoea was observed in adult budgerigers (melopsittacus undulatus) infected with s. gallinarum. the chronic form shows numerous general symptoms such as anorexia, diarrhoea, dyspnoea, lethargy, cachexia, ruffled feathers, subcutaneous granuloma, crop stasis, conjunctivitis, arthritis and panophthalmitis. adult budgerigars with chronic salmonellosis showed loss of condition, unwillingness to fly, inability to perch, and gathering in the bottom of the cage (fig. 2. 3). drop in egg production and increased embryonic mortality rate was observed. in pigeons, in addition to the general syndrome, polyuria, arthritis of the joint, nervous symptoms and dermatitis followed by death was detected. stress associated with environment, season, change of diet and housing, transport, breeding, concurrent infection, and introduction of new birds without quarantine are the major predisposing factors for salmonellosis in pet birds. in acute form of salmonellosis in pet birds, no specific gross lesion is observed. in chronic infection, congestion of lung and intestine, spleenomegaly, hepatomegaly, liver with necrotic foci is observed (fig. 2.4) . the passerines birds infected with s. typhimurium shows the granulomatous lesions like pseudotuberculosis. esophagitis and ingluvitis with necrotic plaques in esophagus and crop, respectively, are consistently detected in wild or free-range passerine birds. in european goldfinch (carduelis carduelis), instead of esophagus lesions are sometimes developed in the subcutaneous tissues. in canaries, congestion of vital organs and necrotic foci on the liver with a nodular and miliary appearance is detected. intestinal content become dark in colour due to haemorrhagic diasthesis. in lories and lorikeets, petechial hemorrhages on the serosal surface of the proventriculus, ventriculus, and cardiac muscle along with atrial dilation are observed. sometimes, bacterial emboli occur in liver, spleen, lung, kidney, and proventriculus. in s. typhimurium infected macaws, pectoral muscle atrophy, fibrinous exudate on intestinal mucosa, white-greyish nodules on intestinal serosa, myocardium, lungs and ingluvies mucosa are commonly observed. in young budgerigars, fibrin deposit is observed as a white, thick layer on the pericardium. petechial haemorrhages are detected on the surfaces of pericardium, myocardium, gizzard, duodenum and ileo-caecum. in garden birds infected with s. typhimurium, esophageal ulcers, granulomata in soft tissues, hepatomegaly and spleenomegaly are observed. microscopic inspection of histopathological sections shows necrosis of parenchymatous organs specially the liver with granulocyte infiltration and fibrin deposition. in young and adult budgerigars, the blood vessel walls become hyalinized in appearance with various-sized microthrombi. the chronic cases are characterized by formation of a granuloma. a typical granuloma consists a necrotic centre which is surrounded by granulocytes and macrophages containing salmonella spp. multinucleated giant cells are found in chronic infection. in passerine birds, epithelial surface of the esophagus is ulcerated and it forms a thick layer of necrotic cellular debris composed of degenerated and intact leukocytes with gram-negative bacteria. infiltration of heterophils, lymphocytes and plasma cells occur into the underlying sub-mucosa. clinical samples include faeces or cloacal swabs, blood/serum of live birds and affected tissues, such as liver, spleen, heart, intestine/caeca, lung, esophagus/crop, brain and kidney in 10% buffered formalin. before collection of cloacal swabs, pericloacal asepsis with iodized alcohol is performed. blood samples are collected from jugular, wing or ulnar vein. the environmental samples, such as pooled faeces, litter and dust from the cages, feed and drinking water should be examined to know about an outbreak, if any. specimens should be collected before antibiotic treatment of the birds. after death, the collection should be done immediately from fresh carcasses. for 'pre-enrichment', swabs should be collected in buffered peptone water. pre-enrichment in buffered peptone water helps in survival of salmonella from freezing, heating and desiccation. the cold chain (4-5°c) should be maintained during transportation of the samples to the laboratory. (a) clinical signs and lesions after necropsy, history of direct contact with infected birds give a tentative diagnosis (b) direct examination: an impression smear prepared from clinical samples such as cloacal swab/faeces/tissues, is stained by gram's method. salmonella appears as gram negative small rods with no distinct characteristics. the tissue samples of heart, lung, liver, spleen, kidney, and intestine are fixed in 10% buffered formalin, embedded in paraffin, sectioned at 3 mm, and stained with hematoxylin-eosin and periodic acid-schiff for direct examination of the bacteria. zoonotic transmission of salmonella spp. to human from parakeet kept as a pet was documented. a salmonellosis outbreak associated with dissection of an owl was reported among the elementary school children. the possibility of children infection (below 5 years age) is more in those families who rear a pet bird (odd ratio: 2.7) or a lizard (odd ratio: 3). in open air aviaries and children's zoos, the transmission of salmonella spp. was reported between wild birds, pet birds and human. special care for designing such aviaries should be adopted. antibiotics against salmonella spp. in infected pet birds can be administered after doing the sensitivity of the bacterial isolates. antimicrobial resistance of salmonella spp. is a global concern at present. successful treatment of infected canaries with 10% (w/v) enrofloxacin solution provided as 200 mg/l in drinking water for 5-7 days was observed. treatment with kanamycin, gentamicin, trimethoprim/sulfamethoxazole suspension along with anti-diarrhoeals such as daolin and pectin combination is recommended. general control and prevention strategies such as isolation of diseased birds from the rest of the flock, cleaning and disinfection of cages, water and feed utensils with 10% (v/v) solution of sodium hypochlorite or commercial disinfectants are recommended. if the feedstuffs are suspected it should be replaced with new batch immediately. 2.1.3 chlamydiosis 2.1.3.1 history halberstaedter and von prowazek (1907) , a czech zoologist, first described chlamydia, although it was actively infecting people for centuries before its discovery. first description of chlamydia associated trachoma in human was found in the ebers papyrus dated around 1550 b.c. in modern times, ritter (1879) first described chlamydia psittaci infection in human acquired from parrots. during 1890-1930, numerous outbreaks of human psittacosis occurred in europe, north and south america, associated with parrots and other pet birds. in 1929-30, pandemic psittacosis outbreaks in human were reported due to import of infected psittacine birds from south america to europe and north america. in 1930, levinthal, coles, and lillie, independently described the properties of the pathogen, and accordingly chlamydia was known as levinthal-coles-lillie (lcl) agent. moulder (1962) first revealed the structural and chemical composition of c. psittaci. hatch (1975) demonstrated the requirement of adenosine-triphosphate (atp) supplementation for growth of chlamydia. in literatures, first description of chlamydiosis in parakeets was reported from germany (strauch and rott 1958) . further study after few years also revealed the presence of chlamydia in parrots and other parakeets in germany (schmittdiel 1966) . human psittacosis outbreaks specially in persons associated with poultry, turkey and duck industry were reported from united states and european countries during 1980-90. all the chlamydiae are placed under the order chlamydiales, and family chlamydiaceae. based on cluster analysis of 16s and 23s rrna genes, the family chlamydiaceae was divided into two genera i.e. chlamydia and chlamydophila. recent genome comparison study of the two genera proposed to unite the chlamydia in a single genus. the latest edition of the bergey's manual of systematic bacteriology also described the single genus of chlamydia. pathogenic species under the genus chlamydia are c. psittaci, c. trachomatis, c. suis, c. muridarum, c. abortus, c. caviae, c. felis, c. pecorum and c. pneumoniae. among the pathogenic species, c. psittaci is mostly associated with avian chlamydiosis (psittacosis) in pet birds, ornithosis in poultry, and zoonotic infection in human. the 16s rrna gene based phylogenetic study indicates the presence of a distinct cluster of c. psittaci strains which are associated with chlamydiosis in psittaciformes (cockatoos, parrots, parakeets, lories etc.) and columbiformes (pigeons) birds. although, other chlamydial species such as c. abortus, c. trachomatis and c. pecorum are occasionally detected in brown skua, parrots, parakeets, and pigeons. three new avian species of chlamydia, namely c. ibidis sp. nov., c. avium sp. nov., and c. gallinacea sp. nov. are proposed. among them, c. ibidis and c. avium are isolated from feral sacred ibis (threskiornis aethiopicus), psittacines, and pigeons. earlier, avian isolates of c. psittaci was divided into six serovars (serotypes) which can infect different species of birds (a-f, table 2 .1). based on major outer membrane protein (ompa) sequence, c. psittaci is currently divided into 15 genotypes. among them, nine genotypes (a-f, e/b, m56, wc) are associated with different species of birds and mammals (table 2 .1). avian strains of c. psittaci are detected in more than 460 species of birds under 30 orders. the pet birds belonged to the order psittaciformes (cockatoos, parrots, parakeets, lories etc.) and columbiformes (pigeons) are most susceptible to c. psittaci infection. in parrots, the worldwide prevalence of c. psittaci varies from 16-81%. the infection in pet birds is reported from europe, brazil, africa, usa, iran and india. free ranging galapagos doves (zenaida galapagoensis) and rock doves (columba livia) in spain; monk parakeets (myiopsitta monachus), amazon parrots, red-tailed amazon (amazona brasiliensis) in brazil; ring necked parakeet (psittacula krameri), alexandrine parakeet (psittacula eupatria), african grey parrot (psittacus erithacus), timneh grey parrot (psittacus erithacus timneh) in iran were reported to be infected with c. psittaci. in india, c. psittaci was isolated from pigeons (columba livh), parrots (psittacula krameri) and crows (corvus splendens). infection of passeriformes birds is not common, although, canaries were detected to be infected with c. psittaci in croatia. c. psittaci was also detected in healthy asymptomatic birds such as in ara macao, and amazona ochrocephala in costarica, and free-living hyacinth macaw (anodorhynchus hyacinthinus) and blue-fronted parrot (amazona aestiva) in brazil. the syndrome was not expressed either due to infection with low virulent strain or resistance of some bird species. the seroprervalence studies revealed the presence of c. psittaci antibodies in macaws (ara macao, ara ambigua), hyacinth macaws (anodorhynchus hyacinthinus), budgerigars (melopsittacus undulatus), lovebirds (agapornis sp.), cockatiels (nymphicus hollandicus), alexandrine parakeets (psittacula eupatria), eurasian siskins (carduelis spinus), oriental skylarks (alauda arvensis), and black-tailed grosbeaks (coccothraustes migratorius) in different countries. the presence of c. psittaci antibodies indicates the exposure of the birds to the organism. in a study in china, highest seroprevalence was observed in cockatiel which was followed by alexandrine parakeets, lovebirds, and budgerigars. it seems that lovebirds and budgerigars among the psittacine birds are relatively resistant against c. psittaci infection, although, the reason is unexplored. among the wild predator birds, white-tailed sea eagle (haliaeetus albicilla) and the peregrine falcon (falco peregrinus) are detected to be infected with c. psittaci. inhalation of contaminated dust, airborne particles from the feathers and ingestion are major ways of c. psittaci transmission in the birds. direct contact during close proximity with the infected birds also helps in transmission. throughout the breeding season, specially during incubation of eggs, male psittacine birds prefer to feed the females by regurgitation. in this process the feeds are often mixed with secreations of the crop, pharynx and nasal cavity. transmission of c. psittaci is observed from parent birds to their nestlings during feeding. asymptomatic carrier birds infected with c. psittaci, excreate the organisms through faeces, nasal and lacrimal discharge, oropharyngeal mucus, crop milk and other secreations. shedding is increased during coexisting infections and stress conditions such as shipment, breeding, crowding, chilling and nutritional deficiencies. when the excreated faecal material dries, the organisms are aerosolized. elementary bodies (infections form) of c. psittaci survive in the dried faeces for several months, in the contaminated feed for up to two months, on glass for 15 days, and in straw for 20 days. mechanical transmission of c. psittaci by biting arthropods such as flies, mites and lice are observed. vertical transmission is infrequently observed in parakeets, seagulls, snow geese and poultry. zoonotic transmission of c. psittaci in human occurs mostly through inhalation of contaminated dust, feathers and aerosolized excreations. direct contact with infected pet birds or their cages, utensils, beddings contaminated with discharges can transmit the bacteria. sometimes, biting of the infected birds also helps in transmission. person to person spread is rarely reported although possible through inhalation. chlamydia follows a unique life cycle with tri-phasic developmental stages. the infectious form (elementary body, eb) is extremely small (250-350 nm in diameter), pear to spherical shaped particle with electron dense irregular nucleoid. it has rigid cell wall with disulphide cross linkage among the cysteine rich amino acids of outer membrane proteins (omp). this form can survive for a prolonged period in the environment. after transmission, infection starts with the attachment of elementary bodies to the host cell membrane. in birds, apical surface of columnar epithelial cells in intestine acts as preferred site for attachment of c. psittaci. primary attachment of eb takes place by electrostatic interactions, most likely with glycosaminoglycan (gag) moieties on the host cell surface. this reversible binding is followed by receptor mediated irreversible attachment. protein disulfide isomerase (pdi), present in the host cell membranes and causing disulphide reduction, helps in attachment of ebs. chlamydial major outer membrane protein (momp) mostly acts as adhesin to bind with the host cellular receptor. following receptor mediated attachment, the eb enters the cell via endocytosis [microfilament dependent/independent process (clathrin mediated)]. some chlamydial strains enter the host cells through cholesterol-rich lipid raft domains. among different pathways, c. psittaci strains prefer to use clathrin-coated vesicles for cellular entry. c. psittaci elementary bodies contain rosette like long projections (matsumoto's projection) on their surface which acts as type three secreation system (t3ss). c. psittaci-t3ss helps in introduction of chlamydial proteins into the host cell cytoplasm. these t3ss-injected proteins interact with host cellular proteins and cause modulation of host cell function. after cellular entry, vesicles containing ebs escape the lysosomes in the host cell cytoplasm and reach near the nucleus within 8-12 h after entry. in c. psittaci infection, incb proteins (t3ss-effector protein) interact with host cell proteins (dynein motor proteins) for intracellular transport of vesicles with ebs into the nuclear zone. the eb is converted into reticulate bodies (rb) in this nuclear zone. the eb loses its electron dense dna core and its cell wall loses its rigidity due to break of disulphide bridges. the reticulate bodies are non-infectious form, larger in size (500-2000 nm diameter) and metabolically active. rbs multiply by binary fission and start genus specific protein synthesis. the structural reorientation started and rbs are transformed into intermediate bodies (ib, 300-1000 nm in diameter). a central electron-dense core with radially arranged nucleoid fibres surrounding the core is observed in the ibs. the ibs are converted into progeny ebs within a vesicle after 30 h of the entry of initial ebs. chlamydial micro colony with 100-500 ebs within the vesicle is called 'inclusion body' and it is generated after 48-50 h. the inclusion bodies move to the golgi apparatus region with the help of host dynein proteins. eb is released to attack new cells by rupturing the vesicles and the cycle is repeated. the signal for release of ebs is yet unknown but it is associated with host cellular apoptosis. suppression of host cellular apoptosis can induce persistent chlamydial infection. intracellular survival of ebs depends on escape from the lysosomal breakdown process. the ebs can induce delayed maturation of lysosomes as an escape mechanism. the intracellular inclusion bodies are covered with a mesh of host cytoskeletal filaments which prevent the exit of the content and consequent activation of the host immune system. close attachment of c. psittaci inclusions with the mitochondria helps in acquisition of atp because they cannot synthesize it. moreover, intracellular survival of the inclusions depends on acquisition of lipids such as sphingomyelin, phosphatidylinositol and phosphatidylcholine. golgi apparatus of the host cells act as major source of lipids for the inclusions and often the golgi apparatus are fragmented to provide the lipid. some non-replicating reticulate bodies persist within the host cytoplasm and produce latent infection. the growth cycle of chlamydia within the body of the host is disrupted due to nutritional deprivation, treatment with antibiotic and activated immune system. in disrupted growth cycle, reticulate bodies are converted into enlarged pleiotrophic 'aberrant' rbs. the aberrant rb contains chromosome but the genes associated with growth (genes encoding membrane proteins, transcription regulators, cell division factors, eb-rb differentiation factors) are not expressed. further, the genes encoding chlamydia protein associated with death domains (cadd) are down regulated which causes suppression of host cell apoptosis and persistence of infection. interaction of host cellular protein (g3bp1) and chlamydial inca (t3ss-effector protein) also suppress host cellular apoptosis. when the inducers of the disrupted growth cycle (antibiotic, immune system products) are removed, the aberrant rb is again converted into normal rb and they can complete the growth cycle. in birds, chlamydiosis has an incubation period of 3-10 days. clinical symptoms are not specific. general syndrome such as loss of condition, anorexia, fever, diarrhoea, respiratory problems, nasal and ocular discharges are observed. expression of syndrome and associated mortality (up to 80%) depends on virulence of c. psittaci strains, age, species, nutritional and immune status of the pet birds. occasionally, sub-clinical c. psittaci infection without visible syndrome is observed in birds. during stress conditions, the sub-clinical infection is activated with increased shedding of c. psittaci. the pet birds with avian chlamydiosis do not show any pathognomonic gross lesion. conjunctivitis, lateral nasal adenitis, sinusitis, fibrinous airsacculitis, lung congestion, fibrinous pneumonia, pericarditis with presence of fibrinous cover, peritonitis, hepatitis with multifocal necrosis and splenitis are observed (figs. 2.5 and 2.6). in pigeons, conjunctivitis, swollen eyelids, rhinitis, presence of fibrinous exudates over peritoneum, air sac and pericardium, enlarged, soft and dark coloured liver and spleen are observed. the budgerigars, infected with c. psittaci and reovirus, showed distinct cachexia, hepatomegaly and spleenomegaly. the livers become enlarged, mottled and tan-brown in colour. other lesions include uric acid atherosclerosis is considered as a well defined ailment specially in aged pet birds. african gray parrots, macaws and amazon parrots are most susceptible to this condition. sudden death without prior symptom is the cardinal sign of atherosclerosis. like human, the risk factors for atherosclerosis include high cholesterol and triglyceride concentrations, sex, age, species, obesity and inactivity, and moreover, c. psittaci infection. arteriosclerotic plaques are observed between the intima and internal elastic lamina of the blood vessels in many species of birds ( fig. 2.7) . the plaques are composed of fibrous tissues and are observed as pale yellowish areas at the thickened portion of intima. in severe cases, the plaques become circumferential lesion which cause narrowing of the lumen and reduced blood flow. clinical specimens from live birds, pharyngeal/choanal slit swabs, conjunctival swabs and nasal swabs can be collected aseptically as ante-mortem samples. faeces or cloacal swabs are less preferred because shedding of chlamydia is not consistent. post mortem samples collected from the dead birds include lungs, spleen, liver and air sacs. chlamydiae are relatively labile organisms and special precautions are required for their detection. samples should be maintained in cold chamber and processed immediately after collection. the tissue samples can be preserved at −80°c for prolonged period. dna extracted from the tissue samples can be stored in stabilization buffer. for successful isolation of chlamydia, the clinical samples should be collected in special chlamydia transport medium such as 2sp (0.2 m sucrose phosphate medium containing 10 lg/ml of gentamicin, 25 u of nystatin and 25 lg/ml of vancomycin) and spg (75 g of sucrose, 0.52 g of kh 2 po 4, 1.22 g of na 2 hpo 4 , 0.72 g of glutamic acid and water in 1litre, ph 7.4-7.6) supplemented with bovine serum albumin, streptomycin, vancomycin and nystatin. broad spectrum antibiotics like tetracycline, chloramphenicol, macrolides, sulphonamides, penicillin should not be added as they have anti-chlamydial effect. (a) direct examination: smears prepared from collected faecal samples, conjunctiva or impression smears of tissue samples can be stained with macchiavello, castaneda, giemsa, giménez, modified gimenez (pvk stain), stamp, modified z-n, and methylene blue for demonstration of chlamydial inclusion bodies. giemsa stain is more useful in the smears prepared from conjunctival scrapings. the inclusion bodies appear purple/blue with giemsa, castaneda and methylene blue stain and red with macchiavello, giménez, stamp, and modified z-n stains. (b) isolation of chlamydia from clinical samples: isolation of chlamydia can be done in the yolk sacs of embryonated hen eggs, laboratory animals and cell culture. fertile chicken eggs (6-8 days old) are inoculated through the yolk sac route. the embryo dying three or more days after incubation is examined for chlamydial inclusions. mice are ideal laboratory animal for isolation of chlamydia. the mice usually die within ten days of intranasal, intracerebral or intraperitoneal inoculation and the ebs can be isolated from viscera and peritoneal exudates. cell lines treated with a metabolic inhibitor (cycloheximide at 2 lg/ml) can be used for isolation of chlamydiae human psittacosis cases are reported in europe, usa, south america, japan and australia. other than the persons who rear the birds in their home, occupational risk groups such as veterinarians, pet shop workers, avian quarantine workers, poultry processing plant workers, bird breeders, and farm workers are most susceptible. even psittacosis outbreak was detected among custom officers in some countries due to their exposure to imported parakeets in the airport. incubation period in human is 5-14 days. clinical syndrome in human includes fever, chills, headache, pneumonia, renal disorders, and miscarriages in pregnant women. all the vital organs are affected with the progression of infection and endocarditis, hepatitis, myocarditis, arthritis and encephalitis are reported. ocular infection with follicular kerato-conjunctivitis is also observed. doxycycline, tetracycline and enrofloxacin were successfully used in budgerigars and psittacine birds to cure avian chlamydiosis. doxycycline is the drug of choice for the birds and the treatment should be continued for 45 days. it may induce toxicity in some bird species and produce signs of depression, inactivity, anorexia, greenish or yellowish urine. use of the drug in those birds should be stopped immediately and supportive symptomatic treatment should be started. recommended dose of doxycycline in feed is 300 mg/kg feed for 45 days. in drinking water, 400 mg of doxycycline hyclate/litre of water will maintain therapeutic concentration in psittacine birds. administration of the drug through the feed or drinking water is suitable for aviaries. for pet bird owners, oral administration of the capsule in individual bird is appropriate. recommended oral dose of the drug is 40-50 mg/kg body weight in every 24 h for cockatiels, senegal parrots, blue-fronted, orange-winged amazon parrots; 25 mg/kg body weight in every 24 h for african gray parrots, blue and gold macaws, green-winged macaws; and 25-50 mg/kg body weight in every 24 h for other psittacine birds. injectable doxycycline is administered at doses of 75-100 mg/kg body weight, intramuscularly (pectoral muscle), in every 5-7 days for the first 30 days and subsequently in every 5 days for the rest of the treatment period. long acting oxytetracycline can be injected sub-cutaneously at the dose of 75 mg/kg body weight in every 3 days in cockatoos, blue-fronted and orange-winged amazon parrots, and blue and gold macaws. the oxytetracycline injection causes irritation at the site. if tetracycline is orally administered or used in feed, dietary calcium sources (mineral block, oyster shell, supplemented pellets) should be reduced. to control the psittacosis infection in aviaries, general precautionary measures, such as quarantine of newly introduced birds for 30 days and periodical testing for c. psittaci infection, separation of birds after return from bird shows or fairs, rodent control, control of exposure to wild birds, regular disinfection of the cages and utensils, proper ventilation to reduce aerosol load within the unit should be followed. use of prophylactic antibiotic is not recommended as it may produce resistant bacteria. recommended disinfectants for c. psittaci infection are 1:1,000 dilution of quaternary ammonium compounds, 70% isopropyl alcohol, 1% lysol, and chlorophenols. use of vacuum cleaner in the aviaries is not preferred as it will aerosolize infectious particles. no vaccine is commercially available for the pet birds against c. psittaci infection. experimental dna vaccination in budgerigars with plasmid dna expressing momp of c. psittaci was found effective. were also carried out by smith in 1918 when similar organisms were isolated from aborted bovine foetuses. in this period, the bacteria were known as vibrio foetus. in 1963, due to certain differentiating characteristics, the bacteria were separated from vibrionaceae family and the new genus campylobacter ('curved rod') under campylobacteriaceae family was proposed. campylobacter spp. is gram negative, comma shaped rods specially in infected tissues and young cultures. when two bacterial cells are found together in a microscopic field, occasionally it looks like 's' or 'wing of gull' ('flying seagull'). they are motile by single unipolar/bipolar unsheathed flagella. motility is darting or corkscrew type, best observed by dark field microscopy. campylobacteriaceae family contains four genera namely campylobacter, arcobacter, sulfurospirillum and thiovulum. currently there are 18 species and 6 sub species of the genus campylobacter. important species and sub species of campylobacter are-campylobacter jejuni ssp. jejuni, c. jejuni ssp. doyeli, c. coli, c. lari, c. fetus ssp. venerealis, c. fetus ssp. fetus. thermotolerant campylobacter (c. jejuni ssp. jejuni, c. coli, c. lari, some strains of c. upsaliensis) is isolated from pet birds with or without clinical syndrome. the thermotolerant species requires higher temperature for their growth (42°c) which is provided by the pet birds due to high body temperature. other than thermotolerant campylobacter species, c. fetus and c. intestinalis have also isolated from parrots. the pet birds such as tropical finches (juvenile estrildidae), canaries, pigeons, parakeets [except red-crowned parakeet (cyanoramphus novaezelandiae), dusky-headed parakeet (aratinga weddellii), orange winged parrot (amazona amazonica), red bellied macaws (ara manilata)], emu, ostriches, waterfowls (mallard duck, shoveler duck, green-winged teal duck) are detected to harbour campylobacter spp. in a study in peruvian amazon, parrots (ara, brotogeris and pionites) were detected to be infected with campylobacter spp. wild and free-living birds, for instance, sparrows, crows, waders, black-headed gull (larus ridibundus), sparrow hawk (accipter nisus), jackdaw (corvus monedula), hooded crow (corvus cornix), dunnock (prunella modularis), yellowhammer (emberiza citrinella), white wagtail (motacilla alba), dunlin (calidris alpina), curlew sandpiper (calidris ferruguinea), bald ibis (geronticus eremita), little stint (calidris minuta), broad-billed sandpiper (limicola falcinellus), ruff (philomachus pugnax), wood sandpiper (tringa glareola), long-eared owl (asio otus), starling (sturnus vulgaris), reed warbler (acrocephalus scirpaceus), winter wren (troglodytes troglodytes), redwing (turdus iliacus), blackbird (turdus merula), song thrush (turdus philomelos), fieldfare (turdus pilaris), blackbirds (turdus merula), thrush (turdus viscivorus) can act as reservoir of c. jejuni in nature. certain clonal lineages of c. jejuni and species of wild birds are positively associated. among the raptors (birds of prey), only hawks were detected to carry c. jejuni in their gut. possession of campylobacter spp. in birds depends on feeding habits. gulls and crows have higher possession rate than the pigeons due to their preference for sewages. the shoveler ducks (spatula clypeata) have higher carriage rate than green-winged teal duck (anas acuta) because they prefer bottom sediments of aquatic environments containing molluscs as a feed. direct and indirect contact with infected birds and vectors (house flies, beetles, cockroaches, mealworms) are major ways of c. jejuni transmission in pet birds. c. jejuni is sensitive to oxygen and cannot grow below 31-32°c temperature. so, they cannot survive in feed and drinking water for a prolonged period. presence of c. jejuni in drinking water acts as an indicator for faecal contamination from wild birds or livestock. sometimes, campylobacter spp. can make a symbiosis with aquatic protozoa and survive in the environmental water. in human, major source of c. jejuni is contaminated poultry and its products, pork (with intact skin), beef, mutton and raw milk. consumption of undercooked meat, milk or their products and handling poultry are the key ways of transmission. direct or indirect contact with infected pet birds may play a role in zoonotic transmission of c. jejuni, although, not recorded in scientific literatures. in poultry, after transmission by faecal-oral route, c. jejuni colonizes at the mucous layer of caecal and cloacal crypts. the colonization is mediated by adhesin proteins like cadf (campylobacter adhesin to fibronectin), peb (periplasmic/ membrane-associated protein), capa (campylobacter adhesion protein a), jlpa (jejuni lipoprotein a), ciab (campylobacter invasin antigen b), flagella, and lipopolsaccharide (lps). occasional invasion of the intestinal epithelium takes place. no gross or microscopic lesions and clinical signs are produced in poultry during c. jejuni colonization or invasion. similar type of c. jejuni colonization takes place in psittacines and canaries and they mostly act as asymptomatic carriers. severe clinical signs and lesions are produced in tropical finches, especially in juvenile estrildidae. the precise mechanism of c. jejuni infection in pet birds is still unexplored. no clinical signs and lesions are detected in canaries, psittacines, free-living (migrating passerines) and wild birds, and they act as asymptomatic reservoir of c. jejuni. in finches [juvenile estrildidae, gouldian finch (chloebia gouldiae)], symptoms include sitting posture with its head under the wings, yellow droppings due to undigested starch (amylum), lethargy, and retarded moulting. high rate of mortality is observed among fledglings. in young ostriches, green coloured urination is the predominant sign. recent study indicates the possible synergistic role of c. jejuni in proventricular dilatation disease (pdd) in parrots caused by avian bornavirus. in tropical finches infected with c. jejuni, distinct cachexia, congestion in gastrointestinal tract, and presence of yellow coloured amylum or undigested seeds in gastrointestinal tract are the lesions. in sub-acute cases, hepatitis with focal necrosis and mucoid haemorrhagic enteritis is observed. fresh droppings (without urine) and cloacal swabs can be collected as clinical specimens. post mortem samples include intestine or intestinal contents and liver. infection with c. jejuni in human causes watery or bloody diarrhoea, elevated body temperature, abdominal pain, nausea, and vomition. septicaemia develops in a few diarrhoeic cases (0.15%) which may cause enlargement of the liver and spleen, endocarditis, arthritis, and meningitis. rarely, a complicated auto-immune response is developed as a sequel, known as guillain-barré syndrome. it is a demyelating disorder which results muscle weakness and neuromuscular paralysis. in mild infection, treatment with antibiotics is not recommended due to possibility of antibiotic resistance development. in severe cases, several antibiotics such as clindamycin, gentamicin, tetracyclines, erythromycin, cephalothin, and fluoroquinolones (nalidixic acid) can be used under the supervision of a qualified veterinarian. choice of antibiotic depends on sensitivity of the c. jejuni isolates, availability in suitable form, and species of the birds. in aviaries or personal collection, implementation of biosecurity practices such as regular cleaning and use of fly repellents in the cages is effective to prevent the introduction of campylobacter spp. no vaccine against campylobacter spp. is currently available for birds. 2.1.5 lyme disease 2.1.5.1 history lyme disease is a tick-borne, multi-system disorder of human and animals and it is characterized by swelling of joints, pain, lameness, fever, lethargy, anorexia, nephropathy with renal failure, myocarditis, cardiac arrest and cns involvement. the etiological agent is maintained in tick and several birds and animals. clinical description of lyme disease was first documented by arvid afzelius, a swedish dermatologist. the disease was first identified in 1975 (or 1976), among the people suffering with suspected juvenile rheumatoid arthritis in the area of lyme, connecticut, united states. hence it is known as 'lyme disease' or 'lyme borreliosis'. the causative agent, borrelia burgdorferi sensu lato (s.l.) was identified in 1982. borrelia spirochete is a gram negative, spiral organism with linear chromosome. the life cycle of borrelia requires arthropod vectors and mammalian hosts. it belongs to the family spirochetaceae under the order spirochaetales. borrelia spirochetes comprise three distinct species groups i.e. lyme borreliosis group (borrelia burgdorferi sensu lato; hard tick transmission), relapsing fever group (b. duttonii, b. hermsii; soft ticks transmission) and a third group phylogenetically similar with relapsing fever group but transmitted by hard ticks (b. theileri, b. lonestari, b. miyamotoi). lyme disease in human and animals (dogs, horses) is mostly caused by borrelia burgdorferi sensu lato (s.l.). borrelia miyamotoi is recently detected to produce lyme disease like syndrome in human. borrelia burgdorferi s.l. can be divided into 15 genomic groups or genospecies table 2 .2. borrelia burgdorferi is maintained in nature through a cycle. in the cycle, hard ticks (ixodes spp, haemaphysalis spp.) and small mammals (birds, rodents) act as vector and reservoir host, respectively (table 2 .2). the serum complement of the reservoir hosts determines host preference of b. burgdorferi. the bird associated genospecies are resistant to the bird complement but susceptible to the rodent complement. ixodes scapularis and i. pacificus in usa and canada, i. ricinus in europe and i. persulcatus in asia (japan) act as major vectors of borrelia burgdorferi s.l. occasionally, other species of ticks such as i. uriae, i. affinis, i. dammini, i. frontalis, i. angustus neumann, i. spinipalpis hadwen and nuttall, i. auritulus neumann, i. pacificus cooley and kohls are also associated. all of these ticks cannot parasitize human to transmit the spirochete, but, they can act as maintenance host (e.g. i. affinis, i. dentatus). ixodes persulcatus in japan and i. scapularis in usa was detected to act as vector of borrelia miyamotoi. different stages of ixodes ticks (larva, nymph and adult) can attach with three different hosts to take the blood meal and after engorgement they drop off the host in the environment. immatured ticks (larva or nymph) prefer to stay in moist areas such as vegetative mat of the forest floor or meadow. ground-feeding birds (passerines, game birds, sea birds), rodents, lizards act as preferred hosts of the imamatured ticks (table 2. 3). although, in comparison to rodents, tick infestation in migratory passerine birds is 20-30 times less, but the birds can transmit the infection for long distances. sometimes, reservoir birds generate mutant and more virulent strains of borrelia. passerine birds in mixed coniferous (evergreen) forest were more infected with b. burgdorferi s.l. than the birds in alder swamp forest. experimentally, mallard ducks (anas platyrhynchos platyrhynchos) are susceptible to b. burgdorferi infection and the ducks shed the organism in the droppings. they may transmit the infection without the help of tick vectors. the study indicated that psittacine birds such as yellow naped amazon parrots (amazona auropalliata) are generally not infected with b. burgdorferi s.l. more studies are needed to explore their resistance status against borrelia infection. the ticks normally attach with eyelid, head, neck and ventral feather of passerine birds during blood meal ( fig. 2 .8). the immature ticks take a blood meal for 2-4 days from their preferred hosts. in adult stage, the ticks attach with the tip of the grasses to get adhere with a large mammalian host. the adult ticks take a blood meal for 5-6 days. the ticks itself have less mobility but they can be carried by their hosts specially the migratory passerine birds across the countries. the seabird tick (i. uriae) is observed to disseminate borrelia burgdorferi s.l. from one hemisphere to another (trans-hemispheric transmission). in canada, passerine birds move northward during spring for breeding and nesting and they disseminate ticks with the pathogens. b. burgdorferi is transmitted to immatured ticks from infected birds, rodents and lizards along with the blood meal. the spirochete after transmission multiplies in the gut of the ticks. when the immature ticks molt into adult stage, the numbers of borrelia spirochete is decreased. during attachment of adult tick with large mammalian hosts, the multiplication of spirochete restarts and the number is increased. the expression of b. burgdorferi outer surface protein (osp) is also changed from ospa to ospc. the ospc helps in transmission of borrelia from the mid gut to the salivary glands of ticks. thus, b. burgdorferi is transmitted transstadially from larva to nymph and from nymph to adult. rarely, within the tick population, b. burgdorferi is transmitted transovarially. when the adult ticks bite a new host, the spirochetes are transmitted from the salivary glands. possibility of b. burgdorferi transmission by the adult ticks is more than the nymph and larvae, because the adult ticks have two blood meals in different hosts. sometimes, a single species of tick is infected with more than one numbers of borrelia genospecies (e.g. b. garinii and b. valaisiana) due to superinfection of the already infected ticks during their consecutive blood meals. occasionally, two different species of ticks (i. scapularis and i. affinis) may attach with the same borrelia burgdorferi infected host. co-transmission occurs between the infected and naive nymphs or larvae. infected larvae. occasionally, transmission of borrelia occurs from the infected ticks to uninfected ticks during their co-feeding from the same birds. the migratory birds not only import the infected ticks in a locality, but also, there is a possibility that local ticks get the infection during attachment with the birds. after a long journey, the birds prefer to take rest in some places for a few days. recently, role of cottontail rabbit in this transmission cycle is also explored. during carriage of b. burgdorferi most of the birds do not show any clinical symptom or lesion. experimental inoculation of b. burgdorferi in canary finches (serinus canaria) produced only a brief episode of diarrhoea. natural infestation of b burgdorferi infected tick (ixodes auritulus) results gasping, lameness and death in fledgling american robin (turdus migratorius). clinical specimens for collection of suspected ticks from the migratory birds, the birds are caught by mist nets and are observed carefully for the presence of ticks in head, neck, and beak by magnifying glasses. the ticks are collected by a blunt forcep and are placed in 70% ethanol. they should be labelled properly indicating species of bird and tick, and date of collection. for identification of bird and ticks up to the species level, expertise is needed. from the birds, suspected for b. burgdorferi infection, heparinized blood and tissues from liver, spleen, kidneys can be collected after post mortem. (a) direct examination: dark field microscopy or giemsa stain can directly demonstrate the borrelia spirochete in the blood film, liver/spleen smears. fat can be used for direct examination of the smears. (b) isolation of bacteria from clinical samples: isolation of borrelia is difficult due to its slow and fastidious growth and microaerophilic requirements. modified bsk (barbour-stoenner-kelly) medium is used for isolation of b. burgdorferi s.l. it is an enriched serum broth containing the antibiotics like kanamycin and 5-flurouracil. the media after inoculation is incubated at 33-34°c for 3 weeks. the collected heparinized blood sample (0.02 ml) or triturated tissue sample (0.1 ml) can be added in bsk medium (7-8 ml). (c) serological tests: elisa based kits for detection of total immunoglobulin, ig g, ig m against b. burgdorferi s.l is available for human. however, studies in animals (dogs), indicated that results of serological tests are inconclusive. the antibodies may be produced due to earlier exposure specially in those areas where infected tick bite is a common phenomenon. such kind of serological studies are not conducted in birds probably due to this uncertainty. (d) molecular biology: the whole blood samples collected from the birds can be used for dna extraction. whereas, from the ticks, dna is extracted by spin column technique. pcr for consensus flab gene of borrelia and the spacer region between the 5s and 23s rrna genes can be carried out to confirm borrelia burgdorferi s.l. zoonotic transmission of borrelia burgdorferi s.l. occurs from the bites of infected ticks. the persons during outdoor recreation, professionals such as wildlife and forest caretakers are at high risk. migratory birds passively maintain the infection in nature. no direct transmission of borrelia burgdorferi s.l. from the birds to human is evidenced. the infection in human initiates with a red coloured allergic pimple (erythema migrans), and it is followed by fever, headache, fatigue, muscle and joint pain. in severe cases, meningitis, unilateral facial nerve palsy and renal failure occur. products that kill or repel ticks (e.g. permethrin) can be used in the habitat to reduce the tick density. however, these acaricides may cause environmental hazard and they are only recommended during epidemic situation. 2.1.6 others 2.1.6.1 yersiniosis yersinia spp. was first isolated by alexandre yersin in 1894 in hongkong. he was sent by louis pasteur to investigate about plague outbreak there. in japan, s. kitasato also independently isolated the bacteria a few days later. previously, it was known as pasteurella pestis in honour of pasteur. later, in memory of yersin, the bacterium was renamed as yersinia pestis. in 1976, yersinia pseudotuberculosis was isolated from a sick palm dove (streptopelia senegalensis) in israel. yersinia enterocolitica was first detected in budgerigars in 1980. yersinia is gram negative, short rods or coccobacilli shaped bacteria. they show bipolar staining characteristics ('safety pin appearance') when stained with leishman's or wright or giemsa stain. the genus yersinia is classified under the family enterobacteriaceae that belongs to the order enterobacteriales. yersinia consists of 11 numbers of species. among them, y. pseudotuberculosis and y. enterocolitica are commonly associated with psittacine and passerine bird infection. mynahs are most susceptible to yersinia spp. infection. yersiniosis is also reported from canaries (serinus canaria), zebra finch (poephila guttata), kaka (nestor meriondalis), rainbow lorikeet (trichoglossus mollucanus), budgerigar (melopsittacus undulatus), new zealand wood pigeons (hemiphaga novaeseelandiae), blue-fronted amazon (amazona aestiva), yellow-headed amazon (amazona oratrix), eurasian collared dove (streptopelia decaocto) and cockatoo (cacatua alba). rodents and wild birds are major reservoir of infection and the feed and water are often contaminated with rodent urine or faeces. ingestion of contaminated feed and water is the key route of transmission. high mortality and non-specific clinical signs such as ruffling of feathers, depression, diarrhoea, and biliverdin in the urine are observed in the birds. the infection is acute and mostly enteric in passerine birds. chronic infection takes place in psittacines and pigeons, and it produces hepatitis, splenitis, pneumonia, nephritis and enteritis. the liver becomes dark, swollen and congested. yellow coloured foci (bacterial granulomata) are found in the liver, spleen, lungs, kidneys, intestines and heart ( fig. 2.9 ). microscopically, these foci are composed of necrosed hepatocytes and splenic pulp with fibrin and yersinia colonies. accumulation of iron in the liver (hepatic haemosiderosis) may act as a predisposing factor for systemic yersinia spp. infection. a smear can be prepared from the collected tissues of liver, spleen, kidney, intestine and stained by leishman's, wright, and giemsa stain. yersinia shows typical 'bipolar characteristics' (safety-pin appearance). yersinia can be isolated in blood agar, nutrient agar, macconkey's agar, brilliant green agar (y. enterocolitica). the selective medium is cin agar which contains antibiotics such as cefsulodin (15 mg/l), irgasin (4 mg/l) and novobiocin (2.5 mg/l). 'cold enrichment' method can be followed for primary isolation of y. pseudotuberculosis and y. enterocolitica from clinical samples. the samples are kept in sterile phosphate buffered saline (pbs) or nutrient broth at 4°c for 3 weeks. subculture in macconkey's or cin agar is done at weekly interval. amoxicillin in drinking water or soft food is the choice of treatment. in unresponsive cases, treatment should be carried out on the basis of antibiotic sensitivity test. albert bernhard frank (1889), a german biologist, first coined the term mycoplasma which is originated from the greek word mykes (fungus) and plasma (formed). earlier mycoplasma was known as pleuropneumonia-like organisms (pplo). adler (1957) first reported isolation of pplo from the air sac of a parakeet bird. mycoplasma is the smallest pathogenic bacteria (0.3-0.8 µm) and is pleomorphic in shape due to absence of the rigid cell wall. in stained smears, they appear as ring, globules, filaments or elementary bodies. the cell membrane is constituted of trilaminar structure enriched with phosphoprotein, lipoprotein, glycolipid, phospholipid and sterol moieties. mycoplasma belongs to the class mollicutes, order mycoplasmatales, and family mycoplasmataceae. the family comprises of two genera i.e. mycoplasma and ureaplasma. among different species under the genus mycoplasma, m. gallisepticum, m. iowae, and m. sturni are associated with pet bird infection. an epidemic of mycoplasmal conjunctivitis was noticed in house finches (carpodacus mexicanus) in usa in 1994. other birds such as budgerigars, cockatiel, canary, yellow-naped amazon parrot, pigeons, pea-fowls (pavo cristatus), fledgling cliff swallows, european starling (sturnus vulgaris), chukar partridges (alectoris chukar), ring-necked pheasants, purple finches (carpodacus purpureus), evening grosbeaks (coccothraustes vespertinus), pine grosbeaks (pinicola enucleator) are also reported to be infected. concomitant mycoplasmal infection with other bacteria and protozoa (cryptosporidium spp.) was detected in amazon parrots and fledgling cliff swallows. experimentally, american goldfinch (carduelis tristis) carried m. gallisepticum for prolonged period without showing any clinical sign. house sparrows (passer domesticus) are transiently infected experimentally with m. gallisepticum for a short period. in united states, tufted titmice (baeolophus bicolor) bird was reported as non-clinical carriers of m. gallisepticum. feeders or any focal point where the birds gather, act as a source of m. gallisepticum infection, because the infected birds excreate their droplets in the feeder. statistical correlation (multivariate analysis) was established between presence of tube style feeders, non-breeding period and low environmental temperature and m. gallisepticum infection in house finches. vertical way is a rare possibility of mycoplasmal transmission in birds. clinically the infected birds show variable symptoms ranging from serous nasal discharge, sinusitis, swollen eyes with discharge, conjunctivitis and blindness ( fig. 2.10 ). in fledgling cliff swallows and european starling (sturnus vulgaris) infected with m. sturni, bilateral conjunctivitis, episcleritis, epiphora, hyperaemia of palpebrae and nictitans are observed. gross lesions in birds include congestion of mucosa and accumulation of exudates in nasal sinus, trachea, bronchi, and air sacs. air sac congestion was also detected in budgerigars experimentally challenged with m. gallisepticum. histological investigation in birds reveals the presence of mucous gland hyperplasia and thickened mucous membrane of the respiratory tract with mononuclear cell infiltration. in european starling birds, ulceration of mucous membrane and absence of epithelial hyperplasia and lymphoplasmacytic infiltration was observed. in fledgling cliff swallows, lymphoplasmacytic conjunctivitis, rhinitis and infraorbital sinusitis with follicular lymphoid hyperplasia were detected. conjuntival swabs and head, lung, and spleen in 10% buffered formalin after post-mortem, can be collected as clinical samples from the suspected birds. the smears prepared from clinical specimens are stained with giemsa. m. gallisepticum appears as coccoid organisms having 0.25-0.5 lm in size. contrast phase microscopy, dark phase illumination techniques can be used for their direct visualization. m. gallisepticum can be isolated in specialized medium supplemented with 10-15% heat-inactivated avian, swine or horse serum. change in broth colour indicates positive growth after incubation at 37°c for 3-5 days. serum plate agglutination test is the rapid serological test for detection of m. gallisepticum antibodies in birds, although, sometimes it produces false positive result due to cross reaction. pcr targeting 16srrna gene and loop-mediated isothermal amplification (lamp) assay based on a gene within the pyruvate dehydrogenase complex (pdha) are developed for rapid detection of m. gallisepticum. in house finches, application of oral tylosin (1 mg/ml drinking water for 21 days) and ciprofloxacin eye drop (for 7 days) successfully treated conjunctivitis associated with m. gallisepticum. doxycycline (40-50 mg/kg body weight, orally) is also recommended for mycoplasmal infection in cockatiels and amazons. 2.1.6.3 pasteurella multocida, gallibacterium spp., volucribacter spp. bollinger (1878) first reported the isolation of pasteurella like organisms from cattle and wild animals. louis pasteur (1880) conducted more comprehensive studies on fowl cholera and its etiological agent. trevisan (1887) coined the name pasteurella for the bipolar organisms described earlier by pasteur and others. lignières (1900) proposed the specific name for each species of pasteurella according to their host preference, such as pasteurella aviseptica for fowls, p. suiseptica for pigs, p. boviseptica for bovines, p. oviseptica for ovines and p. leptiseptica for rabbits. rosenbusch and merchant (1939) proposed a single species pasteurella multocida and it is in use till date. miringa (1975) described pasteurellosis in african grey parrots. p. multocida is a gram-negative, non motile, non spore-forming short rod or coccobacillus bacterium. in fresh cultures and animal tissues, it produces typical bipolar staining characteristics, particularly with leishman or methylene blue stain. pasteurella belongs to the family pasteurellaceae. other avian pathogens such as gallibacterium (pasteurella anatis) and volucribacter are also members of the same family. p. multocida can be classified into 6 capsular types (a-f) and 16 somatic types (1-16). psittacines [parrots, red-fronted conure (aratinga wagleri)], passerine birds, owls, raptors and waterfowls (ducks) suffer from pasteurellosis. p. multocida is also isolated from eye swabs of healthy psittacine birds. unclassified members of the pasteurellaceae family were isolated from lesions in domestic goose (anser anser forma), fischer's lovebird (agapornis fischer), parrots (amazona spp.), macaws (ara macao), rock dove (columba livia), budgerigars (melanopsittacus undulates), and african gray parrot (psittacus erithacus). contaminated environment (e.g. water) is the major source of p. multocida infection. mechanical transmission by blood sucking arthropods and cat-bite is possible. in cat-bite cases, dermatitis and myositis develops rapidly and it is followed by septicaemia and death. in psittacine birds p. multocida serotype 3 and 4 are associated with septicaemia and cutaneous lesions, respectively. in african gray parrots, p. multocida produced obstruction of nares and dyspnoea, due to formation of intranasal caseous and fibrinous plugs along with other bacteria. in p. multocida infected budgerigars, crop inflammation and apathy was observed. gallibacterium melopsittaci are associated with septicaemia and salpingitis in budgerigars and parakeets. volucribacter psittacicida causes respiratory tract infections, septicaemia, crop inflammation, and diarrhoea in psittacine birds. a smear can be prepared from collected blood sample or the nasal swabs and it is stained by leishman or methylene blue or gram's stain. pasteurella appears as gram negative non-sporing coccobacilli with typical bipolar staining characteristics. p. multocida can be isolated in dextrose-starch agar, casein-sucrose-yeast (csy) medium with 5% blood (bovine or sheep). p. multocida specific pcr (pm-pcr) helps in rapid and confirmatory detection from clinical samples. treatment of avian pasteurellosis with ampicillin (150-200 mg/kg body weight for pigeons, amazon parrots) and tiamulin fumarate (25-50 mg/kg body weight, oral) are recommended. escherichia coli was first isolated by theodor escherich in 1885 from the faeces of human infants. it was named in honour of the german pediatrician and its major natural habitat i.e. colon. in 1978, e. coli was detected in faecal samples collected from psittacine birds. raphael and iverson (1980) described e. coli associated coligranuloma in amazon parrot along with psittacosis. e. coli is gram negative, short rods, varying form coccoid shape to long filamentous forms ( fig. 2.11 ). they occur singly, in pair or in short chain. they are non-spore forming and mostly motile by peritrichous flagella. the genus escherichia is classified under the family enterobacteriaceae that belongs to the order enterobacteriales. there are total 6 species under the genus escherichia. among them, escherichia coli are the important pathogen. the gastro-intestinal tract of all vertebrates including birds is the most common natural habitat of e. coli. the studies revealed that healthy parrots (31%), cockatoos (cacatua spp., 60%) and shore birds carried e. coli in their intestine. in healthy passerine birds, e. coli are not considered as a major intestinal flora. psittacines imported or illegally traded from other countries and shore birds act as source of e. coli. majority of these psittacine e. coli isolates possessed antimicrobial resistance due to the exposure of the birds to the prophylactic antibiotics after their capture. in pet birds, e. coli is transmitted by contaminated feed, drinking water, aerosols, and fomites. the stress conditions like transport, dietary change and extreme climate also help to establish the infection. in adult canaries and finches, e. coli are most common secondary pathogens associated with epizootic mortality. non-specific clinical signs and lesions such as lethargy, rhinitis and conjunctivitis are detected. e. coli as a primary pathogen is reported from a hyacinth macaw (anodorhynchus hyacinthinus), died due to septicaemia and enteritis with hemorrhages in different organs, and a kakapo (strigops habroptilus) with exudative cloacitis. recently, attaching-effacing e. coli is detected as a primary pathogen in a captive flock of budgerigars (melopsittacus undulatus). common lesions in budgerigars include hepatitis, enteritis, and attaching and effacing lesions along the intestinal tract. in nestlings of canaries and finches, e. coli is considered as most important cause of diarrhoea, dehydration, cachexia and mortality. appearance of young birds and their mothers became dirty, wet and yellowish ('sweating disease'). isolation of e. coli from the clinical samples is the major diagnostic technique. blood agar, macconkey's agar are choice of the medium for isolation. after overnight incubation in macconkey's agar, characteristic pink coloured colonies are transferred into eosine methylene blue (emb) agar for detection of 'metallic sheen' (fig. 2.12 ). the isolates are further confirmed by different biochemical tests. pathogenicity of the e. coli isolates from clinical samples should be confirmed as they are present as normal bacterial flora within the body. virulence of the isolates can be ascertained by ligated loop assay, cell culture cytotoxicity assay, typing and detection of toxin by serological or dna based methods. e. coli infections can be treated with ampicillin sodium, amoxicillin/clavulanate, cephalexin, oxytetracycline and spectinomycin. in unresponsive cases, antibiotic should be selected after sensitivity test of the etiological e. coli isolates. in nestlings, antibiotics are administered in drinking water and egg food from one day before hatching up to 6 days after hatching. extra drinking water should be provided to prevent dehydration. other bacterial infections of pet birds are described in table 2 .4. malbrant (1942) reported an outbreak in australian parrots and red-headed lovebirds (agaporius pullaria pullaria l.) in africa suspected to be suffered and died off newcastle disease. zuydam (1952) first isolated newcastle disease virus (ndv) from parakeet (psittacula krameri borealis nearn) and ospray birds (pandion haliaetus) in the netherlands. the first major outbreak of newcastle disease among grey parrots (psittacus erithacus l.) occurred in kenya in 1955 (scott et al. 1956 ). subsequently, in 1960, ndv was isolated from grey parrots (psittacus erithacus l.) in kenya (scott and winmill 1960) . in 1970s, pigeon paramyxovirus-1 (ppmv-1), a variant of ndv, was discovered in the middle east countries. ndv belongs to the avulavirus (avian paramyxovirus-1) genus within the paramyxoviridae family in the order mononegavirales. the virus is an enveloped, single-stranded, un-segmented, negative-sense rna virus. the virion has a genome of 15 kb in size and the genome comprises of the genes which encode nucleocapsid protein (np), phosphoprotein (p), matrix protein (m), fusion protein (f), hemagglutinin-neuraminidase (hn) and polymerase enzyme (l). on the basis of virulence, ndv can be classified into velogenic (highly virulent, icpi > 1.5), mesogenic (intermediate, icpi > 0.7 but 1.5) and lentogenic strains (non-pathogenic, icpi 0.7). intracerebral pathogenicity index (icpi) is oie recommended in vivo test for determination of ndv virulence. the strains differ in virulence also differ in amino acid sequence at the cleavage site of precursor fusion protein (f0). velogenic strains have more than two basic amino acids (arginine or lysine at positions 113-116) and phenylalanine at the position 117. on the basis of fusion protein (f) and polymerase enzyme (l) nucleotide sequence, ndv is classified into two major classes (class i and ii) with a single genotype under class i and 18 genotypes under class ii. distribution of ndv genotypes and sub-genotypes in different species of birds is described in table 2 .5. the virulence of class i ndv isolates is low (except one isolate from ireland) and they are mostly maintained by velogenic or mesogenic strains of ndv were detected in psittacine birds (cockatoos, budgerigars, macaw, lory, parrot, love bird, conure, yellow-headed amazon parrots, yellow-naped amazon parrots), pelicans, gulls, kestrels, falcons, white crested laughing thrush, pheasants, swans, robin, peafowl, whooper swan, spotted necked dove, white-cheeked starling, eurasian blackbird, wild little tern, wild village weaver, mynah (gracula religiosa), drongo (dicrurus spp.) and partridges (family phasianidae) ( table 2 .5). these wild and pet birds may act as reservoir or in pet birds, horizontal transmission through direct contact with infected birds or ingestion of contaminated feed and water are the major ways of ndv transmission. sometimes, infection of parrots occurs from live animal market during their direct contact with poultry. use of contaminated or improperly attenuated live vaccines in poultry against ndv is another possible source of infection in nature and pet birds. experimentally, yellow-headed amazon parrot was infected with velogenic ndv strain by nebulization. common houseflies (musca domestica) act as mechanical vector for ndv transmission, although, yet to be validated in pet birds. imported exotic birds including the psittacines may act as reservoir and they excreate the virus in the faecal matter for prolonged period without showing clinical signs. legally or illegally trafficked pet birds and migratory birds thus introduce the virus into the countries which produces a persistent risk for poultry. moreover, the study has also shown the possibility of spreading ndv in a native wildlife population through ndv contaminated illegal wildlife trade. after transmission of the virus into the susceptible host, cellular entry requires activation of fusion protein (f0) present in the viral envelope. the f0 is activated by post-translational cleavage by the host protease enzyme. the post-translational cleavage varies with the amino acid sequence present in the cleavage site and the type of host protease enzyme. in lentogenic ndv strains, cleavage site contains monobasic amino acid sequence at the c-terminus and leucine at the n-terminus ( 112 g-r/k-q-g-r-l 117 ). the cleavage site of velogenic and mesogenic strains contains multibasic amino acid sequence at the c-terminus and a phenylalanine at the n-terminus ( 112 r/g/kr-q/k-k/r-r-f 117 ). the cleavage site of lentogenic strains is cleaved by the protease present in respiratory and intestinal tract only. velogenic strains are cleaved by ubiquitous protease present in all vital organs. moreover, activation of hn (hn0) protein and other viral proteins such as v, np, p, and l also play role in pathogenesis. psittacine birds suffering with ndv mostly show respiratory signs (rhinitis, conjunctivitis), greenish watery diarrhea (green staining around the vent), lethargy, drooping of wing, torticollis, waving movement of head and neck and limb paralysis. experimental inoculation of six species of pet birds (budgerigar, yellow-headed amazon parrot, halfmoon conure, hill mynah, black-headed nun, canary) with velogenic ndv strain produced ruffled plumage, conjunctivitis, ataxia, wing tremors, paralysis of the extremities, and tremors of the head. neurological signs are more common in parakeets. mortality can reach as high as 100%, but typically ranges between 20-80% depending upon the virulence of the virus strain, host species, age, and immune status. petechial haemorrhages are often observed in viscera of pet birds suffering with newcastle disease. in naturally infected cockatiels and parrots, diffuse spongiosis of gray and white matter, neuronal necrosis, perivascular infiltration of mononuclear cells are detected. the lamina propria of the proventriculus shows infiltration of mononuclear cells and ulceration. accumulation of haemosiderin is detected in the cytoplasm of mononuclear cells. depletion of lymphoid cells is observed in spleen and bursa of fabricious. tracheal and cloacal swabs from live birds and the organs such as liver, brain, spleen, kidney after post mortem can be collected as clinical specimens. collection of cloacal swabs from small birds is a complicated process which may causes injury of the vent. fresh faeces collection is an alternative approach. during transport, the samples should be kept in isotonic phosphate buffered saline (ph 7.0-7.4) or brain heart infusion broth with antibiotics such as penicillin (2000 units/ml), streptomycin (2 mg/ml), gentamicin (50 lg/ml), and mycostatin (1000 units/ml). the samples can be preserved at 4°c for four days. (a) clinical signs and lesions after necropsy, history of direct contact with infected birds give a tentative diagnosis. (b) isolation of virus from clinical samples: the clinical samples collected in isotonic phosphate buffered saline with antibiotics are centrifuged (1000 g) and the supernatant fluid is inoculated into 9-11 days old embryonated hen's eggs (specific pathogen free) by allantoic sac route or the cell lines such as chicken embryo kidney (cek) cells, chicken embryo fibroblast (cef) cells. the inoculated eggs are incubated at 37°c for 4-5 days. after incubation, eggs are kept at 4°c. the embryo will die in positive samples and the allantoic fluids are collected to detect the haemagglutination activity of the viral isolate. the isolate is further confirmed by haemagglutination inhibition (hi) test. virulence of the isolate should be detected by intracerebral pathogenicity index (icpi) or amino acid sequencing of fusion protein to confirm a nd outbreak. (c) serological tests: virus neutralization test, plaque neutralization, hemagglutination-inhibition (hi), single radial immunodiffusion, agar gel immunodiffusion (agid), enzyme-linked immunosorbent assay (elisa) are employed for detection of ndv antibodies. however, these tests can not differentiate the infection caused by velogenic and lentogenic viral isolates. serological tests can give a tentative diagnosis of ndv exposure in the birds. (d) molecular biology: real-time reverse-transcriptase polymerase chain reaction (rrt-pcr) can be used for detection of fusion protein, matrix protein and rna-dependent rna polymerase enzyme either from the viral isolate or from the collected tissues and faeces of suspected birds. it can also confirm the virulence of the isolates. in conventional reverse-transcriptase polymerase chain reaction (rt-pcr), cloning and sequencing of pcr products will confirm the pathogenic potentiality of the isolate. zoonotic transmission of ndv is possible which causes acute conjunctivitis, malaise, and sinusitis in susceptible human. the flu like symptoms resolves automatically within 1-3 weeks. direct transmission of ndv into human from pet birds is still not documented possibly due to similarity of symptoms with common flu and its auto recovery. the pet birds as reservoir of ndv become a potential hazard for poultry population where the human may act as intermediate host. human to human transmission of ndv is not documented. no effective treatment for pet birds against ndv infection is documented. to control the infection in pet birds, exposure to live bird market, wild birds, and migratory birds should be restricted. infected birds should be kept separately and general hygiene practices should be followed to avoid the contamination of feed and drinking water. vaccination is an effective measure to control ndv infection in commercial and domestic poultry. vaccination in pet birds is not recommended because it cannot eliminate the carrier birds. vaccination with modified live virus may produce the infection in pet birds. however, experimental use of inactivated ndv vaccine produced a humoral response in wild house sparrows with doses above 0.05 ml per bird. the humoral response was produced within 4-6 weeks after experimental inoculation of the vaccine. in 1878, avian influenza (earlier known as fowl plague) was described for the first time in italy by perroncito. centanni and savonuzzi (1901) first observed the role of filterable agent (virus) as etiology of avian influenza. the definitive etiological correlation with influenzavirus a was established later (1955) . highly pathogenic avian influenza (h5 subtype) was first reported from chickens in scotland (1959) and common terns (sterna hirundo) in 1961 from south africa. during 1972-80, influenzavirus a was reported from exotic birds including budgerigars (melopsittacus undulatus), migratory ducks, and pelagic seabird (shearwater). avian influenza virus (aiv) belongs to the family orthomyxoviridae, genus influenzavirus a. the virions are sensitive to heat (56°c for 30 min exposure), acid (ph 3.0) and lipid solvents. so they are easily destroyed under common environmental conditions. the virions are enveloped and pleomorphic, spherical to filamentous in shape. the virion surface is covered with two types of glycoprotein projections, known as haemagglutinin (ha, rod shaped trimer protein) and neuraminidase (na, mushroom shaped tetramer protein). other constituent viral proteins are nucleoprotein (np), matrix proteins (m1, m2), polymerase basic (pb1, pb2), polymerase acidic (pa), and non structural proteins (ns2). the viral genome is linear, negative sense single stranded rna and it contains eight numbers of segments. during genetic reassortment, these segments are exchanged between two viral strains to generate a mutant one (antigenic shift). the point mutation in ha and na gene causes antigenic drift which can generate a new viral strain. on the basis of ha and na gene sequences, influenzavirus a has 16 ha (h1-16) and 9 na (n1-9) subtypes. genetic reassortment between the subtypes theoretically can produce 144 types of combinations. on the basis of pathogenicity, aiv can be further differentiated into two categories i.e. highly pathogenic avian influenza (hpai, e.g. h5 and h7 subtypes) and low pathogenic avian influenza (lpai, e.g. h9n2). hpai causes 90-100% mortality in birds and lpai infection is mostly restricted within the respiratory system. further molecular variations of ha gene can differentiate hpai (h5n1) into several clades (first order-fourth order clade). other than commercial and domestic poultry, birds belong to the order anseriformes (waterfowls) and charadriiformes (shorebirds, gulls, auks, terns, waders) are considered as major reservoir of avian influenza virus (table 2 .6). h3 and h6 subtypes are common in waterfowls (anseriformes), and h4, h9, h11, and h13 subtypes are widespread in charadriiformes. among the anseriformes, mallard (anas platyrhynchos) and northern pintail ducks (anas acuta) in united states and bar headed geese (anser indicus) in india are considered as major reservoirs. natural h5n1 outbreak among the migratory waterfowls such as bar-headed geese (anser indicus), great cormorants (phalacrocorax carbo), pallas's gulls (larus ichthyaetus), brown-headed gulls (larus brunnicephalus), ruddy shelducks (tadorna ferruginea) is also detected in china. role of passeriformes (canary, finch, starling, sparrow) as aiv reservoir is uncertain. a study in france with large numbers of wild passerine birds failed to detect aiv. although, wild passerine birds such as eurasian tree sparrow (passer montanus) in china and golden crowned kinglet (regulus satrapa), fox sparrow (passerella iliaca), western tanager (piranga ludoviciana), northern waterthrush (seiurus noveboracensis), cassin's finch (carpodacus cassinii) in united states, and flycatchers (family muscicapidae) in central africa is detected to possess aiv infection. experimentally, society finches (lonchura striata domestica), zebra finches (taeniopygia guttata), house sparrows (passer domesticus) and starlings are found susceptible to aiv (h5n1, h7n9, h7n7) . the sialic acid receptors for both avian influenza (a 2, 3) and human influenza (a 2, 6) viruses are present in house sparrows (passer domesticus) and starlings (sturnus vulgaris). in contrast, eurasian tree sparrows contain abundance of sialic acid receptors (a 2, 6) for aiv. detection of aiv is rare in psittacine birds and as such the psittacines are not considered as potential reservoir of aiv. limited reports of viral isolation such as h9n2 subtype from indian ring-necked parakeets and h5n2 subtype (mexican lineage) from red-lored amazon parrot is available (table 2.6). experimentally, parakeets (melopsittacus undulates) are found susceptible to human h7n9 isolate and after inoculation, development of clinical signs and shedding of the virus into water troughs is observed. on the other hand, psittacine isolate can also replicate in chicken, duck and turkeys and transmission into healthy cage mates is observed. water mediated transmission of aiv is possible in waterfowls and shorebirds due to their exposure to the contaminated water. lower temperature maintained in water bodies helps in survival of aiv for prolonged period. possibility of aiv transmission in waterfowl is more during their assembly in water bodies associated with post-breeding and pre-migratory molt. migratory shorebirds and other charadriiformes birds mostly transmit the infection when they congregate to feed and roost at places en route of migration. thus the migratory birds become a possible source of infection in pet birds living in open air aviaries. possibility of water mediated transmission is low in terrestrial and passerine birds. detection of aiv in passerines is possible, when the virus is maintained in local poultry population or the passerines share a common habitat with infected waterfowls. this kind of aiv transmission dynamics was observed during h5n1outbreaks (2005-10) in wild birds in china, russia and mongolia. consumption of infected bird carcass by raptors (bird of prey) is another possible way of transmission. in psittacine birds, aiv infection is transmitted by direct contact with infected birds if kept together after capture or during quarantine. international trade of exotic birds can transmit the aiv infection from one continent to another. further transmission of aiv from wild birds to local poultry population is possible. in hpai infection, most of the wild birds die except in h5n1-hpai (guangdong lineage) infection. the virus of guangdong lineage can persist in wild birds and is transmitted to the poultry. sometimes, lpai maintained in the wild birds is transmitted into poultry as observed during hpai outbreaks in poultry in united states. the source of the virus was confirmed as lpai from wild birds which undergo several mutations to generate hpai strain. pathogenesis of aiv in psittacine and passerine birds is still unexplored. experimental inoculation of hpai (h5n1) in finches, sparrows and budgerigars indicated the existence of variations in pathogenesis of aiv from the gallinaceous poultry. neurotropism of hpai in nongallinaceous birds is identified as the major cause of localization of virus is also detected in other tissues such as heart, pancreas, spleen, nasal epithelium, and reproductive organs of nongallinaceous birds. muti-organ failure or dysfunction is also identified as additional factor for mortality of the affected birds. non-specific clinical symptoms such as neurological signs (head between legs), depression, ruffled feathers, and standing at the bottom of the cage are observed in pet birds with aiv infection (fig. 2.13) . in a natural outbreak of lpai infection (h5n2) in a red-lored amazon parrot (amazona autumnalis autumnalis), lethargy, diarrhoea and dehydration are noted. sudden onset, depression, neurological symptoms are detected in finches and budgerigars experimentally inoculated with a chicken isolate of hpai (h5n1). in wild migratory passerine birds (blackcap, red-billed quelea) experimentally inoculated with hpai (h5n1), sudden death, ruffled feathers, lethargy, and neurological disorders (ataxia) are observed. in experimentally inoculated passerine and psittacine birds (zebra finches, house finches, budgerigars) with hpai (h5n1), carcass dehydration, spleenomegaly with mottling of parenchyma, accumulation of watery faeces in cloaca is observed. in house finches and budgerigars, vents are pasted with faeces and bile tinged urates. in wild migratory passerine birds (blackcap, red-billed quelea) experimentally inoculated with hpai (h5n1), lung congestion, pancreatic necrosis with multiple, white foci on pancreas are major gross lesions. cloacal swabs from live birds and tissue specimens from heart, pancreas, spleen, and brain after post-mortem can be collected as clinical specimens. the samples should be transported in isotonic phosphate-buffered-saline (pbs, ph 7.0-7.4) with antibiotics such as penicillin (2000 units/ml), streptomycin (2 mg/ml), gentamycin (50 lg/ml), mycostatin (1000 units/ml) and protein (5% cattle serum, 0.5% bovine albumen). the specimens can be preserved at 4°c for 4 days and at −80°c for extended period. the laboratory should have at least biocontainment level 3 facilities and official clearance from the concerned authority to handle the aiv suspected samples. in future, rapid isothermal nucleic acid detection assay-lateral flow (rida-lf) and immunoassay-based biosensors will be a better choice due to less dependence on instrumentation and rapid process of a good numbers of samples in less time, respectively. world health organization (who) has reported more than 600 human infections with hpai (h5n1) since 2003, of which 60% infected people died. transmission of h5n1 infection in human occurred during close contact with birds or contaminated environments. keeping pet birds in household also increased the seroconversion of the owners during a h7n7 outbreak. religious ceremonies, such as 'merit release' among buddhists, in which a passerine bird is purchased, kissed and released, may increase the transmission possibility of aiv among the human. no report of human to human transmission of aiv is reported so far. no effective treatment for pet birds against aiv infection is documented. to control the infection in pet birds, exposure to live bird market, wild birds, and migratory birds should be restricted. infected birds should be kept separately and general hygiene practices should be followed to avoid the contamination of feed and drinking water. the cages or aviaries should be cleaned with formaldehyde, gluteraldehyde, beta-propiolactone, binary ethylenimine, quaternary ammonium disinfectants, sodium hypochlorite, dilute acids, and hydroxylamine after an aiv outbreak. vaccination against aiv in birds is a controversial issue. due to high mutating capability of the virus, instead of control, vaccination with live virus may cause more damage to the birds. experimentally, inactivated recombinant h5n3 vaccine was used in several types of zoo birds (anseriformes, charadriiformes, ciconiiformes, columbiformes, coraciiformes, falconiformes, galliformes, gruiformes, passeriformes, psittaciformes) which produced strong antibody titer against h5 subtype in all the birds except in psittaciformes. prime-boost strategy of vaccination (priming with h5n9 and booster with h5n3) produced strong antibody titer in psittaciformes. west nile virus (wnv) was first isolated from a woman suffering with fever and other complications in uganda (1937) . in africa, middle east and european countries, wnv was mostly known to cause sub-clinical and self-limiting infections in horses and human during 1960s. later in 1990s, higher frequency of wnv infection was noticed among human, farm animals, pet animals and birds of prey. in 1999, a fatal outbreak of wnv was detected among birds, horses and human in new york, usa. during 2008-10, wnv was isolated from a clinically infected sun conure (aratinga solstitialis) and green-winged macaw (ara chloropterus) in united states. west nile virus is an arthropod-borne (arbovirus), enveloped virus which belongs to family flaviviridae and genus flavivirus. the virion is 50 nm in diameter with icosahedral symmetry. no spikes or peplomers are present on the virion surface. genome of the virus is positive sense single-stranded rna. the genome (11 kbp) encodes envelope protein (e), membrane precursor protein (prm), capsid protein (c) and non-structural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, ns5). the structural proteins help in formation of virion and the non-structural proteins help in viral replication and evasion of host immune response. seven lineages of wnv have identified and lineages 1 (clade ia and ib) and 2 are considered as major lineages. lineage 1is mostly distributed in europe (clade ia), america (clade ia), the middle east (clade ia), india, africa (clade ia) and australia (clade ib, kunjin virus). lineage 2 is widespread in south africa, madagascar and europe. both the lineages of wnv have neurotropism property, although, viruses belong to lineage 1 (clade ia) are more virulent than the clade ib and lineage 2 viruses. wnv is identified in 326 species of birds with or without clinical symptoms. the most susceptible birds to wnv infection are crows (corvus spp.), ravens (corvus corax), jays (garrulus spp.), magpies (pica spp.), owls (strigiformes spp.), and some raptors (spanish imperial eagle, goshawk, golden eagle, sparrow hawk, gyrfalcon). the passerine birds and the mosquitoes (culex spp.) are considered as major host and vector of wnv, respectively. crows are more exposed to wnv infection due to their communal roosting (perching) behaviour. after sunset, during the communal roosting, the mosquitoes (culex spp.) mostly feed on the birds. migratory passerines (american robins) can transmit the infection in distant places, whereas, resident passerines (house sparrows), crows [american crows (corvus brachyrhynchis)] and other birds act as local amplifying host of wnv. wnv is detected from resident birds such as columbiformes (columbina talpacoti), coraciiformes (melanerpes aurifrons), piciformes (cardinalis cardinalis, molothrus aeneus), and passeriformes (myiozetetes similis, sporophila torqueola, thamnophilus doliatus, tiaris olivaceus, tyrannus melancholicus). seroprevalence of wnv is observed among passerines such as northern cardinals (cardinalis cardinalis) and carolina wren (thryothorus ludovicianus). studies indicated that presence of non-passerine birds (enormous numbers) in a population can reduce the virus amplification and human transmission risk due to diversity of hosts (dilution effect). among the psittacine birds, seroprevalence of wnv is detected in budgerigars (melopsittacus undulatus), cockatiels (nymphicus hollandicus), cockatoos (cacatua spp.), macaws (ara spp.), parrots (amazona, rhynchopsitta, poichephalus, psittacus spp.), pacific parrotlets (forpus coelestis), canary-winged parakeet (brotogeris versicolurus), rosellas (platycercus spp.), lories and lorikeets (eos, lorius, pseudeos, trichoglossus spp.) and blue-crowned conure (thectocercus acuticaudata). red-legged partridges (alectoris rufa) are resistant to natural wnv infection, although they can be experimentally infected with the virus. other than birds, human, horses, sheep, alpacas, dogs, cats, white-tailed deer, reindeer, squirrels, chipmunks, bats, and alligators are susceptible to wnv. human and horses are dead-end-hosts of the virus as sufficient amount of virions are not maintained in the blood to infect the mosquitoes feeding on the hosts. maintenance of wnv throughout the world takes place by an enzootic cycle ('rural cycle'). the susceptible birds and mosquitoes (culex spp.) are two major components of the cycle. the birds maintain the virus by acting as reservoir and the mosquitoes act as vector and spread the virus into new hosts. microfilarial infection of mosquitoes can hasten viral replication and rapid transmission of the virus (microfilarial enhancement of arboviral transmission). when the mosquitoes introduce the virus into the human habitats, the 'urban cycle' begins. in endemic zones, urban cycle begins with mortality of wild birds (summer to autumn) and the cycle ends with human and horse infection (dead-end-hosts). in winter months, when the adult mosquitoes are mostly inactive, vertical transmission of the virus takes place to sustain in the vector population ('overwintering strategy'). sometimes, wnv is re-introduced into the vector population through migratory birds and rarely by human transport (mosquitoes on aeroplanes). other than mosquito bites, wnv is rarely transmitted by oral route (ingestion of infected prey, drinking water) and direct contact in birds, cats and other vertebrates. in psittacine birds, feathers are identified as an important source of wnv. association of testes in psittacine birds suggests the possibility of sexual transmission. following the wnv transmission through the mosquitoes in human and rodents, the virus primarily enters dendritic cells (langerhans cells) via receptor mediated endocytosis. the cell surface proteins (dc-sign, integrin) act as wnv receptors. the dendritic cells carry the virus into draining lymph nodes where viral multiplication occurs. following genomic replication and translation, the progeny virions are matured through er-golgi secreation pathway and are released by exocytosis into the blood circulation. transient viraemia develops and different vital organs such as liver, spleen, kidney are infected. neuroinvasion can take place through direct infection with or without breakdown of blood-brain barrier or virus transport along peripheral neurons. certain host proteins such as drak2 (death-associated protein-kinaserelated 2), icam-1 (intercellular adhesion molecule), mip (macrophage migration inhibitory factor) and mmp-9 (matrix metaloproeinase 9) help in altering blood-brain barrier permeability. sometimes, host innate immune response (tlr3) mediated up regulation of tumor necrosis factor alpha (tnfa) causes capillary leakage and increased permeability of blood-brain barrier. in contrast, in birds, viraemia develops within 30-45 min of mosquito bite without any local virus multiplication in the lymph nodes. positive correlation of peak viraemia and bird mortality is observed. in birds, wnv prefers to replicate primarily in spleen and mononuclear phagocytic cells and are disseminated into vital organs (liver, kidney, heart). different lineages (1 and 2) of wnv have different tissue tropism in avian hosts. lineage 1 virions prefer to infect liver and myocardium, whereas, lineage 2 of wnv mostly infect spleen, kidney and liver in goshawks. depending upon the viral load in blood wnv may infect central nervous system in birds. exact mechanism of neuroinvasion in birds is unexplored. role of endothelial cells and immune cells in neuroinvasion is predicted. death of the birds occurs due to wnv associated lesions and secondary infections with bacteria, fungi and parasites. pathogenicity of wnv infections in birds is influenced by route of viral transmission, host defense, age and species of birds. the virus can persist in different organs of birds such as spleen, kidney, eye, brain and skin. detection of wnv in a hawk (birds of prey) during winter months, when mostly the mosquitoes are inactive and unable to transmit the infection, revealed the possibility of persistent viral infection. experimentally, persistent wnv infection is produced in ducks, pigeons and immunocompromised mice. effect of persistent wnv infection in health status of the birds is indistinct. low level of wnv infection is detected in carcasses of rock pigeons (columba livia) and mourning doves (zenaida macroura), although, wnv is not confirmed as a cause of death. whereas, in a kea (nestor notabilis gould), a large mountain parrot, natural persistence of wnv in central nervous system for more than 6 years is detected. this persistence is associated with death of the bird after prolonged incubation period. non-specific clinical signs such as depression, anorexia, dehydration and ruffled feathers are observed in birds. in complicated cases, neurological signs, for instance, convulsions, ataxia, abnormal head postures and movements, tremors, paresis, and uncoordinated flight are detected. the neurological signs do not always correlate with the lesions in the brain (neuronal necrosis). partial or complete blindness develops in raptors and owls. sequelaes of viral neuroinvasion are observed in long-lived birds (e.g. raptors). in raptors, feather pulp abnormalities and abnormal molt can persist up to 4 years as sequelae of wnv infection. in naturally infected psittacine birds (rosellas, conures, lorikeets, cockatoos, caiques, parakeets), sudden death without any symptoms or non-specific signs like loss of weight, anorexia, lethargy, depression, and weakness are noted. specific neurological signs consisted of rolling over, legs stretched backward, stumbling and disorientation. no pathognomonic lesion of wnv infection is detected in birds. in highly susceptible birds (crows) sudden death without any gross lesion is observed. in passerines, necrosis and mild inflammation in the heart, spleen, liver, kidney, mild encephalitic lesions and absence of neuronal necrosis is detected. in naturally infected psittacines (rosellas, conures, lorikeets, cockatoos, caiques, parakeets), splenomegaly, hepatomegaly, mottled pale liver with multifocal petechiae, diffuse pallor in kidneys, myocardial pallor, petechiae on the gizzard serosa are observed. in long-lived birds (raptors) due to chronic wnv infection, hemorrhages, petechiae and congestion in vital organs, splenomegaly, hepatomegaly, myocardial pallor, pale mottling in the liver, spleen, kidney, cerebral atrophy and malacia are detected. in central nervous system, gliosis, perivascular cuffing and glial nodules are major microscopic findings. from the dead birds after post mortem, brain, heart, liver and kidney can be collected as clinical specimens for laboratory confirmation. the laboratories should have containment level 3 to handle the samples suspected for wnv infection diagnostic techniques real-time rt-pcr can be used for detection of wnv from avian tissues. sporadic outbreaks of wnv occurred in human in the mediterranean region, africa and europe before 1994. severe wnv outbreaks with neuroinvasion took place in human throughout the world after 1994. global warming associated increased temperature and prolonged rainfall helps in breeding of mosquitoes and spreading of arboviral diseases such as wnv. in human, wnv transmission can occur through biting of infected mosquitoes, blood transfusion, organ transplantation, breast milk, and intrauterine route. the clinical presentation ranges from asymptomatic (80% of infections) to encephalitis/paralysis and death (1% of infections). sometimes, flu like symptoms such as fever, headache, malaise, myalgia, fatigue, skin rash, lymphadenopathy, vomiting, and diarrhea are observed. no effective treatment for pet birds against wnv infection is documented. to control the infection in pet birds, exposure to mosquitoes should be restricted. maternal antibodies can protect the house sparrow chicks up to 3 days post hatch. no specific vaccine against wnv is available to use in birds. in some countries, equine vaccine is used, although not licensed. successful use of recombinant subunit wnv vaccine is reported from experimental geese. experimental vaccination in thick-billed parrots (rhynchopsitta pachyrhyncha) produced detectable antibody titer against wnv. usutu virus (usuv) was first detected in a mosquito (culex neavei) in 1959 in south africa. the isolated virus is currently considered as a reference strain of usuv (southafrica-1959) . subsequently, the virus was detected in different bird and mosquito species in africa. in recent decade, usuv was identified in passerine birds in austria (2001) usutu virus belongs to the genus flavivirus (japanese encephalitis serocomplex) under the family flaviviridae. different species of flavivirus such as usuv, west nile virus (wnv), japanese encephalitis virus (jev), murray valley encephalitis virus (mvev) and saint louis encephalitis virus are originated from same ancestral virus. nucleotide and amino acid sequencing revealed that mvev, among japanese encephalitis serocomplex, are the closest relative of usuv. usuv is a small (40-60 nm), spherical, enveloped virus with positive sense single stranded rna genome (11 kbp). the genome has a cap at 5′ end but no poly-a-tail at 3′ end. the genome can encode three structural proteins (core protein, pre-membrane and envelope protein) and eight non-structural (ns1, ns2a, ns2b, ns3, ns4a, 2k, ns4b, ns5) proteins. eurasian blackbirds (turdus merula) mostly suffer from usuv infection. several other families of birds (accipitriformes, anseriformes, caprimulgiformes, charadriiformes, ciconiiformes, columbiformes, coraciiformes, galliformes, passeriformes, piciformes, strigiformes) of different european countries are also susceptible to usuv infection (table 2 .7). migratory birds such as whitethroat (sylvia communis), lesser whitethroat (sylvia curruca), garden warbler (sylvia usuv is maintained in nature by a mosquito-bird cycle in which mosquitoes act as vector and the birds act as amplifying host. the mosquitoes occasionally spread the virus into other hosts (incidental) such as human, horses and rodents. usuv is detected in several mosquitoes such as culex pipiens, culex neavei, culex perexiguus, aedes albopictus, aedes caspius, anopheles maculipennis, culex perfuscus, coquillettidia aurites, mansonia africana. among them, c. pipiens and c. neavei are considered as competent vectors for usuv. although the migratory birds are sometimes infected with usuv the evidence of their role in transmission of infection is still missing. trematode infestation in blackbirds is detected as a predisposing factor for usuv infection. after introduction of usuv into the body of the host, viraemia lasts for a short period (2 days). the tissue tropism of the virus is almost similar to wnv infection. the virus is detected in brain, heart, liver, kidney, lungs, and intestinal tissues of laboratory mice and naturally infected birds. demyelination of neurons and formation of autophagosome are unique features of usuv infection. the process of autophagy helps in incorporation of host cellular components in viral replication. usuv infection in birds produces non specific clinical symptoms such as apathy, depression, anorexia, dehydration, ruffled feathers and moulting. in complicated cases, neurological signs, for instance, convulsions, ataxia, abnormal head postures and movements, tremors, paresis, torticollis and nystagmus are detected. neurological symptoms are often followed by death. the disease in birds is characterized by encephalitis, myocardial degeneration, and necrosis in liver and spleen. degeneration of purkinje cells, accumulation of glial nodules surrounding the degenerated purkinje cells ('glial shrubbery'), perivascular cuffing are characteristic findings in brain of affected birds. hepatomegaly, enlargement and discoloration of the kidneys, necrosis on the sheathed arteries of spleen are detected in great grey owls and boreal owls. in blackbirds, affected liver and spleen contains myriads of small (up to 1 mm) yellowish foci. enlarged gallbladder and intestine, hyperaemic meninges and brain are also found in blackbirds. clinical specimens blood or serum (paired sera collected in two weeks interval) from live bird and liver, spleen, lung, kidney, gizzard, and intestines are collected and fixed in 10% buffered formalin as post mortem specimens. igg-capture elisa is developed for human use. however, the serological tests often produce cross reactivity with other flavivirus infections such as wnv. prnt is more specific than other serological tests but requires specialized laboratory which can handle the virus. detection of acute infection is not possible by serological tests as the birds die before development of antibody titer. (f) molecular biology: reverse transcriptase-pcr can specifically detect usuv in tissues of suspected birds. recently real-time pcr is also developed for detection of usuv in human blood and cerebrospinal fluid samples which can be adapted in avian diagnostics. in 1981, in central african republic, a man with fever and rashes was diagnosed as a first human patient of usuv infection (car-1981) . in italy (2009), usuv infection was detected in two different patients of meningoencephalitis and orthotropic liver transplantation. both the patients were immunosuppressed and received blood transfusion before the infection. the common clinical symptoms were persistent fever, headache and neurological disorders. in 2012-13, a sero-surveillance program in germany and croatia detected low prevalence of usuv antibodies among the human population. no effective treatment for pet birds against usuv infection is documented. to control the infection in pet birds, exposure to mosquitoes should be restricted. water should not be stagnant in the vicinity of the aviaries or bird owner's houses. culex pipiens, the potent vector of usuv do not prefer to fly a long distance and lack of breeding site will significantly reduce their numbers. use of mosquito net (window and door) and repellants help to reduce the mosquito population. n, n-diethyl-meta-toluamide (ddet) is most effective repellant against culex pipiens. no specific vaccine against usuv is available to use in birds. vaccines against flavivirus (japanese encephalitis, yellow fever) are available for human use but no cross protection against usuv infection is reported. borna disease was first detected among animals (horse, sheep) in southeast germany during 19th century. it was named after the german district of borna around the town of borna in saxony where the infection remained endemic for prolonged period. the etiological correlation of borna disease with a virus was established in 1920. proventricular dilatation disease (pdd) was first reported from macaws and conures in usa during 1977. at that time, pdd was known as 'macaw wasting or fading syndrome' and 'gastric distension of macaws' as mostly macaws were associated with the syndrome. actual etiology of pdd remained uncertain for a considerable period. two independent research groups from israel and usa (honkavuori et al. 2008; kistler et al. 2008 ) identified a novel genus of the family bornaviridae, provisionally named as avian bornavirus (abv) as etiological agent of pdd. the virus was identified by molecular techniques such as panviral dna microarray and high throughput sequencing. avian bornavirus (abv) belongs to the family bornaviridae and order mononegavirales. the virion is enveloped, spherical, and 80-100 nm in diameter. the virions replicate in the nucleus of the host cells and use the host cellular splicing machineries for generation of mrnas. the genome is non-segmented, single stranded rna which encodes six major viral proteins such as nucleoprotein (n), regulatory protein (x), phosphoprotein (p), matrix protein (m), membrane-bound glycoprotein (g), and rna-dependent rna polymerase (l). after the discovery of avian bornavirus in psittacine birds, several types of bornavirus were detected in both psittacine and non-psittacine birds. recently it is proposed that the genus should include five species such as mammalian 1 bornavirus, psittaciform 1 bornavirus (avian/psittacine bornaviruses 1, 2, 3, 4, 7), passeriform 1 bornavirus (canary bornaviruses c1, c2, c3, ls), passeriform 2 bornavirus (estrildid finch bornavirus ef) and waterbird 1 bornavirus (avian bornavirus 062cg). till date, 14 abv genotypes have been detected in psittacine (abv-1, 2, 3, 4, 5, 6, 7) and non psittacine birds (abv-c1, abv-c2, abv-c3 in canaries; abv-cg in canada geese; abv-ef in estrildid finch; abv-bf in bengalese finch). avian borna viruses are detected in over 80 species of birds of which more than 70 species belongs to psittaciformes. the members of psittaciformes commonly infected with abv include cacatuidae (cockatoos, cockatiels) and psittacidae (lovebirds, macaws, parakeets, parrots, amazon parrots, conures) ( table 2 .8). among non-psittacine birds, canary, long-wattled umbrella bird, weaver finch, red-tailed hawk, falcon, canada geese, swan, duck, bald eagle are found naturally infected with abv (table 2 .8). pdd is reported from united states, australia, middle east, south america, south africa and japan. the studies suggest about faecal-oral or faecal-oronasal transmission of abv between the captive birds. in a bird with pdd, abv infected cells are detected in the intestinal villi from where they are excreated through the faeces. sometimes abv infected birds remain healthy and act as source of infection for other birds kept in the same aviary. it is observed that birds with high serum antibody titer against abv or viral rna load are prone to become clinically infected with pdd. overcrowding in aviaries, hand-feeding of parrot chicks are detected as predisposing factors for pdd. no gender based predisposition of pdd is observed. transmission route of abv in wild birds is still unexplored. irrespective of transmission route, classical borna disease virus enters central nervous system. in experimentally infected rats, centrifugal spread of virus into peripheral nerves and autonomic nerve fibers and ganglia are detected. in avian borna virus infection in birds, autonomous nervous system of the upper and middle digestive tract, including the esophagus, crop, proventriculus, ventriculus, and duodenum is chiefly infected. further spread of abv in extraneural tissues such as smooth or heart muscle fibers, liver, kidney, spleen, pancreas, lung, gonads, thyroid, and skin is observed. in mammalian borna virus infection, extraneural spread in hepatocytes, kidney epithelial cells, and myocytes of the intestine and heart is associated with immunosuppression of the host. the incubation period of abv infection in birds is highly variable (10 days-years). clinically the birds show gastrointestinal dysfunction or neurological signs or both. the symptoms of gastrointestinal dysfunction are impaction of proventriculus, dysphagia, polyuria, regurgitation, diarrhoea, presence of undigested food (seeds) in faeces, and crop stasis which leads to starvation and death. death due to circulatory collapse or food aspiration is also found. neurological signs are ataxia, seizure, blindness, tremor, abnormal gait, reduced proprioceptive skills, motor deficit and peripheral neuritis in sciatic, brachial and vagal nerves. no gross lesion is observed in sudden death of birds due to pdd. in majority of the birds suffering with pdd (70%), proventriculus is thin walled and distended with seeds. rupture of proventriculus wall releases the food particles and causes peritonitis. sometimes, enlargement of duodenum and adrenal glands, pale area on the epicardium is observed. microscopic lesions consist of lymphocytic infiltration along with plasma cells in the ganglia and nerve plexus (specially myenteric plexus supplying the digestive tract) of proventriculus, intestine, crop, esophagus, adrenal gland, conduction fibers of heart, central nervous system and spinal cord. perivascular cuffing by lymphocytes are detected in cerebral cortex, cerebellum, spinal cord and in peripheral nerves such as sciatic, brachial and vagus nerves. for intra vitam (ante-mortem) diagnosis of abv infection, faeces, blood, swabs of crop and cloaca, tissue biopsies from crops can be collected. left lateral sac of the crop (cranial portion) is preferred site for biopsy collection. the biopsy should be elliptical and it should contain a blood vessel so that the nerve sections can be visualized. after post-mortem, brain, crop, intestine and adrenal glands are collected. (a) clinical signs, haematology: clinical signs are mostly associated with gastrointestinal upset and/or neurological signs. non-regenerative anemia, leukocytosis, heterophilia, decreased total protein and albumin, increased level of muscle enzymes such as lactate dehydrogenase, creatine kinase, aspartate amino-transferase are detected in pdd infected birds. (b) radiography: distended proventriculus, ventriculus, crop and small intestine with ingesta and gas and prolonged gastrointestinal transit time are observed in infected birds by contrast radiography, contrast fluoroscopy and ultrasonography. spontaneous ruptures of the dilated proventriculus are rarely observed. although these findings are not specific for pdd. in healthy neonatal birds, distension of proventriculus and crop is also found. contrast radiography is performed in birds by introducing barium sulfate or iodine-based contrast media (@ 10-15 ml/kg) into the crop by gavage. barium sulfate produces better contrast but causes airway irritation. in psittacine birds, normal gastrointestinal transit time is 90 min-3 h. m and n genes of abv is developed. quantitative real time-pcr for detection of p gene is recently developed. brain, crop, intestine and adrenal glands collected after post-mortem and crop tissue, blood, cloacal swabs, and faeces can be used for abv-rna extraction. however, both false-positive (from asymptomatic bird) and false-negative results can be obtained by rt-pcr. zoonotic potentiality of abv is not established. pdd is a highly contagious infection and it spreads rapidly from one bird to another within a flock. decision to offer long term treatment or euthanasia of the affected bird is crucial. euthanasia is the best policy for management of pdd, although not preferred by most of the owners. management of inflammation, indigestion and secondary bacterial infections are currently considered as line of treatment for pdd. use of nonsteroidal anti-inflammatory drugs (nsaids, e.g. celicoxib, 20 mg/kg body weight, orally) along with antivirals (amantadine hydrochloride, 10 mg/kg po or 20 mg/kg with food) is recommended to treat pdd in birds. use of surfactants (for reduction of gas production), metoclopramide (0.5 mg/kg body weight, intramuscularly) and b complex vitamins are suitable supportive therapy. diet of the pdd infected birds should be easily digestible (preferably formulated diets), and in liquid or pelleted forms because the proventriculus and ventriculus function is adversely affected in pdd. addition of vegetables in the diet will increase intestinal motility. toys and cage accessories should be provided to the birds to avoid ingestion of foreign bodies. for prevention of pdd, new birds should be quarantined and checked for pdd before introduction into aviaries. maintenance of strict biosecurity and hygienic measures should be followed. overcrowding should be avoided in the aviaries. 2.2.6.1 history fist description of beak and feather disease (bfd) was observed in 1907 in an australian journal ('the emu') and the author described about wild red-rumped parrots (psephotus haematonotus) in the adelaide hills being unable to fly due to loss of feathers (ashby 1907) . in 1916, death of a captive sulphur-crested cockatoo (popular by its name 'cocky bennett') at the age of 120 years in sydney was published in local news paper. the bird was suspected for bfd due to loss of feather and presence of elongated beak. psittacine beak and feather disease (pbfd) was first scientifically documented in 1975 in sulfur-crested cockatoos, lovebirds, budgerigars and galahs in australia (pass and perry 1984) . beak and feather disease is caused by beak and feather disease virus (bfdv) which belonged to the genus circovirus and family circoviridae. circoviruses are icosahedral, non-enveloped and the smallest known autonomously replicating animal virus, measuring 15-26 nm in diameter. the viruses have an ambisense, circular, single-stranded dna genome (2000 nt) which can encode a replicase enzyme and capsid protein. the virus possesses highest mutation rate and genetic diversity although it is antigenically conserved. no serotype variation of circovirus is detected. the virus is considered as a model to study host parasite interaction due to its simple genome structure. it is also the representative of ancient viral form and circovirus sequences are detected in fossils of vertebrates, invertebrates, protozoa, plant, fungi, algae and bacteria. bfd virus mostly infects psittacine birds (more than 60 species) besides other bird families such as passeriformes, columbiformes and anseriformes (table 2 .9). a few susceptible bird species are enlisted as endangered or threatened by international union for conservation of nature (http://www.iucnredlist.org). bfdv infection is considered as a significant conservation threat. the infection is more fatal in young birds (0-3 years) due to poor development of immune system and the viral load is more prevalent in parental bird species than the hybrids. cockatoos mostly show chronic viral infection with excreation of virus through the faeces and dystrophic feathers. sometimes, cockatoos do not show any visible symptoms. occasionally, birds other than the common susceptible species are also infected with bfdv ('host switch over'). recently, bfdv infection is detected in rainbow bee-eaters (merops ornatus), a species of coraciiformes, unrelated to psittacine birds. mostly the bfd infection is detected in australia, new zealand, europe (poland, uk, denmark, portugal, germany, italy), united states, africa (zambia, zimbabwe) and asia (japan, china, taiwan, thailand). circumstantial evidence indicated that the infection was originated in australia. the virus was disseminated from australia in early 1970s to european countries with the imported parrots. from europe, the virus is further distributed to africa, new zealand, japan and united states during unregulated parrot trafficking. transmission of bfdv can take place by both horizontal and vertical means. horizontal route via direct contact with infected birds is the major mode of transmission in both wild and captive birds. the virus is excreated in high titers in the environment through feather dust, crop secreations, and faeces of infected birds. in aviaries, the virus once spread is difficult to control due to its high infectious and persistence nature. in the fomites, bfdv can persist for several years and after a long period, the fomites may act as a source of ancestral viral genotype in the host population. in forests, 'host switch over' is facilitated by horizontal transmission. the switch over mostly takes place in the unoccupied nests in the trees where competition exists between psittaciformes and other birds for reproductive opportunities. bfdv depends on host cell machineries for their replication and prefers the actively dividing cells such as basal follicular epithelium, lymphoid tissues, and intestinal epithelium. the virus causes necrosis of basal epithelium in the birds. the necrosis is found to be responsible for feather dystrophy and beak and claw deformities. bfdv also causes lymphoid depletion and associated immunosuppression. secondary infection with bacteria, fungi, parasite takes place due to immunosuppression and it is responsible for 70% bird mortality as observed in ducks, pigeons, geese, black-backed gull and other avian species. more experiments are needed to explore the pathogenesis of bfdv in pet birds. bfdv infection has three clinical forms in birds such as per acute, acute and chronic. sudden death without any symptom or mild symptom (feather dystrophy) occurs in per acute and acute forms, respectively. these forms are common in juvenile birds. in chronic form of the infection, weight loss, lethargy, anaemia, diarrhoea, shedding of developing feathers, abnormal development of new feathers is observed (figs. 2. 14, 2.15 and 2.16). mostly, contour, tail and down feathers are lost symmetrically and they are replaced with dystrophic feathers that fail to grow. deformities of beak and claws are not a constant feature. it depends on species of the bird and other predisposing factors. cockatoos are more susceptible to beak and claw deformities than other psittacine birds. beak elongation, transverse or longitudinal fractures, palatine necrosis are possible beak deformities observed (fig. 2.17) . chronic infection is not always fatal, the birds may survive for several years. sometimes death occurs due to secondary infection. gross lesions in feathers of bfdv infected birds are retention of sheaths, fracture of the proximal rachis, haemorrhage in pulp cavity, short clubbed feathers, curled feathers and circumferential constrictions. in naturally infected cockatoos, vane of feather is ragged with multiple fractures. hooklets, barbules and barbs are poorly developed and fractured ( fig. 2.18 ). hyperkeratotic sheaths are found in affected feathers which results terminal clubbing and mid-shaft constriction. in beaks of infected birds, abnormal elongation, palantine necrosis, transverse to longitudinal fractures is detected. histopathological examinations revealed basophilic intranuclear and/or intracytoplasmic inclusion bodies in feather epithelial cells, follicular epidermal cells and macrophages. clinical specimens feathers (newly grown quill portion is the best specimen), blood or serum, cloacal swabs, pharyngeal swabs can be collected as clinical specimens from the suspected birds. although, feathers produce low amount of viral dna because most of the fully grown feathers are separated from the blood supply. the blood should be collected from vein (not toenail) to avoid environmental contamination of bfdv. zoonotic potentiality of bfdv is not established. currently there is no known treatment for bfdv infection. secondary infection should be diagnosed and treated properly. avian gamma interferon injection (intramuscular) along with quaternary ammonium compound (as nebulizer) has shown success in treatment of bfd. no vaccine is commercially available to control bfdv infection. studies revealed that maternal antibodies against bfdv can protect the young birds. surviving birds sometimes develop long lasting immunity. the only way to control the disease is through maintenance of hygiene, strict isolation or culling of infected birds. pacheco and bier (1930) , a veterinarian from brazil first described an outbreak of acute, fatal hepatitis in psittacine birds. this syndrome became known as 'pacheco's disease'. later in 1975, psittacid herpesvirus type 1 (pshv-1) was confirmed as etiological agent. psittacid herpesvirus type 1 (pshv-1) is closely related with gallid herpesvirus-1 (infectious laryngotracheitis of chicken). pshv-1 has been classified into 4 genotypes (1-4) on the basis of variations in ul16 gene sequence. the genotypes of the virus have preference for different hosts (table 2 .10). pacheco's disease. the infection is common in united states, united kingdom, spain, south africa, kenya and japan. pshv-1 is transmitted by direct contact with the infected birds. persistently infected birds can shed the virus through faeces and pharyngeal secreation. in most of the cases, the infected birds die suddenly without showing any syndrome. if the birds survive, non-specific clinical signs such as depression, anorexia, diarrhoea, tremor and instability are observed. neurological disorder is followed by death. gross lesions are not distinct. intranuclear inclusion bodies (cowdry type a) are observed in liver, kidney, spleen, pancreas and small intestine. cloacal swabs, pharyngeal swabs, faeces, newly emerged feathers (blood/pin feather) can be collected from live bird as clinical specimens. after post-mortem, liver, spleen, kidney, lung, cerebellum can be used for detection of the virus. laboratory confirmation of pshv-1 infection depends on isolation of virus in cell lines, demonstration of virus in clinical samples by electron microscopy, detection of viral dna by pcr or real-time pcr. presence of intranuclear inclusion body in tissues is inconclusive because many other viruses (avian polyoma, psittacine adenovirus) also produce the same. use of antiviral (acyclovir, oral or intramuscular, intravenous injection) in early stage of infection can prevent the outbreak. an inactivated virus vaccine adjuvinated with oil is available for selected psittacine birds against pacheco's disease. the vaccine is recommended to use subcutaneously in smaller psittacines and subcutaneously or intramuscularly in larger psittacines (more than 100 g body weight). maintenance of strict hygiene, quarantine (30 days) of newly procured birds, and regular use of disinfectants in cages can prevent the pshv-1 infection. psittacine adenovirus (psadv) was first reported from senegal parrots (poicephalus senegalus) with acute infection. the virus was confirmed by amplification of hexon gene (l1 variable loop) by pcr (raue et al. 2005) . psittacine adenovirus belongs to the family adenoviridae and genus aviadenovirus. adenovirus is non-enveloped and has an icosahedral capsid with a diameter of 70 nm. the hexon protein is the major capsid protein and it has conserved pedestal regions (p1, p2) and the variable loops (l1-l4). adenovirus infection is reported from pet birds such as budgerigars, macaws, amazon parrots and cockatoos. aviadenoviruses are present in faeces, urine, tracheal and nasal secreations of infected birds. the virus is readily transmitted by horizontal mode. direct faecal contact and aerial spread are major ways of horizontal transmission. fomites, personnels and transport also contribute in horizontal transmission of aviadenovirus. in most of the cases, the infected birds die suddenly without showing any syndrome. if the birds survive, non-specific clinical signs such as depression, anorexia, diarrhoea, ruffled feathers are observed. gross lesions include hepatomegaly, splenomegaly, nephromegaly, dilatation of duodenum and , and congestion of lungs. the livers become enlarged, friable, haemorrhagic, pale or mottled. basophilic intranuclear inclusion bodies are observed. faeces from live birds and organs (kidney, liver, intestine) after post mortem are collected from the suspected birds for confirmatory diagnosis. psadv can be isolated in primary chicken embryo kidney (cek) or chicken embryo liver (cel) cell lines. embryonated chicken eggs can be inoculated by yolk sac, chorioallantoic membrane and allantoic cavity route. the eggs should be free of pathogen (spf) and antibodies against aviadenovirus. replication of the virus can be confirmed by death of embryos and gross microscopic lesions observed in hepatocytes. psittacine embryonated eggs are very expensive and are not readily available for diagnostic purpose. other diagnostic methods for detection of psadv in clinical samples are electron microscopy due to typical morphology of the virus and demonstration of basophilic or eosinophilic, intranuclear inclusion bodies in liver and intestinal epithelium by haematoxylin and eosin staining. psadv in clinical samples can be confirmed by pcr. no specific treatment for psadv infection is documented. secondary bacterial infection can be treated by broad spectrum antibiotics. aviadenoviruses are extremely resistant to the environment (heat, ph 3-9) and common disinfectants (ether, chloroform, trypsin, 50% alcohol) and they can persist for prolonged period in the cages or aviaries. treatment with formalin, aldehydes and iodophors for more than 1 h can inactivate the virus. poxvirus infection was reported for first time from lovebirds (agapornis personata, a. roseicollis) in germany (kraft and teufel 1971) . psittacine poxvirus belongs to the genus avipoxvirus, family poxviridae and subfamily chordopoxvirinae. the viral genome consists of double stranded dna (230-300 kbp). several psittacine and passerine birds, specially amazon and pionus parrots, lovebirds and canaries are susceptible to psittacine poxvirus infection. transmission of poxvirus from infected owners or caretakers is possible into the birds having skin injuries. mechanical transmission is also possible by mosquitoes and mites. in a contaminated cage or aviary, infected aerosols generated from dried scabs and feathers may act as source of infection. the infection in psittacine birds is characterized by ocular discharge, rhinitis, conjunctivitis and ulcerations on the eyelid. typical crusty lesions develop on the eyelid margins, lateral and medial canthi of the eyes and occasionally on face and feet (figs. 2.19 and 2.20) . persistent cutaneous lesion for a period of 13 months is detected in yellow-shafted flicker. diptheretic lesions develop in some birds which is associated with dyspnea and higher mortality rate. diphtheritic form produces necrotic lesions in the trachea, larynx and oral cavity. gross lesions include necrosis of heart, liver, air sac, lungs, peritoneum, and accumulation of necrotic debris on the surface of the alimentary tract. intracytoplasmic inclusion bodies (bollinger bodies) are observed in the mucosa of sinus, trachea, crop, esophagus and throat. presence of typical cutaneous lesions primarily suggests about poxvirus infection. vesicular fluid from cutaneous lesions, faeces and pharyngeal swabs can be collected as clinical specimens. the infection is confirmed by electron microscopy, detection of bollinger body in tissue samples and psittacine poxvirus specific-pcr. no specific treatment and vaccine is available to control psittacine poxvirus infection. infected birds should be separated from the healthy group. cages, fomites and utensils should be properly disinfected because the virus persists for prolonged period in the dried scabs and the aerosols generated from the infected scabs. among the pet birds, polyomavirus was detected for first time in young budgerigars (melopsittacus undulatus) in 1980. the virus was named as budgerigar fledgling disease polyomavirus which was renamed later as avian polyomavirus. the virus belongs to the family papovaviridae. the virion is icosahedral, non-enveloped with a diameter of 45-50 nm. the viral genome is a circular double-stranded dna and has two regions-early and late. the early region encodes tumour protein and the late region encodes four structural proteins (vp1, vp2, vp3, vp4). other than budgerigars, lovebirds, canaries, finches, macaws, eclectus parrots, conures, cockatoos and indian ringneck parakeets are also susceptible to avian polyoma virus infection. the infection is common in canada, china, australia, germany, slovakia and italy. direct contact with infected birds is the major way of transmission. contaminated cages, fomites, utensils, nestboxes, egg incubator may also act as source of infection as the virus is highly stable in the environment. the infection is more fatal in young birds of less than 16 weeks age. in fledgling and young budgerigars, death without symptom, or brief illness showing feather dystrophy, loss of down feathers, presence of 'filoplumes' (feather like projection with a thin rachis and few barbs, figs. 2.21 and 2.22) on head and neck, abdominal distension followed by death, are observed. it is known as 'french molt' or 'budgerigar and fledgling disease'. in cockatoos, two clinical forms i.e. acute death with haemorrhages in the feather shaft and pneumonia with gasping (in young (table 2 .11). gross lesions include distension of heart with hydropericardium, swollen liver, congested kidneys, and hemorrhage in the body cavity ( fig. 2.23 ). intranuclear and basophilic inclusion bodies are detected in spleen, liver and kidney. blood, cloacal swabs from live animals and tissues from spleen, liver and kidney can be collected as clinical specimens. the presence of avian polyomavirus in the clinical samples is confirmed by electron microscopy, virus neutralization test, immunofluorescent antibody staining, in situ hybridization, pcr and real-time pcr. no specific treatment for avian polyomavirus is documented for pet birds. an inactivated and oil adjuvinated vaccine is commercially available. a dose of 0.25 ml is recommended for birds below 200 g of body weight and larger dose (0.5 ml) is given to the birds with more than 200 g of body weight. primary vaccination is done at 5 weeks of age which should be followed by a booster after 2-3 weeks. annual vaccination is recommended. sometimes thickened skin at the vaccination site is observed as adverse reaction. papovavirus infection was detected in budgerigars (melopsittacus undulatus) and splendid parakeets (neophema splendida) which died suddenly without distinct clinical symptoms (graham and calnek 1987; pass et al. 1987) . psittacine papillomavirus belongs to papovaviridae family. pet birds such as amazon parrots, african grey parrots, macaws, finches, budgerigars, canaries and parakeets are most susceptible to psittacine papillomavirus infection. direct contact with infected bird is the major way of transmission. introduction of infected bird into aviary or cage rapidly transmit papillomavirus infection into other birds. the infection is characterized by reddish cauliflower like growth (papilloma) in oral (larynx, crop and upper gastrointestinal tract) and cloacal mucosa. presence of a large mass in larynx causes wheezing and change of voice. papillomas in oral mucosa hinder swallowing and digestion which causes anorexia, chronic weight loss and vomition. in clocal papillomatosis, raised, coalescing mass appears at the cloaca ( fig. 2.24) . presence of fresh blood is noted in the droppings. sometimes cloacal prolapse is detected. gross lesion is characterized by proliferation of epithelial cells on thin fibrovascular stalks. neoplastic growth of bile duct, pancreas and liver is also observed specially in amazon parrots and macaws. presence of typical cauliflower like growth in cloaca or oral mucosa primarily suggests about psittacine papillomavirus infection. tissue biopsy samples from cloaca can be collected by a sterile speculum, moistened cotton-tipped applicator or a gloved hand (fig. 2.25 ). histological examination of biopsy samples, in situ hybridization and pcr can confirm the infection. treatment of papillomas includes chemical cauterization with silver nitrate or surgical removal of the mass. in chemical cauterization, possibility of re-appearance of the papillomas is detected. use of interferon (50,000 iu/kg, intramuscular) in some species of birds can prevent the recurrence of growth. in a few countries, autogenous vaccine is used to prevent the infection. among the members of family reoviridae, orbivirus mostly causes pet bird infection. parrots, budgerigar, cockatiel, duck and american woodcock are susceptible to orbivirus infection. pheasants, pigeons and raptors mostly act as carriers. the carrier birds can shed the virus through faeces and contaminate the environment that can act as a source of infection. biting insects sometimes also help in transmission of infection. in psittacine birds, conjunctivitis, swollen eyelids, enteritis, emaciation, incoordination and other neurological signs are observed. in budgerigars and cockatiels, sudden death without clinical symptom is detected. stunted growth and feather deformity is observed in muscovy ducks. gross lesions include swollen liver, kidney and spleen with necrotic areas. accumulation of fluid in lungs and pericardium and myocarditis are often detected as gross lesions. cloacal swabs and tissues from liver, spleen, and kidney can be collected as clinical specimens. the virus can be isolated in chick embryo liver cells or chick kidney cells. the virus is confirmed by electron microscopy, immunofluorescence staining and pcr. psittacine birds (budgerigar, amazon parrot), pigeons, ostrich, rhea are sometimes infected with infectious bronchitis virus of coronaviridae family. respiratory signs, mucopharyngitis, ulcerated crop and esophagus, swollen kidney, egg peritonitis are commonly observed. in ostriches, thin walled and blood filled proventriculus is detected. nasal swabs, pharyngeal swabs can be collected as clinical specimens in 50% glycerol. the virus can be isolated in chicken embryo liver cells and primary embryo liver cells derived from blue and yellow macaw embryos. presence of virus can be confirmed by electron microscopy and pan-coronavirus reverse transcriptase-pcr. 2.3.1 toxoplasmosis 2.3.1.1 history splendore (1913, brazil) first observed toxoplasma like organisms in blood smear prepared from an infected rabbit. in the same year, nicole and manceaux detected the same organisms at gondi (tunisia) and described them as toxoplasma gondii. in pet birds (pigeons), carini (1911) first observed toxoplasma like parasites in smears prepared from liver and spleen in são paulo, brazil. based on phenotypical detection in smears prepared from blood and tissues, toxoplasma was described subsequently in different species of birds. catar (1974) detected the organisms in mistle thrush (turdus viscivorus), song thrush (turdus philomelos), robin toxoplasma gondii is an obligate intracellular protozoa and a member of suborder eimeriina, phylum apicomplexa. feral and domestic cats (felidae family) are the definitive host of the parasite. it has a wide range of intermediate hosts including different species of birds. t. gondii has three conventional clonal lineages (type i, ii, iii) which has different host preference and virulence pattern. most of the animal strains belong to type iii, whereas, type i and ii strains commonly infect human. occasionally, all the typical genotypes (i, ii and iii) and their variants are isolated from birds such as free-range chickens, raptors etc. progress in this area identified several other genotypes ('atypical') of t. gondii such as type bri, brii, briii, briv, type 12, africa 1 and chinese 1. a classification scheme is adopted to designate each genotype as 'toxo db-pcr-rflp genotype' followed by a specific number (table 2 .12). a total of 189 toxodb pcr-rflp genotypes is identified so far (2012). clinical toxoplasmosis is detected in birds belonged to passeriformes, psittaciformes, columbiformes, strigiformes, galliformes and anseriformes orders (table 2 .13). among the pet birds, passerines (canaries, finches, mynahs), pigeons and partridges are commonly infected with toxoplasma. clinical toxoplasmosis in pet birds is reported from different countries of europe, north america, south america and oceania (table 2 .13). apparently healthy wild and domestic birds belonged to different families (accipitriformes, anseriformes, galliformes, gruiformes, charadriiformes, columbiformes, strigiformes, passeriformes) can also harbour t. gondii without showing clinical symptoms. examples of carrier birds include goshawk (accipiter gentilis), common buzzard (buteo buteo), kestrel (falco tinnunculus), pallid harrier (circus macrourus), black vulture (aegypius monachus), red-tailed hawk (buteo jamaicensis), pheasant (phasianus colchicus), turkey (meleagris gallopavo), mallard duck (anas platyrhynchos), pintail duck (anas acuta), coot (fulica atra), blackheaded gull (larus ridibundus), common tern (sterna hirundo), collared dove (streptopelia decaocto), woodpigeon (columba palumbus), common pigeon (columba livia), ferruginous pygmy owl (glaucidium brasilianum), little owl (athene noctua), chaffinch (fringilla coelebs), house sparrow (passer domesticus), tree sparrow (passer montanus), yellowhammer (emberiza citrinella), starling (sturnus vulgaris), black bird (turdus merula), mistle thrush (turdus viscivorus), song thrush (turdus philomelos), robin (erithacus rubecula), great tit (parus major), treecreeper (certhia familiaris), jackdaw (corvus monedula), rook (corvus frugilegus). toxoplasma gondii has three stages in life cycle-oocyst (with sporozoites), tissue cyst with bradyzoites and rapidly multiplying form or tachyzoites. oocysts are infective stage of the protozoa present in cat faeces (definitive host) . the infected cats can shed millions of oocysts within 3-10 days of infection, regardless of the presence of clinical signs. the oocysts are activated within 1-5 days after faecal excreation and can survive in soil and water for prolonged period (up to 1 year). the feral cats bury the faeces into soil but earthworms and other soil associated focal discoloration of liver and heart, edematous lungs anseriformes hawaiian goose nene goslings (nesochen sandicensis) edematous, consolidated lungs and necrosis in liver, brain, heart and muscles apterygiformes north island brown kiwi (apteryx mantelli) hepatospleenomegaly and swollen lungs insects bring them into the top layer of soil. the oocysts are ingested by intermediate hosts (birds, rodents, sheep, marine mammals and human) from the soil or water. the parasite invades tissues of intermediate hosts and produce bradyzoites within tissue cyst. after ingestion of oocysts by the intermediate hosts, sporozoites prefer to invade most of the vital organs, with predilection for the reticuloendothelial and central nervous systems. the parasite enters the host cell by an active process. after intracellular multiplication, the progeny parasites go into the blood circulation by lysis of the infected cells and finally reach vital organs through the blood circulation. onset of clinical symptoms depends on type of organs invaded by the parasite. the parasitemia (presence of parasite in blood) declines after development of host immunity. the parasites localize and persist in the form of 'tissue cysts'. most of these tissue cysts are benign in nature and do not produce any clinical symptom during persistence. in immunosuppression due to stress, concurrent viral infection or immunosuppressive therapy, the tissue cysts are ruptured. granulomatous lesions develop with invading inflammatory cells into the surrounding tissues of the site where the cyst persisted. in canaries infected with toxoplasmosis, weight loss, diarrhoea, dyspnoea, crusty exudates around eye lids, collapsed eyeballs, inflammation of choroid with or without retinal involvement, cataracts and blindness, head twitch, walking in circles (due to encephalitis) are commonly observed. dull, visionless, closed eyes and encephalitis are consistent features in passerine birds (fig. 2.26) . in psittacine birds, non-specific clinical symptoms are observed. in lories, respiratory distress is distinct. in pigeons infected with t. gondii, anorexia, emaciation, high fever, weakness, conjunctivitis and convulsions are detected. in passerines (canaries), gross lesions include osseous replacement of eye globe, non-suppurative inflammation of optic nerve, anterior and posterior uveitis, swelling of the lenticular fibers in the lens, choroiditis, focal necrosis and detachment of retina and atrophy of periocular tissue (sunken appearance of eye). t. gondii tachyzoites are detected in choroid, retina (nerve fiber layer), vitreous and lens of the affected birds. histological evidence of pneumonia and non-suppurative encephalitis associated with tissue cysts is also observed. gross lesions observed in affected psittacine and other birds are enlisted in table 2 .13. clinical specimens blood, serum, eye suspensions from live birds and organs (spleen, liver, intestine, eye, brain, gizzard, proventriculus) collected after post mortem in buffered neutral 10% formalin can be used as clinical specimens. for bioassay (isolation), collected tissues are homogenized in 0.85% normal saline solution and brain tissue homogenates are digested with acidic pepsin before bioassay. (a) direct examination: preliminary diagnosis is made by demonstration of t. gondii tachyzoites in giemsa stained impression smears prepared from collected organs. in smears, tachyzoites are crescentic to globular in shape ( fig. 2.27 ). (b) histological examination: in formalin fixed tissues, globular to oval shaped t. gondii tachyzoites are detected which are smaller in size than their appearance in impression smear (fig. 2.28) . t. gondii 'tissue cysts' appear as a globular structure with a thin cyst wall and small, slender bradyzoites are present within it (50-500). the bradyzoites can be visualized by periodic acid schiff (pas) staining. immunohistochemical staining with polyclonal antibody raised against whole parasite can confirm the presence of t. gondii in formalin fixed tissues. (c) serological tests: modified agglutination test (mat) is a sensitive, specific and easy to do serological test for detection of t. gondii antibodies in different species of birds. elisa, indirect fat in tissues can also be performed. (d) bioassay: t. gondii can be isolated in laboratory mice by subcutaneous inoculation of eye suspensions or tissue homogenates (liver, brain) collected from suspected birds. in positive cases, inoculated mice will die and t. gondii tachyzoites or tissue cysts are detected from dead mice. (e) molecular biology: the parasite can be confirmed by t. gondii-specific pcr. nested pcr analysis based on t. gondii pppk-dhps gene is recently developed for confirmation. genotyping of t. gondii depends on restriction fragment length polymorphism of surface antigen 2 gene (sag2). human get the infection by ingestion of vegetables, fruits and other foods or water contaminated with t. gondii oocysts. direct transmission of t. gondii from pet birds to human is not documented. feral or domestic cats may ingest the t. gondii infected carcasses of pet birds disposed into the surroundings without proper measures. the cats may become infected with toxoplasmosis and start to shed oocysts into the environment. person to person transmission is rarely possible during organ transplantation or blood transfusion. most of the human infection is aymptomatic but sometimes cervical or occipital lymphadenopathy is observed. in immunosuppressed persons, encephalitis, chorioretinitis and pneumonitis are detected. in pregnant women, abortion and fetal infection causing congenital hydrocephalus, intracranial calcifications and mental retardation is documented. 2.3.1.10 treatment and control strategy pyrimethamine (0.5 mg/kg body weight, orally, 12 h interval) is recommended for treatment of clinical toxoplasmosis in birds. use of tiamulin fumarate (300-400 mg/kg feed for 7 days in different species of birds; 225-250 mg/l drinking water for 3-7 days in pigeons and poultry) and clindamycin (25 mg/kg body weight, oral, 48 h interval) is also observed. in canaries, treatment with trimethoprim (80 mg/ml) and sulfadiazine (400 mg/ml) in drinking water for 14 days was apparently successful. prophylactic use of coccidiostats (monensin, decoquinate) is recommended in calves and lambs to prevent toxoplasmosis. low acute toxicity of decoquinate is observed in avian species and no regulation or evidence is available regarding its prophylactic use. no vaccine is also available for birds to prevent toxoplasmosis. antony van leeuwenhoek (delft, netherlands, 1681) first observed giardia under his self made simple microscope during investigation of his own diarrhoeic stool. he described it as 'animalcule' with 'flattish belly' and 'sundry little paws' (flagella). under microscope, the organisms showed a slow and helical motion with occasional rapid movement by 'paws'. later (dobell 1932) it was concluded as trophozoite stage of giardia spp. in 1859, vilem dusan lambl (prague, czech republic) described the organism in more details and named it as cercomonas intestinalis. in 1915, charles wardell stiles coined the name giardia lamblia in honour of professor alfred mathieu giard (zoologist, france) and dr. vilem dusan lambl (czech) for their contribution in progress of giardia associated knowledge. leibovitz (1962) first described giardia infection in a budgerigar in united states with a history of chronic diarrhoea and debility. in 1977, jones and carroll also observed presence of giardia in the intestine of budgerigars in united kingdom. in 1978, giardia infection causing high mortality in parakeets was reported from united states which was successfully treated by dimetridazole (panigrahy et al. 1978 ). avian giardiasis is caused by giardia spp., an eukaryotic, multicellular, binucleate, flagellated protozoan belonged to sarcomastigophora phylum. six valid species of giardia are recognized based on morphological observations using light and electron microscopy. the valid species are giardia psittaci and g. ardeae in birds; g. duodenalis (syn. g. lamblia, g. intestinalis) in human, livestock and wildlife; g. microti and g. muris in rodents; and g. agilis in amphibians. g. duodenalis has eight identified genotypes (assemblages, a-h). assemblage a and b are common in both human and animals and assemblage c-h are restricted within animals only. giardia spp. has two stages in life cycle-motile 'trophozoite' and 'cyst' (fig. 2.29 ). the cysts are smaller than trophozoites (10 lm â 8 lm), dormant, resistant to adverse environmental condition (like bacterial spore) and infectious form of the protozoa. they are common in streams, lakes and ponds. they can survive for months in cold water also (8°c). after transmission of the cysts into the host, the cysts pass through acidic ph, increased co 2 level and slight alkaline ph (proximal small intestine) consecutively and excystation takes place. one trophozoite from each cyst emerges which undergoes cytoplasmic division to produce two trophozoites. trophozoites are pear shaped (pyriform) structure which measures 12-18 lm in length, 10 lm in breadth and 2-4 lm in thickness (fig. 2.30) . the trophozoites have a concave disc with a raised ridge at the ventral surface of anterior site (broad portion) and eight flagella arranged bilaterally. the trophozoites attach with enterocytes at duodenum and jejunum with the ventral disc ('sucker') for feeding on mucosal secreations. the colonization is followed by binary fission. some trophozoites detach from the enterocytes and move forward (tumbling and skipping) with their flagella to re-attach with a new enterocyte. a few of the trophozoites instead of re-attachment prefer to be excreated through the faeces as cyst. during the process of encystment, the trophozoites stop their active motility, become rounded in shape and covered with a cyst wall. nuclear division takes place and a quadrinucleate, matured cyst is excreated into the environment. giardia is identified in faecal samples of more than 20 species of birds specially in psittacines. giardia psittaci associated clinical infection is fatal in young budgerigars. avian giardiasis is also reported in cockatiels, lovebirds, finches, great blue herons, raptors and gray-cheeked parakeets throughout the world. some psittacines such as blue-fronted amazon (amazona aestiva), blue and yellow macaw (ara ararauna), scarlet macaw (ara macao) act as carrier (transport host) of giardia duodenalis (assemblage a) without showing clinical symptoms. the infection is transmitted by faeco-oral route through ingestion of the food or water contaminated with infective cysts. feeding or watering trough, cage materials, toys or other inanimate objects contaminated or soiled with faeces of the infected birds (captive or wild) may serve as a source of infection. asymptomatic birds generally shed the cyst intermittently and thus serve as a potential source of infection. giardia psittaci mostly reside in the duodenum with manifestation of diarrhoea and malabsorption syndrome in birds. the trophozoites adhere to the intestinal villi with ventral suckers. the adherence results inflammatory cell infiltration, villous atrophy, reduced villous to crypt ratio and reduction in disaccharidase enzymes (e.g. lactase). food absorption is hampered and the food particles are accumulated in the lumen which increases the osmotic pressure and causes diarrhoea. with inhibition of food absorption, chronic weight loss may be noticed in affected birds. there may be deficiency of vitamins and minerals due to absorption failure. unlike mammals where immunity has a direct relation with occurrence of giardiasis, avian immunity rarely shows such association. some risk factors are also noticed in birds for giardiasis such as overcrowding, unhygienic cage condition and inadequate nutrition. the studies revealed that giardiasis may be more common among heavily inbred population of birds. mucoid and persistent diarrhoea with loose, brown or pale coloured and foul smelling faeces, anorexia, depression, hypoproteinemia, weight loss, ruffled feathers are common clinical symptoms in birds infected with giardia spp. stunted growth and high mortality are observed in young budgerigars and cockatiels. in cockatiels, feather picking and pruritus are also detected. feather picking from wings, flanks and legs along with screaming is common. feather damage is sometimes observed in non-cockatiel birds also. secondary bacterial, viral or yeast infection are identified in birds with avian giardiasis. concurrent infection of g. psittaci and polyomavirus, cryptosporidium, chlamydia and macrorhabdus spp. (megabacterium) are observed in budgerigars. no gross lesion or sometimes distended small intestine with mucous, yellowish or creamy fluids is detected in most species of the birds. atrophy of villous, infiltration of inflammatory cells, hyperemia of intestinal mucosal layer and presence of numerous trophozoites throughout the entire length of villi are observed. in cockatiels, thickened skin, haemorrhage and areas of feather loss are observed in patagium (membranous structure that helps in flight) and axillary area. it may progress to squamous cell carcinoma. clinical specimens fresh faeces collected in saline from the suspected birds, blood or serum, intestine (duodenum) in 10% formalin can be used as clinical specimen. (a) direct examination: wet mount examination of collected faecal sample (mixed with warm saline, not tap water) or the content of duodenum can be performed for direct visualization of motile trophozoites (pear shaped) or cysts (fig. 2.31 ). the faecal sample can be concentrated with formalin-ethyl acetate or saf (sodium acetate, acetic acid, formaldehyde) and zinc sulphate. multiple fresh faecal samples (three consecutive samples) should be tested from a single suspected bird due to intermittent shedding of trophozoites and cysts. if the faecal sample is more than 10 min old, possibility of trophozoite visualization is low. if immediate processing is not possible, faecal samples may be preserved in polyvinyl alcohol for trichrome staining. the trophozoites are also destroyed in salt or sugar flotation solutions for faecal sample observation. faecal sample can be stained with lugol's iodine for visualization of giardia cysts. (b) molecular biology: for extraction of dna from giardia, oocysts present in faecal samples are concentrated. faecal suspension prepared with sterile distilled water is kept over sucrose solution (1 m) and is centrifuged at 400 g for 15 min at room temperature. the water-sucrose interface is removed with a pasteur pipette, washed in normal saline and centrifuged at 600 g for 10 min. dna is extracted from the sediment by a standard nucleic acid extraction kit. pcr targeting b-giardin gene, giardia elongation factor 1 alpha gene (ef1a) or giardia glutamate dehydrogenase gene (gdh) can be performed for confirmation. (c) antigen detection tests: elisa (antigen capture) and immunofluorescence tests are available for detection of giardia antigen in clinical samples. human giardiasis is caused by g. duodenalis and it has two phases i.e. acute and chronic. flatulence, belching, abdominal distension with cramps, frequent watery diarrhoea with offensive smell occurs in acute phase. in chronic phase, malabsorption syndrome takes place with chronic weight loss. the stools are usually pale or yellow, frequent and of small volume. the prevalence of giardiasis in developing countries is approximately 20% compared to about 5% in the developed world. transmission of giardia to humans can occur through ingestion of food and water contaminated with infectious cysts. waterborne transmission is associated with contaminated community water systems of municipality or corporation in urban area, and ponds, rivers and streams in rural area. contaminated swimming pool also plays a role in transmission of giardiasis. giardiasis is not specific for any human race and sex but it is more prevalent in children below 4 years of age. the cysts are excreated in the environment through the faeces of infected human, animals and birds. the studies revealed that psittacine birds (blue fronted amazon, blue and yellow macaw, scarlet macaw) can carry giardia duodenalis (assemblage a) cysts which may be disseminated into human. metronidazole (10-20 mg/kg body weight, oral, 12-24 h interval for two days for psittacines and pigeons) is the drug of choice in confirmed cases of avian giardiasis. necrosis at site of injection in all species of birds and toxicity in finches are adverse drug reactions of metronidazole. fenbendazole (50 mg/kg body weight, oral, 24 h interval for 3 days) is an alternative choice for effective treatment. other drugs such as tinidazole (20 mg/25 ml of drinking water for 7-14 days) or paromomycin (100 mg/kg twice daily for 7 days) are also used successfully against avian giardiasis in budgerigars and barred parakeets. in budgerigars, amphotericin b and metronidazole combination is indicated for synergistic infection of giardia, candida, megabacterium, trichomonas and other bacterial infections. with the discontinuation of therapy the infestation may relapse. it is necessary to properly rinse and dry the cage, feeding, watering and other in contact inanimate objects. cleaning and drying reduce both the number of cysts and their viability and helps to prevent reinfestation. feeding of boiled water in clean water bottles also helps to reduce the risk of infestation. dr. ernest edward tyzzer (1929, united states) first described cryptosporidiosis in bird (chicken) although cryptosporidium species was not identified. it resembled cryptosporidium muris as described in mice earlier by same worker (tyzzer 1907 (tyzzer , 1910 . c. meleagridis was first identified in turkeys by slavin (1955) . in 1986, current, upton and haynes isolated another species from chickens and coined the name c. baileyi. cryptosporidium is a protozoon under phylum apicomplexa, class sporozoasida, order eucoccidiorida, and family cryptosporidiidae. three cryptosporidium species such as c. meleagridis, c. baileyi, and c. galli are commonly associated with avian infection. recent molecular epidemiological studies have also identified several genotypes of cryptosporidium causing infection in birds. the genotypes are: avian genotypes (i-v), goose genotypes (i-iv), black duck genotype, and eurasian woodcock genotype. in addition, c. hominis, c. parvum, c. serpentis, c. muris and c. andersoni are detected in a number of birds due to accidental ingestion of cryptosporidial oocysts. among all these species and genotypes, c. baileyi is considered as the most common cause of avian infection. cryptosporidium has a direct life cycle which requires a single host. 'oocyst' with four sporozoites is the infectious form and it is shed in faeces and cough of infected birds, animals and human. the oocyst can enter a new host and after excystation the sporozoites penetrate to epithelial cells of the gastrointestinal or respiratory tract. the sporozoites undergo six developmental stages such as merogony (asexual multiplication), gametogony (gamete formation), fertilization, oocyst formation, and sporogonyv (sporozoite formation). the sporozoites of c. baileyi produce 3 types of meronts (type i, ii, iii) during merogony to generate merozoites. two types of oocysts (thin and thick walled) are observed in c. baileyi infection. thin-walled oocysts are not excreated and they excyst in situ within the host to begin auto-infection. whereas, thick walled (multilayered) oocysts are more resistant to environment and are excreated out to enter new hosts. sometimes, auto-infection takes place after excreation and the excreated oocysts may infect the same host. cryptosporidium is detected in more than 30 bird species (anseriformes, charadriiformes, columbiformes, galliformes, passeriformes, psittaciformes and struthiniformes) in different countries such as australia, argentina, canada, china, czech republic, denmark, egypt, germany, greece, hungary, japan, korea, the netherlands, romania, scotland, spain, south africa, taiwan, turkey, and united states (table 2 .14). avian cryptosporidiosis is transmitted by ingestion of contaminated food and water (faeco-oral) or inhalation of sporulated oocysts. feeding or watering trough, cage materials, toys or other inanimate objects contaminated with faeces of the infected birds may serve as a source of infection. in the same aviary or premises, transmission is possible from one avian species to another through contaminated equipments or personnel. once the oocyst enters the premises possiblity of large scale outbreak increases in the aviaries or zoos. following ingestion of oocysts by susceptible birds, it can enter salivary and esophageal glands, proventriculus, small intestine, caecum, colon, cloaca, and bursa of fabricius and developmental stages of cryptosporidium begins. the intracellular stages of the organism occur within a parasitophorous vacuole. detachment of enterocytes and villous atrophy in small intestine are common lesions which hamper the absorption of nutrients and causes osmotic diarrhoea and malabsorption syndrome. following inhalation of oocysts, primary colonization in upper respiratory tract (sinus) is followed by colonization in lower respiratory tract of the birds (trachea, bronchi, air sacs, lungs). mucoid exudates are detected in sinus, nasal passage, and trachea. the air sacs and lungs become cloudy and mottled grey-red, respectively. the site of predilection also varies with species of cryptosporidium infecting the birds. c. baileyi prefers to invade mucosal epithelium of a number of organs (small and large intestines, caeca, cloaca, trachea, air sacs, urinary system, bursa and conjunctiva), whereas c. meleagridis and c. galli are restricted within the intestine and proventriculus, respectively (table 2 .14). concurrent viral, bacterial or parasitic infections are observed in birds suffering with both clinical forms of cryptosporidiosis due to bursitis and impairment of immunity. escherichia coli and isospora sp. infection are common with c. galli infestation. two clinical forms such as respiratory and gastrointestinal cryptosporidiosis are observed in birds. in respiratory form, increased mortality, depression, lethargy, anorexia, unthriftines, coughing, sneezing, gurgling, dyspnoea, conjunctivitis and sinusitis are the common clinical symptoms. in gastrointestinal form, lethargy, decreased bodyweight gain, lower pigmentation, and diarrhoea with lime-green stool, chronic or intermittent regurgitation, lockjaw, aspiration pneumonia, seizure, egg binding are the common clinical observations. c. galli infection in passerine birds is characterized by chronic apathy and weight loss. in brazil in a cockatiel infected with c. galli lethargy for approximately 1 year before death was observed. lethargy and slow crop emptying was also detected in an indian ring-necked parrot infected with c. meleagridis. in peach-faced lovebirds infected with avian genotype iii, chronic vomiting and weight loss was detected. presence of too much mucoid exudates in conjunctival sacs, nasal passage, sinuses and trachea, chemosis, hyperemia, mucus gland distension or cystic hypertrophy/hyperplasia, mottled grey-red lungs, cloudy air sacs, bursal atrophy, hepatospleenomegaly are common gross lesions in respiratory form of cryptosporidiosis in birds. in gastrointestinal form, distended intestine filled with mucoid contents and gas, detachment of enterocytes, villous atrophy, bursal epithelial cell hypertrophy and hyperplasia and necrosis in the bursa are common gross and microscopic lesions in birds. histopathology demonstrates the presence of cryptosporidial oocysts adhered to the surface of intestinal brush border (fig. 2.32 ). in finches, purulent nephritis with enlarged and pale kidneys is detected. recently, visceral gout with renal and cloacal lesions is detected in mitchell's cockatoo infected with cryptosporidium sp. dilated proventriculus with thickened proventricular wall is common in lovebirds, cockatiels, red-faced aurora finch, australian diamond firetail finch and bronze mannikin finches. histopathology demonstrates the presence of cryptosporidial oocysts adhered to the surface of proventricular glandular epithelial cells along with hyperplasia and necrosis of epithelial cells. the organs affected by different cryptosporidium spp. are enlisted in table 2 .14. fresh faeces or cloacal swabs collected in potassium dichromate solution (2.5-5%) from the suspected birds, blood (collected from wing vein) or serum, and formalin fixed intestine, proventriculus can be used as clinical specimen. the cloacal swabs are filtered through wire mesh (0.3 mm) and the filtrate is centrifuged at room temperature at 1000 g for 10 min. after discarding the supernatant, the concentrated faecal sample is used for further analysis. the faecal samples can be preserved at 4°c in phosphate buffered saline solution (ph 7.2) with antibiotics and antifungals (streptomycin @ 100 mg/ml, penicillin g @100 iu/ml and amphotericin b @ 0.25 mg/ml). (a) direct examination: detection of cryptosporidial oocysts in clinical samples is the most common diagnostic technique used in laboratories. it is a low sensitive method which requires minimum 5000-50000 oocysts/g of faeces for detection. the oocysts are non-refractile, spherical (5-6 µm in diameter) in shape and are often confused with yeast cells. gram's stain, kinyoun acid-fast stains are used in light microscopy. in kinyoun stained smear, oocysts appear as red spherical bodies against blue background whereas yeast cells take blue stain. phase contrast microscopy provides better resolution and the oocysts appear as bright bodies with 1-4 dark granules (granules are absent in yeast cells). faecal samples can be concentrated by centrifugal flotation in high specific-gravity salt or sugar solutions (sheather's sugar flotation technique, discontinuous density sucrose gradient) for detection of oocysts. the sample after concentration should be used rapidly because in presence of sugar solutions oocysts are distorted or collapsed. in histologic sections stained with hematoxilin and eosin (h & e), cryptosporidia appear as basophilic bodies (fig. 2.33 ). (b) molecular biology: cryptosporidium is detected by means of pcr, followed by either restriction fragment length polymorphism (rflp) or sequencing of the amplified fragments. the gene commonly used for determining the species or genotype is 18s rrna. other target genes are actin, heat shock protein (hsp-70) and cryptosporidium oocyst wall protein (cowp). subtyping of c. meleagridis is based on 60-kda glycoprotein (gp60) gene. duplex-real-time pcr is recently developed for confirmation of c. galli and avian genotype iii. (c) serological tests: elisa can be used for detection of anti-cryptosporidium antibodies in serum samples. (d) antigen detection assays: capture enzyme-linked immunoassays (elisa), direct fluorescent antibody (dfa) assay using commercially available antibodies are used for detection of cryptosporidial antigen which is more sensitive and specific than staining. the detection of cryptopsordial species is not possible due to cross reactivity. cryptosporidial oocysts are excreated in the faeces of infected birds and animals which can be transmitted to human by faeco-oral route. human cryptosporidiosis is mostly caused by c. hominis, c. parvum and c. meleagridis. the pet birds and domestic chicken may act as source of c. parvum and c. meleagridis oocysts in the environment. watery diarrhoea, abdominal cramping, and increased gas production are common clinical signs of cryptosporidium infection in human. severe complications such as pancreatitis, cholangitis, respiratory distress and death are observed in immunosuppressed individuals. person to person transmission is possible. macrolide antibiotic (erythromycin) is used successfully against cryptosporidiosis in cliff swallow and owls. azithromycin (40-45 mg/kg body weight, 24 h interval, oral) is recommended for treatment of avian cryptosporidiosis in most of the bird species. united states food and drug administration (fda) have approved nitazoxanide for use in humans suffering with cryptosporidiosis. the cryptosporidial oocysts are highly resistant to common disinfectants used in aviaries and environmental stress such as temperature variation, desiccation, humidity and change of ph. maintenance of strict hygiene and biosecurity in the aviaries to reduce the possibility of oocyst contamination is the control strategy for avian cryptosporidiosis. other parasites affecting pet birds include protozoa, helminths (nematodes, cestodes, trematodes), and arthropods. captive birds kept in small and dirty cages are more susceptible to parasitic infestations. capability of a parasite to survive and reproduce in pet birds ('parasite fitness') depends on host immunity and nutrients for parasite available in the host body. the parasites may avoid the birds with good health condition due to strong immune response, but the parasites also avoid the birds with very poor health due to less possibility to obtain nutrients. the balance between immunity and nutrient availability is crucial for selection of host by the parasites. cockatoos, cockatiels, grey parrots can get the protozoon by the ingestion of insects with sarcocysts. three clinical forms such as acute pulmonary form, muscular form, and neurologic forms are observed in birds. in acute form, death without development of sarcocysts in the muscle occurs. in other two forms, clinical signs for instance dullness, weakness, respiratory signs, yellow urates are detected. pulmonary hemorrhage and edema are the cause of death. presence of sarcocysts in muscles of heart, breast, thigh, neck, and esophagus is the major lesion. hepatosplenomegaly, lung consolidation, pulmonary edema are also observed. diagnosis is usually based on visualization of sarcocysts in the muscles after post-mortem. ante-mortem diagnosis is possible by muscle biopsy, indirect immunofluorescent assay and pcr. successful treatment of birds can be done with pyrimethamine (0.5 mg/kg body weight, 12 h interval for 14-28 days) and sulphadiazine-trimethoprim (30 mg/kg body weight, 8-12 h interval). removal of insects from cages, maintenance of cleanliness, fencing of outdoor aviaries to prevent opossum access can prevent the occurrence of sarcocysticosis in birds. avian sarcocystosis is usually not considered to be a public health hazard. trichomonas sp. is a motile pear shaped protozoon which contains a central, longitudinal rod up to the posterior end (axostyle) and four flagella attached to the anterior end. an undulating membrane extending from the anterior to posterior end is present (fig. 2.34) . the membrane encloses a flagellum which does not have any free end. t. gallinae has direct life cycle without any intermediate hosts or/and vectors. they multiply by longitudinal binary fission. in budgerigars, finches, and cockatiels, transmission of t. gallinae occurs through ingestion of contaminated food and water. young domestic pigeons after entry through the oral route t. gallinae can invade the mucosal surface of the buccal cavity, sinuses, pharynx, esophagus, crop, proventriculus and conjunctiva (rare invasion) depending on the species of affected birds (figs. 2.35 and 2.36) . clinical signs include weight loss, hypersalivation, vomiting, and diarrhoea. death due to starvation occurs with progression of the infection. gross lesion includes formation of diphtheritic membrane or white plaques on gastrointestinal mucosa (oropharynx to esophagus), blockage of esophagus lumen with cheesy material and ingluvitis (inflammation of crop). solid, white to yellow circular masses appears in liver of affected pigeons (fig. 2.37) . buccal cavity and crop can be collected as clinical specimens after post-mortem of the bird. wet mount prepared from fresh crop wash with saline can be observed under microscope for detection of typical pear shaped trichomonas sp. in live birds, wet mount prepared from fresh faeces or crop swabs can be observed under microscope for detection of trichomonas sp. however, diagnosis of avian trichomoniasis is difficult in ante-mortem samples because the parasite is unstable in the environment due to lack of cyst formation capacity. metronidazole (10-20 mg/kg body weight, 12-24 h interval, for 2 days), ronidazole (6-10 mg/kg body weight, 24 h interval, for 6-10 days), carnidazole (100-200 mg/kg body weight, oral, once), dimetridazole [1 tea spoonful/gallon (4.5 l) drinking water] and ipronidazole (500 mg/galon drinking water for 7-30 days) are recommended for avian trichomoniasis. coccidia (eimeria spp., isospora spp.) eimeria spp. and isospora spp. belongs to phylum apicomplexa and they have a direct life cycle without any intermediate host or vector. sporulated oocysts are the infectious form and ingestion of food and water contaminated with oocysts is the major way of transmission. the oocyst wall is crushed in gizzard and the sporozoites are released. the sporozoites enter intestinal mucosa and undergo schizogony or merogony to produce progeny oocysts. the new oocysts leave intestinal mucosa and are excreated through the faeces. the direct life cycle requires 6-8 days time to complete. coccidiosis is common in mynahs, toucans, pigeons, lories, finches, budgerigars and canaries. clinical symptoms do not appear until the birds are immunosuppressed or infected with enormous numbers of coccidial oocysts. during schizogony in intestine, mucosa is damaged and enteritis is produced. lethargy, weight loss, severe haemorrhagic diarrhoea is detected as clinical symptoms within 4-6 days after infection. nonsporulated oocysts are shed with the faeces. diagnosis is made through examination of faeces and smears taken from the suspected lesion. the oocysts or macrogametes are detected in the smears in positive cases. the oocysts of eimeria contain four sporocysts each with two sporozoites, whereas, two sporocysts each with four sporozoites are present in oocysts of isospora. treatment with amprolium (50-100 mg/l drinking water for 5-7 days), trimethoprim and sulphamethoxazole (25 mg/kg body weight, 24 h interval, oral) are recommended for coccidiosis in pet birds. amprolium produces toxicity in falcons (@22 mg/kg body weight, 24 h interval) and trimethoprimsulphamethoxazole is recommended for toucans and mynahs. atoxoplasmosis is a dreadful disease caused by the protozoa atoxoplasma sp. causing significant mortality among the fledgling birds. although atoxoplasmosis is mostly asymptomatic among the adult birds, fatal infection with hepato-spleenomegaly may be noticed among the young birds of canaries and passeriformes. the infected birds usually shed the oocysts via faeces and the healthy one picks up the infection by consuming the oocysts. asymptomatic adults may also shed such oocysts and can be a potent but silent source of infection for the in-contact and prone young birds. interestingly, the atoxoplasma sp. from one host may not be infectious to other species of birds exhibiting certain degree of host specificity. infected birds may periodically shed large number of oocysts. in general, the birds were detected to shed the oocysts up to eight months of infection. the oocysts are environmentally stable and can persist for along period. birds of less than 1 year of age, usually exhibit clinical symptom and adults are usually asymptomatic. clinical findings are usually non-specific and include anorexia, depression, weight-loss, chronic diarrhoea, lethargy and depression. in few cases mortality may reach up to 80%. the enlarged liver, spleen and dilated intestinal loops can be palpated in few cases. during the period of parasitaemia, the protozoa a serini undergoes scizogony in the polymorphomononuclear cells (pmns) and spread throughout the body circulation. thereafter, the parasites reach out to their site of predilection reticuloendothelial cells (re cells) of vital internal organs like liver, spleen, pancreas and intestinal epithelial cells. during this process of multiplication and propagation, the parasite causes heavy damage to these organs resulting in hepatospleenomegaly and enteritis in young birds. when any adult bird is suspected as an asymptomatic carrier, microscopic examination of the fecal samples may confirm the presence of the oocysts (20.1 â 19.2 µm). in the height of severe infection, zoite form of the parasite may be detected in the lymphocytes following processing the buffy coat smear with romanowsky stain. reddish intracytoplasmic inclusion bodies are detected in the pmn cells when stained with giemsa. impression smear prepared from the enlarged liver, spleen or other affected organ may be helpful. pcr or nucleic acid detection method may give more confirmatory diagnosis. there is no effective therapy available for the disease. the birds may remain infected and continues to show periodic clinical symptoms even after therapy up to 4 months. primaquine has been recommended to suppress the tissue form of the parasite where as sulfachlorpyridazine can be used to decrease the faecal shedding of the oocysts. generally, toltrazuril is employed @ 12.5 mg/kg/day, orally for 14 days and sulfachlorpyridazine @ of 150-300 mg in one liter of drinking water. this sulfachlorpyridazine should be given for 5 days in a week for at least 2-3 weeks. ascaridia hermaphrodita, a. columbae, a. nymphii and a. galli have detected in hyacinth macaw, pigeon, australian parrots, budgerigar, cockatiel, and princess parrot. in some ground feeding species of birds (e.g. dove), baylisascaris procyonis (raccoon roundworm) and baylisascaris columnaris (skunk roundworm) infestation is observed due to occasional consumption of food, water and faeces contaminated with baylisascaris eggs. ingestion is the major route of transmission in pet birds for other ascarids also. clinical signs include lethargy, diarrhoea and death. engorgement of duodenal loop with roundworms is major necropsy finding. in baylisascaris procyonis infestation, considerable damage of central nervous system and subsequent ataxia, depression, head tilt, stumbling, recumbancy, torticollis, wing paralysis and death is common (fig. 2.38) . detection of thick shelled ascarid eggs in faecal sample is the major diagnostic approach. during necropsy, presence of roundworm in the intestine and larval stage in tissue section can aid in diagnosis. confirmation can be done by pcr targeting ribosomal dna, internal spacers (its-1, its-2), and mitochondrial gene (mtdna-cox2) of ascarids. treatment with fenbendazole (20-100 mg/kg body weight, once) is recommended for ascaridiasis in most of the birds except finches, marabou storks and vultures due to acute toxicity. the drug is contraindicated during growth of feathers also. other common anthelmentics such as piperazine dihydrochloride and mebendazole are also contraindicated in psittacines, pigeons, cormorants, pelicans, raptors, finches and ostriches due to toxicity and reported death. regular deworming of birds kept in crowded and unclean cages is needed to avoid ascaridiasis. other partasitic infections of pet birds are described in table 2 (sanfelice 1894 ) and subsequently, it was also isolated from a german patient (busse 1894) . two years later, an encapsulated bacilliform yeast, which was named saccharomyces subcutaneous tumefaciens, was detected from an apparently healthy man in france, later identified as cryptococcus gattii (curtis 1896) . vuillemin (1901) examined several of these cultures and due to lack of saccharomyces specific characteristics he placed these species in the genus cryptococcus. in 1905, von hansemann reported the first case of meningoencephalic cryptococcosis. in 1951, emmons isolated c. neoformans from pigeon nests and their droppings. disseminated cryptococcosis in a macaw was described later (clipsham and britt 1983) . cryptococcus belongs to the filobasidiella clade of the tremellales, under the order tremellomycetes, phylum basidiomycota. the genus cryptococcus includes over 37 species majority of which do not cause any infection in mammals. important pathogenic species are c. neoformans-c. gattii species complex, which includes c. neoformans var. neoformans, c. neoformans var. grubii, and c. gattii (c. bacillisporus). among them, c. neoformans var. neoformans and c. gattii are considered as primary pathogens in immunocompetent avian hosts. other cryptococcal species such as c. uniguttulatus, c. albidus and c. laurentii are occasionally detected in droppings and crops of pigeons and psittacine birds although clinical significance is uncertain. cryptococcus life cycle is predominantly divided into two phases i.e. vegetative and sexual growth phase. two major morphological forms (yeast and pseudohyphae) exist in the vegetative growth phase. the predominant form found in the environment and avian and animal hosts is unicellular budding yeast. the yeast cells are thin walled, spherical to oval with varying diameter (2-20 µm) and they reproduce by mitotic division. the buds are present at the narrow base ( fig. 2.45) . the morphological transition from the yeast phase to hyphal phase (pseudohyphae) is noticed during sexual mating. they are not considered as true dimorphic fungi probably due to their predominant existence as yeast form in the environment and hosts and the lack of involvement of this transition in the infection process. further, both at 25 and 37°c they can produce yeast like colonies in the isolation media. recently unusually large yeast like morphological form (30-100 µm) is also detected in clinical samples, known as 'giant' or 'titan' cells. another unique morphological feature of cryptococcus is the presence of capsule. the capsule can be best observed in fresh preparations by staining with diluted india ink or phase contrast microscopy ( fig. 2.46 ). giemsa can also partially stain the capsule. like other eukaryotic organism cryptococcus also possesses mitochondria which serves as source of energy, and is involved in processes of aging, calcium homeostasis, apoptosis, and regulation of virulence. presence of c. neoformans in the faeces of different avian species except raptors (table 2. 16.) is a saprobiotic phenomenon. avian faeces rich in creatine, urea, uric acid and protected from sunlight and ultraviolet light, high flock density and poor sanitary conditions create a microenvironment for c. neoformans. pigeons as mechanical carrier of c. neoformans in their feathers, feet and crop are mostly studied avian species. description of clinical cryptococcosis in birds is relatively rare although reported in pigeons, kiwis, major mitchell's cockatoo, moluccan cockatoos, thick-billed parrot, african grey parrot, green-winged macaw, papua lories, blackcapped lories, goldie's lorikeet, and ring necked parrot. cryptococcus is transmitted primarily through inhalation route in birds, animals and human. the basidiospores are major infectious particles, small in size (2-3 lm) which can easily invade the lung alveoli than the encapsulated yeast (10-60 lm). rarely ingestion of large number of organisms may cause the infection as observed during c. gattii infection of psittacine birds in brazil. psittacine birds have the habit of chewing wooden objects, such as the perches made of eucalyptus spp. in tropical countries, c. gattii is commonly associated with eucalyptus trees. the basidiospores lodge in the lung alveoli of the birds after inhalation. the capsule of the fungi helps in evasion of immune system and survival within the host. the capsule is constituted with mannan (polysaccharide) which is highly hydrophilic. it makes a gelatinous zone surrounding the yeasts that conceals the pattern recognition receptors (prr) of the yeast from the immune system. the capsule also prevents antibody binding and phagocytosis of the yeasts. c. gattii can produce extracellular fibrils which can prevent the phagocytosis by neutrophils and help to establish the primary pulmonary infection. after primary colonization in upper respiratory tract, haematogenous spread of yeast into different organs such as heart, liver, spleen, intestine, kidneys, and central nervous system is observed in different psittacine birds. occasionally, coelomic dissemination occurs between organs within close proximity. superficial colonization of yeast is detected in choanas, sinus, upper beak, and infraorbital sinus of african grey parrot, goldie's lorikeet and beccaris's crowned pigeon. minimal inflammatory response with epithelioid macrophages, multinucleated giant cells and heterophils or absence of inflammatory response is detected in different organs of psittacine birds. and blindness are also detected. in domestic pigeons (columba livia), rare report of clinical cases revealed the central nervous system signs, weight loss, dyspnoea, and infraorbital sinus mass. development of thick crust over the right naris is observed in major mitchell's cockatoo suffering with clinical cryptococcosis. gelatinous myxomatous mass (granulomata) in respiratory tract, abdominal cavity, sinuses and brain is the most common lesion observed in birds. in psittacines, hepatomegaly, multifocal hepatitis, yellow areas in the capsule and parenchyma of liver, congestion of lung, replacement of pulmonary parenchyma with yellow gelatinous material, thickened air sacs, sinuses filled with yellow coloured substance, nephromegaly, tubular and glomerular degeneration, splenomegaly, congested intestine with black coloured material in lumen are observed. cloacal/choanal swabs, dry faeces, blood/serum, aspiration biopsy of the gelatinous mass present in upper beak, infraorbital sinus and choana, visceras collected in 10% formalin after post mortem are considered as clinical specimens of avian cryptococcosis. dry faecal samples (2.5 gm) are suspended in 15 ml sodium chloride solution in tubes containing chloramphenicol (0.1 mg/ml). (a) direct examination: the smear can be prepared from the aspiration biopsy samples and is stained with india ink, nigrosin, or romanowski for demonstration of capsule. the romanowski stain produces clearer capsule against the lightly stained background. the tissue samples can be stained with periodic acid schiff base (pas)-haematoxylin stain which will outline the yeast cell and the capsule will appear as clear zone surrounding the cell (fig. 2.49 ). in mayer's mucicarmine stain, the capsule and cell wall appears as red. another characteristic feature of cryptococcus is narrow base budding ( fig. 2.45 ) in comparison to other yeasts (blastomyces) having broad based budding. sometimes false positive results are produced due to confusion with globules of myelin, lysed cells, lymphocytes and dead yeasts after successful treatment. (b) isolation of cryptococcus spp. from clinical samples: faecal suspension (dry faeces mixed with sodium chloride and chloramphenicol) of the suspected birds can be inoculated into corn meal agar, sabouraud's dextrose agar, blood agar, honey agar, brain heart infusion agar and malt agar. the specific medium is birdseed agar/staib medium (niger seed agar)/sunflower seed extract agar with antibiotics. the plates are incubated at 28-37°c for 2 days to 2 weeks. the colonies are initially small, convex, mucoid, creamy in colour, increases in diameter up to several centimeters after prolonged incubation. in birdseed agar the colonies appear as brown coloured in the center of the plate due to the production of melanin by the action of phenyl oxidase. the optimum capsule production is detected in chocolate agar after incubation at 37°c with 5% co 2 tension. the isolates are confirmed by api system or different biochemical tests such as urea hydrolysis, assimilation of inositol and creatinine, non-fermentation of carbohydrates, and melanin production. the l-canavanine glycine bromothymol blue media can differentiate c. neoformans and c. gattii by the formation of distinctive blue coloration with the growth of c. gattii. (c) detection of cryptococcal antigen: detection of cryptococcal antigen in clinical specimens can be performed by latex particles coated with polyclonal serum and antigen-capture elisa. the detection of antigen is possible from both live and dead organisms. during initial phase of therapy, disintegration of the yeast cells releases the capsule which produces high titer. so, the tests should not be done within 6-8 weeks after initiation of the therapy. the titer can be observed even after successful treatment as the dead organisms also have the intact capsule. (d) molecular biology: multiplex pcr and mating type-pcr are used for determination of molecular and mating types of c. neoformans var. neoformans and c. gattii isolates. in human the patients with suppressed immunity (suffering from aids, lymphoma, haematologic malignancy and using corticosteroids for prolonged period) are mostly susceptible to the cryptococcal infection. it causes meningitis, meningoencephalitis and death in human. inhalation of cryptococcal basidiospores is the major way of transmission in human. cryptococcal infection in patients after exposure to the birds is observed. it was first reported from united states in a renal transplanted patient who developed cryptococcal meningitis transmitted from her pet cockatoo (nosanchuk et al. 2000) . report of cryptococcal meningitis is also documented in immunocompetent patient who acquired the infection from her pet magpie, although, her contact with the bird was limited to passing by the cage when entering home (lagrou et al. 2005 ). fluconazole (5-15 mg/kg body weight, oral, 12 h interval for 15-60 days) and itraconazole (10 mg/kg body weight, oral, 24 h interval for 15-90 days with food) are recommended for treatment of avian cryptococcosis in pet birds. use of itraconazole is contraindicated in african grey parrots. proper ventilation, regular cleaning of droppings and organic debris from the cages can prevent the occurrence of avian cryptococcosis. in tropical countries, wooden perches made of eucalyptus tree should be avoided because psittacine birds like to chew the perches. in 1729, micheli first described aspergillus who found the similarity between the spore chain of the fungi with the brush ('aspergillium') used for sprinkling holy water in the churches. later in 1842, pathogenic aspergillus (a. candidus) was detected in air sac lesion of a bullfinch by rayer and montagene. whereas, a. fumigatus was first detected in lung of a great bustard (otis tardaga) in 1863 by fresenius, who was the first to use the term 'aspergillosis' for this respiratory infection. the major members of aflatoxins were first detected and its association with aspergillus flavus was established in 1961 during investigation of mysterious 'turkey-x disease' causing high mortality in turkey poults (sargeant et al. 1961) . in 1962, the name 'aflatoxin', using first letter from 'aspergillus' and the first 3 letters from 'flavus' was proposed. aspergillus is the fungus which belongs to trichocomaceae family under the order eurotiales and phylum ascomycota. the genus aspergillus contains more than 250 species grouped into sub genera and species complex. there are eight such sub genera i.e. aspergillus, fumigati, circumdati, candidi, terrei, nidulantes, warcupi, and ornati. the important species complexes are a. fumigatus, a. flavus, a. terreus, a. niger, a. nidulans, and a. ustus. among them, a. fumigatus is the most common cause of avian aspergillosis. other species complexes such as a. flavus, a. terreus, a. niger, and a. nidulans are occasionally associated with avian aspergillosis. a. fumigatus is the major cause probably due to smaller size of the spores than other species which helps in easy transmission. all species of domestic, pet and wild birds including chicken, duck, quail, and geese are susceptible to avian aspergillosis due to anatomic and physiologic characteristics of avian respiratory system (table 2 .17). among the wild birds, some species such as goshawks (accipiter gentilis), gyr falcons (falco rusticolus), penguins, and auk (alca torda) are more susceptible to respiratory fungal infections. diving birds (auk, penguin) are more susceptible due to air re-circulation during diving. inhalation of fungal spores (conidia) is the major way of aspergillus transmission in birds. in addition to inhalation route, ingestion of contaminated feed (seed mixture) with the fungal spores is another way of transmission. due to small non-expanding lung and presence of air sacs, the birds are more susceptible to aspergillus infection. primary colonization of the spores takes place in the air sacs because the inhaled air passes through the sacs to reach the lungs. other predisposing factors for aspergillus infection include higher body temperature which helps in fungal growth, lung injury, stress due to malnutrition and vitamin deficiency, use of immunosuppressive drugs, very young or old age and use of hay or straw contaminated with fungal spores in preparation of aviary litter. the conidia of aspergillus spp. reach the lung parenchyma through inhaled air. the conidia are trapped between atrium and infundibulum of parabronchus region of the lungs. conidial sialic acids act as ligand for adherence with the alveolar epithelial cells specially with fibrinogen and fibronectin, commonly found in the wounded epithelial surfaces. so, lung injury acts as major predisposing factor for causation of invasive aspergillosis. gliotoxin, fumagillin, and helvolic acid produced by the fungi causes damage in the respiratory mucosa and slow ciliary movement facilitating the attachment of the conidia. the conidial maturation begins which causes loss of hydrophobic layer and exposure of the inner cell wall. the cell wall is composed of galactomannan, chitin, and b glucan which act as ligand for the soluble and cell associated pattern recognition receptors (prr). the soluble receptors act as opsonin and can bind with fungal cell wall carbohydrate which enhances phagocytosis by the alveolar macrophages. most of the conidia are killed by reactive oxygen species (ros) or acidified phago-lysosome produced within the alveolar macrophages. in immunosuppressed birds having defective alveolar macrophage function the conidia are able to escape the phago-lysosome mediated killing. the conidia which survive the first line of defense can germinate. the germination involves conidial swelling (isotropic growth) followed by protrusion of germ tube (polar growth). they produce a necrotic focus (plaque) without a structured granuloma which can obstruct the trachea or bronchi and fill up the air sacs. hyphae with fruiting bodies can penetrate the air sacs or lungs and produce serositis. fungal growing hyphae also invade the endothelial cell lining of blood vessels (angioinvasion) from the albuminal side to the luminal side. dissemination of infection into vital organs occurs through the haematogenous route. avian aspergillosis is considered as an opportunistic infection in birds with immunosuppression. primary infection in immunocompetent hosts sometimes takes place when numerous spores are inhaled. acute and chronic types of aspergillosis may develop in affected birds. mostly young birds suffer with acute infection in which sudden death of birds occurs without prior symptoms probably due to hepatic damage by aflatoxins released by the fungi. if the birds are alive for a few days, general symptoms such as laboured breathing, anorexia, diarrhoea, polydypsia, and cyanosis develop. chronic aspergillosis is more common in adult birds. it produces non-specific syndrome such as fever, diarrhoea, respiratory distress, change in voice ('sore throat' sound due to tracheal and syringeal inflammation), change in behaviour, emaciation, cheesy deposit in conjuntival sac, and nares, development of rhinolith ('nose-stone') and facial swelling, neurological disorders, dermatitis, wing droop due to humerous involvement ( fig. 2.50 ). death occurs due to obstruction of airways and respiratory failure. congestion and yellow nodules in lungs, thickened air-sacs with small whitishyellow plaques are detected in acute aspergillosis (figs. 2.51 and 2.52). in chronic form, granulomatous lesions (nodules and plaques) are observed in periphery of lungs and air sacs which may occlude the trachea and bronchi. typical granulomas are also detected in kidneys, oviduct, and ovaries. whole blood, tracheal washings, air sac fluids, tissue biopsies from respiratory tract granuloma and vital organs such as lungs, air sacs, kidney, liver collected after post the tissues collected as clinical specimens should be cleared with 10% koh and are observed under microscope. histopathological staining can be performed with periodic acid schiff (pas), grocott's silver or methenamine silver stain for detection of tissue invasion. in the tissue section, aspergillus hyphae are narrow and septate which are not easily distinguishable from other fungi (fig. 2.53 ). different species of aspergillus has characteristic fruiting body structures which can be identified by an expert. confirmation of aspergillosis is also possible by immunohistochemistry. (c) isolation of aspergillus from clinical specimens: aspergillus can be isolated in sabouraud dextrose agar (sda) with or without chloramphenicol (0.05 gm/l) and other common bacteriological media such as blood agar. the cycloheximide can inhibit the growth. the plates are incubated at 37°c for 4-5 days. a. fumigatus is thermophilic which is able to grow at 55°c and can survive at more than 75°c. however, repeated isolation from the clinical specimen is required, along with correlation with history, clinical signs, histopathological observations for proper diagnosis of clinical aspergillosis. (d) blood biochemical tests and radiography: haematological parameters such as leukocytosis (20,000-100,000), heterophilia with a left shift (degenerative shift), monocytosis, lymphopenia, and change in b-globulin concentration indicate about occurrence of infection such as aspergillosis. radiographic changes of pneumonia and airsacculitis (lateral and ventrodorsal views) also suggests respiratory tract infection such as aspergillosis. both these biochemical tests and radiogrpahy cannot confirm the infection. (e) detection of aspergillus antigen: detection of aspergillus antigen is useful in acute infection. galactomannan (gm) is the predominant antigen released by a. fumigatus in the circulation during angioinvasion which can be detected by latex agglutination test, sandwich elisa in blood samples. the detection limit of both the tests is 15 ng/ml and 1 ng/ml, respectively. however, gm detection assay is not specific for aspergillus as it is cross-reacting with other fungi such as penicillium, fusarium, alternaria, and histoplasma. similarly, b-d-glucan (bdg) can be detected for identification of aspergillus. it is also produced by a lot of other fungi such as candida, fusarium, pneumocystis etc. so the test can predict the general fungal infection rather than specifically aspergillosis. (f) serological tests: serological tests such as counter-immunoelectrophoresis, agar gel immunodiffusion and enzyme-linked immunosorbent assays (elisa) can be employed as supportive diagnostic assays to detect the antibodies in chronic infection. in immunosuppressed birds production of antibody is undetectable. (g) molecular biology: confirmation of aspergillus spp. can be done by conventional pcr and real-time pcr from clinical samples. a. fumigatus isolates can be characterized by pcr-rflp using bcci, mspi and sau3ai restriction enzymes. a. fumigatus causes invasive aspergillosis in immunocompromised human which involves lung parenchyma, pleura, trachea and bronchi. it is common in the patients with haematological malignancy, prolonged antibiotic users or stem cell transplant recipients. transmission of clinical aspergillosis from pet birds is not documented so far. removal of granulomatous lesion is essential for effective treatment. it is difficult due to remote location of granuloma within respiratory tract, risk of surgical trauma and anaesthesia. in most of the confirmed cases, treatment is restricted with antifungals. nebulization, oral, intravenous, nasal or air sac flushing, surgical irrigation of the abdominal cavities are ways for drug application. antifungals used in avian aspergillosis with suitable dosage are described in table 2 .18. sometimes, immunostimulants, for example, levamisole and microbial products (b-glucans) can be used for boosting of the immune system. no vaccine is currently available against avian aspergillosis. maintenance of hygiene and nutrition, avoiding mouldy feeds, proper ventilation and spraying of fungistatic agents [nystatin, thiabendazole, copper sulphate (1 g per 2 l of water)] in large aviaries can prevent avian aspergillosis. candida spp. is a common inhabitant of avian enteric tract, although, it can cause infection in young, immunosuppressed and stressed birds and during prolonged use of antibiotics. candida albicans, c. krusei, and c. tropicalis are major cause of avian candidiasis. c. parapsilosis is rarely isolated from the crops of the birds. among the pet birds, clinical candidiasis is reported from peach-faced lovebirds, fisher's lovebirds, pigeons, cockatiels, amazon parakeets, budgerigars and peacocks. in eclectus parrot, concomitant infection of histoplasmosis and candidiasis is detected. healthy cockatiels and some other psittacines are also detected to act as carrier of candida spp. infected birds show whitish, pseudomembranous lesions in oral cavity, crop and esophagus. crop is the most suitable organ for fungal growth due to its pouch like structure and availability of nutrients. general symptoms such as anorexia, depression, delayed crop emptying, and regurgitation is sometimes observed. presence of thickened mucosa (pseudomembranes) and ulcers in oral cavity, esophagus and crop is the major necropsy findings. the smear prepared from the clinical specimens can be observed under microscope either by 10% koh preparation or by staining with gram's stain method. in the tissue section candida can be observed by pas-haematoxylin or methenamine silver stain. they appear as unicellular budding yeast or hyphae and pseudohyphae ( fig. 2.54 ). candida can be isolated in common fungal or bacteriological media such as sabouraud dextrose agar (with penicillin, streptomycin, chloramphenicol to prevent the bacterial growth), potato dextrose agar, blood agar and brain heart infusion agar. the plates are incubated at 25-30°c for 2-3 days. immunohistochemistry is also useful in detection of candida spp. in birds. several types of pcr such as semi nested and nested pcr, real time pcr, multiplex pcr followed by dna sequencing or pyrosequencing are developed for detection of candidal dna. the target gene includes rrna (5.8s, 18s, 28s) gene, internal transcribed spacer (its) and intergenic spacer (igs) region genes. treatment with nystatin (3,00,000-600,000 iu/kg body weight, 8-12 h interval for 7-14 days in psittacines; 1,000,000 iu/kg body weight, 12 h interval in raptors and pigeons) is recommended for pet birds in confirmed cases of avian candidiasis. the drug is not well absorbed from the gastro intestinal tract and it should be mixed with food for better absorption. except vomition in some species the drug is safe and cost-effective. infection) proventriculitis was originally described as megabacterium associated infection in aviaries. due to presence of a eukaryotic nucleus, mycotic staining properties, presence of chitin in cell wall, and finally phylogenetic analysis based on 18s rrna and 26s rrna gene, megabacterium is classified as yeast. it is renamed as macrorhabdus ornithogaster. the infection is reported from captive-bred budgerigars (melopsitticus undulatus), parrotlets (forpus spp.), canaries (serinus canaria), pheasants (phasianus colchicus), red-legged partridges (alectoris rufa), helmeted guinea fowl (numida meleagris) and gray partridges (perdix perdix) as primary pathogen or concomitant with other infections. faecal-oral route is the probable transmission route of macrorhabdus infection in birds. the yeast prefers to colonize isthmus of proventriculus or ventriculus in birds. the infection is chronic and it produces certain non-specific symptoms such as anorexia, continuous loss of body weight, depression, plumage disorder, regurgitation, diarrhoea and dehydration ( fig. 2.55 ). sudden death due to rupture of proventriculus is observed in budgerigars and parrots. swollen and hyperemic proventriculus, covering of isthmus with thick, transparent-to-white mucus, and mucosal erosions in gizzard are specific necropsy findings. fresh faecal samples from live birds and tissues from vital organs (esophagus, crop, proventriculus, gizzard, pancreas, liver etc.) can be collected in 10% buffered formalin after post-mortem. smears prepared from faecal samples can be stained by gram's method. the tissue sections are stained with periodic acid-schiff (pas), grocott's methenamine silver nitrate (gms) and mayer's hematoxylin and eosin. detection of typical gram-positive, large rod shaped organisms (20-60 µm long and 2-3 µm wide) suggests macrorhabdus infection (fig. 2.56) . isolation of macrorhabdus is difficult because it does not grow in commonly used solid media. it can be isolated only in liquid medium (minimum essential media) with 20% foetal bovine serum (fbs) and 5% sucrose and more than two weeks incubation. antifungals for instance nystatin and amphotericin-b can be used for the treatment (figs. 2.57, 2.58 and 2.59). other fungal infections of pet birds are described in table 2 .19. molecular characterization of giardia psittaci by multilocus sequence analysis ascaridia nymphii n. sp. 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japanese encephalitis virus group exudative cloacitis in the kakapo (strigops habroptilus) potentially linked to escherichia coli infection the isolation of west nile virus from man and of usutu virus from the bird biting mosquito mansonia aurites (theobald) in the entebbe area of uganda die halb-acute gehirn-entzündung oder kopf-krankheit der pferde sarcocystis falcatula-associated encephalitis in a free-ranging great horned owl (bubo virginianus) molecular characterization of the cloacal microbiota of wild and captive parrots prevalence of campylobacter jejuni in selected domestic and wild birds in louisiana isolation of newcastle disease virus from the osprey and the parakeet key: cord-003425-c5jdp5jv authors: fu, yangxi; tang, zhengzhen; ye, zhixu; mo, shi; tian, xingui; ni, ke; ren, luo; liu, enmei; zang, na title: human adenovirus type 7 infection causes a more severe disease than type 3 date: 2019-01-09 journal: bmc infect dis doi: 10.1186/s12879-018-3651-2 sha: doc_id: 3425 cord_uid: c5jdp5jv background: human adenovirus type 3 (hadv-3) and 7 (hadv-7) cause significant morbidity and develop severe complications and long-term pulmonary sequelae in children. however, epidemiologic reports have suggested that nearly all highly severe or fatal adenoviral diseases in children are associated with hadv-7 rather than hadv-3. here, we conduct in-depth investigations to confirm and extend these findings through a comprehensive series of assays in vitro and in vivo as well as clinical correlates. methods: a total of 8248 nasopharyngeal aspirate (npa) samples were collected from hospitalized children with acute respiratory infections in children’s hospital of chongqing medical university from june 2009 to may 2015. among 289 samples that tested positive for hadvs, clinical data of 258 cases of hadv-3 (127) and hadv-7 (131) infections were analyzed. all hadv-positive samples were classified by sequencing the hexon and fiber genes, and compared with clinical data and virological assays. we also performed in vitro assays of virus quantification, viral growth kinetics, competitive fitness, cytotoxicity and c3a assay of the two strains. mouse adenovirus model was used to evaluate acute inflammatory responses. results: clinical characteristics revealed that hadv-7 infection caused more severe pneumonia, toxic encephalopathy, respiratory failure, longer mean hospitalization, significantly lower white blood cell (wbc) and platelet counts, compared to those of hadv-3. in cell culture, hadv-7 replicated at a higher level than hadv-3, and viral fitness showed significant differences as well. hadv-7 also exhibited higher c3a production and cytotoxic effects, and hadv-7-infected mice showed aggravated pathology and higher pulmonary virus loads, compared to hadv-3-infected mice. macrophages in balf remained markedly high during infection, with concomitant increase in pro-inflammatory cytokines (tnf-α, il-1β, ifn-γ, and il-6), compared hadv-3 infection. conclusions: these results document that hadv-7 replicates more robustly than hadv-3, and promotes an exacerbated cytokine response, causing a more severe airway inflammation. the findings merit further mechanistic studies that offer the pediatricians an informed decision to proceed with early diagnosis and treatment of hadv-7 infection. human adenovirus is well-recognized as an important pathogen of respiratory tract infection in childhood [1] [2] [3] . more than 70 hadv serotypes are further subdivided into seven species (a-g) [4, 5] ; however, species b (hadv-3,7,11 and 14), c (hadv-1,2,5 and 6), and e (hadv-4) are the ones most associated with respiratory infections [6] . epidemiological studies have reported an approximately 5-10% hadv-positive rate among acute respiratory tract infections in children [7, 8] . adenovirus infections may in fact result in high morbidity and mortality in children, and fatality rates for untreated severe hadv pneumonia or disseminated disease may exceed 50% [9] . severe adenovirus infection in children can be complicated with pleural effusions [10] , acute respiratory distress syndrome (ards) [11] , respiratory failure [2] , myocarditis [12] , and central nervous system dysfunction [13] , leading to either mechanical ventilation or extracorporeal life support, even death. unfortunately, effective adenoviral vaccine for children and specific antiviral drugs against human adenoviruses are currently not available. hadv-3 and hadv-7, belonging to subgenus b1, cause infections that are usually mild and self-limiting in immunocompetent individuals; however, severe, and even life-threatening infections and outbreaks, associated with both type 3 and 7 in children, are increasingly reported [2, 8, 12] . epidemiology of the disease suggests that hadv-3 and hadv-7 are the major types responsible for lower respiratory diseases in children less than 5 years old worldwide [2, 3, 7, 8] . in beijing, china, hadv-3, and hadv-7 were the most common serotypes causing pneumonia from 1958 to 1990 [14] . in chongqing, china, 53.9% of the hadv-7-infected patients were diagnosed as having severe pneumonia, significantly higher than the 26.9% of the hadv-3-infected patients from 2009 to 2014 [3] . in taiwan, hadv-3 and hadv-7, causing complication with respiratory failure, accounted for 53% of infected children; seven cases due to hadv-7 were among ten deaths reported in a single year, between 2010 and 2011 [2] . in korea, both hadv-3 and hadv-7 cause epidemics of severe lower respiratory tract infections in young children. overall, 16 and 39%, of the children infected with hadv-3 and hadv-7, respectively, required mechanical ventilation [15] . certain adenovirus serotypes are associated with particular clinical features and severe illness; however, no study has offered persuasive evidence for in the different degrees of disease severity caused by hadv-3 and hadv-7 infection because it still limits on the basis of clinical observation. in the present study, therefore, we undertook a comprehensive analysis of the comparative clinical features of hadv-3 and hadv-7 infection, as well as a serial of experiments, were performed to better understand the association between severity of the disease and the serotypes of hadvs. participants, demographic data, clinical data analysis patients ranging in age from 1 month to 16 years and requiring inpatient treatment due to acute respiratory tract infections (arti) at the department of respiratory medicine, children's hospital of chongqing medical university between june, 2009 and may, 2015, were enrolled in this study. parents/guardians provided demographic data and medical history during an interview. severe disease was defined as respiratory failure confirmed by an abnormal blood gas analysis result (an oxygen saturation level of approximately 90% or less) or toxic encephalopathy. a total of 8248 nasopharyngeal aspirates (npas) were collected on the day of hospital admission after obtaining permission from the patients or their guardians. imaging and laboratory data on admission and during hospitalization were collected. white blood cell (wbc) count > 15,000/ μl was defined as leukocytosis, whereas that < 4000/μl was defined as leukopenia. the study procedure was approved by the ethics committee of the children's hospital of chongqing medical university. informed consent was obtained from parent or guardian of all participants. hadv isolation, identification, and molecular typing npas from hadv-positive patients were inoculated into hep-2 cell monolayers and cytopathic effect (cpe) was monitored for 10 days. viral dna and rna were extracted from 200 μl aliquots of the npa samples by a qiaampminelute virus spin kit (qiagen, hilden, germany). hadv-positive samples were used to amplify the hexon hypervariable regions (hvrs) 1-6 and fiber genes for subsequent sequencing [16, 17] . the hadvs strains were molecularly typed by pcr amplification and sequencing of all seven hypervariable regions in the hexon gene, described previously [18] . the hadv-3 (cq5291) and hadv-7 strains (cq4411) used in this study were originally isolated from npas of children infected with hadv. the virus was purified using a standard cesium chloride gradient centrifugation procedure, as described [19] . the concentration of viral particles (vps) was determined by spectrophotometry according to the a 260 value. the virus concentration was calculated as vps/ml = a 260 *dilution*10 12 . three cell lines (a549, 16hbe, and hek-293) at 80% confluency in 12-well plates were infected with either hadv-3 or hadv-7 at a multiplicity of infection (moi) of 1 vp/cell. viruses were cultured as previously described [20, 21] . the culture supernatants and cells were sampled at 48 h post-infection. viral loads were quantified by quantitative pcr (qpcr). briefly, 25 μl reaction mixtures were prepared by adding 5 μl of sample nucleic acid extract to 20 μl of iq supermix (bio-rad, hercules, ca), primers and probes. the sequences of primers and probes for qpcr and the amplification conditions have been detailed [22, 23] . the real-time hadv primer sequences were as follows: hadv-3 forward, 5′-ggga gacaatattactaaagaaggtttgc-3′, hadv-3 reverse, 5'-caacttgaggctctggctgata-3′. the sequence of the hadv-3 probe was 5'-cactac"t"-gaaggagaagaaaagcccatttatgcc, which was labeled with 6-carboxyfluorescein (fam) and internally quenched at "t" with black hole quencher-1 on the 5′end and phosphate on the 3′-end. the forward primer sequences of hadv-7 were 5'-gaggagccag atat tgatatggaatt-3′, the reverse primer sequences of hadv-7 were: 5'-aattgacattttccgtgtaaagc a-3′, and probe, 5'-aagctgctgacgctttttcgcc tga-3′. the hadv-7 probe was labeled with 6-carboxy fluorescein (fam) on the 5′-end and 3′-terminally quenched with black hole quencher-1. the thermal cycling condition was as follows: one cycle of 3 min at 95°c; 45 cycles of 15 s at 95°c; and 1 min at 60°c. a549 cells at 80% confluency in 12-well plates were infected with either hadv-3 or hadv-7 (moi = 25 vps/ cell). after 1 h adsorption with rocking every 15 min, the inoculum was removed, 1 ml of fresh dmem-f12 with 10% fbs was added to each well, and the plates were incubated at 37°c in 5% co 2 . the culture media were harvested at 0, 4, 8, 12, 24, 36, 48, 60, 72 and 96 h post-infection. tcid 50 was used to titer the virus at each time point. a549 cells were infected with a mixture hadv-3 and hadv-7 at a ratio of 1:1 and moi of 10 vps/cell in standard 12-well cell culture plates, and incubated as described under 'viral growth kinetics'. media supernatants and cells were harvested at 0, 2, 8, 12, 24, 36, 48, 72 h post-infection. virus copies were quantified at each indicated time point by qpcr described earlier [22, 23] . a549 cells (2.0 × 10 4 ) were seeded in each well of 96-well flat bottom plates and incubated at 37°c in 5% co 2 overnight. thereafter, the media were removed, and cells were infected with hadv-3 or hadv-7 (low moi = 25 vps/cell, or high moi = 250 vps/cell). infected and uninfected cell viability were assayed at 2, 4, 24, 36, 48 and 60 h post-infection and the absorbance was determined as described [20, 21] . cell viability was expressed as the ratio, and the mean (mean ± sem) of 5 replicates of two strains was presented. all samples were collected as described previously, with minor modifications [24] . purified hadvs (0.016 g) in pbs were mixed with a 1:10 dilution of normal human serum (nhs) in an assay volume of 20 μl, and incubated for 2, 4 and 10 min at 37°c. the samples were further diluted 1:500 and tested using an opteia human c3a elisa kit (bd biosciences), according to the standard procedure provided by the manufacturer. female balb/c mice, 6-8-week old, were purchased from the animal laboratory of chongqing medical university and housed in a dedicated pathogen-free facility at chongqing medical university. the use of all experimental animals strictly met the ethical requirements of the animal committee of chongqing medical university (license numbers: syxk(yu) 2012-0001). the mice under anesthesia were infected intranasally (i.n.) with 5 × 10 9 vps/ml of hadv-3 or hadv-7 in 100 μl. mock-infected mice were instilled with 100 μl of phosphate-buffered saline (pbs) under the same conditions. mice were processed after 3, 5, and 7 days of infection. for studies of viral load, total viral nucleic acid was directly extracted from the right lung lobe, using a qiaamp mini-viral dna extraction kit (qiagen, hilden, germany) and following the manufacturer's recommendations. quantification of hadv-3 and hadv-7 copies were performed as described earlier [22, 23] . after 3, 5, and 7 days of infection (4 mice in each time point in every group), the animals were euthanatized by intraperitoneal injection of pentobarbital (150 mg/kg). the left pulmonary hilum was closed by clamping, in order to collect balf from the right side. the specific steps were as described previously [25] . briefly, bronchoalveolar lavage was performed using 0.25 ml of ice-cold sterile pbs for a total of 6 times. balf was centrifuged at 2500 rpm for 5 min in cold (4°c). the supernatant was aliquoted and stored at 80°c for subsequent cytokine measurements. the precipitate was resuspended in 1 ml of pbs, and the total number of cells was counted under a microscope. the cells were centrifuged, smeared onto slides, fixed, and stained with diff quik (baxter healthcare corp, deerfleld, miami, fl) to analyze and classify white blood cells. cell counting was performed using a microscope. a total of 200 random cells was counted from each slide and included macrophages, lymphocytes, and neutrophils. mice were sacrificed at 3, 5, and 7 d after infection for pathological analyses. the left lung lobe was collected, fixed in 4% paraformaldehyde for 24 h, dehydrated, and then embedded in paraffin. lung tissue blocks were cut into 4 μm thick sections that were then stained with hematoxylin and eosin to evaluate lung tissue pathology associated with hadv-3 and hadv-7 infection. the levels of interferon-γ (ifn-γ), tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β), and interleukin-6 (il-6) in balf were all measured using commercial murine elisa reagent kits (neobioscience, shenzhen, china) according to the manufacturer's instructions. descriptive statistics were performed for all variables; the continuous variables were summarized as means and standard deviations (sd) or as medians and ranges, and the categorical variables were summarized as frequencies and proportions. the graph pad prism 5.0 software was used for data analysis. statistical significance was assessed by one-or two-way analysis of variance (anova). one-way anova was used to analyze significant differences between three or more groups. two-way anova was used to analyze significant differences between two variables. if an overall test was significant, the tukey's test was utilized for specific comparisons between individual groups. differences were considered significant at p < 0.05. more severe disease in patients infected with hadv-7 compared to hadv-3 among the 8248 npas, 289 specimens were identified as positive for adenovirus, of which hadv-3 (127) and hadv-7(131) were the most common types. as shown in table 1 , age and gender distribution, underlying disease, and coinfection were comparable between hadv-3 and hadv-7. co-infection with other respiratory viruses were observed in 57.5 and 40.5% of patients infected with hadv-3 and hadv-7, respectively. the most common clinical manifestations of hadv-3 and hadv-7 infections, such as cough, wheezing, croup, dry rales, moist rales and fever, including the peak of febrile body temperature (> 39°c), were not significantly different. in contrast, pneumonia was the most common diagnoses of the patients. among the 131 hadv-7-infected patients, 31.3% were diagnosed as having severe pneumonia, significantly higher than that of the hadv-3-infected patients (31.3% vs 16.5%, p = 0.005). when the median duration of hospitalization was analyzed, we found that the hadv-7 inpatients had a longer mean duration of hospitalization, 8.2 ± 0.6 days (p = 0.031). several severe outcomes, toxic encephalopathy, and respiratory failure were significantly higher in patients with hadv-7 infection compared with those with hadv-3 (p < 0.05), but no statistically significant differences were found in icu admission, pleural effusion, suspicious bo and intracranial infection. notably, one child, infected by hadv-7, exhibited complications of acute respiratory distress syndrome (ards). laboratory findings for the hadv-7-positive inpatients were also significantly different from those infected by hadv-3 (table 2) . specifically, the hadv-7-positive inpatients had lower white blood cell count (10.28 ± 0.51 vs. 11.69 ± 0.46 × 10 9 cells/l; p = 0.041), platelet count (259.9 ± 11.38 vs. 370.2 ± 14.22 × 10 9 cells/l; p = 0.0001). in contrast, hemoglobin and c-reactive protein levels, and the percentages of lymphocytes, neutrophils and positive sputum culture were found to be statistically similar. in the chest radiographs (table 2) , alveolar infiltration, consolidation and pleural effusion were more frequently observed with the hadv-7 patients compared to hadv-3 patients but they did not differ significantly. increased lung texture and interstitial inflammation showed a similar proportion. two patients infected with type 3, and four patients infected with type 7, had atelectasis. notably, two patients infected with adenovirus type 7 had complications with pneumothorax. since type-specific adenovirus infection is known to cause different tissue tropisms and clinical manifestations as indicated before, viral loads and fitness of hadv-3 and hadv-7 were evaluated in several human epithelial cells to determine if there were differences. as shown (fig. 1a) , at 48 h post-infection, hadv-7 replicated to a higher number of viral copies than hadv-3 in all three cell lines tested, namely, a549, hek-293 and 16hbe cells. hadv-7 also exhibited stronger growth than hadv-3 in a549 cells at 24, 36 and 60 h post-infection (fig. 1b) . in comparing the competitive fitness between hadv-3 and hadv-7, however, no significant difference was observed. lastly, hadv-7 loads were significantly higher than those of hadv-3 at 12, 24, 36 and 72 h post-infection (p < 0.05) (fig. 1c) . cytotoxicity assay may serve as a measure of cellular distress induced by viral infection. as shown (fig. 2) , at a low moi of 25 vps/cell, viability of hadv-7-infected cells was slightly higher than that of the hadv-3-infected cells, but there was no statistically significant difference. in contrast, at high moi of 250 vps/cell, hadv-7 induced a remarkably greater decrease in cell viability at both 2 and 4 h post-infection (26.5 and 42.6%, respectively; p < 0.0001) (fig. 2) . to compare complement activations by hadv-3 and hadv-7, the anaphylatoxin c3a, a split fragment of c3 (the step where classical, lectin, and alternative pathways of complement activation merge), was measured in vitro. as shown (fig. 3 ), hadv incubation with serum, compared with nhs incubation alone, resulted in a marked increase of c3a at 2, 4 and 10 mins. importantly, elevation of c3a by hadv-7, compared with hadv-3, was statistically higher at 2 mins (p < 0.05). to better understand the differences in adenovirus-induced airway inflammation, we established a mouse respiratory infection model for human adenovirus. in this model, assays of viral load in the lung tissues revealed that virus copies of hadv-7-infected groups were higher than those of hadv-3-infected groups at 3, 5 and 7 days of infection, particularly on day 3, when virus copies in the hadv-7 group were 1.5-fold higher than the hadv-3 group (p < 0.05) (fig. 4a) . pulmonary inflammation in mice was also observed following hadv-3 and hadv-7 infection at each time point and found to gradually increase from day 3 to day 5, decreasing on day7. compared with hadv-3-infected mice, lung tissue damage of hadv-7-infected mice was also higher on all days of infection (fig. 4b) . consistent with the morphological changes, the total number of cells in balf was higher, compared with those in the control group on all days (p < 0.001) (fig. 4c-e) ; importantly, the hadv-7 group showed a significantly greater increase in these cells, in comparison with the hadv-3 group (p < 0.05) (fig. 4d-e) . specifically, abundant macrophages and smaller number of lymphocytes, neutrophils infiltrated into the balf at all days post-infection. whereas the neutrophils registered a slight rise, only on day 3, followed by dramatic decrease, the lymphocyte and macrophage numbers remained persistently elevated through days 5 and 7. in addition, macrophages in balf of the hadv-7 group were significantly higher than the hadv-3 group after 5 and 7 days of note: data are shown as medians (interquartile range) or number (%) abbreviations: wbc white blood cell, crp c-reactive protein " a ": a significant difference between two groups infection ( fig. 4d-e) , while the lymphocytes exhibited significant difference only on day 5 (p < 0.05) (fig. 4d) . neutrophils in balf of the hadv-7 group was not significantly different compared with that of the hadv-3 group at all time points (fig. 4c-e) . together, these results showed that hadv-7 infection induced a relatively more severe acute airway inflammation. the levels of inflammatory mediators associated with airway inflammation, including il-1β, tnf-α, ifn-γ, and il-6, were also clearly elevated in balf at 3, 5, and 7 days post hadv-3 and hadv-7 infection. nevertheless, the levels of il-1β, il-6, and tnf-α in the hadv-7 group were higher than the hadv-3 group post 3, 5, and 7 days of infection (p < 0.01) (fig. 4f-i ). hadv-3 and hadv-7 have been prevalent throughout the world for decades, and both serotypes frequently play an important role in the etiology of lower respiratory infection in children. in the present study, coinfection with hadv-3 and hadv-7 with other respiratory viruses accounted for half of the pediatric cases, but coinfection per se did not result in a difference in clinical manifestation or severe clinical outcomes. these results and the severe respiratory diseases and complications, which we identified with hadv-7 infection in hospitalized children, were all consistent with previous reports [2, 3, 11, 15] . remarkably, we also found a higher rate (19.1%) of toxic encephalopathy associated with hadv-7 infection. previous studies in japan showed that mild encephalitis/encephalopathy with a reversible splenial lesion (mers) could be triggered by adenovirus infection [26, 27] , which was proposed to be caused by neurotoxins, likely acting as an antigen that induced a strong immune response, leading to a series of symptoms. recently, silkworm chrysalis ingestion was shown to induce toxic encephalopathy [28] ; however, our results constitute the first reported case of toxic encephalopathy associated with adenoviral infection. earlier studies reported that several viruses, including adenoviruses, influenza a virus, parainfluenza virus, and japanese encephalitis virus, can use the olfactory nerve as a shortcut into the central nervous system, causing infection of nerve cells and induction of immune response [29] ; however, the exact mechanisms of such neurotropic infections remain to be elucidated. in the current study, we have compared and correlated the biological characteristics of hadv-3 and hadv-7 with their properties ex vivo and in vitro. first of all, the results of cellular infectivity (fig. 1a) indicated that a549, 16hbe, and hek-293 are susceptible cell lines for the growth of adenovirus ex vivo. furthermore, a difference of viral loads recorded in a549 and 16hbe cell lines between adenovirus 3 and 7 indicated differential inflammatory reactions were produced from infection of host respiratory epithelial cells by hadv stimulated the innate immune system that might partially explain the role of hadv-7 infection in the pathogenesis of a more severe pneumonia. this is likely because the airway epithelial cells not only serve as a passive barrier to infectious particles but also actively participate in the innate immune response to foreign antigens. the immune response of epithelia to infection and antigen exposure involves the release of chemokines and cytokines into the submucosa, which initiates an inflammatory reaction [30] . the results of co-infection of the two viruses indicated that distinctive fitness is type-specific and not interaction. the result of cell viability also provided useful information that was important for understanding type-specific viral virulence (fig. 2) . taken together, we conclude that the differences observed in the replication, fitness, and virulence of the two adenoviruses are dependent on virus type. the complement system plays important roles in innate immunity and is vital in the protection against invading pathogens. the complement cascade can be initiated through three main pathways: classical, lectin, and alternative. the classical pathway is mainly antibodydependent. it is activated by interaction of c1q (a subunit of the first complement protein c1) with the fc portion of the igm or igg immune complex, although activation can also be achieved in an antibody-independent manner by some membrane components of viruses, bacteria and fungi. the lectin pathway is first activated when the mannose-binding lectin, a structural homologue of c1q, binds to carbohydrate moieties on the surface of pathogens, including yeast, bacteria and viruses. the alternative pathway is antibody-independent; it is spontaneously activated on biological surfaces, as well as in plasma and other body fluids, when the level of c3 hydrolysis remains consistently low. this spontaneous cleavage readily initiates amplification of the activation cascades [31] . although every pathway is triggered independently, all of the complement cascades culminate in the central cleavage of c3 and the generation of its active fragments c3a and c3b, which can bind covalently to viral components to aid in opsonization and phagocytosis. furthermore, complement activation also mediates the inflammatory reaction via the generation of anaphylatoxins (c3a and c5a) and recruitment of inflammatory cells to the site of infection [32, 33] . therefore, activation of the complement system leads to inflammation, opsonization, and membrane perturbation. previous studies by johnson et al. and others [24, [34] [35] [36] have shown that the complement is strongly activated by a wide range of negative-strand rna viruses, including parainfluenza virus 5 (piv5), nipah virus (niv), mumps virus ; c-e the number of total cells and the differential counting of cells in balf. f-i concentration of cytokine il-1β, tnf-α, and il-6 in balf; n = 4; *,**,*** indicate p < 0.05, 0.01, and 0.001, respectively, compared with the mock group; #, ##, ### indicate p < 0.05, 0.01, and 0.001, respectively, compared with hadv-3 group implicate the plasma level of c3a as a marker of the early innate response to adenovirus [37] . in previous studies, our group successfully established a hadv-7 pneumonia model in the laboratory mouse, and studies in this in vivo model suggested that hmgb1 is a mediator of hadv-7-induced pulmonary inflammation [38] . our current study has addressed whether different types of human adenovirus infection result in distinct inflammatory reactions in vivo. in this study, we clearly observed robust virus growth, and also documented significantly higher viral load in hadv-7infected mice at 3 days post-infection. these results are different from those reported by kajon et al. in which only low levels of hadv-3 and hadv-7 replication were seen at the early stage of infection in mice [39] . we speculate that the apparent discrepancy is due to the effect of different experimental animal models and detection methods used. adenovirus infection is characterized pathologically by a time-dependent progression in the type of inflammatory cells present. the initial inflammation is a neutrophilic interstitial infiltration with neutrophilic alveolitis. subsequently, monocytes become evident and, finally, predominantly lymphocytic infiltrates appear. during the acute stage of hadv-3 and hadv-7 infection, we also found that the total number and classes of cells in balf increased, primarily accounted for by macrophages. neutrophils only transiently increased at 3 days post-infection. a mixture of mononuclear cells (macrophages and lymphocytes) exclusively became evident at 5 and 7 days postinfection. macrophages were most prominent than neutrophils and lymphocytes at all time points. these results are fully consistent with several studies, including those of kajon et al., which showed that hadv-3 and hadv-7-induced pneumonia mainly produces neutrophil infiltration at 1 and 2 days post-infection, and affects macrophages [39] . apart from phagocytosing and killing the pathogens, macrophages also secrete chemokines to recruit cells to the sites of infection. yoon et al. found that adenovirus type 7 produced a more robust il-6 and il-8 response than adenovirus 3 in human bronchial epithelial cells [1, 30] . diaz et al. compared cytokine responses between type 7 and type 3, and documented significantly higher production of interferon-γ from type 7 [40] . similarly, subsequent production of il-1β, induced by macrophages, also reached the highest levels after 3, 5, and 7 days of hadv-3 and hadv-7 infection. asgari et al. have reported that engagement of the c3a receptor triggers il-1β processing and release via caspase-1 activation [41] . the results are consistent with the immune response of the epithelia to adenovirus infection and antigen exposure, which involves the release of chemokines and cytokines into the submucosa and initiates an inflammatory reaction. lastly, complement activation mediates the inflammatory reaction via anaphylatoxins (c3a and c5a) and recruitment of inflammatory cells to the site of infection. the inflammation then leads to the recruitment of phagocytes that help clear the invaders. collectively, we conclude that these innate immune responses play an important role in the initial defense against adenovirus infection. although our results implicate a novel type-specific relationship between adenoviral infection, it has potential limitations. the data presented herein only interrogated virological differences; however, adenovirus infection is a very complex process that involves not only the structural proteins of the virus, such as fiber, hexon, and penton, but also includes host and environmental factors. nonetheless, our data confirm that severe adenovirus infection was associated with hadv-7 rather than hadv-3. the significance of the study lies in that the pediatrician should be aware of the importance of early diagnosis and treatment of hadv-7 infection in children in clinical practice. the findings should also stimulate further studies of mechanisms of the different pathogenicity among human adenovirus serotypes. the severity of adenoviral infection, as studied in chongqing, china, may be correlated to human adenovirus type 7 (hadv-7) instead of type 3 (hadv-3). overall, strain of the hadv-7 type caused a more severe pneumonia and an exacerbated cytokine response, which also paralleled their more robust replication in cell culture, as compared to hadv-3. while the exact mechanism of the type-specific pathogenicity merits further investigation, these findings may eventually contribute to better control and treatment of adenoviral infection. serum inflammatory markers in patients with adenovirus respiratory infection adenovirus serotype 3 and 7 infection with acute respiratory failure in children in taiwan epidemical features of hadv-3 and hadv-7 in pediatric pneumonia in chongqing human mastadenovirus type 70: a novel, multiple recombinant species d mastadenovirus isolated from diarrhoeal faeces of a haematopoietic stem cell transplantation recipient computational analysis of a species d human adenovirus provides evidence of a novel virus human adenovirus associated with severe respiratory infection prevalence of adenovirus in children with acute respiratory tract infection in lanzhou comprehensive serotyping and epidemiology of human adenovirus isolated from the respiratory tract of korean children over 17 consecutive years (1991-2007) adenovirus: epidemiology, global spread of novel serotypes, and advances in treatment and prevention pneumonia and massive pleural effusion associated with adenovirus type 7 adenovirus pneumonia complicated with acute respiratory distress syndrome: a case report first report of sudden death due to myocarditis caused by adenovirus serotype 3 adenovirus infection associated with central nervous system dysfunction in children molecular epidemiology of adenovirus types 3 and 7 isolated from children with pneumonia in beijing lower respiratory tract infections due to adenovirus in hospitalized korean children: epidemiology, clinical features, and prognosis human adenoviruses in respiratory infections: sequencing of the hexon hypervariable region reveals high sequence variability phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy identification and typing of respiratory adenoviruses in guangzhou, southern china using a rapid and simple method a simple and efficient method for purification of infectious recombinant adenovirus a comparison of viral fitness and virulence between emergent adenovirus 14p1 and prototype adenovirus 14p strains in vitro characterization of human adenovirus type 55 in comparison with its parental adenoviruses, types 11 and 14 pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr quantitative real-time pcr assay panel for detection and type-specific identification of epidemic respiratory human adenoviruses a novel factor i activity in nipah virus inhibits human complement pathways through cleavage of c3b mmp-12-mediated by sarm-trif signaling pathway contributes to ifngamma-independent airway inflammation and ahr post rsv infection in nude mice epidemiology of acute encephalopathy in japan, with emphasis on the association of viruses and syndromes transient hemiparesis and hemianesthesia in an atypical case of adult-onset clinically mild encephalitis/ encephalopathy with a reversible splenial lesion associated with adenovirus infection the effect of hemoperfusion on patients with toxic encephalopathy induced by silkworm chrysalis ingestion the olfactory nerve: a shortcut for influenza and other viral diseases into the central nervous system cytokine induction by respiratory syncytial virus and adenovirus in bronchial epithelial cells complement: a unique innate immune sensor for danger signals complement. first of two parts second of two parts virion-associated complement regulator cd55 is more potent than cd46 in mediating resistance of mumps virus and vesicular stomatitis virus to neutralization incorporation of host complement regulatory proteins into newcastle disease virus enhances complement evasion differential mechanisms of complementmediated neutralization of the closely related paramyxoviruses simian virus 5 and mumps virus adenovirus activates complement by distinctly different mechanisms in vitro and in vivo: indirect complement activation by virions in vivo hmgb1 mediates hadv-7 infection-induced pulmonary inflammation in mice acute inflammatory response and remodeling of airway epithelium after subspecies b1 human adenovirus infection of the mouse lower respiratory tract differential effects of respiratory syncytial virus and adenovirus on mononuclear cell cytokine responses c3a modulates il-1beta secretion in human monocytes by regulating atp efflux and subsequent nlrp3 inflammasome activation we thank professor rong zhou, dr. xingui tian, and colleagues (state key lab of respiratory disease, guangzhou medical university, china) for kind assistance in various areas of the study. we would like to acknowledge the assistance of the patients and their caregivers involved in the study, the staff in the department of respiratory medicine and the department of intensive care unit of children's hospital of chongqing medical university help for nasopharyngeal aspirates collection. this study was supported by the national natural science foundation of china for young scholars (81301424). the funders had no role in the study design, data collection, analysis and interpretation of data and writing the manuscript. the datasets used and analyzed during the current study are included within the article and its tables. additional data may be available from the corresponding author on reasonable request.authors' contributions yxf performed the experiments, analyzed and/or interpreted the data, and wrote the manuscript. zzt, zxy, sm helped to perform the experiments. xgt contributed to the design of the paper and revised the manuscript. kn contributed to the design of the study. lr assisted revision of the manuscript. n z contributed to the design of the study and assisted in the analysis and/or interpretation of data, revised the manuscript. em l contributed to conception, collected clinical information, and revision of the manuscript. all authors read and approved the final manuscript. the study procedure was approved by the ethics committee of the children's hospital of chongqing medical university, chongqing, china. written informed consent was obtained from parent or guardian of all participants. not applicable. the authors declare that they have no competing interests. key: cord-018101-zd4v222b authors: kawashima, kent; matsumoto, tomotaka; akashi, hiroshi title: disease outbreaks: critical biological factors and control strategies date: 2016-05-31 journal: urban resilience doi: 10.1007/978-3-319-39812-9_10 sha: doc_id: 18101 cord_uid: zd4v222b disease outbreaks remain a major threat to human health and welfare especially in urban areas in both developed and developing countries. a large body of theoretical work has been devoted to modeling disease emergence, and critical factors that predict outbreak occurrence and severity have been proposed. in this chapter, we focus on biological factors that underlie both theoretical models and urban planning. we describe the sars 2002–2003 pandemic as a case study of epidemic control of a human infectious disease. we then describe theoretical analyses of disease dynamics and control strategies. an important conclusion is that epidemic control will be strongly dependent on particular aspects of pathogen biology including host breadth, virulence, incubation time, and/or mutation rate. the probability, and potential cost, of future outbreaks, may be high and lessons from both past cases and theoretical work should inform urban design and policy. interdisciplinary collaboration in planning, swiftness of information dissemination and response, and willingness to forgo personal liberties during a crisis may be key factors in resilience to infectious disease outbreaks. infectious diseases pose an ever-present danger to human societies. despite tremendous advances in medical care, roughly one quarter of worldwide human deaths are attributed to infectious and parasitic disease (mathers et al. 2008) . several seemingly unalterable aspects of urban life, including long-distance travel and dense human contact networks, facilitate outbreaks from both known and newly evolved pathogens. epidemics are defined as widespread occurrences of infectious disease in a community at a particular time, and the 14th century bubonic plague, or "black death", was the most devastating epidemic in human history (benedictow 2004) . death rates were as high as 25-60 % in europe, africa, and asia from a disease caused by a bacterial infection (yersinia pestis) that persists in rodent populations and is transmitted by fleas to humans. close contact between humans and rats and worldwide travel contributed to the global impact of bubonic plague which appears to have originated in asia and traveled to europe via trade routes (especially rat-infested ships). the most destructive modern pandemic was the 1918 influenza that infected one third of the world's population (about 500 million) and killed 50-100 million between january 1918 and december 1920 (taubenberger and morens 2006) . "spanish influenza", as the disease was named, is caused by the h1n1 virus which is endemic in pigs and birds and often transitions into human populations. the lethality of the 1918 strain was high and showed an unusual relationship between lethality and patients' age: 50 % of deaths were in the 20-40 age group which is the opposite pattern for milder flu strains (higher mortality among the very young and the aged). this partly reflected the impact of wwi where contagion was passed among troops both in training facilities as well as during warfare. however, the strain also had an unusual and lethal property; virulence was enhanced by a human immune over-reaction called a "cytokine storm" which causes the lungs to fill with liquid. some important aspects of the epidemic were: a deadly pathogen arose from a jump from animal to human (close between-species interactions were important in the origin of the virus), a few mutations were sufficient to confer strong lethality for the virus, and human travel allowed rapid spread (close quarters and massive troop movements helped to spread the virus and allowed new mutations to spread quickly). this chapter will focus on biological factors that are relevant for understanding and controlling epidemics. we will briefly describe some pathogens that cause human disease and their transmission mechanisms before analyzing the sars 2002-2003 epidemic as a case study of a modern urban epidemic. disease models will be discussed with a goal of determining how human societies can prepare to minimize the impact of future disease outbreaks. infectious diseases can be classified into two broad categories based on their pattern of transmission (table 1) . "long-range" infectious diseases are infections that do not require close contact for transmission. for example, water-borne diseases, such as cholera, can rapidly spread throughout the community when the supply of drinking water becomes contaminated with the pathogen vibrio cholerae through poor sanitation or hygiene practices. food-borne infections follow a similar transmission pattern to water-borne diseases. transmission through contaminated food and water is also known as "fecal-oral transmission" because fecal matter is often the source of contamination while oral ingestion is the primary route for infection (mount sinai hospital 2007) . diseases transmitted by an animal vector, such as bubonic plague, are also considered long-range infections because a vector facilitates the spreading of the pathogen and direct contact is not necessary. one interesting aspect of some vector-borne infections is that direct contact with an infected individual cannot transmit the infection without the help of the vector. for example, dengue fever, caused by a mosquito-borne virus, can only be transmitted through the bite of an infected mosquito (us centers for disease control and prevention 2014). in contrast, plague, caused by bacteria living in fleas of rodents, is primarily transmitted through flea bites, but contact with contaminated body fluids like blood can also lead to plague bacterial infection (us centers for disease control and prevention 2015a). in general, fecal-oral and vector-borne diseases are infections transmitted through an environmental (water, food) or a biological (animal) carrier that extends transmission range to large distances, but other routes are also possible depending on the specific pathogen. compared to long-range diseases, "short-range" infectious diseases are infections that transmit over limited distances and may require close or direct physical contact with an infectious individual. examples of short-range infections are pathogens that infect via contaminated airborne particles or expectorated droplets, and diseases that require contact with skin or bodily fluids such as blood or semen. infections capable of airborne transmission have the widest range among short-range infections and are caused by pathogens that spread through minute solid or liquid particles suspended in the air for an extended period of time (mount sinai hospital 2007) . in addition, the pathogen must be resistant to desiccation to remain viable for long periods of time outside its host. respiratory diseases are commonly believed to spread via airborne transmission of contaminated particles expectorated from coughing and sneezing. however, many respiratory pathogens do not have the capacity to withstand dry environments. instead, these pathogens transmit via "droplets"-expectorated moisture particles that are too big to indefinitely remain suspended in the air-to ensure ample moisture while outside the host. transmission occurs when contaminated droplets from an infected individual come in contact with surfaces of the eye, nose, or mouth. this mode of transmission is called "droplet contact". although diseases spreading via droplet contact have a more limited range than truly airborne infections, in the later sections, we will show how environmental factors can extend the range of droplet transmission. finally, diseases that transmit via direct contact generally have the most limited transmission range and some have stringent requirements for transmission. in the case of ebola, the disease is transmissible only via direct exposure of broken skin or mucous membranes with contaminated body fluids like blood, urine and semen, and excretions such as vomit and feces (us centers for disease control and prevention 2015c). sexually transmitted diseases like hiv/aids are a special form of direct contact infection that requires sexual intercourse or sharing contaminated needles for exposure (us centers for disease control and prevention 2015b). thus, short-range infections are characterized by some dependence on distance for infection and can be transmitted directly without a carrier. distinguishing between these two classes is important because measures to alleviate and control the spread of long-range infections are not applicable for short-range cases and vice versa. for instance, targeting the carrier or vector of the disease to control the spread of long-range infections (e.g., decontaminating or blocking off access to contaminated water or food) and reducing exposure to vectors of the disease, are irrelevant for mitigating the spread of short-range infections. in contrast, measures to control short-range diseases such as limiting person-to-person contact and imposing quarantine procedures do little to help alleviate the spread of water-borne or vector-borne illnesses. thus, identifying the mode of transmission is crucial to controlling the spread of any contagious infection. however, we will show that the distinction between long-and short-range transmissions is not always clear-cut. in this chapter, we focus on the emergence and spreading of severe acute respiratory syndrome or sars; the first worldwide pandemic in the age of globalized air travel and telecommunications. through theoretical analyses and data gathered from the epidemic, we examine how globalization exacerbates the problem of containing epidemics and show how urban environments can be especially prone to epidemics. the emergence and control of the sars epidemic is extensively documented. research on both the origin and epidemiology of the outbreak as well as the biological underpinnings of the disease making them excellent cases to determine methods to enhance urban resilience to epidemics. the history of the 2002-2003 global outbreak of severe acute respiratory syndrome (sars) provides key lessons on biological and policy factors that should be of general importance in designing resilient cities. we will summarize the history of the epidemic, with a focus on biological factors, before our discussion of disease models. according to the world health organization (who), over 8000 worldwide sars cases and over 770 deaths occurred in 30 different countries, mostly over a period of about four months (kamps and hoffmann 2003) . the severe, "atypical" pneumonia originated in guangdong province in southern china in mid-november 2002. most of the early cases appear to have occurred among those who kill and sell animals and meat as well as food preparers and servers (breiman et al. 2003) . by mid to late january 2003, the disease began to spread rapidly within the province, but a combination of symptoms difficult to distinguish from pneumonia (fever, dizziness, muscle soreness, coughing) and government policy to discourage coverage delayed the reporting of the epidemic until february 11. the initial communication reported 305 cases (including >100 healthcare workers) and 5 mortalities, but claimed that the epidemic was under control (enserink 2013) . the role of "superspreaders" and amplification in hospitals remained characteristics of sars as it spread to a worldwide epidemic. the first of several superspreading (generally defined as ten or more transmissions from a single infected individual) events occurred in hong kong on february 21, 2003 (braden et al. 2013 ). the index case was a physician from guangdong who stayed at the hotel metropole. the physician had treated sars patients in guangdong (although the disease was still unrecognized) and showed symptoms before his trip. he stayed only one night at the hotel before being hospitalized with severe symptoms but the short stay was sufficient to spread the infection to 13 or more of the guests from the same floor of the hotel as well as a hong kong resident who visited one of the guests. eventually, over 4000 (almost half) of the documented 2003 sars cases could be traced to this "index" case. remarkably, there was no known direct contact in most of the transmissions among the hotel guests and visitors. the hong kong resident who visited a friend in the hotel subsequently infected over 140 others at the prince of wales hospital in hong kong. others were business/holiday travelers who spread the pathogen to canada, vietnam, and singapore. as we will discuss below, this high transmission rate with little close contact in the metropole hotel remains mysterious. rapid recognition of a new epidemic was aided by a who disease expert, carlo urbani, who was asked to examine patients in a hanoi hospital. the affected included one of the metropole guests and roughly 20 hospital staff who became affected not long after his admission. urbani recognized a severe, and possibly new, disease and warned who headquarters as well as the hospital and vietnam government before contracting, and eventually dying from, the disease (bourouiba et al. 2014) . response time is a critical parameter in epidemic control and his efforts played a large role in the effort to subdue the epidemic. who designated a new disease, "severe acute respiratory syndrome" (sars), on march 10 and issued a global health alert on march 12 followed by an emergency travel advisory on march 15. the etiological agent of sars was later discovered to be a novel coronavirus and was named sars-associated coronavirus (sars-cov). this discovery, in late march 2003, came as a surprise to disease experts as previous human coronaviruses were only known to cause mild illness. in animals, related viruses were known to cause fatal respiratory as well as neurological diseases but coronaviruses are usually highly species-specific (kamps and hoffmann 2003) . forensic analysis of the metropole hotel in late april 2003 revealed physical components of sars in the common areas of the 9th floor including the corridor and elevator hall. however, no bacteria were found inside the guest rooms of the infected guests (the ventilation systems employed positive pressure within the guest rooms so that air was not shared among rooms). respiratory droplets, or suspended small particle aerosols generated by the index case-patient, are the most likely transmission mechanism (braden et al. 2013) . sars and other respiratory infections are considered to undergo short-range (approximately 1 m) transmission via pathogen-infected droplets from host coughing or sneezing. such transmission requires "close contact", physical proximity between infected and susceptible individuals who can be infected when large droplets spray enter their bodies via air or touch. however, minute droplets or even solid residues that can arise via evaporation (droplet nuclei) may allow potential indirect and/or long-range transmission (bourouiba et al. 2014) . for example, contaminated gas clouds that form during coughing/sneezing may have carried the pathogen and extended its transmission range, removing the distinction between droplet contact and airborne modes of transmission. aerosol transmission probably caused high infection rates in an airline flight (air china 112) from hong kong to beijing in which a single 73-year old individual infected at least 20 others (olsen et al. 2003) . this feature of the disease may be highly relevant for medical and urban policy. long-range aerosol/nuclei transmission does not require direct contact between infected and uninfected individuals and can greatly elevate the number of "contacts" for a given infected individual. interestingly, genetic analysis showed that several sars strains entered hong kong, but only the hotel metropole index case was associated with the subsequent global outbreak (guan et al. 2004) . a related superspreading event occurred at a crowded high-rise residence, the amoy gardens, in hong kong. many of the infected individuals inhabited vertically placed apartments (in contrast to transmissions on a common floor at the metro hotel case). sanitary drainage fixtures that were malfunctioning and allowing air and sars-contaminated aerosols to flow back into resident bathrooms may have been the main driver of infection spread in the condominium (stein 2011) . the superspreader was likely a medical patient undergoing treatment for a kidney problem including hemodialysis, a medical treatment that inhibits immune capacity (stein 2011) . the index case carried a high viral load and suffered from diarrhea. an important feature of this event was again, a lack of direct contact between the spreader and the individuals he infected, and the "opportunity" for the pathogen to be exposed to a large number of individuals through airborne transmission (yu et al. 2004) . at the amoy gardens, more than 300 individuals showed symptoms of sars almost simultaneously. high rates of hospital (nosocomial) transmission were an important and disturbing characteristic of the sars outbreak. the large fraction of infections among healthcare workers probably reflects a combination of contact from respiratory secretions from patients who were at a highly contagious stage (critically ill individuals also were the most infectious) as well as from medical procedures that inadvertently generated aerosol contamination. a single patient appears to have transmitted infections to over 140 hospital staff in a span of two weeks at the prince of wales hospital in hong kong (see below). two other superspreader events occurred in hospitals in other countries (braden et al. 2013) . one infected patient (the son of one of the hotel metropole guests) infected over 100 cases among patients, visitors, and healthcare workers at the acute care hospital in toronto, canada. finally, although taiwan instituted strict port entry screening and isolation of potentially exposed travelers entering the country, there was an outbreak in the ho ping hospital which spread into the community. in spite of a lock-down quarantine of over 1000 people in the hospital (included a large fraction of uninfected individuals), over 600 cases emerged before the outbreak was contained. the initial rapid spread of sars caused widespread concern and panic and the epidemic seemed unstoppable. however, the disease was eventually contained within several months through efforts coordinated by the who. although advances in biomedical science and cooperative efforts among laboratories played key roles in isolating the infectious agent, "classic" epidemiological practices of patient isolation (separation of infected individuals from the general population), contact tracing, and large-scale quarantine (isolation of non-symptomatic individuals who have had contact with the infectious agent) were the main elements that halted the epidemic ). the 2002-2003 sars pandemic was caused by a moderately transmissible viral infection that produced 2.7 new cases for every infection (riley et al. 2003 ) and yet it spread to over 30 countries across three continents potentially exposing tens of thousands of people in the span of only a few months. several studies have shown that the vast majority of infected cases had very low infectivity and that a few outliers were responsible for a disproportionate number of new infections riley et al. 2003; lipsitch et al. 2003; wong et al. 2004 ). in fact, riley et al. (2003) and lipsitch et al. (2003) found that early in the epidemic, an infected individual would only produce approximately three new infections when outliers are excluded. in singapore, 81 % of the first 201 probable sars cases showed no evidence of transmitting the infection yet 5 cases appeared to have transmitted the disease to 10 or more individuals (lipsitch et al. 2003) . shen et al. (2004) found a similar pattern in beijing where 66 out of the 77 confirmed cases did not infect others whereas four cases were responsible for infecting eight or more. the rapid spreading of sars despite only moderate average infectiousness has revived interest in the concept of superspreading events and heterogeneity in pathogen transmission. the transmission potential of an infectious disease is often described by the parameter r, the average number of new infections that infected cases produce over the course of their infection. r 0 is the transmission potential of an infected individual within an otherwise completely susceptible population (dietz 1993) . however, population-based summary statistics may obscure individual variation of infectiousness and other types of heterogeneities. woolhouse et al. (1997) have shown that heterogeneities in infectiousness exist such that only 20 % of the host population contributes at least 80 % of a pathogen's transmission potential. these individuals who significantly transmit more than the average are called superspreaders. in hong kong, apart from the incident at hotel metropole, at least two large clusters of infection were attributed to superspreading events (riley et al. 2003) . data from the sars pandemic showed the effect that superspreaders and superspreading events could have on the trajectory of the epidemic. given their crucial role in intensifying an outbreak, we review the risk factors that facilitate superspreading events. co-infection and the presence of a comorbid disease could be risk factors for turning infected individuals into superspreaders (stein 2011) . studies on hiv/aids transmission showed that co-infection with another sexually transmitted pathogen increased the urethral shedding of hiv in infected individuals. moss et al. (1995) demonstrated that urethral hiv infection is associated with gonococcal infection and treatment for urethritis may reduce the risk of hiv transmission. in the case of sars, peiris et al. (2003) reported that other viral respiratory pathogens such as human metapneumovirus were detected in confirmed sars cases. in addition, the index case in the prince of wales hospital superspreading event was described to have a "runny nose" , an uncommon symptom for a lower-respiratory tract infection such as sars. these observations have led to the hypothesis that co-infection or presence of a comorbid condition could endow an infected individual with characteristics or behaviors that increases their infectiousness (bassetti et al. 2005) . for example, rhinovirus, the major cause of common colds, can cause swelling of nasal tissues that can elevate airflow speed and contribute to aerosol production (sherertz et al. 1996) . rhinovirus co-infection with more serious, but less transmissible respiratory ailments, such as sars, could be an important factor contributing to high infectivity. environmental factors also play an important role in facilitating superspreading events (stein 2011) . in the sars superspreading event at the prince of wales hospital, the index case was placed on a nebulized bronchodilator four times daily for one week (kamps and hoffmann 2003) . nebulized bronchodilators are often used to deliver drugs to the lungs of respiratory patients but may have inadvertently aerosolized the virus and left infected droplets in the immediate surroundings leading to extensive dissemination of the pathogen (tomlinson and cockram 2003) . tracheal intubation, which involves placing a flexible tube into a patient's windpipe to maintain an airway to deliver drugs, may also have inadvertently spread sars within hospitals. patients often emit respiratory secretions during the procedure. an outdated ventilation system and overcrowding likely also contributed to the spreading of the virus at the prince of wales hospital (riley et al. 2003; tomlinson and cockram 2003) . through a case-control study of hospitals treating sars patients, yu et al. (2007) confirmed overcrowding as one of the general risk factors of hospital-based sars superspreading events. the case-control study performed included 86 wards in 21 hospitals in guangzhou and 38 wards in five hospitals in hong kong and showed that the main risk factors included closely arranged beds (less than 1 m apart), a workload of more than two patients per healthcare worker, hospital staff that continued working despite experiencing symptoms of the disease, and lack of washing or changing facilities for staff. despite the explosive growth and global distribution of the sars outbreak, the pandemic was largely contained through isolation and quarantine, increasing social distance, and social behavioral adjustments (bell and world health organization working group on prevention of international and community transmission of sars 2004). isolation and quarantine were shown to significantly interrupt transmission of sars in several countries including hong kong (riley et al. 2003) , china (pang et al. 2003) , singapore (lipsitch et al. 2003) , taiwan (twu et al. 2003) , and canada (svoboda et al. 2004 ). in general, symptomatic cases were immediately placed in isolation while contacts of confirmed infected cases were placed in some form of quarantine. in some cases, contacts were not immediately confined but instead were monitored for the disease and isolated only when symptoms emerged. confinement was usually at home but designated facilities were available in countries like taiwan (twu et al. 2003) . in some cases, individuals under quarantine were allowed to travel with the permission from the local health authorities provided they wore masks and refrained from using public transportation or visiting crowded places. to further reduce the chance of transmission, hong kong and singapore also closed schools and public facilities, and canceled mass gatherings to "increase social distance". people were also required to wear masks when using public transport, entering hospitals, or in jobs where interacting with numerous people is unavoidable such as in restaurants (bell and world health organization working group on prevention of international and community transmission of sars 2004). the concerted effort has been marginally associated with the rapid reduction of new sars cases in several countries. however, because of the simultaneous introduction of these measures, it is difficult to evaluate the effectiveness of each. several characteristics of the infectious agent were important factors in controlling the sars epidemic. the incubation period from contact with the infectious agent to onset of symptoms was, on average, 4.5 days. importantly, peak infectivity coincided with clinical symptoms and often required an additional 10 days or more ). thus, infectious individuals tended to be hospitalized before peak transmissibility. in addition, the two-week interval from exposure to high infectivity gave epidemiologists critical time to perform contact tracing to identify and quarantine potentially infected individuals before they reached high infectivity. this feature, in combination with moderate transmission rates (except in special cases), contributed to making sars a relatively controllable outbreak. in the next section, we present current theories on the emergence and spreading of epidemics and review the theoretical underpinnings behind control measures used to contain outbreaks. we briefly highlight different mathematical models used to describe epidemic dynamics in populations. we explain the factors that govern the emergence and transmission of diseases as well as the evolution of pathogens that cause them. finally, we examine how control measures such as isolation, quarantine, and vaccination mitigate the spread of infections. mathematical models have played an important role in our understanding of disease propagation. if biological factors can be accurately incorporated, such models may have predictive power to evaluate control strategies and guide policy. a key parameter in epidemic models is the total number of new infections that arise from a single affected host, the reproduction number, r. this value determines the outbreak potential of the infection; if r = 1, the infection will be maintained at a constant level (if we ignore random effects). r > 1 leads to disease spread and r < 1 predicts eventual extinction. however, r is not an intrinsic property of the pathogen. variability of the reproductive number across pathogens, hosts, and environments over time must be understood to accurately model disease. in the following three subsections, we discuss theoretical results on three important aspect of disease outbreak: (1) the effect of "superspreaders" on the probability of outbreak, (2) the impact of control strategies such as isolation and quarantine, and (3) factors that affect the evolution of pathogen virulence. the 2002-2003 sars epidemic was characterized by the large impact of "superspreaders" on disease propagation. in theoretical models, superspreaders can be treated as individuals with large number of connections to other individuals. individual-based simulations incorporating network structures can efficiently address this topic and, in this subsection, we introduce three theoretical studies focused on the effect of network structure on disease outbreak. lipsitch et al. (2003) studied the effect of superspreaders on outbreak probability using the estimated parameters from the sars outbreak in singapore. the authors first estimated the distribution of the parameter r, which expresses the number of new infections from an infected host. probabilities of outbreak (persistence of initially introduced pathogen lineages) were determined for r distributions with a fixed mean but differing in variance. the authors found that large variance in r distribution greatly decreased the probability of outbreak (fig. 1) . contrary to the expectation of the importance of superspreaders, their result showed that distributions strongly clustered around the mean had higher probabilities of outbreak than distributions that included superspreaders (right-hand tail outliers). one reason of this apparent inconsistency might be the assumption of a fixed mean r. under this assumption, increased variance in the r distribution increases both the numbers of individuals with extremely high r and low r. individuals with low r are essentially "dead ends" in disease infection and high numbers of such individuals will decrease outbreak risk. a similar result was obtained in meyers et al. (2005) . this study also focused on the case of sars outbreak in asian countries and used parameters estimated from the case study in individual-based simulations. meyers and co-workers examined differences in the probability of outbreak among three different networks among individuals. in the first network, termed "urban", many individuals have numerous contacts at public places including schools, hospitals, shopping centers and workplaces, and have more limited numbers of contacts at their home. the second network was a power law network, in which the distribution of the number of connections has a long right-hand tail. in such a distribution, a small fraction of people have large numbers of connections but most people have only a few connections. the third network was a poisson network, in which the majority of the people have numbers of connections close to the mean number. if the existence of superspreaders increases the probability of outbreak, then power law networks should show the highest outbreak probability. however, similar to lipsitch et al. (2003) , power law networks showed the lowest probabilities of outbreak. the reason might be similar to what we discussed above; in a power law network, the numbers of individuals with extremely small numbers of connection are elevated compared with the other two networks. pathogens cannot spread if they infect such individuals and will go extinct before they have a chance to infect superspreaders. fig. 1 theoretically estimated probability that a single introduced pathogen persists after infinite time under a markov process with different mean (e) and variance (v) in the r distribution. in lipsitch et al. (2003) this persistence probability is considered as probability of an outbreak. modified from lipsitch et al. (2003, fig. 4a) the two studies above indicated reduced probabilities of outbreak for populations that include superspreaders, but this conclusion may be strongly sensitive to model assumptions. networks with more total connections (including superspreaders) may realistically model urban environments (this relaxes the assumption of constant mean connectedness). fujie and odagaki (2007) modeled superspreaders as individuals with higher infection rates (strong infectiousness model) or more connections including connections with distant individuals (hub model). they calculated the probability of outbreak under different fractions of superspreaders in a population and showed that, as the fraction of superspreaders increases, the probability of outbreak increases greatly (fig. 2) . they also analyzed several features of outbreaks like speed of disease spread and infection path between the two models and suggested that the hub model is consistent with data from the sars outbreak in singapore. these contrasting results highlight the need to validate model assumptions for applications to human society. higher outbreak probabilities with larger numbers of connections may seem obvious but this may be a realistic scenario for human society. a key issue is whether the number of connections of one person statistically affects that of others in human society. if not, the comparison between different fraction of number of superspreaders like fujie and odagaki (2007) would fig. 2 theoretically estimated "percolation" probability of a single introduced pathogen under different fraction of superspreaders and population density in a hub model. in fujie and odagaki (2007) this percolation probability of percolation theory, in which a pathogen that has infected an individual in the bottom of 2 × 2 grid finally reaches an individual in the top of the grid, is considered the probability of an outbreak. as density becomes lower, distance between individuals becomes longer. the results for different fraction of superspreaders (λ) are shown in different markers. modified from fujie and odagaki (2007, fig. 4) more realistically predict the effect of superspreaders on the probability of outbreak. however, if higher number of connections of one person necessitates reduced numbers for others, the results in lipsitch et al. (2003) and meyers et al. (2005) could be more applicable for human society. in either case, models should focus on both outbreak probability as well as the nature (explosiveness) of disease spread. lloyd-smith et al. (2005) demonstrated that many previous human epidemics appear to have spread through superspreaders (although not to the same extent as sars). they showed that, although pathogen extinction probability increases with variance in reproductive number, populations with superspreaders experienced more rapid infection spread in cases of pathogen survival. under their model, host populations may suffer greatly from improbable epidemics. the first step to control the rise of any infectious disease is to understand how it transmits between hosts. often, we imagine these infections as readily communicable illnesses that can be caught by even the most fleeting contact. but as we have shown, exposure and transmission depends on the route the infectious disease pathogen takes. this means that some diseases can be transmitted even without direct or close contact with an infected individual. we have also shown how particular conditions can make a close-range disease transmit over extended distances, as is the case with sars transmission in the amoy gardens condominium complex. aside from mode of transmission, the timing between infectiousness and showing symptoms of the disease is another crucial factor to consider. an infectious disease is an illness caused by the presence of a pathogen within the host as well as the host's response to the invading pathogen. upon entry into the host, the pathogen begins to increase its numbers by redirecting resources to itself. after a certain time, its presence and the damage it has done to the host raises an internal host response to thwart the infection. it is at this stage of the infection that overt symptoms appear and the infection can be observed. the time elapsed between exposure to the pathogen and observing the initial signs and symptoms of the disease is called as the "incubation period" of the disease. the length of the incubation period varies among diseases and is affected by several factors such as dose and route of infection, and host susceptibility and ability to respond to the pathogen. because of these considerations, incubation period is described as a range of values depicting how short or how long it takes before an infection would show symptoms. during this period, the infected individual may or may not be contagious depending on the type of disease and the individual's health state. the disparity between the time we observe the symptoms of the infection and consider an individual ill and the time the individual is contagious are important aspects to consider in modeling as well as in prescribing infection control measures. the timings vary widely depending on the infectious disease (fig. 3) . in the simplest scenario, the entire time an infected individual is contagious occurs after the first symptoms of the disease and ends well before the symptoms disappear. a completely overlapping timing where all symptomatic individuals are infectious would simplify identification and make control measures more effective. this timing pattern can be easily modeled by assuming that newly infected individuals simultaneously start to cause new infections to other individuals. and because the disease spreads specifically through a single class of individuals, control measures can simply identify symptomatic individuals to prevent new infections. in the case of sars, peak infectiousness occurs 7-8 days following the onset of disease symptoms and correlates with viral load over the course of the infection ). many believe this pattern helped contain the sars pandemic (chau and yip 2003; diamond 2003; fraser et al. 2004 ) despite exponential growth of the epidemic that quickly spread to multiple continents. in contrast, diseases such as hiv/aids have completely different infectious and symptomatic periods. the first signs of aids do not appear until the infecting pathogen has significantly damaged the host yet the infected individual is contagious throughout the asymptomatic phase and peak infectivity occurs before the onset of symptoms ). modeling diseases with disconnected infectious and symptomatic periods requires splitting the "infectious" class into "asymptomatic infectious" and "symptomatic infectious" classes to more accurately reflect the clinical characteristics of the disease. though sars and hiv/aids have significantly different timing patterns, the relationship between peak infectivity and symptomatic period is clear. however, some diseases exhibit partially overlapping contagious and symptomatic periods that make their outbreaks more difficult to stop. identifying the precise period that infected individuals are contagious is difficult because the values are affected by anderson et al. (2004) numerous factors such as susceptibility of the host, mechanism of infection, and immune response (baron 1996) . individual variation in incubation periods further complicates the problem. in dealing with diseases that exhibit partially overlapping periods such as pandemic influenza, it is best to rely on conservative measures that consider both exposed and likely infected individuals as targets of containment measures. note that it is possible to harbor an infection yet not show any signs or symptoms of the disease. called a "subclinical infection", this asymptomatic state may be a result of the pathogen infection strategy and the host's ability to tolerate an infection instead of purging it (baron 1996) . asymptomatic cases that are infectious can help spread the contagion despite strict control measures by being misclassified as uninfected individuals. asymptomatic cases are usually discovered by chance or by reviewing epidemiological data after an epidemic (baron 1996) . modeling asymptomatic cases requires adding an "asymptomatic infectious" class that is capable of exposing and transmitting the disease. containing the spread of an infectious disease suspected to have a high proportion of asymptomatic infected individuals is difficult but procedures such as contact tracing may reveal some of these asymptomatic carriers and quarantining of exposed and high-risk individuals can minimize their impact. most emerging infections have no available vaccine or treatment. thus the only way to control the spread of these diseases is to prevent exposure and further transmission. isolation and quarantine are two control measures that help block transmission by isolating the individuals who have, or may have, the contagious disease. "isolation" describes separating sick individuals (symptomatic) from people who are not sick (naïve) while quarantine pertains to the practice of separating and restricting the movement of asymptomatic individuals who may have been exposed to the disease to see whether they become sick. these control measures aim to progressively reduce the number of new secondary infections until the disease is eradicated from the population. formally, we can measure the effect of isolation and quarantine by taking a survey of new infected cases and deriving the basic reproduction number r of the infectious disease for each step of the outbreak. without any intervention, r is expected to eventually decrease as the number of susceptible individuals decreases in a finite population without migration. however, by the time the rate decreases to r < 1, a large proportion of the population has already been infected with the disease. by "removing" potentially infected individuals from the population, isolation and quarantine can more rapidly decrease r below 1 by reducing the incidence of the disease, leading to fewer new infected cases capable of transmitting the infection. isolating symptomatic individuals prevents new cases by separating individuals spreading the pathogen from the host population. given a clearly defined set of symptoms to diagnose the disease, this strategy is intuitive and straightforward to implement from a public health point of view. a precise case definition also reduces misdiagnoses and prevents unnecessary isolation of non-target cases. however, many diseases share symptoms and may occur in combination with other infections so case definitions are not always precise. in the sars epidemic, infected individuals showing atypical symptoms were a major source of transmission, partly because co-infection may have elevated transmission rates (kamps and hoffmann 2003) . modern biomedical research may serve to quickly identify new pathogens and providing diagnostic tests may be the most important function of initial research (vaccines and treatments generally require months or years and may not be helpful for new diseases). isolating symptomatic individuals is most effective if peak infectivity occurs after observing the first symptoms of the disease and transmission only occurs in symptomatic cases ). while diseases like sars have shown such properties, other infections such as influenza appear to be transmissible even prior to showing overt symptoms. when peak infectivity occurs before the onset of symptoms, quarantine for symptomatic individuals may have little impact on dampening the spread of the infection . even for infectious diseases that transmit only after symptoms emerge, infected individuals may not immediately practice self-isolation or report to a healthcare facility. during the lag time between diagnosis and isolation, the pathogen can still spread to susceptible hosts undermining isolation as a way to control the spread of the infection. on the other hand, quarantining individuals that have been exposed to the disease addresses the shortcomings of isolation as a control measure. identifying exposure is dependent on how the pathogen spreads from one host to another. if the pathogen transmits via airborne droplets, then people present in the same room with an infected individual are considered "exposed". however, if the pathogen spreads only through sexual contact, then only individuals who have had sexual relations with the infected case are considered exposed. when the transmission mechanism is unknown, scenarios such as airborne transmission or via physical contact that lead to the most conservative outcome may be used instead. because the criteria to select individuals are independent of disease status, this strategy sacrifices sensitivity but works regardless of timing of infectivity and does not suffer from the lag time problem. such a conservative strategy is well suited for emerging infections, especially when the mechanisms of transmission and pathogenesis have yet to be revealed. in a perfect quarantine, all exposed individuals are expected to undergo quarantine regardless of whether they develop the disease or not, and during the quarantine period, exposed individuals do not transmit the disease. however, tracing all contacts is often problematic especially when an infected individual has traveled to numerous locations and when exposure occurred in public spaces and mass transit. compared to isolation, quarantine sometimes faces more resistance from expected participants especially from those who have been exposed but appear to be in a healthy condition. during the sars epidemic, mass quarantines were implemented in many countries. over 130,000 potentially exposed individuals were quarantined in taiwan, but in retrospect, the action may have spread panic among uninfected individuals and may not have been an effective strategy (university of louisville school of medicine 2003). in reality, quarantines are never perfect. compliance to the procedures is often problematic: quarantined individuals do not reduce their geographical movement or they only abide by the procedure for a short period. formal quarantines have good compliance rates but are costly and difficult to manage for a large number of cases. therefore a majority of quarantines are made voluntarily or with less monitoring than formal quarantines, but these suffer from reduced compliance and are less effective overall. knowledge about potential superspreaders to identify candidates for isolation can greatly enhance the efficacy of quarantines with much lower numbers of required isolations (diamond 2003) . although such knowledge may be rare at the beginning of an epidemic, rapid epidemiological analyses may play a critical role in reducing the costs of epidemic control. a critical aspect of human pathogens is their virulence or extent of damage to host. high virulence infectious disease such as hiv, plague or smallpox can be a great threat to human society, and the number of cases of pathogens that have been reported to have evolved virulence and/or resistance against drugs is alarming (altizer et al. 2003; holden et al. 2009 ). understanding the factors which affect the evolution of virulence in human society is an important issue. if it is possible to control these factors in urban design, human society can be more resilient against serious disease outbreaks. several classic theoretical studies on the evolution of virulence concluded that reduced virulence is generally adaptive and should evolve among pathogens. low virulence allows infected host individuals to survive, and pathogens can have more chance to spread to other host individuals. if pathogens have high virulence, they can propagate within an infected host individual, but risk killing the infected host and limiting their spread to other hosts. trade-offs between reproduction within a host and transmission among hosts is a well-studied explanation for the evolution of reduced virulence (anderson and may 1982; alizon et al. 2009 ). however, the balance (or equilibrium) of this trade-off can differ depending on biological characters of pathogens. ewald (1993) discussed how transmission mechanisms of pathogens can alter the predicted trajectory of virulence evolution. highly virulent diseases tend to immobilize hosts in early stage of infection. therefore, if pathogens are mainly transmitted by contacts between hosts, higher virulence would greatly decrease chances of new transmission. however, if pathogens can survive outside of the host and can be transmitted by air, water or vectors in which they are not virulent, host immobility should have less effect on the chances of new transmission. ewald (1993) noted that such pathogens, such as smallpox, tuberculosis or diphtheria, are often more virulent than pathogens that depend more directly on hosts for transmission. other factors can affect the balance of the trade-off and allow evolution of high virulence (galvani 2003) . for example, if multiple pathogen strains infect simultaneously and compete within individual hosts, high reproduction rate within a host (leading to high virulence) may be favored. in sexually-transmitted diseases, frequent exchange of sexual partners makes transmission of pathogens between hosts easier and as a result cause high virulence. this may be the case of hiv in human society (lipsitch and nowak 1995) . host population structure also affects the transmission of pathogens and therefore, has a large impact on the evolution of virulence. because urban planning and design can create or alter population structure by its use of the the environment, in the following paragraphs, we introduce two studies focused on the effect of host population structure on evolution of virulence. these studies are based on relatively simple models that may yield general insights. boots and sasaki (1999) incorporated a grid-like spatial structure of "sites" at which individuals can exist. each site can have one of three states: empty, occupied by susceptible individual, or occupied by infected individual. in the spatial structure, connections between individuals were divided into two types, those between neighbors and those between randomly chosen individuals. randomly chosen individuals can be in distant sites, and in such cases, pathogens can be transferred to distant locations. they found that pathogen virulence is favored as contact between hosts living in distant places becomes more common. in this model, a site becomes empty after death of an occupant. therefore, higher virulence is more likely to create a situation in which pathogens kill all susceptible hosts around them and can no longer spread. however, long-distance transfer allows pathogens to spread to new locations where they are surrounded by susceptible hosts. long distance transportation in human society allows contact between distant individuals and may be an important factor that facilitates the spread of outbreaks and favors pathogen virulence. boots and sasaki (1999) did not consider host immunity in their model. immune (infection-resistant) hosts can block pathogen spread and may have a large impact on the evolution of virulence. this question was theoretically addressed by the same authors. boots et al. (2004) incorporated the immune state after the recovery assuming a negative correlation between recovery rate and virulence and found that evolutionary trajectories could lead to low, or even extremely high, virulence depending on host population density. in host populations with high density, pathogens can easily find susceptible hosts and therefore, low virulence which increases the opportunity of infection to a new host evolves. on the other hand, in host populations with low density, immune hosts around a newly infected host efficiently block pathogen spread. in this case, highly lethal pathogens which kill infected hosts and make open spaces can spread more efficiently compared with pathogens with low virulence which induce immunity in hosts. even after killing some hosts, pathogens still have a chance to spread by infecting new susceptible hosts that emigrate to the open spaces. in boots and sasaki (1999) , infected hosts are assumed to be susceptible just after they recover and therefore, lower virulence pathogens spread more efficiently. however, in boots et al. (2004) , immune hosts block pathogen spread and create scenarios where highly lethal pathogens evolve. the results in boots and sasaki (1999) and boots et al. (2004) reveal scenarios in which low virulence can evolve to higher virulence depending on the structure of host populations. a key point is that outcomes are sensitive to the scenarios of population structure, transmission mechanisms, and host immunity. because of their short generation times and high genetic mutation rates, pathogens like rna viruses may evolve rapidly, even over the course of an outbreak. influenza virus, norovirus or dengue virus are well known examples of rna viruses that infect humans. because these viruses cause epidemics every year, controlling their impact is a very important aspect of urban resilience. as mentioned above, the models in boots and sasaki (1999) and boots et al. (2004) may be too simple to directly apply for particular diseases. theoretical studies under more realistic conditions based on structures that closely resemble actual human society and biological characteristics relevant to particular pathogens will be valuable to prevent and control outbreaks of high virulent diseases. important points to consider include parameters and assumption sensitivity for aspects of both host populations and pathogens. in addition, the definition of "connection" differs depending on the transmission mechanism of the pathogen. the concept of "network" must take the view of the pathogen and different networks may need to be considered for different diseases in the same human populations. many of the major human infectious diseases are zoonotic infections that have crossed over from animals into humans (wolfe et al. 2007 ). bubonic plague (schmid et al. 2015) , influenza (palese 2004) , hiv (gao et al. 1992) , ebola (marí saéz et al. 2015) , sars (lau et al. 2005; li et al. 2005b ) and mers (memish et al. 2014; wang et al. 2014 ) have all been shown to have originated from animals before infecting humans. wolfe et al. (2007) surveyed 25 major infectious diseases ranked by highest mortality and/or morbidity to identify patterns in their animal origins and geographical spreading. all the diseases they surveyed appeared to have originated from the old world (africa, asia, europe) and a remarkable proportion of causative pathogens arose from warm-blooded vertebrates while the remaining were attributed to birds. interestingly, the purported geographical origin of the disease was correlated with the type of animal to which the pathogen originally infected. for example, many diseases that trace back to tropical regions have come from wild non-human primates whereas diseases attributed to temperate regions often emerged from domestic animals. although the exact reason for this pattern is unknown, wolfe et al. (2007) suggested that, because livestock and pets were domesticated in the old world, ancestral pathogens had more opportunity to infect humans compared to more recently domesticated new world animals. for the disparity between old world and new world monkeys, they believe that closer genetic relatedness between human and old world monkeys may have aided in cross-species transmission. these results stress the importance of considering both environmental and biological factors as key determinants of cross-species transmission of infectious diseases. recent spreading of human population by urbanization exposes us to novel pathogens that were previously isolated from human society. the risk of zoonotic infections may be increasing and it is notable that many novel pathogens appear to have high virulence in human (reads 1994; schrag and wiener 1995) . during their long evolutionary history, pathogens and their original hosts may have been recurrently co-evolving by which hosts evolve to be resistant against the pathogens, and pathogens evolve to evade the resistant system (little 2002; woolhouse et al. 2002) . this means if hosts are exposed to a novel pathogen, it is highly possible that the hosts do not yet have immune resistance against the pathogen and are affected by high virulence (longdon et al. 2015) . there are also cases in which infections of novel pathogens cause inappropriate immune response and as a result, increase their virulence (graham and baric 2010). as introduced above, these highly virulent pathogens can spread in a host population depending on host spatial structure. however, it is important to note that not all novel pathogens have high virulence for human. highly virulent pathogens are more likely to be detected and studied and therefore, the patterns may result from ascertainment bias (alizon et al. 2009; longdon et al. 2015) . in any case, careful surveillance of both human and animal populations in regions of high human-animal contact may be an important component to defending against novel disease (woolhouse et al. 2012) . finding the original animal host of a new human pathogen requires scientific rigor but also guesswork and luck. the search for the animal reservoir of the sars pathogen first identified the himalayan palm civet (paguma larvata) after sars-like coronaviruses (sl-covs) were isolated from civets in live-animal markets in guangdong, china (guan et al. 2003) . however, tu et al. (2004) showed that while civets in live-animal markets were infected with sl-covs, civets on farms did not possess antibodies against the virus, which indicated that they have never been exposed to the pathogen. moreover, palm civets infected with sars-cov showed signs of illness contrary to the expectation that animal reservoirs should be clinically asymptomatic (calisher et al. 2006) . this observation and that other animals in the same live-animal markets were also infected by the virus (guan et al. 2003) indicated that the palm civets were infected in live-animal markets rather than being the ultimate source of the pathogen. surveillance of wild animals in the region later lead to the serendipitous discovery that chinese horseshoe bats (rhinolophus sinicus) are the original animal host of the coronavirus that became sars-cov (lau et al. 2005; li et al. 2005b) . the focus on bats may have been inspired by outbreaks of nipah and hendra virus a decade before that were also traced back to these mammals (normile 2013 ). in addition, li et al. (2005b) stated that the use of bat products in food and traditional medicine in southern china led them to investigate bats as a potential reservoir. interestingly, bats appear to harbor many human pathogens and have been implicated as the animal reservoir of nipah virus, hendra virus, ebola virus, and sars-cov. even mers-cov, initially transmitted from camels, have been traced back to bat through phylogenetic analysis and biochemical studies (wang et al. 2014; yang et al. 2014) . while sars-cov infection primarily affected the respiratory system, high concentrations of coronavirus were observed in bat feces and recovery from the small and large intestines indicate that replication is primarily through the excretory system (drexler et al. 2014) . lau et al. (2005) speculated that the use of bats in traditional medicine, especially bat feces, may have played a crucial role in the cross-species transmission of the virus. bat meat is also considered a delicacy and many chinese believe it possess therapeutic activity, which led to bat trade in live-animal markets such as those in guangdong, china. exposure between the pathogen reservoir and the new potential host species is a key factor dictating the probability of successful cross-species transmission. for example, hiv-1 and -2 seem to have transferred multiple times to humans since 1920 based on phylogenetic analysis, but only after 1970 was there a significant spreading of the infection (heeney et al. 2006) . one explanation suggests that the limited interactions between humans and primates created a barrier for the transference of the virus and insufficient interhuman encounters of infected cases delayed the rise of the epidemic (parrish et al. 2008) . to describe this phenomenon, let us model the underlying host contact network as a network of nodes (individuals) and connections (exposure). assuming a heterogeneously connected network such as human social network and contact networks (eubank et al. 2004) , we find that the probability of a new infection becoming extinct by chance is very high both because the pathogen may be poorly adapted to transmit in the new host (parrish et al. 2008; daszak et al. 2000; dobson and foufopoulos 2001) and because cross-species transmission events tend to occur in sparsely connected rural areas (tibayrenc 2011) . the limited connections inhibit emergence of the disease and only the few that avoid stochastic extinction proceed to produce an epidemic in the host population (lloyd-smith et al. 2005; eubank et al. 2004 ). this may explain why spillover events of animal infections, such as h5n1 avian influenza, fail to take hold in the human population despite the hundreds of human cases and deaths that have been reported (parrish et al. 2008) . while distribution is skewed towards fewer connections in these networks, it is still possible that the cross-species transmission event occurs at a highly connected portion of the network. such an outcome will only make it more likely that the infection will take hold to produce an epidemic due to the presence of highly connected hubs that can spread the disease to a disproportionate number of hosts (rock et al. 2014) . lloyd-smith et al. (2005) expand this concept to show that any type of individual variance, for example infectiousness, produces the same effect. high individual variance increases the probability of extinction of an invading disease regardless of the strength of mean infectiousness. when the host population has a highly heterogeneously connected network, emergence of disease may be rare, but infections that survive stochastic extinction produce "explosive" epidemics similar to the case of sars in 2002. these findings show that host population structure and demography significantly affects the probability of cross-species transmission as well as the subsequent epidemic that may follow. host factors also play a significant role in determining the success of new infections in novel hosts, especially for viruses. to infect a host, a virus must be able to interact with the host's cellular receptors to gain entry into cells and hijack the cell's machinery to replicate itself. at the same time, the pathogen must survive against the host's defense mechanisms. the initial interaction between the virus and host receptors is a critical step that determines host specificity and host range. for example, in sars as well as in other coronaviruses, the viral structure responsible for viral entry is the spike glycoprotein, which also appears to be the key determinant of host specificity (graham and baric 2010) . in humans, the receptor-binding domain on the spike glycoprotein interacts with a cell surface metalloproteinase called human angiotensin-converting enzyme (ace) 2 to gain entry and infect lung epithelial cells (li et al. 2003) . however, ren et al. (2008) showed that sl-covs found in bats do not interact with palm civet or human ace2 receptors implying that changes must have occurred to gain this new interaction. in fact, there appears to be a sizeable difference between coronaviruses isolated from the putative bat reservoir and sars. sl-covs from bats were found to be at most only 92 % similar compared to sars-cov (li et al. 2005b) . later, ge et al. (2013) were able to isolate and characterize a sl-cov that utilizes the ace2 for cell entry in bats, palm civets and humans. this finding argues that ace2 utilization may have evolved prior to any cross-species transmission event. while gaining the ability to bind to a novel receptor appears to be a complicated process, in some instances, even a few amino acid changes may confer the ability to recognize a new species. initial studies comparing sars-cov isolated at different time points in the pandemic revealed the spike protein of viruses taken from palm civets and early human cases bind less efficiently than those from later on in the epidemic (yang et al. 2005) . further genetic and biophysical studies demonstrated that two amino acid changes had an enormous effect on the binding affinity of the sars spike protein to human lung epithelial cells (li et al. 2005a, c; qu et al. 2005) . in most palm civet samples, lysine at position 479 and serine at position 487 of the spike protein were observed whereas asparagine and threonine were present in human samples. li et al. (2005a) found that replacing lysine with asparagine removed the electrostatic interference with the histidine residue on the receptor while replacing serine with threonine provided a methyl group capable of filling in a hydrophobic pocket at the interface of the human ace2 receptor. although the structural changes appear to be subtle, these substitutions caused a thousand-fold increase in binding affinity to the human ace2 and lead to enhanced human transmission. however, li et al. (2005a) also found that some civet specimens have asparagine instead of lysine at position 479 yet this did not affect binding to the civet ace2 receptor. changes that are neutral to the original host but advantageous in the new host may have played a critical role in facilitating cross-species transmission between palm civets and humans. once a pathogen has evolved to reliably infect the new host's cells, the innate immune response is the host's first line of defense against the infection. when a virus successfully infects a cell, cytoplasmic enzymes that detect the production of double-stranded rna, a hallmark of virus replication, activate the expression and release of interferons from the cell. interferons act as an early-warning signal to other cells nearby by activating their intracellular antiviral response to combat viral infection and replication (roy and mocarski 2007) . because of the importance of this immune response against viral infection, many viruses have evolved features to subvert interferon signaling. for example, the influenza virus prevents the infected cell from detecting viral replication by using its ns1 protein to sequester double-stranded viral rna (lu et al. 1995) . another method to interfere with the innate immune response is to prevent interferons from activating antiviral mechanisms. nipah virus produces two proteins that prevent stat1 from translocating into the nucleus as well as another protein that sequesters stat1 present in the nucleus, obstructing the activation of interferon-stimulated genes (shaw et al. 2004 ). in the case of sars, kopecky-bromberg et al. (2007) found that sars-cov nucleocapsid and accessory proteins inhibit both the expression of interferon and associated transcription factors, as well as inhibiting cellular response to interferon by subverting the jak/stat activation of intracellular antiviral mechanisms. while infection with sars-cov did not induce production of interferons, coinfection with another virus did produce interferons. it appears that sars-cov does not induce interferon expression, yet does not shut down the whole pathway as interferon signaling continues to work when other stimuli are present (frieman et al. 2008) . by antagonizing the induction and response to interferons, the pathogen blocks the activation of more than 300 interferon-stimulated genes which prevents the cell from going into an "antiviral state" (de lang et al. 2009 ). under the antiviral state, inhibitors are activated to prevent cell division, enzymes that digest proteins initiate programmed cell death, and proteins that present viral particles to activate the adaptive immune response are upregulated. blocking interferon signaling causes a general decrease of both innate and adaptive immune system response, allowing sars-cov to infect cells unimpeded and potentially cause a more serious disease. the rate at which a pathogen evolves is another biological factor that may determine risk of cross-species transmission. most recent emerging infections have been caused by rna viruses such as hiv (gao et al. 1992) , ebola virus (gire et al. 2014) , dengue virus (gubler 1998) , sars-cov (lee et al. 2003) and mers-cov (de groot et al. 2013) . rna viruses have an extremely high mutation rate because their rna polymerase, the enzyme that copies their genome, lacks proofreading activity which leads to error-prone replication. mutation rates for rna viruses range from 10 −6 to 10 −4 substitutions per nucleotide per cell infection, two orders of magnitude higher, on average, than dna viruses (sanjuán et al. 2010 ). at those rates, about 1 out of 100,000 nucleotide changes every time an rna virus replicates itself. this may not seem high but note that hundreds of millions of viral particles may be produced during a single infection (haase 1994) , which gives the virus numerous opportunities to explore potentially advantageous mutations. although high mutation rates helps rna viruses to rapidly adopt advantageous changes and alter phenotype, deleterious mutations are also produced at an elevated rate. lauring et al. (2012) have demonstrated that viruses mitigate the effects of deleterious phenotypes by outcompeting and quickly purging these low-fitness variants. the ability of viruses to incorporate functional components made by other functional viruses within the same cell appears to also mitigate the negative effects of high mutation rates (makino et al. 1988 ). indeed, studies have shown that raising the mutation rate through mutagens can be used to create large numbers of dysfunctional mutants that rapidly leads to the extinction of the viral population (pathak and temin 1992; loeb et al. 1999; domingo 2000) . interestingly, in the case of sars, the coronavirus that caused the disease did not have a very high mutation rate relative to other rna viruses. coronaviruses have the largest genomes (approximately 30,000 nucleotides) among rna viruses and genome size and mutation rate appear to be negatively correlated (sanjuán et al. 2010) . one reason behind the relative stability of coronavirus genomes could be the presence of proofreading enzymes that guard against mutagenesis. in the case of sars-cov, smith et al. (2013) showed that the exoribonuclease domain in non-structural protein 14 had proofreading activity and was responsible for protecting the viral genome against mutagenesis. although high mutation rates appear to facilitate adaptation of rna viruses to new environments, diseases of animal origin are not always caused by the fastest evolving rna viruses. the discussions above have introduced how several aspects of urban life, including high connectedness of individuals (including connections among distant individuals) and regions of high human/animal contact, are likely to elevate the risk of future epidemics. because these properties may be intrinsic to urban life and difficult to alter or control, monitoring and preparedness are critical for urban resilience to disease outbreak. opportunities for the evolution of new or variant human pathogens are difficult to limit and may, in fact, be increasing in modern societies. each contact between microbe and host can be considered a "trial" for a potential pathogen with random mutations in their genomes that may confer new functions or specificities. thousands of such trials occur daily in many regions and are likely spawning candidate emerging pathogens with the ability to reproduce within humans and possibly also to transmit from person to person. the trajectory of pathogen evolution depends strongly on the numbers of contacts (potential transmissions) among individuals in the host population as well as on chance. the vast majority of potential new pathogens are likely to be lost early in their histories. however, given continuous opportunities, the chance event of pathogen emergence is simply a matter of time. in guangdong province, several recent outbreaks of bird influenza (h7n9) have led to limits on live poultry markets, but consumer preference for freshly slaughtered poultry and wild animals remain an impediment to regulating the high-risk concentration of multiple species (pig, poultry, dog, cat, rabbit, as well as reptiles, fish and numerous wild game) in close contact with one another (and often in poor health) as well as with humans. regions of recent human expansion where wild animal populations are in close proximity to high density human settlements must also be monitored carefully for new zoonotic diseases. new strains of swine and bird influenza are currently monitored as candidates for outbreaks but pathogen emergence is unpredictable and may come from completely unexpected sources. regardless of the source of new infectious agents, a major concern is that future pathogens may have properties that will make control much more difficult than sars. shorter incubation times and pre-symptomatic transmission strongly limit the efficacy of isolation and quarantine and may allow rapid disease spread. in this chapter, we have focused on biological factors that are central to disease emergence and control. policy prescriptions have been discussed extensively (university of louisville school of medicine 2003; beaglehole et al. 2003 ) and we highlight selected topics below. one of the important lessons from the sars crisis was the need for a rapid and organized response, even in the case of a relatively controllable disease. recognition of new epidemics through surveillance and global warnings and travel advisories are obvious critical factors but the necessary infrastructure has been difficult to implement, especially in developing regions. given the likely lag-time between the start of an outbreak and pathogen isolation and development of diagnostics, well-trained physicians and epidemiologists at the frontlines of the epidemic play a critical role in initial response. for an infected individual, the numbers of contacts and possible and actual transmissions increase rapidly with time so diagnosis and contact tracing are time-critical events. finally, communicating with, and educating the public and controlling panic are major concerns especially in the context of false reports and rumors. establishing trusted sources of information prior to emergencies should be a major objective for cities/regional governments. issues with coordination among government agencies or between medical and government agencies were strong obstacles in the response to sars in most affected regions (university of louisville school of medicine 2003). high transmission in medical care settings was one of the prominent features of both the sars and mers outbreaks. because infected individuals with weakened immune systems or co-infections may be the most difficult to diagnose and may show high infectiousness, proper training in pathogen containment is a critical element of epidemic preparation. similar basic techniques (proper use of gloves, gowns, masks, and goggles) were successful for sars and ebola suggesting that many practices will be of general value but specifics for particular transmission mechanisms (e.g. airborne versus vector transmission) are also critical. intervals between outbreak occurrences may be large, so regular confirmation of preparedness is important. low margins in health care are strongly linked to overcrowding and government and private organization incentives (e.g., increased funding for hospitals that rate highly on infection control training and preparedness) can greatly enhance hospital safety (committee on the future of emergency care in the united states health system 2007). the importance of patient isolation in limiting disease spread is clear from recent sars, ebola and mers outbreaks; hospitals must have containment facilities and "surge capacity" to limit superspreading events. although public health measures were sufficient to eventually control sars, additional measures including antiviral drugs and rapid vaccine development and production may be necessary for stronger pathogens. the economic impact of epidemics, roughly 200 billion usd (2 % of regional gdp) for east asia from sars and potentially over 800 billion usd for pandemic influenza (the economist 2005) should help to justify the costs of outbreak preparation. informed sanitation (water purification, sewage treatment) and building regulations/inspections (e.g. airflow control) policies can play a key role in preventing disease emergence and spread. the sars example illustrates the need for extensive interdisciplinary efforts, combining expertise from physics (fluid mechanics), biology (especially understanding mechanisms of disease transmission), and building design for resilience to future outbreaks. isolation and quarantine were critical to controlling sars in hong kong, singapore, taiwan, china, vietnam and canada. compliance rates appeared to be high in all regions (university of louisville school of medicine 2003) perhaps partly because the "cultural" value placed on solidarity and cohesion was relatively high in these regions. more severe movement restrictions may be necessary for more transmissible and/or virulent pathogens. it is unclear whether similar measures can be employed with success in other regions where personal liberties are emphasized and/or government is less trusted. biological studies can help to determine the necessity and guide planning for future epidemics, but social and economic issues may be the more critical limiting factors in developing preparedness for disease outbreaks. understanding the social, psychological, and economic costs of previous and potential disease outbreaks among both citizens and government officials will be central to planning for resilient communities. the ability to overcome economic and psychological barriers (e.g., normalcy bias) to implementing such plans may require a fundamental transformation in human society. virulence evolution and the trade-off hypothesis: history, current state of affairs and the future rapid evolutionary dynamics and disease threats to biodiversity epidemiology, transmission dynamics and control of sars: the 2002-2003 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programs human viruses: discovery and emergence biological and biomedical implications of the co-evolution of pathogens and their hosts receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses evidence of airborne transmission of the severe acute respiratory syndrome virus why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others key: cord-006129-5rog0s98 authors: hemida, maged gomaa; ye, xin; thair, simone; yang, decheng title: exploiting the therapeutic potential of micrornas in viral diseases: expectations and limitations date: 2012-08-16 journal: mol diagn ther doi: 10.1007/bf03256383 sha: doc_id: 6129 cord_uid: 5rog0s98 new therapeutic approaches are urgently needed for serious diseases, including cancer, cardiovascular diseases, viral infections, and others. a recent direction in drug development is the utilization of nucleic acidbased therapeutic molecules, such as antisense oligonucleotides, ribozymes, short interfering rna (sirna), and microrna (mirna). mirnas are endogenous, short, non-coding rna molecules. some viruses encode their own mirnas, which play pivotal roles in viral replication and immune evasion strategies. conversely, viruses that do not encode mirnas may manipulate host cell mirnas for the benefits of their replication. mirnas have therefore become attractive tools for the study of viral pathogenesis. lately, novel therapeutic strategies based on mirna technology for the treatment of viral diseases have been progressing rapidly. although this new generation of molecular therapy is promising, there are still several challenges to face, such as targeting delivery to specific tissues, avoiding off-target effects of mirnas, reducing the toxicity of the drugs, and overcoming mutations and drug resistance. in this article, we review the current knowledge of the role and therapeutic potential of mirnas in viral diseases, and discuss the limitations of these therapies, as well as strategies to overcome them to provide safe and effective clinical applications of these new therapeutics. therapeutic strategies based on mirna technology for the treatment of viral diseases have been progressing rapidly. although this new generation of molecular therapy is promising, there are still several challenges to face, such as targeting delivery to specific tissues, avoiding off-target effects of mirnas, reducing the toxicity of the drugs, and overcoming mutations and drug resistance. in this article, we review the current knowledge of the role and therapeutic potential of mirnas in viral diseases, and discuss the limitations of these therapies, as well as strategies to overcome them to provide safe and effective clinical applications of these new therapeutics. rna interference (rnai) is a system in living cells that regulates the activation and silencing of gene expression. rnai governs the regulation of host cell genes, mainly through two types of rna molecules: small interfering rna (sirna) and microrna (mirna). [1] sirnas are a class of double-stranded rna molecules, 20-25 nucleotides in length, which play different roles in cellular biology. experimental application or targeting of sirnas in various cell types and animal models has shown promise for the potential treatment of diseases induced by viruses such as hepatitis c, influenza, and hiv. [2] [3] [4] the suppression of viral gene expression by sirnas makes them very attractive for antiviral therapy, and some sirnas are already being used in clinical trials. [5] however, sirnas still have a long way to go before being brought to market, because of their potential side effects. one of the major concerns in using sirnas as molecular therapeutics is their induction of a strong immune response. mirnas, which were discovered in 1993, [6] are a group of non-coding, single-stranded rna molecules, ranging in size from 19 to 25 nucleotides. [7] it is now believed that mirnas compose one percent of the total human genome. mirnas are widely expressed in various species, including viruses. the first virally encoded mirna was discovered in the epstein barr virus (ebv) genome. [8] now, there are more than 141 identified mirnas encoded by 15 viruses, with more expected to be identified in the near future as a result of the improvement in online prediction and validation tools. [9] cellular mirnas in animals seem to be conserved, while virally encoded mirnas are highly variable even within the same group of viruses. [10] this may be due to the high frequency of viral mutations relative to eukaryotes. mirnas play an important role in regulation of almost one-third of all known human mrnas. [11] most mirnas have a specific tissue expression profile. their unique expression pattern may explain their roles in different biologic activities, such as cellular differentiation, environment adaptation, oncogenesis, and host-pathogen interaction. [12] the therapeutic potential of mirnas was first realized with the discovery that downregulation of mir-15 and mir-16 is associated with development of b-cell leukemia. [13] shortly after that, the potential for treatment of several other cancers was realized. [14] scientists have aimed to control the expression level of key genes via manipulation of cellular or viral mirnas to treat disease. these approaches include anti-mirna oligonucleotides (amos), peptide nucleic acids, and mirna sponges. [15] initial experiments have obtained promising results in controlling various viral infections. [16] [17] [18] in addition, it has been found that restoring or over-expressing certain mirnas may also be beneficial for reverse pathologic conditions, especially in cancer treatments. [19] the goal of this article is to review the recent progress in the understanding of the roles of mirnas in viral diseases, and to discuss the potential of these molecules in serving as a therapeutic target or as a useful therapeutic tool. we also specifically highlight the major obstacles faced by mirna technology in both therapeutics and vaccine strategies. viruses are obligate intracellular parasites. they lack the essential machinery required for their replication. thus, viruses adopt several clever strategies to ensure the success of their replication in a suitable host, one of which is manipulation of the host mirnas to modify the cellular environment for their own benefit. [20] some dna viruses are capable of encoding their own mirnas to modulate both the viral and cellular protein expression in order to provide a favorable environment for viral replication. [12] answering back, certain host mirnas alter the cell gene expression to defend the cells against the viral infection by interfering with viral proteins or other cellular factors as a type of immune response against these particular viruses. [21] therefore, the relationship between viruses and mirnas is complicated, to say the least. since mirnas play essential roles in viral infections, they are considered to be promising therapeutic targets in infectious diseases. their endogenous nature, small size, and flexible function make mirnas very good candidates, as they may trigger lower immunogenic responses and have fewer side effects than sirnas. [12, 14] in view of the current data regarding the roles of different viral and cellular mirnas in various viral replication cycles, we believe that manipulation of these mirnas will have a promising therapeutic role in infectious diseases. [12, 14, [22] [23] [24] [25] currently, there are more than 700 mirnas encoded by the human genome alone. [12] we will discuss the roles and therapeutic potential of cellular as well as viral mirnas (if any) in the pathogenesis and treatment of different viral diseases. human polyomaviruses (hpyvs) are a group of oncogenic, circular, non-enveloped, double-stranded dna (dsdna) viruses. [26] five polyomaviruses have been found to infect humans. of particular interest are two strains of these viruses, named (according to the initials of the first affected patients) bk virus (bkv) and james canyon virus (jcv). [27] the reservoir species for human infection is the rhesus macaque. humans have also acquired simian virus 40 (sv40) infection from contaminated poliovirus vaccines, and recent studies have reported horizontal transmission between people. [28] hpyvs induce a wide range of tumors affecting almost all body organs (including the brain, bones, colon, pancreas, stomach, and urogenital tract), as well as lymphomas and leukemia. [28, 29] the viral genome of sv40 encodes five proteins; two large t antigen (lt), one small t antigen (st-ag), and three that encode the capsid proteins (vp1, vp2 and vp3). hpyvs are able to encode viral mirnas for their own benefit. both bkv and jcv encode the same mirna, named mir-j1. it is upregulated in the brain in progressive multifocal leukoencephalopathy syndrome, suggesting a major role in this particular disease. [30] sv40 encodes mirnas called v-mirnas during the late stages of infection. [31] they are complementary to the viral mrnas produced at the early stage of viral infection. [31] these v-mirnas slow down the expression of the viral t-antigen genes and lower the level of interferon (ifn)-g produced by cytotoxic t lymphocytes, thus reducing the influx of inflammatory cells and facilitating evasion of the immune response. [31] human papillomaviruses (hpvs) are also oncogenic viruses. [32] they are usually associated with different forms of both benign and malignant tumors, especially those affecting the skin and the genital tract. [32] these viruses are usually classified, on the basis of their virulence, into either low-or high-pathogenic variants. [26] only a few hpv strains can produce mirnas during their replication. [33] for example, hpv-31 encodes viral mirnas at a very early stage of the infection, but these mirnas are usually degraded once latent infection takes place. [34] in another independent study, both hpv-31 and hpv-18 were found to encode viral mirnas, but they are not involved in cell transformation or cancer development. [33] it is well known that host mir-34a is involved in inhibition of abnormal cell growth in tumors. [35] mir-34a inhibits cell cycle progression at the g2 phase and subsequently induces apoptosis. [36] studies have reported success using the tumor suppressor complex (mrna-cellular mirna-34a), which targets downstream genes of tumor protein p53 (tp53). mir-34a is usually downregulated during hpv infection in primary keratinocytes. [36] thus, restoration of the normal expression level of this mirna is a potential strategy for therapeutic intervention. [36] adenoviruses are a group of non-enveloped dsdna viruses. over 50 serotypes have been identified in different clinical diseases, such as respiratory, gastrointestinal, urogenital, and eye diseases. [36] adenovirus usually encodes several small noncoding rna molecules called virus-associated rnas, such as va1 and va2. [37] they facilitate immune evasion by inhibiting dsrna-induced protein kinase r (pkr), which blocks ifn-a activity. [38] one study reported that adenovirus va1-rna interferes with the biogenesis of host mirnas and the function of sirna shrna (short hairpin rna), through inhibition of the nuclear transport of the pre-mirnas and the shrnas and direct inhibitory action of dicer. [37] usually, a small part of the va1 rna is subjected to processing by dicer, and this results in generation of mirna. use of anti-mirna antisense inhibitors (2 0 -o-methyl amo) to downregulate this mirna resulted in inhibition of virus production. [39] herpesviruses are a group of enveloped dsdna viruses, classified into three subfamilies (a, b, and g). they are characterized by induction of latent infections in their target hosts. [40] these virus-encoded mirnas play important roles in the establishment of latent infection, as well as the pathogenesis of virally induced diseases. according to the most recent studies, herpesviruses utilize their encoded mirnas in a wide range of biologic functions, such as inhibition of apoptosis, immune evasion, control of cellular proliferation, and regulation of viral replication. [41] [42] [43] in the following section, we will discuss herpesvirus-encoded mirnas. one of the most important genes encoded by herpes simplex virus (hsv) is called the latency-associated transcript (lat). [44] this gene does not encode proteins but may be involved in the production of mirnas or in cell survival after viral infection. [44] there has been debate around the origin of mir-lat as to whether it is a virus-encoded or cell-encoded mirna. [41] this mirna is believed to act by downregulating transforming growth factor (tgf)-b and smad3. tgf-b plays an important role in cell proliferation and induction of apoptosis. smad3 is a signaling pathway mediator, which is triggered by the action of tgf-b. [41] hsv-1 mir-lat-icp34.5 has been recently discovered during hsv-1 infection. [45] according to bioinformatic analysis, the hsv-1 genome encodes 24 mirnas, eight of which were found to be conserved between both hsv1 and hsv-2, and thus are believed to be functional. [46] six of these mirnas are upregulated in the trigeminal ganglia of mice infected with hsv-1. these mirnas are encoded by lat (table i) . [46] in addition, quantitative reverse transcription pcr showed that both mir-1 and mir-6 were highly expressed in vero cells infected with hsv-1 -as high as 1200 and 300 copies, respectively -whereas the other four mirnas showed downregulation, with only 40 copies per infected cell. [55] the mirna expression profile during hsv-1 infection revealed that several mirnas among the eight candidates mentioned earlier were upregulated. those mirnas were believed to play major roles in induction of the latent phase of viral infection. this assumption was based on comparison between the mirna expression profiles of the latent and active infections. for example, mir-h2 was found to be upregulated in latent infection to a level of 63 000 copies cell, compared with less than 40 copies cell during active infection. [56] furthermore, mirna-2 inhibits the production of the infected cell polypeptide (icp)-0 protein, which is responsible for triggering the active phase of hsv infection. another upregulated mirna, mir-6, is responsible for downregulation of icp4, which is responsible for the increased expression level of many hsv genes during the active phase of hsv-1 infection. [55, 56] human cytomegalovirus (hcmv) is a herpesviruses affecting humans, and can result in acute or latent infections. [57] the form of infection largely depends on the immune status of the affected host. [57] it can be fatal in immune-compromised patients, such as those with aids or recent organ transplants. [57] it may also be responsible for birth defects and congenital abnormalities in pregnant women. [21] as with other herpesviruses, there is evidence supporting the presence of mirnas that modulate viral pathogenesis in different tissues. [21] specifically, hcmv has been recently reported to encode 15 mirnas. [21] the most commonly expressed three mirnas during the active phase of hcmv infection are mir-ul23-5p, mir-ul23-3p, and mir-us24, [48] which target different cellular proteins, such as transcription factors (hfn3 and tgif2), receptors (cd206 receptors for interleukin-18), and other proteins involved in signal transduction pathways, such as rab2l. [48] hcmv is able to induce the latent phase of infection by a cunning immune-evasion strategy through the action of mir-ul112, which targets several cellular proteins such as major histocompatibility complex (mhc) class i associated proteins, especially the mhc class i-related chain b (micb). [58, 59] mir-ul112-1 also regulates early viral protein expression, such as immediate early protein (ie)-72. ie72 is a key mediator in the shift from the latent to the active phase of viral infection, because it suppresses ie1. [58, 59] thus, if the hcmv infection is synchronized with overexpression of the mir-ul112, the ie1 protein expression level is greatly reduced, which mediates the latent phase of infection. [43] it is also thought that mir-ul112-1 targets another viral protein called ul114. [58] downregulation of ul114 protein, using mir-ul112-1, results in inhibition of viral dna replication and subsequently triggers the latent phase of infection, making the virus able to evade the host immune system. [34] exploitation of this mechanism is being considered as a potential therapeutic strategy. [21] ebv is another oncogenic virus affecting humans. it is usually associated with induction of latent infection in more than 95% of affected patients. [60] in most cases, benign tumors develop; in some cases, however, malignant tumors may also develop, such as hodgkin's lymphoma, t-cell lymphoma, nasopharyngeal carcinoma, and gastric tumor. [61] during the active phase of ebv infection, more than 100 genes are usually expressed, whereas only 11 of them are expressed during latent infection. [21] it has been recently reported that ebv encodes more than 20 mirnas. [33] these mirnas are divided into two groups: one group is encoded from the intronic regions of a gene called bart, which is expressed at high levels in epithelial cells, but at lower levels in b cells, in the latent phase of infection; the other group is encoded from the untranslated region of a gene called bhrf1, a viral bcl2 homolog that prevents apoptosis. [8] although the functions of most ebv-encoded mirnas have not been completely identified, it has been found that mirna-bart2 targets balf5 mrna, a viral dna polymerase, and mirna-bart2 induces cleavage in the 3 0 untranslated region (utr) of balf5, resulting in inhibition of the lytic viral infection ( figure 1 and table i) . [63] according to bioinformatic prediction data, ebv mirna-bart5 is believed to target puma, a modulator of apoptosis in the bcl2 protein group regulated by tp53. [64] in some cases, puma can also induce apoptosis through the tp53-independent pathway. therefore, targeting puma with ebv mir-bart5 results in suppression of its action in apoptosis. [64] other ebv-encoded mirnas target the ifng-inducible chemokine cxcl11. [65] without cxcl11, ebv is able to evade the host immune response and subsequently enhance ebv replication. [65] the same group performed bioinformatic analysis and showed that cxcl11 contains a target sequence for mir-bhrf1-3 at its 3 0 utr sequence. targeting cxcl11 using this viral encoded mirna will have an immunomodulatory effect on the viral-induced tumor. [65] therefore, it is believed that targeting bhrf1-3 could be a good therapeutic approach for viral ebv-induced tumors. [65] kshv is one of the gamma-herpes viruses groups and is usually associated with kaposi's sarcoma infection, from which it acquired its name. [47] like other herpesviruses, kshv usually induces latent infections. [47] kshv encodes several mi-rna candidates from the genomic region spanning 4 kilobases between orf12 and orf71. [66] kshv-encoded mirnas target important genes involved in cell proliferation, modulation of the host immune system, apoptosis, and angiogenesis. for example, mir-k5 targets the mrna of the bcl2 (prosurvival gene) interacting protein called bclaf1. [62] this leads to reactivation of the kshv lytic infection. [62] thus kshvencoded mirnas play an important role in the virus host interactions, and silencing of those mirnas using different approaches, particularly the amo, is therefore a promising therapeutic strategy against such virus infection (figure 1). [62] marek's disease virus (mdv) is one of the alpha herpesviruses, characterized by rapid production of t-cell lymphomas in chickens. [67] the viral genome encodes several mirnas, which are mainly encoded from the oncogenes, especially meq and lat. [68] the mirnas of this virus are highly expressed in mdv-transformed cells, suggesting an essential role in mdv oncogenesis. however, their target genes have not been well identified. [68, 69] it is known that several host cell mirnassuch as mir-17, mir-92, mir-21, and let-7i -are involved in the cancer development induced by mdv in chickens. [68] they are most often upregulated in an msb-1 (mdv-transformed cd4+ t-cell line derived from a spleen lymphoma induced by the bc-1 strain of mdv-1) library after mdv infection. [69] this makes for an ideal model for the study of mirnas. comparative analysis of mirna expression profiles during mdv infection, using microarray analysis, may enable identification of specific mirnas involved in neoplastic transformation when compared with non-infected cells. this may become a useful diagnostic approach through examination of mirna signatures profiles. [70] this is supported by a recent report showing an association between downregulation of the host cellular mir-155 expression and upregulation of mdvencoded mirnas (m2-5p, m12-3p, m5-3p, m4-5p, m3-5p, m11-5p, m2-3p, m4-3p, and m3-3p), a potential marker for mdv-induced tumor formation. [69] subsequent identification of the putative target of these mirnas will lead to a better understanding of the molecular mechanism of mdv-induced tumorigeneses. [69] the human immunodeficiency virus (hiv) genome encodes several important genes, such as nef, vef, tat, and vpu. [71] bioinformatic analysis suggests that the hiv genome encodes five pre-mirnas, which are processed into ten mature mi-rnas, but their definite functions are still not well identified. [72] the hiv nef gene is located at the 3 0 end of the hiv genome and is highly expressed in early viral infection. [73] it has been shown to downregulate the expression levels of cd4, cd28, and mhc class i molecules. [73] hiv nef encodes mir-n367, which has a unique function at both the transcriptional and translational levels, rather than at the post-transcriptional level. [51, 74] the mechanism of action of mir-n367 is believed to be suppression of hiv promoter activity; however, further studies are required to explain the exact mechanism of such an action. [51] several studies reported that downregulation of the nef gene results in inhibition of hiv replication. [51, 74] this may lead to production of low-pathogenic strains of hiv or may favor latent hiv infection. [74] targeting of these viral mirnas using different approaches, especially amo treatment, could potentially have a therapeutic effect on hiv. [ hybridizing with corresponding targets of host genes (blue sequences). the viral mirna sequence and the 3 0 untranslated region (utr) sequence of the targeted host and viral genes were obtained from the referenced articles or from the national center for biotechnology information (ncbi) website and then submitted to the rnahybrid software program (http://bibiserv.techfak. uni-bielefeld.de/rnahybrid/). the predicted secondary structure for the hybridization between viral mirnas and their targets are generated by the rnahybrid program. (a) mir-k5 encoded by kaposi's sarcoma-associated herpesvirus (kshv), targeting host gene bclaf1 (bcl2-associated transcription factor 1). [62] (b) mir-bart2 encoded by ebv, targeting the viral dna polymerase gene balf5. [63] (c) mir-ul112 encoded by herpesviruses, targeting host gene micb (mhc class i-related chain b). [59] mfe = minimum free energy. currently, there are more than 170 million people affected by hepatitis c virus (hcv) infection worldwide. [75] drug resistance is one of the major hindrances in treating such viral infection. [76] hcv induces different forms of tumors in humans and is one of the major causes of liver diseases all over the world, resulting in hepatocellular carcinoma and, finally, complete liver failure. [77] it has been recently reported that host cellular mirna-122 has two recognition sites in the 5 0 utr of the hcv genome, resulting in upregulation of hcv infection. [78] further investigation showed that interaction between mirna-122 and the viral genome causes accumulation of viral rna in the liver tissues (table i) . furthermore, the level of viral rna in the liver tissues is usually controlled by mir-122 binding sites. [78] interestingly, hcv infection also modulates cellular mirna expression profiles. following hcv infection, three mirnas (mir-122, mir-100, and mir-10a) are upregulated, while other two mirnas (mir-198 and mir-145) are downregulated. [78] ura et al. [52] found that cyclin g1 acts as a putative target for mir-122. use of a primate model targeting mir-122 with specific antagonists resulted in a reduction in the level of hcv replication in the affected livers, demonstrating the promise of this strategy. [79] in addition, a recent study demonstrated the therapeutic potential of silencing mir-122 in chronic hcv viral infection in primate models, whereby chimpanzees that were positive for hcv infection were treated with a specific lna-modified oligonucleotide (spc3649). [79] these lna-oligomers targeted the complement sequence of mir-122 and resulted in a decrease in the duration of the viremia following acute hcv infection. there were no reports of any side effects or any viral resistance observed after the treatment. [79] this approach provided long-lasting effects in the hcv-infected animals, as well as great improvement in the liver pathology. [79] more clinical trials are needed to further confirm the promising results of this new molecular therapeutic approach. [78] hepatitis b virus (hbv) belongs to the genus orthohepadnavirus in the family of hepadnaviridae. hbv infection progresses into cirrhosis and hepatocellular carcinoma in most cases. [80] it is believed that hbv encodes mirnas that regulate their own gene expression. [81] according to bioinformatic predictions, hbv encodes only one mirna. however, several studies have failed to identify any cellular genes regulated by this virus-encoded mirna, implying alternative gene expression mechanisms. [81, 82] according to the mirna expression profiles of several patients suffering from cirrhosis due to hbv infection, the host hsa-mirna-615-3p is usually upregulated. in recent clinical studies, ura et al. [52] studied the role of different mirnas in the pathogenesis of both hbv and hcv in the context of development of hepatocellular carcinoma in infected liver tissues. [52] in this study, the differential expression levels of 188 mirnas from 12 hbv and 18 hcv patients were tested using qrt-pcr. according to this study, 19 mirna candidates were highly expressed in both hbv and hcv infections, and 31 mirnas served as markers for the severity of liver damage. it is known that hbv infection triggers pathways associated with dna damage, recombination, and signal transduction pathways -whereas hcv infection usually triggers an immune response, antigen presentation, cell cycle progression, proteosome activation, and lipid metabolism. therefore, the overall conclusion was that certain mirnas may act as important mediators in the pathogenesis of both hbv and hcv infections. [52] these studies have paved the way to a new era in molecular antiviral therapy through modulation of the expression levels of those key mirnas. there is now a new antiviral therapy for controlling hbv infection, using artificial mi-rnas. this approach has revealed a dramatic reduction in hbv protein expression levels and a remarkable reduction in viral dna replication in vitro. [83] [84] [85] severe acute respiratory syndrome coronavirus (sars-cov) is a single-stranded rna virus, which belongs to the family coronaviridae. although several trials have been performed to treat this pathogen using conventional drugs such as ribavirin, antibiotics, anti-inflammatory steroids, and different kinds of immune stimulators, these approaches still lack viral specificity. sars-cov infection in bronchioalveolar stem cells (bascs) is a prime example of how mirna modulates the virus-host interaction. sars-cov is unable to replicate in well differentiated cells, so it has to control basc cellular differentiation in order to establish a successful viral infection. [86] this virus usually hijacks cellular mirnas such as mir-17 * , mir-574-5p, and mir-214 for the benefits of its replication and immune evasion. the nucleocapsid and spike glycoproteins downregulate the expression levels of mir-223 and mir-98, respectively. this action enables the virus to hinder basc cellular differentiation and the production of inflammatory chemokines, creating an environment that is optimal for virus replication. restoration of the levels of mir-223 and mir-98 poses a potential novel approach in treating sars-cov infection. [86] the influenza a outbreak of 2009 provided a warning about the urgent need for new alternative molecular therapeutic approaches for both the treatment and prophylaxis of such viral infections. molecular therapy using mirna technology may offer a new therapeutic approach to cope with the continuous changes in virus strains every year. recent bioinformatics tools have paved the way for the discovery of new mirnas and their target sequences for the design of nucleic acid-based therapeutics. for example, there are two human-encoded mirnas that have potential binding sites within both the viral polymerase (pb2) and hemagglutinin (ha) genes (mir-507 and mir-136, respectively) [table i]. [22] the target sequences of these two mirnas are highly conserved among different influenza virus strains. the ha protein is involved in the attachment of the virus to its receptors, and the pb2 protein is an essential component in the ribonucleoprotein complex, needed for rna transcription and replication. the presence of human mirnas that target conserved regions of influenza rna suggests that the human genome has evolved to use this as a defense mechanism against infection. this supports the argument that targeting viral genes with mirnas may be an effective strategy. this may also suggest that it is a futile attempt, since we have the mirnas and yet still succumb to influenza infections. a recent study has been conducted to determine mi-rna expression profiles after avian influenza virus (aiv) infection in chickens. this study showed changes in the cellular mirna profile in response to the aiv infection, suggesting that the mi-rnas play a role in the host-pathogen interaction during aiv infection. [87] specifically, there were alterations in the mirna profiles of mir-146, which had been previously reported to play a role in immune-related signal pathways in mammals. [87] one recent study utilized mirna technology in the development of influenza virus vaccines, whereby a new influenza a virus vaccine was developed using mirna-based gene silencing. the method involves introducing an mirna sequence of non-avian origin, known as a mirna-responsive element (mre), into the viral nucleoprotein gene, resulting in construction of new reassortant h1n1 and h5n1 viruses. with this strategy, the degree of the viral attenuation is controlled by the expression level of mir-124, which targets the introduced mre sequence. this novel strategy offered a very good vaccine that was species specific, offering a high level of protection. [4] the nascent viruses were attenuated for mice, while they still propagated well in embryonated chicken eggs and were able to generate high levels of neutralizing antibodies in animals. this novel approach for influenza vaccine development may be used in combination with the currently available vaccine in order to increase both the safety and the efficacy of influenza virus vaccines in the near future. [4] coxsackievirus, especially coxsackievirus b3 (cvb3), is the most common pathogen of human myocarditis. anti-cvb3 drug development has been recently focused on a nucleic acidbased strategy. our laboratory first reported the successful inhibition (>92%) of cvb3 replication in hela cells by transfection of sirnas targeting viral protease 2a rna. we also found that the antiviral effect was disrupted by mutations in the central strand region, and mismatch was tolerated near the 3 0 end but not near the 5 0 end of the sirna; [88] furthermore, the sirna effect was mediated by the antisense strand to the viral genome, rather than the sense strand complimentary to the viral negative-strand of the replicating intermediate. this finding was further conformed by another report. [89] when applied systemically to mice, sirna targeting 2a had a significant protective effect if applied 6 and 14 hours after infection, including reduced viral replication and tissue injury, as well as an increased survival rate. [90] recently, we tested a packaging rna (prna) vector (a component of the bacterial phage nano-motor) for targeted delivery of sirnas. through conjugation of a folate ligand to the prna vector, we specifically delivered the sirnas targeting cvb3 2a to hela cells and hl-1 cardiomyocytes that expressed folate receptors. [91] in addition to the transfection of mature sirnas, overexpression of shrnas was also effective against cvb3 3d rna polymerase and structural protein vp1, both in cells and in mice, where viral pancreatitis was significantly reduced. [92] schubert et al. [89] used the sidex double expression vector to simultaneously transfect two sirna sequences targeting the cvb3 3d rna polymerase sequence in a green fluorescent protein (gfp) reporter construct. this double expression of both sirnas successfully suppressed reporter expression despite the intentional introduction of an artificial point mutation (simulating an escape mutation or a mirna target) that caused a mismatch with one of the two sirnas. as we have discussed above, there have been some promising results supporting the development of mirnas for the treatment of several viral infections, and some of these mirna-based drugs have reached the clinical trial stage. despite this great progress, their clinical applications are still hampered by several challenges. in the following section, we briefly discuss the current obstacles or limitations facing mirnas-based antiviral therapy. one of the major limitations for the use of mirna-based antiviral therapy is the production of transgene-specific immunity. [93] delivery of mirnas using viral vectors usually results in the development of immune response against the viral vector. basically, the delivery vector will stimulate an innate immune response in the forms of cytotoxic t-lymphocytes, humoral neutralizing antibody against their viral capsid proteins, and cytokine-mediated inflammatory responses in vivo. [93, 94] direct correlation between the immune response to the adenovirus capsid protein and the concentration of the viral vector has been reported; this interaction is usually associated with undesirable side effects in the host, especially if the construct moves from the target tissue into the blood circulation. [95] the targeted delivery of sirna, mirna, and other nucleic acid-based therapies is another major concern of using these molecular therapeutic approaches. in contrast to the great progress in local administration of both sirna-and mirna-based therapies in the eyes, lung, and vagina, systemic delivery to target organs such as the liver, heart, and intestine is still undergoing optimization. [96, 97] it is interesting to note that some studies have shown success in administering sirnas via the intracerebral route; however, the risk is that foreign nucleic acid may be delivered to the central nervous system. [93] several laboratory techniques -such as real-time pcr, microarray analysis, luminex bead arrays, northern blotting, in situ hybridization, formalin fixation, and paraffin embedding -are currently in use in mirna detection and quantification. however, all of these techniques still require further optimization. [98] [99] [100] once they are optimized, a clear choice for sensitivity and specificity will emerge, and this approach will allow early and sensitive detection of mirna expression in different disease syndromes. this will have a great impact on the early tracking of serious viral diseases. [101] the off-target effects of sirnas were one of the major concerns in earlier studies using both sirna and shrna technologies in gene therapy. as a new generation of molecular gene therapy, mirnas would be expected to have a high degree of specificity for their targets. however, since mirna action is based on imperfect base pairing with the target sequence in most circumstances, the specificity will be lower than that of sirna. this prediction has been confirmed by recent clinical trials, followed up by microarray analysis, which revealed possible off-target effects of mirnas. [102] another follow-up study by birmingham et al., [103] using the combination of bioinformatics and microarray analysis, found that using either the sirna or the mirna could result in off-target silencing. in addition, in vivo studies have revealed that one mirna may target several genes at the same time, and the targets are not clearly identified. this suggests diverse modes of action of a given single mirna. on the other hand, one gene may be regulated by several mirnas, [104] indicating that the mode of action is more complicated than expected. since drug therapies must precisely target the virus in question and nothing else, a large undertaking is needed to gather all possible information regarding all targets of each mirna that is being considered for drug development. [104, 105] although the currently used viral vectors in mirna delivery are non-pathogenic, there is always the possibility of mutations within those viral vectors. these mutations may not only result in abnormal gene expression of the viral mirna construct but may also cause possible insertion of vectors into the human genome, increasing the risk of cancer. [23] moreover, the targeted viruses (especially the rna viruses) are prone to mutation, which may drive drug resistance. there are currently two possible approaches to conquer these issues: one is the targeting of cellular factors that are essential for virus replication or use of more than one mirna for the same target gene; the other possible solution is the targeting of several conserved regions of the viral genome by different sirnas or mirnas. viruses are among the most common causes of human diseases. because of the unique biologic properties of viruses, there is no effective and specific antiviral therapy available so far. several vaccines and antiviral drugs have shown a limited degree of efficacy for prophylaxis and treatment of some viral infections. however, high mutation rates enable viral diseases to emerge and re-emerge frequently. thus, new strategies for drug and vaccine development must be devised to fight the threat of viral diseases to human health. recent advances in the understanding of mirna structure, function, and particularly their association with the molecular pathogenesis of a variety of complex diseases, have served as a theoretical basis for drug development. on the one hand, as key factors for viral replication and latency, mirnas are ideal targets for inhibition. in this regard, construction of mrnas that contain multiple tandem binding sites of a given mirna may be useful to produce decoys or 'mirna sponges' to inhibit the function of a specific mirna. in addition, chemically synthesized antisense rna oligomers ('antagomirs') targeting a mirna of interest could be also be a promising approach to inhibit mirna activity. on the other hand, mirna expression vectors can be used to overexpress specific mirnas to achieve a long-term effect of reversing the imbalance of mirna expression caused by infection. further, introduction of pre-mirna mimetics for transient replacement is another option for investigation. in summary, although there 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ability to auto-regulate viral gene expression regulation of human immunodeficiency virus 1 transcription by nef microrna differential expression between hepatitis b and hepatitis c leading disease progression to hepatocellular carcinoma a cellular microrna mediates antiviral defense in human cells human papillomaviruses modulate expression of microrna 203 upon epithelial differentiation to control levels of p63 proteins micrornas expressed by herpes simplex virus 1 during latent infection regulate viral mrnas herpes simplex virus type 1 icp4 promotes transcription preinitiation complex formation by enhancing the binding of tfiid to dna severe cytomegalovirus infection in apparently immunocompetent patients: a systematic review identification and function of human cytomegalovirus micrornas suppression of immediate early viral gene expression by herpesvirus encoded micrornas: implications for latency latent epstein-barr virus (ebv) infection and cytomegalovirus (cmv) infection in synovial tissue of autoimmune chronic arthritis determined by rna-and dna-in situ hybridization epstein-barr virus infection in humans: from harmless to life endangering virus-lymphocyte interactions tandem array-based expression screens identify host mrna targets of virus-encoded micrornas epstein barr virus encoded microrna mir-bart2 down-regulates the viral dna polymerase balf5 the nuclear function of p53 is required for pumamediated apoptosis induced by dna damage ebv micrornas in primary lymphomas and targeting of cxcl-11 by ebv-mir-bhrf1-3 micrornas of kaposi's sarcoma-associated herpes virus marek's disease: a model for herpesvirus oncology analysis of the expression profiles of marek's disease virus-encoded micrornas by real-time quantitative pcr differential expression of micrornas in marek's disease virus-transformed t-lymphoma cell lines micrornas accurately identify cancer tissue origin are viral-encoded micrornas mediating latent hiv-1 infection? hiv-1 encoded candidate micro-rnas and their cellular targets hiv-1 nef suppression by virally encoded microrna challenges in modern drug discovery: a case study of boceprevir, an hcv protease inhibitor for the treatment of hepatitis c virus infection mechanisms of drug resistance to current and future antiviral therapies for hepatitis c virus infection occurrence of hcc in asymptomatic hcv-related chronic hepatitis therapeutic silencing of microrna-122 in primates with chronic hepatitis c virus infection mir-122 regulation of lipid metabolism revealed by in vivo antisense targeting association between hepatitis b virus and pancreatic cancer hbv-encoded microrna candidate and its target targeted deletion of dicer in the heart leads to dilated cardiomyopathy and heart failure expressed anti-hbv primary micro-rna shuttles inhibit viral replication efficiently in vitro and in vivo inhibition of hepatitis b virus gene expression and replication by artificial microrna micrornaome of splenic macrophages in hypersplenism due to portal hypertension in hepatitis b virus-related cirrhosis an animal model of sars produced by infection of macaca mulatta with sars coronavirus oncogenic hpv infection interrupts the expression of tumor-suppressive mir-34a through viral oncoprotein e6 inhibition of coxsackievirus b3 replication by small interfering rnas requires perfect sequence match in the central region of the viral positive strand maintaining inhibition: si-rna double expression vectors against coxsackieviral rnas targeting 2a protease by rna interference attenuates coxsackieviral cytopathogenicity and promotes survival in highly susceptible mice targeted delivery of anti-coxsackievirus sirnas using ligand-conjugated packaging rnas expression of short hairpin rnas against the coxsackievirus b3 exerts potential antiviral effects in cos-7 cells and in mice prospects and obstacles to using small interfering rnas as small molecule drugs progress and prospects: immune responses to viral vectors lethal toxicity, severe endothelial injury, and a threshold effect with high doses of an adenoviral vector in baboons inhibition of respiratory viruses by nasally administered sirna an sirna-based microbicide protects mice from lethal herpes simplex virus 2 infection identification of micrornas and other small regulatory rnas using cdna library sequencing mustering the micromanagers analysis of microrna expression by in situ hybridization with rna oligonucleotide probes transcription and processing of human microrna precursors expression profiling reveals off-target gene regulation by rnai a protocol for designing sirna with high functionality and specificity a pattern-based method for the identification of microrna binding sites and their corresponding heteroduplexes serum response factor regulates a muscle-specific microrna that targets hand2 during cadiogenesis correspondence: professor decheng yang, 166-1081 burrard street, the heart and lung institute, st paul's hospital this work was supported by grants from the canadian institutes of health research and the heart and stroke foundation of bc and yukon. dr. maged gomaa hemida is a recipient of the cihr-impact postdoctoral training fellowship. xin ye is supported by a ugf award from the university of british columbia.the authors have no conflicts of interest that are directly relevant to the content of this review. key: cord-267791-v10eh408 authors: chughtai, abrar ahmad; khan, wasiq title: use of personal protective equipment to protect against respiratory infections in pakistan: a systematic review date: 2019-02-07 journal: j infect public health doi: 10.1016/j.jiph.2019.01.064 sha: doc_id: 267791 cord_uid: v10eh408 like other low-income countries, limited data are available on the use of personal protective equipment (ppe) in pakistan. we conducted a systematic review of studies on ppe use for respiratory infections in healthcare settings in pakistan. medline, embase and goggle scholar were searched for clinical, epidemiological and laboratory-based studies in english, and 13 studies were included; all were observational/cross-sectional studies. the studies examined ppe use in hospital (n = 7), dental (n = 4) or laboratory (n = 2) settings. policies and practices on ppe use were inconsistent. face masks and gloves were the most commonly used ppe to protect from respiratory and other infections. ppe was not available in many facilities and its use was limited to high-risk situations. compliance with ppe use was low among healthcare workers, and reuse of ppe was reported. clear policies on the use of ppe and available ppe are needed to avoid inappropriate practices that could result in the spread of infection. large, multimethod studies are recommended on ppe use to inform national infection-control guidelines. healthcare workers are at the frontline when treating infectious disease cases and at high risk of acquiring influenza and other respiratory infections [1] [2] [3] . several outbreaks of new infectious diseases have occurred in recent decades, such as the outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in 2002-2003 [4] , influenza pandemic (h1n1) in 2009 [5] , middle east respiratory syndrome coronavirus (mers-cov) in 2012 [6] and ebola virus diseases in 2014-2016 [7] . many healthcare workers were infected and died during these outbreaks because of a lack of infection control [4, 8, 9, 10] various infection control strategies are used to protect healthcare workers from respiratory and other infections in healthcare settings [11, 12] . these strategies can be broadly classified as administrative control measures, environmental control measures and the use of personal protective equipment (ppe). administrative control measures include developing policies and procedures, implementing triage protocols and providing health education and trainings. environmental control measures includes ensuring proper ventilation, establishing airborne infection isolation and negative pressure rooms, developing systems for cleaning and waste disposal. [13, 14] . ppe is commonly used in healthcare settings as standard or transmission based precaution to protect healthcare workers from infections and to prevent further spread to patients around them [14, 15] . ppe is generally ranked lowest in the infection control hierarchy due to less effectiveness compared to other control measures and high expenditure in the long run. therefore, most infection control guidelines recommend using ppe together with other administrative and environmental control measures. however, ppe is important during the early stage of an outbreak or a pandemic when drugs, a vaccine and other control measures are not available, or access is limited. commonly used ppe to protect from respiratory infectionsare; face masks, respirators, gloves, and goggles or face shields [16] . face masks (or medical masks) and respirators are the most commonly used ppe to protect from influenza and other respiratory infection in healthcare settings. however, these two products are not the same. face masks are not designed for respiratory protection and are used to avoid respiratory droplet and spray of body fluids on the face. they are also used by sick patients to prevent spread of pathogens to others (referred to as "source control"), or by surgeons in the operating theatre to maintain a sterile operating field. face masks are not fit to the face and have varying filtration capacities [17] . respirators ratory protection and are used to protect from respiratory aerosols [18] . a properly fitted respirator provides better protection again respiratory infections than a face mask. gloves are used to protect hands from blood and body fluids, including respiratory secretions. goggles and face shields are used to prevent transfer of respiratory pathogens into the eyes from contaminated hands and other sources. gowns, coveralls, surgical hoods and shoe covers can also be used where procedures on infectious patients generate aerosols or when a new respiratory virus has emerged [18] . there is an ongoing debate about the selection and use of various types of ppe in healthcare settings. this is mainly because of a lack of high quality studies on the use of ppe. most studies are observational and on the use of masks and/or respirators [18] . to date, only five randomized clinical trials have been conducted on use of ppe in hospital settings and all were on face masks/respirators [18] . moreover, most studies on ppe use were conducted in high/middle income countries and currently there are limited data from lowincome countries where the burden of infectious diseases is high. it is therefore important to examine the use of ppe in low resource countries to inform infection control policies. pakistan has a population of about 200 million. as a low-income country, its gross domestic product is low, as is its expenditure on health [19] . the country has one of highest rates of infant and maternal mortality in the south asia region. infectious diseases are still among the main causes of death, particularly in young children. health and surveillance systems are generally weak and limited data are available on infection prevention and control strategies. the aim of this study was to examine the use of ppe for respiratory infections in healthcare settings in pakistan. a systematic review was conducted using the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines. we searched for studies on the electronic databases medline and embase using selected key words. a combination of keywords were used including: 'face mask' or 'mask' or 'medical mask' or 'surgical mask' or 'cloth mask' or 'respirator' or 'gloves' or 'gowns' or 'coverall' or 'surgical cap/hood' or 'shoe/boot covers' or 'goggles' or 'face shield' or 'eye protection' and ' respiratory infection' or 'respiratory tract infection' or 'respiratory diseases', 'outbreaks' or 'infectious disease' or 'influenza' or 'pandemic influenza' or 'flu' or 'tuberculosis' or 'pneumonia' and 'pakistan' or 'punjab' or 'sindh' or 'balochistan' or 'khyber pakhtunkhwa'. we used an open date strategy up to december 2017. we anticipated that studies published in local journals might not be indexed on the medline or embase, therefore, an additional search was made on google scholar using the same keywords. we set a limit of 20 results per page on google scholar and first three pages were reviewed for each keyword search. after the initial search; we reviewed titles and abstracts and selected studies for full text review (fig. 1) . clinical, epidemiological and laboratory-based studies conducted in any part of pakistan and published in english were included in the review. the focus of this systematic review was on the use of ppe for prevention of respiratory infections. therefore, we only included those studies which examined the use of facemask and/or respirator in healthcare settings, with or without other ppe. we only included those studies where ppe was discussed for respiratory infections. studies where ppe was examined for general infection control were also included, given respiratory protective equipment (face masks and/or respirators) was mentioned. we excluded studies on the use of ppe only for bloodborne infections. conference abstracts and poster presentations were also excluded. a total of 137 studies were found in the initial search. after reviewing titles and abstracts, 46 studies were selected for full text review. finally, 13 articles were included in this review (table 1 ) [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] . we only found observational/cross-sectional studies on the use of ppe for infectious diseases in healthcare settings in pakistan. in all studies data were collected through questionnaires or interviews. no clinical trials or laboratory-based studies on the use of ppe in such settings were found. seven studies examined the use of ppe in hospital [20] [21] [22] [23] 25] and among those, two examined the ppe perceptions among medical students [24] or pharmacy students [32] . two studies were conducted in the laboratory settings [26, 27] while, four in dental settings [28] [29] [30] [31] two studies focused on the use of ppe for influenza [24, 32] , two were for tuberculosis [20, 25] and nine studies were on multiple respiratory diseases, including influenza [21, 22] or general infections [23, [26] [27] [28] [29] [30] [31] . only two studies examined the use of ppe alone [21, 22] , while other studies examined other infection control practices as well [20, [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] . guidelines and standard operating procedures on ppe do not exist in most of the hospitals [23] or laboratories [26, 27] in pakistan. two studies examined the guidelines and current practices on the use of face masks/respirators for influenza, tuberculosis and sars in pakistan [21, 22] . recommendations on the use of masks were reported to be inconsistent and different types of product were recommended and used in various healthcare settings [21, 22] . face masks were the most commonly used ppe to protect from respiratory infections in most hospitals in pakistan. medical masks were generally used to protect from influenza, tuberculosis and other respiratory infections, while the use of respirators was limited to high-risk situations [21, 22] . in a cross-sectional survey among final-year pharmacy students in seven universities of karachi, about 60% of 443 participants highlighted the need to cover the nose or mouth to protect from influenza and about 57% highlighted the use of face masks, gloves and other ppe [32] . laboratory coat and gloves were the most commonly used ppe in the laboratories in pakistan while face masks and eye covers were rarely used [26, 27] . a survey of dentists working in various settings (dental colleges, hospitals and private clinics) showed that face masks and gloves were also commonly used ppe [29] . the use of ppe was also reported to be low among health workers. according to a hospital-based survey, face masks are not provided to patients with tuberculosis and respirators are not provided to the healthcare workers [23] . another survey showed that 25% of participants used ppe for patients with suspected tuberculosis and 56% used ppe for patients with confirmed tuberculosis [25] . a study in a ward for patients with multidrug-resistant tuberculosis reported that 65% of the healthcare workers used n95 respirators and 58% were provided with a mask [20] . a study on 253 biosafety level (bsl) 2 laboratory workers showed that ppe was not used by about half of the staff (46.2%) [27] . a countrywide survey showed that almost one third (31.9%) of bsl-2 laboratory workers did not routinely use ppe [26] . both gloves and laboratory coats were used by only 26.7% of the personnel, while a laboratory coat or gloves alone were used by 30.4% and 8.1%, respectively. less than 1% of all the respondents across pakistan reported using eye covers [26] . in a survey of medical students during pandemic (h1n1) 2009, 75% said that they would use a face mask to protect from infection. students with less risk perception were more hesitant to use face masks [24] . the use of face masks was common in dental practice and according to various surveys, 68-100% of dentists wear masks during dental procedures [28] [29] [30] [31] . across all the studies in dental settings, more than 90% also used gloves. among the ppe, face masks were considered the most bothersome to use by wearers. reuse of ppe was also reported in many studies, mainly because of unavailability of ppe and lack of training. gowns are shared among the healthcare workers in hospital many times [23] . two surveys in dental clinics showed that more than half of the dentist reuse masks during routine work [28, 29] . the availability of ppe was generally low in all healthcare settings [21, 22, 28] and varied according to the type [23] ; gloves and masks were available while gowns and n95 respirators were not available in several wards [23] . a shortage of ppe was also reported during sars and pandemic (h1n1) 2009 [21, 22] . a lack of training was a common issue reported and most healthcare workers were not trained in the use of ppe. most of the studies (11/13) discussed other infection control practices as well, in addition to the use of ppe. other non-standard infection control practices included reuse of syringes, improper waste disposal, a lack of hand hygiene practices, non-isolation of infectious cases and low influenza vaccination among healthcare workers. we reviewed the use of ppe in various healthcare settings in pakistan. a lack of guidelines and standard operating procedures, inconsistent policies and practices, low compliance, and non-availability and reuse of ppe were the main issues highlighted in this study. evidence is lacking on the use of ppe in hospitals and other healthcare settings in pakistan and most studies are of low quality. clinical studies should be conducted to examine the effectiveness of ppe and improve the compliance. reuse of ppe may increase the risk of self-contamination to the wearer and this practice should be discontinued. there is a need to improve the availability of ppe and healthcare workers should be trained. ppe is generally considered lowest in the infection control hierarchy and is generally recommended in combination with other control measures. other infection control practices in such settings should also be examined. different types of ppe are used by healthcare workers in pakistan, which reflects a lack of standard policies and guidelines. the different policies and practices may be because of the different recommendations by the world health organization (who) and the united states (us) centers for disease control and prevention (cdc) [33, 34] . debate continues about the selection and use ppe for different infections, for example, face masks versus respirators, gowns versus coveralls, face shields versus goggles [7, 18, 34, 35] . selection of ppe mainly depends on mode of transmission, however, several individual and organizational factors also contribute the selection and use of ppe, such as risk perception, presence of adverse events, pre-existing medical illness, availability and cost [7] . respiratory infections are generally transmitted through contact, droplet and/or airborne routes. gloves should be used to protect from infections transmitted through contact (e.g. respiratory syncytial virus and adenovirus), face masks should be used for droplet infections (e.g. influenza and coronavirus) and a respirator should be used to protect form airborne infection (e.g. tuberculosis and measles). however, infection transmission is rarely by only one route and most infections are transmitted by more than one route [36] . for example, influenza and sars primarily transmit through droplet and contact routes, but airborne transmission has also been reported [37, 38] . similarly, ebola primarily transmits through direct contact with blood and body fluids [39] , but animal studies have shown that airborne transmission is also possible [7] . the risk of transmission further increases during aerosol-generating and other high-risk procedures [40, 41] . moreover, uncertainty exists about how pathogens transmit during outbreaks and pandemics [14, 34, 42, 43] . therefore, superior ppe should be used where the mode of transmission is uncertain, the case-fatality rate is high and pharmaceutical interventions are not available [7] . infection control guidelines in pakistan need to be updated urgently to reflect these recommendations. given that mers cov is circulating in the eastern mediterranean region (emr), policies and practices on the use of ppe in other countries of the region should also be examined. our study also reported low availability of ppe in hospital, dental and laboratory settings in pakistan. the availability of ppe is a challenge, not only in low-resource counties, but also in highincome countries, particularly during outbreaks and pandemics when the use of ppe greatly increases [46, 47] . this may result in non-standard practices such as reuse and extended use of ppe. shortages of ppe were even reported in many high-income countries during the 2009 influenza h1n1 pandemic and staff had to use various alternatives [46] [47] [48] . the availability of ppe is important to ensure proper use and compliance. low use of ppe among laboratory workers in pakistan may be due to non-availability and a lack of resources. for example, ppe use was relatively higher in laboratory workers in punjab, which is an affluent province, than other provinces [26] . moreover ppe use was reported more in the private sector in pakistan than the public sector which has fewer resources [27] . proper use of ppe depends on several factors such as availability, knowledge, training, risk perception and comfort [7, 44, 45] . this study showed the compliance with the use of ppe was generally low among healthcare workers and was mainly due to unavailability of ppe, discomfort and a lack of training. while the use ppe depends on many factors, a greater perception of risk was positively associated with compliance [24] . continuous use of face masks and respirators may have psychological and physiological effects on the wearer and result in more adverse events [49] [50] [51] . compliance with the use of face masks has been shown to be based on the nature of the disease, infectiousness of patients and the performance of high-risk procedures [52] . previous studies have tested the precede (predisposing, reinforcing and enabling) framework to examine healthcare workers' compliance with universal precautions [53] . the results showed that reinforcing factors, such as availability of ppe and less job hindrance, and enabling factors, such as safety climate and regular feedback, were significant predictors of compliance with ppe [53] . in addition, the health belief model [54] was also used to examine the compliance and use of face mask during the sars outbreak [55, 56] . perceived susceptibility (vulnerability to acquiring sars and close contact with case), perceived benefits (that face masks can prevent infection) and cues to action (someone asked them to use face masks) were significant predictors of protective behaviour and use of face masks [55] . our study showed that most healthcare workers were not trained on the use of ppe in pakistan. the risk of infection can be reduced with proper training and availability of policies and standard operating procedures [57] . however, regular monitoring is also required to make sure that healthcare workers are using ppe according to the protocols. a study in the us reported many deviations from the protocols even though all healthcare workers were trained [15] . this may result in self-contamination to the wearers and the spread of infection to others [58] . training programmes should be arranged for newly recruited staff and then annual refresher courses should be provided. our study had some limitations. the initial search was made on medline and embase but very few studies were retrieved because many papers are not indexed on these databases. therefore, we also searched google scholar but we only reviewed the first 3 pages after each search so some studies could have been missed. however, we checked the references lists of the relevant studies and could not find any other studies. our search was up to 2017 and studies in 2018 were not included. we only considered ppe in this study and did not examine other infection control practices. the use of ppe is generally recommended with other administrative and environmental control measures. the selection and use of ppe vary according to the type of healthcare worker and working environment. face masks and gloves were the most commonly used ppe to protect from respiratory and other infections. overall, compliance with the use of ppe was low, and non-availability and reuse of ppe were reported. most studies were observational and large-scale prospective studies are needed to collect more evidence about the use of ppe in healthcare settings in pakistan. no funding sources. aac tested the filtration of mask samples by 3 min in another study; 3m products were not used in this study. wk declares none. as this was a systematic review of published data, ethics approval was not required. aac devised the structure and topic areas for this review and made the initial search. wk and aac reviewed titles and abstracts and selected studies for full text review. aac prepared the first draft of manuscript and both authors contributed equally to the final manuscript. influenza and rhinovirus infections among health-care workers nosocomial transmission of measles among healthcare workers tuberculosis among health care workers emergencies preparedness, 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self-contamination during doffing of personal protective equipment key: cord-015967-kqfyasmu authors: tagore, somnath title: epidemic models: their spread, analysis and invasions in scale-free networks date: 2015-03-20 journal: propagation phenomena in real world networks doi: 10.1007/978-3-319-15916-4_1 sha: doc_id: 15967 cord_uid: kqfyasmu the mission of this chapter is to introduce the concept of epidemic outbursts in network structures, especially in case of scale-free networks. the invasion phenomena of epidemics have been of tremendous interest among the scientific community over many years, due to its large scale implementation in real world networks. this chapter seeks to make readers understand the critical issues involved in epidemics such as propagation, spread and their combat which can be further used to design synthetic and robust network architectures. the primary concern in this chapter focuses on the concept of susceptible-infectious-recovered (sir) and susceptible-infectious-susceptible (sis) models with their implementation in scale-free networks, followed by developing strategies for identifying the damage caused in the network. the relevance of this chapter can be understood when methods discussed in this chapter could be related to contemporary networks for improving their performance in terms of robustness. the patterns by which epidemics spread through groups are determined by the properties of the pathogen carrying it, length of its infectious period, its severity as well as by network structures within the population. thus, accurately modeling the underlying network is crucial to understand the spread as well as prevention of an epidemic. moreover, implementing immunization strategies helps control and terminate theses epidemics. for instance, random networks, small worlds display lesser variation in terms of neighbourhood sizes, whereas spatial networks have poisson-like degree distributions. moreover, as highly connected individuals are of more importance considering disease transmission, incorporating them into the current network is of outmost importance [4] . this is essential in case of capturing the complexities of disease spread. architecturally, scale-free networks are heterogenous in nature and can be dynamically constructed by adding new individuals to the current network structure one at a time. this strategy is similar to naturally forming links, especially in case of social networks. moreover, the newly connected nodes or individuals link to the already existent ones (with larger connections) in a manner that is preferential in nature. this connectivity can be understood by a power-law plot with the number of contacts per individual, a property which is regularly observed in case of several other networks like that of power grids, world-wide-web, to name a few [14] . epidemiologists have worked hard on understanding the heterogeneity of scalefree networks for populations for a long time. highly connected individuals as well as hub participants have played essential roles in the spread and maintenance of infections and diseases. figure 1 .1 illustrates the architecture of a system consisting of a population of individuals. it has several essential components, namely, nodes, links, newly connected nodes, hubs and sub-groups respectively. here, nodes correspond to individuals and their relations are shown as links. similarly, newly connected nodes correspond to those which are recently added to the network, such as initiation of new relations between already existing and unknown individuals [24] . hubs are fig. 1.1 a synthetic scale-free network and its characteristics those nodes which are highly connected, such as individuals who are very popular among others and have many relations and/or friends. lastly, sub-groups correspond to certain sections of the population which have individuals with closely associated relationships, such as group of nodes which are highly dense in nature, or having high clustering coefficient. furthermore, it is important in having large number of contacts as the individuals are at greater risk of infection and, once infected, can transmit it to others. for instance, hub individuals of such high-risk individuals help in maintaining sexually transmitted diseases (stds) in different populations where majority belong to long-term monogamous relationships, whereas in case of sars epidemic, a significant proportion of all infections are due to high risk connected individuals. furthermore, the preferential attachment model proposed by barabási and albert [4] defined the existence of individuals of having large connectivity does not require random vaccination for preventing epidemics. moreover, if there is an upper limit on the connectivity of individuals, random immunization can be performed to control infection. likewise, the dynamics of infectious diseases has been extensively studied in case of scale-free as well as small-world and random networks. in small-world networks, most of the nodes may not be direct neighbors, but can be reached from all other nodes via less number of hops, that are number of nodes between start and terminating nodes. also, in these networks distance, dist, between two random nodes increases proportionally to the logarithm of the number of nodes, tot, in the network [15] , i.e., dist ∝ log tot (1.1) watts and strogatz [24] identified a class of small-world networks and categorized them as random graphs. these were classified on the basis of two independent features, namely, average shortest path length and clustering coefficient. as per erdős-rényi model, random graphs have a smaller average shortest path length and small clustering coefficient. watts and strogatz on the other hand demonstrated that various real-world networks have a smaller average shortest path length along with high clustering coefficient greater than expected randomly. it has been observed that it is difficult to block and/or terminate an epidemic in scale-free networks with slow tails. it has especially been seen in case the network correlations among infections and individuals are absent. another reason for this effect is the presence of hubs, where infections could be sustained and reduced by target-specific selections [17] . it has been well known that real-world networks ranging from social to computers are scale-free in nature, whose degree distribution follows an asymptotic power-law. these are characterized by degree distribution following a power law, for the number of connections, conn for individuals and η is an exponent. barabási and albert [4] analyzed the topology of a portion of the world-wide-web and identified 'hubs'. the terminals had larger number of connections than others and the whole network followed a power-law distribution. they also found that these networks have heavy-tailed degree distributions and thus termed them as 'scale-free'. likewise, models for epidemic spread in static heavy-tailed networks have illustrated that with a degree distribution having moments resulted in lesser prevalence and/or termination for smaller rates of infection [14] . moreover, beyond a particular threshold, this prevalence turns to non-zero. similarly, it has been seen that for networks following power-law, does not exist and the prevalence is non-zero for any infection rates. due to this reason, epidemics are difficult to handle and terminate in static networks having powerlaw degree distributions. likewise, in various instances, networks are not static but dynamic (i.e., they evolve in time) via some rewiring processes, in which edges are detached and reattached according to some dynamic rule. steady states of rewiring networks have been studied in the past. more often, it has been observed that depending on the average connectivity and rewiring rates, networks reach a scale-free steady state, with an exponent, η , represented using dynamical rates [17] . the study of epidemics has always been of interest in areas where biological applications coincide with social issues. for instance, epidemics like influenza, measles, and stds, can pass through large group of individuals, populations, and/or persist over longer timescales at low levels. these might even experience sudden changes of increasing and decreasing prevalence. furthermore, in some cases, single infection outbreaks may have significant effects on a complete population group [1] . epidemic spreading can also occur on complex networks with vertices representing individuals and the links representing interactions among individuals. thus, spreading of diseases can occur over the network of individuals as spreading of computer viruses occur over the world-wide-web. the underlying network in epidemic models is considered to be static while the individual states vary from infected to non-infected individuals according to certain probabilistic rules. furthermore, the evolution of an infected group of individuals in time can be studied by focusing on the average density of infected individuals in steady state. lastly, the spread as well as growth of epidemics can also be monitored by studying the architecture of the network of individuals as well as its statistical properties [2] . one of the essential properties of epidemic spread is its branching pattern, thereby infecting healthy individuals over a time period. this branching pattern of epidemic progression can be classified on the basis of their infection initiation, spread and further spread ( fig. 1.3) [5]. 1. infection initiation: if an infected individual comes in contact with a group of individuals, the infection is transmitted to each with a probability p, independent of one another. furthermore, if the same individual meets k others while being infected, these k individuals form the infected set. due to this random disease transmission from the initially infected individual, those directly connected to it get infected. if infection in a branching process reaches an individual set and fails to infect healthy individuals, then termination of the infection occurs, which leads to no further progression and infection of other healthy individuals. thus, there may be two possibilities for an infection in a branching process model. either it reaches a site infecting no further and terminating out, or it continues to infect healthy individuals through contact processes. the quantity which can be used to identify whether an infection persist or fades out is defined as basic reproductive number [6] . this basic reproductive number, τ, is the expected number of newly infected individuals caused by a single already infected individual. in case where every individual meets k new people and infects each with probability p, the basic reproductive number is represented as it is quite essential as it helps in identifying whether or not an infection can spread through a population of healthy individuals. the concept of τ was first proposed by alfred lotka, and applied in the area of epidemiology by macdonald [13] . for non-complex population models, τ can be identified if information for 'death rate' is present. thus, considering death rate, d, and birth rate, b, at the same time, moreover, τ can also be used to determine whether an infection will terminate, i.e., τ < 1 or it becomes an epidemic, i.e., τ > 1. but, it cannot be used for comparing different infections at the same time on the basis of multiple parameters. several methods, such as identifying eigenvalues, jacobian matrix, birth rate, equilibrium states, population statistics can well be used to analyze and handle τ [18] . there are some standard branching models that are existent for analyzing the progress of infection in a healthy population or network. the first one, reed-frost model, considers a homogeneous close set consisting of total number of individuals, tot. let num designate the number of individuals susceptible to infection at time t = 0 and m num the number of individuals infected by the infection at any time t [19] . here, here, eq. 1.7 is in case of a smaller population. it is assumed that an individual x is infected at time t, whereas any individual y comes in contact with x with a probability a num , where a > 0. likewise, if y is susceptible to infection then it becomes infected at time t + 1 and x is removed from the population ( fig. 1.4a ). in this figure, x or v 1 ( * ) represents the infection start site, y(v 3 ), v 2 are individuals that are susceptible to infection, num = 0, tot = 11, and m num = 1. the second one, 3-clique model constructs a 3-clique sub-network randomly by assigning a set of tot individuals. here, for individual/vertex pair (v i , v j ) with probability p 1 , the pair is included along with vertices triples here, g 1 , g 2 are two independent graphs, where g 1 is a bernoulli graph with edge probability p 1 and g 2 with all possible triangles existing independently with a probability p 2 ( fig. 1.4b ). in this figure, 9 ) are the three 3-clique sub-networks with tot = 9, and g = g 1 g 2 g 3 respectively [21] . the third one, household model assumes that for a given a set of tot individuals or vertices, g 1 is a bernoulli graph consisting of tot b disjoint b−cliques, where b tot with edge probability p 2 . thus, the network g is formed as the superposition of the graphs g 1 and g 2 , i.e., g = g 1 g 2 . moreover, g 1 fragments the population into mutually exclusive groups whereas g 2 describes the relations among individuals in the population. thus, g 1 does not allow any infection spread, as there are no connections between the groups. but, when the relationship structure g 2 is added, the groups are linked together and the infection can now spread using relationship connections ( fig. 1.4c ). in this figure, tot = 10 where the individuals (v 1 to v 10 ) are linked on the basis of randomly assigned p 2 and b = 4 tot = 10. the fig. 1 .5b-d respectively [23] . thus, it is essential to identify the conditions which results in an epidemic spread in one network, with the presence of minimal isolated infections on other network components. moreover, depending on the parameters of individual sub-networks and their internal connectivities, connecting them to one another creates marginal effect on the spread of epidemic. thus, identifying these conditions resulting in analyzing spread of epidemic process is very essential. in this case, two different interconnected network modules can be determined, namely, strongly and weakly coupled. in the strongly coupled one, all modules are simultaneously either infection free or part of an epidemic, whereas in the weakly coupled one a new mixed phase exists, where the infection is epidemic on only one module, and not in others [25] . generally, epidemic models consider contact networks to be static in nature, where all links are existent throughout the infection course. moreover, a property of infection is that these are contagious and spread at a rate faster than the initially infected contact. but, in cases like hiv, which spreads through a population over longer time scales, the course of infection spread is heavily dependent on the properties of the contact individuals. the reason for this being, certain individuals may have lesser contacts at any single point in time and their identities can shift significantly with the infection progress [25] . thus, for modeling the contact network in such infections, transient contacts are considered which may not last through the whole epidemic course, but only for particular amount of time. in such cases, it is assumed that the contact links are undirected. furthermore, different individual timings do not affect those having potential to spread an infection but the timing pattern also influences the severity of the overall epidemic spread. similarly, individuals may also be involved in concurrent partnerships having two or more actively involved ones that overlap in time. thus, the concurrent pattern causes the infection to circulate vigorously through the network [22] . in the last decade, considerable amount of work has been done in characterizing as well as analyzing and understanding the topological properties of networks. it has been established that scale-free behavior is one of the most fundamental concepts for understanding the organization various real-world networks. this scale-free property has a resounding effect on all aspect of dynamic processes in the network, which includes percolation. likewise, for a wide range of scale-free networks, epidemic threshold is not existent, and infections with low spreading rate prevail over the entire population [10] . furthermore, properties of networks such as topological fractality etc. correlate to many aspects of the network structure and function. also, some of the recent developments have shown that the correlation between degree and betweenness centrality of individuals is extremely weak in fractal network models in comparison with non-fractal models [20] . likewise, it is seen that fractal scale-free networks are dis-assortative, making such scale-free networks more robust against targeted perturbations on hubs nodes. moreover, one can also relate fractality to infection dynamics in case of specifically designed deterministic networks. deterministic networks allow computing functional, structural as well as topological properties. similarly, in case of complex networks, determination of topological characteristics has shown that these are scale-free as well as highly clustered, but do not display small-world features. also, by mapping a standard susceptible, infected, recovered (sir) model to a percolation problem, one can also find that there exists certain finite epidemic threshold. in certain cases, the transmission rate needs to exceed a critical value for the infection to spread and prevail. this also specifies that the fractal networks are robust to infections [11] . meanwhile, scale-free networks exhibit various essential characteristics such as power-law degree distribution, large clustering coefficient, large-world phenomenon, to name a few [16] . network analysis can be used to describe the evolution and spread of information in the populations along with understanding their internal dynamics and architecture. specifically, importance should be given to the nature of connections, and whether a relationship between x and y individuals provide a relationship between y and x as well. likewise, this information could be further utilized for identifying transitivitybased measures of cohesion ( fig. 1.6 ). meanwhile, research in networks also provide some quantitative tools for describing and characterizing networks. degree of a vertex is the number of connectivities for each vertex in the form of links. for instance, degree(v 4 ) = 3, degree(v 2 ) = 4 (for undirected graph (fig. 1.6a) ). similarly for fig. 1 likewise, shortest path is the minimum number of links that needs to be parsed for traveling between two vertices. for instance, in fig. 1 diameter of network is the maximum distance between any two vertices or the longest of the shortest walks. thus, in fig. 1 [15] . radius of a network is the minimum eccentricity (eccentricity of a vertex v i is the greatest geodesic distance), i.e., distance between two vertices in a network is the number of edges in a shortest path connecting them between v i and any other vertex of any vertex. for instance, in fig. 1 .6b, radius of network = 2. betweenness centrality (g(v i )) is equal to the number of shortest paths from all vertices to all others that pass through vertex v i , i.e., is the number of those paths that pass through v i . thus, in fig. 1 similarly, closeness centrality (c(v i )) of a vertex v i describes the total distance of v i to all other vertices in the network, i.e., sum the shortest paths of v i to all other vertices in the network. for instance, in fig. 1.6b, c( lastly, stress centrality (s(v i )) is the simple accumulation of the number of shortest paths between all vertex pairs, sometimes interchangeable with betweenness centrality [14] . use of 'adjacency matrix', a v i v j , describing the connections within a population is also persistent. likewise, various network quantities can be ascertained from the adjacency matrix. for instance, size of a population is defined as the average number of contacts per individual, i.e., the powers of adjacency matrix can be used to calculate measures of transitivity [14] . one of the key pre-requisites of network analysis is initial data collection. for performing a complete mixing network analysis for individuals residing in a population, every relationship information is essential. this data provides great difficulty in handling the entire population, as well as handling complicated network evaluation issues. the reason being, individuals have contacts, and recall problems are quite probable. moreover, evaluation of contacts requires certain information which may not always be readily present. likewise, in case of epidemiological networks, connections are included if they explain relationships capable of permitting the transfer of infection. but, in most of the cases, clarity of defining such relations is absent. thus, various types of relationships bestow risks and judgments that needs to be sorted for understanding likely transmission routes. one can also consider weighted networks in which links are not merely present or absent but are given scores or weights according to their strength [9] . furthermore, different infections are passed by different routes, and a mixing network is infection specific. for instance, a network used in hiv transmission is different from the one used to examine influenza. similarly, in case of airborne infections like influenza and measles, various networks need to be considered because differing levels of interaction are required to constitute a contact. the problems with network definition and measurement imply that any mixing networks that are obtained will depend on the assumptions and protocols of the data collection process. three main standard techniques can be employed to gather such information, namely, infection searching, complete contact searching and diary-based studies [9] . after an epidemic spread, major emphasis is laid on determining the source and spread of infection. thus, each infected individual is linked to one other from whom infection is spread as well as from whom the infection is transmitted. as all connections represent actual transmission events, infection searching methods do not suffer from problems with the link definition, but interactions not responsible for this infection transmission are removed. thus, the networks observed are of closed architecture, without any loops, walks, cliques and complete sub-graphs [15] . infection searching is a preliminary method for infectious diseases with low prevalence. these can also be simulated using several mathematical techniques based on differential equations, control theories etc., assuming a homogeneous mixing of population. it can also be simulated in a manner so that infected individuals are identified and cured at a rate proportional to the number of neighbors it has, analogous to the infection process. but, it does not allow to compare various infection searching budgets and thus a discrete-event simulation need to be undertaken. moreover, a number of studies have shown that analyses based on realistic models of disease transmission in healthy networks yields significant projections of infection spread than projections created using compartmental models [8] . furthermore, depending on the number of contacts for any infected individuals, their susceptible neighbors are traced and removed. this is followed by identifying infection searching techniques that yields different numbers of newly infected individuals on the spread of the disease. contact searching identifies potential transmission contacts from an initially infected individual by revealing some new individual set who are prone to infection and can be subject of further searching effort. nevertheless, it suffers from network definition issues; is time consuming and depends on complete information about individuals and their relationships. it has been used as a control strategy, in case of stds. its main objective of contact searching is identifying asymptomatically infected individuals who are either treated or quarantined. complete contact searching deals with identifying the susceptible and/or infected individuals of already infected ones and conducting simulations and/or testing them for degree of infection spread, treating them as well as searching their neighbors for immunization. for instance, stds have been found to be difficult for immunization. the reason being, these have specifically long asymptomatic periods, during which the virus can replicate and the infection is transmitted to healthy, closely related neighbors. this is rapidly followed by severe effects, ultimately leading to the termination of the affected individual. likewise, recognizing these infections as global epidemic has led to the development of treatments that allow them to be managed by suppressing the replication of the infection for as long as possible. thus, complete contact searching act as an essential strategy even in case when the infection seems incurable [7] . diary-based studies consider individuals recording contacts as they occur and allow a larger number of individuals to be sampled in detail. thus, this variation from the population approach of other tracing methods to the individual-level scale is possible. but, this approach suffers from several disadvantages. for instance, the data collection is at the discretion of the subjects and is difficult for researchers to link this information into a comprehensive network, as the individual identifies contacts that are not uniquely recorded [3] . diary-based studies require the individuals to be part of some coherent group, residing in small communities. also, it is quite probable that this kind of a study may result in a large number of disconnected sub-groups, with each of them representing some locally connected set of individuals. diary-based studies can be beneficial in case of identifying infected and susceptible individuals as well as the degree of infectivity. these also provide a comprehensive network for diseases that spread by point-to-point contact and can be used to investigate the patterns infection spread. robustness is an essential connectivity property of power-law graph. it defines that power-law graphs are robust under random attack, but vulnerable under targeted attack. recent studies have shown that the robustness of power-law graph under random and targeted attacks are simulated displaying that power-law graphs are very robust under random errors but vulnerable when a small fraction of high degree vertices or links are removed. furthermore, some studies have also shown that if vertices are deleted at random, then as long as any positive proportion remains, the graph induced on the remaining vertices has a component of order of the total number of vertices [15] . many a times it can be observed that a network of individuals may be subject to sudden change in the internal and/or external environment, due to some perturbation events. for this reason, a balance needs to be maintained against perturbations while being adaptable in the presence of changes, a property known as robustness. studies on the topological and functional properties of such networks have achieved some progress, but still have limited understanding of their robustness. furthermore, more important a path is, higher is the chance to have a backup path. thus, removing a link or an individual from any sub-network may also lead to blocking the information flow within that sub-network. the robustness of a model can also be assessed by means of altering the various parameters and components associated with forming a particular link. robustness of a network can also be studied with respect to 'resilience', a method of analyzing the sensitivities of internal constituents under external perturbation, that may be random or targeted in nature [18] . basic disease models discuss the number of individuals in a population that are susceptible, infected and/or recovered from a particular infection. for this purpose, various differential equation based models have been used to simulate the events of action during the infection spread. in this scenario, various details of the infection progression are neglected, along with the difference in response between individuals. models of infections can be categorized as sir and susceptible, infected, susceptible (sis) [9] . the sir model considers individuals to have long-lasting immunity, and divides the population into those susceptible to the disease (s), infected (i) and recovered (r). thus, the total number of individuals (t ) considered in the population is the transition rate from s to i is κ and the recovery rate from i to r is ρ . thus, the sir model can be represented as likewise, the reproductivity (θ) of an infection can be identified as the average number of secondary instances a typical single infected instance will cause in a population with no immunity. it determines whether infections spreads through a population; if θ < 1, the infection terminates in the long run; θ > 1, the infection spreads in a population. larger the value of θ, more difficult is to control the epidemic [12] . furthermore, the proportion of the population that needs to be immunized can be calculated by known as endemic stability can be identified. depending upon these instances, immunization strategies can be initiated [6] . although the contact network in a general sir model can be arbitrarily complex, the infection dynamics can still being studied as well as modeled in a simple fashion. contagion probabilities are set to a uniform value, i.e., p, and contagiousness has a kind of 'on-off' property, i.e., an individual is equally contagious for each of the t i steps while it has the infection, where 1 is present state of the system. one can extend the idea that contagion is more likely between certain pairs of individuals or vertices by assigning a separate probability p v i ,v j to each pair of individuals or vertices v i and v j , for which v i is linked to v j in a directed contact network. likewise, other extensions of the contact model involves separating the i state into a sequence of early, middle, and late periods of the infection. for instance, it could be used to model an infection with a high contagious incubation period, followed by a less contagious period while symptoms are being expressed [16] . in most of the cases, sir epidemics are thought of dynamic processes, in which the network state evolves step-by-step over time. it captures the temporal dynamics of the infection as it spreads through a population. the sir model has been found to be suitable for infections, which provides lifelong immunity, like measles. in this case, a property termed as the force of infection is existent, a function of the number of infectious individuals is. it also contains information about the interactions between individuals that lead to the transmission of infection. one can also have a static view of the epidemics where sir model for t i = 1. this means that considering a point in an sir epidemic when a vertex v i has just become infectious, has one chance to infect v j (since t i = 1), with probability p. one can visualize the outcome of this probabilistic process and also assume that for each edge in the contact network, a probability signifying the relationship is identified. the sis model can be represented as removed state is absent in this case. moreover, after a vertex is over with the infectious state, it reverts back to the susceptible state and is ready to initiate the infection again. due to this alternation between the s and i states, the model is referred to as sis model. the mechanics of sis model can be discussed as follows [2] . 1. at the initial stage, some vertices remain in i state and all others are in s state. 2. each vertex v i that enters the i state and remains infected for a certain number of steps t i . 3. during each of these t i steps, v i has a probability p of passing the infection to each of its susceptible directly linked neighbors. 4. after t i steps, v i no longer remains infected, and returns back to the s state. the sis model is predominantly used for simulating and understanding the progress of stds, where repeat infections are existent, like gonorrhoea. moreover, certain assumptions with regard to random mixing between individuals within each pair of sub-networks are present. in this scenario, the number of neighbors for each individual is considerably smaller than the total population size. such models generally avoid random-mixing assumptions thereby assigning each individual to a specific set of contacts that they can infect. an sis epidemic, can run for long time duration as it can cycle through the vertices multiple number of times. if at any time during the sis epidemic all vertices are simultaneously free of the infection, then the epidemic terminates forever. the reason being, no infected individuals exist that can pass the infection to others. in case if the network is finite in nature, a stage would arise when all attempts for further infection of healthy individuals would simultaneously fail for t i steps in a row. likewise, for contact networks where the structure is mathematically tractable, a particular critical value of the contagion probability p is existent, an sis epidemic undergoes a rapid shift from one that terminates out quickly to one that persists for a long time. in this case, the critical value of the contagion probability depends on the structure of the problem set [1] . the patterns by which epidemics spread through vertex groups is determined by the properties of the pathogen, length of its infectious period, severity and the network structures. the path for an infection spread are given by a population state, with existence of direct contacts between the individuals or vertices. the functioning of network system depends on the nature of interaction between their individuals. this is essentially because of the effect of infection-causing individuals and topology of networks. to analyze the complexity of epidemics, it is important to understand the underlying principles of its distribution in the history of its existence. in recent years it has been seen that the study of disease dynamics in social networks is relevant with the spread of viruses and the nature of diseases [9] . moreover, the pathogen and the network are closely intertwined with even within the same group of individuals, the contact networks for two different infections are different structures. this depends on respective modes of transmission of infections. for instance, a highly contagious infection, involving airborne transmission, the contact network includes a huge number of links, including any pair of individuals that are in contact with one another. likewise, for an infection requiring close contact, the contact network is much sparser, with fewer pairs of individuals connected by links [7] . immunization is a site percolation problem where each immunized individual is considered to be a site which is removed from the infected network. its aim is to transfer the percolation threshold that leads to minimization of the number of infected individuals. the model of sir and immunization is regarded as a site-bond percolation model, and immunization is considered successful if the infected a network is below a predefined percolation threshold. furthermore, immunizing randomly selected individuals requires targeting a large fraction, frac, of the entire population. for instance, some infections require 80-100 % immunization. meanwhile, targetbased immunization of the hubs requires global information about the network in question, rendering it impractical in many cases, which is very difficult in certain cases [6] . likewise, social networks possess a broad distribution of the number of links, conn, connecting individuals and analyzing them illustrate that that a large fraction, frac, of the individuals need to be immunized before the integrity of the infected network is compromised. this is essentially true for scale-free networks, where p(conn) ≈ conn − η , 2 < η < 3, where the network remains connected even after removal of most of its individuals or vertices. in this scenario, a random immunization strategy requires that most of the individuals need to be immunized before an epidemic is terminated [8] . for various infections, it may be difficult to reach a critical level of immunization for terminating the infection. in this case, each individual that is immunized is given immunity against the infection, but also provides protection to other healthy individuals within the population. based on the sir model, one can only achieve half of the critical immunization level which reduces the level of infection in the population by half. a crucial property of immunization is that these strategies are not perfect and being immunized does not always confer immunity. in this case, the critical threshold applies to a portion of the total population that needs to be immunized. for instance, if the immunization fails to generate immunity in a portion, por, of those immunized, then to achieve immunity one needs to immunize a portion here, im denotes immunity strength. thus, in case if por is huge it is difficult to remove infection using this strategy or provides partial immunity. it may also invoke in various manners: the immunization reduces the susceptibility of an individual to a particular infection, may reduce subsequent transmission if the individual becomes infected, or it may increase recovery. such immunization strategies require the immunized individuals to become infected and shift into a separate infected group, after which the critical immunization threshold (s i ) needs to be established. thus, if cil is the number of secondary infected individuals affected by an initial infectious individual, then thus, s i needs to be less than one, else it is not possible to remove the infection. but, one also needs to note that an immunization works equally efficiently if it reduces the transmission or susceptibility and increases the recovery rate. moreover, when the immunization strategy fails to generate any protection in a proportion por of those immunized, the rest 1−por are fully protected. in this scenario, it can be not possible to remove the infection using random immunization. thus, targeted immunization provides better protection than random-based [13] . in case of homogenous networks, the average degree, conn, fluctuates less and can assume conn conn, i.e., the number of links are approximately equal to average degree. however, networks can also be heterogeneous. likewise, in a homogeneous network such as a random graph, p(conn) decays faster exponentially whereas for heterogenous networks it decays as a power law for large conn. the effect of heterogeneity on epidemic behavior studied in details for many years for scale-free networks. these studies are mainly concerned with the stationary limit and existence of an endemic phase. an essential result of this analysis is the expression of basic reproductive number which in this case is τ ∞ conn 2 conn . here, τ is proportional to the second moment of degree, which finally diverges for increasing network sizes [15] . it has been noticed that the degree of interconnection in between individuals for all form of networks is quite unprecedented. whereas, interconnection increases the spread of information in social networks, another exhaustively studied area contributes to the spread of infection throughout the healthy network. this rapid spreading is done due to less stringency of its passage through the network. moreover, initial sickness nature and time of infection are unavailable most of the time, and the only available information is related to the evolution of the sick-reporting process. thus, given complete knowledge of the network topology, the objective is to determine if the infection is an epidemic, or if individuals have become infected via an independent infection mechanism that is external to the network, and not propagated through the connected links. if one considers a computer network undergoing cascading failures due to worm propagation whereas random failures due to misconfiguration independent of infected nodes, there are two possible causes of the sickness, namely, random and infectious spread. in case of random sickness, infection spreads randomly and uniformly over the network where the network plays no role in spreading the infection; and infectious spread, where the infection is caused through a contagion that spreads through the network, with individual nodes being infected by direct neighbors with a certain probability [6] . in random damage, each individual becomes infected with an independent probability ψ 1 . at time t, each infected individual reports damage with an independent probability ψ 2 . thus, on an average, a fraction ψ of the network reports being infected, where it is already known that social networks possess a broad distribution of the number of links, k, originating from an individual. computer networks, both physical and logical are also known to possess wide, scale-free, distributions. studies of percolation on broad-scale networks display that a large fraction, fc, of the individuals need to be immunized before the integrity of the network is compromised. this is particularly true for scale-free networks, where the percolation threshold tends to 1, and the network remains contagious even after removal of most of its infected individuals [9] . when the hub individuals are targeted first, removal of just a fraction of these results in the breakdown of the network. this has led to the suggestion of targeted immunization of hubs. to implement this approach, the number for connections of each individual needs to be known. during infection spread, at time 0, a randomly selected individual in the network becomes infected. when a healthy individual becomes infected, a time is set for each outgoing link to an adjacent individual that is not infected, with expiration time exponentially distributed with unit average. upon expiration of a link's time, the corresponding individual becomes infected, and in-turn begins infecting its neighbors [7] . in general, for an epidemic to occur in a susceptible population the basic reproductive rate must be greater than 1. in many circumstances not all contacts will be susceptible to infection. in this case, some contacts remain immune, due to prior infection which may have conferred life-long immunity, or due to some previous immunization. therefore, not all individuals are infected and the average number of secondary infections decrease. similarly, the epidemic threshold in this case is the number of susceptible individuals within a population that is required for an epidemic to occur. similarly, the herd immunity is the proportion of population immune to a particular infection. if this is achieved due to immunization, then each case leads to a new case and the infection becomes more stable within the population [6] . one of the simplest immunization procedure consists of random introduction of immune individuals in the population for achieving uniform immunization density. in this case, for a fixed spreading rate, ξ , the relevant control parameter in the density of immune individuals present in the network, the immunity, imm. at the meanfield level, the presence of a uniform immunity reduces ξ by a factor 1 − imm, i.e., the probability of identifying and infecting a susceptible and non-immune individual becomes ξ(1−imm). for homogeneous networks, one observes that, for aconstant ξ , the stationary prevalence is given by for imm > imm c and for imm ≤ imm c here imm c is the critical immunization value above which the density of infected individuals in the stationary state is null and depends on ξ as thus, for a uniform immunization level larger than imm c , the network is completely protected and no large epidemic outbreaks are possible. on the contrary, uniform immunization strategies on scale-free heterogenous networks are totally ineffective. the presence of uniform immunization elocally depresses the infections prevalence for any value of ξ , and it is difficult to identify any critical fraction of immunized individuals that ensures the eradication of infection [2] . cascading, or epidemic processes are those where the actions, infections or failure of certain individuals increase the susceptibility of others. this results in the successive spread of infections from a small set of initially infected individuals to a larger set. initially developed as a way to study human disease propagation, cascades ares useful models in a wide range of application. the vast majority of work on cascading processes focused on understanding how the graph structure of the network affects the spread of cascades. one can also focus on several critical issues for understanding the cascading features in network for which studying the architecture of the network is crucial [5] . the standard independent cascade epidemic model assumes that the network is directed graph g = (v, e), for every directed edge between v i , v j , we say v i is a parent and v j is a child of the corresponding other vertex. parent may infect child along an edge, but the reverse cannot happen. let v denote the set of parents of each vertex v i , and for convenience v i ∈ v is included. epidemics proceed in discrete time where all vertices are initially in the susceptible state. at time 0, each vertex independently becomes active, with probability p init . this set of initially active vertices are called 'seeds'. in each time step, the active vertices probabilistically infects its susceptible children; if vertex v i is active at time t, it infects each susceptible child v j with probability p v i vj , independently. correspondingly, a vertex v j susceptible at time t becomes active in the next time step, i.e., at time t + 1, if any one of its parents infects it. finally, a vertex remains active for only one time slot, after which it becomes inactive and does not spread the infection further as well as cannot be infected again either [5] . thus, in this kind of an sir epidemic, where some vertices remain forever susceptible because the epidemic never reaches them, while others transition, susceptible → active for one time step → inactive. in this chapter, we discussed some critical issues regarding epidemics and their outbursts in static as well as dynamic network structures. we mainly focused on sir and sis models as well as identifying key strategies for identifying the damage caused in networks. we also discussed the various modeling techniques for studying cascading failures. epidemics pass through populations and persists over long time periods. thus, efficient modeling of the underlying network plays a crucial role in understanding the spread and prevention of an epidemic. social, biological, and communication systems can be explained as complex networks with their degree distribution follows a power law, p(conn) ≈ conn − η , for the number of connections, conn for individuals, representing scale-free (sf) networks. we also discussed certain issues on epidemic spreading in sf networks characterized by complex topologies with basic epidemic models describing the proportion of individuals susceptible, infected and recovered from a particular disease. likewise, we also explained the significance of the basic reproduction rate of an infection, that can be identified as the average number of secondary instances a typical single infected instance will cause in a population with no immunity. also, we explained how determining the complete nature of a network required knowledge of every individual in a population and their relationships as, the problems with network definition and measurement depend on the assumptions of data collection processes. nevertheless, we also illustrated the importance of invasion resistance methods, with temporary immunity generating oscillations in localized parts of the network, with certain patches following large numbers of infections in concentrated areas. similarly, we also explained the significance of damages, namely, random, where the damage spreads randomly and uniformly over the network and in particular the network plays no role in spreading the damage; and infectious spread, where the damage spreads through the network, with one node infecting others with some probability. infectious diseases of humans: dynamics and control the mathematical theory of infectious diseases and its applications a forest-fire model and some thoughts on turbulence emergence of scaling in random networks mathematical models used in the study of infectious diseases spread of epidemic disease on networks networks and epidemic models network-based analysis of stochastic sir epidemic models with random and proportionate mixing elements of mathematical ecology intelligent information and database systems propagation phenomenon in complex networks: theory and practice relation between birth rates and death rates the analysis of malaria epidemics graph theory and networks in biology mathematical biology spread of epidemic disease on networks the use of mathematical models in the epidemiology study of infectious diseases and in the design of mass vaccination programmes forest-fire as a model for the dynamics of disease epidemics on the critical behaviour of simple epidemics sensitivity estimates for nonliner mathematical models ensemble modeling of metabolic networks on analytical approaches to epidemics on networks computational modeling in systems biology collective dynamics of 'small-world' networks unifying wildfire models from ecology and statistical physics key: cord-026005-f2khcjdy authors: lópez, alfonso; martinson, shannon a. title: respiratory system, mediastinum, and pleurae date: 2017-02-17 journal: pathologic basis of veterinary disease doi: 10.1016/b978-0-323-35775-3.00009-6 sha: doc_id: 26005 cord_uid: f2khcjdy nan diseases of the respiratory system (respiratory apparatus) are some of the leading causes of morbidity and mortality in animals and a major source of economic losses. thus veterinarians are routinely called to diagnose, treat, and implement health management practices to reduce the impact of these diseases. in companion animals, diseases of the respiratory tract are also common and, although of little economic significance, are important to the health of the animals and thus to clinicians and pet owners. in the past few years, animal shelters have been recognized as a major risk factor for respiratory diseases in dogs and cats, a comparable situation to what is reported in human beings with nosocomial infections. to facilitate the understanding of the structure and function, it is convenient to arbitrarily divide the respiratory system into conducting, transitional, and gas exchange systems ( fig. 9-1 ). the conducting system includes nostrils, nasal cavity, paranasal sinuses, nasopharynx, larynx, trachea, and extrapulmonary and intrapulmonary bronchi, all of which are largely lined by pseudostratified, ciliated columnar cells, plus a variable proportion of secretory goblet (mucous) and serous cells (figs. 9-2 and 9-3 and efig. 9-1 ). the transitional system of the respiratory tract is composed of bronchioles, which are microscopic structures that serve as a transition zone between the conducting system (ciliated) and the gas exchange (alveolar) system (see fig. 9 -1). the disappearance of cilia in the transitional system is not abrupt; the ciliated cells in the proximal bronchiolar region become scarce and progressively attenuated, until the point where distal bronchioles no longer have ciliated cells. normal bronchioles also lack goblet cells but instead have other types of secretory cells, notably club cells (formerly clara cells) and neuroendocrine cells. club cells, also referred to as secretory bronchiolar cells, contain numerous biosynthetic organelles that play an active role in detoxification of xenobiotics (foreign substances), similar to the role of hepatocytes ( fig. 9-4) . club cells are also critical stem cells in the repair and remodeling of not only the bronchioles but also of most of the respiratory tract. in addition, club cells contribute to the innate immunity of the lung by secreting protective proteins (collectins) and pulmonary surfactant (see b) . in carnivores and monkeys, and to a much lesser extent in horses and human beings, the terminal portions of bronchioles are lined not only by cuboidal epithelium but also by segments of alveolar capillaries. these unique bronchioloalveolar structures are known as respiratory bronchioles ; also see fig. 9 -1). the gas exchange system of the respiratory tract in all mammals is formed by alveolar ducts and millions of alveoli ( fig. 9-6 ; also see fig. 9 -1). the surface of the alveoli is lined by two distinct types of epithelial cells known as type i (membranous) pneumonocytes and type ii (granular) pneumonocytes ( fig. 9-7) . all three-the conducting, transitional, and exchange systems of the respiratory system-are vulnerable to injury because of constant exposure to a myriad of microbes, particles and fibers, and toxic gases and vapors present in the air. vulnerability of the respiratory system to aerogenous (airborne) injury is primarily because of (1) the extensive area of the alveoli, which are the interface between the blood in alveolar capillaries and inspired air; (2) the large volume of air passing continuously into the lungs; and (3) the high concentration of noxious elements that can be present in the air (table 9 -1). for human beings, it has been estimated that the surface of the pulmonary alveoli is approximately 200 m 2 , roughly the area animal and cultured for microbes, yeasts, and fungi, many species of bacteria are recovered, such as mannheimia (pasteurella) haemolytica in cattle; pasteurella multocida in cats, cattle, and pigs; and bordetella bronchiseptica in dogs and pigs. the organisms that constitute the normal flora of the respiratory tract are restricted to the most proximal (rostral) region of the conducting system (nasal cavity, pharynx, and larynx). the thoracic portions of the trachea, bronchi, and lungs are considered to be essentially sterile. the types of bacteria present in the nasal flora vary considerably among animal species and in different geographic regions of the world. some present in the nasal flora are pathogens that can cause important respiratory infections under some circumstances. for instance, mannheimia (pasteurella) haemolytica is part of the bovine nasal flora, yet this bacterium causes a devastating disease in cattle-pneumonic mannheimiosis (shipping fever). experimental studies have established that microorganisms from the nasal flora are continuously carried into the lungs via tracheal air. despite this constant bacterial bombardment from the nasal flora and from contaminated air, normal lungs remain sterile because of their remarkably effective defense mechanisms. the conducting portion of the respiratory system is lined by pseudostratified columnar ciliated epithelium (most of the nasal cavity, paranasal sinuses, part of the larynx, and all of the trachea and bronchi), olfactory epithelium (part of the nasal cavity, particularly ethmoidal conchae), and squamous epithelium (nasal vestibulum and parts of the larynx). the pattern of injury, inflammation, and host response (wound healing) are characteristic for each of these three types of epithelium independent of its anatomic location. pseudostratified ciliated epithelium, which lines most of the nasal cavity and nasopharynx, part of the larynx, and all of the trachea and bronchi, is exquisitely sensitive to injury. when these cells are irreversibly injured, whether caused by viral infection, trauma, or inhalation of toxic gases, the ciliated cells swell, typically lose their attachment to underlying basement membrane, and rapidly exfoliate ( fig. 9-8) . a transient and mild exudate of fluid, plasma proteins, and neutrophils covers the ulcer. in the absence of of a tennis court. the alveolar surface of the equine lung is estimated to be approximately 2000 m 2 . it has also been estimated that the volume of air reaching the human lung every day is approximately 9000 l. lungs are also susceptible to blood-borne (hematogenous) microbes, toxins, and emboli. this fact is not surprising because the entire cardiac output of the right ventricle goes into the lungs, and approximately 9% of the total blood volume is within the pulmonary vasculature. the pulmonary capillary bed is the largest in the body, with a surface area of 70 m 2 in the adult human; this area is equivalent to a length of 2400 km of capillaries, with 1 ml of blood occupying up to 16 km of capillary bed. the respiratory system has its own normal flora (microbiota), as does any other body system in contact with the external environment. if a sterile swab is passed deep into the nasal cavity of any healthy (rhinoviruses), infectious bovine rhinotracheitis (bovine herpesvirus 1), feline rhinotracheitis (felid herpesvirus 1), and viruses of the canine infectious respiratory disease (cird) group such as canine adenovirus 2 (cav-2) and canine parainfluenza virus (cpiv) . if damage to the mucociliary blanket becomes chronic, goblet cell hyperplasia takes place, leading to excessive mucus production (hypersecretion) and reduced mucociliary clearance, and when there is loss of basement membrane, repair is by fibrosis and granulation tissue (scarring). in the most severe cases, prolonged injury causes squamous metaplasia, which together with scarring causes airway obstruction and an impediment to mucociliary clearance. in laboratory rodents, hyperplastic and metaplastic changes, such as those seen in nasal polyps and squamous metaplasia, are considered a prelude to neoplasia. the second type of epithelium lining the conducting system is the sensory olfactory epithelium, present in parts of the nasal mucosa, notably in the ethmoidal conchae. the patterns of degeneration, exfoliation, and inflammation in the olfactory epithelium are similar to those of the ciliated epithelium, except that olfactory complications or secondary bacterial infections, a specific type of progenitor cells known as basal cells or nonciliated secretory cells (preciliated cells) , which are normally present in the mucosa, migrate to cover the denuded basement membrane and undergoes mitosis, eventually differentiating into new ciliated epithelial cells (see fig. 9 -8). cellular migration, proliferation, and attachment are regulated by locally released interleukins (il-1β, il-2, il-4, and il-13), growth factors, integrins and extracellular matrix (ecm) proteins such as collagen, and fibronectin. the capacity of ciliated epithelium to repair itself is remarkably effective. for example, epithelial healing in an uncomplicated ulcer of the tracheal mucosa can be completed in only 10 days. this sequence of cell degeneration, exfoliation, ulceration, mitosis, and repair is typically present in many viral infections in which viruses replicate in nasal, tracheal, and bronchial epithelium, causing extensive mucosal ulceration. examples of transient infections of this type include human colds epithelium. neurons in the olfactory mucosa have the unique ability to regenerate, a fact that is being explored as a potential source of new neurons in the treatment of spinal cord injury. squamous epithelium, located in the vestibular region of the nose (mucocutaneous junction), is the third type of epithelium present in the nasal passages. compared with ciliated and olfactory epithelia, nasal squamous epithelium is quite resistant to all forms of injury. the pharyngeal mucosa, composed of squamous epithelium, has similar patterns of necrosis and inflammation as the oral mucosa (see chapter 7). the patterns of necrosis, inflammation, and repair in intrapulmonary bronchi are similar to those previously described for the nasal and tracheal epithelium. in brief, injury to ciliated bronchial epithelium may result in degeneration, detachment, and exfoliation of necrotic cells. under normal circumstances, cellular exfoliation is promptly followed by inflammation, mitosis, cell proliferation, cell differentiation, and finally by repair ( fig. 9-9 and see . depending on the type of exudate, bronchitis can be fibrinous, catarrhal, purulent, fibrinonecrotic (diphtheritic), and sometimes granulomatous. when epithelial injury becomes chronic, production of mucus is increased via goblet cell hyperplasia (chronic catarrhal inflammation). this form of chronic bronchitis is well illustrated in habitual smokers who continually need to cough out excessive mucus secretions (sputum). unfortunately, in some cases, excessive mucus cannot be effectively cleared from airways, which of the bronchial wall or cylindrical when destruction involves a large segment of a bronchus. grossly, bronchiectasis is manifested by prominent lumps in the lungs (bosselated appearance or having rounded eminences) resulting from distention of bronchi with exudate, which results in a concurrent obstructive atelectasis of surrounding parenchyma ( fig. 9-11 ). the cut surfaces of dilated bronchi are filled with purulent exudates; for this reason, bronchiectasis is often mistaken for pulmonary abscesses. careful inspection, usually requiring microscopic examination, confirms that exudate is contained and surrounded by remnants of a bronchial wall lined by squamous epithelium and not by a pyogenic membrane (connective tissue) as it is in the case of a pulmonary abscess. the squamous metaplasia further interferes with the normal function of the mucociliary escalator. the epithelial lining of the bronchiolar region (transitional zone) is exquisitely susceptible to injury, particularly to that caused by some respiratory viruses (bovine parainfluenza virus 3, bovine respiratory syncytial virus, adenovirus, or canine distemper virus), oxidant gases (nitrogen dioxide [no 2 ], sulfur dioxide [so 2 ], or ozone [o 3 ]), and toxic substances (3-methylindole or paraquat). the precise explanation as to why bronchiolar epithelium is so prone to injury is still not clear, but it is presumably due in part to (1) its high vulnerability to oxidants and free radicals; (2) the presence of leads to chronic obstructive bronchitis and emphysema (see . chronic bronchial irritation causes squamous metaplasia of highly functional but vulnerable ciliated epithelium to nonfunctional, but more resistant, squamous epithelium. squamous metaplasia has a calamitous effect on pulmonary clearance because it causes a structural loss and functional breakdown of portions of the mucociliary escalator. hyperplasia of bronchial glands occurs frequently in chronic bronchitis, which translates to an increase of the reid index (bronchial-gland to bronchial-wall ratio) (efig. 9 -2). this index is less than 30% in the healthy human lung and in the lungs of most domestic species, except for cats, which generally have an index higher than 40%. the term airway remodeling encompasses all the structural changes that accompany chronic bronchitis such as hypertrophy and hyperplasia of smooth muscle, submucosal glands, and goblet cells; fibrosis; and increased bronchial vascularity. bronchiectasis is one of the most devastating sequelae to chronic remodeling of the bronchi. it consists of a pathologic and permanent dilation of a bronchus with rupture of the bronchial wall as a result of obstruction or chronic inflammation. destruction of walls occurs in part when proteolytic enzymes and oxygen radicals released from phagocytic cells during chronic inflammation degrade and weaken the smooth muscle and cartilage (chondromalacia) that help to maintain normal bronchial diameter ( fig. 9-10 ). bronchiectasis may be saccular when destruction affects only a small localized portion . this same type of lesion is seen in viral or mechanical injury to the mucosa of the conducting system. two days after exposure, the basement membrane is lined by rapidly dividing preciliated cells, some of which exhibit mitotic activity (inset). ten days after injury, the nasal epithelium is completely repaired. h&e stain. b, schematic representation of the events of injury and repair in the respiratory mucosa of the conducting system. blue cell, ciliated mucosal epithelial cell; pink cell, goblet cell; red cell, neutrophil. (a from lópez a, prior m, yong s, et al: am j vet res 49:1107 -1111 , 1988 into well-organized, microscopic polyps inside the bronchiolar lumen. the external surface of the exudate eventually becomes covered by ciliated cells. this lesion is referred to as bronchiolitis obliterans, and the polyps may become so large as to cause airflow impairment ( fig. 9 -12 and see fig. 9 -9). in mild but persistent bronchiolar injury, goblet cells normally absent from bronchioles proliferate from basal cells, resulting in goblet cell metaplasia and causing a profound alteration in the physicochemical properties of bronchiolar secretions ( fig. 9-13 ). the normally serous bronchiolar fluid released by club (clara) cells becomes a tenacious material when mucus produced by goblet cells is added. as a result of increased viscoelasticity of the mucus, bronchiolar secretions cannot be removed effectively by ciliary action, leading to plugging and obstruction of distal airways. under such conditions, often grouped as chronic obstructive pulmonary disease, coughing is required to clear mucus from obstructed bronchioles. pulmonary emphysema and atelectasis are further sequelae to bronchiolar metaplasia and mucous hypersecretion blocking or partially blocking the lumens of these bronchioles. these two inflation abnormalities are characteristically present in chronic obstructive pulmonary disease (copd), which is called "recurrent airway obstruction (rao or "heaves") in horses (see recurrent airway obstruction, under disorders of horses). peribronchiolar club (clara) cells rich in mixed function oxidases, which locally generate toxic metabolites (see fig. 9 -4); and (3) the tendency for pulmonary alveolar macrophages and leukocytes to accumulate in this region of the lungs. depending on the types of injury and inflammatory response, bronchiolitis is classified as necrotizing, suppurative, catarrhal (mucous metaplasia), or granulomatous. once injury to bronchiolar ciliated cells becomes irreversible, the cells degenerate and exfoliate into the bronchiolar lumen, leaving a denuded basement membrane. repair in the bronchiolar region is similar to, but less effective than, that in the tracheal or nasal mucosa. under normal circumstances, recruited phagocytic cells remove exudate and cell debris from the lumina of affected bronchioles, thus preparing the basement membrane to be repopulated with new, undifferentiated cells originating from a rapidly dividing pool of club (clara) cells. after several days, these proliferating cells fully differentiate into normal bronchiolar cells. in severe acute injury, such as that caused by aspiration pneumonia or by highly pathogenic microorganisms, exudate attaches and cannot be removed from the basement membrane of bronchioles. the exudate becomes infiltrated by fibroblasts, which form small nodular masses of fibrovascular tissue that develop postviral bronchiolitis is associated with increased expression of tlrs and unusual susceptibility to inhaled endotoxin. hyperreactive animals typically have an increased number of mast cells, eosinophils, and t lymphocytes in the airway mucosa. clinically, airway hyperresponsiveness is characterized by an exaggerated bronchoconstriction after natural exposure to mild stimuli, such as cold air, or after animals are experimentally exposed to aerosols of histamine or methacholine. because of their extremely delicate structure, alveoli are quite vulnerable to injury once the local defense mechanisms have been overwhelmed. the alveolar wall is a thin membrane formed by a core of interstitium supporting an extensive network of alveolar capillaries. fibroblasts (septal cells), myofibroblasts, collagen, elastic fibers, and few interstitial macrophages and mast cells constitute the alveolar interstitium. the wall of the alveolar capillaries facing the airspace is remarkably thin and has three layers composed of vascular endothelium, basal lamina, and alveolar epithelium. these three layers of the alveolar capillaries constitute what is customarily referred to as the blood-air barrier (see fig. 9 -7). the epithelial side of the alveolus is primarily lined by rather thin type i proliferation of lymphocytes (balt hyperplasia) is also a common microscopic lesion seen in chronic bronchiolitis. airway hyperresponsiveness, or hyperreactive airway disease, is another sequela of bronchiolar injury arising from gene-environment interactions. it develops in human beings and animals (experimentally) after a transient and often innocuous viral infection of the lower respiratory tract or from exposure to certain allergens. experimental work has shown that airway hyperreactivity in club (clara) cell edema. alveolar repair is possible as long as the basement membrane remains intact and lesions are not complicated by further injury or infection. within 3 days, cuboidal type ii (granular) pneumonocytes, which are the precursor cells and more resistant to injury, undergo mitosis and provide a large pool of new undifferentiated cells . these new cells repave the denuded alveolar basement membrane and finally differentiate into type i pneumonocytes. when alveolar injury is diffuse, proliferation pneumonocytes, which are arranged as a very delicate continuous membrane extending along the alveolar surface (see fig. 9 -7). type i pneumonocytes are particularly susceptible to noxious agents that reach the alveolar region either aerogenously or hematogenously. injury to type i pneumonocytes rapidly causes swelling and vacuolation of these cells . when cellular damage has become irreversible, type i cells detach, resulting in denudation of the basement membrane, increased alveolar permeability, and alveolar figure 9-15 hyperplasia of type ii pneumonocytes. a, acute alveolar injury, crude oil aspiration, cow. note proliferation of cuboidal epithelial cells (type ii pneumonocytes) (arrows) along the luminal surface of the alveolar wall. during alveolar repair, type ii pneumonocytes are the precursor cell for necrotic and lost type i pneumonocytes. h&e stain. b, chronic alveolar injury, interstitial pneumonia, horse. note entire alveolar membrane lined with cuboidal type ii pneumonocytes (arrowheads). the alveolar interstitium is expanded with inflammatory cells, and the alveolar lumens contain cell debris mixed with leukocytes. h&e stain. ( (2). necrosis of these cells leads to transient alveolar edema (area that is pink) (3), which is followed by hyperplasia of type ii pneumonocytes (4), stem cells that differentiate (5) into type i pneumonocytes as part of alveolar repair and healing (6). (courtesy dr. a. lópez, atlantic veterinary college.) more information on postmortem examination of the lung can be found at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. microbes, toxins, and pneumotoxicants can gain access into the respiratory system by the following routes (table 9 -2; also see table 9 -1): aerogenous, hematogenous, direct extension, and by local production of free radicals and toxic metabolites. pathogens, such as bacteria, mycoplasmas, and viruses, along with toxic gases and foreign particles, including food, can gain access to the respiratory system via inspired air. this is the most common route in the transmission of most respiratory infections in domestic animals. some viruses, bacteria, parasites, and toxins can enter the respiratory system via the circulating blood. this portal of entry is commonly seen in septicemias, bacteremias, and with protozoa and viruses that target endothelial cells. also, circulating leukocytes may release infectious organisms such as retroviruses and listeria monocytogenes while traveling through the lungs. of type ii pneumonocytes becomes so spectacular that the microscopic appearance of the alveolus resembles that of a gland or fetal lung; this lesion has been termed epithelialization or fetalization. although it is part of the normal alveolar repair, hyperplasia of type ii pneumonocytes can interfere in gas exchange and cause hypoxemia. in uncomplicated cases, type ii pneumonocytes eventually differentiate into type i pneumonocytes, thus completing the last stage of alveolar repair (see fig. 9 -14). in some forms of chronic interstitial lung injury, the surface of the alveolar basement membrane could become populated with migrating bronchiolar cells, a process known as alveolar bronchiolization or lambertosis. in severe cases, lambertosis, a metaplastic change, can be mistaken microscopically with alveolar adenomas. type i pneumonocytes are one of the three structural components of the blood-air barrier, so when these epithelial cells are damaged, there is an increase in alveolar capillary permeability and transient leakage of plasma fluid, proteins, and fibrin into the alveolar lumen (see fig. 9-14) . under normal circumstances, these fluids are rapidly cleared from the alveolus by alveolar and lymphatic absorption, and necrotic pneumonocytes (type i) and fibrin strands are phagocytosed and removed by pulmonary alveolar macrophages. when there is persistent and severe injury, fibroblasts and myofibroblasts may proliferate in the alveolar walls (alveolar interstitium), causing alveolar septal fibrosis, whereas in other forms of severe injury, fibroblasts and myofibroblasts actively migrate from the interstitium into the alveolar spaces, causing intraalveolar fibrosis. these two types of alveolar fibrosis are most commonly seen in toxic and allergic pulmonary diseases and have a devastating effect on lung function. endothelial cells are also major players in the normal and abnormal physiology of the alveolus (see . these cells trap and share circulating antigens with intravascular and interstitial macrophages. the junction between alveolar endothelial cells is not as tight as that of the type i pneumonocytes, allowing some movement of fluid and small-size molecular weight proteins into the alveolar interstitium. endothelial cells maintain an intimate cell contact with erythrocytes and leukocytes passing through the lung, since the lumen of alveolar capillaries is slightly smaller (5.0 µm) than the diameter of red and white blood cells. erythrocytes are easily deformable, so their transit time through the alveolar capillaries is shorter than that of leukocytes, which are less deformable cells. this longer transit time of leukocytes and their close cellular contact with alveolar endothelial cells have major impacts in lung inflammation and acute respiratory distress syndrome (ards). on a minute-to-minute basis, the pulmonary defense mechanisms deal effectively with noxious stimuli and mild tissue injury without the need for an inflammatory response. however, if normal defense mechanisms are ineffective or insufficient (overwhelmed), the inflammatory process is rapidly turned on as a second line of defense. postmortem examination of the respiratory tract should always be conducted in a thorough and systematic manner and include the conducting system (trachea, bronchi, and bronchioles), the lungs, and the thoracic cavity and pleura. detailed record keeping and photographic documentation are essential elements of a thorough examination. normal lungs typically have a homogeneous pink color and are slightly deflated from loss of negative intrathoracic pressure. the e-sections that follow describe a systematic approach to this process. 480.e1 chapter 9 respiratory system, mediastinum, and pleurae the respiratory tract should always be examined in a systematic manner. to determine whether negative pressure is present in the thoracic cavity, the diaphragm is punctured through the abdominal cavity before the thoracic cavity has been opened. when the diaphragm is punctured in a fresh carcass, the loss of negative pressure in the thorax causes the diaphragmatic cupola to drop back caudally toward the abdominal cavity, and at the same time, there is an audible sound caused by the inrush of air into the thorax. lack of this movement may be an indication of advanced pneumothorax, pleural effusion, or the presence of uncollapsed lungs caused by pulmonary edema, pneumonia, fibrosis, or emphysema. in carcasses that have been dead for a long time, pulmonary air and gas produced by saprophytic bacteria leak into the pleural cavity, reducing the negative thoracic pressure and collapsing the lung. the rib cage must be removed by cutting along the costosternal joints and along the neck of the ribs (close to the costovertebral joints) in such a way that pleural adhesions and abnormal thoracic contents can be observed and grossly quantified (e.g., 200 ml of clear, yellow fluid). the tongue, pharynx, esophagus, larynx, trachea, and thoracic viscera (lungs, heart, and thymus) should be removed as a unit (often called the pluck) and placed on the necropsy table. the pharynx and esophagus are opened starting at the pharynx by a single cut with scissors along the dorsal midline and are inspected for ulcers, foreign bodies, and neoplasms. the larynx and trachea must be examined by opening both along the dorsal midline from cranial to caudal ends and then extending the incision into the large bronchi of the caudal lung lobes. normal tracheobronchial mucosa has a smooth and glistening pearl-colored surface with empty lumina in airways. the presence of foamy fluid in airways indicates pulmonary edema. feed particles may suggest aspiration; however, careful examination of the mucosa is required because aspiration of ingesta from stomach or rumen into the lungs commonly takes place agonally or can be displaced into these areas when the carcass is moved. the lungs should be examined before incision. normal lungs typically have a homogeneous pink color (see fig. 9 -16). external changes include the presence of rib imprints on the pleural surface when lungs fail to collapse. in addition, the lungs should be inspected for changes in color and texture and distribution of lesions. color changes can be various shades of red, indicating hypostatic congestion, hyperemia (acute pneumonia), and hemorrhage; dark blue collapsed lobules or areas are indicative of atelectasis; pale pink to white lungs indicate notable anemia, fibrosis, or emphysema; and uniformly or patchy yellow-brown lungs indicate chronic passive congestion and pulmonary fibrosis likely secondary to chronic heart failure. lungs from exsanguinated animals are generally paler than the normal pink color because of reduced blood in the pulmonary tissue. lungs with postmortem autolysis show green discoloration, a change that is also seen in other organs (efig. 9 -3). a covering of yellowish material on the pleural surface indicates accumulation of fibrin. because it is impossible to describe the texture of normal lungs, experience in palpation is required to appreciate the actual texture of a normal lung. texture is determined by gently palpating the surface and parenchyma of the lungs. normal texture can change to firm, hard, elastic (rubbery), or crepitus (with a crackling sound or feeling). for a detailed description of lung texture, see the section on classification of pneumonias in domestic animals. palpation of the lungs, which should be gentle, also permits detection of nonvisible nodules or abscesses in the parenchyma. knowing the distribution of a lesion in the lungs also facilitates diagnosis because particular etiologic agents cause lesions with specific distribution. distribution of lesions is generally described as focal, multifocal, locally extensive, or diffuse. according to their topography, pulmo-nary lesions can also be classified as cranioventral, dorsocaudal, and so on. necropsy reports must also contain an estimate of the extent of the pulmonary lesions, preferably expressed as a percentage of the volume of the lungs affected. for instance, a report may read "cranioventral consolidation involving 40% of the lungs." if the lungs have focal lesions, a rough estimate of the number should also be included in the report. for instance, "numerous (approximately 25), small (1 to 2 cm in diameter), hard nodules were randomly distributed in all lung lobes." two methods are used to examine the nasal structures. the first is making a midsagittal cut through the head and removing the nasal septum; the second is making several transverse sections of the nose at the level of the second premolar teeth. this latter method is preferred when examining pigs suspected of having atrophic rhinitis or animals suspected of having nasal neoplasms. microscopic examination of pulmonary tissue is routinely done in diagnostic laboratories. samples of normal and abnormal lungs, along with other appropriate tissue, should always be submitted in 10% buffered-neutral formalin for histopathologic evaluation. a minimum of four lung samples (left cranial, left caudal, right cranial, and right caudal) should be taken for histopathologic examination in animals with a history of respiratory signs. to improve fixation, a paper towel can be placed over the samples of lung floating in fixative. when detailed evaluation of the alveolar walls is required, lungs can be fixed by a gentle intratracheal injection of fixative; however, this technique displaces transudates and exudates and can artificially cause distention of the perivascular and peribronchial spaces. lung biopsy specimens are taken only sporadically because complications often outweigh the diagnostic value. however, the use of new techniques, such as endoscopic-directed biopsies, has notably reduced some of these complications. biopsies of the lungs are recommended in cases of chronic persistent pulmonary disease unresponsive to treatment or intrathoracic masses of undetermined origin. endoscopic-directed biopsies of the nasal and bronchial mucosa are routinely used in clinical practice and generally have a much better diagnostic value. two valuable diagnostic tools in human medicine, bronchoalveolar lavage (bal) and transtracheal wash (ttw), have in recent years become more widely used in veterinary clinical diagnosis of respiratory ailments, particularly in horses, dogs, and cats. the basis of bal and ttw is sampling the lung or trachea of a living animal by infusing sterile fluid into the trachea or deep lung (respectively) and retrieving it to determine the cellular and biochemical composition of this fluid. in other words, the composition of the fluid reflects what is present in the bronchioloalveolar spaces and trachea. these procedures are performed by inserting a tube directly through the larynx into the trachea or bronchus, or transtracheally by inserting a tube through a needle percutaneously into the cervical trachea. microscopic examination of properly collected, stored, and processed samples may reveal many erythrocytes and siderophages in pulmonary hemorrhage or left-sided heart failure; inclusion bodies or syncytial cells in viral pneumonias; increased number of leukocytes in pulmonary inflammation; abundant mucus in asthma or equine recurrent airway obstruction (rao); the presence of pulmonary pathogens, such as parasites, fungi, and bacteria; or tumor cells in cases of pulmonary neoplasia. in the healthy animal, 80% to 95% of the bal cells are pulmonary alveolar macrophages (see fig. 9-19, a) . clearance, or a combination of both is the underlying pathogenetic mechanism in many pulmonary diseases ( fig. 9-17) . the anatomic configuration of the nasal cavity and bronchi plays a unique role in preventing or reducing the penetration of noxious material into the lungs, especially into the alveoli, which is the most vulnerable portion of the respiratory system. the narrow nasal meatuses and the coiled arrangement of the nasal conchae generate enormous turbulence of airflow, and as a result, physical forces are created that forcefully impact particles larger than 10 µm onto the surface of the nasal mucosa ( fig. 9-18 ). although particles smaller than 10 µm could escape trapping in the nasal cavity, these mediumsized particles meet a second barrier at the tracheal and bronchial in some instances, pathogenic organisms can also reach the pleura and lungs through penetrating injuries, such as gunshot wounds, migrating awns, or bites, or by direct extension from a ruptured esophagus or perforated diaphragm. the lungs, particularly the bronchioles and alveoli, are vulnerable to endogenous injury caused by the local generation of free radicals during inflammation or by toxic metabolites generated by club (clara) cells (see fig. 9 -4, b). inflammatory processes in the respiratory system, particularly those caused by infectious organisms, can spread to contiguous or distant tissues. for instance, rhinitis may spread into the sinuses causing rhinosinusitis. similarly, laryngeal inflammation may spread into the lungs when exudate in the larynx is aspirated. lung disease can have profound systemic effects when cytokines, produced locally during necrosis or inflammation, are released into circulation. as a result of the enormous vascular bed present in the lung, sepsis and septic shock often develop when proinflammatory molecules overwhelm the antiinflammatory response during the so-called "cytokine storm." it is axiomatic that a particle, microbe, or toxic gas must first gain entry to a vulnerable region of the respiratory system before it can induce an adaptive immune response or have a pathologic effect. the characteristics of size, shape, dispersal, and deposition of particles present in inspired air are studied in aerobiology. it is important to recognize the difference between deposition, clearance, and retention of inhaled particles. deposition is the process by which particles of various sizes and shapes are trapped within specific regions of the respiratory tract. clearance is the process by which deposited particles are destroyed, neutralized, or removed from the mucosal surfaces. the difference between what is deposited and what is cleared from the respiratory tract is referred to as retention. the main mechanisms involved in clearance are sneezing, coughing, mucociliary transport, and phagocytosis (table 9 -3). abnormal retention of particles resulting from increased deposition, decreased same rate in all levels of a conducting system, a "bottleneck" effect would be created in major airways as the minor but more numerous airways enter the bronchi. for this reason, the mucociliary transport in proximal (rostral) airways is physiologically faster than that of the distal (caudal) ones. ciliary activity and mucus transport increase notably in response to stimuli such as in respiratory infections. the mucociliary blanket of the nasal cavity, trachea, and bronchi also plays an important role in preventing injury from toxic gases. if a soluble gas contacts the mucociliary blanket, it mixes with the mucus, thus reducing the concentration of gas reaching deep into the alveoli. in other words, mucus acts as a "scavenger system," whereby gases are solubilized and subsequently cleared from the respiratory tract via mucociliary transport. if ciliary transport is reduced (loss of cilia) or mucus production is excessive, coughing becomes an important mechanism for clearing the airways. in addition to the mechanical barrier and physical transport provided by the mucociliary escalator, other cells closely associated with ciliated epithelium contribute to the defense mechanism of the conducting and transitional systems. among the most notable are the microfold (m) cells, which are modified epithelial cells covering the bronchial-associated lymphoid tissue (balt), both of which are strategically situated at the corner of the bifurcation of bronchi and bronchioles, where inhaled particles often collide with the mucosa because of inertial forces. from here, inhaled particles and soluble antigens are phagocytosed and transported by macrophages, dendritic cells, and other professional antigen-presenting cells (apcs) into the balt, thus providing a unique opportunity for b and t lymphocytes to enter into close contact with inhaled pathogenic substances. pulmonary lymphocytes are not quiescent in the balt but are in continual traffic to other organs and contribute to both cellular (cytotoxic, helper, and suppressor t lymphocytes) and humoral immune responses. immunoglobulin a (iga), produced by mucosal plasma cells, and, to a lesser extent, immunoglobulin g (igg) and m (igm) play important roles in the local immunity of the conducting and transitional systems, especially with regard to preventing attachment of pathogens to the cilia. chronic airway diseases, especially those caused by infectious agents such as mycoplasmas or retroviruses, are often accompanied by severe hyperplasia of the balt. the mucociliary clearance terminates at the pharynx, where mucus, propelled caudally from the nasal cavity and cranially from the tracheobronchial tree, is eventually swallowed and thus eliminated from the conducting system of the respiratory tract. some respiratory pathogens, such as rhodococcus equi, can infect the intestines after having been removed and swallowed from the respiratory tract into the alimentary system. alveoli lack ciliated and mucus-producing cells; thus the defense mechanism against inhaled particles in the alveolar region cannot be provided by mucociliary clearance. instead, the main defense mechanisms of alveoli (exchange system) are phagocytosis provided by the pulmonary alveolar macrophages and antimicrobial molecules of the alveolar lining fluid ( fig. 9-19 ). pulmonary alveolar macrophages are highly phagocytic cells, which are not to be confused with pulmonary intravascular macrophages, and are derived largely from blood monocytes and, to a much lesser extent, from a slowly dividing population of interstitial macrophages. after a temporary adaptive stage within alveolar interstitium, blood monocytes reduce their glycolytic metabolism and increase their oxidative metabolism to function in an aerobic rather than an anaerobic environment. pulmonary alveolar macrophages contribute to the bifurcations. abrupt changes in the direction of air (inertia), which occurs at the branching of major airways, cause particles in the 2-to 10-µm size range to collide with the surface of bronchial mucosa (see fig. 9 -1). because the velocity of inspired air at the level of the small bronchi and bronchioles has become rather slow, inertial and centrifugal forces no longer play a significant role in the trapping of inhaled particles. here, in the transitional (bronchiolar) and exchange (alveolar) regions, particles 2 µm or smaller may come into contact with the mucosa by means of sedimentation because of gravitation or by diffusion as a result of brownian movement. infective aerosols containing bacteria and viruses are within the size range (0.01 to 2 µm) that can gain access to the bronchiolar and alveolar regions. in addition to size, other factors, such as shape, length, electrical charge, and humidity, play an important role in mucosal deposition, retention, and pathogenicity of inhaled particles. for example, particles longer than 200 µm may also reach the lower respiratory tract provided their mean aerodynamic diameter is less than 1 µm. asbestos is a good example of a large but slender fiber that can bypass the filtrating mechanisms by traveling parallel to the airstream. once in the terminal bronchioles and alveoli, asbestos fibers cause asbestosis, a serious pulmonary disease in human beings. in summary, the anatomic features of the nasal cavity and airways provide an effective barrier, preventing the penetration of most large particles into the lungs. once larger particles are trapped in the mucosa of conducting airways and small particles are deposited on the surface of the nasal, tracheal, or bronchoalveolar mucosa, it is crucial that these exogenous materials be promptly removed to prevent or minimize injury to the respiratory system. for these purposes, the respiratory system is equipped with several defense mechanisms, all of which are provided by specialized cells operating in a remarkably well-coordinated manner. conducting system (nose, trachea, and bronchi) mucociliary clearance is the physical unidirectional movement and removal of deposited particles and gases dissolved in the mucus from the respiratory tract. mucociliary clearance, also referred to as the waste disposal system, is provided by the mucociliary blanket (mucociliary escalator) and is the main defense mechanism of the conducting system (nasal cavity, trachea, and bronchi) (see figs. 9-2 and 9-3). mucus acts primarily as a barrier and a vehicle, and it is a complex mixture of water, glycoproteins, immunoglobulins, lipids, and electrolytes. these substances are produced by goblet (mucous) cells, serous cells, submucosal glands, and fluid from transepithelial ion and water transport. once serous fluid and mucus are secreted onto the surface of the respiratory mucosa, a thin, double-layer film of mucus is formed on top of the cells. the outer layer of this film is in a viscous gel phase, whereas the inner layer, which is in a fluid or sol phase, is directly in contact with cilia (see fig. 9 -3 and see efig. 9 -1). the respiratory system of a healthy human produces approximately 100 ml of mucus per day. each ciliated cell in the conducting system has approximately 100 to 200 motile and chemosensory cilia (6 µm long), beating metachronously (forming a wave) at a ciliary beat frequency of approximately 1000 strokes per minute, and in a horse, for example, mucus moves longitudinally at a rate of up to 20 mm per minute. rapid and powerful movement of cilia creates a series of waves that, in a continuous and synchronized manner, propel the mucus, exfoliated cells, and entrapped particles out of the respiratory tract to the pharynx. the mucus is finally swallowed or, when present in large amounts, is coughed up out of the conducting system. if mucus flow were to move at the activated alveolar macrophages. similarly, inhaled particles, such as dust, pollen, spores, carbon, or erythrocytes from intraalveolar hemorrhage, are all phagocytosed and eventually removed from alveoli by pulmonary alveolar macrophages. most alveolar macrophages leave the alveoli by migrating toward the bronchiolar (transitional) region until the mucociliary blanket is reached. once there, pulmonary macrophages are removed in the same way as any other particle: along the mucociliary flow to the pharynx and swallowed. in the cat, as many as 1 million macrophages per hour move out from the alveoli into the conducting system and pharynx. destruction and removal of inhaled microbes and particles by alveolar macrophages is a well-orchestrated mechanism that engages many cells, receptors (i.e., toll-like receptors [tlrs]), and pulmonary secretions in the lung. the cell-to-cell interactions are complex and involve pulmonary alveolar macrophages, pneumonocytes, endothelial cells, lymphocytes, plasma cells, natural killer (nk) cells, and dendritic cells. antibodies are also important in the protection (acquired immune response) of the respiratory tract against inhaled pathogens. iga is the most abundant antibody in the nasal and tracheal secretions and prevents the attachment and absorption of antigens (immune exclusion). igg and, to a lesser extent, ige and igm promote the uptake and destruction of inhaled pathogens by phagocytic cells (immune elimination). igg is the most abundant antibody in the alveolar surface and acts primarily as an opsonizing antibody for alveolar macrophages and neutrophils. in addition to antibodies, there are several secretory molecules locally released into the alveoli that constitute the alveolar lining material and contribute to the pulmonary defense mechanisms. the most important of these antimicrobial products are transferrin, anionic peptides, and pulmonary surfactant (table 9 -4). to facilitate phagocytosis and discriminate between "self" and "foreign" antigens, pulmonary alveolar macrophages are furnished with a wide variety of specific receptors on their cell surfaces. among the most important ones are fc receptors for antibodies; complement receptors (for c3b, c3a, and c5a); tumor necrosis factor (tnf) receptor; and cd40 receptors, which facilitate phagocytosis and destruction of opsonized particles. toll-like receptors (tlrs) recognize microbial components, and apoptosis stimulating fragment (fas) receptors are involved in apoptosis and in the phagocytosis of apoptotic cells in the lung. "scavenger receptors," which are responsible for the recognition and uptake of foreign particulates, such as dust and fibers, are also present on pulmonary alveolar macrophages. lungs are also susceptible to hematogenously borne microbes, toxins, or emboli. the hepatic (kupffer cells) and splenic macrophages are the primary phagocytic cells responsible for removing circulating bacteria and other particles from the blood of dogs, some rodents, and human beings. in contrast, the cell responsible for the removal of circulating particles, bacteria, and endotoxin from the blood of ruminants, cats, pigs, and horses is mainly the pulmonary intravascular macrophage, a distinct population of phagocytes normally residing within the pulmonary capillaries (see fig. 9 -7). in pigs, 16% of the pulmonary capillary surface is lined by pulmonary intravascular macrophages. in ruminants, 95% of intravenously injected tracer particles or bacteria are rapidly phagocytosed by these intravascular macrophages. studies have shown that an abnormally reduced number of kupffer cells in diseased liver results in a compensatory increase in pulmonary intravascular macrophages, even in animal species in which these phagocytic cells are normally absent from the lung. in some abnormal conditions, such as sepsis, pulmonary innate and adaptive immune response by rapidly attaching and phagocytosing bacteria and any other particles reaching the alveolar lumens. the number of free macrophages in the alveolar space is closely related to the number of inhaled particles reaching the lungs. this ability to increase, within hours, the number of available phagocytic cells is vital in protecting the distal lungs against foreign material, particularly when the inhaled particle load is high. unlike that of tissue macrophages, the life span of alveolar macrophages in the alveoli is notably short, only a few days, and thus they are continuously being replaced by newly migrated blood monocytes. alveolar phagocytosis plays a prominent role in the innate defense mechanism against inhaled bacteria without the need of an inflammatory reaction. bacteria reaching the alveoli are rapidly phagocytosed, and bactericidal enzymes present in lysosomes are discharged into the phagosome containing the bacteria (see b) . except for some facultative pathogens that are resistant to intracellular killing (e.g., mycobacterium tuberculosis, listeria monocytogenes, brucella abortus, rhodococcus equi, and some salmonella spp.), most bacteria reaching the lungs are rapidly destroyed by (ros) not only induce extensive pulmonary injury but also impair the defense and repair mechanisms in the lung. oxygen and free radical scavengers, such as catalase, superoxide dismutase, ubiquinone, and vitamins e and c, are largely responsible for protecting pulmonary cells against peroxidation. these scavengers are present in alveolar and bronchiolar epithelial cells and in the extracellular spaces of the pulmonary interstitium. in summary, the defense mechanisms are so effective in trapping, destroying, and removing bacteria that, under normal conditions, animals can be exposed to aerosols containing massive numbers of bacteria without any ill effects. if defense mechanisms are impaired, inhaled bacteria colonize and multiply in bronchi, bronchioles, and alveoli, and they produce infection, which can result in fatal pneumonia. similarly, when blood-borne pathogens, inhaled toxicants, or free radicals overwhelm the protective defense mechanisms, cells of the respiratory system are likely to be injured, often causing serious respiratory diseases. for many years, factors such as viral infections, toxic gases, stress, and pulmonary edema have been implicated in predisposing human beings and animals to secondary bacterial pneumonia. there are many pathways by which the defense mechanisms can be impaired; only those relevant to veterinary species are discussed. viral agents are notorious in predisposing human beings and animals to secondary bacterial pneumonias by what is known as viral-bacterial synergism. a good example of the synergistic effect of combined virus-bacterial infections is documented from epidemics of human beings with influenza virus in which the mortality rate has been significantly increased from secondary bacterial pneumonia. the most common viruses incriminated in predisposing animals to secondary bacterial pneumonia include influenza virus in pigs and horses; bovine herpesvirus 1 (bohv-1), bovine parainfluenza virus 3 (bpiv-3), and bovine respiratory syncytial virus (brsv) in cattle; canine distemper virus (cdv) in dogs; and felid herpesvirus 1 (fehv-1) and feline calicivirus (fcv) in cats. the mechanism of the synergistic effect of viral-bacterial infections was previously believed to be the destruction of the mucociliary blanket and a concurrent reduction of mucociliary clearance, but in experimental studies, viral infections did not significantly reduce the physical removal of particles or bacteria out of the lungs. now, it is known that 5 to 7 days after a viral infection, the phagocytic function of pulmonary alveolar macrophages and, to a lesser extent, the mucociliary clearance are notably impaired (see fig. 9 -8). other mechanisms by which viruses impair defense mechanisms are multiple and remain poorly understood (box 9-1). immunization against viral infections in many cases prevents or reduces the synergistic effect of viruses and thus the incidence of secondary bacterial pneumonia. certain gases also impair respiratory defense mechanisms, rendering animals more susceptible to secondary bacterial infections. for instance, hydrogen sulfide and ammonia, frequently encountered on farms, especially in buildings with poor ventilation, can impair pulmonary defense mechanisms and increase susceptibility to bacterial pneumonia. the effects of environmental pollutants on the defense mechanisms of human beings and animals living in crowded and polluted cities remain to be determined. excessive release of cytokines by pulmonary intravascular macrophages may result in acute lung injury. existing in an oxygen-rich environment and being the site of numerous metabolic reactions, the lungs also require an efficient defense mechanism against oxidant-induced cellular damage (oxidative stress). this form of damage is caused by inhaled oxidant gases (e.g., nitrogen dioxide, ozone, sulfur dioxide, or tobacco smoke), by xenobiotic toxic metabolites produced locally, by toxins reaching the lungs via the bloodstream (e.g., 3-methylindole and paraquat), or by free radicals (reactive oxygen species) released by phagocytic cells during inflammation. free radicals and reactive oxygen species anomalies 2 localized congenital anomalies of the nasal cavity are rare in domestic animals and are often merely part of a more extensive craniofacial deformity (e.g., cyclops) or a component of generalized malformation (e.g., chondrodysplasia). congenital anomalies involving the nasal cavity and sinuses, such as choanal atresia (lack of communication between the nasal cavity and pharynx), some types of chondrodysplasia, and osteopetrosis, are incompatible with life. examples of nonfatal congenital anomalies include cystic nasal conchae, deviation of the nasal septum, cleft upper lip (harelip and cheiloschisis), hypoplastic turbinates, and cleft palate (palatoschisis) (see fig. 7 -32). bronchoaspiration and aspiration pneumonia are common sequelae to cleft palate. nasal and paranasal sinus cysts are slowly growing and expansive lesions that mimic neoplasia and cause severe cranial deformation in horses. as in other organs or systems, it is extremely difficult to determine the actual cause (genetic vs. congenital) of anomalies based on pathologic evaluation. metabolic disturbances affecting the nasal cavity and sinuses are rare in domestic animals. immunodeficiency disorders, whether acquired or congenital, are often associated with increased susceptibility to viral, bacterial, and protozoal pneumonias. for example, human beings with acquired immunodeficiency syndrome (aids) are notably susceptible to pneumonia caused by proliferation of pneumocystis (carinii) jirovecii. a similar ubiquitous organism, which under normal circumstances is not pathogenic, is also found in the pneumonic lungs of immunosuppressed pigs, foals, dogs, and rodents. pigs infected with the porcine reproductive and respiratory syndrome (prrs) virus frequently develop pneumocystis carinii infection ( fig. 9-20) . arabian foals born with combined immunodeficiency disease easily succumb to infectious diseases, particularly adenoviral pneumonia. combined infections with two respiratory viruses, such as canine distemper virus (cdv) and canine adenovirus 2 (cav-2), are sporadically reported in immunosuppressed puppies. also, large doses of chemotherapeutic agents, such as steroids and alkylating agents, cause immunosuppression in dogs, cats, and other animals, increasing susceptibility to secondary viral and bacterial infections. stress, uremia, endotoxemia, dehydration, starvation, hypoxia, acidosis, pulmonary edema, anesthesia, and ciliary dyskinesia are only some of the many conditions that have been implicated in impairing respiratory defense mechanisms and consequently predisposing animals to develop secondary bacterial pneumonia. the mechanisms by which each of these factors suppresses pulmonary defenses are diverse and sometimes not well understood. for example, hypoxia and pulmonary edema decrease phagocytic function of pulmonary alveolar macrophages and alter the production of surfactant (abnormal head tilt and abnormal gait), which in severe cases may lead to emaciation. based on the nature of exudate, rhinitis can be classified as serous, fibrinous, catarrhal, purulent, or granulomatous. these types of inflammatory reactions can progress from one to another in the course of the disease (i.e., serous to catarrhal to purulent), or in some instances exudates can be mixed, such as those seen in mucopurulent, fibrinohemorrhagic, or pyogranulomatous rhinitis. microscopic examination of impression smears or nasal biopsy, and bacterial or fungal cultures are generally required in establishing the cause of inflammation. common sequelae of rhinitis are hemorrhage, ulcers, and, in some cases, nasopharyngeal polyps (hyperplasia) arising from inflamed mucosa. rhinitis also can be classified according to the age of the lesions as acute, subacute, or chronic; to the severity of the insult as mild, moderate, or severe; and to the etiologic agent as viral, allergic, bacterial, mycotic, parasitic, traumatic, or toxic. serous rhinitis. serous rhinitis is the mildest form of inflammation and is characterized by hyperemia and increased production of a clear fluid locally manufactured by serous glands present in the nasal submucosa. serous rhinitis is of clinical interest only. it is caused by mild irritants or cold air, and it occurs during the early stages of viral infections, such as the common cold in human beings, upper respiratory tract infections in animals, or in mild allergic reactions. catarrhal rhinitis. catarrhal rhinitis is a slightly more severe process and has, in addition to serous secretions, a substantial increase in mucus production by hypersecretion of goblet cells and mucous glands. a mucous exudate is a thick, translucent, or slightly turbid viscous fluid, sometimes containing a few exfoliated cells, leukocytes, and cellular debris. in chronic cases, catarrhal rhinitis is characterized microscopically by notable hyperplasia of goblet cells. as the inflammation becomes more severe, the mucus is infiltrated with neutrophils, giving the exudate a cloudy appearance. this exudate is referred to as mucopurulent. purulent (suppurative) rhinitis. purulent (suppurative) rhinitis is characterized by a neutrophilic exudate, which occurs when the nasal mucosa suffers a more severe injury that generally is accompanied by mucosal necrosis and secondary bacterial infection. cytokines, leukotrienes, complement activation, and bacterial products cause exudation of leukocytes, especially neutrophils, which mix with nasal secretions, including mucus. grossly, the exudate in suppurative rhinitis is thick and opaque, but it can vary from white to green to brown, depending on the types of bacteria and type of leukocytes (neutrophils or eosinophils) present in the exudate . in severe cases, the nasal passages are completely blocked by the exudate. microscopically, neutrophils can be seen in the submucosa and mucosa and form plaques of exudate on the mucosal surface. neutrophils are commonly found marginated in vessels, in the lamina propria, and in between epithelial cells in their migration to the surface of the mucosa. fibrinous rhinitis. fibrinous rhinitis is a reaction that occurs when nasal injury causes a severe increase in vascular permeability, resulting in abundant exudation of plasma fibrinogen, which coagulates into fibrin. grossly, fibrin appears as a yellow, tan, or gray rubbery mat on nasal mucosa. fibrin accumulates on the surface and forms a distinct film of exudate sometimes referred to as pseudomembrane ( fig. 9-22 ). if this fibrinous exudate can be removed, leaving an intact underlying mucosa, it is termed a croupous or pseudodiphtheritic rhinitis. conversely, if the pseudomembrane is difficult to remove and leaves an ulcerated mucosa, it is referred to as diphtheritic or fibrinonecrotic rhinitis. the term diphtheritic was derived from human diphtheria, which causes a severe and destructive inflammatory process of the nasal, tonsillar, pharyngeal, and laryngeal mucosa. nasal amyloidosis. amyloidosis, the deposition of amyloid protein (fibrils with a β-pleated configuration) in various tissues, has been sporadically reported as a localized lesion in the nasal cavity of horses and human beings (see nasal amyloidosis, in disorders of horses). congestion and hyperemia. the nasal mucosa is well vascularized and is capable of rather dramatic variation in blood flow, whether passively as a result of interference with venous return (congestion) or actively because of vasodilation (hyperemia). congestion of the mucosal vessels is a nonspecific lesion commonly found at necropsy and presumably associated with the circulatory failure preceding death (e.g., heart failure, bloat in ruminants in which the increased intraabdominal pressure causes increased intrathoracic pressure impeding the venous return from the head and neck). hyperemia of the nasal mucosa is seen in early stages of inflammation, whether caused by irritation (e.g., ammonia and regurgitated feed), viral infections, secondary bacterial infections, toxemia, allergy, or trauma. hemorrhage. epistaxis is the clinical term used to denote blood flow from the nose (nosebleed) regardless of whether the blood originates from the nasal mucosa or from deep in the lungs, such as in horses with "exercise-induced pulmonary hemorrhage." unlike blood in the digestive tract, where the approximate anatomic location of the bleeding can be estimated by the color the blood imparts to fecal material, blood in the respiratory tract is always red. this fact is due to the rapid transport of blood out of the respiratory tract by the mucociliary blanket and during breathing. hemorrhages into the nasal cavity can be the result of local trauma, can originate from erosions of submucosal vessels by inflammation (e.g., guttural pouch mycosis), or can be caused by neoplasms. hemoptysis refers to the presence of blood in sputum or saliva (coughing or spitting blood) and is most commonly the result of pneumonia, lung abscesses, ulcerative bronchitis, pulmonary thromboembolisms or hemorrhage, and pulmonary neoplasia. inflammation of the nasal mucosa is called rhinitis, and inflammation of the sinuses is called sinusitis. these conditions usually occur together, although mild sinusitis can be undetected. clinically, rhinosinusitis is characterized by nasal discharge. rhinitis. the occurrence of infectious rhinitis presupposes an upset in the balance of the normal microbial flora of the nasal cavity. innocuous bacteria present normally protect the host through a process called competitive exclusion, whereby potential pathogens are kept at a harmless level. disruption of this protective mechanism can be caused by respiratory viruses, pathogenic bacteria, fungi, irritant gases, environmental changes, immunosuppression, local trauma, stress, or prolonged antibacterial therapy. inflammatory processes in the nasal cavity are not life-threatening and usually resolve completely. however, some adverse sequelae in cases of infectious rhinitis include bronchoaspiration of exudate leading to bronchopneumonia. chronic rhinitis often leads to destruction of the nasal conchae (turbinates), deviation of the septum, and, eventually, craniofacial deformation. also, nasal inflammation may extend into the sinuses causing sinusitis; into facial bones causing osteomyelitis; through the cribriform plate causing meningitis; into the eustachian tubes causing otitis media or guttural pouch empyema (eustachitis) in horses; and even into the inner ear causing otitis interna and vestibular syndrome the nasal septum has been removed to expose nasal conchae. the nasal mucosa is hyperemic and covered by yellow-white purulent exudate (arrows). inset, histological section showing submucosal congestion and edema and also large aggregates of neutrophils on the superficial mucosa (asterisk). h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) microscopically, the lesions include a perivascular edema with fibrin, a few neutrophils infiltrating the mucosa, and superficial plaques of exudate consisting of fibrin strands mixed with leukocytes and cellular debris covering a necrotic and ulcerated epithelium. fungal infections, such as aspergillosis, can cause a severe fibrinonecrotizing rhinitis. granulomatous rhinitis. granulomatous rhinitis is a reaction in the nasal mucosa and submucosa that is characterized by infiltration of numerous activated macrophages mixed with a few lymphocytes and plasma cells (figs. 9-23 and 9-24). in some cases, chronic inflammation leads to the formation of polypoid nodules that in severe cases are large enough to cause obstruction of the nasal passages ( fig. 9-25 ). granulomatous rhinitis is generally associated with chronic allergic inflammation or infection with specific organisms, such as fungi (see fig. 9 -24), tuberculosis, systemic mycosis (see section on granulomatous pneumonia), and rhinosporidiosis ; also see fig. 9 -25). in some cases, the cause of granulomatous rhinitis cannot be determined. sinusitis. sinusitis occurs sporadically in domestic animals and is frequently combined with rhinitis (rhinosinusitis), or it occurs as extend into the adjacent bone (osteomyelitis) or through the ethmoidal conchae into the meninges and brain (meningitis and encephalitis). nasal amyloidosis. amyloidosis, the deposition of amyloid protein (fibrils with a β-pleated configuration) in various tissues, has been sporadically reported as a localized lesion in the nasal cavity of horses. unlike amyloidoses in other organs of domestic animals where amyloid is generally of the reactive type (amyloid aa), equine nasal amyloidosis appears to be of the immunocytic type (amyloid al). affected horses with large amyloid masses have difficulty breathing because of nasal obstruction and may exhibit epistaxis and reduced athletic performance; on clinical examination, large, firm nodules resembling neoplasms (amyloidoma) can be observed in the alar folds, rostral nasal septum, and floor of nasal cavity. microscopic lesions are similar to those seen in other organs and consist of a deposition of hyaline amyloid material in nasal mucosa that is confirmed by a histochemical stain, such as congo red. progressive ethmoidal hematoma. progressive ethmoidal hematoma (peh) is important in older horses and is characterized clinically by chronic, progressive, often unilateral nasal bleeding. grossly or endoscopically, an ethmoidal hematoma appears as a single, soft, tumor-like, pedunculated, expansive, dark red mass arising from the mucosa of the ethmoidal conchae ( fig. 9-28) . microscopic examination reveals a capsule lined by epithelium and hemorrhagic stromal tissue infiltrated with abundant macrophages, most of which are siderophages. viral infections. viruses, such as equine viral rhinopneumonitis virus, influenza virus, adenovirus, and equine picornavirus, cause mild and generally transient respiratory infections in horses. the route of infection for these respiratory viruses is typically aerogenous. all of these infections are indistinguishable clinically; signs consist mainly of malaise, fever, coughing, conjunctivitis, and nasal a sequela to penetrating or septic wounds of the nasal, frontal, maxillary, or palatine bones; improper dehorning in young cattle, which exposes the frontal sinus; or maxillary tooth infection in horses and dogs (maxillary sinus). based on the type of exudate, sinusitis is classified as serous, catarrhal, fibrinous (rare), purulent, or granulomatous. paranasal sinuses have poor drainage; therefore exudate tends to accumulate, causing mucocele (accumulation of mucus) or empyema (accumulation of pus) ( fig. 9-27 ). chronic sinusitis may promised horses, particularly in arabian foals with inherited combined immunodeficiency disease. bacterial infections. strangles, glanders, and melioidosis of horses are all systemic bacterial diseases that cause purulent rhinitis and suppuration in various organs. these diseases are grouped as upper respiratory diseases because nasal discharge is often the most notable clinical sign. strangles. strangles is an infectious and highly contagious disease of equidae that is caused by streptococcus equi ssp. equi (streptococcus equi) . it is characterized by suppurative rhinitis and lymphadenitis (mandibular and retropharyngeal lymph nodes) with occasional hematogenous dissemination to internal organs. unlike streptococcus equi ssp. zooepidemicus (streptococcus zooepidemicus) and streptococcus dysgalactiae ssp. equisimilis (streptococcus equisimilis), streptococcus equi is not part of the normal nasal flora. infection occurs when susceptible horses come into contact with feed, exudate, or air droplets containing the bacterium. after penetrating through the nasopharyngeal mucosa, streptococcus equi drains to the regional lymph nodes-mandibular and retropharyngeal lymph nodes-via lymphatic vessels. the gross lesions in horses with strangles (mucopurulent rhinitis) correlate with clinical findings and consist of copious amounts of mucopurulent exudate in the nasal passages with notable hyperemia of the nasal mucosa. affected lymph nodes are enlarged and may contain abscesses filled with thick purulent exudate (purulent lymphadenitis). the term bastard strangles is used in cases in which hematogenous dissemination of streptococcus equi results in metastatic abscesses in such organs as the lungs, liver, spleen, kidneys, or brain or in the joints. this form of strangles is often fatal. common sequelae to strangles include bronchopneumonia caused by aspiration of nasopharyngeal exudate; laryngeal hemiplegia ("roaring"), resulting from compression of the recurrent laryngeal nerves by enlarged retropharyngeal lymph nodes; facial paralysis and horner syndrome caused by compression of sympathetic nerves that run dorsal to the medial retropharyngeal lymph node; and purpura hemorrhagica as a result of vasculitis caused by deposition of streptococcus equi antigen-antibody complexes in arterioles, venules, and capillaries of the skin and mucosal membranes. in severe cases, nasal infection extends directly into the paranasal sinuses or to the guttural pouches via the eustachian tubes, causing inflammation and accumulation of pus (guttural pouch empyema). rupture of abscesses in the mandibular and retropharyngeal lymph nodes leads to suppurative inflammation of adjacent subcutaneous tissue (cellulitis), and in severe cases the exudate escapes through cutaneous fistulas. strangles can affect horses of all ages, but it is most commonly seen in foals and young horses. it is clinically characterized by cough, nasal discharge, conjunctivitis, and painful swelling of regional lymph nodes. some horses become carriers and a source of infection to other horses. glanders. glanders is an infectious world organization for animal health (oie)-notifiable disease of equidae caused by burkholderia mallei (pseudomonas mallei) that can be transmitted to carnivores by consumption of infected horsemeat. human beings are also susceptible, and untreated infection is often fatal. this gramnegative bacterium has been listed as a potential agent for biologic warfare and bioterrorism. in the past, burkholderia mallei was found throughout the world, but today, glanders has been eradicated from most countries, except for some areas in north africa, asia, and eastern europe. there also have been sporadic outbreaks reported in brazil. the pathogenesis of glanders is not fully understood. results from experimental infections suggest that infection occurs discharge varying from serous to purulent. viral respiratory infections are common medical problems in adult horses. equine viral rhinopneumonitis. equine viral rhinopneumonitis (evr) is caused by two ubiquitous equine herpesviruses (ehv-1 and ehv-4) and may be manifested as a mild respiratory disease in weanling foals and young racehorses, as a neurologic disease (myeloencephalopathy), or as abortion in mares. the portal of entry for the respiratory form is typically aerogenous, and the disease is generally transient; thus the primary viral-induced lesions in the nasal mucosa and lungs are rarely seen at necropsy unless complicated by secondary bacterial rhinitis, pharyngitis, or bronchopneumonia. studies with polymerase chain reaction (pcr) techniques have demonstrated that, like other herpesviruses, ehv-1 and ehv-4 persist in the trigeminal ganglia for long periods of time (latency). reactivation because of stress or immunosuppression and subsequent shedding of the virus are the typical source of infection for susceptible animals on the farm. equine influenza. equine influenza is a common, highly contagious, and self-limiting upper respiratory infection of horses caused by aerogenous exposure to type a strains of influenza virus (h7n7 [a/equi-1] and h3n8 [a/equi-2]). equine influenza has high morbidity (outbreaks) but low mortality, and it is clinically characterized by fever, conjunctivitis, and serous nasal discharge. it occurs mainly in 2-to 3-year-old horses at the racetrack. as with human influenza, equine influenza is usually a mild disease, but occasionally it can cause severe bronchointerstitial pneumonia with pulmonary edema. in some horses, impaired defense mechanisms caused by the viral infection are complicated by a secondary bacterial bronchopneumonia caused by opportunistic organisms (streptococcus zooepidemicus, staphylococcus aureus, or bacteroides sp.) found in the normal flora of the upper respiratory tract. uncomplicated cases of equine influenza are rarely seen in the postmortem room. equine influenza virus (h3n8) recently did an equine to canine "host-jump" causing extensive outbreaks of respiratory disease in dogs (see pneumonias of dogs). other equine respiratory viruses. equine picornavirus, adenovirus, and parainfluenza virus produce mild and transient upper respiratory infections (nasopharynx and trachea) in horses, unless complicated by secondary pathogens. in addition to reduced athletic performance, infected horses may have a temporary suppression of cell-mediated immunity leading to opportunistic infections such as pneumocystis carinii pneumonia. fatal adenoviral infections with severe pneumonia or enteritis occur commonly in immunocomdisorders of ruminants (cattle, sheep, and goats) infectious bovine rhinotracheitis. infectious bovine rhinotracheitis (ibr), or "rednose," occurs worldwide and is a disease of great importance to the cattle industry because of the synergism of the ibr virus with mannheimia haemolytica in producing pneumonia. the causative agent, bovine herpesvirus 1 (bohv-1), has probably existed as a mild venereal disease in cattle in europe since at least the mid-1800s, but the respiratory form was not reported until intensive management feedlot systems were first introduced in north america around the 1950s. typically, the disease is manifested as a transient, acute, febrile illness, which results in inspiratory dyspnea caused by obstruction of the airways by exudate only in very severe cases. other forms of bohv-1 infection include ulcerative rumenitis; enteritis; multifocal hepatitis in neonatal calves; nonsuppurative meningoencephalitis; infertility; and in experimental infections, mastitis, mammillitis, and ovarian necrosis. except for the encephalitic form, the type of disease caused by bohv-1 depends more on the site of entry than the viral strain. like other herpesviruses, bohv-1 also can remain latent in nerve ganglia, with recrudescence after stress or immunosuppression. this virus also causes bovine abortion, systemic infections of calves, and genital infections such as infectious pustular vulvovaginitis (ipv) and infectious balanoposthitis (ibp). the respiratory form of ibr is characterized by severe hyperemia and multifocal necrosis of nasal, pharyngeal, laryngeal, tracheal, and sometimes bronchial mucosa ( fig. 9 -29 and see fig. 9 -22). as in other respiratory viral infections, ibr lesions are microscopically characterized by necrosis and exfoliation of ciliated cells followed by repair. secondary bacterial infections of these areas of necrosis result in the formation of a thick layer of fibrinonecrotic material (diphtheritic) in the nasal, tracheal, and bronchial mucosa (see fig. 9 -22). intranuclear inclusion bodies, commonly seen in herpesvirus infections, are rarely seen in field cases because inclusion bodies occur only during the early stages of the disease. the most important sequela to ibr is bronchopneumonia, which is caused either by direct aspiration of exudate from airways or as a result of an impairment in pulmonary defense mechanisms, thus predisposing the animal to secondary bacterial infection, most frequently mannheimia haemolytica (see pneumonic mannheimiosis discussion). postmortem diagnosis of ibr is confirmed by isolation of the virus or its identification by immunohistochemistry or pcr in affected tissues. other causes of rhinitis. nasal granulomas occur in cattle presumably as a result of repeated exposure to an unidentified inhaled antigen. nasal granulomas (atopic rhinitis) are reported mainly in cattle in australia, south africa, and the united kingdom, where affected cattle develop multiple, small, pink or red, polypoid nodules, starting in the nasal vestibule that in time extend into the caudal aspect of the nasal septum (see fig. 9 -23). these nodules are composed of fibrovascular tissue mixed with lymphocytes (granulation tissue) superficially lined by hyperplastic epithelium with abundant mast cells and eosinophils in the lamina propria (nasal eosinophilia). the microscopic features suggest that hypersensitivity type i (immediate), type iii (immune complex), and type iv (delayed) may be involved in nasal granulomas of cattle. bovine (idiopathic) nasal granuloma must be differentiated from nasal mycetomas, nasal rhinosporidiosis, and nasal schistosomiasis, which also cause the formation of nodules in the nasal mucosa of cattle. an eosinophilic material consistent with the splendore-hoeppli phenomenon is occasionally observed in bovine mycotic granulomas. this phenomenon seen in some mycotic or bacterial infections is microscopically via the ingestion of contaminated feed and water and, very rarely, via inhalation of infectious droplets. the portals of entry are presumed to be the oropharynx or intestine, in which bacteria penetrate the mucosa and spread via lymph vessels to regional lymph nodes, then to the bloodstream, and thus hematogenously to the internal organs, particularly the lungs. lesions in the nasal cavity start as pyogranulomatous nodules in the submucosa; these lesions subsequently ulcerate, releasing copious amounts of burkholderia mallei-containing exudate into the nasal cavity (see fig. 4-25, a) . finally, ulcerative lesions in conchal mucosa heal and are replaced by typical stellate (star-shaped), fibrous scars. in some cases, the lungs also contain numerous gray, hard, small (2 to 10 mm), miliary nodules (resembling millet seeds) randomly distributed in one or more pulmonary lobes because of the hematogenous route. microscopically, these nodules are typical chronic granulomas composed of a necrotic center, with or without calcification, surrounded by a layer of macrophages enclosed by a thick band of connective tissue infiltrated with macrophages, fewer giant cells, lymphocytes, and plasma cells. cutaneous lesions, often referred to as equine farcy, are the result of severe suppurative lymphangitis characterized by nodular thickening of extended segments of lymph vessels in the subcutaneous tissue of the legs and ventral abdomen (see fig. 4 -25, c). eventually, affected lymph vessels rupture and release large amounts of purulent exudate through sinuses to the surface of the skin. melioidosis (pseudoglanders). melioidosis (pseudoglanders) is an important, life-threatening disease of human beings, horses, cattle, sheep, goats, pigs, dogs, cats, and rodents caused by burkholderia pseudomallei (pseudomonas pseudomallei). this disease in horses is clinically and pathologically similar to glanders, hence the name pseudoglanders. in human beings, this infection can cause severe sepsis and septic shock and has also been considered to have potential for biologic welfare. melioidosis is currently present in southeast asia and, to a much lesser extent, in northern australia and some european countries where the causative organism is frequently found in rodents, feces, soil, and water. ingestion of contaminated feed and water appears to be the main route of infection; direct transmission between infected animals and insect bites has also been postulated as a possible mechanism of infection. after gaining entrance to the animal, burkholderia pseudomallei is disseminated by the bloodstream and causes suppuration and abscesses in most internal organs, such as nasal mucosa, joints, brain and spinal cord, lungs, liver, kidneys, spleen, and lymph nodes. the exudate is creamy or caseous and yellow to green. the pulmonary lesions in melioidosis are those of an embolic bacterial infection with the formation of pulmonary abscesses, which can become confluent. focal adhesive pleuritis develops where abscesses rupture through the pleura and heal. rhinosporidiosis. the protistan parasite, rhinosporidium seeberi, causes nasal infection in human beings, horses, mules, cattle, dogs, and cats. gross lesions vary from barely visible granulomas to large expansive polypoid nodules that may be mistaken as tumors. these granulomatous nodules are detected by direct observation when present in the nasal mucosa close to the nares or by rhinoscopy when located in the deep nasal cavity. the offending organism, rhinosporidium seeberi, is readily visible in histologic preparations and in impression smears, appearing as a large (400 µm), oval sporangium containing thousands of endospores (see fig. 9 -26). rhinosporidium seeberi was once considered a mycotic agent, but recent phylogenetic investigations suggest that it is an aquatic protistan parasite of the class mesomycetozoea. giant, basophilic, intranuclear inclusion bodies in the nasal epithelium, particularly in the nasal glands ( fig. 9-32 ). immunosuppressed piglets can develop a systemic cytomegalovirus infection characterized by necrosis of the liver, lungs, adrenal glands, and brain with intralesional inclusion bodies. inclusion body rhinitis is clinically a characterized by a deeply eosinophilic homogeneous material surrounded by bacteria or mycelia. it is thought to result from a localized antigen-antibody response in tissue. oestrus ovis. oestrus ovis (diptera: oestridae; nasal bot) is a brownish fly about the size of a honeybee that deposits its first-stage larvae in the nostrils of sheep in most areas of the world. microscopic larvae mature into large bots (maggots), which spend most of their larval stages in nasal passages and sinuses, causing irritation, inflammation, and obstruction of airways. mature larvae drop to the ground and pupate into flies. this type of parasitism in which living tissues are invaded by larvae of flies is known as myiasis ( fig. 9-30 ). although oestrus ovis is a nasal myiasis primarily of sheep, it sporadically affects goats, dogs, and sometimes human beings (shepherds). the presence of the larvae in nasal passages and sinuses causes chronic irritation and erosive mucopurulent rhinitis and sinusitis; bots of oestrus ovis can be found easily if the head is cut to expose the nasal passages and paranasal sinuses. rarely, larvae of oestrus ovis penetrate the cranial vault through the ethmoidal plate, causing direct or secondary bacterial meningitis. other causes of rhinitis. infectious rhinitis is only sporadically reported in goats, and most of these cases are caused by pasteurella multocida or mannheimia haemolytica. the lesions range from a mild serous to catarrhal or mucopurulent inflammation. foreign body rhinitis caused by plant material is sporadically seen cattle, sheep, and goats ( fig. 9-31 ). inclusion body rhinitis. inclusion body rhinitis is a disease of young pigs with high morbidity and low mortality caused by a porcine cytomegalovirus (suid herpesvirus-2) and characterized by a mild rhinitis. this virus commonly infects the nasal epithelium of piglets younger than 5 weeks and causes a transient viremia. because this disease is seldom fatal, lesions are seen only incidentally or in euthanized animals. in uncomplicated cases, the gross lesion is hyperemia of the nasal mucosa, but with secondary bacterial infections, mucopurulent exudate can be abundant. microscopic lesions are typical and consist of a necrotizing, nonsuppurative rhinitis with toxigenic strains of pasteurella multocida. the only lesion associated with infection with bordetella bronchiseptica alone is a mild to moderate turbinate atrophy (nonprogressive atrophic rhinitis), but this bacterium actively promotes the colonization of the nasal cavity by pasteurella multocida. the toxigenic strains of pasteurella multocida produce potent cytotoxins that inhibit osteoblastic activity and promote osteoclastic reabsorption in nasal bones, particularly in the ventral nasal conchae, where abnormal bone remodeling results in progressive atrophy of conchae. the degree of conchal atrophy in pigs with atrophic rhinitis varies considerably, and in most pigs, the severity of the lesions does not correspond to the severity of the clinical signs. the best diagnostic method of evaluating this disease at necropsy is to make a transverse section of the snout between the first and second premolar teeth. in normal pigs, conchae are symmetric and fill most of the cavity, leaving only narrow airspaces (meatuses) between coiled conchae. the normal nasal septum is straight and divides the cavity into two mirror-image cavities. in contrast, the septum in pigs with atrophic rhinitis is generally deviated and the conchae appear smaller and asymmetric ( fig. 9-33 ). conchal atrophy causes dorsal and ventral meatuses to appear rather enlarged, and in the most advanced cases, the entire nasal conchae may be missing, leaving a large, empty space. it may seem logical to assume that after loss of conchae in an obligate nasal breather, such as the pig, the filtration defense mechanism of the nasal cavity would be impaired, thus enhancing the chances of aerogenous infections in the lung. however, the relationship between atrophic rhinitis, pneumonia, and growth rates in pigs is still controversial. osteoclastic hyperplasia and osteopenia of the conchae are the key microscopic lesions in atrophic rhinitis. depending on the stage of the disease, mucopurulent exudate may be found on the surface of the conchae. hyperplastic or metaplastic changes can occur in the nasal epithelium and glands, and infiltrates of lymphoplasmacytic cells can be present in the lamina propria. in summary, atrophic rhinitis is an important disease in pigs worldwide; morphologic characterized by a mild and transient rhinitis, causing sneezing, nasal discharge, and excessive lacrimation. atrophic rhinitis. a common worldwide disease of pigs, atrophic rhinitis (progressive atrophic rhinitis) is characterized by inflammation and atrophy of nasal conchae (turbinates). in severe cases, atrophy of the conchae may cause a striking facial deformity in growing pigs because of deviation of the nasal septum and nasal bones. the etiopathogenesis of atrophic rhinitis is complex and has been a matter of controversy for many years. pathogens historically associated with atrophic rhinitis include bordetella bronchiseptica, pasteurella multocida, haemophilus parasuis, and viral infections such as porcine cytomegalovirus (inclusion body rhinitis). in addition, predisposing factors have included genetic makeup, environment, and nutritional deficiencies. the cause of atrophic rhinitis is currently believed to be a combined infection by specific strains of bordetella bronchiseptica producing dermonecrotic toxin and linguatula serrata. linguatula serrata is a rare but highly specialized pentastomid parasite that shares some morphologic features with arthropods and annelids and causes infection when dogs consume uncooked ruminant meat containing infective larvae. it occurs primarily in carnivores, although sheep and goats may become aberrant hosts. human beings can also acquire the infection by ingesting raw ovine or caprine meat. the adult parasite is found throughout the nasal passages and sometimes can reach the sinuses and middle ear by moving through the exudate in the eustachian tubes. in common with other nasal parasites, linguatula serrata acts as an irritant, causing sneezing, catarrhal inflammation, and epistaxis. the eggs of this parasite leave the host in the exudate, which is coughed up or swallowed and eliminated in the feces. the nasal cavity and paranasal sinuses of dogs can occasionally be infested with other parasites, including mites (pneumonyssus caninum) and rhinosporidium seeberi (see figs. 9-25 and 9-26). allergic rhinitis. allergic rhinitis (hay fever; nasolacrimal urticaria), which is so common in human beings sensitized and reexposed to inhaled pollens or allergens, has been reported only sporadically in dogs and cats. hay fever in human beings and animals is a type i hypersensitivity reaction in which an ige-mediated degranulation of mast cells results in an acute rhinitis and conjunctivitis. microscopically, the nasal mucosa is edematous and infiltrated with numerous eosinophils, neutrophils, and some macrophages. clinically, allergic rhinitis is characterized by profuse serous nasal discharge and lacrimation. other causes of rhinitis. a nonspecific (idiopathic) chronic lymphoplasmacytic rhinitis is occasionally seen in dogs. immotile cilia syndrome (ciliary dyskinesia), a congenital disease, reduces mucociliary clearance and is an important factor in recurrent canine rhinosinusitis, bronchitis, bronchiectasis, and pneumonia. feline viral rhinotracheitis. feline viral rhinotracheitis (fvr) is a common, worldwide respiratory disease of cats caused by felid herpesvirus 1 (fehv-1). the disease causes an impairment of pulmonary defense mechanisms predisposing cats to secondary bacterial pneumonia or to a coinfection with feline calicivirus. the virus also can remain latent in ganglia. the vast majority of cats that recover from fvr become carriers and shed fehv-1, either spontaneously or following stress. susceptible animals, particularly kittens with low maternal immunity, become infected after exposure to a diseased or carrier cat. replication of fehv-1 in the nasal, conjunctival, pharyngeal, and, to a lesser extent, tracheal epithelium causes degeneration and exfoliation of cells. lesions caused by fehv-1 are fully reversible, but secondary infections with bacteria, such as pasteurella multocida, bordetella bronchiseptica, streptococcus spp., and mycoplasma felis, can cause a chronic, severe suppurative rhinitis and also conjunctivitis. intranuclear inclusion bodies are rarely seen in cats with fvr because inclusions are only present during the early stages of infection and have already disappeared by the time the cat is presented for diagnosis. respiratory sequelae to fvr can include chronic bacterial rhinitis and sinusitis with persistent purulent discharge; lysis of nasal bones, which can lead to conchal atrophy; permanent damage to the olfactory epithelium; and secondary bacterial pneumonia. in addition to rhinitis and interstitial pneumonia, fvr also causes ulcerative keratitis, hepatic necrosis, emaciation, abortion, and diagnosis is simple, but additional understanding of the pathogenesis will be necessary before effective preventive measures can be established. atrophic rhinitis is clinically characterized by sneezing, coughing, and nasal discharge. obstruction of the nasolacrimal duct is common and results in accumulation of dust and dried lacrimal secretions on the skin inferior to the medial canthus of the eye. viral infections. dogs have no specific viral infections affecting exclusively the nasal cavity or sinuses. acute rhinitis and sinusitis occurs as part of the canine infectious respiratory disease (cird) group caused by several distinct viruses, such as canine distemper virus, cav-1 and -2, canine parainfluenza virus, reovirus, and canine herpesvirus. the viral lesions in the respiratory tract are generally transient, but the effect of the virus on other tissues and cells can be fatal, as in distemper encephalitis in dogs. bacterial infections. as in other species, secondary bacterial rhinitis, sinusitis, and pneumonia are possible sequelae of respiratory viral infections; bordetella bronchiseptica, escherichia coli, and pasteurella multocida are the most common isolates in dogs with bacterial rhinitis. mycotic infections. aspergillus spp. and penicillium spp. cause mycotic rhinitis and sinusitis in dogs (canine nasal aspergillosis) ( fig. 9-34 ). nasal biopsies reveal extensive necrosis of the nasal epithelium and thick plaques of fibrinopurulent exudate mixed with many fungal hyphae. cryptococcus neoformans and blastomyces dermatitides infections of the nasal cavity occur sporadically in dogs ( fig. 9-35 ). lesions are characterized by mucosal granulomas containing periodic acid-schiff (pas)-positive fungal organisms, and the infection is clinically characterized by mucopurulent nasal discharge. fvr; these two viral infections account for 80% of all cases of feline respiratory diseases. a febrile systemic hemorrhagic syndrome with high mortality (up to 50%) has been reported in cats infected with virulent strains of fcv. feline chlamydiosis. feline chlamydiosis is a persistent respiratory infection of cats caused by chlamydophila felis. infection results in a conjunctivitis (similar to the conjunctivitis seen in human trachoma caused by chlamydia trachomatis) and serous or mucopurulent rhinitis. in the past, chlamydophila felis was incriminated as the agent responsible for "feline pneumonitis," but its role in causing bronchointerstitial pneumonia in cats has been seriously challenged in recent years (see pneumonias of cats). mycotic infections. the most common mycotic infection in the feline nasal cavity is caused by cryptococcus neoformans and cryptococcus gatti, but not all animals exposed to these fungi necessarily develop cryptococcosis unless they are immunosuppressed. stillbirths. clinical signs of fvr infection are characterized by lethargy, oculonasal discharge, severe rhinitis, and conjunctivitis. feline calicivirus. feline rhinitis can be caused by different strains of feline calicivirus (fcv). it is an important infection of the respiratory tract of cats, and depending on the virulence of the strain, lesions vary from a mild oculonasal discharge to severe rhinitis, mucopurulent conjunctivitis, and ulcerative gingivitis and stomatitis. the lesions, in addition to rhinitis and conjunctivitis, include acute, diffuse interstitial pneumonia with necrotizing bronchiolitis (see pneumonias of cats) and in some cases prominent ulcers of the tongue and hard palate. primary viral lesions are generally transient, but secondary bacterial infections (bordetella bronchiseptica, pasteurella multocida, or escherichia coli) are a common complication. some kittens develop lameness after infection or vaccination with calicivirus because of an acute and self-limiting arthritis ("limping kitten syndrome"). carrier state and virus shedding from oronasal secretions and feces are natural sequelae after recovery from the acute phase of the disease. clinical and pathologic features of fcv disease are strikingly similar but not identical to those of hemorrhage, increased lacrimation as a result of obstruction of nasolacrimal ducts, and sneezing. in some instances, it is not possible to clinically or grossly differentiate neoplasms from hyperplastic nodules or granulomatous rhinitis. some neoplasms may infiltrate adjacent bone structures and produce notable facial deformities, loss of teeth, exophthalmus, and nervous signs. large neoplasms also project into the meatuses, narrow the lumen, and interfere with airflow, causing stertorous breathing (see . biopsies, as well as brush and imprint cytology, have proven effective in the antemortem diagnosis of nasal neoplasms, particularly in those of epithelial lineage. a unique group of nasal carcinomas (enzootic nasal tumors, enzootic intranasal tumors, and enzootic nasal carcinoma) of sheep and goats arise from the surface epithelium and glands of the ethmoidal conchae. these types of carcinomas are caused by betaretroviruses in sheep (entv-1) and goats (entv-2). the enzootic nasal tumor has been successfully transmitted to susceptible animals by the lesions vary from discrete nasal granulomas to large confluent masses of mucopurulent exudate filling the entire nasal cavity and paranasal sinuses. microscopic examination of the exudate reveals the typical thick-walled pas-positive organisms (see fig. 9 -35). mycoplasma felis can also cause mucopurulent conjunctivitis and a mild upper respiratory infection, with clinical signs and lesions overlapping those seen with chlamydiosis, fvr, and fcr infections. respiratory infections and bronchopneumonia in cats may also be associated with the immunosuppressive effects of feline retroviruses such as feline leukemia virus (felv) and feline immunodeficiency virus (fiv). nasal aspergillosis and allergic rhinosinusitis are sporadically reported in cats (see disorders of the conducting system: species-specific diseases of the nasal cavity and paranasal sinuses: disorders of dogs: mycotic infections). neoplasms of the nasal cavity and paranasal sinuses may arise from any of the tissues forming these structures, including bone (osteoma or osteosarcoma), cartilage (chondroma or chondrosarcoma), connective tissue (fibroma or fibrosarcoma, myxoma or myxosarcoma), and blood vessels (hemangioma or hemangiosarcoma), and from all the different types of cells of glands and lining epithelium (adenoma, carcinoma, or adenocarcinoma). nasal tumors originating from stromal tissues, such as bone, cartilage, and connective tissue, are morphologically indistinguishable from those seen in other sites. in general, nasal neoplasms are rare in domestic animals, except for enzootic ethmoidal tumor (retroviral) in sheep and goats, which can occur in several animals in a herd (see the next section). in companion animals, nasal neoplasms are most common in dogs, particularly in medium to large breed dogs such as the collie, airedale terrier, basset hound, and german shepherd. the cat and the horse are less frequently affected. the main sites in order of frequency are the nasal passages and sinuses for dogs, the tip of the nose and nasal passages for cats, and the maxillary sinus and nasal passages for horses. the majority of neoplasms in the nasal cavity are malignant. benign nasal neoplasms (papilloma and adenoma) are rare and generally are either solitary or multiple, well-delineated nodules. in contrast, nasal carcinomas and nasal sarcomas are generally larger but vary in size and are often pale and multilobulated masses composed of fleshy to friable tissue (figs. 9-36 and 9-37). malignant neoplasms are locally invasive and tend to infiltrate sinuses, meninges, frontal brain, olfactory nerves, and vessels resulting in epistaxis. carcinomas vary from anaplastic (poorly differentiated) to well differentiated, in which cell and tissue morphology retains some glandular (adenocarcinoma) or squamous cell patterns. because nasal tumors in dogs and cats are usually large and invasive at the time of diagnosis, prognosis is usually poor and survival times are short. sarcomas originating in the nasal cavity and paranasal sinuses are less common than carcinomas. mesenchymal tumors can arise from bone (osteoma or osteosarcoma), cartilage (chondroma or chondrosarcoma), blood vessels (hemangioma or hemangiosarcoma), and connective tissue (fibroma or fibrosarcoma). overall, benign epithelial and mesenchymal tumors are less common than their malignant counterparts. secondary tumors in the nasal cavity are rare, with lymphoma being the most common secondary tumor in the nasal cavity of domestic animals ( fig. 9-38 ). nasal neoplasms become secondarily infected by bacteria, and clinical signs often overlap with those of infectious rhinitis and include catarrhal or mucopurulent nasal discharge, periodic anomalies 3 congenital anomalies of the pharynx, guttural pouches, larynx, and trachea are rare in all species. depending on their location and severity, they may be inconsistent with postnatal life, pose little or no problem, interfere with quality of life, or manifest themselves in later life. if clinical signs of respiratory distress, such as stridor, coughing, dyspnea, or gagging, do occur, they are usually exacerbated by excitement, heat, stress, or exercise. brachycephalic airway syndrome. see disorders of the conducting system: species-specific diseases of the pharynx, guttural inoculation of cell-free tumor filtrates. enzootic nasal tumors are typically invasive but do not metastasize ( fig. 9 -39). in some regions of the world, ethmoid tumors have been reported in horses and pigs, particularly in those farms where the endemic nasal tumors of ruminants are known to occur. nonneoplastic exophytic masses that resemble neoplasms are commonly found in horses, cats, and, to a lesser extent, other species. in horses, polyps tend to form in the ethmoidal region, whereas in cats, polyps are most frequently found in the nasopharynx and eustachian tubes. the pathogenesis of these benign growths is uncertain, although in many cases they follow chronic rhinitis or sinusitis. most recently, lymphatic obstruction secondary to inflammation has been postulated as the main culprit. grossly, polyps appear as firm, pedunculated nodules of various sizes protruding from the nasal mucosa into the nasal passages or nasopharynx ( fig. 9 -40); the surface may be smooth, ulcerated, secondarily infected, and hemorrhagic. microscopically, polyps are characterized by a core of wellvascularized stromal tissue that contains inflammatory cells and are covered by pseudostratified or squamous epithelium (see fig. 9 -40). nasal and paranasal sinus cysts are common idiopathic lesions in horses and are medically important because they clinically mimic table 1 -1 for potential, suspected, or known genetic disorders. tracheal collapse and tracheal stenosis. tracheal collapse with reduction in tracheal patency occurs in toy, miniature, and brachycephalic breeds of dogs, in which the condition is also called tracheobronchial collapse or central airway collapse. the defect also occurs in horses, cattle, and goats. by radiographic, endoscopic, or gross examination, there is dorsoventral flattening of the trachea with concomitant widening of the dorsal tracheal membrane, which may then prolapse ventrally into the lumen ( fig. 9-41 ). most commonly, the defect extends the entire length of the trachea and only rarely affects the cervical portion alone. affected segments with a reduced lumen contain froth and even are covered by a diphtheritic membrane. in horses, the so-called scabbard trachea is characterized allergic reactions. grossly, the mucosa of the epiglottis and vocal cords is thickened and swollen, often protrudes dorsally onto the epiglottic orifice, and has a gelatinous appearance ( fig. 9-43 ). laryngeal and tracheal hemorrhage. hemorrhages in these sites occur as mucosal petechiae and are most commonly seen in coagulopathies; inflammation; septicemia and sepsis, particularly in pigs with classical swine fever (hog cholera); african swine fever or salmonellosis; and horses with equine infectious anemia. severe dyspnea and asphyxia before death can cause congestion, ecchymosis, and petechiae in the laryngeal and tracheal mucosa; this lesion must be differentiated from postmortem imbibition of hemoglobin in autolyzed carcasses (see chapter 1). by lateral flattening so that the tracheal lumen is reduced to a narrow vertical slit. segmental tracheal collapse causing stenosis has been associated with congenital and acquired abnormalities. in severe cases, abnormal cartilaginous glycoproteins and loss of elasticity of tracheal rings causes the trachea to collapse. in some other cases, it is an acquired tracheal lesion that follows trauma, compression caused by extraluminal masses, peritracheal inflammation, and flawed tracheotomy or transtracheal aspirate techniques. other tracheal anomalies include tracheoesophageal fistula, which is most commonly found in human beings and sporadically in dogs and cattle. congenital fistulas can occur at any site of the cervical or thoracic segments of the trachea. acquired tracheoesophageal fistula can be a complication of improper intubation, tracheotomy, or esophageal foreign body. laryngeal hemiplegia. laryngeal hemiplegia (paralysis), sometimes called roaring in horses, is a common but obscure disease characterized by atrophy of the dorsal and lateral cricoarytenoid muscles (abductor and adductor of the arytenoid cartilage), particularly on the left side. muscular atrophy is most commonly caused by a primary denervation (recurrent laryngeal neuropathy) of unknown cause (idiopathic axonopathy) and, to a much lesser extent, secondary nerve damage (see the section on denervation atrophy in chapters 14 and 15). idiopathic laryngeal hemiplegia is an incurable axonal disease (axonopathy) of the cranial laryngeal nerve that affects mostly larger horses. secondary laryngeal hemiplegia is rare and occurs after nerve damage caused by other pathologic processes such as compression or inflammation of the left recurrent laryngeal nerve. the medial retropharyngeal lymph nodes are located immediately ventral to the floor of the guttural pouches. as a result of this close anatomic relationship, swelling or inflammation of the guttural pouches or retropharyngeal lymph nodes often results in secondary damage to the laryngeal nerve. common causes of secondary nerve damage (wallerian degeneration) include guttural pouch mycosis, retropharyngeal abscesses, inflammation because of iatrogenic injection into the nerves, neck injury, and metastatic neoplasms involving the retropharyngeal lymph nodes (e.g., lymphosarcoma). grossly, the affected laryngeal muscle in a horse with laryngeal hemiplegia is pale and smaller than normal (muscle atrophy) . microscopically, muscle fibers have lesions of denervation atrophy (see chapters 14 and 15). atrophy of laryngeal muscles also occurs in dogs as an inherited condition (siberian husky and bouvier des flanders), as a degenerative neuropathy in older dogs, secondary to laryngeal trauma in all species (e.g., choke chain damage), or secondary to hepatic encephalopathy in horses. the abnormal inspiratory sounds (roaring) during exercise in horses with laryngeal hemiplegia are caused by paralysis of the left dorsal and lateral cricoarytenoid muscles, which cause incomplete dilation of the larynx, obstruction of airflow, and vibration of vocal cords. laryngeal edema. laryngeal edema is a common feature of acute inflammation, but it is particularly important because swelling of the epiglottis and vocal cords can obstruct the laryngeal orifice, resulting in asphyxiation. laryngeal edema occurs in pigs with edema disease; in horses with purpura hemorrhagica; in cattle with acute interstitial pneumonia; in cats with systemic anaphylaxis; and in all species as a result of trauma, improper endotracheal tubing, inhalation of irritant gases (e.g., smoke), local inflammation, and animal species is classified as fibrinous, catarrhal, purulent, or granulomatous (figs. 9-44 and 9-45). chronic polypoid tracheitis occurs in dogs and cats, probably secondary to chronic infection. the most common causes of tracheitis are viral infections, such as those causing infectious bovine rhinotracheitis (see fig. 9 -29), equine viral rhinopneumonitis, canine distemper, and feline rhinotracheitis. viral lesions are generally mild and transient but often become complicated with secondary bacterial infections. at the early stages, the mucosa is notably hyperemic and can show white foci of necrosis. in the most severe cases, the affected mucosa detaches from the underlying basement membrane, causing extensive tracheal ulceration. chemical tracheitis is also commonly seen after aspiration (see fig. 9 -45). also, inhalation of fumes during barn fires can cause extensive injury and necrosis of the tracheal mucosa. in forensic cases, the presence of carbon pigment in the mucosal surface of trachea, bronchi, and bronchioles indicates that the burned animal was alive during the fire. parasitic infections of the larynx and trachea can cause obstruction with dramatic consequences, but burdens sufficient to cause such effects are not commonly seen in veterinary practice. besnoitiosis (besnoitia spp.) . besnoitiosis (besnoitia spp.) is caused by several species of this apicomplexan coccidian parasite, whose life cycle is still unknown. this parasite can cause pedunculated lesions on the skin, sclera, mucosa of the nasal cavity, and larynx of horses and donkeys, cattle, goats, and wild animals. besnoitiosis has been reported from africa, central and south america, north america, and europe. grossly, pale, round, exophytic nodules up to 2 cm in diameter can be observed protruding from mucosal surfaces. microscopically, these nodules consist of finger-like projections covered by hyperplastic and sometimes ulcerated epithelium containing numerous thick-walled parasitic cysts with little inflammatory response. cattle. see disorders of cattle. inflammation of the pharynx, larynx, and trachea are important because of their potential to obstruct airflow and to lead to aspiration pneumonia. the pharynx is commonly affected by infectious diseases of the upper respiratory and upper digestive tracts, and the trachea can be involved by extension from both the lungs and larynx. pharyngeal obstruction and perforation. intraluminal foreign bodies in the pharynx, such as medicament boluses, apples, or potatoes, can move down and obstruct the larynx and trachea. also, pharyngeal obstruction can be caused by masses in the surrounding tissue, such as neoplasms of the thyroid gland, thymus, and parathyroid glands. a number of nonspecific insults can cause lesions and clinical signs. trauma may take the form of penetrating wounds in any species: perforation of the caudodorsal wall of the pharynx from the improper use of drenching or balling guns in sheep, cattle, and pigs; choking injury because of the use of collars in dogs and cats; and the shearing forces of bite wounds. the results of the trauma may be minimal (local edema and inflammation) or as serious as complete luminal obstruction by exudate. foreign bodies may be lodged anywhere in the pharyngeal region; the location and size determine the occurrence of dysphagia, regurgitation, dyspnea, or asphyxiation. pigs have a unique structure known as the pharyngeal diverticulum (4 cm long in adult pigs), which is located in the pharyngeal wall rostral and dorsal to the esophageal entrance. it is important because barley awns may lodge in the diverticulum, causing an inflammatory swelling that affects swallowing. the diverticular wall may be perforated by awns or drenching syringes, which results in an exudate that can extend down the tissue planes between muscles of the neck and even into the mediastinum. the pharynx of the dog may also be damaged by trauma from chicken bones, sticks, and needles, resulting in the formation of a pharyngeal abscess. equine inflammation of the trachea (tracheitis). the types of injury and host inflammatory responses in the trachea are essentially the same as those described for the nasal mucosa. although tracheal mucosa is prone to aerogenous injury and necrosis, it has a remarkable capacity for repair. according to the exudate, tracheitis in all palate, are important causes of respiratory problems and reduced athletic performance in horses. an undersized epiglottis is prone to being entrapped below the arytenoepiglottic fold, causing an equine syndrome known as epiglottic entrapment. this syndrome also occurs in horses with lateral deviation and deformity of epiglottis, epiglottic cysts, or necrosis of the tip of the epiglottis. hypoplastic epiglottis also occurs in pigs. dorsal displacement of the soft palate, particularly during exercise, narrows the lumen of the nasopharynx and creates abnormal air turbulence in the conducting system of horses. epiglottic entrapment is clinically characterized by airway obstruction, exercise intolerance, respiratory noise, and cough. subepiglottic and pharyngeal cysts. anomalous lesions, such as subepiglottic and pharyngeal cysts, are occasionally seen in horses, particularly in standardbred and thoroughbred racehorses. these cysts vary in size (1 to 9 cm) and occur most commonly in the subepiglottic area and to a lesser extent in the dorsal pharynx, larynx, and soft palate. cysts are lined by squamous or pseudostratified epithelium and contain thick mucus. large cysts cause airway obstruction, reduced exercise tolerance, or dysphagia and predispose to bronchoaspiration of food. equine pharyngeal lymphoid hyperplasia. equine pharyngeal lymphoid hyperplasia, or pharyngitis with lymphoid follicular hyperplasia, is a common cause of partial upper airway obstruction in horses, particularly in 2-and 3-year-old racehorses. lymphoid hyperplasia is also seen in healthy horses as part of a response to mild chronic pharyngitis, which in many instances tends to regress with age in older animals. the cause is undetermined, but chronic bacterial infection combined with environmental factors may cause excessive antigenic stimulation and lymphoid hyperplasia. the gross lesions, visible endoscopically or at necropsy, consist of variably sized (1 to 5 mm) white foci located on the dorsolateral walls of the pharynx and extending into the openings of the guttural pouches and onto the soft palate. in severe cases, lesions may appear as pharyngeal polyps. microscopically, the lesions consist of large aggregates of lymphocytes and plasma cells in the pharyngeal mucosa. clinical signs consist of stertorous inspiration, expiration, or both. inflammation of guttural pouches. the guttural pouches of horses are large diverticula (300 to 500 ml) of the ventral portion of the auditory (eustachian) tubes. these diverticula are therefore exposed to the same pathogens as the pharynx and have drainage problems similar to the sinuses. although it is probable that various pathogens, including viruses, can infect them, the most common pathogens are fungi, which cause guttural pouch mycosis and guttural pouch empyema in the horse. eustachitis is the term used for inflammatory processes involving the eustachian (pharyngotympanic) tube. because of the close anatomic proximity of guttural pouches to the internal carotid arteries, cranial nerves (vii, ix, x, xi, and xii), and atlantooccipital joint, disease of these diverticula may involve these structures and cause a variety of clinical signs in horses. guttural pouch mycosis occurs primarily in stabled horses and is caused by aspergillus fumigatus and other aspergillus spp. infection is usually unilateral and presumably starts with the inhalation of spores from moldy hay. grossly, the mucosal surfaces of the dorsal and lateral walls of the guttural pouch mucosa are first covered by focal, rounded, raised plaques of diphtheritic (fibrinonecrotic) exudate, which with time can become confluent and grow into a large fibrinonecrotic mass ( fig. 9-46) . microscopically, the lesions are severe necrotic inflammation of the mucosa and submucosa with widespread vasculitis and intralesional fungal hyphae. necrosis of the mammomonogamus (syngamus) spp. mammomonogamus (syngamus) laryngeus is a nematode that is seen attached to the laryngeal mucosa of cattle in tropical asia and south america, and cats (gapeworm: mammomonogamus ierei) in the caribbean and southern united states. occasionally, human beings with a persistent cough or asthma-like symptoms have the parasite in the larynx or bronchi. oslerus (filaroides) osleri. see disorders of dogs. b a c empyema of guttural pouches is a sequela to suppurative inflammation of the nasal cavities, most commonly from streptococcus equi infection (strangles). in severe cases, the entire guttural pouch can be filled with purulent exudate ( fig. 9 -47). the sequelae are similar to those of guttural pouch mycosis except that there is no erosion of the internal carotid artery. it is clinically characterized by nasal discharge, enlarged retropharyngeal lymph nodes, painful swelling of the parotid region, dysphagia, and respiratory distress. guttural pouch tympany develops sporadically in young horses when excessive air accumulates in the pouch from the one-way valve effect caused by inflammation or malformation of the eustachian tube. arabian and german warm-blooded horses are particularly susceptible to develop guttural pouch tympany. it is generally unilateral and characterized by nonpainful swelling of the parotid region. cattle. tracheal edema and hemorrhage syndrome of feeder cattle, also known as the honker syndrome or tracheal stenosis of feedlot cattle, is a poorly documented acute disease of unknown cause, most often seen during the summer months. severe edema and a few hemorrhages are present in the mucosa and submucosa of the dorsal surface of the trachea, extending caudally from the midcervical area as far as the tracheal bifurcation. on section, the tracheal mucosa is diffusely thickened and gelatinous. clinical signs include inspiratory wall of the guttural pouches can extend into the wall of the adjacent internal carotid artery causing hemorrhage into the lumen of the guttural pouch and recurrent epistaxis. invasion of the internal carotid artery causes arteritis, which can also lead to formation of an aneurysm and fatal bleeding into the guttural pouches. in other cases, the fungi may be angioinvasive, leading to the release of mycotic emboli into the internal carotid artery, generally resulting in multiple cerebral infarcts. dysphagia, another clinical sign seen in guttural pouch mycosis, is associated with damage to the pharyngeal branches of the vagus and glossopharyngeal nerves, which lie on the ventral aspect of the pouches. horner's syndrome results from damage to the cranial cervical ganglion and sympathetic fibers located in the caudodorsal aspect of the pouches. finally, equine laryngeal paralysis (hemiplegia) can result from damage to the laryngeal nerves as previously described in the section on laryngeal hemiplegia. pekingese, and others. the defects are a result of a mismatch of the ratio of soft tissue to cranial bone and the obstruction of airflow by excessive length of the palatine soft tissue. secondary changes, such as nasal and laryngeal edema caused by forceful inspiration, eventually lead to severe upper airway obstruction, respiratory distress, and exercise intolerance. tracheal hypoplasia. tracheal hypoplasia occurs most often in english bulldogs and boston terriers; the tracheal lumen is decreased in diameter throughout its length. canine infectious respiratory disease. canine infectious respiratory disease (cird), formerly called canine tracheobronchitis or kennel cough, is a highly contagious group of infectious diseases characterized clinically by an acute onset of coughing notably exacerbated by exercise. the term is nonspecific, much like the dyspnea that can progress to oral breathing, recumbency, and death by asphyxiation in less than 24 hours. necrotic laryngitis. necrotic laryngitis (calf diphtheria, laryngeal necrobacillosis) is a common disease of feedlot cattle and cattle affected with other diseases, with nutritional deficiencies, or housed under unsanitary conditions. it also occurs sporadically in sheep and pigs. necrotic laryngitis, caused by fusobacterium necrophorum, is part of the syndrome termed necrotic stomatitis or laryngeal necrobacillosis, which can include lesions of the tongue, cheeks, palate, and pharynx. an opportunistic pathogen, fusobacterium necrophorum produces potent exotoxins and endotoxins after gaining entry either through lesions of viral infections, such as ibr and vesicular stomatitis in cattle, or after traumatic injury produced by feed or careless use of specula or balling guns. the gross lesions, regardless of location in the mouth or larynx (most common in the mucosa overlying the laryngeal cartilages), consist of well-demarcated, dry, yellow-gray, thick-crusted, and fibrinonecrotic exudate ( fig. 9 -48) that in the early stages is bounded by a zone of active hyperemia. deep ulceration develops, and if the lesion does not result in death, healing is by granulation tissue formation. microscopically, the necrotic foci are first surrounded by congested borders, then by a band of leukocytes, and finally the ulcers heal by granulation tissue and collagen (fibrosis). the lesions can extend deep into the submucosal tissue. numerous bacteria are evident at the advancing edge. there are numerous and important sequelae to calf diphtheria; the most serious is death from severe toxemia or overwhelming fusobacteremia. sometimes, the exudate may be copious enough to cause laryngeal obstruction and asphyxiation or be aspirated and cause bronchopneumonia. the clinical signs of necrotic laryngitis are fever, anorexia, depression, halitosis, moist painful cough, dysphagia, and inspiratory dyspnea and ventilatory failure because of fatigue of the respiratory muscles (diaphragmatic and intercostal). laryngeal contact ulcers. ulcerative lesions in the larynx are commonly found in feedlot cattle. grossly, the laryngeal mucosa reveals circular ulcers (up to 1 cm in diameter), which may be unilateral or bilateral and sometimes deep enough to expose the underlying arytenoid cartilages. the cause has not been established, but causal agents, such as viral, bacterial, and traumatic, have been proposed, along with increased frequency and rate of closure of the larynx (excessive swallowing and vocalization) when cattle are exposed to market and feedlot stresses such as dust, pathogens, and interruption of feeding. contact ulcers predispose a calf to diphtheria (fusobacterium necrophorum) and laryngeal papillomas. ulceration of the mucosa and necrosis of the laryngeal cartilages have also been described in calves, sheep, and horses under the term laryngeal chondritis. laryngeal abscesses involving the mucosa and underlying cartilage occur as a herd or flock problem in calves and sheep, presumably caused by a secondary infection with trueperella (arcanobacterium) pyogenes. anomalies 5 brachycephalic airway syndrome. brachycephalic airway syndrome is a clinical term that refers to increased airflow resistance caused by stenotic nostrils and nasal meatuses and an excessively long soft palate. these abnormalities are present in brachycephalic canine breeds such as bulldogs, boxers, boston terriers, pugs, a "common cold" in human beings or bovine respiratory disease complex (brdc) in cattle. the infection occurs commonly as a result of mixing dogs from different origins such as occurs at commercial kennels, animal shelters, and veterinary clinics. between bouts of coughing, most animals appear normal, although some have rhinitis, pharyngitis, tonsillitis, or conjunctivitis; some with secondary pneumonia become quite ill. the pathogenesis of cird is complex, and many pathogens and environmental factors have been incriminated. bordetella bronchiseptica, canine adenovirus-2 (cav-2), and canine parainfluenza virus-2 (cpiv-2) are most commonly implicated. the severity of the disease is increased when more than one agent is involved or if there are extreme environmental conditions (e.g., poor ventilation). for example, dogs asymptomatically infected with bordetella bronchiseptica are more severely affected by superinfection with cav-2 than those not carrying the bacterium. other agents are sometimes isolated but of lesser significance and include canine adenovirus-1 (cav-1: infectious canine hepatitis virus), reovirus type 1, canid herpesvirus-1 (cahv-1), canine respiratory coronavirus (crcov), and mycoplasma species. depending on the agents involved, gross and microscopic lesions are completely absent or they vary from catarrhal to mucopurulent tracheobronchitis, with enlargement of the tonsils and retropharyngeal and tracheobronchial lymph nodes. in dogs with bordetella bronchiseptica infection, the lesions are suppurative or mucopurulent rhinitis and tracheobronchitis, and suppurative bronchiolitis. in contrast, when lesions are purely viral, microscopic changes are focal necrosis of the tracheobronchial epithelium. sequelae can include spread either proximally or distally in the respiratory tract, the latter sometimes inducing chronic bronchitis and bronchopneumonia. oslerus (filaroides) osleri. oslerus (filaroides) osleri is a nematode parasite of dogs and other canidae that causes characteristic protruding nodules into the lumen at the tracheal bifurcation. they are readily seen on endoscopic examination or at necropsy. in severe cases, these nodules can extend 5 cm cranially or caudally from the tracheal bifurcation and even into primary and secondary bronchi. the disease occurs worldwide, and oslerus osleri is considered the most common respiratory nematode of dogs. the gross lesions are variably sized, up to 1 cm, submucosal nodules that extend up to 1 cm into the tracheal lumen ( fig. 9 -49, a). microscopically, nodules contain adult parasites with a mild mononuclear cell reaction; with the death of the parasite, an intense foreign body reaction develops with neutrophils and giant cells b) . clinically, it can be asymptomatic, although it most often causes a chronic cough that can be exacerbated by exercise or excitement. severe infestations can result in dyspnea, exercise intolerance, cyanosis, emaciation, and even death in young dogs. neoplasms of the guttural pouches occur rarely in horses and are usually squamous cell carcinomas. laryngeal neoplasms are rare in dogs and extremely so in other species, although they have been reported in cats and horses. the most common laryngeal neoplasms in dogs are papillomas and squamous cell carcinomas. other less common tumors are laryngeal rhabdomyoma, previously referred to as laryngeal oncocytoma, and chondromas and osteochondromas. lymphoma involving the laryngeal tissue is sporadically seen in cats. when large enough to be obstructive, neoplasms may cause a change or loss of voice, cough, or respiratory distress with cyanosis, collapse, and syncope. other signs include dysphagia, anorexia, and exercise intolerance. the neoplasm is sometimes visible from the oral cavity and causes swelling of the neck. the prognosis is poor because most lesions recur after excision. tracheal neoplasms are even more uncommon than those of the larynx. the tracheal cartilage or mucosa can be the site of an osteochondroma, leiomyoma, osteosarcoma, mast cell tumor, and carcinoma. lymphoma in cats can extend from the mediastinum to involve the trachea. each lung is subdivided into various numbers of pulmonary lobes (see fig. 9 -16). in the past, these were defined by anatomic fissures. however, in current anatomy, lobes are defined by the ramification of the bronchial tree. following this criterion, the left lung of all domestic species is composed of cranial and caudal lobes, whereas the right lung, depending on species, is composed of cranial, middle (absent in horse), caudal, and accessory lobes. each pulmonary lobe is further subdivided by connective tissue into pulmonary lobules, which in some species (cattle and pigs) are rather prominent and in others are much less conspicuous. from a practical standpoint, identification of the lungs among different species could be achieved by carefully observing the degree of lobation (external fissures) and the degree of lobulation (connective tissue between lobules). cattle and pigs have well-lobated and well-lobulated lungs; sheep and goats have well-lobated but poorly lobulated lungs; horses have both poorly lobated and poorly lobulated lungs and resemble human lungs; finally, dogs and cats have well-lobated but not well-lobulated lungs. the degree of lobulation determines the degree of air movement between the lobules. in pigs and cattle, movement of air between lobules is practically absent because of the thick connective tissue of the interlobular septa separating individual lobules. this movement of air between lobules and between adjacent alveoli (via the pores of kohn) constitutes what is referred to as collateral ventilation. this collateral ventilation is poor in cattle and pigs and good in dogs. the functional implications of collateral ventilation are discussed in the section on pulmonary emphysema. the lungs have an interconnecting network of interstitial stromal tissue supporting the blood and lymphatic vessels, nerves, bronchi, bronchioles, and alveoli. for purposes of simplicity, the pulmonary interstitium can be anatomically divided into three contiguous compartments: (1) bronchovascular interstitium, where main bronchi and pulmonary vessels are situated; (2) interlobular interstitium separating pulmonary lobules and supporting small blood and lymph vessels; and (3) alveolar interstitium supporting the alveolar walls that contain pulmonary capillaries and alveolar epithelial cells (no lymphatic vessels here) (see discussion on the blood-air barrier in the section on alveoli). pulmonary changes, such as edema, emphysema, and inflammation, may affect one or more of these interstitial compartments. anomalies 6 congenital anomalies of the lungs are rare in all species but are most commonly reported in cattle and sheep. compatibility with life largely depends on the type of structures involved and the proportion of functional tissue present at birth. accessory lungs are one of the most common anomalies and consist of distinctively lobulated masses of incompletely differentiated pulmonary tissue present in the thorax, abdominal cavity, or subcutaneous tissue virtually anywhere in the trunk. large accessory lungs can cause dystocia. ciliary dyskinesia (immotile cilia syndrome, kartagener's syndrome) is characterized by defective ciliary movement, which results in reduced mucociliary clearance because of a defect in the microtubules of all ciliated cells and, most important, in the ciliated respiratory epithelium and spermatozoa. primary ciliary dyskinesia often associated with situs inversus has been reported in dogs, which as a result usually have chronic recurrent rhinosinusitis, pneumonia, and infertility. pulmonary agenesis, pulmonary hypoplasia, abnormal lobulation, congenital emphysema, lung hamartoma, and congenital bronchiectasis are occasionally seen in domestic animals. congenital melanosis is a common incidental finding in pigs and ruminants and is usually seen at slaughter (fig. 9-50 ). it is characterized by black spots, often a few centimeters in diameter, in various organs, mainly the lungs, meninges, intima of the aorta, and caruncles of the uterus. melanosis has no clinical significance, and the texture of pigmented lungs remains unchanged. congenital emphysema is sporadically seen in dogs (efig. 9 -4). pulmonary calcification ("calcinosis"). calcification of the lungs occurs in some hypercalcemic states, generally secondary to hypervitaminosis d or from ingestion of toxic (hypercalcemic) plants, such as solanum malacoxylon (manchester wasting disease), that contain vitamin d analogs. it is also a common sequela to uremia and hyperadrenocorticism in dogs and to pulmonary necrosis (dystrophic calcification) in most species. calcified lungs may fail to collapse when the thoracic cavity is opened and have a characteristic "gritty" texture ( fig. 9-51) . microscopically, lesions vary from calcification of the alveolar basement membranes (see by pathologists. recent investigations suggest that excessive lipid originates from the breakdown products of neoplastic cells. bronchial and bronchiolar obstructions such as those caused by lungworms can also cause alveolar lipidosis. the pathogenesis relates to the inability of alveolar macrophages that normally remove part of the surfactant lipids to exit the lung via the mucociliary escalator. exogenous lipid pneumonia. another form of lipid pneumonia occurs accidentally in cats or horses given mineral oil by their owners in an attempt to remove hairballs or treat colic (aspiration pneumonia). to achieve gaseous exchange, a balanced ratio of the volumes of air to capillary blood must be present in the lungs (ventilation/perfusion ratio), and the air and capillary blood must be in close proximity across the alveolar wall. a ventilation-perfusion mismatch occurs if pulmonary tissue is either collapsed (atelectasis) or overinflated (hyperinflation and emphysema). the term atelectasis means incomplete distention of alveoli and is used to describe lungs that have failed to expand with air at the time of birth (congenital or neonatal atelectasis) or lungs that have collapsed after inflation has taken place (acquired atelectasis or alveolar collapse) (figs. 9-52 and 9-53). during fetal life, lungs are not fully distended, contain no air, and are partially filled with a locally produced fluid known as fetal lung fluid. not surprisingly, lungs of aborted and stillborn fetuses sink when placed in water, whereas those from animals that have breathed float. at the time of birth, fetal lung fluid is rapidly reabsorbed and replaced by inspired air, leading to the normal distention of alveoli. congenital atelectasis occurs in newborns that fail to inflate their lungs after taking their first few breaths of air; it is caused by obstruction of airways, often as a result of aspiration of amniotic fluid and meconium (described in the section on meconium aspiration syndrome) (see fig. 9 -52). congenital atelectasis also develops when alveoli cannot remain distended after initial aeration because of an alteration in quality and quantity of pulmonary surfactant produced by type ii pneumonocytes and club (clara) cells. this infant form of congenital atelectasis is referred to in human neonatology as infant respiratory distress syndrome (irds) or as hyaline membrane disease because of the clinical and microscopic features of the disease. it commonly occurs in babies who are premature or born to diabetic or alcoholic mothers and is occasionally found in animals, particularly foals and piglets. the pathetic, gasping attempts of affected foals and pigs to breathe have prompted the use of the name "barkers"; foals that survive may have brain damage from cerebral hypoxia (see chapter 14) and are referred to as "wanderers" due to their aimless behavior and lack of a normal sense of fear. acquired atelectasis is much more common and occurs in two main forms: compressive and obstructive (see fig. 9 -53). compressive atelectasis has two main causes: space-occupying masses in the pleural cavity, such as abscesses and tumors, or transferred pressures, such as that caused by bloat, hydrothorax, hemothorax, chylothorax, and empyema ( fig. 9-54 ). another form of compressive atelectasis occurs when the negative pressure in the thoracic cavity is lost because of pneumothorax. this form generally has massive atelectasis and thus is also referred to as lung collapse. obstructive (absorption) atelectasis occurs when there is a reduction in the diameter of the airways caused by mucosal edema and inflammation, or when the lumen of the airway is blocked by fig. 9 -51) to heterotopic ossification of the lungs (efig. 9 -5). in most cases, pulmonary calcification in itself has little clinical significance, although its cause (e.g., uremia or vitamin d toxicosis) may be very important. alveolar filling disorders are a heterogeneous group of lung diseases characterized by accumulation of various chemical compounds in the alveolar lumens. the most common are alveolar proteinosis, in which the alveoli are filled with finely granular eosinophilic material; pulmonary lipidosis, in which alveoli are filled with macrophages containing endogenous or exogenous lipid; and alveolar microlithiasis, in which the alveoli contain numerous concentric calcified "microliths" or "calcospherites." a similar but distinct concretion is known as corpora amylacea, which is an accumulation of laminated bodies composed of cellular debris, lipids, proteins, and possibly amyloid. for most alveolar filling disorders, there is little host response, and in many cases, it is an incidental finding. most of the alveolar filling disorders originate from inherited metabolic defects in which alveolar cells (epithelial or macrophages) cannot properly metabolize or remove lipids or proteins, whereas others result from an excessive synthesis of these substances in the lung. endogenous lipid (lipoid) pneumonia. endogenous lipid pneumonia is an obscure, subclinical pulmonary disease of cats and occasionally of dogs, which is unrelated to aspiration of foreign material. although the pathogenesis is not understood, it is presumed that lipids from pulmonary surfactant and from degenerated cells accumulate within alveolar macrophages. accumulation of surfactant lipids can occur in metabolic abnormalities of alveolar macrophages or in bronchial obstruction where surfactant-laden macrophages cannot exit the lungs via the mucociliary escalator. the gross lesions are multifocal, white, firm nodules scattered throughout the lungs (efig. 9-6) . microscopically, the alveoli are filled with foamy lipid-laden macrophages accompanied by interstitial infiltration of lymphocytes and plasma cells, fibrosis, alveolar epithelialization, and, in some cases, cholesterol clefts and lipid granulomas. lipid (lipoid) pneumonia occurs frequently in the vicinity of cancerous lung lesions in human beings, cats, and dogs. the reason for this association remains unknown and frequently unrecognized appearance of atelectasis is more common in species with poor collateral ventilation, such as cattle and pigs. the extent and location of obstructive atelectasis depends largely on the size of the affected airway (large vs. small) and on the degree of obstruction (partial vs. complete). atelectasis also occurs when large animals are kept recumbent for prolonged periods, such as during anesthesia (hypostatic atelectasis). the factors contributing to hypostatic atelectasis are a combination of blood-air imbalance, shallow breathing, airway obstruction because of mucus and fluid that has not been drained from bronchioles and alveoli, and from inadequate local production of surfactant. atelectasis can also be a sequel to paralysis of respiratory muscles and prolonged use of mechanical ventilation or general anesthesia in intensive care. in general, the lungs with atelectasis appear depressed below the surface of the normally inflated lung. the color is generally dark blue, and the texture is flabby or firm; they are firm if there is concurrent edema or other processes, such as can occur in ards or "shock" lungs (see the section on pulmonary edema). distribution and extent vary with the process, being patchy (multifocal) in congenital atelectasis, lobular in the obstructive type, and of various degrees in between in the compressive type. microscopically, the alveoli are collapsed or slitlike and the alveolar walls appear parallel and close together, giving prominence to the interstitial tissue even without any superimposed inflammation. pulmonary emphysema. pulmonary emphysema, often simply referred to as emphysema, is an extremely important primary disease in human beings, whereas in animals, it is always a secondary condition resulting from a variety of pulmonary lesions. in human medicine, emphysema is strictly defined as an abnormal permanent enlargement of airspaces distal to the terminal bronchiole, accompanied by destruction of alveolar walls (alveolar emphysema). this definition separates it from simple airspace enlargement or hyperinflation, in which there is no destruction of alveolar walls and which can occur congenitally (down syndrome) or be acquired with age (aging lung, sometimes misnamed "senile emphysema"). the pathogenesis of emphysema in human beings is still controversial, but current thinking overwhelmingly suggests that destruction of mucus plugs, exudate, aspirated foreign material, or lungworms (see fig. 9 -53). when the obstruction is complete, trapped air in the lung eventually becomes reabsorbed. unlike the compression type, obstructive atelectasis often has a lobular pattern as a result of blockage of the airway supplying that lobule. this lobular lungs are extremely well-vascularized organs with a dual circulation provided by pulmonary and bronchial arteries. disturbances in pulmonary circulation have a notable effect on gaseous exchange, which may result in life-threatening hypoxemia and acidosis. in addition, circulatory disturbances in the lungs can have an impact on other organs, such as the heart and liver. for example, impeded blood flow in the lungs because of chronic pulmonary disease results in cor pulmonale, which is caused by unremitting pulmonary hypertension followed by cardiac dilation, right heart failure, chronic passive congestion of the liver (nutmeg liver), and generalized edema (anasarca). hyperemia is an active process that is part of acute inflammation, whereas congestion is the passive process resulting from decreased outflow of venous blood, as occurs in congestive heart failure ( fig. 9-56 ). in the early acute stages of pneumonia, the lungs appear notably red, and microscopically, blood vessels and alveolar capillaries are engorged with blood from alveolar walls is largely the result of an imbalance between proteases released by phagocytes and antiproteases produced in the lung as a defense mechanism (the protease-antiprotease theory). the destructive process in human beings is markedly accelerated by defects in the synthesis of antiproteases or any factor, such as cigarette smoking or pollution, that increases the recruitment of macrophages and leukocytes in the lungs. this theory originated when it was found that human beings with homozygous α 1 -antitrypsin deficiency were remarkably susceptible to emphysema and that proteases (elastase) inoculated intratracheally into the lungs of laboratory animals produced lesions similar to those found in the disease. more than 90% of the problem relates to cigarette smoking, and airway obstruction is no longer considered to play a major role in the pathogenesis of emphysema in human beings. primary emphysema does not occur in animals, and thus no animal disease should be called simply emphysema. in animals, this lesion is always secondary to obstruction of outflow of air or is agonal at slaughter. secondary pulmonary emphysema occurs frequently in animals with bronchopneumonia, in which exudate plugging bronchi and bronchioles causes an airflow imbalance where the volume of air entering exceeds the volume leaving the lung. this airflow imbalance is often promoted by the so-called one-way valve effect caused by the exudate, which allows air into the lung during inspiration but prevents movement of air out of the lung during expiration. depending on the localization in the lung, emphysema can be classified as alveolar or interstitial. alveolar emphysema characterized by distention and rupture of the alveolar walls, forming variably sized air bubbles in pulmonary parenchyma, occurs in all species. interstitial emphysema occurs mainly in cattle, presumably because of their wide interlobular septa, and lack of collateral ventilation in these species does not permit air to move freely into adjacent pulmonary lobules. as a result, accumulated air penetrates the alveolar and bronchiolar walls and forces its way into the interlobular connective tissue, causing notable distention of the interlobular septa. it is also suspected that forced respiratory movements predispose to interstitial emphysema when air at high pressure breaks into the loose connective tissue of the interlobular septa ( fig. 9-55 ). sometimes these bubbles of trapped air in alveolar or interstitial emphysema become confluent, forming large (several centimeters in diameter) pockets of air that are referred to as bullae (singular: bulla) (see efig. 9 -4); the lesion is then called bullous emphysema. this lesion is not a specific type of emphysema and does not indicate a different disease process but, rather, is a larger accumulation of air at one focus. in the most severe cases, air moves from the interlobular septa into the connective tissue surrounding the main stem bronchi and major vessels (bronchovascular bundles), and from here it leaks into the mediastinum, causing pneumomediastinum first, and eventually exits via the thoracic inlet into the cervical and thoracic subcutaneous tissue causing subcutaneous emphysema. note that mild and even moderate alveolar emphysema is difficult to judge at necropsy and by light microscopy unless special techniques are used to prevent collapse of the lung when the thorax is opened. these techniques include plugging of the trachea or intratracheal perfusion of fixative (10% neutral-buffered formalin) before the thorax is opened to prevent collapse of the lungs. important diseases that cause secondary pulmonary emphysema in animals include small airway obstruction (e.g., heaves) in horses and pulmonary edema and emphysema (fog fever) in cattle (see fig. 9 -55) and exudates in bronchopneumonia. congenital emphysema occurring secondary to bronchial cartilage hypoplasia with subsequent bronchial collapse is occasionally reported in dogs. severe and persistent cases of heart failure, the lungs fail to collapse because of edema and pulmonary fibrosis. terminal pulmonary congestion (acute) is frequently seen in animals euthanized with barbiturates and should not be mistaken for an antemortem lesion. hypostatic congestion is another form of pulmonary congestion that results from the effects of gravity and poor circulation on a highly vascularized tissue, such as the lung. this type of gravitational congestion is characterized by the increase of blood in the lower side of the lung, particularly the lower lung of animals in lateral recumbency, and is most notable in horses and cattle. the affected portions of the lung appear dark red and can have a firmer texture. in animals and human beings who have been prostrated for extended periods of time, hypostatic congestion may be followed by hypostatic edema, and hypostatic pneumonia as edema interferes locally with the bacterial defense mechanisms. pulmonary hemorrhage. pulmonary hemorrhages can occur as a result of trauma, coagulopathies, and disseminated intravascular coagulation (dic), vasculitis, sepsis, and pulmonary thromboembolism from jugular thrombosis or from embolism of exudate from a hepatic abscess that has eroded the wall and ruptured into the caudal vena cava (cattle). a gross finding often confused with intravital pulmonary hemorrhage is the result of severing both the trachea and the carotid arteries simultaneously at slaughter. blood is aspirated from the transected trachea into the lungs, forming a random pattern of irregular red foci (1 to 10 mm) in one or more lobes. these red foci are readily visible on both the pleural and the cut surfaces of the lung, and free blood is visible in the lumens of bronchi and bronchioles. rupture of a major pulmonary vessel with resulting massive hemorrhage occurs occasionally in cattle when a growing abscess in a lung invades and disrupts the wall of a major pulmonary artery or vein ( fig. 9-58 ). in most cases, animals die rapidly, often with spectacular hemoptysis, and on postmortem examination, bronchi are filled with blood (see fig. 9 -58). pulmonary edema. in normal lungs, fluid from the vascular space slowly but continuously passes into the interstitial tissue, where it is rapidly drained by the pulmonary and pleural lymphatic vessels. clearance of alveolar fluid across the alveolar epithelium is also a major mechanism of fluid removal from the lung. edema develops when the rate of fluid transudation from pulmonary vessels into the interstitium or alveoli exceeds that of lymphatic and alveolar removal ( fig. 9-59 ). pulmonary edema can be physiologically classified as cardiogenic (hydrostatic; hemodynamic) and noncardiogenic (permeability) types. hydrostatic (cardiogenic) pulmonary edema develops when there is an elevated rate of fluid transudation because of increased hydrostatic pressure in the vascular compartment or decreased osmotic pressure in the blood. once the lymph drainage has been overwhelmed, fluid accumulates in the perivascular spaces, causing distention of the bronchovascular bundles and alveolar interstitium, and eventually leaks into the alveolar spaces. causes of hemodynamic pulmonary edema include congestive heart failure (increased hydrostatic pressure); iatrogenic fluid overload; and disorders in which blood osmotic pressure is reduced, such as with hypoalbuminemia seen in some hepatic diseases, nephrotic syndrome, and protein-losing enteropathy. hemodynamic pulmonary edema also occurs when lymph drainage is impaired, generally secondary to neoplastic invasion of lymphatic vessels. permeability edema (inflammatory) occurs when there is excessive opening of endothelial gaps or damage to the cells that constitute the blood-air barrier (endothelial cells or type i pneumonocytes). hyperemia. pulmonary congestion is most frequently caused by heart failure, which results in stagnation of blood in pulmonary vessels, leading to edema and egression of erythrocytes into the alveolar spaces. as with any other foreign particle, erythrocytes in alveolar spaces are rapidly phagocytosed (erythrophagocytosis) by pulmonary alveolar macrophages. when extravasation of erythrocytes is severe, large numbers of macrophages with brown cytoplasm may accumulate in the bronchoalveolar spaces. the brown cytoplasm is the result of accumulation of considerable amounts of hemosiderin; these macrophages filled with iron pigment (siderophages) are generally referred to as heart failure cells ( fig. 9-57 ). the lungs of animals with chronic heart failure usually have a patchy red appearance with foci of brown discoloration because of accumulated hemosiderin. in this type of edema is an integral and early part of the inflammatory response, primarily because of the effect of inflammatory mediators, such as leukotrienes, platelet-activating factor (paf), cytokines, and vasoactive amines released by neutrophils, macrophages, mast cells, lymphocytes, endothelial cells, and type ii pneumonocytes. these inflammatory mediators increase the permeability of the blood-air barrier. in other cases, permeability edema results from direct damage to the endothelium or type i pneumonocytes, allowing plasma fluids to move freely from the vascular space into the alveolar lumen ( fig. 9 -60 and see fig. 9 -14). because type i pneumonocytes are highly vulnerable to some pneumotropic viruses (influenza and brsv), toxicants (nitrogen dioxide [no 2 ], sulfur dioxide [so 2 ], hydrogen sulfide [h 2 s], and 3-methylindole), and particularly to free radicals, it is not surprising that permeability edema commonly accompanies many viral or toxic pulmonary diseases. a permeability edema also occurs when endothelial cells in the lung are injured by bacterial toxins, sepsis, ards, dic, anaphylactic shock, milk allergy, paraquat toxicity, adverse drug reactions, and smoke inhalation (efig. 9-7) . the concentration of protein in edematous fluid is greater in permeability edema (exudate) than in hemodynamic edema (transudate); this difference has been used clinically in human medicine to differentiate one type of pulmonary edema from another. microscopically, because of the higher concentration of protein, edema fluid in lungs with inflammation or damage to the blood-air barrier tends to stain more intensely eosinophilic than that of the hydrostatic edema from heart failure. grossly, the edematous lungs-independent of the cause-are wet and heavy. the color varies, depending on the degree of congestion or hemorrhage, and fluid may be present in the pleural cavity. if edema is severe, the bronchi and trachea contain considerable amounts of foamy fluid, which originates from the mixing of edema fluid and air ( fig. 9-61 ). on cut surfaces, the lung parenchyma oozes fluid like a wet sponge. in cattle and pigs that have distinct lobules, the lobular pattern becomes rather accentuated because of edematous distention of lymphatic vessels in the interlobular septa and the edematous interlobular septum itself ( fig. 9-62 ). severe pulmonary edema may be impossible to differentiate from peracute pneumonia; (h&e)-stained sections (see fig. 9 -60), particularly if a fixative such as zenker's solution, which precipitates protein, is used. acute respiratory distress syndrome. acute (adult) respiratory distress syndrome (ards; shock lung) is an important condition in human beings and animals characterized by pulmonary hypertension, intravascular aggregation of neutrophils in the lungs, acute lung injury, diffuse alveolar damage, permeability edema, and formation of hyaline membranes ( fig. 9-63) . these membranes are a mixture of plasma proteins, fibrin, surfactant, and cellular debris from necrotic pneumonocytes (see fig. 9-55, b) . the pathogenesis of ards is complex and multifactorial but in general terms can be defined as diffuse alveolar damage that results from lesions in distant organs, from generalized systemic diseases, or from direct injury to the lung. sepsis, major trauma, aspiration of gastric contents, extensive burns, and pancreatitis are some of the disease entities known to trigger ards. all these conditions provoke "hyperreactive macrophages" to directly or indirectly generate overwhelming amounts of cytokines causing what is known as a "cytokine storm." the main cytokines that trigger ards are tnf-α, interleukin (il)-1, il-6, and il-8, which prime neutrophils previously recruited in the lung capillaries and alveoli to release cytotoxic enzymes and free radicals. these substances cause severe and diffuse endothelial and alveolar damage that culminates in a fulminating pulmonary edema (see fig. 9 -63). ards occurs in domestic animals and explains why pulmonary edema is one of the most common lesions found in many animals dying of sepsis, toxemia, aspiration of gastric contents, and pancreatitis, for example. a familial form of ards has been reported in dalmatians. the pulmonary lesions in this syndrome are further discussed in the sections on interstitial pneumonia and aspiration pneumonia in dogs. neurogenic pulmonary edema is another distinctive but poorly understood form of life-threatening lung edema in human beings that follows cns injury and increased intracranial pressure (i.e., head injury, brain edema, brain tumors, or cerebral hemorrhage). this type of pulmonary edema can be experimentally reproduced in laboratory animals by injecting fibrin into the fourth ventricle. it involves both hemodynamic and permeability pathways presumably from massive sympathetic stimulation and overwhelming release of catecholamines. neurogenic pulmonary edema has sporadically been reported in animals with brain injury or severe seizures or after severe stress and excitement. pulmonary embolism. with its vast vascular bed and position in the circulation, the lung acts as a safety net to catch emboli before they reach the brain and other tissues. however, this positioning is often to its own detriment. the most common pulmonary emboli in domestic animals are thromboemboli, septic (bacterial) emboli, fat emboli, and tumor cell emboli. pulmonary thromboembolism (pte) refers to both local thrombus formation and translocation of a thrombus present elsewhere in the venous circulation ( fig. 9-64 ). fragments released inevitably reach the lungs and become trapped in the pulmonary vasculature ( fig. 9-65 and see fig. 9 -64). small sterile thromboemboli are generally of little clinical or pathologic significance because they can be rapidly degraded and disposed of by the fibrinolytic system. larger thromboemboli may cause small airway constriction, reduced surfactant production, pulmonary edema, and atelectasis resulting in hypoxemia, hyperventilation, and dyspnea. parasites (e.g., dirofilaria immitis and angiostrongylus vasorum), endocrinopathies (e.g., hyperadrenocorticism and hypothyroidism), glomerulopathies, and hypercoagulable states can be responsible for pulmonary arterial thrombosis and pulmonary thromboembolism in dogs (efig. 9-8) . pieces of this fact is not surprising because pulmonary edema occurs in the very early stages of inflammation (see efig. 9-7) . careful observation of the lungs at the time of necropsy is critical because diagnosis of pulmonary edema cannot be reliably performed microscopically. this is due in part to the loss of the edema fluid from the lungs during fixation with 10% neutral-buffered formalin and in part to the fact that the fluid itself stains very poorly or not at all with eosin because of its low protein content (hemodynamic edema). a protein-rich (permeability) edema is easier to visualize microscopically because it is deeply eosinophilic in hematoxylin and eosin fig. 9-104) . 1, normal alveolar capillary externally covered by type i and type ii pneumonocytes and internally by vascular endothelium (see fig. 9 -14 for more detail). 2, at the early stages of sepsis, proinflammatory cytokines (interleukin 1 [il-1] and tumor necrosis factor [tnf]) cause circulating neutrophils to adhere to the endothelial surface. following a "cytokine storm," the marginated neutrophils further activated by inflammatory mediators suddenly release their cytoplasmic granules (proteolytic enzymes and elastases myeloperoxidase) into the surrounding milieu (arrows). 3, enzymes released by these neutrophils cause injury to type i pneumonocytes (arrows) and endothelial cells (arrowheads), disrupting the blood-air barrier and causing permeability edema (curved arrows), alveolar hemorrhage (double-headed arrow), and exocytosis of neutrophils into the alveolar space (double-headed arrow). 4, extravasated plasma proteins admixed with surfactant and cell debris form thick hyaline membranes along the alveolar wall. 5, in the unlikely event that the animal survives, the healing process starts with alveolar macrophages removing cellular debris, reabsorption of edema, and hyperplasia of type ii pneumonocytes (double-headed curved arrow) that subsequently differentiate into type i pneumonocytes (see fig. 9 b a recognized in the bovine lung after strong pneumatic stunning at slaughter (captive bolt) ( fig. 9-66, a) . although obviously not important as an antemortem pulmonary lesion, brain emboli are intriguing as a potential risk for public health control of bovine spongiform encephalopathy (bse). fragments of hair can also embolize to the lung following intravenous injections (see fig. 9-66 , b). hepatic emboli formed by circulating pieces of fragmented liver occasionally become trapped in the pulmonary vasculature after severe abdominal trauma and hepatic rupture (see fig. 9-66, c) . megakaryocytes trapped in alveolar capillaries are a common but incidental microscopic finding in the lungs of all species, particularly dogs (see fig. 9-66, d) . tumor emboli (e.g., osteosarcoma and hemangiosarcoma in dogs and uterine carcinoma in cattle) can be numerous and striking and the ultimate cause of death in malignant neoplasia. in experimental studies, cytokines released during pulmonary inflammation are chemotactic for tumor cells and promote pulmonary metastasis. pulmonary infarcts. because of a dual arterial supply to the lung, pulmonary infarction is rare and generally asymptomatic. however, pulmonary infarcts can be readily caused when pulmonary thrombosis and embolism are superimposed on an already compromised pulmonary circulation such as occurs in congestive heart c d thrombi breaking free from a jugular, femoral, or uterine vein can cause pulmonary thromboembolism. pulmonary thromboembolisms occur in heavy horses after prolonged anesthesia (deep vein thrombosis), recumbent cows ("downer cow syndrome"), or in any animal undergoing long-term intravenous catheterization in which thrombi build up in the catheter and then break off (see fig. 9 -65). septic emboli, pieces of thrombi contaminated with bacteria or fungi and broken free from infected mural or valvular thrombi in the heart and vessels, eventually become entrapped in the pulmonary circulation. pulmonary emboli originate most commonly from bacterial endocarditis (right side) and jugular thrombophlebitis in all species, hepatic abscesses that have eroded and discharged their contents into the caudal vena cava in cattle, and septic arthritis and omphalitis in farm animals (see fig. 9 -64). when present in large numbers, septic emboli may cause unexpected death because of massive pulmonary edema; survivors generally develop pulmonary arteritis and thrombosis and embolic (suppurative) pneumonia, which may lead to pulmonary abscesses. bone marrow and bone emboli can form after bone fractures or surgical interventions of bone. these are not as significant a problem in domestic animals as they are in human beings. brain emboli (i.e., pieces of brain tissue) in the pulmonary vasculature reported in severe cases of head injury in human beings have recently been pulmonary macrophages (alveolar, intravascular, and interstitial), which have an immense biologic armamentarium, are the single most important effector cell and source of cytokines for all stages of pulmonary inflammation. these all-purpose phagocytic cells modulate the recruitment and trafficking of blood-borne leukocytes in the lung through the secretion of chemokines (see etable 9 -1). before reviewing how inflammatory cells are recruited in the lungs, three significant features in pulmonary injury must be remembered: (1) leukocytes can exit the vascular system through the alveolar capillaries, unlike in other tissues, where postcapillary venules are the sites of leukocytic diapedesis (extravasation); (2) the intact lung contains within alveolar capillaries a large pool of resident leukocytes (marginated pool); and (3) additional neutrophils are sequestered within alveolar capillaries within minutes of a local or systemic inflammatory response. these three pulmonary idiosyncrasies, along with the enormous length of the capillary network in the lung, explain why recruitment and migration of leukocytes into alveolar spaces develops so rapidly. experimental studies with aerosols of endotoxin or gram-negative bacteria have shown that within minutes of exposure, there is a significant increase in capillary leukocytes, and by 4 hours the alveolar lumen is filled with neutrophils. not surprisingly, the bal fluid collected from patients with acute pneumonia contains large amounts of inflammatory mediators such as tnf-α, il-1, and il-8. also, the capillary endothelium of patients with acute pneumonia has increased "expression" of adhesion molecules, which facilitate the migration of leukocytes from capillaries into the alveolar interstitium and from there into the alveolar lumen. in allergic pulmonary diseases, eotaxin and il-5 are primarily responsible for recruitment and trafficking of eosinophils in the lung. movement of plasma proteins into the pulmonary interstitium and alveolar lumen is a common but poorly understood phenomenon in pulmonary inflammation. leakage of fibrinogen and plasma proteins into the alveolar space occurs when there is structural damage to the blood-air barrier. this leakage is also promoted by some types of cytokines that enhance procoagulant activity, whereas others reduce fibrinolytic activity. excessive exudation of fibrin into the alveoli is particularly common in ruminants and pigs. the fibrinolytic system plays a major role in the resolution of pulmonary inflammatory diseases. in some cases, excessive plasma proteins leaked into alveoli mix with necrotic type i pneumonocytes and pulmonary surfactant, forming microscopic eosinophilic bands (membranes) along the lining of alveolar septa. these membranes, known as hyaline membranes, are found in specific types of pulmonary diseases, particularly in ards, and in cattle with acute interstitial pneumonias such as bovine pulmonary edema and emphysema and extrinsic allergic alveolitis (see pneumonias of cattle). in the past few years, nitric oxide has been identified as a major regulatory molecule of inflammation in a variety of tissues, including the lung. produced locally by macrophages, pulmonary endothelium, and pneumonocytes, nitric oxide regulates the vascular and bronchial tone, modulates the production of cytokines, controls the recruitment and trafficking of neutrophils in the lung, and switches on/off genes involved in inflammation and immunity. experimental work has also shown that pulmonary surfactant upregulates the production of nitric oxide in the lung, supporting the current view that pneumonocytes are also pivotal in amplifying and downregulating the inflammatory and immune responses in the lung (see etable 9 -1). as the inflammatory process becomes chronic, the types of cells making up cellular infiltrates in the lung change from mainly neutrophils to largely mononuclear cells. this shift in cellular composition is accompanied by an increase in specific cytokines, such as failure. it also occurs in dogs with torsion of a lung lobe (fig. 9-67) . the gross features of infarcts vary considerably, depending on the stage, and they can be red to black, swollen, firm, and cone or wedge shaped, particularly at the lung margins. in the early acute stage, microscopic lesions are severely hemorrhagic, and this is followed by necrosis. in 1 or 2 days, a border of inflammatory cells develops, and a few days later, a large number of siderophages are present in the necrotic lung. if sterile, pulmonary infarcts heal as fibrotic scars; if septic, an abscess may form surrounded by a thick fibrous capsule. in the past three decades, an information explosion has increased the overall understanding of pulmonary inflammation, with so many proinflammatory and antiinflammatory mediators described to date that it would be impossible to review them all here (see chapters 3 and 5). pulmonary inflammation is a highly regulated process that involves a complex interaction between cells imported from the blood (platelets, neutrophils, eosinophils, mast cells, and lymphocytes) and pulmonary cells (type i and ii pneumonocytes; endothelial and club [clara] cells; alveolar and intravascular macrophages; and stromal interstitial cells, such as mast cells, interstitial macrophages, fibroblasts, and myofibroblasts). blood-borne leukocytes, platelets, and plasma proteins are brought into the areas of inflammation by an elaborate network of chemical signals emitted by pulmonary cells and resident leukocytes. long-distance communication between pulmonary cells and blood cells is largely done by soluble cytokines; once in the lung, imported leukocytes communicate with pulmonary and vascular cells through adhesion and other inflammatory molecules. the best known inflammatory mediators are the complement system (c3a, c3b, and c5a), coagulation factors (factors v and vii), arachidonic acid metabolites (leukotrienes and prostaglandins), cytokines (interleukins, monokines, and chemokines), adhesion molecules (icam and vcam), neuropeptides (substance p, tachykinins, and neurokinins), enzymes and enzyme inhibitors (elastase and antitrypsin), oxygen metabolites (o 2 •, oh•, and h 2 o 2 ), antioxidants (glutathione), and nitric oxide (e -table 9 -1). acting in concert, these and many other molecules send positive or negative signals to initiate, maintain, and, it is hoped, resolve the inflammatory process without causing injury to the lung. chapter 9 respiratory system, mediastinum, and pleurae ewith several episodes of hemorrhage are characterized by large areas of dark brown discoloration, largely in the caudal lung lobes. microscopically, lesions are alveolar hemorrhages, abundant alveolar macrophages containing hemosiderin (siderophages), mild alveolar fibrosis, and occlusive remodeling of pulmonary veins. recurrent airway obstruction. recurrent airway obstruction (rao) of horses, also referred to as copd, heaves, chronic bronchiolitis-emphysema complex, chronic small airway disease, alveolar emphysema, and "broken wind," is a common clinically asthma-like syndrome of horses and ponies. rao is characterized by recurrent respiratory distress, chronic cough, poor athletic performance, airway neutrophilia, bronchoconstriction, mucus hypersecretion, and airway obstruction. the pathogenesis is still obscure, but genetic predisposition, t h 2 (allergic) immune response, and the exceptional sensitivity of airways to environmental allergens (hyperreactive airway disease) have been postulated as the basic underlying mechanisms. what makes small airways hyperreactive to allergens is still a matter of controversy. epidemiologic and experimental studies suggest that it could be the result of preceding bronchiolar damage caused by viral infections; ingestion of pneumotoxicants (3-methylindole); or prolonged exposure to organic dust, endotoxin, and environmental allergens (molds). it has been postulated that sustained inhalation of dust particles, whether antigenic or not, upregulates the production of cytokines (tnf-α, il-8, and monokine-inducible protein ) and neuropeptides (neurokinin a [nka], neurokinin b [nkb], and substance p), attracting neutrophils into the bronchioloalveolar region and promoting leukocyte-induced bronchiolar injury. summer pasture-associated obstructive pulmonary disease (spaopd) is a seasonal airway disease also reported in horses with similar clinical and pathologic findings. more recently, the term inflammatory airway disease (iad) has been introduced in equine medicine to describe rao-like syndrome in young horses 2 to 4 years old. the lungs of horses with heaves are grossly unremarkable, except for extreme cases in which alveolar emphysema may be present. microscopically, the lesions are often remarkable and include goblet cell metaplasia in bronchioles; plugging of bronchioles with mucus mixed with few eosinophils and neutrophils (see fig. 9 -13); peribronchiolar infiltration with lymphocytes, plasma cells, and variable numbers of eosinophils; and hypertrophy of smooth muscle in bronchi and bronchioles. in severe cases, accumulation of mucus leads to the complete obstruction of bronchioles and alveoli and resultant alveolar emphysema characterized by enlarged "alveoli" from the destruction of alveolar walls. feline asthma syndrome. feline asthma syndrome, also known as feline allergic bronchitis, is a clinical syndrome in cats of any age characterized by recurrent episodes of bronchoconstriction, cough, or dyspnea. the pathogenesis is not well understood but is presumed to originate, as in human asthma, as a type i hypersensitivity (igemast cell reaction) to inhaled allergens. dust, cigarette smoke, plant and household materials, and parasitic proteins have been incriminated as possible allergens. this self-limited allergic disease responds well to steroid therapy; thus it is rarely implicated as a primary cause of death except when suppressed defense mechanisms allow a secondary bacterial pneumonia. bronchial biopsies from affected cats at the early stages reveal mild to moderate inflammation characterized by mucosal edema and infiltration of leukocytes, particularly eosinophils. increased numbers of circulating eosinophils (blood eosinophilia) are present in some but not all cats with feline asthma. il-4, interferon-γ (ifn-γ), and interferon-inducible protein (ip-10), which are chemotactic for lymphocytes and macrophages. under appropriate conditions, these cytokines activate t lymphocytes, regulate granulomatous inflammation, and induce the formation of multinucleated giant cells such as in mycobacterial infections. inflammatory mediators locally released from inflamed lungs also have a biologic effect in other tissue. for example, pulmonary hypertension and right-sided heart failure (cor pulmonale) often follows chronic alveolar inflammation, not only as a result of increased pulmonary blood pressure but also from the effect of inflammatory mediators on the contractibility of smooth muscle of the pulmonary and systemic vasculature. cytokines, particularly tnf-α, that are released during inflammation are associated, both as cause and as effect, with the systemic inflammatory response syndrome (sirs), sepsis, severe sepsis with multiple organ dysfunction, and septic shock (cardiopulmonary collapse). as it occurs in any other sentinel system where many biologic promoters and inhibitors are involved (coagulation, the complement and immune systems), the inflammatory cascade could go into an "out-of-control" state, causing severe damage to the lungs. acute lung injury (ali), extrinsic allergic alveolitis, ards, pulmonary fibrosis, and asthma are archetypical diseases that ensue from an uncontrolled production and release of cytokines (cytokine storm). as long as acute alveolar injury is transient and there is no interference with the normal host response, the entire process of injury, degeneration, necrosis, inflammation, and repair can occur in less than 10 days. on the other hand, when acute alveolar injury becomes persistent or when the capacity of the host for repair is impaired, lesions can progress to an irreversible stage in which restoration of alveolar structure is no longer possible. in diseases, such as extrinsic allergic alveolitis, the constant release of proteolytic enzymes and free radicals by phagocytic cells perpetuates alveolar damage in a vicious circle. in other cases, such as in paraquat toxicity, the magnitude of alveolar injury can be so severe that type ii pneumonocytes, basement membranes, and alveolar interstitium are so disrupted that the capacity for alveolar repair is lost. fibronectins and transforming growth factors (tgfs) released from macrophages and other mononuclear cells at the site of chronic inflammation regulate the recruitment, attachment, and proliferation of fibroblasts. in turn, these cells synthesize and release considerable amounts of ecm (collagen, elastic fibers, or proteoglycans), eventually leading to fibrosis and total obliteration of normal alveolar architecture. in summary, in diseases in which there is chronic and irreversible alveolar damage, lesions invariably progress to a stage of terminal alveolar and interstitial fibrosis. for pneumonia, see section species-specific pneumonia of domestic animals. exercise-induced pulmonary hemorrhage. exercise-induced pulmonary hemorrhage (eiph) is a specific form of pulmonary hemorrhage in racehorses that occurs after exercise and clinically is characterized by epistaxis. because only a small percentage of horses with bronchoscopic evidence of hemorrhage have clinical epistaxis, it is likely that eiph goes undetected in many cases. the pathogenesis is still controversial, but current literature suggests laryngeal paralysis, bronchiolitis, and extremely high pulmonary vascular and alveolar pressures during exercise, alveolar hypoxia, and preexisting pulmonary injury as possible causes. eiph is seldom fatal; postmortem lesions in the lungs of horses that have been affected disease may be known by different names. in pigs, for instance, enzootic pneumonia and mycoplasma pneumonia refer to the same disease caused by mycoplasma hyopneumoniae. the word pneumonitis has been used by some as a synonym for pneumonia; however, others have restricted this term to chronic proliferative inflammation generally involving the alveolar interstitium and with little or no evidence of exudate. in this chapter, the word pneumonia is used for any inflammatory lesion in the lungs, regardless of whether it is exudative or proliferative, alveolar, or interstitial. on the basis of texture, distribution, appearance, and exudation, pneumonias can be grossly diagnosed into four morphologically distinct types: bronchopneumonia, interstitial pneumonia, embolic pneumonia, and granulomatous pneumonia. by using this classification, it is possible at the time of a necropsy to predict with some degree of certainty the likely cause (virus, bacteria, fungi, or parasites), routes of entry (aerogenous vs. hematogenous), and possible sequelae. these four morphologic types allow the clinician or pathologists to predict the most likely etiology and therefore facilitate the decision as to what samples need to be taken and which tests should be requested to the diagnostic laboratory (i.e., histopathology, bacteriology, virology, or toxicology). however, overlapping of these four types of pneumonias is possible, and sometimes two morphologic types may be present in the same lung. the criteria used to classify pneumonias grossly into bronchopneumonia, interstitial pneumonia, embolic pneumonia, and granulomatous pneumonia are based on morphologic changes, including distribution, texture, color, and general appearance of the affected lungs (table 9 -5). distribution of the inflammatory lesions in the lungs can be (1) cranioventral, as in most bronchopneumonias; (2) multifocal, as in embolic pneumonias; (3) diffuse, as in interstitial pneumonias; or (4) locally extensive, as in granulomatous in the most advanced cases, chronic bronchoconstriction and excess mucus production may result in smooth muscle hyperplasia and obstruction of the bronchi and bronchioles and infiltration of the airway mucosa by eosinophils. a syndrome known as canine asthma has been reported in dogs but is not as well characterized as the feline counterpart. few subjects in veterinary pathology have caused so much debate as the classification of pneumonias. historically, pneumonias in animals have been classified or named based on the following: 1. presumed cause, with names such as viral pneumonia, pasteurella pneumonia, distemper pneumonia, verminous pneumonia, chemical pneumonia, and hypersensitivity pneumonitis 2. type of exudation, with names such as suppurative pneumonia, fibrinous pneumonia, and pyogranulomatous pneumonia 3. morphologic features, with names such as gangrenous pneumonia, proliferative pneumonia, and embolic pneumonia 4. distribution of lesions, with names such as focal pneumonia, cranioventral pneumonia, diffuse pneumonia, and lobar pneumonia 5. epidemiologic attributes, with names such as enzootic pneumonia, contagious bovine pleuropneumonia, and "shipping fever" 6. geographic regions, with names such as montana progressive pneumonia 7. miscellaneous attributes, with names such as atypical pneumonia, cuffing pneumonia, progressive pneumonia, aspiration pneumonia, pneumonitis, farmer's lung, and extrinsic allergic alveolitis until a universal and systematic nomenclature for animal pneumonias is established, veterinarians should be acquainted with this heterogeneous list of names and should be well aware that one the parts of the face with the tip of your finger has been advocated by some pathologists. the texture of a normal lung is comparable to the texture of the center of the cheek. firm consolidation is comparable to the texture of the tip of the nose, and hard consolidation is comparable to the texture of the forehead. the term consolidation is frequently used to describe a firm or hard lung filled with exudate. pneumonias ( fig. 9-68) . texture of pneumonic lungs can be firmer or harder (bronchopneumonias), more elastic (rubbery) than normal lungs (interstitial pneumonias), or have a nodular feeling (granulomatous pneumonias). describing in words the palpable difference between the texture of a normal lung compared with the firm or hard texture of a consolidated lung can be a difficult undertaking. an analogy illustrating this difference based on touching changes in the gross appearance of pneumonic lungs include abnormal color, the presence of nodules or exudate, fibrinous or fibrous adhesions, and the presence of rib imprints on serosal surfaces (see fig. 9 -68). on cut surfaces, pneumonic lungs may have exudate, hemorrhage, edema, necrosis, abscesses, bronchiectasis, granulomas or pyogranulomas, and fibrosis, depending on the stage. palpation and careful observation of the lungs are essential in the diagnosis of pneumonia. (for details, see the section on examination of the respiratory tract.) bronchopneumonia refers to a particular morphologic type of pneumonia in which injury and the inflammatory process take place primarily in the bronchial, bronchiolar, and alveolar lumens. bronchopneumonia is undoubtedly the most common type of pneumonia seen in domestic animals and is with few exceptions characterized grossly by cranioventral consolidation of the lungs (fig. 9-69 and see fig. 9 -68). the reason why bronchopneumonias in animals are almost always restricted to the cranioventral portions of the lungs is not well understood. possible factors contributing to this topographic selectivity within the lungs include (1) gravitational sedimentation of the exudate, (2) greater deposition of infectious organisms, (3) inadequate defense mechanisms, (4) reduced vascular perfusion, (5) shortness and abrupt branching of airways, and (6) regional differences in ventilation. the term cranioventral in veterinary anatomy is the equivalent of "anterosuperior" in human anatomy. the latter is defined as "in front (ventral) and above (cranial)." thus, applied to the lung of animals, "cranioventral" means the ventral portion of the cranial lobe. however, by common usage in veterinary pathology, the term cranioventral used to describe the location of lesions in pneumonias has come to mean "cranial and ventral." thus it includes pneumonias affecting not only the ventral portion of the cranial lobe (true cranioventral) but also those cases in which the pneumonia has involved the ventral portions of adjacent lung lobes-initially the middle and then caudal on the right and the caudal lobe on the left side. bronchopneumonias are generally caused by bacteria and mycoplasmas, by bronchoaspiration of feed or gastric contents, or by improper tubing. as a rule, the pathogens causing bronchopneumonias arrive in the lungs via inspired air (aerogenous), either from infected aerosols or from the nasal flora. before establishing infection, pathogens must overwhelm or evade the pulmonary defense mechanisms. the initial injury in bronchopneumonias is centered on the mucosa of bronchioles; from there, the inflammatory process can spread downward to distal portions of the alveoli and upward to the bronchi. typically, for bronchopneumonias, the inflammatory exudates collect in the bronchial, bronchiolar, and alveolar lumina leaving the alveolar interstitium relatively unchanged, except for hyperemia and possibly edema. through the pores of kohn, the exudate can spread to adjacent alveoli until most or all of the alveoli in an individual lobule are involved. if the inflammatory process cannot control the inciting cause of injury, the lesions spread rapidly from lobule to lobule through alveolar pores and destroyed alveolar walls until an entire lobe or large portion of a lung is involved. the lesion tends to spread centrifugally, with the older lesions in the center, and exudate can be coughed up and then aspirated into other lobules, where the inflammatory process starts again. at the early stages of bronchopneumonia, the pulmonary vessels are engorged with blood (active hyperemia), and the bronchi, bronchioles, and alveoli contain some fluid (permeability edema). in cases in which pulmonary injury is mild to moderate, cytokines locally released in the lung cause rapid recruitment of neutrophils and alveolar macrophages into bronchioles and alveoli ( fig. 9-70 and see fig. 9 -69). when pulmonary injury is much more severe, proinflammatory cytokines induce more pronounced vascular changes by further opening endothelial gaps, thus increasing vascular permeability resulting in leakage of plasma fibrinogen (fibrinous exudates) and sometimes hemorrhage in the alveoli. alterations in permeability can be further exacerbated by structural damage to pulmonary capillaries and vessels directly caused by microbial toxins. filling of alveoli, bronchioles, and small bronchi with inflammatory exudate progressively obliterates airspaces, and as a consequence of this process, portions of severely affected (consolidated) lungs sink to the bottom of the container when placed in fixative. the replacement of air by exudate also changes the texture of the lungs, and depending on the severity of bronchopneumonia, the texture varies from firmer to harder than normal. the term consolidation is used at gross examination when the texture of pneumonic lung becomes firmer or harder than normal as a result of loss of airspaces because of exudation and atelectasis. (for details, see the discussion of lung texture in the section on classification of pneumonias in domestic animals). inflammatory consolidation of the lungs has been referred to in the past as hepatization because the affected lung had the appearance and texture of liver. the process was referred to as red hepatization in acute cases in which (1) congestion, (2) red hepatization (liver texture), (3) gray hepatization, and (4) resolution. because of the use of effective antibiotics and prevention, pneumococcal pneumonia and its four classic stages are rarely seen; thus this terminology has been largely abandoned. currently, the term bronchopneumonia is widely used for both suppurative and fibrinous consolidation of the lungs because both forms of inflammation have essentially the same pathogenesis in which the pathogens reach the lung by the aerogenous route, injury occurs initially in the bronchial and bronchiolar regions, and the inflammatory process extends centrifugally deep into the alveoli. it must be emphasized that it is the severity of pulmonary injury that largely determines whether bronchopneumonia becomes suppurative or fibrinous. in some instances, however, it is difficult to discriminate between suppurative and fibrinous bronchopneumonia because both types can coexist (fibrinosuppurative bronchopneumonia), and one type can progress to the other. suppurative bronchopneumonia. suppurative bronchopneumonia is characterized by cranioventral consolidation of lungs (see figs. 9-68 and 9-69), with typically purulent or mucopurulent exudate present in the airways. this exudate can be best demonstrated by expressing intrapulmonary bronchi, thus forcing exudate out of the bronchi (see fig. 9 -69). the inflammatory process in suppurative bronchopneumonia is generally confined to individual lobules, and as a result of this distribution, the lobular pattern of the lung becomes notably emphasized. this pattern is particularly obvious in cattle and pigs because these species have prominent lobulation of the lungs. the gross appearance often resembles an irregular checkerboard because of an admixture of normal and abnormal (consolidated) lobules (see fig. 9 -69). because of this typical lobular distribution, suppurative bronchopneumonias are also referred to as lobular pneumonias. different inflammatory phases occur in suppurative bronchopneumonia where the color and appearance of consolidated lungs varies considerably, depending on the virulence of offending organisms and chronicity of the lesion. the typical phases of suppurative bronchopneumonia can be summarized as follows: 1. during the first 12 hours when bacteria are rapidly multiplying, the lungs become hyperemic and edematous. 2. soon after, neutrophils start filling the airways, and by 48 hours the parenchyma starts to consolidate and becomes firm in texture. 3. three to 5 days later, hyperemic changes are less obvious, but the bronchial, bronchiolar, and alveolar spaces continue to fill with neutrophils and macrophages, and the affected lung sinks when placed in formalin. at this stage, the affected lung has a gray-pink color, and on cut surface, purulent exudate can be expressed from bronchi. 4. in favorable conditions where the infection is under control of the host defense mechanisms, the inflammatory processes begin to regress, a phase known as resolution. complete resolution in favorable conditions could take 1 to 2 weeks. 5. in animals in which the lung infection cannot be rapidly contained, inflammatory lesions can progress into a chronic phase. approximately 7 to 10 days after infection, the lungs become pale gray and take a "fish flesh" appearance. this appearance is the result of purulent and catarrhal inflammation, obstructive atelectasis, mononuclear cell infiltration, peribronchial and peribronchiolar lymphoid hyperplasia, and early alveolar fibrosis. complete resolution is unusual in chronic bronchopneumonia, and lung scars, such as pleural and pulmonary fibrosis; bronchiectasis as a consequence of chronic destructive bronchitis (see bronchiectasis [dysfunction/responses to injury and patterns of injury]); atelectasis; pleural adhesions; and lung abscesses may remain unresolved there was notable active hyperemia with little exudation of neutrophils; conversely, the process was referred to as gray hepatization in those chronic cases in which hyperemia was no longer present, but there was abundant exudation of neutrophils and macrophages. this terminology, although used for and applicable to human pneumonias, is rarely used in veterinary medicine primarily because the evolution of pneumonic processes in animals does not necessarily follow the red-to-gray hepatization pattern. bronchopneumonia can be subdivided into suppurative bronchopneumonia if the exudates are predominantly composed of neutrophils and fibrinous bronchopneumonia if fibrin is the predominant component of the exudates (see table 9 -5). it is important to note that some veterinarians use the term fibrinous pneumonia or lobar pneumonia as a synonym for fibrinous bronchopneumonia, and bronchopneumonia or lobular pneumonia as a synonym for suppurative bronchopneumonia. human pneumonias for many years have been classified based on their etiology and morphology, which explains why pneumococcal pneumonia (streptococcus pneumoniae) has been synonymous with lobar pneumonia. in the old literature, four distinct stages of pneumococcal pneumonia were described as lumen of bronchiole capillary pulmonary defense mechanisms to allow them to colonize the lungs and establish an infection. suppurative bronchopneumonia can also result from aspiration of bland material (e.g., milk). pulmonary gangrene may ensue when the bronchopneumonic lung is invaded by saprophytic bacteria (aspiration pneumonia). fibrinous bronchopneumonia. fibrinous bronchopneumonia is similar to suppurative bronchopneumonia except that the predominant exudate is fibrinous rather than neutrophilic. with only a few exceptions, fibrinous bronchopneumonias also have a cranioventral distribution (fig. 9-72 and see fig. 9-68) . however, exudation is not restricted to the boundaries of individual pulmonary lobules, as is the case in suppurative bronchopneumonias. instead, the inflammatory process in fibrinous pneumonias involves numerous contiguous lobules and the exudate moves quickly through pulmonary tissue until the entire pulmonary lobe is rapidly affected. because of the involvement of the entire lobe and pleural surface, fibrinous bronchopneumonias are also referred to as lobar pneumonias or pleuropneumonias. in general terms, fibrinous bronchopneumonias are the result of more severe pulmonary injury and thus cause death earlier in the sequence of the inflammatory process than suppurative bronchopneumonias. even in cases in which fibrinous bronchopneumonia involves 30% or less of the total area, clinical signs and death can occur as a result of severe toxemia and sepsis. the gross appearance of fibrinous bronchopneumonia depends on the age and severity of the lesion and on whether the pleural surface or the cut surface of the lung is viewed. externally, early stages of fibrinous bronchopneumonias are characterized by severe congestion and hemorrhage, giving the affected lungs a characteristically intense red discoloration. a few hours later, fibrin starts to permeate and accumulate on the pleural surface, giving the pleura a ground glass appearance and eventually forming plaques of fibrinous exudate over a red, dark lung (see fig. 9 -72). at this stage, a yellow fluid starts to accumulate in the thoracic cavity. the color of fibrin deposited over the pleural surface is also variable. it can be bright yellow when the exudate is formed primarily by fibrin, tan when fibrin is mixed with blood, and gray when a large number of leukocytes and fibroblasts are part of the fibrinous plaque in more chronic cases. because of the tendency of fibrin to deposit on the pleural surface, some pathologists use the term pleuropneumonia as a synonym for fibrinous bronchopneumonia. on the cut surface, early stages of fibrinous bronchopneumonia appear as simple red consolidation. in more advanced cases (24 hours), fibrinous bronchopneumonia is generally accompanied by notable dilation and thrombosis of lymph vessels and edema of interlobular septa (see fig. 9-72, b) . this distention of the interlobular septa gives affected lungs a typical marbled appearance. distinct focal areas of coagulative necrosis in the pulmonary parenchyma are also common in fibrinous bronchopneumonia such as in shipping fever pneumonia and contagious bovine pleuropneumonia. in animals that survive the early stage of fibrinous bronchopneumonia, pulmonary necrosis often develops into pulmonary "sequestra," which are isolated pieces of necrotic lung encapsulated by connective tissue. pulmonary sequestra result from extensive necrosis of lung tissue either from severe ischemia (infarct) caused by thrombosis of a major pulmonary vessel such as in contagious bovine pleuropneumonia or from the effect of necrotizing toxins released by pathogenic bacteria such as mannheimia haemolytica. sequestra in veterinary pathology should not be confused with "bronchopulmonary sequestration," a term used in human pathology to describe a congenital malformation in which whole lobes or parts of the lung develop without normal connections to the airway or vascular systems. for a long time. "enzootic pneumonias" of ruminants and pigs are typical examples of chronic suppurative bronchopneumonias. microscopically, acute suppurative bronchopneumonias are characterized by hyperemia, abundant neutrophils, macrophages, and cellular debris within the lumen of bronchi, bronchioles, and alveoli (see fig. 9 -70). recruitment of leukocytes is promoted by cytokines, complement, and other chemotactic factors that are released in response to alveolar injury or by the chemotactic effect of bacterial toxins, particularly endotoxin. in most severe cases, purulent or mucopurulent exudates completely obliterate the entire lumen of bronchi, bronchioles, and alveoli. if suppurative bronchopneumonia is merely the response to a transient pulmonary injury or a mild infection, lesions resolve uneventfully. within 7 to 10 days, cellular exudate can be removed from the lungs via the mucociliary escalator, and complete resolution may take place within 4 weeks. in other cases, if injury or infection is persistent, suppurative bronchopneumonia can become chronic with goblet cell hyperplasia, an important component of the inflammatory process. depending on the proportion of pus and mucus, the exudate in chronic suppurative bronchopneumonia varies from mucopurulent to mucoid. a mucoid exudate is found in the more chronic stages when the consolidated lung has a "fish flesh" appearance. hyperplasia of balt is another change commonly seen in chronic suppurative bronchopneumonias; it appears grossly as conspicuous white nodules (cuffs) around bronchial walls (cuffing pneumonia). this hyperplastic change merely indicates a normal reaction of lymphoid tissue to infection. further sequelae of chronic suppurative bronchopneumonia include bronchiectasis (see figs. 9-10 and 9-11), pulmonary abscesses, pleural adhesions (from pleuritis) ( fig. 9-71) , and atelectasis and emphysema from completely or partially obstructed bronchi or bronchioles (e.g., bronchiectasis). clinically, suppurative bronchopneumonias can be acute and fulminating but are often chronic, depending on the etiologic agent, stressors affecting the host, and immune status. the most common pathogens causing suppurative bronchopneumonia in domestic animals include pasteurella multocida, bordetella bronchiseptica, trueperella (arcanobacterium) pyogenes, streptococcus spp., escherichia coli, and several species of mycoplasmas. most of these organisms are secondary pathogens requiring a preceding impairment of the fulminating hemorrhagic bronchopneumonia can be caused by highly pathogenic bacteria such as bacillus anthracis. although the lesions in anthrax are primarily related to a severe septicemia and sepsis, anthrax should always be suspected in animals with sudden death and exhibiting severe acute fibrinohemorrhagic pneumonia, splenomegaly, and multisystemic hemorrhages. animals are considered good sentinels for anthrax in cases of bioterrorism. interstitial pneumonia refers to that type of pneumonia in which injury and the inflammatory process take place primarily in any microscopically, in the initial stage of fibrinous bronchopneumonia, there is massive exudation of plasma proteins into the bronchioles and alveoli, and as a result, most of the airspaces become obliterated by fluid and fibrin. leakage of fibrin and fluid into alveolar lumina is due to extensive disruption of the integrity and increased permeability of the blood-air barrier. fibrinous exudates can move from alveolus to alveolus through the pores of kohn. because fibrin is chemotactic for neutrophils, these types of leukocytes are always present a few hours after the onset of fibrinous inflammation. as inflammation progresses (3 to 5 days), fluid exudate is gradually replaced by fibrinocellular exudates composed of fibrin, neutrophils, macrophages, and necrotic debris ( fig. 9-73 ). in chronic cases (after 7 days), there is notable fibrosis of the interlobular septa and pleura. in contrast to suppurative bronchopneumonia, fibrinous bronchopneumonia rarely resolves completely, thus leaving noticeable scars in the form of pulmonary fibrosis and pleural adhesions. the most common sequelae found in animals surviving an acute episode of fibrinous bronchopneumonia include alveolar fibrosis and bronchiolitis obliterans, in which organized exudate becomes attached to the bronchiolar lumen (see fig. 9-12) . these changes are collectively referred to as bronchiolitis obliterans organizing pneumonia (boop), a common microscopic finding in animals with unresolved bronchopneumonia. other important sequelae include pulmonary gangrene, when saprophytic bacteria colonize necrotic lung; pulmonary sequestra; pulmonary fibrosis; abscesses; and chronic pleuritis with pleural adhesions. in some cases, pleuritis can be so extensive that fibrous adhesions extend onto the pericardial sac. pathogens causing fibrinous bronchopneumonias in domestic animals include mannheimia (pasteurella) haemolytica (pneumonic mannheimiosis), histophilus somni (formerly haemophilus somnus), actinobacillus pleuropneumoniae (porcine pleuropneumonia), mycoplasma bovis, and mycoplasma mycoides ssp. mycoides small colony type (contagious bovine pleuropneumonia). fibrinous bronchoa b n alveolar epithelium. inhaled antigens, such as fungal spores, combine with circulating antibodies and form deposits of antigen-antibody complexes (type iii hypersensitivity) in the alveolar wall, which initiate a cascade of inflammatory responses and injury (allergic alveolitis). hematogenous injury to the vascular endothelium occurs in septicemias, sepsis, dic, larva migrans (ascaris suum), toxins absorbed in the alimentary tract (endotoxin) or toxic metabolites locally generated in the lungs (3-methylindole and paraquat), release of free radicals in alveolar capillaries (ards), and infections with endotheliotropic viruses (canine adenovirus and classical swine fever [hog cholera]). interstitial pneumonias in domestic animals and human beings are subdivided based on morphologic features into acute and chronic. it should be kept in mind, however, that not all acute interstitial pneumonias are fatal and that they do not necessarily progress to the chronic form. acute interstitial pneumonias. acute interstitial pneumonias begin with injury to either type i pneumonocytes or alveolar capillary endothelium, which provokes a disruption of the blood-air barrier and a subsequent exudation of plasma proteins into the alveolar space (see fig. 9-14) . this leakage of proteinaceous fluid into the alveolar lumen constitutes the exudative phase of acute interstitial pneumonia. in some cases of diffuse alveolar damage, exuded plasma proteins mix with lipids and other components of pulmonary surfactant and form elongated membranes that become partially attached to the alveolar basement membrane and bronchiolar walls. these membranes are referred to as hyaline membranes because of their hyaline appearance (eosinophilic, homogeneous, and amorphous) microscopically (see figs. 9-55 and 9-63). in addition to intraalveolar exudation of fluid, inflammatory edema and neutrophils accumulate in the alveolar interstitium and cause thickening of the alveolar walls. this acute exudative phase is generally followed a few days later by the proliferative phase of acute interstitial pneumonia, which is characterized by hyperplasia of type ii pneumonocytes to replace the lost type i pneumonocytes (see fig. 9 -15). type ii pneumonocytes are in fact progenitor cells that differentiate and replace necrotic type i pneumonocytes (see fig. 9-14) . as a consequence, the alveolar walls become increasingly thickened. this process is in part the reason why lungs become rubbery on palpation, what prevents their normal collapse after the thorax is opened, and why the cut surface of the lung has a "meaty" appearance (see fig. 9 -74). of the three layers of the alveolar walls (endothelium, basement membrane, and alveolar epithelium) and the contiguous bronchiolar interstitium (see fig. 9-7) . this morphologic type of pneumonia is the most difficult to diagnose at necropsy and requires microscopic confirmation because it is easily mistaken in the lung showing congestion, edema, hyperinflation, or emphysema. in contrast to bronchopneumonias, in which distribution of lesions is generally cranioventral, in interstitial pneumonias, lesions are more diffusely distributed and generally involve all pulmonary lobes, or in some cases, they appear to be more pronounced in the dorsocaudal aspects of the lungs (see fig. 9 -68). three important gross features of interstitial pneumonia are (1) the failure of lungs to collapse when the thoracic cavity is opened, (2) the occasional presence of rib impressions on the pleural surface of the lung indicating poor deflation, and (3) the lack of visible exudates in airways unless complicated with secondary bacterial pneumonia. the color of affected lungs varies from diffusely red in acute cases to diffusely pale gray to a mottled red, pale appearance in chronic ones. pale lungs are caused by severe obliteration of alveolar capillaries (reduced blood-tissue ratio), especially evident when there is fibrosis of the alveolar walls. the texture of lungs with uncomplicated interstitial pneumonia is typically elastic or rubbery, but definitive diagnosis based on texture alone is difficult and requires histopathologic examination. on a cut surface, the lungs may appear and feel more "meaty" (having the texture of raw meat) and have no evidence of exudate in the bronchi or pleura (fig. 9-74 ). in acute interstitial pneumonias, particularly in cattle, there is frequently pulmonary edema (exudative phase) and interstitial emphysema secondary to partial obstruction of bronchioles by edema fluid and strenuous air gasping before death. because edema tends to gravitate into the cranioventral portions of the lungs, and emphysema is often more obvious in the dorsocaudal aspects, acute interstitial pneumonias in cattle occasionally have a gross cranioventral-like pattern that may resemble bronchopneumonia, although the texture is different. lungs are notably heavy because of the edema and the infiltrative and proliferative changes. the pathogenesis of interstitial pneumonia is complex and can result from aerogenous injury to the alveolar epithelium (type i and ii pneumonocytes) or from hematogenous injury to the alveolar capillary endothelium or alveolar basement membrane. aerogenous inhalation of toxic gases (i.e., ozone and no 2 ) or toxic fumes (smoke inhalation) and infection with pneumotropic viruses (influenza, herpesviruses, or canine distemper virus) can damage the figure 9 -74 interstitial pneumonia, lung, feeder pig. a, the lung is heavy, pale, and rubbery in texture. it also has prominent costal (rib) imprints (arrows), a result of hypercellularity of the interstitium and the failure of the lungs to collapse when the thorax was opened. b, transverse section. the pulmonary parenchyma has a "meaty" appearance and some edema, but no exudate is present in airways or on the pleural surface. this type of lung change in pigs is highly suggestive of a viral pneumonia. (courtesy dr. a. lópez, atlantic veterinary college.) pneumonia are centered in the alveolar wall and its interstitium, a mixture of desquamated epithelial cells, macrophages, and mononuclear cells are usually present in the lumens of bronchioles and alveoli. ovine progressive pneumonia, hypersensitivity pneumonitis in cattle and dogs, and silicosis in horses are good veterinary examples of chronic interstitial pneumonia. pneumoconioses (silicosis and asbestosis), paraquat toxicity, pneumotoxic antineoplastic drugs (bleomycin), and extrinsic allergic alveolitis (farmer's lung) are well-known examples of diseases that lead to chronic interstitial pneumonias in human beings. massive pulmonary migration of ascaris larvae in pigs also causes interstitial pneumonia ( fig. 9-77 ). there is an insidious and poorly understood group of chronic idiopathic interstitial diseases, both in human beings and in animals, that eventually progress to terminal interstitial fibrosis. these were originally thought to be the result of repeated cycles of alveolar injury, inflammation, and fibroblastic/myoblastic response to an unknown agent. however, aggressive antiinflammatory therapy generally fails to prevent or reduce the severity of fibrosis. now, it is acute interstitial pneumonias are often mild and transient, especially those caused by some respiratory viruses, such as those responsible for equine and porcine influenza. these mild forms of pneumonia are rarely seen in the postmortem room because they are not fatal and do not leave significant sequelae (see the section on defense mechanisms/barrier systems). in severe cases of acute interstitial pneumonias, animals may die of respiratory failure, usually as a result of diffuse alveolar damage, a profuse exudative phase (leakage of proteinaceous fluid) leading to a fatal pulmonary edema. examples of this type of fatal acute interstitial pneumonia are bovine pulmonary edema and emphysema, and ards in all species. chronic interstitial pneumonia. when the source of alveolar injury persists, the exudative and proliferative lesions of acute interstitial pneumonia can progress into a morphologic stage referred to as chronic interstitial pneumonia. the hallmark of chronic interstitial pneumonia is fibrosis of the alveolar walls (with or without intraalveolar fibrosis) and the presence of lymphocytes, macrophages, fibroblasts, and myofibroblasts in the alveolar interstitium (figs. 9-75 and 9-76). in other cases, these chronic changes are accompanied by hyperplasia and persistence of type ii pneumonocytes, squamous metaplasia of the alveolar epithelium, microscopic granulomas, and hyperplasia of smooth muscle in bronchioles and pulmonary arterioles. it should be emphasized that although the lesions in interstitial the term bronchointerstitial pneumonia is used in veterinary pathology to describe cases in which microscopic lesions share some histologic features of both bronchopneumonia and interstitial pneumonia (efig. 9-9 ). this combined type of pneumonia is in fact frequently seen in many viral infections in which viruses replicate and cause necrosis in bronchial, bronchiolar, and alveolar cells. damage to the bronchial and bronchiolar epithelium causes an influx of neutrophils similar to that in bronchopneumonias, and damage to alveolar walls causes proliferation of type ii pneumonocytes, similar to that which takes place in the proliferative phase of acute interstitial pneumonias. it is important to emphasize that bronchointerstitial pneumonia is a microscopic not a gross diagnosis. examples include uncomplicated cases of respiratory syncytial virus infections in cattle and lambs, canine distemper, and influenza in pigs and horses. embolic pneumonia refers to a particular type of pneumonia in which gross and microscopic lesions are multifocally distributed in all pulmonary lobes. by definition, lung injury is hematogenous, and the inflammatory response is typically centered in pulmonary arterioles and alveolar capillaries. lungs act as a biologic filter for circulating particulate matter. sterile thromboemboli, unless extremely large, are rapidly dissolved and removed from the pulmonary vasculature by fibrinolysis, causing little, if any, ill effects. experimental studies have confirmed that most types of bacteria when injected intravenously (bacteremia) are phagocytosed by pulmonary intravascular macrophages, or they bypass the lungs and are finally trapped by macrophages in the liver, spleen, joints, or other organs. to cause pulmonary infection, circulating bacteria must first attach to the pulmonary endothelium with specific binding proteins or simply attach to intravascular fibrin and then evade phagocytosis by intravascular macrophages or leukocytes. septic thrombi facilitate entrapment of bacteria in the pulmonary vessels and provide a favorable environment to escape phagocytosis. once trapped in the pulmonary vasculature, usually in small arterioles or alveolar capillaries, offending bacteria disrupt endothelium and basement membranes, spread from the vessels to the interstitium and then to the surrounding lung, finally forming a new nidus of infection. embolic pneumonia is characterized by multifocal lesions randomly distributed in all pulmonary lobes (see fig. 9 -68 and e-figs. 9-10 and 9-11). early lesions in embolic pneumonia are characterized grossly by the presence of very small (1 to 10 mm), white foci surrounded by discrete, red, hemorrhagic halos ( fig. 9-78 ). unless emboli arrive in massive numbers, causing fatal pulmonary edema, embolic pneumonia is seldom fatal; therefore these acute lesions are rarely seen at postmortem examination. in most instances, if unresolved, acute lesions rapidly progress to pulmonary abscesses. these are randomly distributed in all pulmonary lobes and are not restricted to the cranioventral aspects of the lungs, as is the case of abscesses developing from suppurative bronchopneumonia. the early microscopic lesions in embolic pneumonias are always focal or multifocal ( fig. 9-79) ; thus they differ from those of endotoxemia or septicemia, in which endothelial damage and interstitial reactions (interstitial pneumonia) are diffusely distributed in the lungs. when embolic pneumonia or its sequela (abscesses) is diagnosed at necropsy, an attempt should be made to locate the source of septic emboli. the most common sources are hepatic abscesses that have ruptured into the caudal vena cava in cattle, omphalophlebitis in farm animals, chronic bacterial skin or hoof infections, and a contaminated catheter in all species (see fig. 9-64) . valvular or mural endocarditis in the right heart is a common source of septic emboli and embolic pneumonia in all species. most frequently, bacterial proposed that a genetic mutation alters the cell-cell communication between epithelial and mesenchymal cells in the lung. this aberrant cellular communication leads to an overexpression of inflammatory and repair molecules (i.e., il-4, il-13, tgf-β1, and caveolin), leading to increased apoptosis and interstitial deposition of extracellular matrix (ecm). the chronic interstitial (restrictive) diseases in human medicine include "idiopathic pulmonary fibrosis," "nonspecific interstitial pneumonia," "unusual interstitial pneumonia," and "cryptogenic organizing pneumonia," also referred to as idiopathic bronchiolitis obliterans organizing pneumonia (idiopathic boop). feline idiopathic pulmonary fibrosis is an example of this type of progressive interstitial disease in veterinary medicine. it has been reported that in rare cases, chronic alveolar remodeling and interstitial fibrosis can progress to lung cancer. the lung has numerous circular areas of hemorrhage distributed randomly throughout all lung lobes (embolic pattern [see fig. 9 -68]). these foci arise from injury to the microvasculature in alveolar septa and the visceral pleura secondary to lodgment of bacterial or fungal emboli (septic emboli) from valvular or mural endocarditis in the right heart or from other bacterial or fungal diseases where the bacterium or fungus gains access to the circulatory system as occurs in many bacterial and fungal enteritides or pneumonias caused by salmonella spp., e. coli, or aspergillus spp. the pathogenesis of granulomatous pneumonia shares some similarities with that of interstitial and embolic pneumonias. not surprisingly, some pathologists group granulomatous pneumonias within one of these types of pneumonias (e.g., granulomatous interstitial pneumonia). what makes granulomatous pneumonia a distinctive type is not so much the portal of entry or site of initial injury in the lungs but, rather, the unique type of inflammatory response that results in the formation of granulomas, which can be easily recognized at gross and microscopic examination. as a rule, agents causing granulomatous pneumonias are resistant to intracellular killing by phagocytic cells and to the acute inflammatory response, allowing prolonged persistence of these agents in tissues. the most common causes of granulomatous pneumonia in animals include systemic fungal diseases, such as cryptococcosis (cryptococcus neoformans and cryptococcus gatti), coccidioidomycosis (coccidioides immitis), histoplasmosis (histoplasma capsulatum), and blastomycosis (blastomyces dermatitidis) (see fig. 9 -35). in most of these fungal diseases, the port of entry is aerogenous, and from the lungs the fungi disseminate systemically to other organs, particularly the lymph nodes, liver, and spleen. filamentous fungi such as aspergillus spp. or mucor spp. can also reach the lung by the hematogenous route. granulomatous pneumonia is also seen in some bacterial diseases, such as tuberculosis (mycobacterium bovis) in all species and rhodococcus equi in horses. sporadically, aberrant parasites such as fasciola hepatica in cattle and aspiration of foreign bodies can also cause granulomatous pneumonia (efig. 9-12 granulomatous pneumonia is characterized by the presence of variable numbers of caseous or noncaseous granulomas randomly distributed in the lungs (see fig. 9 -68). on palpation, lungs have a typical nodular character given by well-circumscribed, variably sized nodules that generally have a firm texture, especially if calcification has occurred ( fig. 9-80) . during postmortem examination, granulomas in the lungs occasionally can be mistaken for neoplasms. microscopically, pulmonary granulomas are composed of a center of necrotic tissue, surrounded by a rim of macrophages (epithelioid cells) and giant cells and an outer delineated layer of connective tissue commonly infiltrated by lymphocytes and plasma cells ( fig. 9-81 ). unlike other types of pneumonias, the causative agent in granulomatous pneumonia can, in many cases, be identified isolates from septic pulmonary emboli in domestic animals are trueperella (arcanobacterium) pyogenes (cattle), fusobacterium necrophorum (cattle, pigs, and human beings), erysipelothrix rhusiopathiae (pigs, cattle, dogs, and human beings), streptococcus suis (pigs), staphylococcus aureus (dogs and human beings), and streptococcus equi (horses). granulomatous pneumonia refers to a particular type of pneumonia in which aerogenous or hematogenous injury is caused by organisms or particles that cannot normally be eliminated by phagocytosis and that evoke a local inflammatory reaction with numerous alveolar and interstitial macrophages, lymphocytes, a few neutrophils, and sometimes giant cells. the term granulomatous is used here to describe an anatomic pattern of pneumonia typically characterized by the presence of granulomas. g g but yet unproven that viral infections may also predispose horses to airway hyperresponsiveness and recurrent airway obstruction (rao). equine influenza. equine influenza is an important and highly contagious flulike respiratory disease of horses characterized by high morbidity and low mortality and explosive outbreaks in susceptible populations. it is an oie-notifiable disease. two antigenically unrelated subtypes of equine influenza virus have been identified (h7n7 [a/equi-1] and h3n8 [a/equi-2]). the course of the disease is generally mild and transient, and its importance is primarily because of its economic impact on horse racing. the types of injury and host response in the conducting system are described in the section on disorders of the nasal cavity and paranasal sinuses of horses. uncomplicated lesions in the lungs are mild and self-limiting bronchointerstitial pneumonia. in fatal cases, the lungs are hyperinflated with coalescing areas of dark red discoloration. microscopically, there is a bronchointerstitial pneumonia characterized by necrotizing bronchiolitis that is followed by hyperplastic bronchiolitis, hyperplasia of type ii pneumonocytes, hyaline membranes in alveoli, and sporadic multinucleated giant cells. the microscopic changes are ards in severe and fatal cases. the influenza virus antigen can be readily demonstrated in ciliated cells and alveolar macrophages. clinical signs are characterized by fever, cough, abnormal lung sounds (crackles and wheezes), anorexia, and depression. secondary bacterial infections (streptococcus equi, streptococcus zooepidemicus, staphylococcus aureus, and escherichia coli) commonly complicate equine influenza. equine viral rhinopneumonitis. equine viral rhinopneumonitis (evr), or equine herpesvirus infection, is a respiratory disease of young horses that is particularly important in weanlings between 4 and 8 months of age and to a much lesser extent in young foals and adult horses. the causative agents are ubiquitous equine herpesviruses (ehv-1 and ehv-4) that in addition to respiratory disease can cause abortion in pregnant mares and neurologic disease (equine herpes myeloencephalopathy) (see the section on disorders of the nasal cavity and paranasal sinuses of horses). the respiratory form of evr is a mild and a transient bronchointerstitial pneumonia seen only by pathologists when complications with secondary bacterial infections cause a fatal bronchopneumonia microscopically in sections by pas reaction or grocott-gomori's methenamine silver (gms) stain for fungi or by an acid-fast stain for mycobacteria. viral infections of the respiratory tract, particularly equine viral rhinopneumonitis and equine influenza, are important diseases of horses throughout the world. the effects of these and other respiratory viruses on the horse can be manifested in three distinct ways. first, as pure viral infections, their severity may range from mild to severe, making them a frequent interfering factor in training and athletic performance. second, superimposed infections by opportunistic bacteria, such as streptococcus spp., escherichia coli, klebsiella pneumoniae, rhodococcus equi, and various anaerobes, can cause fibrinous or suppurative bronchopneumonias. third, it is possible equine henipavirus (hendra virus). fatal cases of a novel respiratory disease in horses and human beings suddenly appeared in approximately 1994 in hendra, a suburb of brisbane, australia. this outbreak was attributed to a newly recognized zoonotic virus that was tentatively named equine morbillivirus. now called hendra virus (hev), this emerging viral pathogen is currently classified as a member of the genus henipavirus (includes hendra virus and nipah virus), in the family paramyxoviridae. fruit bats (flying foxes) act as natural reservoirs and are involved in the transmission by poorly understood mechanisms. the lungs of affected horses are severely edematous with gelatinous distention of pleura and subpleural lymph vessels. microscopically, the lungs have diffuse alveolar edema associated with vasculitis, thrombosis, and the presence of multinucleated syncytial cells in the endothelium of small pulmonary blood vessels and alveolar capillaries. the lymphatic vessels are notably distended with fluid. the characteristic inclusion bodies seen in other paramyxovirus infections are not seen in horses; however, the virus can be easily detected by immunohistochemistry in pulmonary endothelial cells and alveolar epithelial cells (pneumonocytes). clinical signs are nonspecific and include fever, anorexia, respiratory distress, and nasal discharge. equine multinodular pulmonary fibrosis. equine multinodular pulmonary fibrosis is a lung disease characterized by well-demarcated fibrotic nodular lesions in the lung (efig. 9-13 ). until recently, the pathogenesis was unclear, but recent studies proposed equine herpesvirus 5 (ehv-5) as the putative etiology. grossly, the lungs show multifocal to coalescing, firm tan nodules scattered in all pulmonary lobes, which resemble pulmonary neoplasia. microscopically, alveolar walls are thickened due to collagen deposition, infiltration of lymphocytes and macrophages, and cuboidal cells lining the alveolar walls. the alveolar lumens contain neutrophils and macrophages, some of which may contain a large eosinophilic intranuclear inclusion body. typical clinical signs include weight loss, low-grade fever, and progressive exercise intolerance. this condition has a poor prognosis. rhodococcus equi. rhodococcus equi is an important cause of morbidity and mortality in foals throughout the world. this facultative intracellular gram-positive bacterium causes two major forms of disease: the first involves the intestine, causing ulcerative enterocolitis, and the second severe and often fatal bronchopneumonia. although half of foals with pneumonia have ulcerative enterocolitis, it is rare to find animals with intestinal lesions alone. occasionally, infection disseminates to lymph nodes, joints, bones, the genital tract, and other organs. because rhodococcus equi is present in soil and feces of herbivores (particularly foals), it is common for the disease to become enzootic on farms ("hot spots") where the organism has been shed earlier by infected foals. serologic evidence of infection in horses is widespread, yet clinical disease is sporadic and largely restricted to young foals or to adult horses with severe immunosuppression. virulence factors encoded by plasmids (virulenceassociated protein a [vapa gene]) are responsible for the survival and replication of rhodococcus equi in macrophages, thus determining the evolution of the disease. this bacterium has also been sporadically incriminated with infections in cattle, goats, pigs, dogs, and cats, and quite often in immunocompromised human beings, for example, those infected with the aids virus, after organ transplantation, or undergoing chemotherapy. it is still debatable whether natural infection starts as a bronchopneumonia (aerogenous route) from which rhodococcus equi reaches the intestine via swallowed sputum or whether infection starts as an enteritis (oral route) with a subsequent bacteremia into the lungs. (streptococcus equi, streptococcus zooepidemicus, or staphylococcus aureus) . uncomplicated lesions in evr are seen only in aborted fetuses or in foals that die within the first few days of life. they consist of focal areas of necrosis (0.5 to 2 mm) in various organs, including liver, adrenal glands, and lungs. in some cases, intranuclear inclusion bodies are microscopically observed in these organs. outbreaks of interstitial pneumonia in donkeys have been attributed to multiple strains of asinine herpesviruses (ahv-4 and -5). clinically, horses and donkeys affected with the respiratory form of evr exhibit fever, anorexia, conjunctivitis, cough, and nasal discharge. equine viral arteritis. equine viral arteritis (eva), a pansystemic disease of horses, donkeys, and mules caused by an arterivirus (equine arteritis virus [eav]), occurs sporadically throughout the world, sometimes as an outbreak. this virus infects and causes severe injury to macrophages and endothelial cells. gross lesions are hemorrhage and edema in many sites, including lungs, intestine, scrotum, and periorbital tissues and voluminous hydrothorax and hydroperitoneum. the basic lesion is fibrinoid necrosis and inflammation of the vessel walls (vasculitis), particularly the small muscular arteries (lymphocytic arteritis), which is responsible for the edema and hemorrhage that explain most of the clinical features. pulmonary lesions are those of interstitial pneumonia with hyperplasia of type ii pneumonocytes and vasculitis with abundant edema in the bronchoalveolar spaces and distended pulmonary lymphatic vessels. viral antigen can be detected by immunoperoxidase techniques in the walls and endothelial cells of affected pulmonary vessels and in alveolar macrophages. clinical signs are respiratory distress, fever, abortion, diarrhea, colic, and edema of the limbs and ventral abdomen. respiratory signs are frequent and consist of serous or mucopurulent rhinitis and conjunctivitis with palpebral edema. like most viral respiratory infections, eva can predispose horses to opportunistic bacterial pneumonias. african horse sickness. african horse sickness (ahs) is an arthropod-borne, oie-notifiable disease of horses, mules, donkeys, and zebras that is caused by an orbivirus (family reoviridae) and characterized by respiratory distress or cardiovascular failure. ahs has a high mortality rate-up to 95% in the native population of horses in africa, the middle east, india, pakistan, and, most recently, spain and portugal. although the ahs virus is transmitted primarily by insects (culicoides) to horses, other animals, such as dogs, can be infected by eating infected equine flesh. the pathogenesis of african horse sickness remains unclear, but this equine orbivirus has an obvious tropism for pulmonary and cardiac endothelial cells and, to a lesser extent, mononuclear cells. based on clinical signs (not pathogenesis), african horse sickness is arbitrarily divided into four different forms: pulmonary, cardiac, mixed, and mild. the pulmonary form is characterized by severe respiratory distress and rapid death because of massive pulmonary edema, presumably from viral injury to the pulmonary endothelial cells. grossly, large amounts of froth are present in the airways, lungs fail to collapse, subpleural lymph vessels are distended, and the ventral parts of the lungs are notably edematous (see fig. 4-40) . in the cardiac form, recurrent fever is detected, and heart failure results in subcutaneous and interfascial edema, most notably in the neck and supraorbital region. the mixed form is a combination of the respiratory and cardiac forms. finally, the mild form, rarely seen in postmortem rooms, is characterized by fever and clinical signs resembling those of equine influenza; it is in most cases transient and followed by a complete recovery. this mild form is most frequently seen in donkeys, mules, and zebras and in horses with some degree of immunity. detection of viral antigen for diagnostic purposes can be done by immunohistochemistry in paraffin-embedded tissues. chapter 9 respiratory system, mediastinum, and pleurae clinically, rhodococcus equi infection can be acute, with rapid death caused by severe bronchopneumonia, or chronic, with depression, cough, weight loss, and respiratory distress. in either form, there may be diarrhea, arthritis, osteomyelitis, or subcutaneous abscess formation. parascaris equorum. parascaris equorum is a large nematode (roundworm) of the small intestine of horses; the larval stages migrate through the lungs as ascarid larvae do in pigs. it is still unclear whether migration of parascaris equorum larvae can cause significant pulmonary lesions under natural conditions. experimentally, migration of larvae results in coughing, anorexia, weight loss, and small necrotic foci and petechial hemorrhages in the liver, hepatic and tracheobronchial lymph nodes, and lungs. microscopically, eosinophils are prominent in the interstitium and airway mucosa during the parasitic migration and in focal granulomas caused by dead larvae in the lung. dictyocaulus arnfieldi. dictyocaulus arnfieldi is not a very pathogenic nematode, but it should be considered if there are signs of coughing in horses that are pastured together with donkeys. donkeys are considered the natural hosts and can tolerate large numbers of parasites without ill effects. dictyocaulus arnfieldi does not usually become patent in horses, so examination of fecal samples is not useful; bal is only occasionally diagnostic because eosinophils (but not parasites) are typically found in the lavage fluid. mature parasites (up to 8 cm in length) cause obstructive bronchitis, edema, and atelectasis, particularly along the dorsocaudal lung. the microscopic lesion is an eosinophilic bronchitis similar to the less acute infestations seen in cattle and sheep with their dictyocaulus species. the results of experimental studies suggest that natural infection likely starts from inhalation of infected dust or aerosols. once in the lung, rhodococcus equi is rapidly phagocytosed by alveolar macrophages, but because of defective phagosome-lysosome fusion and premature lysosomal degranulation, bacteria survive and multiply intracellularly, eventually leading to the destruction of the macrophage. interestingly, rhodococcus equi appears to be easily killed by neutrophils but not macrophages. released cytokines and lysosomal enzymes and bacterial toxins are responsible for extensive caseous necrosis of the lungs and the recruitment of large numbers of neutrophils, macrophages, and giant cells containing intracellular gram-positive organisms in their cytoplasm. depending on the stage of infection and the immune status and age of affected horses, pulmonary lesions induced by rhodococcus equi can vary from pyogranulomatous to granulomatous pneumonia. in young foals, the infection starts as a suppurative cranioventral bronchopneumonia, which progresses within a few days into small variable-size pulmonary abscesses. these abscesses rapidly transform into pyogranulomatous nodules, some of which become confluent and form large masses of caseous exudate ( fig. 9-82 ). microscopically, the early lesion starts with neutrophilic infiltration, followed by an intense influx of alveolar macrophages into the bronchoalveolar spaces. this type of histiocytic inflammation persists for a long period of time because rhodococcus equi is a facultative intracellular organism that survives the bactericidal effects of equine alveolar macrophages. in the most chronic cases, the pulmonary lesions culminate with the formation of large caseonecrotic masses with extensive fibrosis of the surrounding pulmonary parenchyma. pcr analysis of tracheobronchial aspirates has successfully been used as an alternative to bacteriologic culture in the diagnosis of rhodococcus equi infection in live foals. b a rhinotracheitis (ibr)/bovine herpes virus 1 (bohv-1), bovine parainfluenza virus 3 (bpiv-3), and bovine respiratory syncytial virus (brsv); and noninfectious interstitial pneumonias, such as bovine pulmonary edema and emphysema, reinfection syndrome, and many others. bovine enzootic pneumonia. enzootic pneumonia, sometimes simply referred to as calf pneumonia, is a multifactorial disease caused by a variety of etiologic agents that produces an assortment of lung lesions in young, intensively housed calves. the hostmicrobial-environmental triad is central in the pathogenesis of this disease. morbidity is often high (up to 90%), but fatalities are uncommon (>5%) unless management is poor or unless new, virulent pathogens are introduced by additions to the herd. enzootic pneumonia is also called viral pneumonia because it often begins with an acute respiratory infection with bpiv-3, brsv, or possibly with one or more of several other viruses (adenovirus, bohv-1, reovirus, bovine coronavirus [bcov] , and bovine rhinitis virus). mycoplasmas, notably mycoplasma dispar, mycoplasma bovis, ureaplasma, and possibly chlamydophila, may also be primary agents. following infection with any of these agents, opportunistic bacteria, such as pasteurella multocida, trueperella (arcanobacterium) pyogenes, histophilus somni, mannheimia haemolytica, and escherichia coli, can cause a secondary suppurative bronchopneumonia, the most serious stage of enzootic pneumonia. the pathogenesis of the primary invasion and how it predisposes the host to invasion by the opportunists are poorly understood, but it is likely that there is impairment of pulmonary defense mechanisms. environmental factors, including air quality (poor ventilation), high relative humidity, and animal crowding, have been strongly incriminated. the immune status of the calf also plays an important role in the development and severity of enzootic pneumonia. calves with bovine leukocyte adhesion deficiency (blad), which prevents the migration of neutrophils from the capillaries, are highly susceptible to bronchopneumonia. lesions are variable and depend largely on the agents involved and on the duration of the inflammatory process. in the acute phases, lesions caused by viruses are those of bronchointerstitial pneumonia, which are generally mild and transient, and therefore are seen only sporadically at necropsy. microscopically, the lesions are necrotizing bronchiolitis, necrosis of type i pneumonocytes with hyperplasia of type ii pneumonocytes, and mild interstitial and alveolar edema. in the case of bpiv-3 and brsv infection, intracytoplasmic inclusion bodies and the formation of large multinucleated syncytia, resulting from the fusion of infected bronchiolar and alveolar epithelial cells, can also be observed in the lungs (fig. 9-83) . airway hyperreactivity has been described in calves after brsv infection; however, the significance of this syndrome in relation to enzootic pneumonia of calves is still under investigation. the mycoplasmas also can cause bronchiolitis, bronchiolar and alveolar necrosis, and an interstitial reaction, but in contrast to viral-induced pneumonias, mycoplasmal lesions tend to progress to a chronic stage characterized by striking peribronchiolar lymphoid hyperplasia (cuffing pneumonia). when complicated by secondary bacterial infections (e.g., pasteurella multocida and trueperella pyogenes), viral or mycoplasmal lesions change from a pure bronchointerstitial to a suppurative bronchopneumonia (fig. 9-84) . in late stages of bronchopneumonia, the lungs contain a creamy-mucoid exudate in the airways and later often have pulmonary abscesses and bronchiectasis (see fig. 9-11) . note that the same viruses and mycoplasmas involved in the enzootic pneumonia complex can also predispose cattle to other diseases, such as pneumonic mannheimiosis (mannheimia aspiration pneumonia. aspiration pneumonia is often a devastating sequela to improper gastric tubing of horses, particularly exogenous lipid pneumonia from mineral oil delivered into the trachea in treatment of colic. gross and microscopic lesions are described in detail in the section on aspiration pneumonias of cattle. opportunistic infections. chlamydophila (chlamydia) spp., obligatory intracellular zoonotic pathogens, can cause systemic infection in many mammalian and avian species; in horses, they can also cause keratoconjunctivitis, rhinitis, pneumonia, abortion, polyarthritis, enteritis, hepatitis, and encephalitis. serologic studies suggest that infection without apparent disease is common in horses. horses experimentally infected with chlamydophila psittaci develop mild and transient bronchointerstitial pneumonia. there are unconfirmed reports suggesting a possible association between these organisms and recurrent airway obstruction in horses. detection of chlamydial organisms in affected tissue is not easy and requires special laboratory techniques such as pcr, immunohistochemistry, and fluorescent antibody tests. horses are only sporadically affected with mycobacteriosis (mycobacterium avium complex, mycobacterium tuberculosis, and mycobacterium bovis). the intestinal tract and associated lymph nodes are generally affected, suggesting an oral route of infection with subsequent hematogenous dissemination to the lungs. the tubercles (granulomas) differ from those in ruminants and pigs, being smooth, gray, solid, sarcoma-like nodules without grossly visible caseous necrosis or calcification (efig. 9-14) . microscopically, the tubercles are composed of macrophages, epithelioid cells, and multinucleated giant cells. fibrosis increases with time, accounting in part for the sarcomatous appearance. adenovirus infections occur commonly in arabian foals with combined immunodeficiency (cid), a hereditary lack of b and t lymphocytes. in cases of adenoviral infection, large basophilic or amphophilic inclusions are present in the nuclei of tracheal, bronchial, bronchiolar, alveolar, renal, and intestinal epithelial cells. as it occurs in other species, infection with a unique fungal pathogen known as pneumocystis carinii typically occurs in immunosuppressed or immunoincompetent individuals such as arabian foals with cid (see fig. 9 -20). diagnosis of pneumocystis carinii requires microscopic examination of lungs and special stains. idiopathic interstitial pneumonia. interstitial and bronchointerstitial pneumonias of undetermined cause that can progress to severe pulmonary fibrosis have been reported in foals and young horses. the gross and microscopic lesions are reminiscent of those of bovine pulmonary edema and emphysema or ards. the lungs are notably congested and edematous and microscopically are characterized by necrosis of the bronchiolar epithelium, alveolar edema, hyperplasia of type ii pneumonocytes, and hyaline membranes. the cause of this form of equine interstitial pneumonia is not known, but toxic and particularly viral causes have been proposed. bovine respiratory disease complex (brdc) and acute undifferentiated respiratory disease are general terms often used by clinicians to describe acute and severe bovine respiratory illness of clinically undetermined cause. these terms do not imply any particular type of pneumonia and therefore should not be used in pathology reports. clinically, the brd complex includes bovine enzootic pneumonia (multifactorial etiology); pneumonic mannheimiosis (mannheimia haemolytica); respiratory histophilosis (histophilus somni), previously known as respiratory hemophilosis (haemophilus somnus); mycoplasma bovis; respiratory viral infections, such as infectious bovine 528.e1 chapter 9 respiratory system, mediastinum, and pleurae pneumonic mannheimiosis (shipping fever) is the most important respiratory disease of cattle in north america, particularly in feedlot animals that have been through the stressful marketing and assembly processes. mannheimia haemolytica biotype a, serotype 1 is the etiologic agent most commonly responsible for the severe pulmonary lesions. a few investigators still consider that pasteurella multocida and other serotypes of mannheimia haemolytica are also causes of this disease. even after many years of intense investigation, from the gross lesions to the molecular aspects of the disease, the pathogenesis of pneumonic mannheimiosis remains incompletely understood. experiments have established that mannheimia haemolytica a1 alone is usually incapable of causing disease because it is rapidly cleared by pulmonary defense mechanisms. these findings may explain why mannheimia haemolytica, despite being present in the nasal cavity of healthy animals, only sporadically causes disease. for mannheimia haemolytica to be established as a pulmonary infection, it is first required that stressors impair the defense mechanisms and allow the bacteria to colonize the lung (see section on impairment of defense haemolytica). clinically, enzootic pneumonia is usually mild, but fatal cases are occasionally seen even in farms with optimal health management. pneumonic mannheimiosis (shipping fever). shipping fever (transit fever) is a vague clinical term used to denote acute respiratory diseases that occur in cattle several days or weeks after shipment. the disease is characterized by a severe fibrinous bronchopneumonia, reflecting the fact that death generally occurs early or at an acute stage. because mannheimia haemolytica (formerly pasteurella haemolytica) is most frequently isolated from affected lungs, the names pneumonic mannheimiosis and pneumonic pasteurellosis have been used synonymously. it is known that pneumonic mannheimiosis can occur in animals that have not been shipped and that organisms other than mannheimia haemolytica can cause similar lesions. therefore the term shipping fever should be relinquished in favor of more specific names, such as pneumonic mannheimiosis or respiratory histophilosis. irregular areas of coagulative necrosis are typically bordered by a rim of elongated cells often referred to as oat-shaped cells or oat cells that are degenerating neutrophils mixed with a few alveolar macrophages (see fig. 9 -86). in the early stages of necrosis, there is no evidence of vascular thrombosis, suggesting that necrosis is primarily caused by the cytotoxin of mannheimia haemolytica and is not the result of an ischemic change. the interlobular septa become distended with protein-rich edematous fluid, and the lymphatic vessels contain fibrin thrombi. the trachea and bronchi can have considerable amounts of blood and exudate, which are transported by the mucociliary escalator or coughed up from deep within the lungs, but the walls of the trachea and major bronchi may or may not be involved. because of the necrotizing process, sequelae to pneumonic mannheimiosis can be serious and can include abscesses, encapsulated sequestra (isolated pieces of necrotic lung), chronic pleuritis, fibrous pleural adhesions, and bronchiectasis. clinically, pneumonic mannheimiosis is characterized by a severe toxemia that can kill animals even when considerable parts of the lungs remain functionally and structurally normal. cattle usually become depressed, febrile (104° to 106° f [40° to 41° c]), and anorexic and have a productive cough, encrusted nose, mucopurulent nasal exudate, shallow respiration, or an expiratory grunt. hemorrhagic septicemia. pneumonic mannheimiosis should not be confused with hemorrhagic septicemia (septicemic pasteurellosis) of cattle and water buffalo (bubalus bubalis) caused by inhalation or ingestion of serotypes 6:b and 6:e of pasteurella multocida. this oie-notifiable disease does not occur in north america and currently is reported only from some countries in asia, africa, and recently in germany. in contrast to pneumonic mannheimiosis, in which lesions are always confined to the lower respiratory tract, the bacteria of hemorrhagic septicemia always disseminates hematogenously to other organs. at necropsy, typically, generalized petechiae are present on the serosal surfaces of the intestine, heart, and lungs and in skeletal muscles. superficial and visceral lymph nodes are swollen and hemorrhagic. variable lesions include edematous and hemorrhagic lungs with or without consolidation; hemorrhagic enteritis; blood-tinged fluid in the thorax and abdomen; and subcutaneous edema of the head, neck, and ventral abdomen. bacteria can be cultured from blood, and animals have high fever and die rapidly (100% case fatality). respiratory histophilosis (haemophilosis). respiratory histophilosis is part of the histophilus somni (haemophilus somnus) disease complex, which has at least eight different clinicopathologic forms, each one involving different organs. this complex includes septicemia, encephalitis (known as thrombotic meningoencephalitis [tme]), pneumonia (respiratory histophilosis), pleuritis, myocarditis, arthritis, ophthalmitis, conjunctivitis, otitis, and abortion. the portals of entry for the different forms of histophilosis have not been properly established. the respiratory form of bovine histophilosis is the result of the capacity of the bacterium to induce both suppurative and fibrinous bronchopneumonia (efig. 9-15 ). the latter is in some cases indistinguishable from that of pneumonic mannheimiosis. the pathogenesis of respiratory histophilosis is still poorly understood, and the disease cannot be reproduced consistently by administration of histophilus somni alone. like mannheimia haemolytica, it requires predisposing factors such as stress or a preceding viral infection. histophilus somni is often isolated from the lungs of calves with enzootic pneumonia. the capacity of histophilus somni to cause septicemia and localized infections in the lungs, brain, eyes, ear, heart, mammary gland, male and female genital organs, or placenta is perhaps attributable to specific virulence factors, such as immunoglobulin-binding proteins (igbps) and lipooligosaccharide (los). also, histophilus mechanisms). these stressors include weaning, transport, fatigue, crowding, mixing of cattle from various sources, inclement weather, temporary starvation, and viral infections. horizontal transmission of viruses and mannheimia haemolytica occurs during crowding and transportation of cattle. viruses that most commonly predispose cattle to pneumonic mannheimiosis include bohv-1, bpiv-3, and brsv. once established in the lungs, mannheimia haemolytica causes lesions by means of different virulence factors, which include endotoxin, lipopolysaccharide, adhesins, and outer membrane proteins; however, the most important is probably the production of a leukotoxin (exotoxin), which binds and kills bovine macrophages and neutrophils. the fact that this toxin exclusively affects ruminant leukocytes probably explains why mannheimia haemolytica is a respiratory pathogen in cattle and sheep but not in other species. during mannheimia haemolytica infection, alveolar macrophages, neutrophils, and mast cells release maximum amounts of proinflammatory cytokines, particularly tnf-α, il-1, il-8, adhesion molecules, histamine, and leukotrienes. by locally releasing enzymes and free radicals, leukocytes further contribute to the injury and necrosis of bronchiolar and alveolar cells. the gross lesions of acute and subacute pneumonic mannheimiosis are the prototypic fibrinous bronchopneumonia, with prominent fibrinous pleuritis ( fig. 9-85 and see fig. 9 -72) and pleural effusion. lesions are always cranioventral and usually ventral to a horizontal line through the tracheal bifurcation. the interlobular septa are distended by yellow, gelatinous edema and fibrin. the "marbling" of lobules is the result of intermixing areas of coagulation necrosis, interlobular interstitial edema, and congestion ( fig. 9-86) . microscopically, lung lesions are evident 4 hours after experimental infection in which neutrophils fill the bronchial, bronchiolar, and alveolar spaces. within 24 to 48 hours, the cytotoxic effect of mannheimia haemolytica is manifested by necrosis of individual alveolar cells and fibrin begins to exude into the alveoli from increased permeability of the air-blood barrier. these changes are exacerbated by endothelial swelling, altered platelet function, increased procoagulant activity, and diminished profibrinolytic activity in the lungs. by 72 hours, alveolar macrophages start to appear in the bronchoalveolar space. at this time, large and the pulmonary defense mechanisms. lung lesions are typically those of a chronic bronchopneumonia with numerous well-delineated caseonecrotic nodules (fig. 9-87 and e-fig. 9-16) . microscopically, lesions are quite characteristic and consist of distinct areas of pulmonary necrosis centered on bronchi or bronchioles. the lesion is formed by a core of fine eosinophilic granular debris surrounded by a rim of neutrophils, macrophages, and fibroblasts (see fig. 9-87) . although the origin of the caseonecrotic lesions is under investigation, recent studies incriminate reactive oxygen species (ros) and reactive nitrogen species (rns) as the major contributors for cell injury in the lung. the diagnosis is confirmed by isolation or somni has the ability to undergo structural and antigenic variation, evade phagocytosis by promoting leukocytic apoptosis, inhibit intracellular killing, reduce transferrin concentrations, and induce endothelial apoptosis in the lungs of affected calves. mixed pulmonary infections of histophilus somni, mannheimia haemolytica, pasteurella multocida, trueperella pyogenes, and mycoplasmas are fairly common in calves. mycoplasma bovis pneumonia. mycoplasma bovis is the most common mycoplasma sp. isolated from pneumonic lungs of cattle in europe and north america. pulmonary infection is exacerbated by stress or any other adverse factor (e.g., viral infection) that depresses n control programs for infectious disease. it was eradicated from north america in 1892 and from australia in the 1970s, but it is still enzootic in large areas of africa, asia, and eastern europe. the etiologic agent, mycoplasma mycoides ssp. mycoides small colony type, was the first mycoplasma isolated and is one of the most pathogenic of those that infect domestic animals. natural infection occurs in cattle and asian buffalo. the portal of entry is aerogenous, and infections occur when a susceptible animal inhales infected droplets. the pathogenic mechanisms are still inadequately understood but are suspected to involve toxin and galactan production, unregulated production of tnf-α, ciliary dysfunction, immunosuppression, and immune-mediated vasculitis. vasculitis and thrombosis of pulmonary arteries, arterioles, veins, and lymphatic vessels lead to lobular infarction. the name of the disease is a good indication of the gross lesions. it is a severe, fibrinous bronchopneumonia (pleuropneumonia) similar to that of pneumonic mannheimiosis (see figs. 9-72 and 9-85) but having a more pronounced "marbling" of the lobules because of extensive interlobular edema and lymphatic thrombosis. typically, 60% to 79% of lesions are in the caudal lobes (not cranioventrally), and pulmonary sequestra (necrotic lung encapsulated by connective tissue) are more frequent and larger than pneumonic mannheimiosis. unilateral lesions are common in this disease. microscopically, the appearance again is like that of pneumonic mannheimiosis, except that vasculitis and thrombosis of pulmonary arteries, arterioles, and capillaries are much more obvious and are clearly the major cause of the infarction and thrombosis of lymphatic vessels in interlobular septa. mycoplasma mycoides ssp. mycoides small colony type remains viable in the sequestra for many years, and under stress (e.g., starvation), the fibrous capsule may break down releasing mycoplasma into the airways, thus becoming a source of infection for other animals. clinical signs are those of severe sepsis, including fever, depression, and anorexia followed by severe respiratory signs such as opened-mouth breathing, dyspnea and coughing, and crepitation and pleural friction on thoracic auscultation. vaccination is highly effective in preventing the disease. bovine tuberculosis. tuberculosis is an ancient, communicable, worldwide, chronic disease of human beings and domestic animals. it continues to be a major problem in human beings in underdeveloped countries, and it is on the rise in some industrialized nations, largely because of the immunosuppressive effects of aids, immigration, and movement of infected animals across borders. the world health organization (who) estimates that more than 1 million people die of tuberculosis and 8 million new cases appear each year, mostly in developing countries. mycobacterium tuberculosis is transmitted between human beings, but where unpasteurized milk is consumed, mycobacterium bovis from the milk of cattle with mammary tuberculosis is also an important cause of human tuberculosis. mycobacterium bovis infections have also been reported in a number of domestic and wild mammalian species; in some countries, wildlife reservoirs exist and may act as a source of infection for cattle. bovine tuberculosis is primarily caused by mycobacterium bovis, but infection with mycobacterium tuberculosis, the pathogen of human tuberculosis, and mycobacterium caprae (formerly mycobacterium bovis ssp. caprae/mycobacterium tuberculosis ssp. caprae) can occur sporadically. tuberculosis can be acquired by several routes, but infection of the lungs by inhalation of mycobacterium bovis is the most common in adult cattle, whereas ingestion of infected milk is more predominant in young animals. organisms belonging to the mycobacterium avium complex can also infect cattle, but for infection caused by these organisms, the term atypical mycobacteriosis (not tuberculosis) is currently preferred. immunohistochemical labeling of tissue sections for mycoplasma antigens. mycoplasma bovis is also incriminated in arthritis, otitis, mastitis, abortion, and keratoconjunctivitis. contagious bovine pleuropneumonia. contagious bovine pleuropneumonia is an oie-notifiable disease of historic interest in veterinary medicine because it was the object of early national a b c than 90% of bovine cases, a chronic, moist cough can progress to dyspnea. enlarged tracheobronchial lymph nodes can contribute to the dyspnea by impinging on airways, and the enlargement of caudal mediastinal nodes can compress the caudal thoracic esophagus and cause bloating. interstitial pneumonias. atypical interstitial pneumonia (aip) is a vague clinical term well entrenched in veterinary literature but one that has led to enormous confusion among veterinarians. it was first used to describe acute or chronic forms of bovine pneumonia that did not fit in any of the "classic" forms because of the lack of exudate and lack of productive cough. microscopically, the criteria for diagnosis of aip in cattle were based on the absence of obvious exudate and the presence of edema, interstitial emphysema (see the section on pulmonary emphysema), hyaline membranes, hyperplasia of type ii pneumonocytes, and alveolar fibrosis with interstitial cellular infiltrates. at that time, any pulmonary disease or pulmonary syndrome that had a few of the previously mentioned lesions was traditionally diagnosed as aip, and grouping all these different syndromes together was inconsequential because their etiopathogenesis were then unknown. field and laboratory investigations have demonstrated that most of the bovine syndromes previously grouped under aip have rather different causes and pathogeneses ( fig. 9-88) . furthermore, what was "atypical" in the past has become so common that it is fairly routine nowadays to find "typical cases" of aip. for all these reasons, investigators, largely from britain, proposed that all these syndromes previously clustered into aip should be named according to their specific cause or pathogenesis. the most common bovine syndromes characterized by edema, emphysema, hyaline membranes, and hyperplasia of type ii pneumonocytes include bovine pulmonary edema and emphysema (fog fever), "extrinsic allergic alveolitis" (hypersensitivity pneumonitis), "reinfection syndromes" (hypersensitivity to dictyocaulus sp. or brsv), milk allergy, ingestion of moldy potatoes, paraquat toxicity, toxic silo gases, mycotoxins, and others. acute bovine pulmonary edema and emphysema (fog fever). acute bovine pulmonary edema and emphysema (abpee), known in britain as fog fever (no association with atmospheric conditions), occurs in cattle usually grazing "fog" pastures (i.e., aftermath or foggage, regrowth after a hay or silage has been cut). epidemiologically, abpee usually occurs in adult beef cattle in the fall when there is a change in pasture from a short, dry grass to a lush, green grass. it is generally accepted that l-tryptophan present in the pasture is metabolized in the rumen to 3-methylindole, which in turn is absorbed into the bloodstream and carried to the lungs. mixed function oxidases present in the nonciliated bronchiolar epithelial (club) cells metabolize 3-methylindole into a highly pneumotoxic compound that causes extensive and selective necrosis of bronchiolar cells and type i pneumonocytes (fig. 9-89 and see fig. 9 -88) and increases alveolar permeability, leading to edema, thickening of the alveolar interstitium, and alveolar and interstitial emphysema. 3-methylindole also interferes with the lipid metabolism of type ii pneumonocytes. the gross lesions are those of a diffuse interstitial pneumonia with severe alveolar and interstitial edema and interlobular emphysema (see fig. 9-55, a) . the lungs are expanded, pale, and rubbery in texture, and the lesions are most notable in the caudal lobes. microscopically, the lesions are alveolar and interstitial edema and emphysema, formation of characteristic hyaline membranes within alveoli (see fig. 9-55, b) , and in those animals that survive for several days, hyperplasia of type ii pneumonocytes and alveolar interstitial fibrosis. respiratory infection usually starts when inhaled bacilli reach the alveoli and are phagocytosed by pulmonary alveolar macrophages. if these cells are successful in destroying the bacteria, infection is averted. however, mycobacterium bovis, being a facultative pathogen of the monocytic-macrophage system, may multiply intracellularly, kill the macrophage, and initiate infection. from this first nidus of infection, bacilli spread aerogenously via airways within the lungs and eventually via the lymph vessels to tracheobronchial and mediastinal lymph nodes. the initial focus of infection at the portal of entry (lungs) plus the involvement of regional lymph nodes is termed the primary (ghon) complex of tuberculosis. if the infection is not contained within this primary complex, bacilli disseminate via the lymph vessels to distant organs and other lymph nodes by the migration of infected macrophages. hematogenous dissemination occurs sporadically when a granuloma containing mycobacteria erodes the wall of a blood vessel, causes vasculitis, and allows the granuloma to discharge mycobacteria into the alveolar circulation. if dissemination is sudden and massive, mycobacteria are widely disseminated and numerous small foci of infection develop in many tissues and organs and the process is referred to as miliary tuberculosis (like millet seeds). the host becomes hypersensitive to the mycobacterium, which enhances the cell-mediated immune defenses in early or mild infections but can result in host-tissue destruction in the form of caseous necrosis. the evolution and dissemination of the pulmonary infection are closely regulated by cytokines and tnf-α production by alveolar macrophages. unlike abscesses that tend to grow rather fast, granulomas evolve slowly at the site of infection. the lesion starts with few macrophages and neutrophils ingesting the offending organism, but because mycobacterium organisms are resistant to phagocytosis, infected macrophages eventually die, releasing viable bacteria, lipids, and cell debris. cell debris accumulates in the center of the lesion, whereas viable bacteria and bacterial lipids attract additional macrophages and a few lymphocytes at the periphery of the lesion. some of these newly recruited macrophages are activated by local lymphocytes and become large phagocytic cells with abundant cytoplasm resembling epithelial cells, thus the term epithelioid macrophages. multinucleated giant cells (also macrophages) appear at the edges of the lesion, and finally the entire focus of inflammatory process becomes surrounded by fibroblasts and connective tissue (see fig. 9 -81). it may take weeks or months for a granuloma to be grossly visible. bovine tuberculosis, the prototype for granulomatous pneumonia, is characterized by the presence of a few or many caseated granulomas (see fig. 9 -80). the early gross changes are small foci (tubercles) most frequently seen in the dorsocaudal, subpleural areas. with progression, the lesions enlarge and become confluent with the formation of large areas of caseous necrosis. calcification of the granulomas is a typical finding in bovine tuberculosis. single nodules or clusters occur on the pleura and peritoneum, and this presentation has been termed pearl disease. microscopically, the tubercle is composed of mononuclear cells of various types. in young tubercles, which are noncaseous, epithelioid and langhans' giant cells are at the center, surrounded by lymphocytes, plasma cells, and macrophages. later, caseous necrosis develops at the center, secondary to the effects of cell-mediated hypersensitivity and enclosed by fibrosis at the periphery. acid-fast organisms may be numerous but more often are difficult to find in histologic section or smears. clinically, the signs of tuberculosis relate to the dysfunction of a particular organ system or to general debilitation, reduced milk production, and emaciation. in the pulmonary form, which is more grossly, the postmortem lesions vary from subtle, gray, subpleural foci (granulomatous inflammation) to severe lesions, in which the lungs are firm and heavy and have a "meaty appearance" because of interstitial pneumonia (efig. 9 -17) with type ii pneumonocyte hyperplasia, lymphocytic infiltration, and interstitial fibrosis. characteristically, discrete noncaseous granulomas formed in response to the deposition of antigen-antibody complexes are scattered throughout the lungs. chronic cases of extrinsic allergic alveolitis can eventually progress to diffuse fibrosing alveolitis. clinically, it can be acute or chronic; the latter has a cyclical pattern of exacerbation during winter months. weight loss, coughing, and poor exercise tolerance are clinical features. full recovery can occur if the disease is recognized and treated early. reinfection syndrome. hypersensitivity to reinfection with larvae of dictyocaulus viviparus is another allergic syndrome manifested in the lungs that causes signs and lesions indistinguishable from abpee, with the exception of eosinophils and possibly larvae in the alveolar exudate. the hypersensitivity reaction in the lung causes diffuse alveolar damage and edema, necrosis of type i pneumonocytes, and hyperplasia of type ii pneumonocytes. in the later stages of the disease, there is formation of small granulomas with interstitial infiltrates of mononuclear cells. it has been suggested but not confirmed that emphysema with diffuse proliferative alveolitis and formation of hyaline membranes can also occur sporadically in the late stages of brsv infection in cattle. presumably, this disease shares many similarities with "atypical" infections occasionally seen in children with respiratory syncytial virus (rsv human strain), in which a hypersensitivity to the virus or virus-induced augmentation of the immune response results in hypersensitivity pneumonitis (see fig. 9 -88). brsv infection is also known to enhance hypersensitivity to environmental allergens in cattle. other forms of bovine interstitial pneumonia. inhalation of manure ("pit") gases, such as nitrogen dioxide (no 2 ), hydrogen interstitial cell infiltrates, fibrosis, emphysema acute proliferation phase hyperplasia of type ii pneumonocytes clinically, severe respiratory distress develops within 10 days of the abrupt pasture change, and cattle develop expiratory dyspnea, oral breathing, and evidence of emphysema within the lungs and even subcutaneously along the back. experimentally, reducing ruminal conversion of l-tryptophan to 3-methylindole prevents the development of abpee. a number of other agents cause virtually the same clinical and pathologic syndrome as is seen in abpee. the pathogenesis is assumed to be similar, although presumably other toxic factors are specific for each syndrome. one of these pneumotoxic factors is 4-ipomeanol, which is found in moldy sweet potatoes contaminated with the fungus fusarium solani. mixed function oxidases in the lungs activate 4-ipomeanol into a potent pneumotoxicant capable of producing irreversible oxidative injury to type i pneumonocytes and bronchiolar epithelial cells, presumably through lipoperoxidation of cell membranes. similarly, purple mint (perilla frutescens), stinkwood (zieria arborescens), and rapeseed and kale (brassica species) also cause pulmonary edema, emphysema, and interstitial pneumonia. extrinsic allergic alveolitis. extrinsic allergic alveolitis (hypersensitivity pneumonitis), one of the most common allergic diseases in cattle, is seen mainly in housed adult dairy cows in the winter. this disease shares many similarities with its human counterpart known as farmer's lung, which results from a type iii hypersensitivity reaction to inhaled organic antigens, most commonly microbial spores, mainly of the thermophilic actinomycete, saccharopolyspora rectivirgula (micropolyspora faeni), commonly found in moldy hay. this is followed by an antibody response to inhaled spores and local deposition of antigen-antibody complexes (arthus reaction) in the lungs (see fig. 9 -88). because it affects only a few animals of the herd or the sporadic person working in a farm, it is presumed that intrinsic host factors, such as dysregulation of dendritic cells, t lymphocytes, igg, interleukins, ifn-γ, and surfactant, are involved in the pathogenesis of the disease. chapter 9 respiratory system, mediastinum, and pleurae efigure 9 -17 interstitial pneumonia, adult cow. note meaty appearance of the pulmonary parenchyma and mild edematous distention of the interlobular septa. inset, thick hyaline membranes (arrows) lining hypercellular alveolar walls. hypersensitivity pneumonia was suspected. (courtesy dr. a. lópez, atlantic veterinary college.) gases, inhalation of no 2 (silo gas) also causes bronchiolitis, edema, and interstitial pneumonia and, in survivors, bronchiolitis obliterans ("silo filler's disease"). smoke inhalation resulting from barn or house fires is sporadically seen by veterinarians and pathologists. in addition to skin burns, animals involved in fire accidents suffer extensive thermal injury produced by the heat on the nasal and laryngeal mucosa, and severe chemical irritation caused by inhalation of combustion gases and particles in the lung. animals that survive or are rescued from fires frequently develop nasal, laryngeal, and tracheal edema, and pulmonary hemorrhage and alveolar edema, which are caused by chemical injury to the blood-air barrier or by ards caused by the excessive production of free radicals during the pulmonary inflammatory response (see efig. 9-7) . microscopic examination of the lungs often reveals carbon particles (soot) on mucosal surfaces of the conducting system. verminous pneumonia (dictyocaulus viviparus). pulmonary lesions in parasitic pneumonias vary from interstitial pneumonia caused by migrating larvae to chronic bronchitis from intrabronchial adult parasites, to granulomatous pneumonia, which is caused by dead larvae, aberrant parasites, or eggs of parasites. in many cases, an "eosinophilic syndrome" in the lungs is characterized by infiltrates of eosinophils in the pulmonary interstitium and bronchoalveolar spaces and by blood eosinophilia. atelectasis and emphysema secondary to the obstruction of airways by parasites and mucous secretions are also common findings in parasitic pneumonias. the severity of these lesions relates to the numbers and size of the parasites and the nature of the host reaction, which sometimes includes hypersensitivity reactions (see section on reinfection syndrome). a common general term for all of these diseases is verminous pneumonia, and the adult nematodes are often visible grossly in the airways ( fig. 9-90) . dictyocaulus viviparus is an important pulmonary nematode (lungworm) responsible for a disease in cattle referred to as verminous pneumonia or verminous bronchitis. adult parasites live in the bronchi of cattle, mainly in the caudal lobes, and cause severe bronchial irritation, bronchitis, and pulmonary edema, which in turn are responsible for lobular atelectasis and interstitial emphysema. atelectasis is confined to the lobules of the lungs ventilated by the obstructed bronchi (dorsocaudal). interstitial emphysema (interlobular) is caused by forced expiratory movements against a partially obstructed single bronchus. in addition to the inflammation of bronchial mucosa, bronchoaspiration of larvae and eggs also causes an influx of leukocytes into the bronchoalveolar space (alveolitis). verminous pneumonia is most commonly seen in calves during their first summer grazing pastures that are repeatedly used from year to year, particularly in regions of europe that have a moist cool climate. the parasite can overwinter in pastures, even in climates as cold as canada's, and older animals may be carriers for a considerable length of time. at necropsy, lesions appear as dark or gray, depressed, wedgeshaped areas of atelectasis involving few or many lobules usually along the dorsocaudal aspect of the lungs. on cut surface, edematous foam and mucus mixed with white, slender (up to 80-mm long) nematodes are visible in the bronchi (see fig. 9 -90). in the most severe cases, massive numbers of nematodes fill the bronchial tree. microscopically, the bronchial lumens are filled with parasites admixed with mucus because of goblet cell hyperplasia, and there is squamous metaplasia of the bronchial and bronchiolar epithelium because of chronic irritation. there are also inflammatory infiltrates in the bronchial mucosa; alveolar edema; hyperplasia of balt sulfide (h 2 s), and ammonia (nh 3 ), from silos or sewage can be a serious hazard to animals and human beings. at toxic concentrations, these gases cause necrosis of bronchiolar cells and type i pneumonocytes and fulminating pulmonary edema that causes asphyxiation and rapid death (see fig. 9-60) . like other oxidant secretory granules released by club cells contain several proteins, such as surfactantlike protein, antiinflammatory protein (cc10), and bronchiolar lining proteins. b, ros produced by club cells are also absorbed into capillaries within the lamina propria and are transferred by the circulatory system to pulmonary capillaries where they disrupt the air-blood barrier, causing degeneration and necrosis of type i pneumonocytes. this process leads to leakage of plasma fluid (alveolar edema [pink color]) and extravasation of erythrocytes (alveolar hemorrhage) and neutrophils (inflammation). ingested pneumotoxicants can be metabolized by the liver, leading to release of ros into the circulatory system that then disrupts the air-blood barrier in a similar manner. fig. 9 -77). microscopically, there are focal intraalveolar hemorrhages caused by larvae migrating through the alveolar walls. some larvae admixed with edematous fluid and cellular exudate (including eosinophils) may be visible in bronchioles and alveoli. the alveolar walls are thickened because of edema and a few inflammatory cells. clinical signs include cough and expiratory dyspnea to the point of oral breathing. hydatid cysts, the intermediate stage of echinococcus granulosus, can be found in the lungs and liver and other viscera of sheep and to a lesser extent in cattle, pigs, goats, horses, and human beings. the adult stage is a tapeworm that parasitizes the intestine of canidae. hydatidosis is still an important zoonosis in some countries, and perpetuation of the parasite life cycle results from animals being fed uncooked offal from infected sheep and consumption of uninspected meat. hydatid cysts are generally 5 to 15 cm in diameter, and numerous cysts can be found in the viscera of affected animals ( fig. 9-91 ). each parasitic cyst is filled with clear fluid; numerous daughter cysts attach to the wall, each containing several "brood capsules" with protoscolices inside. hydatid cysts have little clinical significance in animals but are economically important because of carcass condemnation. aspiration pneumonias. the inhalation of regurgitated ruminal contents or iatrogenic deposition of medicines or milk into the trachea can cause severe and often fatal aspiration pneumonia. bland substances, such as mineral oil, may incite only a mild suppurative or histiocytic bronchopneumonia, whereas some "home remedies" or ruminal contents are highly irritating and cause a fibrinous, necrotizing bronchopneumonia. the right cranial lung lobe tends to be more severely affected because the right cranial bronchus is the most cranial branch and enters the ventrolateral aspect of the trachea. however, the distribution may vary when animals aspirate while in lateral recumbency. in some severe cases, pulmonary necrosis can be complicated by infection with saprophytic organisms present in ruminal contents, causing fatal gangrenous pneumonia. aspiration pneumonia should always be considered in animals whose swallowing has been compromised-for example, those with cleft palate or hypocalcemia (milk fever). on the other hand, neurological diseases such as encephalitis (e.g., rabies) or encephalopathy (e.g., lead poisoning) should be investigated in animals in which the cause of aspiration pneumonia could not be caused by persistent immunologic stimuli; hypertrophy and hyperplasia of bronchiolar smooth muscle because of increased contraction and decreased muscle relaxation; and a few eosinophilic granulomas around the eggs and dead larvae. these granulomas, grossly, are gray, noncaseated nodules (2 to 4 mm in diameter) and may be confused with those seen at the early stages of tuberculosis. the clinical signs (coughing) vary with the severity of infection, and severe cases can be confused clinically with interstitial pneumonias. expiratory dyspnea and death can occur with heavy parasitic infestations when there is massive obstruction of airways. a different form of bovine pneumonia, an acute allergic reaction known as reinfection syndrome, occurs when previously sensitized adult cattle are exposed to large numbers of larvae (dictyocaulus viviparus). lesions in this syndrome are those of a hypersensitivity pneumonia as previously described. other lung parasites. ascaris suum is the common intestinal roundworm of pigs; larvae cannot complete their life cycle in calves, but the larvae can migrate through the lungs and cause severe pneumonia and death of calves within 2 weeks of infection. infection is usually acquired from the soil on which infested pigs were previously kept. the gross lesions are a diffuse interstitial pneumonia with hemorrhagic foci, atelectasis, and interlobular edema and it also occurs in canada, europe, australia, and probably elsewhere. this disease has two major clinicopathologic forms: one involves the central nervous system of goat kids and young goats and is characterized by a nonsuppurative leukoencephalomyelitis; the other form involves the joints of adult goats and is characterized by a chronic, nonsuppurative arthritis-synovitis. in addition, infection with cae virus can cause chronic lymphocytic interstitial pneumonia. the lentivirus of cae, caprine arthritis and encephalitis virus (caev), is closely related to visna/maedi virus and, in fact, cross infection with cae virus in sheep has been achieved experimentally. similar to maedi, cae infection presumably occurs during the first weeks of life when the doe transmits the virus to her offspring through infected colostrum or milk. horizontal transmission between infected and susceptible goats via the respiratory route has also been described. after coming into contact with mucosal cells at the portal of entry, the virus is phagocytized by macrophages, which migrate to the regional lymph nodes. infected macrophages are disseminated hematogenously to the central nervous system, joints, lungs, and mammary glands. like maedi, there is some evidence that the recruitment of lymphocytic cells results from dysregulation of cytokine production by infected macrophages and lymphocytes in affected tissues. it can take several months before serum antibodies can be detected in infected goats. grossly, the interstitial pneumonia is diffuse and tends to be most severe in the caudal lobes. the lungs are gray-pink and firm in texture with numerous, 1-to 2-mm, gray-white foci on the cut surface. the tracheobronchial lymph nodes are consistently enlarged. microscopically, the alveolar walls are thickened by lymphocytes and conspicuous hyperplasia of type ii pneumonocytes ( fig. 9-93 ). one important difference between the pneumonias of cae and maedi is that in cae the alveoli are filled with proteinaceous eosinophilic material (alveolar proteinosis), which in electron micrographs has structural features of pulmonary surfactant. the pulmonary form of cae can be mistaken for parasitic pneumonia (muellerius capillaris) because these two diseases have lymphocytic interstitial pneumonia and can coexist in the same goat. explained otherwise. depending on the nature of the aspirated material, histopathologic evaluation generally reveals foreign particles such as vegetable cells, milk droplets, and large numbers of bacteria in bronchi, bronchioles, and alveoli (efig. 9-18 ). vegetable cells and milk typically induce an early neutrophilic response followed by a histiocytic reaction with "foreign body" multinucleated giant cells (see efig. 9-12 ). special stains are used for the microscopic confirmation of aspirated particles in the lung (e.g., pas for vegetable cells and oil red-o for oil or milk droplets). maedi (visna/maedi). maedi is an important, lifelong, and persistent viral disease of sheep and occurs in most countries, except australia and new zealand. maedi means "shortness of breath" in the icelandic language, and it is known as graaff-reinet disease in south africa, zwoegerziekte in the netherlands, la bouhite in france, and ovine progressive pneumonia (opp) in the united states. more recently, the disease has also been referred to as ovine lentivirusinduced lymphoid interstitial pneumonia or simply lymphoid interstitial pneumonia (lip). maedi is caused by visna/maedi virus (vmv), a nononcogenic small ruminant lentivirus (srlv) of the family retroviridae that is antigenically related to the lentivirus causing caprine arthritisencephalitis (cae). seroepidemiologic studies indicate that infection is widespread in the sheep population, yet the clinical disease seems to be rare. the pathogenesis is incompletely understood, but it is known that transmission occurs largely vertically, through ingestion of infected colostrum, and horizontally, via inhalation of infected respiratory secretions. once in the body, the ovine lentivirus causes lifelong infections within monocytes and macrophages, including alveolar and pulmonary intravascular macrophages; clinical signs do not develop until after a long incubation period of 2 years or more. pulmonary lesions at the time of death are severe interstitial pneumonia and failure of the lungs to collapse when the thorax is opened. notable rib imprints, indicators of uncollapsed lungs, are often present on the pleural surface ( fig. 9-92 ). the lungs are pale, mottled, and typically heavy (two or three times normal weight), and the tracheobronchial lymph nodes are enlarged. microscopically, the interstitial pneumonia is characterized by balt hyperplasia and thickening of alveolar walls and peribronchial interstitial tissue by heavy infiltration of lymphocytes, largely t lymphocytes (see fig. 9 -75). recruitment of mononuclear cells into the pulmonary interstitium is presumably the result of sustainable production of cytokines by retrovirus-infected pulmonary macrophages and lymphocytes. hyperplasia of type ii pneumonocytes is not a prominent feature of maedi, likely because in this disease there is no injury to type i pneumonocytes, but there is some alveolar fibrosis and smooth muscle hypertrophy in bronchioles. secondary bacterial infections often cause concomitant bronchopneumonia. enlargement of regional lymph nodes (tracheobronchial) is due to severe lymphoid hyperplasia, primarily of b lymphocytes. the virus can also infect many other tissues, causing nonsuppurative encephalitis (visna), lymphocytic arthritis, lymphofollicular mastitis, and vasculitis. maedi is clinically characterized by dyspnea and an insidious, slowly progressive emaciation despite good appetite. death is inevitable once clinical signs are present, but it may take many months. caprine arthritis-encephalitis. caprine arthritis-encephalitis (cae) is a retroviral disease of goats (small ruminant lentivirus) that has a pathogenesis remarkably similar to that of visna/maedi in sheep. it was first described in the united states in the 1970s, but such as pasteurella multocida, pneumonia may progress to fibrinous or suppurative bronchopneumonia. one might expect some specific evidence pointing to the infectious agents (e.g., large intranuclear inclusion bodies in epithelial cells with adenoviral infection), but this is often not the case, either because examination is seldom done at the acute stage when the lesions are still present or because secondary bacterial infections mask the primary lesions. in the late stages, chronic enzootic pneumonia is characterized by hyperplastic bronchitis, atelectasis, alveolar and peribronchiolar fibrosis, and marked peribronchial lymphoid hyperplasia (cuffing pneumonia). ovine pneumonic mannheimiosis. ovine pneumonic mannheimiosis is one of the most common and economically significant diseases in most areas where sheep are raised. it is caused by mannheimia haemolytica and has a pathogenesis and lesions similar to those of pneumonic mannheimiosis of cattle. colonization and infection of lungs are facilitated by stressors such as changes in weather; handling; deworming; dipping; viral infections such as parainfluenza virus 3 (piv3), respiratory syncytial virus (rsv), and adenovirus; and probably chlamydiae and bordetella parapertussis infections. lesions are characterized by a severe fibrinous bronchopneumonia (cranioventral) with pleuritis ( fig. 9-94 and e-fig. 9-19 ). subacute to chronic cases progress to purulent bronchopneumonia, and sequelae include abscesses and fibrous pleural adhesions. a similar form of pneumonic mannheimiosis has been reported with increased frequency in bighorn sheep. septicemic pasteurellosis. septicemic pasteurellosis, a common ovine disease, is caused by bibersteinia trehalosi (formerly pasteurella trehalosi or mannheimia haemolytica biotype t) in lambs 5 months of age or older or by mannheimia haemolytica (biotype a) in lambs younger than 2 months of age. both organisms are carried in the tonsils and oropharynx of clinically healthy sheep, and under abnormal circumstances (particularly under stress from dietary or environmental changes) bacteria can invade adjacent tissues, enter the bloodstream, and cause septicemia. gross lesions include a distinctive necrotizing pharyngitis and tonsillitis; ulcerative esophagitis (efig. 9-20) ; severe congestion and edema of the lungs; focal hepatic necrosis; and petechiae in the mucosa of the tongue, esophagus, and intestine and particularly in the lungs and pleura. clinically, goats are active and afebrile but progressively lose weight despite normal appetite. the encephalitic or arthritic signs tend to obscure the respiratory signs, which are only evident on exertion. secondary bacterial bronchopneumonia is common in affected animals. bacterial pneumonias. in the past, pasteurella haemolytica was incriminated in four major ovine diseases known as (1) acute ovine pneumonic pasteurellosis (shipping fever), (2) enzootic pneumonia (nonprogressive chronic pneumonia), (3) fulminating septicemia, and (4) mastitis. under the new nomenclature, mannheimia haemolytica is responsible for ovine pneumonia resembling shipping fever in cattle (ovine pneumonic mannheimiosis), septicemia in young lambs (younger than 3 months of age), and ovine enzootic pneumonia and sporadic severe gangrenous mastitis in ewes. bibersteinia (pasteurella) trehalosi (formerly pasteurella haemolytica biotype t) is the agent incriminated in septicemia in lambs 5 to 12 months old. chronic enzootic pneumonia. in sheep, this entity is a multifactorial disease complex that, in contrast to ovine pneumonic mannheimiosis, causes only a mild to moderate pneumonia and it is rarely fatal. it generally affects animals younger than 1 year of age. significant costs associated with chronic enzootic pneumonia include reduction of weight gain, labor costs, veterinary fees, and slaughterhouse waste. the modifier "chronic" is used here to avoid any confusion with pneumonic mannheimiosis ("acute enzootic pneumonia"). it is also sometimes called atypical pneumonia, chronic nonprogressive pneumonia, proliferative pneumonia, or other names. chronic enzootic pneumonia is a clinical epidemiologic term and does not imply a single causal agent but is the result of a combination of infectious, environmental, and managerial factors. the list of infectious agents involved in ovine enzootic pneumonia includes mannheimia haemolytica, pasteurella multocida, parainfluenza virus 3 (pi-3), adenovirus, reovirus, respiratory syncytial virus (rsv), chlamydiae, and mycoplasmas (mycoplasma ovipneumoniae). in the early stages of enzootic pneumonia, a cranioventral bronchointerstitial pneumonia is characterized by moderate thickening of alveolar walls because of hyperplasia of type ii pneumonocytes. in some cases, when lungs are infected with secondary pathogens, viviparus of cattle. as seen in cattle with dictyocaulus viviparus, areas of atelectasis secondary to bronchiolar obstruction are present, particularly along the dorsal caudal aspects of the caudal lung lobes. microscopically, affected lungs are characterized by a catarrhal, eosinophilic bronchitis, with peribronchial lymphoid hyperplasia and smooth muscle hyperplasia of bronchi and bronchioles. bronchioles and alveoli can contain edematous fluid, eosinophils, and parasitic larvae and eggs. microscopic granulomas caused by aspirated eggs can be observed in the distal lung. the clinical signs (cough, moderate dyspnea, and loss of condition) and lesions relate mainly to obstruction of the small bronchi by adult worms and filaria. anemia of undetermined pathogenesis and secondary bacterial pneumonia are common in small ruminants with this parasitic disease. muellerius capillaris. muellerius capillaris, also called the nodular lungworm, occurs in sheep and goats in most areas of the world and is the most common lung parasite of sheep in europe and northern africa. it requires slugs or snails as intermediate hosts. the lesions in sheep are typically multifocal, subpleural nodules that tend to be most numerous in the dorsal areas of the caudal lung lobes ( fig. 9-95, a) . these nodules are soft and hemorrhagic in the early stages but later become gray-green and hard or even calcified. microscopically, a focal, eosinophilic, and granulomatous reaction occurs in the microscopically, the hallmark lesion is a disseminated intravascular thrombosis often with bacterial colonies in the capillaries of affected tissues. the alveolar capillaries contain bacteria and microthrombi, and the alveolar lumens have fibrin and red blood cells. mannheimia haemolytica and bibersteinia trehalosi are readily isolated from many organs. affected animals usually die within a few hours of infection, and these animals only rarely have clinical signs such as dullness, recumbency, and dyspnea. contagious caprine pleuropneumonia. a number of mycoplasma spp., often referred to as the "mycoides cluster," can produce respiratory tract infections in goats; however, only mycoplasma capricolum ssp. capripneumoniae is considered to cause contagious caprine pleuropneumonia. this disease is the goat counterpart of contagious bovine pleuropneumonia in cattle; sheep do not have a corresponding disease. this oie-notifiable disease is important in africa, the middle east, and areas of asia, but it is also seen elsewhere. the gross lesions caused by mycoplasma capricolum ssp. capripneumoniae are similar to those of the bovine disease and consist of a severe, often unilateral fibrinous bronchopneumonia and pleuritis; however, distention of the interlobular septa (which are normally not as well developed in goats as in cattle) and formation of pulmonary sequestra are less obvious than in the bovine disease. clinically, contagious caprine pleuropneumonia is similar to contagious bovine pleuropneumonia, with high morbidity and mortality, fever, cough, dyspnea, and increasing distress and weakness. other small ruminant mycoplasmas. pneumonia, fibrinous polyarthritis, septicemia, meningitis, mastitis, peritonitis, and abortion are possible manifestations of disease caused by mycoplasma mycoides ssp. mycoides large colony type and mycoplasma mycoides ssp. capri. the pathogenicity of other mycoplasmas, such as mycoplasma ovipneumoniae, mycoplasma arginini, and mycoplasma capricolum ssp. capricolum, in sheep and goats is still being defined and specific description of the lesions would be premature. these organisms probably cause disease only in circumstances similar to those for enzootic pneumonia, where host, infectious, and environmental factors create a complex interaction in the pathogenesis of the disease. it has been suggested that igg antibodies directed against ovine mycoplasmal antigens cross-react with ciliary proteins, causing inflammation and ciliary dysfunction, a condition in lambs referred to as coughing syndrome. tuberculosis. although tuberculosis has generally been considered uncommon in sheep and goats, caprine tuberculosis has become a significant disease in areas of spain and europe. mycobacterium caprae (formerly mycobacterium bovis ssp. caprae/mycobacterium tuberculosis ssp. caprae) is the most common cause, but infection with mycobacterium bovis or with the mycobacterium avium complex does occur when the disease is prevalent in other species in the locality. the pulmonary form, similar to that seen in cattle, is characterized by a granulomatous pneumonia with multiple, large, caseous, calcified, and well-encapsulated granulomas scattered throughout the lungs. intralesional acid-fast organisms within macrophages are not as abundant as in bovine tuberculosis. staphylococcus aureus. young sheep (2 to 12 weeks old) are susceptible to staphylococcus aureus septicemia (tick pyemia). this bacterium causes disseminated inflammation and abscesses in the joints, heart, liver, kidneys, and cns, and in the lung it can also produce bronchopneumonia and pulmonary abscesses (efig. 9-21 ). dictyocaulus filaria. dictyocaulus filaria, also called the large lungworm, is a serious, worldwide, parasitic disease of the lungs, most commonly of lambs and goat kids but occurring in adults as well. the life cycle and lesions are similar to those of dictyocaulus epithelial cells spreads rapidly throughout the nasal, tracheal, and bronchial mucosa, with the more severe outbreaks reflecting more involvement of intrapulmonary airways and secondary infection with pasteurella multocida, trueperella (arcanobacterium) pyogenes, or haemophilus spp. although uncommon, human beings infected with swine influenza (h1n1) can transmit the virus to pigs; therefore it is important that veterinarians or workers with influenza-like illness stay away from pig farms. natural transmission of h1n1 and h5n1 from human beings to ferrets (mustela putorius furo) and from human beings to cats and dogs has also been reported. pulmonary lesions caused by influenza virus alone are rarely seen in the postmortem room because this disease has a very low mortality rate unless complicated with secondary bacterial infections. grossly, a copious catarrhal to mucopurulent inflammation extends from the nasal passages to the bronchioles, with the volume of mucus being sufficient to plug small airways and cause a lobular or multilobular atelectasis in the cranioventral regions of the lungs. the appearance can be similar grossly, although not microscopically, to that of mycoplasma hyopneumoniae. fatal cases have severe alveolar and interstitial pulmonary edema. microscopically, the lesions in uncomplicated cases are typical of a virus-induced, necrotizing bronchitis-bronchiolitis, which in severe cases extends into the alveoli as bronchointerstitial pneumonia. it is characterized by necrosis of the bronchial/bronchiolar epithelium, thickening and infiltration of the alveolar wall with mononuclear cells and aggregates of macrophages, neutrophils, mucus, and some necrotic cells within the alveolar lumen. if these changes are extensive enough, the lumen of bronchioles can be occluded by exudate, causing lobular atelectasis. viral antigen can be demonstrated in infected epithelial cells by immunoperoxidase techniques. in the later stages of alveolar inflammation, neutrophils are progressively replaced by intraalveolar macrophages, unless the pneumonia is complicated by secondary bacterial infections. recent serologic surveys indicate that infection is also prevalent in wild pigs. clinically, a sudden onset of fever, nasal discharge, stiffness, labored breathing, weakness or even prostration, followed by painful and often paroxysmal coughing, is seen in animals of all age groups and may affect most of the herd. the outbreak subsides virtually without mortality within 1 or 2 weeks; the clinical appearance is much more alarming than the pathologic changes, unless the pigs have secondary infection with bacteria. infection can be confirmed using pcr in secretions collected with nasal swabs. the most important effect of most outbreaks of influenza is severe weight loss, but pregnant sows may abort or give birth to weak piglets. porcine reproductive and respiratory syndrome. a disease originally named mystery swine disease was first recognized in the united states in 1987. in 1990, it was seen in europe, and the disease now occurs worldwide in most major pig-raising countries. in 1991, dutch investigators isolated a virus as the etiologic agent; porcine reproductive and respiratory syndrome virus (prrsv) is currently classified in the genus arterivirus of the family arteriviridae. as its name implies, prrs is characterized by late-term abortions and stillbirths and respiratory problems. the respiratory form is generally seen in nursery and grow/finish pigs. the pathogenesis has not been completely elucidated, but it is presumed that there is a mucosal portal of entry with virus replication in macrophages of the lymphoid tissue, followed by viremia and finally dissemination of infected macrophages to the lungs and other organs, such as the thymus, liver (kupffer cells), spleen, lymph nodes, and intestine. the pulmonary alveolar and intravascular macrophages are the major targets for prrs virus, which induces apoptosis of these cells. the virus also downregulates the innate immune response by subpleural alveoli where the adults, eggs, and coiled larvae reside ( fig. 9-95, b) . clinical signs are usually not apparent. goats differ from sheep by having diffuse interstitial rather than focal lesions, and the reaction to the parasites seen microscopically varies from almost no lesions to a severe interstitial pneumonia with heavy infiltrates of mononuclear cells in alveolar walls resembling cae or mycoplasmal infections. secondary effects of muellerius capillaris infection in sheep and goats include decreased weight gain and possibly secondary bacterial infections. protostrongylus rufescens. protostrongylus rufescens is a worldwide parasite of sheep, goats, and wild ruminants. it requires an intermediate snail as a host. infection is usually subclinical, but protostrongylus rufescens can be pathogenic for lambs and goat kids and can cause anorexia, diarrhea, weight loss, and mucopurulent nasal discharge. the adult parasite lives in bronchioles as dictyocaulus spp., but it causes pulmonary nodules similar to those of muellerius capillaris. porcine pneumonias are unequivocally a major obstacle for the contemporary swine industry. the incidence, prevalence, and mortality rates of pneumonias in pigs depend on a series of complex, multifactorial interactions. among the most commonly recognized elements linked to porcine pneumonias are the following: • host (age, genetic makeup, immune status) • infectious agents (viruses, bacteria) • environmental determinants (humidity, temperature, ammonia concentrations) • management practices (crowding, mixing of animals, air quality, nutrition, stress) because of the nature of these multifactorial interactions, it will become obvious in the following paragraphs that more often than not a specific type of pneumonia frequently progresses to or coexists with another. the term porcine respiratory disease complex (prdc) has been introduced in clinical practice to describe pigs with signs of respiratory infection involving combined bacterial and viral infections. commonly implicated microbes include porcine reproductive and respiratory syndrome virus (prrsv), swine influenza virus (siv), porcine circovirus 2 (pcv2), porcine respiratory coronavirus (prcov), mycoplasma hyopneumoniae, and pasteurella multocida. swine influenza (swine flu). swine influenza is a highly contagious acute respiratory viral disease of swine that is caused by swine influenza virus (siv), a type a influenza virus of the family orthomyxoviridae. it is generally accepted that swine influenza resulted from adaptation of the type a influenza virus that caused the human influenza pandemic during world war i. the most common subtypes of siv currently circulating in pigs are h1n1, h1n2, and h3n2. swine influenza is enzootic worldwide and is known to infect human beings who are in close contact with sick pigs. in 2009, an outbreak of swine-human influenza (h1n1), presumably transmitted from pigs to human beings, emerged in mexico and rapidly spread to many countries throughout the world. this new "pandemic" was attributed to a triple-reassortant of influenza a virus containing gene segments of swine, eurasian avian, and human strains. human infection with this novel strain affected mainly children and young adults, as well as individuals of any age with an underlying debilitating condition. transmission between influenza-infected and susceptible pigs occurs mainly by aerosol or oral route. siv attaches to and replicates within epithelial cells of the upper respiratory tract; the infection of similar inclusions are occasionally seen in bronchial glandular and renal epithelial cells. the lungs show thickening of the alveolar walls because of hyperplasia of type ii pneumonocytes and interstitial infiltrates of mononuclear cells, peribronchiolar fibrous hyperplasia, and necrotizing bronchitis/bronchiolitis. circovirus can be confirmed in affected tissue by immunohistochemical or pcr techniques. dual infections with pcv2 and prrsv frequently occur in pigs, and secondary infections with pneumocystis carinii are commonly seen in pigs with this coinfection. characteristically, alveoli are filled with a distinctive foamy exudate that contains the organism, which is not visible in h&e-stained sections but is easily demonstrated with gomori's methenamine silver stain (see fig. 9-20) . in human beings, pneumocystis (carinii) jirovecii pneumonia (pneumocystosis) is one of the most common and often fatal complications in aids patients. as in aids patients, abnormal populations of cd4 + and cd8 + t lymphocytes have been incriminated as the underlying mechanism leading to pneumocystosis in foals and pigs. nipah virus. nipah virus belongs to the paramyxoviridae family and shares a genus (henipavirus) with the closely related hendra virus (see section on pneumonias of horses). another emerging zoonotic disease, nipah virus caused a major epidemic with significant human mortality in southeast asia in 1998 and 1999. people handling pigs were primarily affected. similar to hendra virus, fruit bats (flying foxes) act as natural reservoir and are involved in the transmission to pigs by poorly understood mechanisms. in pigs, this virus infects the respiratory system resulting in pneumonia with syncytial cells occurring in the vascular endothelium and in the respiratory epithelium at all levels of the lung. disease is spread to human beings via the respiratory route. human-to-human transmission of this virus has been reported in more recent outbreaks. other viral pneumonias of pigs. porcine respiratory coronavirus (prcov) is sporadically incriminated in pneumonia in pigs. this viral pneumonia is generally mild, and most pigs fully recover if the pneumonia is not complicated with other infections. lesions in the lung are those of bronchointerstitial pneumonia with necrotizing bronchiolitis. interestingly, infections with porcine and other respiratory coronaviruses have been used to investigate the pathogenesis of severe acute respiratory syndrome (sars), an emerging and highly contagious condition in human beings that is attributed to a novel human coronavirus (sars-cov). the relationship between sars-cov and animal coronavirus is still under investigation. other viruses rarely incriminated in porcine respiratory disease complex (prdc) include paramyxovirus, encephalomyocarditis virus, hemagglutinating encephalomyocarditis virus, and adenovirus. petechial hemorrhages in the lung and pulmonary edema may be seen with african swine fever, classical swine fever, and pseudorabies virus infections. porcine enzootic pneumonia. porcine enzootic pneumonia, a highly contagious disease of pigs caused by mycoplasma hyopneumoniae, is grossly characterized by suppurative or catarrhal bronchopneumonia ( fig. 9-96 and efig. 9-23 ). when its worldwide prevalence and deleterious effect on feed conversion are taken into account, this disease is probably the most economically significant respiratory disease of pigs. although an infectious disease, it is very much influenced by immune status and management factors, such as crowding (airspace and floor space), ventilation (air exchange rate), concentrations of noxious gases in the air (ammonia and hydrogen sulfide), relative humidity, temperature fluctuations, and mixing of stock from various sources. it has been demonstrated with inhibiting interferons and deregulates the adaptive immune response, thus interfering with the normal defense mechanisms predisposing pigs to septicemia and bacterial pneumonia. the most common opportunistic organisms are streptococcus suis, salmonella choleraesuis, mycoplasma hyopneumoniae, haemophilus parasuis, bordetella bronchiseptica, pasteurella multocida, and pneumocystis carinii. dual viral infections with prrsv and porcine circovirus 2 (pcv2), siv, and porcine respiratory coronavirus (prcov) are commonly found in pigs, and such coinfections increase the severity of disease. on postmortem examination, pulmonary lesions vary from very mild changes characterized by failure of the lung to collapse when the thorax is opened and the presence of rib imprints (see fig. 9 -74) to severe changes manifested by consolidation of the lung in cases that have been complicated with bacterial pneumonia. tracheobronchial and mediastinal lymph nodes are typically enlarged. microscopically, pulmonary changes are those of interstitial pneumonia characterized by thickening of alveolar walls by infiltrating macrophages and lymphocytes and mild hyperplasia of type ii pneumonocytes. necrotic cells are scattered in the alveolar lumens. unlike some other viral infections, bronchiolar epithelium does not appear to be affected. diagnosis of prrs in tissue collected at necropsy can be confirmed by immunohistochemistry and pcr techniques. infected pigs may become carriers and transmit the infection through body fluids and semen. clinically, prrs in nursery and young growing animals is characterized by sneezing, fever, anorexia, dyspnea, cough, and occasional death. some piglets develop severe cyanosis of the abdomen and ears, which explains why this syndrome was named blue ear disease when first described in europe. porcine circovirus-associated disease. another emerging porcine syndrome, characterized clinically by progressive emaciation in weaned pigs, was originally described in the 1990s in canada, the united states, and europe. since then, it has disseminated to many countries, causing economic devastation in pig farms worldwide. because of the clinical signs and lesions in many organs, this syndrome was named postweaning multisystemic wasting syndrome (pmws). porcine circovirus 2 (pcv2) has been incriminated as the etiologic agent and is a member of the circoviridae family. pcv2 has been associated with a number of syndromes in pigs, including systemic pcv2 infection (the preferred term for pmws because it may also affect mature pigs), pcv2-associated pneumonia, pcv2-associated enteritis, porcine dermatitis and nephropathy syndrome (pdns), pcv2-associated reproductive failure, and, most recently, pcv2-associated cerebellar vasculitis. the diseases caused by pcv2 are now collectively known as porcine circovirus-associated disease (pcvad); the most common manifestations are systemic pcv2 infection (pmws) and pcv2-associated pneumonia as part of the porcine respiratory disease complex. all of these manifestations affect more than one organ, and there is substantial overlap between the syndromes. at necropsy, pigs with systemic pcv2 infection (pmws) and pcv2-associated pneumonia are often in poor body condition, and the most remarkable changes, not considering other possible secondary infections, are enlargement of the superficial and visceral lymph nodes and a mild interstitial pneumonia characterized by failure of the lungs to collapse when the thorax is opened. jaundice is occasionally observed. microscopically, the lymphoid tissues show lymphoid depletion, histiocytic replacement of follicles, and notable proliferation of parafollicular histiocytes, some of which fuse and form syncytial cells (granulomatous lymphadenitis); necrosis of the lymphoid follicles is seen less often. in some cases, large basophilic inclusion bodies are present singly or as grapelike clusters (botryoid inclusions) within the cytoplasm of macrophages, particularly in peyer's patches, spleen, and lymph nodes (efig. 9-22 ). chapter 9 respiratory system, mediastinum, and pleurae peribronchial, bronchiolar, and alveolar interstitium. additional virulence factors include the ability of mycoplasma hyopneumoniae to cause immunosuppression, reduce the phagocytic activity of neutrophils in the lung, and change the chemical composition of mucus. all of these functional alterations can predispose the lung to secondary bacterial infections. the lesions caused by mycoplasma hyopneumoniae start as a bronchointerstitial pneumonia and progress to a suppurative or mucopurulent bronchopneumonia once secondary pathogens are involved (commonly seen at necropsy). in most pigs, gross lesions affect only portions of the cranial lobes, but in more severely affected pigs, lesions involve 50% or more of the cranioventral portions of the lungs (see fig. 9 -96). the affected lungs are dark red in the early stages but have a homogeneous pale-gray ("fish flesh") appearance in the more chronic stages of the disease. on cut surface, exudate can easily be expressed from airways, and depending on the stage of the lesions and secondary infections, the exudate varies from purulent to mucopurulent to mucoid. microscopic lesions are characterized by an influx of macrophages and neutrophils into the bronchi, bronchioles, and alveoli, and with time there is also notable balt hyperplasia (see fig. 9-96, b) . in some cases, accumulation of exudate can be severe enough to cause occlusion of bronchioles and atelectasis of the corresponding lobules. the suppurative bronchopneumonia may be accompanied by a mild fibrinous pleuritis, which is often more severe if other organisms, such as mycoplasma hyorhinis, pasteurella multocida, or actinobacillus pleuropneumoniae, are also involved. abscesses and fibrous pleural adhesions are sequelae of chronic complicated infections. clinically, enzootic pneumonia occurs as a herd problem in two disease forms. a newly acquired infection of a previously clean herd causes disease in all age groups, resulting in acute respiratory distress and low mortality. in a chronically infected herd, the mature animals are immune and clinical signs are usually apparent only in growing pigs at times of particular stress such as at weaning. in such herds, coughing and reduced rate of weight gain are the most notable signs. porcine pasteurellosis. porcine pasteurellosis is an infectious disease complex with unclear pathogenesis that includes primary infections by pasteurella multocida alone (primary pasteurellosis) or, more frequently, after the defense mechanisms are impaired and a secondary bacterium colonizes the lung (porcine pneumonic pasteurellosis). in rare cases, pasteurella multocida causes acutely fatal septicemias in pigs (primary septicemic pasteurellosis). it is important to remember that pasteurella multocida serotypes a and d are both part of the normal nasal flora and are also causative agents of bronchopneumonia, pleuritis, and atrophic rhinitis in pigs. pasteurella multocida is one of the most common secondary pathogens isolated from the lungs of pigs with swine influenza virus (siv), porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus 2 (pcv2), pseudorabies (suhv-1), classical swine fever (hog cholera), enzootic pneumonia, and porcine pleuropneumonia. secondary infections with pasteurella multocida notably change the early and mild bronchointerstitial reaction of enzootic and viral pneumonias into a severe suppurative bronchopneumonia with multiple abscesses and sometimes pleuritis. the other important role of pasteurella multocida in porcine pneumonias is as a cause of a fulminating, cranioventral, fibrinous bronchopneumonia (pleuropneumonia) after influenza virus infection or stress from inadequate ventilation resulting in high levels of ammonia in the air. the nature of the lesion and the predisposing factors of poor management or coexisting viral infections suggest that fulminating porcine pasteurellosis has a pathogenesis similar to that of pneumonic mannheimiosis of cattle. pharyngitis with subcutaneous cervical edema, fibrinohemorrhagic polyarthritis, and focal lymphocytic pcr that mycoplasma hyopneumoniae is present in the air of infected farms. the causative agent, mycoplasma hyopneumoniae, is a fastidious organism and very difficult to grow; thus the final diagnosis is frequently based on interpretation of lesions alone or supported by ancillary tests to detect this mycoplasma in affected lungs by immunohistochemistry, immunofluorescence, or pcr. the bronchopneumonic lesions of porcine enzootic pneumonia are in most cases mild to moderate, and thus mortality is low unless complicated with secondary pathogens, such as pasteurella multocida, trueperella (arcanobacterium) pyogenes, bordetella bronchiseptica, haemophilus spp., mycoplasma hyorhinis, and other mycoplasmas and ureaplasmas. although the pathogenesis of porcine enzootic pneumonia is not completely elucidated, it is known that mycoplasma hyopneumoniae first adheres to the cilia of the bronchi by means of a unique adhesive protein, produces ciliostasis, and finally colonizes the respiratory system by firmly attaching to the ciliated epithelial cells of the trachea and the bronchi of the cranioventral regions of the lungs. once attached to the respiratory epithelium, it provokes an influx of neutrophils into the tracheobronchial mucosa; causes extensive loss of cilia (deciliation); stimulates an intense hyperplasia of lymphocytes in the balt; and attracts mononuclear cells into the factors have been identified. these factors allow actinobacillus pleuropneumoniae to attach to cells; produce pores in cell membranes; damage capillaries and alveolar walls, resulting in vascular leakage and thrombosis; impair phagocytic function; and elicit failure of clearance mechanisms. the gross lesions in the acute form consist of a fibrinous bronchopneumonia characterized by severe consolidation and a fibrinous exudate on the pleural surface. although all lobes can be affected, a common site is the dorsal area of the caudal lobes. in fact, a large area of fibrinous pleuropneumonia involving the caudal lobe of a pig's lung is considered almost diagnostic for this disease (fig. 9-97) . on cut surface, consolidated lungs have notably dilated interlobular septa and irregular but well-circumscribed areas of necrosis caused by potent cytotoxins produced by actinobacillus pleuropneumoniae. except for the distribution, pulmonary lesions of porcine pleuropneumonia are identical to those of pneumonic mannheimiosis of cattle. the microscopic lesions are also very similar and include areas of coagulative necrosis surrounded by a thick cluster of "streaming (oat-shaped/oat cell) leukocytes" and notable distention of the interlobular septa because of severe edema and lymphatic thrombosis. bronchioles and alveoli are filled with edematous fluid, fibrin, neutrophils, and few macrophages (see fig. 9 -97). pigs with the chronic form have multiple pulmonary abscesses and large (2 to 10 cm) pieces of necrotic lung encapsulated by connective tissue (sequestra)-changes frequently seen in slaughterhouses. interstitial nephritis are also present in porcine pneumonic pasteurellosis. sequelae of porcine pneumonic pasteurellosis include fibrous pleuritis and pericarditis, pulmonary abscesses, so-called sequestra, and usually death. in contrast to ruminants, mannheimia haemolytica is not a respiratory pathogen for pigs, but in some instances, it can cause abortion in sows. porcine pleuropneumonia. porcine pleuropneumonia is a highly contagious, worldwide disease of pigs caused by actinobacillus (haemophilus) pleuropneumoniae (app), which is characterized by a severe, often fatal, fibrinous bronchopneumonia with extensive pleuritis (pleuropneumonia). survivors generally develop notable residual lesions and become carriers of the organisms. porcine pleuropneumonia is an increasingly important cause of acute and chronic pneumonias, particularly in intensively raised pigs (2 to 5 months old). transmission of actinobacillus pleuropneumoniae occurs by the respiratory route, and the disease can be reproduced experimentally by intranasal inoculation of the bacterium. considered a primary pathogen, actinobacillus pleuropneumoniae can sporadically produce septicemia in young pigs and otitis media and otitis interna with vestibular syndrome in weaned pigs. two biovars and 15 serotypes of the organism have been identified; all serotypes can cause the disease, but differences in virulence exist. the pathogenesis is not yet well understood, but specific virulence factors, such as rtx toxins (hemolytic/cytolytic toxins apx i to apx iv), capsular factors, fimbriae and adhesins, lipopolysaccharide, and permeability tuberculosis. tuberculosis is an important disease in domestic and wild pigs that has a much greater prevalence in pigs than in cattle or other domestic mammals in many countries. porcine tuberculosis is attributed to infection with mycobacterium bovis and porcine mycobacteriosis to infection with mycobacterium avium complex. a common scenario in small mixed-farming operations is the diagnosis of avian tuberculosis at the time that pigs are slaughtered, and the source is ingestion of tuberculous chickens or contaminated litter. as would be expected, granulomas are found in the mesenteric, mandibular, and retropharyngeal lymph nodes; to a lesser extent in the intestine, liver, and spleen; and only in rare cases in the lung. the route of infection in pulmonary tuberculosis and mycobacteriosis of pigs is most often hematogenous after oral exposure and intestinal infection. lung lesions are those of a granulomatous pneumonia. the microscopic lesions are basically those of tubercles (granulomas), but the degree of encapsulation, caseation, and calcification varies with the type of mycobacterium, age of the lesion, and host immune response. other bacterial pneumonias of pigs. septicemias in pigs often cause petechial hemorrhages in the lung and pulmonary edema. salmonellae, escherichia coli, and listeria monocytogenes can cause severe interstitial pneumonia in very young animals. salmonella choleraesuis causes a necrotizing fibrinous pneumonia similar to porcine pleuropneumonia, and salmonella typhisuis causes a chronic suppurative bronchopneumonia. in high health herds, actinobacillus suis may cause fibrinohemorrhagic pleuropneumonia and is easily confused with porcine pleuropneumonia. metastrongylosis. metastrongylus apri (elongatus), metastrongylus salmi, and metastrongylus pudendotectus (lungworms) of domestic and feral pigs occur throughout most of the world and require earthworms as intermediate hosts for transmission. the incidence of disease has therefore decreased with development of confinement housing. the importance of pig lungworms is mainly because infection results in growth retardation of the host. clinical signs include coughing because of parasitic bronchitis. the gross lesions, when noticeable, consist of small gray nodules, particularly along the ventral borders of the caudal lobes. the adult worms are grossly visible in bronchi, and microscopically, the parasites cause a catarrhal bronchitis with infiltration of eosinophils and lobular atelectasis ( fig. 9-99) . ascaris suum. the larvae of ascaris suum can cause edema, focal subpleural hemorrhages, and interstitial inflammation (see fig. 9 -77). along their larval migration tracts, hemorrhages also occur in the liver and, after fibrosis, become the large white "milk spots" seen so frequently as incidental findings at necropsy. it has been reported that ascaris suum may cause immunosuppression in severely affected pigs. pigs can be killed if exposed to an overwhelming larval migration. other causes of pneumonia. foreign body granulomatous pneumonia occurs frequently in pigs after inhalation of vegetable material (starch pneumonia), presumably from dusty (nonpelleted) feed. lesions are clinically silent but are often mistaken for other pneumonic processes during inspection at slaughterhouses. microscopically, pulmonary changes are typical of foreign body granulomatous inflammation in which variably sized feed particles are surrounded by macrophages and neutrophils, and often have been phagocytosed by multinucleated giant cells. feed (vegetable) particles appear as thick-walled polygonal cells that stain positive with pas because of their rich carbohydrate (starch) content (see efig. 9-12) . clinically, porcine pleuropneumonia can vary from an acute form with unexpected death and blood-stained froth at the nostrils and mouth to a subacute form characterized by coughing and dyspnea accompanied by clinical signs of sepsis such as high fever, hypoxemia, anorexia, and lethargy (efig. 9-24) . a chronic form is characterized by decreased growth rate and persistent cough. animals that survive often carry the organism in the tonsils, shed the organism, and infect susceptible pigs. haemophilus pneumonia. in addition to glasser's disease characterized by polyserositis (pericarditis, pleuritis, peritonitis, polyarthritis, and meningitis) (efig. 9-25) , some serotypes of haemophilus parasuis (originally haemophilus influenzae suis) can also cause suppurative bronchopneumonia that in severe cases can be fatal. the causal organism, haemophilus parasuis, is usually carried in the nasopharynx of normal pigs and requires abnormal circumstances such as those following stress (weaning and cold weather) or viral infections (swine influenza or pcv2). specific pathogen-free (spf) pigs seem to be particularly susceptible to glasser's disease (arthritis and serositis) but not to pulmonary infection (bronchopneumonia). streptococcal pneumonia. streptococcus suis is a common cause of porcine disease worldwide and a serious zoonosis capable of causing death by septic shock or meningitis and residual deafness in butchers, veterinarians, and pig farmers. typically, streptococcus suis gains entrance to the susceptible young pig through the oropharyngeal mucosa and is carried in the tonsils, nasal mucosa, and mandibular lymph nodes of healthy animals, particularly in survivors of an outbreak. infected sows can abort or vertically transmit the infection to their offspring. some serotypes of streptococcus suis cause neonatal septicemia, and this can result in suppurative meningitis, otitis, arthritis, polyserositis, myocarditis, valvular endocarditis, and embolic pneumonia ( fig. 9-98 ). other serotypes of streptococcus suis can reach the lung by the aerogenous route and cause a suppurative bronchopneumonia, in combination with pasteurella multocida, escherichia coli, or mycoplasma hyopneumoniae, or in combination with actinobacillus pleuropneumoniae, which causes a fibrinous bronchopneumonia. coinfections of streptococcus suis with pcv2 and prrsv are also frequently seen in some farms. gross lesions in the acute stages include serous to catarrhal to mucopurulent nasopharyngitis and conjunctivitis. the lungs are edematous and have a diffuse interstitial pneumonia ( fig. 9 -100) microscopically characterized by necrotizing bronchiolitis, necrosis and exfoliation of pneumonocytes, mild alveolar edema, and, several hours later, thickening of the alveolar walls because of interstitial mononuclear cell infiltrates and hyperplasia of type ii pneumonocytes. secondary infections with bordetella bronchiseptica and mycoplasmas are common and induce life-threatening suppurative bronchopneumonia. the thymus may be small relative to the age of the animal because of viral-induced lymphocytolysis. microscopically, eosinophilic inclusions are present in the epithelial cells of many tissues, in the nuclei or cytoplasm, or in both (see fig. 9 -100). they appear early in the bronchiolar epithelium but are most prominent in the epithelium of the lung, stomach, renal pelvis, and urinary bladder, making these tissues good choices for diagnostic examination. viral inclusions are rarely seen in the later stages of this disease. the suppurative secondary bronchopneumonias often hinder the detection of viral lesions in the lung, particularly because bronchiolar cells containing inclusion bodies exfoliate and mix with the neutrophils recruited by the bacterial infection. distemper virus antigens can be readily demonstrated in infected cells by the immunoperoxidase technique (see fig. 9 -100), which can also be used in skin biopsies for the antemortem diagnosis of canine distemper. distemper virus also has a tendency to affect developing tooth buds and ameloblasts, causing enamel hypoplasia in dogs that recover from infection. of all distemper lesions, demyelinating encephalomyelitis, which develops late, is the most devastating (see chapter 14). sequelae to distemper include the nervous and pneumonic complications mentioned previously and various systemic infections, such as toxoplasmosis and sarcocystosis, because of depressed immunity. persistent viral infection occurs in some dogs that survive the disease, and they may become carriers and the source of infection for other susceptible animals. clinical signs consist of biphasic fever, diarrhea, vomiting, weight loss, mucopurulent oculonasal discharge, coughing, respiratory distress, and possible loss of vision. weeks later, hyperkeratosis of the foot pads ("hard pad") and the nose are observed, along with nervous signs, including ataxia, paralysis, convulsions, or residual myoclonus (muscle twitches, tremors, and "tics"). in general, inflammatory diseases of the lungs are less of a problem in dogs than in food-producing species and can be subdivided in two major groups, infectious and noninfectious pneumonias. "canine infectious respiratory disease" (cird) is the term currently used by clinicians to describe a heterogeneous group of respiratory infections in dogs; these diseases were previously clustered under the name of infectious tracheobronchitis or "kennel cough." cird is the canine counterpart of brd and prd complexes in cattle and pigs, respectively. the most common viruses in cird include canine parainfluenza virus (cpiv), canid herpesvirus 1 (cahv-1), canine adenovirus-2 (cav-2), canine respiratory coronavirus (crcov), canine distemper virus (cdv), and canine influenza virus (civ). bordetella bronchiseptica, streptococcus equi ssp. zooepidemicus, and mycoplasma spp. are the most frequent bacterial isolates in cird. it has been recently recognized that animal shelters are an important source of viral and bacterial infections for dogs and cats. uremia and paraquat toxicity are perhaps the two most notable noninfectious causes of canine respiratory disease. canine distemper. canine distemper is an important and ubiquitous infectious disease of dogs, other canidae, wild felidae, mustelidae, and marine mammals throughout the world. it is caused by a morbillivirus that is antigenically related to the human measles, rinderpest (officially eradicated in 2011), "peste de petit ruminants," and phocine distemper viruses. canine distemper virus (cdv) is transmitted to susceptible puppies through infected body fluids. the virus invades through the upper respiratory tract and conjunctiva, proliferates in regional lymph nodes, becomes viremic, and in dogs with an inadequate antibody response, infects nearly all body tissues (pantropic), particularly the epithelial cells. distemper virus hampers the immune response, downregulates cytokine production, and persists for a long time in some tissues. cdv can target the lungs either directly as a viral pneumonia or indirectly by its immunosuppressive effects rendering the lungs susceptible to secondary bacterial and protozoal infections, or as a coinfection with other viruses such as canine adenovirus-2 and canid herpesvirus 1. 15:292-294, 2003.) inclusion bodies occur within epithelial cells in early lesions. cahv-1 has also been identified as a cause of ulcerative keratoconjunctivitis in older dogs. canine influenza (canine flu). canine influenza is an emerging contagious respiratory infection of dogs that was first described in the united states and subsequently in other countries. it has a high morbidity (close to 100%), but the mortality, as with most other influenza infections, is relatively low (less than 8%). this disease, first diagnosed in greyhounds, is caused by a novel influenza-a virus (canine influenza virus or civ), a mutation from a previously recognized h3n8 strain of equine influenza virus. dog-to-dog transmission does occur and therefore this infection must be distinguished from other viruses of the canine infectious respiratory disease (cird) group. pulmonary lesions are generally mild and transient, but infected dogs are susceptible to secondary bacterial bronchopneumonia. the most relevant lesions in dogs dying unexpectedly from canine influenza are pleural and pulmonary hemorrhages. microscopically, there is necrotizing tracheitis, bronchitis, and bronchiolitis with exudation of neutrophils and macrophages. in severe cases, hemorrhagic interstitial or bronchointerstitial pneumonia may be accompanied by vasculitis and thrombosis. influenza antigen can be demonstrated by immunohistochemistry in airway epithelium and alveolar macrophages. clinically, dogs with canine influenza are lethargic, inappetent, and hyperthermic and frequently cough and show nasal discharge. these signs resemble those seen in dogs with kennel cough or secondary bacterial pneumonia. in addition, there are confirmed cases of canine influenza caused by the porcine h1n1 presumably transmitted from infected pet owners. bacterial pneumonias. dogs generally develop bacterial pneumonias when the pulmonary defense mechanisms have been impaired. pasteurella multocida, streptococcus spp., escherichia coli, klebsiella pneumoniae, and bordetella bronchiseptica can be involved in pneumonia secondary to distemper or after aspiration of gastric contents ( fig. 9-102 and efig. 9-27 ). streptococcus zooepidemicus can cause acute and fatal hemorrhagic pleuropneumonia with canine adenovirus type 2 infection. cav-2 infection is a common but transient contagious disease of the respiratory tract of dogs, causing mild fever, oculonasal discharge, coughing, and poor weight gain. the portal of entry is generally by inhalation of infected aerosols followed by viral replication in the surface cells of the upper respiratory tract, mucous cells of the trachea and bronchi, nonciliated bronchiolar epithelial cells, and type ii pneumonocytes. pulmonary lesions are initially those of bronchointerstitial pneumonia, with necrosis and exfoliation of bronchiolar and alveolar epithelium, edema, and, a few days later, proliferation of type ii pneumonocytes, mild infiltration of neutrophils and lymphocytes in the alveolar interstitium, and hyperplastic bronchitis and bronchiolitis. large basophilic intranuclear viral inclusions are typically seen in bronchiolar and alveolar cells ( fig. 9-101) . infection with cav-2 is clinically mild unless complicated with a secondary bacterial infection or coinfections with other viruses such as distemper virus. experimental work suggests cav-2 reinfection may lead to hyperreactive airways, a nonspecific condition in which the bronchial mucosa becomes highly "responsive" to irritation such as that caused by cold air, gases, or cigarette smoke. however, it is not clear if this outcome is true in natural infections. canid herpesvirus 1. canid herpesvirus 1 (cahv-1) can cause fatal systemic disease in newborn puppies and is probably a contributing factor in "fading puppy syndrome." hypothermia has been suggested as a pivotal component in the pathogenesis of fatal infections in puppies. many dogs are seropositive, suggesting that transient or subclinical infections are more common than realized; the virus remains latent in the trigeminal and other ganglia and can be reactivated after stress, resulting in asymptomatic transmission of cahv-1 virus to offspring via the placenta, thus resulting in abortion or stillbirths. in puppies, cahv-1 causes ulcerative tracheitis, interstitial pneumonia (efig. 9-26) , and focal necrosis and inflammation in the kidneys, liver, and brain. eosinophilic intranuclear aspiration pneumonia starts as an acute necrotizing bronchitis and bronchiolitis caused by aspiration of irritant materials such as gastric acid or a caustic material administered by mouth. the aspirate also contains potentially pathogenic bacteria, and because the mucociliary apparatus is damaged and these bacteria are not removed, they settle into the ventral portions of the lung (from gravity) and provoke a fibrinosuppurative and necrotizing bronchopneumonia. b, bronchoalveolar spaces are filled with neutrophils, macrophages, and bacteria (arrows). h&e stain. inset, large colonies of bacteria (arrows). h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) a b infection; thus it most frequently affects outdoor and hunting dogs. from the lung, infection is disseminated hematogenously to other organs, mainly bone, skin, brain, and eyes. pulmonary lesions are characterized by multifocal to coalescing pyogranulomatous pneumonia, generally with firm nodules scattered throughout the lungs (fig. 9-103) . microscopically, nodules are pyogranulomas with numerous macrophages (epithelioid cells), some neutrophils, multinucleated giant cells, and thick-walled yeasts (see fig. 9 -35, c). yeasts are 5 to 25 µm in diameter and are much better visualized when they are stained with pas reaction or gomori's methenamine silver stain. nodules can also be present in other tissues, chiefly lymph nodes, skin, spleen, liver, kidneys, bones, testes, prostate, and eyes. this fungus can be easily identified in properly prepared and stained transtracheal washes or lymph node aspirates. clinical signs can reflect involvement of virtually any body tissue; pulmonary effects include cough, decreased exercise tolerance, and terminal respiratory distress. coccidioidomycosis. coccidioidomycosis (san joaquin valley fever), caused by the dimorphic fungus coccidioides immitis, occurs mainly in animals living in arid regions of the southwestern united states, mexico, and central and south america. it is a primary respiratory tract (aerogenous) infection commonly seen at slaughterhouses in clinically normal feedlot cattle. in dogs, coccidioidomycosis also has an aerogenous portal of entry and then a b hemorrhagic pleural effusion in dogs. death is generally a consequence of severe sepsis and septic shock or from β-hemolytic streptococcal bacteremia causing emboli in the lungs, liver, brain, and lymph nodes. the primary source of the infection cannot be determined in most cases. dental disease in dogs may be a source of systemic and pulmonary infection, a concept wellrecognized in human medicine for many years. the role of mycoplasmas in canine pneumonia is still uncertain because these organisms are frequently isolated from normal nasopharyngeal flora. tuberculosis is uncommon in dogs because these animals appear to be quite resistant to infection; most cases occur in immunocompromised dogs or in dogs living with infected human beings. dogs are susceptible to the infection with mycobacterium tuberculosis, mycobacterium bovis, and mycobacterium avium complex, and therefore canine infection presupposes contact with human or animal tuberculosis. the clinicopathologic manifestation is pulmonary after inhalation or alimentary after oral exposure, but in most cases infection is disseminated to lymph nodes and visceral organs. the gross lesions are multifocal, firm nodules with necrotic centers, most often seen in the lungs, lymph nodes, kidneys, and liver. diffuse granulomatous pleuritis and pericarditis with copious serofibrinous or sanguineous effusion are common. microscopically, granulomas are formed by closely packed macrophages but with very little connective tissue. mycotic pneumonias. mycotic pneumonias are serious diseases seen commonly in animals in some regions. there are two main types: those caused by opportunistic fungi and those caused by a group of fungi associated with systemic "deep" mycoses. all of these fungi affect human beings and most domestic animals but are probably not transmitted between species. aspergillosis. opportunistic fungi, such as aspergillus spp. (particularly aspergillus fumigatus), are important in birds, but in domestic animals, they mainly affect immunosuppressed individuals or those on prolonged antibiotic therapy. the pulmonary lesion is a multifocal, nodular, pyogranulomatous, or granulomatous pneumonia. microscopically, there is necrosis and infiltrates of neutrophils, macrophages, and lymphocytes, with proliferation of fibroblasts eventually leading to encapsulation of the granuloma. fungal hyphae are generally visible in the core of the lesion and in the walls of blood vessels. systemic mycoses (dimorphic fungal infections) . systemic (deep) mycoses are caused by blastomyces dermatitidis, histoplasma capsulatum, coccidioides immitis, and cryptococcus neoformans/ cryptococcus gatti (see fig. 9 -35). blastomycosis mainly affects dogs and is discussed here, whereas cryptococcosis is discussed in the section on pneumonias of cats. in contrast to other fungi, such as aspergillus spp., organisms of the systemic mycosis group are all primary pathogens of human beings and animals and thus do not necessarily require a preceding immunosuppression to cause disease. these fungi have virulence factors that favor hematogenous dissemination and evasion of immune and phagocytic responses. systemic dissemination is often exacerbated by the administration of immunosuppressant drugs such as corticosteroids. these fungi are usually detected by cytological evaluation of affected tissues. blastomycosis. blastomycosis occurs in many countries of the north american continent, africa, the middle east, and occasionally in europe. in the united states, it is most prevalent in the atlantic, st. lawrence, and ohio-mississippi river valley states, compared with the mountain-pacific region. blastomyces dermatitidis is a dimorphic fungus (mycelia-yeast) seen mainly in young dogs and occasionally in cats and horses. this fungus is present in the soil, and inhalation of spores is considered the principal route of from the alveolar interstitium associated with larvae or dead worms because little reaction develops to the live adults. crenosoma vulpis. crenosoma vulpis is a lungworm seen commonly in foxes and sporadically in dogs with access to the intermediate hosts-slugs and snails. the adult lungworms live in small bronchi and bronchioles in the caudal lobes, causing eosinophilic and catarrhal bronchitis manifested grossly as gray areas of inflammation and atelectasis. in some animals, crenosoma vulpis causes bronchiolar goblet cell metaplasia and mucous obstruction, resulting in lobular atelectasis due to the valve effect of the mucous plug. eucoleus aerophilus. eucoleus aerophilus (capillaria aerophila) is a nematode parasite typically found in the trachea and bronchi of wild and domestic carnivores. in some cases, this parasite may also involve the nasal passages and sinuses. although generally asymptomatic, some dogs cough because of the local irritation caused by the parasites on the tracheal or bronchial mucosa. paragonimus spp. paragonimus kellicotti in north america and paragonimus westermani in asia are generally asymptomatic fluke infections in fish-eating species. the life cycle involves two intermediate hosts, the first a freshwater snail and the second a freshwater crab or crayfish; in north america, cats and dogs acquire infection by eating crayfish. gross lesions include pleural hemorrhages where the metacercariae migrate into the lungs. later, multifocal eosinophilic pleuritis, and subpleural cysts up to 7 mm long containing pairs of adult flukes, are found along with eosinophilic granulomas around clusters of eggs. like many other parasitic pneumonias, lesions and scars are more frequent in the caudal lobes. pneumothorax can occur if a cyst that communicates with an airway ruptures to the pleural surface. other parasitic infections. angiostrongylus vasorum and dirofilaria immitis are parasites of the pulmonary arteries and right ventricle and, depending on the stage, can produce different forms of pulmonary lesions. adult parasites can cause chronic arteritis that leads to pulmonary hypertension, pulmonary arterial thrombosis, interstitial (eosinophilic) granulomatous pneumonia, pulmonary interstitial fibrosis, congestive right-sided cardiac failure, and eventually caudal vena caval syndrome. other lesions include pleural petechial hemorrhages and, in later stages, diffuse pulmonary hemosiderosis and multifocal pulmonary infarcts. larvae and eggs also cause alveolar injury, thickening of the alveolar walls with eosinophils and lymphocytes (interstitial pneumonia), and multifocal or coalescing granulomas with giant cells (parasitic granulomas). pneumocystis carinii has been reported as a sporadic cause of chronic interstitial pneumonia in dogs with a compromised immune system (see pneumonias of horses; also see fig. 9 -20). aspiration pneumonia. aspiration pneumonia is an important form of pneumonia that occurs in dogs when vomit or regurgitated materials are aspirated into the lungs, or when drugs or radiographic contrast media are accidentally introduced into the airways (efig. 9-28) . as in other animal species, aspiration pneumonia may be unilateral or may more often affect the right cranial lobe ( fig. 9-104 ). the severity of lesions depends very much on the chemical and microbiologic composition of the aspirated material. in general, aspiration in monogastric animals, particularly in dogs and cats, is more severe because of the low ph of the gastric contents (chemical pneumonitis). in severe cases, dogs and cats die rapidly from septic shock and ards (see fig. 9 -63), which is microscopically characterized by diffuse alveolar damage, protein-rich pulmonary edema, neutrophilic alveolitis, and formation of typical hyaline membranes along the alveolar walls (see fig. 9-104 ). in animals that survive the acute stages of aspiration, pulmonary lesions progress to bronchopneumonia. aspiration pneumonia is a common sequela to disseminates systemically to other organs. clinical signs relate to the location of lesions, so there can be respiratory distress, lameness, generalized lymphadenopathy, or cutaneous lesions, among others. the lesions caused by coccidioides immitis consist of focal granulomas or pyogranulomas that can have suppurative or caseated centers. the fungal organisms are readily seen in histologic or cytologic preparation as large (10 to 80 µm in diameter), double-walled, and highly refractile spherules containing numerous endospores (see fig. 9-35, d) . histoplasmosis. histoplasmosis is a systemic infection that results from inhalation and, in dogs, possibly ingestion of another dimorphic fungus, histoplasma capsulatum. histoplasmosis occurs sporadically in dogs and human beings and, to a lesser extent, in cats and horses. bats often eliminate histoplasma capsulatum in the feces, and droppings from bats and birds, particularly pigeons, heavily promote the growth and survival of this fungus in the soil of enzootic areas. pulmonary lesions are grossly characterized by variably sized, firm, poorly encapsulated granulomas and, sometimes, more diffuse involvement of the lungs. microscopically, granulomatous lesions typically have many macrophages filled with small (1 to 3 µm), punctiform, intracytoplasmic, dark oval bodies (yeasts) (see fig. 9 -35, a) that are best demonstrated with pas reaction or gomori's methenamine silver stain. similar nodules or diffuse involvement can be present in other tissues, chiefly lymph nodes, spleen, intestine, and liver. toxoplasmosis. toxoplasmosis is a worldwide disease caused by the obligate intracellular, protozoal parasite toxoplasma gondii. cats and other felidae are the definitive hosts in which the mature parasite divides sexually in the intestinal mucosa. human beings, dogs, cats, and many wild mammals can become intermediate hosts after accidental ingestion of fertile oocysts shed in cat feces or ingestion of undercooked or raw meat containing tissue cysts, and fetuses can be infected transplacentally from an infected dam. in most instances, the parasite infects many cells of different tissues and induces an antibody response (seropositive animals) but does not cause clinical disease. toxoplasmosis is often triggered by immunosuppression, such as that caused by canine distemper virus. toxoplasmosis is characterized by focal necrosis around the protozoan. pulmonary lesions are severe, multifocal necrotizing interstitial pneumonia with notable proliferation of type ii pneumonocytes and infiltrates of macrophages and neutrophils. other lesions in disseminated toxoplasmosis include multifocal necrotizing hepatitis, myocarditis, splenitis, myositis, encephalitis, and ophthalmitis. the parasites appear microscopically as small (3 to 6 µm) basophilic cysts that can be found free in affected tissues or within the cytoplasm of many epithelial cells and macrophages (see efig. 8-8) . similar findings can be seen sporadically in dogs infected with neospora caninum and sarcocystis canis, and immunohistochemistry would be required to differentiate those protozoal organisms from toxoplasma gondii. filaroides hirthi. filaroides hirthi, a lungworm of the alveoli and bronchioles of dogs, has long been known as a cause of mild subclinical infection in large colonies of beagle dogs in the united states. however, it can on occasion cause severe and even fatal disease in individual pets, presumably as a result of immunosuppression. clinical signs may include coughing and terminal respiratory distress. grossly, the lesions are multifocal subpleural nodules, often with a green hue because of eosinophils, scattered throughout the lungs. microscopically, these nodules are eosinophilic granulomas arising 548.e1 chapter 9 respiratory system, mediastinum, and pleurae other pneumonias. idiopathic pulmonary fibrosis is a rare condition of uncertain etiology reported in the west highland white terrier breed that shares similarities with human and feline idiopathic pulmonary fibrosis. microscopically, there is diffuse interstitial pneumonia and progressive alveolar fibrosis with capillary obliteration, hyperplasia of type ii cells, some of which exhibit cellular atypia, and finally hypertrophy and hyperplasia of smooth muscle. the interstitial fibrosis eventually spills over alveolar spaces causing conspicuous intraalveolar fibrosis. although upper respiratory tract infections are common and important in cats, pneumonias are uncommon except when there is immunosuppression or aspiration of gastric contents. viral infections such as feline rhinotracheitis and calicivirus may cause lesions in the lungs, but unless there is secondary invasion by bacteria, they do not usually cause a fatal pneumonia. feline rhinotracheitis. feline rhinotracheitis is an important viral disease of cats caused by the ubiquitous felid herpesvirus 1 (fehv-1). this infection affects primarily young or debilitated cats causing inflammation in the nasal, ocular, and tracheal mucosa and, to a much lesser extent, the lung (see species-specific diseases of the nasal cavity and paranasal sinuses). when lungs are affected, fehv-1 causes bronchointerstitial pneumonia with necrosis of bronchiolar and alveolar epithelium, thickening of the alveolar walls, and extensive permeability edema. eosinophilic intranuclear inclusion bodies may be seen in infected epithelial cells early in infection. feline calicivirus. feline calicivirus (fcv) causes upper respiratory disease, stomatitis, conjunctivitis, and, to a lesser extent, interstitial pneumonia. microscopically, affected lungs exhibit the typical pattern of bronchointerstitial pneumonia with necrotizing bronchiolitis, thickening of alveolar walls, occasionally hyaline membranes, hyperplasia of type ii pneumonocytes, and macrophages admixed with cellular debris in the alveolar lumens. because pulmonary lesions are similar to those caused by fehv-1, isolation or in situ detection is required for final diagnosis. feline infectious peritonitis. feline infectious peritonitis (fip) is caused by fip virus (fipv), a mutated form of feline enteric cleft palate, and in dogs with megaesophagus secondary to either myasthenia gravis or persistent right aortic arch. it is also an important complication of general anesthesia or neurologic diseases affecting laryngeal function. paraquat. paraquat, a broad-spectrum herbicide widely used in gardening and agriculture, can cause severe and often fatal toxic interstitial pneumonia (pneumonitis) in dogs, cats, human beings, and other species. after ingestion or inhalation, this herbicide selectively accumulates in the lung where paraquat toxic metabolites are produced by club (clara) cells. these metabolites promote local release of free radicals in the lung, which causes extensive injury to club cells and to the blood-air barrier, presumably through lipid peroxidation of type i and ii pneumonocytes and alveolar endothelial cells (see fig. 9 -89). paraquat toxicity has been used experimentally as a model of oxidant-induced alveolar injury and pulmonary fibrosis. soon after poisoning, the lungs are heavy, edematous, and hemorrhagic because of extensive necrosis of epithelial and endothelial cells in the alveolar walls. the lungs of animals that survive acute paraquat toxicosis are pale, fail to collapse when the thorax is opened, and have interstitial emphysema, bullous emphysema, and occasionally pneumomediastinum. microscopic findings in the acute and subacute phases include necrosis of type i pneumonocytes, interstitial and alveolar edema, intraalveolar hemorrhages, and proliferation of type ii pneumonocytes. in the chronic stages (4 to 8 weeks later), the lesions are typically characterized by severe interstitial and intraalveolar fibrosis. uremic pneumopathy. uremic pneumonopathy (pneumonitis) is one of the many extrarenal lesions seen in dogs with chronic uremia. lesions are characterized by a combination of pulmonary edema and calcification of vascular smooth muscle and alveolar basement membranes. in severe cases, alveolar calcification prevents lung collapse when the thorax is opened. in the more advanced cases, the lungs appear diffusely distended, pale red or brown in color, and show a rough pleural surface with rib imprints (see fig. 9 -51). on palpation, the pulmonary parenchyma has a typical "gritty" texture because of mineralization of the alveolar and vascular walls, which are best visualized microscopically by using special stains such as von kossa (see fig. 9 -51). because this is not primarily an inflammatory lesion, the term pneumonitis should not be used. fig. 9-63) . a, note that the lungs did not collapse when the thorax was opened (loss of negative pressure) and as a result fill almost the entire thoracic cavity. the cranioventral aspects of the lung are consolidated with hemorrhage. b, alveolar capillary congestion, thick hyaline membranes along the alveolar septa (arrows), and intraalveolar hemorrhage. these microscopic changes are typical of the diffuse alveolar damage seen in lungs with ards. h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) feline calicivirus has removed chlamydophila felis from its previously overstated importance as a lung pathogen. tuberculosis. cats are susceptible to three types of mycobacterial infections: classic tuberculosis, feline leprosy, and atypical mycobacteriosis. classic tuberculosis in cats is rare and generally caused by mycobacterium bovis and mycobacterium microti but also, to a lesser extent, by mycobacterium tuberculosis. nosocomial tuberculosis (mycobacterium bovis) in cats has been reported with increased frequency. the usual route of infection for feline tuberculosis is oral, through infected rodents/meat or unpasteurized milk, so the granulomatous lesions are mainly in the intestine and mesenteric lymph nodes where they may disseminate through infected phagocytes to other organs. the solid and noncaseated appearance of tuberculous nodules is grossly similar to that of neoplasms, so they must be differentiated from pulmonary neoplasms (e.g., lymphoma). classic tuberculosis with dermal lesions in cats should be differentiated from feline leprosy (localized skin granulomas) caused by mycobacterium lepraemurium and other nonculturable species of acid-fast bacilli. atypical mycobacteriosis is caused by contamination of a skin wound with saprophytic and nonsaprophytic mycobacteria such as those of the mycobacterium avium complex. advances in pcr techniques have notably reduced the time required for etiologic diagnosis of mycobacteriosis in veterinary diagnostic laboratories. cryptococcosis. cryptococcosis (pulmonary cryptococcus neoformans or cryptococcus gatti) is the most frequent systemic mycosis in cats, and lesions are akin to those discussed in the section on mycotic pneumonias of dogs. it occurs worldwide in all species but is diagnosed most frequently in cats, horses, dogs, and human beings. some healthy dogs and cats harbor cryptococcus in the nasal cavity and become asymptomatic carriers. clinical infection may occur in immunocompetent cats and in cats that are immunologically compromised, such as by felv, fiv, malnutrition, or corticosteroid treatment. lesions can occur in nearly any tissue, resulting in a wide c coronavirus (fecv), and is one of a few viral infections of domestic animals that result in pyogranulomatous pneumonia. this disease is microscopically characterized by a vasculitis affecting many tissues and organs ( fig. 9-105) . other viral pneumonias. other viruses sporadically incriminated in feline interstitial pneumonia are cowpox virus (cpxv) and influenza a h1n1. pasteurellae. bacteria from the nasal flora such as pasteurella multocida and pasteurella-like organisms are occasionally associated with secondary bronchopneumonia in cats ( fig. 9-106) . pasteurella multocida also causes otitis media and meningitis, but its role as a respiratory pathogen is mainly associated with pyothorax. interestingly, there are reports of pasteurella multocida pneumonia in older or immunosuppressed human beings acquired through contact with domestic cats. mycoplasmas. mycoplasmas are often isolated from the lungs of cats with pulmonary lesions but are not definitively established as primary pathogens in feline pneumonias. feline pneumonitis. the term feline pneumonitis is a misnomer because the major lesions caused by chlamydophila felis (formerly chlamydia psittaci) are severe conjunctivitis and rhinitis (see species-specific diseases of the nasal cavity and paranasal sinuses). the elucidation of the importance of feline viral rhinotracheitis and organism infects erythrocytes in the erythrocytic stage of disease and multiplies in intravascular macrophages/monocytes, including those in the alveolar capillaries (efig. 9-29) , during the leukocytic stage of disease. aspiration pneumonia. aspiration pneumonias are common in cats as a result of vomiting, regurgitation, dysphagia, or anesthetic complication or after accidental administration of food, oral medicaments, or contrast media into the trachea (iatrogenic). pulmonary lesions are similar to those described for dogs, and the type of lung lesion depends on the chemical and bacterial composition of the aspirated material (see the section on aspiration pneumonia of dogs). feline idiopathic pulmonary fibrosis. feline idiopathic pulmonary fibrosis is a rare, progressive, and fatal disease of cats of uncertain etiology characterized by multifocal fibrotic nodules subpleurally and randomly in the lung making the pleural surface resemble nodular cirrhosis of the liver ( fig. 9-108) . microscopically, the affected alveolar and peribronchiolar interstitium is thickened by excessive fibrosis, abundant deposition of extracellular matrix, and hypertrophy of smooth muscle. some investigators suggest an intrinsic cellular defect in type ii pneumonocytes as the underlying cause. the alveolar walls are diffusely lined by cuboidal hyperplastic type ii pneumonocytes, and the alveolar lumens often contain exfoliated cells and necrotic debris. this feline condition has morphologic features similar to "equine multinodular pulmonary fibrosis" and "cryptogenic pulmonary fibrosis" in human beings. fetal pneumonias. pneumonia is one of the most frequent lesions found in fetuses submitted for postmortem examination, particularly in foals and food-producing animals. because of autolysis, lack of inflation, and the lungs being at various stages of development, fetal lesions are often missed or misdiagnosed. in the nonaerated fetal lung, the bronchoalveolar spaces are filled with a viscous, locally produced fluid known as lung fluid or lung liquid. it has been estimated that an ovine fetus produces approximately 2.5 ml of "lung fluid" per kilogram of body weight per hour. in the variety of clinical signs. however, granulomatous rhinitis, sinusitis, otitis media and interna, pneumonia, ulcerative dermatitis, and meningoencephalitis are most common. the pulmonary lesion in cryptococcosis is a multifocal granulomatous pneumonia and, like those occurring in other internal organs, they are small, gelatinous, white foci. the gelatinous appearance is due to the broad mucous capsule around the yeast (see fig. 9-35, b) . microscopically, lesions contain great numbers of fungal organisms (4 to 10 µm in diameter without the capsule) and only a few macrophages, lymphocytes, and multinucleated giant cells. this thick polysaccharide capsule does not stain well with h&e, and thus there is a large empty space or halo around the yeast. feline lungworm. aelurostrongylus abstrusus, known as feline lungworm, is a parasite that occurs in cats wherever the necessary slug and snail intermediate hosts are found. it can cause chronic respiratory disease with coughing and weight loss and, sometimes, severe dyspnea and death, particularly if there are secondary bacterial infections. the gross lesions are multifocal, amber, and subpleural granulomatous nodules up to 1 cm in diameter throughout the lungs. on incision, these nodules may contain viscous exudate. microscopically, the adult parasites, eggs, and coiled larvae are in the bronchioles and alveoli, where they cause catarrhal bronchiolitis, hyperplasia of submucosal glands, and, later, granulomatous alveolitis, alveolar fibrosis, and fibromuscular hyperplasia ( fig. 9-107) . during routine examination of feline lungs, it is quite common to find fibromuscular hyperplasia in bronchioles and arterioles in otherwise healthy cats. it was alleged in the past that this fibromuscular hyperplasia was a long-term sequela of subclinical infection with aelurostrongylus abstrusus. however, this view has been challenged; thus the pathogenesis and significance of pulmonary fibromuscular hyperplasia in healthy cats remains uncertain. in severe cases, fibromuscular hyperplasia is grossly visible in the lungs as white subpleural nodules. other parasitic pneumonias. toxoplasma gondii, paragonimus kellicotti, and dirofilaria immitis can also affect cats (see the section on parasitic pneumonias of dogs). cytauxzoon felis is an apicomplexan hemoparasite that affects domestic and wild felidae. the diseases that cause fetal pneumonia in farm animals. gross lesions in the lungs are generally undetected, but microscopic lesions include focal necrotizing interstitial pneumonia and focal necrosis in the liver, spleen, or brain. fetal bronchointerstitial pneumonia also occurs in some viral abortions, such as those caused by infectious bovine rhinotracheitis (ibr) virus and bovine parainfluenza virus 3 (bpiv-3) in cattle and equine viral rhinopneumonitis (evr) in horses. fetal pneumonias in dogs and cats are infrequently described, perhaps because aborted puppies and kittens are rarely submitted for postmortem examination. with advancements in molecular biology techniques, the etiologic diagnosis of abortions and their association with pulmonary fetal lesions is rapidly improving. neonatal pneumonias and septicemias. these entities are rather common in newborn animals lacking passive immunity because of the lack of either ingestion or absorption of maternal colostrum (failure of passive transfer or hypogammaglobulinemia). in addition to septicemias causing interstitial pneumonia, farm animals with hypogammaglobulinemia can develop bronchopneumonia by inhalation of bacterial pathogens. these include histophilus somni and pasteurella multocida in calves; streptococcus spp. in foals; and escherichia coli, listeria monocytogenes, and streptococcus suis in pigs. meconium aspiration syndrome. meconium aspiration syndrome (mas) is an important but preventable condition in human babies that originates when amniotic fluid contaminated with meconium is aspirated during labor or immediately after birth. the pathogenesis of mas is basically the same as in those of fetal bronchopneumonia (see fig. 9-109) . fetal hypoxia, a common event during dystocia or prolonged parturition, causes the fetus to relax the anal sphincter and release meconium into the amniotic fluid. aspiration of meconium can occur directly from aspirating contaminated amniotic fluid before delivery (respiratory movements with an open glottis) or immediately after delivery when the meconium lodged in the nasopharynx is carried into the lung with the first breath of air. this latter form of aspiration is prevented in delivery rooms by routine suction of the nasopharynx in meconium-stained babies. mas is well known in human babies, but the occurrence and significance in animals remains largely unknown. mas has been reported in calves, foals, piglets, and puppies. although pulmonary lesions are generally mild and transient, aspiration of meconium can be life-threatening for newborn babies and animals because it typically occurs in compromised neonates already suffering from intrauterine hypoxia and acidosis. neonatal acidosis is known to impair colostrum absorption in calves. common mas sequelae are lobular atelectasis, pulmonary hypertension, and possibly airway hyperreactivity. in the most severe cases of mas, focal (patchy) atelectasis can be observed grossly in the lung, indicating failure of the lungs to be fully aerated because of the mechanical obstruction and the chemical effect of meconium on pulmonary surfactant (see fig. 9 -52). microscopically, meconium and keratin exfoliated from skin of the fetus into the amniotic fluid are present in bronchi, bronchioles, and alveoli and accompanied by mild alveolitis characterized by infiltration of leukocytes followed by alveolar macrophages and occasional giant cells (efig. 9-30) . lung cancer in animals is rare, unlike in human beings, in which the incidence is alarming and continues to be the number one cause of death due to cancer in canada, the united states, and europe. interestingly, prostatic and breast cancers, so much feared by men fetus, this fluid normally moves along the tracheobronchial tree, reaching the oropharynx, where a fraction is swallowed into the gastrointestinal tract, and a small portion is released into the amniotic fluid. at the time of birth, the lung fluid is rapidly reabsorbed from the lungs by alveolar absorption and lymphatic drainage. aspiration of amniotic fluid contaminated with meconium and bacteria from placentitis is the most common route by which microbial pathogens reach the fetal lungs. this form of pneumonia is secondary to fetal hypoxia and acidosis ("fetal distress"), which cause the fetus to relax the anal sphincter, release meconium into the amniotic fluid, and, in the terminal stages, inspire deeply with open glottis, resulting in the aspiration of contaminated fluid ( fig. 9-109 ). gross lesions are only occasionally recognized, but microscopic changes are similar to those of a bronchopneumonia. microscopically, bronchoalveolar spaces contain variable numbers of neutrophils, macrophages, epidermal squames, and pieces of meconium that appear as bright yellow material because of its bile content. in contrast to postnatal bronchopneumonia, lesions in fetuses are not restricted to the cranioventral aspects of the lungs but typically involve all pulmonary lobes. in cattle, brucella abortus and trueperella (arcanobacterium) pyogenes are two of the most common bacteria isolated from the lungs of aborted fetuses. these bacteria are usually present in large numbers in the amniotic fluid of cows with bacterial placentitis. inflammation of the placenta interferes with oxygen exchange between fetal and maternal tissue, and the resultant fetal hypoxia induces the fetus to "breathe" with an open glottis and aspirate the amniotic fluid. aspergillus spp. (mycotic abortion) and ureaplasma diversum cause sporadic cases of placentitis, which results in fetal pneumonia and abortion. in addition to the respiratory route (aspiration), pathogens, such as bacteria and viruses, can also reach the lungs via fetal blood and cause interstitial pneumonia. listeriosis (listeria monocytogenes), salmonellosis (salmonella spp.), and chlamydiosis (chlamydophila abortus [c. psittaci]) are the best known examples of blood-borne primary benign neoplasms of the lungs, such as pulmonary adenomas, are highly unusual in domestic animals. most primary neoplasms are malignant and appear as solitary masses of variable size that, with time, can metastasize to other areas of the lungs and to distant organs. it is sometimes difficult on gross and microscopic examination to differentiate primary lung cancer from pulmonary metastasis resulting from malignant neoplasms elsewhere in the body. it is often difficult to determine the precise topographic origin of a neoplasm within the lungs-for example, whether it originates in the conducting system (bronchogenic carcinoma), transitional system (bronchiolar carcinoma), exchange system (alveolar carcinoma), or bronchial glands (bronchial gland carcinoma). according to the literature, pulmonary carcinomas in animals arise generally from club (clara) cells or type ii pneumonocytes of the bronchioloalveolar region, in contrast to those in human beings, which are mostly bronchogenic. tumors located at the hilus generally arise from major bronchi and tend to be a solitary large mass with occasional small metastasis to the periphery of the lung. in contrast, tumors arising from the bronchioloalveolar region are often multicentric with numerous peripheral metastases in the lung parenchyma. because of histologic architecture and irrespective of the site of origin, many malignant epithelial neoplasms are classified by the all-encompassing term of pulmonary adenocarcinomas. dogs and cats are the species most frequently affected with primary pulmonary neoplasms, largely carcinomas, generally in older animals. the mean age for primary lung tumors is 11 years for dogs and 12 years for cats. pulmonary carcinomas in other domestic animals, except for retrovirus-induced pulmonary carcinoma in sheep, are less common, possibly because fewer farm animals are allowed to reach their natural life span. these neoplasms can be invasive or expansive, vary in color (white, tan, or gray) and texture (soft or firm), and often have areas of necrosis and hemorrhage, which result in a "craterous" or "umbilicate" appearance. this umbilicate appearance is frequently seen in rapidly growing carcinomas in which the center of the tumoral mass undergoes necrosis as a result of ischemia. some lung neoplasms resemble pulmonary consolidation or large granulomas. cats with moderately differentiated neoplasms had significantly longer survival time (median, 698 days) than cats with poorly differentiated neoplasms (median, 75 days). dogs with primary lung neoplasms, grades i, ii, and iii, had survival times of 790, 251, and 5 days, respectively. ovine pulmonary adenocarcinoma (ovine pulmonary carcinoma). ovine pulmonary adenocarcinoma, also known as pulmonary adenomatosis and jaagsiekte (from the south african afrikaans word for "driving sickness"), is a transmissible, retrovirus-induced neoplasia of ovine lungs caused by jaagsiekte sheep retrovirus (jsrv). it occurs in sheep throughout the world, with the notable exception of australia and new zealand; its incidence is high in scotland, south africa, and peru and unknown but probably low in north america. this pulmonary carcinoma behaves very much like a chronic pneumonia, and jsrv shares many epidemiologic similarities with the ovine lentivirus responsible for maedi and the retrovirus responsible for enzootic nasal carcinoma in small ruminants. pulmonary adenomatosis has been transmitted to goats experimentally but is not known to be a spontaneous disease in that species. ovine pulmonary adenocarcinoma affects mainly mature sheep but can occasionally affect young stock. intensive husbandry probably facilitates horizontal transmission by the copious nasal discharge and explains why the disease occurs as devastating epizootics with 5% to 80% mortality when first introduced into a flock. differential diagnosis between maedi and pulmonary adenomatosis can prove difficult because both diseases often coexist in the same flock and women, are a distant second. to say that cigarette smoking is responsible for this epidemic of lung cancer is unnecessary. although dogs have been proposed as valuable "sentinels" for environmental hazards, such as exposure to passive smoking, asbestos, dyes, and insecticides, it is not known if the prevalence of canine lung tumors has increased in geographical areas with high contamination. alterations in genes (oncogenes) and chromosomes and changes in biologically active molecules have been linked to lung cancer in recent years. as with many other forms of cancer, epidemiologic studies indicate that the incidence of pulmonary neoplasms increases with age, but there are still insufficient data to confirm that particular canine or feline breeds have a higher predisposition to spontaneous lung neoplasms. a standard nomenclature of pulmonary neoplasms in domestic animals is lacking, and as a consequence, multiplicity of names and synonyms occur in the veterinary literature. some classifications are based on the primary site, whereas others emphasize more the histomorphologic type. the most common types of benign and malignant pulmonary neoplasms in domestic mammals are listed in box 9-2. clinically, the signs of pulmonary neoplasia vary with the degree of invasiveness, the amount of parenchyma involved, and locations of metastases. signs may be vague, such as cough, lethargy, anorexia, weight loss, and perhaps dyspnea. in addition, paraneoplastic syndromes, such as hypercalcemia, endocrinopathies, and pulmonary hypertrophic osteoarthropathy, have been associated with pulmonary neoplasms. primary neoplasms of the lungs. primary neoplasms of the lungs arise from cells normally present in the pulmonary tissue and can be epithelial or mesenchymal, although the latter are rare. any malignant tumor metastatic from another body location (e.g., osteosarcoma in dogs, uterine carcinoma in cows, and malignant melanoma in horses) box 9-2 classification of pulmonary neoplasms 553.e1 chapter 9 respiratory system, mediastinum, and pleurae abundant cytoplasm containing numerous acidophilic granules, which are positive for pas and for s-100 protein using immunohistochemistry. although this tumor can cause bronchial obstruction and respiratory signs, in most cases, it is an incidental finding in older horses submitted for postmortem examination. lymphomatoid granulomatosis. lymphomatoid granulomatosis is a rare but interesting pulmonary disease of human beings, dogs, cats, and possibly horses and donkeys characterized by nodules or large solid masses in one or more lung lobes. these frequently metastasize to lymph nodes, kidneys, and liver. microscopically, tumors are formed by large pleomorphic mononuclear (lymphomatoid) cells with a high mitotic rate and frequent formation of binucleated or multinucleated cells. tumor cells have a distinct tendency to grow around blood vessels and invade and destroy the vascular walls. lymphomatoid granulomatosis has some resemblance to lymphoma and is therefore also referred to as angiocentric lymphoma; phenotypic marking confirms that neoplastic cells are a mixed population of plasma cells, b and t lymphocytes, and histiocytes. cerebral and cutaneous forms of lymphomatoid granulomatosis have also reported in human beings, dogs, and cats. secondary neoplasms of the lungs. secondary neoplasms of the lungs are all malignant by definition because they are the result of metastasis to the lungs from malignant neoplasms elsewhere. because the pulmonary capillaries are the first filter met by tumor emboli released into the vena cava or pulmonary arteries, secondary neoplasms in the lung are relatively common in comparison to primary ones. also, secondary tumors can be epithelial or mesenchymal in origin. common metastatic tumors of epithelial origin are mammary, thyroid ( fig. 9-111) , and uterine carcinomas. tumors of mesenchymal origin are osteosarcoma ( fig. 9-112, a) ; hemangiosarcoma ( fig. 9-112, b) ; malignant melanoma in dogs; lymphoma in cows, pigs, dogs, and cats ( fig. 9-113) ; and vaccineassociated sarcoma in cats. usually, secondary pulmonary neoplasms are multiple; scattered throughout all pulmonary lobes (hematogenous dissemination); of variable size; and, according to the growth pattern, can be nodular, diffuse, or radiating (efig. 9-31) . the appearance of metastatic neoplasms differs according to the type of neoplasm. for example, dark red cystic nodules containing blood indicate hemangiosarcoma, dark black solid nodules indicate melanoma, and hard solid nodules (white, yellow, or tan color) with bone spicules indicate osteosarcoma. the gross appearances of or in the same animal. death is inevitable after several months of the initial onset of respiratory signs, and a specific humoral immune response to jsrv is undetectable in affected sheep. during the early stages of ovine pulmonary carcinoma, the lungs are enlarged, heavy, and wet and have several firm, gray, variably sized nodules that in some cases can be located in the cranioventral lobes mimicking a bronchopneumonic lesion ( fig. 9-110, a) . in the later stages, the nodules become confluent, and large segments of both lungs are diffusely, but not symmetrically, infiltrated by neoplastic cells. on cross section, edematous fluid and a copious mucoid secretion are present in the trachea and bronchi ( fig. 9-110, b) . microscopically, the nodules consist of cuboidal or columnar epithelial cells lining airways and alveoli and forming papillary or acinar (glandlike) structures (see fig. 9-110, a) . because the cells have been identified ultrastructurally as originating from both type ii alveolar epithelial cells and club (clara) cells, the neoplasm is considered a "bronchioloalveolar" carcinoma. sequelae often include secondary bronchopneumonia, abscesses, and fibrous pleural adhesions. metastases occur to tracheobronchial and mediastinal lymph nodes and, to a lesser extent, to other tissues such as pleura, muscle, liver, and kidneys. neoplastic cells stain strongly positive for jsrv using immunohistochemistry. clinically, ovine pulmonary adenocarcinoma is characterized by a gradual loss of condition, coughing, and respiratory distress, especially after exercise (e.g., herding or "driving"). appetite and temperature are normal, unless there are secondary bacterial infections. an important differentiating feature from maedi (interstitial pneumonia) can be observed if animals with pulmonary adenomatosis are raised by their hind limbs; copious, thin, mucoid fluid, produced by neoplastic cells in the lungs, pours from the nostrils of some animals. carcinoid (neuroendocrine) tumor of the lungs. carcinoid tumor of the lungs is a neoplasm presumably arising from neuroendocrine cells and is sporadically seen in dogs as multiple, large, firm pulmonary masses close to the mainstem bronchi. it has also been reported in the nasal cavity of horses. tumor cells are generally polygonal with finely granular, pale, or slightly eosinophilic cytoplasm. nuclei are small, and mitotic figures are absent or rare. granular cell tumor. granular cell tumor is a rare and locally invasive tumor that has been reported mainly in human beings and older horses. the cell origin of this tumor was thought to be the myoblast, but it is currently presumed to be schwann cells, which are normally present in the bronchovascular bundles of the lung. microscopically, neoplastic cells are large, polyhedron-shaped with metastatic carcinomas are generally similar to the primary neoplasm and sometimes have umbilicated centers. proper diagnoses of pulmonary neoplasms in live animals require history, clinical signs, radiographs, cytologic analysis of bal fluid, and, when necessary, a lung biopsy. identification of a specific lineage of neoplastic cells in biopsy or postmortem specimens is often difficult and requires electron microscopy or immunohistochemical techniques. electron microscopy allows identification of distinctive cellular components such as osmiophilic lamellar phospholipid nephritic bodies in alveolar type ii epithelial cells or melanosomes in melanomas. immunohistochemical staining is also helpful in identifying tumor cells. the thoracic wall, diaphragm, and mediastinum are lined by the parietal pleura, which reflects onto the lungs at the hilum and continues as the visceral pleura, covering the entire surface of the lungs, except at the hilus where the bronchi and blood vessels enter. the space between the parietal and visceral pleura (pleural space) is only minimal and under normal conditions contains only traces of clear fluid, which is a lubricant, and a few exfoliated cells. samples of this fluid are obtained by thoracocentesis, a simple procedure in which a needle is passed into the pleural cavity. volumetric, biochemical, and cytologic changes in this fluid are routinely used in veterinary diagnostics. anomalies 7 congenital defects are rare and generally of little clinical significance. cysts within the mediastinum of dogs and, less often, cats in severe cases, the amount of fluid present in the thoracic cavity can be considerable. for instance, a medium-size dog can have 2 l of fluid, and a cow may accumulate 25 l or more. excessive fluid in the thorax causes compressive atelectasis resulting in respiratory distress (see fig. 9 -54). hydrothorax is most commonly seen in cattle with right-sided heart failure or cor pulmonale (hydrostatic) (efig. 9 -32); dogs with congestive heart failure (hydrostatic), chronic hepatic disease (hepatic hydrothorax) ( fig. 9-114) , or nephrotic syndrome (hypoproteinemia); pigs with mulberry heart disease (increased vascular permeability); and horses with african horse sickness (increased vascular permeability). hemothorax. blood in the thoracic cavity is called hemothorax, but the term has been used for exudate with a sanguineous component. causes include rupture of a major blood vessel as a result of severe thoracic trauma (e.g., hit by car); erosion of a vascular wall by malignant cells or inflammation (e.g., aortitis caused by spirocerca lupi); ruptured aortic aneurysms; clotting defects, including coagulopathies; warfarin toxicity; disseminated intravascular coagulation (consumption coagulopathy); and thrombocytopenia. hemothorax is generally acute and fatal. on gross examination, the thoracic cavity can be filled with blood, and the lungs are partially or completely atelectatic ( fig. 9-115 ). chylothorax. the accumulation of chyle (lymph rich in triglycerides) in the thoracic cavity ( fig. 9-116 ) is a result of the rupture of major lymph vessels, usually the thoracic duct or the right lymphatic duct. the clinical and pathologic effects of chylothorax are similar to those of the other pleural effusions. causes include thoracic neoplasia (the most common cause in human beings but a distant second to idiopathic cases in dogs), trauma, congenital lymph vessel anomalies, lymphangitis, dirofilariasis, and iatrogenic rupture of the thoracic duct during surgery. the source of the leakage of chyle is rarely found at necropsy. when the leakage of chyle occurs in the abdominal cavity, the condition is referred to as chyloabdomen. cytologic and biochemical examination of fluid collected by thoracocentesis typically reveals large numbers of lymphocytes, lipid droplets, few neutrophils in chronic cases, and high triglyceride content. can be large enough to compromise pulmonary function or mimic neoplasia in thoracic radiographs. these cysts may arise from the thymus (thymic branchial cysts), bronchi (bronchogenic cysts), ectopic thyroid tissue (thyroglossal duct cysts), or from remnants of the branchial pouches, and they are generally lined by epithelium and surrounded by a capsule of stromal tissue. anomalies of the thoracic duct cause some cases of chylothorax. pleural calcification. pleural calcification is commonly found in dogs and less often in cats with chronic uremia. lesions appear as linear white streaks in parietal pleura, mainly over the intercostal muscles of the cranial part of the thoracic cavity. the lesions are not functionally significant but indicate a severe underlying renal problem. vitamin d toxicity (hypervitaminosis d) and ingestion of hypercalcemic substances, such as vitamin d analogs, can also cause calcification of the pleura and other organs. pneumothorax. pneumothorax is the presence of air in the thoracic cavity where there should normally be negative pressure to facilitate inspiration. human beings have a complete and strong mediastinum so that pneumothorax is generally unilateral and thus not a serious problem. in dogs, the barrier varies, but in general it is not complete, so often some communication exists between left and right sides. there are two main forms of pneumothorax. in spontaneous (idiopathic) pneumothorax, air leaking into the pleural cavity from the lungs occurs without any known underlying disease or trauma. in secondary pneumothorax, movement of air into the pleural cavity results from underlying pulmonary or thoracic wall disease. the most common causes of secondary pneumothorax in veterinary medicine are penetrating wounds to the thoracic wall, perforated esophagus, iatrogenic trauma to the thorax and lung during a transthoracic lung biopsy or thoracoscopy, tracheal rupture from improper intubation, and rupture of emphysematous bullae or parasitic pulmonary cysts (paragonimus spp.) that communicate with the thoracic cavity. pneumothorax and pneumomediastinum caused by high air pressure (barotrauma) are also well documented in cats after equipment failure during anesthesia. clinical signs of pneumothorax include respiratory distress, and the lesion is simply a collapsed, atelectatic lung. the air is readily reabsorbed from the cavity if the site of entry is sealed. pleural effusion. pleural effusion is a general term used to describe accumulation of any fluid (transudate, modified transudate, exudate, blood, lymph, or chyle) in the thoracic cavity. cytologic and biochemical evaluations of pleural effusions taken by thoracocentesis are helpful in determining the type of effusion and possible pathogenesis. based on protein concentration and total numbers of nucleated cells, pleural effusions are cytologically divided into transudates, modified transudates, and exudates. hydrothorax. when the fluid is serous, clear, and odorless and fails to coagulate when exposed to air, the condition is referred to as hydrothorax (transudate). causes of hydrothorax are the same as those involved in edema formation in other organs: increased hydrostatic pressure (heart failure), decreased oncotic pressure (hypoproteinemia, as in liver disease), alterations in vascular permeability (inflammation), or obstruction of lymph drainage (neoplasia). in cases in which the leakage is corrected, if the fluid is a transudate, it is rapidly reabsorbed. if the fluid persists, it irritates the pleura and causes mesothelial hyperplasia and fibrosis, which thickens the pleura. from a perforated esophagus. chronic injury typically results in serosal fibrosis and tight adhesions between visceral and parietal pleurae (see fig. 9 -71). when extensive, these adhesions can obliterate the pleural space. pleuritis or pleurisy. inflammation of the visceral or parietal pleurae is called pleuritis, and according to the type of exudate, it can be fibrinous, suppurative, granulomatous, hemorrhagic, or a combination of exudates. acute fibrinous pleuritis can progress with time to pleural fibrosis ( fig. 9-117 ). when suppurative pleuritis results in accumulation of purulent exudate in the cavity, the lesion is called pyothorax or thoracic empyema ( fig. 9-118) . clinically, pleuritis causes considerable pain, and in addition, empyema can result in severe toxemia. pleural fibrous adhesions (between parietal and visceral pleura) and fibrosis are the most common sequelae of chronic pleuritis and can significantly interfere with inflation of the lungs. pleuritis can occur as an extension of pneumonia, particularly in fibrinous bronchopneumonias (pleuropneumonia), or it can occur alone, without pulmonary involvement ( fig. 9-119 ). bovine and ovine pneumonic mannheimiosis and porcine and bovine pleuropneumonia are good examples of pleuritis associated with fibrinous bronchopneumonias. polyserositis in pigs and pleural empyema, particularly in cats and horses, are examples of pleural inflammation in pleural tissue is readily susceptible to injury caused by direct implantation of an organism through a penetrating thoracic or diaphragmatic wound; by hematogenous dissemination of infectious organisms in septicemias; or by direct extension from an adjacent inflammatory process, such as in fibrinous bronchopneumonia or in contrast to those with the effusive ("wet") form, in which thoracic involvement is primarily that of a pleural effusion. cytologic evaluation of the effusion typically shows a low to moderate cellularity with degenerated leukocytes, lymphocytes, macrophages, and mesothelial cells, and a pink granular background as a result of the high protein content. pleuritis is also an important problem in horses. nocardia spp. can cause fibrinopurulent pneumonia and pyothorax with characteristic sulfur granules. although mycoplasma felis can be isolated from the respiratory tract of normal horses, it is also isolated from horses with pleuritis and pleural effusion, particularly during the early stages of infection. the portal of entry of this infection is presumably aerogenous, first to the lung and subsequently to the pleura. the pleural surface of the lung is often involved in neoplasms that have metastasized from other organs to the pulmonary parenchyma and ruptured the visceral pleura to seed the pleural cavity. mesothelioma is the only primary neoplasm of the pleura. which involvement of the lungs may not accompany the pleuritis. pleural inflammation is most frequently caused by bacteria, which cause polyserositis reaching the pleura hematogenously. these bacteria include haemophilus parasuis (glasser's disease) (see , streptococcus suis, and some strains of pasteurella multocida in pigs; streptococcus equi ssp. equi and streptococcus equi ssp. zooepidemicus in horses; escherichia coli in calves; and mycoplasma spp. and haemophilus spp. in sheep and goats. contamination of pleural surfaces can be the result of extension of a septic process (e.g., puncture wounds of the thoracic wall and, in cattle, traumatic reticulopericarditis) and ruptured pulmonary abscesses (e.g., trueperella pyogenes). in dogs and cats, bacteria (e.g., nocardia, actinomyces, and bacteroides) can cause pyogranulomatous pleuritis, characterized by accumulation of blood-stained pus ("tomato soup") in the thoracic cavity. this exudate usually contains yellowish flecks called sulfur granules ( fig. 9-120 ), although these are less common in nocardial empyema in cats. many species of bacteria, such as escherichia coli, trueperella pyogenes, pasteurella multocida, and fusobacterium necrophorum, can be present in pyothorax of dogs and cats. these bacteria occur alone or in mixed infections. the pathogenesis of pleural empyema in cats is still debatable, but bite wounds or penetration of foreign material (migrating grass awns) are likely. pyogranulomatous pleuritis with empyema occurs occasionally in dogs, presumably associated with inhaled small plant material and penetrating (migrating) grass awns. because of their physical shape (barbed) and assisted by the respiratory movement, aspirated grass awns can penetrate airways, move through the pulmonary parenchyma, and eventually perforate the visceral pleura causing pyogranulomatous pleuritis. cats with the noneffusive ("dry") form of feline infectious peritonitis (fip) frequently have focal pyogranulomatous pleuritis, mesothelioma is a rare neoplasm of the thoracic, pericardial, and peritoneal mesothelium of human beings that is seen most commonly in calves, in which it can be congenital. in human beings, it has long been associated with inhalation of certain types of asbestos fibers (asbestos mining and ship building) alone or with cigarette smoking as a probable cocarcinogen; no convincing association between the incidence of mesothelioma and exposure to asbestos has been made in domestic animals. in animals, there may be pleural effusion with resulting respiratory distress, cough, and weight loss. mesothelioma initially causes a thoracic effusion, but cytologic diagnosis can be difficult because of the morphologic resemblance of malignant and reactive mesothelial cells. during inflammation, mesothelial cells become reactive and not only increase in number but also become pleomorphic and form multinucleated cells that may be cytologically mistaken for those of a carcinoma. grossly, mesothelioma appears as multiple, discrete nodules or arborescent, spreading growths on the pleural surface ( fig. 9-121) . microscopically, either the mesothelial covering cells or the supporting tissue can be the predominant malignant component, so the neoplasm can microscopically resemble a carcinoma or a sarcoma. figure 9 -120 nocardiosis. a, chronic pleuritis (nocardia asteroides), pleural cavity, cat. the pleural cavity is covered with abundant red-brown ("tomato soup") exudate" (syringe). once considered to be pathognomonic of nocardia spp. infection, it is no longer regarded as being diagnostic of nocardiosis. the fluid contains abundant protein, erythrocytes, granulomatous inflammatory cells, and sulfur granules. b, chronic pleuritis (nocardia asteroides), visceral pleura, dog. the thickened pleura has a granular pink-gray appearance because of granulomatous inflammation and the proliferation of fibrovascular tissue of the pleura. c, chronic pleuritis (nocardia asteroides), dog. the pleura has been thrown up into villous-like projections composed of abundant fibrovascular tissue and granulomatous inflammation. leakage from the neocapillaries of the fibrovascular tissue is responsible for the hemorrhagic appearance of the pleural exudate. h&e stain. d, chronic pleuritis (nocardia asteroides), thoracic cage, parietal pleura, cat. large pieces of exudate, which contain yellow sulfur granules, are present on the thickened pleura. although considered malignant, mesotheliomas rarely metastasize to distant organs. secondary neoplasms of the pleura. secondary tumors may also spread into the visceral and parietal pleura. thymomas are rare neoplasms that grow in the cranial mediastinum of adult or aged dogs, cats, pigs, cattle, and sheep. thymomas are composed of thymic epithelium and lymphocytes (see chapter 13). old age, both in human beings and in animals, is known to be a risk factor for pulmonary infections, but the precise mechanisms involved in this increased susceptibility are still under investigation. some studies have shown that in aged individuals the antibacterial properties provided by surfactant proteins, proinflammatory cytokines, and complement are altered. pulmonary hyperinflation (often referred to as senile emphysema) has been reported as an age-related change in human and canine lungs. other age-related changes described in canine lungs include mineralization of bronchial cartilage, pleural and alveolar fibrosis, and heterotopic bone formation (so-called "pulmonary osteomas"). we thank all pathologists at the atlantic veterinary college, university of prince edward island for providing case material. suggested readings are available at www.expertconsult.com. lung section showing a distended and partially occluded blood vessel (center of figure) containing large granular cells. these large cells are macrophages, and their cytoplasm is filled with myriad merozoites isolation of porcine circoviruslike viruses from pigs with a wasting disease in the usa and europe exercise-induced pulmonary hemorrhage effect of mucociliary transport relies on efficient regulation of ciliary beating epidemiology, diagnosis, and treatment of blastomycosis in dogs and cats canine h3n8 influenza virus infection in dogs and mice failure of respiratory defenses in the pathogenesis of bacterial pneumonia in cattle the respiratory system advances in diagnosis of respiratory diseases of small ruminants canine nasal disease transmission of equine influenza virus to dogs dear jd: bacterial pneumonia in dogs and cats acute respiratory distress syndrome in dogs and cats: a review of clinical findings and pathophysiology inflammatory response to infectious pulmonary injury laryngeal paralysis: a study of 375 cases in a mixed-breed population of horses stem cells of the respiratory tract exudative pleural disease in small animals bovine respiratory disease research pulmonary thromboembolism coccidioidomycosis in dogs and cats: a review cousens c: pathology and pathogenesis of ovine pulmonary adenocarcinoma prognosis factors for survival in cats after removal of a primary lung tumor: 21 cases (1979-1994) the acute respiratory distress syndrome: from mechanism to translation endogenous lipid pneumonia in cats: 24 cases (1985-1998) retroviral infections in sheep and goats: small ruminant lentiviruses and host interaction canine and feline nasal neoplasia the acute respiratory distress syndrome equine respiratory medicine and surgery canine pleural and mediastinal effusions: a retrospective study of 81 cases a review of histiocytic diseases of dogs and cats estimation of nasal shedding and seroprevalence of organisms known to be associated with bovine respiratory disease in australian live export cattle polymicrobial respiratory disease in pigs current state of knowledge on porcine circovirus type 2-associated lesions common and emerging infectious diseases in the animal shelter chronic rhinitis in the cat advances in the understanding of pathogenesis, and diagnosis and therapeutics of feline allergic asthma mannheimia haemolytica and bovine respiratory disease detection of respiratory viruses and bordetella bronchiseptica in dogs with acute respiratory tract infections current perspectives on the diagnosis and epidemiology of mycoplasma hyopneumoniae infection mannheimia haemolytica: bacterialhost interactions in bovine pneumonia acute lung injury review rhodococcus equi: the many facets of a pathogenic actinomycete tumors of the respiratory system key: cord-016962-8vjaot6i authors: pantanowitz, liron; leiman, gladwyn; garcia, lynne s. title: microbiology date: 2011-07-04 journal: cytopathology of infectious diseases doi: 10.1007/978-1-4614-0242-8_4 sha: doc_id: 16962 cord_uid: 8vjaot6i in order to render an accurate diagnosis, and correctly identify clinically important microorganisms, a good understanding and knowledge of microbiology is essential. this chapter provides a broad overview of microbiology that is relevant to the practicing cytologist. virology addresses the cytopathic effects caused by viruses and discusses many key infections. bacteriology covers important bacterial causes of infection including those due to mycobacteria and filamentous bacteria. mycology deals with common fungi as well as deep mycoses, particularly those caused by invasive and dimorphic fungal organisms. parasitology highlights the protozoa, apicomplexans, and helminths likely to be seen in cytology samples. algae are also briefly mentioned. cytologists are likely to encounter infectious diseases either because a cytology sample was obtained for diagnostic purposes or incidentally when an infectious process or microorganism is discovered in the material they are reviewing. in order to render an accurate diagnosis, and correctly identify clinically important species or microorganisms, a good understanding and knowledge of microbiology is essential. this chapter provides a broad overview of microbiology that is relevant to the practicing cytologist, but is not intended to replace standard microbiology texts. viruses replicate only inside host cells. their particles (called • • virions) consist of dna or rna and a capsid (coat) that may be surrounded by a lipid envelope. once they attach to and penetrate cells, they uncoat and replicate so that their progeny may be released following host cell lysis. viral infection may cause cell death, proliferation, or neoplastic • • transformation (oncogenesis) ( table 4 .1). tumor viruses may promote cancer by expression of viral oncoproteins (or oncogenes) and/or inactivation of tumor suppressor genes. in general, viruses are too small to be identified directly by • • light microscopy. viruses that remain latent often do not cause apparent changes to infected cells. however, several viruses may cause cytopathic changes (fig. 4 .1) that affect the nucleus (e.g., inclusions, margination, multinucleation), cytoplasm (e.g., koilocytosis, syncytial giant cell formation), and/or entire cell (e.g., cytomegaly, ciliacytopthoria). recognition of these changes can be life-saving as this would initiate confirmatory studies and/or therapy ( fig. 4.2) . superinfection by pyogenic bacteria is a complication of many • • viral infections that may mask subtle viral changes. papillomaviruses (genus) are nonenveloped viruses that contain • • double-stranded circular dna molecules that replicate exclusively in skin and/or mucosal keratinocytes. they belong to the papillomaviridae family. their genome is divided into an early (e) region that encodes • • genes e1-e7, and a late region (l) that encodes the capsid genes l1 and l2. in oncogenic human papillomavirus (hpv) types, the genes e6 (binds to p53) and e7 (binds to retinoblastoma [rb] protein) are key players in transforming cells. hpv infection causes various diseases including warts (ver-• • ruca), anogenital lesions (condylomata acuminata, intraepithelial neoplasia) and cancer, epidermodysplasia verruciformis (genodermatosis), oral and laryngeal papillomas, as well as orpharyngeal and conjunctival cancer. genital hpv infection is covered in greater detail in chap. 5. retroviruses are enveloped viruses that belong to the viral family retroviridae. they are rna viruses that replicate in host cells using the enzyme reverse transcriptase to produce dna from its rna genome. dna is then incorporated into the host genome. that measure 0.5-5.0 mm in length. mycoplasma spp. are among the smallest bacteria. bacteria have a wide range of shapes. most are spherical (cocci) or rod-shaped (bacilli), but they may also be curved or spiral-shaped (e.g., spirochaetes, helicobacter pylori). some bacteria are described as being coccobacilli because they have the ability to exist as a coccus, bacillus, or intermediate form (e.g., haemophilus influenzae, rhodococcus equi, bartonella spp.). bacteria may also form pairs (e.g., diploids), chains (e.g., streptococcus), or clusters (e.g., staphylococcus). some bacteria may also have flagella. anerobic bacteria do not need oxygen for growth. some anerobes • • die when oxygen is present (obligate anerobes), whereas others will utilize oxygen if it is present (facultative anaerobes). they are found in normal flora (e.g., fusobacterium in the mouth, bacteroides fragilis in the large bowel, lactobacillis in the vagina). these bacteria can usually be isolated from abscesses, aspiration pneumonia, empyema, and wounds. material being collected from sites that do not harbor indigenous flora (e.g., body fluids other than urine and fine needle aspirates) should always be cultured for anerobic bacteria. bacteria, along with some fungi (mainly • • candida spp.) and archaea (single-celled microorganisms), make up the normal human flora of the skin, mouth, gastrointestinal tract, conjunctiva, and vagina (lactobacilli). loss of normal flora may permit the unfavorable growth of harmful pathogens that can lead to infection. some bacteria form biofilms, which are bacterial aggregates • • embedded within a self-produced matrix (slime). these bacterial clusters, seen associated with amorphous mucoid material, may be encountered in cytology specimens related to catheter infections, pseudomonas aeruginosa pulmonary infections in cystic fibrosis, middle ear infections, joint prostheses, and dental (gingival) disease. bacteria can generally be divided into gram-positive and mycobacteria are aerobic gram-positive rod-shaped bacilli • • that are acid-alcohol fast (so-called afb) with acid fast stains (fite, ziehl-neelsen, kinyoun, and auramine rhodamine stains). mycobacterium tuberculosis are strongly acid fast positive (stain deep red), thin, and slightly curved bacilli that measure 0.3-0.6 × 1-4 nm ( fig. 4 .5). the bacteria of mai are typically short and cocobacillary like. beading may be seen in some mycobacteria, which represents nonuniform staining of the bacillus. for example, m. kansasii are characteristically long and broad and exhibit a cross-banded or barred appearance. acid-fast staining of morphologically similar bacteria such as nontuberculous mycobacteria (ntm), which include all of the other mycobacteria. these are also known as atypical mycobacteria, mycobacteria other than tuberculosis (mott), or environmental mycobacteria. infections with these mycobacteria are increasingly being seen in immunosuppressed patients. infection causes lung disease but may also disseminate to involve the hematopoietic system, gastrointestinal tract, as well as skin and soft tissue. while most ntm can be detected microscopically with an acid-fast stain, culture and/or molecular studies may be required to identify these species. bacteria can be elongated to form filaments (e.g., actinobacteria, nocardia, rhodococcus, streptomyces, actinomadura). they can sometimes form complex, branched filaments that morphologically resemble fungal mycelia (mass of branching hyphae). these bacteria are usually part of the normal oral flora. most infections are acquired by inhalation of the bacteria or via trauma. • • actinomyces (genus) belong to the actinobacteria (class of bacteria). infection (actinomycosis) with these gram-positive bacteria forms multiple abscesses and sinus tracts that may discharge sulfur granules. actinomycosis is most frequently caused by actinomyces israelii. chlamydia are known to produce human disease: chlamydia pneumonia (also chlamydophila pneumoniae and previously known as the twar agent). infection causes pharyngitis, bronchitis, and atypical pneumonia. less common infections include meningoencephalitis, arthritis, and myocarditis. an association with atherosclerosis and possibly lung cancer has been reported. chlamydia trachomatis (previously called tric agent). this includes three human biovars: trachoma (serovars a, b, ba or c), urethritis (serovars d-k), and lymphogranuloma venereum (lgv, serovars l1, 2 and 3). infection causes inclusion conjunctivitis (trachoma), pneumonia in neonates, and sexually transmitted disease in adults (e.g., cervicitis, urethritis, salpingitis, proctitis, epididymitis). chlamydia psittaci (also called chlamydophila psittaci) causes respiratory psittacosis and is acquired from birds ( fig. 4.8) . on the basis of morphologic forms fungi can be divided into hyphae can produce • • conidia (synonymous with spores). large complex conidia are called macroconidia. smaller more simple conidia are termed microconidia. when these conidia are enclosed in a sac (the sporangium) they are called endospores. a sporangium-bearing hypha is referred to as a sporangiophore. dimorphism ( • • dimorphic fungi) is the condition whereby a fungus can exhibit either the yeast form or the hyphal form, depending on growth conditions (fig. 4.9 ). candida • • candida is a polymorphic fungus that undergoes a yeast-tomycelial transition. in clinical specimens, they produce pseudohyphae (hyphae that show distinct points of constriction resembling sausage links), rarely true septate hyphae, and budding yeast forms (blastoconidia). the yeast-like forms (blastoconidia) are oval and measure • • 3-5 mm in diameter fig. 4.9 . fungal morphology. hyphae may be characterized as (a) pseudohyphae (e.g., candida spp.), (b) septate (e.g., aspergillus) or (c) coenocytic (aseptate) hyphae (e.g., zygomycetes). conidia (spores) develop from asexual fruiting structures such as (d) a conidiophore or (e) enclosed in a sac called a sporangium, in which case they are then called endospores. although typically seen extracellularly, intracellular can mimic other small fungi such as histoplasma. candida usually exhibit variably sized yeast cells, lack a pseudocapsule, and elicit more of a suppurative reaction than a granulomatous response. • • candida yeasts form part of the normal flora on the skin and mucous membranes of the respiratory, gastrointestinal, and female genital tracts. they often contaminate cytology samples from these sites. they may also colonize tissue (e.g., after prolonged antibiotic use, prolonged skin moisture, and in patients with diabetes). infection may result from overgrowth or when introduced into • • the body (e.g., intravenously). superficial infections include oropharyngeal and vulvovaginal candidiasis (thrush). candidiasis may also become a systemic illness causing widespread abscesses, endocarditis, thrombophlebitis, endocarditis, eye infections, or involve other organs. • • candida albicans is clinically the most significant member of this genus. candida glabrata (previously known as torulopsis glabrata) is a nondimorphic species (only has a yeast form) (fig. 4.10 ). cryptococci are small (5-15 • • mm) pleomorphic (ovoid to spheroid) yeasts that are characterized by often having a thick gelatinlike capsule and demonstrating narrow-based (teardrop-shaped) budding. they have thin walls and are occasionally refractile. their capsules may have a diameter of up to five times that of the fungal cell, and form a halo on diff-quik, pap, and india ink stains. smaller (2-5 • • mm) capsule-deficient cryptococci can resemble other organisms with similar microforms (e.g., histoplasma, candida, and immature spherules of coccidioides immitis). in such cases, with careful examination some weakly encapsulated yeasts can still be detected. loss of capsular material usually elicits an intense inflammatory reaction characterized by suppuration and granulomas. yeasts usually produce single buds, but multiple buds and even • • aspergillus genus consists of many mold species. pathogenic species include aspergillus fumigatus and aspergillus flavus. these fungi consist of septate hyphae that branch at 45° angles. other dichotomous hyphae that may mimic aspergillus include the hyalinohyphomyces (e.g., fusarium, penicillium) and dermatophytes. species specific conidiophores called fruiting bodies have swollen the zygomycetes belong to the phylum zygomycota ( (zygospores) and a vegetative mycelium. they have broad, ribbon-like, aseptate hyaline hyphae (coenocytic hyphae) with wide-angle branching. these morphological features are helpful in differentiating the zygomycetes from other fungal agents of infection that may be seen in cytologic specimens (table 4 .5). in cytology samples hyphal forms may be twisted, collapsed, or • • wrinkled making them hard to evaluate. moreover, in tissue sections from biopsies or cell block material, folds and creases in the section may cause the hyphae to appear as if they have septae. in respiratory samples, the zygomycetes can be distinguished dimorphic fungi can exist both as a mold form that consists of • • hyphae (when grown at room temperature outside the host) and as yeast (when grown at body temperature in the host). therefore, in clinical samples obtained from patients the cytologist will encounter yeasts from these organisms (table 4 .6). several such fungal species are potential pathogens. the most well-known species of this genus is blastomyces dermatitidis, endemic to the united states (especially the southeastern, south central, and midwestern states) and canada. infection (blastomycosis) occurs by inhalation of the fungus from its natural soil habitat. infection may involve virtually any organ including the lungs, skin, bones, and brain ( fig. 4.15 ). • • coccidioides. this fungus presents with endospores contained within thick-walled spherules that vary in size (20-150 mm). endospores measuring 3-5 mm may be seen scattered singly if the spherule ruptures. when free endospores occur within macrophages, they can imitate other intracellular yeasts. the causative agents of infection (coccidioidomycosis) are c. immitis and c. posadasii. these fungi are endemic in american deserts. infection causes granulomatous and miliary disease affecting largely the lungs. in endemic regions, fungus balls may develop within lung cavities. • • paracoccidioides. these yeasts measure 5-30 mm in size and are round to oval. budding is characterized by a central yeast with multiple surrounding daughter buds, that morphologically resembles a "ship's wheel." infection (paracoccidioidomycosis) is caused by paracoccidioides brasiliensis, typically found in brazil and elsewhere in south america. primary infection (valley fever) is usually mild and self-limiting, but may progress into a systemic mycosis producing oral lesions, generalized lymphadenopathy, and miliary pulmonary lesions. infection can also spread to bones, meninges, and the spleen. • • histoplasma. this budding yeast is round to oval, on average 1-5 mm in size and observed mainly within macrophages (fig. 4.16 ), but sometimes also within neutrophils. narrow based round to oval budding may be noted, but because of their small size buds are often not seen. intracellular yeasts are usually surrounded by a clear zone (halo). however, with cell disruption organisms may be spilled extracellularly. this fungus is usually found in bird and bat ( • • penicillium. these fungi produce penicillin. penicillium marneffei is the only known thermally dimorphic species. in cytology specimens, penicillium present in the mold phase with septate and branched hyphae represent a contaminant. however, in immunosuppressed patients p. marneffei is an opportunistic infection that causes penicilliosis. there is a particularly high incidence of penicilliosis in aids patients from tropical southeast asia. infection after inhalation spreads from the lungs to involve the hematopoietic system and skin. yeast-like cells are present within macrophages and extracellularly. they are not true yeast cells, but rather arthroconidia. intracellular "yeasts" measure 2-3 mm in diameter and are round to oval. because they divide by binary fission, budding is not observed. the extracellular organisms tend to be more elongated, sometimes up to 13 mm, and can have "septae" (crosswalls from binary fission). • • pneumocystis jirovecii (previously called pneumocystis carinii) is a yeast-like fungus of the genus pneumocystis, which is the causative organism of pneumocystis pneumonia (or pneumocystosis, formerly referred to as pcp). the cysts often collapse forming crescent-shaped bodies. all stages of the life cycle are found within the lung alveoli. once inhaled, unicellular trophozoites (1-4 mm, giemsa positive) undergo binary fission to form a precyst (difficult to distinguish by light microscopy) and ultimately develop thick walled cysts (5-8 mm, gms positive). spores (eight) form within these cysts, which are eventually released on rupture of the cyst wall. this organism is often seen in the lungs of healthy individuals, • • but is an opportunistic pathogen in immunosuppressed people, especially those with aids. extrapulmonary disease may be seen with advanced hiv infec-• • tion presenting with involvement of the lymph nodes, spleen, liver, bone marrow, gastrointestinal tract, eyes, thyroid, adrenal glands, kidneys, and within macrophages in pleural effusions (fig. 4.17 ). the dematiaceous (naturally pigmented) group of fungi pro • • mycosis (also called chromomycosis) and phaeohyphomycosis (or phaeomycotic cyst). dermatophytes cause infections of the skin and hair (ringworm • • or tinea) as well as the nails (onychomycosis). the three genera that cause these diseases include microsporum, epidermophyton, and trichophyton. a rapid scraping of the nail, skin, or scalp can be used to iden-• • tify characteristic hyphae and sometimes spores associated with squamous cells or within broken hairshafts. hyalohyphomycosis is the term used to group together inva-• • sive mycotic infections caused by hyaline septate hyphae. this includes species of aspergillus, penicillium, paecilomyces, acremonium, beauveria, fusarium, and scopulariopsis. they may represent contamination or cause invasive disease in the immunosuppressed host. • • fusarium hyphae are similar to those of aspergillus, with septate hyphae that branch at acute and right angles. sporulation may also occur in tissue with infection (fusariosis). their macroconidia are crescent-shaped, orangeophilic, and septate structures that measure 80-120 × 3-6 mm in size. protozoa are unicellular motile organisms. they are traditionally divided according to their means of locomotion such as amebae, flagellates, and ciliates. their life cycle often alternates between trophozoites (feeding-• • dividing stage) and cysts (dormant stage able to survive outside the host). their characteristics (particularly the nuclei and cytoplasmic inclusions) help in species identification. ingested cysts cause infection by excysting (releasing trophozoites) in the alimentary tract. intestinal amebae. entamoeba histolytica causes amebiasis that may manifest with dysentery, flask-shaped colon ulcers, a colonic ameboma, and possible extraintestinal abscesses that contain anchovy paste-like material within the liver, and infrequently spleen or brain. several of the amebae such as entamoeba dispar are harmless and some may be relatively common, such as entamoeba gingivalis that is usually found in the mouth. unlike other amebae, the cytoplasm of pathogenic e. histolytica contains ingested red blood cells. free-living amebae. these include acanthamoeba spp., balamuthia mandrillaris, and naegleria fowleri. acanthamoeba cause granulomatous amebic encephalitis (gae), contact lensassociated acanthamoeba keratitis, and skin lesions. balamuthia also causes gae. n. fowleri ("brain-eating" ameba) is associated with rapidly fatal primary amebic meningoencephalitis (pam), most often seen in children swimming in fresh water ponds and rivers during which amebae enter the nasal passages and migrate to the brain via the olfactory nerve. these trophozoites can be seen in csf specimens, but culture on nonnutrient agar plates seeded with escherichia coli and/or a flagellation test is required for confirmation. • • flagellates (mastigophora) are organisms that have one or more flagella. giardia. there are approximately 40 species described, but the species that infects humans is giardia lamblia (also called g. intestinalis or g. duodenalis). this parasite is the most common cause of protozoal gastroenteritis (giardiasis). trophozoites (9-21 mm long and 5-15 mm wide) are kite (or pear)-shaped and have two nuclei, four pairs of flagella, and two central axonemes running down their middle. their cysts (8-14 × 7-10 mm) seen in stool specimens are oval, thick walled, and contain four nuclei and multiple curved median bodies. trichomonas. there are several trichomonad species such as the intestinal pentatrichomonas hominis which is a nonpathogenic organism. trichomonas vaginalis is the anaerobic, flagellated protozoan that causes trichomoniasis (fig. 4.18) . details of this sexually transmitted infection are covered in chap. 5. apart from urogenital infections, t. vaginalis has also been reported to cause pneumonia, bronchitis, and oral lesions. pulmonary trichomoniasis is usually caused by aspirated trichomonas tenax (mouth commensal) and less often t. vaginalis infection. an association between flagellated protozoa and asthma has been reported. leishmania. these parasites are acquired from the sandfly. depending on the species, they may cause cutaneous (e.g., oriental sore), mucocutaneous (espundia or uta), or visceral (kala-azar) leishmaniasis (table 4 .7). there are two morphological forms: promastigote (with a flagellum) found in the insect host and an amastigote (without flagella) present in the human host ( fig. 4.19 ). diagnostic samples may be procured from skin lesions (cutaneous or mucocutaneous) or bone marrow aspirates (visceral). the morphologic hallmark is the presence of multiple small (2-5 mm) intracellular amastigotes within macrophages (leishman-donovan bodies). amastigotes are spherical to ovoid in shape and have both a nucleus and ovoid or rod-shaped kinetoplast. the differential diagnosis for multiple small organisms within histiocytes includes h. capsulatum (with budding and only intracellular) and toxoplasmosis (more curved and mostly extracellular). trypanosoma. the major human diseases caused by trypanosomatids are african trypanosomiasis (sleeping sickness) caused by trypanosoma brucei and american trypanosomiasis (chagas disease) caused by t. cruzi. trypomastigotes (30 mm in length) are usually found in peripheral blood. they have an undulating membrane, central nucleus, and kinetoplast at the anterior end. • • ciliates (ciliophora) include the parasitic species balantidium coli, the only member of this phylum known to be pathogenic to humans. the trophozoite is relatively large (50-70 mm), has a ciliated surface, and contains a kidney bean-shaped macronucleolus. the cyst form may occasionally also have cilia. cilicytophthoria may be sometimes mistaken for these ciliated organisms. the apicomplexa are a diverse group of protists that includes the microsporidia, at one time a separate group (not coccidian), are now classified with the fungi. • • cryptosporidium spp. c. parvum is the causative agent of cryptosporidiosis in humans and animals, a major cause of protracted diarrhea in patients with aids. other species that cause human disease include c. hominis. these small (8-15 mm) oval parasites are identified within the brush border of the intestinal epithelium, and are discussed in the chapter on gastrointestinal infections. ultrastructural studies have shown them to be intracellular but with an extracytoplasmic localization in enterocytes. modified acid-fast thick-walled oocysts (4-6 mm) may be detected in stool samples. cryptosporidium can disseminate beyond the intestine, especially in patients with aids, to involve the biliary tract, stomach, lungs, middle ear, and pancreas. • • microsporidia include enterocytozoon bieneusi and encephalitozoon intestinalis (previously septata intestinalis). although these organisms are now considered fungi, parasitologists still maintain them in most books. in cytology specimens obtained from the small intestine, microsporidia appear as numerous small intracellular organisms within the apical portion of enterocytes. they stain well with gram and silver stains (e.g., warthin-starry). their spores (1-1.5 mm) may be found with a modified trichrome stain in stool samples as well as urine. • • i. belli and sarcocystis infections very rarely have trophozoites that are detected. their oocysts, however, may be seen when excreted in feces. • • t. gondii belongs to the genus toxoplasma. although infection can be acquired from the accidental ingestion of infective oocysts from cat feces (definitive host), most infections are acquired from eating infected rare or raw meats. disease ranges from mild flu-like illness to fatal fetal infections. latent infection may reactivate in immunosuppressed patients. organisms may be found in samples from the brain, heart, eye, hematopoietic system, and lungs. specimens may contain free (extracellular) tachyzoites which are small (3-5 mm), curved (banana-shaped) forms (fig. 4.20) . when parasites accumulate within macrophages (so-called "bag of parasites") they form a pseudocyst (parasitophorous vacuole) containing bradyzoites. helminths (parasitic worms) are categorized into three groups: • • cestodes (tapeworms), nematodes (roundworms), and trematodes (flukes) ( (fig. 4.21) . infections are usually diagnosed by the characteristics of these different developmental stages. diagram showing a macrophage with a pseudocyst containing multiple bradyzoites: note that some of these microorganisms are still crescent shaped. the cysts are usually round in brain tissue, more elongated in muscle, and often very small and hard to identify in lung tissue. (bottom right) an infected macrophage contains a cluster of bradyzoites (pap stain, high magnification). parasites the host response to these parasites includes eosinophilia, acute containing larvae located in several sites such as the brain (neurocysticercosis causes seizures), eye, as well as muscles and subcutaneous tissue (causes painful nodules). these cysts may cause eosinophilia, an inflammatory reaction and eventually they may calcify. echinococcus. infection from the accidental ingestion of food or drink contaminated with tapeworm eggs results in hydatid disease, also known as echinococcosis. the larval cysts that develop can be found in virtually any site, grow slowly, and persist for many years until they cause symptoms or are discovered incidentally. disruption of a cyst containing highly antigenic fluid may result in anaphylactic shock, and for this reason it has been recommended that they should not be biopsied. however, on the basis of published data adverse reactions are rare. use of a fine gauge needle by a skilled operator is important to prevent fluid leakage during aspiration. (table 4 .7), but those likely to be seen in cytology specimens are enterobius vermicularis, strongyloides stercoralis, and the microfilariae. e. vermicularis (pinworm, also known as the threadworm in the united kingdom) causes intestinal infestation (enterobiasis). the adult female worm migrates out onto the perianal area at night, and once exposed to oxygen she lays her eggs. this causes perianal pruritis and possibly vaginitis. occasionally subcutaneous perineal nodules may develop. in such cases, pap tests may contain an adult worm and/or eggs. the worm has a characteristic pointed tail (fig. 4.23) . their eggs are colorless, oval, thin walled, and flattened on one side, s. stercoralis. this worm causes strongyloidiasis. after penetrating the skin, infective larvae pass through the lung (loeffler syndrome). they are then coughed or swallowed and infest the duodenum. here new autoinfective larvae may develop (autoinfection) leading to chronic infection. in immunocompromised patients, larvae may penetrate the intestinal wall and the pattern of nuclei in their tail are the main features used to distinguish the various species. occult filariasis has been diagnosed by many bloody fna procedures containing microfilariae, worms, or even eggs. filarial morphology is best appreciated with a giemsa stain. the background tissue response in cytology aspirates may include eosinophils, neutrophils, chronic inflammation, and even granulomas. • • trematodes. the flukes are oval or worm-like helminthes that are parasites of molluscs and vertebrates. the liver flukes include fasciola hepatica and clonorchis sinensis that result in infestation of the bile ducts and subsequent biliary fibrosis. infection with c. sinensis is a risk factor for cholangiocarcinoma. fasciolopis buskii is an intestinal fluke that infests both the bile ducts and duodenum. paragonimus westermani, the lung fluke, causes lung infestation with pulmonitis. also included are the schistosomes (blood flukes). schistosomes. infection by these trematodes causes schistosomiasis (bilharzia). there are three human schistosome pathogens: schistosoma mansoni from africa (nile delta) which causes intestinal schistosomiasis, s. haematobium from africa and the middle east that infests the urinary bladder, and s. japonicum from china and southeast asia that primarily involves the liver. s. haematobium infection can lead to squamous cell carcinoma of the bladder. microscopic identification of eggs in stool or urine specimens, as well as tissue biopsies, can provide a rapid diagnosis. the spines present on schistosome eggs help distinguish these different species. the egg of s. haematobium (oval) has a terminal spine, s. mansoni (oval) has a lateral spine, and s. japonicum (round) has a small knob-like lateral spine. algae are ubiquitous and include a diverse group of simple • • organisms that range from unicellular to multicellular forms. the main algal groups include the cyanobacteria, green algae, and red algae (e.g., dinoflagellates). most algae present in cytology samples are from contamination, • • discussed in greater detail in chap. 15. organisms may be seen within macrophages or extracellularly. they form spherical sporangia that range in size from 3 to 10 mm, have thick walls, and show no budding. these morular forms often contain endospores arranged symmetrically ash & orihel's atlas of human parasitology cytologic diagnosis of adenovirus bronchopneumonia amoebiasis: diagnosis by aspiration and exfoliative cytology left) diagram of protothecae demonstrating variable morula formation: (right) a sporangium of p. wickerhamii is shown in which endospores have a moruloid (daisy-like) pattern (pap stain, high magnification) (image courtesy of rafael martinez girón diagnostic medical parasitology histoplasmosis: cytodiagnosis and review of literature with special emphasis on differential diagnosis on cytomorphology role of fine needle aspiration cytology in diagnosis of filarial infestation kala-azar: liver fine needle aspiration findings in 23 cases presenting with a fever of unknown origin medically important fungi. a guide to identification disseminated nocardiosis diagnosed by fine needle aspiration biopsy: quick and accurate diagnostic approach challenges and pitfalls of morphologic identification of fungal infections in histologic and cytologic specimens: a ten-year retrospective review at a single institution the study of cytopathological aspects induced by human cytomegalovirus infection atypial cytomorphologic appearance of cryptococcus neoformans: a report of five cases key: cord-017252-88b3preq authors: morgan, carrie i.; shah, samir s. title: pneumonia date: 2014-02-20 journal: pediatric critical care medicine doi: 10.1007/978-1-4471-6356-5_6 sha: doc_id: 17252 cord_uid: 88b3preq respiratory diagnoses continue to make up a large number of admissions to the pediatric intensive care unit (picu), most notably lower respiratory infections including pneumonia. this chapter will focus on pediatric community-acquired pneumonia (cap), immunocompromised pneumonia, and aspiration pneumonia. the pathogenesis for developing pneumonia varies; it can occur by direct inhalation of infectious particles in the air or aspiration, direct extension from the upper airways, and hematogenous spread. there are multiple levels of defense against pathogen invasion including anatomic barriers, as well as innate and adaptive immunity, which may be compromised in picu patients. the etiologies of pediatric pneumonia vary depending on age, host condition, and environmental factors like time of year and location. viruses remain the most common form of lower respiratory tract infection in children, especially in neonates. community-acquired bacterial pneumonia continues to be most prevalent in younger children as well, most often affecting children less than 5 years of age who are otherwise healthy. despite immunizations and public health initiatives, the most common bacterial causes of cap have remained largely unchanged over the last several decades and include: streptococcus pneumoniae, staphylococcus aureus, haemophilus influenzae (including non-typable strains) and moraxella catarrhalis. pulmonary infection in an immunocompromised host provides a much broader differential and must be aggressively treated without delay. this chapter will also address various imaging modalities and typical findings with pediatric pneumonia. methods for pathogen identification are broad and range from non-specific markers of illness to invasive techniques for culture. the mainstay of therapy continues to be antibiotics tailored to the patient and presumed etiology; more novel therapies may include corticosteroids or macrolide antibiotics for immune modulation. in those patients with pneumonia with effusion or empyema, drainage therapies with thoracostomy tubes or a vats procedure may be indicated. respiratory diagnoses continue to make up a large number of admissions to the pediatric intensive care unit (picu) [ 1 ] . lower respiratory tract infections are considered to be any infection beneath the anatomic level of the vocal cords, including bronchitis, bronchiolitis, tracheitis, and pneumonia [ 2 ] . pneumonia remains an important cause of pediatric morbidity and mortality. there are nearly two million pneumoniarelated deaths worldwide each year among children 5 years of age and younger [ 3 , 4 ] . in the u.s., pneumonia causes over three million outpatient visits and more than 150,000 hospitalizations each year [ 5 , 6 ] . in the developed world, early recognition and availability of antimicrobial therapies and respiratory support have lessened the mortality of pneumonia, but its morbidities remain. while widespread use of the heptavalent pneumococcal conjugate vaccine in 2000 was associated with fewer pneumonia-associated complications in infants <1 year of age, complications remained unchanged or increased in school-age children and adolescents [ 5 ] . thus, despite our best efforts at prevention through vaccination, morbidities continue to plague our patients and pneumonia remains a common cause of pediatric hospital admission. this chapter will focus on pediatric community-acquired pneumonia (cap), immunocompromised pneumonia, and aspiration pneumonia. hospital acquired pneumonia is an important type of lower respiratory infection found in the picu, but it is discussed extensively in the chapter on hospital-acquired infections elsewhere in this textbook. the defi nition of pneumonia is generally accepted to be a lower respiratory illness with fever, respiratory symptoms including tachypnea, and often, radiologic evidence of parenchymal infi ltrates [ 7 ] . the world health organization (who) has defi ned pneumonia solely based on clinical fi ndings due to the lack of radiologic studies in many parts of the world [ 8 ] . determining the type of pneumonia can help guide clinical management. previously healthy children presenting with the signs and symptoms of a lower respiratory tract infection are generally considered to have cap. aspiration involves inhaling foreign material beyond the vocal cords, often causing aspiration pneumonitis (chemical pneumonitis) or pneumonia (an infectious process secondary to the aspiration) [ 9 , 10 ] . commonly aspirated materials in children include oropharyngeal secretions, gastric contents, water, hydrocarbon, lipid, and foreign bodies [ 11 ] . guidelines for admission to the icu are available for both young children and adults, and are summarized in table 6 .1 [ 12 , 13 ] . pneumonia can occur by direct inhalation of infectious particles in the air or aspiration, direct extension from the upper airways, and hematogenous spread. anatomic and cellular protection serves as the fi rst line of defense against potential pathogens. airway mucus traps inhaled toxins and microbes and helps to transport them up and out of the respiratory tract via ciliary beating and cough, a mechanism referred to clinically as mucociliary clearance [ 14 ] . when the microbe burden or virulence of the organism surpasses the abilities of these simple mechanical protections, the innate immune response is activated. the innate immunity is responsible for immediate recognition and control of microbial invasion. in mammals, conserved receptors enable rapid recognition of pathogens to begin elimination of the infection as well as initiate the adaptive immune response. activating the innate immune receptors in the airway epithelium leads to mobilization and activation of dendritic cells, t cells, and b cells that amplify antigen recognition, antibody production, and further cellular recruitment and infl ammation [ 15 ] . the specifi cs of these interactions and signaling cascades are beyond the scope of this chapter, but are further discussed in other chapters within this text. the lower respiratory tract remains generally clear of pathogens [ 2 ] . the mechanisms by which microbes are able adapted from refs. [ 12 , 13 ] to overwhelm defensive measures and result in pneumonia vary and depend on host conditions. the most common mechanism of pathogen entry is via inhalation of infectious particles, particularly in the case of specifi c organisms that spread via respiratory droplets such as mycobacterium tuberculosis. many viruses that cause lower respiratory tract infections are also spread utilizing aerosolized modes of transmission, including respiratory syncytial virus (rsv), infl uenza, and rhinoviruses. due to their smaller size compared with bacteria, viruses consolidate more effi ciently on smaller particles [ 16 , 17 ] . hematogenous spread results in pneumonia when bacteria in the bloodstream directly deposit in lung tissue. pulmonary aspiration can occur as a result of swallowing dysfunction, gastroesophageal refl ux, anatomic anomalies such as tracheoesophageal fi stulas, or an inability to protect the airway from oropharyngeal secretions. in the picu, many patients have neurologic diseases that coexist with one, if not several, of these aforementioned mechanisms. furthermore, impaired consciousness, as may occur with head injury, intoxication, sedation, and tracheal intubation, can also impair the ability to protect the airway, diminish the cough refl ex, and exploit the patency of the anatomical connection between the larynx and trachea [ 9 , 10 , 18 ] . direct aspiration of a large inoculum of infectious organisms can result when there is impairment of the host's anatomic defense, usually the gag and cough refl ex. this most commonly occurs in children with profound neurologic impairment or during tracheal intubation [ 19 , 20 ] . viruses still remain the most common cause of lower respiratory tract infection, especially in infants [ 21 ] . the occurrence of primary viral infections and co-infections with bacterial pneumonia are receiving more attention in recent years due to advances in detection methods to improve the reliability and sensitivity in diagnosis [ 22 ] . viruses have been found in approximately 50 % of sampled patients with a range of 43-67 %, although this prevalence is diffi cult to compare across studies that utilize different identifi cation techniques [ 22 -28 ] . the most commonly noted infectious viruses were rhinovirus, human bocavirus, human metapneumovirus (hmpv), and respiratory syncytial virus (rsv). human metapneumovirus causes signifi cant respiratory infection, accounting for 5-8 % of viral pneumonia cases [ 29 , 30 ] . human bocavirus, fi rst described in 2005, is detected in up to 10 % of children with respiratory infections [ 31 ] . however, co-infection with another virus occurs in more than half of human bocavirus infected children, making its role as a predominant respiratory pathogen unclear. one possible explanation for the high prevalence of viral coinfection with human bocavirus is that this virus is shed in respiratory tract secretions for a longer period of time than other viruses [ 32 -34 ] . other important respiratory tract pathogens include adenovirus, parainfl uenza viruses, and infl uenza a or b, all of which vary in prevalence based on season and epidemic periods. the most common complication of viral pneumonia is a secondary bacterial infection. bacterial co-infection occurs in about 15-33 % of pediatric patients hospitalized with a lower respiratory tract infection [ 23 ] . the most often occurring combination was rhinovirus and streptococcus pneumoniae , though it remains diffi cult to interpret the causal role of rhinovirus in lower respiratory tract infections [ 23 , 25 ] . rsv remains an important cause of bronchiolitis in infants and can often progress to pneumonia. a recent study noted that 40 % of children admitted to the picu with rsv bronchiolitis had bacterial co-infection [ 35 ] . community-acquired bacterial pneumonia continues to be most prevalent in younger children as well, most often affecting children less than 5 years of age who are otherwise healthy. despite immunizations and public health initiatives, the most common bacterial causes of cap have remained largely unchanged over the last several decades and include: streptococcus pneumoniae , staphylococcus aureus , haemophilus infl uenzae (including non-typable strains) and moraxella catarrhalis [ 7 , 8 , 21 , 23 ] . in developing countries, other bacterial and viral etiologies must be considered, including mycobacterium tuberculosis, h. infl uenzae type b (in unvaccinated areas of the world), and the measles virus [ 8 ] . in infants under 3-4 weeks of life, the most common etiologic agents include group b streptococcus, listeria monocytogenes, and gram-negative enteric bacteria. mycoplasma pneumoniae and chlamydophila pneumoniae (formerly chlamydia pneumoniae ), once considered to occur primarily among adolescents and young adults, are increasing being recognized as a cause of cap in younger children, including those less than 5 years of age [ 21 ] . there are many causes of immunodefi ciency in pediatrics including congenital, acquired (hiv/aids), or iatrogenic (during chemotherapy or after solid organ or stem cell transplant). these states can result in defi ciencies in humoral immunity, cellular immunity, and neutrophil availability or function, making the host susceptible to not only typical pneumonia etiologies, but many opportunistic agents. thus, the approach to an immunocompromised patient must be altered to consider the type and severity of immunodeficiency, as well as the temporal pattern after chemotherapy or transplant. other considerations that are important in immunocompromised patients include neutropenia, where a low white blood cell count can hinder the patient's ability to exhibit cxr fi ndings and the lack of infl ammation can alter the clinical presentation, and environmental factors and exposures that can cause geographic and temporal clustering of pathogens [ 11 ] . the causes of pneumonia following solid organ and stem cell transplant may follow a predictable temporal relationship. in the early post-transplant period (<1 month), infections from nosocomial or iatrogenic sources are most common. in the middle post-transplant period (1-6 months), donor-associated and opportunistic infections, including reactivation of latent infections, predominate; specifi c causes include cytomegalovirus (cmv), epstein-barr virus (ebv) or human herpes virus 6 (hhv6). late post-transplant period (>6 months) etiologies include community-acquired infections as well as infections associated with profound immunosuppression [ 36 , 37 ] . in an effort to diminish the risk associated with post-transplant immunosuppression, immunosuppressive agents (e.g., calcineurin inhibitors, high-dose corticosteroids) are used sparingly when possible and most protocols include anti-viral (especially cmv), anti-fungal, and pneumocystis jiroveci (pcp) prophylaxis [ 36 ] . still, many common infections continue to pose a great risk. for example, viral infections (e.g., rsv, infl uenza, adenovirus) cause greater virulence following solid organ or stem cell transplantation immediately after transplant when cellular immunity is profoundly low. later in the course of transplantation, fungi such as aspergillus spp. and candida spp. become more prevalent causes of pneumonia with long-term steroid therapy [ 11 , 37 ] . thus, when a pulmonary process is suspected, aggressive treatment with broad-spectrum antibiotics, antifungals, and antivirals must be employed. immunocompromised patients with pulmonary infi ltrates may rapidly progress to respiratory failure and, thus, often require icu care. infection must be aggressively treated without delay, but other conditions must also be sought including pulmonary hemorrhage, malignancy, idiopathic pneumonitis, or cardiac disease [ 11 , 38 ] . the clinical presentation of aspiration pneumonitis or pneumonia can vary and like other pneumonia etiologies, aspiration can result in acute lung injury (ali) or acute respiratory distress syndrome (ards) manifested by severe pulmonary infl ammation and alveolar-capillary permeability injury. it is estimated that approximately one-third of patients with aspiration pneumonitis develop ali/ards [ 39 ] . etiologies of aspiration pneumonia depend if the aspiration is community acquired or hospital acquired. bacteriologic studies in aspiration patients have shown that community acquired aspiration pneumonias are generally the same bacterium as cap, including h. infl uenzae , s. pneumoniae , s. aureus , and enterobacteriaceae species. in those patients who aspirated in a hospital setting, the most common organisms cultured were gram-negative enteric bacteria including pseudomonas aeruginosa . these recent studies failed to grow any anaerobic organisms, refuting the prior studies that endorsed anaerobes as common etiologies [ 10 ] . the role for imaging in pediatric pneumonia is to detect the presence of pneumonia, determine the location and extent, and identify complications such as effusion or empyema. modalities include chest radiographs (cxr), ultrasound (us), and computed tomography (ct) [ 11 ] . the presence of an infi ltrate on cxr, combined with clinical and other laboratory fi ndings can aid in the diagnosis of pneumonia. however, these modalities are not suffi ciently sensitive or specifi c to reliably differentiate between viral, bacterial, and atypical bacterial causes [ 40 ] . the main use for us is to identify and characterize a parapneumonic effusion or empyema and provide image guidance for chest tube placement. this modality is limited by availability of equipment and operators. chest ct is helpful to further evaluate diffi cult cases, particularly immunocompromised children with ill-defi ned infi ltrates on cxr, complex empyema or effusion, or recurrent or chronic pneumonia [ 11 ] . imaging fi ndings in pneumonia can be non-specifi c, but when combined with other factors such as patient age, immune status, and historical information, they may help to narrow the differential diagnosis. in viral pneumonia, the most common fi ndings are bilateral symmetrical parahilar and bronchial opacities with or without atelectasis and air trapping; pleural effusions are rare ( fig. 6.1 ). this is in contrast to bronchopneumonia, a form of bacterial pneumonia that begins as peribronchiolar infl ammation and spreads to the lung parenchyma. bacterial pneumonia is characterized by consolidation and fi lling of the alveolar air spaces with exudate, infl ammation, and fi brin. bronchopneumonia is typical of many bacteria including s. pneumoniae , h. infl uenzae , s. aureus , and gram-negative enteric bacteria. the cxr often reveals fl uffy lobar consolidation or diffuse bilateral opacities extending peripherally, with or without associated pleural effusion. in aspiration pneumonia, the cxr may reveal ground-glass or consolidative opacities predominantly involving the middle and lower (dependent) lobes [ 41 ] . finally, atypical pneumonia etiologies include mycoplasma pneumoniae , chlamydophila pneumoniae and, less commonly, legionella species. the cxr fi ndings for these atypical causes are varied. diffuse interstitial infi ltrates are characteristic though other fi ndings include lobar consolidation, small bilateral pleural effusions, perihilar and peribronchial opacities that resemble butterfl y wings, or a bi-lobar reticular pattern ( fig. 6. 2 ) [ 42 , 43 ] . the etiology of pneumonia in the immunocompromised patient can be diffi cult to determine though further imaging can help elucidate the cause. respiratory failure in an immunocompromised child frequently necessitates a chest ct to better visualize the pattern and extent of disease, aid in diagnosis of the etiology, determine the need for more invasive procedures, and to increase the sensitivity of assessing treatment response [ 11 ] . fungal infections are more diffi cult to diagnose; classic fi ndings include pulmonary nodules on chest ct (fig. 6.3 ). the "gold standard" diagnosis of pneumonia is microbiological identifi cation of a pathogen from the lower respiratory tract [ 2 ] . obtaining a lrt specimen can be diffi cult, especially in children, as it may require an invasive procedure and can be contaminated with oropharyngeal bacteria. most children younger than 8 years of age cannot produce a suffi cient sputum sample, defi ned as <10 squamous or epithelial cells and >25 polymorphonuclear white blood cells per low power fi eld. therefore, most samples are obtained through either an endotracheal tube via aspiration or bronchoalveolar lavage [ 44 ] . other laboratory tests helpful in identifying the causative agent in cap can include blood cultures, viral polymerase chain reaction (pcr) tests, and bacterial serologies. commonly used diagnostic methods available for an individual microorganism may be found in table 6 .2 [ 8 ] . the clinician may also be limited by the capabilities of the laboratory in their institution for performing these tests. because of the diffi culties in determining the etiology of pneumonia, non-microbiologic approaches have been sought to differentiate serious bacterial infections from nonbacterial pneumonia [ 21 ] . many studies have evaluated markers including serum c-reactive protein (crp), blood white cell count (wbc), serum procalcitonin (pct), and erythrocyte sedimentation rate (esr), attempting to fi nd a test, or combination of tests, that would differentiate viral pneumonia from serious bacterial pneumonia necessitating antibiotic therapy [ 8 , 45 -49 ] . all of the aforementioned tests have limited utility in reliably differentiating viral from bacterial pneumonia, but when one or more of the markers are signifi cantly elevated, a bacterial etiology is more likely. thus, taken together with the clinical examination and radiologic fi ndings, these tests can aid the clinician in deciding which patients require antibiotic therapy. pct levels appear to be more sensitive than wbc, esr, and crp in identifying children with bacterial pneumonia and have been used to identify children who may benefi t from a longer duration of antibiotic therapy [ 50 ] . when non-invasive identifi cation techniques are inadequate, or when identifying the cause is especially important, such as when treating an immunocompromised host, invasive diagnostic procedures may be necessary. fiberoptic bronchoscopy with bronchoalveolar lavage (bal) is the preferred diagnostic procedure in an immunocompromised host with an unknown pathogen [ 51 ] . the sensitivity for diagnosis varies and depends on the host, pathogen, and the post-collection microbiologic detection methods employed. while many atypical organisms may be diffi cult to culture, p. jiroveci and mycobacterium infections are more easily detected in bal because of high organism burden in the lungs. the diagnosis of aspiration pneumonia is mainly clinical, often based on historical or witnessed events or conditions, and thus can be diffi cult to ascertain. if a bal is performed in suspected aspiration, the presence of lipid-laden macrophages can help diagnose the aspiration of lipophilic foods such as formula [ 52 ] . a lipid-laden macrophage index can be obtained using the oil red o stain and when high, can be very sensitive and specifi c for aspiration [ 53 ] . other invasive procedures include transbronchial biopsy if diffuse infi ltrates are present but the bal is negative, or ct-guided needle biopsy of a focal lesion. the improved diagnosis with these invasive procedures must be balanced against the risks to critically ill patients [ 54 ] . important noninfectious etiologies to rule out with these invasive procedures include lung rejection (if transplanted), post-engraftment syndrome, idiopathic pneumonitis, graft versus host disease, and bronchiolitis obliterans. children with severe pneumonia requiring admission to the picu are likely to receive intravenous antimicrobial therapy even if only until the possibility of bacterial infection can be excluded. in critically ill children with respiratory failure from pneumonia, prompt initiation of broad-spectrum antimicrobials is crucial. one study in pediatric patients with cap showed that longer delays in receipt of antibiotics were independently associated with adverse outcomes [ 55 ] . however, antibiotic resistance is increasing and the principles of appropriate antibiotic utilization must be adhered to: use of drug with narrowest spectrum, aiming for high tissue penetration, short half-life, and abiding to a short, intense duration of therapy [ 7 ] . the duration of therapy is typically 7-14 days, with 10 days being the best studied. a 7-day course may be reasonable in non-severe cases of pneumonia [ 12 ] . the choice of antimicrobial agent is based on many things including the patient's age, the type of pneumonia, and clinical and epidemiologic factors. recent guidelines published by the pediatric infectious diseases society and the infectious diseases society of america offer guidance for empiric antibiotic selection in children hospitalized with cap (table 6. 3 ) [ 12 ] . pneumonia causes a profound infl ammatory response in the lungs and it has long been postulated that regulating this infl ammation with steroid therapy may help to modulate local tissue damage and accelerate recovery for the patient. in addition, steroids are frequently utilized in other pulmonary infl ammatory conditions such as reactive airway a b the fi nding of at least a quadrupling of serum antibody levels between the acute phase and convalescence the fi nding of igm antibody in serum late in the acute phase or early in convalescence is helpful, as is a positive pcr assay of secretions from a throat or a nasopharyngeal swab rapid igm assays can provide results within 10 min. in younger children, an elevated igm titer is often diagnostic; in older children, the fi nding of at least a quadrupling of serum antibody levels between the acute phase and convalescence is diagnostic. cold agglutinin titers lack sensitivity and specifi city and thus are no longer recommended separate serum specimen because some agents (e.g., piperacillintazobactam) may cross-react with the assay. if invasive aspergillosis is suspected in high-risk patients, serial sampling is recommended. the false positive rate is higher in children than adults [ 91 , 92 ] adapted from mcintosh [ 6 ] . with permission from massachusetts medical society disease (rad) and acute respiratory distress syndrome (ards) [ 56 ] . the infl ammatory responses in pneumonia and ards are similar with increases in pro-infl ammatory cytokines concurrent with illness severity; severe pneumonia can often progress to acute lung injury (ali) or ards [ 57 -59 ] . while preclinical data support the use of steroids, current studies have not demonstrated a reduction in mortality among corticosteroid recipients compared with non-recipients. several trials, however, have shown some secondary benefi ts of steroids, including reduced length of hospital stay and reduced infl ammatory markers [ 60 , 61 ] . in contrast, a multi-center, retrospective cohort study using administrative data found that among patients not receiving concomitant beta-agonist therapy (used as a proxy for wheezing), corticosteroid recipients had a longer los and higher readmission rate compared with non-recipients [ 62 ] . at present, the lack of high quality data supporting the effi cacy of corticosteroids prevents the recommendation for the use of steroids in most patients with severe pneumonia. however, corticosteroids may provide benefi t to certain subgroups of patients such as those with acute onset of wheezing and those who meet the criteria for ali/ards [ 59 ] . macrolide antibiotics have important anti-microbial as well as anti-infl ammatory properties, though the relative importance of these two mechanisms in children with pneumonia is unknown. in adult studies, macrolides have recently been touted for their immunomodulatory effects and clinical benefi t in multiple chronic pulmonary conditions such as asthma, chronic obstructive pulmonary disease (copd), and cystic fi brosis (cf). the specifi c immunomodulatory effects are vast and include inhibition of intracellular signaling to suppress the production of transcription factors such as nf-îºb and decrease production of infl ammatory cytokines that recruit neutrophils [ 63 , 64 ] . several recent studies in adult patients with severe cap and sepsis have shown a benefi t in survival in patients treated with macrolide antibiotics in addition to the recommended antibiotics based on pathogen [ 63 , 65 -68 ] . the role of macrolides in children with pneumonia is unclear. in pediatrics, several small retrospective studies have shown that among children with atypical cap, those treated with macrolides were less likely to have persistence of signs and symptoms after 3 days of therapy [ 69 , 70 ] . among children with m. pneumoniae infection, lu et al. found a shorter duration of fever among macrolide recipients compared with non-recipients [ 71 ] . finally, a large multi-center study of 690 patients with m. pneumoniae infection defi ned by discharge diagnosis codes, the median length of hospital stay was 3 days (interquartile range, 2-6 days); macrolide recipients had a 32 % shorter length of stay compared with non-recipients [ 72 ] . pneumonia-associated complications such as empyema affect 7.5-15 % of children hospitalized with pneumonia [ 5 , 73 -76 ] . the progression from simple parapneumonic effusion to empyema occurs in stages that represent a continuous spectrum (table 6 .4 ) [ 77 ] . in the fi rst stage, there is a rapid infl ux of exudative fl uid into the pleural space as a result of increased pulmonary interstitial fl uid traversing the pleura and an increase in vascular permeability due to pro-infl ammatory cytokines. the pleural fl uid is marked by the absence of bacteria, fl uid ph >7.20, normal glucose, and ldh <3 times the upper limit of normal. at this stage, drainage is not generally required for resolution but if the effusion becomes large and piperacillin-tazobactam if concern for gram negative enteric bacteria iv cefotaxime if >20 days of age adapted from refs. [ 8 , 12 ] impairs respiratory mechanics, drainage might become necessary. the fl uid in the pleural space can fl ow freely and often layers along the lateral chest wall in decubitus fi lms or along the posterior chest wall in supine fi lms [ 37 , 78 ] ( fig. 6.4a , b) . if left untreated, exudative effusions can progress to fi bropurulent effusions characterized by the new presence of bacteria or positive microbial cultures. cellular lysis and phagocytosis in the fl uid can result in ph < 7.20, higher ldh, and low glucose. loculations begin to develop, causing these effusions to now be referred to as "complicated." a chest radiograph may be diffi cult to interpret with respect to evidence of complicated effusions. thoracic us is more accurate than chest radiographs in distinguishing simple from complicated pleural effusions. complicated effusions are associated with fl oating debris and echogenic material or septations. ultrasound is also useful in guiding pleural aspiration and drainage. chest computed tomography (ct) may be indicated to better defi ne pulmonary and pleural anatomy. thickening of the parietal pleura on a contrasted ct scan is suggestive of empyema, even if the effusions are small in size ( fig. 6.4c ) . finally, stage three is the organizing phase where fi broblasts grow into the pleural space and eventually results in a pleural peel, restricting chest mechanics. this stage often necessitates surgical decortication, especially if there is restrictive impairment [ 78 ] . the typical organisms responsible for the development of an empyema include s. pneumoniae and s. aureus . pleural fl uid cultures identify an organism in only 20-30 % of children with empyema. blood cultures are positive in 13-30 % of children with empyema [ 79 -82 ] . s. aureus is most often identifi ed in pleural fl uid culture. however, molecular identifi cation techniques reveal that most culture-negative cases are attributable to s. pneumoniae [ 83 , 84 ] . regardless of the type of effusion present, antibiotic coverage based on treatment guidelines for pneumonia are essential. a recent study on the impact of early antibiotic therapy on the laboratory analysis of pleural fl uid found that pre-treatment signifi cantly hindered a bacterial diagnosis but did not alter the biochemical parameters of the fl uid [ 85 ] . however, delaying antibiotic treatment for a thoracentesis would not be recommended in a critically ill child with respiratory failure secondary to pneumonia. the treatment of complicated effusions and empyema remains controversial but recent studies have better defi ned protocols. a complete list of the available treatments for effusions and empyema is found in table 6 .5 . small, uncomplicated pleural effusions do not routinely require drainage. moderate or large pleural effusions as well as those with evidence of septations or loculations usually require drainage. the medical options include appropriate antimicrobials and chest tube insertion with or without fi brinolytic therapy. surgical options include video-assisted thoracoscopic surgery (vats) or open thoracotomy and decortication. recent guidelines concluded that chest tube drainage with the addition of fi brinolytic agents and vats are equivalent methods of treatment and emphasize the importance of local expertise in determining the optimal approach for individual patients [ 12 , 86 ] . vats has gained popularity over conservative medical therapy as a way to directly visualize the pleural space, mechanically disrupt the adhesions, and strategically place the chest tube for optimal drainage [ 73 , 87 ] . the higher cost and risk of anesthesia with vats must be balanced against the more frequent requirement for additional drainage procedures for those undergoing primary chest tube placement. thoracotomy and decortication are rarely needed. the argument of medical management versus surgical management remains controversial. to date, at least two prospective trials in pediatrics have been completed directly comparing these methods. both trials failed to show any outcome superiority with surgical management [ 80 , 88 ] . certainly children who have a very high white blood cell count in their pleural fl uid (> 15,000), poor output drainage by chest tube, low pleural ph, the presence of bacteria in the pleural fl uid and/or bloodstream, or failure of medical therapy alone may benefi t from early vats [ 86 ] . patients who underwent vats required fewer adapted from refs. [ 12 , 78 , 93 ] additional drainage procedures, but had no difference in hospital length of stay [ 74 ] . however, one study of adults with empyema found that patients treated with a combination of tpa and recombinant human dnase required fewer surgical interventions and had a shorter length of hospital stay [ 89 ] . cost-effectiveness, balance of risks, and availability of resources also plays a role in considerations for surgical management. a comparison of multiple strategies for pediatric empyema noted that the most cost effective method was insertion of a chest tube with fi brinolytic therapy [ 90 ] . abscesses develop in localized areas of parenchymal infection that becomes necrotic and cavitates ( fig. 6.5a , b ) . primary lung abscesses can develop either in previously healthy children or in children with underlying lung disease such as congenital cystic lesions, cystic fi brosis, or immunodefi ciency. mechanisms for abscess development can include direct aspiration of infectious material, embolic phenomena, hematogenous spread from septicemia, or local extension from abdominal or oropharyngeal processes. the most common organisms include gram-positive bacteria such as streptococci, staphylococcus aureus or anaerobes. most abscesses resolve with intravenous antibiotics alone, but aspiration or drainage with a pigtail catheter may be necessary [ 37 ] . vaccines against specifi c bacteria that predominantly cause pneumonia in children, specifi cally pneumococcal conjugate vaccine (pcv-7) and h. infl uenzae vaccine (hib) have drastically lowered the prevalence of infections causes by these strains. since the introduction of pcv-7, several studies have documented its effi cacy, and the decrease in cases of h. infl uenzae are equally striking [ 7 , 21 ] . however, while pcv-7 has decreased the prevalence of invasive pneumococcal disease, the incidence of empyema is rising, the reason for which is unclear [ 76 ] . the licensure of pneumococcal conjugate vaccines that include even more serotypes (e.g., 13-valent) may further change the epidemiology of childhood pneumonia. other vaccines, such as for measles (mmr) and infl uenza, can also aid to reduce these viral infections that so commonly lead to secondary bacterial pneumonia. while vaccines appear to be our greatest effort toward preventing pneumonia in children, more work needs to be done to increase their microbial coverage and availability throughout the world. operative technique in which a small camera and instruments are inserted into the pleural space through 2-3 small (1-2 cm) incisions of the skin and muscle on the lateral chest wall to mechanically remove purulent material and pleural adhesions. a thoracostomy tube is placed through one of the existing incisions following completion of the procedure open thoracotomy operative technique where instruments are inserted into the pleural space through a single 5-8 cm incision of the skin and muscle on the posterolateral chest wall to mechanically remove purulent material and pleural adhesions. a thoracostomy tube is placed through a second smaller 1-2 cm incision following completion of the procedure general anesthesia reprinted from swami and shah [ 43 ] . with permission from mcgraw-hill three decades of pediatric intensive care: who was admitted, what happened in intensive care, and what happened afterward defi ning pneumonia in critically ill infants and children world health organization (who) childhood pneumonia mortality-a permanent global emergency national hospitalization trends for pediatric pneumonia and associated complications ambulatory visit rates and antibiotic prescribing for children with pneumonia pneumonia and other respiratory infections community-acquired pneumonia in children pulmonary complications of pediatric neurological diseases aspiration pneumonitis and aspiration pneumonia pneumonia in normal and immunocompromised children: an overview and update the management of community-acquired pneumonia in infants and children older than 3 months of age: clinical practice guidelines by the pediatric infectious diseases society and the infectious diseases society of america infectious diseases society of america/ american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults airway mucus function and dysfunction beyond infl ammation: airway epithelial cells are at the interface of innate and adaptive immunity the role of particle size in aerosolised pathogen transmission: a review distribution of airborne infl uenza virus and respiratory syncytial virus in an urgent care medical clinic aspiration lung disease the impact of tracheal intubation on host defenses and risks for nosocomial pneumonia feeding the disabled child community-acquired pneumonia: a review and recent advances viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection viruses and bacteria in sputum samples of children with community-acquired pneumonia viral pneumonia induced sputum in the diagnosis of childhood community-acquired pneumonia etiology of community-acquired pneumonia in 254 hospitalized children etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections etiology of community-acquired pneumonia in hospitalized children based on who clinical guidelines the changing face of pediatric respiratory tract infections: how human metapneumovirus and human bocavirus fi t into the overall etiology of respiratory tract infections in young children population-based incidence of human metapneumovirus infection among hospitalized children human bocavirus human bocavirus in children with acute lymphoblastic leukemia frequent and prolonged shedding of bocavirus in young children attending daycare human bocavirus: passenger or pathogen in acute respiratory tract infections? high incidence of pulmonary bacterial coinfection in children with severe respiratory syncytial virus (rsv) bronchiolitis infection in solid-organ transplant recipients respiratory infections: pneumonia, lung abscess, and empyema approach to the immunocompromised host with infection in the intensive care unit aspiration-induced lung injury community-acquired pneumonia in children: what's old? what's new? lipoid pneumonia: spectrum of clinical and radiologic manifestations pulmonary infections pediatric practice: infectious diseases murray and nadel's textbook of respiratory medicine value of the c-reactive protein test in the differentiation of bacterial and viral pneumonia serum procalcitonin, c-reactive protein and interleukin-6 for distinguishing bacterial and viral pneumonia in children differentiation of bacterial and viral communityacquired pneumonia in children white blood cells, c-reactive protein and erythrocyte sedimentation rate in pneumococcal pneumonia in children non-specifi c host response markers in the differentiation between pneumococcal and viral pneumonia: what is the most accurate combination? procalcitonin measurements for guiding antibiotic treatment in pediatric pneumonia role of fl exible bronchoscopy in immunocompromised patients with lung infi ltrates chronic pulmonary aspiration in children lipid-laden macrophages in induced sputum are a marker of oropharyngeal refl ux and possible gastric aspiration open lung biopsy in pediatric bone marrow transplant patients timing of correct parenteral antibiotic initiation and outcomes from severe bacterial community-acquired pneumonia in children activation and regulation of systemic infl ammation in ards: rationale for prolonged glucocorticoid therapy understanding the infl ammatory cytokine response in pneumonia and sepsis: results of the genetic and infl ammatory markers of sepsis (genims) study infl ammatory markers at hospital discharge predict subsequent mortality after pneumonia and sepsis steroids in severe pneumonia: a literature review dexamethasone and length of hospital stay in patients with community-acquired pneumonia: a randomised, double-blind, placebo-controlled trial hydrocortisone infusion for severe community-acquired pneumonia: a preliminary randomized study adjunct corticosteroids in children hospitalized with community-acquired pneumonia immunomodulatory agents in the treatment of community-acquired pneumonia: a systematic review mechanisms of action and clinical application of macrolides as immunomodulatory medications combination antibiotic therapy improves survival in patients with community-acquired pneumonia and shock monotherapy may be suboptimal for severe bacteremic pneumococcal pneumonia impact of macrolide therapy on mortality for patients with severe sepsis due to pneumonia combination antibiotic therapy with macrolides improves survival in intubated patients with communityacquired pneumonia role of mycoplasma pneumoniae and chlamydia pneumoniae in children with community-acquired lower respiratory tract infections characteristics of streptococcus pneumoniae and atypical bacterial infections in children 2-5 years of age with community-acquired pneumonia macrolide use shortens fever duration in mycoplasma pneumoniae infection in children: a 2-year experience macrolide therapy and outcomes in a multicenter cohort of children hospitalized with mycoplasma pneumoniae pneumonia primary operative versus nonoperative therapy for pediatric empyema: a meta-analysis comparative effectiveness of pleural drainage procedures for the treatment of complicated pneumonia in childhood primary early thoracoscopy and reduction in length of hospital stay and additional procedures among children with complicated pneumonia: results of a multicenter retrospective cohort study empyema hospitalizations increased in us children despite pneumococcal conjugate vaccine parapneumonic effusions and empyema parapneumonic pleural effusion and empyema blood cultures in the emergency department evaluation of childhood pneumonia thoracoscopic decortication vs tube thoracostomy with fi brinolysis for empyema in children: a prospective, randomized trial impact of the pneumococcal conjugate vaccine on pneumococcal parapneumonic empyema an epidemiological investigation of a sustained high rate of pediatric parapneumonic empyema: risk factors and microbiological associations the changing face of pleural empyemas in children: epidemiology and management molecular analysis improves pathogen identification and epidemiologic study of pediatric parapneumonic empyema impact of antibiotic therapy on laboratory analysis of parapneumonic pleural fl uid in children management of parapneumonic empyema pediatric respiratory diseases: 2011 update for the rogers' textbook of pediatric intensive care comparison of urokinase and video-assisted thoracoscopic surgery for treatment of childhood empyema intrapleural use of tissue plasminogen activator and dnase in pleural infection cost-effectiveness of competing strategies for the treatment of pediatric empyema diagnostic aspects of invasive aspergillus infections in allogeneic bmt recipients diagnostic potential of nested pcr, galactomannan eia, and beta-d-glucan for invasive aspergillosis in pediatric patients medical and surgical treatment of parapneumonic effusions : an evidence-based guideline key: cord-009193-244ii7e2 authors: giancane, gabriella; swart, joost f.; castagnola, elio; groll, andreas h.; horneff, gerd; huppertz, hans-iko; lovell, daniel j.; wolfs, tom; herlin, troels; dolezalova, pavla; sanner, helga; susic, gordana; sztajnbok, flavio; maritsi, despoina; constantin, tamas; vargova, veronika; sawhney, sujata; rygg, marite; k. oliveira, sheila; cattalini, marco; bovis, francesca; bagnasco, francesca; pistorio, angela; martini, alberto; wulffraat, nico; ruperto, nicolino title: opportunistic infections in immunosuppressed patients with juvenile idiopathic arthritis: analysis by the pharmachild safety adjudication committee date: 2020-04-07 journal: arthritis res ther doi: 10.1186/s13075-020-02167-2 sha: doc_id: 9193 cord_uid: 244ii7e2 background: to derive a list of opportunistic infections (oi) through the analysis of the juvenile idiopathic arthritis (jia) patients in the pharmachild registry by an independent safety adjudication committee (sac). methods: the sac (3 pediatric rheumatologists and 2 pediatric infectious disease specialists) elaborated and approved by consensus a provisional list of oi for use in jia. through a 5 step-procedure, all the severe and serious infections, classified as per meddra dictionary and retrieved in the pharmachild registry, were evaluated by the sac by answering six questions and adjudicated with the agreement of 3/5 specialists. a final evidence-based list of oi resulted by matching the adjudicated infections with the provisional list of oi. results: a total of 772 infectious events in 572 eligible patients, of which 335 serious/severe/very severe non-oi and 437 oi (any intensity/severity), according to the provisional list, were retrieved. six hundred eighty-two of 772 (88.3%) were adjudicated as infections, of them 603/682 (88.4%) as common and 119/682 (17.4%) as oi by the sac. matching these 119 opportunistic events with the provisional list, 106 were confirmed by the sac as oi, and among them infections by herpes viruses were the most frequent (68%), followed by tuberculosis (27.4%). the remaining events were divided in the groups of non-oi and possible/patient and/or pathogen-related oi. conclusions: we found a significant number of oi in jia patients on immunosuppressive therapy. the proposed list of oi, created by consensus and validated in the pharmachild cohort, could facilitate comparison among future pharmacovigilance studies. trial registration: clinicaltrials.gov nct 01399281; encepp seal: awarded on 25 november 2011. conclusions : we found a significant number of oi in jia patients on immunosuppressive therapy. the proposed list of oi, created by consensus and validated in the pharmachild cohort, could facilitate comparison among future pharmacovigilance studies. trial registration: clinicaltrials.gov nct 01399281; encepp seal: awarded on 25 november 2011. keywords: infections, opportunistic, juvenile idiopathic arthritis, immunosuppressive therapy, biologics with the advent of biologic disease-modifying antirheumatic drugs (dmards), in a chronic condition like juvenile idiopathic arthritis (jia), regulatory authorities such as the food and drug administration (fda) and the european medicines agency (ema) have demanded from pharmaceutical companies and clinical researchers to evaluate the long-term safety of drugs used in children enrolled in phase ii-iii clinical trials [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] . due to the limited number of patients enrolled in these trials [17] , clinical researchers have devoted their work to the implementation of national and international registries [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] or to the analysis of insurance claim data [29] [30] [31] . during their development, all children experience a natural rate of infections compared to adults. treatments in jia with synthetic and biologic dmards are expected to increase the frequency of common infections and the risk of serious and opportunistic infections (oi) [23, [30] [31] [32] [33] [34] , including especially tuberculosis in some geographic areas [35] [36] [37] . in order to tackle the long-term safety and efficacy evaluations, the paediatric rheumatology international trials organization (printo) started in 2011 the "pharmacovigilance in juvenile idiopathic arthritis patients" (pharmachild), an observational international registry supported by a european union grant [38, 39] . recent literature seems to confirm the high incidence of infections among jia patients treated with immunosuppressants [21] , but conclusive data are not available, yet. in particular, little evidence exists about the role of jia or its immunosuppressive therapy in acquiring oi. several studies in the literature have the objective to define and classify oi, for example in hiv or in cancer patients [40] [41] [42] [43] . in rheumatology, winthrop and colleagues [32] were the first to convene a consensus meeting in 2015 to review the published literature on oi in patients with immune-mediated diseases treated with biologic dmards, in order to provide consensus recommendations for their evaluation in the context of clinical trials and observational studies. primary objectives of the present study were to derive a consensus-based list of opportunistic pathogens for use in children with jia and confirm its role in identifying oi through the evaluation of the infectious events reported in pharmachild registry by an independent safety adjudication committee (sac). the pharmachild registry (project number 260353) involves 86 participating printo (www.printo.it) centers in 32 countries [38] and the paediatric rheumatology european society (pres at www.pres.eu), with the aim to (1) monitor children with jia for disease activity and comorbidity; (2) compare the long-term incidence rates of moderate, severe, and very severe adverse events (ae) and serious ae (sae); and (3) assess the long-term efficacy of biologic and synthetic dmards in jia. the pharmachild registry contains both a retrospective and a prospective cohort. in brief, the retrospective cohort includes data from patients under treatment or previously treated with dmards obtained by one-time clinical chart review for safety events and complete drug exposure since disease onset to last available follow-up; the prospective cohort includes all cases newly treated with dmards since enrollment in the registry and cases still under treatment with any drug. in case of repeated events (e.g., infection with multiple reporting in the registry for the follow-up evaluation), only the initial event was considered. full details of the registry methodology are available elsewhere [39] . the study was divided into 5 main steps (additional figure 1). step 1: provisional listing of opportunistic pathogens/ infection presentations the study steering committee (sc) included two phd medical doctors (gg and js), two certified medical dictionary for regulatory activities (meddra) coders (cp, lv), 3 biostatisticians (ap, fb, fb), and two senior researchers (nw, nr). the sac was organized as an independent group of 5 physicians: 2 pediatric infectious disease specialists (ec and ag) and 3 pediatric rheumatologists (gh, hih, dl), who have experience and expertise in the diagnosis and treatment of children with infectious or rheumatic diseases. the sc starting point was the prior work by winthrop et al. [32] , an international consensus committee (infectious disease, public health, and pulmonary physicians and rheumatologists) that recommended a list of definite and probable oi after systematic review of literature on immune-mediated disorders (including jia), and a consensus process. this list was discussed, modified, and approved by consensus by the sac, through three subsequent delphi web rounds, with the final result of a list of opportunistic pathogens/presentations for use in immunosuppressed children with jia. in the first round, sac members worked independently from each other, while during the second round, they could also revise their responses based on the review of comments from the other members. final consensus was reached through a dedicated teleconference (moderator nr). the sc then integrated the review of the literature with more recent evidence on oi in jia [31, 44, 45] and prepared a provisional list of oi pathogens, then matched them with the meddra preferred terms (pt) in order to retrieve correctly the cases from the pharmachild database. this provisional list was not shared with the sac members as it was used only for data retrieval (see next step). for the pharmachild study, the treating physicians reported online in the registry database all aes from the disease onset to the last available follow-up visit. all terms contained in the meddra system organ class (soc) "infections and infestations" were considered in pharmachild as events of special interest (esi) and classified in two different esi sub-groups, named "tuberculosis" and "targeted infections (epstein-barr virus, cytomegalovirus, papilloma virus, herpes zoster primary and reactivation, and opportunistic infections)." according to the pharmachild protocol, all events (aes and esis) of at least moderate intensity and all saes were collected. aes and esis were coded initially by the treating physicians during data entry using the meddra dictionary, then recoded, if needed, by printo-certified meddra coders and revised by the printo medical monitor (js), based on the most current version of meddra. all infectious events (both initial and follow-up) in the meddra system organ class (soc) (additional figure 2) "infection and infestations" in pharmachild as of january 2017 were retrieved (fig. 1) . step 3: adjudication of infections by the sac a standard operating procedure (sop) described the work to be done by the sac. in brief, the sac adjudication process included all the opportunistic events in the provisional list of oi derived by step 1 (any grade of severity) plus the non-oi infections of at least severe intensity and all serious infections from both retrospective and prospective charts. the list of the events to be adjudicated by the sac was provided in a dedicated external area of the printo/pharmachild website, with access through secure personal username and password. the sac members who reviewed all eligible cases (presented in numerical order by patient's code) did not participate in the data collection in pharmachild. the complete patients' data were available for the sac members: (1) demographic characteristics of the patient (with personal data encrypted), (2) ilar category of jia, (3) laboratory and clinical information, (4) complete drug therapy with whole drug exposure for synthetic and biologic dmards since disease onset to the last available observation, (5) concurrent medications at the time of the infectious event, and (6) full ae report plus esi-specific form for infections. in addition, jia disease activity and a damage measure were available for prospective visits. the sac members had the possibility to access the complete clinical information in a read-only mode, with no possibility to modify the original data. a numeric code, without any patient or center identifier and no a priori categorization of ae as oi, was provided to decrease potential bias during the adjudication exercise. the sac mandate was to evaluate each infectious case, based on the whole patient's history available in pharmachild, by answering 5 questions: (1) based on the information provided, do you confirm that this patient had an infection?; (2) is this infection common?; (3) is this an opportunistic infection?; (4) was the treatment appropriate for the infection?; (5) could the event be possibly related to any of the drug(s) taken at the time of the event? the study sc was available to provide any additional information related to the event and required by the sac at any time. the consensus among the sac members was defined as an agreement of at least 3 out of 5 (60%) members, on the first 3 out of 5 adjudication questions ("is this an infection?," "is it common?," "is it opportunistic?"). initially, the sac members worked independently from each other, while in the next phase, for all cases without consensus, each member could access the evaluations of the other sac members. step 4: analysis of the pharmachild registry step 4 was designed to evaluate, in an evidence-based fashion, the frequency of those events in the pharmachild registry classified as infections by consensus among the sac and to assign a final meddra code (high level term (hlt)/pt) to the event. in case of discrepancies in the categorization, after printo and medical monitor (js) check, a third independent examiner (gg) reevaluated the individual case and assigned the final med-dra code (hlt/pt). step 5: final evidence-based listing of opportunistic pathogens/infection presentations in this step, all the infectious events adjudicated by the majority of the sac in pharmachild were matched by meddra pt term with the provisional list of oi (see step 1) and divided in three groups: "confirmed oi," if there was full agreement between the sac and the provisional list of oi; "confirmed non-oi," for the events adjudicated as non-oi by the sac and missing in the provisional list; "possible/patient and/or pathogenrelated oi," for the remaining events in pharmachild that could be possibly considered opportunistic depending on the physician's evaluation of the patient history and by the detection of the specific pathogen causing the disease. descriptive statistics were reported in terms of absolute frequencies and percentages for qualitative data. quantitative data were described in terms of median values and inter-quartile range (iqr) values due to their nonnormal (gaussian) distribution. step 1: provisional list of opportunistic pathogens/ presentations after the three web delphi rounds, the probable and definite definitions of oi were agreed with one major change by 5/5 (100%) of the sac [32] . in particular, the definition of definite oi was confirmed, while for probable infections, it was integrated with the following: "in case of the unusually severe course of infection due to a common pathogen with usually mild disease the pathogen might tentatively be considered opportunistic in a patient with impaired immune function." two definite categories of pathogens/presentations were modified by the sac, while twelve were added in the provisional list of probable oi from the literature and matched with the hlt/pt meddra dictionary; none of the infections already included in the list by winthrop et al. [33] was removed. table 1 shows the provisional list of pathogens/presentations, with the corresponding hlt terms according to meddra dictionary. step 2: retrieval of infections in pharmachild among the 8274 patients enrolled in the pharmachild registry as of january 2017, 895 (10.8%) patients had experienced 1585 infections. a total of 772 events (48.7%) in 572 patients ( fig. 1 and step 3 of the "methods" section) were eligible for the evaluation by the sac, of which 437 were defined as preliminary oi, as per the provisional list of opportunistic pathogens/presentations, and 335 as very severe/severe or serious non-oi events (fig. 1) . the baseline characteristics of the 572/895 (63.9%) adjudicated patients are reported in table 2 in comparison with those who were not adjudicated. among the 895 patients with infections, about 85% were from europe, specifically 29.3% from italy and 23.6% from the netherlands, while the remaining patients were distributed among russia (8%), south america (4%), middle east, and india (3%). the adjudicated group was represented by younger patients, with longer disease duration, higher frequency of systemic jia, and more frequent use of systemic glucocorticoids. step 3: adjudication of infections by the sac a total of 689/772 (89.2%) events achieved consensus (3/5 sac members) on the first 3 adjudication questions, and of these, 682 (99.0%) were considered as infections by the sac and included in the analysis ( table 3 ). the majority of the 682 infections were considered common (88.4%), with 119 (17.4%) classified as opportunistic by the sac after evaluation of the whole patient's history. the consensus on the last 2 questions was more difficult to reach. regarding the fourth question on the appropriateness of the treatment for the infection, consensus was achieved for 484 (77.1%) events, while for 140 (22.3%) of the cases, it was impossible to determine the suitability of the anti-infective treatment. similarly, for the fifth question about the possible relationship between the infection and the related treatment(s) for jia, the lack of consensus increased up to 279 (41%). for 307/403 (76.2%) cases for which there was consensus, the sac considered the drug(s) possibly related to the event. when we considered the drugs administered at the time of infection, the association of 1 biologic (more commonly etanercept or adalimumab) plus 1 synthetic dmard was the most frequently reported (32% of the cases), followed by methotrexate alone (21%) and etanercept alone (20.3%), and finally by the association of either 1 biologic plus 1 synthetic dmard plus systemic glucocorticoids (9%) or 1 synthetic dmard plus systemic glucocorticoids (3.7%). step 4: analysis of the infections according to meddra dictionary the evaluation of the pharmachild registry conducted by the sac led to the adjudication of the 682 infections corresponding to 53 hlts and 153 pts. for 92 (60%) pts, the expert committee confirmed the same pt used by the pharmachild medical monitor, while for the remaining 40%, discrepancies were solved by the study sc after re-evaluation of the individual cases. the final number of hlts was 50, with corresponding 149 pts, showed in details with the frequency of the events in the additional table 1. step 5: final evidence-based listing of opportunistic pathogens/infection presentations after matching the adjudicated events with the provisional list of oi, among the 682 events, 106 (15.5%) for 22 pt were classified as "confirmed oi," 274 (40.2%) for 89 pt were classified as "confirmed non-oi," and 302 (44.3%) for 38 pt were classified as "possible/patient and/or pathogen-related oi." table 4 shows the frequency of the 106 "confirmed oi" by hlt/pt in 93 patients. regarding pathogens, herpes viral infections resulted the most frequent hlt/ pt category, with 72 events (68% of the total confirmed oi), mostly represented by herpes zoster infection (66/ 72, 91.6%). among the 64 patients with 72 confirmed herpes zoster infections, 35/64 (54.7%) had varicella in the past history and later developed herpes zoster (34 patients) and herpes zoster oticus (1 patient). one out of 35 patients, who had been vaccinated against varicella zoster had varicella in the past, and later zoster infection. two additional patients had been vaccinated for varicella among those who developed zoster infection without having varicella reported in the history. tuberculosis, candida, papilloma, and pneumocystis followed with a frequency higher than 3%. among the 4 papilloma viral infections, affecting 2 patients in their history, no one was preceded by hpv vaccination. of all the 29 mycobacterium tuberculosis infections in pharmachild (additional table 1), only 11/29 (38%) were "confirmed oi," mostly in female patients (70%), at a median age of 5.2 years, not previously vaccinated with bcg, with published data is currently lacking, but expert opinion believes that risk is likely elevated in the setting of dmard therapy. in case of the unusually severe course of infection due to a common pathogen with usually mild disease the pathogen might tentatively be considered opportunistic in a patient with impaired immune function. below there is a non-exhaustive list of possible pathogens list of probable pathogens and/or presentations of specific pathogens pulmonary or disseminated presentations. the remaining were either latent tuberculosis or not wellspecified contact with the pathogen, classified by the sac as "possible/patient and/or pathogen-related oi." the majority of the "confirmed oi" was reported in europe (75.5%), while 11.3% was reported in russia, 9.4% in brazil, and 1.9% in india and israel. these events occurred after a median period of 5.3 years from disease onset (iqr 3.4-9.2). scanty data were reported on the immune status of the patients with "confirmed oi" at the moment of infection and soon afterwards. for 17.8% (10/93) of the patients with "confirmed oi," there was evidence of lymphocytes below 500/î¼l only in 2 patients with cytomegalovirus and herpes zoster infection. no other immunological abnormalities could be observed (data not shown). when we considered the most frequent "confirmed oi," namely herpes zoster infections, candida infections, and hpv infections, we noticed that patients were mostly female, with a median age at event onset between 5 and 6 years, except for hpv infection, with a median age at the event onset during adolescence (median 14.5 years, iqr 11.9-17.1). the most frequent "confirmed oi," herpes zoster and tuberculosis, occurred in the majority of the cases, during treatment with biologics (70.8% and 90.9%, respectively) and methotrexate (56.9% and 90.9%, respectively), followed by systemic glucocorticoids (19.4% and 27.3%, respectively). for candida, glucocorticoids were reported in half of the cases, followed by biologics. by excluding one patient who got one steroid pulse at high dose and developed disseminated tuberculosis, the remaining patients with "confirmed oi" received a median dose of prednisone of 15 mg/day concomitantly to the infection. details on the remaining infections can be found in the additional table 2. table 5 reports the frequency of "confirmed non-oi" and "possible/patient and/or pathogen-related oi," after removing 218 infections for which pts did not include a specific pathogen (the complete list of "confirmed non-oi" and "possible/patient and/or pathogen-related oi" is presented in additional table 1). among the 274 infections classified as "confirmed non-oi," only 59 (21.5%) were related to a specific pathogen, most frequently influenza virus, streptococcus, staphylococcus, and escherichia. conversely, almost all the infections classified as "possible/patient and/or pathogen-related oi" (299/302, 99%) were related to a specific pathogen. most of the herpes virus infections (193/299, 64.5%) were classified as "possible patient-and/or pathogenrelated oi" with a different clinical presentation compared to the previous group of "confirmed oi." in particular, varicella was the most common herpetic manifestation in this group, with 155/299 (51.8%) cases, followed by herpes simplex infections. epstein-barr virus infections were reported in 38/299 cases (12.7%), [32] . pvan polyomavirus-associated nephropathy, bal bronchoalveolar lavage, cns central nervous system, csf cerebrospinal fluid, dmard diseasemodifying anti-rheumatic drug, cmv cytomegalovirus classified as infectious mononucleosis in 13 cases (4.3%). latent tuberculosis accounted for 12/299 (4.1%) cases, followed by a few cases of tuberculosis, also with lymph node involvement included in this group. the remaining events of "possible/patient and/or pathogen-related oi" affected less than 3% of the cases. an evidence-based list of opportunistic pathogens with the related meddra classification in immunosuppressed children with jia has been derived by the combination of consensus among a panel of pediatricians with expertise in rheumatology and infectious diseases, and the analysis of the pharmachild international registry in jia data are n (%) or medians with iqr range. drugs refer to their administration at any time during the patient's history and are sorted by their descending frequencies. *the adjudicated patients are represented by those with opportunistic infections as per the provisional list of opportunistic pathogens/presentations (step 1), and very severe/severe or serious non-opportunistic infections. the remaining ones represent the not adjudicated patients. jia juvenile idiopathic arthritis, fu follow-up, rf rheumatoid factor, dmards disease-modifying anti-rheumatic drugs [39] . the final list of opportunistic infections/presentations could constitute a future reference for researchers, pharmaceutical companies, and regulatory authorities dealing with pharmacovigilance issues. the introduction of biologics in the 2000s for the treatment of jia has dramatically changed the prognosis of children affected by jia, but has also raised concerns on the possible risk of infections and other safety events in these patients. despite the widespread use of these drugs, there is still a lack of knowledge regarding the assessment of the long-term safety of the biologics in jia. in this context, the role of national and international registries becomes an important source of data [39, [45] [46] [47] . the pharmachild international registry has the advantage of combining information from different countries based on real clinical data. in pharmachild, infections occurred in about 11% of patients with jia [39] , and among them, it is of primary importance to identify the opportunistic infections that may impose a serious threat to the immunocompromised child. this is not an easy task, because apparently there is a large gap between what treating pediatric rheumatologists feel can be considered as an oi and what a panel of experts adjudicates as such. while most serious infections also occur in the healthy population, some events are more frequent or severe in case of immunosuppression. conversely, some infections, such as tuberculosis, more common in immunocompromised children, may affect also the general population, although usually less severely [48] . considering these difficulties in correctly defining oi, we made an effort to produce a document defining oi specifically in children with jia on immunosuppression. something similar was recently developed by a specialized committee convened in the adult setting to define oi in adults and children with immune-mediated diseases on biologics [32] . with the same approach, our panel of specialists voted, through a three-step delphi procedure, for a correct definition of definite and probable oi and subsequently produced a list of oi by cross-matching the provisional list produced by consensus with the pharmachild data. in a first phase of our study, among the pharmachild patients, a considerable percentage of infections (119/682, 17.4%) was adjudicated as opportunistic. when we matched the provisional list of oi with the patients' clinical information, it became clear that other than events with full agreement between the sac and the list, which could be considered either "confirmed oi" (106/682, 15.5%) or "non-confirmed oi" (274/682, 40.2%), there was a considerable number (299/682, 43.8%) of debatable infections due to the specific patient's history and/or the pathogen presentation, and classified as "possible/patient and/or pathogen-related oi." the best example is represented by herpes zoster (tables 4 and 5 ). varicella zoster presentation was included among the "confirmed oi," as suggested in the majority of the literature in this issue [49] [50] [51] . however, primary varicella infection, frequently observed in our population (155/682, 22.7%), was included among the "possible/patient and/or pathogen-related oi" rather than "definite oi" due to the high incidence in healthy non-vaccinated children and its usually non-complicated presentation. this group of patients highlights the difficulties in defining oi in children with jia on treatment, but also the critical importance of providing a reference document listing those infections that should always be considered as opportunistic in these patients, with possible implications for treatment or prophylaxis. half of the patients with herpes zoster infections had varicella in their history indicating a possible subsequent reactivation of the virus due to a transient immunosuppressive condition. one patient developed varicella despite vaccination while 2 patients had herpes infection despite previous vaccination, without manifesting primary varicella. this observation may give rise to speculations about a possible increase in zoster infections in jia population under immunosuppressive therapy through varicella infection as well as herpes zoster reactivation. limited data are available on vaccinations for other infections such as papillomavirus, which occurred only in 2 patients not previously vaccinated. therefore, it would be worthwhile to develop further studies focused specifically on this topic, in order to understand if vaccinations may maintain a protective immune status in jia patients under treatment or not. it would also be interesting to investigate how to identify patients with jia at risk for developing oi, but this would require further comparative studies on the immune status in jia patients receiving different immunosuppressive treatments. per definition, a definite oi can occur in patients without recognized forms of immunosuppression but its presence indicates a potential or likely alteration in host immunity. therefore, it is worthwhile to consider each oi as relevant and representing a potential risk for the jia patient's life, thus requiring a prompt treatment. in fact, to the best of our knowledge, there are no studies indicating who is at major risk of complications due to an opportunistic pathogen among jia patients on immunosuppressive therapy. the use of an immune screening to help primary care practitioners who may care for, diagnose, and manage infections is already consolidated in clinical practice [45] . in our study, we found no specific level of immunosuppression indicating an increased frequency of infections such as pneumocystis jirovecii, although too little data are available on this issue and further analysis is needed to understand the correlation between immune status and oi in patients with autoimmune diseases. biologics and methotrexate were often seen at the time of a "confirmed oi." nevertheless, a comparative study about the role of immunosuppressive drugs would require a larger population and a deeper analysis, which was not the aim of the present manuscript. besides those pathogens confirmed as oi and non-or possible oi by the panel on the basis of the pharmachild real patients' data, there are also pathogens (e.g., nocardia) that have been included in the list of definite/possible oi (table 1) by consensus, although they were not identified in pharmachild. these infections, apparently uncommon since there was none in such a large database, should be considered potential indicators of alterations in host immunity when present in jia patients and deeply investigated by the physician of the center in order to prevent possible complications in these patients. the current literature provides similar evidence, but remains controversial for the majority of oi. beukelman et al. in 2012 reviewed us medicaid data comparing the incidence of bacterial infections requiring hospitalization in children with and without jia [1, 30] . the infection rate was already twice as high in patients with jia not exposed to treatments, compared to children with attention-deficit hyperactivity disorder (adhd) used as controls [30] . the same author 1 year later re-analyzed the same data by comparing the incidence rate of selected oi among children with and without jia. coccidioides, salmonella, and herpes zoster were more common among children with jia [31] . among the 15 pathogens they used to define their list of oi, all in our provisional oi list (table 1) , only herpes zoster, tuberculosis, pneumocystis, and aspergillus were confirmed in table 4 frequency of the 106 infections classified as "confirmed oi" by the sac. opportunistic infections (oi) were classified as "confirmed oi" after the evaluation of the cases available in pharmachild with full agreement between the safety adjudication committee (sac) and the list of provisional pathogens/presentations. data are presented as per the meddra high level and preferred term and sorted by frequencies in descending order clinical presentations were removed because of the lack of the specified pathogen. data are presented as per the meddra high level term and preferred term and sorted by frequencies in descending order. only pathogens with hlt % â�¥ 2% are presented in details. the full listing is available in additional table 1. sac safety adjudication committee our final list of "confirmed oi." the remaining cases were included in the "possible/patient and/or pathogenrelated oi" list. interestingly, the authors included primary varicella infection in the oi only if the affected patient received critical care services during the hospitalization. an increased risk of herpes zoster infection was confirmed in many studies, both in jia [49] and in adult rheumatoid arthritis [52] . [39] . a limitation of our study is that pharmachild is mainly a european registry, although it includes countries worldwide. this means that our results mainly depict the european scenario of oi. a future manuscript will focus on those factors increasing the risk of oi through appropriate modeling to identify the risk factor for oi infection including disease duration, drugs, comorbidities, etc. in conclusion, almost 1/5 of all severe and/or serious infections in jia patients on immunosuppressive therapy are opportunistic. the most frequent opportunistic pathogens were herpes virus (excluding non-complicated primary varicella), mycobacterial, and candida infections. we provided with our work a list of "confirmed oi" in children with jia on immunosuppressive therapy that could be used as possible reference document for future works on pharmacovigilance in children with jia on immunosuppressive therapy and a list of infections that could possibly display an opportunistic nature related to the patient's history and/or the pathogen presentation. more clarity in the understanding of oi in children with jia on immunosuppressant will help in deciding on immunosuppressive treatment and prophylaxis in this group of patients. authors' contributions gg and nr made substantial contributions to the conception of the work and drafted the first and subsequent versions of the manuscript. js, gg, fb, ap, nr, nw, and am contributed to the planning of the study. gg, nr, ec, ahg, gh, hih, djl, and tw interpreted the data. th, pd, hs, gs, fs, dm, tc, vv, ss, mr, sko, and mc made substantial contributions to the acquisition of the data. all authors have approved the submitted version and have agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. pharmachild has been supported by a grant from the european union (grant 260353) and by funding from the irccs g. gaslini. pharmachild registry is registered at clinicaltrials.gov (nct01399281) and at the european network of centres for pharmacoepidemiology and pharmacovigilance (encepp; http://www.encepp.eu/encepp/viewresource. htm?id=19362). ethics approval and consent to participate all registries and participating centers obtained approval from their respective ethics committee and obtained consent/assent based on national existing regulations. competing interests gg declares that she has no competing interests. jfs has received sponsorship for a meeting by sobi (< $10,000 usd). ec declares that he has no competing interests. ahg has received research grants from gilead, merck, sharp & dohme, and pfizer; is or has been a consultant to amplyx, astellas, basilea, f2g, gilead, merck, sharp & dohme, and pfizer; and served at the speakers' bureau of astellas, basilea, gilead, merck, sharp & dohme, pfizer, and schering-plough. all of the above is < 10.000 per entity. gh has received consultancies, speaking fees, and honoraria from abbvie, chugai, pfizer, novartis, roche, and sanofi (< $10,000 usd each). hih declares that he has no competing interests. djl has served on speaker bureaus for genentech and bristol-meyers squibb and served on a data and safety monitoring boards for forest research and the nih-niams; the cincinnati children's hospital medical center has received consulting fees for the work of dr. lovell from abbvie, astrazeneca, bristol-myers squibb, centocor, genentech, hoffman-la roche, lilly, janssen, novartis, pfizer, regeneron, r-pharm and ubc. each activity is less than $10,000. tw declares that he has no competing interests. th declares that he has no competing interests. pd has received speaker's fees or consultancies or travel grants (all < 10,000 usd) from medac, novartis, abbvie, roche, sobi, and lilly. hs declares that she has no competing interests. gs has received honoraria as a sub-investigator in a pfizer trial (> 10,000 usd). fs declares that he has no competing interests. dm declares that she has no competing interests. tc has received consultancies, speaking fees, and honoraria from roche and abbvie < $10,000. vv has received consultancies, speaking fees, and honoraria from pfizer, abbvie, and sobi (< $10,000). ss declares that she has no competing interests. mr declares that she has no competing interests. sko has received teaching honoraria from pfizer (< $10,000). mc declares that he has no competing interests. fbo received teaching honoraria from novartis (< $10,000) and consulting fees from biogen (< $10,000). fba declares that she has no competing interests. ap declares that she has no competing interests. am: starting from 1 march 2016 to december 2018 prof. alberto martini did not have any conflict of interest to declare since he was the scientific director of irccs istituto gaslini and this role did not allow him to render private consultancies resulting in personal income. he performed consultancy activities on behalf of the gaslini institute for the following companies: abbvie, biogen, boehringer, bristol-myers and squibb, emd serono, janssen, novartis, pfizer, and r-pharm. the money received for these activities was directly transferred to the gaslini institute's bank account. since january 2019, prof. alberto martini is no longer the scientific director of irccs istituto gaslini; therefore, he can perform private consultancy services. currently, he has active consultancy agreements with janssen, novartis, and pfizer (< 10.000 usd each). nw has received an institutional research grant from abbvie (> 10,000 usd) for an e-health project. consultancies: bms (< 10,000 usd) on e-health developments. nr has received honoraria for consultancies or speaker bureaus (< 10.000 usd each) from the following pharmaceutical companies in the past 3 years: ablynx, abbvie, astrazeneca-medimmune, biogen, boehringer, bristol-myers and squibb, eli-lilly, emd serono, glaxo smith and kline, hoffmann-la roche, janssen, merck, novartis, pfizer, r-pharma, sanofiservier, sinergie, sobi, and takeda. the gaslini hospital, where nr works as full-time public employee, has received contributions (> 10.000 usd each) from the following industries in the last 3 years: bms, eli-lilly, glaxosmithkline, f hoffmann-la roche, janssen, novartis, pfizer, and sobi. this funding has been reinvested for the research activities of the hospital in a fully independent manner, without any commitment with third parties. ingrida rumba-rozenfelde department of clinical and molecular medicine, faculty of medicine and health sciences 21 instituto de puericultura e pediatria martagao gesteira (ippmg) unitã  di immunologia e reumatologia pediatrica etanercept in children with polyarticular juvenile rheumatoid arthritis safety and efficacy of up to eight years of continuous etanercept therapy in patients with juvenile rheumatoid arthritis effects of long-term etanercept treatment on growth in children with selected categories of juvenile idiopathic arthritis efficacy and safety of open-label etanercept on extended oligoarticular juvenile idiopathic arthritis, enthesitis-related arthritis and psoriatic arthritis: part 1 (week 12) of the clipper study two-year efficacy and safety of etanercept in pediatric patients with extended oligoarthritis, enthesitis-related arthritis, or psoriatic arthritis disease status, reasons for discontinuation italian children with juvenile idiopathic arthritis treated with etanercept longterm efficacy and safety of infliximab plus methotrexate for the treatment of polyarticular-course juvenile rheumatoid arthritis: findings from an openlabel treatment extension adalimumab with or without methotrexate in juvenile rheumatoid arthritis abatacept in children with juvenile idiopathic arthritis: a randomised, double-blind, placebo-controlled withdrawal trial longterm safety and efficacy of abatacept in children with juvenile idiopathic arthritis longterm safety, efficacy, and quality of life in patients with juvenile idiopathic arthritis treated with intravenous abatacept for up to seven years randomized trial of tocilizumab in systemic juvenile idiopathic arthritis efficacy and safety of tocilizumab in patients with polyarticular-course juvenile idiopathic arthritis: results from a phase 3, randomised, doubleblind withdrawal trial two randomized trials of canakinumab in systemic juvenile idiopathic arthritis subcutaneous golimumab for children with active polyarticular-course juvenile idiopathic arthritis: results of a multicentre, double-blind, randomised-withdrawal trial fda. fda: cancer warnings required for tnf blockers impact of the european paediatric legislation in paediatric rheumatology: past, present and future board of registry. the swedish paediatric jia-registry biologic treatment response among adults with juvenile idiopathic arthritis: results from the british society for effectiveness and long-term retention of anti-tumour necrosis factor treatment in juvenile and adult patients with juvenile idiopathic arthritis: data from reuma.pt biologic-associated infections in pediatric rheumatology experience with etanercept, tocilizumab and interleukin-1 inhibitors in systemic onset juvenile idiopathic arthritis patients from the biker registry risk of serious infection in juvenile idiopathic arthritis patients associated with tumor necrosis factor inhibitors and disease activity in the german biologics in pediatric rheumatology registry report on malignancies in the german juvenile idiopathic arthritis registry development of a web-based register for the dutch national study on biologicals in jia: www.abc-register.nl longterm follow-up on effectiveness and safety of etanercept in juvenile idiopathic arthritis: the dutch national register factors associated with treatment response to etanercept in juvenile idiopathic arthritis disease-modifying antirheumatic drug use in the treatment of juvenile idiopathic arthritis: a cross-sectional analysis of the carra registry rates of malignancy associated with juvenile idiopathic arthritis and its treatment rates of hospitalized bacterial infection associated with juvenile idiopathic arthritis and its treatment brief report: incidence of selected opportunistic infections among children with juvenile idiopathic arthritis opportunistic infections and biologic therapies in immune-mediated inflammatory diseases: consensus recommendations for infection reporting during clinical trials and postmarketing surveillance initiation of anti-tnf therapy and the risk of optic neuritis: from the safety assessment of biologic therapy (saber) study risks of serious infections in children treated with biologic response-modifying drugs tuberculosis in pediatric patients treated with anti-tnfã¡ drugs: a cohort study american college of rheumatology recommendations for the treatment of juvenile idiopathic arthritis: recommendations for the medical therapy of children with systemic juvenile idiopathic arthritis and tuberculosis screening among children receiving biologic medications risk of tuberculosis in children with juvenile idiopathic arthritis: a nationwide population-based study in taiwan networking in paediatrics: the example of the paediatric rheumatology international trials organisation (printo) pharmacovigilance in juvenile idiopathic arthritis patients treated with biologic or synthetic drugs: combined data of more than 15,000 patients from pharmachild and national registries ecil-4): guidelines for diagnosis, prevention, and treatment of invasive fungal diseases in paediatric patients with cancer or allogeneic haemopoietic stem-cell transplantation guidelines for the prevention and treatment of opportunistic infections among hiv-exposed and hiv-infected children: recommendations from cdc, the national institutes of health, the hiv medicine association of the infectious diseases society of america, the pediatric infectious diseases society, and the american academy of pediatrics ecil guidelines for preventing pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients a systemic review of tuberculosis with hiv coinfection in children invasive fungal infections in pediatric patients treated with tumor necrosis alpha (tnf-alpha) inhibitors infectious complications with the use of biologic response modifiers in infants and children the safety profile of biologic therapies for juvenile idiopathic arthritis using registries to identify adverse events in rheumatic diseases targeted tuberculin testing and treatment of latent tuberculosis infection incidence of herpes zoster infections in jia patients herpes virus infections during treatment with etanercept in juvenile idiopathic arthritis clinical course and therapeutic approach to varicella zoster virus infection in children with rheumatic autoimmune diseases under immunosuppression real-world comparative risks of herpes virus infections in tofacitinib and biologic-treated patients with rheumatoid arthritis risk of serious infections associated with biologic agents in juvenile idiopathic arthritis: a systematic review and meta-analyses publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank all printo centers which contributed to the data collection. supplementary information accompanies this paper at https://doi.org/10. 1186/s13075-020-02167-2.additional file 1 figure. flowchart of the process.additional file 2 figure. hierarchy of meddra clinically-validated international medical terminology.additional file 3 table 1 . key: cord-023367-ujflw19b authors: newcomer, benjamin w.; cebra, chris; chamorro, manuel f.; reppert, emily; cebra, margaret; edmondson, misty a. title: diseases of the hematologic, immunologic, and lymphatic systems (multisystem diseases) [image: see text] date: 2020-04-17 journal: sheep, goat, and cervid medicine doi: 10.1016/b978-0-323-62463-3.00025-6 sha: doc_id: 23367 cord_uid: ujflw19b nan in this chapter, multisystemic diseases are discussed in small ruminants (sheep, goats, and cervids). these include diseases of the hematologic, immunologic, and lymphatic systems. in general, species will be discussed together, but when pertinent data are available, each species will be considered separately. the terms "cervid" and "deer" have been used interchangeably in parts of this chapter by the authors. an adequate volume of blood for hematologic and biochemical analysis is best obtained from the jugular vein. a docile animal may be restrained in a standing position or tipped up (sheep only) with the head turned away from the jugular vein to be used. wilder ones, such as some cervids, may require restraint devices or chemical sedation. ideally, the animal should be restrained by someone other than the blood collector, although the same person may be able to both restrain a sheep and collect blood if the animal is tipped up or a halter is used (see chapter 1) . the animal should be at rest, with minimal excitement. the collector parts or clips the wool or hair to visualize the jugular vein and then uses the hand not holding the needle to apply digital pressure proximally just above the thoracic inlet to block blood movement through the vein. the vessel may take a second or more to distend after pressure is applied. the collector may then use the needlebearing hand to "strum" the vessel and cause the blood to oscillate. if in doubt about whether the distended vessel is the jugular vein, the collector can release the hand placing pressure on the vessel and observe whether the distended vessel disappears; if it does, the distended vessel was probably the jugular vein. the collector should avoid vessels that pulsate because these are likely to be the carotid arteries. the area should be cleaned with alcohol or other disinfectant, water, or a clean, dry gauze sponge. an 18-or 20-gauge, 1-to 1.5-inch needle is usually adequate to collect blood from an adult, whereas a 22-gauge needle may be used in a neonate. the skin of adults or males may be thicker and more difficult to penetrate with the needle. a syringe or evacuated tube attached to a vacutainer (becton dickinson inc., rutherford, nj) can be used to collect blood. the needle should be plunged through the skin into the vein at an approximate 30-degree angle. the blood should not come out of the vessel in pulsatile waves; this is suggestive of an arterial stick. after aseptically obtaining an adequate volume of blood, the collector removes the needle and releases the pressure on the vessel near the thoracic inlet. pressure should be applied to the site of puncture for a minute or more to prevent extravascular leakage of blood and hematoma formation. the blood should be carefully transferred to a vial containing the appropriate anticoagulant to prevent red blood cell (rbc) rupture. goat erythrocytes are small and particularly prone to hemolysis. to minimize this problem, goat blood should be collected with a needle and syringe, not a vacutainer. white blood cell (wbc) differential distribution, individual blood cell staining characteristics, and morphology may be assessed by microscopic examination of a stained blood film. the differential distribution provides more information than total wbc count because inflammatory conditions in artiodactyls often result in a shift in neutrophil populations toward more degenerate, toxic, or immature forms without changing the overall wbc count. 1 the preferred anticoagulant for a complete blood count (cbc) is ethylenediaminetetraacetate (edta), and tubes should be filled to ensure the proper blood-to-anticoagulant ratio. blood samples should be processed as soon as possible after collection. if a delay is anticipated, the blood sample should be refrigerated (4° c) and an air-dried blood smear should be made because prolonged contact of blood with edta causes changes in wbc morphology and the separation of some rbc parasites. blood can be refrigerated for 24 hours and still yield an accurate cbc. a reference range for hematologic data for sheep and goats is provided in table 16.1 (see appendix 2, tables 1 and 2) . goats tend to have a low mean corpuscular volume (mcv) because of their small erythrocytes. sheep and goats younger than 6 months old tend to have lower hematocrit, rbc count, hemoglobin, and plasma protein concentrations, as well as a higher total wbc count. neonates often have a high hematocrit at birth that decreases with colostral ingestion. lactating animals may have decreased hematocrits, rbc counts, and hemoglobin concentrations. animals grazing at high altitude (mountain goats and bighorn sheep) tend to have increased rbc counts, hematocrits, and hemoglobin concentrations. interpreting hematologic changes in cervids is more complex. restraint method affects a variety of parameters in non-acclimated individuals. physical restraint yields red cell counts and hematocrit and hemoglobin concentrations that are 20 to 40% higher than animals immobilized chemically. 2, 3 neutrophil, lymphocyte, monocyte, and total white cell counts are also 70 to 100% higher in physically restrained cervids (see appendix 2, tables 1 and 2) . adult deer also have seasonal variations in their hemogram. red cell numbers and related values are highest during midsummer and late winter. 3 white cells, especially neutrophils, are also highest in midsummer, and platelet counts are highest in spring and fall. these changes may relate to diet or to seasonal activities, such as antler growth and rutting conflicts, which increase the chance of trauma. red cell stickling has also been reported in a variety of deer species. this appears to relate to a mutation in hemoglobin's b-globin component, similar to the disorder in people, but no pathologic role has been described. 4 bone marrow aspirates and core biopsy samples taken from sites of active erythropoiesis can be useful to evaluate erythrocyte production and determine the cause of anemia and other hemogram abnormalities. the sites of biopsy include the sternebrae, femur, and ileum. the procedure should be done under chemical sedation or anesthesia (see chapter 18) . the area over the biopsy site is clipped and surgically prepared; the sampler should wear sterile gloves to maintain asepsis. aspirates can be obtained by inserting a sterile needle attached to a 3-or 6-cc syringe containing one or two drops of edta through the bone and into the bone marrow. drawing back on the syringe plunger several times may aid in the procurement of an acceptable sample; such a sample may consist of as little as 0.5 ml of bone marrow. if the sample is going to be processed immediately, no anticoagulant is required. core biopsies are obtained using a jamshidi or westerman-jensen biopsy needle. the skin is incised with a scalpel and the biopsy needle is inserted into the bone and turned several times to obtain a core sample. more than one site may be used. the sampler then closes the skin with sutures or staples. biopsy samples are preserved by placing them in 10% neutral buffered formalin solution. impression smears can be made from these samples by gently rolling them on a clean glass slide before placing them in the formalin solution. information obtained from bone marrow samples includes subjective data regarding cell density, megakaryocyte numbers, abnormal cells, maturation patterns of rbcs and wbcs, and the ratio of erythroid to myeloid cells. prussian blue stain can be used on bone marrow to demonstrate iron stores. bone marrow aspirates and biopsies are painful and invasive procedures. therefore, animals should be placed on antibiotics and antiinflammatory drugs prophylactically. blood cultures can be useful in diagnosing bacteremia in an intermittently or persistently febrile animal or one with numerous sites of organ infection. ideally, the clinician should obtain the sample before instituting antimicrobial therapy. however, if this is impossible, antimicrobial therapy should be discontinued 48 to 72 hours before sampling. samples should be taken before and during febrile episodes. the jugular vein is most commonly used to attain a blood culture. as described previously, the skin over the jugular vein should be clipped and surgically prepared. the person collecting the blood sample should wear sterile gloves and use a sterile needle and syringe. blood samples should be placed immediately in a blood culture flask. the chances of attaining a positive culture from bacteremic animals increase with the size of the sample up to about 30 ml, but adding more than the recommended amount to any single culture vial may overwhelm the capacity of the specialized antibiotic-absorbing resins within the flasks. the clinician should change the needle on the sample syringe after collecting the blood and before putting the sample in the culture medium. samples should be refrigerated until they can be sent to a diagnostic laboratory, where aerobic and sometimes anaerobic cultures are made. as an alternative to hematologic testing, comparing conjunctival color to swatches on a standardized famacha chart has been used as a rapid and inexpensive assessment of anemia in whole flocks, primarily to assess the impact of haemonchus contortus and other blood-sucking parasites. 5, 6 results from a number of trials have yielded fair to good sensitivity to packed cell volume and h. contortus load in both sheep and goats. similar to body condition scoring systems, it is essential to calibrate assessors to ensure consistency when using this system. 7 also, some breeds hematocrit (%) 27-45 22-36 hemoglobin (g/dl) 9-15.8 [8] [9] [10] [11] [12] red blood cell count (310 6 /ml) 9-17.5 [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] mean corpuscular volume (fl) 28-40 15-26 mean corpuscular hemoglobin concentration (g/dl) 31-34 [29] [30] [31] [32] [33] [34] [35] platelet count (310 5 /ml) 2.4-7.0 2.8-6.4 total white blood cell count (/ml) 4000-12,000 4000-13,000 segmented neutrophils (/ml) 1500-9000 1400-8000 band neutrophils (/ml) 0 0 lymphocytes (/ml) 2000-9000 2000-9000 monocytes (/ml) 0-600 0-500 eosinophils (/ml) 0-1000 0-900 basophils (/ml) 0-300 0-100 total plasma protein (g/dl) 6.2-7.5 6.0-7.5 fibrinogen (mg/dl) 100-600 read differently on the cards, and use of an electronic color analyzer, while more expensive and less field-friendly, may detect anemia earlier (see chapter 6, figure 6 .4a, b, and chapter 19) . 8 easy use of this technique in deer is limited by their intractability and has not been reported. the most common and significant abnormality of the hemogram is anemia. anemia occurs most commonly after blood loss, hemolysis, or chronic disease. blood loss is usually covert and commonly caused by gastrointestinal or external parasites. overt blood loss is usually caused by major trauma such as that caused by dog bites, severe lacerations, male rivalry fighting, or complications of castration or dehorning. cbc values appear normal immediately after acute blood loss. however, after a few hours of fluid redistribution, anemia and hypoproteinemia are evident. evidence of red cell regeneration (macrocytosis, reticulocytosis, and nucleated red cells) should appear within a day or two of the blood loss. hemolysis occurs most commonly after ingestion of toxic plants, rbc parasitism, intravenous (iv) injection of hypotonic or hypertonic agents, contact with bacterial toxins, water intoxication, or immune-mediated destruction of opsonized erythrocytes. ingested toxins include sulfur compounds from onions and brassica plants (kale and canola), [9] [10] [11] [12] nitrates, nitrites, and copper. [13] [14] [15] [16] except for that caused by copper, hemolysis usually occurs within a day or two after ingestion. copper toxicosis can occur after acute overingestion but more commonly is seen in animals that are chronically overfed copper and suffer some stressful event. goats are more tolerant of excess copper than sheep are, and certain breeds of sheep, particularly the suffolk, are highly sensitive to copper toxicosis (see chapters 2 and 5) . hemolytic bacterial toxins include those from clostridium perfringens type a, clostridium haemolyticum, and leptospira interrogans. 17, 18 intraerythrocytic parasites include anaplasma species, mycoplasma (eperythrozoon) species, and babesia species. [19] [20] [21] [22] [23] immune-mediated rbc destruction is very uncommon except with parasitemia, the administration of certain drugs (penicillin), or bovine colostrum to small ruminant neonates. 24 rapid reduction of plasma osmolality can lead to osmotic lysis of erythrocytes. this can occur locally as a sequela to rapid iv injection of hypotonic substances or after ingestion of a large quantity of water following a period of water deprivation and dehydration (water intoxication). selenium and copper deficiency have also been associated with heinz body anemia. 25 parasite infestation, opsonization, and ingestion of toxic plants typically cause extravascular hemolysis. in these cases, damaged erythrocytes are removed by cells of the reticuloendothelial system, resulting in anemia, pallor, weakness, depression, icterus, and dark urine. bacterial toxins, changes in plasma osmolality, and copper toxicosis cause intravascular hemolysis, resulting in the additional signs of hemoglobinemia and hemoglobinuria. other signs such as fever, neurologic symptoms, and sudden death may be seen with specific diseases. signs of regeneration should be seen on the hemogram 1 to 2 days after the onset of hemolysis. anemia that is not related to the loss or destruction of erythrocytes usually results from a lack of production and thus are nonregenerative. although mild forms may exist in pregnant sheep and goats and those deficient in vital minerals (e.g., iron, selenium, copper, and zinc), the most common cause of nonregenerative anemia is chronic disease. under these conditions, iron is sequestered in an unusable form in the bone marrow; staining a marrow sample with prussian blue stain reveals large iron stores, differentiating this disease from iron-deficiency anemia. the causes of anemia of chronic disease are numerous and include infectious conditions (e.g., pneumonia, foot rot, and caseous lymphadenitis), malnutrition, and environmental stressors. 1 most anemia does not require treatment. unless loss of rbc mass is rapid and severe, the animal is usually able to compensate to the decreased oxygen-carrying capacity by decreasing activity. it is important to remember in this regard that anemia often first becomes apparent to the manager of a flock or herd when animals appear overly stressed or die during movement or handling. if possible, the cause of the anemia should be addressed. this can involve trying to control internal and external parasites, changing the diet, and treating infectious diseases. maintaining adequate hydration is essential in animals with intravascular hemolysis to avoid hemoglobin-induced renal tubular damage. specialty compounds such as molybdenum salts, such as ammonium molybdate, and sulfur or penicillamine for copper toxicosis 16 and methylene blue (15 mg/kg in a 4% solution in 5% dextrose or normal saline intravenously) for nitrate toxicity are usually too expensive or difficult to be used on a flock-wide basis but may be useful in valuable individual animals. veterinarians should be aware that methylene blue is no longer approved for use in food-producing animals. animals with severe acute blood loss or hemolysis may benefit from a whole blood transfusion. because transfusion reactions are rare and strong erythrocyte antigens have not been identified in small ruminants (including cervids), almost any donor of the same species is acceptable for a first transfusion. cross-matching can be done to ensure compatibility, which becomes more important if the animal receives more than one transfusion. blood should be withdrawn aseptically from the donor and collected by a bleeding trocar into an open flask or by a catheter into a special collection bag. blood should be mixed at a 7.5:1 ratio with acid-citrate dextrose, or 9:1 with 2% sodium citrate, or another suitable anticoagulant and administered through a filtered blood administration set. if the jugular vein is not accessible, blood may be infused into the peritoneal cavity, but the slower absorption from that site makes it less effective for treating acute blood loss. the first 15 to 30 minutes of administration should be slow. if no reaction is seen (fever, tenesmus, tachypnea, tachycardia, and shaking), the rate may be increased. transfused erythrocytes may only survive a few days, and therefore, the original cause of the anemia must be addressed. 1 peripheral wbcs include granulocytes (neutrophils, eosinophils, and basophils) and mononuclear cells (lymphocytes and monocytes). immature forms of neutrophils and lymphocytes may be seen during severe inflammatory diseases. abnormalities of the neutrophil line are usually the best cellular evidence of inflammation in small ruminants, and inflammation is almost always a sequela of infection. an increase in neutrophil numbers and their proportional contribution to the total wbc count is usually seen in mild gram positive, subacute, or chronic bacterial infections. animals with more severe disease may exhibit high or normal counts, but a greater proportion of the neutrophils will have toxic changes or be immature forms (band cells, metamyelocytes, or myelocytes). in severe, acute inflammation and many diseases caused by gram negative bacteria, a temporary reduction in neutrophil numbers is observed, often with a concurrent shift toward more toxic or immature forms. if the animal survives the peracute disease, neutropenia should resolve over 3 to 4 days, first through an increase in immature cells, and later through a mature neutrophilic response. another important cause of increased total and relative neutrophil counts is stress (or glucocorticoid administration), which inhibits neutrophil margination and extravasation and thereby increases the number of these cells in the midstream blood. increases in eosinophil counts are usually related to exposure to eukaryotic parasites. decreases are rarely of clinical significance and may be part of the stress response. idiopathic allergic-type reactions also are indicators of pathology but are very rare. increases in basophils are rarely clinically significant. increases in lymphocyte counts often reflect chronic inflammatory disease such as that seen with internal abscesses. in rare cases, lymphocytosis may consist of abnormal, blast-type cells and indicate a lymphoproliferative neoplasm. lymphopenia is an important part of the stress response; nevertheless, the clinician must keep in mind that many diseases stimulate a stress response. therefore, lymphopenia and neutrophilia may represent either stress or inflammation, and an examination of neutrophil morphology and plasma fibrinogen concentrations may be useful in distinguishing the two situations. a high fibrinogen concentration, toxic changes, and high counts of immature neutrophils indicate inflammation under those circumstances. blood monocyte counts also may indicate stress or chronic inflammation. the difficulties in interpreting individual cell count abnormalities highlight the importance of obtaining a differential wbc count and description of cellular morphology in assessing sick sheep and goats. leukogram abnormalities are rarely given specific treatment. it is far more common and useful to use the information from the leukogram to develop a plan to treat the disease responsible for the abnormality. palpation of external lymph nodes is part of the thorough physical examination. lymph nodes that can be found in normal sheep and goats include the submandibular, prescapular, and prefemoral nodes. none of these should be prominent or painful on palpation. additional nodes that may be palpated occasionally in normal animals include the parotid, retropharyngeal, supramammary, perirectal, and popliteal nodes. internal lymph nodes that may be identified during specialized diagnostic procedures include the mediastinal, mesenteric, and other abdominal nodes. enlargement of lymph nodes may be focal, multifocal, or generalized. identification of a single enlarged superficial node does not always rule out a multifocal or generalized disorder because the status of the internal nodes often cannot be determined. enlargement generally indicates either inflammation or neoplasia. inflammatory enlargement is generally related to an associated disease with an infectious component. small ruminants are particularly sensitive to lymph node-based infections (e.g., caseous lymphadenitis), so the search often does not extend beyond aspirating or draining the lymph node itself. neoplastic enlargement almost always results from lymphosarcoma. lymphosarcoma pathogenesis. neoplastic transformation of a member of the lymphocyte cell line leads to unregulated clonal expansion of that cell. the cause of transformation is usually unknown; in rare cases, especially in flock outbreaks in sheep, it can be linked to exposure to the bovine leukemia virus, which has occurred experimentally and as a result of the administration of whole blood anaplasma vaccines. whether the bovine leukemia virus can induce lymphosarcoma in goats and cervids is still unclear. multicentric lymphosarcoma has been reported sporadically in white-tailed deer (odocoileus virginiatus) and other deer, but bovine leukemia virus infection has not been diagnosed in cervids. 26 in one study of neoplastic diseases affecting goats from 1987 to 2011, lymphoma was identified as the most common neoplasm, accounting for 17.7% of the assessed tumors. 27 in contrast to other species such as cattle, sheep, and horses, lymphomas in goats are predominantly t-cell lymphomas affecting the mediastinum. a recent study attempted to classify the type of lymphoma affecting 15 goats. using immunohistochemistry (ihc), it was determined that 73% (n 5 11) of affected goats had t-cell lymphoma and only 27% (n 5 4) had b-cell lymphoma. 28 proliferation of t or b lymphocytes leads to mass lesions and infiltration of viscera. these changes cause physical obstruction (to breathing, blood flow, urination, defecation, etc.), ulceration of mucosal surfaces (blood loss, bacterial invasion), immune system dysfunction, organ failure, and generalized malaise and cachexia. tissue masses may be internal or visible on external examination. clinical signs. clinical signs in affected animals vary according to the type of lymphoma (t-or b-cell) and the location of the masses. t-cell lymphomas in goats are usually localized in the thoracic cavity and/or neck, suggesting thymic origin or homing. 27 in contrast, b-cell lymphomas tend to have a multicentric distribution. 27 lymphoma in small ruminants usually presents with non-specific signs that can mimic other respiratory or gastrointestinal conditions. slowly progressive weight loss is the most common finding. in some cases, generalized peripheral lymphadenopathy and expansile masses are noted 29 ; at first, they usually are presumed to be caseous lymphadenitis abscesses. progressive chemosis and exophthalmos have been reported in a sheep and a goat with multicentric b-cell lymphoma. 29, 30 most masses form at the sites of internal or external lymph nodes. in sheep, masses in the brain, skin, joint, and lymphoid tissue have been reported. 30 leukemia is rare. the most common abnormalities are those of chronic disease and cachexia and include nonregenerative anemia and hypoalbuminemia. bone marrow examination may reveal clonal expansion of lymphoid precursor cells. in cervids, lymphadenopathy and multifocal masses affecting the heart, blood vessels, kidney, urinary bladder, and peritoneum have been reported. 31 a more recent report described a subcutaneous maxillary mass in a 13-year-old captive-born, female whitetailed deer. 26 the mass was diagnosed as focal lymphosarcoma with local metastasis. diagnosis. history and clinical signs are important in the diagnosis of lymphoma in small ruminants. age of affected animals ranges from 2 to 18 years and no gender or breed predisposition has been reported. 29 final diagnosis of affected animals is achieved through necropsy, histopathology, and ihc. lesions seen at necropsy include homogeneous white to tan masses that bulge on the cut surface. they may be small or large. less commonly, diffuse paleness of the reticuloendothelial organs is noted. microscopic examination of these tissues reveals infiltrates of abnormal cells of the lymphocyte line. prevention. avoiding exposure to the bovine leukemia virus and restricting the use of instruments to one animal between cleaning procedures may help prevent the spread of lymphosarcoma. in most animals, however, this neoplasm appears to develop spontaneously. pathogenesis. lambs, kids, and fawns are born with functional lymphocytes that can produce endogenous immunoglobulin. these cells develop the ability to respond to foreign antigens in the fetus during mid to late gestation. because of a lack of in utero exposure, however, basal concentrations of immunoglobulin are very low at birth. these cells therefore are naïve to foreign antigens and unable to develop protective immunity through specific cellmediated and immunoglobulin production. additionally, as with other ruminants, no transplacental passage of maternal immunoglobulin to fetal sheep, goats, and fawns occurs. lambs, kids, and fawns depend exclusively on intestinal absorption of maternally derived colostral antibodies, immune cells (t-lymphocytes), and other immune factors to provide a ready supply of specific immunity and allow opsonization of pathogens for the first months of life. adequate passive transfer requires delivery of a sufficient quantity of good-quality colostrum (immunoglobulin g [igg] concentration in mg/ml) into the gastrointestinal tract, as well as adequate absorption of antibodies (timely) from the colostrum into the blood. however, the amount of maternal colostrum produced by the dam, and its composition, as well as the ability of the newborn to stand and nurse in a timely manner, can be affected by several factors. colostrum igg concentration and volume of production can be influenced by breed, age, nutrition, body condition score (bcs) at parturition, and vaccination status of the dam. the igg concentration in colostrum samples from ewes of different breeds can vary between 60 and 125 mg/ml. 32 one study demonstrated that primiparous ewes with low bcs (, 2.75) at lambing produced less colostrum compared with multiparous ewes with similar bcs. additionally, ewes with higher bcs (. 2.75) tended to produce higher volumes of colostrum compared with ewes with lower bcs. 32 another study suggested that undernutrition of ewes during late gestation can affect colostrum quality and immune development and function in newborn lambs. 33 it has been suggested that at least 30 g of total igg should be fed to newborn lambs and kids during the first 24 hours of life to reach adequate transfer of passive immunity. adequate transfer of passive immunity in small ruminant neonates has been suggested as serum igg levels at 24 hours of life of  15 mg/ml. one study indicated that lambs that nurse low-quality colostrum (igg , 30 mg/ml) had lower serum igg concentrations compared with lambs that that nurse colostrum of higher quality (igg . 110 mg/ml), indicating that the concentration of igg in colostrum is a determining factor for the presentation of failure in the transfer of passive immunity. 34 other factors such as pregnancy toxemia, gastrointestinal parasitism, excess of iodine intake during pregnancy, and inadequate vaccination of the dam can result in poor colostrum synthesis and quality. 35 timely consumption of maternal colostrum during the first hours of life is essential to achieve adequate transfer of passive immunity. in small ruminants, cells of the small intestine are able to internalize and transfer igg into the blood during the first 24 hours of life; however, the absorption efficiency of igg is higher during the first 6 to 12 hours of life. 32, 36 factors associated with the neonate, such as weakness, inability to stand, and congenital abnormalities, will prevent timely nursing of maternal colostrum and lead to failure of passive transfer (fpt). litter size and body weight (bw) of the kid(s) have also been correlated with inadequate absorption of igg from colostrum. one study demonstrated that litter sizes of three light goat kids (, 2.8 kg bw) or more had significantly lower mean serum igg levels at 24 hours of life when compared with litter sizes of one or two heavier kids (9.85 versus 18.30 mg/ml, respectively). 37 this suggests that special attention and monitoring should be paid to multiple fetus gestation as the risk of fpt under these circumstances at kidding is higher; however, the quality of colostrum, amount ingested, and adequacy of absorption are rarely monitored by small ruminant producers in natural or artificial rearing systems. the use of monitoring tools to evaluate colostrum quality and igg absorption is common in modern dairy cattle operations, and these tools are readily available for small ruminant production systems. recent reports have presented the use of %brix in maternal colostrum and neonate serum and its positive correlation with serum total proteins (stps) at 24 hours as effective monitoring tools of fpt in lambs and goat kids. [38] [39] [40] the use of stp has also been used to monitor colostrum deficiency intake in mule deer fawns 41 ; however, adequate values of serum igg for cervid neonates have not been established yet. inadequate colostrum intake and low serum igg at 24 to 48 hours of life have been consistently associated with higher morbidity and mortality rates in lambs, goat kids, and fawns. one study reported that 46% of lamb mortality between 24 hours and 5 weeks of age can be attributed to fpt. 42 another study suggested that colostrum deficiency and low serum igg in goat kids resulted in higher mortality rates at weeks 10 and 12 of life due to chronic infections with pasteurella multocida and escherichia coli. 43 other reports demonstrated that 45% of lambs with a serum igg of , 6 mg/ml at 24 hours died before 3 weeks of age compared with only 5% of the lambs with a serum igg of . 6 mg/ml at 24 hours. 34 in a previous report, mule deer (odocoileus hemionus) fawns with a stp of # 5 g/dl between days 1 and 7 of age developed diarrhea and died before 17 days of age compared with fawns with stp . 5 g/dl. 41 in a more recent report, a 7-day-old formosan sambar deer (rusa unicolor swinhoei) with a history of colostrum deprivation died due to severe suppurative meningitis caused by e. coli infection. 44 in addition to immunoglobulins, colostrum also contains large quantities of fat-soluble vitamins that do not cross the placenta. the most important of these are vitamins a, d, and e, which are important in bone development and the immune or inflammatory response. neonates that have not ingested enough colostrum are likely to be deficient in these vitamins. diagnosis. history of dam dystocia, inadequate colostrum nursing, complete colostrum deprivation, and signs of undernourishment or sepsis in the first few days after birth are usually a presumptive indication of failure in the transfer of passive immunity. a high prevalence of diarrhea and respiratory disease in neonates should prompt investigation and evaluation of passive transfer of immunity in affected herds or flocks. owners occasionally evaluate lambs or kids for adequate intake by picking up the animal and holding it at ear level, while carefully cradling the head and neck, and then shaking the abdomen to hear milk in the abomasum; however, this is not a reliable indication of adequate transfer of passive immunity. a definitive diagnosis of fpt can be made by direct laboratory measurement (single radial immunodiffusion [srid]) of igg in serum at 24 hours of life. although some practitioners use the value of igg used in dairy calves (10 mg/ml), others have suggested an igg value , 15 mg/ml to establish the presence of fpt in small ruminants. 45 numerous semiquantitative methods of estimating igg are available and are easy to use in sheep, goats, and cervids. the most common is the measurement of serum total solids or stp values at 24 hours of life through an optical refractometer. the stp at 24 hours of life in a well-hydrated animal has demonstrated correlation with serum igg in calves, lambs, and goat kids. studies in goat kids indicated that an stp between 5.3 and 5.4 g/dl was associated with adequate transfer of passive immunity. 39, 40 another study demonstrated fpt in lambs with stp values , 4.5 g/dl at 24 hours of life. 32 a study in mule deer suggested that fawns with an stp # 5 g/dl had inadequate colostrum intake and fpt. recently, the measurement of %brix in maternal colostrum and serum with a digital brix refractometer has become an alternative method to evaluate colostrum quality and fpt in dairy operations. colostrum %brix . 22% and serum %brix . 8.4% have been associated with adequate transfer of igg in calves and goat kids. 39 other qualitative methods to assess the transfer of passive immunity in large animals include various agglutination (glutaraldehyde), precipitate assays (sodium sulfate), and measurement of g-glutamyl transferase (ggt) in serum. these methods may be relied on to give an overall flock assessment of adequacy of passive transfer, but they are rarely accurate enough to provide definitive information on individual animals. treatment. fpt is not in itself pathologic, but it greatly increases the neonate's susceptibility to infectious diseases. the amount of colostrum absorbed across the gut decreases with time, especially in animals that have been ingesting other proteins (e.g., the casein in milk); it also decreases with illnesses that decrease gastrointestinal function. neonatal small ruminants should receive at least 4 g of igg/kg of bw or ideally 30 g of total mass of igg from a good-quality colostrum source (. 50 mg/ml of igg) during the first hours of life. 32 other authors recommend an intake of 180 to 210 ml of colostrum/kg during the first 18 hours of life. 46 in artificial rearing systems or lamb feedlots, feeding of colostrum every 6 hours until 24 hours of life is recommended. 47 when same species' maternal colostrum is unavailable, goat colostrum or bovine colostrum/colostrum replacers or are a good alternative; however, hemolysis has been reported in lambs receiving cattle colostrum. 48 one study demonstrated that there was no difference in serum igg levels of lambs that received the same volume of sheep or goat colostrum at birth. 47 another study demonstrated that lambs that received 250 ml of a bovine colostrum replacer at birth in addition to 250 ml of stored sheep colostrum at 6 hours of life had higher serum %brix values at 24 hours and had less incidence of disease during the preweaning period compared with lambs that received the same volume of stored sheep colostrum at birth and at 6 hours of life. 38 since igg absorption cannot be extended more than 24 hours after birth, administration of an oral colostrum source is the best treatment in the immediate postpartum period in still-healthy neonates. after the window for immunoglobulin absorption has closed, plasma, serum, or whole blood administered by the iv or intra-peritoneal route is the best way to raise the neonate's blood immunoglobulin concentrations. adult donor plasma contains approximately 2.5 to 3.5 g of immunoglobulin/dl, so administration of a volume equivalent to 10% of bw or a dose of 20 to 40 ml/kg has been recommended for the treatment of large animal neonates. if plasma is used instead of colostrum, administration of vitamins a, d, and e also may be beneficial. if colostrum and plasma are unavailable or cost-prohibitive, "closing" the gut as quickly as possible with milk, maintaining high standards of hygiene, and possibly administering prophylactic antibiotics offer the greatest prospects for preventing infectious disease. vaccination of the neonate or the administration of antitoxin hyperimmune serum should not be considered protective but may be of value. prevention. prevention of fpt should be based on the establishment of an adequate colostrum program managing the previously mentioned factors that affect production, quality, and absorption of maternal colostrum components in lambs, goat kids, and fawns. ensuring colostral quality is best done through good nutrition, health care, and vaccination of dam (see chapters 2 and 19) . administration of vaccines 6 weeks before parturition, followed in 2 weeks with a booster, provides the highest quantity of protective immunoglobulin in the colostrum. antepartum leakage is rarely the problem in small ruminants that it is in horses and cattle. however, in a flock or herd environment, still-pregnant dams may steal babies from other sheep or goats. to prevent such theft and the resultant loss of colostrum by the "adopted" neonate, owners may choose to keep pregnant animals separate from those that have already delivered. if complete separation is not possible, the dam and her offspring should be allowed to bond with each other in a private pen ("jug" or "crate") for at least 24 hours before being placed back with the flock. clipping excessive wool or mohair from around the perineal area and udder before lambing or kidding, expressing the teats to ensure they are not plugged, and having extra colostrum available when pregnant females are placed in jugs or crates are other good preventive measures. etiology and pathogenesis. uncomplicated diarrhea in lambs, goat kids, and fawns may be caused by infectious agents such as viruses, bacteria, and protozoa. in goat kids and elk calves, metabolic causes of diarrhea have been described. 49, 50 group b and a rotavirus, enterotoxigenic e. coli k99, cryptosporidium parvum, and other cryptosporidium spp. have been commonly identified as causal agents of diarrhea in small ruminant neonates. [51] [52] [53] [54] with recent advances in diagnostics and metagenomics of the enteric environment of large animals, novel viruses have been identified as potential causal agents of diarrhea in lambs and goat kids. adenovirus, astrovirus, calicivirus, coronavirus, and picornavirus have been identified in feces of diarrheic lambs and goat kids 55 ; however, their role in the pathogenesis of neonatal diarrhea is still uncertain. these organisms differ from the agents of complicated diarrhea in that they do not invade beyond the gut wall or result in systemic toxemia (see chapter 5) . additional causes of diarrhea reported in goat kids and elk include lactose intolerance and hypernatremia, respectively. 49, 50 less frequently, bacteria such as c. perfringens, clostridium difficile, and attaching and effacing e. coli have been associated with complicated diarrhea in small ruminant neonates. 54, 56 the net result of such an infection is that a large volume of water and electrolytes are lost into the bowel due to malabsorptive, hypersecretory, or hyperosmolar processes. if enough fluid and electrolytes are lost, dehydration and metabolic acidosis arise, inducing systemic clinical signs of depression and weakness in association with diarrhea. in goats, this clinical entity is one component of the floppy kid syndrome. clinical signs. profuse, watery, yellowish-green to brown diarrhea without fever is the hallmark clinical sign. with severe dehydration and acidosis, affected lambs, kids, and fawns become weak and dull and lack appetite. [50] [51] [52] excessive salivation and loss of suckle reflex have also been reported in affected lambs and kids. 51, 52 mucous membranes become tacky, and skin tenting times are prolonged. shock signs may develop. physical assessment often must take the place of clinicopathologic analysis in affected neonates. mild, nonclinically complicated diarrhea is characterized by profuse diarrhea with minimal systemic signs. the affected animal is bright and alert, with minimal skin tenting, and can stand and eat readily, with a strong suckle reflex. it is less than 5% dehydrated, with a blood ph of 7.35 to 7.50, and bicarbonate deficit is minimal. moderate uncomplicated diarrhea is characterized by profuse diarrhea in a dull but responsive animal. skin tenting is prolonged, but eye luster is normal. the affected animal is able to stand and eat but eats slowly and has a weak suckle reflex. the head typically is held down. it is 5 to 7% dehydrated, with a blood ph of 7.10 to 7.25 and a bicarbonate deficit of 5 to 8 meq/l. severe uncomplicated diarrhea is characterized by profuse diarrhea. the affected animal is dull and minimally responsive, with a very long skin tent time and dull, sunken eyes. it can stand only with assistance and prefers to stay in sternal recumbency with its head up. the animal eats very slowly, if at all, and has a minimal suckle reflex. it is 8 to 10% dehydrated, with a blood ph of 6.90 to 7.10 and a bicarbonate deficit of 10 meq/l. very severe uncomplicated diarrhea is characterized by profuse diarrhea and profound weakness. the animal's skin remains tented for more than 1 minute, and its eyes are very sunken and dull. it is nonresponsive with no suckle response. it is unable to maintain sternal recumbency, lying on its side instead. the animal is 10 to 12% dehydrated, with a blood ph of 6.8 to 7.0 and a bicarbonate deficiency of 15 to 20 meq/l. epidemiology. morbidity and mortality of uncomplicated diarrhea in small ruminants and fawns vary depending on the cause. reports of rotaviral diarrhea in newborn lambs indicate morbidity rates between 50% and 100% and mortality rates between 0 and 10% 51,52 ; however, one study reported a 50% case fatality rate in lambs affected with types b and a rotavirus diarrhea. 52 another study reported mortality rates between 10% and 30% in lambs and kids affected with c. parvum diarrhea. 57 most of infectious agents associated with uncomplicated neonatal diarrhea in small ruminants are shed by adult animals and older lambs/kids around stressful events such as lambing/kidding and extreme weather conditions. one study reported that pregnant does shed 7 to 10 times more oocysts during the 3 weeks around kidding compared with other time periods. 58 additionally, poor husbandry/hygiene of lambing/kidding sheds, fecal soiling, flock size (. 200 animals), lambing/kidding season (winter/spring), and the presence of c. perfringens type a in feces have been suggested as potential risk factors for uncomplicated diarrhea in small ruminant neonates. [58] [59] [60] clinical pathology. the leukogram should be normal or show abnormalities compatible with stress. serum biochemical or blood gas analysis may reveal evidence of intestinal malabsorption, electrolyte loss, metabolic acidosis (hypoglycemia, hyponatremia, hypochloremia, hyperkalemia, low bicarbonate, and increased anion gap), and dehydration (hyperalbuminemia and increased blood urea nitrogen [bun] and creatinine). in contrast with the common leukogram and biochemical abnormalities found in calves, lambs, and goat kids with uncomplicated diarrhea, elk calves with diarrhea develop leukocytosis, hyperchloremia, and hypernatremia (serum na . 153 meq/l). 50 additionally, increased anion gap, bun, creatinine, and albumin concentrations have been reported in affected elk calves. 50 a presumptive diagnosis may be based on the characteristic history and clinical signs. response to conservative treatment also is supportive of this diagnosis. identification of the specific causative agent is less important than proper treatment of affected animals; however, feces or intestinal contents from affected animals can be submitted for electron microscopy, reverse-transcription polymerase chain reaction (pcr), and cell culture immunofluorescent assays to identify viruses. [51] [52] [53] additionally, intestinal tissue can be submitted for ihc for rotavirus and c. parvum. 53, 57 feces of affected animals can also be submitted for enzyme-linked immunosorbent assay (elisa), ziehl-neelsen staining technique, light or fluorescence microscopy, sugar flotation, and auramine or fluorescent antibody staining for the diagnosis of c. parvum infection. 60 fecal culture to determine a bacterial cause is recommended. treatment. the immediate goals of treatment are rehydration, replacement of lost electrolytes, and restoration of acid-base balance as these are usually the leading causes of death in affected neonates. less immediate goals are provision of nutrition and replacement of ongoing losses. the aggressiveness of treatment is dictated by the severity of the condition, as well as economic considerations. 61 1. rehydration: calculate the percent dehydration and use to calculate fluid requirements for a 24-hour period. example: 10% dehydration in a 3-kg lamb: dehydration: 0.1 3 3 kg 3 1 kg/l 5 0.3 l or 300 ml. maintenance: 100 ml/kg/day 5 0.3 l or 300 ml. total fluids to replace in 24 hours 5 0.6 l or 600 ml fluid loss due to dehydration (300 ml in this case) should be replaced during the first 4 hours and the rest can be replaced in the next 20 hours. 2. replace lost electrolytes: sodium, chloride, and bicarbonate are lost roughly in proportion to extracellular fluid (ecf) in the acute phase of diarrhea (1-2 days) in untreated animals. potassium tends to be increased in this phase due to the presence of metabolic acidosis and care should be taken when selecting fluids containing potassium to treat affected animals at this time. in chronic cases of diarrhea, and especially in cases where the owner/producer has given oral milk replacer or electrolyte supplements/replacements to affected animals before veterinary evaluation, the serum concentration of sodium, potassium, and bicarbonate might be variable or increased. special care should be taken in these cases when selecting fluids to treat affected animals as the risk of causing hypernatremia is higher. 61 in cases of diarrhea in elk calves, hypernatremia is common, and fluids should be selected accordingly. 50 in the majority of cases, initial replacement of sodium, chloride, and bicarbonate with fluids containing proper composition is recommended. 61 example: assessment suggests a bicarbonate deficit of 16 meq bicarbonate in a 3-kg, comatose lamb with prolonged skin tenting (0.6 is the multiplier for ecf in a neonate): 0.6 3 (16 meq) 3 3 kg 5 29 meq bicarbonate. commercial iv 8.4% sodium bicarbonate solutions contain 1 meq of bicarbonate per milliliter and could be added directly to iv fluids in severely dehydrated and acidotic animals. 61 therefore, the immediate goal is to provide 300 ml of fluid and 29 meq of bicarbonate to this lamb in a formulation that resembles normal ecf. fluids can be given by various routes. selection of route of administration of fluids depends on degree of dehydration, presence or not of a strong suckle reflex, and degree of depression. neonates with advanced degrees of dehydration, depression, and absence of suckle reflex will benefit from iv fluid therapy. in contrast, neonates with mild dehydration and active suckle reflex can be effectively treated with oral electrolytes 61 ; however, if oral fluids have not produced an improvement within 2 to 4 hours, iv treatment should be strongly considered. other routes such as subcutaneous, intra-peritoneal, and intra-osseous can also be used for fluid administration to neonates. • advantages: oral fluids are inexpensive (nonsterile) and easy to give. they are less likely to cause fatal arrhythmias or neurologic disease than iv fluids. • disadvantages: an animal receives a maximum of its gastric volume (5% of bw), and good gastric motility is required. oral fluids may not be well absorbed by a damaged gut. absorption also is slow. intravenous • advantage: this method allows rapid correction of all deficits, even in moribund animals. • disadvantages: it is expensive (sterile), requires venous access, and can rapidly lead to overcorrection. subcutaneous • advantages: this method does not require venous access or good gut motility. • disadvantages: it is expensive (sterile), and the fluids may not be well absorbed in very dehydrated animals. absorption is not as quick as by iv administration. animals should be given only hypotonic or isotonic fluids. intra-peritoneal • advantages: this method does not require venous access or gut motility. fluids are absorbed quickly by this route. • disadvantages: it is expensive (sterile) and can cause peritonitis. isotonic fluids are best used in this route. only a limited volume can be given. many commercial oral electrolyte solutions for neonatal ruminants are available; however, not all of them fulfill the requirements to adequately replace fluids and electrolytes in neonatal ruminants with diarrhea. oral electrolyte solutions must contain enough sodium , provide agents that increase absorption of water (glycine, glucose, and acetate), provide an alkalinizing agent (bicarbonate, propionate, acetate, and citrate; acetate has demonstrated best results), and an energy source (glucose). 61 the amount of carbohydrates might vary and is usually higher in "high-energy" solutions specifically used for severely affected neonates that are not eating and develop negative energy balance. less carbohydrate is needed in less severely affected animals because they are usually eating some and are less likely to have severe negative energy balance. fluids to be avoided include medicated milk replacers and unbuffered saline solutions. iv treatment should be provided with a sterile commercial product. such preparations typically contain 25 to 30 meq/l of base. additional sodium bicarbonate solution or sterile powder can be added to fluid therapy based on the bicarbonate deficit (1 meq/ml of 8.4% solution and 12 meq of bicarbonate/g of powder, respectively). the bicarbonate deficit should be over the first 4 hours. after deficits are replaced, the following continued treatments and adjuncts may be considered: 1. continued administration of fluids (oral rather than iv, if possible) to replace ongoing losses: • oral electrolytes at a volume equal to 5% of the bw per feeding can be given; the number of feedings can be increased from two (normal) to three to six per day. • iv fluids can be continued at twice the maintenance fluid rate until appetite is restored. • more bicarbonate may be necessary. 2. consideration of addition of milk to the treatment regimen: • milk or milk replacers should be added to the therapy of neonates with diarrhea. they provide nutrition to the affected neonate, preventing negative energy balance and promoting intestinal healing. • care should be taken to not mix oral electrolyte solutions with milk or milk replacers in the same container as the concentration of sodium and overall osmolarity of the solution can dramatically increase, leading to hypernatremia or other metabolic abnormalities. • milk or milk replacers should be given in small volumes (,20% of total requirements) but at a higher frequency (every 3-6 hours) to avoid overloading the abomasum and intestine of affected animals. lambs fed milk lose less weight with scours. • free water helps prevent hypernatremia. • milk is a good potassium source (see chapter 3). elk deer calves. elk deer calves commonly develop diarrhea with hypernatremia (serum na . 153 meq/l) compared with other large animal neonates, where hyponatremia is more common. 50 therefore, administration of oral electrolyte solutions designed for other ruminants (calves, lambs, and kids) should be avoided in these animals. a dilution (1:2 or 1:4) of commercially available bovine calf electrolyte solutions to reduce sodium content is recommended for the treatment of elk calf diarrhea. 50 the use of lactated ringer's solution, which has a low sodium concentration in addition to a very low reduction rate of serum sodium (, 1.7 meq/l/hour) has been advocated in the fluid therapy of hypernatremic elk calves with diarrhea. 50 additional therapy. dextrose (2.5-5%) solutions can be added to the fluid therapy of hypoglycemic animals. the use of nonsteroidal antiinflammatory drugs (nsaids) in neonatal ruminants with diarrhea is controversial due to the risk of renal damage and abomasal ulceration; however, in cases of diarrhea complicated by septicemia or endotoxemia, nsaids should be used to reduce the effects of systemic inflammation. flunixin meglumine at a dose of 1.1 to 2.2 mg/kg is the only nsaid approved for food animal use. similarly, the use of oral or systemic antibiotics in cases of uncomplicated diarrhea is controversial due to its potential effect on the intestinal microbiota and development of bacterial resistance; however, their use is warranted in the presence of septicemia or endotoxemia in addition to diarrhea. in these cases, b-lactams such as oral amoxicillin or systemic ceftiofur are usually good choices. the effect of mucosal protectants and probiotics in cases of diarrhea is unknown in small ruminant neonates, and their use is left to practitioners based on their own experiences (see appendix 1) . prevention. prevention of uncomplicated diarrhea in small ruminant neonates is based primarily on the timely feeding of adequate amounts of good quality maternal colostrum or colostrum replacer (see "failure of passive transfer" section). vaccination of dams with antigens of common infectious agents associated with uncomplicated neonatal diarrhea before parturition has demonstrated to be effective increasing colostrum immunity and prevention of diarrhea in lambs. 62 maintenance of adequate husbandry and hygiene conditions in lambing/kidding sheds or barns is necessary to reduce neonatal exposure to infectious agents normally shed in feces of dams during parturition such as rotavirus and c. parvum. ruling out infectious causes of depression and weakness is difficult, and clinicians often do well to assume that an infectious disease is contributing to clinical signs when making treatment decisions. however, several noninfectious systemic disturbances also can depress neurologic and muscular function. successful treatment often requires identification and correction of each of these disturbances. among the more common abnormalities leading to depression in neonates are hypoxemia, metabolic or respiratory acidosis, hypothermia, hyperthermia, hypoglycemia, dehydration, azotemia, and some electrolyte imbalances. hypothermia and hyperthermia can easily be diagnosed by measuring body temperature with a rectal thermometer. hypothermia is far more common and can result from weakness, shock, and environmental stress. cold, windy weather or tube feeding with cold milk replacer or fluids can lead to a rapid drop in core body temperature, especially in neonates that are small or weak or have been inadequately licked off or were rejected by their dams. strong, vigorous neonates usually are protected by heat produced during muscular activity and are able to seek food and shelter. clinical signs appear when the rectal temperature drops to 98° f (36.7° c) or below. protection from wind and cold such as with an individual ewe jug or pen, heat lamps (positioned far enough away so as not to burn the neonate), hot water bottles, blankets, and administration of warm fluids is helpful in treating and preventing hypothermia. shearing the ewe before lambing is of value because it forces the ewe to seek shelter. if this management technique is used, care should be taken to avoid inducing severe hypothermia in the dam. environmental hyperthermia is much less common than fever in neonates. therefore, treatment for infectious diseases in young animals with high temperatures usually is warranted. providing cool shelter with good ventilation, minimizing stressful events, ensuring adequate fluid intake, and shearing the adults are the best defenses against environmental heat stress. hypoglycemia also is easy to diagnose with the aid of an inexpensive, portable glucose meter. lambs and kids typically develop hypoglycemia under the same circumstances as those leading to hypothermia. administering 50 ml/kg of dextrose (approximately 3.5 fl oz/lb, or 5% of bw) in warm milk replacer or 1 ml/kg of 50% dextrose, by either the iv or oral route (diluted to 5% dextrose), should provide ample energy to correct hypoglycemia. iv administration may be necessary if gut motility is absent. follow-up treatment may be necessary if the neonate does not regain its appetite. except during severe conditions, normal lambs and kids should be able to maintain normal body core temperature. they should therefore be examined for an underlying disorder if they exhibit signs of hypothermia or hyperthermia. clinicians and owners should not assume that warming and feeding a cold, weak neonate will always correct the problem. hypoxemia is much more difficult to diagnose. portable blood gas meters for arterial analysis and radiography units for thoracic imaging are available but are still not in common use in small ruminant practice. for those reasons, hypoxemia usually is underdiagnosed. hypoxemia can result from prematurity or dysmaturity, infection, depression or weakness (decreased ventilation), meconium aspiration, bullous emphysema, hernias, and other thoracic fluid or tissue masses. it is likely to be a contributing factor in illness and death in most weak neonates younger than 3 days of age. such animals benefit from the provision of supplemental oxygen, either through a nasal insufflation tube or by oxygen tent. in addition to its direct effect on general wellbeing and behavior/ attitude, hypoxemia at birth leads to poor gut function and subsequent poor colostral absorption. many animals that exhibit fpt and subsequent sepsis had a previous bout of hypoxemia. azotemia, metabolic acidosis, and electrolyte imbalances are difficult to diagnose without clinicopathologic analysis. therefore, these problems are best treated in animals showing signs of dehydration with the administration of a balanced, physiologic electrolyte solution. metabolic acidosis usually is accompanied by either obvious evidence of bicarbonate loss (diarrhea) or severe dehydration. however, neither of these conditions is present with floppy kid syndrome. this descriptive title is applied to muscle weakness, anorexia, and depression in kids observed in the first 2 weeks of life. by its strictest definition, floppy kid syndrome refers to metabolic acidosis with a high anion gap without dehydration or any known cause in young kids that were normal at birth. a variety of disorders and conditions have been proposed as the cause of metabolic acidosis without dehydration, including intestinal fermentation of milk in well-fed kids with subsequent absorption of volatile fatty acids, transient neonatal renal tubular acidosis, and lactic acidosis secondary to toxic impairment of cardiovascular function. overgrowth of c. perfringens type a often is suggested as a source of the toxin. with a high anion gap, a pathologic condition that leads to overproduction of an organic acid is more likely than one that leads to bicarbonate loss. the disease can occur in individual animals or in outbreaks; although parity of the dam and number of offspring have not been associated with this metabolic disturbance, aggressively feeding kids are more likely to suffer from milk fermentation or clostridial overgrowth. an infectious etiology appears to be more likely in herds displaying an increased incidence of this metabolic disturbances as the kidding season progresses. the disease also is reported to be more common in meat goats than in dairy goats. the prevalence can vary tremendously from year to year in a single flock or region. a similar disease has been reported in calves and llama crias, and lambs are likely to be susceptible under the right conditions. because blood gas analysis and exclusion of other diseases often are impractical, the term floppy kid syndrome frequently is used by owners to refer to any kid that is weak and does not have an overt, organ-specific sign (e.g., diarrhea). different pathologic processes are grouped together by their common clinical endpoint (as with "thin ewe syndrome"), and the veterinarian is charged with determining the etiology in a specific flock. most possible causes are found in the previous list of conditions that cause weakness and depression in neonates. among these entities, sepsis and hypoxemia are the most important items and therefore must also be considered possible causes of floppy kid syndrome. treatment and prevention of floppy kid syndrome currently follow the same lines as for treatment and prevention of neonatal sepsis or enteritis. spontaneous recovery of animals with floppy kid syndrome may occur. however, in valuable kids, quick assessment of blood chemistry and base deficits will allow requisite correction of electrolyte and blood ph abnormalities with 1.3% sodium bicarbonate. 63 tissue-invading clostridia are large, straight, gram positive rods that are 3 to 10 mm in length. c. perfringens and c. haemolyticum are smaller bacteria, and clostridium novyi, clostridium chauvoei, and clostridium septicum are larger. the bacteria grow best under anaerobic conditions and produce waste gases. clostridia bear spores, which may be the only viable form in the environment (soil and decomposed organic matter). identification of these spores within bacteria on microscopic examination is useful to identify clostridia, but it is not diagnostic of disease. spores in c. perfringens are central and do not affect the shape, whereas most other species have the spore toward one end and appear slightly club shaped. clostridia cause infectious, noncontagious disease. the bacteria inhabit the intestinal tract and are present in the feces of ruminants. small numbers of organisms in their dormant spore form also may reside in tissues such as liver and skeletal muscle. they can be isolated from soil, where most are thought to have short life spans. soil concentrations are highest in locations recently contaminated with ruminant feces, especially crowded, overused facilities such as feedlots and lambing sheds. environmental contaminations are associated with cool, damp times of the year such as late winter and spring. the concentration of organisms and their toxins found in the feces, gut contents, and internal organs of most adult ruminants usually is small. competition and peristalsis prevent overgrowth in the gut, and aerobic conditions prevent overgrowth in other tissues in live animals. however, rapid overgrowth and tissue invasion ensue after death, making rapid postmortem examination essential to ascertain whether clostridial organisms are responsible for the death. pathogenic clostridial organisms all produce heat-labile protein exotoxins. most make a variety of toxins, and the relative contribution of each toxin to the disease state is not known. c. perfringens is a normal commensal of the intestinal tract of clinically healthy large animals, including cervids; however, the number of bacteria and their toxin production within the intestine usually remain low due to peristalsis and normal homeostasis. 64 c. perfringens is classified into five biotypes (a, b, c, d, and e) based on the production of four major exotoxins, namely alpha (cpa), beta (cpb), epsilon (etx), and iota (itx); however, the production of more than 16 different exotoxins in various combinations has been associated with these bacteria, including perfringolysin o (pfo), enterotoxin (cpe), and beta2 toxin (cpb2). 64 the different biotypes of c. perfringens cause different diseases in relation with the exotoxins they produce. the major effect of the phospholipase/ sphingomyelinase cpa, produced by all c. perfringens biotypes, is cell lysis and hemolysis, and its role on intestinal disease of large animals is not well understood. however, this toxin has been associated with hemolytic disease and hemorrhagic enteritis in large animals; cpb, produced by c. perfringens types b and c, is a trypsinlabile toxin associated with necrotizing enteritis and enterotoxemia in large animal neonates; etx, produced by c. perfringens types d and b, is a trypsin-activated necrotizing toxin associated to vasculitis, edema, and necrosis of the cns and enterotoxemia; and itx, another trypsin-activated necrotizing toxin produced by c. perfringens type e, has also been associated to intestinal disease in small ruminants. 64,65 c. perfringens types c and d are considered the most important types in veterinary medicine as they can cause disease in most farm animals. 66 severe clinical disease due to bacteria sporulation and massive toxin production only occurs when the normal intestinal environment and microbial balance are disrupted in affected individuals. decreased peristalsis and poor ruminal and abomasal function have also been proposed as factors that contribute to disease presentation. weather and handling stresses, feed changes, and an overabundance of high-energy feeds such as milk, bakery products, and cereal grains might promote bacteria overgrowth and exotoxin synthesis and release. additionally, other enteric infections that disrupt the mucosal border may increase systemic absorption of toxins and promote severe disease. c. perfringens type a is a normal inhabitant of the intestinal tract of large animals and is ubiquitous in the environment (soil). one study reported c. perfringens type a as the most common isolate among other clostridia from healthy young lambs. 67 c. perfringens type a has been associated with a fatal hemolytic syndrome in younger lambs and cattle but not goats ("yellow lamb disease"), 66, 68 acute hemorrhagic enteritis and hemolytic enterotoxemia in cattle (hemorrhagic bowel/jejunal syndrome) and goats, 66, 69, 70 and intestinal hemorrhage and splenomegaly in farmed deer. 71, 72 risk factors for infection have not been established; however, high soluble carbohydrate diets and high bcss have been associated with clinical disease. 69, 70, 72 this disease occurs most commonly in lambs 2 to 6 months old. under favorable conditions, the organisms proliferate and cause a corresponding increase in alpha toxin production. the alpha toxin (cpa), in synergy with the beta2 toxin (cpb2), is responsible for hemolytic crisis, vasculitis, and gastrointestinal lesions. the clinical course usually is less than 24 hours. clinical signs. in most cases, sudden death or history of found dead is common. clinical signs observed usually include weakness, depression, fever or hypothermia, icterus, anemia, hemoglobinuria, tachypnea, colic, hemorrhagic diarrhea or absence of feces, and terminal recumbency. 66, 69, [70] [71] [72] adult animals also are susceptible to hemolytic disease and vasculitis caused by c. perfringens type a infection. 66 fatal abomasitis and rumenitis in neonates and juveniles also have been blamed on c. perfringens type a, but the rapid postmortem proliferation of the organism makes substantiation of this claim difficult. 73 morbidity in a flock is lower than for many of the other enteric clostridial diseases, but the mortality rate is very high. diagnosis. the most characteristic clinicopathologic change is neutrophilic leukocytosis with a left shift. other evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. laboratory evaluation reveals evidence of intravascular hemolysis. necropsy in sheep, goats, and cervids usually reveals evidence of hemolysis, pallor, jaundice, hemoglobinuria, hyperemic and edematous intestines, splenomegaly, gastrointestinal serosal and mucosal hemorrhage, and multifocal internal petechial hemorrhages. 66, 69, [70] [71] [72] the isolation of c. perfringens type a from necropsied animals is not itself diagnostic. definitive diagnosis can be made based on identification of the alpha toxin and the absence of other toxins by elisas or older, live animal assays. more recently, multiplex pcr techniques are replacing immunodiffusion assays for the identification of a specific toxin-producing gene isolate, typing of bacteria, and demonstration of toxins or toxin genes. 74 gut content and intestinal samples collected from freshly dead animals make the most meaningful samples for diagnosis. 74 treatment. administration of high doses (. 30,000 iu/kg bid) of penicillin and clostridium antitoxin (10-20 ml subcutaneously [sc] or orally [po] ) is the mainstay of treatment, although animals may die acutely before therapies can be instituted. prevention. a conditionally licensed toxoid against the clostridial alpha toxin is available for cattle in the united states. a recent report demonstrated that a new vaccine including recombinant cpa, cpb, and etx was effective at inducing protective antibodies to c. perfringens biotypes in cattle, sheep, and goats. this could be an alternative for the prevention of morbidity and mortality caused by c. perfringens type a. prevention efforts should focus on environmental hygiene and avoiding gut conditions favorable for proliferation of the organism (high content of soluble carbohydrates in the diet). because this type appears to survive better in soil than other types, preventing ingestion of soil may be important in preventing disease. c. perfringens types b and c occur in the soil and the animals' housing environment and can be shed by asymptomatic individuals. the reported geographic range of both diseases is limited (type b to the united kingdom and south africa and type c to the united kingdom, australia, and north america), even though infection with c. perfringens type c appears to occur worldwide. these organisms cause very similar diseases called lamb dysentery and hemorrhagic enterotoxemia, respectively. very young lambs and kids (1-4 days to 2-3 weeks of age) are usually affected due to the presence of trypsin inhibitors in colostrum. 75 older animals may become susceptible as a result of overwhelming infection or trypsin inhibition by some soy and sweet potato products or temporary suppression of pancreatic trypsin production (struck in adult sheep). with both diseases, the beta toxin (cpb) is a required pathophysiologic factor, and inactivation of this toxin after maturation of pancreatic trypsinogen secretion is what commonly limits the susceptible population to neonatal animals. the cytolytic and necrotizing effects of the beta toxin (cpb), in synergy with the beta2 toxin (cpb2), cause necrosis and ulceration of the intestinal mucosa and are translocated into circulation, causing severe toxemia and death. 75 the diseases initially affect lambs and kids younger than 3 days of age, with illness occasionally occurring in older lambs. the incidence of disease in lambs and kids can be around 15 to 30%, with a case fatality rate of 100%. high stocking density in lambing areas, cold weather, single-born lambs, and high milk production of dams have been suggested as potential risk factors for type b and c enterotoxemia. 76 because of management practices in young animals and age-related vulnerability, fecal contamination of teats, hands, and equipment that enter the mouths of the neonates (orogastric tubes and nipples) is a major cause of infection. clinical signs. severely affected animals or those at the beginning of an outbreak usually are found dead. less acutely affected animals expel initially yellow, fluid feces that progressively become brownish and/or hemorrhagic. feces may also contain flecks of blood and show splinting of the abdomen, especially when handled, along with signs of colic and feed refusal. the clinical course usually is short, and the disease is almost always fatal. one study reported acute abdominal pain, hemorrhagic diarrhea, and death within 24 hours of experimental oral inoculation of three goat kids with a field strain of c. perfringens type c. 75 dehydration, anemia, and severe weakness are also common clinical signs in affected animals. terminal convulsions and coma occasionally are noted, especially in outbreaks in the united states. c. perfringens type c in older sheep causes the disease known as "struck." affected animals usually are found dead or with signs of toxemia. specific antemortem signs of gastrointestinal disease are rare. specific antemortem signs of gastrointestinal disease are rare. clinical pathology changes observed in these animals include neutrophilic leukocytosis with a left shift. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. necropsy findings. postmortem examination reveals focal hemorrhagic ulcers (up to 2.5 cm in diameter) in the small intestine (mostly in the ileum) with type b infection and diffuse reddening with hemorrhage and necrosis of the abomasum and the entire segments of the intestine with type c infection. type c infections in ruminants can also present with generalized peritonitis, subendocardial and subepicardial hemorrhages, and hemorrhagic lymph nodes. animals that die very rapidly may exhibit minimal or no gross abnormalities of the intestine. a similar syndrome of type c enterotoxemia has been previously reported in a sika deer (cervus nippon). 72 sudden death, severe hemorrhagic gastritis including forestomach and abomasum, and catarrhal enteritis was observed in the affected animal. diagnosis. diagnosis of these diseases is made by identification of characteristic history, clinical signs, postmortem lesions, and positive toxin assays. because the beta toxin is very labile, negative toxin assays are less significant than negative assays for presence of other tissue-invading clostridia. the isolation of c. perfringens type b or c from necropsied animals is not itself diagnostic. immunodiffusion assays or multiplex pcr of intestinal contents for specific isolate and beta toxin (cpb) identification are recommended to obtain final diagnosis (see "diagnosis" in "c. perfringens type a" section). treatment. if the infection is identified early in the disease course, high doses of oral and parenteral penicillin and c. perfringens c and d antitoxin may be of benefit. iv fluids and antiinflammatory agents may be indicated as well. usually, the condition is not recognized early enough, and animals are found dead or dying. prevention. a beta toxoid is available in the united states and other countries. it usually is packaged with an epsilon toxoid. the best protection is achieved by vaccinating pregnant dams twice, with the second dose administered approximately 3 to 4 weeks before lambing or kidding and annual booster. deer does should receive double the dose of sheep as low antibody responses to clostridia have been reported in these animals. 77, 78 vaccination of pregnant dams is directed to increase specific colostrum antibodies to protect neonates. juveniles also should be vaccinated twice or three times at 2 months, 3 months, and 4 months. adults, including males, should receive an annual booster. in the face of an outbreak, the lambing area should be moved to a different place. additionally, vaccination of dams and newborns with a beta toxoid and administration of c. perfringens c and d antitoxin can be carried out in the face of an outbreak to reduce morbidity and mortality. c. perfringens type d produces epsilon toxin (etx), which is responsible for causing type d enterotoxemia in sheep, goats, calves, and deer. 79, 80 other common names for the disease include "overeating disease" or "pulpy kidney disease." the disease has a worldwide distribution and occurs primarily in suckling lambs of 1 to 10 weeks of age, although it has also been reported in weaned lambs up to 10 months of age and adult sheep. the disease is also common in grazing goats and deer. the prevalence of disease has been reported from 1.49 to 3.14%, with a 100% case fatality rate in feedlot lambs. 81 one study on proportional distribution of goatherd mortality in the province of quebec, canada, reported a 17.1% mortality of goats to c. perfringens type d enterotoxemia. 82 the disease is more common in feedlot lambs after they enter the lot. tail docking, castration, and other management interventions are thought to decrease the incidence of this disease by temporarily decreasing appetite. the disease also affects unvaccinated adult sheep, even without any history of stressors or feed changes. sudden changes in the diet are the main predisposing factor in goats. the disease can occur in vaccinated goats, as vaccination has not demonstrated to be completely protective in this species. 83, 84 c. perfringens type d is normally found in the gastrointestinal tract of healthy ruminants, but the acid environment of the abomasum and continuous peristalsis help to keep numbers of bacteria and levels of toxin production low. however, under specific conditions such as overingestion of high-energy feeds (milk, grain, and lush pasture), excess of fermentable starches in the intestine, and intestinal stasis, the organism proliferates rapidly, producing lethal quantities of epsilon toxin. these conditions are usually triggered in well-conditioned, fast-growing animals that are on a highly nutritious diet. the epsilon toxin, once produced, acts locally, causing increasing gut permeability and widespread tissue damage. epsilon toxin and other exotoxins are then absorbed through the intestinal tract into systemic circulation and transported to the brain, lungs, and kidneys, causing increased endothelial permeability, perivascular edema, and generalized necrosis. 79, 83 the characteristic increased vascular permeability and perivascular edema in the kidney and brain are responsible for the name of "pulpy kidney disease" and "focal symmetric encephalomalacia." clinical signs. the course of the disease is usually very short (0.5-12 hours), so sudden or spontaneous death is a common clinical sign across affected small ruminant species. 80,84-86 natural disease caused by c. perfringens type d differs between sheep and goats, possibly because of a difference in relative local and systemic actions of the epsilon toxin, although experimental models have demonstrated that both species develop similar lesions. 84, 87, 88 in sheep, systemic actions of the toxin leads to mostly neurological signs such as dullness, depression, ataxia, trembling, stiff limbs, opisthotonus, convulsions, frothy mouth, and rapid death. in goats, actions of the toxin appear to be more localized to the intestinal tract, causing enterocolitis, colic, diarrhea, dehydration, and occasional neurological signs. 85, 86 necropsy findings. postmortem findings in sheep are characterized by edema of the brain, lungs, and heart in addition to hydropericardium. 89 edema of the kidneys (pulpy kidney lesion) is inconsistent. sheep usually demonstrated minor and inconsistent intestinal changes. 89 other lesions reported in cattle and deer include hemorrhages on the epicardium, thymus, and diaphragm and petechial hemorrhages in the jejunal mucosa. 80, 90 necropsy lesions reported in goats include pseudomembranous enterocolitis with mucosal ulceration, as well as fibrin, blood clots, and watery contents in the bowel lumen. evidence of systemic toxemia, including multifocal petechial and ecchymotic hemorrhage, proteinaceous exudates in body cavities, pulmonary edema, hydropericardium, and cerebral malacia with perivascular cuffing, have also been reported in goats and affected deer. 80, 84, 87, 88, 91 clinical pathology. characteristic clinicopathological changes include pronounced hyperglycemia and glucosuria, which are considered a hallmark of c. perfringens d enterotoxemia. 86 additionally, neutrophilic leukocytosis with a left shift and evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. treatment. in general, the course of disease is too acute for the establishment of any treatment. however, as with infections with types b and c, if the disease is identified early in the disease course, high doses of oral and parenteral penicillin in addition to clostridium c and d antitoxin may be of benefit. iv fluids and antiinflammatory agents may be indicated as well. prevention. vaccination of pregnant ewes with two doses of toxoid, with the second dose given 3 to 4 weeks before lambing, and adequate ingestion of colostrum are the best methods of protecting newborn lambs. vaccination of older lambs should occur before exposure to diets rich in carbohydrates (grain-feedlot settings) or lush pastures. in these cases, lambs should be vaccinated twice or three times around 2, 3, and 4 months of age. males and adult females that are not part of the breeding program may be vaccinated annually. vaccination has been shown to protect goats from experimental disease, but clinical evidence suggests that well-vaccinated goats are still susceptible to developing clostridial enteritis. the toxoids may not protect against local action of the toxins in the goat, which appears to play a greater role in their disease than it does in the sheep. 84, 87, 88 more frequent vaccination (every 6 months) in goats is suggested to increase protection. the adjuvant present in some multivalent clostridial vaccines may cause subcutaneous reactions that may lead to abscess formation. in the face of an outbreak, immediate mass administration of c and d antitoxin (200 iu/kg) in addition to vaccination is recommended. 92 nonenteric clostridial infections c. novyi, c. septicum, c. chauvoei, and c. sordelli have been identified as causal agents of severe muscle, liver, and abomasal necrosis in small ruminants and cervid species. 66, [93] [94] [95] these organisms are usually present in the soil and environment and in the gastrointestinal tract and liver of healthy ruminants. 66 pathogenesis is usually facilitated by trauma of affected tissues, local multiplication of the organism, local and systemic damage by exotoxin production, and ultimately death. 66, 96 four types of c. novyi have been described, a, b, c, and d. c. novyi type c is considered nontoxigenic and therefore is not associated with disease. c. novyi type a produces alpha toxin and is associated with wound infections and myonecrosis in cases of "bighead" and "malignant edema." c. novyi type b produces alpha and beta toxins and is associated with infectious necrotic hepatitis or "black disease." 97, 98 the temporal and geographic distributions of black disease resemble those of fascioliasis, with the highest incidence of disease in milder, moister months in many countries. black disease is less common in sheep than in cattle and is rare in goats. 66, 96 c. novyi type d (c. haemolyticum) produces beta toxin and is associated with bacillary hemoglobinuria (red water disease). c. septicum produces alpha toxin and is associated with malignant edema and necrotic abomasitis (braxy). c. chauvoei produces alpha and beta toxins and is associated with severe myonecrosis observed in blackleg and c. sordelli produces a hemolytic toxin associated with myonecrosis in cases of malignant edema and blackleg. 96, 97 pathogenesis. spores of the organism shed in feces of carrier animals contaminate the environment and are ingested with feed/ grass and stored within kupffer cells. 97, 98 liver damage caused by migrating liver fluke larvae (fasciola hepatica, fasciola gigantica, and cysticercus tenuicollis) create perfect ischemic conditions that induce germination of c. novyi type b spores and toxin synthesis and production. 97, 98, 99 the alpha toxin is necrotoxic and causes liver necrosis and diffuse damage of the vascular system. 98 the beta toxin is produced in smaller amounts and contributes to vascular damage and systemic toxemia. infective organisms also may be brought into the liver by the flukes. clinical signs. the course of disease from first illness to death is short and never lasts more than a few hours in sheep. therefore, peracute or sudden death is not uncommon in this species. wellnourished adult sheep between 2 and 4 years are more commonly affected. the disease course is a little longer (1-2 days) in cattle and deer. 66, 95 affected sheep are debilitated, fail to keep up with the flock, and exhibit generalized weakness, sternal recumbency, separation, and anorexia. tachypnea and tachycardia may be seen; high fever (105-107° f) occurs early in the disease. clinical signs observed in cattle, goats, and deer are similar and may include severe depression, anorexia, abdominal distention, colic, ruminal stasis, and lateral recumbency. 66, 94, 95, 99, 100, 101 a report of black disease in a forest reindeer (rangifer tarandus fennicus) described serosanguinous discharge from mucocutaneous orifices (nostrils and anus), periorbital edema, and nystagmus in addition to other clinical signs. 95 necropsy findings. necropsy might be difficult due to rapid autolysis of tissues in affected animals. severe venous congestion usually darkens the underside of the skin of affected animals, giving this disease its common name of "black disease." fluid in the pericardial sac, pleural space, and peritoneal cavity is usually present. 66 endocardial and epicardial hemorrhages are a common finding. the liver is swollen and congested and on its diaphragmatic surface presents pale foci of coagulation necrosis; however, solid organs such as liver and kidneys could be in an advanced state of autolysis. characteristic lesions of black disease in the liver are single or multiple yellow to white areas (1-2 cm in diameter) of necrosis surrounded by a bright hyperemic zone. 102 a recent report of black disease in a reindeer described moderate amounts of dark red thoracic and pericardial fluid, edema of the lungs and upper respiratory tract, swollen spleen, and several well-circumscribed areas of black discoloration in the liver. 95 diagnosis. the most characteristic clinicopathological change is neutrophilic leukocytosis with a left shift. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen; however, diagnosis of black disease is based on characteristic history (endemic liver fluke areas), clinical signs, and postmortem findings and testing. an impression smear of the margins of the liver might reveal large numbers of gram positive rods, but this is not definitively diagnostic. anaerobic culture of c. novyi from typical liver lesions, in addition to demonstration of the alpha/beta toxins from peritoneal fluid or liver (fresh-refrigerated), through elisa or pcr is required to establish final diagnosis. 98, 100 the use of fluorescent antibody and ihc for the identification of c. novyi on liver impression smear samples or other liver (formalin-fixed) samples have also been described. 95, 100 treatment and prevention. treatment is rarely possible because of the fulminant clinical course of the disease; however, if treatment is attempted, high doses of penicillin g sodium (20,000-40,000 iu/kg) iv every 6 hours or oxytetracycline 10 mg/kg iv every 12 hours should be initiated. supportive care, including iv fluids, nutritional support, and stress reduction, may be beneficial. in the face of an outbreak, vaccination of the whole herd/flock should be initiated immediately. efforts to control fluke infestation constitute the most effective approach to prevention of this disease. administration of multivalent clostridial vaccines containing c. novyi is highly effective. animals should be vaccinated every 6 months starting around 2 to 3 months of age and before parturition as protective immunity is short lived. in flocks at high risk for developing this disorder, a booster vaccine given 1 month before expected fluke exposure may provide additional protection. 99, 100 deer should be vaccinated in the same fashion as sheep but double the vaccine dose for sheep should be used for these animals as they do not develop a strong antibody response to commercially available multivalent vaccines. 77, 78 efforts to eliminate the organism from soil and environment are usually unrewarding but carcasses of animals dying from the disease should be burned, deeply buried, or removed from the premises. pathogenesis. c. novyi type d (c. haemolyticum) is the etiologic agent associated with red water disease. c. haemolyticum is similar to other clostridial species in its life cycle and appears to thrive on alkaline soils and pastures with standing water. the disease tends to be seasonal occurring at times of high larval fluke migration. similar to c. novyi b, c. haemolyticum colonizes the livers of healthy animals and proliferates after liver damage, including damage caused by migrating flukes (f. hepatica, fascioloides magna, dicrocoelium dendriticum, and c. tenuicollis), liver abscessation (fusobacterium necrophorum or trueperella pyogenes), or damage incurred during liver biopsy. 100, 103 under ischemic conditions of the liver, spores of c. haemolyticum germinate and produce high amounts of beta toxin. the beta toxin causes localized hepatic necrosis and after reaching circulation induces severe intravascular hemolysis and damage of the capillary endothelium. 103 intravascular hemolysis leads to rapid anemia and death due to anoxia. the disease is seen worldwide and is more commonly reported in sheep than in goats. bacillary hemoglobinuria has been reported in a free-ranging elk calf (cervus elaphus roosevelti) found dead in the southwest of the state of washington, united states. 104 clinical signs. bacillary hemoglobinuria usually affects wellnourished animals older than 1 year of age. 105, 106 in most cases, the disease is per-acute and sudden dead or found dead is the only sign. 103 in cases where signs are recognized antemortem, affected animals appear weak, depressed, and febrile (104-106° f); blood or blood-tinged froth may be present in the nostrils; rectal bleeding and bloody feces may be present; and severe hemoglobinuria (dark red, port wine-colored urine) is usually observed. 105, 106 blood appears thin and watery and mucous membranes are pale and icteric. heart and respiratory rates are high and become much higher with any sort of effort or stress. other terminal signs include bloat and the presence of blood in the nostrils, mouth, vagina, and rectum. death occurs within hours to a few days after onset of clinical signs. 100 necropsy findings. gross lesions include jaundice of mucous membranes and tissues and subcutaneous petechial/ecchymotic hemorrhages, edema, and emphysema. marked autolysis of internal organs might prevent identification of typical lesions. dark red urine is present in the bladder. 102 lymph nodes and spleen are congested and hemorrhagic. hemorrhagic abomasitis and enteritis might occur, as well as the presence of hemoglobin-stained transudate in pleural and peritoneal cavities and pericardial sac. pulmonary edema is common. the pathognomonic lesion is the ischemic hepatic infarcts ranging from 5 to 30 cm in diameter with a hyperemic interface with healthy liver tissue. 100, 102 diagnosis. clinicopathological abnormalities usually include anemia, leukocytosis with mature neutrophilia, and degenerative left shift (immature forms of neutrophils and toxic changes) often is present. 106, 107 serum biochemical evaluation may reveal increased levels of liver enzymes such as sorbitol dehydrogenase, ggt, aspartate aminotransferase, and increased indirect total serum bilirubin. [105] [106] [107] presumptive diagnosis can be made on history, clinical sigs, clinicopathological abnormalities, and postmortem findings; however, similar to black disease, final diagnosis should be based on anaerobic culture of c. novyi from typical liver lesions in addition to demonstration of the beta toxins from peritoneal fluid or liver (fresh-refrigerated) through elisa or pcr techniques. 98, 100, 104, 106 the use of fluorescent antibody and ihc for the identification of c. novyi on liver impression smears or other liver (formalin-fixed) samples has also been described. 95, 100 more recently, a pcr assay for the detection of c. novyi type d in cattle has been reported. 107 treatment and prevention. treatment is rarely possible because of the fulminant clinical course of the disease; however, if treatment is attempted, high doses of penicillin g sodium (20,000-40,000 iu/kg) iv every 6 hours or oxytetracycline 10 mg/kg iv every 12 hours should be initiated. supportive therapy should include the administration of iv fluids, blood transfusions, and antiinflammatory agents. efforts to control liver flukes and prevent other causes of liver damage are most important. administration of multivalent clostridial vaccines containing c. novyi is highly effective. animals should be vaccinated every 6 months starting around 2 to 3 months of age and before parturition as protective immunity is short lived. in flocks at high risk for developing this disorder, a booster vaccine given 1 month before expected fluke exposure may provide additional protection. 100 deer should be vaccinated in the same fashion as sheep, but double the vaccine dose for sheep should be used as these animals as they do not develop a strong antibody response to commercially available multivalent vaccines. 77, 78 efforts to eliminate the organism from soil and environment are usually unrewarding but carcasses of animals dying from the disease should be burned, deeply buried, or removed from the premises. pathogenesis and clinical signs. fecal and soil contamination of wounds received during fighting (head-butting) or dehorning (disbudding) leads to proliferation of c. novyi type a in damaged head and neck tissues. 100 accumulation of secreted toxins leads to swelling, edema, serohemorrhagic exudates, and local tissue necrosis. wounds appear and smell gangrenous. systemic toxemia may affect internal organs, leading to the death of the animal. c. sordelli causes identical disease. diagnosis. laboratory analysis may reveal an increase in enzymes of muscle or liver origin as well as neutrophilic leukocytosis with many immature and toxic neutrophils. postmortem findings include local necrosis around the injury site. diagnosis usually is made by characteristic clinical signs and lesions. treatment. wound management (disinfection, debridement) and administration of high doses of penicillin g sodium (20,000-40,000 iu/kg) iv every 6 hours are important treatment considerations. prevention. ram management may aid in the prevention of head-butting wounds. vaccination with multivalent clostridial toxoids starting around weaning time (3-6 months of age) and with annual boosters also may be helpful. in flocks with a high prevalence of this disorder, a booster vaccine given to rams 1 month before the breeding season and to ewes/does before parturition may provide additional protection. 100 pathogenesis. c. septicum is the most important agent in the pathogenesis of malignant edema and braxy. in the case of malignant edema, other tissue-invasive clostridia (c. chauvoei, c. sordelli, and c. perfringens a) have also been associated with this disease, and mixed infections are common. the pathogenesis of infection is often similar to that seen with bighead and blackleg: soil or fecal clostridial invasion of a contaminated wound. in sheep and goats, this disease has been reported following lambing/kidding, after shearing of tail docking. 100 c. septicum can also invade the abomasal lining of lambs, causing severe hemorrhagic, necrotic abomasitis known as braxy. 108 activation of dormant bacteria in previously damaged tissue (myositis/abomasitis) similar to that seen in clostridial necrotic hepatitis also occurs. 108 in both cases (malignant edema and braxy), bacterial toxins precipitate local tissue necrosis and systemic toxemia. the alpha, beta, gamma, and delta toxins produced by c. septicum are lecithinase, deoxyribonuclease, hyaluronidase, and hemolysin, respectively. commonly affected sites of malignant edema include castration, dehorning, and injection sites; the umbilicus; and the postpartum uterus. 100 factors that promote braxy have not been identified, although it usually affects weaned and yearling lambs in the winter after ingestion of frozen feedstuffs implicated as initial causes of abomasitis. 100, 108 both forms of the disease have worldwide distribution and are described more in sheep than in goats. 100, 108 clinical signs. malignant edema is characterized by local lesion (wound) or regional pain characterized by swelling and edema that progressively becomes tense and dark (skin). high fever, signs of shock/toxemia, and frothy exudation of the wound are usually present. evidence of subcutaneous gas production is less common in this infection than in blackleg. uterine infection may cause a fetid vaginal discharge. death occurs within hours to a few days after onset of clinical signs. 100 braxy usually causes death before any abnormalities are noted. on rare occasions, signs of sudden onset of illness with high fever, abdominal distention, depression, colic, and recumbency may be seen before death. 100 diagnosis. characteristic clinicopathologic changes include neutrophilic leukocytosis with a left shift. a decrease in wbc and rbc counts also is possible because of the leukocidal and hemolytic effects of the toxins. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. examination of a gram-stained smear from the edematous swelling(s) or wound swabs could give an early diagnosis. one study reported the successful use of a pcr assay for the identification of bacteria associated with malignant edema in cattle, sheep, and other ruminants. 109 postmortem changes with malignant edema include dark red, swollen muscle filled with hemorrhagic, proteinaceous exudate and little or no gas. with braxy, the abomasal wall is hemorrhagic and necrotic. both diseases are associated with rapid postmortem decomposition of the carcass. treatment and prevention. wound management and the rapid administration of high doses of penicillin (penicillin g sodium at 20,000-40,000 iu/kg iv every 6 hours) are important in treating malignant edema. local treatment consists of surgical incision of the affected area to provide drainage and irrigation with peroxide. injection of penicillin directly into or in the periphery of the lesions may help. ancillary treatments such as iv fluids, antiinflammatory agents (e.g., flunixin meglumine, 2 mg/kg iv), and nutritional support may be necessary. maintenance of good hygiene during procedures such as lambing, tail docking, shearing, castration, obstetric manipulation, and administering injections is helpful in preventing malignant edema. multivalent clostridial toxoids may provide some protection and should be given annually to animals at risk for the disorder. 110 pathogenesis. several species of clostridial organisms can cause myonecrosis in small ruminants. 93, 111, 112 the disease is acute to per-acute, has a short course of duration, and is usually fatal. c. chauvoei, c. septicum, and c. sordelli are commonly involved with clostridial myonecrosis in ruminants. [111] [112] [113] blackleg can be enzootic in some areas or farms because of increased bacterial contamination and occurs more commonly in the warm months of the year. [113] [114] [115] animals between 4 months and 3 years of age can be affected. 111, 112 c. chauvoei is the most important cause of blackleg. c. sordelli tends to be involved in the myonecrosis of older feedlot animals. 112 these organisms are found in the soil and can gain access to muscles after translocation from the gastrointestinal tract and liver into systemic circulation. additionally, direct inoculation of the organisms by penetrating wounds or intramuscular injections has been suggested. local tissue trauma, wounds, unsanitary procedures (i.e., shearing, tail docking, and castration), umbilical infection (neonates), or vaginal trauma from lambing can create perfect conditions for the germination of clostridial spores inducing rapid toxin synthesis and production. 113 in some cases, bacterial proliferation appears to occur in a site distant from the original wound (i.e., fetal infections after shearing of a ewe and myocardial necrosis in cattle and sheep). 113 bacterial toxins cause severe local tissue necrosis, systemic toxemia, and ultimately death. as with braxy, several other strains of tissue-invasive clostridia can cause this disease and mixed infections are common. clinical signs. clostridial myonecrosis usually progresses rapidly and sudden death or history of found dead is not uncommon. [111] [112] [113] clinical signs in animals who are still alive include local to regional painful, edematous swelling most commonly in the limbs or trunk muscles. skin of the affected area can become discolored and crepitus; however, in affected sheep, subcutaneous edema and gaseous crepitation are uncommon and cannot be felt before death. 111 other signs might include stiff gait, lameness, fever, and signs of shock. in cases where the infection occurred through a wound, there is extensive local damage and malodorous serosanguinous fluid discharge. c. chauvoei also causes uterine infection and severe gangrenous mastitis in postparturient ewes. 111, 113 in these cases, uterine and mammary infections may cause fetid vaginal and mammary discharge, respectively. death often occurs within 12 to 36 hours after onset of clinical signs. necropsy findings. rapid tissue autolysis is not uncommon in animals that succumb to clostridial myonecrosis. bloodstained fluid and froth can be observed discharging from nostrils and anus. in small ruminants and especially sheep, affected muscle areas are more localized and deeper, brown to black discoloration is present, the subcutaneous edema is not as severe, and, although there is gas present, is not in such large amounts as in cattle. in cases of infection from skin wounds, the area demonstrates subcutaneous edema, swelling, and underlying muscle discoloration. in cases of infection through the urogenital tract, typical lesions are found in the perineal area, vagina, uterus, and fetus. lung congestion, fibrinohemorrhagic pleuritis, pericarditis, myocardial damage, and bloat are also common findings. 111, 112 diagnosis. it is rarely possible to obtain samples for clinicopathological analysis due to the per-acute course of the disease. if samples can be obtained, common findings include neutrophilic leukocytosis with a left shift. a decrease in wbc and rbc counts also is possible because of the leukocidal and hemolytic effects of the toxins. additional evidence of systemic toxemia-metabolic acidosis, azotemia, and increases in liver and muscle enzymesalso may be seen. presumptive diagnosis can be made from history, characteristic clinical signs, and gross pathology findings; however, aspirates or tissue specimens from affected muscles for direct smear examination, fluorescent antibody testing, or anaerobic culture are required for definitive diagnosis. 111, 112 a multiplex pcr is available for identification of pathogenic clostridia on fluid and tissue samples. 109 treatment and prevention. aggressive antibiotic therapy (e.g., penicillin g sodium or potassium penicillin at 20,000-40,000 iu/kg iv every 6 hours), in combination with surgical debridement of affected tissues (fasciotomy), and supportive care (nutritional support, iv fluids, and antiinflammatory agents) are important within the treatment plan for clostridial myositis. prognosis for treatment of all types of clostridial myositis cases is usually guarded to poor and depends on the duration and extension of the lesions. maintaining excellent hygiene during invasive procedures such as castration, obstetric manipulation, shearing, tail docking, and administering injections is helpful in preventing blackleg. multivalent clostridial toxoids may provide some protection and should be given to all animals starting at weaning time, before parturition, and annually. 114, 115 both tetanus and botulism are important diseases in small ruminant medicine. these two diseases are covered elsewhere in this book (see chapters 5, 11, 13, 19, and 20) . older animals are generally more resistant to sepsis than neonates because they have larger amounts of circulating antibodies. however, this resistance can be overwhelmed by aggressive bacteria, or loss of immune function can allow invasion by opportunistic bacteria. malnutrition, parasitism, transport, overcrowding, other diseases, extreme weather conditions, and other stressors are the major causes of immune suppression. sepsis may produce peracute, acute, or chronic disease signs. peracute signs include fever, injected mucous membranes (including the sclera), tachycardia, tachypnea, dyspnea, swollen joints, lameness, splinting of the abdomen, weakness, depression, anorexia, recumbency, seizures, coma, and sudden death. acute signs are similar, except that they persist for a longer period and therefore are more likely to be noticed. chronic signs usually result from the partial clearance of infection after an acute episode, which may be clinical or inapparent. pathogenesis. gram negative bacteria and their toxins gain access to the blood from a site of proliferation or destruction. the most important toxin is endotoxin, a group of lipopolysaccharide molecules that reside within the wall of the bacteria. bacteria or endotoxins incite a systemic inflammatory response, chiefly through activation of host macrophages and stimulation of host cytokine release. these cytokines cause inflammation, produce leukocyte recruitment, increase capillary permeability, induce fever through stimulation of the hypothalamus, and have regional or diffuse vasomotor effects. because the ruminant gut has a plentiful population of gram negative bacteria, it is implicated as the source of most cases of gram negative sepsis. grain overload causes a die-off of the normal gram negative ruminal flora, ulcerative enteric disease allows invasion of bacteria or absorption of their toxins, and ingestion of pathogens provides a suitable place for proliferation and route for invasion of the body. gram negative sepsis caused by opportunistic organisms is best recognized in immunocompromised neonates but also can be seen in stressed or immunocompromised animals of all ages. e. coli is commonly found in fecal material, klebsiella pneumoniae is found in feces and wood products, f. necrophorum lives in the gastrointestinal tract and in soil and invades through compromised gastric mucosa or foot-rot lesions, and pseudomonas aeruginosa is commonly found in water and wash solutions. primary pathogens are most common in adults. although some coliform bacteria may fit into this category, by far, the most important genus is salmonella. sources of salmonella infection are numerous and include carrier animals of the same species, cattle, rodents, birds, other animals, environmental contamination, and possibly feedstuffs. only one serotype of salmonella is specifically adapted to sheep (salmonella abortus ovis), and it is not found in north america. no strain is known to be host-adapted to goats or cervids. therefore, all infections in sheep, goats, and cervids have the potential to spread to and from other species, including humans. serotypes of salmonella that have caused important infections in sheep or goats include salmonella typhimurium, salmonella dublin, and salmonella montevideo. most of these infections lead to bacteremia with mild systemic signs, followed by abortion. s. dublin and s. typhimurium tend to cause more illness in adults because of fibrinonecrotic enteritis. clinical signs. affected animals can exhibit anything, from mild depression with a low-grade fever to shock. common signs include fever, tachycardia, tachypnea, depression with slow or absent eating and drinking, weakness or recumbency, and injection or cyanosis of mucous membranes. organ-specific signs may betray the source or at least the primary location of the infection. fetid discharge may be seen with metritis or abortion; dyspnea and abnormal lung sounds may be seen with pulmonary infection; and bloat, ruminal atony, abdominal distention, and diarrhea may be seen with gastrointestinal infections. diagnosis. the most common abnormality identified on a cbc with peracute gram negative sepsis is panleukopenia. over the course of several days, this condition may resolve, first through an increase in immature neutrophils and later through an increase in mature neutrophils and restoration of lymphocyte counts. very immature cells, severe toxic changes, and persistence of neutropenia suggest a poor prognosis. serum biochemical changes often reflect the severity of the condition. the greater the evidence of shock or tissue damage, the worse the prognosis. metabolic acidosis with a large anion gap and azotemia suggest advanced disease. necropsy findings include diffuse evidence of inflammation, including pulmonary congestion, and polyserositis with body cavity exudates. hemorrhagic pneumonia or fibrinonecrotic enteritis may be seen and reflect the source of bacterial invasion. in all cases, diagnosis is best confirmed by bacteriologic culture of body tissues or fluids. in the live animal, culture of blood, feces, or tracheal fluid yields the best results. when several animals are infected, environmental samples (including feed, water, and bedding) should be tested for the presence of the bacteria. bacteriologic culture of aborted fetuses or placentas frequently yields heavy growth of the organism. prevention. maintaining overall good health and hygiene is the best means of preventing gram negative sepsis. antiendotoxin bacterins are available for cattle in the united states, but their use in small ruminants has been too limited to assess their efficacy. during a flock outbreak, the use of autogenous bacterin may help prevent the spread of disease on a farm. actinobacillus seminis is a gram negative bacillus or coccobacillus that affects primarily the male and female reproductive tracts. infection causes posthitis, epididymitis, and orchitis in rams and metritis and abortion in ewes. other sites of infection, including rare occurrences of chronic sepsis, also are possible. serologic tests are much more useful for identifying infected flocks than infected individuals within flocks. definitive diagnosis depends on bacteriologic culture of the organism and differentiation of it from brucella ovis. the bacillus is common in sheep in some parts of the world but is uncommon in north american sheep and goats. t. pyogenes is best known as an abscess-forming bacterium because of the thick pus formed in response to infection by it and the fibrinous response it elicits. it occasionally also causes sepsis. its association with chronic sepsis lends credence to the belief that trueperella is often a secondary invader that colonizes tissues damaged by another bacterium (see chapter 10) . bacillus anthracis is a large, gram positive, anaerobic bacillus that causes anthrax. it forms spores under aerobic conditions (such as on culture plates) but rarely does so when oxygen tensions are low, as in carcasses. the organism affects most mammals, with herbivores being most susceptible. it is usually carried from one area to another by shedding or dying animals and also can multiply in alkaline, nitrogenous soils. periods of heat and intermittent flooding promote overgrowth of the organism. b. anthracis spores may be inhaled or ingested; in rare cases, the bacillus itself may be spread by biting flies. after local replication, the organism gains access to the blood, where it multiples readily. large numbers of the organisms colonize the spleen. b. anthracis secretes a holotoxin made of edema factor), protective antigen, and lethal factor. this toxin impairs phagocytosis, increases capillary permeability, and inhibits clotting. splenic engorgement, generalized edema, circulatory shock, and bleeding diathesis are the most common lesions and signs of anthrax. generalized infection should be considered uniformly fatal. death may occur before or within hours of initial recognition that the animal is sick. prophylactic antibiotic treatment of healthy animals (oxytetracycline 10 mg/kg iv sid) may decrease spread and mortality during outbreaks. the disease is reportable in many areas. local forms of anthrax also occur, most commonly after transmission through a skin wound or fly bite. local heat, pain, swelling, and necrosis are seen first, and the generalized syndrome often follows. bacterial organisms are rarely identified before important treatment decisions must be made. therefore, treatment should follow general principles and have a wide spectrum of efficacy. antimicrobial drugs are the cornerstone of treatment. in meat-or milkproducing animals, the veterinarian must be careful to use drugs within label directions or have a rational plan for extra-label drug use. the issue of extra-label drug use is especially important in small ruminants and cervids because very few pharmaceutical products have been licensed for them in north america. unless a specific organism is suspected (clostridiosis or anaplasmosis), a single antibiotic or combination of antimicrobial drugs to provide a broad spectrum of coverage should be selected. penicillins, macrolides, tetracyclines, and cephalosporins all provide effective coverage against gram positive pathogens. the newer third-generation cephalosporins are effective against many systemic and enteric gram negative pathogens. the gram negative pathogens of the respiratory tract are often sensitive to other classes of antibiotics. macrolides and tetracyclines also are effective against mycoplasma species and rickettsial organisms. nsaids are almost always beneficial in severe infectious conditions because of their antiinflammatory, antipyretic, and antiendotoxic effects. they are likely to be more effective than corticosteroids because they provide benefits without suppressing the immune response. all such drug use should be considered extralabel and administered accordingly with appropriate withdrawal times established. specific antisera are available for some of the clostridial diseases and may be beneficial if given before widespread tissue necrosis has occurred. severely compromised animals should be treated with fluids for shock (see chapter 3). the most common zoonotic disease risk posed by exposure to small ruminants is orf, also known as contagious ecthyma in animals (see chapter 10) . the disease is caused by an epitheliotropic poxvirus and is transmitted to humans by direct contact with infected animals. skin trauma is a significant risk factor for transmission in both humans and animals. in humans, erythematous macules or papules appear at the site of infection 2 to 3 days following exposure. the infection is generally self-limiting in immunocompetent individuals with complete healing occurring within 8 weeks. brucella melitensis. apart from contagious ecthyma, the greatest risk of zoonotic disease from small ruminants is due to pathogens typically found in the reproductive tract that are transmitted to humans through contact with aborted fetuses, the placenta, or birthing fluids or through the consumption of raw or improperly pasteurized dairy products. b. melitensis is more common in goats than sheep (see chapter 8) . swine, cattle, and other ruminants are common hosts. infection in animals usually causes inapparent mammary infection and abortions; infection in humans is characterized by undulant fever, myalgia, and fatigue. coxiella burnetii. c. burnetii is a rickettsial organism that is an important cause of abortion in sheep and goats (see chapter 8) . wildlife and farm-raised deer may serve as reservoir hosts for infection in other ruminants and humans. 116 infection is a documented cause of reproductive failure in farmed deer and prolonged shedding of the organism is an important source of environmental contamination. 117 in addition to abortion, newly infected sheep and goats occasionally have mild, transient fevers. c. burnetii is far more important as the cause of q fever in humans, who become infected after inhaling particles, handling contaminated animals, or coming into contact with contaminated body fluids (uterine fluid, milk) from infected animals. infection in humans may be asymptomatic, present with flu-like symptoms, or, in the chronic form, present as granulomatous hepatitis, osteomyelitis, or bacterial endocarditis. chlamydophila spp. chlamydophila abortus (previously chlamydia psittaci) is an obligate intracellular parasite and the cause of enzootic abortion of small ruminants (see chapter 8) . chlamydophila pecorum may cause polyarthritis and keratoconjunctivitis (see chapter 14) in sheep and goats. transmission between animals and to humans most commonly occurs through direct contact with infected tissues or materials. infection in humans results in an acute febrile syndrome or respiratory symptoms. chlamydial diseases are more commonly reported in sheep than in goats. chlamydial diseases are suspected to cause disease in other species, including deer. recent serologic evaluation of wild ungulates identified multiple species of deer with antibodies against several chlamydial species. 118 the clinical significance of serological infection in these species remains undetermined. francisella tularensis. f. tularensis is more common in sheep than goats. the organism has many hosts, of which the most important are wild rabbits and rodents. it can contaminate water sources. transmission to sheep is usually through biting arthropods that have previously fed on an infected wild mammal. acute or chronic sepsis may be seen, with more widespread and severe disease occurring in sheep with poor immune function. at necropsy, the disease is characterized by military foci of necrosis in the liver, and less commonly in the lymph nodes, spleen, and lungs. most cases in humans result in acute onset of flu-like symptoms a few days after exposure. l. interrogans. pathogenesis. leptospira spp. are spirochete bacteria that live in moist environments. their survival time outside of hosts is usually short, so their most important reservoirs are the kidneys of infected animals, especially rodents. infected animals shed the organisms through urine and most other body fluids. organisms enter new hosts through mucous membranes and skin breaks and cause bacteremia. signs of sepsis range from inapparent to severe, with more severe signs predominating in neonates. intravascular hemolysis may result. in animals that survive the acute stage, infection may localize in sites such as the kidneys, eyes, and fetoplacental unit. abortion may occur a month or more after acute signs first become evident while renal shedding may occur for several months. leptospirosis is zoonotic. in most cases, infections in humans are asymptomatic and selflimiting. however, in approximately 10% of cases, severe, and potentially fatal, systemic disease may develop, including jaundice, renal failure, and pulmonary hemorrhage. clinical signs acute leptospirosis causes signs of sepsis, including fever, depression, dyspnea, exercise intolerance, weakness, and death (see chapter 12) . additionally, many affected animals show signs of intravascular hemolysis such as anemia, icterus, and hemoglobinuria. diagnosis evidence of intravascular hemolysis such as anemia, hyperbilirubinemia, hemoglobinuria, and hemoglobinemia is suggestive of this disease. in chronic infection, non-specific inflammatory changes and azotemia may be seen. animals dying in the acute hemolytic stage are likely to have dark, discolored urine, bladder, and kidneys. spirochetes can be identified on dark-field microscopy of fresh urine or plasma from infected animals and may be cultured with special techniques. in animals with less severe infection, a rise in antibody titers can be used to support a diagnosis of leptospirosis. prevention numerous vaccines are available for sheep. because protection is serotype specific, it is important to vaccinate against common serotypes in the area. leptospira pomona is the most consistent isolate from sheep and goats; leptospira hardjobovis is the predominate serovar in deer. 119 vaccination immunity is thought to be short lived; boosters should be given at least twice a year in endemic areas. vaccination of deer against serovars hardjobovis and pomona has been associated with decreased urine shedding and increased growth rate in young animals. 120 pathogenesis. l. monocytogenes causes disease with similar frequency in sheep and goats (see chapter 13) . the organism is a common soil and fecal contaminant. it also proliferates in silage that is not properly acidified and in rotting, woody debris. risk of exposure depends on the feed and environment of the animals. environmental and fecal contamination is a more common source than silage in small ruminants overall because most sheep and goats throughout the world are not fed silage. infection in humans almost always results from ingestion of contaminated food products or unpasteurized milk. clinical signs. nervous system dysfunction and abortion are the most common manifestations of the disease. animals with the brainstem form of the disease display signs reflective of cranial nerve dysfunction, including drooped ears or eyelids, decreased facial sensation, and deviated nasal septum. a head tilt and circling may be present; in advanced cases of the disease, the animal is recumbent. clinical signs are mainly unilateral, occasionally bilateral, according to the nerve nuclei affected. diagnosis. antemortem diagnosis of listeriosis is difficult. a presumptive diagnosis is made based on history, clinical signs, and potential response to treatment. histopathologic identification of microabscesses in the brainstem and culture of the organism from affected tissues can be used to confirm the diagnosis. pathogenesis. p. multocida is a small, gram negative, bipolar, ovoid rod that inhabits the pharynx of healthy ruminants. it can survive in soil and water for varying amounts of time after contamination with ruminant nasal secretions. healthy ruminants shed p. multocida much more frequently than mannheimia haemolytica. disease occurs when bacteria colonize the lower respiratory tract or enter the blood. risk factors for pulmonary and systemic infection include viral or mycoplasmal respiratory diseases, temperature extremes, respiratory tract irritants, transport, overcrowding, changes to higher-energy feeds, and handling stress. these factors are thought to both increase bacterial replication in the airway and suppress mechanisms to clear the infection. pasteurellosis is a major problem in feedlot sheep but less common in small breeding or hobby flocks. pasteurellosis also is a significant disease in certain wild small ruminants such as bighorn sheep. direct spread of the organism between animals occurs with nasal contact, and indirect spread occurs after contact with infected nasal secretions. the organism persists in the environment for longer periods during warm, moist weather. p. multocida produces a polysaccharide capsule that inhibits phagocytosis and an endotoxin that contributes to clinical signs. the major disease caused by p. multocida is pneumonia (see chapter 7). however, pasteurella spp. also are capable of entering the blood to cause septicemia in young lambs and hemorrhagic septicemia in adults. occasionally, focal infections such as septic arthritis and mastitis are found. clinical signs. clinical signs of pneumonic and septicemic pasteurellosis include severe depression, bilateral purulent nasal discharge, coughing, diarrhea, anorexia, high fever, and edema of the head, neck, and brisket. the disease course can be short with septicemic pasteurellosis and is usually more insidious with p. multocida pneumonia. pasteurella mastitis is characterized by the bluebag condition or gangrene of the udder. diagnosis. inflammatory changes in the leukogram and hyperfibrinogenemia are the most frequent abnormalities. with severe disease and in the septicemic form, immature neutrophils may predominate over mature cells. inflammation of the intestine and abomasum also may be seen. hemorrhage and fibrin are usually absent or less prominent than in pneumonia caused by m. haemolytica. samples for bacteriologic culture are usually obtained postmortem. blood or tracheal fluid may be obtained before death if the value of the animal warrants it. m. haemolytica is a gram negative rod that is a common commensal inhabitant of the tonsils of young animals. disease is much more frequently described in sheep than in goats and occurs when the organism gains access to the lower respiratory tract. clinical signs and diagnosis. the most common syndrome is enzootic pneumonia, which is seen in young lambs and their dams (see chapter 7) . hemorrhagic bronchopneumonia is the major lesion and respiratory signs predominate. gangrenous mastitis (bluebag) is seen in some of the dams, presumably after they have been nursed by infected offspring. factors that promote respiratory disease, including viral infections, airborne irritants, high stocking density, and stress, are thought to promote invasion of the lower airway by these bacteria. b. trehalosi is a gram negative rod that is a commensal inhabitant of the upper respiratory tract (see chapter 7) . disease is much more frequently described in sheep than in goats and occurs when the organism gains access to the lung or blood. replication occurs in the lung and systemic toxemia or bacteremia resulting in septicemic pasteurellosis. septicemic pasteurellosis is a significant cause of mortality in young lambs and in some farms is the leading cause of death in the age group. clinical signs. septicemic pasteurellosis occurs most commonly in weaned lambs, often following some form of stress such as transport, marketing, or weaning itself. the course of the disease is relatively rapid, and animals may be found dead within 6 hours without showing premonitory clinical signs. when observed, clinical signs include depression, recumbency, and signs of toxemia. diagnosis. septicemic pasteurellosis should be suspected when presented with a dead, recently weaned, sheep with a recent history of stress. diagnosis is best confirmed by typical lesions at necropsy and culture of the organism from bodily tissues. demonstration of b. trehalosi in nasal swabs is of limited value due to the high prevalence of upper respiratory tract colonization in healthy lambs. at necropsy, there may be no evidence of pneumonia, but blood-stained foam can be found in the upper respiratory tract. ulceration of the pharynx and esophagus is commonly present as is subcutaneous hemorrhage of the neck and thorax. prevention. treatment is difficult due to the rapid course of disease. efforts should be made to minimize stressors, particularly during and following weaning, and to manage management factors that may contribute to the disease. vaccination with pasteurella bacterins is rarely effective at controlling natural outbreaks of disease. pathophysiology. abscess-forming bacteria are usually able to survive phagocytosis and thereby avoid destruction by cells of the immune system. alternatively, they invoke such an inflammatory response that the host body "walls off" the entire region with fibrous tissue. abscesses may occur locally, frequently after a wound infection, or at numerous or distant sites from the point of infection. for abscesses to occur at the latter sites, the organism must travel either by way of the blood or within leukocytes. disease characterized by multifocal or internal abscesses usually results from a low-grade, transient event of bacteremia. the best known and most important abscess-forming bacterium in small ruminants is corynebacterium pseudotuberculosis, the gram positive, facultative anaerobic coccobacillus that causes caseous lymphadenitis. infection is usually maintained in a flock by infected animals that spread the organism to others through purulent material draining from open abscesses. the organism is very hardy, so infection can occur through direct contact or indirect contact with contaminated common instruments and facilities. infection is usually introduced into a flock through acquisition of an infected animal, although it also can occur when a naive flock is moved into a contaminated area. horses, cattle, and humans also are minor hosts. infection is thought to occur after ingestion, inhalation, or wound contamination. except for lower respiratory tract invasion, a surface break is thought to be necessary. contaminated shears, tail-docking knives, and emasculators readily spread the organisms through a flock. abscesses can form at the site of invasion or more commonly at the site of the local lymph node. clinical signs. clinical signs of external abscesses include surface swellings and draining lesions. drainage may be intermittent and usually consists of thick, yellow-white purulent material. internal abscesses are more difficult to diagnose. thoracic masses may cause inspiratory dyspnea or occlude venous return to the heart. abdominal lesions may cause tenesmus, stranguria, and occasionally colic. the most common sign of internal abscesses is weight loss with or without intermittent fever. common external sites include the submandibular or retromandibular space and the preinguinal, prefemoral, and supramammary lymph nodes. head and neck lesions are more common in goats, whereas sheep have a more even distribution of cranial and caudal lesions, presumably as a result of shearing wounds. external infections rarely cause clinical illness beyond the draining abscess, although some degree of cachexia may be present. diagnosis. diagnosis is often made by the characteristic lesions with their thick, nonmalodorous pus. bacteriologic culture provides a definitive diagnosis, which may be important for flock management. serologic tests have been developed to identify carrier animals and may be useful if the manager wishes to eliminate infection from the flock. treatment. treatment is often unrewarding: antibiotic sensitivity profiles do not reflect the degree of protection afforded the organisms within the abscesses. long-term treatment with antibiotics and drainage of any compromising masses may lead to some degree of resolution, but internal abscesses are likely to persist. prevention. prevention through the use of vaccines has been attempted. vaccines appear to reduce the severity of the disease but do not completely prevent infection. moreover, live attenuated bacterins lead to de facto infection of all vaccinated animals and therefore should not be used in naïve flocks. 121 other abscess-forming bacteria are most important as differential diagnoses for caseous lymphadenitis. t. pyogenes is another wound contaminant that affects focal areas or regional external lymph nodes. it also commonly colonizes damaged internal tissues such as postpneumonic lungs, postacidotic livers, and damaged feet and heart valves. it is thought to be ubiquitous and poorly invasive in ruminants and therefore does not have the same flock significance as c. pseudotuberculosis. flocks with outbreaks of this infection often have suboptimal management. f. necrophorum causes similar disease and often coinfects with t. pyogenes. it is generally more necrotizing and leads to greater systemic signs of acute illness, including death. f. necrophorum also produces fetid pus, whereas t. pyogenes usually does not. rhodococcus equi is a rare cause of pulmonary abscesses in sheep. numerous small, coalescent, nodular skin abscesses may result from pseudomonas pseudomallei infection (melioidosis). infection usually occurs after the sheep or goat is bitten by an insect that previously fed on an infected rodent. this organism is found in many subtropical regions, including the caribbean, but is not reported in north america. f. necrophorum causes or is associated with a variety of diseases in sheep and is likely to cause many similar diseases in goats. it is best known as a cause of foot rot and hepatic abscesses and appears to be important in lip-leg ulceration. it is an enteric gram negative anaerobe and as such can cause gram negative sepsis after entrance of the bacteria or its toxins into the circulation. f. necrophorum has a poor ability to invade healthy tissue. however, it readily colonizes regions damaged by trauma, persistent moisture, and infection. in addition to endotoxin, the bacterium produces leukocidal and cytolytic toxins that form zones of necrosis around bacterial colonies. this tissue necrosis and the foul-smelling waste gases produced by the bacteria are characteristic of necrobacillosis, or f. necrophorum infection. clinical signs include necrotic, fetid lesions, usually of the mouth or feet, that can cause ingestion or lameness problems. efforts to maintain good hygiene are helpful in preventing fecal contamination. additionally, preventing trauma to foot and mouth tissues through good surface choices and proper pasture drainage is important. pathogenesis. yersinia spp. are gram negative bacteria. yersinia enterocolitica and yersinia pseudotuberculosis both have many mammalian and avian hosts, including humans, and cause clostridial enteritis-like disease in goats. rodent and bird hosts may be important reservoir populations for infections in domestic animals. kids younger than 6 months develop enteritis, bacteremia, and diarrhea that is watery but not bloody. severe toxemia and sudden death can occur. older kids and flocks with chronic exposure tend to have less severe acute disease. instead, chronic diarrhea and weight loss are seen, usually in association with gut wall and abdominal abscesses. sheep, deer, and wild ungulates are rarely affected. clinical signs. signs of enteritis or sepsis predominate in acute disease, whereas signs of wasting are more common in chronic disease. diagnosis. evidence of acute or chronic inflammation is provided by blood work. characteristic necropsy lesions include numerous microabscesses in the gut wall and mesenteric lymph nodes, as well as other evidence of enteritis or sepsis. culture of lesions and demonstration of a rising antibody titer are diagnostic. prevention. avoiding exposure to sources and maintaining overall flock health are helpful in preventing losses due to yersiniosis. pathogenesis. mycobacteria are small, aerobic, straight or curved pleomorphic rods with thick lipid cell walls. they can be stained with acid-fast stains and are usually gram positive. the bacteria live within infected animals of many mammalian species and survive for several years in warm, moist environments. infection occurs after ingestion or inhalation. an identifying characteristic of the mechanism of infection by mycobacteria is the bacteria's ability to survive within macrophages by preventing fusion of phagosomes and lysosomes. the organisms are carried to local lymphatic vessels or lymph nodes, where they form granulomas. as they enlarge, granulomas may develop necrotic or mineralized centers surrounded by macrophages and giant cells. disease can be local, regional, or generalized, depending on the distance the organism is carried from the original site of infection. granulomatous pneumonia, enterocolitis, and lymphadenitis are the most common local and regional forms of the disease. organisms from ruptured granulomas may be spread in contaminated respiratory secretions and feces. mycobacterial infections of all types are uncommon in north american sheep, goats, and cervids, and these species are considered to be relatively resistant to infection. mycobacterium bovis is the most common organism associated with ovine tuberculosis in other countries (see chapter 7), but mycobacterium avium is more common in the united states. the most common mycobacterial infection is johne's disease (paratuberculosis) caused by the etiologic agent m. avium subsp. paratuberculosis (see chapter 5) . mycobacterium tuberculosis is rare in the united states. mycobacterial infections are reportable in most parts of the united states. some debate is ongoing about human susceptibility to m. avium subsp. paratuberculosis; the other organisms are known to be pathogenic in people. clinical signs. the most common clinical sign is emaciation. diarrhea may be seen terminally in both tuberculosis and paratuberculosis. the disease is insidious, with signs becoming more apparent over several weeks to months. respiratory signs may be seen, especially with infection by m. bovis or m. avium subsp. diagnosis. reports of clinicopathologic abnormalities are rare. hypoalbuminemia and hypoproteinemia are likely to be common with chronic enterocolitis caused by either tuberculosis or paratuberculosis. the most common necropsy lesions seen in tuberculosis are nodular lesions of the lung, liver, lymph nodes, spleen, and intestines. histologic evaluation reveals the nodules to be granulomas with giant cells and acid-fast organisms. frequently, the center of the lesion is necrotic and mineralized. intestinal lesions appear to be more common than pulmonary lesions in goats. the lesions of paratuberculosis are centered around the ileocecocolic junction and the adjacent mesentery. the regions may appear normal or be notably thickened. thickening of bowel or nodular infiltrates of lung or liver may be detected antemortem using imaging modalities, such as ultrasonography or computed tomography. postmortem diagnosis is made by identifying characteristic lesions and culturing the organisms. antemortem diagnosis of tuberculosis is best achieved by observing the reaction to intradermal injection of tuberculin with or without comparative injection of purified protein derivatives of m. bovis and m. avium subsp. paratuberculosis. all tuberculosis testing should be done in accordance with local regulations. antemortem diagnosis of johne's disease can be achieved by fecal culture of the organism, but this test takes several weeks to months to complete and is far less reliable in sheep or goats than cattle, with a sensitivity as low as 0.08. serologic tests (e.g., elisa) appear to be sensitive and specific for johne's disease in animals demonstrating clinical disease rather than preclinical infection. serologic detection of clinical johne's disease in cervids has been shown to be highly sensitive and specific while the sensitivity of fecal culture is low in both sheep and goats. the recommended organism detection method in both species is fecal pcr. 122 fecal or milk pcr can be used on pooled samples for flock identification and to type the organism. prevention. tuberculosis should not be endemic in flocks in the united states because positive animals are quarantined or destroyed. preventing exposure to wild ruminants and other possible sources is crucial. except in goat flocks raised for the production of milk that is to be sold unpasteurized, testing is uncommon, so animals are usually not identified until they develop overt disease. paratuberculosis is much more common and may be maintained in flocks by carrier animals. no effective treatment is available for either disease, nor should any be encouraged because efforts should be concentrated on eliminating infection from the flock or herd. vaccination of sheep is used extensively in australia to control paratuberculosis. prolonged vaccination has been shown to decrease fecal shedding in infected animals over time. 123 pathogenesis. mycoplasma spp. are very small, simple bacteria that parasitize cells of higher species. they are common inhabitants of mucous membranes and can have either a commensal or pathogenic relationship with the host. transmission between animals is most likely through direct or indirect contact with body fluids from infected animals, inhalation of respiratory droplets, and arthropod vectors. common sites for superficial infection include the ocular membranes, lung, mammary gland, and female reproductive tract. the organisms can also enter the blood and cause septicemia, abortion, pleuritis, and polyarthritis. flare-ups often occur during times of crowding and during parturition, when neonates can spread the organisms from the mother's mouth to her udder and in turn become infected by ingesting contaminated milk. the most important mycoplasma species in the united states are mycoplasma conjunctivae, mycoplasma capricolum, and the less pathogenic mycoplasma ovipneumoniae. they are most commonly associated with keratoconjunctivitis, acute or chronic sepsis, and pneumonia, respectively. m. conjunctivae and c. abortus are the most common causes of pinkeye in north american small ruminants. mycoplasma spp. are thought to inhibit tracheal ciliary function and thus may have a role similar to viruses in "shipping fever pneumonia" in facilitating lower respiratory tract invasion by primary bacterial pathogens. many of the major pathogenic serotypes found in other countries (some of which cause severe pleuropneumonia without the participation of another bacteria), including mycoplasma mycoides subsp. mycoides, mycoplasma mycoides subsp. capri, mycoplasma agalactiae, and strain f38, are not found in or have been eradicated from north america clinical signs. keratoconjunctivitis, mastitis, exudative vulvovaginitis, fever, cough, dyspnea, exercise intolerance, abortion, lameness, swollen joints, neonatal death, and depression may all be seen with mycoplasma infections. diagnosis. no specific clinical pathologic findings occur with these diseases. mycoplasma infection should be suspected in sheep and goats with severe exudative pleuropneumonia in some parts of the world. mycoplasma can be identified by bacteriologic culture or staining of exudates. examiners must take care in interpreting positive cultures from body surfaces because nonpathogenic mycoplasma are common. prevention. vaccines against mycoplasmal infections are available in some parts of the world, but not in the united states. providing fly control, preventing stress and overcrowding, and isolating sick animals from healthy ones may help prevent the spread of disease. anaplasma ovis, mycoplasma ovis, and babesia spp. a. ovis and m. ovis are small bacteria that lack cells walls and parasitize erythrocytes. these and similar organisms have undergone recent reclassification following molecular analysis. other species of hemotropic mycoplasmas may affect sheep and cervids. 116 the organisms are spread from animal to animal by insect or mechanical vectors. known arthropod vectors for a. ovis include ticks and horseflies; other biting flies may be more important with m. ovis infection. hypodermic needles and equipment used for tail-docking, castrating, or disbudding animals may be important in iatrogenic transmission. after being introduced into a naive host, the organisms proliferate, and the number of red cells infected increases rapidly until an effective immune response begins 1 to 2 weeks later. a similar proliferation of organisms may occur in chronically infected animals after temporary immune suppression. the humoral and cellular immune responses against a. ovis lead to opsonization of parasitized erythrocytes and their removal by cells of the reticuloendothelial system; m. ovis infection is thought to cause more intravascular hemolysis. the result in both cases is hemolytic anemia. 117 the protozoon parasites babesia ovis and babesia motasi have similar life cycles and cause similar diseases, but they have been eradicated and are reportable in the united states. babesia spp. affecting small ruminants are generally less pathogenic than are their bovine counterparts. animals surviving acute hemolytic crisis reduce the parasites to low numbers but rarely clear the infection completely; they serve as sources of infection for other animals. sheep and goats are susceptible to infection by either organism; goats generally appear to be more resistant to the development of severe parasitemia and clinical signs. clinical signs. signs present during hemolytic crises include fever, weakness, pale mucous membranes, and pigmenturia. urine discoloration results from increased amounts of bilirubin in most cases, although hemoglobinuria may be seen in some sheep with m. ovis infection. icterus is usually present only after the acute hemolytic crisis. clinical signs are exacerbated during times of stress, and infection is often first noted when the animals are moved or handled. chronically infected animals may appear clinically normal, may have recrudescence of infection after stress, or may display signs of ill-thrift such as poor body condition and fleece. babesiosis occasionally causes concurrent central neurologic signs. diagnosis. the major clinical laboratory finding is regenerative anemia with detection of the intraerythrocytic bodies. chronically infected sheep often have high counts of nucleated erythrocytes. because m. ovis consumes glucose, hypoglycemia and metabolic acidosis may be detected, especially in blood samples that are not processed immediately. diagnosis is by identification of the organisms on blood smears. special stains are available to make the organisms more visible. postmortem lesions include pallor or icterus of membranes and splenomegaly. some evidence of vasculitis, including edema or exudates in body tissues or cavities, may be seen with m. ovis infection. treatment. mycoplasma spp. and anaplasma spp. are sensitive to tetracycline antibiotics. babesiosis is more difficult to treat. effective drugs include diminazene, pentamidine, and imidocarb dipropionate. supportive care for all blood parasite infections includes whole blood transfusions, nutritional support, and administration of fluids. prevention. prevention in most cases involves maintaining low levels of parasites rather than eliminating them entirely. this method ensures continual stimulation of the immune response, whereas eradication often leaves the animal susceptible to another bout of acute infection. vector control can also be important in management of the disease. pathogenesis. two organisms belonging to the anaplasmataceae family, ehrlichia ovis and anaplasma phagocytophilum, infect ovine wbcs, causing fever, immune suppression, and some organ damage. a. phagocytophilum is the causative agent of tick borne fever in sheep and granulocytic anaplasmosis in horses, dogs, and humans. the organism is transmitted by ticks (ixodes spp.) and maintained in the environment by asymptomatic carrier animals. the distribution and incidence of disease is seasonal with the life cycle of the tick. the organism infects cells of the granulocytic lineage, leading to severe persistent neutropenia and acute lymphopenia. fever occurs 1 to 2 weeks after infection, lasts as long as 2 weeks, and occasionally relapses. chronic infection is common. spleen, lung, liver, and kidney tissue may show some damage because of immune destruction of infected cells, but organ-specific signs are usually the result of secondary infection. secondary bacterial joint infections in lambs infected with a. phagocytophilum develop debilitating lameness known as tick pyemia. e. ovis causes fever (benign ehrlichiosis) 1 to 2 weeks after infection. because of this organism's predilection for mononuclear cells, the degree of immunosuppression and subsequent importance of this disease are much less than for a. phagocytophilum infection. diagnosis. specific diagnosis is best made by identifying darkly stained bodies at the periphery of granulocytic cells, as well as occasional large bodies deep within the cytoplasm of some cells. stained bodies also can be seen on the periphery of mononuclear cells from a blood smear during the acute febrile stage or in tissues during chronic infection. serologic tests are available for detection of anaplasmosis. the available celisa is incapable of distinguishing species of anaplasma and serologic results must be interpreted appropriately, and the species confirmed by pcr. both infections affect sheep and goats (a. phagocytophilum also affects many other ruminants, including white-tailed deer), but neither has been reported in north america. a recent study demonstrated that sheep are capable of being experimentally infected with a human isolate a. phagocytophilum. interestingly, the sheep did not develop clinical disease. 118 such findings suggest that sheep could serve as asymptomatic carriers and potential reservoirs for humans. a. phagocytophilum is widespread in northwestern europe, including the united kingdom, scandinavia, and india, and e. ovis is found mainly in countries bordering the indian ocean. in spite of documented seropositive status of animals, there have been no reports of sheep or goats naturally infected with a. phagocytophilum in the united states developing clinical disease. treatment and prevention. treatment and prevention efforts should focus on reducing vectors and bacterial counts during vector season. both organisms are susceptible to treatment with tetracycline. people and animals can become infected with trypanosome protozoa. the trypanosomes can complete their developmental cycle only in tsetse flies (glossina species). trypanosomes multiply in blood, tissues, and body fluids of their vertebrate hosts and are transmitted between vertebrate hosts in the saliva of blood-sucking flies as they feed. the trypanosome species that are known to infect goats and sheep include trypanosoma congolense, trypanosoma vivax, trypanosoma brucei subsp. brucei, trypanosoma evansi, and trypanosoma simiae. pathogenesis. after entering through the skin, trypanosomes reach the bloodstream by way of the lymphatic system. the parasites multiply, and the prepatent period lasts for 10 to 14 days after infection. the infection is characterized by periods of parasitemia, followed by the absence of parasites. this pattern of infection occurs because of antigenic variation: trypanosomes vary the antigenic nature of their glycoprotein surface coat to evade the host's immune system. this immune system-evasive maneuver prolongs infection and is responsible for chronic disease. some trypanosomes tend to invade extravascular spaces, such as the ocular aqueous humor and cerebrospinal fluid. the pathogenicity of trypanosomes varies with the different host species. trypanosomes may produce a hemolysin early in the course of the disease that causes anemia in the host. later, increased phagocytic activity results in massive erythrocyte destruction. clinical signs. the clinical signs are variable and non-specific and depend on the speed of onset of anemia and the degree of organ impairment. entire herds may be affected. all aspects of production are impaired-fertility, birth weight, lactation, weaning weight, growth, and survival. trypanosomiasis may predispose the animal to the development of other diseases that mask the underlying trypanosome infection. trypanosomiasis may be acute, subacute, or chronic, with chronic infection occurring most commonly. acute disease often causes abortion. dairy goats may show a sudden drop in milk production. depression, anorexia, and a stiff gait may be present. physical examination reveals tachycardia, tachypnea, and a slight fever. hyperemic mucous membranes and excessive lacrimation may be noted. affected animals often become recumbent and anorexic and die within 1 to 3 weeks of onset of clinical signs. if the animal survives, progression to the subacute phase, characterized by listlessness, weight loss, enlargement of superficial lymph nodes, and a dull, dry hair coat, may occur. in such cases, auscultation findings are similar to those in other forms of acute cardiac disease, as well as pale mucous membranes and a pronounced jugular pulse. the animal may linger for several weeks or months, or the chronic form of the disease may develop. affected animals show ill-thrift: dull and dry hair coat, inelastic skin, lethargy, emaciation, peripheral lymphadenopathy, pale mucous membranes, and exercise and stress intolerance. death may occur many months or even years after infection and usually results from congestive heart failure. subclinical trypanosomiasis causes acute episodes when animals are stressed by inadequate nutrition, increased production demands, or concurrent disease. diagnosis. diagnosis is difficult because the parasitemia is intermittent, clinical signs are non-specific, and infection is not always synonymous with disease. a pcr assay is gaining acceptance as the most sensitive diagnostic modality, but not all infected animals exhibit clinical disease. although a tentative diagnosis of pathologic trypanosomiasis can be made on the basis of history, clinical signs, and the presence of appropriate vectors, a definitive diagnosis requires identification of trypanosomes on a fresh blood smear, a giemsa-stained blood smear, or less commonly, a lymph smear. examination of the buffy coat of centrifuged blood with darkfield phase-contrast spore illumination is the most sensitive direct microscopic method and is useful when parasite numbers are low. pathogenic trypanosomes must be distinguished from more ubiquitous, nonpathogenic species particularly common in cattle, such as trypanosoma theileri. repeated blood sampling in individual animals often is necessary, because as noted, parasitemia is intermittent. the diagnosis is supported by evidence of anemia on a cbc. indirect diagnostic methods include an indirect fluorescent antibody test and the elisa. these tests are less helpful for diagnosis of a single clinical case but are useful in assessment for herd infection. both t. congolense and t. brucei readily infect rats and mice, and detection of these pathogens can be used to diagnose the infection indirectly. treatment. treatment consists of the use of trypanocidal agents and supportive care. animals with acute, subacute, and subclinical disease respond better to treatment than those with chronic disease because of the irreversible damage to hematopoiesis associated with chronic infection. with most trypanocides, the therapeutic index is low and varies with the host species. trypanocide efficacy also varies with the species of trypanosome present; resistance to agents is common. some trypanocides are irritating to the skin and may cause severe inflammation at the injection site. in sheep and goats with t. brucei infection, the trypanocide of choice is diminazene aceturate, which should be used at a higher dosage rate (7 mg/kg given intramuscularly [im] or sc) than that recommended for cattle. protection after trypanocide use usually lasts 2 to 4 months, depending on the season. animals must be rested before and after treatment. supportive care consists of providing fluids, an environment conducive to rest, good nutrition, and possibly blood transfusions. prevention. vector control, stress and nutrition management, and selection of trypanosome-tolerant breeds of sheep and goats all help control or prevent trypanosomiasis. no vaccine is available. animals can be treated with insecticides (pyrethroids) to prevent bites by tsetse flies and other flies. control is accomplished by strategic use of trypanocides during the peak season. continued parasitologic and clinical surveillance is essential to determine the efficacy of control measures. pathogenesis. sarcocystis spp. are protozoon parasites that have a two-host life cycle. sexual reproduction occurs in the bowel of a carnivore (mainly dogs and wild canids) after the carnivore ingests cysts in the muscles of sheep, goats, and cervids. sporocysts are passed in the carnivore's feces and later ingested by a sheep, goat or cervid. the sporocysts hatch in the ruminant gut and invade the vascular endothelium during three phases of asexual reproduction. after the third phase (approximately 8 to 10 weeks after ingestion), merozoites enter the ruminant's muscle tissue and encyst. clinical signs are uncommon but can occur during the stages of reproduction and muscle invasion of the host. n. caninum has a similar life cycle and causes similar disease, except that it appears more likely to cause abortion and affect the central nervous system. clinical signs. most infections are asymptomatic. however, if a large number of sporocysts are ingested, tissue damage may occur during the intestinal, vascular, and muscle stages of the sarcocystis life cycle. fever, lameness or a stiff gait, reluctance to move, and diarrhea may be seen. central neurologic signs (blindness, changes in mentation, and seizures) may occur if the organisms invade the brain or interrupt blood flow to it. abortion can occur as early as 4 weeks after ingestion. with severe chronic infections, emaciation and anorexia are seen. diagnosis. the most characteristic abnormality is an increase in muscle enzyme activity in the blood. anemia is common and may result from extravascular hemolysis. cerebrospinal fluid may show mild mononuclear pleocytosis or may appear normal. on necropsy, muscles may display pale streaks or macroscopic cysts throughout. other evidence of vasculitis includes hemorrhagic serosal surfaces, body cavity fluids, and lymphadenopathy. microscopic or ultrastructural examination of affected tissues should reveal the presence of organisms. specific antibody tests are available and do not cross-react with t. gondii antibodies. blood antibody titers often peak around the onset of clinical signs and should be markedly higher than baseline values. antibody preparations also are available for identification of organisms in tissue preparations. treatment. sheep infected with sarcocystis species can be treated with salinomycin (200 ppm in complete feed), monensin (0.5-1 mg/kg po), or amprolium (25-40 mg/kg po). drugs such as sulfadiazine or trimethoprim (25-44 mg/kg im sid), pyrimethamine (0.5-1 mg/kg po sid), and clindamycin have shown some success in treating neospora infections. these treatments are off-label and thus are governed by regulations regarding extra-label drug use. prevention. preventing contamination of feedstuffs with the feces of infected carnivores and preventing ingestion of raw meat by carnivores are most important, but these measures may not be possible in flocks handled with dogs or those living on range land. anticoccidial drugs appear to decrease the chance of clinical disease. 119 pathogenesis. t. gondii is a protozoon parasite with a life cycle very similar to sarcocystis, except that the definitive host is the cat and that a wider range of mammalian and avian species, including humans, appear to be capable of acting as intermediate hosts. sporocysts are infective a few days after passage in cat feces, and most ruminants are infected by eating feed contaminated with cat feces. people can become infected by ingesting raw meat or milk from infected animals. abortion, stillbirth, and neonatal death are the most common forms of clinical disease in sheep and goats, and toxoplasma should be considered one of the most common causes of perinatal losses in small ruminants (see chapter 8) . abortion usually occurs during the final month of pregnancy. fever, vasculitis-induced disease, and neurologic disease are less common manifestations. clinical signs. beyond abortion, clinical disease is rare in adults and resembles systemic sarcocystosis. clinical signs include fever, dyspnea, depression, and anorexia. neurologic signs are more common than with sarcocystis infection, especially in lambs and kids infected in utero. diagnosis. no specific laboratory abnormalities are associated with toxoplasmosis. nodular lesions similar to sarcocysts may be seen in various tissues, including the brain. aborted or stillborn fetuses may appear normal except for histologic lesions in the brain, liver, or lung, but more commonly fetuses are macerated. the placenta is usually abnormal, with gross and microscopic evidence of necrosis of the cotyledons. microscopic identification of the organism in body tissues is the most common means of diagnosis. serologic tests also are available. treatment and prevention. drugs similar to those used to treat neospora may be effective against toxoplasma. preventing contamination of feeds with cat feces and preventing ingestion of dead animals by cats are the most important ways of stemming the spread of this organism. both methods are likely to be difficult in most flocks. direct spread from one animal to another is rare. clinical signs. bluetongue disease has two different manifestations-reproductive problems (see chapter 8) and acute vasculitis of several organ systems. with vasculitis, a spiked fever often precedes depression, anorexia, and rapid weight loss. leukopenia is present. affected animals may develop edema of the lips, tongue, throat, ears, and brisket. other signs include excessive salivation and hyperemia or cyanosis of the oral mucosa, including the tongue (hence the name bluetongue). affected sheep often produce profuse serous nasal discharge that soon becomes mucopurulent and produces crusts and excoriations around the nose and muzzle. oral lesions progress to petechial hemorrhages, erosions, and ulcers. pulmonary edema is often severe, and pneumonia may develop. skin lesions can progress to localized dermatitis. affected sheep may exhibit stiffness or lameness because of muscular changes and laminitis. cyanosis or hemorrhagic changes of the skin of the coronet can extend into the horny tissue. after recovery, a definite ridge in the horn of the hoof may be present for many months. in severe cases, the hoof sloughs. mortality varies widely. in africa, the virus is much more virulent than in the united states, and mortality ranges from 2 to 30%. the reproductive or teratogenic form of the disease varies greatly with strain, host, and environmental factors. teratogenic effects include abortions, stillbirths, and weak, live "dummy lambs." congenital defects may include hydranencephaly. diagnosis. in parts of the world where the disease is common, the diagnosis is usually based on clinical signs alone. the virus can be isolated from blood, semen, or tissues (spleen and brain from aborted fetuses). viral isolation from blood obtained during the viremic state is the most definitive means of diagnosis. serologic evaluation involves two types of viral antigen groups called p7 and p2. the former is found in all bluetongue viruses, and the latter determines the serotype. sera are commonly tested with complement fixation, agar gel immunodiffusion (agid), or one of several elisa tests. a competitive elisa is considered the best serologic test for detecting group antibodies to bluetongue virus. a direct fluorescent antibody test is available. molecular tests (e.g., pcr) for bluetongue have recently become available and are extremely sensitive and specific. they can be useful for distinguishing serotypes. other clinicopathologic signs that aid in diagnosis include leukopenia during the early febrile stage of the disease and an increase in serum ck corresponding to the latter phase of muscle stiffness and lameness. treatment. treatment is non-specific and consists of nursing care. because of the reluctance of animals to eat, they should be fed a gruel of alfalfa pellets by stomach tube or encouraged to eat soft feeds and green grass. broad-spectrum antimicrobials are often used to treat secondary pneumonia and dermatitis. animals should be kept on soft bedding with good footing. water and shade should be readily available. nsaids are commonly used. prevention. the culicoides vector is difficult to eliminate, so animals should be kept indoors during periods of peak gnat activity (dusk and early evening). owners should attempt to eliminate gnat breeding grounds such as overflowing watering troughs and shallow septic systems and should limit exposure of sheep to gnats with the use of repellent sprays. modified live vaccines based on local strains and serotypes are available in some parts of the world. some cross-protection among serotypes does occur. the vaccine should be administered at least 2 weeks before breeding season to prevent teratogenic effects. vaccinated breeding rams may have a slight risk of decreased fertility. lambs can be vaccinated in the face of an outbreak. pregnant animals cannot be vaccinated with modified live vaccines. sheep that have recovered from an attack of bluetongue are solidly resistant for months to infection by the same viral strain and to some other viral types. active immunity in sheep requires both humoral and cellular immunity. etiology. epizootic hemorrhagic disease virus (ehdv) is an orbivirus belonging to the family reoviridae. the virus is structurally related to bluetongue virus, and the pathogenesis and clinical signs of disease resulting from these two viral infections are very similar. at least seven distinct serotypes of ehdv are recognized, although formal classification of serotypes has yet to be finalized. only two serotypes (ehdv1 and ehdv2) have historically circulated throughout north america, and those serotypes are largely considered to be endemic in almost all areas of the united states, with the exception of the northeast and arid areas of the southwest. however, in 2006, ehdv6 was isolated from surveillance efforts in dead white-tailed deer. 124 since then, ehdv6 has been increasingly identified from both surveillance samples and clinical cases and is also believed to be endemic in several regions. 125 pathogenesis. epizootic hemorrhagic disease (ehd) is a noncontagious disease that is transmitted by the culicoides biting midges. culicoides sonorensis is the primary vector of ehdv in the united states, although other species are also suspected to transmit the disease based on the geographic distribution of clinical cases, although this has yet to be formally shown. due to the vector-borne route of transmission, peak incidence of the disease is closely associated with peak vector population, namely, in the late summer and fall of the year. although capable of infecting a wide range of wild and domestic ruminants, ehdv is largely a pathogen of wild cervids, particularly white-tailed deer. episodes of clinical disease are less common in mule deer, pronghorn antelope, and bighorn sheep and have lower morbidity and mortality. sheep are only rarely infected with the virus and goats appear to be resistant to the virus. cattle are commonly infected based on seroprevalence surveys, but overt clinical disease is uncommon. as a rule, infection in livestock is usually asymptomatic except for periodic epidemics. the last major ehd epidemic in the united states occurred in 2012 and affected a variety of captive and wild ruminant species. 126 in endemic areas, seroprevalence in cervids and other ruminants is high, but clinical disease is not commonly seen. conversely, where seroprevalence is low, introduction of the virus results in widespread infection, where morbidity and mortality can reach 90% and 60%, respectively. following transmission of the virus by biting midges, ehdv replicates in the endothelial cells of the lymphatics surrounding the site of the bite. a primary viremia allows for systemic spread of the virus and secondary replication in lymph nodes throughout the body and the spleen. viremia is important for disease propagation and generally lasts no more than 3 weeks following infection, although the virus can occasionally be isolated from deer infected 50 days previously. antibodies to ehdv are first detected 10 to 14 days following infection but are not always capable of completely neutralizing the infection. thus, it is possible to find both neutralizing antibodies and live virus in the same animal. passive antibodies in fawns can be found up to approximately 4 months of age. as in adults, antibodies in fawns may not protect from infection but generally protect from severe clinical signs. clinical signs. clinical disease in white-tailed deer can be peracute, acute, or chronic. the course of the peracute syndrome of diseaseis relatively short, with death often occurring within 36 hours of infection, with or without the presence of clinical signs. when present, clinical signs include severe edema of the head and neck, swelling of the tongue and conjunctiva, anorexia, fever, weakness, and respiratory distress. hemorrhagic diatheses are not present antemortem but may occur after death. in contrast, in the acute form of the disease, the clinical signs of the peracute form are accompanied with bleeding throughout body tissues (figure 16.1a, b) . ulcers may be evident in the oral cavity and throughout the upper gastrointestinal tract, forestomachs, and abomasum. case fatality rates are high for both the peracute and acute forms. deer that recover after several weeks of illness are said to suffer from the chronic form of the disease. signs of previous illness may include breaks or rings in the hoof horn due to interrupted growth and synthesis leading to lameness, sometimes severe. ulceration and scarring of the rumen and gastrointestinal tract may result in loss of body condition despite a seemingly normal appetite and ample nutrition. widespread evidence of vasculitis may be observed histopathologically. diagnosis. the gold standard for ehdv diagnosis is virus isolation. demonstration of neutralizing antibodies to ehdv reference strains is evidence of previous infection but may be of limited value in endemic areas where seroprevalence levels are expected to be high. also, all potentially suspected serotypes must be used when testing the sample, thereby increasing the time and cost involved with the test. continued research and refinement of molecular techniques, including pcr, are ongoing and are attractive due to the short turnaround times and the potential for high throughput of samples. however, it is important to remember that a positive result using molecular techniques does not equate to the presence of infectious virus, and thus, interpretation of results must be done with caution. control. control of ehd is difficult and relies on a combination of disease surveillance, vector control, and potentially, vaccination. eradication of vector-borne diseases from endemic areas is difficult and time-consuming, and thus, disease control is likely more attainable than strict eradication. vector control is more important in the late fall and summer, when populations are at peak levels and viral transmission is more likely. midge-proofed housing and the treatment of animals with pyrethroid insecticides have been attempted but may be logistically challenging and have yet to have been demonstrated efficacious. vaccine availability in north america is limited, but inactivated autogenous vaccines have been developed from isolates obtained from ill or recently diseased animals. autogenous vaccines are tested for purity but not necessarily for efficacy. vaccine usage must be approved by the u.s. department of agriculture prior to administration. etiology. peste des petits ruminants (ppr) is an acute or peracute, febrile, often fatal disease of ruminants caused by a virus in the family paramyxoviridae and genus morbillivirus. sheep are less susceptible than goats and white-tailed deer. cattle are only subclinically infected, and some wild ungulates, as well as camels, appear to suffer the occasional epizootic. the virus (pprv) is serologically related to the virus that causes rinderpest. geographically, the virus is found throughout northern africa, the middle east, and adjacent regions of asia, with possible movement into southern africa and europe noted. pathogenesis. the main route of infection is respiratory, and ppr is spread by airborne droplets. all secretions and excretions of infected animals are contagious throughout the course of the disease, but no carrier state exists. the virus targets lymphoid tissue. lymphocytes are destroyed in germinal centers in lymph nodes, peyer's patches, tonsils, splenic corpuscles, and cecal lymphoid tissue. immunosuppression results from lymphoid destruction. lymphocytes are partially replaced by plasma cells, macrophages, an eosinophilic acellular matrix, and occasionally neutrophils. the epithelial lining of the mouth and digestive tract is highly vulnerable to the pprv. with the loss of the alimentary tract mucosa, weight loss and diarrhea become severe. the incubation period is usually 2 to 6 days, with up to 10 days possible. clinical signs. the clinical disease produced by pprv in sheep and goats closely resembles that of rinderpest, but the course is much more rapid. with the acute form, sheep and goats typically display an abrupt rise in temperature to 104° to 106° f (40°-41° c). within a few days, infected animals develop nasal and lacrimal discharge, depression, thirst, anorexia, and leukopenia. congestion of the conjunctival and other mucous membranes occurs, followed by serous and mucopurulent exudates. sheep and goats develop oral erosions with necrotic foci, which results in excessive salivation. diarrhea that may be profuse but rarely hemorrhagic develops within 2 to 3 days and is accompanied by abdominal pain, tachypnea, emaciation, and severe dehydration. bronchopneumonia, particularly that caused by pasteurella spp., may be a terminal • fig. 16.1 a. the lungs of the adult pen-raised, white-tailed deer, have been retracted to reveal to ecchymoses on the ventral surface of the "ribcage." petechiae and ecchymoses can occur anywhere within the carcass in cases of epizootic hemorrhagic disease (ehd), but common locations are on the epicardium, on the pleural surface the ribs, subcutaneously, and on the surface of the spleen. b. ecchymoses over the surface of the reticulum (bottom right of photo) and the surface of the rumen (left side of photo). in addition to ehd, this deer also had bronchopneumonia (fibrin overlying consolidated lung can be seen in the far right of photo). (courtesy dr. kelley steury, auburn, al.) a b sequela. death usually occurs 5 to 10 days after the onset of fever. pregnant sheep or goats with ppr may abort. diagnosis. a presumptive diagnosis of ppr can be made on the basis of clinical, pathologic, and epizootiologic findings. the diagnosis can be confirmed by isolating the virus from blood or tissues, including lymph nodes, tonsils, spleen, and lung. immunocapture elisa or pcr may be used to detect infection several days before the development of clinical disease. most serologic tests (complement fixation or agid) cannot differentiate between ppr and rinderpest. characteristic postmortem findings include necrotic stomatitis that is generally confined to the inside of the lower lip and adjacent gum, the cheeks near the commissures, and the ventral surface of the free portion of the tongue. abomasal erosions are often present. in the small intestine, peyer's patches are markedly affected, particularly in the first portion of the duodenum and terminal ileum. the large intestine may be severely affected. lesions occurring near the ileocecal valve, at the cecocolic junction, and in the rectum are often described as zebra stripes that indicate areas of congestion along the folds of the mucosa. treatment and prevention. infection with pprv has no specific treatment. mortality can be reduced by supportive care, including the administration of antimicrobial and antiinflammatory agents, as well as nutritional support. in the united states, state and federal veterinarians should be notified if pprv is suspected. methods used to eradicate rinderpest are useful in the eradication and control of ppr. all sick sheep and goats and those exposed should be slaughtered and disposed of by burning, burying, or rendering. the premises should be decontaminated, and the area quarantined. sheep and goats can be protected against ppr by immunization with rinderpest vaccines or by the simultaneous administration of ppr hyperimmune bovine serum and virulent pprv. 127 pathogenesis. louping ill is a tickborne disease caused by a flavivirus. it affects mainly lambs but occasionally also affects other livestock species and infrequently affects deer, camelids, and humans. transmission is most common during tick season, and ixodes ricinus is thought to be the most important infective host. many sheep clear the infection after a few days of fever and viremia, but others develop severe, fatal viral encephalitis. the virus is shed in many secretions, including milk, which is an important source of infection for other animals (and humans). the severity of the disease depends on herd immunity because previous exposure gives long-lasting immunity. colostrum from immune females is protective for the neonate. high antibody titers also appear to shorten the duration and level of viremia and thereby prevent invasion of the central nervous system. naïve flocks may have fatality rates as high as 60%. clinical signs. high biphasic fever, anorexia, and depression are seen in most infected sheep. lambs may die quickly before illness is noted. some sheep also develop central neurologic signs, including hyperexcitability, muscle tremors, and rigidity. abnormal coordination and muscle activity may cause sheep to move with a bounding gait (hence the name louping ill). diagnosis. the condition has no characteristic gross lesions. microscopic examination of animals with neurologic signs reveals evidence of viral meningoencephalitis. diagnosis is made by history (based on location, signs, and time of year), the identification of characteristic lesions, virus isolation, or fluorescent antibody staining of fresh brain tissue. a demonstrated increase in specific antibody titers in survivors strongly suggests the presence of this infection. prevention. vaccines are available in endemic areas to control infection. vector control during tick season also is important. lambing season should also be timed so that lambs have high colostral antibody protection at the time of exposure to ticks. pathogenesis. foot-and-mouth disease is caused by a highly contagious picornavirus and has been eradicated from the united states. vesicular stomatitis is caused by a rhabdovirus and is intermittently eradicated from the united states. both diseases are highly contagious, nearly indistinguishable from each other clinically, and reportable. foot-and-mouth disease has a broad host range that includes most hoof stock (including pigs but not horses) and several other mammalian species. vesicular stomatitis also affects many species of hoof stock, including both pigs and horses. sheep and goats are relatively less susceptible than cattle, particularly to vesicular stomatitis. the viruses are spread by aerosol and mechanical vectors and primarily colonize skin or mucous membranes. milking machines, flies, birds, and humans all may be important mechanical vectors. vesicular stomatitis tends to remain at the site of infection, and colonization is facilitated by damage to the skin. oral mucous membranes, coronary bands and interdigital skin, and teat-end skin are common sites of lesions. vesicular stomatitis outbreaks in the united states tend to occur in the summer or fall and end with the first killing frost. viremia plays more of a role with foot-and-mouth disease. the virus is present in most body tissues and fluids in infected animals and can be transmitted through milk, meat, bone, and hide products; semen; equipment that pierces the skin; and biting arthropods. it also tends to spread through the circulation from the site of infection to other susceptible tissues, including the sites of vesicular stomatitis, as well as to the nasal cavity, mammary glandular epithelium, and ruminal pillars. the basic lesion for both diseases are the vesicles that form in the oral cavity and on the teats and coronary band. the vesicles quickly rupture and may not be visualized before forming erosions. ruptured vesicles leave deep erosions on the skin or mucous membranes and appear to cause pain. tissue damage and inflammation are often compounded by secondary bacterial infection, which can cause greater morbidity and mortality than the original viral infection. morbidity is related to feed refusal, increased recumbency, and secondary infections of the mouth, udder, and feet. clinical signs. sheep and goats usually develop minor lesions, if any, and are more important in many outbreaks as transport or multiplying hosts than as primary clinical cases. however, identification of lesions should raise suspicion of this disorder. in the worst cases, vesicles, erosions, and ulcers are seen at target sites. they may appear mildly inflamed and erythematous; if they are infected, they may appear severely inflamed with hemorrhage and necrosis. other signs vary according to the location and severity of the lesions. lingual and buccal lesions cause salivation, dysphagia, and feed refusal. foot lesions, which are the most common clinical manifestation in small ruminants, cause lameness and recumbency. teat lesions cause reluctance to be milked or nursed and a decrease in production. fever also may be seen early in the disease, when vesicles are most apparent. the fever then usually abates, and vesicles are replaced by erosions or ulcers. abortion may occur, especially with foot-and-mouth disease, and is probably related to the fever rather than to fetal infection. the disease is usually self-limiting; most animals recover within 2 to 3 weeks. shedding of the virus causing vesicular stomatitis is thought to subside soon after healing of lesions. foot-and-mouth disease virus may be shed for as long as 6 months, and all body secretions and tissues should be considered contagious, including milk, semen, meat, and offal. both viruses have zoonotic potential and cause a disease in humans that resembles mild influenza. the diseases are self-limiting, but people can shed the viruses in sufficient quantities to infect other animals. diagnosis. no characteristic clinicopathologic changes are reported for either virus. gross lesions resemble those seen before death and include vesicular, erosive, and ulcerative lesions of the mouth, feet, and teat ends; foot-and-mouth disease also causes lesions of the mammary gland and ruminal epithelium. microscopic findings include hydropic degeneration of cells of the stratum spinosum of the epidermis without inclusion bodies. secondary bacterial infection may lead to deeper ulcers and complicate identification of the viral etiology of these lesions. myocarditis lesions may be seen with some forms of foot-and-mouth disease. a presumptive diagnosis may be made by identifying characteristic lesions during a season and in an area at risk for one of these infections. in north america, bluetongue should be considered as an important differential diagnosis for ulcerative oral lesions in sheep. a confirmed diagnosis of foot-and-mouth disease is achieved by a combination of virus isolation (from vesicles), ihc, and serology by regulatory officials. identifying the source of infection also is very important. diagnosis of vesicular stomatitis is achieved by complement fixation or fluorescent antibody staining of virus in vesicular fluid or detection of a rise in antibody titers. flocks with either of these diseases in the united states are subject to quarantine and possible destruction (especially for foot-and-mouth disease). prevention. meticulous personal hygiene and avoidance of contact with new animals are important during outbreaks to prevent spread between flocks. vaccines against foot-and-mouth disease are available in many parts of the world, but not in the united states. most nations slaughter or quarantine affected animals. vaccines against vesicular stomatitis are available and are most commonly used if the risk of outbreak is high, but vaccination does not prevent infection or shedding. good hoof and teat care and soft feeds may help prevent spread of the virus by providing a healthy, intact barrier against invasion. pathogenesis. sheep and goat pox are caused by two closely related poxviruses. some strains are infective to both sheep and goats; most are species specific. they are maintained in populations by infected animals, and transmission occurs by aerosol or direct or indirect contact. flies may play an important role as mechanical vectors in some flocks. viruses remain infective in the environment for as long as 6 months. after infection, viremia and inflammation of the oral, nasal, and ocular mucous membranes occur. erythematous papular pox lesions appear a few days later. severity varies according to strain pathogenicity, breed susceptibility, and immune status. mild infections are characterized by lesions concentrated in the non-wooled or hairless regions of the skin. severe infections produce lesions throughout the oral cavity, respiratory tract, and peritoneal cavity. secondary infection is common with the severe form and mortality is high. if the animal survives, lesions heal in 3 to 4 weeks. both diseases have been eradicated from the united states and are reportable. people can develop mild disease on exposure to these viruses. clinical signs. fever, inappetence, conjunctivitis, and upper respiratory signs are seen in the initial stages. pox lesions are visible shortly thereafter. secondary infection can lead to a variety of more serious signs indicative of respiratory disease, sepsis, and shock. diagnosis. characteristic pox lesions are highly suggestive of this disease. microscopic analysis reveals eosinophilic intracytoplasmic inclusion bodies, acantholysis, and pustule formation within the epidermis and occasionally the dermis. viral particles may be seen on ultrastructural examination. gross and microscopic lesions are characteristic with the severe form, but mild disease may produce mild lesions that are difficult to differentiate from other viral diseases that cause oral proliferative or ulcerative lesions. virus can be isolated from blood or tissues (mainly skin) during the acute viremic stage and identified by antibody staining of more chronic lesions. serologic tests are available to detect rising titers in convalescent animals. treatment and prevention. no specific treatment is available for sheep or goat pox. antibacterial drugs may be useful to treat secondary infection. judicious use of insecticides and confinement of affected animals may prevent spread. vaccines are available in some countries, but not in the united states. infected flocks are placed under quarantine or destroyed in regions where the diseases are not endemic. these viruses are difficult to eradicate from flocks because of their environmental persistence and the constant supply of susceptible hosts. caprine arthritis-encephalitis virus (caev) is an enveloped, singlestranded retrovirus in the lentivirus genus. like other retroviruses, caev integrates into the host chromosomal dna before replicating. the virus is able to remain latent or undergo sporadic bouts of productive viral replication. caev is closely related to ovine lentiviruses. clinical signs. clinical disease may be evident in only 10% of goats from a caev-infected herd at any given time. as many as 85% of seropositive goats may be clinically normal. caev produces four clinical syndromes: encephalomyelitis, arthritis, interstitial pneumonia, and indurative mastitis. the pattern of disease usually varies with age. arthritis is generally seen in sexually mature goats, whereas encephalomyelitis is generally seen in kids 2 to 4 months old. interstitial pneumonia and indurative mastitis are more common in adult goats. some goats suffer from a wasting disorder characterized by poor body condition and rough hair coat. diagnosis. a presumptive diagnosis of caev can be made on the basis of history and clinical signs suggestive of one or more of the syndromes. in general, elisa tests are better for detecting disease in an individual animal because the sensitivity of the test is higher than that of the agid, whereas the agid is better for herd screening that requires high specificity. with the agid test, false negatives may occur in goats that have not yet seroconverted to recent infection. individual goats may take months or years to seroconvert or may never do so. parturition or advanced stages of disease also may contribute to a false-negative result. false positives may occur in goats younger than 90 days old that have colostral antibodies. for this reason, it is often suggested that kids be at least 6 months old before they are tested. pcr testing has high specificity and sensitivity and can detect infection within a day of exposure. other less commonly used tests include a western blot to detect antibodies and a northern blot to look for mitochondrial rna. because of the limitations in interpreting serologic results, caev-induced disease can only be definitively diagnosed by identification of characteristic lesions from examination of biopsy specimens or postmortem viral isolation. treatment. no specific treatments are available for any of the syndromes associated with caev. young goats suffering from encephalomyelitis may benefit from physical therapy if they are recumbent, and bottle feeding may help maintain hydration and caloric intake. antibiotics may be beneficial to goats affected with interstitial pneumonia or mastitis if secondary bacterial infection is present. generally, the prognosis is poor for the encephalitic form and guarded for the other forms. prevention. prevention of caev is crucial because infection is lifelong. infected colostrum and milk are the most important sources of infection. newborn kids should be prevented from ingesting colostrum from infected does and should instead be fed pasteurized goat's milk or milk from caev-negative goats. all goats in a herd should undergo serologic testing twice yearly; seropositive goats should be segregated or culled to prevent direct contact between infected and uninfected animals. ovine progressive pneumonia (opp) is an ultimately fatal retroviral disease that causes chronic, progressive, debilitating inflammatory conditions of the lungs (united states) and central nervous system (other parts of the world). it also is called maedi-(maeði is icelandic for "shortness of breath") visna (meaning "wasting"). the virus is a member of the lentivirus genus of retroviruses and is closely related to caev. recombination between opp and cae viruses has been observed. 128 the virus primarily affects sheep and rarely goats and has been identified worldwide, except in australia and new zealand. the disease has a long incubation period and protracted clinical course. pathogenesis. only sheep older than 2 years of age are affected by opp virus (oppv). the virus is spread by direct contact, probably in respiratory and salivary secretions, and by excretion in the milk and colostrum. transplacental transfer is of minor importance. virus is excreted by animals that exhibit clinical signs and asymptomatic animals. infection is established in the monocyte and macrophage cell line and spread by these cells to the lungs, lymph nodes, choroid plexus, spleen, bone marrow, mammary gland, and kidneys. like caev, oppv evades the cellular and humoral immune system of the host by incorporation of its provirus in host dna, low-grade replication of virus only when monocytes differentiate into macrophages (restricted replication), and production of antigenic variants that are not neutralized by existing antibodies. continual antigenic stimulation of the host by low-grade replication of oppv results in chronic inflammation and resultant lymphoid proliferation in various target tissues. the virus may prevent b lymphocytes from differentiating into plasma cells in lymph nodes and may thereby impair immunoregulation. seroconversion occurs within 2 to 3 weeks after infection. clinical signs. in the united states, serologic surveys reveal infection rates of between 30 and 67% but rarely is more than 5% of a flock lost to oppv. icelandic, texel, border leicester, and finnish landrace appear to be susceptible sheep breeds. more resistant sheep breeds include rambouillet, suffolk, and columbia. various clinical syndromes are associated with oppv and include wasting (thin ewe syndrome), dyspnea occasionally with a dry cough, pneumonia, mastitis ("hard bag"), posterior paresis, arthritis, and vasculitis. in north america, pneumonia and indurative aseptic mastitis are common sequelae of infection. coinfection with the jaagsiekte virus (the cause of pulmonary adenomatosis) worsens respiratory signs. visna, the neurologic form, is more common in goats. over the course of up to a year, subtle signs such as a head tilt or hindlimb weakness progress to gross incoordination, whole body tremors, and rarely more profound cranial nerve signs. diagnosis. a presumptive diagnosis can be made on the basis of clinical signs, poor response to treatment, characteristic postmortem findings, and serologic testing. definitive diagnosis requires pcr or isolation of the virus from wbcs (buffy coat of whole blood sample) or tissues. less expensive and faster serologic tests include agid, elisa, and an indirect immunofluorescence test. the agid test is frequently used as a flock screening test, but the elisa is more sensitive on an individual basis and can detect antibodies earlier in the course of the disease. as with caev, false negatives and false positives are possible. characteristic postmortem lesions include generalized wasting and firm, noncollapsing lung or firm, mottled mammary glands, both with regional lymphadenopathy. microscopic evaluation of those tissues reveals interstitial non-septic, mononuclear cell infiltrates, although these may be complicated by secondary infections. histopathology of nervous tissue reveals meningoleukoencephalitis. treatment. no effective treatment is available for oppv. supportive therapy that includes appropriate husbandry and control of secondary infection with antibiotics may prolong life for a few weeks or months but, ultimately, the disease is fatal. because of the poor prognosis and risk of exposure of naive animals to clinical disease, long-term treatment is not recommended. prevention. the only known method of preventing oppv infection in a flock is to prevent exposure to the virus. management practices that help decrease the incidence of horizontal transmission include disinfection of milking equipment, dehorning instruments, and tail docking and castration tools before use and between animals. contaminated feed and water also are potential routes of infection and should not be shared among infected and uninfected animals. serologic testing and separation or culling of seropositive animals may help reduce infection. although oppv can readily be isolated from ewe colostrum, colostral transmission of oppv has not been definitively established. however, many prevention guidelines recommend that offspring from infected dams be separated from the dam before they nurse and then be fed cow colostrum and artificially reared. quarantine and serologic testing of flock additions before placing them with the current flock and purchase of sheep only from oppv-free flocks are important to prevent the introduction of new infections. because of the potential cross-species spread, all precautions taken for sheep also apply to contact goats. serologic testing should be performed at least annually in a flock until two consecutive negative test results are obtained. border disease virus (bdv) is in the genus pestivirus and family flaviviridae, which also includes the two genotypes of bovine viral diarrhea virus (bvdv) and classical swine fever virus. it rarely causes disease in adults and is most important as a cause of in utero infection of lambs and kids. the condition gets its name from the fact that it was first reported in sheep along the welsh border of the united kingdom. other names such as "hairy shakers" and "fuzzy lamb disease" refer to some of the clinical signs seen in affected newborns. it is important to recognize that although bdv is genetically distinct from the two types of bvdv, sheep and goats also are susceptible to some strains of bvd. pathogenesis. horizontal transmission of bdv occurs through contact with secretions and excretions of body fluids and tissues from infected animals. the virus crosses intact mucous membranes and can spread rapidly through a flock. the major reservoir is the persistently infected sheep or goat. these reservoirs are usually asymptomatic, congenitally infected, and often seronegative animals that shed large quantities of virus. these may be residents of a flock with an ongoing problem or bought in as replacement animals to a naïve flock. some cross-infection from other species is possible, particularly from cattle. adult, immunocompetent sheep rarely show any signs of acute infection. however, if a pregnant ewe or doe is infected, the virus may be transmitted vertically to the embryo or fetus. depending on the stage of gestation, embryonic or fetal infection may have different outcomes ranging from embryonic reabsorption to normal birth. these infections are the most important aspect of border disease. the major organ system targeted by bdv is the fetal central nervous system. the hallmark lesion is hypomyelination, or degeneration of oligodendroglial cells. three factors contribute to this lesion. the first is direct viral damage. the second is viral-induced inhibition of the thyroid gland that causes decreased secretion of thyroid hormones. in the absence of these hormones, a resultant lowered concentration of a specific nucleotide in the central nervous system also contributes to the hypomyelination. the third factor is altered immune function. the virus causes the host to produce a virus-specific delayed hypersensitivity reaction that causes inflammation in the central nervous system. it also causes immunosuppression. death often results from opportunistic conditions such as parasitism, diarrhea, and bronchopneumonia. clinical signs. clinical signs depend on the time during gestation when the fetus or embryo is exposed to the virus. clinical signs also may vary in severity from animal to animal because different fetuses develop competent immune systems at different times. if the fetus or embryo is exposed to the virus within 45 days of conception, it dies and is resorbed or aborted. these losses are not usually noticed by the flock manager. the principal manifestation in the flock is a large number of open ewes and a small lamb crop. infection of the fetus between days 45 and 80 of gestation causes damage to rapidly growing systems such as the skin and nervous, lymphoid, thyroid, and skeletal systems. congenital malformations are seen at birth. lambs have abnormal fleece characteristics (hairy rather than woolly in consistency), small stature, domed heads, shortened legs, and dark pigmentation of the skin, particularly on the dorsal aspect of the neck. the lamb may exhibit tonic-clonic tremors ("hairy shakers") when awake, which may prevent standing or suckling. most of these lambs die within a few days of birth. if they survive, the hair changes disappear in 9 to 12 weeks and the central nervous system signs resolve by 20 weeks. goats infected at this time have similar symptoms except that they rarely exhibit hair coat changes. if kids are infected before day 80 of gestation and are still viable, they may become persistently infected and immunologically compromised. they are small at birth and generally weak. typical outbreaks of border disease cause abortions and birth of weak lambs in the first year as the virus rapidly spreads throughout a susceptible flock and then insignificant losses in the succeeding years as adult sheep develop immunity. however, if new naïve ewes are introduced in the flock, substantial losses may occur in perpetuity. diagnosis. border disease viral antigens can be demonstrated in abomasum, pancreas, kidney, thyroid, skin, and testicle tissues from aborted fetuses and persistently infected animals using fluorescent antibody tests. however, ihc on ear notch samples is not considered as reliable for detecting persistently infected small ruminants as it is for cattle. the virus can be isolated, or viral antigen detected by elisa, from serum, heparinized whole blood, and tissue taken from brain, spinal cord, spleen, and bone marrow from affected lambs. whole blood is better than serum if colostral antibodies are likely to be high; serum is an adequate sample in neonates and juveniles that have not suckled. antibodies to the virus may be quantified by serum neutralization, agid, and complement fixation with hyperimmune bvd antiserum. serologic tests are useful to detect exposure in lategestation (after day 80) neonates and unvaccinated animals but may be confounded by colostral antibodies in suckling neonates, previous exposure, and vaccination in older animals. any titer in a presuckling neonate indicates in utero exposure, whereas a serum neutralization titer of 1:20 to 1:320 suggests infection in adults. the presence of specific antibodies in the cerebral spinal fluid suggests bdv infection. negative presuckling serologic tests do not rule out exposure because persistently infected lambs tend to be immunotolerant to the bdv and therefore are born without an antibody titer. these animals may subsequently develop a titer that is indistinguishable from that of a normal animal. although persistently infected animals do not respond immunologically to the strain of the virus they carry, they may respond to other strains of the virus, including vaccine strains. as with bvd, pcr assays are gaining popularity for the detection of bdv in fluids and tissue samples. these assays appear to be superior to other techniques, except in autolyzed tissues. realtime pcr may also be used to differentiate bdv from bvd and to type isolates. gross postmortem findings include hydranencephaly, porencephaly, microcephaly, cerebellar hypoplasia, abnormal rib curvature, brachygnathia, doming of the frontal bones of the skull, narrowing of the distance between the orbits, shortening the crown-to-rump length, shortening of the diaphyseal length, retention of secondary hair fibers, and abnormal skin pigmentation. the major histopathologic changes include hypomyelination and hypercellularity of the white matter. glial cells appear normal. treatment. no treatment is available for border disease infection. supportive care may include assistance in nursing and standing for affected lambs, provision of good bedding and solid footing, and treatment of secondary opportunistic infection. prevention. control is primarily achieved by eliminating persistently infected carrier animals from the flock and preventing the addition of new carrier animals. this is easiest in a closed flock but especially difficult in small ruminant flocks because of the frequent desire to import new genetics. to identify carriers, virus isolation must be performed on every animal in the flock; carrier animals must be culled. additionally, all unborn animals must be considered potential carriers and should be tested at birth. an alternative solution in hobby flocks is to arrest breeding activity until all animals have been shown to be free of infection. new animals should be quarantined and tested before admission to the flock. herd screening with the ear skin biopsy test using fluorescent antibody staining to detect virus is less expensive and more convenient than the whole blood virus isolation test. the role of vaccination in preventing infection is still unclear. no vaccine against bdv is available, but some reports suggest that bvdv vaccines for cattle may be helpful for sheep at risk. however, these vaccines have proven to be more effective at preventing clinical disease in vaccinated animals than in preventing in utero infection because they do not prevent transient viremia. vaccination decreases viremia and fetal infection but does not eliminate them. therefore, vaccines play a role in decreasing economic loss but do not replace culling of carrier animals as the major method of control. another member of the slow infection group of diseases of small ruminants is scrapie. it is an afebrile, chronic, progressive degenerative disorder of the central nervous system of sheep and occasionally of goats (see chapter 13) . scrapie is caused by a prion and, as such, is one of the transmissible spongiform encephalopathies. sheep (and goats and mouflon to a lesser degree) are the natural hosts for scrapie. clinical signs often do not usually appear until animals are 2 years old, and animals as old as 5 years may exhibit clinical disease. both vertical and horizontal transmission have been demonstrated experimentally in sheep and goats. abnormal scrapie protein has been identified in milk, urine, and seminal plasma of sheep up to 20 months prior to the development of clinical signs. also, new evidence from deer with chronic wasting disease, a similar disorder, suggests that infective prions are excreted in the saliva and feces well before the development of clinical signs. these new revelations may help explain horizontal transmission of infection. clinical signs. the onset of scrapie is insidious. initially, sheep show subtle changes in behavior such as mild apprehension, staring or fixed gaze, failure to respond to herding dogs, and boldness around humans. several months later, the animals become intolerant of exercise and develop a clumsy, unsteady gait and floppy ears. later, the sheep develop itchy skin that causes them to rub themselves excessively against firm, immobile objects (origin of the name scrapie). this leads to excoriations and wool damage. there is a general decline in body condition and coordination. diagnosis. histologically, the only consistent lesions are degenerative changes in the central nervous system consisting of bilaterally symmetric vacuolation of the neurons in the brainstem and spinal cord with accompanying spongy degeneration. as a preclinical test, ihc may be performed in lymphoid tissue from the tonsils, third eyelid, or rectoanal mucosa, but none of these methods is foolproof. cwd is discussed in chapters 13, 19, and 20. testing for clinical anaemia caused by haemonchus spp. in goats farmed under resource-poor conditions in south africa using an eye colour chart developed for sheep validation of the fama-cha eye color chart for detecting clinical anemia in sheep and goats on farms in the southern united states validation of the famacha © eye colour chart using sensitivity/ specificity analysis on two south african sheep farms is the famacha chart suitable for every breed? correlations between famacha scores and different traits of mucosa colour in naturally parasite infected sheep breeds rumen bacteria are involved in the onset of onion-induced hemolytic anemia in sheep the role of free radicals in brassicainduced anaemia of sheep: an esr spin trapping study kale poisoning: the brassica anaemia factor hemolytic anemia in sheep fed wild onion (allium validum) copper toxicosis in sheep: a case report chronic copper poisoning in sheep. i. the relationship of methaemoglobinemia to heinz body formation and haemolysis during the terminal crisis chronic copper toxicity of ruminants copper poisoning in a flock of sheep. copper excretion patterns after treatment with molybdenum and sulfur or penicillamine evaluation of mechanisms of leptospiral hemolytic anemia fatal hemolytic anemia attributed to leptospirosis in lambs studies on eperythrozoon ovis-infection in sheep eperythrozoon ovis-a blood parasite of sheep experimental eperythrozoon ovis infection of sheep mycoplasma ovis comb. nov. (formerly eperythrozoon ovis), an epierythrocytic agent of haemolytic anaemia in sheep and goats molecular characterization of two different strains of haemotropic mycoplasmas from a sheep flock with fatal haemolytic anaemia and concomitant anaplasma ovis infection bovine colostrum as a cause of hemolytic anemia in a lamb heinz body anaemia in lambs with deficiencies of copper or selenium maxillary lymphosarcoma in a white-tailed deer (odocoileus virginianus) large animal internal medicine effect of physical restraint and xylazine sedation on haematological values in red deer (cervus elaphus) seasonal variations in red deer (cervus elaphus) hematology related to antler growth and biometrics measurements the genetic basis and evolution of red blood cell sickling in deer one hundred two tumors in 100 goats lymphoma classification in goats exophthalmos due to multicentric b-cell lymphoma in a goat ocular involvement of multicentric malignant b-cell lymphoma in a ewe. a case report diseases and parasites of white-tailed deer, miscellaneous publication no. 7. tall timbers research station colostrum composition of santa inês sheep and passive transfer of immunity to lambs effects of maternal undernutrition during late gestation and/or lactation on colostrum synthesis and immunological parameters in the offspring failure in passive transfer of immunoglobulin g1 to lambs: measurement of immunoglobulin g1 in ewe colostrums iodine supplementation of the pregnant dam alters intestinal gene expression and immunoglobulin uptake in the newborn lamb short communication: apoptosis regulates passive immune transfer in newborn kids effects of newborn characteristics and length of colostrum feeding period on passive immune transfer in goat kids a field trial evaluating the health and performance of lambs fed a bovine colostrum replacement use of a digital brix refractometer to estimate serum immunoglobulin in goat kids field methods for estimating serum immunoglobulin concentrations in newborn kids colostrum deficiency in mule deer fawns: identification, treatment and influence on neonatal mortality passive transfer of colostral immunoglobulins from ewe to lamb and its influence on neonatal lamb mortality transfer of maternal passive immunity to kids in goat herd suppurative meningitis in a 7-day-old formosan sambar deer (cervus unicolor swinhoei) caused by escherichia coli factors affecting igg concentration in day-old lambs effects of maternal nutrition on udder development during late pregnancy and on colostrum production in scottish blackface ewes with twin lambs the effect of colostrum source (goat vs. sheep) and timing of the first colostrum feeding (2h vs. 14h after birth) on body weight and immune status of artificially reared newborn lambs bovine neonatal pancytopenia and anaemia in lambs caused by feeding cow colostrum secondary lactose intolerance in a neonatal goat hypernatremia in neonatal elk calves: 30 cases (1988-1998) group b rotavirus associated with an outbreak of neonatal lamb diarrhea rotaviruses associated with neonatal lamb diarrhea in two wyoming shed-lambing operations novel group a rotavirus g8 p[1] as primary cause of an ovine diarrheic syndrome outbreak in weaned lambs role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in spain enteric viral infections in lambs or kids suspected clostridium difficile-associated hemorrhagic diarrhea in a 1-week-old elk calf observations and immunohistochemical detection of coronavirus, cryptosporidium parvum and giardia intestinalis in neonatal diarrhoea in lambs and kids giardia duodenalis and cryptosporidium parvum infections in adult goats and their implications for neonatal kids case control study of diarrhoea and faecal soiling in two-to six-month-old lambs comparison of two techniques for diagnosis of cryptosporidiosis in diarrhoeic goat kids and lambs in cyprus fluid therapy in calves passive immunisation of neonatal lambs against infection with enteropathogenic escherichia coli via colostrum of ewes immunised with crude and purified k99 pili floppy kid syndrome (metabolic acidosis without dehydration in kids clostridium perfringens toxins involved in mammalian veterinary diseases first isolation of clostridium perfringens type e from a goat with diarrhea clostridial enteric diseases of domestic animals isolation and molecular characterization of clostridium perfringens from healthy merino lambs in patagonia region lamb losses associated with clostridium perfringens type a hemorrhagic bowel syndrome in dairy cattle: 22 cases clostridium perfringens type a and beta2 toxin associated with enterotoxemia in a 5-week-old goat investigation of a syndrome of sudden death, splenomegaly, and small intestinal hemorrhage in farmed deer gastric mucormycosis in a sika deer (cervus nippon) associated with proliferation of clostridium perfringens the relationship between the presence of helicobacter pylori, clostridium perfringens type a, campylobacter spp, or fungi and fatal abomasal ulcers in unweaned beef calves multiplex pcr method for genotyping clostridium perfringens the effect of clostridium perfringens type c strain cn3685 and its isogenic beta toxin null mutant in goats beta toxin is essential for the intestinal virulence of clostridium perfringens type c disease isolate cn3685 in a rabbit ileal loop model clostridial diseases vaccines for control, prevention and eradication of disease in farmed deer development and application of an oral challenge mouse model for studying clostridium perfringens type d infection enterotoxaemia caused by clostridium perfringens type d in farmed fallow deer rates of diseases and their associated costs in two colorado sheep feedlots (1985-1986) proportional mortality: a study of 152 goats submitted for necropsy from 13 goat herds in quebec, with a special focus on caseous lymphadenitis the pathology of experimental clostridium perfringens type d enterotoxemia in sheep enterotoxaemia in goats: a review diagnosis of clostridium perfringens intestinal infections in sheep and goats clinico-pathological findings of clostridium perfringens type d enterotoxaemia in goats and its hemolytic activity in different erythrocytes experimental clostridium perfringens type d enterotoxemia in goats clinical signs, treatments, and postmortem lesions in dairy goats with enterotoxemia: 13 cases epsilon toxin is essential for the virulence of clostridium perfringens type d infection in sheep, goats, and mice clinicopathologic features of experimental clostridium perfringens type d enterotoxemia in cattle ulcerative enterocolitis in two goats associated with enterotoxin-and beta2 toxin-positive clostridium perfringens type d the passive protection of lambs against clostridium perfringens type d with semi-purified hyperimmune serum blackleg in deer bacterial diseases of farmed deer and bison black disease in a forest reindeer bovine vaccines and herd vaccination programs toxigenic clostridia characterization of the catalytic domain of clostridium novyi alpha-toxin first report of infectious necrotic hepatitis (black disease) among nubian goats in sudan clostridium novyi (myonecrosis, black disease, and bacillary hemoglobinuria) and clostridium septicum (braxy) infections first report of infectious necrotic hepatitis (black disease) among nubian goats in sudan liver and biliary system bacillary hemoglobinuria: induction by liver biopsy in naturally and experimentally infected animals bacillary hemoglobinuria in a free-ranging elk calf bacillary hemoglobinuria in dairy cows an outbreak of bacillary haemoglobinuria in sheep in india successful treatment of bacillary hemoglobinuria in japanese black cows acute abomasitis due to clostridium septicum infection in experimental sheep rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the 16s-23s rdna spacer region suppurative abomasitis associated with clostridium septicum infection clostridial myocarditis in lambs outbreak of clostridial myocarditis in calves clostridial myositis in cattle: bacteriology and gross pathology clostridial vaccination efficacy on stimulating and maintaining an immune response in beef cows and calves failure of clostridium chauvoei vaccines to protect against blackleg prevalence of coxiella burnetti infection in wild and farmed ungulates coxiella burnetii shedding by farmed red deer (cervus elaphus) high prevalence of antibodies against chlamydiaceae and chlamydophila abortus in wild ungulates using two regional seroprevalence of leptospirosis on deer farms in new zealand growth response and shedding of leptospira spp. in urine following vaccination for leptospirosis in young farmed deer corynebacterium pseudotuberculosis paratuberculosis (johne's disease) in cattle and other susceptible species efficacy of a killed vaccine for the control of paratuberculosis in australian sheep flocks detection of a novel reassortant epizootic hemorrhagic disease virus (ehdv) in the usa containing rna segments derived from both exotic (ehdv-6) and endemic (ehdv-2) serotypes the first 10 years (2006-15) of epizootic hemorrhagic disease virus serotype 6 in the usa review of the 2012 epizootic hemorrhagic disease outbreak in domestic ruminants in the united states peste des petits ruminants demonstration of coinfection with and recombination by caprine arthritis-encephalitis virus and maedi-visna virus in naturally infected goats key: cord-021413-1ht1xm88 authors: kraft, lisbeth m. title: viral diseases of the digestive system date: 2013-10-21 journal: diseases doi: 10.1016/b978-0-12-262502-2.50016-x sha: doc_id: 21413 cord_uid: 1ht1xm88 this chapter discusses three virus infections affecting the digestive system of mice and their properties: (1) epizootic diarrhea of infant mice (edim), (2) reovirus 3 infection, and (3) murine hepatitis virus infection (mhv). all three infections may cause serious, debilitating, and sometimes fatal diarrheal disease in nursling and weanling mice. mice of all ages can be infected by the edim virus but overt disease is restricted to animals up to about 12–13 days of age at the time of first exposure. the edim virus is worldwide in distribution. its prevalence is difficult to estimate because serologic tests have not been readily available, and it is not customary to sacrifice animals for the purpose of examining the appearance of their intestinal tract or for electron microscopic visualization of fecal contents. the acute disease of reovirus 3 infection affects mainly sucklings and weanlings, whereas the chronic disease is encountered in animals over 28 days of age. the mhv virus, on the other hand, has been found to affect cotton rats, rats, and hamsters. epidemic diarrhea of infant mice, it is common knowledge that virtually every colony of conventional mice suffered that infec tion to a greater or lesser extent. in recent years, with the advent of measures such as cesa rean derivation, barrier-sustained breeding and maintenance procedures, routine serologic surveillance, laminar flow hoods or rooms, and filter-top cages, the problems associated with these diseases have become somewhat less critical. neverthe less, they are at times and under certain circumstances still troublesome. all three infections may cause serious, debilitating, and sometimes fatal diarrheal disease in nursling and weanling mice. thus, the economic impact on commercial mouse col onies can be severe. furthermore, newborn mice, pooled from many dams and then redistributed to them at random, are used for the isolation and identification of certain viruses in diagnos tic and epidemiological studies, for example arboviruses (shope, 1980) , coronaviruses , and reoviruses (stanley, 1977) . clearly, indigenous infection with related or identical agents in only a few such infants could confound and compromise the validity of observations follow ing the inoculation of test materials. it should be pointed out, too, that collins and parker (1972) demonstrated both reovirus 3 and murine hepatitis virus as contaminants in some murine leukemia and transplantable tumor specimens. whereas it is mainly from these standpoints that the diseases in question derive importance, they should not be dismissed without considering their intrinsic value as models for elucidat ing the pathogenesis and control of related infections in man and other animals as well as their utility for the molecular biologist. cheever and mueller (1947, 1948) , pappenheimer and en ders (1947) , and pappenheimer and cheever (1948) were the first to describe the pathological changes and epidemiology of edim. runner and palm (1953) and cheever (1956) also con tributed to knowledge concerning the epidemiology and etiol ogy of the disease. thereafter kraft (1957 kraft ( , 1958 kraft ( ,1961 kraft ( ,1962b kraft ( , 1966 reported on studies regarding the etiology, mode of transmission, carrier state, immune response, pathogenesis, and control of the disease. serologic studies were also under taken by blackwell et al (1966) . kraft (1963, 1967) first demonstrated the agent in electron micrographs of infected infant mouse intestinal epithelium. banfield et al (1968) enlarged on those findings, comparing the virions to those of the reoviruses. particles of similar morphology were subsequently observed in the diarrheal feces of many species, primarily in the young: cattle (mebus et al, 1969) , man (flewett et al, 191 a) , horse (flewett et al, 1975) , pig (rodger et al, 1975) , sheep , rabbit , deer (tzipori et al, 1976) , goat (scott et al, 1978) , and dog (england and poston, 1980) . in addition, one virus, sa-11, was isolated from a nondiarrheal monkey, and another, oa (offal agent), was recovered from intestinal washings of sheep in an abattoir (els and lecatsas, 1972) . much and zajac (1972) purified edim virus from diarrheal infant mice and further characterized it. based on its moφhology and other known characters, edim virus has been placed into the genus rotavirus in the family reoviridae (matthews, 1979) . during the past decade, knowl edge of the rotaviruses as agents of diarrheal disease of young mammals has burgeoned, especially with regard to those af fecting children, calves, and piglets. reviews concerning the genus have been published by wyatt et al (1978 ), mcnulty (1978 , flewett and woode (1978) , andrewes et al (1978) , and holmes (1979) . additional comments may be found in the american veterinary medical association panel the young (anonymous, 1978) . the classification of edim virus in the genus rotavirus, family roeviridae, derives from its moφhology and mode of replication as seen in electron micrographs (adams and kraft, 1967; banfield et al, 1968) , which were later shown to re semble those observed in ultrathin sections in human intestinal material containing ''reovirus-like" particles (bishop et al, 1973) . the term rotavirus (l. rota, wheel) was proposed by flewett et al (1974) because of its moφhology as viewed in negative-contrast electron micrographs. it has become widely used in preference to duovirus, which is synonymous (david son et al, 1975) . there is no evidence that antigenic or pathogenetic variants of edim virus exist. analysis of rna segment and struc tural polypeptide variation, however, as has been accom plished for other rotaviruses derbyshire and woode, 1978; rodger and holmes, 1979) , may reveal differences among strains. further, several serotypes have been described a m o n g the h u m a n rotaviruses by m e a n s of the serum neutralization test (beards et al., 1980) . this technique may also prove useful in future studies of e d i m virus strains. a. morphology, size, and composition. kraft (1962b) , using millipore filters, determined the size of the infective e d i m virion to be between 33 and 100 n m . in electron micro graphs, a d a m s and kraft (1967) mined the m e a n diameter of 100 virions of purified e d i m virus to be 5 4 . 4 ± 2 n m . o n the other h a n d , melnick (1979) gives about 50% is lost at 4x for 24 hr or at 37t for 1 hr. although some infectivity (>0.05%) remains at either 56°c or 60°c for 30 min, it is abolished at 70t for 15 min (cheever and muel ler, 1947; kraft, 1957 kraft, , 1962b . much and zajac (1972) found that the purified virus is unstable at both 4° and -24''c for 2 weeks but found that at -70°c infectivity is retained for at least 4 weeks. c. ejfect of chemicals. in an intestinal filtrate, edim virus titer is not significantly reduced when held in ether or 0.1% sodium deoxycholate at 4°c for 24 hr (kraft, 1962b) . according to much and zajac (1972) , the purified virus is stable in 20% ether, 5% chloroform, or 0.1% sodium deoxycholate at 4°c for 1 hr and, as is true for other rotaviruses, is resistant to pancreatin. ward and ashley (1980a,b) examined the effects of the anionic detergent sodium dodecyl sulfate and of the chelating agent ethylenediaminetetraacetate on purified simian virus and determined that low concentrations and mild temper ature conditions readily inactivated the agent. both chemicals modified the viral capsid to prevent adsoφtion of the inacti vated virions to cells. indeed, this study was the outgrowth of a need to determine the survival of enteric viruses in wastewater. it had been determined that wastewater sludge reduced the heat necessary for simian rotavirus inactivation. ionic detergents in the sludge were identified as the active components. nonionic detergents did not destabilize the virus; further, these com pounds protected the virus from the destabilizing effect of sodium dodecyl sulfate. destabilization by both cationic and anionic detergents was found to be dependent on the ph of the medium. a systematic study of the stability of edim virus to extremes of ph has not been reported. much and zajac (1972) kraft (1966) replication of edim virus occurs in epithelial cells of the villi of the small intestine. in electron micrographs, kraft (1967), banfield etal. (1968) , and holmes etal. (1975) demonstrated replication by budding in distended cistemae of the endoplasmic reticulum. the significance of intracytoplas mic and intranuclear tubular structures encountered is not clear. using direct immunofluorescence, wilsnack et al. (1969) found that edim virus antigen in infant mice is limited to the cytoplasm of the villous epithelium from the duodenum to the colon and is detectable within 48 hr after peroral inoculation. stainable virus was also seen in the intestines of contact infant mice without necessarily causing clinical signs to appear. the virus could not be stained in the stomach or liver of mice infected either naturally or experimentally. kraft (1958) studied the distribution of infectious edim virus in 3-day-old mice following peroral inoculation. at 3 hr, virus was detected in the stomach and small and large intes tines (the cecum was not tested) and could not be recovered from the lungs, liver, spleen, kidney, or blood. at 22 hr, all those tissues were positive except for the kidney. the bladder and urine, as well as the brain, were also devoid of the agent, but at 30 hr, these too were positive. at 72 hr, the liver, spleen, kidney, and intestines yielded virus, but the brain was negative. the blood, stomach, lungs, bladder, and urine were not tested at that interval. at 6 days, blood, liver, and intestines, the only tissues examined, still contained virus. ingested virus from a diarrheal litter brings about an active infection in the nursing dam that previously had been nondiarrheal herself and had had only nondiarrheal litters (kraft, 1958) . blood, liver, spleen, and feces all contain infectious virus in the dam 1 week after her litter is given virus perorally as an intestinal filtrate. later, kraft (1961) determined that adult male mice can also be intestinal carriers for at least 17 days after a single peroral exposure. with regard to the mechanism of cell penetration. holmes et al. (1976) proposed that lactase of the villous brush border of the intestine may be the receptor that uncoats the rotavirus virion by attacking the glycolysated polypeptides of the outer capsid. in addition, pancreatic enzymes within the lumen may also be instrumental in the infectious process (see section ii,b,6). the question of cofactors that might enhance pathogenicity or of interfering substances, in addition to antibodies, that might inhibit edim virus replication is open. kraft (1958) found increased numbers of clostridium tertium in the intes tine of diarrheal animals, and pappenheimer and enders (1947) remarked on the persistent presence of ''coccoid bodies" in the intestines of diarrheal animals as seen by light microscopy. in both instances, these may be considered opportunistic or ganisms. their effect on the severity of the clinical disease is unknown, however. one report (labonnardiere and devaurieux, 1979) the use of standard methods to grow edim virus in tissue culture has resulted for the most part in failure. habermann (1959) (thiel et al., 1978) . further, babiuk et al. (1977) and al meida et al. (1978) state that calf rotavirus production is mark edly enhanced when passaged in the presence of trypsin. clark et al. (1979) produced high-titer calf rotavirus in a variety of continuous cell lines, also by utilizing trypsin, and matsuno et al. (1977) were successful in producing plaques with bovine rotavirus in monkey kidney cell monolayers when trypsin was incorporated in the overlay medium. estes and graham (1979) found that simian virus titers were enhanced by both trypsin and elastase in vero as well as ma 104 cells. ramia and sattar (1980) studied additional proteolytic enzymes utilizing plaque formation by simian virus sa-11 in ma 104 cells as the endpoint. elastase, a-chymotrypsin, subtilase, pronase, and pan creatin were as effective as trypsin, whereas pepsin, papain, and thermolysin were ineffective. another approach with human rotavirus has been to incoφorate the agent into cells by means of low-speed centrifugation of the two together (banatvala et al., 1975; bryden et al., 1977; schoub et al., 1979; wyatt et al., 1980) . it remains to be seen if similar treatments will lead to suc cessful cultivation of edim virus in tissue culture. chick embryos appeared unaffected when the chorioallantois was inoculated. the agent did survive up to 5 days, but an increase in infectious titer could not be demonstrated (kraft, 1958) . moon (1978) of each disease appears to be reflected not only in the particular group of cells but also in the numbers of host cells involved. lucid descriptions of the natural clinical disease are given by cheever and mueller(1947) , seamer (1967) , and mcclure et al. (1978) . the experimental disease does not differ signifi cantly (kraft, 1957 (kraft, , 1962b . overt illness is confined to pretypical appearance of infant mice infected with enterotropic mhv (pair of mice at left) or rotavirus (pair at right) compared with normal mice of the same age (center pair). note the evidence of inanition and dehydration in the mhv-infected animals, whereas those infected with rotavirus show mainly a somewhat prominent abdomen and only a slightly smaller size than the normal control mice. weanling mice. t h e first signs usually appear at 7 -8 days of age. in mild cases diarrhea is manifested by a minimal amount of pasty fecal material about the perineum. in severe cases the amount is copious, the entire infant becoming soiled. rectal impaction may occur at about 1 2 -1 6 days of a g e , and death can ensue if the impacted mass is not removed spontaneously or deliberately. death does not seem to be the result of the virus infection per se, but rather as a consequence of protracted obstipation. in pure e d i m virus infections, mice continue to nurse throughout their illness (in the absence of impaction). they may be slightly stunted (fig. 2 ), but those with mild cases soon attain the weight of their nondiarrheal peers. especially when diarrhea is mild, morbidity in the natural disease m a y be difficult to ascertain, but one could quite cor rectly assume that, if diarrheal signs are observed in a few litters, it is likely that all infants in a colony will sooner or later be affected. concerning mortality, cheever and mueller (1947) state that there was both 100% recovery and 100% lethality in their outbreaks. based on present k n o w l e d g e , the first is suggestive of e d i m virus infection and the latter of another disease, perhaps murine (mouse) hepatitis. at no time do a d u h mice exhibit signs of illness ascribable to e d i m virus infection. in the experimentally induced dis ease, the absence of external clinical signs cannot be relied upon for a negative diagnosis (kraft, 1957) . necropsy of each animal is essential. in this w a y , the appearance of the colonic contents can be observed, which in normal infant mice are burnt orange in color and semisolid but formed in consistency. t h e colon is not distended. in diarrheal m i c e , however, even in those with no external soiling, the colonic contents are fluid or m u c o i d , bright lemon yellow to amber, or gray-green, with no formed feces in evi dence. g a s is often seen in the colon and c e c u m , which are distended. t h e stomach, t o o , is distended with curdled milk (except in terminal cases with anal impaction). all other or gans appear normal. gross change is not seen in the intestines of adults exposed perorally. it is noteworthy that in germ-free infant m i c e , ex perimental e d i m virus infection appears in the gross as it does in conventional mice (kraft, 1966) . the histologic picture has been confirmed by others (adams and kraft, 1967; mcclure et al, 1978) . there is no inflam matory reaction in the intestines. inclusions may indeed be found in enterocytes near the tips of villi, especially in the jejunum, and infrequently in the sloughed cells seen in the lumen. epithelial cells near and at the tips of villi are fre quently vacuolated. acres and babiuk (1978) have pointed out that in all species studied, the rotaviruses cause diarrhea by attacking and de stroying the columnar epithelium of the small intestine. middleton (1978) considers that cell migration from the crypts is speeded in response to diarrhea and that the infected cells are relatively immature. enzyme levels, high thymidine kinase and low sucrase, are similar in such cells to those of normal crypt cells and thus support this view. the microscopic appearance belies the severity of the gross and clinical findings of edim. in this regard, d-xylose absorp tion has been studied in calves in which 60-90% reduction occurred during the acute illness with the calf rotavirus . aberrations in sodium transport have also been investigated in other animals and in humans (middleton, 1978) , but such studies have not been carried out in mice using the mouse agent. cheever and mueller (1947) and kraft (1957) demonstrated that the agent is transmissible perorally. kraft (1957) showed further that dissemination in a mouse colony is mediated prin cipally by the airborne route. kraft (1961) examined the neutralization test in edim and found that antibodies were not always formed following infec tion, and when they were present, they tended to be low in titer. sera from adult mice having had lifelong contact with the agent (in the form of consistently diarrheal litters) were more apt to neutralize the virus than those of mice exposed for the first time as adults. on the other hand, hyperimmunization of mice has resulted in the production of significant serum titers of both complement-fixing and neutralizing antibodies (blackwell etal, 1966) . antibody response measured by other serologic tests has not been investigated for edim, nor have immune substances in lacteal secretions been measured directly. suggestive of their presence, however, are data showing that infants of primiparae who themselves had been diarrheal in infancy resisted about 10 id50 more of edim virus than did offspring of previously nondiarrheal dams (kraft, 1961) . antirotaviral immunoglobu lins have been found in both colostrum and milk in man (yolken et al, 1978c; simhon and mata, 1978; thouless et al, 1977a) , bovines (acres and babiuk, 1978) , and lambs (snodgrass and wells, 1976) . indeed, local immunity afforded by lacteal immune substances is regarded as crucial for protection against any intestinal infection in the young (welliver and ogra, 1978; snodgrass and wells, 1976 as indicated, mice of all ages can be infected, but overt disease is restricted to animals up to about 12-13 days of age at the time of first exposure. there seems to be no predilection for a particular sex. the observation that first litters are more apt to show copious and protracted soiling than those of later parity has been reported by cheever and mueller (1947, 1948) and by runner and palm (1953 edim virus is probably worldwide in distribution. whether it occurs in wild mouse populations is unknown. its prevalence is difficult to estimate, since serologic tests have not been readily available, and it is not customary to sacrifice animals for the purpose of examining the appearance of their intestinal tract or for electron microscopic visualization of fecal contents. as has been shown experimentally, latent carriers can exist. the carrier rate in a diarrheal colony is not known, nor is the frequency of viral shedding under colony conditions. cheever and mueller (1948) examined seasonal variations in the weaning percentage in their mouse strains and found that there was a significant effect in only the cfw mice. they experienced the lowest weaning rate in the late fall and winter. runner and palm (1953) , studying c3h mice, indicated that there was a higher incidence of diarrhea in december/january (kraft, 1961; blackwell et al., 1966) , complement fixation (wilsnack et al., 1969; kapikian et al, 1976; thouless et al., 1977b) , direct immunofluorescent staining or precipitin (wilsnack et al., 1969; spence et al., 1975; foster α/., 1975; peterson α/., 1976) , immune electron microscopy (kapikian et al., 1974; bridger and woode, 1975) , immunoelectroosmophoresis (tufvesson and johnsson, 1976; middleton et al., 1976) , enzyme-linked im munosorbent assay (elisa) (scherrer and bernard, 1977; el lens etal., 1978; yolken etal., 1978a,b,c) , radioimmunoas say (acres and babiuk, 1978; kalica et al., 1977; middleton et al., 1977) , immunodiffusion (woode et al., 1976) , hemagglutination inhibition (fauvel et al., 1978) , enzymelinked fluorescence assay (elisa) (yolken and stopa, 1979) , an unlabeled soluble enzyme peroxidase-antiperoxidase method , plaque reduction test (estes and graham, 1980) , serologic trapping on antibody-coated electron microscope grids (nicolaieff et al., 1980) , a solid phase system (space, solid phase aggregation of coupled erythrocytes) for detection of rotaviruses in feces (bradbume et al., 1979) , and immune electron microscopy with serum in agar diffusion (lamontagne et al., 1980) . more recently, sheridan and aurelian (1981) have described an elisa test for edim virus which should prove beneficial for both practical (serologic) purposes and for investigations of the antigenic structure of the virion. in the absence of a reliable tissue culture system, edim virus isolation is generally impractical. bacteria-free filtrates of intestinal suspensions can, of course, be given to diarrheafree animals (gnotobiotes or axenics), but this is expensive and inefficient. on the other hand, such filtrates may be concen trated by ultracentrifugation and examined in the electron mi croscope for characteristic virus particles flewett, 1978) . the particles may also be identified by immune electron microscopy. a practical approach to a presumptive diagnosis would be to kill selected animals in order to examine the appearance of the colonic contents. the inclusion of sentinel dams with litters in a breeding colony should be considered. these might be sac rificed at intervals to determine the presence of edim in the colony. together with the clinical history of the mouse colony, this practice may provide a fairly reliable, although not pathognomonic, indication of the presence of edim. his topathologic examination could also be of value. based on experimental results, kraft et al. (1964) proposed the use of air-filter devices, essentially dust caps for each cage, for the practical control of airborne transmission of edim in a commercial mouse colony. it was subsequently shown (kraft, 1966 ) that 43% of first litters and 79% of all other litter parities were weaned from cages without filters, whereas in cages pro vided with filters, weaning percentages were 96 and 99%, respectively, during the same observation period. it should be pointed out that both edim and mouse hepatitis virus (livim) were present simultaneously in that colony. although the filter devices did not eliminate the disease(s), they probably reduced the pathogenic microbial load in the immediate environment of the susceptible animals, but the precise mechanism by which this control method succeeds to the extent that it does is obscure. since that time (1964), various devices based on a filter cage or filter-top design have been utilized. some of these are de scribed by simmons and brick (1970) . woods et al. (1974) evaluated them from the viewpoint of environmental factors within the cages. they found that dry bulb temperature dif ferentials, comparing environments inside and outside the cage, were not significantly different (about 2°c) between fil tered and unfiltered cages, but that dew point differentials were significantly greater in the filtered cages (about 5°c) than in unfiltered cages (about 3°c). however, they concluded that a suitable cage size for a particular species and number of ani mals could compensate for the higher wet bulb readings under filters to maintain acceptable conditions for the animals. cur rently, several types of filter covers or bonnets are available commercially. vaccination as a method of control has not been attempted. judging from the lack of reports to the contrary, caesarian derivation together with barrier maintenance apparently elimi nates and controls the infection. kunstyf (1962) attempted to control edim by decontamina tion of air by the use of triethylene glycol but was unable to do so. the use of antibiotics, too, is without value, although there seems to be amelioration of signs for a short period as secon dary organisms are temporarily reduced in number. as indicated by kraft (1966) , it may be relatively easy to establish a colony of mice free from edim virus infection. this may be accomplished by eliminating those breeding pairs whose first litter is diarrheal. filter devices are required for this, and they and the animals must be handled with the aid of a transfer or laminar flow hood using sterile techniques during observation and handling of the animals. the method is suita ble for small colonies, but it is impractical for commerce where caesarian derivation and barrier maintenance are the methods of choice. reovirus 3 was first isolated from the feces of an australian child manifesting a cough, fever, vomiting, hypertrophic tonsils, and bilateral bronchopneumonia. it was named hepatoencephalomyelitis virus by stanley et al. (1953) , who originally recovered the agent. sabin (1959) proposed the name reovirus for a group of agents associated with the respi ratory and enteric tracts of humans. they were found to be ether resistant, about 70 nm in size by membrane filtration but of unknown shape, and caused distinctive cytopathic effects in monkey kidney tissue cultures. one of the echo group of viruses, echo 10, now synonymous with reovirus, became a member of the new group on that basis. (echo is the acronym for enteric cytopathic /zuman 6>φhan, agents isolated in tissue culture from asymptomatic humans-so-called viruses in search of disease.) stanley (1961) then demonstrated that the hepatoencephalomyelitis virus was serologically identical to reovirus 3. additional strains have since been recovered from humans, other mammals, marsupials, birds, insects, and reptiles (for review, see stanley, 1974) , and from mouusks (meyers, 1979; meyers and hirai, 1980) . reoviruses have been divided into three serotypes on the basis of hemagglutination inhibition and neutralization tests (sabin, 1959; rosen, 1960) . reovirus 3 was established as an indigenous murine virus by hartley et al. (1961) and by cook (1963) . reviews concerning the biological and clinical aspects of disease caused by this agent have been prepared (stanley, 1974 (stanley, , 1977 . when gomatos et al. (1962) and discovered that reoviruses possess double-stranded rna, a unique characteristic among viruses, molecular biologists were inspired to study them in minute detail. currently, knowledge of reovirus replication on biochemical and biophysical planes is as extensive as that available for any other virus and is, for the most part, outside the scope of this chapter. interested readers are therefore referred to reviews by shatkin (1969) and joklik (1974) for extended literature coverage and detailed dis cussion and to andrewes et al. (1978) for a condensed over view. or reovirus, in the family reoviridae (melnick, 1979) . wild-type strains of reovirus 3 include: dearing, the pro totype strain (sabin, 1959) , isolated from a child with diarrhea; abney (rosen, 1960) , isolated from a child with a febrile upper respiratory infection; can 230, from a case of burkitt's lymphoma (bell et al., 1964) ; and several strains obtained from naturally infected cattle (rosen, 1960) . mutant, temperature-sensitive (ts) strains have been developed in the laboratory (fields and joklik, 1969) and have been used for studying the synthesis of viral rna and peptides (cross and fields, 1972; as well as for examining problems of pathogenesis. a neurotropic strain was also de-rived from the m o r e hepatotropic original isolate (stanley et al, 1954) . t h e reovirus 3 virion (fig. 3) has a m e a n diameter of about 6 0 -7 6 n m and is icosahedral in shape, with 5:3:2 symmetry. particles have a core, an inner layer or shell containing a n u m b e r of capsomers formalin at 56°c. ether treatment was ineffective. rozee and leers (1967) determined that although cholorform does not affect infectivity, it does destroy the hemagglutinin. wallis et al. (1964) found that mg^^ enhances the titer of reovirus at 50°c. the infective titer increased four to eight times by heat ing for 5-15 min in 2 μ mgcl2. other divalent cations and nacl were ineffective. hemagglutinin was not affected. it is thought that the high temperature and mg^+ caused activation of reovirus particles that were inactive in the original prepa rations. as with rotavirus. ward and ashley (1978) found that reovirus was sensitive to anionic detergents in wastewater sludge, i.e., these chemicals decreased the temperature needed to inactivate the virus. cationic detergents were more active than anionic, and nonionic detergents were inactive in decreas ing reovirus thermal stability. mutagens (nitrous acid, nitrosoguanidine, and proflavine) have been applied to reovirus 3 (fields and joklik, 1969) . the resulting mutants are of interest not only to the molecular biologist but to the clinical virologist as well, since some of them produce altered disease pictures (fields and raine, 1972) . for example, when inoculated into rats, wild-type virus produced a necrotizing encephalitis, whereas a mutant gave rise to a slowly progressive communicating hydrocephalus. the effects of enzymes are described below (see section iii,b,5). in phosphate-citrate buffers, stanley et al. (1953) ascertained that the virus is stable between ph 2.2 and 8.0. e. antigenic determinants. sabin (1959) demonstrated that the mammalian reoviruses known at that time could be divided into three serologic groups by neutralization tests. they could also be differentiated by hemagglutination inhibi tion (rosen, 1960 ), hull et al. (1956 having discovered that echo 10 virus possessed a hemagglutinin for human o eryth rocytes. later, reported that reovirus 3, but not reovirus 1 or 2, agglutinated ox erythro cytes. the reovirus 3 hemagglutinin was inhibited by nonspecific substances such as normal mouse, rabbit, or rat serum, and by vibrio cholerae filtrate. weiner et al. (1978) were able to show that the 57 rna segment, which is associated with type specificity, encodes the polypeptide that determines the hemagglutinating properties of the virion. complement-fixing antigens were prepared by stanley et al. (1953, 1954) and by j. c. parker et al. (1965, 1966) . these are group specific. leers et al. (1968) determined that reovirions display at least one type-specific and one to two group-specific antigens when studied by immunodiffusion. with the more sensitive im munoelectrophoresis technique, however, these authors en countered two type-specific and four group-specific precipitin lines. the agent is regarded as pantropic in mice. in neonates, stanley et al. (1953) (wolf et al., 1981) . papadimitriou (1965 papadimitriou ( , 1966 papadimitriou ( , 1968 also studied viral replica tion by electron microscopy in the mucosa of the common bile duct and in the liver. reovirus 3 has been reported to be oncolytic for a mouse ascites tumor by bennette (1960) , bennette et al. (1967,a,b) , and nelson and tamowski (1960) . most of the studies dealing with replication of the reoviruses have been accomplished in tissue culture, frequently in plaque assays in l-cell monolayers. other cells that have been suc cessfully employed are primary kidney monolayers of rhesus, patas, and capuchin monkeys, as well as those of pigs, cats, and dogs. continuous lines, such as fl human amnion, bs-c-1, and kb, have also been used (hsiung, 1958; cook, 1963; mcclain etal., 1967; rhim and melnick, 1961; harford et al., 1962) . harford et al. (1962) (1973a,b,c, 1974, 1979) . stanley et al. (1953, 1954) reported the development of pocks on the chorioallantois of 12-day-old chick embryos in oculated with infectious brain and liver. the embryos appeared unaffected, and with succeeding passages, the pocks could no longer be observed, although oral inoculation of suckling mice with chorioallantoic suspensions resulted in active disease. essentially the same results were found following amniotic inoculation. the experimental and natural diseases appear identical ex cept for variations in intensity of signs, perhaps due to dif ferences in infectious dose. up to 16 days after intraperitoneal inoculation, the mice appear emaciated and uncoordinated. the hair is oily and matted-the so-called oily hair effect (ohe)-an effect that can be demonstrated in contact animáis as well. however, in these mice it disappears as soon as the diseased animal is removed from the healthy ones. the effect was ultimately traced to a high proportion of fat in the intesti nal contents (steatorrhea): 12.9% in infected animals as com pared with 4.6% in normal mice. feces may contain as much as 29% fat in infected mice (stanley et al., 1953 (stanley et al., , 1954 . the thymus and other organs appeared normal. in the central nervous system, neuronal degeneration began about the ninth day and was most prominent in the brain stem and cerebral hemispheres. by the tenth day, perivascular cuf fing as well as neuronal satellitosis was evident. the meninges were infiltrated with round cells and netrophilic leukocytes. by the fourteenth day encephalitis was severe and widespread, with small hemorrhages occurring in necrotic regions. in suckling rats inoculated intracerebrally with reovirus types 1, 2, or 3, viral cytoplasmic inclusions and intranuclear bodies corresponding to cowdry type β inclusions have been ob served (margolis et al., 1975) . the latter were seen in cells that were free from intracytoplasmic inclusions; they were readily found in weanlings, were unassociated with inflam and their microvilli swollen. the common bile duct is dilated, and the ampullary region becomes obstructed with debris. it is this obstruction that leads to both hepatic and pancreatic dys function, for after studying the pancreatic lesions further, papadimitriou and walters (1967) concluded that, even though virions were seen in pancreatic acinar cells, the principal cause of acinar degeneration is ductal obstruction. based on the neutralization test, determined that the si genome segment is linked to type speci ficity, and finberg et al (1979) found that the same genome segment is also responsible for the production of cytolytic τ lymphocytes after reovirus infection. tytell et al (1967) , working with reovirus 3 rna, found that it was highly active in inducing interferon in rabbits and tissue culture, and lai and joklik (1973) showed that coreless virions as well as those lacking the outer capsid shell induce no interferon. the question of the role of interferon in protection of mice from either the acute or chronic infection remains an intriguing problem. in an effort to permit a precise definition of the host cellular immune response to viral antigens, weiner et al (1980b) and greene and weiner (1980) which also determined serotype-specific humoral (neutraliz ing) and cytolytic t-cell responses. it is clear that the agent can be transmitted by the oral route as well as by parenteral inoculation. mice respond to the natural infection with neutralizing, hemagglutination-inhibiting, and complement-fixing an tibodies. as indicated earlier (section iii,b,3,^), precipitating antibodies can also be demonstrated. there is no evi dence that any mouse strain is more or less susceptible to reovirus 3 infection than any other, provided the animals come from a colony that is free of the infection. the host range is broad. stanley (1974) cited at least 60 species that may be infected with reoviruses, and, as men tioned above, it is thought that the prevalence of antibodies in otherwise normal mammals is related to this fact and that mosquitoes or other insects may be operational in the spread of the infection. the acute disease affects mainly sucklings and weanlings, whereas the chronic disease is encountered in animals over 28 days of age. there is no indica tion that either sex is more or less susceptible than the other. in an epizootic described by cook (1963) , 130 of 800 first litters were affected. later-parity litters were involved hardly at all. in view of the absent or low complement-fixing antibody titers in the presence of significant hemagglutination-inhibiting titers that follow natural infection, prevalence estimation by immunologic means may be difficult to assess. the data cited by parker et al. (1966) , 82% of 34 colonies positive, and by descoteaux et al. (1977) , 100% of five colonies posifive, may be typical incidences for conventional mouse colonies. as already indicated, reovirus 3 infection is regarded as worldwide in distribution. this aspect of reovirus 3 infection has been covered above (section iii,b,l,2a,¿?). munoperoxidase method (enzyme-labeled antibody) has been employed for reovirus 1 (ubertini et al., 1971 ) and may find application for reovirus 3 diagnosis as well. although ohe may not be absolutely pathognomonic for reovirus 3 infection, it seems distinctive enough so that a provisional diagnosis may be made when it is seen. stronger evidence is afforded if the animals are also jaundiced and wasted. necropsy coupled with histopathologic examination is never a mistake and is to be encouraged. the placement of sentinel animals at strategic locations in an animal colony should be considered. such animals may be regarded as expendable for sacrific, necropsy, and virus isolation as well as for the acquisition of serum for antibody determinations. there is no evidence that epizootics occur preferentially at a particular time of the year. j. c. parker et al. (1965, 1966) discussed the serologic diagnosis of reovirus 3 infection, concluding that for these puφoses the hemagglutination inhibition test was the most reliable. in preparing type-specific antisera for standardizafion and controls, behbehani et al. (1966) found that the ubiquity of inhibitory substances (antibodies included) in most mamma lian species precluded accurate work. they therefore used domestic geese, in which virtually no hemagglutinafion inhibi tion or neutralizing antibodies could be detected prior to im munization. in any event, for routine surveillance, the hemagglutination inhibition test is currently utilized. comments similar to those expressed for edim virus recov ery and visualization apply. although it is possible to perform these procedures, they are inefficient for routine diagnostic puφoses. stanley (1977) has outlined methods for this pur pose. in brief, infectious material can be inoculated into tissue cultures (primary rhesus monkey or human kidney) or newbom mice (from reovirus 3 free colonies!), or the material may be subjected to immunofluorescent methods. an imcesarean derivation and barrier maintenance are believed to be suitable techniques for control and prevention of reovirus 3 infection. although no experimental evidence has been found, it is possible that the use of filter devices in conventional colonies might also be helpful in preventing the spread of infection. in the absence of information on the vertical trans mission of the agent, it is impossible to evaluate the influence of that route on successful control of the endemic disease. although therapy is impractical from the standpoint of con trolling epizootics of reovirus infection, it is nonetheless of considerable interest that willey and ushijima (1980) found that thymosin given intraperitoneally to 7-day-old mice that had been neonatally infected with reovirus 2 (2 ld50) signifi cantly increased their mean survival time, provided it was ad ministered at 2200 hr. when given at 0800 hr, significantly increased survival time was not observed, but when inoculated at 1200 hr, there was an apparent decrease in mean survival time. reviews on the subject include those by piazza (1969) , mcintosh (1977) , kapikian (1975) , andrewes et al (1978) , holmes (1979) , garwes (1979) , and robb and bond (1980) . based on their own electron microscopic studies and on those of david-ferreira and manaker (1965) , becker et al (1967) considered that mhv might be an 'tbv-like" (infecti ous bronchitis viruslike) agent. virions of similar moφhology seen in electron micrographs and also ether sensitive, as is ibv, were being isolated at that time from human cases of colds (almeida and tyrell, 1967) . the following year, the term coronavirus was proposed for the group (tyrell et al, 1968) , and in 1975 the coronaviridae became an official fam ily with a single genus, coronavirus (tyrell etal, 1975 additional agents may be added to this group, e.g., ''runde" virus (traavik et al, 1977) , a coronavirus causing car diomyopathy in rabbits (small et al, 1979) . as noted, many strains of mhv have been recovered from mice under various circumstances. in addition to jhm, these include: mhvl, from "white mice" (p or parkes strain), dur ing attempts to adapt human hepatitis virus to animals (the strain was originally isolated as a dual agent consisting of the virus and a protozoon parasite, eperythrozoon coccoides (gledhill and andrewes, 1951) ; mhv2, from mice used to propagate murine leukemia virus (nelson, 1952a,b) ; mhv3, also found during studies on adaptation of human hepatitis virus to mice (dick et al, 1956) ; mhv-b (ehf-120), from mice used for human epidemic hemorrhagic fever (hehf) adaptation attempts (buescher, 1952) ; an unnamed strain from mice undergoing murine leukemia chemotherapy trials (braunsteiner and friend, 1954) ; h747, following intracere bral inoculation of suckling mice with hehf materials (morris, 1959) ; mhv-a59, during transfer of moloney leukemia virus in mice (manaker et al, 1961) ; mhv-s, from cesareanderived mice that had been barrier-maintained before exposure to conventional mice ; mhv-c (mhv-balb/c), isolated during passage of spleens from leukemic mice (nelson, 1955) ; four addiiional strains from spleens of leukemic mice or from natural outbreaks (mhv-srl, -sr2, -sr3, -sr4) (nelson, 1963 (nelson, , 1965 ; lethal intestinal virus of infant mice (livim), from infant mice dying of a spontaneous infection (kraft, 1962a) , identified as an mhv strain by broderson et al (1976) and hierholzer et al (1979) ; and nuu, nua, nu66, from nude mice with hepatitis and wasting syndrome . other isolates have also been derived from nude mice (sebesteny and hill, 1974; ward et al, 1977) , and fox et al (1977) have described a strain that appeared during passage of an ascites myeloma cell line in balb/c mice. *hev may also denote the hepatoencephalomyelitis virus of stanley et al. (1954) , which is identical to reovirus 3. a s reviewed by mcintosh (1974) , coronaviruses are p l e o m o φ h i c , enveloped, and variable in size, measuring about 8 0 -1 5 0 n m in diameter. peplomers are 1 2 -2 4 n m in length (fig. 4 ) . using various techniques, a n u m b e r of workers have con firmed the diameter of m h v virions to fall within the range of the coronaviruses (gledhill et al., 1955; miyazaki et al., 1957; kraft, 1962a; starr etal., 1960) . svoboda etal. (1962) further described the virion as consisting of a nucleoid sepa rated from an outer m e m b r a n e by an electron-lucent space. david-ferreira and manaker (1965) , working with m h v -a 5 9 in tissue culture, found that the virions had a m e a n diameter of 75 nm with an electron-dense inner shell, 55 nm in diameter, separated from the outer double m e m b r a n e by an electronlucent space 8 nm wide. peplomers were 1 6 . 6 -2 3 . 4 n m long. not only was the virion significantly smaller than that of i b v , but the peplomers dif fered from those of both i b v and h c v 2 2 9 e , being conerather than club-shaped. mallucci (1965) in general, coronaviruses are inacti vated at 56°c in 10-15 min, at 37°c in several days, and at 4°c in several months (tyrell etal., 1968; kapikian, 1975) . wildtype mhv isolates also fall into this range of sensitivity gledhill and andrewes, 1951; gledhill et al., 1955; kraft, 1962a) . hirano et al. (1978) , however, indicated that mhv2 is not completely destroyed at 56°c for 30 min and is stable at 50t for 15 min in 1 μ mgclg or mgs04 but not in water. freezing and thawing or sonication at 20 kc for 3 min does not affect the virus titer. c. effect of chemicals. all coronaviruses are sensitive to ether when exposed overnight at 2°-4°c. chloroform also de stroys or reduces infectivity (mcintosh, 1974) . fifty percent glycerol inactivates mhvl after 6 weeks at 2°c (gledhill and andrewes (1951) . sodium deoxycholate reduces the titer of livim significantly (kraft, 1962a) , but calisher and rowe (1966) regard mhv virus as moderately resistant. according to hirano et al. (1978) , mhv2 is completely inactivated by ether, chloroform, sodium deoxycholate, and /3-propiolactone, but it is completely resistant to trypsin. mutagenesis has been reported by means of nmethyl-/v'-nitroguanidine or 5-fluorouridine and by 5-azacytidine or 5-fluorouracil (haspel et al., 1978) . the ph stability of all known murine hepatitis viruses has not been reported. for coronaviruses in general, acid sensitivity is regarded as variable (mcintosh, 1974) . hirano et al. (1978) found that mhv2 is stable be tween ph 3 and 9 at 37°c for 30 min. calisher and rowe (1966) although they have been sought, hemagglutinins have not been demonstrated for mhv strains (see, e.g., hirano et al., 1978; kraft, 1962a; bradbume, 1970; miyazaki etal., 1957) , but two human coronavirus strains do agglutinate human 0 erythrocytes (kaye and dowdle, 1969 cheever et al. (1949) found jhm virus to be unrelated to other neurotropic viruses, including gd vii, pseudorabies, lansing poliomyelitis, and mengo virus. kraft (1962b) found no relationship between livim, edim, and reovirus 3. with regard to other coronaviruses, the picture is somewhat different, for as a group, coronaviruses display complex serologic variability (bradburne, 1970; mcintosh et al., 1969) . mhv is serologically closely related to rcv and sdav in complement fixation tests and distantly related to rcv in cross-neutralization tests (parker et al., 1970; bhatt et al., 1972) . several strains of mhv are closely related to human coronaviruses oc 38 and oc 43 , and mhv3 is related to hcv-229e (bradbume, 1970) . antibody to mhv strains commonly found in human sera is probably present because of endemic human infection with related coronavimses (hartley et al., 1964) . electron micrographs and studies using fluorochrome stains indicate that coronaviruses develop exclusively in the cyto plasm of infected cells, that the virions collect in cytoplasmic vesicles of diverse size, that particles may also be seen in the matrix outside of the endoplasmic reticulum as well as in the golgi apparatus, and that they are not observed in the nucleus. in the main, replication involves budding into cytoplasmic cis temae, but tubular structures have also been seen within the cytoplasm during virus formation (ruebner et al., 1967; stan di α/., 1960; watanabe, 1969a,b) . wilsnack (1971) , confirming the work of boss and jones (1963) , elicited immunofluorescent antigen staining in sinusoidal lining cells in necrotic liver foci of weanling mice within 24 hr after intraperitoneal inoculation of the a59 strain. stainable antigen in intestinal impression smears of mice in fected by cage contact was also demonstrated. piazza et al. (1967) examined the fate of mhv3 after in travenous inoculation. the agent was not demonstrable be tween 40 min and 3.5 hr, when it appeared first in the spleen, then in the liver (4 hr) and blood (4.5 hr). at 5 hr it was recoverable from brain and kidney. high titers were then reached in all organs. barinsky and dementiev (1968) thereafter, the cells of the olfactory bulb and other brain re gions are affected. a number of cell systems have been successfully employed for in vitro growth of mouse hepatitis viruses: mhv-c in mouse embryo explants (mosley, 1961) ; mhvl in newborn mouse kidney explants (starr and pollard, 1959) ; mhv-s in mouse embryo explants (compels, 1953) and in liver (gallily et al, 1964) ; mhv-b in liver cell monolayers (paradisi and piccinino, 1968) ; mhv3 in liver explants (vainio, 1961) ; mhv-b in liver cells (miyazaki et al, 1957) ; mhv2 and mhv3 in dbt cells (hirano et al, 1978; takayama and kim, 1978) ; and various strains in nctc 1469 cells (david-ferreira and manaker, 1965; wilsnack etal, 1971; hartley and rowe, 1963) . mallucci (1965) , seamer (1965) , and lewis and starr (1972) described syncytium formation by mhv in mouse macrophage cultures, and a plaque assay for mhv2 in primary peritoneal macrophage cultures was described by shif and bang (1966) . laufs (1967) also described multinucleated giant cells with as many as 200 nuclei per cell in macrophage cultures infected with mhv3. using autoradiography, he ascertained that there was no dna synthesis in them and that they originated from cell fusion. macrophages derived from either liver or peritoneal wash ings are of enormous interest for the question of host cell-vims interactions. bang and warwick (1959, 1960) macrophages from the mice mirror these changes in resistance as the animals age. kantoch et al. (1963) determined that temporary susceptibility could be induced in resistant cells in culture if they were exposed to homogenates of susceptible cells, and gallily et al. (1964) showed that macrophages from genetically resistant mice treated with cortisone to enhance susceptibility behave in culture as if they were from susceptible animals. macrophages from mice susceptible to mhv2 virus can be converted to resistance by the intraperitoneal inoculation of concanavalin a in the donor mice. this enhanced resistance is also expressed in vivo (weiser and bang, 1977) . cheever etal. (1949) , nelson (1952b) , and kraft (1962a) in suckling mice, rapid wasting with or without neurologic signs may take place, accompanied in some cases by diarrhea, inanition, and dehydration (fig. 2) . mortality and morbidity are variable, ranging between al most 0 and 100% depending on factors like those affecting clinical signs. of importance to users and breeders of mice alike is the fact that a number of agents and procedures are known to modify the reactivity (and therefore the clinical signs) of mice to both spontaneous and experimental infection. examples of these, together with pertinent references, are presented in table i . leprévost et al. (1975a,b) have taken the view that there are three types of sensitivity to mhv3 infection in mice: resis tance, full susceptibility, and semisusceptibility. these are re flected in the susceptibility of their macrophages response of nuinu mice to sheep erythrocytes dba/2 mice, on the other hand, are regarded as fully suscep tible since deaths begin 4-6 days after infection, even when the mice are 90 days old, whereas the a/j strain is resistant, being susceptible to the acute disease only up to 3-4 weeks of age. although c3h mice are partially susceptible to the acute phase of the disease, they are fully susceptible to the chronic stage. in nude (nu/nu) mice, mhv takes on special significance. indeed, perhaps the best description of the clinical signs may be found in the original report describing this mutant mouse (flanagan, 1966) , published before runt disease, as the wast ing syndrome was called, came to be recognized as something other than a genetic effect. mhv-infected nude mice lose weight slowly or rapidly. they move stiffly with a stilted gait, and their faces assume a pointed, anxious appearance. partial paralysis may develop first in the hindlimbs and then in the forelimbs, resulting in almost total immobility. nu/+ heterozygotes are not affected in this way. flanagan (1966) found that at weaning, the nude animals were much smaller than controls (heterozygotes), that 55% died within 2 weeks of birth, and that 100% were dead by 25 weeks of age, whereas only 6% of controls died in the same period. it was not until sebesteny and hill (1974) uv-inactivated virus did not elicit the effect. the authors be lieve that fixed macrophages in athymic mice may be acti-vated, as was indicated by nickol and bonventre (1977) and by cheers and waller (1975) in certain bacterial infections in nude mice. t a m u r a et al (1979) (see, e . g . , r u e b n e r and bramhall, 1960; gledhill etal., 1952; nelson, 1953) . in liver regeneration m a y take place as early as 1 0 -1 4 days after infection (ruebner and bramhall, 1960) , ranging from complete healing to chronic scarring with intermediate gradations. kupffer cells were examined by r u e b n e r and miyai (1962) and r u e b n e r et al. (1967) . t h e y m a y undergo nuclear pyknosis and karyorrhexis 24 hr after intravenous inoculation of m h v 3 . in infection with neurotropic variants, such as j h m , the principal lesions appear in the central nervous system . meningitis is present but varies in degree and loca tion. in the brain, lesions m a y be found in all regions, but the hippocampus and its connections, the olfactory lobes, the periependymal tissues, and the brain stem seem to b e affected most often. necrotizing lesions predominate in the olfactory lobes and hippocampal regions, whereas demyelination is the major change in the brain stem. s o m e exudate m a y be found around blood vessels associated with lesions, and at about 5 days after infection, proliferating pericytes and scant lym phocytic cuffing can be seen. peripheral nerves show n o change. in sucklings, j h m virus produces extensive lesions in the brain and cord at 6 -8 days. meningitis is present, and large regions of necrosis with m a n y giant cells occur throughout the brain. in the cord, the lesions consist of spongy necrosis of the central gray matter. ganglion cells appear unaffected. powell and lampert (1975) resolved into small foci of fibrillary gliosis with an increased size and n u m b e r of astrocytic processes. t h e importance of this finding for investigations into the cause of multiple sclerosis and other demyelinating diseases in m a n is obvious and has been addressed b y , a m o n g others, lucas et al. (1977) and lampert (1978) . in sucklings infected with enterotropic strains (kraft, 1962a; broderson et al., 1976; ishida et al., 1978; ishida and fuji wara, 1979; r o w e et al., 1963; hierholzer et al., 1979) strains (dick et al., 1956; r u e b n e r and bramhall, 1960; hirano and ruebner, 1966) , and biggart and ruebner (1970) attribute the change to virus replication in lymphocytes. in other organs, minute superficial necrotic foci m a y be found in the stomach. n o changes are seen in the heart, lungs, pancreas, kidney, adrenals, voluntary m u s c l e , femoral o r ver tebral bone m a r r o w , or pituitary gland, although virus m a y be isolated from some of those organs. occasional giant cells are found in peripancreatic lymph nodes and in p e y e r ' s patches. flanagan (1966) islands of hepatocytes, markedly hypertrophied, remained. changes in other organs were not described. the liver lesions observed in other nude mice infected with mhv were similar. sebesteny and hill (1974) noted central nervous system lesions in their nude mice. ward et al. (1977) also encountered central nervous system lesions in addition to vascular changes, giant cell peritonitis, ascites, and giant cells in the villous epithelium of the intestines. since virus can be transmitted perorally and intranasally, it may be assumed that these are the principal routes of natural infection. transmission may be mediated by both the airborne and contact modes. feces, nasopharyngeal exudates, and perhaps urine would be sources of infection. evidence of vertical transmission is at hand, but reports are conflicting. piccinino et al. (1966) with or without booster, showed no antibody in the same colony. in a conventional colony of ddd mice, furthermore, seropositivity increased from 0 to 12% and in ddy mice from 6.3 to 45% as a result of the booster technique. in barrier-maintained animals, the booster did not elicit antibodies where there had been none before. presumably, these animals were free from mhv infection. from the foregoing, it is evident that the classic humoral immune response to mhv seems to be weak, and that a colony in contrast to the findings of bang and warwick (1960) , who concluded that one gene (or factor) was responsible for host susceptibility in mhv2 infection, stohlmann and freiinger (1978) showed that two genes are required for resistance of the central nervous system in sjl mice to fatal disease due to mhv-jhm. further, stohlmann et al. (1980) reported that there is an age-related change in resistant mice that protects them from acute central nervous system disease. they iden tified this change as due to a maturing adherent spleen or peritoneal exudate cell population. in extensive genetic studies, levy-leblond et al. (1979) found no correlation between the h-2 locus and either the acute or chronic disease in c57bl (susceptible) or a/j (resistant) animals. using congenie c3h lines, however, they were able to show that the h-2^ allele enables both heterozygous and homozygous animals to resist the development of the chronic disease. they believe, therefore, that mhv sensitivity appears to be influenced by at least two major genes: one for the acute disease, and the other, linked to the h-2 gene complex, for the chronic disease. mice also produce interferon as a result of mhv infection virelizier and gresser, 1978) , and taguchi et al (1979a) ascribe the greater susceptibility of suckling c3h/hejms mice to serum levels of interferon that are considerably lower than in weanling and aduh mice, as well as to greater macrophage sensitivity in the neonates. mhv-induced interferon may be regarded as the principal mechanism by which the virus modifies the immune respon siveness of mice to sheep red blood cells, for example. (see also (1949) infected cotton rats, rats, and hamsters intracerebrally, but rabbits and guinea pigs failed to respond. sebesteny and hill (1974) at tempted to infect infant wistar rats and hamsters using virus recovered from nude mice. all survived at least 21 days with out signs of illness. of considerable importance is a report by taguchi et al (1979c) concerning asymptomatic mhv-s infection in suckl ing rats following intranasal inoculation. the agent multiplied mainly in the nasal epithelium without any clinical signs. neu tralizing antibodies were produced, however, and could also be demonstrated in adult rats following infection. necrotic changes took place in the nasal mucosa, and cytoplasmic im munofluorescence was demonstrated in the nasal epithelium 2 days after intranasal inoculation of 10-day-old rats. age susceptibility has been amply addressed above in the consideration of virus growth in mice and tissue culture and in the discussion of the clinical picture. there appears to be no difference in susceptibility between the sexes (taguchi et al., 1976) . parker et al. (1966) found that the incidence of complement-fixing antibodies was greater in females than in males under colony conditions, as cribing this difference to continual exposure to virus-infected litters. there appears to be no evidence that first litters are significantly more susceptible than later ones. as noted above (section iv,β, 2 and c,l), the susceptibility of various mouse strains depends on the age of the host at the time of infection, the virus strain, and the host genotype. a completely resistant mouse strain has not been reported. prevalence of mhv infection in an animal colony is often difficult to ascertain. in addition to examples given earlier, descoteaux et al. (1977) found a low prevalence of hepatitis antibodies in three of five colonies studied in canada. complement-fixing antibodies ranged in titer from 8 to 32, 8 being considered positive. fewer than 20% of the animals, which were 6-9 months old at the time of testing, were posi tive. in distribution, mhv is regarded as occurring worldwide. these have been defined as occurring more than 2 months after intracerebral, intraperitoneal, intranasal, or intravenous inoculation (as reviewed in robb and bond, 1980) . the chronic clinical manifestations range from none to porenceph aly, paralysis, hepatitis, immunodeficiency manifestations, encephalitis, lymph node adenopathy, and vasculitis. virus may or may not be isolated. cells stainable by immunofluores cence may be found. demyelination with or without remyelination may occur. occasional scattered mononuclear cell infil trates may be evident. there is no evidence that seasonal changes influence the oc currence of epizootics of mhv infection. as indicated above, serologic testing is routinely carried out by means of the complement fixation test. cross-reactions with other coronaviruses must be taken into account when inteφreting the results, however. neutralization tests performed in tis sue culture systems are also possible. employing virus strain a59 as antigen in the elisa, peters et al. (1979) found a high prevalence of mhv antibodies in colonies with a low incidence of both complement-fixing and neutralizing antibodies. hierholzer and tannock (1977) have used the single radial hemolysis test for human coronavirus serodiagnosis. they then applied it to some mhv strains (hierholzer et al, 1979) . these techniques are helpful under certain circumstances, e.g., experimental investigations, but they are inefficient for field conditions. necropsy of dead or sick animals is always useful, although a definitive diagnosis in the absence of serologic evidence cannot be made. nevertheless, wherever possible, gross and microscopic pathologic examination should be undertaken. sentinel animals, especially gnotobiotes, could be ιηοοφοrated into a program of colony health surveillance. these ani mals can then be checked at predetermined intervals for clini cal, serologic, and histopathologic evidence of endemic dis ease in the colony. control of mhv is difficult in mouse colonies unless caesa rian derivation coupled with barrier maintenance is under taken. with the finding that vertical transmission is possible, however, barrier maintenance alone may not be adequate if, for example, such transmission is frequent. using small number of animals for experimental puφoses, kraft (1962a) tamura et al (1976) found that nu/nu mice could resist mhv infection when they previously received thymocytes from weanling nu/+ littermates. they were then not only able to produce antibody but survived a challenge infection as well. studies on rotaviral antibody in bovine serum and lacteal secretions, using radioimmunoassay epidemic diarrhea of infant mice. identification of the etiologic agent electron microscopic study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (edim virus) the morphology of three pre viously uncharacterized human respiratory viruses that grow in organ culture the effect of trypsin on the growth of rotavirus viruses of verte brates panel report of the colloquium on selected diarrheal diseases of the young rotavirus isolation and cultivation in the presence of trypsin a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. ii in vitro detection of human rotaviruses further observa tions on the virus of epizootic diarrhea of infant mice. an electron microscopic study genetics of resistance of animals to viruses: i. introduc tion and studies in mice macrophages and mouse hepatitis mouse macrophages as host cells for the mouse hepatitis virus and the genetic basis of their susceptibility virological and cytogenetic studies on the involvement of bone marrow of mice in some hepatoencephalotropic viral infections rotavirus serotypes by serum neutralisation moφhogenesis of avian infectious bronchitis virus and a related human virus (strain 229e) preparation of type-specific antisera to reoviruses isolation of a reovirus from a case of burkitt's lymphoma isolation of a non-pathogenic tumour-destroying virus from mouse ascites characteristics of a newborn runt disease induced by neonatal infection with an oncolytic strain of reovirus type 3 (reo3mh). ii. immunological aspects of the disease in mice characteristics of a newborn runt disease induced by neonatal infection with an oncolytic strain of reovirus type 3 (reo3mh). i. pathological investigations in rats and mice characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group lymphoid necrosis in the mouse spleen produced by mouse hepatitis virus (mhv3): an electronmicroscopic study lethal intestinal virus infection of mice (livim). an important new model for study of the response of the intestinal mucosa to injury virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis serological studies with an agent of epizootic diarrhea of infant mice pathogenic murine coronaviruses. ii. characterization of virus-specific proteins of murine coronaviruses jhmv and a59v specific monovalent cation effects on modification of reovirus infectivity by chymotrypsin digestion in vitro ex traordinary effects of specific monovalent cations on activation of reovirus transcriptase by chymotrypsin in vitro new intermediate subviral particles in the in vitro uncoating of reovirus virions by chymotrypsin reovirus transcriptase activation in vitro: involvement of an endogenous uncoating activity in the second stage of the process two modes of entry of reovirus particles into l cells hepatic localization of infectious agent in murine viral hepatitis antigenic relationships amongst coronaviruses a solid-phase system (space) for the detection and quantification of rotavirus in faeces small intestinal epithelial brush border enzymatic changes in suckling mice infected with reovirus type 3 reovirus type 3 infection in a suckling mouse: the effects on pancreatic structure and enzyme content viral hepatitis associated with trans plantable mouse leukemia. i. acute hepatic manifestations following treatment with urethane or methylformamide neonatal calf diarrhoea: identifica tion of reovirus-like (rotavirus) agent in faeces by immunofluorescence and immune electron microscopy lethal en teritis in infant mice caused by mouse hepatitis virus the laboratory diagnosis of epizoo tic diarrhoea of infant mice a rabbit rotavirus. vet. ree. 99 diagnosis of rotavirus infection by cell culture a hepatitis virus of mice two coronaviruses isolated from central nervous system tissue of two multi ple sclerosis patients mouse hepatitis, reo-3, and the theiler viruses activated macrophages in congenitally athymic "nude" mice and in lethally irradiated mice epidemic diarrheal disease of suckling mice epidemic diarrheal disease of suckling mice. i. manifestations, epidemiology, and attempts to trans mit the disease epidemic diarrheal disease of suckling mice. iii. the effect of strain, litter, and season upon the incidence of the disease a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. i. isolation and biological prop erties of the virus serological inter relationships of murine hepatitis viruses production of high-titer bovine rotavirus with trypsin murine virus contaminants of leukemia viruses and transplantable tumors reovirus type 3 infection in laboratory mice temperature-sensitive mutants of reovirus type 3: studies on the synthesis of viral rna effect of corticosteroids on mouse hepatitis virus infection an electron microscope study of the development of a mouse hepatitis virus in tissue culture cells importance of a new virus in acute sporadic enteritis in children comparison of the morphology of three coronaviruses classification of rotaviruses: report from the worid health organization/food and agriculture or ganization comparative virology program serologic study on the prevalence of murine viruses in five canadian mouse colonies a virus related to that causing hepatitis in mice (mhv) immunopathology of mouse hepatitis virus type 3. ii. effect of im munosuppression in resistant mice the appearance of a hepatotrophic virus in mice thymectomized at birth comparison of five diagnostic methods for the detection of rotavirus antigens in calf faeces morphological studies on simian virus sa 11 and the "related" 0 agent electron microscopic identification and subsequent isolation of a rotavirus from a dog with fatal neonatal diarrhea enhancement of rotavirus infectivity by trypsin and elastase identification of rotaviruses of different origins by the plaque-reduction test hemagglutination and hemagglutination-inhibition studies with a strain of nebraska calf diarrhea virus (bovine rotavirus) isolation and preliminary genetic and biochemical characterization of temperature-sensitive mutants of reovirus altered disease in rats due to mutants of reovirus type 3 temperature-sensitive mutants of reovirus type 3: studies on the synthesis of viral peptides generation of cytolytic τ lymphocytes after reovirus infection: role of the si gene nude", a new hairless gene with pleiotropic effects in the mouse electron microscopy in the diagnosis of infectious diarrhea the rotaviruses relation between viruses from acute gastroen teritis of children and newborn calves virus diarrhoea in foals and other animals. vet. ree. 96 fluorescent virus precipitin test adverse effects of mouse hepatitis virus on ascites myeloma passage in the balb/cj mouse problems in checking inapparent infections in laboratory mouse colonies: an attempt at serological checking by anamnestic re sponse implication pancréatique chez la souris infectée avec le virus de l'hépatite murine immunisation de la souris "nude" contre le virus de l'hépatite murine par transfert de lymphocytes sensibilisés carrier state of anti body and viruses in a mouse breeding colony persistently infected with sendai and mouse hepatitis viruses effect of cortisone on genetic resistance to mouse hepatitis virus in vivo and in vitro ontogeny of macrophage resistance to mouse hepatitis in vivo and in vitro structure and physicochemical properties of coronaviruses comparison of an enzyme-linked immunosorbent assay for quantitation of rotavirus an tibodies with complement fixation in an epidemiological survey enhancement of the pathogenicity of mouse hepatitis virus (mhvl) by prior infection of mice with certain leukaemia agents a hepatitis virus of mice production of hepatitis in mice by the combined action of two filterable agents mouse hepatitis virus and its pathogenic action reactive sites of reovirus type 3 and their interaction with receptor substances reovirus type 3: physical characteristics and interaction with l cells the propagation of s virus of mouse hepatitis in tissue culture comparison of methods for immunocytochemical detection of rotavirus infections delayed hypersensitivity in mice infected with reovirus. ii. induction of tolerance and suppressor τ cells to viral specific gene products spontaneous diseases and their control in laboratory animals electron microscopic examination of cells infected with reovirus tissue culture cytopathic and plaque assays for mouse hepatitis viruses recovery of reoviruses from wild and laboratory mice antibodies to mouse hepatitis viruses in human sera temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination mouse hepatitis virus-induced recurrent demyelination quantitation of antibody to non-hemagglutinating viruses by single radial hemolysis: serological test for human coronaviruses new strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice isolation of low-virulent mouse hepatitis virus from nude mice with wasting syndrome and hepatitis studies on the mechanism of destruc tion of lymphoid tissue in murine hepatitis virus (mhv3) infection. i. selective prevention of lymphoid necrosis by cortisone and puromycin physico-chemical properties of mouse hepatitis virus (mhv-2) grown on dbt cell culture viral gastroenteritis infantile enteritis viruses: morphogenesis and morphology is lactase the receptor and uncoating enzyme for infantile enteritis (rota) viruses? some distinctive biological characteristics of echo-10 virus new viral agents recovered from tissue cultures of monkey kidney cells. i. origin and properties of cytopathogenic agents svj, sv2, sv4, sv5, svg, svn, sv12, and sv15 pathology of diarrhea due to mouse hepatitis virus in the infant mouse isolation of mouse hepatitis virus from infant mice with fatal diarrhea some aspects on the transmission of hepatitis β antigen; model experiments by mosquitoes with murine hepatitis virus the molecular biology of reovirus studies on the effect of chymotrypsin on reovirions reproduction of reoviridae the effect of a murine hepatitis virus on the liver the fine structure of reovirus, a new member of the icosahedral series a microtiter solid phase radioimmunoassay for detection of the human reovirus-like agent in stools detection of differences among human and animal rotaviruses using analysis of viral rna the cellular nature of genetic susceptibility to a virus the coronaviruses reoviruslike agent in stools: association with infantile diarrhea and development of serologic tests antigentic relationships among five reovirus-like agents by complement fixation vertical transmission of mouse hepatitis virus infection in mice some characteristics of hemaggluti nation of certain strains of "ibv-like" virus studies on the etiology and transmission of epidemic diarrhea of infant mice observations on the control and natural history of epidemic diarrhea of infant mice (edim) responses of the mouse to the virus of epidemic diarrhea of infant mice. neutralizing antibodies and carrier state two viruses causing diarrhea in infant mice an apparently new lethal virus disease of infant mice epizootic diarrhea of infant mice and lethal intestinal virus infection of infant mice practical control of diarrheal disease in a commercial mouse colony reovirus infection in suckl ing mice: immunofluorescent and infectivity studies discussion of kraft, l. m. two viruses causing diarrhea in infant mice in vivo interference between heterologous rotaviruses the induction of interferon by temperature-sensitive mutants of reovirus, uv-irradiated reovirus, and subviral reovirus particles diagnosis of rotavirus, adenovirus, and heφes virus infections by immune electron microscopy using a serum-in-agar diffusion method autoimmune and virus-induced demyelinating dis eases mechanism of demyelination of jhm virus encephalomyelitis untersuchungen über die entstehung von riesenzellen in mäusemakrophagenkulturen nach infektion mit dem mäusehepatitisvirus (mhv-3) differential growth of mhv(pri) and mhv(c3h) in genetically resistant c3h rendered susceptible by eperythrozoon coccoides relationship of phagocytic activity to pathogenicitiy of mouse hepatitis virus as affected by triolein and cor tisone immunodiffusion and immunoelectrophoretic studies of reovirus antigens immunopathology of mouse hepatitis virus type 3 infection. iii. clinical and virologic observation of a persistent viral infection immunopathology of mouse hepatitis virus type 3 infection. i. role of humoral and cell-mediated immunity in resistance mechanisms neonatal susceptibility to mhv3 infection in mice. i. transfer of resistance genetic study of mouse sensitivity to mhv3 infection: influence of the h-2 complex polykaryocytosis and replication of mouse hepatitis virus in peritoneal macrophages in vivo and in vitro models of demyelinating diseases: tropism of the jhm strain of murine hepatitis virus for cells of glial origin an ultrastructural study of virions and cores of reovirus type 3 infectivity assay of reoviruses: comparison of immunofluorescent cell count and plaque methods the digestive system the nature of the polypeptide encoded by each of the 10 double-stranded segments of reovirus type 3 trans-stadial maintenance of reovirus type 3 in the mosquito culex pipiens fatigans weidmann and its implications coronaviruses: a comparative review coronaviruses as causes of diseases: clinical observa tions and diagnosis growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease antigenic relationships among the coronaviruses of man and between human and animal coronaviruses the polypeptides of human and mouse coronaviruses rotaviruses-a review moφhology and chemical composition of rotaviruses observations on the growth of mouse hepatitis virus (mhv-3) in mouse macrophages a. hepatitis virus complicating studies with mouse leukemia identity of cowdry type β inclusions and nuclear bodies: observations in reovirus encephalitis an antigenic subunit pre sent in rotavirus infected faeces plaque assay of neonatal calf diarrhoea and the neutralising antibody in human sera the classification and nomenclature of viruses calf diarrhea (scours) reproduced with a virus from a field outbreak taxonomy of viruses a reo-like virus isolated from juvenile american oys ters (crassostrea virginica) moφhology of a reo-like virus isolated from juvenile american oysters (crassostrea virginica) pathogenesis of rotaviral infection counter-immunoelectro-osmophoresis for the detection of infantile gastroenteritis virus (orbi-group) antigen and antibody solid phase radioimmunoassay for the detection of rotavirus experimental studies on hepatitis virus of mice in tissue culture mechanisms in the pathogenesis of diarrhea: a review a new member of hepatoencephalitis group of murine viruses multiplication and cytopathogenicity of mouse hepatitis virus in mouse cell cultures purification and characterization of epi zootic diarrhea of infant mice virus acute hepatitis associated with mouse leukemia. i. etiology and host range of the causal agent in mice acute hepatitis associated with mouse leukemia. i. pathological features and transmission of the disease acute hepatitis associated with mouse leukemia. iv. the relationship of eperythrozoon coccoides to the hepatitis virus of prince ton mice acute hepatitis associated with mouse leukemia. v. the neurotropic properties of the causal virus recovery and behavior of hepatitis virus from swiss mice injected with ascites tumor pathogenicity of murine hepatitis virus recovered from infant swiss mice an oncolytic virus recovered from swiss mice during passage of an ascites tumour anomalous high native resistance of athymic mice to bacterial pathogens detection of rotavirus by serological trapping on antibody-coated electron micro scope grids further light on mouse hepatitis defective virions of reovirus virusinduced diabetes mellitus: reovirus infection of pancreatic /3-cells in mice electron micrographic features of acute murine reovirus hepatitis ultrastructural features of chronic murine hepatitis after reovirus type 3 infection an electron microscopic study of murine reovirus-3 encephalitis the biliary tract in acute murine reovirus 3 infection studies on the exocrine pancreas. π. ultrastructural investigation of reovirus pancreatitis epidemic diarrheal disease of suckling mice. iv. cytoplasmic inclusion bodies in intestinal epithelium in relation to the disease an epidemic diarrheal dis ease of suckling mice. ii. inclusions in the intestinal epithelial cells propagation of mouse hepatitis virus (mhv-3) in monolayer cell cultures from liver of newborn mice virus studies with germfree mice. i. preparation of serologic diagnostic rea gents and survey of germfree and monocontaminated mice for indige nous murine viruses prevalence of viruses in mouse colonies rat coronavirus (rcv): a prevalent, naturally occurring pneumotropic virus of rats the isolation of reovirus type 3 from mosquitoes and a sentinel infant mouse enzyme-linked immunosorbent assay for detection of antibodies to murine hepatitis virus detection of neonatal calf diarrhea virus, infant reovirus-like diarrhea virus, and a coronavirus using the fluorescent virus precipitin test chronic obstructive jaundice induced by reovirus type 3 in weanling mice experimental viral hepatitis the fate of murine hepatitis virus (mhv-3) after intravenous injection into susceptible mice lack of transplacental transmissibility of mhv-3 virus oligodendrocytes and their myelin-plasma membrane connections in jhm mouse hepatitis virus encephalomyelitis proteolytic enzymes and rotavirus sa-11 plaque formation a genetic map of reovirus. ii. assignment of the double-stranded rnanegative mutant groups c, d, and ε to genome segments plaque formation by reoviruses cytochemical, fluorescent-antibody and electron microscopic studies on the growth of reovirus (echo 10) in tissue culture pathogenic murine coronaviruses. i. characterization of biological behavior in vitro and virus-specific in tracellular rna of strongly neurotropic jhmv and weakly neurotropic a59v viruses pathogenic murine coronaviruses. iii. biological and biochemical characterization of temperature-sensitive mutants of jhmv demonstration of reovirus-like particles in intestinal contents of piglets with diarrhoea comparison of the genomes of simian, bovine, and human rotaviruses by gel electrophoresis and detec tion of genomic variation among bovine isolates serologic grouping of reoviruses by hemagglutinationinhibition mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection of mice chloroform inactivation of reovirus hemagglutinins the growth of the virus of epidemic diarrhea of infant mice (edim) in organ cultures of intestinal epithelium pathology of experimental hepatitis in mice the kupffer cell reaction in murine and human viral hepatitis with particular reference to the origin of acidophilic bodies electron microscopy of the hepatocellular and kupffer-cell lesions of mouse hepatitis, with par ticular reference to the effect of cortisone factors associated with the incidence of infantile diarrhea in mice reoviruses. a new group of respiratory and enteric viruses formerly classified as echo type 10 is described application d'une technique immunoenzymologique (elisa) á la detection du rotavirus bovin et des anticorps diriges contre lui enhancement of antigen incorporation and infectivity of cell cultures by human rotavirus rotavirus in goats. vet. ree. 103 mouse macrophages as host cells for murine viruses some virus infections of mice hepatitis and brain lesions due to mouse hepatitis virus accompanied by wasting in nude mice a genetic map of reovirus. i. correlation of genome rnas between serotypes 1, 2, and 3 replication of reovirus mouse hepatitis virus (mhv) infection in thymectomized c3h mice rotavirus infection of neonatal mice: characterization of the humoral immune response plaque assay for mouse hepatitis virus (mhv-2) on primary macrophage cell cultures in vitro interaction of mouse hepatitis virus and macrophages from genetically resistant mice. i. adsorption of virus and growth curves arboviruses the mechanisms of reoviris uncoating and gene activation in vivo anti-rotavirus antibody in human colos trum the laboratory mouse. selection and management rabbit cardiomyopathy, associated with a virus antigenically related to human coronavirus strain 229e gel electrophoresis of rotavirus rna derived from six different animal species rotavirus infection in lambs: studies on passive protection a rotavirus in lambs with diarrhoea test for reovirus-like agent enhancement of reovirus infectivity by extracellular removal or alteration of the virus capsid by proteolyitic enzymes relationship of hepatoencephalomyelitis virus and reoviruses the reovirus murine models diagnosis of reovirus infection: comparative aspects murine infection with reovirus type 3 and the runting syndrome studies on the pathogenesis of a hitherto undescribed virus (hepatoencephalomyelitis) producing unusual symptoms in suckling mice studies on the hepato-encephalomyelitis virus (hev) murine infection with reovirus. ii. the chronic disease following reovirus type 3 infection propagation of mouse hepatitis virus (gledhill) in tissue culture electron and fluorescence microscopy of mouse hepatitis virus resistance to fatal central nervous system disease by mouse hepatitis virus, strain jhm. i. genetic analysis resistance to fatal central nervous system disease by mouse hepatitis virus, strain jhm. ii. adherent cell-mediated protection postsplenectomy viral hepatitis characterization of a coronavirus. i. structural pro teins: effects of preparative conditions on the migration of protein in polyacrylamide gels an electron microscopic study of viral hepatitis in mice difference in response to mouse hepatitis virus among susceptible mouse strains factors involved in the age-dependent resistance of mice infected with low virulence mouse hepatitis virus pathogenesis of mouse hepatitis infection. the role of nasal epithelial cells as a primary target of low virulence virus asymptomatic infection of mouse hepatitis virus in the rat in vitro growth characteristics and heterogeneity of mouse hepatitis virus type 3 igm and igg response to sheep red blood cells in mouse hepatitis virus-infected nude mice response of nude mice to a mouse hepatitis virus isolated from a wasting nude mouse persistent infection with mouse hepatitis virus of low virulence in nude mice the role of macrophages in the early resistance to mouse hepatitis virus infection in nude mice enhanced phagocytic activity of macrophages in mouse hepatitis virus-infected nude mice neonatal susceptibility to mhv3 infection in mice. ii. role of natural effector marrow cells in transfer of resistance techniques for rotaviral propagation rotavirus neutralization by human milk serological relation ships between rotaviruses from different species as studied by comple ment fixation and neutralization potentiating effect of k-virus on mouse hepatitis virus (mhv-s) in weanling mice runde" virus, a coronavirus-like agent associated with seabirds and ticks immunoelectroosmophoresis for de tection of reo-like virus: methodology and comparision with electron microscopy inducers of interferon and host resistance. iii. double-stranded rna from reovirus type 3 virions (reo 3-rna) isolation of rotavirus from deer use of horseradish peroxidase labelled antibody for light and electron microscopic localiza tion of reovirus antigen studies on murine hepatitis virus (mhv3) in vitro the moφhology of reovirus new inteφretation of reovirus struc ture effect of x radiation and cortisone on mouse hepatitis virus infection in germfree mice correlation of persistent mouse hepatitis virus (mhv-3) infection with its effect on mouse macrophage cultures role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. v. protective role in mouse hepatitis virus type 3 infection of susceptible and resistant strains of mice neuropathological effects of persistent infection of mice by mouse hepatitis virus the role of circulating interferon in the modifications of immune responsiveness by mouse hepatitis virus (mhv-3) reovirus activation by heating and inactivation by cooling in mgcl2 solutions effects of pancreatin on the growth of reovirus murine infection with reovirus: i. pathology of the acute phase naturally occurring mouse hepatitis virus infection in the nude mouse identification of detergents as compo nents of wastewater sludge that modify the thermal stability of reovirus and enteroviruses effects of wastewater sludge and its detergents on the stability of rotavirus comparative study on the mechanisms of rotavirus inactivation by sodium dodecyl sulfate and ethylenediaminetetracetate electron microscopic studies of experimental viral hepatitis in mice. ii. ultrastructural changes of hepatocytes associated with virus multiplication electron microscopic 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the genetic resistance of c3h mice to mouse hepatitis vims the circadian rhythm of thymosin therapy during acute reovirus type 3 infection of neonatal mice immunofluorescent detection of murine virus anti gens identification of an agent of epizootic diarrhea of infant mice by immunofluorescent and complement-fixation tests intestinal μ cells: a pathway for entry of reovirus into the host moφhological and an tigenic relationships between viruses (rotaviruses) from acute gastroen teritis of children, calves, piglets, mice, and foals intestinal damage in rotavirus infected calves assessed by d-xylose malabsoφtion heat and moisture transfer in fllter-top rodent cages reovirus-like agents (rotavimses) associated with diarrheal illness in animals and man human rotavirus type 2: cultivation in vitro enzyme-linked fluorescence assay: ultrasensitive solid-phase assay for detection of human rotavirus enzyme-linked immunosorbent assay for identification of rotaviruses from different animal species measurement of rotavirus antibody by an enzyme-linked immunosorbent assay blocking assay secretory antibody directed against rotavirus in human milk-measurement by means of enzyme-linked immunosorbent assay key: cord-268758-2o2dwulc authors: daniel, krupa; goli, kiran; sargent, anita title: repeat cesarean section in a covid-19 positive mother in the united states date: 2020-10-22 journal: sage open med case rep doi: 10.1177/2050313x20945536 sha: doc_id: 268758 cord_uid: 2o2dwulc in our report, we present a case of repeat cesarean section in a 29-year-old ecuadorian mother who contracted covid-19 and traveled to the united states during her last trimester of pregnancy. we assembled a multidisciplinary team to safely deliver the mother by cesarean section. she received supportive care for her covid-19 infection. infection prevention procedures were based on early available data, and the baby was delivered without complications. the novel coronavirus (2019-ncov) has become a rapidly disseminating global pathogen. as more people are infected, we encounter perinatal covid-19 infections. in a review of the literature we have found evidence that may support the possibility of vertical transmission of covid-19 infection. in peru, a 41-year old covid-19 positive patient in her third trimester of pregnancy required mechanical ventilation prior to her cesarean section (c/s). she delivered a neonate that was immediately separated from the mother, foregoing delayed cord clamping and skinto-skin contact. nasopharyngeal swab of the neonate, taken 16 h after delivery was positive for the novel coronavirus (2019-ncov) by reverse-transcriptase-polymerase-chainreaction (rrt-pcr). 1 also, a recent case report from switzerland describes a second trimester miscarriage in a covid-19 positive patient. the placental submembrane and cotyledon were found to be positive for severe acute respiratory syndrome coronavirus 2 (sars-cov-2). 2 we cannot positively determine if vertical transmission of covid-19 is possible. initial data from china suggested low levels of viremia in serum. 3 case reports of pregnant women diagnosed in the third trimester of pregnancy show negative rrt-pcr testing for 2019-ncov in the amniotic fluid, cord blood, and neonatal throat swabs. 4, 5 some case reports published show adverse neonatal outcomes such as fetal distress, pre-mature labor, and abnormal liver function, but again all neonatal testing for covid-19 remained negative. 4, 6 as there is limited data published on covid-19 in the perinatal period and the majority of this available data does not show evidence of vertical transmission of covid-19, it was commonly thought to be less likely. [4] [5] [6] as a consequence, optimal measures for early diagnosis, treatment, and preventing transmission in the perinatal period are still being explored. we describe a case of covid-19 in a pregnant female who underwent c/s without any complications or known procedural transmission of the illness. a 29-year-old spanish-speaking female, g2p1 with no significant past medical history, presented to our hospital at 35 weeks plus 5 days gestation inquiring about prenatal care. she had arrived at jfk airport in new york on 3 march 2020, from ecuador, where she was receiving medical care for her pregnancy. her initial evaluation revealed normal non-stress test and laboratory findings (table 1) . she returned to the prenatal clinic on 18 march and complained of severe headache, nasal congestion, swollen legs, and epigastric pain. she denied shortness of breath, cough, chest pain, or fever. she was referred to the emergency department (ed) where she was placed on enhanced droplet precautions. her temperature was 98.5 degrees fahrenheit, blood pressure (bp) was 100/60 mmhg, heart rate (hr) was 110 beats/min, and oxygen saturation was 89%-91% on room air. she was ill-appearing with facial flushing and cyanotic lips. episodes of fetal tachycardia were noted. her white blood cell (wbc) was normal. her aspartate aminotransferase (ast) was 42 u/l, and her urine protein to creatinine ratio was 0.31. a nasopharyngeal nucleic acid amplification test (naat) for common viral respiratory pathogens including influenza a/b and respiratory syncytial virus (rsv) was negative. rrt-pcr for novel coronavirus (2019-ncov) was sent to the lab. she received 1 l of lactated ringers, with improvement of maternal and fetal tachycardia. her oxygen saturation also improved to 96%. she was discharged and went home to quarantine for 14 days. her rrt-pcr for 2019-ncov returned positive on 20 march. she reported persistent nasal congestion at the time, and she denied any fever, cough, or shortness of breath. repeat covid-19 testing 1 week later remained positive. the patient remained clinically stable having received no treatment for covid-19. on 3 april, she underwent a repeat low transverse c/s at 39 weeks plus 5 days due to her prior history of c/s. she underwent spinal anesthesia without difficulty. the main operating room (or) was fitted with a high efficiency particulate air (hepa) scrubber to create a makeshift negative airflow room. or staff wore n-95 masks underneath their surgical masks. the patient delivered a vigorous male infant, weighing 6 lbs and 2 oz, with an apgar score of 9, and 9 at 1 and 10 min. no perinatal testing for covid-19 was performed on the baby as he was asymptomatic and we had limited access to covid-19 testing supplies at the time. to our knowledge, this is the first reported case of a c/s delivery with maternal covid-19 infection in the united states. a normal vaginal delivery was recently reported by iqbal et al. 7 the mother in our case recovered clinically without receiving any antiviral medication. chest x-ray and computerized tomography (ct) scan of the chest were not performed, even though radiation exposure for imaging below 610 mgy is not associated with any teratogenic effects. a ct scan is associated with a fetal radiation dose of 0.01-0.66 mgy of radiation. 8 an important aspect of her care was distinguishing covid-19 infection from hellp syndrome and preeclampsia, which can present with abdominal pain, elevation of liver enzymes, proteinuria, and fetal distress. the patient's only respiratory complaint was nasal congestion; she had no cough or fever. but noting her hypoxia and travel history, we had a high index of suspicion for covid-19. therefore, we initiated appropriate isolation and testing strategies, and formed a multidisciplinary team comprising obstetrics, anesthesia, and infectious disease to devise effective infection control measures perioperatively. throughout the perinatal course, the mother had a normal wbc count. mild leukocytosis is usually seen in the second trimester of pregnancy. 9 her differential showed a normal lymphocyte count and percentage. previously documented hematologic abnormalities during covid-19 infection include lymphopenia and neutrophilia. 10 it is unclear if her wbc parameters were normal variants or a reflection of the multiple complex factors affecting her hematopoiesis. the patient and delivered baby had a favorable outcome, as do most mothers and neonates with covid-19 infection, as noted in available literature. 11, 12 in the present covid-19 pandemic situation, it is vital to assess patients in all medical settings for the possibility of covid-19 infection. current guidance on infection control methods in the operative and perinatal setting are not based on high quality data. more studies are needed to better understand transmission risks and appropriate measures for transmission prevention in the perinatal and perioperative setting. severe covid-19 during pregnancy and possible vertical transmission second-trimester miscarriage in a pregnant woman with sars-cov-2 infection detection of sars-cov-2 in different types of clinical specimens clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records a case of 2019 novel coronavirus in a pregnant woman with preterm delivery. clin infect dis. epub ahead of print 28 clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia an uncomplicated delivery in a patient with covid-19 in the united states expert consensus for managing pregnant women and neonates born to mothers with suspected or confirmed novel coronavirus (covid-19) infection leukocytosis in labor: what are its implications clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan coronavirus in pregnancy and delivery: rapid review coronavirus disease 2019 among asymptomatic and symptomatic pregnant women: two weeks of confirmed presentations to an affiliated pair of new york city hospitals debra goldberg ma assisted with editing and formatting this article. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. this case study is exempt from lehigh valley health network internal review board review. the author(s) received no financial support for the research, authorship, and/or publication of this article. dr sargent obtained verbal and written consent from the patient over the telephone and via email using a spanish translator. krupa daniel https://orcid.org/0000-0001-7168-7362 key: cord-014540-27hnlu5v authors: sutthiruk, nantanit; botti, mari; considine, julie; driscoll, andrea; hutchinson, ana; malathum, kumthorn; cucunawangsih, cucunawangsih; wiwing, veronica; puspitasari, vivien; shanmugakani, rathina kumar; akeda, yukihiro; kodera, takuya; santanirand, pitak; tomono, kazunori; yamanaka, takayuki; moriuchi, hiroyuki; kitajima, hiroyuki; horikoshi, yuho; lavrinenko, alyona; azizov, ilya; tabriz, nurlan; kozhamuratov, margulan; serbo, yekatherine; yang, dahae; lee, woonhyoung; bae, il kwon; lee, jae hyun; lee, hyukmin; kim, jung ok; jeong, seok hoon; lee, kyungwon; peremalo, thiba; madhavan, priya; hamzah, sharina; than, leslie; wong, eng hwa; desa, mohd nasir mohd; ng, kee peng; geronimo, marionne; tayzon, maria fe; maño, maria jesusa; chow, angela; hon, pei-yun; win, mar-kyaw; ang, brenda; leo, yee-sin; chow, angela; hon, pei-yun; see, tina; ang, brenda; marin, rocio alvarez; de sousa, marta aires; kieffer, nicolas; nordmann, patrice; poirel, laurent; laochareonsuk, wison; petyu, sireekul; wanasitchaiwat, pawin; thana, sutasinee; bunyaphongphan, chollathip; boonsomsuk, woranan; maneepongpermpoon, pakpoom; jamulitrat, silom; sureshkumar, dorairajan; supraja, kalyanaraman; sharmila, soundararajan; cucunawangsih, cucunawangsih; setiawan, benny; lumbuun, nicolaski; nakayama, haruo; ota, toshiko; shirane, naoko; matuoka, chikako; kodama, kentaro; ohtsuka, masanobu; bacolcol, silverose ann andales; velmonte, melecia; alde, allan; chavez, keithleen; esteban, arlene joy; lee, aisa jensen; hsieh, tai-chin; shio-shinjean; huang, huey-jen; huang, shu-ju; huang, yu-huan; cheng, pei-chen; yu, su-fang; tsao, shih-ming; lee, yuan-ti; li, chien-feng; lu, min-chi; pruetpongpun, nattapol; khawcharoenporn, thana; damronglerd, pansachee; suwantarat, nuntra; apisarnthanarak, anucha; rutjanawech, sasinuch; cushinotto, lisa; mcbride, patty; williams, harding; liu, hans; hang, phan thi; anh, dinh pham phuong; le, ngai; khu, dung; nguyen, lam; castillo, roel beltran; sureshkumar, dorairajan; gopalakrishnan, ram; ramasubramanian, venkatasubramanian; sreevidya, subramanian; jayapradha, ranganathan; umetsu, atsushi; noda, tetsuhiro; hashimoto, kenyuu; hayashi, akihiro; kabashima, mikie; jadczak, ursula; elvelund, knut; johnsen, marit; borgen, bente; lingaas, egil; mao, chia-hua; chang, fu-chieh; liu, chang-pan; chao, ru-hui; chang, fu-chieh; liu, chang-pan; pawapotako, junpen; prasertpan, chadanan; malaihuan, wantanee; uirungroj, phisit; prasertpan, chadanan; saenjum, chalermpong; ouirungrog, teerapat; uirungroj, phisit; borrell, sue; bass, pauline; worth, leon; xian-li, zhao; xiao-long, li; xue-hua, yao; wei, ren; zeng, zhang xia; kong, man ying; lai, christopher koon chi; lee, suet yi; tsang, ngai chong; o’donoghue, m. m.; boost, m. v.; suen, l. k. p.; siu, g. k.; mui, k. w.; lai, c. k. c.; tsang, d. n. c.; sato, yuka; tateishi, mariko; mihashi, mutsuko; flor, jose paulo; bautista, marko; de roxas, v. jay; vergara, justine; añonuevo, nicolo andrei; kwek, marion; acuin, jose; sanchez, anna josea; bathan, avel; jantan, jamilah binte; guek, chua chor; kian, eu chiow; pirido, pampe anak; aron, nur fadilah binte mohd; estacio, leah may; palana, francis alvarez; gracia, michelle; shamsuddin, nur syafiqah binte; castro, kersten timbad; baloria, madonna; adam, faezah binte; wei, zhang; fong, poh bee; kalisvar, marimuthu; chow, angela; ang, brenda; chuang, i-ju; yi-chuncho; chiu, yu-fen; chen, lung-chih; lin, yi-chun; dong, shao-xing; lee, yi-chieh; kuan, hui-chen; lin, hsin-hua; chi, chia-chun; lu, chin-te; chang, fu-chieh; liu, chang-pan; ya-fen, tang; li-hsiang, su; jien-wei, liu; chao, hsuehlan; changchien, pinru; chen, weifang; lai, chunghsu; ara, lutfe; mowla, syed mohammad niaz; vashkar, shaikh mahmud kamal; chan, wai fong; chunyau, mabel yin; lingchong, karen kam; onli, tze; kaur, rajwinder; yan, ng po; chiu, gloria chor shan; cheung, christina w. y.; ching, patricia t. y.; ching, radley h. c.; lam, conita h. s.; kan, c. h.; lee, shirley s. y.; chen, c. p.; chan, regina f. y.; leung, annie f. y.; wong, isadora l. c.; lam, s. s.; chan, queenie w. l.; chan, cecilia; kaur, rajwinder; nematian, seyed sadeq seyed; palenik, charles john; askarian, mehrdad; nematian, seyed sadeq seyed; palenik, charles john; hatam, nahid; askarian, mehrdad; nakamura, itaru; fujita, hiroaki; tsukimori, ayaka; kobayashi, takehito; sato, akihiro; fukushima, shinji; matsumoto, tetsuya; flor, jose paulo; añonuevo, nicolo andrei; bautista, marko; vergara, justine; de roxas, v. james; kwek, marion; flor, jose paulo; bautista, marko; vergara, justine; de roxas, v james; andreiañonuevo, nicolo; kwek, marion; ho, yeng may; kum, jia qi; poh, bee fong; marimuthu, kalisvar; ang, brenda; liu, tzu-yin; chu, sin-man; chen, hui-zhu; chen, tun-chieh; chen, yichun; tsao, ya-ching; skuntaniyom, sumawadee; malathum, kumthorn; tipluy, pirawadee; paengta, sangwan; wongsaen, ratchanee; thanomphan, sutthiphun; tariyo, samettanet; thongchuea, buachan; khamfu, pattama; thanomphan, sutthiphan; songtaweesin, wipaporn natalie; anugulruengkit, suvaporn; samransamruajkit, rujipat; sosothikul, darintr; tansrijitdee, ornanong; nakphunsung, anry; srimuan, patchareeyawan; sophonphan, jirachaya; thanyaweeputhanakit; payuk, kunyanut; picheansathian, wilawan; viseskul, nongkran; denardo, elizabeth; leslie, rachel; cartner, todd; barbosa, luciana; werner, heinz-peter; brill, florian h. h.; kawagoe, julia yaeko; de nardo, elizabeth; wilson, sarah edmonds-; macinga, david; mays-suko, patricia; duley, collette; hang, phan thi; hang, tran thi thuy; hanh, tran thi my; gordon, christopher; sureshkumar, dorairajan; durairaj, roopa; rohit, anusha; saravanakumar, saujanya; hemalatha, jothymani; hirano, ryuichi; sakamoto, yuichi; yamamoto, shoji; tachibana, naoki; miura, miho; hieda, fumiyo; sakai, yoshiro; watanabe, hiroshi; velmonte, melecia; bacolcol, silverose ann; alde, allan; chavez, keitleen; esteban, arlene joy; lee, aisa jensen; chow, angela; lim, jia-wei; hon, pei-yun; hein, aung-aung; tin, grace; lim, vanessa; ang, brenda; chow, angela; hein, aung-aung; lim, jia-wei; hon, pei-yun; lim, vanessa; tin, grace; ang, brenda; chow, angela; tin, grace; hein, aung-aung; lim, vanessa; lim, jia-wei; hon, pei-yun; ang, brenda; chao, huwi-chun; yeh, chiu-yin; lo, mei-feng; chao, huwi-chun; piwpong, chonlada; rajborirug, songyos; preechawetchakul, ploypailin; pruekrattananapa, yada; sangsuwan, tharntip; jamulitrat, silom; wongsaen, ratchanee; paengta, sungwan; nilchon, napatnun; thanompan, sutthipun; tariyo, samattanet; le, ngai; khu, dung; kolesnichenko, svetlana; azizov, ilya; lavrinenko, alyona; tishkambayev, yerbol; lavrinenko, alyona; azizov, ilya; tishkambayev, yerbol; alibecov, asylkhan; kolesnichenko, svetlana; serbo, yekaterina; nam, youngwon; park, jae hyeon; hong, yun ji; kim, taek soo; park, jeong su; park, kyoung un; kim, eui-chong; aziegbemhin, samuel abumhere; enabulele, onaiwu; tung, yao-shen; chen, an-chi; huang, shen-min; yang, yui-yein; wu, li-hung; lin, chin-cheng; chang, fu-chieh; liu, chang-pan; lien, tzu hao; chang, jia hao; huang, yu shan; chen, yi shun; saenjum, chalermpong; sirilun, sasithorn; ouirungrog, teerapat; ouirungroj, phisit; trakulsomboon, suwanna; prasajak, patcharee; kwok, maryanne w. n.; ng, lady s. h.; wong, lindy m. t.; poon, lenina s. l.; lai, mary k. l.; cheng, holly h. s.; fong, s. k.; leung, cindy f. y.; hasegawa, jumpei; shirakawa, hiroki; wakai, sachiko; mieno, makiko; hatakeyama, shuji; tateishi, mariko; mihashi, mutsuko; sato, yuka; saenjum, chalermpong; deeudom, manu; tharavichitkul, prasit; ouirungrog, teerapat; ouirungroj, phisit; chinniah, terrence; tan, jackson; prabu, kavitha; alam, sartaj; wynn, aung kyaw; ahmad, rashidah; sidek, amalina; samsuddin, dg azizah; ajis, noraini; ahmad, aliyah; magon, susylawathi; chu, boon; kuang, jiqiu; gao, yan; wang, shoujun; hao, yunxiao; liu, rong; li, dongmei; wang, hui; yan, ng po; nishio, hisanori; mori, hitomi; morokuma, yoshiko; yamada, takaaki; kiyosuke, makiko; yasunaga, sachie; toyoda, kazuhiro; shimono, nobuyuki; babenko, dmitriy; turmuhambetova, anar; cheşcă, antonella; toleman, mark a.; babenko, dmitriy; turmuhambetova, anar; cheşcă, antonella; toleman, mark a.; babenko, dmitriy; turmuhambetova, anar; azizov, ilya; cheşcă, antonella; toleman, mark a.; akhmaltdinova, lyudmila l.; turmuhambetova, anar; cheşcă, antonella; babenko, dmitriy; magsakay, mark albert; macatibag, angelo; tayzon, maria fe; lerios, jeannica kriselle; azizov, ilya; lavrineko, alyona; babenko, dmitry; sheck, eugene; edelstein, mikhail; liu, tzu-yin; li, lih-yue; chan, chiung-wen; pan, hui-chuan; chen, tun-chieh; vanishakije, wipa; jaikampun, warisra; cheng, pei-chen; huang, huey-jen; huang, shu-ju; huang, yu-huan; li, su-yin; yu, su-fang; li, jian-feng; wu, yu-ping; lee, yuan-ti; lin, chiao-hui; chang, ping-chin; tariyo, samatanet; paengta, sangwan; wongsaen, ratchanee; thanompan, suttsiphan; skuntaniyom, sumawadee; malathum, kumthorn; sukkra, suchada; zaman, khalequ; zaman, sheikh farzana; zaman, farzana; aziz, asma; faisal, sayeed-bin; traskine, magali; ruiz-guiñazú, javier; borys, dorota; zaman, khalequ; zaman, sheikh farzana; zaman, farzana; aziz, asma; faisal, sayeed-bin; traskine, magali; ruiz-guiñazú, javier; borys, dorota; lam, wendy wai yee; chow, may; choy, lucy; kam, joseph; salleh, sharifah azura; yacob, razila; yusof, siti rokiah; jalil, nordiah awang; flor, jose paulo; añonuevo, nicolo andrei; bautista, marko; de roxas, v. jay; vergara, justine; millan, maria lourdes; kwek, marion; acuin, jose lito; lee, aisa jensen; velmonte, melecia a.; bacolcol, silverose ann a.; alde, allan; chavez, keitleen; esteban, arlene joy; ting, ching-i; dissayasriroj, sunisa; chinniah, terrence rohan; prabu, kavitha; ahmad, rashidah; magon, susylawathi; dinisuhaimi, jauharatud; mirasin, aizzuddin; morni, nurul; chu, boon; samsuddin, azizah; ahmad, aliyah; sidek, amalina; ajis, noraini; abubakar, amalina; shafiee, amanie; safar, julaini; yan, ng po; annie, leung; ling, fung yuk; edna, lau; kristine, luk; shinomiya, satoshi; yamamoto, kumiko; kjiwara, kayoko; yamaguchi, mitsuhiro; chow, angela; tin, grace; zhang, wei; hon, pei-yun; poh, bee-fong; marimuthu, kalisvar; ang, brenda; chan, ming-chin; wang, chih-chien; huang, shu-ju; huang, huey-jen; yu, su-fang; huang, huan-yu; cheng, pei-chen; li, jian-feng; lee, yuan-ti; lai, chiung-ling; lu, min-chi; kosol, sajeerat; sakolwirat, wantana; paepong, patchanee; jansanga, sawalee; jaisamoot, pattarin; thongnuanual, nuttha; srithong, chittima; somsakul, somporn; malathum, kumthorn; plongpunth, sutima; punpop, mukkapon; malathum, porntip; malathum, kumthorn; thanomphan, sutthiphan; wongsaen, ratchanee; peautiwat, kulada; boon kirdram, nattawipa; picheansathian, wilawan; klunklin, pimpaporn; samethadka, geetha; suzuki, naoko; asada, hitomi; katayama, masao; komano, atsushi; sato, akihiro; nakamura, itaru; watanabe, hidehiro; matsumoto, tetsuya; seo, hye kyung; hwang, joo-hee; shin, myoung jin; kim, su young; kim, eu suk; song, kyoung-ho; kim, hong bin; un, lai-si; vong, choi-ian; flor, jose paulo; añonuevo, nicolo andrei; bautista, marko; de roxas, v. james; vergara, justine; kwek, marion; koh, jocelyn; agustinus, sherly; hassan, rozita bte abu; thinn, yin phyu; ng, benjamin; tun, soe pyae; ha, su mon thi; xiaoting, xue; li, lin; chuang, leyland; niroshika, attanayaka mudiyanselage chulani; perera, kaluarachchige anoma kaluarachchi; fernando, dimingo kankanamalage diana grace; hemamala, bodhipakshage rohini; yeh, chiu-yin; chao, huwi-chun; yang, hui-chun; chiu, hsiang-ju; shih, ya-ling; chien, yu-shan; lin, wan-yi; pan, chia-yun; chang, ying-yun; yea, chiu-yuch; chu, ming-hsien; lee, li-chu; chiu, hsiang-ju; shih, ya-ling; yang, hui-chun; yu-hsiu, lin; siao-pei, guo; pak-on, leung; mei-fe, sie; jyh-jou, chen; yu-hsiu, lin; yong-yuan, chang; kuo, shu-yuan; lin, yu-hsiu; zhang, ji-sheng; leung, pak-on; sie, mei-fe; chen, jyh-jou; chen, yan-ru; lin, yu-hsiu; chen, ying-ling; taou, chi-fen; chen, hsiao-shan; tang, hung-jen; chen, shin yu; chen, yin yin; der wang, fu; shih, tzu-ping; chen, chin-yu; chen, su-jung; wu, mei-chi; yang, wan-ju; chou, mei-ling; yu, man-ling; li, li-chu; chu, cheng-wei; tsou, wen-hao; wu, wen-chih; cheng, wen-chi; sun, cho-ching; shih, tzu-ping; chen, chin-yu; lu, shu-hua; chen, su-jung; yang, hsin-ling; lu, cheng-yu; yu, man-ling; li, li-chu; chu, cheng-wei; tsou, wen-hao; wu, wen-chih; cheng, wen-chi; sun, cho-ching; hirunprapakorn, nitchawan; malathum, kumthorn; apivanich, sirilux; pornmee, ttipakorn; beowsomboon, chonnikarnt; rajborirug, songyos; pruekrattananapa, yada; sangsuwan, tharntip; jamulitrat, silom; kumkoom, itthaporn; kasatpibal, nongyao; chitreecheur, jittaporn; kasatpibal, nongyao; whitney, joanne d.; saokaew, surasak; kengkla, kirati; heitkemper, margaret m.; apisarnthanarak, anucha; muntajit, thanomvong; apivanich, siriluk; malathum, kumthorn; somsakul, somporn; phan, hang thi; dinh, anh pham phuong; nguyen, tuyet thi kim title: abstracts from the 8th international congress of the asia pacific society of infection control (apsic): bangkok, thailand. 12-15 february 2017 date: 2017-02-22 journal: antimicrob resist infect control doi: 10.1186/s13756-017-0176-1 sha: doc_id: 14540 cord_uid: 27hnlu5v nan antimicrobial resistance (amr) is a major problem worldwide. antimicrobial stewardship (ams) has the vital aim of ensuring optimal use of antimicrobial medicines to minimize amr. new strategies are needed to reduce amr. it is vital to ensure that key stakeholders are involved in the development of these strategies. this study aimed to examine key stakeholders' perspectives on the underlying causes of amr in thailand. semi-structured interviews were conducted with 15 key multidisciplinary clinicians, heads of department and healthcare administrators who were involved in ams programs in a 1,000-bed university hospital in bangkok thailand. qualitative content analysis was used to analyze the interview data. one of the key themes that emerged was lack of regulatory control resulting in widespread antibiotic availability and use both in health and agriculture in thailand, including over-the-counter availability of antibiotics. this ease of accessibility combined with poor consumer knowledge was considered one of the most important contributors to the increasing prevalence of amr. the development and implementation of more effective infection prevention and control strategies was identified as a priority, particularly in healthcare. three major concerns related to the perception that many patients admitted to hospital already have amr infections, that staff prescribing behaviors are not ideal, and that the lack of resources to develop and implement ams programs is an important barrier to decreasing the overuse of antibiotics. participants recognized that amr is a major problem in thailand and in healthcare. there was agreement that what is required is better regulatory control of antibiotics and medical engagement in ams. background antimicrobial resistance is a major problem of post-operative neurosurgical infection over the recent years. this study aimed to evaluate an increasing trend of infection in neurosurgical patients and susceptibility pattern of the causative pathogen. material and methods over a period of five years (june 2010 to june 2015), 216 cerebrospinal fluid and pus samples derived from clinically suspected cases of post-operative neurosurgical infection were processed using the standard procedures for culture and antibiotic susceptibility testing. of these 216 patients, causative pathogens were identified in 55 patients (25.5%). majority of infections were caused by multidrugresistant gram-negative bacilli (mdrgnb) including pseudomonas aeruginosa (n = 7, 12.7%), acinetobacter baumannii (n = 6, 10.9%), sphingomonas paucimobilis (n = 5, 9.0%), escherichia coli(n = 4, 7.3%), aeromonas salmonocida (n = 3, 5.4%), and klebsiella pneumoniae (n = 3, 5.4%). the common isolates showed a high susceptibility to tigecycline (86.7%) and amikacin (90%), ceftriaxone (76.9%) and ceftazidime (70%). all gram-positive bacteria isolates were susceptible to tigecycline and vancomycin. although multidrug-resistant (mdr) gram-negative bacilli (gnb) become a global concern, the disease burden of mdr gnb in children has not been reported yet in japan. we elucidate the impact of invasive mdr gnb infections among japanese children in the hospital setting. materials and methods a primary questionnaire was sent to 520 pediatric training facilities. a secondary questionnaire was sent to determine whether any cases showed a positive blood or cerebral spinal fluid culture for extended spectrum beta-lactamase (esbl) producing gnb, ampc β-lactamases producing gnb, or carbapenem-resistant enterobacteriacae (cre) between april 2012 and march 2015.the following data were collected; demographic data pertaining to both the care facilities and patients, clinical diagnosis, and outcomes. the response rate for the primary questionnaire was 57%. among facilities that responded, 66 facilities were eligible for the secondary questionnaire. the response rate for secondary questionnaire was 48%. a total of 92 pediatric patients had invasive mdr gnb infection. the median age was 2.5 years old (interquartile range 3 months-10 years old). the number of patients with bacteremia caused by esbl gnb, ampc gnb, and cre were 66 (72%), 22 (24%), and 4 (4%), respectively. the clinical diagnosis of esbl and ampc gnb showed 53 cases of sepsis. the clinical diagnosis of cre showed 2 cases of catheter related blood stream infection and 2 cases of sepsis. mortality at 30 days for esbl, ampc and cre bacteremia was 6%, 9% and 0%, respectively. the most common mdr gnb bacteremia was esbl gnb among children in this survey. tuberculosis is often complicated by the addition of non-specific inflammation, which changes not only the clinical manifestation of tuberculosis, but the course and outcome of disease. this study aimed to study the spectrum of non-specific microflora from patients with active tuberculosis and to evaluate its susceptibility to antimicrobial agents. the study was conducted in 2014-2015; 343 sputum samples were investigated. identification of microorganisms was carried out by maldi-tof methods using mass-spectrometer microflex (bruker daltonics, germany). the sensitivity of microorganisms to antibiotics was determined by disk-diffusion methods (clsi 2012). statistical processing and data analysis was performed using whonet 6.3 program. the growth of non-specific microflora in patients with tuberculosis was obtained in 21% of cases. the predominant etiologic role in non-specific inflammation belonged to s. aureus (22%), k. pneumoniae were isolated in 12.5%, a. baumanniiin 11.1%. remaining microorganisms were isolated in individual cases. 12.5% staphylococci were mrsa, to other anti-staphylococcal drugs s. aureus has kept a high sensitivity. isolated k. pneumoniae strains were resistant to cephalosporins of the 3rd generation in 12.5%, the resistance to meropenem marked in 11.1%. a. baumannii was characterized by a high resistance to antibiotics -85.7% esbl-producing strains, 37.5% and 62.5% strains were resistant to imipenem and meropenem, 80% a. baumannii strains were resistant to fluoroquinolones. conclusions according to antibioticogram data, the isolated microorganisms obtained from non-specific microflora of patients with tuberculosis, may adversely affect the course of the disease and impede the selection of antibacterial drugs and affect the outcome of the disease. rising number of candidiasis significantly contribute towards resistance of commonly used antifungal agents. lately, candida species such as c. rugosa and c. pararugosa have emerged as fungal pathogens that cause invasive infections. material and methods clinical isolates were from two tertiary referral hospitals in malaysia. test for antifungal susceptibility, biofilm, protease and phospholipase activities, all of which contribute to their virulence were performed. biofilms were quantified using crystal violet (cv) and tetrazolium (xtt) reduction assays in 96-well microtiter plates. time point reading was done on all strains incubated at 6, 12, 24, 48 and 72 hours. there were seven isolates of c. rugosa and one isolate of c. pararugosa in this study. e-test antifungal tests showed that all candida rugosa strains were susceptible-dose dependent towards voriconazole and resistant to fluconazole, amphotericin b and caspofungin based on clinical and laboratory standard institute guidelines. highest biomass was observed in one of the c. rugosa strains, followed by c. pararugosa at 12 hours of incubation. however, highest bioactivity was observed in the atcc at 24 hours, followed by c. pararugosa at 48 hours and the same c. rugosa train at 24 hours. virulence was also contributed by secretion of protease enzymes by all the clinical strains. none of the c. rugosa and c. pararugosa trains showed any phospholipase activity. conclusions c. rugosa and c. pararugosa clinical isolates should be considered pathogenic species because of their resistance against commonly used antifungal drugs and their contributing virulence factors. in response to an antimicrobial resistance "apocalypse" the medical city, a private tertiary hospital in the philippines, conducts microbiologic surveillance and has an existing "prior approval of restricted antibiotics" wherein release of identified broad spectrum antibiotics were done only upon approval of id consultants. in june 2015, a second tier of asp was introduced. the "drug duration, audit and feedback" (ddaf) program monitors and audits the duration of empiric antibiotics prescribed by clinicians. sticker reminders are being placed on the chart on day 3 as a reminder to de-escalate and on day 10 as a reminder to consider stopping the antibiotics. this study aimed to present a comparative study of the antimicrobial resistance of the top 3 bacteriologic agents from respiratory isolates in a tertiary care hospital in the philippines, from january to june 2015 versus january to june 2016, as a surrogate marker to the success of the second tier of asp recently introduced materials and methods most prevalent organisms from sputum, endotracheal aspirate and bronchoalveolar lavage were determined through laboratory surveillance comparing their resistance pattern from january to june 2015 versus january to june 2016. the top 3 respiratory pathogens in the icu were identified. some decrease in the resistance data of the most common isolate, klebsiella pneumoniae were as follows: 12% decrease in resistance to ceftriaxone, 9% to levofloxacin and 4% to piperacillin-tazobactam. similar decreases in resistance were seen with pseudomonas aeruginosa and other organisms. the study showed decrease in resistance of most common bacteriologic agents from respiratory isolates upon introduction of ddaf program. methicillin-resistant staphylococcus aureus (mrsa) is a growing clinical problem in subacute wards where patients have a longer length of stay than in acute wards. universal antiseptic baths could be added to the armamentarium for mrsa prevention and control. our study aimed to assess for the baseline antiseptic susceptibilities in mrsa, prior to the institution of universal antiseptic baths in subacute wards. we conducted a cross-sectional study, testing for susceptibilities to chlorhexidine and octenidine in mrsa isolates obtained from inpatients of two subacute wards from may-july 2013. minimum inhibitory concentrations (mics) of chlorhexidine and octenidine were determined by the modified clinical and laboratory standards institute (clsi) methods, with microbroth dilution susceptibility testing for a range of 0.125-8 ug/ml. results a total of 43 mrsa isolates were tested: 10 in may, 14 in june, and 19 in july. for chlorhexidine, all except for one had mic = 2. the remaining with mic = 4 had occurred in july 2013. in comparison, the majority (90.7%) of the isolates had mic = 0.5 when tested for octenidine susceptibility, with the remaining having an mic = 1. a higher proportion of mrsa isolates with the higher mic level (mic = 1) to octenidine was observed in june 2013 than in the other months, although statistical significance was not achieved due to the small sample size (or 7.64, 95%ci 0.72-81.54, p = 0.092). conclusions mrsa isolated from patients from subacute wards were highly susceptible to chlorhexidine and octenidine. universal antiseptic baths could be implemented in subacute wards, with follow-up studies conducted to monitor for any development of antiseptic resistance. antimicrobial activity of octenidine against multidrug-resistant gram-negative pathogens rocio alvarez marin 1,2 , marta aires de sousa 3 , nicolas kieffer 1 , patrice nordmann 1,4 , laurent poirel 1 multidrug-resistant gram-negative (mrgn) pathogens pose a major and growing threat for health care systems, as therapy of infections is often limited due to the lack of available systemic antibiotics. well tolerated antiseptic molecules may be a very useful implementation in infection control,not only to reduce the dissemination of methicillin-resistant staphylococcus aureus (mrsa), but also mrgn. as decolonization strategies with regard to mrsa are already implemented in high risk areas (i.e. icus), this study aimed to investigate, if the same protocol might be concomitantly efficient against mrgn. a series of 5 different species (escherichia coli, klebsiella pneumoniae, enterobacter cloacae, acinetobacter baumannii, pseudomonas aeruginosa) was studied to prove efficacy under clinically relevant conditions according to an official test norm (en13727). we used 5 clonally-unrelated isolates per species, including a single wild-type strain, and four mrgn isolates, corresponding either to the 3 mgrn or 4 mgrn definition of multidrug resistance. octenidine (oct, schuelke&mayr gmbh, germany) susceptibility was evaluated with and without organic load. results a contact time of 30 seconds or 1 minute was fully effective for all isolates by using different oct concentrations (0.01% and 0.05%), with a bacterial reduction factor of >5 log systematically observed. growth kinetics were determined with two different wild-type strains (a. baumannii and k. pneumoniae), proving a time-dependent efficacy of oct, mirroring what has been previously observed for mrsa. these results highlight that oct, besides being a very effective agent against mrsa, may also be extremely useful to eradicate emerging highly resistant gram-negative pathogens associated with nosocomial infections. acinetobacter baumannii is an important opportunistic nosocomial pathogen causing a variety of infections. the intrinsic virulence of drug-resistant a. baumannii has remained controversial. we compared mortality rates and sepsis score of patients with a. baumannii bacteremia caused by different level of drug resistance. materials and methods a retrospective study was conducted in adult patients (age > 15 years) admitted to songklanagarind hospital during 2009 and 2015 and blood culture positive for a. baumannii after 3 days of admission. antimicrobial resistance was categorized into four levels comprising of non-multidrug resistance (non-mdr), multidrug-resistant (mdr), extensively drug-resistant (xdr), and possible pandrug-resistant (possible-pdr). severity of underlying disease of the patients immediately before onset of bacteremia was determined by sequential organ failure (sofa) score and american association of anesthesia (asa) score. virulence of a. baumannii was assessed in terms of sepsis score and in hospital mortality rate. the study identified 38, 110, 168, and 14 cases of bacteremia caused by non-mdr, mdr, xdr, and possible pdr, respectively. after adjusting for confounding effect by using cox proportional hazard model, mortality rates attributable to a. baumannii was significantly associated to levels of drug resistance. using non-mdr as a reference, the incidence rate ratios and corresponding 95% confidence intervals (95% c.i) of mdr, xdr, and possible pdr were 2.3 (95% c.i = 0.9-4.9), 3.1(95% c.i = 1.4-7.0), and 1.9(95% c.i = 0.6-5.5) respectively. the virulence of a. baumannii did not loss with drug resistance. most of the antibiotic stewardship programs (asp) in the developing world measure antibiotic consumption, adherence to antibiotic guidelines and antibiotic resistance. however, antibiotic associated diarrhea (aad) is a common medical problem of antibiotic treatment and important quality monitor of asp was not monitored commonly in india. this study aimed to measure the prevalence of aad in hospitalized patients receiving antibiotics. materials and methods a point prevalence study was conducted in a 300-bed tertiary care cardiac hospital in chennai, south india. all hospitalized patients in cardiology wards and intensive care units (icus) receiving at least one dose of either oral or intravenous antibiotic were audited by physician assistant for the symptoms of diarrhea and cross checked by interviewing patients. during the study period, 107 eligible patients had available records for analysis. there were 58 patients (54.20%) receiving antibiotics. of these, there were 34 receiving single antibiotic, 17 receiving two antibiotic combinations, 4 receiving three antibiotic combinations and 3 were taking four antibiotic combinations. the details of diarrhea was missing in 3 patients' medical records. only 2 patients (3.44%) developed diarrhea and they received two antibiotics combination. conclusions although more than half of the patients received antibiotics, aad was not common in our hospital. however, regular monitoring of aad along with other parameters were required for better implementation of asp in the hospital. antimicrobial resistance and infection control 2017, 6(suppl 2):as2 background infections caused by antibiotic-resistant bacteria have led to increase burden on the healthcare system. effective antimicrobial stewardship control program (ascp) requires the clinician acceptance of program recommendation. we evaluate the antibiotic consumption and antibiotic susceptibility after ascp implementation in 2013 in a teaching hospital in tangerang, indonesia. our ascp restrict the prescription of carbapenems, fourth generation cephalosporins, and tigecycline. antibioticsusceptibility and consumption of restricted antibiotics were extracted from database. antibiotics use was measured by the number of ddds per 100 bed-days results the proportion of susceptible bacteria against; cefpirome increased from 57% to 73%, cefepime 63% to 64%, imipenem 78% to 83%, and tigecycline 74% to 75% during 2013 to 2015. the proportion of meropenem susceptibility remained the same at 70% in 2013 and 2015. the defined daily doses (ddds) per 100 bed-days was significantly reduced in all restricted antibiotics from 2013 to 2015 except tigecycline. the consumption of 1.0 g cefepime was 2190, 1035 and 107, 1.0 g cefpirome was 313, 473 and 140, 1.0 g meropenem was 16047, 11271 and 5281, 0.05 g teicoplanin was 40.4, 58.8 and 0, 0.5 g vancomycin was 716, 731 and 212.5, and 0.6 g linezolid was 129.6, 72 and 0 in 2013, 2014 and 2015, respectively conclusions our ascp was effective in terms of lowering broad spectrum antibiotic consumption and improving the antibiotic susceptibility. utilization of diagnosis-procedure combination data for advancing the antimicrobial stewardship program haruo nakayama, toshiko ota, naoko shirane, chikako matuoka, kentaro kodama, masanobu ohtsuka toho university ohashi medical center, tokyo, japan background infection with antibiotic-resistant bacteria results in increased morbidity, mortality and economic burden. antimicrobial stewardship program (asp) has been widely implemented to guide appropriate antibiotic use, in order to minimize antibiotic resistance. however, establishment of asp is not always possible due to lack of interest. we examined the application of diagnosis-procedure combination (dpc) data as an incentive for achieving the target of asp. the toho university ohashi medical center inpatient initiated asp focusing on reduction of inappropriate perioperative antibiotics and anti-mrsa drugs. the dpc data was extracted for antibiotic consumption and duration in each patient from april 2013 to march 2016. the consumption of the first-generation and second-generation cephalosporins as perioperative antimicrobial agents was 62% during observation period. this proportion was below the initial benchmark. on the other hand, the consumption of anti-mrsa agents was 2.26% higher than the benchmark. more than half of patients undergoing surgery received perioperative antibiotics only one day. the majority was cardiovascular surgery patients used intraoperatively. our study shows that utilization of the dpc database for advancing the asp is possible. it is convenient process to measure outcome of asp at the group or organizational level. background extensively drug-resistant acinetobacter calcoaceticus-baumannii complex (xdr-abc) pneumonia is an important cause of healthcareassociated pneumonia. although tigecycline was not approved for treatment of healthcare-associated pneumonia, it has been used offlabel for xdr-abc pneumonia. we evaluated whether the clinical efficacy of tigecycline combined with aerosolized colistin methanesulfonate (cms) is superior to aerosolized cms alone. this is a retrospective case-control study, conducted in wan-fang medical center, taipei medical university, taipei, taiwan from november 2014 to february 2015. the definition of xdr-abc pneumonia was pneumonia caused by abc with susceptibility only to colistin and tigecycline. cases were patients who received aerosolized cms in combination with intravenous tigecycline for at least 5 days to treat xdr-abc pneumonia. controls were those who received inhaled cms alone and were selected based on the following matching criteria to cases; age (±5 years), acute physiology and chronic health evaluation (apache) ii score (±4 points). there were 53 patients in each group. the mean age of patients was 80 years old. the proportion of patients underwent mechanical ventilation were 35.8% and 28.3% in cases and controls, respectively (p = 0.45). the median apache ii score was 17.5 (15.3-19.6) in cases and 17.3 (15.3-19.2) in control group (p = 0.9). the mean length of hospital stay was 32 days (p = 0.524), 30-days mortality rate was 34% and 22.6%(p = 0.02), and overall mortality was 46.2% and 33.3% (p = 0.19)in cases and controls, respectively. conclusions despite the active in-vitro susceptibility of tigecycline against xdr-abc, combination therapy with tigecycline and aerosolized cms for xdr-abc pneumonia showed no additional clinical benefit. the effectiveness of reducing the amount of antibiotic resistant strains by promoting antibiotic stewardship program of a medical center huey-jen huang 1 , shu-ju huang 1 ,yu-huan huang 1 ,pei-chen cheng 1 , su-fang yu 1 , shih-ming tsao 1,2 , yuan-ti lee 1,2 ,chien-feng li 1,2 , min-chi lu 3 in 2013, the amount of antibiotics in this medical center accounted for 8.4% of its total drug amount. the calculated did dosage of inpatient antibiotics was 917 and the rate of antibiotic-resistant strains has been on the rise. to cope with the development of drug resistance, from 2014 to 2015, the antibiotic stewardship program (asp) was executed to promote proper use and therefore to decrease the volume of antibiotics, and to reinforce mdro isolation. materials and methods a multi-discipline team for antibiotic stewardship was reformed. id doctors and infection control practitioners developed regulations for antibiotic use, conducted training programs, reviewed antibiotic uses and gave feedback. the compliance and accuracy of hand hygiene, and isolation precaution and protection were strengthened by nursing personnel. the pharmacist team division provided daily antibiotic assessment and statistics. microbiology laboratory was responsible for drug-resistant data. the expenditure of consumed antibiotic, as a ratio of total drug, declined from 8.4% in 2013 to 5.6% in 2015. from 2013 to 2015, the did from 917 to 824, carbapenems from 53.4 to 47.4, quinolones from 95.6 to 81.9, and glycopeptides from 28.4 to 21.9. furthermore, crpa from 16.7% to 5.7%, crab from 55.7% to 26.2%, mrsa from 52.2% to 43.5%, vre from 60% to 45%. however, a little elevation of crkp from 9.62% to 11.3% was observed. employing asp, we have enhanced the cooperation among antibiotic team members. as a result, the correct use of antibiotics was improved, the amount of antibiotics was less consumed, and the ratios of most mdro declined. antimicrobial resistance and infection control 2017, 6(suppl 2):as8 background increased antibiotic resistance among escherichia coli has led to inappropriate empirical antibiotic use (iau) for associated infections. limited data exists for iau among cases with acute uncomplicated cystitis (auc). we conducted a prospective observational study at a general practice (gp) clinic from december 2014 to february 2016. eligible participants included women aged 15-60 years with auc. all participants' urine cultures were sent before empirical antibiotics were prescribed at the gp physicians' discretion. the rate of iau was subsequently identified by the investigators. strategies to minimize iau were then determined based on the relevant data of auc treatment in this study. eighty participants were enrolled. e. coli was the most common pathogen isolated (78.3%) with resistance rates to trimethoprimsulfamethoxazole, fluoroquinolone, ceftriaxone, amoxicillin-clavulanate and ertapenemof61.7%, 42.6%, 21.3%, 2.1% and 0%, respectively. extended-beta-lactamase production was confirmed in 12.5% of e. coli isolates. the rate of iau was 91.3%. ciprofloxacin use was the only independent risk factor for iau (adjusted odds ratio, 6.471; 1.089-38.461; p =0.04). based on the study results, including the in-vitro susceptibility data and the risk factors for acquisition of antibiotic-resistant e.coli, a specific algorithm for auc treatment was created. if this algorithm was used along with education about iau and antibiotic stewardship program focusing on ciprofloxacin use, the rate of iau would have decreased to 3.8%. our findings suggest the high rate of iau in auc treatment in a gp setting and underlie the need for multifaceted interventions to reduce iau. background infection control/prevention (ic) programs seek to prevent infections via surveillance and outbreak investigation, hand hygiene, isolation precautions, environmental disinfection, evaluating ic products, and policy development. antimicrobial stewardship programs (asp) try to optimize antibiotic use for better outcomes and less toxicity. both reduce antimicrobial resistance in pathogens, educate healthcare staff, patients, and families, and optimize resource use. our study objectives were to define areas of synergy between ic and asp efforts with specific examples and to determine how best to promote these. bryn mawr hospital: 250-bed community-teaching hospital with 3 ic practitioners; asp team has one infectious diseases (id) physician and an id-trained pharm.d. it is part of a 5 hospital system with microbiology laboratory. asp has been in placed for 5-1/2 years. synergy between asp and ic: (1) collaborative identification of outbreaks (e.g., regional babesiosis in 2015, ongoing c. difficile cases), (2) monitoring antimicrobial resistance via complementary computer surveillance systems, (3) coordinating presentation of microbiology antibiograms, (4) collaboration on healthcare staff educational programs, and (5) joint presentations to system wide committees and accrediting organizations. from 2011 to 2015 dosage days for selected antibiotics decreased 58-90% per 1000 patient-days and total antibiotic cost decreased $577,680 (54%) per year. conclusions ic and asp programs should work together to the benefit of both and the institution and health system as a whole. this can be facilitated by regular communications and meetings, ongoing review of microbiological pathogen and susceptibility trends, and collaboration on research and educational programs. healthcare workers (hcws) play an important role to be a consultant about antibiotic use for patients. this would be helpful to reduce antibiotic overuse and prevent emergence of antimicrobial resistance in the hospital and public settings. we conducted across sectional study by using a self-assessment validated questionnaires, to determine current awareness and common habits related to antibiotic usage and antimicrobial resistance among hcws in hung vuong hospital; an obstetrics and gynecology hospital in vietnam. a total of 161 hcws were enrolled in the survey. although 99% of hcws responded correctly "many infections are becoming increasingly resistant to treatment by antibiotics", 77% of them thought that "antibiotic resistance occurs when your body becomes resistant to antibiotics". a total of 19% (95% ci: 0.13 -0.26) and 8% (95% ci: 0.04 -0.13) of hcws have correct knowledge about antibiotic usage and antimicrobial resistance. a total of 16% of hcws answered that they have to prescribe antibiotic because they cannot follow up the patients' condition. twenty-two percent of hcws answered that it is necessary to take antibiotic when people have fever. antimicrobial resistance and infection control 2017, 6(suppl 2):ds1 the global community demands standardization to approaches in patient safety. protocols differ to meet the specific demands of health care facilities; patient safety has always been the common end. an introduction and understanding of compliance under the australian setting would reaffirm this common end. our similarities and differences in achieving the purpose are interesting to note.this study aimed to share information between infection control professionals, how australia kept pace with the technological evolution of reusable medical devices (rmds) and caters to specific reprocessing requirements to ensure patient safety. how rmds are reprocessed to minimize, control and prevent healthcare-associated infections (hais) materials and methods the australian commission on safety and quality in healthcare requires health service organizations to comply with 10 standards ensuring patients get the quality of care they truly deserve. all cssd throughout australia are responsible for: standard 3. 16 antimicrobial resistance and infection control 2017, 6(suppl 2):ds2 background fumigation of operating rooms (or) with high concentration of toxic chemicals is an age old tradition practiced in most of the developing world to control hospital acquired infections. this approach lost favor in the developed world due to questionable efficacy and toxicity concerns. however, most of the hospitals in developing world continue to use fumigation practices with variable frequency. here we report our experience of fumigation free or in india. this quasi-experimental before and after intervention study was conducted in a 50-bed tertiary care referral women and children hospital in chennai (south india) between january 2015 and september 2016. the practice of or disinfection using quaternary ammonium compounds fumigation was allowed in addition to standard cleaning methods in before-intervention phase (jan 2015 to dec 2015). in after-intervention phase (jan 2016) onwards the fumigation practice was stopped and standard cleaning methods alone followed. the monthly environmental microbiological surveillance cultures and surgical site infection (ssi) rates were compared and analyzed. in the before-intervention phase there were 715 surgical procedures were carried out with 2 ssis and 156 environmental samples tested all were within acceptable limits as per defined standards. in the after-intervention phase 535 surgeries were carried out with no ssis and all 117 environmental samples collected were within acceptable limits. the standard cleaning methods alone without chemical fumigation is sufficient for operating rooms disinfection in india. however, this finding should be confirmed in large multi-site studies before universal recommendation. antimicrobial resistance and infection control 2017, 6(suppl 2):ds3 background it is reported that skin antiseptic with a chlorhexidine-alcohol concentration of more than 0.5% usedduring blood cultures lowers contamination rate more effectively than povidone-iodine skin antiseptic. as an approach to enhance precision of blood cultures, i heldcampaigns for appropriate sterilization methods in 2010 also changed 10% povidone-iodine to 1% chlorhexidinealcohol antiseptic during blood cultures in 2012, and reported results. material and methods study periods:april,2010-march,2011(a), august,2011-july,2012 after the sterilization methods campaign(b), and august,2012-july,2013 after the antiseptic change(c). i investigated and compared each period. the contamination was calculated by dividing the number of cases in which there was only one positive result of two sets or more of blood culture specimens submitted on the same day by the total of all paired sets collected. the contaminants were defined as cons, bacillus spp., corynebacterium spp., micrococcus spp., propinonibacterium spp. which a doctor took as causative organism of the infection were excluded. the contamination of the blood culture for periods a: 2.25%, b: 0.98%, and c: 1.05%. there's significant decrease in the contamination during blood cultures after sterilization methods campaign (a-b)(p < 0.05). it suggests contamination decreased by performing appropriate sterilization methods. there were no contamination differences in b-c after changing disinfectant (p = 0.86). it was suggested that an equal skin sterilization effect was provided when i performed the sterilization with1% chlorhexidine-alcohol and10% povidone-iodine, which was appropriate.the 1% chlorhexidine-alcohol has an immediate effect and durability in comparison with povidone-iodine, shorting the time for drawing blood after sterilization. reflecting importance for busy on-site blood cultures. antimicrobial resistance and infection control 2017, 6(suppl 2):ds4 background lovisenberg diakonale hospital has olympus edt machines for cleaning of endoscopes. in the standard program the wash time is 3 minutes with special detergent and the disinfection time is 5 minutes. periodical testing of final rinse-water is performed regularly with good results. microbiological control of the final rinse-water from the washer-disinfector for endoscopes (ewd) is the most widely used methods for detecting growth of bacteria. the validation process is a comprehensive procedure that requires both resources, time, knowledge, special equipment and access to the microbiological laboratory. the hospital wanted to assure the quality of decontamination of their endoscopes. we have established a partnership with the department of infection control at oslo university hospital in order to validate the ewd. the decontamination of flexible endoscopes must be tested and validated according to the standard en iso 15883. to validate the disinfection process and secure a repeatable method, we used a surrogate endoscope (spypach). this is equipped with biological indicators, temperature sensors and pressure-and flowmeasures. microbiological control of final rinse-water: satisfactory results according to standard.cultivation of biological indicators: unsatisfactory results according to standard. discovery of protein and fibrin in surrogate endoscope: unsatisfactory results according to standard. measurement of temperature, pressure and flow: no deviation conclusions the endoscope was not adequately cleaned during decontamination. final rinse-water had satisfactory quality but remaining biological indicators and protein residues showed that the decontamination was not satisfactory. alternative solutions may include: increasing the wash time and/ or modify the contents of detergent. antimicrobial resistance and infection control 2017, 6(suppl 2):ds5 background as we know, the biological indicators are the most accepted means of the qc in sterilization. because the biological indicators contain geobacillus stearothermophilus that have spore inside the cell, if the sterilization were successful, the g.sterothermophilus should be killed, and the biological indicator can't detected by color or machine. so, in this study, we aimed to make sure whether the super rapid biological indicator can replace the traditional biological indicator or not. according to the instruction of the super biological indicator, after 60 minutes, the data will show pass or not. so we using the culture method to make sure the result are the same or not. in this study, we collected 214 super rapid readout biological indicators from feb.2016 to mar. 2016, and all the result showed that was no bacteria growth after 1 hour incubated. then we used thioglycolate and tsa to make sure the results . according to the experiment's result, all of the culture data showed that 214 super rapid readout biological indicators were the same. the accuracy were 100% and the sensitivity and specificity were 100% and 100%. antimicrobial resistance and infection control 2017, 6(suppl 2):ds6 background sterilization wrap is commonly used for instrument trays or cassettes. there are many different types and sizes of wraps available. typically, two sheets are needed to provide an effective barrier and a specific technique is recommended [cdc, aami st79] to allow for aseptic opening. wrapped instruments should be secured with sterilization tape that also serves as an external indicator. before closing, a multiparameter chemical indicator should be included inside along with the instruments. before this study, we used cotton of wraps. and the problem we focused was the expiry date. according to the taiwan cdc suggestion, the cotton's expiry date is 7 days. but it's too short for us. this study aimed to find some types that can replace the cotton. in this study, we used the crepe paper of wraps to replace cotton. otherwise, we want to elongate the expiry date. so we collected the instruments that cover by crepe paper and that storage in csr (hepa level: 100000) for 1, 4, 8, 12, 16, 20, 24, 28 weeks. then we used the broth methods to make sure whether the instruments were contamination or not. in this study, we found all of the instruments were clean on 20 weeks. after 20 weeks, the culture result showed that some bacteria were growth on the instruments. according to the experiment's results, we suggested that the crepe paper can replace the cotton of wraps, and it could elongate the expiry date for 20 weeks in our csr. antimicrobial resistance and infection control 2017, 6(suppl 2):ds7 in 2014, 47 pieces of the mouth mirrors from dental unit were broken as they were packed and mixed with the other tools that were heavy, shape, and without protection. it broke the mouth mirrors from a process of packaging. the important is to prevent the broke of mouth mirror in order to have the good quality and enough instruments to service patients.this study aimed to determine the result of development of sterilization process toprevent the broke of mouth mirror in cssd of wanonniwas hospital. the sample group was composed of 1) mouth mirror and 2) personnel from the cssd 8 persons.the process included 3 steps: 1) prepare process is to learn the reason of the breaking of mouth mirror and create the system in every process from caring, washing, packaging, sterilization, keeping, sending to dental unit, 2) methods process is inform the personnel about the reason, why mouth mirror were broken, change the methods of sterilization of mouth mirror, and 3) evaluation : this study were collected during fiscal year 2014 through 2016. the breaking of mouth mirror in year 2014, 2015, and 2016 (may) were 48 pieces, 33, and 4 pieces, respectively. it has a clearly decrease. the sterilization to prevent the broke of mouth mirror that caring keep the mouth mirror into the box with a lid. the process of washing and drying, sorting the mouth mirror. packing cloth wrapped. sterilization, distribution and storage kit for the side impact protection. antimicrobial resistance and infection control 2017, 6(suppl 2):ds8 the medical instrument cleaning and disinfection procedures are important process to remove organic and inorganic matter, due to concerns about contamination risks especially protein residue, to prevent cross-contamination and ensure the safety. this study aimed to determine the cleaning efficacy of two different cleaning methods, manual cleaning process and an automatic washer disinfector machine, using the protein residue check test in the central sterile supply department (cssd), maharaj nakorn chiang mai hospital. the 35, 40, and 25 samples of medical instrument washed and cleaned by manual cleaning process, the automatic washer disinfector machine, and re-sterile (without washing process), respectively, were collected from july to august, 2014 to determine the protein residue. the protein residue was detected with two different detections, the protein residue check test (pose health care co., ltd., thailand) and fluorescence-based protein detection test (lab focus co., ltd.). the 27 (77.14%), 33 (82.50%), and 11 (44.00%) samples of manual cleaning process, the automatic washer disinfector machine, and re-sterile produced undetectable protein residues, respectively (p-value = 0.002). the 8 (22.86%), 7 (17.50%), and 14 (56.00%) samples of manual cleaning process, the automatic washer disinfector machine, and re-sterile produced detectable protein residues, respectively. the results indicated that, the medical instrument using the re-sterile process should pre-clean instrument with enzymatic detergent to remove gross soil immediately and must be thoroughly washed and cleaned before being sterile. moreover, the manual cleaning process and an automatic washer disinfector machine must be optimized to eliminate protein contamination and minimize the cross-contamination. background different medical device cleaning and disinfection procedures are used on a large scale. it is an important procedure to remove organic and inorganic from medical device to prevent cross-contamination. this study aimed to determine the cleaning efficacy of an alkaline detergent used in an automatic washer disinfector machine in the central sterile supply department (cssd), maharaj nakorn chiang mai hospital. an alkaline detergent was developed by pose health care co., ltd., thailand. the programs were designed with different concentrations of alkaline detergent 50, 60, 80, and 100 ml (0.15, 0.18, 0.24, and 0.30 v/v, respectively) and two different temperatures (60 and 65°c). the cleaning efficacy was monitored using a tosi (en iso 15883) and brown stf loaded check strips. each program used three tosi test kits and five brown stf strips to evaluate the cleaning efficacy. additionally, protein residue was detected with the pyromol® test, protein residue check test, and fluorescence-based protein detection test. moreover, biofilm and microorganisms were determined using a scanning electron microscope (sem). the optimum concentration of the alkaline-based cleaning detergent and temperature were 60 ml (0.18 v/v) and 60°c, respectively. this program (60 ml and 60°c) produced undetectable protein residues, biofilm, and microorganisms on medical instruments after the cleaning process. comparable to the recent condition of 100 ml (0.30 v/v) concentration and at 65°c, the optimum condition of 60 ml concentration and at 60°c reduced the cleaning costs by 40% per each cleaning process. background carbapenemase-producing enterobacteriaceae (cpe) infections are an emerging threat in specific regions of south east asia, europe, and the united states. cpe occurs sporadically in australia with isolated genomic clusters. different classes of carbapenemases are prevalent by region worldwide, and in australia, imipenemase-producing cpe (bla imp-4) is endemic at low levels. in our healthcare facility, carbapenemases other than imp demonstrate epidemiological links to recent overseas healthcare or residency. following retrospective recognition of a cluster of cpe cases linked to a single healthcare facility in melbourne, the local health authority, the victorian department of health and human services, released comprehensive consensus guidelines for the management of cpe in december 2015. based on a "search and destroy" strategy, instruction is provided regarding identification of cpe, centralised reporting, contact tracing and screening, the need for alerts, clearance protocols for contacts, cleaning validation and 6-monthly point-prevalence surveys. oversight of outbreaks is provided by a state incident management team and a single state reference laboratory performs genomic analysis of all isolates. this presentation will outline the experience and lessons learned in implementing these cpe guidelines in our healthcare facility. examples include the development of new systems to identify and communicate cpe cases, electronic alerts for isolation and screening, a cpe staff education program and methods for conducting pointprevalence surveys in high-risk wards. early identification and screening of patients hospitalised abroad and the need for a functional it platform to facilitate electronic flagging/ alerts represent challenges likely to be faced by many healthcare facilities in our region. in recent years, we found rising in trends of measles infections, even in a small local epidemic. it may because of the measles virus' genes per se, antigenic variation or other factors. infected with measles virus can cause temporarily decline in human's immune, especially cellular immunity. lack of effective immune reactions can lead to secondary bacterial and multiple infections. we investigated measles-infected patients and evaluated susceptible factors for multiple drug-resistant bacteria infections. we also examined hospital infection prevention and control protocol in controlling measles outbreaks in our hospital. during 2013 to july 2015, 492 cases of patients with measles were detected in our hospital. we retrospectively reviewed and analyzed 51 measlesinfected cases who had multiple drug-resistant bacteria infection data. we found that the main risk factors to be infected with multiple drug-resistant bacteria included: 1) age under 8 months, 2) abnormal cardiac functions, 3) having malnutrition and 4) having encephalitis. the hospital should focus on patients under 8 months of age, with abnormal cardiac functions, having malnutrition or encephalitis to avoid multiple drug resistant bacteria infections and decrease mortality by strengthening treatment and implementation of infection prevention and control protocols. the global spread of carbapenemase-producing enterobacteriaceae (cpe) is a major challenge for infection control practitioners.we adopted a proactive approach that all cpe carriers were isolated in a designated ward with strict contact precautions. here, we investigated if cpe can survive terminal disinfection in hospital environment. our study aimed to evaluate the extent of cpe contamination in patient care environment after terminal disinfection using microbiological sampling. microbiological samples for cpe were taken from the general wards' environment whenever a patient newly identified with cpe was removed for isolation. environmental samplings were collected by trained personnel. high-touch and wet surfaces were sampled using sterile polywipe sponge. chromid carba agar was used for selective cultivation of cpe. suspicious colonies grown after overnight incubation at 35°c were further examined for carbapenemase production using carba-np. we confirmed carbapenemase production using genexpert carba. between 7 april 2014 and 4 october 2016, 468 environmental samples were collected from 23 wards. we found 1.92%tested positive for cpe that included7 hand-wash basins, one sink and one hospital curtain. these isolates were imp-producing cpe. six affected basins/ sinks were cleared from cpe after cleaned with detergent followed by disinfection with 5.25% sodium hypochlorite solution (1,000 ppm) daily for one week. the cpe in the remaining two hand-wash basins survived for 21 days after daily decontamination and the basins need to be replaced. our results highlighted hand-wash basins may serve as a potential environmental reservoir for cpe. as standardized decontamination regimen for sinks was lacking, we recommended hand-wash basins should not be used for the disposal of body fluids. terminal cleaning of isolation rooms is an essential step in infection control. however, traditional cleaning and disinfection may be inadequately performed by time limitation. ultra-violet (uv) devices are effective in environmental decontamination but their cost can be prohibitive. recently, a more economical version of a uv-c sterilizing unit has become available but its effectiveness for environmental decontamination has not yet been independently evaluated. our study aimed to evaluate the effectiveness of the spectra 1000 uvc light system to reduce viability of healthcare-associated pathogens in a ward environment. four organisms (staphylococcus aureus, enterococcus faecalis, acinetobacter baumannii and klebsiella pneumoniae) were coated onto designated areas of formica that were then attached to eight locations in the room. the room was irradiated for 15 minutes and the lamp was moved to a second position then treatment was repeated. cultures were performed and resulting colonies were enumerated. surviving numbers were compared with non-irradiated controls. all organisms were rendered non-viable in areas receiving direct irradiation or substantial reflected light. at two sites where heavily shaded (rear of bedside lockers and armchairs), a 2-log reduction in viability of e. faecalis was observed. conclusions spectra 1000 uvc light offered an effective adjunct to conventional terminal disinfection of isolation rooms. although two irradiation periods using two positions of the lamp were needed, to ensure that shaded areas would receive adequate treatment, the total time for disinfection was only 30 minutes. as uv treatment does not produce residues, there is no down time after use. the current status of cross-infection risks in hotels yuka sato, mariko tateishi, mutsuko mihashi kurume university, kurume, fukuoka, japan in japan, the ministry of health, labour, and welfare published the "guidelines for the prevention of new strains of influenza infections in employers and employees [1] ". however, our survey, which investigated infection control for new strains of influenza in 2014, revealed that recognition of these guidelines was low, showing that infection prevention control had been insufficiently implemented in hotels. our study aimed to identify the current status of cross-infection risks in hotels, and use the results to improvethe control of infectious diseases in such areas. the study ran from march 17-18, 2016. the study volunteers were members of the all japan ryokan hotel association. to assess environmental contamination levels in hotel settings, the atp and amp swab test kits (kikkoman lumitester pd-20) were used. frequently touched surfaces were measured for contamination. measurement sites included 30 that were measured intermittently, and 39 that were measured before and after cleaning. there were 5 intermittently measured monitoring sites that exceeded 5,000 rlu including the open/close buttons inside the kitchen elevator and first floor handrails. there were 5 monitoring sites measured before and after cleaning that exceeded 5,000 rlu even after cleaning, including the inner sides of restaurants' sliding doors and the inner washroom door knobs of guest rooms. conclusions atp values of more than 5,000 rlu were detected at some monitoring sites, suggesting the need to reconsider methods and frequency of cleaning by taking risk of cross-infection into account. the purpose of this study was to test the effectively of the infection control risk assessment (icra) monitoring tool developed by the infection prevention and control unit (ipcu) of asian hospital and medical center with the aim to increase the compliance of construction workers to recommended infection prevention and control measures during construction, renovation and demolition in the hospital. materials and methods indicated in the icra monitoring tool were the details of the activity and the infection risk level (class i,ii,iii and iv). the design used wasa quasi-experimental designwhich was conducted among all construction projects in the hospital within a 1-year period. the percent compliance was computed by number of compliant projects per month over total number of monthly projects which thenmultiplied by 100. there were a total of 151 construction projects monitored by direct observation which utilized the icra tool. other interventions included orientation of construction workers to the tool, acknowledgment and accountability of recommended infection prevention and control measures by signing the tool and lastly, making use of the tool to provide feedback. results show an improvement in the compliance to infection prevention and control interventions from average of 84% during pre-intervention to 91% post intervention. having an icra tool paved the way for construction workers to be pro-active and be involved in preventing infections brought by construction, renovation and demolition. national kidney foundation, singapore, singapore; 2 jurong west1 dialysis centre, singapore, singapore; 3 kolam ayer dialysis centre, singapore, singapore; 4 woodlands1 dialysis centre, singapore, singapore; 5 teck whye dialysis centre, singapore, singapore; 6 yishun1 dialysis centre, singapore, singapore; 7 hougang1 dialysis centre, singapore, singapore; 8 kim keat dialysis centre, singapore, singapore; 9 tampines2 dialysis centre, singapore, singapore correspondence: jamilah binte jantan (jamilah.jantan@nkfs.org) antimicrobial resistance and infection control 2017, 6(suppl 2):e6 in the dialysis centre, there is potential for cross transmission of infectious agents through contaminated devices, hands, equipment, supplies and environmental surface during haemodialysis (hd) treatment. to reduce the risk of acquiring infections, staff routinely clean and disinfect medical equipment and high-touch areas after each patient's hd treatment at the national kidney foundation (nkf). this study aim of the study is to assess environmental cleaning of hightouch areas and develop intervention program to achieve compliance ≥85%. materials and methods this is a quantitative study involving 29 infection control link nurses (iclns) at the community-based dialysis centres (cb-dcs), nkf from october 2015 to april 2016. in november 2015, iclns conducted an environmental cleaning assessment of high-touch areas using a checklist and glo germ kits, to ascertain the efficacy of environmental cleaning at 29 cb-dcs. pre-study data showed an overall average of 67% compliance. rca revealed the absence of an audit tool for high-touch areas, a lack of training leading to knowledge deficit, poor cleaning techniques and staff incompetency. interventions included a checklist (audit tool) for environmental cleaning assessment of high-touch areas, a "train the trainer" programme for the 29 iclns, an annual competency assessment and video tutorials on environmental hygiene to standardise practice. following the interventions, environmental cleaning assessment of high-touch areas showed an overall average of 86% compliance, with 17 cb-dcs achieving ≥85% compliance in environmental cleaning of high-touch areas. this study illustrated that the intervention programme increased staff awareness, thereby improving compliance. besides promoting positive outcomes, it enhanced the internal monitoring system at nkf. antimicrobial resistance and infection control 2017, 6(suppl 2):e7 in march 2016, a surge of 34 mrsa acquisitions (30 from screening and 4 from urine cultures) was noted in rehabilitation ward (rehward) at tan tock seng hospital, singapore. our objective was to investigate if fomite transmission could be a cause of these acquisitions. we conducted one-day surveillance screening of the gym equipment and reh-ward's environment. samples were collected by rolling swabs moistened with sterile saline five times on the surfaces of gym equipment before and after use. in the ward, selected patients' beds and common items or equipment in shared area were also sampled. samples were cultured for mrsa using selective chromogenic media. in the gym, all 156 samples collected from equipment pre-use were negative for mrsa. however, 7.3% (6/85) of the samples collected after use were mrsa positive. in the wards, all swabs (55) that were taken from the common shared area such as computers, case notes carts, were negative. two out of 12 beds (16.7%) occupied by mrsa carriers and 3 out of 53 beds (5.7%) occupied by non-mrsa carriers were contaminated with mrsa (or 3.33, 95%ci 0.24-32.46, p = 0.23). overall rates of mrsa-positive swabs were comparable between the wards and gym (2.5% vs. 2.2%, p = 0.82). gym equipment was not more likely than the ward environment to contribute to mrsa acquisition. the importance of environmental cleaning in all areas including rehabilitation facilities cannot be overemphasised. with the widespread use of antibiotics in the treatment of human bacterial infections, the multidrug-resistant microorganisms also appear to threaten human health. environmental cleaning to avoid the spread of bacteria and healthcare-associated infection is an important part of healthcare infection control practice. through this program, our hospital aimed to improve the environmental cleaning, reduce the bacterial antibiotics resistance, and further reduce the use of antibiotics. we reformed program of environmental cleaning and measured incidence of multidrug-resistant bacteria and consumption of designated antibiotics. our results were shown below. the unqualified rate in environmental cleanliness of our cleaners was 39.1% before this program was implemented and 21.7% after this program that demonstrated 44.5% reduction. the numbers of healthcare-associated infections with multidrugresistant bacteria was 30 before this program, and 8 after this program (73% reduction). the consumption of anti-methicillin-resistant staphylococcus aureus was 142.0 defined daily dose (ddd) /1000 bed-days before this program, and 101.5 ddd /1000 bed-days after this program (28.5% reduction). the consumption of glycopeptides was 120.0 ddd /1000 bed-days before this program, and 83.2 ddd /1000 bed-days after this program (30.8% reduction). the consumption of carbapenems was 164.5 ddd /1000 bed-days before this program, and 98.1 ddd /1000 bed-days after this program (40.4% reduction). according to our results, environmental cleaning may effectively reduce the number of healthcare-associated infections with multidrugresistant bacteria and used of broad-spectrum antibiotics. if it can be promoted consistently, in accordance to the qualified data, the use of antibiotics could be reduced and the prevention of bacterial resistance occurred. antimicrobial resistance and infection control 2017, 6(suppl 2):e9 the invention related to an antibacterial treatment process of a curtain fabric material with functions of free washing and environmental protection. they were used for the antibacterial treatment of a nonwoven fabric made from polypropylene fibers and as the curtain fabric material with functions of free washing and environmental protection. before our hospital use this production, we aimed to investigate the expiry date of antimicrobial curtain to establish our protocol. we conducted this experiment from august to october 2016. in this study, we used three companies' antimicrobial curtains. we used the c.difficile, mrsa and a. baumannii as study models. then we putted the antimicrobial curtain on the agar then see the bacteria growth or not. according to our study, c. difficile, mrsa and a. baumannii were not detected in the antimicrobial curtains. after 11 weeks, there were no bacteria growths on the curtain. the curtain fabric material produced by the invention has the advantages of good air permeability, easy maintenance of dry curtain fabric surfaces, dirt resistance, free washing, no toxicity and irritative peculiar smell, easy recycling, environmental protection, good antibacterial property and low antibacterial treatment cost. antimicrobial resistance and infection control 2017, 6(suppl 2):e11 environmental contamination is the important source for bacterial spread causing hospital-acquired infections. environmental cleaning with an established standard operation procedure (sop) and cleaning staff's strict adherence to the sop are therefore extremely important. however, evaluation of the effects of environmental cleaning in general has not been fully reported in the literature. this study aim to elucidate the effects of environmental cleaning with the established sop and by the well trained cleaners in kaohsiung chang gung memorial hospital (kscgmh). environmental cleaning was based on the sop which followed the principle of cleaning from higher locations to lower ones, and from the contaminate areas to the comparatively cleaner ones in rooms where patients were staying. before and after daily environmental cleaning routine, environmental surfaces were swabbed for sampling specimens for bacterial culture and for bacterial count evaluation in case of culture positive. data showed that before and after cleaning environmental, bacterial burdens in environmental surfaces of the bedroom were significantly reduced (p < 0.0001). indicating the environmental cleaning with the current cleaning sop and by these well trained cleaners has been effective. hospital-wide environmental cleaning monitoring program reduces healthcare-associated infections related to multidrugresistant organism hsuehlan chao, pinru changchien, weifang chen, chunghsu lai e-da hospital, kaohsiung, taiwan the evidence-based policies to clean hospital environment can reduce the colonization and infection of multidrug-resistant organisms (mdros). the purpose of this study is to measure the effectiveness of environmental cleaning policies. (1) the effectiveness of policies was examined by adenosine triphosphate (atp) and microbial cultures (mcs) before and after the implementation of policies at both the general wards (gws) and the intensive care units(icus). (2)the study audited selected 20 points related to mdros, including bed rails, bed button beds (the desktop corner), etc. (3)the standard values: atp less than 250 rlu at icu and less than 500 rlu at gw. bacterial colonies count < 100 cfu. (1)20/49 atps (41%) had been detected over 500rul before policies, but 28/67 (42%) after policies without statistical difference (p = 0.45) in gws. however, in icus, before policies 40/81 atps (49%) over250 rlu and after policies 30/67 atps over 250 rlu, no statistical difference (p = 0.29). (2) 50% selected points were over 500rul both before and after policies at gws. only 16.6% selected points were over 250rul in icus. (3) mcs had statistically significant (p = 0.01) before and after policies at gws, including oxacillin-resistant staphylococcus aureus (orsa), enterococcus faecium (vre), acinetobacter baumannii, (xdr).but icus were not dirty over standard both before and after policies. this survey helps us understand how much dirty and contamination in the environment. especially, bed rails, button, isolation unit, car-related equipment, room telephone, curtains. establishing good environmental policies are very important to prevent healthcare-associated infections. icus environment is cleaner than gws in this study. antimicrobial resistance and infection control 2017, 6(suppl 2):h1 background contaminated hands are the foremost source of spreading infections in healthcare facilities. wellness and safety of patients and healthcare workers (hcws) can be achieved by promoting best practices in infection control through education and advocacy. we aimed to develop effective hand hygiene (hh) practices among hcws by improving their knowledge, attitude and practices through implementing standard hh guidelines. a year-long project was conducted at two hospitals of bangladesh. this included a baseline survey, intervention by implementing standard hh guidelines through classroom and hands-on training, and a post intervention survey. pretest-posttest was conducted with preformed questionnaire and observation checklist during pre and post intervention surveys. total of 600 physicians and nurses were trained on standard hh practices. at the institute of child and mother health, rate of hh compliance before patient contact improved from 4% to45.32%(p < 0.0001) among physicians and 2.07% to 60% (p < 0.0001) among nurses. after patient contact, it increased from 4.8% to 50.36% (p < 0.0001) among physicians and 5.93% to 59.51% (p < 0.0001) among nurses. at general hospital, sirajgonj, rate of compliance before patient contact increased from 2.25% to 49.18% (p < 0.0001) among physicians and 3.10% to 53.57% (p < 0.0001) among nurses. after patient contact, it increased from 2.25% to 58.20% (p < 0.0001) among physicians and 5.31% to 60.71% (p < 0.0001) among nurses. the project outcomes signify that implementing standard hh guidelines improves the knowledge, attitude and practices of the hcws. the results emphasize the necessity of continuous education and advocacy in improving hh compliance to promote excellence in infection prevention and control. antimicrobial resistance and infection control 2017, 6(suppl 2):h2 the world health organization multi-modal intervention to increase hand hygiene compliance was adopted in a rehabilitation hospital since 2008. however, the effectiveness of the individual measure was unknown. this study was conducted for evaluating the effectiveness of hand hygiene promotion activities used in the hospital. a cross-sectional survey was applied in 2012. a 16-item selfadministered questionnaire was adopted to collect the opinion on the effectiveness of measures previously used on hand hygiene promotion. nurses and healthcare assistants working in the inpatient settings of hospital were invited to participate in the survey. sevenpoint likert-type scale from "extremely ineffective" to "extremely effective" was used for ratingindividual items. rasch measurement was employed for data analysis by winsteps version 3.92.1. one hundred and seventy-nine questionnaires were returned contributing 97.2% of the response rate. the categories in the rating scale were collapsed into 4-point scale before further analysis. thirtyone misfitting persons and three misfitting items were removed after examination in quantitative and qualitative manners. no differential item functioning was found between subgroups. the final scale was considered as unidimensional with reliabilities ranged at 0.95-0.96 and 0.91-0.92for persons and items respectively. "placing the alcoholbased handrub" was identified as the most effective measure on hand hygiene promotion and "set up an annual target of hand hygiene compliance" was considered as the least effective. the survey identified and located the effective measures on hand hygiene promotion. for a more efficient approach, the hospital may prioritize the most effective items in hand hygiene promotion. antimicrobial resistance and infection control 2017, 6(suppl 2):h3 background adenosine-triphosphate (atp) bioluminescence assay has been popularly adopted in clinical and catering industry due to its ease of use and immediate results. atp bioluminescence assay picks up cellular discharged atp, which can also be found on cellular debris or organic components that are not microbial in nature.its measurement on animate objects can be misleading. this study was developed for the catering crew of a private hospital as a hand hygiene (hh) practice monitoring. microbial viable count was used as a validating reference for the atp relative light units (rlus) as a control measure of hh effectiveness. a set of basal microbial values was developed for each staff member. this provided a convenient but reliable protocol for atp luminometry users. swab sampling was collected from crews' hand for bacterial culture. selective media and serological tests were used for pathogen screenings, which included staphylococcus aureus, coliform and salmonella. standard curves to demonstrate the correlation between the viable microbial counts and the corresponding rlus of the atp measurements were developed. the study showed the actual viable microbial density of individuals after handwashing did not correlate positively with rlus. each individual had his/her own confidence regarding to the limitation of rlus. however, the hh compliance could be reflected by the viable microbial counts.individual skin condition played a role in this association. the measurement instilled a positive effect on the crew. hand hygiene compliance can be reflected with the bench marking standard technique. hand hygiene compliance was increased and microbial load was significantly reduced. hand hygiene is the single and most effective way to prevent the spread of microorganisms in hospital. when health care workers (hcws) have own sense and aware of the importance of hand hygiene, it yields twice the result with half the effort in infection control. the study aimed to promulgate hand hygiene is the responsibility of every hcw and maintain hand hygiene as the standard of care in the daily work of hcws materials and methods on the international hand hygiene day 5th may, a hand hygiene campaign was held in cmc. hcws were invited to take photo and pledged on hand hygiene compliance. hand hygiene technique was also taught personally by infection control nurses and return demonstration was needed during the activity. an instant photo was taken when they pledged. these photos were shown on a board during the hand hygiene promotion activity and returned to each colleague as a souvenir afterward. hospital managers, frontline doctors and nurses, allied health professionals and supporting staff, total over 100 colleagues in cmc were pledged on hand hygiene on that day. this pledge motivated other colleagues to compile in hand hygiene. the hand hygiene compliance rate in cmc maintained over 90%. motivate health care workers to perform hand hygiene by a soft commitment is another way to promote and raise their awareness of hand hygiene. to introduce the "who hand hygiene save life campaign" and enhance the awareness of public and healthcare workers for the importance of hand hygiene. hong kong infection control nurse association (hkicna) has joined actively in the community events such as "world health day carnival" since 2008. in these few years, there were over a thousand public to participate in hh and infection control related educational game booths and talks. further to extend the engagement of public and healthcare workers, a poster design competition for promotion of hand hygiene was organized in 2012; the winning poster was used as "talking wall" in community and healthcare settings and the background of hkicna's souvenirs. in 2014, a creative reminder-hand-held electric fan with visual lit up "hand hygiene" was distributed. in addition, another innovative ideatwo hh dances were designed to continuously promote the hh; they stress that hh practice should start from children to adulthood, from healthcare worker to all in the community. both of them were used as a tool for promoting in hospitals and schools and assessable in youtube which was gained thousands of 'likes' conclusions hkicna is working hard on introducing hand hygiene concept for infections prevention and control in healthcare settings and community. the concept of "clean hand save lives" will continue be emphasized. antimicrobial resistance and infection control 2017, 6(suppl 2):h6 hand hygiene (hh) is recognized as the most effective measure to prevent the spread of micro-organisms through hand contact during patient care. besides yearly mandatory infection control training through on-line learning management system, matilda international hospital (mih) also provides relentless training to staff emphasizing the importance of proper hand hygiene and embracing who 5 moments. in june 2016, educational booth game -"unforgettable 5 moments and 7 steps" was held to assess knowledge and techniques of both clinical and non-clinical. this study aimed to evaluate the techniques of staff's hand hygiene practice, compliance to rubbing time and accuracy in reiterating the 5 moments. observational methods were used to evaluate hh technique and rubbing time. staff were required to accurately call to remembrance the 5 moments through direct questioning and demonstrate 7 steps of hand hygiene technique with at least 20 seconds of rubbing time. immediate feedbacks were supplemented. the overall accuracy of all assessed criteria's was 90.53%. majority of hh steps achieved greater than 90% compliance rate except "between finger" and "back of finger". "before clean aseptic procedure" demonstrated to be the most difficult to recall. the audit allowed for gaps and in-depth understanding of staff hh practices to be more accurately identified with subsequent staff training strategies to be drawn and implemented. antimicrobial resistance and infection control 2017, 6(suppl 2):h7 background alcohol-based hand rubs (abhrs) are the preferred methods for performing routine hand hygiene (hh) in healthcare facilities. however, soap-and-water hand washing is still popular. this study measured the hh knowledge and self-reported practices of shiraz nemazee hospital nurses. this study employed two questionnaires. a six-question survey covered hh knowledge, (19 possible points), while hh practices were monitored in a second survey containing four multi-part self-reporting inquiries (37 possible points) in 2016. surveys were voluntarily completed at work. responses were analyzed anonymously. results 342 nurses completed the questionnaire.54.4%had formal hh training in the past year.55.6%reported using abhrs for more than a year. 53.8% preferred traditional soap-and-water hand washing. eleven nurses never used abhrs. nursing experience varied -15.1% (>ten years), 10.8% (5-10 years), 35.2% (2-5 years) and 28.9% (<2 years). knowledge scores ranged from 11-15 (high score was17). selfreported hh compliance scores ranged from 24-31 (high score of 37). a positive, but weak correlation existed between knowledge and self-reported practice scores (r =0.28, p <0.001). no correlation existed between years of experience and knowledge (p =0.85, r = 0.011) or self -reported practice scores (p = 0.86, r = 0.01). also, no correlation was found between age and self-reported practices (p =0.4, r = -0.048) and/or knowledge scores (p =0.85, r = -0.011). antimicrobial resistance and infection control 2017, 6(suppl 2):h9 background simulation healthcare education is widely used in medical education and has great potential. however, scenario-based simulation healthcare education for preventing nosocomial infections has not been described. this study aimed to determine the effectiveness of scenario-based simulation education to improve hand hygiene. a single-centre, prospective, cohort study was conducted at tokyo medical university hospital (1015 beds), an acute-care teaching hospital, from january 2011 to december 2014. each infection-control training course (ictc) was held every month and lasted 2 hours. trainees put on and removed personal protective equipment under scenarios of standard precaution (two scenarios) and contact precaution with methicillin-resistant staphylococcus aureus (one scenario), while considering timing of hand hygiene. we determined the correlations between the participation rate in the simulation education and use of alcohol-based hand disinfection and reduction of catheter-related blood stream infection (crbsi). there were 1077 trainees. the total participation rate for hospital staff was 76% by the end of the study. the overall correlation between use of alcohol-based hand disinfection in the hospital and the course participation rate was significant (correlation coefficient, 0.97). an inverse correlation (−0.94) was observed for the relation between the ictc participation rate and the incidence of crbsi. with participation in the ictc, crbsis due to staphylococcus spp. and enterobacteriaceae were significantly lower than those due to candida spp. our ictc had a positive effect on hand hygiene and reducing crbsi. this study is the first effective scenario-based simulation healthcare education to hand hygiene and control of nosocomial infection. antimicrobial resistance and infection control 2017, 6(suppl 2):h10 background the purpose of this study is to increase and sustain hand hygiene compliance through evidence-based approach and to relate compliance with the trend of healthcare associated infections in the hospital. the study was conducted over a 1 year period from september 2015 to august 2016. the methods used in monitoring hand hygiene compliance are "direct observation" which includes self-reporting and by the use of secret shoppers. another is "electronic monitoring" through the use of radio frequency identification device. surveillance of healthcare associated infection (hai) is conducted in the general nursing units, telemetry and intensive care units. interventions to increase hand hygiene compliance were implemented such as training of nurse linc representatives in monitoring hand hygiene compliance, use of social media (facebook) in promoting hand hygiene, recognition of individuals and department with high compliance. after the implementation of interventions, result showed an increase in the hand hygiene compliance from below 50%, during the start of the period monitored, to above 80% during the succeeding months. comparison of hand hygiene compliance versus healthcare associated infection rates was shown through a graph, this information was cascaded to the different departments during unit meetings. correlation showed a contrasting trend between hand hygiene compliance and healthcare associated infection rates. it was therefore concluded that an increase in the compliance to hand hygiene can decrease the healthcare associated infections among patients. in addition, feedback methods and other evidencedbased interventions can increase hand hygiene compliance. would also like to correlate the trend of hand hygiene rate with healthcare associated infections in the msicu. the data collected is generated by an automated system through the use of radio frequency badges worn by the healthcare worker and sensors attached to the hand rubs and soap dispensers. badges provide real time feedback by means of an alarm system. a quasi-experimental design was used to test the effectiveness of the interventions implemented. the utilization of visual boards in providing feedback, converting the door entrance into a giant hand hygiene poster, use of social media, and reward system were included in the interventions. total number of opportunities captured is 121,252. post intervention data showed an increase in the compliance from below 50% to above 80%. it was therefore concluded that the multiple strategic approach, through the use of electronic monitoring, helped in increasing compliance of healthcare workers to hand hygiene. moreover, when hand hygiene trend was compared to the healthcare associated infection, a contrasting trend was evident. antimicrobial resistance and infection control 2017, 6(suppl 2):h16 hand hygiene (hh) is a known effective measure for the prevention of healthcare-associated infections and spread of antimicrobial resistant organisms. unfortunately a compliance rate with healthcare workers in emergency department is very low because of rushed working environment. the objective of this study focused on the interventions at the point of care (poc) to improve the compliance, or "system change" according to the who hand hygiene strategies, which key aspect was the provision of alcohol based hand rub on a new design bottle holder that optimized the acceptance and usage. the first phase was the baseline period set on 3 months baseline (january to march 2015) observation of hh compliance and continue the second phase using the bottle holder (april to june 2015). an anonymous, self-administered questionnaire, had distributed to 126 emergency healthcare workers (hcws) to assess their behaviors and attitudes toward hand hygiene compliance and their satisfaction. overall, 86.5% (109 of 126)of hcws (36 nurses, 19 physicians) satisfied with the device and 87.3% believed they could improve hh compliance.overall compliance significantly increased from 19.7% to 59% (p-valve = .045), 0 to 27.3% before patient contact, 11.5% to 57.7% after patient contact, 27.3% to 60.0% before clean/aseptic procedures, 48.1% to 64.0% after body fluid exposure/risk, and 17.1% to 61.9% after touching patient surroundings. the device successfully provide easy access to the bottle holder, which is critical for its success in improving hh compliance in one of the busiest area in the hospital. antimicrobial resistance and infection control 2017, 6(suppl 2):h17 hand hygiene compliance among hospitalized personnel is a one of safety indicator. in the past, our hospital had used the paper-based system for auditing hand hygiene compliance. the majority of the data entry, collection and analysis were performed manually which was complicated and time-consuming.we aimed to create an electronic hand hygiene auditing tool to measure hand hygiene compliance. the study was conducted in a tertiary-care hospital in thailand. a development process using five steps of the program development life cycle(pdlc). 1) requirement gathering and analysis 2.)design on google docs access methods transition.3.)program testing. 4.) implementation on mobile phones. 5.) maintain the program/system. all steps were conducted in a month (april 2016). the electronic hand hygiene auditing tool is the result of this study. both methods of data collection were compared. the results demonstrated that the duration of the processes decreased from 5 months to 1 month. questionnaires return rate increased from 86% to 100%.the assessors' satisfaction rate was 80%. antimicrobial resistance and infection control 2017, 6(suppl 2):h19 background good hand hygiene (hh) practices are a simple and cost-effective strategy to limit pathogen transmission between patients. this study explores the effect of a multimodal hand hygiene promotion program on hh compliance amongst healthcare workers. a prospective study was conducted at the pediatric intensive care unit (picu) and pediatric immunocompromised ward at king chulalongkorn memorial hospital, bangkok, thailand. interventions performed were: hh promotion videos sent to staff via mobile phone, hand hygiene signs at the bedside, distribution of portable alcohol gel bottles, and hh promotion culture led by senior staff members. all interventions were tailored according to pre-intervention opinion surveys with staff. hh compliance was assessed by direct observation using the who 5-moments for hand hygiene (who5hh)before touching patients, before clean/aseptic procedures, after body fluid exposure risk, after touching patients, and after touching patient surroundings. 200 opportunities in total were observed monthly. in december 2015, pre-intervention, overall hh compliance rates were 50%. between january and june 2016, post-intervention, overall hh compliance increased to 72%. when divided into the five moments for hand hygiene, hand washing prior to touching patients significantly improved following intervention from 43.8% to 85.1% (p < 0.001) on the immunocompromised ward and from 44.4% to 88.9% (p < 0.001) on the picu. hand hygiene after touching patient surroundings remained low. hh compliance was highest amongst nurses. a multimodal hh promotion campaign tailored towards the local population was effective in increasing hh compliance overall. however, hh after touching patient surroundings remained low post campaign. antimicrobial resistance and infection control 2017, 6(suppl 2):h20 the world health organization (who) has developed multimodal hand hygiene improvement strategies to support hospitals to improve hand hygiene and thus reduce hospital-acquired infection. the aim of the study was to study the implementation of these multimodal strategies, obstacles, and supports to promote hand hygiene in government hospitals including university hospitals, regional hospitals, and general hospitals. the samples were 59 infection control nurses who implement multimodal hand hygiene improvement strategies. the data collection instrument was the hand hygiene self-assessment framework questionnaire developed by the who. the questionnaire also asked about obstacles and support. most of the samples (61.02%) implemented multimodal hand hygiene improvement strategies at an intermediate level, followed by basic and advanced levels (22.03% and 16.95%, respectively). fifty percent of the university hospitals had an advanced level. most of the regional hospitals and the general hospitals (57.14% and 67.57% respectively) had an intermediate level. the highest score for implementation was the system change for hand hygiene (median score, 90.00), followed by reminders in the workplace, training and education, evaluation and feedback, and institutional safety climate for hand hygiene (median score, 72.50, 60.00, 60.00 and 50.00, respectively). all samples encountered obstacles and needed support when implementing hand hygiene improvement programs. personnel were the first obstacle, followed by management, facilitative equipment, and budget, respectively. the greatest need was for environmental and equipment support, followed by support from staff and management support. the government hospitals should improve implementation of multimodal hand hygiene strategies by addressing obstacles and providing support in order to promote hand hygiene and reduce hospital-acquired infection. additionally, using e1174, the efficacy of these formulations was compared against seven commercially available abhrs and who recommended formulations containing alcohol from 60% to 90% at a more realistic 2-ml volume application. the novel abhr formulations met efficacy requirements for both hcphw and en 1500 when tested at volumes typically used in these methods. moreover, these formulations met hcphw requirements when tested at 2-ml. in contrast, the commercial abhrs and world health organization formulations failed to meet hcphw using a 2-ml application. importantly, product efficacy did not correlate with alcohol concentration and format. conclusions abhr are complex formulations, combining alcohol with various ingredients that can influence the overall antimicrobial efficacy. formulation as a whole and is more important than alcohol concentration alone for efficacy. product format has the same efficacy. when selecting abhr, ask for the in vivo efficacy results based on standard protocols and the volume of the product used to meet the criteria for hcphw test. antimicrobial resistance and infection control 2017, 6(suppl 2):he3 after the earthquake in kumamoto on april 14th, 2016, disaster medical assistance team (dmat) and japan medical association team (jmat) immediately supported the shelters and medical facilities in the disaster area. however, the arrivals of dmat and jmat to nursing homes were delayed due to lack of information and no transportation by landslides. we started supporting nursing homes as jmat members 16 days after the quake. this study aimed to report about our infection control intervention in the disaster area. we conducted a survey on the occurrences of infectious gastroenteritis and influenza at nursing homes, and interviewed the healthcare workers about problems on infection control. then, we offered guidance on infection control necessary to each facility. we visited eleven nursing homes between april 30th and may 2th, 2016 to find two residents with symptoms of influenza and one with symptoms of infectious gastroenteritis. the most frequent questions from the healthcare workers at nursing homes were how to clean and sterilize medical devices, and how to clean their hands appropriately. antimicrobial resistance and infection control 2017, 6(suppl 2):he8 as taiwan is situated in the high risk subtropical region, dengue fever has virtually become a seasonal infectious disease. this study aimed to determine risk factors and causes of death in dengue fever. analysis was conducted on the 93 confirmed severe cases of dengue fever or dengue hemorrhagic fever reported to this hospital over the period between july 20 and september 30, 2015 in terms of gender, age, history of chronic diseases, warning signs and diagnostic criteria for severe conditions. retrospective case study was also conducted to identify risk factors in dengue fever and dengue hemorrhagic fever as well as predictors of death among dengue fever cases for statistical analysis. antimicrobial resistance and infection control 2017, 6(suppl 2):he9 the importance of influenza is its rapid spreading of the epidemic in a wide range and serious complications, especially bacterial and viral pneumonia. taiwan medical and public health system experienced huge impacts of such epidemics. our study integrated epidemiological results to improve the effectiveness of influenza surveillance and assess the effectiveness of health policies for influenza prevention. we retrospectively reviewed recorded data of patients admitted in the intensive care unit who were diagnosed as severe influenza with respiratory failure during january 1, 2015 to december 31, 2015. we also recorded their clinical outcomes and history of influenza vaccine administration. we analyzed their prognosis, predictors of death and their possible correlations. sensitivity analysis was performed to assess the severity of patients admitted in the icu. mechanical ventilation was estimated for epidemiological week of hospitalization. these patients had low proportion of treatment with antiviral medication in the first period of the study. influenza deaths were increased. it may contributed from aging of the population, underestimated the needs for better prevention modalities, including more effective vaccines and vaccination programs for elderly persons. ) . moreover, the length of stay in the hospital of the study group was significantly higher than those in the control group (p < .05). controlling and preventing cre infection in medical ward and surgical ward were important and necessary. especially among patients who were admitted in the hospital for longer than 4 weeks. antimicrobial resistance and infection control 2017, 6(suppl 2):he13 background newborn could be seen as patients who are potentially exposed to ventilator associated pneumonia (vap) at high risk. identify the incidence as well as risk factors for vap in newborn would be much necessary for prevention application. this study aimed to identify incidence and risk factors of vap in nicu at the national children's hospital (nch) in vietnam. this cohort prospective study was conducted between january and december in 2012. after univariate analysis, multivariate regression analysis was used to handle all statistically significant risk factors. and bactec fx were used to identify the total blood volume that was obtained. an average blood volume was 3.80 ml. and the positive rate of blood culture was 10.4%. the emergency room had a lowest mean blood volume (2.20 ml) with a positive blood culture rate of 10.4%. the surgical intensive care unit had a highest mean blood volume (5.70 ml) with a positive blood culture rate 15.6%. in addition to a number of blood culture set, amount of blood taken for blood culture plays an important role for the positive rate. according to the clsi m47 blood culture guidelines, the recommended blood volume taken from adult is at least 10 ml per bottle. the recommended blood volume from the blood bottle company is 8-10 ml. adherence to the clsi recommendation could improve the positive rate, which would result in reduction of antibiotic overuse, reduction of hospital expenditure as well as improvement of quality of care. this study was conducted between march and october 2016. a total of 30 stool samples were transferred using 95% etoh as a transport buffer and were processed by using the chromagar as a selective media. toxin screening was performed by using the multiple step eia kit as. finally, we used the pcr methods to validate the final result. before this process, the culture positive ratio was very low (7%) in our hospital. the cdi ratio is only 69/100,000 patients while the cdi ratio of other medical in taiwan is more than 100/100,000 patients. after implementation the new diagnostic scheme, the culture positive ratio increased to 26% and the cdi ratio was higher than before. we also performed the gdh eia test which revealed the similar results to the culture methods. our study confirmed that the gdh eia may be an alternative option for diagnosis of cdi. furthermore, use of 95% etoh as a transport media would improve the cdi ratio. antimicrobial resistance and infection control 2017, 6(suppl 2):m9 background chlorhexidine gluconate (chg) is a cationic bis-biquanide biocide with low mammalian toxicity and broad-spectrum antibacterial activity. positively charge of chg molecules bind to negatively charge of phospholipid in bacterial cell wall coursing membrane disruption. incompatible of anionic thickener in lubricating gel formulation can negate the efficacy benefits of chg. this study was carried out to investigate the in vitro chemical and biological incompatibilities of marketed lubricating gels with chg solution. five marketed lubricating gels containing anionic thickener (carbomer) and two marketed lubricating gels containing nonionic thickener were collected to investigate for in vitro kinetic chemical and biological incompatibilities with 2%w/v chg in water. the contact time was 15, 30, 45, 60, 90, and 120 seconds. the results demonstrated that, five marketed lubricating gels containing carbomer reduced the amount of chg (32.5 -45.5%), which was measured by reversed-phase hplc technique and other two marketed lubricating gels containing nonionic thickener maintained the amount of chg. the antimicrobial activity revealed that 2%w/v chg solution achieved the 5.69 and 5.45 log 10 reduction for staphylococcus aureus atcc25923 and escherichia coli atcc25922, respectively. the antimicrobial activity was dramatically reduced to the 1.40 -2.70 log 10 reduction for s. aureus atcc25923 and 1.02 -2.11 log 10 reduction for e. coli atcc25922 after 30 seconds mixed with five marketed lubricating gels containing carbomer. two marketed containing nonionic thickener maintained the antimicrobial activity of 2%w/v chg solution. it can conclude here that, anionic thickeners in lubricating gel formulation may neutralize positively charge and reduce the antibacterial activity of chg. mobile phone serves as a major reservoir of pathogenic microbes and may act as major carriers for transmission of pathogens from laboratories. the aim of the study is to determine pathogenic bacterial contamination on mobile phones using by medical technology students in clinical microbiology laboratory practices. bacterial contamination were isolated and identified from overall surface of 50 mobile phones after touching by medical technology students before and during clinical laboratory microbiology practice. species of pathogen contaminated on mobile phones used by 54% (27 from 50) of students were identical with the pathogenic organisms that were given for practice in each student. a total of 105 bacterial isolates were found on student's mobile phones collected before practice. these were s. epidermidis 92%, bacillus spp. 90%, mssa 12%, gnf 10%, s. saprophyticus 2%, micrococus spp. 2%, enterococcusspp. 2%. sample collected during clinical microbiology practice found 122 bacterial isolates these isolates were identified as bacillus spp. 90%, s. epidermidis 88%, mssa 22%, gnf 16%, p. vulgaris 4%, k. pneumoniae 4%, e. cloacea 4%, e. aerogenes 4%, s. rubidaeae 4%, s. saprophyticus 2%, s. marcescens 2%, p. agglomerans 2%, c. freundii 2%, s. paratyphi a 2%. significant higher numbers of bacterial isolates (p < 0.05) obtained from student's mobile phones during clinical microbiology laboratory practice were observed in comparison with those obtained before practice. these results indicated that mobile phone can act as a mobile reservoir for laboratory and hospital infection. therefore, mobile phone using should be prohibited during clinical laboratory practice. in the additional; mobile phone decontamination and hand washing; should be strictly included as essential tools for laboratory safety and infection control. antimicrobial resistance and infection control 2017, 6(suppl 2):nt2 background universal prophylaxis and preemptive therapy are used to prevent cytomegalovirus (cmv) disease after transplantation. however, the optimal strategy has not been identified. this study aimed to evaluate optimal prevention strategies, particularly in high-risk recipients (donor-positive/recipient-negative [d+/r-] cmv serostatus pairs). we studied 118 recipients (21 high-risk, 97 intermediate-risk; median follow-up >1,100 days). asymptomatic cmv infection and cmv disease developed significantly more frequently in high-risk than in intermediate-risk patients (38.1% vs. 16.5%, p = 0.04; 28.6% vs. 3.1%, p < 0.01, respectively). a higher peak cmv antigenemia titer (112 vs. 19 cells, p < 0.01) and a longer duration of antigenemia (28 vs. 16 days, p = 0.02) were observed among high-risk patients. among high-risk patients, all cmv infection and cmv disease cases developed within the first 3 months and 9 months, respectively, after transplantation. survival analysis showed no significant difference in mortality rates (log-rank p = 0.63) and graft loss rates (log-rank p = 0.50) between the groups. graft rejection occurred more frequently among high-risk patients, but the difference was not significant (log-rank p = 0.24). graft rejection was significantly more common in patients who developed cmv infection/disease than in those who did not (logrank p < 0.0001), regardless of cmv serostatus. further studies comparing universal prophylaxis and preemptive therapy, particularly in high-risk patients, are required to identify the optimal strategy to reduce graft rejection rates. background japan is a natural disaster-prone archipelago. however few guidelines for public health activities have focused on infectious disease outbreaks during disasters. we have created the risk management guideline for infectious diseases during disasters to develop a comprehensive support system for prevention of infectious disease outbreaks in disasters. interviews were conducted with fifteen nurses who worked in any disasters occurred since the great hanshin-awaji earthquake in 1995. the interview data as well as published documents were analyzed. factors influencing infectious disease outbreaks were extracted as categories by six researchers. furthermore, measures were extracted while universalizing these measures. following categories were extracted: "restroom", "water management", "hygiene products", "hygiene behavior to be clean" "environment", "food hygiene", and "situations of infectious diseases". there were 38 measures in "restroom", 31 measures in "water management", 36 measures in "hygiene products", 30 measures in "hygiene behavior to be clean", 28 measures in "environment", 39 measures in "food hygiene", 52 measures in "situations of infectious diseases". measures could be visualized as a risk management guideline for infectious diseases outbreaks. this guideline is regarded as useful for preparing future disasters. however, it is necessary to consider that measures vary according to what kind of, where, and when disasters occur as the guideline was extracted from disaster experiences in japan. the rapid emergence of resistant bacteria is occurring worldwide. the prevalence of antibiotic resistance among the aforementioned bacteria has been increasing. this study was carried out to develop novel formulation against antibiotic-resistance microbes using for cross-transmission and cross-contamination control. the novel formulation was developed using quaternary ammonium compounds as major active ingredient and synergistic with three minor active ingredients. the antimicrobial activity against antibioticresistance microbes, methicillin-resistant s. aureus it can conclude here that, the novel formulation exhibited the potent antimicrobial activity against antibiotic-resistance microbes and also showed the safety in animal study. microbiology laboratory isolated ralstonia picketti in blood cultures from four patients who underwent hemodialysis on the same day at a same center, similar antibiogram of all these isolates lead to a suspicion of an outbreak. repeated blood cultures and the jugular venous catheter tip from one of the patient grew the same pathogen leading to the suspicion of this patient as the index case of the outbreak. routine cleaning, disinfection and sterilization of the water treatment system did not stop further patients from experiencing rigors and infection with the same pathogen. no new patient was identified after the cessation of dialysis reprocessing in the center. this confirms ralstonia pickettii has infected the dialysis reprocessing system from the index case that was dialyzing via a jugular catheter, and subsequently, infecting other patients in the center. ralstonia pickettii can be encountered in dialysis facility through contamination of water treatment and reprocessing system, resulting in outbreaks. early identification of pathogen is essential to prevent escalation of outbreaks in dialysis centers. antimicrobial resistance and infection control 2017, 6(suppl 2):oi2 klebsiella pneumoniae are important pathogen in healthcareassociated infections. therefore, carbapenem-resistant klebsiella pneumonia (crkp) causes infections with high mortality rates, which the ratio of increases in the recent years. this study aimed to investigate the epidemiological characteristics of crkp in a tertiary hospital in beijing and seek for control strategy. retrospective survey and temporal-spatial distribution analysis were used to crkp cases newly diagnosed from jan to april, 2016 and pulsed-field gel electrophoresis (pfge) was used to analyze the homology of crkp isolates. in the 25 crkp cases, the median duration of hospitalization within 90 days before detected was 27 days (5-90 days), the duration of using carbapenems was a median of 12 days (0-43 days), 10 cases had history inpatient of other hospitals before admission. the major sources of specimens with crkp positive were blood, respiratory tract and urinary tract, with a positive rate of 32%, 32% and 20%, respectively. homology test was performed in 20 crkp cases from 11 wards. mlst analysis showed that 19 cases were all st11 subtype producing kpc-2 enzyme; pfge results showed that 8 cases were type 1, 10 cases were type 2, with 2 unclassified. temporal-spatial distribution of type 1 and type 2 cases suggested that there might be contact transmission between cases. it is necessary to establish regional multidrug-resistant organisms monitoring and tracking network and information should be shared between departments and hospitals during patient referral, which was the key strategy for crkp prevention and control. cgmlst, wgmlst and pangenome typing showed ultimate discriminatory power. concordance between different typing methods were poor at type level, excluding wgmlst-cgmlst. on cluster level, the agreement was better achieving 85% and more. antimicrobial resistance and infection control 2017, 6(suppl 2):oi8 background multilocus sequence typing (mlst) as a genotyping methods has been extensively used for study of evolution and population dynamics of s.aureus. the knowledge about genetic distances of genomes within and between mlst types estimated on wgmlst can be useful for understanding the population structure.we explored the variation within and between mlst types of global collection of s.aureus using wgmlst typing. materials and methods 6645 s.aureus genomes from genbank were analyzed with seqsphere + to determine mlst and wgmlst types. data with more than 10% missing values were excluded from analysis. genetic distance was calculated with nei formula (neim., 1972) using wgmlst data with 2103 loci. eight mlst types including 5591 s.aureus were determined: st5 (n = 1673), st8 (n = 1392), st22 (n = 987), st398 (n = 632), st105 (n = 534), st36 (n = 161), st239 (n = 128) and st609 (n = 84). the mean of variation within mlst type was 2.9% (95%ci;0.9-5.0). in some sts, such as st239, st5, st8, the genetic distance reaches 15.8%, 17.4% and 21%, respectively. in general, the difference between isolates from different sts ranged 65-92%. however, in some cases, the difference between s. aureus from different mlst types were much lower -28% (st239-st609), 14% (st8-st239), 11% (st8-st609) and 5% (st5-st105). analysis of global collection s.aureus isolated from different sources and places demonstrated high genome difference between mlst types differing by 6 alleles. while genetic distances within and between closest sts might be overlapped what makes impossible to find appropriate cut-off for s.aureus clustering using wgmlst data to differentiate mlst with slv adequately, at least in some cases. antimicrobial resistance and infection control 2017, 6(suppl 2):oi11 in june 2015, three nurses had flu-like symptoms in our medical, surgical and neurological wards within couple days. we started outbreak investigation in medical, surgical and neurological wards. we identified 9 nurses with influenza a infections. after investigation, we found that compliance of wearing surgical masks while caring patients among healthcare providers is adequate but the compliance of hand hygiene is inadequate. furthermore, they neglected their flulike symptoms and did to practice good respiratory hygiene or cough etiquette. although these healthcare providers are able to follow the infection control standards while taking care a patient, they fail to follow the rule while contacting with staff members. these resulted in outbreak among healthcare providers. the outbreak was successfully controlled by introducing fever surveillance, advocating respiratory hygiene and cough etiquette and reinforcing environmental clearness. of these findings, enhanced hand hygiene and associated infection control strategies should be implemented to effectively reduce the transmission of type a influenza. background this research and development was a cohort study from 2013 to 2015. firstly, varicella disease had impacts on hospital administration: 1) unable the ward having patient(s) who had varicella infections to admit new patients, 2) infected non-immunized hospital staff. secondly, the disease interfered normal hospital services: 1) the affected ward was unable to admit new patients for 4 months because it contained the infected patients 2) an affected ward increased amount of new patients in others under limited resources of staff and facilities. in consequences, it increased patients' complains on hospital services. this study aimed to develop a guideline for controlling an outbreak of chickenpox in in-patient department and to evaluate the use of this guideline. this study was separated into 3stages: 1) isolating patients having varicella infections, prohibiting admission of new patients in the units and evaluated contact persons by testing for varicella immunoglobulin g (igg), 2) isolating patients having varicella infections but allowed admission of new patients into the units, evaluated 275 contact persons by testing for varicella igg and used protective isolation room for patients who were at risk for acquiring the disease and 3)isolating patients having varicella infections, allowed admission of new patients, evaluated 143 contact persons by testing for varicella igg, used protective isolation room for high-risk patients and vaccinated individuals who had negative results for varicella igg. no additional patients having varicella infection were found even though the hospital did not stop accepting new patients. the study is in progress to create screening criteria to identify patients having varicella infection before hospitalization. infection control personnel had identified this event through routine surveillance, and implemented some infection control measures immediately. the interventions included the following points: (1) cohort care for the infected patients was introduced; (2) daily environmental cleaning with 5000 ppm bleach was implemented; (3) the other patients were followed with active screening for crkp colonization; (4) environment and healthcare workers were screened for crkp colonization; (5) healthcare professionals were educated for contact precautions and hand hygiene. among 138 swab cultures from rcc environment, crkp were isolated from a bedside table and a physiological monitor of rcc05 and an infusion pump of rcc15. we collected 12 samples of hcws' hands and found crkp from one nurse, who was a key nursing staff of rcc05 patient. anal swab screening in contact patients showed two of crkp colonization that contacted withrcc05 and rcc15 patients. the study showed that poor compliance of hand hygiene among staff and wards' environment contamination was the reasons for this outbreak. the implementation of hand hygiene and ward environment cleaning and disinfection along with other infection control interventions were very important. however, this incident has not yet been ceased. ongoing monitoring and enforcement of infection control measures are still required. background incidence of clostridium difficile infection (cdi) in taiwan has not been widely studied, because the yield of culture based diagnostic test was low and the clinical characteristics of cdi had limited studies in the past. however many studies found the incidence of cdi increased in recent years. this study aimed to analysis on patients clostridium difficile infection to prevent cross infection. the following data was retrospectively reviewed; clinical evaluation, operational definition/diagnosis, diagnostic test using rapid molecular detection of toxigenic c. difficile. the medical records was reviewed and collected in case assessment form, which included underlying chronic diseases, past medical history, history of hospitalization, recent operation, past and current medication, particularly antibiotics use, and nutritional status. one-year incidence in our hospital was 18.1%. of the patients with cdi, 4 patients underwent chemotherapy, 1 patient had intestinal surgery, 2 patients were on hemodialysis, and 1 patients had diabetes mellitus and long-term use of steroids. all patients were hospitalized within 90 days, and received antibiotics. the medication duration was 1-2 weeks. the hospitalization time was 8-14 days. the mean age of patients was 60 years old. the development of rapid and reliable diagnostic tools is important to identify cdi. effective treatment, prevention strategies are needed to control this rising infection. antimicrobial resistance and infection control 2017, 6(suppl 2):oi15 in september 2015, the infection control nurse at a tertiary care hospital, northern thailand was notified of an influenza-like illness (ili) outbreak among the health care workers (hcws) in a medical record department. we conducted an outbreak investigation to determine the etiologic agent, identify additional cases, and implement control measures. an outbreak investigation of respiratory infections among hcws in a medical record department was promptly initiated. hcws with influenza ili were enrolled during august -september 2015. respiratory swabs were collected fromthe hcws with ili andwere tested by realtime reverse transcriptase polymerase chain reaction (rt-pcr) for respiratory pathogens. mycoplasma pneumoniae is a significant cause of respiratory disease in this outbreak. the infection control team alerted hcws to the outbreak and recommended prevention measures. mycoplasma pneumoniae should be considered as a possible cause of respiratory disease in hcws in outbreak setting. morbidity from outbreaks in hospital might be minimized if facilities closely monitor for respiratory disease clusters, promptly report outbreaks to infection control team, prioritize diagnostic testing in outbreak situations, and timely implement strict infection control measures to halt transmission. background surgical site infections (ssi) following coronary artery bypass graft (cabg) procedures is a devastating complication with significant morbidity, mortality, and extra-cost. this study aims to describe the incidence of ssi and causative pathogens following cabg surgery in a university hospital during 2013-2015. hospital records and post-discharge surveillance data between 2013 and 2015 were reviewed. ssi was diagnosed according to the us-cdc's surveillance definitions. there were 682 patients undergone cabg surgery. of these, 54 (7.92%) had ssi; 23 at sternal sites and 31 at leg harvest sites; 52 classified as superficial ssi and 2 as deep ssi. this prevalence is higher than what have been reported. in this study, proportion of grampositive and gram-negative bacteria was 11% each. of 54 cases with ssi, 3 cases had polymicrobial infection. gram-positive bacteria included coagulase negative staphylococci 18.2%, enterococcus spp. 13.6%, and staphylococcus aureus 9.1%. gram-negative bacteria were escherichia coli 13.6%, klebsiella pneumoniae 4.5%, enterobacter cloacae 4.5% and pseudomonas aeruginosa 27.3%. there was no difference in the prevalence of infection among surgeons. one-third of patients received prophylactic antibiotic very closely to operation, (≤30 minutes) and it was discontinued mostly within 48 hours after surgery, while 8.1% of all patients received antibiotic for longer than 48 hours. post cabg ssi was high in this study, probably related to inappropriate administration of prophylactic antibiotic. in-depth study is needed to delineate other risk factors which could affect the implementation of preventive strategies. the 4-dose vial (with preservative) of pneumococcal non-typeable haemophilus influenzae protein d-conjugate vaccine (phid-cv, gsk vaccines) was developed to improve logistics of and adherence to immunization programmes. the aim of this study was to assess reactogenicity and safety of the investigational phid-cv 4-dose vial presentation in infants. in this phase iii, mono-centre, observer-blind study (nct02447432) conducted in bangladesh, 6-10-week-old infants, randomized 1:1, received primary vaccination with either phid-cv 4-dose (4-dose group) or phid-cv 1-dose vial (preservative-free, 1-dose group) at 6/ 10/18 weeks of age. dtpw-hbv/hib and polio vaccines were (co)-administered at 6/10 weeks of age. solicited and unsolicited adverse events (aes) within 4 and 31 days post-vaccination, respectively, and serious aes (saes) throughout the study were assessed. the total vaccinated cohort comprised 160 infants in each group. the most commonly reported phid-cv injection site and general solicited aes were pain (after 13.1% and 13.4% of doses in 4-dose and 1-dose groups, respectively) and irritability/fussiness (after 23.3% and 26.6% of doses, respectively). the overall/dose incidences of other injection site (redness and swelling; 2.4%-5.9%) and general aes (drowsiness, loss of appetite and fever [axillary temperature ≥37.5°c]; 10.0%-15.6%) were within similar ranges between groups. unsolicited aes were reported after 2.1% and 3.7% of doses, and saes were reported for 4 and 9 infants in 4-dose and 1-dose groups, respectively, none was considered as vaccination-related. two infants died (sepsis, 4-dose; septic shock, 1-dose). phid-cv 4-dosevial presentation (with preservative) had an acceptable reactogenicity and safety profile, similar to phid-cv 1-dose vial (preservative-free) in infants. immunogenicity of 4-dose vial presentation of pneumococcal nonantimicrobial resistance and infection control 2017, 6(suppl 2):o2 to facilitate multi-dose use, gsk vaccines developed the pneumococcal non-typeable haemophilus influenzae protein d-conjugate vaccine (phid-cv) 4-dose vial, which contains preservative. the aim of this study was to demonstrate non-inferiority of the immunogenicity of investigational phid-cv 4-dose versus licensed 1-dose vial presentation in infants. in this phase iii, observer-blind study (nct02447432) conducted in bangladesh, 6-10-week-old infants, randomized 1:1, received phid-cv primary vaccination at ages 6/10/18 weeks with either 4-dose (4-dose group) or 1-dosevial (preservative-free, 1-dose group). dtpw-hbv/hib and polio vaccines were (co)-administered at ages 6/10 weeks. immune responses (antibodies against pneumococcal serotypes [22f-elisa] and opsonophagocytic activity [opa]; anti-protein d antibodies [elisa]) were measured. non-inferiority of phid-cv 4-dose versus 1-dose for each vaccine pneumococcal serotype (vt) and vaccine-related serotype 19a (confirmatory objectives) in terms of antibody geometric mean concentration (gmc) ratios was assessed. of 320 vaccinees, 154 (4-dose) and 146 (1-dose) were included in the according-to-protocol cohort for immunogenicity. non-inferiority criterion (upper limit of 2-sided 95% confidence interval of the antibody gmc ratios [1-dose/4-dose] <2-fold) was met for each vt and 19a. for each vt, ≥97.9% of infants in each group had antibody concentrations ≥0.2 μg/ml, except for 6b (84.4%, 4-dose; 84.9%, 1-dose) and 23f (89.0%, 4-dose; 94.5%, 1-dose); for 19a, ≥80.1% of infants in both groups. opa for each vt and 19a, and anti-protein d responses were within similar ranges between groups. immunogenicity of phid-cv 4-dose vial (with preservative) was noninferior to 1-dose vial (preservative-free) in terms of antibody gmc ratios for each vt and 19a post-primary vaccination in infants. percutaneous injuries resulting from contaminated sharp devices among healthcare workers (hcws) is a major concern of staff safety in hospitals, with increased risk hiv, hepatitis b and c infections. percutaneous injuries were at top five risks in our hospital in 2010-2013, with >60% of injuries related to operating theatre (ot). we aimed to study the impact of a multi-interventional strategy on incidence of percutaneous injuries at a non-profit private care hospital in hong kong. canossa hospital (caritas) is a 148-bed non-profit private care hospital treating approximately 10,000 patients yearly with 400 hcws. between 9-10 incidents of percutaneous injury occurred annually in 2010-2013. from 2013, a multi-interventional strategy was introduced to reduce incidence of percutaneous injuries, including: identifying risks of bloodborne infections exposure, formulation of ward policy guidelines, staff education and training to create safe workplace environment, enhancement of reporting system, individual counseling by infection control nurse, provision of safety-engineered sharps devices (e.g. needle counter in ots), verbal-visual reminder (meetings, posters). compliance audits were performed in ots after implementation. number of percutaneous injuries declined from between 9-10 incidents in 2010-2013 to 5 and 3 incidents in 2014 and 2015 respectively. the proportion of injured staff showed an initial decrease of 50% (p = 0.1, chi-square test) in 2014 and then significant reduction of 70% (p < 0.05) in 2015. we achieved reduction in percutaneous injuries using multiinterventional strategy. implementation of guideline formulation, education, individual counselling after incident, verbal-visual reminders, safety devices and performance audits led to an improvement in hospital staff safety. surgical ward (30 cases). during this study period, 19.6% cases occurred sharp injury in relation to blood taking procedure (n = 61). waste collection is the second commonest situation for sharp injury at 13.8% of reports (n = 43) and 35 cases reported during disposing sharps into sharp bins (11.2%). doctors reported the highest incident with 126 cases (40.4%) while nurses reported 62 cases (19.9%) and various groups of hcws constitutes the remaining 39.7%. all hcws are at risk of sharp injury however certain groups are at higher risk. the rate of sharp injury is persistently high despite the ongoing education program for hcws in our institution. background influenza can be a serious disease, since health care workers may care for or live with people at high risk for influenza-related complications. this study aimed to increase staff vaccination rates in 2015 and to know whether the additional cost is serving its worth, which shows the incremental cost -benefit ratio of increasing vaccine coverage. in 2015, the team conducted unit by unit information campaigns through floor to floor dissemination of information about free vaccination in the employee health clinic, and lastly "i am asian" jackets were provided to staff who underwent the vaccination. in from 2015 to 2016, the infection prevention and control office conducted an outbreak investigation on the cases of vzv among healthcare workers. this involved contact tracing and review of medical history of the healthcare workers. a survey on the immunization history of all healthcare workers (hcw) was done. outbreak control was also recommended through vaccination of all exposed that have no active immunity on the virus. the calculated cost of the treatment for hcws was done. result of the investigation revealed that 18% of the cases are cross transmission from one healthcare worker to his other co-workers. most of the healthcare workers who are on the prodromal stage of the virus still reported to work. the cost of the treatment per personnel who have vzv was calculated at 23,400 php (479 usd) while the cost of the vzv is at 4,000 php (83 usd). among all hcws 68% have no active immunity to vzv while 60% have no vaccination to vzv. there is a need to strengthen vzv information campaign among hcw. mandatory immunization of all susceptible staff to vzv must be recommended and barriers to vaccination uptake among hcws must be studied. reiteration of the sick leave policy must be emphasized. o7 nursing experience with one postoperative oral cancer patient using transdisciplinary care model ching-i ting (alice3030301@yahoo.com.tw) chi-mei medical center intensive care unit, tainan, taiwan antimicrobial resistance and infection control 2017, 6(suppl 2):o7 oral cancer patients are susceptible to postoperative structural defects of tissues such as in the orofacial region and jaw bones, which cause changes in the facial appearance, defects of physical functions such as dysphagia, slurred speech and eating difficulties and consequent negative impacts on quality of life. each of these patients, therefore, represents a highly complicated case with multiple problems and requires collaboration between a wider range of specialty departments on case discussion and provision of care to reduce their postoperative complications and improve their quality of life. the healthcare provided to the patient and the family through the transdisciplinary care model included: (1) alleviation of wound pain for the patient; (2) planning of training sessions for rehab from dysphagia; and (3) continuing coaching for the patient's mother on caregiving for the patients at home. this paper, based on the reason for and the definition of transdisciplinary care, presents for the reference of clinical practitioners leaders of medical institutions a specifically addressed strategy of evidence-based transdisciplinary practice, integrating the care plans suggested by all the teams and providing healthcare and rehab plan that reduced and improved the patient's wound pain and dysphasia. the evidence-based clinical practice of transdisciplinary care is a key trend of care in medical environments. using transdisciplinary evidence-based care as the foundation helps healthcare professionals across disciplines to understand and respect the spirit of collaborative care and obtain best treatment strategies through communications, thereby providing good quality of care. the introduction of asean economic community (aec) and thai government's policy in creating an international center for medical treatment, there has been a significant increase in number of patients travelling into thailand for treatments. patients are travelling on commercial aircrafts as well as air ambulance when their conditions are urgent or critical. air ambulance is a well-recognized method of transportation by the insurance company which medical wings has been serving patient from around the world since 1999. at medical wings, we focus on safety, quality care and infection control. on occasion, we receive patient from facility which is not able to adequately assess infectious disease so our medical team are the first group to meet patient and preparation is the utmost important in a successful mission. we aimed to prevent the exposure of patients, visitors and healthcare workers to communicable or infectious diseases by stressing maintenance of sound habits in personal hygiene and individual responsibility in infection control, monitoring and investigating potentially harmful infectious exposures, providing care to personnel for work-related illnesses, identifying infection risks related to employment and instituting appropriate preventive measures. this study was performed between october 1st, 2015 and march 31st, 2016. the imu had 20 beds, 2 were single-bed isolation room but the remaining was in a common hallway divided into three 6-bed sections. space between each bed was only 90 cm. a total of 396 patients were observed. after the first three months to determine the baseline prevalence, we implemented the control measures including cohorting patients with xdr-ab in the same area, encouraging hand hygiene by providing alcohol hand rub solution at each bed and common areas in the ward, mandatory isolation gown and gloves for caring of patient with xdr-ab, improving environmental cleaning technique and frequency. the prevalence of xdr-ab was gradually decreased, from 16.47 episodes /1,000 patient-days in the first phase to 3.98 episodes /1,000 patient-days in the second phase. this study suggests that cohorting along with stringent infection prevention technique was effective in controlling spread of drugresistant organisms and it might be an option for the crowded patient care areas. descriptive statistics were used to analyze the data. the adherence rate to gloving was highest, followed by gowning and hand hygiene (81%, 56% and 51% respectively). hand hygiene was performed more after than before giving care to patients. although they wore gowns and gloves before patient care, hcws immediately removed gown and gloves infrequently afterward. forty percent of the hcws adhered to the guidelines in every step, whereas 14% did not adhere at all. nurses adhered most to the guidelines (72%). hcws agreed with the guidelines but were unable to adhere to the guidelines at every step because of the crowded environment and perceived work overload. results of this study suggested that adherence to the guidelines for contact precautions needs more cooperation among hcws and barriers to the adherence should be minimized to prevent widespread multi-drug resistant organisms, and thereby improving the quality of care. the gloves compliance rate was highest than gown and hand hygiene. overall compliance rate 40%. the study finding needs to be concerned by healthcare workers for improving the quality of care. the methods of study were used development and empowerment program (dep), which followed role and competency of advanced practice nurse (apn). data were gathered from medical and surgical departments from 1 january to 30 september 2015. there were 3 purposive selected groups: 1) case manager team, 2) medical and surgical nursing teams and 3) xdr infected cases. the research tools were: 1) dep, 2) data questionnaires, 3) the xdr ic practice observational forms and 4) the evaluation quality of xdr cases forms. data were analyzed using descriptive statistics and t-test. the results of study suggested that a model for nursing care should consisted of 3 components: 1) providing xdr infection control activities in process of care, 2) developed and empowered case managers in role and competency of apn and 3) feedback outcomes of caring and supporting system. after using dep, the xdr infection rate significantly decreased from 4.1 to 1.6 per 100 patient-days (p < 0.05). this study showed that using dep in case managers and nurses promoted participants learning in teams. the dep also aligned xdr caring processes with ic standard practices that resulted in decreased xdr infection rate in hospital efficiency. background transmission of multidrug-resistant organisms in hospitals has a direct impact on patients, health care personnel, the hospital, community, and the nation. this developmental research aimed to develop a clinical pathway for prevention and control of multidrug-resistant organism transmission in the medical department at a regional hospital. study samples included 131 health care personnel who worked in the medical department and related including: hemodialysis, echocardiogram, radiology and ultrasound, ct scan, mri, and stretcher. ten patients infected or suspected of multidrug-resistant organism infection at the medical department were included in the test. the process for developing was based on cheah's (2000) framework. three development steps were thus included: assessment and situation analysis, designing, and testing the implementation. the data collection instruments consisted of a demographic data record form and questionnaire assessing the opinions of health care personnel towards. the content validity of the questionnaire was examined by 5 experts and the content validity index was 0.90. data were analyzed using descriptive statistics and data categorization. the we demonstrated that our education-based intervention program was effective to reduce the rate of lcbi 2 among clbsi. this is primarily due to the lowering the risk of contamination of common commensal in the blood specimen. improved cauti data collection methods idc reminder system nurse-led protocol to empower nurses to remove catheters in simple cases using s hook to keep urine bag below bladder level during ambulation daily maintenance audit of idc care frontline staffs were regularly engaged and feedback gathered. the changes were modified, tested and improved after (plan-do-study-act) pdsa cycles. preliminary results in the pilot wing showed a reduction in cauti rate although it is still early to tell if results are sustainable. results on compliance to idc care have also been encouraging. we plan to test and spread the changes to other wards to reduce cauti rate in the hospital. getting feedback and buy-in from the ground helps in designing sustainable changes. antimicrobial resistance and infection control 2017, 6(suppl 2):p10 background ventilator-associated pneumonia (vap) with vap bundles, should result in dramatic reductions in the incidence of vap. compliance with the vap bundle has been most successful when all elements are executed together as a \"all or none\" strategy. vap increases patient mortality, hospital stay and medical expenses. according to the latest vap criteria of the american disease control agency, there are many difficulties and blind spots in the diagnosis of vap, and the low rate of vap in this hospital is expected to be improved by the study of vap low reporting rate. the study of the hospital vap low reporting rate of the underlying causes: first, the clinical symptoms and chest x-ray interpretation of the lack of uniform standards, lack of education and training and advocacy. second, the existing receiving process can not immediately grasp the patient condition. third, to strengthen the quality of x-ray film and rules to track. the team developed the criteria and reporting process for the diagnosis of high-risk vap patients by the centers for disease control, in order to facilitate vap bundle in the care of patients with high-risk vap infection. the rate of vap in our hospital increased from 1‰ to 2‰, but it was significantly lower than that of foreign vap. difficulty in diagnosing patients and blind spots in vap are also a major limitation for clinicians. of vaps have a low reporting rate. therefore, the proposed medical institutions to standardize the hospital medical behavior and infection control measures to cohesion team consensus. the cvc bundle was implemented on all patients admitted to the icu starting april 2013. data of crbsi rates from 2012 and 2015 were pooled to compare the differences between observed values and predicted values before and after the intervention. cvc bundle care was implemented since april 2016. our study showed that the central catheter bloodstream infection rate was 1.4‰ (p = .039), the ventilator-associated pneumonia (vap) infection rate was 2.0‰ (p = .72), the catheter associated urinary tract (ca-uti) infection rate was 2.35‰ (p = .78), and the icu infection rate was 5.10‰ (p = .025). the overall central catheter utilization rate was 58.88% (p = .086); the mean length of icu (intensive care unit) stay was 15.72 days, and the average length of a hospital stay was 43.57 days. the time sequence analysis model was adopted to analyze the catheter-related bloodstream infection rate in 2013. although it did not decrease, but only slowed down the trend of increase the crbsi incidence. to effectively reduce the catheter-related bloodstream infection rate is to persistently support the combination of cvc bundle care and to enhance the compliance of the policy. the clinical benefit of chlorhexidine gel in severe units was the use of chlorexidine gel containing chlorhexidine mouthwash compared with the use of oral disinfection can be reduced by about 42,250 yuan. nursing care hours, about 78.5 hours per month can be reduced. the density of vap was maintained at 0.64‰. the use of chlorhexidine gel for mouth disinfection 54% less than the cost of mouthwash and save 33% of the time, not only reduce the cost of medical expenses also save the number of clinical nursing work, effectively improve the medical quality. to investigate the feasibility and compliance of the male catheterization in clinical practice yan-ru chen, yu-hsiu lin chi mei medical center,liouying, tainan, taiwan correspondence: yan-ru chen (c89040507@yahoo.com.tw) antimicrobial resistance and infection control 2017, 6(suppl 2):p17 background urinary tract infections (utis) are one of the most common sites of health care-associated infection, accounting for about 40%, of which about 70% are related to the use of catheters. in our subacute respiratory care unit, 62% of the cauti cases were men, whereas those who performed the technique clinically were nurse practitioner, through clinical monitoring and found that nurse practitioner to implement this technology compliance rate of only 25%. the aim of this study was to assess the feasibility and compliance of catheterization among male patients with competition and questionnaires. the standard operating procedures of "male catheterization" were developed and practiced in clinical practice by means of practical operation and questionnaires. antimicrobial resistance and infection control 2017, 6(suppl 2):p19 nebulizers are widely used in clinical for treating the disease of respiratory tract, which can aerosolize medicine to 0.5-10 μm to reaching the lower respiratory tract, so as to induce expectorant action or treat the inflammatory of respiratory tract. however, nebulizers can be contaminated by the exogenous route owing to improper care, so that increase risks of oral colonization or respiratory tract infection. the previous study showed that suggestion about the frequency of replacing nebulizers and nebulizers practices were vary variable and inconsistent. the aim of this study was to evaluate the effect of replacing nebulizers (at 24, 48 or 72 hours) in contamination. a randomized controlled trial study is conducted from july 9, 2014 to february 2, 2015 in a surgical ward of a medical center in taipei. during the study period, include above 20 years old people with steam in halation treatment. after participants agree to join the trail, randomise cases into 24, 48 or 72-hour group. in the protocol, researcher collect culture of nebulizers after used. total 525 specimens of nebulizes are collected. the rate of contaminated nebulizers at 24 hours is 14.1%, which is significantly higher (p = 0.008) in 48 hours (24.3%) and 72-hour group (25.6%). multivariate statistical analysis show that steam inhalation increase by one day with the contaminated risk of nebulizers increased by 12% (95% confidence interval: 1.03-1.21, p = 0.003). the replacing nebulizers more than two days will increase contaminated rates significantly. therefore, health care workers should understand and comply with guidelines of steam inhalation clinical care to reduce the contaminated risk of nebulizer. educational programs increase the compliance of bundle care: experience in a regional hospital in taiwan antimicrobial resistance and infection control 2017, 6(suppl 2):p20 taipei veterans general hospital yuanshan branch is a 471 bed regional hospital that provides single veterans acute and chronic care. there is a team of care providers helping the nursing staff manage patient's daily care. urinary tract infections (utis) are common hospital-acquired infections accounting for 15% of hais. recent studies and guidelines suggested that multidisciplinary care bundle could effectively reduce the incidence of catheter-related urinary tract infections (cautis). the aim of this study was to evaluate the impact of educational programs for care providers on compliance of cautis bundles. the intervention study was conducted over a period of 3 years, between 2012 and 2015. during a pre-intervention phase, parameters of compliance including the correct rate of had hygiene and correct rate of aseptic techniques were collected and compared with those during the post-intervention phase. the educational programs included (1) posters reminders on the walls of bedside, (2) aseptic urine sampling and cleaning technique, (3) correct draining bag positioning, (4) frequent tests and feedbacks to supervisors, (5) hand hygiene. during the study period,a total of 21 care providers received the training programs. the pre-intervention phase was between 2012 and 2014, the post-intervention phase was 2015. the correct rate of hand hygiene increased from 72.7% to 95.2%; the correct rate of aseptic techniques increased from 54.5% to 92.8%. our investigation showed that implementing educational programs for care providers increased the compliance of bundle care of cau-tis. however, whether the improvement reduced the incidence of cautis needed further investigations. impact of implementing vap bundle: experience in a veteran's regional hospital in taiwan tzu antimicrobial resistance and infection control 2017, 6(suppl 2):p21 experience in a veteran's regional hospital is a 471 bed (in intensive care unit is a 10 bed), many critically ill patients need to rely on ventilator for survived. although preventable, ventilator-associated pneumonia (vap) is a main cause of death due to healthcare-related infection in the intensive care unit (icu). recent studies and guidelines suggested that multidisciplinary care bundle could effectively reduce the incidence of catheter-related infections. the aim of this study was to assess implementation of vap bundle, can be effective in preventing ventilator-associated pneumonia occurred. materials and methods vap bundle care was implemented in a medical icu of our hospital from march 2015 to march 2016. the contents of vap bundle checklist including daily (1) readiness-to-wean assessment daily, (2) diary "sedation vacation", (3) hand hygiene, (4) oral care every 12 hours, (5) head-of-bed elevation above 30 degrees. education, training, and regular feedback from external auditing units can encourage the clinical care team to improve implementation rates. the results showed a daily goal checklist implementation rate increase from 38% to 100%. the ventilator bundle compliance rate, assessed by external audit, increased from 62% to 98%. the ventilator usage rate decreased 8.1% after implanted vap bundle. the implementation of the vap bundle during 1 year. a record of 12-month period of zero vap case from march 2015 to march 2016 had achieved. our experience has shown that the implementation of vap bundle, can be effective in prevention ventilator-associated pneumonia occurred. in addition, staff education and regular feedback from external audit data can improve implementation rates. antimicrobial resistance and infection control 2017, 6(suppl 2):p22 background ventilator-associated pneumonia (vap) is a major healthcareassociated infection worldwide. the incidence of vap in our hospital was around 3 episodes/1000 ventilator-days for many years. this study aimed to reduce vap rates among patients in icus. this prospective study was done among mechanically ventilated adult patients in every icu between january 1st, 2014 and december 31st, 2015. a total of 40,601 ventilation days were observed. a 5element care bundle including daily assessment of readiness for ventilator weaning, hand hygiene, aspiration precautions, prevention of contamination and oral care using 0.12% chlorhexidine mouthwash were introduced to all icu. infection control nurses regularly visited and supervised the practice and periodic meeting among icu nurses were arranged. the adherence was monitored. the incidence of vap was gradually decreased, from 3.1 in 2013, to 2.8 and 2.5 per 1,000 ventilator-days in 2014 and 2015, respectively. the adherence rate to hand hygiene before tracheal suctioning was 44.7% at the beginning and 66.3% during the last period of the study while adherence to other measures were 93.4% throughout. this study suggests that participation and experience sharing among nurses, regular monitoring, and supervision significantly contributed to the reduction of vap. surveillance of data for post-craniotomy infection were retrieved from infection control unit of songklanagarind hospital. which surveillance system use the former national nosocomial infections surveillance system (nnis) risk index for ssi risk adjustment. the data included 2437 patients underwent 3250 brain surgeries in the hospital from september 2004 to june 2016. medical records were reviewed for additional information including emergency operation, traumatic brain injury, brain cancer, and duration of steroid administration. the predictive performance of the former nnis risk index, new nhsn model, and our proposed model was then compared by mean of area under receiver operating curve (auc). the study identified 157 operations with post craniotomy ssi while the former and new model predicted 64.3 and 174.9 ssis respectively. auc of the new model (56.3%; 95%c.i = 52.1-60.4) was significantly (p-value = 0.02) less than the former one (50.7%; 95%c.i = 46.2-55.2). we analyzed the data and construct a model which included brain cancer, duration of operation more than 3 hours, and contaminated or dirty/infected wound class. auc of our model was 59.8% (95%c.i = 55.7-63.8). the new cdc nhsn model is poor for risk stratification postcraniotomy ssi and may be improved by adding brain cancer variable into the model. antimicrobial resistance and infection control 2017, 6(suppl 2):p24 background catheter-associated urinary tract infections (cautis) are a common infection among elderly in long-term care facilities (ltcfs). nursing personnel play an important role in prevention of cautis. this study aimed to examine the effects of coaching on nurses' knowledge and practice regarding cautis prevention in elderly in ltcfs. a quasi-experimental study was performed during october 2014 to january 2015. the participants were 16 nursing personnel working in ltcfs. all samples attended the four steps of coaching intervention including 1) preparation; 2) discussion; 3) active coaching; and 4) follow-up. the research tools consisted of a demographic data questionnaire, a knowledge test, an observation form, a coaching plan, and a manual for prevention of cautis. data were analyzed using descriptive statistics, paired t-test, and chi-square test. all of participants were female. the mean age was 31.9 years old, and the mean working experience was 8.7 years. most participants had no experience in training of cauti prevention in elderly patients (87.5%). after coaching, average knowledge scores on prevention of cauti among nursing personnel increased significantly from 13.3 points to 19.6 points (p < .001) and the proportion of correct practices on prevention of cautis in nursing personnel was statistically higher than the pre-coaching rising from 74.3% to 98.0% (p-value < 0.001). the findings of this study suggest that coaching could enhance knowledge and practice skills in prevention of cautis among nursing personnel in the ltcfs. application of coaching may improve knowledge and practices in prevention of cautis among nursing personnel in other ltcfs. microbiome-directed therapies are increasingly used preoperatively and postoperatively to improve postoperative outcomes. recently, the effectiveness of probiotics, prebiotics, and synbiotics in reducing postoperative complications (pocs) has been questioned. this systematic review aimed to examine and rank the effectiveness of these therapies on pocs in adult surgical patients. we searched for articles from pubmed, embase, cochrane, web of science, scopus, and cinahl plus. from 2002 to 2015, thirty-one articles meeting the inclusion criteria were identified in the literature. risk of bias and heterogeneity were assessed. network meta-analyses (nma) were performed using random-effects modelling to obtain estimates for study outcomes. risk ratios (rrs) and 95% confidence intervals (cis) were estimated. we then ranked the comparative effects of all regimens with the surface under the cumulative ranking (sucra) probabilities. a total of 2,952 patients were included. we found that synbiotic therapy was the best regimen in reducing surgical site infection (ssi) (rr = 0.28; 95%ci, 0.12-0.64) in adult surgical patients. synbiotic therapy was also the best intervention to reduce pneumonia (rr = 0.28; 95%ci, 0.09-0.90), sepsis (rr = 0.09; 95%ci, 0.01-0.94), hospital stay (mean = 9.66 days, 95%ci, 7.60-11.72), and duration of antibiotic administration (mean = 5.61 days, 95%ci, 3.19-8.02). no regimen significantly reduced mortality. this systematic review suggests that synbiotic therapy is the best regimen to reduce ssi, pneumonia, sepsis, hospital stay, and antibiotic use. surgeons should consider the use of synbiotics as an adjunctive therapy to prevent pocs among adult surgical patients. increasing use of synbiotics may help to reduce the use of antibiotics and multidrug resistance. antimicrobial resistance and infection control 2017, 6(suppl 2):p26 the incidence of catheter-associated urinary tract infection (cauti) is a major healthcare-associated infection in ramathibodi hospital. the incidence was more than 10 episodes/1000 urinary catheter-day for many years. we studied the effect of the intervention programs to reduce the incidence of catheter-associated urinary tract infection. a quasi-experimental research was conducted in the surgical intensive care and medical step-down units. the baseline and intervention period was from january to june 2012; and from september 2012 to may 2013, respectively. the cauti bundle according to the cdc's guidelines recommendation was employed. we found non-significant reduction in the rates of cauti (15.83 versus 13.12 episodes/1000 urinary catheter-day) and urinary catheter utilization ratio (0.76 versus 0.67). removal of catheter was not successful in patients with urinary retention, which were major population in our study. multiple logistic regression revealed that duration of hospitalization >10 days (or 2.84, 95% ci = 1.35-5.97), duration of indwelling catheter >7 days (or 6.03, 95% ci = 2.85-12.75), and female gender (or 1.97, 95% ci = 1.12-3.49) were associated with a higher incidence of cauti in the medical step-down unit, while >8 days of hospitalization was associated with cauti (or 7.42,95% ci = 1.39-39.64) in the surgical intensive care unit. the most common organisms identified in both units were fungus and e. coli. prolonged catheterization is associated with higher incidence of cauti and early removal of catheter may not be achievable in certain group of patients. further study is needed to determine the optimum care of patients who need indwelling catheters. background surgical site infection (ssi) is the most common hospital acquired infection at obstetrics and gynecology hospital. ssi prolonged hospital stay and increased treatment costs. we aimed to identify incidence and causative organism of surgical site infection among cesarean section and gynecology surgery cases. materials and methods a prospective cohort study was conducted at hung vuong hospital, viet nam from january 2015 to may 2016. women who underwent caesarean and gynecological surgery were included in the study. cdc criteria (2009) were used to diagnose ssi. results 29,681 women underwent caesarean and gynecological surgery. ssi rate was 1.1% (95% ci: 0.98 -1.22%), in which 0.96% ssi after caesarean section (95% ci: 0.84 -1.08%) and 1.7% ssi after gynecological surgery (6.26% ssi after hysterectomy with 95% ci: 4.91 -7.79%). average number of treatment days that with ssi is 9 ± 4.5 days. staphylococcus aureus and coagulase-negative staphylococci were the most common organism caused ssi after caesarean section (50%) and escherichia coli was the main organism for ssi after gynecology operation (66.7%). the incidence of ssis was limited because we only follow patients with ssis who were treated in hospital. the incidence of ssis after hysterectomy surgery was higher than other types of gynecologic surgery. it's necessary to promote infection control to reduce ssi in hysterectomy surgery. guidelines for the prevention of new strains of influenza infections in employers and employees (update glaxosmithkline biologicals sa. acknowledgements glaxosmithkline biologicals s.a. a total of 93 participants were involved in this competition, with a rate of 96.88%. among them, 9 of them (9.68%) were not up to 60 points, and 3 of them were below 80% according to the technical project contents, respectively:1. pre-hand hygiene. 2. the first disinfection and wait disinfectant dry. 3. the second disinfection.after the competition, the questionnaire is investigated and analyzed as follows:1. professional nurse practitioner years: more than 5 years (36%) accounted for the highest. 2. 72% felt it was suitable for clinical practice. 3. difficult to implement technology: the proportion of the first disinfection accounted for the highest (52%), followed by wearing sterile gloves to no bacteria were connected to the catheter and urine bag (20%), and again the hand hygiene (16%). this technology is standardized and it is recommended to incorporate the new training content of the nurse practitioner. the impact of bundle care intervention reduce catheter-associated urinary tract infections (cautis): experience in a medical center in taiwan key: cord-022380-49oti4zg authors: panlilio, adelisa l; gerberding, julie louise title: occupational infectious diseases date: 2009-05-15 journal: textbook of clinical occupational and environmental medicine doi: 10.1016/b978-0-7216-8974-6.50026-9 sha: doc_id: 22380 cord_uid: 49oti4zg nan infections acquired in the work setting represent an eclectic group that is seldom, if ever, considered together as a single category. occupational infections involve several organ systems, respiratory, enteric, and skin infections being particularly common. transmission involves not only casual person-to-person contact, but also a variety of other routes in special work environments. it is thus important to consider what unique features characterize the infectious diseases that can be considered occupational. perhaps one useful way of thinking about these diseases is that, as a group, they tend to be transmitted during work schedules or practices that are systematized. therefore, they can be anticipated, and to the extent that unsafe infectionprone practices can be identified and modified, they can be systematically prevented. another common feature is that many of the occupational infectious diseases can be regarded as behavioral. to the extent that unsafe practices have been defined, and practice policies modified to reduce infection risk, continued transmission often represents failure to follow accepted standards. although certain occupational infections can be prevented by vaccines (e.g., hepatitis b), prevention often depends on simple behavioral changes, such as hand hygiene, use of gloves, and not working while ill. finally, the anthrax cases during the fall of 2001 in the united states demonstrate the possibility of intentional (and criminal) exposure to infectious agents in the workplace, in this instance among those handling mail. 1 these intentional exposures, fortunately, are rare events, but should be considered in assessing sources of exposure for unexpected illnesses. prevention depends primarily on defining risky occupational practices or environments, clearly articulating policies for preventing communicable disease acquisition; removing structural barriers to compliance with policies (e.g., providing soap, hand cleansers, and gloves; allowing time away from work during periods of illness); and promoting healthy practices through behavioral change. because infectious diseases may represent the most common cause of time lost from work, it is important for the clinician concerned with occupational medicine to understand the relationship of specific infections to specific work environments and practices, and to give at least as much attention to prevention as to diagnosis and treatment. occupationally acquired infections have historically been associated with animal exposures and unsanitary work environments. modernization of agrarian techniques and improvement in sanitation have markedly decreased the incidence of these infections in the developed world, though they remain problematic in developing countries. while nearly all infectious diseases could conceivably be transmitted in the workplace, the emphasis here is on those that can be transmitted by casual contact or by specific workrelated exposures, with emphasis on diseases that are most common, most serious, or most readily prevented. healthcare settings pose a unique challenge because of the proximity of infectious patients, susceptible patients, and healthcare personnel. infections transmitted from personnel may have devastating effects on certain groups of patients, particularly the immunosuppressed. likewise, certain infections transmitted to personnel, such as multidrug-resistant tuberculosis, may have serious or even fatal, consequences. table 22 .1 summarizes the microbial etiology, sources, routes of infection, categories of workers at risk, and clinical manifestations of selected infectious diseases that have occupational predilections. a detailed discussion of waterborne microbial diseases is also provided in chapter 54. because the treatment of occupationally acquired infections does not differ from that of infections acquired non-occupationally, 2 the emphasis of this chapter is on the recognition and prevention of these infections. etiologic category, as well as the potential for recurrences, account for the high incidence of the common cold, even among healthy adults. colds are more common in the fall, winter, and early spring, perhaps because of increased crowding among children during the colder seasons. workers are most apt to acquire colds from exposure to young children in the home. secondary cases among coworkers may then develop. adults experience two to four colds each year, although the incidence among adult women exceeds that of men by a small margin, and smokers have a substantially increased risk. the modes of transmission of cold viruses are not entirely elucidated. for rhinoviruses, transmission among experimental subjects occurs most readily by direct handto-hand contact, with a case followed by autoinoculation of the mucous membranes of the eye or nose. such finger-tomucous membrane contact is ubiquitous and unavoidable. other viruses are transmissible by aerosolized droplets. the importance of fomites (such as drinking glasses, telephone receivers, and shared office equipment) as vectors of transmission has not been determined. clinical manifestations typical cold symptoms include nasal congestion, coryza, non-productive cough, sneezing, pharyngitis, and laryngeal irritation. fever is often low grade, or may be absent. viral upper respiratory infections usually resolve within 7-10 days, but longer durations are not uncommon. treatment, prevention, and control treatment for uncomplicated infections is symptomatic. decongestants are more useful in relieving symptoms than are antihistamines. expectorants, saline gargles, and other nonprescription remedies are useful in some cases. antibiotics should not be prescribed for the treatment of colds. colds are difficult to prevent. a policy of work restriction until symptoms improve may prevent the spread of colds but is likely to be impractical (table 22. 2). the cost-benefit analysis of such an approach could be useful, especially in childcare and healthcare settings. hand washing after contact with nasal secretions may be helpful. care should be taken to use tissues when coughing or sneezing and to dispose of soiled tissues after use. epidemiology influenza is a self-limited respiratory illness caused by types a and b influenza virus. epidemics of influenza occur annually in the winter months. adults remain susceptible to the illness despite prior episodes of infection because the antigenic structure of influenza viruses changes frequently, leading to new epidemics. influenza is spread from person to person, primarily by the coughing and sneezing of infected persons or sometimes by direct contact, either with infected persons or a contaminated surface. the disease is easily transmitted, and a single index case may transmit to a large number of susceptible persons in a short period of time. adults and children typically are infectious from 1-2 days before through 5-6 days after the onset of symptoms. clinical syndromes an attack of influenza starts abruptly with fever, malaise, myalgia, and headache. respiratory symptoms mimicking those of the common cold and lower respiratory symptoms including dry cough also are frequent. fever resolves in uncomplicated cases in 48-72 hours, but other symptoms may persist for days to weeks. influenza pneumonia, associated with hypoxemia, cough, and interstitial infiltrates, is not common in healthy adults. elderly patients and those with underlying immunodeficiencies and chronic pulmonary diseases are at high risk for secondary bacterial pneumonias, often caused by streptococcus pneumoniae and less often by haemophilus influenzae and staphylococcus aureus. the diagnosis of influenza frequently is made on the basis of clinical symptoms and signs. however, influenza is very difficult to differentiate from respiratory illnesses caused by other pathogens on the basis of clinical symptoms alone. other pathogens that can cause similar symptoms include, but are not limited to, mycoplasma pneumoniae, adenovirus, respiratory syncytial virus (rsv), rhinovirus, parainfluenza viruses, and legionella species. many pathogens, including influenza, rsv, and parainfluenza, cause outbreaks in a seasonal pattern. laboratory confirmatory tests can be performed to differentiate influenza from other illnesses. appropriate patient samples to collect for laboratory testing can include a nasopharyngeal or throat swab from adults, or nasal wash or nasal aspirates, depending on which rapid test is used. samples should be collected within the first 4 days of illness. rapid influenza tests provide results within 24 hours; viral culture provides results in 3-10 days. most of the rapid tests are more than 70% sensitive for detecting influenza and more than 90% specific. because as many as 30% of samples that would be positive for influenza by viral culture may give a negative rapid test result, negative rapid tests should be followed by viral culture in a sample of the swabs collected. viral culture can also identify other causes of influenza-like illness when influenza is not the cause. serum samples can be tested for influenza antibody to diagnose acute infections. two samples should be collected per person: one sample within the first week of illness and a second sample 2-4 weeks later. if antibody levels increase from the first to the second sample, influenza infection likely occurred. because of the length of time needed for a diagnosis of influenza by serologic testing, other diagnostic testing should be used for rapid detection of possible outbreaks. during community outbreaks, specific virologic or serologic diagnosis is not necessary once the type(s) of influenza virus causing the outbreak have been identified. treatment, prevention, and control persons at high risk for serious morbidity (persons aged 65 and older, persons with chronic underlying diseases) should receive influenza vaccine annually. immunization also is recommended for healthcare personnel and others at risk for transmitting influenza to high-risk patients ( had been associated with reduced work absenteeism and fewer deaths among nursing home patients. 3, 4 most employers do not provide influenza prevention programs for workers outside the healthcare field. 3 amantadine and rimantadine can reduce the duration of uncomplicated influenza a illness when administered within 2 days of onset of illness in otherwise healthy adults. zanamavir and oseltamivir can reduce the duration of uncomplicated influenza a and b illness by approximately 1 day compared with placebo. none of these antiviral agents has been shown to be effective in preventing serious influenza-related complications. to reduce the emergence of antiviral drug-resistant viruses, the duration of therapy should typically be no longer than 5 days. both amantadine and rimantadine are indicated as prophylaxis for influenza a, but not for influenza b infection. oseltamivir has been approved as prophylaxis for influenza a and b. zanamivir has not been approved for prophylaxis, but has been shown to be as effective as oseltamivir in preventing febrile, laboratory-confirmed influenza illness. they are approximately 70-90% effective in preventing illness from influenza a. chemoprophylaxis can be a component of influenza outbreak control programs. 3 epidemiology the incidence of measles (rubeola) has steadily declined in the united states during the last decade and is no longer considered endemic. measles is a major cause of morbidity and mortality worldwide. the majority of cases in the united states in recent years have been imported or secondary cases epidemiologically linked to imported cases. 5 infected persons are highly contagious by the air-borne route during the viral prodrome, and when cough and coryza are prominent, until about 2 days after the rash appears. infection confers lifelong immunity. although most adults born prior to 1957 experienced childhood infection and are no longer susceptible, up to 10% may lack natural immunity. in recent epidemics, cases occurred among unimmunized children, as well as children and young adults who had received a single vaccination with live virus, and among older adults. 5 clinical syndromes measles progresses in several phases. initial virus replication occurs in the respiratory tract and leads to a primary viremic phase, which usually is asymptomatic. release of virus from infected reticuloendothelial cells produces secondary viremia and infection of the entire respiratory system, accompanied by symptoms of coryza, cough, and in some, bronchiolitis or pneumonia. koplik's spots, a bluish-gray enanthem most prominent on the buccal mucosa, precede development of the rash. in a typical case of measles, the rash begins on the face, then progresses to the trunk and distal extremities, and disappears in the same sequence after 5-6 days. treatment, prevention, and control live-attenuated vaccine for prevention of measles became available in the early 1960s. all healthy children should receive the vaccine at age 15 months. because 10% do not respond to a single dose of vaccine, a second dose is now recommended to improve vaccine efficacy. 7 all healthy adults born after 1957 who have not received two doses of live virus vaccine or have not experienced measles also are advised to receive vaccine (table 22. 3). persons who received killed vaccine have a risk of developing atypical measles, and require re-immunization with live virus vaccine. live virus vaccine is contraindicated in infants, pregnant women, and immunosuppressed persons. passive immunization with γ globulin is available for unimmunized persons exposed to infected individuals, but is not routinely recommended for adults. measles rarely can exacerbate tuberculosis and cause a temporary inhibition of delayed hypersensitivity. vaccine administration should be delayed for 1 month after tuberculin testing, and until treatment is under way in persons with active tuberculosis. epidemiology mumps is a viral illness transmitted by the oral or respiratory route during contact with contaminated fomites or aerosolized droplet secretions. mumps is less contagious than measles or rubella but produces significant morbidity, especially among adults. the incubation period ranges from 2 to 3 weeks. virus is detectable for 6 days prior to and 9 days after the appearance of symptoms. most adults are immune to mumps, but 10-20% of unimmunized adults have no serologic evidence of prior infection and are considered susceptible. mumps incidence is now very low in all areas of the united states. the substantial reduction in mumps incidence during the past few years likely reflects the change in the recommendations for use of measles mumps rubella (mmr*) vaccine. 6 clinical syndromes parotitis typically is bilateral, but unilateral disease and involvement of other salivary glands occurs in some persons. localized parotid tenderness and swelling, fever, and painful swallowing suggest the diagnosis. aseptic meningitis is common but benign. encephalitis is a rare but serious manifestation. about 25% of affected postpubescent men develop orchitis, epididymitis, or both, which is bilateral in 15% of cases and may be the sole manifestation of mumps infection. about 50% of cases of mumps orchitis result in testicular atrophy, but neither sterility nor impotence are common sequelae. oophoritis occurs in about 5% of women with mumps. vaccine separated by at least 1 month (i.e., a minimum of 28 days), and administered on or after the first birthday, are recommended for all children and for certain highrisk groups of adolescents and adults. 6 adult men and healthcare personnel with no history of mumps or mumps immunization should be screened for immunity and vaccinated if they are susceptible. immunization is contraindicated in persons with immunosuppression and in pregnant women (table 22. 3). passive immunization with mumps immune globulin decreases the incidence of orchitis and is recommended for mumps in adult men with a single testis. individuals with active mumps should be excluded from work until 9 days after the onset of parotitis to avoid transmission to others in the workplace (table 22. 2). epidemiology fifth disease, also called erythema infectiosum or 'slapped cheek disease', is an infection caused by parvovirus b19. it is a common rash illness that is usually acquired in childhood, but can be an occupational risk for school and childcare personnel. it has been transmitted to personnel in healthcare settings. clinical syndromes symptoms begin with mild fever and symptoms of fatigue. after a few days, the cheeks take on a flushed 'slapped' appearance. there may also be a lacy rash on the trunk, arms, and legs. not all infected persons develop a rash. the child is usually not very ill, and the rash resolves in 7-10 days. most persons who get fifth disease are not very ill and recover without any serious consequences. an adult who is not immune can be infected with parvovirus b19 and either have no symptoms or develop the typical rash of fifth disease, joint pain or swelling, or both. usually, joints on both sides of the body are affected. the joints most frequently affected are the hands, wrists, and knees. the joint pain and swelling usually resolve in a week or two, but they may last several months. about 50% of adults, however, have been previously infected with parvovirus b19, have developed immunity to the virus, and cannot get fifth disease. fifth disease is believed to be spread through direct contact, fomites, or large droplets. the period of infectivity is before the onset of the rash. once the rash appears, a person is no longer contagious. the incubation period is 4-14 days but may be as long as 20 days. treatment, prevention, and control symptomatic treatment for fever, pain, or itching is usually all that is needed for fifth disease. adults with joint pain and swelling may need to rest, restrict their activities, and take anti-inflammatory medications to relieve symptoms. transmission can be prevented by careful attention to hygiene, especially hand washing. no special precautions are necessary. excluding persons with fifth disease from work, childcare centers, or schools is not likely to prevent the spread of the virus, since people are contagious before they develop the rash. epidemiology pertussis, or whooping cough, is an acute infectious disease caused by the bacterium bordetella pertussis. pertussis continues to be an important cause of mortality in the united states. a dramatic decline in the incidence followed the widespread use of whole-cell pertussis vaccines in the mid-1940s. however, since the early 1980s, the reported pertussis incidence has increased cyclically with peaks occurring every 3-4 years. 7, 8 contributing to this increase in incidence is the waning of immunity over time following vaccination, particularly in older age groups. transmission most commonly occurs by contact with respiratory secretions or large aerosol droplets from the respiratory tracts of infected persons and less frequently by contact with freshly contaminated articles of an infected person. analysis of national surveillance data for pertussis during 1997-2000 indicates that pertussis incidence continues to increase in infants too young to receive three doses of pertussis-containing vaccine and in adolescents and adults. 7 clinical syndromes the incubation period of pertussis is commonly 7-10 days. pertussis begins insidiously with non-specific upper respiratory symptoms including coryza, sneezing, low-grade fever, and a mild, occasional cough, similar to the common cold. the cough gradually becomes more severe, and after 1-2 weeks, the second, or paroxysmal stage, begins. characteristically, the patient has paroxysms of numerous, rapid coughs generally with a characteristic high-pitched whoop, commonly followed by vomiting and exhaustion. the patient usually appears normal between attacks. older persons (i.e., adolescents and adults), and those partially protected by the vaccine may become infected with b. pertussis, but usually have milder atypical disease. pertussis in these persons may present as a more persistent cough of greater than 7 days duration, and may be indistinguishable from other upper respiratory infections. inspiratory whoop is uncommon. b. pertussis is estimated to account for up to 7% of cough illnesses per year in older persons. even though the disease may be milder in older persons, these infected persons may transmit the disease to other susceptible persons, including unimmunized or underimmunized infants. the medical management of pertussis cases is primarily supportive, although antibiotics are of some value, with erythromycin being the drug of choice. this therapy eradicates the organism from secretions, thereby decreasing communicability and, if initiated early, may modify the course of the illness. there is no pertussis-containing vaccine (including dtap) currently licensed for persons 7 years of age or older, and vaccination with dtap currently is not recommended after the 7th birthday. vaccine reactions are thought to be more frequent in older age groups, and pertussis-associated morbidity and mortality decrease with increasing age. studies are currently under way to determine if a booster dose of acellular pertussis vaccine administered to older children or adults may reduce the risk of infection with b. pertussis. this may in turn reduce the risk of transmission of b. pertussis to infants and young children who may be incompletely vaccinated. studies among older children, adolescents, and adults examining pertussis disease burden and transmission of disease to infants might guide future policy decisions on the use of acellular pertussis vaccines among persons more than seven years of age. currently, vaccination of children more than 7 years of age, adolescents, and adults is not recommended either routinely or as an outbreak control measure. in the future, licensure of pertussis vaccines for adolescents or adults may lead to new recommendations for the use of vaccines in outbreaks. 9 epidemiology most epidemics of bacterial pneumonia in the workforce are due to community-acquired infections. however, legionellosis is one type of pneumonia which can be transmitted in the workplace. legionella pneumophila is an important cause of both epidemic and endemic adult pneumonia, and it can be associated with outbreaks in the workplace. this organism colonizes aquatic ecosystems and potable water, and it is transmitted to humans by the air-borne route. contaminated air conditioners, humidifiers, and shower heads have been implicated in outbreaks among workers and hospital patients. outbreaks of legionellosis have occurred after persons have breathed mists that come from a water source (e.g., air conditioning cooling towers, whirlpool spas, showers) contaminated with legionella bacteria. persons may be exposed to these mists in homes, workplaces, hospitals, or public places. legionellosis is not passed from person to person. a careful occupational history should be obtained from all adults who present with pneumonia, because occupational exposures cause many otherwise rare pneumonias. public health authorities should be notified if an occupational source is suspected so that an epidemiologic investigation to identify transmission routes and other susceptible individuals may commence. clinical pneumonia syndromes community-acquired bacterial pneumonia usually is exhibited acutely, with fever, chills, productive cough, and often, pleurisy. chest examination demonstrates signs of consolidation that may be confirmed radiologically. sputum examination may aid implementation of empiric therapy by suggesting the etiologic pathogen. blood cultures should be obtained when invasive disease is suspected, and lumbar puncture to evaluate meningeal fluid is indicated when symptoms or signs of meningitis are present. patients with legionnaire's disease usually have fever, chills, and a cough, which may be dry or productive. some patients also have muscle aches, headache, tiredness, loss of appetite, and, occasionally, diarrhea. chest x-rays often show pneumonia but are not pathognomonic. it is difficult to distinguish legionnaire's disease from other types of pneumonia by symptoms alone; other tests are required for diagnosis. the definitive test is culture isolation of the organism in sputum, bronchoalveolar fluid, or pleural fluid. other useful diagnostic tests detect the bacteria in sputum by specialized stains, identify legionella antigens in urine samples, or compare antibody levels to legionella in two blood samples obtained 3-6 weeks apart. the time between the patient's exposure to the bacterium and the onset of illness for legionnaire's disease is 2-10 days. treatment, prevention, and control empiric ambulatory therapy of acute community-acquired bacterial pneumonia, not requiring hospitalization, should include coverage for pneumococcus and h. influenzae, if the patient has a history of chronic obstructive lung disease. amoxicillin, trimethoprim-sulfamethoxazole and cefixime are reasonable choices, unless atypical pneumonia caused by m. pneumoniae or c. pneumoniae is suspected, in which case erythromycin is preferred. erythromycin is the antibiotic currently recommended for treating persons with legionnaire's disease. in severe cases, a second drug, rifampin, may be added. preventing bacterial pneumonia is a difficult challenge. workers at risk for pneumococcal and haemophilus infections should be immunized, although the efficacy of this approach among patients at highest risk is debated (table 22. 3). influenza immunization could eliminate a major risk factor for both primary and secondary bacterial pneumonias. occupational exposures to potential pathogens should be minimized with proper ventilation. prevention of legionellosis is achieved by maintaining an environment that is not conducive to survival or multiplication of legionella. the necessary preventive measures may involve water treatment or modification of air conditioning and ventilation systems. 10 these preventive steps, which may be costly, should be directed at healthcare facilities, and occupational settings where cases have been identified. 11 epidemiology rubella (german measles) virus is transmitted person to person by mucosal exposure to infected droplets of respiratory secretions. since 1969, children in the united states have been routinely immunized against rubella at age 15 months, so that the majority of recognized cases today occur in adults and unimmunized children. since 1992, reported indigenous rubella has continued to occur at a low but relatively constant endemic level with an annual average of less than 200 rubella cases. 7 recent data indicate that the rate of rubella susceptibility and risk for rubella infection are highest among young adults. no large epidemics have occurred since the vaccine was licensed for use in 1969. however, outbreaks continue to occur among groups of susceptible persons who congregate in locations that increase their exposure, and among persons with religious and philosophic beliefs against vaccination. several recent outbreaks have occurred in workplaces where most employees are foreign born, particularly from latin america. 6 reinfection can occur following natural or acquired immunity, but it is usually asymptomatic and only rarely accompanied by viremia. rubella virus is shed from the respiratory tract of infected persons beginning 10 days before the development of rash and for several days after the rash appears. the onset of the rash coincides with the period of maximal contagiousness, and infected persons are not considered infectious for more than 7 days after the rash appears. infected infants shed virus for several months despite the presence of antibody. clinical syndromes adult rubella is often asymptomatic. symptoms occur 12-23 days after exposure. following a prodrome of fever and malaise, adults exhibit a maculopapular rash that begins on the face and extends downward, persists for 3-5 days, and often is accompanied by regional lymphadenopathy of the head and neck, which persists for days to weeks. one-third of adult women may develop arthritis in the fingers, knees, and wrists during the exanthematous phase of illness. children develop hemorrhagic complications more often than adults. in contrast, encephalitis, albeit rare, is more common in adults and is fatal in 20-50% of cases. maternal rubella infection acquired in the first 20 weeks of gestation frequently results in congenital rubella. the earlier in pregnancy rubella occurs, the more severe the fetal consequences. infection in the first trimester results in deafness, congenital heart disease, cataracts or glaucoma, endocrine abnormalities, and mental retardation in up to 60% of newborns. spontaneous abortion also occurs commonly. treatment, prevention, and control immunization of children and susceptible adults with live attenuated rubella virus effectively prevents rubella and accounts for the dramatic decline in the incidence of this disease in the united states. however, many adult women of childbearing age remain susceptible to rubella and require immunization prior to conception to prevent congenital rubella. the hemagglutination-inhibition serologic assay detects natural or acquired immunity. the advisory committee on immunization practices (acip) recommends screening of healthcare personnel who have not been vaccinated, and immunization of susceptible individuals. 7 complications of rubella vaccine occur among adults and include lowgrade fever, symmetric polyarthralgias, distal paresthesias, lymphadenopathy, and rash. vaccine is contraindicated in immunosuppressed persons and pregnant women. pregnancy should be avoided for 3 months after vaccination (table 22. 3). susceptible household contacts of infected adults and children pose a transmission risk in the workplace during the period of virus shedding, beginning about 10 days before the development of rash (about 1 week after exposure) until 7 days after rash appears. therefore, susceptible individuals should not report to work during this time interval (table 22. 2). epidemiology tuberculosis (tb) is caused by mycobacterium tuberculosis and, rarely today, by m. bovis. the incidence of tuberculosis (tb) in the united states declined steadily until the mid-1980s, but then sharply increased, especially in urban areas. the resurgence of tb in the united states in the late 1980s and early 1990s was associated with the emergence of multidrug-resistant tb (mdr-tb) and the hiv/aids epidemic. with this resurgence of tb in the united states came several high-profile nosocomial outbreaks associated with lapses in infection control practices and delays in diagnosis and treatment of persons with infectious tb, as well as the appearance and transmission of mdr-tb strains. since 1992, the declines in the overall number of reported tb cases, including the level of mdr-tb, appear to reflect successful efforts to strengthen tb control following the resurgence of tb and the emergence of mdr-tb. 12 activities emphasizing the first priority of tb control (i.e., promptly identifying persons with tb, initiating appropriate therapy, and ensuring completion of therapy) have been the most important factors in achieving this improvement. such activities reduced community transmission of m. tuberculosis, particularly in areas with a high incidence of aids. improvements in implementation of infection control measures in healthcare settings, concurrent with mobilization of the nation's tb control programs, succeeded in reversing the upsurge in reported cases of tb, and case rates have declined to their lowest levels to date. the threat of mdr-tb is decreasing, and the transmission of tb in healthcare facilities continues to abate due to implementation of infection controls and reductions in community rates of tb. nevertheless, some healthcare personnel are at risk for acquiring tb. pulmonary tb is most commonly transmitted in healthcare settings by inhalation of aerosolized droplet nuclei derived from the respiratory secretions of patients with active respiratory tb. close contact usually is required. most other categories of workers generally are not at risk without close and sustained workplace contact with a person who has active untreated disease. ingestion of unpasteurized milk from cows infected with m. bovis is no longer an important source of tb in most industrialized countries. clinical syndromes primary infection usually is asymptomatic in adults. teenagers and young adults are at higher risk for rapid progression to active disease, usually characterized by apical cavitary disease, than are older adults. primary infection in the elderly usually is exhibited as lower lobe consolidation with hilar adenopathy. primary tuberculosis in persons with advanced hiv infection is commonly symptomatic and progressive. once infection occurs, the organism may disseminate from the lungs to other sites, including the gastrointestinal and genitourinary tracts, and bone. normally, the infection is contained by the host's immune response at this stage. the risk for reactivation is highest in the first year after exposure and declines thereafter. however, aging and stressors such as immunosuppression, intercurrent illness, and chronic malnutrition may increase the risk for reactivation or dissemination of the disease later in life. clinically, reactivation tuberculosis usually is exhibited as upper lobe pulmonary cavitary disease, but virtually any organ system may be involved. treatment, prevention, and control tuberculin skin testing allows determination of prior exposure to tb in immunologically healthy adults, by assessing delayed hypersensitivity to tuberculin antigens using purified protein derivative. the tuberculin skin test (tst) is the only proven method for identifying infection with m. tuberculosis in persons who do not have tb disease. although the available tst antigens are neither 100% sensitive nor specific for detection of infection with m. tuberculosis, no better diagnostic methods have yet been devised. the preferred skin test for diagnosing m. tuberculosis infection is the mantoux test. it is administered by injecting 0.1 ml of 5 tuberculin units (tu) ppd intradermally into the dorsal or volar surface of the forearm. tests should be read 48-72 h after test administration, and the transverse diameter of induration should be recorded in millimeters. there are three cut-off levels recommended for interpretation of the tst results. 14 in hiv-infected persons, any reaction resulting in an induration larger than 5 mm is read as positive. among others, the presence of 15 mm or more of induration always indicates a positive test, 5-10 mm indicates a positive result in persons at risk for tb, and less than 5 mm is negative. a positive tst means an individual has been exposed to tb in the past and is at risk for reactivation. a baseline chest radiogram should be performed on all persons with newly diagnosed tst positivity. if the x-ray study suggests active disease, sputum samples should be obtained, stained for acid-fast bacilli, and cultured for mycobacteria. treatment should be implemented immediately if the index of suspicion is high. public health officials should be notified to institute case management and evaluation of contacts in the home and work environment. if the x-ray study is negative, treatment with isoniazid to suppress or eradicate latent organisms may be recommended, especially in persons younger than age 35 and for those who have recently converted to positive tsts. 13 although bcg vaccine is the most widely administered of all vaccines in the world, and has the highest coverage of any vaccine in the who expanded programme on immunization, it appears to have had little epidemiologic impact on tb. despite its shortcomings, and because of its beneficial effect in children and against leprosy, bcg vaccine likely will remain a component of childhood vaccination strategies in developing countries. however, because of questions about the vaccine's efficacy, and because it induces dermal hypersensitivity to purified protein derivative (ppd) tuberculin in most recipients, bcg has never been recommended for programmatic use in the united states. 14 healthcare providers should follow appropriate infection control procedures, including use of isolation rooms and respiratory protection, when caring for patients with active tuberculosis. 15, 16 varicella/zoster epidemiology varicella virus, the causative agent of chickenpox and zoster, is a highly contagious herpes virus spread by the respiratory route from person to person. the incubation period is about 14 days, and the period of infectivity begins a few days prior to the onset of the rash to about 6 days after the first crop of vesicles appears. immunosuppression usually prolongs the period of infectivity, especially if varicella zoster immune globulin (vzig) has been administered. zoster represents reactivation of varicella virus that is latent in sensory nerve ganglia, and it is not a manifestation of primary infection except in newborns infected in utero. the incidence of zoster increases with age and immunosuppression. susceptible persons in direct contact with zoster lesions risk developing primary varicella. in the prevaccine era, varicella was endemic in the united states, and virtually all persons acquired varicella by adulthood. as a result, the number of cases occurring annually was estimated to approximate the birth cohort, or approximately 4 million per year. this incidence has likely decreased since licensure of the vaccine in 1995. varicella is not a nationally notifiable disease, and surveillance data are limited. 17 clinical syndromes varicella in otherwise healthy children usually is a benign, self-limited disease characterized by low-grade fever and vesicular rash, often preceded by a viral prodrome. varicella vesicles of primary infection appear first on the scalp and trunk and disseminate in crops showing various stages of development over the next 3-4 days. healing results in crusting accompanied by intense pruritus. manifestations of varicella are more severe in adults than in children. about 15% of adults with varicella show radiographic evidence of pulmonary involvement, but this is rarely clinically significant. however, cough, tachypnea, and impaired gas exchange can occur and persist for months after infection. varicella during pregnancy can produce congenital varicella. in its most severe form, this infection can result in mental retardation, blindness, growth retardation, deafness, chorioretinitis, and a peculiar dermatomal lesion of the upper or lower extremity associated with limb atrophy. zoster, the most common manifestation of varicella infection among adults, characteristically produces unilateral vesicular eruptions preceded by pain in one to three dermatomes. disseminated zoster, which is more likely in immunosuppressed patients, probably poses the same risk of infection transmission as primary varicella infection. the major complication of zoster is postherpetic neuralgia, which is especially common in the elderly and may be extremely debilitating. zoster frequently produces cerebrospinal fluid pleocytosis and occasionally encephalitis. immunologically healthy persons may experience recurrences of zoster, usually in the same dermatome as the initial outbreak. zoster is a marker of deteriorating cellmediated immunity among hiv-infected patients, and it may disseminate. treatment, prevention, and control passive immunization with vzig is recommended for immunosuppressed susceptible persons, children with leukemia and other malignancies, and neonates exposed in utero within 5 days before delivery. 18 several antiviral drugs are active against varicella zoster virus, including acyclovir, valacyclovir, famciclovir, and foscarnet. 18 famciclovir and valacyclovir are approved for use only in adults. clinical studies indicate that these drugs may be beneficial if given within 24 hours of onset of rash, resulting in a reduction in the number of days new lesions appeared, in the duration of fever, and in the severity of cutaneous and systemic signs and symptoms. antiviral drugs have not been shown to decrease transmission of varicella, reduce the duration of absence from school, or reduce complications. oral acyclovir can be considered in otherwise healthy adolescents and adults or secondary cases in the household, because of the increased risk of severe illness in these groups. antiviral therapy may also be considered for persons with chronic cutaneous or pulmonary disorders, persons receiving long-term salicylate therapy, and for children receiving short, intermittent or aerosolized courses of corticosteroids. antiviral drugs are not recommended for routine postexposure prophylaxis. systemic steroids in older adults (more than 60 years old) may reduce the incidence and severity of postherpetic neuropathy if started early (within 5 days of skin manifestations). varicella has been difficult to prevent because of the high degree of contagion in households, schools, and healthcare settings. live attenuated virus vaccines have demonstrated efficacy in preventing primary infection, and one was licensed for use in the united states in 1995. routine immunization is now recommended for children less than 18 months of age. 17 varicella vaccination should be given to susceptible adolescents and adults who are at high risk of exposure to varicella. this group includes persons who live or work in environments in which there is a high likelihood of transmission of varicella, such as teachers of young children, residents and staff in institutional settings, and military personnel. varicella vaccination is also recommended for susceptible adolescents and adults who will have close contact with persons at high risk for serious complications of acquired varicella, including healthcare personnel and susceptible family contacts of immunocompromised individuals. the acip recommends that all healthcare personnel be immune to varicella, either from a reliable history of prior varicella infection or vaccination, to reduce the risk of infection and its complications, and to decrease the possibility of transmission of varicella zoster virus to patients (table 22. 3). susceptible adults exposed to children with varicella or with disseminated zoster pose a risk of transmitting varicella to non-immune coworkers, and they should not work until the incubation period is over or, if they become ill, until all lesions are crusted (table 22. 2). dermatomal zoster is not spread efficiently by the air-borne route and otherwise healthy adults afflicted with this illness may be allowed to work if they can avoid touching the lesions and contaminating the work environment. the role of vaccine in the postexposure management of susceptible employees needs to be elucidated. data from the united states and japan in a variety of settings indicate that varicella vaccine is effective in preventing illness or modifying the severity of illness if used within 3 days, and possibly up to 5 days, of exposure. acip recommends vaccine be used in susceptible persons following exposure to varicella. 17 personnel should be excluded from work who have onset of varicella until all lesions have dried and crusted (table 22. 2). following exposure to varicella, personnel who are not known to be immune to varicella (by history or serology) should be excluded from duty beginning on the 10th day after the first exposure until the 21st day after the last exposure (28th day if vzig was given). epidemiology acute gastrointestinal infection follows upper respiratory illness as the next leading category of infectious diseases causing absenteeism among adult workers. a wide array of pathogens, including viruses, bacteria, and protozoa, can result in acute infections of the stomach, small bowel, or colon. a comprehensive discussion of enteric pathogens is provided in chapter 54 (waterborne microbial diseases). most of the etiologic agents are acquired by the fecal-oral route; produce mild, self-limited diseases; and resolve without specific therapy. agents of dysentery (e.g., shigella spp.) often are highly transmissible through low-inoculum exposures. occupational transmission of food-borne or water-borne illnesses occurs; person-to-person transmission has propagated outbreaks of many of these illnesses in healthcare settings, daycare and nursery schools, and institutions where sanitation is poor. instances of such transmission have generally involved food handlers, who are often poorly trained and short-term employees, serving as sources of transmission to others. occupations requiring travel to countries with poor sanitation present a major risk for gastrointestinal infections. poultry workers are frequently exposed to salmonella infections. avoidance of oral contact with sources of fecal contamination is the most important strategy for preventing transmission of pathogens associated with intestinal infections. maintaining good personal hygiene, including careful hand hygiene after using restrooms and before food preparation; proper cooking and storage of foods; and avoidance of contaminated foods and water when traveling are essential prevention strategies. food handlers with diarrheal illnesses should not work until symptoms have resolved, and cure of bacterial infections should be documented by obtaining negative stool cultures more than 48 hours after antimicrobial therapy is completed (table 22. 2). the only vaccines for any of the etiologic agents for acute enteric infections are for typhoid and hepatitis a, which are recommended for personnel in laboratories who frequently work with salmonella typhi or hepatitis a virus (table 22. 3). epidemiology cytomegalovirus (cmv) is a ubiquitous herpes virus transmitted by direct inoculation with infected body fluids (including blood, blood products, respiratory secretions, saliva, and urine) and through sexual contact with infected partners. at least 40% of healthy adults have serologic evidence of prior cmv infection. infection can be acquired perinatally, in utero during maternal primary infection, during birth by passage through infected vaginal secretions, or through ingestion of infected breast milk. cmv is known to be highly transmissible in daycare centers and nursery schools. sexually active adults and recipients of blood products are also at high risk for infection. infants and young children excrete cmv in their urine, saliva, and respiratory secretions for several months after infection. virus is much less readily detected in healthy adults, but intermittent shedding has been documented. like all herpes viruses, cmv remains latent in the host after initial infection. previously infected persons may be reinfected with new strains of cmv. occupational transmission of cmv has been documented in childcare settings, where person-to-person spread through exposure to infected secretions and urine is believed to provide an efficient mode of transmission. up to 50% of seronegative workers in preschool daycare centers have acquired cmv infection in some studies, indicating a potentially serious risk to women of child-bearing age, because of the adverse effects of primary maternal cmv infection on the fetus. at one time, employment in healthcare settings also was believed to pose a high risk for cmv acquisition. however, epidemiologic investigations suggest that most infections in healthcare personnel are acquired sexually, or from exposure to young children in the home, and not from work-related contact. is asymptomatic in healthy persons. a self-limited mononucleosis-like illness occurs in a minority, which may be complicated by hepatitis, pneumonitis, hematologic abnormalities, and myocarditis. immunosuppressed children and adults with primary cmv infection, reactivation, or reinfection may develop severe sequelae. organ transplant recipients, hiv-infected patients, and persons with malignancies have a risk of developing cmv viremia, pneumonia, hepatitis, pancreatitis, enteritis, and retinitis. primary cmv infection at any stage of pregnancy carries a greater risk to the fetus than does recurrent cmv infection during pregnancy. symptoms of congenital cmv infection may be present at birth, and are due to the consequences of active virus replication and resultant end-organ damage. congenital cytomegalic inclusion disease, the most severe form of this entity, includes central nervous system disease, respiratory distress, hepatitis, hepatosplenomegaly, rash, and multi-system failure. infection acquired from exposure to cervical cmv during birth usually is asymptomatic and detected by the onset of virus shedding 4-8 weeks postpartum. with ganciclovir or foscarnet for cmv infection is reserved for immunosuppressed persons at high risk for severe complications. the safety and effectiveness of these agents in preventing congenital cmv have not been established. avoidance of mucosal contact with infected body fluids is the best strategy for preventing cmv transmission. hand washing after contact with secretions and fomites is essential, especially in nurseries and daycare settings. the presence of persons at risk for cmv shedding in the workplace does not pose a hazard to other employees unless direct contact with infected secretions is anticipated. isolation of infected neonates or children is not essential if hand washing is performed after contact with secretions, blood, and urine. pregnant healthcare providers compliant with hand washing protocols can generally safely care for patients with cmv infection. [19] [20] [21] no work restriction is necessary for individuals with cmv infection (table 22 .2). direct exposure to blood and other infected body fluids. children born to infected mothers are at high risk for hbv infection. persons parenterally exposed to blood, including multi-transfused patients, hemophiliacs, dialysis patients, and injection drug users, also are at significant risk. sexual contact with infected partners is another efficient mode of hbv spread. in most industrialized countries, adult infections usually are acquired sexually or by injection drug use. hbv is a relatively hardy virus capable of surviving on environmental surfaces and fomites. transmission in households is well documented and may, in part, be attributable to mucosal contact with fomites contaminated with secretions or blood from infected persons. healthcare personnel and others at risk for occupational blood exposure through percutaneous, mucosal, or dermal routes can acquire hbv infection. the risk associated with accidental needle-stick inoculation of infected blood to susceptible healthcare personnel varies between 5% to 35%, depending on the hepatitis b e antigen (hbeag) status, and hence the viral titer of the source. in up to 50% of occupational infections, a discrete exposure cannot be identified. hepatitis b has an incubation period of 40-180 days. the period of infectivity precedes the development of jaundice by 2-7 weeks and correlates with the presence of hepatitis b surface antigen (hbsag) in the serum; 5-10% of persons with acute (but often clinically silent) infection develop chronic antigenemia. in the united states, up to 1% of adults are carriers of hbv, and provide a reservoir for maintenance of the disease in the population. apparent hepatitis in about one-third of acutely infected adults. clinical hepatitis may be preceded by a prodrome of fever, malaise, urticarial or maculopapular rash, and arthralgias for several days. fever usually resolves before the onset of jaundice. jaundice, dark urine, and scleral icterus usually are present by the time patients seek medical attention. right upper quadrant tenderness, mild hepatic enlargement, and occasionally, splenomegaly are signs that should suggest the diagnosis. the most striking laboratory abnormality is the finding of extreme elevations in the aminotransferase enzymes. alanine aminotransferase (alt) and aspartate aminotransferase (ast) may be elevated to more than 10 times the normal levels, whereas the bilirubin and alkaline phosphatase levels are increased to a much lesser extent. fulminant liver involvement occurs in about 1% of adults and may be complicated by more serious abnormalities, including hypoglycemia, coagulopathy, and hypoalbuminemia. hepatic encephalopathy, hepatorenal syndrome, and bleeding diatheses are life-threatening complications seen in these patients. about 10% of adults with clinically apparent acute hbv infection proceed to chronic hbs-antigenemia, and are at risk for chronic hepatitis, postnecrotic cirrhosis, and primary hepatocellular carcinoma. patients with asymptomatic primary hbv infection are at higher risk for chronic infection than those with symptomatic infection. while chronic persistent hepatitis, a benign illness of little clinical consequence except for the potential for hbv transmission to susceptible individuals, may occur, the major health concern is chronic active hepatitis, which eventually may produce cirrhosis, liver failure, and hepatoma. hepatitis b is differentiated from other causes of hepatitis by serologic assays. 23 a positive hepatitis b surface antigen (hbsag) test identifies patients with current infection and correlates with infectivity during acute and chronic infection. titers of hbsag in the chronic phase of illness may wax and wane and occasionally fall below the limits of laboratory detection, so sequential testing should be performed if chronic hbv is suspected. the presence of hbeag correlates with active virus replication and is a marker of high infectivity and high titer of hbv in the liver and blood. antibody to hepatitis b surface antigen (hbsab) appears when hbsag is cleared and is positive in individuals with immunity after recent or prior infection or immunization. persons with hbsab are not susceptible to acute infection or chronic hepatitis b, except in the very rare case in which reinfection occurs with a strain of hepatitis b against which the normal antibody response does not provide cross-protection. hepatitis b core antibody (hbcab) appears before hbsab and just after hbsag is cleared from the serum, and this is a useful test for diagnosing acute hepatitis b in the window period before hbsab appears. high titers of hbcab persist in chronically infected persons and obviously do not predict immunity from further liver disease. there currently is no treatment for acute hepatitis b. alpha interferon and lamivudine have been licensed for the treatment of persons with chronic hepatitis b. these drugs are effective in up to 40% of cases. hbv infection is largely preventable. inoculation with recombinant vaccines containing hbsag components is safe and highly immunogenic, and appears to confer protection from infection for at least 12 years (table 22. 2). postvaccination testing should be done 1-2 months after completion of the three-dose series to document an appropriate response (i.e., > 10 miu/ml). 23 more than 90% of persons immunized with three properly timed doses (e.g., 0, 1, and 6 months) of vaccine administered intramuscularly in the deltoid region develop protective hbsab levels. factors associated with a lack of response include improper vaccination (improperly stored vaccine, gluteal inoculation, subcutaneous injection), obesity, older age, and smoking. persons who do not respond to the primary vaccine series should receive a second three-dose series or be evaluated for hbsag positivity. since 1982, substantial progress has been made toward eliminating hbv transmission in children and reducing the risk for hbv infection in adults. 24 recommendations of acip have evolved from universal childhood vaccination, to prevention of perinatal hbv transmission, vaccination of adolescents and adults in high-risk groups, and catch-up vaccinations for susceptible children in high-risk populations. 25, 26 the acip vaccination strategies for children and adolescents have been implemented successfully in the united states, and routine immunization of all children is now recommended. the occupational safety and health administration's (osha's) blood-borne pathogen standard mandates provision of vaccine at no cost to all healthcare employees and others at occupational risk for blood exposure. 27 substantial declines in the incidence of acute hepatitis b have occurred among highly vaccinated populations, such as young children and healthcare personnel. vaccine should also be provided to susceptible individuals before sexual maturity, particularly to teenagers in those settings (e.g., inner cities, concentration of poverty) where hbv is highly prevalent, and to all adults at risk for sexual or occupational exposure. preimmunization screening for evidence of prior or persistent infection usually is not cost effective. however, postimmunization testing for antibody response is recommended 1-2 months after the 3 rd dose to detect nonresponders among persons at high risk for exposure. titers of hbsab fall over time and may be undetectable after 5-10 years. the duration of vaccine protection is under investigation. most data suggest that protection persists even when hbsab titers fall below the level of detection, and routine screening and boosting are not recommended. the need for prophylaxis for persons sustaining accidental percutaneous or mucosal exposures to blood should be based on several factors, including the hbsag status of the source, and the hepatitis b vaccination and vaccineresponse status of the exposed person. such exposures usually involve persons for whom hepatitis b vaccination is recommended. any blood or body fluid exposure to an unvaccinated person should lead to initiation of the hepatitis b vaccine series. a summary of prophylaxis recommendations for percutaneous or mucosal exposure to blood according to the hbsag status of the exposure source and the vaccination and vaccine-response status of the exposed person is shown in table 22 .4. 23 when hepatitis b immune globulin (hbig) is indicated, it should be administered as soon as possible after exposure (preferably within 24 hours). the effectiveness of hbig when administered more than 7 days after exposure is unknown. when hepatitis b vaccine is indicated, it § hepatitis b immune globulin; dose is 0.06 ml/kg intramuscularly. ¶ a responder is a person with adequate levels of serum antibody to hbsag (i.e., anti-hbs ≥10 miu/ml). ** a non-responder is a person with inadequate response to vaccination (i.e., serum anti-hbs <10 miu/ml). † † the option of giving one dose of hbig and reinitiating the vaccine series is preferred for non-responders who have not completed a second three-dose vaccine series. for persons who previously completed a second vaccine series but failed to respond, two doses of hbig are preferred. § § antibody to hbsag. should also be administered as soon as possible (preferably within 24 hours) and can be administered simultaneously with hbig at a separate site (vaccine should always be administered in the deltoid muscle). for exposed persons who are in the process of being vaccinated but have not completed the vaccination series, vaccination should be completed as scheduled, and hbig should be added as indicated (table 22 .4). persons exposed to hbsag-positive blood or body fluids who are known not to have responded to a primary vaccine series should receive a single dose of hbig and reinitiate the hepatitis b vaccine series with the first dose of the hepatitis b vaccine as soon as possible after exposure. alternatively, they can receive two doses of hbig, one dose as soon as possible after exposure, and the second dose 1 month later. the option of administering one dose of hbig and reinitiating the vaccine series is preferred for non-responders who did not complete a second three-dose vaccine series. for persons who previously completed a second vaccine series but failed to respond, two doses of hbig are preferred. 23 states. it is estimated that 1.8% of americans have been infected with hcv. hcv-associated end-stage liver disease is the most frequent indication for liver transplantation among us adults. 28 the incubation period for acute hcv infection ranges from 2 to 24 weeks (averaging 6-7 weeks). hcv transmission occurs primarily through exposure to infected blood, such as through injection drug use, blood transfusion, solid organ transplantation from infected donors, unsafe medical practices, occupational exposure to infected blood, and birth to an infected mother (i.e., vertical transmission). hcv may also be acquired through sexual contact, but the importance of this mode of transmission in the united states is not well characterized. hcv is not transmitted efficiently through occupational exposures to blood. healthcare personnel who are parenterally exposed to infected blood through needlestick injuries may acquire hcv infection, but the magnitude of risk (approximately 2 in 100 hcv needlesticks) is less than that associated with hbv exposure. one epidemiologic study indicated that transmission occurred only from hollow-bore needles compared with other sharps. transmission rarely occurs from mucous membrane exposures to blood, and no transmission in hcv has been documented from skin exposures to blood. data are limited on survival of hcv in the environment. in contrast to hbv, the epidemiologic data for hcv suggest that environmental contamination with blood containing hcv is not a significant risk for transmission in the healthcare setting, with the possible exception of the hemodialysis setting where hcv transmission related to environmental contamination and poor infection control practices have been implicated. the risk for transmission from exposure to fluids or tissues other than hcv-infected blood also has not been quantified but is expected to be low. hcv is not known to be transmissible through the airborne route, through casual contact in the workplace, or by fomites. clinical syndromes hepatitis c virus infection produces a spectrum of clinical illness similar to hbv and is indistinguishable from other forms of viral hepatitis based on clinical symptoms alone. serologic tests are necessary to establish a specific diagnosis of hepatitis c. most adults acutely infected with hcv are asymptomatic. after acute infection, 15-25% of persons appear to resolve their infection without sequelae as defined by sustained absence of hcv rna in serum and normalization of alt levels. 28 chronic hcv infection develops in most persons (75-85%), with persistent or fluctuating alt elevations indicating active liver disease developing in 60-70% of chronically infected persons. no clinical or epidemiologic features among patients with acute infection have been found to be predictive of either persistent infection or chronic liver disease. moreover, various alt patterns have been observed in these patients during follow-up, and patients might have prolonged periods (greater than or equal to 12 months) of normal alt activity even though they have histologically confirmed chronic hepatitis. thus, a single alt determination cannot be used to exclude ongoing hepatic injury, and long-term follow-up of patients with hcv infection is required to determine their clinical status and prognosis. the course of chronic liver disease is usually insidious, and progresses slowly without symptoms or physical signs in the majority of patients during the first two or more decades after infection. chronic hepatitis c frequently is not recognized until asymptomatic persons are identified as hcv positive during blood donor screening, or elevated alt levels are detected during routine physical examinations. most studies have reported that cirrhosis develops in 10-20% of persons with chronic hepatitis c over a period of 20-30 years, and hcc in 1-5%, with striking geographic variations in rates of this disease. however, when cirrhosis is established, the rate of development of hcc might be as high as 1-4% per year. longer follow-up studies are needed to assess lifetime consequences of chronic hepatitis c, particularly among those who acquired infection at young ages. although factors predicting severity of liver disease have not been well defined, recent data indicate that increased alcohol intake, being aged greater than 40 years at infection, and being male are associated with more severe liver disease. in particular, among persons with alcoholic liver disease and hcv infection, liver disease progresses more rapidly; among those with cirrhosis, a higher risk for development of hcc exists. in addition, persons who have chronic liver disease are at increased risk for fulminant hepatitis a. screening enzyme immunoassay (eia) and supplemental confirmatory immunoblot tests are licensed and commercially available to detect antibodies to hcv (anti-hcv). anti-hcv may be detected within 5-6 weeks after the onset of infection but a single anti-hcv test cannot distinguish between acute, chronic, or past infection. hcv rna can be detected within 1-2 weeks of exposure to the virus and several weeks before elevations of alt and detection of anti-hcv. testing for anti-hcv by eia is recommended 4-6 months after an exposure to detect infection; testing for hcv rna may be performed 4-6 weeks after exposure if earlier detection of infection is desired. 23 treatment, prevention, and control hcv-positive patients should be evaluated for the presence and severity of chronic liver disease. initial evaluation for presence of disease should include multiple measurements of alt at regular intervals, because alt activity fluctuates in persons with chronic hepatitis c. patients with chronic hepatitis c should be evaluated for severity of their liver disease and for possible treatment. alpha interferon (with or without ribavirin) treatment of hcv appears to prevent hcv replication, decrease hepatic inflammation, and improve symptoms among chronically infected persons. persons with chronic hcv have undergone successful liver transplantation, although recurrences have been documented in this setting. antiviral therapy is recommended for patients with chronic hepatitis c who are at greatest risk for progression to cirrhosis. these persons include anti-hcv-positive patients with persistently elevated alt levels, detectable hcv rna, and a liver biopsy that indicates either portal or bridging fibrosis or at least moderate degrees of inflammation and necrosis. therapy for hepatitis c is a rapidly changing area of clinical practice and consultation with a knowledge specialist (e.g., hepatologist) is recommended. 29 no clinical trials have been conducted to assess postexposure use of antiviral agents (e.g., interferon with or without ribavirin) to prevent hcv infection, and antivirals are not fda approved for this indication. available data suggest that an established infection might need to be present before interferon can be an effective treatment. 23, 29 because there is currently no postexposure prophylaxis (pep) for hcv, the intent of recommendations for postexposure management is to achieve early identification of infection and, if present, referral for evaluation of treatment options. in addition, no guidelines exist for administration of therapy during the acute phase of hcv infection. however, limited data indicate that antiviral therapy might be beneficial when started early in the course of hcv infection. when hcv infection is identified early, the person should be referred for medical management to a specialist knowledgeable in this area. 23 at present, avoidance of parenteral exposure to blood is the only available strategy for preventing hcv infection. epidemiology it is estimated that more than 60 million persons worldwide had been infected by hiv and that 40 million were living with hiv/aids by the end of the year 2000. 30 in the united states, almost 1 million persons are living with hiv. 30 most individuals with hiv infection are active adults employed in the workforce. the primary means of acquiring infection among adults is either through behaviors such as unprotected homosexual or heterosexual intercourse with an infected partner, involving the exchange of body fluids, or injecting drug use involving shared needles and syringes. the virus also is perinatally transmitted to approximately 20-40% of children born to infected mothers, (e.g., vertical transmission). breastfeeding is a bidirectional mode of transmission, to nursing infants of infected mothers and, rarely, to mothers of nursing infants when nipple maceration and biting occur. since 1985, all donated blood in the united states has been screened for hiv infection. the risk of hiv infection due to transfusion of blood products screened by current methods is estimated to be 1 in 1,900,000 units transfused. 32 screening does not completely eliminate the potential for a seronegative but infected unit from a recently infected donor to escape detection. hiv is not transmitted by the air-borne route, by household or workplace contact with infected persons, by exposure to contaminated environmental surfaces, or by insect vectors. the virus is easily inactivated by most common disinfectants, including household bleach (diluted 1:100). commercial sex workers are at the greatest risk of acquiring hiv infection occupationally. the other group of workers at risk for acquiring hiv infection occupationally is healthcare personnel. healthcare providers and other workers in contact with blood or other body fluids who sustain accidental percutaneous or mucosal inoculations with virus-infected material are at risk for infection. the magnitude of risk depends on the severity of exposure, but on the average, about 1 in 300 hiv needlesticks results in infection. the risk for infection following mucosal exposures is estimated to be lower at approximately 0.09%. in the absence of direct exposure, healthcare providers are not at occupational risk for hiv infection. in the united states, through december 2002, there have been 57 cases of occupationally acquired hiv infection reported with an additional 139 possible cases. 32 clinical syndromes the clinical course of hiv infection is variable and changing with the advent of antiretroviral therapy, as well as treatment and prophylaxis for infectious complications. early after infection, within a few weeks to months, an acute febrile illness characterized by malaise, pharyngitis, lymphadenopathy, maculopapular rash, and headache may occur. the frequency of this mononucleosis-like illness has varied widely in reports of seroconverting individuals. at initial presentation of such patients, hiv antibody screening tests (enzyme immunoassay (eia)) may be negative, but viral antigen (p24 antigen) and serologic reactivity to one or more viral components (western blot test) allows the diagnosis to be established at this stage. hiv infection should be suspected in any person with a mononucleosis syndrome lacking a positive heterophil antibody response (monospot test). following initial infection, most persons have generalized asymptomatic lymphadenopathy and appear well. however, laboratory tests document a gradual decline in the number of circulating t-helper lymphocytes (cd4 cells), beginning soon after infection and continuing over the next several years. t-helper cells are essential components of the immune system and mediate aspects of both cellular and humoral immunity. symptoms, signs, and illness suggestive of mild to moderate immunodeficiency appear after about 5 years, when cd4 cells decrease by about 50%, to less than 500 cells/dl. intermittent fever, oral thrush, bacterial pneumonia, enteric infections, and reactivated tb are typically diagnosed at this time. signs suggestive of more rapid deterioration include oral hairy leukoplakia (a wart-like white growth in the oral cavity), shrinking lymphadenopathy, fever, weight loss, and elevated erythrocyte sedimentation rate. when cd4 cell counts fall below 200, serious opportunistic infections can be anticipated. pneumocystis pneumonia (pcp) was the most common index diagnosis in the first 5 years of the epidemic, but the advent of effective pcp prophylaxis has altered the picture. other opportunistic infections and malignancies, including kaposi's sarcoma, lymphoma, disseminated tb, toxoplasmosis, and cryptococcal meningitis, now account for the majority of index hiv diagnoses. with the exception of tb, the infectious complications of hiv infection generally are not transmissible to healthy individuals and pose no risk in the workplace. indeed, the causative organisms are ubiquitous and most adults have already been exposed. opportunistic infections in hivinfected patients usually represent reactivation of dormant organisms when the immune system can no longer keep them inactive. be offered to all patients with symptoms ascribed to hiv infection. 34 recommendations for offering antiretroviral therapy in asymptomatic patients require analysis of many real and potential risks and benefits. information regarding treatment of acute hiv infection from clinical trials is very limited. ongoing clinical trials are addressing the question of the long-term clinical benefit of potent treatment regimens for primary infection. in general, treatment should be offered to individuals with fewer than 350 cd4 t cells/mm or plasma hiv rna levels exceeding 55,000 copies/rnl (by rt-pcr or bdna assay). once the decision has been made to initiate antiretroviral therapy, the goals should be maximal and durable suppression of viral load, restoration and/or preservation of immunologic function, improvement of quality of life, and reduction of hivrelated morbidity and mortality. 33 hiv-infected individuals found to have latent tb infection should be treated with antituberculous therapy to prevent activation of disease. 13 persons at risk for direct contact with blood and other potentially infected materials should receive specific instruction in universal/standard precautions for infection control, as recommended by the centers for disease control and prevention 34, 35 and mandated by the occupational safety and health administration. 27 for most environments outside healthcare settings, common sense and attention to personal hygiene are adequate to protect workers. gloves should be worn to clean up visible sites of blood contamination. environmental surfaces can then be decontaminated with disinfectant solutions or household bleach (diluted 1:100). 34, 36 individuals sustaining accidental parenteral exposures to hiv should be counseled to undergo baseline and followup testing for 6 months after exposure (e.g., 6 weeks, 3 months, and 6 months) to diagnose occupational infection. postexposure chemoprophylaxis with antiretroviral agents has been recommended by the us public health service since 1996 after certain exposures to hiv-infected sources which pose a risk of infection transmission, such as needlesticks, mucous membrane, and non-intact skin exposures (tables 22.5 and 22.6). data from animal models of prophylaxis with these agents suggest that antiviral activity is diminished when treatment is delayed for more than 24 hours. for this reason, immediate reporting and access to chemoprophylaxis is recommended. occupational exposure is a frightening experience. consultation with clinicians knowledgeable about hiv transmission risks who can provide supportive counseling to the worker is essential during the follow-up interval. cdc recommends that occupationally exposed workers refrain from unsafe sexual practices, pregnancy, and blood and organ donation for 6 months after exposure to minimize the risk of transmission. zoonoses are infections that are maintained in nature by transmission between vertebrate animals, and they can be transmitted from other vertebrates to humans or from humans to other vertebrates. zoonotic pathogens can be divided into two major groups: (1) those transmitted primarily among wild animals (e.g., yersinia pestis, rabies), and (2) those transmitted primarily among domestic animals (e.g., sporothrix schenkii, non-typhoid salmonella spp.). other infections not properly classified as zoonoses can result from working directly with animals (e.g., infected wounds resulting from animal bites) or with animal products (e.g., anthrax in carpet weavers). many zoonotic infections present occupational risks, not only to those who work with live or dead vertebrate animals or animal products but also to workers exposed to certain environments contaminated by animals or animal products. thus, workers in veterinary medicine, animal husbandry, and animal research are at risk for acquiring a host of zoonotic infections specific to the type of live animal exposure, just as those involved in healthcare work with humans are at risk for infections acquired from humans. examples of such zoonotic infections include q fever in veterinarians, psittacosis (caused by chlamydia psittaci) in duck farmers, orf (contagious ecthyma) in shepherds, lymphocytic choriomeningitis (e.g., leptospirosis) in laboratory workers who handle rodents, fatal herpes virus simiae infection in primate handlers, and more recently, monkeypox in veterinarians and pet store owners. 37, 38 influenza a (h5nl) (avian flu) infection was shown to have been transmitted from ducks and chickens to poultry workers in hong kong and has become an important source of epidemic infection in various international settings; 39 lyssavirus (related to rabies virus) infections have been transmitted from bats to humans in australia, 41 and a large outbreak of febrile encephalitic and respiratory illnesses among workers who had exposure to pigs was shown to be due to infection with a previously unrecognized paramyxovirus (formerly known as hendra-like virus, now called nipah virus). 41 brucellosis is an example of a zoonotic infection in abattoir workers exposed to live or dead animals or animal products. examples of zoonotic infections acquired by workers exposed to environments harboring or contaminated by contagious animals include leptospirosis in rice field workers, and argentine hemorrhagic fever typically acquired by adult males harvesting corn in cornfields inhabited by rodents, which serve as the reservoir for junin or machupo virus. it is beyond the scope of this chapter to review the large number of zoonoses (about 200 have been described) that could pose a risk to workers in unique jobs that involve contact with various animals or environments. for each type of occupation that involves regular animal contact, it is important to recognize the types of infectious disease risks involved, consider baseline studies and storage of serum for future serologic tests if risks are high, plan preventive measures when possible, and prepare for early diagnosis and treatment of such infections when illness occurs. 42 some of the zoonoses, the occupational groups they affect, and their clinical presentations are included in table 22 .1. infectious diseases continue to emerge, posing threats to the health of workers in numerous settings. a prime example of such a threat is severe acute respiratory syndrome or sars, first identified in early 2003 and responsible for illness and death primarily among exposed healthcare personnel. 43 emerging infectious issues which may prove to be challenges for occupational health include those posed by bioterrorism, biotechnology, 44 and emerging and reemerging infections. these emerging infections emphasize the need for continued vigilance and for careful history taking about occupational exposures when evaluating individuals for illnesses that could possibly be occupationally acquired. timeliness in identification and reporting of cases assists in the accurate estimation of the magnitude of the infectious disease problem and in the development of additional preventive and therapeutic measures. screening of employees for infection with or susceptibility to infectious diseases is an important part of healthcare maintenance, especially when the occupational setting poses a significant risk of transmitting or acquiring infections. screening also is warranted if specific interventions are available to prevent disease transmission among workers. assessment of behaviors, such as smoking, that increase the risk of acquiring infections also is valuable so that employees can be provided educational and other interventions to modify risks. preventing infectious diseases in workers can decrease absenteeism and financial costs associated with disability, sick leave, and health insurance, even if the primary source of infection is non-occupational. attending to these issues at the time of employment obviates the need for ongoing surveillance of many infections and simplifies outbreak investigations by documenting the pre-exposure immune status of contacts. tst screening for active disease identifies those persons who would benefit from prophylaxis (tables 22.5 and 22.6). 14 tb vaccination with bacillus of calmette-guerin (bcg) vaccine, a live attenuated strain of m. bovis, is provided for children and some workers in most european countries, but it is not recommended in the united states because of its unproven efficacy when used in adults and because it induces dermal hypersensitivity to purified protein derivative (ppd) tuberculin in most recipients, impeding the usefulness of tst as a screening tool. persons age 65 years and older, persons with chronic diseases or pulmonary disorders, and healthcare personnel should be offered pneumococcal vaccine and annual influenza vaccine (table 22. 3). 45 rubella immune status should be ascertained and men and women immunized in settings where women of childbearing age are employed (table 22. 3). even though rubella is not often transmitted in the workplace, outbreaks can occur among susceptible individuals and vaccination is an important public health intervention. medical personnel should demonstrate proof of rubella immunity or vaccination prior to patient contact. measles vaccine should be provided to all workers born after 1957 with no documented history of measles who have not received two injections of live virus vaccine (table 22. 3). screening for immunity to varicella and mumps is not routinely recommended, except for healthcare providers and adults with no history of infection with these agents who are exposed to young children. all adults require tetanus immunization. tetanus diphtheria toxoid (td) boosters should be administered every 10 years to adults who have completed primary immunization (table 22. 3). employees with no prior history of tetanus diphtheria immunization or with uncertain histories should receive a series of three primary vaccine injections. 45 similarly, adults with no history of polio immunization should undergo primary immunization with inactivated polio vaccine, especially if they are employed in healthcare settings or when travel to endemic areas is anticipated. persons employed in occupations that pose a risk for parenteral contact with blood and other body fluids should be offered hepatitis b immunization (table 22. 3). healthcare personnel, laboratory workers, animal handlers, first responders, and personal service workers such as barbers, tattooists, and cosmetologists are included in this category. adults with multiple sexual partners also should be encouraged to undergo immunization. serum banking to allow documentation of baseline serostatus is useful for laboratory and healthcare personnel at risk for other bloodborne infections such as hiv or more exotic infections. laboratory workers and animal handlers may be at risk for unusual infectious diseases. q fever, a rickettsial disease transmitted by the air-borne route, is a special risk encountered by handlers of sheep and similar animals. serologic testing for q fever titers prior to occupational exposure is important to document baseline status and to detect seroconversion at follow-up testing. although smallpox vaccine is no longer recommended routinely, genetically engineered vaccines prepared from vaccinia may pose a risk to researchers and clinicians treating patients enrolled in vaccine trials. laboratory workers who handle vaccinia or recombinant vaccinia preparations in culture or in animals should receive vaccinia vaccine. healthcare personnel caring for patients immunized with vaccinia or other orthopoxviruses or tissues and specimens from patients with these infections also should be immunized. a program for smallpox vaccination for selected individuals who may be in the frontline for responding to a bioterrorist attack has recently been initiated in the united states. 46 consultation should be obtained to determine the need for screening, immunization, and testing for other exotic infections. some animal handlers are at risk of acquiring rabies through bites or exposure to infected secretions and tissues. immunization with human diploid cell vaccine (three 1-ml intradermal doses on day 0, 7, and 21 or 28) should be provided to workers at risk for rabies and for persons traveling for more than 1 month to areas where rabies is endemic (table 22. 3). booster injections should be provided every 2 years for those with continuing exposure. surveillance of infectious diseases is conducted to detect increased occurrence of disease so that preventive interventions can be initiated. surveillance can be passive (based on employee health consultations or reports from contractual providers or supervisors) or active (actual monitoring of disease occurrence). active surveillance for infectious diseases is not required in most occupational settings. in work environments where exposure to m. tuberculosis may occur -such as healthcare settings, residential care facilities, shelters, and correctional facilities -active tst surveillance among susceptible individuals is indicated. periodic tsts are especially important in the wake of several recent outbreaks associated with drug-resistant strains of m. tuberculosis in hospitals, adult care settings, and home healthcare settings. 16 skin testing should be performed at least annually in these settings, and perhaps as often as every 6 months, for personnel at high risk for exposure to active tb. surveillance of teachers, travelers to endemic areas, and employees in other institutional settings where close contact with infected individuals is possible also may be warranted, depending on the local prevalence of tb. 14 surveillance for infections among laboratory workers and animal handlers exposed to specific pathogens should be individualized in accordance with standard guidelines for biosafety in microbiologic and biomedical laboratories. 47 maintaining standardized records of reportable infectious diseases is an important component of passive surveillance in the workplace. centralized collection and assessment of these records at regular intervals may allow early detection of outbreaks of occupational infections amenable to specific control interventions. geographic or temporal clusters of cases or clustering among persons with similar attributes or occupational tasks suggest a common source of exposure and infection and warrant investigation. local public health officials and regulatory agencies should be consulted promptly when an outbreak is initially suspected. reporting of occupationally acquired infections permits public health agencies to identify clusters of old and emerging illnesses and ultimately prevent them. these events should be reported as mandated by state and local regulations. 48, 49 return-to-work criteria employees diagnosed with communicable infectious diseases should not return to work until the period of infectivity is past. specific guidelines should be consistent with local public health regulations. some workers, for example food handlers with certain diarrheal illnesses, cannot resume their duties until culture evidence of cure is obtained. employees should be advised of the return-towork policies at the time of employment and when illness is diagnosed. a table for length of work restriction for healthcare personnel can be used to guide return-to-work policies for the workplace (table 22. 2). 19 common sense dictates attention to personal hygiene among all workers. hand washing after using the bathroom and before handling food is essential. the mouth should be covered while sneezing or coughing, and soiled tissues and dressings should be disposed of in trash containers. employers have a responsibility to minimize crowding in the work setting. facilities for hand washing should be available in bathrooms and food preparation areas. proper ventilation also is important. trash should be emptied at regular intervals, and work areas should be clean and free of pests. smoking should be prohibited in common work areas. spills of blood, body fluids, and other potentially infectious substances should be removed with disposable paper towels or other suitable procedures. contaminated areas should then be disinfected with commercial products or with a solution of household bleach (diluted 1:100). 34, 36 infection control in healthcare settings infection control programs in healthcare settings are necessary to prevent transmission of healthcare-related infections to patients and healthcare personnel. the cdc has established a two-tiered system of infection control precautions. 16 the first tier consists of 'standard precautions' which are precautions recommended for delivery of care to all patients regardless of diagnosis or presumed infection status. they are designed to limit exposure to blood or other body substances and include elements such as hand hygiene and use of appropriate protective barriers, e.g., masks, eye protection, and gloves, as needed to prevent direct contact. the second tier of precautions recommended by cdc are 'transmission-based precautions', designed for the management of patients known or suspected to be infected with pathogens whose transmission can be limited by the adoption of additional measures beyond those which are part of standard precautions. they apply to pathogens transmitted by the air-borne or aerosol routes, droplets, and by direct and indirect contact. respiratory precautions are employed for patients with infections communicable by the air-borne route. such patients are housed in private rooms with special ventilation and should wear surgical masks when leaving their rooms. respiratory protection (i.e., n-95 respirators) also are advised for providers in close contact with patients on respiratory precautions. however, the re-emergence of epidemic and mdr-tb has led to a re-emphasis of other fundamentals for prevention of transmission of tuberculosis in healthcare and other settings. early identification of tb allows early indication for therapy, and requires alertness in considering tb in high-risk patients with pulmonary symptoms, especially those with hiv infection. special ventilation measures and respiratory protection are especially important for cough-inducing procedures, such as sputum induction and aerosolized pentamidine administration. healthcare personnel who have the potential for being exposed to m. tuberculosis should be screened on employment and at least annually thereafter by ppd skin testing, comparing previous test results to current results to identify those who have converted to skin test positivity. 15 procedures for disposal of infectious wastes have been developed by the cdc. 36 needles and other sharp objects should be sterilized prior to disposal. liquid and laboratory wastes may be dumped into sewage systems. materials heavily contaminated with bacteria or blood should be placed in special bags that are specifically labeled for infectious waste and should be disposed of according to community standards for such materials. 36 employers have a responsibility to educate employees about infection control. barriers to prevent exposure, including masks, gowns, eye protection, and gloves, should be readily available to workers at risk. hand washing facilities and hand hygiene supplies are essential. where access to sinks or running water is not feasible, alcohol hand rubs or packaged towels containing disinfectants should be provided. impervious containers for the disposal of needles and other sharp objects are essential. 50 such containers should be made available on ambulances and provided to home health aides and other visiting healthcare personnel. personal service workers who use needles, razors, and other sharp objects also should have access to safe disposal units. persons receiving care at home who require injections or other procedures that demand the use of needles also should be provided with impervious disposal containers and instructed in proper disposal methods to protect sanitation workers and others in contact with waste. despite improvements in engineering controls, work practices, and personal protective equipment, laboratory personnel are nevertheless at risk for occupationally acquired infections. laboratory personnel may acquire infection by aerosolization of specimens, mouth pipetting, or percutaneous injury or mucocutaneous contact. methods of infection control applicable to laboratory settings are described in the cdc document entitled 'biosafety in microbiological and biomedical laboratories'. 47 by 1995, it was estimated that 14.5 million children were attending out-of-home daycare in a variety of settings including licensed child daycare centers, regulated daycare homes, and unregulated family daycare homes. 51 many serious infections occur as endemic or microepidemic problems in the daycare setting. these include h. influenzae type b, hepatitis a, cytomegalovirus, parvovirus b 19, and enteric infections (shigella, giardia, rotavirus, clostridium difficile, campylobacter, cryptosporidium, calcivirus, salmonella, enteric adenovirus, astrovirus, and several types of e. coli infections). in addition, the high rate of acute respiratory infections leads to early onset of otitis media, frequent antibiotic use, and emergence of multidrug-resistant enteric pathogens. thus, workers in close contact with children risk exposure to a wide variety of communicable pathogens contained in secretions, urine, and stool. all such personnel, as well as all children in schools and daycare centers, should be screened for immunity to common childhood infections and vaccinated if immunity is not present (table 22. 2). regulation of childcare facilities is essential to reduce risks to children and workers. national standards for infection control in childcare facilities were promulgated in 1990. 52 hand washing facilities and policies are the most important component of disease prevention in school and daycare settings. hands should be washed after contact with mucous membranes and potentially infected body fluids. older children should be instructed in personal hygiene. children with fever or diagnosed infections should be excluded from attending daycare or school until transmission risk is no longer present, and policies for such exclusion should be in place. prompt reporting of disease outbreaks and prompt involvement of public health authorities are essential. employees should be instructed in common-sense first aid procedures for handling wounds, bites, and other situations in which exposure to infected blood or tissues is possible. barrier protection is rarely required in schools, but gloves should generally be available for emergencies requiring first aid. infection with a variety of agents during pregnancy has the potential to cause fetal damage, especially when primary infection occurs. while a number of these infections can be community acquired, the likelihood of exposure to certain of these pathogens can be greater in healthcare settings. infections with as rubella, cmv, and parvovirus are among the infectious agents which may be of special concern to pregnant healthcare personnel. in general, adherence to standard precautions as well as preexposure immunizations when available and appropriate are the best way of preventing the devastating effects of such infections (table 22. 2). 19, 21 immunodeficient workers are at increased risk of devastating infections, particularly with opportunistic agents. the greatest risk for such workers is likely in the healthcare setting where there can be ample opportunity for exposure to these agents. many immunocompromising illnesses would be viewed by the us legal system as disabilities and therefore, individuals with those conditions would be covered under the provisions of the americans with disabilities act of 1990 (see chapter 57.1). 53 such persons should be informed about their risks and furthermore, their employers should make reasonable accommodations to allow their employees to continue to perform their jobs, taking into consideration the provisions of applicable federal, state, and local regulations. occupational infectious diseases encompass a large variety of infections which can involve many organ systems. they include some common infections, such as influenza, that pose a special problem in the workplace because of close interpersonal contact and crowding, and that taken together account for a large proportion of time lost from work. many of these infections are preventable by policies that promote hygiene and provide exclusion from work during periods of contagion. in addition, a variety of less common, but sometimes serious, infections are particularly associated with specific occupations. recognition of the types of infection risk associated with specific occupations can, in most cases, lead to effective, often simple steps for primary prevention, as well as opportunities for early diagnosis and treatment. bioterrorismrelated inhalational anthrax: the first 10 cases reported in the united states principles and practice of infectious diseases centers for disease control and prevention. prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) influenza vaccination in long-term-care hospitals reduces the mortality of elderly patients measles, mumps, and rubella -vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of mumps: recommendations of the advisory committee on immunization practices (acip) changing epidemiology of pertussis in the united states: increasing reported incidence among adolescents and adults guidelines for the control of pertussis outbreaks ashrae guideline 12-2000: minimizing the risk of legionellosis associated with building water systems guideline for prevention of nosocomial pneumonia. the hospital infection control practices advisory committee progressing toward tuberculosis elimination in low-incidence areas of the united states: recommendations of the advisory council for the elimination of tuberculosis centers for disease control and prevention. targeted tuberculin testing and treatment of latent tuberculosis infection the role of bcg vaccine in the prevention and control of tuberculosis in the united states: a joint statement by the advisory council for the elimination of tuberculosis and the advisory committee on immunization practices guidelines for preventing the transmission of tuberculosis in health-care facilities guideline for isolation precautions in hospitals. the hospital infection control practices advisory committee prevention of varicella: update recommendations of the advisory committee on immunization practices (acip) epidemiology and prevention of vaccine-preventable diseases guideline for infection control in healthcare personnel guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force infection control precautions for the pregnant healthcare worker. bailliere's risk and management of bloodborne infections in healthcare workers updated us public health service guidelines for the management of occupational exposures to hbv, hcv, and hiv and recommendations for postexposure prophylaxis achievements in public health: hepatitis b vaccination -united states hepatitis b virus: a comprehensive strategy for eliminating transmission in the united states through universal childhood vaccination: recommendation of the immunization practices advisory committee (acip) protection against viral hepatitis: recommendations of the immunization practices advisory committee (acip) occupational safety and health administration, department of labor. 29 cfr part 1910.1030, occupational exposure to bloodborne pathogens; final rule recommendations for prevention and control of hepatitis c virus (hcv) infection and hcv-related chronic disease national institutes of health consensus development conference panel statement. management of hepatitis c an unequal epidemic in an unequal world the risk of transfusion-transmitted viral infections surveillance of healthcare personnel with hiv/aids, as of panel on clinical practices for the treatment of hiv infection. guidelines for the use of antiretroviral agents in hiv-infected adults and adolescents. us department of health and human services recommendations for prevention of hiv transmission in healthcare settings update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis b virus, and other bloodborne pathogens in health-care settings guideline for environmental control in healthcare facilities centers for disease control and prevention. multistate outbreak of monkeypox -illinois risk of influenza a (h5n1) infection among poultry workers, hong kong, 1997-1998 emerging viral diseases: an australian perspective outbreak of hendra-like virus -malaysia and singapore, 1998-1999 risks and prevention of nosocomial transmission of rare zoonotic diseases sars -looking back over the first 100 days occupational standards for the protection of employees in biotechnology centers for disease control and prevention. general recommendations on immunization: recommendations of the advisory committee on immunization practices (acip) and the american academy of family physicians (aafp) recommendations for using smallpox vaccine in a pre-event vaccination program: supplemental recommendations of the advisory committee on immunization practices (acip) and the healthcare infection control practices advisory committee (hicpac) us department of health and human services centers for disease control. case definitions for public health surveillance mandatory reporting of infectious diseases by clinicians. mandatory reporting of occupational diseases by clinicians selecting, evaluating, and using sharps disposal containers. dhhs (niosh) publication no. 97-111 child care arrangements for preschoolers by family characteristics: fall national standards for infection control in out-ofhome child care americans with disabilities act of 1990, 104 stat. 327, 42 u.s.c. sec. 12101 et seq immunization of health-care workers: recommendations of the advisory committee on immunization practices (acip) and the hospital infections control practices advisory committee (hicpac) known responder ¶ known non-responder** antibody response unknown hbig § × 1 and initiate hepatitis b vaccine series no treatment hbig x 1 and initiate revaccination or hbig × 2 † † test exposed person for anti-hbs: § § if drug resistance is a concern, obtain expert consultation. initiation of postexposure prophylaxis (pep) should not be delayed pending expert consultation, and, because expert consultation alone cannot substitute for face-to-face counseling, resources should be available to provide immediate evaluation and follow-up care for all exposures. § source of unknown hiv status (e.g., deceased source person with no samples available for hiv testing). ¶ unknown source (e.g., splash from inappropriately disposed blood). ** small volume (i.e., a few drops). † † the designation 'consider pep' indicates that pep is optional and should be based on an individualized decision between the exposed person and the treating clinician. § § if pep is offered and taken, and the source is later determined to be hiv negative, pep should be discontinued. ¶ ¶ large volume (i.e., major blood splash). key: cord-017021-n6rpuvwd authors: marriott, deborah j.; orla morrissey, c. title: common infections following lung transplantation date: 2018-08-31 journal: essentials in lung transplantation doi: 10.1007/978-3-319-90933-2_15 sha: doc_id: 17021 cord_uid: n6rpuvwd the lungs are the only transplanted organ in direct contact with the ‘outside world’. infection is a significant cause of morbidity and mortality in lung transplantation. early accurate diagnosis and optimal management is essential to prevent short and long term complications. bacteria, including mycobacteria and nocardia, viruses and fungi are common pathogens. organisms may be present in the recipient prior to transplantation, transmitted with the donor lungs or acquired after transplantation. the degree of immunosuppression and the routine use of antimicrobial prophylaxis alters the pattern of post-transplant infections. infection accounts for around 35% of all deaths in the first year after transplantation with bacterial pathogens responsible for approximately half of all infections [1] . the risk of infection following lung transplantation is determined by a number of factors including: • physical factors such as denervation of the allograft resulting in a reduced cough reflex and anastomotic site stenosis with distal infection • the 'net state of immunosuppression'-the result of all factors including host immune system, anti-rejection immunosuppressive therapy and concomitant viral infections such as cytomegalovirus that contribute to a patient's risk of infection • epidemiological exposure to organisms, including donor-derived infections, community acquired infections, travel related infections and healthcare associated infections • the use of prophylactic antimicrobial agents in the post-transplant period bacteria are defined by their morphology or shape and size. most pathogenic bacterial species are spherical (cocci) or rod-shaped (bacilli) and may exist as single cells (for example many of the common bacilli such as pseudomonas and stenotrophomonas) or in a variety of characteristic patterns such as s. pneumoniae (pairs of lancet shaped cocci), s. aureus (large clusters of cocci forming 'bunches of grapes') and streptococci (long chains of cocci). whilst molecular diagnostic techniques such as polymerase chain reaction (pcr) are increasingly important the basis of much microbiological diagnosis remains the characteristic appearance of the organism on a glass microscope slide when stained with dyes under a variety of conditions. common stains include the gram stain, first described by hc gram in 1884 but still in everyday use, the ziehl-neelsen or acid-fast stain for mycobacteria and the modified ziehl-neelsen stain for nocardia. the gram stain divides bacteria into gram positive or gram negative depending on the ability of the cell wall to prevent decolourisation after staining with crystal violet. it is important to remember that bacteria such as s. aureus and pseudomonas species are not stained by the ziehl-neelsen stain and conversely mycobacteria cannot be seen on a gram stain. culture techniques also differ with mycobacteria often unable to grow on conventional agar plates, requiring special growth media and prolonged culture periods. therefore if mycobacterial infection is suspected the request form for the sample must specify 'mycobacterial culture' so the appropriate investigations are performed by the laboratory. the laboratory diagnosis of important bacteria in the setting of lung transplantation is summarised in s. aureus is a common colonizer of the upper respiratory tract and skin, and is isolated with increased frequency from the sputum of patients with cystic fibrosis although the frequency decreases with age [2] . s. aureus can be acquired from the donor, the recipients own bacterial flora or the hospital environment as a healthcare associated infection, and is responsible for a wide range of health care-associated infections such as ventilator-associated pneumonia, bactereamia, and surgical site infections. isolates of s. aureus are characterised according to their susceptibility to methicillin, an anti-staphylococcal penicillin. methicillin susceptible s. aureus (mssa) is more common in community acquired infections whereas methicillin resistant s. aureus (mrsa) occurs with greater frequency in hospital acquired infections. the largest study of s. aureus following lung transplantation was a retrospective single centre study conducted over a 5 year period [3] . s aureus infection developed in 109 of 596 lung transplant (18%) recipients within 90 days of transplantation. mssa (62%) was more common than mrsa (38%) but the proportion of mrsa infections increased over time. pneumonia (48%) was the most common infection, followed by tracheo-bronchitis (26%), bacteremia (12%), intrathoracic infections (7%), and skin/soft tissue infections (7%). infected patients required longer hospital and intensive care unit stays (p < 0.0001 for both) but the 30-and 90-day mortality rates were low (7% and 12%, respectively). however infected patients had higher rates of rejection (both acute and chronic) at 1 (p = 0.048) and 3 years (p = 0.002), and higher mortality at 1 (p = 0.058) and 3 years (p = 0.009). • dicloxacillin or flucloxacillin • cefazolin or cephalothin for penicillin allergic patients (note-there is 5-10% risk of anaphylaxis in patients with documented penicillin anaphylaxis) • clindamycin is often prescribed for deep infections because it exhibits good tissue penetration. however it is a bacteriostatic antibiotic and should only be administered to patients with s. aureus bacteraemia following specialist advice • vancomycin with appropriate therapeutic drug monitoring (tdm) • teicoplanin-tdm not available in most centres. standard dosing may be inadequate, especially for bacteraemia • linezolid-superior to vancomycin for mrsa pneumonia. toxicity may occur with long-term administration unless tdm is undertaken • some isolates may be susceptible to clindamycin, cotrimoxazole and doxycycline. however these agents should not be used to treat bacteraemia infection control mssa: no specific measures required. mrsa: patients are usually placed on contact precautions (gown or apron, glove and careful hand hygiene as per 5 moments for hand hygiene) and may be isolated in a single room or cohorted with other colonised patients to prevent spread to other non-identified colonised patients. haemophilus influenzae is an important respiratory pathogen. in patients with cystic fibrosis it often causes infection early in life but is replaced by other organisms such as pseudomonas spp. over time [4] . in contrast, patients undergoing lung transplantation for other indications may be colonized with h. influenzae at any stage of life. post-transplant infection with h. influenzae is relatively uncommon. this is at least in part because of the wide spread practice of the administration of azithromycin and trimethoprim/sulphamethoxazole as prophylactic agents in the post-operative period. both these antimicrobial agents have activity against h. influenzae thereby reducing the frequency of infection. • approximately 25% of h. influenzae isolates are susceptible to ampicillin • ampicillin resistant isolates are generally susceptible to augmentin, cefuroxime and third generation cephalosporins (cefotaxime, ceftriaxone) • cephalexin is ineffective no specific infection control measures required other than standard precautions and hand hygiene. like h. influenzae s. pneumoniae is an important respiratory pathogen which is uncommon in the setting of lung transplantation, again in part because of the impact of antimicrobial prophylaxis with trimethoprim/sulphamethoxazole and azithromycin. after lung transplantation a reduction in an important component of the immune system, serum immunoglobulins, is common occurring in up to 63% of lung transplant recipients [5] . it is likely that this increases the risk and frequency of severe pneumococcal infection. • s. pneumoniae is generally susceptible to penicillin • penicillin resistant s. pneumoniae pulmonary infection can usually be successfully treated with penicillin as the concentration achieved in the lung is sufficient to exceed the threshold for efficacy • alternative treatment options for penicillin resistant s. pneumoniae causing meningitis or blood-stream include third generation cephalosporins and vancomycin no specific infection control measures required other than standard precautions and hand hygiene. pseudomonas aeruginosa is a gram negative bacillus which commonly colonises the airways of patients with cystic fibrosis but is also found in other patients proceeding to lung transplantation, for example those with chronic obstructive pulmonary disease. in many centres pseudomonas is the most common cause of post-transplantation bacterial infection. prolonged pre-transplant therapy with a variety of antibiotics frequently results in highly resistant organisms colonizing the patient at the time of transplantation. laboratory reports may refer to 'mucoid pseudomonas' isolated from a specimen. mucoid pseudomonas develops under certain environmental conditions following infection with non-mucoid species. the thick polysaccharide capsule gives the organism a 'wet' appearance when growing on an agar plate in the laboratory but more importantly renders the organism more resistant to immunological defense mechanisms such as phagocytosis and to standard anti-pseudomonas therapy. • guided by laboratory susceptibility testing, especially in patients with extensive prior antibiotic exposure • susceptibility testing of mucoid strains is less reliable than standard strains • commonly used antibiotics include aminoglycosides (gentamicin, tobramycin, amikacin), antipseudomonal beta-lactams (piperacillin-tazobactam, ceftazidime, cefepime), ciprofloxacin and meropenem. colistin may occasionally be required for extremely resistant organisms contact precautions are generally reserved for patients with multi-drug resistant pseudomonas aeruginosa. stenotrophomonas maltophilia is a gram negative bacillus which is increasingly recognized as an important pathogen of the airways in the setting of lung transplantation. the organism is widespread in the environment, found in soil, water and animal and plant material. treatment is complicated by the multi-drug resistance. • trimethoprim-sulphamethoxazole is the treatment of choice although resistance is increasingly described • ciprofloxacin is active against approximately 50% of laboratory isolates infection control • no specific infection control requirements other than standard precautions and hand hygiene. burkholderia species are gram negative bacilli closely related to pseudomonas species (in fact they were previously called pseudomonas cepacia and you will sometimes see this referred to in older literature). in the setting of cystic fibrosis and lung transplantation, the clinically important species belong to the burkholderia cepacia complex (bcc), a group of 17 genetically closely related organisms. however it has been recently recognised that not all bcc are equally pathogenic. the most important organisms include b. cenocepacia (previously named bcc genomovar iii) and burkholderia multivorans (previously bcc genomovar 2) which account for up to 97% of all burkholderia cepacia complex isolates from patients with cystic fibrosis [6] . one of the most feared organisms is burkholderia cenocepacia which can be an aggressive pathogen that is transmissible between patients and can cause epidemics. recent studies have suggested that b. cenocepacia is associated with poor outcome and is a contraindication to transplantation in many centres. therefore accurate detection and identification of burkholderia species prior to transplantation is absolutely essential: a false positive result can lead to exclusion from the transplantation waiting list whereas a false negative result can lead to poor transplantation outcome and possible cross infection between patients if appropriate infection control measures are not put in place. there is no standard treatment that can eliminate bcc. eradication of bcc is extremely difficult as many species of bcc, particularly b. cenocepacia, are intrinsically resistant via a variety of resistance mechanisms to numerous antimicrobial agents including the aminoglycosides (gentamicin, tobramycin), most antipseudomonal beta-lactam antibiotics (piperacillin-tazobactam, cefepime, ceftazidime) and colistin. rapid development of resistance may occur during therapy [6] . in a study of a large number of bcc isolates, 2621 strains of burkholderia cepacia complex isolated from 1257 cystic fibrosis patients were tested. resistance to all available antimicrobial agents was demonstrated in 18% of isolates with the most active agents, minocycline, meropenem, and ceftazidime inhibiting 38%, 26%, and 23% of strains, respectively [7] . the use of combination antimicrobial therapy to overcome these issues has not usually been successful. bcc can be spread to susceptible patients by: • person to person contact • contact with contaminated surfaces or objects • exposure to bcc in the environment contact precautions and isolation (see mrsa) may be implemented in hospital. alternately, patients colonised with bcc should not be housed next to an immunosuppressed patient. mycobacteria are bacteria forming their own genus within the phylum actinobacteria. over 190 species have been identified but not all are pathogenic (that is have the potential to cause infection in humans). mycobacteria are slender, curved rods that, unlike most bacteria, are acid fast (see preceding section). in addition, they are resistant to alkalis and dehydration meaning they can survive for long periods in the environment. the cell wall contains complex waxes and glycolipids. they multiple very slowly on special media and some clinical isolates can take 4-6 weeks to grow. based on their growth rate, catalase and niacin production and pigmentation in light or dark conditions mycobacteria are classified as mycobacterium tuberculosis complex (m. tuberculosis, m. bovis, m. africanum, m. microtii) and non-tuberculous mycobacterium (ntm). molecular techniques (e.g. pcr) can now readily differentiate between them. m. tuberculosis is transmitted from person to person. the incidence in transplant recipients is much higher than in the general population [8] . the most common cause in the transplant population is reactivation of latent infection but other causes include unrecognised transmission in the donor lungs (that is donor-derived), especially in countries where tb is endemic, and primary infection after transplantation [9] . the median time to infection from lung transplantation is 3.5 months (earlier than in renal transplant recipients) but donor-derived infections usually occur earlier, often within the first month post-lung transplant [8] . risk factors include prior residence in an endemic country, history of untreated tb, a chest x-ray which shows evidence of old healed tb, augmented immunosup-pression for rejection, use of t-cell depleting agents for immunosuppression and recipient age. the lung is the most common site of infection but in up to 33% extra-pulmonary or disseminated tb can occur with unusual presentations (e.g. skin ulcers, abscesses, tenosynovitis) [9] . fever is a very common presenting complaint as are night sweats and weight loss [9] . instead of the classical cavity that is seen on chest x-ray in immunocompetent patients, in lung transplant recipients focal infiltrates, miliary pattern, nodules or pleural effusions are more common (fig. 15 .1) [9] . the diagnosis of active tb can be challenging in lung transplant recipients with sputum samples commonly stain and culture negative. bronchoalveolar lavage (bal) with the fluid sent for acid fast bacilli (afb) staining ( fig. 15 .2) and culture is ideal. pcr testing is useful to decrease the time to diagnosis given cultures are slow to grow. biopsy of skin lesions, abscesses, soft tissue lesions or other accessible extra-pulmonary sites for afb staining, culture, histology and/or pcr can assist in the diagnosis of extra-pulmonary tb. guidelines exist for the treatment of active tb; however, there are a few specific things to note in the lung transplant setting [10] [11] [12] . • a rifamycin-based regimen (rifampicin is the most common drug used in this group) is strongly preferred because of its sterilizing capacity and ability to prevent the emergence of resistance • rifamycins interact with immunosuppressant agents. dose adjustments will be required at initiation and cessation with close monitoring of levels of immunosuppressant drugs whilst receiving a rifamycin • some centres prefer rifabutin for use in the transplant setting as it has less impact on drug metabolism than rifampicin • for localised non-severe infection and no suspicion of isoniazid resistance a fluoroquinolone could be substituted for the rifamycin with the duration extended to 12-18 months depending on the number of drugs used. otherwise a rifamycin agent should be used in the regimen. • the minimum duration is 6 months but some experts prefer a minimum of 9 months in the transplant setting. longer treatment is required for severe or disseminated infection or for infection involving the central nervous system and/or bone and joint and in pulmonary disease with ongoing afb detectable in sputum (>2 months) • streptomycin should not be used in the lung transplant setting because of the associated high-risk of nephrotoxicity. • immunosuppressive agents used to prevent rejection may only require minimal or no dose reduction. this is because immune reconstitution inflammatory syndrome (iris) can occur even when the immunosuppressant agents are not dosereduced as the anti-tb treatment can reverse some of the immunosuppressive effects of tb. screening for latent tb (prior exposure to m. tuberculosis which can reactivate and cause clinical disease) needs to be performed pre-transplant in all lung transplant candidates. two tests are available, namely, the tuberculin skin test (tst) and the interferon-gamma release assay (igra). the igra is used in most centres. screening algorithms are available [10, 13] . as the risk of reactivation and severe infection is increased in transplant recipients and the annual risk of active tb with a positive tst is 7.4%, there is a good argument for latent tb treatment. the optimal timing for latent tb treatment is pre-transplant. latent tb should be treated if: • the initial or boosted tst produces induration of ≥5 mm or a positive igra; • prior history of untreated latent tb; or • receipt of an organ from a donor known to have untreated latent tb. isoniazid (with oral pyridoxine) is the treatment of choice and has a low risk of toxicity. rifampicin for 16 weeks or isoniazid in combination with rifapentine for 12 weeks are alternative regimens, but only pre-transplantation because of drug interactions. as already stated person-to-person transmission of tb can occur. the major route is by inhalation of airborne particles. there are a number of factors that increase the risk of transmission of airborne particles including presence of untreated active pulmonary or laryngeal tb, cavitary disease, smear positivity and short time to positive m. tuberculosis culture. a number of procedures can also increase the risk of dispersal of airborne particles including intubation and bronchoscopy. patients with extrapulmonary tb are not contagious; however, concomitant pulmonary or laryngeal tb needs to be excluded firstly. immunocompromised patients with extra-pulmonary tb should be presumed to have pulmonary tb until proven otherwise. there are numerous international and national tb control guidelines on which hospitals base infection control programs for tb [14] . if tb is suspected or untreated: • the patient must be managed in airborne isolation rooms with negative pressure ventilation • masks must be worn by health-care workers when in contact with the patient and by the patient when he/she leaves the room • when tb is excluded the patient can be removed from isolation • for patients with confirmed tb isolation can be discontinued when the patient is receiving treatment, demonstrates a clinical response and has three negative afb smears from sputum • close liaison with the institutional infection control team is essential in cases of suspected or untreated tb. common ntm affecting lung transplant recipients include m. avium complex, m. kansasii and m. abscessus. these are environmental organisms so infection usually occurs via acquisition from an environmental reservoir and not person to person transmission. healthcare-associated infection from contaminated medical devices can occur and person-to-person transmission has been described with m. abscessus [15, 16] . risk factors for infection include cystic fibrosis as an underlying disease, the isolation of a ntm (particularly m. abscessus) pre-transplant and the use of rabbit anti-thymocyte globulin. median time to onset is later when compared with tb (1 year). the lungs are most commonly affected but cutaneous, soft tissue and disseminated infection can be seen, especially with m. abscessus, m. chelonae and m. kansasii [17] . with disseminated disease constitutional symptoms (e.g. sweats, tiredness, weight loss) predominate [18] . the most common radiological features seen are fibrocavitary and cavitary, nodules, bronchiectasis, tree-in-bud, and large opacities (>2 cm) [19] . diagnosis is very challenging as these are environmental organisms and it is difficult to determine whether isolation of these organisms reflects contamination/ colonization or true infection. guidelines for diagnosis exist for ntm [20] . factors such as organism burden, specific species, clinical signs and symptoms and radiological features all need to be considered when determining infection category and whether or not to treat. treatment is similar to the immunocompetent population. a multi-drug regimen is used (see table 15 .2); however, similar to tb a few specific points need to be considered in the transplant setting. • susceptibility testing should be performed to direct initial and maintenance regimens. • clarithromycin can increase serum levels of calcineurin inhibitors and rapamycin agents via the cytochrome (cyp) 3a4 pathway so with the initiation and cessation of clarithromycin the immunosuppressant agents may need dose adjustment. close monitoring of immunosuppressant concentrations is required. • the issues outlined above for rifamycin use in tb treatment also apply to the treatment of ntm. • the duration of treatment is longer than for the immunocompetent population. the minimum is usually 12 months after last positive culture; but lifelong suppressive therapy may be needed in some patients. • reduction of immunosuppression needs to be considered. • surgical resection may be required if: -large abscesses are present -there is a large burden of disease -focal disease not responding to therapy -the patient cannot tolerate therapy. m. abscessus is a particular problem in the lung transplant setting. it is increasing in incidence and can cause disseminated infection post-lung transplant which can be very difficult to eradicate. it is also resistant to many of the available antimicrobial agents and drug-related toxicity has been detected in up to 44% post-lung transplantation [21, 22] . treatment is complicated and prolonged. in some centres isolation of m. abscessus in a lung transplant candidate is considered as a strong relative contra-indication to transplantation [23] . other centres have determined that transplantation of patients with pre-transplant isolation of m. abscessus is possible with the precautions outlined in table 15 .3 [24, 25] . currently, expert opinion indicates that transplantation in those with pre-transplant isolation of m. abscessus should be decided on a case-by-case basis. as ntm are ubiquitous in the environment, transmission is usually from an environmental source. in addition, ntm are resistant to chlorine and have the ability to form bio-films. as a result infection control measures are directed at ensuring adequate disinfection of hospital equipment, rigorous and repeated surface cleaning and high-quality water supply. ongoing environmental surveillance in the hospital setting and close liaison with institutional infection control and engineering teams is critical to prevent outbreaks of ntm, particularly in the setting of construction. there are some evidence in the literature that m. abscessus has been associated with person-to-person transmission but other studies have indicated that this may not be the case [26, 27] . careful assessment of each institution's epidemiology will assist in deciding if patients with m. abscessus require airborne isolation or simply rigorous cleaning of the environment [28] . recently m. chimera contamination of heater-cooler units used in cardiac surgery has been reported resulting in cases of surgical-site and disseminated infection worldwide. new enhanced decontamination strategies have been developed and ongoing surveillance is required to ensure that these remain effective [29] . nocardia are ubiquitous, saprophytic, gram-positive bacteria that belong to the aerobic actinomycetes group. they are partially acid-fast rods that grow slowly in branching chains resembling fungal hyphae. there are more than 80 species but most infections in humans are caused by nocardia asteroides sensu stricto, n. farcinica, n. nova, and n. brasiliensis. infections with nocardia are increasing in lung transplant recipients [30] . whilst widespread throughout the world infections with nocardia have the highest frequency in dry windy climates which facilitate aerosolisation and dispersal. infections mostly occur in the first year after lung transplantation but are rare within the first month unless it is donor-derived infection. risk factors include corticosteroids (particularly in the preceding 6 months), and augmented immunosuppression (high median calcineurin inhibitor levels in the preceding 30 days) [30] . rituximab use and hypogammaglobulinaemia have also been associated with an increased risk of developing nocardia infection as has the use of alemtuzumab for treatment of allograft rejection [31] [32] [33] . inhalation is the most common route of infection therefore the lungs are most commonly affected. dissemination to other organs, particularly the skin and central nervous system (cns) has been reported in 50% of cases. the skin can also be infected by direct inoculation, especially if the lung transplant recipient is involved in outdoor activities. the most common signs and symptoms are fever, weight loss, cough, pleuritic chest pain and dyspnoea. chest imaging frequently shows irregular nodular lesions which may be cavitary (fig. 15. 3) [34] . other features include diffuse infiltrates or consolidation with associated pleural effusions. diagnosis is by microscopy, culture and histological examination of respiratory specimens (most particularly bronchoalveolar lavage fluid [bal]) or biopsy tissue (e.g. skin or brain tissue). nocardia grows on non-selective media forming characteristic white and chalky colonies. if there is a suspicion that the infection may be nocardia inform your diagnostic laboratory as the specimens require longer incubation for the growth of nocardia and in samples with mixed growth (that is multiple organisms [particularly sputum]) nocardia may be obscured. selective media can be used to improve the yield of nocardia growth (e.g. thayer-martin). nocardia has characteristic features on gram stain (see fig. 15 .4). in tissue nocardia appears as gram positive branching and beaded rods with surrounding pyogenic inflammatory reaction. it is important to determine the species and susceptibility profiles as different species have different susceptibility profiles. this information is very useful in determining the treatment regimen. antibiotics are the mainstay of treatment. the site(s) and burden of infection, the species and the potential drug-drug interactions all determine the antimicrobial regimen to be used for treatment [35] . • mild pulmonary infection-trimethoprim-sulfamethoxazole (tmp-smx) for 6-12 weeks • severe pulmonary infection (no cns involvement)-parenteral treatment with tmp-smx plus amikacin • cns infection-parenteral treatment with tmp-smx plus imipenem • multi-organ infection including the cns-intravenous (iv) amikacin added to the regimen of iv tmp-smx and imipenem. • meropenem may be used instead of imipenem as the former is less likely to precipitate seizure activity. sensitivity to meropenem must be demonstrated in the laboratory before use [36] . • linezolid has excellent in vitro activity against nocardia and has been used with success in treatment; therefore linezolid may be used as part of a multi-drug regimen [37, 38] . • if the patient has a tmp-smx allergy desensitisation should be performed if possible. parenteral treatment is continued for 3-6 week followed by oral therapy for 6-9 months. oral agents that are commonly used include tmp-smx, minocycline and/or amoxicillin-clavulanate. surgery may be required in cases of cerebral nocardiosis or large soft tissue abscesses not responding to treatment, empyema or mediastinal fluid collections and for pulmonary nocardiosis that is complicated by pericarditis. consideration should be given to reducing immunosuppression especially in cases with severe disease or those progressing on anti-microbial treatment. indefinite secondary prophylaxis is also recommended as the immunosuppression cannot be fully reversed. there are no reports of person-to-person transmission of nocardia in the literature. as nocardia are ubiquitous environmental organisms, acquisition is mostly from an environmental source. similar to ntm infection control measures in the hospital setting for nocardia are directed at disinfection of equipment and surfaces and ensuring high-quality water supply. ongoing surveillance is required to prevent outbreaks, particularly in the setting of construction. fungal infections are a significant problem in lung transplant recipients occurring in 8.6% and causing death in up 39.5% of those infected [39, 40] . the majority of infections are caused by aspergillus and candida species. cryptococcus is the third most common cause of fungal infection. the fungi that cause mucormycosis (e.g. rhizopus species), scedosporium, and fusarium are emerging and are associated with very high mortality rates (60.5%); thus, increasing emphasis is placed on early recognition, diagnosis and treatment [40] . histoplasma, coccidioides and blastomyces species are important for those who live in or have previously resided in or visited endemic areas. pneumocystis jirovecii, whilst infrequent, can cause significant morbidity and mortality. the risk factors for infection are very similar to those described above for bacterial infection. in addition, fungal infections have been implicated in triggering the development of chronic rejection (that is, chronic lung allograft dysfunction [clad]) [41] . fungi are a major problem in lung transplant recipients. the importance of thinking about fungi in any lung transplant recipient suspected of having infection cannot be over-estimated. early diagnosis and treatment is critical to optimising outcomes. prophylaxis may reduce the impact of fungal infections in lung transplant recipients but issues such as drug intolerance and drug-drug interactions and the emergence of resistance may complicate treatment and reduce overall efficacy. fungi can be a single cell or complex multicellular organisms. fungi are mainly found in soil or on dead plant matter. they can be divided up into yeasts, multicellular filamentous moulds and dimorphic fungi. yeasts are small, lemon-shaped single cells that are around the size of red blood cells. they multiply by budding a daughter cell off from the original parent cell. multicellular filamentous moulds are made up of very fine threads known as hyphae. they grow from the hyphal tips and divide repeatedly along their length creating long and branching chains. some of the hyphal branches grow into the air and spores form on these aerial branches. these spores can be carried by the wind, rain or insects to new habitats where they can germinate to start growing and producing new hyphae. the process of infection is mimicked in immunosuppressed individuals where the conidia (spores) are inhaled and with impaired immune defence mechanisms the conidia (spores) can germinate and uncontrolled hyphal growth can occur. dimorphic fungi are fungi that can exist as yeast or mould. a prime example of a dimorphic fungus is penicillium marneffei, a human pathogen that exists as a mould at room temperature but as yeast at human body temperature. aspergillus fumigatus is the most common of all aspergillus species [42] . other species that can cause infection in the lung transplant setting include a. flavus, a. terreus, a. niger and a. nidulans [42] . the importance of identifying a. terreus is that it has a different susceptibility profile to the other aspergillus species. it is resistant to amphotericin b [43] . aspergillus species commonly cause 4 types of infection in lung transplant recipients: • aspergillus colonisation • tracheobronchial aspergillosis • invasive pulmonary aspergillosis (ipa) (also known as aspergillus pneumonia) • disseminated invasive aspergillosis (ia). aspergillus colonization is defined as the detection of aspergillus in respiratory secretions by culture, pcr or by the detection of aspergillus galactomannan (a cell wall protein) in the absence of any symptoms, lesions in the airways seen on bronchoscopy or new changes seen on chest x-ray or computed tomography (ct) scan [44, 45] . aspergillus colonization has been detected pre-transplant in 8-59% of patients (most commonly in cystic fibrosis [cf] patients) and is a risk factor for post-transplant ipa and clad [3, 7] . post-transplant colonization is found in 30-40% [45] . some centres give antifungal agents to all lung transplant recipients (immediately post-transplant for 4-6 months) to minimize aspergillus colonization and its complications [45, 46] . with the use of universal prophylaxis the time to aspergillus colonisation has lengthened from 3.2 months to 6.8 months post-lung transplant [47, 48] . other centres only give antifungal treatment (for 3 months) once aspergillus is detected [45, 46] . this is known as the pre-emptive strategy. it is not known which strategy is best. tracheobronchial aspergillosis is defined as the detection of aspergillus in respiratory secretions by culture, pcr or the detection of aspergillus galactomannan with new lesions demonstrated on bronchoscopy including patches of redness (erythema), ulceration, necrosis or pseudomembranes but with no changes detected on chest x-ray or ct scan [44, 45] . the patient may be asymptomatic or may present with symptoms such as fever, cough, wheeze and/or hemoptysis [49] . it occurs in the majority of patients in the first 3 months post-lung transplant [47] . the importance of tracheobronchial aspergillosis is that the lung transplant recipient is at risk of progressing to ipa or disseminated ia [47] . • the treatment of choice is voriconazole. alternative agents include amphotericin b, posaconazole and itraconazole • combine with nebulized amphotericin b for a direct local effect [50] . • repeated bronchoscopic debridement particularly in those with large amounts of necrotic debris [51] • stenting occasionally required to maintain a patent airway the duration of treatment is dependent on the severity of the initial infection, degree of immunosuppression and response to therapy but should be given until the lesions have completely healed and potentially life-long in those with bronchial anastomotic involvement. proven ipa is defined as evidence of parenchymal (lung tissue) invasion by aspergillus hyphae or positive culture from sterile lung tissue alone or with signs/ symptoms such as fever, abnormal white cell count, new onset purulent sputum or change in the character or quantity of sputum or respiratory secretions, new onset or worsening cough, dyspnoea, tachypnoea, pleural rub, crackles or bronchial breath sounds. probable ipa is defined as signs/symptoms (as above) and new or progressive and persistent infiltrate, consolidation, cavitation or nodules and detection of aspergillus in respiratory secretions by culture, pcr or the detection of aspergillus galactomannan (single positive for bronchoalveolar lavage [bal] or 2 positives for sputum) (fig. 15 .5) [44, 45] . average time to development is 6 months [52] . in disseminated ia, respiratory disease can be associated with infection in the sinuses, orbits and central nervous system (cns). other sites where aspergillus can rarely cause infection include skin, bones, eyes (endophthalmitis), in the intra-abdominal cavity or retroperitoneum (e.g. abscess) and in the pericardium [42, 47] . • voriconazole is the treatment of choice [53] • an echinocandin (anidulafungin, caspofungin, micafungin) can be added for synergy in those with extensive disease or who are very unwell (e.g. hypoxic at presentation) [53] • treatment of disseminated disease is the same as for ipa and as for ipa treatment continues until complete resolution it is important to remember that when giving voriconazole (or other azole antifungal agents) in lung transplant recipients there are significant interactions with the immunosuppressant (e.g. tacrolimus, cyclosporine and sirolimus). dose adjustments of the immunosuppressants are required at initiation and cessation of voriconazole (or other azole) and regular monitoring of serum immunosuppressant levels is required. no specific infection control measures required. the most common infection type seen with candida species is candidaemia (infection in the bloodstream; fig. 15.6 ). this is most common during the first month post-transplant and is usually related to the recent surgery, intensive care unit stay and broad-spectrum antibiotic use peri-transplant. tissue infections can also occur and include infected pleural effusion, pleural space infection, infection of the incision sites and bronchial anastomotic site infections [50, 54] . candida species are frequently isolated from the mouth, pharynx, sputum and bal specimens but almost never spread to invade the lung tissue. universal prophylaxis targeting candida species during the first month post-transplant have been shown to be effective [55] . however, universal prophylaxis may be associated with the emergence of resistant candida strains [56] . candidaemia can manifest as fever or as severe sepsis (e.g. hypotension, tachycardia, requirement for inotrope support). invasive candidiasis is related to the site of the infection. for example, if disseminated to the skin invasive candidiasis cause skin pustules or to the eye results in endophthalmitis. blood cultures are still the gold-standard for the diagnosis of candidaemia; therefore a blood culture is required for all patients in whom candidaemia is suspected. in patients with invasive candidiasis a biopsy of the relevant tissues for staining, culture and histological examination is useful. some centres have access to beta-d-glucan testing. this non-culture based assay detects a cell wall protein of candida species and is a useful as an additional test (in addition to blood cultures and biopsy) in some patients, particularly those with intra-abdominal candidiasis. • echinocandin or liposomal amphotericin b for the treatment of candidaemia and serious candida infection [45] . • once the candida is detected and the sensitivity profile is known antifungal therapy can be altered [45] . if the isolate is sensitive to fluconazole then a change to this agent is recommended [45] • if candida is causing symptomatic infection of the urinary tract an echinocandin is not recommended as it has poor penetration into the urinary tract [45] . in this setting, fluconazole (if the isolate is sensitive) or amphotericin b and 5-flucytosine in combination (if the isolate is fluconazole-resistant) is recommended [45] . no specific infection control requirements. cryptococcus causes infection in 2% of lung transplant recipients. the most common site of cryptococcal infection is the lung (fig. 15 .7) but disseminated infection can also occur with a predilection to the central nervous system. skin involvement including cellulitis [57] and infection transmitted in the donor lungs has also been described. the median time to infection onset is 190 days. in addition to the usual diagnostic tests of culture and biopsy cryptococcal antigen assay is very useful as it is sensitive and specific and can be used to monitor disease treatment response. pre-transplant cryptococcosis has been described and is not a contra-indication to transplantation so long as disease control has been achieved with no positive cultures and cryptococcal antigen level is declining. fluconazole is continued throughout the transplant procedure and for a minimum of 6 months post-transplantation. immune reconstitution inflammatory syndrome (iris) is common with treatment of cryptococcal infection (5-14%) [58] and manifests as an apparent flare of the antifungal agents used depend on the site and burden of infection, indicating that diagnosis/exclusion of cns disease by ct scan or mri scan of head, a lumbar puncture for culture and cryptococcal antigen testing and ct of chest to determine extent of disease is critically important. • cns infection, disseminated infections or severe lung disease-liposomal amphotericin b and 5-flucytosine for a minimum of 2 weeks followed by fluconazole at high dose for 8 weeks and fluconazole at lower doses from 6 months to 1 year is recommended. • small volume pulmonary disease-fluconazole alone for 6-12 months is recommended [59] . no specific infection control requirements. the most common manifestation is pulmonary infection or infection of the cns and sinuses but gastrointestinal infection (likely through ingestion) has also been described [60] . the cumulative incidence is 0.07% and it accounts for 2% of all fungal infections. risk factors for infection include renal failure, diabetes and prior voriconazole and/or caspofungin use [61] . mucormycosis is particularly associated with tissue infarction and necrosis due to invasion of the tissue blood vessels with the growing hyphae ( fig. 15.8) . the fungi also spread rapidly along tissue planes. both these factors contribute to the high mortality rates of up to 87% seen with this infection. in view of the aggressive nature of the fungus and high mortality rate treatment requires a multi-pronged approach. • anti-fungal therapy -first-line therapy is liposomal amphotericin b. -caspofungin can be added if the infection is severe. -posaconazole or isavuconazole can be given as maintenance therapy or if the patient in intolerant of liposomal amphotericin b. • surgical debridement of all the necrotic tissue ( fig. 15.8 ), • reduction of immunosuppression • reversal of underlying factors (e.g. diabetes mellitus) no specific infection control measures required. scedosporium is an environmental organism that is recognised worldwide but has a higher incidence in specific geographical areas such as spain, the middle east and australia. it is a common fungus in floods, tsunamis and tornados resulting in a risk for transmission if the donor drowned [62] . it is commonly isolated from cf patients pre-transplant. risk factors for invasive scedosporium disease post-transplant include pre-transplant colonisation, prior receipt of amphotericin b and augmented immunosuppression. scedosporium is prone to disseminate and can be detected in blood cultures unlike other moulds such as aspergillus. in lung transplant recipients scedosporium mainly causes colonisation with invasive infection occurring in about 25%. the most common clinical manifestations of invasive disease include pneumonia, mediastinitis, fungaemia or disseminated disease [63] . progression to invasive disease is more likely in those with pre-transplant isolation; thus, if scedosporium is isolated prior to transplantation it should be treated [63] . • scedosporium is innately resistant to many of the available antifungal agents including amphotericin b. • s. apiospermum is sensitive to some of the azole antifungal agents, particularly voriconazole • a combination of voriconazole and terbinafine may be the only option against s. prolificans (now known as lomentospora prolificans) [63] . no specific infection control measures required. fusarium accounts for <1% of all invasive fungal disease in solid organ transplant patients with lung transplant recipients most commonly affected. like scedosporium whilst fusarium occurs worldwide it has a higher incidence in some countries such as in brazil, where the incidence of fusarium is second only to aspergillus. infection usually occurs within a year of transplantation and most commonly affects the lungs. outcome is poor with a 67% mortality rate. voriconazole is the most effective agent. no specific infection control measures required. histoplasma is endemic to the states bordering the ohio river valley and the lower mississippi river, usa but it has also been detected in montana and idaho. other countries and regions where it has been isolated include canada, mexico, central and south america, parts of eastern and southern europe, africa, eastern asia and australia. pulmonary and disseminated infections are the most common manifestations post-transplant. infection can range from asymptomatic to severe. the diagnosis is made by using a combination of serology (antigen and antibody), culture of respiratory secretions and biopsy with histological examination of the affected tissue [64] . routine screening pre-transplant is not recommended. serial monitoring or the administration of prophylaxis is recommended in those who had active infection prior to transplantation [65] . • mild disease-itraconazole • more severe infection-amphotericin b [66] infection control no specific infection control measures required. coccidioides are fungi that endemic to the southwest of the united states particularly the san joaquin valley, and the sonoran desert of southern california, arizona and northern mexico. in the lung transplant recipient these fungi can cause severe pneumonia or disseminated infection. disseminated infection is most commonly characterized by skin, bone and joint lesions and/or meningeal involvement. diagnosis is established by serological testing, culture or histopathology. pre-transplant assessment is required and includes a detailed past history [65] . any history of residence or travel to an endemic area requires evaluation with serological testing and chest x-ray [65] . any transplant candidate with past infection requires assessment by a specialist infectious diseases physician for clearance for transplantation [65] . in the case of active infection transplantation is deferred until the infection is quiescent (on radiology, clinically and serologically) [65] . • focal pneumonia can be treated with fluconazole • diffuse disease is treated initially with amphotericin b until clinical response followed by fluconazole or itraconazole • coccidioidal meningitis is treated with fluconazole no specific infection control measures required. blastomyces is endemic to parts of eastern north america, particularly northern ontario, south-eastern manitoba, quebec, south of the st. lawrence river, parts of the appalachian mountains and the interconnected eastern mountain chains, the west bank of lake michigan, the state of wisconsin and the entire mississippi river including the valleys of the major tributaries (e.g. ohio river). it also occurs in africa, the arabian peninsula and the indian subcontinent. similar to histoplasma and coccidioides it causes pneumonia and skin involvement (with verrucous or wartlike lesions) and is also common in the transplant recipient. diagnosis is made by culture of sputum, bal or tissue or by histopathological examination of biopsy tissue. pre-transplant assessment includes symptom assessment and chest radiography for those who live in endemic areas [65] . prophylaxis is given on a case by case basis. • liposomal amphotericin b until clinical improvement followed by oral itraconazole no specific infection control measures required. pneumocystis jirovecii was previously classified as a protozoan but with modern molecular techniques it has recently been reclassified as a fungus [67] . it was previously named p. carinii (which infects rats) but has been renamed p. jirovecii as this is the species that infects humans [68, 69] . if prophylaxis is not universally administered 5-15% of all solid organ transplant recipients develop p. jirovecii pneumonia (pjp) with the highest incidence occurring in the lung and heart-lung transplant recipients [70] . most centres administer pjp prophylaxis and as a result very few cases are now seen. the most important risk factor for pjp is corticosteroid use in combination with other immunosuppressive agents [71] . there are no good data as to a dose and duration of corticosteroids to decide when to give prophylaxis. the period of highest risk is the first 6 months post lung transplantation but most centres recommend indefinite prophylaxis [72] . previously, patients presented in respiratory failure with fever and a dry cough but as the awareness of the significance of the infection has increased and as more sensitive diagnostic tests have been developed diagnosis is made earlier when the disease in mild or indolent (that is less severe cough and dyspnoea). chest x-ray or ct scan of thorax usually demonstrates diffuse bilateral infiltrates. it is important to make a microbiological diagnosis so obtaining a respiratory specimen (induced sputum or ideally a bal) is best. a lung biopsy is rarely required. the best test is pcr although it is very sensitive so false positive results can occur. serum beta-d-glucan testing may be a useful adjunct if available [73] . like the treatment of pjp the first-line agent for prophylaxis is tmp-smp but at lower doses (1 double-strength tablet 3 times a week or a single-strength tablet daily). alternatives include dapsone, atovaquone or aerosolized pentamidine. several clusters or outbreaks of pjp have been reported, particularly in renal transplant patients. in some of these clusters or outbreaks person-to-person transmission was postulated as the cause [74] . consequently hospitalised patients with pjp should not be placed in the same room as other immunocompromised patients. otherwise standard precautions apply [75] . this review clearly illustrates that fungi are a major problem in lung transplant recipients. the importance of thinking about fungi in any lung transplant recipient suspected of having infection cannot be under-estimated. early diagnosis and treatment is critical to optimising outcomes. prophylaxis may reduce the impact of fungal infections in lung transplant recipients but issues such as drug intolerance and drug-drug interactions and the emergence of resistance may complicate and reduce its overall efficacy. further multicentre research is required to determine the optimal prophylactic strategies for lung transplant recipients. viruses are organisms that are much smaller than bacteria and are unable to be detected on routine microscopy. they are only able to survive and replicate within a living cell, using the chemical machinery of that cell to reproduce. viruses contain either deoxyribonucleic acid (dna) or ribonucleic acid (rna). important dna viruses in the setting of transplantation include the herpesvirus family whilst the rna viruses include most significant respiratory pathogens. viral infection, either primary or following reactivation of latent virus, remains an important cause of morbidity and mortality following lung transplantation. viral culture is extremely laborious and difficult and is restricted to specialist laboratories. increasingly the diagnosis of viral infection is made by pcr of peripheral blood or affected tissue with pcr available for all the members of the herpesvirus family listed below. the members of the herpesvirus family are: hsv-1 and hsv-2 cause oral and genital ulceration but occasionally cause disseminated infection, particularly in the immunocompromised host. as a rule, hsv-1 causes 80% of oral infection and 20% of genital ulceration whereas hsv-2 is responsible for 20% of oral infection and 80% of genital infection. infection may be primary, which can be severe, or reactivation from the site of latency in the neurons. the incidence of prior infection increases with age and varies according to socio-economic status, race and country of residence, with 50-96% of the general population having antibodies to hsv-1 and therefore at risk of reactivation [76, 77] . the most common manifestation of hsv-1 and hsv-2 in lung transplant recipients are mucocutaneous ulcers involving either the oral cavity or genital tract. less commonly, pneumonia, hepatitis or encephalitis may result from viral reactivation. the introduction of acyclovir in the 1980s marked the first highly effective antiviral therapy and resulted in a significant reduction in morbidity and mortality from post-transplant hsv infections. the incidence of hsv-1 and hsv-2 has fallen dramatically since ganciclovir, an anti-cmv agent with activity against hsv, has been widely used as prophylaxis against cmv infection in the transplant setting. • acyclovir/valaciclovir/famciclovir • suppressive therapy may be appropriate for frequent recurrences • the development of resistant virus is uncommon standard precautions apply to patients with active hsv lesions; however contact precautions may apply in healthcare settings if lesions are not covered and for 3 days post initiation of treatment or until crusting occurs. if hsv is disseminated contact precautions required until lesions are dried and crusted. immunocompromised staff should not care for patients. infected staff in high risk clinical areas require urgent review for leave/ redeployment. vzv primary infection results in chicken pox. the virus then lays dormant in neural tissue prior to reactivating as shingles, in particular during periods of immunosuppression. shingles may follow a single nerve pathway or dermatome, may involve multiple dermatomes or the virus may disseminate involving a variety of organs including the liver, lungs, brain and spinal cord. approximately 90% of adults in australia and the united states have antibody against vzv indicating prior infection. however, the incidence of antibody positivity varies between geographic areas with the incidence lower in tropical regions. patients who do not have antibodies to vzv should be considered for vaccination prior to transplantation. as the vaccine is a live vaccine it should not be administered after transplantation as there is insufficient safety data in immunosuppressed transplant recipients [78] . • high dose acyclovir/valaciclovir/famciclovir • ganciclovir • potential role for zoster immune globulin contact precautions for patients with active vzv lesions and for 3 days postinitiation of treatment or until crusting occurs. ebv is the causative agent of infectious mononucleosis (glandular fever), a common infection in the general population. it is also associated with the development of two cancers, nasopharyngeal carcinoma and burkitt's lymphoma. like other herpesviruses, ebv is associated with latent infection; in the case of ebv, b lymphocytes in the blood and lymphoid tissue which sets the scene for lymphoproliferative disorders. in the setting of transplantation, ebv has a clearly established role in the pathogenesis of post transplantation lymphoproliferative disorder (ptld) with up to 90% of cases associated with ebv latent infection. ptld is a spectrum of disease caused by the abnormal proliferation of lymphoid cells, with clinical manifestations varying from asymptomatic to tissue infiltration and/or focal masses in a variety of organs. figure 15 .9 is a pet-ct scan from a patient with ptld and demonstrates the widespread involvement that can occur. diagnosis is made by excisional biopsy and histological examination. high levels of ebv dna measured by pcr in peripheral blood provide supportive evidence. • reduce the level of immunosuppression • no good data to support a role for antiviral therapy (acyclovir, ganciclovir) • immunomodulatory agents such as anti cd20 (rituximab) • resection of localised lesions no specific precautions are required. cmv infection is defined as the detection of cmv replication (usually by pcr to detect cmv dna or rna in plasma or whole blood) regardless of the clinical presentation or symptoms. as with other herpesviruses, cmv infection may be the effects of cmv infection may be due to either direct tissue damage to a variety of organs (e.g. colitis) or indirect effects on the graft and the immune system (e.g. induce clad). figure 15 .10 demonstrates the 'owl's eye' appearance of cmv inclusion bodies in the bowel of a patient with cmv enteritis. there are two approaches to the prevention of cmv disease. • prophylaxis strategy-prescribing anti-cmv drugs for a defined period after transplantation (usually 6-12 months). • pre-emptive therapy-treatment with anti-cmv drugs only when the plasma or blood cmv pcr becomes positive during regular monitoring the choice of strategy varies between transplant centres and will in part be determined by the ability to rapidly and regularly perform cmv pcr on blood or plasma. in the setting of lung transplantation the prophylaxis strategy is the most frequent approach. in addition, high risk d+/r− patients are more likely to receive prolonged cmv prophylaxis. despite the various approaches to prevent cmv disease, active infection occurs in up to 30% of transplant recipients [79] . treatment options include: hhv-6 is very common in the community with approximately 95% of the general population demonstrating serological evidence of prior infection [80] . as with other herpesviruses it remains latent after primary infection and frequently reactivates after transplantation. however the significance of reactivation is uncertain as it is not reliably associated with any specific clinical syndrome. infection is most commonly asymptomatic but encephalitis, hepatitis, gastro-duodenitis and pancytopenia have been described. cmv prophylaxis does not appear to prevent hhv-6 reactivation [81] . there is limited clinical treatment data available but ganciclovir, valganciclovir and foscarnet appear to have activity against hhv-6 in laboratory testing. there may be a role for reduction of immunosuppression. no specific infection control measures required. like hhv-6, hhv-7 infection is very common in the community and reactivation can occur following transplantation. however the clinical importance of this is uncertain with no syndromes regularly associated with this virus. for this reason most laboratories do not perform pcr for hhv-7. there is minimal anecdotal data and no controlled trials for the treatment of hhv-7 although anti-cmv drugs such as ganciclovir, foscarnet and cidofovir may be effective. there are no specific infection control procedures required. along with ebv, hhv-8 is an oncogenic or cancer-causing herpesvirus. clinical manifestations include kaposi's sarcoma (ks), body cavity lymphoma and castleman's disease, a rare lymphoproliferative disorder. the prevalence of hhv-8 varies greatly, from 0 to 5% in north america and northern europe to up to 70% in regions of sub-saharan africa and the southern mediterranean where the virus is endemic [82] . previously recognized as an uncommon malignancy of elderly mediterranean men, african children, and ashkenazi jews, ks became the most common neoplasm of patients with hiv infection with an incidence >20,000 times that of the general population [83] . seropositive transplant recipients have a small risk of reactivation of latent virus and donor-derived infection has been infrequently reported. ks is the most common manifestation and body cavity lymphoma and castleman's disease are rare presentations of hhv-8. diagnosis of hhv-8 reactivation is generally by pcr whilst ks, body cavity lymphoma and castleman's disease require histological diagnosis. treatment antiviral drugs do not appear to be clinically effective. the mainstay of treatment includes: • reduction of immunosuppression or reversal of underlying immune deficiency • chemotherapy • rituximab for castleman's disease no specific infection control procedures required. respiratory viruses circulate within the community with seasonal and geographic variability. serious complications are uncommon in the non-immunocompromised host but in the setting of lung transplantation respiratory virus infections are associated with secondary bacterial infections, acute rejection and chronic graft dysfunction. increased susceptibility to respiratory viruses in lung transplant recipients is multifactorial and includes immunosuppression, impaired cough reflex, poor mucociliary clearance, altered lymphatic drainage and the direct exposure of the lung allograft to the environment. a prospective study compared 50 lung transplant recipients with respiratory virus infection with 50 uninfected recipients and demonstrated that those with a respiratory virus infection had a greater risk of acute rejection, bronchiolitis obliterans syndrome and death [84] . important respiratory viruses include: respiratory viral infections are common in lung transplantation. a recent study of 112 lung transplant recipients over a 2 year period found an infection rate of 19.3% with 61% having one or more viral infections over the study period [85] . asymptomatic carriage was uncommon (<10%) and was mainly associated with coronavirus/rhinovirus. the hospitalisation rate was 50% for influenza and parainfluenza and 16.9% for other viruses. infection control precautions for respiratory viruses include droplet precautions (single room, mask, gown and gloves for room entry) until asymptomatic and hand hygiene as per 5 moments. staff should not come to work if they have a respiratory illness and unwell visitors should not be allowed patient contact. chemoprophylaxis may be administered to patients following exposure where appropriate (see below for specific viruses). many microbiology laboratories perform a respiratory pathogen pcr diagnostic panel which includes the common respiratory viruses such as influenza a, influenza b, enterovirus, rhinovirus, coronavirus, hmpv, parainfluenza, adenovirus, rsv and non-viral organisms including bordetella pertussis, bordetella parapertussis, mycoplasma pneumoniae and pneumocystis jirovecii. testing is generally performed on nose and throat swabs (both required), a nasopharyngeal aspirate or bronchial washings. like many respiratory viruses, rsv is seasonal with a winter predominance. in healthy adults rsv is usually associated with mild, self-limited infection but in lung transplant recipients rsv can cause bronchiolitis, pneumonia and respiratory failure with a significant acute mortality up to 20% [86] and decline in lung function associated with the subsequent development and progression of bronchiolitis obliterans syndrome (bos) [87] . rsv has also been associated with acute rejection but a recent prospective study failed to confirm this finding [85] . ribavirin is a nucleoside analogue with broad range of activity against many rna viruses and, despite a lack of randomised trial data, is the cornerstone of treatment for rsv. ribavirin can be administered in 3 ways: • oral • intra-venous • aerosolised (negative pressure room and specific equipment required) advice regarding administration and dosing regimen should be sought as ribavirin has significant toxicity (primarily haematological), is teratogenic and has a very long half-life. standard and droplet precautions required. patients should be managed in a single room. influenza is a seasonal virus with the greatest incidence of infections during the winter months, although it is detectable year-round in the community. the two most frequent types are influenza a followed by influenza b. influenza viruses characteristically undergo 'antigenic drift' or minor annual changes in the surface glycoprotein that allows reinfection due to inadequate immunity. every 10 years or so 'antigenic shift' occurs secondary to the reassortment of genes between species. major outbreaks of influenza occur at this time as there is little immunity present in the community. the rate of influenza is higher after lung transplantation than other solid organ transplants [88] and may be community acquired, nosocomial or donor-derived infection. complications such as viral and bacterial pneumonia occur more frequently than in the general population. annual vaccination of transplant recipients, transplant candidates and their families is strongly recommended although the antibody response may be impaired in immunosuppressed patients [89] . treatment should be initiated in all transplant patients with suspected or proven influenza and ceased if an alternative diagnosis is made. therapeutic options include: • oseltamivir (oral, influenza a and b) • zanamivir (inhaled, influenza a and b) • amantadine (oral, influenza a only) • rimantadine (oral, influenza a only) chemoprophylaxis should be offered to patients known to be exposed to influenza virus either in the hospital or the community setting. droplet and standard precautions for duration of symptoms or until 3 days of active influenza treatment. parainfluenza viruses (piv) consist of a group of 4 serotypes, piv 1-4, which circulate year-round in the community and cause a variety of clinical presentations from the 'common cold' to bronchiolitis and pneumonia. piv 3 has been associated with large hospital outbreaks of infection due to person-to-person transmission, especially in haematology wards, with mortality rates up to 30% in outbreaks [90] . in lung transplant recipients piv infection can lead to loss of lung function and bronchiolitis obliterans syndrome. figure 15 .11 shows extensive interstitial pneumonia in a patient with severe piv infection. there are no randomised studies of antiviral therapy. however there are reports published primarily in the haematology setting suggesting ribavirin, either orally or intravenously administered, may be effective treatment. a small single centre study of rsv and piv in lung transplant recipients indicated that 33% of lung transplant patients with lower respiratory tract paramyxoviral infections who were treated with inhaled ribavirin died or did not return to baseline lung function [91] . droplet and standard precautions. hmpv was first described as recently as 2001 and has been increasingly recognised as a seasonal (predominantly late winter) respiratory pathogen causing both upper and lower respiratory tract infection. about 100% of school aged children have antibodies to this virus indicating the widespread nature of hmpv [92] . hmpv is closely related to rsv and in lung transplant recipients is thought to result in graft dysfunction. however there was mainly anecdotal data to support this until a recent review by dosanjh [93] who conducted a literature search to identify cases of both hmpv and allograft rejection within 6 months of the initial infection. 1007 lung transplantation recipients, with a total of 2883 samples, were identified. of these, 57 had hmpv without co-infection with other agents. the results of the study indicated that 35% of acute hmpv infections without co-infection were associated with acute cellular rejection within 3 months and 9.4% of the cases subsequently developed chronic allograft dysfunction/bronchiolitis obliterans syndrome suggesting that hmpv is an important pathogen in the lung transplant setting. ribavirin has been shown to have activity against hmpv in vitro [94] and in animal models of infection [95] . however no human studies in hmpv infection have been performed and the use 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the name really been changed? has the name really been changed? it has for most researchers opportunistic infections in patients with and patients without acquired immunodeficiency syndrome prevention of infection due to pneumocystis spp. in human immunodeficiency virus-negative immunocompromised patients risk factors for pneumocystis carinii pneumonia in kidney transplant recipients: a case-control study accuracy of β-d-glucan for the diagnosis of pneumocystis jirovecii pneumonia: a meta-analysis an outbreak of pneumocystis jiroveci pneumonia with 1 predominant genotype among renal transplant recipients: interhuman transmission or a common environmental source? guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings seroprevalence of hsv-1 and hsv-2 in the general french population the burden of infection with hsv-1 and hsv-2 in england and wales: implications for the changing epidemiology of genital herpes update on immunisations in solid organ transplant recipients: what clinicians need to know meta-analysis: the efficacy of strategies to prevent organ disease by cytomegalovirus in solid organ transplant recipients a prospective survey of human herpes virus 6 infection in solid organ transplant recipients human herpesvirus-6 and -7 after lung and heart-lung transplantation principles and practice of paediatric infectious diseases kaposi's sarcoma among persons with aids: a sexually transmitted infection? clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study community respiratory viral infection in adult lung transplant recipients human metapneumovirus in lung transplant recipients and comparison to respiratory syncytial virus influenza virus in solid organ transplant recipients influenza vaccine responses in lung transplant recipients molecular epidemiology of two outbreaks of parainfluenza 3 in a bone marrow transplant unit clinical features and outcomes of paramyxoviral infection in lung transplant recipients treated with ribavirin pediatric human metapneumovirus infection: epidemiology, prevention and therapy respiratory metapneumoviral infection without co-infection in association with acute and chronic lung allograft dysfunction comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro effect of ribavirin and glucocorticoid treatment in a mouse model of human metapneumovirus infection rhinovirus as a cause of fatal lower respiratory tract infection in adult stem cell transplant patients: a report of two cases chronic rhinoviral infection in lung transplant recipients invasive adenoviral infection in t-cell depleted hematopoietic stem cell transplantation: high mortality in the era of cidofovir adenovirus infection in the lung results in graft failure after lung transplantation there are no randomised studies of treatment. anecdotal case reports suggest cidofovir may have a role but results are mixed. droplet and standard precautions. key: cord-007362-pjpkz6wv authors: bielefeldt-ohmann, helle title: the pathologies of bovine viral diarrhea virus infection: a window on the pathogenesis date: 2016-01-06 journal: vet clin north am food anim pract doi: 10.1016/s0749-0720(15)30461-8 sha: doc_id: 7362 cord_uid: pjpkz6wv pathologic lesions caused by bovine viral diarrhea virus (bvdv) infections comprise a wide spectrum of type, degree, and, by implication, pathogenesis, including congenital defects, necrotic-erosive lesions in mucosal epithelia and skin, and reactive as well as degenerative changes in lymphoid tissues. at least some of the pathology may not be solely due to bvdv replication per se, but rather caused by a host response to the virus, particularly the production of pro-inflammatory cytokines. the pathologic lesions caused by bovine viral diarrhea virus (bvdv) infections comprise a wide spectrum of both types and degrees, and are, like the clinical manifestations,6, 102, 114 a result of the interactions of factors such as strain and biotype of the virus (see the article by donis, this issue), 35, 36, 51-53, 60,77,113,116,121 ,122 the host genotype, age, and immune status of the animal at the time of infection, the immune response induced, and intercurrent infections or other stress factor(s). 2, 29, 44, 45, 96 some of the lesions are not specific for bvdv, reflecting the limitations in tissue reactions to noxae or parallels in pathogenic mechanisms. in other cases, only microscopic or submicroscopic changes are present, and, thus, not apparent in a routine postmortem examination. other lesions, or rather simultaneous occurrence of a set of lesions, however, may be near pathognomonic for bvdv, and when combined with clinical manifestations and epidemiologic data, they can have conclusive diagnostic value. the latter may be the case for "typical" mucosal disease (md) and for the occurrence of congenital defects of epidemic proportions. with the improvements in and wider application of virus diagnosis in clinical practice, however, it has become apparent that bvdv may be the cause of a much wider spectrum of clinical and pathologic phenomena, some of which in the past probably went undetermined. 96 traditionally, the pathologic (as well as clinical) manifestations of bvdv infection are presented under the categories (1) fetal infections, (2) acute virus diarrhea, (3) md. this categorization will largely be maintained, although it should be emphasized that these groupings are only truly useful for didactic purposes, and that extensive overlap can be expected in the occurrence and severity of any particular type of lesion, between animals or outbreaks,i4, 18, 36, 113, 114 and that pathologic lesions can only be suggestive of a bvdv infection. a final diagnosis must rest on isolation of the virus and/or detection of specific viral antigen in appropriate samples. in this section, special emphasis will be given to the histopathologic features and immunocytochemical analysis in persistently infected (pi) animals, whether clinically normal or suffering from md, as a basis for a final discussion of pathogenic mechanisms, which, in addition to the virus replication per se (see articles by donis and bolin, this issue), may contribute to the varied picture of lesions in bvdv infections. transplacental spread of bvdv during a clinically inapparent or apparent viremia of the dam may be the most crucial event in the entire bvdv complex (see articles by moennig and bolin, this issue). considering the difficulties often encountered in field cases in retrospectively establishing the exact time for the fetal infection, much emphasis has been given to the experimental evidence of the gestational agedependency of particular types of pathology. although this temporal categorization will not be disputed and is used here, it should be remembered that the possibility that some bvdv strains may have relatively low pathogenicity; therefore, their effect may be more insidious or slow, resulting in morphologic and/or clinical manifest changes only after a prolonged effect on the germinal tissues. 14 , 17, 111, 146 additionally, lesions that have been examined and seem to be similar in the perinatal period of a full-term calf may be the end stage of basically different processes, e.g., interference with normal stem cell differentiation and tissue development versus repair processes after an inflammatory process. 12 , 14, 41, 72 conclusive differentiation between these possibilities based on morphologic criteria may not be possible. 14 fetal infections in the first trimester of gestation may result in embryo/fetal death followed by absorption, mummification, or abortion, with expulsion occurring at any time up to several months later. pathologic and virologic examinations, therefore, are rarely informative if performed on isolated cases at the time of expulsion. lo , 105 as previously suggested, some of the congenital defects observed in full-term calves that often are ascribed to infections in the second trimester actually may have been caused by infections at the time of organ blastems differentiation in the first trimester, whereas an infection in the second trimester is more likely to provoke an inflammatory response and result in reparatory rather than regenerative processes and, thus, organ defects. 14 , 40, 41 the teratogenic lesions recognized as possible consequences of a intrauterine infection with bvdv are listed in table l . the ocular and central nervous system (cns) lesions have attracted special attention, both in studies of field cases 14 , 34,58 and in experimental work. 40 , 41, 72, 127 the chorioretinopathy often consists of variable depigmentation and loss of neurons as well as cone-and rod-cells (fig. 1) . the lens may be affected by capsular cataract and degenerative changes in the lens fibers,40 and there may be signs of inflammation of the cornea and gliosis in the optic nerve. 14 the cerebellar hypoplasia may be constituted by subtle changes such as reduction of the molecular layer and granule cell numbers, as well as reduction and displacement of purkinje cells,14, 144 frank loss of overall structure, and total loss of purkinje cells. 41 ,58 in the latter cases, the remains of an inflammatory process such as leukocyte infil tra tion may still be evident. 41 in other cases, demyelination seems to be the most prominent histopathologic finding. 34 ,58 so far studies of virus distribution in cns lesions have been inconclusive with respect to pathogenic mechanisms. 14 , 72, 144 it also remains unresolved whether these variations are the result of differences in virus strains (pathogenicity and tissue tropism), gestational stage at initial infection, host genotype, a combination of these, or some other factor(s), including the purely technical. 144 the fact that only one or a few of the possible teratogenic lesions previously listed are seen in anyone field case/outbreak could be explained by several of these factors. the thymus hypoplasia/atrophy has attracted much attention and speculation in the context of the purported immunosuppressive effect of bvdv (see the article by potgieter, this issue).5 alternative results can be found elsewhere. 76, 87 although bvdv antigen may be present in the thymus at the time of birth (fig. 2) , it does not always result in hypoplasia, as discussed in later sections. although thymocytes seem to be responsive (or susceptible) to signals for apoptosis such as antigen at inappropriately high levels or presented in the absence of appropriate costimulatory signals, corticosteroids, or certain cytokines,47, 90, 92,97,141 it may be questioned whether the bvdv infection is the cause of the atrophy or whether other events (parturition stress, neonatal infection, insufficient nutrition) around the time of delivery are the (additional?) cause(s) of an acute involution, which could have been reversible and inconsequential given time and appropriate care. l1 , 68 the generalized intra-uterine growth retardation, most often noted in pi calves (see section on persistent infection without overt clinical disease) may have significant clinical implications in relation to the "weak-calf syndrome," although the extent of the problem is not known because of the defect's subtle character (see the article by baker, this issue). 114 intra-uterine infection in the last trimester is not usually thought to cause serious consequences for the fetus. at this stage, the fetus has attained immunocompetence/o, 126 although still with some deficits,67,125 and will respond to the infection with bvdv-specific antibody production. 9 , 10, 126 whether the virus is eliminated in all cases,lo, 14,30 and whether an infection at this stage could potentially contribute to the problem of growth-retardation and/or weak calves does not seem to have been subjected to thorough investigation. despite being the first described clinical picture of bvdv infection,l1o acute virus diarrhea remains one of the two least well-characterized bvdv-associated syndromes (the other being respiratory disease, see later) mainly because of its perceived negligible long-term effect and mortality. primarily described as a disease of cattle aged 6 to 24 months,6 it remains unclear exactly what role bvdv plays in neonatal disease. 83, 84, 107 in either case the pathologic lesions usually are subtle and rather nonspecific, whether the infecting virus is of the noncytopathic (ncp) or cytopathic (cp) biotype,107, 143 comprising flaccid, edematous intestines, occasionally with ecchymotic hemorrhages, and mild reaction in the mesenteric lymph nodes. oral and esophageal lesions characterized by small erosions or shallow ulcerations have also been described. 61 occasionally, lesions resembling those seen in md (see section on mucosal disease and figs. 3 and 4a) and consisting of linear erosions and hemorrhages, peyer's patch-atrophy and hemorrhage, and erosion of the proximal colon occur. 36 ,43 virus antigen may be detected in the intestinal epithelium, as well as in tonsils, lymph nodes, and lungs. the importance of bvdv in bovine respiratory disease (brd) remains another contentious subject. 3 , 6, 113, 116 clinical signs of respiratory distress are common in animals with both acute virus diarrhea 43 , 59 and in md. in many cases, however, these do not seem to be accompanied by gross pathologic changes, and are thought to be a consequence of high fever and/or abdominal pain. 1l4 in other cases, particularly in young animals, either pi or infected postnatally, bvdv is seen as a predisposing factor for bacterial infection, similarly to that of bovine herpesvirus-i (bhv-i), parainfluenza-3, and others. 3, 22, 29 in pi calves, bvdv can be isolated from lung tissue, and virus antigen is widespread in bronchiolar and alveolar epithelial cells, however, without accompanying histopathologic changes (see section on persistent infection without overt clinical disease and fig. 5 ).17 conversely, a proportion (the size varying with study) of cattle succumbing to brd are positive for bvdv, by virus isolation from or antigen detection in lung tissue, but the ensuing pathologic changes cannot be distinguished from those of other viral pathogens in the brd complex (perhaps with the exception of bhv-p5), and usually are dominated by the pathology caused by the secondary bacterial agent. 3 , 29, 96 it is quite conceivable, although it remains to be shown experimentally, that the pathogenic mechanisms involved in the respiratory tract pathology of bhv -i plus bacteria also occur in bvdv -associated respiratory disease. 19 , 22,94,115,116 whether strain differences exist with respect to pneumopathogenicity as it does for other aspects of the bvdv complex remains unresolved. 43 , 113, 115, 116 although of considerable interest in its own right and as a model-system for host-virus interactions,2° an elucidation of this aspect may require an extensive experimentation. despite the generally accepted notion that infection of immunocompetent animals with either ncp or cp bvdv results in no apparent or figure 5 . demonstration of bvdv antigen in tissues of persistently viraemic, clinically normal calves. a, lung. bronchiolar epithelial cells appear to label intensely for viral antigen (original magnification, x 100). b, esophageal epithelium. despite intense virus antigen labeling of cells in the basal layer, no inflammatory reaction is seen, and the epithelium seems to be morphologically intact (original magnification, x 100). a relatively innocuous disease (see section on acute virus diarrhea), more severe outbreaks of diarrhea preceded by severe thrombocytopenia and widespread hemorrhages have been described. 52 , 53, 96,120 this syndrome, so far only described in the united states and canada, is mainly, but not exclusively, seen in young calves. 96 , 114,120 at this stage it remains unclarified whether cases of hemorrhagic diathesis seen in cattle in england 56 and continental europe,137 as well as north american outbreaks of severe peracute-acute bvd with pneumonia, diarrhea, and abortions 43 , 59 represent the same syndrome. peracute death with an overall lack of gross lesions or with hemorrhages of the peyer's patches as the only grossly apparent lesion may be a frequent event in this syndrome? other gross pathologic findings may consist of petecchial and ecchymotic hemorrhages throughout the body, including on the sclera of the eyes, the margins and inner surfaces of the eyelids, the mucosal surfaces of the cheeks and gingiva, the tip of and ventrally on the tongue and soft palate, in the esophageal and ruminal epithelium and the mucosal surface of the abomasum and intestines, on the epicardium and subperiosteally on the calvaria. additionally, some calves may have secondary bacterial pneumonia. 52 , 53, 96,120 although only likely to be diagnosed under clinic and experimental conditions, the cardinal sign seems to be megakaryocytosis, detected in bone marrow aspirates and core biopsies. 52 although the syndrome has some superficial resemblance to dengue hemorrhagic syndrome in humans 68 and hog cholera lesions,47 similarities in the pathogenic mechanisms have been ruled out,53 leaving the possibility that it is a virus-strain specific phenomenon 36 , 113 to be further explored. pi calves born as a result of an intra-uterine infection most likely in the first trimester of gestation, may be indistinguishable from noninfected animals,17, 100 or may seem to be growth retarded, at or soon postpartum. 6 ,58 a large serologic survey of healthy adult cattle showed that up to 1 0 /0 of such animals may be pi virus carriers.loo it is not known how many of these animals would have had microscopic evidence of the infection. however, in other, more limited studies of pi animals (natural or experimental infections), histopathologic lesions were detected in some, but not all, 17, 18 clinically healthy animals, and comprised local interstitial infiltrations of mononuclear cells (mnc) in kidneys and local thickening of the glomerular basement membrane,15, 54, 70 mild accumulation of mnc in liver triads, increased frequency of peribronchiolar lymphonoduli, and microfocal necrosis in the epithelium of the tongue and esophagus accompanied by moderate subepithelial mnc accumulation. 15 conspicuously, in these same studies the lymphoid tissues of pi animals were indistinguishable from those of normal animals of comparable age and environmental exposure. 1 8 ,54 in animals with obvious growth retardation, both macroscopic and microscopic evidence of skin lesions may be present. macroscopically, the changes may resemble those seen in chronic md (see section on the gross pathology of acute and chronic md) and consist of varying degrees of scurfiness and shallow erosive lesions around orifices and in the interdigital cleft. microscopically, the changes may vary from slight attenuation of the rete pegs and mild subepithelial mnc infiltration (mainly macrophages) to more pronounced changes of parakeratotic and hyperkeratotic character, as seen in md (see section on mucosal disease). similar changes may be found in the keratinized epithelia of esophagus, rumen, reticulum, and omasum. frank epithelial necrosis, however, is not common. 15 mild retina lesions, of a type described in the section on congenital infections and not diagnosed clinically, may also occasionally become evident on histologic examination. despite the absence or very limited extent of histopathologic changes, virus occurrence seems to be widespread, with little variation between animals, regardless of the clinico-pathologic manifestations l7 , 18,91 ( table 2 , and see figs. 2 and 3). this distribution is similar to what is found in cases of md, but differs with respect to extent, i.e., number of cells infected and/or virus antigen-labeling intensity, potentially reflecting virus-replication level and/or the biotype present. 66 this phenomenon is most notable in the gut in which in the fundi of the crypts of pi healthy cattle only few, often scattered, cells were found to be infected in comparison to more uniform infection in md calves (figs. 2 and 6).13,17,91 moreover, epithelial infection was not detected beyond the ileocaecal orifice. below this anatomical site the alimentary infection was confined to intraepitheliallymphocytes and mnc in the propria. 17 before euthanasia, virus was not or was only intermittently isolated from fecal samples, whereas ncp bvdv was readily isolated from blood and nasal swabs. is the clinically healthy pi animals also may differ from md cases with respect to virus distribution in the cns, although some discrepancies have been noted between published reports. in several studies, widespread occurrence of viral antigen in neurons, especially in the deep lamina of the cerebral cortex, was described,54, 64, 71, 144 whereas other studies have found that only microglia (macrophage-like cells) may be infected in pi clinically normal cattle, and large neurons only became virus-antigen positive in animals with md.15 likewise, discrepancies exist regarding the presence of virus antigen in endothelial cells of small vessels, although the latter may be explained by the use, in some studies, of antibodies with nonspecific affinity for endothelia and/or adventitial cells. 27 as an extension of pi cells of the lymphoid tissues, a proportion of peripheral blood mnc seems to contain bvdv antigen and replicating virus 16 , 17, 24, 31, 37, 95, 132 with no or only minor apparent consequence for total leukocyte numbers, relative frequencies, or the fine structure of the cells, perhaps with the exception of slight "activation" of the blood monocytes. 24 whereas there is agreement between reports as to the virus-carrier state of blood monocytes and t lymphocytes, there seems to be some discrepancy with regard to the b lymphocytes. 16, 31, 37, 95, 132 whether this is ascribable to differences in methodology (including sensitivity and specificity of reagents), host genotype or virus strains involved remains unclarified, as does the importance of the infection of the leukocyte types to the pathogenic processes (see the section on multifactorial nature of md pathology).13, 102 although md is a relatively uncommon form of the bvd spectrum of diseases, it has attracted much attention and continues to challenge the veterinary research community and virologists because of its elusive nature. the clinical manifestations and gross pathologic lesions of md can be extremely varied, although this is mostly in degree rather than in type. 6 , 9,114 this has led a number of authors to distinguish an acute and a chronic form of md.6, 119 whether this is entirely justified in terms of clinical entities must await clarification of the roles played by the previously mentioned factors such as the virus strains involved, host genotype, and the degree of "tolerance" and, therefore, host-reaction to the infecting virus(es) in the pathogenic processes (also see the section on multifactorial nature of md pathology).17, 18, 42, 102 classically, the gross pathologic lesions comprise necrotizing and erosive/ulcerative lesions on the muzzle and lips (see fig. 3 ), the buccal mucosa, gingiva and the tongue, in the latter case particularly on the latero-posterior surfaces. minor erosions may also occur on the external nares and in the nasal cavity. elongated (.5-2 mm in width, 1-2 cm in length) necrotic-erosive lesions are common in the esophagus (see fig. 3a ), as are erosions on the rumenal pillars (see fig. 3c ), in the reticulum and omasum. it should be emphasized, however, that especially in the chronic (protracted) cases of md gross lesions in the upper alimentary tract may be completely absent or subtle, although histologic examination usually will show micro-lesions similar in type to those described in the section on pi without overt clinical disease (see fig. 7 a) . erosive edematous and hemorrhagic lesions are seen in the abomasum. the enteritis can vary from catarrhal to hemorrhagic, fibrinonecrotic, and erosive/ulcerative. the peyer's patches as well as the lymphoid tissue in the proximal colon may seem hemorrhagic and necrotic (see fig. 4 ). the thymus is usually atrophic, and in many cases, only leaves a stringy fibro-epithelial, partly fat-infiltrated tissue. peripheral lymph nodes may be enlarged, edematous, or hypoplastic, depending on degree of inflammation in the drainage bed, length of clinical disease course, and/ or virus-host cell interactions in the organ. most animals will have apparent skin lesions, although with considerable individual variations in degree and extent of distribution. lesions are most common and most pronounced in the neck, shoulder, and perineal region. the changes present as an eczema resulting from crustlike dermatosis and hyperkeratosis and parakeratosis accompanied by hypotrichosis or alopecia. underneath the dry scales, the corium seem hyperemic and occasionally thickened. where the crustous formations are well-delineated, a hyperemic zone can be observed in the edge as well as local alopecia. in some cases, the dermatitis is of a generalized seborrhagic nature with hyperkeratinization and alopecia. erosive lesions may be found in the perineal area, around the preputial opening or vulva. erosive lesions with or without lichenification and exudation are also common in the interdigital cleft and around the skin-hoof junctions. suppuration may occur due to secondary bacterial infection, although this is less common than might be expected. generally, the skin lesions may be more pronounced in the protracted (chronic) cases, but there seems to be considerable variation between published descriptions-perhaps due to differences in the clinical categorization of acute versus chronic. conjunctivitis is an almost consistent finding. bronchopneumonia and fibrinous pleuritis may occur, but can then usually be ascribed to secondary bacterial infection (often actinomyces bovis or pasteurella species). despite the variation in the gross-pathologic appearance, the histologic picture in anyone particular organ or organ-system is one of uniformity of type with variations ascribable to intensity and progression of the lesions, and perhaps modified by mechanically or, more rarely, bacterially induced secondary changes. the initial changes in keratinized epithelia (skin, the muzzle, and upper alimentary tract including rumen, reticulum, and omasum) is a focal infiltration of mnc in the papillar and reticular strata in propria/ dermis, often with a tendency to periarteriolar and pericapillary localization, but never developing into distinct "cuffing," thus, probably reflecting migration in progress, rather than a reaction to vascular changes (see fig. 7 a) . a thickening of propria occurs by widening and lengthening of the dermal papillae, often at the expense of the epithelial basal folds, which may appear lamella-like. the tissue may be hyperemic with diapedesis hemorrhage. in the epithelium degeneration and eosinophilic necrosis of scattered cells in stratum spinosum seems to constitute the initial changes, which progress to widespread cellular necrosis with disruption of the intercellular junctions. at the same time, mnc infiltration of the epithelium occur, often without apparent relation to the necrotic epithelial cells, but perhaps directed toward cells inapparently modified by virus infection. is as this process progresses, well-delineated foci with a reticulated appearance arise in the stratum spinosum. they may remain covered by an intact stratum corneum. however, eventually this layer also loses integrity and erodes (fig. 7b) . the basal layer usually remains intact while the previously mentioned processes are in progression, but eventually necrobiotic and metaplastic changes may also occur. the basal cells may become squamous, but unless ulceration and/or secondary bacterial infection occur, the layer will remain uninterrupted. in other areas, especially in the skin in the neck and shoulder regions, the epithelial changes may be dominated by hyperkeratotic and parakeratotic processes, with formation of thick keratinoid masses, often 50 to 100 (cell) layers thick, interspersed with varying amounts of necrotic cell debris, keratin, and infiltrating mnc. the hair follicles and sweat glands seem to become involved only relatively late in the processes of the skin, with initial periglandular and perifollicular mnc-infiltration and later degenerative and necrotizing changes of the follicles similar to those previously described. it is presumably at this stage that hypotrichosis and alopecia develops. in the intestines and abomasum pathologic changes are selectively localized in the fundi of the crypts. the initial changes occur in scattered single or small groups of neighboring cells, and consist of cytoplasmic condensation followed by vacuolation, rounding off, and desquamation. electron microscopic examinations, supported by immunocytochemistry for virus antigen (see below), have shown that this cellular reaction occurs in cells with extensive viral replication, presumably cp virus. 9 ,23, 66, 91 cell debris and mucus accumulate in the crypt lumina that may become occluded and dilated with flattening of the epithelium and disappearance of microvilli. 23 especially over the peyer's patches these changes progress to a state of complete atrophy of crypts and villi, leaving a flat epithelium covering a lamina propria of fibrotic, almost acellular tissue (see fig. 4 ). although such areas seem especially prone to ulceration, it is worth noting that the unecrosis" described macroscopically in many, if not most, cases corresponds to areas with complete epithelial atrophy/metaplasia as seen in figure 4 . in some cases, or some areas/parts of the intestine mucus cell metaplasia may dominate the crypt changes, and is accompanied by excessive mucus production and accumulation. the lamina propria reaction is usually dominated by massive infiltration of mnc, especially macrophages. other changes may include edema in the subepithelial layers, hyperemia, and diapedesis. although there may be general agreement in the literature that clinical md is accompanied by changes in the lymphoid tissues, there seems to be a conspicuous lack of attempts at staging the changes and correlating, if possible, these with functional characteristics of the "immune tolerance" and "suppression," presumed to be central to the pathogenesis of md.42,102 as previously mentioned, no or very subtle morphologic changes occur in lymphoid tissues (thymus, spleen, lymph nodes, peyer's patches) of pi clinically healthy animals despite widespread virus infection of all or most of the cellular elements. 17 changes may occur (thymus atrophy, lymph node reactivity) in growth-retarded, weak calves, but may be caused by intercurrent infections and stress rather than as an antecedent to secondary infections. 67 , 125 unfortunately, no thorough description of changes in lymphoid tissues of experimentally reproduced md cases has yet appeared. the descriptions of changes in the thymus and peyer's patches to follow, therefore, are based on natural cases of md in which a staging was attempted based on clinical appearance and duration of the disease. 9 in acute cases of brief duration (1-4 days) the overall structure of the thymus is retained, but a clear reduction occurs primarily affecting the cortex, which may be dominated by apoptotic and disintegrating thymocytes (fig. sa) , prominent epithelial cells and large, actively phagocytosing macrophages. by electron microscopy degenerative changes are apparent in the epithelial cells, in particular in the cortical zone. variable thymocyte depletion characterizes the medullary changes, but signs of frank cell death are rarely seen. in subacute to distinctly chronic cases of md, the thymus is characterized by progressive loss of zonal organization (fig. sb) . the cortex may remain as a thin layer of "collapsed" epithelium, surrounding a fibro-reticular tissue with prominent hassall's corpuscles and myoid cells. few lymphocytes may persist in scattered islets, and fat infiltration may become prominent, especially in older animals. the peyer's patches already seem to be severely depleted of lymphoid cells during the peracute to acute clinical phases, which especially affects the follicles as well as the interfollicular and subepithelial zones. the follicular changes comprise apparent hypertrophy followed by degeneration of the follicular dendritic cells (fdc) and replacement with a homogenous fibroid material (fig. 9 ). the structure of the peyer's patches may be distorted further by extension of crypts into the submucosa and crypt-dilation followed by degeneration. in more protracted (chronic) cases of md, the peyer's patches are characterized by atrophy. often only a condensed/retracted (hyalinized) fibrotic tissue with small lymphoid islets remain (fig. 9b) , which are surrounded by concentric layers of fibrous tissue. the interfollicular zones comprise a narrow zone under the muscular layer with few lymphocytes, monocytes, and macrophages between fibroblasts and col-lagen. the high-endothelial postcapillary venules (hev) are no longer distinguishable. in contrast to the thymus and peyer's patches, a staging based on clinical criteria has not been possible for the parietal and mesenteric lymph nodes,9 which not only vary in appearance between animals, but also vary within an animal. the most conspicuous changes comprise lymphocyte-depletion of the peripheral subsinusoid zone and regressive changes in primary and secondary follicles, the numbers of which seem to be comparable to that which occurs in conventionally raised noninfected or pi healthy calves. in secondary follicles, the germinal center may be replaced by a lightly stained eosinophilic, homogenous (structureless) cytoplasmic stroma, consisting of large dendritic cells, identified by electron microscopy as fdc. distinct fibrosis, sometimes with a pericapillary origin, may be present. scattered macrophages with large phagolysosomes may surround this area, partly embedded in an irregular and ill-defined lymphocyte corona. the central fibrosis may progress to a state in which the fdc have disappeared and the germinal center is completely replaced by an acellular, condensed hyalin fibroid mass that is surrounded by few scattered lymphocyte islets. the paracortex may be depleted of lymphocytes to varying degrees; directly related to this event is a flattening of the endothelial cells of the hev and decreased transendothelial migration of lymphocytes. the number of interdigitating cells may either remain within the normal range or decrease. the medulla may be relatively or actually expanded. this is partly the result of a prominent sinus reaction characterized by accumulation of lymphocytes and typical monocytes, as well as large ii activated" macrophages ( fig. 10) . this is especially true for the more protracted cases and in the parietal lymph nodes, in which these cells may attain epithelioid cell characteristics, inclusive electron microscopically detectable birbeck granules and micropinocytosis vermiformis (fig. 11) . changes in the bronchial and retropharyngeal lymph nodes may often be compounded by reactions to secondary respiratory bacterial infections and are not examined in this article. changes in the tonsillar lymphoid tissue resemble those described for the peyer's patches and lymph nodes, with a progression similar to that described for peyer's patches. in the spleen, lymphocyte depletion of the periarteriolar lymphoid sheath and follicle changes that are similar to, albeit less pronounced, than those in the lymph nodes are characteristic findings. generally, bvdv antigen distribution in cattle with md corresponds to that described for pi clinically healthy calves (see section on pi without overt clinical disease, table 2 ; figs. 6 and 12). a number of notable variations, however, should be examined. as the thymus involution progresses, the amount of detectable virus antigen also decreases and eventually is limited to mnc in the adventitia of blood vessels and scattered macrophages. the same occurs to the secondary follicles in peripheral lymphoid organ. as the germinal centers become depleted of fdc and are replaced by acellular matrix, the virus antigen disappears. in contrast, the occurrence of virus antigen in lymph node medullary cord and sinus cells increases with protraction of the clinical course. most of these cells seem to be macrophages, as determined by doublelabeling for virus antigen and phenotypic (cd)-markers. the degeneration of hev is notably not accompanied by virus antigen presence in the endothelial cells. in the skin, virus antigen-positive cells occur as fairly evenly scattered cells in the basal lamina, inclusive of hair follicles. they may be more concentrated and the labeling more intense in areas of early and progressed subepithelial inflammatory reactions. additionally, some of the infiltrating macrophages and t cells are virus antigen positive (see fig. 6 ). 18 in the oral cavity, esophagus, and keratinized fore stomachs, the antigen distribution tends to be more focal, even in areas without visible inflammatory reactions or frank erosion, although considerable variation between different anatomical sites of the same animal is usual. 13 in the intestines of cattle with md, more widespread occurrence of virus antigen-positive cells occur in the crypts compared with pi healthy calves, and the cells tend to label more intensely (compare figs. 2 and 6). 13, 17, 18, 66, 91 this is the case in the epithelium associated with submucosal lymphoid tissues (peyer's patches in ileum and in the proximal colon). additionally, many macrophages in the lamina propria are virus antigen positive (see fig. 6 ), although it remains unknown whether this is due to productive infection in all cases, or if it reflects extensive uptake of cellular debris from surrounding infected and lysed cells in some cells. in animals succumbing to md, the previously described hyperkeratotic and parakeratotic changes, as well as necrotizing lesions, are accompanied by massive infiltration of mhc class ii antigen-positive macrophages and cells with dendritic morphology. t lymphocytes (predominantly cd8 + cells) also occur, whereas b lymphocytes are rare. using macrophage differentiation markers as immunocytochemical target antigens, it was determined that the majority of macrophages in the infiltrates may be relatively undifferentiated but highly activated,18, 28, 33 and this may reflect a highly dynamic process with continuous influx from the blood circulation and either premigratory or postmigratory activation. 33 macrophages of all maturation stages as well as t lymphocytes occur in the lamina propria of the digestive tract (inclusive in peyer's patches) with occasional focal concentration of cd8+ cells perivenularly. from the findings in natural cases of md and with the use of only a limited set of lymphocyte surface markers, detection of consistent selective losses of anyone particular subset of lymphocytes from lymphoid tissues, or significant changes over time in the composition of cell infiltrates in other tissues has not been possible.i 8 with the availability of a larger panel of cd-specific reagents and the possibility of reproducing md experimentally, however, it would now be feasible to conduct such studies in a controlled and sequential manner. the role of cytokines in the pathogenesis and pathology of bvdv infections in general and in md in particular, so far has only attracted limited attention/i, 25, 33 but deserves thorough investigation. lesions comparable to those seen in the intestines, lymphoid tissues, and cutaneous epithelia can be provoked by either overexpression, overproduction, or gene-disruption of several cytokines, including tnfa/ 6 ,73 gm-csf, 85 tgffj,129 and il-6.32, 74 the lesions characteristic of bvd-md are likely to have a multifactorial cause: by a combination of virus-cpe and specific and nonspecific immune responses, with the latter comprising local cytokine production by nonlymphoid cells as well as macrophages and perhaps cds + t cells and "null" cells in response to cell injury or other cytokines. in the unveiling of such mechanisms, however, it will be crucial to take into account the temporal relationships.22, 57,112 liebler et al have begun such studies, but much remains to be done. 91 another aspect of the pathogenesis of md that needs clarification is the tissue/cell location for the events leading to a biotype change.i, 98, 99, 136 a number of studies have documented that flavivirus genome mutations and selection of variants only occur in certain cell types. 50, 93 it is notable that the cp virus seems to have a somewhat restricted distribution,66,91 and it is even more notable that this includes fdc, tissue macrophages, and other dendritic cells. a similar, although not identical, distribution was described in lymphocytic choriomeningitis virus (lcmv) infections in which specific types of mutations in the viral genome caused the virus to become either macrophage-tropic, lymphocytotropic, or both, i.e., amphotropic. 1 ,80 likewise, in visna virus infection tissue tropism is determined by a specific genome segment. 131 although the noted distribution for cp-bvdv cannot be taken to imply that these are the types of cells in which the change(s) occurs, the preferential replication of cp-bvdv in professional antigen presenting cells (apc) makes it possible that the cleavage products of the 125kda protein, after genomic changes, are processed and presented differently to the immune system than ncp-virus products. 106 the tolerant state may be "broken" with the subsequent development of immunopathologic, perhaps even autoimmune-like, disease.109, 123 that a measurable antibody (ab) response does not always occur,11, 15,62 which might other-wise be expected,65, 147 does not contradict this hypothesis, because such a response would not only depend on a time-factor, i.e., survival time, but also on the type of t-cell response induced. 4 , 103, 104, 112, 142 if this is preferentially a cd8+ t-cell response, which might be inferred from the predominance of cd8 + to cd4 + cells in inflammatory foci, submucosae, and lymphoid tissues,18 and is followed by destruction of antigen-positive cells, including the fdc (see figs. 9b and 12),108 the b-cell response would be abrogated?9, 117 such mechanisms have been demonstrated to occur in lcmv infection,108 and are thought to take place in hiv infections. 63 if at the same time as the apc are presenting "new" virus-protein epitopes to the immune system one or more of their accessory functions, such as expression of co stimulatory surface molecules or production of cytokines, have been altered directly or indirectly due to the virus infection, 1 apoptosis may be the outcome rather than stimulation of a proliferative t-cell response. 89 , 141 such a scenario could be invoked to explain the apparent "acuteness" of thymocyte depletion (see fig. 8a ), as well as lymphocyte depletion of peripheral lymphoid tissues. 117 in addition, factors such as corticosteroids and sudden excessive release of certain cytokines may contribute to the depletion by directly inducing apoptosis in lymphocytes. 48 ,90 the resulting disruption of lymph node, peyer's patches, and tonsil architecture would result in an abnormal (even if in some cases subtle) distribution of lymphocytes within these tissues. this reaction combined with the productive virus infection of both lymphocytes, macrophages, and dendritic cells (see figs. 2 and 12) may profoundly impair normal cell-cell interactions. 16 , 17, 31, 66 such mechanisms may help to explain why neither t nor b lymphocytes are particularly prominent in the inflammatory processes in epithelial lesions. 18 furthermore, the absence of de novo expression of mhc class ii antigens on endothelia and epithelia suggest that either ifn-y is not produced locally to any significant extent/ 2 ,25 or the ifn-y effect is antagonized by other cytokines. because inflammatory macrophages in the lesions express high levels of mhc class ii antigen, however, this would suggest that cytokines such as il-10 and tgf~ may not play major roles in the inflammatory reactions,55, 139, 140 despite at least the latter's known involvement in skin pathology.38, 129 instead, to explain the inflammatory process in the cutaneous epithelia the role of il-1u and il-1~ may be invoked. keratinocytes are known to express large amounts of intracellular pro-il-1u and pro-il-~.82, 101 cell injury, due to virus-cpe or a cell-mediated cytotoxic response, would result in release of the cytokines. because pro-il-1u is biologically active, its release could initiate the inflammatory reaction by induction of adhesion molecules on endothelia and the production, by keratinocytes and macrophages, of chemokines and other proinflammatory cytokines such as csfs, il-6, tnfu, and fibroblast and keratinocyte growth factors. 81 the process may start with the damage of only a few keratinocytes, and thereby create the impression of mnc-infiltration preceding the epithelial lesions (see section on histopathology of md and see fig. 7 a). furthermore, it is possible that such a process could become self-perpetuating. 81 an alternative, although not a mutually exclusive possibility, is that the cutaneous lesions in md are initiated by cp-bvdv replication in langerhans cells. these cells are potent apc and cytokine producers,86 and the latter function could be envisioned to be upregulated on infection, in particular of such pro-inflammatory mediators as il-l, il-6, tnfa, and chemokines, which subsequently could stimulate the keratinocytes as well as attract macrophages. another possibility is virusinduced depletion of langerhans cells, directly or indirectly,l28 with subsequent loss of an important immunoregulatory component of the skin. 4, 8, 124, 133 thus the question of distribution and density variation of epidermal and mucosal langerhans cells as a determining factor in the distribution and/or severity of the lesions in cutaneous epithelia, such as has been described in other dermatologic reactions,39, 130, 138, 145 becomes pertinent. so far the cutaneous lesions characteristic for md have received comparatively little attention; further study in this area is needed. despite the impressive progress in the unveiling of the molecular phenomena involved in the biotype-change, from ncp to cp, of bvdv98, 99,118,135,136 (see the article by donis and bolin, this issue), the biological significance of the pbo protein at the level of host cell-virus interaction(s) remains unresolved.134 it is by no means clear that the pbo protein is the cytopathogenic factor, and the possibility remains that this protein represents only an epiphenomenon or convenient marker for other more crucial events in the host-pathogen relationship. thus, before jumping to (easy) conclusions about virus-cpe as the main, if not sole, cause of tissue pathology and ultimate death of the animal,42,98, 136 it might be prudent to remember a number of characteristic features of the md pathology and to place these in context of what is currently known about inflammatory processes and induction of pathomorphologic changes in lymphoid tissues, as well as the possible role of target cell distribution. it also should be remembered that many of the lesion types characteristic of md can also be found in acute outbreaks of bvd in which the causative strain is ncp.43, 56, 96, 114 a broader and more integrated approach may be needed to succeed in unraveling the enigmas of the pathogenesis of md and of bvdv infections in general. molecular basis of organ-specific selection of viral variants during chronic infection bovine coronavirus as the causative agent of winter dysentey: serological evidence serological titers to bovine herpesvirus 1, bovine viral diarrhea virus, parainfluenza 3 virus, bovine respiratory syncytial virus and pasteurella haemolytica in feedlot calves with respiratory disease promethean viruses? suppression of in vitro immunoglobulin biosynthesis in bovine spleen cells by bovine viral diarrhea virus bovine viral diarrhea virus: a review reduced langerhans' cell ia antigen and atpase activity in patients with the acquired immunodeficiency syndrome bovine viral diarrhoea virus-infections. 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bovine viral diarrhoea virus p80 protein in subpopulations of bovine leukocytes skin-associated lymphoid tissues (salt): origins and functions rna-stimulated ntpase activity associated with the p80 protein of the pestivirus bovine viral diarrhea virus processing of poly-ubiquitin in the polyprotein of an rna virus pathogenesis of mucosal disease: a cytopathogenic pestivirus generated by an internal deletion kasuistischer beitrag zu hemorrhagischen diathesen bei kalbern mit bvd-virusinfektion epidermal langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with dnfb interleukin-lo treatment can suppress stromal keratitis induced by herpes simplex virus type 1 interleukin 10 (il-i0) and viral il-i0 strongly reduce antigen-specific human t cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class ii major histocompatibility complex expression differential activation of antigen-stimulated suicide and cytokine production pathways in cd4 + t cells is regulated by the antigen-presenting cell intracerebral cytokine mrna expression during fatal and nonfatal alpha virus encephalitis suggests a predominant type 2 t cell response experimental primary postnatal bovine viral infections in six-month-old calves distribution of bovine virus diarrhoea viral antigens in the central nervous system of cattle with various congenital manifestations role of epidermal langerhans cells in resistance to herpes simplex virus infection altered growth, differentiation, and responsiveness to epidermal growth factor by persistent rubella virus infection virus-induced autoantibody response to a transgenic viral antigen key: cord-016127-tbot0fc9 authors: hurtado, f. j.; buroni, m.; tenzi, j. title: sepsis: clinical approach, evidence-based at the bedside date: 2009-11-19 journal: intensive and critical care medicine doi: 10.1007/978-88-470-1436-7_25 sha: doc_id: 16127 cord_uid: tbot0fc9 sepsis is a common disease in intensive care medicine representing almost one third of patient admissions. its incidence has substantially increased over the past decades and overall mortality has declined during this period of time. it was reported that sepsis incidence increased from 82.7 to 240.4 per 100,000 population between 1979–2000. at the same time, sepsis global mortality decreased from 27.8 to 17.9% [1–3]. however, the absolute number of deaths significantly increased from 21.9 to 43.9 per 100,000 population. male gender, some chronic diseases like diabetes, immunosuppressive states, human immunodeficiency virus infections, and malignancies are factors that increase the risk for sepsis. some particular conditions like progressive number of organ dysfunctions, in-hospital-acquired infections and increasing age are associated with higher risk of death [1,4]. on the other hand, septic shock mortality only diminished from 61.6 to 53.1% [5]. this slight decline in mortality observed during recent decades could be attributable to improvements in supportive care and/or avoidance of iatrogenic complications. for example, the instrumentation of early goal resuscitation protocols not aiming at supranormal targets for cardiac output and oxygen delivery, and the use of lung protective strategies could explain at least in part this favorable change. other strategies directed to treat the pathophysiological mechanisms involved in the septic process like recombinant human-activated protein-c (rhapc), have also contributed to improve survival. however, mortality remains unacceptably high and further improvement in sepsis management is needed. sepsis is a common disease in intensive care medicine representing almost one third of patient admissions. its incidence has substantially increased over the past decades and overall mortality has declined during this period of time. it was reported that sepsis incidence increased from 82.7 to 240.4 per 100,000 population between 1979-2000. at the same time, sepsis global mortality decreased from 27.8 to 17.9% [1] [2] [3] . however, the absolute number of deaths significantly increased from 21.9 to 43.9 per 100,000 population. male gender, some chronic diseases like diabetes, immunosuppressive states, human immunodeficiency virus infections, and malignancies are factors that increase the risk for sepsis. some particular conditions like progressive number of organ dysfunctions, in-hospital-acquired infections and increasing age are associated with higher risk of death [1, 4] . on the other hand, septic shock mortality only diminished from 61.6 to 53.1% [5] . this slight decline in mortality observed during recent decades could be attributable to improvements in supportive care and/or avoidance of iatrogenic complications. for example, the instrumentation of early goal resuscitation protocols not aiming at supranormal targets for cardiac output and oxygen delivery, and the use of lung protective strategies could explain at least in part this favorable change. other strategies directed to treat the pathophysiological mechanisms involved in the septic process like recombinant human-activated protein-c (rhapc), have also contributed to improve survival. however, mortality remains unacceptably high and further improvement in sepsis management is needed. novel therapeutic approaches are under investigation and will probably be incorporated in the clinical practice in the near future. since 2002 the surviving sepsis campaign was introduced with the initial goal of increasing clinicians' awareness about severe sepsis mortality and to improve outcome in this patient population. it was pursued to generate a change in the standard of care that could finally result in a significant mortality reduction. a consensus committee from several international organizations was created and evidence-based guidelines were elaborated [6] . despite the fact that most of these recommendations were not supported by high levels of evidence, they represented the international consensus on the best available standards of care for the management of sepsis. these guidelines were recently updated and continue to be the core of the surviving sepsis campaign [7] . the clinical practice needs clear and concise recommendations based on the best available level of evidence. sepsis is defined as the host response to infection. in other terms, it is the clinical syndrome that results from the inflammatory response to infection. in the clinical setting, sepsis is diagnosed when an evident or suspected infection courses with a systemic response called the systemic inflammatory response syndrome (sirs). according to the 1991 north american consensus conference, sirs was defined by the presence of at least two of the following signs: body temperature >38ºc or <36ºc, heart rate >90 beats/min, respiratory rate >20 breaths/min (or paco2 <30 torr), and/or white blood cells count >12,000 or <3,000/mm 3 [8] . however, these signs are too sensitive and nonspecific for sepsis and could occur in many other different situations not related to infection. in an attempt to better reflect the systemic response to infection, the clinical manifestations described by bone et al. were expanded by the 2001 consensus conference [9] . other possible signs, symptoms, and laboratory findings were summarized (table 25.1) . again, most of them are also nonspecific for sepsis. it is well known that infection and sepsis are sometimes difficult to confirm. in an attempt to improve diagnostic capabilities, some biological markers were developed. procalcitonin (pct) and c-reactive protein (c-rp) have been proposed but it is considered that there is still no ideal biological marker for sepsis diagnosis [10, 11] . none of the mentioned biomarkers are absolutely specific, meaning that diagnosis or prognosis cannot be made solely on this basis. an infection probability score (ips) was also proposed to be calculated from several variables: body temperature, heart rate, respiratory rate, white blood cells count, c-reactive protein, and sequential organ failure assessment score (sofa). the potential role of such an index was recently evaluated [12] . some concepts and definitions remained unchanged after the last consensus conferences and should be emphasized. the following terms are widely accepted. infection is the pathologic process caused by the invasion of normally sterile tissues, fluids, or cavities by pathogenic microorganisms. sepsis is the clinical syndrome defined by the presence of infection and a systemic inflammatory response syndrome. severe sepsis relates to the presence of sepsis and one or more related organ dysfunctions. septic shock should be diagnosed when severe sepsis courses with acute circulatory failure. cardiovascular compromise becomes evident when arterial hypotension remains after adequate fluid infusion or there is need for vasopressor therapy. systemic hypotension is defined when arterial systolic pressure remains <90 mm hg, mean arterial pressure <60 mm hg, or there is a decrease in blood pressure >40 mm hg from previous values. different processes could occur during severe sepsis and septic shock at the same time. hypovolemia, maldistribution of blood flow within or between organs, vasoreghowever, cytokine release from the inflammatory reaction or prolonged tissue hypoxia is followed by severe microcirculatory abnormalities that become a central protagonist of organ dysfunction/failure [13] . the main significance of microvascular dysfunction has been studied during sepsis and major changes like decrease of both capillary density and microvascular blood flow were documented in vivo by video microscopy [14] . thus, peripheral gas exchange becomes impaired and tissue dysoxia ensues. these abnormalities also occur despite normal or even supranormal hemodynamic variables. in terms of peripheral oxygen metabolism, severe heterogeneity of oxygen distribution within the tissues is characteristic during septic shock. under-and overperfuse areas coexist within the same tissue resulting in an inhomogeneous tissue oxygen partial pressure distribution (pto2). in these conditions, metabolic demands are not met by microvascular oxygen delivery, making peripheral shunting and tissue dysoxia the cause of organ failure. however, metabolic abnormalities also occur at the cellular level. mitochondria dysfunction is secondary to oxidative and nitrative stress initiated by the inflammatory reaction. energetic failure develops and less high energy compounds are available for cellular function. this situation was referred to as cytopathic hypoxia and leads to multiple organ failure and death. mitochondria dysfunction and decreased atp production was documented during the course of sepsis in experimental and clinical situations. efforts should be made to preserve mitochondrial functioning and improve the cellular energetic state [15] [16] [17] . early and accurate recognition of the signs and symptoms of sepsis is mandatory after patient admission. risk factors like age, gender, race, immunocompromised states, presence of invasive instrumentation maneuvers, or any other condition that could represent a via for bacterial colonization. clinical presentation and laboratory findings are essential. fever is the hallmark of infection, but hypothermia is also possible in some patients. other nonspecific signs like tachycardia, tachypnea, and hypotension should also be documented. when looking for the source of infection a careful physical examination should be complemented with x-rays images, ct scans, ultrasound, etc. finally, it is necessary to investigate the presence and severity of organ dysfunction. in the vast majority of the cases this information is easily collected and diagnosis becomes straightforward. however, this is not always the case. it is important to realize that the septic patient is always at risk of death and some clinical signs may be indicative of disease severity. clinical demonstration of acute respiratory and/or circulatory failure, or any other organ dysfunction are indicative of the aggressive host response to the septic insult [8, 9] . since organ failure is an integral part of severe sepsis, a brief summary of major organ dysfunctions will follow. pulmonary or extrapulmonary ali is present in about 60-70% of severe sepsis. it is defined by pulmonary infiltrates in the chest x-ray and the absence of left ventricular failure (pulmonary wedge pressure <18 mm hg). pulmonary gas exchange is impaired showing a pao2/fio2 ratio under 300 for ali or below 200 for ards. most of the time, the severity of ali/ards determines mechanical ventilation. while mechanical ventilation will restore pulmonary gas exchange and decrease systemic metabolic demands, detrimental effects should be avoided by a rational application of protective ventilatory strategies. when the focus of infection is located outside the central nervous system (cns), the neurologic compromise could be attributable to septic encephalopathy. some other conditions may add secondary effects such as hypoxemia, metabolic and electrolytical disorders, and cerebral hypoperfusion during shock states. symptoms may vary from agitation, confusion, delirium, and coma. no focal neurologic deficits are present but myoclonias and seizures are possible [18] . severe cns derangement requires airway protection and ventilatory support. liver dysfunction is characterized by some degree of hepatomegalia and total bilirubin plasma levels >2 mg/dl. higher conjugate bilirubin concentration is characteristic and increased gamma glutamil transferase is frequently observed. moderate levels of aminotransferases generally <200 ui can also be found. decreased red blood cells without bleeding evidence and platelets <100,000/mm 3 are frequent findings. coagulation cascade has been widely studied. sepsis enhances coagulation and impairs fibrinolysis. endogenous-activated protein c that prevents microvascular thrombosis is decreased during sepsis. when small and medium microvessels become occluded, the disrupted microcirculation generates tissue dysoxia. given the context of severe sepsis, rhapc could contribute to ameliorate coagulation disorders [19] . renal dysfunction could course with normal or decreased urine output. increase in creatinine level >0.3 mg/dl from previous values or a percentage increase >50%, or a reduction in urine output (oliguria <0.5 ml/kg/h for more than 6 h) defines acute renal injury and is associated with poor outcome. arterial hypotension unresponsive to volume expansion defines septic shock. variable degrees of hemodynamic dysfunction may vary from hypodynamic to hyperdynamic shock. mortality increases according to the presence of shock, and metabolic markers like arterial lactate are useful to characterize disease severity and the response to treatment [8] . despite the fact that lactate concentration depends on the balance between tissue production and metabolism, a plasma level >4 mmol/l should be considered as indicative of circulatory failure. some other organ compromise could also be part of the multiple organ dysfunction syndrome. splanchnic ischemia and intramucosal acidosis ensue early during the course of sepsis. clinical expression includes changes in smooth muscle function like ileum or diarrhea. gastrointestinal bleeding because of stress ulcer or acute gastritis may also be a manifestation of sepsis. gastric intramucosal ph monitoring was used to identify and guide resuscitation therapy. increased levels of intraluminal pco2 are associated with tissue ischemia and mucosal acidosis. skeletal muscles are also affected by inflammatory mediators and reactive oxygen species. there is simultaneous decrease in protein synthesis and proteolysis. in conjunction, these factors explain decreased muscular force. respiratory muscles are involved and respiratory pump failure may aggravate or precipitate an acute respiratory failure. multiple organ dysfunction is part of the severe sepsis syndrome. poor prognosis is related to increased number of organ failures. technical resources for the management of organ dysfunction have improved in recent years and consume a substantial part of the therapeutic effort. most of the described dysfunctions are reversible as long as the infectious disease becomes controlled. however, the additive effects of the different failures may initiate a series of independent processes that may aggravate the patient status and be the cause of death. prognostic scores are helpful to predict mortality and some organ failure scores were proposed to evaluate severity and to follow the evolution of septic patients. the multiple organ dysfunction score (mods) and the sequential organ failure assessment (sofa) are frequently used for this purpose [20, 21] . to identify the source of infection and the microbial agent is crucial during sepsis. microbiological investigation is mandatory and adequate antibiotic therapy must be initiated as early as possible [7] . most of the time the diagnosis results from a correct anamnesis and clinical examination. suspicion of sepsis must be followed by complete bacteriological cultures, taking samples from blood and other possible foci of infection. some other special exams should not be deferred and may add complementary information. positive blood cultures are only confirmed in about 50% of the cases [22] . no bacterial etiology is identified in 20-30% of septic patients. almost 45% of the initial antibiotic selection should be changed or adjusted after blood cultures are informed. decreased mortality is related to prompt bacteriological identification [23] . despite the fact that infection is generally caused by bacterial agents, virus and fungal agents are possible, especially in immunocompromised patients. epidemiological data coming from each icu or hospital could be helpful when hospital-acquired infections are under study. infection is a frequent complication in polytrauma and in the critically ill patient who was subject to invasive procedures. increased life expectancy and special situations like organ transplant create further opportunities for microbial invasion and sepsis development. as mentioned before, biochemical markers of infection could be helpful in particular situations where diagnosis is not straightforward. procalcitonin (pct), c-reactive protein (crp), and some interleukins like interleukin-6 (il-6) have been proposed to contribute to diagnosis. however, further evidence is needed before these biomarkers are incorporated in the clinical practice. actually, some authors have considered pct as a good indicator for severe sepsis and septic shock [24, 25] . research on new biomarkers continues with the aim of early detection of patients at risk of severe sepsis [26] . the current management of severe sepsis and septic shock aims to control infection, achieve hemodynamic stabilization, modulate the immune response, and provide metabolic and organ support. evidence-based medicine has become the cornerstone of medical practice but it is difficult to apply in patients with sepsis. the ssc is a global initiative that involves several international organizations with the common objective of elaborating evidence-based guidelines and recommendations for the management of severe sepsis and septic shock. lack of high-level evidence coming from large rct is a severe limitation in sepsis. to accomplish these goals, experts determined that improving patient care was a possible task and could lead to a significant decrease in mortality. despite the fact that only a few of the guidelines were supported by high levels of evidence, it was agreed that they represent the best available evidence for the management of the septic patient. during the last consensus conference a grade system was agreed upon by the participants. guidelines were classified from a to d based on the levels of evidence; however, at the same time a strong or weak recommendation was introduced by the panel of experts [7] . the international guidelines of the surviving sepsis campaign will be briefly discussed below. this group of measurements should be accomplished within the first 6 h of patient admission. this could probably happen in the emergency department before icu admission. early identification and comprehensive resuscitation of septic patients will have a significant impact on outcome. the first 6 ''golden hours'' constitute a critical opportunity for the patient [27, 28] . resuscitation should be started immediately when hypotension or elevated serum lactate (>4 mmol/l) are detected and treatment should not be delayed until icu admission. initial resuscitation not only includes hemodynamic stabilization but also simultaneous administration of empiric antimicrobial drugs and actions directed toward the control of infection [7] . early resuscitation is initially based on aggressive volume expansion. it could be administered via a peripheral vascular access while a central venous line and central venous pressure (cvp) measurement are instrumented somewhat later within the initial hours. when fluid therapy does not restore arterial blood pressure or lactate remains elevated, administration of vasopressors becomes mandatory. the resuscitation goals are based on easily obtainable physiologic variables. treatment targets cvp pressures between 8 and 12 mm hg, mean arterial pressure ≥65 mm hg, urine output ≥0.5 ml/kg/h, and superior cava vein oxygen saturation ≥70% or mixed venous oxygen saturation ≥65% [7, 29] . no difference between crystalloids and colloids fluid was demonstrated [30] . however, it is mentioned that resuscitation with crystalloids is less expensive but requires more fluid to achieve the same end points and may result in more edema formation. fluid challenges of 1,000 ml of crystalloids or 300-500 ml of colloids over 30 min are recommended, but larger volumes or infusion rates could be required [7] . when resuscitation goals are not rapidly achieved vasopressor therapy must be started. there is no high-quality primary evidence to recommend norepinephrine over dopamine. however, norepinephrine could be more effective in reversing hypotension in patients with septic shock. the selected vasopressor, either norepinephrine or dopamine, should be titrated until map ≥65 mm hg. epinephrine is another alternative vasoactive agent when blood pressure is poorly responsive to norepinephrine or dopamine. low-dose dopamine should not be used for renal protection [31] . in patients requiring vasopressors, an arterial catheter and continuous arterial pressure monitoring must be instrumented. if central venous oxygen saturation remains <70% further fluid infusion and/or packed red blood cells transfusion should be considered. hematocrit ≥30% is desirable to assure systemic oxygen delivery. increase in cardiac index by the effect of dobutamine infusion to a maximum 20 μg/kg/min is recommended. dobutamine is the first line inotrope for patients with measured or suspected low cardiac output and adequate or high left ventricular filling pressures. a combination of inotropes/vasopressors, such as norepinephrine and dobutamine, is recommended if cardiac output is not directly measured [7] . antibiotics should be administered during the first hour of the initial resuscitation. the time taken to initiate effective antimicrobial therapy is one of the strongest predictors of outcome in septic shock [32] . initial antimicrobial selection should be wide enough to cover likely pathogens. there is evidence that failure to initiate appropriate antimicrobial therapy within this period of time correlates with increased mortality [33] . source control includes an appropriate diagnosis of the specific site of infection within the first 6 h. surgical procedures aimed at abscess draining, debridement of infected necrotic tissue, or removal of potentially infected devices should be instrumented without delay [7] . these practices are believed to be important for infection control but no randomized trials support them [34] . most of the measurements initiated in the previous stage will continue during the following hours. at the same time, some other therapeutic interventions could be started earlier, during the initial resuscitation phase. two big trials of patients with vasopressor-unresponsive septic shock showed a significant and faster resolution of shock when steroid therapy was associated [35, 36] . thus, low-dose intravenous hydrocortisone (≤300 mg/day) should be considered for adult septic patients when hypotension is poorly responsive to fluid resuscitation and vasopressors. on the other hand, an adrenocorticotrophic hormone (acth) stimulation test is not recommended. steroid therapy must be weaned once vasopressors are no longer required [7] . the importance of lung-protective strategies for patients with ali/ards is supported by clinical trials and has been widely accepted [37] . low tidal volume (6 ml/kg) and upper limit plateau pressure ≤30 cm h2o are desirable in patients with ali/ards. this respiratory pattern may result in paco2 increase above normal or permissive hypercapnia. a prone position should be considered when potentially injurious levels of fio2 or plateau pressure cannot be controlled. titration of positive end expiratory pressure (peep) should be made according to bedside measurements in an attempt to reach optimal levels of respiratory system compliance [7] . several randomized, observational clinical trials showed reductions in icu mortality when intensive insulin therapy was utilized [38, 39] . a large randomized trial recently showed that intense glucose control increased mortality. these authors found that a blood glucose target <180 mg/dl resulted in lower mortality than a target between 81 to 108 mg/dl. higher episodes of hypoglycemia were reported in the tight glucose control group [40] . based on this report, intense insulin therapy is questionable. however, the ssc recommendation is to maintain blood glucose levels below 150 mg/dl [7] . recent studies reported nonsignificant mortality reduction after rhapc administration in patients with a low risk of death or in the pediatric population. furthermore, rhapc administration is associated with increased risk of bleeding. the evidence concerning rhapc use in adults is primarily based on two rcts: the prowess and the address (stopped early for futility) [41, 42] . additional information comes from an open-label observational study, the enhance that suggested that early administration of rhapc was associated with better outcomes [43] . as a result, the latest recommendation of the ssc is to consider rhapc only for adult patients at high risk of death (apache ii ≥25 or multiple organ failure). during patient selection, possible contraindications for rhapc administration should be discharged. red blood cell transfusion should be administered when haemoglobin decreases below 7.0 g/dl. a hemoglobin target between 7.0 to 9.0 g/dl in adult septic patients is recommended. do not use fresh frozen plasma to correct laboratory clotting abnormalities unless there is bleeding evidence or planned invasive procedures. administer platelet concentrates when platelet counts are <5,000/mm 3 regardless of bleeding. platelet counts between 5,000 to 30,000/mm 3 do not call for platelet administration unless there is a significant risk of bleeding [7] . • sedation and analgesia in sepsis. it is recommended to use sedation protocols with daily interruption/lightening to produce awakening [7] . • renal replacement therapy. current evidence is insufficient to draw strong conclusions regarding the best replacement therapy method for ari in septic patients [44, 45] . it is not clear whether high doses of renal replacement may influence patient outcome [46] . intermittent hemodialysis and continuous veno-venous hemodiafiltration (cvvh) are considered equivalent for septic patients. however, cvvh and sustained low-efficiency dialysis will probably offer a safer and easier management in hemodynamically unstable patients. • bicarbonate therapy. sodium bicarbonate infusion must not be used for the purpose of improving hemodynamics or reducing vasopressor requirements when treating hypoperfusion-induced lactic acidemia with a ph ≥7.15 [7] . • deep vein thrombosis prophylaxis. use either low-dose unfractioned heparin (ufh) or low-molecular weight heparin (lmwh), unless contraindicated. use a mechanical prophylactic device when heparin is contraindicated [7] . • stress ulcer prophylaxis. stress ulcer prophylaxis based on h2 blockers or proton pump inhibitors could be used for septic patients [7] . • nutritional support. it is very important to initiate early nutritional support in critically ill patients. enteral nutrition is generally safer and more effective than total parenteral nutrition. immunonutrition needs to be further studied before clear recommendations can be made. as mentioned before, these guidelines were based on the best available evidence. ongoing and future studies will provide further valuable information and changes in these recommendations will become necessary. the use of these guidelines is not easy in clinical practice. it was an objective of the ssc to facilitate the instrumentation of these recommendations [47] . in the last phase of the ssc the concept of sepsis bundles was introduced. this idea aimed to create a series of simple recommendations easily applicable as packages of measurements for the clinical setting [48] . conceptually, a bundle is a group of interventions that when implemented together will result in better outcomes. the bundles were developed in conjunction with the institutes for health care improvement (ihci). different tools were created to assist clinicians at the bedside. these clinical tools and databases are available on the ssc web page. treatment of severe sepsis can be organized into two groups of interventions known as the initial resuscitation bundle (initial 6 h) and the management bundle (24 h bundle) [49] . table 25 .2 summarizes this particular approach. some recent works have studied bundles compliance and new favorable results on outcomes are coming. it has been shown that bundles compliance is associated with a reduction in icu mortality and length of stay [50, 51] . the epidemiology of sepsis in the united states from 1979 through 2000 epidemiology of severe sepsis in the united states: analysis of incidence, outcome, and associated cost of care is the mortality rate for septic shock really decreasing? epidemiology of sepsis and infection in icu patients from an international multicentre cohort study has the mortality of septic shock changed with time surviving sepsis campaign: guidelines for management of severe sepsis and septic shock surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis sccm/esicm/accp/ats/sis. international sepsis definitions conference procalcitonin as a marker of bacterial infection in the emergency department: an observational study c-reactive protein levels correlate with mortality and organ failure in critically ill patients a prospective evaluation of the infection probability score (ips) in the intensive care unit the microcirculation is the motor of sepsis bench-to-bedside review: sepsis is a disease of the microcirculation cytopathic hypoxia. is oxygen use impaired in sepsis as a result of an acquired intrinsic derangement in cellular respiration? cellular dysfunction in sepsis septic diaphragmatic dysfunction is prevented by mn(iii)porphyrin therapy and inducible nitric oxide synthase inhibition 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resuscitation colloid use for fluid resuscitation: evidence and spin use of dopamine in acute renal failure: a meta-analysis duration of hypotension prior to initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock the influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the icu setting approach to the patient with sepsis effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with septic shock hydrocortisone therapy for patients with septic shock ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome intensive insulin therapy in critically ill patients intensive insulin therapy in the medical icu intensive versus conventional glucose control in critically ill patients (2009) the nice-sug-ar study investigators efficacy and safety of recombinant human activated protein c for severe sepsis drotrecogin alfa (activated) for adults with severe sepsis and a low risk of death drotrecogin alfa (activated) treatment in severe sepsis from the global open-label trial enhance: further evidence for survival and safety and implications for early treatment continuous versus intermittent renal replacement therapy: a meta-analysis acute renal failure in the intensive care unit: a systematic review of the impact of dialytic modality on mortality and renal recovery the va/nih acute renal failure trial network: intensity of renal support in critically ill patients with acute kidney injury sepsis change bundles: converting guidelines into meaningful change in behavior and clinical outcome the impact of compliance with 6-hour and 24-hour sepsis bundle on hospital mortality in patients with severe sepsis: a prospective observational study implementation of the surviving sepsis campaign guidelines for severe sepsis and septic shock: we could go faster key: cord-023463-vr6uaw3a authors: liu, wei; tang, fang; fang, li‐qun; de vlas, sake j.; ma, huai‐jian; zhou, jie‐ping; looman, caspar w. n.; richardus, jan hendrik; cao, wu‐chun title: risk factors for sars infection among hospital healthcare workers in beijing: a case control study date: 2009-06-05 journal: trop med int health doi: 10.1111/j.1365-3156.2009.02255.x sha: doc_id: 23463 cord_uid: vr6uaw3a objective to evaluate possible severe acute respiratory syndrome (sars) infection associated risk factors in a sars affected hospital in beijing by means of a case control study. methods fifty‐one infected and 426 uninfected staff members were asked about risk behaviours and protective measures when attending to sars patients. univariate and multivariate logistic regression analyses were performed to identify the major risk and protective factors. results multivariate analysis confirmed the strong role of performing chest compression (or intubation, which is highly correlated), contact with respiratory secretion, and emergency care experience as risk factors to acquire sars infection. for the studied protective measures, wearing 16‐layer cotton surgical mask, wearing 12‐layer cotton surgical mask, wearing multiple layers of mask, taking prophylactic medicine, taking training and nose washing turned out to be protective against infection. conclusions this study highlighted activities associated with increased and decreased risk for sars infection during close contact with sars patients. these findings may help to guide recommendations for the protection of high‐risk occupational groups. severe acute respiratory syndrome (sars) is caused by a novel coronavirus, transmitted from human to human by droplets or by direct contact. airborne spread of the virus also accounted for certain community outbreaks of sars (yu et al. 2004) . healthcare workers (hcw) were at the highest risk of having the disease, accounting for one fifth of the global total (world health organization 2003) . in hong kong, 23% of the cases of sars were healthcare workers. in canada and singapore, the proportions were higher (40% and 41%, respectively), as they had fewer sars cases in the community than hong kong. risk factors for infection in hcws have been studied extensively, and a review on sars infection and healthcare workers disclosed a number of risk and protective factors (chan-yeung & yu 2003; lau et al. 2004) . for example, lack of awareness and pre-paredness when the disease first struck, poor institutional infection control measures, lack of training in infection control procedures, poor compliance with the use of personal protection equipment (ppe), exposure to high-risk procedures such as intubation and nebulisation, and exposure to unsuspected sars patients were associated with sars infection. measures to prevent nosocomial infection included establishing isolation wards for triage sars patients; training and monitoring hospital staff in infection-control procedures; active and passive screening of hcws; enforcement of droplet and contact precautions; and compliance with the use of ppe. in mainland china, 1 021 hcws became infected, accounting for 19.2% of the 5 327 sars cases in the whole country (feng et al. 2009, this issue) . several hospitals in beijing reported that nosocomial transmission occurred among hcws, one of which (referred to hereafter as afh (armed forces hospital)) suffered one of the most serious outbreaks: 67 confirmed cases and 16 deaths among hcws. several studies were conducted during the outbreak. one case control study was designed to understand how hcws contracted the disease and how to prevent their infection (ma et al. 2004) . it was conducted shortly after the epidemic, but the data have never been prepared for an english language publication. we therefore made a systematic analysis based on the data to investigate possible risk and protective factors associated with infection of sars among the hcws in afh. the retrospective case control study was conducted in afh hospital in beijing where the nosocomial outbreak was reported. the case group were all hcws who were diagnosed as probable sars cases admitted between 5 march and 17 may 2003, were recruited as cases. diagnosis was based on who's criteria of documented fever (temperature >38°c), presence of cough, shortness of breath or breathing difficulty, and a significant history of exposure to a sars patient not more than 10 days prior to onset of symptoms, plus radiographic evidence of infiltrates consistent with pneumonia or respiratory distress syndrome (rds) on chest x-ray (cxr) (world health organization 2003) . two cases that were suspected of contracting the infection outside their hospital stay were excluded. eligible uninfected hcws who worked in the same hospital and had self-reported exposure to sars patients between march and may 2003 were identified as controls. they were subsequently confirmed following who vigorous criteria for close contacts: the exposure was only deemed to be definite, where there was a history of being within close physical proximity (1 m) of a patient subsequently confirmed with sars (world health organization 2003). all cases and controls included were subsequently tested for ig g antibody against sars-cov using elisa method as previously described (liu et al. 2006) . for the case group, only one sars patient was detected as igg antibody negative, which was thereafter excluded from the analysis. for the control group, none of them was detected as serologically positive, thereby supporting that they had not been infected with sars. interviews with cases and controls were carried out using a pretested questionnaire by a trained epidemiological group between june and july 2003. information collected included demographic data (age, gender and ethnic group), personal medical history, coexisting conditions, work unit and ward, job description, sars-related work behaviours, protection measures and training activities. all study participants gave oral informed consent before the interviews were conducted. between 14 june and 19 june, the completed questionnaires were retrieved from all participants, after which they were immediately checked for validity and completeness. data were analysed using spss (version 13.0, spss inc, chicago, il, usa) and s-plus software (version 6.0. insightful corp., seattle, wa, usa). logistic regression was performed to estimate odds ratios (ors) and their 95% confidence intervals (ci). univariate analyses were conducted to determine the effect of each variable separately. for variables with missing value of >5%, we performed logistic regression with being missing as binary outcome and age, sex and occupation as predictors to test their non-deviance of the possibility of having missing values against the important background variables. a multivariate logistic regression was fitted using a stepwise-forward procedure with all variables that were marginally significant (p < 0.10) in the univariate analyses as candidates for selection. for the multivariate analysis we performed a stepwise-forward selection procedure and took care that at each step to select a new variable we only removed observations with missing values for the variables in the model. as a result, in the final model, cases were removed with missing values for selected variables only instead of missing values for all variables that were investigated. a stepwise-forward (or backward) procedure has a disadvantage that it does not show in how far predictors are chosen at random. thereafter we calculated all correlations between predictors and selected from the significant pairs the ones with one predictor in the final multivariate model and one not in the model. if swapping of the chosen predictor by its correlated counterpart does not seriously deteriorate the model, we conclude that we cannot tell whether one or the other predictor played a role. for all the analyses, statistical tests were based on two-tailed probability. a p-value < 0.05 was considered statistically significant. altogether 477 completed questionnaires were successfully retrieved from infected staff (51) and uninfected controls (426). the cases represent 76% (51 ⁄ 67) of all infected survived staff in the hospital. sixteen infected hcws could not be accessed or refused to be studied. the control group represents over 90% of all employees who were exposed to sars in the hospital. the demographic and epidemiological characteristics of the two groups are listed in table 1 . the mean age was 29.5 ± 9.6 years for the cases, and 31.4 ± 8.8 years for the control group. the statistical analysis showed that the two groups were comparable in almost all of the demographic information we obtained, including age, gender, marital status, ethnicity, co-morwork load was expected to be an important factor for infection occurrence and was measured by the maximum (or average) number of contacts with sars patients per day, and the maximum (or average) working hours in the sars-designated isolation ⁄ contagious area per day. statistical analysis showed that none of the factors mentioned above was significantly different between the two groups (table 1) . altogether we included 28 variables to test their association with disease occurrence, of which 17 gave significant results (p < 0.10), including seven risk factors and 10 protective factors. for example, among the healthcare workers who reported emergency care experience (i.e. over a mean of 1 h) 21% got sars infection, while only 8% of those without emergency care experience acquired sars, reflecting a highly significant difference between both groups. detailed information on the contact with sars patients were identified. contact with respiratory secretions, sputum, pathological specimens and deceased significantly increased the ors of sars infection. but this was not seen for other types of contact, i.e. with faeces, blood, urine, pulmonary lavage, medical waste, nursing contact or equipment contacts ( table 2 ). the previously reported high-risk activities were also identified in the studied subjects: endotracheal intubation and chest compression were behaviours significantly associated with high risk for infection. the other activities, including post-mortem, intensive care unit (icu) nursing, sample collection and chest physiotherapy, patient transferring etc. did not achieve significant results, all with p > 0.10. two kinds of personal protective equipment were studied, face protection and body protection. six categories of most often used masks were identified from the participating staff: disposable mask, surgical mask, 12-layer cotton surgical mask, 16-layer cotton surgical mask, n95, and higher-level protective respirator. when evaluated individually, 12-layer cotton surgical mask and 16-layer cotton surgical mask displayed significant difference of distribution between the two groups. healthcare workers who wore glasses had about half the risk of sars compared to those not wearing glasses (p = 0.006) ( table 2) . a similar reduction in ors was achieved by wearing a protective goggle when attending to patients (p = 0.046), and washing the nose after attending to patients showed an even stronger effect (p = 0.0002) (the nose wash was performed as a nasopharyngeal rinse for cleansing). wearing gloves and multiple layers of protective gowns also each reduced the ors of infection significantly (p = 0.011 and p = 0.052, respectively). among the administrative factors, taking training in infection control before contact with patients and taking prophylactic medicine (including anti-viral medicine and supplemental nutrition to enhance immunity) were shown to be protective (p < 0.001 and 0.003, respectively). five significant univariate variables (contact date, wearing glasses, layers of protective clothes, wearing gloves and taking training) had missing values of >5%. the only significant difference between individuals with and without missing values is that the probability for not answering the question about wearing gloves is smaller for nurses (data not shown). so it is not very likely that missing of values will have caused important bias. the final multivariate model (table 3 ) contains a total of nine variables, three variables concerning masks and none concerning glasses, gloves etc. we have tried to exchange the mask-variables for the other protection variables, but this dramatically worsened the model, so we conclude that wearing masks, of whatever design, is of the utmost importance to prevent infection. also, emergency care experience and not taking training were selected as important predictors of getting sars infection. nose wash and taking prophylactic medicine turned out to be useful. regarding contacts with patients, we found respiratory secretion and performing chest compression as important predictors. the correlations between seven predictor pairs are listed in table 4 . training and contact date were shown to be correlated. supplementary analysis showed that given taking training, contact date is not significant (p = 0.11), and given contact date, taking training is not significant. we therefore conclude that we cannot distinguish between these two variables. contact with respiratory secretion was correlated with contact with sputum, but here we found that given secretion, sputum is not significant (p = 0.63), but given sputum, contact with secretion is clearly significant (p = 0.027). we therefore conclude that these variables are non-interchangeable: contact with respiratory secretion is the dangerous event. chest compression and intubation were also highly correlated and we found that distinction between those two is not possible. the investigation was carried out in a hospital with a nosocomial outbreak of sars. a total of 477 staff was recruited into the study, representing about 90% of hcws exposed to sars patients. the multivariate logistic regression finally disclosed three factors to be significantly associated with disease occurrence (i.e. performing chest compression or intubation, contact with respiratory secretion, emergency care experience), whereas six actions our study was subjected to a number of limitations. first, as in all retrospective surveys, recall bias was a concern. however, it is unlikely that ors of this magnitude could result primarily from recall bias, since the associations shown are clear. the study was conducted shortly after the outbreak, thus minimising the information bias to which retrospective studies are otherwise susceptible. another possible bias is that the case group attributed their infection to some high risky performance (e.g. performing intubation) and less efficient protection (wearing only one layer of mask while attending patients), while the control group did the opposite. during sars epidemic in mainland china, all healthcare staff working in hospitals had been required to follow the recommended personal protection procedures. however, the risk of sars infection, the level of precaution taken and the compliance with the standard largely depended on multiple factors, including the different time phases of patient contacts, different working areas and the various types of procedures performed. the outbreak in the hospital could be divided into two phases. in the early phase, there was a general lack of familiarity and training regarding infection-control measures among hospital staff. medical wards and equipment were not adequately set up for the strict infection control standard. while in the later phase, after a super spreading event (sse) occurred in the hospital, hcws became more vigilant about protecting themselves from sars transmission, which together with adequate training and infection control measures led to significant differences in the infection rate between subjects from before and after 2 april 2003, when the institutional infection control measures were established and enforced. we defined two phases of contact with sars using the date of 2 april as the divide. as found in univariate analysis, early contact with sars patients increased the ors of sars infection significantly. for the different working areas the univariate analysis did not show significant difference of the distributions between the case and control group. in multivariate analysis, history of contact with respiratory secretion from an infected patient was associated with 3.27-fold increased ors of infection for the attending hcws. this is consistent with previous findings (teleman et al. 2004) , thus adding to the evidence that contact with respiratory secretions is an important risk factor. certain types of procedures have been shown to be high-risk because they can lead to extensive spreading of droplets from the patient, for example, the use of the jet nebulizer and intubation and assisted ventilation (cdc 2003a; ofner et al. 2003; fowler et al. 2004 ). our study found a significant association of infection with performing chest compression; while performing intubation showed significant correlation with the former, we cannot distinguish between these two variables given multivariate logistic regression, so both might act as factors that enhance the probability of sars infection. however, lau et al. (2004) showed that performing particular high-risk procedures on sars patients was not considered to increase the disease risk. this might be due to the different types of procedures considered and inadequate sample size. severe acute respiratory syndrome (sars)-cov infection is thought to occur primarily by either contact or large respiratory droplet transmission (cdc 2003b; ruan et al. 2003). the effectiveness of protective measures of using masks, gloves, gowns, and hand-washing, recommended under 'droplet precautions' when caring for sars patients, was also investigated for their protection effect. some studies show that use of any mask was associated with lower ors of infection in healthcare-related clusters (ruan et al. 2003; seto et al. 2003; nishiura et al. 2005) . one study showed that consistent use of an n95 mask was more protective than not wearing a mask or consistent use of a surgical mask (loeb et al. 2004) . in our study, wearing a 16-layer cotton surgical mask or 12-layer surgical mask, as well as multiple layers of masks, reduced the risk of infection. however, this does not mean that the higher-level protective masks, e.g. the n95 mask, cannot cause effective protection. the counting of n95 mask use was not large enough to give a significant p-value. this might pose an important limitation leading to the failure to detect an effect, even though it may be there. the factor using more than one layer of masks was shown to be more protective than a single layer, which presents strong evidence that necessary and appropriate face protection should be advised to diminish the risk of droplet infection. the adoption of 16-layer and 12-layer cotton surgical masks individually was shown to be highly effective on risk reduction and therefore should be recommended accordingly. wearing glasses is not intended as protection, yet it indeed showed significant protective effect in univariate analysis. this, together with the significant results of wearing goggles as shown in univariate analysis, might confirm the crucial role of eye protection, although both factors were not confirmed in the subsequent multivariate analysis. these findings fit well with droplets transmission because droplets are generated at the face level, making the mask and eye protection necessary for protection. the finding that wearing of gowns and gloves achieved statistical significance in univariate, but not in multivariate analysis, differs from the previous studies, where neither gloves nor gowns were found to be protective (varia et al. 2003; teleman et al. 2004 ). in the study by seto et al. (2003) in hong kong, however, gowns were found to be protective, although protection from gloves also failed to achieve statistical significance. in our final multivariate model, we explicitly tested the hypothesis that inclusion of mask over gowns and gloves was due to change but were strengthened in confidence by the fact that exchange of one set of predictors for the other seriously distorted the model. so the failure of inclusion of gowns and gloves was due to their insufficient contribution to the multivariate logistic model. however, wearing of gloves or gowns should never be downplayed because of the insignificant p-value in multivariate analysis. it should be noted that the effect of protective measures might be enhanced by beforehand training on infection control knowledge. in both univariate and multivariate analysis, the infection control training was significantly associated with lower risk of infection and also showed significant interaction with wearing multiple layers of masks. the latter finding had also been suggested in a previous study (lau et al. 2004) . although several risk factors and protective measures have been identified, we should judge them in relation to the two exposure phases. during the early phase, hcws in the hospital did not fully acknowledge the risk of the exposure to patients, and adequate personal protective equipments were not applied. the performance of endotracheal intubation and chest compression also helped to disseminate infected aerosol widely to a large number of patients and staff on the ward. this happened a lot during emergency care. during the latter phase, when the workers realised that they were dealing with a high risk infectious disease, they started to use personal protective equipment; however, due to general lack of familiarity and training regarding infection-control measures among the working staff, the protective measures were not administered adequately while dealing with sars patients. this might explain to some extent that even after protective measures were implemented, there remained hospital workers that contracted the infection. the high degree of correlation in the risky and protective procedures makes it difficult to ascertain which type of activity is most important for sars infection, e.g. performing chest compression and performing intubation cannot be distinguished for their contribution to the multivariate model. the same applies to taking training and contact data (after 2 april). thus, we should be aware that these types of personal risk and protective activities definitely played role in the disease occurrence, although they could not be identified as such in the stepwise multivariate model. we also believe that, whereas this study accepted the current factors as significant using a p-value < 0.05, other risk and protective factors for disease infection exist, which we failed to elucidate in this study. in summary, this study identified exposure to high-risk procedures (such as chest compression), and contact with respiratory secretions to be significant risk factors for sars infection among hcws in a hospital in beijing. these results also provide confirmation that personal protective measures against droplet spread, such as wearing multiple layers of mask, are effective against the nosocomial spread of sars. this knowledge may help prepare public health officials and clinicians for a reappearance of sars, should it occur, or for the emergence of another comparable infectious disease. all hcws should remain vigilant to evaluate and improve their infection-control practices to limit possible future outbreaks of sars and sars-like other nosocomial outbreaks). from the administrative point of the hospital, teaching and training of the medical profession in infectious diseases and the capacity of the public health sector to deal with these diseases must be strengthened. cluster of severe acute respiratory syndrome cases among protected health-care workers-toronto, canada outbreak of severe acute respiratory syndrome-worldwide outbreak of severe acute respiratory syndrome in hong kong special administrative region: case report the sars epidemic in mainland china: bringing together all epidemiological data transmission of severe acute respiratory syndrome during intubation and mechanical ventilation sars transmission among hospital workers in hong kong two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome sars among critical care nurses a case-control study on the risk factors of severe acute respiratory syndromes among health care workers rapid awareness and transmission of severe acute respiratory syndrome in hanoi french hospital cluster of severe acute respiratory syndrome cases among protected health care workers-toronto comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) factors associated with transmission of severe acute respiratory syndrome among health-care workers in singapore investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto case definitions for surveillance of severe acute respiratory syndrome (sars). who evidence of airborne transmission of the severe acute respiratory syndrome virus the authors have declared no conflicts of interest. key: cord-015922-5wwy0m2k authors: marty, francisco m.; baden, lindsey r. title: infection in the hematopoietic stem cell transplant recipient date: 2008 journal: hematopoietic stem cell transplantation doi: 10.1007/978-1-59745-438-4_19 sha: doc_id: 15922 cord_uid: 5wwy0m2k nan there are three key elements of the hsct procedure that determine the type and timing of the infectious risk profile after transplantation [1, 7] : 1, 7 a. the duration of neutropenia and mucosal injury which is a function of the conditioning regimen selected (myeloablative or not) and the stem cells' procurement (cord or adult; peripheral or bone marrow acquisition among adult donors). b. the strategy chosen to prevent gvhd among allogeneic recipients. t cell depletion and other t cell manipulation procedures lead to delayed recovery of lymphocyte function and provide a specific immune deficiency profile. c. the occurrence and severity of acute and chronic gvhd and its treatment [1, 7] . the temporal course of infection following hsct can be divided into three time periods (fig. 19-1 ) [1, 3, 4, 7] : 1. conditioning to engraftment the duration of this period has become dynamic and depends on the conditioning regimen itself, the source and dose of stem cells infused and whether growth factors are used. it usually ranges from five (with nonmyeloablative transplants) to 30 days (with bone marrow or umbilical cord blood transplants). the combination of profound granulocytopenia and mucositis with myeloablative conditioning makes the patient particularly vulnerable to bacterial and candidal infections. in addition, infection present in the transplant recipient pre-transplant may be amplified by the granulocytopenic state and deficiencies of t and b-cell numbers and function. thus, control of pre-transplant infection is needed before initiating the conditioning regimen. prior to engraftment (both with autologous and allogeneic transplants), approximately 50 percent of patients will have fever of unknown origin, with bloodstream infection in ~12.5 percent and pneumonia in ∼10 percent. the risk of an invasive mold infection is related to the duration of neutropenia and the environmental strategy used in a transplant center. 2. engraftment to post-transplant day 100 during this time period viral infections, particularly cytomegalovirus (cmv) and the other herpes group viruses, are the major concerns. the occurrence, severity and treatment modalities selected for acute gvhd further modulates and increases the risk of herpesvirus infections, especially cmv and epstein-barr virus (ebv), and invasive mold infections [8] [9] [10] [11] . 3. more than 100 days post-transplant in the absence of gvhd, the incidence of infection decreases significantly, with varicella zoster virus (vzv), pneumocystis jiroveci (formerly carinii) pneumonia (pcp) and pneumococcal infection being the primary problems of this time period. routine use of prophylaxis, such as with trimethoprim/sulfamethoxazole and acyclovir, significantly decreases the occurrence of pcp and herpesvirus infections, respectively. in addition, late or relapsing cmv infection may manifest during this time. if gvhd is present, it is typically treated with significant augmentation of immunosuppressive therapy such as with high-dose corticosteroids and monoclonal antibodies. patients in this last category (gvhd under treatment) are at particular risk for invasive mold infection, cmv reactivation, pcp and other common and opportunistic pathogens. there are four modes in which antimicrobial therapy can be administered to the hsct patient [4] : 1. a therapeutic mode, in which antimicrobial therapy is prescribed for the treatment and eradication of identified microbes causing clinical illness. 2. a prophylactic mode, in which antimicrobial therapy is prescribed to an entire population before an event to prevent clinically important infection. for such a strategy to be successful, the infection(s) being targeted must be important enough to justify the intervention, and the antimicrobial therapy prescribed must be nontoxic and inexpensive enough to justify the intervention. by far the most effective antimicrobial prophylactic strategy is low-dose trimethoprim-sulfamethoxazole, which has virtually eliminated the occurrence of pneumocystis jiroveci, listeria monocytogenes, nocardia sp, and toxoplasma gondii in patients who adhere to the regimen. other prophylactic strategies commonly utilized in hsct patients include acyclovir to prevent herpes simplex virus (hsv) and vzv reactivation, fluoroquinolones [5] to prevent gram-negative sepsis and fluconazole to prevent yeast infection. 3. an empiric mode, in which antimicrobial therapy is administered in response to a symptom complex. in this context, empiric antimicrobial therapy is initiated during the period of profound granulocytopenia in response to fever +/− rigors or subtle signs of sepsis (unexplained hypotension, tachypnea, an ongoing volume requirement, or acidosis). in the patient deemed not to be a therapeutic emergency, initial therapy is usually aimed at aerobic gramnegative bacilli (e.g., the enterobacteriaceae and pseudomonas aeruginosa). a variety of drugs have been utilized for this purpose, depending in part on the nature of particular problem organisms found at a given medical center. advanced spectrum beta-lactams (e.g., ceftazidime, piperacillin or imipenem), either alone or together with an aminoglycoside or a fluoroquinolone, are the mainstays of this approach. thus, empiric therapy is based on an algorithm rather than on microbiologic or other studies. 4. a preemptive mode, in which antimicrobial therapy is prescribed to a proportion of patients deemed to be at particularly high risk because of clinical/epidemiologic information or the isolation of microbial pathogens. examples of preemptive therapy in hsct are the molecular surveillance of cmv linked to deployment of ganciclovir or, more recently, the use of galactomannan monitoring for initiation of anti-aspergillus antifungal treatment [12] . given the nature, duration and severity of host defense defects present in hsct patients, it is not surprising that bacterial infection is a regular feature of the post-transplant course. the most common involved sites include blood stream (often catheter-related), lung, gastrointestinal tract and skin/soft tissue. the greatest rate of bacterial infections occur during the period prior to engraftment; this rate is a product of granulocytopenia, mucositis that permits the translocation of bacteria and yeast from the oral cavity and gut into circulation, and the presence of vascular access devices that traverse the skin and serve as direct conduits into the systemic circulation. thus, the primary mucocutaneous barriers to infection are compromised, and the absence of granulocytes only amplifies the susceptibility of the patient [1, 4, 7] . in an attempt to decrease bacterial infections during the neutropenic period, especially those due to gram-negative bacilli, strategies of prophylactic antimicrobial use have been studied, including the use of trimethoprimsulfamethoxazole and fluoroquinolones. some studies, most recently with levofloxacin, have demonstrated benefit in decreasing the occurrence of fever and microbiologically-confirmed bacterial infections [13] [14] [15] . however, significant concerns regarding this approach have been raised given that no mortality benefit has been demonstrated, the emergence of resistant organisms, and the impairment this widespread antimicrobial approach has on the use of quinolones in future oral outpatient management. thus, in many transplant centers, an empiric antibacterial regimen targeting pseudomonas and other enterobacteriaceae in response to fever or other infectious syndromes remains a preferred approach. whereas gram-negative bacteremia was the major cause of blood stream infection 15 to 30 years ago, today gram-positive organisms are the most frequent cause of positive blood cultures. the possible reasons for this shift are many: the widespread use of fluoroquinolones, with their potent activity against gram-negative bacteria, as prophylaxis during this period; the presence of indwelling central venous catheters for prolonged periods; and the widespread use of systemic anti-gram-negative therapy all contribute to the gram-positive predominance. the bacteria isolated during the preengraftment period, then, include staphylococci (especially coagulase-negative staphylococcus), viridans streptococci, enterococci and corynebacteria, with fewer isolates of enterobacteriaceae or pseudomonas aeruginosa being identified. an increasing problem in the hsct population is antibiotic resistant organisms, particularly vancomycin-resistant enterococci, methicillin-resistant staphylococcus aureus, and resistant gram-negative bacilli (such as extended spectrum β-lactamase producing klebsiella and chromosomal inducible βlactamase producing enterobacter species) [1, 4, 6, 7, [16] [17] [18] [19] [20] . the typical approach for the severely granulocytopenic patient at present is the initiation of empiric antibacterial therapy in response to an unexplained fever or other signs of sepsis. what remains controversial is what the regimen should be. since clinical deterioration can occur rapidly with untreated gram-negative sepsis in the granulocytopenic patient, anti-gramnegative therapy is always employed. the traditional approach of a β-lactam (e.g., piperacillin) plus an aminoglycoside is still favored by some experts, although nephrotoxicity from the aminoglycoside has led to the trial of other approaches, including the substitution of a fluoroquinolone for the aminoglycoside, or the prescription of a single advanced spectrum drug such as ceftazidime, cefepime, imipenem or meropenem. if fluoroquinolone prophylaxis has been utilized, then its use as a therapeutic agent may be diminished. empiric fluoroquinolone monotherapy is inferior to other regimens, and if pure aerobic gram-negative agents are utilized, (e.g., aztreonam, aminoglycosides) due to confirmed severe beta-lactam hypersensitivity, then the addition of empiric gram-positive coverage that targets aerobic and anaerobic streptococci of the gastrointestinal tract should be considered. use of extended interval (once-daily dosing) aminoglycoside administration may be safer and as effective. the second area of controversy is whether empiric gram-positive treatment should be initiated at the same time, given the preponderance of grampositive infection. as there is typically time to evaluate culture data and deploy targeted gram-positive antimicrobial therapy rather than empiricism, vancomycin should rarely be required empirically. furthermore, empiric gram-positive coverage is not associated with better outcomes [21] . indications for the immediate initiation of vancomycin as part of the empiric therapy regimen include the following [6, 16, [18] [19] [20] [21] : catheter-related sepsis is likely because of evidence of infection at the insertion site (or within the tunnel), severe illness such as shock and/or respiratory distress are present, the patient is at particular risk for seeding of a prosthetic device (e.g., a prosthetic valve, a hip prosthesis, etc.), or the empiric gram-negative coverage exclusively covers aerobic gram-negative rods -such as the combination of aztreonam and gentamicin. vancomycin or other anti-staphylococcal agents should be started if cultures become positive for gram-positive cocci. in our experience, vancomycin can be discontinued safely in patients in whom vancomycin was started empirically, but in whom blood cultures remain negative after 48 to 72 hours and there is no specific syndrome, such as cellulitis, that requires treatment with vancomycin. on the other hand, empirical treatment against gram-negative organisms should be continued until resolution of neutropenia, whether fevers resolve or not [22] . the emergence and persistence of multidrug-resistant organisms should guide local practice in a dynamic fashion. indwelling long-term catheters remain a feature of the early post-transplant period to provide chemotherapy, nutritional and blood product support until stable engraftment. routine anti-gram-positive antimicrobial therapy is not required just because a central catheter is in place for the prevention and management of catheter-related infections [21, 23] . the use of antimicrobial-coated catheters should be studied in this population, especially when non-tunneled catheters need to be used. nonantimicrobial-based strategies to prevent bacterial infections during the neutropenic period include the systematic use of hand hygiene and the use of mask and gloves by health care personnel and family members. other nonantimicrobial strategies which may be beneficial in preventing infections, but have not been tested in hsct, include the use of palifermin to prevent mucositis [24] in patients undergoing myeloablative conditioning. after engraftment, the risk of bacterial infections depend on the community exposures to common and opportunistic bacteria (e.g., nocardia, rhodococcus, listeria), the presence of acute and chronic gvhd, the degree of b-cell reconstitution and the use of trimethoprim-sulfamethoxazole prophylaxis. patients with chronic gvhd are at risk for invasive infection from encapsulated organisms, particularly streptococcus pneumoniae, haemophilus influenzae and neisseria meningitidis. it is postulated that the combination of b lymphocyte dysfunction secondary to the conditioning regimen and the effects of gvhd and its treatment have resulted in the loss and failure to develop an opsonizing antibody to these organisms, particularly streptococcus pneumoniae. in addition, for at least one to two years post-transplant, hsct patients have an inadequate response to pneumococcal vaccine. as igg levels are often low for some time after hsct, they should be routinely monitored, with replacement being considered when the igg level falls below 500 mg/ml [25, 26] . in addition, antimicrobial prophylaxis, such as with low-dose trimethoprimsulfamethoxazole (one single strength tablet daily for pcp prophylaxis), may afford further protection against this problem [1, 3] . there are several classes of viral infection of particular importance in the hsct recipient: those due to herpesviruses (cmv, ebv, hsv, vzv and human herpesvirus-6 [hhv-6]); those due to hepatitis viruses (e.g., hepatitis b [hbv]); those due to respiratory viruses (e.g., influenza, rsv, parainfluenza, adenoviruses, and others), and those due to polyoma viruses. the human herpesviruses share a number of characteristics that make them particularly successful pathogens in hsct recipients [1, 4, 27] : 1. latency once infected with a herpesvirus, one is infected for life, with a circulating antibody (seropositivity) in the absence of active viral replication being the classic marker for latent infection. reactivation from latency may be triggered by tumor necrosis factor (tnf), with the catecholamines epinephrine and norepinephrine and proinflammatory prostaglandins also playing a role. thus, the virus may be reactivated by such processes as sepsis, gvhd, allogeneic reactions, okt3 and antilymphocyte globulin. once a replicating virus is present, medications such as cyclosporine, tacrolimus and prednisone may significantly amplify the viral replication. 2. cell association these viruses are highly cell-associated, meaning that transmission occurs through intimate person-to-person contact, or transfusion or transplantation of latently or actively replicating cells from a seropositive donor. humoral immunity is, hence, less important than cell-mediated immunity. indeed, the key host defense is accomplished by major histocompatibility complex (mhc)-restricted, virus-specific, cytotoxic t cells, just that component of host defense most affected by gvhd and its treatment. 3. oncogenesis herpesviruses, such as ebv and hhv-8, play a direct role in oncogenesis-causing post-transplant lymphoproliferative disease (ptld) and kaposi's sarcoma, respectively. herpesviruses may also play an indirect role in oncogenesis with symptomatic cmv disease, increasing the incidence of ebv-associated ptld severalfold. 4. indirect effects in addition to the direct causation of infectious disease syndromes, human herpesviruses, particularly cmv, have indirect effects that are clinically important. it is believed that cytokines, chemokines and growth factors produced in response to viral replication may be responsible for these effects. they include, in addition to the modulation of oncogenesis, increasing the net state of immunosuppression so that the risk of opportunistic infection is increased. this last point is particularly important, as a variety of experiments have shown that gvhd and infection are closely linked by the production of these mediators. that is, there is a bidirectional trafficking of mediators between these two processes. the clinically most important direct effects of cmv in the hsct recipient are pneumonia and gastrointestinal disease. before effective antiviral treatment became available, cmv pneumonia occurred in 20 to 30 percent of seropositive recipients and had an associated mortality around 80 percent [28] . cmv commonly causes fever in the absence of preemptive treatment, and end-organ disease (hepatitis, bone marrow dysfunction, retinitis, and encephalitis) may occur. among allogeneic hsct recipients, the risk of cmv reactivation (60-80%) and end-organ disease is greatest in the seropositive recipient who receives a graft from a cmv seronegative donor (cmv d−/r+), likely due to the loss of native immunity during the transplant process and immune reconstitution with a cmv naïve allograft [29, 30] . patients who are cmv d+/r+ have cmv reactivation (50-60%) and disease risk that is similar to or slightly lower than that of the cmv d−/r+ patient. patients who are cmv d+/ r− have a lower risk of cmv infection (10-30%) and disease, but higher than cmv d−/r− patients (<5%). the risk of cmv infection in this latter group has been greatly decreased by use of leukoreduced blood products or by exclusive use of cmv negative products when available [31] . the risks of cmv reactivation and disease among autologous hsct recipients is minimal (<1%) [1, 4] . another major risk factor for the development of cmv reactivation and disease is the occurrence, severity and treatment of acute gvhd [29, 30] . other potential factors associated with an increased risk of cmv reactivation and disease are reception of t cell-depleted or cord blood allograft, whether the donor is unrelated or mismatched, or donated bone marrow (instead of peripheral stem cells), and whether the conditioning regimen was myeloablative [29, 32] . the most widely used therapy for clinical cmv disease is ganciclovir, which can be administered either intravenously or orally in the form of a prodrug, valganciclovir, with an acceptable bioavailability profile (~50-60%). typically, the parenteral form is administered until the patient is able to tolerate oral therapy. gastrointestinal absorption of valganciclovir, even in the setting of mild to moderate gi gvhd, has been demonstrated to be adequate [33, 34] . duration of treatment depends on the clinical response and the nature of the recovery of native immune function. in the case of serious illness, particularly pneumonia, anti-cmv hyperimmune globulin can be considered as adjunctive therapy. despite these efforts, the mortality from cmv pneumonia remains high. the major toxicity of ganciclovir is myelosuppression, so that great effort is placed in monitoring these patients closely and adjusting doses appropriately [1, 4] . occasionally, g-csf support may be required to preserve an acceptable neutrophil count and to allow adequate therapy of a serious cmv infection. while certain medications, such as atg and okt3, are likely to induce cmv reactivation, others like sirolimus may inhibit this [29] . current strategies are based on preventing cmv disease through prophylaxis or preemption. prophylaxis with ganciclovir from the time of engraftment until at least day 100 post-transplant has been studied in randomized trials [35, 36] . although cmv viremia and disease were prevented, there was no overall benefit of this strategy due to secondary bacterial and fungal infections related to ganciclovir-induced neutropenia. alternatively, a preemptive strategy is employed in which patients are monitored weekly for viremia through either a pcr assay for cmv dna or an antigenemia assay. positive results are linked to initiating ganciclovir or other antiviral drugs active against cmv. typically, these assays turn positive several days to weeks prior to the onset of clinical disease, permitting the use of effective preemptive therapy [1, 4, 29, 33, [37] [38] [39] [40] [41] . a preemptive approach significantly decreases the amount of prophylactic medication used, thus minimizing medication-associated toxicity. in the pre-ganciclovir era, cmv disease typically occurred during the first three months post-transplant. increasingly, with the widespread use of a prophylactic or preemptive antiviral strategy, breakthrough occurs much later, typically one to three months after the cessation of the antiviral therapy. risk factors for late cmv disease include chronic gvhd, low cd4-t cell counts, and cmv infection before day 100. relapse or the emergence of ganciclovirresistant virus also can occur, particularly in the face of high viral loads and inadequate courses or dosing of ganciclovir. foscarnet is the preferred drug in this setting or when further potential myelosuppression with ganciclovir is not advisable. the experience with cidofovir use in the hsct population is limited. both foscarnet and cidofovir are potentially nephrotoxic and should be administered with caution [1, 4] . studies are examining the emerging strategies for the management of cmv infection and the use of cmv vaccines in donors and recipients, adoptive immunotherapy for patients with refractory or relapsing cmv infection and the use of maribavir for prophylaxis. the major recognizable clinical effect of ebv in the hsct patient is in the pathogenesis of ptld. following the recovery from primary ebv infection (>95% of the adult population), ongoing lytic infection of b-cells occurs in the oropharynx, with latent infection of b-cells in the peripheral blood and lymphoid tissues. these latently infected cells can be transformed and immortalized, resulting in polyclonal proliferation. in the normal seropositive individual, these cells are kept in check by a specific cytotoxic t cell response. in the presence of immunosuppressive therapy, this surveillance system is inhibited in a dose-related fashion, thus permitting continued b-cell proliferation. such ongoing proliferation results in particular clones being favored and the potential for developing cytogenetic abnormalities, which leads to the development of a truly malignant process-ptld [1, 4, 27, 42] . the spectrum of clinical disease seen with ptld is quite broad, ranging from a mononucleosis-like process or a polyclonal proliferation of lymphocytes that usually responds to decreasing immunosuppressive therapy, to a monoclonal, highly malignant b-cell lymphoma. the mononucleosis-like process is seen particularly in children with primary post-transplant ebv infection. the clinical presentation is one of fever, sore throat, cervical adenopathy and tonsillar hypertrophy and inflammation. unlike b-cell lymphoma in the normal host, in the transplant patient, particularly the adult, the process can be extranodal. thus, presentations may include central nervous system (cns) invasion (from involvement of the meninges to focal cerebral lesions), liver, lung and bone marrow diseases. not uncommonly, involvement of the gut (particularly the small bowel) may lead to recognition of the ptld, with a clinical presentation of small bowel obstruction, perforation, or occult gastrointestinal bleeding. disseminated, multi-organ disease is quite common in the hsct patient [1, 4, 41, 42] . risk factors for developing ptld include: primary ebv infection in association with high-dose immunosuppression; interventions such as t cell depletion, umbilical cord blood transplant and the systemic administration of anti-thymocyte globulin increase the risk significantly; and intensive immunosuppression that results in suppression of the key host defense against ebv-transformed cells (mhc-restricted, ebv-specific, cytotoxic t cells) significantly increases the risk of ptld. in addition to the host characteristics mentioned, high ebv viral loads correlate with an increased risk of ptld. it has been suggested that ebv viral load surveillance in peripheral blood be carried out in high risk patients (those with primary ebv infection, anti-t cell antibody therapy for gvhd, hla-mismatched or t cell-depleted hsct recipients), with decreased immunosuppression +/− antiviral therapy (acyclovir or ganciclovir) carried out in the setting of high viral loads [1, 4, 41, 42] . treatment of ptld remains controversial. all patients with diagnosed ptld should have a significant decrease in immunosuppressive medications. many centers also prescribe antiviral therapy. patients not responding to these measures are usually treated with an anti-b-cell monoclonal antibody (rituximab, an anti-cd20 monoclonal antibody) [43, 44] . after that, therapies have ranged from anti-lymphoma chemotherapy to alpha-interferon and intravenous gamma globulin. hsv infection prior to the introduction of acyclovir was a major problem in the hsct recipient. occurring in the preengraftment period, hsv infection greatly exacerbated the severity of mucositis. not only were ulcers observed in the oral cavity and anogenital areas, ulcerations of the esophagus, stomach and intestine were also observed. hsv pneumonia was also noted, with rare cases of cutaneous dissemination and encephalitis. the current standard of care is to test all candidates for hsct for an antibody to hsv, with seropositive individuals then placed on antiviral prophylaxis, beginning prior to hsct. effective agents for hsv prophylaxis include acyclovir (intravenous or oral), valacyclovir or famciclovir. recurrence of hsv may occur later in the course, and should again be treated with an acyclovir regimen, with repeated episodes justifying long-term prophylaxis. acyclovir resistance is uncommon in this situation, but can occur, and requires treatment with foscarnet [1, 4] . all patients and donors should have serologic testing for vzv prior to transplant. seronegative individuals post-transplant should avoid exposures to vzv, but if such an exposure occurs, valacyclovir or varicella hyperimmune globulin should be promptly initiated. before universal prophylaxis with acyclovir became standard, an estimated 40 percent of hsct patients developed active vzv, with a median time of onset being five months post-transplant. the great majority of these patients had zoster, but approximately 20 percent had a more generalized process resembling primary varicella. a significant concern was visceral involvement in the setting of disseminated disease as well as neurologic complications such as myelitis or encephalitis [1, [45] [46] [47] [48] . prophylaxis with acyclovir in the early period post-transplantation substantially decreases the occurrence of herpesvirus infections, including vzv, and is rarely, if ever, seen during acyclovir prophylaxis. prophylaxis is typically given for the first year post-allogeniec transplantation. vzv reactivation is often seen three months post-discontinuation of prophylaxis. as the vzv vaccine is a live attenuated viral vaccine, its use is contraindicated for at least two years posttransplantation, and unless a research study or close follow-up is involved, should be omitted. hhv-6 is a β-herpesvirus (as is cmv) whose role in post-transplant complications is being defined. in the great majority of instances, hhv-6 primary infection occurs by the third year of life, with a seroprevalence rate of 90 percent at one year, and close to 100 percent at three years [49, 50] . the clinical effects associated with primary hhv-6 infection include exanthem subitum (roseola), and a form of encephalitis. in hsct patients, bone marrow suppression, especially delayed platelet engraftment, and encephalitis have been associated with hhv-6 type b. the encephalitis typically occurs one to two months after transplantation and is associated with profound memory loss, especially short-term memory, and mri changes in the mesial temporal lobes (limbic encephalitis) [51, 52] . the highest risk patients for this complication are male, umbilical cord blood recipients for whom the attack rate may be as high as 10 to 20 percent. detecting hhv-6 dna in the blood of allogeneic hsct recipients is a common phenomenon occurring transiently in 40 to 60 percent of patients, yet encephalitis is a rather infrequent occurrence (1-2%). as obtaining brain biopsies is not usually feasible early after transplantation, the diagnosis of hhv-6 encephalitis is currently achieved by developing an acute limbic encephalitis syndrome, confirmed with mri imaging of the brain and by the detection of hhv-6 in the csf [52] . it remains unclear what the treatment of choice for this virus is. one approach that we currently favor is to use foscarnet. it is possible that anti-cmv preventative strategies with ganciclovir may have a beneficial effect on this virus as well [1, 53] . hsct recipients are at significant risk for infection with respiratory viruses circulating in the community. these infections can occur at any time in the post-transplant course, and can be acquired in the community or during hospitalization from infected staff, family and friends. overall, an estimated 10 to 20 percent of hsct patients will become infected in the first year posttransplant, with the potential for this figure to rise significantly in the setting of a community-wide outbreak [54] . the dilemma for the clinician is how to prevent these infections, as there is a far higher rate of progression to pneumonia (viral and/or bacterial or fungal superinfection), which carries a far higher morbidity and mortality than what is observed in the general population. in addition, antiviral therapy for these agents is in its infancy. it is important to attempt to make an etiologic diagnosis. avoiding exposure to infected individuals by systematic infection control measures in both family members and friends, but most importantly in health care workers, is the best preventative strategy available [55] [56] [57] [58] . although rsv can be acquired by inhaling an aerosol, direct contact with infected secretions is the usual mode of spread between individuals. in the hsct patient, both adult and pediatric, rsv is a cause of significant morbidity and mortality. the illness begins with the signs and symptoms of a viral upper respiratory tract infection (rhinorrhea, sinus congestion, sore throat and/or otitis media), that may progress to pneumonia, especially if the virus is acquired in the preengraftment phase. as with influenza, pneumonic syndromes can be due to rsv itself, but in our experience it is more frequently due to secondary bacterial and fungal infections. the advent of rapid rsv diagnosis by antigen detection in nasopharyngeal swabs has resulted in the recognition that rsv is a significant pathogen for both adults and children, particularly in immunosuppressed patients. optimal antiviral management, however, remains unclear. there are reports that aerosolized ribavirin +/− anti-rsv polyclonal or monoclonal antibody may have therapeutic benefit, but this remains unproven. there is also interest in prophylaxis with an anti-rsv antibody, although there have been no trials in hsct patients [55] [56] [57] [58] [59] . as with rsv, the incidence of influenza infection in hsct patients reflects the level of influenza activity in the community. the impact of this virus on infected hsct recipients is demonstrated by the following statistics: ~60 percent of the patients with influenza develop pneumonia and ~25 percent of patients with influenza pneumonia die of progressive respiratory failure. when influenza is identified as a pathogen, use of a neuraminidase inhibitor (oseltamvir or zanamavir) or an amantadate (amantadine or rimanatine) should be considered. the neuraminidase inhibitors are attractive in this setting as they are effective against both influenza a and b and antiviral resistance occurs more slowly compared with amantadine use. annual influenza vaccination should be considered, but its benefit is attenuated; indeed, it is probably fair to say that maximal benefit from vaccination occurs when the vaccine is administered to health care workers, family, friends and other contacts of the patient. when an infection is diagnosed, early treatment should be considered [58, 60] . there are more than 50 serotypes of adenovirus and nearly all have been described to cause human disease. adenovirus disease post-transplantation is likely due to both a newly acquired virus and viral reactivation. the most common adenovirus-associated illness post-transplantation is hemorrhagic cystitis which has been described in a recent report to occur in up to 42 percent of patients in the first year post-transplantation [61] . the overwhelming majority of cases are asymptomatic and require no intervention [62] . occasionally the severity of hemorrhage or bladder-associated pain is so great that intervention is required. other important adenovirus-associated syndromes include hepatitis and pneumonitis which may be fatal in the early post-transplant period. in the late post-transplant period adenovirus gastroenteritis may occur which is often a self-limited illness; however, severe disease has been described especially in patients requiring significant levels of immunosuppression for gvhd. therapeutic options for adenovirus are limited. the role of the antiviral cidofovir is controversial with mixed results having been reported [63] . decreasing immunusuppression and attempting reconstitution of the native host immune response is critical. the role for other adjunctive therapies, such as ivig, is unproven, but can be considered in severe cases. avoiding exposure to new infection, as with all community-acquired pathogens, is central to optimal care. parainfluenza, rhinoviruses, metapneumovirus and coronaviruses are all capable of causing lower respiratory tract infection in hsct recipients. of these many viruses, parainfluenza virus type iii is especially associated with a high mortality [64, 65] . again, specific therapy is not available, emphasizing infection control strategies in the hospital setting and avoiding individuals with respiratory tract complaints at home. when upper respiratory tract complaints occur in hsct patients, a diagnosis should be made, utilizing rapid diagnostic techniques (e.g., antigen detection assays or nucleic acid testing). preemptive therapy, when available, should be initiated, while immunosuppressive therapy diminished and isolation from other hsct patients should be accomplished. bk and jc viruses are the two important species in this family of viruses with a genitourinary and cns predilection, respectively. approximately 60 to 80 percent of adults have been infected with one or both of these viruses, typically in childhood. with immunosuppression, reactivation occurs which may lead to disease. bk virus is associated with hemorrhagic cystitis in the early post-transplant period. this virus is commonly found in the urine and rarely requires any therapeutic intervention. jc virus is the etiologic agent of progressive multifocal leukoencephalopathy (pml) which is a rare, but severe post-transplant complication. pml involves the white matter and presents with focal neurologic symptoms associated with the specific area of the cns where the lesion(s) occur. diagnosis requires correlating the clinical presentation, radiographic findings (typically by contrast-enhanced mri imaging) and csf pcr results for jc virus. control of jc virus is associated with an intact cell-mediated immune response. therapy for polyomavirus infection is quite limited with minimizing immunsuppression, when possible, being critical. the role of cidofovir is controversial with mixed results being reported. the use of quinolones for bk viruria is controversial at best and we do not recommend this practice [66] . although the use of gatifloxacin was advocated by some authors, this drug is no longer available. leflunomide administration has been used by some for treatment of bk in renal transplant patients, but no randomized trial data exists to support or recommend its use in either kidney or hsct recipients at this time. hepatitis b and c viruses may cause chronic infection which often leads to eventual significant liver dysfunction. given the high global prevalence of these viruses, it is prudent to screen for past or current infection prior to transplantation. when ongoing infection is found, careful assessment of liver function and a pre-transplant liver biopsy should be considered to assess for occult cirrhosis, as this may influence peri-transplant management [67] . hbv infects approximately 350 million people worldwide chronically, and substantially more have had prior resolved infection. the use of the hbv vaccine as a routine childhood immunization will likely decrease the number of chronically infected individuals over the next several decades. the advent of nucleic acid detection technology has allowed a more precise mechanism to detect active hbv replication compared with antigen-(for surface and e) only methods. for patients with evidence of prior hbv exposure (hbv core antibody positive), it is important to consider hbv reactivation in the setting of post-transplant liver dysfunction and to differentiate this from other causes such as hepatic gvhd or medication toxicity, although reactivation initially occurs in the setting of normal liver tests. the best strategy for surveillance post-transplantation remains to be defined. some recommend routine surveillance for hbv reactivation post-transplantation, whereas others would suggest antiviral prophylaxis. it is important to be aware that old resolved infections, including those with hepatitis core and surface antibody, but without antigen or hbv dna detected, are at risk for reactivation (seroreversion) post-transplantation, especially in the setting of high levels of immunosuppression [68, 69] . several therapeutic options have become available over the last several years and include lamivudine [70] [71] [72] , adefovir [73] , entecavir [71, 72] and telbivudine. other agents such as tenofovir and emtricitabine also have excellent anti-hbv activity. use of these agents requires careful consideration to minimize the risk for the emergence of resistant virus, which may be as high as 10 percent per year for lamivudine, but is less than 1 percent for adefovir and entecavir. epidemiologic studies suggest that more than 170 million people worldwide have been infected with hepatitis c virus (hcv) and the majority (approximately 85%) are chronically infected [67] . over several decades, chronic hcv infection is associated with progressive hepatic fibrosis, liver failure, and hepatoma. this process is accelerated in certain immunocompromised patients including hsct recipients [74, 75] . it is important to assess patients for seropositivity to hcv prior to transplantation and in those who are found seropositive, to assess the hcv viral load, genotype and liver pathology. the presence of elevated liver enzymes in the setting of hcv before allogeneic hsct has been associated with an increased incidence of vod [76] . a more precise profiling of the hcv-infected patient, including liver biopsy, should be considered to better define the extent of the hcv-induced liver disease, and to optimize the conditioning regimen and frequency of surveillance posttransplantation. treatment of hcv is limited and typically requires use of an interferon and ribavirin which are likely to be poorly tolerated in the early post-transplant setting. in patients who are infected, it is prudent to counsel them to avoid hepatotoxins, receive the hepatitis a and b vaccines, and minimize the risk of transmission to close contacts. there are three categories of fungal pathogens that can infect the hsct patient: a) the classic opportunistic fungi, which cause >90 percent of the invasive fungal infections that occur in the hsct patient -candida, aspergillus and cryptococcus being the most important of these infections; b) the geographically restricted systemic mycoses caused by blastomyces dermatitidis, coccidioides immitis and histoplasma capsulatum; and c) invasive infection due to the so-called "newly emerging fungi" -fusarium, the zygomycetes and such dematiaceous fungi as scedosporium, scopulariopsis and dactylaria [4] . candida is a major cause of fungal bloodstream infection during the preengraftment phase of hsct. although there is the possibility that the portal of entry can be vascular access catheters, it is believed that translocation of candida species across gut mucosa damaged by the pre-transplant conditioning regimen is the major route of access to the bloodstream in the granulocytopenic patient [77, 78] . in the past, c. albicans and c. tropicalis accounted for virtually all of the candida bloodstream infections. the incidence of candidemia was ∼11 to 16 percent (with a median time to onset of two weeks post-transplant), resulting in a high rate of tissue invasion and an attributable mortality of nearly 40 percent [79, 80] . with the introduction of empirical antifungal therapy or fluconazole prophylaxis (400 mg/day) during the preengraftment period, the incidence of candidemia has been significantly decreased, hepatosplenic candidiasis has become quite rare, and the attributable mortality has been significantly decreased. fluconazole-resistant candida sp, c. krusei and c. glabrata, have emerged as not uncommon causes of candidemia in hsct patients, as have the other non-albicans candida species [1, 4, [81] [82] [83] [84] [85] [86] [87] . it is also important to recognize that other species of yeast (e.g., trichosporon sp, blastoschyzomyces capitatus, saccharomyces cereviseae and rhodotorula sp.) can cause clinical syndromes identical to those observed with invasive candidiasis (bloodstream infection, infection metastatic to the skin and subcutaneous tissues, as well as other sites, including hepatosplenic disease identical to that caused by candida species) [88] . in an era of increased use of echinocandins for prophylaxis [89] and empirical antifungal treatment [90] , these organisms [88, 91] and echinocandin-resistant candida sp, especially c. parapsilopsis, have become emerging causes of fungemia in the hsct units [1, 4, 82] . invasive fungal disease has been most commonly caused by aspergillus sp, with a. fumigatus, a. flavus, a. terreus, a. niger and a. nidulans being the most common causes of invasive aspergillosis. the portal of entry for 90 percent of cases of invasive aspergillosis is the lungs, with the nasal sinuses and the skin accounting for virtually all of the remaining cases. there are two major host defenses that are mobilized in response to inhalation of the aspergillus spores -granulocytes and cell-mediated immunity, specifically cytotoxic t cells. the importance of these mechanisms is demonstrated by the clustering of cases of invasive aspergillosis at two timepoints in the posttransplant course: preengraftment when profound granulocytopenia is present, with the incidence of invasive aspergillosis increasing steadily as the period of granulocytopenia is extended, and after the diagnosis of gvhd and the treatment of this adverse event. indeed, these late cases of invasive aspergillosis have become more common than the preengraftment cases. mortality rates have traditionally been high in patients who developed invasive aspergillosis in either time period [1, 4, 92, 93] . the clinical syndromes caused by aspergillus invasion reflect the pathologic consequences of the vasculotropic nature of this mold. the three major consequences of the vascular invasion that characterizes aspergillus invasion include hemorrhage, infarction and metastatic disease. initial clinical complaints include persistent fever, chest pain, tachypnea, hypoxemia and hemoptysis, as well as symptoms related to metastases. before the availability of noninvasive fungal markers (galactomannan and β-glucan) and aggressive imaging with spiral chest computerized tomographic (ct) scanning, 50 percent or more of patients experience disseminated infection at the time of first diagnosis, accounting for the high mortality observed in allogeneic hsct recipients. a particular problem is infection in the cns, where mortality historically has approached 100 percent. metastases can present any site, but particularly important is the skin, as innocent appearing skin lesions can lead to early recognition of the disease, and should be aggressively biopsied [4] . definitive diagnosis of invasive mold infections, including invasive aspergillosis, is usually accomplished by biopsying the site of abnormality. early diagnosis is the key to effective therapy [94] . sputum or bronchoscopic samples rarely yield mold on culture. in recent years, considerable effort has been made to find other technology that will lead to an earlier and timely diagnosis. the ones that have been incorporated into practice are the systematic measurement of aspergillus antigens and serial chest ct imaging. monitoring the serum of hsct patients for galactomannan or β-glucan is now commercially available and has been incorporated into the current diagnosis guidelines [95, 96] . the detection of circulating fungal dna in the blood by pcr [97] remains experimental. findings on chest ct, in particular the halo sign ( fig. 19-2) , are associated in the neutropenic patient with invasive aspergillosis (although other pathogens can cause the same radiologic finding: fusarium and other vasculotrophic molds and nocardia asteroides being examples of this). european groups have been advocating protocol serial chest ct scans to find such pathology as a guide to early diagnosis [98] . if prevention fails, then early diagnosis is the key to the patient's survival [4, 92, 93] . given the limitations of current diagnostic techniques and the significant morbidity associated with invasive fungal infection, two strategies of antimicrobial use are commonly deployed in the hsct patient. the first is prophylactic fluconazole use during the initial transplant period, which has been shown to decrease fungal infections [80] in one study, and overall mortality fig. 19-2 . computerized tomographic scan of the chest in a patient with a "halo sign" due to invasive aspergillosis. note that halo signs most commonly occur in granulocytopenic hsct recipients with invasive aspergillosis. however, it must be emphasized that a halo sign is occasionally seen in patients with nocardia, scedosporium, fusarium and other forms of pneumonia. the patient was treated with voriconazole monotherapy during neutropenia during consolidation chemotherapy for aml and his treatment was continued through allogeneic transplantation. [79] in another, when started on day 0 until engraftment [80] or day +75 [79] . it is important to note that a high background rate of candida infections was noted in both of these reports and may not represent the experience of other transplant centers. echinocandins may be an alternative to fluconazole prophylaxis during this risk period [89] . the second common strategy is empiric antifungal therapy in neutropenic patients with persistent fever without a source, despite broad-spectrum antimicrobial therapy for >96 hours [99, 100] . in this setting the primary concern is both candida and invasive mold infection, especially aspergillus [2] . the traditional antifungal therapy utilized as empiric therapy is an amphotericin product [101, 102] . caspofungin use in this setting has become common because of the favorable side effect profile of this class of agents, but at the expense of a more limited fungal spectrum [90] . other echinocandins (micafungin, anidulafungin) are likely to be similarly effective, but no randomized comparisons with these latest drugs have been performed. the role of voriconazole in this setting is controversial [102] . when treating invasive aspergillosis several approaches should be considered simultaneously: 1) antifungal therapy, 2) reverse or minimize the host immune defects (decrease corticosteroids, increase neutrophils), 3) control permissive viral infections (e.g., cmv) and 4) consider surgical excision, if possible. voriconazole has become a cornerstone of therapy for invasive aspergillus infections, though the management of potential side effects is substantial [103] [104] [105] . whether the combination of therapeutic agents (polyenes, azoles and echinocandins) increases the therapeutic benefit has yet to be determined [106] . increasing experience suggests that voriconazole alone is sufficient in most cases for a successful outcome in invasive aspergillosis and has decreased the morbidity and mortality of this infection [11, 106] . another significant risk period for invasive fungal infections (ifi) is in the setting of significant gvhd, such as grade iii or iv, and its therapy [9] . in this setting, posaconazole (versus fluconazole) prophylaxis has recently demonstrated some benefit in preventing ifi compared to fluconazole (5.3% versus 9.0%, p = 0.07) and in preventing probable or proven invasive aspergillosis (2.3% versus 7.0%, p = 0.006 -interestingly, these results were largely driven by results from galactomannan assay testing) [107] [108] [109] . posaconazole has activity against the zygomycetes as well as aspergillus sp [110] [111] [112] . when an azole is used in this patient population, careful assessment of drug interactions, both with the initiation and cessation of therapy, is critical. therapy for the emerging fungi fusarium and scedosporium should be guided by in vitro sensitivity testing done locally or at regional reference laboratories, but voriconazole use should be considered. when therapy for the endemic mycoses is indicated, initial treatment (induction therapy) with an amphotericin preparation should be considered, followed by a prolonged course of consolidative therapy with an oral azole. cryptococcal disease should be treated initially with an amphotericin preparation, cns involvement should be excluded by cerebrospinal fluid sampling, and the use of flucytosine should be considered if present. pneumocystis jiroveci, formerly carinii, is a ubiquitous environmental organism which is an important cause of pneumonia in patients who are immunosuppressed, such as those who have undergone an hsct, on chronic prednisone (typically >20mg per day) or with advanced hiv infection. pcp infection typically presents as an interstitial pneumonitis with marked hypoxemia. severe infection can be life threatening. fortunately, universal prophylaxis of high risk patients during the high risk periods with trimethoprim-sulfamethoxazole has markedly decreased this complication. however, intolerance to prophylaxis, use of second line prophylaxis agents (e.g., dapsone, pentamidine, or atovaquone), poor medication compliance or failure to re-institute prophylaxis in the setting of augmented immunosuppression (e.g., treatment of gvhd) are common reasons why cases still occur. several important issues must be addressed after successful hsct to minimize infectious complications. first, it is important to avoid exposure to pathogens, especially when the immunosuppressive therapy to prevent gvhd is the highest. this includes avoiding gardening and soil exposures, mold exposures such as cleaning out damp basements or smoking marijuana, individuals with active respiratory infections, especially children, and avoiding enteric pathogens. second, optimal treatment or monitoring for latent infections such as herpesviruses, hepatitis viruses and prior granulomatous diseases (e.g., mycobacterium tuberculosis). those patients with a positive test for latent tuberculosis should receive secondary prophylaxis, which typically is begun within one month post-transplantation, after the acute regimen toxicities associated with transplantation have subsided, when screening and preventive treatment have not occurred previous to hsct. the first line therapy for secondary prophylaxis is isoniazid for nine months. however, in patients with significant hepatic dysfunction or peripheral neuropathy alternative regimens need to be considered. rifamycin-based regimens are difficult given the potential hepatotoxicity, as well as the significant drug interactions, especially with concomitant use of a calcineurin or an azole. a quinolone, such as levofloxacin, with ethambutol may be considered. when a mycobateriologically static regimen is chosen, the duration of therapy often must be extended with some using this combination for 18 months as secondary prophylaxis (table 19-2) . third, optimizing vaccinations for routine pathogens such as diphtheria, tetanus, pertussis, influenza and pneumococcus (table 19-3) . this optimal timing of re-vaccination depends on the nature of the transplant, with earlier re-vaccination schedules being considered in the nonmyeloablative setting. fourth, prophylaxis for pcp, which is typically continued for approximately one year or until the immunosuppressive medications are tapered off. the optimal medication to use for pcp prophylaxis is trimethoprimsulfamethoxazole which offers some protection for a variety of other important pathogens including pneumococcus, hemophillus influenza, nocardia sp., toxoplasma, listeria, salmonella sp., and other enteric bacterial pathogens. if trimethoprim-sulfamethoxazole is not tolerated due to significant renal dysfunction or bone marrow suppression, then alternative agents for pcp prophylaxis include dapsone, atovaquone or aerosolized pentamidine; however, none of these second line agents afford the broad microbial protection which trimethoprim-sulfamethoxazole affords. and lastly, herpes group viral prevention which should include acyclovir to prevent hsv and vzv and systematic monitoring for cmv, in the allogeneic setting, with early use of a cmv active antiviral if evidence for cmv activation or disease is observed. prophylaxis must be re-assessed in the setting of persistent or augmented immunosuppression, such as in the setting of clinically significant gvhd, regardless of time since hsct. medication doses may need to be adjusted for renal dysfunction. * trimethoprim-sulfamethoxazole affords modest protection for a broad array of potential environmental and community pathogens including: nocardia sp, toxoplasmosis, pneumococci, h influenza, listeria, shigella, and slamonella sp. ** alternatively preemptive monitoring with serial viral antigen or viral load assays can be considered. *** for those with evidence of prior hbv infection (e.g., hepatitis b core antibody positive), consider monitoring hbv viral load for evidence of reactivation periodically. if reactivation is detected then consider treatment if persistent hbv viremia detected. specific hbv antiviral therapy is discussed in the text. **** pre-transplant secondary prophylaxis for mtb is preferred. ***** decision for systemic azole prophylaxis should be based on local epidemiology of invasive fungal infections. consider posaconazole prophylaxis in the setting of significant gvhd (e.g., grade 3 or 4) and its therapy. drug interactions must be carefully managed both with initiation and cessation of azole therapy. an important aspect of antimicrobial therapy in the hsct patient is the management of drug interactions, especially between antimicrobial agents (e.g., azoles, macrolides) and the immunosuppressive medications (e.g., calcineurin inhibitors, sirolimus) used to prevent and treat gvhd. there are three important categories of interaction to pay particular attention to, two of which are related to the major route of drug metabolism for the calcineurin inhibitors, hepatic cytochrome p450 enzymatic metabolism. these interactions are as follows: 1) certain antimicrobial agents (most notably the macrolides [eryth romycin>clarithromycin>azithromycin] and the azoles [ketoconazole>itraco nazole>voriconazole>fluconazole]) will downregulate the metabolism of the calcineurin inhibitors, resulting in elevated blood levels of active drug, and an increased risk of nephrotoxicity, as well as over-immunosuppression and an increased incidence of opportunistic infection; 2) certain antimicrobial agents (such as rifampin and rifabutin) upregulate metabolism of the calcineurin inhibitors, leading to a fall in blood levels and an increased risk of gvhd, and 3) therapeutic blood levels of the calcineurin inhibitors, when combined with such drugs as amphotericin b, aminoglycosides and vancomycin, can cause significant renal toxicity. hsct has become one of the great success stories of modern medicine. it is the therapy of choice for an increasing number of conditions, including a variety of cancers, bone marrow failure states, congenital immunodeficiencies, metabolic disorders and even as a means for introducing new genes. the major hurdle in most of these attempts, however, remains infection. bacterial and fungal sepsis, as well as herpes group viral infection and community-acquired respiratory virus infection threaten the well-being of these patients. there are two phases of the post-transplant course when the patient is at particular risk: preengraftment with profound granulocytopenia and mucositis, and post-engraftment when gvhd and its therapy render the patient vulnerable to both fungal and viral infection. new preventative strategies are being formulated involving both prophylaxis and preemptive therapy. similarly, new non-culture diagnostic approaches that rely on antigen detection or pcr detection of microbial dna are being developed. new therapies, both antiviral and antifungal, have emerged. these should prompt much more effective prevention and therapeutic 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patients who are refractory to or intolerant of conventional therapy: an externally controlled trial we would like to thank robert h rubin, m.d. for the many years of advice and teaching, and for critically reviewing this work. key: cord-007013-tlvgyzft authors: chan, kok fei; carolan, louise a; korenkov, daniil; druce, julian; mccaw, james; reading, patrick c; barr, ian g; laurie, karen l title: investigating viral interference between influenza a virus and human respiratory syncytial virus in a ferret model of infection date: 2018-08-01 journal: j infect dis doi: 10.1093/infdis/jiy184 sha: doc_id: 7013 cord_uid: tlvgyzft epidemiological studies have observed that the seasonal peak incidence of influenza virus infection is sometimes separate from the peak incidence of human respiratory syncytial virus (hrsv) infection, with the peak incidence of hrsv infection delayed. this is proposed to be due to viral interference, whereby infection with one virus prevents or delays infection with a different virus. we investigated viral interference between hrsv and 2009 pandemic influenza a(h1n1) virus (a[h1n1]pdm09) in the ferret model. infection with a(h1n1)pdm09 prevented subsequent infection with hrsv. infection with hrsv reduced morbidity attributed to infection with a(h1n1)pdm09 but not infection, even when an increased inoculum dose of hrsv was used. notably, infection with a(h1n1)pdm09 induced higher levels of proinflammatory cytokines, chemokines, and immune mediators in the ferret than hrsv. minimal cross-reactive serological responses or interferon γ–expressing cells were induced by either virus ≥14 days after infection. these data indicate that antigen-independent mechanisms may drive viral interference between unrelated respiratory viruses that can limit subsequent infection or disease. viral interference is a phenomenon whereby infection with one virus limits or delays infection with a second virus. it has been described in human epidemiological studies observing viral epidemic peaks [1] [2] [3] [4] , vaccine efficacy studies [5] , studies assessing virus infections in clinical samples [6] [7] [8] , animal studies [9] [10] [11] [12] [13] and in vitro infectivity studies [14] . viral interference has been observed between a range of viruses, including between arboviruses, such as yellow fever and dengue virus [15] ; between different respiratory viruses [9, 13, 16] ; and between influenza viruses of different types [10] and subtypes/lineages [10, 11] . at a population level, respiratory virus infections may display distinct epidemic peaks. observational studies from the netherlands, france, and hong kong showed that emergence of 2009 pandemic influenza a(h1n1) virus (a[h1n1]pdm09) delayed infections with human respiratory syncytial virus (hrsv) [1, 3, 4] . influenza a virus infections also interrupted peak incidences of hrsv infections in japan during 2000-2002 [2] and in the netherlands during 2003-2012 [3] . negative associations between respiratory viruses have been reported when analyzing the proportion of coinfections with different respiratory viruses, using swab specimens from patients [6, 7, 17] . a(h1n1)pdm09 was least likely to be detected with any of the other respiratory viruses tested, including hrsv, in samples from all age groups [8, 18] . taken together, these data suggest that interference may occur between a(h1n1)pdm09 and hrsv. the ferret provides an ideal model of human influenza because animals can be directly infected with virus without adaptation and display similar disease symptoms to those in humans [19, 20] . historically, the ferret has also been used to study hrsv infection [21] [22] [23] , with recent studies assessing the pathogenesis, immunity, and transmission of hrsv [24, 25] . clinical symptoms are mild in ferrets infected with hrsv strains described to date [24, 25] . previously, we used the ferret model to demonstrate that viral interference can occur following infection with human influenza a and b viruses and will prevent, delay, or limit subsequent infection with an influenza virus of a different type, subtype, or lineage [10, 11] . notably, this effect depends on the virus combinations and the order and timing of sequential infections [10, 11, 26] . we have established complementary influenza viral dynamics models that explain these observations via the innate immune response [27] and cross-reactive adaptive immune responses [28] . ecological data suggest that infection with a(h1n1)pdm09 can prevent or delay infection with hrsv. using our ferret models of influenza and hrsv, we have systematically investigated this hypothesis. adult ferrets were housed at the peter doherty institute for infection and immunity bioresources facility. experiments were conducted with approval from the university of melbourne microbiology and immunology animal ethics committee, in accordance with the australian national health and medical research council code of practice for the care and use of animals for scientific purposes. all ferrets were seronegative for antibodies to currently circulating influenza viruses and hrsv (long and a2 strains) before use in experiments. a/tasmania/2004/2009 (a[h1n1]pdm09) virus was passaged allantoically in embryonated hen's eggs and stored at −80°c. the infectious influenza virus titer was measured by a 50% tissue culture infectious dose (tcid 50 ) assay [29] , read by hemagglutination with turkey red blood cells. hrsv long and a2 strains were passaged [24] . infectious hrsv titers were determined by plaque assay [24] . ferrets were infected intranasally with 10 3.5 tcid 50 a(h1n1) pdm09 in 500 µl and 10 5 plaque-forming units (pfu) of long hrsv or 10 6 pfu of long or a2 hrsv in 500 µl and monitored [24, 26] . ferrets were housed in pairs, by infection group. nasal wash specimens were collected and stored [24] . on the day of collection, viral rna was extracted from 140-µl nasal wash specimens for quantitative polymerase chain reaction (qpcr) analysis. blood samples were obtained from ferrets before primary virus infection and immediately before and 14 days after challenge, and serum was isolated. the proportional change in weight was calculated as the percentage difference from the weight on the day of challenge. four microliters of viral rna [24] was assayed by rt-qpcr with a(h1n1)pdm09 hemagglutinin-specific primers/probes from the cdc influenza virus rt-qpcr influenza a (h1/h3/h1pdm09) subtyping panel, obtained from the influenza reagent resource (available at: http://www.influenzareagentresource.org/) and hrsv n-specific primers/probes [24] . copy numbers for a(h1n1) pdm09 viral rna were calculated relative to plasmid phw2000-a/ tasmania/2004/2009 hemagglutinin; copy numbers for rsv rna were calculated relative to a hrsv rna standard [24] . mrna was isolated from nasal wash samples [30] . mrna expression of cytokines, chemokines, and housekeeping genes was quantified by qpcr [30, 31] . infectious hrsv in nasal wash samples was measured using the vs assay [24] . interferon γ (ifn-γ) enzyme-linked immunospot (elispot) assay ifn-γ-producing cells were detected by a ferret ifn-γ elispotplus assay (mabtech). single cell suspensions were prepared from ferret retropharyngeal lymph nodes [31] . a total of 5 × 10 4 lymph node cells were cultured with or without live influenza virus, hrsv, or 5 µg/ml concanavalin a (sigma) for 48 hours at 37 o c in 5% co 2 [11] . titers of antibodies to a/tasmania/2004/2009 were measured using hi assays [31, 32] . titers were expressed as the reciprocal of the highest dilution of serum for which hemagglutination was prevented. geometric mean titers (gmts) were calculated, with undetectable titers expressed as having a value of "5." seroconversion was defined as a titer of ≥40 at the end of the experiment and at least a 4-fold rise from baseline. titers of antibodies that neutralize hrsv long and a2 were measured using vs mn assays [24] . seroconversion was defined as a titer ≥160 at the end of the experiment and an increase of at least 4-fold from the baseline titer. antibodies that bind to the f glycoprotein of hrsv were detected by an elisa [24] . viral kinetics were assessed in viral rna from nasal wash specimens. for a(h1n1)pdm09, >10 6 copies of hemagglutinin/100 µl of nasal wash were positively correlated with replicating virus, based on the tcid 50 assay [10] and the level of infectious virus as measured by transmission in ferrets [33] . for hrsv, 10 3.8 copies of n/100 µl of nasal wash corresponded to a 50% chance of a sample being positive by the virospot assay, as determined using a probit regression model (supplementary figure 1) . accordingly, samples were considered to be infectious for hrsv when the amount of viral rna exceeded 10 3.8 copies/100 μl nasal wash and infectious for a(h1n1)pdm09 when viral rna exceeded 10 6 copies/100 µl of nasal wash for at least 1 measurement. clinical signs (ie, weight loss and fever) were assessed daily, and seroconversion was measured 14 days after challenge. statistical analysis was conducted using prism, version 6.0g, unless otherwise indicated and is described in the figure legends. ferrets were first infected with a(h1n1)pdm09 virus then challenged with hrsv 3, 7, or 11 days later, or vice versa ( figure 1a ). the intervals between inoculations spanned the times of peak titer and clearance of both virus infections [24, 30] and induction of humoral immunity ( figure 2a ). primary infection with a(h1n1)pdm09 prevented subsequent infection with hrsv in 3 of 4 ferrets when primary infection and challenge were separated by 3 days. shedding of hrsv was minimal in the single ferret infected, compared with control animals ( figure 1b and 1c) . no ferrets in this group seroconverted to hrsv (figure 2bi and 2bii ). primary infection with a(h1n1)pdm09 prevented infection with hrsv in 2 of 4 ferrets when infections were separated by 7 days ( figure 1d ). ferrets that did not shed virus did not seroconvert (figure 2bi and 2bii), while ferrets that shed virus seroconverted to hrsv (figure 2bi and 2bii). prior infection with a(h1n1)pdm09 did not prevent infection with hrsv 11 days later ( figure 1e ), with all ferrets showing a similar pattern of virus shedding ( figure 1b ) and antibody titers to control animals that received hrsv alone (figure 2bi and 2bii). the kinetics of hrsv shedding was examined in animals not protected from hrsv challenge. the peak of hrsv shedding was delayed in ferrets infected with a(h1n1)pdm09 followed by hrsv as compared to control animals infected with hrsv alone (median, 8 vs 6 days; p = .0091 by the mann-whitney test; figure 2ci ). there was no change in the duration of virus shedding ( figure 2cii ). clinical signs following hrsv challenge were minimal (supplementary figure 2) , consistent with our previous study [21] . all ferrets, except 1 control ferret infected with hrsv, maintained or gained weight (supplementary figure figure 2biv ). the median duration of a(h1n1)pdm09 shedding was increased in ferrets infected with hrsv followed by a(h1n1)pdm09 as compared to control animals infected with a(h1n1)pdm09 alone (8 vs 7 days; p = .0196 by the mann-whitney test; figure 2civ ). there was no change in the peak day of shedding (figure 2ciii ). prior infection with hrsv did reduce disease following infection with a(h1n1)pdm09. the mean maximum weight loss (±sd) among 8 control ferrets infected with a(h1n1)pdm09 was 10.6% ± 3.7% (supplementary figure 3a and 3d) . the mean maximum weight loss (±sd) for ferrets in all test groups (n = 12) was 4.1% ± 2.3% (supplementary figure 3b , 3c, and 3e). thus, prior infection with hrsv significantly reduced morbidity, as measured by weight loss, after challenge with a(h1n1) pdm09 (p = .0002 by the mann-whitney test). no fever was detected following a(h1n1)pdm09 infection (supplementary figure 3f -j). ferrets were infected (1) with an increased viral dose of the same hrsv strain, long, or (2) with an alternate hrsv strain, a2 (also at an increased viral dose), then challenged with a(h1n1) pdm09 3 days later. a2 is a laboratory-adapted strain that is shed at similar levels to long in ferrets and transmits between cohoused animals [24] . infection of ferrets with a 10-fold higher inoculum (ie, 10 6 pfu) of hrsv long led to a small increase in virus shedding on days 2-6 after infection, compared with animals infected with 10 5 pfu of hrsv long, although these differences were not significant (supplementary figure 4) . primary infection with 10 6 pfu hrsv long or a2 did not prevent infection with a(h1n1)pdm09 when infections were separated by 3 days (figure 4 ). all animals seroconverted to a(h1n1)pdm09 at similar levels ( figure 2bv and 2bvi). most ferrets lost weight after a(h1n1)pdm09 infection. the mean maximum weight loss (±sd) among 4 ferrets infected with a(h1n1)pdm09 alone was 7.1% ± 3.0%, whereas the mean maximum weight loss (±sd) for ferrets (n = 8) that received primary infection with hrsv prior to a(h1n1)pdm09 challenge was 4.9% ± 3.2% (p = .2828 by the mann-whitney test; supplementary figure 5a -c). there was no difference in fever (supplementary figure 5d -f) and no change to the kinetics of infection between animals that received a prior hrsv infection, compared with those that did not (data not shown). inflammation induced by viral infection may contribute to viral interference [10, 11] . we investigated the localized immune response following infection with hrsv or a(h1n1)pdm09. nasal wash specimens were collected early (day 2) and later (day 6/7) after infection, because the pattern of inflammatory mediators changes throughout h(h1n1)pdm09 [30] and hrsv [24] infections. expression of influenza virus matrix (m) mrna was highest on day 2 after infection, whereas expression of hrsv nucleoprotein (n) mrna was highest on day 6 ( figure 5a ). two days after infection, animals infected with a(h1n1)pdm09 had significantly higher levels of interferon β (ifn-β), granzyme b, ifn-γ, interleukin 6 (il-6), monocyte chemoattractant protein 1 (mcp-1), and tumor necrosis factor α (tnf-α) mrna as compared to animals infected with hrsv ( figure 5d, 5g-i, 5n , and 5o). expression of ifn-α and granzyme a mrna was increased but not significantly (ifn-α, p = .097; granzyme a, p = .18; figure 5e and 5f). on day 6/7 after infection, levels of granzyme closed circles and open circles indicate animals that did or did not, respectively, shed detectable challenge virus, as determined by quantitative reverse-transcription polymerase chain reaction analysis of viral rna from nasal wash (nw) samples. for statistical analysis, titers or fold changes were compared between test and control groups, using 1-way kruskal-wallis analysis of variance with the dunn multiple comparison test. *p < .05 and **p < .01. c, the kinetics of shedding was analyzed for all ferrets that shed challenge virus in figures 1 and 3 . data from ferrets obtained at the 3-day, 7-day, and 11-day intervals were pooled into the test group. the number of days from challenge inoculation to the peak level of challenge virus shedding (ci and ciii) and the number of days the challenge virus was shed (cii and civ) was determined for each ferret in the indicated groups. horizontal lines indicate median values. the number of days of virus shedding were compared between test and control groups, using the mann-whitney test. *p < .05 and **p < .01. a, granzyme b, ifn-γ, interleukin 17, and mcp-1 mrna were significantly higher in animals infected with a(h1n1)pdm09 as compared to those infected with hrsv ( figure 5f -h, 5m, and 5n). there was significant increase in expression of interleukin 8 (il-8) mrna 2 days after infection and of interleukin 1β, il-6, and il-8 mrnas 6/7 days after infection in ferrets infected with hrsv as compared to a(h1n1)pdm09 ( figure 5c, 5i, and 5j ). this suggests a localized inflammatory response was induced after hrsv infection, which coincided with the increase in hrsv virus replication ( figure 5a ). to directly compare the magnitude of expression of cytokines and chemokines induced by both virus infections, we assessed mrna expression on the day after infection at which the level of virus shedding was highest (ie, day 2 for a(h1n1) pdm09 and day 6 for hrsv). when assessed at these times, an equivalent fold change in mrna expression was observed for rsv n and influenza virus m ( figure 5a ). infection with a(h1n1)pdm09 induced significantly higher levels of ifn-β (p = .00021; figure 5d ), il-6 (p = .0088; figure 5i ), interleukin 12p40 (p = .0137; figure 5l ), mcp-1 (p = .0018; figure 5n ), and tnf-α (p = .00078; figure 5o ) mrna expression in ferrets, compared with hrsv. these data suggests there is increased inflammation in nasal tissues of animals infected with a(h1n1)pdm09 as compared to hrsv. there was no difference in expression of any cytokines or chemokines between ferrets infected with 10 5 or 10 6 pfu of hrsv long (data not shown). we have demonstrated that cross-reactive ifn-γ cellular responses can be detected between influenza b virus lineages and may contribute to viral interference [11] . thus, we assessed whether cellular immunity induced by infection with a(h1n1) pdm09 showed any cross-reactivity to hrsv. whereas retropharyngeal lymph node cells from a(h1n1)pdm09-infected ferrets were restimulated with a(h1n1)pdm09 ( figure 6b ), few cells produced ifn-γ when stimulated with hrsv ( figure 6a ). lymph node cells from hrsv-infected ferrets were restimulated with hrsv in vitro, although at much lower levels ( figure 6a ), and were not restimulated by a(h1n1)pdm09 ( figures 6b) . responses to concanavalin a were similar for all ferrets regardless of infection ( figure 6b ). moreover, there was limited serological cross-reactivity. animals infected with a(h1n1)pdm09 had high levels of influenza virus-specific neutralizing antibodies ( figure 6c ), yet minimal total serum or neutralizing antibodies to hrsv ( figure 6d and 6e) . similarly, infection with hrsv induced total serum and neutralizing antibodies to hrsv but few antibodies that were reactive with a(h1n1)pdm09 ( figure 6c-e) . discussion we have demonstrated that infection with a(h1n1)pdm09 can prevent infection and replication of hrsv in a ferret model of human disease for up to 7 days. infection with hrsv did not prevent subsequent infection with a(h1n1)pdm09; rather, animals were coinfected, albeit with reduced morbidity. infection with a(h1n1)pdm09 leads to increased levels of proinflammatory cytokines in the respiratory tract as compared to infection with hrsv. overall, these data support the ecological observation that viral interference induced by a(h1n1)pdm09 infection delayed infection with hrsv in the winter of 2009-2010. infection with a(h1n1)pdm09 induced higher expression of mcp-1, il-6, type i ifns, tnf-α, ifn-γ, and granzyme a/b mrnas as compared to hrsv infection. mcp-1 and tnf-α regulate the migration of macrophages/monocytes and natural killer (nk) cells into the respiratory tract. macrophages produce mcp-1, tnf-α, and il-6; thus, upregulation of these genes suggests an influx of macrophages and nk cells into the respiratory tissues [34] . nk cells produce ifn-γ, which activates macrophages and neutrophils and promotes t-cell proliferation and killing of virus-infected cells [34] . because cytotoxic t lymphocytes and nk cells also produce granzymes a/b, increased expression of ifn-γ and granzyme a/b mrnas on day 6 after infection suggests recruitment/activation of these cells to the site of infection. il-6 and type i ifns are produced by respiratory epithelial cells, monocytes/macrophages, and dendritic cells [34, 35] . il-6 is a proinflammatory cytokine, whereas type i ifns induce an antiviral state that may also limit replication and spread of hrsv [34, 35] it would be useful to explore the cellular infiltrate following a(h1n1)pdm09 and hrsv infections to gain further insight . ferrets were infected with 10 5 plaque-forming units (pfu) of hrsv strain long or 10 3.5 50% tissue culture infectious doses of a(h1n1)pdm09 (n = 4 ferrets/virus). nasal wash specimens were collected after challenge from ferrets on days 2 and 6 after infection, and mrna was assayed for the indicated genes, using quantitative polymerase chain reaction (qpcr) assays. for each graph, qpcr data are expressed as fold changes relative to values for nasal wash specimens from uninfected animals and normalized to atf4 and gapdh housekeeping genes. in panel a, expression of n is shown for hrsv-infected ferrets, and expression of m is shown for influenza virus-infected ferrets. for statistical analyses, inflammatory mediators were compared between (1) hrsv-infected and a(h1n1)pdm09-infected animals sampled on day 2 after infection, (2) hrsv-infected and a(h1n1)pdm09-infected animals sampled on day 6/7 after infection, and (3) a(h1n1)pdm09-infected animals sampled on day 2 and hrsv-infected animals sampled on day 6 after infection. fold changes were compared between viruses, using the mann-whitney u test. ifn, interferon; il-1, interleukin 1; il-6, interleukin 6; il-8, interleukin 8; il-10, interleukin 10; il-12p40, interleukin 12p40; il-17, interleukin 17; mcp-1, monocyte chemoattractant protein 1; tnf-α, tumor necrosis factor α. *p < .05, **p < .01, ***p < .001, and ****p < .0001. into potential differences in the level and cellular composition present in local inflammation. infection with a(h1n1) pdm09 induced a 7-fold higher cellular ifn-γ recall response as compared to infection with hrsv in our study. because there was no significant difference in ifn-γ responses to concanavalin a between the groups, this observation was not due to a difference in overall t-cell numbers but, instead, was due to an increase in the reactivation of a(h1n1)pdm09-specific cells. taken together, these data suggest that infection with a(h1n1)pdm09 induces a robust cytokine and chemokine response that strongly stimulates the adaptive and memory immune responses. conversely, infection with hrsv elicited a weaker and more limited cytokine and chemokine response that led to a reduced antigen-specific cellular response. however, it is possible that hrsv may not infect the ferret respiratory tract as efficiently as a(h1n1)pdm09 does, and this could result in reduced inflammatory responses. yet, infected animals seroconverted at titers consistent for sterilizing immunity, indicating a productive infection (data not shown) [24] . furthermore, increasing the inoculum of hrsv did not significantly affect the pattern or amount of virus shedding nor the expression of inflammatory mediators, suggesting that the hrsv level was already maximal in this ferret model. notably, increased expression of inflammatory mediators following infection with influenza virus as compared to hrsv has been observed in studies assessing human clinical samples and in vitro airway epithelial cell cultures [36] [37] [38] [39] . what is the mechanism of viral interference induced by a(h1n1)pdm09? the increased antiviral state and inflammation observed after a(h1n1)pdm09 infection has the potential to prevent subsequent infection or delay shedding of hrsv, as was observed here. both viruses predominantly infect ciliated airway epithelial cells, and we have shown that a(h1n1)pdm09 and hrsv long replicated in the upper and lower respiratory tracts of ferrets [24, 30] . infection with a(h1n1)pdm09 can also prevent infection with an influenza b/yamagata virus [10] . there are minimal shared epitopes between influenza a and b viruses [40] , and we showed that minimal cross-reactive ifn-γ-producing cells were induced between hrsv and influenza virus. these data suggest that short-lived mechanisms drive this effect, as no effect was detected in ferrets after one week or, as shown by others, in mice, when infections were separated by 35 days [41] . the timing of interference indicate that interactions between different viruses may also be important. it is possible that different mechanisms act on different virus combinations. gene expression analysis of early markers of the immune response (cona; b) . the number of interferon γ (ifn-γ)-producing cells was determined by an enzyme-linked immunospot (elispot) assay. c-e, sera were tested for antibodies to a(h1n1)pdm09, by a hemagglutination inhibition (hi) assay (c); for total serum antibody binding to hrsv f protein, by an enzyme-linked immunosorbent assay (elisa; d); and for neutralizing serum antibody to hrsv long or a2, by a virospot microneutralization (mn) assay (e). data were obtained from 2 ferrets per group. of respiratory epithelium infected with the virus strains used in these studies may provide further insight. it is notable that infection with hrsv reduced morbidity induced by a(h1n1)pdm09 infection. although virus loads were not decreased in nasal wash specimens, virus shedding may be reduced in the lower respiratory tract, limiting clinical disease. il-8 mrna expression was elevated in nasal wash samples of ferrets infected with hrsv as compared to a(h1n1) pdm09. increased il-8 expression has been associated with milder disease in ferrets infected with pathogenic influenza virus strains, potentially mediated by rapid recruitment of neutrophils, which assist in clearing virus [42] . analysis of the lung influenza virus loads in animals that have been infected with hrsv prior to challenge with a(h1n1)pdm09 would be of interest. epidemiological data reported in france described a 3-4week delay in the peak incidence of hrsv infections following the emergence of the a(h1n1)pdm09, compared with previous years [1] . similarly, a delay of 2-4 weeks of the expected peak of hrsv was reported following an early influenza a season in the netherlands. [3] . these population-level observations of viral interference arise from the interplay between (1) immunodynamics (ie, host-level viral interference), (2) heterogeneity between hosts (ie, differences in immunity to virus strains between individuals), and (3) transmission dynamics (ie, within or between different age groups) [43] . for influenza, these processes have been investigated in some detail. others have demonstrated that a short period (ie, days rather than weeks) of viral interference at the host level may result in substantial separation between epidemic waves at the population level [43] . our results provide the first hostlevel immunodynamic evidence in support of these processes driving the epidemiological interactions observed previously in europe [1, 2] . our study has limitations. we used a circulating strain of a(h1n1)pdm09 from early 2009 and laboratory strains of hrsv, long and a2. the long and a2 strains induce consistent infections and disease in ferrets, with 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parainfluenza virus infections plasticity and virus specifcity of airway epithelial cell immune response during respiratory virus infection interferon in nasal secretions from infants with viral respiratory tract infections cross-reactive human b cell and t cell epitopes between influenza a and b viruses influenza virus lung infection protects from respiratory syncytial virus-induced immunopathology severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction does homologous reinfection drive multiple-wave influenza outbreaks? accounting for immunodynamics in epidemiological models supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.notes key: cord-006819-sxz1s6kz authors: daniel givens, m.; marley, m.s.d. title: infectious causes of embryonic and fetal mortality date: 2008-05-27 journal: theriogenology doi: 10.1016/j.theriogenology.2008.04.018 sha: doc_id: 6819 cord_uid: sxz1s6kz the purpose of this review is to summarize bacterial, fungal, protozoan, and viral causes of reproductive dysgenesis in cattle, sheep, goats, pigs, horses, dogs, and cats. the clinical presentations of disease due to reproductive pathogens are emphasized, with a focus on assisting development of complete lists of causes that result in abortion and infertility in these species. clinicians are encouraged to assess clinical presentation, create complete lists of differential diagnoses, obtain appropriate diagnostic samples, maximize diagnostic laboratory support, and avoid zoonotic infections resulting from reproductive pathogens of animals. the foundation of an accurate diagnosis of reproductive loss due to infectious pathogens facilitates the prudent use of immunization and biosecurity to minimize reproductive losses. the purpose of this review is to summarize infectious causes of reproductive dysgenesis that compromise viability of domestic animals prior to parturition. for sake of clarity, the embryo is defined as the part of the conceptus that gives rise to the neonate, from fertilization to completion of organogenesis (day 42 of pregnancy in cattle). furthermore, the fetus is defined as that part of the conceptus that gives rise to the live neonate from completion of organogenesis to completion of the second stage of parturition. although more deaths occur at and around birth than between 1 week of age and weaning, embryonic losses are usually higher than perinatal losses. notably, maternal infections during pregnancy may or may not impact fetal development. the impact on fetal development may be caused directly by infectious agents or their toxins, or indirectly by placentitis. furthermore, detection of a reproductive pathogen should not preclude further diagnostic testing, as dual infections are not uncommon. embryonic or fetal death may result in resorption, mummification, maceration, or abortion. factors that impact the outcome of embryonic and fetal death include gestational age, cause of death, and source of progesterone for pregnancy maintenance (table 1) . aborted fetuses may be autolytic due to death 24-48 h before abortion. fetal mummification occurs most commonly in polytocous species (e.g. swine). bacterial infections are usually not involved in mummification; however, viruses are a common cause of mummification in pigs, dogs, and cats. fetal maceration results when abortion or parturition fails to occur following fetal death and cl regression (occasionally in bovine www.theriojournal.com available online at www.sciencedirect.com theriogenology 70 (2008) 270-285 table 1 infectious causes of infertility and abortion in domestic animals fungal protozoan viral trichomoniasis and vibriosis). the majority of abortions are not epizootic but sporadic, with <5% of pregnant animals in a group aborting. severe maternal illness resulting in high fever (e.g. mastitis, pneumonia), hypoxia (e.g. anaplasmosis, severe anemia), or endotoxemia may result in abortion. although very few lesions are pathognomonic, gross fetal lesions may be characteristic for mycotic abortion (fresh dysmature fetus with dermatitis) and epizootic bovine abortion (fresh fetus with a nodular liver). when multiple fetuses are present in utero, infectious pathogens may result in different outcomes in different fetuses. sows physiologically terminate pregnancies comprised of less than four embryos and subsequently exhibit a regular interestrus interval. thus, litters of four or less liveborn piglets suggest embryonic or fetal death, even if stillbirths or mummified fetuses are not clinically observed. numerous bacterial, viral, protozoan and fungal pathogens have been associated with infertility and abortion in cattle. these pathogens can result in substantial economic losses, indicating the need for control measures to prevent infection or disease. leptospira spp. are zoonotic spirochetes. cattle are the maintenance hosts for leptospira interrogans serovar hardjo (type hardjoprajitno) and leptospira borgpetersenii serovar hardjo (type hardjo-bovis) and incidental hosts for serovar pomona which is maintained in swine [1] . transmission among maintenance hosts is through contact with infected urine, milk, placental fluid, transplacentally, or venereally. transmission to an incidental host occurs via contact with an environment contaminated with infected urine. the bacteria gain access through the mucous membranes of the eyes, nose, vagina, or abraded skin. infection in pregnant animals can lead to abortion, stillbirth, or birth of weak calves. abortion following infection with serovar pomona occurs in the last trimester, whereas abortion caused by serovar hardjo occurs from 4 months of gestation to term. abortion rates within a temporal ''outbreak'' are generally higher following infection with serovar pomona compared with hardjo. infertility is also considered to occur with serovar hardjo infections. campylobacter fetus subsp. venerealis is a gramnegative rod transmitted venereally among cattle [1] . it might also be transmitted through contact with contaminated bedding or instruments or other infected animals. infection of the vagina, cervix, endometrium and placenta can occur. infected animals can experience signs of infertility due to early embryonic death and abortion between 4 and 7 months of gestation. infected cows usually clear the organism within 3-6 months. histophilus somnus is part of the normal bacterial flora of the reproductive tract, but has rarely been associated with bovine abortion [1] . the organism might also lead to infertility by binding to the zona pellucida of embryos [2] . transmission occurs via contact with materials contaminated by infected respiratory or vaginal discharges; the bacteria then spread hematogenously to the fetus. brucella abortus is a zoonotic, gram-negative coccobacillus, transmitted via ingestion of fetus, placenta, uterine discharge, or materials contaminated by these products [1] . through hematogenous spread, the bacteria localize in the trophoblasts. abortions generally occur after the fifth month of gestation. retained fetal membranes and metritis often occur. listeria monocytogens is a zoonotic, gram-positive coccobacillus, transmitted through ingestion of spoiled feed or feed contaminated with infected fetus, placenta or uterine discharge [1] . the organism spreads hematogenously to the placenta and fetus. abortion generally occurs in the last trimester. infected cows can show clinical signs of fever, weight loss, endometritis, and retained fetal membranes. although ureaplasma diversum and mycoplasma spp. are part of the normal flora of the reproductive tract, they can also cause reproductive failure [1, 3] . transmission can occur through direct contact, environmental contamination with infected urine, and venereally. infection with u. diversum can lead to granular vulvitis, infertility, abortion, and birth of weak calves. mycoplasma bovigenitalium infection can cause granular vulvovaginitis, infertility, and endometritis. experimentally, mycoplasma bovis can cause endometritis, salpingitis, infertility and abortion. chlamydophila abortus and chlamydia-like waddlia chondrophila can infect cattle [1, 4, 5] . infection with c. abortus can result in abortion (6-8 months of gestation) or birth of weak calves. w. chondrophila also causes abortion. transmission occurs through ingestion or inhalation of feces, urine, or contaminated discharges (feces, nasal, ocular, vulvar, uterine, placenta). the organism causes endometritis and can multiply in cotyledons. these pathogens are zoonotic and may cause abortion in women. abortions can also be caused by other bacterial pathogens that are part of the normal microflora or present in the environment [1, 6] . these bacteria might include: arcanobacterium pyogenes, bacillus spp., escherichia coli, pasteurella spp., pseudomonas spp., salmonella spp., staphylococcus spp., and streptococcus spp. bacteria are able to spread hematogenously to the fetoplacental unit and cause abortion, usually late in gestation. bovine herpesvirus 1 is an alpha herpesvirus that can lead to respiratory and genital infections, as well as abortion [7, 8] . transmission occurs through contact with upper respiratory, conjunctival or genital tract mucous membranes, aborted fetuses, or through venereal transmission. abortions are most commonly associated with the respiratory form of the disease and not the genital form. cows can have fever, anorexia, red nasal mucosa, coughing, and conjunctivitis, followed by abortion in 15-64 d. abortion generally occurs between 4 and 8 months of gestation. infection also can result in early embryonic death [9] . bovine herpesvirus 1 establishes latent infections in the trigeminal and sacral ganglia; following immunosuppression, the virus can become reactivated. therefore, these animals serve as a source of infection for unexposed cattle. bovine viral diarrhea virus is a pestivirus that is transmitted transplacentally or through inhalation or ingestion of material contaminated with infected secretions [8, 10] . animals with acute infection present with fever, nasal discharge, enteritis, and leukopenia. pregnant animals infected up to 45 d of gestation can have decreased fertilization rates and embryonic death. infection between 45 and 175 d of gestation can result in abortion; however, fetuses that survive infection with a noncytopathic strain of bvdv between 70 and 150 d of gestation usually become persistently infected (pi). animals that are pi shed large amounts of bvdv and generally do not produce antibodies to bvdv. these animals can be stunted in growth or appear normal. fetal infection occurring at 100-150 d of gestation can result in congenital abnormalities, including cerebellar hypoplasia, microencephalopathy, cataracts, microopthalmia, and thymic aplasia. fetuses infected between 150 and 285 d of gestation are usually able to clear the virus, develop normally, and exhibit precolostral neutralizing antibodies to bvdv. bluetongue virus is an orbivirus that is transmitted by a midge (culicoides variipennis) [8] . fetuses infected during the first 100 d of gestation, resorb or abort. infections between 75 and 100 d of gestation can result in stillbirths, birth of weak calves, or birth of calves with cerebral abnormalities. infection after day 150 of gestation does not generally have a negative effect on the fetus. epizootic bovine abortion is a disease that results in late-term abortion [11, 12] . abortion does not generally occur the next year. the disease is possibly caused by an unnamed bacteria in the delta subdivision of the class proteobacteria and in the order myxococcales. the organism is transmitted by the tick ornithodoros coriaceus. the disease occurs in california, nevada, oregon, and southern idaho. akabane disease is caused by a bunyavirus [13] . the disease occurs in australia, japan, the middle east, south africa, and turkey, and affects cattle, sheep and goats. in japan, the virus is transmitted by mosquitoes and in australia transmission occurs via the midge (culicoides brevitarsis). fetuses infected in the first trimester usually die shortly after birth or have sensory, motor and optical nerve damage. infection during the second trimester can result in arthrogryposis, neurogenic torticollis, kyphosis, and scoliosis. tritrichomonas foetus is a flagellated protozoa that causes venereal disease in cattle [14] . infected cows can experience early embryonic death, infertility, and abortion in the first half of gestation. some cows develop postcoital pyometra. generally, the infection is cleared within 90 d. neospora caninum is a protozoa that can cause abortion early in the second trimester [15] . infected cows can abort repeatedly. the definitive host for the organism is the dog that ingests tissue cysts. the cow then ingests sporulated oocysts in feed, water or soil contaminated with the dog feces. tachyzoites can then be transmitted through the placenta to infect the fetus. infected cows are asymptomatic. mycotic abortions can be caused by various molds and yeasts [6, 16, 17] . aspergillus fumigatus is the most common pathogen, with morteriella wolfii being common in the southern hemisphere. other pathogens include other aspergillus spp., absidia spp., rhizomucor pusillus, rhizopus arrhizus, pseudallescheria boydii, species of penicillium, candida, and torulopsis. the disease occurs sporadically, usually occurring when animals are housed indoors or in small pens and fed hay. the fungal pathogens might enter the respiratory or gastrointestinal tract and then gain access to the systemic circulation and spread to the placentomes. abortion generally occurs between 6 and 8 months of gestation. placentitits and retained placenta are frequent occurrences. other clinical signs are generally not seen. however, cows infected with morteriella wolfii can have postabortion pneumonia and death within 72 h after abortion. many of the bacterial, viral, and protozoan causes of ovine abortion are zoonotic. thus, attendants should employ protective measures to prevent human infection. campylobacter is the most important cause of abortion in sheep in north america [18, 19] . campylobacter jejuni is responsible for sporadic abortions, whereas campylobacter fetus subsp. fetus is associated with large outbreaks of recurring abortion. the zoonotic, gram-negative rods are transmitted by ingestion and shed in feces, aborted fetuses, placentae, and vaginal discharges of ewes that abort. ewes are asymptomatic and abort in the third trimester, have stillbirths or give birth to weak lambs. some ewes are persistently infected and continue to shed the organism in their feces. infection with listeria monocytogenes occurs through ingestion of the organism in poor-quality alkaline silage or pastures contaminated with feces containing the zoonotic organism [19] . ewes can abort 7-30 d after infection. prior to aborting, ewes might show signs of fever and decreased appetite. following abortion, ewes often develop metritis. as well, listeria ivanovii has also been shown to cause abortion in sheep. abortion in sheep can be caused by brucella melitensis or rarely b. ovis [18, 19] . brucella can be transmitted to the ewe through mucous membrane (vaginal, preputial, and conjunctival) contact with infected rams. ewes are generally asymptomatic, but can abort in the third trimester, have stillbirth or give birth to a weak lamb. ewes clear the bacteria within a few weeks following an abortion. b. melitensis has zoonotic potential, whereas b. ovis does not. abortion in ewes can follow infection with salmonella abortus-ovis, salmonella montevideo, or salmonella arizonae [18, 19] . overcrowded conditions and shipping can predispose flocks to abortion storms. affected ewes can be asymptomatic prior to aborting, or have clinical signs of fever, depression and diarrhea. metritis and retained placenta can occur after aborting. these pathogens are zoonotic. c. abortus is responsible for causing ovine enzootic abortion [19] . the organism is transmitted through ingestion or exposure to aborted materials, vaginal discharge, or contaminated environment. when the infected ewe becomes pregnant, the organism is hematogenously spread to the trophoblast cells and by 95 d of gestation, has infected the cotyledons and intercotyledonary areas. infection of the fetus and abortion may result. prior to 95 d, stillbirth or resorption can occur. if the ewe is not pregnant at the time of infection, the organism can affect the subsequent pregnancy. once abortion occurs, the immune system will prevent subsequent abortions, but the organism can continue to be shed in vaginal secretions. c. abortus can cause abortion in humans. coxiella burnetti is transmitted through inhalation, mucous membrane contact, ticks, and possibly semen [19] . infected ewes might develop placentitis and abort late in gestation, although abortion is not as common as in does. abortion is not likely to occur the next lambing season due to development of immunity within the flock. humans can become infected when handling infected placentas or lambs, and from inhalation of contaminated dust. the clinical signs of infection in people may include prolonged illness, undulant fever, atypical pneumonia, hepatitis, myalgia, or endocarditis. tick-borne fever occurs in europe and is caused by anaplasma phagocytophilum [20] . clinically, the disease results in fever, inappetance and abortion of sheep, cattle and goats. the bacteria causes flu-like symptoms in humans. yersinia pseudotuberculosis [21] and yersinia enterocolitica (o serotype) [22] have been isolated from ovine abortion cases. infection of ewes with y. pseudotuberculosis can lead to abortion, stillbirth or birth of weak or healthy lambs. infection with y. enterocolitica resulted in placentitis and abortion, with subsequent normal pregnancies. acute mesenteric lymphadenitis with fever, anorexia, vomiting, and diarrhea are common in people infected with these organisms. border disease virus is a non-zoonotic pestivirus similar to bovine viral diarrhea virus and hog cholera virus [19] . ewes infected before days 60-85 of gestation have fetal resorption, abortion, maceration or mummification. if the fetus survives, it will be persistently infected with the virus, have potential cerebellar damage, shortened facial and long bones, and produce hair rather than wool. if infection occurs after day 85 of gestation, abortion, birth of weak or normal lambs, or birth of antibody positive lambs can occur. no clinical signs are seen in the ewe. bluetongue virus, an orbivirus, is transmitted by a midge (culicoides variipennis) [18, 19] . infected ewes can abort, have mummified fetuses or deliver lambs with congenital defects (hydranencephaly, porencephaly, cerebellar dysgenesis, skeletal deformities) [23] . the ewe can show clinical signs of fever, lameness, oral and nasal ulcers, and swollen tongue, ears or face [18, 19] . akabane virus can cause abortion, stillbirth, mummified fetuses, and congenital deformities (arthrogryposis, hydranencephaly) in sheep [18] . cache valley virus, a bunyavirus, is transmitted by mosquitoes [19] . infected ewes have fever, depression, and a reluctance to move. infection at 28-32 d of gestation results in early embryonic death and mummification. infection at 32-37 d of gestation causes cns and musculoskeletal deformities (arthrogryposis, brachygnathia, hydranencephaly, agenesis of the spinal cord), whereas infection at 37-48 d leads to milder musculoskeletal deformities, with nonsuppurative encephalitis and encephalomyelitis. more than one clinical syndrome can be present in a litter. ewes are infected by toxoplasma gondii through ingestion of feed contaminated with sporulated oocysts [19] . the fetus can then be infected approximately 14 d after ingestion of the parasite. whereas ewes are asymptomatic, infection occurring <40 d of gestation results in embryo resorption. infection at 40-120 d of gestation leads to maceration, mummification or abortion, whereas infection after 120 d can result in stillbirth or birth of weak or healthy lambs. different stages of fetal involvement can be seen within the flock and within the same litter. t. gondii is a zoonotic agent that can affect nonimmune individuals. similar to ovine reproductive pathogens, caprine pathogens include many zoonotic organisms. infection with l. monocytogenes early in gestation can result in abortion, whereas infection late in gestation results in stillbirth or birth of weak kids [18] . prior to abortion, does may have fever, decreased appetite, and reduced milk production. the organism can be shed in the milk from the aborting doe. generally, the encephalitic form of the disease does not occur simultaneously with abortions. l. monocytogenes can survive in soil and feces, and grows in poorly fermented silage. abortions are reported after grazing on boggy, high-ph soils. listeria is zoonotic and can cause neurologic disease in humans. c. abortus is the most common cause of infectious abortion in goats in the usa [18, 24] . it is responsible for abortions, stillbirths, birth of weak kids, and neonatal pneumonia. the organism does not proliferate until 90 d of gestation, at which time fetal death and abortion can occur. does can show anorexia, fever, and a bloody vaginal discharge 2-3 d before aborting. following abortion, uterine clearance of c. abortus takes 3 months. the organism can reside in pigeons or sparrows and be transmitted via ticks or insects. the organism is shed in uterine discharge, fetus and placenta. c. abortus is zoonotic and can result in flu-like symptoms and abortion in pregnant women. brucellosis is present in africa, mexico, the middle east, india, pakistan and parts of south america [18, 24] . b. melitensis is transmitted to goats through ingestion of contaminated feed or water. in pregnant does, the bacteria can infect the placenta with resultant late-term abortion. does might show clinical signs of fever, depression, weight loss, diarrhea, mastitis, lameness, and birth of weak kids. the bacteria is shed in milk, urine, feces, and for 2-3 months in vaginal discharge. the organism causes undulant fever (synonymous with malta fever, gibraltar fever, and mediterranean fever) in humans that consume unpasteurized contaminated milk or cheese. coxiella burnetti is the causative agent of q fever [25] . this gram-negative coccobacillus causes disease in sheep, cattle, goats, and other mammals. affected animals might have no clinical signs of disease, but serve as a source of infection, or they can abort late in gestation and have stillbirths. the organism is transmitted in placenta, uterine fluids, milk, urine, feces, and aerosol droplets. transmission occurs through contact, inhalation, or possibly ingestion. as well, the organism is zoonotic and can be transmitted to humans by dogs and cats. affected individuals might have no clinical signs, or they might show fever, headaches, myalgia, coughing, fatigue, vomiting, respiratory disease, and rarely endocarditis. mycoplasma can cause late-term abortion, mastitis, vulvovaginitis, arthritis and conjunctivitis [24, 26, 27] . following abortion, does can shed the organism in milk, amniotic fluids, and the placenta [18] . leptospirosis can result in abortion in the last trimester, anorexia, fever, jaundice, anemia, and nervous signs in affected does [18, 24] . transmission occurs through exposure to environments contaminated with infected urine. due to the zoonotic potential, humans should avoid contact with aborted tissues and infected urine. s. abortus-ovis infection of does can cause abortion, metritis, retained placenta, diarrhea, and systemic illness [18, 24] . although abortion can occur throughout gestation, it is more common at the end of gestation. the bacteria are transmitted through ingestion. salmonella infections are zoonotic and can cause enteritis, abdominal pain and abortion in humans. although campylobacterosis is a common cause of abortion in sheep, it is less prominent in goats [18, 24] . does may exhibit late abortions, stillbirths, birth of weak kids, and a mucopurulent vaginal discharge. the placenta is edematous, with necrosis of the cotyledons. the does do not show evidence of systemic illness, but may have diarrhea. as well, campylobacter jejuni causes mild gastroenteritis in humans. y. pseudotuberculosis is transmitted by fecal-oral route [24] . abortion and early neonatal death have been reported in goats [28] . the bacteria is zoonotic. akabane virus is transmitted by mosquitoes and midges (culicoides brevitarsis) [13, 18, 24] . it occurs in australia, japan, the middle east, south africa, and argentina. nonpregnant goats do not show clinical signs, whereas pregnant does can appear healthy but abort, have stillbirths or mummified fetuses. kids have arthrogryposis and/or hydrencephaly that can result in dystocia. caprine herpesvirus is an alpha herpesvirus that can cause late-term abortion without any prior clinical signs [29] [30] [31] [32] . the virus can also cause vulvovaginitis and respiratory disease. subsequent pregnancies are not affected by the virus [32] . like other herpesviruses, caprine herpesvirus has a latent state that can be reactivated following stress, immunosuppression or possibly estrus in does. following reactivation, the virus can be shed via the respiratory or genital route. toxoplasmosis can cause abortion, stillbirth, fetal death, fetal resorptions, birth of a weak kid, or birth of a healthy kid [24, 33] . infection early in gestation (30-90 d) generally results in fetal resorptions or mummification, whereas infection in the last half of gestation results in asymptomatic does aborting 2-3 weeks prior to parturition. abortion occurs due to necrosis of the cotyledons. the protozoa is transmitted to cats through ingestion of infected rodents or birds. does ingest food or water that is contaminated with cat feces containing resistant oocytes; the organism then enters the bloodstream and spreads to the placenta and fetus. toxoplasmosis has zoonotic potential. sarcocystosis is caused by a cyst-forming protozoan [24] . inoculation of pregnant does (75-105 d of gestation) with sporocysts of sarcocystis resulted in abortion and neonatal death [34] . unlike reproductive pathogens in other species, porcine reproductive pathogens are predominantly viruses. this is hypothesized to be due to the density of the swine population within many production units. brucella suis is transmitted through direct contact with aborted fetuses and secretions, as well as venereally [35] . infected gilts or sows might present with infertility. in addition, abortion can occur in the first trimester, if infection occurs at breeding, and during late gestation if infection occurs after day 35 of gestation. the organism has zoonotic potential. swine suspected of having brucellosis should be reported to state animal health authorities. leptospirosis is caused by a gram-negative spirochete [35, 36] . transmission occurs through contact of oral, nasal or ocular mucosa with contaminated urine. pigs are maintenance hosts for serogroups pomona, australis and tarassovi, whereas incidental infections occur with strains of the canicola, icterohaemorrhagiae, and grippotyphosa serogroups. acute infection of leptospirosis is generally asymptomatic. however, chronic leptospirosis may manifest as abortions, stillbirths, infertility, and birth of weak piglets. leptospirosis is an important occupational zoonosis for farmers and abbatoir staff in contact with pigs. erysipelothrix rhusiopathiae is a gram-positive rod that causes erysipelas [35] . the bacteria is transmitted through oronasal exposure, followed by septicemia. the disease is characterized by abortion, fever, diamondshaped skin lesions, arthritis, vegetative endocarditis, and sudden death. abortion can occur at any stage of gestation, additionally, some piglets may become mummified. as well, embryo resorption can occur. the bacteria is zoonotic, causing erysipeloid in humans. although streptococci are part of the normal flora of the vagina, they have also been associated with abortion, stillbirths, vaginitis, postparturient agalactia, and early neonatal death [35, 37] . chlamydiae are bacteria that can be transmitted by inhalation of aerosols, ingestion of contaminated feed, and venereally [38] . the species involved are chlamydophila psittaci, chlamydophila pecorum, and chlamydia trachomatis. chlamydial infections can be inapparent or have clinical signs of fever, dyspnea, pneumonia, conjunctivitis, pericarditis, and polyarthritis. in addition, gilts or sows may exhibit lateterm abortions, birth of weak piglets, or mummified piglets. actinobacillus suis, actinobacillus ross, and actinobacillus equuli have been suggested to cause abortion, metritis, and decreased litter sizes [35] . mauch and bilkei reported late-term abortions during the first and second parities due to a. suis [39] . a. suis and a. ross are normal flora of the vagina and upper respiratory tract of healthy sows, and the bacteria can be isolated from healthy herds [35, 39] . transmission of a. suis occurs via inhalation of aerosols or through abrasions in the skin and mucous membranes. infected animals might demonstrate fever, erythematous skin lesions, and anorexia. mycoplasma suis is the causative organism of eperythrozoonosis [40] . m. suis attaches to and replicates on the red blood cell. it causes anemia, icterus, anorexia, immune suppression, and fever. furthermore, infertility, abortion, stillbirth, and birth of weak piglets can also result from infection. porcine reproductive and respiratory syndrome virus (prrsv) can be shed in nasal secretions, feces and urine; infected pigs can be long-term carriers [41] [42] [43] . the virus might be spread by contact, oral transmission and aerosol transmission. experimentally, the virus is transmitted in semen [44] . additionally, the virus can be transmitted transplacentally [41] , most commonly in the last trimester. during the first phase of the disease, reproductive signs include abortions and irregular returns to estrus. pulmonary edema or nephritis also can result in sows. during the second phase of the disease, pregnant animals can give birth to a combination of normal or weak piglets and stillborn, autolytic, or mummified fetuses. porcine parvovirus is endemic in most herds, with most gilts and sows exhibiting active immunity against the virus [45] . gilts that do not have immunity to porcine parvovirus before conception are at a high risk of infection and reproductive disease. pseudorabies is caused by an alpha herpesvirus [46, 47] . the pig is the only natural host of pseudorabies, whereas infection in cattle, sheep, goats, dogs, and cats is fatal. transmission is primarily through direct contact with nasal and oral secretions, but can occur by aerosols, venereally, or transplacentally. like other herpesviruses, pseudorabies can establish latent infections. in naã¯ve herds, the clinical signs might be severe. infection during the first trimester results in resorption and a return to estrus, whereas infection during the second and third trimester results in abortion, stillbirth, or birth of weak piglets. neonatal pigs may have 100% morbidity and mortality rates. the dam's immunity against pseudorabies determines the clinical signs seen in piglets. piglets of dams lacking immunity have nervous signs (trembling, hypersalivation, ataxia, circling, paddling) and die within 1-2 d, whereas piglets of immune dams usually do not show clinical signs. classical swine fever, previously known as hog cholera, is caused by a pestivirus [48] . pigs are also susceptible to two other pestiviruses, bovine viral diarrhea virus and border disease virus. pigs are the only natural host of classical swine fever virus. transmission occurs through oronasal contact with infected pigs, ingestion of contaminated feed, airborne spread over short distances, indirectly by fomites, and potentially through semen [48, 49] . clinical signs include fever, anorexia, conjunctivitis, diarrhea, and respiratory signs. transplacental infection can occur at any stage of gestation resulting in abortions, mummies and stillbirths. infection at 50-70 d of gestation can result in the birth of persistently viremic piglets. these piglets appear normal initially, but subsequently develop congenital tremors and lose weight. they serve as a continual reservoir of classical swine fever virus. porcine enterovirus and teschovirus are picornaviruses [50] . transmission is through fecal-oral route, but transmission by fomites might also occur. sows and gilts may experience infertility, embryonic death, stillbirths, and mummies, without any other clinical signs. encephalomyocarditis virus is a picornavirus that is isolated from several species [51] . in swine, the disease is primarily transmitted through feed or water contaminated by infected rodent feces, urine, or carcasses. pregnant females might not show clinical signs, or they might experience abortion, mummies, or stillbirths [52] . porcine cytomegalovirus is a beta herpesvirus that is transmitted oronasally and congenitally [53] . pigs >3 weeks of age are asymptomatic, whereas younger pigs might show coughing, nasal discharge, rhinitis, neurological signs, or death. pregnant animals can have mummies and stillbirths without showing any other clinical signs. as well, preweaning mortality can be 25%. rubulavirus is a paramyxovirus that causes blue eye disease in swine in mexico [54] . transmission of the virus occurs through nose-to-nose contact or contact with fomites. infected pigs of all ages develop corneal opacity. pregnant animals show reproductive signs for approximately 4 months. these signs consist of stillbirths, mummies, decreased number of live births, increased number of animals returning to estrus, and an increase in weaning-to-service interval. in closed herds, the disease is self-limiting. menangle virus is a paramyxovirus that has caused disease in new south wales, australia [54] . the virus causes disease in pigs, humans and fruit bats, which are the reservoir host. transmission is suspected to be through fecal-oral or urinary-oral routes. the disease caused reproductive failure in sows [55] . stillbirths and mummified fetuses were present, as well as stillborn piglets with congenital deformities such as arthrogryposis, brachygnathia, kyphosis, and degeneration of the brain and spinal cord. many sows had a delay in return to estrus. additionally, two farm workers were affected. they experienced fever and headaches, but fully recovered. porcine circovirus type 2 has been associated with reproductive disease, respiratory disease, postweaning multisystemic wasting syndrome, and porcine dermatitis and nephropathy syndrome [56] . the virus is thought to be transmitted by oronasal exposure. reproductive disease is manifest as late-term abortion, stillbirths, and mummified fetuses [57] . japanese encephalitis virus is a mosquito-borne virus that causes disease in humans and animals [58] . swine are reservoir hosts that allow mosquitoes to transmit the virus to humans (dead-end hosts). the disease is manifest in pregnant sows and gilts as abortions, stillbirths, mummified fetuses, and birth of weak piglets that can have hydrocephalus and subcutaneous edema. infection after 60-70 d of gestation does not appear to affect piglets. soft ticks (ornithodorus moubata and ornithodorus erraticus) are the reservoir and vector for african swine fever virus [42, 59] . as well, the virus can be spread among pigs via oronasal secretions. the clinical signs of disease can vary from sudden death (with no previous clinical signs) to inapparent infection. acute infections may consist of fever, anorexia, cyanosis, hemorrhages in internal organs and skin, and abortion. toxoplasmosis occurs through ingestion of food, water or soil contaminated with sporulated oocysts or through ingestion of meat containing tissue cysts [60] . whereas most infections are asymptomatic, abortion may occur [60, 61] . additionally, piglets may be born premature, dead, weak, or die soon after birth [60] . prevention of toxoplasmosis in pigs is important to prevent infection of humans through ingestion of undercooked pork. pathogens that cause sporadic reproductive loss in mares may be due primarily to host factors, e.g. impaired reproductive defense mechanisms. notably, other pathogens may cause disease, depending on variables of the environment (e.g. movement of horses that predisposes naã¯ve mares to novel infections). streptococcus zooepidemicus is one of the most common bacteria isolated from mares with uterine disease [62] . whereas the uterus of a healthy mare is more likely to clear the infection, the uterus of a mare with impaired defense mechanisms or poor genital conformation might become inflamed or develop an infection leading to infertility. as well, streptococcus zooepidemicus can cause abortion via ascension through the cervix and infection of the fetus and placenta, or possibly through a maternal systemic infection [62, 63] . bacterial abortions usually occur at 5-10 months of gestation [62] . in addition to bacterial culture, diagnosis of infection is coupled with evidence of autolysis and inflammation of the fetus and placenta. other bacteria implicated in infertility or abortion are e. coli, pseudomonas aeruginosa, klebsiella pneumoniae var genitalium, staphylococcus aureus, streptococcus equisimilis, leptospira spp., s. abortus equi, and nocardioform actinomycetes. although no longer very common, s. abortus equi has caused abortion outbreaks [64] ; in one of these, 21 of 26 mares aborted between 5 and 10 months of gestation [64] . taylorella equigenitalis is a gram-negative coccobacillus that causes contagious equine metritis (cem) [62] . taylorella equigenitalis is transmitted venereally, highly contagious, and can cause a copious vulvar discharge [65] . diagnosis is by bacteriologic culture of the organism from the clitoral fossa, clitoral sinuses, and endometrium or cervix. as well, polymerase chain reaction can be used to identify the organism. serologic tests are also available to detect antibodies against taylorella equigenitalis in the serum. most mares recover spontaneously from cem over a period of several months. imported horses used for breeding should be quarantined and extensively tested. any animals suspected of having cem should be reported to state or federal animal health authorities. equine herpesvirus 1 can cause abortion, neonatal death, respiratory disease, and neurologic disease [66] . the virus is transmitted through inhalation of infectious aerosol droplets, or by direct contact with infectious secretions. like other herspesviruses, equine herpesvirus 1 persists in a latent state that can be reactivated. most abortions are sporadic and occur between 7 months of gestation and term. the abortions can occur up to 4 months after respiratory disease, which may have been clinical or subclinical. equine viral arteritis can cause sporadic abortions between 5 and 10 months of gestation [67] . the virus is transmitted by inhalation of infectious aerosol droplets or venereally. if clinical signs of disease are apparent, they can consist of fever, hind limb edema, nasal and ocular discharge, urticarial rash, anorexia, coughing, and dyspnea [68] . the virus can also cause uterine disease, with subsequent abortion in pregnant mares [69] . trypanosoma equiperidum is a venereally transmitted protozoa that causes dourine [65] . although it has been eradicated from north america and most of europe, the disease is still found in africa, south and central america, and the middle east [70] . clinical signs include a mucopurulent vulvar discharge, fever, and cutaneous raised plaques followed by ataxia, depression, severe anemia, and death [65, 70] . mortality can reach 50-70%. strict quarantine, testing, and eradication of positive animals are recommended for control of the disease. fungal pathogens account for up to 10% of reported sporadic abortions [71] . aspergillus fumigatus is the most common fungal pathogen, but mucor spp. and allescheria boydii have also been reported. infection of the uterus may occur via ascension through the cervix or hematogenously. abortions usually occur at 8-11 months of gestation, and may be associated with a purulent vulvar discharge. the affected chorioallantois is thickened, yellowish, and covered with a mucoid exudate. infectious causes of canine reproductive losses include some pathogens with a zoonotic potential, as well as viruses that can quickly spread throughout a kennel if bitches are not adequately vaccinated. brucella canis is a small, gram-negative intracellular coccobacilli. infection can cause infertility, early embryonic death, fetal resorptions, and late-term abortion [72, 73] . the bitch might not show any clinical signs prior to aborting. following abortion, a serosanguinous vaginal discharge may be present for 1-6 weeks. large numbers of bacteria can be present in the aborted material and postabortion vulvar discharge; therefore, caution should be exercised to prevent other dogs from contacting the fluid or aborted tissue. whereas the zoonotic potential of b. canis is less than most brucella sp., immunosuppressed or pregnant individuals should avoid contact with aborted fluid or tissue. streptococci are gram-positive cocci that are commensal microflora of the genital tract [74] . although the bacteria have been associated with abortion, infertility and neonatal septicemia, its isolation does not confirm the cause of the reproductive problem. campylobacter is a gram-negative curved rod. although campylobacter jejuni has been isolated from the feces of normal dogs [75] , it has also been associated with abortions [76] . if campylobacter is suspected, the diagnostic laboratory should be contacted to obtain instructions for sample submission and to be informed of the suspected pathogen [74] . also, owners should be informed that the bacteria can cause enteric disease in humans. salmonella is a gram-negative bacilli that is transmitted through the gastrointestinal tract via contaminated water, food, or fomites [77] . infection with salmonella can cause abortion, stillbirth and birth of weak puppies. it is important that owners by notified of the zoonotic potential of salmonella. 7.5. escherichia coli e. coli is commonly isolated in cases of metritis and pyometra [74] . it has also been isolated in the case of a partial abortion [78] . mycoplasma and ureaplasma can be isolated from the genital tract of both healthy and infertile bitches [79] . although these organisms have been associated with infertility, fetal resorption, abortion, stillbirth and birth of weak puppies [74] , it is recommended that culture results be interpreted in the context of other diagnostic findings to determine if antibiotic treatment should be instituted [80] . when submitting specimens to the diagnostic laboratory, samples should be placed in amies medium (without charcoal) or modified stuart bacterial transport medium and the laboratory informed of the suspected pathogen [80] . canine herpesvirus can lead to abortion, stillbirth and embryonic resorptions [81] . a pregnant bitch can become infected through direct contact with mucosal secretions (respiratory or genital) [82, 83] . as well, a latent infection might be reactivated during pregnancy with resultant virus shedding. neonatal infection usually occurs at birth; however, transplacental infection can occur and lead to mummified or dead fetuses, stillbirth, or birth of weak puppies. canine distemper is caused by a morbillivirus [84] . the virus has been shown to cause abortion, stillbirth and congenital infections in puppies [84, 85] . abortion can follow systemic infection of the bitch or transplacental infection [74] . puppies infected transplacentally can develop neurologic signs within 6 weeks of birth [84] . canine parvovirus type 1, the causative agent of minute virus of canines, can cause embryo resorptions, stillbirth, or birth of weak puppies [86] . toxoplasmosis can cause placentitis with spread of tachyzoites to the fetus [87] . experimental infection of bitches caused congenital infection and abortion. 7.11. neospora caninum n. caninum has been shown experimentally to be transmitted transplacentally [88] . neosporosis can result in early fetal death, mummification, resorption and birth of weak puppies [87] . however, it has not been shown to cause abortion. feline reproductive loss is more commonly attributed to a viral etiology than to bacterial or protozoan pathogens. although uncommon, abortions can occur when normal vaginal flora (e. coli, staphylococcus sp., streptococcus sp.) ascend into the uterus and cause fetal infection [89] . the queen might show clinical signs of infection, such as anorexia, pyrexia, abdominal discomfort, and vaginal discharge [90] . feline herpesvirus 1 is an alpha herpesvirus that causes rhinotracheitis [91, 92] . experimental infection caused abortion and intrauterine fetal death [89] ; however, the virus has not been isolated from the aborted fetal tissue [92] . hickman reported that in a herpesvirus outbreak in a specific pathogen-free colony, only 1 of 51 queens pregnant at the initial time of the outbreak aborted part of her litter [91] . however, there was a 62% mortality rate within 1 week of birth among the kittens born to queens acutely infected during the perinatal period. feline immunodeficiency virus (fiv) is a lentivirus, similar to human immunodeficiency virus [93] . the virus can be transmitted in utero resulting in abortion, stillbirth, arrested fetal development, and birth of viable, virusinfected kittens [94] . weaver et al. reported that experimental inoculation of specific pathogen-free queens with fiv resulted in 60% of kittens being resorbed or having arrested fetal development [93] . feline infectious peritonitis is caused by a coronavirus [90] . the virus is associated with late-term abortion, stillbirths, fetal resorptions, endometritis, and high mortality in kittens the first week of life. some purebred cats have a genetic predisposition for fip (heritability of 50%), and thus should not be used as breeding animals [95] . feline leukemia is a retrovirus that can lead to abortion, infertility and fetal resorptions [90] . generally, queens are asymptomatic prior to aborting. feline panleukopenia virus is a parvovirus that can cause abortion, stillbirth, and cerebellar hypoplasia in kittens [90] . these signs are not always associated with the classical gastrointestinal disease in the queen. although not common, t. gondii is reported to cause abortion and congenital infections [89, 90] . infection can occur transplacentally or postnatally via milk [87] . preparation is essential to maximize the potential for accurate diagnosis of reproductive pathogens. whenever possible, entire fetuses, placental tissues, and serum and urine from the dam should be sent promptly, along with a complete case history, to the diagnostic laboratory. in many cases, listing the pathogens of concern on submission forms can improve diagnostic test selection. effective communication with your diagnostic laboratory to ensure appropriate sample handling and prudent test selection can maximize your diagnostic support. it is noteworthy that appropriate precautions should be taken to avoid zoonotic infection of personnel (in clinical or diagnostic settings) with reproductive pathogens. finally, appropriate immunization or biosecurity to prevent infection often can reduce reproductive losses in domestic animals. bacterial causes of bovine infertility and abortion adherence of haemophilus somnus to bovine embryos after in vitro exposure bovine genital mycoplasmosis chlamydia-related abortions in cattle from graubunden parachlamydia spp. and related chlamydia-like organisms and bovine abortion infectious diseases causing bovine abortion and fetal loss bovine herpesvirus 1 infection and infectious bovine rhinotracheitis viral diseases of the fetus experimentally induced infectious bovine rhinotracheitis virus infection during early pregnancy: effect on the bovine corpus luteum and conceptus reproductive consequences of infection with bovine viral diarrhea virus the geographic distribution of the putative agent of epizootic bovine abortion in the tick vector, ornithodoros coriaceus epizootic bovine abortion (foothill abortion) veterinary virology current therapy in large animal theriogenology protozoal abortion in cattle mycotic bovine abortion fungi associated with bovine abortion in the northern plains states (usa) philadephia: w.b. saunders company abortion in sheep: diagnosis and control anaplasma phagocytophilum-the most widespread tick-borne infection in animals in europe ovine abortion and stillbirth due to purulent placentitis caused by yersinia pseudotuberculosis experimental yersinia enterocolitica placentitis in sheep observations on transplacental infection with bluetongue virus in sheep infectious causes of abortion caprine abortion following exposure to mycoplasma capricolum subsp. capricolum isolation of mycoplasma mycoides, mycoides (lc variant), from two naturally aborted caprine fetuses abortion and early neonatal death of kids attributed to intrauterine yersinia pseudotuberculosis infection multiple abortions associated with caprine herpesvirus infection in a goat herd abortion and ulcerative posthitis associated with caprine herpesvirus-1 infection in goats in california caprine herpesvirus-1 abortion storm in a goat herd in quebec serologic and reproductive findings after a herpesvirus-1 abortion storm in goats toxoplasma gondii-induced abortion in dairy goats abortion and death in goats inoculated with sarcocystis sporocysts from coyote feces bacterial, rickettsial, protozoal, and fungal causes of infertility and abortion in swine diseases of swine streptococcus suis type ii-associated diseases in swine: observations of a one-year study miscellaneous bacterial infections actinobacillus suis, a potential cause of abortion in gilts and low parity sows exploratory study of mycoplasma suis (eperythrozoon suis) on four commercial pig farms in southern brazil porcine reproductive and respiratory syndrome virus (porcine arterivirus) viral causes of porcine reproductive failure-part i porcine reproductive and respiratory syndrome detection of porcine reproductive and respiratory syndrome virus in boar semen by pcr diseases of swine aujeszky's disease (pseudorabies) viral causes of infertility and abortion in swine classical swine fever and other pestiviruses transmission of classical swine fever virus by artificial insemination porcine enteric picornaviruses encephalomyocarditis virus reproductive failure in sows following experimental infection with a belgian emcv isolate diseases of swine rubulavirus, menangle, and nipah virus infections reproductive disease and congenital malformations caused by menangle virus in pigs porcine circovirus diseases myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus 2 japanese encephalitis and west nile viruses afrcian swine fever coccidia and other protozoa isolated, spontaneous toxoplasma abortion in a young sow bacterial causes of subfertility and abortion in the mare abortion in mares an outbreak of abortion in mares associated with salmonella abortus equi infection sexually transmitted (venereal) diseases of horses equine herpesvirus infections equine viral arteritis equine viral arteritis pathology of maternal genital tract, placenta, and fetus in equine viral arteritis disease transmission in horses current therapy in large animal theriogenology clinical signs and diagnosis of brucella canis infection canine brucellosis: outbreaks and compliance canine pregnancy factors influencing fecal shedding of campylobacter jejuni in dogs without diarrhea abortion in the dog due to campylobacter species infectious diseases of the dog and cat partial abortion associated with genital escherichia coli infection in a bitch the genital mycoplasma and ureaplasma flora of healthy and diseased dogs infectious diseases of dogs and cats canine herpesvirus-1 (chv-1): clinical, serological and virological patterns in breeding colonies infectious diseases of the dog and cat clinical considerations of canine herpesvirus infection infectious diseases of the dog and cat experimental and naturally occurring transplacental transmission of canine distemper virus pathogenicity of minute virus of canines (mvc) for the canine fetus infectious diseases of dogs and cats transplacental neospora caninum infection in dogs canine and feline theriogenology infectious causes of infertility, abortion and stillbirths in cats an epizootic of feline herpesvirus, type 1 in a large specific pathogen-free cat colony and attempts to eradicate the infection by identification and culling of carriers herpesviral abortion in domestic animals placental immunopathology and pregnancy failure in the fiv-infected cat vertical transmission of feline immunodeficiency virus the inheritance of susceptibility to feline infectious peritonitis in purebred catteries key: cord-269652-t7ghng17 authors: santos, roberto parulan; tristram, debra title: a practical guide to the diagnosis, treatment, and prevention of neonatal infections date: 2015-04-30 journal: pediatric clinics of north america doi: 10.1016/j.pcl.2014.11.010 sha: doc_id: 269652 cord_uid: t7ghng17 neonatal infections continue to cause morbidity and mortality in infants. among approximately 400,000 infants followed nationally, the incidence rates of early-onset sepsis infection within 3 days of life are 0.98 cases per 1000 live births. newborn infants are at increased risk for infections because they have relative immunodeficiency. this article provides evidence-based practical approaches to the diagnosis, management, and prevention of neonatal infections. neonatal infections continue to cause morbidity and mortality in infants. among approximately 400,000 infants followed nationally, the incidence rates of early-onset sepsis (eos) infection within 3 days of life were 0.98 cases per 1000 live births. 1 more than two-thirds of the frequently isolated organisms were associated with group b streptococcus (gbs) (43%) and escherichia coli (29%). although 20% of the term infants were treated in the newborn nursery, 77% of the infected infants required intensive care management. of those who survived beyond 3 days of life, about 21% had an episode of late-onset sepsis (los) infection after 3 days of life. the overall mortality rate of infected infants was 16%. newborn infants are at increased risk for infections because they have relative immunodeficiency. this may be due to decreased passage of maternal antibodies in preterm infants and to immaturity of the immune system in general. 2, 3 the innate immune functions in infants are impaired with decreased production of inflammatory markers (interleukin 6 and tumor necrosis factor) 4 and with decreased dendritic and neutrophil functions. 5 the adaptive immune system is less than optimal with decreased cytotoxic functions, 2 decreased cell mediated immunity, 6 and delayed or lack of isotype switching. 2, 3 complement is important in opsonization and bacterial killing. in term infants, complement levels are approximately half compared with adults. 2 taken together, these predispose infants to severe, prolonged, or recurrent infections associated with bacterial, viral, or fungal infections. suspected sepsis, presumed infection, and ruling out sepsis remain the most common diagnoses in the nursery intensive care unit (nicu). the american academy of pediatrics (aap) committee on fetus and newborn 7 has published a clinical report extensively discussing clinically relevant challenges: identifying newborns with signs of sepsis with high likelihood of eos requiring antimicrobial regimen and identifying healthy-appearing newborns with high likelihood of eos requiring antimicrobial regimen. the committee concluded that, although these guidelines are evidencebased, they may be modified by the clinical judgment of the provider. the primary reason is that the clinical presentation of neonatal infection may be subtle and nonspecific, and may overlap with noninfectious causes. 7, 8 many clinicians empirically start broad spectrum antimicrobial regimen for infants considered at risk for sepsis but antibiotics are occasionally continued despite a negative blood culture. this practice may be detrimental to the infant 8 because it increases the risk of invasive fungal infections, 9 necrotizing enterocolitis (nec), or death, 10, 11 which increases the pressure for selecting multidrug-resistant organisms 12 and even the risk of los. 11 the purpose of this article is to provide evidence-based practical approaches to the diagnosis, management, and prevention of neonatal infections. the timing of transmission is one of the factors contributing to the cause of neonatal infections. different pathogens may be acquired during pregnancy (prenatal), during delivery (perinatal), or after delivery (postnatal). table 1 shows the different periods of transmission of various neonatal pathogens. the introduction of new molecular-based assays, such as quantitative real-time polymerase chain reaction (pcr), 13 has paved the way for increasing recognition of respiratory viral infections contributing to ruling out sepsis in late-onset infections. 14 table 1 includes respiratory viral infections (coronavirus, enterovirus, human metapneumovirus, influenza, parainfluenza virus, respiratory syncytial virus [rsv], and rhinovirus) as possible causes of postnatal infections in infants. [14] [15] [16] clinical presentations early-onset infections eos is arbitrarily defined as infection within the first 3 days of life. the most common organisms associated with eos include gbs and e coli. 1, 19 in general, the risk of bacterial infection in a healthy-appearing newborn remains relatively low. 20 the most common clinical findings include hypoglycemia (<40 mg/dl, 22%) and hypothermia (<36.5 c, 20%), followed by hyperglycemia (>140 mg/dl, 19%) and apnea (18%). 19 edwards and baker 21 summarized that newborn infants with sepsis manifest similar clinical signs as those with meningitis, including hyperthermia; hypothermia; respiratory distress; anorexia or vomiting; jaundice; and lethargy. hypotension may be more frequently found in infants with sepsis, whereas irritability, convulsions, and bulging or full fontanel is found in those with meningitis. however, they cautioned that absence of any of the aforementioned signs do not exclude central nervous system involvement. furthermore, it was suggested to evaluate infants for various foci of infections such as acute otitis media, conjunctivitis, osteomyelitis, pyogenic arthritis, and skin soft-tissue infections. los is arbitrarily defined as infection after 3 days of life. the most common organisms isolated with los include coagulase-negative staphylococci in more than a third of the cases, which may or may not be associated with a medical device. 19 yeast or candida spp infection is another important pathogen. 19 also, there is increasing recognition of viral respiratory infections as a possible cause in los. 14 the most common clinical findings include hypothermia (41%), hyperglycemia (38%), apnea (38%), and bradycardia (30%). 19 there are several factors that may increase the risk for los. there are significantly more infants with los who have an indwelling central vascular catheter at the time of infection than those infants with eos (78% vs 10%, p<.0001). 19 additionally, there are more infants with los who had a surgical procedure before infection (8% vs 1%, p<.0001). the clinical presentations of infections may overlap with noninfectious causes in newborns. it has been previously demonstrated that relying on symptoms alone may not be sufficient in diagnosing neonatal infections. 22 bacteremia has been reported in infants without clinical signs of sepsis. 23 there are several diagnostic tests and principles that may guide clinicians in evaluating infants with infections. the aap committee on fetus and newborn have published a clinical report on the evaluation of asymptomatic infants (<37 and !37 week gestation) with risk factors for sepsis. 7 evaluation of asymptomatic preterm infants (<37-week) with risk factors for sepsis is shown in fig. 1 . 7 similar algorithms for the evaluation of asymptomatic term infants (!37 week gestation) are available from the aap committee on fetus and newborn. (http://www.ncbi.nlm.nih.gov/pubmed/22547779). 7 additional principles in the evaluation of infants with risk factors for sepsis 7 follow: major risk factors for neonatal sepsis include chorioamnionitis, prolonged rupture of membrane 18 or more hours, and colonization of gbs with inadequate intrapartum antimicrobial prophylaxis (iap). chorioamnionitis usually presents as maternal fever greater than 38 c (100.4 f) and its diagnosis should be discussed with the obstetric providers. maternal fever may be the only abnormal finding in chorioamnionitis. adequate iap means maternal treatment with penicillin, ampicillin, or cefazolin at or earlier than 4 hours before delivery. at least 1 ml of blood may be sufficient for a single blood culture from a peripheral vein. blood culture from umbilical artery catheter or umbilical vein may be a reliable alternative following aseptic techniques screening blood cultures have not been proven of value and are not recommended. complete blood count with differential has poor positive predictive value and it is suggested waiting 6 to 12 hours after birth to avoid falsely normal values at birth. platelet counts remain low days to weeks after sepsis; thus this cannot be used in following response to treatment. the sensitivity of c-reactive protein (crp) improves if done 6 to 12 hours after birth. bacterial sepsis is unlikely if crp remains normal. lumbar puncture may be indicated in infants whom sepsis is highly suspected, those infants with bacteremia, and in infants who fail to respond to antimicrobial therapy. urinary tract infection in newborns is associated with episodes of bacteremia; thus urine culture should not be part of routine sepsis workup. microbiologic evaluation using gastric aspirates, tracheal aspirates, or superficial body sites cultures are of limited value and are not routinely recommended for sepsis. several acute-phase reactants or biomarkers (neutrophil cd64 [ncd64], procalcitonin [pct], or crp) may be used adjunctively in the evaluation and management of neonatal infection. the diagnostic usefulness of the various surrogate markers depends on the phases of neonatal sepsis: early phase or 2 to 12 hours (ncd64), mid phase or 12-24 hours (pct), and late phase or greater than 24 hours (crp). 24 ncd64 is a high-affinity fc receptor that increases with exposure to bacterial or fungal agents. 2, 24 the usefulness of ncd64 is related to its high negative predictive value as well as decreasing concentration on serial determinations on infants undergoing antimicrobial treatment of bacteremia. 24 however, there is a scarcity of medical evidence to recommend ncd64 for routine evaluation of neonatal infection and this may not be readily available. procalcitonin released from tissues increases with infection at around 2 hours and peaks at 12 hours. 7 it may also increase with noninfectious causes such as in respiratory distress syndrome and a physiologic increase during the first 24 hours of birth. 7 pct may not be readily available and the turnaround time varies in different institutions from 20 minutes to 5 hours. 24 crp increases around 6 hours associated with an inflammatory response with release of interleukin-6 and peaks at 24 hours. 7 crp has been used in the algorithm-based guideline from the aap committee on fetus and newborn for the evaluation of asymptomatic term and preterm infants with a risk factor for sepsis. 7 it is best used as part of a group of diagnostic tests 2 together with blood culture and white blood cell with differential in the evaluation of neonatal infection. 7 however, there is not enough medical evidence at this time to recommend serial determinations of crp in guiding duration of antimicrobial therapy in infants. 7, 24 further studies are needed to evaluate the usefulness of sequential determination of crp and biomarkers for an antimicrobial stewardship program (asp) in the nicu setting. in 2013, the infectious disease society of america, in collaboration with the american society for microbiology, affirmed the importance of close collaboration and positive working relationships between clinicians and microbiologists 25 to better serve patients. the most up-to-date edition of the red book provides contact information for expert advice and national collaborative study groups that give guidance on diagnostic assays regarding specific agents causing mother-to-child transmission. it is important to know the various microbiologic resources available locally, which include but are not limited to pcr and matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof). rapid antigen tests for respiratory viruses may lack sensitivity, 25 which is important in the nicu setting in controlling local outbreaks. there are several nucleic acid amplification test platforms currently available that differ in the number of analytes detected. 25 it is important to obtain adequate specimens and to use suitable viral transport media following manufacturer instructions. maldi-tof is a valuable alternative to the conventional microbiologic assays; however, it may not be a readily available resource for diagnostic testing in most institutions. however, if it is available, it has several practical applications that may benefit clinical management even in the nicu settings: earlier and accurate diagnosis of neonatal sepsis due to various bacteria 26 rapid identification of highly virulent gbs that causes meningitis and los in infants 27 identification of maternal-to-child transmissions (chorioamnionitis and neonatal infections) of opportunistic pathogen 28 accurate identification of bloodstream infection associated with fungal infections in the nicu 29 identification and monitoring the spread of nosocomial outbreak (eg, methicillinresistant staphylococcus aureus [mrsa] 30 and candida parapsilosis 31 in the nicu). when appropriate specimens for diagnostic evaluations are collected in clinically stable patients, then empirical antimicrobial therapy should be initiated for neonatal sepsis. it is recommended to discuss complicated cases, such as multidrug resistant organisms and infants not improving while on therapy or those requiring unconventional dosing regimens and antimicrobial agents, with pediatric infectious disease specialists. ampicillin and gentamicin remains the cornerstone of initial antimicrobial regimen for early-onset neonatal infections. the combination of such broad-spectrum antibiotic regimens cover the most common cause (gbs and e coli in more than 70%) 1 of eos and has synergistic activity (against gbs and listeria monocytogenes). 7, 32 the dosing regimen for ampicillin may change over time based on the chronologic age of the infant and body weight. 32 for example, an 8-day-old infant weighing greater than 2000 g may need dosing adjustment of ampicillin from 150 mg/kg/d intravenous (iv) divided every 8 hours to 200 mg/kg/d iv divided every 6 hours. once-daily dosing of gentamicin (4 mg/kg iv qd) 32 has been used in the term newborn for more than a decade. the pharmacodynamic characteristics of aminoglycosides that allow the use of once-daily dosing include concentration-dependent killing (peak concentration to minimal inhibitory concentration [peak/mic] ratio), 33, 34 postantibiotic effect with leukocyte enhancement, 35, 36 and prevention of adaptive resistance. 37 third-generation cephalosporins should be used judiciously. there is significant association between the use of third-generation cephalosporins and invasive candidiasis in preterm infants. 9 cefotaxime has excellent penetration to the cerebrospinal fluid and its therapeutic use should be limited to gram-negative meningitis. 7 routine use of cefotaxime for eos may lead to rapid development of drug-resistant organisms. 38 ceftriaxone is contraindicated in neonates for 2 reasons: (1) it is highly protein bound and may displace bilirubin progressing to hyperbilirubinemia 7 and (2) concurrent administration with calcium-containing solutions may produce insoluble precipitates (ceftriaxone-calcium salts) leading to cardiorespiratory complications. 39 the aap periodically updates the dosing regimens and recommended therapy for selected neonatal infections through nelson's pediatric antimicrobial therapy. 32 it provides various antimicrobial regimens (antibiotic, antiviral, and antifungal agents) based on body weight of infants and their chronologic age or gestational and postnatal age. between new editions, a monthly update of short and interesting reports related to pediatric antimicrobial therapy is posted at www.aap.org/en-us/aap-store/nelsons/ pages/whats-new.aspx. suggested durations of antibiotic therapy for eos adapted from 2014 nelson's pediatric antimicrobial therapy 32 and the aap committee on fetus and newborn 7 are shown in table 2 . there are several antiviral agents that can be used for the treatment of neonatal viral infections. acyclovir (60 mg/kg/d iv divided every 8 hours) is the treatment of choice for term infants with herpes simplex virus (hsv) and varicella-zoster infections. 32 there are several topical agents (0.15% ganciclovir ophthalmic gel, 0.1% iododeoxyuridine, or 1% trifluridine) that may be added to systemic antiviral regimen if there is eye involvement. 32 after parenteral therapy with acyclovir, it is recommended to give hsv suppressive regimen (300 mg/m 2 /dose po tid), which improves neurodevelopmental outcomes of infants with central nervous system involvement. 40 there is currently no dosing regimen for valacyclovir in infants younger than 3 months of age. 32 the aap committee on infectious diseases and the committee on fetus and newborn recently published an algorithm-based guideline on the evaluation and treatment of asymptomatic infants born to mothers with active herpes lesions 41 (http:// www.ncbi.nlm.nih.gov/pubmed/23359576). oral valganciclovir (16 mg/kg/dose po bid) is the drug of choice for infants with symptomatic congenital cytomegalovirus (cmv) disease with or without central nervous system involvement. 32, 42 the treatment of congenital cmv should be initiated in the first month of life. kimberlin and colleagues 42 concluded from the phase iii randomized double-blind placebo-controlled multinational study that 6 months of valganciclovir regimen for symptomatic congenital cmv disease significantly improves hearing and neurodevelopmental outcomes. there is significant improvement in language and receptive communication at 2 years of age. there was less grade 3 to 4 neutropenia at 6 weeks oral valganciclovir (w19%) compared with 6 weeks of iv ganciclovir (63%) reported previously. iv ganciclovir (6 mg/kg/dose bid) can be used initially for infants with symptomatic congenital cmv disease if oral valganciclovir is contraindicated due to extreme prematurity or nec. 32 the same dosing regimen is the treatment of choice for perinatally or postnatally acquired cmv disease associated encephalitis, hepatitis, pneumonitis, or persistent thrombocytopenia. oral oseltamivir (3 mg/kg/dose bid) remains the treatment of choice for term infants with influenza infections. 32, 43 oral suspension formulation is available (6 mg/ml) and should be offered to young infants with suspected or confirmed influenza infection regardless of severity because they are at higher risk for complications. 43 limited data are available for the weight-based dosing regimen for preterm infants using postmenstrual age (ie, gestational age plus chronologic age): less than 38 weeks postmenstrual age, 1 mg/kg/dose po bid 38 to 40 weeks, 1.5 mg/kg/dose po bid greater than 40 weeks, 3 mg/kg/dose po bid. there is currently no dosing regimen for inhalational zanamivir for young infants. suggested durations of antiviral therapy, prophylaxis, and suppressive regimen for congenital and perinatal or postnatally acquired viral infections adapted from 2014 nelson's pediatric antimicrobial therapy 32 and the aap committee on infectious diseases and the committee on fetus and newborn 41 are shown in table 3 . fig. 2 shows an extensive cutaneous aspergillosis on a preterm infant who was a poor surgical risk successfully treated with combination antifungal agents. fluconazole prophylaxis (6 mg/kg/d twice a week) may be indicated in high-risk infants with birth weight of less than 1000 g from institutions with high incidence of candidiasis (ie, above 10%). 32 fluconazole prophylaxis (25 mg/kg once weekly) may be offered to young infants younger than 4 months old on extracorporeal membrane oxygenation. suggested durations of antifungal treatment of candidiasis and aspergillosis adapted from 2014 nelson's pediatric antimicrobial therapy 32 are shown in table 4 . surgical interventions may be indicated for the source control of neonatal infections. in a single-center 20-year retrospective study, nec-associated blood stream infection (bsi) occurred within 3 days of nec diagnosis and was noted in approximately 43% (69 out of 158 infants with one episode of bsi). infants with nec-associated bsi had higher odds (adjusted odds ratio 3.51; 95% ci 1.98-6.24) of having surgical interventions compared with those without bsi. 44 it is of utmost importance to correspond with pediatric surgery regarding source control of infection if clinically indicated because nec-associated bsi had higher odds of death (adjusted odds ratio 2.88; 95% ci 1. 39-5.97 ). 44 the following includes disease-specific conditions that may require surgical interventions for adequate source control of infections if the infant is clinically stable. pediatric providers are encouraged to discuss with their surgical colleagues the following surgical treatment options 32 : early debridement of cutaneous lesions with disseminated aspergillosis surgical drainage of peritonitis with bowel rupture wound cleaning and debridement rapidly spreading cellulitis (s aureus), necrotizing fasciitis (group a or b streptococci), tetanus neonatorum surgical drainage of pus in osteomyelitis and suppurative arthritis thoracostomy drainage of empyema surgical drainage of breast abscess may be needed to minimize damage to breast tissue. surgical interventions for primary diseases in infants may also increase the risk for neonatal infections. higher rates of surgical site infection defined as superficial, deep, and organ infections within 30 days of surgical procedures were noted among infants following closure of gastroschisis. 45 it is important to closely monitor infants with surgical site infection because they require significantly longer hospital stay. 45 there are various measures that can be used, depending on the availability of local resources, to prevent neonatal infections. these include but are not limited to gbs prophylaxis, hand hygiene, immunization and immunoprophylaxis, asp, probiotics and prebiotics, and care bundles. iap is the only preventive strategy that substantially reduces the incidence of earlyonset gbs. 7, 46 the following are indications for iap: previous infant with invasive gbs disease gbs bacteriuria during the current pregnancy positive gbs vaginal-rectal screening (at 35-37 week gestation) except for cesarean delivery without labor or ruptured membrane unknown maternal gbs status with delivery at less than 37 weeks, rupture of membrane at or before 18 hours, or fever equal to or greater than 100.4 f (!38 c). adequate iap means receiving penicillin, ampicillin, or cefazolin for at least 4 hours before delivery. cefazolin may be used if with nonserious b-lactam allergy. if there is history of serious b-lactam allergy (anaphylaxis, angioedema, respiratory insufficiency, or urticarial rash) and if gbs isolate is susceptible, clindamycin may be used. otherwise, vancomycin is an alternative. because of high resistance rates, erythromycin is not recommended. the center for disease control and prevention has an extensive online resource on gbs for clinicians, including the algorithm-based guidance on secondary prevention of eos in newborns. 47 the web page also provides an application, prevent group b strep, which includes guidance on various patient scenarios in collaboration with different medical societies, such as the aap and the american college of obstetricians and gynecologists (http://www.cdc.gov/groupbstrep/guidelines/index.html). there is no doubt that hand hygiene remains the cornerstone in decreasing health care-associated infections in different hospital settings, including the nicu. in fact, there are various educational programs, multidisciplinary quality-improvement teams, and guidelines on the proven effectiveness of hand hygiene in decreasing infection; however, this is significantly affected by compliance. 48, 49 the center for disease control and prevention has a web site (http://www.cdc.gov/handhygiene/) containing resources for hand hygiene in health care settings including an application, iscrub, for monitoring hand hygiene compliance using an iphone or ipod touch. 50 thus, hand hygiene guidelines are effective in reducing infections only if we use it. soap and water is recommended for decontaminating visibly soiled hands by rubbing hands together vigorously for 15 seconds. 48, 49 alcohol-based gel or foam or an antiseptic soap may be used for routine hand hygiene if not grossly contaminated. 48, 49 hand hygiene compliance is improved if with available alcohol-based products at the infant's bedside. 48 antimicrobial-impregnated towelettes or wipes are considered alternatives but not substitutes for washing with soap and water or alcohol-based gel or foam. 49 the development of a safe and effective vaccine is arguably one of the greatest medical interventions in the last century. 51 hepatitis b vaccine is the only agent in the united states recommended to be administered at birth. the various brands available in the united states have an efficacy of 90% to 95% in preventing hepatitis b virus infection and disease. 52 additional information regarding recommended dosages of hepatitis b vaccines are available in the most recent edition of the red book 52 and in the annual publication from the advisory committee on immunization practices. 53 the first dose in the primary series of the subsequent vaccines (diphtheria and tetanus toxoids and acellular pertussis vaccine [dtap], haemophilus influenza type b, inactivated polio, pneumococcal, or rotavirus vaccine) can be administered at a minimum age of 6 weeks. 53 care givers at home should be advised on the importance of immunizing family members to protect infants who are too young to be vaccinated. this is called cocooning 54 and prevents vaccine-preventable diseases, such as pertussis and influenza, in young infants. educational materials on cocooning for parents and clinicians are available at the aap web site http://www2.aap.org/immunization/families/ cocooning.html. in october 2012, the use of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (tdap) during every pregnancy was recommended because of increasing cases of pertussis in young infants in the recent years. 55 the mother's protective antibodies against pertussis are short-lived and a dose of tdap in a previous pregnancy may not be protective to the infants of subsequent pregnancies. 56 young preterm infants (<1 year of age born at <29 weeks and 0-day gestation) should receive palivizumab during the rsv season for immunoprophylaxis. 57 five monthly doses of palivizumab at 15 mg/kg given intramuscularly will provide adequate protection for 6 months. other indications during the rsv season include preterm infants (<1 year of age born at <32 weeks and 0-day gestation) with chronic lung disease of prematurity who required greater than 21% oxygen for at least the first 28 days of life and young infants (<1 year of age) with certain hemodynamically significant heart disease. 57 clinicians should consult the most current guidelines or policy statement from the aap regarding palivizumab prophylaxis among young children at increased risk for hospitalization for rsv infection. injudicious use of antibiotics can alter the neonates' microflora that increases exposure and pressure that leads to antimicrobial resistance. the nicu milieu and interventions are permissive for the development of antibiotic-resistant organisms. 48, 58 the aap committee on fetus and newborn 48 have listed asp strategies that may be useful in the nicu setting based on the guideline from the infectious diseases society of america and the society for healthcare epidemiology of america (box 1). 59 there is some medical evidence supporting the use of probiotics in the prevention of nec in preterm infants. probiotic is an oral supplement containing sufficient amount of viable microorganisms that alters the host microflora with potential for health benefits. 60 a meta-analysis based on 9 randomized control trials involving approximately 1400 infants born before or at 37 weeks gestation and/or weighing less than or equal to 2500 g at birth showed that enteral use of probiotic significantly decreased the incidence of severe nec and mortality. 61 there were no severe adverse events or systemic infections directly related to the probiotics used were reported. the aap committee on nutrition, 60 however, cannot recommend the use of all probiotics in young infants until further studies are done to resolve problematic issues. they noted the large heterogeneity of the studies included in the review, the different mixture of probiotics used, and that the combinations of probiotics used in the studies are not available in the united states. further, there remains some gap in knowledge on which probiotic bacteria species to use, the microbial dose, as well as the duration of administration. in 2014, an updated review of the aforementioned meta-analysis of randomized controlled trials continues to support a change in practice of supplementing preterm infants with probiotics. the review provided similar results involving more than 5000 infants in whom probiotics significantly reduced severe nec and mortality. 62 however, the previously mentioned gap in knowledge remains, as well as the need for comparative studies. there is scarcity of medical evidence to recommend the addition of prebiotics such as oligosaccharides in infant formula. prebiotics are nondigestible food ingredients that occur naturally or as dietary supplements that enhance growth of probiotic bacteria such as bifidobacterium spp. 60 several studies had reported that the addition of prebiotics in infant formula significantly increased the bifidobacteria counts in their stool without adverse effects noted. however, clinical efficacy as well as cost-benefit analyses regarding the addition of oligosaccharides to infant formulas is lacking. 60 for infants, human milk remains the best source of naturally occurring prebiotics and probiotics, and immunoprotective compounds known to decrease the incidence of respiratory and gastrointestinal infections. 48, 60 nursery intensive care unit care bundles there are invasive procedures that may increase the infant's risk of health care-associated infections in the nicu setting. these infections include central line-associated bsis (clabsis), pneumonia, skin, and soft tissue infections; and, occasionally, vaccine-preventable diseases and outbreak of respiratory viral infections. care bundles are sets of interventions aimed at reducing health care-associated infections in the nicu. 48 the most common cause of clabsi 48 and los 8 are coagulase-negative staphylococci. several randomized clinical trials on the use of low-dose vancomycin in parenteral solutions in preterm infants did not show significant decrease in the length of stay and mortality. 48 there is an antibiotic-lock therapy done in neonates that significantly decrease clabsi however it was not powered to answer whether vancomycin resistance occurred. both are currently not recommended because of the lack of long-term efficacy evidence as well as concern for development of drug-resistant organisms. infection control intended to decrease clabsi in the nicu should include measures to decrease extraluminal and intravascular catheter-related infections. various techniques and guidelines in the prevention of clabsi in infants adapted from the aap committee on fetus and newborn are shown in box 2. 48 there are specific practices that may be adapted in the local setting for preventing vaccine-preventable diseases and outbreaks of respiratory viral infections. these include but are not limited to vaccination of health care providers against influenza and pertussis (tdap), visitation guidelines to screen ill or symptomatic visitors, and cohorting in cases of clustering of infections or in outbreak situations. 48 cohorting may only be possible if early screening procedures, such as the use of pcr-based assays, are in place if available in cases of clustering of respiratory viral infections. [14] [15] [16] further, appropriate isolation (eg, contact precautions for mrsa, droplet precautions for influenza, and airborne precautions for measles) should be observed if the infant is clean the umbilical insertion site using an antiseptic such as povidone-iodine before catheter insertion. avoid using topical antibiotic ointment or creams on insertion sites to prevent fungal infections and antimicrobial resistance. use low doses of heparin (0.25-1.0 u/ml) to the fluid infused through umbilical arterial catheter. remove umbilical catheters as soon as no longer needed or if signs of vascular insufficiency to the lower extremities (for umbilical artery access) are present; they may be replaced if malfunctioning. umbilical artery catheters should not be left in place for more than 5 days. umbilical venous catheters may be used up to 14 days if managed aseptically. colonized or infected with a pathogen requiring additional protection beyond standard precautions. 63 neonatal infections continue to cause morbidity and mortality in infants. gbs and e coli are the most common agents of eos, whereas coagulase-negative staphylococcus is the predominant cause for los. there is increasing recognition of respiratory viral infections contributing to ruling out sepsis in very young infants whose presentations are similar to bacterial infections. blood culture at birth and white blood cell with or without crp has been used in the algorithm-based guideline for the evaluation of asymptomatic term and preterm infants with risk factors for sepsis. ampicillin and gentamicin remains the cornerstone of initial antimicrobial regimen for neonatal infections. third-generation cephalosporins should be used judiciously. the use of antiviral (acyclovir, ganciclovir, valganciclovir, and oseltamivir) and antifungal (fluconazole, amphotericin b, and voriconazole) treatment and prophylactic regimens may reduce mortality and morbidity to specific viral and fungal disease in infants. there are various strategies, such as gbs prophylaxis, hand hygiene, immunization, and immunoprophylaxis, asp, probiotics, and prebiotics, and nicu care bundles, which may be used in preventing infections in infants. early onset neonatal sepsis: the burden of group b streptococcal and e. coli disease continues neonatal infectious diseases: evaluation of neonatal sepsis the many faces of b cells: from generation of antibodies to immune regulation neonatal innate tlr-mediated responses are distinct from those of adults innate immunity of the newborn: basic mechanisms and clinical correlates the neonatal adaptive immune system committee on fetus and newborn. management of neonates with suspected or proven early-onset bacterial sepsis antibiotic use and misuse in the neonatal intensive care unit the association of third-generation cephalosporin use and invasive candidiasis in extremely low birth-weight infants prolonged duration of initial empirical antibiotic treatment is associated with increased rates of necrotizing enterocolitis and death for extremely low birth weight infants prolonged initial empirical antibiotic treatment is associated with adverse outcomes in premature infants duration of empiric antibiotics for suspected early-onset sepsis in extremely low birth weight infants clinical utility of pcr for common viruses in acute respiratory illness viral respiratory tract infections in the neonatal intensive care unit: the virion-i study unrecognized viral respiratory tract infections in premature infants during their birth hospitalization: a prospective surveillance study in two neonatal intensive care units nosocomial rhinovirus infection in preterm infants principles and practice of pediatric infectious diseases successful medical treatment of cutaneous aspergillosis in a premature infant using liposomal amphotericin b, voriconazole and micafungin seventy-five years of neonatal sepsis at yale: 1928-2003 neonatal sepsis workups in infants >/52000 grams at birth: a population-based study principles and practice of pediatric infectious diseases culture negative sepsis and systemic inflammatory response syndrome in neonates utility of complete blood count and blood culture screening to diagnose neonatal sepsis in the asymptomatic at risk newborn effective biomarkers for diagnosis of neonatal sepsis a guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2013 recommendations by the infectious diseases society of america (idsa) and the american society for microbiology (asm)(a) laboratory medicine in neonatal sepsis and inflammation group b streptococcus st-17 and emerging st-1 clones by maldi-tof mass spectrometry capnocytophaga species and perinatal infections: case report and review of the literature bloodstream infections by malassezia and candida species in critical care patients first outbreak of pvl-positive nonmultiresistant mrsa in a neonatal icu in australia: comparison of maldi-tof and snp-plus-binary gene typing maldi-tof mass spectrometry and microsatellite markers to evaluate candida parapsilosis transmission in neonatal intensive care units nelson's pediatric antimicrobial therapy introduction to paediatric and perinatal drug therapy the therapeutic monitoring of antimicrobial agents in vitro postantibiotic effect and postantibiotic leukocyte enhancement of tobramycin once-daily versus multiple-daily dosing of aminoglycosides pharmacodynamic factors of antibiotic efficacy gentamicin vs cefotaxime for therapy of neonatal sepsis. relationship to drug resistance intravenous ceftriaxone and calcium in the neonate: assessing the risk for cardiopulmonary adverse events oral acyclovir suppression and neurodevelopment after neonatal herpes committee on infectious diseases, et al. guidance on management of asymptomatic neonates born to women with active genital herpes lesions six months versus six weeks of oral valganciclovir for infants with symptomatic congenital cytomegalovirus (cmv) disease with and without central nervous system (cns) involvement: results of a phase iii, randomized, double-blind, placebo-controlled, multinational study. 2013 id week. infectious disease society of america committee on infectious diseases. recommendations for prevention and control of influenza in children concurrent bloodstream infections in infants with necrotizing enterocolitis surgical site infections in infants admitted to the neonatal intensive care unit prevention of perinatal group b streptococcal disease-revised guidelines from cdc group b strep (gbs) strategies for prevention of health careassociated infections in the nicu healthcare infection control practices advisory committee, et al. guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hic-pac/shea/apic/idsa hand hygiene task force hygiene in healthcare settings how to communicate with vaccine-hesitant parents recommended childhood and adolescent immunization schedule-united states committee on practice and ambulatory medicine, et al. immunizing parents and other close family contacts in the pediatric office setting updated recommendations for use of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (tdap) in pregnant women-advisory committee on immunization practices (acip) importance of timing of maternal combined tetanus, diphtheria, and acellular pertussis (tdap) immunization and protection of young infants updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection neonatal sepsis: the gut connection infectious diseases society of america and the society for healthcare epidemiology of america guidelines for developing an institutional program to enhance antimicrobial stewardship probiotics and prebiotics in pediatrics probiotics for prevention of necrotizing enterocolitis in preterm infants probiotics for prevention of necrotizing enterocolitis in preterm infants transmission-based precautions key: cord-010233-772e35kx authors: monto, arnold s.; fendrick, a.mark; sarnes, matthew w. title: respiratory illness caused by picornavirus infection: a review of clinical outcomes date: 2002-01-03 journal: clin ther doi: 10.1016/s0149-2918(01)80133-8 sha: doc_id: 10233 cord_uid: 772e35kx background: respiratory infections result from invasion of the respiratory tract, mainly by viruses, and are the leading cause of acute morbidity in individuals of all ages worldwide. during peak season, picornaviruses cause 82% of all episodes of acute nasopharyngitis (the common cold), the most frequent manifestation of acute respiratory infection, and produce more restriction of activity and physician consultations annually than any other viral or bacterial source of respiratory illness. objective: this article reviews the clinical impact and outcomes of picornavirus-induced respiratory infections in specific populations at risk for complications. it also discusses the potential economic impact of the morbidity associated with picornavirus-induced respiratory infection. methods: relevant literature was identified through searches of medline, ovid, international pharmaceutical abstracts, and lexis-nexis. the search terms used were picornavirus, rhinovirus, enterovirus, viral respiratory infection, upper respiratory infection, disease burden, economic, cost, complications, asthma, copd, immunocompromised, elderly, otitis media, and sinusitis. additional publications were identified from the reference lists of the retrieved articles. conclusions: based on the clinical literature, picornavirus infections are associated with severe morbidity as well as considerable economic and societal costs. future research should focus on identifying patterns of illness and the costs associated with management of these infections. new treatments should be assessed not only in terms of their ability to produce the desired clinical outcome, but also in terms of their ability to reduce the burden of disease, decrease health care costs, and improve productivity. respiratory infections are recognized as the leading cause of acute morbidity in individuals of all ages worldwide. in developing countries, the morbidity associated with respiratory infections may be at least as severe as that in industrialized countries, and these infections are the leading cause of death in children under 5 years of age.',* the health interview survey quantifies illnesses that result in disability and/or physician consultation in the us population. based on this survey, the annual incidence of acute respiratory infections in the united states in 1996 was 79%, exceeding the rates of digestive conditions, injuries, and infective/parasitic conditions combined. these infections may result from invasion of the respiratory tract by bacteria, viruses, or, in rare cases, other infectious agents; however, viruses are the most frequently identified pathogens. the viral pathogens primarily associated with acute respiratory infections include picomaviruses, coronaviruses, adenoviruses, paraintluenza viruses, influenza viruses, and respiratory syncytial viruses' (figure 1 3) . picornaviruses are a large group of rna viruses. in terms of causing acute respiratory infection in humans, the most important picornaviruses are the rhinoviruses and enteroviruses. picornaviruses contribute significantly to the incidence of acute respiratory infections. in a study conducted during the autumn,5 picornaviruses were found to cause 82% of all episodes of acute nasopharyngitis, which is the most common manifestation of acute respiratory infection. individually, the incidence of acute nasopharyngitis in adults ranges from 2 to 4 cases per year and in children from 6 to 8 cases per year.6,7 although picornavirus-induced nasopharyngitis is often referred to as the common cold, the morbidity associated with these infections should not be trivialized. in fact, because rhinovirus-induced illnesses are so common, they produce more restriction of activity and physician consultations annually than respiratory illnesses caused by other viruses or bacteria.s this article reviews the clinical impact and outcomes associated with picomavirusinduced respiratory infection in specific populations at risk for complications secondary to these infections. it also discusses the potential economic impact of the morbidity associated with picornavirusinduced respiratory infections. relevant literature was identified through searches of medline, ovid, international pharmaceutical abstracts, and lexis-nexis. the search terms used were picnrnuvirus, rhinovirus, enterovirus, vim1 respiruto~ infection, upper respiratory infection, disease burden, economic, cost, complicutions, asthma, copd, immunocompromised, elderly, otitis media, and sinusitis. additional publications were identified from the reference lists of the retrieved articles. rhinovirus infections are associated with more pronounced clinical manifestations than respiratory infections of other viral etiologies. as many as 70% to 88% of human rhinovirus infections result in symptomatic respiratory episodes characterized by rhinorrhea, nasal obstruction, cough, and hoarseness." the median duration of illness in young adults is 7 days; however, symptoms may last up to 2 weeks in one quarter of cases, and the duration of illness may be even longer in children and the elderly.9 there is a peak in the incidence of rhinovirus infection in early fall and again in mid-to late springjo ( figure 24 ). younger children appear to be more susceptible to viral respiratory infections, as demonstrated by the fact that preschool children can have 5 to 9 respiratory infections per year. i ' in fact, during an outbreak of a new virus strain, over three fourths of the children in a nursery school were infected.12 also, secondary attack rates in family members have been observed to range from 25% to 70%, depending on the immune status of the exposed individual.r2 the high incidence of viral respiratory infections is most likely due to the large number of rhinovirus types and the fact that immunity is type specific and of short duration. for these reasons among others, the role of rhinoviruses has overshadowed that of enteroviruses, which also contribute to the morbidity of viral respiratory infections. it is estimated that enteroviruses infect between 10 and 15 million people annually. episodes of enterovirusinduced respiratory infection are seen throughout the year; however, the most prominent respiratory syndrome caused by these pathogens occurs in children during the summer months.'" these enteroviral infections typically result in a nonspecific febrile illness complicated by respiratory symptoms. i4 although viral respiratory infections due to picornaviruses appear to be selflimiting in most healthy adults, they are increasingly implicated in acute infectious exacerbations of illnesses in high-risk populations. technological advances such as polymerase chain-reaction testing have made virus identification more sensitive and readily available. this, in turn, has made it possible to identify specific patient populations (eg, the elderly, infants, and immunocompromised persons), who are particularly susceptible to picornavirus infection and in whom infectious episodes typically significantly increase the use of health care resources. 15 in addition, these advances in technology have confirmed the results of earlier studies, further demonstrating the significance of rhinoviruses in causing or predisposing patients to otitis media and sinusitis and exacerbating other chronic respiratory diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (copd). asthma is estimated to affect >17 million people (6% of the population) in the united states, resulting in over i00 million days of restricted activity and 470,000 hospitalizations annually. 16.17 in 1990, the estimated cost of treating asthma was $6.2 billion; 43% of those costs were associated with emergency department visits, hospitalizations, and death. is respiratory infections caused by picornaviruses are believed to be a major contributor to asthma exacerbations, particularly in children. asthmatic children appear to experience a significantly greater number of viral respiratory infections compared with nonasthmatic children. 19 up to 45% of acute asthma exacerbations in children are thought to be related to viral respiratory infections20 the most common cause of these infections are the rhinoviruses, which have been recovered from the respiratory tract at the onset of an asthma attack, suggesting that the virus may have been an initiating factor in the attack.*' several studies have identified rhinovirus as the pathogen most commonly associated with asthma in those >2 years of age.r5 rakes et a122 conducted a crosssectional study examining the prevalence of respiratory viruses in children presenting to the emergency department because of wheezing episodes. they found that respiratory viruses were present in 82% of children <2 years of age and in 83% of children between 2 and 16 years of age. rhinovirus was the predominant pathogen isolated in children >2 years of age (7 i%), indicating that the majority of wheezing episodes in this age group may be associated with rhinoviruses. the prevalence of rhinoviruses in asthmatic children was further investigated by johnston et a1.2" in a 13-month trial, these authors evaluated 108 children between the ages of 9 and 11 years with the respiratory symptoms of wheezing and/or cough and a decrease in peak expiratory flow rate (pefr). respiratory viruses, two thirds of which were rhinoviruses, were detected in 77% of patients. viral respiratory infections were implicated in 85% of episodes with upper respiratory symptoms and 80% of episodes with reductions in pefr. the median reduction in pefr was 81 l/min, and the median duration of decline in pefr was 14 days. based on their results, the authors reported that 80% to 85% of asthma exacerbations in schoolaged children appear to be associated with viral respiratory infections, primarily with rhinoviruses. in only 10% to 21% of adult asthmatic patients.*(' however, beasley et a124 found the prevalence of viral respiratory infections to be 36% in adults with severe asthma and 10% in those with mild exacerbation of asthma. nicholson et a125 conducted a longitudinal trial in asthmatic adults between the ages of 19 and 46 years, evaluating 3 15 episodes of respiratory illness over 2 years. the average duration of asthma in the patient population was -20 years. thirty-eight percent of the patients had previously been hospitalized for asthma. the investigators found that 80% of subjective asthma exacerbations occurred with symptomatic colds and 89% of symptomatic colds were associated with symptoms of asthma. these findings suggest that acute respiratory infections may be as commonly linked to exacerbations of asthma in adults as they are in children. in this study, 84 clinical episodes of respiratory illness were associated with objective evidence of an asthma exacerbation (decrease in pefr 250 l/min). sixty-three percent of laboratory-confirmed acute respiratory infections were found to be caused by rhinoviruses. of the 84 clinical episodes of respiratory illness associated with an objective asthma exacerbation, patients consulted a general practitioner in 49% of episodes, required oral steroids in 38%, required a nebulizer in 2 1 %, and were prescribed antibiotics in 33%. in addition to the clinical impact of rhinoviruses in asthma, their impact on medical resource use has also been demonstrated. teichtahl et al*" compared the incidence of respiratory infection in 79 patients admitted to the respiratory medical unit for acute asthma with that in 54 control subjects admitted to the surgical unit for elective surgery. twenty-nine (37%) of the patients who were admitted for asthma exacerbation had evidence of a respiratory infection, compared with 5 (9%) of the control group (p < 0.001). of the 23 asthma patients with confirmed evidence of a viral infection, 9 (39%) had rhinovirus infections. significant mortality resulting from viral respiratory infections is routinely observed in the elderly. although mortality resulting specifically from rhinoviral respiratory infections has not been demonstrated, considerable morbidity is associated with these infections. in a study by nicholson et a1, 26 533 adults aged between 60 and 90 years were monitored for episodes of viral upper respiratory tract infections. the annual incidence of viral respiratory infections was found to be 1.2 per patient. a total of 23 1 pathogens were identified in 2 11 (42%) of the 497 episodes for which specimens were available. of the pathogens identified, 121 (52%) were rhinoviruses. of the 96 (45%) patients in whom rhinovirus was the sole pathogen isolated, 60 (63%) patients had lower respiratory tract involvement. the median duration of illness in these patients was 16 days. of the 60 patients with lower respiratory tract involvement, 25 (42%) had to restrict their normal activities (eg, cleaning, grocery shopping), and 16 (27%) were bedridden. a total of 4 i (68%) patients sought medical attention, and antibiotics were prescribed for 76%.27 altogether, 3 patients were hospitalized, 1 of whom died from exacerbation of copd by rhinovirus infection. the authors reported that the overall burden of rhinovirus infection was greater than that of the viruses typically associated with significant morbidity and mortality, such as influenza. 26 a second study assessing the impact of rhinovirus infection on the elderly focused on an outbreak of respiratory illness among patients in a long-term care facility.'x specimens from the throat and nasopharynx of 67 patients were obtained and cultured. of the 67 cultures, 33 (49%) were positive for rhinovirus. each of the patients with rhinovirus-positive cultures had upper respiratory tract symptoms, 23 (70%) had systemic symptoms, 22 (67%) had gastrointestinal symptoms, and 1 i (33%) had lower respiratory tract symptoms. more severe disease progression was found in 17 (52%) rhinovirus-infected patients with concomitant copd. five of 17 (29%) patients with copd required pharmacologic bronchodilation, 2 (12%) had to be transferred out of the facility because of declining respiratory function. and 1 (6%) died of respiratory failure. these findings indicate that outbreaks of rhinovirus infections in nursing homes should be considered potentially serious. a study from the 1960s indicated that 2.0% of community respiratory diseases were complicated by otitis media and 0.5% by sinusitis.7 although such percentages may seem insignificant, the high frequency of respiratory infections means that large numbers of patients are affected. a conservative estimate places the inci-dence of viral respiratory infections in the united states at 0.7 infection per person per year." based on these data, an estimated 196 million people in the united states experience a viral respiratory infection each year, with -4 million of these infections complicated by otitis media and almost 1 million complicated by sinusitis. new technologies that permit more accurate identification of viral pathogens will probably reveal that the frequency of virally induced infections is even higher and that viral pathogens are a precursor in more cases of otitis media and sinusitis than originally estimated; however, further research is necessary. it has been estimated that >90% of patients with otitis media also have symptoms of upper respiratory tract infections that are probably the result of a primary viral infection.29 this correlation is supported by the fact that 40% to 50% of patients with otitis media have detectable virus in the nasopharynx, and 17% have detectable virus in the middle ear.29 viruses can also complicate the course of recovery for patients with otitis media. in 2 recent studies,"o,"' viruses in the middle ear have been implicated in the apparent failure of antibiotic treatment by complicating the response to treatment. in a study in 71 patients with combined viral and bacterial otitis media,"' the presence of rhinovirus was associated with a higher failure rate of antibiotic therapy than was respiratory syncytial virus, parainfluenza virus, or influenza virus. rhinovirus has been identified as a potential pathogen in -40% of cases of community-acquired sinusitis.32 although there is evidence that these viruses contribute to the development of sinusitis, their exact role remains undefined. in a study by turner et al,"" 34 healthy adults were experimentally infected with rhinovirus. on magnetic resonance imaging, changes were observed in the paranasal sinuses of 33% of subjects. the relationship between viral infections and sinusitis was further investigated in a study evaluating naturally acquired acute nasopharyngitis.'4 in this study, sinus abnormalities were detected by computed tomography in over 85% (29) of adult patients. after 2 weeks of observation, spontaneous resolution of these abnormalities occurred in almost 80% (27) in some patient populations. is although the role of picornaviruses in these more serious diseases of the lower respiratory tract cannot be determined definitively without more extensive sampling of the lower respiratory tract, recent studies have pointed to the importance of picornavirus infection in these disease processes in particular patient populations.'" infants with bronchopulmonary dysplasia are at a significantly increased risk for acute episodes of severe viral respiratory infection. in a study by daily, 35 27 of 41 (66%) preterm infants with bronchopulmonary dysplasia required rehospitalization, the most common cause being upper respiratory tract infection. whereas some studiesi0,i5 have indicated that respiratory syncytial virus is the pathogen most commonly isolated in these patients, oth-er@.s7 have demonstrated that rhinovirus makes a substantial contribution. in fact, because none of these studies used assays suitable for identifying rhinoviruses, the role of these agents may have been underestimated. chidekel et alx7 studied 40 patients (44 cases) with a history of bronchopulmonary dysplasia for an average of 16 months and identified 8 (18%) cases of severe lower respiratory tract infection that were associated with rhinoviruses. of the 7 patients in these 8 cases, 5 (7 1%) required hospitalization for a mean duration of 11 days, and 4 (57%) were admitted to the intensive care unit. patients with rhinoviral respiratory infections required additional long-term medical therapy, suggesting that rhinoviruses can have lasting clinical implications. patients with cystic fibrosis are also at increased risk for complications of viral respiratory infections. in fact, 18% to 38% of pulmonary exacerbations are preceded by viral respiratory infections. collinson et ais recorded 147 episodes of acute nasopharyngitis over 17 months in 37 children with cystic fibrosis aged between 2 months and 18 years, translating into an annual incidence of 2.7%. cultures were available for 1 19 of the 147 episodes, and the causative pathogen was identified as rhinovirus in 2 1 (18%) episodes. these viral respiratory infections were associated with acute decreases in pulmonary function, increased rates of disease progression, and a predisposition to bacterial pulmonary infection. lower respiratory tract infections are the most common complication of viral respiratory infection in immunocompromised patients.s9 in one study,"o the com-monly identified causes of viral respiratory infection in immunocompromised patients included cytomegalovirus, herpes simplex virus, and varicella-zoster virus; however, viruses such as the picornaviruses were also implicated. in this and other studies, it is not certain that the techniques used were capable of detecting rhinovirus and whether these viruses were accurately represented in the study findings. immunocompromised patients are at risk for developing serious or life-threatening disease as a result of a viral infection of the respiratory tract. in the study by rabella et a14" both rhinoviruses and enteroviruses were identified as viral pathogens in 785 immunosuppressed patients (ie, patients infected with hiv, bone marrow transplant recipients, organ transplant recipients, patients with hematologic malignancies) with a suspected respiratory infection between january 1991 and december 1995. picornavirus infections were associated with respiratory failure in 2 patients, 1 of them requiring mechanical ventilation and the other having a clinical course resulting in pneumonia. in a study conducted at the university of texas m.d. anderson cancer center (mdacc), " 130 cases of picornavirus infection were identified in adult patients with leukemia and bone marrow transplant recipients. more than 90% of the picornavirus infections tested were found to be caused by rhinoviruses. in several patients, the infection progressed to pneumonia that appeared to be bacterial or fungal in origin. however, some patients had a clinical course consistent with viral pneumonia and died of unexplained interstitial pneumonia. in a 5-year retrospective follow-up study of 23 blood and bone marrow transplant recipients who had symptoms of up-per respiratory tract infection,42 rhinovirus was identified as the causative pathogen in all patients. eight (35%) patients developed progressive pneumonia that resulted in death. autopsies on 5 of 6 patients demonstrated interstitial pneumonitis and/or changes of acute respiratory distress syndrome. the deaths were attributed to progressive viral pneumonia on the basis of histologic features and ante mortem isolation of rhinovirus from lower respiratory tract sites. in a prospective study conducted at mdacc4" an evaluation of adult bone marrow transplant recipients hospitalized for an acute viral respiratory infection demonstrated that 18% of the 2 17 identified infections were due to picornaviruses. of 12 patients in whom rhinovirus had been implicated as the causative pathogen, 7 (58%) developed complicating pneumonia, and 3 (25%) died as a result. findings from autopsies performed on 2 of the patients were consistent with progressive viral pneumonia. a third trial at mdacc investigating acute respiratory illnesses in hospitalized patients with leukemia found that 18% of viral infections were caused by picornaviruses. 41 together, the results of these studies demonstrate that immunocompromised hosts are at increased risk from serious picornavirus infections, which have the potential to result in death. although the economic impact of picornavirus infections is most pronounced in the at-risk populations described, these infections have a significant economic impact on the general population as well. picornavirus infection is manifested primarily as acute nasopharyngitis. according to estimates from the national center for health statistics3 >62 million cases of the common cold required medical attention and resulted in 148 million days of restricted activity in 1996. even more significant from the economic and productivity perspectives, acute nasopharyngitis caused -20 million lost workdays in adults aged 218 years and 21 million lost school days in children aged <18 years. also important from an employer's perspective is the fact that employees miss days of work not only when they themselves are ill but when they stay home to care for sick children. productivity is also diminished when employees come to work with a picornavirus-induced respiratory infection. the compromised quality of life associated with these infections in turn has a negative effect on productivity. furthermore, many of the medications currently used for the palliative treatment of respiratory symptoms cause drowsiness, which may further diminish efficiency and productivity in the workplace. finally, because respiratory picornavirus infections are easily transmitted to persons in proximity to infected individuals, other employees are likely to become infected, resulting in additional losses in productivity. because factors affecting productivity and quality of life are increasingly important in today's health care environment, further analyses are needed to better quantify the impact of respiratory picornavirus infections on the overall health care population. to date, the direct use of health care resources associated with viral respiratory illness in the general population has not been investigated comprehensively. the current literature focuses primarily on atrisk populations. these data indicate that use of emergency department and hospital services for exacerbations or compli-cations caused by picomavirus infections is the major factor in the economic impact of respiratory infections within such atrisk subpopulations as asthma patients, the elderly, high-risk patients with diseases of the lower respiratory tract, cystic fibrosis patients, and immunocompromised patients. patients with sinusitis or otitis media are an exception, with physician office visits and antibiotic use accounting for the majority of the economic impact. the economic impact of picornavirus infections is illustrated by findings that asthma exacerbations are linked to rhinovirus infections in 57% of children and 15% of adults hospitalized for asthma exacerbations.2",2" if these statistics are applied to the average 463,500 asthmarelated hospitalizations per year (34.6% of which involve children),ls rhinovirus infections are responsible for an estimated 136,800 hospitalizations annually. based on these figures and an average cost of $3000 per hospitalization for an asthma exacerbation, the cost of hospitalizations due to rhinovirus-induced asthma exacerbations would be ~$410 million annually. a prospective economic analysis of hospitalizations due to respiratory infections in the asthmatic population remains to be conducted. other markers of the economic impact of rhinoviral respiratory infections in the asthmatic population are not as well defined as the costs associated with hospitalization. factors such as the use of other health care resources (medications and physician office visits), productivity, quality of life, and work and school absenteeism have not been measured to a significant extent, and further evaluation of these variables will be crucial to establishing the true economic burden of viral respiratory infections in the asthmatic population. patients with picornavirus infections who are at high risk for complications because of age (infants, the elderly), lower respiratory tract involvement (patients with copd), or immune status (cystic fibrosis patients, immunocompromised patients) constitute additional populations in which hospital and emergency department use are the greatest contributors to overall costs. again, the literature described in this review illustrates a definite link between picornavirus infections and increased health care use in these populations; however, the overall patterns of resource use and costs of illness have not been defined. in addition to the significant morbidity associated with picornavirus infections in the clinical literature, these infections also have substantial economic and societal implications. because the magnitude of these implications is not yet well defined, future research should focus on identifying the patterns of illness and costs associated with the management of these infections. the ability to determine the costs associated with viral respiratory infections will be particularly important when evaluating new treatments, which should be viewed not only in terms of their ability to produce the desired clinical outcome, but also in terms of their ability to reduce the burden of disease, decrease health care costs, and improve productivity. acknowledgment support for this article was provided by viropharma inc, exton, pennsylvania. the clinical impact of human respiratory virus infections acute respiratory infection in children of developing countries: challenge of the 1990s current estimates from the national health interview survey, 1996. national center for health statistics rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes frequency and natural history of rhinovirus infections in adults during autumn rhinovirus infections in an industrial population. i. the occurrence of illness illness in the home: study of 25,000 illnesses in a group of cleveland families viral respiratory infections in the community: epidemiology, agents, and interventions principles and practice oj' infectious diseases the common cold respiratory disease in group day care acute respiratory illness in nursery school children: a longitudinal study of the occurrence of illness and respiratory viruses viral isolation rates during summer from children with acute upper respiratory tract disease and healthy children temporal and geographic patterns of isolates of nonpolio enterovirus in the united states rhinoviruses: important respiratory pathogens surveillance for asthma-united states 1960-1995 centers for disease control and prevention. forecasted state-specific estimates of self-reported asthma prevalence-united states an economic evaluation of asthma in the u.s greater frequency of viral respiratory infections in asthmatic children as compared with their nonasthmatic siblings the incidence of respiratory tract infection in adults requiring hospitalization for asthma viruses as precipitants of asthmatic attacks in children rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses community study of role of viral infections in exacerbations of asthma in l i year old children viral respiratory tract infection and exacerbations of asthma in adult patients respiratory viruses and exacerbations of asthma in adults acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study a rhinovirus outbreak among residents of a long-term care facility report of a workshop on respiratory viral infections: epidemiology, diagnosis, treatment, and prevention bacteriologic failure of amoxicillinclavulanate in treatment of acute otitis media caused by nontypeable haemophilus influenzae association of rhinovirus infection with poor bacteriologic outcome of bacterial-viral otitis media. c/in infecr dis hayden fg. detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-pcr physiologic abnormalities in the paranasal sinuses during experimental rhinovirus colds computed tomographic study of the common cold home oxygen therapy for infants with bronchodysplasia: growth and development rhinovirus infection associated with serious respiratory illness in patients with bronchopulmonary dysplasia effects of upper respiratory tract infections in patients with cystic fibrosis community respiratory virus infections in immunocompromised patients with cancer respiratory viral infections in immunocompetent and immunocompromised persons rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients conventional respiratory viruses recov community respiratory virus infections among hospitalized adult bone marrow transplant recipients key: cord-021966-5m21bsrw authors: shaw, alan r.; feinberg, mark b. title: vaccines date: 2009-05-15 journal: clinical immunology doi: 10.1016/b978-0-323-04404-2.10092-2 sha: doc_id: 21966 cord_uid: 5m21bsrw nan vaccines represent one of the most effective and cost-effective medical and public health achievements of all time. 1 worldwide, vaccination programs are currently estimated to save over 3 million lives each year. in addition to having such a major beneficial impact on vaccine-preventable disease morbidity and mortality, the direct and indirect impacts of vaccination programs translate into economic savings of many billions of dollars each year. in what is considered to be one of the most significant medical successes of all time, a collaborative and comprehensive vaccination campaign against smallpox resulted in the global eradication of the disease in 1979. 2 similarly, efforts to eradicate poliomyelitis have made tremendous progress in reducing the global disease burden, and will hopefully soon overcome certain residual societal and programmatic obstacles to provide the second successful example of elimination of a major health threat by vaccination. concerted global efforts to provide measles vaccine have resulted in the control and elimination of the disease in many countries, including substantial reductions in mortality in a number of developing countries where the residual disease burden is greatest. these and other examples provide clear evidence of the power of vaccines in favorably manipulating host immunity to confer dramatic public health benefits, at both the individual and population level. as vaccines are administered to healthy individuals (often to entire age cohorts or populations), to prevent diseases caused by infectious agents to which they might be exposed in the future, they differ in important ways from pharmacologic agents that are used to treat individuals in whom a disease process is already manifest (or who display predispositions to disease). for this reason, vaccines are unique in the way that they impact on societies and in the way that societal commitment to vaccination determines their ultimate impact. as a result, vaccination efforts provide an informative window on challenges that need to be successfully navigated at the interface between scientific opportunity and societal capacity and commitment. indeed, current limitations in realizing the full global potential of available vaccines relate more to existing inadequacies in health care financing and infrastructure (especially as they are manifest in developing countries), and the relative value that societies place on disease prevention, than they do to any inherent biological limitations of vaccines themselves. fortunately, recent acceleration of new vaccine introductions in developing countries through public and private initiatives to build immunization infrastructure and provide funding of vaccine purchase offers hope that vaccines will one day be equitably available to all who need them. 3 the importance of vaccines extends beyond their use as public health tools to include their role as drivers of immunologic discovery. the history of vaccine development is rich with immunologic insights that emerged from careful observations of how diseases spread in populations and how such spread differs in disease-naïve and experienced populations, as well as of how innovative experimental approaches revealed fundamental aspects of immune system function. the general concept of immunity induced by prior exposure to a disease (including its specificity and potential lifelong duration) was appreciated by the ancient greeks. use of the word 'immunity' itself dates to the 14th century when it was applied to describe the relative susceptibility and resistance of populations to plague. the subsequent successes of edward jenner and louis pasteur in the development of effective smallpox and fowl cholera immunization strategies, respectively, provided a foundation for modern immunology; pasteur himself coined the term 'vaccine' in recognition of jenner's use of vaccinia virus. jenner's smallpox immunization studies also provided early experimental support for the concept of immune memory. pasteur's efforts provided the first demonstration of the attenuation of pathogens by their propagation in culture (or by passage in nonnatural animal hosts), while robert koch demonstrated that killed pathogens could also engender immunity. the discovery of bacterial exotoxins by emile roux and alexandre yersin facilitated the discovery of antibodies and their potential use in passive immunotherapy with antitoxin antibodies by emil von behring and shibasaburo kitasato. these discoveries enabled the development of active immunization against diphtheria and tetanus using toxin-antitoxin mixtures. paul ehrlich's development of accurate methods for antibody quantitation made passive immunotherapy and active toxin-antitoxin immunization far more reliable and effective, and provided a stimulus for significant advances in immunologic theory. in each of these instances, vaccine development illuminated central mechanisms of immune system biology. vaccine development today has transitioned from an approach that was once largely empirical to one that is based on the hypothesis-driven application of techniques in molecular biology and immunology. evidence for this synergy can be seen in recent studies of vaccine-elicited immune responses to illuminate primary and memory t-and b-cell responses in humans, as well as the strong discovery stimulus provided by ongoing efforts to develop new vaccines for major infectious diseases for which vaccines are not currently available. vaccine development today faces a number of significant challenges. there exist tremendous public health needs to address major well-known pandemic diseases, including acquired immunodeficiency syndrome (aids), tuberculosis, and malaria, for which no vaccines currently exist and for which natural immunity does not provide a helpful guide for vaccine development. furthermore, there exists a need to confront effectively newly emerging and re-emerging diseases, ranging from the well-known, but constantly changing, threats from influenza pandemics to the appearance of previously unknown zoonotic infections such as the coronavirus that causes severe acute respiratory syndrome (sars). with changes in population density, mobility, and social constructs, along with alterations in the global climate, ecological circumstances, and the proximity of humans to animal reservoirs for previously confined infectious agents, the concept of new infectious agents entering human populations and spreading rapidly around the world is no longer novel. in confronting prevalent or newly emerging diseases, vaccines are looked to as the most promising line of defense. however, the speed at which new infectious disease threats have been shown to emerge and spread, and the fact that the pathogens that now need to be confronted may display tremendous genetic variability (e.g., human immunodeficiency virus (hiv)) or an identity that cannot be predicted in advance (e.g., avian influenza or agents like sars) places unprecedented demands on the vaccine development process. in addition to these new challenges, there remain unmet needs in the derivation of vaccines that can achieve the greatest public health benefit. these needs include the development of new ways to achieve more effective vaccine-elicited immune responses in neonates whose immune systems are immature (or are impacted by maternal antibodies) (chapter 32) and in the elderly whose immune system function may be waning as a result of immune senescence (chapter 33). fortunately, the scientific foundation provided by basic and applied immunology and the use of new methods for pathogen identification, antigen discovery, vaccine production, adjuvant development, and novel vector derivation afford important opportunities for vaccine development and additionally present the possibility of improving on natural immunity. success in vaccine development will be predicated on continuing the historical synergy between advances in vaccine technology and basic immunologic discovery. toward that end, this chapter focuses on preventive vaccines for infectious diseases and how they are developed. although current routine vaccine recommendations are reviewed, given the active state of new vaccine introduction and evolving vaccine recommendations, as well as differences in recommendations in different countries, readers are encouraged to refer to up-to-date national resources for the most current information. while vaccine approaches are being actively explored to modify beneficially malignant and immunologic diseases (autoimmunity and allergy), these are beyond the scope of the current discussion. n impact of vaccination programs n unlike other medical interventions, vaccines confer benefits to both individuals and populations. 4, 5 while individuals may be protected from infection or disease by vaccine-induced immune responses, decreasing the number of susceptible hosts in a population also helps break the chain of transmission that pathogens require to spread and persist in human populations by induction of 'herd immunity. ' the benefits of herd immunity depend on achieving sufficiently high immunization rates in a population to impact pathogen transmission dynamics (including the potential for extinction of ongoing interhost transmission). the requisite level of vaccination coverage of a population needed to compromise pathogen spread significantly varies between pathogens, and is influenced both by vaccine efficacy (and its duration) and by the reproductive characteristics and infectiousness of the pathogen. analysis of the impact of vaccination programs in the usa provides an example of the beneficial impact of vaccines when used routinely and when high coverage levels are achieved. 6 as shown in tables 92.1 and 92.2, vaccination programs in the usa dramatically decreased the annual morbidity of many vaccine-preventable diseases. in many instances, the disease burden from several vaccine-preventable diseases of childhood has been reduced by over 99% since vaccine introduction (e.g., diphtheria, tetanus, measles, mumps, rubella, and polio). the somewhat lower rate of decline of pertussis (the annual morbidity of which has been reduced by a nonetheless impressive 83%) relates to the limited duration of vaccine-induced immunity, which is estimated to wane within 5-10 years after childhood vaccination. it is anticipated that recent availability of pertussis booster vaccines for use in adolescents and adults will lead to significant further declines in pertussis morbidity. even for diseases targeted by vaccines that have been in widespread use for less time (< 10 years), impressive decreases in disease morbidity have been seen (e.g., varicella, hepatitis a, and pneumococcal disease). in a notable recent demonstration of the population benefits of vaccines, introduction of the 7-valent pneumococcal conjugate vaccine resulted in a decrease of 73% in disease morbidity in children under 5 years of age within the first 5 years of its introduction. interestingly, the rate of meningitis and bloodstream infections caused by antibiotic-resistant streptococcus pneumoniae also fell by 81% in this age group. in a striking related finding illustrating how vaccines can impact pathogen transmission dynamics, rates of antibiotic-resistant pneumococcal infections also declined by 49% in individuals over the age of 65 who had not received the vaccine. thus, direct protection by vaccination of children who represent a reservoir of infection provided, via herd immunity, significant indirect benefits to those who did not themselves receive the vaccine. in addition to their benefits in preventing disease morbidity and mortality, routine vaccination programs are also impressively cost-effective. evaluation in the usa of the impact of ten vaccines routinely given as part of the childhood immunization schedule (diphtheria, tetanus, pertussis, haemophilus influenzae b (hib), polio; measles, mumps, rubella, hepatitis b and varicella) found that more than 14 million cases of disease and more than 33 500 deaths were averted over the lifetime of the immunized birth cohort of children. 7 when the cost of the vaccination program was compared to the economic impact of diseases prevented, these vaccines alone are estimated to save nearly $10 billion each year. when including indirect economic benefits (such as the time parents take off from work to care for sick children), the annual savings to society exceed $40 billion. when 30 preventive services were ranked based on clinically preventable disease burden and cost-effectiveness, childhood immunization received the highest score. 8 progress in the development of new vaccines accelerated significantly towards the end of the 20th century, with the development of vaccines against diseases that were not previously preventable by vaccination, but also with the development of improved versions of existing vaccines. thus, the number of diseases that can be prevented by vaccines included in the us centers for disease control and prevention's (cdc) routine childhood and adolescent immunization schedules grew from seven in 1985 to 16 in 2007 (table 92 .3 and fig. 92.1) . moreover, in the past several years, new vaccines have been introduced for adolescents and young adults (e.g., pertussis booster (tdap), meningococcal conjugate, and human papillomavirus (hpv) vaccines), and older adults (e.g., tdap and zoster vaccines) have shown that the value of vaccines extends across the human lifespan (figs 92.1 and 92.2). new combination vaccines have been developed to increase the simplicity and acceptability of vaccination regimens, as well as to improve overall compliance with the recommended series of vaccines. such combinations include either those that contain multiple inactivated or recombinant antigens (such as a combination diphtheria, pertussis, tetanus, hib, and hepatitis b vaccine) or multiple live attenuated viruses (such as a combination measles, mumps, rubella, and varicella vaccine (mmrv)). the development of a combination vaccine is often more complicated than simply combining individual antigens, for when antigens are administered in combination, immunologic interference is sometimes seen. this necessitates titration of antigen combinations (and in the case of combinations of inactivated and/or recombinant antigens, adjuvant selection) to achieve immune responses that are not inferior to each of the antigens administered individually. despite their readily demonstrable public health impact, the value of vaccines is often not appreciated, for when vaccine programs are successful the diseases that they cause become less prevalent and may disappear. however, to prevent resurgence of an infectious disease that has been brought under control, vaccination programs need to be continued. the difficulties facing current efforts to eradicate poliomyelitis have demonstrated that failure to maintain high immunization coverage rates can lead to prompt re-emergence and spread of the disease. even in developed countries, maintenance of strong immunization programs with high degree of coverage is needed where infectious diseases can travel with remarkable speed -and do so even before the extent of spread is evident. n principles of immunization n the terms vaccination and immunization are often used interchangeably. however, vaccination specifically refers to efforts to induce protective immune responses by administration of a vaccine, whereas immunization more generically refers to interventions -either active or passive -that seek to confer immune protection. active immunization describes the induction of immune responses by administration of a specific antigen or antigens, while passive immunization involves the administration of exogenous immunologically active substances (historically, antibodies present in sera obtained from immune individuals or animals) to confer temporary protection from an infectious pathogen or toxin. although the approaches for passive immunization waned in the later half of the 20th century, the advent and increasing robustness of monoclonal antibody technology have led to a resurgence of interest in passive immunization. vaccines seek to engender immune responses similar to those that confer immunity to re-infection in individuals who experience (and survive) natural infection with a given pathogen. in lieu of formal demonstration of a specific type of antibody or cellular immune response that contributes to prevention or accelerated clearance of an infection, most often vaccine efficacy is demonstrated first in the course of a placebo-controlled trial. in some instances, specific immune effector mechanisms, such as a specific level or type of antibody response, can be identified that correlate with immune protection. in this case, the 'correlate of immunity' provides a benchmark against which similar vaccines can be compared. in the case of most inactivated vaccines, subunit vaccines, and recombinant vaccines that produce antibody responses, but generally meager cd8 t-cell responses, it is likely that humoral immune responses are the primary or sole protective immune mechanism. in the case of live attenuated vaccines that induce both cellular and humoral immune responses against the pathogen, it is likely that both arms of the immune system act in concert to confer immunity. however, the actual mechanisms of immune protection induced by either a natural infection or a vaccine are generally not understood in detail for many infectious diseases. similarly, although vaccines depend on the induction of immunologic memory, the magnitude, character, and duration of immune memory differ between vaccines, as can the actual mechanism of immune protection. for certain vaccines, such as those that protect against bacterial diseases induced via production of toxins (e.g., diphtheria or tetanus), protection induced by toxoid-based vaccines is clearly dependent on persistent antibody (igg) and memory b-cell responses, ensuring that sufficient antitoxin antibodies are present at the time of toxin exposure to inactivate and clear the toxin. in other cases, such as long-lived protection against hepatitis b, if sufficient levels of antibodies are achieved in the initial immunization period, even hosts who may with time lose detectable levels of antibody responses remain protected. 9 in this instance, given the relatively long incubation period of hepatitis b, memory antiviral b-cell responses induced by the vaccine can be activated, facilitating neutralization and clearance of the infection before clinical disease is manifest. although it is popularly believed that vaccines confer protection by inducing 'sterilizing immunity' -wherein an infectious agent is blocked from even infecting one cell in an exposed host -this is clearly not the case for a number of vaccines. for example, the inactivated poliovirus and live attenuated rotavirus vaccines do not prevent some degree of local replication of their pathogenic counterparts in the gastrointestinal tract of exposed hosts. however, they are both effective in preventing clinical disease. in the case of poliovirus vaccine, this is mediated by elicitation of antibody responses that block dissemination of the infection to the central nervous system; while in the case of rotavirus, as yet unidentified immune effectors limit local virus replication so that significant gastrointestinal damage does not occur following infection. 10, 11 the major types of vaccines licensed for use include live attenuated organisms, killed or inactivated organisms, subunit vaccines consisting of purified (or partially purified) components of an organism, and subunit vaccines produced by recombinant dna technologies. the use of live attenuated vaccines dates back to the early work of jenner and pasteur on smallpox and fowl cholera vaccines, respectively. 12, 13 the fundamental concept of live attenuated vaccines is to mimic the effective host immune responses that follow natural infections. most live attenuated vaccines currently in use were derived by propagation of initially pathogenic organisms in culture on cells from different (nonhuman) species, or at nonphysiologic temperatures, for prolonged periods. driving pathogen evolution in culture to select for variants adapted to growth in heterologous cell types ex vivo often leads to the derivation of pathogen variants that grow poorly in vivo in humans and are unable to cause clinical symptoms. vaccines developed via this approach include those used to prevent a number of viral and bacterial infections, including yellow fever, measles, mumps, rubella, polio (the 'sabin vaccine'), varicella-zoster (used both for the prevention of chickenpox and shingles) and rotavirus (one version of the available vaccines), tuberculosis, and cholera. more recent technologies being applied to live attenuated vaccine development include the application of reverse genetic strategies ( fig. 92 .3) and those involving genetic reassortment with attenuated viral variants, as have been used to develop polyvalent live attenuated vaccines against influenza and rotavirus ( fig. 92.4) . 10, 11 the live attenuated vaccines currently in use are highly efficacious (> 90%) and protection is frequently durable. the efficacy of many live attenuated vaccines likely reflects the ability of the attenuated vaccine to replicate within vaccinated hosts, and to expose the immune system to pathogen-derived antigens in a manner that closely resembles the nature, location, and effects of natural infection. because live attenuated vaccines replicate within immunized individuals, they can induce both cellular (cd4 and cd8) and humoral (b-cell) effector responses and immunologic memory. in addition, as the live attenuated vaccines likely activate the host innate system in a manner similar to their pathogenic parents, they provide inherent adjuvant effects in augmenting adaptive immune responses. a key consideration in the development of any live attenuated vaccine relates to the relative balance between the ability to induce sufficient immune responses in vivo to confer protection (often associated with level of preserved replicative ability in vivo), and the ability to cause symptoms (which may also relate to the extent of in vivo replication). as such, an effective but also safe and well-tolerated vaccine needs to strike a specific balance between level of attenuation and level of immunogenicity. in addition, depending on the nature and number of genetic mutations responsible for the attenuated phenotype, a potential risk of reversion to a pathogenic form exists for certain vaccines. for most live attenuated vaccines, this has not been observed to be a problem in clinical practice -likely because the attenuating mutations are sufficiently numerous or genetically stable. one vaccine where reversion to pathogenic form was seen involved specific components of the live attenuated oral poliovirus vaccine (opv; the 'sabin vaccine'). in this instance, vaccine reversion to wild-type was shown to lead rarely to cases of paralytic polio (approximately one case per million doses administered). 14 based on these observations and the elimination of endogenous polio transmission in many developed countries, the inactivated polio vaccine (ipv; the 'salk vaccine') was substituted for opv. however, in light of a favorable cost-benefit ratio, high degree of efficacy, and ease of administration, opv continues to be the mainstay of polio vaccination efforts in developing countries. the use of physical or chemical methods to kill or otherwise inactivate a pathogenic organism represents a second major approach to vaccine production. 15, 16 in most cases, treatment with chemical agents such as β-propiolactone and formaldehyde is used to eliminate pathogen infectivity. while this approach has the benefit of presenting most of a pathogen's antigenic repertoire to the immune system of the immunized host, it can only be used in instances where the inactivated pathogen does not possess constituents that would confer significant toxicity. vaccines based on killed pathogens are believed to exert their protective effects via elicitation of pathogen-neutralizing antibodies and the induction of memory b-cell responses (likely in concert with cd4 t-cell memory). however, because inactivated pathogens cannot accomplish de novo synthesis of pathogen-derived gene products in antigen-presenting cells (apcs), they do not typically induce cd8 t-cell responses (chapter 6). in addition, killed vaccines are generally less immunogenic than live attenuated vaccines. as a result, they are commonly administered with an adjuvant (most often alum: see section on adjuvants, below) to augment their immunogenicity. a number of viral and bacterial vaccines currently in use are killed/inactivated vaccines, including whole-cell bordetella pertussis vaccine and the influenza virus, rabies virus, and hepatitis a virus vaccines. a number of bacteria produce toxins that represent the major pathogenic components responsible for disease in infected humans. examples include corynebacterium diphtheriae and clostridium tetani. detoxified versions of these toxins are referred to as 'toxoids,' and represent the purified components of vaccines preventing diphtheria and tetanus, respectively. toxoids have historically been produced by chemical inactivation of toxins, but more recently, genetic inactivation via targeted mutagenesis has been employed. the acellular pertussis vaccine is also a purified subunit vaccine composed of a defined set of protein constituents prepared from cultured bordetella pertussis. the mechanism of immune protection conferred by purified subunit vaccines is the antibody response elicited by vaccination. antibodies directed against the capsular polysaccharides present on encapsulated bacteria also confer protective immunity in a number of important instances by inducing antibodies that exert opsonophagocytic effects (promoting phagocytosis of antibody-coated bacteria) and, in some instances, bactericidal effects. 17 initial successful vaccine efforts against streptococcus pneumoniae and neisseria meningitidis utilized purified preparations of capsular polysaccharides. although such purified polysaccharides can induce protective levels of antibody responses in adults, they are poorly immunogenic in children under 2 years of age (as a function of the relative immaturity of their immune systems). in addition, t-independent antibody responses elicited by purified capsular polysaccharides are less durable than those that are produced in the presence of cd4 t-cell help. as a means of both augmenting antibody responses against polysaccharide antigens in young children and facilitating their persistence, the development of conjugate vaccines represented an important advance. 18 in this approach, purified polysaccharides are chemically conjugated to a carrier protein (such as diphtheria toxoid or an outer-membrane protein complex (ompc) derived from n. meningitidis). the carrier protein augments cd4 t-cell helper responses to the polysaccharide antigens, and enables elicitation of durable protective antibody responses even in young children. polysaccharideconjugate vaccines have been produced that protect against haemophilus influenzae b, streptococcus pneumoniae, and n. meningitidis infections. if two such segmented viruses with different genetic characteristics are used to infect one cell, the progeny viruses from this mixed infection will carry a range of mixtures of the genes of the two parent viruses. using either genetic or immunologic screening methods, reassorted viruses carrying the precise gene composition of interest can be selected. this approach has recently been employed to generate live attenuated vaccines against rotavirus and influenza virus. the strategy for generation of the pentavalent bovine-human reassortment rotavirus vaccine is shown above. rotaviruses have a segmented double-stranded rna genome comprising 11 independent rna elements. the outer shell of the virus comprises two proteins vp4 and vp7 that are involved in cell binding and entry and that specify the viral serotype (p type for vp4 and g type for vp7). vp4 and vp7 also represent the targets of virus-neutralizing antibodies. the pentavalent bovine-human rotavirus vaccine was generated by a 'modified jennerian' approach in which the bovine rotavirus wc3 (which is attenuated in humans as a result of host range restriction) serves as the gene donor for the backbone on to which gene segments encoding four common human rotavirus g types (g1-4) as well as one very common p type (p8) (derived from individual rotavirus isolates) were reassorted via a process of cell co-infection and subsequent selection of the recombinant viruses with the desired composition of bovine and human gene segments. an analogous genetic reassortment approach has also been used to generate live attenuated influenza vaccines. in this instance, three attenuated 'cold-adapted' viral strains (two a types and one type b) are used in co-infections in tissue culture with recent circulating wild-type influenza strains to derive vaccine strains that include the two relevant hemagglutinin (ha) and neuraminidase (na)-encoding gene segments admixed with the six 'backbone' genes from the attenuated master donor virus for use in annual influenza vaccines. the first recombinant vaccine developed, the recombinant hepatitis b surface antigen (hbsag) prepared in yeast, was developed in hopes of avoiding safety concerns related to the plasma-derived hbsag vaccine. 19 the knowledge that immune sera could provide protection by passive immunization of naïve hosts, and that purified inactivated plasmaderived hbsag vaccine could elicit protective antibodies, laid the groundwork for development of this recombinant vaccine. 20 the recombinant vaccine, when combined with adjuvant (alum), elicits favorable immune responses, is highly efficacious and is well tolerated -all features that recombinant vaccines are now expected to deliver. the second recombinant vaccine developed targeted prevention of borrelia burgdorferi infection (the cause of lyme disease), and was based on a purified recombinant version of the ospa protein. this vaccine, although conferring some degree of efficacy, faced implementation challenges, and was not widely embraced. as a result, it was withdrawn from the market. more recently, recombinant technology-derived purified subunit vaccines have been developed that consist of virus-like particles (vlps) that self-assemble when the l1 protein of hpv is produced in isolation of other viral proteins ( fig. 92 .5). 21 the l1 protein is the target of virusneutralizing antibodies and vaccines consisting of a mixture of types 16 and 18 (the cause of ~70% of cases of cervical cancer) and 6 and 11 (the cause of ~90% of cases of genital warts) or of hpv types 16 and 18 alone have been shown to be highly efficacious and well tolerated. 22 interestingly, hpv vlps induce antibody responses that exceed those that follow natural hpv infections. 23 in light of these successes, and the power and versatility of recombinant antigen production methods, a major proportion of new vaccine development efforts involves the use of protein subunit vaccines produced by recombinant technologies. vaccines produced by this method are those that depend largely or exclusively on the induction of antibodies against individual or a selected subset of pathogen proteins. because a number of proteins produced in isolation by recombinant methods have been observed to elicit lower immune responses than do natural infections or live attenuated vaccines, the development and use of adjuvants to optimize recombinant vaccine immunogenicity represent an important parallel area for future exploration. n vaccine development and evaluation n as a necessary prelude to clinical evaluation of candidate vaccines in humans, extensive preclinical research and development activities are undertaken to establish that the vaccine candidate has the desired properties. toward this end, a number of key issues need to be addressed. first, animal studies must show that the vaccine candidate raises the desired type and magnitude of immune response against the infectious agent. second, the vaccine needs to protect animals against death or disease in an appropriate challenge model, when feasible. ideally, in the course of these studies, a specific type or level of immune response, referred to as a correlate of immune protection, can be identified. third, the vaccine should be relatively free of serious discernible toxicities and side effects in animals when administered by the route intended for humans. fourth, it is necessary to demonstrate that the vaccine can be produced in a consistent manner by a process that is consistent with the current good manufacturing practices (cgmp) process by which the first clinical trial materials will be produced (www.fda.gov/cber/gdlns/indcgmp.pdf ). bioengineered l1 proteins (5) l1 pentamer self-assembled virus-like particle in specific instances, vlps can be produced via a process of self-assembly of individual viral capsid proteins produced by recombinant dna methods in cell culture systems. this approach has a number of attractive aspects, including the ability to produce vlps that accurately display conformationally correct epitopes recognized by neutralizing antibodies and the absence of pathogen-derived nucleic acids. in addition, recombinant vlps have been employed to derive safe and effective vaccines for pathogens, such as hepatitis b virus (hbv) and human papillomavirus (hpv), that cannot be grown in culture (and are thus refractory to standard vaccine approaches of attenuation or inactivation). the generation of the vlps that comprises newly developed hpv vaccines is shown. the hpv l1 proteins (which represent the major capsid protein and target of virus-neutralizing, protective antibodies), derived from hpv types of interest (e.g., types 16, 18, 6, and 11) are produced via recombinant methods. under appropriate conditions, individual bioengineered l1 proteins first self-assemble into pentamers, and then into vlps that are comprised of 72 pentamers and that are almost identical, both morphologically and antigenically, to infectious hpv virus particles. vlps prepared from individual hpv types are then combined with specific adjuvants to prepare the final vaccine products. even before preclinical studies are completed, vaccine developers typically begin an initial dialog with regulatory authorities (such as the food and drug administration (fda) or the european medicines agency (emea)) to set expectations about what will be necessary and sufficient for advancement to clinical studies in humans (www.fda.gov/cber/ genetherapy/isct092506sh.pdf ). phase i studies primarily focus on detailed assessment of the safety and tolerability of a vaccine, but evaluation of its immunogenicity is also frequently conducted. generally, a phase i study includes fewer than 100 healthy volunteers divided unequally between those who receive vaccine or placebo (2 or 3 vaccinees per placebo recipient). phase i studies typically employ escalating doses of the candidate vaccine, with a dose range progressively increasing in steps of three-to fivefold often being used. blood samples are taken at prescribed intervals and analyzed for laboratory evidence of potential toxicity, as well as for evidence of vaccine-elicited immune responses. a phase i study is considered successful if it demonstrates that the candidate vaccine is well tolerated or identifies any immediate safety concerns that will need to be closely monitored in potential future clinical studies. ideally, phase i studies also provide an initial indication of the optimal dose level and number of doses required. a phase ii study typically includes several hundred to a few thousand volunteers (randomized between vaccine and placebo) and can assume two general design types. phase iia studies provide additional safety data on a larger number of individuals of the intended age who receive the intended vaccine dose (who are more representative of the general population intended for vaccine use than the very healthy individuals included in the phase i study), as well as provide additional data on vaccine immunogenicity. even larger phase iib studies can provide additional data on vaccine safety and immunogenicity in subjects generally representative of those for whom the vaccine might be recommended, but importantly, also provide the first opportunity to address to answer the question, 'does this vaccine work in humans?' the size of a phase iib study needed to detect a signal of vaccine efficacy depends on the attack rate of the infection being targeted by the vaccine. the development of new vaccines depends on the convergence of public health need, biological plausibility, and practical feasibility. vaccine development programs are influenced by multiple considerations, including: >> what are the major unmet medical and public health needs today? >> what is known about the natural history and pathogenic mechanisms of the infection of interest? >> is immunity to a given antigen associated with protection against disease following re-exposure in the context of natural infection? >> if natural immunity capable of preventing re-infection follows an initial infection with the pathogen, can a specific host immune effector mechanism (e.g., antibody, cd8 t-cell) be identified as the likely agent (or 'correlate') of immune protection? if so, can a threshold level of this specific immune correlate needed for protection from re-infection be defined? >> can the pathogen be grown in culture? if so, does the pathogen cause such a life-threatening disease that an attenuated version of the virus would face an impossible barrier for demonstration of safety? >> can a specific antigen (or antigens) be identified that represents the target of protective host immune responses? >> if the protective immune response is mediated by antibodies, can the target antigen (be it a protein or polysaccharide) be produced in scalable quantities in a form that mimics its native structure so that it can effectively elicit antibody responses that can block the key functional role(s) of the target molecule in the pathogen lifecycle or otherwise lead to the clearance of an incipient pathogen infection? >> having chosen an antigen and presentation system, what is the best way to produce it on a large scale? choices will be limited by the nature of the antigen and delivery system, but definition of an optimal system for producing the vaccine (prokaryotes like escherichia coli, or diverse eukaryotic hosts including yeast, insect cells, plants, or cultured plant cells, mammalian cells) is a central consideration. >> what is the most effective way to present the antigens of the pathogen of interest to the immune system? modern molecular biology and biochemistry have provided numerous options for vaccine immunogen presentation, including recombinant proteins (and recombinant virus-like particles (vlps)), synthetic proteins, protein-polysaccharide conjugates, and gene delivery systems (recombinant viral vectors, or dna vaccines) >> is the antigen of interest sufficiently immunogenic on its own, or is augmentation of the desired immune response by conjugation to a specific carrier or addition of an adjuvant necessary to elicit a sufficient and sufficiently durable immune response in individuals in the target population for vaccination? >> what types of potential safety concerns can be anticipated for the vaccine in question? >> what is the attack rate of the infection in the general population? if the infection occurs relatively rarely in an overall population, can a subset of the population be identified that has a higher risk of infection so as to accelerate the achievement of statistically significant protection? is this subset sufficiently similar to the rest of the population to enable extrapolation of the clinical results to the broader target population as a whole? >> what tests to evaluate vaccine immunogenicity will need to carried out on clinical samples obtained from participants in the clinical trials? will measurement of antibody titers, t-cell responses, pathogen presence and quantity, pathogen serotype, and any other parameter peculiar to the disease in question represent the primary criteria for vaccine effect? development and validation of theses tests represent an essential component for the feasibility and success of a vaccine clinical study phase ii studies also present the first opportunity to identify a potential laboratory immunological correlate of protection from disease -if nature and prior experience have not already done so. in order to do so, the placebo recipients in the phase ii trial must experience a sufficient number of cases of disease while vaccine recipients need to exhibit significant evidence of decreased risk of infection or disease. in addition, immunological measurements in the vaccinees need to capture the relevant protective immune responses (e.g., the type and level of antibody and/or cellular immune response that predict protection) and measure them with sufficient precision and reliability. if laboratory measurements of immunity correlate with vaccine protection, subsequent refinements of the vaccine, its adjuvant, its manufacturing process, or its regimen may be assessed by simple immunogenicity studies, rather than repeating efficacy studies. once efficacy is established for a vaccine, it is very difficult to carry out a double-blinded, placebo-controlled efficacy study. vaccines that have been shown to be immunogenic and well tolerated in phase ii studies can then advance to pivotal phase iii studies required for vaccine licensure by regulatory authorities. phase iii studies are intended to expand further the safety database in a larger number of individuals (who are representative of the specific populations for which the vaccine will ultimately be used), establish definitive evidence of protective efficacy, and to establish clinical consistency of the vaccine made by the process run in the facility intended for licensure and commercialization (www.fda.gov/cber/genetherapy/ isct092506jcr.htm). typically, phase iii studies include 10 000 or more subjects in a blinded, placebo-controlled design. this size trial allows the identification of less frequent safety events. it also provides an opportunity to capture data on health care utilization, cost, and impact of the vaccine on these parameters. as a new vaccine will ultimately be included in a vaccine program where multiple vaccines may be administered at the same time, it is also necessary to conduct concomitantuse studies. the developer of the new vaccine must show that the new vaccine does not impact on the immunogenicity of the existing vaccines, and that the existing vaccines do not impact on the immunogenicity of the new vaccine. in contrast to drugs, where licensure by the fda is the primary determinant of how a new product is implemented in medical practice, vaccine use in the usa includes an additional process that evaluates how best to employ a new vaccine to optimize its implementation and public health impact. the us cdc has responsibility for making recommendations about the use of licensed vaccines, and it relies on its advisory committee on immunization practices (acip) for guidance. the acip considers several aspects in addition to a vaccine's safety and efficacy, including the anticipated costeffectiveness and practical feasibility of potential alternative vaccine deployment strategies and consideration of how a new vaccine may be successfully implemented in clinical practice to achieve the greatest public health impact. once the cdc has received, reviewed, and accepted the recommendation of the acip, the recommendation is published in its final official form in morbidity and mortality weekly report (mmwr; www.cdc.gov/nip/publications/acip-list.htm). the recommended immunization schedule for children and adolescents ( fig. 92.1) is updated on an annual basis and can be accessed at www.cdc. gov/nip/recs/child-schedule.htm. the recommended adult immunization schedule (fig. 92. 2) is also updated on an annual basis and can be accessed at www.cdc.gov/nip/recs/adult-schedule.htm. the recommended adult immunization schedule includes information concerning use in special populations (such as health care workers and pregnant women) and individuals with specific conditions associated with altered or impaired immune function (such as individuals with congenital and acquired immunodeficiency syndromes, recipients of immunosuppressive therapies, malignancies, asplenia, liver disease, and renal disease). readers are encouraged to check to ensure that they are following current recommendations. pregnancy registries currently exist for four vaccines in the usa. health care professionals are encouraged to report exposures of pregnant women to the appropriate registry: hbv vaccine (800-670-6126), hpv vaccine (800-986-8999), meningococcal vaccine (800-822-2463), and varicella vaccine (800-986-8999). n vaccine safety n unlike drugs that are utilized to treat individuals suffering from a given disease state, vaccines are administered to normal, healthy infants, adolescents, and adults. consequently, standards for the safety and tolerability of vaccines are set at a very high level. when developing a new vaccine, a graded process of clinical studies is employed that involves increasingly larger numbers of volunteers and that typically progresses from individuals who are selected to be free of any identifiable health problems to those who are selected to be representative of the overall population for whom the vaccine is being developed. if phase i studies reveal no evidence of safety concerns and the desired evidence of immunogenicity, a major focus of the series of larger randomized double-blind, phase ii placebo-controlled studies that are then conducted is to explore the safety and tolerability of a vaccine in increasingly vulnerable populations (such as those who may have identified pre-existing health problems or asymptomatic abnormalities detected on screening laboratory studies). reflecting the importance of documenting the safety of a new vaccine, phase iii studies to assess the safety and efficacy of a new vaccine now typically involve large numbers of volunteers. indeed, as a result of needing to provide evidence for safety, it is now common to have the size of the phase iii trial be significantly larger than would be necessary to document vaccine efficacy. the ability of a study to identify an increased risk of any given adverse event with sufficient statistical power is directly related to the size of the population in the study. as a general rule, a study of 300-400 subjects is needed to measure the risk of an event that happens in one out of 100 individuals. for one in 1000, 3000-4000 subjects are needed. even in studies of this size, very rare events may not be identified, and if a specific safety concern exists substantially larger trials may be needed. the recent experience with the development of rotavirus vaccines provides an illustrative example of the importance placed on documenting vaccine safety. 24 rotavirus is an important cause of serious gastroenteritis in infants and young children, and the associated diarrhea and vomiting can lead to life-threatening dehydration. in developing countries where health care resources and effective rehydration options are limited, over 600 000 infants die of rotavirus gastroenteritis each year. 25 given the global importance of rotavirus gastroenteritis, the first licensure of an orally administered rotavirus vaccine in 1998 was a very welcome advance. however, as the vaccine entered routine pediatric practice, it was recognized that a low, but increased incidence of intestinal intussusception was seen after the first and second doses (with about one case of intussusception seen per 10 000 vaccinees.) 26 upon recognition of this association, the vaccine was withdrawn from the market. 27 with the evident public health need for a safe and effective rotavirus vaccine, it was hoped that alternative rotavirus vaccines then in development (both oral vaccines based either on a combination of bovine-human reassortant viruses (fig. 92.4) or an attenuated human rotavirus strain) might differ from the first licensed rotavirus vaccine and not result in an increased rate of intussusception. however, to demonstrate that these alternative rotavirus vaccines were safe, and that an increased risk of intussusception was not inherent to rotavirus vaccines as a class, very large-scale safety studies were required. toward this end, the safety of each of these vaccines was evaluated in studies involving about 70 000 infants -just to evaluate whether the rate of intussusception in vaccinees was discernibly increased compared to the normal background rates seen in the placebo recipients. 10, 11 fortunately, both vaccines were found to be well tolerated and no increase in intussusception was observed in vaccine as compared to placebo recipients. in light of the documented efficacy of these vaccines determined in earlier and significantly smaller phase iii trials, both have now been licensed in a number of countries. however, even with the large phase iii studies conducted for these newer rotavirus vaccines, they will still be studied in large postlicensure active surveillance safety studies and closely monitored in active and passive vaccine safety surveillance systems (see below). following vaccine licensure, safety is tracked via a number of means, including both active and passive surveillance studies of adverse events. active surveillance includes phase iv postmarketing studies of vaccine safety in larger populations in real-world use. formal postmarketing studies can include tens of thousands of individuals or more. an alternative type of postmarketing safety study is carried out by the us fda and the cdc within the context of the vaccine adverse event reporting system (vaers) database (www.vaers.hhs.gov or by telephone: 800-822-7967). the vaers database accepts spontaneous reports of adverse experiences from health care providers, patients, parents, vaccine manufacturers, and other sources. 28 the best use of the vaers database is to identify signals in a population that may appear following the introduction of a new vaccine. a newer vaccine safety surveillance system, known as the vaccine safety database (vsd), has been developed by the cdc in cooperation with seven large health maintenance organizations (hmos) around the usa. 29 the vsd contains the complete medical records of all the members from the participating hmos, and the information used to populate the database is entered by health care professionals using relatively consistent terminology, improving the quality, uniformity, and usefulness of the data. particularly important is that the vsd construct allows comprehensive epidemiological analyses to determine if the incidence rate of a specific adverse event is higher among vaccinees than nonvaccinees. in addition to vaers and the vsd, the cdc has also created a clinical immunization safety assessment network that reviews patterns of clinical syndromes that may follow vaccination. while the safety profile of a vaccine can be relatively well defined through the efforts described above, confidence in vaccination programs has often been challenged by public perceptions, either real or unsubstantiated, about vaccine safety. in some instances, specific vaccines have been associated with increased incidence of a specific adverse experience, such as the association between the first-generation rotavirus vaccine and an increased risk of intussusception following vaccination. however, a number of other safety concerns that have emerged are not supported by scientific evidence. an example of this can be found in the case of concerns about the association of whole-cell pertussis vaccines with permanent brain damage -concerns that were later shown to be unfounded. nevertheless, public concerns about the safety of the whole-cell pertussis vaccine resulted in decreased levels of pertussis vaccination coverage that were soon followed by epidemics of whooping cough in the uk and japan. 30 another example is the allegation that certain vaccines, such as the combination measles, mumps, rubella (mmr) vaccine, are associated with autism. highlighting how perceptions of temporal association can give rise to public concerns, mmr vaccines are generally given around 1 year of age, and autism is generally diagnosed in the second year of life. although the alleged causal association between mmr and autism has been refuted by thorough scientific analyses, reports in the popular media in the uk resulted in a dramatic drop in vaccination rates, followed by an increased rate of new infections. 31, 32 n vaccines not yet available n although an impressive armamentarium of vaccines is now available, safe and effective vaccines have yet to be developed for a number of very important infectious diseases. the reasons underlying the lack of effective vaccines for an array of important pathogens include biological considerations, safety concerns, and practical constraints. of these, the biological considerations are often the most important barrier. as discussed above, vaccines have been successfully developed for pathogens whose natural infections give rise to natural immunity wherein the infected host (at least those who survive initial infection) is no longer susceptible to re-infection (such as measles, yellow fever virus, or smallpox) or who experiences significantly less severe clinical sequelae upon re-infection (such as rotavirus). in instances where natural immunity follows natural infection, not only is a precedent for immune protection established, but the nature of protective host responses can be studied, providing a correlate of protection to guide vaccine development efforts. however, for many of the pathogens for which vaccines remain elusive, natural immunity does not follow natural infection. in the absence of natural immunity, not only is a precedent for successful immune containment lacking, but no potential correlates of protection are available to inform vaccine development. in some instances where natural immunity does not follow natural infection, persistent infections are established and maintained by active virus replication that cannot be controlled or cleared by host immune responses (such as hiv and hepatitis c). alternatively, other pathogens are able to persist in the host through establishment, via diverse mechanisms, of latent infections that are resistant to host immune clearance (such as tuberculosis or herpes viruses (such as herpes simplex virus (hsv) or epstein-barr virus (ebv))). in other instances, even when the host is cleared of an infection via drug vaccines not yet available treatment, the host remains susceptible to re-infection and disease in the future (such as malaria). although different pathogens have evolved diverse strategies for evasion of host immune responses -ranging from manifestation of tremendous genetic diversity and propensity for immune escape; to sequestration of critical structural domains that might be susceptible to antibody neutralization; to the utilization of specific mechanisms to evade host innate and adaptive immune effectors -the common end result is frustration of vaccine development. while failure of host clearance of an infection is a common theme underlying the lack of vaccines, additional obstacles to vaccine development include other immunologically related considerations as well as both practical and safety considerations. examples of immunologically related obstacles include instances where prior exposure to a given pathogen predisposes the host to more severe disease manifestations upon re-infection (as has been proposed in the case of dengue virus) or where earlier vaccine development efforts inadvertently lead to severe adverse events following infection with the targeted pathogen (such as respiratory syncytial virus (rsv )). in each of these cases, the adverse events that follow a secondary immune exposure are believed to be the result of immunopathologic responses that result from the nature of the immune response elicited by the initial exposure to pathogen-derived antigens (by either infection or vaccination). given that the mechanisms underlying these immunopathologic processes are incompletely understood, the development of vaccines that are highly immunogenic but not similarly inclined to elicit immunemediated adverse consequences represents a substantial challenge (especially given the very high expectations for vaccine safety). an additional immunologically related challenge relates to the observation that certain organisms encode antigens that resemble constituents of the human host. for example, in the case of neisseria meningitidis group b, the bacterial polysaccharide resembles those found on certain human cell lineages, thus raising concerns about whether polysaccharide-based vaccines successfully developed for group b n. meningitidis might yield undesirable autoimmune responses. 33 an additional distinct, but important, practical barrier to new vaccine development relates to the prevention of diseases that are threats to pregnant women or their offspring (where immunization of the pregnant woman might be able to protect the neonate). although a number of inactivated vaccines are either routinely recommended for use in pregnant women (e.g., inactivated influenza vaccine) or can be used in pregnant women for pre-or postexposure prophylaxis for those at risk of infection (e.g., inactivated hepatitis a vaccine and recombinant hbsag vaccine), the development of new vaccines specifically for use in pregnant women or the study of new vaccines in pregnant women has been impeded by concerns arising from potential litigation that might follow the appearance of a congenital abnormality in a child born to a mother who was vaccinated while pregnant. 34 given the 2-3% prevalence of congenital abnormalities, the practical difficulties in proving the safety of a new vaccine specifically administered to pregnant women, and the current litigious environment surrounding vaccines, the development of new vaccines to address important infections of pregnant women and their neonates (e.g., group b streptococcus: gbs) faces significant challenges. there remain a number of important infectious diseases for which no effective preventive vaccines exist. below, we list the major 'missing' vaccines, comment on why they are not yet available, and highlight the major approaches currently being explored to develop them. at the end of 2006, an estimated 40 million people were living with hiv infection, and in the preceding year approximately 4.5 million people became newly infected, and approximately 3 million individuals died of aids. as the most promising biomedical intervention to contain the aids pandemic, the development of an hiv vaccine is a top global health priority. yet, hiv infection represents a vexing challenge to vaccine development. 35, 36 hiv infection does not result in clearance of the virus due to a host immune response. following infection of target cells, the genome of hiv -a retrovirus -is transcribed into a dna copy via the action of reverse transcriptase. the newly formed dna copy of the hiv genome then integrates into the host cell chromosomes (referred to as a provirus) as a requisite step in the viral lifecycle. once integrated into the chromosome of an infected cell, the hiv provirus can alternatively be actively transcribed, leading to the synthesis of viral mrnas and subsequently to production of new virus particles, or it can remain in a transcriptionally silent, functionally latent state in a small percentage of infected cells. as infected cells harboring latent hiv proviruses do not produce hiv protein antigens, they cannot be recognized by host antiviral immune responses and can thereby persist undetected. upon subsequent activation of latently infected cells at some later time, viral rna transcription can be coincidently activated leading to production of progeny virions. as hiv targets activated cd4 t cells for infection and consequent depletion, the host's ability to mount both hiv-specific and non-hivspecific immune responses is progressively impaired. the ability of the host to clear hiv infection is further complicated by the extensive genetic diversity of virus populations that emerge, and progressively diverge, within infected individuals as a function of a replicative cycle that is accomplished by the inherently error-prone reverse transcriptase and the numerous cycles of replication that occur in infected individuals. as a result of these influences, genetically diverse populations of hiv variants are established in infected persons that facilitate the outgrowth of genetic variants that can escape from selective pressures -be they effective host cellular or humoral immune responses, or the inhibitory effects of antiretroviral drugs. 37 an extraordinary degree of genetic diversity is also manifest in the hiv variants seen in different individuals and in different geographic regions. as successful vaccines for other infectious agents have historically had to protect against pathogens exhibiting only limited genetic diversity, hiv represents an unprecedented challenge. as many successful vaccines protecting against viral infections are predicated on the induction of neutralizing antibody responses against the viral surface proteins that mediate attachment to and entry into target cells, significant efforts have focused on the potential of the hiv surface envelope (env) glycoprotein, gp120, to elicit infectionneutralizing antibodies. 38 unfortunately, hiv gp120 is highly resistant to the action of antibodies by virtue of its heavy glycosylation and its native conformation that shields functionally critical structural domains from antibody binding. as a result, candidate gp120-based vaccines have failed to elicit meaningful levels of neutralizing antibodies in immunized human volunteers and have not protected from hiv infection in two large phase iii studies. given the inability, to date, of candidate hiv env-based vaccines to elicit appreciable levels of neutralizing antibodies, current vaccine strategies are largely focused on the induction of cd8 cytotoxic t-cell responses against the more constrained and conserved antigens, such as vaccines not yet available gag, pol, and nef. it has been hypothesized that induction of high levels of hiv-specific cd8 t-cell responses prior to infection may not prevent infection, but may enable infected individuals to control virus replication better. should this hypothesis be valid, individuals immunized with such vaccines may exhibit lower levels of ongoing hiv replication, progress to aids more slowly, and potentially be less likely to transmit hiv infection to others. much of this work involves vectored gene delivery systems (such as adenoviral vectors, described below). however, the recently announced results of a phase iib 'test of concept study' 39 failed to demonstrate a beneficial effect on either prevention of infection or reduction of viral load among volunteers who received the vaccine despite the induction of appreciable levels of hiv-specific ctl responses by the recombinant adenovirus-based vaccine employed. while this study result does not, in and of itself, refute the 'ctl hypothesis', it represents a significant disappointment for the aids vaccine research effort, and raises important questions about the ability of vaccine-elicited cell mediated immune responses to favorably alter the outcome of hiv infection. 39a there are also efforts under way to utilize the recently solved three-dimensional structure of the hiv env glycoprotein to guide the derivation of nonnative structures that might serve as better immunogens to elicit broadly cross-reactive neutralizing antibodies. in addition, relatively conserved and functionally essential sequences of the extracellular domain of the hiv transmembrane env protein, gp41, are being explored as immunogens to elicit broadly neutralizing antibodies. malaria is the world's most common vector-borne disease -estimated to cause approximately 500 million clinical cases and 2 million deaths annually. 40, 41 the disease hits hardest in africa, and is especially severe in children under 5 years of age. in addition to direct morbidity and mortality, malaria is responsible for debilitating illness with enormous social and economic consequences. of the four malaria-associated protozoal species, plasmodium flaciparum and p. vivax represent the two major agents. these parasites have a three-stage lifecycle taking place both within the mosquito, and in the liver and blood of the infected host, and each cycle is largely distinct from the others from an immunological perspective. as a result of the multiple strategies for evasion of host immune response that the parasite has evolved, parasite replication proceeds at high levels despite active host immune responses. 42, 43 either as a result of these specific immune evasion strategies or the inability of the infected human host to mount immune responses that clear the parasite, prior infection does not protect an individual from repeated subsequent infections. although the severity of disease is often attenuated following repeated infection, the mechanism of disease modulation is incompletely understood, and the limited relative immunity engendered by prior infection is easily lost if an individual leaves a malaria-endemic region. as such, the limited impact and duration of host immune responses to malaria parasites suggest that any successful vaccine strategy will need to do far better than natural immune responses -a high bar for efforts to develop an effective vaccine. roughly two dozen antigens have been cloned and tested as potential vaccine immunogens, and with a few exceptions the results have been disappointing. 44 one antigen, the circumsporozoite antigen, presented as a fusion with hbsag (rts,s), has shown modest promise in human studies. 45 this vaccine is now undergoing larger-scale clinical efficacy testing to determine if the magnitude of protection would justify largescale implementation efforts. should 'proof of concept' be supported in these studies, but the absolute magnitude of efficacy be insufficient, future efforts will likely focus on the identification of an appropriate adjuvant to improve the magnitude and duration of immune responses. alternative approaches include immunization with irradiated or genetically attenuated sporozoites. 46 definition of novel antigens expressed at specific stages of the parasite lifecycle, and evaluation of combinations of multiple parasite antigens. mycobacterium tuberculosis is an intracellular mycobacterial pathogen that represents one of the world's most common and most serious infectious diseases. 47 over 2 billion people are believed to harbor latent m. tuberculosis infections, and approximately 8 million active cases of tuberculosis and over 2 million deaths occur each year. furthermore, the interface of hiv infection and its attendant immune system damage both increases the severity of m. tuberculosis infection and increases the infectiousness of infected individuals. the emergence and dissemination of m. tuberculosis isolates that are resistant to multiple antimicrobial drugs represent a growing public health threat. however, while the need for a vaccine to prevent tuberculosis is clear, a significant number of challenges face vaccine development efforts. 48 most individuals infected with m. tuberculosis can control the acute phase of mycobacterial replication, and mount vigorous innate and adaptive immune responses to the infection. however, the infection is often not cleared by the host's immune response, and the mycobacteria are able to persist and multiply within vacuoles inside macrophages. longterm latency is established in fibrotic cysts in the lung. the recrudescence and dissemination of m. tuberculosis occur at a later time in a number of infected individuals, likely as a result of waning host immune control. although the ability of m. tuberculosis to persist despite active innate and adaptive immune responses represents a major challenge to vaccine development, the fact that most individuals can contain (if not clear) m. tuberculosis infection suggests that a vaccine that can alter the course of the natural infection by limiting early dissemination and decreasing the risk of later recrudescence could provide major public health benefits. efforts to develop a vaccine against tuberculosis date back many decades. bacille calmette-guérin (better known as bcg), based on mycobacterium bovis, was first introduced in 1921. 49 currently, bcg is provided as a component of the routine expanded programme for immunization (epi) schedule and is administered to a significant majority of the world's children. although some protective efficacy (50-80%) has been reported against miliary infection and m. tuberculosis meningitis in children, conflicting results have been obtained in different studies regarding the ability of bcg to protect against pulmonary tuberculosis in adults. one explanation for the overall limited efficacy of bcg emerges from formal genome sequencing studies that have disclosed significant differences between m. tuberculosis and of the vaccine strain of bcg. the variability in the results of bcg efficacy studies in different populations and geographies may derive from variations in the geographic prevalence of cross-reactive mycobacterial species (that may themselves confer partial protection), or the fact that bcg vaccines used throughout the world do not represent a homogenous preparation -with the root strain of bcg having been widely distributed and passaged extensively under diverse conditions. vaccine efforts against tuberculosis have primarily focused on the evaluation of specific mycobacterial antigens (e.g., esat6, ag85, and hsp60) that have been tested as vaccines in animal models with variable success. 50, 51 some of these strategies are now being advanced into human clinical trials. an alternative strategy is based on improving the performance of the bcg vaccine by insertion of genes encoding specific potential protective antigens that it normally lacks. in addition, the development of auxotrophic mutants of m. tuberculosis is being explored as a potential immunogenic and specifically attenuated live vaccine. the determination of the sequence of the m. tuberculosis genome nearly a decade ago helped identify numerous previously unknown gene products, and increased the repertoire of antigens to be evaluated for their ability to induce protective immune responses. 52 the pathogen sequence is also being used to elucidate virulence determinants and thereby help guide efforts to attenuate m. tuberculosis rationally. together with influenza virus, rsv and piv account for a substantial majority of pediatric upper respiratory illness and consequent acute otitis media. a variety of influenza vaccines are licensed for pediatric use, but vaccines to prevent infection with the paromyxoviruses rsv and piv remain elusive. a significant impediment to vaccine development for rsv and piv traces back to unanticipated untoward results obtained in clinical studies of inactivated rsv vaccines in the early 1960s. 53 these early-generation rsv vaccines -based on cultured virus that had been inactivated with formalin -raised a potent antibody response in immunized children. however, on subsequent natural exposure to rsv, vaccine recipients exhibited more frequent and significantly more severe lower respiratory tract rsv infections than did unimmunized children. as a similar phenomenon was also seen with a formalin-inactivated measles vaccine in the same era, a common immunopathologic mechanism may be operative. 54 while the mechanism of exacerbation of rsv disease by the early inactivated vaccines is incompletely understood, it has been suggested that chemical inactivation of rsv and measles resulted in modification of a critical neutralizing structure on the surfaces of these viruses, thereby limiting the induction of the most potent neutralizing antibodies and favoring nonneutralizing and potentially immunopathologic antibody responses. (passive protection against rsv is available for premature infants in the form of monoclonal antibodies that target the rsv f protein (one of the viral envelope glycoproteins); certain anti-rsv antibody responses can clearly mediate protective as opposed to deleterious effects. 46 ) alternatively, or in addition, it has been proposed that inactivated rsv vaccines may have preferentially induced a th2-type immune response when a th1-type response may be needed to effect protection of the lower respiratory tract from rsv infection and damage. while excellent live attenuated measles vaccines have been developed, rsv and piv have so far resisted the approach used for measles and mumps (these are all members of the paramyxoviridae family of viruses). based on the successful precedent provided by the live attenuated measles vaccine, an attenuated or reverse genetics-engineered rsv is considered the most promising approach. however, stable attenuation of rsv has been difficult to achieve and vaccine safety concerns result in their cautious advancement through clinical evaluation. 55 effective vaccines for meningococcus types a, c, y, and w135 are available as straight capsular polysaccharides and as conjugated polysaccharides. 56 the group b polysaccharide shares chemical similarity with a shorter sugar found on the surface of neuronal tissue. 57 while it is possible to make highly immunogenic conjugates with the group b polysaccharide, theoretical concerns about cross-reactivity with self antigens has impeded the development of this type of vaccine. current work centers on a handful of relatively well-conserved surface proteins of meningococcus. gbs is a common component of the flora of the female genital tract, and transfer to the neonate is the cause of severe infections that are fatal or have serious sequelae. 58 short-course intrapartum antibiotics are recommended for culture-positive women, and this approach has cut the incidence of neonatal infections by about two-thirds, thus reducing somewhat the urgency of vaccine development. however, short-course antibiotics could ultimately drive the emergence of antibiotic-resistant gbs. candidate vaccines have been shown to elicit a protective response. 34 however, aside from a reduced market, the main impediment to development of a gbs vaccine is concern over vaccination of pregnant women or women of childbearing age. any birth defect might be attributed to the vaccine, and in a litiginous society, this would be problematic for a vaccine producer. prior to the advent of effective polymerase chain reaction methods for screening blood donations, hcv was a significant cause of transfusionrelated hepatitis. currently, transmission of hcv among the normal population is quite low; transmission among injection drug users remains high. hcv is another pathogen where infection does not typically result in an immune response that clears the infection. however, a minority of hcv patients do spontaneously clear their infection, suggesting that an appropriate immune response could do the job. current vaccine work is concentrated on vectored gene delivery vaccines, primarily adenoviruses, intended to raise antiviral cytotoxic t-cell responses. 59 with the exception of the live attenuated varicella-zoster virus (vzv) vaccine used for the primary prevention of chickenpox and reactivation of latent vzv infections (the cause of shingles and postherpetic neuralgia in older individuals), there are no other vaccines available for use in humans to prevent infection with members of the herpes virus family. 60 hsv types 1 and 2 cause recurrent vesicular eruptions "above or below the belt," respectively. like other herpes viruses, hsv infections are not cleared by the immune system and the virus can persist, remaining in a latent state that is functionally inaccessible to immune recognition and clearance. in addition, like other herpes viruses, hsv encodes a number of gene products that promote evasion of host immune responses. recent attempts to make hsv2 vaccines have used virus glycoproteins produced by recombinant dna methods. a recent clinical efficacy trial of this vaccine approach showed partial protection of women, but not men, who were seronegative for hsv1. 61 the reasons for this curious result are not clear, but efforts to develop this type of vaccine continue. in addition, a number of preclinical studies are exploring the ability of cell-mediated immune responses to hsv antigens induced by recombinant vaccine vectors (e.g., adenoviruses: see novel vaccine vectors, below) to prevent or ameliorate hsv infections. genetically engineered attenuated hsv variants have also been studied in experimental animal models. it is not clear when these new strategies may advance to clinical evaluation in humans. another herpes virus, cmv is a very common infection in humans, with 50-80% of individuals being infected by adulthood. cmv is a cause of severe infections in neonates, causing debilitating neurological sequelae. following initial infection, cmv persists in infected humans, despite the fact that anti-cmv antibodies are present and that a very sizeable proportion of the overall host cd4 and cd8 immune responses are specific for cmv antigens. ongoing virus persistence and replication in the face of active host immune responses are likely explained by cmv's sophisticated repertoire of host immune evasion functions (including those that inhibit antigen presentation mechanisms and immune effector responses). for these reasons, to be successful, vaccine development efforts will need to elicit immune responses that are significantly more effective than the quantitatively impressive, but functionally limited, immune responses that are generated in the course of natural cmv infections. live attenuated vaccines have been investigated sporadically since the 1970s. 62 an attenuated strain, the towne strain, showed some effect, but was judged to be insufficiently immunogenic. hybrids of the attenuated towne strain and the virulent toledo strain remain in development. recent work has included recombinant dna (rdna)-derived proteins (via either dna vaccine approaches or recombinant viral vectors, such as attenuated poxviral vectors). 63, 64 ebv is a herpes virus that represents the causative agent of infectious mononucleosis and is widespread among the human population. in concert with incompletely understood environmental (and perhaps additional host) factors, ebv is also etiologically associated with burkitt's lymphoma. the ability of ebv to establish persistent infections in humans (along with latent infections at the cellular level) despite readily detectable antiviral immune responses suggests that, like other herpes viruses, the development of effective ebv vaccine will likely be challenging. ebv vaccines have been in development since the 1980s with the coat protein, gp220/350, as the most common vaccine antigen studied. 65 dengue fever virus is a mosquito-borne flavivirus (the virus family that includes japanese encephalitis virus and yellow fever virus -for which successful vaccines exist). dengue virus is endemic in a substantial portion of tropical and subtropical areas and causes febrile disease as well as hemorrhagic fever. there are four distinct serotypes of dengue fever virus. prior infection with one serotype has been implicated in predisposing for more severe disease following infection with a second dengue fever virus serotype, although the evidence supporting this concept has been questioned and the underlying pathogenic mechanisms are incompletely understood. 66 the processes of vaccine development have changed significantly in recent years -a process facilitated by substantial improvements in understanding of human immune system function, as well as the advent of powerful new technologies for vaccine development. as a result of these advances, vaccine development is now commonly pursued in a hypothesis-driven manner and is a far less empiric pursuit than in the past. however, at the same time, the infectious diseases for which no effective vaccines currently exist represent more challenging targets than those diseases that have yielded to vaccine development efforts in the past. furthermore, global changes that influence the emergence and rate of spread of infectious diseases place unprecedented challenges on the productivity and pace of new vaccine development efforts. vaccines not yet available >> the challenge of responding rapidly and effectively, with powerful new technologies, to newly emerging infectionsincluding those that haven't been seen in humans before (e.g., severe acute respiratory syndrome (sars)) or for which novel antigenic variants are anticipated but cannot be predicted (e.g., pandemic influenza) >> maximizing the value of innovative new approaches while ensuring the safety of new vaccines so derived one hypothesis proposes that antibodies against the initial infecting serotype bind to the surface of virus particles of the novel infecting serotype, but do not neutralize the infection. in a process referred to as "immune enhancement" of infection, still infectious complexes of antibody virus particles are then envisioned to be preferentially taken up by cells of the reticuloendothelial system that represent primary target cells for virus replication. although the veracity of this hypothesis in not established, it does present certain theoretical concerns about what type of antibody responses will need to be induced by vaccines to exert beneficial rather than detrimental effects. the general belief is that a vaccine providing equivalent immunity against all four serotypes will be required. vaccines based on inactivated virus, engineered chimeric viruses based on the yellow fever virus vaccine platform, engineered deletion mutant viruses, and rdna-derived proteins are in various stages of development. 67 n new antigen discovery methods n historically, vaccine antigens were not discovered in the literal sense. rather, whole organisms were inactivated by either heat or chemistry or organisms were attenuated by forcing growth in nonphysiological conditions. the entire antigenic repertoire of the organism was delivered to the immune system. the isolation of tetanus, diphtheria, and pertussis toxins, along with chemical detoxification schemes, allowed the production of more refined vaccines. the isolation and purification of polysaccharide capsules from a range of important bacterial pathogens enabled the development of additional vaccines. with the advent of molecular biology in the late 1970s, a new set of tools allowed a more directed approach for the discovery of pathogen virulence factors, vaccine antigen discovery, and vaccine development. the tools of molecular biology enabled for the first time the development of vaccines against pathogens that could not be propagated in culture, including the successful development of recombinant hbv and hpv vaccines. development of these vaccines was enabled by clinical and animal model studies showing that antibodies directed against a specific viral target antigen (e.g., the hbv surface antigen or the hpv l1 protein) were implicated in protection. in addition, molecular biologic approaches enabled the derivation of fully recombinant vaccine antigens (such as those developed using a limited set of defined antigens of bordetella pertussis), including genetically modified versions of bacterial toxins that maintain their proper antigenic structures but are no longer toxic. however, for many of the pathogens for which vaccines do not currently exist, application of these recombinant dna technology-enabled strategies are insufficient due to incomplete understanding of the pathogen antigens that would elicit a protective host immune response. as such, the development of additional techniques to discover protective antigens was needed. fortunately, several important technological advances that facilitate discovery of previously unknown protective antigens from even very complex microorganisms have opened a new era in vaccine development. the earliest rdna technology-enabled methods of antigen discovery involved the expression of individual pathogen-derived gene products (or fragments thereof ) in bacterial hosts (typically escherichia coli) using rdna expression vectors. here, the genome of a pathogen is broken up, and the fragments are inserted into a plasmid or a viral vector, typically a lambda bacteriophage. 68 colonies, or plaques, are spread on a membrane, allowed to grow, and hopefully express the cloned gene fragments. the ability of these recombinant gene products (now isolated in individual colonies) to react with antibodies present in the serum of individuals who had recovered from infection with that pathogen could then be directly assessed. antibodies present in the immune sera are assessed for their ability to identify antigens by immunochemical reactivity. in this way, the entire genome of a given pathogen could be scanned for potential immunoreactivity. 69 such reactivity would both indicate the in vivo expression of that gene product, as well as document its antigenicity. however, additional studies are needed to demonstrate whether antibody responses against a newly defined antigen have any protective potential. to document the ability of an antigen to elicit protective immune responses, it is necessary to immunize an experimental animal (most commonly, mice) and then, following experimental pathogen challenge, evaluate infection outcomes in immunized versus nonimmunized animals. as this approach has most often been used to identify antigens recognized by host humoral responses, sera from animals immunized with a candidate antigen can then be transferred to a naïve host to provide evidence that the antibody response to the antigen represents the relevant agent of immune protection. more recently, as dna sequencing became more efficient and scaleable, determining the entire sequence of the genomes of viruses, bacteria, and parasites has become routine, 70, 71 allowing identification of previously unknown genes (and predicted gene products) that can be evaluated as vaccine immunogens. scanning the entire pathogen genome via specific computer analysis programs, genes that exhibit specific characteristics can be identified (e.g., predicted expression on the cell surface by virtue of possession of a leader sequence for secretion or membrane anchor sequences). 72 in addition, the relative conservation of the gene within the pathogen population can be determined by assessment of gene sequences from multiple distinct isolates. once potential vaccine antigens are identified, each candidate gene is expressed in an appropriate rdna system, and the protein product is tested in an animal model. 73 the first bacterial genome sequenced in its entirety was that of haemophilus influenzae, marking the beginning of a new approach to vaccine antigen discovery. 74 since this initial bacterial genome sequence determination, genomic sequencing of pathogens has advanced exponentially. over 300 bacterial genomes have now been sequenced, and hundreds more are currently in process. genome-based antigen discovery is being applied to a wide range of bacteria, including streptococci, pneumococci, staphylococci and chlamydia, as well as nonbacterial pathogens such as plasmodium falciparum. 75 an alternate, promising approach to novel antigen discovery has been built on technological advances in proteomics. 76, 77 these advances include development of high-resolution two-dimensional gel electrophoresis techniques and mass spectrometry methods that enable separation, identification, and purification of individual proteins from the complex mixture of proteins expressed by a pathogen. in proteomic analyses, a small culture of bacteria, preferably taken directly from an infected person, or otherwise grown in physiologically similar conditions, is subjected to physical or enzymatic treatment with specific proteases to generate peptide fragments that are then fractionated by a micro-high-performance liquid chromatography method and sequenced by molecular mass-by-mass spectrometry. an overlapping set of peptides of approximately 8-10 amino acids is sufficient to identify an antigen and provides the means to find the gene. although proteomic analysis is, in some ways, more involved than genomic analysis, it provides an important new approach to antigen identification, and offers a direct way to document that the specific protein identified is actually expressed by the pathogen (including, for example, demonstration that a protein of interest is expressed on the external surface of the pathogen). 78, 79 a combination of proteomic and serologic methods to select potential novel vaccine immunogens, called serological proteome analysis, or serpa, 80, 81 can be used to screen the pathogen proteome for expressed proteins that are recognized by antibodies present in sera obtained from individuals who have recovered from an infection with the pathogen. the proteomic approach to antigen discovery has been applied to identify novel vaccine candidates for a number of human pathogens, including helicobacter pylori, chlamydia pneumoniae, staphylococcus aureus, bacillus anthracis, haemophilus influenzae, and plasmodium falciparum. as in new genomic methods for antigen discovery, having identified a gene encoding a candidate antigen by proteomic methods, it is then necessary to show that an immune response of the desired type can be raised against the protein. in addition, it is necessary to show that immune responses elicited following immunization with the candidate antigen engender some degree of protection. often, pure protein antigens produced by recombinant methods are not very immunogenic. as such, many of the emerging recombinant vaccines so produced will likely require enhancement of their immunogenicity by means of an adjuvant. the term 'adjuvant' (derived from the latin adjuvare, to help) refers to any substance added as a component of a vaccine preparation -in addition to the vaccine antigens themselves -that improves the immunological response to the antigen. as such, 'adjuvant' is a catch-all term including a broad range of molecular entities that act via diverse -and, in a number of instances, yet to be elucidated -pathways. until recently, most adjuvants were derived empirically and the mechanisms by which they augmented immune responses were unknown . as a result, there were few, if any, principles available to guide the improvement of known adjuvants or the development of new ones. however, recent advances in understanding of the mechanisms by which dendritic cells sense the presence of pathogens and their constituents, and translate this information to shape the quantity, quality, and durability of host cellular and humoral adaptive immune responses, have transformed adjuvant discovery and optimization. what was once a process of trial and error now represents an area of hypothesis-driven research and mechanism-based discovery. a particularly promising advance emerged from the discovery that pathogen sensing by the innate immune system is mediated by recognition of specific pathogen-associated molecular patterns (pamps) by pathogen recognition receptors (prrs) such as the toll-like receptors (tlrs) that are expressed on dendritic cells (dcs) and other hematolymphoid and some epithelial cells 82 (chapter 3). the pathogen-derived pamps recognized by tlrs consist of structures that are found only in or on pathogens (including bacteria, viruses, and parasites) and are not part of normal vertebrate biology. following binding of a specific pamp to a specific prr, a specific cellular activation and response cascade is triggered that can directly confront an intruding pathogen and/or lead to the activation of specific host adaptive immune response mechanisms. these breakthroughs in basic immunology have been readily translated into what can now be considered the science of adjuvant biology. 83, 84 such progress has occurred at an especially opportune time as new vaccine development strategies have transitioned from traditional approaches using attenuated or killed pathogens to highly defined and purified recombinant proteins (so-called "subunit" vaccines) or nonreplicating vectored antigens. although these newer approaches are promis-ing from the perspective of vaccine safety and the opportunity they afford to design the structures of vaccine immunogens, recombinant or synthetic vaccines are often inherently less immunogenic than traditional vaccines based on attenuated live viruses or intact killed organisms. in the context of current vaccine development and regulatory approval processes, an adjuvant is developed as part of a vaccine, not as an independent product. consequently, there are currently no adjuvants licensed by regulatory authorities as stand-alone products. most contemporary efforts to develop novel adjuvants are focused on the targeted activation of tlrs that are expressed on specific cells critical for the generation of innate and adaptive host responses to specific pathogens. 85 a family consisting of 10 distinct tlrs has been identified to date in humans (chapter 3). tlrs are expressed in a number of innate immune cells, including dcs, macrophages, neutrophils, endothelial cells, and fibroblasts. given the importance of dcs as critical antigen-presenting cells, most studies of the biology of tlr signaling have focused on these cells. different tlrs are expressed on distinct subpopulations of dcs, and, depending on the tlr, in distinct cellular compartments. tlrs expressed on the surface of human myeloid dcs include tlr2 (which is heterodimerized with tlr 1 or 6), as well as tlrs 4, 5, 6, and 10 ( fig. 92.6 ), while these same cells express tlrs 3 and 8 within endoplasmic reticulum (er) and phagolysosomes. plasmacytoid dcs (pdcs) express tlr7 and 9 within er/phagolysosomes. the tlrs expressed on the cell surface are primarily activated by pamps encountered in the extracellular environment, while tlrs expressed in the er/phagolysosomes are activated by pamps (including viral pathogen-derived rna or dna) that tend to be routed through these endosomal compartments. activation of tlrs on innate immune cells leads to their production of specific cytokines, as well as their expression of co-stimulatory molecules, leading to induction of adaptive immune responses. given that different dcs express different tlrs, and that signaling via different tlrs results in the expression of a distinct pattern of cytokines, it is believed that activation of specific tlrs can variously favor the induction of th1-or th2-biased immune responses, or can differentially augment either direct or cross-presentation pathways for antigen presentation ( fig. 92.6 ). although most data on induction of specific types of immune responses by engagement of specific tlrs have emerged from murine studies (and have not yet been validated in humans), the ability to tailor an adjuvant preparation to achieve a desired type of immune response with a specific vaccine immunogen is a promising notion. naturally occurring ligands for tlrs include lipopolysaccharide (lps) from bacterial cell walls (recognized by tlr4), triacyl lipopeptides (recognized by tlrs 1 + 2), diacyl lipopeptides (recognized by tlrs 1 + 6), peptidoglycan (recognized by tlr2), flagellin (the monomer that makes up flagella, recognized by tlr5), singlestranded rna (recognized by tlr7), double-stranded rna (recognized by tlr3), and unmethylated dna containing the dinucleotide pair cpg 86 (recognized by tlr9). based on these insights, a variety of approaches to develop adjuvants predicated to activation of specific tlr pathways are being actively pursued. one interesting aspect of adjuvant development is how it is revealing the mechanisms of action of adjuvants that were originally identified via a process of trial and error, as well as delineating important aspects by which certain empirically derived vaccines are able to induce high-level, long-lasting immune responses. one illustrative example can be found in the case of complete freund's adjuvant (cfa), which has long served as the benchmark for laboratory studies of adjuvants. cfa is a mixed emulsion of mineral oil, mannide monooleate, and killed mycobacteria. however, it is far too reactogenic for use in humans, causing significant pain and abcesses at the site of injection -reactions that would be exacerbated if cfa were to be used repeatedly. an alternative preparation termed incomplete freund's adjuvant (ifa) lacks the mycobacterial component, but it too is associated with injection site reactions that are severe enough to limit its use to experimental therapeutic cancer vaccines. although cfa's toxicity precludes its use as a vaccine adjuvant in humans, many of its constituents (including liposaccharides, dna, and specific bacterial cell wall components) are now understood to exert their adjuvant effects on vaccine-induced immune responses via engagement of specific tlrs. similarly, the live attenuated mycobacterium bovis strain, bcg, long widely employed as a vaccine for the prevention of tuberculosis, includes cell wall, peptidoglycan, and dna components that activate specific tlrs. interestingly, the highly effective yellow fever vaccine 17d has been shown to activate multiple tlrs as part of its induction of antiviral immune responses. 87 it is quite likely that other live attenuated viruses that transiently replicate in immunized hosts also activate innate immune responses via engagement of tlrs. in yet another example involving a nonreplicating vaccine immunogen, one version of the hib polysaccharide conjugate vaccines now licensed for use in children for the prevention of invasive hib disease includes the meningococcal outer-membrane protein complex (ompc) as its protein carrier. ompc conjugates have favorable immunogenic properties that correlate with the ability of ompc to activate dcs via tlr2. 88 hundreds of different adjuvant formulations have been tested in animal models, and a few have been advanced into human studies. with a few α α (7) toll-receptor agonists. alum, the classical adjuvant most often used in vaccines in humans, includes a range of salts of aluminum precipitated under basic conditions, usually aluminum sulfate mixed with sodium or potassium hydroxide plus a variable amount of phosphate. 89 the relative proportions will determine the size, charge, and solubility of alum. the composition of alum used as an adjuvant varies in currently available vaccines and may influence vaccine immunogenicity. alum is utilized as an adjuvant in many of the currently available vaccines composed of inactivated toxins or recombinant proteins (live attenuated vaccines do not include alum or other adjuvants). alum serves two main purposes as an adjuvant. first, it acts as an antigen depot. vaccine antigens adsorb to alum and elute from it following injection into the host. second, alum acts a mild irritant, causing the recruitment of leukocytes necessary for generation of an immune response to the site of injection. adsorption of antigens on to alum routinely improves immunogenicity, particularly the antibody response. alum does not typically enhance cd8 t-cell responses. alum has been a component of many vaccines for decades and has an excellent safety record. as new adjuvants are developed, alum may remain as a component of combination adjuvant mixtures (as is the case with some newer adjuvants now approaching clinical use), or it may eventually be supplanted by other agents that more effectively provide favorable depot and local inflammatory responses to accentuate host immune responses. using lipids with polar head groups (e.g., triglycerides) and differing types of hydrophobic tails, one can form either micelles (spheres) or multilamellar sheets in aqueous environments. 90 under the right conditions, antigens can be incorporated into the spheres or between layers of the sheets, providing a potential slow-release depot system. immunopotentiators such as qs21 or detoxified lps derivatives (such as monophosphoryl lipid a (mpl)) may be added to the lipid mix. 45 iscoms are a proprietary form of liposomes made of cholesterol, saponins from quillaia bark (various members of the qs-x family of triterpene glycosides), and phospholipids that form cage-like structures into which antigens can be entrapped or intercalated. 86 iscom complexes may provide a depot function, as well as facilitate the delivery, uptake, and processing of vaccine immunogens by apcs. purified influenza virus hemagglutinin (ha) and neuraminidase mixed with phosphatidyl choline and phosphatidyl ethanolamine (polar lipids) will form empty particles that have the surface properties of influenza virus. adding an antigen in solution before mixing the lipids results in the incorporation of the antigen inside the particle. this provides a vehicle for delivering antigens to the interior of a cell, via the influenza ha membrane fusion process, thereby enabling antigen processing and presentation via both major histocompatibility complex (mhc) class 1 and 2 pathways. 91 emulsions numerous oil-in-water and water-in-oil emulsions have been tested as adjuvants. one such emulsion, mf59, is used in a licensed influenza vaccine. mf59 consists of squalane, a metabolizable shark oil and two surfactants, polyoxyethylene sorbitan monooleate and sorbitan trioleate, in an oil-in-water emulsion. 92 cytokines are host-produced immunomodulators that regulate immune cell action (chapter 10). several cytokines are being tested as potential vaccine adjuvants, including granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin-2 (il-2), and il-12. of the defined tlr agonists being explored as vaccine adjuvants, lps and its partially detoxified form, mpl, which activate tlr 4, have been most thoroughly explored in clinical trials. with evidence of enhanced ability to increase the percentage of individuals responding with protective antibody levels to hepatitis b as compared to a standard hepatitis b vaccine, one hepatitis b vaccine that employs an adjuvant formulation (termed as04) consisting of a combination of alum and mpl 93 has been licensed for use in high-risk individuals. in addition, a vaccine against hpv that uses the same adjuvant formulation may be licensed soon, and candidate hsv-1 and malaria vaccines currently being studied in latestage clinical trials also include this alum-mpl combination adjuvant. a wide variety of tlr9-specific agonists consisting of oligodeoxynucleotides containing unmethylated cpg motifs (cpg-odn) are being evaluated in preclinical studies. these cpg-odns resemble bacterial dna, modified to include a phosphorothioate backbone to increase their stability. two cpg-odn adjuvants have been evaluated in recent phase i and ii trials and shown to increase the timing and magnitude of induction of protective antibody levels, as well as the proportion of responding individuals, to recombinant hbsag vaccine as compared with the current commercially available version of the vaccine. 94 one of these cpg-odn adjuvants also elicits protective antibody responses in immunized hiv-infected individuals who had previously failed to respond to the hepatitis b vaccine. this approach is now being studied as a way of inducing protective immune responses to hepatitis b earlier after initiation of the vaccination regimen or with fewer doses of the vaccine. in addition to the cpg-odn-based tlr9 adjuvants described above, small chemical compounds with structures that resemble nucleic acid bases have been identified that activate tlr7 (e.g., imiquimod) or both tlr7 and 8 (e.g., resiquimod). these compounds are being evaluated as vaccine adjuvants in preclinical studies. flagellin, a tlr5 agonist, is also being explored as an adjuvant. recently, attention has also been focused on coupling, rather than mixing, tlr agonists to antigens. cpg oligonucleotides conjugated to antigens have been tested in preclinical studies of hepatitis b vaccines 95 and in human clinical trials for treatment of allergy. 96 ligands for tlr7/8 have been coupled to hiv antigens, 97 and the ligand for tlr5 (flagellin) has been fused to a variety of antigens. 98, 99 in some instances, coupling a tlr ligand to an antigen resulted in a substantial improvement of the immune response compared to mixtures -potentially the result of enabling the antigen and the tlr ligand to co-locate in the same dc compartments. numerous preclinical studies have confirmed that many natural and synthetic tlr agonists possess adjuvant activity. importantly, early human clinical trials of tlr-predicated adjuvants have supported the promise of this approach to mechanism-based strategies to augment vaccine immunogenicity. an important challenge is to define the most potent and best-tolerated variants, and to define rules by which activation of specific tlr pathways might translate into predictable augmentation of desired types of immune responses. it is hoped that general rules will emerge to suggest which of an increasing number of novel adjuvants in development performs best with which type of vaccine immunogen, and if results obtained with a specific type of immunogen-adjuvant combination can be extrapolated to predict the likelihood of enhanced immunogenicity with other vaccines. although beyond the reach of available experimental results, the ability to tailor, titrate, and otherwise optimize immune responses to vaccines by manipulation of specific tlr pathways appears a realistic future possibility. however, important challenges remain. in particular, a primary challenge for nextgeneration adjuvant development is finding a combination that retains immunopotentiating action while minimizing vaccine-associated adverse experiences. short-term adverse experiences, such as local injection site reactions, represent undesirable side effects that may disqualify candidate adjuvants early in clinical development. however, given that vaccines are administered to healthy people to prevent potential future infectious diseases, the potential for rarer adverse experiences (such as autoimmunity) that may only be manifest with much longer latency from the time of vaccine adjuvant administration will undoubtedly be important considerations for use in prophylactic vaccines. as induction of cell-mediated immune responses is considered an important component of vaccine strategies for many diseases for which no vaccines are currently available (many of which are caused by intracellular pathogens), there is a need to develop safe and readily scalable approaches to elicit durable cd8 t-cell responses in immunized humans. further, given the critical role that cd4 t cells play in induction, differentiation, and maintenance of cd8 t-cell responses, any such novel vaccine strategy will likely also require appropriate cd4 tcell responses. as elicitation of cd8 t-cell responses against a foreign antigen usually depends on the de novo expression of the antigen within a host cell and its subsequent processing and presentation via class i mhc pathways (chapter 6), most novel vaccine strategies are predicated on the need to achieve synthesis of pathogen-derived antigens within apcs of immunized human hosts. with an increasing appreciation of the role that cross-presentation pathways can play in elicitation of class i-restricted cd8 t-cell responses, such de novo antigen synthesis may not need to occur within apcs themselves (which may be an advantage for potential vaccine delivery strategies that do not directly target apcs). one of the many attractive attributes of effective live attenuated vaccines is their ability to recapitulate (to various degrees) many of the processes that lead to the generation of potent immune responses following natural infection. these processes include the fact that replication of all viruses depends on gaining access to host cells for genome replication and for the synthesis of essential components of virus particles that permit further propagation of the infection within and between hosts. one immunologic benefit of this requirement is that de novo synthesis of viral gene products within infected cells provides a key opportunity for viral antigen presentation (via mhc class i pathways) and elicitation of antiviral cellular immune responses. along with the processing and presentation of intact virus proteins via mhc class ii pathways leading to production of antiviral antibody responses, live attenuated viral vaccines have a strong track record for induction of broad cellular and humoral immune responses that likely both contribute to conferring protective immunity. however, despite their track record of success, it is likely that few, if any, new live attenuated viral vaccines will be derived in a manner that resembles previous successful efforts (e.g., the empiric derivation of live attenuated polio, yellow fever, or varicella-zoster vaccines). important reasons for this change include the desire for safe and well-characterized vaccines whose mechanisms of attenuation are defined and that can be monitored in the course of vaccine production and use. indeed, most of the recently developed live attenuated vaccines were derived using new approaches for genetic reassortment (fig. 92.4) wherein genome segments encoding pathogen-derived antigens of interest are recombined with a common set of genome segments that carry attenuating mutations (derived either by use of attenuating viral passage under specific conditions in cell culture (e.g., the cold-adapted influenza vaccine or use of a virus obtained from a nonhuman host that is itself inherently unable to replicate to high levels in humans, e.g., the reassortant rotavirus vaccine prepared via genetic reassortment between human and bovine rotavirus strains (fig. 92.4) 100 ). although such approaches have proven successful, they are limited in that they can only be applied to homologous viruses (e.g., those derived from the same virus type) whose genomes are segmented and capable of ready genetic reassortment in culture, or to viruses that can be manipulated by reverse genetics. in response to the desire to produce vaccines that can safely and reliably elicit desired immune responses, especially t-cell responses, several approaches are being explored to develop novel vector systems that permit the expression of pathogen-derived antigens. as many of these approaches are based on viruses distinct from the viral pathogen targeted for induction of host immune responses, the inserted pathogen-derived gene products are expressed via recombinant methods as heterologous antigens. alternatively, in nonviral expression systems, such as dna vaccines, the pathogen-derived antigen is expressed in isolation and does not depend on virus-mediated antigen delivery to apcs following host inoculation. collectively, such recombinant heterologous expression systems are commonly referred to as 'vaccine vectors.' in some instances, such recombinant vectors express only a specific antigen (in the case of dna vaccines or certain viral vectors, e.g., adenovirus), while in others both the inserted pathogen-derived antigens and antigens encoded by the viral vector 'backbone' are expressed (e.g., poxvirus vectors). most new approaches employ expression systems that are inherently nonreplicating (e.g., dna vaccines) or that employ viral vectors that can replicate at high levels in tissue culture but not in vivo (e.g., complemented adenovirus deletion variants or host range-restricted poxviruses). while numerous approaches are being pursued to develop novel vaccine vectors, they will all need to meet certain common criteria to emerge as vaccine approaches applicable for widespread use. in particular, any successful approach must be safe in healthy and immunodeficient humans (given their increased representation in the population as a result of hiv infection and therapeutic immunosuppression), desirably immunogenic (including in individuals who may have been previously exposed to the virus from which the vector was derived, e.g., vaccinia or adenovirus), and able to be produced in large quantities and in a stable manner. a limited number of vaccine vector approaches now being pursued are likely to meet these criteria. should successful approaches emerge, there will likely be interest in applying them for use in vaccines targeting diverse pathogens. thus, while definition of promising, broadly applicable, vaccine vector approaches may help simplify certain aspects of vaccine regulatory review and manufacture, they may also present challenges to prioritize use for specific applications should administration of a given vaccine vector on one occasion compromise or preclude successful administration at a later time. nevertheless, the development of novel vaccine vectors is laying essential groundwork for the development of next-generation vaccines. several novel vaccine vectors currently being studied in preclinical studies and human clinical trials are described below, all of which depend on the delivery and expression of a candidate pathogen-derived gene sequence. in a number of ways, dna vaccines represent the simplest approach to deliver pathogen-derived genes. viral vectors similarly serve to deliver pathogen gene sequences to host apcs, either directly or indirectly, but do so in a manner that depends on and takes advantage of the lifecycle and tropism of the virus that is being adapted to express the exogenous pathogen gene products. the ability of purified plasmid dna containing heterologous antigens expressed under the control of eukaryotic transcriptional regulatory and rna processing signals to elicit immune responses when injected into experimental animals was discovered serendipitously. 101 however, since the initial, quite surprising, description, the development of so-called dna vaccines has become an active area of preclinical and clinical vaccine development. 102 reasons for this enthusiasm include the attractive simplicity and facile preparation of vectors that encode only the defined antigen of interest (which can itself be manipulated via recombinant methods to assume a desired configuration), a reasonably straightforward method for vaccine production, and the inherent stability to temperature (which is much greater than most currently live or subunit vaccines). although the dna vector is most commonly injected intramuscularly, the generation of specific immune responses depends on the uptake of the vector dna by apcs followed by the expression, processing, and presentation of vector-encoded antigens. as tissue and tissue fluids present a hostile environment for purified dna, and the process of dna uptake by apcs appears to be relatively inefficient, much of the dose of injected dna is degraded before it can be reached by an apc that can initiate the desired immune response. most dna vaccine research has been pursued in mice, although studies have now been performed in numerous animal species. studies have usually utilized intramuscular injection of vaccine vector dna, but various intradermal and transdermal approaches have also been explored. murine studies have shown that administration of antigen-encoding plasmid dna can elicit appreciable cellular and humoral immune responses that may confer protection against experimental challenge. however, translation of these promising results in animal models to humans has proven frustrating. while dna vaccines have been generally well tolerated in immunized volunteers, in most human studies of dna vaccines, administration of even substantial quantities of dna vaccine vectors has elicited relatively low-level immune responses. it is not yet known whether these disappointing results reflects fundamental differences in the immunogenic behavior of dna vaccines in humans and mice, or the fact that the dna doses administered to humans do not match those administered to mice (dna per weight of the immunized host). given the substantial size differences between humans and mice, it would likely be impractical (for reasons of both vaccine supply and the actual process of administration of sufficiently high doses of dna) to administer the relative murine dose to humans. as such, a variety of approaches are being explored to prolong dna survival in tissue, promote more efficient targeting of dna to apcs, or to develop novel adjuvants that might specifically amplify immune responses to dna vaccines. 103, 104 dna vaccines are currently being used as candidate preventive vaccines for a wide variety of infectious diseases, including hiv, tuberculosis, malaria, and cmv. poxviruses represent the family of viruses that are physically the largest viruses and that possess the largest genomes. much of the poxvirus genome encodes gene products that serve to evade host immune responses, and that are not required for virus replication in tissue culture. further, facile techniques for the insertion and deletion of specific viral genes have been developed. the ability to accommodate sizeable foreign gene inserts is, in part, a function of the large size of the poxvirus genome (and the large packaging capacity of poxvirus virions). as a result of these favorable attributes, poxviruses have been utilized extensively in laboratory studies of virus biology, recombinant protein production, and host immune responses. 105 although poxviruses encode multiple gene products that help the virus evade host immune responses, they are, nevertheless, potent immunogens. studies of individuals immunized decades ago with vaccinia virus (in the course of smallpox eradication efforts) have shown that this virus induces long-lasting memory t-and b-cell immune responses. in contrast to most of the other viral vectors currently being developed, poxviruses can replicate readily in culture and do not require an engineered host cell to support propagation ex vivo. one important limitation of all poxvirus vectors developed to date is that, given the large size of the poxvirus genomes and the multitude of gene products they naturally express, even large inserts derived from foreign pathogens of interest will present only a minority of the vaccine vector antigens delivered to and recognized by the host immune system. to be effective, approaches to focus immune responses on the antigen of interest will need to be developed. toward this end, a variety of so-called 'prime-boost' 106 approaches are being explored where the host immune response is primed with one type of recombinant vaccine vector (such as a dna vaccine or adenovirus vector) and then boosted with subsequent delivery of poxvirus vectors encoding the same antigen. in this manner, immune responses to antigens of interest have been significantly augmented in a number of preclinical studies. vaccinia virus represents the prototypic vaccine vector. this virus is the same one that was employed in the successful smallpox eradication campaign, and has been used as a laboratory tool for decades. however, given current high expectations for vaccine safety, and the increased number of immunodeficient individuals present in the population (as a result of the new antigen discovery methods emergence of the hiv pandemic and the increased use of immunosuppressive therapies in clinical medicine) at high risk of serious adverse events, and potentially fatal consequences, from vaccinia immunization, the original vaccinia strains used in smallpox eradication efforts are not considered safe for general use. however, studies of vaccinia-based vaccine vectors have provided a strong basic foundation for research on other more highly attenuated poxvirus variants. modified vaccinia ankara (mva) is an attenuated vaccinia virus that was originally derived by prolonged passage of a vaccinia virus isolate on chicken embryo fibroblasts in culture. in the course of extensive passage in culture, a viral variant emerged that had fortuitously deleted large sections of the viral genome, including those that encode important poxvirus immune evasion genes and those that determine the ability of the virus to replicate on cells obtained from different animal species. specifically, while mva grows well on chicken cells, it cannot replicate in human cells in culture or in vivo, conferring an inherent safety feature. mva was safely administered to over 100 000 individuals at high risk of adverse consequence for vaccinia immunization toward the end of the smallpox eradication effort. more recently, it has garnered renewed interest as a potential safer smallpox vaccine in the wake of concerns about bioterrorism threats. even though mva cannot replicate in mammalian cells, the virus demonstrates favorable immunogenic properties. mva has been used as a vector expressing genes for a wide variety of genes, including hiv and malaria antigens either alone or, as described above, in 'prime-boost' regimens, where mva has been administered following initial priming immunizations with other vaccine vectors. a concerted effort is under way to improve further the performance of mva by manipulating a series of poxvirus genes that dampen the human immune response to the virus (and to any antigens inserted in it). 107 avipox is a family of poxviruses that infect birds and cause respiratory diseases in poultry. canarypox, a member of the avipox group, has been adapted as a vaccine vector. canarypox replicates well on avian cells in culture but cannot replicate on human cells in culture or in humans in vivo. as a result, canarypox, like mva, provides an interesting vector system with inherent safety features. 108 canarypox vectors carrying hiv genes have been tested in several clinical studies, either alone, or in 'prime-boost' regimens following priming with adenovirus vectors and recombinant protein antigens. in a large ongoing phase iii hiv vaccine trial, a recombinant canarypox vector is being used as a priming vector, followed by boosting with a recombinant version of the hiv gp120 surface env protein. to date, the results from human clinical trials of canarypox vectors have been disappointing, with only low-level specific immune responses generated in human volunteers. 109 adenoviruses, one of the common causes of upper respiratory and gastrointestinal infections, have seen extensive use in clinical trials and were one of the first gene therapy vectors. 110 most adenovirus vectors currently being studied in preclinical and clinical settings are disabled by deletion of the early e1 genes that are necessary for replication in an immunized host. most adenovirus vaccine vectors developed have used the well-characterized and readily produced adenovirus serotype 5 (ad5) as the vector 'backbone.' disabled adenovirus vectors are grown in cells that express the e1 genes artificially inserted into the cell's genome. 111 once these disabled vectors, encoding a heterologous pathogen-derived antigen of interest, enter a cell, the pathogen gene product is expressed, processed, and presented by host apcs. as adenoviruses can directly infect dendritic cells, they promise to provide efficient vaccine vectors. robust antibody and cd8 t-cell responses to heterologous antigen genes expressed by adenovirus vectors have been observed in preclinical animal models. furthermore, in early-phase human clinical trials, adenovirus vectors have been generally well tolerated, and proven to be the most effective of any recombinant vector system studied to date in eliciting high-level cd8 t-cell responses. the main potential drawback to widespread use of adenovirus vectors in humans is that, depending on the adenovirus type and the geographic location, variable levels of pre-existing immunity are found in humans as a result of prior naturally acquired adenovirus infections. high levels of antibody against the adenovirus vector might blunt the immunogenicity and efficacy of an adenovirus vector-based vaccine, but it remains to be seen if this will be a significant limitation. 112 should pre-existing immunity to adenovirus vectors derived from epidemiologically prevalent serotypes (e.g., ad5) limit vaccine immunogenicity, current efforts to develop vaccine vectors based on serotypes that are rare in human populations or novel adenovirus vectors specifically designed to avoid preexisting antibody responses may yield effective alternative approaches. adenovirus vectors are currently used in clinical trials for vaccines against hiv, 113 malaria, influenza, and a range of other pathogens. alphaviruses are rna viruses that cause zoonotic diseases, such as venezuelan equine encephalitis. these viruses do not normally circulate in humans, so immunity to these viruses is quite rare in humans. alphaviruses have a strategy for overexpressing the proteins that make up the virion by making a separate subgenomic rna specifically encoding these gene products. current recombinant alphavirus vaccine vector strategies take advantage of this subgenomic transcript, replacing the viral genes with selected genes for other antigens, but maintaining the signals for translation and protein production. in addition, through use of genetic complementation, it is possible to generate virus particles that only contain this heterologous antigen-encoding expression cassette. such virus particles can efficiently mediate infection of host cells, but because they lack other alphavirus genes needed for virus replication cannot spread beyond the initial target cell infected. 114, 115 alphavirus vectors rival the adenoviruses in efficiency of protein production in tissue culture and have induced robust antibody and t-cell responses in preclinical studies. 116 one current limitation of the alphavirus vector system is the difficulty of scaling the production system; however, this is a technical matter that should be addressable. in addition, ample safety data will be needed before widespread use of alphavirus vaccines achieves endorsement by regulatory authorities for use in healthy populations. adeno-associated viruses (aav ) belong to a family of single-stranded dna viruses (parvoviruses) that include the b19 parvovirus that causes a rash in children known as 'fifth disease' (measles, mumps, rubella, and varicella make up the first four). aav is transmitted in conjunction with adenovirus infection, and is not known to cause any significant disease. it is poorly immunogenic in the course of natural infections. 117 aav can integrate into the genome of the infected cell, usually in a particular place on chromosome 19, although integration does not appear to be efficient or site-specific when replicationdefective adenoviruses of the type being developed as vaccine vectors are used. the propensity for chromosomal integration and poor immune response to the virus made aav a good candidate for gene therapy; cells with an integrated viral genome could deliver a gene product for a long time without the immune system killing the infected cell. recently, efforts have been made to adapt replicationdefective aav as a vaccine vector. although encouraging results have been reported in preclinical studies, phase i studies in humans have demonstrated disappointing immunogenicity. n summary n the challenges to optimizing the full public health potential of existing vaccines largely relate to programmatic considerations. in contrast, the terrible impact of infectious diseases that cannot now be prevented by vaccines (such as the 'big three' killers of hiv, tuberculosis, and malaria) pose direct challenges to the scientific community to develop new generations of vaccines that overcome the largely biological obstacles to control and elimination of these diseases. the nature of the challenges posed by such pathogens necessitates that future vaccine efforts will not simply recapitulate the immune responses engendered by natural infection (as has been the premise of traditional vaccine development efforts), but rather, substantially improve upon them. as the development of vaccines to prevent infections with the so-far refractory pathogens is pursued, improved understanding of the immune response to natural infection, as well as delineation of the reasons why host immune responses fail either to clear incipient infections or prevent future new ones, will be essential. fortunately, early empiric approaches have now been replaced with hypothesis-driven strategies enabled by improved insight into the functioning of the human immune system, as well as new technologies, including higher-resolution tools to describe and quantitate pathogen-specific immune responses; novel methods for antigen discovery and targeted optimization of immunogenicity; the development of new, mechanism-based adjuvants; and the advent of innovative methods for vaccine vector-mediated antigen delivery. thus, although the challenges may be vexing, the scientific and technical foundations on which vaccine development efforts rest have never been stronger. n references n for many important infectious diseases for which no vaccines are currently available, successful derivation of effective vaccines will depend on improving upon natural immunity, especially in those instances where natural immunity does not follow natural infection (such as human immunodeficiency virus (hiv) and malaria) or where safety concerns limit the development of specific protective antigens (neisseria meningitidis group b). in addition, for other pathogens that typically manifest significant genetic (and antigenic) diversity (such as influenza, hiv, and bacteria such as streptococcus pneumoniae), a need exists to develop novel vaccines that can protect against a wide range of variants with a limited number of vaccine immunogens. towards these ends, a number of new approaches, enabled by new vaccine technologies, are being pursued, including: >> targeted alteration of protective antigens to increase their ability to elicit protective immune responses (e.g., efforts to alter the structure of the hiv env glycoproteins gp120 and gp41 so that they elicit higher-level, more potent, neutralizing antibody responses than their native counterparts) >> the development of synthetic consensus antigens able to elicit broader immune responses than would sequences obtained from individual pathogen isolates (e.g., efforts to develop consensus immunogens able to elicit cytotoxic t-lymphocyte (ctl) responses against genetically diverse hiv-1 variants) >> techniques for new antigen discovery to identify novel conserved antigens within otherwise genetically diverse pathogens (e.g., streptococcus pneumoniae) or those for which currently known protective antigens cannot be developed as vaccines (e.g., neisseria meningitidis group b) >> the use of novel adjuvants or vaccine vectors to enable generation of higher-level and/or more functional immune responses to pathogen antigens by vaccination than are seen following natural infection, and to enable high-level, fully functional memory immune responses to be activated at the time of initial infection (e.g., efforts to elicit high-level hivspecific or hepatitis c virus-specific ctls by recombinant viral vectors) >> use of novel methods to shift relative immunodominance of specific pathogen gene products to increase the immunogenicity of conserved antigens from otherwise diverse pathogen genomes that are typically poorly immunogenic in the course of natural infections (e.g., efforts to augment the antibody response to the influenza a virus m2 protein via the use of potent adjuvants or conjugation to immunogenic carrier proteins) ten great public health achievements -united states principles and lessons from the smallpox eradication programme advance market commitments for vaccines against neglected diseases: estimating costs and effectiveness mass vaccination: when and why immunisation and herd immunity standards for immunization practice for vaccines in children and adults economic evaluation of the 7-vaccine routine childhood immunization schedule in the united states priorities among recommended clinical preventive services development of an anti-core lipopolysaccharide vaccine for the prevention and treatment of sepsis safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis safety and efficacy of a pentavalent human-bovine (wc3) reassortant rotavirus vaccine the history of the smallpox vaccine grease, anthraxgate, and kennel cough: a revisionist history of early 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surveillance using large linked databases: opportunities, hazards and proposed guidelines permanent brain damage and pertussis vaccination: is the end of the saga in sight? absence of detectable measles virus genome sequence in blood of autistic children who have had their mmr vaccination during the routine childhood immunization schedule of uk the mmr vaccination and autism controversy in united kingdom 1998-2005: inevitable community outrage or a failure of risk communication? molecular mimicry of host structures by lipooligosaccharides of neisseria meningitidis: characterization of sialylated and nonsialylated lacto-n-neotetraose (galbeta1-4glcnacbeta1-3galbeta1-4glc) structures in lipooligosaccharides using monoclonal antibodies and specific lectins prospects for prevention of childhood infections by maternal immunization an hiv vaccine -evolving concepts prospects for an aids vaccine: three big questions, no easy answers update of the drug resistance mutations in hiv-1: 2004 hiv 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immunization. i. a field trial of two inactivated respiratory virus vaccines; an aqueous trivalent parainfluenza virus vaccine and an alum-precipitated respiratory syncytial virus vaccine sensitizing versus immunizing properties of inactivated measles vaccine recombinant bovine/ human parainfluenza virus type 3 (b/hpiv3) expressing the respiratory syncytial virus (rsv) g and f proteins can be used to achieve simultaneous mucosal immunization against rsv and hpiv3 the new meningococcal conjugate vaccine. a profile of its safety, efficacy, and indications for use antigenic similarities between brain components and bacteria causing meningitis. implications for vaccine development and pathogenesis prevention of perinatal group b streptococcal disease. revised guidelines from cdc current status of a hepatitis c vaccine: encouraging results but significant challenges ahead prophylactic vaccine strategies and the potential of therapeutic vaccines against herpes simplex virus clinical trials 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of the live attenuated yellow fever vaccine 17d haemophilus influenzae type bouter membrane protein complex glycoconjugate vaccine induces cytokine production by engaging human toll-like receptor 2 (tlr2) and requires the presence of tlr2 for optimal immunogenicity structure and properties of aluminum-containing adjuvants immunoactivating potential of multilamellar liposome vesicles (mlv) in murine popliteal lymph node (pln) test adjuvant and antigen delivery properties of virosomes safety and immunogenicity of nonadjuvanted and mf59-adjuvanted influenza a/h9n2 vaccine preparations new hepatitis b vaccine formulated with an improved adjuvant system cpg 7909 adjuvant improves hepatitis b virus vaccine seroprotection in antiretroviral-treated hivinfected adults testing of cpg-optimized protein and dna vaccines against the hepatitis b virus in chimpanzees for immunogenicity and protection from challenge immunostimulatory sequences of dna and conjugates in the treatment of allergic 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alphaviruses as vectors for anti-tumour and anti-microbial immunotherapy immune responses to adeno-associated virus and its recombinant vectors key: cord-009507-l74c9x0n authors: singh, amandeep; moayedi, siamak title: clinicopathological conference: fever, productive cough, and tachycardia in a 22‐year‐old asian male date: 2008-01-08 journal: acad emerg med doi: 10.1111/j.1553-2712.2004.tb01429.x sha: doc_id: 9507 cord_uid: l74c9x0n nan were appreciated. his abdomen was soft, nontender, and nondistended, with normal bowel sounds. no abdominal masses were palpated. he had no peripheral edema. he was alert and oriented, without any focal neurologic deficits. his skin was mildly diaphoretic, without any rashes or lesions. upon arrival to the ed, the patient was triaged to a telemetry bed. intravenous (iv) access was established and an electrocardiogram (ecg) was obtained ( figure 1) . a 1-l bolus of iv normal saline was started, and appropriate blood work was collected. a portable chest radiography (figure 2 ) was obtained. after reviewing the radiograph, a bedside echocardiogram was requested ( figure 3) . laboratory values returned as follows: sodium, 134 meq/l; potassium, 3.7 meq/l; chloride, 99 meq/l; bicarbonate, 22 meq/l; bun, 10 mg/dl; creatinine, 1 mg/dl; glucose, 98 mg/dl. white blood cell count was 5.2 (310 9 /l), hemoglobin was 12.6 g/dl, and platelets were 393 (310 9 /l). creatine kinase was 141 u/l with a normal mb fraction, and troponin i was .2 ng/ml. additionally, blood cultures were pending, and a urine drug screen was negative. the patient's heart rate remained around 150 beats/ min despite the iv fluid bolus. after reviewing the echocardiogram, appropriate consultation was made and the patient was admitted to the intensive care unit. a 22-year-old man who recently entered the united states from indonesia presented to the ed with four weeks of intermittent fever and a productive cough. his pcp saw him three weeks earlier and treated him with cephalexin for seven days, but his symptoms persisted despite treatment. upon presentation to the ed, he was found to have a heart rate of 150 beats/min, a temperature of 100.78f, and a pulse oximetry reading of 97% on room air. his physical examination revealed a well-developed and wellnourished male in mild respiratory distress, with bibasilar pulmonary rales, distant heart sounds, and mild diaphoresis on examination. no dermatologic, ophthalmic, gastrointestinal, genitourinary, musculoskeletal, or neurologic signs or symptoms were reported. his electrolytes, renal function, and cardiac markers were normal, as well as his leukocyte count and hemoglobin. no information regarding his prior immunization status was reported. a chest radiography ( figure 2 ) was ordered, presumably to see if an infiltrate was present, and serendipitously showed enlargement of the cardiopericardial silhouette. additionally, a 12-lead ecg ( figure 1 ) was obtained and revealed a sinus tachycardia, diffuse t-wave flattening, and low qrs-wave voltage. on the ecg, there was no st-segment elevation, pr-segment depression, or t-wave inversion to suggest early or resolving acute pericarditis. an echocardiogram was performed to evaluate the enlarged cardio-pericardial silhouette and electrocar-diographic findings, and revealed a large hypoechoic fluid collection surrounding the heart indicative of a pericardial effusion ( figure 3 ). to generate the appropriate differential diagnosis in this case, there are several key features of the patient's history and physical examination that aid in the understanding of his pathologic process: 1) symptom development occurred soon after emigration from indonesia, 2) the patient's primary symptoms were pulmonary in origin, and 3) secondary cardiac involvement resulted in a large, initially asymptomatic pericardial effusion. although several disease processes are possible explanations for this patient's pathology, one likely diagnosis will become clear through our discussion. travel from southeast asia. with over 500 million people crossing international borders each year, the potential for emerging pathogens to be spread from other geographic regions is greater than ever. 1 over a decade ago, the institute of medicine identified travel as one of six major factors related to the emergence and re-emergence of tropical disease. 2 several years later, the centers for disease control and prevention (cdc) released addressing emerging infectious disease threats: a prevention strategy for the united states; in this doctrine international travelers, refugees, and immigrants are targeted as a priority group for infectious disease surveillance. 3 the updated 1998 document from the cdc entitled preventing emerging infectious diseases: a strategy for the 21 st century continues to identify international travelers as a high-risk population that has contributed to the spread of these diseases. 4 the cdc recommendations are in part the result of the concern that many tropical and developing countries contain infectious pathogens not common to the united states. these diseases, such as malaria, plague, dengue fever, and yellow fever, have explosive potential if introduced in this country in sufficient quantity. for symptomatic patients who recently have immigrated to the united states, the potential for diagnosis of these unusual pathogens is significant. new emigrants from developing countries are at particular risk for infectious disease. this population frequently does not utilize appropriate pretravel medical care, may not be appropriately or completely vaccinated, and may experience living or working conditions that place them at higher risk for virulent infectious diseases. 5 additionally, genetic variations in the immune system of immigrants and lack of exposure to antigens on indigenous infectious organisms can contribute to the development of an infection soon after entering a new country. a significant number of immigrants who develop medical complaints will seek primary medical care through the ed. these patients are particularly challenging because of language barriers, atypical presentations of diseases, self-medication with atypical treatments before arrival, and a lack of physician familiarity of indigenous infections from the country of origin. upto 3% of individuals will complain of fever in the period immediately after travel. 6 the evaluation of the febrile immigrant begins with a thorough pretravel history regarding the living conditions in the country of origin, the patient's vaccination history, and any pretravel health care. specific questions regarding medical history and risk factors for common enteric, respiratory, neurologic, dermatologic, and hematologic infections should be made. in the post-travel period, fever most often indicates an underlying infection; however, other causes of fever should be simultaneously explored (table 1 ). many infectious diseases will have characteristic clinical features that distinguish them from other illness. our patient entered this country from indonesia five months before presentation to the ed. assuming that he became symptomatic with intermittent fever and productive cough three to four weeks before his presentation (i.e., when seen and treated by his pcp), one can assume that one of two possibilities occurred. he either became infected with an organism in the united states four months after entering this country or he harbored an occult, slow-growing infection with a long incubation period from indonesia. in this type of patient, it is useful to generate differential diagnoses for each of these possibilities. a listing of potential pulmonary pathogens is seen in table 2 . pulmonary infection. if we assume that this patient developed his infection in the united states, then we are most likely dealing with a common pulmonary pathogen such as those on the left side of table 2 . the clinical presentation and description of these organisms can be found in most emergency medicine textbooks. it is more likely that this patient harbored a pulmonary infection from indonesia. many of the common pulmonary pathogens from indonesia are similar to those found in the united states; however, many unusual pathogens are found with increasing frequency in this developing country (see right side of table 2 ). mycobacterium tuberculosis is a slow-growing aerobic rod with characteristic acid-fast staining properties. infection with tuberculosis continues to be a worldwide problem, with approximately one-third of the world population currently infected. 7 indonesia has the dubious distinction of having the third largest tuberculosis organism burden in the world. individuals throughout portions of asia are given bacille-calmette-guerin (bcg) vaccination as children; however, the overall efficacy and duration of protective immunity using the bcg vaccine remain unclear. 8 tuberculosis is transmitted primarily through inhalation of aerosolized bacilli. initially, patients often are asymptomatic following primary infection, although the organism may remain viable and dormant for years. in these individuals, the only indication of primary infection is a positive tuberculin purified protein derivative (ppd) skin test. reactivation of the disease is highest in the first two years following exposure, and is highest in young adults. typically, reactivation occurs in the lungs and should be considered in any patient who presents with a cough of more than three weeksõ duration, intermittent fever, night sweats, hemoptysis, weight loss, and anorexia. although any patient may be at risk for developing tuberculosis, patients with an immunocompromising illness such as hiv infection, prior institutionalization, travel to an endemic region, a positive ppd placed in the past, or known exposure to tuberculosis are especially at increased risk. 9 this patient came from a region known to be endemic for tuberculosis and presented with typical signs and symptoms of reactivation of the disease. the failure of his symptoms to respond to cephalexin also is consistent with a diagnosis of tuberculosis because this antibiotic has little effect against these slowgrowing bacilli. infection with tuberculosis may exhibit extrapulmonary manifestations of this disease that can occur during primary infection or during reactivation of the disease. dissemination can result in infection spreading to any organ system, including the heart and pericardium. q fever is a self-limited infection seen in about 50% of people infected with the obligate intracellular rickettsial pathogen coxiella burnetii. transmission is primarily through inhalation and is generally seen in farmers as a result of exposure to livestock, through ingestion by drinking unpasteurized milk, or uncommonly via a tick bite. 10 acute infection begins with sudden onset of one or more of the following: high fevers (up to 104-1058f), severe headache, general malaise, myalgia, confusion, sore throat, chills, sweats, nonproductive cough, nausea, vomiting, diarrhea, abdominal pain, and chest pain. 11, 23 fever usually lasts for one to two weeks, and abnormalities on liver function testing can be seen. symptoms of pneumonia occur in 30% to 50% of symptomatic patients. the chest radiograph typically shows rounded segmental lower-lobe densities, but may show lobar consolidation. 12 in general, most patients will recover to good health within several months without any treatment. only 1% to 2% of people with acute q fever die from the disease. the chronic form of q fever is much more serious, and as many as 65% of persons with chronic q fever may die from the disease. endocarditis, most commonly involving the aortic valve, can occur in up to twothirds of patients with chronic q fever. 13 our indonesian patient reported no farm-exposure history and did not complain about the typical headache seen in c. burnetii infection. additionally, the typical incubation period of q fever ranges from 14 to 39 days, much longer than the four-month asymptomatic period our patient would have had to experience if he was infected in indonesia. psittacosis and tularemia are caused by infection with chlamydia psittaci and francisella tularensis, respectively. pet shop workers and bird handlers, especially parrot and pigeon handlers, are especially at risk for acquiring psittacosis. the clinical presentation of psittacosis includes high fever associated with a relative bradycardia, severe headache, nonproductive cough or hemoptysis, and hepatic and spleen enlargement. chest radiography reveals patchy perihilar or lower-lobe infiltrates. 14 hunters and trappers of rabbits and those exposed to ticks and rodents contaminated with f. tularensis are at risk for development of tularemia. depending on the route of exposure, the tularemia bacteria may cause skin ulcers, swollen and painful lymph glands, inflamed eyes, sore throat, oral ulcers, or pneumonia. an acute infection presents with the abrupt onset of fever, chills, headache, muscle aches, joint pain, dry cough, and progressive weakness. persons with pneumonia can develop chest pain, difficulty breathing, bloody sputum, and respiratory failure. chest radiography reveals bilateral patchy infiltrates. treatment with streptomycin or gentamycin is highly effective and reduces the mortality rate from 30% to \1%. 15 although uncommon, pericarditis can be seen in both psittacosis and tularemia. our patient had no avian or rabbit exposure and did not have any of the laboratory or physical examination findings consistent with psittacosis or tularemia. infection with bordetella pertussis is characterized by three distinct phases: the catarrhal phase, the paroxysmal phase, and the convalescent phase. the catarrhal phase begins after an incubation period of seven to ten days. symptoms last one to two weeks and are indistinguishable from an upper respiratory tract infection with cough. the paroxysmal phase lasts two to four weeks and is characterized by paroxysms of coughing followed by a forceful inspiration producing the characteristic ''whoop'' sound. a residual, irritating cough lasting weeks to months is seen in the convalescent phase. whooping cough has seen an increase in worldwide incidence as a result of a reduction in the use of its vaccine. 16 it is unlikely that our patient had whooping cough. although pertussis is seen in any age, it is predominately a pediatric illness. additionally, pericardial effusion is not a known complication of whooping cough. although diphtheria has been nearly eradicated in the united states, endemic infection still occurs in areas throughout the world, including southeast asia. corynebacterium diphtheriae is a gram-positive, club-shaped bacillus that presents as an infection involving the respiratory tract or skin. systemic involvement of the cardiovascular and nervous system may occur. following a short incubation period, patients present with signs of a typical upper respiratory tract infection. on oropharyngeal examination, a grayish-white pseudomembrane adherent to the posterior pharynx can be seen in infected individuals. c. diphtheriae releases a powerful exotoxin that directly injures myocytes, producing a myocarditis, congestive heart failure, and conduction blocks. the exotoxin's disruption of protein synthesis produces a peripheral neuropathy manifesting as muscle weakness. 17 the typical symptoms and signs of diphtheria infection were absent in our patient. hantavirus and the pneumonic plague are endemic in many parts of southeast asia, including indonesia. whereas hantavirus occurs with exposure to rodent excrement, infection with yersinia pestis is transmitted by flea bites from infected rodents. hantavirus infection occurs after a one-to five-week incubation period and initially presents with fever and myalgias. symptoms of cough and shortness of breath herald the development of a rapidly aggressive bilateral pneumonia, often requiring mechanical ventilation within 24 hours. 18, 22 hematogenous spread of y. pestis leads to a highly contagious and rapidly fatal pneumonia. 19, 22 without a history of rodent or flea exposure and as a result of the slowly developing symptoms seen in our patient, it is unlikely that our patient was infected with hantavirus or pneumonic plague. lassa virus and lymphocytic choriomeningitis virus are members of the arenaviridae viruses. lassa fever is transmitted person to person and through contact with infected rodent urine. a gradual onset of fever and malaise begin after an incubation period lasting up to three weeks. severe headache and retrosternal chest pain may accompany the development of pneumonitis and respiratory distress. lymphocytic choriomeningitis virus begins as an influenza-like illness after a one-to three-week incubation period. aseptic meningitis may ensue, but even severe cases are associated with good recovery. 20 melioidosis, or whitmore's disease, is caused by infection with burkholderia pseudomallei. a handful of cases are confirmed in the united states each year, seen exclusively in travelers and immigrants, especially from southeast asia, where it is endemic. transmission occurs through either direct person-to-person contact, direct contact with contaminated soil and surface water, or inhalation. acute infection with melioidosis can produce fever and general muscle aches, and may progress rapidly to infect the bloodstream. the acute, localized form of infection presents as a nodule and results from inoculation through a break in the skin. the pulmonary form of the disease presents with symptoms consistent with a mild bronchitis to severe pneumonia. the onset of pulmonary melioidosis is typically accompanied by a high fever, headache, anorexia, and general muscle soreness. chest pain is common, but a nonproductive or productive cough with normal sputum is the hallmark of this form of melioidosis. bacteremic spread of this organism leads to septic shock with microabscesses found throughout the body, including pus-filled skin lesions. 21 numerous fungal and parasitic organisms endemic to southeast asia have potential pulmonary involvement. the acute phase (invasion and migration) of paragonimus westermani may be marked by diarrhea, abdominal pain, fever, cough, urticaria, hepatosplenomegaly, pulmonary abnormalities, and eosinophilia. during the chronic phase, pulmonary manifestations include cough, expectoration of discolored sputum, hemoptysis, and chest radiographic abnormalities. echinococcus granulosus infections remain silent for years before the enlarging cysts cause symptoms in the affected organs. hepatic involvement can result in abdominal pain, a mass in the hepatic area, and biliary duct obstruction. pulmonary involvement can produce chest pain, cough, and hemoptysis. rupture of the cysts can produce fever, urticaria, eosinophilia, and anaphylactic shock, as well as cyst dissemination. ascaris lumbricoides, strongyloides stercoralis, schistosoma haematobium, and several other less common parasites also can present with pulmonary findings. it is unlikely that one of these organisms infected our patient, because the majority of symptoms listed were not present in our patient. additionally, our patient lacked information regarding eosinophilia, which is classically seen in parasitic infections. with these possibilities in mind, let us turn to the last piece of critical information in this case: the development of a large pericardial effusion. pericardial effusion. the normal pericardium is composed of two layers and a potential space that exists between them. the two layers of the pericardium include a thin, visceral layer closely applied to the epicardium and a dense, outer parietal layer. the parietal layer is attached to the sternum, diaphragm, and mediastinum by fibrinous extensions and adventitia. between 15 and 60 ml of fluid normally is contained in the space between the visceral and parietal pericardium. the pericardium is thought to maintain the heart's position, lubricate the heart's surface, prevent the spread of infection from adjacent thoracic structures, prevent cardiac overdilatation, augment atrial filling, and maintain the normal pressurevolume relationships of the cardiac chambers. a minimum of 250 ml is needed to fill the pericardial reserve volume sufficiently to detectably increase the cardio-pericardial silhouette by chest radiography. 24 our patient had multiple imaging findings consistent with a large pericardial effusion. his chest radiograph showed enlargement of the cardio-pericardial silhouette. common etiologies for cardiopericardial enlargement include pericardial effusion, valvular heart disease, cardiomyopathy, and congenital heart disease. our patient did not carry a congenital heart disease diagnosis and did not have a cardiac murmur to signify valvular heart disease. an echocardiogram is needed to investigate these possibilities and in this patient showed a large hypoechoic area surrounding the myocardium. finally, his 12-lead ecg showed sinus tachycardia, low voltage in the precordial leads, and diffuse t-wave flattening, all of which are consistent with a pericardial effusion. notably, there was no st-segment elevation, prsegment depression, or t-wave inversion to suggest the early or resolving stages of acute pericarditis. the causes of pericardial effusion are numerous and parallel the etiologies of acute pericarditis (table 3) . large pericardial effusions are most common with tumors, tuberculosis pericarditis, cholesterol pericarditis, myxedema, vasculitis/connective tissue disease, uremic pericarditis, and parasitoses. 25 an effusion is often asymptomatic but should be suspected in the appropriate clinical setting. pericardial effusions can present with vague chest symptoms such as a feeling of chest pressure and chest ache. a very large effusion can manifest as dyspnea on exertion (compression of lung parenchyma), dysphagia (compression of esophagus), cough (compression of pulmonary bronchi), hiccups (compression of vagus and phrenic nerve), or hoarseness (compression of recurrent laryngeal nerve). classic physical examination findings of distant heart sounds and jugular venous distension are generally unreliable and difficult to detect in the ed. cardiovascular changes occur as fluid within the pericardium accumulates. tachycardia occurs commonly, but many patients may have heart rates of 90 to 100 beats/min or lower in hypothyroidism or uremic patients. significant cardiac tamponade produces absolute or relative hypotension. chest radiography and 12-lead ecg can suggest the diagnosis of pericardial effusion, but are neither sensitive nor specific enough to confirm the diagnosis. the diagnostic criterion standard is two-dimensional echocardiography in the diagnosis of pericardial effusion. in our patient, the most likely cause of the pericardial effusion was infection with tuberculosis. tuberculous pericarditis is estimated to occur in 1% to 2% of patients with pulmonary tuberculosis 26 and is one of the leading causes of pericarditis in nonindustrialized countries. associated pericardial effusions typically are slowly accumulating, and several hundred milliliters of fluid may develop before symptoms become apparent. in many patients, the chest radiography film shows an enlarged cardiac silhouette, but a pulmonary infiltrate often is absent, as was seen in this patient. special cultures of pericardial fluid are needed to diagnose tuberculous pericardial effusion. yields may be increased with biopsy of the pericardium or culturing the precipitant after centrifugation of pericardial effusion. in addition to the development of a constrictive pericarditis, or myocarditis, complications of tuberculous pericarditis includes impairment of cardiac function either directly or through cardiac tamponade. probable diagnosis: large pericardial effusion secondary to tuberculosis infection. to summarize, this patient's recent immigration, symptoms of intermittent fever and of chronic cough that failed to respond to outpatient antibiotics, and development of large pericardial effusion all are consistent with a diagnosis of tuberculosis. suggested management includes admission to a monitored setting and ap the ecg revealed sinus tachycardia and low voltage. the chest radiography revealed a large cardiac silhouette consistent with either cardiomegaly and or a pericardial effusion. the echocardiogram confirmed a large pericardial effusion and global hypokinesis. cardiovascular surgery was consulted and the patient underwent a pericardial window. six hundred milliliters of bloody fluid was drained from the pericardium. a biopsy of the thickened pericardium revealed necrotizing granulomas consistent with tuberculous pericarditis. m. tuberculosis was isolated from the pericardial specimen. postsurgical serial echocardiograms revealed persistent moderate-to-severe global hypokinesis. five days following surgery, the patient remained stable and was transferred to a telemetry unit. an asymptomatic tachycardia persisted throughout his hospital stay. the patient was discharged 12 days after admission with a four-drug tuberculosis regimen (pyrazinamide, isoniazid, rifampin, and ethambutol). follow-up was arranged with cardiology and infectious disease specialists. tuberculosis is a lethal infectious disease with diverse manifestations. the incidence of tuberculosis has been rising over the past decades. according to the world health organization, approximately 4 million new cases occur annually. 27 it is reported that more than 95% of these cases are in developing countries such as indonesia. 28 in the united states, more than 22,000 cases are reported annually. 29 tuberculous pericarditis is thought to occur in 2% of all instances of pulmonary tuberculosis. 29 worldwide, tuberculosis is the leading cause of pericarditis. in the united states, it is the leading cause of immunodeficiency-related pericarditis. 30 the typical symptoms of this disease are cough, dyspnea, and chest pain. additionally, night sweats, orthopnea, and weight loss are common. cardiomegaly, pericardial rub, fever, and tachycardia are frequent signs. 31 the mycobacterium spreads to the pericardium either by direct extension from the lungs or by a hematogenous route. the resultant effusion is thought to be caused by a hypersensitivity reaction to the tuberculoprotein. proinflammatory cytokines are implicated as the etiology of symptoms such as fever, weakness, and weight loss. 31 cardiomegaly caused by pericardial effusion frequently is evident on chest radiography. however, fewer than half of the patients may have radiographic evidence of pulmonary tuberculosis. 32 characteristic ecg findings of tuberculous pericarditis are lowvoltage qrs waves and inverted t waves. however, these findings are present only in a minority of cases and are not diagnostic. 29 similarly, echocardiogram findings are nonspecific. evidence of pericardial effusion with possible pericardial thickening is suggestive of the diagnosis. rarely, there may be evidence of cardiac tamponade. 31 definitive diagnosis of tuberculous pericarditis requires isolation of m. tuberculosis. a positive tuberculin skin test may increase the suspicion for the diagnosis; however, a negative test does not exclude it. given the difficulty of isolating the bacteria, pericardial fluid culture is neither reliable nor timely. in fact, culture of the fluid reveals the diagnosis in only 30% of the cases. 27 a pericardial tissue specimen has a higher yield for isolation of the bacteria. when a large pericardial effusion is present, an open biopsy along with a pericardial window serves as both a diagnostic and a therapeutic procedure. 31 antibiotic therapy mimics the same dose and length as that of pulmonary tuberculosis. resolution of the pericarditis is expected within three months in 80% of patients. 32 before the advent of modern antituberculosis drug therapy, mortality rates of up to 85% were noted. with current therapy, the mortality rate has decreased to 50%. 29 constrictive pericarditis develops in 30% to 50% of patients despite medical therapy. 32 thus, the placement of a pericardial window generally is recommended in the treatment of tuberculous pericarditis. travel and the emergence of infectious diseases d www.aemj.org 2. institute of medicine. emerging infections: microbial threats to health in the united states addressing emerging infectious disease threats: a prevention strategy for the united states centers for disease control and prevention. preventing emerging infectious disease threats: a strategy for the 21 st century travel medicine illness after international travel the global tuberculosis situation and the new control strategy of the world health organization efficacy of bcg vaccine in the prevention of tuberculosis. meta analysis of the published literature rosen's emergency medicine concepts and clinical practice rosen's emergency medicine concepts and clinical practice coxiella burnetii (q fever) pneumonia the chest film findings in ''q'' fever-a series of 35 cases chlamydia psittaci (psittacosis) francisella tularensis (tularemia) bordetella pertussis and chronic cough in adults viral hemorrhagic fevers and hantavirus infections in the americas pseudomonas species (including melioidosis and glanders) emerging infectious diseases and risk to the traveler fever in the returned traveler heart disease: a textbook of cardiovascular medicine should pericardial drainage be performed routinely in patients who have a large pericardial effusion without tamponade? rosen's emergency medicine: concepts and clinical practice harrison's principles of internal medicine, 15 th edition cardiac tamponade as a manifestation of tuberculosis tuberculous pericarditis human immunodeficiency virus-associated pericardial effusion: report of 40 cases and review of the literature tuberculous pericarditis: optimal diagnosis and management tuberculous pericarditis: ten years of experience with a prospective protocol for diagnosis and treatment key: cord-007445-2folsh35 authors: tuffaha, amjad; gern, james e.; lemanske, robert f. title: the role of respiratory viruses in acute and chronic asthma date: 2000-06-01 journal: clin chest med doi: 10.1016/s0272-5231(05)70267-7 sha: doc_id: 7445 cord_uid: 2folsh35 respiratory tract infections caused by viruses, 24, 70 chlamydia, 18, 19, 43, 55, 116 and mycoplasma(61) have been implicated in the pathogenesis of asthma. viruses have been demonstrated to be associated with asthma epidemiologically in at least two ways (fig. 1). first, during infancy, certain viruses have been implicated as potentially being responsible for the inception of the asthmatic phenotype. second, in patients, particularly children, with established asthma, viral upper respiratory tract infections play a significant role in producing acute exacerbations of airway obstruction that may result in frequent outpatient visits or hospitalizations. 24, 55, 56, 57 this article reviews these two areas by focusing first on mechanisms by which virus infections may lead to the development of asthma in infants and children and, second, on mechanisms by which virus infections may produce acute asthmatic symptoms in patients who already have established disease. respiratory tract infections caused by viruses,24, 70 chlamydia,'*, 19, 43* 55* 116 and myco-plasma6i have been implicated in the pathogenesis of asthma. viruses have been demonstrated to be associated with asthma epidemiologically in at least two ways (fig. 1) . first, during infancy, certain viruses have been implicated as potentially being responsible for the inception of the asthmatic phenotype. second, in patients, particularly children, with established asthma, viral upper respiratory tract infections play a significant role in producing acute exacerbations of airway obstruction that may result in frequent outpatient visits or hospitalization^.^^, [55] [56] [57] this article reviews these two areas by focusing first on mechanisms by which virus infections may lead to the development of asthma in infants and children and, second, on mechanisms by which virus infections may produce acute asthmatic symptoms in patients who already have established disease. supported by nih grants a134891, hl56396, and lrolhl61879. infections with respiratory syncytial virus (rsv) or parainfluenza virus (piv) have received much attention because of their predilection to produce a pattern of symptoms termed bronchiozitis that parallels many of the features of childhood and adult asthma.67 respiratory syncytial virus causes about 70% of these episodes and it is estimated that, by age 1 year, 50% to 65% of children will have been infected with this virusn and 40% of these infections involve the lower respiratory tract.92 by age 2, nearly all children will have been infected with rsv at least once. children aged 3 to 6 months are most prone to develop lower respiratory tract symptoms, suggesting that a developmental component (e.g., lung or immunologic maturation) may be involved as 83 the relationship between rsv infections during the first few years of life and the subsequent development of the asthmatic phenotype has been the subject of much interest as well as controversy. variations in reporting longitudinal outcomes (e.g., recurrent wheezing, measurements of airway hyperresponsiveness, diagnosis of asthma) appear to be influenced mostly by the criteria used to define "bronchiolitis." these criteria include the type of virus producing the symptoms (in addition to rsv, viruses that may contribute to the development of bronchiolitis in this age group could be piv, coronavirus, influenzavirus, and rhin~virus~~); the age at the time of infection; the nature and severity of symptoms required for inclusion; and, finally, the characteristics of both the study population (community versus hospital-based) and the study design (retrospective versus prospective). a number of long-term prospective studies of children admitted to a hospital with documented rsv-induced bronchiolitis have shown that about 75% experience wheezing in the first 2 years after the initial illness, more than 50% still wheeze 3 years later, and approximately 40% continue to wheeze after 5 years?,, 49, 733 91, 117, additional insight into these areas recently was provided by the results of an ll-year prospective study involving 880 children who were enrolled at birth, followed for the development of lower respiratory tract illnesses (lris) in the first 3 years of life, and then evaluated for the presence or absence of physician-diagnosed asthma or a history of current wheezing at ages 6 and 11 years.14 most importantly, lung function was evaluated in the first few months of life in a subset of these children prior to the development of a documented lri. during the first 3 years of life, 7.4% had pneumonia documented radio-graphically and 44.7% had a significant lri without pneumonia. respiratory syncytial virus and piv were identified in 36.4% and 7.3%, respectively, in the subjects with pneumonia, and in 35.6% and 15.2%, respectively, of the subjects with a lri. at age 6, physiciandiagnosed asthma was present in 13.6% (or = 3.3), 10.2% (or = 2.4), and 4.6% of the subjects with pneumonia, lri, and no lri, respectively. by age 11, these values increased to 25.9% (or = 2.8), 16.1% (or = 1.6), and 11%, respectively. mean maximum volume at functional residual capacity values before any lri were lower in children with pneumonia and with lris than in children with no lris. these values continued to be lower at age 6 and by age 11, when forced expiratory volume in 1 second (fevl) and fef, , were recorded, similar group relationships persisted. interestingly, despite the persistence of lowered baseline lung function in both the pneumonia and lri groups, many of these deficits were markedly (but not completely) reduced following administration of albuterol. in a second report, further follow-up of this large cohort of children demonstrated that the risk for both frequent (more than three episodes of wheezing per year) and infrequent (three episodes of wheezing per year) wheezing in relation to rsv lower respiratory illnesses decreased markedly with age and became nonsignificant by age 13.1°4 these data suggest that, although rsv infections contribute substantially to the expression of the asthmatic phenotype, other factors (e.g., genetic, environmental, developmental) ap-pear to contribute as well, either in terms of its initial expression or the modification of the phenotype over time. in addition to premorbid lung function, the influence of atopy on the development of the asthmatic phenotype in relationship to viral infections has also been evaluated. interactions between these two factors appear to be bidirectional and dynamic, in that the atopic state can influence the lower airway response to viral infections? 71 viral infections can influence the development of allergen sensitization,28, 29, 99 and interactions can occur when individuals are exposed simultaneously to both allergens and viruses.1z, 68, atopy can be defined as the genetic predisposition to the preferential development of an immunoglobulin (1g)e antibody response to a variety of environmental allergens. as stated previously, atopy has been considered to be a risk factor for the development of childhood asthma and its influence on the pattern of responses following viral infections has been of interest to many investigative groups. it has also been suggested that atopy could be a significant predisposing factor for the development of acute bronchiolitis during rsv epid e m i c~.~~ although some have found that children most likely to have persistent wheezing were those born to atopic parents,64, 91, lz1 others have not.14, 73, 84 some have found that personal atopy is not more prevalent in symptomatic children after br~nchiolitis'~, 73; others have found that documented rsv bronchiolitis significantly increases a child's chances (32% versus 9% in controls) of subsequently developing ige antibody% or lymphocyte proliferative responses75 to both food and aeroallergens. respiratory syncytial virus infections may interact with immunoinflammatory mechanisms involved in immediate hypersensitivity responses in a number of ways.18 first, it has been suggested that viruses capable of infecting lower airway epithelium may lead to enhanced absorption of aeroallergens across the airway wall, predisposing to subsequent sensitizati~n.~~, 94 second, rsv-specific ige antibody formation may lead to mast-cellmediator release within the airway, resulting in the development of bronchospasm and the ingress of eo~inophils.~~, 60, 85, 115 respiratory syncytial virus belongs to the family paramyxoviridae, the genera pneumovirus, and can be differentiated into two serologic subgroups, a and b.m, it has 10 genes, with 12 potential gene products. the g (attachment) and f (fusion) proteins are the major su$ace glycoproteins against which neutralizing antibody is directed. interestingly, in both murine2 and hurnan5l in vitro experiments, it has been noted that the g protein elicits a predominant th2 response, whereas the f protein produces a predominant thl response. in mice, to test the activities of t cells recognizing individual rsv proteins in vivo, virus-specific t-cell lines have been produced using recombinant vaccinia viruses that express either the g or f proteins. following passive transfer of these cell lines to naive recipients and subsequent intranasal inoculation with rsv mice receiving g-specific cells have more severe illnesses, characterized by lung hemorrhage, pulmonary neutrophil recruitment, and intense pulmonary eosinophi1ia.l these experiments are of interest based on the adverse clinical response noted in many infants who received a formalin-inactivated rsv vaccine and subsequently became infected with rsv? these intriguing observations regarding rsv and its influence on thl /th2 responses have recently been expanded. roman et a1 evaluated 15 hospitalized infants (1-15 months) with an acute lower respiratory tract infection caused by rsv. compared with control infants, peripheral blood cells from infected children had suppressed ifn-?/ production ex vivo and, although il-4 production was also decreased, the il-4/ ifn-y ratio was significantly increased. renzi et a p prospectively followed 26 infants hospitalized with bronchiolitis by obtaining blood samples at the time of illness and 5 months later, and found that immune responses during the acute infection correlated with long-term pulmonary outcomes. blood lymphocytes, obtained during the time of bronchiolitis, produced less ifn-?/ ex vivo in response to il-2 and more il-4 in response to d. furinue antigen in children who went on to develop a pattern of recurrent wheezing.8s finally, lower ifn-y production at the time of bronchiolitis has been demonstrated to be an indicator of reduced pulmonary function and increased responsiveness to histamine 5 months after bronchiolitis, and was related to the development of asthma after bronchiolitis in infants. 87 in contrast, other groups have noted increased levels of ifn-y respiratory tract secretions during rsv illnesses in infants and children with bronchiolitis and recurrent wheezing compared with those with upper respiratory tract symptoms unfortunately, in all of the studies reported thus far, the pattern of cytokine response these infants had prior to infection was not evaluated, begging the question as to which of the observed results may be cause and which effect. to more comprehensively evaluate the relationships among virus infection, atopy (cytokine dysregulation of thl / th2 imbalance), and immune system or lung developmental components, a rat model of virus-induced airway dysfunction has been studied extensively.'l' in this model, infection with piv type 1 during a critical developmental time period (when the animals are weaning [ 3 4 weeks of age] as opposed to when they are neonates [4-5 days] or adults) produces chronic (8-12 weeks fol-lowing infection), episodic, reversible airway inflammation and remodeling with associated alterations in airway physiology (increased resistance and rnethacholine responsiveness) that resemble human asthma in high (brown norway strain) but not low (f344 strain) igeantibody producing rats.62 the temporal progression of this asthma-like syndrome is associated with a thl / th2 imbalance within the lung, and its development can be significantly attenuated by the exogenous administration of ifn-8 just prior to and during the viral infection in the brown norway responder strain.lo2 this model further supports the concept of both genetic (atopy; cytokine dysregulation or imbalance) and environmental factors (virus infection) being important in the inception of the asthmatic phenotype, as well as a developmental component contributing. respiratory viruses are common causes of asthma exacerbations in asthmatic subjects of different age 74, 86 serology or culture detection methods of viruses initially indicated an association during asthma exacerba-tionss2 despite the fact that these detection methods are relatively insensitive for viruses such as rhinovirus (rv). the use of reverse transcription polymerase chain reaction (rt-pcr) assays that are more sensitive for detection of rv have confirmed and expanded these initial observation^.^^ indeed, johnston et a157 found that 80% to 85% of school-aged children with acute wheezing episodes tested positive for a virus using rt-pcr and other standard virologic techniques. the virus most often detected was rv. seasonal patterns of upper respiratory virus infections correlate closely with hospital admissions for asthma, particularly in pediatric age these studies indicate that rv infections are the most common cause of asthma exacerbations in children, especially during spring and fall. similar studies, performed in found that about half of asthma exacerbations were associated with rv infection. as discussed previously, in infancy, atopy may define a susceptibility of the host to wheezing with respiratory infections. duff et a1,= for example, studied children who presented to an emergency department with wheezing. children over 2 years of age were more likely to have respiratory allergies or a confirmed respiratory viral infection compared with children with no wheezing. children with the highest risk for wheezing were those who had respiratory allergies and respiratory viral infection, implying that respiratory viral infections and respiratory allergies may have synergistic effects on lower airway physiology and enhance the likelihood of wheezing with respiratory infection. in children less than 2 years of age, wheezing was also noted, but risk factors for wheezing were quite different. these infants were not allergic, had rsv as the major viral isolate, and had passive tobacco smoke exposure as a major risk factor for wheezing. available epidemiologic data in children and adults have shown that episodic drops in peak flow measurements are associated with rv infections. this was found to correlate with an increase in asthma symptoms and nonspecific airway hyperresponsiveness following experimentally infecting asthmatic subjects with rv.15, 41 further studies by griinberg et a140 demonstrated that experimental rv16 infection leads to a transient drop in daily home recordings of fev, in subjects with asthma. this variable airway obstruction correlated significantly with cold symptoms, asthma symptoms, and the increase in airway hyperresponsiveness to histamine. such daily variability in fev, reflects the inflammatory changes within the airway wall, which can be induced by the natural rv infection. following infections with rv8 and influenza a.@, 72 in a study by cheung et all5 14 subjects with mild asthma were inoculated with rv16 or placebo. the maximal contractile response to inhaled methacholine was significantly greater during the rv16 infection and remained elevated for up to 15 days after the acute infection. this study indicates that an upper respiratory viral infection can enhance the reactivity of the lower airway and the magnitude of bronchonstriction changes, which can persist for weeks after the acute infection. respiratory viral infections' effect on lower airway responses are also influenced by host factors. in particular, allergic subjects experience greater changes in airway responsiveness after viral infection than nonallergic control s~b j e c t s .~, 34 furthermore, subjects with lower fev, values tend to have greater changes in airway responsiveness during viral infecti0n.3~ these studies suggest that effects of pre-existing conditions such as allergy and intrinsic lower airway function on caliber are likely to contribute to airway hyperresponsiveness during respiratory viral infection. potential mechanisms through which viral infections could potentially cause bronchoconstriction and increased airway responsiveness include enhancing parasympathetic bronchoconstrictive responses, stimulation of airway sensory nerves, and interference with the bronchodilatory functions of the nonadrenergic, noncholinergic neurons (table 1) . because of difficulties in assessing dysfunction of pulmonary neural regulation in humans, most data that support these proposed mechanisms were derived in animal models of acute respiratory viral infection. further definition of these pathways in humans will depend upon the development of new experimental techniques or inhibition of specific neural pathways. increased bronchial responsiveness has been found in normal and asthmatic subjects changes in small airways structure and function may also contribute significantly to the severity of hyperinflation and gas exchange abnormalities noted in acute asthma exacerbations. the maximal airway contractile response to methacholine in mild asthmatic subjects is increased during a cold, which is probably secondary to excessive airway narrowing attributable to airway wall thickening, airway parenchymal uncoupling, or abnormalities in smooth muscle ~0ntraction.l~ significant changes in airway morphology are noticed in animals with acute viral respiratory illness that leads to marked bronchiolar narrowing and plugging. these changes include bronchiolar airway edema and cell infiltration, epithelial hyperplasia, and folding and sloughing of airway epithelial surfaces. in addition, rats with mild increases in pulmonary resistance and methacholine sensitivity during acute viral respiratory illness have evidence of air trapping and ventilationperfusion mismatches.101 these latter findings indicate that viruses can induce significant changes in the peripheral airways that have significant functional outcomes in the absence of marked changes in measurements of airway obstruction and hyperresponsiveness. respiratory viruses can cause inflammation and injury to healthy airways and can worsen injury in airways that are already inflamed, as demonstrable in asthma. respiratory viruses can induce an inflammatory process by direct cytopathic effects on the airway epithelium (e.g., rsv bronchiolitis) and can induce an immune response to stop viral replication and eradicate the virus. the immune re-sponse to viral infection may be a doubleedged sword, however, as virus-induced inflammation can also contribute to airway obstruction and respiratory symptoms. indeed, although many common cold viruses (e.g., rv) do not produce significant cytopathic effects, possibly because few cells are infected, the immunoinflammatory response to the virus is probably the major cause of respiratory symptoms. in this section, the association between virus-induced immune responses and respiratory symptoms is explored. respiratory viruses replicate primarily in airway epithelial cells. in addition to serving as host cells, it is now well documented that epithelial cells also initiate the immune response to infections through the secretion of cytokines and chemokines. in vitro studies of epithelial cells or cell lines have demonstrated that respiratory viruses such as rv, rsv, and parainfluenzavirus can induce the secretion of many different proinflammatory cytokines (il-1, tnf-a, gm-csf, il-6, il-11) and chemokines (rantes, il-8, mip-la).lo, 20, 23, 96, 98, lo5 epithelial-derived chemokines are likely to be an important signal in initiating antiviral responses through the recruitment of leukocytes to the airway. in support of this concept, il-8, a potent neutrophil chemoattractant, is found in high levels in nasal secretions of children with virus-induced asthma, and levels of il-8 correlate with the number of airway neutrophils and neutrophil myeloperoxidase levels (suggesting neutrophil activation).lo8 there is also evidence, however, that enhanced airway inflammation caused by chemokine secretion may also disturb normal air-way physiology. chemokine levels in nasal secretions correlate closely with cold symptoms,"o for example, and il-8 levels correlate with virus-induced changes in airway respon-~i v e n e s s .~~ levels of epithelial-derived cytokines such as il-6 and il-11 also correlate with respiratory syrnpt0ms,2~ and animal studies indicate that overexpression of il-11 can cause bronchial hyperresponsi~eness.~~~ io7 in addition to stimulating cytokine production, rv can upregulate epithelial cell surface expression of intercellular adhesion molecule-1,79 which, in addition to facilitating cell-cell adhesion, is the receptor for the major group of rv3" lo3 this enhanced expression of adhesion proteins may contribute to the persistence and severity of inflammation in asthmatic subjects and, possibly, the greater susceptibility of asthmatic children to colds compared with nonasthmatic children. mechanisms for the activation of cytokine genes in epithelial cells and adhesion molecules are under investigation. it is known that nuclear factor-lc b activation is important in virus-induced transcriptional regulation of il-650 and, possibly, for the synthesis of a variety of inflammatory cyt~kines.~ in addition, nitric oxide may regulate virus-induced chemokine production through a posttranscriptional mechanism and by inhibiting viral repli~ation;~ although a clinical study did not find a relationship between il-8 and nitrate levels in nasal secretion^.^^ granulocyte recruitment and activation seem to have an important role in the pathogenesis of virus-induced asthma exacerbations. griinberg et a1y1 for example, experimentally inoculated 35 atopic asthma subjects with either rv16 or placebo and found that neutrophil counts in the peripheral blood correlated with the cold and asthma symptom scores and cold-induced changes in airway hyperresponsiveness. in addition, eosinophil granular proteins and leukotriene c, have been detected in the nasal secretions of infants and children with virus-induced wheezing i l l n e~s e s .~~~ 86, 97, increased concentrations of sputum eosinophil cationic protein found during the acute phase of rv infection corre-lated with increases in airway responsiveness in a group of adults with asthma after experimental inoculation with rv16.39 in vitro experiments indicate that rv does not activate eosinophils dire~tly,~; it is more likely that inflammatory mediators and cytokines, secreted by virus-activated cells in the lung, contribute to eosinophil activation. finally, guinea pigs infected with piv develop airway eosinophils and airway hyperresponsive-nessz5 and this outcome is blocked if the guinea pigs are pretreated with il-5neutralizing antibody.l12 most respiratory viruses replicate quickly and, within a few days of inoculation, the quantity of viruses and viral proteins is sufficient to activate mononuclear cells in the airway. in vitro infection of human monocytes with respiratory viruses, for example, leads to a potent proinflammatory cytokine response by release of il-8, il-1, and tnf-cx.~~, 54, 90 interleukin-1 and tnf-a can increase cell recruitment into the airway by enhancing adhesion molecule expression on endothelial cells. in addition, tnf-a has been associated with wheezing illnesses in infancy6 and the development of late-phase allergic reaction and asthma.3, 37 monocytes and macrophages also produce interferon (inf), and its appearance in nasal secretions coincides with the onset of the recovery process. in addition to cytokine production, macrophages incubated with rsv or piv produce lipid mediators such as prostaglandin e, platelet-activating factor, and thromboxane b248, 78, 114 that can augment airway inflammation. lymphocytes, including natural killer cells, cd8+ cytotoxic t cells, and cd4+ t cells, are involved in limiting viral replication and viral clearance. to test the possibility that variations in lymphocyte responses might account for variability in the ability to clear viral infections, parry et apo measured in vitro lymphocyte responses in a group of allergic subjects who were then inoculated with rv 16. vigorous virus-induced responses (lymphocyte proliferation or ifn--y secretion) before inoculation correlated with reduced viral shedding after inoculation. these results sug-gest that factors related to the host cellular response help determine the degree of viral replication during respiratory viral infections. further characterization of these host factors may lead to new therapeutic strategies for respiratory infections, a goal that is particularly important for people with asthma. several studies have shown that viral infections activate a wide range of t cells. evidence for this comes from experiments in mice, in which most of the t cells found in the lung after an acute viral infection are not virus-specific,2l and in vitro studies, in which 25% to 50% of human peripheral blood t cells express the early activation marker cd69 after 24 hours in culture with rv.36 rantes, induced by respiratory viruses, at high concentrations can also induce antigen-independent t-cell activation? these studies suggest that respiratory viruses can induce early, nonspecific t-cell activation and recruitment that could significantly increase the intensity of airway inflammation, resulting in airway dysfunction and respiratory symptoms. this hypothesis is supported by studies of volunteers infected with rhinovirus. respiratory virus infections usually cause peripheral lymphopenia and increased numbers of lymphocytes in the upper and lower airways, for example. the degree of peripheral blood lymphopenia and lymphocytic infiltration of the airway epithelium has been correlated with the increases in airway responsi~eness.~~~ 26 although viral infections cause similar upper respiratory symptoms in allergic and nonallergic individual^,^^, loo there is evidence of interactions between virus-and allergen-induced responses in the lower airway. lemanske and colleag~es,~~ for example, identified 10 patients with allergic rhinitis and experimentally infected them with rv16. the viral infection increased airway reactivity to both inhaled allergen and histamine, and also increased the frequency of a late allergic reaction to inhaled antigens. moreover, calhoun and colleag~es'~ used bronchoscopy to study the inflammatory response to allergen in indi-vidual lung segments before, during, and 1 month after rv16 infection. rv infection enhanced the immediate antigen-induced release of histamine, and also increased eosino-phi1 recruitment of eosinophils to the lung. respiratory infections can have dual effects related to asthma. first, there is increasing evidence that severe infections with rsv and piv in infancy can alter lung development and physiology to increase the risks of subsequent wheezing and asthma. second, infections with common cold viruses and influenza commonly precipitate wheezing symptoms in children and adults who already have established asthma, and rv appears to be the most important virus in producing exacerbations of the disease. the principal mechanisms by which this occurs appears to be viral replication in epithelial cells, triggering a cascade of inflammation involving granulocytes, macrophages, t cells, and secreted cytokines and mediators. the inflammatory process, although essential to clear the infection, augments pre-existing airway inflammation in asthma, leading to increased airway obstruction and lower respiratory tract symptoms. greater understanding of virus-induced changes in inflammation and corresponding changes in airway physiology may lead to new therapeutic approaches to the treatment and prevention of virus-induced airway dysfunction. association of radiologically ascertained pneumonia before age 3 years with asthma-like symptoms and pulmonary function during childhood. a prospective study. openshaw pjm distinct types of lung disease caused by functional subsets of antiviral t cells phenotypic and functional characterization of t-cell lines specific for individual respiratory syncytial virus proteins induction of human airway hyperresponsiveness by tumour necrosis factor-a cytokine (il-8, il-6, tnf-a) and soluble tnf receptor-i release from human peripheral blood mononuclear cells after respiratory syncytial virus infection virusinduced alterations in macrophage production of tumor necrosis factor and prostaglandin-e2 rhinovirus infection induces expression of its own receptor intercellular adhesion molecule 1 (icam-1) via increased nf-kappab-mediated transcription rhinovirusinduced peripheral blood mononuclear cell responses and outcome of experimental infection in allergic subjects interleukin-la mediates the enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus viruses as precipitants of asthma symptoms. i. epidemiology development of allergen-specific t-cell memory in atopic and normal children wheezing, asthma, and pulmonary dysfunction 10 years after infection with respiratory syncytial virus in infancy increase in cd23+ cells in infants with bronchiolitis is accompanied by appearance of ige and igg4 antibodies specific for respiratory syncytial virus rhinovirus and respiratory syncytial virus in wheezing children requiring emergency ca-ige and eosinophil analyses reduced interferon-y production in infants with bronchiolitis and asthma cellular immunity is activated and a th-2 response is associated with early wheezing in infants after bronchiolitis different effects of influenza virus, respiratory syncytial virus, and sendai virus on human lymphocytes and macrophages interleukin-1 and interleukin-1 inhibitor production by human macrophages exposed to influenza virus or respiratory syncytial virus the relationship between proved viral bronchiolitis and subsequent wheezing ruuskanen 0, ogra pl: respiratory syncytial virus enhancement by parainfluenza 3 infection of contractile responses to substance p and capsaicin in airway smooth muscle from the guinea pig effect of influenza virus infection on allergic sensitization to aerosolized ovalbumin in mice nitric oxide inhibits rhinovirus-induced cytokine production and viral replication in a human respiratory epithelial cell line rhinovirus replication causes rantes production in primary bronchial epithelial cells increased levels of eosinophil major basic protein in nasal secretions in rhinovirus infection [abstract respiratory syncytial virus-induced release of rantes and mip-1 by bronchial epithelial and peripheral mononuclear cells asthma and immunoglobulin-e antibodies after respiratory syncytial virus bronchiolitis: a prospective cohort study with matched controls lower airway responses to influenza a virus in healthy allergic and nonallergic subjects virusinduced airway obstruction and parasympathetic hyperresponsiveness in adult rats prevention of chronic post-bronchiolitis airway sequelae with interferon-y treatment in rats a cell adhesion molecule, icam-i, is the major surface receptor for rhinoviruses respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years infection of a human respiratory epithelial cell line with rhinovirus. induction of cytokine release and modulation of susceptibility to infection by cytokine exposure respiratory syncytial virus infection of neonatal monocytes stimulates synthesis of interferon regulatory factor-1 and interleukin-lp (il-lp)-converting enzyme and secretion of il-1p targeted expression of il-11 in the murine airway causes lymphocytic inflammation, bronchial remodelling, and airways obstruction role of nasal interleukin-8 in neutrophil recruitment and activation in children with virus-induced asthma respiratory syncytial virus-induced cytokine production by neonatal macrophages association between interleukin-8 concentration in nasal secretions and severity of symptoms of experimental rhinovirus colds parainfluenza virus-induced persistence of airway inflammation, fibrosis, and dysfunction associated with tgf-pi expression in brown norway rats antibody to interleukin-5 inhibits virus-induced airway hyperresponsiveness to histamine in guinea pigs jncreased production of ifn--y and cysteinyl leukotrienes in virus-induced wheezing respiratory syncytial virus infection of human mononuclear phagocytes stimulates synthesis of platelet-activating factor the release of leukotrienes in the respiratory tract during infection with respiratory syncytial virus: role in obstructive airway disease asthma, atopy and chlamydia pneumoniae antibodies in adults continuing respiratory problems three and a half years after acute viral bronchiolitis immunologic mechanisms of virus-induced wheezing and asthma predictive value of respiratory syncytial virus-specific ige responses for recurrent wheezing following bronchiolitis the development of respiratory syncytial virus-specific ige and the release of histamine in nasopharyngeal secretions after infection patterns of allergic respiratory disease in children with a past history of bronchiolitis key: cord-002757-upwe0cpj authors: sullivan, kathleen e.; bassiri, hamid; bousfiha, ahmed a.; costa-carvalho, beatriz t.; freeman, alexandra f.; hagin, david; lau, yu l.; lionakis, michail s.; moreira, ileana; pinto, jorge a.; de moraes-pinto, m. isabel; rawat, amit; reda, shereen m.; reyes, saul oswaldo lugo; seppänen, mikko; tang, mimi l. k. title: emerging infections and pertinent infections related to travel for patients with primary immunodeficiencies date: 2017-08-07 journal: j clin immunol doi: 10.1007/s10875-017-0426-2 sha: doc_id: 2757 cord_uid: upwe0cpj in today’s global economy and affordable vacation travel, it is increasingly important that visitors to another country and their physician be familiar with emerging infections, infections unique to a specific geographic region, and risks related to the process of travel. this is never more important than for patients with primary immunodeficiency disorders (pidd). a recent review addressing common causes of fever in travelers provides important information for the general population thwaites and day (n engl j med 376:548-560, 2017). this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. . this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. physician be familiar with emerging infections, infections unique to a specific geographic region, and risks related to the process of travel. this is never more important than for patients with primary immunodeficiency disorders (pidd). a recent review addressing common causes of fever in travelers provides important information for the general population [1] . this review covers critical infectious and management concerns specifically related to travel for patients with pidd. this review will discuss the context of the changing landscape of infections, highlight specific infections of concern, and profile distinct infection phenotypes in patients who are immune compromised. the organization of this review will address the environment driving emerging infections and several concerns unique to patients with pidd. the first section addresses general considerations, the second section profiles specific infections organized according to mechanism of transmission, and the third section focuses on unique phenotypes and unique susceptibilities in patients with pidds. this review does not address most parasitic diseases. reference tables provide easily accessible information on a broader range of infections than is described in the text. emerging infectious diseases are a result of a convergence of numerous factors and comprise complex interactions among multiple variables. some of those factors are human movement, land use change, encroachment and wildlife translocation, rapid transport, and climate change. several studies demonstrate that for a pathogen to persist in a population, a minimal host population size that is specific for the type of pathogen and host population. of particular relevance to emerging infections is the pattern of rapid population growth. in the tropics, before wwii, most regional ecosystems consisted of few large cities and scattered human settlements separated by large areas of cropland, pastureland, or undisturbed forest. today, the pattern is the opposite: many large cities have developed with only patches of undisturbed forest or grassland. domestic vectors have therefore expanded their population with increasing urbanization and this markedly impacts the interactions between vectors and pathogens [2] . human activities such as deforestation, use of pesticides, pollution, etc. lead to the loss of predators that naturally regulate rodent and insect populations. this contributes to emerging zoonotic diseases and explains why they occur more frequently in areas recently settled. in today's global economy and affordable vacation travel, it is increasingly important that visitors to another country and their the southern, with a reduction in the number of cold days per year, changes in rainfall (more winter precipitation and summer droughts) [4] , and together these changes increase the risk of several vector-borne diseases in new areas. climate change involves not only global warming but also changes in precipitation, wind, humidity, and the location and frequency of extreme weather events like floods, droughts, or heat waves. changes in climate produce changes in pathogens, vectors, hosts, and their living environment. increases in precipitation can increase mosquitoes for example, but heavy rainfalls may cause flooding that temporarily eliminates larval habitats and decreases mosquitoes. flooding may force rodents to look for new habitats in houses and increase the opportunities of vector-human interactions, as occurs for example in the case of epidemic leptospirosis. humidity is another very important factor of climate change in the development of vector-borne diseases. mosquitoes and ticks do not survive well in dry conditions. therefore, weather impacts infectious pathogen distribution in complex ways that are not predictable by the forecast. extreme weather events can precipitate outbreaks of infection. an increase in the frequency and intensity of natural disasters like hurricanes and tsunamis, in relation to the el niño/southern oscillation phenomenon, may result in more flooded grasslands, which favor the breeding of aedes and culex mosquitoes [5] , and impact water sanitation fostering outbreaks such as cholera. flooded areas can displace rodents leading to plague. tornados and other severe weather can stir up soil leading to infections with soil fungi leading to episodic outbreaks of invasive fungal disease such as mucormycosis such as apophysomyces trapeziformis [6] . malaria is a common disease that can vary dramatically depending on weather, and extreme weather can alter the very landscape, providing new bodies of water to support larval development. if the melting of glaciers and the polar ice caps bring coastal cities underwater, or if overpopulation and waste cause drinkable water shortages in certain regions of the world, we can expect mass migrations. these could change the patterns of infection and drive outbreaks. migrants traversing tropical forests, or feeding with meat from game or carcasses, are but two scenarios that could be envisioned for the emergence of zoonotic infectious diseases [7] . several predictive models have been developed to evaluate the impact of climate change on the emerging infectious diseases: climex, dymex, miasma models. nevertheless, it remains difficult to predict when and where pathogen behavior will deviate from its typical pattern. climate change primarily affects vector-borne diseases by increasing rates of reproduction and biting and by shortening the incubation period of the pathogen they carry. ticks have gained spread from the mediterranean basin to northern and eastern europe, as well as appearing at higher altitudes. increased survival, density, and activity have also been reported following shorter, milder winters. climate change has also resulted in more days of activity per year for mosquitoes. as temperatures rise, more parasites are viable within regions ranging from the mediterranean and tropical zones, up to the balkans, russia, scandinavia, and the uk. for some ticks and fleas, temperatures over 25°c with relative humidity of over 85% are optimal for their proliferation and activity throughout the year [4, 8, 9] . for example, dengue fever is usually limited to a tropical latitude and a low altitude, since mosquito eggs and larvae lose viability with sustained low temperatures. during unusually warm summers, however, dengue has been reported as high up as 1700 m above sea level. warmer temperatures also result in smaller adult mosquitoes, which need to bite more frequently to feed themselves and be able to lay eggs, thus increasing the rate of transmission [8] . in contrast, the incidence of malaria has followed mixed patterns of increase and decrease along recent decades, and computer models have failed to predict the spread. the explanation for this is, in part, that climate change also results in diminished survival of the vectors (warming over 34°c affects the survival of both parasites and vectors), and in part, that the effect of climate change is non-linear and complex [10] . the frequency of emerging vector-borne infections varies per changes in land use, human activity, intervention maneuvers to eradicate the vector or prevent transmission to humans, drug treatment, and vaccines. both ecologic and economic changes may bring together rodents and humans. hunting activities may change vector distribution and large-scale animal movements can impact disease distribution. impoverishment of cities and overcrowding in slums, but also reforestation, golf club development, and the urbanization of rural suburbs facilitate exposure to ticks and rodents [11] . many patients with pidd require immunoglobulin replacement. immunoglobulin products have been demonstrated to improve outcomes in hepatitis a and measles [12, 13] . at least some neutralizing antibody is present directed to rsv and group b streptococci [14, 15] . this raises three distinct issues for patients: (1) difficulty in the diagnosis of infections in travelers because locally produced immunoglobulin may have antibody titers to local infections that are not typical for other countries, (2) safety concerns about locally produced immunoglobulin, if the patient resides in the location long enough to require immunoglobulin from a local provider, (3) the decision to use locally produced immunoglobulin products to provide superior prevention of infection compared to the patient's usual product. there are limited data on each of these subjects. serologic diagnostic testing in patients on immunoglobulin therapy will be addressed below in terms of issues related to lack of specific antibody produced by the patient (potentially) after infection. the converse can also be an issue. patients arriving from countries with significant occurrences of infections unusual in their current country may have igg antibodies to those infections simply through their immunoglobulin product and not reflecting a true infectious event in the patient. this can lead to diagnostic confusion when serologic testing demonstrates the presence of antibodies due to the infusion product. patients will often ask if immunoglobulin products from other countries are held to the same rigorous standards as their home country. today, commercially produced immunoglobulin is safe and tightly regulated. all commercially produced immunoglobulin around the world has one of the time-tested viral inactivation procedures such as nanofiltration, caprylate absorption, pasteurization, solvent/detergent, vapor heating, and low ph treatment. these procedures uniformly inactivate enveloped viruses. many emerging viruses are specifically tested for their ability to withstand the purification process. much has been learned since the transmission of hepatitis c viruses through immunoglobulin products in the early 1990s [16] . nevertheless, vigilance is important. in 2009, counterfeit immunoglobulin was identified. therefore, patients should ensure that they receive only brand name products while traveling. the subject of whether a patient's interests would be best served by using a local immunoglobulin product, with antibodies to pathogens that are prevalent in the community, or have their home physician ship their usual product, for which the patient has a known tolerance is hotly debated. patients with a history of intolerance to immunoglobulin products should not switch unless necessary. however, there are compelling reasons to consider a locally produced product when patients are in a foreign country for an extended period. it is known that antibodies to west nile virus have tracked with the distribution of the virus as it has emerged in several areas [17, 18] . titers in products using donors from the usa have higher neutralizing titers to west nile virus than those using donors from europe, although there is a 400-fold difference in titers between lots from the usa [18] . similarly, protective antibodies to concerning pathogens may be optimal in locally produced immunoglobulin. it is critical to inquire where the plasma source is derived. in most countries, the utilized immunoglobulin is produced in europe or the usa. having a different name does not ensure that the plasma pool comes from a different country. most lots of immunoglobulin are produced from 3000 to 60,000 plasma donors. the infrastructure to support such an endeavor is not easy to establish in each geographic area. serologic testing is commonly used for the diagnosis of infection. this approach relies upon detection or quantitation of antibodies made by the host against specific pathogens. the presence of igm antibodies against a specific pathogen indicates recent infection, while igg antibodies against a specific pathogen indicate past infection. importantly, serologic testing can only be applied for the diagnosis of infection if the host can mount a specific antibody response to the pathogen. conversely, serology cannot be relied upon to diagnose infection in the setting of immune deficiency where there is impairment of the specific antibody response, such as in the case of primary antibody deficiencies, cellular immune deficiencies, combined immune deficiencies, and other secondary immune deficiencies affecting t and/or b cell function. in these situations, the causative pathogen must be identified by alternate means such as culture of the organism, antigen detection, or molecular approaches (nucleic acid hybridization, nucleic acid sequencing, or oligonucleotide probe arrays). molecular approaches are particularly relevant for the diagnosis of infection in patients with pidd. signal and target amplification techniques can be coupled with nucleic acid hybridization or probe assays to allow detection of pathogen dna or rna that is present in very small amounts in clinical samples. in patients with pidd, vaccines could play an important role in preventing infections with vaccine-preventable diseases. even pidd patients may generate some protective responses [19, 20] . the decision to immunize such patients or not depends on the type and severity of pidd as well as the type of vaccine to be administered (live or inactivated) ( table 1 ). in general, inactivated vaccines are safe for pidd patients while immunization with live attenuated vaccines is a known hazard to patients with serious immunodeficiencies of t cell, b cell, and phagocytic cell origin (table 1 ). in less severe pidd, the vaccine can induce adequate protection as in healthy individuals or the efficacy may be reduced [20] [21] [22] . of note, immunoglobulin replacement therapy induces passive immune protection to some vaccine-preventable infections, such as measles, mumps, rubella (mmr), and varicella. in addition, live viral vaccines have greatly reduced efficacy while on immunoglobulin replacement. therefore, vaccine administration in patients receiving regular immunoglobulin replacement treatment should be withheld until at least 3 to 11 months (depending on dose) after cessation of such treatment, if cessation and vaccination are safe. in addition, pidd patients who have received hematopoietic stem cell transplantation but have incomplete immune reconstitution or are under immunosuppression should not receive live attenuated vaccines. in general, the decision of administering live viral vaccines should be made by clinical immunology experts [23] . in developing countries where polio is still endemic and oral polio vaccine is essential for eradicating the disease, it is of utmost importance that all pidd patients and family members should not receive live oral polio (opv) because of the reported prolonged excretion of the virus for months and even years [24] . in addition, vaccine-associated paralytic polio is a real risk for some with pidd. these patients and family members should receive inactivated polio vaccine (ipv) instead of opv. similarly, the hazards of administering bacillus calmette-guerin (bcg) vaccine to pidd patients have been documented. in a series of 349 bcg, vaccinated severe combined immunodeficiency (scid) patients from 28 centers in 17 countries, 34% of scid patients developed disseminated bcg infection and had the worst outcome [25] . patients with chronic granulomatous disease vaccinated with bcg also developed local and disseminated bcg infection. recently, vaccine strains of rubella virus were found to be associated with skin granulomas in pidd patients [26] [27] [28] . siblings and household contacts of patients with suspected or diagnosed pidds should receive all the national immunization scheduled vaccines. ipv should be substituted for op in families where an antibody-deficient patient exists. yearly influenza vaccination of family members is recommended in order to reduce the risk of household-social transmission [20, 22, 29] . diseases where routine vaccination has reduced incidence can occasionally experience a resurgence in times of economic hardship with reduced attention to vaccination. diphtheria is currently seen in venezuela for this reason. war and disruption of health infrastructure are other common reasons for resurgence in vaccine-preventable diseases. in other settings, antivaccination sentiment has led to outbreaks of diphtheria, measles, and mumps. an additional consideration is that not all countries provide the same level of vaccination, and therefore, vaccine-preventable illness can still be seen regionally. these outbreaks represent a significant risk to patients with pidd. a universal consideration for patients with pidd is the concern about antibiotic resistance, which varies dramatically around the world. for certain high impact infections, the emerging antibiotic resistance patterns will be discussed below. antimicrobial resistance occurs naturally, but misuse and overuse of antimicrobials are accelerating this process. in nearly every country, antibiotics are overused and misused in people and animals leading to antibiotic resistance in every country. key resistance patterns to common bacteria include emergence of carbapenem-resistant klebsiella pneumoniae around the world with high frequency of resistance (due to different mechanisms) in the mediterranean, with greece, italy, and turkey having endemic spread of this pathogen [17] . carbapenem-resistant strains among other genera of enterobacteriaceae have also been recognized. they are particularly common in greece, but have been found widely distributed [30] . the new delhi metallo-beta-lactamasemediated resistance, which is endemic in the indian subcontinent but becoming increasingly spread worldwide, is a growing concern [30, 31] . as a common cause of urinary tract infections, colistin is the only recourse when carbapenemresistant enterobacteriaceae, and colistin resistance has recently emerged in small outbreaks throughout the world [32] . in these cases, the infection is essentially untreatable. fluoroquinolone-resistant escherichia coli, a common cause of urinary tract infections, now accounts for over half of the isolates in some asian countries [33, 34] . t he emergence of resistance to antibiotics in grampositive pathogens has become a major international problem as there are fewer, or even sometimes no effective, antimicrobial agents available for infections caused by these bacteria. methicillin-resistant staphylococcus aureus is common in many countries and in fact has spawned a nomenclature recognized by the general public: mrsa. several studies have reported resistance to the newer antimicrobial agents like linezolid, vancomycin, teicoplanin, and daptomycin [35] . thus far, these isolates appear to be uncommon and have been found in <1% of isolates in brazil, china, ireland, and italy, with nearly undetectable rates elsewhere. vancomycinresistant enterococcus (vre) is growing in frequency and can now be a cause of primary bacteremia in immunocompromised individuals [36] . a key message is that antibiotic resistance is increasing (generally) and travelers should be alerted to resistance to commonly encountered organisms, and if antibiotic prophylaxis is required, their prophylaxis is adjusted. neisseria gonorrhoeae is a specific organism for which resistance has become especially problematic. it has progressively developed resistance to virtually all antimicrobial agents since introduction of sulfonamides in mid-1930s. the current treatment guidelines recommend dual antimicrobial therapy (ceftriaxone 250-500 mg × 1 plus azithromycin 1-2 g × 1) as first-line regimen [37, 38] . although dual therapy is very effective, development of concomitant ceftriaxone and azithromycin resistance is likely to emerge [39] . the risk of untreatable n. gonorrhoeae demands better global antimicrobial surveillance system, clinical trials on combined therapy of existing drugs as well as novel agents in monotherapy, and development of a gonococcal vaccine. for pidd patients, guidance on barrier methods for the prevention of sexually transmitted diseases should be a part of any pre-travel counseling. mycobacteria tuberculosis (mtb) is an age-old pathogen with emerging resistance. drug-resistant tb, fueled by the hiv epidemic, is a global threat. in 2015, who estimated 480,000 new cases of multidrug-resistant tb (mdr-tb) and an additional 100,000 new cases of rifampin-resistant tb (rr-tb) who would also require mdr-tb treatment. treatment of mdr-tb and mycobacterium bovis disease is beyond the scope of this text and reader is referred to recent who mdr treatment guidelines [40] . regions of the world with >6% mdr tb include regions of azerbaijan, kazakhstan, russia, uzbekistan, china, georgia, and eastern europe. extensively drug-resistant tb (xdr tb) refers to mtb resistant to isoniazid, rifampin, any fluoroquinolone and at least one second-line drug. xdr tb has been reported in 105 countries. on average, 10% of patients with mdr tb have xdr tb. as is true for all types of mtb, xdr tb is contagious and small outbreaks related to person-person transmission have been reported. non-tuberculous mycobacteria (ntm) cause significant systemic infections in patients with defects of the il-12/ ifnγ/stat1 axis as well as in gata2 deficiency and can cause significant pulmonary infections in pidd patients with bronchiectasis. compared to tb, ntm is acquired from the environment and not from person-to-person transmission; therefore, acquisition of antibiotic-resistant strains is less common. however, in these individuals with pidd, ntm disease is often chronic and can be difficult to eradicate, and resistance can then easily develop during therapy. using combination of antibiotics is essential, and consultation with those familiar with treatment of treatment refractory ntm disease is recommended. aspergillus species are ubiquitous inhaled molds with worldwide distribution that cause opportunistic infections in immunocompromised patients [41] . aspergillosis also occurs in pidds associated with quantitative and/or qualitative phagocyte defects; it develops most frequently in chronic granulomatous disease (cgd) patients (prevalence,~40%), while it is seen less often in patients with gata2 deficiency, card9 deficiency, and congenital neutropenia syndromes [42, 43] . upon inhalation, aspergillus species cause invasive pulmonary disease in susceptible hosts with the exception of card9 deficiency, where aspergillosis has a predilection for extrapulmonary tissues with sparing of the lungs [44] . diagnosis is established by fungal culture and/or histopathology showing acute-angle septate hyphae and/or detection of galactomannan, an aspergillus cell wall component released during invasive infection, in serum or bronchoalveolar lavage fluid [41] . while azole-susceptible aspergillus fumigatus is still the most common infecting species in pidd patients, the emergence of azole-resistant a. fumigatus and nonfumigatus aspergillus species underscores the importance of a high index of suspicion in patients who do not respond to front-line voriconazole treatment [45] . the advent of fungal molecular diagnostics has demonstrated that patients with pidds are more prone to infections by uncommon low-virulence aspergillus species with intrinsic resistance to azole antifungal agents that do not infect patients with iatrogenic immunosuppression. these primarily include aspergillus viridinutans, aspergillus tanneri, and neosartorya udagawae, which pose major diagnostic and therapeutic challenges due to their impaired sporulation and propensity for contiguous and distant tissue spread, respectively. in addition, acquired azole resistance in a. fumigatus can be seen in patients on long-term exposure to azole drugs used as treatment and/or prophylaxis [46] . azole resistance in these strains is predominantly caused by point mutations in the lanosterol 14α-demethylase gene that encodes the cyp51a protein, the primary target of azole drugs. importantly, infection by azole-resistant a. fumigatus strains without prior exposure of patients to azole antifungals has recently emerged as an important global health concern due to the widespread use of sterol demethylase inhibitor fungicides in agriculture that results in cross-resistance with the triazole antifungals used in clinical practice [47] [48] [49] . fungicide-driven azole-resistant environmental aspergillus strains were first observed in the netherlands in 2007 and have since then been documented in other parts of europe, south and north america, the middle east, australia, africa, and asia. the prevalence of these azole-resistant aspergillus strains among clinical aspergillus strains recovered from patients in 19 european countries was reported to be 3.2%, while alarmingly in some areas >20% of recovered strains exhibited azole resistance [50] . the emergence of such aspergillus environmental strains poses serious threats to the treatment of immunosuppressed patients. mortality rates as high as 88% have been seen due to delays in diagnosis and suboptimal treatment with azole antifungals [51] . although no prospective data exist for the treatment of patients with such resistant fungi, the use of amphotericin b-and echinocandin-based regimens are preferred over azoles [52] . in the absence of azoles, the lack of alternative oral antifungal agents is particularly challenging for pidd patients such as those with cgd who require long-term suppressive antifungal treatment. candida species are commensal yeast fungi that colonize the mucosal surfaces of~60% of healthy individuals [53] . when perturbations in immunity and/or microbiota occur, candida causes opportunistic mucosal or systemic infections that depend on clearly segregated immune responses for host defense. specifically, t cells of the th17 program are critical for mucosal and phagocytes for systemic immunity [54] . indeed, a proportion of patients with cgd and complete myeloperoxidase deficiency develop systemic, but not mucosal candidiasis [42] , whereas patients with monogenic syndromes of chronic mucocutaneous candidiasis due to mutations in the il-17 signaling pathway (il17ra, il17rc, il17f, act1) or in other genes that adversely affect th17 differentiation (rorc, stat3, stat1, aire, dock8, stk4, irf8) do not develop systemic candidiasis. the only known pidd to date that results in combined mucosal and systemic candida infection susceptibility is card9 deficiency. systemic candidiasis in card9-deficient patients has a predilection for the central nervous system, associated with brain-specific impaired recruitment and effector function of neutrophils [55] [56] [57] . diagnosis of candida infections is established by culture. azole-susceptible candida albicans is still the most common infecting species in pidd patients; however, emergence of azole-resistant c. albicans is not uncommon during chronic azole use, making long-term therapy challenging due to lack of alternative oral antifungal treatment options [58] . beyond c. albicans, non-albicans candida species can rarely infect pidd patients, some of which have intrinsic resistance to azole antifungals, including candida glabrata and candida krusei [59] . most recently, candida auris has emerged as a global public health concern due to its resistance to antifungal drugs, high virulence potential, propensity for health careassociated horizontal transmission and outbreaks in health care settings, persistence in the human skin and hospital environment, inherent resilience to antiseptics, and misidentification by routine biochemical methods as other yeasts (most often candida haemulonii, but also candida famata, rhodotorula glutinis, or saccharomyces cerevisiae). c. auris was first recovered from the ear canal of a patient in japan in 2009 and has since then been reported to cause life-threatening infections and hospital outbreaks in europe, asia, africa, the middle east, and south and north america [60] [61] [62] [63] . most of the reported strains of c. auris have intrinsic resistance to fluconazole and other triazole antifungal agents, while a significant proportion of strains has elevated minimum inhibitory concentrations to amphotericin b and echinocandins, with some strains reportedly resistant to all three classes of azoles, polyenes, and echinocandins [64] . avoidance of azole antifungals is important in c. auris-infected patients, and echinocandinor amphotericin b-based regimens are preferred, guided by strain-specific in vitro susceptibility patterns. this section on vector-borne infections is a major focus of this review because the infections are often more severe in immunocompromised individuals and because there are mitigation strategies that should be considered even in the absence of defined medical treatments for infection. prevention of mosquito bites depends somewhat on the endemic species but there are generalizations. the use of a mosquito repellant such as deet, oil of lemon eucalyptus, ir3535, or picaridin is as important as using long sleeves and long pants while in an affected area. deet and picaridin are safe in pregnancy, and some data support their greater efficacy [65] . air conditioning and fans tend to keep mosquitoes away but netting at night is essential in mosquito-prone areas. light-colored clothing is less attractive to mosquitoes than dark clothing, and scented detergents and use of dryer sheets tend to attract mosquitoes, hence should be avoided. aedes species prefer to bite indoors and thrive in urban areas with small puddles of water. they bite most frequently around dawn and dusk. anopheles species have very similar behaviors. culex mosquitoes, in contrast, bite primarily at night. tick and fly bite prevention is focused on physical and chemical prevention. for ticks, physical inspection for biting ticks should also be incorporated. arthropod-borne viruses (arboviruses) are transmitted to humans through the bites of infected insects: mosquitoes, ticks, sand flies, or midges. some arboviruses can be transmitted through blood transfusion, organ transplantation, perinatal transmission, consumption of unpasteurized dairy products, or breastfeeding. there are >100 arboviruses causing human disease. most arboviral infections are asymptomatic. infectious manifestations range from mild febrile illness to severe encephalitis. arboviral infections are often categorized into two primary groups: neuroinvasive disease and non-neuroinvasive disease. tables 2 and 3 list the encephalitigenic viruses and the febrile/hemorrhagic disease causing viruses. in this section, we will highlight west nile virus, the most common of the encephalitogenic arboviruses. west nile virus is a single-stranded mosquito-borne rna virus of the family flaviviridae. the natural transmission cycle of the virus occurs in culex mosquitoes and birds. humans and horses are dead-end hosts for the virus. the most common mode of transmission to humans is through the bite of infected mosquitoes. other less common modes of transmission include blood transfusions, organ transplantations, occupational exposure in laboratories and mother-to-child transmission during pregnancy, childbirth, and breastfeeding. west nile virus has been diagnosed in >2000 people in the usa with slightly more than half having neuroinvasive disease. since 1999, >40,000 people in the usa have been infected. it is also common in africa, europe, and asia [66] . infection with west nile virus is asymptomatic in most individuals [67, 68] . the incubation period lasts for 2 to 6 days. however, it can be significantly longer in immunocompromised hosts. clinical manifestations following infection develop in 20-40% of those infected and include fever, headache, myalgia, arthralgia, vomiting, and rash. severe neuroinvasive disease leading to meningitis, encephalitis, and flaccid paralysis develops in less than 1% of infected individuals. the overall case fatality is approximately 10% which is a disproportionately high mortality in patients with encephalitis and myelitis. diagnosis of west nile virus rests on demonstration of specific antibody responses especially specific igm antibodies in the serum or csf of infected individuals by enzyme immunoassays. plaque reduction neutralization tests can differentiate cross-reactive antibodies. detection of virus in csf, blood, or tissue specimens by culture or pcr is particularly useful in immunosuppressed individuals who may have impaired serological responses. west nile virus has been reported in the context of both primary and secondary immunodeficiency. severe neurological manifestations have been reported in hiv-positive individuals, individuals receiving immunosuppressive therapy including rituximab, and individuals with pidd. infection with wnv has been reported in individuals with common variable immunodeficiency, idiopathic cd4 lymphopenia, gata2 deficiency, and a case of probable good's syndrome [69] [70] [71] . individuals with antibody defects, neutropenias, and impaired t cell responses are potentially at an increased risk of severe manifestations of wnv disease including severe neurological involvement. this section highlights the four important non-neuroinvasive arboviruses based on current geographical distribution: dengue, yellow fever, zika, and chikungunya (table 3) . approximately 100 countries/territories have reported local transmission for both chikungunya and dengue viruses [72] . dengue is due to infection with one of four dengue virus serotypes, transmitted by a mosquito (typically aedes aegypti). this febrile illness affects all ages with symptoms appearing 3-14 days after the infective bite. symptoms range from mild to high fever, with severe headache, musculoskeletal pain, and rash. severe dengue (also known as dengue hemorrhagic fever) occurs in 0.5-5% of cases and is characterized by fever, abdominal pain, persistent vomiting, bleeding, and breathing difficulty and is a potentially lethal complication [73] . paradoxically, the main risk factor for dengue hemorrhagic fever is pre-existing antibodies. early clinical diagnosis and comprehensive management by experienced clinicians increase survival. dengue is ubiquitous throughout the tropics with the highest infection rates in the americas and asia. dengue is now endemic in 100 countries, causing up to 50 million infections a year and 22,000 deaths, mainly among children. over half of the world's population inhabits areas at risk for dengue infection [74] . the presence of a. aegypti in over 125 countries potentially puts almost the whole world at risk of becoming infected with this virus. pcr is widely used as serologic methods to diagnose dengue. immunity to one serotype does not confer protection against the other three serotypes, and heterologous antibody may be a risk factor for hemorrhagic dengue [73] . the natural history of dengue has been studied in hiv patients where hiv did not appear to increase severity. there have been no reports of patients with pidd having dengue; however, dengue infection after solid transplantation has been reported [75] [76] [77] [78] with some patients having severe complications suggesting that t cell compromise in pidd could be a risk for severe disease. there are no antiviral medications utilized for dengue virus. care of patients with hemorrhagic disease requires meticulous approach to fluids and coagulation status. one dengue vaccine has been registered in several countries (cyd-tdv) for individuals from 9 to 45 (or 60) years old. it is a live attenuated recombinant tetravalent vaccine with backbone of the attenuated yellow fever 17d virus genome with the prm and e genes that encode the proteins from the four wild-type dengue viruses. the who has suggested its use in regions where seroprevalence of dengue virus of any serotype is 70% or greater, but has not recommended it to hiv-infected, immunocompromised individuals, nor pregnant or lactating women [79] . most people infected with the yellow fever virus have no illness. symptoms of yellow fever include sudden onset of fever, chills, headache, musculoskeletal pain, nausea, vomiting, fatigue, and weakness. the incubation period is typically 3-6 days, and symptoms may appear after return from travel. most people improve after the initial presentation, but 15% of cases progress to develop a more severe form of the disease, usually after a day of presumed recovery. the severe form is characterized by high fever, jaundice, bleeding, and eventually shock and failure of multiple organs [80] . yellow fever virus is an rna virus that belongs to the genus flavivirus. it is transmitted from mosquitoes after biting an infected primate. it is widely distributed in the equatorial tropics [80] . aedes species of mosquitoes are primarily responsible for transmission. large epidemics of yellow fever occur when the infection enters heavily populated areas with a high mosquito density and where most people have little or no immunity. west africa has undergone a large-scale vaccination campaign with impressive results and yellow fever is now uncommon in west africa [81] . serologic testing for yellow fever is the diagnostic standard. pcr can be performed on tissue samples. there are no published studies of yellow fever in immunocompromised people, but the elderly, very young, people with autoimmune disease, or who are post-thymectomy are at risk from the attenuated vaccine strain. thus, it seems likely that any immunodeficiency would be associated with more severe wildtype disease. currently, no specific antiviral drug for yellow fever exists. treatment of dehydration, liver and kidney failure, and fever improves outcomes. the yellow fever vaccine is highly effective; however, immunodeficient patients should not receive it. infection with zika virus is often asymptomatic. it represents a mild infection for those who have any symptoms [82] . the zika virus has been detected in urine, semen, and saliva of infected individuals, and transmission from transfusion and sexual relations has been reported. it is also detectable in breast milk, but breastfeeding-associated transmission has not been reported so far [83] [84] [85] . contact with highly infectious body fluids from patients with severe zika infection has also been suggested as a possible mode of transmission [86] . of tremendous importance is the presence of prolonged shedding of zika virus in a congenitally infected newborn [87] . the main public health risk of zika virus is microcephaly in newborns from infected mothers [88] . zika virus is capable of infecting human neural progenitor cells in vitro. infection results in disruption of cell cycle, increased cell death, and attenuated neuron growth [89] . zika is not thought to be a major risk for people with pidd (based on the experience with hiv patients, but our recognition of zika is very recent. there is no known specific treatment for zika; however, there is an important effort to develop a vaccine. chikungunya fever is an acute febrile illness caused by the alphavirus, chikungunya virus. the incubation period is usually 3-7 days after the bite of an infected aedes mosquito. there is abrupt onset of high fever, and the fevers can be biphasic [90, 91] . severe polyarthraligias develop after the onset of fever. the joint pains can affect any joint, but the pattern is usually symmetric and a true acute arthritis is not uncommon. the proportion of infected people with rash has varied across studies. when seen, the rash appears after the fever as a truncal maculopapular type of rash [92] . cervical adenopathy is another common feature of infection. death is uncommon in chikungunya, but serious complications such as myocarditis have been seen. over half of the patients report continued joint symptoms 1 year after acute illness and 12% have long-term joint symptoms [93] . chikungunya originated in central/east africa. in forests, the virus circulates in aedes mosquitoes and non-human primates. in urban centers, the virus circulates between humans and mosquitoes similar to the pattern of dengue. there have been periodic urban outbreaks in asia and africa since 1960 with an acceleration in spread since 2004 [94] . an important consideration is the periodic outbreaks with high attack rates in naïve populations. areas at risk currently are east africa, central africa, la reunion, india, and southeast asia. diagnostic testing utilizes pcr or serology. the threat to immunodeficient patients is not entirely clear. there are a few provocative cases where the immunocompromised appears to have been associated with fewer joint symptoms, but there were two patients, medically immune compromised, who had very severe disease [95, 96] . this suggests that the presentation may be atypical and the course may be severe in immunodeficient people. treatment is supportive, although chloroquine, acyclovir, ribavirin, interferon-ɑ, and steroids have limited preclinical data to support clinical trials. babesia microti (the main species in the usa) infection can be asymptomatic, but many people develop fever, chills, headache, myalgias, anorexia, nausea, or fatigue [97] . babesiosis often causes hemolytic anemia. b. microti is spread by ixodes scapularis ticks in the usa and babesia divergens (the main species in europe) is spread by ixodes ricinus. symptoms begin 1-3 weeks after a bite from an infected tick with b. divergens having a higher mortality rate and greater symptomatology compared to b. microti. the main geographic areas involved are the coastal eastern usa and cattle breeding areas throughout europe. the diagnosis is usually by inspection of a blood smear or through serology. a pcr test has just been developed. immunodeficiency, asplenia, and older age are recognized risk factors for severe disease and even death [98] [99] [100] . thus, congenital asplenia would be considered a major risk for severe disease. a combination of atovaquone and azithromycin is generally used for therapy, although clindamycin and quinine have been used with success. patients with severe illness have been treated with exchange transfusions. five different types of plasmodium (plasmodium falciparum, plasmodium vivax, plasmodium ovale, plasmodium malariae, and plasmodium knowelsi) infect humans. malaria is transmitted primarily by female anopheles mosquitoes. symptoms vary depending on the type of plasmodium involved but usually include high fever, chills, and headache. in some cases, the illness can progress to severe anemia, kidney and respiratory failure, and death. the incubation period typically ranges from 9 to 14 days for p. falciparum, 12 to 18 days for p. vivax and p. ovale, and 18 to 40 days for p. malariae. in p. vivax and p. ovale infections, relapses can occur months or even years without symptoms. p. vivax and p. ovale have dormant liver stage parasites that must be specifically eradicated through medical therapy. malaria has been a global health concern throughout history and is a leading cause of death and disease across many tropical and subtropical countries. over the last 15 years, new control measures have reduced malaria by over half [101] . the democratic republic of the congo and nigeria account for over 40% of the estimated total of malaria deaths globally. high rates of malaria are seen in india as well. nevertheless, malaria exists in most tropical regions of the americas, africa, and asia [101] . the diagnosis of malaria depends on the demonstration of parasites in the blood, usually by microscopy. the threat to immunodeficient patients is not entirely clear, but patients with hiv seem to have no additional burden of disease other than an increase in placental malaria, suggesting that t cells are not central to the defense of malaria [102, 103] . asplenia is a known risk factor for severe malaria [104] . antibodies appear to be both protective and pathologic [105, 106] . treatment and prophylaxis depend on the region of the world because the parasites and resistance are highly variable and highly dynamic. therefore, it is best to consult an infectious disease specialist familiar with the prophylaxis before travel and for treatment of acute cases. leishmaniasis is due to infection with an obligate macrophage intracellular protozoa of the genus leishmania. it causes a spectrum of disease ranging from a cutaneous ulcer to mucosal disease and the most severe form, visceral leishmaniasis (vl). the liver, spleen, and bone marrow are major sites of parasite growth and disease pathology in vl [107] . purely cutaneous leishmaniasis is most often caused by leishmania major, leishmania. tropica, leishmania aethiopica, leishmania infantum, and parasites belonging to the leishmania mexicana complex, the leishmania braziliensis complex, and the leishmania guyanensis complex. mucocutaneous disease is most often due to l. braziliensis complex, leishmania panamensis, leishmania amazonensis, and rarely by leishmania guyanensis. vl is most often caused by leishmania donovani and leishmania infantum (previously l. chagasi) [108] . cutaneous leishmaniasis can have many variations but is most often an ulcer that develops after an indolent papule. the incubation period ranges from weeks to months. the ulcer usually heals within months to years, and there can be mild adenopathy. mucocutaneous leishmaniasis follows a cutaneous ulcer and is only caused by l. braziliensis parasites. oral and respiratory mucosa are most often involved with granulomatous lesions that may be extremely destructive. vl is associated with fever, lymphadenopathy, hepatosplenomegaly, wasting, hypoalbuminemia, and pancytopenia. this picture evolves over months to years. there can be secondary immune deficiency due to the pancytopenia. the epidemiology has changed dramatically and has been impacted by climate change [109] . the sand flies that spread the parasite are affected by temperature and rainfall. in most endemic regions, leishmania has a patchy distribution due to micro-ecologic factors. poverty has been demonstrated to be a major risk factor for leishmaniasis [110] . it has been estimated that up to half a million new cases of vl occur every year, but the majority are in resource-poor countries such as bangladesh, nepal, india, sudan, ethiopia, and brazil. emergence of resistance to antimony-based drugs has also led to a major resurgence of disease. the primary reservoir for leishmania is forest rodents, but dogs are increasingly important. the growing spread of leishmania is due to a combination of factors, and now 88 countries have reported cases. immunodeficient patients are more susceptible to infection, and relapse occurs more frequently [111] . the risk of developing vl is estimated to be between 100 and 2300 times higher in hiv-infected than in non-hiv-infected individuals [112] , and these patients have higher rates of treatment failure with the illness often taking a prolonged chronic course and higher mortality rates [113] . a similar picture has been seen in patients with vl-infected post-kidney transplantation [114] . dendritic cells, t cells, and the generation of reactive oxygen species have been shown to be essential for parasite control [115] [116] [117] . pidd with impaired il-12 production have been associated with severe disease [118] . a patient with cd40l deficiency, associated with poor il-12 production, had chronic leishmania and died in spite of aggressive treatment. vl has been reported in cgd patients [119] . six cgd patients were infected by leishmania, and they developed hemophagocytic syndrome with a poor outcome for one of them [120] . the diagnosis of leishmaniasis is usually by visual inspection for parasites. immunofluorescence microscopy, direct agglutination, skin test, and pcr have been used. treatment is long-term and difficult. emerging resistance to first-line treatment is increasingly problematic. pentavalent antimonials are the mainstay of treatment in most countries, but liposomal amphotericin is widely used where resistance occurs. newer drugs with more favorable side effect profiles have been used in certain geographic settings: miltefosine, paromycin, and sitamaquine. rickettsiae are small gram-negative bacteria. they are obligate intracellular parasites, and the primary target in humans appears to be endothelial cells with subsequent thrombosis and clinical presentation of vasculitis [121] . the rickettsiaceae family, originally defined by non-specific phenotypic characteristics, was reclassified into different strains and subspecies based on gene sequencing and genetic phylogeny ( table 4 ). the clinical presentation of rickettsial disease can vary, but the classic triad of fever, rash, and headache still provides major clues for the diagnosis [122] [123] [124] . however, rash is not an obligatory sign, and the incidence of rash can range between 100% for rickettsia conorii infection,~90% for rickettsia rickettsii, 30% for rickettsia africae, and less than 10% in the case of anaplasma phagocytophilum infection. therefore, fever in patients with exposure to a potential vector should raise a concern for a rickettsial disease, especially if there is also evidence of rash, inoculation eschar, or localized lymphadenopathy. additional supporting laboratory findings can include neutropenia, thrombocytopenia, and increase in liver transaminases. the geographic distributions of rickettsioses and ehrlichioses are mostly dependent on their vector distribution [125] . as such, louse-borne and flea-borne are worldwide, reflecting the worldwide distribution of lice and fleas, with a tendency to parasite poor people in cold places and, characteristically, during wars. ticks, on the other hand, depend on their environment and most do not have a worldwide distribution. with the exception of the dog tick, vector for r. conorii in asia and north africa, r. rickettsii in the usa, rickettsia massiiae and erhlichia canis worldwide, most other tick-borne diseases are restricted to areas of the world correlating with the distribution of their vector [126] . for that reason, it should be anticipated that climate and environmental changes will affect vector distribution and its reservoir host and, hence, the geography and epidemiology of tick-borne diseases [10, 127, 128] . diagnosis presents a challenge, as it is extremely difficult to grow these organisms in culture. immunohistochemistry and pcr can be helpful. the severity of rickettsial disease varies with the causative agent and the host. r. rickettsii, rickettsia prowazekii, and orienta tsutsugamushi are considered most pathogenic. as for host factors, although severe and fatal cases have been described in healthy immunocompetent hosts [129, 130] , there is evidence to suggest that children under the age of 10 [130] and immunocompromised hosts either secondary to hematologic malignancies, immunosuppressant treatment for organ transplantation, or hiv infection are at a greater risk to develop more severe disease with higher case fatality rates [131, 132] . all rickettsiaceae are intracellular pathogens, and one could expect an increased risk for severe disease in pidds with abnormal t cell function. five to 7 days of doxycycline is the preferred treatment for non-pregnant adults and children. treatment should not be delayed while awaiting diagnostic testing [133] and can be given to children despite a minimal risk for dental staining. alternative treatments include azithromycin for mild disease [134] and chloramphenicol for pregnant women. anaplasma is an intracellular bacterium that infects wild and domestic mammals, including man. a. phagocytophilum was formerly known as human granulocytotropic ehrlighiosis but is now known as human granulocytotropic anaplasmosis [135] . e. chaffeensis infects monocytes and causes human monocytic ehrlichiosis [136] . anaplasma and ehrlichia have historically cycled within non-human enzootic hosts, and man has become infected through increasing interactions with the environment. ehrlichia and anaplasma are transmitted by ixodes species of ticks, and their ranges include the eastern usa, south central usa, and scattered regions of europe, as far north as sweden. these infections have not been seen in humans in the southern hemisphere, but there are reports of organisms being identified [137] . a less common mode of transmission is through transfusions. the symptoms of ehrlichia and anaplasma infections are similar [136] . abrupt onset of an influenza-like illness occurs about 12 days after a tick bite. ehrlichia can cause a mild rash (30% of adults and 60% of children), but rash is uncommon in anaplasma infections. highly suggestive laboratory features are leukopenia and thrombocytopenia. mortality in the general populations appears to be <5%, but icu admission is not uncommon. hemophagocytosis has been described with anaplasma [138] and ehrlichia [139] . both infections are more severe in any setting of immune compromised, including asplenia [129, 135] . the diagnosis is typically made by pcr, and doxycycline is the recommended treatment. intracellular inclusions can be seen on cbc smears (more often in anaplasma than ehrlichia). an uncommon but well described facet of these infections is that the tick vector can also transmit borrelia burgdorferi and babesia microti, and simultaneous infection with multiple organisms can occur. c. burnetii is a highly pleomorphic gram-negative coccobacillus and the causative agent of q fever. q fever is a zoonosis, and the most common reservoirs are cattle, sheep, and goats but many other animals can be infected by c. burnetii [140, 141] . when infected, these domestic animals can shed the organism in urine, feces, milk, and especially birth products. the pathogen survives within the phagolysosome of host cells, and a spore stage has been described. this spore stage explains the ability of c. burnetii to survive in unfavorable environmental conditions, and it can be an environmental risk for months to years after shedding from an infected animal. q fever is considered an occupational disease affecting people with direct contact with infected animals; however, indirect contact through exposure to contaminated animal products has also been described to cause disease outbreaks. humans are infected by inhalation of contaminated aerosols. following an average incubation period of 20 days, infected patients can present with severe headache, fever, chills, fatigue, and myalgia. other signs and symptoms depend on the organs involved. in contrast to rickettsial diseases described above, rash rarely occurs in the early stages of the disease. c. burnetii can cause a range of clinical symptoms. a self-limited febrile illness is probably the most common form of q fever. pneumonia, either atypical or severe, is also common and can be a part of acute q fever syndrome. in contrast, a variety of manifestations can be recognized in chronic q fever, including endocarditis, endovascular infection, osteomyelitis, hepatitis, interstitial pulmonary fibrosis, prolonged fever, and purpuric vasculitic rash. q fever diagnosis is based on serologic testing with indirect immunofluorescence being the best for differentiating between acute and chronic q fever (high antiphase i antigen titer). the treatment of choice for acute q fever is doxycycline, with co-trimoxazole, chloramphenicol, or rifampin being an accepted alternative. there is no agreement on the treatment for q fever endocarditis, and a combination of doxycycline with either fluoroquinolone or hydroxychloroquine is recommended. there is also controversy regarding the duration of treatment, ranging from 2 years to indefinite treatment. old evidence suggests that q fever is more common in immunocompromised patients. a french study showed higher incidence of antibodies to c. burnetii in hiv positive compared to hiv-negative patients (10.4 vs 4.1%). in addition, 5 out of 68 hospitalized patients were hiv positive (7.3%), suggesting a more frequent symptomatic disease [142] . a smaller similar study performed in central africa failed to show increased incidence of seropositivity in hivpositive patients [143] . two case reports describe severe disease in immunocompromised patients. the first was a case of fatal q fever disease in an 11-year-old male with cgd [144] . the patient was treated with broad spectrum antibiotics, but without coverage for q fever. the second case was a 53-yearold asplenic male who presented with fever, jaundice, and encephalopathy and was diagnosed with acute q fever [145] . the patient was successfully treated, but the two case reports could suggest susceptibility in cases of phagocytic disorders. the bartonellaceae are fastidious, facultative intracellular gram-negative bacilli (table 5 ). most species infect primarily non-human animals, and in most cases, human are considered incidental hosts. the documented common human pathogens include bartonella bacilliformis, bartonella henselae, and bartonella quintana, and it is believed that humans are the primary mammalian reservoir of b. quintana. infection occurs through inoculation of bartonella-infected arthropod feces into breaks in the skin. the epidemiology of bartonella infection in humans follows the distribution of the vector. as such, infection with b. bacilliformis follows the distribution of the sand fly vector (lutzomya) and is confined to the andes mountain in peru, ecuador, and colombia at heights of between 500 and 3200 m. the human body louse pediculus humanus is the vector of b. quintana, which explains the global distribution of this pathogen and worldwide outbreaks of trench fever, mostly in conditions of poor sanitation and upon exposure to body lice. trench fever was responsible for over a million infections during world war i. fever, either abrupt or indolent in onset, with a maculopapular rash, conjunctivitis, headache, myalgias (most often affecting legs), and splenomegaly was described. urban b. quintana infections occur most often among homeless and have a distinct clinical picture with fever as the most common manifestation. endocarditis occurs in many [146] . bartonella henselae is globally endemic, and domestic cats are a major reservoir. the major arthropod vector of b. henselae is the cat flea, which is responsible for cat-to-cat transmission. human infection, called cat scratch disease, is assumed to involve inoculation of bartonella-infected flea feces into the skin during a cat scratch. b. henselae causes primarily adenopathy and neurologic symptoms [147] . b. bacilliformis causes a condition with two phases: the acute phase with fever, anemia, and transient immunosuppression followed by nodular dermal eruption [148] . recently appreciated are the ongoing systemic features during the eruptive phase such as arthralgias, adenopathy, and anorexia [149] . diagnosis of bartonella-associated diseases can be achieved by direct examination of clinical material, bacteriologic culture methods, serologic and immunocytochemical studies, pcr-based assay, or combination of these methods. bartonella infection can present differently in immunocompromised hosts [150] . in addition to higher prevalence of bartonella infection in hiv patients [151] , both b. quintana and b. henselae can induce neovascular proliferation which might involve the skin, lymph nodes, and a variety of internal organs including the liver, spleen, bone, brain, lung, bowels, etc. these neovascular lesions, known as bacillary angiomatosis/peliosis (ba/bp), were initially described in hiv-infected patients with advanced disease and was later described in other immunocompromised hosts secondary to immunosuppressant treatment for solid organ transplantation or hematologic malignancy [152] [153] [154] [155] . cutaneous ba lesions can vary in presentation and can be subcutaneous, dermal nodules, single or multiple papule that can be flesh colored, red, or purple. lesions may ulcerate and bleed. they can change in number and size (millimeters to centimeters; few to hundreds) and can involve mucosal surfaces or deeper soft tissues. similar variation can be seen with visceral involvement. histologically, lesions consist of lobular proliferation of small blood vessels and neutrophilic predominant cell infiltration. the term bacilliary peliosis is used to describe bloodfilled cystic spaces mostly involving the liver, spleen, and lymph node. pathogenic bacteria can be isolated from vascular lesions. while both pathogens were associated with cutaneous lesions, only b. henselae was associated with visceral bp [156] . based on hiv literature, it is reasonable to expect an unusual presentation of bartonella infection especially in pidds involving t cell dysfunction. bartonella infection was described as a cause for hepatitis in a single cd40l deficiency patient [157] . in addition, since cases of granulomatous disease due to bartonella infection [158] [159] [160] have been described, it should be considered in the differential diagnosis of pidd with granulomatous inflammation. borrelia spp. the genus borrelia belongs to the spirochaetaceae family. it includes b. burgdorferi which causes lyme disease and species that cause relapsing fever. the latter are further divided into tick-borne species and louse-borne species. louse-borne relapsing fever (lbrf) is caused only by borrelia recurrentis and is spread by human body louse. the disease was epidemic in the early twentieth century, and it is estimated that more than 50,000 died of lbrf during world war ii. with sanitation improvement lbrf is now found only in the horn of africa and among homeless people in europe. more recently, cases of lbrf in refugees and migrants were described [161, 162] . tick-borne relapsing fever (tbrf) is caused by a group of pathogens which are maintained by and survive in softticks (orinthodoros genus). each tbrf borrelia species depends on one specific species of soft-body tick. except for australia and antarctica, tbrf species can be found in all continents. the animal reservoir includes small animals and rodents. since the spirochetes persist in the tick salivary gland, disease transmission occurs when humans intrude the tick's environment. tick bites are painless, and history of a tick bite is often missing. therefore, a history of potential exposure can be valuable. the major clinical symptom is relapsing fever. after a median incubation period of 7 days, patients present with febrile episode that can last 2-7 days, followed by afebrile period of 4-14 days. patients with tbrf can have up to 30 febrile relapses, while lbrf is usually associated with only one relapse. other symptoms include myalgia, arthralgia headaches, and vomiting, and physical findings include lymphadenopathy and splenomegaly with rash occurring only in third of the patients. a range of neurologic complications as well as systemic inflammatory response syndrome also have been described [163] . diagnosis is based on identifying the spirochetes on blood smear. sensitivity of blood smear is higher during febrile period (about 70%), and a negative blood smear does not exclude rf. in lbrf, the spirochete load can be low and specific stains can be helpful. other diagnostic methods include serologic testing, pcr, and mouse inoculation. doxycycline, tetracycline, and penicillin are the preferred treatment, with most patients treated with 7-10 days of doxycycline. jarisch-herxheimer reactions with high fever and leukopenia occur in half of the patients following the first antibiotic dose and can develop into a severe reaction with hypotension, respiratory distress, and death [163] . without treatment, tbrf carries a mortality rate of up to 10% with even higher 40% mortality rate for untreated lbrf. two cases of meningoencephalitis with borrelia miyamotoi in heavily treated immunocompromised patients have been described [164, 165] . lyme borreliosis is the most common vector-borne disease in the northern hemisphere caused by a group of at least 13 genospecies. lyme disease is a multisystem illness affecting the skin, joints, nervous system, and heart. human infection is caused mainly by three species: b. burgdorferi is the most common cause in north america but also found in europe, and borrelia afzelii and borrelia garinii which cause the disease in europe and asia. emerging infections in the mid-western usa with borrelia mayonii cause a condition similar to lyme disease. most tick species do not carry borrelia species. the vectors of all borrelia species are the ixodid tick species; this includes the deer tick, i. scapularis, in the northeast and midwest of the usa, ixodes pacificus in the west, the sheep tick, ixodes ricinus, in europe and the taiga tick, ixodes persulcatus, in asia. the ixodid tick demonstrates a complex vector ecology with preferences for different hosts in different geographical regions and at different stages of its development. more than 300 different species, including deer, rodents, birds, and reptiles, have been described. infection rates also show seasonal variation with highest rates during lyme disease follows several stages starting with localized disease at the site of inoculation, followed by dissemination stage and, later, persistent infection [166] . however, an individual patient can show highly variable disease progression with different patterns of organ involvement and disease severity. erythema migrans (em) is often seen at the site of the tick bite after 3-30 days of incubation. regional lymphadenopathy can be seen. secondary skin lesions represent hematogenous dissemination. at this stage, constitutional symptoms of general fatigue, fever and headaches, migratory musculoskeletal pain, conjunctivitis, and cardiac involvement occur. in total, 15% of untreated patients can develop frank neurologic manifestations of meningitis, encephalitis, and variable forms of neuritis with fluctuating symptoms. persistence can occur in untreated (on inadequately) patients. antibiotic refractory arthritis is well described. however, even without treatment, intermittent or persistent attacks usually resolve completely within several years. co-infection with a. phagocytophilum and b. microti can cause diagnostic confusion [167, 168] . the diagnosis of lyme disease is established based on clinical symptoms, history of potential exposure, and serologic studies. although positive culture can confirm the diagnosis, it can usually be obtained only from early em lesions. pcr testing is superior to cultures and can be performed on joint fluid samples [169] . cdc recommendations for the diagnosis of lyme disease are based on serology which might be impossible in pidd patients with abnormal humoral response. cdc guidelines require both an elisa (or comparable test) to be positive and a western blot (2 out of 3 bands (23, 39, or 41 kd) on the igm or 5 out of 10 bands on the igg (18, 23, 28, 30, 39, 41, 45, 58, 66, 93 kd) . most lyme manifestations can be treated with oral antibiotics, while patients with neurologic abnormalities and some patient with lyme arthritis require intravenous therapy [170] . doxycycline is the treatment of choice for early and disseminated disease, with amoxicillin as the second-line choice. jarisch-herxheimer-like reactions can appear in the first 24 h in about 15% of the patients. for patients with clear neurologic symptoms, 2-4 weeks of iv ceftriaxone is the most commonly used therapy. few cases of neuroborreliosis and hiv have been described with a good response to treatment. descriptions of lyme disease in pidd patients are lacking. zoonoses are infectious diseases that pass between animals and humans and span the spectrum of pathogens including viruses, bacteria, fungi, and parasites. zoonoses are very common and range from mild such as certain forms of tinea to lifethreatening infections such as rabies. some of the zoonoses that are vector-borne will be covered in other sections. risk mitigation strategies for zoonoses include patient education, proactive advice about risk scenarios, and avoidance of infected animals. several zoonoses are associated with contact with mammals such as rodents or domestic farm animals through direct contact or through contact with their feces. for instance, hantavirus infections are often associated with exposures to mouse droppings when staying in cabins in the western usa. occupational exposures can occur with buffalopox or parapoxvirus (causing orf infection) through direct contact with buffalo and goats/sheep, respectively [171] [172] [173] [174] . in general, there are very few cases of pidd with zoonotic infections acquired from mammals. however, there are a few special considerations. for instance, lymphocytic choriomeningitis virus is acquired through exposure to house mice primarily, with hamsters being a less common source of infection. both domestic and wild mice can carry the infection without exhibiting symptoms. although infection is rare, there have been severe cases in patients with t/ nk cell dysfunction, such as a case in xlp1 and cases in solid organ transplant recipients [175] . therefore, in patients with severe t/nk defects, consideration should be given to whether small rodents are appropriate household pets. tularemia is a disease of animals and humans caused by the bacterium francisella tularensis. rabbits, hares, and rodents are the main reservoirs. humans become infected through direct contact, ingestion of contaminated water, or inhalation of organisms. ticks and deer flies can also transmit the disease through bites. fever is universal, but other features depend on the mode of transmission. a patient with cgd had a complex course suggesting myeloid defects are a risk for more severe disease [176] . rabies is an almost universally fatal infection caused by contact with oral secretions from infected mammals, typically raccoons, bats, or foxes, and there is no suggestion that pidd or immune compromised modifies the prognosis. for individuals with high-risk exposures, such as those working with wildlife or traveling in endemic areas, pre-exposure prophylaxis is given with vaccination, and if an exposure occurs, rabiesspecific immunoglobulin is provided as well as vaccination. however, for those with humoral immunodeficiencies who cannot respond to the typical pre-exposure vaccination, there needs to be counsel on the additional risk without vaccination. in table 6 , several of the bacterial and viral zoonoses are summarized with their typical endemic areas, which is somewhat limited by diagnostic abilities and reporting, as well as the typical clinical scenarios, known cases in immunodeficiency and an approach to diagnosis and therapy. nipah virus causes a range of infectious phenotypes ranging from asymptomatic infection to acute respiratory distress and encephalitis. nipah virus was identified in 1999 on pig farms in malaysia, leading to identification of 257 human cases, including 105 human deaths and the culling of one million pigs [177] . the natural host is the fruit bat: pteropodidae pteropus. symptoms of infection from the malaysian outbreak were primarily encephalitic in humans, but later, outbreaks have caused respiratory illness, increasing the likelihood of human-to-human transmission. fever, headache, cough, abdominal pain, nausea, vomiting, weakness, problems with swallowing, and blurred vision were common. seizures were seen in 25% and coma in 60%. relapses of encephalitis have been described [178] . increasing infections due to nipah virus is thought to be due to an increasing overlap between bat habitats and pig sties in malaysia. all outbreaks thus far have been in india, bangladesh, or malaysia. the diagnosis of nipah virus relies on pcr of fluid samples, serology in convalescent samples, and immunofluorescence of tissue. there have been no infections of immune compromised patients reported. therapy is largely supportive, although preliminary reports of ribavirin use have been encouraging. a vaccine is under development. severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) are two zoonotic coronaviruses. the sars pandemic in 2002-2003 resulted in 8096 reported cases in 27 countries. no further sars cases were reported after the pandemic except isolated cases linked to laboratory accidents. patients usually presented with fever and respiratory symptoms, but occasionally had diarrhea and vomiting. about 20-30% of sars patients required mechanical ventilation, with a case fatality rate of about 9% [179] [180] [181] . mers was first noted in saudi arabia in 2012, and countries around the arabian peninsula are now endemic for mers-cov. patients usually present with fever, cough, chills, sore throat, myalgia, and arthralgia rapidly progressing to pneumonia with over 50% of patients requiring intensive care. about one-third of patients present with diarrhea and vomiting, and acute renal impairment is a striking feature of mers. risk factors for poor outcome include diabetes, hypertension, and renal and lung disease. cases have been exported to at least 26 countries with travel occasionally causing cluster of secondary outbreaks. one such example is the mers-cov outbreak involving 82 patients in south korea, and the median incubation period was estimated to be 7 days with a range of 2 to 17 days [182] . at the end of 2015, there were 1621 confirmed mers with a 36% mortality rate [179] [180] [181] . bats are the natural reservoirs of both sars-cov and mers-cov. sars-cov crossed the species barrier into palm civets and other animals in live animal markets in china, which then infected human, while a mers-cov ancestral virus crossed species barrier into dromedary camels. abundant circulation of mers-cov in camels results in continuous zoonotic transmission of this virus to human, while sars-cov was not found to circulate in the intermediate reservoirs, explaining sars being a one-off outbreak and mers a continuing zoonotic disease [179] . aerosolgenerating procedures such as intubation were associated with increased viral transmission of both covs resulting in nosocomial outbreaks [179] . super-spreaders are responsible for large and prolonged outbreaks [181] . the diagnosis for sars and mers include both serological tests and pcr assays that can quantify viral loads [183] . functional genetic polymorphisms leading to low serum mannose binding lectin (mbl) are associated with susceptibility to but not severity of sars in both southern and northern chinese [184] [185] [186] . mbl was shown to bind to sars-cov and inhibit the infectivity [184] , suggesting its role as first-line defense against sars-cov. although no patients with primary immunodeficiency infected with sars-cov or mers-cov were identified, likely due to the limited number of such infections, patients with t cell defect and type 1 interferon pathway defects could suffer a more severe disease course [187, 188] . virus-based and host-based treatment strategies are largely experimental with uncertain benefits. ribavirin, type 1 interferons, small molecules, and monoclonal antibodies that block covs entry have been explored [183] . passive immunotherapy and multiple candidate vaccines have been tested in various animal models. convalescent plasma immunotherapy has been considered, but clinical trials are lacking in mers [189] , while for sars a systematic review concluded convalescent serum may reduce mortality and appear safe [190] . the filoviridae family contains three known genera, the ebolaviruses, marburgviruses, and cuevavirus. ebolavirus and marburgvirus cause hemorrhagic fever syndromes in primates and humans, with high fatality rates. cuevavirus infects only bats. the ebolavirus genus contains five species, with two of the species (zaire ebolavirus and sudan ebolavirus) being responsible for the majority of cases of human disease, while marburgviruses contain two species (marburg virus and ravn virus). filoviruses are capable of replicating in a number of cell types (with the exception of neurons and lymphocytes). upon entry into the body of the host (via breaks in the skin, parenterally, or through mucosal surfaces), filoviruses employ a variety of mechanisms to evade the activity of the immune system [191] . the incubation period (interval from infection to onset of symptoms) varies from 2 to 21 days. symptoms begin abruptly, with high fever, severe headache, malaise, myalgia, diarrhea, nausea, and vomiting. a rash can occur between 2 and 7 days after onset of symptoms. hemorrhagic manifestations occur between 5 and 7 days, and fatal cases usually have some form of active bleeding. in an outbreak setting, the symptoms are unmistakable but confusion with malaria can occur early in the disease or in sporadic cases. since their original descriptions in 1967 and 1976, respectively, for marburg and ebola, there have been a number of sporadic cases and several major outbreaks. the largest marburg virus outbreak occurred in angola in 2005 (with a fatality rate of >80%), while the largest ebola epidemic happened between 2014 and 2016 in west africa (sierra leone, guinea, and liberia) and claiming over 11,000 lives (fatality rate > 40%). although not definitively proven in the case of ebola, bats are believed to be the natural animal reservoir for these viruses [192, 193] . these viruses are transmitted via contact with blood or body fluids from an infected host; notably, certain body fluids can harbor virus for weeks to months after resolution of disease. given the recent outbreak in west africa, there has been renewed interest in understanding the pathogenesis of filovirus infections and possible therapies. literature regarding how the pathogenesis of disease may be altered in patients with pidd is lacking. however, the assumption is that in the absence of an intact cellular and/or humoral immune response, the patient with a pidd may be at increased risk of mortality in the setting where mortality is already high. these viruses induce apoptosis of lymphocytes and macrophages, and there is therefore a profound secondary immune compromised [194, 195] . filoviruses can be detected in multiple body fluids via pcr. although practiced for decades, a study in guinea in 2015 failed to show a decrease in mortality among patients receiving convalescent plasma from previously infected donors [196] . a number of additional compounds (e.g., tkm-ebola, bcx4430, and gs-5734) and biologics (zmapp) have been shown to offer protection in animal models of ebola, but to date, no controlled and appropriately powered clinical trials have addressed their efficacy in humans. finally, a number of vaccines for ebola are undergoing clinical studies (including four in phase iii trials). importantly, in late 2016, the rvsv-zebov vaccine was shown to have displayed high efficacy in protecting immunized adults during the 2015 guinea ebola outbreak, and the data also suggested that the vaccine may even offer bherd immunity^to unimmunized persons in proximity to recipients of the vaccine [197, 198] . hepatitis e virus is a single-strand rna virus of the hepeviridae family. it is an important zoonotic disease in asia and africa, and fecal-oral spread is the usual route of transmission [199] . handling of pig or boar meat is a risk factor, and 2-10% of pig livers sold in grocery stores in japan and the usa are infected [200, 201] . swine represent the major reservoir, although antibodies to the virus have been found in many species [199] . the incubation period is 2 weeks to 2 months, and viremia disappears with the onset of symptoms. the mortality rate is 1-4% and can reach 20% in pregnancy [202] . acute hepatitis usually resolves but can lead to liver failure in severe cases. patients with hepatitis e posttransplant have had severe courses in some cases [203] . in immune compromised patients, the course can become chronic [204] [205] [206] . in these cases, cirrhosis develops. the diagnosis is by serology or pcr, and the treatment is supportive. prevention modalities for infections transmitted by humans are conceptually different than infection prevention for vector-borne infections. hand hygiene is extremely important, and avoidance of clearly infected people can be helpful. recognition of infections with fecal-oral transmission and the importance of water purity are critical for patients with pidd. in contrast, infections transmitted by aerosols require prevention strategies related to droplet precautions. in outbreak scenarios, if the risk to the patient is high, specific chemoprophylaxis may be considered. influenza viruses type a and b cause annual epidemic influenza, while type c causes sporadic mild influenza-like illness. patients present with sudden onset of fever, chills, and myalgia, followed by sore throat and cough. other less common features include diarrhea, acute myositis, and encephalopathy [207, 208] . co-infection with bacteria such as pneumococci results in more severe disease [209, 210] . influenza pandemics occur yearly around the world. influenza viruses infect 5 to 15% of the global population, resulting in~500,000 deaths annually [211] . the viruses circulate in asia continuously and seed the temperate zones, beginning with oceania, north america, and europe, then later seeding into south america [212] . diagnosis of influenza includes direct/ indirect immunofluorescent antibody staining for antigens in nasopharyngeal aspirates and pcr. a patient with compound heterozygous null mutations of the gene encoding irf7, a transcription factor for amplifying ifn-α/β, was reported to have life-threatening influenza during primary infection [213] . fatal influenza-associated encephalopathy in both chinese and japanese children has been reported to be associated with genetic variants of thermolabile carnitine palmitoyltransferase ii [214] . patients with scid will have prolonged viral shedding [215] . severe pandemic influenza a virus (h1n1) infection has been associated with igg2 and igg3 subclass deficiency [216, 217] . in addition, influenza infection can be more severe in pidd patients with underlying lung disease, such as bronchiectasis, and antibiotic coverage of chronic colonizing bacteria (such as pseudomonas) in this setting may be helpful. inactivated seasonal influenza vaccine should be given to pidd patients even those with humoral deficiencies as their t cell response to influenza could be normal and offer protective immunity against severe influenza [218, 219] . antiviral drugs include neuraminidase inhibitors (oseltamivir and zanamir) and adamantanes (amantadine and rimantadine), but resistance to adamantanes is widespread. measles is a single-stranded, negative-sense, enveloped (nonsegmented) rna virus of the genus morbillivirus. measles is highly communicable, transmitted by droplets, and less commonly by airborne spread. patients present with fever, cough, coryza conjunctivitis rash, and koplik spots. complications include pneumonia, acute encephalitis, and subacute sclerosing panencephalitis (sspe) [220] . diagnosis of measles includes serological tests, virus isolation, and pcr. in an outbreak, the clinical features may be sufficient for diagnosis. measles vaccine has caused severe measles in children with stat2 and ifn-α/β receptor deficiency [187, 188] , demonstrating the importance of type 1 interferon pathway in controlling measles. immune compromised of nearly any type is associated with severe disease and higher mortality [221] . t cell deficiency states are the most strongly associated with the development of giant cell pneumonia and inclusion body encephalitis, the most feared complications of measles. sspe is a slow encephalitis due to persistence of replication defective measles virus in the cns. it is most frequently seen when young infants are infected with measles and 6-10 years later, sspe becomes evident. there are case reports supporting the immune compromised as increasing the risk of sspe [222] . treatment of sspe with ribavirin has shown some improvement, but the prognosis in general with sspe is very grave. patients with cgd have defective memory b cell compartment, resulting in lower measle-specific antibody levels and antibody-secreting cell numbers, but severe disease has not been reported [223] . pidd patients may harbor the virus latently for longer than usual, leading to complications at the time of transplant [224] . specific antiviral therapy is lacking, but ribavirin has been given to severely ill and immunocompromised children. for measles post-exposure prophylaxis, intravenous immunoglobulin (ivig) is recommended for severely immunocompromised patients without evidence of measles immunity [225] . this would likely include patients with scid and hypogammaglobulinemia who are not yet on regular ivig. measles vaccine, given in a two-dose regimen, has brought down incidence enormously worldwide and the who is planning for eradication globally. enteroviruses (evs) are among the most common viruses infecting humans worldwide. evs are small non-enveloped, single-stranded rna viruses of the picornaviridae family. human evs are categorized into seven species that include hundreds of serotypes, such as polioviruses (pv), coxsackie viruses a, and b (cv-a and b), echoviruses, and human rhinoviruses (hrvs). of these species, many important serotypes are known to infect human such as pv1-3, cv-a16, cv-b3, ev-a71, ev-d68, and hrv (table 7) . non-polio enteroviruses (npevs) have a worldwide distribution. infants and young children have higher incidence of infection and a more severe course of illness than adults. the mode of transmission is mainly through fecal-oral and respiratory routes. infection occurs all around the year in tropical and subtropical regions, while in temperate climates the peak incidence of infection is during summer and fall months [226] . npevs are associated with diverse clinical manifestations ranging from mild febrile illness to severe, potentially fatal conditions. most cases are asymptomatic or have mild symptoms including fever with or without rash; symptoms of hand, foot, and mouth disease; herpangina; acute hemorrhagic conjunctivitis; upper respiratory infection; and gastroenteritis. more severe symptoms occur in infants and young children [227, 228] . acute flaccid paralysis [229] , neonatal enteroviral sepsis [230] , myocarditis/pericarditis [231, 232] , hepatitis, pancreatitis, pneumonia, and atypical hemolytic uremic syndrome [233] are severe manifestations seen in immunocompetent people. chronic infections have been seen in immunocompromised patients [234] . each virus may produce one or more of the aforementioned manifestations; however, some serotypes are often associated with particular features ( table 7) . the definitive diagnosis of npev infection relies on pcr or virus isolation from the cerebrospinal fluid, blood, stools, urine, or throat swab [229, 235] . treatment of npevs is mainly supportive since most infections are self-limited. high doses of intravenous immunoglobulin (ivig) are recommended in patients with severe symptoms. the efficacy of some new antiviral drugs (pleconaril, vapendavir, and pocapavir) is still under investigation [236] . no vaccine has been licensed yet for npevs. however, phase 3 clinical trials of inactivated monovalent ev-a71 vaccines manufactured in china showed high efficacy against ev-a71 in infants and young children [237] . patients with a variety of pidds are unusually susceptible to ev [238] . the most susceptible groups are patients with primary antibody deficiency such as xla, cvid, and hyper-igm syndrome (higms) as well as those having scid and major histocompatibility class ii deficiency [239, 240] . the most severe form of infection has been described in patients with xla due to the profound deficiency of immunoglobulins essential for viral neutralization during infection. affected patients usually present with indolent but relentlessly progressive non-necrotizing meningoencephalitis. regression of cognitive skills, flaccid quadriplegia, and deafness has been described. the reported non-neurologic presentations in xla include septicemia, dermatomyositis-like disease, hepatitis, and/or arthritis [238, 241] . the incidence of npev meningoencephalitis in large registries of xla cases is 1-4% [242] . unpublished data from the kuwait national pidd registry, which includes 271 pidd patients, showed that nine patients had documented npev infections and two died from these infections. the two deaths were seen in scid patients (personal communication with prof. waleed al-herz, md). in addition, npev meningoencephalitis and/or septicemia were reported in few cases with either primary b cell deficiency such as b cell linker (blink) protein deficiency [243] or acquired b cell deficiency following the administration of anti-cd20 (rituximab) [244, 245] . in all reports, better outcome was attributed to the early administration of high doses of ivig during npev viremia [246] . npev infection in pidd diseases remains a major threat to patients. also, the possible prolonged viral excretion and the emergence of resistant strains runs the risk of spreading infection to the surrounding community. oral polio vaccine (opv) consists of a mixture of three live attenuated poliovirus serotypes. opv induces production of neutralizing antibodies against all three serotypes, in addition to a local intestinal immune response. opv can result in vaccine-associated paralysis (vap) secondary to reversion of the vaccine strain to the neurovirulent wild-type strain. an example for such an event was demonstrated by the 2000-2001 outbreak in the dominican republic and haiti [247] , believed to be driven at least in part by undervaccination of the population, which allowed the spread of the reverted vaccine strain [248] . although rare, patients with pidd have a higher risk to develop vap. reports have shown that pidd patients with antibody deficiency can have prolonged viral replication which can persist for years and therefore theoretically increase the risk for a spontaneous reversion within the immunodeficient host [249] [250] [251] [252] . cases of vap were shown in patients with antibody deficiency and combined immunodeficiency syndromes [248, 253, 254] . therefore, opv is contraindicated in patients with pidd, and this contraindication extends to their household contacts [22] . beyond the obvious risk for the pidd patient, prolonged virus shedding also increase the risk for spreading vaccine-derived paralytic strain in the general population. bacterial infections have molded human behavior and altered societies over human history. today, largely ignored due to antibiotic susceptibility, they continue to cause misery and disease around the world. three infections are highlighted, and additional commonly encountered infections are listed in table 8 . pertussis is a respiratory infection caused by bordetella pertussis that begins after a 7-to 10-day incubation period as a minor upper respiratory infection that progresses with cough. initially intermittent, it evolves into paroxysmal coughing spells usually followed by vomiting in infants and young children. it lasts 6 to 10 weeks and can have many complications such as syncope, weight loss, rib fracture, and pneumonia. infants under 6 months are more severely affected, developing pneumonia, pulmonary hypertension, hypoxia, subdural bleeding, and seizures. death can occur, especially in young infants [255, 256] . adults typically have a prolonged cough with fewer complications [257] . it is transmitted via aerosolized droplets during close contact. people are most contagious during the catarrhal stage and the first half of the paroxysmal phase, totaling 5 to 6 weeks [258] . the introduction of whole-cell pertussis vaccine (dpt) in the 1940s in the usa reduced the incidence of the disease from 250,000 cases to around 1000 cases per year in the 1970s. a resurgence in 2012 was associated with the substitution of the whole-cell vaccine by the acellular pertussis vaccine (dtap) [258] . new strategies such as boosters with acellular pertussis for adolescents and adults with tdap and use of tdap during pregnancy seem to be effective in partially reducing the incidence of the disease [259] ; however, pertussis cases in the usa remain higher than the 1970s. the lack of persistence of antibody in the adult population means adults not only represent a reservoir for the disease but also do not provide sufficient titers to immunoglobulin products prepared from adult plasma pools. a relatively recent requirement in some countries is vaccination of adults every 10 years to maintain immunity. this should, over time, improve titers in immunoglobulin products. culture of specimens obtained by nasopharyngeal swabs is the gold standard of laboratory diagnosis due to the 100% specificity, but polymerase chain reaction (pcr) is gaining prominence due to its higher sensitivity and speed of results; serodiagnosis can be used in the late stages of the disease [259] . filamentous hemagglutinin (fha) and pertussis toxin (pt) antibodies were detected at peak measurements in pidd patients on regular ivig, although some of them had pt antibodies below the protective level as trough measurements [260] . severe pertussis cases have not been reported in pidd patients, but severe disease has been seen in malignancies [261] . antimicrobials such as azithromycin, erythromycin, and clarithromycin, if given during the catarrhal stage, may ameliorate the disease and shorten the contagious period. to avoid cases of pertussis, it is also worth emphasizing the importance of good vaccine coverage rate among the whole population, but especially among healthcare workers and family members of patients with pidd. neisseria meningitidis the onset of neisserial meningitis is associated with sore throat, headache, drowsiness, fever, irritability, and neck stiffness [262, 263] . purpuric lesions are very characteristic. this pathogen can also present with sepsis which has a 20% mortality rate as opposed to 11% mortality with a meningitic presentation. this bacterium can also cause a chronic condition referred to as chronic meningococcemia. this condition is characterized by intermittent fevers lasting 1 week to 3-4 months [264] . a non-purpuric rash is common which may evolve into purpura. arthritis, similar to that seen with gonococcus, is common. meningococcal disease primarily affects children under 5 years of age. n. meningitidis is a global pathogen [265] . there are 12 serogroups, but the majority of invasive meningococcal infections are caused by organisms from the a, b, c, x, y, or w serogroups. the annual number of invasive disease cases worldwide is estimated to be at least 1.2 million, with 135,000 deaths related to invasive meningococcal disease. serogroups b and c are responsible for most infections in europe. serogroup a has historically been the major organism in africa; mass vaccination has led to some improvement, but the emergence of group x disease is worrisome. the hajj in the middle east has seen epidemics of w-135, and vaccination is now required for hajj travelers. b and c serogroups are the most common through the americas. n. meningitidis cases occur at a rate of about 1 case per 100,000 people throughout the world [266] , but across the sahel of africa and in china, epidemics can lead to case rates of 500 per 100,000 [267] . the bacterium is a natural human commensal, with carriage rates of about 10%. diagnosis can be by clinical examination in epidemics or by gram stain and culture. complement-deficient individuals have an increased risk of neisserial disease, but not necessarily increased mortality. hiv is associated with increased disease, suggesting that t cells are also important for defense. thirdgeneration cephalosporins are typically used for treatment. penicillin, ampicillin, aztreonam, and chloramphenicol are alternatives. there is great inter-individual variability in the development of tb disease. roughly, 5% of infected individuals develop clinical disease within 2 years of infection (mostly during childhood). about 90% became latently infected without clinical disease, and the remaining 5 to 10% develop pulmonary tb later in life, either from reactivation of latent infection or reinfection. molecular epidemiology studies from high burden areas suggest more disease results from recent transmission than from reactivation of latent tb, particularly in people living with hiv [268] . acquired or inherited host factors may at least partially account for the variable disease course, resulting in increased susceptibility to mycobacteria infections [269] . pidd associated with tb and ntm infections include t cell deficiencies, gata2 deficiency, cgd, anhidrotic ectodermal dysplasia with immunodeficiency, x-linked (xl) recessive cd40 ligand deficiency, autosomal recessive (ar) stat1 deficiency, ar irf8 deficiency, and ar tyk2 deficiency. in addition, a group of disorders with a strong susceptibility to ntm, named mendelian susceptibility to mycobacterial diseases (msmd), have been recognized since the 1990s. these are rare inborn errors of ifn-γ signaling pathway that present with isolated predisposition to infections caused by weakly virulent mycobacteria such as bcg vaccine and environmental ntm, in otherwise healthy patients. the genetic defects involve impairment in the production of interferons (ar il12rβ1, ar il12p40, autosomal dominant (ad) irf8, ar isg15, xl recessive nemo) or response to interferons (ifn-γr, ad stat1, ad irf8, cgd) [270] . an acquired form exits: adults with ntm infection in thailand and taiwan were found to have high-titer anti-interferongamma antibody [271] . these individuals from southeast asia were found to have hla-drb1*1502/16:02 and dqb1*05:01/05:02. patients suspected of having pulmonary tb should have acid-fast bacilli (afb) smear microscopy and culture performed in three sputum samples. pcr for mtb can be performed [272] . the use of rapid tests facilitates early diagnosis, and the who has recently recommended their use. the only recommended rapid test for detection of tb with and without rifampicin resistance is the xpert mtb/rif assay (cepheid, sunnyvale, ca). the who recommends the xpert test for those suspected of having drug-resistant tb or in hiv; however, culture is still the mainstay and is not replaced by the xpert test. tb skin testing (mantoux testing) uses a purified protein derivative injected under the skin. its advantages are that it can be used for large-scale screening and it is cost effective. skin testing does have several disadvantages when used as a diagnostic test. reading the induration requires training and immunodeficiencies can alter the magnitude of the induration. immunosuppressed patients (hiv, organ transplant) are considered positive if the induration is ≥5 mm. some immunodeficiencies may completely ablate the response. other causes of false-negative tests are malnutrition, concurrent infection, recent live viral vaccine administration, renal failure, malignancy, medical stress, very elderly, young infants, or with a very recent infection with mtb. conversely, the results may be falsely positive if bcg has been administered. interferon gamma release assays can be used in any setting where skin testing would be done but are considered superior in settings where the patient has had bcg vaccination and, in some cases, where skin testing has been sown to have high false-negative rates. in general, tb treatment for patients with impaired immune response, including pidd, hiv infection, and immunosuppressive therapy, is based on the standard 6-month regimen consisting of a 2-month intensive phase of isoniazid, rifampin, pyrazinamide, and ethambutol, followed by 4 months of isoniazid and rifampin. decisions regarding treatment duration can be individualized, taking into account disease severity, organs involved, and response to treatment. significant pharmacological interaction can occur between rifampin-based mtb regimens and immunosuppressive drugs, such as calcineurin inhibitors or rapamycin, requiring strict monitoring of drug plasma concentrations [273] . therapy for ntm is complex with highly variable drug resistance patterns and a need for biological augmentation to effectively clear the organism. aspergillus fumigatus (see above), cryptococcus gattii, histoplasma capsulatum, coccidioides immitis (or c. posadasii), blastomyces dermatitidis, paracoccidioides brasiliensis, emmonsia pasteuriana, and penicillium (talaromyces) marneffei are environmental fungi that are endemic in certain parts of the world (table 9 ). with the exception of penicillium marneffei and emmonsia pasteuriana that only cause disease in profoundly immune compromised individuals, these fungi can cause infection in healthy individuals, ranging from mild, self-limited pulmonary disease to infection that requires antifungal therapy for eradication. on the other hand, patients with acquired defects in cell-mediated immunity such as those infected with hiv, and patients with specific monogenic disorders, particularly those involving the il-12/ ifn-γ/stat signaling pathways, scid, and gata2 depends on underlying state of immunosuppression and magnitude of environmental exposure icl idiopathic cd4 lymphocytopenia, aids acquired immune deficiency syndrome, stat1 signal transducer and activator of transcription 1, gata2 gata binding protein 2, scid severe combined immunodeficiency, cvid common variable immune deficiency, pidd primary immunodeficiency, il12rb1, interleukin-12 receptor subunit beta 1, ifngr1 gamma interferon receptor 1 deficiency, are at high risk of developing life-threatening disseminated infections by these endemic fungi following environmental exposure [42, [274] [275] [276] [277] [278] . diagnosis relies on culture of the corresponding fungus, histopathological demonstration of the fungus-specific characteristic morphologies, and/or surrogate serological and fungal antigen tests. treatment of clinical disease (as opposed to colonization) typically involves an initial induction phase with amphotericin b, followed by long-term azole maintenance therapy and secondary prophylaxis, and prognosis varies significantly depending on the fungal pathogen and underlying pidd. melioidosis is caused by b. pseudomallei, a gram-negative bacteria found in soil and water, in tropical climates of southeast asia and northern australia [279, 280] . melioidosis is an emerging, potentially fatal disease (20% mortality). b. pseudomallei can be transmitted by inhalation, ingestion, or direct contact (through open skin) with contaminated soil or water. animals (sheep, goats, swine, horses, cats, dogs, and cattle) are also susceptible to infection and cases of zoonotic transmission through direct contact of skin lesions with infected animal meat or milk have been described [280] . b. pseudomallei infections are endemic in northern australia and southeast asia. approximately 75% of reported infections occur during the rainy seasons. cases have also been reported in south pacific, africa, india, and the middle east. in temperate areas, infection is extremely rare and is predominantly imported by travellers or immigrants [281] . the incubation period of melioidosis is variable from 1 day to years, although common symptoms develop between 2 and 4 weeks after exposure. melioidosis presents most frequently in adults 40-60 years of age, but can occur in all ages, with one study reporting 5% of cases occurred in children [282] . in australia, the average annual incidence in 2001-2002 was reported as 5.8 cases per 100,000 people [279] . the incidence in indigenous australians was higher at 25.5 cases per 100,000. a case-cluster in an australian community was associated with post-cyclonic flooding. a recent review suggests that b. pseudomallei is increasingly prevalent in the americas, with a mortality rate of 39% [283] . infection in healthy individuals is uncommon, and more than 70% of cases occur in the setting of underlying conditions such as chronic renal disease, diabetes, chronic lung disease, and alcoholism. a recent review of melioidosis in travelers found that 46% of cases were acquired in thailand. symptoms usually started at 23 days (range 1-360 days) after leaving the endemic area. traveller infections were less often associated with predisposing risk factors (37.5%), diabetes mellitus being the most common (21%). melioidosis in travelers had lower mortality (17%) than infection in autochthonous cases in southeast asia [284] . pneumonia (~50-55%) is the most common presentation in adults. there is usually high fever, headache, anorexia, and myalgia. b. pseudomallei infection may also present as localized skin infection, septicemia, or disseminated infection. localized infection results in an ulcer, nodule, or skin abscess. this usually occurs from the bacterium breaching through a break in the skin. patients with renal disease and diabetes are more susceptible to sepsis. in disseminated infection, abscesses may develop in the liver, spleen, lung, and prostate. in children, primary cutaneous melioidosis is the commonest presentation (60%). bacteremia is less common in children than in adults, but brainstem encephalitis has been reported [282] . difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes [285] . diagnosis of melioidosis is primarily by isolation of the organism. identification of b. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. although various serological tests have been developed, they are generally unstandardized bin house^assays with low sensitivities and specificities. pcr assays have been applied to clinical and environmental specimens but are not widely available and sensitivity remains to be evaluated. cases of melioidosis have been reported in patients with acquired or inherited immune deficiency. melioidosis was the presenting complaint in several patients with cgd. bacteremic melioidosis was recently reported in two patients with prolonged neutropenia, who succumbed despite appropriate antibiotics [286] . it is likely that there is increased susceptibility in situations where innate or adaptive immunity is compromised. treatment is with intravenous antimicrobial therapy for 10-14 days, followed by 3-6 months of oral antimicrobial therapy. intravenous therapy with ceftazidime or meropenem is usually effective. oral therapy may continue with trimethoprim-sulfamethoxazole or doxycycline. free-living amoebas (fla) are protozoa found worldwide that do not require hosts to survive. fla do not employ vectors for transmission and are not well adapted to parasitism in humans. however, there are four genera/species that can cause human disease: naegleria (n. fowleri), acanthamoeba (multiple species), balamuthia (b. mandrillaris), and sappinia (s. pedata). all of these amoebae are capable of inducing cns disease in humans, but acanthamoeba species also cause various extra-cns infections, especially in immunocompromised hosts. the fla that are pathogenic in humans are reviewed below. naegleria are a diverse group of fla flagellate protozoans with a large number of distinct species. only one species, n. fowleri, has been shown to cause infection in humans. n. fowleri has a multi-stage life cycle with amoeboid and trophozoite-infective forms as well as a cyst form [287] . n. fowleri is found commonly in warm freshwater around the world including lakes, rivers, and hot springs. humans become can become infected when swimming or diving in contaminated water. in rare circumstances, infections have also been attributed to exposure from contaminated tap water sources when utilized for religious cleansing of the nose or irrigation of the sinuses. thus, tap water should not be used for nasal and sinus irrigation. it is not possible to become infected from drinking contaminated water or from contact with an infected person, and the amoeba is not found in salt water. after entry to the nasal cavity, the amoeba travels through the cribiform plate to the olfactory bulbs and migrates to the cerebellum, resulting in primary amoebic meningoencephalitis (pam), a rapidly fatal brain infection characterized by the destruction of brain tissue. in its initial presentation, pam can mimic bacterial meningitis, further delaying accurate diagnosis and initiation of therapies that may save the patient. overall, n. fowleri infections are rare. worldwide, most cases are reported in the usa, australia, and europe; however, in developing countries, it is suspected that a large number of cases go unreported. between 2006 and 2015, there were only 37 infections reported in the usa with 33 of the cases attributed to contaminated recreational water, 3 infections following nasal irrigation with contaminated tap water, and 1 case where a person was infected following use of a backyard slipn-slide utilizing contaminated tap water [288] . the fatality rate associated with n. fowleri infection is over 95%, and between 1962 and 2015, only 3 of the 138 infected persons in the usa have survived infection. initial symptoms of pam start 1 to 9 days after infection and can include headache, fever, nausea, or vomiting [288] . progressive symptoms can include stiff neck, confusion, lack of attention, loss of balance, seizures, and hallucinations. cardiac arrhythmias have also been observed. the infection progresses rapidly after initial onset and causes death within 1 to 12 days after exposure (mean of 9.9 days). since infection often progresses rapidly to death, there is often insufficient time to mount a robust immune response. however, both the innate (neutrophils, macrophages, and complement system) and the adaptive (both t and b cells) arms of the immune system have been shown to participate in the immune response to n. fowleri [289] . patients with pam have csf with elevated pressure that is often cloudy or hemorrhagic, with neutrophil-predominant pleiocytosis, elevated protein levels, and very low glucose. wet mounts from centrifuged csf will show motile mono-nucleated trophozoites measuring~10-25 μm in size. additionally, trophozoites can be identified with giemsa and wright stains of csf smears combined with an enflagellation test [289] . confirmation can be achieved via a variety of timeconsuming methods including an immunofluorescence assay [290] , culture of csf [291] , or pcr-based methods [292] . the optimal therapy for n. fowleri pam is still debated. the use of intravenous amphotericin b and fluconazole followed by oral administration of rifampin resulted in survival of a 10year-old child with pam [293] . another child was shown to survive following intravenous and intrathecal amphotericin b and miconazole as well as oral rifampin [294] . most recently, an adolescent girl was successfully treated with the combination of azithromycin, rifampin, fluconazole, and miltefosine [295] . prevention is critical for this highly fatal infection and warning pidd patients not to use tap water for nasal irrigation is important. since its original description in 1986, over 200 cases of b. mandrillaris infections have been described worldwidewith most cases occurring in south america and the usa. balamuthia are found in soil, and acquisition of disease has been associated with agricultural activities, dirt-biking, gardening, and swimming in contaminated water sources. b. mandrillaris is thought to enter the body of the host through breaks in the skin and or via inhalation. the organism is believed to access the cns through hematogenous spread, resulting in a chronic, insidious, but often fatal granulomatous amoebic encephalitis (gae), which has been documented in both immunocompetent and immunocompromised hosts [291, 296] . the incubation period from exposure to development of clinical symptoms is not well established and experts believe that this may occur between 2 months and 2 years. finally, an alternative mode of transmission via solid organ transplantation has also gained attention [297] [298] [299] . in many cases, gae is diagnosed post-mortem, due to delayed diagnosis or unawareness of the clinical entity. following infection by b. mandrillaris, two clinical patterns of presentation have been described. in the first pattern, patients initially develop a skin lesion that may resemble a painless plaque that may evolve into subcutaneous nodules and rarely ulcerations [300] . these patients may develop neurologic manifestations weeks to months later. histopathologic examination of these lesions typically reveals granulomatous reactions in the reticular dermis, associated with lymphocytic and plasma cell infiltrates as well as multinucleated giant cells, without distinct epidermal changes. skin lesions will harbor trophozoites, but these are scarce and often easily overlooked as they resemble histiocytes. it is believed that early diagnosis of b. mandrillaris infections in those presenting with skin lesions may prevent subsequent development of cns disease, but there have also been cases in which patients presenting with skin lesions have progressed to developing gae despite treatment. in the second pattern, patients present with cns involvement without a previously recognized skin lesion. patients presenting with gae may initially display fever, malaise, headache, nausea and vomiting, and frank lethargy. later, these symptoms evolve into visual abnormalities, cranial nerve palsies, seizures, focal paresis; as intracranial pressure builds, coma, and eventually death with tonsilar or uncal herniation ensue within 2-3 weeks [301] . upon infection with b. mandrillaris, brain endothelial cells produce the proinflammatory cytokine il-6, thereby initiating an inflammatory response [302] . moreover, the amoebic trophozoites infiltrate blood vessel walls. degradative enzymes, vessel wall infiltration, and the host inflammatory responses result in tissue necrosis and infarctions in the cerebral hemispheres, cerebellum, and the brainstem. in a mouse model of b. mandrillaris infection, cd4+ t cells were shown to be protective [303] , suggesting that patients with lowered number or dysfunction in cd4+ t cells may be more susceptible to disease by this amoeba. however, b. mandrillaris infections have been described in a variety of human hosts [304] , ranging from the young, healthy, and presumably immunocompetent to the elderly, and those with hiv, chronic corticosteroid exposure, on post-transplant immunosuppression and even patients with cvid. as such, further research is necessary to fully delineate the susceptibility of pidd patients. in patients who develop the characteristic skin lesions, recognition, testing, and treatment for b. mandrillaris may prevent subsequent gae. as such, obtaining tissue and looking for granulomas and trophozoites is quite helpful. skin biopsies can be stained via immunofluorescence or immunoperoxidase techniques to identify b. mandrillaris [305] . additionally, a pcr technique that identifies mitochondrial 16s ribosomal rna from b. mandrillaris is also available through the cdc [306] . in patients in whom the diagnosis is confirmed via skin biopsy, wide resection and medical treatment appears to prevent development of cns disease in at least a proportion of patients. in patients presenting with cns involvement, lumbar punctures reveal csf with lymphocytic pleiocytosis, low-to-normal glucose, and mildly to significantly elevated protein levels. trophozoites are not typically found in the csf, but pcr analysis may be performed. ct or mr imaging may show multiple nodules with ring enhancement; some of these nodules may also contain focal areas of hemorrhage. biopsies of brain tissues typically reveal granulomas and foamy macrophages and multinucleated giant cells surrounded by lymphocytic infiltrates. additionally, there will be areas of necrosis filled with neutrophils, multinucleated giant cells, and lymphocytes, with balamuthia trophozoites and cysts interspersed with macrophages [16] . as with the skin biopsies, immunofluorescent and immunoperoxidase stains may aid diagnosis and should be performed. unfortunately, the optimal medical management of cns disease is unknown. in the usa, a few patients have been successfully treated with a combination of fluconazole, flucytosine, pentamidine, a macrolide antibiotic (either clarithromycin or azithromycin), and one of the following agents: liposomal amphotericin b, miltefosine, sulfadiazine, or thoridazine [307] [308] [309] ; others in peru have been treated successfully with fluconazole (or itraconazole), albendazole, and miltefosine [307] . based on these case reports, most experts recommend treatment with a combination of medications (along with partial or complete resection of nodules) for a prolonged period of time to prevent further deterioration and death [307] [308] [309] [310] . acanthamoeba spp. the genus acanthamoeba contains at least 24 morphologically distinct species that live in a diverse array of habitats, including soil, salt, brackish, and fresh water. acanthamoeba spp. have also been found in humidifiers, heating and cooling unit components, jacuzzis, hot water tanks, bathrooms and drains, eye wash stations and dentistry irrigation systems, and more. acanthamoeba spp. have been isolated from reptiles, birds, and other non-human mammals, suggesting a broad distribution in the environment. acanthamoeba trophozoites feed on bacteria, but have also recently been shown to harbor a number of bacteria (including legionella and burkholderia spp., e. coli, listeria monocytogenes, vibrio cholerae, mycobacteria spp., chlamydophila, and others) and at least one virus (mimivirus) as endosymbionts. acanthamoeba infections in humans can present in a variety of ways. of primary importance are cns infections. like b. mandrillaris, acanthamoeba spp. can induce gae (described above). there is a high predilection for gae in those with hiv/aids, patients on chemotherapy, and those receiving broad spectrum antibiotics [301] . acanthamoeba are rarely found in csf, but some case reports indicate isolation of amoebae by culturing csf on bacterized agar plates. similar to gae seen with b. mandrillaris, cns histopathology may reveal edema, multiple areas of necrosis and hemorrhage, and occasional findings of angitis and blood vessel cuffing by inflammatory cells, as well as occasional trophozoites or cysts. cns disease treatment is not standardized, but several patients have been successfully treated with pentamidine, fluconazole, flucytosine, sulfadiazine, as well as miltefosine. acanthamoeba can rarely cause cutaneous infections; these lesions, like gae, are also predominantly seen in immunocompromised hosts. these lesions can start as nodules or papules on the lower extremities and develop into necrotic ulcers. histopathologic examination may reveal granulomatous dermal lesions in immunocompetent hosts, with histiocytes, as well as neutrophils and plasmacytes; trophozoites are typically visible [311, 312] . the optimal management of cutaneous disease is not known, but typically involves combinational therapy with topical (e.g., chlorhexidine, gluconate, or ketoconazole) and systemic (miltefosine, sulfadiazine, flucytosine, liposomal amphotericin b, azole antifungals, etc.) drugs. additionally, nasopharyngeal and sinus infections have been seen in people with severe compromise in immunity [313, 314] . patients typically present with purulent nasal discharge, and examination may reveal erosion of the nasal septum. nasopharyngeal disease can present concomitantly with cutaneous disease. treatment of nasopharyngeal or sinus disease is difficult and involves surgical debridement and combinations of systemic drugs. disseminated disease is also seen in immunocompromised hosts and typically involves concomitant pulmonary and cutaneous disease in the presence or absence of cns infection. keratitis readily occurs in immunocompetent hosts-with the major risk factor being contact lens wearing without proper adherence to recommended cleansing protocols. this infection less commonly presents as a result of direct inoculation with trauma. one of the most common reasons for contact lens wearers to acquire disease is due to the use of non-sterile tap water in preparing contact lens saline solutions [315] , although contaminated solutions from manufacturers have also been identified. patients will have pain and photophobia. physical exam reveals conjunctival injection and epithelial abnormalities (including pseudodendritic lesions) and stromal infiltrates [316] . the proper diagnosis can be made by staining corneal scrapings with calcofluor or wright-giemsa stains and examined by confocal microscopy, culture, or pcr analysis. prompt therapy with a combination of polyhexamethylene biguanide (or biguanide-chlorhexidine) and propamidine or hexamidine [317, 318] is indicated, but misdiagnosis and delayed therapy are common. more severe cases may also require debridement. the use of topical steroids before administration of combinational therapy may result in worse outcomes and should be avoided; however, if scleritis ensues, it may be necessary to use immunosuppressants to reduce the need for enucleation. severe and/or refractory cases may result in the need for cornea transplantation. phenotypes seen in pidd like hsv-1 and candida [337] . patients with ifnar2 deficiency seem highly susceptible to cns disease caused by mmr vaccine, an otherwise extremely rare phenomenon [187] . recently, a case of noroviral cns disease was described associated with a novel, yet unpublished pidd, suggesting that some pidds may lead to susceptibility of the cns to viruses that normally do not exhibit neurotropism (casanova jl, personal communication) . this again favors metagenomic approaches in the study of cns sequelae in pidd patients. in pidds, the cns is also more vulnerable to virally induced immunodysregulation [325] . conditions like primary hemophagocytic lymphohistiocytosis (hlh) may present as isolated cns disease or relapse only in the cns [338] [339] [340] . almost 20% all human malignancies are associated with chronic infections by hbv, hcv, hpv, ebv, hhv8/kshv, htlv-i, hiv-1, hiv-2, jcv, merkel cell polyomavirus (mcpv), helicobacter pylori, schistosomes, or liver flukes [341] . accordingly, pidd patients' malignancies are often associated with chronic infections. mcpv-associated merkel cell carcinoma has now been described in gata2 and tmc8 (ever2) deficiencies as well as other forms of pidd [330, [342] [343] [344] [345] [346] . large follow-up cohorts are needed to refute or confirm associations between novel pidds and malignancies, such as hyperactivating pik3cd and ovarian dysgerminoma or gata2 deficiency and leiomyosarcoma [329, 347] . recently, hymenolepis nana was found to have driven malignant transformation in an hiv patient. likely, other novel pidd-and pathogen-associated malignancies will be found in the future by those with an open and inquisitive mind [348] . understanding the specific infection susceptibility for each pidd allows not only a better understanding of host defense, but also allows the clinician to collaborate with the microbiology laboratory to make definitive diagnoses and provide the best therapy. reviewing all of the infections for each pidd is not within the scope of this article, but there are several infections that are unique for specific pidds and require special attention from the microbiology laboratory. three examples are provided below. g. bethesdensis is a gram-negative bacterium that was identified to cause disease in patients with cgd in 2006 [349] . g. bethesdensis is a member of the methylotroph group of bacteria, which are able to use single-carbon organic compounds as their only source of energy. they are widespread in the environment, but are rare human pathogens, and infections with g. bethesdensis have been limited thus far to patients with cgd. the organism was first detected in an adult patient with indolent and recurrent necrotizing lymphadenitis [349] . subsequently, g. bethesdensis was isolated from nine patients with cgd, primarily causing lymphadenitis, but there have been two fatalities [350] . treatment has been most effective with intravenous ceftriaxone. the microbiology laboratory should be alerted when there is concern for g. bethesdensis infection to allow for proper culture media. charcoal yeast extract (cye) agar and lowenstein jensen (lj) media are appropriate culture media. mycoplasma and ureaplasma spp. as molecular techniques are becoming more widely used to detect pathogens, the spectrum of infections that were previously only detected through serologic assays and research laboratories will increase. this is important especially for patients with pidd who have unique susceptibility to infection and may not have the ability to mount a serologic response. examples of infections in this group are those caused by mycoplasma and ureaplasma [351, 352] . these pathogens have been known to cause osteoarticular infections for those with antibody deficiency, such as xla and cvid. recently, mycoplasma orale, typically an oral commensal, has been isolated from two patients with defects in the activated pi3k delta syndrome, as chronic lymphadenitis in one and chronic splenic abscess in the other (sm holland personal communication). defects in pi3kcd and pi3kr are frequently associated with hypogammaglobulinemia and therefore would fit in the pattern of mycoplasma infections in those with humoral immunodeficiency. mycoplasma orale has also previously been reported as causing bone disease in a patient with cvid [353] . in patients with xla, helicobacter, camplyobacter, and the related flexispira bacteria that are typically isolated to the gi tract can disseminate and often lead to chronic bacteremia, ulcers, and bone infections [354] [355] [356] . xla patients have higher susceptibility than other humoral pidd and are thought to be due to the role that igm is playing in controlling the dissemination of these pathogens and potentially iga in providing mucosal immunity. these bacteria can be fastidious to grow, and therefore, when there is suspicion, identification needs collaboration with the microbiology laboratory. for instance, the blood culture media may allow growth (although with a longer incubation period), but then the organisms may need molecular techniques for identification, such as 16 s sequencing, as they will not grow on the agar plates. treatment is often difficult, requiring combination antimicrobials for prolonged periods (such as 1 year), and relapse is common. this review provides an important perspective for practicing immunologists, namely that we are a part of a global community as are our patients. this overview of emerging infections and infectious concerns for travelers serves as a foundation for practical considerations for clinicians and patients. using prevalence data, an estimation of the number of infected patients with pidd (table 10) can be developed [25, 130, [357] [358] [359] [360] . thus, the concerns addressed in this review are not theoretical but impact a considerable number of patients already. the landscape of emerging infections is by its nature highly dynamic. during the preparation of this manuscript, a mumps outbreak in the usa occurred, a new bunyavirus outbreak causing an hlh picture was reported (severe fever with thrombocytopenia syndrome), a new outbreak of the hantavirus seoul virus occurred, and an enlarging demographic of the h7n9 influenza virus was reported. this review lists several resources, many of which are updated in real time to support efforts to provide information to patients. 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to medical knowledge. conflict of interest the authors declared that they have no conflict of interest. pidds display wide genetic and phenotypic heterogeneity [319] . similar disease phenotypes may be caused by multiple genes, while patients' phenotypes caused by the same gene and even by the same mutations vary between individuals. importantly, after a novel pidd has been described, subsequent reports often reveal a wider variation in associated infections and cellular findings, often without clear genotype-phenotype correlations [320] [321] [322] [323] [324] . variation may be caused by mechanisms such as other contributing genes or geographical variation in infectious exposures. geographic differences seem most pronounced in intracellular and often chronic infections. while the numbers of described pidd patients increase, at first seemingly rarely pidd-associated infections turn out to be found in a significant subset of pidd patients [325] [326] [327] . for example, patients with cd40l deficiency living in endemic areas display susceptibility to bartonellosis and paracoccidioidomycosis, infections not described in european and us cohorts [157, 328, 329] .often, an infectious phenotype previously only described in secondary immunodeficiencies may reveal the possibility of an underlying primary immunodeficiency [327, 330, 331] . increasing numbers of genetic defects causing early-onset, severe, and recurrent susceptibility to commonly circulating pathogens like pneumococci, tuberculosis, herpes simplex, and influenza viruses as well as endemic protozoans like trypanosomes and fungi are being recognized, and thus, infections with unusual pathogens require a high index of suspicion for pidd [332] . in contrast, pidds may also manifest as suspected infection but sterile inflammation. for example, in inflammatory lesions like granulomas and necrotizing fasciitis where no clear pathogens are found, one needs to rule out aberrant host responses due to pidd [333] .chronic viral and fungal infections may also display novel phenotypes never or rarely seen in secondary immunodefic i e n c i e s . i n f e c t i o n s l i k e d e r m a t o p h y t o s i s a n d phaeohyphomycosis deeply infiltrating the skin and lymph nodes, occasionally extending to bones and central nervous system (cns) as well as predisposition to primary cns candidiasis and extrapulmonary aspergillus slowly revealed the full phenotypic spectrum of card9 deficiency [44, 324, 334] . chronic skin ulcers caused by hsv-1 and severe molluscum contagiosum suggest dock8 deficiency or gain-of-function mutations of stat1 [320, 323] . chronic mucocutaneous candidiasis has revealed a large group of monogenic diseases (il17ra, il17rc, il17f, stat1 (gof), rorx, act1), which may also be associated with recurrent bacterial infections or syndromic features [335] . while novel diseases by newly described viruses are being discovered, one needs awareness to suspect these in pidd patients [336] .interestingly, most novel forms of infectious disease in pidd patients have been described either in easily accessible sites like the skin or in immunologically privileged, normally sterile sites like the cns. this suggests that with the increasing use of invasive sampling and sensitive metagenomic approaches, we might find more novel infectious phenotypes. pathogens highly suggestive of certain pidds, like chronic enteroviral cns infections in xla patients are reviewed above. in hypomorphic mutations, cns seems to be especially vulnerable to chronically active and/or recurrent novel infectious bsmoldering^focal encephalitis lesions by pathogens key: cord-016572-6fu5s89c authors: hage, chadi a.; knox, kenneth s.; sarosi, george a. title: endemic mycosis date: 2005 journal: tropical and parasitic infections in the intensive care unit doi: 10.1007/0-387-23380-6_12 sha: doc_id: 16572 cord_uid: 6fu5s89c nan histoplasmosis, blastomycosis, and coccidioidomycosis are the three major endemic fungi in north america. although histoplasmosis is found in all continents except antarctica, coccidioidomycosis in south america, and blastomycosis in africa, only in north america are these illnesses common. these three fungal diseases share many characteristics. the causative agents are mycelial soil organisms. illness is acquired by inhaling aerosolized spores. in the infected host, the organisms change their form, a characteristic called dimorphism. histoplasma capsulatum and blastomyces dermatitidis convert to a yeast form at 37 degrees c (thermal dimorphism), whereas coccidioides immitis converts in tissue to a spherule that replicates by forming endospores (tissue dimorphism). the endemic areas are large. most of the midwest and south central united states is endemic for both histoplasmosis [1] and blastomycosis [2] , and a large area in the southwest united states and an adjacent area of mexico are endemic for coccidioidomycosis [3] . all three illnesses occur in normal hosts, although histoplasmosis and coccidioidomycosis are also major opportunistic mycoses in patients with depressed cell-mediated immunity, and especially in patients with acquired immunodeficiency syndrome (aids) [4] , [5] . histoplasmosis, blastomycosis and coccidioidomycosis are major t-cell opportunistic infections, as demonstrated by the very aggressive course seen in patients with aids, in whom t-cell deficiency is most severe. in the united states h. capsulatum var. capsulatum is responsible for the majority of the cases of histoplasmosis. h. capsulatum var. duboisii is predominantly found in south africa and europe (6). the spectrum of disease ranges from the asymptomatic acquisition of a positive histoplasmin skin test reaction to a rapidly fatal pulmonary or disseminated illness. it is the balance between the net immune status of the subject and the load of the infecting inoculum that determines the severity of the illness. during the past two decades, histoplasmosis has emerged as a common opportunistic infection in patients with aids especially those residing in endemic areas. most of our current knowledge about this disease is derived from outbreak investigation in the midwestern united states. histoplasma is a thermally dimorphic fungus. it has two forms; a mycelia phase and a yeast phase. mycelia are the forms found in the environment, they are considered to be the infectious form. mycelia display macro-and micro-conidia. yeasts are what are found in the infected individuals. in the laboratory these two forms are inter-convertible by altering the temperatures and nutrients of the growth medium. the disease is highly endemic in the midwestern and southeastern parts of the us. it is estimated that 50-80% of people living in the ohio and mississippi river valleys have evidence of remote infection with histoplasma [7] human and animal infections occurred after inhalation of aerosolized micro-conidia, the infecting particle, which can reach the alveolar space due to its very small size of 2 to 5 mm. extensive skin test surveys suggest that as many as 50 million people in the united states have been infected by h. capsulatum and that there are up to 500,000 new infections yearly [8] . for decades most outbreaks have occurred in urban settings. most are associated with construction projects that disturbed contaminated soil. the most recent (and largest ever) outbreak occurred in indianapolis, indiana [9] , associated with downtown construction of a swimming pool complex. mini-outbreaks also still occur. activities such as cutting up fallen trees or cleaning large bushes have been linked to smaller outbreaks. histoplasmosis occurs in 2 to 5% of patients with aids who reside in endemic areas and up to 25% in selected areas during periods of outbreaks [10] . pathogenesis. the lung is the portal of entry in almost every case. due to their small size, microconidia can reach the alveolar space where they convert to the yeast form, a key step in the pathogenesis. the initial tissue response to the organism is predominantly neutrophilic, followed by an increase in alveolar macrophages [11] . after being phagocytosed, yeast survive and actually proliferate inside the macrophages, which serve then as a carrier throughout the reticuloendothelial system. shortly after infection the yeast forms can be identified in the mediastinal lymph nodes. the fungus then gains access to the circulation. it is likely that transient self-limited fungemia occurs in most, if not all, patients. later the yeast disseminates throughout the body to establish foci of infection in many organs, such as the liver, spleen and the adrenal glands [5] . about two weeks after infection, specific cellular immunity begins to develop. activated lymphocytes secrete cytokines that stimulate macrophages in an attempt to boost their fungicidal activity. in mice, interleukin-12 is an important signal, leading to increased interferongamma production that confers protection against primary infection [12] , [13] , [14] . tumor necrosis factor-alpha seems to be an important element in this scheme. inhibition of has been shown to alter the adaptive immune response to histoplasma infection and may predispose patients disseminated infection [12] , [15] , [16] . with the advent of t-lymphocyte-mediated cellular immunity, fungal replication is checked and granuloma formation begins. healing of these lesions is accompanied by peripheral fibrosis. central areas of encapsulated, necrotic material frequently calcify. these calcified foci manifest on chest roentgenogram as single or multiple calcified nodules. calcified lesions are often seen in the liver and the spleen [17] . if the cell-mediated immune response is poor, the yeast continues to multiply. more macrophages are recruited, which in turn become parasitized and eventually disrupted, perpetuating the cycle [5] . a severe systemic illness develops, which invariably leads to death unless treated promptly and aggressively. clinical manifestations. most normal persons who are infected by the fungus remain asymptomatic. when present, symptoms vary widely from brief periods of malaise to severe, life-threatening illness. it is classically a flu-like illness with abrupt onset of fever, chills, and substernal chest discomfort. a harsh, nonproductive cough develops along with headache, arthralgias, and myalgias. in immunocompetent individuals severe pulmonary illness develops rarely, only when the infective dose is unusually high. it may progress rapidly to the acute respiratory distress syndrome, and may lead to death from respiratory insufficiency if not treated promptly [1] . immunocompromised individuals are more likely to progress to disseminated disease even after an infection with a smaller inoculum. the vast majority of progressive disseminated histoplasmosis is seen in patients with advanced aids with cd4 counts below 200 cells/ml [10]. most patients with progressive disseminated histoplasmosis present with fever, chills, weight loss, cough and progressive dyspnea [18] . on physical examination, patients are febrile and acutely ill. hepatosplenomegaly may be present. laboratory evaluation shows anemia, leukopenia, and thrombocytopenia. in extremely ill patients the syndrome of disseminated intravascular coagulation may be seen. up to 20% of patients present with severe septic shock, respiratory failure and progress to multi-organ failure. this syndrome represents an advanced stage of the illness and is usually seen when diagnosis and appropriate therapy were delayed. mortality is very high with this scenario. the chest roentgenographic findings are variable, ranging from normal to diffusely abnormal, with reticulonodular pattern being the most frequently reported finding. pleural effusions are rarely seen [19] . the radiographic findings are very similar to those seen with pneumocystis carinii pneumonia [4] ( figure 1 ). chest x-ray of a hiv + patient with progress disseminated histoplasmosis. peripheral blood smear may show phagocytoses yeast in some of patients with severe disease. biopsy material from the bone marrow and other involved tissue shows collections of macrophages full of intracellular yeast or, in the most severe instances, widespread necrosis with large numbers of organisms lying loose in the extracellular debris. there is little, if any, evidence of granuloma formation [20] . virtually all patients with this form of the illness have some degree of t-cell defect. before the modern era of widespread use of cytotoxic agents and glucocorticoids, many patients had underlying hodgkin's disease, a well-known example of naturally occurring t-cell immune deficiency [5] , [4] . the most severe form of progressive disseminated histoplasmosis (pdh) occurs in patients with aids with profound t-cell dysfunction [21] . in fact, most cases of pdh now occur in aids patients, and most occur in highly endemic areas [22] , [23] . newer biological therapies for rheumatoid arthritis and other auto-immune disorders have added a new pool of immunosuppressed patients at risk for tuberculosis and also histoplasmosis and other t-cell opportunistic fungal infections. in most instances, exposure of an immunosuppressed person to an infected aerosol is the antecedent event preceding pdh. in the recent large outbreak in indianapolis, most patients who were immunocompromised when they developed primary histoplasmosis progressed to pdh [23] . in particular, patients with aids nearly always progressed to pdh [10] . in other cases, the onset of pdh is temporally related to intense immunosuppression, most commonly progression of aids or therapy with high doses of glucocorticoids. in some of these cases, reactivation of dormant histoplasmosis may be the mechanism of infection [4] . patients with aids who develop pdh after long residence in new york city or san francisco are clear examples of such endogenous reactivation because primary infections are never seen in these cities. some patients with pdh present with a subacute to chronic illness. they may have a chronic wasting disease with anorexia, weight loss, and lowgrade fever. mucosal and mucocutaneous junction ulcers may occur in the mouth, oral pharynx, rectum, and glans penis. adrenal involvement may cause addison's disease [24] . biopsy material from involved tissues shows well-formed epithelioid granulomas, and only a diligent search will reveal rare organisms. demonstration of organisms almost always requires special stains [5] , [20] . the disease may be systemic or involve only one organ. this more chronic form of pdh generally occurs in patients who are less immunosuppressed than the patients who develop more fulminant pdh. central nervous system histoplasmosis is rare and may present as chronic meningitis or intracranial histoplasmoma [25] . endocarditis can also occur, involving either the aortic or mitral valves. vegetations are usually large, and emboli are common. endocarditis may occur on prosthetic or previously normal valves. recently, histoplasma involvement of abdominal aortic aneurysms has been reported in a few patients with the chronic form of pdh [26] . the gold standard of diagnosis is culture of the fungus from biologic material. cultures are time consuming and one cannot wait for them in the management of cases with severe pdh. delay in diagnosis while awaiting results of fungal cultures may lead to a fatal outcome in more severe cases isolation of h. capsulatum may occur within 1 week in a minority of patients but usually takes several weeks. serologic studies of histoplasmosis are seldom useful in the management of pdh. positive serology does not predict development of pdh in patients undergoing bone marrow transplantation [27] . the complement fixation test is negative in up to 50% of patients with pdh. immunodiffusion fails to identify up to 50% of patients with acute histoplasmosis and usually does not reach maximum positivity for 4 to 6 weeks after exposure [28] . their main drawbacks are imperfect sensitivity and lack of timeliness. several weeks must pass before they become positive. by that time, most patients have either recovered or have required other more invasive methods of diagnosis because of rapidly worsening disease there are two ways to make a rapid diagnosis of pdh, sampling and examination of likely infected tissue with the use of special stains and the use of the ultrasensitive assay for fungal antigens. bronchoscopy is an important diagnostic tool, especially for pdh. specimens obtained from bronchoscopy have a high but poorly defined yield in severe primary histoplasmosis with progressive ards and especially in pdh in aids, when diffuse infiltrates are one of the clinical features. in highly selected series the diagnostic yield of bronchoscopy for diagnosis of histoplasmosis in an endemic area is about 60% in patients with infiltrates [29] . in a strictly aids population in indianapolis, indiana, fungal stains performed on bronchoalveolar lavage fluid provided a rapid diagnosis in 70% of patients; diagnostic yield increased to 89% when culture results were included. in that series, 22% of patients had co-infections or alternative diagnoses that were detected by bal and would not have been detected if histoplasma antigen testing had been the sole diagnostic test [30] . use of cytologic examination without special fungal staining (silver, pas) may explain the lower yield of bal reported in series from non-endemic areas [31] . it is likely best to do a battery of stains including a silver stain. although transbronchial biopsy is not mandatory at the time of bronchoscopy and bal, histopathology does appear to enhance the diagnostic yield [31] . the fungus is difficult to see on standard hematoxylin and eosin stains; special stains (usually a silver stain) are needed. special stains are particularly important when well-formed granulomas are present because of the paucity of organisms in such cases. in patients with suspected pdh, sampling of the reticuloendothelial system is often effective for diagnosis. bone marrow biopsy is likely the best and safest method [20] . in heavily parasitized samples, a direct smear of the bone marrow, stained with a supravital stain such as the giemsa stain, usually gives a rapid diagnosis ( figure 2 ). on permanent histologic sections, the fungus is difficult to see on standard slides prepared with hematoxylin and eosin stain. it is best to go directly to special stains, usually one of several modifications of the silver stain or the pas stain. recently, the role of blood cultures in diagnosis of pdh has expanded. the lysis-centrifugation system increases sensitivity. in aids patients with pdh, the density of organisms is higher than in other immunosuppressed patients, and blood cultures are particularly useful, yielding a diagnosis in up to 90% of cases. in fact, in aids patients with pdh typical intracellular organisms can be seen directly on peripheral blood smears (buffy coat preparations) up to 50% of the time. bronchoalveolar lavage also has a very high yield, both by direct smear and by culture in aids patients with high burden of organisms. bronchoalveolar lavage offers the additional advantage of ready diagnosis of other opportunistic infections that are usually in the differential diagnosis, including p. carinii pneumonia. antigen detection. another approach to diagnosis of fungal infections is the use of the ultrasensitive assays for fungal antigens. this test is very useful when the burden of the infection is high. detection of the histoplasma antigen in body fluids permits rapid diagnosis of pdh. the specificity of antigen detection is greater than 98%. there is, however, known cross reactivity with the other endemic fungi such as african histoplasmosis, blastomycosis, paracoccidioidomycosis, and penicillium marneffii infection [32] . sensitivity is lower in patients who are moderately immunosuppressed. however, the high density of organisms in aids patients with pdh makes antigen testing extremely useful in that setting. antigen testing is more sensitive on urine than serum. it is positive in either urine or serum in up to 95% of patients with pdh complicating aids [30] . levels of histoplasma polysaccharide antigen in urine and serum also are useful for following the course of treatment and for predicting relapses [33] , [34] . the test is done reliably in a single reference laboratory in indianapolis, which makes it a "send out" for many institutions. severe cases of pdh in non-aids patients are best treated promptly and aggressively with amphotericin b to a total dose of 40 mg/kg. itraconazole (400 mg/day in a single daily dose) can be used successfully for patients with mild to moderate disease [35] . sequential therapy for severely ill patients with amphotericin b to clinical improvement followed by 6 months of itraconazole is also being used but is not well studied. aids patients are treated differently from other pdh cases. relapse is expected if treatment is stopped. all patients require induction therapy to control symptoms and then maintenance therapy. amphotericin b is used initially for all moderately and severely ill patients. after clinical response, treatment is changed to itraconazole (400 mg daily for 6 or more weeks, then 200 mg daily until sustained recovery of the immune system) [36], [37] . there is emerging data that support the discontinuation of maintenance itraconazole in patients with hiv who recover their immune system [38] . itraconazole can be used from onset for mild cases [39] . other maintenance strategies for those intolerant of itraconazole include weekly amphotericin b infusions or fluconazole at doses of at least 400 mg/day. ketoconazole therapy is ineffective for maintenance therapy, and fluconazole even at high daily doses (400 to 800 mg) is less effective than itraconazole [40] . in a recent double blind, randomized trial liposomal amphotericin b (l-amb) was somewhat more effective than amb in both time of response and survival, although the differences were not statistically significant [41] . in this study both types of amb were used for fourteen days before switching to oral itraconazole therapy. blastomycosis is an illness caused by the thermal dimorphic fungus blastomyces dermatitidis. the spectrum of disease ranges from the asymptomatic acquisition of the fungus to a rapidly progressive and lifethreatening respiratory or disseminated illness. blastomycosis is most common in the central and south-central united states [42] . the proposed endemic area includes much of the central, south central, and southeastern united states, beginning near the minnesota-north dakota border and extending eastward and southward. the southeastern limit extends to south carolina but not to florida. this area overlaps most of the endemic area of histoplasmosis [1]. the northern limit, however, extends further. northern minnesota and northern wisconsin and also the adjacent canadian provinces of ontario and manitoba are endemic for blastomycosis but are free of histoplasmosis [43] , [44] . similar to h. capsulatum, b. dermatitidis is a soil-dwelling fungus. infection occurs by inhalation of airborne spores. infected individuals develop a positive blastomycin skin test reaction or the in vitro correlate of delayed hypersensitivity. as with histoplasmosis, isolated microfoci of high infectivity exist in a large endemic area. outbreaks often occur during activities such as hunting, camping, or canoeing in wooded or swampy environments [45]; these are most common when soil temperatures have been increasing for several days and when there is rain on the day of exposure. for sporadic cases, residence close to water in a highly endemic area and recent excavation activity are risk factors [46] . dogs are also susceptible to blastomycosis. canine blastomycosis is a well-recognized entity in veterinary practice in the endemic areas. the recognition of canine cases in a community should alert physicians that human blastomycosis may be present in their geographic area [47] . at ambient temperatures, the fungus grows as an aerial mycelium. when foci of actively growing blastomyces are disturbed, small 2-to spores become airborne and an infective aerosol is formed. these infecting particles then may be inhaled by humans or by other mammals disturbing the site. some spores may escape the nonspecific defenses of the lung and reach the alveoli. the initial inflammatory response is neutrophilic. as the organism converts to the parasitic yeast form and begins to multiply, large numbers of yeast are seen, surrounded by neutrophils. following this macrophages increase in number. eventually, as specific cellular immunity develops, there are giant cells and well-formed epithelioid granulomas. in contrast to histoplasmosis, the neutrophilic component of the inflammatory response does not disappear completely, and the histopathologic examination often shows a mixed pyogenic and granulomatous response, even in chronic cases [2] . it would be misleading, however, to think that there is a "characteristic" tissue response in blastomycosis. occasionally, the neutrophilic component is minimal and the granulomas are noncaseating, producing a picture similar to that of sarcoidosis. in contrast, granulomas are sometimes entirely absent in overwhelming infections. the entire inflammatory reaction consists of neutrophils, and the histopathologic picture mimics that of bacterial infection. the histopathologic response in cutaneous blastomycosis is striking. the stratified squamous epithelium becomes markedly hyperplastic, with exaggerated downgrowth of the rate pegs. within these fingerlike projections are a number of microabscesses. the same hypertrophic tissue response is seen when the disease involves the oropharynx or the larynx. the histopathologic appearance may superficially resemble carcinoma. the characteristic organisms, seen best with special stains, provide a diagnosis. the initial inflammatory and immune response may confine the infection to the lungs and the hilar lymph nodes. it is likely (but not proven) that selflimited early fungemia does not occur as often as it does in histoplasmosis. in some instances, however, the organism spreads beyond lung and the hilar nodes. dissemination is usually to skin, bones, prostate, and meninges but can be seen in any organ [2], [48] . the incubation time for blastomycosis is longer than for histoplasmosis and more variable. in the eagle river outbreak, in which time of exposure was short and precisely defined, the median incubation period was 45 days, with a range of 21 to 106 days [49], [50] . the portal of entry is almost always the lung, and the primary illness is a lower respiratory infection. some patients have an acute illness that resembles bacterial pneumonia, in contrast to acute pulmonary histoplasmosis, which more closely mimics influenza. the onset of symptoms is abrupt, with high fever and chills, followed by cough that rapidly becomes productive of large amounts of mucopurulent sputum. pleuritic chest pain may occur. this acute onset is common in an outbreak setting, but also may be seen in sporadic cases. in most sporadic cases, however, the onset of clinical symptoms is more gradual. the patient presents with a low-grade fever, productive cough, and weight loss [48] [51] . lung cancer or tuberculosis are highest in the differential diagnosis, rather than bacterial pneumonia, depending on the roentgenographic findings. physical examination is usually unremarkable except for fever. auscultation of the chest in patients who have segmental or lobar infiltrates may show crackles and focal consolidation; more often the physical examination is negative. skin lesions are highly variable in appearance, ranging from subcutaneous nodules and abscesses to papules to ulcers with heaped up borders mimicking squamous cell cancers. perhaps the most characteristic lesion has irregular borders and a crusted surface, varying in size from 1 to 10 or more centimeters. skin lesions may be single or multiple and may occur in crops of several new lesions daily or every few days if the disease is rapidly disseminating. routine laboratory tests are seldom helpful. in cases resembling acute bacterial pneumonia, the white blood cell count is elevated, and frequently there is a shift to the left toward earlier forms in the granulocyte series. there is no "characteristic" chest roentgenographic pattern in blastomycosis [52] . lesions may vary from single or multiple round densities throughout both lung fields to segmental or lobar consolidation. severe pulmonary infections can present with diffuse infiltrates, nodular, interstitial, or even alveolar (figure 3) . the diffuse alveolar infiltrates are identical to acute lung injury (as in acute respiratory distress syndrome) of diverse cause. mass-like perihilar infiltrates, especially on the right side, are common and are often misinterpreted as neoplastic. on the lateral chest roentgenogram, the mass-like infiltrate is usually behind the hilum, in the apical-posterior segment of the lower lobe. hilar lymph node involvement may occur but is not nearly as common as in histoplasmosis. cavities may occur during the acute phase of the illness and usually close during successful treatment. unlike histoplasmosis, calcification due to healed blastomycosis is rare. figure 3 47 year old man who failed antibiotic therapy for a community acquired pneumonia, presenting with a rapidly progressive respiratory failure. bronchoalveolar lavage recovered blastomyces dermatitidis. extrapulmonary spread of the fungus may occur during the acute, symptomatic phase of the illness. in some instances, only the distant lesion (usually skin or bone) is symptomatic. the skin and the bony skeleton are the most common sites of symptomatic extrapulmonary spread. the prostate gland, meninges, oral pharynx, larynx, and abdominal viscera, including the liver and the adrenal glands, are involved less frequently [2] . blastomycosis can present as a progressive infection in patients with t-cell defects, including organ transplant recipients and other patients being treated with high-dose glucocorticoids and other immunosuppressive therapy for malignant and nonmalignant disorders. as with histoplasmosis, the disease can often be cured with amphotericin b in patients with intermediate degrees of immunosuppression. blastomycosis is much less common in aids and other t-cell-deficient conditions than are histoplasmosis and coccidioidomycosis. this is probably because exposure to this fungus, while immunosuppressed, is less common and because there is a smaller reservoir of patients with remote healed infection waiting to relapse should t-cell function markedly decline [53] . blastomycosis can also occur in aids, usually with a cd4 count below infection is particularly severe, and cure is not likely. maintenance therapy is required for those who respond to initial treatment. patients with aids are likely to progress with widespread dissemination of the infection with multi-organ involvement. patients may present with sepsis like picture. meningitis or brain abscess are common in this setting and it is associated with a high early mortality [54]. the easiest and most rapid method of diagnosis is examination of expectorated sputum or aspirated pus after 10% potassium hydroxide digestion [2] . the characteristic large (8-to organism is easily identified. the yeast is single budding, with a broad neck of attachment between the parent and the daughter cells. the wall is thick and is double refractile, and there are multiple nuclei. other direct fungal stains including periodic acid schiff (pas), calcoflour white, and silver stains are more sensitive. another sensitive technique for rapid diagnosis of blastomycosis on direct sputum smears is cytologic analysis with the standard papanicolaou stain (figure 4) . the direct techniques are probably complementary and examining multiple sputum samples increases diagnostic yield [55] . papanicolaou stain of a sputum specimen from a patient with rapidly progressive respiratory failure after a community acquired pneumonia, showing the large budding yeast with broad neck of blastomyses. bronchoscopy is useful when patients are unable to expectorate adequate sputum, and when urgent diagnosis is needed because of rapid pace of the illness. in one study, bronchoscopy (specimens obtained included bronchoalveolar lavage (bal) in 64% and bronchial washings in all) was diagnostic in 92% of patients when culture results were included in the final analysis [56] . for patients who are acutely ill or have an ards-like picture, rapid diagnosis is crucial and can only be achieved by direct examination of respiratory secretions. if direct sputum smears are negative or not possible, then bronchoscopy should be done urgently with bal and bronchial washings sent for both direct fungal stains (some combination of koh, calcofluor white, silver stain, pas and cytology preparation) and for culture [57] . the cytology laboratory should be informed whenever there is high clinical suspicion of infection. cellblocks of concentrated bal fluid can be done to maximize the yield of the submitted specimens. histopathologic examination of biopsy material is also an excellent way to establish the diagnosis. the decision whether or not to perform transbronchial biopsies at the time of initial bronchoscopy will likely depend on contraindications in any given patient that give added risk beyond risk of bal. this is particularly true in critically ill patients. if blastomycosis is being considered in this setting, bronchoscopy with bal can be the initial procedure, reserving transbronchial biopsy for cases with no diagnosis from a safer and easier first procedure. standard hematoxylin and eosin stains do not stain the fungus and special stains are required. the periodic acid-schiff stain preserves morphological detail, but silver stains are more commonly used and likely have better sensitivity. identification of the fungus by culture is not difficult, but it is slower. growth may occur as early as 5 to 7 days but often takes several weeks. exoantigen testing can provide positive identification as soon as good growth is established. formerly, positive identification required conversion of the mycelial culture to the yeast phase of growth, adding 1 or more weeks of delay. serologic testing is of limited value in diagnosis of blastomycosis. they are positive in about a quarter of cases. most cases of blastomycosis are diagnosed by smear, culture, and histopathology rather than by serological tests. severely ill patients with pulmonary blastomycosis require immediate and aggressive treatment with amphotericin b. itraconazole is highly effective for blastomycosis and is the treatment of choice for most patients with pulmonary and nonmeningeal disseminated disease; oral therapy with 400 mg/day for 6 months successfully treats most patients with mild to moderate pulmonary disease, skin disease, and bone disease. amphotericin b (dosing as described for histoplasmosis; total cumulative dose 2000 mg) is preferred for a small minority of severely ill patients, including all patients with diffuse infiltrates and severe gas exchange abnormalities. patients with edematous lobar pneumonia (bulging fissures), extremely toxic patients, and patients who are rapidly disseminating should all receive amb. for these severe infections, sequential therapy with amb to clinical improvement (usually 500 to 1000 mg total dose) followed by 6 months of oral itraconazole is often used and is effective though not well studied. this approach is also used for aids patients. patients with aids and blastomycosis are not permanently cured. life-long maintenance therapy is needed after induction therapy to improve symptoms. meningeal blastomycosis is always treated with systemic amphotericin b therapy in standard doses. l-amb achieves higher brain tissue levels in animals and may be preferred. fluconazole is overall less potent for blastomycocis than itraconazole but penetrates cns much better. high dose fluconazole (often in combination with l-amb) has been used for central nervous system blastomycosis. voriconazole is theoretically attractive but has not been studied. intracisternal amphotericin b has been used anecdotally in addition to systemic therapy for selected patients, but it is uncertain whether there is additional benefit [58], [59] . intracisternal therapy is used less often now that there is a wider range of therapy. coccidioidomycosis is the illness caused by the tissue-dimorphic fungus coccidioides immitis. although most infections are mild and self-limited, the spectrum of illness includes life-threatening pulmonary disease and widely disseminated systemic disease with a high mortality rate. differences between it and histoplasmosis and blastomycosis include different endemic areas, higher frequency of meningeal infections, and poorer response to all antifungal therapy, including amphotericin b. the endemic area for coccidioidomycosis in north america is the southwestern united states and the contiguous areas of northern mexico. the endemic area of the united states includes central and southern california and extends eastward to arizona, new mexico, and western texas. in nature, the fungus grows as an aerial mycelium with septate hyphae. alternating cells form thick-walled barrel-shaped structures called arthroconidia, with empty cells in between. when a natural site is disturbed, the mature arthrospores easily detach and become airborne, producing an infective aerosol. the risk of infection is greatest during the hot dry summers. strong winds can carry the arthrospores for long distances. a huge wind storm that blew north from the san joaquin valley in 1977 caused a major outbreak of coccidioidomycosis in sacramento, far north of the usual endemic area [60] . not surprisingly, occupations and activities with exposure to the soil carry the greatest risk for infection, including construction work, farm labor, and working on archeological digs [61]. after inhalation, some arthrospores evade the nonspecific lung defenses and reach the alveoli, where germination begins. the arthrospores develop into spherules, the tissue phase of the fungus. spherules are large, round, thick-walled structures that vary in diameter between 10 and reproduction of the fungus occurs within the spherule. the cytoplasm of the spherule undergoes progressive cleavage, forming numerous endospores within the spherule. once a spherule matures, it bursts and releases the endospores into the surrounding tissues. each endospore can become a new spherule and thus repeat the process. the initial inflammatory response to inhaled arthrospores is neutrophilic. resident alveolar macrophages also phagocytose the arthrospores and prime specific t lymphocytes, which multiply, recruit more macrophages, and arm the macrophages, engaging specific cell-mediated immunity. even though many well-formed granulomas are seen, the neutrophilic inflammatory exudate does not disappear. histopathologically, there is a mixed granulomatous and suppurative reaction more similar to blastomycosis than to histoplasmosis [3] . granuloma formation is important for successful limitation of the infection. good outcome correlates with preponderance of well-formed granulomas. most primary infections are asymptomatic or relatively mild. the fungus usually remains localized to the lung and hilar lymph nodes. dissemination occurs in less than 1% of patients. hematogenous spread can affect many tissues, including the skin, bones, lymph nodes, visceral organs, and meninges [62] . meningitis is the most feared clinical syndrome with an ominous prognosis [63] . except for rare instances of inoculation coccidioidomycosis, the portal of entry is the lung. about 60% of individuals with primary pulmonary infection remain totally asymptomatic. in the remaining 40%, the spectrum of disease ranges from a mild, influenza-like respiratory illness to a severe, life-threatening pneumonia [62] . the clinical symptoms and their severity are variable. common symptoms include cough, fever, and pleuritic chest pain. cough may be nonproductive, or there may be small amounts of mucopurulent sputum. true rigors are not common. headache, common during the acute phase of the illness, is nonspecific. severe headache is always worrisome, however, because coccidioidal meningitis often becomes clinically apparent during the early part of the illness. if meningitis is suspected, a lumbar puncture should be performed immediately. several dermatologic aspects of acute coccidioidomycosis are important. a mild nonspecific so-called toxic rash occurs in many patients [61], [64] . it is an erythematous macular rash that occurs early during the illness, before the skin test turns positive. erythema nodosum and erythema multiforme are other skin manifestations of primary coccidioidal infection. together with fever and arthralgias, these skin lesions are part of a variable symptom complex first recognized by locals in the san joaquin valley of south central california and labeled "valley fever". more than 75% of patients with primary coccidioidomycosis have an abnormal chest roentgenogram. the most common roentgenographic abnormality is a single or multiple areas of patchy pneumonitis. the ipsilateral hilar nodes are enlarged in about 25% of patients [65] . hilar adenopathy may also be seen without recognizable parenchymal disease ( figure 5 ). this primary complex usually heals rapidly. necrosis in the center of a pneumonic lesion may produce cavitation [66] . this is often accompanied by minor hemoptysis, which may be alarming but is seldom lifethreatening. hemoptysis as a late complication is uncommon but can be life-threatening, in contrast to the minimal bleeding that is often seen as the cavity forms. another complication is rupture of the cavity with development of a pyopneumothorax. in rare instances, primary pulmonary coccidioidomycosis is not self-limited but progresses within the lung. symptoms are fever, cough, and weight loss. the chest roentgenogram shows progression of the infiltrate and variable involvement of the hilar nodes [65] . this form of pulmonary coccidioidomycosis is dangerous and augers impending dissemination. most of the patients with progressive pulmonary disease are either immunosuppressed or belong to groups at high risk for dissemination. the primary pulmonary infection usually either resolves completely or stabilizes. rarely does a patient die when the disease is restricted to the lung. in some individuals, however, the fungus spreads widely throughout the body, resulting in a systemic infection known as disseminated coccidioidomycosis. patients receiving glucocorticoid or cytotoxic or newer immune modulating therapy for malignant or nonmalignant diseases are at risk of dissemination. this is especially true for recipients of renal and other organ transplant and patients with aids [67], [68] . the excess risk of coccidiodomycosis in organ transplant recipients in highly endemic areas has led to targeted prophylaxis to prevent re-activation whenever there is a history of coccidiodal infection or positive serologic results on pretransplant screening [69] . there are other well-recognized risk factors for dissemination. race and ethnicity are important. disseminated coccidioidomycosis is more likely in blacks, filipinos, and native americans than in whites. male gender is also a risk factor, as is diabetes mellitus. the very young and the very old are more likely to have dissemination [62] . there is much anecdotal information suggesting that coccidioidomycosis during the third trimester of pregnancy may be a severe illness with rapid dissemination. dissemination from the primary pulmonary focus tends to occur early, usually within a few months after a symptomatic pulmonary infection. in some patients, however, the findings of disseminated disease are the first manifestations of coccidioidomycosis, presumably because the preceding pulmonary infection was sub-clinical. dissemination may involve any organ in the body. the skin is one of the most common sites of dissemination and is involved in most patients some time in the course of the disease. involvement of the bones is the next most common manifestation of disseminated coccidioidomycosis. osteomyelitis may be either the sole evidence of extrapulmonary spread or part of a more widespread dissemination. bone disease is usually restricted to one or two sites, but occasionally as many as eight separate lesions may be present. meningitis is the most dreaded complication of coccidioidal dissemination. between one third and one half of all patients with disseminated disease have meningitis, frequently as the only obvious extrapulmonary site. the onset of meningitis may be subtle, with only mild headache and minimal alteration of mental functions. striking boardlike nuchal rigidity, as in purulent meningitis, is seldom seen [63] . in fact, the findings of meningitis can be so minimal that all patients with dissemination at other sites should have a diagnostic lumbar puncture to exclude meningitis. involvement of the base of the brain is characteristic. as the disease progresses, an exudate frequently obstructs the aqueduct of sylvius and the foramina of the fourth ventricle, producing hydrocephalus. when obstruction occurs, the patient's clinical condition suddenly worsens, with diminished level of consciousness and the development of papilledema. the cerebrospinal fluid shows characteristics of chronic meningitis: predominantly mononuclear cell pleocytosis, increased protein, and decreased glucose. occasionally, eosinophils are present in the cerebrospinal fluid. if present, they are a valuable clue to the possible coccidioidal nature of the chronic meningitis. when coccidioidomycosis complicates hiv infection, the severity depends on the residual immune competence of the host. with near-normal cd4 lymphocyte counts, coccidioidomycosis is not significantly different from the disease seen in normal hosts. when the cd4 count falls below 250 cells/ml, disseminated disease tends to be severe and rapidly progressive. patients usually have high fever, complain of dyspnea, and are hypoxemic; chest roentgenograms often show diffuse reticulonodular infiltrates with nodules 5 mm or greater in diameter ( figure 6 ). diffuse macronodular pulmonary infiltrates are present in less than one per cent of non-aids patients with disseminated coccidioidomycosis, but in up to 50% of advanced aids patients with this condition. meningeal disease is present in up to 25% of the patients [70], [71] . mycologic studies. direct examination of sputum and other respiratory specimens (or pus from a non-pulmonary site) may reveal the diagnostic spherules. direct smears have highest utility in patients who produce copious sputum or have multi-lobar infiltrates [72] . bronchoscopy is often performed in selected cases. in one study bronchoscopy was diagnostic in 69% of patients (compared to 32% for sputum stains and cultures) when patients with solitary pulmonary nodules on chest radiograph were excluded from analysis [73] . this study also showed usefulness of a postbronchoscopy sputum and equivalent sensitivity for papanicolaou and silver staining. the airway can be examined at the time of bronchoscopy and may be abnormal, providing clues to the diagnosis [74]. bronchoscopy is typically performed in patients who are immunosuppressed and severely ill, especially if they have diffuse infiltrates on chest radiograph. multiple infections often co-exist, adding additional value to diagnostic bronchoscopy early in the course of illness [75] , [76] . bronchial washings and bronchoalveolar lavage fluid should be sent for cytology, fungal stains, and culture. in a recent study of an aids patient in phoenix, arizona, the papanicolaou stain was the most useful direct test (when compared to koh and calcofluor white) for rapid diagnosis of pulmonary coccidioidomycosis and was even positive in two patients with negative cultures [77] . histopathologic examination of biopsy material is extremely helpful. when mature spherules (visible on standard hematoxylin and eosin stained tissue sections) are seen, the diagnosis is secure. more commonly, only endospores, immature spherules or spherule fragments are present. therefore fungal stains such as a silver stain should always be used in addition to hematoxylin and eosin staining. in one study, transbronchial biopsy yielded a specific tissue diagnosis of coccidioidomycosis in eight of eight patients. cultural identification of the fungus is not difficult but is hazardous to laboratory personnel. isolation should be attempted only under rigid biohazard protection. traditional laboratory methods for identifying culture isolates require conversion of mycelial-phase cultures to the tissue phase either by animal inoculation or directly by the use of slide cultures. now immunodiffusion tests are performed directly on the supernatants of liquid mycelial-phase cultures. this method of identification (called exoantigen testing) is safer, simpler, and faster [78] . positive identification of a coccidioidal isolate can sometimes be made by day 5, although it usually takes longer. because cultural identification is slow and even somewhat dangerous, serologic tests have been developed that facilitate rapid diagnosis [3] [79] . a tube precipitin tests for detection of ig m antibodies is positive in 90% of patients by the third week (negative only in very mild infections). because the test usually reverts to negative within 3 months, it is quite specific for recent infection [3] . currently, an immunodiffusion test for igm has largely replaced the tube precipitin test. the immunodiffusion test measures the same antibodies, but it is easier to perform. the most important serodiagnostic test is the complement fixation (cf) test. cf antibodies are of the igg class and appear later than igm antibodies. in most symptomatic patients, the cf test is positive by 2 months and remains positive for several months or longer [79] . the test is highly specific but is not sensitive. most asymptomatic skin test converters never have cf titers over 1:8, which is the threshold for a positive result. most symptomatic patients have titers of 1:8 or 1:16. titers of 1:32 or higher are generally associated with more severe infections and poorer prognosis. in the classic studies of smith and colleagues [79], many patients with these high titers either had already undergone or were about to undergo dissemination. however, other patients with disseminated coccidioidomycosis did not have high titers. also the cutoff of a 1:32 cf titer as a harbinger of dissemination never transferred perfectly to other laboratories that did not use the same method or the same antigen. a single cf titer, no matter how high, should never be used to make a diagnosis of disseminated coccidioidomycosis. nonetheless, a steadily rising titer should raise the suspicion of disseminated coccidioidomycosis and prompt further tests (including bone scan, spinal tap, or both when appropriate) to better define the extent of disease. because dissemination is more likely in immunosuppressed patients, in diabetics, and in certain racial and ethnic groups, it may be prudent to treat patients in high-risk groups during the primary infection, before dissemination takes place. in the past some authorities recommended a treatment course to a total dose of 500 to 2000 mg of amphotericin b [80] . similarly, many experts believed that all patients with pulmonary disease that is severe or persists beyond a few weeks should receive amphotericin b to approximately the same total dose to prevent local pulmonary progression and to prevent dissemination. in current practice, many such patients (and also less symptomatic patients with pulmonary coccidioidomycosis of shorter duration) are often given fluconazole for 3-6 months, reserving amb for patients with diffuse infiltrates and women in the third trimester of pregnancy. these recommendations are based on expert opinion and observational studies. amphotericin b is likely the best treatment for persistent pulmonary coccidioidomycosis. because of their lesser toxicity, oral azoles are often tried. about two thirds of patients have clinical improvement with azole therapy, but many relapse when the course of treatment is finished. ketoconazole was used first. currently fluconazole and itraconazole are being used. voriconazole will likely be evaluated in the future. disseminated coccidioidomycosis requires prompt and aggressive treatment. unfortunately, amphotericin b is not as effective for disseminated coccidioidomycosis as it is for disseminated histoplasmosis or blastomycosis. the standard dose of amphotericin b is 2500 to 3000 mg given over many weeks or months. if necessary, much larger total doses may be given [81] . daily doses of amphotericin b (usually 40 to 50 mg) are given while the patient is acutely ill. when the patient stabilizes, frequency should be reduced to three times weekly. currently disseminated disease without cns involvement should be treated with fluconazole or itraconazole first, especially in mild to moderate cases. amb should be reserved for severe disease or treatment failure. fluconazole and itraconazole are now azoles of choice for nonmeningeal disseminated coccidioidomycosis. neither is perfect for difficult cases for which even amphotericin b is often only suppressive. long-term therapy is often required, extending to years or even indefinitely. fluconazole has the advantage of better absorption, less gastrointestinal upset, and better penetration of the central nervous system. in a recently published randomized controlled trial, oral fluconazole and itraconazole were compared for treatment of non-meningeal coccidioidomycosis. soft tissue dissemination responded best. overall, itraconazole was somewhat more effective than fluconazole, producing response in 63% of the patients vs. 50% response in fluconazole treated patients (p = 0.08). among patients with skeletal infections, itraconazole was clearly superior, (p=0.05) [82] . some difficult cases of bone, lymph node, and soft-tissue coccidioidomycosis may be best managed with surgical drainage of focal abscesses, a 1000 to 2000 mg course of amphotericin b, and a prolonged course of itraconazole or fluconazole. as might be expected, the treatment of disseminated coccidioidomycosis in aids is particularly difficult. because of the rapid tempo of the disease, amphotericin b should be used initially, especially if the patient is severely ill. if the clinical course stabilizes, it is reasonable to switch to fluconazole for long-term suppression. prognosis is poor. even with prompt diagnosis and treatment, up to 40% of severely immunosuppressed patients die during the initial hospitalization. other patients, usually with lesser degrees of immunosuppression, respond well to treatment[70], [71] . meningeal coccidioidomycosis is a major therapeutic challenge. the standard therapy in the past included a course of 2000 to 3000 mg systemic amphotericin therapy plus intensive and lengthy intrathecal (by lumbar or cisternal route) amb therapy [63] . intrathecal (or, less commonly, intraventricular via surgically placed reservoir [83] ) amb in doses between 0.25 and 1 mg was injected two to three times weekly until symptoms and cerebrospinal fluid pleocytosis resolve. even after the patient had apparently recovered fully and cerebrospinal fluid pleocytosis had resolved, most authorities recommended continued injections of amphotericin to prevent relapse, first weekly and then at longer intervals. relapses were common, but, with careful management, lengthy remissions could be obtained. because of the toxicity of this once standard approach to coccidioidomycotic meningitis, fluconazole has been evaluated as primary therapy for stable patients and as suppressive therapy after initial response to amphotericin b for more severely ill patients. most patients respond favorably to fluconazole and maintain good clinical function. dosage is 400 to 600 mg/daily or even higher. therapy has to be continued long term, likely indefinitely [84] . recently anecdotal reports have shown favorable response to voriconazole and this agent will undoubtedly be tried in various forms of coccidioidomycosis, including meningitis. a drug with potency and wide spectrum of itraconazole but with tissue penetration like fluconazole seems especially attractive for an treatment resistant illness with high incidence of meningeal spread. however clinical data is sparse. severely ill patients with both nonmeningeal and meningeal disease were previously treated with intravenous and intrathecal amphotericin b. now they are sometimes treated with intravenous amphotericin b for faster, more effective initial therapy of the nonmeningeal disease and with fluconazole to control the central nervous system infection. amphotericin b is continued to clinical improvement and fluconazole indefinitely. newer antifungal agents are being developed; their potential role in coccidioidomycosis is uncertain. as mentioned voriconazole has some promise because it has better cns penetration than itraconazole -and yet may retain the potency advantage of itraconazole over fluconazole which has been demonstrated in non-meningeal disseminated disease. state of the 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of bronchial washings in patients with suspected coccidioidomycosis immunological procedure for the rapid and specific identification of coccidioides immitis cultures pattern of 39,500 serologic tests in coccidioidomycosis practice guideline for the treatment of coccidioidomycosis chemotherapy of systemic mycoses (first of two parts) comparison of oral fluconazole and itraconazole for progressive, nonmeningeal coccidioidomycosis. a randomized, double-blind trial. mycoses study group a subcutaneous reservoir for intrathecal therapy of fungal meningitis is it ever safe to stop azole therapy for coccidioides immitis meningitis key: cord-009144-3slh1nbk authors: jacobs, j.w.; peacock, d.b.; corner, b.d.; caul, e.o.; clarke, s.k.r. title: respiratory syncytial and other viruses associated with respiratory disease in infants date: 1971-05-01 journal: lancet doi: 10.1016/s0140-6736(71)92440-8 sha: doc_id: 9144 cord_uid: 3slh1nbk diagnosis by virus isolation and serology was attempted in 377 cases of respiratory-tract infection in infants under one year of age admitted to hospital during two winters. a diagnosis of infection with respiratory syncytial (r.s.) virus was made in 40%, rhinovirus in 6·1%, adenovirus in 3·7%, parainfluenza in 2·1%, enterovirus in 1·9%, and influenza in 1·3%. r.s.-virus infections were more severe than others and occurred mostly in the first five months of life, with a peak at two months. rhinovirus infections occurred at all ages, and often involved the lower respiratory tract. of the 12 deaths, only 1 (due to r.s. virus) was not associated with a contributory cause. maternal antibody to r.s. virus did not notably affect the incidence or severity of r.s.-virus infections. there have been few intensive studies of respiratoryvirus infections of infants.1-5 to prevent these infections, it is necessary to know which viruses cause the most severe illness and whether maternal antibody plays any part in their prevention. we report here the results of a survey of respiratory-virus infections in infants under one year of age in hospital. all infants under one year of age in southmead hospital and bristol children's hospital who had respiratory infections during a period covering two winters (nov. 1, 1965, to july 2, 1966 , and sept. 10, 1966, to april 30, 1967) were included. 360 had respiratory infections on admission and 17 developed respiratory infections in hospital. soon after admission (or onset) nose and throat swabs were taken by one of us (j. w. j.) from each infant. the throat swabs were taken with a gagging technique, so that the infants coughed over the swab. at the same time blood was taken from most of the mothers. paired serum samples were obtained from some of the infants with an interval of [4] [5] [6] weeks. one throat swab from each infant was inoculated into bristol hela-cell tissue cultures at the bedside, and was examined thereafter in the university of bristol department of bacteriology. a second throat swab and the nose swab were put together into 50% milk-saline transport medium and inoculated in the public health laboratory into hela cells and primary monkey-kidney tissue culture within 90 minutes. they were also inoculated (often after storage at &mdash;40&deg;c, and if not already yielding a virus) into human embryo kidney and wi-38 human embryo fibroblast tissue cultures and into suckling mice. the methods used have already been described. 3, 6 serology sera were tested by complement fixation (c.f.) against respiratory syncytial (r.s.) virus antigen but not against other viruses. the methods of preparing the antigen and conducting the test have already been described.7,8 e this was graded retrospectively according to a scheme developed during the survey. signs in order of severity were: (1) upper-respiratory-tract infection. (2) specific treatment (excluding nasal drops but including humidified air, physiotherapy, or antibiotics) or temperature >38-80c. (3) chest signs found by radiography, auscultation, or aspiration for less than three days. (4) chest signs for three days or more unless the patient died. (5) oxygen therapy for one or more days, or respiration-rate 70 or more per minute, or peripheral cyanosis. (6) digoxin therapy or heart-rate of 170 or more per minute. (7) additional treatment (cortisone, intubation, or tracheostomy). (8) death. the grade was the most severe sign the patient showed, providing that all the less severe signs were also present. for example, an infant showing signs 1, 2, 3, and 6 was said to have a severity grade of 3. in practice it was rare for an infant not to show an intermediate sign. in 272 cases routine bacteriological examination was undertaken. staphylococcus aureus was isolated from 13%, and streptococcus pneumoniae from 7%. several &bgr;-haemolytic streptococci were isolated, but none were group a. haemophilus influen. was isolated from 8 (3%), in 6 of which r.s. virus infection was also diagnosed. twenty of the infants were admitted with respiratory infections twice, and two were admitted three times. from one infant, two different m rhino viruses were isolated-type 1b at the age of two months and type 2 at the age of ten months. one infant, a premature baby, was admitted seven times over a period of four months (table 11). 4 different viruses were isolated. there was no evidence of hypogammaglobulinsemia. the infant's twin sister was in hospital once with a chest infection due to r.s. virus at the same time as her sister's first admission. subsequent infections of the twin were less severe and frequent, and she was not readmitted. fold or greater rise in titre between acute and convalescent specimens, or a change from passive maternal (secondary) type of antibody to an active (primary) type of antibody in a chequerboard c.f. test with a potent antigen.9 9 of the fifty-four infants in whom r.s.-virus infection was diagnosed and both isolation and serology attempted, the diagnosis was made in twenty-four by serology alone (nineteen infants showed a fourfold rise and five a change from secondary to primary antibody), six by isolation alone, and in twenty-four by both methods (twenty-two showed a fourfold rise and two a change from secondary to primary antibody). r.s. virus was isolated more frequently from throat swabs inoculated direct into tissue culture at the cotside (115/127) than from swabs put into transport medium and inoculated within ninety minutes (69/127 isolates). r.s. virus was isolated more frequently from specimens taken three to six days after the onset of the illness (43% of 189 specimens) than from those taken before three days (25% of 72 specimens) or after six days (26% of 93 specimens) from onset. age table i shows virus infections diagnosed in infants of different ages. two-thirds of the specimens came from infants under six months of age. the rate of virus diagnosis was higher under six months of age, with a peak at three months. this was because most infections were caused by r.s. virus, and the peak for this virus was in the age range one to five months. parainfluenza and influenza viruses were found only over four months of age; and rhino viruses, enteroviruses, and adenoviruses were found at all ages. the viruses recognised in the first month of life were r.s. (5), rhinovirus m, and coxsackie b5. of the 377 respiratory infections, 212 (56%) were in boys and 165 (44%) in girls (p<0-02). there was no significant difference between the two sexes in the severity of the illness either in all the infants, or in those in whom r.s.-virus infections were diagnosed. rhinoviruses were often associated with lower-respiratory-tract illnesses. an eight-month-old boy was admitted with a ten-day history of wheezing getting worse two days before admission. coarse crepitations in right and left lower lobes. intercostal recession. pneumonia confirmed radiologically. chest signs persisted four days. respiration and heart rates up to 70 and 180 per minute. temperature up to more than 40-5 &deg;c. oxygen and cortisone given. discharged after ten days. nose and throat swabs taken on the second day in hospital yielded rhinovirus h. there was no evidence of r.s.-virus infection by either serology or isolation. no bacteria implicated. enteroviruses also were often associated with lowerrespiratory-tract illness. a four-month-old girl was admitted with a three-day history of worsening coryza. inspiratory wheeze and coarse rales. bronchopneumonia confirmed radiologically. no fever. respiration and heart rates up to 65 and 145 per minute, respectively. liver enlarged. cyanosis. chest signs lasted two days. discharged after nine days. echo-22 virus isolated from swabs taken on the second day in hospital. there was no evidence of infection with r.s. virus by serology or isolation. no bacteria implicated. the grade of severity was calculated for all illnesses in which definite viruses were implicated and in 129 others (table iv). infants with r.s.-virus infection were more severely ill than other infants. illnesses due to rhinoviruses were of moderate severity. the small numbers of parainfluenza, influenza, and enterovirus infections were mild. there was a contributory cause of death in eleven; ten had congenital defects and one had gastroenteritis due to escherichia coli 0119. the remaining infant was first admitted aged four months with severe bronchiolitis. discharged after twenty-five days, but readmitted two days later with bronchopneumonia. coarse rales in upper anterior chest and right axilla. temperature slightly raised. respiration and heart rates up to 80 and 200 per minute, respectively. no viruses or other organisms were implicated during the first admission, but the throat swab taken on the day of second admission yielded r.s. virus. no other organisms implicated. died three days after admission. at necropsy, muco-pus in larynx, trachea, and bronchi. generalised congestion of lungs. normal aeration in only a few small peripheral areas. the microscopical appearance resembled hyaline-membrane disease of newborn. no other abnormalities. in our hands the c.f. test was a more sensitive method than isolation for diagnosis of r.s.-virus infections in the infants where both methods were attempted. 7 r.s.-virus infections were diagnosed by demonstrating a change from passive to active antibodies,9 for which it is necessary to do a cumbersome chequerboard titration, but most showed straightforward rises in titre when a large amount of antigen was used in the c.f. test. berglund and strahhnann 12 conclude that isolation is better than serology. much will depend on the potency of the c.f. antigen, the number of antigen units used, and the time of taking the convalescent serum 13; and the sensitivity of the cell line used for isolation and the timing of inoculation are also important. other respiratory pathogens the diagnosis-rate for all respiratory pathogens of 53% might have been higher if paired sera had been obtained from all the infants, and if serology for viruses other than r.s. virus had been performed. we did not look for mycoplasma pneumoniae & o a c u t e ; or coronaviruses 14 or cytomegaloviruses, all of which are known to cause respiratory infections, especially cytomegalovirus in the first year of life. 15 there is no convincing evidence that the bacteria we isolated (staph. aureus, str. pneumoniae, and hcemophilus infiuenzae) are a frequent primary cause of respiratory illness in infants. all are common in healthy infants. thiese were found on 9 occasions. in 5 double infections an adenovirus was isolated, but here the second virus (r.s. and influenza) probably caused the illness, the adenovirus being carried in the tonsils. the findngs in one patient (table 11) support this. 19 are subclinical. however, when one of these viruses is isolated from a child with a respiratory infection, it is probable that it is the cause of the illness. it is even more probable that illnesses associated with r.s. and parainfluenza viruses are due to these viruses. 5 type of illness we found that rhinoviruses often produced a severe lower-respiratory-tract illness, as did hamparian et al. 20 the finding of lower-respiratory-tract illness in association with echo 22 virus is interesting in view of similar associations described by berkovich and smithwick. 21 of the twelve infants who died with respiratory infections, eleven had a contributory cause of death. it seems, therefore, that respiratory viruses are not frequent killers of otherwise healthy infants in bristol. in newcastle only a quarter of infants who died with respiratory infections had congenital defects. 2 r it seems that viruses, especially r.s. virus, are more important as a cause of severe illness and death among infants in newcastle than among infants in bristol. there may be socioeconomic reasons for this. in this survey, as in others, r.s. virus was the commonest cause of respiratory illness requiring admission at this age (40&deg;0), and the illnesses were more severe than those associated with other viruses (table iv). rhinoviruses were the next most important (6%) and often caused severe illnesses. in this age-group, adenoviruses, influenza, and parainfluenza viruses were relatively unimportant. this is supported by work with respiratory viruses in the bristol public health laboratory over the past nine years (unpublished). we found a heightened susceptibility of males to respiratory infections, previously noted by moss et al. 23 effect of maternal antibody the few parainfluenza virus infections observed in this survey occurred only in infants more than four months of age. over the past nine years in the bristol public health laboratory, among large numbers of parainfluenza viruses isolated, parainfluenza types 2, 3, and 4 have only occasionally been isolated from infants under four months of age, and type 1 never. this is not a new observation, and there is evidence that maternal antibody protects against these viruses. 24 our experience with influenza virus is similar, and in this survey there were no influenza virus infections below the age of four months. maternal antibodies probably played their role here also. on the other hand, rhinovirus infections were detected from a very early age, and this is to be expected because mothers would not have antibodies against all rhinovirus types at the time of delivery. indeed maternal antibody may protect against infections with this group of viruses. the position with regard to r.s.-virus infections is less clear-cut. for instance it is widely accepted that the majority of severe infections occur in the first three months of life. in our series the peak of incidence was at two months, and infections have been recorded as early as ten days after birth. 25 there is no multiplicity of antigenic types to account for this early incidence as there is for rhinoviruses. on the other hand, the incidence of antibodies to r.s. virus in the adult population-and by implication exposure to r.s. virus-is very high. one would expect, therefore, the epidemiological features of this infection in infants to resemble influenza more closely than the common cold. the series we investigated may have been a selected group, in that for some reason we saw principally infants of mothers who lacked antibodies to r.s. virus. unfortunately a retrospective survey such as this cannot distinguish between maternal-antibody levels present before birth and those acquired at the time of, or just before, the infant's illness; and it is far more likely that the mother of an infected baby has just had an antigenic stimulus from the same virus. the high levels of antibody we observed in mothers of infected infants are thus meaningless in terms of protection afforded to the population studied. the vaccination studies carried out by some american workers, using killed virus vaccines 26-29have often been quoted in support of the idea that antibody to r.s. virus actually increases the severity of a subsequent infection. 10 however, other types of r.s.-virus vaccines prepared differently may not have the same effect.30,3! neither is there any concrete evidence in table vil to support the hypothesis of antibody potentiation, for although at two months of age the geometric mean titre of antibody is higher in the group with r.s.-virus infections, as is the percentage of acute sera with antibody present, this trend is reversed at one month of age. similarly there is no correlation between high antibody titre and mean severity grade. although the numbers are extremely small, there was evidence in table v that in the first month of life infants had less severe r.s. infections than older infants. this could be taken as evidence that maternal antibody may actually have a protective effect. chanock et al. 3 found that the titre of r.s.-neutralising antibody was two times lower in the acute sera of infants with r.s. infections than in those without, and suggests that antibody-antigen complexes in the lung may lead to depletion of antibody in the serum. gardner et al. 3 present persuasive evidence in support of the postulate that the development of immediate hypersensitivity plays a dominant role in the pathogenesis of acute bronchiolitis in r.s. infections. however, they favour the idea of a gell and coombs 33 type-i hypersensitivity response, and to sustain this argument they postulate a previous exposure to r.s.virus antigen. again we find no evidence to support this hypothesis, since, where antibody was detected in the acute-phase sera, it behaved like adult antibody in the c.f. test 9 and was thus, presumably, maternal in origin. we thank the other clinicians for permission to study the infants under their care and dr. m. 0. roebuck for typing the rhinoviruses. financial support for one of us (j. w. j.) by the medical research council is gratefully acknowledged. requests for reprints should be addressed to j. w. j., department of pathology, royal veterinary college, royal college street, london n.w.i. one of the major problems in clinical organ transplantation is the detection and reversal of rejection episodes. centres differ in their methods of overcoming such episodes, but most administer an increased dose of oral steroids.1-4 actinomycin c,1-3,5 antilymphocyte globulin 6 and local graft irradiation 1, 2, 7 have been used to the same end. at the start of our clinical renal-transplant programme we decided to use prednisolone given as a single 1 g. intravenous dose over a period of 2 hours for the treatment of acute rejection crises. this decision was made for several reasons. firstly, prednisolone at this dose level is lympholytic and has a short half-life of 60-90 minutes, 8,9 giving maximum lymphocyte damage with relatively little in the way of chronic side-effects. secondly, we were impressed by the results published by kountz and cohn, 11 who used large doses of intra-arterial steroids, but we felt that this method was potentially dangerous and, perhaps, unnecessary. thirdly, we hoped to avoid the high oral doses of steroids normally used to control rejection and thereby avoid serious complications in the first few months after transplantation. the ability of a high dose of intravenous prednisolone to reverse acute rejection episodes was also investigated using the heterotopic rat-heart-transplant model. we present here the clinical and experimental results of this type of antirejection treatment. sixteen patients were transplanted over a 24-month period. three patients received kidneys from sibling medical research council working party on acute respiratory virus infections. ibid. 1965, ii acta p&oelig;diat. scand "... we need to concentrate less on the reduction of infant mortality as a goal in itself than on assuring that children who survive are whole and healthy; and that it is fallacious to argue about whether the quality of medical care or the child's environment is the more important factor in relation to infant mortality. these cannot be separated. no matter how good the medical care system is, mortality rates cannot be lowered below a certain point unless certain changes are made in the social environment. the ability of single large (1 g.) doses of intravenous prednisolone to reverse rejection episodes has been investigated clinically and experimentally. sixteen renal-transplant recipients have been treated in this way. the oral dose of prednisone was not increased during these episodes and no additional treatment was given. this therapy reversed 86% of rejection crises without any toxic effects. one patient has died from infection, 1 month after transplantation. using the heterotopic rat-heart-transplant model the ability of intravenous prednisolone, antilymphocyte serum (a.l.s.), intraperitoneal prednisolone, and azathioprine to reverse rejection in recipients immunosuppressed with a single dose of a.l.s. at the time of transplantation were compared. intravenous prednisolone was the only successful agent and prolonged survival by 9&plusmn;3 days. key: cord-016223-nk8xwa0t authors: andersen, bjørg marit title: strict isolation date: 2018-09-25 journal: prevention and control of infections in hospitals doi: 10.1007/978-3-319-99921-0_19 sha: doc_id: 16223 cord_uid: nk8xwa0t strict isolation: suspected highly infectious and transmissible virulent and pathogenic microbes, highly resistant bacterial strains and agents that are not accepted in any form of distribution in the society or in the environment. examples are completely resistant mycobacterium tuberculosis, viral haemorrhagic fevers like ebola and lassa, pandemic severe influenza and coronavirus like sars, mers, etc. in most countries, strict isolation is a rarely used isolation regime but should be a part of the national preparedness plan. for instance, in norway, strict isolation has not been used for the last 50–60 years, except for one case of imported ebola infection in 2014. patients in need of strict isolation should be placed in a separate isolation ward or building. infection spread by contact, droplet and airborne infection, aerosols, re-aerosols, airborne microbe-carrying particles, skin cells, dust, droplets and droplet nuclei. at the same time, it is always contact transmission (contaminated environment, equipment, textiles and waste). the source of infection is usually a patient but may also be a symptomless carrier or a zoonotic disease. to prevent transmission from an infectious patient to other patients, personnel, visitors and the environment and to protect patients with impaired immune defence against infection [1] [2] [3] [4] [5] [6] [7] [8] . • all patients having contagious disease that can easily be transferred directly or indirectly via contact, blood and body fluids, air/droplets or via equipment, textiles and surfaces. • all patients with significant reduced infection defence or otherwise infection vulnerable and who should be protected against infection. hospital management should ensure necessary capacity and type of isolating units: contact-and air-droplet isolates and protective isolates. updated isolation routines, adequate protective equipment-including ppe-routines for disinfection of rooms and surfaces and disinfectants and hand hygiene facilities should be available. department management should implement isolation procedures, train the use of ppe, control the use of routines and provide sufficient stock and capacity of ppe and means for disinfection and hand hygiene. the staff should follow current guidelines for treatment of patients with infections and for patients that should be extra protected against infections. the isolation unit is usually located in a separate isolation ward or building and has defined as negative air pressure systems (in pascal), separate ventilation with disinfection of air extraction, a properly interlock function and also direct access from the outside. the waste water is decontaminated (autoclave). the patient room has sluice systems and bathroom with through-put decontaminator or autoclave with direct entrance from the patient room. personnel involved in treating high-risk infections should be specialized in isolation work and be healthy, not immunosuppressed, and if possible should be vaccinated, if vaccine is available. the staff should be bound to the ward/unit while isolation treatment takes place. fewest possible should participate in the isolation work. the staff should come and go directly to the high-risk unit and should not stay, work or visit other wards during the isolation period. the infection ward should have restricted admission with registering (name, address, date, time, etc.) and follow-up of all staff and visitors that attended the department. [1, 7, [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] see chap. 21. isolation units for airborne infections should be safe enough for high-risk cases, if used properly [1, 9, [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] . there should be no offices, laboratories, other patient wards or other regular human activities under, over or around this isolation unit. the unit should be located in a separate ward, preferably in a separate building with direct access via an external sluice and internal access through a negative air pressure sluice with sufficient areal for donning and doffing and for a safe treatment of infectious equipment and waste. • each isolate should be 35-40 m 2 , including sluice (6 m 2 ), decontamination room/bathroom (5-6 m 2 ) and patient room (20-25 m 2 ). there should be a minimum of 2-m free zone around the patient's bed on both long sides and at the foot end. • there must be direct access from the patient room to the decontamination room/ bathroom. • interlocked doors must be included in the sluice system. opening of the doors should be in direction from negative pressure to the positive pressure room to avoid air leaks. if the negative pressure increases in the room, the door will close even tighter. • no through-put cabinet from the disinfection room/bathroom, because it can create imbalances in the air pressure. • through-put autoclave/decontaminator from disinfection room to the sluice may be recommended if secured and controlled against air leakage. • graduated negative air pressure is quality ensured by measurements, pressure manometer and control of ventilation. • exhaust from the isolation unit must be disinfected in a satisfactory manner so that it does not expose personnel, patients or passers-by to infection. exhaust is usually sent out from the disinfection room, via a separate disinfection unit where the used air is treated with uv-irradiated hepafilters, and sent out over the roof (see below). • waste water, sewage and other waste should be disinfected/autoclaved before it is discharged outside the isolation area. if the isolate is not a unit for collecting and treating infectious waste/liquid, the faeces, urine and other body fluids are treated locally with 5% chloramine in 1 h before being discharged to the public sewage system. [1, 9, [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] there should be written guidelines for air pressure and air flow. negative pressure should be defined in relation to adjacent rooms and to outside pressure. pressure conditions are checked outside of the patient unit, before entering the unit. there should be an increasing negative pressure from the sluice through the patient room to the disinfection room. the following negative pressure in pascal is recommended in the unit: the negative air pressure must withstand fluctuations in the pressure outside the building, for example, during high winds with increased pressure against the building. it should not be related to pressure fluctuations within buildings. the unit must be tight to prevent air leakage and pressure variations. • no air leaks. all ventilation ducts must be completely sealed without any air leaks (e.g., all-welded steel pipe) and with a separate (over roof) air input and outlet for each isolation unit. disinfection systems for air extraction/exhaust should be placed as close as possible to each isolation unit so that contaminated air does not go far in the piping on the way out. exhaust air must be sterile after the disinfection process. externally placed pipes for air inlet and outlets from isolates must be protected from strong winds that can create unfavourable pressure conditions. the air inlet/outlet to other parts of the hospital must also be protected against contamination from the isolates. therefore, separate isolate buildings are the best solutions. • air change. minimum of six changes of fresh air per hour in the patient room. this involves the replacement of all air in approximately 70 min. avoid short circuit of the air currents. • clean inlet air-hepa-filtered and backlash secured-is introduced from the ceiling or top of the wall of the patient room in such a way that the least possible turbulence occurs during normal use. • exhaust air is contaminated and should be disinfected and hepa-filtered closest possible to the isolation unit. all air from the isolation unit goes out via the separate exhaust system; from the patient's room through grates on lower end of the door to the disinfection room. the exhaust air is disinfected close to-or in the isolate to avoid contaminated air in the piping. exhaust air is never drawn directly out from the patient room or from the sluice because of risk of unfavourable air currents and increased infection burden on personnel. exhaust air must be sterile after treatment and sent out via pipe over roof. • gassing of ventilation pipes with disinfecting gases (formaldehyde, chlorine dioxide, hydrogen peroxide, etc.) must be implemented in a regular manner. assuming the ventilation duct is completely closed and separately for each unit, various types of gas could be used. if leakages are suspected, it may be difficult to use formaldehyde gas (allergy) and chlorine dioxide gas if other part of the building is to be used. the type of gas used must be chosen according to documented effect of the infectious agent, for instance, ebola infection. chlorine gas and hydrogen peroxide dry gas are among the safest gases used today. there is currently no gas which disinfects tubercle bacilli. • continuous electricity supply must be ensured in the isolation unit and the ventilation and negative pressure system. all fixtures and equipment must be disinfected with strong disinfectants, including disinfecting gases, and must be cleaned easily and satisfactorily. note! disinfectant gas can affect instruments and equipment. there should not be wood, tile or other building materials that are easily chipped or cracked, with storage niches for infectious agents. there should be handwashing and suspension of automatic dispensers of disinfectants and paper towels in the sluice, patient room and disinfection room, to avoid unnecessary contamination of doorknobs, etc. thermal disinfection/autoclaving of waste water from the unit should be done the closest possible to the unit to prevent other wards/departments be charged with infectious agents. the container, tank and piping must be absolutely tight and robust for heat/cold/corrosive agents and for ground movements. the drainage system must be shutdown, if needed. air systems connected to the drain/sewer/water must be connected to isolation unit's exhaust system for full inactivation of infectious materials and must be completely sealed. in isolation units with collection of infectious waste water in tank for further disinfection/autoclaving, it is particularly important to ensure safe and risk-free isolation conditions. this means that infected air, liquid and waste should not be spread to nearby areas, even under accidents in the decontamination process. all supply pipes to collection tanks must be sealed, controllable and withstand disinfectants. the sluicing function to such special areas for collecting and decontamination must be ensured in accordance with regulations for the airborne infection isolation units. in case of technical accidents in the collection and treatment rooms, the air should be disinfected with gases before technical personnel enters. technical staff, handling medical equipment, including collection tanks and autoclaves, shall be specially trained in infection control and must be able to use a sluice function with the use of ppe, when entering these disinfection areas. the sluices should be roomy with plenty of space for larger equipment, handwashing/hand disinfectant, through-put disinfection machine/autoclave, etc. there should be a separate out sluice for doffing (undressing), bags for contaminated waste/equipment and opportunities to disinfect/autoclave used ppe-or parts of it-and other equipment. room for a separate shower in the out sluice system should be considered and a room for disinfection of equipment to be reused (e.g. goggles, shoes, etc. that can be disinfected in chloramine 5% in 1 h or autoclaved). the sluice system should be adapted for separate gas disinfection via sealed and tight pipes and interlocked system that can be implemented systematically daily or as needed, without affecting the patient in the isolation room. inter-locking. one of the doors in the sluice/anteroom must always be kept closed. preferably use controlled closures for all doors. [1, 9, [16] [17] [18] [19] [20] [21] [22] • a quarantine unit should include some large patient rooms where it is a place for intensive treatment, acute surgery, dialysis and other invasive treatments, x-ray, microscopy of infectious materials, some important biochemical analyses, etc. [1, 9, [16] [17] [18] [19] [20] [21] [22] • waste, reusable equipment, etc. from the patient or disinfection room should be autoclaved in the through-put autoclave (or disinfection machine) from the disinfection room or patient room to a clean sluice where it can be taken out and treated as noninfectious. all equipment from patient unit must be autoclaved or disinfected in an appropriate manner. equipment can be decontaminated at high temperature (>90° c) in an instrument washing/decontamination machine or submerged in a bath of chloramine 5% 1 h (or peracetic acid or household bleach 10%), depending on the agent in question. s. • in-sluicing and donning (dressing) is done in a clean sluice before entering the unit, with clean, new clothes and clean, new ppe every time. reuse of disposal ppe is not recommended. • out sluicing needs plenty of space for washbasin and suitable hand disinfectant, for doffing (undressing) and for the use of at least three waste bags (disposal, textiles and reusable equipment). ppe must be removed carefully, following guidelines, and placed in a waste bag which is then placed directly in through-put autoclave. reusable equipment (goggles, shoes, etc.) that can be decontaminated/autoclaved are placed in separate containers for reuse. then shower with, for instance, chlorhexidine (hibiscrub) disinfectant in disposable packages, optionally soap in disposable packages. the shower head is set low and with weak force to avoid aerosols against the face. after the shower is finished, the entire cabinet interior, including the floor, is treated with hot water (>75 °c). self-disinfecting shower unit (after use) is advantageous (gas or disinfectant liquid). used towels are put in the textile bag. new, clean clothes are taken on in a clean wardrobe. only the hospital's clothing and shoes are used. [1, 9, [16] [17] [18] [19] [20] • gown-disposable, with tight-fitting cuffs and discarded after each use. it should have long sleeves, be tight around the neck, long and liquid tight. the opening should be on the back; it should be tied back, never on front. option: disposable coveralls with hood or full and tight costume. • cap/hood-surgical-covering all the hair and ears. keeps the hair in place and covers the ears. respiratory protection is set on the outside of the cap. • respiratory mask, p3-level, check leakage with leak test (to be learned). in special cases, a turbo equipment (papr, powered air-purifying respirators) or fresh air/compressed air system is used. remember that battery and reusable parts should be disinfected between each use. • "phantom hood" (surgical large disposable hood) is placed gently over the head and covers the neck and cheeks. • face protection. tight-fitting goggles if high risk. face shield with danger of splatter of infectious material. • gloves with long "cuff", double gloves to make work in isolation easier. latex gloves are denser than vinyl gloves. • shoe covers/room-bound shoes that may be autoclaved after use or boots that are submerged in chloramine bath or other disinfectant after each use. • special leg covering up to the knee at high risk, serious infection. for out sluicing: [1, 9, [16] [17] [18] [19] [20] follow the hospital's procedure that must be learned, trained and supervised. check if waste bags and equipment for undressing are available in the sluice out. all work in the sluice out, like assistance with undressing, takes place only with full dressed ppe. as a rule, there are two people when undressing; a person who can advise and provide assistance, double pack infection bags and take care of equipment to be autoclaved. the assistant uses full, clean ppe. gloves and hand disinfectant must be used before any handling of the equipment. all undressing must occur very carefully and quiet to minimal re-aerosols of microbes from the ppe. remove the individual infection equipment in such a way that you do not contaminate yourself or the environment. carried out in this order: 1. hand disinfection while the gloves are on. 2. room-bound shoes/boots are tilted off and put foot straight into clean shoes. reusable shoes or boots are put in a separate container for autoclaving or disinfection. shoe covers-if used-are carefully removed one after another and placed gently in the waste bag. rinse the shoes on chloramine inserted diaper lying on the floor. 3. hand disinfection while the gloves are on. 4. gloves-double gloves are removed simultaneously-learn the method. grab the left glove at the wrist (not higher up) and gently turn inside out when doffing. the inside of this glove is usually clean and can be used as a "cloth" placed on the right hand to grab through to remove (roll off) the other glove. learn the technique. for left-handed do the opposite. place gently in the waste bag. 5. hand disinfection. 6. put on clean gloves for further undressing-not over the cuff. 7. gown-learn the method, open closure on the back (neck first). pull the cuffs over gloves and roll the gown gently along from the side and from above downwards without touching the outer side. place gently in the waste bag. 8. hand disinfection while the gloves are on. 9. goggles: take back and bend the body forwards to avoid contact with the skin, hair and cloths while taking off the goggles carefully. place in container for autoclaving of reuse. 10. face shield/visor-loosen from behind as mask and take off bending forward. 11. gloves-learn the method to take off without recontamination (see above). placed gently in the waste bag. 12. hand disinfection. 13. "phantom hood"; take back on the lower part of the hood that has been covered by the gown, and with both hands tear it up along the seam behind while bending the body forwards to avoid contact with the skin, hair and cloths; take off the hood slowly, and put it in the waste bag. 14. hand disinfection. 15. respiratory protection mask-grasp the elastic back on the head; bend the body forwards to avoid contact with the skin, hair and cloths; take off carefully, and place the mask in the waste bag. 16. hand disinfection. 17. the surgical cap/hood-which was under masks and phantom hood; take off from behind, bend the body forwards to avoid contact with the skin, hair and cloths and place the cap in the waste bag. 18. hand disinfection. 19 . showers and dressing of new clothes as described above. autoclaved or heat disinfected (85-90 °c for 10 min) or submerged in disinfectants (chloramine 5%, household bleach 10% or peracetic acid) before normal processing. option: all textiles are treated as infected and put in a container for used textiles. contaminated/wet fabrics are packed in plastic before placing in waterproof textile bag. the outside of the bag is decontaminated and washed with 5% chloramine before double packed with a new clean bag in the sluice. the outside will then be clean during transport for further treatment. the type of the infectious agent and local possibility determine further treatment of textiles (heat washing, chemicals, autoclaving, burning). excess equipment should be removed before the patient arrives. used equipment should be autoclaved in a through-put autoclave before the normal treatment. option: disinfected in the isolation unit's disinfection room. equipment that can withstand heat is heat disinfected in decontaminator or instrument washing machine. heat-sensitive components like thermometers are disinfected in 5% chloramine or other disinfectants for 1 h. used equipment should be autoclaved in a through-put autoclave before normal treatment. option: decontaminated in the isolation unit's disinfection room (85-90 °c) or double packed and brought to the decontamination room in the ward for decontamination-and then processed as usual. disposable equipment is treated as infectious waste. infectious waste is autoclaved in through-put autoclave, before processing as ordinary waste. option: all waste is double packed and treated as special infectious waste. the outside of the bag is decontaminated and washed with 5% chloramine before double packed with a new clean bag in the sluice. the outside will then be clean during transport for further transport and treatment. infectious waste is brought directly to incineration without intermediate storage. follow general guidelines. the boxes may be autoclaved in through-put autoclave, before processing as ordinary waste. option: boxes are double packed and treated as special infectious waste. the box must not be filled more than about ¾ full. see labelling for filling level. the outside of the bag is decontaminated and washed with 5% chloramine before double packed with a new clean bag in the sluice. the outside will then be clean during transport for further special transport and destruction. infectious waste is brought directly to incineration without intermediate storage. in most cases it is not recommended to bring the patient out of isolation during the period of active infection. special solutions should be made for these. if transport out of the isolate is necessary, everything must be planned carefully in advance. death bodies are wrapped in body bag, double packed in the sluice and sent directly to the pathologist or burial. remember information in advance! follow general guidelines and contact serving microbiological laboratory or the national institute of public health before transport of sampled biological materials form the patient! sampling/preparation for transfer of infectious sample material should be provided in the patient room or its disinfection room. sample vials and outside of the package must be clean before transport. blood samples from patients with blood-carrying infection are packaged in special cases (sleeves) before transport. the outer package is put on in the sluice. nb! a good cleaning and disinfection of the workplace! note on the outer bag suspected high-risk agent with red ink! special transportation! contact receiver in advance, and ask how to bring the samples to the laboratory. send no samples as post in pneumatic tube! only essential transport is allowed, depending on infectious agents. patient, cloth and stretcher/bed should be clean during transport. bandages must be clean, and drains, etc. must be covered and free of leaks. staff transporting and receiving the patient must be informed about the infection in advance and guided on individual infection prevention and the use of ppe. patients with respiratory infection should wear surgical mask or respiratory mask (p1-p3 level) outside the isolate. the transport should not go through wards or crowded areas and avoid the use of a lift. internal transport in hospitals is not recommended in certain highly infectious and severe illnesses. in agreement with the patient library, the patient can borrow books that can be discarded. the books are kept in isolation. all books are treated as infectious waste by termination of the isolation. restricted admittance and use the same guidelines and ppe (donning and doffing) as for the staff. handwashing is required when the sluice is left. limit visits to a minimum, especially with highly infectious diseases such as sars. the cleaning and disinfection is performed by trained personnel with required attire and use of ppe. regular detergent, water and clean equipment are used if no other routine is ordered. bucket and squeegee are disinfected in the decontaminator of the isolation unit; shaft is disinfected chemically in 5% chloramine (or household bleach) in 1 h and kept in the unit during the entire treatment. use only disposable mops and cloths placed in yellow plastic bag in yellow infectious waste bag and autoclaved in the isolation unit. discard as infectious waste after appointment. option if not autoclave/decontaminator in the isolation unit: infectious waste bag is treated exterior with 5% chloramine and double packed in new yellow thick plastic bag in the sluice. special infectious waste is brought directly for burning/autoclaving. in case of spills, the nursing staff should immediately remove the spill and disinfect the area with 5% chloramine 1 h (covered by plastic) before cleaning. before regular disinfection of the isolation unit after terminated isolation, it is recommended to treat the unit with hydrogen peroxide dry gas, three cycles, using a spore control. only trained personnel are participating in the work with disinfection, and they use mandatory infection control equipment. the nurse, in cooperation with infection control personnel, is responsible for informing the staff about what should be disinfected and how. bedding, curtains, drapes, etc. should be autoclaved, if possible, before sending it to laundry or to incineration. floors, walls, ceilings, all horizontal surfaces, ceiling suspension, lighting in ceilings, etc., handles, dial string (changed), levers, buttons, switches, bed, waterproof mattresses, bedside tables and other fixtures and equipment (tv, telephone, computer, etc.) are disinfected with 5% chloramine (or household bleach 10%) for 1 h. textiles, reusable equipment and waste: follow described above recommendations. unused disposal equipment that has been inside the isolation unit is disposed of as infectious waste. after manual disinfection, all rooms and ventilation systems are gassed once more. this is done in collaboration between the ward, housekeeping/technical and infection control personnel. to treat with gas disinfection before and after manual disinfection is recommended to reduce the infectious airborne agent (aerosols and re-aerosols) in the unit. after the recommended effect period of the disinfectant, the rooms are cleaned with ordinary detergent, water and clean equipment. there are many discussions concerning the degree of airborne transmissions and how to protect personnel, especially against dangerous infections; see the background information [1, 9, [16] [17] [18] [19] [20] (fig. 19.1) . isolation regimens should not prevent treatment but be included in the diagnosis and treatment. nb! good hand hygiene is important! it prevents the spread of infection! handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control no. 55 of protection against infectious diseases regulations in infection control in health facilities -hospital infections, established by the health and social affairs action plan for infection control in norwegian hospitals, health directorate's guidance series 2-92. directorate of health use of isolation to prevent spread of infection in hospitals. health directorate's guidance series: directorate of health on the protection of workers from the risks related to exposure to biological agents at work cdc draft guideline for isolation precautions in hospital in: handbook of hygiene and infection control in hospitals. oslo: ullevål university hospital cdc draft guideline for isolation precautions in hospital guideline for isolation precautions in hospitals cdc draft guideline for preventing the transmission of mycobacterium tuberculosis in health care facilities the use of adult isolation facilities in a uk infectious disease unit guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings. cdc isolation of dangerous infections. in: handbook of hygiene and infection control in hospitals. oslo: ullevål university hospital scenario pandemic influenza and pandemic avian influenza. in: handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control avian influenza with pandemic potential handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control the design of isolation rooms hospital and community acquired infection and the built environment-design and testing of infection control rooms door-opening motion can potentially lead to a transient breakdown in negative-pressure isolation conditions: the importance of vorticity and buoyancy airflows isolation rooms for highly infectious diseases: an inventory of capabilities in european countries patient care in a biological safety level 4 (bsl-4) environment evidence-based design of health care facilities: opportunities for research and practice in infection prevention the role of facility design in preventing the transmission of healthcare-associated infections: background and conceptual framework the role of the hospital environment in preventing healthcare-associated infections caused by pathogens transmitted through the air the airborne transmission of infection in hospital buildings: fact or fiction? role of ventilation in airborne transmission of infectious agents in the built environment-a multi-disiplinary systematic review virus diffusion in isolation rooms review of aerosol transmission of influenza a virus detection of bordetella pertussis and respiratory syncytial virus in air samples from hospital rooms aerial dispersal of methicillin-resistant staphylococcus aureus in hospital rooms by infected and colonised patients air and surface contamination patterns of methicillinresistant staphylococcus aureus on eight acute hospital wards air contamination around patients colonized with multidrug-resistant organisms international infection control guidelines may not protect against ebola. hospital health care infection control guidelines that did not work against ebola in 2014 textbook of hygiene. oslo: fabritius & sønner forlag violent expiratory events: on coughing and sneezing the potential for airborne dispersal of clostridium difficile from symptomatic patients virus associated with skin rash and children's diseases. in: handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control bordetella pertussis -whooping cough. in: handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control aiborne dispersal as a novel transmission route of coagulase-negative staphylococci, interaction between coagulase-negative staphylococci and rhinovirus infection possible role of aerosol transmission in a hospital outbreak of influenza protecting staff against airborne viral particles: in vivo efficiency of laser masks droplet fate in indoor environments, or can we prevent the spread of infection pneumonic plague outbreak multi drug-resistant tuberculosis outbreak in gaming centres health care response to cchf in us soldier and nosocomial transmission to health care providers key: cord-007575-5ekgabx5 authors: luby, james p. title: southwestern internal medicine conference: pneumonias in adults due to mycoplasma, chlamydiae, and viruses date: 2016-01-14 journal: am j med sci doi: 10.1097/00000441-198707000-00007 sha: doc_id: 7575 cord_uid: 5ekgabx5 pneumonias in adults due to mycoplasma, chlamydiae, and viruses are a common clinical problem. these microorganisms contribute to the etiologies in 6–35% of all cases of pneumonia and are the sole pathogens in 1–17% of hospitalized cases. important trends and developments in the field include (1) the emergence of a chlamydia psittaci strain (twar) that is passaged from human to human, causes a mycoplasma-like illness, and that is relatively resistant to erythromycin, (2) the recognition of respiratory syncytial virus as a pathogen in nursing home outbreaks and in immunosuppressed adults, the continuing high lethality of fully developed influenza pneumonia, (4) the efficacy of acyclovir and adenine arabinoside in limiting the complications of varicella-zoster virus infections, and (5) the increasing frequency of pneumonia caused by cytomegalovirus and the severity of this disorder in highly immunosuppressed patients. developments in the rapid diagnosis and therapy of respiratory syncytial virus infections with an aerosolized antiviral drug in children may pave the way for comparable advances in difficult pneumonias in adult patients. southwestern internal medicine conference: pneumonias in adults due to mycoplasma, chlamydiae, and viruses by james p. luby, md abstract: pneumonias in adults due to mycoplasma, chlamydiae, and viruses are a common clinical problem. these microorganisms contribute to the etiologies in 6-35% of all cases of pneumonia and are the sole pathogens in 1-17% of hospitalized cases. important trends and developments in the field include (1) the emergence of a chlamydia psittaci strain (twar) that is passaged from human to human, causes a mycoplasma-like illness, and that is relatively resistant to erythromycin, (2) the recognition of respiratory syncytial virus as a pathogen in nursing home outbreaks and in immunosuppressed adults, (3) the continuing high lethality of fully developed influenza pneumonia, (4) the efficacy of acyclovir and adenine arabinoside in limiting the complications of varicella-zoster virus infections, and (5) the increasing frequency of pneumonia caused by cytomegalovirus and the severity of this disorder in highly immunosuppressed patients. developments in the rapid diagnosis and therapy of respiratory syncytial virus infections with an aerosolized antiviral drug in children may pave the way for comparable advances in difficult pneumonias in adult patients. key population-based studies on the incidence ofpneumonia have been performed infrequently. in one study, during an 8-year interval from december 1, 1963 , through november 30, 1971 , foy and her colleagues determined the incidence of pneumonia in a prepaid medical health insurance plan comprising more than 100,000 members in seattle, washington. 1 they found that total pneumonia rates varied yearly and ranged between 7.2 and 16.8 cases/1,000 population/year. only 15% of the cases seen by the physicians caring for this group were hospitalized; 85% of the total number of cases were managed as outpatients. in adults, 35% of all cases of pneumonia were associated with cultural and/or serologic evidence of mycoplasma and/or viral infection. total rates for all cases of pneumonia increased during influenza a2 epidemic years. the highest rates were generally found in the winter quarter, followed by rates occurring during the spring quarter ( figure 1 ). the major viral and mycoplasma agents contributing to the etiology of pneumonia in this study were influenza a virus, and mycoplasma pneumoniae, followed by a smaller number of cases due to adenoviruses, influenza b virus, respiratory syncytial virus (rsv), and parainfluenza viruses. most of the parainfluenza virus infections were caused by types 2 and 3, but no attempt was made to ascertain the exact contribution of specific agents involved because of antigenic overlap in the complement fixation test. it was recognized that the majority of pneumonias associated with influenza a were related to bacterial suprainfection. rates for all pneumonia were highest in young children, followed by a peak in pneumonia due to m. pneumoniae in the 30-40-year age group. pneumonia due to influenza a virus increased in incidence above the age of 60 years. of interest is their finding that sometimes pneumonia was associated with laboratory evidence of infection with more than one respiratory nonbacterial agent. the severity of disease did not appear different between patients with a single infection and those with multiple infections, as measured by duration of illness and hospitalization rates. in individual reports, however, it has been suggested that in certain individuals, multiple infections can sometimes lead to a more severe course than would have been predicted by infection with a single agent. 2 overall rates for pneumonia as determined in this study were similar to those observed in the national health survey. in houston, texas, during the years 1975-1978, adult hospitalizations for pneumonia increased sharply during influenza a epidemics but did not change much during influenza b epidemics, a finding that was also seen in the seattle study (figure 2) . 3 although hospitalizations for pneumonia did not increase in houston during the 1976-1977 influenza b epidemic, an increased number of patients hospitalized with complications due to influenza b virus infection was seen in dallas, texas, at this time. 4 the etiology of community-acquired pneumonia in 54 adult outpatients in sweden has been determined. 5 using rises in antibody titer between acute and convalescent sera to determine etiology, these investigators found evidence of mycoplasma pneumoniae infection in 37%; streptococcus pneumoniae in 9%; hemophilus in{luenzae in 12% (6% type band 6% nontypeable); influenza a virus in 6%; chlamydia psittaci in 4%; and influenza b virus, parainfluenza 3 virus, respiratory syncytial virus, and adenovirus in 2% each. multiple infections occurred in several patients and there was no serologic evidence of infection with a particular microorganism in 41 %. the etiologic agents of community-acquired pneumonia in adult patients hospitalized for their disease can be examined (table 1) . six recent studies were selected for analysis because they had a worldwide authorship and because an attempt had been made to estimate the contribution made by both bacterial and nonbacterial agents.6-11 bacterial 46 etiologies contributed most significantly to the problem of community-acquired pneumonia in adult hospitalized patients. the most frequent microorganisms were streptococcus pneumoniae followed by staphylococcus aureus, haemophilus in{luenzae, legionella pneumophila, and other gram-negative bacteria. the role of anaerobic bacteria in the etiology of pneumonia was not studied systematically in five of the series. a nottingham, england, study accentuated the role of l. pneumophila in the etiology of pneumonia and showed the propensity of this microorganism to be associated with specific geographic sites. the major nonbacterial agents implicated in these studies included m. pneumoniae, influenza a and b viruses, adenoviruses, rsv, parainfluenza viruses, varicella, and c. psittaci. all of these latter agents could be seen as single pathogens but influenza a and b viruses, adenoviruses, rsv, parainfluenza viruses, and varicella virus also were associated with bacterial pneumonia. nonbacterial agents contributed from 6 to 35% to the etiologies of all cases. an indication of the approximate incidence of nonbacterial pneumonia without bacterial suprainfection can be ascertained in five of the series and ranged from 1 to 17% of the cases. the association of influenza a and b viruses, rubeola virus, and varicella virus with bacterial suprainfection has been well established. recently, bacterial suprainfection has been shown to occur in adults who have evidence of infection with adenoviruses and rsv. the frequency of bacterial suprainfection in association with m. pneumoniae infections is difficult to ascertain. it has been considered the concept of primary atypical pneumonia was set forth in an article by hobart a. reimann in 1938. 12 the major bacteria-causing pneumonia were known at that time with the exception of l. pneumophila. the clinical entity of psittacosis had been elucidated. influenza a virus had been grown in ferrets by laidlaw, andrews, and smith. reimann described eight cases of what he called atypical pneumonia, which he thought was due to a filterable virus. the description of cases allows a view of untreated primary atypical pneumonia. it now seems probable that m. pneumoniae was the etiologic agent in most of his cases. the illness often began in-the american journal of the medical sciences sidiously with fever, headache, and pharyngitis. with descent of the disease into the respiratory tract, the larynx became involved and hoarseness was present. finally, laryngotracheobronchitis and pneumonia occurred. a troublesome cough developed in the patient that could not be alleviated and was only slightly productive. in some patients, a pulse-temperature dissociation occurred. during the course of the disease, which often lasted several weeks, patients became dyspneic and cyanotic. two of the patients became delirious and had central nervous system dysfunction during the course of the infection. on physical examination, the patient was flushed and had evidence of pharyngitis. the physical examination of the chest usually revealed scattered rales without striking evidence of consolidation; in one patient a large pleural effusion was present. the white blood count was only modestly elevated. chest radiographs revealed mottled or diffuse areas of infiltration. attempts to isolate pathogenic bacteria and influenza a virus were unsuccessful in establishing an etiology for this syndrome. reimann considered diagnoses such as typhoid, psittacosis, and epidemic influenza, but the history in none of these cases was consistent and influenza virus could not be recovered. to summarize reimann's words, "the infection occurred in adults and began as a mild infection of the respiratory tract; this was followed by severe diffuse atypical pneumonia and in two cases by the symptoms of encephalitis. dyspnea, cyanosis, hoarseness, cough without sputum, drowsiness, and profuse sweating were the chief characteristics. the disease lasted several weeks." in 1943, finland found elevated cold agglutinin titers in cases of atypical pneumonia. eaton later isolated the agent in embryonated eggs, and chanock and colleagues were able to grow it on defined media and demonstrate it to be a mycoplasma. the entity of primary atypical pneumonia became well known and later was defined as pneumonia that did not clear with penicillin or sulfonamides, or nonbacterial pneumonia, or pneumonia with no sputum or a mucoid sputum without a predominant organism on gram's stain. we recognize today that the clinical entity of primary atypical pneumonia has multiple etiologies, particularly m. pneumoniae, but also c. psittaci, the twar strain ofc.psittaci, chlamydia trachomatis, q-fever, and viruses such as adenoviruses, rsv, influenza viruses, and para-48 influenza viruses. l. pneumophila infections often are considered in the differential diagnosis. ls early bacterial pneumonia also should be considered when the patient is first seen. in retrospect, persons with this diagnosis are often excluded from series of cases to focus specifically on the nonbacterial nature of the problem. in pertinent geographic areas, acute histoplasmosis and coccidioidomycosis may present like primary atypical pneumonia. major attempts to identify the etiologic agent on clinical grounds have been made, but the exact diagnosis usually depends on laboratory determination of the offending agent. in one study of 150 patients of all ages, 50 with viral pneumonia, 50 with mycoplasma pneumonia, and 50 with bacteremic i:meumococcal pneumonia were compared. 14 the best discriminating variables were the c-reactive protein determination, the presence or absence of predisposing disease or previous antibiotic treatment, the erythrocyte sedimentation rate, the presence of lymphocytosis, and the band neutrophile count. signs of an upper respiratory tract infection and the presence or absence of auscultatory abnormalities also aided significantly in the discrimination. determinations favoring bacteremic pneumococcal pneumonia included predisposing disease, a short duration of illness before hospitalization, alcoholism, the absence of signs of an upper respiratory tract infection, high c-reactive protein determinations and erythrocyte sedimentation rates, no prior antibiotic treatment, total leukocyte counts exceeding 15,000, relative lymphocyte counts less than 35%, relative band neutrophile counts greater than 20%, abnormal auscultatory findings, and the presence of lobar consolidation on chest radiograph. differentiation between viral and mycoplasma pneumonia could not be made easily. however, symptoms of mycoplasma pneumonia before hospitalization lasted a longer time and these patients were more likely to have received antibiotic treatment in the interval before hospitalization. patients with mycoplasma pneumonia were more likely to have lobar consolidation on chest radiograph than those with viral pneumonia, but in this study no distinction could be made between mycoplasma pneumonia and bacteremic pneumococcal pneumonia on the basis of roentgenographic findings alone. in another study comparing community-acquired pneumonias, mycoplasma pneumonia tended to occur at an earlier age than legionnaire's disease, pneumococcal pneumonia, or psittacosis.l 5 homogeneous shadowing on chest radiograph was more common in legionnaire's disease and pneumococcal pneumonia than mycoplasma pneumonia. pleural effusions were uncommon in all groups but occurred most commonly in bacteremic pneumococcal pneumonia as did multilobe disease on presentation. hilar lymphadenopathy occurred only in mycoplasma pneumonia. roentgenographic resolution was fastest in mycoplasma pneumonia, intermediate in psittacosis and nonbacteremic pneumococcal pneumonia, and slowest in legionnaire's disease and bacteremic pneumococcal pneumonia. deterioration on chest radiograph after hospital admission characterized legionnaire's disease and bacteremic pneumococcal pneumonia. because the differential diagnosis of primary atypical pneumonia at a clinical level includes pneumonia due to m. pneumoniae, chlamydial species, q fever, adenoviruses, rsv, influenza viruses, parainfluenza viruses, as well as l. pneumophila infections and early bacterial pneumonia, therapy should include an antibiotic to which the majority of these the american journal of the medical sciences luby microorganisms are susceptible. chlamydial species are more susceptible to tetracycline than erythromycin. tetracycline is effective against rickettsiae but not for l. pneumophila infections. up to 4% of pneumococcal isolates are resistant to tetracycline. a reasonable antibiotic choice is erythromycin at an equivalent dose of 30 mglkg of erythromycin base per day for 10-14 days. if legionnaire's disease is diagnosed, a higher dose of erythromycin may be necessary. if a chlamydial or rickettsial etiology is recognized, tetracycline at a dose of 2 gmiday should be given. occasionally patients with proven m. pneumoniae have been treated with erythromycin, failed to respond to therapy, but subsequently responded to a course of tetracycline therapy.ls conversely, some patients with m. pneumoniae infections have responded to erythromycin after a suboptimal response to tetracycline therapy. viruses may cause primary atypical pneumonia; however, antibiotic treatment in these instances is useless, does not prevent suprainfection, and may actually change the nature of the bacterial species suprainfecting the patient. antibiotic therapy seems reasonable in this syndrome, however, because it is usually impossible to differentiate clinically between mycoplasma pneumonia and an entity such as adenovirus pneumonia in the adult. advances in rapid laboratory diagnosis may be able in the future to influence treatment options but these techniques are still under development, are expensive, and are not widely available. mycoplasmas, the smallest free-living microorganisms, are cell-wall deficient, but have no relationship to cell-wall deficient bacteria with which they were once confused. m. pneumoniae attaches to the mucosal epithelium of the respiratory tract through a specific protein that enables the microorganism to adhere to neuraminic acid residues on respiratory epithelial cells. if mycoplasmas cannot attach, there is no damage to the host. upon adherence, mycoplasmas are able to generate hydrogen peroxide and superoxide anion, resulting in injury to epithelial cells. because infection occurs commonly in children younger than 5 years of age, although disease is rare at this time of life, mycoplasmas may induce disease primarily by immunopathologic mechanisms. l7 . lb in experimental animals not primed by prior mycoplasma exposure, inflammatory changes occur only after a long interval. with reinfection, inflammatory changes occur more briskly. the extrapulmonic manifestations of my coplasma infection have never been explained completely, but there are reports demonstrating m. pneumoniae in sites such as cerebrospinal fluid and blood. l9 alternatively, immunopathologic reactions may be the primary mechanism involved. path-ologically, the disease in man is characterized by tracheobronchial, bronchiolar and septal lymphoplasmocytic infiltrates, luminal exudates rich in polymorphonuclear leukocytes, bronchiolar and alveolar cell metaplasia, and occasionally diffuse alveolar injury.2o the bronchiole appears to be the major site of attack. the microorganisms colonize the nasopharynx and transmission of infection occurs only by close contact. especially conducive to the transmission of m. pneumoniae are situations in which persons are housed in closed quarters, such as military platoon barracks or family unit dwellings. in families, there is a high attack rate and cases continue to occur over a 3-4-month interva1. 21 . 22 the cumulative attack rate of mycoplasma infections in families may approach 90% (figure 3 ).21 mycoplasma carriage is not affected by antibiotic therapy, thereby allowing the family epidemic to continue. mycoplasma disease occurs throughout the year but is particularly frequent during fall and winter. increased numbers of cases occur with a 3-5-year periodicity. although pneumonia may occur soon after infection, the disease is usually manifested as an upper respiratory tract infection progressively descending into the lung. pharyngitis progresses into laryngitis followed by tracheobronchitis, and, finally, pneumonia. hoarseness and dysphonia may be present. middle ear involvement may occur with bullous myringitis, which usually heals without scarring. occasionally, otitis may lead to tympanic membrane perforation. sinus involvement is frequent but usually asymptomatic. the cough is often intractable and usually only slightly productive of a mucoid sputum that contains mainly polymorphonuclear leukocytes but no predominant bacterial microorganism on gram's stain. when pneumonia develops, the patient has an elevated temperature and, occasionally, a 'temperature-pulse dissociation. headache, irritation, a flushed facies, myalgias, and arthralgias are common. 2 3-26 on physical examination, the patient is febrile, appears flushed, and usually has physical evidence of pharyngitis. hemorrhagic bullous myringitis may be present in up to 5% of cases. physical findings on chest examination usually are limited to scattered rales, wheezes, and rhonchi and are often localized to the lung bases. evidence of consolidation is not striking, although m. pneumoniae infections can cause lobar pneumonia. 26 the white count is usually elevated with a shift to the left, but rarely exceeds 15,000 white blood cells/mm 3 and the neutrophile band count is usually less than 10%.14 chest radiograph reveals peribronchial infiltrates with accentuation of interstitial markings in adjacent lung segments, patchy alveolar infiltrates usually localized to the lower lobes, especially on the left, and occasionally hilar lymphadenopathy. 27 more than one lobe may be involved and a confluent lobar infiltrate may be present in some patients ( figure 4 ). less commonly, there is a diffuse interstitial infiltrate and rarely an x-ray picture indistinguishable from the adult respiratory distress syndrome. 28 . 29 without therapy, the disease course usually lasts approximately 3 weeks, but may extend up to 7 weeks. 12 extrapulmonic manifestations of mycoplasma infection often are a clue to the diagnosis and include bullous myringitis; neurologic disturbances suggesting encephalitis or aseptic meningitis and, rarely, transverse myelitis; arthritis; myopericarditis; hepatic dysfunction; splenomegaly; and skin eruptions.~u a stevens-johnson syndrome may occur. japanese workers have described typical cases of pityriasis rosea that followed mycoplasma infection. 32 the cerebrospinal fluid (csf) may be abnormal with an increased number of cells and an elevated protein concentration. hemolytic anemia may be present resulting from antibody directed against the i antigen on the red-cell membrane. 31 almost all patients recover completely after mycoplasma infection but cigarette smokers may have prolonged abnormalities in diffusion capacity.33 individual case reports have described pulmonary fibrosis, bronchiolitis obliterans, and bronchiectasis following m. pneumonia. 3~6 glomerulonephritis with continuing renal dysfunction also has been reported. 37 • 38 the diagnosis is established by culture of the microorganism or the demonstration of a fourfold rise in antibody by complement fixation or other serologic test. a single high complement fixation test antibody titer (~1: 128) may be used as presumptive evidence of infection. cold agglutinin antibody titers at low level are norispecific but very high values (~1 : 128) also can be used to support the diagnosis. treatment consists of the administration of either erythromycin or tetracycline as outlined in the therapy of primary atypical pneumonia. the patient usually responds, but it should be remembered that there are reports of inadequate resolution of the disease and the necessity to switch to the alternate drug to achieve more rapid clinical improvement. antibiotic therapy does not eliminate the carrier state. immunity is relatively short-lasting and documented episodes of repeated mycoplasma infection have been reported. a vaccine against m. pneumoniae, given present priorities, appears only a hopeful future development. mycoplasma pneumonia. an 18-year-old man was well until 8 days before admission into the hospital, at which time he developed fever, a sore throat, and a nonproductive cough. his oral temperature reached 40° c. a "pounding" headache developed. the cough persisted and became productive of a mucoid sputum. oral penicillin was prescribed but did not alleviate his symptoms. physical examination on admission into the hospital revealed a young man who was confused about time and uncertain about recent events. the oral temperature was 38.8° c and the pulse rate was 100. the pharynx was described as normal. chest examination revealed harsh breath sounds with bilateral inspiratory rales, especially on the right, anteriorly and inferiorly. there was no egophony or decreased fremitus. rhonchi were present more on the right than left. hepatosplenomegaly was present. laboratory examination revealed 12,200 white blood cells with 70% polymorphonuclear cells, 28% lymphocytes, one monocyte, and one eosinophile. the serum aspartate aminotransferase was 140 (normal <40). arterial blood gases on room air showed a ph of 7.55, pc02 of29, and p02 of 45. a lumbar puncture was performed that showed 31 white blood cells, 95% of which were mononuclear cells. the csf glucose was 69 mg/dl and the simultaneous plasma glucose 155 mg/dl. the patient was treated initially with intravenous penicillin for presumed pneumococcal pneumonia and partially treated bacterial meningitis. his condition deteriorated but finally he was placed on erythromycin therapy at the advice of a consultant. mycoplasma complement fixation test titers rose from less than 1: 8 to 1: 64. comment. encephalitis, hepatosplenomegaly, and mild hepatic dysfunction were the extrapulmonic manifestations of mycoplasma disease. typical of mycoplasma pneumonia were the long duration of illness before admission into the hospital, prior antibiotic administration, presence of a sore throat, physical examination of the chest, and characteristics of the sputum. psittacosis was first described by ritter in switzerland in 1879 as a disease of the lungs in patients in contact with sick psittacine birds. later, in 1929-1930, a pandemic of psittacosis occurred involving psittacine birds exported from south america. the clinical manifestations were described fully and the epidemiology was established, leading to control measures that have kept psittacosis or the better, more inclusive term, ornithosis, at a low level of occurrence. occasionally migrant birds can carry c. psittaci, and persons dealing with them may develop ornithosis. more importantly now, ornithosis is an occupational hazard to the farmer who manages poultry such as ducks and turkeys.17,lb,40,41 clinically, patients with ornithosis have headache, fever, pulse-temperature dissociation, pneumonia, hepatic function abnormalities, and hepatosplenomegaly. intra-alveolar inflammatory changes predominate in ornithosis with interstitial changes being secondary and less prominent. the chest radiograph reflects this and lobar consolidation may be seen. when lung involvement is minor, the disease can be diagnostically confusing, and present as a fever of undetermined origin. granuloma formation can be found in both the liver and the bone marrow and may be a diagnostic clue. ornithosis can be diagnosed by serologic tests with a chlamydial common group antigen by either complement fixation or the enzyme-linked immunoabsorbent assay (eia). treatment is with tetracycline for 10-14 days. chlamydia trachomatis can cause an afebrile pneumonia-like syndrome in young infants beginning at the age of 1-3 months, and is characterized by an afebrile state, failure to gain weight, and a staccato-like cough. on examination, there are rales, expiratory wheezing, and evidence of hyperaeration of the lungs. chest radiograph usually reveals diffuse interstitial pneumonia and hyperaerated lung fields. laboratory determinations show a modest eosinophilia and hyperglobulinemia. upon this identification, the infants can be treated with oral erythromycin syrup for 3 weeks with benefit. recently, c. trachomatis has been isolated from the lower respiratory tract of immunosuppressed patients with pneumonia, although four of the six patients reported and the only ones tested did not show a serologic response to that microorganism. 42 cases of community-acquired pneumonia in normal adults also have been reported with serologic evidence of infection with c. trachomatis. 43 fifty-two patients were studied and seven were found to have definite or suggestive serologic evidence of infection. 52 the seven ranged in age from 22 to 77. the chest radiographs of these patients have been analyzed and the infiltrates were found to be patchy and characteristically streaky with areas of plate atelectasis. there was no particular localization to a single lobe and three patients had radiographic evidence ofmultilobar involvement. 44 further studies need to be done to corroborate these reports and determine the frequency with which lung involvement occurs. in finland, an epidemic of mild pneumonia has been related to a newly described strain of c. psittaci, capable of being passaged from human to human. this epidemic occurred in adolescents and young adults and had a point prevalence of pneumonia of 15-19 cases/1,000 students at the time of x-ray survey. 45 the contribution of this particular strain of c. psittaci, designated the twar strain from tw-183 and ar-39, the first two isolates, has been examined best during a 2.5-year study at the university of washington. infected students usually presented with a mild pneumonia that simulated mycoplasma infection and was often associated with pharyngitis and laryngitis. 46 in this study, the twar strain of c. psittaci caused 12% of the pneumonias in the student population. the twar strain of c. psittaci was isolated from the students, and serial sera showed conversion to the common chlamydial group antigen by complement fixation tests. microimmunofluorescence tests revealed specific reactions to the twar strain of c. psittaci. the microorganisms isolated from the students formed typical inclusion bodies in tissue culture, were not stained by iodine, and were considered typical of c. psittaci. the clustering of cases had an epidemiology that suggested human-to-human transmission. bird-to-human transmission could not be demonstrated in any of the cases. treatment with tetracycline shortened the course, but, occasionally, patients did not respond to 1 gm of erythromycin given for 5-10 days. this new strain of c. psittaci was isolated from the patients and serologic reactions to specific antigens were demonstrated. the evidence linking c. trachomatis to lung disease has either been by isolation alone or just by serologic testing. further studies similar to the one in seattle need to be performed to link c. trachomatis to lung disease. it is clear, however, that a new strain of c. psittaci exists and can cause disease commonly. the disease due to this microorganism can be diagnosed by complement fixation or eia tests using chlamydial group antigen. specialized laboratories can isolate the organism and also perform microimmunofluorescence tests. a major new development in the evaluation of patients with primary typical pneumonia is the emergence of this c. psittaci strain that is capable of being passaged from human to human, and that may not have the desired response to erythromycin treatment. adenoviruses are ubiquitous nonenveloped dna viruses that colonize the human nasopharynx and are transmitted to other persons by close contact. types 4 and 7 are recognized for their capacity to produce epidemics in military recruit populations. because the transmission of this group of viruses is dependent upon close human contact, disease is often produced in the home or the military recruit barracks. pathogenetically, lung infection usually follows pharyngitis and a movement of the disease process down the respiratory tract. although most cases of pneumonia are not severe, cases coming to autopsy show that the tracheobronchial mucosa is denuded of the normal epithelial structures down to the basal layer. squamous metaplasia occurs along with interstitial space thickening due to the presence of chronic inflammatory cells. alveolar edema and mononuclear cell infiltrates are present. as with m. pneumoniae infections, infiltrates are often peribronchial or peribronchiolar in distribution. nuclear inclusion bodies or nuclei with a smudged appearance may be found in epithelial cells. clinically, the disease often begins with pharyngitis associated with fever and anterior cervical lymphadenopathy with or without conjunctivitis, then involves the tracheobronchial tree, and, finally, the parenchyma of the lung. pneumonia is most common in infants, young children, and military recruits. in military recruit populations, mycoplasma and adenovirus pneumonia have been found to be indistinguishable clinically except for an increased luby frequency of exudative pharyngitis with adenovirus infection. physical examination reveals pharyngitis and rhinitis and scattered rales and rhonchi. 47 evidence of consolidation is infrequent but occasionally lobar consolidation can occur, as can a pleural effusion 48 ( figure 5 ). virus rarely has been isolated from pleural fluid. 2 fatal cases of adenovirus pneumonia can occur in infants, immunosuppressed patients, and rarely in normal persons. 4 9-53 in these cases, the pneumonia is progressive with the development of diffuse bilateral alveolar infiltrates and hypoxemia requiring ventilator assistance for its correction. as the infiltrates progress, leukopenia ensues with marked lymphocytopenia. rhabdomyolysis occurs along with evidence of disseminated intravascular coagulation and renal failure. terminally, the patient becomes obtunded. bacterial suprainfection can be associated with adenovirus pneumonia. suprainfecting species of. bacteria include s. pneumoniae, group a strepto-· cocci, h. influenzae, s. aureus, and group y neisseria meningitidis. 54 in military recruits, an increased frequency of group y meningococcal suprainfection has been observed because these microorganisms commonly colonize the nasopharynx in this population. administration of antibiotics during the course of the adenovirus pneumonia does not prevent bacterial suprainfection. although most persons recover uneventfully from adenovirus pneumonia, occasional patients have residual abnormalities such as restrictive lung disease, bronchiectasis, or bronchiolitis obliterans. 55 ,56 extrapulmonic manifestations of adenovirus infection include pharyngitis, conjunctivitis, pericarditis, arthritis, skin rashes, and hepatic dysfunction. reye's syndrome has been described during the course of adenovirus pneumonia. 57 the occurrence of one or more of these manifestations during the course of pneumonia can lead the physician to order appropriate diagnostic tests to make a specific etiologic diagnosis. virus can be isolated from the nasopharynx, sputum, or endotracheal secretions. antigen can be detected by immunofluorescence tests, eia, or dna hybridization within epithelial cells derived from the respiratory tract. 58 these latter tests are specific but at the present time less sensitive than viral culture. there is no specific therapy for the infection. oral live attenuated vaccines are available against types 4 and 7 adenoviruses, and these are used now in the military to prevent epidemic disease. fatal adenovirus pneumonia. a 32-year-old man with an unremarkable past history except for hypertension was controlled on medication. two weeks before admission he developed a nonproductive, hacking cough and began to have dyspnea, which increased to the time of admission. on physical examination he appeared in moderate respiratory distress. oral temperature was 38.1° c, pulse rate was 100/min, blood pressure was 1701106, and respiratory rate was 30/min. the oropharynx was described as normal. scattered rhonchi and rales were heard diffusely through the lungs. a summation gallop was heard at the cardiac apex. laboratory examination revealed a white blood count of 5,100 with 82% polymorphonuclear cells, 11% band forms, 3% lymphocytes, and 4% monocytes. arterial blood gases on room air showed a ph of 7.44, pco. of 31, and po. of 56. ekg showed left ventricular hypertrophy. chest radiograph revealed an enlarged cardiac silhouette with patchy alveolar infiltrates ofthe entire right lung and left lower lobe. the patient was started on erythromycin 500 mg every 6 hours intravenously. he continued to spike temperatures to 40· c. cefamandole and tobramycin were added to his antibiotic therapy. two days after admission, the creatine phosphokinase value rose to 16,420 and the next day was 37,576. his creatinine rose to 4.8 mg/dl. his heart was enlarged on radiograph and the pulmonary infiltrates continued to increase. his mental status gradually deteriorated and he was transferred to the intensive care unit. the white blood count was 3,700 with 12% lymphocytes. he developed evidence of disseminated intravascular coagulation and died on the eighth hospital day of respiratory insufficiency. postmortem examination revealed changes of viral pneumonia, with some epithelial cells showing intranuclear inclusions and the appearance of "smudged" nuclei, an enlarged heart due to idiopathic myocardial disease, only minimal pathologic evidence of hypertension, and findings of disseminated intravascular coagulation. electron microscopy oflung sections revealed adenovirus. the adenovirus complement fixation test titer rose from less than 1:8 to 1:64. comment. the patient had a 2-week febrile period before admission, and pulmonary infiltrates progressed on antibiotic therapy. he developed leukopenia, lymphocytopenia, rhabdomyolysis, disseminated intravascular coagulation, and acute renal failure. his illness occurred in the setting of idiopathic myocardial disease, and it is possible that mild, chronic, left ventricular failure might have predisposed him to severe adenoviral pneumonia similar to the manner in which cardiac failure augments influenza pneumonia. pneumonia due to respiratory syncytial virus. respiratory syncytial virus is the predominant respiratory tract viral pathogen of infancy and young childhood. infection in adults usually results in no symptoms or a mild upper respiratory tract illness such as the common cold. it is now recognized that immunosuppressed patients and elderly persons can develop pneumonia because of rsv and that it can be severe and complicated by bacterial suprainfection. 5 9-61 furthermore, since immunosuppressed and elderly persons may aggregate in hospitals and nursing homes, these institutions are often sites of acquisition of infection. an epidemic of pneumonia and febrile respiratory illness took place in los angeles in february-march, 1979. 62 forty of 101 residents were affected, 22 having pneumonia. eight persons died for a case-fatality rate of 20%. other such outbreaks have been recorded. nosocomial acquisition ofrsv is very difficult to prevent. hospital personnel become colonized and may have no or mild respiratory tract symptoms. transfer of virus can occur by patient to personnel to patient transmission or directly from the personnel themselves. hands and fomites become contaminated by respiratory secretions and virus is spread to patients by direct contact with these sources. the pathology of pneumonia due to rsv is similar to that of other viral pneumonias; however, epithelial cells with intracytoplasmic inclusion bodies can be seen. the x-ray appearance of the pneumonia can be that of a diffuse interstitial process or have interstitial and patchy alveolar infiltrates in the lower lobes, or have an appearance indistinguishable from the adult respiratory distress syndrome. pneumonia due to rsv in immunosuppressed and elderly persons is a newly described phenomenon, but one that may be of increasing importance. it is also important because rsv infections can be diagnosed early by antigen detection techniques (immunofluorescence or eia) and because effective therapy has been developed recently. antigen detection tests for rsv now equal or exceed the efficacy of viral cultures for diagnosis of infection. respiratory syncytial virus infections in infancy now have been treated successfully with aerosolized ribavirin. 63 • 64 this therapy is indicated for infants and children with lower respiratory tract involvement with rsv who are exceptionally ill or who may have congenital heart disease or bronchopulmonary dysplasia. with aerosol delivery by oxygen tent, hood, or mask, concentrations of ribavirin are quite high in the upper and lower respiratory tract and exceed the minimum inhibitory concentration necessary to inhibit the growth of the virus in tissue culture. to achieve this concentration by oral administration of the drug, unacceptable toxicity would be en-countered. this toxicity would include bone marrow depression and particularly anemia related both to maturation arrest and, to a lesser extent, hemolysis. this latter event occurs because ribavirin triphosphate can accumulate in erythrocytes, having a halflife greater than 40 days, and interferes with the formation of guanosine triphosphate. aerosolized ribavirin therapy is expensive, but is presently approved by the food and drug administration for the therapy of complicated rsv infections in infants and young children. it represents the first example of an effective drug for treating a significant lower respiratory tract viral infection. it is conceivable that this technology could be applied to influenza infections. influenza and rubeola will be covered in detail but it is now recognized that other rna viruses can cause lower respiratory tract involvement in adults. these viruses include the parainfluenza viruses, respiratory enteroviruses such as coxsackie b viruses and coxsackie virus a21, rhinoviruses, and coronaviruses. 65 the magnitude of the problem, however, appears to be limited. documented instances of severe lower respiratory tract infection due to parainfluenza ii and iii viruses have occurred, however. there is presently no accepted therapy for these latter infections, although aerosolized ribavirin has been used successfully to control persisting parainfluenza virus infections of the lower respiratory tract in immunodeficient children. influenza a virus is the cause of pandemics and epidemics that occur every or every other year. all influenza a viruses possess a common group complement fixation test antigen, the nucleoprotein antigen. influenza a viruses differ in the antigenic character of the hemagglutinin and neuraminidase. the h1n1 strain of influenza a circulated in the world from 1918-1919 through 1957, when asian influenza strains (h2n2) became predominant. these strains circulated until 1968-1969, when hong kong influenza (h3n2) appeared; strains of this virus continue to be transmitted. h1n1 strains again began to circulate during 1976 and they continue to do so. influenza b strains have the same common complement fixation test antigen, and this differs from that of influenza a virus. the hemagglutinin and neuraminidase of influenza b virus are less prone to change; pandemic disease due to this virus does not occur and the interepidemic interval is longer than that of influenza a, namely, every 3-4 years. serious morbidity due to influenza a virus occurs because of host factors such as age, underlying disease, and immunosuppression; because immunity wanes with time; and because influenza a viruses are constantly changing their antigenic character. in pandemic years, when both the hemagglutinin and neuraminidase change concomitantly, the american journal of the medical sciences luby there is a tendency for more serious disease to occur than if just one of the surface proteins changes. this is well illustrated by the 1918-1919 and 1957 pandemics. influenza a viruses may, on certain occasions, be more virulent. in the 1918-1919 epidemic the pneumonia rate in persons from the ages of 25 to 40 years was approximately 10% of those who had influenza. 66 this and other facts have been cited to indicate the virulence and striking pneumotropism of the virus that led to 20 million deaths occurring throughout the world during the pandemic. influenza b viruses are more likely to cause disease in younger persons and only occasionally do epidemics occur in which there is excess mortality. pathogenetically, influenza virus attaches to cells of the respiratory epithelium and enters by a process termed "receptor-mediated endocytosis." the virion is uncoated in the endosome by fusion with the membrane of this structure, a process requiring an acidic ph. the particle then undergoes a cytoplasmic and a nuclear stage of replication. virion rna is capped and polymethylated in the nucleus so that the rna message now can be recognized by the cell and translated at the ribosome. 67 in the process of replication, the virus rapidly destroys respiratory tract epithelial structures in order to compromise natural defense mechanisms of the lung, such as mucous production and ciliary activity. in cases of severe pneumonia the epithelium of the trachea and bronchi are destroyed down to the basal layer and then metaplasia occurs, leaving the respiratory tract coated with a layer of squamous cells. there is involvement of bronchiolar structures and an intense peribronchiolar inflammatory process. in uncomplicated influenza, small airways are commonly affected, producing diffuse dysfunction in these structures, mild hypoxemia, and a compensated respiratory alkalosis. 68 . 69 in severe influenza pneumonia, there is alveolar cell destruction and disruption of the alveolar-capillary membrane resulting in hemorrhage into the alveoli along with edema, a mononuclear cell infiltrate, and the presence ofhyaline membranes. thickening of the interstitium occurs with a chronic inflammatory cell infiltrate. the process can be fulminant, occurring coincident with the onset of illness, or it can be more protracted, leading to the occurrence of progressive infiltrates over 5-7 days. when an adult respiratory distress syndrome-like picture is produced, influenza pneumonia has a high case-fatality rate, which may approximate 75%.70.71 not all influenza a pneumonia is this severe, however, and there are cases in which only an interstitial or bronchopneumonic process is apparent and the disease simulates m. pneumonia, except that in influenza the leukocyte count tends to be normal or decreased. 72.73 influenza pneumonia can coexist with bacterial suprainfection or bacterial suprainfection can occur alone. the offending bacterial pathogens may vary between pandemics; in 1889-1890, h. in{luenzae evidently was a major pathogen. in the 1918-1919 pandemic, the group a streptococcus was considered a major pathogen; more recently, s. pneumoniae has been the most common offending agent followed by s. aureus andh. in{luenzae. occasionally, other gram-negative bacteria may be involved. influenza b virus can cause a similar spectrum of pulmonary disease, but the number of patients involved is fewer. the hospitalization rate for lower respiratory tract disease nearly always increases during influenza a epidemics. this rate tends not to increase during influenza b epidemics, although total hospitalizations may be increased during this period; like influenza a virus, influenza b virus can cause a variety of disease processes outside the lung. these include myopericarditis, rhabdomyolysis, disseminated intravascular coagulation, nervous system disturbances such as encephalitis, reye's syndrome, the landry-guillan-barre : syndrome, the stevens-johnson syndrome, and others. 4 • 74 . clinically, the patient with influenza virus pneumonia has the sudden onset off ever, prostration, and myalgias followed shortly by dyspnea. blood-tinged sputum may be produced. the dyspnea progresses until hospitalization and ventilatory support are required. the illness can also assume a more protracted course leading to progressive interstitial and alveolar infiltrates over a week ( figure 6 ). some pa-tients simply have viral pneumonia with pulmonary dysfunction but do not need ventilator assistance. complicating bacterial suprainfection may coexist with viral pneumonia or more commonly presents after an afebrile interval, during which the patient appears to be recovering from the primary infection. morbidity and mortality are greatest in elderly persons, in those with chronic disease states such as chronic obstructive pulmonary disease, chronic congestive heart failure or diabetes mellitus, and in immunosuppressed patients. morbidity due to influenza a and b viruses is not limited to these groups, however. women in the third trimester of pregnancy also may have an increased rate of developing influenza pneumonia and death due to this disease process. 75 in renal transplant recipients who contract influenza a, illnesses are often prolonged, with the development of viral pneumonia, bacterial suprainfection, and myopericarditis. there may be loss of the renal allograft due · to the combination of these disease processes. 76 influenza a and b virus infections are diagnosed by serial titer rises in a suitable serologic test such as the complement fixation or the hemagglutination inhibition test. a single complement fixation test titer :::1: 128 has been shown to correlate highly with recent influenza b infections. 4 virus can be grown from the nasopharynx or endotracheal secretions by inoculation of the specimen into rhesus monkey kidney or madin-darby canine kidney tissue culture. embryonated eggs sometimes need to be used for figure 6 . influenza pneumonia. the radiograph on the lett shows combined influenza pneumonia with lett lower lobe consolidation indicating bacterial supra infection. the radiograph on the right shows diffuse alveolar infiltrates in the course of serologically documented influenza a infection. bacterial cultures from endotracheal secretions consistently showed no growth. optimal recovery of virus. virus may be able to be identified within 72 hours by using immunofluorescence. direct detection of antigen by immunofluorescence or eia can be applied to appropriate secretions, but these tests are not yet as sensitive as viral culture. 77 amantadine and rimantadine are two compounds that have both prophylactic and therapeutic efficacy against influenza a but not influenza b virus. they act by preventing uncoating of influenza a virus, perhaps by preventing the development of an acidic ph so that the envelope of the virion cannot fuse with the endosomal membrane. at the dosage given, 100 mg twice a day, amantadine has more central nervous system side effects and the dose has to be adjusted with renal failure. 78 the dose of rimantadine does not have to be adjusted with renal dysfunction because the compound is metabolized in the body. a study sponsored by the national institutes of health is underway evaluating whether rimantadine can be used effectively in the therapy of hospitalized patients with influenza a, and would include patients with influenza a virus pneumonia. ribavirin has in vitro efficacy against both influenza a and b viruses. it has multiple sites of action including interference with the formation of guanosine triphosphate and deoxyguanosine triphosphate and prevents placement of the polymethylated cap structure on the influenza a viral rna message. with the aerosolization of ribavirin, high concentrations of the drug can be produced within the respiratory tract but serum levels are low. 79 ,8o attempts most likely will be made in the future to treat influenza a virus pneumonia with aerosolized ribavirin or a combination ofribavirin and rimantadine. influenza b virus pneumonia may be able to be treated with aerosolized ribavirin. vaccines exist for both influenza a and b viruses, and standard medical care necessitates yearly immunization of elderly patients or those with underlying medical conditions. a recent emphasis of the public health service is to have medical personnel also immunized yearly, since they are. exposed to persons with influenza, may develop that illness themselves, and may then transmit the infection to sick patients within the hospital. nosocomial influenza pneumonia. a 55-year-old alcoholic man was admitted to the hospital on january 30 with alcoholic liver disease, macrocytic anemia, and symptoms of bladder neck obstruction. he was a heavy smoker and had evidence of chronic obstructive pulmonary disease. 'lwelve days after admission into the hospital, he developed fever to 39.2° c while awaiting a urologic procedure. he "felt terrible" with myalgias and developed a cough, mild diarrhea, and dyspnea. chest examination revealed diffuse rales and rhonchi. chest radiograph showed new interstitial infiltrates, more prominent on the right. the sputum was mucoid. the diagnosis of pneumonia was made and the diag-the american journal of the medical sciences luby nosis of congestive heart failure with pulmonary edema considered. however, his heart examination revealed no gallop sounds and his neck veins were not distended. he was placed on ampicillin, became afebrile after 5 days, and his dyspnea improved with low flow oxygen by face mask. influenza a complement fixation test titer on a single convalescent serum specimen was 2::1: 256. comment. influenza virus pneumonia occurred during hospitalization. the pneumonia cleared with symptomatic therapy. the public health service now recommends widespread immunization of medical personnel in an attempt to prevent nosocomial acquisition of influenza. although predictions were made that rubeola would be eradicated in this country during the early 1980s, this has not been achieved. in dallas, texas, during 1986 more than 150 cases of rubeola occurred. this marked a resurgence of cases after a relatively disease-free interval after 1971, when a large epidemic occurred in dallas, causing more than 1,000 cases, including three deaths. as a consequence ofthis and other epidemics, in 1971 texas adopted a law requiring the compulsory immunization of children against measles, mumps, rubella, poliomyelitis, diphtheria, and tetanus. present rubeola vaccines are at least 95% effective, but universal immunization of the preschool child is not practiced, particularly in lower socioeconomic class population groups. furthermore, rubeola virus has been found to violate the concept of herd immunity, a major principle on which eradication was based, because outbreaks occur in high schools and colleges in which a large percentage of the population has been immunized. an inactivated vaccine was available from 1962 through 1965. children who received the inactivated vaccine may have developed atypical measles on exposure to rubeola virus. following this, an attenuated, live strain of measles virus was used as vaccine but the high sidereaction frequency necessitated the concomitant administration of 'v-globulin. it is now recognized that the concurrent use of 'v-globulin sometimes rendered immunization ineffective. many children were immunized before the age of 12 months; for effective immunization to occur, vaccine must be given after the age of15 months. as a consequence of the lack of universal preschool immunization and difficulties related to the vaccine, there now exist two population groups who may be nonimmune with respect to rubeola, ie, preschool children, and adolescents and young adults. a few years ago rubeola epidemics were common in military recruits. following the occurrence of these outbreaks, recruits now routinely undergo serologic testing, and if antibody to rubeola virus is not detected by either hemagglutination inhibition or indirect immunofluorescence tests, live attenuated vaccine is given. this practice essentially has stopped the occurrence of these outbreaks in the military. persons who received the inactivated vaccine can develop atypical measles. the first cases of this new syndrome were misdiagnosed as rocky mountain spotted fever. they were confused with this disease because the rash began on the extremities and spread inward to involve the trunk. the rash could be maculopapular, vesicular, or petechial. in atypical measles, pulmonary involvement consists of nodular infiltrates, lobar consolidation, and the occurrence of pleural effusions. hilar lymphadenopathy may also present in these patients. s1 • s2 they have an anamnestic response in antibody production with the infection. mild eosinophilia may also be present and the virus cannot be recovered from the nasopharynx. in young adults with typical measles, approximately 5% develop clinical evidence of pneumonia. radiographic evidence of pneumonia, however, may be seen in up to 50% of the patients. the pneumonia is usually characterized by diffuse bilateral interstitial or fine reticulonodular infiltrates, particularly affecting the lower lobes. bacterial suprainfection occurs in as many as 30% of cases of recognized viral pneumonia. the types of bacteria causing infection may be determined by the circumstances in which disease occurs, such as military recruit populations. recognized pathogens include h. influenzae, s. pneumoniae, group a streptococci, group y meningococci, and s. aureus. bacterial suprainfection generally occurs between the fifth and tenth days after the rash and is heralded by clinical worsening, new or different lung infiltrates, or changes in sputum characteristics or the white blood count. antibiotic treatment of viral pneumonia does not prevent bacterial suprainfection. in immunodeficient children, measles pneumonia occurred without a rash and pathologically was called giant-cell pneumonia. it is now recognized that these children lacked cell-mediated immunity and had depressed and delayed antibody production. s3 intact cell-mediated immunity is essential for rash production. in fatal rubeola pneumonia, the entire tracheobronchial tree may be denuded of cells down to the basal layer; squamous metaplasia of the cells occurs; there is widening of the interstitial space with edema and inflammatory cells; and alveoli are filled with edema, hyaline membranes, and mononuclear cells. in addition, giant cells containing multiple nuclei are found within the tracheobronchial epithelium. extrapulmonic manifestations of measles occur and include otitis media, sinusitis, encephalitis, and the common presence of hepatic dysfunction in young adults, mostly consisting of mild elevations of the serum aspartate aminotransferase and lactic dehydrogenase. there presently is no specific therapy. some public health authorities now think that reimmunization with measles, mumps, and rubella vaccines should be 58 given before or when the child enters high school. medical personnel not sure of their rubeola immunity should have that status assessed by determination of specific antibody. varicella-zoster virus can produce pneumonia during the course of varicella or disseminated herpes zoster and can be a severe disease that can lead to mortality. varicella in childhood is not usually associated with viral pneumonia, but bacterial suprainfection can occur, necessitating appropriate antibiotic treatment. in immunosuppressed children, however, pure viral pneumonia can occur in association with varicella. in adults, there is a tendency for the virus to affect the lung relatively commonly during varicella. fifteen to twenty percent of all adults with varicella may have x-ray evidence of pneumonia but only about 5% require hospitalization. most frequently in adults, varicella pneumonia is not complicated by bacterial suprainfection; however, this can occur, particularly when patients require intubation. the virus reaches the lung both by passage down the respiratory tract and by hematogenous seeding because the rash is occurring at the same time as the pneumonia. initially, the pneumonic process appears as nodular infiltrates, 1-4 mm in diameter associated with an interstitial inflammatory infiltrate. a4 the lesions are more dense toward the hilum and are the counterpart in the lung of the pox occurring on the skin (figure 7) . peribronchial inflammatory infiltrates, hilar adenopathy, and pleural effusions may occur. the reticulonodular interstitial infiltrate can progress to widespread alveolar damage and diffuse pulmonary parenchymal infiltrates. pathologically, the pneumonia resembles influenza pneumonia except that areas of coagulative necrosis can be seen. although these necrotic areas generally clear during the course of clinical disease, they can become calcified, and the radiograph shows a picture of miliary calcifications. it has been shown that this area of coagulative necrosis can become surrounded by an inflammatory infiltrate and resemble a granuloma. a5 fibrous tissue envelopes the necrotic granulomatous process and the lesion eventually calcifies. another process occurring in varicella pneumonia is destruction of the epithelium of the trachea and bronchi. in cases in which the illness is protracted, the development of a thick, fibrinopurulent crust may occur over the lower pharynx, larynx, and upper trachea. this thick crust can cause respiratory embarrassment and can pose a problem for intubation. disseminated herpes zoster can cause the same processes in the lung. most normal adults recover from varicella pneumonia without difficulty, but there can be substantial respiratory morbidity and morfigure 7 . varicella pneumonia. the chest radiograph on the lett is from the case report described in the protocol. the close-up film on the right in another pallent shows peribronchial infiltrates and multiple 1-4-mm rounded opacities in the right lung field. tality in immunosuppressed patients or in women during the third trimester ofpregnancy. 86 clinically, the patient with varicella-zoster virus pneumonia presents with a rash followed by cough and dyspnea. the sputum is initially white and modest in amount, but can become hemorrhagic. the process can be complicated by the development of chest pain and pleural effusions, which are often blood tinged and related to the presence of pox on the pleural surface. extrapulmonic manifestations of varicella occur and consist of the characteristic skin rash, otitis media with bacterial suprainfection, myopericarditis, hepatic dysfunction, and encephalitis. reye's syndrome can complicate the course ofvaricella. there can be an associated glomerulonephritis and varicella virus can occasionally induce frank arthritis. in caring for patients with varicella-zoster virus pneumonia, it is important to realize that the external appearance of the patient or his apparent well-being may disguise underlying hypoxemia. if efforts are not made to diagnose and correct the hypoxemia, the patient may become confused, perform inappropriate activity, and become more hypoxemic. ventilator support may become necessary. deaths in varicella pneumonia occur because of respiratory insufficiency, the development of tension pneumothoraces, bacterial suprainfection, or progressive pulmonary fibrosis. two antiviral compounds, adenine arabinoside (ara-a) and acyclovir (acv), have been proven to be efficient in the treatment of significant, complicated varicella-zoster virus infections. adenine arabinoside inhibits viral dna polymerase and is given at a dosage of 10 mglkg over a 12-hour period for at least 5 days. the dosage could be increased to 15 mglkg the american journal of the medical sciences but the majority of experience with varicella-zoster virus infections is with 10 mglkg/day. the drug is sparingly soluble so that 2 ml of vehicle are required for each milligram of drug administered to the patient. at a dose of 10 mglkg, bone marrow suppression does not usually occur. if the dose is not decreased in the setting of hepatic and renal dysfunction, central nervous system disturbances, which consist of insomnia, hallucinations and tremulousness, may occur. these central nervous system manifestations usually fade with stopping the drug and rarely lead to death, but can persist for a protracted period after the drug has been discontinued. acyclovir also has been used to treat varicella-zoster virus infections. it inhibits viral dna polymerase and also acts as a chain terminator. its dosage is 500 mg/m 2 every 8 hours for at least 7 days. the only significant problem with the administration with acyclovir in this setting is the production of an obstructive nephropathy, due to salting out of the drug in the collecting tubules ofthe kidney. this is usually easily managed by administration of a fluid bolus, a diuretic, or mannitol. a comparison of the two drugs in complicated varicella-zoster virus infection has been made. 87 . 88 one group found that acyclovir was more efficacious; the other study determined that ara-a was equally as effective. because the administration of ara-a requires an increased volume of fluid and can be associated with central nervous system side effects, some authorities now consider acyclovir the drug of choice in the treatment of complicated infections due to varicella-zoster virus. oral acyclovir is absorbed poorly by the gastrointestinal tract and its efficacy in uncomplicated herpes zoster is apparent only when 800 mg are given 5 times a day for at least a 5-day period. there has been no experience with oral acv in treating varicella pneumonia. future modes of therapy may include combining ara-a with acv or administering one or other of the drugs with an interferon preparation. alpha-interferon also has been shown to be effective as therapy in complicated varicella-zoster virus infections but its use has been superceded by acv and ara-a. case report varicella pneumonia. a 39-year-old man was exposed to his two children with chicken pox. two days before admission he developed a rash, then dyspnea. on physical examination, a typical varicella rash was present. he was in severe respiratory distress. rales were present diffusely over both lung fields. the chest radiograph revealed bilateral extensive alveolar infiltrates. arterial blood gases showed a ph of 7.42, pc02 of 32, and a p02 of 24. he was intubated and begun on positive end-expiratory pressure (peep) with a fi0 2 of 70%. he was started on intravenous acyclovir 500 mg/m2 every 8 hours. he improved and was able to be extubated after 5 days. comment. severe varicella pneumonia responded to intravenous acyclovir while his oxygenation was maintained on peep with a high fi02. herpes simplex virus can cause a necrotizing bronchopneumonia in neonatal infections and can also cause pneumonia in severely immunosuppressed adult patients. the largest series of patients with herpes simplex virus pneumonia was reported from seattle in bone-marrow transplant recipients 'and consisted of 20 patients with either a focal pneumonia (12 patients) or a diffuse interstitial pneumonia (eight patients). 89 the focal pneumonia was found associated with herpetic esophagitis and tracheitis and probably resulted from contiguous spread of herpes virus to the lung parenchyma. diffuse interstitial pneumonia most probably resulted from hematogenous dissemination of virus to lung. pathologically, the process can be one of a necrotizing bronchopneumonia or of a widening of the interstitial space in the lung associated with diffuse alveolar injury. in these highly immunosuppressed patients, both bacterial and fungal suprainfection occurred and it was difficult to sort out which process was responsible for what proportion of lung damage. acyclovir has been shown to be an effective treatment of complicated herpes simplex virus infections in immunosuppressed patients. its dose in the usual patient is 250 mg/m 2 every 8 hours for at least 7 days, but if the process has been ascertained to be a rapidly progressive herpetic pneumonia, the dose could be increased to 10 mglkg every 8 hours until a therapeutic response had been obtained. with the development of potent antiviral chemotherapy, there is a need to consider and diagnose herpetic pneumonia. specific diagnosis can only be accomplished readily by lung biopsy. 60 although involvement of the lung in infectious mononucleosis due to epstein-barr virus must be considered a rare occurrence, recent studies and case reports demonstrate that it probably can happen. 90 . 91 careful attention should be given to possible coexisting mycoplasma and other viral infections, particularly since the former can be treated. radiographic abnormalities may consist of hilar adenopathy, strand-like parenchymal infiltrates, diffuse bilateral pneumonia, and a picture consistent with primary atypical pneumonia. although epstein-barr virus is susceptible to acyclovir, there are no reports treating lung involvement with this drug. cytomegalovirus (cmv) rarely causes pneumonia in normal adults as part of the cmv mononucleosis syndrome. 92 however, it is more common for cmv to induce pneumonia in normal hosts than epstein-barr virus. of 443 patients with communityacquired pneumonia, 18 had virologic,2 pathologic,2 or serologic 14 evidence of cmv infection. 93 ten of these 18 patients were not immunosuppressed. in five of the ten, cmv was the only pathogen. the remaining five patients had one or more coexisting infections; c. trachomatis in two, m. pneumoniae in one, epstein-barr virus in one, and bacteria in three, both aerobic and anaerobic. cytomegalovirus more commonly causes pneumonia in immunosuppressed patients. it occurs particularly in renal, heart, liver, and bone-marrow transplant recipients. it is now becoming an increasing problem in patients with aids. in the transplant recipient experiencing a primary cmv infection, the virus most probably reaches the lung parenchyma through the hematogenous route, and the first finding is that of a reticulonodular infiltrate and the presence of 1-4 mm opacities ( figure 8 ). pathologically, these focal areas usually consist of necrotic tissue, hemorrhage and alveolar damage with edema, a mononuclear infiltrate, and typical cytomegalic cells. the process can extend leading to diffuse interstitial and alveolar infiltrates. an attempt has been made to separate the foregoing process from that of an insidiously developing interstitial pneumonia that occurs more commonly in reactivated infections and has a better prognosis. 94 in bone marrow transplant recipients, diffuse interstitial pneumonitis due to cmv is much more common in patients receiving allogeneic transplants, and the case-fatality rate approximates 90%. 95 in some renal transplant recipients, the pneumonic process can be focal and does not have to be exceptionally severe. 96 small pleural effusions can occur occasionally. in other renal transplant recipients, however, cmv pneumonia can progress rapidly and lead to death as part of a widely disseminated infectious process. 97 in cardiac transplant recipients a variety of pulmonary opportunistic suprainfections has been well documented to occur in the course of cmv pneumonia. 98 typical microorganisms causing suprainfections include p. carinii and nocardia species. in patients with aids, there may be co-existing infection with pneumocystis. cure of the pneumocystis can be effected by drugs, leaving cmv as the major pulmonary pathogen. in patients with aids, rapid development of cmv pneumonia can occur and lead to the death of the patient. extrapulmonic manifestations of cmv in the renal transplant recipient include fever, malaise, hepatic dysfunction, splenomegaly, leukopenia, and an increase in serum creatinine. 96 with extensive cmv dissemination in heavily immunosuppressed patients, including those with aids, extrapulmonic manifestations include gastrointestinal ulceration with bleeding and perforation, hepatic dysfunction, adrenal cortical involvement, and central nervous sytem dysfunction. cytomegalovirus can be thought of as an immunosuppressive viral agent, and infection with this microorganism may lead to further immunosuppression with consequent bacterial, fungal, and parasitic suprainfection. clinically, patients with cmv pneumonia complain of dyspnea with a nonproductive cough. there can be an associated pleurisy. the process can be transient or can extend to respiratory insufficiency necessitating ventilatory support. therapy of fully developed cmv pneumonia has been shown not to be effective and this includes the use of the adenine arabinoside, acyclovir, ganciclovir (dhpg), and combinations of interferon with all the above. ganciclovir has been successful in achieving an antiviral effect in the lung, yet has not improved outcome in the american journal of the medical sciences bone marrow transplant recipients with cmv pneumonia. 99 in an occasional renal transplant recipient who has the potential of a good immune response to the virus, the cmv illness that can include localized pneumonia might be benefited by the judicious use of ganciclovir. attempts at preventing cmv pneumonia have included donor selection, avoidance of white blood cell transfusions, and prophylactic administration of alpha-interferon or 'y-globulin preparations given before and through the first 60 days after transplantation. alpha-interferon does prevent cmv viremia in the renal transplant recipient; 'y-globulin protects partially against cmv pneumonia if white blood cell transfusions have not been given. studies are in progress trying to make this latter effect more consistent, and consist of determining whether total antibody content is the necessary component or whether the effect necessitates the presence oflarge quantities of neutralizing antibody. on a priority basis, live, attenuated cmv vaccine development has been curtailed for the immediate future. a 40-year-old homosexual man presented to the hospital with fever, cough, and an erythematous rash. he had been followed in clinic with aids-related complex with lymphadenopathy, thrush, lymphopenia, anergy, diarrhea, and a positive antibody test to human immunodeficiency virus. at the time of his acute terminal illness he had a temperature of 38.5° c and had a diffuse, erythematous pruritic rash over the trunk and upper legs. the admission chest radiograph was interpreted as normal. on the second hospital day, the patient became delirious, had a worsening cough, and developed severe dyspnea. arterial blood gases on room air revealed a ph of 7.50, pco. of 30, and a po. of 44. chest radiograph now revealed bilateral diffuse reticulonodular infiltrates. he was started on sulfatrimethoprim but had a respiratory arrest and died. postmortem examination revealed cmv pneumonia without evidence ofpneumocystis. lung viral cultures rapidly grew cmv, with the cytopathic effect being present the second day. comment. explosive illness in a patient with arc revealed only cmv at autopsy and on viral culture of the lung. viral and mycoplasmal pneumonia in a prepaid medical care group during an eight-year period primary atypical pneumonia in a family due to concomitant mycoplasma pneumoniae and adenovirus type 7 infection viral pneumonia as a cause and result of hospitalization severe illness with influenza b strannegard 0, trollfors b: etiology of community-acquired pneumonia in out-patients etiologies and characteristic features of pneumonias in a municipal hospital etiology of community-acquired pneumonia in patients requiring hospitalization adult community-acquired pneumonia in central london virological investigations in adults with acute pneumonia hospital study of adult community-acquired pneumonia acute community-acquired pneumonias an acute infection of the respiratory tract with atypical pneumonia. a disease entity probably caused by a filtrable virus causes of atypical pneumonia: results of a 1-year prospective study differential diagnosis of viral, mycoplasmal and bacteraemic pneumococcal pneumonias on admission to hospital comparative radiographic features of community acquired legionnaires' disease, pneumococcal pneumonia, mycoplasma pneumonia, and psittacosis mycoplasma pneumonia: failure of erythromycin therapy lung infections caused by viruses, mycoplasma pneumoniae, and rickettsiae pneumonias due to rickettsiae, chlamydiae, viruses and mycoplasma neurologic disease associated with mycoplasma pneumoniae pneumonitis. demonstration of viable mycoplasma pneumoniae in cerebrospinal fluid and blood by radioisotopic and immunofluorescent tissue culture techniques open lung biopsy in myco-62 plasma pneumoniae pneumonia mycoplasma pneumoniae infection in families pulmonary involvement in mycoplasma pneumoniae infection in families the clinical spectrum and diagnosis of mycoplasma pneumoniae infection clinical features of mycoplasmal pneumonia in adults mycoplasmal pneumonias in the community hospital. the "unusual" manifestations become common lobar pneumonia caused by mycoplasma pneumoniae radiographic appearances of mycoplasma pneumonia mycoplasmal pneumonia and adult respiratory distress syndrome: a complication to be recognized acute respiratory failure due to atypical pneumonia the protean manifestations of mycoplasma pneumoniae infection in adults weekly clinicopathological exercises: case 39-1983 pityriasis rosea gibert and mycoplasma pneumoniae infection abnormalities in lung function following clinical recovery from mycoplasma pneumoniae pneumonia van der straeten m: mycoplasma pneumonia with fulminant evolution into diffuse interstitial fibrosis bronchiolitis obliterans due to mycoplasma pneumoniae mycoplasma pneumonia complicated by bronchiectasis mycoplasmal pneumonia associated with mesangiocapillary glomerulonephritis type ii (dense deposit disease) mycoplasma pneumoniae pneumonia associated with iga nephropathy new york city medium for enhanced recovery of mycoplasma pneumoniae from clinical specimens principles and practice of infectious diseases psittacosis. a diagnostic challenge isolation of chlamydia trachomatis from the lower respiratory tract of adults chlamydia trachomatis infection in adults with community-acquired pneumonia chlamydia trachomatis pneumonia in adults: radiographic appearance an epidemic of mild pneumonia due to an unusual strain of chlamydia psittaci a new chlamydia psittaci strain, twar, isolated in acute respiratory tract infections clinical features of adenoviral pneumonia in air force recruits lobar pneumonia associated with adenovirus type 7 fatal pneumonia associated with adenovirus type 7 in three military trainess fatal adenovirus pneumonia in a young adult associated with adv-7 vaccine administered 15 days earlier fatal disseminated adenovirus infection in a renal transplant recipient adenovirus infections in patients undergoing bone-marrow transplantation fatal disseminated adenovirus 11 pneumonia in an agammaglobulinemic patient bacterial pneumonia complicating adenoviral pneumonia bronchopneumonia with serious sequelae in children with evidence of adenovirus type 21 infection diffuse pneumoniti~ due to adenovirus type 21 in a civilian association of adenovirus type 16 with reye's-syndrome-like illness and pneumonia rapid diagnosis of respiratory adeno-vi~us infections in young adult men fatal haemorrhagic pneumonia in an adult due to respiratory syncytial virus and staphylococcus aureus respiratory syncytial virus pneumonitis in adults case report: presumed respiratory syncytial virus pneumonia in three immunocompromised adults an outbreak of respiratory syncytial virus pneumonia in a nursing home for the elderly ribavirin treatment of respiratory syncytial viral infection in infants with underlying cardiopulmonary disease ribavirin aerosol treatment of bronchiolitis due to respiratory syncytial virus infection in infants coronavirus infections of man associated with diseases other than the common cold influenza: the newe acquayantance orthormyxo-and paramyxoviruses and their replication, in fields bn sanfordjp: pulmonary function in uncomplicated influenza pulmonary mechanics after uncomplicated influenza a infection studies on influenza in the pandemic of 1957-1958. ii. pulmonary complications of influenza severe influenza virus pneumonia in the pandemic of 1968-1969 mycoplasma and influenza pneumonia in a series of 214 adults the leukocyte response during viral respiratory illness in man acute myocarditis in influenza a infections. two cases of non-bacterial myocarditis, with isolation of virus from the lungs fatal influenza a pneumonia in pregnancy epidemic renal transplant rejection associated with influenza a victoria rapid diagnosis of primary influenza pneumonia pharmacokinetics of amantadine hydrochloride in subjects with normal and impaired renal function ribavirin small-particle aerosol treatment of influenza ribavirin small-particle aerosol treatment of infections caused by influenza virus trains anictoriaj7/83 (h1n1) and bltexas/1/84 measles pneumonia. bacterial suprainfection as a complicating factor measles pneumonia in young adults. an analysis of 106 cases isolation of measles virus at autopsy in cases of giant-cell pneumonia without rash report of seven cases and a review of literature persistent pulmonary granulomas after recovery from varicella pneumonia varicella pneumonia complicating pregnancy treatment of varicellazoster virus infection in severely immunocompromised patients comparative trial of acyclovir and vidarabine in disseminated varicella-zoster infections in immunocompromised patients coreyl: herpes simplex virus pneumonia. clinical, virologic, and pathologic features in 20 patients diffuse pneumonia and acute respiratory failure due to infectious mononucleosis clinical, virologic, and serologic evidence of epstein-barr virus infection in association with childhood pneumonia pneumonia associated with rising cytomegalovirus antibody titres in a healthy adult does cytomegalovirus playa role in communityacquired pneumonia? cytomegalovirus pneumonia in bone marrow transplant recipients: miliary and diffuse patterns nonbacterial nonfungal pneumonia following marrow transplantation in 100 identical twins disease due to cytomegalovirus and its long-term consequences in renal transplant recipients. correlation of allograft survival with disease due to cytomegalovirus and rubella antibody level clinical characteristics of the lethal cytomegalovirus infection following renal transplantation diagnosis of cytomegalovirus pneumonia in compromised hosts activity of 9-[2-hydroxy-1-(hydroxymethyl)-ethoxymethyl]guanine in the treatment of cytomegalovirus pneumonia key: cord-021499-up5vftj4 authors: brayton, cory; mähler, michael; nicklas, werner title: viral infections date: 2007-09-02 journal: the laboratory mouse doi: 10.1016/b978-012336425-8/50076-5 sha: doc_id: 21499 cord_uid: up5vftj4 nan in interpreting the microbiological status of laboratory animals, it must be understood that infection and disease are not synonymous. infection refers to the invasion and multiplication of microorganisms in body tissues and may occur with or without apparent disease. disease refers to interruption or deviation from normal structure and function of any tissue, organ, or system. many of the infections with which we are concerned may not cause discernable disease in many strains of mice. however, they may cause inapparent or subclinical changes that can interfere with research. such interference often remains undetected, and therefore modified results may be obtained and published. the types of interference of an agent with experimental results may be diverse. there is no doubt that research complications due to overt infectious disease are significant and that animals with clinical signs of disease should not be used for scientific experiments. but also clinically inapparent infections may have severe effects on animal experiments. there are numerous examples of influences of microorganisms on host physiology and hence of the interference of inapparent infections with the results of animal experiments. many microorganisms have the potential to induce activation or suppression of the immune system or both at the same time but on different parts of the immune system, regardless of the level of pathogenicity. all infections, apparent or inapparent, are likely to increase inter-individual variability and hence result in increased numbers of animals necessary to obtain reliable results. microorganisms, in particular viruses, present in an animal may contaminate biological materials such as sera, cells, or tumours (collins and parker, 1972; nicklas et al., 1993) . this may interfere with in vitro experiments conducted with such materials and may also lead to contamination of animals (lipman et al., polymerase chain reaction (pcr) testing of biologics to be inoculated into mice is an important component of a disease prevention programme. finally, latent infections may be activated by environmental factors, by experimental procedures, or by the combination and interaction between various microorganisms. for all these reasons, prevention of infection, not merely prevention of clinical disease, is essential. unfortunately, research complications due to infectious agents are usually considered artefacts and published only exceptionally. information on influences of microorganisms on experiments is scattered in diverse scientific journals, and many articles are difficult to detect. to address this problem, several congresses were held on viral complications on research. the knowledge available was summarized in conference proceedings (melby and balk, 1983; bhatt et al., 1986b; hamm, 1986) and has later repeatedly been reviewed (lussier, 1988; national research council, 1991; baker, 1998; nicklas et al., 1999) . this chapter covers only viral infections of laboratory mice. viral infections of mice have been studied in detail, and comprehensive information on their pathogenic potential, their impact on research, and the influence of host factors such as age, genotype, and immune status on the response to infection is available. bacterial agents may be similarly important, but with few exceptions (e.g. helicobacter species) little is known about their potential to influence host physiology and experiments. even less is known about most parasites in this regard. among fungal agents, only pneumocystis carinii can be expected to play a significant role in contemporary mouse colonies. the nomenclature and taxonomy of viruses described are based on recent nomenclature rules by the international union of microbiological societies (2000) and the universal virus database of the international committee on the taxonomy of viruses (http://www.ictvdb.iacr.ac.uk). retroviruses are not covered in this chapter because they are not included in routine health surveillance programmes and cannot be eradicated with presently available methods. this is because most of them are incorporated in the mouse genome as proviruses and thus are transmitted via germline. the ability to accurately determine whether or not laboratory animals or animal populations have been infected with virus depends on the specificity and sensitivity of the detection methods used. most viral infections in immunocompetent mice are acute or short-term, and lesions are often subtle or subclinical. the absence of clinical disease and pathological changes has therefore only limited diagnostic value. however, clinical signs, altered behaviour, or lesions may be the first indicator of an infection and often provide clues for further investigations. serology is the primary means of testing mouse colonies for exposure to viruses, largely because serological tests are sensitive and specific, are relatively inexpensive, and allow screening for a multitude of agents with one serum sample. they are also employed to monitor biological materials for viral contamination using the map test. serological tests detect specific antibodies, usually immunoglobulin g (igg), produced by the host against the virus and do not actually test for the presence of virus. an animal may have been infected, mounted an effective antibody response, and cleared the virus, but remains seropositive for weeks or months or forever, even though it is no longer infected or shedding the agent. active infection can only be detected by using direct diagnostic methods such as virus isolation, electron microscopy, or pcr. meanwhile, pcr assays have been established for the detection of almost every agent of interest. they are highly sensitive and depending on the demands, they can be designed to broadly detect all members of a genus or only one species. however, good timing and selection of the appropriate specimen is critical for establishing the diagnosis. in practice, combinations of diagnostic tests are often necessary including the use of sentinel animals or immunosuppression to get clear aetiological results or to avoid consequences from false-positive results. reports on the prevalence of viral infections in laboratory mice throughout the world have been published frequently. in general, the microbiological quality of laboratory mice has constantly improved during the last decades, and several agents (e.g. herpes-and polyomaviruses) have been essentially eliminated from contemporary colonies due to advances in diagnostic methodologies and modern husbandry and rederivation practices (jacoby and lindsey, 1998; zenner and regnault, 2000; livingston and riley, 2003) . they may, however, reappear, since most have been retained or are still being used experimentally. furthermore, the general trend towards better microbiological quality is challenged by the increasing reliance of biomedical research on genetically modified and immunodeficient mice, whose responses to infection and disease can be unpredictable. increasing numbers of scientists are creating genetically modified mice, with minimal or no awareness of infectious disease issues. as a consequence, they are more frequently infected than 'standard' strains of mice coming from commercial breeders, and available information on their health status is often insufficient. frequently, they are exchanged between laboratories, which amplifies the risk of introducing infections from a range of animal facilities. breeding cessation strategies that have been reported to eliminate viruses from immunocompetent mouse colonies may prove to be costly and ineffective in genetically modified colonies of uncertain or incompetent immune status. it must also be expected that new agents will be detected, although only occasionally. infections therefore remain a threat to biomedical research, and users of laboratory mice must be cognizant of infectious agents and the complications they can cause. two members of the family herpesviridae can infect mice (mus musculus). mouse cytomegalovirus 1 (mcmv-1) or murid herpesvirus 1 (muhv-1) belongs to the subfamily betaherpesvirinae, genus muromegalovirus. murid herpesvirus 3 (muhv-3) or mouse thymic virus (mtv) has not yet been assigned to a genus within the family herpesviridae. both viruses are enveloped, doublestranded dna viruses that are highly host-specific and relatively unstable to environmental conditions such as heat and acidic ph. both agents are antigenically distinct and do not cross-react in serological tests, but their epidemiology is similar (cross et al., 1979) . seropositivity to mcmv-1 was reported in less than 5% of specified pathogen-free (spf) mouse colonies in the usa in 1996 (jacoby and lindsey, 1998) , and some institutions reported to have mice 'on campus' that were positive for mtv. in a more recent study, a low rate (0.1%) of samples was found to be positive for mcmv-1 whereas no sample tested positive for mtv (livingston and riley, 2003) . the data available suggest that the prevalence of both viruses in contemporary colonies and thus their importance for laboratory mice is negligible. however, both mcmv-1 and mtv are frequently found in wild mice, which may be coinfected with both viruses (national research council, 1991; singleton et al., 2000) . natural infection with mcmv-1 causes subclinical salivary gland infection in mice. the virus persists in the salivary glands (particularly in the submaxillary glands) and also in other organs (osborn, 1982; kercher and mitchell, 2002; lenzo et al., 2002) . most information concerning the pathogenesis of mcmv-1 infection is based on experimental infection studies. these results are very difficult to summarize because the outcome of experimental infection in laboratory mice depends on various factors such as mouse strain and age, virus strain and passage history, virus dose, and route of inoculation (osborn, 1982) . in general, newborn mice are most susceptible to clinical disease and to lethal infection. virus replication is observed in newborn mice in many tissues (for details, see osborn, 1982) and appears in the salivary glands towards the end of the first week of infection when virus concentrations in liver and spleen have already declined. resistance develops rapidly after weaning between days 21 and 28 of age. experimental infection of adult mice results in mortality only in susceptible strains and only if high doses are administered. not even intravenous or intraperitoneal injections of adult mice usually produce signs of illness in resistant strains (shanley et al., 1993) . mice of the h-2 b (e.g. c57bl/6) and h-2 d (e.g. balb/c) haplotype are more sensitive to experimental infection than are mice of the h-2 k haplotype (e.g. c3h), which are approximately 10-fold more resistant to mortality than are those of the b or d haplotype (osborn, 1986) . subclinical or latent infections can be activated by immunosuppression (e.g. with cyclophosphamide or cortisone). reactivation of mcmv-1 occurs also after implantation of latently infected salivary glands into prkdc scid mice (schmader et al., 1995) . immunodeficient mice lacking functional t cells or natural killer (nk) cells, such as foxn1 nu and lyst bg mice, are more susceptible than are immunocompetent animals. experimental infection in prkdc scid mice causes severe disease or is lethal, with necrosis in spleen, liver, and other organs, and multinucleate syncytia with inclusion bodies in the liver (reynolds et al., 1993) . similar to aids patients infected with human cytomegalovirus, athymic foxn1 nu mice experimentally infected with mcmv also develop adrenal necrosis (shanley and pesanti, 1986) . the virus also replicates in the lungs leading to pneumonitis whereas in heterozygous (foxn1 nu / ϩ ) littermates replication and disease are not seen (shanley et al., 1997) . the most prominent histological finding of cytomegaloviruses is enlarged cells (cytomegaly) of salivary gland epithelium with eosinophilic nuclear and cytoplasmic inclusion bodies. the inclusion bodies contain viral material and occur in other organs such as liver, spleen, ovary, and pancreas (osborn, 1982) . depending on inoculation route, dose, strain, and age of mice, experimental infections may result in inflammation or cytomegaly with inclusion bodies in a variety of tissues, pneumonitis, myocarditis, meningoencephalitis, or splenic necrosis in susceptible strains (national research council, 1991; osborn, 1982; percy and barthold, 2001) . virus is transmitted oronasally by direct contact and is excreted in saliva, tears, and urine for several months. wild mice serve as a natural reservoir for infection. the virus is most frequently transmitted horizontally through mouse-to-mouse contact but does not easily spread between cages. it is generally assumed that mcmv-1 has a very low prevalence in contemporary colonies of laboratory mice. the risk of introduction into facilities housing laboratory mice is very low if wild mice are strictly excluded. monitoring is necessary if populations of laboratory mice may have been contaminated by contact with wild mice. as for other viruses, enzyme-linked immunosorbent assay (elisa) and indirect immunofluorescence assay (ifa) are the most appropriate tests for detecting antibodies. as the virus persists, direct demonstration of mcmv-1 in infected mice is possible by pcr (palmon et al., 2000) or by virus isolation using mouse embryo fibroblasts (3t3 cells). although mcmv-1 does not play a significant role as a natural pathogen of laboratory mice, it is frequently used as a model for human cytomegalovirus infection (bolger et al., 1999) . however, the virus is known to influence immune reactions in infected mice and may therefore have impact on immunological research (osborn, 1986; national research council, 1991; baker, 1998) . mouse thymic virus was detected during studies in which samples from mice were passaged in newborn mice. unlike other herpesviruses, the virus can not yet be cultured in vitro and is propagated by intraperitoneal infection of newborn mice. the thymus is removed 7-10 days later, and thymus suspensions serve as virus material for further studies. the prevalence of mtv is believed to be low in laboratory mice, and for this reason and also due to the difficulties in virus production for serological assays, it is not included in many standard diagnostic or surveillance testing protocols. limited data are available indicating that it is common in wild mice, and it is also found in laboratory mice (osborn, 1982; morse, 1987; national research council, 1991) . further, mtv obviously represents a significant source of contamination of mcmv-1 (and vice versa) if virus is prepared from salivary glands since both viruses cause chronic or persistent salivary gland infections and can coinfect the same host. all mouse strains are susceptible to infection, but natural or experimental infection of adult mice is subclinical. gross lesions appear only in the thymus and only if experimental infection occurs at an age of less than about 5 days. virus is present in the thymus but may also be found in the blood and in salivary glands of surviving animals. salivary glands are the only site yielding positive virus isolations if animals are infected as adults. mouse thymic virus also establishes a persistent infection in athymic foxn1 nu mice, but virus shedding is reduced compared to euthymic mice and virus recovery is possible only in a lower percentage of mice (morse, 1988) . pathological changes caused by mtv occur in the thymus, and reduced thymus mass due to necrosis in suckling mice is the most characteristic gross lesion (percy and barthold, 2001) . lymphoid necrosis also may occur in lymph nodes and spleen (wood et al., 1981) , with necrosis and recovery similar to that in the thymus. in mice infected during the first 3 days after birth, necrosis of thymus becomes evident within 3-5 days, and its size and weight are markedly reduced at day 12-14. intranuclear inclusions may be present in thymocytes between days 10-14 post infection. the thymus and the affected peripheral tissues regenerate within 8 weeks after infection. regardless of the age of mice at infection, a persistent infection is established in the salivary glands, and infected animals shed virus for life. several alterations of immune responses are associated with neonatal mtv infection. there is transient immunosuppression, attributable to lytic infection of t lymphocytes, but activity (e.g. response of spleen cells to t cell mitogens) returns to normal as the histological repair progresses (wood et al., 1981) . selective depletion of cd4 ϩ t cells by mtv results in autoimmune disease (morse and valinsky, 1989; morse et al., 1999) . information about additional influences on the immune system is given by osborn (1982 ), national research council (1991 ), and baker (1998 . in experimentally infected newborn mice, oral and intraperitoneal infections similarly result in thymus necrosis, seroconversion, and virus shedding suggesting that the oral-nasal route is likely to be involved in natural transmission (morse, 1989) . the virus spreads to cage mates after long periods of contact. it is transmitted between mice kept in close contact, and transmissibility from cage to cage seems to be low. mouse thymic virus is not transmitted to foetuses by the transplacental route, and intravenous infection of pregnant mice does not lead to congenital damage, impairment in size or development, or abortion (st-pierre et al., 1987) . mouse thymic virus and mcmv-1 do not crossreact serologically (cross et al., 1979) . serological monitoring of mouse populations for antibodies to mtv is possible by ifa testing, which is commercially available; elisa tests have also been established (morse, 1990b) . elisa and complement fixation yield similar results . it must be noted that the immune response depends on the age at infection. antibody responses are not detectable in mice infected as newborns whereas adult mice develop high titres that are detectable by serological testing. if neonatal infection is suspected, homogenates of salivary glands or other materials can be inoculated into pathogen-free newborn mice followed by gross and histological examination of thymus, lymph nodes, and spleens for lymphoid necrosis (morse, 1987) . alternatives to the in vivo infectivity assay for detecting mtv in infected tissues include a competition elisa (prattis and morse, 1990) and map testing, although this is slightly less sensitive than infectivity assays (morse, 1990a) . very little experience exists on eradication methods for mtv due to its low prevalence in contemporary mouse colonies. methods that eliminate other herpesviruses likely will eliminate mtv. procurement of animals of known negative mtv status is an appropriate strategy to prevent infection. strict separation of laboratory mice from wild rodents is essential to avoid introduction into laboratory animal facilities. mousepox (ectromelia) virus (ectv) is a member of the genus orthopoxvirus belonging to the family poxviridae. it is antigenically and morphologically very similar to vaccinia virus and other orthopoxviruses. poxviruses are the largest and most complex of all viruses with a diameter of 200 nm and a length of 250-300 nm. mousepox (ectromelia) virus contains one molecule of double-stranded dna with a total genome length of 185,000 nucleotides. it is the causative agent of mousepox, a generalized disease in mice. experimental transmission to young rats (up to 30 days of age) is possible (jandasek, 1968; buller et al., 1986) . the virus is resistant to desiccation, dry heat, and many disinfectants. it is not consistently inactivated in serum heated 30 min at 56њc (lipman et al., 2000b) and persists for 26 weeks when maintained at 4њc in foetal bovine serum (bhatt and jacoby, 1987a) . effective disinfectants include vapour-phase formaldehyde, sodium hypochlorite, and iodophores (small and new, 1981; national research council, 1991) . historically, ectv has been an extremely important natural pathogen of laboratory mice. the virus was widespread in mouse colonies worldwide and can still be found in several countries. between 1950 and 1980 almost 40 individual ectromelia outbreaks were reported in the usa. the last major epizootic in the usa occurred in 1979-80 and has been described in great detail (e.g. wagner and daynes, 1981) . severe outbreaks were also described in various european countries (deerberg et al., 1973; owen et al., 1975; osterhaus et al., 1981) . a more recent outbreak in the usa, which resulted in the eradication of almost 5000 mice in one institution, was described by dick et al. (1996) . the most recent well-documented case of mousepox was published by lipman et al. (2000b) . few additional but unpublished cases of ectromelia have been observed thereafter. in a recent survey conducted in the usa, one population was reported to be seropositive for mousepox (jacoby and lindsey, 1998) . natural infections manifest differently depending on many factors. mousepox may occur as a rapidly spreading outbreak with acute disease and deaths, or may be inconspicuous with slow spreading and mild clinical signs. the mortality rate can be very low in populations in which the virus has been present for long periods. the infection usually takes one of three clinical courses: acute asymptomatic infection, acute lethal infection (systemic form), or subacute to chronic infection (cutaneous form ; fenner, 1981 fenner, , 1982 manning and frisk, 1981; national research council, 1991; dick et al., 1996) . the systemic or visceral form is characterized clinically by facial oedema, conjunctivitis, multisystemic necrosis, and usually high mortality. this form is less contagious than the cutaneous form because the animals die before there is virus shedding. the cutaneous form is characterized by typical dermal lesions and variable mortality. the outcome of infection depends on many factors including strain and dose of virus; route of viral entry; strain, age, and sex of mouse; husbandry methods; and duration of infection in the colony. while all mouse strains seem to be susceptible to infection with ectv, clinical signs and mortality are strain-dependent (fenner, 1982; wallace and buller, 1985, 1986; brownstein et al., 1989b) . acute lethal (systemic) infection occurs in highly susceptible inbred strains such as dba/1, dba/2, balb/c, a, and c3h/hej. immunodeficient mice may also be very susceptible (allen et al., 1981) . outbreaks among susceptible mice can be explosive, with variable morbidity and high mortality (ͼ80%). clinical disease may not be evident in resistant strains such as c57bl/6 and akr, and the virus can be endemic in a population for long periods before being recognized. furthermore, females seem to be more resistant to disease than males, at least in certain strains of mice brownstein et al., 1989b) . the mechanisms determining resistance versus susceptibility are not fully understood but appear to reflect the action of multiple genes. the genetic loci considered to be important include h-2d b (termed rmp-3, resistance to mousepox, on chromosome 17; o'neill et al., 1983) , the c5 genes (rmp-2, on chromosome 2), rmp-1, localized to a region on chromosome 6 encoding the nk cell receptor nkr-p1 alloantigens (brownstein and gras, 1997) , the nitric oxide synthase 2 locus on chromosome 11 (karupiah et al., 1998) , and the signal transducer and activator of transcription 6 locus on chromosome 10 (mahalingam et al., 2001) . clearance of the virus by the immune system is absolutely dependent upon the effector functions of cd8 ϩ t cells while nk cells, cd4 ϩ t cells, and macrophages are necessary for the generation of an optimal response (niemialtowski et al., 1994; delano and brownstein, 1995; karupiah et al., 1996) . mousepox (ectromelia) virus usually enters the host through the skin with local replication and extension to regional lymph nodes (fenner, 1981 (fenner, , 1982 wallace and buller, 1986; national research council, 1991) . it escapes into the blood (primary viraemia) and infects splenic and hepatic macrophages resulting in necrosis of these organs and a massive secondary viraemia. this sequence takes approximately 1 week. many animals die at the end of this stage without premonitory signs of illness; others develop varying clinical signs including ruffled fur, hunched posture, swelling of the face or extremities, conjunctivitis, and skin lesions (papules, erosions, or encrustations mainly on ears, feet, and tail; figure 23 .1). necrotic amputation of limbs and tails can sometimes be seen in mice that survive the acute phase, hence the original name of the disease 'ectromelia' (meaning absent or short limbs; figure 23 .2). common gross lesions of acute mousepox include enlarged lymph nodes, peyer's patches, spleen, and liver; multifocal to semiconfluent white foci of necrosis in the spleen and liver; and haemorrhage into the small intestinal lumen (allen et al., 1981; fenner, 1982; dick et al., 1996; percy and barthold, 2001) . in animals that survive, necrosis and scarring of the spleen can produce a mosaic pattern of white and red-brown areas that is a striking gross finding. the most consistent histological lesions of acute mousepox are necroses of the spleen (figure 23 lymph nodes, peyer's patches, thymus, and liver (allen et al., 1981; fenner, 1982; dick et al., 1996; lipman et al., 2000b; percy and barthold, 2001) . occasionally, necrosis may also be observed in other organs such as ovaries, uterus, vagina, intestine, and lungs. the primary skin lesion, which occurs about a week after exposure at the site of inoculation (frequently on the head), is a localized swelling that enlarges from inflammatory oedema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop 2-3 days later as the result of viraemia (figure 23 .1). they are often multiple and widespread and can be associated with conjunctivitis. the skin lesions also can ulcerate and scab before scarring. mucosal and dermal epithelial cells may have characteristic intracytoplasmic eosinophilic (cowdry type a) inclusion bodies ( figure 23 .4). basophilic (cowdry type b) inclusions may be found in the cytoplasm of all infected cells, especially in hepatocytes. natural transmission of ectv mainly occurs by direct contact and fomites (fenner, 1981; wallace and buller, 1986; national research council, 1991) . the primary route of infection is through skin abrasions. faecal-oral and aerosol routes may also be involved (werner, 1982) . in addition, the common practice of cannibalism by mice may contribute to the oral route of infection (bhatt and jacoby, 1987b) . intrauterine transmission is possible at least under experimental conditions (schwanzer et al., 1975) . virus particles are shed from infected mice (mainly via scabs and/or faeces) for about 3-4 weeks, even though the virus can persist for months in the spleen of an occasional mouse (bhatt and jacoby, 1987b; national research council, 1991) . cage-to-cage transmission of ectv and transmission between rooms or units is usually low and largely depends on husbandry practices (e.g. mixing mice from different cages). importantly, the virus may not be transmitted effectively to sentinel mice exposed to dirty bedding (lipman et al., 2000b) . various tests have been applied for the diagnosis of ectromelia. previous epidemics were difficult to deal with because of limited published data and information on the biology of the virus and the lack of specific and sensitive assays (wallace, 1981) . in the 1950s, diagnosis relied on clinical signs, histopathology, and animal passages of tissues from moribund and dead animals. culture of the virus on the chorioallantoic membrane of embryonated eggs was also applied. serology is currently the primary means of testing mouse colonies for exposure to ectv. the methods of choice are elisa and ifa; they are more sensitive and specific than the previously used haemagglutination inhibition (hi) assay (collins et al., 1981; buller et al., 1983; aclad, 1991) . both tests detect antibodies to orthopoxviruses and do not distinguish between ectv and vaccinia virus. vaccinia virus is commonly used as antigen for serological testing to avoid the risk of infection for mice. thus, false-positive serological reactions may be found after experimental administration of replication-competent vaccinia virus. it has been shown that even cage contact sentinels may develop antibodies, and vaccinia virus leading to seroconversion may even be transmitted by dirty bedding (gaertner et al., 2003) . confirmation of positive serological results is important before action is taken because vaccinia virus is increasingly prevalent in animal facilities as a research tool (e.g. for vaccination or gene therapy). as observed in different outbreaks, serological testing is of little value in the initial stages of the disease. for example, in the outbreak described by dick et al. (1996) depopulation was nearly completed before serological confirmation was possible. for this reason, negative serological results should be confirmed by direct detection methods (pcr, immunohistochemistry, virus isolation) or by histopathology, especially when clinical cases suggestive of mousepox are observed. polymerase chain reaction assays to detect different genes of poxviruses in infected tissues have been described by dick et al. (1996) , neubauer et al. (1997) , and lipman et al. (2000b) . the key to prevention and control of mousepox is early detection of infected mice and contaminated biological materials. all institutions that must introduce mice from other than commercial barrier facilities should have a health surveillance programme and test incoming mice. perhaps even more important than living animals are samples from mice (tumours, sera, tissues). the virus replicates in lymphoma and hybridoma cell lines (buller et al., 1987) , and such cells or material derived from them may therefore be a vehicle for inadvertent transfer between laboratories. the last two published outbreaks of ectromelia were both introduced into the facilities by mouse serum (dick et al., 1996; lipman et al., 2000b) . lipman et al. (2000b) found that the contaminated serum originated from a pooled lot of 43 l that had been imported from china. because mouse serum commonly is sold to the end user in small aliquots (few millilitres), it has to be expected that aliquots of the contaminated lot are still stored in numerous freezers. both cases provide excellent examples of why map or pcr testing should be performed on all biological materials to be inoculated into mice. eradication of mousepox usually has been accomplished by elimination of the affected colonies, disinfection of rooms and equipment, and disposal of all infected tissues and sera. while culling of entire mouse colonies is the safest method for eradication of mousepox, it is not a satisfactory method due to the uniqueness of numerous lines of genetically modified animals housed in many facilities. several studies indicate that mousepox is not highly contagious bhatt and jacoby 1987a,b ) and that it may be self-limiting when adequate husbandry methods are applied. therefore, strict quarantine procedures along with cessation of breeding (to permit resolution of infection) and frequent monitoring with removal of clinically sick and seropositive animals are a potential alternative. the period from the last births before the break until the first matings after the break should be at least 6 weeks (bhatt and jacoby, 1987b) . sequential testing of immunocompetent contact sentinels for seroconversion should be employed with this option. in the past, immunization with live vaccinia virus was used to suppress clinical expression of mousepox. vaccination may substantially reduce the mortality rate, but it does not prevent virus transmission or eradicate the agent from a population bhatt and jacoby, 1987c) . after vaccination, typical pocks develop at the vaccination site, and infectious vaccinia virus is detectable in spleen, liver, lungs, and thymus (jacoby et al., 1983) . vaccination also causes seroconversion so that serological tests are not applicable for health surveillance in vaccinated populations. it is therefore more prudent to control mousepox by quarantine and serological surveillance than by relying on vaccination. mortality and clinical disease are the major factors by which ectv interferes with research. severe disruption of research can also occur when drastic measures are taken to control the infection. the loss of time, animals, and financial resources can be substantial. murine adenoviruses (madv) are non-enveloped, double-stranded dna viruses of the family adenoviridae, genus mastadenovirus. two distinct strains have been isolated from mice. the fl strain (madv-1) was first isolated in the usa as a contaminant of a friend leukaemia (hartley and rowe, 1960) ; the k87 strain (madv-2) was first isolated in japan from the faeces of a healthy mouse (hashimoto et al., 1966) . both strains are now considered to represent different species (hamelin and lussier, 1988; jacques et al., 1994a,b) . in laboratory mice, seropositivity to adenoviruses was reported in 2% of spf colonies and in 8% of non-spf colonies in the usa (jacoby and lindsey, 1998) . antibodies were also detected at a low prevalence rate in french colonies (zenner and regnault, 2000) , but the virus strain used as antigen is not mentioned. a similar range of positive samples was reported by livingston and riley (2003) . antibodies to madv were also found in wild mice and in rats (otten and tennant, 1982; smith et al., 1986) . both viruses are not known to cause clinical disease in naturally infected, immunocompetent mice. however, madv-1 can cause a fatal systemic disease in suckling mice after experimental inoculation (hartley and rowe, 1960; heck et al., 1972; wigand, 1980) . disease is characterized by scruffiness, lethargy, stunted growth, and often death within 10 days. experimental infection of adult mice with madv-1 is most often subclinical and persistent (richter, 1986 ) but can cause fatal haemorrhagic encephalomyelitis with neurological symptoms, including tremors, seizures, ataxia, and paralysis, in susceptible c57bl/6 and dba/2j mice (guida et al., 1995) . balb/c mice are relatively resistant to this condition. athymic foxn1 nu mice experimentally infected with madv-1 develop a lethal wasting disease (winters and brown, 1980) . similarly, prkdc scid mice succumb to experimental infection with madv-1 (pirofski et al., 1991) . gross lesions in response to natural madv infections are not detectable. occasional lesions observed after experimental infection with madv-1 include small surface haemorrhages in the brain and spinal cord of c57bl/6 and dba/2j mice (guida et al., 1995) , duodenal haemorrhage in foxn1 nu mice (winters and brown, 1980) , and pale yellow livers in prkdc scid mice (pirofski et al., 1991) . histologically, experimental madv-1 infection of suckling mice is characterized by multifocal necrosis and large basophilic intranuclear inclusion bodies in liver, adrenal gland, heart, kidney, salivary glands, spleen, brain, pancreas, and brown fat (heck et al., 1972; margolis et al., 1974; national research council, 1991; percy and barthold, 2001) . in experimentally induced haemorrhagic encephalomyelitis, multifocal petechial haemorrhages occur throughout the brain and spinal cord, predominantly in the white matter, and are attributed to infection and damage to the vascular epithelium of the central nervous system (cns; guida et al., 1995) . histopathological manifestations in madv-1-infected prkdc scid mice are marked by microvesicular fatty degeneration of hepatocytes (pirofski et al., 1991) . in contrast to madv-1, the tissue tropism of madv-2 is limited to the intestinal epithelium. naturally or experimentally infected mice develop intranuclear inclusions in enterocytes, especially in the ileum and caecum (takeuchi and hashimoto, 1976; otten and tennant, 1982; national research council, 1991; percy and barthold, 2001) . transmission of madv primarily occurs by ingestion. madv-1 is excreted in the urine and may be shed for up to 2 years (van der veen and mes, 1973). murine adenovirus-2 infects the intestinal tract and is shed in faeces for only a few weeks in immunocompetent mice (hashimoto et al., 1970) ; immunodeficient mice may shed the virus for longer periods (umehara et al., 1984) . murine adenovirus infections are routinely diagnosed by serological tests. however, there is a one-sided cross reactivity of madv-1 with madv-2 (wigand et al., 1977) . serum from mice experimentally infected with madv-1 yielded positive reactions in serological tests with both viruses while serum from mice infected with madv-2 reacted only with the homologous antigen . smith et al. (1986) reported that sera may react with madv-1 or madv-2 or both antigens. occasional reports of mice with lesions suggestive of adenovirus infections and negative serology (with madv-1) indicate that the infection may not be detected if only one virus is used as antigen (luethans and wagner, 1983) . it has therefore become standard practice to test sera for antibodies to both madv-1 and madv-2. the common methods are ifa and elisa, and both are more sensitive than the previously used complement fixation test. the low prevalence in colonies of laboratory mice indicate that madv can easily be eliminated (e.g. by hysterectomy derivation or embryo transfer) and that barrier maintenance has been very effective in preventing infection. the low pathogenicity and the low prevalence in contemporary mouse populations are the main reasons why adenoviruses are considered to be of little importance. however, immunodeficient mice are increasingly used and candidates for natural infections and wasting disease (richter, 1986) , and the viruses might easily be spread by the exchange of genetically modified mice and therefore re-emerge. only few influences on research attributable to madv have been published. for example, it has been shown that madv-1 significantly aggravates the clinical course of scrapie disease in mice (ehresmann and hogan, 1986) . natural infections with madv could also interfere with studies using adenovirus as a gene vector. polyomaviridae are enveloped, double-stranded dna viruses. two different agents of this family exclusively infect mice (mus musculus), and both belong to the genus polyomavirus. murine pneumotropic virus (mptv) has formerly been known as 'newborn mouse pneumonitis virus' or 'k virus' (named after l. kilham who first described the virus). the second is murine polyomavirus (mpyv). both are related but antigenically distinct from each other (bond et al., 1978) . they are enzootic in many populations of wild mice but are very uncommon in laboratory mice. even older reports indicate that both have been eradicated from the vast majority of contemporary mouse colonies, and their importance is negligible (national research council, 1991) . seropositivity to these viruses was not reported in a survey conducted in the usa (jacoby and lindsey, 1998) . in a retrospective study in french facilities, antibodies to mpyv were found in 1 of 69 colonies, and all samples tested for mptv were negative (zenner and regnault, 2000) . comparable data were reported by livingston and riley (2003) . due to their low prevalence, both viruses are not included in the list of agents for which testing is recommended on a regular basis by felasa (nicklas et al., 2002) . although polyomavirus genes, especially those of sv40 are used widely in gene constructs for insertional mutagenesis, very few reports have been published on spontaneous or experimental disease due to mpyv or mptv in the last 10-15 years. the reader is therefore referred to previous review articles for details (eddy 1982; parker and richter, 1982; richter, 1986; shah and christian, 1986; national research council, 1991; orcutt, 1994; porterfield and richter, 1994) . natural infections with mptv are subclinical. the prevalence of infection is usually low in an infected population. the virus may persist in infected animals for months and perhaps for life depending on the age at infection and is reactivated under conditions of immunosuppression. virus replicates primarily in endothelial cells, but renal tubular epithelial cells are the major site of viral persistence (greenlee et al., 1991 (greenlee et al., , 1994 . clinical signs are observed only after infection of infant mice less than 6-8 days of age. infected pups suddenly develop respiratory symptoms after an incubation period of approximately 1 week, and many die within a few hours of onset of symptoms with an interstitial pneumonia caused by productive infection of and damage to pulmonary endothelium. endothelial cells in other organs are involved in virus replication also (ikeda et al., 1988; greenlee et al., 1994) . in older suckling mice, mptv produces a more protracted infection, and the virus or viral antigen can be detected for as long as 4 months. in adult animals, the virus produces a transient asymptomatic infection. even in immunodeficient foxn1 nu mice, experimental infection of adults is clinically asymptomatic although virus is detectable for a period of several months (greenlee, 1986) . in vitro cultivation of mptv is difficult. no susceptible permanent cell line is known to support growth. it can be cultured in primary mouse embryonic cells, but viral titres are not sufficient for use in serological assays (greenlee and dodd, 1987) . for this reason, the hi test using homogenates of livers and lungs of infected newborn mice is still frequently used, but ifa and elisa tests are also available (groen et al., 1989) . furthermore, a pcr test for demonstration of mptv in biological samples has been published (carty et al., 2001) . murine polyomavirus was first detected as a contaminant of murine leukaemia virus (mulv) when sarcomas developed in mice after experimental inoculation of contaminated samples. it has later been shown to be a frequent contaminant of transplantable tumours (collins and parker, 1972) . natural infection of mice is subclinical, and gross lesions including tumours are usually not found. tumour formation occurs if mice are experimentally infected at a young age or if they are inoculated with high virus doses. development of tumours may be preceded by multifocal necrosis and mortality during the viraemic stage (percy and barthold, 2001) . parotid, salivary gland, and mammary tumours are common, and sarcomas or carcinomas of kidney, subcutis, adrenal glands, bone, cartilage, teeth, blood vessels, and thyroid occur also. virus strains vary with regard to the tumour types or lesions that they induce, and mouse strains vary in their susceptibility to different tumour types. those of c57bl and c57br/cd lineage are considered to be the most resistant strains; athymic foxn1 nu mice are considered to be most susceptible; c3h mice are particularly susceptible to adrenal tumours and a mice tend to develop bone tumours. immunosuppression or inoculation into immunodeficient strains (e.g. foxn1 nu ) also support the growth of tumours. on the other hand, experimental infection of adult immunocompetent mice does not result in tumour formation because the immune response suppresses tumour growth, and newborn immunocompetent mice develop runting only if inoculated with high virus doses (atencio et al., 1995) . after experimental intranasal infection, mpyv initially infects the respiratory tract followed by a systemic phase in which liver, spleen, kidney, and the colon become infected (dubensky et al., 1984) . the virus is shed in faeces and in all body fluids, and transmission occurs rapidly by direct contact between animals, but also between cages in a room. further, intrauterine transmission has been documented after experimental infection (mccance and mims, 1977) . murine polyomavirus persists in all organs in prkdc scid mice while viral dna is detectable in immunocompetent mice after experimental infection for only a limited period of about 4 weeks (berke et al., 1994) . however, virus may persist and can be reactivated by prolonged immunosuppression (rubino and walker, 1988) or during pregnancy, at least in young mice (mccance and mims, 1979) . biological materials of mouse origin are likely to be the most common source of contamination of laboratory mice emphasizing the importance of map or pcr screening of biological materials to be inoculated into mice. the most frequently used tests for health surveillance of mouse colonies are elisa and ifa (aclad, 1991) ; in addition, the hi test is still used. latent infections can be detected by intracerebral inoculation of neonate mice or by map testing, but direct demonstration of virus in biological samples is also possible by pcr testing (porterfield and richter, 1994; carty et al., 2001) . parvoviruses are non-enveloped small viruses (approximately 20 nm in diameter) with a single-stranded dna genome of approximately 5000 nucleotides. murine parvoviruses are members of the family parvoviridae, genus parvovirus. they are remarkably resistant to environmental conditions like heat, desiccation, acidic and basic ph-values. two distinct serotypes infect laboratory mice: the mice minute virus (mmv) and the mouse parvovirus 1 (mpv). nonstructural proteins (ns-1 and ns-2) are highly conserved among both viruses whereas the capsid proteins (vp-1, vp-2, vp-3) are more divergent and determine the serogroup (ball-goodrich and johnson, 1994) . both viruses require mitotically active cells for replication. severe infections are therefore not found in mature animals due to the lack of a sufficient number of susceptible cells in tissues. general aspects of rodent parvovirus infections and their potential effects on research results have been reviewed (tattersall and cotmore, 1986; national research council, 1991; jacoby et al., 1996) . already in the mid-1980s mouse colonies were identified that gave positive reactions for mmv by ifa but not by hi tests. it was subsequently shown that these colonies were infected with a novel parvovirus, initially referred to as 'mouse orphan parvovirus'. the first isolate of mpv was detected as a contaminant of cultivated t-cell clones interfering with in vitro immune responses (mckisic et al., 1993) and was named 'mouse parvovirus'. it does not replicate well in currently available cell cultures, and sufficient quantities of virus for serological tests are difficult to generate. hitherto, only very few isolates of mpv have been cultured and characterized on a molecular basis (ball-goodrich and johnson, 1994; besselsen et al., 1996) . at present, mpv is among the most common viruses in colonies of laboratory mice. the prevalence of sera positive for parvoviruses was nearly 10% in a study from livingston et al. (2002) , with the majority of sera being positive for mpv. this is consistent with a recent survey conducted in the usa showing that almost 40% of non-spf colonies were seropositive (jacoby and lindsey, 1998) . similar results were obtained for genetically modified mice in japan (yamamoto et al., 2001) , in contrast to earlier studies indicating that the infection was rare in japan (ueno et al., 1998) . clinical disease and gross or histological lesions have not been reported for mice naturally or experimentally infected with mpv. infections are subclinical even in newborn and immunocompromised animals . in contrast to many other viruses infecting mice, viral replication and excretion is not terminated by the onset of host immunity. tissue necrosis has not been observed at any stage of infection in infected infant or adult mice . humoral immunity to mpv does not protect against mmv infections and vice versa (hansen et al., 1999) . serological surveys have indicated that mpv naturally infects only mice. differences in mouse strain susceptibility to clinical mpv infection do not exist. however, seroconversion seems to be strain-dependent. after experimental infection, seroconversion occurred in all c3h/hen mice, fewer balb/c, dba/2, and icr mice, and seroconversion could not be detected in c57bl/6 mice (besselsen et al., 2000) . diagnosis of mpv infection by pcr testing of small intestine and mesenteric lymph nodes also depended on the mouse strain. mpv dna was detected in all mouse strains evaluated except dba/2 even though seroconversion was detected in these mice. after oral infection, the intestine is the primary site of viral entry and replication. the virus spreads to the mesenteric lymph nodes and other lymphoid tissues, where it persists for more than 2 months , and seems to be excreted via the intestinal and the urinary tract. after experimental inoculation of weanling mice, mpv is transmitted to cagemates by direct contact for 2-4 weeks , and transmission by dirty bedding is also possible. these results implicate a role for urinary, faecal, and perhaps respiratory excretion of virus. another study showed that naturally infected mice may not transmit the virus under similar experimental conditions (shek et al., 1998) . serology is a useful tool to identify mpv infections in immunocompetent hosts, but reaching a diagnosis based on serological assays may be difficult and requires a good knowledge of the available techniques. neither the virion elisa nor hi are practical screening tests for mpv because they require large quantities of purified mpv which is difficult to obtain. diagnosis of mpv infections has long been made on the basis of an mmv hi-negative result coupled with an mmv ifapositive result. a generic rodent parvovirus elisa using a recombinant ns-1 protein as antigen has been developed , but mpv ifa and mpv hi assays are more sensitive techniques than the ns-1 elisa and the mmv ifa (besselsen et al., 2000) . recently, elisa tests have been described that use recombinant vp-2 and provide sensitive and serogroupspecific assays for the diagnosis of mpv infections in mice (ball-goodrich et al., 2002; livingston et al., 2002) . in immunodeficient mice that do not generate a humoral immune response, pcr assays can be used to detect mpv (besselsen et al., 1995; redig and besselsen, 2001 ) and other parvoviruses. mpv has been shown to persist for at least 9 weeks in the mesenteric lymph nodes . this tissue is considered the best suited for pcr analysis, but spleen and small intestine can also be used with good success (besselsen et al., 2000) . the virus persists sufficiently long in mesenteric lymph nodes so that pcr assays may also be used as a primary screening tool for laboratories that do not have access to specific mpv antigenbased serological assays. polymerase chain reaction is further a good confirmatory method for serological assays and has also been described for the detection of parvoviruses in cell lines and tumours (yagami et al., 1995) . in addition, the map test has been reported as a sensitive tool to detect mpv (shek et al., 1998) . given the high environmental stability of the virus and the potential fomite transmission together with the long virus persistence in infected animals, spontaneous disappearance from a mouse population (e.g. by cessation of breeding) is very unlikely. eradication of infection is possible by elimination of infected animals and subsequent replacement with uninfected mice, and the agent can be eliminated from breeding populations only by embryo transfer or by hysterectomy. although there are few published reports of confounding effects of mpv on research, it is lymphocytotropic and may perturb immune responses in vitro and in vivo. infections with mpv have been shown to influence rejection of skin and tumour grafts (mckisic et al., , 1998 . mice minute virus is the type species of the genus parvovirus. the virus was formerly called 'minute virus of mice' (mvm) and was renamed recently (international union of microbiological societies, 2000) . it was originally isolated by crawford (1966) from a stock of mouse adenovirus, and this prototype isolate was later designated mvmp. its allotropic variant was detected as a contaminant of a transplantable mouse lymphoma (bonnard et al., 1976) and designated mvmi because it exhibits immunosuppressive properties in vitro. both variants have distinct cell tropisms in vivo and in vitro. the mmvp infects fibroblast cell lines and does not cause clinical disease (kimsey et al., 1986 , brownstein et al., 1991 . the mmvi grows lytically in t cells and inhibits various functions mediated by these cells in vitro. both strains are apathogenic for adult mice, but the immunosuppressive variant is more pathogenic for neonatal mice than is mmvp. serological surveys show that the mouse is the primary natural host (parker et al., 1970; smith et al., 1993b; singleton et al., 2000) , but the virus is also infective for rats, hamsters (garant et al., 1980; ward and tattersall, 1982) , and mastomys (haag et al., 2000) during foetal development or after parenteral inoculation. natural infections are usually asymptomatic in adults and infants, and the most common sign of infection is seroconversion. kilham and margolis (1970) observed mild growth retardation a few days after experimental infection of neonatal mice with mmvp. studies of transplacental infection yielded no pathological findings in mice (kilham and margolis, 1971 ). the immunosuppressive variant but not the prototype strain is able to produce a runting syndrome after experimental infection of newborn mice (kimsey et al., 1986) . depending on the host genotype, experimental infections of foetal and neonatal mice with mmvi produce various clinical presentations and lesions. infection in c57bl/6 mice is asymptomatic, but the virus causes lethal infections with intestinal haemorrhage in dba/2 mice. infection of strains such as balb/c, cba, c3h/he, and sjl is also lethal and mice have renal papillary haemorrhage (brownstein et al., 1991) . the mmvi also infects haematopoietic stem cells and mediates an acute myelosuppression (segovia et al., 1991 (segovia et al., , 1995 . due to their dependency on mitotically active tissues, the foetus is at particular risk for damage by parvoviruses. mice minute virus and other parvoviruses may have severe teratogenic effects and cause foetal and neonatal abnormalities by destroying rapidly dividing cell populations, often resulting in foetal death. adult prkdc scid mice develop an acute leukopenia 1 month after experimental infection with mmvi and die within 3 months. the virus persists lifelong in the bone marrow of these mice (segovia et al., 1999) . mice minute virus is shed in faeces and urine. contaminated food and bedding are important factors in viral transmission because the virus is very resistant to environmental conditions. direct contact is also important and the virus does not easily spread between cages. routine health surveillance is usually conducted by serological methods. unlike mpv, mmv can easily be cultured in cell lines so that antigen production for hi and elisa (using whole purified virions) is easy. haemagglutination inhibition is a highly specific diagnostic test whereas ifa always exhibits some degree of cross reactivity with mpv and other closely related parvoviruses. enzyme-linked immunosorbent assay is probably the most frequently used test, but depending on the purity of the antigen preparation, cross reactions with mpv may occur due to contamination with nonstructural proteins that are common to both viruses. this problem can be avoided by the use of recombinant vp-2 antigen (livingston et al., 2002) . viral detection is also possible by pcr in biological materials and in organs (intestines, kidney, spleen) from infected animals (yagami et al., 1995; chang et al., 1997; redig and besselsen, 2001) . in contrast to mpv, pcr is not appropriate as a confirmatory method for serology because mmv has not been shown to persist in immunocompetent animals for sufficiently long periods. the virus can be eliminated from infected breeding populations by caesarean derivation or by embryo transfer. in experimental colonies, elimination of infected animals and subsequent replacement with uninfected mice is practical if careful environmental sanitation is conducted by appropriate disinfection procedures. it is important that reintroduction is avoided by exclusion of wild mice and by strict separation from other infected populations and potentially contaminated materials in the same facility. admission of biological materials must be restricted to samples that have been tested and found free from viral contamination. both allotropic variants of mmv have been used as models for molecular virology, and their small size and simple structure have facilitated examination of their molecular biology and expedited understanding of cell tropism, viral genetics, and structure. the significance for laboratory mouse populations was considered low or uncertain because natural infections are inapparent. however, various effects on mouse-based research have been published (tattersall and cotmore, 1986; jacoby et al., 1996; baker, 1998; nicklas et al., 1999) . due to their predilection for replicating in mitotically active cells, they are frequently associated with tumour cells and have a marked oncosuppressive effect (rommelaere and cornelis, 1991) . special attention is also necessary for immunological research and other studies involving rapidly dividing cells (embryology, teratology). in addition, mmv is a common contaminant of transplantable tumours, murine leukaemias, and other cell lines (collins and parker, 1972; nicklas et al., 1993; garnick, 1996) . lactate dehydrogenase-elevating virus (ldv) is a single-stranded rna virus of the genus arterivirus belonging to the family arteriviridae. lactate dehydrogenase-elevating virus has repeatedly been detected in feral mice (mus musculus), which are considered to be a virus reservoir (rowson and mahy, 1975; li et al., 2000) . only mice and primary mouse cells are susceptible to infection with ldv. after infection, virus titres of 10 10 -10 11 particles per ml serum are found within 12-14 h after infection. the virus titre drops to 10 5 particles per ml within 2-3 weeks and remains constant at this level for life. lactate dehydrogenase-elevating virus replicates in a subpopulation of macrophages in almost all tissues and persists in lymph nodes, spleen, liver, and testes tissues (anderson et al., 1995a) . the virus can be stored in undiluted mouse plasma at ϫ70њc without loss of infectivity, but it is not stable at room temperature and is very sensitive to environmental conditions. lactate dehydrogenase-elevating virus was first detected during a study of methods that could be used in the early diagnosis of tumours (riley et al., 1960) . it produces a persistent infection with continuous virus production and a lifelong viraemia despite ldv-specific immune reactions of the host ( van den broek et al., 1997) . lactate dehydrogenase-elevating virus has been found in numerous biological materials that are serially passaged in mice such as transplantable tumours including human tumours (nicklas et al., 1993; ohnishi et al., 1995) , monoclonal antibodies or ascitic fluids (nicklas et al., 1988) , or infectious agents (e.g. haemoprotozoans, k virus, clostridium piliforme). these materials are contaminated after passage in an infected and viraemic animal. contamination with ldv leads to the infection of each sequential host and to transmission of the virus by the next passage and remains associated with the specimen. it is therefore the most frequently detected contaminant in biological materials (collins and parker, 1972; nicklas et al., 1993) . infection with ldv is usually asymptomatic, and there are no gross lesions in immunocompetent as well as in immunodeficient mice. the only exception is polyomyelitis with flaccid paralysis of hind limbs developing in c58 and akr mice when they are immunosuppressed either naturally with aging or experimentally (anderson et al., 1995b; monteyne et al., 1997) . it has been shown that only mice harbouring cells in the cns that express a specific endogenous mulv are susceptible to poliomyelitis (anderson et al., 1995c) . the characteristic feature of ldv infection is the increased activity of lactate dehydrogenase (ldh) and other plasma enzymes (brinton, 1986; national research council, 1991) , which is due to the continuous destruction of permissive macrophages that are responsible for the clearance of ldh from the circulation. as a consequence, the activity of plasma ldh begins to rise by only 24 h after infection and peaks 3-4 days after infection at 5-10-fold normal levels, or even be up to 20-fold in sjl/j mice. the enzyme activity declines during the next 2 weeks but remains elevated throughout life. antigen-antibody complexes produced during infection circulate in the blood and are deposited in the glomeruli (brinton, 1986; national research council, 1991) . in contrast to other persistent virus infections (e.g. lymphocytic choriomeningitis virus lcmv), these complexes do not lead to immune complex disease and produce only a very mild glomerulopathy. the only gross finding associated with ldv infection is mild splenomegaly. microscopically, necrosis of lymphoid tissues is visible during the first days of infection. in mouse strains that are susceptible to poliomyelitis, ldv induces lesions in the grey matter of the spinal cord and the brain stem (brinton, 1986) . lactate dehydrogenase-elevating virus is not easily transmitted between mice, even in animals housed in the same cage. fighting and cannibalism increase transmission between cage mates most likely via blood and saliva. infected females transmit the virus to their foetuses if they have been infected few days prior to birth and before igg anti-ldv antibodies are produced, but developmental and immunological factors (e.g. gestational age, timing of maternal infection with ldv, placental barrier) are important in the regulation of transplacental ldv infection (haven et al., 1996; zitterkopf et al., 2002) . maternal immunity protects foetuses from intrauterine infection. immunodeficient prkdc scid mice transmit virus to their offspring also during chronic infection (broen et al., 1992 ). an important means of transmission is provided by experimental procedures such as mouse-to-mouse passage of contaminated biological materials or the use of the same needle for sequential inoculation of multiple mice. in principal, serological methods such as ifa may be used for detecting ldv infection (hayashi et al., 1992) but they are not of practical importance. circulating virus-antibody complexes interfere with serological tests (aclad, 1991) , and sufficient quantities of virus for serological tests are difficult to generate because ldv replicates only in specific subpopulations of primary cultures of murine macrophages and monocytes for one cell cycle (brinton, 1986) . therefore, diagnosis of ldv infection is primarily based on increased ldh activity in serum or plasma of mice. lactate dehydrogenase-elevating virus activity in serum or plasma can be measured directly, or samples (e.g. plasma, cell or organ homogenates) are inoculated into pathogen-free mice and the increase in ldh activity within 3-4 days is measured. an 8-10-fold increase is indicative of ldv infection. detection of infectivity of a plasma sample by the induction of increased ldh activity in the recipient animal is the most reliable means of identifying an infected animal. however, it is important to use clear nonhaemolysed samples because haemolysis will (falsely) elevate activities of multiple serum or plasma enzymes, including ldh. while this assay may be included in a commercial 'map test', it does not involve antibody detection. persistent infection makes ldv an ideal candidate for pcr detection in plasma or in organ homogenates (van der logt et al., 1994; chen and plagemann, 1997) . however, reports exist that pcr may produce false-negative results and should be used cautiously (lipman et al., 2000a) . similarly important as detecting ldv in animals is its detection in biological materials. this may be done by assay for increased ldh activity after inoculation of suspect material into pathogen-free mice (collins and parker, 1972; nicklas et al., 1993) or by pcr (goto et al., 1998; bootz and sieber, 2002) . lactate dehydrogenase-elevating virus spreads slowly in a population because direct contact is necessary. therefore ldv-negative breeding populations can easily be established by selecting animals with normal plasma ldh activity. embryo transfer and hysterectomy derivation are also efficient. the presence of ldv in experimental populations is indicative of contaminated biological materials. in such cases, it is essential that the virus is also eliminated from these samples. this is easily achieved by maintenance of cells by in vitro culture instead of by animal-to-animal passages (plagemann and swim, 1966) . due to the extreme host specificity of the virus, contaminated tumour samples can also be sanitized by passages in nude rats or other animal species. lactate dehydrogenase-elevating virus is a potential confounder of any research using biological materials that are passaged in mice. once present in an animal, the virus persists lifelong. the most obvious signs are increased levels of plasma ldh and several other enzymes. lactate dehydrogenase-elevating virus may also exhibit numerous effects on the immune system (thymus involution, depression of cellular immunity, enhanced or diminished humoral responses, nk cell activation, development of autoimmunity, and suppression of development of diabetes in nod mice; cafruny and hovinen, 1988; nicklas et al., 1988; takei et al., 1992; markine-goriaynoff et al., 2002; gomez et al., 2003) and enhance or suppress tumour growth (brinton, 1982; baker, 1998; nicklas et al., 1999) . lymphocytic choriomeningitis virus is an enveloped, segmented single-stranded rna virus of the genus arenavirus, family arenaviridae. its name refers to the condition that results from experimental intracerebral inoculation of the virus into adult mice and is not considered to be a feature of natural infections. mice (mus musculus) serve as the natural virus reservoir (salazar-bravo et al., 2002) , but syrian hamsters are also important hosts (ackermann, 1977) . additional species such as rabbits, guinea pigs, squirrels, monkeys, and humans are susceptible to natural or experimental infection. infection in hamsters is considered to be asymptomatic (national research council, 1991) . natural infection of callitrichid primates (marmosets and tamarins) leads to a progressive hepatic disease that is known as 'callitrichid hepatitis' (montali et al., 1995; asper et al., 2001; lukashevich et al., 2003) . antibodies to lcmv have been found in wild mice in europe (ackermann et al., 1964) , africa (el karamany and imam, 1991), asia (morita et al., 1991 (morita et al., , 1996 , australia , and america (childs et al., 1992) . thus, it is the only arenavirus with worldwide distribution. infection with lcmv is rarely found in laboratory mice (smith et al., 1984) . seropositivity to lcmv was reported in approximately 5% of non-spf mouse colonies in the usa in 1996 (jacoby and lindsey, 1998) and in 4% of french colonies in 1996-97 (zenner and regnault, 2000) . recent studies confirm that only a small percentage of mice tested are positive for lcmv (livingston and riley, 2003) . in addition to laboratory mice and other vertebrate hosts, the virus has frequently been found in transplantable tumours and tissue culture cell lines from mice and hamsters (bhatt et al., 1986a; nicklas et al., 1993) . despite the low prevalence in laboratory mice, seropositivity to this zoonotic agent should raise serious concern for human health. lymphocytic choriomeningitis virus is frequently transmitted to humans from wild mice (childs et al., 1991) and is also endemic to a varying degree in the human population (childs et al., 1991; marrie and saron, 1998; lledo et al., 2003) due to contact with wild mice. lymphocytic choriomeningitis virus is further transmitted to humans by domestic syrian hamsters rousseau et al., 1997) . in addition, infected laboratory mice (dykewicz et al., 1992) and contaminated biological materials are important sources of infections for humans, and several outbreaks of lcm among laboratory personnel have been traced to transplantable tumours biggar et al., 1977; mahy et al., 1991) . in mice, clinical signs of lcmv infection vary with strain and age of mouse, strain and dose of virus, and route of inoculation (lehmann-grube, 1982; national research council, 1991) . two forms of natural lcmv infection are generally recognized: a persistent tolerant and an (acute) nontolerant form. the persistent form results from infection of mice that are immunotolerant. this is the case if mice are infected in utero or during the first days after birth. this form is characterized by lifelong viraemia and shedding. mice may show growth retardation, especially during the first 3-4 weeks, but appear otherwise normal. infectious virus is bound to specific antibodies and complement, and these complexes accumulate in the renal glomeruli, the choroid plexus, and sometimes also in synovial membranes and blood vessel walls. at 7-10 months of age, immune complex nephritis develops with ruffled fur, hunched posture, ascites, and occasional deaths. this immunopathological phenomenon is called 'late onset disease' or 'chronic immune complex disease'. the incidence of this type of disease varies between mouse strains. gross lesions include enlarged spleen and lymph nodes due to lymphoid hyperplasia. kidneys affected with glomerulonephritis may be enlarged with a granular surface texture or may be shrunken in later stages of the disease process. microscopically, there is generalized lymphoid hyperplasia and immune complex deposition in glomeruli and vessel walls, resulting in glomerulonephritis and plasmacytic, lymphocytic perivascular cuffs in all visceral organs (percy and barthold, 2001) . the nontolerant acute form occurs when infection is acquired after the development of immunocompetence (in mice older than 1 week). these animals become viraemic but do not shed virus and may die within a few days or weeks. natural infections of adults are usually asymptomatic. surviving mice are seropositive and in most cases clear the virus to below detection levels of conventional methods. however, virus may persist at low levels in tissues (particularly spleen, lung, and kidney) of mice for at least 12 weeks after infection as determined by sensitive assays such as nested reverse transcriptasepolymerase chain reaction (rt-pcr) or immunohistochemisty (ciurea et al., 1999) . such nonlethal infection leads to protection against otherwise lethal intracerebral challenge. protection from lethal challenge is also achieved by maternally derived anti-lcmv antibodies through nursing or by the administration of anti-ldv monoclonal igg2a antibodies (baldridge and buchmeier, 1992) . in experimentally infected animals, the route of inoculation (subcutaneous, intraperitoneal, intravenous, intracerebral) also influences the type and degree of disease (lehmann-grube, 1982; national research council, 1991) . intracerebral inoculation of adult immunocompetent mice typically results in tremors, convulsions, and death due to meningoencephalitis and hepatitis. neurological signs usually appear on day 6 postinoculation, and animals die within 1-3 days after the onset of symptoms or recover within several days. the classic histological picture is of dense perivascular accumulations of lymphocytes and plasma cells in meninges and choroid plexus. while infection following subcutaneous inoculation usually remains inapparent, reaction of mice to intraperitoneal or intravenous inoculation depends on the virus strain and on the mouse strain. infection by these routes primarily causes multifocal hepatic necrosis and necrosis of lymphoid cells. athymic foxn1 nu mice and other immunodeficient mice do not develop disease but become persistently viraemic and shed virus. as a general rule, all pathological alterations following lcmv infection are immune-mediated; and mice can be protected from lcmv-induced disease by immunosuppression (gossmann et al., 1995) . lymphocytic choriomeningitis virus disease is a prototype for virus-induced t-lymphocyte-mediated immune injury and for immune complex disease. for detailed information on the pathogenesis of lcmv infection, the reader is referred to a recent review article by oldstone (2002) . extensive information on the clinical and pathological features of lcmv infection in mice has been assembled by lehmann-grube (1982) . in nature, carrier mice with persistent infection serve as the principal source of virus. intrauterine transmission is very efficient, and with few exceptions all pups born from carrier mice are infected. furthermore, persistently infected mice and hamsters can shed large numbers of infectious virions primarily in urine, but also in saliva and milk. the virus can replicate in the gastric mucosa after intragastric infection (rai et al., 1996 (rai et al., , 1997 . gastric inoculation elicits antibody responses of comparable magnitudes as intravenous inoculation and leads to active infection with lcmv indicating that oral infection is possible, e.g., by ingestion of contaminated food or cannibalism. a self-limiting infection frequently results from infection of adult mice. the virus does not spread rapidly after introduction in populations of adult mice, and the infectious chain usually ends. however, if the virus infects a pregnant dam or a newborn mouse, a lifelong infection results, and soon a whole breeding colony of mice may become infected if the mice live in close proximity (which is the case under laboratory conditions). lymphocytic choriomeningitis virus is most commonly diagnosed by serological methods. methods of choice are ifa and elisa, which have replaced the relatively insensitive complement fixation test. it is important that bleeding of mice is done carefully because of a potential risk due to viraemic animals. historically, direct viral detection was performed by inoculating body fluids or tissue homogenates into the brain of lcmv-free mice or by subcutaneous injection into mice and subsequent serological testing (map test). more recently, pcr assays have been developed for the direct detection of viral rna in clinical samples or animals (park et al., 1997 . both map test and pcr can also be used to detect contamination of biological materials (bootz and sieber, 2002) . vertical transmission of lcmv by transuterine infection is efficient so this virus cannot reliably be eliminated by caesarean rederivation. caesarean derivation may be effective if dams acquired infection after the development of immunocompetence (nontolerant acute infection) and subsequently eliminated the virus, but such a strategy is difficult to justify in light of lcmv's zoonotic potential. in breeding colonies of great value, virus elimination might be possible soon after introduction into the colony by selecting nonviraemic breeders. this procedure is expensive and time consuming and requires special safety precautions. fortunately, infections of laboratory mice with lcmv are very uncommon. however, once lcmv has been detected in animals or in biological materials, immediate destruction of all contaminated animals and materials is advisable to avoid risk of human infection. foxn1 nu and prkdc scid mice may pose a special risk because infections are silent and chronic (mahy et al., 1991) . cages and equipment should be autoclaved, and animal rooms should be fumigated with disinfectants such as formaldehyde, vaporized paraformaldehyde, and hydrogen peroxide. appropriate precautions are necessary for experiments involving lcmv, or lcmv-infected animals or materials. biological safety level (bsl) 2 will be considered to be sufficient in most cases. biological safety level 3 practices may be considered when working with infected animals owing to the increased risk of virus transmission by bite wounds, scratching, or aerosol formation from the bedding. animal biosafety level (absl) 3 practices and facilities are generally recommended for work with infected hamsters. appropriate precautions have been defined for different bsls or animal biology safety levels by cdc (1999) . lymphocytic choriomeningitis virus is an important zoonotic agent. it has been transmitted to humans working with infected animals or with contaminated biological materials and can cause mild to serious or fatal disease in humans (dykewicz et al., 1992; barton et al., 1995; barton and hyndman, 2000) . congenital infection in humans may result in hydrocephalus, or foetal or neonatal death (barton et al., 2002) . lymphocytic choriomeningitis virus is also frequently utilized as a model organism to study virus-host interactions, immunological tolerance, virus-induced immune complex disease, and a number of immunological mechanisms in vivo and in vitro (slifka, 2002; zinkernagel, 2002) . accidental transmission may have a severe impact on various kinds of experiments (for details, see lehmann-grube, 1982; bhatt et al., 1986b; national research council, 1991; baker, 1998; nicklas et al., 1999) . mammalian orthoreoviruses (mrv) are nonenveloped, segmented double-stranded rna viruses of the family reoviridae, genus orthoreovirus. they have a wide host range and are ubiquitous throughout the world. the designation reo stands for respiratory enteric orphan and reflects the original isolation of these viruses from human respiratory and intestinal tract without apparent disease. the term 'orphan' virus refers to a virus in search of a disease. mammalian orthoreovirus can be grouped into three serotypes (1, 2, 3). mammalian orthoreovirus-3 (synonyms: hepatoencephalomyelitis virus; echo 10 virus) infection remains prevalent in contemporary mouse colonies and has been reported in wild mice barthold, 1997a) . seropositivity to mrv-3 was found in less than 5% of spf colonies and in approximately 20% of non-spf mouse colonies in the usa in 1996 (jacoby and lindsey, 1998) . a study in france reported antibodies to mrv-3 in 9% of mouse colonies examined (zenner and regnault, 2000) . more recently, a study in north america found a low rate (0.2%) of mouse sera to be positive for antibodies against this virus (livingston and riley, 2003) . in addition, contamination of mouse origin tumours and cell lines by mrv-3 has been reported many times (national research council, 1991; nicklas et al., 1993; barthold, 1997a) . experimentally, mrv-3 infection of infant mice has been used to model human hepatobiliary disease, pancreatitis, diabetes mellitus, and lymphoma (kraft, 1982; national research council, 1991; fenner et al., 1993) . the literature on mrv-3 infections in mice is dominated by studies on experimentally infected animals. the virus can cause severe pantropic infection in infant mice (kraft, 1982; tyler and fields, 1986; barthold, 1997a) . after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following oral inoculation, reoviruses gain entry by infecting specialized epithelial cells (m cells) that overlie peyer's patches. the virus then becomes accessible to leukocytes and spreads to other organs by way of the lymphatic system and the bloodstream. neural spread to the cns has also been well documented (morrison et al., 1991) . the mechanisms of viral pathogenesis and their interactions with the host cell are reviewed in detail by and . natural infection by mrv-3 in a mouse colony usually is subclinical although diarrhoea or steatorrhoea and oily hair effect in suckling mice may be noted (kraft, 1982; tyler and fields, 1986; national research council, 1991; barthold, 1997a; percy and barthold, 2001) . the latter term has been used to describe the matted, unkempt appearance of the hair coat that results from steatorrhoea due to pancreatitis, maldigestion, and biliary atresia. in addition, runting (attributed to immune-mediated destruction of cells in the pituitary gland that produce growth hormone), transient alopecia, jaundice (due to excessive bilirubin in the blood, which is attributed to the liver pathology, especially biliary atresia), and neurological signs such as incoordination, tremors, or paralysis may develop. when present in natural infections, clinical signs and lesions are similar to but milder than in experimental neonatal infections. early descriptions of naturally occurring disease may have been complicated by concurrent infections such as mhv or murine rotavirus a (murv-a)/epizootic diarrhoea of infant mice (edim) virus that contributed to the severity of the lesions especially in liver, pancreas, cns, and intestine. the outcome of mrv-3 infection depends on age and immunological status of mouse, dose of virus, and route of inoculation. adult immunocompetent mice typically show no clinical signs and have no discernible lesions even in experimental infections. mucosal and maternally conferred immunity are considered to be important in protection from or resolution of disease (cuff et al., 1990; barthold et al., 1993b) . experimental infection of adult prkdc scid mice is lethal (george et al., 1990) . depending on the route of inoculation, experimental infection of adult foxn1 nu mice is subclinical or results in liver disease (carthew, 1984; george et al., 1990) . histological findings reported to occur after experimental mrv-3 infection of neonatal mice include inflammation and necrosis in liver, pancreas, heart, adrenal, brain, and spinal cord; lymphoid depletion in thymus, spleen, and lymph nodes; and hepatic fibrosis with biliary atresia (papadimitriou and robertson, 1976; tyler and fields, 1986; barthold et al., 1993b; barthold, 1997a; percy and barthold, 2001) . transmission of reoviruses probably involves the aerosol as well as the faecal-oral route (national research council, 1991) . fomites may play an important role as passive vectors because reoviruses resist environmental conditions moderately well. serological screening with elisa or ifa is in widespread use for detection of antibodies to mrv-3 in diagnostic and health surveillance programmes. both elisa and ifa detect cross-reacting antibodies to heterologous mrv serotypes that can infect mice (aclad, 1991) . the hi test does not detect such cross-reacting antibodies but is prone to give false positive results due to nonspecific inhibitors of haemagglutination (kraft and meyer, 1986; van der logt, 1986; aclad, 1991) . reverse transcriptase-polymerase chain reaction methods for the detection of mrv-3 rna (steele et al., 1995) or mrv rna (leary et al., 2002) are also available. reports on contamination of mouse origin tumours and cell lines by mrv-3 and its interference with transplantable tumour studies (bennette, 1960; nelson and tarnowski, 1960) emphasize the importance of screening of biological materials to be inoculated into mice by map test or pcr. natural seroconversion to mrv-3 without clinical disease is also observed in laboratory rats, hamsters, and guinea pigs (national research council, 1991; barthold, 1997a) . caesarean derivation and barrier maintenance have proven effective in the control and prevention of mrv-3 infection (kraft, 1982; national research council, 1991) . the virus may interfere with research involving transplantable tumours and cell lines of mouse origin. it has the potential to alter intestinal studies and multiple immune response functions in mice. in enzootically infected colonies, protection of neonates by maternal antibody could complicate or prevent experimental infections with reoviruses. it could further complicate experiments that require evaluation of liver, pancreas, cns, heart, lymphoid organs, and other tissues affected by the virus. the term murine hepatitis virus (mhv; commonly referred to as 'mouse hepatitis virus') designates a large group of antigenically and genetically related, singlestranded rna viruses belonging to the family coronaviridae, genus coronavirus. they are surrounded by an envelope with a corona of surface projections (spikes). murine hepatitis virus is antigenically related to rat coronaviruses and other coronaviruses of pigs, cattle, and humans. numerous different strains or isolates of mhv have been described. they can be distinguished by neutralization tests that detect strain-specific spike (s) antigens. the best studied strains are the prototype strains mhv-1, mhv-2, mhv-3, jhm (mhv-4), a59, and s, of which mhv-3 is regarded as the most virulent. murine hepatitis virus, like other coronaviruses, mutates rapidly, and strains readily form recombinants, so that new (sub)strains are constantly evolving. strains vary in their virulence, organotropism, and cell tropism (homberger, 1997) . based on their primary organotropism, mhv strains can be grouped into two biotypes: respiratory (or polytropic) and enterotropic. however, intermediate forms (enterotropic strains with tropism to other organs) exist. murine hepatitis virus is relatively resistant to repeated freezing and thawing, heating (56њc for 30 min), and acid ph but is sensitive to drying and disinfectants, especially those with detergent activity (national research council, 1991) . mus musculus is the natural host of mhv. it can be found in wild and laboratory mice throughout the world and is one of the most common viral pathogens in contemporary mouse colonies. while polytropic strains have historically been considered more common, this situation is thought to have reversed. a survey conducted in the usa in 1996 reported antibodies to mhv in more than 10% of spf mouse colonies and more than 70% of non-spf colonies (jacoby and lindsey, 1998) , though very recent monitoring results for research institutions across north america indicate that the prevalence of mhv has decreased during the past few years (livingston and riley, 2003) . a retrospective study in france covering the period from 1988 to 1997 reported antibodies to mhv in 67% of mouse colonies examined (zenner and regnault, 2000) . suckling rats inoculated experimentally with mhv had transient virus replication in the nasal mucosa and seroconversion but no clinical disease . similarly, deer mice seroconverted but showed no clinical disease after experimental infection (silverman et al., 1982) . murine hepatitis virus is also a common contaminant of transplantable tumours (collins and parker, 1972; nicklas et al., 1993) and cell lines (sabesin, 1972; yoshikura and taguchi, 1979) . the pathogenesis and outcome of mhv infections depend on interactions among numerous factors related to the virus (e.g. virulence and organotropism) and the host (e.g. age, genotype, immune status, and microbiological status; kraft, 1982; barthold, 1986; national research council, 1991; compton et al., 1993; homberger, 1997; percy and barthold, 2001) . murine hepatitis virus strains appear to possess a primary tropism for the upper respiratory or enteric mucosa. those strains with respiratory tropism initiate infection in the nasal mucosa and then may disseminate via blood and lymphatics to a variety of other organs because of their polytropic nature. respiratory (polytropic) strains include mhv-1, mhv-2, mhv-3, a59, s, and jhm. infection of mice with virulent polytropic mhv strains, infection of mice less than 2 weeks of age, infection of genetically susceptible strains of mice, or infection of immunocompromised mice favour virus dissemination. virus then secondarily replicates in vascular endothelium and parenchymal tissues, causing disease of brain, liver, lymphoid organs, bone marrow, and other sites. infection of the brain by viraemic dissemination occurs primarily in immunocompromised or neonatal mice. additionally, infection of adult mouse brain can occur by extension of virus along olfactory neural pathways, even in the absence of dissemination to other organs. in contrast, enterotropic mhv strains (e.g. livim, mhv-d, and mhv-y) tend to selectively infect intestinal mucosal epithelium, with no or minimal dissemination to other organs such as mesenteric lymph nodes or liver. all ages and strains are susceptible to active infection, but disease is largely age-related. infection of neonatal mice results in severe necrotizing enterocolitis with high mortality within 48 h. mortality and lesion severity diminish rapidly with advancing age at infection. adult mice develop minimal lesions although replication of equal or higher titres of virus occurs compared with neonates. the age-dependent decrease in severity of enterotropic mhv disease is probably related to the higher mucosal epithelium turnover in older mice, allowing more rapid replacement of damaged mucosa. another factor that is of considerable importance to the outcome of mhv infections is host genotype. for example, balb/c mice are highly susceptible to enterotropic mhv disease while sjl mice, at the other end of the spectrum, are highly resistant (barthold et al., 1993a) . unlike in polytropic mhv infection where resistance is correlated with reduced virus replication in target cells (barthold and smith, 1987) , enterotropic mhv grows to comparable titres in sjl and balb/c mice at all ages (barthold et al., 1993a) . therefore, the resistance of the sjl mouse to disease caused by enterotropic mhv seems to be mediated through an entirely different mechanism than resistance to polytropic mhv. furthermore, mouse genotypes that are susceptible to disease caused by one mhv strain may be resistant to disease caused by another strain (barthold, 1986) . it is therefore not possible to strictly categorize mouse strains as susceptible or resistant. the genetic factors determining susceptibility versus resistance in mhv infections are as yet poorly understood. both polytropic and enterotropic mhv infections are selflimiting in immunocompetent mice. immune-mediated clearance of virus usually begins about a week after infection, and most mice eliminate the virus within 3-4 weeks (barthold, 1986; barthold and smith, 1990; barthold et al., 1993a) . humoral and cellular immunity appear to participate in host defences to infection, and functional t cells are an absolute requirement (williamson and stohlman, 1990; kyuwa et al., 1996; lin et al., 1999; haring and perlman, 2001) . therefore, immunodeficient mice such as foxn1 nu and prkdc scid mice cannot clear the virus (barthold et al., 1985; compton et al., 1993) . similarly, some genetically modified strains of mice may have deficits in antiviral responses or other alterations that allow the development of persistent mhv infection (rehg et al., 2001) . recovered immune mice are resistant to reinfection with the same mhv strain but remain susceptible to repeated infections with different strains of mhv (barthold and smith, 1989a,b; . similarly, maternal immunity protects suckling mice against homologous mhv strains but not necessarily against other strains . however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine . therefore, the susceptibility of young mice to infection significantly increases at weaning. most mhv infections are subclinical and follow one of two epidemiological patterns in immunocompetent mice (national research council, 1991; homberger, 1997) . enzootic (subclinical) infection, commonly seen in breeding colonies, occurs when a population has been in contact with the virus for a longer period (e.g. several weeks). adults are immune (due to prior infection), sucklings are passively protected, and infection is perpetuated in weanlings. epizootic (clinical) infection occurs when the virus is introduced into a naive population (housed in open cages). the infection rapidly spreads through the entire colony. clinical signs depend upon the virus and mouse strains and are most evident in infant mice. typically, they include diarrhoea, poor growth, lassitude, and death. in infections due to virulent enterotropic strains, mortality can reach 100% in infant mice. some strains may also cause neurological signs such as flaccid paralysis of hind limbs, convulsions, and circling. adult infections are again usually asymptomatic. as the infection becomes established in the colony, the epizootic pattern is replaced by the enzootic pattern. in immunodeficient (e.g. foxn1 nu and prkdc scid ) mice, infection with virulent polytropic mhv strains often is rapidly fatal while less virulent strains cause chronic wasting disease (compton et al., 1993) . in contrast, adult immunodeficient mice can tolerate chronic infection by enterotropic mhv, with slow emaciation and diarrhoea, or minimal clinical disease (barthold et al., 1985; barthold, 1986) . subclinical mhv infections can be activated by a variety of experimental procedures (e.g. thymectomy, whole body irradiation, treatment with chemotherapeutic agents, halothane anaesthesia) or by co-infections with other pathogens (e.g. eperythrozoon coccoides, k virus; reviewed by kraft, 1982; national research council, 1991) . in most natural infections, gross lesions are not present or are transient and not observed. gross findings in neonates with clinical signs include dehydration, emaciation, and in contrast to edim, an empty stomach (ishida et al., 1978; barthold et al., 1982; kraft, 1982) . the intestine is distended and filled with watery to mucoid yellowish, sometimes gaseous contents. haemorrhage or rupture of the intestine can occur. depending on the virus strain, necrotic foci on the liver (ishida et al., 1978; kraft, 1982; percy and barthold, 2001) and thymus involution godfraind et al., 1995) may also be seen in susceptible mice. liver involvement may be accompanied by jaundice and haemorrhagic peritoneal exudate. splenomegaly may occur as a result of compensatory haematopoiesis (fox et al., 1977) . histopathological changes in susceptible mice infected with polytropic mhv strains include acute necrosis with syncytia in liver, spleen, lymph nodes, gut-associated lymphoid tissue, and bone marrow (kraft, 1982; barthold, 1986; national research council, 1991; percy and barthold, 2001) . neonatally infected mice can have vascular-oriented necrotizing (meningo)encephalitis with demyelination in the brain stem and peri-ependymal areas. lesions in peritoneum, bone marrow, thymus, and other tissues can be variably present. mice can develop nasoencephalitis due to extension of infection from the nasal mucosa along olfactory pathways to the brain, with meningoencephalitis and demyelination, the latter of which is thought to be largely t cell-mediated (haring and perlman, 2001) . this pattern of infection regularly occurs after intranasal inoculation of many mhv strains but is a relatively rare event after natural exposure. syncytia arising from endothelium, parenchyma, or leukocytes is a hallmark of infection in many tissues including intestine, lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow. lesions are transient and seldom fully developed in adult immunocompetent mice, but they are manifest in immunocompromised mice. highly unusual presentations can occur in mice with specific gene defects. for example, granulomatous peritonitis and pleuritis were found in interferon-␥-deficient mice infected with mhv (france et al., 1999) . histopathological changes caused by enterotropic strains of mhv are mainly confined to the intestinal tract and associated lymphoid tissues (kraft, 1982; barthold, 1986; national research council, 1991; percy and barthold, 2001) . the most common sites are terminal ileum, caecum, and proximal colon. the severity of disease is primarily age-dependent, with neonatal mice being most severely affected. these mice show segmentally distributed areas of villus attenuation, enterocytic syncytia (balloon cells), and mucosal necrosis accompanied by leukocytic infiltration. intracytoplasmic inclusions are present in enterocytes. erosions, ulceration, and haemorrhage may be seen in more severe cases. lesions can be fully developed within 24-48 h, but are usually more severe at 3-5 days after infection. surviving mice may develop compensatory mucosal hyperplasia. mesenteric lymph nodes usually contain lymphocytic syncytia, and mesenteric vessels may contain endothelial syncytia. pathological changes in older mice are generally much more subtle and may only consist of transient syncytia. an occasional exception seems to occur in immunodeficient animals such as foxn1 nu mice, which can develop chronic hyperplastic typhlocolitis of varying severity (barthold et al., 1985) , but other agents such as helicobacter species may have been involved. in general, enterotropic mhv strains do not disseminate, but hepatitis and encephalitis can occur with some virus strains in certain mouse genotypes. murine hepatitis virus is highly contagious. it is shed in faeces and nasopharyngeal secretions and appears to be transmitted via direct contact, aerosol, and fomites (kraft, 1982; national research council, 1991) . vertical (in utero) transmission has been demonstrated in experimental infections (katami et al., 1978) but does not seem to be of practical importance under natural conditions. diagnosis during the acute stage of infection can be made by histological demonstration of characteristic lesions with syncytia in target tissues, but clinical signs and lesions can be highly variable and may not be prominent. suckling, genetically susceptible or immunocompromised mice are the best candidates for evaluation. active infection can be confirmed by immunohistochemistry (brownstein and barthold, 1982) or by virus isolation. virus recovery from infected tissues is difficult but can be accomplished using primary macrophage cultures or a number of established cell lines such as nctc 1469 or dbt (aclad, 1991) . these cells, however, may not be successful substrates for some enterotropic mhv strains. virus in suspect tissue can also be confirmed by bioassays such as map testing or infant or foxn1 nu mouse inoculation (de souza and smith, 1989; aclad, 1991) . amplification by passage in these mice increases the likelihood of detection of lesions and antigen, or virus recovery. other direct diagnostic methods that have been successfully utilized to detect mhv in faeces or tissue of infected mice include monoclonal antibody solution hybridization assay (casebolt and stephensen, 1992 ) and a number of rt-pcr assays (homberger et al., 1991; kunita et al., 1992; yamada et al., 1993; besselsen et al., 2002) . because of the transient nature of mhv infection in immunocompetent mice, serology is the most appropriate diagnostic tool for routine monitoring. enzyme-linked immunosorbent assay and ifa are well established and sensitive, and all known mhv strains cross-react in both tests (smith, 1983; aclad, 1991) . the magnitude of antibody response depends on mhv strain and mouse genotype (nakanaga et al., 1983; barthold and smith, 1987) . dba/2 mice are poor antibody responders whereas c57bl/6 mice produce a high antibody titre and are therefore good sentinels. antibody titres remain high over a period of at least 6 months (barthold and smith, 1989b; . infected mice may not develop detectable antibodies for up to 14 days after initial exposure (smith, 1983 ). in such cases, a direct diagnostic method as discussed above may be useful. another drawback of serology is that mice weaned from immune dams can have maternal antibodies until they are 10 weeks of age (homberger, 1992) . this may impact serological monitoring because the possibility must be considered that low positive results are due to maternally-derived passive immunity. because the virus can be transmitted by transplantable tumours and other biological materials from mice, including hybridomas (holmes et al., 1986) and embryonic stem cells (okumura et al., 1996; kyuwa, 1997) , these materials should also be routinely screened for mhv contamination. mouse inoculation bioassay, map test, and rt-pcr can be used for this purpose. the best means of mhv control is to prevent its entry into a facility. this can be accomplished by purchase of mice from virus-free sources and maintenance under effective barrier conditions monitored by a welldesigned quality assurance programme. control of wild mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbour virus are also important measures to prevent infection. if infection occurs, the most effective elimination strategy is to cull the affected colony and obtain clean replacement stock. however, this is not always a feasible option when working with valuable mice (e.g. genetically modified lines, breeding stocks). caesarean derivation or embryo transfer can be used to produce virus-free offspring, and foster-nursing also has been reported to be effective (lipman et al., 1987) . quarantine of an affected colony with no breeding and no introduction of new animals for approximately 2 months has been effective in immunocompetent mice (weir et al., 1987) . the infection is likely to be terminated because mhv requires a constant supply of susceptible animals. this method works best when working with small numbers of mice. large populations favour the development of new mhv strains that may result in repeated infections with slightly different strains (adami et al., 1995) . it may be practical to select a few future breeders from the infected population and quarantine them for approximately 3 weeks (compton et al., 1993) . this can be achieved in isolators, or in individually ventilated cages if proper handling is guaranteed. after this interval, breeding can resume. the 3-week interval should permit recovery from active infection, and the additional 3-week gestation period effectively extends the total quarantine to 6 weeks. it is advisible to select seropositive breeders because the possibility of active infection is lower in such animals. the breeding cessation strategy may not be successful if immunodeficient mice are used because they are susceptible to chronic infection and viral excretion (barthold et al., 1985) . genetically engineered mice of unclear, unknown or deficient immune status pose a special challenge because they may develop unusual manifestations of infection or may be unable to clear virus. rederivation likely is the most cost effective strategy in such situations. along with the measures described, proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. careful testing with sentinel mice should be applied to evaluate the effectiveness of rederivation. if transplantable tumours are contaminated with mhv, virus elimination can be achieved by passage of tumours in athymic whn rnu rats (rülicke et al., 1991) . murine hepatitis virus is one of the most important viral pathogens of laboratory mice and has been intensively studied from a number of research perspectives (e.g. as a model organism for studying coronavirus molecular biology or the pathogenesis of viral-induced demyelinating disease). numerous reports document the effects of natural and experimental infections with mhv on host physiology and research, especially in the fields of immunology and tumour biology (reviewed by barthold, 1986; national research council, 1991; compton et al., 1993; homberger, 1997; baker, 1998; nicklas et al., 1999) . murine pneumonia virus, commonly referred to as 'pneumonia virus of mice' (pvm), is an enveloped, singlestranded rna virus of the family paramyxoviridae, genus pneumovirus. it is closely related to human respiratory syncytial virus (hrsv). the virus name is officially abbreviated as 'mpv' according to the international union of microbiological societies (2000); however, the former designation 'pvm' will be used in this chapter to avoid confusion with the official abbreviation of mouse parvovirus 1 (mpv). 'pneumonia virus of mice' infection is relatively common in colonies of mice and rats throughout the world. seropositivity to pvm was reported in less than 5% of spf mouse colonies and in approximately 20% of non-spf mouse colonies in the usa (jacoby and lindsey, 1998) . a serological survey in france demonstrated antibodies to pvm in 16% of mouse colonies examined (zenner and regnault, 2000) . in a more recent study in north america, such antibodies were found in only 0.1% of mice monitored (livingston and riley, 2003) . antibodies to pvm have also been detected in hamsters, gerbils, cotton rats, guinea pigs, and rabbits (parker and richter, 1982; richter, 1986; national research council, 1991) . experimentally, pvm infection of mice is used as a model for hrsv infection (domachowske et al., 2000) . in immunocompetent mice, natural infection with pvm is transient and usually not associated with clinical disease or pathological findings (parker and richter, 1982; national research council, 1991; brownstein, 1996b) . however, natural disease and persistent infection may occur in immunodeficient mice (carthew and sparrow, 1980; richter et al., 1988; weir et al., 1988) . in particular, athymic foxn1 nu mice seem to be susceptible to pvm infection, which can result in dyspnoea, cyanosis, emaciation, and death due to pneumonia (richter et al., 1988; weir et al., 1988) . similar clinical signs have been reported for experimentally infected, immunocompetent mice (cook et al., 1998) . necropsy findings in naturally infected foxn1 nu mice include cachexia and diffuse pulmonary oedema or lobar consolidation (weir et al., 1988) . pulmonary consolidation (dark red or grey in colour) also has been found after experimental infection of immunocompetent mice (brownstein, 1996b) . histologically, natural infection of foxn1 nu mice with pvm presents as interstitial pneumonia (richter et al., 1988; weir et al., 1988) . experimental intranasal inoculation of immunocompetent mice can result in rhinitis, erosive bronchiolitis, and interstitial pneumonia with prominent early pulmonary eosinophilia and neutrophilia (brownstein, 1996b; domachowske et al., 2000) . hydrocephalus may result from intracerebral inoculation of neonatal mice (lagace-simard et al., 1980) . susceptibility to infection is influenced by age of mouse, dose of virus, and a variety of local and systemic stressors (parker and richter, 1982; national research council, 1991) . pneumonia virus of mice is labile in the environment and rapidly inactivated at room temperature (parker and richter, 1982; national research council, 1991) . the virus is tropic for the respiratory epithelium (carthew and sparrow, 1980; cook et al., 1998) , and transmission is exclusively horizontal via the respiratory tract, mainly by direct contact and aerosol (parker and richter, 1982; national research council, 1991) . therefore, transmissibility in mouse colonies is low, and infections tend to be focal enzootics. serology (elisa, ifa, or hi) is the primary means of testing mouse colonies for exposure to pvm. immunohistochemistry has been applied to detect viral antigen in lung sections (carthew and sparrow, 1980; weir et al., 1988) , however, proper sampling (see chapter on health monitoring) is critical for establishing the diagnosis due to the focal nature of the infection. an rt-pcr assay to detect viral rna in respiratory tract tissues has also been reported . however, the use of direct methods requires good timing because the virus is present for only up to about 10 days in immunocompetent mice (brownstein, 1996b) . embryo transfer or caesarean derivation followed by barrier maintenance can be used to rear mice that are free of pvm. because active infection is present in the individual immunocompetent mouse for only a short period, strict isolation of a few (preferably seropositive) mice with the temporary cessation of breeding might also be successful in eliminating the virus (richter, 1986; national research council, 1991) . pneumonia virus of mice could interfere with studies involving the respiratory tract or immunological measurements in mice. in addition, pvm can have devastating effects on research using immunodeficient mice because they are particularly prone to develop fatal disease (richter et al., 1988; weir et al., 1988) or become more susceptible to the deleterious effects of other agents such as pneumocystis carinii (roths et al., 1993) . murine rotavirus-a/edim (commonly referred to as 'mouse rotavirus' or 'epizootic diarrhoea of infant mice virus') is a nonenveloped, segmented double-stranded rna virus of the family reoviridae, genus rotavirus. it is antigenically classified as a group a rotavirus, similar to rotaviruses of many other species that cause neonatal and infantile gastroenteritis (fenner et al., 1993) . murine rotavirus-a/edim infection remains prevalent in contemporary mouse colonies and appears to occur worldwide. seropositivity to murv-a/edim was reported in approximately 5% of spf colonies and in almost 30% of non-spf mouse colonies in the usa in 1996 (jacoby and lindsey, 1998) . more recently, livingston and riley (2003) found a low rate (1%) of mouse sera to be positive for antibodies against murv-a/edim. experimentally, murv-a/edim infection in mice is used as a model for human rotavirus infection, especially in investigations on the mechanisms of rotavirus immunity and in the development of vaccination strategies (ward and mcneal, 1999) . clinical symptoms following murv-a/edim infection range from inapparent or mild to severe, sometimes fatal, diarrhoea. 'epizootic diarrhoea of infant mice' describes the clinical syndrome associated with natural or experimental infection by murv-a/edim during the first 2 weeks of life (kraft, 1982; sheridan and vonderfecht, 1986; national research council, 1991; barthold, 1997b; percy and barthold, 2001) . diarrhoea usually begins around 48 h after infection and persists for about 1 week. affected suckling mice have soft, yellow faeces that wet and stain the perianal region. in severe instances, the mice may be stunted, have dry scaly skin, or are virtually covered with faecal material. morbidity is very high but mortality is usually low. gross lesions in affected mice are confined to the intestinal tract. the caecum and colon may be distended with gas and watery to paste-like contents that are frequently bright yellow. the stomach of diarrheic mice is almost always filled with milk, and this feature has been reported to be a reliable means to differentiate diarrhoea caused by rotavirus from the diarrhoea caused by mhv infection. histopathological changes may be subtle even in animals with significant diarrhoea. they are confined to the small intestine and are most prominent at the apices of villi, where rotaviruses infect and replicate within epithelial cells. hydropic change of villous epithelial cells is the hallmark finding of acute disease. the villi become shortened, and the cells that initially replace the damaged cells are less differentiated, typically cuboidal instead of columnar, and lack a full complement of enzymes for digestion and absorption, resulting in diarrhoea due to maldigestion and malabsorption. undigested milk in the small intestine promotes bacterial growth and exerts an osmotic effect, exacerbating damage to the villi. intestinal fluid and electrolyte secretion is further enhanced by activation of the enteric nervous system (lundgren et al., 2000) and through the effects of a viral enterotoxin called nsp4 (for nonstructural protein 4; ball et al., 1996) . it is hypothesized that nsp4 is released from virusinfected cells and then triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. susceptibility to edim depends on the age of the host and peaks between 4 and 14 days of age (kraft, 1982; sheridan and vonderfecht, 1986; national research council, 1991; barthold, 1997b; percy and barthold, 2001) . mice older than about 2 weeks can still be infected with murv-a/edim, but small numbers of enterocytes become infected, there is little replication of virus, and diarrhoea does not occur. the exact reason for this age-related resistance to disease is unknown. pups suckling immune dams are protected against edim during their period of disease susceptibility (rosé et al., 1998) . in general, the infection is selflimiting and resolves within days. successful viral clearance is promoted by an intact immune response (feng et al., 1997; mcneal et al., 1997; rosé et al., 1998) , and some immunodeficient mice (e.g. prkdc scid and rag2 tm1fwa mice) may shed virus for extended periods or become persistently infected (riepenhoff-talty et al., 1987; franco and greenberg, 1995) . protection against murv-a/edim reinfection is primarily mediated by antibodies (feng et al., 1997; rosé et al., 1998) . murine rotavirus-a/edim is highly contagious and transmitted by the faecal-oral route (kraft, 1982; sheridan and vonderfecht, 1986; national research council, 1991) . dissemination of the virus occurs through direct contact or contaminated fomites and aerosols. murv-a/edim is stable at ϫ70њc but otherwise tends to be susceptible to extreme environmental conditions, detergents, and disinfectants. enzyme-linked immunosorbent assay and ifa are in widespread use for detection of serum antibodies to murv-a/edim in diagnostic and health surveillance programmes; other assay systems such as those using latex agglutination are also utilized (ferner et al., 1987) . rotazyme ii is a commercially available elisa for detection of rotavirus antigen in faeces; however, great care must be used in interpreting the results because some feeds have been reported to cause false positive reactions (jure et al., 1988) . electron microscopy of faeces of diarrheic pups should reveal typical wheelshaped rotavirus particles, 60-80 nm in diameter. reverse transcriptase-polymerase chain reaction also can be used to detect rotavirus rna in faecal samples (wilde et al., 1990) . good timing is critical for establishing the diagnosis from faeces because virus is shed for only a few days in immunocompetent mice. embryo transfer or caesarean derivation followed by barrier maintenance is recommended for rederivation of breeding stocks (kraft, 1982; national research council, 1991) . in immunocompetent mice in which infection is effectively cleared, a breeding suspension strategy combined with excellent sanitation, filter tops, and conscientious serological testing of offspring may also be effective. murine rotavirus-a/edim has the potential to interfere with any research utilizing suckling mice. it may have a significant impact on studies where the intestinal tract of neonatal or infant mice is the target organ. the infection also poses a problem for infectious disease and immune response studies, particularly those involving enteropathogens in infant mice (newsome and coney, 1985) . in addition, runting could be interpreted erroneously as the effect of genetic manipulation or other experimental manipulation. sendai virus (sev) is an enveloped, single-stranded rna virus of the family paramyxoviridae, genus respirovirus. it is antigenically related to human parainfluenza virus 1. the virus was named for sendai, japan, where it was first isolated from mice. infections of mice and rats are relatively common and occur worldwide. in addition, there is evidence that hamsters, guinea pigs, and rabbits are susceptible to infection with sev (machii et al., 1989; aclad, 1991; national research council, 1991; percy and palmer, 1997) ; however, some apparently seropositive guinea pigs may in fact be seropositive to other parainfluenza viruses instead of sev. seropositivity to sev was reported to be absent from spf mouse colonies and to be approximately 20% in non-spf mouse colonies in the usa (jacoby and lindsey, 1998) . a study in france reported antibodies to sev in 17% of mouse colonies examined (zenner and regnault, 2000) . a low rate of seropositive mice (0.2%) was found in a recent survey in north america (livingston and riley, 2003) . furthermore, sev can contaminate biological materials (collins and parker, 1972) . sendai virus is pneumotropic and the leading cause of viral respiratory disease in mice. the pneumotropism is partially a consequence of the action of respiratory serine proteases such as tryptase clara, which activate viral infectivity by specific cleavage of the viral fusion glycoprotein (tashiro et al., 1999) . in addition, the apical budding behaviour of sev may hinder the spread of virus into subepithelial tissues and subsequently to distant organs via the blood. two epidemiologic patterns of sev infection have been recognized, an enzootic (subclinical) and epizootic (clinically apparent) type (parker and richter, 1982; national research council, 1991; brownstein, 1996a) . enzootic infections commonly occur in breeding or open colonies, where the constant supply of susceptible animals perpetuates the infection. in breeding colonies, mice are infected shortly after weaning as maternal antibody levels wane. normally, the infection is subclinical, with virus persisting for approximately 2 weeks, accompanied by seroconversion that persists for a year or longer. epizootic infections occur upon first introduction of the virus to a colony and either die out (self-cure) after 2-7 months or become enzootic depending on colony conditions. the epizootic form is generally acute, and morbidity is very high resulting in nearly all susceptible animals becoming infected within a short time. clinical signs vary and include rough hair coat, hunched posture, chattering, respiratory distress, prolonged gestation, death of neonates and sucklings, and runting in young mice. breeding colonies may return to normal productivity in 2 months and thereafter maintain the enzootic pattern of infection. factors such as strain susceptibility, age, husbandry, transport, and copathogens are important in precipitating overt disease. dba and 129/j strains of mice are very susceptible to sev pneumonia whereas sjl/j and c57bl/6/j strains and several outbred stocks are relatively resistant. a/j, balb/c, and swr/j are among the strains that show intermediate susceptibility. there is no evidence for persistent infection in immunocompetent mice, but persistent or prolonged infection may occur in immunodeficient mice and can result in wasting and death due to progressive pneumonia (ward et al., 1976; iwai et al., 1979; percy et al., 1994) . clearance of a primary sev infection is mediated by cd8 ϩ and cd4 ϩ t cell mechanisms (kast et al., 1986; hou et al., 1992) . heavier than normal, consolidated, plum-coloured or grey lungs are a characteristic gross finding in severe sev pneumonia (parker and richter, 1982; national research council, 1991; brownstein, 1996a; percy and barthold, 2001) . lymphadenopathy and splenomegaly reflect the vigorous immune response to infection. histologically, three phases of disease can be recognized in susceptible immunocompetent mice: acute, reparative, and resolution phases (brownstein, 1996a; percy and barthold, 2001) . lesions of the acute phase, which lasts 8-12 days, are primarily attributed to the cell-mediated immune response that destroys infected respiratory epithelial cells and include necrotizing rhinitis, tracheitis, bronch(iol)itis, and alveolitis. epithelial syncytiae and cytoplasmic inclusion bodies in infected cells may be seen early in this phase. alveoli contain sloughed necrotic epithelium, fibrin, neutrophils, and mononuclear cells. atelectasis, bronchiectasis, and emphysema may occur as a result of damage and obstruction of airways. the reparative phase, which may overlap the acute phase but continues through about the third week post infection, is indicated by regeneration of airway lining epithelium. adenomatous hyperplasia and squamous metaplasia (with multilayered flat epithelial cells instead of normal columnar cells) in the terminal bronchioles and alveoli are considered to be a hallmark of sev pneumonia. mixed inflammatory cell infiltrates in this phase tend to be primarily interstitial rather than alveolar as they are in the acute phase. the resolution phase may be complete by the fourth week post infection and lesions may be difficult to identify subsequently. residual, persistent lesions that may occur include organizing alveolitis and bronchiolitis fibrosa obliterans. alveoli and bronchioles are replaced by collagen and fibroblasts, foamy macrophages, and lymphoid infiltrates, often with foci of emphysema, cholesterol crystals, and other debris, which represent attempts to organize and wall off residual necrotic debris and fibrin. lesions are more severe and variable when additional pathogens such as mycoplasma pulmonis are present (national research council, 1991) . otitis media has also been reported in natural infections with sev although some of these studies have been complicated by the presence of other pathogens (ward, 1974) . sendai virus has been detected in the inner ear after experimental intracerebral inoculation of neonatal mice (shimokata et al., 1977) . sendai virus is extremely contagious. infectious virus is shed during the first 2 weeks of infection and appears to be transmitted by direct contact, contaminated fomites, and respiratory aerosol (parker and reynolds, 1968; parker and richter, 1982; national research council, 1991) . serology (elisa, ifa, or hi) is the approach of choice for routine monitoring because serum antibodies to sev are detectable soon after infection and persist at high levels for many months, although active infection lasts only 1-2 weeks in immunocompetent mice. the short period of active infection limits the utility of direct methods such as immunohistochemistry (carthew and sparrow, 1980) and rt-pcr (hayase et al., 1997; wagner et al., 2003) . although sev is considered to be highly contagious, studies have shown that dirty bedding sentinel systems do not reliably detect the infection and that outbred stocks may not seroconvert consistently (dillehay et al., 1990; artwohl et al., 1994) . mouse antibody production test and rt-pcr can be used to detect sev in contaminated biological materials. sendai virus infection in mouse colonies has proven to be one of the most difficult virus infections to control because the virus is highly infectious and easily disseminated. depopulation of infected colonies probably is the most appropriate means to eliminate the virus in most situations. embryo transfer followed by barrier maintenance has also been used successfully in eliminating the virus (national research council, 1991) . a less effective alternative is to place the infected animals under strict quarantine, remove all young and pregnant mice, suspend all breeding, and prevent addition of other susceptible animals for approximately 2 months until the infection is extinguished and then breeding and other normal acitivities are resumed (parker and richter, 1982; national research council, 1991) . vaccines against the virus have been developed (brownstein, 1986; national research council, 1991) , but these probably do not represent a practical means to achieve or maintain the seronegative status of colonies that is in demand today. sendai virus has the potential to interfere with a wide variety of research involving mice. reported effects include interference with early embryonic development and foetal growth; alterations of macrophage, nk cell, and t and b cell function; altered responses to transplantable tumours and respiratory carcinogens; altered isograft rejection; and delayed wound healing (reviewed by national research council, 1991; baker, 1998; nicklas et al., 1999) . pulmonary changes during sev infection can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other agents. they could also affect the response to anaesthetics. in addition, natural sev infection would interfere with studies using sev as a gene vector. theiler's murine encephalomyelitis virus (tmev) or murine poliovirus is a member of the genus cardiovirus in the family picornaviridae. members of this genus are nonenveloped viruses with singlestranded rna. the virus is rapidly destroyed at temperatures above 50њc. it is considered to be a primary pathogen of the cns of mice and can cause clinical disease resembling that due to poliomyelitis virus infections in humans. antibodies to tmev have been identified in mouse colonies and feral populations worldwide, and mus musculus is considered to be the natural host of tmev (lipton et al., 2001) . the most well-known and most frequently mentioned tmev strain is gdvii, which is virulent for mice. infant or young hamsters and laboratory rats are also susceptible to intracerebral infection. the original isolate is designated to (theiler's original) and represents a group of tmev strains with low virulence for mice. many additional virus strains have been isolated and studied, and they all fall in the broad grouping of to and gdvii. a similar virus strain has also been isolated from rats, but in contrast to mouse isolates this virus is not pathogenic for rats and mice after intracerebral inoculation (hemelt et al., 1974) . recently, another rat isolate has been characterized and shown to be most closely related to but quite distinct from other tmev viruses (ohsawa et al., 2003) . antibodies to tmev (strain gdvii) have been detected in guinea pigs and are considered to indicate infection with another closely related cardiovirus (hansen et al., 1997) . seropositivity to tmev was reported in approximately 5% of spf mouse colonies and approximately 35% of non-spf mouse colonies in the usa (jacoby and lindsey, 1998) . zenner and regnault (2000) reported a prevalence rate of 9% in french mouse colonies in a retrospective study, and it has been one of the most common virus infections in rodent colonies. in a recent study, antibodies were found in 0.2% of mice monitored (livingston and riley, 2003) indicating that tmev, like most viruses, has meanwhile been eliminated from the majority of mouse colonies. theiler's murine encephalomyelitis virus is primarily an enteric pathogen, and virus strains are enterotropic. in natural infections, virus can be detected in intestinal mucosa and faecal matter, and in some cases it is also found in the mesenteric lymph nodes. however, histological lesions in the intestine are not discerned. virus may be shed via intestinal contents for up to 22 weeks, sometimes intermittently (brownstein et al., 1989a) , and transmission under natural conditions is via the faecal-oral route by direct contact between mice as well as by indirect contact (e.g. dirty bedding). the host immune response limits virus spread, but it does not immediately terminate virus replication in the intestines. virus is cleared from extraneural tissues, but it persists in the cns for at least a year. clinical disease due to natural tmev infection is rare, with a rate of only 1 in 1000-10,000 infected immunocompetent animals (percy and barthold, 2001) . in immunodeficient mice, especially in weanlings, clinical signs may be more common and mortality may be higher (rozengurt and sanchez, 1993) . this group of viruses usually causes asymptomatic infections of the intestinal tract. they may spread to the cns as a rare event where they cause different neurological disease manifestations. the most typical clinical sign of tmev infection is flaccid paralysis of hind legs. the animals appear otherwise healthy, and there is no mortality. experimental infection in mice provides models of poliomyelitis-like infection and virus-induced demyelinating disease including multiple sclerosis (mcgavern et al., 2000) . after experimental infection, tmev causes a biphasic disease in susceptible strains of mice. the acute phase is characterized by early infection of neurons in the grey matter. encephalomyelitis may develop during this phase and may be fatal, but most animals survive and enter the second phase of the disease at 1-3 months after the acute phase. this phase is characterized by viral persistence in the spinal cord white matter, mainly in macrophages, and leads to white matter demyelination. persistence and demyelination occur only in genetically susceptible mouse strains while resistant strains clear the infection after early grey matter encephalomyelitis through a cytotoxic t lymphocyte response. for this reason, the nude mutation (foxn1 nu ) confers susceptibility on mice with an otherwise resistant background. the severity and nature of disease depend on virus strain, route of inoculation, host genotype and age (downs, 1982; lipton and rozhon, 1986; national research council, 1991; percy and barthold, 2001) . in general, virus isolates with low virulence produce persistent cns infection in mice whereas virulent strains are unable to cause persistent infection. intracerebral inoculation results in the most severe infections, but the intranasal route is effective also. experimental intracerebral infections with virulent fa and gdvii strains of tmev are more likely to cause acute encephalomyelitis and death in weanling mice 4-5 days after inoculation ('early disease'). death may be preceded by neurological manifestations of encephalitis such as hyperexcitability, convulsions, tremors, circling and rolling, and weakness. animals may develop typical flaccid paralysis of hind limbs, and locomotion is possible only by use of the forelimbs. interestingly, the tail is not paralysed. experimental infections with low virulence virus strains (e.g. to, da, ww) are more likely to cause persistent infection with development of mild encephalomyelitis followed by a chronic demyelinating disease after a few months ('late disease'). these virus strains infect neurons in the grey matter of the brain and spinal cord during the acute phase of viral growth, followed by virus persistence in macrophages and glial cells in the spinal cord white matter. sjl, swr, and dba/2 strains are most susceptible to this chronic demyelinating disease. cba and c3h/he are less susceptible strains, and strains a, c57bl/6, c57bl/10, and dba/1 are relatively resistant (lipton and dal canto, 1979) . differences in humoral immune responses play a role in resistance to tmev infection (pena rossi et al., 1991a) , but genetic factors are also important. several genetic loci implicated in susceptibility to virus persistence, demyelination, or clinical disease have been identified, including the h-2d region of the major histocompatibility complex (brahic and bureau, 1998) . furthermore, the age at infection influences the severity of clinical disease. in infant mice, intracerebral infection with low virulence virus strains (e.g. to) is often lethal. young mice develop paralysis after an incubation period of 1-4 weeks while adult mice often show no clinical signs of infection (downs, 1982) . the only gross lesions are secondary to the posterior paralysis and may include urine scald or dermatitis due to incontinence of urine and trauma to paralysed limbs, or wasting or atrophy of the hind limbs in long term survivors. theiler's murine encephalomyelitis virus infects neurons and glial cells, and histological changes in the cns include nonsuppurative meningitis, perivasculitis, and poliomyelitis with neuronolysis, neuronophagia, and microgliosis in the brainstem and ventral horns of the spinal cord (percy and barthold, 2001) . demyelination in immunocompetent mice is considered to be immune-mediated. susceptible strains develop a specific delayed-type hypersensitivity response which is the basis for inflammation and demyelination. this reaction is mediated by cytotoxic t lymphocytes (lindsley et al., 1991; pena rossi et al., 1991b) and by the activation of cytokines as a consequence of infection of macrophages and other cells of the cns (rubio and capa, 1993; sierra and rubio, 1993; palma et al., 2003) . protection from chronic demyelinating disease is possible by vaccination with live virus given previously by subcutaneous or intraperitoneal inoculation (crane et al., 1993; kurtz et al., 1995) . early immunosuppression at the time of infection, e.g. by treatment with cyclophosphamide or antithymocyte serum, inhibits or diminishes demyelination. immunosuppression in mice chronically infected with tmev leads to remyelination of oligodendrocytes (rodriguez and lindsley, 1992) . further details related to the pathogenesis of tmev infections and the role of immune mechanisms have been reviewed by yamada et al. (1991) . experimental infection of foxn1 nu mice results in acute encephalitis and demyelination. demyelination associated with minimal inflammation and neurological signs including the typical hind limb paresis develop 2 weeks after inoculation, and most animals die within 4 weeks. in foxn1 nu mice, demyelination is caused by a direct lytic effect of the virus on oligodendrocytes (rosenthal et al., 1986) . demyelination and lethality are reduced after administration of neutralizing antibodies (fujinami et al., 1989) . histopathological changes in prkdc scid mice are very similar to those in foxn1 nu mice (rozengurt and sanchez, 1992) . young mice born in infected populations usually acquire infection shortly after weaning and are almost all infected by 30 days of age. intrauterine transmission to foetuses is possible during the early gestation period, but a placental barrier develops during gestation and later prevents intrauterine infection (miyamae, 1990; abzug et al., 1991) . all tmev isolates are closely related antigenically and form a single serogroup as determined by complement fixation and hi (lipton and rozhon, 1986) . hemelt et al. (1974) demonstrated cross reactions among four strains used in experimental infections, but differences were evident in homologous and heterologous titres. the viral strain most commonly used as antigen for serological testing is gdvii. this strain agglutinates human type 0 erythrocytes at 4њc, and hi has been the standard test for routine screening of mouse populations. meanwhile, hi has been replaced by elisa or ifa, both of which are more sensitive and specific. virus isolation is possible from brains or spinal cords of mice with clinical disease or from the intestinal contents of asymptomatic mice. pcr techniques also are available to test for virus-specific nucleotide sequences in biological samples (trottier et al., 2002) . mice that have been shown to be free from tmev by serological testing can be selected for breeding populations. if the virus is introduced into a mouse population, depopulation of infected colonies may be the most appropriate means to eliminate tmev. embryo transfer or caesarean derivation are the methods of choice for eliminating virus from valuable breeding populations. foster-nursing has been reported to be effective in generating virus-free offspring (lipman et al., 1987) although transplacental transmission has been demonstrated with experimental infection early in gestation. lesions of demyelination in cns of mice with clinically inapparent chronic infection may interfere with investigations that require evaluation of the cns (krinke and zurbriggen, 1997) . conceivably, such lesions also could affect neuromuscular responses or coordination, and affect neurological and behavioural evaluations. viral and mycoplasmal infections of laboratory rodents: effects on biomedical research monographs on pathology of laboratory animals: digestive system monographs on pathology of laboratory animals: digestive system viral and mycoplasmal infections of laboratory rodents: effects on biomedical research the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research monographs on pathology of laboratory animals: respiratory system monographs on pathology of laboratory animals: respiratory system biosafety in microbiological and biomedical laboratories (bmbl), 4th edn. u.s. department of health and human services proc. natl. acad. sci. usa 96 the mouse in biomedical research the mouse in biomedical research the mouse in biomedical research veterinary virology complications of viral and mycoplasmal infections in rodents to toxicology research and testing viral and mycoplasmal infections of laboratory rodents: effects on biomedical research virus taxonomy. seventh report of the international committee on taxonomy of viruses the mouse in biomedical research the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research proc. natl. acad. sci. usa the importance of laboratory animal genetics, health, and the environment in biomedical research proc. natl. acad. sci. usa national research council, committee on infectious diseases of mice and rats manual of microbiologic monitoring of laboratory animals the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research the mouse in biomedical research the mouse in biomedical research pathology of laboratory rodents and rabbits proc. natl. acad. sci. usa manual of microbiologic monitoring of laboratory animals viral and mycoplasmal infections of laboratory rodents: effects on biomedical research lactic dehydrogenase virus viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasma infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research handbook of animal models of infection the mouse in biomedical research key: cord-022520-ebj51v9o authors: marini, robert p.; otto, glen; erdman, susan; palley, lori; fox, james g. title: biology and diseases of ferrets date: 2007-09-02 journal: laboratory animal medicine doi: 10.1016/b978-012263951-7/50016-8 sha: doc_id: 22520 cord_uid: ebj51v9o nan ferrets (mustela putorius furo) belong to the ancient family mustelidae, which is believed to date back to the eocene period, some 40 million years ago. the taxonomic groups in the family mustelidae, as recognized by corbet and hill (1980) , include 67 species from north, central, and south america, eurasia, and africa. no other carnivore shows such diversity of adaptation, being found in a wide variety of ecosystems ranging from earlier references to ferrets are probably the basis of the belief that ferrets originated in north africa (thomson, 1951) . evidently they were bred specifically for rabbiting (rabbit hunting) and were muzzled before being sent into rabbit burrows. this practice was later introduced into europe, asia, and the british isles, where the sport is still practiced today. although the ferret has been historically used for hunting, more recently it has been increasingly used in biomedical research and is popular in north america as a pet. it is most likely a domesticated version of the wild european ferret or polecat (m. putorius or m. furo) (thomson, 1951) . alternatively, it may be related to the steppe polecat (m. eversmanni), which it closely resembles in skull morphology (walton, 1977) . the domesticated ferret, although introduced to north america by the early english settlers some 300 years ago, has not established feral colonies on this continent. the ferret was not recognized as having potential as an animal model for biomedical research until the 1900s. early studies utilized the ferret in classic experiments with influenza virus pathogenesis (pyle, 1940) . its use was cited infrequently; an article published in 1940, detailing the use of ferrets in research, cited only 26 publications (pyle, 1940) . literature reviews undertaken in 1967, 1969, 1973, and 1985 , however, revealed an increasing appreciation for the ferret's usefulness and versatility in the study of human physiologic, anatomic, and disease mechanisms (hahn and wester, 1969; marshall and marshall, 1973; shump et al., 1974; frederick and babish, 1985) . in 1991, a bibliography containing "selected" literature citations on the ferret and its use in biomedical research was published (clingerman et al., 1991) . the document was designed to serve as a reference tool for individuals involved in the care or use of ferrets in the laboratory setting. although not comprehensive, the document provides extensive coverage of ferret biology, diseases, and use as an animal model. the domesticated ferret has been and continues to be used extensively in studies involving virology, reproductive physiology, anatomy, and endocrinology, as well as other areas of biomedical research (morgan and travers, 1998) . the ferret is also being used to replace the cat in some types of neuroendocrinology, neuroanatomy, and cardiology experiments. the ferret's increasing popularity in research and as a pet is mainly a result of large-scale commercial production. for example, commercial farms have been raising ferrets for almost 50 years. biomedical researchers in the united states can request animals of a specific sex, weight, and age for individual experiments. investigators in other countries may acquire fer-rets from fur operations or may make arrangements with commercial vendors in the united states. even though the ferret is nonstandardized with regard to exact genotype and pedigree, its routine availability in a clinically healthy state has aided immeasurably its acceptance as a research animal. readily available commercial stocks, based on coat color, are albino, sable (or fitch), siamese, silver mitt, and siamese-silver mitt (siamese with white chest and feet) (mclain et al., 1985) . the fitch or so-called wild coat color is the most common, recognized by yellow-buff fur with patches of black or dark brown, particularly on the tail and limbs (andrews and illman, 1987) . the production of ferrets by large commercial operations has raised concern by some that inbreeding of these animals has made the ferret more susceptible to diseases, e.g., endocrinerelated disorders. this topic is covered in more detail in chapter 21. housing of ferrets in a research facility is similar to that of other small carnivores such as cats (fox, 1998c) . ferrets tolerate low temperatures well and high temperatures poorly; the recommended temperature range for juvenile and adult animals is 4-18~ (hammond and chesterman, 1972) . ferrets less than 6 weeks of age should be housed at > 15 ~ c. kits under this age require a heat source if separated from the dam; older kits that are group-housed do not. elevated temperatures (>30~ cannot be tolerated by ferrets, because they have poorly developed sweat glands and are susceptible to heat prostration. signs of hyperthermia include panting, flaccidity, and vomiting. the preferred humidity is 40-65%. for nonbreeding animals that will remain in the facility for a short time, a conventional dark-light cycle at 12:12 hr is adequate. lighting may be altered to control breeding cycles. breeding and lactating jills should be exposed to 16 hr of light daily. ferrets that are maintained for breeding or for use beyond 6 months should be exposed to "winter" lightw6 weeks per year of 14 hr of dark dailywto maintain physiologic normalcy. it is also essential that researchers receiving time-pregnant jills preserve the photoperiod to which jills were exposed prior to shipment. failure to do so may cause inappetence, with subsequent negative energy balance and pregnancy toxemia. similar to other laboratory animal species, ferrets should be housed with 10-15 air changes per hour (usdhhs, 1996) . it is important to use nonrecirculated air because of the strong odor of ferrets and the susceptibility to respiratory tract infections. the ferret odor should not overlap into any rodent housing areas, because rodents have an instinctive fear of ferrets, and the ferret scent can disrupt rodent breeding and physiology (fox, 1998c) . female ferrets can be housed singly or in groups, but estrous females that are cohoused may become pseudopregnant (beck et al., 1976) . males should be housed individually after 12 weeks of age. molded plastic caging used to house rabbits works very well for ferrets. the solid bottom is perforated with holes and is readily sanitizable. an absorbable paper liner may be used in the pan beneath the cage to facilitate daily disposal of urine and feces. in a research setting, the plastic caging should be washed weekly to avoid excessive soiling. the spacing of grid walls should be 1.0 x 0.5 inches apart, or 0.25 inch if using wire mesh. ferrets like to lick and bite at their enclosures, so sharp edges and galvanized metal should be avoided. zinc toxicosis has been reported from licking galvanized bars from which metals had leached during steam sterilization (straube and walden, 1981) (table i) . ferrets can be trained to use a litter box because they repeatedly urinate or defecate in one corner of the cage. clay litters have been reported to cause chronic upper respiratory irritation parameter (jenkins and brown, 1993) . ferrets prefer sleeping in a soft isolated area, and in a research facility this can be accomplished by providing a washable "snooze tube" (fox, 1998c) . the thorax of the ferret is narrow and elongated, and as a result the trachea is proportionally long. this makes the ferret an ideal species for studies of tracheal physiology. the tracheal size and laryngeal anatomy make endotracheal intubation somewhat challenging, and as a result the ferret has been advocated as a species suitable for use in pediatric intubation training (powell et al., 1991) . the lungs are relatively large, and the total lung capacity is nearly 3 times that which would be predicted based on body size, as compared with other mammals. this characteristic, together with a higher degree of bronchiolar branching and more extensive bronchial submucosal glands (as compared with the dog), makes the ferret an attractive model for pulmonary research studies (vinegar et al., 1985) . although a previous report (willis and barrow, 1971) commented that the carotid arterial branching pattern in the ferret is unusual, it is actually typical for a carnivore. as is the case in the dog and the cat, the paired common carotid arteries arise from the brachiocephalic trunk (sometimes called the innominate artery) at the level of the thoracic inlet (andrews et al., 1979b) . the ferret's gastrointestinal tract is specialized to fit its carnivorous nature. the simple monogastric stomach is similar to that of the dog. there is no cecum present, and the indistinct ileocecal transition makes it difficult to identify the junction of the small and large intestines during a gross examination. the overall length of the alimentary tract is very short relative to the body size, resulting in a gastrointestinal transit time as short as 3 hr (bleavins and aulerich, 1981) . as in other mustelids, the paired anal scent glands of the ferret are well developed. although not as potent as those of the skunk, the secretions of the ferret are sufficiently odoriferous that many pet or research ferrets are descented. surgical techniques for this procedure have been described (creed and kainer, 1981; mullen, 1997) . ferrets, especially intact males and estrous jills, may possess a distinctive musky odor even after a successful descenting, because of normal sebaceous secretions. ferrets lack well-developed sweat glands for use in thermal regulation, and as a result they are predisposed to heat prostration when ambient temperatures reach 32~ (90 ~ f) (ryland et al., 1983) . extramedullary hematopoiesis is commonly found during histological examination of the spleen, and in some cases it may result in a grossly evident splenomegaly (erdman et al., 1998) . this must be differentiated from splenomegaly that can arise from a variety of pathologic conditions or from isoflurane administration (see section iii,e). experimental evidence suggests that ferrets have no naturally occurring antibodies against unmatched erythrocyte antigens, and that none develop even in the face of repeated transfusions . ferrets are seasonal breeders, and the resulting pronounced physiological variations in body weight, behavior, and gametogenesis are utilized in scientific studies of photoperiod responses and neuroendocrine control. prolonged estrus in unbred females can cause an aplastic anemia, an effect that can be reproduced with exogenous estrogen administration . the male has a radiographically evident os penis, and, contrary to some earlier reports, a prostate gland is present in males (evans and an, 1998) . newborn ferret kits weigh 6-12 gm at birth and will grow to 400 gm by the time they are weaned at 6-8 weeks (shump and shump, 1978) . in sexually intact populations, males (1.0-2.0 kg) can be twice the size of females (0.5-1.0 kg). the adult weight of nonobese male and female ferrets that have been gonadectomized prior to weaning and raised in captivity will generally fall between 0.8 and 1.2 kg (brown, 1997a) . adult animals (especially those that are sexually intact) may be subject to seasonal fluctuations in body fat percentage, which can cause body weight to fluctuate by 30-40% (fox and bell, 1998) . the approximate life span for the ferret is 6-8 years, but on rare occasions they may live as long as 11 years (table ii) . normal hematology and serum chemistry values have been reported for the ferret (thornton et al., 1979; lee et al., 1982; fox, 1998e) . these values are not greatly dissimilar from those of other domestic carnivores. one distinctive hematological characteristic of the ferret is the presence of a relatively robust erythron, characterized by hematocrit, hemoglobin, and total erythrocyte and reticulocyte counts that are generally higher than those of the dog or cat. reported neutrophil-lymphocyte ratios range from 1.7:1 to 0.7:1. representative hematology and chemistry ranges from one of our studies (fox et al., 1986b) are shown in tables iii and iv, but for diagnostic purposes any laboratory that evaluates ferret samples should develop its own set of specific normal ranges. a low-grade proteinuria may be identified by urinalysis in normal, healthy ferrets (thornton et al., 1979) (table v) . ferret diets have been formulated both empirically and based upon the nutrient requirements of other mustelids (fox and mclain, 1998) . specific requirements for various life-cycle stages have not been determined experimentally. available commercial diets are certainly capable of supporting growth, reproduction, and maintenance in conventional settings. in the (fox et al., 1986b) . bfour-to 8-month-old ferrets (loeb and quimby, 1999) . cnd, not done. absence of careful analysis, however, it is uncertain whether the proportion and quantity of ingredients in these diets is optimal. ferrets are strict carnivores with a high requirement for dietary fat and protein. their short digestive tract and rapid gastrointestinal transit time (3-4 hr) require protein to be readily digestible. there is general agreement that ferrets should not be given diets high in complex carbohydrates or fiber. diets that are high in fish products are also not recommended for ferrets (fox and mclain, 1998) . the use of any raw chicken, beef, or other meats is strongly discouraged because of the potential contamination by campylobacter, salmonella, listeria, mycobacterium, and streptococcus (fox, 1998a) . daily maintenance energy consumption for ferrets is 200-300 kcal/kg body weight. calorie-percent protein ratios have been determined for mink (mustela vison) kits up to and after 16 weeks of age (sinclair et al., 1962; allen et al., 1964) . a ratio of 13 and a caloric density of 550 kcal/100 gm of feed, corresponding to 42% protein, provided optimum growth for male kits up to 16 weeks. after 16 weeks, ratios of 17 and 21, corresponding to 36% and 26% protein, respectively, were recommended. diets containing 9-28% fat and 22-42% carbohydrate have been used successfully to maintain ferrets. one author recommends 30-40% protein and 18-20% fat for adult, nonbreeding animals and a minimum of 35% protein and 25% fat for reproductively active animals and those that have notreached sexualmaturity (brown, 1997a) . the long-term impact of diets containing high levels of fat and protein are unknown. ferrets have been used to investigate the absorption, metabolism, and interaction of the dietary micronutrients [3-carotene and vitamin e. ferrets, like humans, convert [3-carotene to vitamin a in the gut and absorb ~-carotene intact (fox and mclain, 1998) . in intestinal perfusion experiments in ferrets, it was demonstrated that [3-carotene, retinol, and retinyl esters are absorbed intact into lymph and that cleavage products, including [3-apo-12'-carotenal, [3-apo-10'-carotenal, and retinoids, accumulate in the intestinal mucosa (wang et al., 1992) . the intestinal mucosa is capable of converting [3-carotene into retinoic acid and other polar metabolites, which are then transported via the portal vein to the liver (wang et al., 1993) . [3-carotene absorption is enhanced by co-perfusion with a-tocopherol, and the perfusion of the latter is unaltered by the presence of [3-carotene. the conversion of [3-carotene into retinol is also enhanced by the presence of a-tocopherol (wang et al., 1995) . these and other findings have established the ferret as an important model for the study of these antioxidants. adult ferrets drink 75-100 ml of water daily, depending on the dry-matter content of the feed (andrews and illman, 1987) . fresh water can be provided ad libitum in stainless steel bowls or water bottles with sipper tubes. ferrets are playful and will overturn bowls or water bottles that are not well secured. features of ferret reproduction may be found in table vi for males, corresponding temporally to increasing day length. ferrets born in the late spring or early summer and maintained under natural lighting will not assume an adult pattern of gonadal activity (i.e., puberty) until the following season (baum, 1998) . under artificial illumination, jills that are maintained at 8 hr light-16 hr dark reach puberty at 10-12 months. stimulatory photoperiods may be used, however, in the laboratory or intensive production setting, as a method of breeding ferrets out of the natural season. however, the transfer from short to long photoperiods should not occur prior to 90 days of age, because jills that are prematurely transferred will remain anestrous (hammond and chesterman, 1972) . management practices in one breeding facility are such that jills commence breeding at 7-10 months, average 3.7 litters a year, and are cycled out of reproduction after 6 litters. in another strategy, ferrets are exposed to a 16:8 hr photoperiod at 12 weeks of age, are bred at 16 weeks during their first estrus, and whelp at 5v2 months. vulvar swelling is the hallmark of estrus in jills. the ease with which estrus is detected in the ferret, as well as the size of the ferret and ease of its maintenance in captivity have made the ferret a model for study of neuroendocrine events and their gonadal correlates. along with the hamster, the ferret has contributed extensively to an understanding of the photoperiodic influences on the hypothalamic-pituitary-gonadal axis (baum, 1998) . as in females of other species, estradiol concentrations are responsible for controlling the development of the female reproductive tract and secondary sexual characteristics, and the tonic inhibition of luteinizing hormone (lh) secretion by the anterior pituitary during both prepubertal life and anestrus. the sensitivity of the hypothalamic gonadostat to negative feedback inhibition by estradiol changes at the time of puberty, and under the influence of increasing light exposure, lh levels rise despite estradiol (ryan, 1984) . similarly, age differences in the sensitivity of negative feedback inhibition of the hypothalamic secretion of gonadotropin-releasing hormone (gnrh) by testosterone, or to estrogenic compounds derived from the aromatization of testosterone, appear to be essential in determining puberty and seasonality of reproduction in the male (baum, 1998) . estrus in jills is characterized by dramatic vulvar swelling from an anestrous diameter of 5-16 mm to an estrous diameter of 17-33 mm. changes in vaginal cytology have also been described for the ferret and other mustelid species, but these changes are seldom used to determine onset of estrus or to schedule breeding (williams et al., 1992) . after a 2-to 3-week proestrus, estrus occurs. estrus onset is not associated with elevated serum fsh in the ferret, as it is in the rodent. once estrus has occurred, it may terminate in coitus-induced ovulation and pregnancy, pseudopregnancy after infertile mating, pharmacologic termination (by injection of human chorionic gonadotropin (hcg) or gnrh), death due to estrogen-induced aplastic anemia, or spontaneous remission and anestrus due to reduced photoperiod. waves of follicular development occur in estrus, and 5-13 ova are ovulated approximately 30-40 hr after coitus. female ferrets are brought to the male approximately 14 days after vulvar enlargement. females and males copulate many times and for prolonged periods of time; they are typically left together for 2 days. both intromission and neck restraint by the male are apparently required for induction of ovulation (baum, 1998 ). an lh surge accompanies coitus in females, but the same is not true of males (carroll et al., 1987) . implantation occurs 12 days after mating; both a functional corpus luteum and the anterior pituitary are required for implantation and maintenance of pregnancy. placentation is typical of carnivores and is zonary and endotheliochorial (morrow, 1980) . pregnancy may be detected by ultrasonographic demonstration of 3-5 discrete nonechogenic structures as early as day 12 (peter et al., 1990) , by palpation as early as day 14, or by radiographic demonstra-tion of calcified fetal skeletons at approximately 30 days of gestation. jills within 2 weeks of parturition should be singly housed and provided with a secluded place in which to deliver their kits. when rabbit cages are used for housing, nest boxes may take the form of polypropylene rat cages or other plastic boxes (cat litter box or dishpan). nest boxes should have bedding provided for warmth and comfort. materials suitable for bedding include pieces of fabric (towels), ripped cageboard, shredded paper, or cotton batting. the nest box should be at least 6 inches deep and should prevent the kits from wandering from the jill. entrance to the nest box should be smooth, to avoid injury to the teats and mammary gland. at our institution, jills are provided a stainless steel rectangular box with a smooth-surfaced plastic entrance ( fig. 1) . a retractable steel roof panel and a guillotine side panel exposing a plexiglas sidewall allow access to the jill and permit observation with minimal disturbance. one major supplier of ferrets uses sunken tubs filled with bedding to promote a sense of security and isolation of the jill. most jills will leave the nest box to eat and drink. if the jill will not leave, however, low-sided food bowls should be placed within the nest box. parturition occurs rapidly in ferrets and may last as little as 2 -3 hr. primiparous jills typically deliver on day 41 of gestation whereas multiparous jills deliver on day 42. there are few signs of impending parturition, although abdominal enlargement and mammary development do occur in the last week or two. small litters (fewer than three) may result in inadequate stimulus for parturition. jills that pass their due date without delivery should be palpated for fetuses. kits remaining in utero beyond the 43rd day typically die; kits with congenital malformations such as cyclopia and exencephaly may also delay the initiation of labor. dystocia is common in ferrets because of positional abnormalities and fetal oversize and should be treated by cesarean section. jills tolerate cesareans well and will nurse kits delivered in this way. if small littel: size is responsible for delayed parturition, prostaglandins (0.5-1.0 mg lutalyse) may be used, followed by 0.3 ml oxytocin (6 u) after 3 hr (fox and bell, 1998) . failure to deliver within 8 hr of administration of prostaglandin is an indication for cesarean section. jills should be provided heat, energy, hydration, and analgesia following cesarean. kits will attempt to nurse soon after parturition, but jills experiencing difficult labor may not allow them to nurse until all kits are delivered. jills that are not attentive to their kits should be palpated for the presence of additional, undelivered kits. oxytocin may be used to facilitate delivery of remaining kits. offering the jill regular chow mixed with warm water may promote maternal acceptance. kits should be kept warm pending acceptance by the jill. jills should be left undisturbed for the first several days postpartum to avoid their cannibalizing the litter. cross-fostering to other jills may be successfully accomplished, provided that the kits are warm and that the foster jill has kits of similar age. kits to be fostered should be allowed to mingle with the foster jill's own kits while their dam is absent so that rejection due to olfaction will not occur. kits are born in an altricial state, covered by lanugo hair and with their eyes closed. by 3 days of age, albino ferrets retain their white hair whereas pigmented ferrets acquire a gray coat. they are completely dependent on the jill for the first 3 weeks of life. defecation and urination are stimulated by jills through anogenital licking of the kits. kits are born weighing 6-12 gm, double their weight in 5 days, and triple it in 10 days to a weight of 30 gm. the 3-week-old male kit should weigh at least 100 gm. sexual dimorphism in size is apparent by week 7 and persists into adulthood. developmental landmarks include ability to hear at 32 days, opening of the eyes at 34 days, eruption of deciduous teeth at 14 days, eruption of permanent canines at 47-52 days, and displacement of deciduous canines by 56-70 days (fox and bell, 1998) . gender may be distinguished in neonatal ferrets, as in other species, by anogenital distance, with the distance being much shorter in females than in males. in males, the urogenital opening is seen just caudal to the umbilicus. the prominent midline raphe penis overlying the palpable os penis is also a distinctive feature in the male. ferrets are typically weaned at 6 weeks of age. early weaning may be encouraged by making a slurry of the jill's chow available at 3-4 weeks; fat may be added to achieve a fat content of 30%. the fatty acid supplement linatone (lambert kay, cranberry, new jersey) is recommended by one author (brown, 1997a) . the diet should contain approximately 30% fat and 40% protein. the slurry should be fed twice daily for a restricted time and then removed to avoid having kits walking through and defecating in the diet. unthrifty kits over 14 days of age may be supplemented with canine or feline milk replacers administered per os by tygon-tipped pasteur pipette . weaned ferrets are best housed in groups until sexually mature. males over 12 weeks old may begin to fight if exposed to greater than 12 hr light per day. jills may return to estrus during the second or third week of lactation if they have fewer than 5 kits or 2 weeks after weaning if the litter is of normal size. jills should be rebred or administered hcg to terminate estrus, even if still lactating. a highquality, calorie-dense diet is required for lactation and to maintain pregnancy. if maintained on a stimulatory photoperiod and adequate nutrition, jills may have 2-3 liters of 6 or more kits yearly until they are 5 years old (fox and bell, 1998) . a nonstimulatory photoperiod should be used 6 weeks per year to rest the ferret and preserve maximum fertility; a maintenance diet can be given at this time. jills return to estrus approximately 3 weeks after reinstitution of the longer photoperiod. artificial insemination is not commonly performed in ferrets but has been studied in the context of providing strategies for species perpetuation of the endangered black-footed ferret (wildt et al., 1989) . synchronization of estrus as practiced in rodent production is not used as a tool of reproductive management in the ferret. synchronization ofjills may be approximated, however, by manipulation of photoperiod. with natural illumination in outdoor housing, jills all come into estrus within a 1-to 2-week period (baum, 1998) . in the laboratory setting, when jills are maintained in a nonstimulatory photoperiod (8 hr light-16 hr dark) for 6-8 weeks, followed by reversal of the cycle (16 hr light-8 hr dark), estrus will follow in 4 weeks (immature jills) or 3 weeks (mature jills) after the change (carroll et al., 1985) . this correlates with follicular development and increased plasma estradiol. the occurrence of infectious disease affects animal health and well-being and may complicate research efforts. a program combining good animal husbandry, optimal nutrition, health monitoring practices, and clinical care is essential to maintaining a healthy ferret colony. etiology. the etiologic agent is clostridium perfringens type a (clostridium welchii). epizootiology and transmission. clostridium perfringens is ubiquitous and is present in the intestinal contents of humans and animals. clostridium perfringens type a has been associated with the occurrence of acute abdominal distension, dyspnea, and cyanosis in weanling ferrets (field and laboratory service veterinary staff, 1984) and an outbreak of gastroenteritis in weanling black-footed ferrets (schulman et al., 1993) . the exact cause of these conditions is uncertain, but predisposing factors such as overeating, sudden changes in diet, the prolifer-ation of c. perfringens type a, and the production of overwhelming amounts of toxins are suspected (field and laboratory service veterinary staff, 1984; schulman et al., 1993) . the alpha toxin is the principal lethal toxin. it is hemolytic and necrotizing and possesses the ability to split lecithin or lecithinprotein complexes, leading to destruction of cell membranes and subsequent necrosis. reported cases have involved weanling animals exclusively. clinical signs. ferrets may present with acute abdominal distension, dyspnea, and cyanosis or may be found dead and bloated (field and laboratory service veterinary staff, 1984; schulman et al., 1993) . diagnosis. isolation of c. perfringens type a from gastric and small-intestinal contents is required. toxin identification may be performed by the use of a mouse protection assay (smith, 1975) . necropsy findings. gross findings include markedly distended stomachs and intestines containing a large amount of gas and a moderate amount of brown, semiliquid ingesta, and subcutaneous emphysema with minimal or no putrefaction (field and laboratory service veterinary staff, 1984; schulman et al, 1993) . histologic findings observed in weanling black-footed ferret cases included the observation of abundant gram-positive bacilli in smears of gastric and intestinal contents. other findings included varying degrees of gastrointestinal mucosal necrosis, numerous gram-positive bacilli lining the denuded mucosal surface and extending into the gastric glands and intestinal crypts; lymphoid necrosis of lymph nodes, spleen, and thymus; mild to moderate dilatation of central hepatic sinusoids with mild, acute, centrilobular hepatocellular dissociation and multifocal aggregates of small numbers of necrotic neutrophils within portal areas (schulman et al., 1993) . and feeding practices is the primary means of control. in the reported cases of c. perfringens type a-associated gastroenteritis in black-footed ferret weanlings, supportive care and gastric trocharization were unrewarding. the occurrence of the condition was eliminated by restricting feeding of weanlings to twice a day instead of 3 times daily. etiology. campylobacteriosis is caused by infection with campylobacter jejuni. epizootiology and transmission. campylobacter jejuni is a gram-negative, spirally curved microaerophilic bacterium that is recognized as a significant cause of human enteritis and is as-sociated with diarrheic illness in several animal species, including dogs, cats, cows, goats, pigs, mink, ferrets, and sheep (carter et al., 1995) . it also known to cause mastitis in cows, infectious hepatitis of chickens, and abortion in cattle, sheep, goats, dogs, and mink (carter et al., 1995) . the organism may also be cultured from the feces of normal asymptomatic dogs, cats, and ferrets carter et al., 1995) . transmission occurs by ingestion of organisms through direct contact with feces or contaminated food and water (carter et al., 1995) . there have been reports linking the disease in humans to pets. many of these outbreaks were associated with dogs, puppies, and kittens recently obtained from animal shelters or pounds and displaying diarrhea before the human illness occurred . isolation of campylobacter jejuni from asymptomatic ferrets also implies a potential for zoonotic transmission (fox et al., 1982 . clinical signs. experimental oral inoculation of ferret kits with various strains of c. jejuni produced a self-limiting diarrhea that ranged in character from very mild to watery manning, 1990a, 1991) . the presence of mucus and/or blood was also noted in the feces of affected animals. anorexia, dehydration, and tenesmus with watery diarrhea were also observed. intravenous inoculation of 4 pregnant mink and 7 pregnant ferrets resulted in reproductive failure, ranging from fetal resorption to expulsion of dead or premature living kits . oral inoculation resulted in abortion in a majority of the infected animals . diagnosis. diagnosis is based on history, clinical signs, and culture of affected animals. reports of spontaneous cases in ferrets require diagnostic confirmation and differentiation from cases of proliferative bowel disease and other infectious and noninfectious causes of diarrhea. campylobacter jejuni grows slowly and has specific culture requirements that involve the use of selective media or filtration techniques, and a requirement for thermophilic (42~176 and microaerophilic conditions (fox, 1998a) . cultures should be examined every 48 hours for round, raised, translucent, and sometimes mucoid colonies (fox, 1998a) . campylobacter jejuni revealed small focal neutrophilic infiltrates in the lamina propria of the colon of relatively few infected animals . bell and manning (1991) noted mild to moderate enterocolitis with neutrophilic infiltration of the lamina propria, which was most severe in kits with concurrent cryptosporidiosis. placentitis was the most notable histologic finding in pregnant ferrets and mink after experimental inoculation of a strain of an abortion storm-associated isolate of c. jejuni . erythromycin is the drug of choice for treatment of human campylobacteriosis (fox, 1998a) . in a study to eliminate the carrier state in ferrets, erythromycin was ineffective even though in vitro isolates of c. jejuni were sensitive to the antibiotic . according to the author, reasons for therapeutic failure included dose selection, interspecies differences in pharmacokinetics and possible reinfection. supportive care should be instituted, and choice of antibiotic therapy in confirmed diarrheic cases should be based on culture and sensitivity. in addition, because of its zoonotic potential, isolation of affected animals and good hygienic practices are recommended. reculture of animals after treatment to ensure elimination of the organism is recommended. epizootiology and transmission. in 1985, a gastric helicobacter-like organism was isolated from the margins of a duodenal ulcer of a ferret and named helicobacter mustelae (fox et al., 1986a (fox et al., , 1989a . subsequently, in the united states, gastritis and peptic ulcers have been routinely reported in ferrets colonized with h. mustelae (fox et al., 1988b (fox et al., , 1991a . every ferret with chronic gastritis is infected with h. mustelae, whereas specific pathogen-free (spf) ferrets not infected with h. mustelae do not have gastritis, gastric ulcers, or detectable igg antibody to the organism (fox et al., , 1991a . helicobacter mustelae has also been isolated from the stomachs of ferrets living in england, canada, australia and, most recently, from ferrets in new zealand (forester et al., 2000; tompkins et al., 1988) . koch's postulates have been fulfilled: by oral inoculation of h. mustelae into naive ferrets uninfected with h. mustelae, the infection induced a chronic, persistent gastritis similar to that observed in ferrets naturally infected with h. mustelae (fox et al., 1991b) . it is now known that h. mustelae colonizes nearly 100% of ferrets shortly after weaning. feces from weanling and adult ferrets have been screened for the presence of h. mustelae to determine whether fecal transmission could explain the 100% prevalence observed in weanling and older ferrets (fox et al., 1988b (fox et al., , 1992b . helicobacter mustelae was isolated from the feces of 8 of 74 nine-week-old and 3 of 8 eight-month-old ferrets. ferrets placed on proton pump inhibitors, which raise gastric ph, have a statistically higher recovery of h. mustelae from feces when compared with age-matched untreated control ferrets . clinial signs and pathology. helicobacter mustelae-infected ferrets examined in our laboratory are usually asymptomatic. ferrets with gastric or duodenal ulcers can be recognized clinically by vomiting, melena, chronic weight loss, and lowered hematocrit. clinical signs in ferrets with h. mustelae-associated gastric adenocarcinoma have consisted of vomiting, anorexia, and weight loss, signs that may be confused with gastric foreign body. diagnosis. gastric and duodenal ulcers are observable endoscopically. it is interesting that the ferret is the only domesticated animal to date that has naturally occurring helicobacterassociated ulcer disease. the h. mustelae isolated from ferrets has similar but not identical biochemical features to those of h. pylori, particularly in regard to the production of large amounts of urease. gastric samples collected by endoscopy or necropsy are minced with sterile scalpel blades and inoculated onto blood agar plates supplemented with trimethoprim, vancomycin, and polymixin b (remel, lenexa, kansas). the plates are incubated at 37~ or 42~ in a microaerobic atmosphere (80% n2, 10% h2, and 10% co2) in vented jars for 3-7 days. bacteria are identified as h. mustelae on the basis of gram-stain morphology; production of urease, catalase, and oxidase; resistance to cephalothin; and sensitivity to nalidixic acid. necropsy and findings. the histopathological changes occurring in the stomach closely coincided in topography with the presence of h. mustelae . a superficial gastritis present in the body of the stomach showed that h. mustelae was located on the surface of the mucosa but not in the crypts. inflammation occupied the full thickness of the distal antral mucosa, the so-called diffuse antral gastritis described in humans (fig. 2a,b) . in this location, h. mustelae was seen at the surface, in the pits, and on the superficial portion of the glands. in the proximal antrum and the transitional mucosa, focal glandular atrophy, a precancerous lesion, and regeneration were present, in addition to those lesions seen in the distal antrum. also, deep colonization of h. mustelae was observed focally in the affected antral glands. animals infected with helicobacter spp. may also be susceptible to gastric cancer yu et al., 1995) . there is recent documentation of the presence of argyrophilic bacteria, compatible in location and morphology to h. mustelae, within the pyloric mucosa of 2 male ferrets with pyloric adenocarcinoma . in humans, epidemiologic data strongly support the association between h. pylori and development of gastric adenocarcinoma. similarly, we have recently documented a series of h. mustelae-infected ferrets with gastric mucosa-associated lymphoid tissue (malt) lymphoma that parallels the same syndi'ome found in humans. lymphoma was diagnosed in the wall of the lesser curvature of the pyloric antrum, corresponding to the predominant focus ofh. mustelaeinduced gastritis in ferrets. gastric lymphomas demonstrated characteristic lymphoepithelial lesions, and the lymphoid cells were igg positive in all ferrets (erdman et al., 1997) . these findings and their parallels in h. pylori-infected humans implicate the involvement of h. mustelae in the pathogenesis of gastric cancer in ferrets. treatment. studies in ferrets indicate that triple therapy consisting of oral amoxicillin (30 mg/kg), metronidazole (20 mg/ kg), and bismuth subsalicylate (17.5 mg/kg) (pepto-bismol original formula, procter and gamble) 3 times a day for 3-4 weeks has successfully eradicated h. mustelae . clinical improvement, including increased appetite and resolution of melena, may occur within 48 hr of initiation of triple therapy. a new treatment regimen being used to eradicate h. pylori in humans has also been used successfully for eradication of h. mustelae from ferrets . ferrets received 24 mg/kg ranitidine bismuth and 12.5 mg/kg clarithromycin per os 3 times daily for 2 weeks. culture of tissue collected by gastric endoscopic biopsy at 16, 32, and 43 weeks after termination of treatment indicated that long-term eradication was achieved in all 6 ferrets. eradication was associated with decrease in anti-h, mustelae igg antibody titers, results that are consistent with findings in humans after h. pylori eradication. omeprazole in ferrets at an oral dose of 0.7 mg/kg once daily effectively induces hypochlorhydria and may be used in conjunction with antibiotics to treat h. mustelae-associated duodenal or gastric ulcers. cimetidine at 10 mg/kg tid per os can also be used to suppress acid secretion. acute bleeding ulcers must be treated as emergencies, and fluid and blood transfusions are essential. etiology. proliferative bowel disease is caused by intracellular campylobacter-like organisms, closely related to desulfovibrio spp., that are now classified as lawsonia intracellularis in proliferative enteropathy of swine (fox, 1998a) . the organisms are gram-negative, comma-to spiral-shaped bacteria. epizootiology and transmission. proliferative bowel disease is a common clinical disease observed in young ferrets. fecaloral spread is suspected. the disease typically involves the large bowel, although it has been observed to affect the small bowel (rosenthal, 1994) . campylobacter species, coccidia, and chlamydia have been isolated from some cases of proliferative bowel disease in ferrets (li et al., 1996b) . the role, if any, of copathogens in this disease is unclear. clinical signs. clinical signs include chronic diarrhea, lethargy, anorexia, weight loss (which is often marked), and dehydration. diarrhea may be blood-tinged, may contain mucus, and is often green in color. rectal prolapse may be observed in affected animals. ataxia and muscle tremors have also been observed (fox et al., 1982) . diagnosis. diagnosis is based on clinical signs, a palpably thickened colon, and colonic biopsy. it is important to rule out other causes of diarrhea and weight loss through diagnostic tests that include but are not limited to a complete blood count, chemistry profile, radiographs, and fecal analysis and culture. necropsy findings. gross findings include a segmented, thickened lower bowel, usually the terminal colon but occasionally including the ileum and rectum (fox et al., 1982; fox, 1998a) . histologic examination consistently reveals marked mucosal proliferation and intracytoplasmic l. intracellularis demonstrated with silver stain within the apical portion of epithelial cells in the hyperplastic epithelial cells (fox et al., 1982; fox, 1998a) (fig. 3a,b) . other common histologic changes observed include the presence of a mixed inflammatory infiltrate that is variable in severity, reduced goblet cell production, hyperplasia of the glandular epithelium, glandular irregularity with penetration of the mucosal glands through the muscularis mucosa, and an increase in thickness of the tunica muscularis (fox et al., 1982; fox, 1998a) . translocation of proliferating glandular tissue to extraintestinal sites, including regional lymph nodes and liver, has been described in two ferrets (fox et al., 1989b) . differential diagnosis. proliferative bowel disease should be differentiated from other diseases that may cause diarrhea and wasting, including dietary changes, eosinophilic gastroenteritis, gastric foreign bodies, lymphoma, aleutian disease, and gastric ulcers (bell, 1997b) . a complete physical exam that includes palpation of the abdomen should reveal a palpably thickened intestine in cases of proliferative bowel disease. treatment and control. supportive care, including fluid therapy and nutritional support, should be provided. treatment with chloramphenicol (50 mg/kg bid po, sq, im) or metronidazole (20 m/kg bid po) for 2 weeks is reported to be effective (krueger et al., 1989; bell, 1997b) . clinical improvement may be apparent within 48 hr. etiology. tuberculosis can be caused by a variety of mycobacteria, including mycobacterium bovis, m. avium, and m. tuberculosis. epizootiology and transmission. mycobacteria are aerobic, gram-positive, nonbranching, non-spore-forming, acid-fast rods. natural infections with mycobacterium bovis and m. avium have been reported in the ferret. ferrets are also susceptible to experimental infection with human tubercle bacillus. most reports of tuberculosis in ferrets are in animals that were used for research in england and the rest of europe between the years of 1929 to 1953 and were likely related to the feeding of raw poultry, raw meat, and unpasteurized milk to ferrets during this time (fox, 1998a) . the feeding of commercially prepared diets and widespread tuberculosis testing and elimination in livestock and poultry have resulted in the reduced incidence of the disease in ferrets. mycobacterium avium-infected wild clinical signs and necropsy findings. clinical signs and lesions are dependent on the infective strain. systemic infection with the bovine strain in ferrets results in disseminated disease with weight loss, anorexia, lethargy, death, and miliary lesions involving the lungs and other viscera (fox, 1998a) . progressive paralysis has also been reported in a case of spontaneously occurring bovine tuberculosis in a ferret (symmers and thomson, 1953) . mycobacterium bovis lesions contain numerous acid-fast bacilli within macrophages with little cellular reaction (fox, 1998a) . in contrast, infection of ferrets with the human tubercle bacilli results in localized infection, often confined to the site of injection and adjacent lymph nodes; microscopically few organisms are observed. an impaired cell-mediated response may account for the large number of organisms observed in m. bovis lesions. vomiting, diarrhea, anorexia, and weight loss were observed in a pet ferret with granulomatous enteritis caused by m. avium (schultheiss and dolginow, 1994) . granulomatous inflammation characterized by large numbers of epithelioid macrophages containing numerous acid-fast bacilli were present in the lamina propria and submucosa of the jejunum and pylorus. other sites of granulomatous inflammation included peripancreatic adipose tissue, mesenteric lymph nodes, spleen, and liver. a source of infection was not identified in this report. pulmonary infection with m. avium has also been reported in 3 ferrets in a zoo in france (viallier et al., 1983) . diagnosis. definitive diagnosis of tuberculosis requires isolation and identification of the organism from suspect tissue specimens. great care should be exercised in handling suspect clinical specimens, and an appropriately equipped laboratory should be identified for culture and identification of the organism. although there has been some experimental work in the area of the intradermal tuberculin skin-test response in ferrets and its apparent use in controlling tuberculosis in a breeding colony of ferrets, a tuberculin skin-testing regimen, including dose and type, has not been definitively characterized for clinical use in ferrets (kauffman, 1981) . treatment and control. because of the zoonotic risk, ferrets infected with m. bovis and m. tuberculosis should be euthanized (fox, 1998a) . recurrent m. bovis infection involving the palmar aspect of the wrist of a 63-year-old man, which developed after he was bitten by a ferret at the age of 12, was reported and demonstrates the zoonotic potential (jones et al., 1993) . mycobacterium avium infection is not reportable but may pose a risk to immunocompromised patients (fox, 1998a) . personnel at risk should be followed up by a physician for appropriate diagnostic testing (fox, 1998a) . etiology. salmonellosis is caused by infection with organisms of the genus salmonella. epizootiology and transmission. salmonella is a gram-negative, non-spore-forming, facultative anaerobic rod in the family enterobacteriaceae (carter et al., 1995) . infection is by the oral route. transmission may be direct from infected carrier animals or humans or through contaminated food products or water (carter et al., 1995) . several salmonella serovars have been isolated from mink with gastroenteritis and abortion (gorham et al., 1949) . contaminated raw meat products were suspected as the source in one outbreak. salmonella typhimurium was isolated in ferrets in an outbreak of clinical disease (coburn and morris, 1949) and several serotypes including s. hadar, s. enteritidis, s. kentucky, and s. typhimurium were isolated from the feces of ferrets surveyed in a research colony (fox et al., 1988a) . clinical signs and necropsy findings. clinical signs of an outbreak of s. typhimurium in ferrets included conjunctivitis, rapid weight loss, tarry stools, and febrile temperature fluctuations (coburn and morris, 1949) . gross findings in 2 ferrets 10 days after inoculation with s. typhimurium of ferret origin included marked tissue pallor, petechiae in the gastric mucosa, and the presence of melena in one and a dark-colored fibrinous exudate in the large intestine of the other ferret (coburn and morris, 1949) . studies involving experimental inoculation with s. enteritidis, s. newport, and s. choleraesuis via the oral route to healthy, distemper-infected, and feed-depleted ferrets and mink showed a fairly high resistance to infection (gorham et al., 1949) . only 2 animals of 29 in the diet-restricted group--1 ferret and 1 minknshowed clinical signs of infection after feeding s. newport culture. signs included lethargy, anorexia, trembling, and fecal blood. the gastrointestinal tract showed a large amount of mucus containing red blood cells; bits of desquamated epithelium and few mononuclear cells overlying the gastric mucosa; an exudate in the small intestine consisting of mu-coid material, red blood cells, and desquamated small intestinal villi; edematous villi in the ileum; and a diffuse infiltrate of the small intestinal mucosa with lymphocytes and macrophages. necrotic foci in the liver, spleen, and, less commonly, the kidney, as well as splenomegaly and visceral lymphadenopathy, were observed in chronic fatal infections (coburn and morris, 1949) . abortion and gastroenteritis have been reported in mink (gorham et al., 1949) . diagnosis. diagnosis is based on history, clinical signs, and isolation of the organism. the organism can be cultured on enrichment and selective media and then characterized serologically. samples of blood, feces, exudates, tissues, and intestinal material may be cultured. treatment and control. coburn and morris (1949) treated 6 of 12 ferrets experimentally infected with s. typhimurium with sulfathalidine in the feed (coburn and morris, 1949) . salmonella typhimurium was isolated in 4 of 6 control animals and none of the treated animals 3 days after the administration of the last dose. sulfathalidine was administered by the same authors to a colony of 77 ferrets in which an outbreak of salmonella occurred. the group was surveyed 2 days after sulfathalidine treatment and showed weight gain, improvement in condition, and a reduction in the number of salmonella-infected ferrets (coburn and morris, 1949) . salmonella spp. isolated from ferrets may show resistance to a number of antibiotics (fox, 1998a) . treatment includes appropriate use of antimicrobials and supportive care, which may include fluid therapy, nutritional support, maintenance of electrolyte balance, treatment of concurrent diseases, recognition of and attention to shock, and reduction of stress (fox, 1998a) . etiology. streptococcus zooepidemicus and other group c and g streptococci, escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, and bordetella bronchiseptica have been reported as primary and secondary bacterial pathogens in pneumonia in ferrets (fox, 1998a) . epizootiology and transmission. bacterial pneumonia may occur secondary to megaesophagus in the ferret. an influenza virus-bacteria synergism has been the subject of several studies in ferrets (fox, 1998a) . debilitated and immunosuppressed animals and animals with concurrent diseases such as influenza may be more susceptible to bacterial pneumonias (fox, 1998a) . clinical signs. clinical signs may include nasal discharge, dyspnea, lethargy, anorexia, increased lung sounds, cyanosis, and fever (rosenthal, 1997) . fulminant pneumonia may progress to sepsis and death (fox, 1998a) . diagnosis. diagnosis is based on history, clinical findings, a complete blood count, culture and cytology of a tracheal wash or lung wash, and radiographs (rosenthal, 1997) . differential diagnosis. diagnostic rule-outs include dilatative cardimyopathy, heartworm disease, mycotic pneumonia, pneumocystis pneumonia in immunosuppressed animals, neoplasia, and influenza. treatment and control. treatment should consist of appropriate antimicrobial therapy and supportive care, which may include the administration of oxygen, fluid therapy, and force feeding (rosenthal, 1997) . etiology. a variety of bacteria have been associated with abscesses and localized infection of the lung, liver, uterus, vulva, skin, mammary glands, and oral cavity. these include staphylococcus spp., streptococcus spp., corynebacterium spp., pasteurella, actinomyces, hemolytic escherichia coli, and aeromonas spp. (fox, 1998a) . epizootiology and transmission. abscesses in ferrets may result from wounds that are inflicted secondary to biting during fighting, playing, mating, or chewing sharp objects. clinical signs. localized or subcutaneous abscesses present as swellings with or without draining tracts. the swelling may be fluctuant. in most cases, the abscess is walled off and does not result in systemic signs (fox, 1998a) . abscesses or infection involving visceral organs may give rise to organ-specific and/or systemic signs. diagnosis. cytologic and gram staining of an aspirate of a suspect subcutaneous swelling will aid in the definitive diagnosis. culture and sensitivity of the aspirate should also be performed to identify the causative organism and guide appropriate antibiotic therapy. differential diagnosis. differential diagnosis of a subcutaneous swelling in a ferret should include myiasis, granuloma, hematoma, and neoplasia. treatment and control. prevention of ferrets from exposure to sharp objects in the cage and feed, and limiting the exposure of male and female during breeding, can minimize the occurrence of abscesses. treatment of localized abscesses should include appropriate antibiotic therapy and establishment of drainage and debridement if necessary. bacterial culture and sensitivity of the exudate should be performed. a broad-spectrum antimi-crobial may be used pending results of culture and sensitivity (orcutt, 1997) . etiology. gram-positive cocci such as streptococcus spp., staphylococcus aureus, and coliforms such as hemolytic e. coli are the most frequently associated organisms (bernard et al., 1984; bell, 1997a) . although the exact pathogenesis of mastitis in ferrets is not clear, a number of factors may play a role and include the stress of lactation, injury to mammary glands by the kits' teeth, environmental contamination, and the virulence of the organism. in one report, the causative organism, hemolytic e. coli, was cultured from the feces of mastitic and healthy ferrets and the oral cavity of suckling kits (liberson et al., 1983) . the high level of perineal contamination and the presence of the organism in the oral cavity of suckling kits may enhance transmission and introduction of this organism into mammary tissue. in another outbreak, the causative organisms were cultured from bovine meat fed prior to the outbreak, and the meat was suspected as a possible source. clinical signs. mastitis occurs in nursing jills and has been characterized as acute or chronic (bell, 1997a) . the acute form is reported to occur soon after parturition or after the third week of lactation. examination of affected jills reveals swollen, firm, red or purple, and painful glands. affected glands may quickly become gangrenous. the chronic form, which may occur when kits are 3 weeks old or as a sequela to the acute form, is characterized by glands that are firm but not painful or discolored. diagnosis. diagnosis is based on history, clinical signs, physical examination findings, and isolation of the causative organism. necropsy findings. in acute mastitis, grossly affected glands are swollen, and the skin overlying the gland may be discolored. surgical biopsies and necropsies of 8 ferrets with mastitis caused by hemolytic e. coli (liberson et al., 1983) revealed extensive edema, hemorrhage, and coagulative and liquefactive necrosis involving the glandular tissue as well as surrounding subcutaneous tissue. other findings included the presence of a mixed leukocytic infiltrate composed primarily of polymorphonuclear leukocytes; large numbers of bacteria; and thrombosis and necrosis of vessels within and immediately adjacent to areas of inflammation (liberson et al., 1983) . in an outbreak of mastitis in mink due to staphylococcus aureus and escherichia coli, histologic examination of affected glands revealed an acute suppurative mastitis with desquamation of alveolar epithelium, edema of the connective tissue stroma, alveoli filled with neutrophils and cellular debris, and lactiferous ducts filled with purulent exudate and mats of bacteria within lobules (trautwein and helmboldt, 1966) . treatment. broad-spectrum antibiotic therapy may be instituted pending culture and sensitivity results of the milk. enrofloxacin (2.5 mg/kg bid po after a loading dose of 5.0 mg/kg im) is often effective. jills may require aggressive care, because acute mastitis may progress rapidly and animals may become septicemic and moribund (liberson et al., 1983) . oral antibiotic administration to kits nursing affected jills is recommended (bell, 1997a) . surgical resection and debridement of affected glands and supportive care may be necessary for jills with acute mastitis. supplementation of kits with milk replacer may also be necessary, because jills with acute mastitis are reluctant to nurse, and jills with the chronic form have diminished lactation as milk-producing tissue is replaced by scar tissue (bell, 1997a) . maintaining thorough personal hygiene practices when handling affected jills is important in minimizing spread to other lactating jills. cross-fostering kits may be required; however, kits may spread infection to healthy jills. it is reported that jills with the chronic form of mastitis should be culled (bell, 1997a ). etiology. canine distemper (cd) is caused by a paramyxovirus of genus morbillivirus that is related to measles and rinderpest (budd, 1981) . there are several strains, including a ferret-adapted strain of canine distemper virus (cdv), that vary in incubation, clinical signs, and duration . the virus can be inactivated by heat, light, and various chemicals, including phenol, roccal, sodium hydroxide, and formalin (shen and gorham, 1980; budd, 1981) . infectious virions have been recovered from fomites after 20 min at room temperature. canine distemper is the most serious viral infection of ferrets. mortality approaches 100%, making appropriate husbandry and vaccination imperative. the disease has a catarrhal phase and a neurological, or central nervous system (cns), phase. the catarrhal phase is 7-10 days postinfection and involves anorexia, pyrexia, photosensitivity, and serous nasal discharge. an erythematous pruritic rash spreads from the chin to the inguinal region. it is suspected that the rash results from cell-mediated immunity to infected endothelial cells, similar to the response seen in humans with measles (norrby and oxman, 1990) . hyperkeratosis of footpads, called hard pad, is an inconsistent feature. secondary bacterial infections result in mucopurulent ocular and nasal discharge and possibly bacterial pneumonia. the cns phase, with ataxia, tremors, and paralysis, may or may not be preceded by the catarrhal phase. death occurs in 12-16 days from ferret strains of cdv and up to 35 days with canine strains. infection is uniformly fatal. epizootiology and transmission. virus is shed from infected hosts from conjunctival, nasal, and oral exudates, urine, feces, and sloughed skin (gorham and brandly, 1953) . transplacental infection is not reported in ferrets. attenuated cdv vaccine strains have not been recovered from the body secretions of ferrets following vaccination (shen et al., 1981) . unvaccinated dogs and other canids, mustelids, and procyonids may serve as reservoirs of infection. viremia is detectable 2 days postinfection and persists until the ferret dies or mounts a neutralizing antibody response (liu and coffin, 1957) . the primary site of replication is the respiratory and lymphatic systems, and cdv has been recovered from the nasal secretions of ferrets 5-13 days postinfection. a decrease in lymphocyte subsets is detectable 5-30 days postinfection. clinical signs and necropsy findings. histologically, intracytoplasmic and intranuclear inclusion bodies may be observed in tracheal, bronchial, epithelia, and bile duct as well as transitional epithelium in the bladder (liu and coffin, 1957) (fig. 4) . the eosinophilic (hematoxylin-eosin) inclusions appear orange using pollack's trichrome stain. diagnosis and differential diagnoses. presumptive diagnosis is based on clinical observation, questionable vaccination history, and exposure. a fluorescent antibody test can be used on peripheral blood and conjunctival mononuclear cells to detect infection. reverse transcriptase-polymerase chain reaction (rt-pcr) has also been used to detect experimental infection (stephensen et al., 1997) . differential diagnoses should include infection with influenza virus or bordetella bronchiseptica. influenza does not rapidly progress to mucopurulent ocular and nasal discharge as cd does. during an outbreak, clinically affected ferrets should be isolated and the remainder of the colony vaccinated. distemper infection can be prevented by vaccination with modified live vaccine of chicken embryo tissue culture origin (cetco) administered subcutaneously or intramuscularly. kits should be vaccinated every 2-3 weeks, starting at age 6 weeks, until 14 weeks and annually thereafter . it is important to adhere to the prescribed vaccination protocol, because ferret deaths have been reported following double-dose vaccination (carpenter et al., 1976) . inactivated distemper vaccines do not elicit consistent, effective immunity and are not recommended. it is important to know the vaccination schedule of your ferret supplier and to vaccinate supplementally as appropriate. new ferrets should be held in quarantine for 2 weeks prior to introduction into the resident colony. ferrets have been experimentally infected with feline panleukopenia, canine parvovirus, canine parainfluenza virus, mink enteritis virus, respiratory syncytial virus, transmissible mink encephalopathy, and pseudorabies, but natural infection with these viruses has not been reported . etiology. aleutian disease virus (adv) is a parvovirus with strains of varying virulence and immunogenicity. mink-derived strains are more virulent to mink than are ferret-derived strains . epizootiology and transmission. aleutian disease (ad) is a chronic progressive illness that was first described in mink (oxenham, 1990) . it was originally named hypergammaglobulinemia (hgg) because of this remarkable finding. infection may be subclinical for years. because the immunomodulation associated with adv infection is disruptive to biomedical research, it is important to seek sources of adv-free ferrets . transmission between ferrets may be direct or via aerosol of urine, saliva, blood, feces, and fomites (kenyon et al., 1963; gorham et al., 1964) . vertical transmission is established in mink but is unproven in ferrets. clinical signs. ferrets infected with adv as adults develop persistent infection but rarely disease, although chronic progressive weight loss, cachexia, malaise, and melena have been described (porter et al., 1982) . ad may also cause ataxia, paral-ysis, tremors, and convulsions (oxenham, 1990; welchman et al., 1993) . the lesions are typically immune-mediated, and there is elevation of the gammaglobulins to generally greater than 20% of the total proteins (porter et al., 1982; fig. 5 ). the precise mechanism of immunomodulation is unknown, but in mink there is depression of b-and t-cell responses. necropsy. ferrets may have no lesions upon necropsy, or infrequently they may have hepatosplenomegaly and lymphadenopathy. the most consistent histological finding is periportal lymphocytic infiltrates (fig. 6 ). bile duct hyperplasia and periportal fibrosis have also been reported. membranous glomerulonephritis has been described (ohshima et al., 1978) . although lesions are subtle, use of adv-infected ferrets in biomedical research is contraindicated because histological lesions interfere with the interpretation of study results . diagnosis and differential diagnoses. presumptive diagnosis is based on hgg and chronic weight loss. diagnosis is confirmed by immunofluorescent antibody (ifa) or counterimmunoelectrophoresis (ciep) for antibody to adv antigen (palley et al., 1992) . pcr-based assays have also been used (erdman et al., 1996b; saifuddin and fox, 1996; erdman et al., 1997) . differential diagnoses include the neurotropic form of cd, as well as chronic wasting diseases such as neoplasia, malabsorption, maldigestion, and bacterial enteritis . vaccination against adv would be contraindicated because of the immune-mediated reaction, and a vaccine is not available. chemical disinfection may be achieved tp -10.6 g/dl 9 /globulin = 5.75 g% , .. with formalin, sodium hydroxide, and phenolics (shen et al, 1981) . there is no treatment for ad, and infected ferrets should be culled from the colony. etiology. influenza is caused by an orthomyxovirus that is transmissible from humans to ferrets and ferrets to humans (smith and stuart-harris, 1936) . human influenza viruses a and b are pathogenic to ferrets . ferrets are also susceptible to avian, phocine, equine, and swine influenza, although only porcine influenza causes clinical signs. because the viruses can be readily transmitted from humans to ferrets, handling precautions such as wearing masks and gloves should be in place to minimize transmission. epizootiology, transmission, and clinical signs. influenza virus generally remains localized in nasal epithelium in ferrets but may cause pneumonia. clinical signs appear 48 hr postinfection and include anorexia, fever, sneezing, and serous nasal discharge. conjunctivitis, photosensitivity, and otitis are also sometimes seen . secondary bacterial infection by streptococcus sp. and occasionally bordetella bronchiseptica may prolong recovery. transmission occurs via aerosol and direct contact. diagnosis. diagnosis is based on typical clinical presentation and recovery within 4 days, unlike with cd, which progresses to more severe disease and death. hemagglutination inhibition antibody titers on acute and convalescent sera are rarely needed. treatment and control. antibiotic therapy may be instituted to preclude secondary bacterial infection. animal technicians and investigators suffering from influenza should avoid contact with ferrets. ferrets have been used extensively as a model for influenza research because the biological response to infection is similar to that in humans . ferrets have been used in influenza a research to study pathogenesis, to investigate reye's syndrome, and to evaluate vaccine trials (deshmukh, 1987; sweet et al., 1987) . etiology. rabies is caused by a rhabdovirus. rabies infection is infrequently reported in ferrets, and until recently, research on rabies in ferrets was lacking . ferrets in a well-managed facility would have low risk of exposure to rabies virus. a usda-approved, killed rabies vaccine given subcutaneously at ages 3 months and 1 year and annually thereafter is recommended to protect ferrets against rabies (rupprecht et al., 1990) . modified live vaccine (mlv) is not recommended, because there is at least one case of rabies in a ferret that was vaccinated with mlv rabies vaccine . there is no treatment for rabies. clinical signs and pathogenesis. clinical signs of rabies infection in ferrets may include anxiety, lethargy, and posterior paresis. in one experimental infection, 11 of 40 ferrets died, and negri bodies were seen in the brain of only 2 of the 11 (blancou et al., 1982) . there is conflicting data on the isolation of rabies virus from the salivary glands following experimental infection. in one study using raccoon variant of rabies for infection, more than half of the ferrets had rabies isolated from the salivary glands . ferrets at risk for exposure to rabies virus that bite or scratch a human should be placed under quarantine for not less than 10 days of observation. veterinarians and facility managers should seek assistance from state public health officials. diagnosis and differential diagnoses. differential diagnosis includes the neurotropic form of cd. diagnosis is based on direct ifa of brain tissue. because rabies in ferrets is poorly understood, the head from ferrets that exhibit signs compatible with rabies and that have exposure histories that raise concerns about rabies should be shipped to the state public health authority for confirmation. etiology. rotaviruses cause diarrhea in young of many species, including humans, calves, pigs, sheep, and rats. diarrhea in ferret kits is thought to be caused by a poorly characterized, atypical rotavirus that has not been cultivated in vitro (torres-medina, 1987) . atypical rotaviruses lack the rotavirus common antigen. epizootiology, transmission and clinical signs. clinical disease may occur in kits as young as 1-4 days old or in older animals up to 6 weeks of age. diarrhea soils the perineum and possibly the fur and nest material. mortality rates are agedependent, with high mortality occurring in young kits and lower mortality occurring in kits over 10 days of age (bell, 1997a; fox et al., 1998b) . secondary bacterial infection may influence the severity of diarrhea. necropsy andpathogenesis. lesions are restricted to the gastrointestinal tract. yellow-green liquid or mucous feces may be seen in the terminal colon on necropsy. subtle small-intestinal villous atrophy and epithelial cell vacuolation are detectable histologically. diagnosis and differential diagnoses. clinical diagnosis can be confirmed by using clarified and ultracentrifuged fecal pel-lets for electron microscopy. the ferret rotavirus does not crossreact with commercially available enzyme immunoassays (torres-medina, 1987) . it is desirable to avoid sources that are known to be infected with ferret rotavirus. affected kits may be supplemented with kitten milk replacer, using a medicine dropper. mortality is reduced if the kits continue nursing. treatment of secondary bacterial infections may reduce severity of the diarrhea, and supportive care, including subcutaneous fluid administration for young kits, may be required . jills develop immunity to rotavirus infection, and subsequent litters are protected. infectious bovine rhinotracheitis (ibr) was isolated from the liver, spleen, and lung of clinically normal ferrets (porter et al., 1975) . raw beef was suspected as the source of infection, reinforcing the need to exclude raw meat products from the diet of ferrets used for research. ibr does cause significant respiratory pathology in experimentally infected ferrets (porter et al., 1975) . a transmissible diarrhea, referred to as epizootic catarrhal enteritis, has been observed in adult ferrets several days after direct contact and fomite exposure to affected ferrets . clinically the diarrhea is green and bile-tinged, and the ferrets become rapidly dehydrated. mortality is low. some ferrets develop elevated liver enzymes. treatment involves aggressive oral and systemic fluid therapy. a recent study implicates a coronavirus as the cause of this disease (williams et al., 2000) . a. protozoa i. enteric coccidiosis etiology. three species of the genera isospora and eimeria have been reported to infect the ferret: isospora laidlawi, eimeria furonis, and e. ictidea (blankenship-paris et al., 1993) . tion of sporulated oocysts. clinical signs. coccidiosis in ferrets is usually subclinical but has been reported to be associated with diarrhea, lethargy, and dehydration in one ferret (blankenship-paris et al., 1993) . clinical signs are often seen in young, newly acquired ferrets and are more common after a stressful event (rosenthal, 1994) . rectal prolapse can also develop in association with coccidial infection (rosenthal, 1994) . diagnosis. diagnosis is generally made by any of the fecal flotation methods commonly used in veterinary practice or by direct wet mount of feces and microscopic examination for sporulated or unsporulated oocysts. because coccidial oocysts are small, slides should be examined under higher magnification. necropsy findings. diagnosis is usually performed antemortem. pathologic lesions associated with enteric coccidiosis in a laboratory-reared ferret that was euthanized were described in one published report (blankenship-paris et al., 1993) . microscopic lesions were confined to the jejunum and ileum and consisted of villous and epithelial thickening. parasitic cysts and microorganisms within epithelium, and a mild granulomatous inflammation in the villar lamina propria, were also observed. a recent report documents clinical and anatomic pathology associated with biliary coccidiosis in a weanling ferret (williams et al., 1996) . differential diagnosis. diarrhea may be observed in ferrets that present with gastroenteritis secondary to gastrointestinal foreign bodies and dietary indiscretion, as well as other nutritional, inflammatory, infectious, or other systemic diseases. infectious causes such as proliferative colitis, salmonellosis, giardiasis, rotavirus, and campylobacteriosis should be considered. diarrhea may also be seen in eosinophilic gastroenteritis, an uncommonly reported condition in ferrets. good husbandry practices that include sanitation and frequent disposal of feces reduce the number of oocysts in the environment. cleaning cages with a strong ammonium hydroxide solution is reported to be effective (kirkpatrick and dubey, 1987) . heat treatment of surfaces and utensils may also be effective (kirkpatrick and dubey, 1987) . treatment of ferrets with sulfadimethoxine at 50 mg/kg orally once and then 25 mg/kg orally every 24 hr for 9 days is recommended (rosenthal, 1994) . as in dogs and cats, the complete elimination of a coccidial infection requires an immunocompetent host. ii. cryptosporidiosis etiology. cryptosporidiosis is caused by infection with cryptosporidium spp. epizootiology and transmission. cryptosporidium is a protozoan in the class sporozoa, subclass coccidia, that inhabits the respiratory and intestinal epithelium of birds, reptiles, mammals, and fish (regh et al., 1988) . it is known to cause gastrointestinal tract disease in many species, including rodents, dogs, cats, calves, and people (hill and lappin, 1995) . it has a life cycle similar to other coccidian parasites and is transmitted by ingestion of sporulated oocysts. autoinfection is also a characteristic of the life cycle. transmission may occur through consumption of contaminated food or water. cattle, dogs, and cats, shedding oocysts, are reported to be potential sources of human infection (hill and lappin, 1995; fox, 1998g) . immunosuppressed people are at greatest risk of developing severe fulminating gastrointestinal disease (hill and lappin, 1995) . the finding of cryptosporidiosis in two ferrets that died from unrelated causes in one animal facility resulted in a survey of the existing ferret population and new arrivals into the facility to determine the prevalence and incidence of infection (regh et al., 1988) . findings indicated that 40% of the resident population and 38-100% of new arrivals had oocysts in their feces but showed no clinical signs. clinical signs. only subclinical infection has been reported in both immunocompetent and immunosuppressed ferrets (regh et al., 1988) . diagnosis. diagnosis is based on the identification of the organism in feces. the oocysts are small when compared with other coccidia and may be overlooked or mistaken for yeasts (kirkpatrick and dubey, 1987) . yeasts are oval, whereas cryptosporidium oocysts are spherical or ellipsoidal. additionally, yeasts will stain with iodine and are not acid-fast, whereas cryptosporidium has the opposite staining characteristics. the oocyst residuum is seen as a refractive dot under phase-contrast microscopy, a structure lacking in yeast (kirkpatrick and dubey, 1987) . sugar-solution centrifugation and fecal sedimentation using formalin-ether or formalin-ethyl acetate are effective di-agnostic concentration techniques (hill and lappin, 1995) . oocysts may then be viewed with phase-contrast or bright-field microscopy of specimens stained with an acid-fast method. a direct fecal smear may be methanol-or heat-fixed and stained with an acid-fast method (hill and lappin, 1995) . necropsy findings. histologic evaluation reveals the presence of organisl3ns, spherical to ovoid in shape and from 2 to 5 ~tm in diameter, associated with the brush border of the villi. a mild eosinophilic infiltrate was observed in the lamina propria of the small intestine in most animals. the ileum was the most common and heavily infected section of small intestine (regh et al., 1988) . there is no known definitive treatment for cryptosporidiosis (fox, 1998g) . supportive and symptomatic care should be provided in clinical cryptosporidiosis. infections are self-limiting in immunocompetent patients (fox, 1998g) . control is aimed at eliminating or reducing infective oocysts in the environment and avoidance of contact with known sources. because of the potential for zoonotic transmission, restricting contact of children and immunosuppressed individuals with infected ferrets and practicing good hygiene may help reduce the potential for infection. drying, freeze-thawing, and steam cleaning inactivate the organism (hill and lappin, 1995) . there are few effective commercial disinfectants. i. sarcoptic mange etiology. sarcoptic mange is caused by infection with sarcoptes scabiei. epizootiology and transmission. transmission occurs through direct contact with infected hosts or contact with fomites. this parasitic infection is rare under research conditions. clinical signs. infection of ferrets with s. scabiei may occur in a generalized or a pedal form (bernard et al., 1984) . in the generalized form, lesions consist of focal or generalized alopecia with intense pruritus. in the pedal form, lesions are confined to the toes and feet, which become swollen and encrusted with scabs. nails may be deformed or lost if the condition is left untreated. diagnosis. diagnosis is made by finding the mites in skin scrapings or removing crusts, breaking them up, and clearing with 10% koh for microscopic examination (phillips et al., 1987) . false-negative results are possible; multiple scrapings may be necessary. differential diagnosis. differential diagnosis should include other pruritic external parasitic conditions, including flea infes-tation. demodicosis has been reported to cause mild pruritus and alopecia in ferrets (noli et al., 1996) . in the pedal form, treatment consists of trimming the claws and removing the scabs after softening them in warm water (bernard et al., 1984) . treatments that have been used include ivermectin, 0.2-0.4 mg/kg, administered subcutaneously and repeated every 7-14 days until mites are gone; shampoos or soaks to reduce the pruritus; and topical or systemic antibiotic administration for treatment of secondary bacterial dermatitis (hillyer and quesenberry, 1997b) . alternatively, weekly dips in 2% lime sulfur until 2 weeks after clinical cure have been shown to be effective (fox, 1998a) . treatment of all affected animals as well as contact animals, and decontamination of enclosures and bedding, are recommended. ii. demodicosis etiology. demodicosis is caused by infection by demodex spp. epizootiology and transmission. the parasite is found in normal skin of almost all dogs and is not considered contagious. predisposing factors such as immunologic or genetic conditions have been suggested (kwochka, 1986) . one clinical report describes demodicosis in two adult ferrets that had been treated with an ear ointment containing triamcinolone acetonide for recurrent ear infections daily for 3 periods of 3 months each during the course of a year (noli et al., 1996) . clinical signs. in the report mentioned above, the ferrets presented with alopecia, pruritus, and orange discoloration of the skin behind the ears and on the ventral surface of the abdomen and an accompanying seborrhea (noli et al., 1996) . diagnosis. deep skin scrapings should be performed to demonstrate mites. finding a large number of live adult mites or immature forms and eggs is necessary to confirm the diagnosis. in very chronic cases, the skin may be so thickened that scrapings may be unrewarding. in these cases, a skin biopsy may be diagnostic (kwochka, 1986) . necropsy findings. histologic evaluation of skin biopsies obtained in the case report described above revealed mites with a short, blunted abdomen similar to that of demodex criceti and located in the infundibulum of hairs. the epidermis was slightly hypertrophic, and there was a mild superficial orthokefatotic hyperkeratosis. a very mild superficial and perivascular mixed cellular infiltrate was also observed in the dermis. differential diagnosis. generalized demodicosis should be differentiated from sarcoptic mange and flea infestation. primary or secondary bacterial dermatitis or pyoderma should also be considered. treatment and control. the ferrets in the above-mentioned clinical report were treated initially with a suspension of 0.0125% amitraz applied as a dip 3 times at 7-day intervals for 3 treatments. two drops of the same solution were applied in each ear every other day. after the initial treatment, the ferrets were reexamined, and treatment was continued with the same concentration of solution applied once every 5 days, while the tail was washed with a higher concentration of amitraz (0.025%) once every other day. thereafter, 3 final treatments with 0.0375% amitraz every 5 days for the body, and every other day for the ears and tail, were administered. the ferrets were evaluated and skin scrapings were performed regularly during treatment and posttreatment to monitor response to therapy. treatment of any associated pyodermas, systemic illnesses, or management problems should also be included as part of the therapeutic regimen. iii. ear mites etiology. the ear mite, otodectes cynotis, which commonly infects dogs and cats, is also a common clinical problem in ferrets (fox, 1998g) . epizootiology and transmission. ear mites are transmitted through direct contact with infested ferrets, dogs, or cats (fox, 1998g) . the entire life cycle is completed in 3 weeks. clinical signs. ear mite infestation in the ferret is usually asymptomatic (orcutt, 1997) . however, clinical signs may include head shaking; mild to severe pruritus with inflammation and excoriation; secondary otitis interna with ataxia; circling; torticollis; and horner's syndrome (orcutt, 1997; fox, 1998g) . a brownish black waxy discharge is often present. diagnosis. diagnosis is based on direct observation of mites via otoscopic examination or microscopic identification of the ear mite or any of the life-cycle stages of the mite in exudate from the ear canal. treatment and control. several treatment regimens, including topical and injectable mitocidal treatments, have been recommended (orcutt, 1997; fox, 1998g) . a recent study using three treatment regimens--two topical and one injectable--revealed that topical treatments were more efficacious than the injectable in reducing or eradicating ear mites (patterson et al., 1999) . efficacy was evaluated by microscopic evidence of ear mites in debris from aural swabs taken weekly for an 8-week period. topical 1% ivermectin (ivomec, merck agvet division, rahway, new jersey), diluted 1" 10 in propylene glycol at a dosage of 400 ~tg/kg body weight divided equally between the two ear canals and administered on days 1 and 14 of the study, was the most effective treatment. all susceptible animals in a household should be treated. ears should be gently cleaned prior to initiating treatment (orcutt, 1997) . high doses of injectable iver-mectin (0.2 ml of 1% ivermectin) administered to jills at 2-4 weeks of gestation resulted in high rates of congenital defects (orcutt, 1997) . iv. fleas etiology. ctenocephalides species can infest ferrets. epizootiology and transmission. transmission requires direct contact with another infested animal or a flea-infested environment. clinical signs. flea infestation may be asymptomatic or may cause mild to intense pruritus and alopecia of the dorsal thorax and neck (timm, 1988) . differential diagnosis. sarcoptic and demodectic mange should be included in the differential diagnosis of pruritic skin disease in the ferret. close examination of the pelage for fleas or flea excrement should be performed. skin scrapings may be indicated. as with flea infestation in dogs and cats, concurrent treatment of the environment, as well as all animals in the household, is essential for effective flea control. compounds approved for flea control in cats such as rotenone or pyrethrin powders or sprays may be used in ferrets (hillyer and quesenberry, 1997a) . ferrets may develop systemic disease from blastomyces, coccidioides, cryptococcus, and histoplasma. the reservoir of most of these fungi is the soil, however, making infection unlikely in a research facility. in production facilities, exposure can be minimized through careful selection of source animals, appropriate sanitation, and control of pests, particularly birds. pneumocystis carinii has been recently reclassified as a fungus. although p. carinii inhabits the lungs of many different species, recent transmission studies suggest that these fungi are highly species-specific (gigliotti et al., 1993; fox et al., 1998b) . clinical disease is evident only in immunocompromised ferrets and can be induced using high doses of exogenous steroids (stokes et al., 1987) . lesions include interstitial pneumonitis with mononuclear cell infiltrates; cysts and trophozooites are evident with gomori methanamine-silver nitrate and giemsa on bronchoalveolar lavage. treatment with trimethoprim sul-famethoxazole probably controls but does not eliminate infection . ferrets are susceptible to secondary fungal infection of the outer ear canal with absida corymbifera or malassezia spp. (dinsdale and rest, 1995; fox, 1998d) . the fungi are widespread in the environment and will cause a secondary fungal infection in the ears of ferrets infested with otodectes cynotis. the yeasts can be visualized by impressions of ear exudates. treatment involves eradication of the underlying mite infestation followed by oral and topical ketoconazole, miconazole, and polymyxin b. dermatomycoses in ferrets are caused by microsporum canis and trichophyton mentagrophytes. dermatophytes are transmissible to humans and are a zoonosis; thus affected animals should be quarantined and removed from the facility to minimize risk (dinsdale and rest, 1995; scott et al., 1995; fox et al., 1998b) . control of infection includes general disinfection and destruction of contaminated bedding. lesions are circumscribed areas of alopecia and inflammation, which begin as small papules that spread peripherally in a scaly inflamed ring. the yellow-green fluorescence of m. canis under ultraviolet light helps distinguish it from t. mentagrophytes. skin scrapings digested with 10% potassium hydroxide reveal characteristic arthrospores. treatment with griseofulvin causes clinical remission but may not clear infection. other ectoparasitic infections observed to occur in ferrets include cutaneous myiasis and tick infestation. granulomatous masses in the cervical region caused by the larval stage of hypoderma bovis have been reported in ferrets (fox, 1998g) . cuterebra larvae, although uncommonly observed in ferrets, may cause subdermal cysts found in the subcutis of the neck (orcutt, 1997) . infestation with the flesh fly has been reported as a problem in commercially reared mink and ferrets housed outdoors (fox, 1998g) . ticks may be found on ferrets housed outdoors or on those used for hunting rabbits (fox, 1998g) . ticks should be removed carefully with hemostats or tweezers, ensuring that the entire head and mouthparts are removed from the skin. appropriate caution should be exercised in tick removal, because ticks are responsible for transmission of various zoonotic pathogens; gloves should be worn. etiology. the ferret is susceptible to natural and experimental infection with dirofilaria immitis. epizootiology and transmission. dirofilaria immitis is a filarial parasite that is transmitted by mosquitoes, which serve as the intermediate host and vector. microfilaria are ingested by mosquitoes and, after two molts, become infective third-stage larvae. infective larvae are deposited onto the skin when mosquitoes feed, and larvae find their way into the body of the final host through the bite wound and migrate subcutaneously to the thorax and eventually to the heart (knight, 1987) . the primary reservoir of infection is dogs, but heartworm may be found in a variety of mammals, including humans. all other species except wild and domestic canids, domestic felines, ferrets, and the california sea lion are considered aberrant hosts (knight, 1987) . clinical signs. the following clinical signs have been reported in clinical reports describing cases of d. immitis in the ferret: weakness, lethargy, depression, dyspnea, cyanosis, anorexia, dehydration, cough, and pale mucous membranes (miller and merton, 1982; parrott et al., 1984; moreland et al., 1986) . moist lung sounds and/or muffled heart sounds were revealed by thoracic auscultation in many of these cases. pleura/or abdominal effusion may be observed radiologically. the ferrets described in these cases were housed outdoors and either died or were euthanized. diagnosis. diagnosis of heartworm is based on clinical signs, radiographic findings, and testing for circulating microfilariae and heartworm antigen. microfilaremia is not consistently observed in naturally occurring and experimental cases of heartworm infection in ferrets (fox, 1998g) . testing for heartworm antigen appears to be more diagnostically useful (stamoulis et al., 1997) . in a study to determine the minimum oral dose of ivermectin needed for monthly heartworm prophylaxis in ferrets, the use of an antigen test (uni-tec canine heartworm test, pitman-moore co., mundelein, illinois) detected infection in more untreated control animals than did the modified knott test for detection of circulating microfilaria in the same ferrets (supakorndej et al., 1992) . necropsy findings. cardiomegaly, pleural and/or abdominal fluid, and pulmonary congestion are common findings at necropsy. grossly, adult worms have been observed in the right atrium, right ventricle, pulmonary artery, and cranial and caudal vena cava. microscopically, microfilaria may be seen in small and large vessels of the lung. differential diagnosis. differential diagnosis should include primary cardiac diseases, such as dilatative cardiomyopathy, and other systemic or pulmonary diseases. control is best directed at prevention through the administration of heartworm preventative and it is recommended that ferrets in heartworm-endemic areas receive monthly oral ivermectin throughout the year (stamoulis et al., 1997; fox, 1998g) . the dosage recommended for ferrets by the american heartworm society is 0.006 mg/kg body weight monthly (fox, 1998g) . housing ferrets indoors, particularly during the mosquito season, would help minimize exposure. successful adulticide treatment in ferrets has been described and includes the administration of thiacetarsemide, with the same precautions used in dogs: antithrombotic therapy, treatment for heart failure, and strict cage confinement (stamoulis et al., 1997) . one should follow up with heartworm antigen tests until negative and resume heartworm prevention 1 month after adulticide treatment (stamoulis et al., 1997) . ferrets are also susceptible to infection with the following nematodes: toxascaris leonina; toxocara cati; ancylostoma spp.; dipylidium caninum; mesocestoides spp.; atriotaenia procyonis; trichinella spiralis; filaroides martis; and spiroptera nasicola (rosenthal, 1994; fox, 1998g) . a day of their due date should include cesarean section and intensive postoperative support, including force-feeding a gruel of high-quality cat food and ferret chow, nutritive pastes, intravenous fluids containing glucose, and supplemental heat. cesarean section should be performed under isoflurane anesthesia because hepatic dysfunction prolongs the metabolism of injectable agents. agalactia is common after cesarean section, and kits may require hand feeding with kitten or puppy milk replacers, administered per os by fine-tipped syringe 6 times daily for the first 24 hr. cross fostering is an effective method of enhancing kit survival; hand rearing of kits if the jill fails to nurse within a day postoperatively is energy-consuming and generally unrewarding. for jills that develop pregnancy toxemia before day 40 of gestation, fluids and nutritional support must be provided until viable kits can be delivered by cesarean. pregnancy toxemia may be avoided by close monitoring of appetite of jills in late gestation, provision of a highly palatable diet with >20% fat and > 35 % crude protein, and avoidance of stress and dietary change. water should be made available in both bowls and water bottles, and food should be provided ad libitum in several bowls. pregnancy toxemia in the ferret occurs predominantly in primiparous jills carrying large litters. an inadvertent fast in late gestation is sometimes implicated. at least 75 % of jills carrying more than 8 kits will develop pregnancy toxemia if subjected to 24 hr of food withdrawal in late gestation (bell, 1997a; batchelder et al., 1999) . any jill with 15 or more kits may develop pregnancy toxemia because abdominal space is not adequate for both the gravid uterus and the volume of food required to support it. pregnancy toxemia of the ferret is of the metabolic type and shares features with similar conditions in pregnant sheep, obese cattle, pregnant camelids, obese guinea pigs, and starved pregnant rats, as well as with the condition feline idiopathic hepatic lipidosis. it is characterized by abnormal energy metabolism with consequent hyperlipidemia, hypoglycemia, ketosis, and hepatic lipidosis. in this condition, energy demand exceeds intake, leading to excessive mobilization of free fatty acids and a chain of metabolic events that culminates in a shift from fatty acid metabolism and export to ketosis and hepatic lipidosis. clinical signs include anorexia, lethargy, melena, dehydration, and easily epilated hair. differentials include dystocia, metritis, pyometra, septicemia, renal failure, and helicobacter mustelae-induced gastric ulcer. in a recent study of ferrets with pregnancy toxemia, consistent clinical chemistry abnormalities included azotemia (100%), hypocalcemia (83 %), hypoproteinemia (70%), and elevated liver enzymes (100%) (batchelder et al., 1999) . anemia was found in 50% of ferrets tested. necropsy findings include tan or yellow discolored liver, gastric hemorrhage, and gravid uterus. treatment forjills within ferrets are induced ovulators and may remain in persistent estrus if they are not bred or if estrus is not terminated chemically or via ovariohysterectomy (bell, 1997a) . jills that remain in estrus for more than 1 month are at risk for developing estrogeninduced anemia. hyperestrogenism from persistent estrus causes bone marrow hypoplasia of all cell lines in approximately half of ferrets in prolonged estrus (ryland et al., 1983) . clinical signs include vulvar enlargement, bilaterally symmetric alopecia of the tail and abdomen, weakness, anorexia, depression, lethargy, weight loss, bacterial infection, and mucopurulent vaginal discharge. hematology findings may vary from an initial neutrophilia and thrombocytosis early in the disease course to lymphopenia, thrombocytopenia, neutropenia, and anemia. the anemia begins as normocytic normochromic but progresses to macrocytic hypochromic (sherrill and gorham, 1985) . coagulopathy associated with hepatic dysfunction and thrombocytopenia combine to produce extensive manifestations of bleeding, pallor, melena, petechiation or ecchymosis, subdural hematoma, and hematomyelia (hart, 1985; fox and bell, 1998) . at necropsy, tissue pallor, light tan to pale pink bone marrow, hemorrhage, bronchopneumonia, hydrometra, pyometra, and mucopurulent vaginitis may be seen. histopathology may reveal cystic endometrial hypoplasia, hemosiderosis, diminished splenic extramedullary hematopoiesis, and mild to moderate hepatic lipidosis (sherrill and gorham, 1985; bell, 1997a) . treatment consists of terminating estrus while supporting the animal with antibiotics, blood transfusion, b vitamins, and nutritional supplementation. estrus may be terminated by injection with 50-100 iu of human chorionic gonadotropin (hcg) or 20 ~tg of gonadotropin-releasing hormone (gnrh), repeated 1 week after initial injection if required. ovariohysterectomy may be considered for ferrets that are stable and have adequate numbers of platelets and red cells. ferrets with a packed cell volume (pcv) of 25% or greater have a good prognosis and require only termination of estrus for resolution of aplastic anemia. jills with a pcv of 15-25% may require blood transfusions and have a guarded prognosis. ferrets with a pcv of less than 15% have a poor prognosis and require aggressive therapy with multiple transfusions. the lack of identifiable blood groups in ferrets makes multiple transfusions uncomplicated by potential transfusion reactions . estrogen-induced anemia may be avoided by ovariohysterectomy. of nonbreeding females, use of vasectomized hobs, or pharmacologic termination of estrus initiated 10 days after estrus onset. a 40-to 45-day pseudopregnancy then follows, except in the case of ovariohysterectomy. repeated administration of hcg may result in sensitization and anaphylaxis. after several administrations, hcg is unlikely to be effective in termination of estrus. anaphylaxis is manifest as incoordination, tremor, vomiting, and diarrhea and may be reversed by prompt administration of diphenhydramine. arginine-free diets are unlikely to be fed in the laboratory setting, but administration of such a diet to young ferrets fasted for 16 hr leads to hyperammonemia and encephalopathy within 2-3 hr (thomas and desmukh, 1986) . exacerbation of signs may be achieved by challenging young ferrets with influenza virus and aspirin (desmukh et al., 1985) and constitutes a model of reye's syndrome in children. lethargy and aggressiveness yield to prostration, coma, and death in affected ferrets. hyperammonemia presumably occurs because of the inability of ferrets to produce adequate amounts of ornithine from non-arginine precursors. detoxification of ammonia is thereby compromised. ferrets more than 18 months old are unaffected by arginine-free diets. ferrets of all ages are susceptible to zinc toxicosis, and the condition has been documented in two ferret farms in new zealand (straube and walden, 1981) . leaching of zinc from steam-sterilized galvanized food and water bowls was implicated. clinical signs included pallor, posterior weakness, and lethargy. definitive diagnosis requires demonstration of elevated concentrations of zinc in kidney and liver. at necropsy, kidneys are enlarged, pale, and soft; livers are orange, and gastric hemorrhage may be seen. histopathology reveals glomerular collapse, tubular dilation, tubular proteinaceous debris, focal cortical fibrosis, hepatic periacinar infiltration, and depression of the erythroid series. avoidance of galvanized materials precludes the development of zinc toxicosis. umbilical entanglement may occur in ferrets on the day of parturition and has been associated with fine-particle bedding, large litters, and short kit-birth intervals (bell, 1997a; fox et al., 1998a) . jills may neglect to clean placentas from their kits, or kits may be born so rapidly that there is not adequate time for the jill to clean the kits of placental membranes, thereby predisposing to entanglement. entangled kits may succumb to dehydration, hypothermia, and hypoglycemia because they are unable to nurse and the jill cannot curl around them. detailed dissection with fine scissors and forceps under a heat lamp or on a heated surface can free the kits. occasionally, kits may need to be rotated on their umbilical pedicle to achieve adequate clearance to cut the cord; cords should be cut as far from the umbilicus as possible. the use of warm saline or water may help soften the mass. some kits in the tangle may present with dark, swollen extremities or prolapsed umbilical cords and may require euthanasia. parturition should be supervised, if possible, to avoid umbilical entanglement. hydronephrosis may occasionally occur in the ferret and is most commonly associated with inadvertent ligation of the ureter during ovariohysterectomy. ovarian remnants are another potential sequela to ovariohysterectomy. ovarian remnants in ferrets may be associated with estrus, vulvar enlargement, and alopecia. appropriate diagnostic procedures include ultrasonography and plain and contrast radiography for hydronephrosis and ultrasonography and serum hormone concentrations for ovarian remnants. exploratory celiotomy confirms the diagnosis, and unilateral nephrectomy or ovariectomy is indicated if the remaining kidney is normal and the ferret is otherwise healthy. over the last few decades, increasing numbers of ferrets have been used in research or kept as pets, and as these animals have received veterinary care, it has become evident that ferrets are subject to a wide variety of neoplastic conditions . however, four categories of cancer account for the majority of ferret neoplasms: pancreatic islet cell tumors, adrenocortical cell tumors, lymphoma, and skin cancers. functional pancreatic islet cell tumors (insulinomas) are the most common neoplasm diagnosed in ferrets . disease may be evident in ferrets as young as 2 years old, but later onset (at 4-5 years of age) is typical (caplan et al., 1996; ehrhart et al., 1996) . nonspecific presenting signs include weight loss, vomiting, and ataxia. weakness is often evident, ranging from lethargy to posterior paresis or outright collapse (caplan et al., 1996) . hypoglycemia caused by excess production of insulin by neoplastic 13 cells may cause tremors, disorientation, or seizures (fox and marini, 1998) . excessive salivation (ptyalism) or pawing at the mouth is a frequent finding. clinical signs are often intermittent or episodic. other common findings include splenomegaly and lymphocytosis. presumptive diagnosis is made based on clinical signs in conjunction with the demonstration of hypoglycemia. blood glucose determinations for the diagnosis of insulinoma are most useful when taken after a 4 hr fasting period. fasting glucose concentrations below 60 may be diagnostic for the condition (quesenberry and rosenthal, 1997) , whereas values between 60 and 85 are suspect and the test should be repeated (fox and marini, 1998) . other potential causes for hypoglycemia should be ruled out, including anorexia, starvation, hepatic disease, sepsis, and nonpancreatic neoplasia (antinoff, 1997) . demonstration of concurrent hyperinsulinemia aids the diagnosis (caplan et al., 1996) . medical management using prednisone and/or diazoxide along with dietary modification such as frequent feeding of high-protein meals can minimize or control clinical signs but will not affect the underlying tumor (quesenberry and rosenthai, 1997) . surgical exploration of the pancreas and tumor excision are recommended for animals that are healthy enough to be subjected to anesthesia and surgery. histological examination of the tissue removed can provide a definitive diagnosis, and although the effect may be transient, clinical signs are often reduced or eliminated after surgical debulking (figs. 7 and 8) (ehrhart et al., 1996) . histologically, these tumors reveal ma-lignant proliferation of pancreatic 13 cells, and local recurrence or metastasis to lymph nodes, mesentery, spleen, or liver may occur (caplan et al., 1996) . adrenocortical cell tumor is the second most common type of neoplasia in ferrets and is generally diagnosed between 3 and 6 years of age. if clinical signs are present, they often include weight loss and a bilateral, symmetric alopecia. pruritus is a variable finding (quesenberry and rosenthal, 1997) . although ferrets with this syndrome have been called "cushingoid," it is rare to diagnose elevated resting levels of glucocorticoids or an abnormal response to adrenocorticotropic hormone (acth) stimulation or dexamethasone suppression testing. elevation of adrenal sex hormones (e.g., androstenedione, 17-hydroxyprogesterone, and/or estradiol) is more likely, and these may lead to characteristic changes such as estruslike vulvar swelling in spayed females and prostatic changes in males coleman et al., 1998) . rule-outs for enlarged vulva include estrus in an intact female or functional ovarian remnants in a spayed female. abdominal palpation may reveal cranial abdominal masses, and ultrasound may be useful (barthez et al., 1998) . serum assay for abnormal levels of the sex hormones listed above should be considered (lipman et al., 1993; wagner and dorn, 1994; rosenthal and peterson, 1996) . in many cases the alopecia begins as a seasonally intermittent partial hair loss that becomes more severe as time goes on (fig. 9) . even severe manifestations of this endocrine alopecia can spontaneously reverse in the absence of specific therapy, as demonstrated in a group of 5 ferrets referred to our facility for diagnostic workup. in each of these 5 ferrets, near total alopecia resolved within a few months of being housed in a research environment. despite being asymptomatic at the end of the study, all 5 were shown to have histologic evidence of adrenocortical neoplasia. although this phenomenon is mediated by hormonal effects, anecdotal reports such as this suggest that the alopecia may be significantly modulated by environmental factors (e.g., photoperiod or diet). surgical exploration and removal of enlarged adrenals are commonly performed to establish the diagnosis and to remove hyperfunctional tissue. unilateral adrenalectomy early in the disease may be curative, but because bilateral neoplastic involvement is not uncommon, full or partial removal of both glands may be required. adrenolytic agents such as mitotane have been used with limited success (quesenberry and rosenthai, 1997) . histologically, adrenocortical adenomas are generally 1 cm or less in diameter and are composed of well-differentiated cells with a granular or vacuolated cytoplasm. adrenal cell carcinomas are less commonly found and are larger, with a more pleomorphic and invasive character . metastasis to nearby tissues can occur. in our experience, adrenal cortical hyperplasia with or without neoplasia is an extremely common finding in aging ferrets, even in those not showing clinical signs. in one retrospective survey of our necropsy records it was found that more than 90% of ferrets greater than 4 years of age had hyperplastic or neoplastic adrenal changes when examined (data not shown). for this reason, careful considerations of other possible disease processes should be made before attributing clinical signs solely to adrenal enlargement. lymphoma can affect ferrets of almost any age. ferrets younger than 2 years of age often present with mediastinal lymphoma and/or leukemia, whereas those older than 3 years of age often develop multicentric solid tumors . the early age of onset in some ferrets and reports of case clustering have led to investigation into potential infectious etiologies for lymphoma in the ferret (erdman et al., 1996b) . earlier reports of feline leukemia virus (felv) seroconversion in affected animals have not been substantiated. however, experimental and epidemiological evidence suggests that a retrovirus that is distinct from felv may be involved . in one study, whole or filtered lymphoma cells from a 3-year-old ferret with spontaneous lymphoma were injected ip into 6 recipient ferrets . two of the 6 ferrets were euthanized after 14 months, but the remaining 4 developed splenomegaly, lymphocytosis, and lymphoma. one ferret that received cell-free materials developed multicentric lymphoma with prominent cutaneous lymphoma nodules. elevated reverse transcriptase activity and retrovirus-like particles evident by electron microscopy were seen in the donor and all of the affected recipient ferrets. other potential etiologies that have been considered include two infectious agents that are known to cause chronic immune stimulation in affected ferrets, the aleutian disease virus (adv) and helicobacter mustelae. a link with adv has not been proven, but h. mustelae seems to be responsible for the development of a very specific type of gastric b-cell lymphoma (erdman et al., 1997) . affected ferrets may exhibit localizing signs (e.g., dyspnea in a ferret with mediastinal involvement or peripheral lymphadenopathy in an animal with a multicentric distribution) but as is the case in many species, lymphoma is a "masquerader," and affected ferrets often present with chronic, nonspecific signs. weight loss, anorexia, and lethargy are often reported. splenic and/or hepatic enlargement may be evident. cutaneous involvement has been documented (li et al., 1995; rosenbaum et al., 1996) . although hematological examination typically reveals anemia and lymphopenia, lymphocytosis may be found, especially in younger ferrets. atypical lymphocytes are identified in the circulation in some cases. antemortem definitive diagnosis of lymphoma can be made by cytological examination of specimens obtained via fine-needle aspiration or excisional biopsy. tan-colored masses involving lymph nodes, spleen, liver, or other organs are commonly found at necropsy (fig. 10) . diffuse involvement may lead to uniform enlargement of these organs or to a thickening of the wall of the stomach or intestines. as in other species, histological evaluation reveals neoplastic lymphocytes in affected tissues, generally evident as a monomorphic population (fig. 11) . although surgery and radiation therapy may be useful in certain cases, most attempts to treat ferret lymphoma have utilized chemotherapeutic regimens with dosages extrapolated from other domestic animals or humans. treatment generally results in a remission that may last from 3 months to 5 years (brown, 1997b; erdman et al., 1998) . mast cell tumors are among the most commonly reported integumentary tumors in ferrets (parker and picut, 1993; . cutaneous mastocytomas may occur anywhere on the body and present as firm, nodular skin lesions 2-10 mm in size that are often associated with alopecia or crusty ulceration of the overlying skin. pruritis is common (stauber et al., 1990) . histologically, they are composed of well-differentiated mast cells with metachromatic cytoplasmic granules that may be difficult to detect in sections stained with hematoxylin-eosin, but are more evident in toluidine bluestained sections. a variety of tumors of epithelial origin occur in ferrets, and they can appear at any site on the body. the most common are the basal cell tumors, which present as firm plaques or pedunculated nodules that are white or pink (parker and picut, 1993) . they may grow rapidly and become ulcerated. the percentage of basiloid cells present in these tumors, and the degree of associated squamous or sebaceous differentiation can vary, resulting in a spectrum of tumor subtypes and associated histological diagnoses (orcutt, 1997) . however, as is the case with mastocytomas, most are benign and will not recur after excision. resected tumors should be examined histologically to rule out less common tumors that might have a more guarded prognosis, such as squamous cell carcinoma or apocrine gland adenocarcinoma. chordomas are not epithelial tumors, but they often present as readily evident firm masses on the tail that may cause ulceration of the overlying skin. these neoplasms arise along the axial skeleton from notochord remnants and are typically slow-growing (dunn et al., 1991) . tumors involving the tail generally do not recur after amputation of the affected region, but a wide surgical margin should be maintained by removing several vertebrae proximal to the tumor. the prognosis is guarded for those rare chordomas that arise in the cervical region, and metastasis has been documented (williams et al., 1993) . congenital defects identified in ferrets include a variety of neural tube defects, gastroschisis, cleft palate, amelia, corneal dermoids, cataracts, and supernumerary incisors (willis and barrow, 1971; ryland and gorham, 1978; mclain et al., 1985; besch-williford, 1987) . cystic or polycystic kidneys have been observed (andrews et al., 1979a; dillberger, 1985) . cystic genitourinary anomalies associated with the prostate, bladder, and/or proximal urethra most likely develop secondary to aberrant hormone secretion by adrenocortical tumors coleman et al., 1998) . newborn ferrets are normally born with a closed orbital fissure and are prone to developing subpalpebral conjunctival abscesses. treatment involves surgically opening the lids (a minor procedure) to establish drainage and to allow topical antibiotics to be administered (bell, 1997a) . cardiomyopathy is a common cause of disease in aging ferrets. the dilatative form of disease is most commonly diagnosed. affected animals commonly present with lethargy, weight loss, and anorexia. physical examination may reveal signs of congestive heart failure such as hypothermia, tachycardia, cyanosis, jugular distension, and respiratory distress (lipman et al., 1987) . auscultation may reveal a heart murmur and/or muffled cardiac sounds. hepatomegaly and splenomegaly are often identified. radiographs may reveal an enlarged cardiac silhouette and evidence of pulmonary edema or pleural effusion (greenlee and stephens, 1984) . electrocardiography and echocardiography can help make the definitive diagnosis. medical therapy (supportive care, diuretics, and inotropic drugs) may relieve clinical signs and improve the qual-ity of life for a period of months (stamoulis et al., 1997) . the long-term prognosis for survival is guarded to poor. splenomegaly is a common finding in ferrets. in many cases the enlarged spleen appears to be a secondary manifestation of another disease (e.g., insulinoma, cardiomyopathy, or adrenal tumor) and is of unknown significance (stamoulis et al., 1997) . histologic examination of affected organs has revealed that the most common cause for splenic enlargement (in the absence of a neoplastic infiltrate) is extramedullary hematopoiesis (emh) (erdman et al., 1998) . this may be an incidental finding, but it has been suggested that in some cases a pathologically enlarged spleen may play a role in chronic anemia that may respond to splenectomy, a syndrome known as hypersplenism (ferguson, 1985) . splenomegaly can also be commonly found in conjunction with lymphoma, with or without intrasplenic neoplastic lymphoid accumulations. in anesthetized ferrets, splenomegaly may be caused by splenic sequestration of erythrocytes (marini et al., 1994 (marini et al., , 1997 . because this is a transient effect, the normalization of splenic size upon recovery from anesthesia can help in the differentiation of anesthetic-induced splenomegaly from that due to other causes. eosinophilic gastroenteritis is an idiopathic disorder characterized by peripheral eosinophilia (10-35% of circulating leukocytes), hypoalbuminemia, and diffuse infiltration of the gastrointestinal tract with eosinophils (fox et al., 1992a) . presenting signs for this syndrome generally include chronic weight loss, anorexia, diarrhea, and occasionally vomiting. eosinophilic granulomas have been found in the mesenteric lymph nodes of most affected ferrets, and in some cases other organs (e.g., lung or liver) may be involved. an interesting finding in many ferrets is the presence of splendore-hoeppli material in the inflamed lymph nodes, a histological phenomenon that has been associated in other species with helminths, bacteria, fungi, and foreign bodies (fig. 12 ). an etiological agent has not been identified; consequently, therapy consists largely of supportive care to treat the chronic enteritis (fox, 1998b) . based on the biology of eosinophils, however, the use of corticosteroids or ivermectin has been attempted and may be beneficial (bell, 1997b) . megaesophagus has been diagnosed in ferrets presenting with a variety of signs, including weight loss, anorexia, difficulty in eating, or repeated regurgitation. the cause is generally unknown, and the prognosis is poor, despite efforts at supportive care (blanco et al., 1994) . gray, yellow, or white small raised lesions may be found on the surface of ferret lungs at gross examination. histologically, these lesions are composed of a superficial thickening of the lung tissue with mononuclear cell infiltration and 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with killed influenza a virus vaccines observations on tuberculosis in the ferret (mustela furo l.) effect of arginine-free diet on ammonia metabolism in young and adult ferrets the ferret, mustela putoriusfuro, as a new species in toxicology pruritis in rabbits, rodents, and ferrets the characterization and pathological significance of gastric campyiobacter-like organisms in the ferret: a model of chronic gastritis? isolation of atypical rotavirus causing diarrhea in neonatal ferrets mastitis in mink due to staphylococcus aureus and escherichia coli guide for the care and use of laboratory animals place de mycobacterium avium dans l'epidemiologies mycobacterienne actuelle chez les animaux domestiques et savages. sci pulmonary physiology as a model for inhalation toxicology evaluation of serum estradiol concentrations in alopecic ferrets with adrenal gland tumors the ferret intestinal uptake and lymphatic absorption of [3-carotene in ferrets: a model for human [3-carotene metabolism intestinal perfusion of [3-carotene in the ferret raised retinoic acid level in portal blood vitamin e enhances the lymphatic transport of [3-carotene and its conversion to vitamin a in the ferret aleutian disease in domestic ferrets: diagnostic findings and survey results semen characteristics and testosterone profiles in ferrets kept in a long-day photoperiod, and the influence of hcg timing and sperm dilution medium on pregnancy rate after laporoscopic insemination biliary coccidiosis in a ferret (mustela putoriusfuro) cervical chordoma in two ferrets (mustela putorius furo) coronavirus-associated epizootic catarrhal enteritis in ferrets comparative vaginal cytology of the estrous cycle of black-footed ferrets (mustela nigripes) the ferret (mustela putorius furo) as a laboratory animal effect of helicobacter mustelae infection on epithelial cell proliferation in ferret gastric tissues key: cord-007797-toam6r5y authors: franquet, tomás; chung, johnathan h. title: imaging of pulmonary infection date: 2019-02-20 journal: diseases of the chest, breast, heart and vessels 2019-2022 doi: 10.1007/978-3-030-11149-6_7 sha: doc_id: 7797 cord_uid: toam6r5y the spectrum of organisms known to cause respiratory infections is broad and constantly increasing as new pathogens are identified, and an increasing number of patients have impaired immunity due to disease or medications. the radiographic manifestations of a given organism may be variable depending on the immunologic status of the patient and the presence of preor coexisting lung disease. moreover, the clinical data and radiographic findings often fail to lead to a definitive diagnosis of pneumonia because there are an extensive number of noninfectious processes associated with febrile pneumonitis. this chapter describes and illustrates the characteristic imaging manifestations of the most common communityacquired pneumonias, nosocomial pneumonias, and the various infections seen in both immunocompetent and immunocompromised patients. community acquired pneumonia refers to an acute infection of the lung in patients who did not meet any of the criteria for hcap, presenting select clinical features (e.g., cough, fever, sputum production, and pleuritic chest pain) and accompanied by an acute infiltrate on a chest radiograph. pulmonary opacities are usually evident on the radiograph within 12 h of the onset of symptoms [3] . although the imaging findings do not allow a specific etiologic diagnosis, cap diagnosis and disease management most frequently involve chest radiography, and other imaging modalities are not usually required [4] . the spectrum of causative organisms of cap includes gram-positive bacteria such as streptococcus pneumoniae (pneumococcus), haemophilus influenzae, and staphylococcus aureus, as well as atypical organisms such as mycoplasma pneumoniae, chlamydia pneumoniae, or legionella pneumophila and viral agents such as influenza a virus and respiratory syncytial viruses [5] . however, many community-acquired pneumonias are still commonly caused by s. pneumoniae and are lobar in appearance. the radiographic patterns of cap are often related to the causative agent. infection of the lower respiratory tract, acquired by way of the airways and confined to the lung parenchyma and airways, typically presents radiologically as one of three patterns: (a) focal nonsegmental or lobar pneumonia, (b) multifocal bronchopneumonia or lobular pneumonia, and (c) focal or diffuse "interstitial" pneumonia. microorganisms responsible for vap may differ according to the population of patients in the icu, the durations of hospital and icu stays, and the specific diagnostic method(s) used. the spectrum of causative pathogens of vap in humans is staphylococcus aureus, pseudomonas aeruginosa, and enterobacteriaceae [6] . chest radiograph is most helpful when it is normal and rules out pneumonia. however, pulmonary opacities were detected by computed tomography (ct) scan in 26% of cases with a normal portable chest x-ray. when infiltrates are present, the particular pattern is of limited value for differentiating among cardiogenic pulmonary edema, noncardiogenic, pulmonary edema, hemorrhage, atelectasis, and pneumonia. when pneumonia is associated with healthcare risk factors such as prior hospitalization, dialysis, residing in a nursing home, and immunocompromised state, it is now classified as a healthcare-associated pneumonia (hcap). the number of individuals receiving healthcare outside the hospital setting, including home wound care or infusion therapy, dialysis, nursing homes, and similar settings, is constantly increasing [7] . a clinical diagnosis of pneumonia can usually be readily established on the basis of signs, symptoms, and chest radiographs, although distinguishing pneumonia from conditions such as left heart failure, pulmonary embolism, and aspiration pneumonia may sometimes be difficult. differentiation of etiologies based solely on the radiograph is not reliable, yet the pattern of abnormalities should be very useful in formulating a differential diagnosis of the nature of disease [8] . chest radiographs are of limited value in predicting the causative pathogen but are of good use to determine the extent of pneumonia and to detect complications (i.e., cavitation, abscess formation, pneumothorax, pleural effusion), to detect additional or alternative diagnoses, and, in some cases, to guide invasive diagnostic procedures. the most common radiographic manifestations of respiratory infection are foci of consolidation, ground-glass opacities, or reticulonodular opacities. other less common radiographic findings include hilar and mediastinal lymphadenopathy, pleural effusion, cavitation, and chest wall invasion [8, 9] . computed tomography, particularly high-resolution ct (hrct), has been shown to be more sensitive than the radiograph in the detection of subtle abnormalities and may show findings suggestive of pneumonia up to 5 days earlier than chest radiographs. ct is recommended in patients with clinical suspicion of infection and normal or nonspecific radiographic findings, in the assessment of suspected complications of pneumonia or suspicion of an underlying lesion such as pulmonary carcinoma. pneumonias are usually divided according to their chest imaging appearance into lobar pneumonia, bronchopneumonia, and interstitial pneumonia. in lobar pneumonia the inflammatory exudate begins in the distal airspaces adjacent to the visceral pleura and then spreads via collateral air drift routes (pores of kohn) to produce uniform homogeneous opacification of partial or complete segments of the lung and occasionally an entire lobe. • pneumonia is the leading cause of death due to infectious disease. • a variety of organisms that may present with similar clinical symptoms result in similar radiographic manifestations. • the radiographic manifestations of a given organism may be variable depending on the immunologic status of the patient. as the airways are not primarily involved and remain patent, there is little to no volume loss, and air bronchograms are common. some pneumonias present as spherical-or nodularshaped consolidations. bronchopneumonia (lobular pneumonia) is characterized histologically by peribronchiolar inflammation manifesting radiologically as patchy airspace nodules with poorly defined margins. radiologically a bronchopneumonia is characterized by large heterogeneous, scattered opacities which only later, with worsening of disease, become more homogeneous. an air bronchogram is usually absent. the most common causative organisms of bronchopneumonia are s. aureus, h. influenzae, p. aeruginosa, and anaerobic bacteria [8] . characteristic manifestations of bronchopneumonia on hrct include centrilobular ill-defined nodules and branching linear opacities, airspace nodules, and multifocal lobular areas of consolidation [10] . the term atypical pneumonia (interstitial pneumonia) was initially applied to the clinical and radiographic appearance of lung infection not behaving or looking like that caused by s. pneumoniae [11] . today, when new diagnostic techniques such as direct antigen detection, polymerase chain reaction, and serology (elisa) have moved beyond the initial diagnostic methods, a debate with regard to the appropriate use of the term "atypical pneumonia" is open [12] . radiographically focal or diffuse small heterogeneous opacities are seen uniformly distributed in the involved lung. frequently these opacities are described as reticular or reticulonodular. the usual causes of interstitial pneumonia are viral and mycoplasma infections [9] . streptococcus pneumoniae, a gram-positive coccus, is the most common bacterial cause of cap among patients who require hospitalization. risk factors for the development of pneumococcal pneumonia include the extremes of age, chronic heart or lung disease, immunosuppression, alcoholism, institutionalization, and prior splenectomy. the characteristic clinical presentation is abrupt in onset, with fever, chills, cough, and pleuritic chest pain. the typical radiographic appearance of acute pneumococcal pneumonia consists of a homogeneous consolidation that crosses segmental boundaries (nonsegmental) but involves only one lobe (lobar pneumonia) ( fig. 7.1) . occasionally, infection is manifested as a spherical focus of consolidation that simulates a mass (round pneumonia). complications, such as cavitation and pneumatocele formation, are rare. pleural effusion is common and is seen in up to half of patients. pneumonia caused by s. aureus usually follows aspiration of organisms from the upper respiratory tract. risk factors for the development of staphylococcal pneumonia include underlying pulmonary disease (e.g., copd, carcinoma), chronic illnesses (e.g., diabetes mellitus, renal failure), or viral infection. the clinical presentation of staphylococcal pneumonia is changing and of particular importance is the dramatic increase of the incidence of methicillinresistant staphylococcus aureus (mrsa) infections in recent years. increasingly, previously healthy young people without traditional risk factors for s. aureus disease are presenting with severe necrotizing infection and high mortality. fever, cough, and purulent sputum are prominent symptoms in cases of post-aspirative staphylococcal pneumonia. severe pneumonia caused by community-associated methicillin-resistant staphylococcus aureus (mrsa) carrying genes for panton-valentine leukocidin has been described in immunocompetent young adults. the characteristic pattern of presentation is as a bronchopneumonia (lobular pneumonia) that is bilateral in 40% of patients. the radiographic manifestations usually consist of bilateral patchy areas of consolidation. air bronchograms are uncommon. other features are cavitation, pneumatoceles, pleural effusions, and spontaneous pneumothorax ( fig. 7. 2). pneumatoceles are seen especially in children [13] . thoracic actinomycosis is a chronic suppurative pulmonary or endobronchial infection caused by actinomyces species, most frequently actinomyces israelii considered to be a gram-positive branching filamentous bacterium. pulmonary infection is characterized pathologically by bronchopneumonia with focal or multifocal abscess formation. actinomycosis has the ability to spread across fascial planes to contiguous tissues without regard to normal anatomic barriers. on ct, parenchymal actinomycosis is characterized by airspace consolidation with cavitation or central areas of low attenuation and adjacent pleural thickening. endobronchial actinomycosis can be associated with a foreign body (direct aspiration of a foreign body contaminated with actinomyces organisms) or a broncholith (secondary colonization of a preexisting endobronchial broncholith by aspirated actinomyces organisms). nocardia is a genus of filamentous gram-positive, weakly acid fast, aerobic bacteria that affects both immunosuppressed and immunocompetent patients. nocardia asteroides is responsible of 80% of infections by this organism in man. pulmonary nocardiosis can be an acute, subacute, or chronic disease. nocardiosis usually begins with a focus of pulmonary infection and may disseminate through hematogenous spread to other organs, most commonly to the cns. imaging findings are variable and consist of unifocal or multifocal consolidation and single or multiple pulmonary nodules [14] . nocardia asteroides infection may complicate alveolar proteinosis. gram-negative pneumonias are chiefly caused by klebsiella pneumoniae, enterobacter sp., serratia marcescens, escherichia coli, proteus sp., and pseudomonas aeruginosa. patients affected are invariably debilitated by a chronic medical or pulmonary disease. the lower lobes contrast-enhanced ct image shows a mixed opacity of consolidation (arrow) and ground-glass opacity (small arrows) consistent with lobar pneumonia tend to be affected, and the radiographic pattern is similar to that seen with s. aureus infections in adults. klebsiella pneumoniae is among the most common gramnegative bacteria accounting for 0.5-5.0% of all cases of pneumonia. these features are bulging fissures, sharp margins of the advancing border of the pneumonic infiltrate and early abscess formation. ct findings consist of ground-glass attenuation, consolidation, and intralobular reticular opacity, often associated with pleural effusion. complications of klebsiella pneumonia include abscess formation, parapneumonic effusion, and empyema. axial contrast-enhanced ct shows a cavitary mass within the right lower lobe (arrow) and a mass in the left upper lobe demonstrating surrounding ground-glass opacity (halo sign) (arrows) escherichia coli accounts for 4% of cases of cap and 5-20% of cases of hap or hcap. it occurs most commonly in debilitated patients. the typical history is one of abrupt onset of fever, chills, dyspnea, pleuritic pain, and productive cough in a patient with preexisting chronic disease. the radiographic manifestations usually are those of bronchopneumonia; rarely a pattern of lobar pneumonia may be seen. pseudomonas aeruginosa is a gram-negative bacillus that is the most common cause of nosocomial pulmonary infection. it causes confluent bronchopneumonia that is often extensive and frequently cavitates (fig. 7.3) . the radiologic manifestations are nonspecific and consist most commonly of patchy areas of consolidation and widespread poorly defined nodular opacities [9] . chlamydia pneumoniae is the most commonly occurring gram-negative intracellular bacterial pathogen. it is frequently involved in respiratory tract infections and has also been implicated in the pathogenesis of asthma in both adults and children. symptoms include sore throat, headache, and a nonproductive cough that can persist for months if treatment is not initiated early. chest radiographs tend to show less extensive abnormalities than are seen with other causes of pneumonia. on ct, c. pneumoniae pneumonia demonstrates a wide spectrum of the most common rickettsia lung infection is sporadic or epidemic q-fever pneumonia caused by coxiella burnetii, an intracellular, gram-negative bacterium. infection is acquired by inhalation from farm livestock or their products and occasionally from domestic animals. imaging findings consist of multilobar airspace consolidation, solitary or multiple nodules surrounded by a halo of "groundglass" opacity and vessel connection, and necrotizing pneumonia. tularemia is an acute, febrile, bacterial zoonosis caused by the aerobic gram-negative bacillus francisella tularensis. it is endemic in parts of europe, asia, and north america. primary pneumonic tularemia occurs in rural settings. humans become infected after introduction of the bacillus by inhalation, intradermal injection, or oral ingestion. chest radiographic findings are scattered multifocal consolidations, hilar adenopathy, and pleural effusion. haemophilus influenzae is a pleomorphic, gram-negative coccobacillus that accounts for 5-20% of cap in patients in whom an organism can be identified successfully. factors that predispose to haemophilus pneumonia include copd, malignancy, hiv infection, and alcoholism. the typical radiographic appearance of haemophilus influenza pneumonia consists of multilobar involvement with lobar or segmental consolidation and pleural effusion. legionella is a pathogenic gram-negative bacterium with at least 50 species. it is one of the most common causes of severe community-acquired pneumonia in immunocompetent hosts. human infection may occur when legionella contaminates water systems, such as air conditioners and condensers. risk factors for the development of l. pneumophila pneumonia include immunosuppression, posttransplantation, cigarette smoking, renal disease, and exposure to contaminated drinking water. patients with legionella pneumonia usually present with fever, cough, initially dry and later productive, malaise, myalgia, confusion, headaches, and diarrhea. thirty percent of patients develop pleuritic chest pain. imaging findings include peripheral airspace consolidation similar to that seen in acute s. pneumoniae pneumonia. in many cases, the area of consolidation rapidly progresses to occupy all or a large portion of a lobe (lobar pneumonia) or to involve contiguous lobes or to become bilateral. cavitation is uncommon in immunocompetent patients, and pleural effusion may occur in 35-63% of cases. moraxella catarrhalis (formerly known as branhamella catarrhalis) has emerged as a significant bacterial pathogen of humans over the past two decades. it is an intracellular gram-negative coccus now recognized as one of the common respiratory pathogens [15] . m. catarrhalis causes otitis media and sinusitis in children and mild pneumonia and acute exacerbation in older patients with copd. it is currently considered the third most common cause of community-acquired bacterial pneumonia (after s. pneumoniae and h. influenzae). the majority of patients with pneumonia (80-90%) have underlying chronic pulmonary disease. chest radiographs show bronchopneumonia or lobar pneumonia that usually involves a single lobe. m. pneumoniae is one of the most common causes of community-acquired pneumonia. it accounts for up to 37% of cap in persons treated as outpatients. patients with copd appear to be more severely affected with m. pneumoniae than normal hosts. the radiographic findings in m. pneumoniae are variable and in some cases closely resemble those seen in viral infections of the lower respiratory tract. chest radiograph shows fine linear opacities followed by segmental airspace consolidation [16] . • a "tree-in-bud" pattern is a characteristic ct manifestation of infectious bronchiolitis. • focal areas of consolidation secondary to infection in immunocompromised patients are most commonly due to bacterial pneumonia. • interstitial and/or mixed interstitial and airspace opacities in cap are typically due to viruses or m. pneumoniae. mycobacterium tuberculosis accounts for more than 95% of pulmonary mycobacterial infections. other mycobacterial species, m. kansasii and m. avium-intracellulare complex (mac), account for the remainder. factors that contribute to the large number of cases seen worldwide are human immunodeficiency virus (hiv) infection, inner city poverty, homelessness, and immigration from areas with high rates of infection. other predisposing conditions are diabetes mellitus, alcoholism, silicosis, and malignancy. this form is seen in infants and children. with improved control of tuberculosis in western societies, however, more people reach adulthood without exposure, and primary patterns of disease are being seen with increasing frequency in adulthood and represents about 23-34% of all adult cases of tuberculosis. although primary tuberculosis typically presents with radiographic manifestations, chest radiograph may be normal in 15% of cases. lymphadenopathy is the most common manifestation of primary tuberculosis in children and occurs with or without pneumonia. in adults hilar or mediastinal lymphadenopathy is less common declining to about 50% of cases in the older population. pleural effusion occurs in children, who usually have parenchymal or nodal disease, or in teenagers and young adults, when it is frequently isolated. most cases are due to reactivation of quiescent lesions, but occasionally a new infection from an exogenous source occurs. pathologically, the ability of the host to respond immunologically results in a greater inflammatory reaction and caseous necrosis. the radiological manifestations may overlap with those of primary tuberculosis, but the absence of lymphadenopathy, more frequent cavitation, and a predilection for the upper lobes are more typical of postprimary tuberculosis. a rasmussen aneurysm is a rare life-threatening complication of cavitary tuberculosis caused by granulomatous weakening of a pulmonary arterial wall. endobronchial spread can occur with or without cavitary disease and is similar to that seen with primary tuberculosis leading to the appearance of the typical images of "tree-inbud" [17, 18] . after antituberculous treatment healing results in scar formation. the fibrosis produces well-defined, upper lobe nodular and linear opacities, often with evidence of severe volume loss and pleural thickening. residual thin-walled cavities may be present in both active and inactive disease. although classically a manifestation of primary disease, miliary tuberculosis is now more commonly seen as a postprimary process in older patients. multiple small (1-2 mm) discrete nodules are scattered evenly throughout both lungs. a tuberculoma may occur in the setting of primary or postprimary tuberculosis and represents localized parenchymal disease that alternately activates and heals. it usually calcifies and frequently remains stable for years [19] . as mentioned above, 1-3% of pulmonary mycobacterial infections are caused by agents other than m. tuberculosis: usually m. avium-intracellulare complex (mac) and less commonly m. kansasii. patients are often predisposed by reason of underlying debilitating disease, immune compromise, chronic airflow obstruction, previous pulmonary tuberculosis, or silicosis and following lung transplantation [20] . clinically, mac may be an indolent process with symptoms of cough, with or without sputum production. more commonly, mac presents with a radiological pattern that does not resemble that of postprimary tuberculosis. it consists of multiple nodules, with or without small ring opacities, showing no specific lobar predilection and bronchiectasis particularly in the lingula and right middle lobe. the most typical form of pulmonary nontuberculous mycobacteria (ntmb) infection is frequently associated to elderly men with underlying lung disease and to elderly white women without underlying lung disease (lady windermere syndrome). radiological findings consist of mild to moderate cylindrical bronchiectasis and multiple 1-3 mm diameter centrilobular nodules. fungi involved in pulmonary infections are either pathogenic fungi, which can infect any host, or saprophytic fungi, which infect only immunocompromised hosts. pathogenic fungi include coccidioidomycosis, blastomycosis, and histoplasmosis. saprophytes include pneumocystis, candidiasis, mucormycosis, and aspergillosis. pulmonary fungal infections may be difficult to diagnose, and a definitive diagnosis of pulmonary fungal infections is made by isolating the fungus from tissue specimen. aspergillosis is a fungal disease caused by aspergillus species, usually a. fumigatus that can take different forms depending on an individual's immune response to the organism. classically, pulmonary aspergillosis has been categorized into saprophytic, allergic, and invasive forms [21, 22] . aspergillus mycetomas are saprophytic growths which colonize a preexisting cavity in the lung (e.g., from sarcoidosis or tuberculosis). most cavities and thus mycetomas are in the upper lobes or superior segments of the lower lobes. allergic bronchopulmonary aspergillosis (abpa) describes a hypersensitivity reaction which occurs in the major airways. it is associated with elevated serum ige, positive serum precipitins, and skin reactivity to aspergillus. the radiographic appearances consist of nonsegmental areas of opacity most common in the upper lobes, lobar collapse, branching thick tubular opacities due to bronchi distended with mucus and fungus, and occasionally pulmonary cavitation. the mucus plugs in abpa are usually hypodense, but in up to 20% of patients, the mucus can be hyperdense on ct (fig. 7.4) . angioinvasive aspergillosis is seen in immunocompromised hosts with severe neutropenia. this form is characterized by invasion and occlusion of small-to-medium pulmonary arteries, developing necrotic hemorrhagic nodules or infarcts. the most common pattern seen in ct consists of multiples nodules surrounded by a halo of ground-glass attenuation (halo sign) or pleural-based wedgeshaped areas of consolidation [21] . candida species has been increasingly recognized as an important source of fungal pneumonia in immunocompromised patients, particularly in those with underlying malignancy (acute leukemia and lymphoma), intravenous drug abuse, and acquired immune deficiency syndrome a b c fig. 7.4 (a, b, c) allergic bronchopulmonary aspergillosis: (a) posteroanterior chest radiograph demonstrates basilar branching opacities suggestive of mucus-filled bronchiectasis (arrows). (b) axial ct image confirms the presence of left basilar bronchiectasis, mucus-filled airways (arrows). (c) hyperdensity of mucus-filled airways on axial mip image (arrows) from chest ct is diagnostic of allergic bronchopulmonary aspergillosis (aids), and following bone marrow transplantation. the most common thin-section ct findings of pulmonary candidiasis consist of multiple bilateral nodular opacities often associated with areas of consolidation and groundglass opacity [23] . pneumocystis jiroveci a unique opportunistic fungal pathogen that causes pneumonia in immunocompromised individuals such as patients with aids, patients with organ transplants, and patients with hematologic or solid organ malignancies who are undergoing chemotherapy. in 90% of patients with pneumocystis jiroveci, pneumonia chest radiographs show diffuse bilateral infiltrates in a perihilar distribution ( fig. 7.5) . the most common high-resolution ct finding in pneumocystis jiroveci pneumonia is diffuse ground-glass opacity [24] . mucormycosis is an opportunistic fungal infection of the order mucorales, characterized by broad, nonseptated hyphae that randomly branch at right angles. the most common radiographic findings consist of lobar or multilobar areas of consolidation and solitary or multiple pulmonary nodules and masses with associated cavitation or an air-crescent sign [25] . viruses can result in several pathologic forms of lower respiratory tract infection including tracheobronchitis, bronchiolitis, and pneumonia. viral infections predispose to secondary bacterial pneumonia. organizing pneumonia, a nonspecific reparative reaction, may result from a variety of causes or underlying pathologic processes including viral infections [26] . influenza type a is the most important of the respiratory viruses with respect to the morbidity and mortality in the general population. in recent years, both influenza and parainfluenza viruses have been recognized as a significant cause of respiratory illness in immunocompromised patients, including solid organ transplant recipients. the predominant high-resolution ct findings are ground-glass opacities, consolidation, centrilobular nodules, and branching linear opacities. adenovirus accounts for 5-10% of acute respiratory infections in infants and children but for less than 1% of respiratory illnesses in adults. swyer-james-macleod syndrome is considered to be a post-infectious bronchiolitis obliterans (bo) secondary to adenovirus infection in childhood. respiratory syncytial virus (rsv) is the most frequent viral cause of lower respiratory tract infection in infants. the major risk factors for severe rsv disease in children are prematurity (< 36 weeks gestation), congenital heart disease, chronic lung disease, immunocompromised status, and multiple congenital abnormalities. ct findings consist of small centrilobular nodules, airspace consolidation, ground-glass opacities, bronchial wall thickening, and "tree-in-bud" opacities ( fig. 7.6 ). primary infection with ebv occurs early in life and presents as infectious mononucleosis with the typical triad of fever, pharyngitis, and lymphadenopathy, often accompanied by splenomegaly. mild, asymptomatic pneumonitis occurs in about 5-10% of cases of infectious mononucleosis. the ct manifestations of ebv pneumonia are similar to those of other viral pneumonias. the findings usually consist of lobar consolidation, diffuse and focal parenchymal haziness, irregular reticular opacities, and multiple miliary nodules or small nodules with associated areas of ground-glass attenuation ("halo"). varicella is a common contagious infection in childhood with increasing incidence in adults. clinically presents in two forms: varicella (chickenpox) representing a primary disseminated disease in uninfected individuals and zoster (shingles) representing reactivation of latent virus (unilateral dermatomal skin eruption). pneumonia, although rare, is the most serious complication affecting adults with chickenpox. the thin-section ct appearances in varicella pneumonia reflect the multicentric hemorrhage and necrosis centered on airways. common findings include numerous nodular opacities measuring 5-10 mm in diameter, some with a surrounding halo of ground-glass opacity, patchy ground-glass opacities, and coalescence of nodules. cytomegalovirus pneumonia is a major cause of morbidity and mortality following hematopoietic stem cell (hsct) and solid organ transplantation and in patients with aids in whom cd4 cells are decreased to fewer than 100 cells/mm 3 . this complication characteristically occurs during the post-engraftment period (30-100 days after transplantation) with a median time onset of 50-60 days posttransplantation. ct features of cmv pneumonia consist of lobar consolidation, diffuse and focal ground-glass opacities, irregular reticular opacities, and multiple miliary nodules or small nodules with associated areas of ground-glass attenuation ("halo"). severe acute respiratory distress syndrome (sars) caused by sars-associated coronavirus (sars-cov) is a systemic infection that clinically manifests as progressive pneumonia. severe acute respiratory distress syndrome was first detected in the guangdong province of china in late 2002, with major outbreaks in hong kong, guangdong, singapore, and toronto and vancouver, canada. over 8000 people were affected, with a mortality rate of 10%. the typical clinical presentation consists of an incubation period of 2-10 days, early systemic symptoms followed within 2-7 days by dry cough or shortness of breath, the development of radiographically confirmed pneumonia by day 7-10, and lymphocytopenia in many cases. histologically, acute diffuse alveolar damage with airspace edema is the most prominent feature. the imaging features consist of unilateral or bilateral ground-glass opacities, focal unilateral or bilateral areas of consolidation, or a mixture of both [27, 28] . mers is a viral disease caused by a coronavirus (mers-cov), with most of the infections believed to have originated in saudi arabia and the middle east. most patients develop a severe acute respiratory illness. ct may depict groundglass opacities, consolidation, interlobular thickening, and pleural effusion. during the subsequent weeks, other findings may be present, such as centrilobular nodules, a "crazypaving" pattern, obliterative bronchiolitis, peribronchial air trapping, and organizing pneumonia [29] . in the spring of 2009, an outbreak of severe pneumonia was reported in conjunction with the concurrent isolation of a novel swine-origin influenza a (h1n1) virus, widely known as swine flu, in mexico. on june 11, 2009 , the world health organization declared the first pandemic of the twenty-first century caused by swine-origin influenza virus a (h1n1). the predominant ct findings are unilateral or bilateral ground-glass opacities with or without associated focal or multifocal areas of consolidation. on ct, the ground-glass opacities and areas of consolidation have a predominant peribronchovascular and subpleural distribution, resembling organizing pneumonia [30] . • primary tb occurs most commonly in children. • the most typical form of pulmonary ntmb infection is frequently associated to elderly non-smoking white women without underlying lung disease (lady windermere syndrome). • with respect to the morbidity and mortality, influenza type a is the most important of the respiratory viruses in the general population. vap: the diachronic linguistics of pneumonia community-acquired pneumonia admission chest radiograph lacks sensitivity in the diagnosis of community-acquired pneumonia community-acquired pneumonia re-evaluation of the etiology and clinical and radiological features of community-acquired lobar pneumonia in adults ventilator-associated pneumonia health care-associated pneumonia (hcap): empiric antibiotics targeting methicillin-resistant staphylococcus aureus (mrsa) and pseudomonas aeruginosa predict optimal outcome imaging infection imaging of 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use key: cord-023528-z9rc0ubj authors: wilkins, pamela a. title: disorders of foals date: 2009-05-18 journal: equine internal medicine doi: 10.1016/b0-72-169777-1/50021-4 sha: doc_id: 23528 cord_uid: z9rc0ubj nan before the 1980s, intensive management of the compromised neonate was unusual and little was known regarding many of the problems of this special patient population. although some specific conditions had been described by astute clinician-researchers, most notably the "dummy" foal syndrome 1 and respiratory distress syndrome caused by primary surfactant deficiency, 2 little information regarding the diagnosis and management of conditions of the foal during the neonatal period was available, although at least one active group was investigating fetal and neonatal physiology of the horse in great britain. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] when treatment of compromised foals was undertaken, the approach most commonly resembled treating them as small adults with little understanding of the different physiology of the equine neonate. the advent of improved management of reproductive efficiency of mares led naturally to increased interest in preservation of the conceptus to parturition and the foal thereafter. interested clinicians, taking their lessons from the field of human perinatology/neonatology and sometimes working hand-in-hand with their counterparts in the human field, pioneered investigations into these small patients and created the fields of equine perinatology and equine neonatal intensive care. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] because of the foresight and energy of these early investigators, the field of veterinary perinatology/neonatology exploded in the 1980s, leading to the creation of equine neonatal intensive care units throughout the united sates and the world. from these units information about the normal and abnormal physiology of foals, the medical conditions affecting them, and methods for treatment and management of these problems has been developed through observational, retrospective, and prospective studies. this veritable explosion of information over the last 20 years has improved greatly the ability of all practitioners to provide appropriate care for these patients, whether in the field or at an equine neonatal intensive care unit. the ability not only to save the lives of these patients but also to treat them in such a manner as to allow them to fulfill their purposes, whether as pleasure animals or racing athletes, has improved almost exponentially from those early days. [20] [21] [22] [23] this chapter aims to provide the clinician with some of the most current information regarding the management of these patients, recognizing that much still remains unknown and that advances will continue to be made in this dynamic field. the reader is cautioned that much of this chapter is flavored by the experiences of the author and that variation in approach and treatment of specific problems exists between neonatal intensive care units (nicus) and between clinicians in the same nicu and that each year results in change. in some cases, information that is presented has been gleaned from human nicu studies, essentially using the critically ill infant as the experimental model. many of the problems of the newborn foal have their genesis in utero. identification of high-risk pregnancies is an important component of prenatal care of the foal, and some of the most commonly encountered problems of the dam resulting in abnormal foals include previous or concurrent disease, poor reproductive history, poor perineal or pelvic conformation, poor general health, poor nutritional condition, prolonged transport, history of previous abnormal foals, placental abnormalities, and twins. 24 some of the more common causes of abortion can result in the birth of severely compromised foals of variable gestation lengths (box 19-1). these include pa m e l a a . wi l k i n s infectious causes such as equine herpesvirus (ehv) types 1 (most commonly) and 4 (rarely), equine infectious anemia, equine arteritis virus, bacterial and fungal placentitis, leptospirosis, equine ehrlichiosis, and gram-negative septicemia/endotoxemia. [25] [26] [27] noninfectious causes of abortion include twinning and noninfectious placental abnormalities such as extensive endometrial fibrosis, body pregnancy, and abnormal length (long or short) of the umbilical cord. 24, 28 to the equine neonatologist opportunities for intervention may appear limited, and in the case of many of the aforementioned causes of fetal loss, this is true. however, one can do much in an attempt to preserve the pregnancy and in effect treat the fetus. when one is faced with a threatened pregnancy, one has various ways of evaluating the fetus and its environment and may use many potential therapies. once one identifies a pregnancy as high risk, one should evaluate the fetus for viability. evaluation should include as thorough an evaluation as possible of the reproductive tract, placenta, and fetal fluids. prepartum disorders in the mare usually are readily recognizable, but disorders of the fetus and placenta can be more subtle and difficult to determine. the first step is to take a thorough history of the mare. of particular interest is any history of previous abnormal foals, but the history taking should include questions regarding transportation; establishment of an accurate breeding date (sometimes more difficult than one would suspect); any pertinent medical history including any diagnostic testing performed for this pregnancy such as culture, endometrial biopsy, and cytologic results; and any rectal and ultrasound examination results. additionally, one should obtain information regarding possible ingestion of endophyte-infected fescue or exposure to potential infectious causes of abortion. 29, 30 a complete vaccination and deworming history is requisite, as is a complete history of any medications and supplements administered during pregnancy. after obtaining a history, one examines the mare per rectum. this examination should include palpation of the cervix, uterus, fetus, and all palpable abdominal contents. one should note any abnormalities. the cervix should be tight throughout gestation; the late gestation uterus will be large and distended with fluid and usually pulled craniad in the abdomen. palpation of the fetus frequently results in some fetal movement; however, one should interpret lack of movement with caution, for some normal fetuses do not respond. ultrasonographic evaluation of the uterus and conceptus per rectum can provide valuable information, particularly regarding placental thickness if placentitis is a concern. one may evaluate fetal fluids and estimate fetal size from the size of the eye later in gestation. 31 in the author's hospital the practitioners choose not to perform vaginal examinations or speculum examinations because of an association between these examinations and the subsequent development of placentitis. unless placentitis is recognized with ultrasonograhic evaluation per rectum and culture is desirable, these types of examinations are generally not necessary. following examination per rectum, one performs transabdominal ultrasonographic evaluation of the uterus and conceptus. 28 one can generate a biophysical profile of the fetus from this examination in the late-term fetus and readily determine viability. 32, 33 one also readily can determine the presence or absence of twins in the late pregnant mare in this manner. one performs the sonogram through the acoustic window from the udder to the xiphoid ventrally and laterally to the skinfolds of the flank. imaging of the fetus usually requires a lowfrequency (3.5-mhz) probe, whereas examination of the placenta and endometrium requires a higher-frequency (7.5-mhz) probe. a complete description of this examination is beyond the scope of this chapter, but the reader will find several complete descriptions of the technique and normal values for specific gestation lengths within the relevant veterinary literature. 33 the utility of this examination lies in its repeatability and low risk to the dam and fetus. sequential examinations over time allow the clinician to follow the pregnancy and to identify changes as they occur. a companion to transabdominal ultrasongraphy is evaluation of the fetal electrocardiogram (ecg). one can measure fetal ecgs continuously using telemetry or can obtain them using more conventional techniques several times throughout the day. 24, 28, 34 one places electrodes on the skin of the mare in locations aimed at maximizing the magnitude of the fetal ecg. because the fetus frequently changes position, multiple sites may be needed in any 24-hour period. to begin, one places an electrode dorsally in the area of the sacral prominence with two electrodes placed bilaterally in a transverse plane in the region of the flank. the fetal ecg maximal amplitude is low, usually 0.05 to 0.1 mv, and can be lost in artifact or background noise, so one commonly must move electrodes to new positions to maximize the appearance of the fetal ecg. the normal fetal heart rate during the last months of gestation ranges from 65 to 115 beats/min, a fairly wide distribution. the range of heart rate of an individual fetus can be narrow, however. bradycardia in the fetus is an adaptation to in utero stress, most commonly thought to be hypoxia. by slowing the heart rate, the fetus prolongs exposure of fetal blood to maternal blood, increasing the time for equilibration of dissolved gas across the placenta and improving the oxygen content of the fetal blood. the fetus also has altered the distribution of its cardiac output in response to hypoxia, centralizing blood distribution. 35, 36 tachycardia in the fetus can be associated with fetal movement, and brief periods of tachycardia should occur in the fetus in any 24-hour period. persistent tachycardia is a sign of fetal distress and represents more severe fetal compromise than bradycardia. the author has recognized dysrhythmias in the challenged fetus, most commonly as atrial fibrillation but also apparent runs of ventricular tachycardia. the ability to monitor the fetus in a high-risk pregnancy inevitably has led to questions of whether, how, and when to intervene. most equine neonatologists would agree that removal of the fetus from the uterus before its attainment of readiness for birth is not desirable. one of the difficulties in determining fetal preparedness for birth is that prediction of parturition is difficult in these mares. many of the parameters used in normal mares are unreliable in the high-risk pregnant mare. one must have an accurate history of any previous gestation length in terms of days for the specific mare in question to allow a more accurate estimate of her usual gestational length. evaluation of the usual mammary gland parameters, including size, the presence of "wax," and alteration of electrolyte concentrations, is not generally predictive in the high-risk mare, for in the author's experience many of these mares have changes predictive of parturition for weeks before actual parturition. 37, 38 this circumstance may be related to the observation that many high-risk pregnant mares, particularly those with placentitis, are presented for a primary complaint of early onset lactation. although pulmonary system maturity in human beings can be assessed with some degree of accuracy using measurement of lecithin/sphingomyelin ratios, this measurement-along with sphingomyelin, cortisol, and creatinine concentrations in the amnionic fluid-has proved to be of no benefit in the horse. [39] [40] [41] amniocentesis carries a high risk of abortion in the horse, even with ultrasound guidance, and is not a clinically useful technique at this time. 41 currently, no clear-cut guidelines are available as to when to intervene, but the presence of persistent fetal tachycardia or prolonged absence of fetal movements, including breathing movements, as determined by transabdominal ultrasound evaluation, should initiate discussion regarding the appropriateness of induction of parturition or elective cesarean section. the goal of induction or cesarean section is to remove a pregnancy that is threatening the survival of the dam with no thought to fetal survival or to remove the fetus from a threatening environment to improve its likelihood for survival. preterm induction is ill advised if fetal survival is desirable because of the limited ability to treat severely immature neonates. timing of intervention in these circumstances remains an art, not a science. the approach to management of the high-risk pregnancy is dictated to some degree by the exact cause for concern, but for many mares therapy is similar. many high-risk mares have placentitis, primarily caused by ascending bacterial or fungal infections originating in the region of the cervix. these infections can cause in utero sepsis or compromise the fetus by local elucidation of inflammatory mediators or altered placental function. 42, 43 premature udder development and vaginal discharge are common clinical signs. treatment consists of administration of broad-spectrum antimicrobial agents and nonsteroidal antiinflammatory drugs (table 19 -1). in the author's clinic, trimethoprim-sulfonamide drugs have been the antimicrobial of choice based on unpublished studies performed at the facility demonstrating increased concentration of these agents in the fetal fluids compared with penicillin and gentamicin. however, if culture and sensitivity results are available, one should institute directed therapy. nonsteroidal antiinflammatory agents such as flunixin meglumine are useful to combat alterations in prostaglandin balance that may be associated with infection and inflammation. although the efficacy of these agents is best when administered before the development of clinical signs, to date no detrimental effects have been reported in the fetus or dam when chronically used at low doses in well-hydrated patients. tocolytic agents and agents that promote uterine quiescence have been used and include altrenogest, isoxuprine, and clenbuterol. [44] [45] [46] [47] [48] altrenogest usually is administered, although its need in late gestation has been challenged. the efficacy of isoxuprine as a tocolytic in the horse is unproven, and bioavailability of orally administered isoxuprine appears to be highly variable. 48 the long-term use of clenbuterol is inadvisable because of receptor population changes associated with chronic use and its unknown effects on the fetus at this time. clenbuterol may be indicated during management of dystocia in preparation for assisted delivery or cesarean section. 46 the intravenous form of clenbuterol is not currently available in the united states. one can use three additional strategies in managing high-risk pregnancy patients. in mares with evidence of placental dysfunction, with or without signs of fetal distress, the author provides intranasal oxygen supplementation in the hope of improving oxygen delivery to the fetus. intranasal oxygen insufflation of 10 to 15 l/min to the mare significantly increases pao 2 and percent oxygen saturation of hemoglobin. 49 because of the placental vessel arrangement of the horse, improvement of these two arterial blood gas parameters should result in improved oxygen delivery to the fetus. blood gas transport is largely independent of diffusion distance in the equine placenta, particularly in late gestation, and depends more on blood flow. information from other species cannot be extrapolated to the equine placenta because of its diffuse epitheliochorial nature and the arrangement of the maternal and fetal blood vessels within the microcotyledons. 50,51 umbilical venous po 2 is 50 to 54 mm hg in the horse fetus, compared with 30 to 34 mm hg in the sheep, whereas the maternal uterine vein to umbilical vein po 2 difference is near 0. also unlike the sheep, the umbilical venous po 2 values decrease 5 to 10 mm hg in response to maternal hypoxemia and increase in response to maternal hyperoxia. [52] [53] [54] vitamin e (tocopherol) is administered orally to some high-risk mares as an antioxidant. administration of large doses of vitamin e before traumatic brain injury improves neurologic outcome in experimental models and has been examined as possible prophylaxis for human neonatal encephalopathy. [55] [56] [57] extrapolation of that information to the compromised equine fetus suggests that increased antioxidant concentrations in the fetus may mitigate some of the consequences of uterine and birth hypoxia, but no evidence is available to date demonstrating that protection occurs or that vitamin e accumulates in the fetus in response to supplementation of the mare. finally, many high-risk mares are anorectic or held off feed because of their medical condition. these mares are at particularly great risk for fetal loss because of their lack of feed intake, which alters prostaglandin metabolism. 58 therefore one should administer 2.5% to 5% dextrose in 0.45% saline or water (5% dextrose) intravenously at maintenance fluid rates to these patients. perhaps the most important aspect of managing high-risk pregnancy mares is frequent observation and development of a plan. one should observe mares at least hourly for evidence of early-stage labor and should put them under constant video surveillance if possible. depending on the primary problem, the team managing the mare should develop a plan for handling the parturition once labor begins and for fetal resuscitation following delivery. any equipment that might be needed should be readily available stallside, and a call sheet, listing contact numbers for all involved, should be posted on or near the stall. the plan should include a decision as to how to handle a complicated dystocia, should it occur, with permission for general anesthesia and cesarean section obtained before the event so that time is not wasted. an important question to be posed to the owner at the outset is which is most important to the owner, the mare or the foal, for the answer may dictate the direction of the decision tree once labor begins. 59 early recognition of abnormalities is of utmost importance for successful management of critically ill foals. to recognize the abnormal, one must know the normal. immediately following birth, foals effect several important physiologic and behavioral changes. chief among these changes is the adaptation of the cardiovascular and respiratory systems to extrauterine life. the normal transition of the respiratory tract involves opening closed 1384 part ii disorders of specific body systems alveoli and absorption of fluid from the airway, accomplished by a combination of breathing efforts, expiration against a closed glottis (grunting), and a change in sodium flux across the respiratory membrane from net secretion to net absorption. [60] [61] [62] [63] [64] the transition from fetal to neonatal circulatory patterns requires resolution of the pulmonary hypertension present in the fetus, normally shunting blood flow through the lower resistance ductus arteriosus in the fetal state, to direct cardiac output to the pulmonary vasculature for participation in gas exchange. this change is achieved by the opening of alveoli, decreasing airway resistance and providing radial support for pulmonary vessels, functional closure of the ductus arteriosus, and increasing the oxygen tension in the lung, reversing pulmonary vasoconstriction mediated by hypoxia. 65, 66 pulmonary tree vasodilators (prostacyclin, nitric oxide [no] ) and vasoconstrictors (endothelin-1, leukotrienes) play apparently well-coordinated, but as yet not fully elucidated, roles. in the normal newborn this change is smooth and rapid. these critical events are undermined by factors such as inadequate lung development, surfactant deficiency (primary or secondary), viral or bacterial infection, placental abnormalities, in utero hypoxia, and meconium aspiration. spontaneous breathing should begin in the neonate within 1 minute of birth, many foals attempt to breathe as their thorax clears the pelvic canal. during the first hour of life, the respiratory rate of a healthy foal can be as high as 80 breaths per minute but should decrease to 30 to 40 breaths per minute within a few hours. similarly, the heart rate of a healthy newborn foal has a regular rhythm and should be at least 60 beats/min at the first minute. 67, 68 one usually can auscultate a continuous murmur over the left side of the heart, although its loudness may vary with position. this murmur is thought to be associated with some shunting through the ductus arteriosus. one may auscultate variable systolic murmurs, thought to be flow murmurs, during the first week of life. 69 one should investigate more thoroughly murmurs that persist beyond the first week of life in an otherwise healthy foal, along with any murmur associated with persistent hypoxia. auscultation of the thorax shortly after birth reveals a cacophony of sounds as airways open and fluid is cleared. end-expiratory crackles are consistently audible in the dependent lung during and following lateral recumbency. for a normal newborn foal to appear slightly cyanotic during this initial adaptation period is not unusual, but this should resolve within minutes of birth. the equine fetus, as do all fetuses, exists in a moderately hypoxic environment, but the equine fetus has a greater partial pressure of oxygen, around 50 mm hg. 70 because the fetus is well adapted to low oxygen tensions, cyanosis is rarely present in newborn foals once adaption occurs, even those with low oxygen tensions. although in many species the fetal blood oxygen affinity is greater than the maternal blood, in the equine fetus the oxygen affinity of its hemoglobin is only about 2 mm hg greater than the maternal blood because of decreased levels of 2,3-diphosphoglycerate compared with other species. 71 the result is enhanced oxygen unloading in the equine fetus compared with others. 2,3-diphosphoglycerate concentration increases after birth in the foal and reaches mature levels by 3 to 5 days of age. the major blood adaptation of the equine fetus to chronic hypoxia is an increase in packed cell volume of up to 20%, increasing the oxygen content of the blood as compensation for decreased oxygen delivery at the placenta. 72 a larger than expected packed cell volume in any newborn foal should alert the clinician for possible sequelae from chronic hypoxia. the presence of significant cyanosis that persists should prompt the clinician to evaluate the foal thoroughly for cardiac anomalies resulting in significant right-to-left shunting or separated circulations, such as transposition of the great vessels. the chest wall of the foal is compliant, facilitating passage through the pelvic canal during parturition. this compliance requires that the foal actively participate in inspiration and expiration with several potential consequences. first, restriction of the thorax or the abdomen can result in impaired ventilation, which can occur easily when one restrains a foal and may result in spuriously abnormal arterial blood gas values (see the discussion on arterial blood gas evaluation, respiratory diseases associated with hypoxemia in the neonate). second, foals with primary pulmonary parenchymal disease resulting in poorly compliant lungs develop paradoxical chest wall motion, with the thorax moving inward during inspiration. [73] [74] [75] [76] the work of breathing can increase greatly, resulting in respiratory failure because of respiratory muscle fatigue. a foal that appears suddenly to improve a previously abnormal respiratory rate and pattern may in fact be in greater respiratory difficulty because of fatigue. one can observe a reduction in respiratory rate or abnormal breathing pattern in premature/dysmature foals or foals subjected to peripartum hypoxia/asphxia. although the genesis of these patterns is not understood fully, cheyne-stokes (lengthy periods of apnea interrupted by short breaths that wax and wane in depth), cluster (short periods of apnea interspersed with long periods of breathing), and biot's breathing (periods of apnea and breathing with no discernible pattern) may occur in these cases. foals attempting to maintain an adequate lung volume expire against a partially closed glottis, called valsalva's maneuver, producing an audible grunt. foals are normally nonresponsive while in the birth canal but should respond to stimulation immediately after birth. 67 the lack of responsiveness while in the birth canal has lead to presumption of fetal death during dystocia. because of this, one should attempt other tests before determining that a foal is dead intrapartum. one possibly may detect pulses in the tongue, neck, or any presented limbs or palpate the thorax for a heartbeat. in the author's facility, nasotracheal intubation of the foal combined with measurement of co 2 tensions in the exhaled gas aids practitioners in cases where they can reach the nose. nasotracheal intubation of foals under these circumstances actually can be performed readily with minimal practice. having long endotracheal tubes available of several different diameters (7 to 12 mm outer diameter) with an inflatable cuff is important. one can pass the tube blindly using a finger in one nostril for guidance and can check the position frequently by palpation of the throatlatch region. one inflates the cuff and begins manual ventilation with 100% oxygen or room air using an ambu-bag or equivalent. one can obtain continuous measurement of co 2 tension using a capnograph or single-use disposable end-tidal co 2 monitor attached to the ambu-bag or the nasotracheal tube. in a dead foal the end-tidal co 2 measurement will be negligible after the first 10 to 20 breaths. one must ensure tube placement and seal integrity and allow for multiple breaths. some co 2 will "wash out" with the first few breaths and can result in false hope initially. end-tidal co 2 varies in living intrapartum foals, depending on cardiac output and ventilation frequency, but should be consistently greater than 20 mm hg and is usually closer to 30 mm hg. once one establishes manual ventilation of a living foal, one must continue ventilation until the foal is delivered satisfactorily. the author has resuscitated and maintained many foals successfully in this manner throughout induction of general anesthesia in the mare and cesarean section delivery of the foal. the nasotracheal tube also provides a convenient site for administration of intratracheal medications such as epinephrine used for extrauterine intrapartum resuscitation of the foal. the reader is cautioned that intratracheal epinephrine increases endtidal co 2 measurements transiently, even in a dead foal, because of local actions on tissues. one should allow a washout period after intratracheal administration of epinephrine. the righting reflex is present as the foal exits the birth canal, as is the withdrawal reflex. cranial nerve responses are intact at birth, but the menace response may take as long as 2 weeks to develop fully. one should not consider lack of a menace reflex diagnostic of visual deficits in the newborn foal. within an hour of birth the normal foal will demonstrate auditory orientation with unilateral pinna control. the normal pupillary angle is ventromedial in the newborn foal; this angle gradually becomes dorsomedial over the first month of life. foals should begin attempting to stand shortly after birth and should be able to achieve this on their own within 2 hours of birth. 67 the normal newborn foal has a suck reflex shortly after birth and should be searching for an udder even before it stands. the expectation is that a normal foal will be sucking from the dam unaided by 3 hours post partum; many foals are overachievers and will be sucking well before this time. the normal foal may defecate shortly after standing but may not attempt defecation until after it first successfully sucks from the dam. urination varies more, with filly foals usually urinating before colt foals, but both usually do not urinate for several hours following birth, up to 12 hours for some colts. 67 for colt foals to fail to drop their penises when urinating over the first few days of life is not unusual. the gait of the newborn foal is hypermetric and the stance is base wide. extreme hypermetria of the forelimbs, usually bilateral but occasionally unilateral, has been observed in some foals and is associated with perinatal hypoxic/ischemic insults, but this gait abnormality usually resolves without specific therapy within a few days. spinal reflexes tend to be exaggerated, whereas the crossed extensor reflex may not be fully present until 3 weeks of age. 77 foals also exhibit an exaggerated response to external stimuli (noise, sudden visual changes, touch) for the first few weeks of life. foals are not bonded strongly to their mother for the first few weeks of life and will follow any large moving object, including other horses and human beings. orphan foals bond with surrogate mothers until they are several months of age; their primary motivation appears to be appetite. conversely, mares strongly bond with their foals shortly after parturition; the process begins once the chorioallantois ruptures and is driven more by olfaction and taste than by vision or hearing. interference with this process, by medical intervention or excessive owner manipulation of the foal, can disrupt normal bonding and result in foal rejection by the dam. 78 most newborn foals make the transition to extrauterine life easily. however, for those in difficulty, recognition of the condition immediately and institution of appropriate resuscitation is of utmost importance. a modified apgar scoring system has been developed as a guide for initiating resuscitation and assessing probable level of fetal compromise (table 19 -2). 79 one also must at least perform a cursory physical examination before initiating resuscitation, for issues of humaneness are associated with with serious problems such as severe limb contracture, microophthalmia, and hydrocephalus, among others. the initial assessment begins during presentation of the fetus. although the following applies primarily to attending the birth of a foal from a high-risk pregnancy, one can perform quiet and rapid evaluation during any attended birth. the goal in a normal birth with a normal foal is to disturb the bonding process minimally. this goal also applies to high-risk parturitions, but some disruption of normal bonding is inevitable. the lead clinician should control tightly the number of persons attending, and the degree of activity surrounding, the birth. one should evaluate the strength and rate of any palpable peripheral pulse and should evaluate the apical pulse as soon as the chest clears the birth canal. bradycardia (pulse <40 beats/min) is expected during forceful contractions, and the pulse rate should increase rapidly once the chest clears the birth canal. persistent bradycardia is an indication for rapid intervention. the fetus is normally hypoxemic compared with the newborn foal, and this hypoxemia is largely responsible for the maintenance of fetal circulation by generation of pulmonary hypertension. the fetus responds to conditions producing more severe in utero hypoxia by strengthening the fetal circulatory pattern, and the neonate responds to hypoxia by reverting to the fetal circulatory pattern. 80 during a normal parturition, mild asphyxia occurs and results in fetal responses that pave the way for a successful transition to extrauterine life. if more than mild transient asphyxia occurs, the fetus is stimulated to breathe in utero; this is known as primary asphyxia. 81 if the initial breathing effort resulting from the primary asphyxia does not correct the asphyxia, a second gasping period occurs in several minutes, known as the secondary asphyxia response. if no improvement in asphyxia occurs during this period, the foal enters secondary apnea, a state that is irreversible except with resuscitation. therefore the first priority of neonatal resuscitation is establishing an airway and breathing pattern. one should assume that foals not spontaneously breathing are in secondary apnea and should clear the airway of membranes as soon as the nose is presented. if meconium staining is present, one should suction the airway before delivery of the foal is completed and before the foal breathes spontaneously. one should continue to the trachea if aspiration of the nasopharynx is productive. overzealous suctioning worsens bradycardia as it worsens hypoxia. one should stop suctioning once the foal begins breathing spontaneously, as hypoxia will worsen with continued suction. if the foal does not breathe or move spontaneously within seconds of birth, one should begin tactile stimulation. if tactile stimulation fails to result in spontaneous breathing, one immediately should intubate the foal and manually ventilate the foal using an ambu-bag or equivalent. one can use mouth-to-nose ventilation if nasotracheal tubes and an ambu-bag are not available. the goal of this therapy is to reverse fetal circulation, and hyperventilation with 100% oxygen is the best choice for this purpose. however, recent evidence suggests that no clinical disadvantages are apparent in using room air for ventilation of asphyxiated human neonates rather than 100% oxygen. 82, 83 human infants resuscitated with room air recovered more quickly than those resuscitated with 100% oxygen in one study as assessed by apgar scores, time to the first cry, and the sustained pattern of breathing. 84 in addition, neonates resuscitated with 100% oxygen exhibited biochemical findings reflecting prolonged oxidative stress, present even after 4 weeks of postnatal life, which did not appear in the group resuscitated with room air. thus the current accepted recommendations for using 100% oxygen in the resuscitation of asphyxiated neonates needs further discussion and investigation. 85, 86 almost 90% of foals requiring resuscitation respond to hyperventilation alone and require no additional therapy. one can initiate nasotracheal intubation while the foal is in the birth canal if the foal will not be delivered rapidly, such as with a difficult dystocia. this technique is "blind" and requires some practice but may be beneficial and lifesaving. once spontaneous breathing is present, one apgar score in the foal should provide humidified oxygen via nasal insufflation at 8 to 10 l/min. one should initiate cardiovascular support in the form of chest compression if the foal remains bradycardic despite ventilation and a nonperfusing rhythm is present. one should make sure the foal is on a hard surface in right lateral recumbency with the topline against a wall or other support. approximately 5% of foals are born with fractured ribs and an assessment for the presence of rib fractures is in order before initiating chest compressions. 87 palpation of the ribs identifies many of these fractures, which usually are multiple and consecutive on one side of the thorax and located in a relatively straight line along the part of the rib with the greatest curvature dorsal to the costochondral junction. unfortunately, ribs 3 to 5 frequently are involved, and their location over the heart can make chest compression a potentially fatal exercise. auscultation over the ribs during breathing results in a recognizable click, identifying rib fractures that may have escaped detection by palpation. one should initiate drug therapy if a nonperfusing rhythm persists for more than 30 to 60 seconds in the face of chest compression. epinephrine is the first drug of choice (table 19-3) . practitioners pose various arguments regarding the best dose and the best frequency of administration for resuscitation. however, most of the data are acquired from human cardiac arrest studies and are not strictly applicable to the equine neonate because the genesis of the cardiovascular failure is different. 88, 89 vasopressin is gaining attention as a cardiovascular resuscitation drug, and although the author has used this drug in resuscitation and as a pressor, experience is limited at this time. 90 the author does not use atropine in bradycardic newborn foals because the bradycardia usually is caused by hypoxia, and if the hypoxia is not corrected, atropine can increase myocardial oxygen debt. 89 the author also does not use doxapram because it does not reverse secondary apnea, the most common apnea in newborns. because birthing areas are generally cold, one should dry the foal and place it on dry bedding once resuscitation is complete. the fetus has some homeothermic mechanisms, but its size in relation to its mother and its position within her body means that it is in effect a poikilotherm. the body temperature of the foal generally reflects that of its environment, namely its mother, although the human fetal temperature directly measured at cesarean section, induction of labor, or during labor is approximately 0.5â°c higher than the mothers. 91, 92 adaptation from poikilothermy to homeothermy normally takes place rapidly following birth. the fetus is capable of nonshivering thermogenesis, primarily through the oxidation of brown fat reserves, but this type of thermogenesis is inhibited in utero, probably by placental prostaglandin e 2 and adenosine. 93, 94 immediately after birth the foal must adapt to independent thermoregulation. local physical factors, including ambient temperature and humidity, act to induce cold stress, and the newborn must produce heat by metabolic activity. in response to the catecholamine surge associated with birth, uncoupling of oxidative phosphorylation occurs within mitochondria, releasing energy as heat. this nonshivering thermogenesis is impaired in newborns undergoing hypoxia or asphyxiation and in those that are ill at birth. infants born to mothers sedated with benzodiazepines are affected similarly, a consideration in the choice of sedative and preanesthetic medications in mares suffering dystocia or 1388 part ii disorders of specific body systems undergoing cesarean section. [95] [96] [97] heat losses by convection, radiation, and evaporation are high in most areas where foals are delivered, resuscitated ,and managed, and one must take care to minimize cold stress in the newborn and the critically ill foal. supplementary heat, in the form of radiant heat lamps or warm air circulating blankets, may be required. one should use fluid therapy conservatively during postpartum resuscitation, for the neonate is not volume depleted unless excessive bleeding has occurred. some compromised newborn foals are actually hypervolemic. fluid therapy of the neonate is discussed in more detail later in this chapter. because the renal function of the equine neonate is substantially different from the adult, one cannot simply scale down fluid therapy from adult therapy. [98] [99] [100] if intravenous fluids are required for resuscitation and blood loss is identified, administration of 20 ml/kg of a non-glucose-containing polyionic isotonic fluid over 20 minutes (about 1 l for a 50-kg foal) once intravenous access is established can be effective. the author stresses non-glucose-containing polyionic intravenous fluids because hyperglycemia, but not hypoglycemia, immediately after fetal or neonatal asphyxia interfered with the recovery of brain cell membrane function and energy metabolism in neonatal piglets in one recent study. 101 these findings suggest that post-hypoxic-ischemic hyperglycemia is not beneficial and might even be harmful in neonatal hypoxic-ischemic encephalopathy. indications for this shock bolus therapy include poor mentation, poorly palpable peripheral pulses, and the development of cold distal extremities, compatible with hemorrhagic shock. one should reassess the patient after the initial bolus and administer additional boluses as necessary. ideally, one should follow up on blood pressures and ecg readings and initiate appropriate pressor therapy if needed. again, these procedures are discussed in detail later in the chapter. one can administer glucose-containing fluids after resuscitation at a rate of 4 to 8 mg/kg/min (about 250 ml/hr of 5% dextrose or 125 ml/hr of 10% dextrose) to the average 50-kg foal, particularly in the obviously compromised foal. this therapy is indicated to help resolve metabolic acidosis, to support cardiac output because myocardial glycogen stores likely have been depleted, and to prevent postasphyxial hypoglycemia. under normal conditions, the fetal-to-maternal blood glucose concentration gradient is 50% to 60% in the horse, and glucose is the predominant source of energy during fetal development. 102, 103 glucose transport across the placenta is facilitated by carrier receptors (glucose transporter [glut] receptors), and a direct relationship exists between maternal and fetal blood glucose concentration when maternal glucose is in the normal range. 102 the glut receptors in the placenta are stereospecific, saturable, and energy independent. 104 although the enzyme kinetics for glut isoform 1 suggest that they are not saturable under conditions of euglycemia, equine maternal hyperglycemia results in increased fetal glucose concentration to a plateau point, likely caused by glut saturation. at term, the net umbilical uptake of glucose is 4 to 7 mg/kg/min, with most of the glucose being used by the brain and skeletal muscle. [105] [106] [107] the fetus only develops gluconeogenesis under conditions of severe maternal starvation. a certain percentage of the delivered glucose is used to develop large glycogen stores in the fetal liver and cardiac muscle in preparation for birth, and at birth the foal liver produces glucose at a rate of 4 to 8 mg/ kg/min by using these stores. fetal glycogen stores also are built using the substrates lactate, pyruvate, and alanine; fetal uptake of lactate across the placenta is about half that of glucose. 102, 108 the transition to gluconeogenesis, stimulated by increased circulating catecholamine concentration from birth and by stimulation of glucagon release at the time the umbilical cord breaks takes 2 to 4 hours in the normal foal, and glycogenolysis supplies needed glucose until feeding and glucose production are accomplished. 109 in the challenged foal, glycogen stores may have been depleted and gluconeogenesis delayed, so provision of glucose at rates similar to what the liver would normally produce during this period is requisite. persistent pulmonary hypertension (pph) also is known as reversion to fetal circulation or persistent fetal circulation, and its genesis lies in the failure of the fetus to make the respiratory and cardiac transition to extrauterine life successfully or reversion of the newborn to fetal circulatory patterns in response to hypoxia or acidosis. differentiating this problem from other causes of hypoxemia in the newborn requires some investigation, and multiple serial arterial blood gas analyses are necessary to confirm suspicion of this problem (see the section on arterial blood gas analysis, respiratory diseases associated with hypoxemia in the neonate). however, one should suspect the condition in any neonate with hypercapnic hypoxemia that persists and worsens; these foals are in hypoxemic respiratory failure. the fetal circulatory pattern, with pulmonary hypertension and right-to-left shunting of blood through the patent foramen ovale and ductus arteriosus, is maintained in these cases. pulmonary vascular resistance falls at delivery to about 10% of fetal values, while pulmonary blood flow increases accordingly. 110 early in the postnatal period these two changes balance each other, and mean pulmonary and systolic pressures remain increased for several hours. systolic pulmonary pressures can remain equivalent to systemic pressure for up to 6 hours of age in human infants, although diastolic pulmonary pressures are well below systemic diastolic pressures by 1 hour. 111 mean pulmonary artery pressures fall gradually over the first 48 hours. 112 the direct effects of lung expansion and increasing alveolar oxygen tension probably provide the initial stimulus for pulmonary arteriolar dilation and partly result from direct physical effects, but vasoactive substances are released in response to physical forces associated with ventilation, for example prostacyclin. 110 other vasoactive mediators thought to play a role in regulating pulmonary arteriolar tone include no, prostaglandins d 2 and e 2 , bradykinin, histamine, endothelin-1, angiotensin ii, and atrial natriuretic peptide. the increase in alveolar and arterial oxygen tensions at birth is required for completion of resolution of pulmonary hypertension. much of this increase is thought to be mediated by no, evidence for this being the parallel increase during gestation of the pulmonary vasodilation response to hyperoxia and the increase in no synthesis. 113 however, inhibition of no synthesis does not eliminate the initial decrease in pulmonary artery resistance occurring because of opening of the airways. 114 when these mechanisms fail, one can recognize pph. right-to-left shunting within the lungs and through patent fetal conduits occurs and can result from many factors, including asphyxia and meconium aspiration, but in many cases the precipitating trigger is unknown. inappropriately decreased levels of vasodilators (no) and inappropriately increased levels of vasoconstrictors (endothelin-1) currently are being examined as potential mechanisms. chronic in utero hypoxia and acidosis may result in hypertrophy of the pulmonary arteriolar smooth muscle. 115 in these cases, reversal of pph can be difficult and cannot be achieved rapidly. treatment of pph is twofold: abolishment of hypoxia and correction of the acidosis, for both abnormalities only bolster the fetal circulatory pattern. initial therapy is provision of oxygen intranasally at 8 to 10 l/min. some foals respond to this therapy and establish neonatal circulatory patterns within a few hours. failure to improve or worsening of hypoxemic respiratory failure following intranasal oxygen administration should prompt intubation and mechanical ventilation with 100% oxygen. this serves two purposes, one diagnostic and one therapeutic. ventilation with 100% oxygen may resolve pph and, if intrapulmonary shunt and altered ventilation-perfusion relationships are causing the hypoxic respiratory failure, arterial oxygen tension (pao 2 ) should exceed 100 mm hg under these conditions. failure to improve pao 2 suggests pph or large right-to-left extrapulmonary shunt caused by congenital cardiac anomaly. the vasodilators prostacyclin and telazoline (an î±-blocking vasodilator) cause pulmonary vasodilation in human infants with pph, but the effects on oxygenation vary and the sideeffects (tachycardia, severe systemic hypotension) are unacceptable. 116 recognition of no as a potent dilator of pulmonary vessels has created a significant step forward in the treatment of these patients, for inhaled no dilates vessels in ventilated portions of the lung while having minimal effects on the systemic circulation. 117 based on evidence presently available, use of inhaled no in an initial concentration of about 20 ppm in the ventilatory gas seems reasonable for term and near-term foals with hypoxic respiratory failure and pph that fails to respond to mechanical ventilation using 100% oxygen alone. 117, 118 the author has used this approach in the clinic, administering a range of 5 to 40 ppm no with success. hypoxic ischemic encephalopathy (hie), currently referred to as neonatal encephalopathy in the human literature, is one systemic manifestation of a broader syndrome of perinatal asphyxia syndrome (pas), and management of foals with signs consistent with a diagnosis of hie requires the clinician to examine other body systems fully and to provide therapy directed at treating other involved systems. 119 although pas primarily manifests as hie, the gastrointestinal tract and kidneys frequently are affected by peripartum hypoxia/ischemia/ asphyxia, and one should expect complications associated with these systems. hypoxic ischemic encephalopathy also may affect the cardiovascular and respiratory systems, and one also may encounter endocrine disorders in these patients. hypoxic ischemic encephalopathy has been recognized as one of the most common diseases of the equine neonate for generations. 1, 10, 12 in the past hie has been known as dummy foal syndrome and as neonatal maladjustment syndrome. the designation hie, although not perfect, attempts to describe the syndrome in terms of the suspected underlying pathophysiology. a wide spectrum of clinical signs is associated with hie and can range from mild depression with loss of the suck reflex to grand mal seizure activity. typically, affected foals are normal at birth but show signs of central nervous system abnormalities within a few hours after birth. some foals are obviously abnormal at birth, and some do not show signs until 24 hours of age. hypoxic ischemic encephalopathy commonly is associated with adverse peripartum events, including dystocia and premature placental separation, but a fair number of foals have no known peripartum period of hypoxia, suggesting that these foals result from unrecognized in utero hypoxia (box 19-2). severe maternal illness also may result in foals born with pas. in human beings, ascending placental infection now is suspected of being a major contributor to neonatal encephalopathy in infants, and the incidence of neonatal encephalopathy increases with the presence of maternal fever, suggesting a role for maternal inflammatory mediators. 120 the underlying pathophysiologic details of hie in the foal are unknown, and to date accurate experimental models of hie and pas in the foal have not been described. however, a great deal of attention has been paid to peripartum hypoxia/asphyxia by human counterparts because the effects of adverse peripartum events in the human neonate have far ranging implications for the affected human neonate and for society. therefore equine neonatologists have long looked to human studies and models of the human disease for understanding of the syndrome in the equine neonate. perinatal brain damage in the mature fetus usually results from severe uterine asphyxia caused by an acute reduction of uterine or umbilical circulation. the fetus responds to this challenge by activation of the sympathetic adrenergic nervous system, causing a redistribution of cardiac output that favors the central organs: brain, heart, and adrenal glands. 121, 122 if the hypoxic insult continues, the fetus reaches a point beyond which it cannot maintain this centralization of circulation, cardiac output falls, and cerebral circulation diminishes. 122 the loss of oxygen results in a substantial decrease in oxidative phosphorylation in the brain with concomitant decreased energy production. the na + /k + pump at the cell membrane cannot maintain the ionic gradients, and the membrane potential is lost in the brain cells. in the absence of the membrane potential, calcium flows down its large extracellular/intracellular concentration gradient through voltage-dependent ion channels into the cell. this calcium overload of the neuron leads to cell damage by activation of calcium-dependent proteases, lipases, and endonucleases. protein biosynthesis is halted. calcium also enters the cells by glutamate-regulated ion channels as glutamate, an excitatory neurotransmitter, is released from presynaptic vesicles following anoxic cellular depolarization. once the anoxic event is over, protein synthesis remains inhibited in specific areas of the brain and returns to normal in less vulnerable areas of the brain. loss of protein synthesis appears to be an early indicator of cell death caused by the primary hypoxic/anoxic event. 123 a second wave of neuronal cell death occurs during the reperfusion phase and is thought to be similar to classically described postischemic reperfusion injury in that damage is caused by production of and release of oxygen radicals, synthesis of no, and inflammatory reactions. 124 additionally, an imbalance between excitatory and inhibitory neurotransmitters occurs. 123 part of the secondary cell death that occurs is thought to be caused by apoptosis, a type of programmed cell death termed cellular suicide. secondary cell death also is thought be caused by the neurotoxicity of glutamate and aspartate resulting again from increased intracellular calcium levels. 125, 126 in human infants the distribution of lesions with hypoxic-ischemic brain damage following prenatal, perinatal, or postnatal asphyxia falls into distinct patterns depending on the type of hypoxia-ischemia rather than on postconceptual age at which the asphyxial event occurs. 126 periventricular leukomalacia was associated with chronic hypoxia-ischemia, whereas the basal ganglia and thalamus were affected primarily in patients experiencing acute profound asphyxia, providing direct evidence that the nature of the event determines the severity and distribution of neurologic damage in human beings. these remarkably selective patterns of injury in children, with differential variability in the damage caused to regions anatomically located within millimeters of each other, resulted in the hypothesis that location within neurotransmitter-specific circuitry loops is important. this hypothesis has important implications in the design of neuroprotective strategies and therapies for neonates experiencing hypoxic-ischemic-asphyxial events. now the evidence is overwhelming that the excitotoxic cascade that evolves during hie extends over several days from the time of insult and is modifiable. 125, 126 in brain injury, traumatic or hypoxic, the mechanisms underlying delayed tissue injury still are understood poorly. many believe that neurochemical changes, including excessive neurotransmitter release, are pivotal in the pathophysiology of secondary neuronal death. excitatory amino acid neurotransmitters and magnesium are known to play at least a minimal role in secondary cell death following brain injury; a fair body of literature regarding these factors has been generated over the last 10 years. the activation of the n-methyl-d-aspartate (nmda) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury and is suspected to play a role in hie. [125] [126] [127] mechanically injured neurons demonstrate a reduction of voltage-dependent mg 2+ blockade of nmda current that can be restored partially by increasing extracellular mg 2+ concentration or by pretreatment with calphostin c, a protein kinase c inhibitor. 128 this finding suggested that administration of mg 2+ to patients with brain injury could lead to improved outcome. subsequently, magnesium sulfate solution was shown to improve dramatically the immediate recovery of rats from hypoxia. 129 however, although pretreatment with magnesium sulfate protected against hypoxic ischemic brain injury, postasphyxial treatment worsened brain damage in 7-day-old rats, suggesting an age-related response in the rat. 130 delayed magnesium treatment of mature rats following severe traumatic axonal brain injury improved motor outcome when administered up to 24 hours after injury, with early treatments providing the most benefit. 131 maternal seizure in rats is associated with fetal histopathologic changes that are abolished by administration of magnesium sulfate to the mother, and magnesium sulfate has been demonstrated to protect the fetal brain from severe maternal hypoxia. 132 clinical trials investigating the efficacy of magnesium treatment following hypoxia in infants are under way, with few reports currently in the medical literature. magnesium sulfate was used to treat nine infants after perinatal asphyxia in one study (no control group), and all children were neurologically normal at 1 year of age. seizures did not occur in any of these children, nor were any adverse side effects noted. 133 magnesium sulfate administration failed to delay the global impairment in energy metabolism after hypoxia ischemia, characteristic of severe brain damage, in newborn piglets; at 48 hours after hypoxia ischemia, no difference could be found in the severity of injury in piglets treated with magnesium compared with piglets treated with placebo, suggesting magnesium may not be protective with severe acute injury. 134 in developing countries, birth hypoxia frequently is associated with hie, and although this finding is attributed most frequently to inadequate obstetric care, poor nutrition also may play a role. red blood cell magnesium levels were measured in more than 500 women in labor at a teaching hospital in south africa. 135 fifty five of the women delivered infants with hie and had significantly lower levels of magnesium than controls; the infants with hie also had significantly lower magnesium levels than controls. the large majority (54 of 55) of the women giving birth to hie infants were from poor social circumstances, suggesting nutrition might play a role in some cases of hie, with maternal magnesium levels affecting outcome in the infants. the authors suggested an early pregnancy intervention study may help determine the role of magnesium in the pathogenesis of hie in human infants born to at-risk mothers. therapy for the various manifestations of hypoxiaischemia involves control of seizures, general cerebral support, correction of metabolic abnormalities, maintenance of normal arterial blood gas values, maintenance of tissue perfusion, maintenance of renal function, treatment of gastrointestinal dysfunction, prevention and recognition and early treatment of secondary infections, and general supportive care. control of seizures is important because cerebral oxygen consumption increases fivefold during seizures. one can use diazepam for emergency control of seizures (table 19 -4). if diazepam does not stop seizures readily or one recognizes more than two seizures, then one should replace diazepam with phenobarbital given to effect. the half-life of phenobarbital can be long in the foal (100 hours), and one should keep this in mind when monitoring neurologic function in these cases after phenobarbital administration (j.e. palmer, personal communication, 1998). 136 earlystage, preseizure administration of phenobarbital has been advocated by some investigators for prevention of neonatal encephalopathy. however, one recent study in asphyxiated human infants demonstrated that early phenobarbital treatment was associated with a threefold increase in the incidence of subsequent seizures and consequently a trend toward increased mortality. seizures per se were associated with almost a twentyfold increase in mortality. their findings suggest that early phenobarbital administration may produce adverse rather than beneficial effects following asphyxia. because this was an observational study; the results need to be confirmed by appropriate randomized trials in similar clinical settings. 137 if phenobarbital fails to control seizures, one may attempt phenytoin therapy. in cases of hie, one should avoid ketamine and xylazine because of their association with increased intracranial pressure. one must protect the foal from injury during a seizure and also ensure the patency of the airway to prevent the onset of negative pressure pulmonary edema 138 or aspiration pneumonia. probably the most important therapeutic interventions are aimed at maintaining cerebral perfusion, which is achieved by careful titration of intravenous fluid support, neither too much nor too little (see fluid therapy in neonates) and judicious administration of inotropes and pressors to maintain adequate perfusion pressures (see pressor and inotrope therapy in neonates). cerebral interstitial edema is only truly present in the most severe cases 139, 140 ; in most cases the lesion is intracellular edema and most of the classic agents used to treat cerebral interstitial edema (e.g., mannitol) are minimally effective treating cellular edema. occasionally the author uses thiamine supplementation in the intravenous fluids to support metabolic processes, specifically mitochondrial metabolism and membrane na + ,k + -atpases, involved in maintaining cellular fluid balance. 141, 142 this therapy is rational and inexpensive but unproven in efficacy. only if cellular necrosis and vasogenic edema are present are drugs such as mannitol and dimethyl sulfoxide indicated, and again these cases are usually the most severely affected. in the author's clinic, practitioners rarely have used dimethyl sulfoxide in neonates for the last several years and have recognized no change in outcome by discontinuing its use. when the practitioners use intravenously administered dimethyl sulfoxide, they do so within the first hour after an acute asphyxial insult and use it primarily for its hydroxyl radical scavenging effects and its theoretical modulation of postischemic reperfusion injury. 143 naloxone has been advocated for treating hie in human beings and in foals, [144] [145] [146] perhaps based on a study suggesting that postasphyxia blood-brain barrier disruption was related causally to poor neurologic outcome in a lamb model of hie and that naloxone prevented disruption and neurologic dysfunction among those survivors with an intact blood-brain barrier. 145 however, other studies have demonstrated that naloxone exacerbates hypoxic-ischemic brain injury in 7-day-old rats subjected to unilateral common carotid artery ligation and hypoxia. moreover, systemic acidosis and cellular edema were no different in naloxone-treated animals compared with animals treated with saline solution. the authors concluded that high doses of naloxone in fact may reduce the resistance of the fetus to hypoxic stress. 146 the use of naloxone in human neonatal resuscitation remains controversial, for whether the contradictory effects are related to a reduction in acute neuronal swelling by osmotic effects or by a more direct receptor-mediated mechanism is currently unknown. 147 naloxone is most effective in resuscitation of compromised human infants born to mothers addicted to drugs. some practitioners are using î³-aminobutyric acid adrenergic agonists to manage hie in foals, based on evidence showing neuroprotection when used in ischemia alone and combined with nmda antagonists. [148] [149] [150] the author currently has no experience with these compounds and cannot comment regarding their efficacy in foals. regional hypothermia also is being investigated as a potential therapy for global hypoxia/ischemia; published data are consistent with the theory that cooling must be continued throughout the entire secondary phase of injury (about 3 days) to be effective. 151 experimentally, this approach has resulted in dramatic decreases in cellular edema and neuronal loss; its practical application remains to be demonstrated. despite a lack of consensus regarding the use of magnesium to treat infants with hie, the author has used magnesium sulfate infusion as part of the therapy for selected foals with hie for the past several years. the rationale is based primarily on the evidence demonstrating protection in some studies and a failure of any one study to demonstrate significant detrimental effects. the clinical impressions of the author to date suggest that the therapy is safe and may decrease the incidence of seizure in patients. the author administers magnesium sulfate as a constant rate infusion over 1 hour after giving a loading dose. the author has continued the infusion for up to 3 days without demonstrable negative effect beyond some possible trembling. given the current evidence, a 24-hour course of treatment may be effective and all that is necessary. postasphyxial treatment certainly may be beneficial in foals with hie, and maternal magnesium therapy may be beneficial in certain high-risk pregnancy patients. foals with pas often have a variety of metabolic problems including hypo-or hyperglycemia, hypo-or hypercalcemia, hypo-or hyperkalemia, hypo-or hyperchloremia, and varying degrees of metabolic acidosis. although one needs to address these problems, one should not forget the normal period of hypoglycemia that occurs postpartum and should not treat aggressively so as to avoid worsening the neurologic injury. foals suffering from pas also have frequent recurrent bouts of hypoxemia and occasional bouts of hypercapnia. intranasally administered oxygen is generally needed in these cases as a preventative therapy and as direct treatment, for the appearance of the abnormalities can be sporadic and unpredictable. additional respiratory support, particularly in those foals with centrally mediated hypoventilation and periods of apnea or abnormal breathing patterns, include caffeine (per os or per rectum) and positive pressure ventilation. caffeine is a central respiratory stimulant and has minimal side effects at the dosages used (10 mg/kg loading dose; 2.5 mg/kg as needed). 152 the author purchases whatever oral form of caffeine is available at the local convenience store or drug store and administers it dissolved in warm water per rectum. foals treated with caffeine have an increased level of arousal and are more reactive to the environment. adverse effects generally are limited to restlessness, hyperactivity, and mild to moderate tachycardia. mechanical ventilation of these patients can be rewarding and generally is required for less than 48 hours. one must monitor and maintain blood ph within the normal range. metabolic alkalosis can develop in some of these foals and requires clinician tolerance of some degree of hypercapnia. ph is important in evaluation and consideration of alternatives for treatment. if the respiratory acidosis is not so severe as to affect the patient adversely (generally >70 mm hg), and the ph is within normal limits, the foal may tolerate hypercapnia. 153 the goal is to normalize ph. foals with respiratory acidosis as compensation for metabolic alkalosis do not respond to caffeine. metabolic alkalosis in critically ill foals frequently is associated with electrolyte abnormalities, creating differences in strong ion balance. one handles this ph perturbation best by correcting the underlying electrolyte problem. maintaining tissue perfusion and oxygen delivery to tissues is a cornerstone of therapy for pas to avoid additional injury. one should maintain the oxygen-carrying capacity of the blood; some foals require transfusions to maintain a packed cell volume greater than 20%. adequate vascular volume is important, but one should take care to avoid fluid overload in the foal. early evidence of fluid overload is subtle accumulation of ventral edema between the front legs and over the distal limbs. fluid overload can result in cerebral edema, pulmonary edema, and edema of other tissues, including the gastrointestinal tract. this edema interferes with normal organ function and worsens the condition of the patient. one maintains perfusion by supporting cardiac output and blood pressure by judicious use of intravenous fluid support and inotrope/pressor support. the author does not target therapy to a specific systolic, mean, or diastolic pressure but monitors urine output, mentation, limb perfusion, gastrointestinal function, and respiratory function as indicators that perfusion is acceptable. for these patients to require pressor therapy is not unusual, but in some cases the hypoxic damage is sufficiently severe to blunt the response of the patient to the drugs. the kidney is a target for injury in patients with pph, and for renal compromise to play a significant role in the demise of these foals is not unusual. clinical signs of renal disease are generally referable to disruption of normal control of renal blood flow and tubular edema leading to tubular necrosis and renal failure. these foals have signs of fluid overload and generalized edema. one must balance urine output and fluid therapy in these cases to prevent additional organ dysfunction associated with edema. although evidence has accumulated that neither dopamine nor furosemide play a role in protecting the kidney or reversing acute renal failure, these agents can be useful in managing volume overload in these cases. [154] [155] [156] the aim is not to drive oliguric renal failure into a highoutput condition but rather to enhance urine output. overzealous use of diuretics and pressors in these cases can result in diuresis requiring increased intravenous fluid support and can be counterproductive. the author's approach is more conservative. low doses of dopamine administered as a constant rate infusion of 2 to 5 âµg/kg/min are usually effective in establishing diuresis by natriuresis. one should avoid large doses of dopamine (>20 âµg/kg/min) because high doses can produce systemic and pulmonary vasoconstriction, potentially exacerbating pph. 157 one can administer a bolus (0.25 to 1.0 mg/kg) or constant rate infusion (0.25 to 2.0 mg/kg/hr) of furosemide, but once furosemide diuresis is established, one must evaluate electrolyte concentrations and blood gas tensions frequently because potassium, chloride, and calcium losses can be considerable and because significant metabolic alkalosis can develop from strong ion imbalances. the author does not aim for urine production rates of 300 ml/hr, as has been presented by other authors as a urine output goal for critically ill equine neonates. 158 rather the author looks for urine output that is appropriate for fluid intake and does not attempt to drive urine output to an arbitrary goal by excessive fluid administration or pressor use. although the average urine output for a normal equine neonate is about 6 ml/kg/hr (~300 ml/hr for a 50-kg foal), these values were obtained from normal foals drinking a milk diet with a large free water component. [98] [99] [100] the urine of normal newborn foals is dilute, reflecting the large free water load they incur by their diet. expecting critically ill foals to produce such large volumes of urine, particularly those on restricted diets or receiving total parenteral nutrition, is an exercise in futility, and manipulating fluid, pressor, or diuretic therapy in attempt to meet an artificial goal is inappropriate. fluid therapy in the critically ill neonate is discussed later in this chapter. one final caveat regarding renal dysfunction in pas is that one should perform therapeutic drug monitoring when it is available. many antimicrobial agents used to manage these cases, most notably the aminoglycosides, depend on renal clearance. aminoglycoside toxicity occurs in the equine neonate and exacerbates or complicates the management of renal failure originally resulting from primary hemodynamic causes. the author monitors aminoglycoside concentrations for 30-minute peak and 23-to 24-hour trough values in these cases and adjusts dosage and frequency of drug administration based on these results. the author considers a trough value of less than 2 âµg/dl as desirable for gentamicin and amikacin. foals with pas suffer from a variety of problems associated with abnormalities within the gastrointestinal tract. 159 commonly they have ileus, recurrent excessive gastric reflux, and gas distention. these problems are exacerbated by constant feeding in the face of continued dysfunction and continued hypoxia. frequently, enteral feeding cannot meet their nutritional requirements, and partial or total parenteral nutrition is required. one must give special attention to passive transfer of immunity (see failure of passive transfer) and glucose homeostasis in these cases. although some practitioners use prokinetic agents as therapy for ileus in these cases, the author's approach is again more conservative. appearance of damage to the gastrointestinal tract can be subtle and lag behind other clinical abnormalities for days to weeks. low-grade colic, decreased gastrointestinal motility, decreased fecal output, and low weight gain are among the most common clinical signs of gastrointestinal dysfunction in these case, but more severe problems, including necrotizing enterocolitis and intussusception, have been associated with these cases. the return to enteral feeding must be slow in many of these cases. a currently debated topic is constant versus pulsed enteral feeding. [160] [161] [162] the author uses pulsed feeding through an indwelling small-gauge feeding tube. in many foals these tubes stay in place for weeks and cause no problems as the foals are returned to their dams for sucking or are trained to drink from a bottle or bucket. foals with pas are also susceptible to secondary infection. treatment of recognized infection is covered under sepsis in this chapter. if infection is recognized in these patients after hospitalization, one should give attention to the likelihood of nosocomial infection and should direct antimicrobial therapy based on known nosocomial pathogens in the nicu and their susceptibility patterns until culture and sensitivity results become available. one should make repeat determinations of immunoglobulin g (igg) concentration; additional intravenous plasma therapy may be required. nosocomial infections are often rapidly overwhelming, and acute deterioration in the condition of a foal with pas should prompt a search for nosocomial infection. the prognosis for foals with pas is good to excellent when the condition is recognized early and aggressively treated in term foals. up to 80% of these neonates survive and go on to lead productive and useful athletic lives. [20] [21] [22] [23] the prognosis decreases with delayed or insufficient treatment and concurrent problems such as prematurity and sepsis. in human nicus the survival rates of low-gestationlength infants has increased dramatically since the 1980s concurrent with improvements in obstetric and neonatal care. the now routine, well-validated use of antenatal steroid and artificial surfactant therapies has contributed greatly to the enhanced survival of this patient population, although the use of these particular therapies is not common or frequently indicated in the equine nicu. 163, 164 however, with improved care, outcomes in the equine nicu population have improved also, with survival of premature patients in many nicus exceeding 80%. 21 in the equine population, gestation length is much more flexible than in the human population; however, the definition of the term prematurity needs reexamination. traditionally, prematurity is defined as a preterm birth of less than 320 days of gestation in the horse. given the variability of gestation length in the horse, ranging from 310 days to more than 370 days in some mares, a mare with a usual gestation length of 315 days possibly could have a term foal at 313 days, whereas a mare with a usual gestation length of 365 days may have a premature foal at 340 days, considered the normal gestation length. foals that are born postterm but are small are termed dysmature; a postmature foal is a postterm foal that has a normal axial skeletal size but is thin to emaciated. dysmature foals may have been classified in the past as small for gestational age and are thought to have suffered placental insufficiency, whereas postmature foals are usually normal foals that have been retained too long in utero, perhaps because of an abnormal signaling of readiness for birth, and have outgrown their somewhat aged placenta. postmature foals become more abnormal the longer they are maintained, also may suffer from placental insufficiency, and are represented best by the classic foal born to a mare ingesting endophyteinfested fescue. 165 box 19-3 compares the characteristics of premature/dysmature foals with those of postmature foals. the causes of prematurity/dysmaturity/postmaturity include the causes of high-risk pregnancy presented in box 19-1. additional causes include iatrogenic causes such as early elective induction of labor based on inaccurate breeding dates or misinterpretation of late-term colic or uterine bleeding as ineffective labor. most causes remain in the category of idiopathic, with no discernible precipitating factor. despite lack of an obvious cause, premature labor and delivery does not just happen, and even if undetermined, the cause may continue to affect the foal in the postparturient period. all body systems may be affected by prematurity, dysmaturity, and postmaturity, and thorough evaluation of all body systems is necessary. respiratory failure is common in these foals, although the cause usually is not surfactant deficiency. immaturity of the respiratory tract, poor control of respiratory vessel tone, and weak respiratory muscles combined with poorly compliant lungs and a greatly compliant chest wall contribute to respiratory failure in these cases. most require oxygen supplementation and positional support for optimal oxygenation and ventilation. one must extend effort to maintain these "floppy foals" in sternal recumbency. some foals may require mechanical ventilation. these foals also require cardiovascular support but are frequently unresponsive to commonly used pressors and inotropes: dopamine, dobutamine, epinephrine, and vasopressin. careful use of these drugs and judicious intravenous fluid therapy are necessary. the goal should not be one of achieving specific pressure values (e.g., mean arterial pressure of 60 mm hg) but of adequate perfusion. renal function, reflected in low urine output, is frequently poor initially in these cases because of delay in making the transition from fetal to neonatal glomerular filtration rates. 166 the delay can result from true failure of transition or from hypoxic/ischemic insult. one should approach fluid therapy cautiously in these cases; initial fluid restriction may be in order to avoid fluid overload. many premature/dysmature/postmature foals have suffered a hypoxic insult and have all of the disorders associated with pas, including hie. treatment is similar to that of term foals with these problems. these foals also are predisposed to secondary bacterial infection and must be examined frequently for signs consistent with early sepsis or nosocomial infection. the gastrointestinal system of these foals is not usually functionally mature, which may result from a primary lack of maturity or from hypoxia. dysmotility and varying degrees of necrotizing enterocolitis are common. one commonly encounters hyperglycemia and hypoglycemia. hyperglycemia generally is related to stress, increased levels of circulating catecholamines, and rapid progression to gluconeogenesis, whereas hypoglycemia is associated with diminished glycogen stores, inability to engage gluconeongenesis, sepsis, and hypoxic damage. 167 immature endocrine function is present in many of these foals, particularly regarding the hypothalamic-pituitary-adrenal axis, and contributes to metabolic derangements. 168, 169 one should delay enteral feeding when possible until the foal is stable regarding metabolic and cardiorespiratory parameters. on intiating enteral feeding, one should provide small volumes initially and slowly increase the volume over several days. one frequently encounters musculoskeletal problems, particularly in premature foals, that include significant flexor laxity and decreased muscle tone. postmature foals frequently are affected by flexure contracture deformities, most likely because of decreased intrauterine movement as they increase in size. premature foals frequently exhibit decreased cuboidal bone ossification that predisposes them to crush injury of the carpal and tarsal bones if weight bearing is not strictly controlled. physical therapy in the form of standing and exercise is indicated in the management of all these problems, but one should take care to ensure that the patient does not fatigue or stand in abnormal positions. bandaging of the limbs is contraindicated because this only increases laxity, although light bandages over the fetlock may be necessary to prevent injury to that area if flexor laxity is severe. the foals are predisposed to angular limb deformity and must be observed closely and frequently for this problem as they mature. 170 the overall prognosis for premature/dysmature/ postmature foals remains good with intensive care and good attention to detail. many of these foals (up to 80%) survive and become productive athletes. 21 complications associated with sepsis and musculoskeletal abnormalities are the most significant indicators of poor athletic outcome. the last 20 years have seen an explosion of new therapeutic agents purportedly useful for treating sepsis. unfortunately, clinical trials investigating these new therapies have failed to demonstrate a positive effect, have shown negative results, or have resulted in diametrically opposed study results, one showing a benefit and another showing no benefit or a detrimental effect. on a positive note, the survival rate of foals being treated for sepsis has improved. work was done regarding foal diseases and their treatment in the 1960s, but the field did not attract much serious attention until the 1980s. since that time almost every major veterinary college and many large private referral practices have constructed nicus or their equivalent. next to hypoxic ischemic asphyxial syndromes, sepsis is the number one reason for presentation and treatment at these facilities. neonatal septicemia of the horse has been the subject of three international workshops, 171-173 and a perinatology lecture covering some aspect of neonatal sepsis has been presented at almost every large continuing education meeting attended by equine veterinarians. concensus criteria conferences 1 in the early 1990s defined sepsis and septic shock for human beings. 174, 175 sepsis was defined as the systemic response to infection manifested by two or more of the following conditions as a result of infection: a) temperature >38â°c or <36â°; b) heart rate >90 beats/min; c) respiratory rate >20 breaths per minute or paco 2 <32 torr; and d) white blood cell count >12,000 cell/âµl, <4,000 cell/âµl, or >10% immature (band) forms. septic shock was defined as sepsis induced hypotension or the requirement for vasopressors/ionotropes to maintain blood pressure despite adequate fluid resuscitation along with the presence of perfusion abnormalities that may include lactic acidosis, oliguria, or acute alteration in mental status. these definitions are broadly acceptable and applicable to neonatal sepsis in foals, and many of the treatment modalities in human medicine have been applied in some manner to the equine neonatal patient. additional definitions that have come into vogue that are actually useful at times, include the following: sirs, the systemic inflammatory response system; mods, multiple organ system dysfuction; and mofs, multiple organ failure syndrome. (sirs is sick, mods is sicker, and mofs is dying.) the compensatory response syndrome (cars) ideally balances sirs and keeps it from becoming detrimental. if balance is achieved, recovery is possible. imbalance progresses to septic shock, mods, and mofs. in horses, mods is manifested most commonly as renal failure, hepatic failure, central nervous system dysfunction, and disseminated intravascular coagulation. managing the septic patient involves early recognition of all the potential alphabet combinations and supporting the patient or intervening in the face of multiple clinical consequences, termed chaos (cardiovacular compromise; homeostasis; apoptosis; organ dysfunction; suppression of the immune system). 176 inflammatory mediators are involved in all these processes and can be beneficial or detrimental, depending on timing and opposing responses. neutrophils, platelets, lymphocytes, macrophages, and endothelial cells are involved, and the implicated inflammatory molecules grow daily in numbers. sepsis in the foal initially can be subtle, and the onset of clinical signs varies depending on the pathogen involved and the immune status of the foal. for the purposes here, the discussion is limited to bacterial sepsis, but the foal also is susceptible to viral and fungal sepsis, which can appear similar to bacterial sepsis. failure of passive transfer (fpt) of immunity can contribute to the development of sepsis in a foal at risk. 177, 178 testing for and treating fpt has received attention in the veterinary literature. it remains true, however, that foals presented to nicus that have an ultimate diagnosis of sepsis have fpt. 16, 19 the current recommendation is that foals have igg levels greater than or equal to 800 mg/dl for passive transfer to be considered adequate. other risk factors for the development of sepsis include any adverse advents at the time of birth, maternal illness, or any abnormalities in the foal. although the umbilicus frequently is implicated as a major portal of entry for infectious organisms in the foal, the gastrointestinal tract may be the primary site of entry. 179 other possible portals of entry include the respiratory tract and wounds. early signs of sepsis include depression, decreased suck reflex, increased recumbency, fever, hypothermia, weakness, dysphagia, failure to gain weight, increased respiratory rate, tachycardia, bradycardia, injected mucous membranes, decreased capillary refill time, shivering, lameness, aural petechia, and coronitis. if sepsis is recognized early, patients with sepsis may have a good outcome, depending on the pathogen involved. gram-negative sepsis remains the most commonly diagnosed, but increasingly gram-positive septicemia is being recognized. 180 foals in intensive care units and at referral hospitals have an additional risk of nosocomial infection. an attempt to isolate the organim involved early in the course of the disease becomes important. if possible, one should obtain blood cultures, and if localizing signs are present, one should obtain samples as deemed appropriate. cultures should be aerobic and anaerobic. recently, work has been done evaluating real-time polymerase chain reaction technology in sepsis in the foal as a means of identifying causative organisms. 181, 182 until one obtains antimicrobial sensitivity patterns for the pathogen involved, one should initiate broad-spectrum antimicrobial therapy (table 19 -5). intravenously administered amikacin and penicillin are good first-line choices, but one should monitor renal function closely. other first-line antimicrobial choices might include high-dose ceftiofur sodium or ticarcillin/clavulanic acid. one should treat failure of passive transfer if present. one should provide intranasal oxygen insufflation at 5 to 10 l/min even if hypoxemia is not present to decrease the work of breathing and provide support for the increased oxygen demands associated with sepsis. 183 should arterial blood gas analysis reveal significant hypoventilation, one may administer caffeine orally or per rectum to increase central respiratory drive. mechanical ventilation may be necessary in cases of severe respiratory involvement such as with acute lung injury or acute respiratory distress syndrome. if the foal is hypotensive, one may administer pressor agents or inotropes by constant rate infusion (table 19-6) . inotrope and pressor therapy generally is restricted to referral centers where these drugs can be given as constant rate infusions and blood pressure can be monitored closely. some practitioners use nonsteroidal antiinflammatory agents and, in specific circumstances, corticosteroids. use of these drugs should be judicious because they may have several negative consequences for the foal including renal failure and gastric/dunodenal ulceration. [184] [185] [186] nursing care is one of the most important aspects of treating septic foals. foals should be kept warm and dry. they should be turned at 2-hour intervals if they are recumbent. feeding septic foals can be a challenge if gastrointestinal function is abnormal, and total parenteral nutrition may be needed. if at all possible, foals should be weighed daily and blood glucose levels monitored frequently. some foals become persistently hyperglycemic on small glucose infusion rates. these foals may benefit from constant rate low-dose insulin infusions (table 19-7) . recumbent foals must be examined frequently for decubital sore development, the appearance of corneal ulcers, and for heat and swelling associated with joints and physis. the prognosis for foals in the early stages of sepsis is fair to good. once the disease has progressed to septic shock the prognosis decreases, although short-term botulism is a neuromuscular disease of foals characterized by flaccid paralysis. 187 although the disease is discussed in detail elsewhere in this text, the form most commonly observed in foals, the toxicoinfectious form, deserves some specific comments. the causative organism is clostridium botulinum, an anaerobic organism. although affected adults usually acquire the disease by ingestion of preformed toxin elucidated from the organism, in the foal less than 8 months of age the organism can survive and multiply in the gastointestinal tract and produce necrotic foci within the liver, giving the foal constant exposure to newly formed toxin. the horse is exquisitely sensitive to the toxin, and only small quantities of toxin are required to produce clinical signs and death in affected animals. the îµ-toxin of c. botulinum binds to the presynaptic membrane of motor neurons and prevents transmission of impulses by blocking the release of acetylcholine from the presynaptic vessicles. this block produces the clinical signs of muscle weakness, manifested in foals as trembling (shaker foals) or acute recumbency. 188 pupillary dilation, dysphagia, tremors, recumbency, and terminal respiratory distress caused by respiratory muscle paralysis occur. foals can be found acutely dead. in endemic areas (the northeast and mid-atlantic regions of united states), for these foals to be evaluated first as having colic is not unusual. treatment aims to neutralize the toxin by administration of botulinum antitoxin and to provide antimicrobial treatment of the infection with penicillin, metronidazole, and/or oxytetracycline. 189, 190 at a minimum, feeding of milk replacer via indwelling nasogastric tube at 20% of the body weight of the foal per day divided into every 2-hour meals is required. many of these foals require respiratory support (in the form of intranasal oxygen insufflation), because of respiratory muscle paralysis. respiratory acidosis is present on arterial blood gas analysis in most of these foals because of hypoventilation and lateral recumbency, but they can tolerate some degree of hypercapnia (paco 2~7 0 mm hg) if the ph is normal and oxygenation (pao 2 >70 mm hg; percent oxygen saturation of hemoglobin, >90%) is adequate. metabolic alkalosis can accompany the respiratory acidosis, but this is a compensatory change and resolves once gas exchange is normalized. some of these patients require mechanical ventilation, which may be lifesaving. one may discontinue mechanical ventilation as clinical signs resolve and the respiratory muscles gain strength. nursing care is important, and these foals should be turned every 2 hours. they should be maintained in sternal recumbency if possible and kept warm and dry. with good nursing care, good nutritional support, and adequate respiratory support, the prognosis for these foals is good. the limiting factor in the prognosis for life is often financial. 190 foals that recover from the acute stage of this disease eventually fully recover. botulism is an expensive disease to treat and is also an entirely preventable disease. 189, 190 all pregnant mares in endemic areas should be vaccinated against c. botulinum. vaccination does not prevent all cases of botulism, particularly if the foal has failure of passive transfer or acquires the disease after maternal immunity wanes and before its own vaccination. nutritional muscular dystrophy or white muscle disease is a vitamin e/selenium-responsive muscle disease of horses of all ages probably caused by a dietary deficiency of selenium and vitamin e. 191 the condition occurs most commonly in geographic areas with low selenium levels in the soil, generally the northeastern, northwestern, great lakes and mid-atlantic regions of the united states. two forms of the disease are described in foals: the fulminant form, in which the foal is found acutely dead, and the subacute form. in the fulminant form, death usually is attributed to myocardial lesions resulting in cardiovascular collapse. the subacute form is characterized by dysphagia and gait abnormalities primarily caused by stiffness of the muscles of locomotion. paralysis, if present, is not flaccid as in botulism. abnormal function of respiratory muscles may complicate the clinical situation. aspiration pneumonia may be present following problems associated with swallowing; the tongue and pharyngeal muscles frequently are affected in the early stages of disease. 191 foals with severe disease may have widespread muscle necrosis leading to hyperkalemia, which can be severe and result in death of the foal. serum activities of the muscle enzymes creatine kinase and aspartate aminotransferase may be greatly increased. diagnosis is confirmed at necropsy or ante mortem by determination of decreased vitamin e, selenium, and glutathione peroxidase concentrations in the blood of the foal before supplementation. myoglobinuria and acute renal failure are not uncommon in these foals. treatment of foals with nutritional muscular dystrophy is primarily supportive. one should address all metabolic abnormalities. some foals require intranasal oxygen insufflation. affected foals are unable to suck effectively, and one should provide enteral (via an indwelling nasogastric tube) or parenteral nutritional support. because of the high likelihood of aspiration pneumonia, one should administer broad-spectrum antimicrobial therapy parenterally. the patient should be kept quiet and should be stimulated minimally. affected foals should receive parenteral (intramuscular) vitamin e and selenium supplementation. selenium is toxic in large doses. the prognosis for severely affected foals is guarded. for less severely affected foals the prognosis is good with appropriate treatment. the disease is preventable by ensuring that mares receive sufficient vitamin e and selenium while pregnant and by supplementing foals with parenteral injections of vitamin e and selenium at birth in endemic areas. a more complete discussion of the pathophysiology of this disease and the nutritional management is presented elsewhere in this text. primary liver disease is uncommon in the foal and occurs primarily as a sequela to sepsis. clinical signs of severe liver disease may include depression, ataxia, and seizures. in affected foals, increases in serum liver enzyme activities and concentrations of ammonia and bile acids frequently can be identified. the mechanism(s) underlying hepatoencephalopathy are not delineated clearly, although increased excitatory neurotransmitters, or compounds that mimic their activity, are implicated. hepatoencephalopathy is discussed in more detail elsewhere in this text. tyzzer's disease (clostridium piliformis infection) rarely causes primary liver disease in foals from 4 to about 40 days of age. this disease is almost uniformly fatal. the incubation period is short, and the mare is thought to be the carrier. [192] [193] [194] [195] [196] clinical signs range from acute death to depression, fever, and pronounced icterus. the feces of affected foals may appear white to grey because of the lack of bile. clinicopathologic abnormalities include leukopenia, hyperfibrinogenemia, metabolic acidosis, and hypoglycemia. 197, 198 liver lesions at postmortem are characterized microscopically by multiple foci of necrosis. one usually can demonstrate variable numbers of elongated, slender intracytoplasmic bacilli within hepatocytes bordering the necrotic foci. infiltration of the portal triads with inflammatory cells and biliary duct hyperplasia and degeneration are observable. the bacillus also occurs in association with myocardial lesions. lesions in the intestine are characterised by mucosal necrosis with inflammatory cell infiltration, increased mucus production, submucosal lymphoid hyperplasia, and submucosal hemorrhage. necrosis of lymphoid follicles, congestion, and hemorrhage can be present in the spleen and mesenteric lymph nodes. 196 affected foals may have a profound metabolic acidosis that is unresponsive to treatment. the clinical course is short, and most affected foals die within a few hours of developing neurologic signs. primary liver disease has been reported in association with ferrous sulfate administration in a probiotic compound. 199 the lesion was massive hepatocellular necrosis and liver failure. the product is no longer commercially available. portosystemic shunt is rare in the foal but has been reported in foals as young as 3 months of age. [200] [201] [202] most infectious causes of neurologic abnormalities in foals are associated with sepsis. although rarely reported, halicephalobus gingivalis (deletrix) infection has been reported in three foals; in one case the foal was 3 weeks of age. 203, 204 possibly transmission in these cases was transmammary; the dam in one case died 1 year later with confirmed h. deletrix infestation of her udder. listeria monocytogenes has been reported as a cause of neurologic disease in foals. 205 recently, sarcocystis neurona was identified as the causative agent of central nervous system disease in a foal, and equine herpes myeloencephalitis has been diagnosed in individual foals and in herd outbreaks involving foals. 206, 207 neospora also was reported in one foal recently. 208 rhodococcus equi abscesses can form in the central nervous system or cause neurologic signs associated with compression, as with vertebral body abscesses. [209] [210] [211] cerebellar hypolasia, occipitoatlantoaxial malformation, and agenesis of the corpus callosum with cerebellar vermian hypoplasia have been reported in foals. [212] [213] [214] [215] [216] [217] ivermectin toxicity and moxidectin toxicity have been reported. 218, 219 electrolyte abnormalities such as extreme hypo-or hypernatremia may result in neurologic manifestations of disease. 220, 221 cervical stenotic myelopathy and degenerative myelopathy also have been reported in foals, although the age at onset is usually more than 4 months. 222 idiopathic epilepsy of arabian foals usually is associated with another infectious disease and is thought to be temporary and self-limiting. causes, diagnosis, and treatment of fpt of immunity are covered in detail elsewhere in this text. failure of passive transfer occurs when a foal fails to ingest a significant quantity of good-quality colostrum. failure of passive transfer may occur by several mechanisms: failure of the foal to suck from the dam for any reason and failure of the dam to produce sufficient quantity of quality colostrum. box 19-4 presents causes of fpt. several methods are available for measuring igg concentration in blood; the most reliable are enzyme-linked immunosorbent assay and single radial immunodiffusion technology-based tests. [223] [224] [225] [226] [227] [228] [229] foals usually are tested at 24 hours of age, but one may test the foal earlier if colostrum ingestion has occurred and a concern exists regarding the passive transfer of immunity status of the foal, recognizing that additional increases in igg concentration may occur with additional time. 230, 231 the concentration of igg in the blood of the foal has been used as an indicator of the adequacy of passive transfer, but the actual blood concentration at which fpt is diagnosed has been challenged in recent years. [232] [233] [234] foals with sepsis commonly have a serum igg concentration of less than 800 mg/dl. 16, 19 foals with fpt are more likely to die from sepsis. 177, 178, [235] [236] [237] one should consider the igg concentration only as a marker for adequacy of colostral absorption. all the measured igg is unlikely to be directed against the specific pathogen affecting any particular neonate, and igg is not the only immune protection afforded the foal by colostrum. many factors that confer local and more general immunity to the newborn are present in colostrum; these include growth factors, cytokines, lactoferrins, cd14, leukocytes, and other yet to be described proteins. [240] [241] [242] [243] [244] by considering igg a marker of adequacy for passive transfer, similar to î³-glutamyltransferase in calves, the clinician can make choices for replacement that are more beneficial to the patient. 245 after one identifies fpt in a foal, treatment depends on the current condition of the foal and its local environment. foals not presently ill and on well-managed farms with low population density and low prevalence of disease may not require treatment if their igg concentration is between 400 and 800 mg/dl. critically ill neonates with fpt in an equine nicu are by definition ill and in an environment with high disease prevalence. these patients require immediate treatment of fpt and frequent reassessment of their passive immunity status. critically ill foals often fail to demonstrate the expected increase in blood igg concentration based on grams of igg administered per kilogram of body mass compared with healthy, colostrum-deprived foals. 235, 246, 247 sick foals also demonstrate a more rapid decline in igg concentration than do healthy foals because they use and catabolize available protein. one may treat foals with fpt by oral or intravenous administration of various products containing igg. one can attempt oral administration of additional colostrum or igg-containing products such as plasma, serum, or lyophilized colostrum in foals less than 12 to 24 hours of age. [248] [249] [250] depending on the age of the foal and the maturity and function of the gastrointestinal tract, this treatment may be effective. many nicus and large breeding farms maintain colostrum banks for this purpose. one should administer plasma intravenously if the foal is not expected to absorb additional colostrum or if the enteral route is unavailable. commercially available hyperimmune plasma products designed for use in foals are available and can be stored frozen. plasma and banked colostrum should be stored in a non-frost-free freezer to minimize protein loss associated with freeze-thaw cycling. 251 one should administer plasma through special tubing with an in-line filter and should monitor patients closely for transfusion reactions. 252 one may use serum and concentrated igg products, but the practitioner should be aware that many of these products focus on igg retention and not on other factors associated with passive transfer of immunity. one should measure igg concentration after transfusion and provide additional plasma as necessary. administration of plasma to critically ill foals without fpt may be beneficial through provision of other factors present in the plasma. in these situations, fresh frozen plasma or fresh plasma may be best, particularly if transfusion of clotting proteins is desired. neonatal isoerythrolysis is a hemolytic syndrome in newborn foals caused by a blood group incompatibility between the foal and dam and is mediated by maternal antibodies against foal erythrocytes (alloantibodies) absorbed from the colostrum. the disease most often affects foals born to multiparous mares and should be suspected in foals less than 7 days of age with clinical signs of icterus, weakness, and tachycardia. a primiparous mare can produce a foal with neonatal isoerythrolysis if she has received a prior sensitizing blood transfusion or has developed placental abnormalities in early gestation that allowed leakage of fetal red blood cells into her circulation. many are the causes of jaundice in newborn foals, including sepsis, meconium impaction, and liver failure, but these usually can be differentiated readily from neonatal isoerythrolysis by measuring the packed cell volume, which is usually less than 20% in foals with neonatal isoerythrolysis. foals with neonatal isoerythrolysis are born clinically normal then become depressed and weak and have a reduced suckle response within 12 to 72 hours of birth. the rapidity of onset and severity of disease are determined by the quantity and activity of absorbed alloantibodies. affected foals have tachycardia, tachypnea, and dyspnea. the oral mucosa is initially pale and then becomes icteric in foals that survive 24 to 48 hours. hemoglobinuria may occur. seizures caused by cerebral hypoxia are a preterminal event. the salient laboratory findings are anemia and hyperbilirubinemia. most of the increased bilirubin is unconjugated, although the absolute concentration of conjugated bilirubin generally is increased well above normal. urine may be red to brown and is positive for occult blood. the natural development of neonatal isoerythrolysis has several prerequisites. first, the foal must inherit from the sire and express an erythrocyte antigen (alloantigen) that is not possessed by the mare. blood group incompatibility between the foal and dam is not particularly uncommon, but most blood group factors are not strongly antigenic under the conditions of exposure through previous parturition or placental leakage. factor aa of the a system and factor qa of the q system are highly immunogenic, however, and nearly all cases of neonatal isoerythrolysis are caused by antibodies to these alloantigens. the exception is in the case of mule foals in which a specific donkey factor has been implicated. [253] [254] [255] mares that are negative for aa or qa or both are considered to be at risk for producing a foal with neonatal isoerythrolysis. the risk involves approximately 19% and 17% of thoroughbred and standardbred mares, respectively. second, and perhaps most important, the mare must become sensitized to the incompatible alloantigen and produce antibodies to it. the mechanism for this is not known in many instances but generally is believed to result from transplacental hemorrhage during a previous pregnancy involving a foal with the same incompatible blood factor. 255 sensitization via transplacental contamination with fetal erythrocytes earlier in the current pregnancy is possible, but an anamnestic response is generally necessary to induce a pathogenic quantity of alloantibodies. 256 ten percent of thoroughbred mares and 20% of standardbred mares have antibodies to the ca blood group antigen without known exposure to erythrocytes. 255 some common environmental antigen is postulated possibly to lead to production of anti-ca antibodies. data suggest that these natural antibodies may suppress an immune response to other blood group antigens because mares negative for aa that have anti-ca antibodies often do not produce antibodies to aa of the erythrocytes in their foals that also contain ca antigen. this antibodymediated immunosuppression is thought to result from the destruction of fetal cells before the dam mounts an immune response to other cell surface antigens. natural alloantibodies have not been associated with neonatal isoerythrolysis in horses. after the mare becomes sensitized to the erythrocytes of her foal, alloantibodies are concentrated in the colostrum during the last month of gestation. unlike the human neonate, which acquires alloantibodies in utero and thus is born with hemolytic disease, the foal is protected from these antibodies before birth by the complex epitheliochorial placentation of the mare. thus the final criterion for foal development of neonatal isoerythrolysis is ingestion in the first 24 hours of life of colostrumcontaining alloantibodies specific for foal alloantigens. immunoglobulin-coated foal erythrocytes are removed prematurely from circulation by the mononuclear phagocyte system or are lysed intravascularly via complement. the rapidity of development and severity of clinical signs are determined by the amount of alloantibodies that was absorbed and their innate activity. alloantibodies against aa are potent hemolysins and generally are associated with a more severe clinical syndrome than antibodies against qa or other alloantigens. the highest alloantibody titers are likely to be produced by mares that were sensitized in a previous pregnancy and then subsequently reexposed to the same erythrocyte antigen during the last trimester of the current pregnancy. prior sensitization of a mare by blood transfusion or other exposure to equine blood products may predispose to neonatal isoerythrolysis. 256 one can make a tentative diagnosis of neonatal isoerythrolysis in any foal that has lethargy, anemia, and icterus during the first 4 days of life. blood loss anemia caused by birth trauma is attended by pallor. icterus caused by sepsis or liver dysfunction would not be associated with anemia. one must base the definitive diagnosis of neonatal isoerythrolysis on demonstration of alloantibodies in the serum or colostrum of the dam that are directed against foal erythrocytes. the most reliable serodiagnostic test for neonatal isoerythrolysis is the hemolytic cross-match using washed foal erythrocytes, mare serum, and an exogenous source of absorbed complement (usually from rabbits). 5 although this test is impractical in a practice setting, a number of qualified laboratories routinely perform this diagnostic service. the direct antiglobulin test (coombs' test) may demonstrate the presence of antibodies on foal erythrocytes; however, false negatives occur frequently. most human or veterinary hematology laboratories can perform routine saline agglutination cross-match between mare serum and foal cells. because some equine alloantibodies act only as hemolysins, agglutination tests may be falsely negative. most field screening tests of colostrum have not proved to be reliable enough for practical use. if one recognizes neonatal isoerythrolysis when the foal is less than 24 hours old, one must withhold the dam's milk and feed the foal an alternative source of milk during the first day of life. one can accomplish this by muzzling the foal and feeding it via nasogastric tube. the minimum necessary amount of milk is 1% of body mass every 2 hours (e.g., a 50-kg foal should receive 500 ml or 1 pint of mare's milk or milk replacer every 2 hours). the udder of the mare should be stripped regularly (at least every 4 hours) and the milk discarded. in most instances, clinical signs are not apparent until after the foal is 24 hours old, when colostral antibodies have been depleted or the absorptive capacity of the foal's intestine for immunoglobulin has diminished. withholding milk at this point is of minimal benefit. supportive care to ensure adequate warmth and hydration is paramount. the foal should not be stressed and exercise must be restricted. confining the mare and foal to a box stall is a best. intravenous fluids are indicated to promote and minimize the nephrotoxic effects of hemoglobin and to correct any fluid deficits and electrolyte and acid-base imbalances. antimicrobials may be necessary to prevent secondary infections. one should monitor foals carefully for the necessity of blood transfusion, although transfusion should be used only as a lifesaving measure. when the packed cell volume drops below 12%, blood transfusion is warranted to prevent life-threatening cerebral hypoxia. erythrocytes from the dam are perfect in terms of nonreactivity with the blood of the foal; however, the fluid portion of the blood of the mare has to be removed completely from the cells to prevent administration of additional harmful alloantibodies to the foal. one can pellet the erythrocytes of the dam from blood collected in acid-citrate-dextrose solution by centrifugation or gravity and then aseptically draw off the plasma by suction apparatus or syringe and replace it with sterile isotonic (0.9%) saline. one thoroughly mixes the cells with the saline and then repeats the centrifugation or sedimentation, followed by aspiration and discarding of the saline. one should perform this washing process at least three times. one then can suspend the packed erythrocytes in an equal volume of isotonic saline for administration. erythrocyte washing by centrifugation is more desirable than gravity sedimentation because antibody removal is more complete and packed cell preparations can be prepared more quickly (each gravity sedimentation requires 1 to 2 hours). packed red blood cells are advantageous in overcoming the problem of volume overload. when equipment or conditions do not allow the safe use of dam erythrocytes, an alternative donor is necessary. because the alloantibodies absorbed by the foal generally are directed against aa or qa and because the latter are highly prevalent among most breeds of horses, a compatible blood donor is difficult to identify. the odds of finding a donor without aa or qa are higher in quarter horses, morgans, and standardbreds than in thoroughbreds and arabians. previously blood-typed individuals negative for aa and qa and free of alloantibodies are optimal. one should give 2 to 4 l of blood or 1 to 2 l of packed erythrocytes over 2 to 4 hours. these allogeneic cells have a short life span and represent a large burden to the neonatal mononuclear phagocyte system, which may cause increased susceptibility to infection. in addition, these cells sensitize the foal to future transfusion reactions. one must measure all potential harm against the benefit in each situation. if a mule foal is the patient, one should not use blood from a female previously bred to a donkey. in cases in which transfusion will be delayed, one cannot identify a compatible donor, or the packed cell volume is so low as to be life-threatening (hemoglobin <5 mg/dl), one may administer polymerized bovine hemoglobin products at a dose of 5 to 15 ml/kg. 257 one may use dexamethasone (0.08 mg/kg) to treat peracute neonatal isoerythrolysis if the packed cell volume is less than 12% and transfusion may be delayed or is not fully compatible, but dexamethasone has detrimental effects on blood glucose regulation in the neonate, and because the antibody in question is of maternal origin, corticosteroid therapy in immunosuppressive doses probably is not indicated. intranasal oxygen insufflation (5 to 10 l/min) may be beneficial. most foals with neonatal isoerythrolysis have adequate passive transfer of immunity, but antimicrobial therapy is indicated to protect against secondary sepsis resulting from the compromised condition of the foal. supportive care and good nursing care, including keeping the foal warm and quiet are essential. one should expect the packed cell volume to decline again 4 to 7 days after transfusion. 258 the prognosis for neonatal isoerythrolysis in foals depends on the quantity and activity of absorbed antibodies and is indirectly proportional to the rate of onset of signs. in peracute cases the foal may die before the problem is recognized, whereas foals with slowly progressive signs often live with appropriate supportive care. like most diseases, neonatal isoerythrolysis is much more effectively prevented than treated. 259 any mare that has produced a foal with neonatal isoerythrolysis should be suspect for the production of another affected foal; thus one should provide all subsequent foals with an alternative colostrum source and discard the colostrum of the dam unless she is bred to a stallion with known blood type compatibility. mares negative for aa and qa alloantigens are most at risk of producing affected foals, thus they should be identified by blood-typing. subsequently, breeding of these mares may be restricted to aa-and qa-negative stallions, thus eliminating the possibility of producing an affected foal. in breeds with a high prevalence of aa or qa alloantigens (e.g., thoroughbreds and arabians), a stallion negative for these and suitable based on other criteria may be difficult to identify. if these "at risk" mares are bred as desired, their serum should be screened in the last month of pregnancy for the presence of erythrocyte alloantibodies. one must test mares with low or equivocal titers closer to the time of parturition. if one detects alloantibodies, the colostrum of the dam should be withheld and the foal then should be provided with an alternative colostrum source. maternal alloantibodies to ca do not appear to mediate neonatal isoerythrolysis in foals and actually may be preventive by removing potentially sensitizing cells from the circulation 56 ; therefore one should not deprive foals of colostrum from mares possessing anti-ca antibodies, even when ca is present on their erythrocytes. rarely, the antigens de, ua, pa, and ab have been associated with neonatal isoerythrolysis in foals; however, to consider mares without these alloantigens to be at risk for neonatal isoerythrolysis is not practical. these syndromes recently have been recognized and described within the veterinary literature, although they have been recognized widely in human neonatology for many years. [260] [261] [262] [263] affected foals demonstrate these hematologic abnormalities within the first week of life, and the mechanism is similar to neonatal isoerythrolysis following ingestion of maternal antibody directed against the platelet or the neutrophil. in general, affected foals are healthy but may demonstrate bleeding tendencies if thrombocytopenia is severe or they may be more susceptible to sepsis. one confirms the diagnosis by appropriate testing for platelet-and neutrophil-associated antibody. 264 one must rule out other causes of neonatal thrombocytopenia and neutropenia, particularly sepsis. foals born to the mare in the future seem likely to be at risk for developing similar problems, and one should treat future foals as one treats neonatal isoerythrolysis foals: prevent sucking from the dam and provide an alternate source of passive immunity in the form of banked colostrum or intravenous plasma. one should provide an alternative nutritional source, such as foal milk replacer, to the foal for the first 48 hours of life and should muzzle the foal while it is in the company of its dam for that period of time. treatment is primarily supportive, but in the case of severe thrombocytopenia, transfusion of platelet-enriched fresh plasma may be indicated. granulocyte colony-stimulating factor has been used in foals with neutropenia, but substantial efficacy has yet to be demonstrated. broad-spectrum antimicrobial therapy may be prudent in cases of alloantibody-associated neutropenia. treatment with immunosuppressive doses of corticosteroids is probably unwarranted, given the increased risk of infection, because the antibody in question is of maternal origin. other specific diseases of the immune system of foals, severe combined immunodeficiency, selective igm deficiency, transient hypogammaglobulinemia, agammaglobulinemia, and other unclassified immunodeficincies are covered in detail elsewhere in this text. the neonate can experience respiratory distress immediately after birth because of several congenital respiratory tract or cardiac anomalies. chief among these causes are bilateral choanal atresia, stenotic nares, dorsal displacement of the soft palate caused by anatomic deformity or neurologic impairment, accessory or ectopic lung lobes, lung lobe hypertrophy, lung lobe dysplasia, cardiac anomalies with right-to-left shunting, and miscellaneous causes such as subepiglotic cysts and severe edema of the larynx. [264] [265] [266] [267] [268] [269] [270] [271] one must evaluate and treat these situations immediately and should consider them true emergencies. one readily can recognize foals with airway occlusion by the lack of airflow through the nostrils despite obvious attempts to breathe and by respiratory stridor. these foals may demonstrate open-mouth breathing and their cheeks may puff outward when they exhale. one foal with congenital bilateral choanal atresia was recognized during extrauterine intrapartum resusucitation because of an inability to pass a nasotrancheal tube. one can establish an effective airway by orotracheal intubation in these cases under most circumstances, but some foals require an emergency tracheostomy. one diagnoses the underlying problem by endoscopy or radiography in most cases. treatment of choanal atresia and cystic structures is surgical, whereas severe laryngeal edema and laryngeal paralysis frequently respond to medical management. until the underlying problem is resolved in these cases, one should administer broad-spectrum antimicrobial therapy and feed the foal by intubation or total parenteral nutrition. one can give colostrum, but these foals frequently develop aspiration pneumonia if allowed to suck from their dams, so intravenously administered plasma also may be necessary to provide sufficient passive immunity. arterial blood gas determinations are the most sensitive indicator of respiratory function readily available to the clinician. the most readily available arteries for sampling are the metatarsal arteries and the brachial arteries. portable arterial/venous blood gas analyzers now are making arterial blood gas analysis more practical in the field, and the technique is no longer reserved for large referral practices. managing a critically ill equine neonate without knowledge of arterial blood gas parameters is veritably impossible. pulse oximetry is useful, but these monitors only measure oxygen saturation of hemoglobin. desaturation can occur rapidly in critically ill neonates. the utility of these monitors in the foal has yet to be demonstrated clearly, particularly in cases of poor peripheral perfusion. 272 the most common abnormalities recognized with arterial blood gas analysis are hypoxemia with normo-or hypocapnia and hypoxemia with hypercapnia. hypoxemia is defined as decreased oxygen tension of the arterial blood (decrease pao 2 ), and hypoxia is defined as decreased oxygen concentration at the level of the tissue, with or without hypoxemia. hypoxia results from hypoxemia, decreased perfusion of the tissue bed in question, or decreased oxygen-carrying capacity of the blood resulting from anemia or hemoglobin alteration. five primary means by which hypoxemia may develop are (1) low concentration of oxygen in the inspired air such as in high altitude or in an error mixing ventilator gas; (2) hypoventilation; (3) ventilation/perfusion mismatch; (4) diffusion limitation; and (5) intrapulmonary or intracardiac right-to-left shunting of blood. hypoxemia is not an uncommon finding in neonates but must be evaluated in terms of the current age of the foal and its position. 15, [273] [274] [275] [276] one also must consider the difficulty encountered in obtaining the sample because severe struggling can affect the arterial blood gas results. table 19 -8 presents normal arterial blood gas parameters for varying ages of foals. the normal foal has a small shunt fraction (~10%) that persists for the first few days of life and contributes slightly to a blunted response to breathing 100% oxygen compared with the adult. hypoxemia frequently occurs in foals with prematurity, pas, and sepsis, although other conditions also result in hypoxemia in the neonate. in the early stage of sepsis associated hypoxemia, paco 2 may be within normal limits or decreased if the foal is hyperventilating for any reason. if the lung is involved significantly in the underlying pathologic condition, such as with severe pneumonia, acute lung injury, or acute respiratory distress syndrome, increased paco 2 may well be present, representing respiratory failure. 277 hypoxemia usually is treated with intranasal humidified oxygen insufflation at 4 to 10 l/min. hypercapnia is not a simple matter to treat. one must try to distinguish between acute and chronic hypercapnia. acute hypercapnia usually is accompanied by a dramatic decrease in blood ph of 0.008 ph units for each 1 mm hg increase in paco 2 . this acidemia can promote circulatory collapse, particularly in the concurrently hypoxemic and/or hypovolemic patient. the effects of more chronic co 2 retention are less obvious because the time course allows for adaptation. the ph change is less, about 0.003 ph units per 1 mm hg increase in paco 2 , because it is balanced by enhanced renal absorption of bicarbonate by the proximal renal tubule. most foals with acute respiratory distress are in the acute stages of respiratory failure, but chronic adaptation begins to occur within 6 to 12 hours and is maximal in 3 to 5 days. one will note an increase in bicarbonate, particularly if the acidemia is primarily respiratory in origin. intravenous administration of sodium bicarbonate to correct respiratory acidosis/ acidemia should be done cautiously in these foals because co 2 retention may only be increased. also, one should remember that 1 meq of sodium is administered with each meq of bicarbonate and hypernatremia has been seen in foals treated exuberantly with sodium bicarbonate. foals with hypercapnia of several days' duration also may develop a blunted respiratory drive to increased co 2 . in these foals, oxygen administration, although essential to treat hypoxemia, may further depress ventilation and further decrease ph. this effect is caused by a loss of hypoxic drive following oxygen therapy. one should consider these foals candidates for mechanical ventilation if the paco 2 is greater than 70 mm hg or is contributing to the poor condition of the foal, such as causing significant ph changes. if hypercapnia is caused by central depression of ventilation, as frequently occurs in foals with pas, one can administer caffeine (10 mg/kg loading dose; then 2.5 mg/kg as needed) per rectum or orally in foals with normal gastrointestinal function. other clinicians may recommend continuous rate infusions of doxapram hydrochoride (dopram; 400 mg/total dose at 0.05 mg/ kg/min) for these foals. if this therapy fails, one should consider mechanical ventilation. mechanical ventilation of foals with central respiratory depression is rewarding and may be necessary only for a few hours to days. a special category is the foal with botulism exhibiting respiratory failure caused by respiratory muscle paralysis. these foals do well with mechanical ventilation, although the duration of mechanical ventilation is more prolonged, frequently more than 1 week. foals with primary metabolic alkalosis usually have compensatory respiratory acidosis. treatment of hypercapnia is not necessary in these cases because it is in response to the metabolic condition. these foals do not respond to caffeine, and they should not be ventilated mechanically if this is the only disorder present. in the neonate, bacterial pneumonia usually results from sepsis or aspiration during sucking. foals with sepsis can develop acute lung injury or acute respiratory distress syndrome as part of the systemic response to sepsis, and this is frequently a contributor to the demise of foals in septic shock. the best way to diagnose bacterial pneumonia is by cytologic examination and culture of a transtracheal aspirate, but blood culture may aid in early identification of the causative organism and allow for early institution of directed antimicrobial therapy. a second frequent cause of bacterial pneumonia in the neonate is aspiration caused by a poor suck reflex or dysphagia associated with pas, sepsis, or weakness. one must take care to ensure that aspiration is not iatrogenic in foals being bottle fed. auscultation over the trachea while the foal is sucking helps identify occult aspiration. one should suspect occult aspiration pneumonia in any critically ill neonate that is being bottle fed or is sucking on its own that has unexplained fever, fails to gain weight, or has a persistently increased fibrinogen level. older foals develop bacterial pneumonia, frequently following an earlier viral infection. 278 bacterial pneumonia is discussed in depth elsewhere in this text, but a few comments specific to the foal are necessary. one should auscultate and percuss the thorax of the foal, but results may not correlate closely with the severity of disease. the most commonly isolated bacterial organism in foal pneumonia is streptococcus zooepidemicus, and one may isolate it alone or as a component of a mixed infection. [278] [279] [280] transtracheal aspirate for culture and cytologic examination is recommended because mixed gram-positive and gram-negative infections are common, and antimicrobial susceptibility patterns can be unpredictable. one should split the obtained aspirate and submit samples for bacterial culture, virus isolation, and cytologic examination. additional diagnostics include radiography, ultrasonography, and serial determination of white blood cell counts (with differential) and blood fibrinogen concentrations. treatment includes administration of appropriate antimicrobial therapy. some foals may benefit from nebulization with saline or other local products. ascarid larval migration through the lung can mimic bacterial pneumonia. 281 in these cases the foal may not respond to antimicrobial therapy and should be dewormed with ivermectin. deworming the mare within 1 month of parturition and frequent deworming of the foal prevent ascarid migration pneumonia in most foals. a special category of bacterial pneumonia in foals is rhodococcus equi bronchopneumonia. this pneumonia of young foals was described first in 1923. 282 the organism originally was known as corynebacterium equi and is a gram-positive pleomorphic coccobacillus usually less than 1 âµm in diameter and 2 âµm in length. the organisms frequently are associated in l-and v-shaped clusters that have been termed chinese character formations. r. equi has an acid-fast staining characteristic under some growing circumstances because of the presence of mycolic acid in its cell wall, similar to mycobacterium and nocardia species. mycolic acid promotes granuloma formation. the organism is able to multiply in and destroy macrophages as it prevents phagosome lysosome fusion. 283, 284 much attention has been paid to this organism in recent years, given its propensity to produce enzootic and epizootic outbreaks of disease. the organism is thought to be primarily an opportunistic pathogen, and it lives in the soil of most geographic areas. foals are affected most frequently between the ages of 1 and 6 months, when maternally derived immunity has begun to wane. the disease is insidious, and foals may have significant pulmonary involvement before developing noticeable clinical signs. phagocytosis of r. equi by equine macrophages is not associated with a functional respiratory burst and, at least in human beings, the l-arginine-no pathway is not required for intracellular killing of this organism. 285, 286 optimal binding of r. equi to mouse macrophages in vitro requires complement and is mediated by mac-1, a leukocyte complement receptor type 3 (cr3, cd11b/ cd18). 287 opsonisation of r. equi with specific antibody is associated with increased phagosome-lysosome fusion and enhanced killing of r. equi, suggesting that the mechanism of cellular entry is important. 283 neutrophils from foals and adult horses are fully bactericidal, and killing of r. equi is enhanced considerably by specific opsonizing antibody. 288 the ability of r. equi to induce disease in foals likely depends on host and microbial factors. knowledge of the virulence mechanisms of r. equi was speculative until the discovery of the virulence plasmid. 289 as opposed to most environmental r. equi organisms, isolates from clinically affected foals typically contain 85-to 90-kb plasmids encoding an immunogenic virulence-associated protein (vapa) that is expressed on the bacterial surface in a temperature-regulated manner. 290 plasmid-cured bacteria lose their ability to replicate and survive in macrophages and are cleared from the lungs within 2 weeks of intrabronchial challenge without producing pneumonia. 291 however, expression of vapa alone is not sufficient to restore the virulence phenotype. six other genes have approximately 40% overall amino acid identity with vapa, and the identification of multiple genes with considerable homology suggests these genes constitute a virulence-associated gene family in r. equi. 292 other candidates for virulence factors include capsular polysaccharides and cholesterol oxidase, choline phosphohydrolase, and phospholipase c exoenzymes ("equi factors"), but their roles have not been defined clearly. the primary manifestation of disease caused by r. equi infection is severe bronchopneumonia with granuloma, abscess formation, or both. up to 50% of foals diagnosed with bronchopneumonia also have extrapulmonary sites of infection. 293 as the pneumonia progresses, clinical signs may include decreased appetite, lethargy, fever, tachypnea, and increased effort of breathing characterized by nostril flaring and increased abdominal effort. cough and bilateral nasal discharge are inconsistent findings. a smaller percentage of affected foals may have a more devastating, subacute form. these foals may be found dead or have acute respiratory distress with a high fever and no previous history of clinical respiratory disease. hyperfibrinogenemia is the most consistent laboratory abnormality in foals with r. equi pneumonia. neutrophilic leukocytosis (>12,000 cells/âµl), with or without monocytosis, is common. 294 thoracic radiography is a useful diagnostic aid, frequently revealing a prominent alveolar pattern with poorly defined regional consolidation and/or abscessation. ultrasonography is a helpful diagnostic tool when the disease involves peripheral lung tissue. although a number of serologic tests have been described, serologic diagnosis of r. equi infections is controversial and difficult because exposure of foals to this organism at a young age leads to production of antibody without necessarily producing clinical disease. 295, 296 serologic tests may be more useful at the farm level to detect overall exposure than at the individual level. bacteriologic culture combined with cytologic examination of a tracheobronchial aspirate remains the most definitive method for accurate diagnosis of r. equi pneumonia. however, foals without clinical disease exposed to contaminated environments may have r. equi in their tracheae from inhalation of contaminated dust; therefore one should interpret culture results in the context of the overall case presentation. 297 culture results in one study were as sensitive as polymerase chain reaction-based assays and offered the advantage of allowing in vitro antimicrobial susceptibility testing. 298 however, polymerase chain reaction is likely to be a useful tool, and results from a second trial suggest the assay is more sensitive and specific than culture of tracheobronchial aspirates for diagnosis. 299 the combination of erythromycin and rifampin has become the treatment of choice for r. equi infections in foals, and the combination reduces the likelihood of resistance to either drug. the recommended dosage regimen for rifampin is 5 mg/kg every 12 hours or 10 mg/kg every 24 hours orally. the recommended dose of estolate or ethylsuccinate esters of erythromycin is 25 mg/kg every 8 or 12 hours orally. 300 recently, azithromycin has been recommended for treatment of r. equi infection at a dosage of 10 mg/kg orally every 24 hours for 5 to 7 days and then every other day. 301 alternatively, clarithromycin at 7.5 mg/kg every 12 hours orally, in combination with rifampin, may be therapeutically effective. severely affected foals may require intranasal oxygen insufflation, intravenous fluid support, and nutritional support. treatment generally continues for 4 to 10 weeks until all clinical and laboratory evidence of infection is resolved. although well tolerated by most foals, erythromycin can result in soft feces. this diarrhea is generally self-limiting and does not require cessation of therapy, but one should monitor affected foals carefully. an idiosyncratic reaction characterized by severe hyperthermia and tachypnea has been described in foals treated with erythromycin during periods of hot weather. 302 affected foals should be moved to a colder environment and treated with antipyretic drugs and alcohol baths if necessary. clostridium difficile enterocolitis has been reported in the dams of nursing foals treated with erythromycin given orally. 303 the dam is exposed to active erythromycin by coprophagy or by drinking from a communal water source where the foal has "rinsed" its mouth. prevention of r. equi pneumonia on farms with recurrent problems is problematic. the most clearly demonstrated prophylactic measure to date has been the administration of plasma that is hyperimmune to r. equi to foals within the first week of life and then again when maternal immunity begins to wane at around 30 days of age. [304] [305] [306] [307] [308] [309] [310] [311] no effective vaccination protocols for the dam or foal have been described to date. farm management is important in preventing disease, and control measures include frequent manure removal, avoidance of overcrowded conditions, and planting of dusty or sandy soils. 304 the prognosis for r. equi bronchopneumonia is fair to good in foals with the more chronic form of the disease. foals with acute respiratory distress have a more guarded prognosis, as do foals with sites of significant extrapulmonary infection. the long-term prognosis for survival for foals with r. equi bronchopneumonia is good, and many foals perform as expected as athletes. 312 the most commonly identified causes of viral pneumonia in foals are equine herpesviruses 1 and 4 (ehv-1 and ehv-4), equine influenza, and equine arteritis virus (eva). equine herpesvirus 1 is probably the most clinically important, but outbreaks of eva in neonates have occurred and are devastating. 27, [313] [314] [315] [316] [317] [318] adenovirus is reported sporadically and as a problem in arabian foals with severe combined immunodeficiency. [319] [320] [321] in the neonate, infection with ehv-1 or eva is almost uniformly fatal and antemortem diagnosis is difficult, even once an outbreak on a particular farm is identified. several factors appear common to foals with ehv-1, including icterus, leukopenia, neutropenia, and petechial hemorrhage, but these problems also are identified in foals with severe sepsis. 315, 322, 323 the antiviral drug acyclovir (10 to 16 mg/kg orally or per rectum 4 to 5 times per day) has been used in cases of ehv-1 in neonates, with some evidence of efficacy in mildly affected foals or foals affected after birth. 323 if viral pneumonia is a possibility, one should collect blood and tracheal aspirates at presentation for bacterial and virus isolation. the lungs of foals with ehv-1 or eva are noncompliant, and pulmonary edema may be present. mechanical ventilation of these cases may prolong life, but death is generally inevitable because of the magnitude of damage to the lungs. foals suspected of having ehv-1 or eva should be isolated because they may be shedding large quantities of virus and pose a threat to other neonates and pregnant mares. foals with eva generally are born to seronegative mares, and intravenous treatment with plasma with a high titer against eva may prove beneficial because passive immunity appears to have a large role in protection against this disease in neonates. 318, 324 older foals and weanlings may be affected by herpesviruses. disease is usually mild, although a fatal pulmonary vasculotropic form of the disease has been described recently in young horses. 325, 326 the clinical signs of disease are indistinguishable from influenza and include a dry cough, fever, and serous to mucopurulent nasal discharge, particularly if secondary bacterial infection occurs. rhinitis, pharyngitis, and tracheitis may be present. treatment of affected foals is primarily supportive. foals also may become infected with ehv-2. the predominant clinical signs are fever and lymphoid hyperplasia with pharyngitis. 327, 328 diagnosis is by virus isolation. rib fractures have been recognized in 3% to 5% of all neonatal foals and can be associated with respiratory distress. 87 potential complications of rib fractures include fatal myocardial puncture, hemothorax, and pneumothorax. rib fractures frequently are found during physical examination by palpation of the ribs or by auscultation over the fracture sites. one can confirm the diagnosis by radiographic and ultrasonographic evaluation. often multiple ribs are affected on one side of the chest. specific treatment is generally unnecessary, but direct pressure on the thorax should be avoided in all cases. some specific patients may benefit from surgical stabilization of some fractures, particularly those fractures overlying the heart. pneumothorax can occur spontaneously or following excessive positive pressure ventilation 329 or following tracheostomy surgery or trauma. any foal being ventilated mechanically that suddenly has respiratory distress and hypoxemia should be evaluated for pneumothorax. diagnosis is by auscultation and percussion of the thorax, but one can confirm the diagnosis with radiographic and ultrasonographic evaluation of the thorax. needle aspiration of air from the pleural space also confirms the diagnosis. treatment is required in cases in which clinical signs are moderate to severe or progressive and involves closed suction of the pleural space. subcutaneous emphysema can complicate treatment of this problem. idiopathic or transient tachypnea has been observed in clydesdale, thoroughbred, and arabian breed foals. in human infants, transient tachypnea can be related to delayed absorption of fluid from the lung, perhaps because of immature sodium channels. 330 in foals, tachypnea generally occurs when conditions are warm and humid and is thought to result from immature or dysfunctional thermoregulatory mechanisms. clinical signs of increased respiratory rate and rectal temperature develop within a few days of birth and may persist for several weeks. treatment involves moving the foal to a cooler environment, body clipping, and provision of cool water or alcohol baths. these foals frequently are treated with broad-spectrum antimicrobial drugs until infectious pneumonia can be ruled out. a syndrome of bronchointerstitial pneumonia and acute respiratory distress has been described in older foals and appears to be a distinct entity from acute respiratory distress syndrome in neonatal foals in association with sepsis. 331 the underlying cause has not been identified, but the genesis is probably multifactorial with several potential pathogens being implicated. affected foals have acute respiratory distress with significant tachypnea, dyspnea, nostril flare, and increased inspiratory and expiratory effort. auscultation reveals a cacophony of abnormal sounds including crackles and polyphonic wheezes in all lung fields. loud bronchial sounds are audible over central airways, and bronchovesicular sounds are lost peripherally. affected foals are cyanotic, febrile, and unwilling to move or eat. foals may be found acutely dead. laboratory abnormalities include leukocytosis, hyperfibrinogenemia, and hypoxemia with hypercapneic acidosis. foals can be dehydrated severely and have coagulation changes consistent with disseminated intravascular coagulation. hypoxic injury to other organs, primarily the kidneys and liver, can occur. chest radiographs reveal a prominent interstitial pattern overlying a bronchoalveolar pattern that is distributed diffusely throughout the lung. this syndrome is a respiratory emergency. treatment is broad-based and includes administration of oxygen, nonsteroidal antiinflammatory agents, broad-spectrum antimicrobial therapy, nebulization, judicious intravenous fluid therapy, nutritional support, and corticosteroid therapy. one must manage hyperthermia in the foal. corticosteroid therapy appears to have been lifesaving in most of the reported surviving foals. because this syndrome is associated with high environmental temperatures in some areas, prevention involves control of ambient temperatures, not transporting foals during hot weather, and keeping foals out of direct sun on hot days, particularly foals being treated with erythromycin for suspected or confirmed r. equi infection. 332 uroperitoneum has been recognized as a syndrome in foals for more than 50 years. 333, 334 classically, affected foals are 24 to 36 hours old at the time clinical signs first are recognized. [334] [335] [336] previous reports had a proportionately larger affected male than female population. 334, 335, 337 the hypothesis was that colts were more at risk because their long, narrow, high-resistance urethra was less likely to allow bladder emptying, resulting in rupture of a full bladder during parturition when high pressures were applied focally or circumferentially around the bladder. 333 more recent reports suggest that such extreme sex bias may have been an artifact of small case numbers in the early reports. rupture or disruption of any structure of the urinary tract can occur. the dorsal wall of the bladder has been reported to be a frequent disruption site, with the ventral wall less likely to be involved. 336 the urachus appears to be the next most commonly affected structure. a few cases of ureteral and urethral defects have been reported. 336, 337 sepsis does not appear to favor one site over the others. 338 the pathophysiology of uroperitoneum is not yet understood fully. the high pressure exerted on a full bladder during parturition once was thought to be the main cause. full bladder and obstruction caused by a partial umbilical cord at parturition, strenuous exercise, and external trauma have been reported as causes. 339 a few reports describe smooth and noninflamed edges of torn tissue, suggesting the possibility of congenital bladder wall defects. 338, 340, 341 sepsis leading to urinary tract rupture and uroperitoneum may occur in foals hospitalized for a variety of unrelated problems. the onset of clinical signs of uroperitoneum may be insidious in these foals, and diagnosis may be less obvious. 338 clinical signs associated with uroperitoneum in the neonatal foal typically include straining to urinate, dribbling urine, and a stretched-out stance. weakness, tachycardia, tachypnea, and not sucking well are also common. a distended abdomen may be evident, and one may feel a fluid wave on ballottement of the abdomen. occasionally, urine accumulates in the scrotum and should not be confused with hernia. foals also may show signs of sepsis, including fever, injected mucous membranes, diarrhea, and disease of other body systems. laboratory findings vary depending on the duration of the uroperitoneum and on the presence and severity of sepsis. classic findings include hyperkalemia, hyponatremia, and hypochloremia arising from equilibration of urine electrolytes and water with blood across the peritoneal membrane. [335] [336] [337] the usual foal diet of milk, which is high in potassium and low in sodium, promotes the electrolyte abnormalities. foals that develop uroperitoneum while receiving intravenous fluids may not have classic electrolyte imbalances at the time clinical signs are recognized. 338 increased serum creatinine concentration is often present, whereas blood urea nitrogen concentrations occasionally, but not consistently, are increased. [335] [336] [337] metabolic acidosis and hypoxemia may be present. some patients also have serum hypoosmolality. 335 one should test foals for failure of passive transfer. one of the most sensitive laboratory tests for uroperitoneum is the ratio of peritoneal to serum creatinine. a ratio greater than or equal to 2:1 is considered diagnostic of uroperitoneum. one should collect peritoneal fluid and test it for creatinine concentration, as well as for cytologic findings, culture, and sensitivity. cytologic evaluation of peritoneal fluid is necessary to identify concurrent peritonitis or other gastrointestinal compromise. one should perform an electrocardiogram on initial evaluation of a foal with suspected uroperitoneum because hyperkalemia may result in bradycardia, increased duration of the qrs complex, a shortened q-t interval, increased p-wave duration, prolonged p-r interval, or atrioventricular conduction disturbances. other possible cardiac sequelae to hyperkalemia include cardiac arrest, third-degree atrioventricular block, ventricular premature contractions, and ventricular fibrillation. 337, 340 for any foal exhibiting signs of dypsnea, tachypnea, or hypoxemia, one should have thoracic radiographs taken before induction of anesthesia to rule out pleural effusion, pneumonia, or acute respiratory distress syndrome, which could complicate ventilation and oxygenation during anesthesia and the postoperative period. ultrasonography has become the tool of choice in the diagnosis of uroperitoneum and is a useful tool available to the practitioner. 342 one can image free peritoneal fluid readily, and tears within the bladder are readily visible. the empty bladder with a significant defect, in a fluid-filled abdomen, will collapse on itself and often have a u shape. one also can visualize urachal and urethral lesions. six of eight foals in one study had urinary tract lesions identified sonographically, and all 31 foals of another study underwent sonographic evaluation, and a significant correlation between ultrasonographic findings and location of the lesion at surgery existed. 336, 338 initial treatment aims to stabilize the patient and correct any electrolyte and acid-base abnormalities and provide fluid volume replacement. one should use 0.9% or 0.45% saline with 5% dextrose until laboratory data are available. a potassium concentration of greater than 5.5 meq/l can be life threatening. one can manage hyperkalemia by peritoneal drainage to decrease whole-body potassium stores using teat cannulae, foley catheters, large-gauge (16 or 14) intravenous catheters, or human peritoneal dialysis catheters. fluid replacement at least should equal the amount of fluid removed from the abdomen to prevent acute hypotension caused by expansion of previously collapsed capillary beds. abdominal drainage also helps ventilation and decreases the work of breathing by decreasing pressure on the diaphragm. one may administer calcium gluconate, glucose, sodium bicarbonate, or insulin intravenously to decrease serum potassium concentrations. these maneuvers do not correct the whole-body potassium overload, however, and once therapy is discontinued, hyperkalemia can reappear until the urine is removed from the abdomen. one should correct hyponatremia slowly. because of the real possibility of concurrent sepsis, one should obtain blood cultures before preoperative administration of antimicrobials. broad-spectrum coverage (penicillin and amikacin or ceftiofur sodium) is recommended until culture results become available. one should perform therapeutic drug monitoring when using aminoglycoside therapy. however, the peak value may be depressed because of the increased volume of distribution represented by the volume of urine in the abdomen, so one should not make dose adjustment based on a low peak until obtaining a new peak after surgical correction of the uroperitoneum. one should treat foals with failure of passive transfer with adequate volumes of intravenously administered plasma. after one has addressed the metabolic abnormalities, one may consider surgical management. medical management using an indwelling foley catheter has been described. 343 preoperative medical stabilization reduces anesthetic risk. safer inhalant agents such as isoflurane also have decreased risk. removal of the internal umbilical remnant at the time of surgery is usual. one should consider culturing any removed umbilical remnant and submitting the remnant for histopathologic evaluation. recurrence of urinary tract rupture can occur. sepsis, hypoxemia, pneumonia, peritonitis, and acute respiratory distress syndrome complicate the management of uroperitoneum. many affected foals are persistently oxygen dependent for several days following surgical correction, and one should perform serial arterial blood gas analyses before discontinuing intranasal oxygen supplementation. prognosis is associated closely with concurrent illness, especially septicemia. uncomplicated uroperitoneum from a defect in the bladder has a good prognosis. if the location of the lesion is other than the bladder, the prognosis is not as favorable. 337 foals with septicemia have a much poorer prognosis. 338, 339 acute renal failure most often occurs as a complication of prenatal asphyxial syndrome, sepsis, or aminoglycoside therapy. acute renal failure also has been reported following oxytetracycline administration in foals. 344 the dose of oxytetracycline commonly used to treat flexural deformities in foals is approximately 10 times the antimicrobial dose. many foals treated in this manner also have suffered some degree of perinatal asphxia, which also damages the kidney, because of prolonged parturition precipitated in part by the flexural deformity. evaluation of renal function in these foals before the administration of the first dose of oxytetracycline and continued monitoring of serum creatinine concentrations before administering subsequent doses of this nephrotoxic compound would seem reasonable. hemodialysis has been used as therapy in one of these cases, but prevention is important because these foals may fail to respond to usual therapy for oliguric renal failure and are euthanized. 344 the most commonly reported congenital deformity of the kidney of the foal is renal hypoplasia and dysplasia, which may have a heritable component. 345, 346 renal arteriovenous malformations have been reported also. 347 ectopic ureters and fenestrated ureters have been described in the foal. [348] [349] [350] congenital renal defects, among others, were reported in three weak, recumbent neonatal foals born to mares being treated for equine protozoal myeloencephalitis. 351 mares received sulfadiazine or sulfamethoxazoletrimethoprim, pyrimethamine, folic acid, and vitamin e orally. the foals were anemic, leukopenic, azotemic, hyponatremic, and hyperkalemic. serum folate concentrations were lower than those reported in the literature for clinically normal brood mares. treatment was unsuccessful. necropsy revealed lobulated kidneys with thin cortices and a pale medulla. the authors postulated that oral administration of sulfonamides, 2,4-diaminopyrimidines (pyrimethamine with or without trimethoprim), and folic acid to mares during pregnancy is related to congenital defects in newborn foals. the umbilicus serves as the conduit for nutrition and gas exchange between the dam and the fetal foal. the urine from the foal is expelled via this structure into the allantoic cavity. the author has recognized cases of in utero bladder distention in the fetus that were associated with multiple twists decreasing urine flow or focal stenosis creating the same effect. foals born with this condition did not have bladder rupture associated with parturition but did have other severe abnormalities that eventually resulted in their demise, primarily premature delivery with failure to adapt to extrauterine life (p.a. wilkins, j.e. palmer, and f.t. bain, unpublished data). at birth the umbilicus breaks, leaving a small external remnant and a large internal remnant. the umbilicus long has been regarded as the primary site of entry of pathogens into the neonate, although this has been challenged recently. treatment of the umbilicus after birth involves dipping it (preferably just the most distal component) with various caustic compounds. the most current recommendation is to treat the umbilicus with dilute chlorhexidine, povidone-iodine, or dilute iodine solutions for just a few times following birth. exhuberant treatment of the umbilical stump with caustic solutions can lead to scalding of the ventral abdomen and may promote patency of the urachus. the ultrasonographic appearance and measurements of the umbilical arteries, urachus, and umbilical vein of foals from 6 hours to 4 weeks of age have been described in detail. 342 a 7.5-mhz sector scanner transducer placed across the midline of the ventral portion of the abdominal wall of the foal works best because of the superficial location of these structures. the mean (â± sd) diameter of the umbilical vein was 0.61 â± 0.20 cm immediately cranial to the umbilical stalk, 0.52 â± 0.19 cm midway between the umbilicus and liver, and 0.6 â± 0.19 cm at the liver. the urachus and umbilical arteries of normal foals have a mean total diameter of 1.75 â± 0.37 cm at the bladder apex. the umbilical arteries scanned along either side of the bladder have a mean diameter of 0.85 â± 0.21 cm. one can use these measurements and the ultrasonographic appearance of the internal umbilical structures from clinically normal foals as references to diagnose abnormalities of the umbilical structures in neonatal foals. 352, 353 the most common abnormalities of these structures are focal abscess formation, hematoma, and urachal tear. herniae traditionally have been thought to develop from failure of closure at the umbilical stump after birth. however, the closure of the body wall defect at the umbilicus was studied in relation to the development of umbilical herniae in a large group of normal foals followed from birth until 5 months of age or from birth until 11 months of age. 354 at birth, approximately half of these foals had a defect in the body wall at the umbilicus that was termed a palpable umbilical ring. in 18 foals this defect disappeared within 4 days, but in one foal the ring did not close and a hernial sac with abdominal contents was palpable. this foal was considered to be the only foal to have a truly congenital umbilical hernia. twelve foals developed an umbilical hernia between 5 and 8 weeks of age. the prevalence of umbilical herniae was much higher than in other studies, possibly because of the prospective nature of the study. based on this study, the large majority of umbilical herniae would appear not to result from failure of closure but rather to be acquired after birth. one should consider the palpable ring structure within the body wall at the umbilicus a variant of normal in the foal and should not call it a hernia until the foal is at least 1 month of age. in one study of 147 horses treated for umbilical herniae over a 13 1 / 2 -year period, only 8.8% developed complications in association with umbilical defects. 355 six horses had intestinal incarceration; the incarceration was reduced manually in 3 horses before admission and resolved without treatment in 2 others. the hernia was surgically reduced in 1 horse. herniorrhaphy was performed on 4 of the 5 horses in which the incarceration did not require surgical reduction, and the fifth was managed conservatively. the study confirmed that complications of umbilical herniae are rare in horses; however, when they do develop, they may be one of various forms, some of which are insidious in onset. the primary differential diagnosis for an external swelling in the umbilical stump region is an external abdominal abscess, which will be firm, variably painful, warm, and nonreducible. ultrasonographic evaluation readily can confirm either possibility. one report describes a 3-day-old foal that died from intestinal strangulation caused by a remnant of vitelline vein that extended between the umbilicus and the portal vein. 356 patent urachus frequently is recognized in the abnormal neonate, probably because of the increased recumbency and decreased movement of these patients. cauterization of a patent urachus is no longer recommended except in cases that persist for long periods of time (>1 month) after the foal becomes more active. surgical resection may provide relief in some foals, but most cases resolve without treatment if given enough time. foals with a patent urachus may posture and strain frequently to urinate, some of this may be associated with irritation or local infection of the urachus. one can alleviate this by administration of broad-spectrum antimicrobial therapy such that the drug has a high concentration in the urine (e.g., trimethoprim-sulfa drug combinations) and by oral administration of phenazopyridine hydrochloride (pyridium), a dye that anesthetizes the urinary tract epithelial surfaces (see table 19 -7). this dye turns the urine orange and stains everything yellow-orange that it or the urine touches but can provide a great deal of relief to foals with this problem. the umbilicus has been considered the traditional point of entry of bacteria into the septic neonate, and septic foals have been referred to as having "navel ill" and "joint ill" in the past. although current thought suggests that the gastrointestinal tract may be the route of entry in most septic neonates, infection of the umbilicus-termed omphalitis, or omphalophlebitis if the vessels are involved-still occurs as a single focus of infection or along with more generalized infection. external signs, such as swelling, heat, pain, ventral edema, or purulent discharge may be present in some foals, but more usually external signs are minimal and one suspects infection because of infection in another site (e.g., an infected joint), fever, or otherwise unexplained increased blood fibrinogen concentration. one confirms the diagnosis by ultrasonographic evaluation of the internal umbilical remnant. any of the umbilical structures may be involved. a complete description of the evaluation is available within the relevant veterinary literature, but the examination is performed best with the foal standing using a 7.5-mhz probe with a standoff. 353 the usual finding is that the affected structure is larger than expected. a fluid-filled core and echogeneic shadows consistent with gas may be apparent in some cases. interpretation requires some experience, and the examiner should be familiar with variants of normal, such as gas shadows associated with a patent urachus and enlarged vessels caused by hematoma formation, so that treatment is not initiated inappropriately. two options for treatment are surgical and medical. medical treatment is preferable in cases in which the lesion is well localized and small and in foals with a medical condition that is not amenable to anesthesia and surgical intervention. one should institute broad-spectrum antimicrobial therapy, and one may need to continue therapy for 2 to 3 weeks. most affected foals respond to medical therapy. frequent reevaluation of the abnormality is necessary, every 5 to 7 days initially, and one should measure blood fibrinogen concentrations at reevaluation because they should stabilize and decrease with effective treatment. failure to respond to therapy within 10 days to 2 weeks suggests that an empiric change in the antimicrobial used may be necessary. in foals that are refractory to medical management or where the lesion is large, surgical excision of the entire umbilical remnant may be desirable. colic in the foal can be difficult to diagnose accurately because one cannot perform an examination per rectum. however, many diagnostic aids, most importantly ultrasonography, are available to help differentiate medical from surgical causes of abdominal discomfort in the foal. intestinal accidents of all types described in adult horses, with the possible exception of enteroliths, occur in foals. intussusception, volvulus, displacement, diaphragmatic hernia, and intra-and extraluminal obstruction have been reported in foals. abdominal ultrasonographic and radiographic evaluation greatly aids diagnosis. treatment is primarily surgical. foals with pas and intestinal dysmotility are at increased risk of intussusception and displacement, and miniature breed foals appear to be at increased risk for fecolith and enterolith formation. meconium retention or impaction is a common cause of abdominal discomfort in newborn foals. most foals defecate shortly after their first meal. the usual practice for most owners or veterinarians attending the birth of a foal is to administer an enema to aid this process. in the past, phosphate-based commercially available enemata (fleet) were used frequently, but if used excessively these types of enemata can create problems of their own, including rectal irritation and hyperphosphatemia. the best enema is warm soapy water made with a mild soap such as liquid ivory soap that can be administered through soft rubber tubing using gravity flow. foals with significant meconium retention become colicky within the first few hours of life as gas accumulates within their bowel. frequently, one can palpate the meconium through the abdominal wall. additional diagnostics can include abdominal ultrasonography and radiography, particularly if one must rule out other, more serious types of colic. these foals assume a classic stance with an arched back. one must differentiate this stance from the stance assumed by foals with uroperitoneum, which is more extended. foals with meconium retention have had simultaneous ruptured bladder, however, so the clinician must be sure to evaluate the foal fully for both problems. foals that do not respond rapidly to enema administration need additional treatment, which can include giving mineral oil (2 to 4 ounces) by nasogastric tube. one can treat persistent meconium retention resulting in significant abdominal distention by muzzling the foal to prevent further milk intake and administering intravenous fluids at an appropriate maintenance rate. if continuous rate infusion is possible, 5% to 10% dextrose is the preferred fluid to use to provide calories to the foal. one should not use dextrose as a bolus fluid. more aggressive treatment would include administration of retention enemata made using acetylcysteine, which serves as an irritant and increases secretion. extreme cases of meconium retention may require surgical intervention, but this is usually not necessary and most cases resolve with medical management alone within 12 to 24 hours. some foals require pain managment. one should avoid nonsteroidal antiinflammatory drugs in the neonate because of their effects on renal function and gastric mucosal blood flow (see gastric ulcers). many foals respond well to butorphanol administered intramuscularly at a dose of 3 to 5 mg to an average 50-kg foal. intranasal oxygen insufflation is beneficial in foals with significant abdominal distention. one should evaluate foals with meconium impaction/ retention for evidence of pas because intestinal dysmotility is common in pas. colostrum is a laxative, and these foals also may suffer from failure of passive transfer, with meconium retention resulting from the lack of adequate colostrum. these foals are also at risk of sepsis because the mucosal intestinal barrier probably has been disrupted and translocation of bacteria can occur. one should obtain blood cultures on these foals and should monitor them closely for signs of sepsis. atresia within the gastrointestinal system of the foal occurs infrequently, but clinical signs are characteristic. 357 acute colic occurs within the first few hours and is accompanied by abdominal distention similar to meconium retention. three primary types of atresia are described in the foal: membrane atresia, cord atresia, and blind-end atresia. antemortem diagnosis of atresia, short of abdominal exploratory surgery, is aided by the lack of meconium staining of the rectum or any administered enema fluids. additional diagnostic tests may include administration of a barium enema for a radiographic study, colonoscopy, and abdominal ultrasonography. abdominocentesis is usually normal until bowel rupture is imminent or has occurred. one can make affected foals more comfortable by muzzling them to prevent further milk intake and by supplying them with fluids and nutrition intravenously. if one attempts surgical correction, one first should initiate broad-spectrum antimicrobial therapy and determine passive transfer status. frequently, these foals are hypoxemic because of the abdominal distention, and oxygen supplementation is desirable. solid white foals born to overo-overo matings of american paint horses may suffer from congential aganglionosis of the ileum, cecum, and colon. these foals present similarly to foals with meconium impaction or atresia in that colic develops shortly after birth and involves progressive abdominal distention with feeding. the inherited defect is in the endothelin receptor gene. [358] [359] [360] [361] no effective treatment exists, but the clinician should be aware that not all white foals of this mating are affected, and some simply may have meconium retention, so a short period of treatment may be warranted. necrotizing enterocolitis is considered the most common acquired gastrointestinal emergency of human infants. 362, 363 the 1500 to 2000 infants that die every year from this disease in the united states and the large number of infants who develop short gut syndrome from this disease only represent the tip of the iceberg of the problems necrotizing enterocolitis causes. the widespread fear of necrotizing enterocolitis among neonatologists and pediatric surgeons has contributed in large part to the use of the intravenous route rather than the gastrointestinal tract for nourishing these infants for long periods. the pathogenesis of necrotizing enterocolitis is unknown but may result from a disturbance of the delicate balance among gastrointestinal perfusion, enteric organisms, and enteral feeding. risk factors for necrotizing enterocolitis in human infants include prematurity, hypoxic-ischemic insult, and formula or breast milk feedings. the clinical spectrum of necrotizing enterocolitis is multifactoral and ranges from temperature instability, apnea, lethargy, abdominal distention, bilious residuals, septic shock, disseminated intravascular coagulation, and death. medical management is usually adequate treatment for necrotizing enterocolitis. in the neonatal foal, necrotizing enterocolitis is probably one of the most underrecognized causes of gastrointestinal dysfunction and in the past has been attributed only to infection with anaerobic organisms including clostridium perfringens type c and c. difficile. 364 although a specific form of enteritis is associated with intestinal infection by these organisms, most necrotizing enterocolitis is associated with prematurity or pas in the infant and the foal. one should suspect necrotizing enterocolitis in any foal that is having difficulty tolerating oral feeding, demonstrating signs of ileus, or having episodes of colic and in any foal with occult blood or frank blood in the stool. foals exhibiting any of these clinical signs should not be fed orally if possible and should receive parenteral nutrition until gastrointestinal function returns to near normal. the mucosal barrier of the intestine is unlikely to be fully intact, and these foals are at risk for sepsis from bacterial translocation. one should institute broadspectrum antimicrobial therapy in these foals and, if any evidence of coordinated gastrointestinal motility is apparent, should administer sucralfate orally as a protectant. gastric ulcer disease has been recognized in foals, and lesions vary in anatomic distribution, severity, and cause. in clinically normal neonatal foals (<30 days of age), gastric ulcers and mucosal desquamation have been documented. [365] [366] [367] [368] because of these reports and other early reports of death following ruptured clinically silent ulcers in neonatal foals, for years many clinicians felt it necessary to treat critically ill neonates with antiulcer medication prophylactically. [369] [370] [371] recently, this paradigm has been challenged. the pathophysiology of gastric ulcer disease is described most reasonably as an imbalance in protective and aggressive factors. [372] [373] [374] these protective factors are responsible for maintaining a healthy gastrointestinal tract by promoting adequate mucosal blood flow, adequate mucus and bicarbonate production, prostaglandin e 2 production, epithelial growth factor production, gastric afferent innervation, epithelial cell restitution, and gastroduodenal motility. probably the most important factor is maintenance of mucosal blood flow. hypoxia, no, prostaglandins, and gastric afferent innervation influence mucosal blood flow. the aggressive factors include gastric acid, bile salts, pepsin, and enzymes. few specific causes have been found for gastric ulcer disease in foals. excessive administration of nonsteroidal antiinflammatory drugs can result in ulceration of the glandular and squamous epithelium because of an inhibition of prostaglandin production, which leads to a decrease in mucosal blood flow and an increase in acid production. nonsteroidal antiinflammatory drugs also can impair the healing of lesions and rarely are indicated in neonatal equine medicine. 372, 373 in the critically ill neonate the suspected cause of gastric ulcers has shifted away from an excessive amount of intraluminal gastric acid toward gastric mucosal ischemia caused by hypoxia, low blood flow conditions, or both. 375 perforating gastric ulcers are more likely a manifestation of necrotizing enterocolitis than of excessive gastric acid. shock, sepsis, or trauma can result in gastric mucosal ischemia, allowing for the disruption of epithelial cell integrity and permitting damage by aggressive factors or providing an environment suitable for the establishment of bacteria colonization. 375,376 impairment of mucosal blood flow also may result in reperfusion injury, allowing the formation of gastric ulcers. in the sick neonatal foal (<7 days of age) a wide variability in the intragastric ph has been documented depending on the type of disease, severity, and milk intake frequency and volume, suggesting that in the critically ill equine neonate, ulcer prophylaxis using histamine antagonists or proton pump inhibitors is not only unnecessary but unlikely to work. 377 clinically significant gastric ulcers can occur in the squamous, glandular, or both portions of the stomach as a primary problem or resulting from another problem. clinical signs include diarrhea, abdominal pain, restlessness, rolling, lying in dorsal recumbency, excessive salivation, and bruxism. in the neonatal foal the only clinical signs present may be depression or partial anorexia until a more catastrophic event, such as perforation, occurs. some lesions in the gastric mucosa extend from the pylorus into the proximal duodenum and can result in stricture of the pylorus and proximal duodenum. these foals are usually older (>1 month of age) and have a greater volume of reflux. bruxism and ptyalism are also more prominent in these older foals. the most sensitive and specific method for diagnosing gastric ulcers is visualization by endoscopic examination. 365 unfortunately, the use of gastric endoscopy has led to recognition of relative nonlesions and ulcers resulting from other problems and of clinically significant disease states. the clinician should not stop simply when ulceration of the stomach is recognized with endoscopy but should examine that patient fully for other potential sources of the clinical signs. other diagnostic tests may help in determining the severity of the ulcers, including fecal occult blood or gastric blood assessments, contrast radiography, abdominal ultrasound, and abdominocentesis. endoscopy of the foal stomach carries an additional risk of exacerbating colic in the short term, unless the examiner ensures that as much introduced air as possible is evacuated from the stomach at the end of the procedure. the presence of a brown gastric reflux fluid may indicate the presence of bleeding ulcers. blood in the feces of the neonate is more consistent with a diagnosis of necrotizing enterocolitis, which can be associated with gastric ulcers. contrast radiography is useful if one suspsects delayed gastric emptying or pyloric or duodenal stricture in older foals. if a stricture has occurred, one will note a delay in complete emptying of barium from the stomach (>2 hours). 367 abdominal ultrasound may be useful to visualize free abdominal fluid and gastric or small intestinal distention if one suspects a perforation. one can visualize portions of the descending duodenum, and a thickened duodenum should increase the index of suspicion for duondenal stricture. abdominocentesis also may confirm perforation. traditional therapy for gastric ulceration includes mucosal adherents, histamine type 2 receptor antagonists, proton pump inhibitors, and antacids. 378 the most widely used mucosal adherent is sucralfate, which is a hydroxy aluminum salt of sucrose. the main therapeutic action of sucralfate is to bind to the negatively charged particles in the ulcer crater. 378, 379 at a ph less than 2, sucralfate is converted to a sticky viscous gel, which adheres to the ulcer crater and remains adhered for 6 hours, but at a higher ph, sucralfate remains in a suspension. sucralfate is still effective because it inhibits pepsin and buffers hydrogen ions. other important actions of sucralfate include stimulating production of prostaglandin e, which maintains mucosal blood flow; increasing bicarbonate secretion; stimulating mucous secretion; decreasing peptic activity; and binding epidermal growth factor. the histamine type 2 receptor antagonists include cimetidine, ranitidine, and famotidine. these compounds block the interaction of histamine with the histamine type 2 receptor on the parietal cell, resulting in inhibition of gastric acid secretion. clinically normal neonatal foals have a highly acidic gastric fluid that is influenced by sucking. intravenous and oral administration of ranitidine increases intragastric ph in normal foals but critically ill neonatal foals have a blunted response to ranitidine administration. 377, 380 one possible conclusion reached from these studies is that in critically ill neonatal foals, gastric ulcers may not be caused by an increased intraluminal gastric acidity. the most commonly used proton pump inhibitor is omeprazole. this drug has not as yet been approved for use in foals under 30 days of age. omeprazole inhibits the secretion of hydrogen ions at the parietal cell by irreversibly binding to the h + ,k + -atpase proton pump of the cell. most of the lesions in older foals were healed after daily administration of omeprazole for 28 days according to one report. 381 table 19 -9 summarizes the therapeutic agents for treating gastric ulcers in foals. prophylactic treatment of critically ill neonates for gastric ulcers has been standard therapy for years because of the evidence of clinically silent ulcers. this approach may not be appropriate for several reasons. an increased incidence of nosocomial pneumonia and systemic sepsis is associated with high gastric ph in human patients in intensive care. [382] [383] [384] patients in intensive care units treated prophylactically with histamine type 2 receptor antagonists are more likely to develop pneumonia during ventilation therapy and gastric colonization with potentially pathogenic bacteria or yeast. 382, 385 an acidic environment appears to protect against airway colonization by bacteria of intestinal origin and bacteria translocated across the gastrointestinal tract. pathogenesis of ulcers in the neonatal foal most likely does not involve increased intraluminal gastric acid but instead may be caused by decreased mucosal perfusion associated with shock, hypoxia, and hypoxic/ischemic insult to the gastric mucosa. a recent report revealed that gastric ulcer disease in equine nicu patients is independent of pharmacologic prophylaxis. 386 in this study, despite decreased treatment, the incidence of gastric ulcers found in these foals at necropsy had decreased significantly. the decrease was attributed to overall improvement in management of these cases. similarly, in a human intensive care unit, the incidence of stress ulcers decreased independent of the use of prophylaxis. 375, 387 early treatment of sepsis, sufficient oxygenation, improved monitoring, institution of enteral feedings, and improved nursing care may contribute to the reduction in gastric ulcers in the neonatal patient. use of histamine type 2 receptor antagonist and proton pump inhibitors apparently may not be necessary; however, in some instances sucralfate may be useful. sucralfate reduced the rate of bacterial translocation in a rat model during hemorrhagic shock and also may prohibit the generation of acute gastric mucosal injury and progression to ulcer formation induced by ischemia-reperfusion. 388, 389 in a human medical intensive care unit, airway colonization by new pathogens occurred more frequently in patients receiving agents that increased gastric ph than in those receiving sucralfate. 382, 390 in the critically ill neonatal foal, risk factors for gastric ulceration have not been identified clearly, although foals treated routinely with nonsteroidal antiinflammatory drugs may be at increased risk for gastric lesions. prophylactic treatment for gastric ulcers in critically ill neonates may not be necessary, and one should consider carefully the pros and cons of their use before their administration. foal heat diarrhea is a mild, self-limiting form of diarrhea that occurs in foals between 5 and 14 days of age, about the time of the "foal heat" in the dam. the definitive cause of foal heat diarrhea has yet to be described, but the condition may be associated with dietary changes or changes in gastrointestinal function that occur around that time. this form of diarrhea is not caused by stongyloides westeri infestation as previously thought. 391 foals with foal heat diarrhea are not systemically ill and should not require therapy. one should evaluate fully any foals with diarrhea at this time for other possible causes of diarrhea, particularly if they are unwell or exhibit anorexia or dehydration. viral diarrhea occurs most commonly in large groups of mares and foals that are housed together. rotavirus is an isolate from the feces of up to 40% of foals with diarrhea worldwide, alone or with another pathogen. 392, 393 the virus infects and denudes the microvilli, resulting in increased secretion combined with decreased absorption. the virus interferes with disaccharidase function and alters the function of the intestinal sodium-glucose cotransport proteins. the initial clinical signs are anorexia and depression, with profuse watery diarrhea occurring shortly thereafter. severely affected foals may become significantly dehydrated and have electrolyte abnormalities, primarily hyponatremia and hypochloremia with metabolic acidosis. these foals generally require intravenous fluid support, whereas less severely affected foals may require only symptomatic therapy. definitive diagnosis is by detection of the virus in the feces of foals with diarrhea. however, none of the available tests are particularly sensitive, and the virus also may be found with other intestinal pathogens. recently, vaccination of pregnant mares has been suggested as a means of prevention, with preliminary results suggesting efficacy. 394,395 although a definitive role for adenovirus has not been established in the foal, adenovirus is a common co-isolate from foals with rotaviral diarrhea. 396 a specific equine coronavirus recently has been identified from an immunocompetent foal with diarrhea, and a second report of cornavirus diarrhea was published recently. 397,398 one case report suggests a parvovirus caused diarrhea in the foal. 399 treatment of viral diarrhea in foals is primarily supportive. intravenous fluid and parenteral nutritional support may be necessary in severe cases. very young foals may benefit from intravenous plasma administration and broad-spectrum antimicrobial coverage to limit bacterial translocation. one can administer sucralfate orally in these cases as a gastrointestinal protectant and to discourage bacterial translocation. foals with moderate to severe metabolic acidosis may benefit from sodium bicarbonate administration if their ventilatory function is normal. one administers sodium bicarbonate at half the calculated deficit (0.5 ã� standard base excess ã� body mass in kilograms) as an isotonic solution at the maintenance fluid rate. one should reevaluate sodium and bicarbonate (or standard base excess) concentrations regularly. nonspecific therapy of diarrhea is discussed elsewhere in this text. diarrhea is frequently the primary presenting complaint in foals with sepsis, so one should rule out this differential diagnosis in foals less than 1 week of age. one should evaluate all neonatal foals with diarrhea for possible sepsis and should include a blood culture whenever possible. clostridium perfringens and c. difficile are recognized increasingly as serious pathogens of the foal. 400-403 foals with either pathogen generally have abdominal pain, dehydration, and profuse watery diarrhea. some foals may have red-tinged or frankly bloody feces, which carries a poorer prognosis. most foals with this type of diarrhea require intensive care or, at the minimum, intravenous fluid administration. outbreaks of this type of diarrhea on farms occasionally occur, and the suggestion is that the dam has a role in transmission of the bacteria. diagnosis is by recognition of the offending organism by gram stain of the feces, by bacterial isolation from the feces, and by detecting the presence of toxins associated with the organisms. specific treatment includes oral administration of metronidazole and broad-spectrum antimicrobial coverage as prophylaxis for bacterial translocation associated sepsis in younger foals. foals with severe blood loss in their feces may require transfusion of whole blood. salmonella spp., escherichia coli, bacteroides fragilis, and aeromonas hydrophila have been implicated in diarrhea in foals. salmonella generally is associated with septicemia in foals, and although some convincing evidence exists for a role for e. coli in foal diarrheal disease, the extent of e. coli as a pathogen of the gastrointestinal tract in foals has yet to be described fully. 371, [404] [405] [406] [407] proliferative enteropathy is a transmissible enteric disease caused by lawsonia intracellulare. 408,409 most foals have been weaned before the appearance of clinical signs of depression, rapid and significant weight loss, subcutaneous edema, diarrhea, and colic. poor body condition with a rough hair coat and a pot-bellied appearance are common in affected foals. clinicopathologic abnormalities included hypoproteinemia, leukocytosis, anemia, and increased serum creatine kinase concentration. postmortem reveals characteristic intracellular bacteria within the apical cytoplasm of proliferating crypt epithelial cells of the intestinal mucosa. antemortem diagnosis of equine proliferative enteropathy is based on clinical signs, hypoproteinemia, and the exclusion of other common enteric pathogens. fecal polymerase chain reaction analysis may be positive for the presence of l. intracellulare, and affected foals develop antibodies against l. intracellulare. 410 treatment with erythromycin estolate alone or combined with rifampin for a minimum of 21 days is recommended with additional symptomatic treatment when indicated. cryptosporidium spp. cause gastroenteritis and diarrhea in many animal species and are not host-specific. cryptosporidium has been implicated as the casual agent of diarrhea in foals, but the organism is isolated from the feces of diarrheic foals and normal foals with the same frequency and concentration, making a clear role for the organism difficult to elucidate. 411-413 diarrhea caused by cryptosporidium in other species and that described for foals is generally self-limiting, with a clinical course of between 5 to 14 days. immunosuppressed patients, including foals compromised by concurrent disease, are thought to be at increased risk for complications resulting from infection with this organism. 411,412 treatment is symptomatic. cryptosporidiosis is a disease with zoonotic potential, and one should take appropriate precautions, including use of gloves and frequent hand washing, if organisms are identified in the feces of any patients so as to prevent spread to other patients and personnel. eimeria leukarti, trichomonas equi, and giardia equi have been identified in the feces of normal horses and horses with diarrhea. transmission studies have failed to produce reliable clinical signs, and the prevalence and significance of these organisms in the genesis of foal diarrhea remain unknown. strongyloides westeri is a common parasitic infection of foals. 392, 414 transmission is transmammary, and patent infection is recognizable in the foal by 8 to 12 days of age. this nematode previously was associated anecdotally with foal heat diarrhea, but the association has not been demonstrated clearly. the diarrhea is generally mild and is treated effectively by deworming with benzimidazole or ivermectin anthelmintics. 391 strongylus vulgaris fourth-stage larvae cause diarrhea in young foals during migration through the arterioles of the cecum and descending colon. clinical signs may resemble thromboembolic colic. 414 the prepatent period is about 6 months, and diagnosis is based on clinical examination, clinicopathologic changes, and farm deworming history. patients with diarrhea associated with this parasite may have peripheral leukocytosis, neutrophilia, eosinophilia, and hypoproteinemia. appropriate deworming with ivermectin (label dose), fenbendazole (10 mg/kg/day orally for 5 days), or thiabendazole (440 mg/kg/day orally for 2 days) is recommended, with the last two drug dosages being larger than the label dose. cyathostomiasis, or diarrhea resulting from the sudden emergence of encysted cyathostome larvae, is an unusual cause of diarrhea in the foal. the clinician managing critically ill neonates must recognize that intravenous fluid therapy simply cannot be scaled down from adult management approaches. fluid management of the ill neonate, particularly over the first few days of life, must take into consideration that the neonate is undergoing a large transition from the fetal to the neonatal state and that important physiologic changes are taking place. 166 these transitions include shifts in renal handling of free water and sodium and increased insensible losses because of evaporation from the body surface area and the respiratory tract. the newborn kidney has a limited ability to excrete excess free water and sodium, and the barrier between the vascular and interstitial space is more porous than that of adults. water and sodium overload, particularly in the first few days of life, can have disastrous long-term consequences for the neonate. 416, 417 in the equine neonate, excess fluid administration frequently manifests as generalized edema formation and excessive weight gain, frequently equivalent to the volume of excess fluid administered intravenously. in cases in which antidiuretic hormone secretion is inappropriate, as in some foals with pas, generalized edema may not form, but the excess free water is maintained in the vascular space. this syndrome of inappropriate antidiuretic hormone secretion is recognized in the foal that gains excessive weight not manifested as edema generally, with decreased urine output and electrolyte abnormalities such as hyponatremia and hypochloremia. 418 the foal manifests neurologic abnormalities associated with hyponatremia. the serum creatinine concentration varies in these cases, but urine always is concentrated compared with the normally dilute, copious amounts of urine produced by foals more than 24 hours of age on a milk diet. if measured, serum osmolarity is less than urine osmolarity. the treatment for this disorder is fluid restriction until weight loss occurs, electrolyte abnormalities normalize, and urine concentration decreases. if the clinician is unaware of this differential diagnosis, the neonate can be assumed mistakenly to be in renal failure, and the condition can be exacerbated by excessive intravenous fluid administration in an attempt to produce diuresis. the problem of appropriate fluid management in critically ill neonates has been recognized by medical physicians for years and has resulted in changes in fluid management of these patients. the approach taken has been one of fluid restriction, in particular sodium restriction but also free water restriction, and has resulted in improved outcome and fewer complications, such as patent ductus arteriosus and necrotizing enterocolitis. 416, 417 the calculations used for maintenance intravenous fluid support in these patients takes into consideration the ratio of surface area to volume and partially compensates for insensible water losses. maintenance fluids are provided as 5% dextrose to limit sodium overload and provide sufficient free water to restore intracellular and interstitial requirements. the calculation for maintenance fluid administration is as follows: first 10 kg body mass 100 ml/kg/day second 10 kg body mass 50 ml/kg/day all additional kilograms of body mass 25 ml/kg/day as an example, the average 50-kg foal would receive 1000 ml/day for the first 10 kg of body mass, 500 ml/day for the next 10 kg of body mass, and 750 ml/day for the remaining 30 kg of body mass for a total of 2250 ml/day. this translates to an hourly fluid rate of about 94 ml/hr. one should adjust the fluid and sodium requirements for ongoing losses exceeding the maintenance requirements. these losses can take the form of diarrheal losses and excessive urine output, such as those with glucose diuresis and renal damage resulting in an increased fractional excretion of sodium. the normal fractional excretion of sodium in neonatal foals is less than that of adult horses, usually less than 1% (j.e. palmer, unpublished data). in the critically ill foal the sodium requirement can be met with as little as 140 meq of sodium per day, about that administered in a single liter of normal equine plasma. one can address sodium deficits by separate infusion of sodium-containing fluids, although this may not be necessary if one considers the sodium being administered in other forms, including drugs administered as sodium salts and any constant rate infusions (pressors, inotropes, etc.) that are being provided as solutions made with 0.9% sodium chloride. the author has used this approach to fluid therapy in her nicu for the last few years and believes that the percentage of foals suffering from generalized edema and related problems has decreased. if one takes this approach to fluid therapy, one should take the weight of the patient once daily, or even twice daily, and monitor the fluid intake and output as closely as practical. one should evaluate any larger than anticipated weight gains or losses. one should not expect urine output to approach the reported normal of 300 ml/hr for a 50-kg foal because the free water administered is limited, unless the patient is experiencing diuresis (glucosuria, resolution of the syndrome of inappropriate antidiuretic hormone secretion, resolution of previous edematous state, renal disease). one should obtain the urine specific gravity several times daily and should determine fractional excretion of sodium at regular intervals. if the volume of urine produced by the patient is measured accurately, one can determine sodium losses accurately and can obtain creatinine clearance values. one should obtain blood pressure measurements at regular intervals throughout the day because hypotension can be a problem in these patients, particularly in septic foals and foals suffering from pas, and one may need to increase fluid therapy to maintain adequate vascular volume. patients with hypotension may need inotrope and pressor support. inotrope and pressor therapy generally is restricted to referral centers where these drugs can be administered as constant rate infusions and blood pressure can be monitored closely. blood pressure can be monitored directly or indirectly by the use of cuffs placed on the base of the tail. both techniques have advantages and disadvantages. although direct blood pressure measurements are considered the gold standard and are generally more accurate, the difficulty in placing and maintaining arterial catheters and lines in these patients severely restricts the utility of this method. indirect techniques can be inaccurate and are affected by cuff size and placement. however, indirect techniques are easier to use in the nicu and can be useful if trained staff are using the equipment. in the author's nicu, once practitioners identify the appropriate cuff size, they dedicate that cuff to that patient for the duration of the hospitalization to decrease variability caused by using different cuffs. one should monitor the blood pressure of all recumbent patients at regular intervals, and trends upward or downward should prompt the clinician to make necessary adjustments. foals suffering from pas and sepsis are the patients most at risk for significant hypotension and perfusion abnormalities. perfusion is maintained by supporting cardiac output and blood pressure with judicious use of intravenous fluid support and inotrope/pressor support. the author does not aim for any specific target systolic, mean, or diastolic pressure. instead the author monitors urine output, mentation, limb perfusion, gastrointestinal function, and respiratory function as indicators that perfusion is acceptable. for nicu patients to require inotrope and pressor therapy is not unusual, but in some cases hypoxic and septic damage is sufficiently severe to blunt the response of the patient to the drugs. one must approach each patient as an individual, and no single inotrope/pressor protocol will suffice for all patients. dobutamine is a î²-adrenergic inotrope that is frequently used as first choice therapy in nicu patients. its effects are î² 1 at the lower dose range. neonates have a limited ability to increase stroke volume in an effort to maintain cardiac output, and one may observe tachycardia in these patients as heart rate increases to maintain cardiac output and vascular pressure. dobutamine is useful after patients are volume replete for support of cardiac output. the dose range is between 2 to 20 âµg/kg/min provided as a constant rate infusion. dopamine has dopaminergic activity at low doses, î² 1 and î² 2 activity at moderate doses, and î± 1 activity at high doses. dopamine causes norepinephrine release, which has lead to the suggestion that this is its major mode of action at higher doses. at doses greater than 20 âµg/kg/min, intrapulmonary shunting, pulmonary venous vasoconstriction, and reduced splanchic perfusion may occur. dopamine also produces natriuresis at lower doses through a direct effect on renal tubules. for these reasons, dopamine has fallen out of favor at some referral institutions. norepinephrine has î± 1 and î² 1 activity but variable î² 2 activity, resulting in potent vasopressor effects; it has inotropic and chronotropic effects, but its chronotropic effect usually is blunted by vagal reflexes slowing the heart rate induced by the increase in blood pressure. in many critical care units, norepinephrine has become a pressor of choice and frequently is used along with dobutamine. evidence suggests that splanchic perfusion is maintained better with norepinephrine than with some other pressors. 419 the dose range is 0.2 to 2.0 âµg/kg/min, although larger doses have been used when necessary in certain patients. epinephrine has î± 1 , î± 2 , î² 1 , and î² 2 activity; î² activity predominates and results in increased cardiac output and decreased peripheral resistance at low doses. epinephrine has been associated with hyperglycemia, hypokalemia, lipolysis, increased lactate concentration, and increased platelet aggregation. the effect on renal function is controversial. use of epinephrine usually is limited to those patients not responding to other pressors. vasopressin (antidiuretic hormone) is a pressor gaining a great deal of attention in the critical care literature. vasopressin appears to be depleted from the neurohypophysis in septic shock, 420 and short-term administration of vasopressin spares conventional vasopressor use, in addition to improving some measures of renal function. 421 low-dose vasopressin infusion increases mean arterial pressure, systemic vascular resistance, and urine output in patients with vasodilatory septic shock that are hyporesponsive to catecholamines. these data indicate that low-dose vasopressin infusions may be useful in treating hypotension in patients with septic shock. 422 the author has been using low-dose vasopressin in patients in her nicu for the past few years and has the clinical impression that blood pressure is defended more readily using this agent in concert with other management strategies. the author commonly uses low-dose vasopressin constant rate infusion with dobutamine constant rate infusion as the initial inotrope/pressor therapy in cases requiring pressure defense, although no prospective studies are yet available regarding this drug in veterinary medicine. 20 for quality health care for animals, advances in medical science, and in some breeds the increasing value of the juvenile equine athlete. equine veterinarians that encounter pediatric orthopedic problems are only beginning to get the information needed to make appropriate treatment decisions. the equine neonate has specific differences in structure and physiology from adults that one must consider when designing an optimal therapeutic or management strategy. few investigations have focused on the equine neonatal musculoskeletal system, 1-6 but a large body of clinical information exists, and one can make cautious extrapolations from work in other species. 7 neonatal equine bones have accelerated modeling and remodeling processes 5 that result in accelerated fracture healing and an increased susceptibility to deformation caused by excessive loading. contralateral limb varus deformities of the growth centers (most commonly distal radius and metacarpus/ metatarsus) are common in overloaded limbs. the increased plasticity of the skeletal structure also is mirrored in the soft tissue support system, for these units become flaccid within 2 weeks of immobilization. 4 this laxity is important, because it further compromises the use of the fractured limb and can last as long as the coaptation was in place. additional divergences from adult physiology include musculoskeletal immaturity (generalized or focal) and immune system differences. finally, foals are lighter and can tolerate and will assume recumbency more readily than adults. the net results of these differences are that one must consider the use of external coaptation carefully, fractures heal quickly, one must consider damage to the contralateral limb from overstress, reducing weight bearing is possible, and infection is always lurking. stresses can affect the musculoskeletal system of the foal at any time, including in utero. although rare, reports describe in utero fractures (k. sprayberry, personal communication, 2003) that result in foal locomotor problems and even maternal uterine damage from sharp bone ends. the cause is presumably from vigorous muscular activity of the foal, but one cannot rule out direct trauma. the fractures result in foal lameness and can increase the likelihood of dystocia and caused colic in one mare when the broken bones damaged the uterus. treatment depends on how long the fracture has been present and on the fracture location and configuration, but if the fracture is repairable, internal fixation probably is necessary. fractures occurring during foaling result from aggressive obstetric manipulation (mandibles) or the advances in medical care of equine neonates in the last 20 years have resulted in the survival of many foals that previously would have died from sepsis, asphyxia, and prematurity; and the successful management of their musculoskeletal system can be a major challenge. major factors adding to the challenge are the immaturity of components of the musculoskeletal system and the demands placed on them by a growing and active foal. additional pressures to treat orthopedic conditions in foals have come from an overall increase in the demand chest compression. one should stabilize unstable mandibular fractures. appendicular fractures usually do not occur during parturition because of the robust character of the bones of the foal. after birth, foals are susceptible to external trauma from many sources. the dilemma is that younger foals with fractures are more likely to heal but also are more likely to develop contralateral limb problems because of excessive weight bearing and affected limb flexor tendon laxity if the limb is immobilized fully. as a result, internal fixation is often the best choice for neonatal fractures to keep the fractured limb in use. proximal sesamoid bone fractures result from hyperextension of the fetlock joint. foals are lame after the fracture, but the lameness can be mild and often diminishes quickly. soft tissue swelling occurs over the sesamoids. fractures are usually simple, can occur uniaxially or biaxially, and can be apical, midbody, or basilar. fractures can occur in any joint and can affect multiple sesamoids in one foal. however, they most commonly are single forelimb fractures 8 and in thoroughbreds are most frequent in the left front medial proximal sesamoid (j.p. morehead, personal communication, 2003). of particular interest to neonatologists is that proximal sesamoids fractures often occur in recovered neonatal patients that are allowed too much exercise too soon. foals from the nicu need a gradual introduction to pasture turnout to allow their musculoskeletal system to adjust. mares are often in need of turnout, but in the interest of their foals, they must wait. treatment of proximal sesamoid fractures in foals is stall confinement with support bandaging. healing occurs, albeit with some distortion of the shape of the sesamoid. severely displaced fragments result in large and misshapen sesamoids, and surgery may be considered for these foals, because restriction of fetlock flexion can occur after conservative therapy. third phalangeal fractures are also common in foals. these foals have a lameness that worsens with hoof compression. hoof abscesses are uncommon in young foals but should be considered. most commonly, radiographs reveal nonarticular small fractures on the wings on the third phalanx. the fractures are associated with hard ground and exercise. the fractures heal with stall confinement, and unlike adults, leave no discernable radiographic fibrous union. avulsion fractures of the proximal insertion of the peroneus tertius and the origin of the long digital extensor tendon have been reported. 9,10 both soft tissue structures attach to the extensor fossa of the distal femur. the two affected foals had lameness of a hindlimb associated with swelling, pain, and crepitation. radiographs revealed multiple avulsion fractures of the extensor fossa. because of the intraarticular fragments in the femoropatellar joint, and the fear of later degenerative joint disease, fragments were removed arthroscopically. both foals were juveniles at last follow-up; one foal was considered normal, and one had a mild residual lameness. tendon and ligament damage is uncommon in neonates probably because of their low body weight. extensor tendon damage following flexural deformities is the most common tendon problem and is discussed in congential flexural deformities of foals. gastrocnemius ruptures are one of the most devastating problems and have occurred after forced extraction because of a breech presentation, severe flexor tendon laxity, and tarsal contracture. loss of gastrocnemius function usually results in a non-weight-bearing limb, although an intact superficial digital flexor tendon may make some weight bearing possible. complete loss of support is difficult to treat successfully. coaptation of the limb is logical but difficult to obtain. schroeder-thomas splints have been used but are difficult to manage. tube casts also are used but must be changed frequently, and cast sores are inevitable (l.r. bramlage, personal communication, 2003) . the prognosis for athletic function is guarded. treatment for ligamentous injuries is usually some form of coaptation, although surgical repairs have been performed when coaptation was unworkable. 11 coaptation in proper limb alignment allows the ligaments to heal and should be used if the injury will destabilize a joint and cause damage to growing epiphyses or cuboidal bones. one can achieve coaptation with casts or splints under a bandage. casts are initially a greater expense, and cast sores and their resulting white hairs are a risk, but the rigid immobilization and the lack of the requirement for daily adjustment makes them preferable. important to musculotendinous health is some measure of weight bearing to avoid laxity after coaptation removal, which one can achieve by using tube casts and splints that allow weight bearing. following coaptation, bandaging and a gradual return to exercise are recommended for ligamentous injuries. patellar luxation can affect foals in one or both hindlimbs, and the luxation can vary from a laxity in the medial attachments to complete luxations that cannot be replaced in the patellar groove of the distal femur. 12, 13 medial luxations have not been reported. clinical signs vary from a slight discontinuous motion during stifle flexion to an inability to stand. many foals have a crouching stance on the affected limb because of an inability to extend the stifle. the pathophysiology of patellar luxations is unknown. congenital bilateral luxations are common in miniature horse foals and are believed to be genetic. luxations are rarer in other breeds and are occasionally traumatic. the affected limbs are usually not grossly abnormal except for effusion of the femoropatellar joint and the luxation. a shallow trochlear groove has been reported to be a cause of patellar luxation, but objective evidence is lacking. one should evaluate foals for the ability to stand. once the appropriate supportive care is provided, if a foal cannot stand, euthanasia is recommended. most bilateral luxations in horses fit in this category. however, miniature horse foals often can stand sufficiently to nurse despite bilateral luxations, and one may consider treatment. treatment consists of replacing and stabilizing the patella and sometimes surgically deepening the patellar groove. delaying surgical repair until the foal is approximately 30 days old is recommended to avoid neonatal problems, allow the musculoskeletal system to mature, and provide good anchors for suture. some surgeons worry that delay may cause further femoropatellar developmental abnormalities, but in a small number of cases, this has not been an issue. the prognosis for miniature horse foals appears to be good because of their low body weights and modest performance expectations. too few reports about the correction of unilateral luxations in light horses exist to make a definitive statement about prognosis except that success and failure have been experienced. 12, 13 congenital flexural deformities in foals can be classified as severe (rarely correctable), moderate (correctable with therapy), or mild (self-correctable). examples of severe flexural deformities include arthrogryposis (deformities of multiple limbs and often the head and neck), severe carpal deformities (flexor angle of the carpus less than 90 degrees), and tarsal contractures (rare). extraordinary methods have been used to correct severe deformities 14 but are often unsuccessful. mild flexural deformities are those that result in an upright conformation to the limb, but the foal can bear weight on the limb and load the flexor structures. these foals require no specific treatment and will self-correct with controlled exercise. moderate flexural deformities are those that make bearing weight on the limb and loading the flexor structures and ligaments difficult for the foal. when these deformities occur bilaterally (most common), the foals cannot rise to suckle or does so with great difficulty, and the lack of weight bearing worsens the flexural deformity. examples of moderate flexural deformities include carpal and forelimb fetlock flexural deformities that usually occur together, hindlimb fetlock flexural deformities with coronopedal flexion or hyperextension, and the uncommon coronopedal flexural deformity alone. treatment of moderate flexural deformities aims to place the solar surface of the foot on the ground so that the weight of the foal can stretch the flexor structures. splints are useful for restoring the limb to normal orientation but require attention to detail because the splints often exert an extreme amount of tension on the soft tissues, and the skin of the foal is thin. pressure sores are easy to create and at a minimum result in an extended convalescence. the first step in splint application is to apply a separate heavy bandage to the limb, which should be reapplied as necessary because the bandage can slip and cause focal constriction. commercial gauze over cotton bandage material works better than sheet cotton as a bandage. the splint is made of polyvinyl chloride pipe cut in half or thirds. using 50% of the diameter of the pipe results in less splint rotation but is bulkier and leaves more splint exposed to cause trauma. one cuts off the corners of the splint and pads the ends with gauze or roll cotton covered with tape. palmar or plantar placement of the splint is preferable, but severe deformities may require initial dorsal placement. as the limb straightens, one can bend the splint to tape the fetlock into the bend to extend it. one can tape the splint tightly to the limb over the bandage with nonelastic (white or duct) tape. this procedure requires at least two persons, one to extend the limb firmly and hold the limb and one to tape. one should leave the splint on for 8 to 12 hours and then remove it for 8 to 12 hours. one can reapply splints as necessary. in addition to splints, some medications are of value for treating flexural deformities. oxytetracycline (40 to 50 mg/kg) given intravenously appears to relax the soft tissues. 15 the mechanism of action is unknown, and the drug is most efficacious when given in the first 3 days of life. this dose is high but appears to be safe for healthy foals and can be repeated at 24-hour intervals. foals should be normovolemic during tetracycline administration. one should use the drug with caution in foals with renal impairment. foals should be urinating and have reasonable urinary parameters (serum urea nitrogen, creatinine, and urinalysis) before tetracycline use. diarrhea is an uncommon sequela to tetracycline use. one should monitor the unaffected limbs closely because all limbs experience a relaxation of the palmar/plantar support. 15, 16 discontinuation of tetracycline therapy before affected limbs are normal but after they can bear weight is common because of worsening laxity in the "normal" limbs. one also can use phenylbutazone (4 mg/kg) for a short time when the splints are used. some analgesia appears to help the foals use the limbs and stretch the soft tissues. one should not use phenylbutazone for long periods of time because of the potential of inducing gastric ulcers. surgical treatment of congenital flexural deformities rarely is indicated. severely affected foals rarely respond favorably to surgery, and mildly affected foals do not need it. surgery is most appropriate for foals with moderate flexural deformities that are neglected or have not responded to splinting and tetracycline. the most common surgical therapy performed for congenital flexural deformities is the inferior check ligament desmotomy for fetlock or coronopedal flexural deformities. ruptures of the extensor tendons commonly occur with congenital flexural deformities and result from the foal overloading the extensor tendons. no specific therapy for the ruptures is necessary. if the rupture is extensive, it can interfere with the ability to extend the fetlock and to place the foot flat. these foals then tend to knuckle over, even after correction of the flexural deformity. a firm fetlock bandage extends the digit and assists in foot placement until the extensor tendons heal. foals commonly are born with hyperextension deformities of the fetlock of varying degrees of severity. all but the worst deformities self-correct as muscle tone improves. a deeply bedded stall is all that is usually necessary to protect the soft tissues, but one can apply a light bandage to the coronary band and pastern if trauma is a problem. severe deformities are more problematic but rare, so therapeutic recommendations are not available. hyperextension of the carpus occasionally occurs and usually is treated conservatively. however, a tube cast to align the limb may be necessary to protect the dorsal surface of developing carpal bones. neonatal foals exhibit three categories of forelimb conformational deviations: angulation, rotation, and carpal offset. angular deviations most commonly are centered in the metaphysis and epiphysis, but their location is described by the closest joint, usually the carpus and fetlock. when the deviation of the distal limb is lateral to the long axis, the deviation is valgus, and when the deviation is medial, the deviation is varus. more than one joint can be affected, and although rare in neonates, valgus and varus can occur in different joints in one limb. rotational deformities appear to originate most commonly in the diaphysis or metaphysis of the radius or the metacarpus. in neonates the direction of rotation of the distal limb at both sites is almost exclusively outward. associated angular and rotational deviations occur. 17 in neonates, limb deviations occur in foals with narrower chests and less developed pectoral muscles than in straight foals, and they appear to have an initial greater overall weakness in the musculoskeletal system because it first interacts with gravity, body mass, and ground reaction forces. however, after the first few days of life, the asymmetric loading of the growth centers does affect limb deviations. angulation results from a compressive load that is asymmetric in a frontal plane but is uniform in the sagittal plane, and rotation occurs when the compressive load is asymmetric in both planes and the limb develops around an overloaded axis point. considered this way, valgus and outward rotation deviations in young foals are coupled, as are varus and inward rotation in older foals. the loading asymmetry for valgus/outward rotation foals is accentuated as foals assume a base-wide posture that is more stable side-to-side but promotes a lateralization of the limb load. the specific effects of intermittent versus static loads, strain magnitude versus strain rate, and shear and hydrostatic stress on growing bones is only beginning to be understood. however, clinical experience supports the general observation that excessive cartilage compression is deleterious to bone growth. offset carpal conformation describes a joint that appears to deviate outwardly and then inwardly, all within the carpus. the deformity is thought to be centered at the radiocarpal joint, but the specific structural cause of offset has not been determined. this conformation is more common in older foals but occasionally occurs in neonates. the deviation is particularly common when incomplete ossification of the carpal bones is present. the causes of conformational deviations are a matter of some debate. as always, the major factors are genetics or environment. genetic influences include the assortment of alleles that controls bone form and growth and the assortment that modulates bone remodeling. many in the horse industry believe that genetics is a strong determiner of limb conformation. environmental influences are many and include the intrauterine environment, the postnatal limb load, nutrition, and bad luck. suffice to say, the situation is complex, but one must consider biologic and mechanicobiologic influences when evaluating the growth of long bones. 18 several factors may contribute to the common occurrence of deviations in the carpus. first, the carpus is in the middle of the limb and is subject to the greatest bending forces. second, the carpal anatomy is complex and perhaps is not understood completely. the carpus has seven cuboidal bones, two long bones, and two epiphyses (distal radial and lateral styloid); and cartilage surrounds all. the ligamentous support includes collateral ligaments, innumerable intracarpal ligaments, and a palmar carpal soft tissue ligament. the distal radial physis is not flat transversely, but undulates in the frontal and sagittal planes. 3 a separate center of ossification for the lateral styloid process is found at its palmar-lateral aspect. because of this separate center of ossification, more cartilage and less bone are in the lateral aspect of the distal radial growth center, suggesting it may be more susceptible to growth alterations from load. less common conformation deformities in young foals include hindlimb deformities, windswept conformation, diaphyseal deviations (usually of the metacarpus/ metatarsus), gross congenital malformations such as agenesis and polydactyly, and acquired varus deformities of the carpus and fetlock. hindlimb conformational deviations can manifest as tarsal and fetlock angular deformities and external limb rotation, usually centered above the tarsus. windswept foals have limbs (usually both forelimb or both hindlimbs) that are curved in the same direction in the frontal plane. diaphyseal deviations, agenesis, and polydactyly are rare and have various presentations. acquired varus deformities are caused by excessive loading, which appears to be focused medially on the growth plates. one should evaluate the limbs to determine the location, extent, and potential cause of the deviation. evaluation consists of observation and then palpation for heat, swelling, or ligament laxity. ligamentous laxity of the medial carpal ligaments is an important cause of carpal valgus and should be evaluated carefully. lameness is not a characteristic of uncomplicated angular limb deformities and suggests further evaluations are necessary. radiography is indicated for foals with severe deviations (all tarsal valgus), ligamentous laxity, lameness, or joint effusions. ultrasonography may be valuable for selected soft tissue evaluations. conservative therapy is by far the most commonly used therapy in foals less than 30 days of age. 19 mild to moderate carpal valgus and external rotation of the carpus and fetlock are common and normal in neonates, particularly light breed horses. most congenital limb deviations improve with age, if the developing musculoskeletal system is protected from overuse and abnormal loads. approximately 90% of thoroughbred foals with congenital carpal valgus self-correct. those foals that do not most often have abnormal bone (incomplete ossification) with normal stress or normal bone with abnormal stress (ligamentous laxity or contralateral limb lameness). correction continues for several months, and on average, foals reach their straightest conformation (regarding angulation) at approximately 10 months of age (e.m. santschi, unpublished data). determination of the appropriate treatment for foals with angular limb deformities is based on the age of the foal, the severity and location of the deviation, and its causes. one must evaluate the entire foal and the affected limb. if the carpal collateral ligaments have no laxity and carpal incomplete ossification is not suspected, one may use an exercise program such as in table 19 -10, assuming that the foal has no contradicting additional problems. exercise is essential for the robust development of almost every body system for neonates, and fresh air and good ventilation reduce the occurrence of respiratory disease. appropriate limb loading along with growth and maturity is what straightens limbs, but excessive amounts of loading can be deleterious. for example, one should use exercise cautiously in foals with very asymmetric deviations. when one limb is much more deviated than the other, it appears to be loaded excessively and compromised more than if both limbs were affected similarly. and finally, limb deviations are additive. foals with external rotation and carpal valgus improve more slowly than those with one type of deviation. incomplete ossification of cuboidal bones and focal ligamentous laxity are complicating matters of great potential impact on adult conformation. they generally manifest as a moderate to severe limb deviation. physical examination indicates laxity because angular limb deviations are reducible. radiographs are the best way to evaluate the extent of carpal bone ossification. incomplete ossification of the cuboidal bones can be focal or widespread. focal immaturity is not common but can result in severe angulation. generalized immaturity is more frequent and initially often manifests as an offset conformation with valgus angulation. when the foal becomes heavier, assumes a base-wide stance, and is allowed exercise, crushing of the bones of the lateral carpus (usually the lateral styloid process of the radius, the ulnar, the fourth and the intermediate facet of the third carpal bone) results in a permanent intracarpal valgus deviation. the same result occurs when significant medial carpal ligament laxity goes untreated. in the forelimb, foals with collateral ligamentous laxity and moderate to severely immature cuboidal bones should have external coaptation placed on the affected limb to maintain axial orientation. tube casts that allow weight bearing on the digit are preferred to splints. ligamentous laxity in the carpus usually responds to tube casting for 7 to 10 days followed by bandaging and cautious exercise. the duration of similar coaptation necessary for immature carpal bones depends on the degree of immaturity and the speed with which the bones mature. because casts cannot be left on neonatal limbs for more than 7 to 10 days because of their fast growth, more than one cast may be necessary. treatment of tarsal valgus and rotational deformities is much less common than in the forelimbs because deviations are less common than in the forelimb, because some breeds prefer an outward position to the hindlimb, and perhaps because owners recognize it less frequently. 20 hindlimbs generally are unaffected by ligamentous laxity, but tarsal incomplete ossification is common and often is associated with tarsal valgus. treatment of tarsal incomplete ossification is important because tarsal crushing results in an unfavorable prognosis for athletic performance. 20, 21 hindlimbs require a slightly different approach to coaptation than forelimbs because of their anatomy. foals can rise to stand if their forelimbs are fixed in extension but cannot do so if their hindlimbs are extended. the multiple bony protuberances of the hock make cast sores more likely than in the forelimb, so casts are problematic. gutter splints are not useful because of the angle of the hock. severely limiting exercise is part of allowing the tarsus to mature without cartilage crushing, but foals cannot always be recumbent. extra small articulated anterior cruciate ligament splints for human beings (playmaker wraparound, dj orthopedics, vista, california) have given the best results. for small foals, a padded bandage is necessary under the splint, which is reversed to conform to the angle of the hock. the splints allow enough flexion in the hock for the foal to rise but appear sufficient when combined with stall rest to protect the cartilage from crushing. splints are left on the hocks until the cuboidal bones have ossified as shown by radiography. fetlock conformational deviations in neonates that are treated best conservatively are rare. outward rotation is the most common deviation but is thought to have minimal effect on the performance and improves with maturity. the only therapy used is to rasp the toe square to promote central breakover. severe outward rotation can promote a fetlock valgus conformation, so one can use a medial hoof wall extension of epoxy to bring the limb load medially. the most commonly treated fetlock deviations are inward but usually occur in foals older than 30 days. however, if the deviation is noticed in neonates, one can use small lateral hoof wall extensions that generally are made of epoxy with fiberglass cloth embedded to prevent chipping. windswept foals are born with multiple deviations. evaluating the foal as a whole is best rather than focusing on individual joints. most of these foals become straight over time with conservative therapy. no surgical procedures are commonly accepted for direct treatment of rotational or carpal offset deviations, so angular deviations are described. surgical procedures to correct carpal and fetlock valgus include periosteal transection and elevation and transphyseal bridging. periosteal elevation is thought to accelerate growth on the concave side of the metaphysis, and transphyseal bridging is used to restrict the growth on the convex side of the physis. studies indicate an approximately 80% improvement of carpal valgus foals after periosteal transection and elevation, but unfortunately they do not compare foals that had surgery with controls that did not. 22, 23 recently, some have suggested that most of the correction was unrelated to the surgery, 24 and one experimental study supports that conclusion. 25 as a result, at this time making firm recommendations about the indications for periosteal transection and elevation is difficult. however, periosteal transection and elevation has a low likelihood of complications and may be effective. the procedure is inexpensive and can be done in the field and therefore may be an option for clients with foals with carpal valgus in which a transphyseal bridging is undesirable or unnecessary. one indication is the very young foal born with a notably asymmetric epiphysis that results in a severe carpal valgus. this distal radial appearance is not particularly common, but the lack of ossification in the epiphysis can make a firm hold with a transphyseal bridging difficult to achieve. however, one can use distolateral radial periosteal elevation at an early age in an attempt to accelerate correction of the valgus and protect developing carpal bones. often a degree of anxiety exists about correction of fetlock angulations because of the much shorter time period for physeal growth. most fetlocks are in their final conformation by 60 days of age, so correction is best accomplished with earlier treatment, usually by 4 weeks of age. one can perform periosteal elevation on the medial (for varus deviations) or lateral (for valgus deviations) aspect of the distal metacarpus/metatarsus. the definitive treatment of limb angulation at a growth plate is transphyseal bridging. one should consider using the procedure at about 3 weeks of age for all moderate to severe fetlock deviations, at about 4 weeks for severe carpal deviations, and 6 to 8 weeks for mild fetlock deviations, moderate carpal deviations, and any worsening angular deformities. one must perform bridge removal when the limb straightens to prevent overcorrection. diaphyseal deviations are rare but can occur in varying degrees of severity. if the foal can bear weight on the limb, a conservative approach is indicated. one can consider periosteal elevation of the length of the concave surface of the long bone. if the foal cannot bear weight on the limb because of the severity of deviation, euthanasia is probably the best option. however, a revision osteotomy and internal fixation may be appropriate for selected foals. 26 polydactyly is also rare and sometimes can be corrected surgically. the outcome is based on the degree of articular involvement. bacteria may invade the foal musculoskeletal system and cause orthopedic infection after delivery by the circulation, by direct extension from another system, or by direct inoculation. hematogenous delivery is by far the most common and results in infection of synovial structures (joints, tendon sheaths, bursae) and bone. extension from another site without hematogenous delivery is rare. direct inoculation almost exclusively results from traumatic rather than surgical wounds. much is still to be learned about the pathophysiology of orthopedic infection, including the source of the infecting bacteria. the umbilicus commonly is accepted as a possible source of bacteria, 27 but many believe that the gastrointestinal and respiratory tracts are at least equally responsible. associated conditions in foals with septic arthritis include failure of passive transfer, pneumonia, and enteritis. 28 the classification of orthopedic sepsis in foals into infection of bones and joints is probably irrelevant because most foals with septic arthritis also have infectious osteitis or osteomyelitis. 27, 29 septic arthritis is more readily recognizable because the reactivity of the synovium to the bacteria causes joint effusion and lameness and because early radiographic signs of bone infection in foals are equivocal. also unclear are the reasons for the apparent site predilection for orthopedic infection in foals. the femoropatellar joint and the tarsocrural joint are affected most frequently, followed by the carpal and fetlock joints, and finally an assortment of miscellaneous joints such as the elbow, shoulder, and hip. 28 the common association of osteomyelitis of the distal femoral, tibial, and metacarpal/metatarsal physes with a newly recognized septic arthritis suggests that the infection in that area started at the growth center (epiphysis, physis, or metaphysis). the localization of the apparent initial site of infection to the growth center has been suggested to result from "looping" metaphyseal vessels with sluggish blood flow that allow pathogens more time to escape the circulation. 29, 30 however, transmission electron microscopy indicates that osteogenic cells and the vascular endothelium are a continuous network in developing embryos, 31 indicating that the relationship between circulation and bone is more intimate than previously suspected. a possible association between osteomyelitis and thickened or traumatized cartilage exists. focal osteomyelitis lesions occur commonly at the bone cartilage junction 27, 29 and particularly in areas where cartilage is attached at an angle to the long axis or where thickened. 29 an association also exists between incomplete ossification of the central and third tarsal bones and osteomyelitis. 32 trauma to the metaphysis is a known predisposing cause of osteomyelitis in young bacteremic rabbits. 33 a trend exists for foals with more than one joint affected to be affected bilaterally in the same joint, rather than in random joints. this trend suggests that a "window" exists when a joint may be more susceptible to infection and that trauma to the developing cartilage may be a contributing factor. in neonates, cartilage is vascular, 34 and possibly small traumatic cartilage lesions with associated hemorrhage and exposure of bacterial binding sites might be the inciting cause for the location of infection. the pathogens most commonly associated with septic arthritis in young foals are also those that frequently are implicated in neonatal sepsis. the most commonly isolated gram-negative organisms are escherichia coli and other enterobacteriaceae, actinobacillus equuli, and salmonella spp. frequently isolated gram-positive organisms include streptococcus spp., staphylococcus spp., and rhodococcus equi. 28 anaerobic bacteria and fungi are rare but should be considered in refractory cases. the diagnosis of orthopedic sepsis can be challenging. the most common clinical sign is lameness, followed by swelling around a joint or metaphysis. joint effusion alone may cause the swelling, but edema is also common, especially if metaphyseal osteomyelitis is present. but effusion and edema can be difficult to detect because of the tissue surrounding the focus of infection in the shoulder, elbow, hip, and coffin joints. one should evaluate lame foals carefully by palpation to localize pain and swelling. if one can find no pain or swelling, one should obtain a complete blood count and fibrinogen level. although a complete blood count is not always abnormal in foals with septic arthritis, abnormalities should raise the index of suspicion of infection. elevations in fibrinogen are fairly common in septic arthritis, 28 and fibrinogen almost always is elevated if the infection involves bone. if hematologic values are normal, the lameness could be caused by trauma, but the foal should be monitored closely for improvement, and closer evaluation is indicated if improvement is not rapid. an arthrocentesis is the diagnostic test of choice for confirmation of septic arthritis. one should perform joint puncture in a sterile fashion, and sedation is indicated to get an atraumatic tap. short-term anesthesia is preferable when joints have effusion because one may perform joint lavage at the same time. normal joint fluid should be clear to slightly yellow, should be viscous, and should contain less than 2500 nucleated cells per deciliter. the cell ratio should be roughly 50:50 polymorphonuclear and mononuclear. the total protein content should be less than 2.5 mg/dl. one should consider joints to be infected if the nucleated cell count is greater than 10,000 cells/dl. for joints falling between 2500 and 10,000 cells/dl, if the polymorphonuclear cell count is >90%, one should consider the joints infected. cytologists are often reluctant to diagnose infection when nuclear degeneration or bacteria are not visible. this is overly conservative and results in delay in treating infections because bacteria and nuclear degeneration are rare in early cases of joint infection. out of an abundance of caution, one should treat lame foals with suspicious joints as infected unless they are clearly normal. one should always culture joint fluid in an attempt to identify the offending organisms, but because of difficulties in culturing pathogens from joint fluid samples, absence of growth does not mean absence of infection. one obtains the best culture results if the foal has not been treated with antimicrobial agents beforehand. one should obtain as much joint fluid as possible for culture and should incubate it overnight in blood culture media before plate inoculation. as always in potentially septic foals, blood culture may assist in the isolation of the organism. other orthopedic infections that do not involve the joint may be more difficult to detect. often these are not apparent until infection breeches the joint and causes lameness. however, astute caretakers may notice early clinical signs such as mild lameness, fever, or edema centered at a growth center. radiography and advanced imaging modalities such as magnetic resonance imaging are the best diagnostic tools for the localization of areas of osteitis and osteomyelitis. one should examine the area of concern carefully, giving particular attention to the growth centers and subchondral bone. interpretation of radiographs may be difficult because these areas are complex and normally have irregular bone margins in the growing foal. if a normal contralateral joint is available, comparison radiographs may be useful. because of the high metabolic turnover in growing foal bone, changes occur faster than with adults, so radiographs at the earliest sign of potential infection of bone and joint are recommended. if evidence of osteolysis is clear, aspiration of the area may yield material for culture. the goals of treatment are to eliminate infection immediately and then resolve inflammation. bacteria and products of inflammation elicited by infection are responsible for destruction of bone and cartilage. the ultimate aim of treatment is to protect the structures critical to athletic performance such as subchondral bone and cartilage in weight-bearing areas. advances in the treatment of sepsis have resulted in hospital discharge rates of 78% for foals with septic arthritis, but their rate of high performers is 30%, 28 indicating a need for improvement. equine veterinarians cannot replace what has been destroyed, so early identification and aggressive therapies are presently the best methods to improve performance rates. one achieves the goals of treatment by physical removal of bacteria, products of inflammation, and debris and by medications to kill the bacteria and reduce inflammation. one should optimize the physiology and general health of the foal to assist this process; one should include other treatments and supportive therapies for septic foals, especially treatment of failure of passive transfer, in the therapeutic plan. intravenous administration of antimicrobials (see chapter 4) is the cornerstone of treatment of orthopedic infection, and if the drug is administered early in the course of infection and bacteria are susceptible, intravenous administration may be sufficient to eliminate the organisms. however, treatment of many foals does not begin until disease is advanced. if treatment begins after bacteria have had a chance to establish themselves, one should bring all appropriate methods to bear to end the infection. additional therapies for septic arthritis include joint lavage, arthrotomy (for drainage), 35,36 debridement (arthroscopically or arthrotomy), 37 intraarticular administration of antimicrobials, intravenous regional perfusion, 38 and antimicrobial beads. 39, 40 one can use any sterile isotonic solution to flush a joint, and additives do not appear to give significant additional benefit. if radiographs do not indicate osteomyelitis, lavage, intraarticular antibiotics, and if possible, regional perfusion are recommended. if osteitis or osteomyelitis is present, debridement is indicated arthroscopically or via arthrotomy (one should culture the debris if the pathogen is unknown). if the joint is closed, one may use antibiotics intraarticularly. if the joint is left open to drain, regional perfusion is useful. antimicrobial beads theoretically are best to use if the wound is closed, but they appear to give benefit even if the wound is open under a bandage. because of concerns about the use of beads in a joint, 41 beads often are used in tissue defects and the surrounding tissues. the major goal is to remove material that is compromising healthy tissues and to obtain high concentrations of antimicrobials in infected tissues. high antimicrobial concentrations are necessary because adhered bacteria are difficult to kill and may require many times the in vitro bacterial minimum inhibitory concentration. intraarticular administration of antimicrobials has been used for many years and has great value. 35 regional perfusion of diluted antimicrobials recently has come into use and may be administered intraosseously 42 or intravenously. intravenous perfusion is preferable because no special equipment is needed, but intraosseous perfusion may be valuable where intravenous access is impossible. the concept behind both procedures is to fill the venous vasculature in the area of the infection with antimicrobials diluted by a sterile balanced electrolyte solution. one isolates the anatomic area of interest using one or two tourniquets. the perfusate diffuses into all tissues and achieves much higher concentrations than are possible using intravenous therapy. this technique has shown excellent results as an adjunct therapy for orthopedic infection. 43 for foals, 12 to 20 ml total of perfusate containing 250 mg amikacin is useful for most single joint sites. amikacin has given consistently good results without complication and is a good choice based on its concentration-dependent activity. one may use a higher volume for the stifle, but the thigh musculature makes an effective tourniquet difficult to achieve. because of concerns that perfusion might dislodge bacteria and renew systemic sepsis, high concentrations of systemic antimicrobials are recommended at the time of the perfusion. if joint lavage and intraarticular administration of antimicrobials are not sufficient to resolve infection, one may perform arthrotomy to assist the joint to drain. passive and active drains add foreign material and so are not useful. maintaining the joint under a sterile bandage is critical and can be difficult to do in proximal joints such as the stifle and elbow. tie-over bandages can be useful in this application. the best measure of success is the resolution of lameness and local inflammation. radiographs may be helpful, but the most common sign of success is a failure of the infection to progress, rather than radiographic healing. one should continue intravenously administered antimicrobials for at least 1 week after the resolution of lameness. if an appropriate drug is available, one should give foals antimicrobials orally for at least 2 weeks more. a total of at least 4 weeks of antimicrobials is recommended for most foals with orthopedic infection. treatment failures usually result from an inability to kill bacteria adhered to isolated tissue (usually dead bone). sometimes this failure is caused by incomplete debridement or an inability to access a known site of infection, but more frequently it is because infection has flourished in an unknown site. for this reason, multiple imaging modalities (radiographs, ultrasound, computed tomography, and magnetic resonance imaging) used multiple times are recommended for all refractory cases of septic arthritis. osteomyelitis not associated with a joint still involves a growth center. the ideal treatment for these infections is surgical debridement, systemic antimicrobial therapy, and some form of local antibiotic delivery. 44, 45 even in the face of large initial osseous defects, infection may resolve, the defect may heal, and the foal may regain normal limb anatomy and 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gentamicin-impregnated polymethylmethacrylate beads use of antibioticimpregnated polymethyl-methacrylate in horses with open or infected fractures or joints: 19 cases (1987-1995) the effect of implanting gentamicin-impregnated polymethylmethacrylate beads in the tarsocrural joint of the horse regional perfusion of the equine carpus for antibiotic delivery how to perform equine digital intravascular perfusion surgical management of rhodococcus equi metaphysitis in a foal intraosseous regional perfusion for treatment of septic physitis in a 2-week-old foal key: cord-007417-az8xd66p authors: hansbro, nicole g.; horvat, jay c.; wark, peter a.; hansbro, philip m. title: understanding the mechanisms of viral induced asthma: new therapeutic directions date: 2008-01-29 journal: pharmacol ther doi: 10.1016/j.pharmthera.2007.11.002 sha: doc_id: 7417 cord_uid: az8xd66p asthma is a common and debilitating disease that has substantially increased in prevalence in western societies in the last 2 decades. respiratory tract infections by respiratory syncytial virus (rsv) and rhinovirus (rv) are widely implicated as common causes of the induction and exacerbation of asthma. these infections in early life are associated with the induction of wheeze that may progress to the development of asthma. infections may also promote airway inflammation and enhance t helper type 2 lymphocyte (th2 cell) responses that result in exacerbations of established asthma. the mechanisms of how rsv and rv induce and exacerbate asthma are currently being elucidated by clinical studies, in vitro work with human cells and animal models of disease. this research has led to many potential therapeutic strategies and, although none are yet part of clinical practise, they show much promise for the prevention and treatment of viral disease and subsequent asthma. 1. introduction asthma is thought to affect at least 300 million people of all ages and ethnic backgrounds worldwide (global strategy for asthma management and prevention, 1995) . between 1 in 5 and 1 in 10 people are affected in western societies and the prevalence has doubled since 1980 (umetsu et al., 2002; aihw, 2005) . it is now considered to be an epidemic and results in a massive economic burden to communities. exacerbations are typically caused by exposure to environmental factors to which the individual is allergic. although asthma is clearly recognised as an inflammatory condition, our understanding of the mechanisms of pathogenesis remains rudimentary. clinically asthma is characterised by airway obstruction, wheezing and episodic breathlessness in association with increased sensitivity of the airways to non-specific stimuli (termed airway hyperresponsiveness (ahr)) (bousquet et al., 2000) . wheezing is a high-pitched whistling or squeaking, which originates from the chest and is made during breathing (michel et al., 2006) . a predominant feature of disease is the acute-on-chronic infiltration of pro-inflammatory activated cd4+ th2 cells and eosinophils into the airways, which are critical regulators of pathogenesis (robinson et al., 1993; kay, 2005) . typical pathogenic features include: ige production; airway smooth table 1 important factors released by respiratory epithelium upon viral infection (dakhama et al., 2005a) nitric oxide muscle (asm) and goblet cell hypertrophy/hyperplasia; mucus hypersecretion; eosinophil, neutrophil and mononuclear cell infiltration into submucosal layer of the airways; mast cell and macrophage activation; sloughing of airway epithelial cells; and ahr (foster et al., 1996; kumar, 2001; cohn et al., 2004) . th2 cells and activated inflammatory cells release a range of mediators that damage the mucosal epithelial lining and promote an exaggerated repair response that leads to airway remodelling and chronic disease. remodelling is the result of structural changes of the epithelium, submucosal layer, asm and vasculature (angiogenesis) (bousquet et al., 2000; vignola et al., 2003) . it is thought to be a major contributing factor to the development of ahr, and its progression may lead to fixed airflow obstruction and irreversible loss of lung function (li & wilson, 1997; vignola et al., 2003) . thus, airway inflammation is closely linked to ahr and airflow obstruction and recurring inflammatory insults may result in changes that lead to airway remodelling. the mechanisms responsible for the generation of inflammation and remodelling remain poorly understood but may be induced or exacerbated by respiratory viral infection. respiratory infections by rsv, rv, influenza and parainfluenza and metapneumovirus (mpv) have all been implicated in the development of asthma as well as exacerbations. infection with rsv and rv are by far the most widely and commonly associated with bronchiolitis and childhood wheeze and the induction and exacerbation of asthma (papadopoulos et al., 2002a; xepapadaki et al., 2004) . rsv may cause earlier and more severe exacerbations and is more frequently linked to the induction of asthma whereas rv is the most common cause of exacerbations in later life zhao et al., 2002) . whether an infection induces disease depends on viral (type (e.g. rsv, rv)), host (genetic susceptibility, age, immune responses) and environmental (allergen exposure, season) factors. initial infection occurs by inhalation and spreads to the lower respiratory tract (lrt). infection is largely restricted to the respiratory epithelium, which induces the release of a wide range of mediators (table 1 ) (dakhama et al., 2005a ) that drive subsequent immune and physiological responses specific for each virus (fig. 1) . rsv and rv are the most important causes of lrt infections in infants under 2 years, causing bronchiolitis that results in wheezing, and breathing difficulties, which may in severe cases result in hospitalization (sigurs et al., 2000; kotaniemi-syrjanen et al., 2003; henderson et al., 2005; sigurs et al., 2005) . asthmatics may be more susceptible to viral infections, which lead to more severe lrt symptoms and are associated with increased hospitalization (corne et al., 2002) . notably the commonest cause of asthma related-death is respiratory viral infection (mccann & imani, 2007) . severe bronchiolitis resulting in hospitalization has been shown to be associated in fig. 1 . rsv, rv, asthma and therapy. rsv attaches to and invades the respiratory epithelium through the attachment (g) and fusion (f) proteins. major and minor group rvs bind to icam-1 and ldlprs on respiratory epithelium, respectively, which induces viral internalisation and upregulation of additional receptors. upon invasion viral proliferation leads to the induction of inflammatory cells and mediators that enhance allergen penetrance and hallmark features of asthma including inflammation, wheezing, airway obstruction and ahr. this may induce the development and exacerbation of asthma. various processes in virus-associated asthma may be targeted therapeutically. some case control studies with a history of recurrent wheeze and a diagnosis of asthma in later childhood (sigurs et al., 2000 (sigurs et al., , 2005 . rsv is the most widely implicated precipitant but recent studies suggest that rv may also be important, particularly after the age of 2 (heymann et al., 2004) . methods of detection are constantly been improved which will facilitate the elucidation of the role of these viruses in asthma (pierangeli et al., 2007) . the mechanisms that underpin virus-induced induction or exacerbation of asthma or the factors responsible for predisposing an individual remain poorly understood. it is unclear whether virus-induced bronchiolitis promotes the development of asthma or if those individuals that suffer more severely from infection are also more susceptible to asthma development as a result of genetic susceptibility (including atopy) or aberrant lung function. understanding the mechanisms of pathogenicity of respiratory viral infections and their association with asthma will be pivotal in the development of prevention and treatment strategies for asthma. indeed it may be possible to identify at risk individuals and delay infection until later in life and to develop novel therapeutic agents or targets. in asthma triggers of chronic inflammatory processes (airway inflammation, mucus hypersecretion) are overzealous responses of the asthmatic immune system to normally innocuous antigens or infections and recurrent stimulation leads to airway remodelling. respiratory viral infections induce immune responses that may have the potential to both initiate and exacerbate asthma. collectively evidence strongly implicates respiratory viral infections in the development of an asthmatic phenotype in children, although a directly causative role has still not been proven. it is possible that either 1. respiratory infection in early life induces the development of chronic airway inflammation and airway wall remodelling, resulting in persistent wheeze or 2. that some infants have pre-existing th2 responses that induces susceptibility to virus-induced wheeze (legg et al., 2003; stensballe et al., 2006) . the evidence for virus-induced asthma exacerbations is stronger than for causation and infections are responsible for the majority of acute exacerbations (80% in children, 50-76% in adults) inducing worsened airflow obstruction and symptoms wark et al., 2001; murray et al., 2004; tan, 2005) . to determine if virus infection directly influences disease in acute asthma exacerbations wark et al., recruited adults presenting to emergency with acute asthma and determined that 76% of these subjects had evidence of a viral infection (wark et al., 2002) . when compared to subjects with noninfective acute asthma, those with acute asthma and infection had evidence of more severe clinical disease, had a lower mean forced expiratory volume (fev 1 % predicted), were more likely to be admitted to hospital and had a longer median length of stay. an acute neutrophilic infiltrate was observed in induced sputum, with evidence of neutrophil degranulation, unlike the eosinophilic inflammation associated with non-infective asthma (wark et al., 2002) . in addition lactate dehydrogenase was measured as a marker of asthmatic airway necrosis. those with virus-associated acute asthma had significantly elevated lactate dehydrogenase activity, which correlated closely with the degree of neutrophil influx and airflow obstruction and was an independent predictor of the severity of the acute illness. these results strongly implicate viral infection as a trigger of exacerbations with increased severity and indicate an important role for viruses in modifying inflammation in acute asthma (wark et al., 2002) . there are many different categories and phenotypes of asthma including mild, moderate and severe as well as clinical, allergic and pathophysiologic phenotypes (wenzel, 2004) . recently simpson et al., described different inflammatory (neutrophilic, eosinophilic and paucigranulocytic) subtypes of asthma based on the predominant granulocytic cell in induced sputum (simpson et al., 2006) . the precise roles of respiratory viral infection in the development and exacerbation of the different phenotypes of asthma remain largely unknown, however, there is the potential that viral infection may be particularly important in certain phenotypes. different and customised prevention and treatment strategies may be required for different phenotypes depending on their causes. targeted anti-viral strategies may be more effective in certain asthma phenotypes for example in severe and neutrophilic asthma, where infectious agents may play a substantial role in pathogenesis (wark et al., 2002) . many factors may be involved in susceptibility to virusinduced asthma particularly virus and host factors. virus infection is the commonest cause of wheeze in children that may lead to the development of asthma (heymann et al., 2004) . the time of year also plays a role with winter the dominant period for viral infections and wheeze (heymann et al., 2004) . genetic predisposition may be important and many genes are implicated in susceptibility to asthma including those involved in inflammatory responses, ige regulation, cytokine and chemokine production, airway function and remodelling (umetsu et al., 2002) . atopy may lead to adverse responses to infection and childhood wheezing is linked to elevated ige and sensitivity to at least one inhaled allergen. however, the genetics of western populations that are experiencing the asthma epidemic remain unchanged and the focus of this review will be on other important factors that are associated with rsvand rv-associated asthma. these include the age of infection and timing of infection relative to allergen exposure, increased innate susceptibility and adaptive immune, cytokine and chemokine responses, as well as environmental conditions and inhaled bacterial endotoxin. rsv is a lipid-enveloped single stranded (ss) negative sense rna pneumovirus and is a member of the paramyxoviridae family. the virus causes the majority of cases of bronchiolitis in 317 n.g. hansbro et al. / pharmacology & therapeutics 117 (2008) to examine effect of different clinical characteristics and treatments on hospitalization of infants for bronchiolitis in outpatient clinic infants (320) b2 years presenting with 1st episode of wheezing retrospective analysis of medical records 38% of patients with rsv were hospitalized vs 10% without rsv. children exposed to tobacco smoke hospitalized more often (24%) vs not exposed (12%). treatment with oral corticosteroids associated with fewer hospitalizations in those with family history of asthma/allergic rhinitis (9.7% vs 24%) and without rsv (2.5% vs 16.7%). alshawwa and rao, 2007 early life, which may induce wheeze that may develop into asthma and is also a major precipitant of asthma exacerbations. epidemiology -rsv is the most important respiratory pathogen of children under the age of 2 years and primary infections are a common cause of lrt disease (hall, 1999; henrickson et al., 2004) . the majority of infants are infected during the first year of life, and the incidence of exposure approaches 100% by age 3 (parrott et al., 1973; glezen et al., 1986; hall et al., 1995) . these infections are the most frequent cause (50-90%) of bronchiolitis and also induce pneumonia and tracheobronchitis (10-30%) and there are annual epidemics, primarily in infants (hall, 2001) . around 100,000 children are hospitalized as a result of bronchitis annually in the usa with 50% less than 6 months and 80% less than 1 year of age with an estimated cost of $ud300 million per year (shay et al., 1999) . hospitalization for bronchiolitis has dramatically increased over the last 20 years (shay et al., 2001) , which may result from changes in childcare practises or a generalized decrease in th1 immunity in the population. mortality rates from primary infection are 0.005-0.02% for healthy or 1-3% for hospitalized children (ruuskanen & ogra, 1993; chanock et al., 1957) . most children that contract severe rsv disease have no identifiable risk factors, with the exception of premature birth. infections also cause severe disease in the elderly and immunocompromised and mild upper rt (urt) symptoms (rhinorrhea, nasal blockage, pharyngitis and cough) can occur at any age (falsey & walsh, 1998; englund et al., 1988) . re-infection is also common, occurring every 2-3 years throughout life usually resulting in mild urt symptoms. importantly this results from the lack of development of long-term resistance to rsv infection by the immune system (bont et al., 2002) . the majority of symptoms result from the host's immune and inflammatory responses to infection (openshaw, 1995) and re-infection induces sustained and exacerbated inflammatory reactions. pathogenesis -upon rsv infection and interaction of the virus with the respiratory mucosal surface the viral g protein mediates attachment and the f protein induces the fusion of the viral envelope with the cytoplasmic membrane of the host cell resulting in internalisation (fig. 1) . after invasion the viral ssrna is released into the cytoplasm and induces the production of viral rna and proteins that induce inflammatory responses. the rsv proteins and their functions have recently been reviewed by meyer et al. (2007) . the outcome of these inflammatory responses is the development of symptoms of pathogenic infection. typically, rsv infections in humans are restricted to the mucosal epithelial cells of the urt, causing runny nose, nasal congestion and cough (hall et al., 1978) . during severe rsv infections, the virus spreads to the lrt resulting in more severe symptoms. in vitro studies of human infection, as well as autopsy samples from infants and children with acute rsv infections, show that viral replication in airway epithelial cells, particularly in the superficial layer of the bronchiolar epithelium, as well as types 1 and 2 pneumocytes. infection induces the generation of inflammatory mediators and a mononuclear inflammatory response, plugging of the bronchioles with mucus, cellular debris and fibrin strands, as well as necrosis of the bronchiolar epithelium (piedra et al., 1997; johnson et al., 2007) . the lack of cytopathology during infection implicates inflammatory responses as the pivotal driver of rsv-induced disease (zhang et al., 2002) . inflammatory cells consist mainly of monocytes, t cells and neutrophils and accumulate around bronchial and pulmonary arterioles, airways and parenchyma and are associated with edema, mucus production, wheezing, airway obstruction and ahr . the induction of these disease processes may be involved in the development and exacerbation of asthma. clinical and epidemiological studies have shown that rsv infections are associated with a rapid increase in the incidence of asthma in paediatric and adult populations worldwide (wang & forsyth, 1998; tan, 2005) . these infections are also one of the commonest causes of asthma exacerbations. recent epidemiological studies that link rsv with asthma are shown in table 2 and older studies are reviewed in ogra (2004) . rsv infections are the most important risk factor for the development of bronchiolitis leading to recurrent wheezing and respiratory symptoms (decreased lung function, recurrent wheezing, allergic rhinoconjunctivitis). however, it has not yet been conclusively demonstrated that these virus-induced symptoms then progress to the development of asthma. a link between infection, atopy and sensitization to common inhaled allergens (skin prick tests (spts), ige) has also been investigated but the results are inconclusive. asthmatics may be prone to more severe infections, which may be a prognostic indicator of allergic susceptibility. between 50 and 90% of all cases of childhood hospitalizations for bronchiolitis have been attributed to rsv infections (holberg et al., 1991) . collectively studies show that rsv-induced bronchiolitis and diseases of the small airways often lead to wheezing which frequently progresses to asthma (reviewed in heymann et al. (2004) ). many studies have reported that up to 75% of subjects with rsv bronchiolitis suffer from subsequent recurrent wheeze or respiratory symptoms years later (reviewed in ogra (2004) ). an important prospective study by stein et al., showed that children with more than one lrt rsv infection were 4 times more likely to have frequent wheeze by ages 6 and 11, however the association decreased thereafter and was non-significant by age 13 (stein et al., 1999) . another smaller prospective study demonstrated that severe rsv bronchiolitis was linked to current wheezing (38%) and reactive airway disease (rad, 30%) compared to uninfected controls (2% and 3%, respectively) matched for age, sex family, history and environment (sigurs et al., 2000) . controlled retrospective and prospective studies indicate a link between rsv and bronchial obstruction and decreased lung function, particularly for children with severe rsv disease that results in hospitalization (reviewed in sigurs (2002b) ). indeed bronchiolitis is linked to chronic reductions in lung function for at least 10 years after infection (pullan & hey, 1982) and children hospitalized with rsv infection before 2 years of age have reduced lung function (but not asthma) 20 years later (korppi et al., 2004b) . furthermore airway obstruction and ahr is increased in those with rsv bronchiolitis compared to controls (sigurs, 2002b) . a nested case-controlled study also showed that rsv-induced hospitalization correlated with wheezing, lrt infections and asthma during first 4 years of life. the correlation decreased with age and was not significant at 5 years but the association held for increased respiratory symptoms and chronic productive cough at 5-8 years in alaska native children (singleton et al., 2003) . in the stein study wheezing subjects were significantly more responsive to bronchodilators, which indicates that reduced lung function results from an abnormality in airway tone (stein et al., 1999) . rsv bronchiolitis, particularly in early life, is strongly linked to the development of asthma. indeed 50-92% of children with bronchiolitis develop wheezing and asthma 3 (23% versus (vs) 1%), 5, 7.5 (30% vs 3%) or even 10 or more years later (sly & hibbert, 1989; sigurs et al., 1995; larouch et al., 2000; sigurs et al., 2000; ogra, 2004) . recent studies have found that children hospitalized with rsv have an increased risk of recurrent wheeze up to the age of 13 years, independent of atopy and other asthma risk factors, identifying rsv infection as a potential inducer of asthma. indeed these subjects have significantly more respiratory symptoms, (43% vs 8% for asthma/recurrent wheezing), sensitization to common allergens (50% vs 28% spt, 45% vs 26% serum ige), airway obstruction and ahr at age 13 years compared with controls (sigurs et al., 2005) . other recent reports have also shown that rsv infection is associated with concomitant respiratory symptoms (wheezing) and subsequent asthma at 2 years of age and that high rates of rsv are isolated from children with recurrent wheeze or asthma (lazzaro et al., 2007; lee et al., 2007) . the association was not confirmed in other longitudinal or cohort studies and may depend on individual airway structure, genetic predisposition and environmental factors (reviewed in martinez (2003) ). there may be a link between atopy, rsv infection and wheezing (sigurs et al., 2000) and risk factors may include the severity of rsv infection encountered. atopic children have low th1 responses in cord blood and elevated th2 responses compared to non-atopics. atopic adults also have th2 responses to allergens whereas non-atopics have low-level th1 responses (ogra, 2004) . elevated expression of th2 responses may promote susceptibility to rsv infection and may also lead to exaggerated airway inflammation and reduced lung function. however, the link between rsv infections and atopy is controversial and was not found in other studies (stein et al., 1999; kneyber et al., 2000) . early rsv infections (first 2 years) may induce allergic sensitization to unrelated antigens in genetically predisposed individuals (sigurs et al., 1995; forster et al., 1996) . indeed rsv bronchiolitis in the first year of life leads to increased ige levels (33% vs 2% in controls) and is the most important risk factor for allergic sensitization and recurrent wheeze (16% vs 4% controls) (schauer et al., 2002) . furthermore allergic sensitization and asthma are significantly more common in all age groups in children with a prior rsv bronchiolitis (23% vs 2% and 41% vs 22% of controls, respectively) (sigurs, 2002a ). it appears that the severity of infection may be important and severe infection has been associated with the development of allergic sensitization 3, 6 or 7.5 years after hospitalization (sigurs et al., 1995 (sigurs et al., , 2000 . mild infection, does not appear to be a risk factor for allergic sensitization (stein et al., 1999; sigurs et al., 2000) . however, the data are conflicting and severe infection has been shown not to induce sensitization (at age 2-10 years) by other investigators (pullan & hey, 1982; carlsen et al., 1987; noble et al., 1997) . pre-existing th2 or impaired th1 responses and asthmatic predisposition may promote susceptibility to acute bronchiolitis and hospitalization for rsv infection (legg et al., 2003; stensballe et al., 2006) . furthermore, individuals with damaged airway epithelium or that are otherwise immunologically predisposed may be additionally susceptible to infection enhancing epithelial damage and causing a vicious cycle of disease perpetuation. although rsv infection has been extensively linked with inducing asthma and reduced lung function it is possible that infection is also a marker of susceptibility to allergic and/or infectious respiratory disease. it has been shown that although rsv infection is the greatest risk factor for wheezing and asthma in subjects with a family history of asthma, asthma does not develop in those without infection or a family history (sigurs et al., 2000; sigurs, 2001) . this indicates that rsv bronchiolitis is a marker of increased risk of asthma susceptibility. more recently it has been suggested that the host response to infection rather than the nature of the infection itself is the best prognostic indictor for subsequent allergic disease (everard, 2006a (everard, , 2006b . atopy may also be a risk factor for susceptibility to more severe rsv infections and exacerbations, which result in higher rates of mortality and hospitalization (jhawar, 2003) . a large nested case-controlled study demonstrated that maternal atopic dermatitis and maternal and paternal asthma (rr 1.72 and 1.23, respectively) were risk factors for rsv hospitalization in infants b1.5 years (stensballe et al., 2006) . taken together studies show that rsv infection in early life results in bronchiolitis that leads to wheezing and decreased lung function, which may progress to asthma. these processes may be enhanced in atopic individuals with elevated th2 responses. moreover there is evidence, although not conclusive, that severe infection may also promote sensitization to allergens, which may further increase the risk of wheeze and asthma. asthmatics may be susceptible to more severe infections, which may be used as an indictor of susceptibility to the development of asthma. experimental rsv infection of humans has been used to investigate the mechanisms of pathogenesis of infectious disease and the association with asthma. these studies have demonstrated that the virus persists in nasal washes for 5-14 days (noah & becker, 2000) and that infection induces bronchiolitis and airway inflammation. this promotes epithelial sloughing, mucus hypersecretion and an increase in viscosity and edema which in turn lead to hyperinflation of the lungs, airflow obstruction, cough and wheeze (holt & sly, 2002a) . symptoms may persist and resolution of tissue damage may take several weeks or results in structural remodelling of the airway wall and airway narrowing (pare et al., 1997; holt & sly, 2002a) . the mechanisms of rsv pathogenesis that lead to wheezing, ahr, allergy and asthma are still not well understood and many studies have implicated a range of different factors. these include: the age of first infection; type of innate and adaptive responses elicited; mucosal damage and repair involving remodelling (including angiogenesis) and; enhanced neurogenic stimulation leading to asm spasm and bronchoconstriction. according to the hygiene hypothesis (strachan, 2000) , the th2-biased immune system of the newborn must encounter th1-inducing agents during childhood in order to develop the ability to mount a th1 response. however, whether infections in infancy induce beneficial th1 responses depend upon the type of infection and the mechanisms responsible for these effects have not been characterised. thus the immaturity of the developing immune system during early life and the nature of immune responses to infections may be significant determining factors in the development of persistent wheeze and asthma. whether rsv infection induces immunological and pathological processes that may lead to asthma may depend on the age at which an individual is first infected. the immune phenotype of early life may lead to enhanced viral replication and th2-dominated inflammation induced by infection or allergen exposure. increased levels of il-4 were detected in rsv infected infants less than 3 months old compared to more than 3 months but the converse was true for eotaxin and there was no difference in mip-1î² or eosinophil cationic protein (ecp) (kristjansson et al., 2005) . in mothers infection induced high levels of ifn-î³ and low levels of il-4 whereas newborn infants produced 4-7 times less ifn-î³ and higher levels of il-4 but at age 3 these levels approached those of their mothers (mbawuike et al., 2001) . the immune response in early life may be involved in the induction of more severe lrt pathology in response to primary infection but infection of older children is not as severe and involves largely urt symptoms. an additional pathological consequence is that viral infections in early life may generate pulmonary inflammation during the development of the lung, small and large airways and immune, inflammatory and neuronal programing (reviewed in gern et al. (2005) ). this may result in altered pulmonary structure and immune responses leading to enhanced pro-inflammatory and th2-biased immune responses that may precipitate deleterious changes in lung structure and function. furthermore, the small size of neonatal bronchioles determines that they may become obstructed more readily, which may result in reduced clearance and confer enhanced severity of pathogenic infection in this age group. the combination of these events may have longterm effects on lung function, chronic respiratory inflammation, remodelling, alveolarization and epithelial dysfunction (gern et al., 2005) and consequently may promote the development of asthma. however, whether rsv infection induces th1 or th2 responses in humans in early life is debateable. another alternative explanation for the association of rsv infection in infancy with asthma is that early life infection induces th1 memory and therefore cd8+ ctl responses, which have the potential to induce pathologic immune responses to reinfection. rsv infection induces viral specific cd8+ cells in infants and the levels of cytotoxic lymphocytes (ctls) are inversely proportional to il-4 responses. ctl responses are directly linked to protection against infection, ctl memory is initiated upon reinfection and mhc i cd8+ levels correlate directly with ifn-î³ levels (mbawuike et al., 2001) . immune responses induced to rsv enhance viral clearance but are also implicated in disease pathogenesis. ineffective or aberrant innate and adaptive immune responses against rsv have been widely linked to more severe and recurring infections and the development and exacerbation of asthma in both adults and children (glezen et al., 1986; hall et al., 1991) . primary infection, particularly early in life, leads to an incomplete immune response, which does not elicit the development of sustained memory immunity. with respect to allergy rsv infection might only trigger defective immunity in genetically susceptible individuals or that allergic inflammatory and immune responses may promote the influx of virus-specific cells into the airways increasing inflammation and ahr (schwarze et al., 1999c) . elucidation of the pivotal immune responses that are protective against rsv will lead to a better understanding of the processes that result in bronchiolitis, wheezing and progression to asthma. innate responses to rsv infection have not been widely studied but may play an important role in rsv-induced asthma. rsv infection of the respiratory epithelium induces innate cellular and cytokine responses, which have substantial effects on adaptive t cell development and cell-mediated immune responses. neutrophils are the main immune cell in the bronchoalveolar lavage fluid (balf) of patients with severe rsv lrt disease and increased il-8 levels, which is a potent chemoattractor of neutrophils, are also a prominent feature. macrophages internalise and process virus and present antigens to adaptive immune cells and also release il-12 and il-10. monocytes release il-1, -6, -8, -10, platelet-activating factor (paf) and pge2 on exposure to rsv that further promote proinflammatory responses (schaller et al., 2006) . eosinophils are also recruited to the airways during primary rsv infection, which may contribute to the development of allergic airways disease (schwarze et al., 1999a) . infected patients also have more plasmacytoid dendritic cells (pdcs) and myeloid dcs (mdcs) in the rt and reduced numbers of these cells circulating in blood (gill et al., 2005) . this suggests that they are recruited to the lung during infection and may be an important target in rsv vaccine development. understanding the interplays between these different cells types will be crucial in elucidating the effects of infection on the development of asthma. ifn-î± is an important type i ifn innate cytokine that is released upon viral infection and induces innate and adaptive cellular responses. blood cultures of asthmatic children and adults release significantly reduced amounts of ifn-î± upon rsv infection, which indicates a systemic innate deficiency in asthmatics that may lead to heightened susceptibility to infection (gehlhar et al., 2006) . 2.2.4.1. th1/th2 responses. rsv has several t cell epitopes and infection induces cd4+ and cd8+ and th1 and th2 adaptive cellular responses as well as pro-and anti-inflammatory cytokines and chemokines in humans both in vivo and in vitro (meyer et al., 2007) . established infections are primarily cleared by a combination of th1 and th2 cell responses and the balance between the two may be crucial in determining the outcome of infection, the severity of rsv-induced disease and predisposition to asthma. aberrant responses of either of these subsets may induce pathology. nevertheless most studies suggest that th1 responses may result in viral clearance and mild symptoms whereas an aberrant bias towards a th2 phenotype may lead to more intense rsv-induced disease and promote the development of asthma . indeed individuals with elevated th2 responses are predisposed to reduced viral clearance and more severe disease compared to subjects with only urt infection independently of age or viral load (roman et al., 1997; aberle et al., 1999; legg et al., 2003; gern et al., 2006) . early life rsv bronchiolitis results in enhanced il-4 and ifn-î³ responses to rsv in later childhood (pala et al., 2002) and il-4 is the most important stimulus for th2 development and ige production. th2 responses are enhanced during severe disease that develops upon natural infection of rsv-vaccinated infants (kim et al., 1969; kapikian et al., 1969) and severe rsv bronchiolitis correlates with elevated humoral th2 responses, and may be linked with atopy, increased il-4:ifn-î³ and reduced th1 (ifn-î³, il-12 and il-18) responses. rsv infections may also have persistent immunological effects and induce long-term th2 memory responses during sensitization to inhaled allergens in childhood (holt & sly, 2002a ; van rijt et al., 2005) . other studies have demonstrated that mild bronchiolitis is associated with a shift towards th1 responses and is usual in most individuals, and also that there is no increase in th2 responses in severe bron-chiolitis . these contrasting results may be attributable to differences in the timing of sampling during infection, the lack of definitive detection of rsv or determination of virus load, age and atopic status of individuals. 2.2.4.2. cytokines and immunomodulatory molecules. cytokines of the th1, th2 or regulatory type are implicated in rsvinduced asthma and the effect of pro-inflammatory cytokines and chemokines released in response to infection may have particularly important roles. th2 (il-5) cytokines are upregulated in children with acute asthma and children with acute bronchiolitis or il-5 levels have higher numbers of eosinophils (martinez, 2003) . these responses may play key roles in the progression from bronchiolitis to asthma, however, the importance of eosinophils and il-5 in rsv-induced asthma has not yet been confirmed. il-12 is a th1 cytokine that promotes the development of th1 cells, the release of ifn-î³ and il-2 from th1 and natural killer (nk) cells and suppresses th2 responses. levels of il-12 may be reduced in subjects that are more susceptible to infection. non-specifically stimulated whole blood cultures from patients with rsv disease released significantly less il-12 than controls and il-12 levels inversely correlated with disease severity (bont et al., 2000b) . il-12 and ifn-î³ responses were suppressed during acute rsv disease but returned to control levels during convalescence and ifn-î³ (and il-4) levels were not different in subsequently wheezing infants (bont et al., 2000a) . reduced il-12 levels may lead to elevated susceptibility to th2 responses to rsv infection and predisposition to asthma. il-10 is a regulatory cytokine, which may promote an asthma phenotype by suppressing th1 cytokine production and antigen presentation promoting enhanced susceptibility to infection and th2-dominated responses that induce wheezing and pro-asthmatic responses to subsequent antigen challenge (bont et al., 2000a (bont et al., , 2000b . il-10 levels did not change during acute rsv disease but increased during convalescence and were significantly higher in subsequent wheezers and those that went on to develop recurrent wheeze and asthma. thus, the enhancement of il-10 and inhibition of il-12 production upon rsv infection may suppress immune function and permit more severe infection and disease progression. it has also been suggested that rsv infection may change and enhance the profile of pro-inflammatory cytokine and chemokine release that promotes more severe infection and alters the nature of the t cell response promoting allergy and inflammation. patients with rsv-induced bronchiolitis had elevated levels of mip-1î± (but not rantes), which correlated with disease severity in nasopharyngeal secretions and rantes, icam-1, il-4 and -5 and ige in serum (sung et al., 2001; garofalo et al., 2001 garofalo et al., , 2005 . furthermore treatment of human epithelial cells with tgf-î², which plays a pivotal role in airway remodelling and asthma, increased rsv replication and tnf-î± secretion and p38 mitogen-activated protein kinase activation. this may contribute to the elevated inflammatory responses in virus-associated asthma (mccann & imani, 2007). by contrast rsv-induced wheezing does not correlate with il-8 levels in nasal lavage fluid but is elevated with influenza or rv infection (gern et al., 2002) . thus it is possible that infection induces regulatory mechanisms that suppress immune responses allowing viral replication resulting in increased inflammatory responses. alternatively infection may induce inflammatory responses that enhance infection and allergic inflammation. this may subsequently promote the induction of regulatory mechanisms to limit inflammation-induced damage of host tissue. 2.2.4.3. other immune factors. in vivo and in vitro studies have shown that rsv infection of the respiratory epithelium causes the release of other factors that have immune functions including arachidonic acid metabolites and lts, mediators released by eosinophils and chemokines (reviewed in ogra (2004)). cell adhesion and homing molecules such as cd11b, icam-1 and e-selectin and antigen-presenting molecules including human leucocyte antigen classes i and ii are also upregulated upon infection. several transcription factors are also activated, which may induce the expression of a range of genes such as nk-il-6 and nuclear factor (nf)-kb. these factors regulate immunomodulatory mediators (tnf-î±, il-1î², il-2, -6, -11 and gm-csf), adhesion molecules (icam-1, vcam-1 and e-selectin) and chemokines (il-8, mip-1î±, mcp-1, eotaxin and rantes) (garofalo et al., 1996; oh et al., 2002; john et al., 2003; makela et al., 2003; schaller et al., 2006) . in combination these mediators and molecules induce the influx of inflammatory cells and may contribute to the development of infection-induced inflammatory and immune responses, ahr and asthma. ineffective or aberrant humoral responses may also have a role in rsv-induced asthma. infection induces increases in b cell numbers (roman et al., 1997) and the production of serum and mucosal igm, iga and igg antibodies. these are important in protection and not disease but occur at lower levels in infants. antibody responses to primary infection are ineffective and involve the production of partially neutralising antibodies against the g and f proteins but these responses are reinforced (especially igg and iga) upon reinfection. rsv-specific ige antibodies are also produced (welliver et al., 1980) in the majority of children and increased amounts and persistence promote the development of wheezing (ogra, 2004) . increased vascularity (angiogenesis) surrounding the airway wall is associated with chronic and fatal asthma but also with mild-to-moderate asthma in both children and adults (li & wilson, 1997; vrugt et al., 2000; barbato et al., 2006) , suggesting a pathogenetic role at all stages of asthma development. angiogenesis is regulated by a balance between pro-angiogenic (vascular endothelial growth factor (vegf), basic fibroblast growth factor (bfgf), angiogenins, chemokines) and antiangiogenic (endostatin, canstatin, tumstatin, arresten) factors (cohen, 2002) . vasodilation of the increased number of blood vessels in response to inflammatory stimuli may lead to edema and inflammation of the bronchial wall (influx of inflammatory cells, including eosinophils and release of mediators), airway narrowing and ahr (black & page, 1994; wilson, 2000; salvato, 2001; nomura et al., 2005) . elevated levels of vegf, bfgf and angiogenins occur in the airways of asthmatics and correlate with increased vascularity, vessel permeability and ahr (hoshino et al., 2001; kanazawa et al., 2004; nomura et al., 2005; feltis et al., 2006) . vegf influences vascular permeability through the formation of blood vessel fenestrations and vasiculo-vasculo organelles (dvorak et al., 1996; esser et al., 1998; neufeld et al., 1999) . vegf also stimulates endothelial cell proliferation and migration, matrix remodelling, and vasodilation, as well as inhibiting endothelial cell apoptosis and all of these processes are involved in angiogenesis (neufeld et al., 1999) . vegf also enhances sensitization of the rt to allergens and promotes th2 inflammation . rsv infection may contribute to the development of asthma by inducing the production of vegf and angiogenesis. vegf has been detected in nasal washings from rsv infected patients, indicating that infection stimulates vegf production, which may have a role in disease (lee et al., 2000) . furthermore, features of rsv bronchiolitis and pneumonia such as submucosal, adventitial, and interstitial edema are due to alterations in blood vessel permeability brought about by vegf activity (dvorak et al., 1996; esser et al., 1998; neufeld et al., 1999) . the specific role of vegf in rsv infection has not been intensively investigated. it is released from airway epithelial cells, th2 cells and mucosal fibroblasts upon infection (lee et al., 2000) but is also produced by monocytes, macrophages, t cells, keratinocytes, granulocytes, eosinophils and smooth muscle cells (gaudry et al., 1997; horiuchi & weller, 1997; neufeld et al., 1999) . thus a novel mechanism may be involved in rsv-induced asthma whereby the induction of vegf upon infection may lead to the development of angiogenesis that can enhance the inflammatory response. furthermore, rsv-induced secretion of vegf may contribute to exacerbations by increasing vascular permeability, and recurring infections may contribute to cycles of vegf production that promote angiogenesis and remodelling. neurological and immunological interactions that occur as a result of rsv infection have also been linked to the generation of airway inflammation, ahr and rad in children. excitatory non-adrenergic non-cholinergic nerves (nance) release neurotransmitters including substance p that participate in the early phase of the inflammatory response and also have an immunomodulatory role (piedimonte, 2002) . sloughing of the epithelium during infection may lead to exposure of neurogenic receptors (nk1), which enhances the pro-inflammatory effects of substance p leading to asm spasm and bronchoconstriction and contributing to respiratory symptoms. nerve growth factor participates in neuronal development and has beneficial effects on inflammation, repair and remodelling. however, it has been suggested that these effects may become pathogenetic during rsv infection and allergic inflammation and exacerbate inflammation and ahr (nassenstein et al., 2006) . the effect of co-infection of with rsv and other viruses has been little studied, however, notably the risk of developing bronchiolitis is 5 times greater in infants with co-infection with rv (papadopoulos et al., 2002a) . animal models of infection and allergic airways disease (aad) have been developed and used extensively to substantially contribute to the understanding of the mechanisms that underpin rsv-induced asthma and exacerbations. in particular rodent (mouse and to a lesser extent rat) and bovine models have been used to elucidate the mechanisms of the associations and to trial therapeutic agents and vaccines. chimpanzees are permissive to human rsv and are the best animal model but their availability and cost limits all but the most advanced clinical tests (whitehead et al., 1998) . using these models important factors have been identified that may play key roles in rsv-induced asthma and include; age of first infection, timing of infection relative to allergen exposure, induction of asthma, endotoxin exposure, innate factors and adaptive immunity, suppression of immunity, angiogenesis, neural networks and latent infections. the importance of the different viral proteins in infection has also been investigated. the use of animal models enables experimental protocols to be conducted that are not possible in humans. in particular the precise investigation of the different ages of infection, combinations of the timing of infection relative to allergic sensitization and collection of invasive tissue samples can only be achieved in animals. although there are problems with these models of rsv challenge including that rsv does not replicate in mice and does not induce the recruitment of granulocytes that is observed in human disease, such models have been used extensively to gain valuable insights into the mechanisms of rsv-induced disease. in most mouse models rsv infection induces significant acute respiratory inflammation and changes in lung function. high doses (10 7-8 plaque forming units (pfu)) induce severe alveolitis and pneumonia and low doses (10 5-6 pfu) result in bronchiolitis without these effects (dakhama et al., 2005a) . five days after infection mice develop acute airway obstruction, which correlates with the progression of inflammation and histopathology that is characterised by intense perivascular and peri-bronchial/bronchiolar influx of monocytes/ macrophages and some neutrophils and lymphocytes. these cells also occur in the alveoli during the peak of inflammation but leakage into the airway lumen is absent (jafri et al., 2004) . infection peaks at days 4-5 and resolves between days 7 and 9 and cytokines and chemokines (il-8, mips and rantes) are released by the respiratory epithelium and alveolar macro-phages in response to infection. after 3 days nk cells influx into the balf and are replaced between 4 and 8 days by cd4+ and cd8+ cells which return to pre-infection levels at 21 days (hussell & openshaw, 1998) . ctls induced in response to infection contribute to extensive peri-bronchiolar and -alveolar inflammation and are associated with disease symptoms (ostler et al., 2002) . ifn-î³ is important in viral clearance and contributes to pathology and is primarily released by nk cells with levels peaking at day 4. only low levels of il-4 and il-5 are present, however, il-4, -5, -10, -12, -13 and ifn-î³ are produced in the lungs within the first few days and their relative levels determines the course of disease (kalina & gershwin, 2004) . il-10 plays a pivotal role in the development of infectioninduced ahr in the absence of allergen exposure (makela et al., 2002) . acute infection develops into chronic disease with features of chronic inflammation (histopathology score) and ahr (in terms of enhanced pause (penh)) after the clearance of virus and up to 22 weeks after infection . penh is a noninvasive method of whole body plethysmography and uses a single exposure to a spasmogen to determine the overall level of ahr in all airways. jafri et al., used penh to show that airway obstruction and ahr remained for 42 and 154 days after infection, respectively, and that ahr correlated with chronic inflammation which persisted but not in alveoli (jafri et al., 2004) . this supports the concept that rsv disease may induce long-term respiratory changes in children (stein et al., 1999; sigurs et al., 2005) . however, these studies need to be confirmed by the measurement of lower airway resistance using invasive methods that precisely measure changes in airway and tissue specific function and employ dose responses to spasmogens. studies using these methods (measurement of respiratory impedance and airway resistance) have shown a lack of longterm effects of rsv infection on lung function (dakhama et al., 2005c; collins et al., 2007) . infection also induces mucus hypersecretion in the central and peripheral airways in the acute and chronic phases and severe and progressive pneumonia develops with increases in histopathology and chronic inflammatory changes. these processes contribute to airway obstruction (penh) that develops in the acute phase and progresses but does not correlate with viral load in balf, and this agrees with observations made in children (jafri et al., 2004) . the intensity of inflammation declines over time but remains around airways and vessels. neutralising monoclonal antibodies (mabs) against rsv substantially decrease inflammation and disease severity . using mouse models it has been shown that the age of first infection plays a key role in shaping dominant immune responses later in life (reviewed in hansbro et al. (2004) ) and establishes the subsequent pattern of th cell responses and the nature and severity of ensuing respiratory diseases (culley et al., 2002; holt & sly, 2002a; horvat et al., 2007) . primary rsv infection in neonatal mice has the same profile as in adults, however, it is associated with a slower and diminished ifn-î³ response and the development and persistence of th2 cytokine release by cd4+ t cells. these effects are substantial and can reverse the protective effect of mycobacterial exposure on aad . neonatal rsv infection results in early tnf-î± release and has long-term adverse effects on the respiratory system. these effects include the induction of ahr, peri-vascular and -bronchial inflammation and subepithelial fibrosis, which are exacerbated by subsequent allergen exposure and involve persistent il-13 expression and mucus hypersecretion (you et al., 2006) . the generation of th2 responses during immunological development has profound modulatory effects on the balance of subsequent th1/th2 responses and promote a marked increase in the th2 phenotype in adulthood (chen et al., 2001; walzl et al., 2001; culley et al., 2002) . the neonatal th2 bias may result from the types of t cells or dendritic cells present, their environment or a combination of these effects (nelson et al., 1994; goriely et al., 2001; white et al., 2002; bartz et al., 2003) . subsequent re-infection later in life may reinforce aberrant th2 responses (cytokines (il-13) and cd4+/cd8+ cells) and alter responses to allergens resulting in enhanced inflammation, mucus hypersecretion, ahr and allergy (enhanced weight loss and th2 cell and eosinophil recruitment to the airways) (chen et al., 2001; walzl et al., 2001; culley et al., 2002; dakhama et al., 2005b) . this suggests that aad of adults primed with an infection as neonates is likely to be caused by factors that promote th2 responses (culley et al., 2002; holt & sly, 2002a) . if initial infection is delayed until mice are 3 weeks of age ifn-î³ production increases and upon reinfection, although airway inflammation still occurs, there is a subsequent reduction in the severity of disease with no mucus production or ahr (culley et al., 2002; dakhama et al., 2005b) . thus delaying the age of infection until later in life may be an effective strategy for the prevention of rsv-associated asthma. animal models have been used to determine if rsv can induce the development of asthma by triggering pro-asthmatic immune responses that lead to variable airflow obstruction and airway inflammation. chavez-bueno et al. (2005) demonstrated that rsv induces acute and chronic disease with features of aad independently of allergic sensitization or genetic background in mice. rsv infection of balb/c or c57bl/6 mice in the absence of allergic sensitization led to similar levels of acute airway inflammation, airflow obstruction and ahr and the degree of airway inflammation correlated with ahr. the immune response was surprisingly similar between the two strains and was characterised by virus-dependent release of ifn-î³. importantly infection and inflammatory responses were not short lived. acute infection developed into persistent infection that correlated with airway inflammation, which were present 77 days after infection. while virus load and airway inflammation were greatest during the acute phase, chronic infection correlated with chronic inflammation and airflow obstruction. thus rsv infection can initiate acute events that result in airflow obstruction and possibly ahr and these changes can persist beyond the initial inflammatory response. other investigators have also shown that rsv infections in early life act synergistically with atopy to drive the development of allergic asthma (holt & sly, 2002b; kusel et al., 2007) . however, the immunological processes involved in the induction of allergic sensitization by rsv infection are not likely to be the same as those involved in exacerbation of allergic asthma by rsv. the effect of rsv infection on aads may be critically dependent on the relative timing of infection, allergic sensitization and challenge (peebles et al., 2001; barends et al., 2002 barends et al., , 2004 . the majority of studies in mice show that rsv augments aad, however, conversely some investigators suggest that rsv infection prevents atopy. indeed rsv induces a strong th1 and ifn-î³ mediated response that may modulate responses to allergens (peebles et al., 2001; juntti et al., 2003) . these contrasting observations can be explained as two competing immune responses are occurring simultaneously, which may subtly differ in different protocols. the development of allergy may depend on the phenotype of the immune response to allergens and rsv at the time of exposure. 2.3.4.1. allergen exposure prior to infection. prior exposure of the airways to allergen independently of allergen type predisposes to increased severity of virus-induced aad in mice and is likely to play a role in humans (peebles et al., 2001; makela et al., 2003; kalina & gershwin, 2004) . infection enhances th2 cytokine responses, mucus secreting cell hypertrophy, eosinophil influx into the lung and ahr in response to allergen and increases the severity of aad. the potency of the th2 responses elicited overrides the counterregulatory effects of th1 responses that are typically induced by infection (randolph et al., 1999) . 2.3.4.2. infection prior to allergen exposure. exposure of rsv infected mice or rats to allergens increases inflammation, mucus production and ahr and prolongs rsv replication (peebles et al., 1999; kalina & gershwin, 2004; hassantoufighi et al., 2007) . in particular cd4+ and cd8+, inflammatory responses in the lung are enhanced (lukacs et al., 2001; barends et al., 2004; schaller et al., 2006) . blocking il-13 during infection reduces chemokine expression and ahr and blocking rantes removes the effects of infection upon later allergen sensitization and challenge (lukacs et al., 2001) . the absence of the cc chemokine receptor 1 (ccr1) in deficient mice leads to reductions in t cells, il-13, eosinophils, mucus and ahr (john et al., 2005) . these observations suggest that chemokines and their receptors play roles in rsv-induced aberrant responses to allergens and may be important in mobilisation of virus-and allergen-specific t cells and allergic inflammation. it is possible that rsv infection may contribute to the development of allergy by damaging the respiratory mucosa, which exposes apcs and t cells to allergen, which may break tolerance and induce systemic th2 sensitization. excessive infection-induced respiratory damage may occur in predisposed individuals as a result of defects in t cell mobilisation, activation or activity. if allergen challenge of sensitized mice occurs concomitant with infection the inflammatory response is again enhanced and leads to th2 responses to rsvand promotes chronic infection (makela et al., 2002) . il-5 production leads to eosinophilia, il-13 to the release of mcp and further th2mediated inflammation but il-10 does not further enhance ahr. if rats are sensitized to extracts of the common household mould aspergillus fumigatus, which induces eosinophilia and th2 cytokine release, rsv infection before allergen challenge exacerbates the inflammatory response and ahr that is dependent on viral replication. infection causes increased expression of mhc ii on alveolar macrophages, which may be involved in initiating immune responses to allergens. persistence of infection is induced and is related to reduced ifn-î³ expression again suggesting that allergic sensitization can affect the progression of rsv infection (kalina & gershwin, 2004) . treatment of infected a. fumigatus challenged animals with recombinant ifn-î³ reduced allergic responses and th1 and th2 cytokines but not ahr. this suggests that pathological and physiological factors of disease are independent (hassantoufighi et al., 2007) . taken together these studies suggest that in general rsv induces increased severity of th2-mediated aad and that the development of aad increases the severity and persistence of rsv infection. some reports have suggested that exposure to endotoxin or environmental pollution can affect the progression of rsv infection (gurkan et al., 2000; monick et al., 2003) . exposure to such factors alters the relative proportions of cytokines produced upon infection and affects disease progression, however, the cellular and molecular processes involved are not understood. tlr-4 expression may provide a link between rsv, endotoxin and asthma. tlr-4 expression is not usual in resting airway epithelium and requires high endotoxin exposure to be upregulated. rsv infection induces increased expression of tlr-4 and responsiveness to endotoxin and initiates potent inflammatory responses (monick et al., 2003) . this may be linked to human asthma as endotoxin also induces asthma exacerbations in children with rsv-induced asthma (park et al., 2001) . animal models have been used extensively to elucidate the host immune responses that are induced by rsv rt infection and how these responses may contribute to the development and exacerbation of asthma. infection may inhibit or modulate the activity of both innate and adaptive (particularly t cell) immune responses during the development of disease, which may play a crucial role in the induction of pathology and aad. primary rsv infection induces innate responses that involve eosinophil and neutrophil influx into the lung that results in ahr and a cytokine response that is dominated by ifn-î³ (schwarze et al., 1999b) . eosinophil infiltration is il-5 but not il-4 or ifn-î³ dependent and is critical for the development of ahr. the innate response (first 3 days) is also characterised by an influx of nk cells producing ifn-î³, which are replaced by adaptive cd4+ and cd8+ cells and the release of il-12 with low levels of il-4, -5 and -13 (openshaw, 1995; boelen et al., 2000; van schaik et al., 2000; openshaw, 2001) . t cell responses facilitate viral clearance but also induce host tissue damage and pathology. infection induces innate responses involving tlrs, cytokines, chemokines and dcs that ultimately direct the development of adaptive t cell and antibody responses (durbin & durbin, 2004; krishnan et al., 2004) . 2.3.7.1. tlrs. rsv may use a variety of host cell factors to bind to and enter cells including tlr-4, cx3r1, heparin and caveolin (kalina & gershwin, 2004) . binding to tlr-4 initiates the production of il-6, -8 and -1î² and tnf-î± and may be responsible for the initial response to infection. however, the role of tlrs in virus-induced asthma is controversial. some studies report that the innate immune response to rsv infection in mice is dependent on the expression of cd14 and tlr-4 (kurt-jones et al., 2000; haeberle et al., 2002) , which may interact with the viral f protein (openshaw et al., 2003) . another report argues the opposite saying there is no significant role for tlr-4 in infection (ehl et al., 2004) . tlr-3 recognises double stranded (ds) rna and is constitutively expressed on respiratory epithelial cells and dcs. tlr-3 signals independently of myeloid differentiation factor 88 to induce nf-îºb activation and the expression of ifnî² activation of tlr-3 leads to apoptosis and elimination of infected cells and virus. recently it has been shown that tlr-3 and protein kinase r (pkr) are upregulated in human airway epithelial cells by rsv infection, which enhances epithelial responsiveness by activation of nf-îºb and il-8 and may sensitize these cells to subsequent viral or bacterial infection (groskreutz et al., 2006) . 2.3.7.2. chemokines. chemokines are produced by stromal, epithelial and immune cells and regulate immune responses (chemoattraction of leukocytes into the lung), inflammation, mucus production and angiogenesis. although cellular inflammation is often similar in response to different viral infections the types of chemokines and the levels that are released that drive subsequent immune responses differ substantially (schaller et al., 2006) . primary rsv infections induce the expression of chemokines belonging to the cxc (il-8, mip-2 and ip-10), cc (rantes, eotaxin, mip-1î±, mcp-1 and t cell activation gene-3) and c (lymphotactin) families in the lung. mip-1î± expression is high and mip-1î± deficient mice have reduced lung inflammation, but rsv titres that are the same as in wildtype mice. thus rsv-associated lung inflammation may be mediated by early production of inflammatory chemokines (haeberle et al., 2001) . 2.3.7.3. dcs. dcs are the most important antigen-presenting cell (apc) and take up viral antigens, traffic to local lymph nodes and present antigen to naã¯ve t cells causing their differentiation into effector cells. dcs direct innate and adaptive immune responses to viruses and allergens and are essential for allergic sensitization. different subsets of dcs exist with different functions. mdcs present viral antigens and promote th2 cell expansion to a great extent than pdcs, which are more important in the development of tolerance (van rijt et al., 2005) . dc networks are less active in infant animals but can be enhanced and mdcs are increased upon viral infection (holt & sly, 2002b; zuniga et al., 2004) . it is possible that the reduction of pdcs by conversion into mdcs during early life viral infection may inhibit the development of tolerance and exacerbate allergic responses. the outcome of rsv infection may depend on the nature of the adaptive response and the balance of th1 and th2 immunity. primary infection of balb/c mice induces a mixed th1/th2 response with an early burst of ifn-î³ release that is important in determining the phenotype of the subsequent response openshaw & tregoning, 2005) . other adaptive responses involving cd8+ cells and b cell/antibody responses also play significant roles in responses to rsv. 2.3.8.1. th1 cd4+ t cell responses. rsv infection typically induces a robust th1 response with elevated levels of ifn-î³ and il-12 in mice. ifn-î³ is the archetypal th1 cytokine and its release and signalling through the ifn-î³ receptor during infection are pivotal in controlling the th1/th2 response to infection. these processes are essential in moderating eosinophil migration and ifn-î³ and cd8+ cells are crucial for viral clearance. in the absence of ifn-î³ a dominant th2 response induces eosinophil influx of the lung and ahr (barends et al., 2003) . by contrast, the absence of il-12 and il-18 has little effect . il-12 does, however, induce th1 and suppresses th2 responses, promotes ifn-î³ production from nk and cd8+ cells, reduces il-4 and il-5 production from cd4+ and cd8+ cells and can prevent but is not essential for inhibiting virus-induced eosinophil influx to the lung (hussell & openshaw, 2000) . il-12 does not function through cd4+ or b cells and exacerbates disease in mice sensitized to allergen (openshaw et al., 2003) . the removal of cd4+ or the induction of cd8+ cells also eliminates eosinophil influx (hussell et al., 1997) . tnf-î± is another th1 cytokine and is over-produced during viral rt infections, which exacerbates inflammation by promoting neutrophil and eosinophil influx. anti-tnf-î± treatment of mice leads to ablation of weight loss and illness without affecting viral clearance and does not induce adverse side-effects, which indicates a potential for use in therapy (hussell et al., 2001 ). 2.3.8.2. th2 responses. th2 responses are induced by rsv infection in a variety of animal models or in the absence of ifn-î³ , which may contribute to the development of aad. these responses are potent and are similar to th2 responses observed after allergen exposure of allergic individuals (braciale, 2005) . rsv-induced ifn-î³ and il-12 responses do not diminish the th2 response during the development of rsv-induced allergy although the response is even greater in the absence of ifn-î³ (barends et al., 2003) . it is a specific set of t cells, a cd4+vî²14+ subpopulation, that induces a superantigen type response to rsv and is pivotal in inducing th2 mediated pathology (varga et al., 2001) . t1/st2 is a surface receptor of the il-1 family expressed on th2 but not th1 cells. t1/st2 was present on a subset of cd4+ t cells from mice with rsvinduced eosinophilia and t1/st2 mab treatment reduced th2 but not th1 pathology (walzl et al., 2001) . these studies suggest that under some circumstances infection may induce strong th2 responses but the mechanisms of how this leads to aad are unknown. il-4, -5, -10, -11 and -13 are th2 cytokines that are released during th2 responses and are likely to be involved in rsv-induced aad. in mice, primary rsv infection results in increased il-4 levels, however, it's importance in the development of th2 responses and aad is unclear. il-4 deficient mice or mice treated with a neutralising anti-il-4 and immunized with vaccinia virus expressing protein g had no reduction in pulmonary eosinophils or th2 cytokine secretion. furthermore infection of il-4 deficient mice resulted in enhanced numbers of eosinophils in the lung and ahr johnson et al., 2003) . by contrast il-5 release may be pivotal in rsv-induced th2 responses and aad. infection of mice results in il-5-mediated ahr and eosinophil influx into the lung in association with strong ifn-î³ responses. il-5 but not il-4 or ifn-î³ is the critical mediator of eosinophil influx, ahr and allergy (schwarze et al., 1999a) . il-5 deficiency leads to a reduction in pulmonary eosinophils and ahr, which can be reversed by replenishment with il-5. treatment with anti-very late antigen-4 prevents the influx of eosinophils into the lubg and ahr in response to rsv infection or il-5 replenishment. rsv infection induces the expression of il-10 in mouse pulmonary t cells (hussell et al., 1996) . the absence of il-10 inhibits the development of ahr in response to allergen sensitization and challenge. rsv infection overcomes this deficiency and induces eosinophil infiltration of the lung, airway mucus production and ahr, which are associated with increased th2 responses (makela et al., 2002) . il-11 is associated with the development of rsv-induced ahr and may promote the release of other th2 cytokines (einarsson et al., 1996) . although il-13 is required for the development of mucus hypersecretion and ahr in mouse models of aad and after secondary rsv infection of infected neonates (kuperman et al., 2002; dakhama et al., 2005b) it does not appear to play an important role in the induction of these responses after primary infection . this has not yet been investigated in humans. it is likely that as well as inducing a th2 phenotype rsv may take advantage of th2 responses that predominate in asthmatics that are ineffectual against infection. however, surprisingly rsv clearance can occur under th2 conditions whereby eosinophils take up rsv and inactivate the virus with ecp (soukup & becker, 2003) . il-10 increases fasl expression on macrophages and cd8+ cells and therefore also has an anti-viral effect (ruan et al., 2001) . cd8+ t cells target several rsv proteins and are sufficient to clear rsv from infected mice (cannon et al., 1988; cherrie et al., 1992) , however, clearance and immunopathology still occurs in cd8-deficient mice (graham et al., 1991) . adoptive transfer of virus-specific cd8+ ctls which home to the lung eliminates rsv. these cells induce viral clearance through perforin/ granzyme-mediated lysis of virus-infected cells (aung et al., 2001) . however, perforin (which is also produced by nk cells), cd95l and tnf are not necessary but ifn-î³ release is crucial for cd8+ t cell-mediated clearance. rsv was eliminated with unchanged kinetics from perforin deficient mice (aung et al., 2001; ostler et al., 2002) , whereas treatment of mice with neutralising antibody to ifn-î³ or transfusion of ifn-î³-deficient effector ctls abolished virus control and induced cd8+ t cellmediated pathology (ostler et al., 2002) . by contrast, high dose primary infection in ifn-î³ deficient mice led to attenuated immunopathology, but only slightly delayed clearance. this suggests that other cells and molecules can partially substitute for ctl-derived ifn-î³ driven virus clearance and further implicates ifn-î³ as a pivotal immune factor in rsv-induced immunopathology and cd8+ t cell-mediated control (ostler et al., 2002) . cd8+ cells may also suppress the development of virusspecific th2-dominated immune responses and eosinophilia although the mechanism of these effects remains unknown. hussell et al., showed that early ifn-î³ release by cd8+ cells in response to rsv infection of mice resulted in suppression of th2 responses (hussell et al., 1997) and the suppression of th2 responses by cd8+ cells is by other studies (srikiatkhachorn & braciale, 1997a) . interestingly the th2-inducing effects of the rsv g protein can be nullified by incorporating a cd8+ epitope onto the g protein (srikiatkhachorn & braciale, 1997a ). 2.3.10. cd4+/cd8+ interactions cd4+ and cd8+ t cells are exposed to presented viral antigens in the lymph nodes of the respiratory tract, which induces differentiation, activation and mobilisation of both effector and memory t cells. memory cd4+ t cells move to the rsv infected rt and proliferate and differentiate into cytokine releasing effector cells and induce their effects in situ (varga et al., 2000 (varga et al., , 2001 . during differentiation the cells are subject to infectioninduced modulation and it is likely that the cd8+/cd4+ interaction is occurring concurrently. similar interactions may take place during allergen provocation in the rt of asthmatics that have the potential to substantially affect t cell phenotype, an effect that may be enhanced upon infection. thus the complex interactions of cd4+ and cd8+ with infectious stimuli and allergens may be pivotally important in the development of rsvinduced aad. these processes may be targeted therapeutically to suppress allergen specific th2 responses in the lung. differential immune responses are induced by different rsv proteins, which may be important in the development or exacerbation of asthma. protein g vaccination of mice induces cd4+ but not cd8+ memory populations and leads to reduced nk cell influx and ifn-î³ production. this results in the induction of th2 responses, in the absence of a ctl response, with il-4 and il-5 release by th2 cells which promotes eosinophilia upon subsequent infection (alwan et al., 1994; srikiatkhachorn & braciale, 1997a; walzl et al., 2001; openshaw et al., 2003) . g protein defective rsv has been used to demonstrate that this protein is crucial in promoting airway inflammation and reduced lung function (schwarze & schauer, 2004) . f or m2 protein immunization induces a mixture of nk, th1 type cd4+ and mhc i cd8+ cells, leading to ifn-î³ production and reduced disease (alwan et al., 1994; srikiatkhachorn & braciale, 1997a; openshaw et al., 2003) . nk cells and the ifn-î³ they release regulate the induction and proliferation of cd8+ cells, which then clear the virus. primary rsv infection induces the expansion of activated m2 82-90 (h-2k d restricted peptide epitope in rsv m2 protein)-specific cd8+ t cells in lung. this implies that activation and proliferation of m2-specific cd8+ t cell precursors is normal. taken together these results suggest that natural infection that induces th2 responses may be dominated by responses to the g protein exacerbates infectious and allergic disease, whereas responses to the f and m2 proteins may be th1 mediated and reduce disease. antibody responses may have several roles in rsv-associated aad in mice. rsv infection may induce the development of proallergic ige antibodies and their receptors in the lung which may induce mast cell degranulation and ahr (dakhama et al., 2004) . the activation of anti-viral protein kinase upon infection may lead to isotype switching of b cells to produce ige (rager et al., 1998) . other studies have shown that exposure to allergen during acute rsv infection of mice results in the production of antigen specific igg1 responses, which are characteristic of th2 immunity (o'donnell & openshaw, 1998) . moreover non-neutralising antibody produced in response to formalin-inactivated (fi) virus may induce immune complex formation in the lung (openshaw et al., 2003) , which may be involved in pathogenesis. infants have poor t cell independent antibody responses and produce different types of antibodies compared to adults. early life infection with rsv may fix the nature of antibody production as well as t cell responses to subsequent infection throughout life and promote the development of ahr (openshaw et al., 2003). studies in mice have suggested that rsv may promote its own infection by suppressing host immunity resulting in enhanced inflammation. the rsv g protein may attenuate innate responses by binding to tnf-î± (valarcher & taylor, 2007) and the induction of cytokine production from monocytes and macrophages through the inhibition of tlr-4-nf-îºb mediated signalling (polack et al., 2005) . rsv-specific t cells may play a crucial role in viral clearance and limited evidence suggests that infection may suppress the activation and activity of t cells leading to enhanced viral replication and disease (harcourt et al., 2006) . during suppression the f protein of rsv may downregulate the activity of t cells and cytokine production including the release of ifn-î³ (kondo et al., 2004; schauer et al., 2004) . rt infection also suppresses ctl immunity through the rapid loss of virus-specific cd8+ memory cells and ifn-î³ release (chang & braciale, 2002) , which is mirrored in humans by the lack of the induction of immunological memory. this effect may occur during antigen receptor signalling and varies to different extents in different t cell functions (ctl activity, cytokine release). this may result in reduced ctl activity and allow viral persistence but may also enable the maintenance of cytokine responsiveness facilitating further virus-or allergen-associated inflammation promoting enhanced disease. despite this evidence immune suppression during infection has not yet been observed in humans. these effects may only be present in the lung and are not detectable in the blood (braciale, 2005) and it is unknown if cd8+ t cells with this phenotype exist in the rt of humans with severe rsv infection. rsv infection may also attenuate protective immune responses by suppression of type i ifn (ifn-î±,î²), modulation of dc activity, g protein mimicry of the cx3c chemokine, which may inhibit t cell migration and by producing viral variants that are not recognised by neutralising antibodies (tripp, 2004; meyer et al., 2007) . the viral elements responsible for dysregulated immune responses are not known. persistent rsv, other viral or bacterial infections, which are local or systemic, or underlying chronic lung disease may promote susceptibility to rsv-induced asthma. emerging evidence from both mouse and human studies suggests that rsv infections may persist at low levels, by mechanisms involving suppression of immunity and avoidance of immune detection and total clearance (seemungal et al., 2001; . this occurs in mice with functional neutralising antibody and ctl responses even though the virus possesses a cd8+ epitope and productive infection can be reactivated by depletion of cd4+ and cd8+ cells . in mouse models rsv is detectable by culture up to just 7 days after infection but by pcr for up to 77 days independently of genetic background and viral copy number correlates with ahr (tripp, 2004; chavez-bueno et al., 2005) . this association has not yet been proven but treatment with neutralising antibody reduces viral numbers and disease severity . however, it is possible that viral detection by pcr under these conditions may represent the persistence of viral debris following the administration of high viral inocula and may not be part of the disease process. it remains unknown whether viral persistence occurs in children following bronchiolitis and the development of wheezing and long-term pulmonary abnormalities. if persistent infection does occur chronic inflammation and altered immune (cytokine/ chemokine) responses may be induced in individuals with rsvinduced aad. thus persistent infection may be important in long-term morbidity and may provide a new therapeutic target. limited mouse and primate studies of acute and chronic asthma also link vegf with pathophysiological features of rsv infection and asthma suzaki et al., 2005; avdalovic et al., 2006; lee et al., 2006) . over-expression of vegf in the lungs of mice to levels found in asthma or during rsv infection, induces angiogenesis, oedema, inflammation, vascular remodelling, mucus cell hyperplasia/metaplasia and ahr . rat models of rsv-induced bronchiolitis have also been developed, where rats rapidly clear the virus in a similar manner to the self-limiting infection of human infants (piedimonte et al., 1999) . this model has been used to investigate the alteration of neural networks by rsv infection and infection of infant rats has enabled the analysis of the effects of early life infection (king et al., 2001) . a stronger neurogenic inflammatory response develops in the lrt of infant rats than in adult rats during infection, which may explain why bronchiolitis presents in infants but as an urt infection in older subjects. induction of inflammation involves the upregulation of nk1 in infected lungs (but not nk2 that is expressed on asm fibres) that are activated by nance nerves and mediate substance p induced immunomodulation and neurogenic and cellular inflammation that may lead to edema and obstruction (king et al., 2001; piedimonte, 2001; tripp et al., 2002) . nk1 receptors are also upregulated on t cells in bronchus-associated lymphoid tissue in response to infection, which may then be attracted into the airways and release pro-inflammatory cytokines in response to neurogenic stimulation by airborne irritants. recurrent stimulation may lead to persistent cycles of airway inflammation and obstruction. inhibition of nk1 or the administration of cgrp prevents rsv-induced ahr and may be potential therapeutic targets (dakhama et al., 2005c) . neurogenic stimulation by rsv infection also induces the release of lts from mast cells that induce mucus secretion (wedde-beer et al., 2002) . another alternative is that infection-induced damage may expose nerve endings and substance p and neurokinin a may then mediate asm contraction (jacoby, 2002) . other investigators have shown that rsv infection of rats and ferrets results in increases in cholinergic mediated contraction of asm and reduced inhibitory nance responses (larsen & colasurdo, 1999) . despite the vast array of literature describing studies of rsv infections in animal models, there is no single model which duplicates the pathological features of disease observed in human infection. the major drawback in studies of rsvinduced disease in mice is that rsv is not a natural mouse pathogen. symptoms induced upon infection are minimal compared to those observed in human infants, the virus has limited replication and infected animals show few if any signs of respiratory illness (domachowske et al., 2001) . furthermore, large doses of the virus are required and primary infection is rapidly aborted . such discrepancies between human disease and mouse models have severely hampered the investigation of rsv-induced disease. the pneumonia virus of mice (pvm) is the closest genetic relative of rsv and is a natural mouse pathogen (easton et al., 2004) . unlike rsv, pvm infection of mice reproduces many of the acute inflammatory responses described for rsv infection in humans. pvm replicates rapidly inducing inflammation leading to mucus plugging of the airways and overt signs of disease from urt symptoms to fatal pneumonia, which is dependent on the administered dose easton et al., 2004) . virus replication is associated with an influx of granulocytes and severe inflammatory bronchiolitis. we have recently established mouse models of rsv-like disease using pvm infection of neonates and adults . importantly neonatally infected mice also exhibit many of the symptoms observed during rsv disease of human infants harrison et al., 1999; rosenberg et al., 2005) . these models are now being utilised to more precisely elucidate the host pathogen relationships that result in rsvinduced asthma. the importance of inflammation in inducing disease has been demonstrated and inflammatory responses remain active after the cessation of viral replication. mip-1î± and its receptor ccr1 play pivotal roles in these inflammatory responses. a recent study using pvm in wild-type and tlr-4 deficient mice showed that there is no difference in the clinical, functional, histological and virological parameters investigated indicating that pvm infection is independent of tlr-4 signalling (faisca et al., 2006) . anti-viral therapy with ribavirin alone has little effect, however, treatment with ribavirin in combination with the anti-inflammatory agent and the ccr1 antagonist met-rantes substantially reduces morbidity and mortality following infection (rosenberg et al., 2005) . bovine rsv, pathogenesis and vaccine development have recently been reviewed (meyer et al., 2007; valarcher & taylor, 2007) . bovine rsv is closely related to human rsv and is the most common cause of lrt disease and the major single health problem in calves worldwide, particularly in winter (stott et al., 1980; valarcher & taylor, 2007) . indeed 60-70% of epizootic respiratory diseases in the first year of life are attributable to bovine rsv with mortality typically between 2 and 3% but reaches 20% in some outbreaks (meyer et al., 2007) . models of bovine lrt infection have been developed and used to investigate responses to infection and evaluate bovine vaccines. bovine rsv is a natural pathogen and pathogenic infection of cattle shares many similarities with rsv infection in humans (van der poel et al., 1994) . infection is largely restricted to respiratory epithelial cells but causes little cytotoxicity and pathology is mediated by inflammatory responses to infection (viuff et al., 2002; valarcher & taylor, 2007) . these responses also involve innate (neutrophil and macrophage recruitment) and adaptive (pro-inflammatory cytokine and chemokine release) immune responses that result in respiratory damage. the mechanisms of pathology have been investigated using genetic manipulation of the virus and have implicated the g and sh proteins in infection and the f protein and non-structural (ns) proteins in inflammatory responses (reviewed in valarcher & taylor (2007) ). 2.3.18.1. pathogenesis. bovine rsv is transmitted by direct contact, in airborne droplets or is possibly transferred passively by humans (hall et al., 1980; mars et al., 1999) . pathogenic infection of the rt is much more common in calves than adults (stott et al., 1980) , which results from a lack of specific immunity in naã¯ve animals. maternally-derived antibodies afford some protection but primary infection induces the most effective immunity (kimman et al., 1987) . infectious disease takes 2-5 days to develop and has similar clinical features to humans and may possibly result in persistent infection (valarcher et al., 2001) . disease of the urt induces mild symptoms of coughing and mucus production. in the lrt bronchiolitis and bronchopneumonia occur in association with edema, wheezing and dyspnea (verhoeff et al., 1984; belknap, 1993) . infection of cattle induces inflammation involving the influx of mononuclear cells, neutrophils, cd4+ (of mixed th1/th2 phenotype) and cd8+ cells and sometimes eosinophils into peri-bronchial regions along with necrosis and apoptosis of epithelial cells (viuff et al., 2002; antonis et al., 2006) . cd8+ ctls have a major role in the clearance of primary infection in calves and lymphocyte proliferation is attenuated by infection in vitro (keles et al., 1998; antonis et al., 2006) , whereas antibody-mediated responses are important in protection against secondary infection. the lumen of the airways become occluded with mucus and inflammatory debris and remodelling events occur that lead to breathing difficulties (kimman et al., 1989; viuff et al., 2002) . it is likely that similar immune responses are elicited as in human infection with rsv and involve the induction of nf-kb and the generation of pro-inflammatory cytokines and chemokines. the interaction of viral components (f protein and dsrna) with tlrs is known to drive the development of the immune response in cattle (reviewed in valarcher & taylor (2007) ). as in humans and mice there are age-related differences in immune responses to infection in cattle with reduced protective and enhanced inflammatory responses in younger animals. in response to primary infection younger calves have enhanced fever, virus-specific tnf-î±, il-6 and ifn-î³ release from pbmcs and reduced peripheral blood mononuclear and b cells and virus-specific iga and neutralising antibody responses (grell et al., 2005) . the immunology and pathogenesis of bovine rsv infection has been intensively studied using reverse genetic engineering of the virus, which is less variable that human rsv and the results may be extrapolated to human disease (collins et al., 1995) . the importance and roles of different bovine rsv proteins in the induction of pathogenesis and immune responses have been elucidated using these genetically manipulated viruses. the g protein of rsv is the major attachment protein and deletion mutants do not replicate in the absence of the g proteins in vivo, although replication is unaffected in vitro (karger et al., 2001; schmidt et al., 2002) . bovine viruses expressing only the secreted segment of the g protein also have 100 fold attenuated replication, which is 10 fold higher than mutants missing the entire g protein (teng et al., 2001) . mutants expressing the membrane-anchored g protein segment have no impairment of replication in the urt but were attenuated at least 10 fold in the lrt and did not induce pathogenic responses in the lung (maher et al., 2004) . this suggests that both fragments of the g protein are important in infection and pathogenesis with the secreted portion playing a particularly significant role. cleavage of the bovine f protein is required for activation and results in the production of the peptide and tachykinin, virokinin that induces the production of nks, substance p and other neurogenic pro-inflammatory mediators. virokinin itself induces asm contraction and therefore directly contributes to airflow restriction but does not have chemotactic properties (zimmer et al., 2003) . by contrast cleavage of human rsv does not produce tachykinins (valarcher et al., 2006) . deletion of various parts of the bovine f protein has shown that it is not necessary for proliferation in vivo but deficient mutants promote substantially reduced airway inflammation and eosinophil influx (valarcher et al., 2006) . the f protein is responsible for suppressing lymphocyte proliferation (schlender et al., 2002) , however, there is no effect on the expression of the eosinophil attractants rantes or mip-1î± in the absence of the f protein suggesting that other mechanisms may be involved in the suppression of eosinophil influx in cattle (valarcher et al., 2006) . ns proteins, particularly ns2, of bovine rsv downregulates the ifn-î±/î² response involving the attenuation of irf3 and stat-2 (schlender et al., 2000) , which has subsequently also been shown with human rsv (spann et al., 2004 (spann et al., , 2005 lo et al., 2005) . the lack of ns proteins results in highly attenuated replication and a lack of pathogenesis in vitro and in vivo (whitehead et al., 1999; valarcher et al., 2003) . the sh protein is not required for replication of bovine rsv in vitro (karger et al., 2001) but replication is attenuated in the lrt of chimpanzees and calves in vivo (whitehead et al., 1999; valarcher & taylor, 2007) . epidemiological studies have widely linked rsv infection to the development of bronchiolitis, wheeze, decreased lung function and possibly asthma in childhood. this association is equivocal later in life but may depend on the severity of the infection. there may also be a role for rsv in the induction of sensitization to allergens and allergy may predispose to more severe infection. enhanced severity of symptoms that result from infection may be a useful prognostic marker for the future development of allergic disease. both human and animal studies have investigated the mechanisms of how infection induces pathogenesis and how this is associated with asthma. the age of first infection is crucial in determining clinical outcomes and early infection may promote the development of a pro-allergic phenotype. this indicates that delaying the age of infection until later in life may be an effective therapeutic strategy. infection induces both innate and adaptive immune responses that promote th2 immunity and eosinophil influx into the lung that may contribute to the development of ahr. the timing of infection relative to allergen exposure and the immune phenotype of the individual may be significant in determining the effects of infection. an interesting novel concept is that rsv infection may induce the development of angiogenesis, which might exacerbate inflammatory exudation, edema and bronchial obstruction in asthma. other new hypotheses are that rsv may induce immune suppression allowing viral persistence and latent infections that can be reactivated. neural networks also have the potential to be involved through the induction of neurogenic inflammation. rvs are small non-enveloped ssrna viruses of the picornaviridae family and to date over 100 different serotypes have been identified (savolainen et al., 2002) . epidemiology -rvs are responsible for the majority of cases of urt infectious disease in humans particularly colds and have recently also been implicated as the cause of significant numbers of lrt infections. rv is also the most common respiratory viral pathogen that induces wheeze at all ages (kusel et al., 2006) . a third of all episodes of acute respiratory illness involve wheezing in both children and adults and rv infections are implicated in 3 times as many cases as other viruses. wheezing may then progress to the development of asthma. the mechanisms of pathogenesis of rv are much less well understood than for rsv. pathogenesis -rvs are divided into major and minor classes based on receptor binding properties. major group rvs bind to icam-1 while minor group viruses bind to very lowdensity lipoprotein receptors (ldlpr) (fig. 1) (dreschers et al., 2007) . binding of rv to ldlprs induces a conformational change in the capsid that is required for viral uptake. intracellular internalisation occurs through the activation of sphingomyelinase that generates ceramide-rich membrane rafts, which facilitate uptake. rv is not thought to replicate in lrt, however, it is possible that rv can infect the lrt to a certain extent or alternatively that immune responses to urt infection has knock-on effects on inflammation in the lrt. infection of the urt typically causes coryza and pharyngitis and virus can be detected in fluids from the nasopharynx, tonsils and the middle ear (holgate, 2006) . recent epidemiological studies correlating rv with asthma are shown in table 3 . rv is more strongly linked with exacerbations of asthma although it is emerging that rv may also be important in asthma induction. there is a strong association between rv infection and childhood wheeze and exacerbation of asthma that may lead to hospitalization (el-sahly et al., 2000; jartti et al., 2006) . rv is implicated in wheeze in 80% of children n3 years of age and 45-80% of adults and is responsible for 60% of cases of asthma exacerbations nicholson et al., 1997; atmar et al., 1998; freymuth et al., 1999; tan, 2005) . interestingly the peak of severe asthma exacerbations in children occurs shortly after their return to school after breaks and in adults one week later and this coincides with peaks in rv infections in the spring and fall (longini et al., 1984) . indeed in the fall rv was present in 29% and 52% of children that were asymptomatic or were having asthma exacerbations, respectively . while rsv may be an important factor in early childhood wheeze, the majority of children become infected with rsv, but most do not go on and develop asthma, suggesting that other factors are important in this process. to investigate this lemanske et al. (2005) prospectively recruited a cohort of children (285) at risk of asthma based on family history and followed them for 3 years to determine the relationship between viral infections and symptoms of asthma, confirming infective episodes and etiology by pcr on nasopharyngeal aspirates. at 3 years non-infective factors such as exposure to environmental tobacco smoke, the presence of asthma in an older sibling and ige-mediated food allergies were associated with persistent wheeze. rsv infections were also linked with an increased risk, however the strongest independent predictor of persistent childhood wheeze was an episode of rv infection associated with wheeze (or = 10, 63%). this suggests that rv infection in lemanske et al., 2005 infancy is associated with the development of asthma later in life. others agree and have shown that rv infection is the primary risk factor for wheezing in children under 3 years but that atopy is more important thereafter (heymann et al., 2004) . other recent studies have also indicated that recurrent wheezing in infants is promoted by rv and possibly to a greater extent than rsv (lehtinen et al., 2007) . a pathophysiological link between rv and asthma is indicated by the tendency of children infected with rv to be older than those infected with rsv (13 months vs 5 months) and an increased frequency to present with atopic dermatitis and blood eosinophilia (korppi et al., 2004a) . in the study by korppi et al., children (81) hospitalized for wheezing before the age of 2 were assessed by bronchoscopy and had a prior median symptomatic period of 6 months. of these children 40% were found to be asthmatics by the age of 3 years, and 90% of these were atopic. early predictors of asthma were atopic dermatitis, specific ige and inhalant allergy and rv was common during wheezing (58%). another recent study of infants (192) under the age of 3 hospitalized with bronchiolitis identified rv and rsv in 21% and 30% of cases, respectively (jacques et al., 2006) . it is unknown if asthmatics are more likely to develop colds, or if they are at a greater risk of more severe colds. corne et al., attempted to investigate this by recruiting couples, one of whom had asthma and determining if the asthmatics were more at risk of developing colds. they found that those with asthma were no more likely to develop colds (10% vs 9% of controls), but they were substantially more likely to develop lrt symptoms associated with these colds (43% vs 17%). this implies that viral infections in asthmatics are more likely to increase lrt inflammation (corne et al., 2002) . other investigators have shown that asthmatic infants hospitalized with wheezing have a greater chance of testing positive for rv than non-asthmatics, which suggests that asthma sufferers may also be more susceptible to infection (xatzipsalti et al., 2005) . furthermore, rv infection may be more long lasting in asthmatics and may persists for up to 6 weeks after hospitalization for acute asthma exacerbation and rv-induced ahr may also last for several weeks (kling et al., 2005) . experimental rv infection of human volunteers has enabled the study of the effects of infection on immune responses, lung function and asthma as well as the role of atopy and timing of infection on disease outcomes . infection upon binding of rv to icam-1 (greve et al., 1989) not only contributes to pathogenesis but also to the induction of allergic inflammation (canonica et al., 1995) . rv also induces the upregulation of icam-1 by activation of nf-îºb, which promotes further infection (papi & johnston, 1999) . rv infects the bronchial epithelial layer and some mononuclear cells and fibroblasts and increases airway inflammation, with changes observed in induced sputum and endobronchial biopsy. the bronchial epithelium of asthmatics is particularly susceptible to the cytotoxic effects of viruses (papadopoulos et al., 2000; mosser et al., 2005) . initial infection initiates rv-induced exacerbations and cytotoxicity leads to increases in the penetrance and effects of allergens. 3.2.1.1. immune responses. in both asthmatics and non-atopic non-asthmatics inflammatory changes involving almost all types of inflammatory cells are observed upon rv infection. in mild to moderate asthma, infection induces increased levels of il-8 and neutrophils in sputum, promotes the influx of eosinophils, mast cells, cd4+ and cd8+ cells into the airway wall and enhances ahr (fraenkel et al., 1995; grunberg et al., 1997a; van benten et al., 2001) . de kluijver et al., showed the inflammation upon infection may be less intense in the nonasthmatic, although these differences were small and no direct comparison with asthmatics was made at the time (de kluijver et al., 2002) . nevertheless reduced ifn-î³ responses in subjects with asthma may promote susceptibility to infection and there is an inverse correlation between ifn-î³ levels and rv persistence and symptoms in asthmatics . this deficiency may result from either the lack of recruitment of appropriate inflammatory cells or a reduced response from these cells. other differences in immune responses to rv infection have also been observed that may be involved in increased susceptibility of asthmatics to infection or enhanced inflammation. in subjects with acute rv-induced asthma wark et al., found that increased serum ip-10 levels but not rantes or il-8 was specifically associated with infection and correlated with the degree of airflow obstruction (wark et al., 2007) . these subjects also had less bronchodilatory response (increase in fev 1 ) to salbutamol compared to non-infective acute asthma, which correlated with levels of ip-10 in serum. ip-10 is a chemokine that binds to cxcr3 and is important in the recruitment of t cells to infected tissues. increased levels of ip-10 can activate mast cells and cause their migration. mast cells that are resident in the asm are specific for asthma and also express cxcr3. therefore, rv infection of the bronchial epithelium may lead to an early release of ip-10 that sets off a chain of events that enhance pre-existing asthmatic airway inflammation and promotes the migration and activation of mast cells into the asm thereby worsening bronchoconstriction and reducing the response to bronchodilators. nasal secretions of experimentally infected healthy volunteers contain increased levels of il-1î², a pro-inflammatory cytokine (proud et al., 1994) , while rv infection of subjects with allergic rhinitis or asthma, or in children with virusinduced asthma, resulted in elevated levels of g-csf and il-8 as well as increased blood and nasal neutrophilia (teran et al., 1997; gern et al., 2000) . have demonstrated that rv infection worsens airway inflammation and obstruction and enhances ahr to non-specific bronchoconstrictors in subjects with asthma or atopy. however, despite showing an ability to infect these subjects and recovery of virus, significant exacerbations of disease are not observed (grunberg et al., 1997a) . while treatment with inhaled corticosteroids (icss) effectively reduces ahr and even airway eosinophilia, it has no effect on virus-induced airway inflammation. this suggests that in the acute phase there is a disparity between these features of disease. epidemiological studies also link rv infection with reduced lung function. for example, a recent study detected rv in the respiratory epithelium in 45% of infants with recurring respiratory symptoms (201, 3-26 months) which was associated with abnormal lung function (decreased airways conductance, 86%) compared with rv negative infants (58%) (malmstrom et al., 2006) . it is possible that the serotype of rv involved may be important and some strains such as rv16 are particularly associated with reduced lung function and increased ahr and symptoms of asthma and rhinitis (peebles & hartert, 2000) . to determine whether atopy influences infection and worsening of ahr, xepadaki et al., studied a group of asthmatic children dividing them by atopy (xepapadaki et al., 2005) . in both groups ahr increased 10 days after a cold and remained elevated for 5-11 weeks. where the groups differed was that atopic children experienced more symptomatic colds and acute exacerbations, leading to a cumulative affect that resulted in the persistence of ahr. therefore, increased susceptibility of atopics to infection was demonstrated and recurring inflammatory insults were implicated as an indirect cause of more severe ahr. 3.2.1.4. timing of infection. as with rsv the timing of infection relative to allergen exposure may also be important in determining the outcome of rv infection in asthma. the severity of asthma exacerbations increases if infection occurs simultaneously with allergen exposure in asthmatics (green et al., 2002) . however, infection after allergen exposure has little effect on ahr (de kluijver et al., 2003) . numerous important studies have used a variety of in vitro cell culture systems to provide valuable insights into the pathogenesis of rv and its association with asthma. specifically the role of cytotoxicity, immune responses, ahr and remodelling involving angiogenesis have all been implicated. importantly it has now become clear that viral infection directly influences lower airway inflammation in asthma and that asthmatics may have a defective innate response in bronchoepithelial cells (becs) to rv. the bec is the first point of contact between infecting viruses and the host and plays an important early role in initiating innate and adaptive immune responses and inflammation (bals & hiemstra, 2004 ). there is conflicting evidence surrounding the nature of the cpe of rv infection on epithelial cells. while some studies have found that rv induces considerable cpe albeit with serotype dependent variation (schroth et al., 1999; papadopoulos et al., 2000) , others report none or very little cpe in vitro or in vivo (winther et al., 1984 (winther et al., , 1990 ). the magnitude of immune responses as well as differential regulation of different innate and adaptive components has been implicated in the increased susceptibility of asthmatics to rv and in rv-induced asthma and exacerbations. upon rv infection of asthmatics more intense inflammatory reactions involving mucus production, the influx of neutrophils, eosinophils, activated macrophages, cd4+ and cd8+ t cells into the rt and pro-inflammatory mediator release are associated with more severe and persistent lrt involvement and symptoms and delayed repair edwards et al., 2006; inoue et al., 2006) . although there are similarities in the cellular immune responses to rsv and rv infection there are significant differences in the levels and types of cytokines and chemokines released and the cell types that produce them. ineffective presentation of viral antigens also occurs in asthmatics, which may impair immunity and lead to asthma symptoms (parry et al., 2000) . many authors have used epithelial cell culture models to demonstrate that rv induces the release of pro-inflammatory mediators and it is possible that differential regulation of these factors may be important in rv-associated asthma. rv infection promotes the production of oxygen radicals in the bronchial epithelium and leads to the increased expression of nf-îºb and enhanced pro-inflammatory responses (contoli et al., 2005) . the activation of p38 mitogen-activated protein kinase by rho a has been shown to play an important role in these processes (dumitru et al., 2006) . rv infection of human cell lines relevant to the rt and asthma has demonstrated that the production of many different pro-inflammatory cytokines, chemokines and molecules is induced including; eotaxins 1 and 2, il-1î², -4, -6, -8, -16, ena-78, ip-10, gm-csf, tnf-î±, rantes, mcp-1, mip-1î± and icam-1 (subauste et al., 1995; grunberg et al., 1997a; johnston et al., 1998; yamaya et al., 1999; gern et al., 2000; papadopoulos et al., 2000; suzuki et al., 2001; papadopoulos et al., 2001; hosoda et al., 2002; konno et al., 2002; gern et al., 2003; hall et al., 2005; edwards et al., 2006) . of particular importance is the increased production of il-6, -8 and -16 and eotaxin, ip-10 and rantes, which are released upon rv infection of becs . il-6 and il-16 participate in the maturation of b and t cells and eosinophil and macrophage chemotaxis, respectively. rantes and eotaxin are also powerful chemoattractors of eosinophils and drive airway eosinophilic inflammation . il-8 and ena-78 and rantes and ip-10 are potent neutrophil and t cell chemoattractants, respectively . il-8 is also linked with reduced lung function in experimental rv infection, suggesting that chemokines may be involved in asthma exacerbations (grunberg et al., 1997b) . importantly ip-10 and rantes are the chemokines released in the greatest quantities from bec infected with rv (wark unpublished). moreover, when becs were treated with dexamethasone ip-10 was only inhibited at the maximum dose, although were inhibited at all doses. this is in keeping with experimental findings that moderate doses of icss are effective in reducing ahr and infiltration of eosinophils, but do not prevent the accumulation of cytotoxic t cells. therefore rv infection may induce neutrophil and t cell influx that may exacerbate allergic inflammation and which may be refractive to ics treatment. 3.2.2.4. ifns. the release of type i and iii ifns from becs are important innate responses to infection. they have anti-viral properties and play roles in apoptosis of infected cells and directing adaptive immune responses to clear the virus (takaoka et al., 2003; wark et al., 2005) . asthmatics have recently been shown to be particularly susceptible to the development of rv lrt infections as a result of reduced ifn-î± responses. this was shown in vitro by demonstrating that peripheral blood mononuclear cells (pbmcs) from both children and adults with asthma responded to rv inoculation with a reduced release of ifn-î± (corne et al., 2002; gehlhar et al., 2006) . wark et al. , have established a model of acute rv infection of the airway epithelium, using primary becs (pbecs) acquired by bronchoscopy and cultured in vitro. asthmatic pbecs allow a significantly greater replication of rv compared to controls. the innate response of asthmatic pbecs to rv is deficient in the release of ifn-î² (wark et al., 2005) , which promotes susceptibility to infection. this defect directly impairs the ability of the infected host cell to undergo early apoptosis and allows increased virus replication and ultimately cytolysis of infected cells. the defect is the result of altered intracellular signalling and not icam-1 expression or viral internalisation. the administration of exogenous ifn-î² induces apoptosis and suppresses viral replication, which identifies the potential for therapeutic intervention (wark et al., 2005) . as ifn-î² is released mostly by becs, little ifn-î² was detected in the balf of individuals experimentally infected with rv. further confirmation of the importance of these pathways was provided when microarray analysis of rv-infected differentiated becs identified 48 genes that were upregulated and most of which are involved in type i ifn pathways. the expression of these genes could be blocked by anti-ifn-î² mab or a dsrna inhibitor (chen et al., 2006) . rv infection of pbecs also induces the release of the novel immune mediator and type iii interferon, ifn-î», which also has anti-viral effects on rv replication (contoli et al., 2006) . again this group demonstrated that asthmatic pbecs had a markedly deficient ifn-î» response to infection with rv compared to healthy volunteers. the importance of the early response to infection was confirmed in vivo by experimental rv infection of volunteers; those with the greatest impairment of early release of ifn-î» to rv had significantly increased virus replication, severity of cold symptoms, airway inflammation and a more severe fall in lung function (contoli et al., 2006) . it has been suggested that defective innate immune responses result in deficient adaptive th1 responses. in support of this pbmcs from asthmatics have been shown to have deficient ifn-î³ responses to rv (wark et al., 2005) . others have made similar observations and demonstrated time and dose dependent increases in ifn-î³, il-12 and il-10 following rv infection of pbmc (papadopoulos et al., 2002b) . cells from normal subjects produced higher levels of ifn-î³ and il-12, while those from atopic asthmatics predominantly produced il-10 (papadopoulos et al., 2002b) . 3.2.2.5. tlrs. tlr-3 recognises rv and is upregulated upon infection, which leads to nf-îºb expression (groskreutz et al., 2006) . if tlr-3 is blocked anti-viral responses are suppressed and rv replicates and is released (hewson et al., 2005) . pkr is released and promotes the generation of pro-inflammatory il-6, -8 and rantes. asthmatics may be deficient in tlr-3, which may predispose to reduced immune responses and increased and persistent rv infection that may lead to enhanced viral cytotoxicity (hewson et al., 2005) . 3.2.2.6. th2 responses. th2 responses that are typically elevated in asthmatics including those involving il-4, increase the expression of icam-1 on airway epithelium, which promotes increased rv infection (bianco et al., 1998) . icam-1 may participate in eosinophil and t cell influx into the lrt in asthma. elevated th2 along with reduced th1 responses may promote susceptibility to rv infection and more frequent viremia in subjects with acute asthma exacerbations (xatzipsalti et al., 2005) . 3.2.2.7. ahr/remodeling. while viral infections initiate lower airway inflammation and asthmatics appear more susceptible these observations do not provide a link that infections increase the severity of ahr or long-term airway remodelling. infection of pbecs clearly will initiate the release of pro-inflammatory mediators that will enhance the recruitment and trafficking of inflammatory cells to the airways. however, it is unknown whether this will affect remodelling, either directly by influencing asm or indirectly by affecting other features of remodelling in chronic asthma. to determine if rv infection could directly influence remodelling, in vitro culture models of pbecs and fibroblasts from subjects with mild or moderate asthma and healthy controls have been employed. fibroblasts have the potential to play a critical role in remodelling and changes in the airway matrix (holgate et al., 2004) . infection of pbecs and fibroblasts from these subjects does not lead to the release of the known mitogenic factors tgf-î² or endothelin-1. culture of fibroblasts with media taken from pbecs that had been infected with rv also did not induce the release of these mediators. there was also no increase in the expression of the contractile protein alpha smooth muscle actin, which may be expressed if these cells develop a contractile phenotype and there was no change in cellular morphology. these results suggest that rv infection does not induce the release of proremodelling factors from these cells. however, exposure of fibroblasts to both rv directly and rv conditioned pbec media did promote inflammation with the induction of inflammatory mediators, similar to those observed in becs. this observation agrees with the results of oliver et al., who demonstrated that asthmatic asm cells released significantly more il-6 upon infection even though the virus does not replicate in these cells (oliver et al., 2006) . limited data suggest that rv may be involved in the generation of angiogenesis and remodeling. infection induces the release of vegf and fgf and other fibrosis and angiogenesis inducing factors from primary airway epithelial cells, epithelium and fibroblast cell lines and these factors have also been detected in rv-infected nasal secretions (ghildyal et al., 2005; de silva et al., 2006; psarras et al., 2006; volonaki et al., 2006) . psarras et al., demonstrated that rv infection of becs led to the induction of vegf and that when endothelial cells were treated with the medium of infected becs they began to form tubules and to proliferate. these effects were inhibited by anti-vegf and enhanced when the endothelium was exposed to th2 cytokines . rv research and the development of therapeutic strategies have been severely hampered by the lack of a small animal model with which to investigate disease pathogenesis and the induction and exacerbation of asthma by rv. mice do not express icam-1 that can be used as a receptor by major group human rvs and these viruses do not establish infections in mice. minor group rvs, however, can infect airway epithelial cells of mice by binding to ldlprs (dreschers et al., 2007) . a recent report (newcomb et al., 2006) , describes a mouse model of minor group rv infection with rv1b. virus persists in mouse lungs and induces neutrophilic inflammation in the airways involving mip-2 expression whereas major group rv does not. this model has since been used to demonstrate that rv infection induces mucin production in vivo and that mucin production in a model of murine aad is also increased by rv infection (bartlett et al., 2007) . rvs are the most common cause of common colds and urt illness and have recently been implicated in many lrt episodes. rv infections are also the commonest cause of wheezing and asthma exacerbations, particularly in individuals n 3 years of age. rv-induced wheezing may lead to the development of asthma and recent evidence suggests that rv may be the most important risk factor for wheeze and asthma in early life. as is the case with rsv, asthmatics may be more susceptible to rv and are more likely to suffer from more severe rv disease. in vivo and in vitro studies have been used to demonstrate that rv infection exacerbates airway inflammation, obstruction and ahr and atopic status and timing of infection may have important effects. these studies show that the magnitude and phenotype of the immune response to rv has a major impact on asthmatic outcomes. importantly in vitro studies have identified defects in innate (type i and iii ifns) and adaptive responses from cells from asthmatics to rv infection. these defects may promote susceptibility to infection and contribute to the asso-ciation between rv infection and the induction and exacerbation of asthma. in order to prevent virus-induced asthma and exacerbations we require a better understanding of predisposing factors for viral diseases and their association with the causation and exacerbation of asthma. the development of effective anti-viral agents and the conduct of large-scale prospective and intervention trials are also required to assess the potential of such strategies to prevent these diseases (wennergren & kristjansson, 2001) . in asthmatics rsv and rv infection directly enhanced airway inflammation and worsened airflow obstruction. much of this occurs as a result of the immune responses, which develop rapidly upon infection. for example, responses to naturally occurring colds due to rv infection results in the development of symptoms within 12 h which peak at 2-3 days but have resolved within a week (gwaltney et al., 1996) . the rapid response to infection, the often relatively mild symptoms that occur and brisk resolution determine that it is difficult to devise interventions that will be of substantial clinical benefit. in the case of asthmatics where acute exacerbations are triggered by virus infection the clinical consequences are more severe with increased asthma symptoms, medication use, emergency presentations and hospital admissions (johnston et al., 1996) . wark et al., have shown that virus-induced acute asthma worsens airway inflammation and the degree of this inflammatory response is directly related to the severity of acute clinical symptoms (wark et al., 2002) . therefore, effective therapeutic interventions would need to alter the natural history of virus-induced asthma, prevent the worsening of airway inflammation and lead to significant clinical improvement. potential therapeutic strategies to this problem have been discussed but the approaches with the most promise include: prevention of infection from occurring; anti-viral agents that eliminate infection while reducing the host's immune response; treatments that specifically target enhanced inflammatory responses that are induced during virus-induced asthma exacerbations and supplementation of protective anti-viral responses. anti-oxidants and angiogenesis inhibitors may also have beneficial effects (fig. 1) . research into the inhibition of rsv and rv infection may have important implications for the prevention of lrt infections and the development and exacerbations of asthma. there are no human vaccines available and further research is required to develop effective anti-viral preventative strategies, specifically anti-viral mabs, passive immunization and vaccines. although the evidence that anti-viral regimes reduce the risk of developing rad later in life is scarce, the use of antivirals to delay rsv infection in particular to later in life may reduce subsequent virus-related illnesses including asthma (culley et al., 2002) . genetic factors are fixed but investigations of such interventions may be developed to reduce asthma incidence have become areas of intense interest. mabs have demonstrated efficacy in preventing rsv infection and inflammation in animal models and a humanised mab (palivizumab) against the rsv f protein is effective against rsv and wheezing in children and reduces hospitalization in high-risk individuals. studies in mice show that palivizumab reduces viral load, acute disease and long-term lung function abnormalities . in the rat model palivizumab inhibits the invasion of virus particles into the lrt epithelium and the upregulation of the nk1 receptor. this prevents acute neurogenic lrt inflammation and longterm susceptibility to inflammation that is induced by infection (piedimonte et al., 2000; piedimonte, 2002) . a randomised controlled trial with palivizumab in children achieved a 55% reduction in rsv hospitalization. reductions were observed in children with (39%) and without (78%) chronic lung disease (impact study, 1998) . a recent large prospective casecontrolled study of at risk pre-term infants showed that palivizumab also protected against recurrent wheezing (13% vs 26% controls) and physician-diagnosed recurrent wheezing (8% vs 26% controls) independently of confounding variables (simoes et al., 2007) . one drawback is that escape mutants have been detected both in vitro and in vivo (zhao et al., 2004; zhao & sullender, 2005) , indicating that preventative strategies should be targeted at multiple epitopes, because palivizumab is effective in preventing hospitalization in high-risk groups, optimisation of its pharmacokinetic profile, extension of its half-life and increasing its neutralising abilities would produce an even more efficacious preventative strategy for rsv-associated disease. motavizumab is a derivative of palivizumab and is now in clinical trials. this mab has a 20 fold increase in neutralising abilities in vitro and its use reduces viral titres by 100 fold and inhibits viral replication in the rt compared to palivizumab in rats (wu et al., 2007) . further studies are required to establish the application of such mabs for the prevention of asthma. other mabs have also been used in mice as treatments of features of rsv-induced disease. rsv infection of mice leads to eosinophil and neutrophil influx into the lung and ahr, which are inhibited by anti-il-5 (schwarze et al., 2000) . administration of fi-rsv and anti-il-4 leads to a decrease in disease features and il-4 production following challenge, but ifn-î³ release is increased (tang & graham, 1994) . t1/st2 mab treatment reduced th2 but not th1 pathology and may also be a good treatment (walzl et al., 2001) . all of these mabs require further assessment. currently the only option for preventative treatment of rsv is passive immunization, which has been in use for many years. a randomised placebo controlled trial investigated the applicability of rsv immunoglobulin to prevent rsv-associated hospitalization (prevent study, 1997) . treatment reduced hospitalizations for lrt infections but required monthly administration of high levels of fluids and proteins. in another small study, children with chronic lung disease who were treated with immunoglobulin 7-10 years previously had improved lung function, reduced atopy and rad events (wenzel et al., 2002) . this indicates that prophylaxis may be able to prevent rad in children. studies in rats also show that passive transfer of antibodies against rsv induces protection in the lrt but not the urt and that serum titres of â�¥ 1:380 are protective whereas titres of â�¤ 1:100 are not (prince et al., 1985) . infants who are the most susceptible to virus infections are protected by maternal anti-viral antibodies (largely igg1) that are passively transferred, however these antibodies inhibit the induction of protective responses against primary infection or vaccination. as a result primary or recurrent infections do not induce protective immunity until later in life (henderson et al., 1979; glezen et al., 1986) . natural protection against viral disease results from a combination of antibody, cell-mediated and t-helper cell immunity. this combination of responses target the virus before infection of host cells as well as destroying cells that have already been infected with virus. therefore, potentially an ideal vaccine would need to stimulate humoral, cellular and t-helper responses similar to those induced by natural infection. vaccine development has been hampered by the requirement for early-life vaccination, the confounding effects of poor neonatal immune responses, the presence of maternal antibodies, the difficulties in balancing immunogenicity and attenuation and the risk of vaccineinduced disease. mucosal immunization may overcome the immune suppressive effects of maternal antibodies. 4.1.3.1. fi-vaccines. the widely recognised need for an effective rsv vaccine has also been held back by the adverse events following the implementation of the fi-rsv vaccine. this vaccine was developed in the 1960s but not only was it poorly effective it infamously induced more severe rsv-related disease upon natural infection in clinical trials (kapikian et al., 1969; kim et al., 1969) . the vaccine induced high serum antibody titres that were of low neutralising activity, lymphocytes with an enhanced rsv-specific proliferative response and blood eosinophilia . these observations were repeated in mice given the same vaccine or the g protein of rsv. enhanced disease involved eosinophilia of the lung (srikiatkhachorn & braciale, 1997b; tripp et al., 2000) and aberrant cd4+ t cells releasing th2 cytokines (il-4, -5, -10 and -13) (waris et al., 1996; srikiatkhachorn & braciale, 1997b; tripp et al., 2000) . reduced il-12 and cd8+ responses were also detected (waris et al., 1996; aung et al., 1999) . fibovine rsv has also been tested in calves and also induces the development of immunopathology, ineffective neutralising antibodies and induced inflammatory responses leading to eosinophil influx into the lung and enhanced ige production upon subsequent infection (gershwin et al., 1998; antonis et al., 2003; kalina & gershwin, 2004) . as is the case with rsv in mice the fi-bovine rsv did not induce long-term t cell memory . thus it has been proposed that vaccines did not possess sufficient neutralising antibody or ctl responses and on subsequent infection the virus was not cleared but induced a potent th2 response that exacerbated disease. 4.1.3.2. subunit vaccines. subunit vaccines are not associated with enhancing disease and may have potential applicability. however, the variability of human viruses is an issue that must be taken into account in the development of subunit vaccines. for rsv, vaccination with the f protein confers cross-protective immunity whereas only group-specific immunity is developed when the g protein is used (sullender et al., 1990; simard et al., 1997) . intranasal followed by parenteral immunization with the rsv f protein protects against both upper and lower rt infection in mice but may induce pulmonary pathology (murphy et al., 1990; connors et al., 1992; walsh, 1994) . a chimeric parainfluenza viral vaccine expressing the f protein has also been developed and is protective against infectious challenge in primates (tang et al., 2004) . in a variety of different human studies f protein vaccines have now been shown to be immunogenic and safe in children with or without chronic respiratory disease and pregnant women and reduce the prevalence of infections but not lrt infections (groothuis et al., 1998; munoz et al., 2003; piedra et al., 2003; simoes & carbonell-estrany, 2003; meyer et al., 2007) . largescale trials are now required to confirm efficacy. protein g-based subunit vaccines have also been developed but may initially induce detrimental th2 responses. immune responses involving il-4 and il-13 activity following immunization with vaccinia virus expressing rsv-protein g or fi-rsv must be attenuated to modulate protein g-specific responses resulting in severe rsv-induced disease. however, inhibition of il-4 or -13 alone has minimal impact on disease (johnson et al., 2003) . the co-administration of cytokines or th1-inducing adjuvants may abolish initial th2 responses (neuzil et al., 1997) . other novel subunit vaccines in development include the n protein, other chimeric f and g fusion proteins, synthetic peptides, recombinant proteins, recombinant vaccinia and parainfluenza viruses expressing other viral components and dna vaccines (reviewed in meyer et al. (2007) ). subunit vaccines require co-administration with adjuvants to enhance immunogenicity and induce the most desirable immune response. currently quillaja saponi or its component fractions or cpg oligodeoxynucleotides administered with whole or subunit vaccines induce potent neutralising antibodies and desirable immune responses in mice and reduce disease and infection in calves. this occurs even in the presence of maternal antibodies and particularly when presented as immunostimulating complexes (meyer et al., 2007) . however the applicability of these approaches to humans remains unknown. 4.1.3.3. live attenuated vaccines. natural infection does not predispose to enhanced disease upon subsequent infection. therefore, experimental live attenuated viruses are being developed for rsv (karron et al., 2005) for intranasal administration during the first days of life but the immunology underlying their activity is poorly understood. intranasal delivery would induce systemic and local responses and has the potential to promote protective responses in both the upper and lower airways. infants (6-9 months) have limited immune responses to viral glycoproteins and only highly attenuated virus strains with minimal adverse side-effects can be used in this age group which are particularly susceptible to vaccine-related illnesses. these agents do however induce protective immunity even in the presence of passively acquired antibodies in mice and chimpanzees, which is dependent on cd4+ and cd8+ responses (crowe et al., 2001) . several cold passaged, temperature sensitive attenuated viruses have been developed that protect against challenge in chimpanzees and induce protective local and systemic immunity in children (crowe et al., 1995; karron et al., 1997) . these viruses have since been genetically engineered so that they are sufficiently attenuated for use in infants, although their efficacy against infection has not yet been determined (karron et al., 2005) . genetic engineering is also currently in use to delete non-essential viral genes to produce other live attenuated vaccines or to insert genes to enhance immune responses, for example the insertion of gm-csf to increase the production of pdcs and macrophages (reviewed in mayer et al. (2007) ). the assessment of attenuated viruses that induce potent protective immune responses with negligible side-effects requires extensive time consuming and expensive clinical trials. novel prime-boost vaccination strategies with live attenuated and subunit vaccines could be used in combination to further enhance protective immunity. 4.1.3.4. bovine vaccines. the same problems that apply to human rsv vaccines may also occur in the development of vaccines for calves in the farming industry, but the level of risk of trials is more acceptable. vaccine development is also less complex as bovine rsv is less variable than human rsv and there is only 1 major antigenic group (prozzi et al., 1997) . this has led to the commercialization of several vaccines, which are protective against infection in calves and their development may have applicability to human vaccines. further, cattle may be used as the animal model in preclinical human vaccine studies. however, different bovine models have been used with different concentrations of viral inocula and routes of inoculation with passaged virus that does not induce severe disease. thus results are often not comparable or significant between experimental and control groups (mayer et al., 2007) . inactivated bovine rsv vaccines have been available for many years and reduce the prevalence and severity of subsequent infection without enhancing disease (west et al., 1999) . nevertheless, the longevity of induced protection and efficacy in the background of maternal antibodies could be improved. these vaccines are co-administered with cd8+ and th1 stimulating adjuvants in order to dampen th2 responses that may be induced by the vaccine and lead to protective th1 responses (ellis et al., 2005) . live attenuated bovine rsv vaccines have been developed by passaging virus in cell culture and have recently been commercialized (vangeel et al., 2006) . unlike fi-vaccines, live attenuated vaccines induce a primed memory t cell response and reduce pulmonary inflammation upon subsequent infection (miao et al., 2004) . they are administered parenterally, have equivalent efficacy as inactivated vaccines and induce protective immunity in calves ellis et al., 2007) . these vaccines are now used in quadrivalent therapies to protect against a number of bovine viruses (salt et al., 2007) . although effective in cattle inactivated or attenuated bovine rsv are not suitable as vaccines for human rsv as they do not induce protection in chimpanzees . thus there are currently no safe and effective human vaccines for rsvor rvand it is yet to be determined if delay or prevention of infection with neutralising antibodies or vaccination can reduce wheezing, long-term lung function abnormalities and asthma. nevertheless there are many promising approaches and candidates are being used to develop such vaccines, some of which are in human clinical trials to prevent infection, which if successful may be used in attempts to inhibit the development of asthma. the balance of th1/th2 immunity to infection determines the severity of disease where th1 responses mediate clearance and recovery but th2 responses induce eosinophilia and more severe symptoms. strategies to augment th1 responses during vaccination in early life may be useful in preventing the development of virus-induced asthma. recent efforts have concentrated on the development of the combination of subunit vaccines with th1inducing adjuvants and on the development of live attenuated vaccines. the most promising approaches for developing vaccines in young infants are the use of live attenuated vaccines and recombinant viruses expressing components of target viruses. there are no effective treatments for rsv/rv-induced disease and currently the optimal therapy for bronchiolitis involves intensive fluid and nutritional support, while the immune response of the subject clears the infection (jhawar, 2003) . in order to maximize the chances of successful treatment an anti-viral agent would need to be taken early during the course of infection in order to modulate the development of the host's immune response. it would have to be highly effective at controlling infection and the virus would need to have no or at least a limited ability to develop resistance. finally the agent must be acceptable to patients in terms of ease of administration, have a reasonable medication frequency and limited side-effects. currently the agents of most interest are those that inhibit viral attachment, including those that bind to viral capsids, and inhibitors of viral proteases, which are required for viral replication. to cause infection all respiratory viruses need to enter the host's becs and replicate. ninety percent of rvs use icam-1 as a receptor to enter cells. a soluble icam-1 molecule (tremacamra) has been developed and tested as a competitive binding inhibitor in 4 randomised controlled trials of experimental rv (rv39) colds, used as either a dry powder or nasal spray (turner et al., 1999) . treatment results in small reductions in symptoms, virus replication and the development of clinical colds. there were no serious adverse events, but medication had to be taken six times a day and was only effective if used within 12 h of infection. it has not been used as a therapy for acute asthma. the rv outer capsid consists of 4 viral proteins (vp1-4) (rossmann et al., 1985) . several agents have been devised that bind to vp1 and prevent virus attachment to the host cell. the first agents that were produced, the oxazolinyl isoaxoles, have small clinical effects but unacceptable sideeffects . further developments led to pleconaril, an agent with broad spectrum activity against rv as an oral preparation to be taken twice daily (hayden et al., 2003) . pleconaril has been assessed in two phase ii clinical trials to determine its efficacy against natural colds (hayden et al., 2003) . subjects began treatment 1-1.5 days after cold symptoms commenced. those taking pleconaril had reduced cold symptoms and duration of colds (0.5-1.5 days) with few side-effects. an application in the us for fda approval for use in clinical colds was unsuccessful as it was considered that there was a lack of substantial clinical benefit and concerns about the development of resistant rv mutants. at this stage there have been no clinical trials specifically assessing the efficacy of pleconaril in acute asthma. rv relies upon proteases to cleave the viral polyprotein for replication to occur. rupintrivir is a 3c protease inhibitor that targets this process (dragovich et al., 2002) and has high-level anti-viral activity against all rvs (dragovich et al., 2002; binford et al., 2005) . unfortunately in a natural infection study ruprintrivir failed to improve symptoms or reduce viral load and clinical development was ceased (patick et al., 2005) . anti-inflammatory treatments have therapeutic potential in virus-associated diseases including asthma by moderating inflammatory responses in response to infection. long-term treatment of asthma with icss controls airway inflammation (juniper et al., 1990) , improves lung function and symptoms and reduces the risk of acute exacerbations (adams et al., 2005) . while icss are effective at controlling chronic asthma they do not completely prevent acute exacerbations even in those who achieve good control and are compliant with therapy, with the majority of episodes triggered by viral infection (reddel et al., 1999) . in an attempt to prevent the recurrence of virusinduced wheeze, infants were treated intermittently with ics or placebo when these symptoms occurred (bisgaard et al., 2006) . treatment had no clinical effect on acute episodes of wheeze and did not influence whether children went on to develop persistent wheeze (bisgaard et al., 2006) . another study showed that treatment of acute asthma by increasing ics dose also did not prevent worsening of disease (fitzgerald et al., 2004) . the incomplete effect of ics in controlling virus-induced asthma was demonstrated best in adult asthmatics using experimental rv infection (grunberg et al., 2001) . treatment reduced ahr and airway inflammation prior to infection but had no effect on the development of inflammatory changes that were associated with infection (grunberg et al., 2001) . in a systematic review glucocorticoids were suggested to have little effect in rsv disease (black, 2003) and they also enhance viral replication and mortality in pvm infection (rosenberg et al., 2005) . oral corticosteroids have been used to treat acute viral bronchiolitis in children. however, meta-analysis of seven randomised controlled trials found that there was no effect on length of stay or clinical symptoms and complications (patel et al., 2004) . in asthmatics with persistent symptoms despite ics use, treatment with combination therapy with icss and labas effectively controls chronic symptoms and reduces the frequency of exacerbations (walters et al., 2003) . it has recently been shown that early treatment of symptoms of worsening asthma with ics/ laba, where the laba has a rapid onset of action and is used to relieve symptoms, does prevent deterioration leading to severe exacerbations (o'byrne et al., 2005) . while these studies did not identify the cause of the acute triggers it is likely that the majority of these were related to virus infection and the combination of ics/labas appear more effective than icss alone in preventing exacerbations. in vitro studies demonstrate that treatment of airway epithelial cells with ics/labas has synergistic and additive effects in reducing the release of inflammatory mediators . these observations may explain the efficacy of combination therapy and indicate an important role for their use. this may be particularly relevant to children where their value in controlling chronic asthma is not well defined but where ahr is known to persist following viral infection (xepapadaki et al., 2005) . it remains unknown what effect ics/laba treatments have on specifically preventing or treating virus-induced asthma. it has recently been recognised that macrolide antibiotics in addition to their anti-microbial action have important immune modulating effects and reduce the release of inflammatory mediators from airway epithelial cells (takizawa et al., 1997) . epithelial cells infected with rv and treated with erythromycin, showed reduced expression of icam-1, il-6 and -8 (jang et al., 2006) . initial clinical studies were, however, not promising; using experimental rv infection, treatment of healthy subjects with clarithromycin had little or no effect on the development of cold symptoms or nasal inflammation (abisheganaden et al., 2000) . however, unlike colds where the host inflammatory response in the airways is relatively mild, in conditions characterised by more intense neutrophilic inflammation, such as cystic fibrosis, macrolides appear to be effective (ferrara et al., 2005) , which has encouraged their use in asthma. in a small randomised controlled trial of infants with rsv bronchiolitis treatment with clarithromycin reduced systemic inflammation acutely and led to few wheezing episodes in the following 6 months (tahan et al., 2007) . in a large multicentre study of adults (278) with asthma, subjects were randomised to receive the ketolide, telithromycin (800 mg/d) or placebo (johnston, 2006) . those treated with telithromycin had a greater reduction in asthma symptom scores, but there was no difference between the groups in terms of improvement in pef rate. the trial did not document the presence of virus infection but the results suggest the effect is due to an ability to reduce inflammation in acute asthma. further studies are required to determine if there is a specific effect on virus-induced acute asthma. type i ifns are known to play crucial roles in defence against viruses by inducing anti-viral proteins such as pkr and rnase l in infected cells, apoptosis preventing virus replication and adaptive immune responses to infection (malmgaard, 2004) . the recent observations by wark et al., that asthmatic becs are more susceptible to infection with rv, which involves a deficient ifn-î² response, suggests that this may be an important therapeutic target to limit the affects of virus-associated acute asthma (wark et al., 2005) . several trials have also assessed the role of ifn-î±2 treatment (1.5 ã� 10 6 -4.5 ã� 10 7 iu/d intranasally) in experimental rv colds in healthy volunteers (scott et al., 1982; hayden & gwaltney, 1984) . these studies consistently showed a dose dependent improvement in symptom scores and reduced virus shedding, but treatment was associated with nasal bleeding and a lymphocytic nasal infiltrate. a similar response was observed with recombinant ifn-î² (sperber et al., 1988) . when used to treat natural colds ifn-î±2 reduced the frequency of colds but had at best modest effects on symptoms (monto et al., 1986) , while serine ifn-î² failed to reduce colds or improve symptoms. these modest improvements provide a proof of concept but the clinical benefit is difficult to justify in healthy individuals with mild colds. however, as is the case with the use of macrolides in asthma where the clinical effects of colds are much greater (corne et al., 2002) , efficacy may be more evident. a single case series exists for the use of ifn-î± (3 ã� 10 6 iu/d) to treat 10 stable but corticosteroid resistant asthmatics (simon et al., 2003) . treatment resulted in improved lung function and allowed a reduction in oral corticosteroid use. however, side-effects were also common, in the first 4 weeks all subjects experienced a 'flu-like' illness, 9 had nausea and 5 developed headaches. by 5-10 months side-effects were less though 1 subject developed leukopenia severe enough to necessitate temporarily ceasing treatment. the benefits of type i ifns now need to be assessed in the context of treatment of virusassociated acute asthma. important issues will need to be resolved including the optimum delivery of medication, whether administration should be to the nose or the airway, how soon treatment needs to commence following the development of cold symptoms and how this may influence airway inflammation and ahr in acute asthma. oxidative stress may plan an important role in asthma but it is unknown whether it is important in virus-induced disease. the anti-oxidant butylated hydroxyanisole has been used in a mouse model of rsv lung infection to reduce rsv-induced oxidative stress, disease symptoms, weight loss and ahr. the effects were mediated by suppression of neutrophil infiltration and cytokine and chemokine release in the lung (castro et al., 2006) . thus anti-oxidants may prevent rsv-induced inflammation and have long-term beneficial effects, which may ameliorate the consequences of infection in asthma. the use of vegf and angiogenesis inhibitors may have the potential to elucidate the specific roles of these factors in asthma and identify their potential as therapeutic targets. no efficacious vaccines yet exist for the prevention of rsv or rv infections in humans. effective bovine rsv vaccines are available and the formulation of these vaccines may be applicable to the development of human vaccines. combination therapy with icss and labas effectively controls persistent symptoms and numbers of exacerbations. this is the current best treatment option but does not treat the cause of the disease and cannot be used to prevent the development of disease in the first instance. a variety of novel options for the prevention of virus-induced asthma need to be fully assessed for their efficacy and applicability. rsv and rv infection of respiratory epithelium may play an important role in the development and exacerbation of asthma, however the pivotal mechanisms underpinning these relationships remain unresolved. infection is associated with persistent wheeze and decreases in lower lung function and worsens airway inflammation and airflow obstruction acutely in asthma, with other factors contributing to severity. asthmatics are more predisposed to th2 responses, may be more susceptible to infection and experience more severe lrt symptoms upon infection. the development of more severe disease may identify those people more at risk of developing wheeze and asthma. recurring infections may lead to a cycle of inflammation and repair that worsens airway remodelling. rsv infections are strongly linked to both development and exacerbation of asthma. early-life rsv infections, particularly those that induce severe disease, induce recurrent wheeze and bronchial obstruction and predispose to rad and potentially asthma that persists into later life. it is possible that persistent infection affects the developing lung and immune system and predisposes to recurring rsv infections, ahr, reduced lung function and respiratory symptoms. another alternative is that children with reduced lung function, genetic susceptibility or aberrant immune responses may be at increased risk of infection and that rsv disease is a marker of susceptibility to increased respiratory symptoms. both of these processes may occur under different circumstances. the association with rsv is inconclusive for allergic sensitization, which may involve igemediated allergy and randomised intervention studies are required to confirm this link. the mechanisms involved in rsv-induced asthma include; age of first infection, the timing of infection relative to allergic sensitization, the nature of innate and adaptive immune responses induced upon infection, latent infections and the potential involvement of pathogenetic processes such as angiogenesis and neurogenic inflammation. a combination of human studies and mouse models has been widely used to elucidate the mechanisms of how rsv infection are linked with asthma and novel models of pvm infection may be particularly useful in the future. rvs are widely recognised as the commonest cause of clinically significant rt infections and are strongly implicated in the exacerbation of asthma and more recently in the induction of asthma. the mechanisms of these associations have been investigated in human studies both in vivo and in vitro and have been shown to involve deficient immune responses, which may be different to those associated with rsv, that may be influenced by atopy and reduced lung function, ahr and remodelling. nevertheless more work is needed to elucidate role of rv in lrt diseases. it remains unknown if rv induces the development of wheeze and asthma or if asthmatics are more susceptible to rv infection. recently a novel mouse model has been described which may be used to substantially contribute to our understanding of rv and rv-associated asthma pathogenesis. despite the scope of the problem caused by respiratory viruses in acute asthma no specific vaccines or treatments exist that have been shown to modify the clinical outcome of rsv or rv infection. the optimal approach would be to develop safe and effective prevention strategies, such as efficacious vaccines, which induce immune responses that prevent viral infection. in the absence of a vaccine the different processes involved in the generation of virus-associated asthma and exacerbations could be targeted by specific treatments for individual patients. several therapeutic options are available and more are emerging. studies should focus on how established treatments such as with ics/laba may modify virus-associated acute asthma and assess novel anti-inflammatory strategies including macrolides or the use of ifns either alone or in combination to influence the course of disease. the benefits of these strategies now need to be assessed in the context of treatment of virusassociated acute asthma. important issues will need to be resolved including the optimum delivery of medication, including whether this should be intranasally or directly to the airway. it will be necessary to determine how soon treatment needs to commence following the development of cold symptoms and how this may influence airway inflammation and ahr in acute asthma. in summary, although rsv and rv have been implicated in initiating inflammation and asthma, whether previous infections modulate the immunological response or damage the airway epithelium, and promote the progression to chronic disease, or susceptibility to later development of exacerbations, remains unknown. asthmatics may have impaired anti-viral innate responses and defective interactions between innate and adaptive immune responses may promote infection and enhance allergic inflammation and ahr or decrease lung function. further studies are required to elucidate the links between infection, immune responses and susceptibility to chronic respiratory diseases and why some individuals but not others develop persistent wheeze and asthma. it is 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protein of respiratory syncytial virus (rsv) confers protection from rsv infection in african green monkeys contribution of the respiratory syncytial virus g glycoprotein and its secreted and membranebound forms to virus replication in vitro and in vivo role of nasal interleukin-8 in neutrophil recruitment and activation in children with virus-induced asthma the brume surrounding respiratory syncytial virus persistence cd40 ligand (cd154) enhances the th1 and antibody responses to respiratory syncytial virus in the balb/c mouse substance p receptor expression on lymphocytes is associated with the immune response to respiratory syncytial virus infection efficacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection: a randomized clinical trial asthma: an epidemic of dysregulated immunity bovine respiratory syncytial virus infection persistent infection of b lymphocytes by bovine respiratory syncytial virus role of alpha/beta interferons in the 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respiratory outcomes in high-risk children 7 to 10 years after prophylaxis with respiratory syncytial virus immune globulin the effect of formalin-inactivated vaccine on respiratory disease 352 n differential patterns of methylation of the ifn-gamma promoter at cpg and non-cpg sites underlie differences in ifn-gamma gene expression between human neonatal and adult cd45ro-t cells recombinant respiratory syncytial virus (rsv) bearing a set of mutations from cold-passaged rsv is attenuated in chimpanzees recombinant respiratory syncytial virus bearing a deletion of either the ns2 or sh gene is attenuated in chimpanzees the bronchial microcirculation in asthma histopathologic examination and enumeration of polymorphonuclear leukocytes in the nasal mucosa during experimental rhinovirus colds respiratory virus infection of monolayer cultures of human nasal epithelial cells comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract rhinovirus viremia in children with respiratory infections human metapneumovirus as a causative agent of acute bronchiolitis in infants duration of postviral airway hyperresponsiveness in children with asthma: effect of atopy infection of human respiratory submucosal glands with rhinovirus: effects on cytokine and icam-1 production exposure of neonates to respiratory syncytial virus is critical in determining subsequent airway response in adults respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology in vivo selection of respiratory syncytial viruses resistant to palivizumab altered eosinophil levels as a result of viral infection in asthma exacerbation in childhood respiratory syncytial virus escape mutant derived in vitro resists palivizumab prophylaxis in cotton rats virokinin, a bioactive peptide of the tachykinin family, is released from the fusion protein of bovine respiratory syncytial virus bone marrow plasmacytoid dendritic cells can differentiate into myeloid dendritic cells upon virus infection key: cord-006466-e1phpqes authors: nan title: 2018 cis annual meeting: immune deficiency & dysregulation north american conference date: 2018-04-23 journal: j clin immunol doi: 10.1007/s10875-018-0485-z sha: doc_id: 6466 cord_uid: e1phpqes nan mutations in the genes encoding proteasome subunits (psmb8, psmb4, psma3 and psmb9) have been identified as the cause of candle syndrome. these mutations lead to malfunction of the proteasome, which results in buildup of cellular waste products. it is hypothesized that dysregulation in the interferon (ifn) signaling pathway in response to this waste is the driving mechanism of the inflammatory response, and may serve as a therapeutic target in these patients. objectives: to describe a case of suspected candle syndrome successfully treated with tofacitinib. methods: retrospective chart review was conducted with respect to diagnosis, treatment and response. results: a 16-month old caucasian male was admitted to the hospital for evaluation of profound anemia. his medical history was significant for extreme prematurity (born at 22 weeks from premature labor), intraventricular hemorrhage grade iv that resulted in hydrocephalus needing ventriculoperitoneal shunting, and developmental delay. he was noted to have a hemoglobin of 6.2 g/dl during a neurosurgical evaluation for routine shunt revision. he developed hemodynamic decompensation and required hospital admission for packed red-blood cell transfusion. review of systems was remarkable for intermittent pruritic macular rash, daily temperature fluctuations (fever to hypothermia), joint pain/swelling/ stiffness of multiple sites, poor weight gain, irritability, irregular breathing, abdominal distention, and regression of gross motor milestones (no longer rolling over, sitting without support, or pulling up to stand). workup excluded infections and lymphoproliferative malignancies, and he met clinical criteria for systemic juvenile idiopathic arthritis. his initial laboratory studies showed systemic inflammation (wbc 14x 10^3/ul, hgb 6.0 g/dl, platelets 558 x10^3/uul, crp 14.2 mg/dl, sedimentation rate 70 mm/h, ferritin 2370 ng/ml). imaging studies revealed serositis with right-sided pleural and pericardial effusions. he also had myositis supported by imaging and elevation of muscle enzymes (ast 52 u/l, aldolase 30.6 u/l). the patient was started on pulse iv methylprednisolone 30mg/kg daily x3 days, followed by oral prednisolone 2mg/kg/day and anakinra 2mg/kg/day with partial improvement and he was discharged home. he was readmitted 3.5 weeks later due to concerns for macrophage activation syndrome (ferritin 12,362 ng/ml) in the setting of a gastrointestinal infection and anakinra was increased to 4.5mg/kg/day. however, he continued to have persistently elevated inflammatory markers and so the dose was increased again to 7mg/kg/day. three months after initial presentation, he had an upper respiratory and ear infection and became ill with generalized rash, increased work of breathing, and poor perfusion. anakinra was considered a treatment failure at that time. he required several doses of pulse steroids and initiation of tocilizumab 12mg/kg iv every 4weeks with improvement on systemic symptoms. methotrexate 15mg/m² weekly was added soon after for persistent arthritis and inability to wean systemic steroids. he continued to have abnormal inflammatory indices, including ferritin (1,586 ng/ml) and il-18 levels (35,588 pg/ml, normal 89-540). proband only whole exome sequencing revealed a single heterozygous mutation in the psmb4 gene (c.-9g>a), a published pathologic variant. based on this finding, the patient was started on tofacitinib 2.5mg orally twice a day with a dramatic response. laboratory markers of inflammation normalized, and he was able to walk within the first month of treatment. further genetic testing to detect an additional proteasome subunit variant, as well as functional testing on a research basis to demonstrate an interferon signature are being pursued. conclusions: this case highlights the value of early genetic studies in patients with autoinflammation so that initiation of targeted therapy is not delayed in efforts to achieve control of symptoms and evade future complications. this case also illustrates the challenges in diagnosing monogenic autoinflammatory disorders in young patients that present with recurrent fevers, generalized rash, arthritis, and systemic inflammation that mimic systemic juvenile idiopathic arthritis. our experience contributes to the understanding of janus kinase inhibition in type i interferonopathies. of his recurrent infections and etiology of myasthenia gravis. results of the ct chest are notable for a thymoma. thymectomy with biopsy reveals benign pathology with a mixture of type a and b cells. he continues to have persistent fatigue, generalized weakness, diplopia, diarrhea and recurrent respiratory infections after thymectomy. immunoglobulin and lymphocyte subset panels reveal hypogammaglobulinemia with absent b cells. endoscopy reveals villous atrophy and blunting without evidence of celiac disease, inflammatory bowel disease or infection suggesting autoimmune enteropathy. the constellation of clinical and laboratory features are consistent with good syndrome with evans syndrome, seronegative myasthenia gravis and autoimmune enteropathy. the patient is started on immunoglobulin replacement therapy and pyridostigmine with resolution of recurrent infections and improvement of fatigue, generalized weakness and diplopia. three years later his fatigue and evans syndrome recur with new onset loss of appetite and a thirty pound weight loss. repeat immunologic labs were notable for elevated cd3, borderline low cd4 and highly elevated cd8 cells with low absolute number and fraction naïve cd4 and cd8 cells suggesting worsening combined immunodeficiency with peripheral t cell expansion. a bone marrow biopsy reveals large granular lymphocytic (lgl) leukemia and he is started on methotrexate. serum antibodies targeting ifn, and il-12 are negative four years after removal of thymoma. conclusions: this case is consistent with a classic presentation of good syndrome represented by thymoma, t and b cell-mediated immunodeficiency, increased susceptibility to infections and autoimmune manifestations of evans syndrome, myasthenia gravis and autoimmune enteropathy. in this case the combination of evans syndrome, autoimmune enteropathy and lgl leukemia as malignancy further worsen prognosis and is typically not seen together in good syndrome. this case depicts well the crossroad of infection, autoimmunity and malignancy in late onset immunodeficiencies. introduction/background: dedicator of cytokinesis 8 (dock8) deficiency is a known cause of autosomal recessive hyper-ige syndrome with a combined immunodeficiency. most of the mutations in dock8 are lossof-function homozygous or compound heterozygous point mutations or deletions. dock8 deficiency has been associated with low lymphocyte counts with impaired antibody responses, as well as eosinophilia, recurrent bacterial and cutaneous viral infections, malignancies, and severe atopy. we report the case of a 47 year old man with history of hyper-ige syndrome, severe atopy, eosinophilia, and antibody deficiency, phenotypically atypical for dock8, who was noted to have two variants of unknown significance in the dock8 gene. objectives: we report the case of a 47 year old man with history of hyper-ige syndrome, severe atopy, eosinophilia, and antibody deficiency, phenotypically atypical for dock8, who was noted to have two variants of unknown significance in the dock8 gene. methods: a 47 year-old man presented to us for evaluation of known hyper-ige syndrome. he had a long history of elevated ige, peripheral eosinophilia, severe atopic dermatitis, food allergies, asthma, severe eczema since early childhood which failed to respond to methotrexate, mycophenolate, cyclosporine, and omalizmuab, but ultimately responded to intravenous immunoglobulin (ivig). his infectious history included mrsa skin infections and one episode of pneumonia, and he reported a history of fungal skin infections but the history was unclear. initial immune workup revealed eosinophilia of 2898, ige level 3540 (as high as 20,000), igg level 603, igm level 106, and iga level 124. he had no random antibodies to streptococcus pneumonia 13 serotypes, but he had protective antibodies to diphtheria and tetanus. lymphocyte subsets showed cd3 1874, cd4 1597, cd8 277, cd19 85. he had normal mitogen stimulation to pha but decreased mitogen stimulation to candida. dna testing for a stat3 mutation was negative. results: we found two missense variants of uncertain significance in the dock8 gene (1.p.v194i, nm_203447.3:c.580g>a and 2.p.l1330v,nm_203447.3:c.3988c>g ). the first variant had previously been reported in the clinvar database as a variant of uncertain significance, and the second variant had not been previously reported in the literature to our knowledge. our assay could not determine if the two dock8 variants were on the same allele or on different alleles. dock8 protein expression testing is currently pending. conclusions: our patient presented with history of elevated ige, eosinophilia, atopy, severe eczema, and cutaneous mrsa and fungal infections. he was noted to have variants of uncertain significance in the dock8 gene. homozygous or compound heterozygous pathogenic variants in dock8 are associated with an autosomal recessive hyper-ige syndrome and combined immunodeficiency with clinical features of recurrent bacterial infections, cutaneous viral infections, severe atopic disease, as well as susceptibility to malignancy. our patient does not have all the typical features of dock8 deficiency and he seems to have a less severe phenotype. notably, he does not have the cutaneous viral infections or malignancy often seen in dock8 mutation hyper-ige cases. our case demonstrates new missense mutations, which have not previously been described in the literature, possibly causing a milder phenotype of dock8 deficiency. a case of igm deficiency and adult-onset still's disease negative. previous biopsy of her cervical and thoracic lymphadenopathy was unremarkable for malignancy. during her hospitalization, serum immunoglobulins were performed, which demonstrated normal levels of igg and iga, with igm level of <10 mg/dl (reference range 40-230 mg/dl), consistent with selective igm deficiency. liver function tests revealed an elevated aspartate aminotransferase (ast) of 177 u/l and an alanine aminotransferase (alt) of 177 u/l with a total bilirubin of 1.7 mg/dl and an alkaline phosphatase of 146 u/l. her ferritin was elevated at 349 g/l. the patient fulfilled yamaguchi criteria for aosd with three major criteria of evanescent rash, intermittent fevers in a quotidian pattern, bilateral arthralgias in the hips, knees, and ankles. she also met two minor criteria of liver abnormalities and lymphadenopathy. conclusions: selective igm deficiency is an uncommon immunodeficiency disorder associated with increased risk for autoimmune disorders. the recognition of co-morbid autoimmune illnesses in an immunodeficient patient is often complicated by a paucity of examples in the literature and potential confounding of laboratory serology analysis. we report the first case of a patient with selective igm deficiency and aosd. introduction/background: anaphylaxis to protamine is an uncommon but life-threatening complication of cardiac surgery and insulin therapy. here we present a case of recurrent protamine hypersensitivity during vascular surgery. objectives 1. recognize clinical signs of protamine hypersensitivity 2. recognize recurrent hypersensitivity to protamine as a serious complication of anesthesia methods: a 63 year old man with a history of diabetes, previously on nph insulin, hypertension, hyperlipidemia, chronic smoking, and peripheral artery disease with multiple vascular interventions was admitted to undergo a right lower extremity saphenous vein graft bypass. three years earlier during a similar intervention, the patient had developed intraoperative hypotension after protamine sulfate administration. protamine was subsequently held for additional surgeries, however the patient was able to tolerate protamine with slower infusion one year later. for the current vascular surgery, the patient was pretreated the day of surgery with diphenhydramine and dexamethasone, and a test dose of protamine was infused prior to full dosing. the patient initially appeared to tolerate the full protamine dose, but quickly developed facial erythema and angioedema. due to concern for laryngeal edema he remained intubated and was transferred to the surgical intensive care unit, where he received additional diphenhydramine and dexamethasone. his symptoms resolved and he was successfully extubated the next morning. results: anaphylaxis to protamine is an uncommon but lifethreatening complication of cardiac surgery and insulin therapy. protamine sulfate is a polypeptide used widely to neutralize heparin anticoagulation during cardiac and vascular surgeries, and in nph insulin. severe anaphylactic or anaphylactoid reactions caused by injection of protamine sulfate are well documented in literature, and the product contains a black box warning for such. the pathophysiologic mechanisms underlying these reactions are not clear, but ige-mediated hypersensitivity appears to play a role in many reactions, and prior sensitization or cross-sensitization (eg, to fish) have been suggested. type b adverse drug reactions are idiosyncratic drug reactions and are often unpredictable, as in our patient who previously tolerated protamine but subsequently developed an adverse reaction. hypersensitivity reactions during anesthesia should be thoroughly studied to identify the responsible drug and minimize exposure in recurrent surgeries. conclusions: this case illustrates the potential for severe reactions even with newer protamine formulations, and highlights the unpredictable nature of type b adverse drug reactions. it is important for clinicians to exhibit awareness of the potential adverse effects of protamine sulfate in such situations. introduction/background: x-linked lymphoproliferative syndrome type 2 (xlp-2) is a rare primary immune deficiency caused by loss of function in the x-linked inhibitor of apoptosis protein (xiap). common reported manifestations include recurrent hemophagocytic lymphohistiocytosis, splenomegaly, crohns-like inflammatory bowel disease, and transient hypogammaglobulinemia without reductions in major t cell or b cell repertoires, with the exception of inkt cells and mait cells. however, with only~100 known cases worldwide, we are likely only beginning to understand the phenotypic spectrum of this disease. objectives: to describe additional manifestations of xlp-2 that expand our current understanding of its phenotype. methods: a 26 year-old male with adult-onset, treatment refractory ulcerative colitis was evaluated in the immunology clinic for a history of recurrent sinopulmonary infections, skin abscesses, and recurrent ebv and vzv infections. extensive laboratory testing was performed in the course of his evaluation, including lymphocyte immunophenotyping, lymphocyte proliferation and cytotoxicity studies, quantification of total immunoglobulin levels and specific antibody function, hiv testing, and genetic testing. results: laboratory testing was significant for persistent cd4 lymphocytopenia ranging from 380-459 cells/mcl (rr: 5031736 cells/mcl). total b cell count was normal but b cell subsets showed an elevation in the percentage of naïve b cells (range: 85. .7%), low non-switched memory b cells (range: 4.7-6.0%, rr: 7.0-23.8%), and low to low-normal switched memory b cells (range: 7.2%-8.3%, rr: 8.3-27.8%), a pattern that has been seen in some autoimmune diseases. genetic testing with a commercial immune deficiency panel (invitae corp) showed a pathogenic mutation in xiap [exon 2, c.664c>t (p.arg222*)]. this mutation has previously been reported to cause a premature stop codon and reduced xiap function. the patient was referred for hematopoietic stem cell transplant and is currently awaiting transplant with a matched unrelated donor. conclusions: xlp-2 is typically reported as having normal t cell, b cell, and nk cell counts, but the presence of persistent cd4 lymphocytopenia in this patient illustrates that this is not always the case. our patient also had abnormalities in his b cell repertoire that have not been previously reported in xlp-2. additionally, xlp-2 has been associated with crohns disease and celiac-like bowel diseases, while our case indicates that the phenotype may also include ulcerative colitis. (9) submission id#420740 a case report: enteroviral encephalitis as a consequence of partial humoral immunodeficiency in a chronic lymphocytic leukaemia patient treated with rituximab hadeil morsi, st3 immunology st3, oxford university hospitals introduction/background: enteroviral (ev) infections are prevalent and usually self limited or cause mild gastrointestinal manifestations. however , in the context of primary antibody deficiency , rare cases has been reported to develop meningoencephalitis and been linked to poor outcome with fatality or chronic course. ev meningoencephalitis is even far rare reported in the era of rising secondary humoral immunodeficiency as a consequence of b cell depleting therapy e.g. rituximab and lymphoproliferative malignancies. limited treatments for ev encephalitis are available to date, apart from intravenous immunoglobulin replacement which has variable efficiency. objectives: studying such rare cases of ev meningoencephalitis as a consequence of antibody deficiencies would help to develop guidelines for intravenous immunogobulin replacement for treating these infections to improve outcome as well as predicting patients at higher risk who should be considered for prophylactic immunoglobulin therapy. methods: herein, we report a rare case of proven enteroviral meningoencephalitis following rituximab based therapy for b-cell chronic lymphocytic leukaemia and an uneventful six months period of follow up. he was found to have persistent absent b cells six months after completing six cycles of fludarabine, cyclophosphamide and rituximab therapy. interestingly, he had partial pneumococcal igg serotypes deficiency, whilst his total igg, igm and iga were all within normal limits throughout the course of the disease. the patient was treated empirically with intravenous immunoglobulin when his subtle confusion progressed to overt behavioural changes. initially his level of consciousness continued to deteriorate and he was not communicating. results: fortunately enough, the patient did have a remarkable improvement of gcs within couple of days and a slower recovery of higher mental functions e.g. memory and calculations in the next couple of months. conclusions: early suspicion and detection of entervorial meningoencephalitis in patients at risk of secondary antibody deficiency is crucial for timely ivig replacement and better outcome. patients with haematological malignancies and those on b cell depleting immunotherapy should be screened for pneumococal igg serotypes as part of secondary immunodeficiency workup. further studies on enteroviral neurological meningo/encephalitis are required to optimise ivig therapy and prognostication. furthermore, such studies provide an important asset to reveal the underlying mechanisms for humoral/b-cell mediated protective response against ev compared to other t-cell mediated viral immunity, whilst highlighting the mechanisms of immunodeficiency in cll and immunotherapy. (10) submission id#418733 a comparison of immune reconstitution following human placenta-derived stem cells (hpdsc) with umbilical cord blood transplantation (ucbt) vs. ucbt alone in pediatric recipients with malignant and non-malignant diseases introduction/background: ucbt is a safe and effective treatment in children (geyer/cairo et. al bjh, 2011) . however, due to a limited concentration of hematopoietic progenitor cells (cd34+) in ucb, ucbt has been associated with delayed hematopoietic reconstitution and a higher incidence of engraftment failure. hpdscs contain a rich population of hpcs, are low in hla class i/ii expression and t-cells, and have regenerative, anti-inflammatory, and immunosuppressive properties (cairo et al bmt, 2015) . objectives: to determine whether ucbt + hpdsc (vs. ucbt alone) is associated with enhanced hematopoietic and immune cell reconstitution in children with malignant and non-malignant diseases. methods: immune cell reconstitution at days +100, 180, 270 and 365 was assessed in children who received ucbt with hpdscs at nymc (nct01586455, ind#14949). minimum tnc was 5 x 10^7/kg (4/6 hla match) or 3.5 x 10^7/kg (5-6/6 hla match). immune cell subset counts at these time points were compared to those from a historical population of pediatric recipients of ucbt alone (geyer/cairo et. al bjh, 2011) . results: twenty four patients 18 years were enrolled. mean age was 6 (range, 0. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] years. malignant diseases =14, non-malignant diseases =10. fourteen patients received myeloablative conditioning (mac) and ten patients received reduced toxicity conditioning (rtc). there were no severe adverse events associated with hpdsc infusion. two patients with non-malignant disease receiving rtc using alemtuzumab experienced primary graft failure. probability of neutrophil engraftment was 91.6 %, median day 22 . of evaluable patients at day 100, the probability of platelet engraftment in neutrophil engrafted patients was 100%, median day 43.5 (20-98) . at days 30, 60, 100 and 180, mean percent donor chimerism in whole blood was 94, 98, 95, and 99%, respectively. average percent of whole blood hpdsc chimerism was 1% at day 30 and <1% at beyond day 60. one patient with malignant disease relapsed. 12 month overall survival was 83.3%. there was no significant difference in cd3, cd4, cd8, cd19 and cd56 immune cell reconstitution following ucbt + hpdsc vs. ucbt alone (image 1). conclusions: these results suggest that ucbt ± hpdsc results in similar immune cell reconstitution. a larger cohort with extended follow-up would be required to confirm these preliminary findings. supported by a grant from celgene cellular therapeutics. a decade of disseminated abscesses due to mycoplasma faucium in a patient with activated pi3k syndrome 2 (apds2) introduction/background: pik3r1 monoallelic mutations are known to be responsible for apds-like 2 syndrome, a rare form of primary immunodeficiency presenting as combined immunodeficiency or hyper-igm like phenotype. this study reports a patient carrying heterozygous pik3r1 mutation with early onset and long-term disseminated abscesses due to mycoplasma faucium in both peritoneal abscess and skin, with generalized involvement in neck and both upper extremities. objectives: we describe clinical management of retroperitoneal and skin abscesses before molecular diagnosis was available in a patient with a primary immunodeficiency. identification by rdna 16s in so-called sterile abscesses may confirm the clinical suspect of an oportunistic infection. furthermore, this study offers insight on the pik3r1 suspicion even in the absence of higm-like phenotype. methods: a 16-year-old girl with 2-year history of recurrent peritoneal effusion, which had been drained repeatedly was admitted in our institution for a 10-year history of multiple supurative cutaneous and lymph node-abscesses (fig1&2a-c). she had prior diagnosis of agammaglobulinemia under standard subcutaneous immunoglobulin replacement therapy and subcutaneous interferon-gamma treatment. on physical examination at the age of 16 years, she was stunted (weight and height below the 3rd percentile), with facial, arm skin abscesses and right fistulized axillary lymphadenopaties, 5-6cm hepatomegaly and giant splenomegaly results: she had her first immunological work up at the age of 6 years during one isolated episode of knee arthritis and first episode of skin abscesses. serum immunoglobulins revealed panhypogammaglobulinemia (igg<7.3 mg/kg, iga <5.8 mg/kg, igm <4.3 mg/kg) with low b cell count. on her back, there was a 10x15cm, elastic, neither painful nor tender mass. after proper assessment by ct scan and mri she had her retroperitoneal abscess drained percutaneously, and healed with sclerotherapy (percutaneous alcohol and polidocanol instilation) by the interventional radiologist ( fig 2b) . analysis of drained pus as well as pus of skin abscesses was made by 16s rdna pcr, having coincidence of 99.9% with mycoplasma faucium . combination antibiotic therapy (doxycycline and ciprofloxacin) was started with favourable response. unfortunately skin abscesses then relapsed. t-cell phenotype only showed t-cell lymphopenia with senescent (tem & temra expansion) phenotype. whole exome sequencing revealed a heterozygous mutation, previously reported (c.1425+1g>t) conclusions: in summary, this report emphasizes the suspicion of a combined immunodeficiency in the presence of multiple abscesses by mycoplasma, the usefulness of rdna 16s in order to achieve proper objectives: we describe a 15-year-old male patient with novel heterozygous mutation of ep300 gene; his first manifestations were initially characterized by infections, cytopenia and hypogammaglobulinemia suggesting a common variable immunodeficiency (cvid), but later on, persisting lymphopenia was suggestive of a combined immunodeficiency. methods: the patient was born to unrelated healthy italian parents at 34 weeks gestation with adequate weight for gestational age. shortly after birth, he underwent several surgical procedures due to interventricular defect, aortic coarctation, double outlet right ventricle, open botallis duct, and gastroesophageal reflux. at the age of four, he came to our attention due to stomatitis. clinical examination revealed dysmorphisms (microcephaly, wide forehead, sparse eyebrows, high nasal root, low-hanging columella, thick lips, micrognathia), splenomegaly (spleen diameter 11.6 cm at abdominal ultrasound), and severe developmental delay. in the course of the infectious episode, blood tests showed leukopenia associated with neutropenia (white blood cells 2.210/mm3; neutrophils 20/ mm3) and thrombocytopenia (platelets 1.000/mm3). analysis of bone marrow aspirate revealed normal differentiation of both myeloid and erythroid lineages. treatment with high doses immunoglobulin resulted in increase of platelet counts (up to 44000/mm3 after 1 month), while neutrophil counts spontaneously returned to normal when the infection resolved. however, thrombocytopenia relapsed (2000/mm3) after 2 months and intravenous high-doses of corticosteroids did not achieve normal platelets count. despite oral corticosteroid treatment started at the age of six, two episodes of autoimmune hemolytic anemia occurred. during the following six years of follow-up, the patient experienced recurrent infections (stomatitis, upper respiratory tract infections, and skin abscesses), but none of the episodes has required hospitalization. but, at the age of ten, he was admitted to the hospital because of severe cultures negative diarrhea. despite immunoglobulin replacement therapy was started at the age of fourteen, he was admitted twice due to bilateral pneumonia requiring continuous positive airway pressure and, a few months later, acute respiratory failure with evidence of mycoplasma pneumoniae and rhinovirus infections. immunological evaluation under chronic corticosteroid treatment at different time points showed persisting lymphopenia, with lymphocyte counts ranging from 350/mmc3 to 2100/mm3 , thrombocytopenia (platelets ranging from 0/mm3 to 308000/mm3), undetectable anti-diphteria and anti-tetanus toxoid antibodies, and splenomegaly. interestingly, analysis of isohemoagglutinins, revealed low titers of anti-a (1:8 ) at 4 years of age, but normal immunoglobulins (igg 685 mg/dl, iga 73 mg/dl, and igm 83 mg/dl). at the age of seven, reduced mitogen proliferation, hypogammaglobulinemia (igg 274 mg/dl; iga 17 mg/dl, igm 185 mg/dl), increased cd3+tcr+cd4cd8 t-cell counts (2.7/3.6%) and impaired fas mediated apoptosis as measured in two separate assays (table 1 ) . at the age of fourteen, evaluation of b-cell subsets showed increase of cd21locd38lo cells and reduction of switched memory b-cells. analysis of t-cell compartment unveiled a decreased proportion of cd31+ccr7+cd45ra+ recent thymic emigrants (rte) cells and ccr7+cd45ra+ naive cells, with prevalence of effector memory t-cells (ccr7-cd45ra-) ( table 1) . interferon signature gene expression showed borderline levels of ifi27 (data not shown). because of the decrease of igg and the infectious episodes ivig treatment was started at age of fourteen. a molecular investigation performed by whole exome sequencing (wes) revealed a novel heterozygous missense mutation (nm_001429. 3:c.4763t>c , p.met1588thr) in the exon 29 of the gene ep300 encoding the histone acetyltransferase (hat) protein p300. results: few immunological reports are available in rsts patients1-4. in keeping with previous data1,3,4, our patient presented with progressive b-and t-cell lymphopenia, hypogammaglobulinemia with poor antibody response but also reduced naïve t cells, evans syndrome, splenomegaly, and defective lymphocyte apoptosis with increased dnt. at the age of seven, the patient presented the features of cvid. flow cytometry revealed expansion of cd19hicd21locd38lo b cells, that is frequently associated with splenomegaly in cvid patients7, and reduced switched memory b-cell, previously reported in a rsts patient with crebbp mutation4 (fig. s1 ). lougaris et al. reported expansion of cd21locd38lo b-cells in nf-kb1 haploinsufficiency 8, and this suggests in our opinion that alterations in the nf-kb pathway due to ep300 mutations may affect b-cell differentiation. compared to healthy controls and cvid patients with predominant infectious complications, upregulation of interferon responsive genes in cvid subgroup with noninfectious complications (i.e hematologic autoimmunity, lymphoproliferation) and lymphopenia with reduced total b cells and switched-memory b cells has been demonstrated9. borderline levels of ifi27 expression under corticosteroid treatment represent a novel finding in rsts. interferon signature may identify and better characterize subgroup of rsts patients with autoimmune cytopenias and lymphopenia. at the age of fourteen, analysis of lymphocyte subsets revealed decreased total cd3+, cd4+, cd8+, and of both naïve cd4+ and cd8+ cells. elevated and persisting igm levels were also observed (fig. s2 ). according to our data, increased igm levels may be related to high proportion of terminal differentiated igm+ cells. these data (infections requiring hospitalization, immune dysregulation, lymphopenia, reduced naïve t cells, and reduced proliferation to mitogen) together with clinical history (fisher evans syndrome and lymphoproliferation) and exclusion of known syndromic immunodeficiencies, suggested a diagnosis of combined immunodeficiency10. conclusions: our case underlines the value of wes in patients with difficult phenotype-genotype correlation. no rsts typical traits were present and, prior to wes, several syndromes and immunodeficiencies were excluded. our report expands the phenotypic spectrum of ep300 mutations, thus in syndromic patients with clinical and immunological overlap between cvid and cid ruling out ep300 mutation should be advisable. furthermore, immunological work-up should be taken into consideration in rsts patients, in order to early identify immunological abnormalities that may lead to severe immune-hematological complications. introduction/background: interferon gamma receptor (ifngr1)-related disorders are rare variants of mendelian susceptibility to mycobacterial diseases. although hematopoietic stem cell transplantation (hsct) is curative, it is complicated by high rates of delayed or failed engraftment thought to be due to high concentrations of interferon (ifn)-gamma. umbilical cord blood transplantation additionally increases risk of graft failure. objectives: describe a pediatric patient with non-functional ifngr1 who successfully underwent umbilical cord blood transplantation. methods: direct clinical care of described patient with additional electronic medical record chart review. results: the patient is a 19-month-old boy of yemeni descent who initially presented with significant hepatosplenomegaly and extensive lymphadenopathy, including a large mediastinal mass. he then developed salmonella enteritidis sepsis requiring numerous antimicrobials, vasopressor support, intubation and continuous renal replacement therapy. his evaluation showed a hyperinflammatory state with elevations in ferritin, ifn-gamma, scd25, il-10, il-13, il-17, il-6 and il-8 levels. maximal ferritin and ifn-gamma levels reached 12065.4 ng/ml and 463 pg/ml (normal <5 pg/ml), respectively. flow cytometry revealed normal expression of ifngr1 and il12r but absent ifn-gammastimulated stat1 phosphorylation, suggesting defective ifngr1 signaling. genetic testing showed a previously unreported homozygous mutation in ifngr1 (c.373+2t>c) which affects a donor splice site in intron 3 and is predicted to cause absent protein function. dexamethasone and a single dose of alemtuzumab (0.2 mg/kg) were given to decrease inflammation. he then underwent allogeneic hsct using a 5/6 human leukocyte antigen matched umbilical cord unit following a reduced-toxicity conditioning regimen of alemtuzumab (0.6 mg/kg), fludarabine (180 mg/m2) and busulfan (auc 55 mg/l*h). plasma ifn-gamma was undetectable prior to starting conditioning and on the day of transplant. neutrophil engraftment occurred on day +14 with day +30 posttransplant chimerism analysis of peripheral blood myeloid cells showing the presence of donor cells only. conclusions: these early results suggest that umbilical cord blood transplantation may be feasible in patients with ifngr1-related disorders provided adequate control of inflammation is gained prior to transplant. introduction/background: introduction: btk is a cytoplasmic tyrosine kinase that activates phospholipase c2 (plc2) via phosphorylation, which ultimately leads to the activation of nfk, which is essential for b cell development and survival. mutations in btk lead to x-linked agammaglobulinemia (xla). in addition to the pleckstrin homology and tyrosine kinase domains, btk contains two src homology domains, sh2 and sh3, which are essential for btk function. we describe a novel btk mutation (c.1197a>t) resulting in v335a substitution in the sh2 domain that results in aberrant xla function with nearly normal btk protein expression. objectives: case report/results: the male proband presented with recurrent otitis media, persistent fevers and neutropenia beginning in the first year of life with an igg level of 46 mg/dl and a lack of b cells (0 cell/mm3), as demonstrated by flow cytometry. btk protein expression in monocytes, also determined by flow cytometry, was equivalent to controls. family history is significant for a maternal uncle with history of recurrent sinus infections and pneumonias with low iga and igm, low to normal ige, and an absent vaccine response. flow cytometry also showed an absence of b cells and essentially normal btk protein expression in his monocytes compared to controls. targeted high throughput sequencing of both proband and the uncle revealed a previously unreported missense mutation in exon 12, leading to the substitution of an aspartic acid residue for a valine (v335d.) the mutation is in the highly conserved sh2 domain of btk (conservation phylop conservation score 2.8.) the probands mother and his sister were shown to be carriers of the same mutation and had normal serum immunoglobulin levels and normal numbers of b cells. methods: we hypothesized that if the btk v335d mutant protein was non-functional, the female carriers of the mutation would only express wild type (wt) btk in their b cells while their monocytes would express both wt and mutant btk. to test this hypothesis cd19+ b cells and cd14+ monocytes were purified from pbmc by fluorescence activated cell sorting (facs) from the sister, cdnas were generated from the respective populations of cells, and the btk cdna was sequenced using high-throughput sequencing. results: at a read-depth greater than 16,000, cd19+ b cells demonstrated btk expression only from the wt allele (~99% wt btk) whereas both the wild type and mutant allele of btk were expressed at approximately equal levels in monocytes. conclusions: discussion: these results define a novel mutation in btk that nominally affects protein expression, but alters function. the v335a substitution is found in the d structural element of the sh2 domain that is part of a hydrophobic phosphotyrosine binding pocket. a mutation in the adjacent residue, y334s, has been shown to alter protein conformation and decrease binding affinity to plc2 by roughly 4-fold resulting in xla. our study, using a carrier harboring the c.1197 a>t mutant btk, demonstrated that in contrast to mononuclear cells, b cells only expressed the wt allele. this is consistent with the loss of function of v335a btk protein, thereby causing xla in both the proband and affected uncle. introduction/background: pyoderma gangrenosum (pg) is often associated with systemic autoimmune diseases but it has rarely been reported with common variable immune deficiency (cvid). while genetic analysis has been increasing in both disease domains, there has been little investigation into the genetic components associated with the cooccurrence of these entities. heterogeneous nfkb1 mutations have recently been identified in familial cases of cvid, though rarely have they been associated with pg. objectives: this case describes a novel nfkb1 mutation that may link both cvid and pg, and bolsters the recent identification of heterogeneous nfkb1 mutations in cvid. methods: a 24-year-old woman with a history of frequent skin infections in childhood presented with persistent, infected wounds following cholecystectomy. upon admission, she was started on broad spectrum antibiotics but continued to have fevers and leukocytosis. labs were also notable for elevated crp (340; normal (n):0.1-3) and esr (120; , with low c3 (60; n:81-145), c4 (<10; n:16-39) , and ch50 (<10; n:>74). wound cultures grew multi-drug resistant coagulase negative staphylococcus. despite broad spectrum antibiotics, the wounds failed to heal. dermatology was consulted and punch biopsy revealed a dense neutrophilic infiltrate and no identifiable pathogens, which supports the diagnosis of pg. the patient was started on high dose steroids (1mg/kg/ day) and had a rapid response with decreased skin inflammation and lesion expansion. unfortunately, the patient developed posterior reversible encephalopathy syndrome (pres) on steroids and therefore pg treatment was changed to infliximab as recommended by dermatology. further laboratory testing found that the patient also had low igg (499; n: 700-1600) and iga (34; , and a diagnosis of cvid was made given her clinical history of recurrent skin infections. genetic testing was pursued to evaluate additional pg therapy options and this revealed a heterozygous mutation in nfkb1 (c.a2415g; p.q805q), located 2 base pairs upstream of the splice donor site for exon twenty-one. while this mutation has not been previously identified as a pathogenic variant, similar mutations in this gene have been linked to autosomal dominant cvid. the patients father also carried a similar mutation but without any evident clinical phenotype. results: nfkb1 plays a crucial role in both immune and inflammatory responses. this case highlights a novel mutation in nfkb1 that has not been previously described as a disease-causing change. other mutations resulting in nfkb1 haploinsufficiency have been associated with cvid and rarely with concurrent pg, as in this case. based on the location of the mutation, it is expected that the variant causes obliteration of the normal splice site and therefore results in defective mrna that encodes p50/ p105. interestingly, studies on p50 knockout mice show decreased levels of igg, iga, and ige but not igm and our patient similarly had low levels of igg and iga but normal igm. conclusions: further studies are needed to determine if there are other links to this novel nfkb1 mutation in patients with cvid and pg. (17) submission id#412594 a rapid flow cytometric analysis of dna repair proteins reveals a radiosensitive phenotype in bcl11b deficiency associated with severe combined immunodeficiency (scid). introduction/background: we present the second report in the literature of a patient with immunodeficiency, dysmorphic features, growth retardation, and a homozygous variant in the dna ligase i (lig1) gene with associated absence of full-length lig1 protein. results: this is a now 2 year old girl who was the fourth child of parents who are first cousins. she has one healthy older brother, a second older brother who died within 12 hours of birth of meconium aspiration, and an older sister who died at six months of age of an upper respiratory illness / pneumonia after a history of congenital anemia, poor weight gain, cardiomegaly and hepatomegaly. she was born at 37 weeks and spent the first five weeks of life in a neighboring hospital neonatal intensive care unit (nicu) for hepatomegaly, mild cardiomegaly (previously identified on fetal ultrasound) and congenital anemia (requiring transfusions). her exam was and remains notable for weight, height, and head circumference below 3rd percentile, prominent forehead, hypotelorism with epicanthal folds, downslanting palpebral fissures, and low set, posteriorly rotated and prominent ears. after discharge to home from the nicu, her course was subsequently complicated by poor weight gain, chronic diarrhea beginning after her first rotavirus vaccine, and multiple deep vein thromboses. she then became critically ill at 6 months of age with respiratory failure, and was transferred to our institution for respiratory oscillator support. absolute lymphocyte count on admission to our institution was 0.3 k/μl (total wbc 4.0 k/μl, anc 2.7 k/μl) with agammaglobulinemia. she was diagnosed with and treated for pneumocystis jirovecii pneumonia, gradually weaned from oscillator to room air, and was discharged home five weeks later on 0.4 mg/kg every other week igg replacement. she has had no serious infections requiring hospitalization in the 18 months since. alc has remained persistently below 1.0 k/μl with a corresponding uniform deficiency of t, b, and nk cells and no detectable trec positive t-cells. whole exome sequencing identified homozygosity for a c.914g>a coding region variant in the lig1 gene not previously reported in the literature. a fibroblast cell line was successfully established and western blot shows an absence of full-length lig1 protein. further molecular characterization is in progress. conclusions: this second reported case provides further evidence for dna ligase i deficiency as a distinct clinical entity comprising immunodeficiency, dysmorphic features, and growth retardation. introduction/background: chronic mucocutaneous candidiasis (cmc) is associated with a heterogeneous group of primary immunodeficiencies. autosomal dominant stat1 gain-of-function (gof) mutations have been identified in up to 50% of patients with cmc. these mutations lead to impaired il17a/f t cell immunity although the underlying mechanism is unclear. there seems to be no genotype-phenotype correlation. recently, jak inhibitor therapy has been reported to improve cmc and autoimmunity in patients with stat1 gof mutation. objectives: we describe an infant with cmc associated with a novel stat1 gof mutation. results: a 7-month-old girl was referred to our immunodeficiency clinic with chronic diaper rash since 2 weeks of life, failure-to-thrive, and history of labial abscess complicated by rectolabial fistula. she was subsequently diagnosed with food protein-induced enterocolitis syndrome triggered by cows milk-based formula. laboratory evaluation revealed normal cbc with differential, lymphocyte subsets, mitogen response, immunoglobulin levels, antigen response to candida, and neutrophil oxidative burst assay. whole exome sequencing identified a de novo heterozygous variant in stat1 (c. 1627t >c, p.cys543arg) . further evaluation of this mutation revealed increased gas (gamma activation sequence) reporter activity in response to ifng stimulation suggesting that this is a gain-of-function mutation. the patient later developed significantly elevated liver enzymes while on fluconazole treatment, candida parapsilosis sepsis, granulomatous lesions in the liver, splenic lesions, intermittent thrombocytopenia and n o r m o c y t i c a n e m i a . s e p s i s a n d l i v e r l e s i o n s r e s o l v e d on amphotericin treatment but other findings, including tpn dependency persisted. we are planning to initiate a jak inhibitor therapy, ruxolitinib. conclusions: this case is a possible genotypic and phenotypic expansion of cmc due to stat1 gof. professor, the university of british columbia introduction/background: b cell cll/lymphoma 11b (bcl11b) is a zinc finger protein transcription factor with a multitude of regulatory functions in the integumentary, central nervous, cardiac, and immune systems. it is critical for t cell lineage commitment, development, differentiation, survival, and function. in addition, it also specifies the identity and function of innate-like lymphocytes, including t cells, innate lymphoid cells (ilcs), and invariant natural killer t cells (inkt). however, little is known about its function in the human immune system, especially in the context of immune disorders. objectives: to understand the immunopathogenesis of a novel p.c826y bcl11b variant. methods: research study protocols were approved by our institutional research ethics board. two members of the family were enrolled (the index patient and her father). written informed consent for genetic testing and participation was provided by the parents for the child. genetic, bioinformatic, proteomic, and biochemical analyses were performed. results: we have identified the second described case of immune disease caused by a de novo heterozygous damaging variant of bcl11b (p.c826y). this young girl presented with intellectual disability, microcephaly, severe atopy, eczema, alopecia totalis, and brittle nails. extensive clinical immunophenotyping of patient blood showed initially unremarkable b and t cell populations. however, the patient possessed abnormal rare innate-like lymphocyte populations (inkt, dn t cells). using mass cytometry (cytof), a technique capable of concurrently analyzing 40 parameters in a single cell, we were able to examine various innatelike lymphocyte populations, including t cells, ilc1-3, and nk cells. we found that the patient possessed severely compromised numbers of t cells, thus potentially implicating the p.c826y variant in t cell development and function. conclusions: the identification of decreased t cells in a patient with a p.c826y variant of bcl11b suggests that bcl11b is important for human t cell development and provides novel insights into the roles of both bcl11b and t cells in regulating atopy and autoimmunity. introduction/background: adenosine deaminase 2 deficiency caused by mutations in ada2 gene is a newly recognized disorder. it is associated with a spectrum of vascular and inflammatory phenotypes, ranging from early onset recurrent stroke to systemic vasculopathy or vasculitis. objectives: we describe a 13 year old female patient with features of early onset immune thrombocytopenia (itp), autoimmune hemolytic anemia (aiha), chronic splenomegaly and variable abdominal lymphadenopathy. she was diagnosed with evans-syndrome and treated with rituximab at 19 and 27 month of age. from 3 years of age she developed recurrent infections, hypogammaglobulinaemia with specific antibody deficiency, progressively decreasing class-switched memory b cells, and increased cd3+cd4-cd8-//t cells (4%). differential diagnosis included common variable immunodeficiency (cvid) or autoimmune lymphoproliferative syndrome and therefore a broad search for causative genetic defect was initiated. the parents are first cousins of middle-eastern origin suggesting an autosomal recessive inheritance. patient was stable on long-term mycophenolate mofetil (mmf) and immunomodulatory dose (1g/kg/ month) ivig treatment. methods: genomic dna of the patient was sequenced with next generation sequencing technology. a panel of 250 genes linked to primary immunodeficiency was analyzed. the identified variant was confirmed by sanger sequencing. results: genetic testing revealed a homozygous pathogenic mutation in the ada2 gene with one base pair duplication in exon 2 (c.144dup. p.arg49alafs*13) that creates a frame shift starting at codon arg49. the new reading frame ends at a stop codon 13 positions downstream, likely resulting in a truncated protein. plasma ada2 activity of the patient was markedly reduced (0.2 mu/ml, normal 4.8-27.2) and confirmed the diagnosis of ada2 deficiency. the parents of the patient are heterozygous carriers of the same mutation. unlike most previously reported cases, this patient had an extended phenotype with no neurological evidence of vascular pathology, however brain mri revealed two silent lacunar infarct or vasculitis related changes. we speculate whether the long-term mmf or ivig therapy might be protective against vasculitis. conclusions: ada2 deficiency may present with a wide spectrum of clinical phenotypes beyond classical vasculopathy. the diagnosis should be considered in patients with hematological autoimmune disease, splenomegaly and/or cvid like presentation. better understanding of pathophysiology of ada2 deficiency may help diagnosis and targeted treatment. professor, university of california, los angeles ca introduction/background: adenosine deaminase (ada) deficiency as a cause of severe combined immunodeficiency (scid) is distinct from other forms of scid in several ways. historically, survival and clinical outcome of infants with ada scid have been inferior compared to infants with other scid genotypes. there are multiple treatment modalities available for ada-scid, including enzyme replacement therapy (ert), allogeneic hematopoietic cell transplant (hct) and experimental autologous transplant of gene corrected cells (in recent years preceded by low dose busulfan), designated as gene therapy (gt). in addition, there is a growing body of evidence for effects of ada deficiency on non-immunologic organ systems that may contribute to the historically poorer outcomes of these infants. therefore, it is important to evaluate the cohort of patients with ada scid separately from other scid cases. objectives: to capture incidence and treatment trends and to compare outcomes following available treatments for this rare inborn error of metabolism and other forms of scid, the primary immune deficiency treatment consortium (pidtc), a network of 45 north american immunology and transplant centers, has collected standardized data for analysis. methods: 118 ada scid patients, first treated between 1977 through 2016, were enrolled from 26 centers (range 1-25 subjects/site). ada accounted for 13% of the 926 total pidtc scid patients treated during that time. patients were entered into either a retrospective protocol (pidtc 6902, n=96) or a prospective protocol starting in 2010 (pidtc 6901, n=22) . ada-scid patients who received an hct as first therapy entered either of two strata, as with other scid patients in pidtc studies, based on whether their initial presentation met definitions for typical (n=46) or leaky scid (n=6); in contrast, patients initially treated with either ert or gt were entered into a separate stratum (n=66). results: sixty-four patients (54% of all ada-scid enrollees) had ert as first therapy, but only 6 in this cohort received ert as sole therapy; 58 went on to have subsequent hct (n=30) or gt (n=28). there were various combinations of treatment cycles among these ada-scid patients. most received hct {+/-subsequent treatments} (44%), ert followed by hct {+/-subsequent treatments} (25%), or ert followed by gt {+/-subsequent treatment} (24%); several patients received multiple successive treatment modalities, representing either failure of initial treatment or planned progression from ert to cellular therapy. two-year survival has improved over time from 69% in (1977-2000 to 92% 2001-2016 (p=0.007) (figure 1 ). the survival for all other non-ada scid patients registered by pidtc over these two eras were: 78% (1977-2000) and 81% (2001-2016) . hct (either as sole therapy or after ert) accounted for >95% of cellular therapies between 1986 and 2007; in contrast, since 2007, gt was used as commonly as hct (n=27 vs. n=29, respectively). there was a trend toward better two-year survival for patients receiving gt as first cellular therapy since 2001 (100%, n=28, all after initial ert) compared to those receiving hct over the same time period (87%, n=40, either as first therapy or after ert), although this did not achieve statistical significance (p=0.073). conclusions: this study reveals the improved prognosis for patients with ada scid in recent years and the emergence of gt as a new treatment modality. further analyses are investigating the impacts of prior infection and treatment modality, including effects of conditioning, on outcomes (survival, event free survival, clinical outcomes and completeness of immune reconstitution) for ada-scid in successive eras. this study may identify optimal treatment approaches for future ada scid patients. sponsored by the pidtc, a member of the rare diseases clinical research network (rdcrn) and funded by u54 ai 082973 (niaid and ordr, ncats, nih). dbk has potential financial conflict of interest as a member of the scientific advisory board for orchard therapeutics and an inventor on intellectual property which ucla has licensed to orchard therapeutics related to gene therapy for ada scid. jp discloses that her spouse is employed at invitae, a dna sequencing company. intern, imam abdulrahman bin faisal university introduction/background: patients with diabetes mellitus are immunologically vulnerable population to develop different types of microbial infections. immunization has an important role in infection prophylaxis. in fact, vaccines containing thymus-dependent antigens protect patients with diabetes as they produce massive and complex immune response and feature immunologic memory. the recommended vaccinations for patients with diabetes mellitus are influenza vaccination yearly and pneumococcal vaccination. in observational studies, influenza vaccine has been shown to be similarly effective in adults <65 years of age with diabetes as in older patients with or without diabetes [1] . among immunocompetent elderly, vaccine efficacy of the 13-valent pneumococcal conjugate-vaccine (pcv13) was modified by dm with higher vaccine efficacy among subjects with dm [2] . the hepatitis b vaccination should be given to unvaccinated adults with diabetes mellitus who are ages 19 to 59 years. for older patients administration only after assessment of benefits and risks of acquiring hepatitis b virus (hbv). in fact, one review suggests that dm is associated with the progression of severe liver outcomes in adults with hbv [2] . on the other hand, tetanus and diphtheria vaccinations should be updated. in addition to, vaccinations such tick-borne encephalitis, meningococcal infections and other infections that put in risk diabetic patients travelling abroad. accordingly, theres a variability of vaccines that can offer a preventive method to reduce morbidity, mortality, and medical expense. in our multicenter study among eastern province saudi arabia evaluated the degree of adherence of the physicians to the immunization recommendations for adult patients with diabetes mellitus type 1 and type 2 to increased awareness of the immunization importance in diabetic patients. objectives: to increased awareness of the immunization importance in diabetic patients. in fact, patients with diabetes mellitus are immunologically vulnerable population to develop different types of microbial infections. immunization has an important role in infection prophylaxis. methods: this is a cross sectional study involving 500 adult patients with type 1 and type 2 diabetes mellitus using a questionnaire. patients will be recruited from outpatient clinics including the primary care clinics and inpatient words of king fahd hospital-al khobar and other centers in the eastern province saudi arabia. after an informed consent, baseline data will be collected. patients will be then asked if they received the recommended vaccines and who was the provider. their knowledge regarding the needed immunization will be also tested. they will then be asked about frequency of upper respiratory tract infections and pneumonia they had over the last 2years. results: we are expecting to find low adherence to the recommended immunizations by the physician. we may also find that patients who received the vaccines has low incidence of related infections. conclusions: increased awareness of the immunization importance in diabetic patients and adherence to immunization as part of standard care of adult patients with diabetes mellitus that offer a preventive method to reduce hospitalizations, mortality, and medical expense. 1-10, 11-20, and 21-30 bases. repeatability and reproducibility of the assays were 0.994 and 0.998, respectively. 99.5% of the target regions were covered with over 15x sequencing depth. we showed that the assays had 0.748 sensitivity to detect single-exon deldups and 1.000 sensitivity to detect copy number aberrations covering two or more exons. using acmg guidelines for variant classification a diagnosis was established in 20% patients that were sent for comprehensive panel analysis. conclusions: conclusions: our results demonstrate the analytic validity of the developed tests and show that the technology is well-suited for clinical diagnostics of inherited eye disorders. it also demonstrated a cost-effective diagnostic tool to simultaneously diagnose various types of mutations from snvs to copy number variations. introduction/background: loss of function (lof) and null mutations in orai1 and stim1 cause a rare autosomal recessive immunodeficiency by abolishing calcium release-activated calcium (crac) channel function and store-operated ca2+ entry. the clinical presentation is characterized by scid-like disease, dental enamel defects, muscular hypotonia and anhidrotic ectodermal dysplasia. objectives: here we present the outcome of calcium assessments performed on lymphocytes from an adult patient with unusual infections and a purported novel single pathogenic variant in orai1. methods: the ca2+ response in lymphocytes following activation by varying concentrations of non-cross-linked anti-cd3 was assessed by flow cytometry. results: the patient was a 47 year old female with a history of seasonal and medication allergies and recurrent sinus infections, who had recently developed an acute infection of her right first metatarsal joint. cultures from the joint space grew neisseria gonorrhea and atypical mycobacteria at two different time points. hiv screening was negative. follow-up testing with the mantoux test and quantiferon gold suggested that she also had latent tuberculosis infection, for which she was started on rifampicin therapy. immunologic evaluation revealed normal complete blood count and differential, normal t, b and nk cell counts, normal immunoglobulin g, a, and m levels, as well as normal responses to polysaccharide vaccines and normal t cell proliferative responses to mitogen and antigen stimulation. however, given the identification of atypical organisms from joint fluid cultures as well as latent tuberculosis (despite the lack of significant risk factors), genetic testing was recommended by her local physicians to rule out an underlying primary immunodeficiency. an invitae primary immunodeficiency panel identified a single novel pathogenic variant in orai1. she was then referred to our institution for further evaluation. so far, individuals identified as heterozygous for lof or null mutations in either orai1 or stim1 have lacked any phenotype associated with crac channelopathy. however, there have been reports of abnormalities in calcium response in parents of a number of these patients, who are heterozygous for the disease-causing mutation. to examine the functional effect of the observed mutation in our patient, her freshly isolated pbmcs were loaded with indo-1 and the ca2+ response of her lymphocytes were assessed by flow cytometry. this showed a dose-dependent decrease in the patients t cell response to non-cross-linked anti-cd3 in comparison to the normal control, i.e. there was a clear decrease in the patients ca2+ response at 1 microgram/ml in comparison to the normal control, but the decrease was rectified upon stimulation with 5 microgram/ml or more of anti-cd3. conclusions: these findings provide functional support for the identification of a new pathogenic mutation in orai1. nevertheless, it is not yet clear if the mutation has any mechanistic role in the patients recent clinical presentation. introduction/background: patient 1: 10 years old boy, born to nonconsanguineous parents, with hypothesis of autoimmune encephalitis (vasculitis), which was not characterized. csf has already been routed to barcelona 2 times and to vienna 1 time. refractory epilepsy remains (uses 5 drugs yet). he had continuous fever during the entire hospitalization. he had adhd (took ritalin for 2 years) and central auditory processing deficit. one year ago he began to have fever for 3 days, improving later but evolving with bilateral otitis, predominantly on the left ear, accompanied by sinusitis. soon after that he began presenting epilepsy that worsened severely. he was interned and took acyclovir ev, associated with hydantoinate (had stevens-johnson by the drug, suspending and improving quickly). in march had uti + amo, several infections requiring meropenem + vanco, among others. received flebogamma 500 mg/ day/5 days, twice; he also received weekly rituximab for a few months. in use of carbamazepine, clobazan, phenobarbital, levotiracetam and vigabatrin 3 times a day. personal antecedents: allergic rhinitis, bronchial asthma, recurrent otitis media, iga deficiency. patient 2: male, 9 years old, born to non-consanguineous parents, with history of repeated infections in the upper respiratory tract from one year of age with prolonged dry cough and sore throat in all episodes, treated with dexamethasone without improvement, followed by antibiotics with resolution of the condition. at age five, in february 2012, he had a new episode of sore throat and cough that lasted three months, improving spontaneously thereafter. in june 2012, new episode of sore throat with elevated fever and whitish plaques in the tonsils, being prescribed benzetacil, without improvement in three days. he returned to the same emergency room, the antibiotic was replaced by zinnat (axetil-cefuroxime), but within the hospital he began to convulsionate, entering into an epileptic crisis, being hospitalized for 33 days in this service and being transferred to another hospital specialized in pediatrics, intensely investigated and treated with partial improvement of the condition. he remained in coma, not walking and talking for some months, recovering slowly with physical therapy and speech therapy. of relevant exams have: reduced iga, but before it was normal (probably induced by anticonvulsants); full-body magnetic resonance imaging demonstrates generalized lymphadenomegaly and hepatosplenomegaly; pet-ct showing signs of hypoperfusion in temporal (right), occipital and cerebellum regions (suggestive of hypoperfusion -vasculitis?) my first impression was of possible mevalonate kinase (mvk) or autoimmune lymphoproliferative syndrome (alps) deficiency. with these tests described above, i believe that the first diagnostic suspicion is that the epilepsy was triggered by hemophagocytosis in the central nervous system and consequent extremely severe epilepsy, triggered by ebv infection. objectives: to compare the clinical and genetic similarities and differences of both patients. methods: both patients wer submitted to whole exome sequencing looking for the genetic alterations associated to the disease of these patients. results: wes of patient 1 showed an allelic variant in the gene of rai1 (c.4625g.a; p.arg1542gln) possibly pathogenic and that could be related to the clinical features of the patient. wes of patient 2 showed an allelic variant in the gene of cd27 (c.281g>a; p.arg94his) heterozygous and of uncertain significance. another allelic variant was found in the gene of btk (c.707g>a; p.arg236gln) classified as of uncertain significance and hemizygous (as btk is in chromosome x). the expression of both proteins evaluated by flow cytometry is normal, decreasing but not abolishing the possibility of pathogenicity. conclusions: the similarity of the clinical presentations is striking, but the genetic alterations are totally different, leading to the presentation of this abstract. (inf-) have been associated with adult onset immunodeficiency in patients of asian origin. pathogens that cause infections in these patients include mycobacterium avium-intracellularae (mai), non-typhoidal salmonella, cytomegalovirus, penicillium marneffei, and varicella zoster virus. methods: chart review of one patient results: we present a thirty-two-year-old filipino female, with sjogrens syndrome, penicillin and vancomycin allergy, and shellfish allergy suffering from recurrent mai spinal osteomyelitis. after three months of conservative management of back pain, mri showed an abscess at l4/l5 and l5/ s1 vertebrae, which was diagnosed as acid fast bacilli on biopsy. she was treated for mycobacterium tuberculosis with rifampin, isoniazid, pyrizinamide, and ethambutal with subsequent change in antibiotic therapy after six weeks once cultures grew mai. mri showed spread of abscess to l3-s2 vertebrae. three months into treatment, she was found to have a new abscess at a different spinal site, and antibiotics were again changed. two months later, she had recurrence of disease with multiple large iliopsoas abscesses, cutaneous fistulas, insufficiency fractures of the sacrum bilaterally, and osteonecrosis of the l4 vertebra, requiring extensive surgical debridement. medications were adjusted and she was referred to immunology eight months after initial presentation to infectious disease. laboratories were notable for elevated igg (2580 mg/dl) and iga (474 mg/dl) and decreased cd4 (425 cell/ul) and cd16/56 (49 cell/ul) cells. serum electrophoresis showed low albumin, elevated gamma fraction, and polyclonal gammopathy. specific antibody titers, lymphocyte proliferation assay, ch50, and immunofixation were within normal limits. cytokine panel was significant for elevated il-2 receptor cd25 (3860 pg/ml), il-6(15 pg/ml), and il-13 (12 pg/ml), and normal tnf, inf, il-1, il-2, il-4, il-5, il-8, il-10, il-12, and il-17. further serologic testing was positive for autoantibodies to inf-. patient continues treatment with iv antibiotics and is awaiting enrollment in a rituximab trial. conclusions: autoantibodies to inf-should be considered in patients of asian origin presenting with adult onset immunodeficiency, particularly those with severe or recurrent infection with mai. a high level of suspicion is required to make this diagnosis: failure to consider this disease entity leads to delay in diagnosis with potentially significant consequences for the patient. allergy/immunology, university of south florida at johns hopkins all childrens hospital introduction/background: autoimmune and inflammatory conditions are common in cvid. these have been associated with increased morbidity and mortality. objectives: we sought to further understand and evaluate the prevalence of autoimmune and rheumatologic manifestations in patients with common variable immunodeficiency (cvid). methods: we performed a retrospective analysis of cvid patients with rheumatologic/autoimmune complications in the partners healthcare cvid cohort. we evaluated baseline patient characteristics as well as autoimmune and rheumatologic complications in this cohort of patients. results: in the partners cvid cohort, 120/210 (57%) had autoimmune or rheumatologic disease. autoimmune cytopenias were reported in 37/210 (18%) patients, including coombs positive autoimmune hemolytic anemia (n=18), idiopathic thrombocytopenic purpura (n=30), and autoimmune neutropenia (n=10). autoimmune thyroid disease was reported in 43/212 (20%) patients, including hypothyroidism (n=25) and hashimotos thyroiditis (n=18). inflammatory arthritis was present in 35/210 (17%), most commonly seronegative rheumatoid arthritis (ra) (n=18), followed by inflammatory arthritis (n=8), seropositive ra (+rf or +ccp antibody) (n =6), psoriatic arthritis (n=2), and juvenile idiopathic arthritis (n=1). systemic autoantibody disease was diagnosed in 26 patients (12%), with diagnoses including vasculitis (n=7), systemic lupus erythematosus (n= 6), polymyalgia rheumatica (n=4), antiphospholipid syndrome (n=3), mixed connective tissue disease (n=2), crest/ scleroderma (n=2), myositis (n=2), sjogrens syndrome (n=2), and discoid lupus erythematosus (n=1). inflammatory neuropathy was diagnosed in 21 patients, with small fiber polyneuropathy (n=10), uveitis (n=3), myasthenia gravis (n=2), bells palsy (n=2), and multiple sclerosis (n=1). autoimmune skin conditions were diagnosed in 24 patients with diagnoses including psoriasis (n=13), alopecia (n=8), and vitiligo (n=3). while the mean igm was higher in the patients with autoimmune/rheumatologic manifestations than in other cvid patients (83 vs 57mg/dl), this difference did not reach statistical significance (p=0.15). conclusions: autoimmune and rheumatologic complications are present in over half of patients with cvid. increased vigilance for autoimmune and rheumatologic complications is important as survival outcome are worse in cvid patients with non-infectious complications as previously described. further evaluation of these patients to understand the mechanism of immune dysregulation is essential, as this may promote targeted therapies and improve clinical outcomes. introduction/background: immunoglobulin concentrates have been successfully used for decades to treat patients with primary or secondary immunodeficiency disorders. this treatment has substantially decreased the frequency of life-threatening infections in these patients. octanorm is a newly developed maltose-formulated subcutaneous immune globulin (human) 16.5% liquid for the treatment of patients with primary immune deficiency (pid) and secondary immune deficiency (sid). objectives: biochemical and physico-chemical properties were investigated. methods: molecular size distribution of monomers, dimers, polymers and fragments were determined (according to european pharmacopeia (ep) monograph 8.0) by size exclusion chromatography (sec). igg and igg subclass concentrations were quantified by respective nephelometric methods. functionality of the igg was demonstrated by measurement of fc function, opsonophagocytosis and fc gamma receptor binding assays. dynamic light scattering measurement and size exclusion chromatography were used to characterize the integrity of the igg molecule. measurement of potential procoagulant activity was done by natem and tga (fxia-like activity). the capacity of the octanorm manufacturing process to robustly inactivate/remove pathogens was investigated in spiking experiments with prions and viruses. results: octanorm contains more than 96 % of human igg and is characterized by an especially low content of polymers and aggregates, low viscosity, low isoagglutinin titres, low iga and igm contents with a broad spectrum of antibodies against infectious agents. it has a distribution of immunoglobulin g subclasses closely proportional to that in native human plasma. in the final product, potential procoagulant activity is not detectable. functionality and physico-chemical properties of the igg molecules were demonstrated by state-of-the-art methods. virus safety of octanorm is obtained via a combination of three validated orthogonal methods as part of the manufacturing process: cold-ethanol fractionation, solvent/ detergent (s/d) and ph 4 treatment. a substantial depletion of prions during the manufacturing process was demonstrated. conclusions: octanorm is a state-of-the-art subcutaneous immunoglobulin. based on the excellent stability the intended shelf life of octanorm is 24 months stored at +2°c to +8°c protected from light. within its total shelf life the product can be stored at room temperature up to +25°c for up to six months. efficacy and very good tolerability of this new subcutaneous normal immune globulin 16.5% were shown in a clinical phase iii study performed in 18 centers in north america and europe. professor, sapienza university of rome introduction/background: primary antibody deficiencies (pad) are characterized by defective ig production resulting in high susceptibility to bacterial infections, especially caused by s. pneumoniae and h. influenzae. there is a limited evidence on the rate of microbial airway epithelial colonization and on the role of bacterial carriage on the development of recurrent respiratory tracts infections in such populations. objectives: the aim of this study was to investigate the prevalence of s. pneumoniae and haemophilus influenzae colonization in pad adults in italy and its clinical and immunological correlates. methods: nasopharyngeal and oropharyngeal swabs were obtained from 93 cvid and 16 patients with idiopathic primary hypogammaglobulinemia (iph) over 18 years of age and under ig replacement treatment during the period october 2016-april 2017. presence of s. pneumoniae and h. influenzae was investigated using conventional cultural methods and rt pcr. s. pneumoniae isolates were serotyped by the quellung reaction; capsular type of h. influenzae isolates was determined by pcr. the pattern of associations between the two species and potential risk factors were investigated. respiratory infections rate was recorded over 12 months of follow up. results: among cvid prevalence of carriage assessed by traditional culture was 11% and 27% for s. pneumoniae and h. influenzae, respectively. rt pcr allowed to identify a higher rate of carriage of s. pneumoniae and h. influenzae compared to standard culture. cvid and iph had not different rate of pneumococcal colonization, whereas cvid had higher rate of h. influenzae carriage identified by a culture methods and rt pcr. no synergistic association between s. pneumoniae and h. influenzae colonization was observed. among cvid, s pneumonia and h. influenzae carriage were associated to low iga and igm levels. cvid under antibiotic prophylaxis did not have an increased prevalence of carriage. no association was found between carrier status detected by culture and having chronic lung disease, bronchiectasis or the rate of infections during the follow up. rt pcr identified merely the association between igm levels and h. influenzae carriage. antibiotic resistance from isolated stains was also assessed. conclusions: this is the first study assessing the prevalence of s. pneumonia and h. influenzae carriage in cvid. objectives: to discuss clinical challenges in a subject with congenital hair hypoplasia detected on newborn screening with preserved t-cell mitogen responses but declining naive t-cell counts. parents are jehovah's witnesses, which adds complexity to clinical decisionmaking. methods: immune evaluation included complete blood count, lymphocyte subsets, flow cytometry to define t-cell subsets, immune globulins, repeat trec assay from peripheral blood, human immunodeficiency virus (hiv) and cytomegalovirus (cmv) dna pcr, screen for maternal engraftment, chromosome microarray analysis, lymphocyte proliferation to mitogens, t-cell proliferation to interleukins, t-cell receptor v-beta diversity analysis by spectratyping, and thymic ultrasound. results: repeat trec assay from peripheral blood on day 17 confirmed undetectable trecs. cd4+ thymic emigrants were low at 147 (reference 733-3181 cells/ul). naïve cd4+ and cd8+ t cell compartments declined as did naïve cd4+ t cells from 48 to 17 cells/ul and naïve cd8+ t cells from 33 to 5 cells/ul. lymphocyte proliferation to mitogens was preserved except for the cd19+ response to pokeweed mitogen. interleukin proliferation of cd45+ lymphocytes was slightly decreased after stimulation with anti-cd28 (14%, normal >38%). however, t-cell proliferation with other interleukins (il-2, anti-cd3+il-2, anticd28 as %cd3) was preserved. the t cell receptor repertoire had intermediate diversity. antibody replacement therapy was prescribed for declining igg with no history of severe infections except for rhinovirus prior to discharge from the nicu at 8 weeks, during which she required continuous positive airway pressure (cpap) therapy. the viral load for cmv and hiv dna were undetectable. breastfeeding was discontinued early because the mothers cmv serology revealed positive cmv igg. the child has had intermittent anemia, which is common in patients with chh. [2] tests for autoimmune hemolytic anemia were not performed because the patient required ivig from an early age. the parents of the child are jehovahs witnesses, complicating requests for blood transfusions. conclusions: this case highlights challenges in clinical decision making for a newborn with chh identified on nbs. t-cell function is preserved, but declining naïve t cell counts and absent trecs lead us to consider hematopoietic stem cell transplant (hsct). indication and timing of elective hsct is unclear and may depend on the natural progression of disease, including infections and anemia division of immunology and allergy, the hospital for sick children introduction/background: cartilage-hair hypoplasia (chh), caused by mutations in the ribonuclease mitochondrial rna-processing (rmrp) gene, is associated with diverse immune abnormalities including combined immune deficiency (cid). most patients with chh are managed with supportive measurements, while few have received allogeneic hematopoietic stem cell transplantations (hsct). the progression of the immune abnormalities and the impact of hsct in patients with chh and cid have not been well characterized. objectives: to characterize the progression of the immune abnormalities and the impact of hsct in patients with chh and cid methods: the clinical and laboratory findings of 2 siblings diagnosed in infancy with chh and cid due to the common 70 a>g mutation in rmrp, including the effects of hsct performed in 1 of them, were compared. results: both patients suffered from recurrent respiratory infections at early age with reduced t cells numbers and responses. patient #1 immune function continued to deteriorate leading to hsct from an hla-matched sibling at 4.5 years of age. the patient suffered acute and chronic graft versus host disease of the skin with residual mild joint contractures and scleroderma-like skin changes. seven years after hsct patient #1 has normal immune function. immune evaluations of patient #2 in the first years of life indicated mild improvement. the patient did not have a suitable related hsct donor and the family elected to continue with supportive care. at 7 years of age, patient #2 is clinically well and thriving with persistent t cell abnormalities. conclusions: close monitoring of immune function in early life for patients with chh and cid as well as the availability of suitable donors assists in determining management, including hsct introduction/background: leukocyte adhesion deficiency (lad) represents a group of distinct inherited disorders, which inhibit the normal extravasation of neutrophils and their recruitment to sites of infection or inflammation. objectives presentation of case: the patient is a girl of 8 years old with no history of primary inmunological disease (pid) in family members, including a healthy brother (currently 12 years old). she received all the immunization schedule until 1 year old (according to peruvian schedule). she had several admission to the hospital since newborn; the 1st hospital admission was at 15 days of life with diagnosis of: sepsis, pneumony, onphalitis (leukocytes count: 70 000), she had 3 more hospital admission and the leukocytes count were always above 25000. in summary, she presented other infections besides the ones admitted at hospital like: 2 episodes of sepsis, 1 episode of pneumony, 5 episodes of cellulitis (left eye, left elbow, right thigh and vaginal (2), 27 episodes of otitis, 7 episodes of tonsillitis, 6 episodes of diarrhea, 5 episodes of rhinoadenoiditis, 5 episodes of sinusitis, 3 episodes of gingivitis and 1 episode of whooping cough. she received different antibiotics for treatment, even broad-spectrum as vancomycin, meropenem and cefepime. the diagnosis of leukocyte adhesion deficiency (lad) was made at 1 year 9 months by clinical features like delaying in separation of umbilical cord, recurrent infections and persistent leukocytosis>25000. flow citometry was taken at 3 years old, resulting cd11b/cd18: 0,21%. results: discussionf: or the severe phenotype, in which leukocytes express <1% of normal levels of cd18, death occurs at an early age because of severe infection unless patients receive bone marrow transplant. however, it does not happened with her so we suspected in a possible reversion in lad1 so we took a second flow citometry at 5 years old and the results were: total leukocyte: 27870; linfocyte: 6777 cd11b+/cd18+: 0,04%; granulocyte:17438 cd11b+/cd18+: 1,41% and the conclusion was c3 receptor (cd11b/cd18) absent in linfocyte, monocyte and granulocyte. molecular study was taken at 7 years old, resulting a homozygous substitution c.562c>t identified in exon 5, causing a nonsense mutation: p.arg188, confirming lad1 diagnosis. she is, currently, receiving profilactic antibiotic and antimicotic which has reduced considerably recurrent infections. it seems that our patient may have a mixed phenotype due to a clinical expression, which may not require hematopoietic cell transplantation (hct) despite features of the severe type. conclusions conclusion: we conclude that the clinical evolution of this patient is unsual because she has severe lad1, she has not transplanted yet, profilaxis treatment has improved to decrease frecuency of infections, she exceeded life expectancy and last flow citometry confirmed that lad1 was not reverted. , ph.d. 5 , luigi d. notarangelo, md 6 , troy r. torgerson, m.d. ph.d. 7 , ph.d. 8 , hans d. ochs, m.d. 9 , m.d., ph.d. 10 introduction/background: patients with x-linked hyper-igm syndrome (x-higm) due to cd40 ligand (cd40l) deficiency often present with low blood neutrophil counts. however, even when not neutropenic and despite immunoglobulin (ig) replacement therapy, cd40l-deficient patients are susceptible to life-threatening infections by opportunistic pathogens, suggesting impaired function of phagocytes, and requiring novel therapeutic approaches. objectives: to analyze whether peripheral neutrophils from cd40l-deficient patients display functional defects and to explore the in vitro effects of recombinant human interferon (rhifn)-on such cells. methods: we investigated the microbicidal activity, respiratory burst and transcriptome profile of neutrophils from cd40l-deficient patients. in addition, we evaluated whether the lack of cd40l in mice also affects neutrophil responses. results: neutrophils from cd40l-deficient patients exhibited defective respiratory burst and microbicidal activity which were significantly improved in vitro by rhifn-. similar to humans with cd40l deficiency, cd40l-deficient mice were found to have defective neutrophil responses. moreover, neutrophils from cd40l-deficient patients showed reduced cd16 protein expression and a dysregulated transcriptome profile suggestive of impaired differentiation. conclusions: our data suggest a non-redundant role of cd40l-cd40 interaction in neutrophil development and function that could be improved in vitro by rhifn-, indicating a potential novel therapeutic application for this cytokine. methods: in this study, through the use of 23 healthy livers, paired peritumoural tissues (pt) and intratumoural tissues (it) from 236 hcc patients. results: increased expression of cd96 on nk cells was observed in intratumoral but not peritumoral regions, along with increased expression of its ligand cd155 and a poor prognosis. human cd96+ nk cells exhibited functional exhaustion, showing decreased ifn-and tnf-productions, impaired cytolysis in response to in vitro stimulation, and high gene expression of il-10 and tgf-1 with low expression of t-bet, il-15, perforin and granzyme b by global transcriptomic analysis of sorted cd96+ and cd96-nk cells. blocking tgf-1 specifically inhibited cd96 expression and reversed the dysfunction of nk cells. in addition, we compared other two receptors, cd226 and tigit, which share common ligand cd155 with cd96, and found cd96 plays a more important role in nk exhaustion. conclusions: these findings indicate that human cd96+ nk cells have features of functional exhaustion, suggesting that cd96-cd155 blockade has the potential to restore immunity against liver tumors by reversing nk cell exhaustion. introduction/background: mutations in ncf1 (encoding protein p47phox of the nadph oxidase) result in an autosomal recessive form of chronic granulomatous disease (cgd), a rare genetic disease with impaired phagocyte production of reactive oxygen species and recurrent infections. diagnosis of p47phox cgd is based on abnormal dihydrorhodamine assay and absence of p47phox protein by immunoblotting; however, these assays fail to diagnose carriers of ncf1 mutations. instead, carrier status is inferred after the birth of a child with p47phox cgd. furthermore, identification of the specific genetic defect in patients with p47phox cgd is complicated by two highly conserved (>98%) pseudogenes. the ncf1 gene has a gtgt at the start of exon 2, while the pseudogenes (ncf1b and ncf1c) delete one gt (gt). in p47phox cgd, the most common mutation in ncf1 is gt causing c.75_76delgt; p.tyr26fsx26. sequence homology between the wild type gene and pseudogenes precludes using standard sanger sequencing to identify specific mutations in ncf1. objectives: to identify phenotypic and genotypic differences that facilitate the diagnosis of patients and carriers with p47phox cgd. methods: expression of p47phox in neutrophils is determined by fixing and permeabilizing whole blood with intraprep, and then incubating with either anti-p47phox antibody or its corresponding isotype. alexafluor 488-conjugated secondary antibody is used to detect the target antigen. expression of p47phox is based on the mean fluorescence intensity of cells within the neutrophil population gated using forward and side light scatter. differential expression of p47phox by flow cytometry is validated using quantitative immunoblotting. to screen for the gt mutation, a droplet digital polymerase chain reaction (ddpcr) with two distinct probes recognizing either the wild-type gtgt sequence or the gt sequence was used to quantitate the ratio of gtgt vs. gt copies. a second ddpcr reaction established copy number by comparing one probe for an invariant region of ncf1/ncf1b/ncf1c and a second probe for the single-copy telomerase reverse transcriptase gene, tert. the results of these two assays were combined to determine the total number of gtgtcontaining and gt-containing ncf1 copies. results: analysis of p47phox expression in permeabilized neutrophils determined that neutrophils from p47phox cgd patients had negligible p47phox expression; neutrophils from p47phox cgd carriers exhibited~60% of p47phox expression compared to healthy volunteers independent of the mutation in ncf1. of all p47phox cgd patients tested by ddpcr, 83.2% (109/ 131) exhibited 0 copies of gtgt, 6.9% (9/131) exhibited 1 copy of gtgt (compound heterozygotes with 1 non-gt mutation), and 9.9% (13/131) exhibited 2 copies of gtgt (two non-gt mutations). moreover, ddpcr can identify the carriers among kindreds within the gt p47phox cgd families. unexpectedly, among normal subjects tested, only 78.8% exhibited the expected 2 copies of gtgt per 6 total ncf1/ncf1b/ncf1c copies, designated 2/6; a significant number exhibited more than two copies of gtgt (14.6% with 3/6, 1.9% with 4/6); others exhibited ncf1/ncf1b/ncf1c copy number variation (2.8% with 2/7 and 1.9% with 2/5). conclusions: flow cytometric analysis of neutrophil intracellular p47phox staining provides a quick method to identify patients and carriers of p47phox cgd. droplet digital pcr can be used to identify patients and carriers of p47phox gt, the most common mutation in p47phox cgd copy number variation is observed at the ncf1 locus among normal subjects tested. introduction/background: defects in the cd3 subunits of the tcr/cd3 complex account for a small percentage of the scid presentations. cd3 deficiencies are characterized by profound and t-cells lymphocytopenia, and normal numbers of b-and natural killer (nk) cells in the peripheral blood (t-b+nk+ scid). thymocyte development from double negative stage to double positive stage results arrested. affected individuals typically present with severe and opportunistic infections in the early infancy. objectives: to evaluate possible underlying immunodeficiency disorder in the setting of chronic ebv infection. methods: here we report our experience of a patient with an atypical presentation of cd3 deficiency. results: a 7-year-old girl previously healthy, first generation americanborn of gambia immigrants was referred to our clinic for further evaluation of chronic ebv infection. one year before presentation, she developed bilateral parotid enlargement, cervical, axillar, and hilar lymphadenopathy, bronchiectasis, and pulmonary nodules. relevant previous studies included: ebv viremia (pcr quant 63,000 copies), ebv igg and ea positive whereas igm and ebna were negative. peripheral blood phenotyping was not suggestive for malignant process and cervical lymph node biopsy was consistent with a reactive process without evidence of a clonal lymphoproliferative disorder and bal studies positive for ebv only. hiv and tb both negative. she was otherwise thriving with no history of recurrent infections and family history was negative for consanguinity or immunodeficiency. our evaluation revealed: normal blood counts, ebv pcr quant 206,677 copies, normal immunoglobulin levels and vaccine titers. she had a normal lymphocyte subsets, but skewed cd45 ra/ro consistent with low thymic output (very low cd4 naïve t cells (5.8%), low cd31+ naïve t cells (36.7%), and elevated temra (42.6%)), poor mitogen, and antigen responses. all these findings were suggestive of a scid like phenotype; therefore, scid next generation sequencing panel was pursued, and showed a novel homozygous splice site mutation (c.55 +1 g>t) in the cd3 gene. conclusions: a homozygous mutation in the cd3 gene might not necessarily imply profound t-cell lymphopenia. though this patient did not present with classic clinical course, and immune findings; her inability to clear ebv with persistent significant viremia does support a t-cell immune deficiency. she remains at risk for ebv-associated lymphoproliferative disorder, infections, and autoimmunity. bmt will be a curative treatment option. however, it is difficult to predict evolution of clinical phenotype given the atypical presentation. this case illustrates the importance of contextual interpretation of clinical findings, laboratory data, and genetic analysis for treatment approach. introduction/background: pol is multi-subunit polymerase that includes both pole1 with catalytic activity and additional pole2, 3 and 4. this holoenzyme plays a key role in proofreading damaged dna and is required for proper dna replication in proliferating cells, such as lymphocytes. germline mutations are linked to rare cause of primary immunodeficiencies whereas somatic mutations are described in colon cancer. primary immunodeficiencies are reported pole-1 deficiency in members of a large consanguineous french kindred and a palestinian female with fils (facial dysmorphism, immunodeficiency, livedo, and short stature). all reported cases with pole1 deficiency have homozygous intronic splice site variant (c.4444+3a>g) that result in a deletion of exon 34 which lead to subsequent frame shift (from p.s1483v onwards) and a premature stop codon at position 1561; this transcript results in a degraded product. the proportion of the pole1 transcript in t lymphoblasts is significantly lower (10%) in patients then carriers or healthy individuals. objectives: hereby we describe the clinical progression and treatment challeneges of the palestinian female with pole-1 deficiency secondary to homozygous g.g4444+3 a>g substitution. results: initially patient presented with viral and recurrent ear infections and cmv viremia. with age, patient had less episodes of infections even with intermittent pause in immunoglobulin replacement therapy (igrt), however had multiple admissions for fever of unknown origin with negative cultures but increased ferritin level (3300 ng/ml) and low platelet count (46,000 count/ml). autoimmune and inflammatory complications were not reported among the french kindred. her skin also worsened with poor wound healing and scarring throughout that hinders igrt via subcutaneous route. furthermore, igrt with intravenous administration has resulted in symptoms of aseptic meningitis likely related to the underlying inflammatory state. in addition, patient shows decline in immune dysfunction. initially, she had normal immunoglobulin g (igg) however by 5 years of age, she developed hypogammaglobulinemia with low igm unlike in the french family with patients. also, pneumococcal, diptheria, tetanus titers were non-protective off of immunoglobulin replacement therapy (igrt). beyond low switched memory b cells, patient also developed low b cell by 5 yo age. t cell dysfunction continued to decline from decreased lymphocyte proliferation to antigens at 2 yo age to fully absent lymphocyte proliferation to antigens and mitogens. naïve cd4 and cd8 compartments continue to be preserved. currently management challenges include treatment strategies for thrombocytopenia, inflammation and progressive skin disease that complicates the proper selection of route for optimal igrt. conclusions: beyond progression of immunological decline, our patient developed inflammatory phenotype with age. the progression of her immunological decline may be related to further decrease in the proportion of the wild type pole1 transcript and yet to be examined. overall, clinical follow up is essential in patients with pole-1 deficiency as phenotype can change with age and may pose new challenges. longitudinal follow up studies are needed to uncover the potential role of germline pole1 and pole2 pathogenic variants in cancer susceptibility. introduction/background: x-linked hyper-igm syndrome (xhigm) is one type of primary immunodeficiency diseases, resulting from defects in the cd40 ligand/cd40 signaling pathways. objectives: here, we retrospectively reviewed clinical, laboratory and genetic characteristic of xhigm in chinese population, thus further improving diagnosis and treatment for xhigm. methods: we collected and analyzed 47 chinese patients, who were diagnosed and followed up in hospitals affiliated to shanghai jiao tong university school of medicine from 1999 to 2016. targeted gene capture combined with next-generation sequencing technology and sanger sequencing were used to find out related gene mutation. results: the median onset age of these patients was 7 months (range: 20 days66 months). thirty-six percent of them had positive family histories, with a shorter diagnosis lag. the most common symptoms were recurrent sinopulmonary infections (40 patients, 85%), neutropenia (22 patients, 47%), protracted diarrhea (21 patients, 45%), and oral ulcer (19 patients, 40%). ten patients had bcgitis. six patients received hematopoietic stem cell transplantations and four of them had immune reconstructions and clinical remissions. twenty-seven unique mutations in cd40l gene were identified in these 47 patients, with 18 novel mutations. conclusions: to our knowledge, this report provides the largest cohort of patients with xhigm in china. mutation analysis is an important tool for xhigm diagnosis. infants with scid are asymptomatic at birth and unless prompt diagnosis of the disease is made they may horrifically be vaccinated. simultaneous appearance of two live vaccine associated infections in one person is rarely reported. objectives: in this study we present two infants with scid, who received bcg and oral polio vaccines early in life before the diagnosis of immune deficiency was made. both patients developed localized and disseminated infections originating from the bcg vaccine (bcgitis and bcgiosis, respectively) and in addition were diagnosed with chronic fecal secretion of vaccine-derived polio virus (vdpv); alarmingly, in both cases, the vdpv underwent reverse mutation of attenuated sites to the neurovirulent genotype. the rarity of concomitant infection from two live vaccines in one recipient, together with the multiple complexities originating from these infections in immunodeficient infants, led us to report these cases and to inquire the pathogenesis that underlies this unique condition. methods: immunological evaluation:: cell surface markers of peripheral blood mononuclear cells (pbmcs) were measured by immunofluorescent staining and flow cytometry serum concentrations of immunoglobulins were measured using nephelometry. quantitative analysis of the tcr v repertoire was performed by means of flow cytometry. quantification of t cell receptor excision circles (trecs) was determined by real-time quantitative (rq)-pcr. genetic analysis: genetic diagnosis of scid was made for patient 1 by direct sanger sequencing of candidate genes and retrieval of mutation in rag2 , and for patient 2 by wes (whole exome sequencing), followed by validation of the dna cross-link repair 1c (dclre1c) mutation using sanger sequencing. poliovirus detection and characterization: stool samples were collected monthly and transported to the national poliovirus laboratory located at israel central virology laboratory (icvl) for polio detection and characterization. results: in both patients, immunological workup revealed undetectable serum iga and igm levels with normal igg levels (table 1) , lymphocyte immune-phenotyping using flow cytometry revealed complete absence of t and b cells with presence of nk cells (table 2) . trecs, a dna marker of naive t cells and thymic output, were absent in both patients. the diagnosis of scid was made. we initiated prophylactic antibiotic (trimethoprim-sulfamethoxazole) and anti-fungal (fluconazole) treatment, as well as monthly intravenous immunoglobulin (ivig) infusions. genetic workup: the t-b-nk+ scid phenotype in patient 1 led us to search for a mutation in the rag complex genes. indeed, a sanger sequencing of the rag2 gene, revealed a g104t homozygous mutation which predicts an amino acid substitution from glycine to valine in position 35 (fig.1 ). for patient 2, who had a similar t-b-nk+ scid phenotype, we identified a homozygous mutation in the dclre1c gene (del.4bp, c.817-cttt) (fig. 2) , using wes. the genetic evaluation confirmed the diagnosis of scid due to rag2 deficiency in patient 1 and artemis deficiency in patient 2. clinical course: during hospitalization patient 1 developed disseminated bcg related disease with skeletal lesions involving the phalanx, tibia and maxillary bones, as well as involvement of the spleen, liver and pancreas. anti-tubercular therapy with isoniazid, rifampicin, ethambutol and ciprofloxacin was initiated. due to lack of response, empirical trial of g-csf (granulocyte colony-stimulating factor), in order to enhance macrophage activity . the patient showed good response to this combination therapy. patient 2 developed a palpable rigid mass on her left shoulder with surrounded redness at the site of the bcg vaccine at the age of 5 months. the clinical diagnosis of bcgitis was established and triple antitubercular therapy with isoniazid, rifampicin and ethambutol was initiated with good response. throughout their hospitalization, both infants suffered from intermittent diarrhea. pcr for enterovirus was performed and detected the presence of type 3 and type 2 vaccine-derived poliovirus (vdpv) in patient 1 and 2, respectively. during the follow-up, stool samples collection revealed accumulation of several polio virus mutations and some of the neuro-virulence attenuation sites were reverted to the neuro-virulent genotype .fortunately, both patients did not show any signs of flaccid paralysis. precautionary measures of isolation were taken to prevent spread of the vdpv. eventually, both patients underwent allogeneic bone marrow transplantation (bmt): patient 1 had bmt without pre-conditioning, from a matched sibling donor. due to engraftment failure, a second bmt was repeated, this time successfully. on follow up examination her t cell repertoire showed a normal tcr v polyclonality and trec was detected, indicating the emergence of new t cells. due to ongoing low immunoglobulin levels this patient is still on regular ivig-infusions and prophylactic antibiotic treatment, as well as isoniazid. currently, she is well, her bcgitis is not active and her stool specimens are negative for polio. patient 2 underwent an urgent haplo-identical bmt with alpha-beta t cell depletion without pre-conditioning, due to her unstable medical condition. flow cytometry analysis six months post bmt revealed lymphopenia of 7.7% (1334/mm³) with low mature t cells (cd3 32%) and absent b cells. trecs were barely detected. microsatellite analysis as a marker for engraftment revealed a stable donor chimerism of 10%. the patient is still on immunosuppressive therapy doing well, her bcgitis is not active and her stool specimens are negative for polio. conclusions: these cases highlight the importance of early recognition of scid by neonatal screening or thorough family anamnesis, and the need to further defer the timing of administration of live vaccines. introduction/background: measurement of b cells, b cell subsets and specific antibodies produced in response to vaccination are key tests used to investigate immune system function. specific antibody production may indicate b cell functionality. objectives: in this study the correlation between the different b cell subsets and antibody responses to pneumovax® in an immunocompromised population was investigated. methods: b cell subsets were assessed by flow cytometry and pneumococcal responses measured using the vacczyme pneumococcal capsular polysaccharide (pcp) igg, iga and igm elisas (the binding site group, birmingham, uk) in 39 primary immunodeficiency patients (pid) vaccinated with pneumovax®. lower limits of normal were defined as follows: b cells: 6.6%; naive b cells: 65.6%; non-switched memory b cells: 7.4%; switched memory b cells: 7.2%; pcp igg: 50 mg/l; pcp iga: 125 u/ml; and pcp igm: 140 u/ml. results: the correlation coefficients between percentage of b cells/b cell subsets and igg, iga or igm pneumovax® responses ranged from -0.61 to 0.39. the percentage of the cohort achieving a normal b cell and normal igg, iga or igm response to pneumovax® was 45%, 29% and 40% respectively. b-cell responses were measureable in the remaining patients but they did not produce normal concentrations of pcp igg, iga or igm. further stratification of patients who achieved normal percentage of switched or un-switched b cells but who failed to achieve normal igg, iga or igm responses to pneumovax® were 32%, 55% and 50%, respectively, for un-switched and 22%, 47% and 33%, respectively, for switched b cells. conclusions: the combined measurement of b cells and response to vaccination are required to provide a detailed insight into these disorders. introduction/background: this is a 2-year-old boy of african american heritage with multiple congenital malformations who was diagnosed with very early-onset inflammatory bowel disease, which proved to be resistant to treatment. subsequent testing revealed a heterozygous mutation in exon 1 of ctla4. this variant has not been previously reported in the literature in individuals with ctla4-related disease. heterozygous mutations in ctla4 cause a disease of immune dysregulation. clinical presentation is variable and may be characterized by enteropathy, hypogammaglobulinemia, granulomatous lymphocytic interstitial lung disease, lymphocytic organ infiltration in non-lymphoid organs, autoimmune cytopenias, and recurrent infections. early onset colitis has been reported with ctla4-related disease and is unique to our patient's initial clinical presentation. objectives: this is a 2-year-old boy with cloacal exstrophy of the urinary bladder, omphalocele, imperforate anus, polydactyly, and sacral agenesis who was diagnosed with very early-onset inflammatory bowel disease at six months of age. he underwent cloacal exstrophy closure, omphalocele repair, and colostomy placement in the first week of life. at six months of age, he presented with dark tarry stools. upper endoscopy and colonoscopy revealed polyps, ileitis, and colitis. he was p-anca positive and started on sulfasalazine. unfortunately, he continued to have symptoms suggestive of active colitis, prompting a change to prednisone and azathioprine. despite therapy, his colitis persisted leading to chronic bloody diarrhea and growth failure. initial immune evaluation consisted of a normal complete blood count, serum immunoglobulin, lymphocyte subsets, and neutrophil oxidase burst assay. foxp3 analysis by flow cytometry showed a moderately elevated percentage of foxp3+cd25+ cells in the cd4+ t cell population, but the regulatory t cell immunophenotype was normal. because suspicion was high for a monogenic immunologic disease to explain his symptoms, genetic sequencing was performed. a candidate gene panel was sequenced by next generation sequencing, and a heterozygous mutation in exon 1 of ctla4 (c.23g>a; p.arg8gln) was found. this variant has not been previously reported but is predicted to be pathogenic in exac and polyphen databases. results: the patients diagnosis of ctla-4 haploinsufficiency associated with very early-onset inflammatory bowel disease has provided opportunity for targeted treatment of his specific molecular defect. given his poor response to treatment thus far, the patient will be started on abatacept. abatacept is fda approved for the treatment of rheumatoid arthritis but has been used successfully for the treatment of disease-related manifestations of ctla-4 haploinsufficiency. abatacept is a ctla-4 fusion protein formed by the igg1 fc region linked with the extracellular domain of ctla-4; it replaces the defective protein in ctla-4 haploinsufficiency. in addition, given other manifestations of ctla-4 haploinsufficiency including lymphoproliferative disease in non-lymphoid organs, particularly the brain and lung, we have initiated further evaluation of these organs to evaluate for disease-specific manifestations. conclusions: the protein cytotoxic t lymphocyte antigen-4 (ctla-4) is an essential negative regulator of t cells. heterozygous mutations in ctla4 cause a disease of immune dysregulation. clinical presentation is variable and may be characterized by enteropathy, hypogammaglobulinemia, granulomatous lymphocytic interstitial lung disease, lymphocytic organ infiltration in non-lymphoid organs, autoimmune cytopenias, and recurrent infections. inflammatory bowel disease may be associated with certain variants in ctla4, but the literature remains limited, both by number of papers published as well as by ethnic subsets studied. clinicians who are presented with children who have early-onset colitis, and particularly inflammatory bowel disease that is difficult to treat, should consider possible genetic abnormalities, such as ctla-4 haploinsufficiency, as these can impact therapeutic decision-making and outcomes. introduction/background: dock8 deficiency is an autosomal recessive combined immunodeficiency syndrome associated with recurrent infections, eczema and other atopic diseases. the infections are usually viral, bacterial and fungal resulting in predominantly cutaneous and sinopulmonary manifestations. homozygous or compound heterozygous deletions or mutations in the dock8 gene (9p24) lead to abnormal cytoskeletal organization and impaired function of dendritic cells and lymphocytes. an aniline derivative belonging to the group of synthetic sulfones, dapsone has been employed in the treatment of chronic skin diseases characterized by an accumulation of neutrophils and eosinophils. methods: chart review of one patient results: we present a four-year-old male with severe eczema, persistent asthma, allergic rhinitis, as well as peanut and egg allergy suffering from recurrent skin abscesses and prurigo nodularis. abscesses began at age 18 months and required prolonged courses of antibiotics, eight in total prior to presentation. other infectious history included otitis media and lymphadenitis. there was no history of pneumonia or other severe infections. skin abscesses responded to oral antibiotics, but recurred shortly after completing extended courses of treatment. laboratory results including quantitative immunoglobulins, specific antibody titers, myeloperoxidase staining, neutrophil oxidative burst and complement were within normal limits. laboratories were notable for elevated ige (10080 iu/ml) and eosinophilia (1200 eosinophils/microl). lymphocyte immunophenotype was significant for mild elevations in cd3 and cd4. dock8 genetic sequencing by genedx revealed a heterozygous missense mutation in exon 17 (c.1979 c>a, amino acid change p.ala660asp). abscess cultures grew methicillin sensitive staphylococcus aureus (mssa) and enterococcus faecalis. mssa was sensitive to ampicillin/sulbactam, cefazolin, gentamycin, moxifloxacin, oxacillin, rifampin, tetracycline, and vancomycin. isolate was resistant to ciprofloxacin, clindamycin, erythromycin, levofloxacin, penicillin, and trimethoprim/ sulfamethoxazole. the patient was initially treated with emollients, mupirocin washes, topical steroids, anti-histamines, bleach baths, and cephalexin three times a day. he improved clinically but was unable to tolerate cephalexin for more than ten days secondary to abdominal pain. cephalexin, with addition of probiotic, was attempted several months later and again had to be discontinued because of abdominal pain and vomiting. due to limited antibiotic options, dapsone was started after ruling out glucose-6-phosphate dehydrogenase deficiency. dapsone was initiated at a dose of 1.5mg/kg, and the patient was monitored weekly for hemolytic anemia. after two weeks of treatment, anemia was noted and the dose was decreased to 1mg/kg. he has continued on dapsone 1mg/kg once a day, with significant improvement in abscess number and severity. he has not required other systemic antimicrobials since starting dapsone. conclusions: dapsone may be considered as a treatment option for children with heterozygous dock8 mutation and recurrent abscesses, particularly those requiring prophylaxis, long term treatment and lacking antibiotic options. introduction/background: autosomal dominant hyper-ige syndrome (ad-hies) is a rare complicated primary immunodeficiency disease (pid). signal transducer and activator of transcription 3 (stat3) gene mutation is found to cause ad-hies. tlr7/9 signaling plays multiple roles in b cell proliferation, activation, class-switch recombination, and cytokine and antibody production. however, little is known about b cell response to tlr7/9 agonist in patients with ad-hies. objectives: here, we aim to study the response of b cells from ad-hies patients to the tlr7/9 agonist. methods: pbmcs were isolated from peripheral blood of 5 ad-hies patients and 10 age matched healthy controls. pbmcs were stimulated with tlr7 and tlr9 agonist (r848 and cpg odn 2006, respectively), then b cells were analyzed for proliferation, the expression of certain surface markers (cd40, cd80 and cd86), intracellular immunoglobulin levels (igm and igg) and intracellular cytokine levels (il-6 and il-10) by flow cytometry. results: in response to tlr7/9 agonist, proliferative capabilities of b cells were reduced in ad-hies patients compared with those in age-matched healthy controls. besides, defective costimulatory molecule cd80 expression was observed in b cells from ad-hies patients. furthermore, significantly lower igm and igg levels, and il-10 production was detected in b cells from ad-hies patients. however, there was no significant difference in b cell apoptosis between ad-hies patients and healthy controls. conclusions: these data demonstrated that stat3 gene mutations in ad-hies patients contributed to impaired b cell tlr7/9 signaling, and further affected b cell proliferation, activation, cytokine secretion and antibody production. introduction/background: antibody function is most commonly measured by a rise in antibody titers in response to antigen introduced by vaccination or natural infection. specific antibody deficiency is defined as normal serum levels of immunoglobulins with reduced or absent antibody response to antigens, often after administration of the pneumococcal vaccine polyvalent (pneumovax® 23). a paucity of information exists about measurement of antigen-antibody binding, or avidity, as a measure of antibody function, including persons with recurrent sinopulmonary infections who have normal response to immunization with pneumococcal vaccine polyvalent. objectives: the aims of this study are to identify and evaluate children with recurrent sinopulmonary infections who had appropriate rise in pneumococcal antibody titers following immunization with pneumococcal vaccine polyvalent but low response by avidity, and to assess response with igg replacement therapy in these patients. methods: a retrospective chart review involved eight children with recurrent sinopulmonary infections with discordant pneumococcal antibody and avidity results following vaccination with pneumococcal vaccine polyvalent. these eight children subsequently received igg replacement therapy. results: the mean age of subjects was 9.75 (range 2 -15) years. the mean number of serotypes with a normal antibody response (>1.3 ug/ml) among 8 children following immunization with pneumococcal vaccine polyvalent was 18.1 (range 12-22) of 23 serotypes while the mean number of serotypes with a normal avidity response (1.0) was 4.4 (range 2-7) of 23 serotypes. igg replacement was administered subcutaneously in 8 children. the mean igg level was 720 mg/dl. local reactions were all mild and observed in 4/8 (50%) children. no serious adverse events were reported. all 8 children experienced a marked reduction in respiratory illnesses while on igg replacement therapy. conclusions: discordance between pneumococcal antibody titers and pneumococcal avidity titers was identified in eight children with recurrent respiratory illnesses. in children with recurrent sinopulmonary infections despite normal antibody response to pneumococcal vaccine polyvalent, measurement of pneumococcal avidity may identify patients with poor pneumococcal antibody function. igg replacement in these children was well tolerated and associated with a decrease number of respiratory infections. introduction/background: exome sequencing (es) is a powerful genomic tool that can be used to identify novel molecular causes of disorders with multiple etiologies. immunologic disorders are clinically and genetically heterogeneous, and therefore present unique diagnostic challenges both in the clinic and in the laboratory. objectives: the objective of this study was to assess the utility of es for determining the genetic etiology of immunological disorders and describe diagnostic yield and outcomes of exome sequencing (es) for patients with immunologic disorders and immunologic phenotypes. methods: a retrospective review was performed of 315 individuals referred for clinical es for primary abnormalities of the immune system and individuals with additional phenotypes where multiple immunologic features were reported as part of the clinical picture, as determined during internal curation. analysis of clinical es data was performed by boardcertified clinical geneticists and all variants reported were confirmed by a secondary methodology. positive es outcomes required a pathogenic or likely pathogenic variant in a gene with autosomal dominant or x-linked inheritance, or compound heterozygous or homozygous pathogenic or likely pathogenic variants in a gene with recessive inheritance. results: the most common clinical indications for es in this cohort were hypogammaglobulinemia (16%), neutropenia (14%), immune dysregulation (10%), lymphopenia (9%), and combined immunodeficiency (8%). the gender distribution was 59% male (n=185) and 41% female (n=130); 76% of cases were pediatric (<18 years, n=240) and 24% were adult (>=18 years, n=75). positive results were reported in 83 cases (26%), comparable to the overall diagnostic yield of es at our laboratory (retterer et al., 2016) . this included 20/90 (22%) of cases submitted as proband-only, 47/178 (26%) submitted as a trio, and 16/47 (34%) submitted as duo, quad or alternative family structure. diagnostic results spanned 70 different genes and recurrently reported genes with identified pathogenic variants included flg (n=10), rag1 (n=5), sbds (n=4), lrba (n=3), stat1 (n=3), and pik3cd (n=3). variants possibly associated with the phenotype, but not considered diagnostic, were reported in 58% of cases (n=183), while 13% of cases (n=41) had reportable findings in a candidate gene. conclusions: these results support that exome sequencing for individuals with immunologic-based phenotypes has similar diagnostic utility as the overall rate for clinical es. immunologic es cases with trio family structures have a higher diagnostic yield than proband-only cases, as inheritance information improves confidence in classification of variants as pathogenic or likely pathogenic. genetic heterogeneity, as demonstrated by the large number of distinct genes represented in this cohort of diagnostic es cases and rapid candidate gene discovery make es a valuable tool for genetic diagnosis in patients with immunological disorders. introduction/background: vaccination response to the 23-valent polysaccharide vaccine (ppsv23) is often used in the diagnosis of common variable immunodeficiency (cvid). unfortunately, ppsv23 titers are often difficult to interpret and many cvid patients are started on igg replacement therapy (igrt) before adequate evaluation. unlike ppsv23 titers, the enzyme-linked immunosorbent spot assay (elispot) is independent of igrt and can provide an ex vivo functional measurement of specific antibody production on the b cell level. objectives: develop and test an elispot assay to better determine vaccination response to ppsv23 compared to ppsv23 titers in cvid patients on igrt, healthy controls, and igrt patients without immunodeficiency. methods: an elispot assay was successfully optimized and used to evaluate the ppsv23-specific b cell response in 8 healthy adult controls. elispots were performed on day 1, and day 7 (when plasmablasts are best evaluated). ppsv23 titers were measured on day 1, day 7, and day 30. for igrt patients, flow cytometry for b cell subpopulations will be performed to further validate the assay. results: normal controls demonstrated a significant increase in ppsv23 antibody spot forming units (sfu) between day 1 and 7 after ppsv23 vaccination. ppsv23 titers showed generally robust initial titers at day 1, and no significant change at day 7, with day 30 results pending. conclusions: here we optimized an elispot assay that functionally measures the specific antibody response to ppsv23 in normal controls with ppsv23 titer results pending. we are actively recruiting patients on igrt (both with cvid and without immunodeficiency) for comparison. we hope to validate our assay as a useful alternative to ppsv23 titers that may be particularly useful when patients are on igrt. previous studies have demonstrated bal may be a sensitive diagnostic method for treatment failures of clinically diagnosed pneumonias, even if performed under treatment with empiric antibiotics, and can lead to a culture-directed change in antimicrobial therapy in the majority of cases. however, it has also been reported that at least in one piddchronic granulomatous disease (cgd)the diagnostic yield of bal was inferior to that of other diagnostic methods (marciano et al., 2015) . further information on the diagnostic yield of bal and other invasive procedures to obtain a specific organism diagnosis in pidd patients with suspected pulmonary infection is needed. objectives: to characterize the yield of diagnostic procedures used in pidd patients with pneumonia or other suspected pulmonary infections at the ucsf benioff childrens hospital. methods: we screened our database of pidd patients (encompassing patients seen from september 1, 1998 to september 1, 2017 cared for by the pediatric immunology service at the university of california, san francisco to identify patients with history of at least 1 bal or other invasive diagnostic procedure (fna or open lung biopsy) for etiologic diagnosis of suspected pulmonary infection. if multiple bals were performed during a single episode of illness, only the first was used for this analysis. results: we identified 22 pidd patients with history of at least one bal or other invasive diagnostic procedure, for a total of 55 events. most procedures (n=53) were performed at our institution, with 2 documented in outside hospital records. patient diagnoses included cgd (11), nemo deficiency (4), cvid (3), stat3-deficient ad-hies (2), dock8 deficiency (1) , and mhc class ii deficiency (1) . of 49 bals, 24/49 (49%) grew a predominant organism, but only 18/49 (37%) were positive for an organism believed to be causative by providers and/or for which the overall result of the bronchoscopy affected antimicrobial treatment. bal yield was highest in patients with a clinical and/or radiologic diagnosis of pneumonia (13/27, 48%). yield was poor (3/16, 19%) in minimally or chronically symptomatic patients referred for bal for interval changes on chest ct (i.e. suspected fungal infection). our nemo deficiency cohort had the highest rate of positive organism isolation by bal (6/11, 66%) for diagnosis of pulmonary infection. in our cgd cohort, 11/ 26 (42.3%) bals grew a causative organism. lung biopsy yielded positive organism isolation in 2/3 cases (2/2 in cgd, burkholderia cepacia and nocardia cyriacigeorgica; 0/1 in cvid) and fna in 1/2 cases of cgd (aspergillus fumigatus). fna or biopsy was done concurrently or after bal in 5 cases; in 4/5 (80%), fna or biopsy, but not bal, was positive for a causative organism. conclusions: at our institution, bal overall had a 37% rate of causative organism isolation in pidd patients, but had up to a 50% rate of organism isolation in those with clinical and/or features of pneumonia. our rate of causative organism isolation was slightly higher than in previous reports. however, in specific instances, biopsy was still required to make a definitive diagnosis. bal may have limitations in certain populations of pidd patients, such as in cgd, but it may be a reasonable starting point in the diagnosis of pneumonia or worsening pulmonary disease in pidd. prospective research is needed to evaluate whether fna or lung biopsy, though more invasive, could result in overall shorter time to institution of appropriate directed therapy and shorter hospitalizations for specific pidd patients. introduction/background: prior to the introduction of newborn screening, cases of severe combined immunodeficiency often presented with severe or disseminated infections. herein, we report an infant from india who presented for evaluation and treatment of a periorbital mass, presumed to be malignant. however, he was found to have disseminated bacille calmette guerin (bcg), as well as multiple other infections, and was eventually diagnosed with x-linked scid. results: a 4-month old boy was born to unrelated parents in india. he initially presented with growing periorbital mass and fever for two weeks. pet scan showed hypermetabolic areas in the bone marrow, spleen, mesenteric lymph nodes, and left shoulder, along with the periorbital mass. biopsy of the mass revealed numerous b lymphocytes without malignant transformation. there was no evidence for malignancy on bone marrow biopsy, though numerous granulomas and a significant decrease in t lymphocytes were seen. peripheral blood flow cytometry showed a complete lack of t cells. the bone marrow biopsy was reexamined, and innumerous acid-fast bacilli were found. cultures grew mycobacterium bovis, and he was diagnosed with disseminated bcg disease. genotyping revealed a novel splice site variant in il2rg, consistent with x-linked scid. further evaluation revealed multiple other infections. these included extended-spectrum beta lactamase-produce e. coli bacteremia, human metapneumovirus, cytomegalovirus, and pneumocystic jiroveci. he was also tested for vaccine-associated poliovirus because he had received the oral polio vaccine, but this was negative. four-drug therapy was started for the bcg, and the periorbital lesion completely resolved within several weeks. additionally, he was treated with intravenous ribavirin for the human metapneumovirus, and sulfamethoxazole-trimethoprim for the p. jiroveci. although the cmv was initially treated with ganciclovir, the virus eventually developed resistance and required treatment with foscarnet. due to his many infections, he underwent haploidentical stem cell transplant plus donor lymphocyte infusion from mom. this transplant failed to engraft, but a matched unrelated donor was eventually found, and our patient received a second bone marrow transplant. he currently is showing signs of engraftment, and is continuing to be treated for his multiple infections. conclusions: disseminated mycobacterial disease due to the bacille calmette-guérin (bcg) vaccination has been noted in cases of xlinked scid previously, often consisting of lymphatic, skin, and pulmonary manifestations. however, there is a paucity of published cases presenting with multiple other co-infections. nor are there reports of disseminated bcg presenting as a localized mass. this case highlights the unique considerations when evaluating a patient with immunodeficiency from another country, where vaccination practices and epidemiology differ. specifically, unusual presentations of infections or masses may warrant investigation for severe immunodeficiency. prototypic t-b+nk+ immune phenotype is caused by mutations in the iil7ra gene. the il7 signaling has an important role during t-cell development in the thymus, contributing to cell proliferation and survival. in addition, in mouse models, the rearrangement of the t cell receptor genes, specifically the gamma locus (trg), has been shown to be regulated by il7 signaling. similar to other scid phenotypes, patients with il7ra deficiency are predisposed to acquire opportunistic infections early in life and to display poor outcome and death, unless their immune system is restored by mean of hematopoietic stem cell transplantation (hsct). while most patients with il7ra mutations have full il7ra deficiency resulted in a severe t cell depletion, some have a partial deficiency with residual cells or leaky phenotype, or even present symptoms later in life. objectives: here we report two non-related infants detected by the israeli national newborn screening program for scid. despite having similar il7ra missense mutation (f40l) they displayed distinct clinical and immunological course, resulted in a completely different treatment approaches; observation in one patient with an unusual recovery, and hsct in the other patient methods: patient lymphocytes were examined for subset counts, thymic output (via excision circles), t cell receptor repertoire diversity (tcrb) and il7ra expression and function. the pathogenic il7ra mutation was found by whole exome sequencing (wes). high throughput immunesequencing was performed to characterize the trg repertoire. results: we established patients' diagnosis by validating the pathogenic il7ra mutation, and showing profoundly impaired t cell immune work up and abnormal il7ra expression and function, determined by stat5 phosphorylation assay. all these measurements improved over time for the patient with less severe clinical presentation, while remained low in the patient with the severe phenotype. characterization of their trg immune repertoire using high throughput immune-sequencing revealed restriction of t cell receptor repertoires of both patients upon their initial diagnosis, compared to healthy controls. however, skewed usage of variable (v) gene segments and abnormalities of the cdr3 length distribution were more prominent in the patient with the severe phenotype. conclusions: these studies illustrate the gap that exists in our understanding of other non-genetic parameters that may influence disease course and severity in patients harboring a similar genetic defect. furthermore, the results reinforce the role of il-7 signaling not only in cell proliferation but also in trg rearrangements introduction/background: dock8 immunodeficiency syndrome is primary immunodeficiency disease caused by loos of function mutations in the dock8 gene, which was known to play a critical role in the survival, proliferation and function of several types of immune system, especially lymphocyte. dock8 immunodeficiency syndrome is the most common cause of autosomal recessive hyper-immunoglobulin e syndromes (hies) and mainly expressed as recurrent infections and severe allergic disease affecting the skin. in addition, autoimmune features including systemic lupus erythematosus, hemolytic anemia or idiopathic thrombocytopenic purpura may be presented in dock8 immunodeficiency syndrome. objectives: we report a case of 16-month-old boy diagnosed with dock8 immunodeficiency syndrome, which was initially expressed as sle without recurrent skin infections. methods: a child with atopic dermatitis was admitted to another hospital because of fever lasting more than 3 days accompanied by swelling of the hands and foot. he developed whole body edema, perioral purpura and oliguria. complete blood count was normal and blood urea nitrogen , creatinine and albumin levels were normal on his laboratory findings. however, c-reactive protein level was high at 9.85 mg/dl, coagulation parameters were abnormal (prothrombin time 14.8 sec; activated partial thromboplastin time 67.0 sec, d-dimer 5.82 ug/ml). so he was transferred to our hospital for further examination and treatment on the 5th day of fever. additionally, ulceration of the tonsil and maculopapular rashes on the abdomen and both legs were observed in physical examination. we suspected a meningococcal infection and administered antibiotics. however, no bacterial isolates were identified in the blood and csf culture test. fever persisted despite the administration of antibiotics. we checked immunoglobulin level, complement level and autoantibodies based on fever of unknown origin. immunoglobulin g, m and a were normal, complement fractions c3, c4, and ch50 were low at 80.0 mg/ dl, 6.7 mg/dl and 13.3 u/ml, respectively. antinuclear antibodies were positive at 1:128 with homogenous fluorescence. anti-ds dna antibody was positive at 84.4. the tests for anti-ssa, anti-ssb, anti-ribonucleoprotein, anti-scleroderma 70, and anti jo1 antibodies were all negative. he developed leukopenia and thrombocytopenia over time. results: he was treated with steroid satisfied diagnostic criteria for systemic lupus erythematosus (sle) and fever subsided. he was confirmed lupus nephritis by renal biopsy later. because of onset of sle at the young age, we performed diagnostic whole exome sequencing and multiplex ligation-dependent probe amplification assays. conclusions: he was confirmed dock8 gene deletion (a deletion on one allele and point mutation on the other allele). he is preparing for hematopoietic stem cell transplantation due to autoimmunity and nonreversible parenchymal organ damage form infections although he has not yet experienced a life-threatening infection. introduction/background: during immunological investigation, it is important to distinguish those individuals who may hav e hypogammaglobulinemia (hypo) without fulfilling the criteria for the severe antibody deficiency common variable immunodeficiency (cvid) e.g. unspecified hypogammaglobulinemia from those with cvid. objectives: since low igg3 concentrations may support a diagnosis of cvid, we sought to investigate whether the measurement of additional igg subclass antibodies (iggsc) may provide further discrimination between patients with cvid and those with hypo. methods: iggsc concentrations were measured in serum samples from cvid patients (n=15, 1:1.3 m:f, median age 41.5 years, range 20-78) and hypo patients (n=19-21, 1:1.3 m:f, median age 41.5 years, range 20-78). results: cvid patients had lower median iggsc concentrations for all iggsc: igg1 290mg/dl (range 46-478) vs 365mg/dl (range 174-601); igg2 -88mg/dl (range 8-190) vs 116mg/dl (range 13-546); igg3 -15mg/dl (range 7-58) vs 30mg/dl (range 15-62); igg4 6mg/dl (range 0.2-25) vs 11mg/dl (range 0.31-108). this was significantly lower for igg1 and igg4 (p=0.04 and 0.01 respectively). a higher percentage of cvid patients had iggsc below the lower limit of the normal range compared to hypo patients: igg1 (80 vs 57.9%); igg2 (100 vs 90.5%); igg3 (60 vs 28.6%); igg4 (26.7 vs 10.5%). 100% of cvid patients had low concentrations of 2 or more iggsc vs only 68.5% of hypo patients (p=0.02). 5.2% and 26.3% of hypo patients had low levels of 0 or 1 iggsc, respectively. conclusions: igg subclass measurements may have some utility in distinguishing cvid patients from hypogammaglobulinemia patients. introduction/background: the thymus is often removed during cardiac surgery for repair of congenital heart disease, but the extent of tissue removed varies between procedures and surgeons. previous studies have shown decreased t cell counts after thymectomy but there is limited data on the effect of thymectomy on t cell receptor excision circle (trec) levels and infection risk. objectives: to determine the effect of partial and complete thymectomy during cardiac repair surgery on trec levels and infection risk. methods: a retrospective study of electronic medical records was performed on children who received cardiac surgery before age one at new york presbyterian/morgan stanley childrens hospital between 7/1/2013 and 3/31/2017. patients with heart transplant or primary immunodeficiency were excluded. data was recorded on trec levels (abnormal trec < 200 copies/μl on new york state newborn screen), number of positive cultures, viral pcr panels, and infiltrates on chest x-ray. patients were followed for a minimum of six months after cardiac surgery. study was irb approved. results: cardiac surgery was performed on 256 patients and data was available for 133 patients. of patients included, 65 had a partial and 68 had a complete thymectomy. trec levels after surgery were recorded for 79 patients. only 4% of patients had an abnormal trec level on newborn screen. there was no difference between partial and complete thymectomy on risk of abnormal trec (p = 0.58) and mean total number of infections at 6 months (p =0.42). conclusions: thymectomy rarely causes low trec levels in children undergoing cardiac surgery. complete thymectomy does not significantly increase infection rates in these children compared to partial thymectomy. these findings are possibly due to presence of ectopic thymic tissue or thymic regeneration and are reassuring for children undergoing complex cardiac surgeries. however, long-term follow-up of these children will be necessary to determine residual function of the thymus and clinical response. introduction/background: new sequencing techniques have revolutionized the identification of the molecular basis of primary immunodeficiency disorders (pid), not only by establishing a gene-based diagnosis, but also by facilitating defect-specific treatment strategies, improving quality of life and survival, and allowing factual genetic counseling. because these techniques are generally not available for physicians and their patients residing in developing countries, collaboration with overseas laboratories has been explored as a possible, albeit cumbersome, strategy. objectives: we sought to determine whether blood collected by guthrie cards could be shipped across continents by regular airmail to a cliaapproved laboratory for confirmatory testing. methods: blood was collected and blotted onto the filter paper of guthrie cards by completely filling three circles. we enrolled 20 male patients with presumptive x-linked agammaglobulinemia (xla) cared for at the vietnam national children's hospital, their mothers and several sisters for carrier analysis. dbs were stored at room temperature until ready to be shipped together, using an appropriately sized envelope, to a cliacertified laboratory in the us for sanger sequencing. the protocol for sanger sequencing was modified to account for the reduced quantity of gdna extracted from dbs. results: high-quality gdna could be extracted from every specimen. btk mutations were identified in 17 of 20 patients studied, confirming the diagnosis of xla in 85% of the study cohort. type and location of the mutations were similar to those reported in previous reviews. the mean age when xla was suspected clinically was 4.6 years, similar to that reported by western countries. two of 15 mothers, each with an affected boy, had a normal btk sequence, suggesting gonadal mosaicism. conclusions: dbs collected on guthrie cards can be shipped inexpensively by airmail across continents, providing sufficient high-quality gdna for sanger sequencing overseas. using this method of collecting gdna we were able to confirm the diagnosis of xla in 17 of 20 vietnamese patients with the clinical diagnosis of agammaglobulinemia. introduction/background: znf341 is a positive regulator of stat3 expression. it has recently been described that nonsense mutations in znf341 account for the stat3-like phenotype in four autosomalrecessive kindred. patients presented with reduced stat3 expression and diminished th17 cell numbers, in absence of stat3 mutations. objectives: here, we decribed a turkish case having nonsense mutation in znf341 developed dual malignancy in 2 years. results: a 26-year-old female patient presented with severe eczema, recurrent cold skin abscesses, herpetic skin lesions, sinopulmonary infection, otitis media and hearing loss. the parents are cousins. two younger sisters of the index case had eczema and recurrent skin infections since their infancy period. physical examination of the patient revealed severe eczema, high palate, micrognathia, maxillary hypoplasia and hearing loss. laboratory findings showed reversed cd4/cd8 ratio, high serum ige level (13.916 u / l) and low igg2 level (186 mg / dl). the patient was diagnosed as hyper ige syndrome and ivig therapy (400 mg / kg, every 3 weeks) was initiated because of igg subgroup deficiency and recurrent sinopulmonary infections. at the age of 32, a polypoid mass filling the left nasal cavity was detected in her examination. paranasal sinus ct revealed a mass obliterating the left nasal cavity, left ethmoid sinus and frontal sinus. immunohistochemical stains showed a small round cell malignant tumor in the nasal cavity. she was treated with chemoradiotherapy successfully. a homozygous nonsense mutation has been detected at exon 8 in the znf341 gene (c.1156c> t) (kindly provided by grimbacher's lab) very recently. she developed papillary thyroid carcinoma two years after completing the cancer therapy. conclusions: the relationship between znf341 defect and cancer development is unknown. the development of a second malignancy in this patient for a short time of completing the therapy might imply us a tendency for malignancy in znf 341 patients. additionally, the likelihood of increased radiosensitivity in these patients should be taken into consideration. introduction/background: prometic 10% igiv contains purified igg, 95% as monomer; with a distribution of igg subclasses proportional to that in native human plasma. we report the interim results from a phase 3 trial in the usa of prometic 10% igiv in adults and children with pidd. objectives: this was a phase 3, single-arm, open-label, multicenter trial to evaluate the safety, tolerability, and efficacy of prometic 10% igiv in adults and children with pidd. methods: adults and pediatric subjects with pidd on a stable dose of igg replacement therapy (200-900 mg/kg) for at least 3 months with serum igg trough levels > 500 mg/dl were included. subjects received prometic 10% igiv every 3 to 4 weeks for approximately 1 year at the same dose and schedule as their previous igg replacement therapy. results: an interim analysis was conducted when data were available on 50 adult subjects who received at least one dose of prometic 10% igiv (total of 461 infusions), with 15 subjects receiving at least 6 months of treatment (exposure = 27.72 subject years). at this time, pediatric exposure was only 4.29 subject years. there were no serious bacterial infections (sbis) reported, and rate/yr of infections other than sbis was 2.49 which was comparable to rate while on commercial product (2.80) all subjects achieved an igg trough level > 500 mg/dl. there were no deaths, and no subject had a study drug-related serious adverse event or an adverse event that resulted in permanent discontinuation of study drug. a total of 145 adverse reactions (ar) (0.315/infusion) occurred in 32 subjects (64.0%), with 92 infusions (20.0%) associated with an ar. most infusions (97.0%) were completed without a rate reduction. most ars were mild or moderate in severity, with 6 severe ars (0.013/infusion) occurring in 3 subjects (6.0%). the most frequent ars were headache (20.0% subjects or 0.056/infusion) and fatigue (14.0% subjects or 0.017/infusion). conclusions: in adults treated with prometic 10% igiv, there were no sbis and infusions were well tolerated. director, sean n. parker center for allergy and asthma research at stanford naddisy foundation, professor of medicine and pediatrics, stanford university introduction/background: desensitization to food allergies is being studied in clinical trials using oral immunotherapy (oit). there are limited data regarding the immune changes associated with successful oit. epigenetics involves heritable changes in gene function without modification of the underlying dna sequence. this is mediated by methylation, histone modification, or changes in microrna. objectives: to study methylation changes in the loci of four key genes of immune cells involved in allergy, interleukin 4 (il-4), interferon gamma (ifn-g), forkhead box protein 3 (foxp3), and interleukin 10 (il-10), comparing baseline to post-oit. methods: we completed a phase 2, randomized, placebo-controlled, multi-food oit trial using omalizumab, an anti-ige biologic, to facilitate desensitization for 48 multi-food allergic individuals. double-blind, placebo-controlled food challenges (dbpcfcs) to multiple foods were conducted at entry and after 36 weeks of treatment, the primary endpoint. omalizumab (n=36) or placebo (n=12) was administered for 16 weeks, with oit for 2-5 foods starting 8 weeks after the beginning of omalizumab or placebo. after 36 weeks (28 weeks of oit), participants underwent dbpcfcs to their offending foods. treatment failures (n=11) were offered open-label omalizumab. pyrosequencing of bisulfite treated genomic dna purified from pbmcs from each participant at baseline and post-oit was undertaken to investigate changes in methylation. results: forty-four participants achieved successful desensitization, defined as passing dbpcfcs to 2 or more foods following oit. we found that the -48 cpg site in the il-4 promoter region is hypermethylated over time during successful multi-food oit (fdr-adjusted p < 0.001 by wilcoxon signed-rank test). the median % of methylation at baseline was 81.6 (interquartile range 2.1%) and was 84.5 post oit (interquartile range 2.05%). there were no statistically significant (with a significance level of fdr adjusted p value of 0.001) changes in the il-10, foxp3, or ifn-g loci in the cpg sites we studied. conclusions: these preliminary results suggest that one immune mechanism involved in successful desensitization may involve suppression of th2 function by hypermethylation of il-4 in immune cells in the peripheral blood. introduction/background: atopic dermatitis (ad) is a common chronic inflammatory skin disorder afflicting from infancy to adults with itching, scratching, and lichenification. objectives: we investigated the effects of esculetin from fraxinus rhynchophylla on atopic skin inflammation. methods: for induction of atopic skin inflammation, we exposed the ears of female balb/c mice with house dust mite (dermatophagoides farinae extract, dfe) and 2,4-dinitrochlorobenzene (dncb) during 4 weeks. results: oral administration of esculetin reduced dfe/dncbinduced atopic skin inflammation symptoms based on ears swelling and scratch numbers. the immunoglobulin (ig) e, igg2a, and histamine levels in serum were decreased and inflammatory cell infiltration in skin tissue was reduced by the esculetin. it suppressed th1, th2 and th17 responses by inhibiting the production of inflammatory cytokines such as tumor necrosis factor (tnf)-, interferon (ifn)-, interleukin (il)-4, il-13, il-31 and il-17 in the ear tissue. further, we investigated the effects of escueltin on activated keratinocytes, one of the most representative cells for studying pathogenesis of acute and chronic atopic skin inflammation. as results, esculetin suppressed gene expression of th1, th2 and th17 cytokines and activation of nuclear factor-b and signal transducer and activator of transcription 1 in tnf-/ifn-stimulated keratinocytes. conclusions: taken together, the results imply that esculetin attenuated atopic skin inflammation, suggesting that esculetin might be a potential therapeutic candidate for the treatment of ad. introduction/background: autoimmunity is often seen in common variable immune deficiency (cvid) with immune thrombocytopenic purpura (itp) being the most frequent manifestation at a prevalence of 8-14% in cvid patients. in such patients, itp is often recognized and treated long before cvid, the implications of which are unknown. primary itp is a clinicopathologic diagnosis which includes an evaluation of other conditions that may mimic it including cvid. currently, it is unknown how frequently cvid is evaluated during the diagnostic workup of itp and what percentage of those patient actually have cvid. objectives: the two main objectives of this study were to determine the number of itp patients that had an igg level checked during their clinical course and if the globulin fraction can be used as a marker for hypogammaglobulinemia in itp patients at the time of diagnosis. methods: a retrospective chart review was undertaken at a large academic medical center of patients with a new diagnosis of itp between january 2009 and january 2017. igg levels were collected and globulin fractions were calculated as the difference between serum total protein and albumin within 30 days of the initial itp diagnosis. results: six hundred and twenty-three patients were found to have a new diagnosis of itp in the given timeframe. of these, only 61 (9.79%) had igg levels checked at any point during their clinical course. twelve of the 61 (19.7%) had hypogammaglobulinemia with only 3 of the 12 (25%) having a formal immunologic follow-up evaluation. two were diagnosed with a primary immunodeficiency (1 cvid and 1 ctla4 deficiency). globulin fractions were calculated on 52 patients at the time of itp diagnosis. mean calculated globulin fraction in hypogammaglobulinemic patients was 2.64 (range 2.0 3.7), versus 3.92 (range 2.4 7.5) in patients without hypogammaglobulinemia (p=0.0004). conclusions: the diagnosis of cvid is often delayed from the onset of symptoms which can include autoimmune conditions such as itp. our data indicate that clinicians do not routinely check igg levels at the time of itp diagnosis which should be considered standard of care based on the current guidelines. our data suggest that although calculated globulin fractions were significantly lower in hypogammaglobulinemic patients, the variability was substantial and hypogammaglobulinemic patients would be missed using this as an indicator of low immunoglobulins. future directives include a prospective study using igg levels checked at the time of itp diagnosis, with formal evaluation for cvid in any hypogammaglobulinemic patients to evaluate the true prevalence of cvid among itp patients. introduction/background: autoimmune lymphoproliferative syndrome (alps) is a disorder characterized by immune dysregulation due to the rupture of lymphocyte homeostasis, which occurs as a result of mutations in the apoptotic pathway mediated by fas. this disease is sometimes misdiagnosed due to its variable phenotypic expression and the overlapping of symptoms with many other hematological and immunological disorders. (1) a patient diagnosed with evans-fisher syndrome (sef) was referred to the laboratory for immunity studies. this is a patient who from infancy had multiple admissions for severe sepsis, with severe anemia and severe thrombocytopenia. in the evaluation of the history of the disease by the work team, a series of clinical data was observed that suggested the possibility of the patient presenting an alps, so it was decided to incorporate as part of the study, the quantification of the cells t cd3 + cd4-cd8-(double negative t cells or dnt) that express tcr +. the differentiation pathways of dnt tcr + and the role of fas in this process are not clear, some authors hypothesize that they may be represented by direct descendants of chronically activated positive simple t cells, with deregulated co-receptors, in a state of differentiation in which they were destined to perish by fas-mediated apoptosis. (1) (2) (3) (4) the population of tcr + dnt cells required for diagnosis must be derived from the particular study of each laboratory in their populations, but several working groups agree that the pathological limit values are 1,5% in total lymphocytes or 2,5% in cd3 + lymphocytes. (4) evidence shows that dnt alps cells are not simply accumulated in senescent withdrawal; they and their precursors remain active and proliferate under the influence of activation signals. (5) although numerous genetic deficiencies lead to lymphoproliferation of t cells, only that caused by a defective fas pathway is dominated by negative double t cells. (2) in clinical immunology, the distinction between cd45ra + and cd45ro + cells is particularly useful for determining the state of the "naive" cell compartment in relation to its thymic origin. primary immunodeficiency disorders are characterized by a decrease / absence of thymus performance, and often involve a decrease in cd45ra + t lymphocytes. scientific evidence suggests the important role of the "naive" subpopulation in the origin and maintenance of self-reactive effectors in the periphery. (5) regulatory t cells are able to effectively control autoreactive t cells, especially when negative thymic selection is defective. its differentiation and function is controlled by foxp3. the decrease or absence of regulatory t cells leads to autoimmune diseases, specifically those mediated by cd4 + t cells, and to lymphoproliferation characterized by multiorgan inflammation and other autoimmune disorders. (4, 5) objectives general: establish the diagnosis of a pediatric patient with suspected primary immunodeficiency due to dysregulation, an autoimmune lymphoproliferative syndrome (alps). specific: evaluate the tcr + dnt population, relevant in the diagnosis of alps. quantify cell populations that exhibit markers of activation, differentiation and regulation of the relevant t cells in the study of this disease. methods: monoclonal antibodies (mab) cd4fitc / cd8pe / cd3pc5 triple labeled (beckman coulter), tcrapc, cd4pe, cd45rapercp, cd45rofitc, cd8fitc, cd45rope, cd4apc, cd25f, cd56pe, cd19percp, cd3pe, cd5pe, cd45percp, cd4fitc, cd38fitc were used , hla dr and the cd4fitc / cd25pe / foxp3apc regulatory t cell kit, all from miltenyi biotec. the trial included the use of a healthy control. both samples were processed in unison to ensure reproducibility. the samples were acquired in a beckman coulter gallios cytometer, with the use of the kaluza program, version 1.2. the analysis strategy included the formation of overlapping "gate" windows for the quantification of dnt tcr + and regulatory t cells. results: the patient studied showed 11.60% of tcr + negative double t cells, in relation to 1.45% of the healthy control. the presence of this population of t cells in patients diagnosed with evans syndrome, even in the absence of lymphoproliferation, is consistent with alps. (5) (graph 1) the presence of increased tcr + dnt and lymphoproliferationexpressed as lymphadenopathies and splenomegaly of noninfectious or malignant cause, of at least 6 months of evolution; plus the typical immunohistological findings found in the patient's lymph node biopsy (paracortical hyperplasia), the presence of autoimmune cytopenias (hemolytic anemia and thrombocytopenia) and hypergammaglubulinemia, led to the "probable diagnosis" of alps in the patient studied. the accumulation of dnt in the lymph nodes and other peripheral organs is accompanied by qualitative changes in the composition of the t cell repertoire, so that the immunophenotype performed included markers of activation, differentiation and regulation. the evaluation of the activation on t lymphocytes and nk cells showed that there was a slight decrease in the expression of the receptor for il-2, cd 25. the cd19 + cd25 + population was expressed in greater percentalthough discretely-in the patient than in the healthy control, which was directly related to the presence of hypergammaglobulinemia, elevated iga and the presence of autoantibodies. it was also highlighted by the high expression of the cd19 + cd5 + autoreactive population in the patient studied (11.73% vs 0.39%). in order to know the impact on cell differentiation, the subpopulations of effector and memory cells for cd4 + and cd8 + t lymphocytes were evaluated by combining the cd45ra and cd45ro isoforms. cd45ra is expressed in naive t lymphocytes. particularly, the cd4 + cd45ra + population has an essential function as a suppression inducer, and it is diminished in the patient studied, in relation to the control. (29.11% vs. 62.75%, respectively) the same behavior was shown by the cd4 + cd45ro + memory and effector cells, 53.38% vs73.37%. in the cd4 + population itself, it was possible to confirm the presence of a clone that expressed both receptors (cd45ra and cd45ro), probably a temra population (terminally differentiated effector memory cells). when comparing the results, it was unexpectedly found that there was a slight decrease between patient and control (2.70% vs 4.0%). as the characteristic phenotype of this cell population could not be corroborated, conclusive assessments could not be made. the combination of cd45 isoforms was also used to study cd8 + t lymphocytes, but similar behavior between patient and healthy control was observed. (the lab results are shown in table 1 ) the analysis of the regulation of the immune response showed a decrease in the cd4 + cd25 + population and the expression of the foxp3 transcription factor in the patient with respect to the control. (11.16% vs. 6.30%, 0.00% vs. 1.05%, respectively) (graph 2) conclusions 1. the markedly high quantification of cd4-cd8-tcr + t lymphocytes allowed to define the "probable diagnosis" of autoimmune lymphoproliferative syndrome in the patient under study. 2. the increased activation of cd19 + cells and the presence of the cd19 + cd5 + self-reactive population, largely responsible for autoimmunity and lymphoproliferation in the alps, could be confirmed. 3. the decrease in the cd4 + cd45ra + t-cell suppressor-inducing population evidenced corroborated its involvement in this disorder by primary immunodeficiency, in relation to the thymus dysfunction that originates and maintains self-reactive effectors in the periphery. 4. the decrease in the expression of the foxp3 transcription factor observed, points towards a low regulation of the response that leads to autoimmunity and to lymphoproliferation, specifically mediated by cd4 + t cells. allergy and immunology arnp, nicklaus children's hospital 6 allergy and immunology division director, nicklaus children's hospital introduction/background: specific antibody deficiency syndrome is characterized by a weak antibody response to bacterial polysaccharide antigens when no other immune system abnormalities can be found. low titers to pneumococcal vaccine have become one of the most frequently recognized immune abnormalities in pediatric patients with recurrent sinopulmonary infections. nonetheless, insufficient data and lack of consistent testing of the response to pneumococcal polysaccharides continues to affect the optimal diagnosis and management of this specific antibody deficiency. objectives: to characterize the pre-and post-immunization igg antibody trend for each specific serotype included in the pneumococcal 13-valent conjugate vaccine (pcv13), as well as others that are routinely tested, in a cohort of pediatric patients with recurrent sinopulmonary infections. secondarily, to understand differences in the immune response to the vaccine booster between age groups. methods: this retrospective review identified 182 patients with recurrent sinopulmonary infections. in this cohort, 131 required an immune workup, and 99 were found to have low pneumococcal titers needing a pcv13 vaccine booster. baseline pneumococcal serotype-specific antibody titers at initial visit and 6 weeks after the vaccine booster were obtained. patients were categorized by age: 2 years, 3-5, 6-10, and 11-18. an adequate response to the pneumococcal conjugate vaccine was deemed to be a 4-fold increase over baseline and/or a post-immunization titer of 1.3 μg/ml or greater. results: overall, pcv13 booster provided a significant improvement in the number of protective titers, increasing from 3.6 (95% ci: 3.2-3.9) serotypes at baseline to 11.1 (95% ci: 10.7-11.5) serotypes at 6 weeks (p < 0.001). this increase correlated with improved clinical outcomes81% showed no signs of recurrent infection after the first booster and 94% after a second dose. all those who did not improve clinically suffered from co-morbidities (genetic abnormalities and rheumatologic diseases). post-immunization antibody concentrations were significantly higher than at baseline for all serotypes (p < 0.05) and only 8, 9n, and 12f did not exhibit a greater than 4-fold increase (p > 0.05) 6 weeks following the booster. across age groups, only 1, 7f, and 9v showed pre-immunization differences in titers. there were no differences between ages in post-immunization titer levels for all serotypes. similarly, all age groups had a comparable number of baseline titers (p = 0.63) and at follow-up (p = 0.10). conclusions: in pediatric patients with recurrent sinopulmonary infections, an additional pneumococcal booster proved to be effective in the protection of these children from further infections. the pcv13 booster substantially increased titer levels and concentrations, and significantly improved clinical outcomes, independent of age. this investigation has provided us with a better understanding of the response after booster vaccination, and its role in the protection of patients from recurrent sinopulmonary infections. further studies are needed to elucidate whether a fifth dose of pcv13 should be optional as part of the vaccination schedule of patients with recurrent sinopulmonary infections. introduction/background: many primary immunodeficiencies (pids) share overlapping presentations, complicating clinical diagnosis. due to the ability to include many genes in one assay and the rapid turnaround time that these panels allow, expanded next-generation sequencing (ngs) panels are valuable in facilitating the diagnosis of patients with pids. objectives: we aimed to determine the clinical utility of an expanded ngs panel for the genetic diagnosis of patients with suspected pids. methods: we performed a retrospective analysis of the clinical utility of a 207-gene pid ngs panel used in a clinical diagnostic laboratory. from april to october 2017, 260 panels were ordered for patients with suspected or known pids. results: seventy-four pathogenic or likely pathogenic (p/lp) variants were identified in 63 (24.2%) patients. eight (10.8%) of the p/lp variants were copy number variations (6 deletions, 2 duplications). fifty-two patients (20%) had 1 p/lp variant, and 11 patients (4.2%) had 2 p/lp variants. of positive patients, 31.7% (n = 20) were heterozygous carriers for autosomal recessive conditions where a second variant was not identified. twenty-two patients had p/lp heterozygous variants in genes with autosomal recessive and autosomal dominant inheritance patterns, in which the positive findings may or may not explain the patient's phenotype. for example, 13 variants in this category were heterozygous variants in tnfrsf13b (taci). genetic diagnoses were established or likely in 30% of patients with p/lp variants (7.3% of all patients). five patients were heterozygous for a single p/lp variant and a variant of unknown significance in the same autosomal recessive gene. four patients in whom a genetic diagnosis was determined were also heterozygous carriers for a second, unrelated condition. one patient was found to have two distinct genetic diagnoses. variants of uncertain significance (vus) were identified in most (94.2%) patients. the average turnaround time from test requisition to return of results was 18 days. in total, 63% percent of genetic diagnoses were for conditions that are treatable with hematopoietic cell transplantation. conclusions: these results illustrate the utility of broad ngs panels for the diagnosis of patients with pids. introduction/background: severe combined immunodeficiency (scid) is a life-threatening immune deficiency manifest by extreme susceptibility to infection. early diagnosis and definitive treatment with either hematopoietic cell transplant (hct) or, in select cases, gene therapy (gt) has been shown to significantly improve survival. newborn screening for scid has allowed for the opportunity to promptly identify these patients before significant infections occur. at ucsf, newly diagnosed infants with scid are admitted to the hospital for management and remain in isolation for definitive treatment and until adequate immune reconstitution occurs. previous work by our group demonstrated up to 60% of these parents experience psychosocial trauma manifested by depression and post-traumatic stress disorder (ptsd). the psychosocial challenges that contribute to depression and ptsd in these parents have not been qualitatively described. objectives: to understand the range of experiences and feelings of parents/caregivers of infants with scid diagnosed by newborn screening throughout their prolonged hospitalization and isolation in order to better support patients, parents, and their families. methods: voluntary participation was elicited from parents of children with scid who were status post hct/gt for one year or longer. semi-structured, in-person interviews lasting approximately 45-60 minutes were conducted with 11 parents; interviews were recorded and transcribed. parents were asked to discuss their experiences from first notification of an abnormal screening result through discharge after hct or gt. emerging themes were identified from the transcribed interviews. results: we interviewed 6 mothers and 5 fathers of 7 infants with scid. six infants received hct, while one underwent gt for ada-scid. all children were alive and well at the times when interviews were conducted. overall, once admitted, parents reported feeling well supported by the medical team and support staff. however parents identified a number of stressful events. uniformly reported key stressors included: receiving the first phone call regarding their childs abnormal newborn screening results, preparing for hct, coping with prolonged isolation, and transitioning from hospital isolation to care with ongoing isolation at home. other challenges described reflected the additional stressors of caring for a newborn, including coping with postpartum depression. overall, we identified three major themes encompassing the challenges faced by parents of hospitalized scid patients: (i) loss of normalcy and control over multiple aspects of life; (ii) prolonged waiting periods (especially the wait between diagnosis and hct and between hct and evidence of t cell engraftment); and (iii) perceived lack of guidance on realistic expectations during the hospital stay . parents sought and relied on peer support from other scid parents to learn about coping with the nuances of daily life as a parent of an infant with scid. conclusions: we identified multiple psychosocial stressors and challenges uniquely faced by parents and caregivers of infants diagnosed with scid by nbs. parents described barriers to caring for their own physical and mental health, which can be especially harmful to parents experiencing postpartum depression. recognizing these challenges allowed for the identification of opportunities to improve both healthcare delivery to their children and institutional support for future families affected by scid (table i) . emphasis should be placed on providing parents with scid-specific resources as early as the time of diagnosis, connecting parents with scid support networks, and facilitating access to psychosocial and mental health services for caregivers introduction/background: chronic granulomatous disease (cgd) is caused by a genetic defect that impairs phagocyte function. this disease results in recurrent infections and granuloma formation. rarely do patients develop cutaneous symptoms, unless associated with autoimmune disorders such as systemic erythematous lupus. previously described cutaneous findings include granulomas, abscesses, photosensitivity, malar rash, discoid lupus, vasculitis, and rarely vesicular rashes. here we describe two term infants diagnosed with x-linked cgd who present, in addition to frequent infection, with a unique papulopustular skin rash initially diagnosed as non-classic appearing eczema refractory to usual eczema treatment and antibiotics. objectives: to characterize cutaneous findings in x-linked cgd and emphasize the importance of considering further work in patients who present with similar rashes in conjunction with concerning features for primary immunodeficiency. methods: each infant was diagnosed with cgd based on abnormal dhr testing and/or genetic evaluation. after obtaining consent from both families, we have documented photographs of the development of rash in two newly diagnosed infants with cgd. one infant underwent cutaneous biopsy with resulting pathologic evaluation. results: our first patient presented at 8 months of age with episodic fever with chronic leukocytosis, iron deficiency anemia, thrombocytosis, elevated inflammatory markers, proctocolitis with elevated calprotectin, and rash. egd with flexible sigmoidoscopy showed no signs of inflammatory bowel disease, the left colon had macroscopically raised erythematous lesions but was microscopically normal with no signs of active colitis. he was initially diagnosed with fpies and eczema in the setting of diagnosis of various infections (otitis, upper respiratory illness). the skin rash was described as non-pruritic, generalized pink-purple papulopustular lesions with a pink base, most prominent on upper and lower extremities, mostly sparing the trunk. skin biopsy histopathology revealed an essentially unremarkable appearing epidermis and within the dermis, there was a superficial perivascular lymphohistiocytic infiltrate. eosinophils and plasma cells were not abundant. the histologic changes were thought to be non-specific, but could be seen in a drug reaction or urticaria. a viral exanthem could also demonstrate these changes. giemsa stain and a stain for mast cell tryptase revealed normal numbers of mast cells in the biopsy specimen. he was not on any oral medications at the time and did not respond to treatment with topical moisturizers, oral antihistamines, topical steroids, or any standard for eczema care. he had a maternal uncle who passed away in infancy from infection. heightened clinical suspicion for primary immunodeficiency led to obtaining a neutrophil respiratory burst assay which was consistent with cgd and genetic testing was positive for x-linked cgd. pathogenic mutation nucleic acid change was c. 469c>t; hemizygous; amino acid alteration was p.157arg>stp within the cybb gene locus. he had no other features of auto-immune disease including negative ana obtained later in his clinical course and his colitis diagnosed as cgd-associated colitis. his rash did not respond to systemic treatments for his colitis, oral antibiotics, and during hsct. our second patient presented at 14 months of age with persistent fevers, inguinal lymphadenopathy, leukocytosis, elevated inflammatory markers, non-bloody diarrhea and rash. there was a family history of autoimmune gi disease. he presented to the hospital with fever of unknown origin with concern for infection, atypical kawasakis, and drug rash. his rash was described as a blotchy, pink, papular rash most prominent on the upper arms but also present on lower arms, legs, and chest, faint on face. there is some induration and thickness to the rash in some areas (confluent on the arms with more erythema and induration) and more faint pink papules (scattered on legs) elsewhere. a biopsy of his inguinal lymph node showed granulomatous lymphadenitis with neutrophilic abscess formation, and culture was positive for serratia marcescens. neutrophil respiratory burst assay was consistent with cgd and genetic testing was positive for x-linked cgd, mutation with the cybb gene. pathogenic nucleic acid change was c. 141+5g>a; hemizygous; amino acid alteration was p. deletion of exon 2. the rash remained unchanged with treatment for infection, initiation of antifungal and bacterial prophylaxis, along with topical steroid therapy. conclusions: in patients who present with frequent infections and who have unusual cutaneous findings that do not fit with common infant rashes, consideration of work up for cgd should be pursued. specifically, generalized papulopustular rash with a negative skin biopsy can be misdiagnosed as atopic dermatitis, and in the right clinical context cgd should be considered. introduction/background: chronic lung disease in common variable immunodeficiency (cvid) is heterogeneous, and it is a leading cause of morbidity and mortality in this population. currently, the diagnosis and monitoring of chronic lung disease in cvid rely on radiographic findings, biopsy and/or pulmonary function tests. fractional exhaled nitric oxide (feno) is a noninvasive biomarker of airway inflammation, and it has been widely utilized to aid the in-office diagnosis, characterization, and management of inflammatory airway disease, such as asthma. however, exhaled nitric oxide in cvid patients with chronic pulmonary complications has not been examined. objectives: we aimed to determine fractional exhaled nitric oxide levels in cvid patients. methods: we measured exhaled nitric oxide in cvid patients with or without chronic lung disease. results: feno measurements were obtained in 13 cvid patients (mean age: 53, range 34-68). five patients had no known lung disease; 8 patients had chronic lung disease (bronchiectasis, n=3; lung granulomas, n=4; hepatopulmonary syndrome, n=1). four patients were on inhaled steroid (bronchiectasis, n=3; lung granulomas, n=1), 2 patients were on systemic steroid (lung granulomas, n=1; hepatopulmonary syndrome, n=1), and 1 patient was on oral budesonide (no known lung disease). feno was elevated (> 25 parts per billion [ppb]) in 9 of 13 patients, 6 of whom had known chronic lung disease. patients with granulomatous lung disease had higher feno (mean 50.8 ppb, range 41-59 ppb) compared to patients without known lung disease (mean 31 ppb, range 23-38 ppb, p=0.005), patients with bronchiectasis (mean 18.3 ppb, range 8-25, p=0.005), and the patient with hepatopulmonary syndrome (18 ppb). feno levels remained elevated in granulomatous lung disease in patients with inhaled or systemic steroid use. conclusions: this is the first report of feno measurements in cvid patients. feno is elevated in a subgroup of cvid patients, and it may differ according to the underlying lung pathology. further investigation is warranted to determine the utility of feno in the diagnosis and management of chronic lung disease in cvid. professor of pediatrics, university of british columbia introduction/background: whole exome sequencing (wes) has revolutionized the discovery, diagnosis, and treatment of primary immune deficiency diseases (pids), a group of disorders with high genetic and phenotypic heterogeneity. current bioinformatic analysis approaches for wes rely heavily on population databases to exclude variants present in the populations represented by these databases. this approach may confound the ability to detect true disease causing variants, and conversely, flag variants that are absent from population databases only by virtue of originating from minority populations. ultimately, the pathogenicity of novel variants can only be mechanistically established through rigorous biochemical and functional validation. objectives: to evaluate the pathogenicity of a novel homozygous variant in caspase recruitment domain family member 11 (card11) (c.1798g>t, p.c600y), in a patient with features of combined immunodeficiency (cid). methods: research study protocols were approved by our institutional review board. six members of one family (the affected child, healthy siblings and their parents) were enrolled. written informed consent for genetic testing and participation was provided by the parents for their children. genetic, bioinformatic, biochemical and immunological investigations were performed. results: targeted sanger sequencing confirmed the presence of the homozygous card11 variant c.1798g>t, p.c600y in the index patient (and subsequently in one apparently healthy sister). each parent was found to be a heterozygous carrier of the variant. nfkb activation in vitro was found to be normal for both the index patient and sister, and was indistinguishable from unaffected family members. conclusions: although multiple in silico tools predicted the c.1798g>t, p.c600y card11 variant to be pathogenic, the patients b and t cells did not have aberrant nfkb activation in vitro, as would be predicted given the central role of card11 in nfkb activation. this practical experience highlights how imperative it is to functionally characterize novel variants found by wes, even when variants are predicted to be damaging. chief, clinical immunology -faculdade de medicina do abc introduction/background: gata2 is a zinc finger transcription factor essential for embryonic and definitive hematopoiesis. heterozygous mutations leading to gata2 deficiency were first described in 2011. the age of clinical presentation ranges from early childhood to late adulthood, with most of them in adolescence to early adulthood. patients present clinical findings as monocytopenia, nontuberculous mycobacterial infections, myelodisplasia, viral (mainly hpv and ebv), fungal and bacterial infections. patiens may arise with many phenopytes, showing how complex is the effect of this transcription factor. objectives: we report a patient with gata2 mutation. methods: report: a 34 year old female patient was referred due to a mycobacterial non-tuberculosis pneumonia. she was a healthy child until puberty. at the age of 15 she presented genital herpes and recalcitrant vulvo-vaginal warts (hpv). at 18, she complained of lower back pain with no diagnosis for months, medication was unnefective, and she developed ischemic stroke. hemiparesia was mild and temporary. endocarditis with no agent identified was also diagnosed. therapy with acetilsalicilic acid was mantained until the age of 28. five years later, vaginal hpv spread through vulve and anal region evolving to neoplasia. she was submitted to surgery, radio and chemotherapy until october 2016. one year later, profound cytopenias led to hospitalization. during that time, she had cough and fatigue and pulmonary tuberculosis was diagnosed. despite therapy with rifampin, isoniazid and pyrazinamid, there was no improvement. bronchoalveolar lavage was positive for mycobacterium avium. main immunological evaluation showed: monocytes 162 (6%), t cells /mm3; cd4+: 28 (1,5%), cd8+: 74 (4%); b cells 2/mm3 (0,1%); nk: 9/mm3 (0,5%); normal immunoglobulin levels; incomplete response to pneumococcus serotypes; igm and igg positive for cmv; negative serologies for hiv, ebv and htlv1/2. molecular analysis identified an heterozygous mutation c.1061c>tp.(thr354met) in gene gata2. results: we report a patient with gata2 mutation. the same gata2 mutation was previously described in national institute of health nih-usa (n=5) and in australia (n=3). conclusions: it took almost 20 years for diagnosis suspicion. gynecologists should be warned about this diagnosis in order to improve patients prognosis. early diagnosis is crucial with adequate prophylaxis, prompt treatment of infection, surveillance of malignancy, and, moreover, family screening and genetic counseling. this is the first report of this mutation in latin america. introduction/background: severe combined immunodeficiency (scid) due to adenosine deaminase (ada) deficiency was the first human monogenic disease to be approached with gene therapy, and ongoing research advances over 25 years led to the approval by the european medicines agency of a stem cell gene therapy product for its treatment using a gammaretroviral vector (gv), strimvelis®. despite the high success rate using gvs for ada gene transfer without vector-related complications, the development of leukoproliferative complications from the use of gvs in gene therapy of other disorders led us to develop lentiviral vectors (lvs) to deliver the corrective ada sequence/cdna (corrigan-curay et al, mol ther 2012; modlich et al, mol ther 2009.). lvs, typically derived from hiv-1 and devoid of all viral genes, can be produced in a self-inactivating (sin) configuration, in which the viral long-terminal repeat enhancers are absent, eliminating the major identified cause of insertional oncogenesis due to gvs. we developed a lv (efs-ada) that carries a normal human ada cdna (codon-optimized) and demonstrated in pre-clinical studies its efficacy to transfer and express the ada protein, with evidence of significantly decreased potential for insertional mutagenesis compared to gammaretroviral vectors using the immortalization (ivim) assay and in murine bone marrow transplant models (carbonaro et al, mol ther, 2014) . this new lv was evaluated for safety and efficacy in parallel clinical trials of gene therapy for ada scid performed at sites in the u.s (university of california, los angeles and the national institutes of health {nih} clinical center) and u.k. (university college london/great ormond street hospital) enrolling subjects between 2012 and 2016. we report here results from the 20 patients treated in the u.s. between 2013-2016. objectives: this is a prospective, non-randomized phase i/ii clinical study to assess the safety and efficacy of efs-ada lentiviral vector stem cell gene therapy in ada-scid subjects older than 1 month. methods: 20 subjects with ada-scid were enrolled, screened to document eligibility and underwent bone marrow harvest (15-20 cc/kg). bone marrow was processed to isolate cd34+ cells, which were pre-stimulated by overnight culture in serum-free medium containing c-kit ligand, flt-3 ligand, and tpo followed by culture with the efs-ada lentiviral vector overnight. subjects received a single dose of busulfan (4 mg/kg) iv for reduced intensity conditioning. cells were removed from culture, washed, formulated and administered by iv infusion at least 24 hours after busulfan administration. enzyme replacement therapy (ert) with pegylated bovine ada (peg-ada) was continued for one month post-transplant and then stopped. subjects were followed over 24 months to assess safety and efficacy end-points. results: with 17-54 months follow-up, overall survival is 100%. eventfree survival has also been 100%, as all subjects are alive, remain off peg-ada ert, and none have required a second transplant. successful engraftment of gene-corrected cells was observed in all subjects at 6 months, and persisted over the 24 months of observation, based on vector gene marking in granulocytes and peripheral blood mononuclear cells (pbmcs), changes from baseline in rbc ada enzyme activity, and levels of metabolic detoxification of deoxyadenosine nucleotides. immune reconstitution was observed in all subjects and was sustained over the two years of observation, based on improvement of peripheral blood absolute lymphocyte counts and lymphocyte subsets (t, b and nk cells, and naïve cd4+ t cells). eighteen of 20 patients have been able to stop receiving immunoglobulin replacement therapy. subjects who had routine infections all recovered with standard of care treatment, and there were no severe or opportunistic infections. conclusions: conclusions: gene therapy using the efs-ada lv has a favorable safety profile and was efficacious in this trial. a current followon trial at ucla is using a cryopreserved formulation of the cell product and pharmacokinetic-adjusted busulfan dosing, sponsored by the california institute for regenerative medicine (clin2-09339) and orchard therapeutics. acknowledgements: this study was supported by research grants from the national institutes of health (u01 ai100801; 2p01 hl073104-01; nhlbi gtrp rsas #1101 and 1129) and the california institute for regenerative medicine (cl1-00505; fa1-00613); support from the ucla david geffen school of medicine human gene and cell therapy program and the ucla eli & edythe broad center of regenerative medicine and stem cell research; and funding from the nhgri intramural program. dbk has potential financial conflict of interest as a member of the scientific advisory board for orchard therapeutics and as an inventor on intellectual property which ucla has licensed to orchard therapeutics related to gene therapy for ada scid. introduction/background: x-linked severe combined immune deficiency (scid-xl) is a rare monogenic primary immunodeficiency disorder (pid) where male infants are born without an adaptive and innate immune system. it is a life-threatening disease due to patients inability to fight viral and bacterial infections. scid-xl is driven by any of the two hundred known pathogenic mutations in the interleukin 2 gamma receptor (il2rg) gene, which function is required for proper development of t, b and nk cells. the most effective treatment for scid-xl, if performed in the first few months of life, is allogeneic hematopoietic stem cell transplantation (allo-hsct). this treatment is limited by absence of match donors, incomplete immune reconstitution, graft versus host disease and the need for long-term immunosuppression. an alternative curative treatment for scid-xl would be genome editing-based gene therapy by ex vivo genome correction of the patients long-term hematopoietic stem cells (lt-hscs) prior to autologous stem cell transplantation (auto-sct). objectives: here we report a proof-of-concept genome editing-based approach for correcting scid-xl disease. methods: using a crispr/cas9-raav6 platform, we deliver a fulllength codon optimized il2rg complementary dna (cdna) at the endogenous start site in cd34+ hematopoietic stem and progenitor cells (hspcs). results: using an optimized genome editing protocol we achieved >80% genome editing as early as 48h and a median of 45% genome targeting while retaining >80% viability and promoting greater than 70% ex vivo expansion of cd34+ hspcs from healthy donors. we demonstrate that our approach retains proper il2rg signaling function in t-cells derived from healthy male donors and rescues the lymphopoietic defect from a patients derived mobilized cd34+ hspcs both in vitro and in vivo. we further show robust in vivo primary human engraftment potential and multi-lineage hematopoietic reconstitution of il2rg gene targeted hspcs: a median of 26% (bone marrow), 47% (spleen) and 37% (liver) of il2rg genome targeted engrafted cells was achieved at four months following primary transplantation into nsg mice and a median of 9.5% -20% was detected at 8 months from secondary transplants of il2rg targeted hspcs, thus achieving clinical levels of editing of lt-hscs. lastly, our observation of (1) an intact hematopoiesis derived from il2rg targeted cd34+ hspcs combined with (2) a normal karyotype analysis and (3) our deep analysis of potential off-target activity that showed only 2 off-target sites of no known functional significant with < 0.3% frequency of off-target activity presents strong evidence for the safety of our genome editing approach. conclusions: in sum, this pre-clinical study provides specificity, toxicity and efficacy data supportive of continued development of genome editing to treat scid-xl. objectives: to determine the risk of gvhd in msd hsct for scid patients compared to matched related donor (mrd). methods: retrospective cohort study comparing msd with mrd and the outcome of gvhd in all scid patients who underwent hsct between 1993 and 2013. all statistical analysis was done using ibm spss statistic software. results: 145 scid patients underwent 152 hsct, 82 (54%) received gvhd prophylaxis. gvhd occurred in 48 (31.5%); 20/48 (42%) had gvhd prophylaxis compared to 28/48 (58%) that did not, p value = 0.022. acute gvhd occurred at a higher rate in msd 22/120 (18.3%) compared to mrd 2/32 (6.2%) p value = 0.095. we analyzed outcome also according to period of hsct. first periods was 1993 to 2003; 48 hsct, msd: 43, mrd: 5; all had gvhd prophylaxis and there was no difference in gvhd. the second period was 2004 to 2013:104 hsct, 77 had msd and 27 had mrd, gvhd prophylaxis was used in 22.1% in msd and 63% in mrd, p value = 0.000. gvhd was significantly higher in the msd (40.2%) compared to mrd (18.5%) odds ratio of 2.9 (95ci 1.01 to 8.66) p value = 0.041. conclusions: gvhd prophylaxis in msd transplant may have a role to be considered in scid patients. introduction/background: xiap deficiency (also known as x-linked lymphoproliferative disease type 2 xlp2; mim: 300635) is an x-linked primary immunodeficiency associated with mutations in the gene encoding the x-linked inhibitor of apoptosis (xiap; mim: 300079). the pathophysiology is characterized by immune dysregulation, usually triggered by epstein-barr virus (ebv) infection. primary ebv infection is followed by hemophagocytic lymphohistiocytosis (hlh) with high grade persistent fever, splenomegaly, hematologic cytopenias and hepatitis. most patients die during this acute phase, and those who survive usually evolve with hypogammaglobulinemia, recurrent infections, cytopenias, inflammatory bowel disease (ibd) and low counts of inkt cells. dysbiosis of intestinal microbiota is believed to fuel ibd and possibly contribute to the initiation and/or perpetuation of the disease. experimental studies have provided solid evidence to support a role for the indigenous gut microbiota in the pathogenesis of autoimmune diseases, thereby raising the possibility that an altered gut microbiota is an environmental risk factor for xiap disease. objectives: in this study, we sought to investigate the identity and abundance of the bacteria in gut microbial communities in a 28 years old male patient with xiap deficiency. case presentation: at 15 years of age, the patient presented with positive serology of active ebv infection, hlh, severe hepatitis, encephalitis and myocarditis. after recovery, the patient evolved well with few manifestations for several years. approximately 3 years ago, the patient showed slow progression of hypogammaglobulinemia predisposing him to infections of the upper and lower respiratory system that required intravenous immunoglobulin replacement. immunological evaluation revealed reduction (but not absence) of inkt cells. at that time, the patient presented with intermittent diarrhea and abdominal pain that became more frequent and severe. after evaluation as ibd, the patients had been treated but without much improvement of diarrhea or resolution of his pain. after approximately 18 months, the patient presented noted pain and fistulas lesions at the scrotal and gluteal regions. the exact causes of ibd in xiap deficiency are not known, but the abnormal activation of the mucosal immune system due to exaggerated response to the commensal bacteria associated to the dysregulation of nod1 and nod 2 signaling might play an important role in the development and maintenance of the inflammatory status. methods: the gut bacterial composition was assessed by targeted metagenomic from the patients stool sample, collected before initiation of ibd antibiotics therapy. the 16s rrna amplified and its sequences were analyzed using a bioinformatic pipeline based on mothur software. we determined the bacterial community composition using 100,000 filtered reads using illumina miseq platform. results: according to the ezbiocloud database, the obtained dataset included 548 operational taxonomic units at 3% dissimilarity, distributed among the following groups: bacteroidetes (67.3%) with 37.5% of b. dorei, 18,1% b. vulgates, and 15.3% b. fragilis, firmicutes (24.8%), proteobacteria (7.7%), bacteria_uc (0.06%), actinobacteria (0.04%). conclusions: increased abundance of bacteroides species including b. dorei and b. vulgatus have been implicated in inflammation in several gut diseases such as ulcerative colitis, irritable bowel disease, and celiac disease. although this experience is limited to a single patient, the results of the present study suggest an association between altered gut microbiota and the pathogenesis of ibd in xiap disease and may be of relevance to the future development of novel therapeutic strategies for xiap deficiency. (77) submission id#427718 hematopoietic stem cell transplantation in patients with primary immune regulatory disorders: a primary immune deficiency treatment consortium (pidtc) and inborn errors working party (iewp) study introduction/background: primary immune regulatory disorder (pird) is a newly recognized group of immune-mediated diseases with prominent features of autoimmunity, autoinflammation, and non-malignant lymphoproliferation in addition to immunodeficiency. the clinical manifestations of pirds are frequently difficult to manage and hematopoietic cell transplantation (hct) can be considered as a treatment option, often in those with the most severe disease. we sought to aggregate data from patients who have undergone hct for genetically defined pird or features of immune dysregulation. objectives: we sought to aggregate data from patients with pird who have undergone hct in order to determine the quantity of patients, clinical manifestations, indication and hct, and overall outcome. methods: a questionnaire based survey was sent to all primary immunodeficiency treatment consortium sites and 3 hct referral centers in europe to determine the quantity and characteristics of patients with pirds who have undergone hct. the survey captured clinical manifestations, timing and indication of hct, strategy of hct, and outcomes from 1982-2017. results: 224 patients from 34 centers (31 in north america and 3 in europe) were included with either known genetic defects or considered to have immune dysregulation regardless of gene defect. known genetic defects were identified in 170 subjects, while 54 had symptoms of immune dysregulation but lacked a genetic diagnosis. the mean age of onset of disease was 2 years (range 0-20 years). clinical manifestations included gastrointestinal disorders (69%), failure to thrive (67%), dermatitis (51%), hematologic cytopenias (49%), and lymphoproliferative disease (39%). recurrent infections (66%), immunodeficiency (63%), autoimmunity (48%), and autoinflammation (38%) were also common. organ specific autoinflammation occurred most commonly in the lung (39%) and brain (17%). the median age of hct was 9 years (0-64 years). graft sources included matched unrelated donors (47%), matched related donors (20%), mismatched unrelated donors (16%) and haploidentical donors (3%). reduced or minimal intensity conditioning was used in 44% of transplants. five-year overall survival was 61% and the majority of survivors had resolution of symptoms that led to transplantation. among those patients that died, infection was the most common cause. conclusions: based on our survey data, pird patients commonly develop clinical features of autoimmunity, autoinflammation, and susceptibility to infection at a young age. hct can be successful and lead to disease resolution. however, further studies to define the appropriate patient, timing of hct, donor selection and pre-hct conditioning regimen are necessary to improve outcomes of patients with pird. introduction/background: pathogenic variants in tnfrsf13b (taci) are relatively common (found in about 1% of the population) but have been seen in about 7-10 % of cvid patients, interestingly in both homozygous and heterozygous states. recent articles suggest that the heterozygous state increases the risk of developing autoimmunity due to an effect on autoreactive b cell selection and activation. objectives 1) illustrate the importance of genetic testing in patients with difficulty to treat inflammatory disease 2) present a case report of inflammatory bowel disease associated with a pathogenic mutation in the tnfrsf13b gene results: 16 yo wf was diagnosed with crohns disease due to the chronic abdominal pain, vomiting, and bloody stools since 13 years of age. intestinal biopsy revealed inflammatory changes in the entire intestine with active, submucosal lymphoid hyperplasia, neutrophilic cryptitis with focal areas of crypathic damage, and submucosal epithelioid granulomas more predominantly seen in the colon and rectum. she was started on immunosuppressant medications but developed anaphylactic reaction to both infliximab and adalimumab. she was then treated with azathioprine, mesalamine, methotrexate, and oral budesonide. despite these medications, she continued to have frequent relapses, 4-5 episodes a year and required periodic systemic corticosteroid bursts. other biologics, vedolizumab and ustekinumab, were also tried without success, and she subsequently underwent a colectomy. her postoperative course was complicated by ards, poor abdominal wound healing, and sepsis. due to her complicated clinical course, immune work up was performed which revealed a normal cbc and lymphocyte subpopulations, but hypogammaglobulinemia with low isohemagglutinin titers and specific antibody levels. comprehensive genetic testing ruled out chronic granulomatous disease and other known primary immunodeficiencies but revealed a rare missense mutation in tnfrsf13b (taci). this variant, c310t (p.cys104arg) (rs34557412, exac 0.5%) is likely pathogenic. this heterozygous variant has been seen in both cvid cases and unaffected relatives but significantly more common among cvid patients. moreover, the studies on b cells of these relatives showed impaired function. increased number of autoreactive b cells were also found in the bone marrow of heterozygous individuals and these cells could give a risk of developing autoimmunity. conclusions: in difficult-to-treat autoimmune diseases, identifying the underlying immune defect may aid in the treatment decision. in this case, b cell targeted treatment such as anti-cd20 monoclonal antibody could be beneficial. introduction/background: defects in immunoproteasome caused by biallelic or digenic loss-of-function mutations in proteasome catalytic subunits cause an autoinflammatory disease identified as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (candle) associated to an increased interferon type i gene signature. the proteasome maturation protein (pomp) is a chaperone for both standard and immuno-proteasome assembly and is critical for the incorporation of catalytic subunits. here, we characterize and describe pomp-related autoinflammatory immunodeficiency disease (praid) in two unrelated patients and identify the underlying genetic mechanism of disease. objectives: determine the genetic cause and mechanism of disease in two patients with pomp variants . methods: whole-exome sequencing (wes) was performed to identify a genetic cause of our patients dysregulatory syndrome. proteasome assembly and catalytic function was assessed by sds-page and native gel respectively, using patient derived cell lines. expression of interferon type i-induced genes was measured by rt-qpcr. pomp protein was identified by western blot. results: we identified two unrelated individuals with a unique syndrome characterized by neonatal onset autoinflammation, neutrophilic dermatosis, autoimmunity, and combined immune deficiency with severe systemic viral and bacterial infections. immunologic evaluation for both individuals revealed elevated immunoglobulins, low cd8+ t cell numbers and extremely low b cell counts with persistently high titers of autoantibodies and increased expression of interferon type i-induced genes. in both individuals, truncating heterozygous de novo frameshift variants in pomp were identified by wes and confirmed by sanger sequencing. most mrna transcripts with premature termination codons should undergo nonsense-mediated decay (nmd), however in both of our patients, cdna sequencing revealed these transcripts escaped nmd. the expression wildtype and truncated versions of pomp protein was further confirmed by western blot. transfection of mutant constructs into an otherwise healthy cell line recapitulated an increased interferon signature suggesting a dominant negative mechanism. conclusions: we define praid in two unrelated individuals characterized by neonatal onset immune dysregulation and combined immunodeficiency caused by truncating variants in pomp in which transcripts that escape nmd result in a truncated protein that leads to a dominant negative (i.e antimorphic) allele. to our knowledge, praid is the first inherent defect of immunity mechanistically characterized by nmd escape. introduction/background: parvovirus viremia may occur in pediatric heart transplant patients who underwent thymectomy and developed secondary t cell lymphopenia. high dose intravenous immunoglobulin (hdivig) has been used to treat parvovirus infection in these cases. objectives: we aim to review different routes of immunoglobulin treatment in pediatric cardiac transplant patients with parvovirus viremia and compare the patients immunological phenotype. methods: data from three pediatric heart transplant patients with parvovirus viremia in a tertiary care center was reviewed including t cell counts, parvovirus viral load, route, dosage, and frequency of immunoglobulin treatment. results: all three patients received hdivig. patient 1 and 2 tolerated the treatment and viremia improved. patient 3 developed recurrent aseptic meningitis from hdivig treatment and his viral load remained >1 million copies/ml. compared to the other two cases, patient 3 had a much lower t cell count that likely contributed to the persistence of viremia. to improve his quality of life and reduce healthcare costs, a facilitated subcutaneous immunoglobulin (scig) treatment option was explored. scig treatment was well tolerated and led to a dramatic decrease in parvovirus viral loads in patient 3. conclusions: most pediatric cardiac transplant patients with persistent chronic parvovirus viremia respond well to hdivig, scig may serve as an alternative treatment option in refractory cases, especially in those with severe t cell lymphopenia. severe combined immunodeficiency (scid), by detecting tcell receptor excision circles (trecs) from dried blood spots (dbs) on routine newborn screening (nbs). this has lead to improved estimates of the incidence and prevalence of scid, decreased diagnostic delay, and improved patient outcomes. preliminary studies outside the us have demonstrated that nbs can be adapted to include screening for b-cell deficient infants before symptom onset by quantifying kappa-deleting recombination excision circles (krecs) on dbs. objectives: we report results of the initial characterization of a high throughput triplex trec/krec/rnasep assay run in 9,994 samples in new york state (nys). methods: dbs from 9994 anonymous, de-identified infants were included in the current study. dna from patients with confirmed primary immunodeficiencies (n=32), including, but not limited to, x-linked agammaglobulinemia (n=11) and scid(n=13) were obtained from the centers for disease control and prevention, and clinical immunologists working with the nbs programs in massachusetts, minnesota, wisconsin, and nys, were used as positive disease controls. all dbs were extracted and processed according to current nys nbs protocols. a trec/krec/rnasep triplex assay was designed and optimized to minimize reagent use, and maximize target amplification. cycle threshold (ct) was determined, and error detection cutoffs were identified to optimize sensitivity. results: pcr efficiency, assay quantification, intra-assay reproducibility, and error detection rates all met nys nbs standards. error detection rate for the triplex trec/krec/rnase p assay is 6%, comparable to the current error detection rate of 5% for the current duplex trec/rnase p assay. samples falling into the error detection range are repeated (analysis in process) to determine a receiver operating characteristic curve. conclusions: we show that the high-throughput trec/krec/rnase p triplex assay is feasible in a large, racially and ethnically diverse population in nys. compared to the current duplex assay, this assay has favorable performance characteristics and provides additional immunologic characterization. due to assay optimization, we were able to add the krec test at no additional cost. work is underway to further characterize other assay parameters such as sensitivity and specificity, in preparation for adoption of the triplex assay as part of routine nys nbs. highly accurate wiskott-aldrich syndrome diagnosis via rapid flowbased was protein staining samuel chiang 1 , sue vergamini 2 , ammar husami 3 , marianne ifversen 4 , kejian zhang 5 , jack bleesing, md, phd 6 , yenan bryceson 7 , rebecca marsh, md 8 introduction/background: wiskott aldrich syndrome (was) is a rare xlinked hemizygous disease commonly associated with symptoms of immune deficiency. diagnosis is based on clinical parameters including thrombocytopenia and reoccurring infections, but currently does not include any disease specific marker. as the only permanent treatment for was is hematopoietic stem cell transplantation, it is imperative that a swift and accurate diagnosis be made. absent or lowered was protein (wasp) levels have been reported in sporadic was cases. however, no systemic evaluation exists to date on the accuracy of wasp quantification for was diagnosis. objectives: to determine the accuracy of wasp staining in predicting was genetic abnormalities. methods: we retrospectively evaluated results from a rapid whole blood flow cytometry based assay on a cohort of suspected was patients and compared relative wasp staining levels to was genotype. roc curves as well as accuracy calculations were generated. results: a total of 59 patients with normal and 49 patients with a genetic abnormality in was were collected. missense mutations were most common but insertions, deletions, and gross mutations were also found ( fig a) . comparing was sequencing results to whole blood wasp expression levels provided an 82.6% sensitivity and 100% specificity for a combined accuracy of 95.3% when juxtaposed against genetic sequencing. when 3 variants of unknown clinical significance (vucs) were removed, the sensitivity improved to 89.1% (fig b) . conclusions: staining for wasp is a quick, simple, and accurate assay for the prediction of genetic was defects. introduction/background: cytotoxic t lymphocyte antigen 4 (ctla4) is an inhibitory co-receptor essential for regulatory t cell (treg) function and a central regulator of t cell proliferation and expansion. ctla4 haploinsufficiency is a recently described autosomal dominant disease, in which heterozygous ctla4 mutations result in severe immune dysregulation with variable age of onset and a wide array of clinical manifestations. herein we describe atypical findings in a patient with a novel pathogenic variant in ctla4. objectives 1) to understand whether the novel ctla4 variant identified is pathogenic and 2) to describe eosinophilic gastrointestinal inflammation and exocrine pancreatic insufficiency as possible manifestations of ctla4 haploinsufficiency. methods: next generation sequencing (blueprint genetics ©) was used to identify the ctla4 variant. polyphen and sift (blueprint genetics ©) were used for in silico analysis for the prediction of the effect of this genetic variant on protein structure/function. flow cytometry was used to evaluate ctla4 expression of regulatory t cells. results: a 10-year-old boy with type 1 diabetes mellitus and autoimmune thyroiditis presented with abdominal pain, diarrhea, and weight loss. initial studies revealed markedly elevated peripheral blood eosinophils (>4000 cells/μl) and exocrine pancreatic insufficiency (<50 μg elastase per gram of stool). prominent eosinophilic inflammation was appreciated in biopsies of the stomach, duodenum, jejunum, and terminal ileum. no parasitic infection or inciting drug/food trigger was identified. additional blood studies revealed normal total quantification of t cells but with increased memory t cells (45%, cd3+cd4+cd45ro+) and decreased treg (1%, cd3+ cd4+cd25hifoxp3hi). b cell quantification and serum immunoglobulin (ig) levels were unremarkable, save a modestly elevated ige level (74 iu/ml). comprehensive next-generation sequencing of 232 genes associated with primary immune deficiency revealed a novel heterozygous missense mutation (c.457g>a, p.asp153asn) affecting the last nucleotide in the ligand binding domain (exon 2) of ctla4. subsequent analyses revealed decreased ctla4 expression in the patients t cells compared to healthy controls as well as evolving hypogammaglobulinemia. treatment consisted of methylprednisolone and parenteral nutrition followed by sirolimus and abatacept to which the patient responded favorably. conclusions: we report a 10-year-old boy with a history of type 1 diabetes mellitus and autoimmune thyroiditis presenting with hypereosinophilia, eosinophilic gastroenteritis, and exocrine pancreatic ins uff iciency as unique m anifestat ions o f ctla4 haploinsufficiency. although not previously reported in individuals with ctla4 haploinsufficiency, peripheral blood eosinophilia and eosinophilic inflammation of the gastrointestinal tract have been observed in patients receiving ipilimumab (ctla4 blocking antibody) suggesting a potential mechanism for the aforementioned findings. severe exocrine pancreatic insufficiency is a rare but observed manifestation in individuals with type 1 diabetes mellitus. whether severe exocrine pancreatic insufficiency would be expected t o o c c u r m o r e f r e q u e n t l y i n i n d i v i d u a l s w i t h c t l a 4 haploinsufficiency and type 1 diabetes mellitus is unclear; however, our reported case and surveillance of others with ctla4 haploinsufficiency could elucidate incidence and prevalence of this manifestation. introduction/background: mild asymptomatic hypogammaglobulinemia during pregnancy is a well-described phenomenon due to hemodilution. in patients with known humoral primary immunodeficiency such as common variable immunodeficiency, women require an upwards titration of their immunoglobulin replacement dose. isolated symptomatic hypogammaglobulinemia during pregnancy in a patient is not well described in the literature. objectives 1. understand the physiology of igg during pregnancy. 2. define a rare entity with a likely genetic predisposition that manifests as hypogammaglobulinemia isolated in pregnancy. 3. management of this entity with defining goals of treatment. results: 33 year old gravida 5 para 4 female presented with progressive hypogammaglobulinemia restricted to pregnancy starting with 3rd pregnancy, recurrent otitis media requiring 3 sets of myringotomy tubes, recurrent sinusitis, and streptococcal pharyngitis. her son has common variable immunodeficiency (cvid) requiring immunoglobulin replacement (igrt). prior to conception, her igg was 849 mg/dl. during 13th week of pregnancy, her igg level was 627 mg/dl. during 21 weeks of pregnancy, she complained of fatigue, and developed an episode of sinusitis that required 2 different antibiotic treatments. at that time, her igg was 567 mg/dl. she was started on subcutaneous igrt maintain igg troughs of >650 mg/dl. she remained infection-free during her pregnancy and igrt was stopped a few months after delivery with her serum igg level returning to pre-pregnancy levels. patient was initially evaluated during her 3rd pregnancy with recurrent streptococcal pharyngitis. at that time, her igg was 682 mg/dl. diphtheria, haemophilus influenza, mumps, measles and rubella titers were protective, tetanus titer was non protective with 1/14 antipneumococcal titers protective. she was vaccinated with pneumovax and tdap with development of protective titers and remained infectionfree. igrt was not given and post-delivery, her igg levels improved to 792 mg/dl. she was seen one and half years later, for prenatal counseling for her 4th pregnancy. her immune evaluation included an igg 915. she demonstrated protection to tetanus, diphtheria and streptococcal pneumoniae. at 16 weeks of gestation, she developed recurrent upper respiratory infections requiring antibiotics. her igg was 697 mg/dl. at 28 weeks of gestation, her igg was 537 mg/dl. igrt was recommended, but patient refused at that time. after delivery, igg improved to 849 mg/dl. conclusions: decreased immunoglobulin levels during pregnancy are welldescribed phenomenon which can be attributed to hemodilution of pregnancy. igg transport to fetus generally begins in the second trimester and reaches its pinnacle in the third trimester. patients with known cvid require dose adjustments (higher) during pregnancy as they can be more symptomatic during this time. here we describe a patient who has an almost normal immune evaluation except for mildly low iga during absence of pregnancy, but during pregnancy, develops recurrent infections with significantly low igg level. her family history of a son with cvid, new daughter with low igg levels like to be transient hypogammaglobulinemia of infancy (thi), another daughter with thi suggests a genetic b-cell defect that manifests as cvid with mildly low iga and hypogammaglobinemia during metabolic stress such as pregnancy. there is only one similar report of a single pregnancy of transient symptomatic hypogammaglobulinemia during pregnancy. such patients should be adequately worked up and treated during pregnancy with igrt to decrease maternal and fetal mortality. introduction/background: card11 encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nf-b, jnk, and mtorc1. germline mutations in card11 are known to give rise to distinct primary immune disorders in humans, including scid (null mutations), b cell expansion with nf-b and t cell anergy (benta; gain-of-function mutations), and severe atopic disease (loss-of-function, dominant interfering mutations). objectives: here we report our experience with an expanded cohort of patients harboring novel heterozygous card11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with card11 mutations. methods: cell transfections and primary t cell assays were utilized to evaluate signaling and function of card11 variants. results: we demonstrate that in addition to severe atopy, heterozygous missense mutations in card11 associated with dominant negative activity can present with immunologic phenotypes similar to those observed in stat3lof, dock8 deficiency, common variable immune deficiency (cvid), congenital neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome. evaluation of rare or novel card11 variants found in affected patients showed that dominant negative activity was largely confined to the card or coiled-coil domains, but did not always manifest in atopic disease. conclusions: these results illuminate a broader phenotypic spectrum associated with card11 mutations in humans, and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity. introduction/background: increasing number of states have been screening for severe combined immune deficiency (scid) as part of the expanded newborn screening program for nearly a decade. in the era of newborn screening, patients with scid present often asymptomatically and are prepared for early hematopoietic stem cell transplantation (hsct). with advances in genetic testing, mutations in over 20 genes have been associated with development of the scid or leaky-scid phenotype. objectives: to present 2 unique cases of hypomorphic x-linked scid where no known pathogenic mutations were identified on initial genetic testing, the il2r gamma-chain as protein was expressed, but subsequent testing months later revealed pathogenic il2rg mutation affecting either translation or protein function. to expedite earlier identification of pathogenic il2rg mutation, we propose screening with x-inactivation studies in maternal t lymphocytes and assessing gamma-chain function by evaluating il21r signaling in select male infants with abnormal newborn screen for scid, specifically those with scid phenotype but no identified pathogenic gene mutation on initial genetic testing, and the presence of gamma-chain expression. male patient a presented during the first week of life after his newborn screen was found to be abnormal with undetectable t cell receptor excision circle (trec) count. a phenotype of t-b+nk-scid was established and the patient was sent for bone marrow transplant evaluation. genetic testing revealed a novel hemizygous missense mutation in il2rg (p.glu59gln), which was a variant of unknown significance. the patient had common gamma-chain expression by flow cytometry on b and nk cells. he underwent haploidentical hsct with his father as donor. unfortunately, his transplant was complicated by prolonged neutropenia, slow t cell reconstitution, and eventual graft failure. to review his next treatment options, it was necessary to prove that his il2rg mutation was pathogenic. male patient b presented with concern for an underlying immune deficiency after being hospitalized for peumocystis jirovecii pneumonia. his newborn screening for scid was inadequate at birth and follow up was delayed. retrospective analysis of the newborn screening card at birth confirmed absence of trecs. his phenotype was also t-b+nk-, consistent with x-linked scid. he also proceeded to hsct with a haploidentical parent donor. despite hhv-6 viremia pre-hsct which persisted posttransplant, he has had appropriate t cell engraftment. comprehensive genetic testing on whole exome level did not reveal any known mutations contributing to his phenotype. patient did have expression of gamma-chain by flow cytometry on t, b and nk cells. however, further testing revealed an il2rg 3 utr deletion of aa that, based on similar findings from a prior study can possibly lead to mrna abnormalities.1 to expedite the association of scid phenotype with x-linked disease, implying gamma-chain pathology, we obtained x-inactivation studies in maternal t-cells that showed severe skewing in both cases. furthermore, il21r signaling was impaired on b-cells in each case. the combination of these two assays proved that both patients carry a pathogenic il2rg mutation. conclusions: in the era of newborn screening for scid, we are discovering that phenotypic variability of scid patients can be very broad and caused by hypomorphic mutations in the common chain gene. exonbased genetic testing cannot exclude all variants and novel variants of unknown significance have to be evaluated by additional assays, including functional studies for causal effect. it is important to expedite early proof of association with gamma-chain pathology, especially in the era of gene therapy. we propose that in male infants with abnormal scid newborn screening and no known or previously described pathogenic mutation on genetic screen, evaluation continues for hypomorphic il2rg mutations. the probability of this process can be increased by a simple screening test for x-inactivation of maternal t lymphocytes. allergy / immunology, allergy / immunology associates inc. / case western reserve university introduction/background: igg4-related disease (igg4-rd) is an immunologic disorder with multiple clinical presentations previously thought unrelated. it is characterized by the frequent presence of tumor-like swelling of the affected organs and several histopathological findings including tissue lymphoplasmacytic infiltrates with predominantly igg4-positive plasma cells and lymphocytes, storiform fibrosis and obliterative phlebitis. humoral immunodeficiency is a term that encompasses several disease entities associated with impaired antibody production. it is suspected in patients who present with recurrent, frequently severe, sinopulmonary infections with encapsulated bacteria, which leads to evaluation of quantitative immunoglobulin levels and vaccine responsiveness. despite a few reports of primary immunodeficiency in patients with serum igg4 elevation, no adult case has been reported of igg4-rd in a patient with concomitant humoral immunodeficiency. objectives: to present a unique case with the presence of concomitant igg4-related disease and humoral immunodeficiency. methods: comprehensive chart review of our patient and all performed exams. literature review for igg4-related disease, igg4 elevation in humoral immunodeficiency and concomitance of igg4-related disease with humoral immunodeficiency. results: our patient is an 84-year-old caucasian male with relevant past medical history of chronic bronchitis who was referred to our practice after several episodes of pneumonia in the previous years, with six courses of antibiotics just in the year prior for recurrent sinopulmonary infections. blood tests revealed hypergammaglobulinemia and low level of vaccine responsiveness. chest ct showed multiple bilateral pulmonary nodules and hilar and mediastinal lymphadenopathy. sinus ct showed left maxillary sinus opacification. pulmonary function testing was normal. he later presented with left eye edema, proptosis, diplopia, and painless submandibular salivary gland enlargement. laboratory investigation showed an igg of 1730 mg/dl, igg4 of 771 mg/dl. the patient denied any history of pancreatitis or abdominal pain and abdominal ultrasound was normal. biopsy of a salivary gland was normal. mri of the left orbit was obtained, showing lacrimal gland enlargement. based on the patients recurrent infections, lack of response to tetanus immunization, and limited, non-sustained response to pneumococcal immunization, the patient was started on ivig therapy. the patient was also diagnosed with possible igg4-rd based on his salivary gland enlargement and orbital disease in association with hypereosinophilia and increased plasmablast levels. oral prednisone 40mg daily was started for four weeks, later followed by slow steroid taper (reducing 10mg every two weeks) with considerable improvement in left eye swelling and proptosis. a few months after discontinuation of the steroids, the orbital disease returned to its previous severity. left lacrimal gland biopsy confirmed igg4-related disease, with many areas showing greater than one hundred igg4-positive plasma cells per high-power field. after another course of steroids, oral prednisone was weaned to a maintenance dose of 10mg daily and the patient became asymptomatic from his ophthalmologic complaints with normalization of his ophthalmologic exam. his last checked igg was 1105 mg/dl. igg4 was still elevated at 324.8 mg/dl , but given controlled symptoms the patient was spaced to monthly ivig infusions and continued on that daily steroid dosage. conclusions: our patient, initially diagnosed with a humoral immunodeficiency, was later also diagnosed with biopsy-proven igg4-related disease, which is a novel association of this two diseases in an adult patient. previous rare reports of association between elevated serum levels of igg4 and patients with concomitant humoral immunodeficiency were in the presence of isolated igg4 elevation and not in the presence of igg4related disease. this novel association creates a therapeutic dilemma since the patient in question is hypergammaglobulinemic, yet needs ivig, which can lead to side effects such as thrombosis due to a hyperviscous state. the description of additional concomitant cases of both diseases and further understanding of their pathophysiology will be crucial to create awareness and obtain earlier diagnosis, to refine therapeutic options and design adequate treatment protocols. igm and iga anti-pneumococcal capsular polysaccharides as prognostic tool for common variable immunodeficiency: a longitudinal study. professor, sapienza university of rome introduction/background: the clinical spectrum of cvid ranges from a poorly symptomatic form to severe phenotypes characterized by high susceptibility to infections, autoimmunity, granulomatous inflammation, lymphoproliferative disorders, and malignancies. due to high prognosis heterogeneity, prognostic factors are required. objectives: with the aim to identify additional prognostic factors, we evaluated the anti-polysaccharide iga and igm responses by elisa assay in 75 cvid in a longitudinal study over a 6-year period. methods: patients were immunized at baseline with the 23-valent pneumococcal polysaccharide vaccine (pneumovax®). twenty healthy donors (hd) were also included. results: as expected, cvid patient had lower igm/iga response than hd. for cvid, four immunological phenotypes were identified by postvaccination igm and iga levels: igm and iga responders (11%), igmhigh responders (4%), igm-low responders (20%) and non-responders (61%). to simplify, we analysed igm-high group with igm and iga responders and igm-low with non-responders. during the follow up, concomitant cvid-related conditions, immunoglobulin serum levels, respiratory infections and outcome were recorded by medical files. cvid igm-low/non-responders developed more frequently respiratory, gastro enteric and autoimmune manifestation and malignancies in comparison to igm-high/igm and iga responders (respectively, pneumonia: 62% vs 25% ; chronic diarrhea: 33% vs 18%; autoimmunity 38% vs 9%). autoimmune cytopenias were not found in the igm-high/igm and iga responders group. eleven (15%) patients died during the study time. survival analysis according to the igm/iga responder status showed that the 6-years estimated survival for igm-high/igm and iga responders vs igm-low/non-responders group was respectively: 100% vs 100%, 100% vs 95%, 100% vs 92%, 100% vs 89%, 100% vs 85%, 100% vs 85%, 100% vs 82%. interesting, in our series only two deaths were due to infective complications: five were consequent to malignancies, one to autoimmune cytopenias and three to not-cvid related conditions. conclusions: in conclusion, even if patients could not raise the protective humoral level, in cvid the anti-polysaccharide iga and igm responses could represent a prognostic factor, individuating groups of patients with less immunological impairment, lower risk of comorbidities and better survival. introduction/background: gaucher disease (gd) is a rare autosomal recessive disorder characterized by a defective function of the catabolic enzyme -glucocerebrosidase (gba) leading to a progressive accumulation of its substrate-glucocerebroside (gc) -in various organs in particular in mononuclear phagocite system. hepatosplenomegaly and cytopenia represent the most common features of the disease. moreover, gd patients also show hyperinflammatory features -secondary to machrophages engorgement and actviation-hypergammaglobulinemia, and a immune-dysregulation involving b , t and nk cells. since clinical phenotpye can be subdolous, symtoms can overlap with alps, however, few data are available on specific immunity pattern in these patients. objectives: to evaluate immune-phenotype and other alps parameters in a cohort of patients with gd methods: we evaluated lymphocytes subsets, immunophenotypic and serological features of alps (dnts, tcr alfa/beta b220, b-memory cells, tregs/hla-dr ratio, il-10, il-18), and test of apoptosis in a cohort of patients with gd followed-up at igg. results: 35 patients (28 in treatment, 5 not) were studied. dnts and tcr alfa/beta b220+ resulted to be >1.5% of t-lymphocytes and > 60% in 6/32 (19%) and in 7/32 (22%), respectively. b-memory cells and t-regs/hla-dr ratio were <15% and <1 in 11/32 patients (34%). 3/32 evaluable (9%) had all these parameters concomitantly alterated. 4/19 (21%) evaluable patients were resistant to apoposis. il-18 was pathological in 26/29 (89%) patients. all patients had normal levels of il-10 and sfas. conclusions: this study shows that some patients with gd may present an immune-dysregulation pattern that can overlap with alps features. therefore, the differential diagnosis of gd should be taken into consideration by clinicians during diagnostic work-up of patients with an alpslike phenotype. introduction/background: allogeneic hematopoietic stem cell transplantation (hsct) using unrelated and haploidentical donors is complicated by increased rates of graft-versus-host disease (gvhd) and slow immune reconstitution. selective depletion of alpha/beta t lymphocytes and b cells is a recently developed method of graft manipulation that retains mature natural killer (nk) and gamma/delta t lymphocytes, both of which may exert a graft-versus-leukemia effect and protection against life-threatening infections. objectives: to describe the rate and quality of immune reconstitution, incidence of transplant-related complications, including viral reactivation and gvhd, and overall outcomes following tcr-alpha/beta-and cd19depleted hsct for hematologic malignancy in pediatric patients. methods: forty patients of median age 11.2 years (1.7-21.7) underwent hsct for acute myeloid leukemia (n=25), acute lymphoblastic leukemia (n=12), and myelodysplastic syndrome (n=3). grafts were from unrelated (n=29) and haploidentical (n=11) donors. tcr-alpha/beta and cd19 depletion was performed with the miltenyi clinicmacs plus system. median cd34+ cell dose was 10.1 x 106/kg (4.7-15), and median cd3+ cell dose was 2.3 x 107/kg (0.21-43.3). conditioning was with myeloablative busulfan or total body irradiation, cyclophosphamide, and thiotepa. twenty of 29 unrelated donor hscts and 11/11 haploidentical hscts also included antithymocyte globulin x 3. no patient received post-transplantation gvhd prophylaxis. all but 5 patients received rituximab on day +1 per protocol for recipient positive epstein barr virus (ebv) serology. results: all patients engrafted. median time to neutrophil engraftment was 13 (8-30) days, and median time to platelet engraftment was 16 (12-40) days. one patient experienced graft rejection on day +18, and twelve patients relapsed at a median of 173 (47-625) days. overall survival was 28/40 (70%) at a median of 24.2 (5.8-34) months follow-up. two (5%) patients developed grade iii or higher acute gvhd, and 2 (5%) patients developed extensive chronic gvhd. cumulative incidence of cytomegalovirus (cmv) and adenovirus reactivation were 11/40 (27.5%) and 5/40 (12.5%), respectively. nine (22.5%) patients developed bk hemorrhagic cystitis +/-viremia. ebv reactivation was not observed. median total, myeloid, t cell, and b cell donor chimerism were all 100% (ranges 50-100%, 93-100%, 41-100%, and 99-100%) at 1 year post-hsct. immune reconstitution of all cells lines was rapid ( table 1) . eighteen of 26 (69%) patients had detectable t cell receptor excision circles (trecs) by 4 months with a median trec count of 1226 (0-5713) per 10^6 cd3 t cells, and recovery of the naïve t-cell compartment was observed by 8 months in 19/26 (73%) of patients. t cell function as measured by response to pha was normal by 8 months in 15/23 (65%) patients and continued to increase steadily with time. despite rituximab on day +1 for 35/40 (87.5%) patients, there was rapid b cell reconstitution. nineteen of 23 (82.6%) patients had present switched memory b cells at 8 months, and 22/28 (78.5%) surviving patients are off immunoglobulin replacement at a median of 4 (4-18) months. conclusions: selective tcr-alpha/beta and cd19 depletion of haploidentical and unrelated grafts results in high engraftment and rapid immune reconstitution with low incidence of gvhd in children with hematologic malignancy. cd19 depletion and routine post-hsct rituximab on day +1 are effective at preventing ebv reactivation. introduction/background: hyper-igd syndrome (hids; 260920) is an autosomal recessive disorder characterized by recurrent episodes of fever associated with lymphadenopathy, arthralgia, gastrointestinal disturbance, and skin rash. the diagnostic hallmark of hids is a constitutively elevated level of serum immunoglobulin d (igd), although patients have been reported with normal igd levels. the disease is associated with mutations in the gene of mevalonate kinase. objectives: male patient, 30 years old, born to non-consanguineous parents, with a history of severe diarrhea since childhood, followed by respiratory infections and pneumonias. moreover he presented several episodes of severe abdominal pain, with intestinal obstruction since seven months old, needing surgical intervention due to acute abdomen. this picture repeated several times until 10 years of age, being submitted to new surgeries due to intestinal suboclusion. at 16 and 17 years he had gastroenteritis and then pancreatitis. 2 years ago new severe acute gastroenterocolitis with suboclusion, submitted to laparotomy and resection of a little part of the gut. he has frequent diarrheas, triggered by coffee and tea. usually evacuates 5 times a day. he was evaluated by several pediatric immunologists in childhood, who distrusted alpha heavy chain disease. he had an intense reaction to the bcg vaccine, and then did not make any more vaccines (only opv (sabin) in the campaigns). he was tonsillectomized at 2 years of age. he had measles, chickenpox, and mumps (at 5 years old). cellulitis at 7 years, repeating several times since then. he presented improvement of pneumonias but still has sinusitis and otitis (approximately 8 times a year introduction/background: pitthopkins syndrome is a rare neurological disorder caused by mutations the tcf4 gene on chromosome 18q21. clinical features include severe intellectual disability, constipation, microcephaly, and seizures. features distinguishing it from other neurodevelopmental syndromes such as rett syndrome and angelman syndrome include breathing abnormalities (either apneic episodes or hyperventilation) and atypical facial features. typical facial dysmorphism includes bitemporal narrowing, deep-set eyes, an m shaped upper lip, and widely spaced teeth. although a very rare diagnosis (slightly over 200 reported cases), it is not known to be associated with underlying humoral or cellular immunodeficiency. there is only one report of igm abnormalities described in a patient with pitt-hopkins syndrome. objectives: to present a case of pitthopkins syndrome with humoral immunodeficiency. methods: this is a case presentation of a patient with pitt-hopkins syndrome requiring immunoglobulin replacement therapy. results: 15 year old female with genetically diagnosed pitt-hopkins syndrome who presented to our office for immunological evaluation in the setting of recurrent sinopulmonary infections. she was placed on chronic antibiotics by the department of otholaryngology for approximately two years prior to presentation. she was found to be hypogammaglobulinemic (igg: 398 mg/dl, iga: 21 mg/dl, igm: 40 mg/dl), had non-protective titers to streptococcus igg antibody (6/23 titers protective greater than 1.3 mcg/ml) despite booster vaccination with pneumovax, and had borderline tetanus (0.18 iu/ml) and diphtheria titers (0.11 iu/ml). given this laboratory evaluation and her recurrent illness, immunoglobulin replacement therapy (igrt) was started. whole exome sequencing was completed to assess for any other genetic cause of her immunodeficiency. the only abnormality was her previous known pathologic variant p.q670hfsx40 c.2010_2011delga (gln670his) in exon 19 in the tcf4 gene. she continued to have infections despite therapeutic igrt. chronic antibiotic treatment was initially tapered, however needed to be reintroduced as ivig alone was not stopping her infections, despite a igg level over 1000 mg/dl. conclusions: humoral immunologic deficits are not known to be associated with pitt hopkins syndrome. there has been one case report of a patient with poliomyelitis-like syndrome following an asthma attack in a patient with pitt hopkins syndrome, which was treated with igrt and resulted in a nearly complete recovery. however, igrt was not used for reasons of underlying immunodeficiency. to our knowledge this is the first patient with pitt hopkins syndrome with persistent hypogammaglobulinemia and frequent infections requiring immunoglobulin replacement therapy. it remains unclear why the patient continued to have infections despite igg levels > 1000 mg/dl, yet with the combination of igrt and prophylactic antibiotics the patient remains healthy. introduction/background: gata2 deficiency is a rare disease that typically presents in late childhood or early adulthood with heterogeneous phenotypes including emberger syndrome. emberger syndrome is characterized by lymphedema and predisposition for myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). over 75% of patients with gata2 mutations have immune deficiencies. definitive diagnosis of gata2 deficiency is made by gene sequencing, and treatment includes infection control and potentially hematopoietic stem cell transplant (hsct). objectives: the goal of this report is to contribute data to the small documented cohort of patients with gata2 deficiency to aid in diagnosis and management of this rare, heterogeneous disorder. methods: we present a case series of three siblings with identical gata2 mutations with variable phenotypes. a usidnet query resulted 51 patients with gata2 mutations. results: three siblings (12 year-old female (a), 15 year-old male twin (b), and 15 year-old female twin (c)) and their mother had congenital deafness. clinical symptoms include (a) h1n1 influenza requiring mechanical ventilation, warts, and hypogammaglobulinemia, (b) streptococcus pyogenes neck abscess, warts, acne, and mds, and (c) lymphedema and acne. all three patients had absolute monocyte count (amc) of 0 and lymphopenia without documented lymphoid cell dysfunction. a mutation on exon 5, c.1062delg, confirmed the diagnosis. all three patients were started on mycobacterial prophylaxis with azithromycin and recommended hpv vaccination. the usidnet query average age of symptom onset was 22 years, and average age at diagnosis was 31.5 years. 35.3% (18/51) of patients had a family history of gata2 deficiency, 49% (25/ 51) of patients had warts, 7.8% (4/51) had lymphedema and only 1 patient had sensorineural deafness. 38% (19/50) of patients had amc of 0. functional data was limited. conclusions: life threatening infections as well as hematologic malignancies have been reported in patients with gata2 deficiency, which can be successfully treated with hsct. to our knowledge, the gata2 mutation detected in this family has not been previously reported. the clinical presentation in these three patients was heterogeneous despite identical genotypes, and diagnosis occurred years after initial symptoms. variable phenotypes were found in the usidnet gata2 deficiency cohort as well. a high index of suspicion for the disorder and early recognition of clinical manifestations and laboratory abnormalities may aid in timely diagnosis of gata2 deficiency, with potential for improved outcomes. consulting medical advisor, immune deficiency foundation introduction/background: as individuals with antibody deficiencies age they are susceptible to developing shingles. patients with antibody deficiencies are advised not to receive live viral vaccines such as zostavax, the shingles vaccine. objectives: our survey aimed to determine the frequency of shingles and use of zostavax in common variable immunodeficiency (cvid) and hypogammaglobulinemia patients. methods: 11,533 email invitations delivered to members of the immune deficiency foundation database requesting participation in an online survey about zoster, influenza and varicella experiences. data from 881 individuals age 19 years old or older with cvid (n=760; mean age 54 years old; 83% female; 92% white non-hispanic) or hypogammaglobulinemia (n=121; mean age 54 years old; 83% female; 93% white non-hispanic) were analyzed. results: close to one fifth (18%, n=105) of adults age 50 or older with cvid or hypogammaglobulinemia (n=568) had received shingles vaccination. the majority of those who were vaccinated reported receiving zostavax once (92%, n=97), while 5% (n=5) received a booster vaccination as well. mild side effects (e.g., skin rash and muscle pain) were only reported by 18% (n=18) of vaccine recipients after receiving their first vaccination. no side effects were reported after receiving the booster vaccination and no hospitalizations were reported as a result of receiving zostavax. when comparing shingles diagnosis and shingles vaccination, of the 568 adults age 50 years old and older, 35% (n=196) had been diagnosed with shingles, and of those diagnosed 82% (n=160) did not receive zostavax. of adults age 19 years old or older, 32% (n=279) reported a shingles diagnosis. similarly, the cdc reports almost 30% people in the united states will develop shingles during their lifetime. more than half (56%, n=157) of those ever diagnosed with shingles reported experiencing shingles once, 18% (n=51) had shingles twice and 24% (n=68) had shingles three or more times. respondents with more than three shingles episodes were more likely to report their rash lasted more than two months and their blisters became infected. conclusions: almost 20% of adults with cvid or hypogammaglobulinemia reported receiving zostavax despite recommendations against vaccinations for immunodeficient individuals. however, side effects in those pi patients who received the shingles vaccine appears minimal. though it is possible these individuals were vaccinated prior to diagnosis of their pi; additional patient and physician education on live vaccines and immunodeficiency may be needed as well. the approval of the new non-live virus component varicella zoster vaccine may be of benefit to patients with pi. introduction/background: inclusion body myositis (ibm) is a rare disorder characterized as an inflammatory myopathy with endomysial inflammation and numerous red-rimmed vacuoles seen on biopsy. five cases of ibm have been described in the literature in patients with common variable immunodeficiency (cvid). objectives: to make immunologists aware of ibm as a complication of cvid, that may be incorrectly diagnosed as myositis or autoimmune neuropathy. methods: case description results: we report a 62-year-old man with common variable immunodeficiency on gammaglobulin replacement who presented complaining of progressively worsening lower extremity pain, weakness, and fatigue. he states that over the last couple of years, it has become difficult climbing and descending steps, arising from a seated position, and has begun experiencing frequent falls. his creatinine kinase level was found to be elevated at 293u/l and he continued to be lymphopenic with total lymphocyte counts ranging from 300 to 800k/ul (cd4+ t-cells 34%, cd8+ t-cells 18%, cd19+ bcells 2%). his esr ranged from 25 to 60mm/h. anti-glutamic acid decarboxy antibody (gad) was initially elevated at 1.3u/ml and rose to 2.3u/ml within 6 months suggestive of a neuropathy. based on an electromyography (emg) and a muscle biopsy, he was diagnosed with polymyositis. he was treated with high dose steroids with no improvement. his intravenous gammaglobulin dose was then increased from 45mg every 3 weeks to 45mg per day for 3 straight days every 2 weeks. 4 months later, his creatinine kinase level dropped into the normal range (<200u/l), however, he continued to complain of worsening weakness. physical exam showed decreased muscle bulk in forearms and quadriceps bilaterally, lack of a quadriceps tendon reflex, strength 4/5 in flexor digitorum profundus and 4/5 in hip flexors, and a broad-based gait. histology and electron microscopy of a repeat muscle biopsy identified rimmed muscle vacuoles as typically noted in inclusion body myositis. conclusions: inclusion body myositis is a potential rare complication of common variable immunodeficiency. it can mimic polymyositis and inflammatory demyelinating disorders. high dose steroids and ivig are of no clinical benefit in ibm, despite decreasing serum creatinine kinase levels, which may raise a false impression of a clinical benefit. objectives: in this abstract we present the case of the development of generalized cmv infection in a child with scid. girl n. at the age of 3 months entered the children's infectious clinical hospital with complaints of cough, high febrile temperature for 5 days, refusal to eat. from the anamnesis of life the girl from the 1st pregnancy, 1 birth, was born full term in 40 weeks gestation, birth weight 4640g. for 3 months of life, a bad increase in body weight was noted and at the time of admission, the weight in 3 months was 5400 g. according to the parents, the child had atopic dermatitis. from the anamnesis of the disease on 08.01.2017, the temperature rose to 38.2°c, there was a cough and a mucous discharge from the nose. then the child refused to eat, the body temperature rose to 39.2°c. at this time, the girl's mom was borne by the ari. january 14, 2017 patient was hospitalized in the hospital with a diagnosis: acute respiratory viral infection, acute rhinitis, pharyngitis, acute bronchitis, toxicosis of 1-2 degrees. acute pneumonia? atopic dermatitis, infant form. on january 15, 2017, due to the worsening of the condition associated with the increase in oxygen (o2) -dependence, the child was transferred to the department of anesthesiology and resuscitation. methods: in the general analysis of blood upon admission, leukocytes are 14.2x10 9 / l, hemoglobin is 105 g / l, platelets are 172 x 10 9 / l, esr is 3 mm / hour, stabs are 4% (abs. -0.58 x 10 9 / l), segmented -60% (abs-8.64 x 10 9 / l), lymphocytes -24% (abs 3.46 x 10 9 / l), monocytes -10% (abs -1.44 x 10 9 / l). in a biochemical study, the total protein is 51 g / l, total bilirubin is 4.7 mol / l, urea is 7.4 mmol / l, creatinine is 60 mol / l, lactate dehydrogenase is 2249 u / l, alt is 131 u / l, asat -257 e / l, crp -5.8 mg / l. radiography of the lung from 14/01/2017 -data in favor of interstitial pneumonia. the study of the acid-base ph state is 7.367, pc02 is 34.3 mmhg, po2 is 40.1 mmhg, lactate is 1.4 mmol / l. a blood test was performed using the elisa and pcr method for markers of hsv, cmv, enterovirus and toxoplasmosis. ultrasound of the abdominal cavity revealed moderate hepatomegaly, signs of thickening of bile, splenomegaly. moderate diffuse changes in the renal parenchyma (toxic-inflammatory?). the minimum amount of free fluid in the abdominal cavity. ultrasound of the brain revealed signs of subependimal microcyst on the right. according to the immunogram, a sharp decrease in cd3 + 26% (58-85%) was detected, activated t-lymphocytes (cd3 + hla-dr +) were 19.9% (3-15%), t helper / inducers (cd4 + cd8 -26.6% (30-56%) and t suppressors / cytotoxic (cd8 + cd4-) 0.5% (18-45%), a high ratio of tx / tc (cd4 + cd8 +) was detected 53.2% (0.6-2.3), cytotoxic non-t cells (cd3-cd8 +) -1,2, an increase in the number of b-lymphocytes (cd19 +) -58.9% (7-20%), natural killers (cd16 + cd56 +) -6.6% (5-25%), natural t-killers (cd3 + cd16 + cd56 +) -0.3 (0-5%), leukocyte gates (cd45 + cd14-) -99% (95 -100 %). the absolute content of t-lymphocytes was 0.15 x 10 9 / l, b -lymphocytes -0.35 x 10 9 / l. the number of thymic migrants (cd45 + cd45ra + cd31 +) was not detected (0%). according to the results of the immunogram the diagnosis is made: severe combined immunodeficiency (t-b + nk +). 01/17/2017 ct scan of the chest was diagnosed ct signs of a polysergic two-sided inflammatory process in the lungs (figure 3 ). when blood was sown for sterility on january 19, 2017, staphylococcus epidermidis was isolated in an amount of 10 3, sensitive to linezolid, gentamicin resistant to amoxicillin, amoxicillin / clavulonic acid, and ciprofloxacin. on january 19, 2017, cmv dna was detected in an amount of 7.6 × 10 6 copies / ml. results: since the arrival clarithromycin was administered at a dose of 15 mg / kg per day in 2 divided doses from 14/01/2017 to 15/01/2017. from 16/01/2017 to 17/01/2017. change of antibacterial therapy for azithromycin intravenous at a dose of 10 mg / kg per day once a day.17.01.2017 -01/18/2017 the state of the child is very severe with negative clinical and laboratory dynamics despite the ongoing therapy. antibacterial therapy was changed to meropenem in a dose of 60 mg / kg intravenously every 8 hours, linezolid at a dose of 30 mg / kg per day and oseltamivir at a dose of 6 mg / kg per day from 17/01/2017 to 20/01/2017. 19.01.2017-20.01.2017 substitution therapy with an octagam in a dose of 0.4 g / kg was intravenously dripped. the patient's condition without significant dynamics. based on the results of pcr on cmv, ganciclovir was administered at a dose of 10 mg / kg intravenously drip 2 times a day. 01/23/2017 due to a decrease in platelet count, the platelet mass is transfused and there was a rash all over the body at night, which is associated with the development of the "graft versus host" reaction (gvhr). despite the ongoing therapy, a fatal outcome occurred. the main diagnosis: primary immunodeficiency (severe combined immunodeficiency, t0 b + nk +). complications: sepsis. septic shock. spon: ards, renal failure, dis, thrombocytopenia, anemia 3. two-sided lower-lobe pneumonia. generalized cmv infection. gvhd, acute dermal form. concomitant: atopic dermatitis, infant form. conclusions: the peculiarity of the described clinical case was that the patient's first symptoms of scid developed in the first months of life and were manifested by a bad weight gain, atopic dermatitis and the development of a life-threatening generalized cytomegalovirus infection with the development of bilateral low-grade pneumonia, respiratory insufficiency and acute cutaneous gvhd form, after transfusion of unirradiated platelet mass. an expanded immunological study confirmed the diagnosis of scid. methods: this study included 5 patients (2 boys and 3 girls) aged 2 months to 5 years. the reasons for entering the hospital were manifestations of severe hepatitis (in 2 children), acute respiratory infection (2 children) and 1 patient with symptoms of infectious mononucleosis. all patients were examined according to clinical protocols and given the severity and atypicality of the course of any infectious diseases, patients underwent immunological examination of the blood and they were consulted by an immunologist. all children were diagnosed with congenital immunodeficiency. results: in 3 cases, the trigger for the realization of the immunodeficiency state was infection (e. meningoseptica + kl. pneumonia + b. pertussis; cmv; veb); in 2 patients, giant cell hepatitis occurred. in 2 patients, despite the ongoing therapy, the disease had an unfavorable (lethal) outcome (1 patient with hepatitis and 1 patient with generalized cmv infection). conclusions: thus, it should be noted that timely diagnosis of a congenital defect of the immune system and thus timely therapy will avoid adverse outcomes. interferon gamma (actimmune®) effects on severe burkholderia cepacia pneumonia in variant x-linked chronic granulomatous disease 4 professor of pediatrics, university of utah 5 associate professor of clinical pediatrics, keck school of medicine at the university of southern california 6 professor of medicine, university of utah introduction/background: interferon gamma (ifnγ; actimmune®) has been proven to significantly decrease the overall number of infections in patients with chronic granulomatous disease (cgd) when given prophylactically (nejm 324:408, 1991) . therapy with ifnγ has also been employed to treat severe overwhelming infections in some instances, such as severe aspergillosis with success (jid 613:908, 1991) . we report here, two patients with very severe burkholderia cepacia (b. cepacia) infection, one of whom was placed on a respirator for approximately two weeks and another who was on extracorporeal membrane oxygenation for an extended period of time. both were treated with ifnγ (actimmune®) in addition to appropriate antimicrobial therapy in an attempt to affect these lifethreatening infections. objectives: the objective of this presentation is to describe two very severe variant x-linked cgd patients with b. cepacia pneumonia who were treated with ifnγ. in addition, we measured both super oxide production as well as nitric oxide production in the stimulated or unstimulated phagocytes from these patients in the presence or absence of interferon gamma. methods: case histories of both patients were reviewed in respect to the severity of their infection, the time spent on a respiratory or extracorporeal membrane oxygenation, the antimicrobial therapy administered, and the clinical results following the administration of interferon gamma as adjunctive immunomodulatory treatment. a standardized neutrophil oxidative burst assay was employed using cytochrome c reduction to measure super oxide production. in addition, nitric oxide was measured in the phagoctyes of the patients after stimulation with phorbol myristate acetate (pma) in the presence or absence of ifnγ using daf-2 fluorescence dye to detect the production of intracellular nitric oxide. results: a 2-year-old male developed a left lobar pneumonia and was admitted to primary children's medical center's intensive care unit and treated with iv cefotaxime, clindamycin, later, vancomycin and azithromycin were added. ct scan revealed a left-sided pneumonia and moderate parapneumonic effusion. subsequently, the patient decompensated, was intubated, and placed on respirator therapy. broncheoalveolar lavage and blood grew b. cepacia. neutrophil dihydrorhodamine fluorescence (dhr) demonstrated an intermediate broad peak of fluorescence with a small peak of unactivated cells, while the mother's dhr showed a broad intermediate peak suggesting the carrier state of variant x-linked cgd. both the patient, a 2.5 month old younger brother, and the carrier mother were found to have a g to a splice site mutation in exon 3 of the gp91-phox gene at position c.252 confirming the diagnosis of x-linked variant cgd. after approximately 2 weeks on the respirator, ifnγ (actimmune®) therapy was instituted with significant improvement of the patient's lung function. he was taken off the respirator approximately 3 days after the ifnγ therapy was instituted. following addition of ifnγ to his pma-stimulated neutrophil, there was a 22% increase in superoxide production and a 20 fold increase in nitric oxide in his monocytes. the second patient was a 9-year-old male who presented with fever and cough and was diagnosed with right-sided middle and lower lobe pneumonia with cavitations. bronchoalveolar lavage grew b. cepacia and nocardia. following increasing ventilatory and circulatory collapse he was placed on extracorporeal membrane oxygenation and treated with 4-5 antimicrobial agents. after 9 days of such therapy, ifnγ therapy was initiated and he was weaned from ecmo after 3 days and has remained essentially healthy since then when he is on ifnγ prophylaxis. dhr revealed a broad intermediate peak in the patient and normal and intermediate peaks in the mother suggesting variant x-linked cgd in the patient and the carrier state of x-linked variant cgd in the mother. targeted sequencing revealed a g to a splice site mutation in exom 3 of the cybb gene at position c.266 confirming the diagnosis of x-linked variant cgd. following addition of ifnγ to this patient's pma stimulated phagocytes, there was a 150% increase in superoxide production and a 200% increase in monocyte nitric oxide production. conclusions: two male variant x-linked cgd patients with splice site mutations in the cybb gene and severe life-threatening b. cepacia pneumonia, one on a respirator and one on ecmo were administered ifnγ (actimmune®) and each responded dramatically within 2-3 days recovering their respiratory capacity and coming off of assisted ventilation and ecmo, and have continued to do well when on ifnγ (actimmune®) therapy. introduction/background: hyqvia is a recombinant human hyaluronidase (rhuph20)-facilitated subcutaneous immunoglobulin (ighy) 10% replacement therapy for patients with primary immunodeficiency diseases (pidd). objectives: to acquire long-term safety data on ighy, and assess prescribed treatment regimens and administration in routine clinical practice, a global postauthorization safety study (pass) is being conducted. methods: this is an ongoing prospective, non-interventional, open-label, uncontrolled, multicenter study initiated in the united states in november 2015 to assess local and systemic effects of ighy within a routine clinical setting. patients aged 16 years with pidd who have been prescribed and/ or have started ighy are eligible for enrollment. patients are followed according to standard clinical practice and their treatment regimen is at the discretion of the treating physician. the presence of anti-rhuph20 antibody titers is evaluated on a voluntary basis. results: as of august 2017, 175 patients had been enrolled at 26 us study sites. there were no serious aes which were deemed treatment related. sixteen patients experienced a causally related non-serious local ae (9.1%; 0.43 events/patient-year, 0.07 events per infusion) and 25 patients experienced a causally related non-serious systemic ae (14.3%, 0.88 events/patient year, 0.14 events per infusion). of the 113 patients with immunogenicity data, 7 had 1 positive binding antibody test to rhuph20 (titers 1:160); no neutralizing rhuph20 antibodies were detected. conclusions: this interim analysis of prospectively-collected data of ighy use in routine clinical practice indicates that ighy is well tolerated with no treatment-related saes and has not been associated with neutralizing anti-rhuph20 antibodies in patients with pidd. introduction/background: idiopathic thrombocytopenic purpura (itp) and/or hemolytic anemia accompanied by splenomegaly occurs in up to 25% of patients with common variable immunodeficiency (cvid). treatments include steroids, other immune suppressants and rituximab. however, in some that do not respond, splenectomy may be performed. while splenectomy is known to be associated with an increased risk of infections or thromboembolic events, studies in other conditions (hemolytic disorders, hereditary spherocytosis, etc), also suggest an increased risk of pulmonary arterial hypertension (pah) after this procedure. objectives: while splenectomy is known to be associated with an increased risk of infections or thromboembolic events, studies in other conditions (hemolytic disorders, hereditary spherocytosis, etc), also suggest an increased risk of pulmonary arterial hypertension (pah) after this procedure. methods: we report three cases of pah following splenectomy for cytopenias in patients with cvid. results: the first is a 40 yo female, with long standing cvid complicated by interstitial lung disease, nodular regenerative liver disease and itp post splenectomy. the second is a 48 yo man with severe bronchiectasis, cirrhosis/nodular regenerative liver disease and itp post splenectomy. the third is a 54 yo woman with cvid (taci compound) complicated by cirrhosis/nodular regenerative liver disease, lung nodules and evans syndrome. all developed severe pah requiring chronic medications. pah in these patients is best classified as multifactorial, group v. conclusions: whether due to thrombus formation, continued cytopenias, and/or vascular changes, we suggest that pah may be a long-term complication of splenectomy in complex cvid. connective tissue, skeletal and vascular abnormalities. two isoforms exist, stat3, a 770 amino acid protein, and stat3, a 722 amino acid protein produced by alternative splicing of exon 23 resulting in a frame shift and truncated protein at the c-terminus. objectives: we follow 4 patients from 2 families with stat3 mutations leading to altered c-terminal proteins. the patients have high ige but milder features of ad-hies. methods: clinical data were collected. stat3 sequencing, stat3 functional assays as well as lymphocyte phenotyping were performed. results: patient 1 is a 58 year old man diagnosed with hies as a child due to eczema, recurrent boils and high ige (5000s iu/ml). he has tortuous and dilated coronary arteries, however denies lung infections, cmc, retained teeth, scoliosis, minimal trauma fractures, or hyperextensible joints. as an adult he developed avascular necrosis of both hips. whole exome sequencing revealed a novel splice mutation, c.2144+1g>t at the end of exon 22 causing skipping of the 43 nucleotide exon as well as utilization of the stat3 alternative splice acceptor in exon 23, resulting in a 93 nucleotide deletion. the mutant stat3 protein product has a 31 amino acid, in-frame deletion encompassing both y705 and s727 phosphorylation sites. no stat3 is made from the mutant allele. lymphocyte phenotyping was unremarkable, however total stat3 protein levels were decreased in ebv transformed b cell lines and there was decreased y705 phosphorylation after stimulation. patient 2 (family 2) was healthy until diagnosed with severe, refractory coccidiodies pneumonia complicated by pneumothorax with prolonged bronchopleural fistulae at age 16 years. this led to an immune evaluation in which he was found to have elevated ige (878 iu/ml, ref 0.0-91.0 iu/ ml). he had one perianal abscess, primary teeth requiring extraction and mild scoliosis, but denies cmc, bacterial pneumonias, eczema, or minimal trauma fractures. stat3 sequencing revealed a single base insertion in the transactivation domain, c.2185_2186insc causing a frameshift in the stat3 isoform, p.r729pfsx11, occurring immediately after the s727 phoshporylation site. the deletion occurs within the alternatively spliced region of exon 23, providing an intact, wild-type stat3. stimulation with il-6 or il-21 showed reduced pstat3 at y705, and elevated pstat1which is often seen in other ad-hies patients. lymphocyte phenotyping was unremarkable and th17 cell analysis showed low-normal levels of th17 cells while ebv transformed b cells showed reduced total stat3 levels. his mother and infant sister also share this mutation -the 8 month old infant had normal ige, and intermittent rashes; his mother has high ige (1193 iu/ml), recurrent sinopulmonary infections (complicated by tobacco use) but without bronchiectasis or pneumatocele, and denies cmc with the exception of pregnancy related vaginal candidiasis. conclusions: loss of function and gain of function mutations in stat3 lead to distinct syndromes, but it appears that stat3 mutations affecting the isoform expression, such as those reported here, can also lead to immune dysregulation with incomplete features of ad-hies. these stat3 mutations will allow us to better understand the relative roles of the isoforms stat3 and stat3 in somatic and immune cell signaling. physicians and is associated with immunological defects aswell. mutationsin the kmt2d and kdm6a genes are the most common genetic changes that lead to kabuki syndrome but for many cases the genetic basis remains unknown. objectives: recognize the varied presentation for a unique immunodeficiency syndrome methods: this is a case series. results: this is a case series describing three patients with ks and their clinical presentations which predominantly involve immunodeficiency and autoimmunity. our first patient is a 31-year-old male with autoimmune hemolytic anemia (aiha) at a young age, recurrent respiratory infections and hypogammaglobinemia which led to a diagnosis of cvid at the age of six. in addition, he has dysmorphic facial features, intellectual disabilities, and short stature but it was not until his late twenties where he was found to have a missense mutation in kmt2d (p.arg5048cys) which has been described in patients with ks. the second patient is a 34-year-old female with hypogammaglobinemia, evan's syndrome, short stature, and severe complications which include granulomatous-lymphocytic interstitial lung disease, pulmonary hypertension, and chronic kidney disease. she was also found to have a missense mutation in kmt2d (p.arg5048cys) in her early thirties and passed away from complications of her disease. the third patient is a 27-year-old female with a history of low iga/igg and poor vaccine titers, aiha, neutropenia, pulmonary nodules, and developmental delay who was diagnosed with cvid in her mid-twenties. for her immunodeficiency and autoimmunity, she was treated with immunoglobulin replacement, rituximab and cyclosporine and was found to have a missense mutation in kmt2d (p.cys1471trp). this mutation was a de novo mutation in this patient and has also been reported in another patient with ks. conclusions: these cases highlight that the presentation of ks is varied and frequently includes immunodeficiency and autoimmunity in addition to the characteristic short stature and developmental delay. a diagnosis of ks remains challenging due the diversity of symptoms and disease severity, the need for genetic testing, and due to overlapping clinical presentations with other developmental conditions. thus, often times, like in these cases, the diagnosis of ks is delayed. chief medical officer, rocket pharma introduction/background: lad-i is a rare disorder of leukocyte adhesion, resulting from itgb2 gene mutations encoding for the beta-2 integrin component cd18. cd18 deficiencies prevent integrin dimerization and endothelial leukocyte adhesion, essential for extravasation and antimicrobial activity. severe lad-i (<2% of normal neutrophil [pmn] cd18 levels) is characterized by recurrent serious infections and early mortality unless treated by allogeneic hematopoietic stem cell transplant (hsct). mortality for severe lad-i was reported as 75% by age 2 in an initial 1988 multicenter retrospective study. moderate lad-i (2-30% of pmn cd18 levels) is more indolent; although most patients (pts) survive childhood with recurrent skin and mucosal surface infections; mortality by age 40 can exceed 50%. lad-i is characterized by umbilical cord complications (delayed separation and omphalitis), poor wound healing and leukocytosis. objectives: reports regarding lad-i have been published in recent decades but no recent comprehensive prognostic assessments are available. we sought an updated understanding of severe lad-i with emphasis on prognosis in the absence of hsct, hsct outcomes and association of cd18 expression with clinical features. methods: we created a database of all published lad-i cases via pubmed searches and review of available references. results: three hundred twenty-three lad-i cases were reported between 1975-2017 in 107 publications (68 case-reports; largest series n=36). the nations reporting the most cases were iran (n=65), usa (n=50), and india (n=45); the highest number of publications were from us centers (25). 113 pts were considered to have severe lad-i, 63 moderate and 147 were not classified. pmn cd18 expression levels was reported for 265 cases and was <2% in 135 patients (51%) and >=2% in 130 pts. four pts with cd18 >2% were considered to have severe lad-i (cd18% range 2.4 17.3). gender was noted for 282 pts; 148 (52%) were male. age at presentation was reported for 146 cases. for 63 pts with cd18<2%, median presentation was age 1 m (range 0.03-18m); for 62 pts with cd18 >=2%, median presentation was age 6m (range 0.03-192m). infection details and cd18% were available for 154 (48%) cases. the most frequent infections in pts with cd18 <2% were respiratory tract (39%), sepsis (29%) and otitis media (27%) and for pts with cd18 >=2% they were periodontal (52%), otitis media (365%) and sepsis (25%). perianal skin infections and necrotic skin ulcers were noted in >10%. umbilical complications were more frequent in severe lad-i (92 of 110 pts with cd18<2% [84%] and 47 of 81 with cd18 >=2% [58%; p = 0.0002]). for severe lad-i pts with 2 years of follow-up (or death prior to 2y), there was correlation between absence of umbilical complications and survival to 24 m (p < 0.001). wbcs were reported in 143 cases (median 45 x 109/l; range 10 150 x 109/l). there were limited correlations between cd18 expression and wbc (r < 0.1) and between cd18 and cd11 expression (r < 0.5). mutation analyses were reported in 139 cases with >20 gene locations noted and mutations on exons 5, 6 and 7 accounting for 44% of specified cases. in 18 cases, cd18 expression was >30%; in 8 of 12 cases where cd11 expression was noted, at least one cd11 moiety was reported as <2%. we sought to understand whether prognosis for severe lad-i in the absence of hsct is similar to the initially-reported 25% survival to age 2. there were 66 severe lad-i cases (per investigator assessment or cd18 <2%) for whom survival to 2 years was reported, 40 of whom died prior to age 2 (61% mortality). mortality was similar for the subset of 43 cases reported since 2000 (56%, 24 deaths). early mortality was substantially lower in patients with cd18 >=2% and the majority of pts with cd18 >4% survived to adulthood. outcomes for 101 pts who received hsct were consistent with recent series; phenotypic correction was reported in 83% of pts with hla-matched sibling donors. mortality was 19% overall (11% for hlamatched sibling recipients). for 22 pts receiving haploidentical hsct there was 32% mortality and 55% received 1 subsequent hsct. conclusions: severe lad-i remains a life-threatening condition with limited 2-year survival in the absence of allogeneic hsct. umbilical complications and granulocytosis are frequent early manifestations; respiratory tract, ear, sepsis, oral and skin infections are common. hsct is potentially curative; transplant-mortality and other complications are frequent, especially in haploidentical recipients. diverse itgb2 mutations result in lad-i, and genetic evaluation may be valuable for diagnosis and prognosis. rapid identification of pts with potential lad-i (unusual or severe infections in infancy, granulocytosis and umbilical complications) is essential to enable referral to centers with disease expertise. objectives: to determine the phosophorylation of stat3 in ad-hies patients with known stat3 mutations. methods: peripheral blood mononuclear cells (pbmcs) were collected from ad-hies patients under irb-approved institutional research protocols. pbmcs were then stimulated with il-6 or il-21 for 15, 30 and 60 minutes. cells were surface stained for cd3, cd4, cd8, cd56 and cd19. they were then fixed, permeabilized and stained with anti-y705-stat3 to evaluate for phosphorylation of stat3 at position y705. cells were then washed and data acquired using flow cytometry. results: pbmcs from one ad-hies patient with a stat3 sh2 domain mutation (p.y657c) demonstrated normal y705 phosphorylation after il-6 stimulation. pbmcs from another ad-hies patient with a dbd mutation (p.h437y) exhibited highly reduced stat3 phosphorylation after il-6 stimulation and absent phosphorylation after il-21 stimulation. conclusions: these findings highlight that, in the context of ad-hies, the domain location of the stat3 mutation does not predict stat3 phosphorylation potential following stimulation and challenges the current paradigm. (106) submission id#426423 cheng sun 1 1 associate professor, institute of immunology introduction/background: as the predominant lymphocyte subset in the liver, natural killer (nk) cells have been shown to be highly correlated with the outcomes of patients with hepatocellular carcinoma (hcc). previously, we reported that nk cells were decreased and functional deficiency in hcc. however, the mechanism underline remains unknown. objectives: in this study, through the use of 23 healthy livers, paired peritumoural tissues (pt) and intratumoural tissues (it) from 236 hcc patients, methods: we have evaluated the expression of cd160 and its co-ligand receptor btla on hepatic cd8+ t cells and nk cells. results: decreased expression of cd160 on nk cells was observed in intratumoural but not pt regions, along with nk cell dysfunction, poor prognosis and tumour metastasis. human cd160+ nk cells exhibited functional activated, high capacity of ifn-secretion and nk mediated immunity by global transcriptomic analysis of sorted cd160+ and cd160-hepatic nk cells. blocking tgf-1 specifically reversed the ifn-production of cd160+ nk cells. in addition, this decreased cd160 expression is predominantly on cd56bright nk cells. conclusions: these findings indicate that cd160 expression reduction contributes to nk cell exhaustion and tumour immune escape, suggesting that cd160 has the therapeutic potential for fighting liver cancer. introduction/background: the low number of circulating lymphocytes in the blood is a marker for cellular immunodeficiency in young children. ethnicity also affects the lymphocyte count and ethnicity-specific lymphocyte norms have been used in many countries. this study analyzed the lymphocyte counts in a large cohort of infants and young children from the arabian peninsula. objectives 1-to define the normal lymphocyte counts in arab children 2-to define the possible cutoff lymphocyte count that define lymphopenia. methods: this is a cross-sectional analysis of the lymphocyte counts in 11,237 arab children. the age groups were: 1 day, 1-6 months, 6-12 months, 1-2 years, 2-3 years, 3-4 years, 4-5 years, and 5-6 years, 47% females. we analyzed the first blood count performed during their visit to the abu dhabi seha ambulatory healthcare services between april 2008 and october 2013. the median, 10th percentile and 90th percentile counts were calculated. the 10th percentile lymphocyte count was used to define lymphopenia. the kolmogorov-smirnov test, a non-parametric test, was used to compare lymphocyte counts between groups. statistical significance was defined by a two-tailed p<0.05. results: the median counts were higher during infancy. the variability (disparity) of the counts (reference intervals) progressively decreased from birth to 6 years of life. the 10th percentile lymphocyte counts were relatively constant from birth to 2 years (2.5-3.0 x109/l) and from 2 to 6 years (1.4-1.8 x109/l), table 1 . the lymphocyte counts were similar in boys and girls. the lymphocyte counts were compared to those from five other studies. conclusions: arab children have lower lymphocyte counts (10th percentile) than children in the united states, brazil and south africa, but their counts are similar to children in china and uganda. our study results support the development and use of ethnicity-specific lymphocyte count standards. the implication of our results is that using these lower cutoff values for lymphopenia will prevent a large number of arab children from having unnecessarily investigated for immunodeficiency. introduction/background: chronic granulomatous disease (cgd) is a rare phagocytic defect caused by mutations in the nadph oxidase 2 system leading to reduced or absent reactive oxygen species production. in addition to specific infectious susceptibility, patients with cgd are predisposed to hyperinflammation in response to infectious agents, autoimmunity, colitis, and other forms of autoinflammation. cgd patients with mutations in p47phox are at increased risk of diabetes and cardiovascular disease. hyperinflammatory and auto-inflammation responses are often difficult to predict and manage. intracellular adhesion molecule-1 (icam-1) and e-selectin are endothelial adhesion markers that facilitate the adhesion and transendothelial migration of leukocytes and elevations in these markers have been associated with cardiovascular disease, glomerular injury, and thrombotic events. expression of adhesion molecules is induced by pro-inflammatory cytokines and are associated with a hyperinflammatory state. objectives: to determine if e-selectin and icam-1 are elevated in patients with cgd and to determine of endothelial adhesion markers could serve as a biomarker of inflammatory disease in cgd patients. methods: thirty-eight pediatric and adult subjects with cgd (14 xlinked (xl-cgd), 21 p47phox deficient and 1 p22phox deficient, and 2 x-linked cgd carriers were enrolled. e-selectin (pg/ml) and icam-1 (ng/ml) were measured from the plasma of patients via sandwich elisa. results: all 38 cgd patients had histories of severe infection, active infection, chronic colitis, or other autoimmune disease at time of evaluation. nine p47phox deficient cgd patients had history of diabetes and/or early onset cardiovascular disease. one subject with x-linked cgd had undergone hematopoietic stem cell transplantation (hsct). plasma levels of e-selectin were significantly elevated above healthy controls (median 19,648 pg/ml) in subjects with xl-cgd (median 45,990 pg/ ml, p<0.001), p47phox deficient cgd (median 23,707 pg/ml, p=0.0017) and p22phox deficient cgd (80,108 pg/ml). plasma levels of icam-1 were also elevated above healthy controls in subjects with xl-cgd (median 553.2ng/ml), p47phox deficient cgd (median 241.7ng/ml), and p22phox deficient cgd (383.1ng/ml), although none were statistically significant. plasma quantities of e-selectin and icam-1 increased further in those p47phox deficient patients with diabetes and/or cardiovascular disease (median e-selectin 37,143 pg/ml, icam-1 356.7 ng/ml) but neither reached statistical significance. e-selectin and icam-1 quantities in female carriers of xl-cgd and in 1 xl-cgd cured by hsct were similar to values found in healthy controls. conclusions: immune dysregulatory features and hyperinflammation in cgd can be difficult to predict and manage. the endothelial adhesion markers e-selectin and icam-1 are elevated in patients with xl-cgd and p47phox deficient cgd that worsens with presence of early onset cardiovascular diseases and resolves post-hsct. elevations in e-selectin and icam-1 in the serum of cgd patients may serve as surrogate markers of inflammation and suggest a chronic endotheliopathy in cgd patients. introduction/background: the phenotypic presentation of ctla4 haploinsufficiency was only recently described. the management of these patients in the medical literature is limited to anecdotal case reports. we aimed to detail our experience with short and long term immunomodulatory therapy in treating autoimmune cytopenias in the background of ctla4 impairment. objectives: we aimed to assess the efficacy of mtor inhibitors in treating autoimmune cytopenias in patients with ctla4 haploinsufficiency. methods: we retrospectively identified 7 patients with proven ctla4 mutations and documented refractory autoimmune cytopenias while receiving care at nih clinical center (from july 2011 to august 2017). the complete (cr) and partial (pr) clinical response was assessed after six weeks of treatment and defined as hgb >10 g/dl, platelets >100k/ul and hgb >8g/ dl, platelets >50k/ul, respectively without transfusion requirement. results: all analyzed patients failed or exhibited disease recurrence on at least one prior medical therapy, including: corticosteroids, rituximab, romiplostim and eltrombopag. the initial response rate to evrolimus and/or sirolimus was 71% (3 cr and 2pr). one of the partial responders had recurrence of idiopathic thrombocytopenic purpura at four months while on rapalogs. overall, we used mtor inhibitors in 22 patients for a total of 37 patient years to treatm multiple modalities. the top three most common recorded adverse events were: clostridium difficile colitis (n=9, in 4 patients), lipid abnormalities (n =7, 3 patients required treatment) and bacterial pneumonia (n=6, in 4 patients). conclusions: our limited retrospective data suggests that mtor inhibitors might be efficacious in the treatment of autoimmune cytopenias in ctla4 haploinsufficent patients. further prospective studies are required to assess safety and efficacy of mtor inhibitors in this patient population. association with frequent upper respiratory tract infections and pharyngitis. from 2 years of age she continued to have recurrent febrile episodes in the absence of infection, with fever of 39-40 degrees celsius on a monthly basis, normally lasting 1-2 days. she also developed episodes of urticaria, temporally unrelated to her febrile episodes. the rash was noted to be induced by exposure to the cold, and would generally last 1-3 days with some clinical response to antihistamines and naproxen. her clinical picture then progressed and she developed joint pain and swelling, particularly affecting her hands and feet. over the following years the patient continued to have recurrent episodes of urticaria as well as progressive joint involvement and limitation. there was some initial improvement with naproxen and prednisone. her symptoms were refractory to subsequent treatment with methotrexate, leflunomide, infliximab and tocilizumab and she remained corticodependent. immunological work up including lymphocyte phenotype, immunoglobulins, vaccine responses to both protein and live vaccines and ch50 were normal. there was no evidence of raised inflammatory markers with esr 3-11mm/h, crp <0.2mg/l and ferritin 39ug/l. autoimmune workup including ana, ena, anti-dna, anca and c3 and c4 was normal. the initial differential diagnoses included systemic juvenile arthritis, periodic fever syndrome or cryopyrin associated periodic syndromes (caps). the patient therefore had a periodic fever gene panel that identified a heterozygous mutation in exon 3 of nlrp12, c.466c>t (p.arg156trp). whilst mutations in nlrp12 are known to be associated with familial cold autoinflammatory syndrome, this was reported as a variant of unknown significance. we therefore proceeded with functional in vitro testing to demonstrate pathogenicity. the patients monocytes showed increased secretion of il1b upon stimulation with lps as compared to healthy donors. when tested in a luciferase reporter assay, the mutated nlrp12 partially lost the capacity to inhibit the nfkb pathway. overall these in vitro studies show that this nlrp12 mutation results in defective regulation of the inflammatory response. the patient was commenced on anti-il1 therapy with canakinumab, with no clinical improvement so was therefore discontinued. we report a case of familial cold autoinflammatory syndrome due to a mutation in exon 3 of nlrp12, that to this point has remained refractory to multiple treatment modalities. functional testing was able to demonstrate mutation causality and defective regulation of the inflammatory response. objectives: authors aim to evaluate the spectrum of clinical phenotypes associated with nemo hypomorphic mutations within the usidnet registry. methods: investigators obtained demographic, laboratory, and clinical data on patients with a defect in nemo within the usidnet registry. results: there were 19 male patients within the usidnet registry with a diagnosis of eda-id attributed to nemo hypomorphic mutation. of these, 9 were associated with a known variant in nemo (e391x, f312l, m407v), and 4 were associated with previously unreported variants (d113v, e287del, c.1-16g>c). for 6 patients, a mutation was not specified. most reported having an affected family member (n=14, 74%). median age of symptom onset and diagnosis were 1 year (iqr 0.5-2y) and 2 years (iqr 1-10y), respectively. median age at most recent visit was 11 years (iqr 9-16y). infections, additional clinical features, treatments, and outcomes are summarized in table 1 . skin manifestations (n=13, 68%) and pulmonary complaints (n=10, 53%) were common, with eczema (n=11, 58%) and asthma being most prevalent (n=5, 26%). gastrointestinal conditions (n=9, 47%) were also frequently reported, and included non-specific diarrheal illness, enteropathy, colitis, enteritis, and inflammatory bowel disease. neurologic features (n=8, 42%), including seizures, hearing defect, peripheral neuropathy, and encephalopathy, were unexpectedly common, suggesting a previously unrecognized disease association. conclusions: we observed that allergic diseases, including asthma and eczema, were common in patients with nemo mutation. notably, varied neurologic features were more prevalent than previously reported. this study highlights the potential of cross-institutional registry analysis to deepen our understanding of extremely rare genetic diseases. coordinated effort across institutions is required to better characterize the spectrum of clinical phenotypes associated with hypomorphic mutations in nemo. mycobacterial lysate (ml) and purified protein (ppd) in the diagnosis of patients with mendelian susceptibility to mycobacterial disease (msmd) elimination of this infection depends mainly on the success of the interaction between macrophages and infected t lymphocytes. patients with mendelian susceptibility to mycobacterial disease (msmd) present severe and recurrent infections due to impaired signaling of the ifn/il-12 axis. objectives: our aim was to evaluated the ifn/il-12 axis of patients with clinical history suggestive of msmd using mycobacterial lysate (ml) and purified protein (ppd) by elisa assay. methods: samples of patients (n=43) with a clinical history suggestive of msmd arrived in our laboratory. for the diagnosis, 3 ml of blood diluted (1:2) in rpmi 1640 culture medium supplemented were used. the samples were distributed in two different plates, one used in the dosage of il12 (24 h) and the other in the ifn dosage (48 h). thus, they were stimulated with ml (2 ug/ml for the 24 h plate and 10 ug/ml for the 48 h plate) and with ppd (10 ug/ml for the 24 h plate and 2 ug/ml for plate of 48 h). at the same time, in half of the wells stimulated with lm and ppd, the cytokine ifn (1000 iu/ml) or il12p40 (2 ng/ml) were added in the plate. next, the plates were incubated at 37°c and 5% co² and the supernatant were collected and quantified by the elisa assay. the results were evaluated by the statistical mann whitney u test. results: six of the 43 patients presented alterations in the evaluation of the ifn/il-12 axis. the age of the diagnosis of male patients ranged from 2 to 16 years. the only two female patients diagnosed were 36 and 40 years old. the clinical history was heterogeneous: 4 had lymph node hyperplasia, 2 pneumonia, 1 colitis, 1 herpes zozter, another had a urinary tract infection and 1 bcgitis. the pathogens isolated were the m. tuberculosis, m. abscessus, m. gordonae, m. genavense and m. konsossi species found in 5 different patients. statistical analysis of the ifn-/il-12 axis evaluation by elisa was performed.the results of the dosage of ifn were significant in samples with lm and lm plus il-12 (***). similar results were observed in samples treated with ppd and ppd plus il-12 (**) when compared with healthy controls. in the il-12 dosage, statistical difference was observed in the samples with lm (***) and with lm plus ifn (*). in samples stimulated with ppd, the results did not show statistical differences (ns.) but ppd + ifn (*) were significantly different. conclusions: six patients were diagnosed by the evaluation the il-12/ ifn axis. the use of micobacterial lysate (ml) showed reliable results to the diagnosis of patients with il12/ifn pathway defects. the genetics diagnosis will be performed. introduction/background: invasive infections due to mycobacterial species are a feared complication in patients with t-cell deficiency or phagocyte disorders, and treatment is frequently complicated by antimicrobial resistance. cd4+ th1 t-cell immunity is known to be critical to antimycobacterial defense; accordingly, adoptive t-cell immunotherapy with mycobacteria-specific t-cells (mst) may be a beneficial therapy for combating these infections. objectives: to determine if ex vivo expansion of t-cells targeting common mycobacterial antigens is feasible from healthy donors, and whether the same antigens are recognized by patients with primary immunodeficiency (pid) and invasive mycobacterial infections. methods: peripheral blood mononuclear cells (pbmc) from healthy donors were pulsed with overlapping 15-mer peptide libraries encompassing five mycobacterial antigens (ag85b, ppe68, esat-6, cpf10, adk) and expanded for 10 days with cytokines il-4 and il-7. expanded msts were tested for specificity against the targeted antigens via ifn-g elispot, multiplex cytokine analysis, and flow cytometry. pbmcs from pid patients with invasive mycobacterial infections were similarly tested for presence of t-cells recognizing the tested mycobacterial antigens. a minimum of 20 spots per 1x105 cells above negative control was considered specific on elispot. results: ten healthy donors and eight patients with pid were tested. specificity against 1-5 mycobacterial antigens (median 3) was confirmed in all ten healthy donors, with a mean 6.6-fold cellular expansion during the 10-day culture. msts were predominantly cd4+ t-cells (mean/sd: 55 +/-6%), with both central memory (mean/sd 17.7 +/ -6.4%) and effector memory (mean/sd 77.4 +/-7.6%) populations. there was no clear difference in antigen specificity between bcg immunized (n=4) and bcg naïve (n=6) healthy donors. six of 8 pid patients had no detectable immunity to tested mycobacterial antigens. one patient with combined immunodeficiency has a low detectable specificity to ag85b (mean 47 spot forming colonies[sfc]), and a patient with nfkb1 haploinsufficiency mounted a response against ag85b (mean 388 sfc) and ppe68 (mean 40 sfc). conclusions: mycobacteria-specific t-cells can be rapidly expanded from healthy donors utilizing a protocol that could easily be translated to a good manufacturing practices facility. the majority of tested pid patients lacked immunity to the targeted antigens. adoptive immunotherapy with msts derived from third-party healthy donors may be a beneficial adjunctive therapy for pid patients with invasive mycobacterial infections. is an innate immune deficiency, primarily affecting the phagocytic compartment, and presenting with a diverse phenotypic spectrum ranging from severe childhood infections to monogenic inflammatory bowel disease. dihydrorhodamine (dhr) flow cytometry is the standard diagnostic test for cgd, and correlates with nadph oxidase activity. while there may be partial genotype correlation with the dhr flow pattern, in several patients, there is no correlation. objectives: in such patients, assessment by flow cytometric evaluation of nadph oxidase-specific (nox) proteins provides a convenient and rapid means of genetic triage (table) . methods: we performed dhr flow cytometry and nox flow cytometry on granulocytes and monocytes of cgd patients. results: phenotypic and laboratory patient data shown in table. *p9 had decreased p22phox (% and mfi) monocytes, but not in granulocytes. all other siblings (p6, p7, and p8), and mother (p10) had relatively higher p22phox (%) monocytes, but still lower than the healthy control. p7 had normal %p22phox monocytes, comparable to control. however, p6-p8, and p10 had normal p22phox (mfi) in monocytes and granulocytes. p6-9, and p10 have normal %gp91phox+ granulocytes, while p6and p9 have modestly decreased %gp91phox in monocytes. p6-9, and p10 have all moderately decreased gp91phox protein (mfi) in granulocytes and monocytes compared to healthy control, with a single population for protein expression. p6, p9 and p10 have bimodal populations for gp91phox in monocytes but not in granulocytes, with a larger positive population, and a much smaller negative population. the data from p6-9 suggest that the amount of gp91phox does not necessarily correlate with neutrophil oxidative burst, as measured by dhr. also, not all cybb variants affect p22phox protein expression, though both proteins are membrane-bound. the cyba vus in p8 does not appear to have affected p22phox protein expression in either monocytes or granulocytes. conclusions: the atypical clinical presentation of some cgd patients can make genotype-phenotype correlation with dhr flow data challenging. genetic testing, while necessary, can take several weeks. however, nadph-oxidase specific-protein flow assessment offers a rapid alternative to identification of the underlying genetic defect, and can be utilized as a reflex test to an abnormal dhr flow. further, it can provide insight into correlation between oxidative burst relative to protein expression in granulocytes and monocytes. head, division of immunology and allergy, the hospital for sick children introduction/background: adenosine deaminase (ada) is a ubiquitous enzyme important for purine metabolism. few studies have indicated that ada deficiency, in addition to causing profound lymphopenia and susceptibility to infections, is associated with neutrophils abnormalities. objectives: determine whether ada deficiency directly affects neutrophils and what are the mechanisms involved. methods: peripheral blood (pb) and bone marrow (bm) from 2.5 weeks old ada-deficient (ada-/-) mice that closely recapitulate the phenotype observed in ada-deficient patients, as well as ada+/-littermates were used to study neutrophils development and function. some experiments were supplemented with 6-week old ada-/-mice, maintained until 4 weeks of age with ada enzyme replacement, and littermates. results: the number of neutrophils in pb of ada-/-mice at 2.5 and 6 weeks of age was similar to ada+/-mice. the function of pb neutrophils from ada-/-mice, determined by oxidative burst, was also normal. the percentage of lin-/c-kit+/sca1+ hematopoietic progenitor cells in bm demonstrated significant reduction in ada-/-mice compared to littermates (0.93±0.23% and 1.21±0.33%, respectively, p=0.013). moreover, expansion of bm isolated from ada-/-mice in methylcellulose resulted in significantly less cd11b+/ly-6g+ neutrophils compared to healthy controls (13.6±8.4% compared to 25.3±10.3%, respectively, p=0.003). proliferation of bm cells, determined by brdu incorporation into cells dna, was higher in ada-/-mice than in littermates, possibly contributing to the normal neutrophil numbers in pb of ada-/-mice. conclusions: ada deficiency directly affects neutrophil development. further studies will help understand the significance of these effects and potential therapies for ada-deficient patients. objectives: we present here the clinico-pathologic features of a 10-yearold female with a heterozygous fancd2 gene deletion/mutation with evidence of cellular and humoral immune dysregulation. methods: we evaluated the patient using standard immunology anatomic, cellular, and biochemical functional assays. results: the patient has multiple dysmorphias including total anomalous venous return (repaired), mesomelia, absent ear canal, radial ray dysplasia, and short stature. her medical history is significant for an episode of pneumococcal sepsis despite adequate vaccination. whole exome sequencing demonstrated deletion of exons 2-17 and a pathologic mutation (c.2444g>a, p.arg815gln). repeated blood samples and immunophenotyping demonstrated severe lymphopenia. there were markedly low cd4+ t-cell counts with a low cd4:cd8 ratio (0.42). changes in the composition of the b-cell population included: significantly diminished absolute total b-cells, elevated immature cells, low levels of transitional cells, and undetectable advanced b-cell populations. there was no immunogenic response to pcv-13 or varicella/tetanus/ diphtheria vaccination. the nk-cell count was unaffected and demonstrated normal spontaneous and stimulated cytotoxic response. bone marrow analysis demonstrated hypocellularity without dysplasia. conclusions: we report here a pediatric patient with a novel fancd2 deletion/mutation presenting with severe lymphopenia in two cell compartments (b and t cells) and susceptibility to invasive bacterial infection. the findings are suggestive of combined immune deficiency. the cellular immune profile suggests that fancd2 may be involved in the transition of immature b and t cells to mature cells, a process that requires substantial dna recombination. additional genetic and biochemical evaluation is needed to further characterize this rare clinical finding. introduction/background: familial hemophagocytic lymphohistiocytosis type 2 (hlh) is a rare fatal condition due to a mutation in the pfr1 gene on chromosome 10q21-22 inherited in an autosomal recessive pattern which results in overactivation of the immune system. symptoms usually manifest before 1 year of age. a 17-year-old previously healthy male presented with acute onset of bilateral lower extremity pain and weakness, with subsequent inability to ambulate over a 10-day period. neurological exam demonstrated decreased lower extremity power, sensation, absent patellar and achilles reflexes, and a wide-based gait. objectives: not applicable methods: not applicable results: laboratory data was significant for neutropenia and thrombocytopenia, elevated levels of ferritin and serum cd25, and a positive ebv pcr. patient was initially treated with ivig for possible ebv-driven guillain-barre syndrome with some improvement in neurologic symptoms as well as thrombocytopenia. a mass of retroperitoneal lymph nodes were noted on spinal mri. testing was negative for alps and bone marrow biopsy was negative for leukemia/lymphoma. further work-up revealed absent perforin expression in cytotoxic cells and normal sap protein expression on staining, with poor nk function. genetic testing revealed a pathogenic mutation in the pfr1 gene with 1 additional variant of unknown significance. the patient was treated with rituximab for persistent ebvand returned with worsening lower extremity weakness. on mri, focal enhancements were found in the brain as well as worsening mass compression of the lumbosacral nerve roots. nerve root biopsy showed histiocytes with hemophagocytosis and a dense lymphohistiocytic infiltrate which stained positive for cd3, cd2, and granzyme b, with some loss of cd5 and no perforin. bone marrow biopsy was negative for hemophagocytes. conclusions: the patient was diagnosed with worsening familial hlh with cns involvement. hlh-directed chemotherapy (dexamethasone, cyclosporine, etoposide, intra-ommaya methotrexate/hydrocortisone) was started and hsct was performed. familial hlh is a rare and often lethal disorder that generally presents at a very young age. the acute onset and severity of presentation as occurred in this previously healthy adolescent is uncommon. familial hlh should be considered even in older patients with unexplained overactivation of the immune system. is the most profound form of primary immune deficiency, and is usually fatal in the first year of life without treatment. newborn screening for scid using quantitative analysis of t-cell receptor excision circles (trecs), has become the accepted method to facilitate early diagnosis and treatment in most of the united states, as scid babies typically do not make trecs. ikbkb deficiency is a rare form of autosomal recessive scid found in the northern cree first nations people of canada, where t cells develop normally but are non-functional. trec analysis is expected to be normal in ikbkb scid, and does not identify these cases. objectives: the objective of our study was to determine the feasibility of targeted genetic newborn testing for ikbkb deficiency. methods: we implemented a pilot project of prospective targeted genetic testing for the previously described homozygous ikbkb mutation (c.1292dupg) in newborns from 2 small northern manitoba communities. between 2013 and 2017, dna was extracted from dried blood spots of 724 newborns, and targeted sanger sequencing of the mutationharbouring ikbkb exon 13 was performed. results: all 724 infants born in the 2 selected communities underwent testing. fifty-five infants (7.6%, or 1/13.5) were found to be heterozygous carriers. one affected infant was identified, and underwent hematopoietic stem cell transplant before onset of infections. our findings are consistent with the predicted homozygosity for this mutation (1/13.5 x 1/13.5 x 1/4 = 1/729 births). conclusions: we demonstrated that targeted newborn testing for ikbkb deficiency was feasible, and provided the first prospective estimate of the ikbkb mutation carrier frequency in select manitoba northern cree first nations populations. we suggest that if we are to capture all babies with scid in manitoba, future newborn screening should be universal and include both trecs and direct mutation testing for population-specific mutations, including the first nations ikbkb mutation. high throughput analysis for trecs and targeted mutations will be introduced for universal newborn screening in manitoba. introduction/background: immunoglobulin class-switch recombination (csr) and somatic hypermutations (shm) are prerequisites of antibody and immunoglobulin receptor maturation and diversity within the adaptive immune system. the mismatch repair (mmr) machinery, consisting of homologues of mutsa, mutla, and mutsb (msh2/msh6, mlh1/pms2, and msh2/msh3, respectively) and other enzymes, is involved in csr, e.g. as backup of nonhomologous end-joining repair of activation-induced cytidine deaminaseinduced dna mismatches, and furthermore, in addition to errorprone polymerases, in the repair of shm-induced dna breaks. in line, a varying degree of antibody deficiency, from iga or selective igg subclass deficiency, to common variable immunodeficiency and hyper-igm syndrome have been shown in small numbers of patients with constitutional mmr deficiency (cmmrd) in addition to the known severe cancer predisposition due to genomic instability of patients with biallelic loss-of function mutations in one the mmr components. objectives: to elucidate the clinical relevance of primary immunodeficiency (pid) in cmmrd, we collected history and laboratory data of a novel cohort of 14 consecutive patients from 14 families with homozygous mutations in pms2 (n=7), msh6 (n=5), and mlh1 (n=2) reported to the consortium care for cmmrd (c4cmmrd) between 2014 and 2017, most of whom manifested with typical malignancies during childhood. methods: retrospective chart review according to a specific questionnaire and extended routine immunological analyses were performed with irb approval from the medical university of graz, austria. results: none of the presented patients fulfilled any classical or extended clinical warning signs of pid (infections, immune dysregulation, inflammation). furthermore, analyzing multiple specific laboratory parameters of the humoral and cellular immune system, we could not detect a uniform pattern of abnormalities. importantly, our data do not confirm previous suggestive evidence of iga or igg subclass deficiency, a specific antibody formation, or a b memory cell maturation defect. results of next generation sequencingbased detection of impaired class switch recombination and somatic hypermutations are pending. the t cell subsets and receptor repertoires were unaffected. together, neither clinical nor laboratory parameters were suggestive of pid in the present series of novel cmmrd patients. conclusions: we conclude that patients with cmmrd do not generally show a clinically relevant pid that could facilitate early diagnosis. on the contrary, these data support the prospect of potentially successful immune therapy of malignancies in the context of cmmrd. introduction/background: ada-2 deficiency is immune dysregulation diseases caused by an autosomal recessive mutation on cecr1 gene characterized by polyarteritis nodosa, childhood-onset, early-onset recurrent ischemic stroke and fever. objectives: report a new mutation on the cecr1 gene resulting in ada-2 deficient children. methods: female, 6-year-old, presented history of multiple ischemic strokes at one-year-old associated with recurrent fever and livedo racemosa. she has no siblings and parents are not consanguineous. results: the laboratory evaluation shows red cell=4.91x106/ml, hemoglobin=12.4g/dl, leucocytes=6.1x103/ml, neutrophils=4.3x103/ ml, lymphocytes=1.2x103/ml and plateletes=217 x103/ml. ige<25ui/ml, igg=467mg/dl, igm=13.8mg/dl and iga=21.3mg/dl. subsets lymphocytes shows cd3+=81.2%, cd4/cd8=1.41, cd19+ =10% and cd16/56=8.8%. due the clinical history, we performed cecr1 gene sequence homozygous substitution located at position -2 of acceptor splice site intron 6, c.847-2 g>a. this is a high conservative region with no alteration along phylogenetic studies and predicted to be pathogenic. to confirm the functional alteration, the ada-2 activity was tested in dried plasma spot showing 0.0 mu/g protein confirm the gene loss of function and the mutation pathogenicity. conclusions: the authors presented a novel mutation of cecr1 gene, the first one described in splice site causing gene loss of function and confirmed by extremely reduced ada2 activity. introduction/background: severe combined immunodeficiency (scid) is the most severe form of primary immunodeficiency characterized by severe, life threatening infections during early infancy. scid is a medical emergency associated with significant mortality if hematopoietic stem cell transplantations is not instituted early in the course of the disease. scid is a genetically heterogeneous disease caused by mutations in more than 30 different genes. different genes are implicated in different ethnic populations and geographical locales depending on the rates of consanguinity and endogamy in these populations. however, following the institution of newborn screening of scid in almost all state of the us and the widespread use of next generation sequencing in primary immunodeficiency diseases ar-scid due to mutations in rag1 and rag2 genes are found to be more prevalent than reported earlier. objectives: we performed a retrospective analysis of scid cases diagnosed at our centre and referred to us from other centres to determine the clinical, immunological and genetic basis of the disease in these cases. genetic variants both recurrent and novel were analysed in detail. methods: fifty six (56) of the 70 suspected patients met the esid diagnostic criteria. the clinical features, immunological defects and the gene sequencing results of these patients were analysed. gene sequencing was performed at the our centre and other collaborative centres at dept of pediatrics and adolescent medicine, queen mary hospital, hong kong, national defense medical college, saitama japan, kazusa dna research centre, chiba, japan and duke medical university centre, usa. mutations were detected in 36 of the 56 patients. mutations were classified as recurrent or novel after checking different databases such as exome aggregation consortium (exac), human gene mutation database (hgmd) and other relevant scid databases. the effect of novel, previously unreported mutations was determined using in-silico prediction tools such as sift and polyphen2. functional studies were also performed in few cases to determine the effect of novel mutations. results: mutations were detected in 36 patients. mutations were more common in genes causing autosomal recessive form of scid than the x-linked variant. mutations were detected in il2rg gene in 8 patients followed by mutation in the rag1 gene in 7 patients, dclre1c in 6 patients and rag 2 in 5 patients. mutations were also detected in ada gene (4 patients, 6 mutations), il7r (3 patients), stim1, pnp and nhej1 (1 patient each). nine novel mutations were detected. three in il2rg gene, 2 in rag1, 2 in ada and one each in the nhej1 and il7r gene. conclusions: autosomal recessive form of scid was more common in our cohort compared to x-linked form of the disease. mutations in rag1 and rag2 genes were the commonest (12 patients) followed by mutation in il2rg gene (8 patients). nine novel mutations in 5 different pid genes were detected in our cohort of scid patients introduction/background: rasgrp1 is a guanine-nucleotide exchange factor which phosphorylates ras-gdp to the activated form ras-gtp in response to t-cell receptor stimulation, resulting in ras activation. mutations in the gene coding for rasgrp1 have been recently described in four patients with profound t-cell deficiency, resulting in recurrent bacterial and viral infections, autoimmunity and malignancy. here we describe a two-year-old male presenting with recurrent sino-pulmonary infections, found to have two variants in rasgrp1, one not previously described. objectives 1. to describe a case of combined immunodeficiency with two pathogenic compound heterozygous rasgrp1 mutations. 2. to compare the clinical phenotype of rasgrp1 deficiency of our patient with that of previously described cases. 3. to argue for early hematopoietic stem cell transplantation in view of the increased susceptibility to epstein barr virus (ebv) induced lymphoma in patients with rasgrp1 deficiency. methods: a two-year-old male was referred to seattle children's immunology clinic for recurrent otitis media and two episodes of pneumonia. the diagnosis of combined immunodeficiency was considered based on a profound t-cell deficiency during immune evaluation and he was started on azithromycin and tmp/smx prophylaxis. at age 3 ½ years he was hospitalized with a bladder outlet obstruction and found to have two abdominal masses. biopsies were obtained and he was diagnosed with an ebv driven b cell lymphoproliferative disorder. in addition, his csf and bone marrow were considered positive based on pcr and staining, respectively. treatment with cyclophosphamide, prednisone, rituximab, and intrathecal methotrexate was initiated. due to poor csf ebv clearance, intrathecal therapy was escalated to rituximab. results: initial laboratory evaluation showed elevated igg (2290 mg/dl), normal number of cd19 b-lymphocytes, and adequate response to tetanus, prevnar-13 and varicella vaccine. b-cell phenotyping showed elevated immature/transitional b-cells. a profound t-cell defect was identified with cd4 t-cell lymphopenia (361/mm3), elevated cd8 t-cells (2074/mm3), inverted cd4/cd8 ratio (0.3) and absent proliferation in response to mitogens (pha, anti-cd3) and antigen (tetanus). forty-three percent of peripheral blood t-cells were / positive. t-cell phenotyping revealed decreased cd4 and cd8 naïve t-cells with elevated proportion of cd8+ t-effector memory t-cells. cervical lymph node and retroperitoneal mass biopsies showed atypical lymphoproliferation without malignant transformation. exome sequencing revealed two variants in rasgrp1. the first variant (c.1428+1g>a) is located at a splice site predicting an unstable transcript targeted for degradation. the second variant (c.1780c>t) is a novel mutation resulting in a stop codon. conclusions: recurrent sino-pulmonary infections are often a presentation of antibody deficiency. in this case, further investigation showed a profound t-cell defect, resembling that reported in 2 patients with homozygous nonsense mutations in the catalytic domain of rasgrp1 and 2 patients with homozygous insertion mutations leading to a premature stop codon at the bzip domain. our patient had biallelic mutations in rasgrp1 downstream of the catalytic domain, both leading to unstable transcripts. he developed an ebv induced atypical lymphoproliferative disorder, a complication reported in one rasgrp1 deficient patient whose disease progressed to b cell lymphoma and unsuccessful hsct. another patient developed ebv induced lymphoproliferative disorder after hsct for ebv-positive hodgkin lymphoma. two additional patients presented with recurrent infections, developed b-cell lymphoma and one was successfully transplanted. we are preparing the patient for hsct after chemotherapy for his lymphoproliferative disease to correct the underlying immune defect given that these patients are at high risk of developing lymphoma following ebv infection. introduction/background: immunodeficiency-centromeric instabilityfacial anomaly is a group of rare genetic disorders typically involving agammaglobulinemia. type four is caused by variants in the hells gene. the five patients previously reported with icf4 have fit the phenotype of agammaglobulinemia. here we report a patient with novel phenotype including neutropenia and neuroblastoma. objectives: describe a unique presentation of immunodeficiencycentromeric-instability-facial anomaly syndrome 4 to further expand our understanding of this disease. methods: retrospective chart review results: six-month old male was transferred to our tertiary care facility for ongoing chronic respiratory infection, chronic diarrhea, and failure to thrive. his past medical history was significant for 31-week prematurity due to rupture of membranes requiring a two month nicu stay for bronchopulmonary disease. upon discharge, he was bottle feeding and on room air. he had recurrent congestion for three months with two courses of antibiotics and one and a half weeks of diarrhea leading up to admission for difficulty breathing. he was found to have multiple infections including rhinovirus and parainfluenza virus on nasal wash, pjp pneumonia, norovirus, and pseudomonal cellulitis of his nose causing significant destruction. although previous laboratory studies revealed a normal absolute neutrophil count (anc), his anc quickly dropped to 170 cells/ul. his igg, iga, and igm were undetectable. while his total b cell count (451 cells/ul) was normal, he lacked any switched memory b cells. he had near normal total t cell count (cd3 2579 cells/ul) and cd4 count (2373 cells/ul) and a markedly decreased cd8 count (181 cells/ul) and poor proliferative response to low concentrations of phytohaemagglutinin and pokeweed mitogen. a 0.9cm x 1.6cm x 1.9cm paraspinal mass was found on chest ct, which was subsequently characterized as mibg-avid with a curie score 1 neuroblastoma. metastatic evaluation including bone marrow aspirate and biopsy was negative for malignancy. however, marked granulocytic hypoplasia and maturation arrest were present suggesting severe congenital neutropenia or, less likely, immune-mediated. whole exome sequencing detected homozygous variant of unknown significance in the hells gene (p.m223t). he was treated with intravenous immunoglobulin and g-csf with clinical and laboratory improvement. his neuroblastoma was initially observed, then subsequently removed due to a >50% increase in size. pathology confirmed mycn non-amplified favorable histology. he remains in remission 4 months after resection. he is currently awaiting bone marrow transplant for his immunodeficiency. conclusions: the significance of this case report is the novel presentation of icf4. neutropenia and malignancies have been reported in immunodeficiency-centromeric-instability-facial anomaly syndrome 2 (icf2) but not icf4. this case report thus expands upon the clinical picture of icf4 patients to include neutropenia and malignancies, and further describes the immunodeficiency. associate professor, university of south florida -johns hopkins all childrens hospital introduction/background: identification of newborns with severe combined immunodeficiency using state wide newborn screening (nbs) began in florida in 2012. abnormal results require extensive confirmatory diagnostic testing and prophylactic antimicrobial medications are needed to effectively evaluate and treat the infant. several barriers have been identified within government-sponsored health insurance programs that impede delivery of these evaluations and medications, often resulting in delays and/or inpatient hospitalization in order to provide timely and appropriate care. objectives: determine the cost differential between the initial evaluation and treatment of a scid patient detected by newborn screening in the inpatient versus outpatient setting. methods: the cost utilization of inpatient versus outpatient management of newly identified scid patients from nbs were analysed to include the cost of confirmatory testing and initiation of prophylaxis within the inpatient versus outpatient setting. laboratory tests included assessment of t cell immunity with quantitative and functional assessment, immunoglobulin measurement, genetic testing for scid variants, evaluation for maternal engraftment, and hla typing. medications included ig supplementation, pentamidine, fluconazole, and acyclovir. we compared the actual cost of inpatient stay and inpatient evaluation versus the approximate cost that would have been accrued if the patient were not admitted to the hospital. results: from 2012-2016, 4 infants with government-sponsored health insurance had abnormal nbs and were confirmed to have scid after evaluation at our institution. all 4 infants were admitted into the hospital for initial evaluation and initiation of appropriate medications for an average of 7 days. total average cost of medication administration for 7 days was $1,623, total cost of laboratory testing was $12,297, and average inpatient stay averaged $22,158 per patient. conversely, the cost that would have been accrued in the outpatient setting for medication would have been $1,097 for 7 days. laboratory testing costs would be no different as an outpatient. in total, the cost for inpatient evaluation was $36,708 versus $13,389 as an outpatient. conclusions: standard laboratory assessments and medications are necessary for infants identified with scid by population based nbs. despite government sponsoring of the florida nbs program, unnecessary barriers exist by government sponsored insurers that lead to a delay appropriate care. inpatient admission alleviates these barriers, but significantly increases cost. we advocate that standard ambulatory scid outpatient evaluation and initial treatment be authorized in children identified with scid through nbs without delay. patients with cd3g mutations reveal a role for human cd3g in treg diversity and suppressive function. introduction/background: integrity of the tcr/cd3 complex is crucial for positive and negative selection of t cells in the thymus, and for effector and regulatory functions of peripheral t lymphocytes. genetic defects that reduce, but do not abrogate tcr signaling, are associated with a variable degree of immune deficiency and immune dysregulation. in particular, while cd3d, cd3e, and cd3z gene defects in humans present mainly with severe immune deficiency, cd3g mutations lead to milder phenotypes, mainly characterized by autoimmunity. however, the role of cd3, encoded by cd3g, in establishing and maintaining immune tolerance has not been elucidated. objectives: we aimed to investigate abnormalities of treg cell repertoire and function in patients with genetic defects in cd3g with evidence of clinical autoimmunity. methods: high throughput sequencing (hts) was used to study composition and diversity of the t cell receptor (trb) repertoire in treg, conventional cd4+ (tconv), and cd8+ cells from 6 patients with cd3g mutations and in healthy controls. treg function was assessed by studying their ability to suppress proliferation of tconv cells. results: treg cells of patients with cd3g defects had reduced diversity, increased clonality, and reduced suppressive function. the trb repertoire of tconv cells from patients with cd3g deficiency was enriched for hydrophobic amino acids at position 6 and 7 of the cdr3, a biomarker of self-reactivity. overlap between treg and tconv cell repertoires was observed in cd3g mutated patients. conclusions: the treg and tconv cell repertoire of patients with cd3g mutations is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition. introduction/background: a common concern with b cell-depleting therapies is their potential effect on humoral immunity. although there have been reports of prolonged hypogammaglobulinemia in adult patients receiving rituximab, little is know about this phenomenon in children. objectives: we sought to assess humoral immunity in children receiving rituximab and determine risk factors leading to low immunoglobulin levels and infections. methods: we conducted a retrospective study on all pediatric patients ( 18 years) who received rituximab for the first time between january 2014 to december 2016 in a single tertiary pediatric hospital. charts were reviewed and data was collected prior to rituximab treatment and at 6, 12, and > 12 months after treatment. patients who received rituximab after hematopoietic cell transplantation (hct) or for a malignancy and those with an underlying primary immune deficiency (pid) at the time of treatment were excluded. results: in total, 106 patients received rituximab during the study period. of those, 38 were excluded (hct: n=31, lymphoma: n=6, pid: n=1). sixtyeight patients were eligible. indications for rituximab treatment were renal disease (n=23), neurologic disease (n=18), hematologic disease (n=12), rheumatologic disease (n=10), ebv control (n=3), other (n=1). one patient who died from autoimmune encephalitis 8 days after rituximab was excluded from the follow-up study. at any time after rituximab treatment, low igg was present in 24/62 (38.7%), low iga in 6/61 (9.8%), and low igm in 36/61 (59.0%) of patients. over a year after their last rituximab dose, 12/29 (41.4%) of patients still had low b cell counts for age, and 10/10 (100%) had low memory b cell counts (cd27+ among cd19+ cells: mean value = 2.9% +/-1.9% sd). hospitalisation for infection was required in 13/67 (19.4%) patients in the year following rituximab treatment, which was associated with having either low igg (33.3% vs 15.2%, p=0.03) or low iga (50.0% vs 18.2%, p=0.04), but not with low igm levels (19.4% vs 24.0%, p=0.33). also, receiving a treatment with more than one rituximab cycle was a risk factor for low igg (63.0% vs 20.0%, p=0.0003). conclusions: hypogammaglobulinemia following rituximab treatment was frequent, and the presence of low igg and iga were associated with a higher risk of serious infection in this context. introduction/background: subcutaneous immunoglobulin (scig) replacement therapy for patients with primary immunodeficiency (pid) is usually administered once a week. however, a variety of dosing regimens can be used to provide flexibility for patients. objectives: we used pharmacokinetic (pk) analysis to evaluate the pk characteristics of weekly and biweekly (once every 2 weeks) scig administration in patients with pid. methods: this pk substudy was part of a prospective, open-label, phase 4 study (nct02711228) in patients with pid treated with igpro20 (hizentra®, csl behring, bern, switzerland). a noncompartmental analysis of serum igg concentrations was used to calculate pk parameters and compare pk outcomes on weekly and biweekly dosing. results: of the 17 patients included in the pk substudy, 15 provided samples for both weekly and biweekly regimens. the dose-adjusted area under the concentration-time curve was comparable for both treatment regimens: 0.24 and 0.25 (h*g/l)/mg for the weekly and biweekly regimens, respectively. the igg clearance was also similar, being 4.41 for the weekly and 4.14 ml/h for the biweekly regimen. median peak igg concentrations occurred later with the biweekly regimen (3.02 days) compared to 2.00 days for the weekly regimen. igg trough levels were close for both treatment regimens, with arithmetic means slightly lower for biweekly than for weekly regimens, at 10.13 vs. 10.21 g/l respectively. the minimum igg concentrations within a dosing interval were also comparable, with arithmetic means of 9.60 and 9.83 g/l for the weekly and biweekly treatment regimens, respectively. conclusions: biweekly and weekly hizentra® administration at the same total monthly igg doses resulted in similar igg exposures. pharmacokinetics, efficacy, tolerability and safety of a new subcutaneous human immunoglobulin 16.5% in primary immune deficiency introduction/background: patients with primary immune deficiencies (pid) require life-long replacement therapy with immunoglobulins (ig) to prevent severe infections and irreversible complications. in addition to safety and efficacy, tolerability and convenience of administration of ig products are essential factors in patient acceptance. a new 16.5% ig preparation (octapharma, lachen) was developed for subcutaneous administration (scig) derived from the established manufacturing process of octapharmas intravenous ig (ivig) brand octagam®. objectives: primary outcome was to assess efficacy of a new 16.5% subcutaneous human immunoglobulin preparation in preventing serious bacterial infections. secondary endpoints included evaluating tolerability and safety, determining the pk profile, the number and rate of other infections and changes in quality of life measurements. methods: a prospective, open-label, single-arm phase 3 study involving 61 patients was conducted at 18 centers in north america and europe. pid patients who were stable on ivig treatment for at least 6 months and with igg trough levels 5.0 g/l underwent a 12-week wash-in/wash-out period consisting of weekly scig doses 1.5 times the previous ivig dose (based on published conversion rates for marketed scig products), followed by a 52-week efficacy period (64 scig infusions in total). 22 of 61 patients enrolled had complete pharmacokinetic assessments at different time points: before the switch from ivig to scig (pkiv), after the wash-in/wash-out phase (pksc1) and at week 16 of the efficacy period (pksc2). results: 61 patients (age: 2-73 years; mean age 32.2 years; 54.1% female) receiving a total of 3,497 scig infusions (0.135 g/kg/week in young children (2 years and <5 years of age) and 0.185 g/kg/week in adults; average overall: 0.175 g/kg) were included in the full analysis sets. no serious bacterial infections were recorded. among the 188 other infections observed during the efficacy period only one infection was graded as severe (bronchiolitis due to rsv virus), which led to hospitalization (2 days). all other infections were mild (72.3%) or moderate (27.1%) in intensity. infection rate per person-year was 3.43. of the 233 reported adverse events, only 14 were assessed as being related to the study drug; all of these events were non-serious. five non-study drug related serious adverse events were reported in 4 patients (6.6%). serum igg trough levels were nearly constant during the study with a minimum trough level of 6.1 g/l and mean trough plasma concentrations of 11.4 ± 3.3 g/l and 11.6 ± 3.4 g/l for pksc1 and pksc2. median igg trough levels after scig treatment were 1.9 to 2.6 g/l higher compared to ivig treatment prior to enrollment. a dosing conversion factor (dcf) of 1.37 was determined by auc (area under the curve) measurements, allowing dose adjustment to achieve bioequivalence between ivig and scig dosing. improved quality of life measurements, utilizing sf-36v2, were observed in both physical and mental health parameters when compared from the first to last scig infusion. conclusions: this study demonstrated that the new subcutaneous human normal immunoglobulin 16.5% is well tolerated, safe and effective in patients with pid. introduction/background: atopic dermatitis (ad) is a chronic, relapsing, inflammatory skin disorder with associated pruritus that affects 11 percent of children in the united states. severe atopic dermatitis refractory to conventional therapy can be concerning for an underlying immunodeficiency, especially in infants. increased incidence of hypogammaglobulinemia has been associated severe ad. a handful of cases describe a correlation with transient hypogammaglobulinemia of infancy (thi), but a thorough immunological evaluation is often missing to further understand this relationship between ad and characterization of thi. objectives: define various phenotypes of thi with their clinical presentation and laboratory findings. compare thi vs. thi associated with severe a.d. methods: a case series of six patients was conducted at a single academic center from 2/1/2014 to 12/1/2017 for patients with severe atopic dermatitis with low igg levels. all available immunological laboratory data were retrospectively collected during this time period. descriptive statistical analysis was utilized for data comparison. results: of the six patients, four had no infectious history. of the remaining two, one had recurrent skin abscesses associated with his poorly controlled atopic dermatitis requiring oral antibiotics and one patient had two episodes of staphylococcus aureus superinfection of the eczema. at the time of presentation, the mean age was 7 months with mean igg of 164 mg/dl and mean ige of 2,273 ku/l. iga and igm were within normal age cut offs. all patients had normal protein and polysaccharide specific antibody titers after completion of vaccination series. mean cd19+ count was 2,200/ul with normal cd3+, cd4+, cd8+ and cd1656+ cell counts. three patients were tested for lymphocyte mitogen proliferation and complement function which were normal. two patients, who were tested, had normal phagocyte work up. mean age of igg improvement to igg> 217 mg/dl, was 10 months. patients had total protein and albumin levels with mean 5.1 ku/l and 3.5 ku/l, respectively which eventually normalized. four patients improved with skin care and dietary modification to hypoallergenic formula. one patient improved with extensively hydrolyzed formula and three patients improved with amino acid formula. one patient improved with aggressive skin care. one patient, who was noncompliant with dietary recommendations and aggressive skin care, did not improve. conclusions: one prospective study described an increased incidence of hypogammaglobulinemia in patients with atopic dermatitis compared to controls regardless of the severity. no further prospective studies have characterized hypogammaglobulinemia in this population. according to the primary immunodeficiency practice parameters, children with thi often present with frequent viral and bacterial respiratory illnesses, low igg levels and normal vaccine responses. in one study, the period of hypogammaglobulinemia spontaneously corrects to normal by mean age of 27 months with all patients reaching normal levels by 59 months. these patients may have low igm or iga and decreased t or b cells, which eventually normalize. management is often with antibiotic prophylaxis and if refractory to prophylaxis or unable to tolerate, igg administration (igrt) based on severity of symptoms is recommended. we describe a less severe variant of thi associated with severe atopic dermatitis and characterized by very transiently decreased igg levels with earlier resolution than typical thi, normal igm and iga levels, normal specific antibody levels and normal t, b and nk cells. these patients typically do not manifest recurrent viral or bacterial respiratory illnesses. they also do not require antibiotic prophylaxis or igrt. severe atopic dermatitis maybe a positive prognostic indicator for patients with thi and is associated with an earlier self-resolution of hypogammaglobulinemia, lack of typical infections, normal iga, igm and normal t and b cell numbers. a possible mechanism for thi associated with severe ad may be transdermal loss of protein which was supported with mildly low total protein and albumin levels rather than immaturity of the immune system in patients with true thi. introduction/background: in recent years it was found that heterozygous mutation in pik3cd gene produces an autosomal dominant primary immunodeficiency characterized by onset of recurrent sinopulmonary and other infections in early childhood with defects in both b-and t-cell populations and a special susceptibility to uncontrolled viral infections. many patients develop chronic lymphoproliferation and there is also an increased susceptibility to b-cell lymphomas. here we present a patient assumed as a common variable immunodeficiency with heterozygous mutation in pik3cd gene. objectives: to describe a case of a patient assumed as a common variable immunodeficiency with heterozygous mutation in pik3cd gene. results: 60 year old woman with personal history of severe and recurrent upper and lower respiratory infections, chronic pulmonary disease with bilateral bronchiectasis, chronic diarrhea without diagnosis, mild osteopenia, focal lesion in right hepatic lobe, atopic dermatitis and anemia. she was following up in other center and in 1996 she was diagnosis with common variable immunodeficiency (cvid) and started treatment with intravenous immunoglobulin (ivig), but she referred low adherence to it. she did not referred history of lymphoproliferation nor significant viral infections. she has a daughter with spherocytosis who required esplenectomy and also had bronchiectasis and cvid, she deceased at 28 years old because pulmonary infection. other daughter and 2 sons referred healthy. in our first immunologic studies we found severe hypogammaglobulinemia (igg 428 mg%, no dosable iga and igm) with absent of b cells in peripheral blood. we started with high doses of ivig (800 mg/k/month) and antibiotic prophilaxis with improvement of the functional respiratory test and without new infections. we are planning colonoscopy to study her chronic diarrhea. thinking that her clinical picture could be other than cvid we order a genetic study. a nextera exome capture and next generation sequence with illumina hiseq was made and an heterozygous mutation in pik3cd gene (chr1:9.775.746, p.pro97ala) was found. family and functional studies are still pending. conclusions: due that clinical presentations of primary immunodeficiencies are becoming more complex, its diagnosis is a challenge for immunologist now a days. studies with next generation sequence is a very useful tool in indefinite cases, especially when more than one member in the family are involved. chief, laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health introduction/background: heterozygous gain-of-function mutations in pik3cd as well as heterozygous pik3r1 mutations that affect interaction of p85 with p110 lead to constitutive hyperactivation of the pi3k pathway and cause activated pi3k delta syndrome type 1 or type 2 (apds1, apds2), respectively. we describe a female with apds2 with short stature, diffuse lymphadenopathy, recurrent upper respiratory tract infections, elevated igm, persistent ebvand cmv viremia and disseminated toxoplasmosis who gave birth to a genetically affected daughter with severe congenital toxoplasmosis. objectives: to characterize the molecular and cellular defects underlying severe toxoplasmosis in this family methods: investigation of the molecular basis of the disease was performed through whole exome sequencing, and results were validated by sanger sequencing. functionality of the pi3k pathway was assessed by analyzing akt and s6 phosphorylation in freshly isolate b cells with and without stimulation with anti-igm. an excisional lymph node biopsy from the affected mother was stained with anti-pd1 antibody to detect t follicular helper cells, and with igm and igg specific antibodies to analyze the proportion of isotype-specific b and plasma cells results: whole exome sequencing of maternal dna with targeted analysis of 362 pid genes identified a heterozygous mutation at an essential donor splice site of pik3r1 (nm_181523.2:c.1425+1g> a). sanger sequencing confirmed the presence of this mutation in both the mother and her child. functional studies on b cells freshly isolated from both patients confirmed an increase in baseline akt and s6 phosphorylation, suggesting constitutive activation of the pi3k-mtor signaling pathway. a lymph node biopsied from the mother contained numerous pd-1+ tfh cells and igm+ plasma cells conclusions: toxoplasma gondii is an obligate intracellular parasite that is usually only symptomatic in immunocompromised hosts. severe toxoplasmosis has been reported in the following primary immunodeficiencies: cd40 ligand deficiency, tap deficiency, cvid, nfkb2 deficiency, and immunodeficiency due to anti-ifn-autoantibodies. importantly, toxoplasma infection was previously reported in a 9month-old infant with apds1, and ocular involvement has been described in a 36-year-old patient with apds2. this, however, is the first report of systemic and severe congenital toxoplamosis in a mother and child with apds. to evade innate host defenses, t. gondii induces the activation of the pi3k/akt signaling pathway, reducing intracellular reactive oxygen species and creating an intracellular environment that is hospitable to parasite survival and proliferation. therefore, we postulate that the pik3r1 mutation may lead to a hospitable cellular environment for toxoplasmosis replication. this work was partially supported by the division of intramural research, niaid, nih (protocol # 05-i-0213). additional authors of this work are: ottavia delmonte and kerry dobbs, from the laboratory of clinical immunology and microbiology, niaid, nih. introduction/background: aplaid (autoinflammation and plc-gamma-2-associated antibody deficiency and immune dysregulation) is a term that was proposed for the newly discovered autoinflammatory condition resulting from a pathogenic missense variant, ser707tyr, in the cterminal sh2 (csh2) domain of plc-gamma-2 in order to distinguish it from plaid, a distinct clinical entity that results from intragenic deletions of portions of the csh2 domain of the same protein. plc-gamma-2 is a phosphodiesterase that is predominantly expressed in hematopoietic cell lines and acts on pip2 to produce ip3 and dag in the pkc and ras/raf/ erk pathways. the only formally published case of aplaid described a father-daughter pair with an autoinflammatory clinical syndrome affecting the skin, mucosa, eyes, pulmonary and gastrointestinal systems. objectives: to describe the aplaid phenotype resulting from a novel genetic variant of the phosphodiesterase plc-gamma-2 in two unrelated families methods: clinical review and case presentation results: we report a 33-year-old female with a long-standing history of recurrent pneumonia, cellulitis and cystitis with obstructive lung disease characterized as bronchiolitis and dynamic airway collapse, with negative alpha-1-antitrypsin testing. she had a history of childhood onset granuloma annulare and pressure-induced urticaria, as well as episcleritis. immune testing revealed low igm (37 mg/dl), elevated baff levels and a low percentage of cd27+ memory b-cells, prompting sequencing of plcg2, which identified a c.3422t>a, p.met1141lys variant in the calcium binding c2 domain. her 15-month-old daughter, who has a history of bullous skin lesions, failure to thrive, febrile episodes and recurrent respiratory infections was likewise found to have this plcg2 variant. like her mother, the daughter also has low igm and elevated baff levels. similar symptoms of recurrent sinopulmonary infections and hypogammaglobulinemia, have been described in an unrelated family that shares this same plcg2 variant in a doctoral thesis by rozmus. this work also describes functional analysis, in which this particular variant of plc-gamma-2 was demonstrated to have dysregulated plcgamma-2 activity leading to aberrant intracellular calcium signaling and increased apoptosis of immature b-cell subsets. conclusions: we describe our experience in evaluation and treatment of this family with a previously undiagnosed disorder. together, these new cases add to the expanding body of knowledge regarding plc-gamma-2 and its importance for the development of autoinflammatory and primary immunodeficiency conditions. (133) submission id#427357 present a case.non-celiac gluten sensitivity is an emerging entity with symptoms similar to celiac disease, but without positivity in specific diagnostic tests. it is considered more common than celiac disease patients with sigad have a greater risk of concomitant autoimmune disorders than health individuals. sigad was previously to be associated with celiac disease, but not usually in non -celiac gluten sensitive. objectives: describir a case ataxia non celiac gluten sensitivity, methods: clinic case description. results: he patient has negative serology test for gluten, but clinically respond as celiac disease. conclusions: all patient has negative serology test for gluten, but clinically respond as celiac disease, could has non-celiac disease, this case described here ,it suggest than this entity should to thought before than the celiac disease, since not-celiac disease is it more common. associate professor, federal university of rio de janeiro introduction/background: primary cutaneous actinomycosis is a rare condition caused by gram-positive filamentous bacteria and generally occur after traumatic inoculation. objectives: to report an unusual etiology of skin lesions in a patient under anti-tnf-alpha therapy. methods: we report the case of a 30-years-old female receiving conventional doses of adalimumab for ankylosing spondylitis who presented, two weeks before referral for dermatological assessment, with an erythematous nodule with purulent discharge on right pretibial region and pruritic erythematous plaques with scaling and peripheral pustules in the trunk and nose tip. results: a fungal etiology was suspected and scraping specimens from several cutaneous lesions were submitted to direct microscopic examination and culture, which were negative for fungi. cutaneous biopsy of the pretibial lesion was sent for histopathology and microbial cultures. adalimumab was interrupted and empirical therapy with oral terbinafine (250 mg/day) was started with close clinical follow-up. a folliculitis reaction pattern without granulomas was observed during histopathological examination and gram-positive cocci were isolated from biopsy sample and further identified as saccharopolyspora sp. by molecular typing. sulfamethoxazole+trimethoprim was added and discontinued after one week due to a cutaneous rash. terbinafine (250 mg/day, p.o) was used for 12 months with complete clearing of all skin lesions and very good tolerability. spondyloarthritis signs and symptoms were unremarkable and no anti-inflammatory or immunomodulating treatment was necessary. conclusions: increased risk of skin actinomycotic infections in patients under anti-tnf therapy is not consistently reported in the literature. nevertheless, they should be included as a differential diagnosis of atypical skin lesions in individuals under anti-tnf therapy. where we aim to determine the prevalence, incidence, characteristics, treatment and outcomes of primary immunodeficiency disease (pid) patients in qatar. pids are rare heterogeneous disorders of the immune system that result in an increased susceptibility to infection, immune dysregulation and occasionally, to cancer. objectives: determine the range of pids with important epidemiological data in qatar after analyzing the database and creating a registry. methods: this is a retrospective study of pid patients followed at hamad medical corporation from 1989 to 2017 using medical records. all patients who were diagnosed with a pid irrespective of age were included. patients were classified according to the international union of immunological societies expert committee on pid. the data is captured under 5 sectionsa) patient demographics including age, gender, ethnicity b) clinical presentation, c) immunodeficiency profile including age at diagnosis, type of immunodeficiency, family history d) treatment modality and e) lab/genetic data. results: we registered 150 patients (60 females and 90 males) over a span of four years. mean age at onset, diagnosis and diagnostic delay were 2.6, 4.6 and 2 years respectively. majority of the patients were arabs 77% followed by people from the asian subcontinent 12%. antibody deficiency was seen in 32%, immune dysregulation 28%, well defined immunodeficiency (at, hige, digeorge,wiskott aldrich) 25% and t/b cell cid 12%. rare diagnoses (ipex, msmd) were recorded whereas no cases of toll like receptor and complement deficiency were seen. consanguinity rate was (n = 97, % = 65) and first degree cousin marriage (n = 38, % = 40). family history was positive in 50% (n= 75) of the patients. maximum diagnostic delay was seen in scid (33% >3 months) and agammaglobulinemia (66% >3 months).during the patients life, infection was the most common presenting complaint (47%) followed by sinopulmonary disease (16%) and gi-tract manifestations (7%). the most common infections were pneumonia (46 %), otitis media and conjunctivitis (42 % each) followed by failure to thrive (30%) and sepsis (21%).microbial isolates particularly seen as causative agents of infections were p.aeruginosa and salmonella (14%) each, mrsa and e.coli (9%) each. a genetic defect was confirmed in 57% of ataxic telangiectasia and 65% of scid patients. active infections were treated and prophylactic antibiotics were prescribed in 76 cases (51%). prophylactic antibiotics were prescribed to 34% of patients with immune dysregulation and to 25% of well-defined syndromes. out of 18 patients who had hsct, 72% had successful transplants. ivig was given to 42% of the total pid patients with humoral immunodeficiency patients receiving the most (45%). one patient had gene therapy and two required interferon gamma treatment for msmd. mortality rate at 1st year of life was 4% whereas total mortality rate was 12%, excluding cases that passed away before pid was diagnosed. conclusions: the estimated prevalence of pid in qatar is found to be 6 per 100000. over the years, physicians have become increasingly aware of pid and survival rate has improved. initiation of newborn screening for scid and agammaglobulinemia will lead to earlier diagnosis and initiation of therapy with better outcomes. introduction/background: agammaglobulinemia is typically associated with a near absence of b cells secondary to a developmental block in bone marrow, and, usually but not always, manifests in early life. most of such patients are males with x-linked agammaglobulinemia (btk deficiency). females with agammaglobulinemia, of either autosomal recessive or dominant inheritance, are rare. objectives: we present and discuss the differential diagnosis for the conflicting clinical and immunological phenotypes of two adult females who present with infections, cytopenias, and agammaglobulinemia with low to normal b cell count. methods: retrospective chart review of clinical and laboratory data results case 1: a 22-year-old female who, at age 17 years, had evans syndrome (autoimmune thrombocytopenia and hemolytic anemia) that resolved after steroid and high-dose gammaglobulin treatment. at age 21 years, immunologic workups revealed a complete absence of serum immunoglobulins (igg, iga, igm) and low b cells 43 (3%) (normal range 100-500). however, three years prior, patient had detectable igm (218 mg/ dl). b cell subset analysis showed an expansion of cd19hi21lo b cells (30%, normal 0.2-8.6%), with a marked decrease in switched memory b cells (0.2%, normal 7-61%). additional immunophenotyping revealed a reduced frequency of naïve cd4+ (3.6%, normal >50%), cd8+(42%, normal >50%) t cells, and normal lymphocyte proliferative responses to mitogens and anti-cd3, anti-cd3/anti-cd28, and anti-cd3/il-2. case 2: a 30-year-old female with recurrent upper respiratory tract infection, undetectable igg, iga, igm and ige, and no response to vaccinations (tetanus and pneumococcal). thrombocytopenia (38x103 count/microliter) was noted once during her thirdtrimester pregnancy without evidence of pre-eclampsia. postpartum cd4+ t cell count was low (442 cell/microliter, normal 500-1400 cell//microliter). immunophenotyping revealed normal b cell count with reduced frequency of naïve cd4+ (17.6%, normal >50 %) and cd8+ (18%, normal >50 %) t cells. conclusions: we report two adult females who present in early adulthood with recurrent infections and cytopenias. both had agammaglobulinemia and decreased naïve t cells suggestive of late-onset combined immunodeficiency (locid). the presence of peripheral b cells makes autosomal recessive defects in b cell receptor signaling (lamda5, iga, igb, igm, blnk) less likely. differential diagnosis of locid with cytopenias includes ctla-4 haploinsufficiency, gain-of-function pik3cd mutations, ikaros defects and autosomal recessive rag deficiency. both patients remain at risk for developing autoimmune complications. a molecular diagnosis will facilitate targeted therapy for the underlying defect. professor, director of the laboratory of childhood immunology, ku leuven introduction/background: complement factor properdin (cfp) is a soluble glycoprotein which has a unique known role as a positive regulator of the alternative complement pathway by binding and stabilizing the inherently labile c3/c5 convertase enzymes. mutations in the cfp gene lead to aberrant protein expression or to expression of a dysfunctional protein which results in high susceptibility to pyogenic infections especially neisseria meningitidis. properdin-deficient individuals are at greater risk of fulminant meningococcal disease, with mortality rates as high as 75%. objectives: case report: a 5 year old belgium boy from nonconsanguineous parents presented with achronic purulent cough and recurrent infections of upper and lower airways. he suffered from a severe pneumonia at the age of 5. ct thorax showed bronchiectasis of the right middle lobe and left lower lobe. in addition, he had recurrent acute otitis media since the age of 1 year old. immunological work-up showed an absent ap50 activity, with normal ch50, c3 and c4. results: genetic and functional analysis: sanger sequencing of cfp gene identified a hemizygous c.961t>g, p.y321g (cadd score 8.081, msc_cadd 0.102) mutation in the patient and his mother. properdin elisa (hycult biotech) showed absent and 50% of the normal healthy control value of serum properdin concentrations in patient and the healthy mother, respectively. conclusions: conclusion: we diagnosed properdin deficiency in a 5y old boy presenting recurrent lower and upper respiratory tract infections. ap50 testing, added to ch50, should therefore be part of initial workup for patients with recurrent severe respiratory tract infections. indeed early diagnosis allows for appropriate prolonged antibiotic prophylaxis and immunization to reduce the risk of fatal meningococcal disease, to reduce or prevent organ damage and to allow for genetic counselling in the family. 2-4, 5-7, 8-12 and 13-18) and child depression inventory (cdi) were implemented to both children and parents in addition to sociodemographic data form; beck depression inventory (bdi), beck anxiety inventory and zarit caregiver burden scale were implemented only to parents. results: the depression inventory parent and child form values of the patient group with primary immunodeficiency were significantly higher than the control group (p=0,004 and p=0,032). according to beck depression and anxiety inventories, it was seen that the depression and anxiety inventory scores of the parents of the patient group were higher than the control group (p=0,009 and p=0,022 respectively). it was determined that the cdi parent form scores of the patients with hospitalization history were statistically higher than the patients without hospitalization history (5,4±0,5 and 4,8±0,6; p=0,009). bdi scores were significantly higher in the group which received ivig (p=0,024). while the quality of life of the patients compared to their parents was perceived as worse than the healthy children in all dimensions (p=0,003 p<0,0001 and p<0,0001), the quality of life of the children was worse only in psychosocial and total quality of life fields (p<0,0001 p=0,002 and p=0,158). it was seen that quality of life of children physical health scores of only the patients with hospitalization history were statistically significantly lower (15,5±1,9 and 14,3±2,3; p=0,04) . while no statistically significant difference was found in terms of quality of life of the child scores between the groups which received and did not receive ivig replacement treatment, psychosocial and total qualify of life scores of parents were statistically significantly lower in the group which received ivig replacement treatment (p=0,017 p=0,033). zarit care giver burden scale scores were similar in patient and control groups. although both groups were on the limits of mild to moderate caregiving burden, it was seen that the scores of the group which received ivig were significantly higher than the group which did not receive ivig (p=0,026). conclusions: as a conclusion, we think that it will be appropriate to inform and monitor the entire family in relation to psychosocial difficulties and care giving burden they may experience in time as well as the medical aspects of the disease in order to develop a holistic approach to children with primary immunodeficiency. introduction/background: chronic lung disease is the most common complications of cvid, affecting 30-60% of patients. it includes bronchiectasis affecting 50% of patients and granulomatous lymphocytic interstitial lung disease (glild) in 10 to 55%. both are associated with an increased morbidity and mortality. pulmonary functional studies and ct scans have been proposed as screening procedures for lung involvement in cvid. the definitive diagnosis of glild is established histologically, but it is too invasive in patients with radiological abnormalities and no symptoms. objectives: we aim to describe the pulmonary complications of our cohort of patients with cvid comparing them with subjects affected with other types of hypogammaglobulinemia. methods: we reviewed all clinical records of the patients with a diagnosis of hypogammaglobulinemia of any cause until december 2016. we looked at all pulmonary function tests (pft), 6-minute walk tests and ct scans performed. we classified patients according to the ct scan pulmonary disease pattern. we compared the demographic data and pulmonary characteristics of each group and intended to describe similarities and differences between them. results: we collected 34 patients and 70 ct scans from 26 patients. patients were grouped for further analysis as follows: no ct performed: 8; normal ct: 9; bronchiectasis: 9; glild: 7 and bronchiectasis + glild: 1. eight patients (24%) had no ct including six with cvid, one with x linked lymphoprolipherative disease (xlp) and one had a syndromic deletion in chromosome 11. the median age of symptom onset was 23.5yo, with a median delay to diagnosis of 1 year. nine subjects (26%) had ct scans with no chronic disease, six had cvid, 2 rituximab induced hypogammaglobulinemia (rih) and 1 mgus with hypogammaglobulinemia (mguswh). the median age of symptom onset was 58 years old with a median delay to diagnosis of 2 years. nine patients (26%) presented persistent bronchiectasis, 7 with cvid, 1 with an igg3 deficiency (sigg3d) and 1 with xlp. median age at symptoms onset was 14yo and median delay to diagnosis 13 years. seven patients (21%) had glild; all afected with cvid. median symptoms onset was 30 years old, and median delay to diagnosis was 10 years. one patient (3%) was included in the bronchiectasis and glild group. she was referred with a diagnosis of rih, in the context of a pulmonary malt lymphoma. however, her ct did not show a typical ct lung lymphoma pattern, lymphocyte monoclonality was never shown and she had a reduced pre-treatment low gamma globulin concentration and a history of respiratory infections since adolescence, we believed her diagnosis is cvid. pft with an obstructive pattern identified patients with bronchiectasis (p<0.01) and patients with glild had a significantly lower basal and post 6 minute walk o2 saturation (p<0.01, n=22) conclusions: using a systematic approach, we identified that roughly 50% of patients with hypogammaglobulinemia have chronic pulmonary ct abnormalities. half of them had bronchiectasis, that were associated to hypogammaglobulinemia of any cause, a longer disease course and a reduction in fvc and fev1 with a shorter distance reached in the 6 minute walk test. the other half had glild, spirometries in this group were useless, but o2 saturation was significantly lower, basal and after the 6 minute walk test. we found a high frequency of pulmonary disease in our cohort and a disease progression study is now in place. hies) is a primary immunodeficiency characterized by eczema, sinopulmonary infections, and musculoskeletal and vascular abnormalities. care of patients with this disease is largely supportive with the use of prophylactic antibiotics and topical eczema therapies. the use of replacement immunoglobulin is increasing. hematopoietic stem cell transplant (hsct) is being considered more frequently, but many questions remain regarding which patients should undergo transplant. as pulmonary complications are a leading cause of morbidity and mortality in this disease, and potentially improved with replacement immunoglobulin and hsct, we sought to examine more closely the patients with more frequent pulmonary hospitalizations and structural lung disease. objectives: to determine the rates of pulmonary complications in our large cohort of ad-hies patients, and examine the relationship between immunologic markers and pulmonary disease. methods: we retrospectively reviewed the records of 110 ad-hies patients seen more than one time at nih between 2010 and 2016. there were 36 pediatric patients under the age of 18 years. we reviewed the number of and cause for hospitalizations, and reviewed radiology reports for structural lung disease including bronchiectasis and pneumatoceles. we correlated these findings with specific antibody responses and lymphocyte phenotyping, such as the number of memory b lymphocytes. results: the patients range in age from 2 years to 66 years, with a median age of 23 years. 106 patients had chest cts, with 42 percent having bronchiectasis, and 41 percent having cavitary lesions. 31 percent of patients had both. these abnormalities were more prevalent in patients with low memory b cells. 31 percent of patients receive replacement immunoglobulin. in patients not receiving replacement ig, 42 percent had appropriate response to pneumococcal vaccine. during this time period, there were 190 hospitalizations, the majority of which were associated with pulmonary infections. the rate of overall hospitalization was higher in the group with low memory b cells (p=0.006), but there was no difference associated with age. conclusions: immunologic abnormalities may assist in determining the long-term prognosis of patients with ad-hies, and can be considered in management plans such as the use of immune globulin and consideration for hsct. introduction/background: purine nucleoside phosphorylase (pnp) deficiency is an autosomal recessive disorder affecting the purine salvage pathway causing a (severe) combined immunodeficiency disorder, autoimmunity, and neurological symptoms. patients typically present in the first few years of life with frequent infections and a failure to thrive. decreased plasma levels of uric acid are suggestive, while neutropenia is a rare complication. prognosis is generally poor with few patients making it to adulthood without treatment. treatment options are limited to general supportive care and hematopoietic cell transplantation. methods: we present the cases of three patients, one sister and two brothers, from a consanguineous french canadian family. they presented in early adulthood with nearly identical histories of repeated pneumonias and chronic rhinosinusitis. one patient had necessitated filgrastim to treat a parainfectious neutropenia. they did not report any neurological abnormalities or symptoms of autoimmunity. results: a primary immune deficiency was suspected. initial evaluation showed normal immunoglobulin levels, a lack of response to vaccination, positive ebv ebna iggs, and auto-antibodies. the patients were severely lymphopenic with reduced numbers of t, b and nk cells ( l: 300, cd3+81%, cd4+71%, cd8+5%, cd19+12%, cd3-cd56+7%). in particular, they had a severe depletion of naive t-helper recent thymic emigrant cells (cd4+cd45ra+cd31+2%). additional studies revealed increased urinary inosine, guanosine, deoxyinosine, and deoxyguanosine, severely reduced erythrocyte pnp activity (3% residual activity), and a normal uric acid level. a homozygous, missense mutation, c.769 c>g (p.his257asp) was found and described as pathogenic in silico. the diagnosis of pnp deficiency was made; tmp-smx prophylaxis and ivigs were started. conclusions: theses cases are atypical for a number of reasons. the first is the relatively benign immunological course, the lack of neurological symptoms, and the advanced age at the time of the diagnosis, possibly due to residual enzymatic activity. in addition, pnp deficiency has been classically described as causing a marked reduction of t cells with a relative sparing of b cells. however, b cell lymphopenia have been reported, particularly with latter presentations. while this specific mutation had been identified once previously in the literature, as a compound heterozygote, these are the first confirmed cases of homozygotes. the pathogenicity of the mutation was not only suggested in silico, it was confirmed biochemically. in addition, in vitro functional studies have demonstrated the importance of his257 for the binding of pnp to its substrate, particularly in the formation of the early transition state. indeed, pnp (p.his257asp) has been shown to have a greatly reduced affinity for its substrates and catalytic activity. further study is needed to identify the optimal management for these patients. introduction/background: whole exome sequencing has become an integral part of diagnosis and treatment of rare immunodeficiency diseases. one of these diseases is tetratricopeptide repeat domain 7a(ttc7a) mutations; a rare disorder associated with multiple intestinal atresias and severe combined immunodeficiency. histologic assessment of organ biopsies of patients with ttc7a deficiency suggest it may play a role in multiple organs as it is expressed in the cytoplasma of intestinal, thymus and pancreatic cells. many patients with ttc7a deficiency have moderate to severe combined immunodeficiency. nine patients described in the literature with ttc7a mutations have been treated with hematopoietic stem cell transplant, mostly at a young age. objectives: this case report describes a rare presentation of an adolescent with ttc7a deficiency, her clinical presentation, immune evaluation and treatment with eventual referral to bone marrow transplant at the age of 16. methods: retrospective chart review under irb approval was performed. whole exome sequencing performed at baylor texas childrens hospital and mutations were confirmed by sanger sequencing. extensive immune and nk cell phenotyping were performed by flow cytometry and nk cell function was tested using standard cr51 release cytotoxicity assays. results: a 13 year old female was referred to immunology for severe refractory warts, history of severe diarrhea with epithelial dysplasia but no atresia, failure to thrive requiring parenteral nutrition until 6 years old and multiple infections with staphylococcus aureus and candida. at 16 years of age, she developed rapidly worsening pulmonary function that improved with monthly pulse methylprednisone. her immune phenotype demonstrated a combined immunodeficiency including severely low cd4, cd8, b and nk cells. she had no igd-cd27+ memory b cells and undetectable igg, iga and igm, isohemagglutinins and vaccine titers to diphtheria and tetanus. her mitogen proliferation response was low and she had abnormal tcr v-beta repertoire. she was referred to the texas childrens hospital center for human immunobiology for the nk cell evaluation and reasearch clinic (near) the patient was found to have impaired nk cell lytic function and terminal maturation. this was demonstrated by the decreased frequency of cells that matured to the cd56dim subset and was accompanied by decreased expression of the lytic effector molecule perforin and the fc receptor cd16. this phenotype was conserved when we isolated cd34+ hematopoietic precursors and performed nk cell differentiation in vitro. whole exome sequencing demonstrated a compound heterozygous mutation in the ttc7a gene, including a c.1001+3_1001+6aagt mutation, which has been previously described as a pathologic founder mutation in the french canadian population. the second mutation is a previously unreported c.211g>a (p.e71k), a variant of unknown significance. mutations were confirmed by sanger sequencing, and parents are carriers of the mutations. she was referred for hematopoietic stem cell transplantation with a 10/10 unrelated donor option. conclusions: ttc7a deficiency is a genetic mutation associated with high morbidity and mortality leading to gut atresia and dysfunction in combination with severe immunodysfunction. we have described a unique case of ttc7a deficiency with a novel mutation which is associated with intestinal epithelial dysplasia with no atresias and a late presentation and evolution of combined immune deficiency. nk cell assessments show significantly impaired terminal maturation suggesting a critical role for ttc7a in human nk cell development. introduction/background: ras-associated autoimmune leukoproliferative disorder (rald) is a rare condition with significant overlap of clinical and laboratory findings with malignant disorders, notably juvenile myelomonocytic leukemia (jmml) and chronic myelomonocytic leukemia (cmml), but with much better prognosis without specific therapy. we report a case of rald in a 4month-old male infant originally suspected to have jmml. results a 4-month-old male infant, born to an unrelated ethiopian father and turkish mother, presented with splenomegaly and leukocytosis (26,000/l), monocytosis (7,800/l), and thrombocytopenia (14,000/l); hemoglobin was 10 gm/dl and fetal hemoglobin concentration 1.9%. family history indicated early death of three paternal uncles, two at age 6-7 months and one at age 10 years. jmml was suspected and bone m a r r o w a sp i r a t i o n w a s p e r fo r m e d ; h ow e v e r, r e s u l t s o f immunophenotypic testing with flow cytometry analysis and cytogenetic testing with fish did not support this diagnosis. patient had a normal b12 level. serum igg (1884.8 mg/dl [nl 694.0 -1618.0 mg/dl]) and igm (139.0 mg/dl [nl 35.0 -102.0 mg/dl] levels were elevated and iga was normal. lymphocyte subset analysis showed 37% cd3 [nl 51-74%], 20% cd4 [nl 34-53%], 51% cd19 [nl 17-37%] positive cells with normal percentages of cd8 and cd16/cd56 cells; there were no cd4-/ cd8-t cells detected. given the findings of leukocytosis, monocytosis, splenomegaly, hypergammaglobulinemia, and a dearth of cd4-/cd8-t cells, the patient appeared to meet criteria for rald. mutation testing with next generation sequencing showed an nras missense mutation [c.37g>t; pgly13cys] in 46% of t cells and 48% of myeloid cells. the presence of the nras mutation, in the absence of other typical jmml findings or rasopathy syndrome features, supported the diagnosis of rald. there was no therapeutic intervention and, at one year of age, the patient was reported to remain clinically well; he relocated to ethiopia with his parents. conclusions rald is a nonmalignant clinical syndrome originally classified as a subtype of autoimmune lymphoproliferative syndrome (alps) but subsequently distinguished to be a separate entity due to lack of double negative t-cells, a mutation in fas/fasl/caspase-10, or consistently elevated serum b12 levels. though also present in 25% of jmml patients, a somatic mutation in ras signaling protein (kras or nras), which controls b-cell tolerance and production of autoantibodies, is present in all reported cases of rald. while all previously reported cases of rald had somatic kras or nras mutations, our patient's nras mutation was present in more than 40% of t cells and myeloid cells, suggesting a heterozygous germline mutation; this has not previously been reported in rald and needs to be further confirmed (145) the primary objective of this study was to evaluate hct outcomes in patients with was who underwent hct since 2005 in north america. we hypothesized that survival after hct from alternative donors such as cord blood has improved compared to published results, but that overall survival would be superior in patients who undergoing hct at a young age. methods: patients were enrolled on pidtc protocol 6904, a multicenter retrospective natural history study of patients treated for was in north america since 1990. clinical features, disease status, hct type and post-hct outcomes were analyzed in 129 patients who underwent hct at 29 pidtc centers between 2005-2015. descriptive statistics such as median and range for continuous variables and counts and percentages for categorical variables were used to summarize characteristics of the study population. in addition, kaplan-meier curves were used for estimating survival probabilities results: diagnosis of was was confirmed by an expert review panel for eligibility. mutation in the was gene was available for 118 patients, including nonsense (n=27, 23%), frameshift (n=32, 27%), missense (n=31, 26%), splicing (n=20, 17%), gross deletion (n=5, 4%), in-frame deletion (n=2, 2%), pr complex (indel) (n=1, 1%). donor types included matched sibling (n=22, 17%), unrelated adult volunteer bone marrow or peripheral blood stem cells (n=66, 51%), umbilical cord blood (n=39, 30%) and other related donors (1 each, phenotypically matched related, haploidentical) . median age at time of hct was 13.6 months (range, 2.1 260.5 months). the vast majority of patients received busulfan containing conditioning regimens (87%), with some receiving other myeloablative (2%) or reduced intensity regimens (11%). with a median follow-up of 4.6 years, overall survival was excellent with 1-year and 5-year survival probabilities of 92% (95% ci, 86-96%) and 91% (95% ci, 84-95%), respectively. survival was similar at 1 year for recipients of hct from matched sibling (95%, 95% ci, 72-99%), matched unrelated donor (92%, 95% ci, 82-97%) or umbilical cord blood (90%,95% ci, 75-96% see figure) . importantly we confirmed that survival at 1 year was better in patients who were <5 years old (n=118) compared to those who were 5 years old (n=11) at the time of hct (95% versus 62%, respectively p=0.032). this difference persisted when only unrelated donor recipients were analyzed (97% vs 57%, p=0.036). overall the percentage of patients in our study who underwent hct at a young age was high (91%) compared to the literature (84% in moratto et al, 2011, blood) . the rate of second hct was only 4% (n=5). cumulative incidence of acute grade 2-4 at 100 days, acute grade 3-4 at 100 days and chronic graft-versushost disease at 1 year were 26% (95% ci 18-34%), 15% (95% ci 9-22%) and 14% (95% ci 8-21), respectively. conclusions: outcome of hct for was since 2005 shows excellent overall survival for all donor types, including umbilical cord blood with very low rates of second hct. importantly, hct at a younger age (<5 years old) continued to be associated with superior survival supporting the provision of hct earlier in the course of the disease. further analysis of the complete cohort is planned to determine whether age at hct has decreased in the modern era compared to pre-2005 and to analyze factors associated with platelet and immune reconstitution, donor chimerism and autoimmunity. introduction/background: comel-netherton syndrome is a rare disease hallmarked by congenital ichthyosis, atopy, trichorrhexis invaginata (bamboo hair) and, within the past 10 years, has been defined as a primary immunodeficiency. specific, rare genetic polymorphisms (c.230t>a, pl66h) in the fc receptor iiia (fcgr3a) on natural killer (nk) cells have been shown to decrease nk cell function. patients with these defects present with susceptibility to severe and recurrent viral infections, however, not all fcgr3a mutations result in this clinical phenotype. here we report on a child with a mixed genotype, presenting with congenital ichthyosis, atopy and recurrent bacterial and viral infections. methods: immunophenotyping of lymphocyte subpopulations were evaluated by flow cytometry. genomic dna was sequenced using next generation sequencing. all detected variants were then sequenced using sanger sequencing. cd16 dual epitope assay and evaluation of nk cell maturation was also performed. intravenous immunoglobulin replacement therapy was initiated. results: our patient presented at 9 months of age for evaluation of food allergy. she has congenital ichthyotic erythroderma with pruritic atopic dermatitis-like skin eruptions and a history of hypernatremic dehydration immediately following birth, thin and easily broken hair and a history of failure to gain weight with appropriate catch-up growth following formula fortification. she has had multiple episodes of acute otitis media and recurrent upper respiratory tract infections associated with wheezing. t-, b-and nk cell quantitation by flow cytometry was normal, including naïve and memory b cells. immunoglobulins and vaccine antibody levels to haemophilus influenza type b, tetanus and streptococcus pneumoniae were normal. lymphocyte proliferation to mitogens was normal. natural nk cell cytotoxicity was initially decreased, however, subsequent cytotoxicity testing performed at 13 months of age was normal. whole exome sequencing revealed a homozygous mutation in spink5 (c.795-11a>g) and a homozygous missense mutation in the first immunoglobulin domain of fcgr3a (c.305t>a), both variants of unknown significance and under additional investigation. this homozygous mutation in spink5 was not detected in the patients healthy, unaffected sibling. conclusions: this is a case of an infant with a clinical phenotype consistent with comel-netherton syndrome, however genotyping has revealed homozygous mutations in spink5 and fcgr3a, suggestive of a possible mixed genotype. a recent large study investigating the use of whole exome sequencing to identify variants implicated in primary immunodeficiency found that in 11% of families, more than 1 gene contributed to the immunodeficiency phenotype. it should therefore be kept in mind that variability in an individuals clinical phenotype may be attributable to the presence of a mixed genotype. introduction/background: cartilage-hair hypoplasia (chh) is a rare autosomal recessive disease caused by mutations in the rmrp gene and can manifest with scid. allogeneic hct is a curative therapeutic option for scid associated with chh. bordon et al. reported an overall survival of 62% (10/16 patients) following hct with a predominantly myeloablative conditioning regimen with busulfan and cyclophosphamide. data on outcomes of hct using a ric regimen are limited. objectives: we herein report our experience with allogeneic ric hct for scid associated with chh. methods: we reviewed records of all patients who underwent allogeneic hct for scid associated with chh at our institution, with a ric regimen containing alemtuzumab, fludarabine and melphalan. results: five patients (3 male, 2 female) underwent allogeneic ric hct for chh at median age of 8 months (range, 4 months 4 years). all patients had biallelic mutations in the rmrp gene, and met pidtc criteria for scid, prior to hct. two patients were diagnosed by newborn screening for scid. one patient received serotherapy only (rituximab 375 mg/m2 daily x 3 days, anti-thymocyte globulin 1.5 mg/kg daily x 4 days) conditioning for initial hct but developed graft failure and subsequently received a ric regimen for second hct. all patients received a ric regimen consisting of alemtuzumab 1 mg/kg over 5 days(n=4) or 3mg/kg over 4 days (n=1) and fludarabine 5 mg/kg (weight <10 kg, n=4) or 150 mg/m2 over 5 days (n=1), and single dose of melphalan 4.7 mg/ kg(weight <10 kg, n=4) or 70 mg/2(n=1, dose reduced by 50% for preexisting sclerosing cholangitis with grade 3 liver fibrosis). patients received matched (n=3) or 1-2 allele mismatched (n=2) bone marrow grafts and all but 2 grafts were from unrelated donors. all patients received cyclosporine and steroids for gvh prophylaxis. all patients engrafted with full donor chimerism. three patients developed mixed chimerism, but continue to maintain donor t cell chimerism > 90%. two patients developed vod of the liver (one patient developed mild vod whereas the patient with pre-existing sclerosing cholangitis developed severe vod). one patient developed grade 2 skin gvhd and one patient developed limited chronic skin gvhd. none of the patients developed liver or gi-gvhd. all patients remain alive at a median follow up of 4 years (range 11 months-11 years) with good t-cell and b-cell immune reconstitution. conclusions: our experience suggests that allogeneic ric hct with alemtuzumab, fludarabine and melphalan for scid associated with chh is curative, offers durable t-cell engraftment, low gvhd along with excellent survival and might be preferable over a myeloablative conditioning regimen, to further limit toxicity in young infants, especially in the era of newborn screening. refractory thrombocytopenia in a patient with wiskott-aldrich syndrome despite hematopoietic stem cell transplantation: eltrombopag as a therapeutic option. mauricio chaparro-alzogaray 1 , marcela estupiñan-peñaloza 1 , gisela barros-garcía 2 , oscar correa-jimenez 3 1 pediatric hematologist/oncologist, hsct unit, fundación homi hospital de la misericordia 2 pediatric hematologist/oncologist, fundación homi hospital de la misericordia 3 pediatrics resident, universidad nacional de colombia introduction/background: wiskott-aldrich syndrome (was) is an xlinked disorder characterized by: immunodeficiency, eczema and hemorrhage due to thrombocytopenia [1] . hematopoietic stem cell transplantation is the treatment of choice, with an overall survival of approximately 90% regardless of the source of the stem cells [2] . eltrombopag has been used as a pre-transplant stabilization therapy [3] . in our knowledge, there are no reports of its use for the management of post-transplant autoimmune cytopenias in was. objectives: to present a clinical case in which the complexity of diagnosis and management of wiskott-aldrich syndrome is highlighted, as well as the usefulness of eltrombopag in post-transplant persistent thrombocytopenia. methods: clinical case presentation and review of literature. results: clinical case: 9-month-old infant with a history of thrombocytopenia identified at the third day of life (positive serology and viral load for cmv), bacteremia due to serratia marcescens at 4 months, intermittent diarrhea from 4 months, multiple platelet transfusions, ivig cycles, and ambulatory management with corticosteroids; who consulted the er for a 5-day of bloody diarrheic stools, generalized petechiae and fever. he was irritable with generalized petechiae in the lower extremities, diaper area dermatitis with signs of superinfection. admission cbc: wbc: 20180/mm3, neutrophils 330/mm3, lymphocytes 17250/mm3, monocytes 2360/mm3, hb 11.2 g/dl, ht 34.1% mcv 80.2fl, mch 26.3 g/dl, mcmh 32.8%, platelets 15000/mm3 mpv 8 fl. bone marrow aspiration was performed that ruled out proliferative syndrome. immunological profile with normal immunoglobulins and flow cytometry showed t lymphocytosis with cd4/ cd8 inversed ratio, direct coombs was positive. he progressed to refractory thrombocytopenia, with intracranial bleeding and transfusion platelet requirement every 12h. molecular diagnosis of wiskott-aldrich syndrome was made and it was decided to carry out an allogeneic transplant of unrelated umbilical cord. he showed adequate response, 100% chimerism; however, he persisted with important cytopenias, without response to management with ivig and corticosteroids, so that on day +68 he restarted eltrombopag that he received prior to transplantation. from day +97 he received rituximab for 6 weeks. he continued with eltrombopag 10 months post-transplant. currently without cytopenia and without complications, he completed the post-transplant year without additional complications. conclusions: was represents a great diagnostic challenge in pediatric clinical practice. despite the therapeutic option of transplantation of hematopoietic stem cells, patients may persist with different complications, within these; autoimmune cytopenias will require additional therapies. eltrombopag, in addition to being used as a pretransplant transient measure, is useful for the management of post-transplant persistent thrombocytopenia in these cases. references: 1. immunol allergy clin north am. 2010; 30 (2) methods: retrospective study of medical records in two centers of immunology results: in our center we have 1205 p with pid, with made retrospective study of medical records, up today we have registered 1155p (96%). according to this record, these diseases are distributed in the following way: predominantly antibody disorders: 890p (77%), predominantly t cell deficiencies:88p (7,6%), phagocytic disorders:41p (3.5%), complement deficiencies:26p(2.2), other well pid:53p(4.5), autoimmune and immune dysregulation syndromes:8p(0.6%), unclassified immunodeficien cies:57p(4.9%). predominantly antibody deficiencies are the most common pid, which comprise more than half of our all p. these group is represented by: specific iga deficiency (sad):332p, specific igg deficiency:164p, transient hipogammaglobulinemia:206p, common variable immunodeficiency (cvid):96p, agammaglobulinemia linked x:33p, agammaglobulinemia unknown causes:8p, secondary hipogamma globulinemia:23p, selective igm deficiency:1p, subclasses deficiency:10p, cd40l deficiency:7p, hyper igm unknown causes:32p, other hyogammaglobulinemia:3p. among them, selective iga is the most common pid.in our cohort of unclassified immunodeficiency, we have 25p with auto inflammatory syndrome, such as family mediterranean fever, hyper igd syndrome and candle like-syndrome and 6 p with nk deficiency. in all our pid 244p, are under replacement gammaglobulin (gg)treatment, 171p use intravenous gg and 71p use subcutaneous gg conclusions: the lasid registry model represents a powerful tool to improve health policies, showing that are under diagnosed and should receive more attention. more data are needed to define the exact prevalence of pid to avoid underestimation of these diseases due to under reporting. as different reports in different countries, in our centers predominantly antibody deficiencies are the most prevalent. although the number of patient diagnosed with pid, is growing. many physicians still know little about these disorders. introduction/background: a number of anticonvulsant medications have been shown to cause hypogammaglobulinemia. lamotrigine is a phenyltriazine anticonvulsant medication that is approved to treat seizure disorders and bipolar disorder. objectives: we describe a patient who developed hypogammaglobulinemia secondary to lamotrigine use. methods: we performed a chart review and case-based literature review. results: a-27-year old female presented to immunology clinic for evaluation of panhypogammglobulinemia. she was initially evaluated by general internal medicine for lightheadedness and fatigue and was found to have serum igg of 382 mg/dl (767-1590 mg/dl), igm 26 mg/dl (37-286 mg/dl) and iga 33 mg/dl (61-356 mg/dl). the patient reported a history of recurrent sinus infections occurring around twice per year. she reported resolution with oral antibiotics. she denied any history of pneumonia and was never hospitalized for treatment of infection. she did report increased number of recurrent upper respiratory infections; around 4-5 per year for the last several months. she denied any family history of primary immune deficiency. she was never prescribed corticosteroids or immuno-modulators. her past medical history was significant for bipolar disorder type 2 for which she was prescribed lamotrigine 100 mg oral twice daily seven months prior to her initial visit. the medication was prescribed prior to the onset of recurrent sinus infections. she had protective post-vaccination titers against tetanus, diphtheria, and acellular pertussis and non-protective pre-vaccination pneumococcal titers. post vaccination pneumococcal titers were not obtained due to the cost of the pneumonia vaccine. the patient was started on prophylactic azithromycin three times weekly. lamotrigine was discontinued and the patient switched to lurasidone due to concern for anticonvulsant-induced panhypogammaglobulinemia. her serum immunoglobulin levels increased after 2.5 months off of lamotrigine (igg 416 mg/dl, igm 46 mg/dl, iga 34 mg/dl), and she is currently asymptomatic without further infections. conclusions: lamotrigine is likely responsible for the reversible hypogammaglobulinemia in this patient. serial immunoglobulin levels should be checked in all patients who experience recurrent sinopulmonary infections while on lamotrigine. in two separate reports, lamotrigine induced hypogammaglobulinemia began within 7 months and 13 months of starting therapy respectively. in another study, hypogammoglubinemia was reported in 28% of 74 patients taking lamotrigine. further studies are needed to accurately describe onset and frequency of hypogammgeobulinemia in these patients. currently, it is unclear whether post vaccine titers are protective in patients with lamotrigine induced hypogammaglobulinemia. introduction/background: the association of vaccine strain rubella virus with cutaneous and sometimes visceral granulomatous disease has been reported previously in 19 patients with various primary immunodeficiency disorders (pids). the majority (14/19) of these pid patients with rubella positive granulomas had dna repair disorders, namely ataxia telangiectasia (at) (n=9) or nijmegen breakage syndrome (nbs) (n=3) or rag1 (n=1) and rag2 (n=1) deficiency. objectives: to support this line of inquiry, we provide additional descriptive data on the previously reported nbs patients as well as additional previously unreported patients with rubella virus induced cutaneous granulomas and dna repair disorders as well as additional previously unreported pid patients with rubella virus induced cutaneous granulomas. methods: we provide in-depth descriptive data on the previously reported nbs patients as well as 5 additional previously unreported patients with rubella virus induced cutaneous granulomas and dna repair disorders including at (n=4) and dna ligase 4 deficiency (n=1). we also provide in-depth descriptive data on 4 additional previously unreported pid patients with rubella virus induced cutaneous granulomas, including cartilage-hair hypoplasia (n=1), mhc class ii deficiency (n=1), whim syndrome (n=1), and coronin-1a deficiency (n=1). results: the median age of the patients is 10.5 years (range 3-33). the majority are females (83%). cutaneous granulomas have been documented in all cases while visceral granulomas (spleen and liver) were observed in 3 cases. t cell and b cell lymphopenia as well as hypogammaglobulinemia or impaired antibody formation were present in most patients. all patients had received rubella virus vaccine. the median age at presentation of cutaneous granulomas was 84 months (range 14-377). the median duration of time elapsed from vaccination to the development of cutaneous granulomas was 19 months (range 12-152). the diagnosis of rubella was made by pcr in 83% of patients and by immunohistochemistry in the remainder. one patient was confirmed to have vaccine strain rubella virus. hematopoietic cell transplantation was reported in three patients. rubella associated complications did not contribute to death among those patients who died (16%). conclusions: of the now 28 cases, 19 (67%) share the diagnosis of a dna repair disorder and confirm that chronic rubella virus infection is associated with cutaneous granuloma formation. analysis of patients with dna repair disorders and other pids with this complication will help clarify determinants of rubella pathogenesis, identify specific immune defects resulting in chronic infection and may lead to defect-specific therapies. introduction/background: introduction/background: hizentra® is a 20% liquid igg product approved for subcutaneous administration in adults and children greater than two years of age who have primary immunodeficiency disease (pidd). limited information on use of hizentra® is available for children who have received hematopoietic stem cell transplantation (hsct). objectives: objectives: the aims of this study are to determine the safety and efficacy of hizentra® in pediatric patients post hsct, and to characterize reasons for switch from intravenous igg (ivig) to subcutaneous (scig) delivery following hsct. methods: methods: a retrospective chart review involved 13 pidd infants and children (mean age 6.3 [range 3.1 to 14.5] months) status post hsct who received hizentra®. ten patients received hizentra® by pump administration, and 3 patients by manual push. results: results: hizentra® administered weekly to 13 children included an average of 144 infusions (range 14-416). the mean dose was 722 mg/kg/4 weeks. the mean igg level was 889 mg/dl while on hizentra® in 11 patients compared to a mean trough igg level of 563 mg/dl in 6 patients during immunoglobulin administration prior to hizentra® of most children. four patients naïve to igg therapy were started on hizentra®. average infusion time was 4.7 (range 3-15) minutes for manual entry and 71 (range 60-90) minutes for pump entry, and the average number of infusion sites was 1.9 (range 1 to 2). local reactions were mild and observed in 3/13 (23.1%) children. ten patients had no local reactions. no serious adverse events were reported. the rate of serious bacterial infections (sbi) was 0.12 per patient-year while receiving hizentra®, similar to reported efficacy studies. the reasons for switch from ivig to scig in 13 patients (some patients had multiple reasons) were improved igg serum levels and physician desire for steady state serum igg levels (n=6), loss/lack of venous access (n=2), patient/caregiver preference (n=7), home site of care preference (n=4), and physician preference for scig (n=5). conclusions: conclusions: hizentra® is a safe and effective option in children who have received hsct. reasons for switch from ivig to scig included improved serum igg levels, desire for steady state serum igg levels, and patient/caregiver preference. consulting medical advisor, immune deficiency foundation introduction/background: it is currently unknown how many persons with primary immunodeficiency (pi) receive the seasonal influenza vaccination, and what proportion becomes ill. additionally, the effect of immunoglobulin (ig) replacement therapy on the frequency of influenza diagnosis and severity of symptoms is not known. objectives: the current study sought to measure the prevalence of seasonal influenza vaccination and diagnosis among persons with pi, specifically those with antibody deficiencies: x-linked agammaglobulinemia (xla), common variable immunodeficiency (cvid) and hypogammaglobulinemia. methods: 11,533 email invitations were delivered to members of the immune deficiency foundation database requesting participation in an online survey regarding their zoster, influenza and varicella vaccination experiences. data from 1009 persons with xla (total n=41; children age<18; n=19; adults age>17, n=22; 100% male; 81% white non-hispanic), cvid (total n=824; children age<18, n=57; adults age>17, n=767; 79% female; 92% white non-hispanic), and hypogammaglobulinemia (total n=144; children age<18, n=23; adults age>17, n=121; 77% female; 92% white non-hispanic) were analyzed for the 2016-2017 influenza season. results: overall, 74% (n=749) of the sample received a seasonal influenza vaccination during the 2016-2017 influenza season. persons with xla were less likely to receive an influenza vaccination (54%, n=22), than persons with cvid (76%, n=628) and hypogammaglobulinemia (69%, n=99). a fifth of respondents (21% overall, 34% of children and 19% of adults) who attend school and/or work stayed home at some time to avoid seasonal influenza. though the majority (81%, n=821) were not diagnosed with influenza, when stratified by age, children were twice as likely to be diagnosed than adults. of the 99 children sampled, 30% (n=30) were diagnosed with seasonal flu compared to 15% (n=139) of the 910 adults that were sampled. the role of replacement ig therapy in protection against flu can best be examined in the 41 patients with xla since these patients cannot make specific antibodies to vaccine and only 54% (n=22) received the vaccine. although 59% (n=24) of the individuals with xla were exposed to flu, only 10% (n=4) were diagnosed with influenza; two of whom were not receiving ig therapy at time of diagnosis. concerning influenza severity, individuals receiving ig therapy at the time of flu diagnosis tended to have modestly milder symptoms than those not receiving ig treatment (e.g., less likely to report sore throat (78% versus 92%, p<.05)), but the sample size is too low to draw firm conclusions. conclusions: a high proportion of antibody deficient persons received a seasonal influenza vaccination for the 2016-2017 influenza season. in addition to vaccination, many individuals attempted to avoid influenza infection by remaining home from school and/or work. there was a suggestion that ig replacement therapy may partially protect xla patients from symptomatic flu, although the role of cell mediated immunity in protection against flu is not clear. a prospective study could determine if ig replacement therapy partially protects against clinical flu symptoms in xla and cvid patients using two groups those that receive the current flu vaccine vs. those that do not receive the flu vaccine. introduction/background: fanconi anemia (fa) is an autosomal recesive or x-linked genetic disorder, characterized by cytopenia or bone marrow failure, this will lead to a severe anemia, neutropenia and thrombocytopenia, requiring frequent interventions with diversified therapies, including hematopoietic stem cell transplant (hsct). a complete immune reconstitution is requiere for a success transplant. one of the main upcoming events asociated after the hsct, is the secundary immunodeficiency, which is asociated with a significant morbility and mortality in the patients. to rich a successful alogenenic-hsct, is impending a complete reconstitution of the t cells immunity, for this it is crucial the presence of factors like; thymic activity of the hsct recipient, biological features of the allograft (eg, degree of histocompatibility, number and type of infused donor t cells) and preparative regimens. objectives: describe the kinetics of the immune reconstitution in fa patients after alogenic-hsct, as well as the infections associated during this process and other comorbilities. methods: we decribed the lympocyte population (t, b and nk) in pb measured by flow cytometry in fa patients on days +90, +120, +150, +180, +210 and +360 post alogenic hsct from a match related donor. the conditioning was base on fludarabine 150mg/m2, cyclophosphamide 10mg/kg and antithymocyte globulin rabbit 20mg/kg. infectious-disease survillance for virus was determined by the quantification by dna pcr-rt for cmv, ebv, adenovirus, as well as the presence of galactomannan and candida sp antibodies, or isolation of fungus or bacterial culturing in the cases of infectious disease of known or uknown aetiology. results: the lymphocyte population mesurement was performed in a total of five fa patients who undergo to an allogenic hsct, all of them received stem cells from a match related donor and the source was bone marrow. successful engraftment was observed in all 5 patients, there were no deaths reported. after the hsct was performed, the kinetics of recovering for the distinct lymphocytes subsets was the the following: nk cells (cd16+cd56+) were the first to recover, followed by cd+8 t cells, b lymphocyte and finally cd+4 t cells (figure 1 ) all of them rich normal values and remain stable. three out of five patients presented infectious disease: two of them were cmv positive, one patient has a concurrent detection of adenovirus, and in the other was detected aspergillus, in both of them it was presented at day +90. the third one developed acute gvhd which progressed to a chronic gvhd, at day +153 was diagnosed on him listeria monocytogenes meningitis, at that momento he has just cd+16 +cd56 and cd8+ reconstituded. there is no new infectous disease detected in any of the patients after they reconstituted cd4+ t cells. conclusions: complete immune reconstitution is the decisive for the presence of several morbilities and mortalities, mainly because of oportunistic infecctions and gvhd. we show that the kinetics of recovery of the different populations of lymphocytes follows those patterns also described for patients with other hematological malignancies: early recovery of nk cells, followed by effector cytotoxic t cells and b cells, and finally, cd4+ t-helper cells. the utility of post-transplant monitoring of pb-lymphocyte subsets for improved follow-up of patients undergoing bmt and prevent opportunistic infections. introduction/background: early detection of primary immunodeficiency diseases (pids) before serious infection is of utmost importance and a question of patient survival. one of the main organs involved in pids is skin with mucocutaneous manifestations being one of the common complaints in pids. the presenting skin symptoms may serve as an essential element in early diagnosis of pids. objectives: this study aims to determine characteristics, frequency, nature and incidence of skin manifestation in pid patients seen in qatar. methods: this retrospective study was conducted at hamad medical corporation the only tertiary hospital in qatar from january 2009 to july 2017.the subjects included were pid patients < 14 years old, who had dermatological complaints. information collected included dermatological diagnosis, gender, age at onset of signs and symptoms, age at definite diagnosis, family history of related disease, any lab results such as pathology or viral studies obtained from the clinical medical record. patients were diagnosed and classified according to clinical and laboratory criteria by the international union of immunological societies primary immunodeficiency committee. results: a total of 109 patients were studied and skin/mucocutaneous manifestations were found in 65 patients (59%) with male to female ratio of 1.5:1. age at onset of skin manifestations ranged from 0 to 12 years. skin manifestations were divided in two categories according to presentation before pid diagnosis (primary manifestations n=32, 47%) and during the disease period (secondary manifestations n=35, 53%). the type of pids predominantly having primary manifestation were scid (n=5, 15%) cvid and cgd (n=4, 12%, each), xla, at and griscelli syndrome (n=3, 9%, each).various manifestations which were atypical in their presentation and persistently found in patients who did not respond to any effective treatment (n=13) led to the basis of clinical immunological study and diagnosis including rare diagnosis such as ipex. whereas secondary manifestations were primarily reported in scid (n=9, 25%) cgd, at and digeorge syndrome (n=5, 14%, each). the nature of skin manifestations varied in both groups, primary manifestations notably had 41% cutaneous infections comprising of thrush (30%) stomatitis (17%) viral rashes (26%) bacterial rashes (13%) impetigo (8%) and fungal rash (4%). eczema/atopic dermatitis were 31% and other minor miscellaneous skin alteration cases found were apthous mouth ulcer (5) erythroderma (4) gvhd like rash (1) alopecia (1) psoriasis (1) and scleroderma (1) . secondary manifestations were identified as 53% cutaneous infections 20% eczema and infantile seborrheic dermatitis (1) pruritus (1) gvhd and alopecia (1) each. the causative microorganisms were confirmed in 44% of the cases via lab cultures whereas common infections such as chicken pox and herpes were validated through clinical symptoms. none of the cases were biopsied. overall, highest skin infection was seen in ataxia telangiectasia (11). other pids with prominent cutaneous manifestations included cgd (9), digeorge syndrome (5), hyper ige syndrome (4) and scid (rag 1 or 2, 4 cases). more than two types of skin manifestations were found in 47% patients over the due course of their illness. conclusions: an awareness of various cutaneous and skin disorders associated with pid which are persistent and unresponsive to treatments among dermatologist and family physicians is crucial to raise suspicion for early detection, timely management and prevention of complications. introduction/background: ataxic telangiectasia (at) is a rare neurodegenerative autosomal recessive disease associated with immunodeficiency, poor coordination and disability. ataxia telangiectasia has a diverse clinical heterogeneity which may often lead to an incorrect diagnosis or late detection of the disorder resulting in exposing the patient to unnecessary radiation from various sources. objectives: we present a female child from the asian subcontinent that was seen for the first time with ulcerative skin lesions, failure to thrive and marked lymphopenia. methods: a search of the pubmed database was carried out, using different combination of the terms "ataxia", "telangiectasia", typical , atypical and "presentation" results: four-year-old pakistani girl, product of first degree nuptial presented with generalized vesicular rashes mainly on abdomen and scalp which turned into ulcers with residual hypo pigmented lesions and diarrhea. she had short stature, failure to thrive (weight and height < 5th percentile) and no previous infection or family history of primary immunodeficiency diseases (pid). mild delay in milestones was observed especially in speech. immunizations were up-to-date including bcg. on examination she had mild ocular telangiectasia but no cutaneous involvement. investigations revealed lymphopenia (1.6) and eosinophilia. workup for diarrhea including duodenoscopy revealing subtotal villous atrophy however the biomarkers for celiac disease were negative. immune system workup showed high igg levels, normal iga, ige and igm level. igg subclasses: low igg 2, and 3, antibody titers to h.influenza and pneumococcus vaccine were relatively low. lymphocyte subsets showed low cd4,cd19 and high nk cells (40%), low naïve t cells (2%) and inverted cd4/cd8 ratio.t cell function with post pha was 18.7% but had normal response to cd3. alfa feto protein was requested due to continued low cd4 count and the level was found to be high: 260.9 iu/ml. next-generation sequencing (ngs) showed homozygous mutation for trp1750fs in chromosome 11 of atm gene confirmed by whole exome sequencing. immediate treatment with ivig was started leading to mark improvement of skin lesions, diarrhea and weight gain. conclusions: the index of suspicion of at should be highlighted when deciphering lymphocyte subset. currently there is no neonatal screening for pid diseases and next generation sequences in qatar. once implemented it may prove beneficial in discovering cases from early infancy and reaching upon a definite diagnosis. introduction/background: chronic granulomatous disease (cgd) is a genetic disorder of the nadph oxidase complex in phagocytes which results in impaired production of microbicidal reactive oxygen species that can lead to recurrent life-threatening bacterial and fungal infections. the majority of affected patients in the united states have a x-linked defect in the cybb that encodes gp91phox protein followed by an autosomal recessive defect in the ncf1 gene that encodes p47phox . x-linked female carriers show 2 populations of neutrophils and following lyonization, and severe skewing of x-chromosome inactivation can get cgd type infections. a recent study has shown that the lower dihydrorhodamine (dhr) percent in female carriers predicts a higher infection risk but carrier state on its own predicts autoimmunity results: a 39-year-old female presented for an evaluation of recurrent fevers. she had a history of several lobar pneumonias in childhood. at age 32, she developed erythematous bumps on her abdomen that were initially non-bothersome but later became painful. she then developed recurrent fevers, chills, and rapid weight gain. biopsy of the skin lesions showed subcutaneous panniculitis-like t cell lymphoma. she was treated with chemotherapy and found to be in complete remission. she had no previous family history of recurrent infections. at the completion of her chemotherapy, her 1-year-old son became septic in the hospital and was then diagnosed with cgd from mutated cybb gene. post chemotherapy, the patient had a prolonged course with an atypical lung infection that required bronchoscopy and video thorascopic surgery, however no bacteria was ever identified. she did improve after a long course of ivantifungals. evaluation for immunodeficiency was done after she had a recurrence of low grade fevers, cough, and fatigue. dhr flow cytometry with phorbol myristate acetate (pma) was done which showed 3.6% neutrophil oxidative burst activity after stimulation, consistent with a highly skewed lyonization pattern in x-linked cgd. conclusions: this case demonstrates an interesting lesson in a woman with recurrent infections since childhood whose clinical picture was confounded by her history of malignancy. the abnormal lung infections prior to malignancy were alarming and gave cause to do additional immunology testing. however, it begs the questions, if she did not have a son, would she have been diagnosed with severe x-lined carrier disease. introduction/background: measurement of the specific antibody response following vaccine challenge provides clinicians with a better understanding of the adaptive immune response in individuals undergoing immunological evaluation. objectives: we hypothesised that after classification of primary immunodeficiency (pid) patients based on vaccine response (vr), further division using igg subclass (iggsc) 1-3 measurements may identify additional patients with abnormal b cell function and patients with different frequencies of infection at presentation. methods: vrs and serum iggsc concentrations were quantified using the vacczyme anti-pneumococcal polysaccharide (pcp) igg elisa and human iggsc liquid reagent kits (the binding site group limited, uk) in 23 pid patients (1:1.3 m:f, median age 41.5 years, range 20-78). all patients were immunised with pneumovax®23 (sanofi pasteur msd). the lower limits of published normal iggsc ranges were used as cut-off values (igg1 3.2 g/l, igg2 1.2g/l and igg3 0.2g/l). pcp concentrations equal to or less than 70 mg/l post-vaccination was considered an abnormal response. results: agreement between vr and iggsc measurements was 48% (p=1.00). the frequency of respiratory tract infections at presentation among pid patients with a normal pcp igg vr was 45% and 55% among patients with an abnormal vr. subsequently, four separate groups could be identified by including iggsc measurements. the frequencies of infections at presentation were for the vr+/iggsc+ group (n=4) 36% vs. 48% for the vr+/iggsc-group (n=10); and 33% for the vr-/iggsc+ group (n=2) vs. 63% for vr-/iggsc-individuals (n=7). conclusions: these results confirm that vr and iggsc measurements are independent serum biomarkers of humoral immunity. taken together the vr and iggsc results provide more detailed information about the immune status that may influence diagnosis, treatment and monitoring decisions. introduction/background: hizentra® is a 20% liquid igg licensed for subcutaneous administration in adults and children greater than two years of age who have primary immunodeficiency disease (pidd). subcutaneous immunoglobulin (scig) use in autoimmune conditions is reported. stiff person syndrome (sps), a rare neurologic disorder characterized by fluctuating muscle spasms and rigidity, is mediated by autoantibodies to glutamic acid decarboxylase (gad). symptoms of sps have been shown to improve after administration of intravenous immunoglobulin (ivig) however, there is a paucity of information regarding use of scig in sps. objectives: the aim of this study is to describe the use of hizentra® in patients with sps, including indications for scig, clinical characteristics of patients, and clinical and laboratory response to scig. methods: a multicenter retrospective chart review examined 2 patients with stiff person syndrome treated with hizentra®. results: sps was diagnosed in 2 patients at 12 and 25 years. both patients were started on ivig, steroids, and rituximab prior to initiation of weekly hizentra® (ages 15 and 27 years). the average dose of hizentra was 125mg/ kg weekly. the average igg level while receiving ivig was 1,164.5 mg/dl, similar to the average igg level while on hizentra was 1,250 mg/dl. the number of administration sites ranged from 2-4, and duration of infusions ranged from 60-120 minutes. serum anti-gad antibody levels prior to hizentra® were 845 u/ml and 134 u/ml respectively. anti-gad antibody level during treatment with hizentra (available for one patient) was >5,000 u/ml, and 132,240 u/ml following discontinuation of hizentra®. one of the reasons for switching to hizentra® was lack of response while on ivig. the average number of hizentra infusions were 138. patients reported improvement in spasticity related to sps while on hizentra®, and one patient had improvement in seizures. one patient discontinued hizentra® in favor of intravenous immunoglobulin (ivig) due to physician preference. the most common side effects were local reactions including pain, pruritus, and redness. no serious adverse events were reported. conclusions: hizentra® was associated with improved symptoms in sps in both patients including decreased spasticity, and improved seizure frequency in one patient. serum anti-gad levels did not decrease following administration of hizentra. hizentra® was well tolerated in patients with sps, with most side effects reported as mild. hizentra® may be considered as an alternative to ivig treatment in patients with sps. introduction/background: combined immunodeficiency (cid) has been associated with a spectrum of secondary gastrointestinal manifestations, including infectious and non-infectious causes. abatacept, a soluble ctla4-igg1 fusion protein targeting t cell activation, has demonstrated benefit in the treatment of autoinflammatory manifestations in patients with cid due to underlying heterozygous germline mutations in ctla4 and lrba. objectives: herein, we report two patients with cid characterized by low immunoglobulin levels, low class-switched memory b cell counts, and low peripheral naïve cd4+ t cell counts. both patients suffered from severe and persistent diarrhea complicated by protein-wasting and malnutrition, ultimately diagnosed as biopsy-consistent autoimmune enteropathy. the clinical history of patient 1 was also notable for granulomatous lung disease and autoimmune cytopenias. methods: a t-regulatory cell disorder was considered in both cases, however, ctla4 and lrba were found to be unaffected by whole exome sequencing (patient 1) and/or flow cytometry (patient 2). due to progressive worsening of the autoimmune enteropathy despite management with chronic steroids (both patients), combination rituximab/mycophenolate mofetil (patient 1), and total parenteral nutrition (tpn) complicated by recurrent infections (patient 2), abatacept was trialed at 125 mg sc weekly. results: in both patients, the start of abatacept therapy produced a dramatic improvement as measured by decreased stool frequency, improved weight gain, and decreased protein-wasting. patient 1 has been maintained on this monotherapy for over one year, with improvement in the gastrointestinal as well as pulmonary autoinflammatory complications. the only documented adverse event has been hepatitis b reactivation, managed with tenofovir and abatacept continuation. patient 2 has required azathioprine/abatacept combination therapy for clinical stabilization and is without a significant adverse event to date. conclusions: we conclude that abatacept may be a relatively safe and effective therapeutic in the management of severe autoimmune enteropathy in the background of cid, even when used outside of the classical clinical context of ctla4 or lrba haploinsufficiency. objectives: as increasing the outbreak of allergic diseases, study for treatments comes to the force. in this research, we aimed to assess the effects of sg-sp1, a derivative of gallic acid, on mast cell-mediated allergic inflammation using various animal and in vitro models. methods: ovalbumin-induced systemic anaphylaxis and immunoglobulin e (ige)-induced passive cutaneous anaphylaxis are standard animal models for immediate-type hypersensitivity. oral administration of sg-sp1 hindered the allergic symptoms in both animal models. these inhibitions were deeply related to the reductions of histamine and interleukin-4. results: sg-sp1 reduced degranulation of mast cells and expression of inflammatory cytokines in a dose dependent manner. sg-sp1 showed better anti-allergic effects compared to gallic acid and dexamethasone. down regulations of intracellular calcium level and nuclear factor-b activation by sg-sp1 were causative of the reduction of allergic mediators. to anticipate the exact target of sg-sp1, phosphorylation of proteins involved in mast cell signalling was assessed. sg-sp1 suppressed the activations from lyn and was aggregated with high affinity ige receptor (fcri). conclusions: from these results, we assured that sg-sp1 directly interact with fcri. all together, we propose that sg-sp1 might be a therapeutic candidate for allergic disorders. (162) submission id#425361 systematic assessment of pain in patients with primary immune deficiency using validated pain questionnaires: a prospective study. introduction/background: the number of primary immunodeficiency (pid) patients is rising dramatically because of good medical care as well as increased awareness. around 1 in 1200 live births are affected. more than 300 disorders have been discovered so far and this number is expected to rise in the coming years. as with any other chronic illness, pid patients are also prone to acute and chronic pains. chronic pain is a big challenge and lack of understanding of the etiology and underlying mechanisms limit our ability to diagnose or treat it effectively. objectives: to systematically assess chronic pain in patients with pid using validated questionnaires and to try to understand the underlying mechanisms of neuropathic versus non neuropathic pain. methods: short-form mcgill pain questionnaire (sf-mpq) is a recognized way to ascertain different pain characteristics as well as severity. a validated arabic version of the sf-mpq was used to prospectively assess chronic pain in patients with pid. furthermore, a validated arabic version of the neuropathic pain questionnaire-short form (npq-sf) was also used to assess neuropathic pain and to differentiate it from nonneuropathic pain. a total of 27 patients with pid were included. results: males: females were 33.3%: 66.6% respectively. mean age was 29.73 years. commonest diagnosis was combined immune deficiency in 37% of the patients followed by common variable immune deficiency which was 25.9 %. chronic pain was found in 64% of the patients that participated in the study. 50% of the patients who complained of pain found it to be tiring and exhausting. 35% each had aching or heavy or sharp pain. 28% had cramping pain, 25% had tender pain and 21% characterized their pain as throbbing or burning. 18% felt shooting pain, 14% had splitting pain and 7% had gnawing pain. 28% found it to be frightening and 17% described it as being sickening in itself. the commonest pain complaint was abdominal pain in 14% of the patients followed by headache 11 % and chest pain 11%. pain attributed to neuropathy was present in about 17.8% of the study population. most patients described that they had been experiencing pain for at least 2-3 years. conclusions: this is the first international study to understand the prevalence, duration and severity of chronic pain among pid patients. a significant number of patients reported ongoing pain. this is the first time any kind of pain has been studied systematically in the patients with primary immune deficiency. treating pain should have a major impact on improving the patients quality of life. introduction/background: primary immunodeficiency (pidd) patients, particularly those with severe t cell defects, are at increased risk of infections. bone marrow transplant also contributes to significant t cell lymphocytopenia, which can be associated with similar risks. several prophylactic measures, which vary per institution, are placed on these patients in order to prevent infections. we report on a likely outbreak of rapid growing nontuberculous mycobacteria (ntm) at our institution, with a suspected association to tap water objectives: to recognize nontuberculous mycobacteria as a threat to patients with immunodeficiency disorders methods: case series of consecutive patients admitted to one institution with similar infections results: in a period of 16 months, 4/2016 to 8/2017, 5 patients admitted to our institution were found to have rapid growing ntm species on central line culture or bronchoalveolar fluid. these cultures were obtained due to fever and symptoms of systemic infection. per institutional practices no peripheral blood samples were obtained at the time of initial culture. all had significant t cell dysfunction: wiskott-aldrich syndrome (1), an undefined combined immunodeficiency (1) , and three patients post bone marrow transplantation (bmt), one 6 months (173 days) post an unrelated cord blood transplant for farber syndrome, one 120 days out of a matched related transplant for epstein barr virus (ebv)-associated lymphoproliferative disorder, and one patient with high risk neuroblastoma 106 days out of his second tandem autologous transplant. both allogeneic transplant recipients had received serotherapy. all species were rapid growing, identified as mycobacterium mucogenicum (n=3), immunogenum (n=1), or abscessus-chelonae complex (n=1). the patients had not shared rooms, caregivers, invasive procedures, or medications from the same batch. three (60%) cases met cdc criteria for hospital acquired infections (hai). all patients were in general ward rooms, and were on standard precautions given diagnosis had not been established (n=2) or they were considered outside the typical window of strict bmt precautions (n=3). ntm species were subsequently isolated from hospital tap water. this has resulted in a significant increase in infection precautions, including the use of sterile water only for cares, as well as bottled water for drinking (with no use of ice machines), for all patients being evaluated for pidd or with significant lymphocytopenia. conclusions: ntm are a threat to patients with pidd. tap water is a potential source of mycobacterial infections in pidd patients. minimizing exposure risk to water sources containing ntm is very important in this population. patients with concern for pidd or significant t cell lymphocytopenia should take steps to avoid ntm exposures, including the use of sterile water for cares, and bottled water for drinking. introduction/background: purine nucleoside phosphorylase (pnp) deficiency is a rare autosomal recessive condition leading to severe combined immunodeficiency and neurologic impairment, typically presenting in early childhood. the condition is progressive and typically fatal in the first or second decade of life without hematopoietic stem cell transplantation (hsct). objectives: to illustrate the clinical and laboratory presentation of pnp deficiency with a novel mutation in the pnp gene via a case study. results: case: a four year old female of hispanic descent, with a past medical history of spastic diplegia, initially presented with chronic nasal congestion, recurrent sinusitis, and cough. laboratory studies were significant for an alc 500 cells/mcl (2000-8000 cells/mcl). she was lost to follow-up for two years, before returning to medical attention for pneumonia. evaluation of lymphocyte subsets at age 6 years revealed cd3+ 145 cells/mcl (1200-2600 cells/mcl), cd4+ 119 cells/mcl (650-1500 cells/ mcl), cd8+ 28 cells/mcl (370-1100 cells/mcl), and cd19+ 44 cells/mcl (270-860 cells/mcl). t cell mitogen stimulation to pha measured by flow cytometry was 30% of control. pnp activity was nearly absent and urine guanosine and inosine were significantly elevated. gene sequencing revealed a homozygous c.655a>g mutation in the pnp gene. prophylactic antimicrobials were started. despite strong recommendation for hsct, the patient was again lost to follow up until age 10 years, at which time she was found to have progressive lymphopenia, bronchiectasis, and developmental delay. t cell mitogen stimulation to pha measured by thymidine uptake assay was 9% of control and pnp functional activity was undetectable. the patient underwent a 9/10 matched unrelated hsct six years after her initial presentation. one year post-transplant, the patient continues on immunoglobulin replacement therapy. her course was complicated by cutaneous gvhd, treated successfully with topical corticosteroids. conclusions: this case study illustrates the progressive nature of pnp deficiency in the first decade of life. our patient is notable in that she survived without significant medical intervention to the age of 10 years. her presentation at age 4 years was not unlike those previously reported in the literature, with muscle spasticity, ataxia, and recurring bacterial infections. to the authors knowledge, this case reports a novel mutation in the pnp gene. introduction/background: severe congenital neutropenia (scn) is a primary immunodeficiency disease characterized by early onset recurrent infections, persistent severe neutropenia and congenital genetic defect. severe idiopathic neutropenia (sin) is a rare disease defined by persistent severe neutropenia, in the absence of an identifiable etiology. objectives: here, we aim to find out clinical, laboratory, genetic characteristic and remission status in children with scn and sin in chinese population. methods: in this study, we enrolled 39 chinese children who experienced severe neutropenia longer than 6 months without any virus infection or auto-immune antibodies from june 2008 to july 2017 in hospitals affiliated to shanghai jiao tong university school of medicine. their clinical, laboratory and molecular characteristics were analyzed and the patients were followed up to observe their remission status. targeted gene capture combined with next-generation sequencing technology was used to find out related gene mutation. results: patients in this study had a mean age of 29.21±27.94 months. molecular analysis revealed that 7 patients had associated mutations of scn, including elane and g6pc3. among 26 patients with continuous follow-up, one died for unknown reason. ten patients have recovered from sin (r-sin) with mean neutropenia duration of 19.40±10.91 months. scn patients had more frequent infection (5.86±1.57 times per year) than sin (3.95±1.05 times per year, p=0.008) and r-sin patients (p=0.005, 3.89 ±1.27 times per year). scn patients had significantly higher count of anc and monocytes than sin (p=0.015) and r-sin patients (p=0.029). however, there was no difference in anc and monocytes counts between sin and r-sin patients. bone marrow examinations demonstrated a myeloid maturation arrest at the myelocyte-metamyelocyte stage in scn patients, while most of sin and r-sin patients were normal. conclusions: our study indicated that, patients with mild infection, lower anc, monocytes count and normal bone marrow are likely to be sin. whereas others with relatively more severe infection, higher anc, monocytes count and maturation arrest in bone marrow are inclined to be scn. introduction/background: patients with qualitative and/or quantitative defects of humoral immunity often require immunoglobulin (igg) replacement therapy (igrt). usual starting doses range between 400-600 mg/kg and the dose is adjusted as needed depending on the patient. there is a paucity of information about whether and how extreme bmi (obese or underweight) and route of administration may affect a patients rate of infections. objectives: the objective of the study is to determine whether rate of infection is associated with bmi, dose or route of administration in patients receiving igrt. methods: this is a retrospective chart review from december2000-october 2017. we included patients between the age of 1 month and 100 years old who were evaluated initially and had at least one follow up evaluation. data reviewed included the route of administration, dose, infection history, igg serum levels, height and weight to calculate bmi, gender, age and diagnosis requiring immunoglobulin replacement therapy. participants were excluded if the type of immunodeficiency is unknown or if the participant had incomplete data for the requested data fields. the number of infections between visits was modeled using poisson regression as a function of dose, route of administration and the bmi with the log of the follow up interval as an exposure offset. results: eighty-five patients were eligible, and preliminary results for 50 patients are presented here. the mean infection rate per 100 weeks of follow up was 1.9 in obese patients, 3.0 in overweight patients and 1.5 in underweight/normal weight patients adjusted for route of administration; however these rates do not differ significantly from one another. the mean infection rate per 100 weeks of follow up differed by administration route; 1.4 infections for ivig versus 3.1 infections for scig when adjusting for bmi (p<0.0131). the mean infection rate per 100 weeks of follow up was not associated with dosage. conclusions: overweight patients may experience more infections than obese or underweight patients, regardless of administration route. ivig patients may have a lower rate of infections compared to scig patients regardless of bmi. work is ongoing to complete analyses for the remainder of the eligible patient population. (167) submission id#421931 introduction/background: primary immunodeficiencies (pids) are a rare group of genetic disorders associated with a tendency to infectious diseases and an increased incidence of cancer. there is no data regarding the prevalence of malignancies in patients with pid in turkey. objectives: in this study, we aimed to evaluate patients who diagnosed as pids and developed malignancies, and calculate the estimated frequency of malignancies associated with pids in turkey. methods: forty five patients who were diagnosed with malignancy in the follow-up period of pid at the four tertiary immunology clinics between 1992 and 2017 years were included in the study. the data were obtained retrospectively from the hospital records and the database of esid online patient registry. results: the prevalence of malignancies in patients with pid was found as 1.05% (45/4285). the male to female ratio of the patients was 29/16, the median age was 13.5 years (minimum: 1.5, maximum: 57) and the median age at which patients get diagnosed with malignancy was 9 years (minimum: 1.5, maximum: 51) . there was no cancer history in their family members. the most common type of pid which associated with malignancy was ataxia telangiectasia (n=15, 33.3%). non-hodgkin lymphoma was the most common malignancy (n = 25, 55.5%) in our group (table 1) . ebv quantitative pcr was positive in 6 lymphoma cases (17.6%). the median number of x-rays and ct scans in patients with at and bloom syndrome before malignancy developed were 5 (minimum: 1, maximum: 24) and 1 (minimum: 0, maximum: 3), respectively. two cases had dual malignancies (papillary thyroid cancer and anal adenocancer). twenty cases were treated with chemotherapy, 7 cases with hematopoietic stem cell transplantation, 5 cases with radiotherapy, and 5 cases with surgical treatment, treatment information of 17 patients was not reached. remission was detected in 16 cases, while resistance to therapy in 2 cases and recurrences in two patients were observed. four patients are still on chemotherapy. twenty cases died. conclusions: the tendency of malignancy in patients with pids is due to the deficiency in the immune response that lead to failed surveillance against oncogenic viruses, premalignant/malignant cells, or both. lymphoid malignancies are the most common malignancies associated with pids. pids-associated malignancy incidence has increased in recent years because of that improved survival of the patients. this study is the largest cohort investigating the association of malignancy in patients with pid in turkey. additionally, we first reported tendency to malignancy in a patient with znf 341 deficiency. introduction/background: next-generation sequencing (ngs) has become an integral tool in the evaluation of primary immunodeficiency disorders (pid). we describe a patient with a previously described pathogenic foxp3 variant who met clinical and laboratory criteria for cvid. objectives: describe a patient with a previously described pathogenic foxp3 variant who met clinical and laboratory criteria for cvid. results: case description: an 8-year-old male born premature at 32 weeks presented with a history of recurrent infections. family history was negative for immunodeficiency. the patient developed recurrent acute otitis media beginning at 1 year of age, three episodes of pneumonia beginning at 3 years of age, and recurrent sinus infections requiring treatment on average four times a year beginning at 5 years of age. initial immunologic evaluation at age 8 was notable for: igg 305 mg/dl (reference 673-1734 mg/dl), igm 15 mg/dl (reference 47-311 mg/dl), iga 173 mg/dl (reference 41-368 mg/dl), ige 166 iu/ml (reference 0-90 iu/ml). lymphocyte subpopulations were normal. specific responses to vaccines showed: protective antibody titers to diphtheria, but not to tetanus or pneumococcal antigens. he did not respond to booster vaccination and was started on ivig with significantly reduced frequency of infections. at age 10, while on ivig, he developed oral ulcers (biopsy consistent with ulcerative eosinophilic granuloma), abdominal pain, and recurrent arthralgias involving ankles, elbows, hips and the sacroiliac joint. magnetic resonance imaging (mri) was consistent with sacroilitis. subsequent imaging was consistent with chronic relapsing osteomyelitis (crmo). gastrointestinal biopsies showed severe active chronic pangastritis with antralized oxyntic gastric mucosa with enterochromaffin cell hyperplasia; suggestive of autoimmune gastritis. plasma cells were present throughout the gastrointestinal tract. ngs (bcm-ngs, baylor miraca genetics laboratory, houston tx) identified a hemizygous pathogenic missense variant, c.1190g>a (p.r397q) in the x-linked foxp3 gene, that has been reported previously in patients with immunodysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome. flow cytometry studies showed a decreased percentage of treg cells of total cd4 expressing cells, 2.6% (reference 4.2-9.9%), but normal foxp3 expression within these cells, 68% of treg cells with foxp3 expression (reference 55-81%). interestingly, treg cell subset phenotyping obtained at the same time showed a normal percentage of natural tregs, 3.2% cd4 t cells (reference 0.7-4.0%), as well as normal percentage of naive tregs, 5.6% cd4 t cells (reference 0.6-9.0%). up to this point, he had not had any signs of diabetes, thyroid disease or frank enteropathy involving the small or large intestine. conclusions: we report a pathogenic foxp3 variant, occurring in a patient with a cvid-like phenotype, autoimmune gastritis, and an association with crmo. this case demonstrates the increasing utility of ngs, which can profoundly impact prognostic and therapeutic considerations. (169) submission id#413429 the natural history of patients with profound combined immunodeficiency (pcid): interims analysis of an international prospective multicenter study. immunodeficiency in this patient group. so far, 48 patients were transplanted after enrolment, overall 19 patients died (9 in the hsct group, 10 in the non-hsct group). analysis of the hsct decisions revealed the divergent decisions in patients with similar disease burden, favoring an ongoing prospective matched pair analysis of patients with similar disease severity with or without transplantation. so far, neither the genetic diagnosis nor simple measurements of t-cell immunity emerged as good predictors of disease evolution. conclusions: the p-cid study for the first time defines and characterizes a group of patients with non-scid t-cell deficiencies from a therapeutic perspective. since genetic and simple t-cell parameters provide limited guidance, prospective data from this study will be an important resource for guiding the difficult hsct decisions in p-cid patients. (170) submission id#419587 the plasma contact system and its role in common variable immunodeficiency: an explorative study. introduction/background: a growing body of evidence suggests that the contact system is involved in the activation of various vascular and immunological pathways and acts as an interface to help regulate allergic reactions, coagulation, complement, innate immunity and inflammation. as demonstrated in mice experiments, contact activation and high molecular weight kininogen (hk)-derived peptides increased homing of t and b lymphocytes into lymph nodes, which suggests an important area of research for understanding the contact systems role, specifically fxii, in immune-mediated inflammation and immune dysregulation. this novel mechanism prompted further inquiry into its role of various human disease states characterized by inflammation. plasma hk cleavage has been proposed as a useful and minimally invasive biomarker in various inflammatory disease states. this pathway has not been explored in cvid, in which inflammatory complications are found in one-third of patients with an unidentified genetic cause. characterizing the contact system biomarkers in cvid patients could elucidate a role in pathogenesis. objectives: assess the presence of contact activation at baseline in sera from cvid patients with and without inflammatory complications compared to healthy controls. methods: cvid patients were recruited in the outpatient setting and the measurement of cleaved plasma hk (chk) levels was determined by western blot analysis, under reducing conditions, with quantitation of total and chk bands using an odyssey imaging system (licor). a one-way anova test for differences among the 3 studied groups will be applied. c1 inhibitor levels, c3 and c4 levels and high-sensitivity crp were also measured as comparable biomarkers for inflammation. results: to date, 9 cvid patients were studied, 7 with and 2 without inflammatory complications. repeated determinations of cleaved hk% (chk%) revealed an average of 1.20% (range: 0.46-2.66%) in cvid patients with inflammatory complications and those without complications averaged 1.07% (range: 0.79-1.35%). healthy controls had an average chk of 1.15% (n=10, range: 0.60-2.10%). conclusions: cleaved kininogen detected in the sera of cvid patients was found at similar levels compared to healthy controls (chk<5%). findings suggest that systemic activation of the contact system might be absent in cvid, however, future considerations include developing detection methods for local tissue activation. (171) submission id#428729 the underlying primary immunodeficiencies and lung diseases, and low cd3 and cd4 counts are associated with recurrent pneumonia in hiv negative lymphopenia patients. 1 1 clinical fellow, yale university school of medicine introduction/background: lymphopenia can be considered as primary or acquired immunodeficiency. lymphopenia is associated with a considerable increase in susceptibility to infections and the treatment focus for lymphopenia is mainly consists of prophylaxis and treatment of opportunistic infections. aids is the most well known cause of acquired lymphopenia and the cd4 count serves as an effective surrogate marker for disease progression and guideline for prophylaxis for hiv positive lymphopenia patients (hplp). hiv negative lymphopenia patients (hnlp) have been following the same prophylaxis guideline for hplp patients. however, it is unclear whether same prophylaxis guideline will be appropriate for both groups since the underlying immune mechanisms are different between these two groups. objectives: we aimed to define the optimal treatment and prophylaxis guideline for hnlp. in this study, we compared the clinical phenotypes and absolute counts of lymphocyte subsets between hnlp with recurrent pneumonia (pna), recurrent upper respiratory infection (uri) and no pulmonary infection. methods: electronic medical records of hnlp (n=29) seen at an academic immunology clinic between the year of 2012 and 2017 were reviewed retrospectively. lymphopenia was defined as absolute cd3 count less than 1000/μl. the age, absolute counts of cd3, cd4, cd8, cd19, nkt and nk cell counts, history of antibiotic or antiviral prophylaxis use, autoimmune disease, lung disease, immunosuppressive therapy use, hypogammaglobinemia and ivig therapy use were compared between patients with recurrent pna (n=8), recurrent uri (n=13) and no pulmonary infection (n=8). results: this study showed that patients with recurrent pna had significantly lower absolute cd3, cd4, nk cell counts and age compared to the patients with recurrent uri ( table 1) . none of the clinical phenotype was significantly different between these two groups. when we compared the patients with recurrent pna vs no infection, all lymphocyte subset counts and age were similar between these two groups except the frequency of underlying lung disease which was significantly higher in the recurrent pna group (table 1) . lastly, we grouped the recurrent pna and uri groups together as the pulmonary infection group. when we compared the pulmonary infection group with the no infection group, lymphocyte subset counts were not significantly different, however, infection group showed significantly higher incidence of pid and the trend of lower rate of is therapy use than the no infection group (table 1) . conclusions: the initial study has several limitations. this was a retrospective study from a single clinic and patient population was limited to 29 patients. despite these limitations, we believe that this study provides valuable messages regarding prophylaxis guideline for pulmonary infection in nhlp; the underlying pids including icl and cvid, lung diseases particularly bronchiectasis and the absolute cd3 and cd4 counts less than 500/μl and 300/ μl, respectively, are associated with recurrent pna and the patients with these risk factors likely will benefit from antibiotic prophylaxis. in addition, patients with acquired lymphopenia due to chronic use of is therapies without underlying pid or lung disease less likely develop pulmonary infection despite of low cd3 and cd4 counts. further investigations are crucial to elucidate the clinical significance of our initial observation by increasing the patient population and analysis of detailed immunologic and genetic profile of these patients which will likely reveal immunological markers and genes that are involved in the pathogenesis of both primary and hiv negative acquired lymphopenia. director, neuro-oncology program, division of hematology/oncology, ucsf benioff children's hospital oakland, ca introduction/background: gata2 is a hematopoietic transcription factor, required for the development and maintenance of a healthy stem cell pool. heterozygous mutation of gata2 has been associated with different but overlapping syndromes affecting both myeloid and lymphoid cell lines including aplastic anemia, myelodysplastic syndrome, acute myeloid leukemia, pulmonary alveolar proteinosis and lymphedema. it is also associated with immunodeficiency and susceptibility to mycobacterial, fungal, and viral infections. hematopoietic stem cell transplantation (hsct) is the only available cure which should ideally occur before patients develop neoplasia, severe infections or lung disease. objectives -to describe the clinical presentation of a genetically confirmed case of gata2 mutation -to describe the characteristic hematological and immunological profile of patients with gata2 mutation -to emphasize the importance of high index of suspicion and early diagnosis in improving outcome with hsct methods: case: a 17-year-old female presented with prolonged fever, fatigue, nonspecific rash, and unremarkable clinical exam. laboratory evaluation was significant for mild pancytopenia with profound monocytopenia. bone marrow analysis was remarkable for hypocellularity without evidence of dysplasia or malignancy. peripheral blood flow cytometry showed decreased t-and b-cells and absent nk cells. results: the constellation of pancytopenia, marked monocytopenia and absent nk cells, were suggestive of gata2 deficiency. sanger gene sequencing of gata2 revealed a heterozygous nonsense mutation (p.arg337*). conclusions: mutation of gata2 is the underlying defect in overlapping clinical syndromes and is associated with immunodeficiency and malignant predisposition. incidence of organ dysfunction, infections and neoplasia increases with age. confirming the diagnosis during the phase of marrow hypocellularity/monocytopenia and pursuing hsct prior to malignant transformation may improve patient outcome. introduction/background: reduction in child mortality has kept pace with improved immunization and nutritional status. however, there are children with severe infections who have no identifiable reason. the search for children with pid was initiated in a tertiary care referral hospital from a region with no documentation of the prevalence of this disorder, but with a high rate of consanguinity. financial constraints, poor availability of laboratory facilities or therapeutic options locally, limited awareness among pediatricians and vast distances between premier centres were major obstacles. objectives to identify children with primary immune deficiency and study the spectrum of pid in north kerala to prevent infectious and other complications in these children to provide curative therapy whenever feasible methods 1. acquisition of knowledge and skills to diagnose and manage pids 2. establishment of an immune deficiency clinic 3. liaison with centres across the country offering diagnostic tests and stem cell transplant 4. formation of project team and submission of a project to the central government 5. improvement of diagnostic facilities at home institution results: 180 children with recurrent, severe or persistent infections or with two or more esid warning signs were screened for pid. severe combined immune deficiency was diagnosed in 3 children, wiskott -aldrich syndrome in 5 children, chronic granulomatous disease in 6 children and x linked agammaglobulinemia in 11 children and leucocyte adhesion deficiency in 3 children. prophylaxis with ivig was initiated in 22 children and stem cell transplant was done for 8 children. conclusions: in a country with resource constraints, limited awareness among health care providers and vast distances, it is possible to make a difference to the lives of families of children with pid by networking across centers with expertise in immunological and molecular genetic diagnostic methods and life -saving therapeutic modalities like stem cell transplantation. introduction/background: complete thymic aplasia is a rare cause of scid, and requires thymic transplantation for curative treatment. because thymic transplantation is not widely available, there can be significant delay between diagnosis and curative therapy placing the infant at risk of invasive life threatening infections. objectives: describe modalities of therapy for adenoviremia in a patient with scid due to thymic aplasia. methods: chart review was performed. treatment including hlapartially matched third party cytotoxic t cell therapy and matched related hematopoietic cell transplant were assessed for treatment of life threatening adenoviremia in a scid patient with thymic aplasia. we identified an infant with a mutation in tbox transcription factor 1 (tbx1) (c.1176_1195dup20) by way of state newborn screening for scid. she had severe t cell lymphopenia and abnormal t cell function. at 7 months age, she developed adenoviremia with associated fulminant hepatitis, and an initial viral load of 5 million copies/ml. despite prolonged therapy with cidofovir, viral load increased to as high as 264 million copies/ml. treatment with two infusions of partially hlamatched third-party cytotoxic t cells specific to adenovirus (5/10 and 4/10 hla matched, with hla class ii-mediated antiviral restrictions) led to partial clinical improvement without viral clearance. due to continued severe adenovirus-related hepatic dysfunction, unmanipulated bone marrow from an hla-identical sibling was infused without conditioning (10 million cd34+ cells/kg and 5.2x107 cd3+ cells/kg), at 9 months of age. after an initial surge in adenoviral loads attributed to massive viral lysis, the degree of viremia progressively declined and was <1,000 copies/ml within 7 weeks of marrow infusion. antiviral tcell activity against adenovirus was detected at low level in peripheral blood via ifn elispot at 3 weeks post-marrow infusion. she subsequently developed acute cutaneous and hepatic gvhd responsive to tacrolimus and steroids without recrudescence of viral illness. conclusions: delay in curative therapy in scid substantially increases risk of invasive life threatening infections. one strategy of allogeneic hct can be to eradicate severe infection in scid by providing the necessary t cell directed therapy against infectious agents. antigen-specific partial immune reconstitution can be achieved with hct in patients with thymic aplasia but concern regarding the development of full immunologic t cell diversity in athymic patients remains. senior technician, sanquin bloodsupply amsterdam introduction/background: chronic granulomatous disease (cgd), an inherited disorder of granulocyte function caused by a failure of intracellular superoxide production, normally presents as severe recurrent bacterial and fungal infections in the first years of life. the majority of affected individuals are diagnosed before the age of 2 years, although patients may remain undiagnosed until adulthood despite the early onset of the symptoms objectives: investigation of fungal infection in adult cgd patients methods: nbt and dhr 123 for detection of cgd and molecular analysis for detection of type of mutation in cgd and fungal characterization. results: we report here the detection of causative fungal infections in 2 adult patients with cgd. in the first patients we found paecilomyces formosus infection in an adult patient (18 years old female) with undiagnosed cgd who was referred to the shahid beheshti university hospital (tehran, iran) complaining of cough, dyspnea and fever for 5 weeks prior to admission. microscopic analysis revealed branching solitary phialide with ellipsoidal conidia with long chain arrangement and many chlamydospores which mostly resemble botryotrichum species but during subcultures on potato dextrose agar (pda) and sabouraud dextrose agar (sda), phialides typical of paecilomyces species appeared. typically, paecilomyces spp. rarely cause infections in humans and if these fungi are detected in blood urine or cerebrospinal fluid cultures they are considered as contaminants. the second patient was a 26-year-old man was referred to the hospital with weight loss, fever, hepatosplenomegaly and coughing. he had previously been diagnosed with lymphoadenopathy in the neck at age 8 and prescribed antituberculosis treatment. bal and serum galactomannan tests were negative. low, subnormal levels of ros were produced following stimulation of purified neutrophils with the phorbol ester pma. genomic dna was extracted and gene scan was used to determine the ratio between the number of exon 2 sequences of neutrophil cytosolic factor 1 (ncf1) gene, which encodes p47phox, and the number of -ncf1 exon 2 sequences. in addition, the fungal culture was disrupted with glass beads and dna was extracted. the dna sequence results were blasted using the ncbi genebank database, which showed 99% similarity to an aspergillus terreus isolate in the gene bank fungal library with accession no 1168. conclusions: thus, despite its current relative rarity in older patients, the presence of fungal infection is changing our understanding of the diagnosis, management and outcome of cgd. greater appreciation of the potential of fungal infection in older cgd patients is important in regions, such as iran, where tuberculosis is endemic and that sarcoidosis and cgd are considered as differential diagnosis. the demonstration of the successful patient-orientated treatment after using sequencing to confirm cgd and to identify the presence of the specific infectious agent emphasises the importance of adopting this approach across the region. introduction/background: primary immunodeficiencies (pi) represent a heterogeneous group of over 350 genetically distinct disorders which interrupt normal host-defense mechanisms and predispose to significant morbidity and mortality. presently, we are only able to screen for severe t-cell deficiencies at birth; however, the most common forms of pi often go undetected for years leading to adverse patient outcomes and excessive healthcare spending. given the relatively high incidence of pi in the general population, informatic measures could be useful for determining individual risk for pi and facilitating earlier, correct diagnosis and appropriate treatment across the spectrum of pi. objectives: the purpose of this study was to test the jeffrey modell foundations spirit (software for primary immunodeficiency recognition intervention and tracking) analyzer on the texas childrens health plan. major aims were to identify individuals with medium-high risk for pi, assess the clinical characteristics of at risk patients and determine if risk identification led to diagnosis of pi over a 12 month period. methods: after removing all known pi diagnosed patients from the database, 185,892 individual texas childrens health plan enrollees were screened for risk of pi with the spirit analyzer using relevant, weighted icd9 and icd10 codes. patient characteristics are shown in table 1 . following identification of medium-high risk (mhr) individuals, letters were sent to their primary care physicians to alert them of patient risk. a second analysis of the mhr individuals was performed 12 months later. detailed chart reviews were conducted on 769 mhr individuals to further assess clinical features of this group. the study was approved by the baylor college of medicine institutional review board (study h-38501). results: of the original cohort, 2188 (1.2%) were identified as mhr for pi. from that group, 1068 (0.6%) were accessible for analysis and 769 (0.4%) had electronic health records for review. in the 12 months following the first analysis, 43 (0.02%) were diagnosed with a pi. (figure 1 ) another 61 patients had concerning diagnoses coded warranting further investigation. (figure 2 ) concerning diagnoses included: cellulitis(18), abscess (14), recurrent otitis media(11), recurrent sinusitis (5), osteomyelitis (2), and mastoiditis (1) among others. in total 104 patients had a pi diagnosis or a history concerning for pi (0.06% of main cohort; 13.5% of mhr cohort). conclusions: the spirit analyzer is effective at identifying persons at risk for pi and facilitates diagnosis. potential mhr yield by the analyzer is over 10%. these patients are treatable and will benefit from targeted intervention once identified. the analyzer can also highlight concerning conditions worthy of additional assessment. future work should focus on longitudinal healthcare outcomes for patients diagnosed with pi via spirt and physician perspectives on the utility of the tool. introduction/background: genetic variants in card11 contribute to several diseases caused by dysregulation of the adaptive immune system. dominant-negative, loss-of-function and gain-of-function variants in card11 variants lead to primary immunodeficiency disease. however, primary immunodeficiency disease caused by card11 gain-of-function variants often progresses to b-cell malignancy. the clinical course and treatment options depend on the type of card11 mutation. unfortunately lymphocyte immunophenotyping and traditional proliferation assays can't distinguish the variant effect or predict the likelihood of malignancy. objectives: to use multiplexed functional assays for determining variant effect to distinguish between dominant-negative, loss-and gain-offunction effects in card11. our ultimate goal is to generate a variant function map that can be used to guide diagnoses and treatment of immune dysregulation caused by mutations in card11. methods: we co-delivered crispr/cas9 ribonucleoprotein complexes with libraries of single-stranded oligonucleotide repair templates thus generating lymphoma cell populations containing all possible singlenucleotide variants (~2400 different protein coding changes) in the nterminal 140 amino acids of card11. following culture with and without bcr pathway inhibitors, we used next generation sequencing to quantify variant abundance before and after culture, both with and without bcr pathway inhibitors. results: due a requirement for card11 in these lymphoma cells, those with dominant-negative and loss-of-function card11 variants grow more slowly, whereas those with gain-of-function variants grow faster in the presence b cell receptor pathway inhibitors. by tracking the relative abundance of each variant in the population by next generation sequencing over multiple conditions, we determined the functional effect of each. we assessed the functional effects of thousands of card11 variants in parallel. this enabled us to confirm several previously reported gain-offunction and dominant-negative variants in card11, as well as identify several additional novel variants. finally, we evaluated previously undescribed dominant-negative, loss-of-function and gain-of-function variants during differentiation of primary human b cells and during nf-b signaling. conclusions: the results of our experiments demonstrate the utility of multiplexed functional assays for determining variant effect in proteins where distinguishing between dominant-negative, loss-and gain-offunction effects are required to guide diagnoses and treatment. introduction/background: primary immune-deficiencies (pids) are a heterogeneous group of nearly 300 monogenic inborn errors of immunity. in recent years, whole exome sequencing (wes) became a valuable diagnostic approach for the identification of molecular defects in patients with clinical manifestations suggestive of pid. this approach provides a definitive diagnosis and may help in genetic counseling, prenatal diagnosis and pre-symptomatic identification of patients with a potentially lethal disease. the diagnostic yield using wes was found to be~25% in rare mendelian disorders and~40% in pids. we present here a markedly higher yield, of 65%, in pids with a high percentage of consanguinity (65% in this study). objectives: using the whole exome sequencing (wes) approach in 55 israeli pid patients with high percentage of consanguinity to identify the genetic causes underlying their diseases for better diagnosis and clinical management. methods: wes was performed on genomic dna obtained from wbc of 55 immune deficient patients with potential genetic causes. the sequencing data was analyzed bioinformatically. each of the discovered mutations was validated and the familial segregation was confirmed, using sanger sequencing. results: the 55 probands (32 males and 23 females) ranged in age from 2 weeks to 26 years with a mean of 4.4 years. of them, 20 patients are jewish (36%), 34 palestinians (62%) and 1 (2%) is from a greek ethnicity (cyprus). based on their clinical and immunologic phenotype at the time of initial evaluation, patients were assigned to one of seven pid groups: (i) humoral immunodeficiency 5 patients (9%); (ii) combined immunodeficiency (cid) 16 (29%); (iii) cid with syndromic features 10 (18%); (iv) scid 9 (16%); (v) congenital defects of phagocytes 6 (11%); (vi) immune dysregulation 7 (13%) and (vii) phenocopy of pid 2 (4%). we identified 67 mutated alleles, all in coding regions, that are highly likely to be causative in 36 of the 55 patients, achieving a 65% molecular diagnostic yield. among the 36 patients, the mode of inheritance in 28 patients (78%) is autosomal recessive and in 3 is compound heterozygote (8%). four patients (11%) harbor a non-inherited mutation on one allele, either de novo or somatic. the inheritance of the mutation in one patient (3%) is x-linked. the high rate of bi-allelic inheritance (93% of the alleles) is mainly due to the high frequency (65%) of consanguinity among the studied cohort. twenty eight mutated genes were identified in this study. of them, 6 were found to be novel in causing an inherited disease in man. interestingly, some genetic defects in known genes were found in patients with atypical phenotypes. in 23 patients (42% of the total number of patients and 64% of the wes diagnosed) the discovery of the genetic cause led to a change of therapy, towards a more targeted and personalized one. the revised treatments included bone marrow transplantation, conditioning protocols, reduced intensity of immune suppression, and prevention of unnecessary treatments due to their possible deleterious outcome. conclusions: except of being a useful tool for diagnosing and deciphering novel or atypical forms of pids, our wes study demonstrates an immediate and powerful impact on patient therapy in pids. early intervention with bone marrow transplant or gene therapy is critical and best when the infant in uninfected. newborn screening for scid and t-cell lymphopenia has been implemented in 45 states. the screening test is performed from dried blood spots collected at birth and involves pcr quantification of circular dna byproducts of t-cell receptor gene rearrangement, t-cell receptor excision circles (trec). trecs are generated during t-cell maturation in the thymus and are indicative of naïve t-cell output. assays and protocols to measure trecs vary by state and there is no standardized guideline at this time. washington state is unique in that it is one of few states where all newborns undergo two or more independent newborn screens, the first by which igrt treatment helps in these patients is unreported. here we present the first case of an mbl deficient patient with other complicating conditions (low igg and multiple cf polymorphisms, ciliary dyskinesia) who did not show improvement on igrt, thus refuting current literature. results: a 28-year-old caucasian male patient presented due to recalcitrant warts of the hands and feet. he had childhood otitis media status post tympanoplasty, mild molluscum contagiosum, and eczema. also, history of migraines with a negative brain mri. a trial of weekly pegylated interferon alpha 80mcg for his warts was discontinued due to significant neutropenia and depression. physical exam was notable for bulky skin colored warts on the lateral and dorsal fingers and dorsal hands including periungual skin. clusters of verrucae were noted on the feet. initial laboratory testing was notable for mild hypogammaglobulinemia with protective specific antibodies. lymphocyte subset analysis revealed a predominantly t cell lymphopenia with decreased naïve and memory cd4 and cd8 subsets, normal absolute numbers of b cells but with low memory subsets, and normal numbers and function of nk cells. whole exome sequencing ultimately revealed homozygous r169q mutations in the cecr1, a mutation previously described as causing ada2 deficiency. ada2 activity was about 40%. conclusions: ada2 deficiency is a relatively newly defined genetic defect, with a clinical phenotype that continues to evolve with newly diagnosed cases. our patient had not had evidence of vasculitis or stroke, but had recalcitrant warts with lymphopenia as his primary presentation. approach to therapy for those without vasculitis or significant cytopenias remains unknown. introduction/background: whole exome sequencing has added greatly to our library of primary immune deficiencies. however, interpretation of findings is not always straightforward. clinicians need to understand the limitations of this diagnostic tool and be familiar with the next steps in order to achieve a diagnosis. objectives: a 7 year old male presents for evaluation of bronchiectasis of bilateral lower lobes and poor growth. past medical history was significant for gerd with poor weight gain during infancy and childhood, oral thrush, recurrent respiratory infections and bilateral partial hearing loss. the bal showed abundant neutrophils and was positive for strep pneumoniae and haemophilus influenza. methods: serum immunoglobulins were normal with an elevated iga (369) and mildly elevated ige (361). tetanus igg was protective (0.4 iu/ml) but post-vaccine responses to the pneumococcal polysaccharide vaccine were poor with protective titers achieved to 3/14 serotypes. lymphocyte phenotyping was remarkable for a complete absence of b and nk cells. cd4:cd8 ratio was inverted (0.25) and the majority of the cd45+ t cells were memory phenotype. mitogen proliferation was essentially normal. due to the absence of b cells and despite normal total serum immunoglobulins, the patient was started on sc ig weekly and antibiotic prophylaxis with trimethoprim-sulfamethoxazole and fluconazole. clinically he demonstrated good weight gain and a reduction in cough and respiratory symptoms. results: whole exome sequencing was performed. a single base mutation in ada1 (c.320 t>c) was identified in one allele. this mutation (p.l107p) is known to be deleterious and when homozygous is associated with typical scid. however, this mutation was not present on the other allele. secondary analysis looking for large deletions and duplications failed to identify any abnormality in sequence in the normal allele. but the expression of this allele was markedly diminished causing us to suspect that its function might be impaired. functional testing was performed and demonstrated that there was no ada activity in the patients red blood cells, thus confirming a diagnosis of combined immune deficiency due to ada deficiency. conclusions: null mutations in ada result in an absence of ada function with profound cd3 cell lymphopenia and dysfunction. clinically affected infants have typical scid. hypomorphic mutations may lead to partial function of ada with more variable immune defects. interestingly, most patients with ada deficiency have neurologic complications, frequently hearing loss. we believe that most likely this patient has had some degree of spontaneous reversion of the mutation in the normal allele leading to a less severe phenotype. as reported previously in a case involving a mutation in il2rg chain, normal function was not restored and the patient remains with a combined immune deficiency. this case highlights an important limitation of whole exome sequencing and the need for confirming the impact of a genetic defect with a functional assay. introduction/background: the autosomal dominant hyper-ige-syndrome (ad-hies) is a rare primary immunodeficiency and multisystem disorder resulting from heterozygous loss-of-function mutations in the stat3 gene. ad-hies is characterized by skeletal dysplasia, recurrent pulmonary and skin infections (e.g. staphylococcal abscesses, eczematoid dermatitis) due to an increased susceptibility to bacteria and fungi. since many patients do rather well on anti-infective prophylaxis and supportive care, and early case reports suggested no benefit of allogeneic hematopoetic stem cell transplantation (hsct), ad-hies patients are rarely referred for hsct. the literature still contains only a handful of patients who underwent hsct. all these patients had experienced severe disease related complications before hsct and consequently the benefit of hsct was reported to be variable. objectives: currently, the general consensus is to only consider patients with severe pulmonary disease for hsct. it could however be postulated that transplanting patients earlier in their disease course and correcting their immunodeficiency before permanent organ damage due to infectious complications has occurred may extend life-expectancy and improve the quality of life of ad-hies patients. methods: we report on a 15 year old female ad-hies-patient who presented with bronchiectases following recurrent pneumonias, one pneumatocele, and normal pulmonary function tests. her past medical history also included recurrent skin infections, serious haemoptysis, pathological fractures and atopic eczema. considering that lung infections are the major life-limiting complication in ad-hies and are potentially positively influenced by hsct, our patient had enquired about a transplant. after extensive discussion of the pros and cons and obtaining full informed consent from her and her family, she underwent an elective hsct. she received bone marrow from an hla-matched sibling donor after a reduced-intensity conditioning consisting of alemtuzumab (2x0,2mg/kg), treosulfan (3x14g/ m2), fludarabine (5x30mg/m2) and thiotepa (2x5mg/kg), and graftversus-host disease (gvhd) prophylaxis with cyclosporine a (csa) and mycophenolate mofetil (mmf). results: the peri-transplant course was complicated by acute lower gastrointestinal tract bleeding and renal failure of unknown origin. continued kidney function impairment led to early tapering and discontinuation of csa on day +132 after hsct in the absence of any acute gvhd. neutrophils and platelets engrafted on days +15 and +26 respectively. she is currently on day +150, free of gvhd or infection, exhibiting full donor chimerism and recovered kidney function. in view of the preexisting pulmonary damage she currently remains on antibiotic prophylaxis and inhalation therapy. conclusions: this ad-hies patient who underwent hsct with few pre-hsct disease complications and relatively little permanent organ damage may add to our understanding of whether early hsct will lead to improvement of quality of life and possibly increased life-expectancy in ad-hies patients. it remains to be elucidated whether her rather uncommon peri-hsct complications are connected to her underlying disease. future research should be directed at identifying ad-hies patients at high risk of severe pulmonary complications early, so these could be referred for timely hsct. objectives: this is a case study of a patient who had refractory ibd symptoms and recurrent infections who was found to have xiap deficiency and mefv variant mutations. methods: a 37 year old male presented at age 16 with recalcitrant ibd unresponsive to multiple medications including steroids, mesalamine, azathioprine, infliximab, adalimumab, methotrexate, and vedolibumab. in addition, he developed severe infections from a combination of immune dysregulation and immunosuppressant medications. currently he is on ustekinumab but still has severe abdominal symptoms. patient does not have a history of hemophagocytic lymphohistiocytosis (hlh). family history was significant for ibd in both his mother and sister. he has had persistent lymphopenia which ranged between 210 to 838 cells/ mm3. t/b/nk panel showed decreased cd3 t cells (596 cells/mm3) with normal cd4, cd8, and cd4/cd8 ratios. b and nk cells were normal in quantity. t cell antigen/mitogen assays showed normal response to all mitogens (pha, pwm, con a) and most antigens (tetanus, candida, hsv, vzv, adv) but low response to cmv antigen. igg was elevated at 2,750, but iga, igm, and ige were normal. his ebv dna pcr is negative. results: exome sequencing revealed a novel xiap hemizygous variant at position c.693g>a (p.=?) of unknown significance and a mefv heterozygous variant. functional testing performed at medical college of wisconsin showed no expression of xiap on lymphocytes and a defect in nod2 pathway. given the xiap deficiency, bone marrow transplant was discussed as an option for refractory ibd and prevention of hlh. rituximab was also offered to decrease the possibility of hlh. currently the patient is in the decision making process of both treatment options. conclusions: genetic evaluation with clinical exome in early onset refractory ibd, family history of ibd, and recurrent infections demonstrated a novel mutation in the xiap gene with possible contribution of mefv variant as well. the absence of xiap protein expression and abnormal functional assay of the nod2 pathway confirm the pathogenicity of the mutation. identification of this genetic variant will help guide future therapeutic options and prognosis for this patient. introduction/background: x-linked agammaglobulinemia (xla) is a primary immunodeficiency disease caused by mutations of bruton tyrosine kinase (btk), which is essential in b cell maturation. xla is typically associated with bacterial infections of upper and lower respiratory systems, enteritis and increased risk for malignancies. objectives: to present the evolution and treatment of a complicated flexispira (helicobacter bilis) infection with delayed diagnosis in an xla patient. methods: clinical and laboratory features of a patient with xla evaluated at the national institutes of health. results: a 29-year-old male patient with xla presented for initial evaluation of indurated lower extremity and torso lesions. he was diagnosed with xla at age 2 years secondary to bacterial sepsis pneumonia and empyema. after starting ivig at age 2, he was well without significant infections except for recurrent otitis media. at 12 years of age, he developed right leg edema below the knee, which progressed to patchy skin thickening and discoloration. a tissue biopsy at 15 years of age revealed marked fibrous thickening of the subcutaneous septum with diffuse infiltrate of eosinophils with negative cultures for bacterial, fungal, and mycobacterial infections and thought to be consistent with eosinophilic fasciitis. mri demonstrated infiltration in the superficial and deep muscle compartments with fibrosis. symptoms persisted despite empirical treatment with iv antibiotics and steroids. at 16 years of age his left leg became involved with similar findings, while under treatment including multiple immunosuppressants (dapsone, methotrexate, tacrolimus, hydroxychloroquine, iv cyclophosphamide, remicade, cytoxan, and enbrel) targeting eosinophilic fasciitis. at age 28, he developed ulcerations over the left shin and ankle and was diagnosed with chronic multifocal osteomyelitis based on mri findings. physical exam was notable for bilateral leg swelling below the knees with woody appearance and induration, hyperpigmentation, and tenderness. indurated and nodular lesions without discoloration were noted above the waist line, on right forearm and above right nipple. skin biopsies of right lower extremity and right forearm were positive for numerous spirochetal-like organisms with warthin-starry stain. treatment was initiated with meropenem and gentamicin. gentamicin was discontinued due to vestibular ototoxicity six weeks later and doxycycline was added. initial response was followed by worsening of symptoms and intolerance to treatment, which led to the addition of tigecycline and azithromycin. with continued progression, seven months into initial evaluation, chloramphenicol and nitazoxanide were added to the regimen. due to the persistence of flexispira organisms on skin biopsy warthin-starry stains, fresh frozen plasma (ffp) was introduced four months later (11 months after initial evaluation) as an adjunctive treatment. cultures performed at the cdc were negative; however, pcr and sequencing resulted in identification of helicobacter bilis. 6 units of ffp was infused weekly for over three years, then reduced to every two weeks, with goal igm levels > 40mg/dl. subsequently, labs including cytopenias, inflammatory markers, and immunoglobulins improved as well as a negative warthin-starry stain. symptoms have improved with almost complete resolution of findings with only residual small areas of discoloration over both lower extremities. conclusions: xla immune deficiency is associated with flexispira (helicobacter bilis) infections, with typical appearance of discoloration and induration, which may evolve to osteomyelitis due to delayed treatment. although typically observed over the lower extremities, immunosuppressive treatment may lead to further expansion above the waist line. approach to therapy with weekly to bimonthly ffp infusions in addition to antibacterial treatment has proven to be beneficial in controlling the infection. higher igm levels resulting from the ffp may also provide antibacterial effects. introduction/background: x-linked severe combined immunodeficiency (scid) is a well described primary immunodeficiency associated with mutations in the common gamma chain. patients with x-linked scid classically present with profoundly low or absent t cells and nk cells with a variable number of b cells. the lymphocytes that are present typically have a proliferation index <10% control when stimulated with mitogens and antigens. patients must undergo corrective therapy with bone marrow transplant (bmt) or gene therapy to avoid the life-threatening infections that are associated with the nearly absent adaptive immune system. objectives: a 2-week-old boy presented to childrens national immunology clinic for initial evaluation of critical result on newborn screen. methods: targeted partial exome sequencing was performed on a 7-dayold patient who was picked up via trec assay on the maryland newborn screen. flow cytometry was completed at childrens national and proliferation studies completed at cincinnati childrens hospital diagnostic immunology lab. results: flow cytometry revealed markedly decreased lymphocytes with nearly absent cd8+ t cells and low cd4+ t cells with a r e l a t i v e i n c r e a s e i n c d 4 + c d 4 5 r o t c e l l s ( r a t i o cd45ra:cd45ro: 18%:12%). the b and nk cells were within the reference range for age. mitogen proliferation studies showed a mild decrease to pha and normal responses to pokeweed and cona (35587 cpm, 40924 cpm, and 82651 cpm, respectively). partial exome sequencing revealed a hemizygous nonsense substitution in il2rg (c.982c>t, p.r328) . maternal engraftment accounted for 3% of the t cells. the patient was started on prophylaxis with ivig, bactrim, and fluconazole with the plan to proceed with bone marrow transplant. as patient approached bmt maternal engraftment became absent in whole blood and repeat proliferation studies revealed normalization of the response to pha (stim index) with continued normal responses to cona and pokeweed. the patients flow cytometry values and ratios remained unchanged. patient completed a reduced intensity preparative regimen of busulfan, fludarabine and alemtuzumab prior to receiving his 8/8 matched unrelated donor bone marrow transplant. conclusions: it remains to be determined why initial proliferation studies showed >10% function with improvement over time in a patient with a well-described genetic mutation causing scid (187) director, division of intramural research, nih/niaid introduction/background: the advent of next-generation sequencing (ngs) has led to a proliferation of newly discovered genetic diseases and expanded phenotypes of known immunodeficiencies. availability of specific gene panels or whole exome sequencing (wes) with targeted analysis based on broad phenotypes, coupled with clinicians increasing awareness has led to higher utilization of ngs. published reports from high throughput sequencing labs indicate exome analysis identifies causative mutations in only 20-30% of probands. results: we have seen several patients who underwent high quality ngs in whom causative mutations were not identified. two separate families with multigenerational histories of leukemia, aplastic anemia, myelodysplastic syndrome, and cytopenias suggestive of gata2 deficiency had myeloid gene panel screens at commercial labs without causative mutations identified. targeted gata2 sequencing in the first family identified a novel change in gata2, c.1017g>t, p.l339l. cdna analysis demonstrated this synonymous variant resulted in aberrant splicing leading to a frameshift and premature termination. the wes bioinformatics pipeline failed to recognize the splice mutation. in the second family, pcr amplification spanning the 2 terminal exons revealed a shortened pcr product with a 426 base deletion fully encompassing the penultimate exon and leading to a 42 amino acid in-frame deletion. the deletion spanned all capture probes for the exon resulting in only the wild-type allele being captured and sequenced. additionally, capture kits targeting only coding regions of genes fail to capture deep intronic mutations such as those seen in gata2 (hsu, 2013) or in the 5 untranslated region of ikbkg, encoding nemo, (mooster, 2014; hsu, submitted) . lastly, even with good capture and sequencing, the presence of pseudogenes may confound downstream sequence alignment as seen in ncf1, encoding p47phox preventing recognition of disease causing mutations. conclusions: with ngs becoming more widely available as a clinical diagnostic tool, it is important to remember that wes results, unlike many laboratory tests, are not binary. inadequate bioinformatics pipelines, deletions, intronic or untranslated mutations, and pseudogenes can all mask the presence of causative mutations. targeted panel captures or analysis will miss novel genes. astute clinicians need to recognize the limitations of the current technology and pursue alternate assays when suspicions warrant. a cell based assay for the detection of autoantibodies to il-17 in human serum. matt phillips, phd 1 , vijaya knight, md, phd 2 1 senior scientist, national jewish health 2 director of immunology and complement adx labs, national jewish health introduction/background: patients with chronic candida infections are typically deficient in some aspect of il-17 signaling. one mechanism, which has recently come to light, is through the production of il-17 autoantibodies, particularly in patients who already suffer from specific autoimmune diseases. objectives: our objectives are to develop a diagnostic assay to accurately and easily detect il-17 autoantibodies in patient serum. methods: we developed a cell-based reporter assay using hek blue il-17 cells (invivogen) to detect the ability of patient serum to block il-17 receptor signaling. once stimulated with il-17, the cells secrete alkaline phosphatase (ap) into the surrounding media which is detected by invivogen hek blue media and an absorbance reader. addition of serum containing blocking antibodies inhibits secretion of ap. results: we were able to demonstrate, using a single patients serum with known il-17 autoantibodies, the inhibition of il-17 signaling in hek blue il-17 reporter cells. we further characterized the sensitivity of our assay with a commercially available anti-il-17 monoclonal and found it to be sensitive to between 1.4 x 10-6 m and 6.7 x 10-7 m. conclusions: loss of il-17 signaling can lead to problematic immune deficiencies including difficulty in clearing extracellular pathogens such as candida. some people who have an immune deficiency in the il-17 pathway may have developed autoantibodies to il-17 and thus have difficulty generating an appropriate immune response. we have developed a relatively low maintenance, cost effective, and simple test for detecting il-17 autoantibodies in human serum. alternations in repertoire of t and b cell subsets in patients with partial recombination activating gene (rag) deficiency with autoimmunity and history of viral infections introduction/background: patients with partial deficiency of recombination-activating genes 1 or 2 (rag1/2) can present with a wide spectrum of primary immunodeficiencies including combined immunodeficiency with granuloma and/or autoimmunity (cid-g/ai). prior case reports have highlighted alterations in b and t cell compartments; however comprehensive characterization of t and b cell receptor repertoires of lymphocyte subsets regarding diversity and autoreactivity has not been reported. objectives: defects in v(d)j recombination due to rag deficiency results in a skewed t and b cell repertoire that may be further modified by viral infections and promote inflammatory or autoimmune phenotype. methods: peripheral t and b cell compartments were sorted from two patients with combined immunodeficiency secondary to hypomorphic rag1 and rag2 mutations. b cells were stimulated with cd40l, cpg and il-21 to transition to antibody secreting cells (ascs), mimicking viral infection. repertoire analysis and single cell cloning of bcr heavy and light chain variable regions from sorted b cell populations has been performed. repertoire of t cell subsets (treg and follicular helper) were also examined results: we noted skewing towards proximal j usage in all b cell compartments (mature naïve, marginal zone, cd21-/low and memory) of two rag deficient patients compared to healthy controls. b cell clones with v4-34 v genes with low rate of somatic hypermutation expanded during b cell development. after in vitro stimulation mature naïve b cells from rag deficient patient were capable of transitioning to antibody secreting cells and enriched for polyreactivity to dsdna, insulin, lps and ifn cytokine (25 to 36%) compared to healthy control (5 to 14%). in connection to altered b cell compartments, restricted repertoire of regulatory t cells and an expanded and skewed follicular helper t subset were detected. conclusions: our data indicate that patients with partial rag deficiency have skewed t and b cell subsets that can further be altered towards antibody secretion and polyreactivity after stimulation such as viral infections. chief of the division of allergy and immunology, division of allergy immunology, the childrens hospital of philadelphia, philadelphia, pa usa introduction/background: the clinical features of 22q11.2 deletion syndrome include virtually every organ of the body. t cell lymphopenia, as a consequence of thymic hypoplasia, is the most commonly described immunologic feature and is most prominent in childhood. later in life, t cell exhaustion may be seen and secondary deficiencies of antibody function have been described in patients with 22q11.2 deletion syndrome. objectives: the role of deletion breakpoints in determining 22q11.2 deletion syndrome immunophenotype is unknown. in this study, we examined the effect of 22q11.2 deletions with and without tbx1 on lymphocyte counts. methods: lymphocyte counts were compared between 52 total 22q11.2 patients with tbx1-containing deletion (a-b, a-c, a-d deletions), and a total of 8 patients with tbx1-noncontaining deletion (b-d, c-d, d-e, d-f deletions). lymphocyte counts of patients with 22q11.2 deletions were compared to a set of 6 patients with a 22q11.2 duplication including the tbx1 locus. lymphocyte subset counts for each group were analyzed by t-test. results: cd3 counts were significantly lower in the tbx1-deleted cohort compared to the other two cohorts (mean 2169 cells/mm3 in tbx1 deleted cohort, 3709 cd3 cells/mm3 in the non-tbx1 deleted cohort, and 3657 in the duplication cohort, p<0.01 for all). similarly, cd4 counts were lower in the tbx1-deleted patients compared to the other two cohorts. there were no significant differences in cd8, cd19, and nk cell counts between the three cohorts. conclusions: these represent the first data to examine t cell counts in 22q11.2 deletion syndrome patients with different breakpoints. our data highlights an important role for tbx1 or other genes in the a-b region in regulating t cell production. paracoccidioidomycosis associated with a heterozygous stat4 mutation and impaired ifn-immunity introduction/background: mutations in genes affecting ifn-immunity have contributed to understand the essential role of this cytokine in the protection against intracellular bacteria and fungi. however, inborn errors in stat4, which controls il-12 responses, have not yet been reported. objectives: to determine the underlying genetic defect in a family with a history of paracoccidioidomycosis (pcm) disease. methods: genetic analysis was performed by whole-exome sequencing and sanger sequencing. stat4 phosphorylation and translocation from the cytosol to the nucleus, as well as ifnrelease by patient lymphocytes were assessed. the effect on stat4 function was evaluated by site-directed mutagenesis using a lymphoblastoid b cell line (b-lcl) and u3a cells. microbicidal activity of patient monocytes/macrophages was also analyzed. results: a heterozygous missense mutation, c.1952 a>t (p.e651v) in stat4 was identified in the index patient and her father. patients and fathers lymphocytes showed reduced stat4 phosphorylation and nuclear translocation as well as impaired ifn-production. in accordance, b-lcl and u3a cells carrying the stat4 mutant displayed reduced stat4 phosphorylation. patient's and father's pbmcs and macrophages (alone or in the presence of t cells) displayed impaired fungicidal activity compared with those from healthy controls that improved in the presence of recombinant human (rh) ifn-, but not rhil-12. conclusions: our data suggest autosomal dominant stat4 deficiency as a novel inborn error of il-12-dependent ifn-immunity associated with susceptibility to pcm disease. profound b cell lymphopenia in gof-stat1 that improves post ruxolitinib associate professor, university of south florida -johns hopkins all childrens hospital introduction/background: subjects with gain of function signal transducer and activator of transcription (gof-stat1) mutations have a variable clinical phenotype including combined immunodeficiency (cid). ruxolitinib, a janus kinase 1/2 inhibitor has been successful at treating immune dysregulation in subjects with gof-stat1. two subjects with profound b cell and/or t cell lymphopenia as a major manifestation are described, one of which was successfully treated with ruxolitinib. objectives: to discuss gof-stat1 mutations, their effect on the immune system, and the potential benefit of ruxolitinib in these subjects. methods: retrospective chart review was performed. results: subject 1 (c.494a>g) is a 21 year old male with a history of recurrent shingles, chronic mucocutaneous candidiasis (cmc), pneumocysitis jiroveccii pneumonia, varicella zoster meningitis, severe enteropathy, cerebral aneurysm and lymphoproliferation, and autoimmune hypothyroidism. he has profound lymphopenia predominantly affecting t and b cells (cd3+ 456 cells/ul, cd4+ 104 cells/ul, cd8+ 296 cells/ul, cd56+ 42 cells/ul, cd19+ 0 cells/ul). subject 2 (c. 1053g>t) is a 12 year old male with a history of severe cmc, recurrent pneumonia, enteropathy, and autoimmune thyroiditis. he had severe b cell lymphopenia (cd3+ 2168 cells/ul, cd4+ 1461 cells/ul, cd8+ 589 cells/ul, cd56+ 47 cells/ul, cd19+ 24 cells/ul). treatment with ruxolitinib 12.5mg bid led to clinical improvement of enteropathy and increased b cell counts in subject 2 (cd19+ 124 cells/ul). ruxolitinib has not yet been initiated in subject 1. both subjects were treated with anti-microbial prophylaxis and immunoglobulin supplementation. conclusions: combined immunodeficiency with variable degrees of b and t cell lymphopenia and hypogammaglobulinemia can be profound in subjects with gof-stat1 mutation. despite proper anti-microbial prophylaxis, this immunodeficiency can lead to severe infections. in addition to treating the autoimmune and immune dysregulatory features, treatment with ruxolitinib can improve the cid present and potentially reduce infectious susceptibility. igg4-related disease (igg4-rd), its common mimickers and response to anti-il5-(reslizumab) treatment rachel eisenberg, md 1 , arye rubinstein, md-phd 2 1 fellow in allergy and immunology, montefiore medical center 2 chief division of allergy and immunology, albert einstein college of medicine and montefiore hospital, bronx, ny, usa introduction/background: we describe a complicated case of igg4 related disease (igg4-rd) both in its presentation and novel treatment objectives: to review the common mimickers of igg4-related diseases which often lead to delayed diagnosis and treatment. to discuss novel therapeutic treatments for igg4-related disease results: a 70-year-old woman with a history of thyroid disease, sicca symptoms, lipodystrophy, relapsing parotid enlargement, asthma and erdheim chester syndrome initially presented with recurrent bacterial and fungal sinusitis despite multiple sinus surgeries. immunologic workup was notable for lymphopenia of 300/ml, cd4 count of 132 (677-1401 cells/ul normal range) and elevated igg4 of 511 (4.0-86.0 normal range). imaging was notable for nasal septal perforations and hypoplastic maxillary sinuses. there was high suspicion for igg4 disease however the patient was lost to follow up during which time she developed cachexia, eosinophilic pleural effusions (80% eosinophils), lung mass and a parotid mass with predominant t cell infiltrate misdiagnosed as follicular lymphoma. features consistent with igg4-rd included >50% ratio of igg4/igg and a predominant t cell infiltrate a biopsied lung mass showed igg4 plasma cell >50/hpf also consistent with igg4-rd. bone marrow biopsy was within normal limits. the patient was treated with rituximab, an effective treatment for igg4-rd. on treatment her igg4 levels normalized, however she developed recurrent large eosinophilic lung effusions requiring repeat drainage. fractional exhaled no (feno) was elevated to 86ppb. she was started on reslizumab at a dose of 5mg/ kg resulting in marked improvement in her respiratory status along with normalization of peripheral eosinophilia and reduction of feno to the normal level of 12ppb. conclusions: igg4-rd is a fibro-inflammatory condition which can affect any organ system and is diagnosed via tissue histology showing igg4 positive plasma cells and a typical morphologic pattern. this case outlines the common mimickers of igg4-rd often leading to a delayed diagnosis. before the final diagnosis of ig4-rd was made by us, the patient carried multiple diagnoses including: thyroid disease, recurrent parotid enlargement and seronegative sjogrens. these diagnoses in hindsight may have been mikuliczs syndrome, kuttners tumor and/or riedels thyroiditis which are common manifestations of igg4-rd. chronic sinusitis, atopic diseases, peripheral eosinophilia and destructive osseous lesions as noted in our patient are seen in up to 40% of patients with igg4-rd. destructive bony lesions and eosinophilia can mimic granulomatous polyangiitis, which was ruled out in our patient. her cachectic appearance and diagnosis of lipodystrophy can be explained by destruction of osseous tissue in the craniofacial skeleton which was later confirmed on imaging. lymphoid inflammatory infiltrates are commonly seen in igg4-rd and are can be misdiagnosed as a follicular lymphoma as in our case. a novelty in this case is the successful treatment with reslizumab targeted at the eosinophilic component of the disease. on reslizumab our patients asthma was for the first time controlled, pleuritis improved, fractional exhaled no (feno) normalized and her cachexia is improving. treatment with reslizumab should be considered in patients with igg4-rd who manifest with eosinophilic respiratory disease. introduction/background: ikbkb deficiency (c.1292dupg in exon 13) is a rare autosomal recessive form of severe combined immune deficiency (scid) originally described in canadian infants of northern cree descent. ikbkb scid is characterized by normal lymphocyte development, but impaired t-cell activation, along with innate immune defects. objectives: to report the clinical presentation, immunologic phenotype, and outcomes for patients with confirmed or suspected ikbkb scid due to this founder mutation. methods: we retrospectively reviewed hospital records dating back to 1973 of patients with confirmed homozygous ikbkb mutations, as well as patients suspected to be affected due to their clinical presentation and family relations to molecularly confirmed cases. results: fifteen patients were included. they presented early in life (average age 2 months) with invasive and disseminated viral, bacterial, mycobacterial, and fungal infections. patients had concurrent and multiple infectious organisms, with a notable predilection for candida, gram negative organisms, and mycobacteria. four patients in our cohort received bcg vaccination at birth, resulting in fatal disseminated m. bovis infection in all, and 2 additional patients succumbed to atypical mycobacterial infection following hematopoietic stem cell transplant (hsct). one newborn was identified in 2016 through new initiatives for targeted newborn screening for the mutation. immunologic features at presentation included normal to elevated lymphocyte counts with normal to elevated cd3, cd4, and cd8 t cells. when tested, response to pha varied from absent (1/10), to low (4/10), to normal (5/10). most patients had hypogammaglobulinemia, most often of the igg isotype (10/14). six had assessment of trec levels, and all had values above thresholds for screening programs. eight patients died before they could undergo hsct, and 6 received transplants in the setting of ongoing severe, lifethreatening infections. only 1 patient underwent hsct prior to the onset of infection. in our cohort, there are only 2 long-term survivors. conclusions: ikbkb deficiency is a severe form of scid with early onset of invasive and disseminated multi-organism infection. the immunologic phenotype is characterized by normal to elevated lymphocyte numbers which do not meet pidtc criteria for scid, variable (and sometimes normal) mitogen response, normal trec levels, and low igg levels. the disease is universally fatal without hsct, however, conclusions regarding efficacy and long-term outcomes of hsct are uncertain given the small sample size. immune -dysregulation mimicking systemic lupus erythematosus in a patient with lysinuric protein intolerance introduction/background: lysinuric protein intolerance (lpi) is an inherited aminoaciduria caused by defective amino acid transport in epithelial cells of the intestine and kidney due to bi-allelic, pathogenic variants in slc7a7. the clinical phenotype of lpi includes failure to thrive and multi-system disease including hematologic, neurologic, pulmonary and renal manifestations. individual presentations are extremely variable, often leading to misdiagnosis or delayed diagnosis. here we describe a patient that presented with suspected immunodeficiency in the setting of early-onset systemic lupus erythematosus (sle), including renal involvement, who was subsequently diagnosed with lpi post-mortem. objectives: describe a clinical a patient with lysinuric protein intolerance that presented as early-onset systemic lupus erythematosus (sle), including renal involvement and primary immunodeficiency. methods: after informed consent was obtained, dna samples were obtained from the proband and his parents. trio whole exome sequencing was performed to identify a cause of early onset autoinflammation resembling systemic lupus erythematosus. results: the male proband had a history of failure to thrive starting at 12 months of age, recurrent bacterial otitis media, and one episode of severe bacterial pneumonia requiring hospitalization. he presented at 30 months of age with multifocal pneumonia, anemia (hgb 8.2 mg/dl) and mild thrombocytopenia. initial laboratory studies revealed low albumin (2.8 mg/dl), elevated ldh (718), and mild hepatomegaly. renal and liver function testing was initially normal. immunologic evaluation for suspected primary immune deficiency showed normal immunoglobulin titers, low c3 (60) and low c4 (6.3). lymphocyte phenotyping revealed low b cell counts (0.6% of total lymphocytes) with t cells and nk cells within the normal range. despite antibiotic therapy, the patient worsened, developing fevers, a generalized erythematous rash, edema and nephrotic syndrome with oliguria. renal biopsy uncovered glomeruli with accentuated, global thickening and diffuse, peripheral capillary loops, as well as focal spiculated defects, there was endothelial swelling and other signs of acute damage including epithelial flattening, adluminal irregularity and extensive intraluminal proteinaceous detritus. endocapillary proliferative lesions, extracapillary crecents or tubular atrophy was not observed. immunofluorescence studies were positive for c3, igg and c1q granular deposits, mainly at the mesangium, interpreted as lupus nephropathy. endothelial swelling and massive, subepithelial electron-dense deposits with spike formation from the basement membrane were noted on electron microscopy, mimicking a stage ii membranous pattern of injury. autoantibodies included ana (1:320), anti-dsdna, smith, ssa and rnp were positive in agreement with the diagnosis of sle. immunosuppressive therapy with high dose iv corticosteroids and cyclophosphamide was initiated. despite this, the patient developed pancytopenia, elevated ferritin levels, increased triglycerides, and low fibrinogen. bone marrow biopsy displayed erythrocyte phagocytosis by macrophages, confirming a diagnosis of hemophagocytic lymphohistiocytosis (hlh). the patient subsequently died despite aggressive immunosuppression with high dose methylprednisolone and high dose iv immunoglobulin and dialysis. samples were collected from the deceased patient and his parents for research whole exome sequencing. trio analysis identified compound heterozygous missense variants in slc7a7. ammonia levels were not evaluated during the patients hospitalization. conclusions: lysinuric protein intolerance is a severe metabolic disorder that can present with protean systemic features including primary i m m u n o d e f i c i e n c y. i m p a i r e d l y m p h o c y t e f u n c t i o n , hypocomplementemia, immune-mediated glomerulonephritis, autoantibodies, and hlh are known complications of lpi. exactly how ineffective amino acid transport triggers these systemic inflammatory features is not yet understood. lpi should be considered in the differential diagnosis of early-onset sle, particularly in the absence of response to immunosuppressive therapy. director, national institute of immunohaematology introduction/background: chronic granulomatous disease (cgd) is a primary immunodeciency disorder with recurrent pyogenic infections and granulomatous inflammation resulting from loss of phagocyte superoxide production. mutations in any one of the five structural genes of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase complex viz. cybb and cyba encoding for membrane bound gp91phox and p22phox; and ncf1, ncf2, and ncf4 encoding for cytosolic components p47phox, p67phox, and p40phox respectively, have been found to cause cgd. the relative incidence of these gene defects varies significantly depending on the ethnic background of the population. identification of molecular defect is important for patient management as well as for prenatal diagnosis in the affected families. the present study was aimed at studying the pattern of underlying genetic defects in a cohort of indian patients affected with cgd. objectives 1.to identify the underlying genetic defect in patients with chronic granulomatous disease in india 2. clinical,immunological and molecular characterisation 3. to utilise this information for genetic counselling and prenatal diagnosis of the affected families methods: eighty-seven (n=87) patients with abnormal nbt and dhr were included in this study. in case of male patients, mothers were first screened for carrier status to rule out x-linked cgd (xl-cgd). those patients where mother is not showing mosaic pattern were suspected for autosomal recessive cgd (ar-cgd) and were screened for the ratio of ncf1 gene to pseudo ncf1 gene by genescan analysis. additionally, evaluation of nadph oxidase components expression by flow cytometry also helped us to determine the underlined genetic defect and it is validated by dna sequencing of respective genes. results: eighty-seven patients were molecularly characterized to identify disease causing mutation which includes: 12 novel mutations in cybb, 1 in cyba, 2 in ncf2 gene in our cohort. 29.8% (n=26) of the patients belonged to xl-cgd. 58.6% (n=51) of the patients are suspected to have ncf1 gene defect among which; homozygous delgt mutation was identified in 39 patients. 6.8% and 4.5% patients showed abnormal p22phox and p67phox expression suggesting defect in cyba gene and ncf2 gene respectively. spectrum of mutations involve: 41% of delgt mutations, 15% nonsense, 14% missense, 10% deletion, 2% insertion, 14% other than homozygous delgt mutations. male to female ratio is 1.87:1. consanguinity is noted in 30% of the patients. conclusions: despite the male predominance ar-cgd is more common (70%) as compare to xl-cgd (30%) in this cohort of indian patients, which is distinct from the western data. 17% are the novel mutations suggesting, a wide heterogeneity in the nature of mutations in indian cgd patients. flow cytometric evaluation of nadph oxidase component is used as a secondary screening test to identify cgd sub-group. molecular characterisation of cgd genes was not only used in the confirmation of diagnosis but also in genetic counselling and pre-natal diagnosis in affected families. novel nlrc4 gain-of-function mutation presenting with neonatal enterocolitis and autoinflammation, with positive clinical response to rapamycin and anakinra. senior investigator, viral immunology section, national institute of neurological disorders and stroke 8 senior investigator, translational neuroradiology section, national institute of neurological disorders and stroke 9 staff clinician, neuroimmunology clinic, national institute of neurological disorders and stroke 1 0 staff clinician, laboratory of clinical immunology and microbiolology, niaid, nih introduction/background: cytotoxic t-lymphocyte antigen-4 (ctla-4) is an essential negative regulator of the immune response and its function is critical for immune homeostasis. uni-allelic mutation in the ctla4 gene leading to reduced function or expression of ctla-4, termed ctla-4 haploinsufficiency, can lead to systemic immune dysregulation with wide spread clinical disease, but with variable clinical penetrance. the neurological manifestations of ctla-4 haploinsufficiency are not known. objectives: to perform detailed phenotyping of the neurological manifestations of ctla-4 haploinsufficiency. methods: a retrospective review and prospective collection of clinical, imaging, cerebral spinal fluid, and pathological specimens was performed in a cohort of genetically confirmed patients (n=50) with ctla4 mutations who are followed at the national institutes of health. neurological symptoms and exams were collected on patient visits and from historical records. the data collected included 289 brain mris and 53 spinal cord mris that were visually inspected for evidence of inflammation. cerebral spinal fluid values were obtained from 14 patients including flow cytometry in 10 patients. pathological tissue from brain biopsies of inflammatory lesions was examined from 10 patients. results: central nervous system (cns) inflammation was found in 14/50 (28%) of the cohort. common clinical manifestations from the 14 patients with cns inflammation were headaches 13/14 and seizure 8/14. focal deficits were rare. mri findings included contrast enhancing neuroinflammatory lesions in the brain 14/14, brainstem/cerebellum, and spinal cord 8/12. figure a -c show representative inflammatory lesions. lesions were multifocal in 13/14 patients and 12/14 had recurrent inflammatory lesions on longitudinal follow up. lesions were, at times, extremely large, 5/14 with a lesion > 25 cm^3. leptomeningeal enhancement (lme) was seen in 10/13 patients and clearly preceded intraparenchymal lesion development in 3 patients. figure d shows a site of lme (green chevron) that develops into an intraparenchymal lesion (yellow chevron), mris are separated by 11 days. spinal fluid analysis showed a lymphocytic pleocytosis (mean 32 cells/mm^3) with the presence of oligoclonal bands in 6 patients. pathological features included a mixed cellular infiltrate, predominantly lymphocytes or plasma cells, with little evidence of demyelination or necrosis (figure e). conclusions: the neurological manifestations of ctla-4 haploinsufficiency include recurrent and, at times, severe neuroinflammation. however, even large lesions and lesions in eloquent anatomical locations had little to no focal clinical defects resulting in a striking clinical-radiological dissociation. future studies into the mechanisms of cns-related disease may reveal important information related to peripheral and central immune system functioning. conditioning with anti-cd45 immunotoxin in a mouse model of hypomorphic rag1 deficiency allows complete reconstitution of the immune system with lack of toxicity enrica calzoni, md 1 , cristina corsino, technician 2 , marita bosticardo, phd 3 , yasuhiro yamazaki, md phd 1 , hsin-hui yu, md phd 4 , lisa ott de bruin, md 5 , john manis, md 6 , rahul palchaudhuri, phd 7 , david scadden, md 8 , luigi d. notarangelo, md 9 treated mice and 80% in cd45-sap/200 treated mice. at sacrifice, in the bm we observed strong selective advantage for donor b cells at all developmental stages, but in particular in the most mature subsets, in both cd45-sap and cd45-sap/200 treated mice, rescuing the block in development at pre-b cell stage found in untreated f971l mice. donor engraftment in bm hsc reached levels around 70% and 80% in cd45-sap and cd45-sap/200 treated mice, respectively. donor chimerism in t and b cells in the spleen was also higher than 90% in both cd45-sap and cd45-sap/200 groups. in the thymus, full donor chimerism was achieved in both cd45-sap and cd45-sap/200 treated mice, starting at the dn4 stage and persisting at dp, sp4 and sp8. importantly, in both treatment groups, t cell development was corrected both in terms of subset distribution and absolute numbers to levels comparable to those of wt mice. finally, the thymic epithelial cell compartment was also fully reconstituted, with a normal number, distribution and maturation of both cortical and medullary thymic epithelial cells. conclusions: in conclusion, we show here that conditioning with cd45-sap immunotoxin, alone or in combination with 200rads tbi, is safe and leads to full reconstitution of the immune system in rag1 hypomorphic mice, suggesting that this conditioning regimen should be considered for testing in clinical setting. introduction/background: foxp3 is a key transcription factor for the maintenance of immune tolerance. foxp3 mutations result in dysfunction of foxp3+ regulatory t cells (tregs) causing immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome, a severe early onset autoimmune disease, which can be fatal if not promptly diagnosed and treated. our recent international study analyzing the longterm outcome in 96 i.e. patients of the two currently available treatments, pharmacological immune suppression and allogeneic hematopoietic stem cell (hsc) transplantation, showed poor long-term disease-free survival or overall survival limitations, respectively (barzaghi f. et al, jaci, 2017) . ipex syndrome is a good candidate for gene therapy as it has been demonstrated that reconstitution of wild-type treg cells can control the disease. however, foxp3 expression is highly regulated, and its safe and physiological expression in treg and teffector (teff) cells is challenging. lentiviral-mediated (lv) foxp3 gene transfer successfully converts ipex patients-derived cd4+ t cells into treg-like cells (cd4lv-foxp3 t cells) with stable suppressive capacity (passerini l. et al, sci transl med, 2013) . these ex vivo converted tregs are ideal as a short term cell-based therapy for ipex patients, but this approach does not reestablish regulated foxp3 expression in teff cells, that also likely contribute to the ipex pathology. thus, we are further characterizing cd4lv-foxp3 t cells and, at the same time, developing gene editing strategies for ipex, whereby autologous t cells or hscs are genetically modified or corrected, respectively, and reinfused into the patients. objectives: to provide more effective treatments for ipex patients, we are i) optimizing lv-foxp3 gene transfer in t cells to be suitable for clinical use, and ii) establishing a novel foxp3 gene editing in hscs and testing both approaches in preclinical models. methods: lv-foxp3 gene transfer can be obtained in cd4+ t cells activated polyclonally or in an antigen-specific manner. the vector construct is bidirectional, foxp3 expression is under the ef1a-promoter and the truncated form of ngfr, used as marker gene, is under cmv promoter. foxp3 gene editing is performed using a combination of crispr/cas9, a chemically modified sgrna targeting foxp3, and an aav6 packaged homologous donor dna template and the efficacy and safety of the resulting construct is tested in different cell types in vitro and in humanized mice. results: we demonstrate that cd4lv-foxp3 t cells can successfully be generated specific to different antigens. this result opens to new potential clinical benefit of cd4lv-foxp3 t cells with more safe and specific regulatory effect than polyclonal cd4lv-foxp3 t cells. we are currently adapting the protocol to optimal in vitro production for clinical use and assessing dose, survival and efficacy of the cd4lv-foxp3 t cells using different in vivo models. due to the wide distribution of identified mutations throughout the foxp3 gene, we have designed a gene editing strategy that uses homology directed repair to insert the coding sequence of the foxp3 gene at the start codon of the endogenous mutated foxp3 gene. this strategy permits regulated expression of the inserted wild-type, functional foxp3 protein in patient cells independent of the location of the downstream mutation. using this site-specific gene knock-in, we find that the system effectively targets expression of foxp3 in different cell types, namely tregs, teff cells and primary human cord blood-or bone marrow-derived hscs. gene editing of normal donor and ipex tregs and teff cells allowed us to test for regulated gene expression and for establishment of normal treg suppressor function and t cell proliferation upon activation. additionally, preliminary results demonstrate that gene edited hscs can be transplanted into nsg mice for long-term reconstitution. conclusions: our results show the feasibility of different gene therapy approaches for ipex syndrome. in addition, they suggest that cd4lv-foxp3 t cells, either polyclonal or antigen-specific, could be applied not only in ipex but also in immune mediated diseases of different origins. the results from the foxp3 gene editing support the use of crispr/ cas9 to treat ipex syndrome patients with autologous edited hscs. this gene editing approach may also be applied to treat other pediatric monogenic blood and immune disorders. human pi3kgamma deficiency with humoral defects and lymphocytic infiltration of barrier tissues andrew takeda, bs 1 , william comrie, phd 2 , yu zhang, phd 3 , paul tyler, bs 4 , koneti rao, md 5 , carrie l. lucas, phd 6 4 graduate student, yale university 5 clinician, niaid, nih 6 assistant professor of immunobiology, yale university introduction/background: the phosphatidylinositol 3-kinase (pi3k) signaling pathways play a key role in transducing signals from a diverse array of stimuli by producing the pip3 second messenger. class ib pi3k is primarily activated by g protein-coupled receptors (gpcrs), and this class is comprised of the p110gamma catalytic subunit in complex with the p84 or p101 regulatory subunit. in contrast to the class ia pi3k subunits, inherited mutations in the genes encoding the class ib subunits have not been described. objectives: given the leukocyte-restricted expression pattern of p110gamma, we hypothesized that mutations affecting this kinase may be found in cohorts of patients with rare immunodeficiency disorders. our objective was to identify such mutations and determine molecular, biochemical, and cellular derangements in patients with mutated p110gamma. methods: we used whole-exome sequencing of families to identify inherited gene mutations and determined the mechanistic basis of disease using biochemical assays to assess effects on protein function and cellbased assays to define functional defects with disease relevance. results: we identified a patient (here called a.1) harboring compound heterozygous mutations in pik3cg, the gene encoding p110gamma, who presented in early life with autoimmune cytopenias and eczema and, at the age of 9 years, developed cryptogenic organizing pneumonia and prominent t cell infiltration of the lungs. she also has a history of skin infections, lymphadenopathy/splenomegaly, eosinophilia, defective antibody production, and more recently, lymphocytic colitis. she inherited a frameshift pik3cg mutation from her mother and a missense mutation resulting in an r1021p amino acid substitution from her father. expression of p110gamma protein was lost, and stability of its p101 binding partner was reduced. despite defective t cell signaling responses to chemokines (i.e., gpcr stimulation), chemotaxis of patient t cell blasts in vitro was normal. intriguingly, the frequency of peripheral blood treg cells was low in patient a.1, and her cd4 t cells more frequently expressed the tissue-homing cxcr3 chemokine receptor. consistently, serum levels of cxcr3 ligands were elevated in patient a.1. moreover, we found augmented inflammatory cytokine production from m1-polarized macrophages differentiated from patient a.1 monocytes or from thp1 cells treated with p110gamma inhibitor or stably expressing pik3cg shrna. conclusions: we report the first human with loss of pi3kgamma activity and present her clinical presentation with notable t cell infiltration of barrier tissues. based on our analyses, we propose that loss of p110gamma activity in humans causes t cell-intrinsic effects of reduced tregs and increased tissue-homing propensity and the t cell-extrinsic effect of augmented inflammatory responses in macrophages. together, these consequences of p110gamma deficiency drive aberrant accumulation of t cells in lung and gut. introduction/background: pulmonary disease is a frequent complication across many primary immunodeficiencies (pidds), however its impact on the quality of life (qol) in pidds is not well characterized. objectives: to ascertain the types of infectious and non-infectious pulmonary complications occurring in pidds and to determine how these complications affect qol. methods: we analyzed the pulmonary complications, disability descriptions, and clinical status of 3610 subjects with pidds in the usidnet registry using descriptive statistics. karnofsky or lansky performance indices (n=1267) and promis29 qol data (n=120) were also analyzed. the t-test/mann-whitney test and chi square test were utilized to compare continuous and categorical variables, respectively. results: infectious pulmonary disease was reported in a majority of subjects (52.2%), most commonly pneumonia (42.3%) and bronchitis (17.9%). non-infectious pulmonary disease was reported in 30.8% of all subjects, most commonly asthma/reactive airway disease (21.7%), bronchiectasis (7.0%) and interstitial lung disease (4.2%). pulmonary insufficiency was listed as a cause of disability in 4.3% of all subjects with pidds, with highest rates of this disability in subjects with immune dysregulation (15.6%). lower karnofsky/lansky performance scores were observed in subjects with pneumonia, lung abscess, bronchiectasis, interstitial lung disease, and emphysema/copd as compared to without these disorders (p<0.001). promis29 qol metrics were largely similar among subjects with and without pulmonary disease, although physical function scores were significantly worse in those with copd/emphysema (mean= 37.5 +/-5.1) as compared to without (mean= 43.6 +/-8.4, p =0.007). promis29 physical function scores were also worse in subjects with non-infectious pulmonary disease (mean =40.3 +/-6.81) compared to those with infectious pulmonary disease only (mean =45.2 +/-8.53, p=0.046). a significantly greater percentage of patients with a history of copd/emphysema (24.0% vs. 8.95%) or interstitial lung disease (16.7% vs. 8.9%) were deceased as compared to those without a history of these disorders (p<0.0001). conclusions: both infectious and non-infectious pulmonary disorders cause significant morbidity in pidds and are associated with higher mortality in this population. infectious and non-infectious pulmonary complications were often associated with worse karnofsky/lansky scores while there was limited impact on promis29 qol measures. latin-american consensus on the management of patients with severe combined immunodeficiency, part 1: supportive measures during the time from diagnosis to definitive treatment. juan carlos bustamante ogando 1 , armando partida-gaytán 2 , francisco espinosa rosales 3 , lasid "consensus on scid" study group 4 1 pediatric allergy and clinical immunology specialist, clinical immunologist and researcher at primary immunodeficiency research unit, national institute of pediatrics 2 pediatric allergy and clinical immunology specialist, researcher at primary immunodeficiency research unit, national institute of pediatrics 3 pediatric allergy and clinical immunology specialist, president, fundación mexicana para niñas y niños con inmunodeficiencias primarias (fumeni) result in a majority of patients with late diagnosis, more comorbidities and reduced access to curative treatments. the interventions during such period are vital to keeping optimum health status to improve the probability of success of curative therapies. many interventions are not supported by clinical trials, are based mainly on clinical experience, and there are no clinical guidelines to standardize such treatments. objectives: to generate a consensus on the supportive care of patients with scid, from the diagnosis until a curative treatment is given, under a latin-american perspective taking into account particular challenges for our region. methods: in a first step, we gathered available information about scid diagnostic and therapeutic guidelines from two sources: a) literature search and b) personal communications with pid experts from europe and usa. next, we developed an expert consensus through a modified delphi technique (electronic and anonymous). we used google® forms® to gather the information and microsoft office excel® for the analysis of agreement through kappa coefficient and rounds concordance through repeated measures analysis of variance (anova). results: we gathered an expert panel of 34 subjects from 6 latin-american countries (argentina, brazil, chile, costa rica, mexico, and peru) including the primary centers caring for scid patients. we generated a document with 123 agreed diagnostic and therapeutic interventions grouped in 8 topic-domains (i.e. protective and isolation methods to decrease the risk of infections, antimicrobial prophylaxis, immunoglobulin treatment, immunizations, nutritional aspects, antimicrobial treatment, blood derivatives use, routine laboratory workup, imaging and other studies, conventional multidisciplinary approach). we also included 38 nonagreed interventions, but where relevant arguments are shared, to allow for particular clinical scenario decisions. conclusions: this is the first document of its type, and it intends to standardize clinical care of latin-american patients with scid, reduce disease burden and ultimately improve health outcomes. we see this effort as a starting point for the continuous improvement of our professional care to such patients and is intended to help as a tool not only for immunologists but for primary care physicians and other specialists involved in scid patient's care. this work will hopefully be published during 2018 as a lasid collaborative work, and it will help as a guide for clinicians caring for scid patients not only in latin america but in other world regions. also in the future, this consensus may be improved by collaboration from immunologists worldwide. no significant difference in hospitalizations one year before vs. year after treatment for prophylactic antibiotics (p=0.85) or igrt (p=0.07). baseline igg was higher in prophylactic antibiotics vs. igrt (770.6 vs. 914.4 mg/dl, p=0.03) . sex, severity of sad, igg subclasses deficiency, and lymphocyte counts were not significantly different between treatment groups. conclusions: prophylactic antibiotics are not inferior to igrt in preventing infections in some sad patients. while, clearly some patients with sad will need igrt, our date indicate that larger prospective studies are needed to identify patients who will benefit most from igrt vs prophylactic antibiotics alone. richard and barbara schiffrin presidents distinguished professor of microbiology and director, institute for immunology, university of pennsylvania introduction/background: t cell thymic development is dependent on signals received via the pre-tcr complex and we here report the first case of pre-tcr alpha (ptcra) autosomal recessive t cell immunodeficiency in an infant with a positive scid newborn screen (nbs). objectives: we sought to uncover the mechanistic links between ptcra mutations and immune dysfunction. methods: the patient was tracked clinically, with serial clinical immunophenotyping and t cell function testing. in addition, we performed deep immunophenotyping with mass cytometry and single cell rna sequencing to delineate the molecular circuitry underlying her immune phenotype. results: the patient presented with t cell lymphopenia and impaired response to mitogen stimulation. hsct was considered, but she did not meet clinical criteria and remained healthy, so she was watched closely on prophylaxis while awaiting genetic testing. response to serial mitogen stimulation remained between~20-100%, response to serial cd3/cd28 activation was normal and tcrv spectratyping was normal. whole exome sequencing revealed two mutations in ptcra. no prior human cases of ptcra deficiency have been published, but a mouse model bears a striking resemblance to this case (fehling et al, nature, 1995) , with elevated t cells and decreased t cells. her mitogen stimulation responses became persistently normal around 2 years of age with stable t cell lymphopenia, elevated t cells and normal switched memory b cells. anti-fungal prophylaxis was halted, and she remained on atovaquone alone with persistent t lymphopenia. she was able to mount an antibody response to rabies vaccine at 2.5 years and was weaned off scig replacement and is planned to initiate vaccination. deep immunoprofiling with mass cytometry (cytof) demonstrated a unique immunophenotype. single cell rna sequencing confirmed normal cd4 and cd8 tcr clonotypic diversity but increased clonotype diversity in t cells and increased and transcript levels. in addition, cd4 naïve, cd4 memory and cd8 naive t cells demonstrated both increased numbers of expressed genes and transcriptomic diversity, with altered cytoskeletal and tcr proximal signaling pathways across t cell subsets versus control. this may reflect a peripheral role for ptcra, a durable imprint of thymic signaling events mediated by ptcra, evidence of homeostatic proliferation or a combination of the above. conclusions: scid nbs led to identification of homozygous variants in ptcra causing a novel t cell immunodeficiency characterized by t cell lymphopenia, altered proximal tcr and cytoskeletal signaling and increased number of altered t cells. we will continue to pursue the mechanism of these mutations by developing ipsc and studying their t cell differentiation capacity in vitro, as well as further defining her immunometabolic phenotype. rag1 hypomorphic mouse mutants show partial preservation of thymocyte development but peculiar abnormalities of thymic epithelial cell phenotype introduction/background: the recombination-activating gene (rag) 1 and rag2 proteins are essential for v(d)j recombination. in the absence of these proteins, the development of b and t cells is blocked at early progenitor stages, resulting in severe combined immunodeficiency (scid). hypomorphic mutations in rag1, allowing residual activity, result in delayed-onset combined immunodeficiency with residual development of t and b lymphocytes, associated with autoimmunity and/or granulomas (cid-g/ai). objectives: to study in details the effect of rag1 hypomorphic mutations at the early stages of t cell development, we have generated 3 mouse models carrying mutations described in patients with cid-g/ai (r972q, r972w, f971l) (niaid animal protocol: lcim 6e). methods: we performed an extensive evaluation of the thymic phenotype in the 3 mouse models. results: the number of total thymocytes was found to be drastically reduced in all three models. however, two of these mouse models (r972q and f971l) retained a significant level of rag1 activity, and resulted in the development of mature t cells in the thymus, while the mouse model carrying the r972w mutation had minimal rag1 activity and presented a phenotype more similar to that of complete rag1 knockout mice. in r972w mice, almost all thymocytes were blocked at the double negative (dn)3 stage and there were virtually no mature t cells, as found in rag1 ko mice. on the other hand, r972q and f971l mice presented double positive (dp) and single positive (sp)4 and sp8 cells. the cross talk between t cells and thymic epithelial cells (tec) in the thymus is fundamental for the development and maturation of both types of cells. in rag1-/-mice, and consequently in the absence of mature t cells, tecs cannot complete their maturation, and the mtec subset is virtually absent. these results were also observed in the r972w mouse model. instead, in r972q and f971l mice, the residual rag1 gene activity allows development of a reduced number of mature t cells. although the number of tecs was markedly reduced in r972q and f971l mice, ctecs and mtecs were both present, but with an excess of ctecs. furthermore, mtecs were predominantly mhc-iihigh (mtechi), and only a minority of mtecs were mhc-iilow (mteclo) cells, the opposite of what found in adult wt mice. finally, mtechi cells from rag1 mutant mice were found to express aire to levels and frequencies comparable to those of wild-type (wt) mice. conclusions: our results show that tec in mouse models carrying rag1 hypomorphic mutations are affected both in terms of absolute numbers and in terms of subset distribution and maturation state. to further investigate the functional consequences of impaired cross-talk between thymocytes and tecs in rag1 mutant mice, we have performed rnaseq in sp4 and tecs sorted from r972q, f971l and wt mice. analysis of the gene expression profile in tec may thus provide novel insights in the mechanisms that govern normal and pathologic thymic t cell development. vedolizumab for autoimmune enteropathy in primary immunodeficiency: a case series of outcomes introduction/background: gastrointestinal complications are common in patients with primary immunodeficiency. infections are the leading cause, but autoimmune enteropathies including inflammatory bowel disease (ibd)-like colitis, sprue-like enteropathy, and nodular lymphoid hyperplasia (nlh) have been recognized in a subset of these patients. to date, there is no established treatment for these noninfectious disorders. vedolizumab is a humanized monoclonal antibody that binds to the alpha-4 beta-7 integrin, inhibiting the migration of memory tlymphocytes across the endothelium into inflamed gastrointestinal parenchymal tissue. it is fda approved as first-line therapy for inflammatory bowel disease. the safety and efficacy of treating autoimmune enteropathy with vedolizumab in patients with concurrent primary immunodeficiency (pid) has not previously been reviewed. objectives: to review the outcomes of a series of patients with hypogammaglobulinemia and autoimmune enteropathy following vedolizumab therapy methods: 7 patients (3 male, 4 female) at mount sinai with enteric biopsies demonstrating inflammatory enteropathy with t cell infiltrates have been treated with vedolizumab. results: five of the seven patients completed induction therapy. one patient was recently started on therapy. therapy was aborted in one patient who developed acute hepatitis during induction. another developed severe cytomegalovirus enteropathy, prompting discontinuation. two patients discontinued therapy due to response failure. at present, two patients remain on therapy at 12 months with symptomatic improvement. conclusions: vedolizumab was effective in 2 cases, but had no benefit or deleterious side effects in 4 subjects. its effectiveness in another patient is presently under investigation. introduction/background: ataxia telangiectasia (at) is an immunodeficiency most often associated with t cell abnormalities and abnormalities in serum immunoglobulin levels, primarily iga. there is a subset of patients with a hyper-igm phenotype, some with cutaneous granulomas, which may reflect a distinct clinical phenotype. a 5 yearold female presented for evaluation of concern for immunodeficiency because of frequent illnesses, presumed to be viral. she was found to have an ataxic gait, some speech delay, mild ocular and ear pinna telangiectasia, and an ulcerative rash on the left upper and right lower extremity. initial blood work showed elevated -fetoprotein levels (50 ng/ml), elevated serum igm (719 mg/dl), low igg (<75 mg/dl), and iga (0.9 mg/dl). objectives: to determine if the atm mutations in this patient are associated with perturbations in the frequencies, distributions and functions of b and t cell subsets which account for the observed phenotype. methods: next generation sequencing was used to identify the mutations in the atm gene. b and t cells were purified from the patients peripheral blood by positive selection. intracellular staining for foxp3 and t-bet was performed. b cells were activated in the presence of polyclonal f(ab)2 rabbit anti-human igm, multimeric, soluble, recombinant-human cd40l, gardiquimod (tlr7 agonist), or cpg (tlr9 agonist). the treg suppression assay was carried out by co-culturing cd4+cd25hicd127lo tregs and cd4+cd25cd127+ responder t cells at a 1:1 ratio in the presence of beads loaded with anti-cd2, anti-cd3, and anti-cd28 for 4.5 days. results: next generation sequencing revealed two pathogenic mutations in the atm gene, a novel mutation creating a premature stop codon [c.237dela,(p.lys79asnfs370)], and a nonsense mutation [c.3372c>g, (p.tyr124ter)]. proliferative responses of pbmc to mitogens (pha, cona, pwm) were reduced to roughly half of the control responses; the response to tetanus was normal whereas the response to c. albicans was absent. serum cytokine analyses demonstrated elevations in levels of tnf (14.5 pg/ml) and il-10 (7 pg/ml); levels of ifn, il-2, il-5, il-6, and il-12 were below the limits of detection. b cell abnormalities included markedly increased percentages of cd38locd21lo cells (40%) expressing t-bet and fas. activation of these cd21/low b cells through the b cell receptor, tlr7 and tlr9, and cd40 was decreased in response to all of the stimuli as evidenced by a lower percentage of b cells expressing the activation markers cd69 and cd86 relative to healthy control samples. the frequency of unswitched cd27+igd+ memory b cells was also increased (54%). among the naive b cells, the proportion of cd19+ cd27cd21cd10+igmhi transitional b cells that newly emigrated from the bone marrow (bm) was found to be diminished to 1.1% of the naive b cell compartment. in the t cell compartment, there was a decreased frequency of total cd3+ cells but normal absolute numbers of cd3+cd4+ t cells. there was also a decreased proportion of naive cd3+cd4+cd45rocd62l+ t cells and a striking increase in the cd3+cd4+cd45ro+ memory t cells (90%). this appeared to be largely attributed to the increased proportion of cd3+cd4+cd45ro+cd62l effector memory t cells (63%). the circulating t follicular receptor (ctfh) cell frequency in the patient was 5-fold higher (19%) than the average for healthy donors but icos expression levels were normal. treg frequency was decreased but suppressive capacity was not impaired. conclusions: the mutations in atm described here add to the growing understanding of the heterogeneity in degree and complex nature of the immunodeficiency seen in patients with at. these mutations resulted in perturbations in frequencies and distributions of normal and atypical b and t cell subsets, which can explain some immunologic aspects of the clinical phenotype in this patient. the immunophenotype seen here may also differentiate at patients with granulomas from those without cutaneous lesions. supported in part by grifols, the joanne siegel memorial fund, the dreizessen fund (to ewg), grants from niams t32 ar007107-41 (km) and niaid r01 ai071087 (em). introduction/background: a 10-month-old male presented with pancytopenia, b cell deficiency and developmental delay. he was born at 36 weeks with weight of 2.3kg. he was severely anemic with a hb of 7.4, and transfused on day 2 of life. he received hep b and bcg vaccines without complications. a month later he had a hb of 3.6 with a febrile illness. a bone marrow aspiration performed at 45 days, showed dyserythropoiesis without hemophagocytosis, and normal numbers of precursors. t and b cells were decreased. further evaluation with repeat bone marrow showed decrease in all 3 cell lineages. exome sequencing of the family showed homozygous variant in mysm1 (c.899_902delp. (lys300arg/s*11) omim: *612176) in the patient. both parents and hla-matched sister, were heterozygous for the same variant in mysm1. treatment consistent of replacement immunoglobulin, packed rbcs, and g-csf. there was no history of recurrent viral or severe bacterial infections except for 2-3 episodes of urinary tract infections, which were treated with antibiotics. physical exam revealed low set ears, sunken and wide set eyes, depressed nasal bridge, mild micrognathia, frontal bossing, and 1 cm x 1 cm cafeau-lait spot noted behind left knee. he was pancytopenic with a wbc count ranging 600/mcl to 6300/mcl, and anc ranging from 10/mcl to 4500/mcl (on intermittent g-csf). hb = 7.7 gm/dl requiring transfusions every 3-4 weeks, and platelet count was 91,000/mcl. b cell deficiency was confirmed with total b cell count of 36 cells/mcl. b-cell maturation was essentially normal. t cell counts were normal with ageappropriate distribution of naïve and memory t cells, and t cell function. there was normal t cell receptor repertoire diversity. objectives: to assess defect in dna repair using a flow cytometry-based assay in a patient with mysm1 deficiency. methods: patients with mysm1 deficiency are reported to have increased genomic instability. deb testing, and telomere length analysis revealed normal results. defects in the dna repair pathway were assessed using a flow cytometry-based assay measuring phosphorylation of atm (patm), smc1 (psmc1) and h2ax (gh2ax) without irradiation, or 1h or 24h after low-dose (2gy) radiation using a cs137 source. the analysis was performed in t, b and nk cells. results: the patient had higher patm and psmc1 in t cells compared to the experimental controls (hc) at 1h post-irradiation. also, the amount of gh2ax in nk cells was significantly higher than hc at 1h post-irradiation. interestingly, the patients b cells showed approximately 12% of b cells with constitutive gh2ax even without irradiation, and this subset increased slightly to 16% at 1h after irradiation. the mfi (amount) of gh2ax also increased at this time-point. at 24h post-irradiation, there was normal dephosphorylation in healthy control lymphocyte subsets. however, the patients t cells did not de-phosphorylate completely and showed higher residual patm, and psmc1 in both t and b cells. also, both t and b cells, at 24h, demonstrated a small subset of t cells (1%) with constitutive gh2ax without irradiation, which increased to 4% after irradiation. there was also an increase in gh2ax mfi in the irradiated sample. in b cells, 6% showed constitutive gh2ax without irradiation at 24h, and this increased to 37% after irradiation, with a corresponding increase in mfi. conclusions: in summary, this rapid flow analysis revealed defects in the dna repair pathway, including higher patm, psmc1 and h2ax phosphorylation in t, b and nk cells at 1h post-irradiation. at 24h, only t cells showed a residual subset with patm expression. but, psmc1, a downstream target of atm, revealed higher levels in t, b and nk cells at 24h post-irradiation. this assay, which allows lineage-specific analysis, permitted dissection of dna repair defects, in individual lymphocyte subsets revealing heterogeneity within the cell subset to radiation susceptibility. the practical benefit of this rapid multi-parameter flow assay is selection of appropriate conditioning regimen for hematopoietic transplantation, as was the case with this patient. this has significant practical implications for treatment of patients with radiosensitive immunodeficiencies. ctla-4 haploinsufficiency-associated inflammation can occur independently of t-cell hyperproliferation introduction/background: cd28 and ctla4 provide opposing proliferative signals to t cells. we identified an 18-year-old female subject (s1) with heterozygous deletions of cd28 and ctla4 and multi-organ inflammatory disease characterized by a lack of t cell infiltrates in affected organs. inflammatory disease was remarkably responsive to s1 ctla4-ig therapy. objectives: our goal was to characterize the immunologic consequences of combined deletion of cd28 and ctla4, specifically assessing t cell proliferation and treg function in comparison to patients with alps5associated ctla4 haploinsufficiency. we further sought to explain how this s1s inflammatory diseases could occur without a pathologic t cell infiltrate and why they were amenable to ctla4-ig therapy. methods: we performed phenotypic analyses of subject t cells and innate lymphoid cells (ilcs). we functionally characterized subject t cells. we created serum cytokine profiles. we stained and analyzed tissue biopsies. results: cd28 and ctla4 expression on s1 t cells were half that of control t cells. s1 t cells were hypoproliferative. s1 tregs were scarce and lacked suppressive function similarly to alps5 tregs. s1 tregs could suppress autologous t responder cells, likely due to their poor proliferative capacity. s1 colonic biopsies featured significantly fewer infiltrating intraepithelial lymphoid cells than biopsies from an alps5 patients. unlike alps5 patients whose colonic gland infiltrates were overwhelmingly t cells, s1 intraepithelial lymphoid cells were neither t cells nor b cells, suggesting the presence of ilcs. indeed, a greatly expanded population of type 3 innate lymphoid cells (ilc3) and prototypical ilc3 cytokines were identified in s1 peripheral blood. ilc3 frequency and cytokine levels decreased in response to treatment with ctla4-ig, corresponding with marked improvement in enterocolitis, hepatitis and pericarditis. conclusions: we report a novel genetic syndrome of combined cd28/ctla4 deletion and describe the immunolopathologic correlates of this disease. dual ctla4-and cd28-haploinsufficiency results in a phenotype of multi-organ inflammatory disease characterized by ilc3 expansion in the setting of t-cell hypoproliferation and quantitative and qualitative treg defects. our patients clinical response to ctla4-ig parallels published mouse studies and suggests the existence of additional stimulatory b7 receptor(s) preferentially expressed on ilc3s over conventional t-cell populations. diagnosis of radiosensitivity and dna repair defect in dna ligase iv deficiency with a rapid flow cytometry assay. introduction/background: dna ligase 4 deficiency (lig4-scid) is one of several monogenic defects affecting dna repair, and causing lymphopenia (t-b-nk+) and a radiosensitive scid (rs-scid) phenotype. the assignment of a timely diagnosis is vital in the management of patients with rs-scid. laboratory assessment of radiosensitivity is laborious, and utilizes fibroblasts (non-hematopoietic) or lymphoblastoid cell lines, and can take several weeks to months for results. objectives: we demonstrate for the first time, the application of a flow cytometric-based kinetic analysis of phosphorylated h2ax (h2ax) in lymphocyte subsets, especially nk cells, for the diagnostic assessment of lig4-scid. methods: simultaneous measurement of multiple dna repair markers phosphorylated (p) atm, smc1 and h2ax (h2ax) was performed by flow cytometry to assess dna repair defects in a 3-year-old korean female. the patient was evaluated for recurrent fevers, chronic respiratory tract infections, chronic diarrhea, and rash. genetic testing revealed compound heterozygous variants (nm_001098268, c.1341g>t, p.trp447cys and nm_001098268, c.1103a>t, p.asp368val) in lig4. functional assessment (phosphorylation) was measured in t and nk cells (b cells were absent), before irradiation (background control), or after low-dose (2gy) irradiation (1 and 24 hours). results: we observed maximal h2ax generation at 1 hour post-irradiation, with progressive dephosphorylation at 24 hours post-irradiation in healthy controls. the patient showed normal frequencies (%) of t cells and nk cells positive for h2ax (95.62% and 99.40% respectively); (controls (n=2) t cells = 99.29% and 99%; nk cells = 99.6% and 99.11%), but increased intracellular levels (mean fluorescence intensity, mfi) of h2ax (t cells = 50.67 and nk cells = 52.41) compared to controls (t cells = 24.86 and 19.58; nk cells = 41.33 and 35.03) at 1 hour post-irradiation. however, more importantly, at 24 hours post irradiation there was a lack of dephosphorylation in a substantial proportion of lymphocytes (64% of t cells and 99% of nk cells) compared to healthy controls (t cells= 1.75% and 1.51%; nk cells = 9.27% and 17.83%). further, while there was dephosphorylation of h2ax at 24h in patient lymphocytes as compared to 1h, the amount, as measured by mfi, remained elevated at 24h (t cells = 8.97, nk cells =7.57) compared to controls (t cells =1.71 and 2.25; nk cells = 3.18 and 2.75). the data from patm and psmc1 were uninformative for the evaluation of lig4-scid. conclusions: flow-based kinetic analysis of h2ax is a useful marker for the diagnosis of lig4-scid, and can be performed with a small amount (5cc) of blood, and provides a result in 3-4 days, facilitating rapid assessment of radiosensitivity in this condition. human plcg2 haploinsufficiency results in nk cell immunodeficiency and herpesvirus susceptibility objectives: we aimed to investigate the cause of disease in three patients from two kindreds with recurrent or severe herpesvirus infections and nk cell dysfunction. methods: we used exome sequencing and mass cytometry (cytof), as well as traditional immunologic techniques, to investigate the genetic causes, immune cell subpopulations/signaling, and nk cell function of these patients. we additionally used mouse models, crispr cell lines and in vitro assays to assess the role of plcg2 haploinsufficiency in disease. results: kindred a consisted of two patients presenting with hsv1 susceptibility and autoimmunity. kindred b consisted of one patient with severe cmv myocarditis and adenoviral hepatitis. both kindreds were evaluated for nk cell function and showed reductions in target killing in spite of normal cytotoxic granule degranulation against the same target. microscopy analysis suggested that granule mobility was reduced in at least one kindred. cytof revealed reductions in plcg2 phosphorylation after receptor crosslinking in the nk cells of both kindreds. kindred a also presented with a reduction in naïve b cells without perturbations in immunoglobulin output, b cell memory formation or class switching. trio whole exome sequencing was performed and revealed rare heterozygous plcg2 mutations in both kindreds. functional analysis, as well as mouse and crispr models, support a functional haploinsufficiency as a cause for nkd in these patients. conclusions: heterozygous loss-of-function point mutations in plcg2 have not been previously investigated as a cause of nk cell deficiency or recurrent herpesvirus infection. thus, these patients represent a novel immunodeficiency involving plcg2 haploinsufficiency, nk cell dysfunction, and herpesvirus susceptibility. hypomorphic rag1 mutations alter the pre-immune repertoire at early stages of lymphoid development introduction/background: human rag deficiency is associated with a spectrum of clinical phenotypes. while the most severe forms of rag deficiency manifest with severe combined immune deficiency or omenn syndrome since the first weeks of life, more recently patients have been identified who present to medical attention at a much older age predominantly with symptoms of autoimmunity and/or inflammation. many of the mutations associated with this atypical syndrome are found in the c-terminal domain (ctd) of the rag1 gene and allow residual development of t and b cells. these patients have an abnormal peripheral t and b cell repertoire, but how this is affected by abnormalities in the composition of the pre-immune repertoire vs. antigen-mediated selection and homeostatic proliferation in the periphery is unknown. objectives: in order to investigate whether mouse models with hypomorphic mutations in the rag1 ctd recapitulate the phenotype observed in patients with cid-g/ai, and to study how these mutations affect repertoire composition, cell selection and survival during t and b cell development, we generated three mouse models carrying homozygous rag1 mutations (f971l, r972q, and r972w), corresponding to human mutations (f974l, r975q, r975w) previously reported in patients with late-onset combined immune deficiency with granuloma and/ or autoimmunity (cid-g/ai). methods: mice were generated using crispr/cas9 mediated gene editing. t and b cell development, including apoptosis was studied by flow cytometry. immunoglobulins in naïve mice, baff levels and specific antibody responses were measured by elisa. serum igm autoantibodies were measured using a microarray (utsw). analysis of t cell receptor (trb) repertoire in several sorted t cell populations and immunoglobulin heavy chain (igh) repertoire in pre-b cells was performed by adaptive biotechnologies. in order to be able to detect both dj and vdj rearrangements, pro-b cells and spleen b cells were sequenced using high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (htgts-rep-seq). analysis of vk-jk rearrangements in pre-b cells was performed by pcr amplification. results: immunological characterization showed partial development of t and b lymphocytes, with persistence of naïve cells, preserved serum immunoglobulin, but impaired antibody responses and presence of autoantibodies, thereby recapitulating the phenotype seen in patients with cid-g/ai. by using high throughput sequencing, we identified marked skewing of igh v and trb v gene usage in early progenitors, with a bias for productive rearrangements after selection occurred, and increased apoptosis of b cell progenitors. this suggested that more alleles remained in germline configuration. moreover, in the rearranged igh loci, the distal v gene segments were preferentially rearranged already at the earliest stages of b cell development, a finding that has not been previously reported. in addition, rearrangement at the igh locus was impaired, and polyreactive igm antibodies were detected. conclusions: in conclusion, this study demonstrates that hypomorphic rag1 mutations reported in cid-g/ai cause abnormalities of the primary b and t cell repertoire. these changes may affect survival and selection of t and b cells, and thereby contribute to the immune dysregulation often seen in patients with cid-g/ai. senior clinician, nih/niaid/lcim introduction/background: autosomal dominant hyper ige syndrome (ad-hies) is a primary immunodeficiency due to loss of function stat3 mutations. disease manifestations include recurrent skin and pulmonary infections, eczema, mucocutaneous candidiasis, as well as tion and in vitro studies demonstrated that the enhanced signaling could be controlled by ruxolitinib, an approved jak1/2 inhibitor. informed by these experimental data, the patients were treated with ruxolitinib with remarkable improvement in a variety of clinical end-points, including hematological profiles and growth parameters. conclusions: this characterization of a human jak1 gain-of-function mutation expands our current understanding of the role of jak1 in eosinophil biology, hematopoiesis and immune function. chronic granulomatous disease, ornithine transcarbamylase deficiency and x-inactivation is a primary immune deficiency characterized by defects in the nadph oxidase enzyme complex resulting in a susceptibility to a narrow spectrum of bacteria and fungi. mutations in cybb encoding gp91phox and located at xp21.1 are associated with the most common form of cgd. deletions and rearrangements in this region are associated with other genetic diseases such as mcleod syndrome (xk), retinitis pigmentosa (rpgr), duchenes muscular dystrophy (dmd), ornithine transcarbamylase deficiency (otc), and x-linked mental retardation (tspan7). patient phenotype depends on the extent and position of the deletion, creating a "contiguous x-chromosome gene deletion syndrome. objectives: we report a four-year-old female, who presented with symptoms of x-linked cgd and otc deficiency with a large, contiguous multi-gene deletion on the x-chromosome. methods: we report a four-year-old female, who presented with symptoms of x-linked cgd and otc deficiency with a large, contiguous multi-gene deletion on the x-chromosome. the patient is the second born of a set of non-identical triplets from a clomiphene assisted pregnancy at 35 weeks gestation. (weight at birth: 5lbs 2oz). after a 7 day stay in the nicu, and cpap for one day she was discharged home. by the second month, she began having problems gaining weight and had persistent vomiting. at 3 months, she was admitted with a pneumonia diagnosed as methicillin resistant staphylococcus aureus by lung biopsy. during that hospitalization, the mother noted an enlarging lesion on the infants left hand which was biopsy proven serratia marscecens osteomyelitis and was treated with intravenous cefepime for 6 weeks. at this point a dihydrorhodamine assay (dhr) showed only 15.1% positive cells (nl 80-100%) and she was diagnosed as a carrier of x-linked cgd. bactrim was initiated for antibacterial prophylaxis but it was discontinued due to recurrent diarrhea; she was unable to tolerate antifungal prophylaxis as well due to liver function abnormalities. she continued having frequent vomiting episodes, irritability, failure to thrive and development delay. ulcerations in esophagus were confirmed by egd and colonoscopy, probably related to persistent emesis. diarrhea was unresolved. at 30 months of life she was admitted with acute encephalitis, a serum ammonia level of 218 and elevated urine orotic acid. results: given the demonstrated carrier status for cybb and apparent otc deficiency, comparative genomic hybridization was performed, revealing a deletion from xp21.1 to xp11.4 this 3.9mb loss includes 18 genes that are known to cause disease. since the diagnosis, the patient has been on reduced protein diet, resolving her diarrhea, she has subsequently grown and is on 5th percentile for weight and height. her dhr is now 28% positive. at 3 years she was diagnosed with cone rod dystrophy. currently she is on bactrim for cgd prophylaxis and l-citrulline for the otc deficiency. she continues to gain weight, and has shown great improvement in her development delay and no new cgd-related infections have recurred. conclusions: this case reminds us that x-linked carriers with large deletions may be symptomatic and genetic analysis to determine other affected genes can be important for medical management. introduction/background: wiskott-aldrich syndrome (was) is a rare and severe x-linked disorder with variable clinical phenotypes correlating with the type of mutations in the was gene. the long-term prognosis of this syndrome is generally poor, with hematopoietic stem cell transplantation (hsct) remaining the only curative choice. the syndrome is poorly characterized in china. objectives: we retrospectively reviewed patients with was referred to our hospital from 2004 to 2016, and summarize their clinical manifestations and genetic features. methods: sixty-four children suspected to be was from 62 unrelated families were enrolled in this study. the clinical data of children were reviewed in the present study. distribution of lymphocyte subsets from peripheral blood and was protein (wasp) expression in peripheral blood mononuclear cells was examined by ow cytometry (fcm). wasp mutations were identified by direct sequencing of pcramplified genomic dna results: among 725 patients with primary immunodeficiency diseases (pid), 40 (5.52%) were finally diagnosed as was with gene identified. the mean time of diagnosis was 6.25 months (range, 0.4-9.63). the common onset clinical manifestation was diarrhea, and most patients had recurrent upper respiratory tract infection, otitis media, pneumonia, and skin abscess. one patient had nephrotic syndrome and no patient with malignancy. all patients had classical was phenotype with was clinical scores 3-5. total 36 mutations in wasp were identified, including 14 novel mutations. six patients received hsct, five survived, with one died because of gvhd. compared with the other 24 patients without wasp mutations, was patients had lower numbers of cd4+ t cells and b cells, and higher eos and ige level. there was a negative association between the number of b cells and the was clinical scores. conclusions: in china, diagnosis of was has improved over the last decade, although a much higher number of cases had been expected. establishing more diagnostic centers dedicated to the care of pid will facilitate early, correct diagnosis and better care of was in china. regulatory cells (tregs) are precisely quantified by measuring demethylation of the treg-specific-region of foxp3. more recently, we have identified highly cell type-specific dna regions of demethylation for further cell populations. objectives: this novel technology allows the implementation of differential immune phenotyping into the newborn screening procedure. here, we aimed at epigenetically quantify multiple immune cell types in different biological samples including dried blood spots and samples from patients with pid with immundysregulation, where currently no approach is available. methods: using cell type-specific demethylation sites, we developed epigenetic qpcrs for quantification of t-, treg, b-, nk-, monocyte and granulocyte cell population. epigenetic qpcr is applicable for relative and absolute quantification in whole blood and dried blood spots using isolated, bisulfite converted dna. results: we demonstrated >95% concordance with flow cytometric analyses of the same fresh blood samples from healthy subjects. we have validated the method in 30 children with symptoms of immundysregulation resulting from monogenic defects, including for example foxp3, cd25, stat1, ctla4, and leading to treg/teffector cell imbalance where treg deficiency has been difficult to assess by flow cytometry. furthermore, we tested 250 dried blood spot (guthrie card) samples from healthy newborns and 30 patients with diverse pids and correctly identified 29/30 pid patients, indicating that this method holds promise for newborn screening. conclusions: the method we established for immune cell quantification based on epigenetic cell type-specific markers, is feasible and reliable in biological samples either fresh, frozen or archived, including dried blood spot. the analysis of further immune cell types introduces an innovative opportunity to diagnose a variety of pids and immunodysregulatory disorders as early as newborn screening. pancytopenia and immunodeficiency with mds in an infant due to samd9l mutation we present our data on behalf of the pcid study consortium of the inborn errors working party worth profound combined immunodeficiencies (p-cid) are inherited diseases with impaired t-cell function leading to infections, immune dysregulation or malignancies. genetic, immunologic and clinical heterogeneity make patient specific decisions on indication and timing of hematopoietic stem cell transplantation (hsct) difficult. objectives: since 2011 the pcid study recruits non-transplanted p-cid patients aged 1-16 years to prospectively compare natural histories of age and severity-matched patients with or without subsequent transplantation and to determine whether immunological and/or clinical parameters may be predictive for outcome methods: our prospective/retrospective international observational multicenter study recruits pediatric p-cid patients to identify biomarkers and clinical parameters that are predictive of outcome. results: so far >130 growth hormone deficiency comprised the majority of neuroendocrine cases (65%, 11/16). other notable neurologic diagnoses included headache (17%, 211/1227), seizure (5%, 62/1227), cerebrovascular accident (2%, 25/1227), and neurologic tumors (0.4%, 5/1227). in addition to somatic neurologic conditions, many cvid patients (37.7%, 462/1227) had reported diagnoses of depression, anxiety, and post-traumatic stress disorder. conclusions: our findings suggest that neurologic diagnoses are more common in cvid patients than previously recognized. patients with neurologic autoimmune disease appear to have a more severe phenotype with earlier age at symptom onset. many cvid patients had depression, anxiety clinical outcomes of human herpesvirus 6 reactivation after hematopoietic stem cell transplantation prognostic factors and outcome of epsteinbarr virus dnaemia in high-risk recipients of allogeneic stem cell transplantation treated with preemptive rituximab cytomegalovirus in hematopoietic stem cell transplant recipients daratumumab controls life-threatening post-hsct autoimmune haemolytic anaemia objectives: we present a 17 month old pancytopenic male who was diagnosed with myelodysplastic syndrome (mds) with monosomy 7 due to samd9l mutation. methods: whole exome sequencing was performed on a male, who presented at 8 months of age with findings concerning for a bone marrow failure (bmf) syndrome despite a normal bmf genetic panel. results: the patient presented at 8 months of age, with severe pancytopenia, fevers, e. coli bacteremia, pancolitis, and echtyma gangrenosum. bone marrow showed severe aplasia with occasional macrophages.he was treated with antibiotics, as well as steroids, etoposide and cyclosporine for presumed hemophagocytic lymphohistiocytosis (hlh). a bone marrow failure and hlh genetic panels were normal conclusions: this is one of the first reported cases of samd9l mutations causing mds since its initial discovery earlier this year. samd9l mutation should be considered in patients who present with pancytopenia and monosomy 7 mds. as new defects continue to be identified, further evaluation outside of typical bmf panels may be relevant primary immune deficiency disease in patients over age 60: an analysis from a proprietary immunology patient registry roger ucla school of medicine 2 director objectives: to characterize the prevalence of pidd among older individuals using a patient database maintained by the consortium of independent immunology clinics (ciic), comprised of 17 specialty immunology outpatient practices in the us. methods: patients with pidd were identified in the ciic database using icd-10 codes d80 conclusions: our data suggest that pidd in patients over age 60 may be more prevalent than previously reported introduction/background: the patient is a 6-month-old boy, born at 35+4 weeks gestation to non-consanguineous parents of italian origin. he was admitted to the intensive care unit at 12 days of age with profuse bloody diarrhoea, weight loss, severe metabolic acidosis and acute renal failure. he had a rapid respiratory deterioration necessitating intubation, ventilation and inotropic support. the patient developed features of macrophage activation syndrome with: (i) prolonged fever > 38.5°c, (ii) hepatosplenomegaly, (iii) bicytopenia (anaemia and thrombocytopaenia), (iv) hypertriglyceridemia, (v) high ferritin (14,7000) and (vi) haemophagocytosis on bone marrow smear. he also presented with three interesting features (i) a macular erythematous rash that slowly resolved and was replaced by reticulo-livedoid rash, (ii) a marked hypereosinophilia and (iii) no significant elevation of hladr/cd8+ t cells on lymphocyte immunophenotyping (7-15%). the patient underwent rectal biopsy which confirmed the presence of eosinophils, but without significant inflammation or architectural changes. stool microscopy showed presence of partially necrotic intestinal epithelial cells. immune work up demonstrated global t lymphopaenia without balanced subpopulation and eliminated a familial hemophagocytic lymphohistiocytosis (normal perforin and cd107a expression on cd8+ t-cell and nk cells). circulating foxp3+ cd25+cd127lowcd4+ t cells were within normal range. a dihydrorhodamine reduction assay was normal. 9 chief, laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health introduction/background: hematopoietic stem cell transplantation (hsct) has been used for the treatment of hematologic malignancies and primary immunodeficiencies (pid), for several decades with increasing efficacy. however, toxicity related to conditioning regimens based on the use of chemotherapy and/or irradiation to ensure engraftment of donor cells, remains a significant problem. recently, an alternative, potentially low-toxicity, approach has been proposed, which makes use of an immunotoxin targeting cd45-expressing cells, which include hsc and more mature leukocytes. this approach is particularly attractive for leaky forms of severe combined immune deficiency (scid), with residual production of dysfunctional t and/or b cells, such as atypical forms of rag deficiency. objectives: we have developed a mouse model carrying a hypomorphic mutation in the rag1 gene (p.f971l) resulting in a combined immunodeficiency with signs of autoimmunity, recapitulating the phenotype seen in patients. methods: using this model, we have tested the efficacy of conditioning with an anti-cd45 immunotoxin (cd45-sap) alone or in combination with low irradiation (200rads; cd45-sap/200), and compared these regimens to a myeloablative dose of irradiation (800rads) [niaid protocol lcim 6e]. following conditioning, the mice were transplanted with wild-type (wt) bone marrow (bm) lineage-negative cells and followed over time to evaluate immune reconstitution. results: conditioning with cd45-sap alone or with cd45-sap/200 led to a consistent engraftment of donor t and b cells in the peripheral blood (pb) of f971l that increased overtime, reaching 90% donor chimerism at 16 weeks. myeloid (cd11b+) and nk cells in pb of f971l mice also showed high level of donor engraftment that remained stable at around 70% in cd45-sap around 24 hours of life and the second after one week. there is no data on the utility of one versus two screens for scid screening. here we present our data evaluating whether patients with scid or t-cell lymphopenia were identified on the first or second trec screen. objectives 1. determine the benefit of a second trec screen in identifying scid and t-cell lymphopenia at birth. 2. examine outcomes of trec screening in washington state since implementation. methods: results of scid newborn screening performed in washington state between january 2014 and june 2017 were reviewed retrospectively with the staff of the washington department of health laboratory where the trec assay is performed. trec thresholds (copies/μl) were defined as follows: absent (20), low (21-60), borderline (61-80) and normal (>80). all trec assays were run with a beta-actin control to assure sample adequacy. a screen is considered abnormal if there is one low/ absent trec or two borderline trec. newborns with abnormal trec screening have follow-up diagnostic testing consisting of lymphocyte flow cytometry to evaluate numbers of naïve and mature t cells, b cells, and nk cells, performed at seattle childrens hospital. results: a total of 68 positive trec screens were found in washington state between january 2014 and june 2017. five patients who did not have diagnostic flow cytometry testing were excluded from the analysis (one protocol deviation, one lost to follow-up and three who died before testing could be performed). the first screen was abnormal in forty three patients, while the second screen was abnormal in 16 patients. five patients had an abnormal third or fourth trec drawn for other newborn screen follow-up. three patients with scid were identified, all with abnormal values on first screen. fortyfive patients with t-cell lymphopenia were identified; 30 from the first screen and 11 from the second screen. there was one patient with mhc ii deficiency was missed by both first and second screens because she did not have t-cell lymphopenia. the false positive rate with the first screen was 21% versus 28% with subsequent screens. the false positive rate dropped to 3% with two abnormal trec. the positive predictive value of scid or t-cell lymphopenia with the first abnormal trec was 82% versus 96% with two abnormal trec. average age of collection among infants with a positive screen was 30.5 hours for the 1st nbs and 11.5 days for the 2nd nbs. live viral vaccines were postponed in three patients who had an abnormal secondary screen (one with idiopathic t-cell lymphopenia, one with ectrodactyly-ectodermal dysplasia-clefting (eec) syndrome and one with 22q11.2 deletion). one of these was started on pjp prophylaxis. conclusions: the practice of obtaining a second nbs from all newborns in washington state has led to increased identification of patients with tcell lymphopenia but did not result in identification of additional patients with scid. the false positive rate of the first and subsequent newborn screens was similar and decreased in patients with two abnormal trec. interventions including delaying live viral vaccines and pjp prophylaxis were instituted in patients who had a normal initial trec but abnormal secondary screen and documented t cell lymphopenia. two trec screens in all newborns can result in identification of additional patients with t-cell lymphopenia who may require intervention and additional follow-up. it is not yet clear whether the cost of a second mandatory scid newborn screen is balanced by the additional sensitivity gained by this approach. introduction/background: mannose-binding lectin (mbl) is a multimeric lectin that recognizes a wide array of pathogens independently of specific antibody, initiates the lectin pathway of the complement system, and acts as a proinflammatory mediator. mbl deficiency is reported to increase the frequency of infections in patients with impaired immune systems or cystic fibrosis (cf) patients. mbl replacement is experimental and unavailable. immunoglobulin replacement therapy (igrt) is controversial in the treatment of mbl deficiency; however, there are no reports of its efficacy or role in this condition. we describe a cf carrier patient with mbl deficiency, ciliary dyskinesia and mildly low igg who did not respond to igrt. objectives 1. understand when mbl deficiency can be symptomatic. 2. define the relationship between mbl deficiency and cf. 3. describe the treatment options of mbl deficiency. 4. define the efficacy of igrt and mbl deficiency. methods: a single case report. results: 11 year old girl with mbl deficiency, cf carrier state with polymorphisms (2752-26 a>g cf variant with 7t/7t and m470v polymorphism) and ciliary dyskinesia (diagnosed by ciliary biopsy) who initially presented at eight years of age with recurrent sinusitis, recurrent otitis media status post tympanostomy tube placement, reactive airway disease and tracheomalacia. she had nine episodes of recurrent sinusitis with six negative sinus cultures and one positive for pseudomonas aeruginosa. she required a total of nine courses of antibiotics. labs showed several mbl levels <50 ng/ml on three occasions, normal ch50, ah50, igg 577-590 mg/dl (low normal for age), normal iga, igm, t cells, b cells, nk cells, and robust specific antibody titers. despite adequate pulmonary hygiene including nebulized levalbuterol, budesonide, dornase alfa, ipratropium, hypertonic saline and compression vest twice daily, she continued to have recurrent bronchitis and cough. in addition, even with sinus rinses and intranasal corticosteroid, she continued to have 6-8 episodes of sinusitis yearly. for this reason, she underwent bilateral total ethmoidectomies and maxillary antrostomies with modest reduction in frequency of sinus infections and symptoms. however after two years, her bacterial sinusitis recurred. four episodes were positive for methicillin staphylococcus aureus or pseudomonas aeruginosa. this did not improve significantly with a trial of intranasal mupirocin. due to increased frequency of bacterial sinusitis refractory to traditional therapy, she was started on subcutaneous igrt dosed approximately 350 mg/kg/month dosing maintaining igg troughs around 700 mg/dl. after a six month trial, she did not have improvement in the frequency of sinusitis, bronchitis and otitis media. she required 3-4 courses of antibiotics for bacterial upper respiratory tract infections and igrt was stopped. prophylactic antibiotics and repeat sinus surgery were instituted. conclusions: majority of the patients with low/deficient mbl levels do not manifest significant symptomology due to the redundancy of the innate immunity. the increased susceptibility to infections is thought to be due to additional factors that compromise other components of the immune system. specifically in cf patients, mbl deficiency is associated with earlier colonization with pseudomonas, more rapid decline in lung function and earlier death secondary to end-stage lung disease. there are no reports of combined mbl and cf carrier symptomatic patients similar to this patient. there are no validated age-corrected values for mbl levels in pediatric patients. the clinical relevance of these levels to infection frequency, severity, or treatment of mbl deficiency remains to be proven. it has been proposed that <100 ng/ml is considered deficient in children. mbl therapy is still experimental and not commercially available. management when provided for severe or frequent infections includes prompt treatment with antibiotics, prophylactic antibiotics, appropriate vaccinations, and a trial of igrt. there are no reported cases describing the efficacy of igrt in mbl deficiency, and the mechanism subsequent genetic analysis identified the presence of a de-novo heterozygous mutation in the nucleotide binding domain of nlrc4 (c.1021g>c, p.val341leu). a mutation involving this amino-acid position has already been described but with a different substitution pattern in a boy and his father who presented with mas (p.val341ala) (1) . in order to prove the causality of this mutation, our team generated thp-1 cell lines expressing the two different mutations through gene-editing with the crispr system. in this system the mutation p.val341leu, as well as the p.val341ala mutation, were responsible for spontaneous activation of caspase1 , as evidenced by flica assay. the patient was treated with iv methylprednisone 2mg/kg/day. he continued to have progression of his inflammatory state, and was therefore commenced on anakinra. following confirmation of mutation, he was started on rapamycin, reasoning that (i) through autophagy induction, rapamycin could potentiate the action of anakinra (2, 3) and (ii) through mtor inhibition counteract the effect of il-18 on t-cells (4). with a combinatory therapy of anakinra up to 15mg/kg/day and ramapycin (with trough levels of 10-15 ng/l), the patient showed a marked clinical improvement, allowing weaning of steroids and establishment of enteral feeds. ferritin levels reduced to 800-1000 ng/ml. we observed a significant decrease in il-18 plasmatic level following treatment initiation (pre-vs post-treatment levels of 82844 pg/ml and 10055 pg/ml, respectively). we report a novel nlrc4 gain-of-function mutation, presenting with neonatal enterocolitis and autoinflammation with improvement under combinatory therapy of anakinra and rapamycin. to our knowledge this is the first case to report the use of rapamycin in this disease, with what appears to be encouraging results. further studies are required to elucidate the potential role of rapamycin in the management other inflammasome disorders. introduction/background: allogeneic hematopoietic stem cell transplantation (hct) is currently standard treatment for patients with severe combined immunodeficiency (scid), with 2-year overall survival >90% for typical scid (heimall blood 2017). previous studies revealed that poor clinical outcomes correlated with poor long-term t cell reconstitution, including low cd4 t cell counts and low naïve cd45+ t cell counts (pai nejm 2014) . we hypothesized that t cells developing in a poorly reconstituted immunologic environment would show features of chronically activated t cells with increased expression of inhibitory co-receptors. we further hypothesized that the intensity of the conditioning regimen would correlate with the expression of inhibitory co-receptors. objectives: to characterize the t cell phenotype of scid patients at >2 years post-hct and to investigate the impact of conditioning regimen on the quality of t cell reconstitution and the expression of inhibitory coreceptors methods: we analyzed 34 scid patients 3-28 yrs (median 14 yrs) after hct. we excluded from the analysis patients with chronic graft-versushost disease (gvhd), chronic dna viral infections or patients who had received donor lymphocyte infusion or boost in the 6 months prior to study. scid genotypes (n) included il2rg/jak3 (20), rag1/rag2/ dclre1c (5), ada (2), il7r (1) and other/unidentified (4). nine patients had received a reduced intensity (ric) or myeloablative (mac) conditioning regimen, while 25 had received either no conditioning or immunosuppression only (none/is). poor t cell reconstitution was defined as cd4 t cell counts below 500 cells/mm3 (21 patients). t cell phenotype, including expression of inhibitory co-receptors, was assessed by flow cytometry. results: compared to patients with cd4 counts above 500 cells/mm3, patients with low cd4 counts had low naïve cd45ra+ccr7+ t cells (p=0.0004) and high cd45ra-ccr7-t effector memory (tem) cells (p=0.02), low numbers of naïve thymic cd45ra+ cd31+ t cells, low numbers of trecs and a less diverse t cell repertoire (p<0.0001). additionally, they had an increased frequency of cd8 t cells expressing pd1 (8% vs 3%), ctla4 (5% vs 1.5%), cd160 (25% vs 5%, p=0.01), and 2b4 (63% vs 29%, p=0.0001) inhibitory co-receptors. increased inhibitory receptor expression was associated with a differentiation profile skewed toward a tem phenotype, reduced t cell diversity, increased markers of t cell activation, and the development of a highly exhausted cd39high pd-1high t cell population. more importantly, a fraction of ccr7+ cd45ra+ cd8 t cells expressed pd1 (6% vs 1%, p=0.03) and 2b4 (27% vs 4%, p=0.01) in patients with low cd4 counts, suggesting that some naïve t cells of poorly reconstituted patients were chronically activated. inhibitory receptor expression did not increase with hla disparities between the donor and recipient, a history of gvhd after transplant or the infection status of the patient prior to transplant. however, inhibitory co-receptor expression was correlated with conditioning regimen, with increased frequency of 2b4+ cd8 t cells in unconditioned patients (54% vs 28% in none/is and ric/mac patients respectively, p=0.01). conversely, ric/mac conditioning was associated with higher naïve cd8 t cell numbers (p=0.01), higher naïve thymic cd45ra+ cd31+ t cell numbers (p=0.001), a more diverse t cell repertoire (p=0.001) and low expression of inhibitory co-receptors. ric/mac conditioning was also associated with improved naïve t cell generation and limited expression of inhibitory receptors in il2rg/jak3 patients (23% vs 50% for 2b4, 1% vs 11% for cd160 in ric/mac versus none/is il2rg/ jak3 patients respectively), a genotype permissive to t cell engraftment. conclusions: collectively, our results suggest that the expression of inhibitory co-receptors may be a biomarker of poor t cell reconstitution in transplanted scid patients. further, we propose that lack of conditioning limits t cell reconstitution, which correlates with increased expression of inhibitory receptors on circulating cd8 t cells. antibodies targeting inhibitory receptors are now available in clinical trials to treat cancer and viral infections. it will be necessary to evaluate the relationship between inhibitory receptor expression, t cell function and clinical outcome, to see if a selected group of scid patients could benefit from these immunotherapies. wallace chair, chief of allergy immunology, children's hospital of philadelphia introduction/background: prophylactic antibiotics (abx) and immunoglobulin replacement (igrt) are commonly used to treat specific antibody deficiency (sad), but the optimal therapy is not established. objectives: to compared outcomes (number of infections and hospitalizations) in sad treated with igrt vs. prophylactic antibiotics. methods: two-center, retrospective chart review of sad patients from jan 2012-may 2017. we excluded patients with hypogammaglobinemia and/or other immunodeficiency diagnosis. characteristics and treatment were reported, rates of infections/hospitalizations among treatment groups were compared using linear regression model. results: 78 sad patients included. mean age was 18 years, 54% were females. 22 (28.8%) received prophylactic antibiotics, 45 (57.6%) received igrt, 11 (14.1%) did not receive any specific treatment. number of infections decreased from 8.2 (year before treatment) to 2.0 (year after treatment) in prophylactic antibiotics group (p=0.008), and from 7.2 to 2.3 in igrt group (p<0.001). various musculoskeletal and vascular abnormalities. little is known of gynecologic-obstetric complications, but with improved therapies women are living longer making reproductive health more pertinent. objectives: to learn more about obstetric and gynecological health in women with stat3 loss of function. methods: we prospectively interviewed and retrospectively reviewed medical records of adult women with ad-hies evaluated at the nih between 2000-2017. results: of 61 patients aged 18-66 years (mean 35 years), 47 women were interviewed, and chart reviews were performed on 14. age of menarche in our cohort was consistent with the national average (13.05 vs. 12.5 years). five of 30 patients (16.7%) reported having worsening lung symptoms with menstruation, and 14 of 34 participants (41%) reported worsening eczema during menstruation. with regard to routine health maintenance, 21 of 30 women reported having regular cervical cytology testing; 7 (23%) reported an abnormal result. of these 7, 5 had hpv that responded to treatment or was hpv only not requiring treatment; 2 had reactive changes due to yeast and 1 ascus that was subsequently normal. mastitis and breast abscesses occurred in 14 women. eleven women reported vulvar cysts/abscesses requiring drainage. nine women had progestin-releasing iuds placed, in some to suppress menses-associated vulvar eczema/abscess flares; no infectious complications were reported from progestin iud use. over 30% of women chose not to conceive given underlying disease. of 16 women with pregnancies, 15 women had 26 live births, 6 of 16 (38%) had miscarriages, and 3 of 16 (19%) experienced recurrent pregnancy loss. three women whose pulmonary symptoms worsened during pregnancy were diagnosed with progression of parenchymal lung disease post-partum. one woman experienced worsening of skin manifestations. other reported postpartum complications included one wound infection (after caesarean) and one hemorrhage leading to hysterectomy. conclusions: as women with ad-hies are living longer, significant infectious and disease-related exacerbations related to both menstruation and pregnancy were observed in this patient population. it is important to focus on maintenance of their gynecologic and obstetric health and carefully monitor for these morbidities. finally, while these women may choose to attempt pregnancy, the risk of recurrent pregnancy loss and worsening disease warrants discussion. rna sequencing identifies aichi virus 1 as the cause of chronic infection with lymphoproliferation in a patient with x-linked agammaglobulinemia objectives: we here present an xla patient with a complicated course in whom we detected aichi virus 1 (aiv1). methods: case report: the patient was diagnosed with xla at age 3 years, based on agammaglobulinemia with absent b cells and a known pathogenic mutation in btk (c82c>t, p.r28c). he had been suffering from recurrent respiratory and gastrointestinal infections since the age of 2 months. he was started on immunoglobulin (ig) substitution, which resulted in complete control of infections with igg trough levels at 8 g/l. however, at 6 years of age he developed unexplained fever, refractory temporal epilepsy, hepatitis, progressive nephromegaly with chronic renal failure, splenomegaly, episodic diarrhea and growth failure. ultrasound identified multiple focal lesions in the liver, spleen and kidney. a liver biopsy showed severe chronic hepatitis with initial perisinusoidal fibrosis. serial kidney biopsies showed variable oligoclonal cytotoxic t cell infiltrates, suggestive of chronic viral infection. standard diagnostics failed to reveal a pathogen in blood or stool samples and on biopsies. results: we finally resorted to rna sequencing on a kidney biopsy sample. this technique identified aiv1, a kobuvirus of the family picornaviridae, with a high read number. subsequently pcr confirmed the presence of aiv1 in both liver and spleen. results for cerebrospinal fluid, blood and feces are pending. conclusions: aiv1 is a picornavirus responsible for self-limiting gastroenteritis in humans. confirmatory pcrs on blood, csf and stool samples are ongoing. however, given the unequivocal result of the rna sequencing and confirmatory pcrs in other affected organs, we believe that the complications in this xla patient can be explained by aiv1 chronic infection. these findings confirm the potential of next generation sequencing techniques to identify infectious agents in patients with primary immunodeficiency. characterization and successful treatment of a novel autosomal dominant immune dysregulatory syndrome caused by a jak1 gain-offunction mutation. introduction/background: janus kinase 1 (jak1) plays an essential, nonredundant role in the jak/stat signaling cascade, a key pathway in the control of hematopoiesis and immune function. significant progress has been made in elucidating the role of jak1, but gaps in our knowledge still persist. to date, somatic gain-of-function mutations in jak1 have been linked to t-cell acute lymphoblastic leukemia. objectives: to understand and treat human jak1 gain-of-function mutations. methods: research study protocols were approved by our institutional review board. four members of the family (the affected children and their parents) were enrolled. written informed consent for genetic testing and participation was provided by the parents for their children. genetic, bioinformatic, biochemical and immunological investigations were performed. results: we describe the first known patients carrying a germ-line gainof-function mutation in jak1. the clinical phenotype includes severe atopic dermatitis, markedly elevated peripheral blood eosinophil counts with eosinophilic infiltration of the liver and gastrointestinal tract, hepatosplenomegaly, autoimmunity, and failure to thrive. functional analysis established the gain of function phenotype caused by the mutaintroduction/background: herpesviridae infection after hsct for hematologic malignancies, specifically cytomegalovirus (cmv), epstein-barr virus (ebv) and human herpes virus-6 (hhv-6), have been associated with various outcomes including post transplant lymphoproliferative disorder (ptld), graft-versus-host disease (gvhd) and mortality (1) (2) (3) . patients with primary immunodeficiency may have different incidence and outcomes with respect to herpesviridae given their differences in age at transplant, conditioning choices and underlying disease susceptibility to this viral family. objectives: the objective of this study was to describe the incidence and outcomes of cmv, ebv and hhv-6 post hsct for the primary immunodeficiency population. methods: a single center retrospective chart review of primary immunodeficiency registry patients (research ethics board protocol no. 1000005598) who received hsct from january 2000 december 2016 was undertaken. patients who received gene therapy were excluded. antiviral prophylaxis was given according to institutional protocol. demographic and clinical data were collated and analyzed with microsoft excel . the primary outcome was incidence and time to dnaemia for cmv, ebv and hhv-6. results: sixty one patients who underwent hsct for primary immunodeficiency from january 2000-december 2016 were reviewed. diagnoses are noted in table 1 . the average age of transplant was 18.0 months (range 1.9-93.8). 51 transplant recipients received busulfan and cyclophosphamide conditioning (2 with anti-thymocyte globulin (atg), 2 with alemtuzumab added), 3 transplants received busulfan, fludarabine and either atg or alemtuzumab, and 1 received atg alone. six transplants were unconditioned. 70.5% (43/61) of patients developed gvhd requiring systemic immune suppression. the overall incidence of cmv, ebv and hhv-6 post hsct for primary immunodeficiency was 9.8% (6/61), 59.0% (36/61) and 24.6% (15/61) respectively. in those with severe or profound combined immune deficiency the incidence of cmv was 7.0% (3/43), ebv 53.5% (23/43), and hhv-6 18.6% (8/43). in the 6 patients with chronic granulomatous disease, cmv incidence was 17% (1/6), and ebv and hhv-6 were both 50% (3/6). in recipients with pre-transplant negative pcr for ebv, cmv and hhv-6 (r-), time to dnaemia post transplant is seen in figure 1 . ptld was seen in 2 patients with rag1 and il2r, both of whom were d+r-for ebv status with ebv dnaemia, and one of whom died attributed to ptld. two year mortality for all patients was 23% (14/61), with mortality 24.4% (11/45) for those with either cmv, ebv or hhv-6 dnaemia vs 18.8% (3/16) in those without (p=0.64). disseminated cmv prior to transplant was attributed to cause of death for one case. conclusions: cmv incidence was rare, likely from screening for cmv negative hsct donors. cmv, ebv and hhv-6 dnaemia was not associated with differences in mortality in this cohort. ebv incidence was common, and ptld incidence was similar to previously published outcomes for hsct for other disease (2) . the advent of cytotoxic t cell therapy for ebv may help abrogate this risk. professor, senior physician, ulm university medical center, pediatrics, germany introduction/background: new-onset aiha occurs in 2-6% of pediatric patients post-hsct. incomplete immune recovery may predispose to immune dysregulation following hsct including autoimmune cytopenias. although prednisolone or other immunosuppressive drugs control most episodes, some patients respond incompletely to first or second line therapies including rituximab. objectives: we describe an innovative therapy for post-bmt aiha refractory to proteasome inhibition. three patients responded to anti-cd38 antibody (daratumumab) therapy after failing treatment with bortezomib. methods: we retrospectively evaluated data from three patients treated with daratumumab for post-transplant aiha. patients 2 and 3 were treated according to the positive response reported for patient 1. results: aiha occurred between 4-9 months following hsct. daratumumab was curative in 2 patients, the third one had only transient response and relapsed 5 months after this treatment had been initiated. following daratumumab patients no longer required any prbc transfusions. conclusions: in potentially life-threatening aiha in the context of hsct daratumumab may be an effective rescue therapy in combination with rituximab. early post-natal thymus development is strictly dependent on the level of foxn1 expression in tec these infants present severe t cell lymphopenia early in life, but in most cases their immune system gradually normalizes. however, the role of foxn1 haploinsufficiency in causing this phenotype is unclear. objectives: to analyze t cell development and tec phenotype in nu/+ mice of various age, in order to investigate whether foxn1 haploinsufficiency in mice results in impaired thymic development early in life, followed by progressive normalization, thereby recapitulating the human phenotype. methods: we analyzed 3 groups of nu/+ and +/+ littermates, divided by age: 1 day, 4-7 days, 3 weeks. the number of etp and the distribution of cortical and medullary tecs (ctecs, mtecs) were analyzed by flow cytometry. maturation of mtecs was further assessed by staining for mhc-ii and aire. real-time pcr was used to analyze the expression of ccl25, cxcl12, dll4, and scf, four key foxn1 target genes. the study was performed in accordance to niaid animal protocol lcim 6e. results: at 1 day and 4-7 days of life, nu/+ mice showed a dramatic reduction of etps, both in terms of frequency and absolute numbers, as compared to +/+ mice. however, by 3 weeks of age, the frequency and count of etps were comparable in nu/+ and +/+ mice. a slight but significant reduction in the frequency and absolute count of mtecs was observed in all three groups of nu/+ mice. additionally, the ratio between mtec expressing high levels of mhcii (mtechi) and those expressing low levels of mhcii (mteclo) was always higher in +/+ mice as compared to nu/+ mice. moreover, mtechi cells in nu/+ mice expressed lower levels of aire, the gene crucial for thymic negative selection of autoreactive t cells. finally, as compared to wild-type littermates, nu/+ mice showed reduced thymic expression of ccl25, cxcl12, dll4, and scf at day 1. reduced expression of ccl25 persisted at day 5, but normalized at 3 weeks. conclusions: these data indicate that foxn1 haploinsufficiency in mice leads to impaired thymic colonization by etps and abnormalities of tec differentiation and maturation early in life, followed by progressive normalization, thereby recapitulating what observed in newborns with heterozygous foxn1 mutations. these observations have important implications for the management of these infants, who should be monitored closely without rushing to definitive treatment for scid. introduction/background: foxn1 is the master regulator gene for the development and maturation of the thymic epithelial cells (tecs). by inducing the expression of chemokine receptors such as ccl25 and cxcl12, foxn1 also allows the migration of early thymic progenitor (etp) cells from the bone marrow. lack of foxn1 leads to the nude (nu)/severe combined immunodeficiency (scid) phenotype in humans and mice. recently, a number of newborns have been identified with low t cell receptor excision circles (trecs) at birth, associated with heterozygous foxn1 mutations. these infants present severe t cell lymphopenia early in life, but in most cases their immune system gradually normalizes. however, the role of foxn1 haploinsufficiency in causing this phenotype is unclear. objectives: to analyze t cell development and tec phenotype in nu/+ mice of various age, in order to investigate whether foxn1 haploinsufficiency in mice results in impaired thymic development early in life, followed by progressive normalization, thereby recapitulating the human phenotype. methods: we analyzed 3 groups of nu/+ and +/+ littermates, divided by age: 1 day, 4-7 days, 3 weeks. the number of etp and the distribution of cortical and medullary tecs (ctecs, mtecs) were analyzed by flow cytometry. maturation of mtecs was further assessed by staining for mhc-ii and aire. real-time pcr was used to analyze the expression of ccl25, cxcl12, dll4, and scf, four key foxn1 target genes. the study was performed in accordance to niaid animal protocol lcim 6e. results: at 1 day and 4-7 days of life, nu/+ mice showed a dramatic reduction of etps, both in terms of frequency and absolute numbers, as compared to +/+ mice. however, by 3 weeks of age, the frequency and count of etps were comparable in nu/+ and +/+ mice. a slight but significant reduction in the frequency and absolute count of mtecs was observed in all three groups of nu/+ mice. additionally, the ratio between mtec expressing high levels of mhcii (mtechi) and those expressing low levels of mhcii (mteclo) was always higher in +/+ mice as compared to nu/+ mice. moreover, mtechi cells in nu/+ mice expressed lower levels of aire, the gene crucial for thymic negative selection of autoreactive t cells. finally, as compared to wild-type littermates, nu/+ mice showed reduced thymic expression of ccl25, cxcl12, dll4, and scf at day 1. reduced expression of ccl25 persisted at day 5, but normalized at 3 weeks. conclusions: these data indicate that foxn1 haploinsufficiency in mice leads to impaired thymic colonization by etps and abnormalities of tec differentiation and maturation early in life, followed by progressive normalization, thereby recapitulating what observed in newborns with heterozygous foxn1 mutations. these observations have important implications for the management of these infants, who should be monitored closely without rushing to definitive treatment for scid. introduction/background: immunoglobulin g4rd is an immunemediated disease most commonly seen in middle-aged and older men. clinical features include autoimmune pancreatitis, salivary gland disease, orbital disease and retroperitoneal fibrosis. pathologic features include a lymphoplasmacytic infiltrate enriched in igg4-positive plasma cells and fibrosis in a storiform pattern. laboratory evaluation usually reveals an elevated serum igg4 concentration, and glucocorticoids are often used as treatment in early stages of disease. however, disease may recur off of steroids, and prolonged illness or steroid non-responsive disease is usually treated with rituximab. objectives: the objective of this case presentation is to discuss a presentation of a igg4rd is a young adult male. results: 16 year old male adopted from thailand, initially presented to hospital with five days of intermittent abdominal pain, night sweats and worsening fatigue. ct abdomen and pelvis revealed bronchiectasis in lung bases, hepatic masses and soft tissue infiltration in the porta hepatis extending into the liver, pulmonary nodules (read as possible metastases), bilateral renal masses, retroperitoneal nodes and ileocolic intussusception secondary to possible lymphoma. one year prior to presentation he developed new onset bilateral cervical lymphadenopathy. biopsy revealed a heterogenous lymphoid population and lymphoma was ruled out. he had a repeat lymph node biopsies during the admission, which was all negative for malignant cells, and was compatible with reactive lymphoid tissue with plasmacytosis. immunophenotype showed 59% lymphocytes with normal cd4/cd8 ratio, and 8% cd4-,cd8-, tcr ++ t cells. immunohistochemical stain of a lymph node fine needle biopsy showed a mixture of cd20+ b and cd3+ t cells, abundant cd138+, igg+ plasma cells(pcs) , with some igg+ pcs(ct of the neck and chest was completed for possible staging and revealed cervical lymphadenopathy, nasal polyposis, a soft tissue mass vs. enlarged lateral rectus muscle (left orbit), and mediastinal lymphadenopathy. a diagnosis of autoimmune lymphoproliferative syndrome (alps) was presumed by the hematologic/oncologic team given his elevated igg (6,682 mg/dl) and elevated vitamin b12 (>2000) with multiorgan involvement. alps panel revealed a mutation in faslg (allele 1, c.-2c>t) which is a variant of uncertain clinical significance. alps criteria/scorewas + for 1/4 criteria (cd3+cd25+/hla dr ratio < 1.0). he had been referred to the nih for further management of alps before initial presentation to our office. immunologic work up revealed immunoglobulins of igg: 6,600 mg/dl, iga: 83 mg/dl, igm: 71 mg/dl; 13/23 protective streptococcal titers (> 1.3 mcg/ml); hib: 0.46 mg/l; negative quantiferon gold, and hiv-serology. at that point igg4rd vs. castleman disease was suspected instead of alps. further work up showed a normal il-6 level, and human herpes virus 6 was also negative. igg subsets showed an elevated igg1 (2670 mg/dl), igg2 (1450 mg/dl), igg3 (355 mg/dl) and normal igg4 (20.9 mg/dl). excisional lymph node biopsy was recommended to rule out castleman syndrome or igg4rd. a left salivary gland was removed and showed mainly igg4 positive pcs with no multicentric lymphocytic infiltrates. dilution of the patients serum to determine if there was a prozone affect that minimized the igg4 level showed an elevated igg4 level of 2700.9 mg/dl. this is in the 1st percentile of all cases of igg4rd in terms of serum igg4 concentration. he was diagnosed with igg4rd, a form previously called mikulicz disease, which is comprised of lacrimal and parotid gland enlargement. rituximab was given initially because of the severity of his disease. after two cycles he showed decreased lymphadenopathy, notable weight gain and marked decrease in fatigue. conclusions: igg4rd is a rare and complex immunologically based disease process rarely seen in children or adolescents. patients with igg4rd often undiagnosed at initial evaluation. normal serum igg4 levels are seen in 40% of patients with igg4rd and thus a normal igg4 level should not be used as a biomarker to make the diagnosis of igg4rd or in treating this disease. furthermore, the possibility of having a prozone affect in measuring igg4 needs to be considered and evaluated. meticulous correlation of clinical, pathologic, and imaging findings is required to make the diagnosis. modelling human immune deficiency from novel missense mutations with orthologous heterozygous mutations engineered in mice by crispr/cas9 introduction/background: next generation sequencing has resulted in substantial progress in identification of mendelian immune deficiency syndromes. in some cases, however, putative causal mutations occur in single kindreds, or even individual patients. under these circumstances, functional analysis of patient derived cells combined with in vitro analysis of genetically manipulated cell lines can provide additional evidence in support of genetic causation, but this might not be conclusive. objectives: understanding how genetic defects result in complex syndromes of immune deficiency and immune dysregulation can be impossible to achieve in vitro. one method for overcoming these obstacles is to generate accurate mouse models of human immune deficiency methods: mouse models of human immune deficiency are a valuable tool in which the murine genome is engineered to introduce a mutation orthologous to that discovered in the patient. we have applied this strategy to elucidate causation and mechanism of immunological defect in several mutations affecting the nf-kb pathway. results: so far, defects in both canonical and non-canonical pathways of nf-kb activation have been shown to cause immune deficiency, often associated with immune dysregulation. we describe a known defects and novel putative defect identified in the canonical nf-kb pathway conclusions: crispr-cas9 mouse models can be used to elucidate mechanism of disease and provide compelling evidence that mutations are causative. introduction/background: primary immune deficiencies (pid) with or without immundysregulation are rare diseases resulting from monogenetic aberrations leading to either infections or autoimmune manifestations or both. early diagnosis and treatment are crucial for reducing morbidity and mortality. beside genetic diagnosis not commonly yet performed, current standard methods for early diagnosis are dependent on either fresh samples or limited to certain cell types. to overcome those limitations, especially in newborn screening, a novel technology of methylation-based qpcr can be applied. among the known epigenetic modifications, dna demethylation is the most stable and genomic loci with highly cell type specific demethylation sites can be identified. differential methylation can be measured from blood samples of limited availability, or with suboptimal storage. we previously showed that thymic-derived t population, and determine whether there are unique diagnostic and treatment considerations within this demographic. whitney goulstone 1 , elizabeth tough 2 1 executive director, canadian immunodeficiencies patient organization 2 lpn, alberta health services introduction/background: primary immunodeficiency diseases (pids) represent a significant collection of immune system disorders that increase susceptibility to infection, which in some cases are serious or life-threatening. patients with pid often require immunoglobulin g (igg, commonly referred to as ig) replacement therapy to prevent infections and associated comorbidities. pid treatment, in addition to symptoms and associated social and emotional impacts, has a significant impact on patients quality of life (qol). information available on the real-world diagnosis, management, and outcomes of the canadian pid patient population is limited. objectives: to better understand diagnosis and treatment of canadian patients with pid, we surveyed canadian patients with pid associated with the canadian immunodeficiencies patient organization (cipo) the primary goal of the survey was to gain insight into the canadian pid patient population with regards to demographics, diagnosis, treatment, including regimes, qol, and communication and support . methods: the authors conducted a cross-sectional survey to measure health-related qol in a cohort of patients with pid. eligible participants were identified through the canadian immunodeficiencies patient organization (cipo). the questionnaire consisted of 61 questions that covered patient-reported outcomes including diagnosis, qol, treatment regimes, and communication. results: surveys were returned by 149 patients with pid. participants conveyed significant impact on qol including personal, occupational, financial, emotional, and social impacts as a result of pid symptoms, risks, and treatment logistics, limitations, and side effects. the most common diagnoses were related to b-lymphocyte disorders (60.4%). common treatments include intravenous immunoglobulin (ivig; in hospital) and subcutaneous immunoglobulin (scig; at home), in addition to antibiotics and antifungals as required. respondents reported feeling average before treatment on a scale of 0-10 (mean 5.46 ± 2.23) with increased health after treatment (mean 6.66 ± 2.32). respondents felt current treatment was convenient (mean 8.22 ± 1.94) and were comfortable with self-infusions (mean 6.96 ± 3.46). conclusions: patients with pid are not uncommon in the canadian community, and in these patients pid is associated with a significant impairment in qol. experiences range with regards to a particular treatments advantages and disadvantages, cost, travel, and convenience. respondents hope to achieve improved qol through the following solutions: better treatment, improved infusions, gene modification, more research and clinical trials, a cure, and education and outreach. improved financial, medical, and social supports were also requested. introduction/background: severe combined immunodeficiency (scid), the most severe form of t cell immunodeficiency, is detectable through quantification of t cell receptor excision circles (trecs) in dried blood spots (dbs) obtained at birth. for many professional, humanitarian and financial reasons, newborn screening (nbs) for scid is warranted. implementation of this screening test is highly important where high frequency of consanguinity is known to exist. objectives: since october 2015, israel has conducted national scid nbs. this important, life-saving screening test is available at no cost for every newborn in israel. methods: herein, we describe two years results of the israeli scid newborn screening (nbs) program. validation includes cbc, lymphocyte subsets, trec (in a different method), t cell receptor (tcr) repertoire and response to mitogenic stimulation. whole exome sequence (wes) for genetic detection, and next generation sequencing (ngs) to demonstrate tcr clonality are used as well, in some unsolved cases. results: of 396,159 births screened, 719 (0.18%) had abnormal trec in their first screen (448 terms, 271 pre-terms), 69 (0.016%) had a repeated abnormal trec also in their second screen and were referred for a validation process. fourteen scid patients were diagnosed, so far, through the nbs program in its first years, revealing an incidence of 1:28,000 births in the israeli population. consanguine marriages and muslim ethnic origin were found to be a risk factor in affected newborns, and a founder effect was detected for both il7r and dclre1c deficiency scid. other diagnoses were made as follows: 8 cases were found to have t cell lymphopenia; 16 cases were diagnosed with syndromes; 4 cases were found to have secondary t cell lymphopenia; 10 cases were pre-term infants and 18 cases were considered as false positive with normal evaluation. lymphocyte subset analysis and trec quantification in the peripheral blood appear to be sufficient for confirmation of typical and leaky scid and ruling out false positive results. detection of secondary targets (infants with non-scid lymphopenia) did not significantly affect the management or outcomes of these infants in our cohort. we could also report several perspectives regarding t cell development in non immunodeficient newborns that emerged from the accumulated data. conclusions: already in a short term, trec nbs in israel has achieved early diagnosis of scid and other conditions with t-cell lymphopenia, facilitating management and optimizing outcomes. this program has also enabled gaining insights on t cell development in health babies. (hct) . surprisingly, 76% of these infections were acquired during the interval between confirmation of the scid diagnosis and hct (heimall, blood 2017). to investigate further, in late 2017 we surveyed pre-hct management practices for scid patients at pidtc centers. 51 physicians representing 43 north american centers responded, including 18 immunologists, 25 transplant specialists and 7 who identified as both. 33% of centers lacked a standard procedure for the management of infants with a positive nbs result. to confirm the scid diagnosis, basic t, b and nk cell flow cytometry was performed by most. testing for naïve t cells was generally included, but testing of lymphocyte mitogen proliferation was inconsistent. specialists were notified of a patients positive nbs test at median age 7.5 days (2-30 days) , and management of patients as scid was started at median age 9 days (0-90 days). when unconditioned hct was anticipated, 72% of respondents began planning as soon as possible after diagnosis, while 20% awaited genetic testing results before hct. respondants consistently implemented pre-hct prophylaxis with trimethoprim/sulfamethoxazole (84%), fluconazole (80%) and immunoglobulin infusions (96%), although timing of initiation varied. palivizumab was used by 26%. there was little consensus regarding viral monitoring. although most physicians screened for cmv by blood pcr (85%), only about half routinely screened for ebvor adenovirus. while 44% of physicians started prophylaxis against double-stranded dna viruses in all patients, 46% did so only in selected situations, such as active genital hsv at the time of delivery, or when the mother was cmv-seropositive. although all centers used only acyclovir as antiviral prophylaxis, doses and timing varied widely: from 25-90 mg/kg/day, divided into 2 or 3 doses, continued in most centers until immune reconstitution. finally, 84% of physicians recommended that cmvseropositive mothers stop breast-feeding. there was no consensus on where patients should reside prior to hct. hospital or home were favored equally, although need for a reliable family was indicated by 88% as a criterion for home-based management. for hospitalized patients, 62% of centers required patients to be in a reverseisolation/positive-pressure room and over half required staff to wear gown, gloves, and mask. with regard to visitors, 75% required parents/ relatives to perform hand hygiene. approximately 75% did not require a gown, gloves, or mask for visitors. finally, there was no consensus on allowing siblings or friends to visit, but the majority permitted grandparents or other adult relatives. this survey revealed wide variability in the diagnostic pathway, viral surveillance, and isolation practices for scid patients, although pretransplant prophylaxis with immunoglobulin, fluconazole, and trimethoprim/sulfamethoxazole were utilized consistently. we conclude there is considerable opportunity to develop diagnostic and pre-hct management pathways for scid. prospective tracking of management practices could reveal which are important for avoiding pre-hct infections. evidence-based practice guidance is needed to maximize the potential to bring each scid patient identified via nbs to hct infection-free. ikaros/ikzf1 is an essential transcription factor expressed throughout hematopoiesis. in humans, somatic mutations in ikzf1 are linked to b-cell acute lymphoblastic leukemia (all), and germline heterozygous haploinsufficient mutations cause common variable immunodeficiency-like disorder with incomplete penetrance. herein, we report seven unrelated patients with an earlyonset novel combined immunodeficiency associated with de-novo, fully-penetrant, germline heterozygous dominant negative mutations affecting amino acid n159 in ikzf1 dna binding domain. patients presented with different infections, but pneumocystis jirovecii pneumonia was common to all. one patient developed a t-cell all. additional findings included decreased b-cells, neutrophils, eosinophils and myeloid dendritic cells as well as t-cell and monocyte dysfunction. t-cells exhibited a profound naïve/recent thymic emigrant/ t-helper 0 phenotype and were unable to evolve into effector memory cells; monocytes failed to respond to different stimuli or facilitate t-cell activation. this new defect expands the spectrum of human ikzf1-associated immunodeficiency diseases from haploinsufficient to dominant negative. the beta subunit of the il-2 receptor (il2rb; cd122) is essential for il-2 and il-15 mediated signal transduction in a variety of hematopoietic cell types including t and nk cells. here we report the clinical and immunologic phenotypes of two siblings born to consanguineous parents harboring a variant in il2rb, resulting in autoimmunity, with lymphoproliferation of cd8+ t and cd56hi nk cells, and decreased regulatory t cell frequency. the proband presented with inflammatory enteropathy, failure to thrive, and disseminated cmv infection at 2 months of age, and later developed atopy, lymphocytic interstitial pneumonitis, and red blood cell autoantibodies. his younger sister presented with severe autoimmune hemolytic anemia and cmv viremia at 2 months of age, and later developed lymphocytic interstitial pneumonitis. whole exome sequencing and chromosomal microarray studies revealed a homozygous deletion within the highly conserved wsxws motif of the extracellular domain of il2rb (c.665_673delcctggagcc, p.pro222_ser224del). the deletion results in reduced il2rb expression (cell surface and intracellular) in t and nk cells. the functional consequences of the defect include complete impairment of stat5 phosphorylation through the il-2 receptor but only partial impairment through the il-15 receptor, as well as a compensatory increase in serum il-2 and il-15 levels (>100 pg/ml). cd8+ t cell proliferation responses to in vitro t cell receptor (tcr) stimulation were reduced compared to an age-matched, healthy control, but partially rescued by supraphysiologic levels of il-2 and il-15. despite reduced cd8+ t cell proliferation responses, the proband displayed a t cell population skewed toward cd8+ t cells, with an oligoclonal expansion of effector memory cells. arguing against effects of pervasive cmv infection alone, the sister displayed similar phenotypic and functional abnormalities at birth, prior to her cmv infection. our data suggest that the identified hypomorphic mutation in il2rb results in a survival advantage for those cd8+ t and cd56hi nk cells most sensitive to the exuberant serum il-2 and il-15 levels produced in response to the defect. these surviving cd8+ t and cd56hi nk cells have great lymphoproliferative potential, leading to multisystem autoimmunity and inflammatory complications. therefore, we describe il2rb deficiency as a novel primary immunodeficiency disease with prominent immune dysregulation and selective cd8+ t and cd56hi nk cell lymphoproliferation. key: cord-022203-t2f0vr1w authors: dowers, kristy l; lappin, michael r title: the pyrexic cat date: 2009-05-15 journal: problem-based feline medicine doi: 10.1016/b978-0-7020-2488-7.50024-7 sha: doc_id: 22203 cord_uid: t2f0vr1w nan • temperature > 39.2˚c (102.5˚f). • true fever results from a cascade of events, which starts with activation of leukocytes. pyrogenic factors released from the leukocytes increase the thermoregulatory set point in the hypothalamus. signs that may be associated with fever include: • elevated body temperature. • reluctance to move. • anorexia. • depression. • hyperpnea. • muscle or joint stiffness/discomfort. • shivering. • inflammation anywhere in the body can result in elevation of core body temperature above 39.2˚c (102.5˚f). • the most common etiology for fever in the cat is percutaneous cellulitis or abscess. viral diseases such as fiv, felv and fip are important diseases to consider. conjunctivitis is the predominant sign and is often initially unilateral and becomes bilateral. ocular discharge is serous initially then mucopurulent, but is usually mild. fever, anorexia and lethargy may occur. true fever must be differentiated from hyperthermia, which can be caused by increased muscle activity, increased environmental temperature and stress. true fever results from activation of leukocytes that release factors (pyrogens) such as interleukin-1 and tumor necrosis factor. • these factors cross the blood-brain barrier and increase the thermoregulatory set point in the hypothalamus. • leukocytes are activated by a multitude of infectious agents, neoplasia, tissue necrosis and immune-mediated diseases. fever is defined as systemic elevation of core body temperature above 39.2˚c (102.5˚f). the most accurate measurement of core body temperature is obtained rectally. aural temperature is approximately −17.2˚c (0.5˚f) lower than the rectal temperature. fever is a general clinical sign that can be associated with many different diseases. the most common disease causing fever in the cat is percutaneous cellulites or abscess. many viral and bacterial diseases cause fever because leukocytes are recruited and activated as part of the general immune response. organ inflammation, such as pancreatitis, cholangiohepatitis and myocarditis, can be associated with an elevated temperature even when an infectious agent is not present. classical signs • fever. • anorexia (partial or complete). • reluctance to move, lethargy and depression. • pain, heat or swelling at site of abscess or cellulitis. cellulitis usually precedes an abscess, and if treated appropriately, the abscess may not even form. cellulitis may be the only evidence of a previous abscess. an abscess may rupture spontaneously, and the owner may notice foul-smelling, purulent discharge on the fur. • some abscesses resolve on their own with or without rupture, if they have been present long enough. regional lymphadenopathy may occur near the affected site. cellulitis spreads rapidly with the development of multiple fistulae and a febrile response. • lameness from septic arthritis is a common sequelae to infection with l forms. joints are affected by the hematogenous route and may be distant to the initial site. lower limbs (tarsus and carpus) are most commonly affected. the joints often ulcerate with a grayish mucinous exudate. infection remains confined to subcutaneous tissues and joints without systemic spread to internal organs. history supports access to outdoors or conflict with other cats indoors. palpation reveals a tender area or fluctuant swelling, with or without evidence of puncture wounds. microscopic examination of a fine-needle aspirate of the abscess reveals a heterogeneous population of bacteria, numerous degenerate neutrophils and intracellular bacteria. a complete blood count will generally show neutrophilia. l forms are not visible in tissue samples even with special stains, nor do they grow on culture. on electromicroscopy, organisms are visible intracellularly within phagocytes. diagnosis is often made by response to tetracyclines in a therapeutic trial (doxycycline 10 mg/kg po, q 24 h). response is rapid and evident within 48 h. non-healing abscesses should have histopathology and culture of tissue. causes include nocardia, fungi, mycobacteria, and tumors. see page 1081, the cat with non-healing wounds. in plague-endemic regions, yersinia pestis (plague) must be considered, if the swelling is predominately in the neck region and the cat's fever is in the region of 40.5˚c (105˚f). cautionary measures such as gloves, masks and isolation of the suspect cat should be taken until diagnosis established. (see below for discussion of y. pestis infections). fracture. ligament/tendon injury. neoplasia. clip area looking for evidence of puncture wounds. drainage of the purulent material is the key to treatment. surgical drainage can be done under sedation or general anesthesia with a #15 blade. make a 1/4-1/2" incision over the dependent area, or the area most likely to allow for continued drainage. flush the wound thoroughly with sterile saline or a saline/betadine mixture. explore the wound with a sterile cotton swab or hemostats to assess the extent of dead-space and to look for a possible foreign body. leave the wound open to allow drainage of further purulent material. do not suture incision closed, as this will only allow the abscess to reform. a penrose drain may be placed for 2-3 days to allow maximum drainage for abscesses that close too early. antibiotic therapy for 7-10 days directed against anaerobes: penicillins, cephalosporins, clindamycin and metronidazole are reasonable choices. most abscesses respond extremely well to drainage and amoxicillin at 10-20 mg/kg po q 12 hours for 7 days or amoxicillin/clavulonic acid (12.5 mg/kg po q 12 hours). l-forms and mycoplasma spp. respond to doxycycline or tetracycline within 48 hours, but not other antibiotics. if the wound is not healing well, or the cat has had recurrent abscesses, felv/fiv testing is recommended to rule out an underlying immunodeficiency. further considerations are inappropriate antibiotics (consider culture and sensitivity testing) or the presence of an undetected foreign body (consider surgical exploration of the area) or involvement of underlying bone (osteomyelitis). prognosis is good unless there is an underlying immunodeficiency. restrict the cat to an indoor environment only; although less effective, confine cat indoors at least from dusk to dawn. neuter male intact animals to decrease territorial behavior. felv and fiv serology should be repeated 2-4 months following bite wounds. classical signs acute onset of sneezing followed by oculonasal discharge. discharge progresses from serous to mucoid to mucopurulent. severe conjunctivitis with tearing, photophobia and chemosis. hypersalivation may occur as an initial sign before the classic signs of upper respiratory tract appear. punctate corneal ulcers that may coalesce to larger ulcers or perforation. fever of 1-2 days duration, anorexia and depression. retching or coughing may occur. cats with anterior uveitis have occasionally have herpesvirus 1 in the aqueous humor. presumptive diagnosis can be made on the basis of history and clinical signs because treatment for feline herpes virus-1 and calicivirus are similar. ocular ulcerations and chemosis are more suggestive of fhv-1. definitive diagnosis is by direct ifa of cells obtained from conjunctival or nasal scrapings, or by viral isolation or polymerase chain reaction assays from oropharyngeal or nasal swabs. sudden onset of serous ocular discharge and mild conjunctivitis; these signs may begin unilaterally, but often progress bilaterally. initial signs are rapidly followed by sneezing, which are not paroxysmal and are less prominent than in herpesvirus. nasal discharge is primarily serous to mucoid and rarely progresses to purulent. oral ulcerations are common, especially on the tongue, and may be associated with drooling or hypersalivation. ulcers may also occur at the mucocutaneous junction, hard palate and nose. fever generally spikes initially after infection prior to onset of signs, and returns with onset of clinical signs. viral pneumonia occurs occasionally with certain strains, and may produce significant mortality. death is often sudden and preceded by laboured respiration. a rare variant strain (fcv-ari) reported from the united states, produces a high fever, facial and paw edema (50% of cats), ocular and nasal discharge, conjunctivitis and ulcerative stomatitis (50% of cats), hemorrhage from the nose, git, etc. (30-40% of cats), icterus (20% of cats) and rapid death. mortality is high (30-50%). presumptive diagnosis can be made on basis of history and clinical signs because treatment for feline herpes virus-1 and calicivirus are similar. oral ulcerations or clinical signs of pneumonia are more suggestive of calicivirus. definitive diagnosis is by viral isolation or reverse transcriptase polymerase chain reaction assays from swabs taken from the oropharynx, ideally in the first week of illness. demonstration of increasing serum antibody titers to feline calicivirus in paired samples is also useful, whereas measurement of a single titer is not useful because many cats have titers from vaccination. identification of fcv-ari is based on the clinical syndrome, pathology and culture of virus from blood, nasal or ocular discharge, spleen or lungs. clinical signs are often non-specific and include fever, anorexia and weight loss. dyspnea and harsh lung sounds without coughing is common. peripheral and visceral lymphadenopathies are frequently present. pale mucous membranes, icterus, hepatomegaly or splenomegaly may be evident. ocular signs are uncommon, but can occur. gastrointestinal signs are uncommon in cats compared to dogs, and include chronic diarrhea, mesenteric lymphadenopathy and anorexia. osseous lesions produce soft tissue swelling and lameness. diagnosis is by demonstration of the organism in lymph nodes, draining tracts, bone lesions or vitreous humor. the organism has a thin capsule and is intracellular within macrophages. no reliable serologic test available. genetic predisposition appears to play a role. fip is most common in catteries and multi-cat households. there are two clinical forms of fip, effusive or wet form and non-effusive or dry form. both are characterized by a fluctuating fever unresponsive to antibiotics, anorexia, lethargy and weight loss. typical age of onset is 6 months to 2 years, but any age can be affected. the effusive form may have any of the following signs: • abdominal effusion that is non-painful but progressive. the amount of effusion varies from volumes causing abdominal enlargement, to amounts only detectable by abdominocentesis. fluid is straw-colored and highly viscous, like egg white. • pleural effusion resulting in dyspnea occurs in 30% of cats with the effusive form. pericardial fluid may be evident on ultrasound. usually it not associated with clinical signs, but occasionally can produce cardiac tamponade. • male cats may present with scrotal swelling. the non-effusive form may have any of the following signs: • ocular signs result from pyogranulomatous inflammation of the iris and ciliary body. they include bilateral uveitis, perivascular exudates (cuffing), retinal hemorrhage, retinal detachment. • neurologic signs include cerebral and cerebellarvestibular signs such as seizures, personality changes, nystagmus, head tilt, circling, head tremor and hyperesthesia. • dysfunction of any organ system may result from granuloma formation within the tissue of that organ, e.g., liver, kidney, spleen, intestines, lungs, etc., however, organ failure producing clinical signs only rarely occurs, and most dysfunction is only detected on biochemical tests. • granulomatous masses may be palpable in abdominal viscera especially mesentery, mesenteric lymph nodes and omentum as tender, irregular masses. occasionally vomiting or diarrhea results from extensive lesions on the bowel wall. jaundice may occur with either form of the disease. histopathology of affected tissues provides the only definitive antemortem diagnosis. the classic fip lesion is pyogranulomatous infiltration around venules. the following are typical abnormalities associated with fip. all asterisked items must be present for a high likelihood of fip; if any one parameter is not present, fip is unlikely. a negative coronavirus ("fip") titer suggests fip is not the cause of the fever, although a few cats with the effusive form of the disease are titer negative. lymphopenia (< 1.5 × 10 3 cells/μl).* occurs in many cats with fip, and many cats without fip. except where the classical effusive fluid is present, definitive diagnosis of fip requires organ biopsy and demonstration of classical histopathological lesions. various non-specific abnormalities may be evident on laboratory tests, including increased total white cell count, mild to moderate anemia, and increased concentrations of bilirubin, liver enzymes, bun, creatinine, fibrinogen, globulin and mild proteinuria. csf typically has increased protein (> 2 g/l) and cell counts (>100 cells/ml) which are predominantly nonlytic neutrophils. ocular signs: toxoplasmosis, fungal agents. neurologic signs: toxoplasmosis, neoplasia (e.g., lymphoma), trauma, congenital abnormalities in young cats. other clinical signs: rule out other diseases associated with the apparent organ dysfunction. lymphocytic, plasmocytic cholangiohepatitis occasionally produces a high protein abdominal fluid similar to that of effusive fip. fip is a fatal disease with no known treatments. the therapies listed below have been used in an attempt to slow progression and/or to improve quality of life. glucocorticoids at immunosuppressive doses (prednisolone 4 mg/kg/day). cyclophosphamide (200-300 mg/m 2 q 2-3 weeks or 2.2 mg/kg daily for 4 days each week) or chlorambucil (20 mg/m 2 q 2-3 weeks). +/− broad-spectrum antibiotics to control secondary bacterial infections while the cat is immunosuppressed. prognosis is poor. the mortality is > 95%. fecal-oral transmission is most likely; transplacental transmission is rare. fomites, e.g., food bowls and litter trays, may be an important mode of transmission, as some strains of fcov survive in dried secretions for several weeks. a seronegative cat introduced into a household where coronavirus is endemic has a 1 in 6 chance of developing fip; a seropositive cat under the same conditions has a 1 in 12 chance. both young and old animals seem to be most susceptible due to vulnerable immune systems. maternal antibodies that protect kittens wane at approximately 5-6 weeks of age. reduce fecal-oral contamination by providing one litterbox for every 1-2 cats, cleaning litterboxes daily, and placing litterboxes away from feeding areas. minimize stress, especially crowding in catteries. do not introduce fcov-positive cats into a multi-cat household. wean kittens at 5 weeks and remove from the queen's environment if she is seropositive. an intranasal vaccine is available for use in seronegative cats. however, efficacy has not yet been demonstrated against wild strains. classical signs see main reference on page 540 for details (the anemic cat). onset of illnesses occurs over an extended period of time (months to years), although young kittens can become acutely ill. chronic, opportunistic infections occur that do not respond to appropriate antibiotic therapy and are primarily due to immunosuppression. fever may occur in any age cat but is primarily seen initially in the viremic stage or later in response to neoplastic, inflammatory or immunosuppressive effects. chronic fever occurs in later stages of disease. weight-loss/cachexia. non-regenerative anemia. thrombocytopenia. lymphoma is associated with felv-positive cats, especially thymic and multicentric forms. history and clinical signs may be suggestive. complete blood count showing anemia, thrombocytopenia, leukemias, increased mcv and leukopenia are supportive. bone marrow aspirate may show myeloproliferation and arrested erythroid differentiation. a positive felv antigen test (viral core antigen p27) on whole blood using an ifa (can also be done on bone marrow sample) or an elisa test (also on serum, plasma, saliva, tears). see page 543 for interpretation. polymerase chain reaction is available from some laboratories. • pale mucous membranes. see main reference on page 530 for details (the anemic cat). classical signs are pale mucous membranes and/or icterus primarily from extravascular hemolysis due to complement binding of infected erythrocytes. severe, regenerative hemolytic anemia may ensue. anorexia and depression are typical. fever occurs in 50% of cats in the acute phase, and may occur intermittently in chronic infections. history and clinical signs are suggestive, especially if an immunosuppressive disorder is present concurrently. diagnosis is via demonstration of the organism on the surface of erythrocytes. use a marginated blood sample for diagnosis, e.g., ear vein. multiple blood smears over a number of days may be required as most of the organisms are removed from circulation by the time clinical signs are apparent. infected cats may be coomb's positive. a polymerase chain reaction test is available in some laboratories for diagnosis. classical signs gastrointestinal signs, primarily abdominal discomfort and small bowel diarrhea, are due most likely to replication of the organism (tachyzoites) in enteroepithelial cells resulting in necrosis. clinical signs in the acute, fatal form of extraintestinal disease are caused primarily by tissue damage from the rapidly dividing tachyzoites. tachyzoites begin to disappear from tissues approximately 3 weeks after infection. the organism may persist in tissues as tissue cysts containing bradyzoites. chronic disease may be a result of delayed hypersensitivity reactions and tissue reaction to antibody-antigen complex deposition. concomitant illness, such as felv, fiv and immunosuppression with glucocorticoids, has been reported in some cases. gastrointestinal disease. • mild, self-limiting small bowel diarrhea may occur in the definitive host (cats), but only after ingestion of tissue cysts, oocysts or sporulated oocysts. • young kittens are more likely to have gastrointestinal signs, although mild clinical disease has been reported in adult cats as well. all newborn kittens experimentally infected developed severe diarrhea 5-6 days later. • fatal extraintestinal disease is most likely to occur in transplacentally infected kittens. • kittens may be stillborn or exhibit signs that are severe and rapidly progressive and reflect involvement of the lungs, liver and cns tissues. these signs may also be observed in postnatally infected kittens and include: -a distended abdomen from an enlarged liver and/or ascites. -icterus from hepatitis or cholangiohepatitis. -dyspnea is present in most kittens and cats with signs of acute infection. -neurologic deficits; continuous vocalization; excessive sleeping. -fever, anorexia, depression often accompanies the tissue-specific signs. • cats may have a moderate fever, lethargy and depression that waxes and wanes. • hyperesthesia and stiff painful joints or shifting lameness may be evident, presumably due to an immune-mediated process. • unilateral or bilateral anterior or posterior uveitis may occur with possible sequelae of lens luxation, glaucoma or retinal detachment. • seizures and ataxia may be present if cns tissues are involved. • rarely, a toxoplasma granuloma (tissue cyst) forms in the gastrointestinal tract or pancreas causing chronic vomiting. clinical signs consistent with toxoplasmosis are suggestive, especially when other causes of the signs have been ruled out. igm titers > 1:64 and a four-fold increase in igg:igm titers within 2 weeks correlate best with clinical toxoplasmosis. however, some cats do not develop detectable igm titers, and in other cats, positive igm titers can persist for months to years after infection. elevated ocular and csf titers relative to serum titers in cats with ocular or neurologic signs, respectively, are very suggestive. coefficient values > 1.0 are highly suspect and > 8.0 strongly suggest local production of t. gondii antibodies. response to therapy for toxoplasmosis is a useful indicator of infection. definitive diagnosis requires demonstration of the organism in inflamed tissues by histology, immunohistochemistry or polymerase chain reaction assay. rule out diseases associated with affected organs, e.g., fip for neurologic and ocular signs. clindamycin at 10-12 mg/kg orally q 12 hours for 4 weeks is usually effective. • cats should respond within several days of treatment. • if no response is evident after 3 weeks of antibiotic therapy, reconsider the diagnosis. • the chronic form may recur even after successful treatment, as drugs tend to suppress replication rather than kill the parasite. other systemic drugs with potential efficacy include the trimethoprim sulfas combination, doxycycline, minocycline, azithromycin and clarythromycin. cats with ocular lesions should also be treated with corticosteroids, either topically (e.g. topical 0.5% prednisolone acetate drops applied q 6-12 h) or systemically to control inflammation and its sequelae (glaucoma, lens luxation). gastrointestinal disease has a good prognosis, although it may lead to inflammatory bowel disease in rare cases. acute extraintestinal disease has a guarded to poor prognosis. chronic extraintestinal disease has a fair to good. • the placenta or milk with tachyzoites. • ingestion of meat infected with tissue bradyzoites, e.g., rodents. • ingestion of sporulated oocysts in food or water. t. gondii has a zoonotic potential. infection of humans can occur via: • ingestion of undercooked meat containing tissue bradyzoites (most common mode of transmission). • ingestion of sporulated oocysts from the environment. • transplacentally, if first-time exposure to the organism occurs during pregnancy. only cats host the sexual replication that results in oocysts in the feces. • oocysts are shed for 1-2 weeks. • most seropositive cats do not shed oocysts on repeat exposure. oocysts must sporulate to be infectious: • sporulation occurs 1-5 days after environmental exposure, thus handling individual cats rarely results in infection of humans. transplacental transmission occurs in cats and people after primary exposure. discourage cats from going outdoors and hunting behavior. do not feed cats undercooked meat. • cook meat at 80˚c (176˚f) for 15 minutes. • use gloves when gardening or changing the litterbox, and wash hands well. • change litterboxes daily. use litterbox liners or clean with scalding water. • lethargy and anorexia. see main reference on page 272 for details (the cat with depression, anorexia or dehydration). the classical signs are not as well-defined for cats as for dogs for the following reasons: • cats tend to have intermittent bouts of chronic pancreatitis. • diagnostic tests for pancreatitis are not as reliable in cats. • there is poor correlation of biochemical parameters with pancreatitis in the cat. lethargy and anorexia is variable depending on chronicity. vomiting only occurred in 35% of cases in one study. dehydration occurred in 50% of cases in the same study. abdominal pain is quite variable. fever is variably present, and generally mild. in severe acute pancreatitis it may progress to hypothermia, which is a poor prognostic sign. diagnosis is unreliable based on a biochemistry panel. lipase may be increased or normal in pancreatitis. • hyperbilirubinemia and elevated liver enzymes may be present. • hypocalcemia occurs in 40% (total serum calcium) or 60% (plasma ionized calcium concentration) of cats due to soponification of fat. cats with a plasma ionized calcium concentration < 1.00 mmol/l (< 4.00 mg/dl) have a grave prognosis (77% mortality) and aggressive medical treatment is indicated. pancreatic lipase immunoreactivity is probably a more sensitive diagnostic tool for confirming pancreatitis in cats than measurement of plasma lipase concentration or trypsin like immunoreactivity. a feline-specific assay must be used. abdominal ultrasound to visualize an enlarged pancreas or heterogeneous echogenicity in the area of the pancreas is considered by many to be most sensitive. demonstration of higher lipase levels in abdominal fluid compared to those of the serum is suggestive. diagnostic peritoneal lavage may be necessary to obtain a fluid sample. clinical signs may be acute, chronic or intermittent. typically, there is anorexia and depression together with icterus or increased bilirubin and liver enzymes on a biochemistry panel. vomiting and dehydration may be present. fever, especially in the suppurative form occurs in approximately 38% of the cases. chronic cholangiohepatitis may lead to end-stage liver disease and the cat may present with ascites and hepatic encephalopathy. multiple causes include bacterial, protozoal (t. gondii) and immune-mediated disease. complete blood count may show neutrophilia with a left shift, and mild non-regenerative anemia. biochemistry panel shows hyperbilirubinemia, elevated liver enzyme activities (alp, alt, ggt), +/− elevated serum bile acids. • signs of late-stage liver disease are occasionally present, such as decreased bun, glucose and albumin concentrations. abdominal ultrasound should be performed to evaluate the gall bladder and bile duct for cholelithiasis, bile sludging and cholecystitis. liver aspirates/biopsy allows for differentiation of suppurative from non-suppurative forms of cholangiohepatitis. clinical signs are primarily due to immunosuppression, i.e., chronic recurring infections that do not respond to appropriate therapy. gingivitis, stomatitis and peridontitis are more common findings in fiv infections than in felv, although one study suggests that these signs may be to an effect of age, rather than a consequence of fiv infection. fever is chronic and is related to production of tumor necrosis factor and/or il-1 in infected cats. weight loss/cachexia are common in the late stages of fiv, as in human hiv infections. diarrhea resembles a panleukopenia-type syndrome that may be due to actual enterocyte infection by the virus or secondary to inflammation. cats are often thin and scruffy with an unkempt haircoat, and may have miliary dermatitis. diagnosis may be suspected based on history and clinical signs, but requires antibody or antigen tests for confirmation. virus isolation and polymerase chain reaction for virus detection is available at some research facilities. cats infected with fiv can be co-infected with felv. chronic nasal discharge can be unilateral or bilateral and is generally serosanguineous. sneezing and stertorous breathing is often present. facial deformity may occur due to invasion of the surrounding bone. chronic low-grade fever may be present. depression, anorexia and weight-loss are signs of disseminated disease. neurologic signs occur via hematogenous spread or invasion into the cns through the cribiform plate but are uncommon. signs include seizures, blindness, depression and ataxia. the skin form typically produces nodules which often ulcerate. diagnosis is by demonstration of narrow-based budding yeast with a very thick capsule from affected tissue or by culture of affected tissue or csf. demonstration of cryptococcus antigen in serum, urine or csf is also diagnostic. occurs in cats of all ages, with and without outdoor access. progressive clinical signs occur over a period of 1-4 weeks. according to one study, non-supportive meningoencephalitis may be the most common cause of seizures in cats. systemic signs, which are not present in all cats, include fever, anorexia, lethargy, vomiting, diarrhea and lymphadenopathy. the condition, however, does not appear to be contagious to other cats. csf tap can be very useful to rule out other causes of cns signs, specifically toxoplasmosis and fip; csf analysis reveals a normal or mild protein elevation (typically < 1 g/l) and/or an increased white cell count (< 50 cells/μl). complete blood count findings are non-specific and may include leukopenia or leuko-cytosis, eosinophilia and anemia. • uniphasic or biphasic fever. • depression. • lethargy. • mild generalized lymphadenopathy. • +/-signs of cardiac failure. • viral, e.g., fip has been shown to cause cardiac infection. • trypanosoma cruzi, which causes chagas' disease in humans. • streptococcus and borrelia (lyme's disease) in certain geographic areas. no single agent has been identified, and the disease may be multifactorial. fever is biphasic in 50% of the cats; if biphasic: • first fever occurs approximately 10 days after exposure, lasts 1-3 days and peaks at 39.3-40.7˚c (102.7-105.2˚f). • second fever occurs 1-2 weeks after the first fever (at 3-4 weeks post-exposure), lasts 5 days and peaks at 39.9-40.9˚c (103.8-105.6˚f). appetite is mildly decreased in some cats, but most continue to eat and drink. some animals exhibit mild generalized lymphadenopathy. irritable disposition and hyperesthesia may occur, and are most likely due to fever and malaise. in a few case reports, cats have died from peracute cardiac failure, but this outcome is not common. myocarditis/diaphragmitis is a diagnosis of exclusion. biochemistry and complete blood counts are unremarkable, except for a mild to moderate increase in ck in less than 50% of experimentally infected cats. definitive diagnosis can only be made at necropsy. histopathology shows a neutrophilic infiltrate with a foci of myonecrosis in myocardium and diaphragm. any other causes of fever should be ruled out including infectious, inflammatory, immune-mediated, drugs, neoplasia and metabolic. other causes of cardiac failure that should be ruled out include congenital deformities, hypertrophic cardiomyopathy, restrictive cardiomyopathy and dilatative cardiomyopathy. supportive therapy is indicated if dehydration or cardiac disease are present. broad-spectrum antibiotics are indicated if complete blood count supports an infectious cause. fever and depression resolve spontaneously in the majority of cats. prognosis is poor if peracute cardiac failure is present with systemic signs of fever and depression. although an infectious agent is suspected, no single etiologic agent has been identified, making recommendations for prevention difficult. • cardiovascular or respiratory compromise. • external signs of injury. cardiovascular compromise may result in tachycardia, hypovolemia or hypotension. respiratory compromise may produce dyspnea/ tachypnea due to pneumothorax, hemothorax or pyothorax. internal injuries may result in abdominal pain from organ rupture, bone/joint pain or focal swelling. diagnosis is based on clinical signs and history. radiographs of the chest, abdomen and/or limbs may be required to characterize the injury. complete blood count and biochemistry panel is indicated to rule out specific organ injury and primary infection. • alert, febrile cat being treated with antibiotics or antifungal agents. history of treatment with antibiotics or antifungal agents. fever does not correspond to clinical appearance of animal. cats are bright, alert and responsive, despite a temperature in the range of 39.4-40˚c (103-104˚f). onset of fever is idiosyncratic and variable, but the fever is generally present for the duration of the drug treatment. tetracycline is the most common antibiotic cause of drug-induced fever in cats. amphotericin b can cause fever by disrupting cell membranes and releasing pyrogens into circulation. be aware that other drugs (griseofulvin, chloramphenicol and chemotherapeutic drugs) can cause bone marrow suppression leading to a cat with fever, neutropenia and secondary bacterial infection. these cats are obviously sick, whereas the drug-induced fever animals are bright and alert in comparison. • history is of treatment with fever-inducing drugs, especially tetracycline and amphotericin b. • clinical signs are inappropriate, that is, the cat appears bright and alert although febrile. temperature normalizes after drug is discontinued. classical signs • sneezing. • conjunctivitis and ocular discharge. see main reference on page 13 for details (the cat with acute sneezing or nasal discharge). marked conjunctivitis is the predominant sign, which often starts unilaterally, but usually progresses to both eyes. classic triad of upper respiratory infection signs including oculonasal discharge and sneezing. serous ocular discharge accompanied by blepharospasm, chemosis and conjunctival hyperemia are initial signs. discharge becomes mucopurulent over the course of the disease. mild to moderate fever can be seen in the acute phase. pneumonia is rarely associated with this infection. history and clinical signs are highly suggestive. cytology of conjunctival scrapings reveal dark blue inclusion bodies (giemsa stain). immunofluorescent antibody staining or polymerase chain reaction assay to demonstrate the organism in conjunctival scrapings is available from some laboratories. • acute onset of depression. • acute onset of vomiting. rapid onset of depression, anorexia, and vomiting especially in peracute and acute disease. fetid diarrhea (may be hemorrhagic) typically follows 1-2 days after initial onset of signs. severe dehydration and electrolyte abnormalities. initial fever followed by hypothermia as the disease progresses. high mortality rate when signs are severe. the disease should be suspected in cats less than one year of age with no history of vaccination and a rapid clinical course. panleukopenia evident on hematology. parvoviral antigen can be detected in feces using the canine parvoviral antigen tests or electron microscopy. histopathologic changes include denuded intestinal crypts and blunted villi (often a post-mortem diagnosis). classical signs francisella tularensis is a gram-negative coccobacillus. clinical signs are associated with gram-negative endotoxins and bacteremia. there are two main strains of the organism, both of which have been isolated from cats. • associated with tick-rabbit cycle. • found only in north america. • highly virulent for laboratory rabbits. • associated with more severe disease in humans. • associated with a more complex cycle involving rodents, ticks, mosquitoes, mud and water. • found throughout the northern hemisphere. • avirulent for laboratory rabbits. history of contact with rabbits, especially if the cat is a hunter. any age of cat can be infected, but younger cats are more susceptible to developing septicemia. the spectrum of illness varies from severely affected to asymptomatic. fever is generally > 40˚c (104˚f). marked depression, anorexia and lethargy, with or without vomiting are typical. on physical examination, peripheral lymphadenopathy, icterus and palpable splenomegaly and hepatomegaly are reported. oral, lingual or pharyngeal ulcers may be present. clinical signs together with a history of exposure to wild rabbits is highly suggestive. hematologic and serum biochemical abnormalities may include panleukopenia, with severe toxic changes in neutrophils, high band neutrophil count, thrombocyto-penia and hyperbilirubinemia. definitive diagnosis is via identification of the bacterial agent by ifa or bacterial culture, but should only be performed in a qualified laboratory. • samples can be obtained from affected lymph nodes, bone marrow, urine or blood. serum antibody titers > 1:120 or a four-fold increase in serum antibodies in samples collected during acute and convalescent phases (10-14 days) are considered diagnostic. fip, fiv, panleukopenia. toxoplasmosis. multicentric lymphoma. antimicrobial efficacy studies have not been done in the cat, therefore therapy is derived from case reports and/or human therapy regimens. enrofloxacin (5 mg/kg q 12 hours iv or po). tetracycline and chloramphenicol may be effective, but because they are bacteriostatic for f. tularensis, relapses can occur. in humans, the drugs of choice are streptomycin and gentamycin. prognosis is poor to fair as mortality rate varies across case reports. f. tularensis has a serious zoonotic potential if there is contact with infected animal tissue. bites from infected ticks, deer flies or mosquitoes are the most common method of transmission. infection can also occur via ingestion of infected meat. • this is the most common method of transmission to humans in cat-associated cases. • the infected cat may have no obvious signs of illness, but have a history of hunting wild animals, especially rabbits. inhalation of aerosolized organisms may also transmit the disease. care should be taken by veterinary and laboratory personnel handling suspected animals or samples being prepared for ifa or culture. discourage hunting behavior in cats. ectoparasite control, especially tick control. onset of illness occurs 24-72 hours after exposure to the organism. transmission to cats is either via ingestion of infected rodents or a fleabite from infected fleas. rapid multiplication of organism causes tissue damage and necrosis. the host immune response contributes to pathology. three forms of the plague exist: bubonic (local infection), bacteremic/septicemic and pneumonic. bacteremia occurs in many cases, resulting in the septicemic or pneumonic form of plague. endemic regions of the world include the western usa, south america, africa, asia, eastern europe. history of hunting rodents, especially in known endemic areas. current flea infestation is evident. acute onset of fever, anorexia, depression over a period of 2-6 days. the clinical course may last 6-20 days. submandibular or cervical swelling associated with lymph nodes (can be unilateral or bilateral). the inflamed, swollen lymph node is referred to as a bubo. subcutaneous abscessation may occur and appear similar to a cat bite abscess. in the pneumonic form (~10% of cases), upper and lower respiratory signs may be present, including sneezing, nasal discharge, coughing, dyspnea/tachypnea. initially, microscopic examination of a lymph node aspirate, especially a markedly swollen lymph node (bubo) should reveal a homogeneous population of bipolar-staining coccobacilli. • blood should be examined in cats with the bacteremic/septicemic form. fluorescent antibody testing of sample provides a definitive diagnosis. culture of organism should be performed by a qualified laboratory only. a four-fold rise in antibody titers (taken 10-14 days apart) is suggestive of plague. these results must be interpreted carefully, as high titers can persist for up to one year after infection. chest radiographs may reveal patchy, nodular lesions if the pneumonic form is present. be aware that the risk of exposing other staff members to the disease should be weighed against the benefit of the diagnostic test. reactive lymph nodes from a percutaneous abscess or tooth-root abscess. • aspirates of cat bite abscesses contain a mixed bacterial population compared to y. pestis, which is homogeneous. neoplasia, although it is less common in the us for cats with lymphoma to have peripheral lymphadenopathy. respiratory signs may be due to other upper respiratory infections (calicivirus, herpesvirus, chlamydophila) or lower respiratory disease (parenchymal lung disease, pleural disease). other diseases which cause high fever (tularemia, toxoplasmosis, fip, etc.). absolute caution must be practiced in all suspect plague cases. cautionary measures include gloves, mask, isolation of animal and limited exposure to other staff members. doxycycline/tetracycline: (1) doxycycline at 5 mg/kg q 12 hours po for 14-21 days or (2) tetracycline 25 mg/kg q 8 hours po. • begin treatment immediately after samples for diagnosis have been collected. • doxycycline is preferred as tetracycline has been associated with relapse. consider aminoglycosides or enrofloxacin (5 mg/kg im q 8 hours) for the first 3 days to avoid placing hands into the cat's mouth (see transmission section below). prognosis for bubonic plague is fair to good. prognosis for the pneumonic form is guarded to fair. prognosis for septicemic form is guarded. persistent fever > 40˚c (104˚f) despite treatment is associated with a poor prognosis. y. pestis has a serious zoonotic potential, and great care should be taken in suspect cats to prevent transmission to humans and other cats. • infected cats are no longer a zoonotic risk after 3 days of antibiotic therapy. infection can also occur via inhalation of aerosolized organism, either from aspirates of infected tissue or nasal discharge/sneezing of cats with pneumonic form. discourage hunting behavior especially during the peak flea season (april to october). provide effective flea control to prevent flea bites. • anorexia and weight loss. see main reference on page 547 for details (the anemic cat). this disease is uncommonly reported in cats and is difficult to diagnose because of its vague and variable clinical signs. age range of cats with documented disease was 2-10 years of age, with no breed or sex predilection reported. infection has a variable effect on appetite, from mild inappetence to anorexia and mild to moderate weight loss. chronic intermittent fever in the moderate range is common. lymphadenopathy was reported in three of 23 cats. hyperesthesia, joint pain or irritable disposition is common. complete blood counts may show a non-regenerative anemia with a leukopenia or a leukocytosis; thrombocytopenia is present in about 25% of the cats. biochemistry abnormalities are uncommon, except for hyperglobulinemia in about 33% of documented cases. a complete blood count and biochemistry panel consistent with chronic ehrlichia spp. infection is suggestive. diagnosis is by demonstrating e. canis and/or anaplasma phagocytophilum serum antibody titers or a positive ifa test. demonstration of morulae in mononuclear cells, neutrophils or eosinophils (rare) is diagnostic. pcr assays can be positive. • anorexia, lethargy, weight loss. • ± fever. • signs depend on tumor type and organ system involved. anorexia, lethargy and weight loss. poorly groomed coat. some cats have a fever associated with neoplasia, which is generally a secondary neoplastic syndrome. tumors which destroy the bone marrow and result in neutro-penia are classically associated with fever. fever may occur with other tumors via other mechanisms, including antibody stimulation from tumor antigens, and tissue necrosis which activates leukocytes to release pyrogenic factors. signs are specific to the organ system involved. lymphoma (mediastinal, gi, renal) , mammary adenocarcinoma, squamous cell carcinoma (nasal, oral) and mast cell tumor are the most common tumors in cats. hematology, biochemistry panel, radiology, ultrasound and/or bone marrow aspirates may be necessary to provide evidence that a tumor is present, especially if it involves the hematopoietic system (leukemia) or is located internally (splenic mast cell tumor). identification of the tumor type is via fine-needle aspirates and/or biopsies. organ dysfunction due to infectious or degenerative disease process. felv/fiv or other immunosuppressive illness. benign masses (granulomas, abscesses, reactive lymph nodes, benign tumors). treatment involves surgical excision of identifiable masses +/− regional lymph nodes, especially for mammary adenocarcinoma, nasal squamous cell carcinoma, splenic mast cell tumor, etc. chemotherapy may be effective and needs to be based on tumor type, e.g., cop (cyclophosphamide, vincristine, prednisolone) protocol in lymphoma cases. radiation therapy is used for local disease only, and response to radiation therapy is tumor dependent (e.g., squamous cell carcinoma). • radiation therapy is most effective after surgical debulking of the primary mass. the effectiveness of radiation therapy may be enhanced with concurrent chemotherapy. classical signs acute onset of fever and malaise are initial clinical signs. vomiting, diarrhea and abdominal pain may occur, however, approximately 50% do not have gastrointestinal signs. dehydration. shock may occur if septicemia/bacteremia develops. mortality rate approaches 10% and may be higher if the cat is concurrently immunosuppressed. typically, the cat is an outdoor cat with a history of hunting behavior, especially of birds. complete blood count and biochemistry panel supports infectious diarrhea or septicemia, e.g., neutropenia with a left shift, bacterial rods in blood leukocytes if overwhelming sepsis present, hypoglycemia, hypoproteinemia, pre-renal azotemia. blood cultures provide the best definitive diagnosis if positive. three separate samples over a 4-6 hour period should be taken during febrile episodes using aseptic techniques. fecal cultures may isolate salmonella organisms, but because many animals are subclinical carriers, positive culture does not prove that the organism is the cause of the clinical signs. • skin lesions. • respiratory signs. • ocular lesions. • fever, anorexia, depression. the geographical distribution includes south-west usa, central america and south america in areas that have sandy soil with low rainfall and high temperatures. soil is the reservoir for infection, and the highest frequency of cases occur when the soil is dry and dusty, and organisms are disseminated in the wind. most humans and animals in endemic areas become infected, but the majority of infections are subclinical or cause only mild, transient clinical signs. cats are more resistant to infection and signs are less common than in dogs. infection is contracted via inhalation, and only a few organisms are required to produce signs, which occur after 1-3 weeks. initial infection is confined to the respiratory tract, but dissemination may occur resulting in chronic disease over months or years with signs referable to bones, eyes, central nervous system and abdominal organs. localized infection following a penetrating skin wound appears to be rare. cats appear to be resistant to clinical disease. skin lesions are the most frequent types of infection in cats and were reported in 56% of cats in one study. • lesions begin as small bumps and progress to abscesses, ulcers or draining tracts. • in cats, underlying bone involvement is uncommon. systemic signs such as fever, anorexia and depression are commonly reported (44% of cats) and can be seen with skin lesions. respiratory signs such as coughing and wheezing are less common in cats and occur in approximately 25% of cases. musculoskeletal signs such as lameness, with or without painful bone swelling, were reported in 19% of cats. ocular lesions are seen infrequently and include chorioretinitis and anterior uveitis. ocular or cns signs were reported in 19% of cats. most cats have clinical signs for less than 4 weeks prior to diagnosis. hyperproteinemia is present in approximately 50% of cats. definitive diagnosis is by identification of the organism via biopsy of lesions. antibody detection is available using latex agglutination (igm), agid (igm) or elisa (igm or igg). tube precipitin (tp) for igm and complement fixation (cp) for igg were previously thought to be less reliable in cats, but have been subsequently demonstrated to detect feline infections. itraconazole (10 mg/kg po if possible, q 24 h or 5 mg/kg q 12 h) is the treatment of choice. treatment is required for 4-6 months and must be continued for at least 2 months after all signs have resolved. • some cats develop anorexia, and less commonly vomiting or diarrhea. stop the drug for a few days until the cat is eating, and then restart at 1/2 the dose for 7-10 days, before increasing back to the full dose, which is usually then tolerated. amphoteracin b is also effective (0.25 mg/kg in 30 ml dextrose 5% iv over 15 minutes q 48 h or given subcutaneously) -see page 26, for cyptococcosis in the cat with signs of chronic nasal disease. continue amphotericin b therapy until a cumulative dose of 4 mg/kg is given or until bun > 17.9 mmol/l (50 mg/dl). amphotericin has the disadvantage of requiring frequent parenteral or subcutaneous administration and causes significant nephrotoxicity. • because of its quick onset of action, amphoteracin b in combination with itraconazole is useful in cats with severe pulmonary signs that are rapidly deteriorating. if the cat survives, after a few weeks treatment can be continued with itraconazole alone. • lipid-complexed amphoteracin formulations allow higher dosages with less toxicity, and should be used in cats with severe pulmonary signs, although the cost is higher. dose at 2-3 mg/kg iv 3 days per week for a total of 9-12 treatments (cumulative dose of 24-27 mg). dilute to a concentration of 1 mg/ml in dextrose 5% and infuse over 1-2 hours. if the titer has decreased four-fold and there is a similar improvement in physical and radiographic signs, treatment can be stopped after 4-6 months. antibodies may persist for long periods and obtaining a zero titer is not a useful treatment goal. classical signs • fever. • respiratory signs. • ocular signs. • lymphadenopathy. the geographical distribution includes north america, central america and africa. soil is believed to be the reservoir for infection, and living near a lake or river increases the risk of infection in dogs. signs are more common in dogs than in the cats. disseminated disease is primarily contracted via inhalation. respiratory signs include coughing, dyspnea and harsh lung sounds. ocular disease, such as uveitis, glaucoma and retinal detachment, is a frequent finding. fever, anorexia, depression, weight loss and lymphadenopathy are systemic signs associated with disseminated disease. draining skin lesions may occur and are usually a manifestation of systemic disease rather than local disease. neurological signs are associated with cns involvement of the brain or spine and include circling, disorientation, anisocoria, paresis, decreased conscious proprioception, or upper motor neuron signs, hyperesthesia and extensor rigidity. definitive diagnosis is by demonstration of an extracellular, broad-based budding yeast in aspirates or biopsies from lymph nodes, draining tracts, bone lesions or vitreous humor. an antibody detection test is available, but may be negative. itraconazole (10 mg/kg po if possible, q 24 h or 5 mg/kg q 12 h) is the treatment of choice. treatment is required for 4-6 months and must be continued for at least 2 months after all signs have resolved. • some cats develop anorexia, and less commonly vomiting or diarrhea. stop the drug for a few days until the cat is eating, and then restart at 1/2 the dose for 7-10 days, before increasing back to the full dose, which is usually then tolerated. amphoteracin b is also effective (0.25 mg/kg in 30 ml dextrose 5% iv over 15 minutes q 48 h or given subcutaneously) -see page 26, for cyptococcosis in the cat with signs of chronic nasal disease. continue amphotericin b therapy until a cumulative dose of 4 mg/kg is given or until bun > 17.9 mmol/l (50 mg/dl). amphotericin has the disadvantages of requiring frequent parenteral or subcutaneous administration and causing significant nephrotoxicity. • because of its quick onset of action, amphoteracin b in combination with itraconazole is useful in cats with severe pulmonary signs that are rapidly deteriorating. if the cat survives, after a few weeks treatment can be continued with itraconazole alone. • lipid-complexed amphoteracin formulations allow higher dosages with less toxicity, and should be used in cats with severe pulmonary signs, although the cost is higher. dose at 2-3 mg/kg iv 3 days per week for a total of 9-12 treatments (cumulative dose of 24-27 mg). dilute to a concentration of 1 mg/ml in dextrose 5% and infuse over 1-2 hours. classical signs see main reference on page 534 for details (the anemic cat). primarily found in the south-central and southeast united states. the north american bobcat is the natural host. there is usually a history of exposure to ticks in the previous 5-20 days (incubation period is 5-20 days). the clinical course of disease is approximately 1 week and often ends in death. clinical signs are the result of an overwhelming hemolytic crisis. rapid onset of fever, dyspnea, anorexia, pale mucous membranes, icterus and dark-colored urine are typical. collapse and death occur 2-3 days after the fever peak. hypothermia occurs in the terminal stages. there appear to be non-pathogenic strains as well. a complete blood count reveals regenerative anemia, hemoglobinemia and neutrophilia or neutropenia. the biochemistry panel commonly has hyperbilirubinemia. urinalysis may show evidence of hemoglobin and bilirubin. demonstration of the organism in erythrocytes (merozoite stage) is possible only relatively late in the disease, approximately 1-3 days before death. parasitemic cats usually have only 1-2% of rbcs affected, and up to 50% of cats have parasitemias that are very low or undetectable. demonstration of the organism in macrophages from bone marrow, spleen, liver or lymph node aspirates may be possible even when organisms are not evident in blood. serum antibody levels and direct fa test for detection of tissue phase are available through some labs. weight loss in spite of normal to increased appetite. polyuria/polydipsia. behavioral changes which often include hyperactivity and aggression. unkempt, rough hair coat and sometimes overgrown nails. tachycardia accompanied by a "gallop" rhythm and/or a systolic murmur. mild fever which may be intermittent in nature and reflect the increased metabolic rate in this disease. these cats are easily stressed and may present dyspneic and tachycardic with a mildly elevated temperature, usually not greater than 39.4˚c (103.0˚f). enlarged thyroid glands are often evident on palpation of the neck. diagnosis is based on clinical signs and history and confirmed by demonstrating increased thyroid hormone concentrations (total t4, free t4). thyroid glands can be palpated in approximately 80% of cats with hyperthyroidism, and are unilaterally or bilaterally enlarged. • enlarged thyroid glands may not be palpable if the abnormal thyroid tissue is within the thoracic inlet. complete blood count and a biochemistry panel are required to rule out diseases such as diabetes mellitus, renal disease, etc. a trh stimulation test may be necessary when clinical signs are highly suggestive and total and free t4 are in the upper region of the reference range for normal cats. thyroid radionuclide uptake and imaging with pertechnetate ( 99m tc) is also available at some institutions. response to therapy with anti-rickettsial drugs (tetracycline or doxycycline) is highly suggestive. • subclinical or mild fever and occasional ocular signs. bartonella henselae is an intracellular bacterium within erythrocytes. bacteremia is present in many healthy cats in the population, and cats are reservoirs for infection. b. henselae is an important pathogen because of its zoonotic potential in immunocompromised humans. • humans may develop fever, malaise, lymphadenopathy and skin eruptions following cat scratches or bites. • b. henselae causes bacillary angiomatosis, bacillary peliosis and encephalitis in human aids patients. naturally infected cats usually only develop subclinical infection. mild, self-limiting fever lasting 48-72 hours has been documented in some experimentally infected cats. anterior uveitis and fever were documented in naturally exposed cats. lymphadenopathy. atypical seizures occur in some cats. antibody titers are prevalent in healthy cats, but there is a poor correlation with blood culture and pcr assay results. intermittent bacteremia may occur for longer than one year following infection, with 25-41% of healthy cats bacteremic for up to 22 months. the organism is present within erythrocytes, therefore, hemolyzing red blood cells increases the sensitivity of the culture. other causes of mild transient fever need to be considered, such as mild cellulitis following a cat fight. other infectious causes of anterior uveitis need to be ruled out, such as toxoplasmosis, fungal diseases, felv, fiv, cuterebra or dirofilaria. antimicrobial efficacy has not been clearly demonstrated. clinical signs of disease have resolved when the cats are administered doxycycline at 25-50 mg po q 12 h for 28 days. azithromycin is used in humans and is a safe alternative in cats when administered at 10 mg/kg po q 24 h for 28 days fluoroquinolones also may be effective. while clinical signs resolve, bacteremia is usually only temporarily suppressed. b. henselae has very low pathogenicity in cats. once cleared of infection, cats are resistant to re-infection by innoculation, but are still susceptible if transmitted via blood transfusion. transmission is via arthropod vectors. in endemic areas, cats infested with fleas and/or ear mites are more likely to be seropositive. the organism survives in flea feces for at least 9 days. because of the frequency of bacteremia in healthy cats, blood transfusions are a likely route of infection. primary immune-mediated disease is extremely rare in cats. stimulation from primary infectious disease antigens is the most common cause of immune-mediated disease in cats, and is most often associated with hemobartonella (mycoplasma) and calicivirus. systemic lupus erythematosus is rare in cats. a multitude of signs may occur including fever, weight loss and cutaneous lesions. immune-mediated hemolytic anemia is most commonly associated with hemobartonellosis. signs include anemia, icterus, fever and anorexia. cats with immunosuppressive disorders such as felv may be more susceptible. immune-mediated thrombocytopenia is rarely reported in cats. felv-positive cats, however, may have thrombocytopenia that is thought to be the result of an immune-mediated response. immune-mediated polyarthritis is uncommon in cats, but has been documented in kittens and adult cats with post-calicivirus vaccination. 19 -the pyrexic cat 391 a bacteriologic investigation of subcutaneous abscesses in cats clinical, clinicopathologic, and pathologic features of plague in cats: 119 cases (1977-1988) bacterial diseases; fungal diseases; and protozoal diseases feline toxoplasmosis: interpretation of diagnostic test results bartonella spp. antibodies and dna in aqueous humor of cats feline toxoplasmosis and the importance of the toxoplasma gondii oocyst feline infectious peritonitis. part i. etiology and diagnosis feline infectious peritonitis. part ii. treatment and prevention consensus statement of ehrlichial disease of small animals from the infectious disease study group of the acvim feline infectious myocarditis/diaphragmitis diagnostic approach and medical treatment of seizure disorders an appraisal of the value of laboratory tests in the diagnosis of feline infectious peritonitis tularemia in two cats key: cord-011968-abd891ej authors: lai, yen-chung; chao, chiao-hsuan; yeh, trai-ming title: roles of macrophage migration inhibitory factor in dengue pathogenesis: from pathogenic factor to therapeutic target date: 2020-06-12 journal: microorganisms doi: 10.3390/microorganisms8060891 sha: doc_id: 11968 cord_uid: abd891ej dengue virus (denv) infection is the most prevalent mosquito-borne viral infection and can lead to severe dengue hemorrhagic fever (dhf) and even life-threatening dengue shock syndrome (dss). although the cytokine storm has been revealed as a critical factor in dengue disease, the limited understanding of dengue immunopathogenesis hinders the development of effective treatments. macrophage migration inhibitory factor (mif) is a pleiotropic proinflammatory cytokine that mediates diverse immune responses, and the serum level of mif positively correlates with disease severity in patients with dengue. mif is involved in denv replication and many pathological changes, such as vascular leakage, during denv infection. in this paper, the pathogenic roles of mif and the regulation of mif secretion during denv infection are reviewed. furthermore, whether mif is a potential therapeutic target against denv infection is also discussed. dengue virus (denv) infection is the most widespread mosquito-borne viral infection in the tropics and subtropics. it is estimated that denv causes 390 million infections yearly, even though more than 50% of cases of denv infection are asymptomatic or cause mild flu-like illness, including fatigue, headache, myalgia and nausea with sudden onset of fever called dengue fever (df). unfortunately, one in twenty infected individuals may suffer from a more severe illness, which is termed dengue hemorrhagic fever (dhf), and even develop life-threatening dengue shock syndrome (dss) [1] . in 2009, the who issued a new guideline that classifies symptomatic cases as nonsevere dengue or severe dengue according to disease severity. nonsevere dengue cases can be further divided into two subgroups: patients with warning signs and those without warning signs. the warning signs include abdominal pain or tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleeding, lethargy, restlessness, liver enlargement and decreased platelet counts. the criteria for severe dengue include severe plasma leakage, severe bleeding, and severe organ involvement [2] . the mortality of df is less than 1%; however, severe dengue may carry a mortality rate up to 26% [3] . although there is one vaccine (dengvaxia ® ) that has been licensed to prevent dengue in several countries, this vaccine rendered only partial protection against denv2 infection. in addition, the vaccine is associated with an unexplained increased incidence of hospitalization for severe dengue disease in seronegative vaccine recipients [4, 5] . furthermore, due to the limited understanding of the exact pathogenic mechanisms to cause vascular leakage, no effective therapeutic drugs are available, mif, a 12.5 kda protein was first identified as a cytokine that is mainly released from t cells upon antigen stimulation and inhibits the random migration of macrophages [27] . later, mif was found to be widely distributed in various immune and nonimmune cells, including macrophages, platelets, endothelial cells, epithelial cells that mediate several immune responses with pluripotent activity. unlike most cytokines, mif is constitutively expressed and stored in preformed "intracellular pools" during normal homeostasis [28] , which means it can be released from cells upon inflammatory and stress stimulation promptly. accordingly, mif secretion can be found even without de novo synthesis. since mif does not possess an n-terminal secretory sequence, mif is released from cells via nonconventional er/golgi secretory pathways [29] . both intracellular and extracellular mif exhibit pluripotent functions. intracellular mif can modulate ap-1 activity and the cell cycle by interacting with cytosolic jun activation domain binding protein 1 (jab-1) [30, 31] . overexpression of endogenous mif significantly suppresses p53-dependent and oxidative stress-induced apoptosis [32, 33] . on the other hand, secreted mif can bind to some distinct cell receptors, such as cd74 and its coreceptors cd44, cxcr2, cxcr4 and cxcr7, which activate several signaling pathways, such as the erk1/2 and pi3k/akt pathways, which are responsible for cell proliferation, survival, and immune regulation [34] [35] [36] . in addition, mif also has endocrine activity to regulate insulin secretion [37] and tautomerase activity to manipulate cell growth [38] . under normal physiological conditions, mif is constitutively expressed at a low level between 2-10 ng/ml, while the mif concentration in human plasma fluctuates in response to stress stimuli such as sepsis, infection and different inflammatory disorders [39] [40] [41] . high concentrations of mif can activate the synthesis of more proinflammatory cytokines, such as tnf, ifn-γ, il-1β, il-2, il-6, and il-8 [26] , which accelerates inflammatory disease development. furthermore, mif also contributes to the pathogenesis of various viral infections, including human immunodeficiency virus (hiv), respiratory syncytial virus (rsv), west nile virus (wnv) and denv [42] [43] [44] [45] [46] . the first evidence of the pathogenic role of mif in dengue disease was indicated by the positive correlation between the mif level in the sera of dengue patients and disease severity [47] . later, another group also demonstrated that denv2-induced inflammation, thrombocytopenia, viral load and disease severity could be attenuated in mif −/− mice [48] . in 2015, ferreira et al. also showed that the serum level of mif is higher in patients with dhf than df [49] . however, the pathogenic mechanisms and source of circulating mif were not further investigated. here, we propose three main pathogenic roles of mif in the immunoregulatory crosstalk during denv infection: (1) facilitation of denv replication; (2) enhancement of vascular leakage; (3) regulation of the immune response ( figure 1 ). it has been shown that denv2 infection triggers mif expression and secretion, which enhances denv2 replication in huh-7 cells [50] . autophagy is pivotal for denv replication [51] , and mif is required for the induction of autophagy in denv2-infected huh-7 cells. blocking mif with inhibitors or knockdown of mif expression by shrna inhibited both denv-induced lc3 conversion and denv replication. to clarify the correlation between these two parallel effects, we found that incubation with recombinant mif (rmif) only enhanced viral replication in mif knockdown but not lc3 knockdown cells. these results suggest that autophagy is required for mif-induced denv replication in huh-7 cells. given that denv infection induces autophagy, which is required for denv replication, the mechanisms of denv-induced autophagy have been widely studied. the formation of autophagosomes provides a dock for the denv replication complex, which supports viral replication in nonphagocytic cells [51] . denv infection also induces selective autophagy associated with lipid metabolism. denv-induced autophagosomes facilitate mitochondrial β-oxidation associated with host lipid metabolism, which enhances atp production for viral replication [52] . in addition, ns4a expression induces autophagosome formation and inhibits cell apoptosis during denv2 infection, which contributes to prolonged viral replication [53] . previously, mif was found to induce autophagy under starvation conditions. starvation-triggered mif binds to its receptor cd74 in autocrine or paracrine fashions, and then autophagy is induced through reactive oxygen species (ros) generation [54] . cd74-mediated mif endocytosis can also activate erk phosphorylation [55] , leading to autophagy [56] . in another study, it was demonstrated that live denv2-induced endoplasmic reticulum (er) stress is required for autophagy activation, viral replication and pathogenesis in huh-7 and a549 cells [57] . although the direct correlation between denv-induced er stress and mif secretion has not been elucidated, both mif and er stress can induce autophagy through erk1/2 phosphorylation [58, 59] and ros generation [60] . interestingly, mif is able to induce er stress in a liver injury model [61] . therefore, it is possible that mif signal transduction may trigger er stress and erk activation upon denv infection, leading to autophagy induction and viral replication, or vice versa. although further investigation is required to understand the relationship between mif and denv-induced er stress in the future, our previous results showed that denv-induced mif secretion can induce autophagy-facilitated viral replication, which may explain why reduced viremia was found in mif −/− mice than in control mice [48] . in addition to the pro-viral effect in hepatocytes and the lung epithelial cells, pathogenic roles of mif in endothelial cells were also found. mif is crucial in the vasculature, which has been addressed in several studies. mif expression in human endothelial cells is upregulated upon treatment with agonists that can trigger endothelial hyperpermeability, such as thrombin [62] or oxldl [63] . on the other hand, inhibiting mif with either inhibitors or anti-mif antibodies could rescue thrombin-induced vascular leakage [64] , suggesting that mif plays a role in vascular leakage. moreover, treatment with rmif could directly increase permeability within 30 min in the dermal microvascular endothelial cell line hmec-1 and by subcutaneous injection in a mouse model [59] . not surprisingly, mif has also been shown to be involved in vascular hyperpermeability upon denv infection. denv infection can stimulate mif secretion from huh-7 cells. conditioned medium collected from denv-infected huh-7 cells can enhance the permeability of human endothelial cell lines by disrupting the distribution of the endothelial tight junction protein zonula occludens-1 (zo-1) through mif-activated phosphatidylinositol-3-kinase/mitogen-activated protein kinase kinase-extracellular signal-regulated kinase/c-jun n-terminal kinase (pi3k/mek-erk/jnk) signaling pathways [19] . moreover, mif also plays an essential role in denv ns1-triggered endothelial permeability. denv ns1 is a viral protein that is the main reason for vascular leakage during dengue infection [65] . there are at least two mechanisms involved in denv ns1-triggered endothelial permeability, both of which involve mif: enzyme-dependent glycocalyx degradation and autophagy-facilitated disruption of endothelial integrity. denv ns1 can activate mif secretion in endothelial cells, which triggers the release of heparan sulfate-specific heparanase 1 (hpa-1) and endothelial glycocalyx shedding [66] . on the other hand, denv ns1 can cause the disarray of endothelial tight junctions through mif-induced autophagy [67] . taken together, these findings suggest that mif plays important roles in vascular leakage during denv infection. in addition to viral replication and endothelial hyperpermeability, mif may also regulate the immune response of different immune cells during denv infection. in phagocytic cells, mif can upregulate coagulation molecules, such as thrombomodulin (tm), in denv2-infected thp-1 cells [68] . tm can compete with fibrinogen to bind to thrombin and inhibit fibrin formation, thereby contributing to coagulopathy in dengue disease. as a pivotal mediator of inflammatory cytokines, mif inhibition can attenuate denv2 infection-induced tnf-α and il-6 production, and denv ns1-triggered metalloproteinase 9 (mmp-9) production as demonstrated in thp-1 cells [48] . as tnf-α and il-6 are critical proinflammatory cytokines involved in vascular permeability [69, 70] and mmp-9 is a key enzyme that can trim the glycocalyx layer in endothelial cells [71] , upregulation of mif in leukocytes can further contribute to denv-induced vascular permeability. thus, mif can lead to coagulopathy and vascular leakage through denv-stimulated immune cells, which may explain why the increased hematocrit was attenuated in denv2-infected mif −/− mice [48] . on the other hand, neutrophil extracellular traps (nets) induced by activated neutrophils are thought to be a pathogenic feature that amplifies inflammatory responses in dengue disease [72] . mif is required for net formation in immune disorders [73, 74] . therefore, the release of mif from neutrophils may induce net formation and inflammation in denv infection, which contributes to dengue pathogenesis. in addition to these immune cells, activated platelets are also a source of mif. platelet-derived mif is a unique chemokine that leads to monocyte adhesion on endothelial layers [75] . denv ns1 can contribute to platelet activation and aggregation, thus enhancing platelet adhesion to endothelial cells [76] . in addition, extracellular vesicles (evs) have been identified as functional conveyors of mif in obesity [77] , as well as crucial mediators in platelet-leukocyte interactions upon denv2 infection [78] . platelet-derived mif may be involved in denv ns1-induced thrombocytopenia and hemorrhage. although a previous study demonstrated that serum levels of mif are significantly higher in all dhf patients who died than in surviving dhf and df patients [47] , the main source of secreted mif and its release mechanisms are still unclear. here, the possible mechanisms of denv-induced mif secretion and expression are discussed (figure 2 ). as it lacks an n-terminal secretory sequence, mif is known to be released from the intracellular pools very quickly upon stimulation. it has been shown that productive denv2 and denv3 infection can stimulate mif release from human macrophages and hep g2 cells without the requirement of mif rna transcription at 14 h post-infection [48] . similarly, denv2 infection of huh-7 cells can drive two waves of mif secretion [50] . denv2 infection induced the first wave of mif secretion at 3-6 h post-infection. since no obvious lactate dehydrogenase (ldh) release nor a significant increase in mif mrna occurred during this period of time, these results suggest that the first wave of mif secretion was caused by the release of mif from intracellular pools, and this secretion is independent of mif gene transcription and cell death [50] . as it lacks an n-terminal secretory sequence, mif is known to be released from the intracellular pools very quickly upon stimulation. it has been shown that productive denv2 and denv3 infection can stimulate mif release from human macrophages and hep g2 cells without the requirement of mif rna transcription at 14 h post-infection [48] . similarly, denv2 infection of huh-7 cells can drive two waves of mif secretion [50] . denv2 infection induced the first wave of mif secretion at 3-6 h post-infection. since no obvious lactate dehydrogenase (ldh) release nor a significant increase in mif mrna occurred during this period of time, these results suggest that the first wave of mif secretion was caused by the release of mif from intracellular pools, and this secretion is independent of mif gene transcription and cell death [50] . however, mif mrna expression is increased in huh-7 cells at 12 h to 48 h post-denv2 infection, as shown by rt-pcr [50] . although quantitative rt-pcr or real-time rt-pcr should be used to detect changes in the mif rna level more precisely, these results suggest that denv infection can trigger signals to enhance mif transcription. as viral rna and viral proteins can trigger cellular oxidative stress, which is a strong stimulus for mif secretion and expression [79] , various cellular oxidative stresses, such as er stress, the unfolded protein response (upr), hypoxic response and mitochondrial ros production, might be the upstream stimulators for mif production during denv infection [80, 81] . among them, two hypoxia-related transcription factors, hypoxia-inducible factor 1 (hif-1) and camp-response element-binding protein (creb), were found to facilitate denv2 infection [82, 83] . in addition, mif transcription can be driven by hif-1 in response to hypoxia or by creb in response to er stress. therefore, live denv induces a hypoxic response and er stress, followed by mif rna transcription through hif-1 or creb activation, which may lead to the second however, mif mrna expression is increased in huh-7 cells at 12 h to 48 h post-denv2 infection, as shown by rt-pcr [50] . although quantitative rt-pcr or real-time rt-pcr should be used to detect changes in the mif rna level more precisely, these results suggest that denv infection can trigger signals to enhance mif transcription. as viral rna and viral proteins can trigger cellular oxidative stress, which is a strong stimulus for mif secretion and expression [79] , various cellular oxidative stresses, such as er stress, the unfolded protein response (upr), hypoxic response and mitochondrial ros production, might be the upstream stimulators for mif production during denv infection [80, 81] . among them, two hypoxia-related transcription factors, hypoxia-inducible factor 1 (hif-1) and camp-response element-binding protein (creb), were found to facilitate denv2 infection [82, 83] . in addition, mif transcription can be driven by hif-1 in response to hypoxia or by creb in response to er stress. therefore, live denv induces a hypoxic response and er stress, followed by mif rna transcription through hif-1 or creb activation, which may lead to the second wave of mif secretion. however, in another study, mif transcription was only marginally affected by denv3 infection in the human hepatoma cell line hep g2 [48] . a possible explanation for this discrepancy is that different infection time points and virus strains were used in these two studies. we showed that mif transcription was gradually increased at 12 h post-infection and peaked at 24 h post-infection in huh-7 cells, while in another study, the mif rna level was only analyzed within 14 h post-infection. in addition, viral infection-induced mif secretion and production seem to be virus-and cell-dependent. for instance, influenza a virus infection does not induce mif gene transcription but causes the release of preformed mif from lung epithelial cells due to necrotic cell death [84] . however, infection of macrophages with sindbis virus resulted in mif release from intracellular pools without a significant increase in mif transcription or cell death [48] . accordingly, the mechanisms of mif production and the kinetics of mif secretion induced by denv infection in different types of cells require further investigation. compared with the classical pathway of cytokine secretion, mif can be released from preformed pools shortly after stimulation. secretion of preformed mif has been shown to be mediated by the atp binding cassette (abc) transporter [29] , as well as vesicle or exosome secretory pathways [85] . in addition, denv infection may stimulate mif secretion mediated by the golgi-associated protein p115 via golgi vesicle trafficking, which is similar to the effect of bacterial infection [86] . in our study, since uv-inactivated viral particles could not induce mif secretion or expression, it is possible that denv infection triggered rna sensing or pattern recognition receptor (prr) activation, which was followed by the release of preformed mif from the cytosol through the vesicle trafficking secretory pathway. the release of preformed mif may further initiate many cellular signal transduction pathways through binding to membrane-expressed mif receptors, including cd74/cd44, cxcr2, cxcr4 and cxcr7 [87, 88] . initiation of autophagy signal transduction induced by mif binding to its receptors leads to activation of the pi3k/akt/src and mapk/erk pathways [89, 90] , which may explain why the first wave of mif secretion occurs before denv2-induced autophagic flux in huh-7 cells [50] . to reduce the burden of dengue disease, effective drugs to treat denv infection are urgently needed until a satisfactory vaccine becomes available. based on the proposed dengue pathogenesis described above, current denv therapeutic development has focused on two major targets: (1) viral factors and (2) host factors. by targeting viral factors, many approaches to protect against different viral life cycles have been tested, such as inhibition of receptor-dependent viral entry [91] , ph-dependent viral fusion [92] , interaction with the capsid protein [93] and viral particle assembly [93] . neutralizing abs against conserved regions of structural proteins, including e and prm/m, are widely used due to their specificity. however, ab use is often challenging due to the enhancement of viral infection in fcγ receptor-bearing immune cells with subneutralizing doses, as described by the ade theory [9, 94, 95] . in addition to ab therapies, some off-patent drugs and antibiotics have also been tested for repurposing by high-throughput screening, which is beneficial for development time and cost. for instance, prochlorperazine, a dopamine d2 receptor (d2r) antagonist that is used to treat nausea, schizophrenia, migraines, and anxiety, was shown to inhibit viral entry [96] . considering another perspective, aberrant host immunity also plays vital roles in dengue pathogenesis. excessive secretion of proinflammatory cytokines (cytokine storm) induced by denv infection may trigger robust innate and adaptive immune responses, leading to plasma leakage, hemorrhage, and coagulopathy in dhf/dss patients. appropriate regulation of host immunity provides different insights into therapeutic targets, including host restriction factors, host dependency factors, and host-mediated pathogenesis pathways [97, 98] . compared with viral targets, host-targeting antiviral approaches are believed to avoid rapid drug resistance or mutations that arise during viral evolution. since mif is involved in dengue pathogenesis, the therapeutic potential of blocking mif to protect against dengue disease has also been studied. in 2011, assuncao-miranda et al. first used the mif inhibitor iso-1 and a mif neutralizing antibody to test the involvement of mif in denv3-induced macrophage activation in vitro. although blocking mif did not affect viral replication in denv3-infected human macrophages, both the secretion and mrna synthesis of tnf-α and il-6 were reduced. moreover, prostaglandin e2 (pge2), a well-known inflammatory mediator in many inflammatory diseases [99] , was attenuated as well. furthermore, they demonstrated that the concentration of ifn-γ in sera, the level of il-6 in the spleen, and leukocyte infiltration in the lungs of mif −/− mice were significantly lower than those in denv-infected wild-type mice. most importantly, the study showed delayed mortality in denv2-infected mif −/− mice. these results suggest that mif controls amplification of denv-induced inflammatory responses. treatment with mif neutralizing antibodies or inhibitors may provide protection against dengue disease. previously, minocycline, a us food and drug administration (fda)-approved antibiotic, was found to reduce dengue viral output through downregulation of erk1/2 activation and upregulation of interferon stimulated genes (isgs) in hep g2 cells [100] . in our recent study, we found that minocycline can block not only denv2-triggered autophagy but also mif secretion. autophagy could be activated by mif through erk1/2 phosphorylation [59] , and the anti-denv effect of minocycline was abolished in either mif or lc3-deficient huh-7 cells during denv infection. it is possible that the protective effect of minocycline may be due to its ability to block mif secretion, which suppresses the erk1/2-autophagy signaling pathway. in addition, the results showed that minocycline can reduce both mif rna transcription and secretion during denv2 infection, but the mechanism is unclear. given that mif secretion can be triggered by the abc transporter, which is a nonconventional secretory pathway [29] , and minocycline is able to inhibit the function of the abc transporter to block drug-drug interactions at the blood-brain barrier [101] , minocycline may disrupt the efflux of mif via suppression of the abc transporter upon denv infection. moreover, minocycline is reported to reduce the production of tnf-α, il-6, il-12, ifn-γ and ccl2 via suppression of the transcription factor nf-κb in the brain, which confers complete protection against jev in mice [102] . nf-κb binds to the mif promoter and drives mif transcription [103] , and inhibition of nf-b also blocks denv infection-induced mif production in a549 cells [104] ; therefore, attenuation of de novo rna synthesis and secretion of mif from denv-infected cells by minocycline treatment may be due to its inhibition of the nf-κb signal pathway and suppression of the abc transporter, respectively [105] . however, further study is required to clarify these hypotheses. to further understand whether minocycline can protect against denv infection in vivo, we found that minocycline treatment reduced the levels of mif and viremia in sera, as well as attenuated autophagy in murine liver tissue, in ag129 mice. however, the protection of minocycline in ag129 mice was insufficient. to rule out defects in isg-related protection in this model, which lacks type i and type ii ifn receptors, immunocompetent icr suckling mice were further used. minocycline only alleviated denv2-induced neurological symptoms and prolonged the survival rate but did not fully protect against denv2-induced lethality in suckling mice. it is unclear whether the failure of minocycline to fully protect against denv2-induced lethality in suckling mice is due to the mouse-adapted strain ngc-n being too virulent for the suckling mice or the intracerebral challenge of ngc-n inducing irreversible damage in the brains of the suckling mice. however, these results were similar to the outcome in denv2-infected mif −/− mice [48] , which suggests that other pathogenic factors induced by denv infection may also be important for denv-induced pathogenesis. mif plays crucial roles in dengue pathogenesis; however, targeting only mif secretion and expression seems to be insufficient to provide full protection against denv infection. as mentioned above, transcription factors, such as hif-1 and creb, may also be involved in the increase in mif expression during denv infection. it is possible that in addition to mif, these transcription factors may also induce other pathogenic responses that contribute to disease development during denv infection [82, 83] . on the other hand, although mif can induce autophagy and facilitate denv replication in huh-7 cells, autophagy might play different or even opposite roles in denv replication in different cells [106] . it has been reported that autophagy plays pro-viral roles in denv replication in epithelial cells but antiviral roles in immune cells [107] . therefore, the effect of mif on the modulation of autophagy and denv replication should be further systemically investigated in different cells, and the effect of minocycline treatment on denv infection in different cells, such as immune cells, should be compared. taken together, these findings suggest that targeting upstream transcription factors that control mif expression or multiple medication combinations targeting different mif signaling pathways may help to develop better therapeutic strategies against dhf/dss in the future. denv-triggered mif secretion can not only facilitate denv replication through the regulation of autophagy but also worsen the severity of vascular leak by enhancing endothelial permeability. in addition, mif may modulate the interaction of different immune cells, which contributes to dengue pathogenesis. inhibition of mif secretion and transcription by small molecule drugs such as minocycline could attenuate both autophagy and viral replication. however, targeting mif by multiple approaches instead of a single approach may provide a better therapeutic alternative against denv infection for translation from the laboratory to the clinic in the future. the global distribution and burden of 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lipid metabolism flavivirus ns4a-induced autophagy protects cells against death and enhances virus replication macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation β-arrestin1 mediates the endocytosis and functions of macrophage migration inhibitory factor tfeb links autophagy to lysosomal biogenesis dengue virus-induced er stress is required for autophagy activation, viral replication, and pathogenesis both in vitro and in vivo the er stress sensor ire1 and map kinase erk modulate autophagy induction in cells infected with coronavirus infectious bronchitis virus macrophage migration inhibitory factor induces vascular leakage via autophagy endoplasmic reticulum stress and associated ros protection from gao-binge induced liver injury in mif-mice is associated with decreased er stress macrophage migration inhibitory factor is induced by thrombin and factor xa in endothelial cells expression of macrophage migration inhibitory factor in different 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activation and early features of net formation are associated with dengue virus infection in human cystic fibrosis sputum dna has netosis characteristics and neutrophil extracellular trap release is regulated by macrophage migration-inhibitory factor il-16 and mif: messengers beyond neutrophil cell death platelet-derived mif: a novel platelet chemokine with distinct recruitment properties dengue virus nonstructural protein 1 activates platelets via toll-like receptor 4, leading to thrombocytopenia and hemorrhage phenotyping of circulating extracellular vehicles (evs) in obesity identifies large evs as functional conveyors of macrophage migration inhibitory factor extracellular vesicles from clec2-activated platelets enhance dengue virus-induced lethality via clec5a/tlr2 macrophage migration inhibitory factor secretion is induced by ionizing radiation and oxidative stress in cancer cells flaviviridae viruses and oxidative stress: implications for viral pathogenesis dengue-induced autophagy, virus replication and protection from cell death require er stress (perk) pathway activation the role of tissue oxygen tension in dengue virus replication antibody-dependent enhancement infection facilitates dengue virus-regulated signaling of il-10 production in monocytes release of macrophage migration inhibitory factor and cxcl8/interleukin-8 from lung epithelial cells rendered necrotic by influenza a virus infection localization of macrophage migration inhibitory factor (mif) to secretory granules within the corticotrophic and thyrotrophic cells of the pituitary gland the golgi-associated protein p115 mediates the secretion of macrophage migration inhibitory factor mif signal transduction initiated by binding to cd74 rapid and transient activation of the erk mapk signalling pathway by macrophage migration inhibitory factor (mif) and dependence on jab1/csn5 and src kinase activity the role of pi3k/akt/mtor pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration inhibition of mek/erk activation attenuates autophagy and potentiates pemetrexed-induced activity against hepg2 hepatocellular carcinoma cells dengue virus entry and trafficking: perspectives as antiviral target for prevention and therapy progress in the identification of dengue virus entry/fusion inhibitors a novel inhibitor of dengue virus replication that targets the capsid protein cross-reacting antibodies enhance dengue virus infection in humans antibody-dependent enhancement of severe dengue disease in humans repurposing of prochlorperazine for use against dengue virus infection targeting host factors to treat west nile and dengue viral infections contemporary strategies and current trends in designing antiviral drugs against dengue fever via targeting host-based approaches drug repurposing of minocycline against dengue virus infection brain and plasma riluzole pharmacokinetics: effect of minocycline combination regulation of inflammation in japanese encephalitis involvement of nuclear factor-κb in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin-1β in ectopic endometrial cells dengue virus infection induced nf-κb-dependent macrophage migration inhibitory factor production minocycline targets the nf-b nexus through suppression of tgf-1-tak1-i b signaling in ovarian cancer dengue virus and autophagy induced autophagy reduces virus output in dengue infected monocytic cells this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the members of the center of infectious disease and signaling research of ncku for their invaluable input and insights throughout the course of this study. this study is part of yen-chung lai's ph.d. dissertation. the authors declare that they have no competing interests. key: cord-006578-wv3wc0ct authors: stevens, d. l. title: invasive streptococcal infections date: 2001 journal: j infect chemother doi: 10.1007/s101560100012 sha: doc_id: 6578 cord_uid: wv3wc0ct nan in the 15 years since the first description of the streptococcal toxic shock syndrome (streptss), reports have documented the presence of these invasive infections in all corners of the world, in all races, sexes, and age groups. the strains responsible for these infections have been similar, suggesting a global spread of infection. as might be expected, identical strains have also accounted for less serious infections, such as cellulitis, pharyngitis, and even asymptomatic carriage. thus, host factors and co-morbid conditions must account for the different diseases caused by identical strains. the most prevalent m-type associated with streptss has been, and continues to be, m-type 1. interestingly, mtype 3 strains have always been in second place and, together, m-types 1 and 3 account for approximately 50% of cases. recent studies in the netherlands found m-1 and m-3 strains each accounted for 25% of cases of invasive group a streptococcal infection. 1 in some geographical areas, m-1 strains have accounted for 70% of invasive infections. in contrast, in the epidemic that occurred in wannamingo, minnesota, 100% of the strains were m-3. 2 other strains less commonly associated with streptss include m-types 4, 6, 11, 18, 28, and non-typeable strains (reviewed in 3 ). in most geographical settings, a mixture of strains is responsible for sporadic cases. as remarkable as the emergence of invasive group a streptococcal infections has been, the persistence of such infections worldwide is equally unusual. the increased incidence of severe group a streptococcal infections during the later part of the 1990s suggests that the prevalent strains, largely m-1 and m-3 strains, have the ability to persist in the human environment for indefinite periods. in fact, these strains have been widely distributed for decades as causes of simple pharyngitis and asymptomatic carriage. thus, much of the world's population has been exposed to these strains, likely on numerous occasions. despite this exposure and the type-specific immunity that should be generated, the increasing prevalence of m-1 strains in certain regions is troubling. for example, in norway 4 and sweden, 5 the recent prevalence of m-1 strains among invasive streptococcal infections, as well as pharyngeal isolates, has increased to greater than 70%. from the theoretical perspective, the high prevalence and persistence of such strains could be due to an absence of protective immunity in a significant portion of the human population, or could be due to changes in protective epitopes in the organism analogous to antigenic drift among influenza viruses. there is some data supporting the latter hypothesis. although restriction fragment length polymorphism (rflp) patterns of m-type 1 strains suggest that these strains have remained identical over a 10year period, biophysical studies imply that this may not be the case. for example, de malmanche and martin, 6 using functional studies of opsonophagocytosis, demonstrated that immunity may only be strain, and not m-type, specific. in some of these strains, marked differences in the amino acid sequence of m-1 strains was apparent, despite the fact that these strains were serologically typed as m-1. in addition, villasenor 7 has demonstrated that, among 75 strains of m-1 group a streptococci, susceptibility to opsonophagocytosis in the presence of a single polyclonal anti-m-1 sera followed a normal distribution curve, and that some m-type 1 strains were no better opsonized than heterologous, non-m-1 strains. representative strains, selected on the basis of their susceptibility to phagocytosis, had differences in the sequence of m-protein in both the hypervariable region and the a-repeat regions of the molecule. clearly, these changes did not affect the structure enough to alter the serologic m-type, but were sufficient to affect opsonization with convalescent sera. thus, subtle differences in the surface structure of m-1 protein render some strains resistant to immune sera, suggesting that antigenic drift could account for the high prevalence and persistence of m-1 strains for the past 15 years. the increased incidence of invasive group a streptococcal infections also suggests a change in the virulence of the organism. m-protein, streptolysin o (slo), and streptococcal pyrogenic exotoxins a and b (spea, speb) have been implicated as important virulence factors. however, none of these has been associated with all invasive cases. for example, although m-types 1 and 3 have been most common, m-types 12, 28, and non-typeable strains have been also associated with invasive streptococcal infection. 8 similarly, in the united states, m-type 1 strains containing the gene for spea have been commonly associated with streptss; however, only about 50% of these strains produce spea. 9 thus, no single surface component or extracellular toxin has, thus far, been identified that correlates with the recent increase in virulence in group a streptococcus. in 1997, we reported a strong association between isolates from invasive disease, regardless of m-type, and the production of nicotinamide adenine dinucleotide glycohydrolase (nadase). 10 in contrast, isolates that are most commonly associated with rheumatic fever, i.e., mtypes 5 and 18, did not produce nadase. 10 throat isolates associated with uncomplicated symptomatic pharyngitis were most commonly m-types 1, 12, and 4, 8 and 100% of these isolates from the early 1980s to the present time were nadase producers. 10 this is not surprising, because the source of bacteria in invasive streptococcal infections is frequently the throat. it is of special interest that m-type 1 strains isolated from recent (circa 1980) invasive cases were nadase producers, 10 because, historically, m-type 1 group a streptococci have been characteristically non-nadase producers. 11, 12 this observed shift in nadase production has also been reported by karasawa et al. 13 among m-type 1 isolates in japan, and most recently by ajdic et al., 14 at the university of oklahoma, where the group a streptococcal genome has been elucidated. our genetic analysis demonstrated that all strains of group a streptococci, regardless of their ability to produce nadase, possess the nadase gene, nga, and that all isolates examined were more than 96% identical in nga and upstream regulatory sequences. 15 further, because nadasenegative strains did not produce any immunoreactive protein or peptide fragment, we concluded that additional regulatory element(s) control nadase production. 15 taken together, these findings suggest that, in the early 1980s, serotype m-1 underwent a stable genetic change, resulting in the ability to produce nadase. further, acquisition of this trait occurred in temporal association with the emergence of m-type 1 group a streptococcus as the predominant serotype associated with severe invasive infections. 8, 16, 17 nadase's role in pathogenesis is currently under study. until recently, group a streptococcal bacteremia occurred most commonly in the very young and the elderly. 17 among children, predisposing factors included burns, varicella, malignant neoplasm, immunosuppression, and age less than 2 years. 17 patients with septic scarlet fever had complications, such as extension of infection into the sinuses, peritonsillar tissue, or mastoids, 17 yet, bacteremia was uncommon. 18 among the children with varicella studied by bullowa and wishik 18 in 1935, group a streptococcal bacteremia occurred in only approximately 0.5% of patients. in contrast, recently, doctor et al. 19 found a 50% incidence of group a streptococcal bacteremia in patients hospitalized with varicella. in elderly patients, the source of group a streptococcal infection is invariably the skin and is associated with cellulitis or erysipelas. 17 group a streptococcal sepsis in the elderly (mean age, 50-60 years) has also been associated with diabetes, peripheral vascular disease, malignancy, and corticosteroid use. 17 not surprisingly, mortalities of 35%-80% have been described in this patient population. 17 in the united states and europe during the 1850s, the occurrence of group a streptococcal bacteremia was rare in individuals 14-40 years of age, 17 and was primarily associated with puerperal sepsis. recently however, intravenous drug abuse has emerged as a leading cause of group a streptococcal bacteremia in this age group. 17 martin and hoiby 4 have comprehensively demonstrated that the prevalence of group a streptococcal bacteremia in norway in the late 1980s increased in all age groups, but the greatest increase (600%-800%) was in adolescents and young adults. thus, the demographics of invasive group a streptococcal infections, with bacteremia as a marker, have changed dramatically in the past two decades. lymphangitis occurs most commonly during the course of an acute infection of the skin, usually cellulitis. it is most common following hemolytic streptococcal infections caused by groups a, c, and g streptococci. red streaks beginning at the margin of skin infection and extending proximally are easily recognized and may extend several inches per hour. thus, lymphangitis associated with a softtissue infection is likely a marker for a more aggressive strain of streptococci, and its recognition should prompt aggressive antibiotic therapy and close observation for evidence of bacteremia. attributed to streptococci, other organisms, including anaerobic and aerobic mouth flora, commonly contribute. still, the group a streptococcus was found alone or in combination with other organisms in 100% of tonsils removed from 45 different children. 20 in some cases, extension of infection from the tonsil to adjacent cervical nodes, the peritonsillar soft tissues of the neck, or the retropharyngeal space results in necrotizing infections or abscess, which require surgical drainage or debridement. peritonsillar abscesses are readily cured with penicillin plus needle aspiration. 21 like streptococcal pharyngitis, retropharyngeal abscess is most common in children less than 6 years to age. these infections may be caused by group a streptococcus, bacteroides, peptostreptococcus, or staphylococcus aureus. aggressive imaging of the neck, with computerized axial tomography (ct) or magnetic resonance imaging (mri), and emergent surgical consultation, are critically important because of the likelihood of upper airway obstruction, bacteremia, and extension of infection along complex fascial planes. group a streptococcal infections can reach the mediastinum via lymphatics from head and neck infections or soft tissue infection of the thorax, from contiguous spread from pneumonia, empyema, or parapharyngeal infection, or hematogenously from bloodstream infection. mediastinal infections caused by group a streptococcus have a high mortality caused, in part, by the potency of toxins produced, the difficulty in making an early diagnosis, and finally, the difficulty in adequately draining the site of infection because of the abundance of vital structures in this anatomical site. although uncommon among community-acquired infections, pneumonia caused by group a streptococcus is unique for two reasons. first, empyema develops in nearly 40% of cases, and second, pleural fluid accumulates rapidly, and may be as much as several liters per day. in the past, the greatest challenges for physicians treating patients with this infection were adequate drainage of loculated empyema fluid and the management of restrictive lung disease that developed subsequent to fibrosis of pleural surfaces. shock and organ failure were uncommon. in contrast, recent cases of group a streptococcal pneumonia and empyema have been associated with the streptss (author's observations). puerperal sepsis occurs during pregnancy or abortion, when group a streptococcus colonizing the patient invades the endometrium and surrounding structures, as well as the lymphatics and bloodstream. the result is acute endometri-tis and bacteremia complicated by pelvic thrombophlebitis and peritonitis. puerperal sepsis reached epidemic proportions in europe 22 and the united states 23 during the mid-1800s, largely as the result of contamination of women by attending physicians. a small number of cases attributed to group b streptococcus was reported during this time, 24 and this organism remains an uncommon cause of puerperal sepsis. from 1900 to 1970, improvements in infection control practices and the advent of antibiotics significantly reduced the incidence of this infection. in contrast, currently in the united states, 25, 26 europe, [27] [28] [29] and japan, 30 there has been a re-emergence of group a streptococcal puerperal sepsis associated with streptss. while most of these cases have been sporadic, secondary cases associated with rectal or vaginal carriage of group a streptococcus by health-care workers have been reported. 31 clearly, if more than one case occurs in a health facility, searches for a common source infection should be carried out. necrotizing fasciitis is a deep-seated infection of the subcutaneous tissue that results in progressive destruction of fascia and fat, but with sparing of the skin itself. historically, pfanner 32 is credited with the first description of what he called necrotizing erysipelas. several years later, meleney 33 described 20 patients with hemolytic streptococcal gangrene in china, and he argued that this entity was different from erysipelas and should have a different name. these cases were probably caused by group a streptococci as we now know them, although at that time they only characterized the organism as hemolytic streptococci. subsequently, "necrotizing fasciitis" has become the term that is preferred, because bacteria other than streptococci, such as clostridium perfringens, clostridium septicum, staphylococcus aureus, and mixed aerobic-anaerobic bacteria can produce a similar pathologic process. meleney's descriptions of the progression are clinically useful and are summarized as follows: characteristically streptococcal gangrene begins at the site of trivial or inapparent trauma. within 24 hours of the initial lesion, there is aggressive development of swelling, heat, erythema and tenderness with rapid spreading proximally and distally from the original focus. during the next 24-48 hours, the erythema darkens, changing from red to purple and then to blue, and blisters and bullae form which contain clear yellow fluid. on the fourth or fifth day, the purple areas become frankly gangrenous. from the seventh to tenth day, the line of demarcation becomes sharply defined and the dead skin begins to separate at the margins revealing extensive necrosis of the subcutaneous tissue. up until this time, the patient continues to be febrile, prostration increases and the patient becomes more emaciated. in more severe cases, the process advances rapidly until several large areas of skin have become gangrenous, and the intoxication renders the patient dull, unresponsive, mentally cloudy or even delirious. subsequently, the patient may develop metastatic abscesses, bronchopneumonia or lung abscess. (adapted from reference 33 in should be remembered that meleney's description of hemolytic streptococcal gangrene was published in 1924well before the advent of antibiotics. in fact, he was the first to advocate aggressive "bear scratch" fasciotomy and debridement. 33 using this approach, as well as dakan's solution for irrigation, mortalities of as low as 20% were achieved. 33 in contrast, more modern series suggest that mortalities of 30%-60% are common, even with antibiotics and aggressive surgical debridement. in addition, the progression of modern group a streptococcal infections is much more rapid than meleney described, and frequently patients may die within 48-96 h of the onset of symptoms. 16 further, these necrotizing processes are more likely to destroy skin, subcutaneous fat, fascia, and underlying muscle. for example, in one study, 16 myonecrosis was present in about 50% of patients who had necrotizing fasciitis. these data suggest that modern strains are more virulent. group a streptococcal myositis has been an extremely uncommon infection. adams et al. 34 documented only 21 cases that had been reported from 1900 to 1985, and svane 35 found only 4 cases among over 20 000 autopsies. in our series of 20 patients with invasive group a streptococcal infection, 16 1 had myositis alone, 3 had myositis as well as necrotizing fasciitis, and 5 had necrotizing fasciitis alone. recently, deep soft-tissue infections, such as necrotizing fasciitis and myositis, have also been described in reports from norway, 4 sweden, 36 and canada. 37 although group a streptococci can infect viable muscle via a direct penetrating injury or surgical incision, in approximately 50% of cases there is no history of such an event. these cases develop spontaneously, or secondary to very minor blunt trauma or muscle strain, and most patients deny antecedent symptomatic pharyngitis or tonsillitis. 4, 16, [34] [35] [36] 38, 39 thus, translocation of group a streptococci likely occurs from the colonized pharynx to the site of muscle injury via hematogenous or lymphatic spread. severe pain may be the only presenting symptom, and physical findings early may demonstrate only swelling and erythema, although patients may rapidly develop muscle compartment syndromes. 4, 16, [34] [35] [36] [38] [39] [40] in most cases, a single muscle group may be involved; however, because patients are frequently bacteremic, multiple sites of myositis can occur. 16, 34 distinguishing streptococcal myositis from spontaneous gas gangrene caused by clostridium perfringens or clostridium septicum 41 may be difficult, although the presence of crepitus or gas in the tissue would favor clostridial infection. 39 distinguishing necrotizing fasciitis from myositis is easily done anatomically from surgical exploration or incisional biopsy. in streptococcal cases, many patients may have evidence of both necrotizing fasciitis and myositis. 16, 34 in published series, the case fatality rate for necrotizing fasciitis is between 20% and 50%, whereas group a streptococcal myositis has a fatality rate between 80% and 100%. 16, [34] [35] [36] 38, 39, 41 aggressive surgical debridement is of extreme importance, because of the poor efficacy of penicillin that has been described in human cases, 16, 34, 35, 38, 39 as well as in experimental streptococcal models of myositis 42, 43 (see section on antibiotic efficacy). of note, the term "myositis" connotes "inflammation" of muscle; however, in most cases of streptococcal myositis, frank necrosis is present, with only a paucity of infiltrating inflammatory cells. thus, a better term would be "myonecrosis". epidemiology several population-based studies of streptss have documented the annual incidence of 1-5 cases per 100 000 popu44 with most cases being sporadic in nature; however, larger epidemics of invasive group a streptococcal infections have also been described in some settings. in 1994, an epidemic of related invasive infections occurred in wannamingo, minnesota, 2 with an annualized prevalence of 24 cases per 100 000 population. in missoula, montana in 1999, the incidence of invasive infections reached 30 cases per 100 000 population. in addition to community-based infections, invasive group a streptococcal infections have also been described in hospitals, convalescent centers, and among hospital employees and family contacts of patients with invasive infections. 9, 45, 46 some of these studies have documented the same m-type and identical rflp patterns in strains from primary and index cases. 9, [45] [46] [47] in addition, carriage of group a streptococcus by healthcare personnel has been associated with the spread of life-threatening group a streptococcal infections in the obstetrics/gynecology and ear-nose-throat wards of american hospitals. 31 such infections have also originated in outpatient surgical settings and within the home environment. it has been estimated that the risk of secondary cases may be approximately 200 times greater than the risk among the general population. 48 there is ample data from studies conducted over several decades that group a streptococcus is quickly and efficiently transmitted from index cases to susceptible individuals, and that transmission may result in colonization, pharyngitis, scarlet fever, rheumatic fever, or invasive group a streptococcal infections. the risk for secondary cases is likely related to close or intimate contact and crowding, as well as host factors such as: (1) active viral infections such as varicella or influenza; (2) recent surgical wounds and childbirth (author's unpublished observations); (3) absence of type specific opsonic antibody against the group a streptococcus causing the index case; and (4) absence of neutralizing antibody against pyrogenic exotoxin a or b. 1 the portals of entry for streptococci are the vagina, pharynx, mucosa, and skin in 50% of cases. 16 interestingly, a portal cannot be defined in the remaining 50%. 16 rarely, patients with symptomatic pharyngitis develop streptss. surgical procedures, such as suction lipectomy, hysterectomy, vaginal delivery, bunionectomy, and bone pinning provide a portal of entry in some cases. numerous cases have developed within 24-72 h of minor nonpenetrating trauma resulting in hematoma, deep bruise to the calf, or even following muscle strain. 16 virus infections such as varicella and influenza have provided portals in other cases. 16 in some cases, the use of nonsteroidal anti-inflammatory agents may have either masked the presenting symptoms or predisposed to more severe streptococcal infection and shock. 16 most cases of streptss occur sporadically, although outbreaks of severe group a streptococcal infections have been described in closed environments, such as nursing homes, 49, 50 and hospital environments. 45, 46 symptoms and signs twenty percent of patients have an influenza-like syndrome, characterized by fever, chills, myalgia, nausea, vomiting, and diarrhea. 16 fever is the most common presenting sign, although hypothermia may be present in patients with shock. 16 confusion is present in 55% of patients, and in some, coma or combativeness are manifest. 16 pain -the most common initial symptom of streptss -is abrupt in onset, severe, 16 and usually precedes tenderness or physical findings. the pain usually involves an extremity, but may also mimic peritonitis, pelvic inflammatory disease, pneumonia, acute myocardial infarction, or pericarditis. 16 eighty percent of patients have clinical signs of soft-tissue infection, such as localized swelling and erythema, which progresses to necrotizing fasciitis or myositis in 70% of cases, requiring surgical debridement, fasciotomy, or amputation. 16 of the 20% of cases without soft-tissue findings, a variety of clinical presentations were observed, including endophthalmitis, myositis, perihepatitis, peritonitis, myocarditis, and overwhelming sepsis. 16 a diffuse, scarlatinalike erythema is uncommon, occurring in only 10% of cases. the serum creatinine phosphokinase (cpk) level is useful in detecting the presence of deeper soft-tissue infections, and when the level is elevated or rising, there is a good correlation with necrotizing fasciitis or myositis. 16 although the initial laboratory studies may demonstrate only mild leukocytosis, the mean percentage of immature neutrophils (including band forms, metamyelocytes, and myelocytes) is striking, reaching 40%-50%. 16 the presence of hemoglobinuria and elevated serum creatinine values is evidence of renal involvement. it is important to note that renal impairment precedes hypotension in 40%-50% of cases. 16 hypoalbuminemia and hypocalcemia occur early, and become profound within 24-48 h of admission. the rapidity with which shock and multi-organ failure can progress is impressive, and many patients may die within 24-48 h of hospitalization. 16 shock was apparent at the time of admission or within 4-8 h in virtually all patients. in only 10% of patients did systolic blood pressure became normal 4-8 h after the administration of antibiotics, albumin, and electrolyte solutions containing salts or dopamine; in all other patients shock persisted. interestingly, renal dysfunction preceded shock in many cases and was apparent on admission in most patients. renal failure progressed or persisted in all patients for 48-72 h, and several patients required dialysis for 10-20 days. 16 in patients who survived, serum creatinine values returned to normal within 4-6 weeks. acute respiratory distress syndrome (ards) occurred in 55% of patients and generally developed after the onset of hypotension. 16 the severity of ards was such that supplemental oxygen, intubation, and mechanical ventilation was necessary in 90% who developed this syndrome. 16 mortality rates have varied from 30% to 70%; 16, 37, 51 however, morbidity is also high; 13 of 20 patients in one series underwent major surgical procedures, which included fasciotomy, surgical debridement, exploratory laparotomy, intraocular aspiration, amputation, or hysterectomy. 16 characteristics of clinical isolates of group a streptococci many different characteristics have been associated with streptss; however, m-types 1, 3, 6, 12, and 28 account for the majority of isolates. 8, 16 recently, 80% of strains in sweden from all types of group a streptococcal infection have been m-type 1. 36 pyrogenic exotoxin a (spea) and/ or b (speb) were found in the majority of cases of severe infection. in the united states, spea is most frequently associated with these infections, 8, 16, 44, [52] [53] [54] while in sweden and the united kingdom, speb has been most common. 51, 55 recently, streptococcal superantigen (ssa), a novel pyrogenic exotoxin, has been isolated from an m-3 strain, albeit in small concentrations. 56 in addition, mitogenic factor (mf) has been demonstrated in many different m-types of group a streptococcus. 57, 58 pathogenic mechanisms the role of adherence and penetration of pharyngeal epithelial cells has been the subject of intense research recently to determine which surface components of the group a streptococcus are involved, and to determine if penetration of such cells correlates with the ability of a strain to cause invasive infection. lapenta et al. 59 demonstrated that m-1 strains were efficient at adherence and penetration of respiratory epithelial cells. dombeck et al. 60 then demonstrated that a clonal variant of m-1 group a streptococcus linked to invasive infections (designated m1invï©) demonstrated high frequency internalization of cultured hela cells, and that this was dependent upon m-protein. in contrast, others have demonstrated that m-1 strains adhered to and invaded epithelial cells less efficiently than other m-types. 61, 62 in addition, molinari and chhatwal 61 have demonstrated that, regardless of m-type, strains isolated from invasive cases (blood isolates) exhibited poorer attachment and internalization of epithelial cells than strains isolated from throat or skin. kawabata et al. 63 demonstrated that hyaluronic acid capsule decreased the invasion efficiency. tsai et al. 64 investigated the role of speb in the invasion of respiratory epithelial cells (a-549) and found that speb knockout strains demonstrated reduced ability to invade cells, and that exogenous addition of speb increased invasion by the speb knockout mutants. in other studies, speb knockout mutants did not demonstrate reduced invasion of epithelial cells. 65 this discrepancy could, in part, be explained by the recent work of woischnik, buttaro, and podbielski, 66 who demonstrated that inactivation of the speb structural gene (speb) decreased hyaluronic acid capsule production, but did not affect other virulence factors. these findings demonstrate that interpretation of data from experiments using isogenic mutants may not always be straightforward. talay et al. 67 and hanski et al. 68 demonstrated that streptococcal fibronectin binding protein i (sfbi) mediated the binding of group a streptococcus to epithelial cells in the presence of fibronectin. subsequently, okada et al., 69 using strains lacking the prtf1 gene, have conclusively demonstrated that protein f, a fibronectin binding protein (sfbi), mediates the attachment of group a streptococcus to hela cells. molinari and chhatwal 61 demonstrated that the distribution of sfbi was lowest among blood isolates (27%) and highest (95%) among skin and throat isolates. these authors concluded that reduced sfbi expression was responsible for the reduced invasiveness of blood isolates. as described under the discussion on bacterial adherence and penetration, m-protein may serve as a ligand in specific strains of group a streptococcus. recent studies by jadoun et al. 70 suggest that both m-protein (m-6) and sfbi may participate in the adherence and invasion of epithelial cells. this is supported by the observation that type-specific siga and igg antibody prevented the adherence and invasion, respectively, of human pharyngeal cells challenged with m-6 strains. 71 in summary, the adherence and penetration of epithelial cells by group a streptococcus is a very complex process, and it is dependent upon the cell type, state of differentiation (e.g., keratinocyte), strain of group a streptococcus, the immune status of the host, and the inflammatory milieu of the host environment. the importance of these latter points is substantiated by the observation that tumor necrosis factor (tnf) and interleukin-1 (il-1) reduce the adherence of group a streptococcus to keratinocytes, 72 and by the demonstration that sfbi-vaccinated animals showed 80% and 90% protection against homologous and heterologous intranasal challenge with viable group a streptococcus. 73 finally, sfbi and sfbii were found in 64% and 36% of strains in the northern territory in australia, and high levels of igg antibody were found in the sera of 80 subjects with defined streptococcal infections. 74 mechanisms of shock pyrogenic exotoxins induce fever in humans and animals, and also participate in shock by lowering the threshold to exogenous endotoxin. 17 streptococcal pyrogenic exotoxins a and b induce human mononuclear cells to synthesize not only tnfî± 75 but also il-1 76 and il-6, [76] [77] [78] suggesting that tnf could mediate the fever, shock, and organ failure observed in patients with streptss. 16 pyrogenic exotoxin c has been associated with mild cases of scarlet fever in the united states (author's observations) and in england. 79 the roles of newly described pyrogenic exotoxins, such as ssa 56 and mf, 58 in the pathogenesis of streptss have not been elucidated. there is strong evidence suggesting that spea, speb, and spec, as well as a number of staphylococcal toxins (tsst-1, and staphylococcal enterotoxins a, b, and c) act as superantigens and stimulate t-cell responses through their ability to bind to both the class ii mhc complex of antigen-presenting cells and the v region of the t-cell receptor. 80 the net effect is induction of t-cell proliferation (via an il-2 mechanism), with the concomitant production of cytokines (e.g., il-1, tnfî±, tnf , il-6, and interferon [ifn]î³) that mediate shock and tissue injury. recently, hackett and stevens 81 demonstrated that spea induced both tnfî± and tnf from mixed cultures of monocytes and lymphocytes, supporting the role of lymphokines (tnf ) in shock associated with strains producing spea. kotb et al. 82 have shown that a digest of m-protein type 6 can also stimulate t-cell responses by this mechanism. interestingly, quantitation of such v t-cell subsets in patients with acute streptss demonstrated deletion rather than expansion, suggesting that perhaps the lifespan of the expanded subset was shortened by a process of apoptosis. 83 in addition, the subsets deleted were not specific for spea, speb, spec, or mf, suggesting that perhaps an as yet undefined superantigen may play a role in streptss. 83 cytokine production by less exotic mechanisms may also contribute to the genesis of shock and organ failure. peptidoglycan, lipoteichoic acid, 84 and killed organisms 85, 86 are capable of inducing tnfî± production by mononuclear cells in vitro. 17, 86, 87 exotoxins such as slo are also potent inducers of tnfî± and il-1 . speb, a proteinase precursor, has the ability to cleave pre-il-1 to release preformed il-1 . 88 finally, slo and spea together have additive effects in the induction of il-1 by human mononuclear cells. 81 whatever the mechanisms, the induction of cytokines in vivo is likely the cause of shock, and slo, spea, speb, and spec, as well as cell wall components, etc., are potent inducers of tnf and il-1. 9 finally, a cysteine protease formed from the cleavage of speb may play an important role in pathogenesis by the release of bradykinin from endogenous kininogen and by activating metalloproteases involved in coagulation. 89 the mere presence of virulence factors, such as mprotein or pyrogenic exotoxins, may be less important in streptss than the dynamics of their production in vivo. for example, chaussee et al. 9 have demonstrated that, among strains from patients with necrotizing fasciitis and streptss, approximately 40% and around 75% produced spea or speb, respectively. in addition, the quantity of spea, but not of speb, was higher for strains from streptss patients compared to with patients with findings in non-invasive disease. 9 recently, cleary et al. 90 have proposed a regulon in group a streptococcus that controls the expression of a group of virulence genes coding for virulence factors such as m-protein and c5a-peptidase. using dna fingerprinting, cleary et al. 91 showed differences in m-1 strains isolated from patients with invasive disease compared with m-1 strains from patients with non-invasive group a streptococcal infections. such strains of group a streptococci could acquire genetic information coding for spea via a specific bacteriophage. multi-locus enzyme electrophoresis demonstrates two patterns, which correspond to m-1 and m-3 type organisms that produce pyrogenic exotoxin a, a finding that fits epidemiologic studies implicating these strains in invasive group a streptococcal infections 54 in the united states. numerous studies have demonstrated that spea potentiates the action of lipopolysaccharide (lps) in inducing shock. 92 interestingly, this effect is largely the result of tcell activation, and the "potentiating activity" can be passively transferred to rabbits by infusing supernatants from spea-stimulated lymphocytes, 93 and is likely mediated by ifn-î³ and tnf . in contrast, the timing of pre-treatment with spea is crucial, because, as shown by stevens et al., 94 the pretreatment of mice with spea 96 h prior to lps challenge resulted in protection from lps-induced shock, probably due to the production of immunosuppressive cytokines such as il-10. finally, sriskandan et al. 95 have demonstrated that animals immunized with recombinant spea have a higher mortality when challenged with a spea-producing strain of group a streptococcus than animals that are sham immunized. 95 further sriskandan et al. 96 have demonstrated that animals challenged with a spea knockout strain of group a streptococcus had greater mortality and greater tissue destruction than animals challenged with the wild-type parent strain. thus, the roles of the pyrogenic exotoxins in the pathogenesis of severe group a streptococcal infections are complex and remain to be fully elucidated. however, the importance of cytokine induction in group a streptococcal infections cannot be denied. hackett et al. 85 have shown that cell wall components from killed invasive and noninvasive strains of group a streptococci elicit significant tnfî± production by mononuclear cells, exceeding that elicited by spea, speb, or slo. 85 further, viable slodeficient, but not viable wild-type, group a streptococci stimulated high levels of tnfî±, suggesting that slo suppresses the induction of this cytokine, perhaps, in part, because of its cytotoxic effects on host cells. cytokine induction by cell wall components of the group a streptococcus has also been demonstrated by muller-alouf et al. 86 an important role for tnfî± in the shock associated with severe group a streptococcal infections is also suggested by the remarkable efficacy of a neutralizing monoclonal antibody against tnfî± in an experimental streptococcal bacteremia model in non-human primates. 97 the survival of animals was increased by 50%, and markers of organ failure, such as serum creatinine and lactate, were also significantly improved. 97 other mechanisms of shock and organ failure recently, chatellier and kotb 98 demonstrated that the cg rich motifs of emm1 functioned as superantigens and induced t-cell proliferation. sriskandan et al. 99 have also demonstrated that inducible nitric oxide (inos) may play a role in the shock associated with a group a streptococcal necrotizing soft-tissue infection. in that model, nmonomethyl-l-arginine (l-nmma) administration reduced nitrate levels in serum, but did not reduce mortality. 99 the role of streptokinase in invasive group a streptococcal infections has not been extensively studied; however, recent studies demonstrate that certain alleles of streptokinase (ska2) have three energetic folding units which change the tertiary structure of ska into a more lipophilic molecule with high affinity for glomeruli. only ska2 isogenic mutants are capable of causing kidney lesions with polymorphonuclear lencocyte (pmnl) influx into the glomeruli, as well as deposition of c3. 100 streptolysin s (sls) has been known for a long time to be a potent cytotoxin for many different cell types, although it has only recently been implicated as a significant virulence factor. betschel et al. 101 have demonstrated that insertional activation of the gene controlling sls resulted in reduced virulence in a mouse model. recently, nizet et al. 102 have demonstrated that sls production is controlled by a contiguous nine-gene locus, saga to sagi, and that the sls gene product likely has a structure similar to the bacteriocin family of microbial toxins. antibiotic therapy -importance of the mechanism of action streptococcus pyogenes remains exquisitely susceptible to -lactam antibiotics, and penicillin has excellent efficacy in the treatment of erysipelas, impetigo, and cellulitis, as well as in the prevention of acute rheumatic fever. nonetheless, clinical failures of penicillin treatment of streptococcal infection do occur, and penicillin fails to eradicate bacteria from the pharynx of patients with documented streptococcal pharyngitis in 5%-20% of patients. [103] [104] [105] finally, more aggressive group a streptococcal infections (such as necrotizing fasciitis, empyema, burn wound sepsis, subcutaneous gangrene, and myositis) respond less well to penicillin and continue to be associated with high mortality and extensive morbidity. 4, 16, 17, 34, 55, 106, 107 for example, a recent report of 25 cases of streptococcal myositis reported an overall mortality of 85% despite penicillin therapy. 34 studies in experimental infection have demonstrated that penicillin fails when large numbers of organisms are present. 42, 43 for example, in a mouse model of myositis caused by s. pyogenes, penicillin was ineffective when treatment was delayed for 2 h or more after initiation of infection. 43 mice receiving clindamycin, however, had survival rates of 100%, 100%, 80%, and 70% when treatment was delayed 0, 2, 6, and 16.5 h, respectively. 43,108 eagle 42 suggested that penicillin failed in this type of infection because of the "physiologic state of the organism." this phenomenon has recently been attributed to inoculum effects, both in vitro and in vivo. 109, 110 it has also been observed that penicillin and other -lactam antibiotics are most efficacious against rapidly growing bacteria. early in the stages of infection, or in mild infections, organisms grow rapidly in vivo but are present in rather small numbers. with delays in treatment, higher concentrations of group a streptococci accumulate, and growth begins to slow to a stationary phase. that high concentration of s. pyogenes accumulate in deep-seated infection is supported by the data from eagle. 42 why should penicillin lose its efficacy when large numbers of group a streptococci are present or when they are making the transition from logarithmic to stationary growth? because penicillin mediates its antibacterial action against group a streptococci by intimately interacting with the expressed penicillin-binding proteins (pbps), we compared the pbp patterns from membrane proteins of group a streptococci isolated from different stages of growth, i.e., mid-log-phase and stationary-phase. the binding of radiolabeled penicillin by all pbps was decreased in stationary cells. in fact, pbps 1 and 4 were undetectable at 36 h. 109 thus, the loss of certain pbps during stationary-phase growth in vitro may be responsible for the inoculum effect observed in vivo, and may account for the failure of penicillin in both experimental and human cases of severe streptococcal infection. the greater efficacy of clindamycin in severe group a streptococcal infections is due to many factors. first, its efficacy is not affected by inoculum size or stage of growth. 109, 111 secondly, clindamycin suppresses bacterial toxin synthesis. 112, 113 third, clindamycin facilitates the phagocytosis of s. pyogenes by inhibiting m-protein synthesis. 113 fourth, clindamycin suppresses the synthesis of pbps which, in addition to being targets for penicillin, are also enzymes involved in cell wall synthesis and degradation. 111 fifth, clindamycin has a longer post-antibiotic effect thanlactams such as penicillin. lastly, we have recently shown that clindamycin suppresses lps-induced monocyte synthesis of tnfî±. 114 thus, clindamycin's efficacy may also be related to its ability to modulate the immune response to group a streptococcal infection. interestingly, in a recent retrospective analysis of streptss cases, zimbelman et al. 115 demonstrated significantly improved survival in patients receiving clindamycin compared with survival in those treated with -lactam antibiotics. prompt and aggressive exploration and debridement of suspected deep-seated s. pyogenes infection is crucial to limit complications, as well as to prevent extension to vital areas that may be impossible to debride (i.e., the head and neck, thorax, or abdomen). because definite cutaneous evidence of necrotizing fasciitis and myositis may not appear until late in the course, the index of suspicion should be high in patients with fever, excruciating pain, and systemic toxicity. radiographs and ct and mri scans in patients with streptss show soft-tissue swelling and an absence of gas, thus providing clues to the site of the deep infection. prompt surgical exploration through a small incision, with visualization of muscle and fascia, and timely gram stain of surgically obtained material provide the earliest and most definitive etiologic diagnosis. 40 because of intractable hypotension and diffuse capillary leak, massive amounts of intravenous fluids (10-20 l/day) are often necessary, and about 10% of patients have significant clinical improvement. pressors such as dopamine are used frequently, although no controlled trials have been performed in streptss. in patients with intractable hypotension, vasoconstrictors such as epinephrine have been used, but frequently, symmetrical gangrene of digits results (author's unpublished observations). loss of all digits on both hands and feet, or loss of both arms and legs has occurred in this setting. in these cases it is difficult to determine whether the symmetrical gangrene is caused by the pressors or by the infection, or by both. neutralization of circulating toxins would be a desirable therapeutic modality, yet appropriate antibodies are not commercially available in the united states or europe. case reports, 116, 117 and one non-randomized clinical trial, 118 report that commercial intravenous gamma globulin (ivig) has been useful for treating streptss. a doubleblind study using ivig is currently underway in northern europe. because cytokines are important mediators of shock in streptss, strategies to inhibit or neutralize their effects may provide useful treatments. recently, a monoclonal antibody against tnfî± showed promising efficacy in a baboon model of streptss. 97 finally, anecdotal reports suggest that hyperbaric oxygen may be helpful; however, no controlled studies are underway, nor is it clear if this treatment is useful. currently, several group a streptococcal virulence factors are being investigated as potential vaccine candidates. these include: group a polysaccharide, c5a peptidase, speb, spea, the conserved segment of the m-protein molecule, and a cassette of peptides representing the hypervariable regions of m-protein from strains commonly encountered. at the present time, none have been studied in humans. the risk of secondary cases of streptss must be low, based on the low prevalence of this disease despite a high prevalence of "virulent strains of group a streptococci" in the population at large. 2 yet, clusters of group a streptococcal invasive infections have been described in nursing homes 49, 50, 119 in healthcare workers, 46,120 and among family members. 46, 119 finally, patients may acquire group a streptococci from hospital personnel, and this was best demonstrated historically by semmelweis, in vienna, and holmes, in the united states, although even in modern times such transmission is well documented. 46, 120, 121 epidemiologic investigation of clusters of cases is important, and treatment of contacts may be necessary despite the low risk of secondary invasive infection. 122 primary care physicians must consider the extent of exposure, the type of exposure, and the risk factors of the contact. for example, a contact of a case of streptss with risk factors such as chicken pox, leukemia, burns, or recent surgery, or any open skin lesion, should receive prophylaxis with penicillin, erythromycin, or clindamycin. 122 invasive group a streptococcal disease in the netherlands: evidence for a protective role of antiexotoxin a antibodies an outbreak of invasive group a streptococcal disease associated with high carriage rates of the invasive clone among school-aged children streptococcal toxic shock syndrome: spectrum of disease, pathogenesis and new concepts in treatment streptococcal serogroup a epidemic in norway 1987-1988 invasive group a streptococcal infections: t1mi isolates expressing pyrogenic exotoxins a and b in combination with selective lack of toxinneutralizing antibodies are associated with increased risk of streptococcal toxic shock syndrome protective immunity to the group a streptococcus may be only strain specific hetereogeneous opsonic activity of convalescence serum against m-1 strains of group a streptococci (gas) epidemiologic analysis of group a streptococcal serotypes associated with severe systemic infections, rheumatic fever, or uncomplicated pharyngitis genetic and phenotypic diversity among isolates of streptococcus pyogenes from invasive infections production of nadase in clinical isolates of streptococcus pyogenes diphosphopyridine nucleotidase as an extracellular product of streptococcal growth and its possible relationship to leukotoxicity application of a new method for detecting streptococcal nicotinamide adenine dinucleotide glycohydrolase to various m types of streptococcus pyogenes nadï©-glycohydrolase productivity of haemolytic streptococci assayed by a simple fluorescent method and its relation to t serotype the nad-glycohydrolase (nga) gene of streptococcus pyogenes mole-cular epidemiology of nga and nad glycohydrolase/adp-ribosyltransferase activity among streptococcus pyogenes causing streptococcal toxic shock syndrome reappearance of scarlet fever toxin a among streptococci in the rocky mountain west: severe group a streptococcal infections associated with a toxic shock-like syndrome invasive group a streptococcus infections complications of varicella i. their occurrence among 2534 patients group a beta-hemolytic streptococcal bacteremia: historical overview, changing incidence, and recent association with varicella treatment of patients with a history of recurrent tonsillitis due to group a -hemolytic streptococci peritonsillar abscess: incidence, current management practices, and a proposal for treatment guidelines human endothelial cells in culture produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with thrombin the writings of oliver wendell holmes fatal infections by haemolytic streptococcus group b the return of lifethreatening puerperal sepsis caused by group a streptococci an epidemic of "childbed fever life-threatening puerperal sepsis caused by group a streptococci puerperal sepsis due to infected episiotomy wound puerperal sepsis caused by streptococcus group a with a severe form of progression like "toxic shock-like syndrome a new type of fulminant group a streptococcal infection in obstetric patients: report of two cases nosocomial group a streptococcal infections associated with asymptomatic health-care workers -maryland and california zur kenntnis und behandlung des nekrotisierenden erysipels hemolytic streptococcus gangrene streptococcal myositis peracute spontaneous streptococcal myositis. a report on two fatal cases with review of literature aspects of pathogenesis of serious group a streptococcal infections in sweden, 1988-1989 severe invasive group a streptococcal infections in streptococcal necrotizing myositis -a rare entity: a report of two cases spontaneous gangrenous myositis induced by streptococcus pyogenes: case report and review of the literature streptococcal infections in skin and soft tissues spontaneous, nontraumatic gangrene due to clostridium septicum experimental approach to the problem of treatment failure with penicillin. i. group a streptococcal infection in mice the eagle effect revisited: efficacy of clindamycin, erythromycin, and penicillin in the treatment of streptococcal myositis changing epidemiology of group a streptococcal infection in the usa familial transmission of a serious disease-producing group a streptococcus clone: case reports and review spread of serious disease-producing m3 clones of group a streptococcus among family members and health care workers transmission of streptococcus pyogenes causing toxic shock-like syndrome among family members and confirmation by dna macrorestriction analysis invasive group a streptococcal infections in ontario outbreak of invasive group a streptococcal infections in a nursing home. lessons on prevention and control invasive group a streptococcal (gas) serotype m-1 outbreak in a long-term care facility (ltcf) with mortality septic shock induced by group a streptococcal infections: clinical and therapeutic aspects association of exotoxin-producing group a streptococci and severe disease in children molecular analysis of pyrogenic exotoxins from streptococcus pyogenes isolates associated with toxic shock-like syndrome streptococcus pyogenes causing toxic-shock-like syndrome and other invasive diseases: clonal diversity and pyrogenic exotoxin expression correspondence: group a streptococcal infections and a toxic shock-like syndrome a novel superantigen isolated from pathogenic strains of streptococcus pyogenes with aminoterminal homology to staphylococcal enterotoxins b and c cloning, characterization and overexpression of a streptococcus pyogenes gene encoding a new type of mitogenic factor superantigenic properties of the group a streptococcal exotoxin spef (mf) group a streptococci efficiently invade human respiratory epithelial cells high-frequency intracellular invasion of epithelial cells by serotype m1 group a streptococci: m1 protein-mediated invasion and cytoskeletal rearrangements invasion and survival of streptococcus pyogenes in eukaryotic cells correlates with the source of clinical isolates comparison of adherence to and penetration of a human laryngeal epithelial cell line by group a streptococci of various m protein types capsular hyaluronic acid of group a streptococci hampers their invasion into human pharyngeal epithelial cells effect of group a streptococcal cysteine protease on invasion of epithelial cells role of group a streptococcal virulence factors in adherence to keratinocytes inactivation of the cysteine protease speb affects hyaluronic acid capsule expression in group a streptococci fibronectin-binding protein of streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells protein f, a fibronectin-binding protein, is an adhesin of the group a streptococcus streptococcus pyogenes streptococcus pyogenes protein f promotes invasion of hela cells protein f1 is required for efficient entry of streptococcus pyogenes into epithelial cells immunoglobulins to group a streptococcal surface molecules decrease adherence to and invasion of human pharyngeal cells tumor necrosis factoralpha and interleukin-1 alpha decrease the adherence of streptococcus pyogenes to cultured keratinocytes protective immune response against streptococcus pyogenes in mice after intranasal vaccination with the fibronectin-binding protein sfbi distribution and antigenicity of fibronectin binding proteins (sfbi and sfbii) of streptococcus pyogenes clinical isolates from the northern territory, australia toxic shock syndromeassociated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production cytokine production by human mononuclear cells in response to streptococcal exotoxins similar cytokine induction profiles of a novel streptococcal exotoxin, mf, and pyrogenic exotoxins a and b cytokine production by murine cells activated by erythrogenic toxin type a superantigen of streptococcus pyogenes the production of pyrogenic exotoxins by group a streptococci characterization of a superantigen from a pathogenic strain of streptococcus pyogenes (abstract) streptococcal toxic shock syndrome: synthesis of tumor necrosis factor and interleukin-1 by monocytes stimulated with pyrogenic exotoxin a and streptolysin o cellular and biochemical responses of human t lymphocytes stimulated with streptococcal m protein selective depletion of v -bearing t cells in patients with severe invasive group a streptococcal infections and streptococcal toxic shock syndrome gram-positive shock cytokine induction by viable group a streptococci: suppression by streptolysin o comparative study of cytokine release by human peripheral blood mononuclear cells stimulated with streptococcus pyogenes superantigenic erythrogenic toxins, heat-killed streptococci and lipopolysaccharide superantigens associated with staphylococcal and streptococcal toxic shock syndromes are potent inducers of tumor necrosis factor beta synthesis cleavage of interleukin 1 (il-1 ) precursor to produce active il-1 by a conserved extracellular cysteine protease from streptococcus pyogenes activation of a 66-kilodalton human endothelial cell matrix metalloprotease by streptococcus pyogenes extracellular cysteine protease a virulence regulon in streptococcus pyogenes clonal basis for resurgence of serious streptococcus pyogenes disease in the 1980s group a streptococcal pyrogenic exotoxin: pyrogenicity, alteration of blood-brain barrier, and separation of sites for pyrogenicity, and enhancement of lethal endotoxin shock macrophage hyperreactivity to endotoxin induced by streptococcal pyrogenic exotoxin in rabbits pretreatment with either endotoxin (lps), tumor necrosis factor î± (tnfî±) or streptococcal pyrogenic exotoxin a (spea) protects mice from lethal streptococcal infection streptococcal pyrogenic exotoxin a release, distribution, and role in a murine model of fasciitis and multiorgan failure due to streptococcus pyogenes streptococcal pyrogenic exotoxin a (spe a) can augment neutrophil recruitment to sites of inflammation group a streptococcal bacteremia: the role of tumor necrosis factor in shock and organ failure identification of dna cpg sequence motifs in group a streptococcal virulent genes that preferentially stimulate human lymphocytes: evidence for differential host reponsiveness the role of nitric oxide in experimental murine sepsis due to pyrogenic exotoxin a-producing streptococcus pyogenes allele substitution of the streptokinase gene reduces the nephritogenic capacity of group a streptococcal strain nz131 reduced virulence of group a streptococcal tn916 mutants that do not produce streptolysin s genetic locus for streptolysin s production by group a streptococcus association of penicillin tolerance with failure to eradicate group a streptococci from patients with pharyngitis failure of penicillin to eradicate group a streptococci during an outbreak of pharyngitis role of beta-lactamase-producing bacteria in the failure of penicillin to eradicate group a streptococci streptococcal toxic shock syndrome streptococcus pyogenes and the toxic shock syndrome invasive group a streptococcal infection: new concepts in antibiotic treatment penicillin binding protein expression at different growth stages determines penicillin efficacy in vitro and in vivo: an explanation for the inoculum effect the in vitro antibacterial activity of ceftriaxone against streptococcus pyogenes is unrelated to penicillin-binding protein 4 persistent acylation of highmolecular weight penicillin binding proteins by penicillin induces the post antibiotic effect in streptococcus pyogenes effect of antibiotics on toxin production and viability of clostridium perfringens potentiation of opsonization and phagocytosis of streptococcus pyogenes following growth in the presence of clindamycin antibiotic effects on bacterial viability, toxin production, and host response improved outcome of clindamycin compared with beta-lactam antibiotic treatment for invasive streptococcus pyogenes infection intravenous immunoglobulin therapy for toxic shock syndrome necrotising fasciitis (letter) intravenous immunoglobulin therapy for streptococcal toxic shock syndrome. a comparative observational study clusters of invasive group a streptococcal infections in family, hospital, and nursing home settings transmission of 'toxic strep' syndrome from an infected child to a firefighter during cpr wound infections due to group a streptococcus traced to a vaginal carrier the working group on prevention of invasive group a streptococcal infections. prevention of invasive group a streptococcal diseases among household contacts of case-patients: is prophylaxis warranted? acknowledgments this material is based upon work supported by the office of research and development, medical research service, department of veterans affairs. key: cord-022583-9lmudxrh authors: nan title: antimikrobielle und antiinfektiöse maßnahmen date: 2016-07-29 journal: krankenhausund praxishygiene doi: 10.1016/b978-3-437-22312-9.00002-0 sha: doc_id: 22583 cord_uid: 9lmudxrh nan • die transiente hautflora besteht aus nur zeitweise auf der haut vorkommenden bakterien, pilzen oder viren einschließlich nosokomialer infektionserreger. bakterien und hefepilze überleben meist eine stunde oder länger. bei viren reicht die dauer der persistenz von 10 min bis zu mehreren stunden (› tab. 2 .1). • die infektionsflora beinhaltet das vorkommen von ätiologisch an aktuellen infektionen der hand (wie abszessen, panaritium, paronychie, infiziertem ekzem) beteiligten erreger. übertragung nosokomialer infektionen durch hände: verschiedene ni werden über die hände von mitarbeitern übertragen, vor allem ssi, septikämien und pneumonien (kampf, löffler und gastmeier 2009) . gleiches gilt für die ausbreitung von mre. durch händedesinfektion wird daher die anzahl kolonisierter bzw. infizierter patienten reduziert (brown et al. 2003; gagné, bédard und maziade 2010; girou et al. 2006; gordin et al. 2005; johnson et al. 2005; kaier et al. 2009; simor et al. 2002; trick et al. 2003) . ebenso ist die effizienz bei der unterbrechung von ausbrüchen nachgewiesen (armbrust et al. 2009; cheng et al. 2007; fung und cairncross 2007; simor et al. 2002 ). indikationen: schutzhandschuhe dienen sowohl dem eigenschutz als auch der unterbrechung von infektionsketten (johnson et al. 1990 ; tenorio et al. 2001 ). sie müssen immer dann angelegt werden, wenn der kontakt mit erregern vorhersehbar oder wahrscheinlich bzw. wenn eine massive verunreinigung mit körperausscheidungen, sekreten und exkreten möglich ist (trba 531, 1996) . beispiele sind blutentnahmen, die pflege inkontinenter patienten, waschen von mrsa-patienten, umgang mit beatmungsschläuchen, entleerung von wasserfallen, endotracheales absaugen, tracheostomapflege, entsorgung von sekreten, exkreten und erbrochenem sowie die entfernung von drainagen, verbänden oder kontaminierten materialien. da die perforationsrate mit zunehmender tragedauer im pflegeprozess steigt, sollte sie auf etwa 15 min beschränkt werden. dabei sind nitrilhandschuhe den latexhandschuhen überlegen. da die perforationsrate nach patientenwaschung und verbandswechsel signifikant erhöht war, sollte hiernach in jedem fall ein handschuhwechsel durchgeführt werden (hübner et al. 2013) . der einsatz textiler aufbereitbarer unterziehhandschuhe hat durch absorption der feuchtigkeit einen günstigen einfluss auf den hautzustand und wurde für den routineeinsatz in der patientenpflege überwiegend bejaht (hübner et al 2014) . nach beendigung der tätigkeit, ggf. auch zwischen der verrichtung verschiedener tätigkeiten an einem patienten, sind die handschuhe im allgemeinen abzulegen. anschließend ist eine händedesinfektion durchzuführen, da handschuhe durch unbemerkte perforation oder kontamination der hände beim fehlerhaften ausziehen keinen sicheren schutz vor einer kontamination der hände gewähren (doebbeling et al. 1988; korniewicz et al. 1989; tenorio et al. 2001 ). handschuhwechsel erforderlich ist, aber erfahrungsgemäß häufig nicht durchgeführt wird, oder wenn eine notfallsituation zwischen dem kontakt von kontaminierten bedienelementen und dem patienten keinen handschuhwechsel zulässt. dabei sind drei voraussetzungen zu berücksichtigen (kramer et al. 2000b ): • der handschuh muss nachweislich desinfizierbar sein (häufigkeit, materialverträglichkeit, handschuhfabrikat, desinfektionsmittel müssen bekannt sein). • der handschuh weist keine bemerkten perforationen auf und ist nicht mit blut, sekreten oder exkreten kontaminiert. • es besteht keine erhöhte wahrscheinlichkeit einer kontamination mit chemoresistenten viren oder mre. schutzhandschuhe sind wegen des risikos der hautschädigung und erhöhter perforationsgefahr (pitten, herdemann und kramer 2000) nur auf trockenen händen anzulegen. die einfache händewaschung beinhaltet die anwendung einer waschlotion ohne antimikrobielle wirkung mit dem ziel, die hände zu reinigen. die einfache händewaschung ist einmalig zu arbeitsbeginn indiziert, um schmutz und bakteriensporen zu entfernen. risikoabhängig kann sie auch vor essenzubereitung und -verteilung, nach toilettenbenutzung außer bei durchfall und nach dem naseputzen außer bei atemweginfektionen durchgeführt werden. waschlotionen müssen frei von pathogenen sein. wegen der hautverträglichkeit sollte der ph-wert neutral oder schwach sauer sein. nach dem waschen muss die haut abgetrocknet werden, um hautschäden vorzubeugen. anstelle fester seifen ist der einsatz flüssiger seifen zu empfehlen, da erstere häufig kontaminiert waren und nach einführung flüssiger seife die rate von ni abfiel (şenol, çakan und özacar 2011) . die verwendung von einmalflaschen ist zu empfehlen, weil aufbereitung und nachfüllen mit kontaminationsrisiken verbunden sind. im fall eines ausbruchgeschehens sollten auch flüssige seifen in umgebungsuntersuchungen einbezogen werden, da diese vereinzelt quelle für gramnegative bakterien waren (archibald et al. 1997; grohskopf et al. 2001; sartor et al. 2000) . die hautverträglichkeit von seifen ist in allen merkmalen (transepidermaler wasserverlust, entfettung, hautrauhigkeit, schuppung, austrocknung) signifikant schlechter als die anwendung alkoholischer händedesinfektionsmittel (kramer et al. 2003 ). die hygienische händewaschung beinhaltet die anwendung einer antimikrobiellen waschlotion mit dem ziel, die hände zu reinigen und gleichzeitig eine gewisse bakterizide wirkung zu erzielen. die hygienische händewaschung ist im krankenhaus keine alternative zur händedesinfektion (kramer et al. 2000b ). die hygienische händedesinfektion beinhaltet die anwendung eines alkoholischen händedesinfektionsmittels nach tatsächlicher oder fraglicher kontamination der hände bzw. vor bestimmten tätigkeiten. indikationen: vor folgenden situationen wird die hygienische händedesinfektion, angelehnt an die 5 momente der händedesinfektion der who, empfohlen (› abb. 2 durchführung: die hygienische händedesinfektion ist so durchzuführen, dass die transiente flora noch auf den händen weitestgehend abgetötet wird. das alkoholische händedesinfektionsmittel ist über sämtliche bereiche der trockenen hände mit besonderer berücksichtigung der fingerspitzen, daumen, innen-und außenflächen, handgelenke, interdigitalräume und nagelfalze einzureiben. die hautareale sollen für die dauer der deklarierten einwirkzeit feucht benetzt sein. es ist eine einreibetechnik zu wählen, die sicherstellt, dass beide hände möglichst vollständig benetzt sind. für eine akzeptable benetzung der hände ist das verreiben des präparats für 22-28 s erforderlich ). bei mutmaßlicher/wahrscheinlicher viruskontamination muss ein gegen die entsprechenden viren wirksames präparat verwendet werden (valide prüfergebnisse). alkoholische händedesinfektionsmittel sind innerhalb von 30 s hoch wirksam gegenüber bakterien einschließlich mre, hefepilzen und behüllte viren (kampf und kramer 2004) . dagegen benötigen alkoholische gele mit niedrigem alkoholgehalt 1 min (kramer et al. 2002) und waren wegen der geringeren wirksamkeit trotz verbesserter compliance ohne einfluss auf die ni-rate (rupp et al. 2008 ). gegenüber unbehüllten viren sind nur wenige alkoholische desinfektionsmittel innerhalb klinisch vertretbarer einwirkzeit wirksam ). diese präparate weisen eine unterschiedliche einwirkzeit (1 bzw. 2 min) auf und sind aufgrund der zusammensetzung unterschiedlich gut hautverträglich (kampf und reichel 2010) . die effektivität der händedesinfektion ist sowohl anhand der senkung der ni-rate insgesamt (capretti et al. 2008; pitten et al. 2000) als auch für spezielle merkmale nachgewiesen wie senkung von zvk-assoziierten blutstrominfektionen (capretti et al. 2008; larson, quiros und lin 2007) , hwi und ssi (hilburn et al. 2003) , herabsetzung von mrsa-infektionen und der nachweisrate klinischer mre-isolate (gagné, bédard und maziade 2010; harbarth et al. 1999; harrington et al. 2007; johnson et al. 2005; kaier et al. 2009 ; ling und how 2012; macdonald et al. 2004 ). selbst in kommunalen settings war eine präventive wirkung in bezug auf gastrointestinale und respiratorische infektionen nachweisbar (guinan, mcguckin und ali 1997; hammond et al. 2000; hübner et al. 2010; lee et al. 2005; sandora et al. 2005; white et al. 2001) . die compliance der händehygiene liegt im gesundheitswesen bei durchschnittlich etwa 50 %. somit wird die händedesinfektion nur bei etwa der hälfte der situationen mit erforderlicher händedesinfektion durchgeführt. durch die verbesserung der compliance von 48 % auf 66 % konnte gezeigt werden, dass die ni-rate um 41 % sank (pittet et al. 2000) . keine andere einzelmaßnahme der krankenhaushygiene hat einen so großen nachweislichen präventiven nutzen. die compliance kann z. b . durch verwendung besonders hautverträglicher händedesinfektionsmittel, einfachen zugang zum desinfektionsmittel, verbrauchsanalysen, surveillance von ni, schulung und förderung der händehygiene, appell an die vorbildfunktion der vorgesetzten, vermeidung von personalengpässen in der patientenversorgung, automatische spender und standardisierte arbeitsabläufe (z. b. beim legen eines peripheren venenkatheters) verbessert werden (kampf et al. 2013; kampf, löffler und gastmeier 2009; sahud und bhanot 2009) . die chirurgische händedesinfektion wird präoperativ mit dem ziel durchgeführt, die transiente flora der hände zu eliminieren und die residente flora der hände für die dauer der op größtmöglich zu reduzieren. durch die chirurgische händedesinfektion soll das ssi-risiko gesenkt werden, da op-handschuhe in bis zu 40 % bemerkt oder unbemerkt perforieren (harnoss et al. 2009 (harnoss et al. und 2010 und perforierte op-handschuhe mit einem höheren ssi-risiko verbunden sind (cruse und foord 1973; misteli et al. 2009 ). die verwendung einer nichtmedizinischen seife hatte einen ssi-ausbruch zur folge (grinbaum, de mendonç und cado 1995) . indikationen: die chirurgische händedesinfektion ist vor allen operativen eingriffen durchzuführen (krinko 2007) sowie vor sonstigen eingriffen mit gleichen anforderungen an die asepsis. es wird empfohlen, die hände zu dienstbeginn zu waschen, spätestens aber vor anlegen der op-bereichskleidung in der op-schleuse (kramer et al. 2008b ). die hände und fingernägel der mitarbeiter müssen sauber sein, wenn sie den op-trakt betreten. vor dem anlegen der op-bereichskleidung wird eine hygienische händedesinfektion durchgeführt. durchführung: bei optisch sauberen händen ist routinemäßig keine waschung vor der desinfektionsphase erforderlich. die fingernägel sind nur bei verschmutzung mit weicher, thermisch desinfizierter (oder steriler) kunststoffbürste, ggf. zusätzlich mit holzstäbchen oder metallnagelreiniger zu säubern. hände und unterarme sind wegen der wegbereitung von hautirritationen nicht mit der bürste zu behandeln. anschließend werden die hände mit frischem einmaltextil-oder papierhandtuch getrocknet. zur desinfektion werden hände und unterarme nach der vom hersteller an-gegebenen einwirkzeit vollständig mit dem desinfektionsmittel benetzt. anschließend werden die hände an der luft getrocknet, bevor die op-handschuhe angelegt werden (krinko 2007) . alkoholische händedesinfektionsmittel: da alkohole, insbesondere propan-1-ol, gegen die residente hautflora hochwirksam sind, wurde die anwendung von propan-1-ol (60 %) über 3 min zum referenzverfahren für die chirurgische händedesinfektion ausgewählt. durch die referenzdesinfektion lässt sich die koloniezahl der hände um 2,7 log 10 -stufen reduzieren (sofortwirkung). nach 3 h unter dem op-handschuh ist die koloniezahl der hände noch immer um 2,2 log 10 -stufen niedriger (kampf und ostermeyer 2004) . ein präparat zur chirurgischen händedesinfektion darf der referenzdesinfektion weder in der sofort-noch in der langzeitwirkung unterlegen sein. abhängig vom präparat sind auch innerhalb von 1,5 min gleichwertige wirksamkeitsergebnisse erzielbar wie nach einer anwendung über 3 min (kampf, ostmeyer und heeg 2005) . jedoch haben kleine volumina wie 6 ml abhängig von der größe der hände eine schlechtere wirksamkeit, auch wenn die hände über die dauer der einwirkungszeit mit dem präparat benetzt gehalten werden (kampf und ostermeyer 2014) . bei bemerkter intraoperativer handschuhbeschädigung müssen neue sterile op-handschuhe angelegt werden. vor dem anlegen der neuen op-handschuhe ist eine alkoholische händedesinfektion für mindestens 30 s durchzuführen (kampf, ostermeyer und kohlmann 2008) . ist die hand durch blut verschmutzt bzw. hat sich handschuhsaft angesammelt, ist sie vor der desinfektion mit einem sterilen tuch zu reinigen. hat sich die perforation kurz vor op-ende ereignet, kann es ausreichen, einen neuen sterilen handschuh über den perforierten handschuh zu ziehen (arbeitskreis krankenhaus-und praxishygiene der awmf 2008). für das operativ tätige team wird bei eingriffen mit erhöhtem perforationsrisiko das tragen von zwei paar übereinander gezogener op-handschuhe (double gloving) empfohlen (thomas, agarwal und mehta 2001) , da indikatorhandschuhe die perforation nicht mit ausreichender sicherheit anzeigen (partecke et al. 2009 ). für die viszeralchirurgie wird aufgrund des anstiegs der perforationsrate und des ab 90 min nachweisbaren bakterientransfers durch die perforationen ein wechsel der op-handschuhe für operateur und ersten assistenten nach spätestens 90 min, für weitere assistenten und op-pflegepersonal nach 150 min empfohlen (harnoss et al. 2010; partecke et al. 2009 ), sofern kein double gloving bevorzugt wird. handschutz und handpflege sind als berufliche pflicht aufzufassen, weil eine geschädigte haut nicht mehr so gut desinfizierbar ist und in ein irritativ-toxisches kontaktekzem mit berufsunfähigkeit münden kann. eine gesunde haut ist voraussetzung für eine effektive händedesinfektion (mäkela 1993) . um der hautirritation vorzubeugen, müssen hautschutz und hautpflege systematisch und konsequent erfolgen (› kap. 5.20): • hautschutzpräparate werden bereits vor dem kontakt mit wasser und desinfektionsmitteln aufgetragen. • hautpflegeprodukte werden nach dienstschluss und in der freizeit angewandt. der protektive effekt von hautschutzpräparaten wurde in hautirritationsmodellen (fluhr et al. 1999; frosch und korte 1994; gehring 2004 ) und im op-arbeitsbereich (berndt et al. 2001 ) nachgewiesen. für die wirksamkeit war die regelmäßige, häufige und korrekte anwendung rückfettender externa entscheidend, weniger der zeitliche bezug zur wasser-und desinfektionsmittelexposition. hautpflegemittel sollen wegen der kontaminationsgefahr bei der entnahme in spendern oder tuben bereitgestellt werden. bei gefährdung der haut durch arbeiten im feuchten milieu -dazu gehört auch das tragen flüssigkeitsdichter handschuhe > 2 hmuss der arbeitgeber psa bereitstellen, eine betriebsanweisung und einen hautschutzplan erstellen, die möglichkeit zur reduzierung der feuchtigkeitsexposition einschließlich ersatzstoffprüfung überprüfen und die arbeitsmedizinische vorsorge und überwachung gewährleisten (trba 531 von 1996) . im hautschutzplan sind die präparate für reinigung, schutz und pflege der haut festzulegen. bei beginnenden hautschäden ist unverzüglich der betriebsärztliche dienst zu konsultieren. bei der auswahl von hautschutz-und hautpflegepräparaten ist der hauttyp (seborrhoisch oder sebostatisch) zu beachten. wegen des risikos der sensibilisierung und der penetrationsförderung durch harnstoff sind produkte ohne duft-und ohne konservierungszusatz mit einem harnstoffgehalt < 3 % zur bevorzugen. wegen der besseren hautverträglichkeit sind natürliche fettsäuren mineralölderivaten überlegen. üblicherweise werden händedesinfektionsmittelspender mit einmalflaschen bestückt und sollen folgende anforderungen erfüllen (assadian 2012): • bestückung ausschließlich mit nicht wiederbefüllbarem desinfektionsmittelgebinde; bei wiederbefüllen durch "top-up" sind die hygienischen rahmenbedingungen in form einer sop festzuschreiben, deren einhaltung zu dokumentieren ist. • die spender sollen die verwendung von händedesinfektionsmittelgebinden verschiedener hersteller erlauben. • die spender müssen so betrieben und gewartet werden können, dass eine mikrobielle kontamination des pumpkopfs vermieden wird. • das händedesinfektionsmittel muss leicht identifizierbar und der füllstand im spender erkennbar sein. • die außen-und innenteile des spenders müssen wischdesinfizierbar sein. • die spender sowie alle permanenten teile müssen maschinell thermisch bei einem ao-wert von mindestens 60 °c (z. b. 80 °c/1 min) aufbereitbar sein. • spender mit einwegpumpköpfen, die mit dem leeren desinfektionsmittelgebinde zu entsorgen sind, bzw. berührungslos arbeitende spender sind zu bevorzugen. werden die pumpköpfe für nachfolgende gebinde verwendet, muss eine detaillierte aufbereitungsanweisung benannt werden. • aus juristischen gründen ist eine dauerhaft lesbare etikettierung der spender mit einem warnhinweis zu empfehlen, z. b. "händedesinfektionsmittel ausschließlich zum gebrauch auf der hand! kein trinken, verspritzen in die augen oder auftragen auf schleimhäute" • es ist als ideal anzusehen, wenn der spender mechanisch oder elektronisch daten zum desinfektionsmittelverbrauch liefert. rechtlich ist ein umfüllen möglich, sofern das unter der unmittelbaren fachlichen verantwortung des anwendenden arztes oder apothekers erfolgt. der umfüllende haftet für sein hergestelltes produkt. ein umgefülltes desinfektionsmittel darf nicht an andere abgegeben werden. aus medizinischer sicht und aus haftungsrechtlichen gründen müssen hygienische mindeststandards beachtet werden. diese umfassen die reinigung und sterilisation der desinfektionsmittelbehälter vor neubefüllung, das umfüllen unter aseptischen behältnissen (sterile werkbank), dokumentation der chargennummer bzw. umfülldatum und durchführung durch geschultes personal (hengesbach und schneider 2006) . die notwendigkeit für dieses vorgehen kann daraus abgeleitet werden, dass bakteriensporen in alkoholbasierten desinfektionsmitteln überleben können und auf diesem weg z. b . in eine wunde gelangen könnten (theoretisches risiko von gasbrand und tetanus; danchaivijitr et al. 2005; weuffen, berling und hetmanek 1998) . das tatsächliche risiko ist jedoch minimal. so konnten im händedesinfektionsmittel nach längerem stehenlassen der geöffneten flasche in 18 % der proben bakteriensporen gefunden werden, jedoch weniger als eine spore pro 10 ml händedesinfektionsmittel. in keinem fall wurden sporen pathogener bakterienspezies identifiziert ). axel kramer und ojan assadian sir john pringle prägte 1772 den begriff "antisepsis". mit der einführung des karbolwundverbands durch lister wurde die antiseptik zur prävention von ssi etabliert. unter antisepsis (griech. anti = gegen, sepsis = fäulnis) werden alle lokal angewandten maßnahmen zur abtötung oder inaktivierung von mikroorganismen am oder im lebenden gewebe verstanden, die aus prophylaktischer indikation (prophylaktische antiseptik) einer unerwünschten kolonisation oder infektion vorbeugen oder aus therapeutischer indikation (therapeutische antiseptik) diese behandeln. die antiseptik wird in erster linie durch einmalige oder wiederholte topische anwendung von antiseptika realisiert. zur wundantiseptik werden jedoch auch biologische (z. b. madentherapie; daeschlein et al. 2007b ) und physikalische verfahren (z. b. pulsierender gleichstrom und niedertemperatur-plasma; daeschlein et al. 2007a , kramer et al. 2013c ) eingesetzt. zielsetzung der prophylaktischen antiseptik ist die infektionsverhütung durch unterbindung des erregertransfers von kontaminierten bzw. kolonisierten in mikrobiell nicht besiedelte körperbereiche, die sanierung unerwünschter kolonisation, die normalisierung einer dysbiose bzw. die abtötung potenzieller pathogene nach akzidenteller kontamination. • zur prophylaktischen antiseptik, die im allgemeinen einmalig oder kurzfristig wiederholt stattfindet, werden rasch wirksame mikrobiozide (in speziellen fällen auch viruzide) wirkstoffe benötigt. • zur therapeutischen antiseptik sind aufgrund der wiederholten applikation und langfristigen einwirkung ggf. auch mikrobiostatische (bzw. virustatische) wirkstoffe ausreichend. aus therapeutischer indikation eingesetzte antiseptika werden auch als lokale antiinfektiva bezeichnet. die wirkungsanforderungen an antiseptika sind in der testhierarchie der europäischen prüfnormen definiert. bei praxisrelevanter belastung sollen in vitro ≥ 10 3 kbe der für die testung festgelegten mikroorganismenspezies abgetötet werden (kramer 2000) . für die verträglichkeitsprüfung ist bisher kein einheitlicher prüfablauf festgelegt. die irritationspotenz kann zunächst an der chorioallantoismembran des hühnereis geprüft werden (kramer und behrens-baumann 1997) . die gewebeverträglichkeit wird in zell-und gewebekulturen einschließlich dreidimensionaler in-vitro-modelle ermittelt, ggf. danach, falls zwingend erforderlich, tierexperimentell (geerling et al. 2002) oder, wenn der wirkstoff toxikologisch und präklinisch als ausreichend untersucht gilt, für wundantiseptika ohne zwischenstufe direkt an mesh-graft-entnahmestellen (eisenbeiß et al. 2012 sofern der wirkmechanismus auf einer unspezifischen zerstörung der mikroorganismen beruht (z. b. bei alkoholen, pvp-iod, natriumhypochlorit, oct, polihexanid) , ist keine resistenzentwick lung zu befürchten. richtet sich die wirkung gegen mikrobielle enzyme oder transporter, ist eine resistenzentwicklung möglich. so werden zunehmend staphylokokkenisolate mit verminderter invitro-empfindlichkeit gegen chx beschrieben, beruhend auf plasmid-kodierten effluxpumpen in der zellmembran (fritz et al. 2013; ho et al. 2012; horner, mawer und wilcox 2012; lee et al. 2011; mcgann et al. 2011; mcneil et al. 2013; otter et al. 2013; tattawasart et al. 1999) . aufgrund des spezifischen angriffspunkts in der bakterienzelle und der mit der resistenzentwicklung gegen antibiotika vergleichbaren mechanismen bei der resistenzentwicklung gegen triclosan (targetmutation, erhöhte targetexpression, aktiver efflux aus der zelle, enzymatische inaktivierung/abbau) sind laborbefunde zu kreuzresistenzen zwischen triclosan und antibiotika nicht überraschend. die in vitro durch triclosan induzierbare resistenzentwicklung kann mit einer gleichzeitigen resistenzentwicklung gegen antibiotika einhergehen (braoudaki und. hilton 2005 , russel, maillard und fuur 1998 , sanchez, moreno und martinez 2005 . inzwischen wurden auch resistente stämme in der umwelt isoliert (drury et al. 2013) . vor jeder antiinfektiven prophylaxe oder therapie muss anhand folgender kriterien die entscheidung zwischen antiseptik oder antimikrobieller chemotherapie getroffen werden: • erreicht oder übertrifft die lokale anwendung die effektivität einer antimikrobiellen chemoprophylaxe bzw. -therapie? • ist die lokale anwendung ohne risiko von nebenwirkungen? die hautantiseptik ist vor allen durchtrennenden eingriffen der haut notwendig, d. h. vor injektion, punktion, dem legen von gefäßkathetern und zur laufenden antiseptik bei liegendem gefäßkatheter (sog. katheterpflege) sowie präoperativ. als antiseptische körperwaschung dient sie bei einer kolonisation mit mrsa der dekolonisation z. b. vor elektiven operativen eingriffen sowie bei kolonisation/infektion mit anderen mre zur herabsetzung des risikos ihrer weiterverbreitung, z. b. bei its-patienten. mittel der wahl sind alkohole ohne remanenten zusatz. da sich die einwirkungszeit zwischen talgdrüsenarmen und -reichen hautarealen unterscheidet, ist die vom hersteller deklarierte verlängerte einwirkungszeit auf talgdrüsenreicher haut z. b. vor da der talgdrüsenanteil der haut regional unterschiedlich ist und der fettgehalt individuellen schwankungen unterliegt, ist man bei der präoperativen hautantiseptik auf der sicheren seite, wenn auch auf talgdrüsenarmen arealen die präoperative hautantiseptik mit der einwirkungszeit für talgdrüsenreiche haut zugrunde gelegt wird. mit alkoholhaltigen präparaten wird beim auftragen ohne anschließendes mechanisches einreiben für 30 s eine reduktion auf der hautoberfläche nur um etwa 1,2 log 10 erreicht (ulmer et al. 2014 ). außerdem dringt der alkohol nicht in die haarfollikel ein (ulmer et al. 2012 (ulmer et al. , 2013 . wirkstoffauswahl: da in den usa und vielen europäischen staaten bisher überwiegend chx-haltige antiseptika eingesetzt werden, wurden fast alle studien mit diesem wirkstoff durchgeführt. oct übertrifft in vitro chx an wirksamkeit (koburger et al. 2010 ), ist in kombination mit alkoholen vergleichbar effektiv in bezug auf die reduktion der hautflora um die insertionsstelle des zvk, induziert im gegensatz zu chx keine resistenzentwicklung (al-doori, goroncy-bermes und gemmell 2007), nur selten allergische kontaktekzeme (stingeni, lapomarda und lisi 1995) und keine igevermittelten anaphylaktischen reaktionen (hübner und kramer 2010 , pham et al. 2000 . aus diesen gründen und wegen der freisetzung der im chlorhexidinmolekül vorhandenen 4-chloranilingruppen, die als karzinogen eingestuft sind, was bisher nur in der mundhöhle nachgewiesen wurde (below et al. in vorb.) , spricht die nutzen-risiko-analyse zugunsten von oct. die bei anwendung von chx aufgetretenen schweren anaphylaktischen ereignisse betrafen patienten, bei denen ein chx-imprägnierter gefäßkatheter angelegt wurde (faber et al. 2012; guleri et al. 2012; khoo und oziemski 2011). dem bfarm lagen bis 2013 insgesamt 147 berichte aus deutschland über anaphylaktische reaktionen im zusammenhang mit der anwendung von chx vor. es ist zu hoffen, dass durch aussagekräftige endpunktstudien mit oct-haltigen präparaten klarheit über den stellenwert dieses wirkstoffs als ggf. günstigere alternative zu chx geschaffen wird. vor anlage eines zvk ist mit einem alkoholbasierten hautantiseptikums mit zusatz eines remanent wirkenden antiseptikums in ausreichendem abstand um die katheterinsertionsstelle die antiseptik durchzuführen. dadurch wird nicht nur die rekolonisation der haut (reichel et al. 2009 , ulmer et al. 2014 ) und der katheterspitze signifikant verzögert (dettenkofer et al. 2002 (dettenkofer et al. , 2010 mimoz et al. 1996; o'grady et al. 2002) , sondern auch die inzidenz zvk-assoziierter blutstrominfektionen (capsi) reduziert (huang et al. 2011) . wirkstoffauswahl: in internationalen empfehlungen gibt es einen breiten konsens zum einsatz chx-haltiger antiseptika zur hautantiseptik vor anlage eines zvk (burrell et al. 2011 ) oder zur behandlung der eintrittsstelle beim verbandswechsel (marschall et al. 2014; o'grady et al. 2011; tietz, frei und dangel 2005) . die ausschließliche empfehlung von chx beruht darauf, dass in den usa und vielen europäischen staaten entweder chx oder pvp-iod eingesetzt wird und letzteres chx unterlegen ist. durch 2-prozentige wässrige lösung von chx und 0,5-prozentige alkoholische chx-lösung wurde die rate von capsi im vergleich zu 10 % wässriger pvp-iod lösung und 70 % ethanol signifikant reduziert (maki, ringer und alvarado 1991 , valles et al. 2008 ). in einer multizentrischen, prospektiven, randomisierten, kontrollierten studie war dagegen zwischen 0,5 % chx-tinktur vs. 10 % wässriger pvp-iod lösung kein signifikanter unterschied in der kolonisation der katheterspitze und bezüglich der capsi-rate nachweisbar (humar et al. 2000) . im ergebnis eines cochrane review (huang et al. 2011 ) und einer nachfolgenden metaanalyse (maiwald und chan 2012) kann sowohl die katheterkolonisation als auch die cabsi-rate durch alkoholhaltige formulierungen mit chx-zusatz im vergleich zu 10 % wässriger pvp-iod lösung signifikant reduziert werden. als fazit wird in der cdc guideline (o'grady et al. 2011 ) die hautantiseptik mit alkoholischen formulierungen mit zusatz von > 0,5 % chx bzw. in der aktuellen epic guideline (loveday et al. 2014 ) von 2 % chx in kombination mit 70-prozentigem propan-2-ol empfohlen. bei kontraindikationen gegen chx können alternativ iodtinktur, iodophore oder 70 % ethanol verwendet werden. es findet sich kein hinweis auf oct, da dieser wirkstoff in den angloamerikanischen ländern nahezu unbekannt ist. oct kommt aus folgenden gründen als aussichtsreicher remanenter zusatz zu alkoholen in betracht. es ist in vitro mikrobiozid signifikant wirksamer als chx (koburger et al. 2010) . auch in der remanenten wirkung war oct in einem 3d-modell der haut beim vergleich äquimolarer wirkstoffkonzentrationen chx überlegen. für epidermal gebundenes chx war keine oder eine nur sehr geringe remanente mikrobiozide wirksamkeit gegenüber p. aeruginosa nachweisbar, während beim gebundenen oct reduktionsfaktoren zwischen 0,6-1,2 log 10 entstehen ). daher ist da-von auszugehen, dass oct-haltige alkoholische formulierungen auch bei anwendung auf der haut die wirksamkeit von chx erreichen bzw. übertreffen (hübner, siebert und kramer 2010) . auch bezüglich der biokompatibilität war oct überlegen (müller und kramer 2008) . in zwei bisher durchgeführten klinischen studien bei nicht getunnelten zvk wurde die höhere wirksamkeit des oct-zusatzes im vergleich zur analogen alkoholischen formulierung ohne oct-zusatz anhand der reduktion der kolonisation an der insertion sowohl im sofortwert als auch nach 24 h (dettenkofer et al. 2002 ) sowie anhand der anzahl positiver kulturen an der katheterspitze (dettenkofer et al. 2010 ) nachgewiesen. die inzidenz von capsi wurde nur tendenziell reduziert, offensichtlich war die stichprobengröße nicht ausreichend. auf die insertionsstelle aufgebrachte antibiotikahaltige salben besitzen eine unsichere wirksamkeit (zhang et al. 2014 ) und sind wegen des risikos der resistenzentwicklung sowie der schaffung eines feuchten milieus abzulehnen. letzteres trifft auch für mupirocin zu, da bereits highlevel-resistenzen beschrieben sind (zhang et al. 2013) . daher wird analog wie vor der katheterinsertion bei jedem verbandswechsel eine hautantiseptik mit alkohol basierten formulierungen mit zusatz von > 0,5 % chx (maki et al. 2006 ) bzw. mit 2 % chx in 70 % propan-2-ol (loveday et al. 2014 ) empfohlen. zur kontinuierlichen applikation antiseptischer substanzen direkt am kathetereintritt stehen ein chx-getränkter schwamm (z. b. biopatch ™ ; roberts und cheung 1998) in verbindung mit einem semipermeablen folienverband sowie die direkte integration eines durchsichtigen, chx-haltigen gelkissens in einen semipermeablen folienverband (z. b . tegaderm ™ 3m chg) (pfaff, heithaus und emanuelsen 2012; scheithauer et al. 2014 ) zur verfügung. einige präventionsbündel haben antiseptisch wirksame verbände eingeschlossen (hatler et al. 2009; guerin et al. 2010; miller und maragakis 2012; shapey et al. 2009 ). eine metaanalyse (ho und litton 2006) zum einsatz des biopatch ™ am zvk und an epiduralen kathetern zeigte eine signifikante reduktion der kolonisationsdichte im bereich der eintrittsstelle und als trend eine verminderte device-assoziierte infektionsrate. in weiteren studien konnte der infektionspräventive nutzen chx-haltiger verbände am zvk bestätigt werden (camins et al. 2010; levy, katz und solter 2005; ruschulte et al. 2009; timsit et al. 2009 timsit et al. , 2012 . da im michigan-keystone-projekt (berenholtz et al. 2004 (berenholtz et al. , 2014 pronovost, berenholtz und needham 2008; pronovost et al. 2006 pronovost et al. , 2010 safdar, fine und maki 2005) und in anderen initiativen zur senkung der infektionsraten (krein et al. 2010; saint et al. 2010; zingg et al. 2009 zingg et al. , 2014 auch ohne den einsatz dieser kostenintensiven hilfsmittel nachhaltige effekte erreicht wurden, empfehlen die aktuellen britischen und u. s.-amerikanischen empfehlungen mit ausnahme der american pediatric surgical association (huang et a. 2011 ) den einsatz chx-haltiger verbände nur bei hochrisikopatienten oder zur reduktion anhaltend hoher infektionsraten erst nach stringenter implementierung konventioneller präventionsmaßnahmen. • der stellenwert chx-haltiger verbände bei patienten mit gleichzeitiger chx-ganzkörperwaschung ist ungeklärt. • für arterielle katheter liegen bisher nur ergebnisse einer tendenziellen reduktion der infektionsrate vor (timsit et al. 2012 ). • während beim zvk die höhere wirksamkeit von hautantiseptika mit remanentem zusatz im vergleich zu rein alkoholischen formulierungen anhand der capsi-rate gesichert ist, ist beim peripheren venenkatheter bisher nur nachgewiesen, dass durch verwendung von hautantiseptika mit remanentem zusatz die anzahl kolonisierter bzw. kontaminierter katheter reduziert wird (small et al. 2008 ). zielsetzung ist die herabsetzung der erregerlast auf der haut (popovich et al. 2012 ), um abhängig vom endemischen niveau sowohl das risiko der erregertransmission (climo et al. 2009 ) als auch einer ni (climo et al. 2013 , huang et al. 2013 durch vormals die haut des patienten besiedelnde erreger zu senken. darüber hinaus ist die antiseptische ganzkörperwaschung eine additive maßnahme zur prävention von crbsi vor allem dann, wenn die implementierung anderer präventionsstrategien die crbsi raten nicht adäquat senken können. im ergebnis einer 2-jährigen retrospektiven studie mit täglicher ganzkörperwäsche mit oct-haltiger seife auf einer its wurde die besiedlung mit mrsa um 76 % reduziert, allerdings traf das nicht gleichermaßen auch für die rate von capsi zu (spencer et al. 2013) . zum teil erwies sich eine chx-haltige ganzkörperwäsche in unterschiedlichen its-settings auch effektiv zur prävention von trägertum und capsi durch mrsa und vre sowie zur reduktion der körperbesiedlung durch a. baumanii auf its mit endemischer situation dieses erregers bei capsi (borer et al. 2007 ). in einer multizentrischen europäischen studie auf 13 its konnte durch verbesserte händehygiene in verbindung mit chx-körperwaschung die akquisition von mre, speziell von mrsa, signifikant reduziert werden, während aufnahmescreening und isolierung keinen signifikanten einfluss hatten (derde et al. 2014 ). als risiko bei einer zunehmenden anwendung von chx ist zu berücksichtigen, dass mrsa-stämme, die das qaca/b gen tragen, nicht beeinflusst bzw. sogar rascher verbreitet werden (batra et al. 2010; otter et al. 2013) . da intensivpatienten in jedem fall gewaschen werden müssen, kann der einsatz antiseptischer körperwaschlotionen als sinnvolle additive maßnahme der infektionsprävention bei dieser patientengruppe insbesondere zur risikominimierung der weiterverbreitung von mrsa, vre und actinetobacter spp. angesehen werden. außerdem wurde durch tägliches bad mit chx basierter ganzkörperwäsche eine signifikante senkung der rate von crbsi von 5,3 auf 0,7 pro 1 000 kathetertage bzw. kontaminierter blutkulturen von 6.99 auf 4.1 pro 1 000 patiententage erzielt, was sich in mehreren studien (bleasdale et al. 2007; climo et al. 2013; evans und dodge 2010; karki und cheng 2012; munoz-price et al. 2009; o'horo et al. 2012; popovich et al. 2009 ) und im ergebnis eines sys-tematischen reviews (afonso, llauradó und gallart 2013) bestätigt. zugleich wurde die kontamination von personal und umgebung reduziert (afonso, llauradó und gallart 2013) . sogar die sepsisrate konnte reduziert werden (huang et al. 2013) . in einer metaanalyse von 12 studien (o'horo et al. 2012 ) auf internistischen intensivstationen mit anwendung chx-haltiger waschtücher (2 %) oder ganzkörperwaschung (4 %) wurde für beide anwendungen eine signifikante herabsetzung von crbsi gesichert. milstone et al. (2013) untersuchten den einfluss der täglichen ganzkörperwaschung mit chx-haltigen waschtüchern bei pädiatrischen intensivpatienten jenseits des zweiten lebensmonats in einer multizentrischen nicht verblindeten studie mit cluster-randomisiertem crossover-design mit dem ergebnis einer signifikante reduktion der inzidenz von crbsi. 1 % der kinder mussten aufgrund einer unverträglichkeit aus der studie genommen werden. da entgegen der definition der cdc alle, meist über zvk abgenommenen, positiven blutkulturen bei patienten mit infektionszeichen als crbsi gezählt wurden und die inzidenzraten über denen von präventionsbündelstudien ohne chx-ganzköperwaschung lagen, relativiert das nach aussage der autoren die aussagekraft. dagegen wurde die rate von sekundären bsi, c.-difficile-infektionen, vap und katheter-assoziierten hwi nicht beeinflusst (popovich et al. 2009 ). präoperativ sind für die ohrmuschel alkoholhaltige hautantiseptika mit remanentem zusatz zu bevorzugen. da eine ssi der ohrmuschel der op-erfolg infrage stellt, hat es sich bewährt, die einwirkzeit auf mindestens 10 min zu verlängern, z. b. durch auflage die ohrmuschel bedeckender getränkter tupfer. im mittelohr ist chx wegen der neurotoxizität kontraindiziert. infrage kommen wässrig basierte zubereitungen mit gehalt von 0,1 % polihexanid oder 1,25 % pvp-iod. allerdings liegen hierzu keine studien vor. da die details in den jeweiligen klinischen kapiteln behandelt werden, soll ein tabellarischer überblick über wichtige indikationen und infrage kommende wirkstoffe genügen (› tab. 2.2) . für die schleimhautantiseptik ist oct vom grundsatz her wegen der höheren und rascher einsetzenden wirksamkeit polihexanid und chx überlegen. obwohl die wundbehandlung eine herausforderung für die menschheit seit der menschwerdung ist, fehlt bis heute die evidenz für ein allgemein akzeptiertes behandlungskonzept auf naturwissenschaftlicher/molekularbiologischer grundlage, dass durch rcts und metaanalysen verifiziert ist. deshalb müssen die zur verfügung stehenden befunde zur wirksamkeit und verträglichkeit antiseptischer präpa-2 rate von der in-vitro-testung bis zur vereinzelt existierenden rct-studie einschließlich limitierter metaanalysen zu einer plausiblen synopse zusammengeführt werden (kramer et al. 2013c ). wundantiseptika sind nur nach sorgfältiger indikationsstellung und vorausgehender wundkonditionierung anzuwenden. andernfalls können wundheilungsstörungen verursacht werden bzw. können die antiseptika ihre wirkung nicht entfalten. grundsätzlich müssen alle wunden als kontaminiert angesehen werden. das bedeutet jedoch nicht, dass alle kontaminierten wunden eine infektion entwickeln. da die physiologische kolonisation von wunden für den wundheilungsverlauf irrelevant ist (eisenbeiß et al. 2012 ), wurde als hilfestellung für die abschätzung des infektionsrisikos der wounds at risk score entwickelt (› tab. 2.3) . bei der entwicklung dieses risikoscores werden sowohl die wunde exogen belastende faktoren als auch die infektionsanfälligkeit des patienten berücksichtigt. die indikation für den einsatz von antiseptika ergibt sich aus der addition unterschiedlich zu gewichtender gefährdungsursachen, für die punkte vergeben werden. bei > 3 punkten ist eine antiseptische behandlung zu rechtfertigen (dissemond et al. 2011 ). unabhängig von der sich aus dem score ergebenden indikation ist eine dekolonisation von wunden bei nachweis von mre indiziert. verletzungen sind abhängig von der kontamination und dem ausmaß der gewebeschädigung mehr oder stark infektionsgefährdet. aus diesem grund ist die antiseptische primärversorgung ver-schmutzter wunden einschließlich verätzungen und verbrennungen notwendig. bei biss-und stichverletzungen steht die erforderliche tiefenwirkung des antiseptikums im vordergrund. in auswertung des schrifttums zum mikrobiellen spektrum und zu den risikofaktoren bei bissverletzungen wurden folgende emp fehlungen zum management bei bisswunden abgeleitet ): • bei der frischen offenen verletzung ggf. chirurgisches débridement, danach antiseptische spülung der wunde mit einem kombinationsprodukt aus pvp-iod und ethanol (z. b. betaseptic ® ), keine antibiotikaprophylaxe, primärverschluss • bei der nahezu geschlossenen frischen verletzung (z. b. katzenbiss) ggf. chirurgisches débridement, auflage antiseptisch getränkter kompressen für etwa 60 min mit zwischenzeitlicher tränkung (z. b. betaseptic ® ), keine antibiotikaprophylaxe • bei der älteren verletzung nach etwa 4 h ggf. chirurgisches débridement, auflage antiseptisch getränkter kompressen oder verbände für etwa 60 min mit zwischenzeitlicher tränkung (z. b. betaseptic ® ), parallel einmalige iv. oder dosisadaptiert orale gabe von antibiotika (amoxicillin/clavulansäure) • bei der älteren verletzung nach etwa 24 h chirurgisches débridement, danach antiseptische spülung der wunde (z. b. betaseptic ® ). bei klinisch ersichtlicher infektion/entzündung chirurgische revision mit eröffnung und antiseptik sowie antibiotikatherapie gemäß antibiogramm (empirischer beginn mit ampicillin oder amoxicillin/clavulansäure). bei jeder bissverletzung müssen der tetanusimpfstatus und das risiko der tollwutexposition abgeklärt werden. gleiches gilt bei seltenen, doch gelegentlich stattfindenden humanen bissen für die risikoabschätzung für lues, hbv, hcv und hiv. octenidin (› tab. 2.4) oct und pvp-iod erreichen im keimträgertest die antiseptische effektivität rascher (≥ 30 s) als chx (≥ 30 min) und polihexanid (> 30 min) (schedler et al. in vorb) . durch bindung von oct an die zellmatrix wird ein signifikanter postantiseptischer effekt erzielt (müller et al. 2007 ). befunde zur höheren zytotoxizität von oct in der monolayer-zellkultur im vergleich zu iodophoren und polihexanid bedürfen der kritischen interpretation, weil sich oct im wundgewebe an die zellen bindet, wobei die wirksamkeit durch wirkstofffreisetzung in geringen mengen erhalten bleibt, während die zytotoxizität drastisch reduziert wird. durch diese art der "wundversiegelung" dürfte eine nachfolgende wundkolonisation unterbunden werden. oct wird nicht resorbiert, und es sind keine langzeitrisiken einschließlich allergien bekannt. durch oct wird die phagozytose humaner neutrophiler granulozyten gesteigert (steinhauer und goroncy-bermes 2007) , während der tumornekrosefaktor nicht stimuliert wird (menke et al. 2001 ). in konzentrationen von 0,05 % wird sowohl oct als auch pvp-iod von erythrozyten toleriert (wagner et al. 2004 ). dissemond et al . 2011 die relevanz von befunden zur möglichen karzinogenität ist umstritten, weil für den wirkstoff keine genotoxizität nachgewiesen ist. daher bliebe für eine karzinogene wirkung nur die erklärung einer epigenetischen nicht genotoxischen veränderung der dna übrig. im ergebnis der überprüfung wurde weder oxidativer stress induziert, noch waren eine hydroxylierung oder hypermethylierung der dna oder eine signifikante produktion mitogener zytokine und des transkriptionsfaktors nf-κb nachweisbar. auch der status der gap-junctions (gjic) wurde nicht signifikant beeinflusst. damit waren keine eindeutigen epigenetischen einflüsse nachweisbar (creppy et al. 2014 ) und es werden die einschätzungen der epa (2003, 2004, 2005a) und später der australischen behörde ocseh (2011) untermauert. diese leiten aus den tierexperimentellen daten von horner (1996) und milborne (1996) ab, dass kein relevantes gesundheitsrisiko für den menschen erkennbar ist. trotzdem erfolgte 2013 die gefahrstoffrechtliche einstufung des rohstoffs polihexanid im rahmen der europäischen chemikaliengesetzgebung in kategorie 2 "kann vermutlich krebs erzeugen". produkte, die > 1 % polihexanid enthalten, müssen danach als karzinogen klasse 2 gekennzeichnet werden. die europäische chemikalienagentur (echa) kam auf basis der zwei zitierten nagetierstudien zu dem schluss, dass nicht jegliches gesundheitsrisiko für den menschen mit absoluter sicherheit ausgeschlossen werden könnte. zu dieser feststellung muss jedoch die einschränkung getroffen werden, dass das design der beiden studien nicht den aktuellen anforderungen entspricht und die effekte nur bei hoher dosierung (ca. 4 000 ppm/0,4 %), die wahrscheinlich die maximale tolerierbare dosis überschreitet, auftraten. da arzneimittel oder medizinprodukte in der regel 0,02-0,1 % polihexanid enthalten und der wirkstoff nicht resorbiert wird, ist eine gesundheitsgefährdung bei antiseptischer anwendung des wirkstoffs auszuschließen. povidon-iod (› tab. 2.4) pvp-iod bindet elementares iod ohne feste chemische bindung. die eigenschaften des iods bleiben erhalten. abhängig von der umgebenden iodkonzentration wird lediglich die lösungsfähigkeit verändert, wodurch über längere zeit freies iod zur verfügung steht. es wirkt nicht remanent; die wirkung hält nur so lange an, wie die anwesenheit von iod im pvp-trägermolekül gegeben ist. pvp-iod wirkt nicht nur mikrobiozid, sondern bei längerer einwirkzeit sporozid und zusätzlich gegen eine reihe von viren. wegen der allergenen potenz, der resorptionstoxizität für die schilddrüse und der fehlenden remanenz hat pvp-iod zur wundantiseptik in deutschland an bedeutung eingebüßt (kramer et al. 2008c (kramer et al. , 2013c . in einem systematischen review war pvp-iod nicht antiseptisch wirksamen wundauflagen und silbersulfadiazin überlegen, aber in kombination medizinischem honig bezüglich der bakterienelimination und wundheilung unterlegen. im vergleich von 84 studien hält sich die überlegenheit bzw. unterlegenheit im vergleich zur kontrolle etwa die waage (vermeulen, westerbos und ubbink 2010) . iodophore sind besser gewebeverträglich als chx-haltige präparate. in vitro setzt bei scheinbar abgetöteten zellen nach abspülen des wirkstoffs wieder die proliferation ein, sog. revitalisierender effekt (müller und kramer 2006) . in vitro und tierexperimentell ist 0,5 -prozentiges pvp-iod im unterschied zu oct knorpelverträglich. genotoxische, karzinogene und teratogene gefährdungen sind nicht bekannt (kramer et al. 2008c ). als liposomale zubereitung (repithel ® ) ist die gewebeverträglichkeit von pvp-iod bei erhaltener wirksamkeit deutlich verbessert, sodass die liposomale pvp-i-zubereitung einer chx-imprägnierten auflage bei anwendung auf mesh graft an wirksamkeit und verträglichkeit überlegen war (reimer et al. 2000) . bei der anwendung von iodophoren gelten folgende kontra indikationen: hyperthyreose, dermatitis herpetiformis duhring, überempfindlichkeit gegen iod, radioiodtherapie, peritoneallavage. die anwendung ist sorgfältig abzuwägen und die schilddrüsenfunktion ist zu kontrollieren bei blander knotenstruma, gravidität, stillzeit, großflächiger anwendung bei früh-und neugeborenen sowie bei säuglingen bis zum 6. lebensmonat. da bei anwendung am auge (präoperativ und zur prävention der ophthalmia neonatorum) die resorbierte iodmenge unterhalb schilddrüsenkritischer werte bleibt (kramer et al. 2008c ), ist bei dieser anwendung keine schilddrüsenfunktionsbeeinflussung zu erwarten. chlorhexidin (› tab. 2.4) chx und polihexanid unterscheiden sich in der molekülstruktur nur dadurch, dass chx zusätzliche p-chloranilin-reste enthält. daher ist vermutlich die nachgewiesenen abspaltung des p-chloranilins (below et al. in vorb.) für die im unterschied zu polihexanid hohe zytotoxizität, die mutagene potenz (arabaci et al. 2013; fda 2013; grassi et al. 2007; paldy et al. 1984; souza-junior und castro-prado 2005) , die induktion von keratosen und dysplasien (sonis, clark und shklar 1972) sowie die neurotoxizität (aursnes 1981a (aursnes , 1981b bicknell 1971; kramer et al. 2003 kramer et al. , 2008c perez et al. 2000) verantwortlich. das wachstum von peritonealexplantaten wurde in vitro gehemmt (kramer et al. 1998 ); dementsprechend wurde bei tierexperimentellen wunden z. t. eine verzögerte heilung beobachtet (kramer et al. 1993 ). beim menschen fiel bei kontaminierten chirurgischen wunden allerdings keine heilungsverzögerung auf (crossfill, hall und london 1969) . in vitro war chx gegenüber humanen alveolaren knochenzellen zytotoxischer als pvp-lod (cabral und fernandes 2007) . 0,1-prozentig kommt es am auge zum verlust der oberflächlichen schichten des korneaepithels und der mikrovilli der zweiten schicht (dormans und logten 1982) . bei irrtümlicher intraoperativer spülung bei kataraktchirurgie mit chx 1 : 666 bzw. 1 : 1 000 verdünnt kam es zu einer schweren toxischer keratopathie (rij et al. 1995) . nach 8-wöchiger anwendung von augentropfen mit chx 0,02 % + propamidin 0,1 % wurde eine progressive ulzeröse keratitis verursacht, wofür die autoren chx als ursache ansahen (murthy, hawksworth und cree 2002) . daher wird chx > 0,05 % nicht zur ophthalmologischen anwendung empfohlen. nach einer irrigation mit chx 0,02 % bei arthroskopischen eingriffen entwickelte sich als frühantwort eine schwere aggressive destruktive arthritis (van huyssten u. bracey 1999 , van huyssten 2000 . als konsequenz hat chx seine bedeutung zur wundantiseptik verloren und seit 2006 ist in der roten liste kein chx basiertes wundantiseptikum enthalten. in japan ist chx seit 1986 zur schleimhautantiseptik untersagt. 2012 veröffentlichte die medicines & healthcare products regulatory agency (mhra), uk, einen warnhinweis für medizinprodukte und arzneimittel mit gehalt an chx wegen des risikos anaphylaktischer reaktionen mit folgendem hinweis: if a patient experiences an unexplained reaction, check whether chlorhexidine was used or was impregnated in a medical device that was used. ein jahr später warnte swissmedic (swiss agency for therapeutic products) generell von anaphylaktischen reaktion durch anwendung chx haltiger produkte. bei gramnegativen klinischen isolaten wurde eine wirkschwäche nachgewiesen (kramer et al. 2013c ). in vitro ist eine resistenzentwicklung induzierbar (s. o.). natriumhypochlorit (› tab. 2.4) die weltweite verwendung von naocl begann im ersten weltkrieg nach der wiederentdeckung dieses wirkstoffs durch henry drysdale dakin. wegen der ph-abhängigen instabilität der wässrigen lösung verlor die dakin-lösung ihre bedeutung in der wundantiseptik und erlebt erst jetzt ihre renaissance, nachdem mit der entwicklung einer stabilisierten kombination von naocl und hypochloriger säure (hocl) in wässriger lösung eine stabilität von 1 jahr bzw. eine anbruchstabilität von 2 wochen erreicht wurde. diese wirkstoffkombination (naocl/hocl) erfüllt die in-vitro-anforderungen an wundantiseptika nicht nur gegen vegetative bakterien und sprosspilze (aggarwal et al. 2010 ), sondern ist selbst gegen bakteriensporen hoch effektiv (landa-solis et al. 2005 ; tana-ka et al. 1996) . in vitro und bei der behandlung chronischer wunden war die wirkstoffkombination naocl/hocl effektiver und besser verträglich als pvp-iod (abhyankar et al. 2009; dalla paola et al. 2005; kapur und marwaha 2011) . bei der behandlung diabetischer wunden (verblindete rct) wurden die krankenhausverweildauer und die wundheilungsdauer signifikant verkürzt und die wundkategorie signifikant verbessert (hadi et al. 2007 ). die besonderheit von naocl/hocl besteht darin, dass es sich um einen physiologischen wirkstoff handelt, der von phagozyten nach auslösung des respiratory burst durch o 2 -metaboliten mittels myeloperoxidase, eosinophiler peroxidase und superoxiddismutase gebildet und rasch durch taurin zu clund wasser entgiftet wird (kramer et al. 2008e ). daher sind für hypochlorit keine langzeitnebenwirkungen zu befürchten und nicht nachgewiesen (gutiérrez 2006; hasegawa et al. 1986; kurokawa et al. 2010 ; morita, nishida und ito 2011). in übereinstimmung dazu wird die proliferation von fibroblasten im unterschied zum zytotoxischen wasserstoffperoxid (wilson et al. 2004 ) in vitro nicht durch die noch mikrobiozid wirksame konzentration von hypochlorit gehemmt (crabtree, pelletier und pruett 2001) . daraus ergibt sich eine hohe therapeutische breite. zusätzlich wundheilungsbegünstigend ist die antiinflammatorische wirkung durch hemmung der zytokinfreisetzung aus mastzellen ohne beeinträchtigung anderer zellfunktionen (medina-tamayo et al. 2007 octenidin ist der wirkstoff der wahl für akute kontaminierte traumatische einschließlich mit mre kolonisierte wunden (hübner, siebert und kramer 2010) . in kombination mit phenoxyethanol (z. b. octenisept ® ) wird die wirkstoffpenetration in die wunde gefördert. für chronische wunden ist die gelzubereitung mit auf 0,05 % halbiertem octenidingehalt (z. b. octenilin ® ) zu bevorzugen; sie unterscheidet sich in der wundverträglichkeit nicht von ringer-lösung (eisenbeiß et al. 2012) . polihexanid ist der wirkstoff der wahl für chronische wunden, reduziert aber auch bei akuten traumata die ssi-rate signifikant (roth et al. in rev.) . polihexanid erreicht nicht die wirksamkeit von oct, fördert aber als einziges wundantiseptikum die wundheilung (hübner und kramer 2010 • medizinischer honig: er wurde erfolgreich zur behandlung akuter und chronischer wunden, die eine applikation zulassen, eingesetzt, auch dann, wenn diese mit resistenten bakteriellen krankheitserregern kolonisiert oder infiziert waren oder teilweise nekrotische areale aufwiesen (igelbrink et al. 2007; simon et al. 2006 simon et al. , 2008 . für wundauflagen, die silberionen (› tab. 2.4 ) freisetzen, kommen zwei metaanalysen und ein review zu dem schluss der unzureichenden evidenz bezüglich der prävention von wundinfektionen und der damit verbundenen wundheilungsförderung bei chronischen wunden. einige studien mit schlechter evidenz sprechen vielmehr für das gegenteil (beam 2009; storm-versloot et al. 2010; vermeulen et al. 2007 ). in einer rct war bei venösen ulzera weder für den primären noch für den sekundären endpunkt ein positiver einfluss der silberwundauflage nachweisbar (michaels et al. 2009 angesprochen wird in der leitlinie auch der umgang mit mp "kritisch b". nach rki/bfarm-empfehlung (rki 2012) sind diese grundsätzlich maschinell zu reinigen und thermisch zu desinfizieren. diesem grundsatz wurde auch bei der erstellung der leitlinie gefolgt. lediglich in begründeten ausnahmen und nach durchgeführter analyse und bewertung des risikos sind die manuelle reinigung und manuelle chemische desinfektion eine mögliche option. ein nicht beschafftes rdg ist kein grund, auf manuelle verfahren auszuweichen. werden mp mit einem standardisierten verfahren behandelt, sind sämtliche manuellen teilschritte zu dokumentieren; ebenso soll der erfolg überprüft werden. zur überprüfung der reinigung werden i. d. r. bioindikatoren herangezogen, die die qualität der aufbereitung nach dem reinigungsprozess entweder visuell oder mittels proteinbestimmung belegen. da eine visuelle kontrolle erheblichen subjektiven einflüssen unterliegt, wird derzeit im arbeitskreis reinigungsmittel-testung der dg-kh an der optimierung quantitativer nachweisverfahren gearbeitet. dabei wird zurzeit die opa-und bca-proteinbestimmung favorisiert. verfahren zur überprüfung der qualität der reinigung und desinfektion sind in der leitlinie 2013 veröffentlicht (dgkh, dgsv, aki, vah 2013). hände-, flächen-, instrumenten-und wäschedesinfektionsmittel die anforderungen ergeben sich aus den anwendungsgebieten, die entsprechend der normativen vorgaben des cen technical committee 216 "chemische desinfektionsmittel und antiseptika" in der pren 14885 -"anwendung europäischer normen für chemische desinfektionsmittel und antiseptika" in den human-und veterinärmedizinischen bereich sowie den bereich lebensmittel, industrie, haushalt und öffentliche einrichtungen eingeteilt werden (cen 2014a). (bansemir et al. 1996; gebel et al. 2000) , levurozid und begrenzt viruzid sein. sofern keine anschließende sterilisation erfolgt müssen sie zusätzlich fungizid und viruzid wirksam sein. ferner sollen instrumentendesinfektionsmittel für den benutzer ungiftig sein und empfindliche bestandteile der instrumente nicht beschädigen. wäschedesinfektionsmittel: das erforderliche wirkspektrum zur wäschedesinfektion muss bakterien, ggf. einschließlich mykobakterien, dermatophyten, sprosspilze und viren (begrenzt viruzid) umfassen. bei wahrscheinlicher oder bekannter kontamination durch besonders resistente krankheitserreger ist die erregergezielte auswahl zu beachten (rki 1995a). materialien, die nicht gewaschen werden können, müssen mit wasserdampf, formaldehyddampf oder mittels chemischer desinfektion/reinigung desinfiziert werden. nach den deutschen regularien (rki 1995a , hvbg 1997 ) wird die wäsche in 3 gruppen unterteilt: • extrem gefährliche infektiöse wäsche z. b. von patienten, die an pocken oder hämorrhagischem fieber leiden. sie muss innerhalb der stationen desinfiziert und darf erst danach zusammen mit der potenziell infektiösen wäsche gewaschen werden. • infektiöse wäsche stammt von infektionsstationen, mikrobiologischen laboratorien und aus der pathologie und muss mit methoden und verfahren gemäß der rki-liste (rki 2013) desinfiziert werden. textilien und waschwasser müssen vor dem ersten ablassen des wassers desinfiziert werden (alexander et al. 1995; rki 1995a in der vahliste sind verfahren für die routinemäßige und prophylaktische desinfektion zur verhütung von infektionen im krankenhaus, in der ärztlichen und zahnärztlichen praxis, in öffentlichen bereichen (kindertagesstätten, schulen, sportstätten usw.) sowie anderen bereichen, in denen infektionen übertragen werden können, zusammengefasst. für die aufnahme sind gutachten gemäß den standardmethoden der dghm zur prüfung chemischer desinfektionsmittel (› tab. 2.7) und den entsprechenden anforderungen (gebel et al. 2001 , dghm 2002 das rki hat methoden für die flächen-und instrumentendesinfektion beschrieben peters und bräuniger 1997; peters, bräuniger und fischer 1995; rki 1994 rki , 1995a eine reduktion der testorganismen um eine zehnerpotenz bedeutet, dass bei einer ausgangskonzentration von 10 7 eine inaktivierung von 1 000 000 testorganismen, bei 10 5 jedoch nur eine inaktivierung von 10 000 testorganismen erfolgt. wirkungsbeeinflussende faktoren: die wirksamkeit von desinfektionsmitteln wird von den krankheitserregern (kaulfers 1995; mcdonnell und russell 1999; russell et al. 1986; spicher und peters 1976) und deren menge, von umgebungsfaktoren (organische belastung wie blut, sputum usw., vorhandensein protektiver bzw. interagierender substanzen, temperatur, ph-wert, luftfeuchtigkeit; spicher und peters 1981 , von der einbettung in biofilme (exner, tuschewitzki und scharnagel 1987 , donlan 2001 , dem kontaminierten objekt und der anwendungsmethode beeinflusst (spicher 1996 grundsätzlich unterscheidet man zwischen physikalischen und chemischen desinfektionsverfahren. als physikalische verfahren werden filtration, hitze-, plasma-und strahleneinwirkung verwendet. bei der thermischen desinfektion muss in einer bestimmten einzelne wäschestücke können durch einlegen in formaldehydlösung für 5 h (3,0 %) oder 12 h (1,5 %) desinfiziert werden. formaldehydabspaltende substanzen, wie paraformaldehyd, hexamethylentetramin und hexaminiumsalze, werden wegen ihrer unsicheren wirkung nicht für desinfektionszwecke empfohlen. formaldehyd wird ferner zur konservierung von immunseren und kosmetika sowie in endkonzentrationen von 0,05-0,5 % zur virus-und toxininaktivierung bei der impfstoffherstellung eingesetzt. der mak-wert wird derzeit bei 0,3 ml/m 3 angesetzt. in den vergangenen jahren wurden aus tierversuchen mit hohen formaldehydkonzentrationen in der atemluft kanzerogene eigenschaften bekannt, was warnungen vor seinem gebrauch -auch als desinfektionsmittel -nach sich zog (› kap. 2.7.2) . formaldehyd ist ein hinsichtlich seiner umfassenden wirksamkeit und deren nur unbedeutender beeinflussung durch organische belastungen sowie seiner wirkung in der gasphase zumindest in infektiologischen risikosituationen nach wie vor ein bewährter desinfektionswirkstoff. formaldehydbasierte flächendesinfektionsmittel verlieren in gesundheitseinrichtungen vor allem aufgrund der neueinstufung von formaldehyd durch die iarc, wegen der wenig anwenderfreundlichen eigenschaften und des neurotoxischen langzeitrisikos, z. b. in form des sick-building-syndroms, sowie aufgrund geeigneter alternativen zunehmend an bedeutung (schwebke et al., 2007) . in besonderen situationen und im zusammenhang mit außergewöhnlichen infektionskrankheiten kann im rahmen behördlicher desinfektionsmaßnahmen eine desinfektion mit formaldehyd bzw. formaldehydhaltigen desinfektionsmitteln erforderlich sein. hierbei ist durch arbeitsschutz-und organisatorische maßnahmen zu gewährleisten, dass der grenzwert eingehalten und personal sowie dritte nicht gefährdet werden (schwebke et al., 2007) . • formaldehyd ist ein starkes allergen und soll deshalb in konzentrationen ≥ 0,3 % nicht an der haut angewandt werden. • wegen der toxischen risiken, der lokalen reizwirkung und der verfügbarkeit von alternativen ist der einsatz von formaldehyd zur routinemäßigen flächendesinfektion nicht mehr zu empfehlen. das gilt auch für den rettungsdienst. glutaral (glutaraldehyd) wirkt besser sporozid als formaldehyd und wird deshalb in der instrumentendesinfektion eingesetzt. bei alkalischem ph (7,5-8,5) ist seine aktivität am höchsten, seine stabilität aber schlechter (zerfall innerhalb von 2 wochen). die "sterilisation" von thermolabilen geräten (z. b. endoskopen) durch einlegen in eine glutarallösung ist ein unsicheres verfahren, weil nicht alle innenflächen sicher erreicht werden und das anschließend nötige abspülen mit sterilem wasser ein kontaminationsrisiko birgt. glutaral wird auch zur flächendesinfektion eingesetzt, was allerdings häufig zur geruchsbelästigung führt. für bernsteinsäuredialdehyd -zumindest in kombination mit formaldehyd und tensiden -wurde zusätzlich eine viruzide wirkung gegen hbv nachgewiesen. damit ist dieses präparat für die instrumentendesinfektion prädestiniert. glyoxal wird in desinfektionsmitteln nur als wirkungsverstärkender zusatz verwendet. oberflächenaktive stoffe (tenside) senken durch anreicherung an den grenzflächen zwischen zwei medien die grenzflächenspannung. manche dieser netzmittel sind auch antimikrobiell wirksam, sodass sie als "desinfizierende waschmittel" verwendet werden können. tenside lassen sich nach ihrem aufbau in 4 gruppen einteilen: anionische, kationische, amphotere und nicht ionogene tenside (› tab. 2.11 ). antimikrobiell wirksam sind vor allem die kationenaktiven und amphoteren substanzen. quaternäre verbindungen (quats): sie sind durch eine positiv geladene hydrophile gruppe gekennzeichnet, die als ammonium-, sulfonium-, phosphonium-, iodonium-oder arsonium-gruppe vorhanden sein kann. am wichtigsten sind die quaternären ammoniumverbindungen wie benzalkoniumchlorid, cetylpyridiniumchlorid und didecyldimethylammoniumchlorid. die antimikrobielle wirkung dieser substanzen tritt schon in sehr niedrigen konzentrationen auf. sie ist zunächst wachstumshemmend (mikrobiostatisch), bei längerer einwirkzeit oder höheren konzentrationen mikrobiozid. die meisten grampositiven bakterien werden schon durch konzentrationen von 50-100 mg/l, gramnegative erst durch mindestens 200 mg/l oder wie manche pseudomonas oder enterobacteriaceae spp. erst durch noch viel höhere konzentrationen abgetötet. bei diesen kann es sogar vorkommen, dass sie sich in der gebrauchsverdünnung vermehren. amphotenside vereinen elektropositive und -negative gruppen in einem molekül. je nach ph-wert der lösung verhalten sie sich als (negativ geladene) anionische (bei ph 8) oder (positiv geladene) kationische tenside (bei ph < 4). dazwischen sind sie elektrochemisch ausgeglichen. sie weisen den quaternären ammoniumbasen vergleichbare eigenschaften auf, wirken jedoch im gegensatz zu diesen gegen mykobakterien und sind weniger leicht durch eiweiß zu inaktivieren. wegen ihrer guten hautverträglichkeit wären sie als waschende händedesinfektionsmittel prädestiniert, sind aber für die anforderungen im medizinischen bereich zu wenig wirksam. in der lebensmittel-und getränkeindustrie werden sie vielfach eingesetzt. es existieren auch flächendesinfektionsmittel auf basis amphoterer substanzen. die halogene fluor (f), chlor (c), brom (br) und iod (i) sind mikrobiozid hoch wirksam. in der medizin werden nur cl und i zu desinfektionszwecken verwendet. hervorgerufenen oxidationsvorgänge bedingen neben mikrobioziden effekten auch das ausbleichen von farbstoffen. für die mikrobiozide wirkung, die unter geeigneten bedingungen das gesamte spektrum der viren und mikroorganismen einschließt, werden mehrere mechanismen verantwortlich gemacht: freisetzung von naszierendem sauerstoff, verbindung des chlors mit imino-und aminogruppen von im zytoplasma enthaltenen stoffen zu toxischen chloraminen, bildung unterchloriger säure, die ihrerseits oxidierend und chlorierend wirkt. in wässriger lösung ist hauptsächlich unterchlorige säure für die mikrobiozide wirkung verantwortlich. im schwach sauren bereich tritt der desinfektionseffekt wesentlich rascher ein als im alkalischen. eine temperaturerhöhung führt wie bei den meisten desinfektionsmitteln zu einer steigerung der desinfektionswirkung. organische substanzen beeinträchtigen die wirkung von chlor erheblich (chlorzehrung), sulfide, thiosulfat und eisensalze können sie völlig aufheben. chlor wird für die desinfektion gasförmig als cl 2 oder als chlordioxid (clo 2 ), aber auch in form von chlorabspaltenden verbindungen angewandt. die wichtigsten dieser verbindungen sind salze der unterchlorigen säure (hypochlorite) und chloramine. aus anwendungstechnischen gründen werden chlor-und chlordioxidgas nur zur wasserdesinfektion verwendet. wegen der chlorzehrung können für chlorgas keine fixen dosierungsangaben gemacht werden, sondern es muss von der nach verbrauch durch organische substanz zurückbleibenden konzentration des "restchlors" (verfügbares freies chlors) ausgegangen werden, die meist in mg/l (ppm) angegeben wird. für trinkwasser soll sich diese konzentration nach halbstündiger einwirkung um 0,3 mg/l, für schwimmbadwasser um 0,3-0,5 mg/l und für abwässer um 10-30 mg/l bewegen. das anstelle von chlorgas vielfach verwendete chlordioxidgas wirkt stärker bakterizid, ist in seiner wirkung stabiler gegen veränderungen des ph-werts und führt bei anwesenheit von phenolen im trinkwasser, im gegensatz zu chlorgas, nicht zur bildung von haloformen und chloraminen sowie kaum zu geschmacklich und geruchlich unangenehmen chlorphenolen. chlorabspaltende substanzen werden außer zur wasserdesinfektion im kleinen maßstab auch zur desinfektion von wäsche, flächen, händen, ausscheidungen und früchten sowie vor allem im sanitär-und küchenbereich verwendet. zur immer noch manchmal beworbenen desinfektion von fütterungsutensilien für säuglinge sind chlorverbindungen nicht zu empfehlen. mit physikalischen verfahren wie auskochen, dampfdesinfektion oder autoklavieren stehen sicherere desinfektionsverfahren für babyfläschchen und schnuller zur verfügung. hypochlorite führen zu einem rascheren eintritt des desinfektionseffekts als chloramine, zerfallen aber auch schneller. nicht stabilisierte hypochlorit-lösungen müssen deshalb sofort nach zubereitung verwendet werden! am häufigsten wird natriumhypochlo-rit (naocl), das in handelspräparaten mit stabilisatoren angeboten wird, verwendet. der billige chlorkalk, eine mischung aus calciumhypochlorit, -chlorid und -hydroxid, wird meist in krisenzeiten zur trinkwasser-und wischdesinfektion gebraucht. chloramine können als anorganische oder organische substanzen vorliegen. sie spalten chlor langsam ab, wodurch die wirkung zwar protrahiert eintritt, aber länger anhält. zu nennen ist vor allem das als "chloramin t" eingesetzte tosylchloramidnatrium. andere chlorabspalter sind z. b. di-und die antimikrobielle ausstattung von gegenständen des täglichen bedarfs und zunehmend auch im gesundheitsbereich mit metallen in nanokristalliner form hat weltweit in den letzten jahren ein starkes, teils kritikloses ausmaß erlebt. wenn auch je nach eingesetzter technologie und chemisch-physikalischen möglichkeiten der beteiligten komponenten (material, wirkstoff, imprägnierungsverfahren) eine wirksamkeit von objekten durch beschichtung oder imprägnierung mit antimikrobiellen stoffen gegen bestimmte mikroorganismenspezies erzielt werden kann, ist die bezeichnung "antimikrobiell" weder mit einer spezifischen infektionsprävention verknüpft, noch liegen ihr einheitliche kriterien zugrunde. jedes als "antimikrobiell" gekennzeichnete produkt muss neben dem nachweis der antimikrobiellen wirkung auch unter praxisrelevanten bedingungen einen belegten oder zu erwartenden vorteil im sinne der infektionsprävention für den einzelnen und das allgemeinwohl vorweisen können. der nutzen der antimikrobiellen imprägnierung oder beschichtung für den jeweils vorgesehenen anwendungsbereich muss kritisch gegen mögliche risiken und unerwünschte wirkungen für mensch und umwelt abgewogen werden. vor allem ist zu berücksichtigen, ob die wirkung mit üblichen hygienischen maßnahmen (reinigung, desinfektion) wirksamer und ungefährlicher erreicht werden kann . unter diesem aspekt ist auch die aktuelle bewerbung von kupferoberflächen in medizinischen bereichen zu betrachten. metallsalze (silber-und manche zinnsalze) wirken mikrobiozid, quecksilber-und kupfersalze vorwiegend mikrobiostatisch. silbersalze sind, nur bei unsachgemäßer anwendung (gefahr der argyrose) toxisch, können aber in hoher konzentration zu verätzungen führen. sie werden außer zur wasserdesinfektion heute nur noch in geringem ausmaß zur antiseptik verwendet (› kap. 2.2) . nanotechnologische silberapplikationen finden sich zunehmend als mikrobiostatische ausrüstung von kleidung und gebrauchsgegenständen, aber auch von blutgefäß-und hohlraumkathetern. quecksilber und seine anorganischen verbindungen sind stark toxisch, seine organischen verbindungen hingegen sind weniger giftig und besser hautverträglich. ihre verwendung als desinfekti-onsmittel ist obsolet, weil sie fast nur mikrobiostatisch wirken und keine sichere abtötung gegeben ist. mit organischen zinnverbindungen soll in kombination mit rasch wirkenden stoffen ein desinfektionseffekt mit lang anhaltender nachwirkung erzielbar sein. sie finden z. b. als tributylzinnbenzoat in präparaten für die flächen-und wäschedesinfektion anwendung, wobei aber toxikologische risiken nicht ausgeschlossen sind. neben den halogenen existieren einige stoffe, deren mikrobiozide wirkung ebenfalls auf oxidationsvorgänge zurückzuführen ist. es handelt sich dabei um sauerstoffreiche und leicht sauerstoff freisetzende verbindungen wie ozon, anorganische und organische peroxide sowie persäuren. das für den respirationstrakt giftige gas ist noch in verdünnungen von 10 -6 an seinem charakteristischen geruch erkennbar. in wasser und bei hoher relativer luftfeuchtigkeit zerfällt es rasch. in wässrigem milieu umfasst das wirkspektrum bei anwendungskonzentrationen von maximal 5 mg/l sämtliche formen von mikroorganismen. trockene ozon-luft-gemische haben hingegen keinen mikrobioziden effekt. wie die halogene wird auch ozon durch zahlreiche organische und anorganische verbindungen verbraucht (ozonzehrung). unter lichteinwirkung zerfällt es rascher als im dunkeln. bei niedrigen temperaturen ist sein mikrobiozider effekt besser als bei höheren. die wichtigste aussage der prüfung eines desinfektionsverfahrens ist zweifellos die über seine wirksamkeit. zur vollständigen charakterisierung eines desinfektionsverfahrens gehören allerdings auch die untersuchung und beschreibung toxikologischer, allergologischer und sicherheitstechnischer momente sowie sein einfluss auf die qualität des desinfektionsguts und anderen materials. nachfolgend wird nur auf die mikrobiologische prüfung eingegangen. prüfungen von desinfektionsverfahren werden als "typprüfungen" vor ihrer allgemeinen verwendung oder als "praxisprüfung" während und am ort der praktischen anwendung durchgeführt. in den verschiedenen ländern existieren unterschiedliche, meist von den jeweiligen fachgesellschaften für hygiene und mikrobiologie empfohlene prüfanordnungen. desinfektionsverfahren, für die durch gutachten belegbar ist, dass sie den jeweiligen anforderungen genügen, können in die "liste der geprüften und anerkannten desinfektionsverfahren" der jeweiligen körperschaft aufgenommen werden und/oder erhalten ein zertifikat (› tab. 2.12 • nachweis großer mikrobenmengen auf essgeschirr, instrumenten oder behältern, die mit schlecht reinigenden (kontamination durch schmutzige waschflotte!) und mangelhaft desinfizierenden waschmaschinen gewaschen wurden. mikrobiologische kulturen eignen sich nur bedingt für prüfungen, die der anwender selbst und ohne zuhilfenahme von fachleuten durchführt. deshalb ist es äußerst wichtig, dass der anwender seine desinfektionsverfahren mit anderen methoden regelmäßig kontrolliert (s. validierung). die von den fachgesellschaften für hygiene und mikrobiologie oder von regulationsbehörden herausgegebenen listen (› tab. 2.12 ) über gutachterlich geprüfte und für geeignet befundene verfahren (präparate mit anwendungsempfehlung) geben dem anwender eine gute orientierungshilfe für die verfahrensauswahl im krankenhaus. die empfehlungen der entsprechenden liste des rki (rki 2013) orientieren sich an den erschwerten bedingungen der kommunalen seuchenbekämpfung. bei der infektionsübertragung spielen die hände eine doppelte rolle: • sie dienen mikroorganismen als vehikel, indem sie mikrobielle kontaminationen aufnehmen und an anderer stelle deponieren. • sie fungieren als infektionsquelle, wenn sich erreger in den oberen schichten der haut oder auch in infizierten weichgewebeläsionen vermehren und von dort an die kontaktstelle freigesetzt werden. besondere bedeutung kommt den unmittelbaren kontaktflächen zu (bettwäsche, holme im griffbereich). eine manuell durchgeführte exakte reinigung und wischdesinfektion des bettgestells mag den hygienischen anforderungen genügen. die von manchen als unnötig aufwendig befundenen maschinellen verfahren der bettenaufbereitung erbringen, sofern sie richtig betrieben werden, gleich bleibend gute ergebnisse und unterstützen die anliegen der qualitätssicherung. als desinfektionsprinzip werden heißwasser oder wasserdampf, evtl. in kombination mit chemischen desinfektionsmitteln, eingesetzt. für die dampfdesinfektion haben sich insbesondere das dampfströmungsverfahren und das vakuum-dampf-vakuum-verfahren (vdv-verfahren) bewährt und werden vom rki anerkannt (rki 2013). es handelt sich um folgende gegenstände: operationsinstrumente, anästhesiezubehör, endoskope, ess-und trinkgeräte, fütterungsutensilien, auffangbehälter für sekrete, drainageflüssigkeiten, stuhl und urin; atemgas-waschflaschen, blumenvasen, irrigatoren sowie gebrauchsgegenstände der patienten. zur mehrmaligen verwendung bestimmte gegenstände müssen nach jedem gebrauch (gereinigt und) desinfiziert werden. zur einmalverwendung vorgesehene gegenstände behindern durch ihre konstruktion oft eine wirksame reinigung und desinfektion oder/und nehmen durch aufbereitung vielleicht schaden. sie dürfen deshalb nur im ausnahmefall und nur unter streng definierten bedingungen (ausschluss unerwünschter wirkungen, definiertes validiertes aufbereitungsverfahren, zertifiziertes qualitätsmanagement, › kap. 6.1) aufbereitet werden! aus produkthaftungsgründen dürfen nur gegenstände aufbereitet werden, für die geeignete aufbereitungsverfahren definiert sind. dabei ist auch der schutz des personals vor infektionen zu bedenken. die maschinelle reinigung und desinfektion ist manuellen verfahren grundsätzlich vorzuziehen. das hat zwei wesentliche gründe: maschinen mit kombinierten reinigungs-desinfektions-verfahren reinigen undesinfizierte güter ohne personen zu gefährden. das ist wichtig, weil nur nach guter vorreinigung ein gleich bleibend guter desinfektionseffekt mit vertretbarem aufwand sichergestellt werden kann. (verfahren der kommunalen seuchenbekämpfung, die aus epidemiologischen gründen als ersten schritt grundsätzlich eine desinfektion erfordern, bleiben hier außer betracht.) ferner können thermische und chemothermische desinfektionsverfahren wirkungsvoll in maschinelle reinigungsverfahren integriert werden. ausschlaggebend für die desinfektionswirkung solcher maschinen ist die güte ihres reinigungssystems. das gilt insbesondere, wenn der zu beseitigende schmutz stark erregerhaltig ist (z. b. stuhl). in solchen fällen können schon kleinste schmutzreste ein versagen der anschließenden desinfektion zur folge haben. die in der praxis erzielte reinigungswirkung hängt nicht nur von konstruktiven details der maschine ab, sondern auch von ihrer richtigen beschickung (spülgerechte lagerung der güter, keine überladung der maschine, keine behinderung der beweglichen teile des reinigungssystems, keine spülschatten) und ihrer ordnungsgemäßen wartung (reinigung von düsen, schmutzfangsieben usw.) ab. hinweise auf konstruktive details der maschinen und auf die verfahren zur prüfung der reinigungswirkung finden sich z. b. in koller, 2008a und koller, 2008b auch zu hohe temperaturen (> 45 °c) in der reinigungsphase beeinträchtigen das reinigungsergebnis bei organischen verunreinigungen durch koagulation nativer proteine. die in den programmablauf der maschinen integrierten thermischen desinfektionsverfahren bringen meist heißwasser (z. b. 85 desinfizierte güter sollen grundsätzlich möglichst rasch getrocknet und trocken gelagert werden. medizinisch-technische geräte sind gesondert zu besprechen, weil sie meist neben teilen, die mit patienten in direkten oder indirekten kontakt kommen, feinmechanische, optische oder elektronische elemente besitzen, die durch desinfektionsmaßnahmen beschädigt werden können. grundsätzlich sind medizinisch-techni-sche geräte so aufzubereiten, dass sie für die anwendung am nächsten patienten sicher sind. voraussetzung dafür ist eine bauart, die wirksame aufbereitungsverfahren zulässt. viel zu oft blieb dieses gebot in der vergangenheit unbeachtet. oft wurden geräte angeschafft, die eine wirksame und sichere desinfektion nicht zulassen. im folgenden sind wichtige hygienische grunderfordernisse an bauart und beschaffenheit medizinisch-technischer geräte aufgezählt: • reinigbarkeit: teile des geräts, die mit dem patienten oder seinen ausscheidungen in kontakt treten, müssen einfach demontierbar und maschinell zu reinigen sein. die im folgenden angeführten wirkstoffe finden allein oder in kombination in vielen handelsüblichen flächendesinfektionsmitteln anwendung: glutaral, formaldehyd und glyoxal, meist in kombination untereinander und gemeinsam mit tensidischen wirkstoffen. diese produktgruppe weist ein breites wirkspektrum ohne wesentliche lücken auf und ist, insbesondere bei ausreichendem formaldehydanteil auch für situationen mit stärkerer organischer belastung des desinfektionsguts geeignet. wichtige nachteile sind schleimhautreizungen bei großflächigem einsatz und schlechter belüftung sowie hautreizungen bei kontakt mit der gebrauchslösung. die anwendung muss daher in richtiger dosierung erfolgen und nicht in heißem oder warmem wasser (geruchsbelästigung) oder ohne handschuhe. phenolderivate wirken verhältnismäßig rasch, werden durch organische begleitstoffe nur mäßig gehemmt und weisen ein breites wirkspektrum auf. gegen enteroviren ist ihre wirkung nur gering. die kombination mit waschaktiven substanzen ist möglich. wegen der gefahr einer hyperbilirubinämie bei neugeborenen und frühgeborenen werden sie in neonatologischen abteilungen nicht eingesetzt. amphotenside, quats und amine haben eine gute reinigungswirkung und sind wenig aggressiv, besitzen aber ein eingeschränktes wirkspektrum (schwächen gegenüber pilzen, mykobakterien und viren) und einen deutlichen eiweißfehler. in kombination mit guten reinigungsmethoden und bei langen einwirkzeiten ergibt sich eine akzeptable desinfektionswirkung. im krankenhaus kann ihr einsatz nur in niedrigrisikobereichen und nur im zusammenhang mit optimalen reinigungsmethoden akzeptiert werden. natriumhypochlorit und organische chlorabspalter (chloramine, di-und trichlorisocyanurate usw.) wirken rasch und besitzen ein breites wirkspektrum mit guter viruzider wirkung. wegen der starken chlorzehrung durch organische substanzen dürfen sie nur auf reinen oder vorgereinigten flächen verwendet werden. kombiniert mit scheuermitteln eignen sie sich gut zur reinigung und desinfektion im sanitärbereich. ethanol und die beiden propanole sind in konzentrationen wie zur händedesinfektion auch im wischverfahren rasch wirksame flächendesinfektionsmittel mit breitem wirkspektrum bei geringer beeinträchtigung durch organische verschmutzungen. von einer großflächigen aufbringung oder anwendung im sprühverfahren ist wegen der explosions-und brandgefahr abzuraten. peressigsäure und perameisensäure eignen sich zur anwendung an korrosionsbeständigen oberflächen (insbesondere kunststoffen) und finden in situationen einsatz, wo eine sporozide wirkung gewünscht wird (z. b. im rahmen der schutzisolierung bei knochenmarktransplantation). alternativ kommen organische peroxide in betracht. die meisten flächendesinfektionsmittel werden als konzentrate geliefert und sind in gebrauchsverdünnungen, häufig von 0,5 %, anzuwenden. in der praxis macht die richtige einstellung der desinfektionslösung oft schwierigkeiten. bei manueller herstellung müssen vom reinigungspersonal dosierhilfen, z. b. messbecher mit deutlicher markierung, dosierpumpen, die auf den konzentratbehälter aufgeschraubt werden, oder beutel und tuben, die eine auf einen eimer wasser abgestimmte portion enthalten, verwendet werden. nicht akzeptabel ist das zugeben eines "schusses" des konzentrats nach gutdünken. vorsicht ist auch bei der herstellung von konzentrationen, die nur einen oder zwei hübe der dosierpumpe benötigen, geboten, da viele dieser einfachen handpumpen erst ab dem dritten hub richtig dosieren! vielfach werden zur herstellung der gebrauchslösung des flächendesinfektionsmittels automatische zumischanlagen verwendet, die jedoch häufig nicht einwandfrei funktionieren. bei manchen anlagen hängt die konzentration der abgegebenen lösung vom wasserdruck ab oder wird übersehen, dass der konzentratbehälter leer oder die zumischdüse verstopft ist. in perioden ohne anwesenheit von desinfektionsmittel kann es zur ansiedlung von bakterien, vor allem pseudomonas-arten, im leitungssystem der zumischanlage kommen (biofilme). diese mikroben können eine erhöhte chemoresistenz entwickeln. vor allem große zentrale anlagen, die ein ganzes haus versorgen, neigen zu solchen problemen und sind daher nicht zu empfehlen. dezentrale zumischanlagen sind besser kontrollierbar und können dort, wo ein flächendesinfektionsmittel häufig verwendet werden muss, sinnvoll sein. dosieranlagen sollen den technischen anforderungen entsprechen, wie sie z. b. in deutschland (bundesanstalt für materialprüfung und bundesgesundheitsamt 1978) und in österreich (friebes und dosch 1980) in richtlinien festgelegt sind, und müssen regelmäßig kontrolliert werden. wartung der reinigungs-und desinfektionsutensilien › kapitel 2.5. wände, decken und einrichtungsgegenstände in medizinisch genutzten bereichen des krankenhauses müssen abwaschbar sein, um sie reinigen und bei bedarf desinfizieren zu können. wände sollen routinemäßig bis in greifhöhe gereinigt werden. eine desinfektion wird nur nach kontamination (z. b. verspritzen infektiöser sekrete) oder im rahmen der sanierung eines raums nach entlassung eines infektiösen patienten (wischdesinfektion) für nötig erachtet. patientennahe arbeitsflächen, auf denen auch saubere güter und behandlungsbehelfe abgelegt werden, sollen routinemäßig wischdesinfiziert werden. für häufig berührte gegenstände (türklinken, telefonhörer usw.) ist das zumindest in epidemischen situationen ebenfalls angezeigt. die desinfektion von wänden, decken und einrichtungsgegenständen kann die viel wichtigere nichtkontamination (berührungsfreie techniken) und die händehygiene nicht ersetzen, sondern nur ergänzen. die dekontamination der luft soll die entstehung aerogener infektionen verhüten helfen. unter aerogener infektion ist hier nicht die als tröpfcheninfektion bekannte übertragungsart zu verstehen, bei der z. b. husten-oder sprechtröpfchen der infektionsquelle auf das infektionsziel geschleudert werden. gemeint ist die suspension und translokation von mikroorganismen durch luft. mit wenigen ausnahmen (› tab. 2.14) spielt dieser infektionsweg im krankenhaus gegenüber den anderen übertragungsmöglichkeiten eine untergeordnete rolle. sofern aerogene infektionen eine rolle spielen und die streuung der erreger nicht schon an der infektionsquelle blockiert werden kann (wie bei klimaanlagen, raumluftbefeuchtern oder beatmungsgeräten), sollte die blockierung des aerogenen übertragungswegs einen positiven effekt zeitigen. das lässt sich entweder durch filtra tion der luft oder durch physikalische oder chemische inaktivie rung der luftgetragenen mikroorganismen erreichen. schon ausgiebiges lüften eines raums kann eine keimzahlverminderung von bis zu 80 % bewirken. verlässlicher und mit einem besser kalkulierbaren wirkungsgrad werden rlta eingesetzt, z. b. in op-einheiten. eine früher oft übliche form der luftdekontamination ist die durch uvstrahlen. diese methode ist nur unter streng definierten, standardisierten bedingungen zuverlässig und auf sehr umschriebene anwendungen beschränkt (z. b. entkeimung von werkbänken und arbeitsboxen für infektiöse oder infektionsriskante tätigkeiten). eine chemische luftdekontamination während des aufenthalts von personen im raum durch verdampfen oder versprühen von glykolen oder anderen desinfektionsmitteln ist abgesehen vom umstrittenen mikrobioziden effekt solcher maßnahmen aus grundsätzlichen überlegungen abzulehnen. das risiko einer gesundheitsschädigung durch chemisierte atemluft steht in keinem verhältnis zum zu erwartenden nutzen. die formaldehyd-wasserdampf-raumdesinfektion im rahmen der schlussdesinfektion (› kap. 2.5.2) ist nur in sonderfällen indiziert und keinesfalls routinemäßig anzuwenden. zunächst ist es nötig, schwerpunkte festzulegen. die verfügbaren mittel und kräfte müssen auf die wichtigen infektionsüberträger konzentriert werden. anhaltspunkte für kriterien zur beurteilung der gefährlichkeit bestimmter infektionsüberträger und beispiele finden sich in › tab. 2.15 . die desinfektion von händen, instrumenten und ausscheidungsbehältern hat einen hohen stellenwert, die von wänden oder fußböden einen niedrigen. zusätzlich zu desinfektionsmaßnahmen muss in der praxis entschieden werden, ob eine sterilisation nötig ist (bei kontakt mit gewebe, blut oder sterilen körperhöhlen) oder ob einmalware zu bevorzugen ist. bei der auswahl eines desinfektionsverfahrens werden die weichen für den späteren erfolg oder misserfolg gestellt. qualität und kapazität eines verfahrens, seine kompatibilität mit bestehenden systemen, verfügbarkeit und qualität eines kundendienstes, verfügbarkeit des notwendigen personals und verfügbarkeit der nötigen betriebsmittel sind einige faktoren, die vor der anschaffung geklärt sein müssen. jedes desinfektionsverfahren ist nur so gut wie seine gebrauchsan weisung. die wichtigsten bedienungs-, kontroll-und schutzanweisungen müssen dem anwender jederzeit in kurzer, verständlicher und leicht lesbarer form zur verfügung stehen. komplizierte maschinelle verfahren erfordern neben einer schriftlichen betriebsanleitung eine persönliche einführung oder einen eigenen ausbildungskurs für das bedienungspersonal. mikrobiologische kontrollen besitzen zwar eine hohe aussagekraft, sind aber meist zu aufwendig und beschränken sich daher in der regel auf die periodischen behördlichen kontrollen von desin• hygienische anforderungen an räumliche gestaltung und arbeitsabläufe lassen sich leichter realisieren. • leistungsfähige rdg ersetzen eine vielzahl dezentral laufender kleingeräte und werden durch einen begrenzten, gut geschulten personalstab richtig bedient. • wartung der geräte und kontrolle der arbeitsabläufe sind wesentlich besser überschaubar. • der bedarf an spezifisch geschultem personal ist wesentlich kleiner als bei dezentraler ausführung. . 2000) . bei der von flächen ausgehenden risikobewertung ist zu berücksichtigen, dass sich die zur auslösung einer infektion erforderliche infektionsdosis erregerabhängig z. t. deutlich unterscheidet (› tab. 2.19 ). grundsätzlich ist zu berücksichtigen, dass der stel-lenwert der flächendesinfektion zur infektionsprävention mit zunehmender distanz zum patienten abnimmt. folgendes beispiel unterstreicht den beitrag der flächendesinfektion zur aseptik. in einem eingriffsraum konnte durch desinfektion aller flächen einschließlich des inventars nach dem letzten eingriff und anschließende abhängung nicht aus dem raum herausnehmbarer geräte und des inventars mit sterilen op-tüchern die raumluftqualität von reinraumklasse c zu reinraumklasse b verbessert werden (below et al. 2010 ). bei mutmaßlicher oder sichtbarer flächenkontamination mit blut und weiteren sekreten und exkreten ist die sog. gezielte oder anlassbezogene desinfektion durchzuführen. hierbei ist die chemoresistenz des mutmaßlichen erregers zu beachten (z. b. m. tuberculosis, c.-difficile-sporen, noroviren, ggf. prionen 2.20) . bei der entscheidung, ob routinemäßig eine reinigung oder eine desinfizierende reinigung durchgeführt werden soll, müssen auch praktikabilität und sichere durchführbarkeit berücksichtigt werden. in op-einheiten werden zwischen zwei operationen die flächen im arbeitsbereich um den op-tisch, die verkehrswege im op-raum und alle kontaktflächen desinfiziert. die vom vah angegebene möglichkeit, dass mit der vorbereitung der nächsten op begonnen werden kann, sobald die flächen luftgetrocknet sind, d. h. u. u. vor ablauf der vom hersteller angegebenen einwirkungszeit, ist aufgrund der prüfergebnisse im vier-felder-test kritisch zu sehen. in diesem praxistest wird bei einsatz der konzentrationen für lange einwirkungszeiten ≥ 60 min die wirksamkeit z. t . erst nach 60 min oder 240 min erreicht -da sind die flächen aber schon (lange) trocken. daher sollte bei gewählten einwirkzeiten > 30 min mit der vorbereitung der nächsten op die einwirkzeit und nicht nur die trocknung abgewartet werden, solange keine neuen erkenntnisse ein anderes vorgehen ermöglichen. sofern die wände nicht sichtbar kontaminiert sind, entbehrt die noch anzutreffende empfehlung, nach beendigung des op-programms im op-saal eine wischdesinfektion der wände bis zur höhe von 2 m durchzuführen, ihrer experimentellen oder epidemiologischen grundlage. abhängig von der raumlufttechnik, dem op-spektrum und der experimentell ermittelbaren dynamik der mikrobiellen belastung der wände kann der krankenhaushygieniker den rhythmus festlegen (z. b. monatlich). bei nachgewiesenermaßen funktionierender verdrängungslüftung (raumklasse ia) ist die gezielte desinfektion als ausreichend anzusehen, d. h., es kann auf die fußbodenwischdesinfektion zwischen zwei eingriffen -allerdings nur in einem augen-op nachgewiesen und nur bei nicht septischem eingriff vertretbar -verzichtet werden (knochen et al. 2010 ). in op-einheiten/ eingriffsräumen sind umfang und rhythmus der desinfizierenden flächenreinigung im ergebnis des gemeinsamen risk assessments durch den jeweiligen fachvertreter und den krankenhaushygieniker festzulegen. flächen, auf denen aseptische arbeiten ausgeführt werden, sind grundsätzlich desinfizierend zu reinigen. in reinräumen (z. b. apotheke, herstellung von blutprodukten, hornhautbank, stammzellpräparation) sind die dort gültigen spezifischen vorschriften einzuhalten. in küchen und milchküchen gelten die vorgaben des lebensmittelrechts. bei erhöhter infektionsgefährdung entweder aufgrund reduzierter immunabwehr und/oder der hohen wahrscheinlichkeit fortwährender freisetzung kritischer erreger, insbesondere von mre (z. b. its, risikogruppe 2 und 3 immunsupprimierter) in die umgebung sowie bei sichtbarer kontamination sollte zur flächendesinfektion mindestens die konzentration des desinfektionsmittels für den 1-h-wert eingesetzt werden, damit die wahrscheinlichkeit der sicheren erfassung der auf den flächen unterschiedlich verteilten erreger steigt. als anlassbezogene schlussdesinfektion wird die gezielte desinfektion eines raums oder bereichs einschließlich der in ihm vorhandenen oberflächen und gegenstände nach erfolgter pflege oder behandlung eines infizierten bzw. mit hochkontagiösen erregern besiedelten patienten bezeichnet. durch organisation, auswahl der reinigungs-und desinfektionsmittel und -verfahren sowie die häufigkeit müssen im einvernehmen mit dem krankenhaushygieniker/der hfk im hygieneplan festgelegt werden. im alten-und pflegeheim gelten vom grundsatz her die gleichen anforderungen an die desinfizierende reinigung wie im patientenzimmer, wenn patienten mit erhöhter infektionsgefährdung behandelt, bei kolonisation z. b. mit mrsa saniert oder als ausscheider von infektionserregern versorgt werden. die desinfektion muss als prozess betrachtet werden. es sind standards für die reinigung und desinfektion zu erarbeiten, deren sachgerechte umsetzung durch sops, aus-, fort-und weiterbildung sowie durch geeignete auditsysteme sichergestellt wird (gebel et al . 2013 allgemeiner ablauf: reinigungswagen vor dem zu reinigenden zimmer seitlich abstellen und darauf achten, dass die wege frei bleiben. warnschild "rutschgefahr" im flur aufstellen. die reinigung eines zimmers beginnt grundsätzlich mit der müllentleerung, dann erfolgt die reinigung der oberflächen (zuerst zimmer, dann sanitärbereich), erst danach die reinigung des fußbodens. reini-gungstuch immer 4-fach falten; sobald eine seite verschmutzt ist, reinigungstuch wenden und nächste saubere seite verwenden grundsätzlich sind reinigungstätigkeiten von oben nach unten, von hinten nach vorn und von sauber zu schmutzig durchführen. oberflächenreinigung im patientenzimmer: abfallbeutel entleeren, verschließen und am reinigungswagen entsorgen. neuen beutel einsetzen, nicht in abfalleimer hineingreifen, abfall in den behältnissen nicht von hand eindrücken! mit vorgetränktem reinigungstuch folgende oberflächen reinigen: lichtleisten, fensterbank, tische, stühle, wandschmuck, abfalleimer außen, schalter, dosen, stromleisten, türgriffe, türen im griffbereich (desinfektion), fernseher, außenbereiche der schränke im griffbereich. falls waschbecken im patientenzimmer, mit andersfarbigem tuch reinigen. verwendete tücher sind nach jedem patientenzimmer abzuwerfen. oberflächenreinigung in der nasszelle: abfalleimer wie im patientenzimmer entsorgen. wc spülen, dann wc-reiniger in wc-becken, urinal und unter den spülrand spritzen und einwirken lassen, toilettenbürste in das wc-rohr stellen. die reinigung ist immer von oben nach unten durchzuführen. während der einwirkzeit mit reinigungstuch erst den spiegel, dann die ablage und danach waschbecken mit armatur und spritzbereich reinigen, danach alle sonstigen oberflächen, ggf. spiegel mit trockenem gelbem reinigungstuch nachpolieren, anschließend sitzhilfen und abfalleimer mit gelbem reinigungstuch reinigen. danach toilette gründlich nachbürsten und mit wasser nachspülen, dann mit andersfarbigem reinigungstuch wc-brille und becken außen sowie den spritzbereich um das becken reinigen. nach jedem sanitärraum reinigungstücher in das netz am reinigungswagen abwerfen. seife und papier auffüllen. in › tab. 2.21 ist ein muster eines leistungsverzeichnisses für die reinigung und desinfektion ausgewählter räume zusammengestellt, dass der konkreten infektionsgefährdung angepasst werden muss. desinfizierende raumverneblung: wenn sich im rahmen eines ausbruchmanagements herausstellt, dass nach der schlussdesinfektion die ursächlichen erreger noch nachweisbar sind, ist es durch raumverneblung mit wasserstoffperoxid (wpo) sinnvoll, die zeitspanne bis zum erreichen der sicheren flächendesinfektion zu überbrücken. da die qualitätsverbesserung der arbeit des reinigungsteams wochen in anspruch nehmen kann und im rahmen eines vre-ausbruchs die erreger trotz schlussdesinfektion an relevanten stellen im patientenzimmer nachweisbar waren, wurde die verneblung bis zur nachgewiesenen sicheren schlussdesinfektion durchgeführt. mit einführung der verneblung war ein kontinuierlicher rückgang der nosokomialen übertragung von vre bis zur beendigung des ausbruchs nachweisbar (kramer et al. in vorb.) . voraussetzung für die einführung des verfahrens war dessen wirksamkeitsnachweis bei einwirkung auf mit a. brasiliensis kontaminierten prüfkörpern, die in einem versuchsraum wandständig und unter der decke platziert waren, sowie in räumen mit schimmelpilzbefall nach einem wasserschaden (koburger et al. 2011 die sichere aufbereitung ist erforderlich, weil wiederverwendbare tuchspender insbesondere bei einsatz oberflächenaktiver wirkstoffe kontaminiert waren (kampf et al. 2014b ). die aufbereitung im rdg verhinderte ohne und mit zusatz chemischer reinigungsmittel die rekontamination der desinfektionsmittellösung, wenn eine temperatur zwischen 60-70 °c über mindestens 5 min sichergestellt wurde. ebenso verhinderte die vorreinigung mit heißem wasser oder in form eines gründlichen reinigungsschritts mit nachfolgender wischdesinfektion mittels einmaltuch und sauerstoffabspalter die rekontamination (kampf et al. 2014a ), müsste aber als prozess validiert werden. allerdings wurde die wirksamkeit der aufbereitung nicht nach sporenkontamination geprüft, die in praxi jedoch nicht auszuschließen ist. desinfektions-und reinigungsverfahren sowie die aufbereitung der benötigten utensilien sind regelmäßigen kontrollen zu unterziehen. die gewährleistung der qualitätsgerechten desinfizierenden flächenreinigung ist wegen der wachsenden bedeutung für die infektionsprävention ein dauerbrenner sowohl für die leitung des krankenhauses als auch für die patienten und deren besucher (carling, parry und von beheren 2008) . durch hygienisch-mikrobiologische untersuchungen sollte die wirksamkeit von reinigungs-und desinfektionsverfahren kontrolliert werden (krinko 2004) . auch wenn die ermittlung der mikrobiellen belastung trotz mangelnhafter standardisierung von methoden und bewertungskriterien (galvin et al. 2012 ) der goldstandard zur qualitätssicherung ist, eignen sich einfach durchführbare fluoreszenzmethoden zur qualitätskontrolle (blue et al. 2008; boyce et al. 2011; carling et al. 2006; luick et al. 2013; munoz-price et al. 2011 ). als quantitative, standardisierte mikrobiologische beprobungstechnik empfiehlt sich zukünftig die in der en 16615 angegebene methode. während eine sichtkontrolle nicht zielführend ist, ermöglichen audits mit detaillierten checklisten eine realistische bewertung (malik, cooper und griffith 2003) . häufigkeit und umfang der kontrollen werden vom krankenhaushygieniker in zusammenarbeit mit der hfk festgelegt (zu aussagewert, richtwerten und gesamtbeurteilung › kap. 8.10 2.3; jülich et al. 1993; klein und deforest 1983; mahnel 1984; poshni 1968; von rheinbaben und kirschner 1995 in der praxis werden physikalische verfahren oft mit chemischen einflüssen kombiniert. am häufigsten sind chemothermische verfahren. anstelle thermischer einflüsse kann auch ultraschall oder uv-licht (in gegenwart photoinaktivierender substanzen) angewandt werden. selbst wenn bei den vertretern einer virusfamilie deutliche unterschiede in der resistenz gegenüber umwelteinflüssen und desinfektionswirkstoffen auftreten können und sogar innerhalb einer art unterschiede festgestellt wurden, ist es vertretbar, für jede virusfamilie eine zusammenfassende bewertung vorzunehmen. adenoviren ( peters, bräuninger und fischer 1995; rki 1995b; ) wurden parvoviren v. a. wegen ihrer guten trockenstabilität aufgenommen. bei inaktivierungsversuchen an der fläche war parvovirus deutlich stabiler als polio-und adenovirus (eterpi, mcdonnell und thomas 2009 (thomssen et al. 1960 ). die meisten arten sind im ph-bereich 3-9 über viele tage stabil, nicht jedoch rhinoviren, die gegenüber ph-werten im sauren bereich empfindlich reagieren und bei ph 3 in 30 min inaktiviert werden. insbesondere polioviren und offensichtlich auch das hav sowie viele stämme der coxsackie-und echo-viren sind hydrophil. manche unter ihnen, z. b. echo-virus 6 und 18, besitzen aber auch leicht lipophile eigenschaften und reagieren schwach mit lipiden. sie werden deshalb durch lipophile substanzen, v. a. durch längerkettige alkohole, inaktiviert. hydrophile picornaviren, insbesondere das poliovirus, sind stattdessen gegenüber kurzkettigen hydrophilen alkoholen empfindlich. innerhalb der picornaviren liegen die meisten experimentellen erfahrungen bei polioviren vor. in ihrer resistenz gegenüber desinfektionswirkstoffen ähneln sie den parvoviren, zeigen im gegensatz zu diesen aber keine erhöhte thermoresistenz und sind auch gegenüber austrocknen sehr empfindlich. da polioviren neben ihrer hohen desinfektionsmittelresistenz zusätzlich den vorteil einfacher handhabbarkeit bieten, findet man sie als prüfviren in vielen leitlinien und normen (en 14476:2013; rki 2004) . gegenüber methanol und ethanol sind polioviren sehr empfindlich. propan-1-ol zeigt dagegen bei 90 % v/v/5 min keine wirksamkeit und eine exposition gegenüber propan-2-ol führt unter den gleichen bedingungen selbst nach 60 min zu keinem messbaren titerverlust. methanol verursacht bei 60 % v/v/1 min schon bei 5 °c eine titerreduktion von mindestens 3 zehnerpotenzen. für ethanol sind bei raumtemperatur und anwendungskonzentrationen zwischen 60 und 90 % einwirkungszeiten von 1-5 min notwendig, um eine titerreduktion von 3-5 zehnerpotenzen zu erzielen (van engelenburg et al. 2002) . auch hav gehört zu den picornavieren und zeigt eine hohe thermoresistenz und stabilität gegenüber desinfektionswirkstoffen. 56 °c/30 min werden nahezu verlustfrei toleriert, 60 °c reichen auch während 1 h nicht zur sicheren inaktivierung aus. 0,5-prozentiges glutaral führt innerhalb von 3 min zu einer reduktion von 3 zehnerpotenzen. mit 0,1-prozentiger lösung sind dafür 30 min notwendig (passagot et al. 1987) . zur inaktivierung durch peressigsäure sind 2 %/60 min notwendig (rf > 5). bei 1 %/30 min ist dagegen keine ausreichende wirksamkeit zu erwarten (rf 0,5). bei der auswahl von desinfektionsmitteln gegen picornaviren sollten nur mittel verwendet werden, deren wirksamkeit durch untersuchungen gegenüber poliovirus typ 1 belegt wurde. zwar können bei den verschiedenen picornaviren unter gleichen experimentellen bedingungen unterschiede in anwendungskonzentration und/oder einwirkzeit festgestellt werden (sauerbrei et al., 2009) die wichtigste gruppe innerhalb der familie der reoviren (unbehüllt) sind die fäkal-oral übertragenen rotaviren. in der neonatologie und pädiatrie sind sie häufig ursache nosokomialer virusinfektionen, besitzen aber auch als erreger von reisediarrhöen und für alte menschen hohe bedeutung. rotaviren zeigen hohe trockenresistenz und sind im ph-bereich zwischen 3 und 10 stabil (lloyd-evans, springthorpe und sattar 1986). auch temperaturen von 50 °c werden toleriert (baumeister 1981) . ihr komplexes kapsid macht rotaviren nicht nur gegen stark oxidierende desinfektionsmittel, sondern auch gegen lipidlösungsmittel, alkohole und alkoholische chlorhexidinlösungen sowie gegenüber phenolischen wirkstoffen empfindlich (vaughn, chen y-s und thomas 1986) . formaldehyd ist bei 4 %/15 min wirksam, 95-prozentiges v/v ethanol in 15 s (tan und schnagel 1981) . propan-1-ol, propan-2-ol und butanol vermögen in 30-bis 40-prozentiger lösung bovines rotavirus selbst in gegenwart von stuhl in 1 min um 3-4 zehnerpotenzen zu reduzieren (kurtz, lee und parsons 1980) . kurzkettige alkohole wirken aber insgesamt schlechter als längerkettige verbindungen. 20-prozentiges methanol ist unwirksam. ameisensäure inaktiviert rotavirus 0,5-prozentig in 15 min, propionsäure 6-bis 8-prozentig nach dieser einwirkzeit. essigsäure muss 5-prozentig für 30 min angewendet werden. als prüfviren für den humanmedizinischen bereich hat das humane rotavirus (stamm wa) verwendung gefunden. sofern keine unter-suchungen mit reoviren/rotavirus selbst vorliegen, sollten wegen der klinischen bedeutung dieser viren bei der auswahl von desinfektionsmitteln nur viruzide mittel gewählt werden. retroviren (behüllt, lipidhaltig) stellen mit hiv-1 und hiv-2 die zurzeit wichtigsten humanpathogenen viren. zur familie zählen auch die humanen t-zell-leukämieviren. diese viren werden sexuell und durch blut-blut-kontakte übertragen und besitzen nur eine geringe umweltresistenz. das darf aber nicht dazu führen, die stabilität insbesondere in natürlichen begleitmaterialien zu unterschätzen, die unter geeigneten bedingungen mehrere wochen betragen kann. gegenüber desinfektionswirkstoffen sind keine besonderen resistenzen bekannt. trotzdem dürfen bei der behandlung viruskontaminierten materials nur neueste empfehlungen berücksichtigt werden. gerade aus der frühphase der hiv-pandemie existieren angaben, wie z. b. anwendung von 25-prozentigem, ja sogar 15-prozentigem ethanol, die nach heutigem wissen unter praxisbedingungen zu keiner sicheren inaktivierung führen! ebenso werden abhängig von der verwendung von zellfreiem oder zellgebundenem virus z. t. erhebliche unterschiede in der resistenz beschrieben (hanson et al. 1989 unkonventionelle erreger zeichnen sich durch äußerst hohe umwelt-und chemikalienresistenz aus. in der umwelt können sie über jahre persistieren. die üblichen desinfektionswirkstoffe und -verfahren wie alkohole, aldehyde, iod-und phenolhaltige präparate, beta-propiolacton, ethylenoxid und uv-oder radioaktive bestrahlung sind zur inaktivierung nicht geeignet oder zeigen nur eingeschränkte wirksamkeit (danner, 1991) . als sicheres verfahren gilt die dampfsterilisation im autoklaven bei 134 °c (4 h, 4 bar) möglichst unter vorbehandlung von 1 m naoh (riesner 1956 ). in den meisten fällen scheint das autoklavieren bei 13 °c/1 h geeignet zu sein, wenn das ausgangsmaterial nicht mit hoch erregerhaltigem material kontaminiert ist (taylor et al. 1994 ). auch die behandlung mit 1 m naoh über 24 h, 2,5-bis 5-prozentigem natriumhypochlorit über 24 h, kochen in 3-prozentigem natriumdodecylsulfat (sds) für mindestens 10 min sowie 3-6 m guanidiumisothiocyanat (3 m/24 h; 4 m/1 h; 6 m/15 min) zerstören die infektiosität. bei hohem oder erhöhtem cjd-bzw. vcjd-risiko soll zuerst in dieser form desinfiziert, dann maschinell aufbereitet und abschließend bei 134 °c 1 h sterilisiert werden. es gibt allerdings auch alkalische reiniger, die in der lage sind, prionen gegenüber einem dampfsterilisationsverfahren zu sensibilisieren (destabilisieren), sodass eine gute vorreinigung mit diesen nicht nur eine dekontaminationswirkung, sondern auch eine reduktion der kontaktzeit von autoklavierungsverfahren ermöglicht (› kap. 3.3). ausgangspunkt für die in-vitro-untersuchung von präparaten auf viruswirksamkeit ist der quantitative suspensionsversuch. dieser wird nach der aktualisierten leitlinie der dvv und des rki (deutschen vereinigung zur bekämpfung der viruskrankheiten/rki, 2008) oder nach der europäischen norm en 14476 (2013) neben seinem bakteriziden wirkspektrum ist ethanol innerhalb von 15-30 s konzentrationsabhängig viruzid wirksam, propanole hingegen nicht. bakteriensporen werden nicht abgetötet. die bakterizide mindestkonzentration beträgt für n-propanol (propan-1-ol) ca. 55-60 %, für iso-propanol (propan-2-ol) 60 % und für ethanol 60-70 % (v/v). aufgrund der lokalen und systemischen unbedenklichkeit sind alkohole mittel der ersten wahl zur händedesinfektion und hautantiseptik, können aber wegen ihrer raschen wirkung auch auf kleinen flächen angewendet werden. für ethanolhaltige händedesinfektionsmittel ist aufgrund der resorbierten menge kein risiko abzuleiten ). schwierig ist dagegen die risikobewertung bei anwendung von propanolen in der schwangerschaft. bei vergleichsweise geringer exposition wurden bei chirurgen nach einmaliger hygienischer und dreimaliger chirurgischer händedesinfektion im verlauf von 3 operationen als höchste blutspiegelwerte für propan1-ol 4,1 mg/l und für propan-2-ol 2,6 mg/l und als mittlere absorbierte menge 271 mg bzw. 137 mg gemessen (below et al. 2012 aus toxikologischen und allergologischen gründen sind alkohole in kombination mit phenolen und chx nicht zur täglich wiederholten händedesinfektion zu empfehlen, zumal der nachweis der höheren wirksamkeit bisher aussteht. in hinblick auf die umweltverträglichkeit gibt es bei bestimmungsgemäßem gebrauch keine einschränkungen (kramer et al. 2008 f.) . obwohl unverdünnte alkoholkonzentrate brennbar sind, sind entzündungen innerhalb von krankenhäusern eine rarität und ausschließlich fahrlässig durch offenes feuer bzw. aus suizidaler absicht verursacht worden (kramer und kampf 2007 indikationen: mit ausnahme der sporozidie sind alle desinfektionsaufgaben ohne aldehyde mit unbedenklichen substituten realisierbar. voraussetzung hierfür ist aber eine hinreichende kenntnis der anwender über die eigenschaften der substituenten. dies gilt insbesondere, wenn aldehyde durch qav ersetzt werden. • peroxide und hypochlorite sind bei benötigter sporozidie bzgl. der langzeitverträglichkeit bei anwendungen mit wirkstofffreisetzung in die raumluft gegenüber peroxicarbonsäuren zu bevorzugen. • persäuren sind wegen der raschen sporoziden und umfassenden viruziden wirkung sowie der insgesamt höheren wirksamkeit im vergleich zu aldehyden für dialysegeräte und endoskope mittel der wahl. in frankreich wird zum personalschutz bei manueller aufbereitung peressigsäure anstelle von aldehyden zur endoskopaufbereitung empfohlen (hartemann et al. 2010 (dfg, 2000) . diese einstufungen verlangen ein überdenken der bisherigen anwendung von formaldehyd zur flächen-, raumund instrumentendesinfektion (kramer et al. 2008a ). • flächendesinfektion: es ist davon auszugehen, dass die sichere konzentration (bfr 2006) für die raumluft von 0,1 ppm bei der flächendesinfektion auch bei mischpräparaten in der regel überschritten wird (eickmann und thullner 2006) . demzufolge wären, insbesondere in kleinen und wenig belüfteten räumen, aufwendige arbeitsschutzmaßnahmen erforderlich (schwebke et al. 2007 ). sollen bei behördlich angeordneten desinfektionsmaßnahmen ggf. formaldehydhaltige desinfektionsmittel eingesetzt werden, muss durch arbeitsschutzmaßnahmen eine gefährdung ausgeschlossen werden. • raumbegasung: die raumbegasung wurde sowohl im krankenhaus als auch im krankentransport verlassen. nur zur gefahrenabwehr bei außergewöhnlichen seuchengeschehen (fock et al. 2001 ) ist sie noch für transportfahrzeuge vorgesehen. alternativ kommt die verneblung von wasserstoffperoxid in betracht (› kap. 2.5 ameisensäure als wirksamster vertreter ist bakterizid und viruzid wirksam. organische carbonsäuren sind ohne toxische risiken, umweltverträglich und werden zur konservierung, aber auch als kombinationspartner in desinfektionsmitteln, antiseptika und als antiparasitika eingesetzt (kramer et al. 2008d ). (kramer et al. 2008e sie besitzen ein breites bakterizides und fungizides wirkspektrum, sind begrenzt viruzid und z. t. askarizid die wirksamkeit kann je nach molekularmasse und struktur um den faktor 10 variieren (widulle et al. 2008) . qav sind gegen einige erreger bei langsamem wirkungseintritt wirksam, gegen mykobakterien sowie bakteriensporen unwirksam. je größer das molekül und je schlechter die solubilisierenden eigenschaften der qav sind, desto besser ist ihre haut-und schleimhautverträglichkeit. anwendungsabhängig schädigen qav die haut aufgrund ihrer emulgierenden eigenschaften. sie werden dermal resorbiert, allerdings gibt es keine hinweise auf toxische, mutagene und karzinogene risiken sowie auf reproduktionstoxizität. toxikologisch ist die großflächige anwendung von qav nicht ausreichend charakterisiert. bei anwendung auf fußböden kommt es zu sichtbaren auflagerungen (anreicherung), die mit üblichen reinigungsverfahren nicht entfernt werden können. von den angetrockneten auflagerungen können sich beim begehen der fläche partikel ablösen, die eingeatmet werden. aufgrund der hohen oberflächenaktivität der qav ist davon auszugehen, dass eingeatmete staubpartikel das surfactant der lunge angreifen, wodurch die entwicklung einer chronisch obstruktiven lungenerkrankung (copd) verursacht und/oder gefördert werden könnte. hinzu kommt die zytotoxizität speziell für den oberen respirationstrakt. für die inhalative exposition spricht die inhalative auslösung einer dermal bestätigten allergie. bei schweinezüchtern war der gebrauch qav haltiger desinfektionsmittel mit asthma assoziiert. nach großflächiger ausbringung nach feuchteschaden in einem privathaushalt kam es zu massiven innenraumbeschwerden, sodass die wohnung verlassen werden musste. noch nach 4 jahren lag die konzentration des qav im hausstaub 75-fach über dem 95. perzentil (kramer, below und assadian 2012) . auch eine mögliche resistenzentwicklung gegen qav mit kreuzresistenz gegen antibiotika (bragg et al. 2014; buffet-bataillon et al. 2011; hegstad et al. 2010; sidhu, heir und sørum 2001; tezel et al. 2011 ) spricht gegen eine unkontrolliert breite anwendung. der zusatz von benzalkonium-, benzethonium-und didecyldimethylammoniumchlorid zu händedesinfektionsmitteln ist als entbehrlich anzusehen. gegen den einsatz in instrumentendesinfektionsmitteln spricht bei gründlicher abschlussreinigung nichts. bei einsatz in flächendesinfektionsmitteln als hauptwirkstoff oder kombinationspartner ist die unverträglichkeit für einige kautschukbeläge zu beachten. bei großflächiger langfristiger anwendung ist ein inhalatives risiko nicht auszuschließen. da der einsatz der hauptvertreter polihexanid, chlorhexidindiglukonat und octenidinhydrochlorid wegen der günstigen nutzen-risiko-relation vor allem in der antiseptik seine berechtigung hat, wird auf charakterisierung in › kap. 2.2 verwiesen. die mathematischen gesetze für die absterbekinetik sind in den meisten bekannten sterilisationsverfahren gleich, sofern die physikalischen und/oder chemischen parameter während der sterilisation konstant bleiben. unter gleichen sterilisationsbedingungen unterscheidet sich allerdings die resistenz der organismen und kann z. b. durch unterschiedliche kultivierungs-und sporulierungsmethoden um den faktor 10 differieren. unter der bedingung, dass es sich um identische mikroorganismen einer charge handelt und der sterilisationsprozess unter gleichen chemischen und/oder physikalischen bedingungen abläuft, ist die abtötungsgeschwindigkeit i. d. r. nur abhängig von der vorhandenen anzahl von mikroorganismen. das gilt zumindest in den bekannten heißluft-, dampf-, formaldehyd-und eo-sterilisationsprozessen und unter vorbehalt auch für wasserstoffperoxid(wpo)-verfahren. definition der reaktion 1. ordnung: die geschwindigkeit der abtötung wird durch den in gleichung (1) stellt man gleichung 1 um, integriert und wandelt man den natürlichen logarithmus in den dekadischen um, ergibt sich mit der neuen reaktionsgeschwindigkeitskonstanten k: (2) lg(n 0 /n f ) = k x t = if werden die sterilisationsbedingungen nach der o. g. definition so ausgelegt, dass von 1 mio. teilen maximal 1 teil mit einem erreger belastet ist, werden diese produkte in europa als "steril" bezeichnet. der direkte biologische nachweis für diesen wert ist experimentell nicht zu erbringen, er kann nur durch extrapolation der überlebenskurve ermittelt werden. die folgende weitergehende betrachtung erfolgt am beispiel der dampfsterilisation. temperaturabhängigkeit des sterilisationsprozesses: die temperaturabhängigkeit ändert den d-wert und wird durch den z-wert beschrieben. er beschreibt, wie sich die abtötungsgeschwindigkeit der mikroorganismen mit der temperatur verändert. mathematisch ist der z-wert die temperaturdifferenz, die zur änderung des d-werts um den faktor 10 unter sonst gleichen sterilisationsbedingungen führt. werden d-werte bei verschiedenen temperaturen bestimmt und in einer halblogarithmischen skala gegen die temperatur aufgetragen, ergibt sich eine gerade, aus der der z-wert abgelesen werden kann (› abb. 2.7). mithilfe des z-werts lassen sich die d-werte bei unbekannten temperaturen wie folgt berechnen: 1/z = (lgd t1 -lgd t2 )/(t 1 -t 2 ) der f 0 -wert wird bei einer sterilisationstemperatur von 121 °c und einem z-wert von 10 °c definiert und wird in der industrie für viele prozesse als referenz angegeben. weitere f-werte können definiert werden, müssen dann jedoch den zusatz der bezugstemperatur und des z-werts tragen. neuerdings wird im metrischen system der f c -wert bei 120 °c und z = 10 °c angegeben. es werden strahlen-, chemische und thermische sterilisationsverfahren unterschieden (› tab. 2.22) . strahlensterilisationsprozesse (› kap. 2.8.9 ) werden im wesentlichen in der industrie eingesetzt. ihr einsatzbereich ist dort begrenzt, wo energiereiche β-oder γ-strahlen materialeigenschaften verändern. die verpackungen können absolut erregerdicht sein, da kein gasaustausch mit dem innern der sterilisierverpackung notwendig ist. chemische sterilisationsverfahren kommen für temperatursensitive produkte zur anwendung. die industrie verwendet am häufigsten eo (› kap. 2.8.6 ), weil es nicht nur über öffnungen in das innere von hohlkörpern eindringt, sondern sich in vielen (nicht in allen) kunststoffen löst und wände direkt durchdringen kann. nachteilig für die eo-verfahren im gesundheitswesen ist, dass die desorptionszeit bis zur gefahrlosen anwendung zu lang sein kann, wenn die instrumente kurzfristig wiederverwendet werden müssen. daher wird in den letzten jahren für niedertemperatur-steriliinstrumente und andere schwere güter, von denen kondensat abtropfen kann, sind auf den unteren beladeebenen des sterilisators oder beschickungswagens anzuordnen. im besonderen maß gilt das für instrumentencontainer, die so gebaut sind, dass sie viel kondensat abgeben. für gut in "weichen" verpackungen gilt: • es muss in sterilisierkörben sterilisiert, transportiert und gelagert werden. • bei klarsichtverpackungen darf die folienseite nicht nach unten weisen. • es muss so im sterilisator angeordnet sein, dass der dampfzutritt nicht behindert ist, auch nicht zu anderen gütern. • es soll bei der sterilisation nicht auf anderem gut liegen, hingegen können bis zu drei geeignete container mit einem zwischenraum übereinandergestapelt werden. die prozessvariablen müssen grundsätzlich aufgezeichnet werden. in der praxis wird im allgemeinen ein automatisch arbeitendes ge-rät zur registrierung der prozessvariablen (drücke, temperaturen, zeiten) verwendet, der verantwortliche bediener kontrolliert die aufzeichnungen und bestätigt bei der freigabe, dass der prozess korrekt abgelaufen ist. die aufzeichnung darf grafisch oder alphanumerisch erfolgen, sie muss sicherstellen, dass werte außerhalb der zulässigen grenzen identifiziert werden können. rubner (1907) die überhitzungen sind insofern prozessrelevant, als sie bei der validierung gemessene temperaturen in diesem bereich bei der bewertung außerhalb des zulässigen temperaturbandes bringen können. deshalb müssen nicht nur die möglichkeit zur überhitzung trockener, textiler güter bekannt sein, sondern auch die möglichkeiten zum gegensteuern. schwieriger ist die frage zu beantworten, inwieweit die gemessenen überhitzungen zur verminderten abtötung führen. die arbeiten von spicher, peters und borchers (1993 wirkprinzip: die mikrobiozide wirkung von fa beruht auf der reaktion mit aminogruppen in eiweißmolekülen und aminosäuren (kirchhoff 1974 rubner berichtete 1906 über die verbesserung der mikrobioziden wirkung von fa in wasserdampf bei temperaturen < 100 °c. in den 60er-jahren des 20. jahrhunderts wurden in großbritannien erstmals verfahren zur anwendung eines wasserdampf-fa-gemischs als "low-temperature-steam with formaldehyd" beschrieben (adam 1974; alder, brown und gillespie 1966; alder, gingell und mitchell 1971) . in deutschland führten marcy (1974) , in skandinavien handlos (1977a handlos ( , 1977b handlos ( , 1979 und nyström (1983) untersuchungen zur sterilisierenden wirksamkeit des fa-wasserdampf-gemischs durch. durch mecke (1979) wurde in deutschland ein verfahren, das im gegensatz zu bisherigen verfahrenstemperaturen zwischen 76 und 80 °c bei 60 °c arbeitete, beschrieben. grundlagenuntersuchungen an den in deutschland entwickelten sterilisatoren wurden in den 1980er-jahren von spicher und borchers (1984 , 1988 für die praxis ist zu fordern, dass die für das verfahren zulässigen konfigurationen der mp definiert und bei zu erwartenden diffusionsverzögerungen vor allem in hohlkörpern die grenzen durch den hersteller genau benannt werden. der jeweilige mp-hersteller muss ebenfalls angeben, ob und unter welchen voraussetzungen sein produkt mit diesem verfahren sterilisiert werden kann. zur weiteren verfahrensoptimierung sollte die penetration des sterilisiermittels an alle inneren und äußeren oberflächen verbessert werden, da derzeit der einsatz der geräte für das gesundheitswesen noch lücken offenbart. als verpackungsmaterial können nur nicht metallhaltige, hydrophobe materialien verwendet werden. normalerweise wird tyvek-folie bzw. polyprolylen-vlies verwendet. es dürfen keine zellulosehaltigen verpackungen eingesetzt werden. in tyvek-folie verpackte mp werden in offenen kunststoffkästen in die kammer eingebracht. die anforderungen an anordnung und beladungsdichte in siebkorb und kammer entsprechen im wesentlichen denen anderer sterilisationsverfahren. zusätzliche hinweise sind den herstellerangaben zu entnehmen. da die prozessführung automatisch abläuft und redundant überwacht wird, muss davon ausgegangen werden, dass die sterilisation bei ablauf des sterilisationsprozesses ohne störung ordnungsgemäß erfolgt ist. danach kann das sterilgut freigegeben werden. der geräteausdruck ist der dokumentation beizufügen. bezüglich der validierbarkeit der im gesundheitswesen eingesetzten geräte ist festzustellen, dass die hersteller der sterilisatoren fir-meneigene mikrobiologische validierungen in verbindung mit der testung von physikalischen rahmenbedingungen anbieten. ein direkter nachweis am jeweiligen konkreten mp unter praxisbedingungen (performance qualification) erfolgt nicht, d. h., der nachweis, dass vor allem an den inneren oberflächen der mp in jedem fall die physikalischen bedingungen für die aufrechterhaltung der gasphase des sterilisiermittels eingehalten werden, ist derzeit nicht gegeben. im industriellen bereich müssen entsprechende validierungen vergleichsweise am realen mp erfolgen. die vorteile der h 2 o 2 -gas-sterilisation, wie geschwindigkeit, rückstandsarmut, einfachheit für den anwender, können zurzeit im medizinischen alltag nur eingeschränkt genutzt werden. eine verbesserung des penetrationsverhaltens des sterilisiermittels und der penetrationsbedingungen im sterilisationsprozess würden eine überschreitung der o. a. grenzen des verfahrens möglich machen. durch veränderte prozessführung, geeignete, vereinbarte anweisungen und technische hilfsmittel (insbes. solche zur rückstandsfreien vorreinigung der güter und zur verfahrensvalidierung) dürfte es möglich werden, das verfahren weiterzuentwickeln. während bei dampfsterilisationsverfahren den mikroorganismen durch kondensation feuchte zugeführt wird, trocknet man bei der sterilisation mit trockener hitze die mikroorganismen während der erwärmung aus und zerstört die strukturen. dabei ist es besonders wichtig, dass die sterilisiertemperatur während der einwirkzeit an allen stellen im gut gegeben sein muss (› abb. 2.15 eine weitere möglichkeit zur erregerdichten verpackung von mp sind wärmebeständige folienverpackungen. hier ist darauf zu achten, dass das jeweilige folienschweißgerät an das temperaturniveau angepasst ist, da die siegelnähte bei einer temperatur oberhalb der heißluftsterilisationstemperatur verschweißt werden müssen. beladeschema: die kammer ist so zu beladen, dass die luft ungehindert zwischen allen oberflächen der packstücke zirkulieren kann (› abb. 2.17) . der nutzraum darf nicht so überfrachtet werden, dass die zirkulation behindert wird. jedes einzelne teil muss derart eingelegt werden, dass es von allen seiten von heißluft umströmt wird. bei sterilisatoren mit zwangskonvektion ist die richtung des luftstroms zu berücksichtigen. größere gegenstände können einen windschatten verursachen, in dem die erwärmung beträchtlich verzögert werden kann. ein nicht selten zu beobachtender fehler ist die zusammenstellung einzelner objekte zu blöcken. weiterhin ist sicherzustellen, dass die zu sterilisierenden objekte wie bei jedem anderen sterilisationsprozess trocken beladen werden. bei nassen gegenständen wird ein teil der wärme zur verdunstung der feuchtigkeit verbraucht und deren erwärmung dadurch verzögert. die freigabe des sterilguts obliegt der dafür ausgebildeten und benannten person. die freigabekriterien einer erfolgreichen sterilisation werden in den bedienungsanleitungen der geräte benannt. entscheidend sind die stabile einhaltung der prozessabläufe und deren dokumentation. die jeweiligen chargen sind zu bezeichnen und zu dokumentieren. wirkprinzip: in gammaanlagen wird als strahlenquelle fast ausschließlich co-60, sehr selten cs-137 eingesetzt. elektronenbeschleuniger (β-strahler) nutzen beschleunigte elektronen bis zu einer energie von 10 mev. in röntgenanlagen wird ein target mit beschleunigten elektronen bestrahlt und die sekundärstrahlung, die röntgenstrahlen (x-rays), genutzt. die ionisierenden strahlen bewirken bei ausreichend hoher energie ionisierungen, die in der reihenfolge abnehmender empfindlichkeit bakterien, pilze, bakteriensporen und viren abtöten. durch einsatz der strahlen werden zuerst zellteilungsvorgänge beeinträchtigt, danach folgt die verlangsamung des wachstums, ehe es nach ausschaltung der atmung und fermentation zum zelltod kommt. in der regel wird die strahlensterilisation wegen der hohen investitionen, des strahlenschutzes und der hohen kapazität einer anlage nur industriell genutzt und durch serviceunternehmen angeboten. die anlagen unterliegen den bedingungen der strahlenschutzverordnung. verpackung: das sterilisiergut wird in der transport-bzw. endverpackung, z. b. in exotoxine sind gewebeschädigende proteine. sie werden von bakterien, z. b. clostridien und bazillen, aktiv in die umgebung abgegeben, sind durch erhitzung inaktivierbar und lösen typische erscheinungen aus. exotoxine können innerhalb des infizierten wirtsorganismus gebildet werden (z. b. diphtherie-, scharlachtoxin) oder nach bildung außerhalb des körpers z. b. durch nahrungsverzehr aufgenommen werden (z. b. botulinustoxin). letzteres wird seit über 10 jahren bei schweren neurologischen leiden und neuerdings als anti-aging-mittel zur glättung faltiger haut eingesetzt (hacker 2003) . zu den exotoxinen gehören auch die superantigene von strepto-und staphylokokken. diese vernetzen und stimulieren spezifisch makrophagen und cd4-t-lymphozyten, sodass große mengen botenstoffe gebildet werden und eine ähnliche wirkung eintritt wie bei endotoxinen. ein beispiel ist das sog. toxic-shock-toxin. endotoxine sind lipopolysaccharide (lps) der äußeren membran gramnegativer bakterien. sie werden vor allem beim absterben (lyse), aber auch bei der zellteilung vitaler bakterien freigesetzt (rietschel und brade 1993) . ihre wesentliche toxische komponente, das lipid a, ist hitzestabil. die wirkung ist prinzipiell unabhängig von der bakteriellen herkunft, die wirkungsbreite sehr unterschiedlich. endotoxine können als konstanter stimulus den tonus körpereigener immunabwehr aufrechterhalten (leinmüller 2004 es gibt hinweise, dass der regelmäßigen sichtkontrolle und reinigung/desinfektion des wassertanks von kleinsterilisatoren mehr aufmerksamkeit zu schenken ist. strobel (2002) beschreibt postoperativ eine reizung der augenvorderkammer, die durch endotoxineinbringung, ausgehend vom kontaminierten speisewasserbehälter des dampfkleinsterilisators, verursacht sein könnte. dazu bestimmten martin und daily (2001) die erreger-und endotoxingehalte im wasserreservoir eines dampfkleinsterilisators. whitby und hitchins (2002) die ausbildung muss sicherstellen, dass das personal der zsva die mit der aufbereitung eines mp anfallenden arbeiten selbstständig ausführen kann. weiterbildung: die schnellen veränderungen im gesundheitswesen, neue verpackungssysteme und schwieriger aufzubereitende instrumente erfordern die ständige weiterbildung. in allen abteilungen, in denen aufbereitet wird, muss jährlich eine dokumentierte einweisung des personals in den betrieb von sterilisationsgeräten und rdg erfolgen. zur gewährleistung der qualifikation im niedergelassenen bereich ist der erwerb der sachkunde für die instandhaltung von mp in der ärztlichen praxis 2003 als gemeinsame initiative von dgsv, dgkh und dem berufsverband der deutschen hygieniker eingeführt worden. marc thanheiser und martin mielke mit krankheitserregern kontaminierte mp wie z. b. chirurgische instrumente können bei erneuter anwendung zu infektionen führen. auch darf von mp bei der anwendung keine gefahr von gesundheitsschäden durch pyrogenbedingte, allergische oder toxische reaktionen sowie aufgrund veränderter technisch-funktioneller 2 eigenschaften des mp ausgehen. aus diesen gründen müssen mp entsprechend der art der vorherigen und folgenden anwendung sowie der konstruktiven und materialtechnischen eigenschaften vor einer erneuten anwendung aufbereitet werden. die ordnungsgemäße aufbereitung von mp ist in § 4 der mpbetreibv (medizinprodukte-betreiberverordnung, 2009) auch in einer rechtsnorm angesprochen. dort wird unter anderem aufgeführt, dass die aufbereitung von bestimmungsgemäß keimarm oder steril zur anwendung kommenden mp unter berücksichtigung der angaben des herstellers mit geeigneten validierten verfahren so durchzuführen ist, dass der erfolg dieser verfahren nachvollziehbar gewährleistet und die sicherheit und gesundheit von patienten, anwendern oder dritten nicht gefährdet wird. eine ordnungsgemäße aufbereitung wird vermutet, wenn die gemeinsame empfehlung der krinko am rki und des bfarm zu den "anforderungen an die hygiene bei der aufbereitung von medizinprodukten" (krinko und bfarm 2012a) beachtet wird. die in § 4 mpbetreibv (medizinprodukte-betreiberverordnung, 2009) genannte empfehlung, im folgenden als krinko-bfarm-empfehlung bezeichnet, dient als fachliche basis für die nachfolgenden ausführungen. auf den amtlichen originaltext der empfehlung wird ausdrücklich hingewiesen (krinko 2012a). für die korrekte aufbereitung von mp ist der betreiber verantwortlich. basierend auf einer risikobewertung und einstufung (› kap. 2.9.5 ), hat der für die aufbereitung verantwortliche unter berücksichtigung der angaben des herstellers schriftlich festzulegen, mit welchen verfahren (in allen einzelschritten) und unter welchen bedingungen (z. b. räume, arbeitsmittel, qualifikation des personals) seine mp aufbereitet und gelagert werden. die aufbereitung und die stete erfüllung der anforderungen setzt ein qm-system voraus, und es sind vor der aufbereitung von mp die zuständigkeiten für alle schritte der aufbereitung zu regeln und zu dokumentieren sowie die einzelschritte der aufbereitung unter angabe der jeweilig notwendigen prüfungen in standardarbeitsund betriebsanweisungen festzulegen. dabei ist zu beachten, dass der für die verschiedenen prozessschritte jeweils zuständige seine aufgabe aufgrund seiner position und qualifikation (aus-, weiterund fortbildung) auch tatsächlich erfüllen kann (s. hierzu auch die anlage "sachkenntnis des personals" der krinko-bfarm-empfehlung; rki 2012). voraussetzung für die aufbereitung ist, dass die eignung der zur anwendung kommenden aufbereitungsverfahren und die wirksamkeit im rahmen einer produkt-/produktgruppenspezifischen prüfung und validierung belegt wurden (din en iso 17664:2004-07 es ist zweckmäßig, bereits vor der anschaffung eines mp durchführbarkeit und aufwand der aufbereitung zu überdenken und die anwender sowie die für die aufbereitung und für die hygiene zuständigen in die entscheidung über die beschaffung des mp sowie die erforderlichen mittel und geräte für die aufbereitung einzubeziehen. hinsichtlich der art der anwendung und des sich daraus ableitenden risikos können mp eingestuft werden in: (bertram et al., 2004) . einige dieser formulierungen haben auch bakterizide und viruzide eigenschaften (beekes et al., 2010) . von den zur verfügung stehenden sterilisationsverfahren wurde bisher nur für die dampfsterilisation (insbesondere 134 °c, 5-18 min) und für bestimmte wasserstoffperoxid-basierte verfahren eine relevante wirkung auf prionen nachgewiesen (rogez-kreuz et al. 2009 freigabe zur anwendung: die aufbereitung von mp endet mit der freigabe zur anwendung. diese erfolgt auf der basis der übereinstimmung der bei der aufbereitung jeweils ermittelten prozessparameter mit denen der validierungsprotokolle und schließt die durchführung sowie die dokumentation der täglichen routineprüfungen, die überprüfung und dokumentation des vollständigen, korrekten prozessverlaufs (chargenbezogene routineprüfungen und chargendokumentation), die überprüfung der verpackung auf unversehrtheit und trockenheit sowie die überprüfung der kennzeichnung ein. die die aufbereitung beschreibenden sops müssen auch die art und dokumentation der freigabeentscheidung und das vorgehen bei abweichungen vom korrekten prozessablauf enthalten. dokumentation: die im rahmen der aufbereitung erfassten messwerte der prozessparameter und die freigabeentscheidung sind mit bezug auf die freigebende person und die charge zu dokumentieren und mindestens 5 jahre aufzubewahren. sonstige rechtsvorschriften zu aufbewahrungsfristen (z. b. patientendokumentation) bleiben hiervon unberührt. dabei darf weder der ursprüngliche inhalt einer eintragung unkenntlich gemacht werden, noch dürfen änderungen vorgenommen werden, die nicht erkennen lassen, ob sie während oder nach der ursprünglichen eintragung vorgenommen worden sind. die aufzeichnungen und nachweise sind den zuständigen behörden auf verlangen vorzulegen. transport und lagerung: transport und lagerung dürfen die eigenschaften des aufbereiteten mp nicht nachteilig beeinflussen. bei der lagerung von aufbereiteten mp sind die angaben des herstellers des mp und des verpackungsmaterials zu berücksichtigen. die lagerdauer ist abhängig von der qualität des verpackungsmaterials, der dichtigkeit der siegelnähte und den lagerbedingungen. davon abhängig sind auch lagerfristen von über sechs monaten denkbar. keimarme (semikritische) mp müssen so gelagert werden, dass eine rekontamination während der lagerung vermieden wird. 2.9.7 validierung: beleg der reinigungs-, desinfektions-und sterilisationsleistung weder das mit der desinfektion angestrebte ziel der "keimarmut" noch das mit der sterilisation verfolgte ziel der "sterilität" sind an dem aufbereiteten mp unmittelbar erkennbar. auch das ziel "sauberkeit" des reinigungsprozesses ist bei mp mit nicht direkt einsehbaren, z. b. inneren oberflächen, nicht direkt beurteilbar. bei desinfektion und sterilisation, und teilweise bei der reinigung handelt es sich um prozesse, deren effektivität nur durch anwendung validierter verfahren und durch überwachung von relevanten prozessparametern, die im rahmen der validierung definiert werden, belegt werden kann. die validierung soll dem mp und seiner risikobewertung und einstufung angemessen sein. die produktspezifische validierung von aufbereitungsprozessen wird in der regel vom hersteller durchgeführt (din en iso 17664:2004-07) . soweit keine einheitlichen produktchargen gebildet werden können, müssen die dokumentierten prüfungen im rahmen der validierung an produkttypen bzw. prüfmodellen erfolgen, die nachweislich repräsentativ für alle wesentlichen merkmale der zu bildenden gruppe von mp anzusehen sind. die validierung führt zu einem dokument, aus dem hervorgeht, auf welche weise ein zuvor definierter zustand (z. b. sterilität) reproduzierbar erbracht wird. dieses dokument enthält auch angaben darüber, welche daten für die überwachung des prozesses erforderlich sind und wie diese daten zu interpretieren sind. die qualität der maschinellen aufbereitung wird abhängig vom jeweiligen verfahren der reinigung, desinfektion und sterilisation bei reinigungs und desinfektionsverfahren sind speziell maschinelle verfahren validierbar (din en iso 15883) . überwachungs-, kontroll-und warnsysteme der maschinen stellen die voraussetzungen für eine gesicherte reinigung und desinfektion und damit aufbereitung dar. manuelle reinigungs-und desinfektionsverfahren sind schwieriger zu standardisieren und daher weniger zuverlässig reproduzierbar. sofern sie eingesetzt werden, müssen sie stets nach sops und mit auf wirksamkeit geprüften und materialverträglichen mitteln und verfahren durchgeführt werden. auch diese verfahren bedürfen einer validierung und periodischer prüfungen. sterilisationsverfahren sind unter der voraussetzung ihrer anwendung bei rückstandsfrei gereinigten mp vollständig validierbar. marianne abele-horn (mitherausgeberin für dieses kapitel) hintergrund die wirksamkeit von antibiotika ist gefährdet, da resistenzen gegen antibiotika weltweit zunehmen. dies stellt ein ernst zu nehmendes problem für die gesundheit der bevölkerung dar. daher hat die who die antibiotikaresistenz als global threat eingestuft; sie wird auf eine stufe gestellt mit anderen problemen wie umweltverschmutzung oder globaler erwärmung. neue, vielversprechende wirkstoffe sind nicht in aussicht, was ein problem für die therapierbarkeit von bakteriellen infektionen in der zukunft aufwirft. die entwicklung und verbreitung von antibiotikaresistenten erregern wird von einer vielzahl von faktoren beeinflusst. neben hygienemaßnahmen spielt der umsichtige einsatz von antibiotika eine wesentliche rolle. › abb. 2.18 stellt den verbrauch von antibiotika in der humanmedizin in deutschland dar. der verbrauch in der veterinärmedizin in deutschland liegt mit 1 600 tonnen noch höher (stand 2012). die zunahme von antibiotikaresistenzen ist eng gekoppelt an die art und quantität des antibiotikaeinsatzes sowohl in der humanmedizin als auch in tierhaltung und landwirtschaft. so können unter dem durch die antibiotikatherapie entstehenden selektionsdruck sowohl resistente erreger (gegen das verwendete antibiotikum) selektioniert werden als auch resistente mutanten des zu behandelnden erregers einen überlebensvorteil erlangen und sich ungehindert vermehren. vielfach kommt es in abhängigkeit von bakterienspezifischen faktoren und äußeren umständen (z. b. inadäquaten hygienemaßnahmen, horizontalem gentransfer) zur weiterverbreitung in die umgebung (mensch, tier, umwelt wie z. b. wasser). durch die globalisierung insbesondere im hinblick auf den warenverkehr und die mobilität von personen können auch zunächst lokal auftretende resistenzen in kurzer zeit weit verbreitet werden. ein beispiel sind die "neu-dehli-metallo-betalaktamase 1 (ndm1)"-tragenden erreger, die vom indischen subkontinent in zahlreiche länder eingetragen wurden. ndm-1 resistenzgene -um bei diesem beispiel zu bleiben -sind auf mobilen plasmiden lokalisiert, die zwischen unterschiedlichen gramnegativen spezies übertragen werden können (e. coli, klebsiellen, serratien, acinetobacter spp. usw.). die antibiotikaresistenz ist somit ein globales problem, das die ökologische gesamtsituation beeinflusst. es gibt einen zusammenhang zwischen der menge an antibiotikaverordnungen und der resistenzenzwicklung. dieser ist unterschiedlich schnell und nicht unbedingt in linearer dosis-wirkungsbeziehung. daraus folgt, dass eine antibiotikaresistenz oft nicht und vor allem nicht unmittelbar reversibel ist. trotzdem kann ein sorgsamer umgang mit antibiotika den selektionsdruck reduzieren und die resistenzsituation positiv beeinflussen. antibiotic stewardship zielt auf eine verbesserte qualität der antibiotikatherapie: sie soll für den einzelnen patienten bei minimaler toxizität und resistenzentwicklung das bestmögliche klinische behandlungsergebnis erreichen. eine gute antibiotikaverordnungspraxis umfasst z. b. • den einsatz von antibiotika nur dort, wo sie therapeutisch oder prophylaktisch indiziert sind, • die optimierung der antibiotikaregime hinsichtlich der auswahl des antibiotikums, der applikationsart, der dosierung und der dauer der therapie bzw. der prophylaxe. dadurch soll der individuelle nutzen für den patienten verbessert und der selektionsdruck auf die bakterienpopulationen und die kosten für das gesundheitssystem minimiert werden. beispiele für einen nicht rationalen einsatz von antibiotika zeigt› tab. 2.26. unter dem begriff "antibiotic stewardship" (abs) werden alle maßnahmen zusammengefasst, die einer verbesserung der antibiotikaverordnungspraxis sowohl in der stationären wie auch in der ambulanten patientenversorgung dienen (deutsche gesellschaft für infektiologie 2013). antibiotic stewardship erfordert eine systematische herangehensweise, in der verschiedene aktivitäten und maßnahmen in sinnvoller weise miteinander koordiniert werden. mindestens 300 tonnen antibiotika in der humanmedizin in deutschland verbraucht (2007 in der s3-leitlinie "strategien zur sicherung rationaler antibiotikaanwendungen im krankenhaus" werden die wesentlichen eckpunkte von abs bzw. abs-programmen beschrieben (deutsche gesellschaft für infektiologie 2013) . sie umfassen die schaffung und aufrechterhaltung von organisatorischen und strukturellen vor-aussetzungen. unabdingbar dafür ist die finanzielle und personelle unterstützung der klinikleitung für die etablierung eines multidisziplinären abs-teams (› abb. 2.19) . die leitlinie fordert als notwendige personalressource für ein abs team mindestens 0,5 vollzeitstellen pro 250 betten. daten zu antibiotikaverbrauch, infektionenserregern und resistenz sollen mindestens einmal jährlich für das gesamte krankenhaus und aufgeschlüsselt für einzelne fachabteilungen verfügbar sein (vor allem für abteilungen mit hohem verbrauch, z. b. intensivstationen) (› abb. 2.20) . ohne messung der antibiotikaverordnungsdichte ist eine nachhaltige umsetzung intelligenter verordnungsstrategien nicht möglich. in der humanmedizin hat sich für erwachsene patienten als methode der verbrauchsmessung die anzahl der tagesdosen (defined daily dose, ddd nach who atc) pro einwohner oder versicherter für den ambulanten bereich und pro patiententage im stationären bereich durchgesetzt (schweickert et al. 2013 das abs-team erstellt nach aktuellem stand des wissens unter bezugnahme auf vorhandene empfehlungen von fachgesellschaften klinikinterne leitlinien. diese leitlinien zu therapie und prophylaxe müssen regelmäßig aktualisiert werden und sind ein wichtiger bestandteil der kernstrategien jedes abs-programms (› tab. 2.27 die meisten studien zur effektivität der abs belegen eine reduktion von antiinfektivaverordnungen um 10-40 %, eine verkürzte therapiedauern und eine signifikante kostenreduktion trotz initial erforderlicher investitionen. in › tab. 2.29 ist die evidenz von interventionen zusammengefasst (davey et al. 2013) . diese cochrane analyse zeigt auch, dass gezielte abs-interventionen bezüglich mikrobiologischer endpunkte (z. b. anteil von erregern mit speziellen resistenzen und multiresistenzen) meist erst mit einer verzögerung von mindestens 6 monaten effektiv sind, während änderungen in bezug auf den antibiotikaverbrauch oft bereits nach 1 monat erreicht werden. ganz entscheidend zur eindämmung von mre und c. difficile ist die enge strukturell-organisatorische zusammenarbeit von klinischen infektiologen, mikrobiologen und krankenhaushygienikern/ hygienefachpersonal. abs-programme sind nur dann effektiv, wenn sie die verordnungspraxis systematisch und kontinuierlich verbessern. wenn sie zeitlich begrenzt sind und es dem abs-team nicht gelingt, die behandelnden ärzte von der notwendigkeit entsprechender interventionen zu überzeugen, besteht die gefahr, dass die erreichten verbesserungen in der antibiotikaverordnung ohne nachhaltige wirkung bleiben (gerber et al. 2013 (gerber et al. und 2014 szymczk et al. 2014 ). das unterstreicht die eindeutige empfehlung der s3-leitlinie, kontinuierlich ressourcen in form von facharztstellen, arbeitszeit und weiterbildung für ein erfolgreiches abs-programm zur verfügung zu stellen. in deutschland werden jährlich in krankenhäusern etwa 6,4 mio. operationen mit einer durchschnittlichen postoperativen wundinfektionsrate von 2 % durchgeführt (gastmeier et al. 2004 ; siehe auch deutsche fassung der literaturstelle von gastmeier und v. a. bqs portal: hier werden die daten von unseren kollegen in deutschland unter cdc a1-a3 den ssi-definitionen nach dokumentiert). uneinheitlich verwendete definitionen der ssi (barie 2002) und verkürzung der verweildauer erschweren die exakte erfassung, sodass vermutlich mit einer höheren ssi-rate gerechnet werden muss. sie kann nach aseptischen eingriffen bis zu 5 % und nach intraabdominellen eingriffen bis zu 40 % betragen (bratzler und houck 2004; rovera et al. 2005 ). die wundinfektionsrate (ssi-rate, entsprechend der anglo-amerikanischen literatur ssi = surgical site infection) erfasst alle infektionen nach chirurgischen eingriffen (oberflächliche und tiefe infektionen sowie infektionen von organen und körperhöhlen). empfehlungen und leitlinien sowie die einführung von kontrollsystemen können die prophylaxe verbessern, sie aber nicht flächendeckend etablieren (bratzler et al. 2005; forbes et al. 2008; kritchevsky et al. 2008; pan et al. 2009; papaioannidou et al. 2008; rüden et al. 1997; warters et al. 2006 ). vorschläge zur verbesserungen beinhalten z. b. checklisten im rahmen von anästhesieprotokollen und die lückenlose dokumentation der maßnahmen zur perioperativen prophylaxe (fry 2008; haynes et al. 2009; rosenberg et al. 2008; wax et al. 2007; willemsen et al. 2007 ). selbst bei leitlinienkonformer pap werden jedoch nicht in allen fällen ausreichende wirkspiegel erreicht (caffarelli et al. 2006; dalley et al. 2007; koopman et al. 2007 (bratzler et al. 2013 ; sign 2014). • ziel der pap ist das vermeiden postoperativer wundinfektionen und nicht anderer ni (z. b. gefäßkatheter-assoziierte blutstrominfektion, beatmungsassoziierte pneumonie, harnwegskatheter-assoziierte hwi). bei der indikationsstellung sind neben dem grad der bakteriellen besiedlung und der wundklassifikation (cruse und foord 1980; › tab. 2.30 ) je nach wundkategorie ein individuelles infektionsrisiko und patienteneigene sowie op-bedingte besonderheiten zu berücksichtigen. daher können empfehlungen nicht nur auf der basis evidenzbasierter klinischer studien und metaanalysen erarbeitet werden, sondern müssen auch gut ausgewiesene experimentelle und klinische studien, die nachweislich wundunabhängige risikofaktoren für eine ssi oder infektiöse komplikationen (z. b. pneumonie, harnweginfektion, sepsis) anderer art beinhalten, berücksichtigt werden. bei besonders schwerwiegenden infektionskomplikationen (z. b. nach intrakraniellem eingriff) wird die pap unabhängig von risikofaktoren empfohlen. unabhängig von der art des eingriffs wurden risikofaktoren aus unterschiedlichsten patientenkollektiven und studien zusammengetragen und konnten in einzelnen untersuchungen als statistisch signifikante faktoren ausgewiesen werden. es kann eine einteilung in patienteneigene, prä-, intra-und postoperative risikofaktoren vorgenommen werden (› tab. 2.31) . patienteneigene risikofaktoren sind natürliche, nicht änderbare risiken wie alter oder geschlecht, aber auch nicht korrigierbare defizite bei dringlichen eingriffen wie diabetes mellitus, immunabwehrschwäche, reduzierter allgemeinzustand, übergewicht und mangelernährung. patienten mit karzinombedingter chirurgischer intervention besitzen ein signifikant erhöhtes ssi-risiko und sollten grundsätzlich eine pap erhalten. die auswahl des antibiotikums muss die lokalisation des tumors berücksichtigen. wichtige präoperative risikofaktoren sind ergänzend zu › tab. 2 eine generelle pap bei allen aseptischen eingriffen wird nicht empfohlen. es gibt aber zahlreiche hinweise, dass besonders patienten mit infektionsrisiken bei aseptischen eingriffen von einer pap profitieren. bei aseptischen eingriffen mit fremdkörperimplantation ist die pap etabliert. jede pap birgt das risiko einer resistenzentwicklung und der selektion von erregern mit bereits bestehender unempfindlichkeit gegenüber gebräuchlichen antibiotika (ulger et al. 2005 ). initiale empfehlungen zum applikationszeitpunkt einer wirksamen pap gehen auf tierexperimentelle untersuchungen von burke zurück (burke 1977 die effektive periode, in der die pap ssi signifikant reduziert, ist 1 stunde vor bis 2 stunden nach beginn des eingriffs, spätestens jedoch vor wundverschluss (bates et al. 1989 , classen et al. 1992 , weber et al. 2008 ). im klinischen routineablauf bietet sich bei i. v. verabreichung der zeitpunkt der narkoseeinleitung, also etwa 30-60 minuten vor der inzision an. der späteste noch sinnvolle zeitpunkt für eine antibiotikaprophylaxe ist intraoperativ, z. b. beim auftreten von komplikationen. die ssi-rate nimmt mit jeder stunde nach dem hautschnitt signifikant zu, wenn die antibiotikagabe verzögert wird oder die applikation länger als 1 stunde vor op-beginn erfolgt. eine antibiotikagabe nach wundverschluss hat keinen einfluss auf die ssi-rate. da das optimale zeitfenster auch von patientenabhängigen pharmakokinetischen parametern der eingesetzten substanzen und der art der applikation (bolusgabe, kurz-, dauerinfusion) abhängt, ist bei den heute verwendeten moderneren antibiotika mit kürzeren halbwertszeiten und rascher verteilung in die kompartimente eine zur inzision möglichst zeitnahe verabfolgung wünschenswert (zelenitzky et al . 2000) . der nutzen einer dauerinfusion von betalaktamantibiotika wird diskutiert (waltrip et al. 2002 , suffoletta et al. 2008 ). bei der dosierung sollten erhöhte oder erniedrigte verteilungsräume der patienten berücksichtigt werden. einen hinweis können körpermasse, body mass index, einlagerungen, drainagen u. a. geben. eine standarddosierung kann nur unter idealbedingungen erfolgen. bei heute üblichen substanzen sind häufig höhere dosierungen notwendig (hutschala et al. 2007 ). präoperativ intraoperativ postoperativ • alter (zunahme pro dezennium; lizan-garcia, garcia-caballero und asensio-vegas 1997, zelenitsky et al. 2000) • diabetes mellitus (zelenitsky et al. 2000) • immuninkompetenz • reduzierter allgemeinzustand • übergewicht (lofgren 2005 , itani et al. 2008 (centofanti et al. 2007) • frühe re-op wegen blutungen (centofanti et al. 2007 ) • liquorleck, externer shunt (lietard et al. 2008) 2 bei einer op-dauer < 2 stunden ist die einmalige gabe des antibiotikums für eine effektive prophylaxe ausreichend und der mehrmaligen gabe bei eingriffen unterschiedlicher kategorie (kontaminiert bis aseptisch) nicht unterlegen (carignan 2008; fujita et al. 2007; hellbusch et al. 2008; hutschala et al. 2007; su et al. 2005; suehiro et al. 2008 eine antibiotikagabe darüber hinaus gilt als therapie und nicht als prophylaxe. sie kann notwendig werden, wenn infektionsherde operativ nicht vollständig beseitigt werden konnten (z. b. bei septischer cholangitis, eitriger peritonitis, nach appendix-oder divertikelperforation u. a.) und ein anhaltend hohes infektionsrisiko für den patienten besteht. bei eingriffen an extremitäten in blutleere wurden üblicherweise die antibiotikagabe 10 minuten vor anlegen der blutsperre und eine folgedosis nach eröffnen der blutsperre empfohlen. die auswahl erfolgt vorrangig nach dem erwarteten erregerspektrum, das aus der normalen bzw. pathologischen besiedlung des op-gebiets und seiner unmittelbaren haut-und schleimhautumgebung resultiert. falls möglich, sollte sich die auswahl am ergebnis der mikrobiologischen diagnostik orientieren (zutt et al. 2003) . antibiotika zur pap sollten ihre wirksamkeit in klinischen studien bewiesen haben, nebenwirkungsarm und kostengünstig sein. um das zeitfenster optimal für die prophylaktische wirkung des antibiotikums zu nutzen, müssen sich seine applikationsart und dosis nach seinen eigenschaften richten. es gibt nur wenige klinische studien, die pharmakokinetische daten, applikationszeitpunkt und substanzwahl mit ssi-raten korrelieren. betalaktamantibiotika: die mhk für relevante erreger werden bei parenteraler gabe eines betalaktamantibiotikums im serum und gewebe i. d. r. innerhalb weniger minuten erreicht (wittmann, welter und schassan 1982) . die pharmakokinetik der antibiotika im serum korreliert mit der dauer der wirksamkeit im gewebe (novelli 1999) . pharmakokinetische parameter ändern sich mit der substanz und den organfunktionen des patienten. betalaktamantibiotika mit halbwertszeiten von 1-2 stunden wie cefazolin, cefuroxim oder ampicillin-sulbactam (respektive amoxicillin-clavulansäure) sollten möglichst zeitnah zum eingriff gegeben und intraoperativ nach 2 stunden op-dauer wiederholt werden (colombo et al. 1998 ). der vorteil der betalaktamantibiotika mit langer halbwertszeit (z. b. ceftriaxon) liegt in der einmalgabe auch bei länger dauernden eingriffen. aminoglykoside, falls überhaupt eingesetzt, müssen hoch dosiert (gentamicin 4,5 mg/kg körpergewicht) werden (zelenitsky et al. 2000 (zelenitsky et al. , 2002 , um effektive spiegel auch bei wundverschluss zu erreichen. der stellenwert der aminoglykoside in der pap ist heute gering. primäres ziel der pap ist die senkung der ssi-rate, sekundäres ziel die vermeidung lokaler und systemischer postoperativer infektionskomplikationen. die pap sollte risikoadaptiert und individualisiert erfolgen. eine zu frühe gabe des antibiotikums und auch die gabe nach dem wundverschluss sind nutzlos. die fortführung der pap über die op (in der herzchirurgie maximal über die ersten 24 stunden nach op) hinaus bedarf besonderer indikation und kann eigentlich nicht mehr als pap bezeichnet werden (präventive therapie?). bei der auswahl des antibiotikums sind risikofaktoren auf seiten des patienten und ggf. auch die lokale erreger und resistenzstatistik zu berücksichtigen. ein besonderes augenmerk gilt dabei möglichen sekundären infektionen, die v. a. durch gramnegative erreger verursacht werden. es sollten nur substanzen eingesetzt werden, bei denen entsprechende indikationen nachgewiesen sind. die meisten erfahrungen liegen für den einsatz der β-lactam-antibiotika vor. die auswahl der substanzen orientiert sich in erster linie am erregerspektrum und an der pharmakokinetik. für den individuellen patienten ist das risiko der resistenzentwicklung gering. das gilt jedoch nicht für das gesamtkollektiv einer klinik. ökonomische gesichtspunkte sind wichtig, auch wenn die kosten der pap geringer sind als die kosten postoperativer infektionskomplikationen. spätestens seit semmelweis ist klar, dass postoperative wundinfektionen (ssi) nicht "schicksalhaft" auftreten, sondern zumindest in ¼ der fälle als iatrogene komplikationen angesehen werden müssen, die bei entsprechenden hygienischen maßnahmen auf ein mindestmaß reduziert werden können. die immense sozioökonomische bedeutung der sogenannten "surgical site infections" (ssi) wird anhand bundesweit erhobener epidemiologischer daten deutlich: in der nationalen prävalenzstudie (2011) konnte zwar gezeigt werden, dass in deutschland die rate an nosokomialen infektionen (ni) mit 3,4 % im europäischen vergleich stabil niedrig ist (in vergleichsstudien international zwischen 6,1 % und 9,3 %), letztlich aber doch bei 18 mio. stationär behandelten patienten im jahr dementsprechend 400 000 ni auftreten. da ein viertel der ni wundinfektionen sind, bedeutet das jährlich etwa 100 000 wundinfektionen. diese zahlen werden auch durch auswertung der daten des krankenhaus infektions surveillance systems (kiss) bestätigt. eine postoperative wundinfektion verursacht durchschnittlich 3 000 € mehrkosten und verlängert den krankenhausaufenthalt um 6,5 tage. hochgerechnet bedeutet dies eine belastung für die versicherungssysteme von fast 300 000 000 € mehrkosten und 650 000 zusätzliche krankenhausbehandlungstage pro jahr in deutschland. darüber hinaus gibt es hinweise darauf, dass eine postoperative wundinfektion ein unabhängiger risikofaktor für patienten darstellt, im postoperativen verlauf zu versterben oder zumindest intensivmedizinische behandlung zu benötigen. es ist anerkannt, dass ein teil der ssi durch die strikte einhaltung entsprechender präventivmaßnahmen vermieden werden kann. ein wesentlicher beitrag zur vermeidung postoperativer wundinfektionen kann durch die korrekt durchgeführte perioperative antibiotikaprophylaxe (pap) geleistet werden. es gibt zahlreiche empfehlungen zum einsatz der pap. genannt werden sollen hier stellvertretend die empfehlungen der paul-ehrlich-gesellschaft für chemotherapie von 2010 (wacha 2010) und die krinko-empfehlung zur prävention postoperativer infektionen im operationsgebiet von 2007 (krinko 2007 . darüber hinaus wurde im jahr 2013 vom european centre for disease prevention and control (ecdc) eine evidenzbasierte leitlinie zur optimierung der pap veröffentlicht (bratzler et al. 2013; sign 2014) . ziel der pap ist einzig und allein die vermeidung postoperativer wundinfektionen (ssi). es gibt zwar daten, die darauf hinweisen, dass bei korrekt angewandter pap auch postoperative pneumonie und intraabdominelle infektionen (abszesse) positiv beeinflusst werden. diese daten sind aber nicht ausreichend valide und stammen meist aus der nachträglichen auswertungen von studien, deren primärer endpunkt das auftreten von wundinfektionen war. hervorzuheben ist, dass die pap weitere hygienische maßnahmen (krinko 2007) nicht ersetzen kann (also kein ausgleich für unzureichende hygiene ist), sondern lediglich einen mosaikstein im gesamtkonzept aller maßnahmen zur vermeidung postoperativer wundinfektionen darstellt. die indikation zur prophylaxe ergibt sich aus der wundklassifikation nach cruse, (› tab. 2.32 ) und zusätzlichen risikofaktoren (› tab. 2.33 ). demnach ist bei sauberen eingriffen eine prophylaxe nur dann indiziert, wenn ein zusätzlicher risikofaktor vorliegt (› tab. 2.33) . bei sauber-kontaminierten oder kontaminierten eingriffen besteht in jedem fall eine indikation für die pap, bei schmutzigen eingriffen ist eine einmalige prophylaxe nicht ausreichend, hier sollte eine antibiotikatherapie durchgeführt werden. da in einzelfällen die indikation derzeit noch nicht geklärt ist und auch regionale faktoren eine rolle spielen, wird empfohlen, dass ein interdisziplinäres team in der jeweiligen klinik/abteilung die indikationsgruppen für die pap festlegt und jährlich überprüft. dabei sind wundinfektionsraten und das lokale erreger-und resistenzspektrum bei den ssi zu berücksichtigen. das therapeutische fenster, in dem die prophylaxe sinnvoll ist, reicht von 1 stunde vor bis 2 h nach hautschnitt. der ideale zeitpunkt liegt bei 30-60 min vor beginn der operation und sollte demnach am besten im rahmen der narkoseeinleitung durch den anästhesisten erfolgen. die gabe wird durch die abfrage der inzwischen nahezu flächendeckend etablierten op-checklisten im sogenannten ("team time out") überprüft. ein sonderfall ergibt sich beim (selten erforderlichen) gezielten einsatz von glykopeptiden (vancomycin oder teicoplanin) zur pap bei patienten, die mit mrsa kolonisiert sind (bratzler 2013) . vancomycin soll zur besseren verträglichkeit über mindestens eine stunde verabreicht werden. demnach muss mit der präoperativen infusion bereits 2 h vor der op begonnen werden. wegen des schmalen wirkspektrums und der schlechteren wirksamkeit gegen über methicillin-sensiblen s. aureus sollten die glykopeptide nur zusätzlich zur standard-pap gegeben werden (bull, worth, richards 2012; cranny et al 2008 erstaunlicherweise zeigte die prävalenzstudie von 2011, dass die antibiotikaprophylaxe in über 60 % der fälle über den ersten tag hinaus angewandt wurde. in zusammenhang mit einem signifikanten anstieg der c.-difficile-assoziierten erkrankungen (cdad) und zunehmenden resistenzen insbesondere bei den gramnegativen infektionserregern ist eine solche quote inakzeptabel. hochgerechnet könnten in deutschland allein 5 tonnen antibiotika pro jahr eingespart werden, wenn die postoperative "verlängerung" der prophylaxe ausbliebe. die auswahl des antibiotikums richtet sich nach dem erwarteten erregerspektrum. insbesondere muss unterschieden werden, ob eher eine infektion durch hautflora am wahrscheinlichsten ist (v. a. staphylokokken, z. b. bei implantaten in der traumatologie/orthopädie) oder infektionen durch enterobacteriaceae auftreten können (z. b. elektive colonchirurgie). im letzteren fall sollte das zur pap verwendete antibiotikum auch gegen anaerobier wirksam sein. wenn diese durch das eigentliche antibiotikum der wahl nicht erfasst sind (z. b. bei den cephalosporinen), kann diese lücke problemlos durch hinzunahme von metronidazol geschlossen werden. bei verwendung von ampicillin-sulbactam oder amoxicillin-clavulansäure zur pap ist die zusätzliche gabe von metronidazol nicht erforderlich. folgende weitere kriterien sollten bei der wahl des antibiotikums berücksichtigt werden: lokale erreger und resistenzsituation: hier gibt es regional teilweise dramatische unterschiede, die eine allgemeingültige empfehlung im rahmen dieses beitrags verhindern. wirksamkeit des präparats: in einigen richtlinien findet sich noch die empfehlung, zur pap substanzen zu verwenden, die nicht in der therapie zum einsatz kommen. in einigen fällen führt dies dazu, dass bei der prophylaxe substanzen verwendet werden, die aufgrund unbefriedigender resistenzlage nicht mehr zur therapie verwendet werden. die autoren sind der ansicht, dass eine infektionspräventive maßnahme nur dann sinnvoll ist, wenn sie wirksam ist. entsprechend muss das präparat gewählt werden. operationsgebiet: bei einigen richtlinien wird die wahl des antibiotikums im rahmen der prophylaxe abhängig gemacht vom operationsgebiet. so werden untergruppen gebildet wie magen-, ösophagus-, pankreas-, leber-, oder darmchirurgie. die antibiotika, die dann empfohlen werden, unterscheiden sich in den meisten fällen nicht. darüber hinaus ist festzustellen, dass die umsetzung einer empfehlung unmittelbar mit der komplexität korreliert. empfehlungen zur pap sollten übersichtlich und eingängig sein und sich auf ein möglichst schmales spektrum von geeigneten antibiotika beziehen. gemäß empfehlung der peg sind folgende präparate bei der pap in der viszeralchirurgie mittels studien untersucht und sinnvoll: • cephalosporine der gruppe ii + metronidazol • cephalosporine der gruppe iii a + metronidazol • aminopenicilline mit betalaktamaseinhibitor • fluorchinolone der gruppe 2/3 + metronidazol es wird darauf hingewiesen, dass die resistenzen bei e. coli (leiterreger der infektionen mit enterobacteriaceae) gegenüber ampicillin/sulbactam in zahlreichen regionen so weit angestiegen ist, dass dort eine verwendung zur prophylaxe nicht mehr vertreten werden kann. ersatzpräparate bei allergien: bei allergien gegen penicilline können unter berücksichtigung der resistenzlage cephalosporine der 2. (z. b. cefuroxim) oder 3. generation (z. b. ceftriaxon) oder fluorchinolone der gruppe 2/3 jeweils kombiniert mit metronidazol eingesetzt werden. zu erfassendes erregerspektrum: die hauptverursacher von wundinfektionen in der viszeralchirurgie sind enterobacteriaceae (e. coli > klebsiella spp. > pseudomonas aeruginosa > proteus spp.). dies deutet darauf hin, dass der ursprung der wundinfektion bei diesen patienten "aus der tiefe" kommt (d. h. endogen verursacht ist), entweder durch intraoperative kontamination, postoperative translokation oder durch fortleitung eines okkulten intraabdominellen infekts (z. b. abszess) und nicht durch unzureichende hygiene bei der postoperativen wundpflege, wie häufig ver-2 mutet wird. in der traumatologie sind eher kommensalen der hautflora zu finden wie koagulase-negative staphylokokken (kns), ggf. auch s. aureus mit oder ohne methicillin-resistenz. substanzwahl bei vitien: meistens lässt sich die indikation für die endokarditisprophylaxe problemlos mit der antibiotikaprophylaxe durch verwendung eines geeigneten antibiotikums kombinieren. hier wird auf die diesbezüglichen empfehlungen der fachgesellschaften verwiesen. wird bei einem patienten bereits eine antibiotikatherapie mit entsprechendem wirkspektrum durchgeführt, kann von ausreichenden wirkspiegeln ausgegangen werden, sodass eine zusätzliche pap bei diesen patienten in der regel nicht erforderlich ist. in der vergangenheit konnte gezeigt werden, dass die standardisierte durchführung der pap die postoperative wundinfektionsrate senken kann. die pap ist als eine maßnahme im katalog der infektvermeidung anzusehen und ersetzt nicht weitere erforderliche maßnahmen. häufiges problem ist die zeitgerechte anwendung 30-60 min vor hautschnitt, da hier mehrere operationsvorbereitende maßnahmen gleichzeitig ablaufen müssen. in einer exakten interdisziplinären festlegung des präoperativen ablaufs soll auch die zuständigkeiten für die pap eindeutig geregelt sein. ein lösungsansatz besteht in der verwendung präoperativer checklisten, wie sie auch von der weltgesundheitsorganisation (who) ausgearbeitet und empfohlen wurden. essenziell erscheint es, die ungerechtfertigte verlängerung der antibiotikaprophylaxe über den operationstag hinaus in zukunft zu verhindern. peter hinz, axel kramer, matthias frank und axel ekkernkamp die ssi-rate wird für geschlossene frakturen mit 1-5 % angegeben und erreicht bei offenen frakturen abhängig vom ausmaß der gewebezerstörung eine häufigkeit von bis zu 43 %. elektive unfallchirurgische eingriffe zeigen mit bis zu 2 % eine deutlich geringere ssi-rate (seifert et al. 2010 (seifert et al. 2014 bisswunden › kap. 2.2.6. analog wie in der chirurgie ergibt sich die indikation für die pap aus der wundklassifikation und zusätzlichen risikofaktoren (› kap. 2.10.2) . die parenterale single-shot-pap ist indiziert und präventiv wirksam bei sauber-kontaminierten oder kontaminierten eingriffen. bei sauberen eingriffen wird die pap bei folgenden risikoeingriffen empfohlen: osteosynthesen, hep und kep, rückenmarkchirurgie sowie offene reposition und interne fixation von frakturen (prokuski 2008) . in der cochrane-analyse von gosselini, roberts und gillepsie (2004) die pap ist grundsätzlich bei offenen frakturen indiziert (gosselini, roberts und gillepsie 2004) . dauer der pap: gegenstand von analysen ist in der versorgung offener frakturen v. a. die frage einmalige pap oder verlängerte postoperative behandlung, da letztere oft noch als standard angesehen wird . hauser, adams und eachempati (2006) gelangten im ergebnis einer metanalyse zu folgenden schlussfolgerungen: • "die aktuellen pap-standards bei offenen frakturen der röhrenknochen basieren nur auf sehr wenig und in manchen fällen gar keiner evidenz." • "das infektionsrisiko wird durch die kurzzeitige gabe eines cephalosporins der 1. generation möglichst früh nach der verletzung signifikant gesenkt, sofern gleichzeitig ein modernes orthopädisches fraktur-und wundmanagement erfolgt." diese aussagen werden durch folgende rcts bestätigt. bei offenen frakturen der grade 1 und 2 ergab sich kein signifikanter unterschied zwischen der ssi-rate. sie lag bei einmaliger gabe von 800 mg perfloxacin i. v. bei 6,6 % und bei verlängerter gabe von cefazolin über 2 d (4 × 1 g/d, gesamtdosis 8 g) gefolgt von oxacillin über 3 d oral bei 8 % (1 g/d; carsenti-etesse et al. 1999 ). im ergebnis einer weiteren metaanalyse konnte auch bei geschlossenen frakturen der röhrenknochen keine überlegenheit einer mehrfachgabe im vergleich zur single-shot-strategie nachgewiesen werden (slobogean et al. 2008 ). zur auswahl der antibiotika ist die studienlage nicht eindeutig. in einer rct betrug die ssi rate bei frakturen grad 3 nach pap mit ciprofloxacin 31 % und nach pap mit cefamandol (betalaktamasestabiles cephalosporin der 2. generation) und gentamicin 7,7 %. dieser unterschied zeigt aufgrund der kleinen stichprobengröße nur einen trend (p = 0,079). dagegen zeichnete sich bei den frakturen grad 1 und 2 mit einer ssi-rate von 5,8 % bzw. 6 % kein unterschied ab (patzakis et al. 2000 bei hep und kep wird durch die pap im ergebnis zurückliegender und neuerer studien einschließlich einer metaanalyse eine hochsignifikante reduktion von ssi erzielt (al buhairan, hind und hutchinson 2008; henley et al. 1986; hsu und cheng 2009; kuper und rosenstein 2008; lidwell et al. 1987) . entscheidend ist die einhaltung des zeitpunkts der pap, was leider häufig nicht gewährleistet ist (bateman, smith und grimer 2011; bhattacharyya und hooper 2007) . die prophylaktische wirkung antibiotika-freisetzender pmma (polymethylmethacrylat)-knochenzemente hinsichtlich der entstehung periprothetischer infektionen wurde in skandinavischen prothesenregistern sowie in metaanalysen überzeugend nachgewiesen ) mit einer herabsetzung der ssi rate bei primärer hep um durchschnittlich 50 % gesenkt (parvizi et al. 2008 im unterschied zur konventionellen hep ergab sich bei endoprothetischer rekonstruktion nach tumorresektion im ergebnis einer retrospektiven analyse bei verlängerter antibiotikaprophylaxe (im mittel 7,4 d) eine geringere ssi-rate als bei single-shot (hettwer et al. 2015) , womit der trend einer metanalyse bestätigt wird (racano et al. 2013) . hierbei ist zu berücksichtigen, dass bei diesen eingriffen ein deutlich erhöhtes ssi-risiko (ca. 10 %) besteht und die ssi in bis zu 25 % die amputation der extremität zur folge haben kann (hettwer et al. 2015) . aussagekräftige prospektive randomisierte studien liegen jedoch zu dieser frage bisher nicht vor. im unterschied zu den usa (de beer et al. 2009; fletcher et al. 2007; kuong et al. 2009; meehan, jamali und nguyen 2009) und deutschland wird die pap in den niederlanden bei hep nur bei patienten mit eingeschränkter immunabwehr durchgeführt (abrahaminpijn. 2005 inpijn. ). 1994 wurde aus der schweiz berichtet, dass bei hep ein ersatz der pap durch intraoperative antiseptische spülung im operationsgebiet mit polihexanid mit gleicher ssi-rate möglich ist (kramer und willenegger 1994) . auswahl der antibiotika: als antibiotika für die pap werden cefazolin oder cefuroxim empfohlen (bratzler et al. 2013; sign 2014) . clindamycin und vancomycin kommen bei einer allergie gegen betalaktame in betracht (fletcher et al. 2007 ). sofern im ergebnis eines präoperativen screenings eine kolonisation mit mrsa festgestellt wurde und die mrsa-dekolonisation nicht abgewartet werden kann, muss zusätzlich ein gegen mrsa wirksames antibiotikum ausgewählt werden. bei vancomycin ist zu beachten, dass der wirksame spiegel erst nach 2 h gewährleistet ist. da sich die resistenzlage fortlaufend ändert und je nach lokaler resistenzlage ein zunehmender anteil von ssi z. b. durch cefazolin-resistente staphylokokken verursacht wird, muss der auswahl des antibiotikums für die pap durch eine regelmäßige interdisziplinäre überprüfung des hausinternen standards rechnung getragen werden (norton et al. 2014 ). dies ist ein wichtiger bestandteil eines antibiotic-stewardship-programms. nicht traumatisch bedingte neurochirurgische operationen zählen zu den primär sauberen bzw. sauber-kontaminierten eingriffen. transsphenoidale zugangswege gelten als primär kontaminiert. postoperative wundinfektionen (ssi) sind insgesamt in der neurochirurgie selten (0,3-4 %; › tab. 2.34) , jedoch -wenn sie auftreten -mit hoher morbidität, letalität und einem verlängerten krankenhausaufenthalt verbunden (› kap. 5.7). postoperative bakterielle meningitiden sind als spezielle komplikation nach neurochirurgischen eingriffen besonders gefürchtet. neben der physiologischen hautflora des patienten, die vorwiegend aus koagulasenegativen staphylokokken (kns) besteht und als hauptreservoir für postoperative wundinfektionen gilt, können die erreger von der kopfbehaarung stammen oder über kontaminierte instrumentarien und implantate in das op-gebiet gelangen. das bei neurochirurgischen ssi zu erwartende erregerspektrum umfasst vor allem staphylokokken (s. aureus, kns), deutlich seltener p. acnes (insbesondere bei shuntoperationen und in der wirbelsäulenchirurgie) sowie streptokokken der viridansgruppe. in 5-8 % werden enterobakterien, sehr selten auch p aeruginosa oder andere nonfermenter nachgewiesen (z. b. a. baumannii) . bei hirnabszessen finden sich häufig mischinfektionen (felsenstein 2013; mishra 2014). die wichtigsten erreger sind mikroorganismen der oropharyngealen flora (und der nasennebenhöhlen) wie streptokokken der viridansgruppe, angeführt von s. milleri. tab. 2.34 wundinfektionsrate ohne perioperative antibiotikaprophylaxe (petrica et al . 2009 ) kraniotomie, wirbelsäulenchirurgie 1-5 liquorfistel stellenwert der perioperativen antibiotikaprophylaxe (pap) die pap wird in der neurochirurgischen literatur kontrovers diskutiert. sie wird vor allem zur prävention der postoperativen wundinfektion eingesetzt (› tab. 2.35) , deren inzidenz sich mit einer pap um etwa 50 % reduzieren lässt. im unterschied zu einer 2007 publizierten metaanalyse (barker et al. 2007 ) fanden andere studien keinen signifikanten einfluss der pap auf die rate postoperativer meningitiden, die mit und ohne pap 1,5 bzw. 1,6 % beträgt (barker 2007; korinek et al. 2006; ratilal et al. 2008; sharma et al. 2009 ). als eindeutige indikationen gelten aseptische implantationen von fremdkörpern, z. b. eines vp-shunts (prusseit et al. 2009 ) oder einer subkutanen baclofen-pumpe (motta und antonello 2014), eingriffe mit langen op-zeiten (> 2-4 stunden) offene traumata sowie rezidivoperationen innerhalb von 5 tagen nach der erst-op. die pap ist nur eine von zahlreichen weiteren maßnahmen der perioperativen infektionsprophylaxe (krinko 2007; kubilay et al. 2013; prusseit 2009 ). zum beispiel senkt ein wechsel der sterilen op-handschuhe, bevor der ventrikelkatheter erstmals berührt und implantiert wird, das infektionsrisiko bei shunt-operationen (rehmann et al. 2010) . für die wirbelsäulenchirurgie gibt es eine eigene leitlinie amerikanischer fachgesellschaften (watters et al. 2009 ); hier kann bereits der einsatz minimalinvasiver op-methoden das ssi-risiko um den faktor 10 reduzieren (o'toole et al. 2009 ). risikofaktoren für ssi (korinek et al . 2006; lietard et al . 2008 ) liquorfistel ja ja externe ventrikeldrainage ja ja gleichzeitige wundinfektion -ja männliches geschlecht -ja frühzeitige reoperation ja bei neurochirurgischen eingriffen wird die präoperative einmalgabe (single-shot-gabe) der antibiotika favorisiert. betalaktame (z. b. cefazolin, cefuroxim oder ampicillin-sulbactam, in ausnahmefällen auch piperacillin/tazobactam und teicoplanin) werden in den letzten 60 min vor der op gegeben, vancomycin 120 min vorher, weil es über mind. eine stunde infundiert werden muss (bratzler et al. 2013 ; scottish intercollegiate guidelines network 2014). flucloxacillin, cefazolin oder cefuroxim sind mittel der 1. wahl, clindamycin oder vancomycin sind alternativen bei allergie gegen β-lactam-antibiotika. bei vorliegen eines erhöhten anteils von mrsa bzw. von methicllin-resistenten kns an allen postneurochirurgischen wundinfektionen kann der zusätzliche einsatz von glykopeptiden wie vancomycin oder teicoplanin zur pap erwogen werden. alle genannten antibiotika können, z. b. bei implantaten, mit rifampicin kombiniert werden, ob dies einen signifikanten zusätzlichen nutzen hat, ist unklar. je nach dauer der op muss eine zweite gabe des antibiotikums verabreicht werden (cefazolin 4 h, cefuroxim 4 h, ampicillin-sulbactam 2 h, piperacillin-tazobactam 2 h, clindamycin 6 h, vancomycin 8 h) (bratzler et al. 2013) ; dies gilt z. b. auch bei erheblichem intraoperativem blutverlust. bei vp-shunt-operationen und neurochirurgischen eingriffen bei tumorpatienten kann die pap auf maximal 3 gaben in 24 stunden ausgedehnt werden, eine darüber hinaus verlängerte antibiotikagabe bringt jedoch definitiv keinen vorteil (bratzler et al. 2013; rath, costa und sampaio 2008) . patienten mit einer passageren ventrikelsonde (externer ventrikeldrainage; evd), die z. b. zur druckentlastung, zum invasiven monitoring des hirndrucks (hierzu gibt es auch spezielle, ebenfalls invasive drucksonden) oder als passagere lösung bei patienten mit vp-shunt-infektion (nach explantation desselben) eingesetzt wird, haben ein substanzielles risiko für eine menigoventrikulitis. scheithauer et al. (2010) fanden eine inzidenzrate von 7,5/1 000 anwendungstage bei evd. diese patienten erhalten in der praxis häufig nicht nur vor der anlage der evd (leverstein-van hall et al. 2010) , sondern solange die drainage liegt, eine systemische antibiotikaprophylaxe, mit dem ziel, eine evd-assoziierte infektion zu verhindern. dieses vorgehen wird vor dem hintergrund unzureichender daten kontrovers diskutiert (bratzler et al. 2013 ; scottish intercollegiate guidelines network 2014) und von den meisten klinischen infektiologen abgelehnt (mc-carthy et al. 2010) . gerade bei der anwendung dieser devices ist ein streng aseptisches vorgehen nach einem für alle verbindlichen schriftlich festgelegten standard wichtig (camacho et al. 2013; kubilay et al. 2013; leverstein-van hall 2010; lwin et al. 2012 ). die lokoregionäre anwendung von antibiotika wird von einzelnen neurochirurgen favorisiert, ihre wirksamkeit ist jedoch bis heute unbewiesen (alves und godoy 2010). in einer kleinen prospektiven, doppelblind randomisierten studie konnten rozzelle, leonardo und li (2008) zeigen, dass die ssi-rate nach neurochirurgischen shunt-operationen bei verwendung von mit triclosan imprägniertem chirurgischem nahtmaterial niedriger war (4,3 % vs. 21 %), wobei die ssi-rate in der kontrollkruppe in dieser studie sehr hoch ist. zur endgültigen bewertung des klinischen vorteils von antimikrobiell imprägnierten nahtmaterial fehlen in der neurochirurgie größere bestätigungsstudien. in einer prospektiv randomisierten studie an 6 neurochirurgischen kliniken konnte der einsatz von minocyclin/rifampicin-imprägnierten drainagen (n = 149) das risiko der evd-assoziierten meningoradikulitis senken (rate positiver liquorkulturen 1,3 vs. 9,4 %, p = 0,002 (zabramski et al. 2003) ). inzwischen werden vermehrt vp-shunts mit rifampicin-clindamycin imprägnierung implantiert (govender, nathoo und van dellen 2003) von denen die genannten antibiotika nach angaben der herstellers 28 tage lang freigesetzt werden. für den endgültigen nachweis einer signifikant reduzierten ssi-rate (hier v. a. vp-shunt-assoziierte infektionen) stehen jedoch auch hier randomisierte, prospektive multicenterstudien aus (gutiérrez-gonzález und boto 2010) . vereinzelt wurde über nachfolgende infektionen mit rifampicin-resistenten kns berichtet (demetriades und bassi 2011 ). wong et al. (2010 fanden bei 184 patienten mit externer ventrikeldrainage (evd), dass der rifampicin-clindamycin imprägnierte katheter (als evd) einer systemischen antibiotikaprophylaxe in bezug auf den endpunkt der device-assoziierten meningitis nicht unterlegen war. bei den postoperativen wundinfektionen im bereich der eintrittsstelle gab es keinen signifikanten unterschied (wong et al. 2010 ). auch pople et al. konnten bei sehr niedrigen infektionsraten in beiden gruppen (2,3 % vs. 2,8 %) keinen vorteil des rifampicin-clindamycin-imprägnierten katheters gegenüber nicht imprägnierten externen ventrikeldrainagen darstellen (pople et al. 2012) bilal al-nawas invasive eingriffe und vergleichbare maßnahmen, z. t. sogar operationen, werden meist handelt es sich in der zahnmedizin und mkg-chirurgie um eingriffe der gruppen ii und iii, wobei ssi überwiegend durch oropharyngeale pathogene verursacht werden. grundsätzlich kann unterschieden werden zwischen der pap zur vermeidung der negativen folgen einer bakteriämie, wie sie patienten mit gelenkendoprothesen oder endokarditisrisiko betreffen kann, und der prophylaxe von ssi im engeren sinn. auch wenn die antibiotika bezüglich der infrage kommenden erreger oft identisch sind, besteht der unterschied in der konsequenz bei auftreten von problemen. so lässt sich die lokal begrenzte ssi meist gut beherrschen, während eine endokarditis per se vital bedrohlich ist. es empfiehlt sich also, für die indikationsfindung das individuelle risikoprofil des patienten zu grunde zu legen. im vergleich zur humanmedizin finden sich keine systematischen daten zur resistenzentwicklung in der zahnmedizin. berichtet wurde bei unkomplizierten abszessen über geringe resistenzraten für penicillin (eckert et al. 2005a ), aber auch über das auftreten von 15-35 % betalaktamasen bei bakterien aus odontogenen abszessen (kuriyama et al. 2001 kuriyama et al. 2000) . bei schweren weichgewebeinfektionen, die typischerweise schon vorbehandelt sind, muss man demnach mit einer höheren resistenzrate gegen penicillin und clindamycin rechnen eckert et al. 2005b ). aus den vorgenannten daten ergibt sich im odontogenen bereich eine nahezu vollständige wirksamkeit der kombination aus einem aminopenicillin mit einem betalaktamasehemmer (amoxicillin-clavulansäure oder ampicillin-sulbactam). bei der resistenzbeurteilung sollte jedoch bedacht werden, dass die pathogenetische rolle der identifizierten bakterien durchaus nicht geklärt ist (otten et al. 1994 ). da ein erregernachweis in der therapie unkomplizierter odontogener infektionen nicht praktikabel ist, bleibt der wunsch nach validen resistenzdaten in der ambulanten zahnmedizin wohl auch in zukunft unerfüllt. indikationen für die prophylaktische antibiotikagabe bzw. pap indikationen: grundsätzlich ist akzeptiert, dass bakteriämien bei vorgeschädigtem endokard zu einer infektiösen endokarditis führen können. zugleich ist unbestritten, dass bei allen zahnärztlichen behandlungen mit manipulation an der gingiva und bei wurzelka-nalbehandlungen bakteriämien auftreten. aber auch bei routineaktivitäten wie zähneputzen oder kauen sind bakterien im blut nachweisbar. beachtenswert ist, dass im tiermodell 6-8 log bakterien/ml blut zur auslösung einer endokarditis erforderlich sind (bahn et al. 1978) , bei zahnärztlichen behandlungen findet man jedoch nur 1-10/ml (rahn et al. 1987) . in einer richtungweisenden arbeit aus frankreich wurde die effektivität der antibiotikaprophylaxe infrage gestellt (duval et al. 2006) . seitdem hat sich ein paradigmenwechel vollzogen (naber et al. 2007 ). demnach sollen nicht mehr alle patienten mit dem risiko für eine infektiöse endokarditis eine prophylaxe erhalten, sondern nur patienten mit einem hohen erkrankungsrisiko oder einem hohen risiko für einen lebensbedrohlichen verlauf. die auswahl der antibiotika bleibt dagegen unverändert entsprechend den erwarteten oralen pathogenen. für patienten, die bisher eine prophylaxe erhielten und bei denen diese jetzt nicht mehr indiziert ist, gibt es die möglichkeit der individuellen, fakultativen prophylaxe. als risikoprozeduren werden alle eingriffe angesehen, die zu bakteriämien führen können wie manipulationen an der gingiva, der periapikalen zahnregion, perforationen der oralen mukosa. aktuelle daten deuten darauf hin, dass diese gelockerten leitlinien nicht zu einem anstieg der endokarditisinzidenz geführt haben (desimone et al. 2012 ). die einschätzung der prophylaxe von infektionen von endoprothesen ist schwierig. jüngere metaanalysen der spärlichen literatur stellen den sinn dieser prophylaxe bei gesunden patienten infrage (legout et al. 2012) , zumal infektionen von hüft-oder knieendoprothesen als folge von bakteriämien nach oralen eingriffen sehr selten sind (rodgers und richards 2008) . als gute handlungsgrundlage existiert eine methodisch sehr hochwertige interdisziplinäre leitlinie zur prophylaxe aus den usa (watters et al. 2013) , in der die indikation zur prophylaxe kritisch bewertet und die bedeutung der mundhygiene betont wird es besteht konsens, dass für die meisten zahnärztlichen eingriffe bei gesunden patienten keine antibiotikaprophylaxe erforderlich ist (al-nawas 2002), z. b. im rahmen der endodontie (mohammadi 2009 ) und in der einfachen dentoalveolären chirurgie bei gesunden patienten (al-nawas 2002). im gegensatz zur einfachen zahnextraktion wird die pap vor weisheitszahnextraktion (kontaminiertes gebiet gr. iii) auf der basis von 12 studien an über 2 000 patienten empfohlen (ren und malmstrom 2007) . es bestätigte sich, dass die prolongierte prophylaxe keinen zusätzlichen effekt zeigte, wohl aber konnte der negative effekt einer zu späten, ausschließlich postoperativen gabe bestätigt werden. bei der insertion dentaler implantate (sauber-kontaminiertes gebiet gr. ii) sank die implantatverlustrate bei pap in einer meta-analyse um 1,9 % (al-nawas und stein 2010). um diesen effekt zu erreichen, muss jedoch eine hohe anzahl an patienten eine prophylaxe erhalten (number needed to treat 53). ein aktueller cochrane review zu diesem thema bewertet den nutzen einer pap bei der implantatinsertion positiv (esposito et al. 2013) . unbestritten ist bei komplexen implantologischen eingriffen, wie z. b. augmentationen eine pap sinnvoll. auswahl der antibiotika: zur pap empfehlen die meisten autoren penicillin v oder amoxicillin. in hinblick auf gewebespiegel scheint konsens zu bestehen, dass zur prophylaxe eine etwas höhere dosierung (z. b. 1-2 g amoxicillin p. o. als einmalgabe) sinnvoll erscheint und zwar vor dem eingriff (steinberg et al. 2009 ). daher empfiehlt sich, die pap bei der planung ambulanter eingriffe mit dem patienten vorzubereiten. die prolongierte postoperative gabe hat bei einfachen invasiven eingriffen keinen einfluss auf die ssi-rate. immunsupprimierte patienten (z. b. nach radiatio oder bisphosphonattherapie) profitzieren hingegen von einer prolongierten prophylaxe über mehrere tage (grötz 2003) . bei den meist komplexen ops im (sauber-)kontaminierten gebiet wird fast durchgängig die pap empfohlen, z. b. zur versorgung frakturierter gesichtsknochen (knepil und loukota 2010) und für die lappenchirurgie (amland et al. 1995) . bei unterkieferfrakturen wird nicht nur die einmalgabe, sondern eine eintägige gabe diskutiert (andreasen et al. 2006 ); interessanterweise wird das durch daten der kieferorthopädischen chirurgie gestützt (danda et al. 2010 ). deutlich weniger daten liegen für die chirurgie der lippen-, kieferund gaumenspalten vor; dennoch empfehlen die meisten autoren auch hier zumindest die pap (smyth & knepil 2008) . bei komplexen ops im sauberen gebiet (gr. i) wird eine 24 stündige gabe empfohlen; z. b. für die neck dissection (seven, sayin und turgut 2004) . eine pap über mehr als 24 stunden (drei gaben) ist bei sauber-kontaminierten operationen (gr. ii), analog zu daten aus anderen chirurgischen fächern, ohne messbaren effekt und sollte daher vermieden werden (mottini et al. 2014 ). gemäß ifsg ( § 23) sind leiter von einrichtungen für ambulantes operieren verpflichtet, ni fortlaufend aufzuzeichnen und zu bewerten. allerdings bezieht sich der begriff ambulantes operieren auf operationen ( § 115 sgb v) und nicht auf invasive (zahnärztliche) eingriffe. grundsätzlich ist die qualitätssicherung und überwachung der eigenen infektionsraten zu fordern, die systematische prospektive überwachung ist für eingriffe in der kontaminierten mundhöhle jedoch nicht praktikabel. zugleich muss die resistenzentwicklung von den verschreibenden kritisch beobachtet und ein wissenschaftlich nicht gesicherter antibiotikaeinsatz kritisch hinterfragt werden. in allen empfehlungen stellen penicilline die zentrale säule der in der zahnmedizin verwendeten substanzen dar. die verbreitung von mre wird durch ungezielte antibiotikagaben in therapie und prophylaxe gefördert. deswegen ist die streng indizierte pap bei einhaltung aller hygienischen maßnahmen eine wichtige maßnahme zur minderung der resistenzentwicklung. ziel der pap ist die vermeidung von ssi, idealerweise ohne wesentliche beeinträchtigung der normalflora oder induktion eines selektionsdrucks mit der gefahr der ausbildung von antibiotikaresistenzen (peters 1987) . die pap ist kein ersatz für hygienemaßnahmen zur prävention von ssi! gesicherte indikationen im hno-bereich sind tumorchirurgische eingriffe mit eröffnung der schleimhäute von mundhöhle/ pharynx und/oder larynx (johnson, myers und sigler 1984; liu, tung und chiu 2008) sowie gesichtsfrakturen, insbesondere komplizierte unterkieferfrakturen (bratzler et al. 2013; sign 2014) . daneben gibt es akzeptierte indikationen (einbringen von implantaten) wie die kochlearimplantchirurgie. für zahlreiche hno-ärztliche eingriffe der ohr-, nasen-, nasennebenhöhlenchirurgie ist der nutzen einer pap noch ungeklärt! galt eine > 24 stunden liegende nasentamponade als indikation für eine u. u. mehrtägige (ungezielte) antibiotische "prophylaxe" (therapie), zeigen neuere arbeiten keinen vorteil (biswas und mal 2009 in der gynäkologie handelt es sich meist um elektive eingriffe (ausnahmen z. b. akuter unterbauchschmerz, stielgedrehtes ovar, extrauterine gravidität; geburtshilfe: eilige bzw. notfallsektio). neben ambulanter op-vorbereitung und möglichst kurzer krankenhausverweildauer sollten prätherapeutisch vorhandene infektionen wie atemwegs-, harnwegsinfektionen oder infektionen äußerer oder innerer genitalorgane saniert werden. ebenso wichtig sind die internistische abklärung der operabilität, die optimale einstellung eines diabetes mellitus, die stabilisierung von herz-kreislauf-parametern sowie der hämoglobin-und elektrolytausgleich. andere risikofaktoren wie alter, organspezifische komorbiditäten, durchblutungsstörungen, adipositas oder insbesondere ihre kombinationen sind u. u. nicht präoperativ optimierbar. bei vorhandenen und nicht abwendbaren risikofaktoren (asa-kriterien) sollten ggf. konservative therapieoptionen (z. b. bestrahlung von tumoren, primäre chemo-oder antihormontherapie) bzw. eine möglichst kurze op-zeit mit einschränkung der radikalität der op überdacht werden. bei onkochirurgischen, häufig multiviszeralen operationen (insbesondere ovarialkarzinom, darmbeteiligung) erfolgt präoperativ die vollständige darmentleerung. bei kleineren abdominalen eingriffen ist die säuberung des enddarms ausreichend (makroklistier). vorhandene piercings sind präoperativ zu entfernen. rasieren ist nur bei op-technischer notwendigkeit unmittelbar präoperativ durchzuführen. zur verringerung von ssi gehört insbesondere bei vaginalen eingriffen die gründliche reinigung und antiseptik der mikrobiell belasteten anogenitalregion bzw. der bauch-und thoraxwand/ axilla bei abdominalen und mammachirurgischen eingriffen. hierbei ist insbesondere auf die ausreichende antiseptik von umbilikalregion, mamille und submammar-/axillarfalte zu achten. bei präpartalem vaginalem nachweis von streptokokken der sero gruppe b (gbs) sollte prophylaktisch 4-stündlich ab geburtsbeginn penicillin (2. wahl: ampicillin) i. v. verabreicht werden (bei penicillinallergie z. b. clindamycin) (dggh 2006) . auch bei drohender frühgeburt und fehlender gbs-testung wird eine prophylaktische antibiotikagabe empfohlen. die effektivität der gbs-prophylaxe ist eingeschränkt, wenn sie weniger als 4 h vor der geburt begonnen wurde. die infektion des neugeborenen kann eine schwere allgemeininfektion mit pneumonie und schocksymptomatik bzw. neugeborenensepsis zur folge haben, wobei mit neurologischen langzeitschäden und einer letalität von 4 % zu rechnen ist. ein gbs-screening ist daher in der schwangerschaft empfehlenswert. bei hiv1infektion der werdenden mutter beträgt die transmissionsrate bis zu 40 %. das risiko kann durch senkung der viruslast durch antiretrovirale medikation, ggf. primäre sectio, antiretrovirale pep des neugeborenen (oral zidovudin bis zu 6 wochen) und stillverzicht auf < 2 % gesenkt werden. daher sollte jeder schwangeren ein hiv-such-und ggf. hiv-bestätigungstest empfohlen werden. die übertragung von hbv einer akut oder chronisch infizierten schwangeren erfolgt, abhängig von der höhe der viruslast, im letzten schwangerschaftsdrittel (5-12 %) bzw. während der geburt (80 %) bzw. während des stillens (5 %). die peripartale infektion der kindes verläuft oft asymptomatisch und geht in den meisten fällen in einen chronischen hbv-trägerstatus über (bis 90 %); 25 % der kinder sterben an den folgen (leberzirrhose, hepatozelluläres karzinom) (lam, gotsch und langan 2010) . die hbs-antigen-(mutterschaftsrichtlinien) bzw. antikörperbestimmung und weitere antigensuche im letzten schwangerschaftsdrittel ermöglichen die planung der primären sectio, eine frühzeitige aktive und passive immunisierung des neugeborenen (senkung des infektionsrisikos um 95 % bei impfung innerhalb von 12 h post partum). aktiv und passiv immunisierte neugeborene dürfen gestillt werden. die mutter-kind-transmissionsrate von hcv (prävalenz < 1 %) ist gering (1-6 %) und durch sektio oder stillverzicht nicht weiter absenkbar (dgvs 2015) . ein screening bei abwesenheit von koinfektionen oder speziellen mütterlichen risikofaktoren wird in der schwangerschaft wegen fehlender suffizienter medikamentöser therapie nicht empfohlen. gesicherte risikofaktoren für frühgeburten und spätaborte sind bakterielle vaginose, aszendierende oder maternale infektionen (leitich und kiss 2007 (aus et al. 2005) . vorübergehende nebenwirkungen wie örtlicher schmerz, hämaturie, hämospermie, dysurie und rektale blutung werden häufig berichtet (crundwell, cooke und wallace 1999; djavan et al. 2001; loeb et al. 2013) . eine bakteriurie findet sich bei 20 %-53 %, eine transiente bakteriämie bei bis zu 73 % der patienten (lindert, kabalin und terris. 2000; thompson et al. 1980) . fieber in verbindung mit urogenitalen symptomen werden bei 3 %-10 % und eine postinterventionelle sepsis bei bis zu 5 % der patienten beschrieben (crawford et al. 1982; enlund und varenhorst 1997; lindert, kabalin und terris 2000; thompson et al. 1980) . die alleinige rektale instillation von pvp-iod zeigte in einer prospektiv randomisierten studie an 865 männern nach trpb eine nichtsignifikante (wenn auch 42-prozentige) reduktion von infektiösen komplikationen gegenüber der unbehandelten gruppe (abughosh et al. 2013) . eine grundsätzlich andere strategie wäre der ersatz der ultraschall-gesteuerten trbp durch eine perineale prostatabiopsie (wagenlehner et al. 2013) , wobei sich auch hier die frage einer pap zur vermeidung von wundinfektionen stellte. durch die pap kann die inzidenz postinterventioneller infektiöser komplikationen nach trpb verringert werden (aron, rajeev und gupta 2000; aus et al. 1996; crawford et al. 1982; isen et al. 1999; kapoor et al. 1998 ; zani, clark und rodriguez netto 2011), weshalb die perioperative antibiotikaprophylaxe (pap) bei der transrektalen prostatabiopsie als standardvorgehen angesehen werden kann, sofern bestimmte voraussetzungen eingehalten werden. zeitpunkt, dauer und applikationsform der pap sind umstritten. eine metaanalyse der pap bei trpb ergab, dass eine verlängerte prophylaxe nicht wirksamer als die präinterventionelle einmalgabe ist (zani, clark und rodriguez netto 2011) ; dies wurde auch durch eine später publizierte literaturauswertung bestätigt (loeb et al. 2013) . unter welchen umständen eine einmalgabe erfolgreich bzw. nicht indiziert ist, wurde am besten in einer schwedischen studie (lindstedt et al. 2006 ) mit 1 322 prostatabiopsien mit oraler einmalgabe von hochdosiertem ciprofloxacin (750 mg) innerhalb 2 h vor dem eingriff untersucht. mit dieser dosierung können ausreichend hohe urin-und prostatagewebekonzentrationen bis zu 48 h aufrecht erhalten werden (naber, adam und kees 1987; naber 2008; wagenlehner und naber 2003) und in der genannten studie (lindstedt et al. 2006 ) lag die postinterventionelle infektionsrate bei etwa 1 %, was im literaturvergleich niedrig ist (aron, rajeev und gupta 2000; aus et al. 1996; crawford et al. 1982; djavan et al. 2001; enlund und varenhorst 1997; isen et al. 1999; kapoor et al. 1998; raaijmakers et al. 2002) . für die präoperative einmalgabe kommen nur patienten infrage, bei denen zuvor eine harnwegsinfektion (hwi) und eine asymptomatische bakteriurie ausgeschlossen wurden und ferner keine der folgenden risikofaktoren vorliegen: • dauerkatheter wegen harnverhalt, • rezidivierende hwi, prostatitis oder andere fieberhafte genitalinfektion in der anamnese, • immunsuppression. zum ausschluss einer hwi/bakteriurie sollte innerhalb einer woche vor dem eingriff eine urinkultur aus mittelstrahlurin durchgeführt werden. als surrogatparameter kann alternativ auch ein urinstatus mit negativer leukozytenesterase und negativem nitrit im streifentest genutzt werden. nur 12 (0,9 %) der auf diese weise voruntersuchten patienten entwickelte nach der trpb eine fieberhafte urogenitale infektion; davon mussten 5 wegen einer schwerer infektion hospitalisiert werden. die sepsisrate lag bei 0,17 %. bei den 12 patienten, bei denen entweder eine bakteriurie übersehen wurde oder sich trotz negativem urinstatus eine bakteriurie fand, entwickelte sich bei 5 eine postinterventionelle symptomatische hwi, davon in 3 fällen mit sepsis. diese rate ist deutlich höher als bei patienten mit sterilem urin, weshalb auch die asymptomatische bakteriurie als risikofaktor angesehen wird. die infektionsrate lag in etwa gleicher größenordnung wie bei transurethraler resektion der prostata (turp) bei patienten mit bakteriurie (grabe und hellsten 1989) . allerdings hätten von den 12 patienten 8 aufgrund von risikofaktoren von der antibiotika-einmalgabe ausgeschlossen werden müssen. die studie zeigt an einem großen patientenkollektiv, dass eine perioperative antibiotika-einmalgabe auch bei der trpb ausreichend ist, wenn zuvor eine bakteriurie durch urinkultur (oder zumindest durch einen negativen streifentest) weitgehend ausgeschlossen werden kann und keiner der genannten risikofaktoren vorliegt. auf die zusätzliche gabe eines gegen anaerobier-wirksamen antibiotikums wurde in der studie bewusst verzich-tet, obwohl in sehr seltenen fällen auch eine postinterventionelle infektion mit anaerobiern beschrieben worden ist (miura et al. 2008 ). die meisten untersuchungen zur wirksamkeit der pap bei trpb wurden mit fluorchinolonen (fch) zu einer zeit durchgeführt, als die prävalenz der fch-resistenz niedriger als heute war (zani, clark und rodriguez netto 2011) . die antibiotikaauswahl ist heute nicht mehr so leicht, da auch in deutschland bei gramnegativen erregern urogenitaler infektionen eine zunehmende resistenzentwicklung gegen fch zu beobachten ist (kresken, hafner und körber-irrgang 2013) . insofern erscheinen zuletzt häufiger berichte über patienten, die nach prophylaxe mit einem fch eine schwere postinterventionelle infektion bis hin zur sepsis verursacht durch einen fch-resistenten erreger (meist e. coli) erlitten haben (feliciano et al. 2008; miura et al. 2008; nam et al. 2013; shigehara et al. 2008; tal et al. 2003; wagenlehner et al. 2013; young, liss und szabo 2009) . die gastrointestinale kolonisation mit fch-resistenten e. coli prädisponiert für eine solche komplikation (roberts et al. 2014 ). da die prostatabiopsie in der regel transrektal erfolgt, genügt es nicht, lediglich durch eine urinkultur das vorhandensein fch-resistenter erreger in den harnwegen auszuschließen. ein zusätzlicher analabstrich ist hier wahrscheinlich von nutzen, wird jedoch bisher nicht regelmäßig durchgeführt (roberts et al. 2014; taylor et al. 2012) . bereits die prophylaktische einmalgabe eines fch kann die rate fch-resistenter e. coli in der fäkalflora deutlich erhöhen (wagenlehner et al. 2005 ) (medikamentenanamnese!). eine us-amerikanische untersuchung von 865 patienten vor trpb, bei denen ein rektalabstrich auf selektivagar mit einer ciprofloxacin-konzentration von 1 mg/l eingesetzt wurde, fand bei 19 % der patienten ciprofloxacin-resistente coliforme bakterien (81 % e. coli) (taylor et al. 2013) . risikofaktoren für diesen nachweis waren herzklappenersatz und die einnahme von ciprofloxacin innerhalb der letzten drei monate. in der gesamten kohorte erlitten 31 (3,6 %) der patienten infektiöse komplikationen nach trpb, bei 15 (48 %) durch fluorchinolon-resistente coliforme bakterien. die inzidenz von infektionen in der gruppe mit präinterventionellem nachweis fch-resistenter e. coli lag bei 9 % (taylor et al. 2012 ). diese daten zur pap bei trpb sprechen eindeutig dafür, bei patienten mit risikofaktoren fluorchinolone nur noch einzusetzen, wenn durch einen negativen rektalabstrich eine fäkale kolonisation mit fluorchinolon-resistenten coliformen bakterien ausgeschlossen wurde (wagenlehner et al. 2014 ). leider sind andere antibiotika in diesem kontext bisher nicht gut untersucht. zwar zeigte eine 2005 publizierte pap-studie für cotrimoxazol ähnlich gute ergebnisse wie für levofloxacin (wagenlehner et al. 2005) , in den meisten erhebungen (z. b. zur zystitis) liegt 2 jedoch heute der anteil cotrimoxazol-resistenter e. coli über 20 % (naber et al. 2008 ). infrage kämen demnach z. b. cephalosporine der 3. generation, z. b. ceftriaxon 1-2 g, oder piperacillin in kombination mit tazobactam (einzeldosis 4 g/0,5 g); beide währen jedoch gegen einen esbl-bildendes isolat nicht bzw. nicht sicher wirksam (ozden et al. 2009 ). zu oralen betalaktamantibiotika fehlen entsprechende studien. die einmalgabe eines aminoglykosids (nur parenteral, z. b. amikacin) wurde ebenfalls bisher nicht an einem ausreichend großen patientenkollektiv untersucht, gentamicin war oral verabreichtem ciprofloxacin unterlegen (roach et al. 1991) . auch oral verabreichtes fosfomycin (als fosfomycin-trometamol; ft) wurde zur pap bei turp angewendet (di silverio, ferrone und carati 1990; periti et al. 1987) . fosfomycin hat den vorteil, dass fch-resistente, esbl-bildende e. coli nicht gleichermaßen häufig auch gegen fosfomycin resistent sind (keine parallelresistenz) (akyar 2000) . in einer retrospektiven auswertung des klinischen verlaufs bei 620 patienten erhielten 104 patienten 3 g ft, 110 patienten 500 mg levofloxacin (einmalgabe) und 406 patienten 2 × 500 mg/d ciprofloxacin mit beginn vor dem eingriff über 5 d (ongün, aslan und avkan-oguz 2012). insgesamt entwickelten 19 (3 %) patienten eine fieberhafte hwi (3,4 % nach ciprofloxacin, 3,6 % nach levofloxacin und 0,9 % nach ft; unterschiede nicht signifikant). ausreichend abgesichert ist ft zur perioperativen prophylaxe bei trpb damit nicht. falls trotz antibiotikaprophylaxe eine schwere infektion auftritt, was nie vollständig zu vermeiden ist, muss in etwa 50 % der fälle (feliciano et al. 2008 ) mit einem erreger gerechnet werden, der gegen das zur prophylaxe verwendete antibiotikum resistent ist. vor einleitung der empirischen therapie sollte eine urinkultur und -bei sepsiszeichen -auch mindestens eine blutkultur angelegt werden. empirisch sollte dann ein breitspektrumantibiotikum mit guter wirksamkeit gegen enterobacteriaceae aus einer anderen klasse (im vergleich zur pap) zur anwendung kommen. infrage kommt bei schweren infektionen z. b. piperacillin-tazobactam, ggf. auch in kombination mit amikacin bis ein erreger (und dessen in vitro empfindlichkeit) bekannt ist. besteht aufgrund der individuellen anamnese oder der örtlichen resistenzsituation der verdacht auf eine infektion durch einen esbl-bildendenden erreger ist (empirisch) ein carbapenem das mittel der ersten wahl. jörg ringel und markus m. lerch empfehlungen zur antibiotikaprophylaxe in der gastroenterologie beziehen sich im wesentlichen auf endoskopische eingriffe. dabei steht nicht mehr nur die endokarditisprophylaxe im vordergrund, sondern sollen insgesamt interventionsspezifische infektionsrisiken minimiert werden. daneben gibt es leitlinien bzw. studiendaten, die eine antibiotikaprophylaxe bei leberzirrhosepatienten und bei patienten mit pankreaspseudozysten in bestimmten situationen und vor bestimmten eingriffen empfehlen. es besteht keine generelle indikation zur endokarditisprophylaxe vor endoskopischen eingriffen. bei spezifischen patientengruppen mit besonderem risiko für eine endokarditis ist im rahmen unterschiedlicher endoskopischer prozeduren eine antibiotikaprophylaxe indiziert (› tab. 2.36 (allison et al. 2009; rosien 2011) . hochrisikopatienten, die in der vergangenheit eine antibiotikaprophylaxe gut vertragen haben, sollten über die neuen empfehlungen informiert werden und können in absprache mit dem behandelnden arzt weiterhin eine prophylaxe erhalten (rosien 2011) . unabhängig von der endokarditisprävention gibt es für einzelne endoskopische untersuchungsprozeduren in den leitlinien empfehlungen zur prophylaktischen antibiotikagabe (› tab. 2.36 ). das betrifft im rahmen einer endoskopischen retrograden cholangiopankreatikoskopie (ercp) patienten mit cholangitis, patienten, bei denen keine vollständige biliäre drainage erreichbar ist (z. b. bei primär sklerosierender cholangitis oder gallenwegneoplasie), sowie patienten bei z. n. lebertransplantation oder bei denen mit dem gallengangsystem kommunizierende pankreas-oder pseudozysten bestehen (allison et al. 2009 , rosien 2011 . bei schwerer neutropenie (< 0,5 × 10 9 /l) und/oder fortgeschrittenen hämatologischen neoplasien wird bei untersuchungen mit erhöhtem bakteriämierisiko wie z. b. dilatationsbehandlung und sklerosierung eine antibiotikagabe empfohlen (allison et al. 2011 trotz teilweise widersprüchlicher daten empfehlen die internationalen leitlinien vor der anlage einer perkutanen endoskopischen gastrostomie (peg) unabhängig von der methode die einmalgabe eines antibiotikums (fadendurchzugmethode oder direktpunktion) (allison et al. 2009; rosien 2011) . bei leberzirrhosepatienten mit überwundener spontanbakteriel ler peritonitis (sbp) oder mit akuter gastrointestinaler blutung besteht die indikation zur antibiotikaprophylaxe. aufgrund zunehmender resistenzentwicklung sollten bestimmte risikokonstellationen beachtet werden. bei patienten mit leberzirrhose treten gehäuft bakterielle infektionen auf. sie können zu schwerwiegenden beeinträchtigungen der kardiopulmonalen, hepatischen und renalen funktion führen. die sbp stellt eine dieser schwerwiegenden komplikationen dar und ist mit deutlich erhöhter letalität assoziiert (alaniz und regal 2009; chavez-tapia et al. 2009; terg et al. 2008 (lee et al. 2009; terg et al. 2008 ). unter konsequenter diagnostik und sofortiger antibiotikatherapie konnte die letalität in den letzten 20 jahren von etwa 80 % auf 50-30 % gesenkt werden. die entscheidung darüber, welches antibiotikaregime empirisch bzw. kalkuliert eingesetzt werden soll, hängt davon ab, ob die infektion ambulant oder nosokomial erworben wurde (gerbes 2011). das rezidivrisiko liegt ersten jahr nach erfolgreicher behandlung der sbp bei etwa 70 % (alaniz und regal 2009; chavez-tapia et al. 2009 ). deshalb wird in den leitlinien eine prophylaktische antibiotikagabe mit norfloxacin 400 mg/d empfohlen. diese soll bis zur vollständigen aszitesrückbildung, bis zu einer lebertransplantation oder -wenn keines der beiden ziele erreicht wird -lebenslang durchgeführt werden. in diesem zusammenhang haben studien eine zunehmende resistenzentwicklung nachgewiesen, weshalb in der derzeit gültigen aszitesleitlinie eine zeitliche begrenzung empfohlen wird (gerbes 2011). eine nur einmal wöchentliche gabe hat sich als insuffizient herausgestellt (alaniz und regal 2009) . eine aktuelle studie konnte keinen vorteil hinsichtlich des auftretens einer sbp bei einer prophylaktischen gabe von rifaximin bei hospitalisierten patienten nachweisen (lutz 2014) eine erniedrigte aszitesproteinkonzentration (< 1,5 g/dl) wurde als risikofaktor für eine sbp ermittelt. deshalb untersuchten terg et al. (2008) die primärprophylaktische gabe von ciprofloxacin 500 mg/d bei patienten mit erniedrigter aszitesproteinkonzentration, was zu einer reduzierten spb-rate und einer verringerten mor-2 talität in den folgenden 12-monaten führte (alaniz und regal 2009; terg et al. 2008 ). entsprechend der gültigen dgvs-leitlinie kann in diesem fall eine primäre antibiotikaprophylaxe durchgeführt werden. auch bei hochrisikopatienten mit zusätzlicher schwerer leberinsuffizienz (child-pugh score > 9) und niereninsuffizienz (serumkreatinin > 1,2 mg/dl; harnstoff > 25 mg/dl oder natrium < 130 meq/l) besteht die empfehlung zur primären antibiotikaprophylaxe (gerbes 2011; runyon 2013; › tab. 2.37) . patienten mit leberzirrhose, die aufgrund gastrointestinaler blutung stationär aufgenommen werden, stellen eine weitere gruppe für eine antibiotikaprophylaxe dar. studien haben eine um bis zu 45 % erhöhte infektionsinzidenz sowie ein deutlich erhöhtes rezidivblutungsrisiko gezeigt. deshalb ist die sofortige antibiotikagabe (noch vor der endoskopischen diagnostik) mit einem fluorchinolon oder einem cephalosporin der dritten generation für 7 d indiziert (alaniz 2009; allison et al. 2009 ; gerbes 2011; rosien 2011). 2.10.11 antibiotikaprophylaxe in der hämatologie/onkologie (erwachsene) hämatologisch-onkologische patienten können ein erhöhtes bis sehr hohes risiko für bakterielle infektionen haben, verursacht durch die erkrankung selbst und/oder als nebenwirkung der antineoplastischen therapie (donnelly, blijlevens und van der velden 2014; krinko 2010; wilson 2014) (› tab. 2.38) . der wichtigste risikofaktor für die akquisition bakterieller infektionen bei hämatologisch-onkologischen patienten ist die chemotherapie-assoziierte neutropenie (granulozytopenie). wenn eine absolute neutrophilenzahl von < 500/µl oder von < 1 000/µl mit innerhalb von 2 d zu erwartendem abfall auf < 500/µl vorliegt, kann anhand der zu erwartenden dauer der neutropenie eine risikoklassifikation vorgenommen werden (› tab. 2.39) . die gefahr von infektionen ist noch höher, wenn praktisch keine granulozyten (granulozyten < 100 µl) mehr nachweisbar sind (freifeld et al. 2011 ). eine noch übersichtlichere einteilung unterscheidet zwischen einer dauer der granulozytopenie von ≤ oder > 7 tagen (neumann et al. 2013 die mediane dauer der granulozytopenie ist bei patienten nach hochdosistherapie und autologer stammzelltransplantation deutlich kürzer als bei allogener transplantation. eine antibiotikaprophylaxe wird in den jeweiligen behandlungsprotokollen jedoch meist ebenso empfohlen wie die gabe von granulozyten-stimulierendem faktor (g-csf) zur verkürzung der granulozytopenie (kiefer et al. 2006; montemurro et al. 2007 ). standard ist die gabe systemisch wirksamer fluorchinolone (fch; ciprofloxacin, levofloxacin). alternativ kann trimethoprim-sulfamethoxazol (tmp-smz, cotrimoxazol) eingesetzt werden. bei trimethoprim-sulfamethoxazol müssen resistente e. coli, die unzureichende pseudomonas-wirksamkeit, die schlechtere verträglichkeit (gastrointestinal, haut) und die verlängerte granulozytopeniedauer bei prolongierter täglicher einnahme bedacht werden. die orale gabe nicht resorbierbarer antibiotika (aminoglykoside und/oder polymyxin) ist in diesem klinischen kontext inzwischen obsolet. zur antibiotikaprophylaxe bei neutropenischen patienten gibt es umfangreiche studien und metaanalysen sowie leitlinien der fachgesellschaften (krüger et al. 2005; neumann et al. 2013 (imran et al. 2008; neumann et al. 2014) die prophylaktische antibiotikagabe ist mit einem nicht signifikant erhöhten auftreten von nebenwirkungen assoziiert, im ein-zelfall (sehr selten!) können fluorchinolone jedoch auch schwere unerwünschte ereignisse auslösen (z. b. lebertoxizität, neurotoxizität, achillessehnen-ruptur). ungünstige interaktionen mit der chemotherapie sind möglich (z. b. hepatotoxizität; alshammari et al. 2014 gegenwärtig gibt es keinen vollständigen internationalen konsensus zum einsatz von antibiotika zur prophylaxe bakterieller infektionen bei patienten mit chemotherapie-bedingter neutropenie, es liegen jedoch leitlinien der fachgesellschaft hierzu vor (krüger et al. 2005; neumann et al. 2013) . die arbeitsgemeinschaft infektionen in der hämatologie und onkologie (agiho) empfiehlt bei hochrisikopatienten (› tab. 2.40 ) und bei patienten unter allogener stammzelltransplantation die gabe von fch (krüger et al. 2005; neumann et al. 2013) . alternativ kann bei unverträglichkeit cotrimoxazol (tmp-smz) gegeben werden. die fch-prophylaxe ist aber überlegen und sollte bevorzugt werden (cruciani et al. 1996; engels, lau und barza 1998; gafter-gvili et al. 2012; neumann et al. 2013) . bei patienten, die analog eines studienprotokolls behandelt werden, empfiehlt sich die anlehnung an das protokoll, wobei kritische rückmeldungen von klinischen infektiologen an die jeweilige studienleitungen sicher erwünscht sind. die antibakterielle prophylaxe wird entweder nach granulozytärer regeneration oder mit beginn einer antibiotikatherapie, z. b. bei fieber unbekannter ursache, beendet (krüger et al. 2010; link et al. 2003 ). die kombination der fch-prophylaxe mit präparaten die eine bessere wirksamkeit gegen grampositive infektionserreger aufweisen, wird nicht empfohlen (freifeld et al. 2011; neumann et al. 2013) . zwei sonderfälle der antibakteriellen prophylaxe betreffen patienten nach allogener stammzelltransplantation: • die prophylaktische gabe von metronidazol bis zum tag +30 nach transplantation wird in einigen zentren zur prophylaxe einer graft-versus-host-erkrankung durchgeführt (beelen et al. 1999 ). • patienten mit chronischer gvhd und zusätzlicher immunsuppression sind auch besonders anfällig für infektionen mit grampositiven, bekapselten bakterien (atkinson et al. 1979; ochs et al. 1995) . daher kann nach individualmedizinischer abwägung auch hier eine antibiotikaprophylaxe (z. b. mit penicillin) indiziert sein (krüger et al. 2005) . randomisierte studien liegen für diese indikation jedoch nicht vor. ein weiterer ansatz zur reduktion von infektionen im verlauf der chemotherapie-induzierten granulozytopenie ist die verkürzung der granulozytopenie-dauer den einsatz hämatopoetischer wachstumsfaktoren. die aktuelle empfehlung der expertengruppe der agiho (vehrenschild et al. 2014 ) sieht den einsatz von g-csf bereits oberhalb eines fieberrisikos (während der granulozytopenie) von 20 % vor, wobei ein so breiter einsatz von g-csf sicher nicht von allen onkologen befürwortet wird. aussagekräftige studien, die eine fch prophylaxe mit der gabe von g-csf vergleichen, liegen nicht vor. vor mehr als 25 jahren haben stoutenbeek et al. (1983) die sdd als eine methode zur prävention nosokomialer infektionen in die intensivmedizin eingeführt. nosokomiale (beatmungsassoziierte) pneumonien (vap) sind in bestimmten hochrisikogruppen häufige und potenziell lebensbe-drohliche komplikationen und können zur quelle einer sekundären bakteriämie/sepsis werden (› kap. 4.5 das prinzip der klassischen sdd ist die applikation von nichtresorbierbaren antibiotika und antimykotika in den oberen gastrointestinaltrakt. dadurch wird eine lokale, selektive wirkung gegen potenziell pathogene mikroorganismen ohne beeinflussung der anaeroben darmflora erreicht. die these, die dem verfahren zugrunde liegt, ist, dass durch die sdd die kolonisationsresistenz gestärkt wird. der begriff der kolonisationsresistenz geht auf van der waaij zurück und meint, dass verschiedene faktoren von seiten des wirts und der darmflora die kolonisation durch potenziell pathogene mikroorganismen verhindern. demnach könnten durch den einsatz selektiv wirksamer antibiotika die physiologische flora geschont und gleichzeitig potenziell pathogene mikroorganismen eliminiert werden. dieses vorgehen führt nach van der waaij (1983) zur geringeren ausbreitung (multi) resistenter, potenziell pathogener mikroorganismen und zur infektionsprophylaxe bei patienten mit beeinträchtigter immun abwehr (van der waaij, berghuis-de vries und lekkerkerk 1971). nach einer vielzahl tierexperimenteller untersuchungen fand die sdd zunächst bei onkologischen patienten mit granulozytopenie anwendung. entscheidende klinische verbreitung erlangte das verfahren seit mitte der 1980er-jahre bei beatmeten intensivpatienten. zum thema sdd bzw. selektive orale dekontamination (sod) existieren nunmehr mehr als 50 randomisierte klinische studien und mehr als 10 metaanalysen. um die primär endogenen infektionen in der prophylaxe mit zu erfassen, beinhaltet die sdd neben der topischen gabe von tobramycin, colistin und amphotericin b auch die i. v. applikation von cefotaxim über die ersten 4 behandlungstage. medikamente und indikationen für eine antibiotikaprophylaxe bei neutropenie (krüger et al . 2005 (marshall et al. 1993 ). in der annahme, dass beim intensivpatienten durch reduzierte enterale ernährung, subileus und eine vielzahl anderer faktoren die translokation von endotoxin und bakterien aus den gastrointestinaltrakt gesteigert ist, messen viele autoren der enteralen komponente der sdd besondere bedeutung bei. zusätzlich werden die substanzen bei der klassischen sdd den beatmeten patienten als paste oral in die wangentaschen appliziert. dabei soll im falle einer stillen aspiration erregerhaltigen materials an der blockung des tubus vorbei die erregerlast als wesentlicher faktor der entstehung der beatmungsassoziierten pneumonie (vap, › kap. 5.9) reduziert werden. in der klinischen routine der intensivmedizin gibt es eine vielzahl von varianten der sdd, wobei die alleinige selektive orale dekontamination (sod) die am häufigsten untersuchte ist (dombrowski et al. 2013) . ihr ziel ist die reduktion der häufigkeit der vap und die reduktion der erregertransmission von patient zu patient aus dem oropharynx. die sdd ist wahrscheinlich die am besten untersuchte medikamentöse strategie zur infektionsprophylaxe. obwohl in einer vielzahl von studien die wirksamkeit von sod und sdd bei verschiedenen beatmeten patientengruppen nachgewiesen wurde, wird das verfahren bisher kaum in entsprechenden leitlinien empfohlen. grund ist nach ansicht der autoren der leitlinien neben der weiterhin unsicheren studienlage für einzelne erkrankungen, z. b. der sepsis, die potenzielle gefahr der selektion von mre (dellinger et al. 2008; guideline 2005; krinko 2013) . ebenso argumentieren die praktisch tätigen intensivmediziner. bei einer umfrage auf englischen intensivstationen gaben 95 % der befragten an, wegen der unzureichenden evidenz und der gefahr der resistenzentwicklung auf die sdd zu verzichten (bastin und ryanna 2009) . inzwischen konnte in prospektiven rct gezeigt werden, dass durch sdd die sterblichkeit beatmeter intensivpatienten reduziert wird. bei 546 chirurgischen und traumapatienten zeigten sich in der sdd-gruppe weniger infektionen, weniger organversagen und bei patienten mit apache-ii-score von 20-29 bei aufnahme auf die intensivstation eine niedrigere sterblichkeit. surveillancekulturen erbrachten keinen hinweis auf höhere resistenzraten in der sdd-gruppe, was in übereinstimmung zu langzeitbeobachtungen mit sdd bei beatmeten patienten steht (dannemann et al. 2013; leone et al. 2003 , de jonge 2005 , heininger et al. 2006 ). abweichend von den meisten sdd-studien erfolgten die systemische antibiotikaprophylaxe über 4 d mit ciprofloxacin und die topische therapie mit gentamicin und polymyxin ohne ein antimykotikum (krueger et al. 2002) . in einer weiteren studie bei 934 chirurgischen und internistischen intensivpatienten zeigte sich eine signifikant geringere intensiv-und krankenhaussterblichkeit unter sdd. die randomisierung erfolgte stationsbezogen, um effekte durch geringere erregertransmissionsraten zwischen den sdd-und kontrollpatienten zu minimieren (de jonge et al. 2003) . ein ähnliches, allerdings multizentrisches studiendesign an über 6 000 patienten untersuchte zusätzlich die wirksamkeit alleiniger sod ohne systemische antibiotikaprophylaxe. weder die sdd noch die sod zeigten in der studie einen überlebensvorteil. in dieser studie hatten jedoch beide behandlungsgruppen ein primär höheres sterberisiko (höheres alter, höherer apache-ii-score). die logistische regressionsanalyse erbrachte einen signifikanten überlebensvorteil für die patienten der sdd-gruppe (de smet et al. 2009 ). eine metaanalyse mit einschluss von > 8 000 intensivpatienten zeigte, dass mit sdd die rate gramnegativer bakteriämien und die sterblichkeit reduziert werden können. am deutlichsten ist der effekt bei der klassischen sdd. die autoren kommen auf eine number needed to treat (nnt) von 22 für einen geretteten patienten (silvestri et al. 2007 ). in einer weiteren metaanalyse zum nutzen des kompletten regimes der sdd, das neben der antibiotikaprophylaxe ein effektives hygieneregime und surveillancekulturen von rachen und stuhl beinhaltete, ergaben sich bei der analyse von 21 rcts eine signifikante reduktion der mortalität und eine nnt von 18 (silvestri et al. 2009 historie bereits in der bibel wird die absonderung von kranken mit isolierung für 7 d bei weißen hautflecken erwähnt (leviticus 13:4). später wurde in anlehnung an die christliche fastenzeit von 40 d (= quarantina di giorni), eine absonderung über diesen "reinigenden" zeitraum ("quarantenne") durchgeführt. so war eine der schutzmaßnahmen der republik venedig das festhalten von eintreffenden schiffen für 40 d an der isola lazaretto nuovo (isolation), um sicher zu stellen, dass keine krankheiten in den lagunenstaat eingeschleppt werden. das erfolgte jedoch alles ohne kenntnis der hygienischen grundlagen von infektionserkrankungen. die pest im mittelalter ist ein beispiel für eine erfolgreiche empirische isolierung ohne wissenschaftliche grundlage. selbst in der gegenwart unterscheiden sich die reaktionen der bevölkerung und auch der krankenhausmitarbeiter kaum von denen früherer generationen. nach dem ersten ausbruch der sog. schweinegrippe flohen tausende von mexikanern in die usa. hiv-infizierte mitmenschen wurden in den anfängen der aids-pandemie sozial isoliert. die bemühungen anfang des 20. jahrhunderts, die ausbreitung der tbc zu reduzieren, sind ein weiteres beispiel empirischer infektionsmedizin: die meisten sanatorien liegen in den bergen mit einer starken, für mykobakterien letalen uv-strahlung. viele erkenntnisse gingen mangels dokumentation verloren und mussten z. b. im rahmen der tuberkulose-epidemien in den usa teilweise neu erarbeitet werden. 1877 sind aus den usa die ersten medizinischen dokumente zur isolierung von patienten erschienen (garner 1996) . krankenhäusern wurde empfohlen, patienten mit infektionskrankheiten in getrennten gebäuden (sog. pavillonbauweise) zu hospitalisieren. noch heute finden sich beispiele der pavillonbauweise aus jener zeit, z. b. ein teil der charité in berlin oder viele in der gründerzeit (1849-1873) erbaute krankenhäuser in wien. wissenschaftliche grundlagen der isolierung sind ein junges primär aus der pflege hervorgegangenes gebiet. dabei wurde viel an fachwissen eingebracht, das nicht aus randomisierten studien, sondern aus analysen der täglichen praxis resultierte. das erste auf erkenntnisbasis von bakteriellen erkrankungen konzipierte isolierungsmodell wurde 1910 publiziert. danach mussten sich mitarbeiter die hände nach patientenkontakt desinfizierend waschen sowie gegenstände und apparate desinfizieren, bevor sie aus dem bereich des infizierten patienten entfernt wurden. hierfür wurde erstmals der begriff "barrier nursing" verwendet. mit der einführung der sulfonamide und antibiotika wurden bakterielle infektionskrankheiten erstmals kausal behandelbar. diese entwicklung führte in den 50er-jahren des 20. jahrhunderts in den usa und vielen europäischen ländern zur schließung von isolierabteilungen. nach 1960 wurden im gegensatz zu europa, wo zumindest isolierzimmer auf stationen wieder berücksichtigt wurden, die meisten infektionskrankheiten in üblichen krankenzimmern behandelt. 1970 erschien als reaktion vor allem auf die zunahme der ni das erste detaillierte manual der cdc "isolation techniques for use in hospitals to assist general hospitals with isolation precautions". es wurde 1975 und 1996 überarbeitet und erschien zuletzt 2007 als "guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings" der cdc (siegel et al. 2007 ). die regelmäßige überarbeitung belegt die zentrale bedeutung dieser empfehlungen für die infektionsprävention. durch die definition der "standard precautions" (basishygiene) und die einführung der 3 transmissionsbasierten isolierungstypen "airborne precautions" (übertragung über die luft; inzwischen als "airborne infection isolation precautions" bezeichnet), "droplet precautions" (übertragung durch tröpfchen) und "contact precautions" (übertragung durch direkten kontakt) wurde ein klares handlungskonzept von großer praktischer bedeutung eingeführt. die protektive isolierung (umkehrisolierung) ist in die allgemeinen maßnahmen implementiert worden. darauf aufbauend erschienen empfehlun-gen und stellungnahmen zu isolierungsmaßnahmen bei speziellen erregern wie z. b. ebola und enterovirus-d68 (muscarella 2014 die übertragung kann (dominierend) durch direkten oder indirekten kontakt, ferner durch tröpfchen (reichweite der > 5 µm tröpfchen bis ≈ 1 m), aerogen (reichweite der nuklei < 5 µm bis ≈ 3 m, verbleib in der raumluft mehrere stunden), parenteral oder über vektoren (sehr selten nosokomial) erfolgen. die direkte kontaktübertragung erfolgt von mensch zu mensch, die indirekte über die hände nach berühren kontaminierter gegenstände bzw. flächen bei unterlassener oder fehlerhafter händedesinfektion. zur tröpfchenübertragung kann es bei aerosol erzeugenden maßnahmen kommen. zu den über tröpfchen übertragbaren erregern gehören influenza-, adeno-, rhino-und sars-assoziierte coronaviren, b. pertussis, m. pneumoniae, n. meningitidis, legionella spp. und gruppe a-streptokokken. nasale s. aureus-träger können bei vorliegen einer virusinfektion des oberen respirationstrakts diesen erreger über 1 m streuen (sheretz et al. 1996) . die vorstellung, dass durch verdunstung des flüssigkeitsanteils die im tröpfchen enthaltenen erreger als sehr kleine tröpfchenkerne (droplet nuclei) lange in der schwebephase bleiben und damit das risiko einer ansteckung erhöhen, wird für die lungentuberkulose angenommen, lässt sich aber nicht unbedingt auf andere erreger übertragen (cole und cook 1998) . aerogene übertragung: nur bei wenigen erregern ist der nachweis gelungen, dass sie als partikel über die luft übertragen werden. beispiel ist das varicella-zoster-virus, bei dem ansteckungen bis zur distanz von 10 m beschrieben sind und das noch in 5 m entfernung von der infektionsquelle nachweisbar war (sawyer et al. 1994 ). aerogene übertragung findet auch bei masern (bloch et al. 1985) , m. tuberculosis und schimmelpilzsporen (aspergillus spp.) statt (brenier-pinchart et al. 2009; haley et al. 1989 ). die ausbreitung von viren über den luftweg wurde u. a. für influenza-, noro-2 und rotaviren beschrieben (chadwick und mccann 1994) , allerdings nur im bereich des patientenzimmers. bei neu auftretenden krankheitserregern müssen, sofern die eigenschaften nicht genau bekannt sind, je nach der gefährlichkeit u. u. maximale präventionsmaßnahmen ergriffen werden. ein beispiel sind humane bocaviren, die atemwegsinfektionen auslösen, und sich in bezug auf ihre tenazität wahrscheinlich ähnlich wie das humane parvovirus b19 verhalten (schildgen et al. 2008 grundsätzlich können bei der isolierung vorliegende besonderheiten des patienten nicht berücksichtigt werden, weil das eine hohe präsenz der krankenhaushygiene vor ort voraussetzt bzw. einen hohen andauernden aufwand für die praxisanleitung des personals erfordert. deshalb wurden transmissionsbasierte isolierungsmodelle eingeführt, die sich bei gleichzeitiger einhaltung der basishygiene als ausreichend effektiv erwiesen haben. da die meisten krankenhäuser über zu wenige einzelzimmer/isolierzimmer verfügen (haertel et al. 2013) , müssen verschiedene gesichtspunkte bei der unterbringung der patienten berücksichtigt werden. bei begründetem verdacht auf eine übertragbare erkrankung, bei der vermutlich die maßnahmen der basishygiene nicht ausreichen, sollen entsprechend der verdachtsdiagnose auf der basis einer risikoanalyse spezifische schutzmaßnahmen eingeleitet werden. (bruns et al . 2013; diedrich 2012; kiehl 2009 , siegel et al . 2007 bei einer nosokomialen häufung (ausbruch) sollte auch die laufende desinfektion so durchgeführt werden, dass der jeweilige erreger wie bei der schlussdesinfektion erfasst wird. dies kann ggf. mit einer deutlichen raumluftbelastung verbunden sein, daher ist während und kurz nach der ausführung dieser arbeiten auf eine gründliche lüftung der patientenzimmer zu achten. alle potenziell kontaminierten materialien, die aus dem patientenzimmer entfernt werden müssen, müssen so transportiert werden, dass eine freisetzung des jeweiligen erregers außerhalb des patientenzimmers sicher unterbunden wird. bettpfannen oder urinflaschen müssen unmittelbar und direkt in das steckbecken-spülgerät gegeben werden und dürfen keinesfalls im unreinen arbeitsraum "zwischengelagert" werden. dies setzt voraus, dass steckbeckenspülgeräte in ausreichender zahl auf der jeweiligen station und möglichst in der nähe der isolierten patienten vorhanden sind. sabine wicker, axel kramer und frank-albert pitten zur prävention sowohl von ni als auch von arbeitsbedingten infektionen der mitarbeiter ist die enge zusammenarbeit von krankenhaushygiene und betriebsärzten unabdingbar. krankenhaushygiene und betriebsärzte sollten eine kooperierende einheit bilden, um erfolgreich zu sein. in der universitätsmedizin greifswald findet aus diesem grund der monatliche jour fixe des ärztlichen direktors gemeinsam mit der krankenhaushygiene, dem betriebsärztlichen dienst und der apotheke statt, um das gemeinsame vorgehen abzustimmen und zu koordinieren. am universitätsklinikum frankfurt gewährleistet die enge kooperation zwischen krankenhaushygiene und betriebsärztlichem dienst, dass schnell und effektiv auf infektionsgefährdungen reagiert werden kann und dass präventive maßnahmen sowohl für patienten als auch für das medizinische personal implementiert sind. schutzimpfungen gehören zu den wichtigsten präventiven maßnahmen in der medizin, da sie nicht nur individualschutz bewirken, sondern bei erreichen hoher impfquoten die eliminierung und sogar ausrottung (eradikation) einzelner krankheitserreger regional bzw. weltweit ermöglichen. der impfschutz reicht allerdings in der deutschen bevölkerung bei weitem nicht aus, um das auftreten bzw. die weiterverbreitung bestimmter infektionskrankheiten zu verhindern. für medizinisches personal ist die frage nach dem durchimpfungsgrad aufgrund der arbeitsbedingten infektionsgefährdung und der patientengefährdung durch ungeimpftes personal besonders brisant. leider ist die akzeptanz von schutzimpfungen im medizinischen bereich unzureichend. während die hepatitis b-impfung von den medizinischen beschäftigten meist sehr gut akzeptiert und auch aktiv nachgefragt wird, sind die impfquoten z. b. bzgl. influenza und pertussis alarmierend niedrig (› kap. 5.24, › kap. 7.5) . hier liegt die wichtige aufgabe der betriebsärzte, die aufklärung bezüglich der impfpräventablen infektionen zu verbessern. in deutschland besteht keine impfpflicht. impfungen werden von den obersten gesundheitsbehörden der länder entsprechend § 20 abs. 3 ifsg "öffentlich empfohlen". darüber hinaus müssen al-2 le beschäftigten gemäß trba 250/bgr 250 über notwendige immunisierungsmaßnahmen bei tätigkeitsaufnahme und aus gegebener veranlassung informiert werden. der gesundheitsschutz am arbeitsplatz wird primär durch die verordnung zur arbeitsmedizinischen vorsorge (arbmedvv) und ergänzend durch die biostoffverordnung (biostoffv) geregelt. impfungen sind bestandteil der arbeitsmedizinischen vorsorge und den beschäftigten anzubieten, soweit das risiko einer infektion tätigkeitsbedingt und im vergleich zur allgemeinbevölkerung erhöht ist und noch kein ausreichender immunschutz vorliegt; die kosten trägt der arbeitgeber. die nachfolgenden ausführungen orientieren sich an den empfehlungen der stiko (2014), die jährlich aktualisiert werden. zu den impfleistungen des arztes gehören neben der durchführung der impfung die erhebung der anamnese (frage nach kontraindikationen), die feststellung der aktuellen befindlichkeit zum ausschluss akuter erkrankungen, die umfassende aufklärung des impflings über die zu verhütende krankheit und ihre behandlungsmöglichkeiten, den nutzen der schutzimpfung für das individuum und die allgemeinheit, die art des impfstoffs, die durchführung der impfung, beginn und dauer des impfschutzes, verhalten nach der impfung, kontraindikationen, mögliche nebenwirkungen bzw. impfkomplikationen und termine für folge-und auffrischimpfungen (stiko 2014). die aufklärung sollte in den unterlagen des impfarztes dokumentiert werden. die kontraindikationen sind den fachinformationen der impfstoffhersteller zu entnehmen. nicht geimpft werden sollte personal mit akuten schweren erkrankungen (ausnahme postexpositionelle impfung). bei erlittenem impfschaden ist wegen der gesundheitlichen und wirtschaftlichen folgen ein antrag auf versorgung in der regel beim zuständigen versorgungsamt zu stellen. keine kontraindikationen sind z. b. banale infekte, auch mit subfebrilen temperaturen (≤ 38,5 °c), ein möglicher kontakt des impflings zu personen mit ansteckenden krankheiten, eine in der familie bekannte epilepsie und fieberkrämpfe in der anamnese des impflings, chronische erkrankungen, nicht progrediente erkrankungen des zns, ekzeme und andere dermatosen, lokalisierte hautinfektionen, behandlung mit antibiotika oder niedrigen kortikosteroiddosen, angeborene oder erworbene immundefekte bei impfung mit totimpfstoffen, schwangerschaft der mutter des impflings (varizellenimpfung nach risikoabwägung, es gilt jedoch zu bedenken, dass das risiko für ein konnatales varizellensyndrom bei einer seronegativen schwangeren mit kontakt zu ihrem ungeimpften und damit ansteckungsgefährdeten kind höher ist als das risiko einer solchen komplikation durch die impfung und ggf. die übertragung von impfvarizellen durch ihr kind, stiko 2014), neugeborenenikterus und frühgeburtlichkeit (stiko 2014). bei applikation von lebendimpfstoffen sollte bei immundefekten die konsultation des behandelnden arztes eingeholt werden und bei evtl. gegebener indikation eine serologische kontrolle des impferfolgs erfolgen. allergien gegen impfstoffbestandteile (z. b. neomycin, streptomycin, hühnerproteine) sind potenzielle kontraindikationen. in der schwangerschaft sollten möglichst nur dringend indizierte impfungen vorgenommen werden. impfungen mit einem lebendimpfstoff, wie z. b. gegen masern-mumps-röteln (mmr) oder varizellen, sind in der schwangerschaft grundsätzlich kontraindiziert, wobei eine versehentlich in der schwangerschaft durchgeführte impfung mit lebendimpfstoffen jedoch keine indikation für einen schwangerschaftsabbruch darstellt. alle schwangere ab dem 2. trimenon und bei erhöhter gesundheitlicher gefährdung ab dem 1. trimenon sollten sich gegen influenza impfen lassen (stiko 2014). schwangere sind die hauptzielgruppe der who für die influenzaimpfung, da die impfung sowohl die mutter als auch später das neugeborene schützt. in mehreren ländern wird mittlerweile auch empfohlen, schwangere gegen pertussis zu impfen. als optimaler zeitpunkt wird die 27.-36. schwangerschaftswoche angegeben. der plazentare transfer maternaler antikörper kann einen passiven schutz der neugeborenen und säuglinge gegen pertussis für ca. 2-6 monate bewirken. das amerikanische advisory committee on immunization practices (acip) der cdc empfiehlt seit 2013, dass frauen in jeder schwangerschaft unabhängig vom impfstatus eine pertussisimpfung mit tdap erhalten sollen, um die neugeborenen vor schwerwiegenden verläufen einer pertussisinfektion zu schützen. zu den mindestabständen zwischen zwei lebendimpfungen sowie zur möglichkeit der koadministration von impfstoffen sind die fachinformationen des jeweiligen impfstoffs zu beachten. für einen lang dauernden impfschutz ist es von besonderer bedeutung, dass bei der grundimmunisierung der empfohlene mindestabstand zwischen vorletzter und letzter impfung nicht unterschritten wird (stiko 2014). • bei gabe von lebendimpfstoffen ist zu beachten, dass diese simultan oder in der regel in einem mindestabstand von 4 wochen zu verabreichen sind -unter der voraussetzung, dass die impfreaktion vollständig abgeklungen ist und keine komplikationen aufgetreten sind. • bei schutzimpfungen mit totimpfstoffen müssen keine abstände, auch nicht zu lebendimpfstoffen, beachtet werden. • nach gabe von immunglobulinen sollten in einem zeitraum von 3 monaten keine parenteral zu verabreichenden lebendimpfstoffe gegeben werden (stiko 2014). medizinisches personal sollte bereits vor dem ersten patientenkontakt über eine ausreichende immunität gegen impfpräventable arbeitsmedizinisch und/oder hygienisch indizierte impfungen verfügen. zum aufbau eines sicheren impfschutzes sollen die schutzimpfungen nach dem von der stiko empfohlenen impfkalender durchgeführt werden. bei nichteinhaltung empfohlener impfabstände muss mit dem impfschema nicht neu begonnen werden, da jede impfung zählt. so reicht auch nach einer über 10 jahre zurückliegenden grundimmunisierung gegen diphtherie und tetanus eine boosterimpfung aus (cave: pertussis nicht vergessen!). mindestabstände zwischen den impfungen sind jedoch entsprechend fachinformation einzuhalten. die impfung ist einschließlich chargen-nummer und handelsname des impfstoffs im impfausweis und in der dokumentation des impfenden arztes zu dokumentieren. bei nichtvorlage des impfausweises ist eine impfbescheinigung auszustellen. jede ernsthafte gesundheitliche schädigung im zeitlichen zusammenhang mit einer impfung ist, wenn es gleichzeitig keine andere plausible erklärung für die erkrankung gibt, meldepflichtig. in extrem seltenen fällen werden schwere unerwünschte wirkungen beobachtet, die sofort diagnostisch abzuklären sind und umgehend entweder über das gesundheitsamt oder direkt dem bfarm und/oder dem paul ehrlich institut (formular oder online) gemeldet werden müssen. wird der impfende oder behandelnde arzt vom patienten bzw. dessen angehörigen auf den möglichen zusammenhang hingewiesen, ist die im ifsg festgelegte meldepflicht für alle verdachtsfälle einer impfkomplikation einzuhalten. zugleich ist der geschädigte auf die möglichkeit einer antragstellung auf versorgung hinzuweisen. die im einzelfall gebotenen maßnahmen zur immunisierung sind im einvernehmen mit dem arzt, der die arbeitsmedizinische vorsorge durchführt, festzulegen. die immunisierung ist kostenlos zu ermöglichen (› kap. 5.29). empfohlene schutzimpfungen: folgende impfungen werden für das gesamte medizinische personal einschließlich auszubildenden, praktikanten, studenten, reinigungspersonal, hebammen, externen dienstleistern mit patientenkontakt (z. b. fußpflege, physiotherapie) empfohlen, sofern kein impfschutz oder ein unklarer impfstatus vorliegt: tetanus, diphtherie, poliomyelitis, hepatitis a, hepatitis b, virusgrippe (influenza), pertussis, masern, mumps, röteln und varizellen. es wird allen beschäftigten im gesundheitsdienst dringend empfohlen, von der möglichkeit arbeitsmedizinisch und/oder hygienisch indizierter impfungen gebrauch zu machen. impfungen aufgrund eines erhöhten beruflichen risikos sind in den stiko-empfehlungen mit "b" gekennzeichnet. die impfungen gemäß stiko dienen sowohl dem schutz der beschäftigten als auch dem drittschutz (impfungen aus hygienischer indikation). bei medizinischem personal, das immuninkompetente patienten betreut, sollte der nachweis eines effektiven impfschutzes voraussetzung für die beschäftigung in einem risikobereich sein (wicker et al. 2013) für masern z. b. wird das infektionsrisiko von medizinischem personal 2-bis 19-mal so hoch geschätzt wie in der normalbevölkerung (wicker et al. 2013) . daher sollten alle medizinischen beschäftigten über eine sichere masernimmunität verfügen. varizellen: bei krankenhauspersonal mit unklarer varizellenanamnese muss serologisch der antikörperstatus abgeklärt werden. bei mangelndem schutz ist die impfung indiziert, das trifft insbesondere für seronegatives personal in den bereichen pädiatrie, onkologie, gynäkologie/geburtshilfe, intensivmedizin und betreuung immunsupprimierter patienten zu. pertussis: aufgrund der verlagerung der pertussis in das erwachsenenalter steigt die gefährdung für ungeimpfte, sodass wiederholt übertragungen auf mitarbeiter im gesundheitswesen beobachtet wurden (kuncio et al. 2014; wicker und rose 2010; zivna et al. 2007 ). da bei der versorgung erkrankter kinder die exposition weitgehend unvermeidbar ist und das prä-und postexpositionelle management sehr ressourcenintensiv ist, ist die schutzimpfung die kostengünstigste und sicherste alternative (daskalaki et al. 2007) . pneumokokken: mitarbeiter ≥ 60 jahren oder mit erhöhter gesundheitlicher gefährdung infolge einer grundkrankheit sollten gegen pneumokokken-infektionen immunisiert werden (stiko 2014). da die anzahl älterer mitarbeiter im gesundheitswesen und in der pflege in zukunft deutlich ansteigen wird, zeichnet sich erheblicher bedarf ab. die schutzimpfung gegen tbc auf der grundlage der bcg-impfung wird in deutschland nicht mehr empfohlen, da sie keinen sicheren schutz vor der infektion bietet. die schutzimpfung gegen die saisonale influenza wird für das gesamte personal jährlich dringend empfohlen. hierbei ist der jeweils aktuelle impfstoff, der die aktuellen sai-2 sonalen varianten umfasst, anzuwenden. die jährliche impfung wird auch dann empfohlen, wenn die antigenzusammensetzung des impfstoffs gegenüber der vorhergehenden saison unverändert ist. bei etwa 60 % der geimpften im alter von 18-65 jahren wird eine schutzwirkung erreicht (cox, brokstad und ogra 2004; osterholm et al. 2012) . die impfquoten gegen influenza sind in den meisten einrichtungen des gesundheitswesens deutlich zu niedrig. in hessen gaben über 80 % der befragten krankenhäuser influenzaimpfquoten beim medizinischen personal < 20 % an, wobei lediglich rund 70 % der befragten krankenhäuser die impfquote erfasst haben (wicker et al. 2012 ). dabei kommt einer immunisierung des medizinischen personals gerade zum patientenschutz eine besondere bedeutung zu, da gesichert ist, dass influenza-viren bereits in der inkubationszeit übertragen werden können. gewöhnlich wird seitens der betriebsärzte der krankenhäuser jeweils im herbst dem medizinischen personal die influenza-schutzimpfung angeboten. die erfahrung zeigt jedoch, dass das bloße angebot keinesfalls ausreicht, da die influenzaimpfung nur verhältnismäßig selten in anspruch genommen wird. besonders hohe impfquoten können nach eigener erfahrung erzielt werden, wenn der betriebsarzt auf die mitarbeiter zugeht, d. h. die impfung auf den stationen bzw. in den einzelnen pflegeeinheiten und funktionsbereichen (z. b. op, zna) anbietet. wenn auch die effektivität und effizienz der influenzaimpfung nicht optimal und ebenso abhängig vom impfstamm ist und selbst gegenüber dem tatsächlich zirkulierenden stamm u. u. keinen vollständigen schutz gewährleistet, war z. b. die anzahl der an a/h1n1 erkrankten bei geimpftem gesundheitspersonal signifikant geringer (chu et al. 2012 ). personen mit anatomischer oder funktioneller asplenie sollte die impfung gegen h. influenzae typ b, pneumokokken und meningokokken empfohlen werden. meningokokken: bei ausbrüchen oder regionalen häufungen durch n. meningitidis kann von den gesundheitsbehörden in ergänzung zur antibiotikaprophylaxe eine impfempfehlung gegeben werden. bezüglich der anwendungshinweise der schutzimpfungen wird auf die stiko-empfehlungen verwiesen. hinweise zur postexpositionellen impfung bzw. anderen maßnahmen der speziellen prophylaxe übertragbarer krankheiten finden sich in den jeweils aktuellen stiko-empfehlungen. als präventionsmaßnahme werden hier die postexpositionelle impfung, die passive immunisierung durch die gabe von immunglobulinen sowie die chemoprophylaxe aufgeführt (stiko 2014). die jeweiligen indikationen und die anwendungshinweise sind tabellarisch zusammengefasst, um einen schnellen überblick zu gewährleisten (aktuell tab. 3 der stiko empfehlungen 2014). empfehlungen zur postexpositionellen prophylaxe (pep) einer hiv-infektion werden von der deutschen aids-gesellschaft in zusammenarbeit mit der österreichischen aids-gesellschaft herausgegeben und regelmäßig aktualisiert (daig 2013). grundsätzlich sind folgende schutzmaßnahmen einzuhalten (details werden in den speziellen kapiteln erläutert): • anwendung medizinischer schutzhandschuhe und auswahl walter a. maier, weitergeführt durch michael k. faulde bei gesundheitsschädlingen im engeren sinn handelt es sich um gliederoder nagetiere, die krankheitserreger entweder als blutsaugende ektoparasiten direkt übertragen (aktive vektoren) oder als mechanische erregerverschlepper (passive vektoren bzw. hygieneschädlinge) im menschlichen umfeld verbreiten können. bei der aktiven erregerübertragung werden krankheitserreger definitionsgemäß beim hämophagen stech-(z. b. zecken) oder bissakt (z. b. milbenlarven mit paarigen mundwerkzeugen, nagetiere) in den wirtskörper eingebracht (faulde und freisel 2014; faulde und hoffmann 2001) . während bei den nagern bisse in aller regel akzidentell auftreten, fallen bei den arthropoden potenziell alle blutsaugenden arten in diese gruppe (faulde 2004; faulde und freisel 2014; faulde und hoffmann 2001; hoffmann 1992 die zahl der kopflausträger hat mit 3-4 % der gesamtbevölkerung relativ hohe werte erreicht, da sich p. capitis auch bei sehr gepflegten personen halten kann (burgess 2008 hygienemaßnahmen: gem. § 34 abs. 1 ifsg dürfen personen, bei denen ein kopflaus-befall festgestellt wurde, in den in § 33 ifsg genannten gemeinschaftseinrichtungen keine lehr-, erziehungs-, pflege-, aufsichts-oder sonstige tätigkeit ausüben, bei denen sie kontakt zu den dort betreuten haben, bis nach der entscheidung des behandelnden arztes eine weiterverbreitung der verlausung durch sie nicht mehr zu befürchten ist. dieses verbot gilt entsprechend für die in der einrichtung betreuten kinder und jugendlichen mit der maßgabe, dass sie die dem betrieb der gemeinschaftseinrichtung dienenden räume nicht betreten, einrichtungen der gemeinschaftseinrichtung nicht benutzen und an veranstaltungen der gemeinschaftseinrichtung nicht teilnehmen dürfen. gem. § 34 abs. 5 ifsg haben die genannten beschäftigten und die betreuten bzw. deren sorgeberechtigte über eine verlausung der gemeinschaftseinrichtung unverzüglich mitteilung zu machen. nach abs. 6 benachrichtigt darüber die leitung der einrichtung das zuständige gesundheitsamt. gemäß § 17(5) können die landesregierungen zur verhütung und bekämpfung übertragbarer krankheiten rechtsverordnungen über die feststellung und die bekämpfung von gesundheitsschädlingen, kopfläusen und krätzmilben erlassen. sie können die ermächtigung durch rechtsverordnung auch auf andere stellen übertragen. § 36 ifsg bestimmt außerdem, dass neben den in § 33 ifsg genannten einrichtungen auch häuser der stationären pflege und betreuung, wohnheime und massenunterkünfte der infektionshygienischen überwachung durch die gesundheitsämter unterliegen. treten z. b. in krankenhäusern, obdachlosenunterkünften oder gemeinschaftsunterkünften für asylbewerber, flüchtlinge und spätaussiedler läuse auf, gelten die obigen ausführungen in gleicher weise. behandlung: kopfläusekönnen nicht ohne gezielte behandlung des patienten beseitigt werden. hierzu werden primär insektizide eingesetzt, die aus der roten liste (antiparasitika) sowie der § 18 ifsg-mittelliste zu entnehmen sind. ob als langfristige alternative eine systemische oder topische applikation des antihelminthikums ivermectin zur läusebehandlung eingesetzt werden sollte, ist offen (burgess 2008; habedank 2010; ko und elston 2004) . die topische behandlung sollte grundsätzlich nach 8 d wiederholt werden, da die nissen nicht immer zuverlässig abgetötet werden (habedank 2010; maier 2002) . als wichtigster potenzieller vektor ist sie in mitteleuropa nur bei verwahrlosten personen oder personen ohne festen wohnsitz zu finden. im letzten jahrzehnt trat in den industrienationen besonders innerhalb der obdachlosenbevölkerung vor allem das wolhyni-oder 5-tage-fieber wieder vermehrt auf (burgess 2008; habedank 2010) . die kleiderlaus entwickelt sich weitgehend wie die kopflaus, ist aber etwas größer (3,3-3,8 mm) und robuster. sie hält sich im bereich der körperhaare zwischen körperoberfläche und unterwäsche auf, legt aber ihre nissen nicht an körperhaare, sondern an stofffasern (meist an nähten) ab. da die larven erst 1-2 wochen später schlüpfen, verhindert regelmäßiger wechsel der unterwäsche, wenigstens einmal wöchentlich, kleiderlausbefall sie ist mit 1,25-2 mm die kleinste laus des menschen und hat eine gedrungene gestalt. filzläuse sind träger als kopf-und kleiderläuse und lassen ihre mundwerkzeuge oft stundenlang an derselben stelle eingestochen. daher wechseln sie weniger leicht den wirt. ihr auftreten kann dem arzt als hinweis auf möglicherweise vorhandene geschlechtskrankheiten dienen (burgess 2008; habedank 2010; ko und elston 2004) . besonders in alten-und pflegeheimen jedoch ist durch den engen beruflichen kontakt zwischen senioren und deren pflegepersonal eine übertragung durch körperkontakt möglich. neben dem bevorzugten aufenthaltsort, den schamhaaren, findet man sie gelegentlich auch an den groben körperhaaren (bart-, augenbrauen-und achselhaare). bei befall bilden sich an der einstichstelle oft bläuliche hautveränderungen (maculae caeruleae), die diagnostische bedeutung haben können. die behandlung muss auf die empfindlichere haut der genitalregion rücksicht nehmen. insbesondere bei sexuell bedingter transmission müssen geschlechtspartner mitbehandelt werden. behandlung: schamläuse können nicht ohne gezielte behandlung des patienten beseitigt werden. hierzu werden primär insektizide eingesetzt, die aus der roten liste (antiparasitika) sowie der § 18 ifsg-mittelliste zu entnehmen sind. ob als langfristige alternative eine systemische oder topische applikation des antihelminthikums ivermectin zur läusebehandlung eingesetzt werden sollte, ist offen (burgess 2008; habedank 2010; ko und elston 2004 die blinden, fußlosen, etwa 5 mm großen flohlarven sind nicht blutsaugend und ernähren sich von hautschuppen sowie getrockneten blutresten am boden. die larven des menschenflohs (pulex irritans) finden sich außer in schweineställen auch in fußbodenritzen. in fugenlosen und sauberen böden können sie sich nicht halten. flohlarven verpuppen sich nach 2 häutungen und einer entwicklungsdauer von etwa 2 wochen. die puppenruhe beträgt zumindest 1-2 wochen und kann sich bis zu 6 monaten ausdehnen. das schlüpfen erfolgt auf einen außenreiz hin, z. b. durch vibrationen, die einen potenziellen wirt ankündigen. daher ist eine hungerquarantäne in befallenen gebäuden oder zimmern nur über sehr lange sperrzeiten möglich! adulte flöhe stechen gern an körperstellen, an denen die kleidung eng anliegt. dort hinterlassen sie oft "perlschnurartige" stichfolgen (die sog. "flohstich-leiter") mit heftigem juckreiz. um das scheinbar unerklärliche, plötzliche auftreten einer flohplage erklären zu können, müssen die flöhe identifiziert werden. mit einem geeigneten bestimmungsschlüssel ist das mit einem einfachen mikroskop zumindest für die gebäudebefallenden floharten in mitteleuropa möglich (weidner 1993 aufgrund ihrer unvollständigen metamorphose saugen alle fünf larvenstadien und adulte tiere blut. ihre wirtsspezifität ist nicht streng auf den menschen begrenzt. die blutmahlzeit dauert etwa 10 min, wonach sich die tiere wieder in ihrem schlupfwinkel verstecken. bettwanzen sind nachtaktiv und halten sich tagsüber in spalten, hinter tapeten, fußleisten, möbeln usw. auf. nach 30-35 d 2 ist ihre entwicklung abgeschlossen. danach leben sie noch etwa 1 jahr. bettwanzen weisen ein ausgeprägtes, wochen-bis monatelanges hungervermögen auf. durch die an den hüften der hinterbeine ausmündenden stinkdrüsen lässt sich ein bettwanzenbefall auch geruchlich feststellen. das wird beim bettwanzenmonitoring mit abgerichteten hunden genutzt, wobei die sensitivität und spezifität dieser methode noch ungeklärt ist (kuhn und van der pan 2014) . besonders in krankenhäusern und gesundheitseinrichtungen ist daher nie auf einen professionellen schädlingsbekämpfer zu verzichten. bei den aus tauben-und schwalbennestern vor allem in der kalten jahreszeit in bewohnte gebäude einwandernden wanzen handelt es sich in der regel um morphologisch sehr ähnliche vogelwanzen, die jedoch auch den menschen stechen können. vogelwanzen rufen keinen stationären gebäudebefall hervor, weshalb immer eine artdiagnostik erforderlich ist. die medizinische bedeutung von bettwanzen liegt vor allem in der teilweise heftigen stichreaktion, die von mensch zu mensch variieren kann, sowie in der teilweise starken psychischen belastung betroffener bei dauerbefall (harlan, faulde und baumann 2008) . extrem hohe befallsraten können in einzelfällen zu anämien führen. obwohl in bettwanzen bis heute etwa 40 erreger nachgewiesen werden konnten, gibt es bislang noch keine konkreten hinweise auf eine aktive erregerübertragung über bettwanzenstiche auf den menschen (doggett et al. 2012; harlan, faulde und baumann 2008) . sie werden bis heute als reine hämophage hygieneschädlingenicht als aktive vektoren -angesehen (harlan, faulde und baumann 2008; kuhn, van der pan 2014) . argas reflexus lebt oft im dachstuhl von altbauten, die von tauben besiedelt werden oder wurden. sie saugen in allen entwicklungsstadien blut, ähnlich wie bettwanzen, und werden gelegentlich mit diesen verwechselt. in befallenen häusern können sie viele jahre ohne nahrung überleben und befallen menschen in den oberen stockwerken, vor allem wenn keine tauben mehr als blutquelle zur verfügung stehen. die taubenzeckenbekämpfung gestaltet sich sehr schwierig und muss fachpersonal vorbehalten bleiben. bei implementierung ausreichender baupräventiver maßnahmen haben taubenzecken in krankenhäusern keine bedeutung (faulde und freise 2014). der erreger der skabies oder krätze ist nur 0,2-0,45 mm groß. die milbe lebt im grunde bereits "endoparasitisch" im stratum corneum der haut. die weibchen graben bohrgänge, in denen sie vom zellsaft beschädigter zellen leben und ihre faeces absetzen. bevorzugt werden stellen, an denen die haut dünn und faltig ist, meist zwischen den fingern und am handgelenk, aber auch an ellenbogen, füßen, penis, skrotum, gesäß und achselhöhlen, bei frauen auch im bereich der mammae und mamillen (stary und stary 2010) . im bohrgang werden die eier abgelegt; die nach 3-8 d schlüpfenden larven häuten sich zweimal, bis nach 4-6 d die adulten milben entstehen. erst wenn die weibchen auf der haut befruchtet wurden, bohren sie sich in die haut desselben oder eines anderen wirts ein. der gesamtzyklus von ei zu ei dauert 14-31 d. ansteckung ist im regelfall nur durch intimen, persönlichen kontakt möglich, z. b. wenn gleichzeitig dasselbe bett benutzt wird. man nimmt an, dass für eine übertragung eine mindestkontaktdauer von 10-15 min notwendig ist. eine übertragung durch bettwäsche kann normalerweise ausgeschlossen werden, obwohl die milben 2-4 d, unter günstigen bedingungen sogar etwa eine woche, fern vom menschen überleben können. es gibt jedoch besonders heftige verlaufsformen der skabies, bei denen wegen der starken milbenvermehrung die ansteckungsgefahr bedeutend höher ist. in solchen fällen muss die wäsche entweder bei über 50 °c gewaschen, 4 d im plastikbeutel gelagert oder mit einem insektizid eingesprüht werden. auch das bügeln der wäsche tötet milben ab. klinik: das klinische bild der skabies ist bei wiederholter infektion ausgepräger als bei erstinfektion. die schwere hautreizung, die dabei entsteht, veranlasst zu heftigem und anhaltendem kratzen, vor allem nachts. sekundärinfektionen sind meist die folge. eine besonders schwere form ist die "norwegische krätze", die durch bildung einer dicken hornschicht über händen und füßen und papulären eruptionen an anderen körperstellen imponiert. obwohl die zahl der milben sehr hoch ist, verspürt der patient in diesem fall kaum juckreiz. epidemisches auftreten ist in asylantenheimen, altenheimen und krankenhäusern nicht selten. bei leukämischen kindern soll sie sich ebenfalls bevorzugt ausbreiten können (gröschel 1981) . die diagnose der skabies ist nur bei mikroskopischem nachweis der milben eindeutig gesichert. dazu sucht man das etwas breitere ende eines bohrgangs in der haut, in dem die weibchen zu vermuten sind, entfernt mit scharfer kanülenspitze die haut und überträgt die milben, die meist an der nadelspitze hängen bleiben, auf einen objektträger, evtl. mit einem tropfen immersionsöl (stary und stary 2010) . die behandlung der krätze muss topisch oder mit oralen antiparasitika durchgeführt werden. wegen der geringen toxizität werden heute zumeist akarizide pyrethroide, z. b. permethrin und allethrin, bevorzugt (reich 1996; stary und stary 2010) . bei aids-patienten hat sich ivermectin in einmaliger oraler dosis offenbar als medikament bewährt (stary und stary 2010; wolff 1998 hygieneschädlingen sind vor allem mechanische verschlepper von krankheitserregern oder um passive vektoren. sie sind nicht hämophag, in ihnen findet kein entwicklungszyklus eines pathogens und i. d. r. keine erregervermehrung statt. 2 lia spp.) bekannt. die larven leben an verwesenden kadavern, exkrementen und in nekrotischem gewebe. daher treten sie vor allem in tropischen regionen sowie unter krisensituationen häufig auch als wundmyiasis-erreger auf; in mitteleuropa gelegentlich während der sommermonate (hogsette und amendt 2010). besonders angelockt werden diese fliegen durch übelriechende, eitrige geschwüre. maden von speziellen l.-sericata-stämmen werden zur behandlung chronischer wunden eingesetzt, da sie diese debridieren (durch alimentäre aufnahme und lytische zersetzung), antiseptisch effektiv sind und die wundheilung durch freigesetzte faktoren fördern (daeschlein et al. 2007b ). sie schmeißen (werfen!) ihre larven im flug auf fleisch und wunden, legen also keine eier ab. im übrigen verhalten sie sich wie aasfliegen. in diese nicht blutsaugende unterfamilie der kleinen (1-5 mm) schmetterlingsmücken (psychodinae) gehören auch die abortmücken der gattung psychoda. sie entwickeln sich vor allem in offenen fäkalien und waren zu zeiten vom wohnhausfernen toilettenanlagen deutlich verbreiteter und als verschlepper von krankheitserregern wichtiger, als heute. nach einschleppung aus dem mittelmeerraum und geografischer ausbreitung von clogmia albipunctata in deutschland änderte sich diese situation. diese vormals in den tropen und subtropen heimische schmetterlingsmücke hat sich derzeit östlich bis berlin und nördlich bis etwa kiel ausgebreitet, entwickelte einen ausgeprägten synanthropismus und ist vor allem in öffentlichen gebäuden und auch in krankenhäusern mittlerweile häufig und ganzjährig anzutreffen (faulde und spiesberge 2012). aus deutschen krankenhäusern wurden an ihr 45 bakterienspezies aus 40 gattungen isoliert. neben a. baumannii, s. maltophilia, k. pneumoniae ssp. pneumoniae wurden noch eine reihe weiterer nosokomialer pathogene identifiziert (faulde und spiesberger 2013) . der erstmalige nachweis von a. baumannii auf einer insektenoberfläche eröffnete völlig neue epidemiologische perspektiven sowie neue übertragungsmöglichkeiten dieses immer wichtiger werdenden nosokomialen pathogens. außer multiresistenten (4mrgn), carbapenemase-produzierenden s. maltophilia konnten bislang keine weiteren besonderen resistenzmuster oder multiresistenzen festgestellt werden. das potenzial als mechanischer verschlepper von krankheitserregern in krankenhäusern wurde dennoch belegt (faulde und spiesberger 2013) . die larven von c. albipunctata entwickeln sich aquatisch inauch kleinsten -wasseransammlungen. in krankenhäusern wurden als brutplätze identifiziert: • toiletten und urinale, insbesondere wenn diese wenig genutzt werden, • duschen-und bodensiphons in duschkabinen, insbesondere, wenn diese unzureichend gereinigt und saniert wurden, (meistens lag eine verstopfung des siphons durch haarknäuel vor.) • in krankenhausküchen sowie in den sanitärräumen des küchenpersonals vorwiegend ungereinigte und verstopfte bodensiphons, • wasseransammlungen, die im zusammenhang mit dauerleckagen von wasserrohren, nicht abfließendem kondenswasser oder abläufen von ve-wasseranlagen stehen (faulde und spiesberger 2012) . die behebung dieser schwachstellen durch ausreichendes hygieneund wassermanagement reduziert oder eradiziert bereits einen befall. c . albipunctata tritt als sensibler indikator für unzureichendes hygieneund wassermanagement in krankenhäusern auf. schaben (blattodea) sind urtümliche insekten, die sich -dorsoventral abgeflacht -gut in ritzen und spalten verstecken können. sie lieben wärme und hohe luftfeuchtigkeit. zentren des nicht seltenen befalls in krankenhäusern sind daher meist zentral-und stationsküchen, toiletten und bäder. bei der nahrungsaufnahme sind schaben nicht wählerisch. sie bevorzugen zwar weiche, zuckeroder stärkehaltige lebensmittel, verschmähen aber auch blut, sputum, exkremente u. ä. stoffe nicht. schon die anwesenheit von schaben kann den gesundungsprozess eines patienten stören, wenn sie mit unberechenbaren bewegungen durch den raum huschen und ihren charakteristischen, unangenehmen geruch verbreiten. außerdem können sie allergien verursachen (pospischil 2010; rust 2010) . von hygienischer bedeutung ist ihr verhalten bei der nahrungssuche. sie laufen wahllos über offen zugängliche speisen; dabei erbrechen sie gelegentlich halbverdautes futter und setzen ihren kot auf speisen, geschirr, möbeln, instrumenten usw. ab. dadurch können die verschiedensten krankheitserreger auf lebensmittel und instrumente gelangen (pospischil 2010; rust 2010) . ob im konkreten fall eine kontamination zustande kommt, hängt davon ab, ob die schaben zuvor kontakt mit nosokomialen erregern aus dem krankenhausumfeld hatten. typischerweise waren in einem klinikum alle kliniken von schaben befallen, nicht aber die zugehörigen institute, sofern sie räumlich getrennt waren, weil über die lebensmittelversorgung der patienten auch die schaben versorgt wurden (maier 1983) . hygienemaßnahmen: schaben, die üblicherweise mit lebensmitteln eingeschleppt werden, können sich in einem sauberen gebäude ohne schlupfwinkel nicht einnisten. ein schlechter erhaltungszustand oder konstruktionsbedingte mängel eines gebäudes gewähren ein reichliches angebot an schlupfwinkeln. schaben verstecken sich tagsüber in spalten und ritzen und kommen erst bei dunkelheit zum vorschein. durch ausbessern von rissen, losen kacheln usw. wird das verhindert (pospischil 2010; rust 2010) . werden schmutziges verbandmaterial, sputum, fäkalien und abfälle ordnungsgemäß sofort beseitigt, nimmt das risiko einer verschleppung von krankheitserregern durch schaben ab. größtmögliche sauberkeit ist ein wesentlicher faktor, um die verbreitung von krankheitserregern zu verhindern und auch die einfachste bekämpfungsmaßnahme, weil die schabenvermehrung wegen des damit verbundenen nahrungsmangels begrenzt wird. neben der konsequenten beseitigung der abfälle nach jeder mahlzeit und ihrer aufbewahrung in gut schließenden behältern, möglichst außerhalb des krankenhauses, muss bedacht werden, dass lebensmittel grundsätzlich schabensicher aufbewahrt werden müssen. exkremente und schmutziges verbandmaterial müssen sofort restlos beseitigt werden. die einhaltung dieser regeln wird zu einer entwicklungshemmung, bei konsequenter durchführung zur beseitigung der schaben führen. als schabenarten sind in krankenhäusern mitteleuropas blattella germanica, blatta orientalis und supella longipalpa von bedeutung (pospischil 2010; rust 2010) . deutsche schabe (blattella germanica): sie ist im adultstadium 10-15 mm lang und hell-bis schmutzigbraun. alle stadien zeigen auf dem thorax zwei schwarze längsstreifen. die adulten können mithilfe besonderer haftlappen über senkrechte, glatte wände laufen. nach der kopulation bildet das weibchen eipakete (ootheken), in denen sich die larven entwickeln und die nach 2-4 wochen abgelegt werden; danach schlüpfen die larven. nach 5 häutungen der männchen bzw. 7 der weibchen wird im günstigsten fall nach etwa 38-63 d das imaginalstadium erreicht. die optimale temperatur dafür liegt bei 30 °c. orientalische schabe (blatta orientalis): sie fällt durch ihre größe (20-27 mm) und fast schwarze oder schokoladenbraune färbung auf. nur das männchen trägt flügel. erst nach 10 häutungen ist das weibchen nach durchschnittlich 282 d, das männchen nach nur 7 häutungen und 164 d ausgereift. die oothek wird nach 1-5 d abgelegt, die larven schlüpfen aber erst nach 44 d (bei 30 °c, bei niedrigeren temperaturen viel später). das temperaturoptimum liegt zwischen 20 und 29 °c. da sie also auch relativ niedrige temperaturen toleriert, findet man b. orientalis auch in kellerräumen. braunbandschabe (supella longipalpa): sie wurde nach dem zweiten weltkrieg mit lebensmitteln aus den usa nach deutschland eingeschleppt. sie ähnelt der deutschen schabe, besitzt aber keine längsstreifen auf dem thorax: stattdessen ist der thorax ist sehr dunkel mit hellem seitenrand. auffallend ist ein braunes band zwischen zwei gelblichen querstreifen auf hinterbrust und abdomen. neben küchen und ähnlichen räumen, wie sie auch von der deutschen schabe besiedelt werden, verschont sie auch wohn-und schlafräume nicht. daher findet man sie u. a. auch in schubladen von schreibtischen und kommoden (möbelschabe), wo sie ihre ootheken verstecken. die pharaoameise (monomorium pharaonis) ist zum ständigen bewohner zentralbeheizter gebäude und vieler krankenhäuser ge-worden (oi 2010) . sie baut ihre staaten in nestern unter fußböden und in mauerritzen. dort legen die königinnen ihre eier ab, bis der staat auf mehrere tausend ameisen angewachsen ist. die nur 1,5-2,4 mm großen arbeiterinnen sammeln nahrung, die in das nest gebracht wird und zur ernährung der königinnen und larven dient. bevorzugt nehmen sie zucker oder honig, aber auch proteine (fleisch, käse) oder fett auf. mit spürsinn finden sie diese nahrung auch in verschlossenen behältern, da sie klein genug sind, um durch engste ritzen zu schlüpfen. hat eine arbeiterin den zugang gefunden, folgen über markierte straßen andere nach. im krankenhaus können pharaoameisen erhebliche probleme verursachen, wenn sie in sterile verbände, geräte, bakteriologische kulturen usw. einwandern und diese kontaminieren. aber auch patienten selbst, vor allem frischoperierte, bewegungsunfähige schwerkranke und neugeborene können befallen werden. die ameisen wandern unter wund-und gipsverbände und benagen die wunden. dabei können sie verschiedenste krankheitserreger wie streptokokken, staphylococcus spp. und clostridium spp. übertragen (oi 2010) . die bekämpfung ist schwierig, da die königinnen im nest nicht durch insektizide erreicht werden. als vorratsschädlinge kommen eine große zahl von insekten (motten, käfer) und milben in betracht. die hygienische bedeutung dieser arthopoden ist gering, da sie normalerweise keine krankheiten verursachen. anders ist es bei synanthropen schadnagern wie ratten und mäusen, die wegen ihrer eigenschaften als mechanischer überträger von krankheitserregern, aber auch als erregerreservoir gleichzeitig eine funktion als gesundheitsschädling besitzen (faulde 2004) . mäuse, ratten: sie können in der vorratshaltung schädlich werden und krankheitserreger übertragen (faulde 2004) . durch vorbeugende bauliche maßnahmen und regelmäßige kontrollen, z. b. anbringen engmaschiger gitter vor kellerfenstern, muss dafür gesorgt werden, dass nager nicht in krankenhausgebäude eindringen können. nagerbefall ist in kellerräumen von krankenhäusern und gesundheitseinrichtungen nicht selten, spielt aber in hygienisch gut geführten krankenhäusern nur eine untergeordnete rolle und wird daher nicht weiter betrachtet. während die topische und orale behandlung von körperungeziefer am patienten mit arzneimitteln aus der gruppe der antiparasitika durchgeführt wird, gestalten sich schädlingsbekämpfungen unter freisetzung biozider substanzen und präparate in die umwelt völlig anders. die verfügbarkeitsbreite von insektiziden und akariziden für nicht sachkundige "laien" ist in den letzten jahren und jahrzehn-2 ten gesetzlich stark eingeschränkt worden (faulde 2010) . zum schutz des verbrauchers und seiner umwelt sind für den "hausgebrauch" derzeit noch fertig formulierte "laienmittel" kommerziell verfügbar. insektizide und akarizide konzentrate in anwendungsüblichen mengen und konfektionierungen sind derzeit z. b. für die anwendung an haustieren gegen zecken-und flohbefall sowie für die bekleidungs-und moskitonetzimprägnierung für die vektorenabwehr in tropischen regionen erhältlich (faulde 2010) . die ausbringung professionell einzusetzender insektizide, akarizide und rodentizide ist allein entsprechend ausgebildetem fachpersonal vorbehalten. aus diesem grund wird auf mittelauswahlkriterien und toxikologische eigenschaften von bioziden nicht eingegangen. diese können für den konkreten bedarf aus der einschlägigen literatur entnommen werden (faulde 2010; maroni et al. 2010 ). zum schutz des menschen vor übertragbaren krankheiten dürfen bei behördlich angeordneten entseuchungen (desinfektion), entwesungen (bekämpfung von gliedertieren) und maßnahmen zur bekämpfung von wirbeltieren, durch die krankheitserreger verbreitet werden können, nur mittel und verfahren verwendet werden, die von der zuständigen bundesoberbehörde in einer liste im bundesgesundheitsblatt bekannt gemacht worden sind. auf ausreichende wirksamkeit geprüfte und anerkannte mittel und verfahren zur bekämpfung von tierischen gesundheitsschädlingen nach § 18 ifsg werden in jeweils aktualisierten fassungen im bundesgesundheitsblatt veröffentlicht (klasen et al. 2014) . dem anwender steht die wahl des mittels bei schädlingsbekämpfungsmaßnahmen grundsätzlich frei, soweit es sich nicht um behördlich angeordnete maßnahmen bei entseuchungen, entwesungen oder maßnahmen gegen wirbeltiere gemäß § 17 ifsg handelt. da bei auftreten von gesundheitsschädlingen in krankenhäusern und anderen gesundheitseinrichtungen in der regel immer der begründete verdacht einer erregerübertragung oder zumindest -verschleppung vorliegt, ist die ausschließliche nutzung dieser behördlich und unabhängig auf ausreichende wirksamkeit geprüften mittel und verfahren dringend zu empfehlen (klasen et al. 2014 ). (indirekte übertragung durch kontaminierte kleidung möglich), cryptococcus spp ifsg hat die zuständige behörde (gesundheitsamt) anzuordnen, dass personen, die an lungenpest oder an von mensch zu mensch übertragbarem hämorrhagischem fieber erkrankt oder dessen verdächtig sind, unverzüglich in einem krankenhaus oder einer für diese krankheiten geeigneten einrichtung abgesondert werden adverse effects of isolation in hospitalised patients efficacy and safety of oxum in treatment of chronic wounds a prospective randomized trial of povidone-iodine prophylactic cleansing of the rectum before transrectal ultrasound guided prostate biopsy versuche mit sporen von b . subtilis in defibriniertem blut zur prüfung der wirksamkeit von gas-sterilisatoren versuche zur kaltsterilisation mit formalindämpfen the value of chlorhexidine gluconate wipes and prepacked washcloths to prevent the spread of pathogens -a systematic review evaluation of microbiocidal activity of superoxidized water on hospital isolates antibiotic resistance rates of extended spectrum beta-lactamase producing escherichia coli and klebsiella spp . strains isolated from urinary tract infections in a private hospital antibiotic prophylaxis for wound infections in total joint arthroplasty: a systematic review emergence of multidrug-resistant gramnegative bacteria during selective decontamination of the digestive tract on an intensive care unit spontaneous bacterial peritonitis: a review of treatment options disinfection of heat-sensitive material by low-temperature steam and formaldehyde disinfection of cystoscopes by subatmospheric steam and steam and formaldehyde at 80 °c low-level exposure of mrsa to octenidinedihydrochloride does not select for resistance anforderungen der hygiene an die wäsche aus einrichtungen des gesundheitsdienstes, die wäscherei und den waschvorgang und bedingungen für die vergabe von wäsche an gewerbliche wäschereien use of a daily disinfectant cleaner instead of a daily cleaner reduced hospital-acquired infection rates clinical experience with a new and stable super-oxidized water in wound treatment on behalf of the endoscopy committee of the british society of gastroenterology . guideline -antibiotic prophylaxis in gastrointestinal endoscopy stellungnahme der dgzmk -einsatz von antibiotika in der zahnärztlichen praxis severe versus local odontogenic bacterial infections: comparison of microbial isolates indikation einer präoperativen antibiotischen prophylaxe bei insertion enossaler implantaten -ein systematisches review clinical and microbiological efficacy of moxifloxacin versus amoxicillin/clavulanic acid in severe odontogenic abscesses -a pilot study risk of hepatotoxicity associated with fluoroquinolones: a national case-control safety study reducing bacterial infectious complications from burn wounds topical antibiotics and neurosurgery: have we forgotten to study it? american academy of orthopedic surgeons . advisory statement: antibiotic prophylaxis for dental patients with total joint replacements a prospective, double-blind, placebo-controlled trial of a single dose of azithromycin on postoperative wound infections in plastic surgery streptococcus pneumoniae meningitis a systematic review of prophylactic antibiotics in the surgical treatment of maxillofacial fractures methods of analysis of aoac international . washington: aoac -association of analytical chemists assessment of cytogenetic and cytotoxic effects of chlorhexidine digluconate on cultured human lymphocytes arbeitskreis krankenhaus-und praxishygiene der awmf . händedesinfektion und händehygiene . awmf-registernummer 029-027 atemschutz bei aerogen übertragbaren krankheiten arbeitskreis krankenhaus-und praxishygiene der awmf . anforderungen an handschuhe zur infektionsprophylaxe im gesundheitswesen . awmf-registernummer 029-021 arbeitskreis krankenhaus-und praxishygiene der awmf . hygienemaßnahmen bei vorkommen von clostridium difficile arbeitskreis krankenhaus-und praxishygiene der awmf . blutübertragbare virusinfektionen: prävention . awmf-registernummer 029-026 multiresistenter erreger (mre): maßnahmen beim auftreten von mre . awmf-registernummer 029-019 prophylaxe in krankenhaus und praxis arbeitskreis krankenhaus-und praxishygiene der awmf . gastroenteritis-ausbrüche durch noroviren: hygienemaßnahmen . awmf-registernummer infektionen mit respiratorischem synzytialvirus (rsv): anforderungen der hygiene arbeitskreis krankenhaus-und praxishygiene der awmf . perioperative antibiotikaprophylaxe bgbl . i s . 2179), die zuletzt durch artikel 4 der verordnung vom 19 serratia marcescens outbreak associated with extrinsic contamination of 1 % chlorxylenol soap norovirus infections in preterm infants: wide variety of clinical courses antibiotic prophylaxis for transrectal needle biopsy of the prostate: a randomized controlled study the role of endoscopy in the diagnosis and the management of cystic lesions and inflammatory fluid collection of the pancreas recommendations and requirements for soap and hand rub dispensers in healthcare facilities analysis of late infections in 89 long-term survivors of bone marrow transplantation determination of the 50 % human infectious dose for norwalk virus cochlear damage in guinea pigs from chlorhexidine vestibular damage in guinea pigs from chlorhexidine infection after transrectal core biopsies of the prostate -risk factors and antibiotic prophylaxis eau guidelines on prostate cancer experimental endocarditis induced by dental manipulation and oral streptococci . oral surg oral med oral pathol effect of predelivery vaginal antisepsis on maternal and neonatal morbidity and mortality in egypt prüfung der wirksamkeit von desinfektionsmitteln gegen mykobakterien im quantitativen suspensionstest surgical site infections: epidemiology and prevention efficacy of prophylactic antibiotics against meningitis after craniotomy: a meta-analysis hepatitis c virus molecular clones and their replication in vivo and in cell culture use of selective decontamination of the digestive tract in united kingdom intensive care units an evaluation of antibiotic prophylaxis in endoprosthetic replacements (epr) at the royal orthopaedic hospital (roh) birmingham timing of prophylactic antibiotics in abdominal surgery: trial of a preoperative versus an intra-operative first dose efficacy and limitation of a chlorhexidine-based decolonization strategy in preventing transmission of methicillin-resistant staphylococcus aureus in an intensive care unit infektionserreger in praxis und krankenhaus . mainz: mhp topical silver for infected wounds fast, broad-range disinfection of bacteria, fungi, viruses and prions influence of intestinal bacterial decontamination using metronidazole and ciprofloxacin or ciprofloxacin alone on the development of acute graft-versus-host disease after marrow transplantation in patients with hematologic malignancies: final results and long-term follow-up of an open-label prospective randomized trial use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants einfluss von flächendesinfektion und steriler abdeckung von mobiliar auf die raumluftqualität in einem kardiologischen eingriffsraum mit raumlufttechnischer anlage (mischströmung, raumklasse ib din 1 .946-4) dermal and pulmonary absorption of propan-1-ol and propan-2-ol from hand rubs prevalence of chlorhexidine digluconate, 4-chloraniline, and 4-chlornitrobenzene in saliva after pre-and postoperative mouth rinse with 0 .2 % chlorhexidine digluconate eliminating catheter-related bloodstream infections in the intensive care unit eliminating central line -associated bloodstream infections: a national patient safety imperative vergleich einer hautschutzcreme und ihrer grundlage bezüglich wirksamkeit gegen das berufsbedingte irritative handekzem bei krankenschwestern inaktivierung und entfernung von prionen bei der aufbereitung von medizinprodukten bgbl . i s . 3777), die zuletzt durch artikel 5 des gesetzes vom 8 antibiotic dosing before primary hip and knee replacement as a pay-for-performance measure sensorineural deafness following myringoplasty operations are systemic prophylactic antibiotics indicated with anterior nasal packing for spontaneous epistaxis? effectiveness of chlorhexidine bathing to reduce catheter-associated bloodstream infections in medical intensive care unit patients measles outbreak in a pediatric practice: airborne transmission in an office setting ist eine schnelldesinfektion von mobilen elektronischen geräten ohne schäden möglich use of a fluorescent chemical as a quality indicator for a hospital cleaning program survival of hepatitis b virus after drying and storage for one week controlling methicillin-resistant staphylococcus aureus: quantifying the effects of interventions and rapid diagnostic testing impact of 4 % chlorhexidine whole-body washing on multidrug-resistant acinetobacter baumannii skin colonisation among patients in a medical intensive care unit role of musca domestica in the transmission of multiresistant bacteria in the centres of intensive care setting in sub-saharan africa fluoroquinolones, antimicrobial resistance and neutropenic cancer patients the bowie and dick autoclave tape test impact of hydrogen peroxide vapor room decontamination on clostridium difficile environmental contamination and transmission in a healthcare setting comparison of fluorescent marker systems with 2 quantitative methods of assessing terminal cleaning practices bacterial resistance to quaternary ammonium compounds (qac) disinfectants mechanisms of resistance in salmonella enterica adapted to erythromycin, benzalkonium chloride and triclosan antimicrobial prophylaxis for surgery: an advisory statement from the national surgical infection prevention project baseline results from the national surgical infection prevention project . use of antimicrobial prophylaxis for major surgery clinical practice guidelines for antimicrobial prophylaxis in surgery untersuchungen zur prüfung der viruziden wirksamkeit von desinfektionsmitteln für die chemische instrumentendesinfektion influence of internal and outdoor factors on filamentous fungal flora in hematology wards use of an alcohol-based hand rub and quality improvement interventions to improve hand hygiene in a russian neonatal intensive care unit laboratory evaluation of selected disinfectants as virucidal agents against porcine parvovirus, pseudorabies virus, and transmissible gastroenteritis virus dgpi handbuch infektionen bei kindern und jugendlichen guide to choice of chemical disinfectants levofloxacin to prevent bacterial infection in patients with cancer and neutropenia bovine viral diarrhea virus as a surrogate model of hepatitis c virus for the evaluation of antiviral agents effect of higher minimum inhibitory concentrations of quaternary ammonium compounds in clinical e . coli isolates on antibiotic susceptibilities and clinical outcomes impact of vancomycin surgical antibiotic prophylaxis on the development of methicillin-sensitive staphylococcus aureus surgical site infections: report from australian surveillance data (vicniss) öffentliches produktregister der zugelassenen desinfektionsmittel . laufend (nur unter nennung eines bestimmten biozidprodukts) richtlinie der bam und des bga für desinfektionsmittel-dosiergeräte verfügbar auf www .bfarm .de bundesinstitut für risikobewertung . toxikologische bewertung von formaldehyd . stellungnahme des bfr nr . 023/2006 vom 30 bundesministerium für umwelt, n . u . r . biozidgesetz -gesetz zur umsetzung der richtlinie 98/8/eg des europäischen parlamentes und des rates vom 16 public health significance of urban pests preventing bacterial infection by coordinating antibiotic and host activity: a time-dependent relationship prevention of excess neonatal morbidity associated with group b streptococci by vaginal chlorhexidine disinfection during labour aseptic insertion of 2132 central venous lines to reduce bacteraemia in vitro comparison of chlorhexidine and povidone-iodine on the long-term proliferation and functional activity of human alveolar bone cells plasma cefazolin levels during cardiovascular surgery: effects of cardiopulmonary bypass and profound hypothermic circulatory arrest impact of an educational intervention implanted in a neurological intensive care unit on rates of infection related to external ventricular drains a crossover intervention trial evaluating the efficacy of a chlorhexidine-impregnated sponge in reducing catheter-related bloodstream infections among patients undergoing hemodialysis impact of a standardized hand hygiene program on the incidence of nosocomial infection in very low birth weight infants risk of clostridium difficile infection after perioperative antibacterial prophylaxis before and during an outbreak of infection due to a hypervirulent strain identifying opportunities to enhance environmental cleaning in 23 acute care hospitals an evaluation of patient area cleaning in 3 hospitals using a novel targeting methodology epidemiology of bacterial infection during management of open leg fractures tackling c difficile with environmental cleaning prüfverfahren und anforderungen (phase 1) (deutsche fassung) . c . t . 216, european committee for standardization (cen) 1997a prüfverfahren und anforderungen (phase 1) (deutsche fassung) . c . t . 216 13704 -chemische desinfektionsmittel und antiseptka -quantitative suspension test for the evaluation of sporicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areastest method and requirements (phase 2/step 1) cen . en 14347 -chemische desinfektionsmittel und antiseptka -sporizide wirkung (basistest)-prüfverfahren und anforderungen (phase 1) (deutsche fassung) . c . t . 216 din-en 14348 -chemische desinfektionsmittel und antiseptika -quantitativer suspensionsversuch zur bestimmung der mykobakteriziden wirkung chemischer desinfektionsmittel im humanmedizinischen bereich einschließlich der instrumentendesinfektion . prüfverfahren und anforderungen (phase 2/stufe 1) . c . t . 216, european committee for standardization (cen) cen . din en 14561 -chemische desinfektionsmittel und antiseptika -quantitativer keimträgerversuch zur prüfung der bakteriziden wirkung für instrumente im humanmedizinischen bereich -prüfverfahren und anforderungen din en 14562 -chemische desinfektionsmittel und antiseptika -quantitativer keimträgerversuch zur prüfung der fungiziden oder levuroziden wirkung für instrumente im humanmedizinischen bereich -prüfverfahren und anforderungen (phase 2/stufe 2) . c . t . 216, european committee for standardization (cen) din en 14563 -chemische desinfektionsmittel und antiseptika -quantitativer keimträgerversuch zur prüfung der mykobakteriziden oder tuberkuloziden wirkung chemischer desinfektionsmittel für instrumente im humanmedizinischen bereich 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anforderungen (phase 2/stufe 2) . c . t . 216, european committee for standardization (cen) cen . din-en 13624 -chemische desinfektionsmittel und antiseptika -quantitativer suspensionsversuch zur prüfung der fungiziden wirkung chemischer desinfektionsmittel für instrumente im humanmedizinischen bereich -prüfverfahren und anforderungen 14885 -chemische desinfektionsmittel und antiseptika -anwendung europäischer normen für chemische desinfektionsmittel und antiseptika . c . t . 216 cen . din-en 16777 -chemische desinfektionsmittel und antiseptika -quantitativer versuch auf nicht porösen oberflächen ohne mechanische einwirkung zur bestimmung der viruziden wirkung im humanmedizinischen bereich -prüfverfahren und anforderungen cen . en 13727 -chemische desinfektionsmittel und antiseptika -quantitativer suspensionsversuch zur prüfung der bakteriziden wirkung chemischer desinfektionsmittel für instrumente im humanmedizinischen bereich -prüfverfahren und anforderungen desinfektionsmittel und antiseptika -quantitative carrier test for the evaluation of virucidal activity in the medical area -test method and requirements european committee for standardization (cen) wi00216068 -chemical disinfectants and antiseptics -quantitative suspension test for the evaluation of sporicidal activity of chemical desinfectants in the medical area -test method and requirements (phase 2, step 1) . c . t . 216, european committee for standardization (cen) 2014e . centers for disease control and prevention (cdc) . checklist for core elements of hospital antibiotic stewardship programs a prospective study of prevalence of 60-days postoperative wound infections after cardiac surgery . an updated risk factor analysis transmission of a small round structured virus by vomiting during a hospital outbreak of gastroenteritis antibiotics for spontaneous bacterial peritonitis in cirrhotic patients . cochrane database syst rev outbreak of human metapneumovirus infection in psychiatric inpatients: implications for directly observed use of alcohol hand rub in prevention of nosocomial outbreaks a surveillance system to reduce transmission of pandemic h1n1 (2009) influenza in a 2600-bed medical center the timing of prophylactic administration of antibiotics and the risk of surgical-wound infection the effect of daily bathing with chlorhexidine on the acquisition of methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococcus, and healthcare associated bloodstream infections: results of a quasi-experimental multicenter trial effect of daily chlorhexidine bathing on hospital-acquired infection characterization of infectious aerosols in health care facilities: an aid to effective engineering controls and preventive strategies a randomized comparison of one-dose versus two-dose antibiotic prophylaxis in gynecologic surgery lasting over two hours primary prophylaxis of invasive fungal infections in patients with hematologic malignancies . recommendations of the infectious diseases working party of the german society for haematology and oncology influenza virus: immunity and vaccination strategies . comparison of the immune response to inactivated and live, attenuated influenza vaccines surgical antisepsis . in: block ss (ed) disinfection, sterilization, and preservation . 5 . aufl . philadelphia: lippincott williams wilkins a systematic review and economic model of switching from non-glycopeptide to glycopeptide antibiotic prophylaxis for surgery prevention of urinary tract infection and sepsis following transrectal prostatic biopsy study of epigenetic properties of poly(hexamethylene biguanide) hydrochloride (phmb) efficacy of conventional endoscopic disinfection and sterilization methods against helicobacter pylori contamination the use of chlorhexidine antisepsis in contaminated surgical wounds prophylactic corticosteroids for preterm birth prophylaxis with fluoroquinolones for bacterial infections in neutropenic patients: a meta-analysis patients' tolerance of transrectal ultrasound-guided prostatic biopsy: an audit of 104 cases a five-year prospective study of 23 649 surgical wounds the epidemiology of wound infection: a 10-year old prospective study of 62 939 wounds antibacterial prophylaxis after chemotherapy for solid tumors and lymphomas rational selection of patients for antibacterial prophylaxis after chemotherapy hygienesicherheit von lamellenvorhängen antibacterial activity of positive and negative polarity low-voltage pulsed current (lvpc) on six typical grampositive and gramnegative bacterial pathogens of chronic wounds in vitro antibacterial activity of lucilia sericata maggot secretions use of dermacyn, a new antiseptic agent for the local treatment of diabetic foot ulcers inadequate antimicrobial prophylaxis during surgery: a study of β-lactam levels during burn debridement hospital cleaning in the 21st century measuring the effect of enhanced cleaning in a uk hospital: a prospective cross-over study microbial contamination of antiseptics and disinfectants single-dose versus single-day antibiotic prophylaxis for orthognathic surgery: a prospective, randomized, double-blind clinical study effect of selective decontamination on antimicrobial resistance in intensive care units: a systematic review and meta-analysis übertragung spongiformer encephalopathien durch arzneimittel resource consumption in the infection control management of pertussis exposure among healthcare workers in pediatrics interventions to improve antibiotic prescribing practices for hospital inpatients antibiotic prophylaxis for total joint replacement surgery: results of a survey of canadian orthopedic surgeons effects of selective decontamination of digestive tract on mortality and antibiotic resistance in the intensive-care unit effects of selective decontamination of digestive tract on mortality and acquisition of resistant bacteria in intensive care: a randomised controlled trial decontamination of the digestive tract and oropharynx: hospital acquired infections after discharge from the intensive care unit decontamination of the digestive tract and oropharynx in icu patients surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock antibiotic resistant infections with antibiotic-impregnated bactiseal catheters for ventriculoperitoneal shunts interventions to reduce colonisation and transmission ofantimicrobial-resistant bacteria in intensive care units: an interrupted time series study and cluster randomised trial incidence of infective endocarditis caused by viridans group streptococci before and after publication of the 2007 american heart association's endocarditis prevention guidelines effect of skin disinfection with octenidine dihydrochloride on insertion site colonization of intravascular catheters skin disinfection with octenidine dihydrochloride for central venous catheter site care: a double-blind, randomized, controlled trial deutsch-österreichische leitlinien zur postexpositionellen prophylaxe der hiv-infektion hiv-therapie in der schwangerschaft und bei hiv-exponierten neugeborenen deutsche gesellschaft für gastroenterologie, verdauungas-und stoffwechselstörungen (dgvs) . hepatitis-c-virus-infektion deutsche gesellschaft für gynäkologie und geburtshilfe (dggh) anforderungskatalog für die aufnahme von chemischen desinfektionsverfahren in die desinfektionsmittel-liste der dghm . wiesbaden: mhp strategien zur sicherung rationaler antibiotika-anwendung im krankenhaus . awmf-registernr empfehlungen für die validierung und routineüberwachung von sterilisationsprozessen mit sattdampf für medizinprodukte leitlinie zur validierung der manuellen reinigung und manuellen chemischen desinfektion von medizinprodukten . wiesbaden: mhp-verlag reinigung in krankenhäusern -eine umfrage der dgkh im jahr stellungnahme der deutschen gesellschaft für pädiatrische infektiologie und des paed ic projektes zur erfassung des antibiotika-verbrauches in kinderkliniken im rahmen eines antibiotic stewardship programmes leitlinie für die validierung des siegelprozesses nach din en iso 11607-2 . zentr steril suppl 2 robert koch-institut . leitlinie zur prüfung von chemischen desinfektionsmitteln auf wirksamkeit gegen viren in der humanmedizin fassung vom 1 . august deutsche vereinigung zur bekämpfung der viruskrankheiten (dvv) . quantitative prüfung der viruziden wirksamkeit chemischer desinfektionsmittel auf nicht-porösen oberflächen liste der nach den richtlinien der dvg geprüften und wirksam befundenen desinfektionsmittel für die tierhaltung liste der nach den richtlinien der dvg (4 . auflage, 2007) geprüften und wirksam befundenen desinfektionsmittel für den lebensmittelbereich prophylactic chemotherapy with fosfomycin trometamol during transurethral surgery and urological manoeuvres verfügbar auf www .rki .de din 58921 -prüfverfahren zum nachweis der eignung eines medizinproduktsimulators bei der dampf-sterilisation -medizinproduktsimulatorprüfung din 58946 -sterilisation -dampf-sterilisatoren din 58948 teil 7 -sterilisation -niedertemperatur-sterilisatoren -teil 7: bauliche anforderungen und anforderungen an die betriebsmittel sowie den betrieb von ethylenoxid-sterilisatoren din 58953 -sterilisation -sterilgutversorgung (teilweise ersetzt durch din en 868) . din en 285 -sterilisation -dampf-sterilisatoren -groß-sterilisatoren din en 1422 -sterilisatoren für medizinische zwecke, ethylenoxid-sterilisatoren din en 14180 -sterilisatoren für medizinische zwecke -niedertemperatur-dampf-formaldehyd-sterilisatoren -anforderungen und prüfung din en 15424 -sterilisation von medizinprodukten -niedertemperatur-dampf-formaldehyd din en 16615 . chemische desinfektion und antiseptika -quantitatives prüfverfahren zur bestimmung der bakteriziden und levuroziden wirkung auf nicht-porösen oberflächen mit mechanischer einwirkung mithilfe von tüchern oder mops im humanmedizinischen bereich (4-felder-test) -prüfverfahren und anforderungen din en 556 teil 1 -sterilisation von medizinprodukten -anforderungen an medizinprodukte, die als "steril" gekennzeichnet werden -teil 1: anforderungen an medizinprodukte, die in der endpackung sterilisiert wurden 010 teil 1 -sicherheitsbestimmungen für elektrische mess-, steuer 010 teil 2-040 -sicherheitsbestimmungen für elektrische mess-, steuer-, regel-und laborgeräte -teil 2-040: besondere anforderungen an sterilisatoren und reinigungs-desinfektionsgeräte für die behandlung medizinischen materials din en 867 teil 5 -nichtbiologische systeme für den gebrauch in sterilisatoren -teil 5: festlegungen von indikatorsystemen und prüfkörpern für die leistungsprüfung von klein-sterilisatoren vom typ b und vom typ s din en 868 teil 2 bis 10 -verpackungen für in der endverpackung zu sterilisierende medizinprodukte din en iso 10 .993 teil 7 -biologische beurteilung von medizinprodukten -teil 135 teil 1 -sterilisation von produkten für die gesundheitsfürsorge -ethylenoxid -teil 1: anforderungen an die entwicklung 138 teil 1 bis 5 -sterilisation von produkten für die gesundheitsfürsorge -biologische indikatoren 140 teil 1, 3 und 4 -sterilisation von produkten für die gesundheitsfürsorge -chemische indikatoren medizinprodukte -qualitätsmanagementsysteme -anforderungen für regulatorische zwecke (iso 13485: 2003 + cor 161 -sterilisation von produkten für die gesundheitsfürsorge -biologische indikatoren -leitfaden für die auswahl, verwendung und interpretation von ergebnissen 937 -sterilisation von produkten für die gesundheitsfürsorge -allgemeine anforderungen an die charakterisierung eines sterilisierenden agens und an die entwicklung medizinprodukte -anwendung des risikomanagements auf medizinprodukte (iso 14971: 2007, korrigierte fassung din en iso 15883 teile 1-7 . reinigungs-desinfektionsgeräte din en iso 17 .664 -sterilisation von medizinprodukten -vom hersteller zu stellende informationen zur wiederaufbereitung von resterilisierbaren medizinprodukten din en iso 17 .665 teil1 und 2 -sterilisation von produkten für die gesundheitsfürsorge sterilisation von medizinprodukten -vom hersteller bereitzustellende informationen für die aufbereitung von resterilisierbaren medizinprodukten sterilisation von produkten für die gesundheitsfürsorge -feuchte hitze -teil 1: anforderungen an die entwicklung sterilisation von produkten für die gesundheitsfürsorge -trockene hitze -anforderungen an die entwicklung 424 (normentwurf) -sterilisation von medizinprodukten -niedertemperatur-dampf-formaldehyd -anforderungen an die entwicklung beschichtungsstoffe -beurteilung von beschichtungsschäden -bewertung der menge und der größe von schäden und der intensität von gleichmäßigen veränderungen im aussehen -teil 1: allgemeine einführung und bewertungssystem classification of wounds at risk and their antimicrobial treatment with polihexanide: a practice-oriented expert recommendation safety and morbidity of first and repeat transrectal ultrasound guided prostate needle biopsies: results of a prospective european prostate cancer detection study removal of nosocomial pathogens from the contaminated glove bed bugs: clinical relevance and control options clinical components and associated behavioural aspects of a complex healthcare intervention: multi-methods study of selective decontamination of the digestive tract in critical care biofilms and device-associated infections the effects of ophthalmic preservatives on corneal epithelium of the rabbit: a scanning electron microscopical study an outbreak of norovirus infection in a bone marrow transplant unit triclosan exposure increases triclosan resistance and influences taxonomic composition of benthic bacterial communities determination of the efficancy of sterile barrier systems against microbial challanges during transport and storage estimated risk of endocarditis in adults with predisposing cardiac conditions undergoing dental procedures with or without antibiotic prophylaxis keimspektren und antibiotika bei odontogenen infektionen -renaissance der penicilline? weichteilinfektionen in der mund-, kiefer-und plastischen gesichtschirurgie -keimspektren und antibiotika reduction of clostridium difficile and vancomycin-resistant enterococcus contamination of environmental surfaces after an intervention to improve cleaning methods berufliche expositionen gegenüber formaldehyd im gesundheitswesen prospective, double-blinded, randomised controlled trial assessing the effect of an octenidine-based hydrogel on bacterial colonisation and epithelialization of skin graft wounds in burn patients chemical disinfectants and antiseptics . virucidal quantitative suspension test for chemical disinfectants and antiseptics used in human medicine . test method and requirements (phase 2, step 1) 2013 . en 16615 efficacy of quinolone prophylaxis in neutropenic cancer patients: a meta-analysis morbidity of ultrasound-guided transrectal core biopsy of the prostate without prophylactic antibiotic therapy . a prospective study in 415 cases poly(hexamethylenebiguanide) hydrochloride (phmb) -case 3122, pc code: 111801 . toxicology disciplinary chapter for the reregistration eligibility decision document . environmental protection agency document guidelines for carcinogen risk assessment (final) reregistration eligibility decision (red) for phmb anthrax spore decontamination using hydrogen peroxide vapor interventions for replacing missing teeth: antibiotics at dental implant placement to prevent complications disinfection efficacy against parvovirus compared with reference viruses opinion of the scientific panel on food additives, flavourings, processing aids and materials in contact with food on a request from the commission related to propan-2-ol as a carrier solvent for flavourings, question number efsa-q-2003-136 simulation and patient safety: evaluative checklists for central venous catheter insertion juni 1993 (abl . eg nr . l 169 s . 1) zuletzt geändert durch artikel 2 der richtlinie influence of biofilms by chemical disinfectants and mechanical cleaning allergy to chlorhexidine: beware of the central venous catheter arthropoden und nagetiere als krankheitsverursacher sowie überträger und reservoire von krankheitserregern vorkommen und verhütung vektorassoziierter erkrankungen des menschen in deutschland unter berücksichtigung zoonotischer hospital infestations by the moth fly, clogmia albipunctata (diptera: psychodinae), in germany role of the moth fly clogmia albipunctata (diptera: psychodinae) as a mechanical vector of bacterial pathogens in hospitals krank durch arthropoden ratten und mäuse -unterschätzte überträger und reservoire gefährlicher infektionskrankheiten? chlorhexidine official fda information, side effects and uses the incidence of fluoroquinolone resistant infections after prostate biopsy -are fluoroquinolones still effective prophylaxis? clinical and microbiologic features guiding treatment recommendations for brain abscesses in children untersuchungen von sterilverpackungsmaterial für die formaldehyd-sterilisation untersuchungen zur sterilisation mit formaldehyddampf im unterdruckverfahren, mikrobiologische und toxikologische aspekte kondensation bei der dampfsterilisation poröser güter prevention of perioperative infection glycerol accelerates recovery of barrier function in vivo rahmenkonzept zur gefahrenabwehr bei außergewöhnlichen seuchengeschehen implementation of evidence-based practices for surgical site infection prophylaxis: results of a pre-and postintervention study the house fly (musca domestica) as a potential vector of metazoan parasites caught in a pig-pen in germany comprehensive study on the occurrence and distribution of pathogenic microorganisms carried by synanthropic flies caught at different rural locations in germany human pathogens in body and head lice clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious diseases society of america tackling contamination of the hospital environment by methicillin-resistant staphylococcus aureus (mrsa): a comparison between conventional terminal cleaning and hydrogen peroxide vapour decontamination richtlinie der österreichischen gesellschaft für hygiene, mikrobiologie und präventivmedizin (öghmp) für apparate zur automatischen dosierung flüssiger chemischer desinfektionsmittel mupirocin and chlorhexidine resistance in staphylococcus aureus in patients with community -onset skin and soft tissue infections the repetitive irritation test (rit) with a set of 4 standard irritants surgical site infections and the surgical care improvement project (scip): evolution of national quality measures randomized, multicenter trial of antibiotic prophylaxis in elective colorectal surgery: single dose vs 3 doses of a second-generation cephalosporin without metronidazole and oral antibiotics how often do you wash your hands? a 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wound complications after major gastrointestinal operations . the surgeon as a risk factor impact of an environmental cleaning intervention on the presence of methicillin-resistant staphylococcus aureus and vancomycinresistant enterococci on surfaces in intensive care unit rooms reduction in nosocomial transmission of drug-resistant bacteria after introduction of an alcohol-based handrub antibiotics for preventing infection in open limb fractures evaluation of an antibiotic-impregnated shunt system for the treatment of hydrocephalus a risk factor in men with bladder outflow obstruction dna damage in multiple organs after exposure to chlorhexidine in wistar rats efficacy of soap and water and alcohol-based hand-rub preparations against h1n1 influenza virus on the hands of human volunteers an outbreak of handscrubbing-related surgical site infections in vascular surgical procedures serratia liquefaciens bloodstream infections from contamination of epoetin alfa at a hemodialysis center infektiöser hospitalismus in einrichtungen für krebskranke und seine bekämpfung (internationaler trend) zahnärztliche betreuung von patienten mit tumortherapeutischer kopf-hals-bestrahlung (stellungnahme der dgzmk und degro) . strahlentherapie und onkologie reduction in central line-associated bloodstream infections by implementation of a postinsertion care bundle guidelines for the management of adults with hospital-acquired, ventilatorassociated, and healthcare-associated pneumonia who washes hands after using the bathroom? anaphylaxis to chlorhexidine coated central venous catheters: a case series and review of the literature the science behind stable, super-oxidized water do antibiotic-impregnated catheters prevent infection in csf diversion procedures? review of the literature hand-to-hand transmission of rhinovirus colds interruption of experimental rhinovirus infection medizinische bedeutung und bekämpfung . in: aspöck h (hsg .) krank durch arthropoden treating infected diabetic wounds with superoxidized water as antiseptic agent: a preliminary experience nosokomiale infektionen bei frühgeborenen -umsetzung der krinko-empfehlungen im deutschen frühgeborenennetzwerk effect of hand sanitizer use on elementary school absenteeism antibiotic stewardship formaldehyde sterilisation, i determination of formaldehyde residuals in autoclavesterilized materials formaldehyde sterilisation, ii formaldehyde sterilisation, the process and the influence on the formaldehyde residuals formaldehyde sterilisation, iii the behaviour of the loaded autoclaves and the permeability of plastic materials to formaldehyde chemical inactivation of hiv on surfaces outbreak of enterobacter cloacae related to understaffing, overcrowding, and poor hygiene practices public health significance of urban pests . world health organization concentration of bacteria passing through puncture holes in surgical gloves wann sollte in operationsräumen ein wechsel chirurgischer handschuhe erfolgen? reduction in hospital-wide incidence of infection or colonization with methicillin-resistant staphylococcus aureus with use of antimicrobial hand hygiene gel and statistical process control charts étude risque-bénéfice de l'usage des aldéhydes comme désinfectants à l'hôpital novel pyrogen tests based on the human fever reaction carcinogenicity study of sodium hypochlorite in f344 rats evaluating central venous catheter care in a pediatric intensive care unit surgical infection society guideline: prophylactic antibiotic use in open fractures: an evidence-based guideline comparison of the microbiological efficacy of hydrogen peroxide vapor and ultraviolet light processes for room decontamination reduction in acquisition of vancomycin-resistant enterococcus after enforcement of routine environmental cleaning measures risk of hand or glove contamination after contact with patients colonized with vancomycin-resistant enterococcus or the colonized patients' environment the safe surgery saves lives study group: a surgical safety checklist to reduce morbidity and mortality in a global population does the wide use of quaternary ammonium compounds enhance the selection and spread of antimicrobial resistance and thus threaten our health? effects of long-term routine use of selective digestive decontamination on antimicrobial resistance nachweis der antibakteriellen aktivität von chitosan single-dose vs multiple-dose antibiotic prophylaxis in instrumented lumbar fusion -a prospective study umfüllen von händedesinfektionsmitteln -hygienische und haftungsrechtliche aspekte prophylaxis with cefamandole nafate in elective orthopedic surgery zur inaktivierenden wirkung organischer säuren auf parvoviren bei verschiedenen temperaturen hessische verwaltung für bodenmanagement und geoinformation (hvbg) low infection rate after tumor hip rthroplasty for metastatic bone disease in a cohort treated with extended antibiotic prophylaxis use of alcohol hand sanitizer as an infection control strategy in an acute care facility hsg .) public health significance of urban pests . world health organization high rate of qaca -and qacb -positive methicillin -resistant staphylococcus aureus isolates from chlorhexidine -impregnated catheter -related bloodstream infections use of chlorhexidine-impregnated dressing to prevent vascular and epidural catheter colonization and infection: a meta-analysis schadwirkungen durch tierische gesundheitsschädlinge, insektizide und akarizide (allergieverursachung, sachgerechte bekämpfung sowie arbeits-und betroffenenschutzmaßnahmen) public health significance of urban pests . world health organization reduced susceptibility to chlorhexidine in staphylococci: is it increasing and does it matter? polyhexamethylene biguanide: two year feeding study in rats . study performed by zeneca central toxicology laboratory practice guideline adherence and health care outcomesuse of prophylactic antibiotics during surgery in taiwan strategies for the prevention of central venous catheter infections: an american pediatric surgical association outcomes and clinical trials committee systematic review risk of acquiring antibiotic-resistant bacteria from prior room occupants targeted versus universal decolonization to prevent icu infection review on the efficacy, safety and clinical applications of polihexanide, a modern wound antiseptic octenidine dihydrochloride, a modern antiseptic for skin, mucous membranes and wounds effectiveness of alcohol-based hand disinfectants in a public administration: impact on health and work performance related to acute respiratory symptoms and diarrhoea survival of bacterial pathogens on paper and bacterial retrieval from paper to hands . preliminary results the durability of examination gloves used on intensive care units einsatz textiler aufbereitbarer unterziehhandschuhe für medizinische tätigkeiten: eine machbarkeitsstudie prospective randomized trial of 10 % povidone-iodine versus 0 .5 % tincture of chlorhexidine as cutaneous antisepsis for prevention of central venous catheter infection in vivo microdialysis to measure antibiotic penetration into soft tissue during cardiac surgery chlorhexidine and chondrolysis in the knee chlorhexidine and chondrolysis in the knee fluoroquinolone prophylaxis in patients with neutropenia: a meta-analysis of randomized placebo-controlled trials international agency for research and cancer . iarc classifies formaldehyde as carcinogenic to humans: press release: n° 153 effect of antimicrobial prophylaxis on the incidence of infections in clean surgical wounds in hospitals undergoing renovation antibiotic prophylaxis for transrectal biopsy of the prostate: a prospective randomized study of the prophylactic use of single dose oral fluoroquinolone versus trimethoprim-sulfamethoxazole 857 -sterilization of health care products -dry heat -requirements for the development, validation and routine control of a sterilization process for medical devices effect of body mass index and ertapenem versus cefotetan prophylaxis on surgical site infection in elective colorectal surgery risk factors for infection after knee arthroplasty . a registerbased analysis of 43 149 cases antimicrobial prophylaxis for contaminated head and neck surgery efficacy of an alcohol/chlorhexidine hand hygiene program in a hospital with high rates of noso-comial methicillin-resistant staphylococcus aureus (mrsa) infection prospective, controlled study of vinyl glove use to interrupt clostridium difficile nosocomial transmission zur viruziden wirksamkeit chemischer und physikalischer desinfektionsmittel und -verfahren epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs small volumes of n-propanol (60 %) applied for 3 minutes may be ineffective for surgical hand disinfection gehäufte hautirritationen durch ein viruzides händedesinfektionsmittel mit hohem phosphorsäuregehalt händehygiene zur prävention nosokomialer infektionen bacterial in-use contamination of an alcohol-based hand rub under accelerated test conditions surgical hand disinfection with a propanolbased hand rub: equivalence of shorter application times bacterial population kinetics on hands during 2 consecutive surgical hand disinfection procedures influence of applied volume on efficacy of 3-minute surgical reference disinfection method pren 12 the impact of antimicrobial drug consumption and alcoholbased hand rub use on the emergence and spread of extended-spectrum β-lactamase-producing strains: a time-series analysis influence of rub-in technique on required application time and hand coverage in hygienic hand disinfection improving patient safety during insertion of peripheral venous catheters: an observational intervention study effective reprocessing of reusable dispensers for surface disinfection tissues -the devil is in the details poorly processed reusable surface disinfection tissue dispensers may be a source of infection single-dose oral ciprofloxacin versus placebo for prophylaxis during transrectal prostate biopsy evaluation of effect and comparison of superoxidised solution (oxum) v/s povidone iodine (betadine) impact of non-rinse skin cleansing with chlorhexidine gluconate on prevention of healthcare -associated infections and colonization with multi-resistant organisms: a systematic review antibiotic prophylaxis and the risk of surgical site infections following total hip arthroplasty: timely administration is the most important factor epidemiologie und ursachen mikrobieller biozidresistenzen comparison of etiology and rate of infection in different surgical wounds chlorhexidine impregnated central venous catheter inducing an anaphylatic shock in the intensive care unit the modification of high-dose therapy shortens the duration of neutropaenia by delay of leucocyte nadir zur bedeutung der listen bekannt gemachter mittel und verfahren für behördlich angeordnete entseuchungen bekämpfung von wirbeltieren aufgrundlage des § 18 infektionsschutzgesetz disinfection, sterilization and preservation, 3 . aufl . philadelphia: lea & febiger outcomes of prophylactic antibiotics following surgery for zygomatic bone fractures einfluss der fußbodendesinfektion auf die mikrobielle und partikulare belastung der raumluft in augen-op-räumen mit verdrängungslüftungsbereichen standardized comparison of antiseptic efficacy of triclosan, pvp-iodine, octenidine dihydrochloride, polyhexanide and chlorhexidine digluconate decontamination of room air and adjoining wall surfaces by nebulizing hydrogen peroxide reinigung und desinfektion von eßgeschirr, instrumenten und ausscheidungsbehältern im krankenhaus wallhäußers praxis der sterilisation, desinfektion, antiseptik und konservierung . 5 . auflage kramer a, assdian o (hrsg,), wallhäußers praxis der sterilisation, desinfektion, antiseptik und konservierung . 5 . auflage . stuttgart kommission für krankenhaushygiene und infektionsprävention am robert koch-institut . anforderungen der hygiene bei operationen und anderen invasiven eingriffen ausbruchmanagement und strukturiertes vorgehen bei gehäuftem kommission für krankenhaushygiene und infektionsprävention (krinko) am robert koch-institut (rki) kommission für krankenhaushygiene und infektionsprävention (krinko) am robert koch-institut . prävention postoperativer infektionen im operationsgebiet kommission für krankenhaushygiene und infektionsprävention (krinko) am robert koch-institut . anforderungen an die hygiene bei der medizinischen versorgung von immunsupprimierten patienten anforderungen an die hygiene bei der aufbereitung von medizinprodukten kommission für krankenhaushygiene und infektionsprävention (krinko) am robert koch-institut . hygienemaßnahmen bei infektionen oder besiedlung mit multiresistenten gramnegativen stäbchen kommission für krankenhaushygiene und infektionsprävention (krinko) am robert koch-institut . prävention der nosokomialen beatmungsassoziierten pneumonie end-of-procedure cefazolin concentrations after administration for prevention of surgical-site infection risk factors for adult nosocomial meningitis after craniotomy role of antibiotic prophylaxis integrity of vinyl and latex procedures gloves hand disinfection and antiseptic of skin, mucous membranes, and wounds kramer a, assadian o, hrsg . wallhäußers praxis der sterilisation, desinfektion, antiseptik und konservierung . qualitätssicherung der hygiene in medizinischen und industriellen bereichen use of biocidal surfaces for reduction of healthcare acquired infections prophylactic use of topical antiinfectives in ophthalmology hand rub-associated fire incidents during 25 038 hospital-years in germany wallhäußers praxis der sterilisation, desinfektion, antiseptik und konservierung . stuttgart: thieme perioperative antibiotikaprophylaxedominierende möglichkeit zu infektionsprophylaxe bei chirurgischen eingriffen? health risks of surface disinfection in households with special consideration on quaternary ammonium compounds (qacs) mycotoxins in indoor and outdoor environments and human health how long do nosocomial pathogens persist on inanimate surfaces? a systematic review klinische antiseptik zielsetzung und möglichkeiten der antiseptik im genitalbereich explantationstest mit haut und peritoneum der neonatalen ratte als voraussagetest zur verträglichkeit lokaler antiinfektiva für wunden und körperhöhlen mitteilung der kommission für krankenhaushygiene und infektionsprävention am robert koch-institut limited efficacy of alcohol-based hand gels toxikologische bewertung für die händedesinfektion relevanter antimikrobieller wirkstoffe konsensusempfehlung zur auswahl von wirkstoffen für die wundantiseptik virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations quantity of ethanol absorption after excessive hand disinfection using three commercially available hand rubs is minimal and below toxic levels for humans kramer a, assadian o (hrsg) wallhäusers praxis der sterilisation desinfektion improving adherence to surgical hand preparation wallhäußers praxis der sterilisation, desinfektion kramer a, assadian o (hrsg) wallhäusers praxis der sterilisation desinfektion, antiseptik und konservierung wallhäusers praxis der sterilisation, desinfektion, antiseptik und konservierung . stuttgart: thieme wallhäusers praxis der sterilisation desinfektion, antiseptik und konservierung gemeinsame stellungnahme der dgkh und der desinfektionsmittelkommission des vah zum stellenwert der antimikrobiellen ausstattung von objekten in der infektionsprävention maintaining health by balancing microbial exposure and prevention of infection: the hygiene hypothesis versus the hypothesis of early immune challenge suitability of tissue tolerable plasmas (ttp) for the management of chronic wounds wound antiseptics today -an overview nationales referenzzentrum für surveillance von nosokomialen infektionen the influence of organizational context on quality improvement and patient safety efforts in infection prevention: a multi-center qualitative study resistenzsituation bei klinisch wichtigen infektionserregern aus dem ambulanten versorgungsbereich gegenüber antibiotika . bericht über die ergebnisse einer multizentrischen studie der paul-ehrlich-gesellschaft für chemotherapie e . v . aus dem jahre for the trape study group: the effect of a quality improvement collaborative to improve antimicrobial prophylaxis in surgical patients: a randomized trial influence of combined intravenous and topical antibiotic prophylaxis on the incidence of infections, organ dysfunctions, and mortality in critically ill surgical patients: a prospective, stratified, randomized, double-blind, placebo-controlled clinical trial antimicrobial prophylaxis in allogeneic bone marrow transplantation . guidelines of the infectious diseases working party (agiho) of the german society of haematology and oncology aminoglycoside-free interventional antibiotic management in patients undergoing haemopoietic stem cell transplantation decreasing ventricular infections through the use of a ventriculostomy placement bundle: experience at a single institution die weltweite ausbreitung von bettwanzen stellt auch in deutschland ein problem dar health care worker exposures to pertussis: missed opportunities for prevention antibiotic prophylaxis after total joint replacements infection prevention in total knee and total hip arthr oplasties past administration of beta-lactam antibiotics and increase in the emergence of beta-lactamase-producing bacteria in patients with orofacial odontogenic infections . oral surg oral med oral pathol oral radiol endod incidence of beta-lactamase production and antimicrobial susceptibility of anaerobic gramnegative rods isolated from pus specimens of orofacial odontogenic infections long-term in vivo carcinogenicity tests of potassium bromate, sodium hypochlorite, and sodium chlorite conducted in japan . environ the action of alcohols on rotavirus, astrovirus and enterovirus caring for pregnant women and newborns with hepatitis b or c microcyn: a novel super oxidized water with neutral ph and disinfectant activity dissemination of the cdc's hand hygiene guideline and impact on infection rates appropriate use of silver dressings in wounds . an expert working group consensus . london: wounds international impact of combined low-level mupirocin and genotypic chlorhexidine resistance on persistent methicillin-resistant staphylococcus aureus carriage after decolonization therapy: a case-control study illness transmission in the home: a possible role for alcoholbased hand gels enterococcus: not an innocent bystander in cirrhotic patients with spontaneous bacterial peritonitis antibiotic prophylaxis to reduce the risk of joint implant contamination during dental surgery seems unnecessary die methoden der praktischen hygiene . wiesbaden: bergmann asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome long-term (6-year) effect of selective digestive decontamination on antimicrobial resistance in intensive care, multiple-trauma patients a bundle approach to reduce the incidence of external ventricular and lumbar drain-related infections chlorhexidine impregnated dressing for prevention of colonization of central venous catheters in infants and children: a randomized controlled study über die ursachen der widerstandsfähigkeit der sporen gegen hohe temperaturen . ein beitrag zur theorie der desinfektion ultraclean air and antibiotics for prevention of postoperative infection . a multicenter study of 8 052 joint replacement operations risk factors for neurosurgical site infections: an 18-month prospective survey -clinical article bacteremia and bacteriuria after transrectal ultrasound guided prostate biopsy single-dose antibiotic prophylaxis in core prostate biopsy: impact of timing and identification of risk factors impact of a hospital-wide hand hygiene promotion strategy on healthcare-associated infections antimicrobial therapy of unexplained fever in neutropenic patients -guidelines of the infectious diseases working party (agiho) of the german society of hematology and oncology (dgho), study group interventional therapy of unexplained fever, arbeitsgemeinschaft supportivmassnahmen in der onkologie (aso) of the deutsche krebsgesellschaft (dkg-german cancer society) preliminary report of associated factors in wound infection after major head and neck neoplasm operations -does the duration of prophylactic antibiotic matter? risk factors for surgical-wound infection in general surgery: a prospective study chemical disinfection of human rotavirus-contaminated inanimate surfaces systemic review of complications after prostate biopsy postoperative infections and antibiotic prophylaxis for hysterectomy in sweden: study by the swedish national register for gynecologic surgery epic3: national evidence -based guidelines for preventing healthcare -associated infections in nhs hospitals in england therapie in der hno-heilkunde diagnostic assessment of different environmental cleaning monitoring methods untersuchungen über die mikrobielle kontamination von außenseiten von sterilgutverpackungen in abhängigkeit von der lagerungsdauer impact of rifaximin on the frequency and characteristics of spontaneous bacterial peritonitis in patients with liver cirrhosis and ascites external ventricular drain infections: successful implementation of strategies to reduce infection rate performance feedback of hand hygiene, using alcohol gel as the skin decontaminant, reduces the number of inpatients newly affected by mrsa and antibiotic costs bacterial translocation studied in 927 patients over 13 years thermoinaktivierung von viren durch mikrowellen virusdesinfektion in labor und tierärztlicher praxis experimentelle ergebnisse über die stabilität von pockenviren unter labor-und umweltbedingungen mögliche auswirkungen von klimaveränderungen auf die ausbreitung von primär humanmedizinisch relevanten krankheitserregern und deren vektoren sowie auf die wichtigen humanparasiten in deutschland die bedeutung von schaben in der krankenhaushygiene the forgotten role of alcohol: a systematic review and meta-analysis of the clinical efficacy and perceived role of chlorhexidine in skin antisepsis gesunde haut als voraussetzung für eine effektive händedesinfektion the risk of bloodstream infection in adults with differentintravascular devices: a systematic review of 200 published prospective studies prospective randomised trial of povidoneiodine, alcohol, and chlorhexidine for prevention of infection associated with central venous and arterial catheters use of audit tools to evaluate the efficacy of cleaning systems in hospitals probleme bei der biologischen testung von gas-sterilisatoren public health significance of urban pests . world health organization strategies to prevent central line-associated bloodstream infections in acute care hospitals: 2014 update the gastrointestinal tract . the "undrained abscess" of multiple organ failure a preliminary investigation of the microbiology endotoxin content in the water reservoirs of bench top non-vacuum autoclaves international and specialty trends in the use of prophylactic antibiotics to prevent infectious complications after insertion of external ventricular drainage devices antiseptics and disinfectants: activity, action, and resistance ad hoc clostridium difficile surveillance working group . recommendations for surveillance of clostridium difficile-associated disease detection of qaca/b in clinical isolates of methicillin-resistant staphylococcus aureus from a regional healthcare network in the eastern united states staphylococcus aureus 2 946 infections in pediatric oncology patients: high rates of antimicrobial resistance, antiseptic tolerance and complications a meta-analysis comparing suprapubic and transurethral catheterization for bladder drainage after abdominal surgery desinfektion und sterilisation thermolabiler instrumente mit einem formaldehyd-unterdruckverfahren (alhydomat 50) medicines healthcare products regulatory agency (mhra) risk factors of surgical wound infection in patients undergoing herniorrhaphy super-oxidized solution inhibits ige-antigen-induced degranulation and cytokine release in mast cells bgbl . i s . 3396), die zuletzt durch artikel 4 des gesetzes vom 29 current concepts review . prophylactic antibiotics in hip and knee arthroplasty ein neues lokales antiseptikum zur oberflächenbehandlung bei schwerstverbrannten the pathogenesis and epidemiology of catheter-related infection with pulmonary artery swan-ganz catheters: a prospective study utilizing molecular subtyping what is the predominant source of intravascular catheter infections? area fumigation with hydrogen peroxide vapor antibiotic consumption and resistance: data from europe and germany randomized controlled trial and cost-effectiveness analysis of silver-donating antimicrobial dressings for venous leg ulcers (vul-can trial) polyhexamethylene biguanide: two year oncogenicity study in mice . study performed by zeneca central toxicology laboratory reducing picu central line-associated bloodstream infections: 3-year results central line-associated bloodstream infection prevention daily chlorhexidine bathing to reduce bacteraemia in critically ill children: a multicentre, cluster-randomised, crossover trial prospective, randomized trial of two antiseptic solutions for prevention of central venous or arterial catheter colonization and infection in intensive care unit patients molecular revolution in the diagnosis of microbial brain abscesses surgical glove perforation and the risk of surgical site infection levofloxacin resistant escherichia coli sepsis following an ultrasound-guided transrectal prostate biopsy: report of four cases and review of the literature prophylactic and local applications of antimicrobials in endodontics: an update review primary central nervous system lymphoma treated with high-dose methotrexate, high-dose busulfan/thiotepa, autologous stem-cell transplantation and response-adapted whole-brain radiotherapy: results of the multicenter ostdeutsche studiengruppe hamato-onkologie osho-53 phase ii study biological toxicity of acid electrolysed functional water: effect of oral administration on mouse digestive tract and changes in body weigth analysis of complications in 430 consecutive pediatric patients treated with intrathecal baclofen therapy: 14-year experience the role of postoperative antibiotics in facial fractures: comparing the efficacy of a 1-day versus a prolonged regimen . the journal of trauma and acute care surgery safety and impact of chlorhexidine antisepsis interventions for improving neonatal health in developing countries nosokomiale sepsis bei sehr kleinen frühgeborenen -diagnostik und therapie residual antimicrobial effect of chlorhexidine digluconate and octenidine dihydrochloride on reconstructed human epidermis comparative study of in vitro cytotoxicity of povidoneiodine in solution, in ointment, or in a liposomal formulation (repithel ® ) and selected antiseptics biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity interaction of octenidine and chlorhexidine with mammalian cells and the resulting microbicidal effect (remanence) of the combinations finding a benchmark for monitoring hospital cleanliness prevention of bloodstream infections by use of daily chlorhexidine baths for patients at a long-term acute care hospital use of uv powder for surveillance to improve environmental cleaning bed bugs in healthcare settings progressive ulzerative keratitis related to the use of topical chlorhexidine gluconate (0 .02 %) isolation precautions for the prevention of the transmission of ebola, enterovirus-d68 and other infectious agents in healthcare settings in vitro activity and concentrations in serum, urine, prostatic secretion and adenoma tissue of ofloxacin in urological patients surveillance study in europe and brazil on clinical aspects and antimicrobial resistance epidemiology in females with cystitis ( aresc): implications for empiric therapy use of quinolones in urinary tract infections and prostatitis increasing hospital admission rates for urological complications after transrectal ultrasound guided prostate biopsy stand und perspektiven der antibiotika-prophylaxe bei patienten mit künstlichem gelenkersatz primary prophylaxis of bacterial infections and pneumocystis jirovecii pneumonia in patients with hematological malignancies and solid tumors : guidelines of the infectious diseases working party (agiho) of the german society of hematology and oncology (dgho) désinfection par voie aérienne : une norme pour sonder la qualité des produits antibiotic prophylaxis in surgery -2005 and beyond orthopedic surgical site infections: analysis of causative bacteria and implications for antibiotic stewardship antimicrobial prophylaxis in surgery: the role of pharmacokinetics late infections after allogeneic bone marrow transplantations: comparison of incidence in related and unrelated donor transplant recipients polihexanide carcinogenicity: analysis of human health risk . prepared for the australian pesticides and veterinary medicines authority guidelines for the prevention of intravascular catheter-related infections . the hospital infection control practices advisory committee, center for disease control and prevention guidelines for the prevention of intravascular catheterrelated infections mediastinal irrigation with superoxidized water after open heart surgery: the safety and pitialls of cardiovascular surgical applications the efficacy of daily bathing with chlorhexidine for reducing healthcare-associated bloodstream infections: a meta-analysis . infect contr hsg .) public health significance of urban pests regulatory action criteria for filth and other extraneous materials . iii . review of flies and foodborne enteric disease the effectiveness of single-dose fosfomycin as antimicrobial prophylaxis for patients undergoing transrectal ultrasound-guided biopsy of the prostate efficacy and effectiveness of influenza vaccines . a systematic review and meta-analysis expertisen-verzeichnis der österreichische gesellschaft für hygiene, mikrobiologie und präventivmedizin hygiene-richtlinien für krankenhauswäsche bearbeitende wäschereien surgical site infection rates after minimally invasive spinal surgery bakterien in sekreten extra-und intraoraler operationswunden kontamination oder infektion? hydrogen peroxide vapor decontamination of an intensive care unit to remove environmental reservoirs of multidrug-resistant gramnegative rods during an outbreak selection for qaca carriage in cc22, but not cc30, methicillin-resistant staphylococcus aureus bloodstream infection isolates during a successful institutional infection control programme the role of contaminated surfaces in the transmission of nosocomial pathogens the survival of influenza a(h1n1)pdm09 virus on 4 household surfaces incidence of acute prostatitis caused by extended-spectrum beta-lactamase-producing escherichia coli after transrectal prostate biopsy mutagene potenz von wofasteril, wofasept, formaldehyd, chlorhexidin und bronopol im knochenmark an der maus adherence to surgical site infection guidelines in italian cardiac surgery units quality of perioperative chemoprophylaxis in obstetrics and gynecology: preliminary results of asppoc in greece and italy incidence of microperforation for surgical gloves depends on duration of wear efficacy of antibiotic-impregnated cement in total hip replacement . a meta-analysis effect of glutaraldehyde on the antigenicity and infectivity of hepatitis a virus an evaluation of environmental decontamination with hydrogen peroxide vapor for reducing the risk of patient acquisition of multidrug-resistant organisms prospective, randomized, double-blind study comparing single-agent antibiotic therapy, ciprofloxacin, to combination antibiotic therapy in open fracture wounds vestibular and cochlear ototoxicity of topical antiseptics assessed by evoced potentials etiology and mortality of spontaneous bacterial peritonitis in liver transplant recipients: a cohort study prophylactic chemotherapy with fosfomycin trometamol salt during transurethral prostatic surgery: a controlled multicenter clinical trial associations for surgical research, angers and colombes, france . risk factors for prediction of surgical site infections in "clean surgery perioperative antibiotikaprophylaxe in der chirurgie vergleichende untersuchungen zur resistenz von mycobacterium terrae untersuchungen zur prüfung der viruziden wirksamkeit von desinfektionsmitteln für die chemische instrumentendesinfektion . 2 . mitteilung: vergleich der ergebnisse von suspensionsversuchen und praxisnaher prüfung zur prüfung der viruziden wirksamkeit von flächendesinfektionsmitteln surgical site infections surveillance in neurosurgery patients use of a 1-piece chlorhexidine gluconate transparent dressing on critically ill patients university of minnesota, department of food science and nutrition and school of public health anaphylaxis to chlorhexidine . case report . implication of immunoglobulin e antibodies and identification of an allergenic determinant the integrity of latex gloves in clinical dental practice effectiveness of a hospital-wide program to improve compliance with hand hygiene learning, techniques, and complications of endoscopic ultrasound (eus)-guided sampling in gastroenterology core elements of hospital antibiotic stewardship programs from the centers for disease control and prevention comparison of infection rate with the use of antibiotic-impregnated vs standard extraventricular drainage devices: a prospective, randomized controlled trial effectiveness of routine patient cleansing with chlorhexidine gluconate for infection prevention in the medical intensive care unit relationship between chlorhexidine gluconate skin concentration and microbial density on the skin of critically ill patients bathed daily with chlorhexidine gluconate chemical inactivation of viruses . dissertation, univ . missouri dictyoptera, blattodea) -ihre bedeutung als überträger von krankheitserregern und als verursacher von allergien . in: aspöck h (hrsg .) . krank durch arthropoden prophylactic antibiotics in orthopaedic surgery translating evidence into practice: a model for large scale knowledge translation an intervention to decrease catheter-related bloodstream infections in the icu sustaining reductions in catheter related bloodstream infections in michigan intensive care units: observational study prevention and management of ventriculoperitoneal shunt infections in children psa-benutzungsverordnung vom 4 . dezember complication rates and risk factors of 5 802 transrectal ultrasound-guided sextant biopsies of the prostate within a populationbased screening program high infection rate outcomes in long-bone tumor surgery with endoprosthetic reconstruction in adults: a systematic review bestimmung der keimzahl und kinetik der keimeliminierung bei bakteriämie nach zahnentfernung carriage by the housefly (musca domestica) of multiple-antibiotic-resistant bacteria that are potentially pathogenic to humans, in hospital and other urban environments in misurata antibiotic prophylaxis for surgical introduction of intracranial ventricular shunts: a systematic review definition der desinfektion a simple method to reduce infection of ventriculoperitoneal shunts ein neues therapiekonzept bei skabies alcohols for skin antisepsis at clinically relevant skin sites an innovative tropical drug formulation for wound healing and infection treatment: in vitro and in vivo investigations of a povidone iodine liposome hydrogel effectiveness of antibiotic prophylaxis in third molar surgery: a meta-analysis of randomized controlled clinical trials richtlinie 2007/47/eg, bekanntmachungen im amtsblatt der europäischen union juni 1993 (abl . eg nr . l 169 s . 1) zuletzt geändert durch artikel 2 der richtlinie richtlinie 93/42/ewg über medizinprodukte, bekanntmachungen im amtsblatt der richtlinie 97/23/eg über druckgeräte, bekanntmachungen im amtsblatt der europäischen union richtlinien des bundesausschusses der ärzte und krankenkassen über die ärztliche betreuung während der schwangerschaft und nach der entbindung ciprofloxacin versus gentamicin in prophylaxis against bacteremia in transrectal prostate needle biopsy richtlinie des robert koch-institutes zur prüfung der wirksamkeit von flächendesinfektionsmitteln bei tuberkulose 3 und 6 .4 der richtlinie für krankenhaushygiene und infektionsprävention -anforderungen der hygiene an die wäsche aus einrichtungen des gesundheitsdienstes, die wäscherei und den waschvorgang richtlinie des robert koch-institutes zur prüfung der wirksamkeit von desinfektionsmitteln für die chemische instrumentendesinfektion bei tuberkulose (stand 1 .9 .1994) richtlinie des robert koch-instituts zur prüfung der viruzidie von chemischen flächendesinfektionsmitteln und instrumentendesinfektionsmitteln, die in die liste gemäß § 10c des bundesseuchengesetzes aufgenommen werden sollen, fassung vom 1 . märz empfehlungen der kommission für krankenhaushygiene und infektionsprävention empfehlung zur prüfung und deklaration der wirksamkeit von desinfektionsmittel gegen viren liste der vom robert koch-institut geprüften und anerkannten desinfektionsmittel und -verfahren biopatch -a new concept in antimicrobial dressings for invasive devices baseline prevalence of antimicrobial resistance and subsequent infection following prostate biopsy using empirical or altered prophylaxis: a bias-adjusted meta-analysis no evidence to link prosthetic joint infections with dental procedures inactivation of animal and human prions by hydrogen peroxide gas plasma sterilization environmental exposure to carbapenem-resistant acinetobacter baumannii as a risk factor for patient acquisistion of a . baumannii ensuring appropriate timing of antimicrobial prophylaxis empfehlungen zur antibiotikaprophylaxe vor gastrointestinalen endoskopien bei patienten mit erhöhtem endokarditisrisiko nosocomial infections after off-pump koronary artery bypass surgery: frequency, characteristics, and risk factors . interact cardiovasc cleaning and disinfection in outbreak controlexperiences with different pathogens arzneimittelverzeichnis für deutschland (einschließlich eu-zulassungen und bestimmter medizinprodukte) . rote liste service gmbh effect of antiseptic wound irrigation of traumatic soft tissue wounds on postinterventional wound infection rates -a longitudinal mono-centre cohort study hospital epidemiology and infection 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antimicrobial-impregnated external ventricular drain catheters: a prospective, randomized, controlled trial antibiotic prophylaxis for transrectal prostate biopsy . cochrane database syst rev a prospective, randomized, double-blind study of single high dose versus multiple standard dose gentamicin both in combination with metronidazole for colorectal surgical prophylaxis antibiotic pharmacodynamics in surgical prophylaxis: an association between intraoperative antibiotic concentrations and efficacy molecular investigation of bacterial communities on the inner and outer surfaces of peripheral venous catheters molecular investigation of bacterial communities on intravascular catheters: no longer just staphylococcus impact of a prevention strategy targeting hand hygiene and catheter care on the incidence of catheter-related bloodstream infections hospital-wide multidisciplinary, multimodal intervention programme to reduce central venous catheter-associated bloodstream infection impact of bordetella pertussis exposures on a massachusetts tertiary care medical system operative sanierung florider venöser ulcera -wundinfektionsrate unter antibiogramm-orientierter perioperativer prophylaxe krankheitsübertragung: von herausragender bedeutung für effektivität der mechanischen erregerverbreitung ist der anpassungsgrad (synanthropiegrad) eines hygieneschädlings an den menschlichen siedlungsbereich, da dieser das ausmaß des kontinuierlichen erregerkontakts zwischen kontaminationsort und behandlungs-, wohn-und/oder arbeitsumfeld bestimmt (faulde und freise 2014) . vor allem in sensiblen bereichen wie krankenhäusern und großküchen ist die mechanische erregerverschleppung bedeutsam (sramova et al. 1992 ). besondere relevanz bei der übertragung von ni haben passive vektoren, wenn sie multi-oder sogar panresistente, fakultativ oder obligat humanpathogene erreger großflächig verbreiten und gleichzeitig die verfügbare erregermenge durch vermehrung z. b. im gastrointestinaltrakt erhöhen (faulde und freise 2014; sramova et al. 1992) .multiresistente humanpathogene bakterien an synanthropen arthropoden konnten innerhalb bzw. in unmittelbarer nähe von krankenhäusern in deutschland, libyen, in afrika südlich der sahara sowie in indien an fliegen, schaben und an der schmetterlingsmücke clogmia albipunctata nachgewiesen werden (boulesteix et al. 2005; faulde und spiesberger 2013; rahuma et al. 2005; tilahun et al. 2012 key: cord-014712-5u4e00q6 authors: nan title: selected abstracts from the 100th j project meeting, antalya, turkey, march 12-14, 2014 date: 2014-08-02 journal: j clin immunol doi: 10.1007/s10875-014-0065-9 sha: doc_id: 14712 cord_uid: 5u4e00q6 nan hans d. ochs the identification of single gene defects involving genes that play crucial roles in adaptive or innate immunity is not only important for confirming a pid diagnosis, but may contribute to optimal therapy, and contribute to genetic counseling, carrier identification and pre-natal diagnosis. to accomplish this, the diagnostician has to consider the inheritance of these disorders: x-linked, autosomal recessive, autosomal dominant and the type of mutation: loss of function, hypomorphic, dominant negative or gain of function. conventional techniques to screen for single gene mutations include flow cytometry to measure disease-specific expression of proteins (cell surface, cytoplasmic or nuclear) or to analyze relevant signaling pathways (e.g. stat5b phosphorylation via the il-2r; pstat3 via the il-10 receptor); and sanger sequencing of mrna or genomic dna using dye-terminator sequencing. next generation sequencing ("by synthesis") has been refined, and is being used increasingly to study families with multiple affected members with an atypical pid phenotype, or to explore consanguineous families with one member affected. whole exome sequencing requires less data analysis, compared with whole genome sequencing, but may miss intronic or regulatory elements. the challenge of whole exome/genome sequencing is to confirm that the multiple variants identified by these techniques are causative for the clinical phenotype of the study patient. however, with increasing experience, next generation sequencing will become a standard procedure for the identification of genetic defects responsible for inherited diseases, including pid. stephen jolles immunodeficiency centre for wales, university hospital of wales, cardiff, uk. immunoglobulin (ig)-replacement therapy represents the mainstay of treatment for patients with primary antibody deficiency and is administered either intravenously (ivig) or subcutaneously (scig). recent developments using a high-purity recombinant human hyaluronidase have allowed the longer term repeated use of this enzyme to facilitate the delivery of immunoglobulin and other molecules including antibiotics, local anesthetics, insulin, morphine and fluid replacement into the subcutaneous space. hyaluronidase facilitated scig (fscig) has helped overcome the limitations on the volume which can be delivered into the subcutaneous tissues by enabling dispersion of scig and its absorption into lymphatics. the rate of facilitated scig infusion is equivalent to that of ivig, and the volume administered at a single site can be greater than 700 ml, an enormous increase over conventional scig, at 20-40 ml. the use of fscig avoids many of the systemic side effects of ivig, and has higher bioavailability than scig. over three years of safety data are now available for this approach though longer term safety data and information on anti-hylauronidase antibodies and their relevance will be required. fscig could aid several areas of patient management in both primary antibody deficiency and immunomodulatory indications. key factors influencing how it will be used in future are long-term safety data and cost-benefit analysis. date after the cd19 deficiency was firstly described in a turkish girl in 2006. the patients with cd19 deficiency had normal b-cell differentiation in bone marrow, normal absolute number of b cells in peripheral blood and normal bcr repertoire. also, the patients had normal stimulation via the bcr, normal proliferation response upon antigen stimulation but reduced memory-b-cell compartment in peripheral blood. the cd19 deficiency leads to hypogammaglobulinemia and impaired antigen-specific humoral immune responses after vaccination. we had described thirty carriers in relatives of our two patients with cd19 deficieny and also showed that the mfi value of cd19 and cd21 expressions were lower in the carriers than in controls. during the last five years, it was also showed that the mutations of the other coreceptors of b cells such as cd81 and cd21 caused antibody deficiency. in conclusion, the description of cd19 deficiency reminds the importance of the molecules on b cells and contribute to identify new genetic defects (cd81, cd21), and it was showed that coreceptors could affect the expressions and the functions of each other. ege university faculty of medicine, dept of pediatric immunology, izmir, turkey ig class switch recombination deficiencies are rare pids (1:500,000 births) with normal or elevated serum igm and low igg, iga and ige levels, defective or normal somatic hypermutation, defective t/b cooperation (50%), intrinsic b cell defect (50%), susceptibility to bacterial infections begining from the first year of age (impaired b cell immunity) and lack of germinal centres in secondary lymphoid organs. we present a cd40l defective case with clinical findings such as recurrent otitis media, recurrent upper and lower respiratory tract infections, sinusitis, arthritis, relapsing polychondritis , ebv-associated cervical lymphoproliferation, cmv infection, bronchiectasis, liver and spleen enlargement, multiple nodules in the liver, chronic diarrhea due to persistent cryptosporidium parvum, fungal pneumoniae, osteoporosis, and schwannoma. this case is remarkable with low igm levels and normal cd40l ezpression on activated t cells although he had a novel mutation in cd40l gene (a novel missense mutation in cd40lg (c. c139t), leading to an a. a. change from histidine to tyrosine at position 47 (h47y) at the start of the extracellular domain). in addition, we present two cases with cd40 deficiency with normal cd40 expression on b cells.both of these cases had homozygous-cd40-mutation leading to a longer protein due to deletion of stop-codon. in conclusion; cd40 molecules although non-functional in b cells, may be normally expressed on cell surface. these cd40 molecules are unable to trigger signal, because cd40l + il4 activation leads to complete lack of proliferation. evaluation of cd40 or cd40l expression by flow cytometry may lead false results. study of cd40l + cytokine (or cd40+ cytokine)induced b cell proliferation appears as a useful tool for these diagnosis. institute for immunology and physiology (ub ras). yekaterinburg, russia 2 regional children clinical hospital №1, yekaterinburg, russia the ural regional center of clinical immunology, which based on children clinical hospital number one (№1) in yekaterinburg, observe patients from different territories of ural region and neighboring areas. it consists of laboratory department, consultative department, vaccination and treatment rooms, beds and boxes in special departments in the regional children hospital. the close collaboration with j-project started in 2009. this is an example of such collaboration. in patient a. an international consilium was diagnosed a progressive neurodegenerative disease as a manifestation of primary immunodeficiency: x-linked agammaglobulinemia with b-cell deficiency. mri results: unspecified leukodystrophya rapidly progressive multifocal brain lesions with demyelinating, generalized cerebral atrophy, iii degree, signs of periventricular leukomalacia in the anterior horns of the lateral ventricles. the brain biopsy was recommended in order to clarify the nature of the defeat of the pathological process and define the role of the immune mechanisms of its development (held in the neurosurgical department with subsequent histological and immunohistochemical studies). histological and immunohistochemical study of the brain tissue of the right frontal lobe: a signs of productive meningoencephalitis in brain tissue with vasculitis, perivascular and focally moderate diffuse infiltration of mononuclear (accumulation of mononuclear cd45rb+, vimentin+), most of which are cd8 + lymphocytes with granules of granzymeb. around -dystrophy and necrobiosis neurons, intracellular edema, small focuses of gliosis -there are isolated myeloid cells (myeloperoxidase +) and plasma cells with cytoplasmic expression of immunoglobulin light chains lambda and kappa; cells and the extracellular matrix of brain tissue expressing cd56 antigen and s100 protein. virological and bacteriological studies of brain tissue and liquor: connection of progressive degenerative changes and infectious process weren't obtained verified acknowledgments of an infectious or autoimmune process has not been received. search for a genesis of cytotoxic process in the brain continues. center for chronic immunodeficiency, university medical center freiburg and university of freiburg, germany the essential role for igg replacement therapy (iggrt) for common variable immunodeficiency (cvid) has been demonstrated in many studies and metaanalyses. while patients with "infection only" reach a nearly normal life expectancy -though still not quality -under iggrt, cvid patients with additional manifestations like inflammatory lung, bowel or liver disease, lymphoproliferative and/or autoimmune disease often require additional immunosuppressive treatment. there is little consensus on the form of immunosuppressive regimen, once steroids have failed, with possibly the one exception of rituximab treatment for autoimmune cytopenia. additional studies are essential to guide therapeutic algorithms. some of these patients suffer from late onset combined immunodeficiency (locid). as in classic forms of combined immunodeficiency, iggrt can be only a part of the treatment strategy, which needs to additionally address the cellular immunodeficiency of the patients. therefore a retrospective survey was performed on patients diagnosed with cvid who underwent hematopoietic stem cell transplantation. the results of this study are currently in revision. in summary, iggrt is the baseline therapy for cvid but does not address sufficiently the immune dysregulation in a subgroup of patients. better predictive markers have to be identified for the selection of patients for additional, potentially even definite forms of treatment in order to prevent the morbidity and mortality associated with these secondary manifestations of cvid. the first department of primary immunodeficiencies in russia was established on the basis of the institute of immunology in 1980, when 3 patients with pid were registered. currently, 327 patients with pid are followed in the department of immunopathology in adults. 65% of pid adult patients have pid with immunoglobulin deficiency. analysis of this group of adult patients showed that the diagnosis of pid, on average has a delay of 10-15 years from the first symptoms. in 88% of cases, there is an infectious clinical phenotype, 32% -combined infectiouslymphoproliferative phenotype, 27% -infectious and enteropathy. the study of immunophenotyping of b-lymphocytes for the degree of maturation in this group of patients was begun. 13 patients are currently included in this study. 2 patients showed complete absence of b-lymphocytes, 11 -the reduction of b-cells, 2 patients of those have a normal amount switched memory b cells (mbc), 9 people -a decrease amount of switched mbc. 8 persons of the group with decreased amount of switched mbc had an expansion of transitional mbc. at present, a clear link of immunophenotypes with specific clinical phenotypes in not found, but this may be due to small sample of patients at the moment, the investigation continues. for the treatment of this category of patients only intravenous immunoglobulins are available in russia. we use drugs in various concentration of russian and foreign production. the availability of immunoglobulins for the adult patients unfortunately is not sufficient in russia, so the recommended pretransfusion level of igg is not achieved in about 70% of patients. our work presents the experiences of our center with the subcutaneous form of immunoglobulin therapy (scig). we have 64 patients on such therapy. the youngest child is 7 months old. the largest group consists of cvid patients, next-xla patients. we also substitute children diagnosed with the dgs and accompanying hypogammaglobulinemia and some children with subclasses deficiency as well as secondary hypogammaglobulinemia. in most cases we start therapy with intravenous preparates, but there have been some children to whom we proposed the subcutaneous form at the initial stage of the therapy. the main factors which made us change the mode of the drug application were adverse reactions to ivig, poor vein access and the parents`wish. the administration of scig is very rarely complicated by severe adverse reactions (the risk of their incidence amounts to about 0.3%). even patients with serious side effects to previous immunoglobulin therapy and/or blood transfusion can be safely treated with scig. the most common side effects are local reactions but their incidence decreases during following substitutions. we can observe swelling, redness, induration, soreness. but we should remember that more severe side effects are also possible, for example: the first cvid patient presented with fever, weakness, difficulties in breathing during the 3 following infusions. changing the brand of the drug turned out to be a sufficient method of getting rid of side effects. the second patient, also with cvid, suffered from nausea, headache, meningismus. we changed the drug brand, slowed down the infusion rate and introduced premedication with an antihistaminic drug. the third patienta girl with dgs and hypogammaglobulinemia, after having been operated on for hypoplastic left heart syndrome, responded to infusions with high fever, muscle and joint pain, skin changes (erythrodermia). we introduced premedication, changed the drug brand and slowed the infusion rate, yet without any positive effects. in two patients we observed adverse reactions after preparates at a concentration of twenty percent. there were: weakness, chills, fever, headache, and very intense pain in the place of injection. during the subcutaneous treatment of xla patients, we observe significant reduction in the number of infections and days of school absence. despite that, all our patients with xla suffer from chronic sinusitis. similar results occurred in cvid patients, but the severity of infections was the same. the use of scig results in more stable and higher igg through levels especially in xla patients. in our practice, we had only a few cases in which iv form appeared to be better than sc one. in the case of two boys with higm syndrome, we observed recurrent enthesitis of the first patient and progression of lung fibrosis of the other. ivig was better to control platelets levels in the girl with cvid and thrombocytopenia. it has also occurred that parents refuse to allow us to start subcutaneous therapy, giving two main reasons: they feel safer under frequent doctor`s control and they are afraid of making mistakes in procedures. as for the youngest children (below 7)their fear of needle is independent of its size, which is the third reason. in conclusion, we would like to emphasize that education programmes implemented by doctors and nurses are essential to make this form of therapy easier, safer and more satisfying for patients and their parents. despite intensive investigation into the nature of cvid, the exact molecular defect(s) and pathogenesis of disease remain unknown. our aim was to evaluate the role of t cells in the mechanisms of cvid development. additionally the impact of some innate and adaptive immunity related genes (hla, cytokine gene polymorphism, mbl genes) was investigated. based on previously observed by us constellation of shared immunogenetic profiles a comparison of t-cell phenotype of cvid patients, and elderly/young healthy individuals was performed. ten patients with cvid were enrolled (4 male, 6 female; average age -42,9 years) presented mainly with pulmonary infections, followed by bronchoectasis and splenomegaly. our study demonstrated multiple t-and b-cell abnormalities in cvid patients such as: decreased cd4+, increased cd8+ t cells and low cd4/cd8 ratio, loss of naïve and early differentiated t cells, expansion of terminal effectors (cd8 + cd45ra + cd62l-) t cells, memory/effectors (cd8 + cd28-cd27-) and terminally differentiated (cd8 + cd57+) t cells. excessive t-cell activation reflecting the prevalence of activated t cell phenotype was also detected, due perhaps to an antigen-driven process. the very low numbers of circulating mature (cd21 + cd24+) and class-switched memory (igm-igd-cd27+) b cells were pathognomic for our patients and could be used as an additional diagnostic criteria in the national guidelines. furthermore high level of nonclass switched (igm + igd + cd27+) b memory cells and suppressed nk cell count was observed. decreased responsiveness to polyclonal stimuli via cd3 and cd28 pathway correlated with the loss of cd28 expression which was more pronounced in the treatmentnaïve cvid patients. these findings were further discussed in the context of the similarities that exist along with markers for immune senescence (lack of cd28 or expression of cd57). increased frequency of ifn-γ polymorphisms associated with low expression level found could indicate genetically predisposition to high activation of th2 lymphocytes in cvid and consequently support the concept of impaired th1-type responses. in conclusion our study provided new insight into the pathogenesis of cvid. this work was partially granted by medical university sofia, grant#55 bcg vaccination at birth is the constant element of vaccination programmes in poland. high reactogenic bcg danish vaccine has been replaced in 1955 , by bcg moreau vaccine. frequency of disseminated bcg infection, in children with primary immunodeficiencies after bcg moreau vaccine manufactured by biomed, poland were estimated. one thousand five hundred sixty three cases of primary immunodeficiencies were diagnosed in the department of immunology, children's memorial health institute in warsaw between 1980 -2013. among patients with t cell predominant deficiency, group high risk of bcg infection, scid was recognized in 62 children. mendelian susceptibility to mycobactarial diseases (msmd) was detected in four patients: ifgr1 deficiency and il12 deficiency -equally in two patients, and nemo -in one. in the group of primary immunodeficiencies regarded to be less prone to mycobacterium infections, cgd was diagnosed in52, hies in 20 patients, and xl-higm in 4 patients. disseminated bcg infection was recognized in 10 scid patients, 2 of them died, because of bcg diseases. all 4 patients with msmd developed bcg infection, one with il-2 deficiency died. during nearly 30-year-follow-up, no case of tuberculosis or disseminated bcg infection have been diagnosed among cgd , hies and xl-higm patients. early anti -tb drug prophylaxis and usage of wide range of antibiotics in therapy is crucial for cleaning of bcg infection. peter čižnár 1 ; julia horáková 2 ; peter švec 2 ; ivana boďová 2 ; sabina šufliarska 2 ; linda libai veghová 1 ;, marieta hricová 1 1 1 st pediatric department, comenius university medical faculty, children`s university hospital, bratislava, slovakia. 2 transplantation unit, department of pediatric haematology and oncology, children`s university hospital, bratislava, slovakia. objectives: severe combined immunodeficiency (scid) is a group of disorders due to more than 15 genetic defects, characterized by increased susceptibility to severe infections and early life death. the diagnosis is supported by the demonstration of low absolute t lymphocyte count variably associated with numerical defects of b and nk cells. patients are very heterogeneous regarding clinical course, immune parameters and clinical outcome. bcg (bacillus calmette-guerin) vaccine, a life attenuated vaccine was the part of slovak immunization program, administered at birth until 2011. a comparison of clinical course of bcg exposed (bcg+) and non-exposed (bcg-) scid patients in slovakia in period of past 10 years are given. results: incidence rate of diagnosed and treated scid in slovakia was calculated to 1:87.000, meaning 1,5 cases per year. in total 10 cases represent 4 ada patients, 2 il2rg deficiencies, 1 case of complete del22q11 and in 3 cases genetic defect was not found by analysis of rag1/2, il2rg, artemis, il7ra, jak3 and ada genes. all patients were confirmed absent trec (t-cell receptor excision circles) copies in a retrospective neonatal guthrie card analysis. seven out of these 10 patients underwent hsct, in 5 the hsc source was a mud. favorite outcome was achieved in 6 of them. half of our patients have been exposed to a live bcg vaccine during neonatal period. patients vaccinated with bcg faced severe complications and organ damage due to generalized skin and organ abscess formation, requiring prolonged (up to 18 months) hospital care and complex antibiotic therapy with more than four types of anti-mycobacterium drugs, for more than 2 years. average length of hospital care for bcg exposed patient was 10,8 months vs. 2,7 months in non-exposed group (p < 0,05). no statistical difference was found between the time of recognized first symptoms, and time of diagnosis in bcg + and bcg-group. the clinical presentation of non-bcg vaccinated patient differs in the initial symptoms when failure to thrive and pneumonia at 4 months was the most common finding. post-transplantational recovery in bcg-group was less complicated. conclusions: two major improvements for the outcome for scid patients in slovakia have occurred in past 10 years. early life vaccination for tuberculosis has been retreated and improvements in diagnostics for severe t cell defects have been made, including flow cytometry phenotyping and genetic testing within the middle european countries cooperation, the j-project. bcg and late diagnosis prolongs time for hospital care, immune reconstitution and carries severe complications, consequently it increase the costs of health care and decrease the quality of patients' life. the perspective of newborn screening for scid would be the next major step in improving the outcome of scid patients. ahmed aziz bousfiha 1 ; leïla jeddane 1 ; nahla erwa 2 ; monika esser 3 ; shereen m. reda 4 in africa, primary immunodeficiencies are still largely undiagnosed, with no cases reported in 40 of 54 countries. though the african society for immunodeficiencies (asid) already organized 3 international meetings and 5 training schools, their impact outside the hosting country is still insufficient. at this time, only a few pid patients are reported in africa (less than 2000 patients), the majority of whom are in north africa and south africa. so, asid propose the a-project, a training program based on the j-project. some issues prevent effective training for pid in africa: diversity of languages, only a few are initiated to pid, lack of resources for travel expenses, difficulty to access to care and shading by the hiv pandemic. a-project is designed as a one-day training by an african pid expert in a small group of motivated caregivers. this project is adapted to the african context, as it only requests minimum funding and can reach more people. each a-project will be co-organized by asid and a local committee, and shall lead to some commitments to be realized by locals, in particular the establishment of a registry, a network between physicians and scientists and creation of a patient association. moreover, each a-project will be done in the medical language used in the country (english, french or portuguese). the first a-project was already done in benin, and five more are already planned for 2014. our goal is to reach all 40 countries where no patient were reported in 3 years. this clinical program will raise pid awareness in africa and can potentially discover new aspects of the immunity. till very recently primary immunodeficiency diseases (pids) were not being frequently recognized in india. however, the scenario has changed over the last 5 years or so. the indian society for primary immune deficiency (ispid) was founded in 2010-2011. over the last 4 years the ispid has organized 2 international conferences (at new delhi and mumbai), 2 national conferences (at chandigarh and varanasi) and 2 continuing medical education programmes (at new delhi and lucknow). these meetings have served to act as catalysts for the cause of pids and have resulted in increasing the awareness about these conditions amongst paediatricians and physicians in our country. several centres now have the clinical skills and the technical wherewithal to perform laboratory investigations for these patients. the repertoire of tests includes nephelometery, elisa based tests and flow cytometry. facilities for molecular diagnosis of pids are also being developed at some of these centres. a lot more, however, needs to be done. the clinical phenotype of several pids in india is likely to be different from that in the west 1 . further, the type of infections in these patients is also likely to be different. this is because of the differences in the micro-and macro-environment to which these patients exposed in developing countries. these differences have been well brought in the recent publication on chronic granulomatous disease from our centre 2 . further, the genetic background of the indian population is diverse and several new mutations are likely to be identified amongst these patients. the indian council of medical research has taken up the lead in this regard and is proposing to set up 2 centres for advanced research (cars) in pids -1 each at the post graduate institute of medical education and research, chandigarh and the institute of immunohematology, mumbai. the foundation for primary immunodeficiency (fpid), usa has also been closely involved in these efforts and has helped facilitate the development of these 2 cars. the field of pid research in india is wide open and we are likely to witness new and exciting scientific developments in the coming years. references: 1. gupta s, madkaikar m, singh s, sehgal s. primary immunodeficiencies in india: a perspective. annals of the new york academy of sciences 2012; 1250: 73-79. the prevention of pid's complications, the improvement of the health care of patients with pid, the creation of the registry of the patients with pid, the implementation of the finance regulations for detection and treatment of patients with pid diseases in public health facilities, the training and professional development of medical professionals in this field. objective: to improve the detection and diagnosis of pid diseases and the life quality of patients with pid in the republic of kazakhstan. undertaken activities for realization of the project: 1. professor. l. marodi visited kazakhstan in october 2012 and the meeting was held in ministry of health of the republic of kazakhstan, presentations were given at the international conference held in national research center for maternal and child health, the negotiations with heads of the national medical holding' clinics and scientific center of pediatrics and pediatric surgery were held. international islamic university, kuantan pahang, malaysia. two hundred and sixty three (263) suspected pid (primary immunodeficiencies) cases were referred to clinical immunologist led clinics in malaysia from 1986 -2013. patients referred were from all states of malaysia and seen at the institute of pediatric, hospital kuala lumpur and university associated hospitals in north and central peninsular malaysia the initial pid patients were seen by -1 pediatric immunologist between 1986-2006 followed by another 2, beginning in 2007 followed by the other in 2012. there were 168 (68.7 %) patients with at least 1 abnormality on the immune prameter recorded and regarded as probable pid. however 153 (62.7%) were recorded as pid based on existing criteria. (who scientific committee 1986, iuis scientific committee, primary immunodeficiency disease.1999) our population were mainly children, 83% below 10 years and 36 % below 1 year. only 3 were above 18 years. pid were classified as; predominant antibody deficiencies 34 %, combined immunodeficiencies 19.3 %, other cellular immunodeficiencies 14.7 %, phagogocytic defect 11.3%, immunodeficiency associated lymphoproliferative disorders 2 %. our data differed from most classification where predominant antibody deficiency is most frequent as high as 65 %, (steihm, 2004) . of the specific 153 pid recorded, x linked a gammaglobulinemia (xla) 19, hyper igm syndrome (higm) 11, common variable immunodeficiencis (cvid 7), selective iga deficiencies 7 severe combined immunodeficiencies (scid) 17, di george syndrome (dgs)12,chronic granlomatous disease (cgd) 14, hyper immunoglobulin e syndrome (hige ) 14, primary cd4 deficiencies 6, ataxia telangiectassi 3 % malaysia comprises of multi ethnic groups with a population of 29.6 million in 2013. pid amongst them showed, malays at 61.4%, chinese 11 %, indians 23.7 % and others 5.2 % whilst the male predominate over female at a ratio of 2.8:1. family history of affected sibling or in first degree relative, or early death with suspected infant dying of infection was positive in 53.3 % which is higher than in most reports eg egypt 23.4 % (reeda 2009). this could be due to high consanguinity in the population. alternatively the symptomatic sibling of affected patients is more likely to be referred to the clinical immunologist. scid records the most varied organism from the positive microbiological isolate viz bacteria 4, fungus 2, virus 1, parasite 2. chromobacterium violaceum was seen in 3 cgd patients in which 2 deteriorated with eventual death. as in many national registries diagnostic delays remains prominent. in our series the mean diagnostic delay was o 3.61 ± 4.82 years. in comparison thailand stands at 2.1 ± 2.6 years, while france, a median of 1.3 years. malaysia remains committed to provide better diagnostic services and improved care of the pid patients through research collaboration with foreign partners with a drive for creating subspecialty training. patient groups aligned to ipopi is now closer to its formation with the creation of its protem committee ensuring that patients' interest will always be guarded aknowledgement. the authors thank all who had contributed directly or indirectly, especially medical officers, nurses, consultant pediatricians especially dr kamarul azhar, institut of pediatrics hkl, laboratory scientists especially dr shanaz murad of the imr kuala lumpur university of manchester, uk although the concept of grouping mendelian disorders associated with an upregulation of type i interferon (ifn) has not been previously recognised in the medical literature, our past and current work argues that this concept has scientific validity and clinical utility. i will discuss the possibility that such conditions can usefully be considered to represent a novel set of inborn errors of immunity, and that the recognition of diseases as type i interferonopathies will have significance for the development of targeted therapies, as well as informing our understanding of viral and retroelement biology, and the pathogenesis of some forms of autoimmunity. 1 classic kaposi sarcoma (ks) is exceedingly rare in children from the mediterranean basin, despite the high prevalence of hhv-8 infection in this region. we hypothesized that rare single-gene inborn errors of immunity to hhv-8 might underlie classic ks in childhood. we report here autosomal recessive ox40 deficiency in an otherwise healthy adult with childhood-onset classic ks. ox40 is a costimulatory receptor expressed on activated t cells. its ligand is expressed on various cell types, including endothelial cells. the mutant ox40 protein was poorly expressed on the cell surface and failed to bind ox40 ligand, resulting in complete functional ox40 deficiency. the ox40-deficient patient had a low proportion of effector memory cd4 + t cells in the peripheral blood, consistent with impaired cd4 + t-cell responses to recall antigens in vitro. the proportion of effector memory cd8 + t cells was less diminished. the proportion of circulating memory b cells was low, but the antibody response in vivo was intact, including to a vaccine boost. together, these findings suggest that human ox40 is important for cd4 + t-cell memory, but redundant for immunity to most common pathogens, with the notable and surprising exception of hhv-8. the chronic mucocutaneous candidiasis disease (cmcd) is characterized by persistent or recurrent infection of skin, nails, oral, or genital mucosae with candida albicans. il-17-mediated immunity has been concerned in host defense against candida on body surfaces. we have investigated nine patients with chronic mucocutaneous candidiasis disease (cmcd) and signal transducer and activators of transcription 1 (stat1) mutations. the novel c.537c > a (n179k) and c.854a > g (q285r) mutations in the coiled-coil domain (ccd) and the c.1154c > t (t385m) mutation in the dna-binding domain (dbd) of stat1 are gain-of-function (gof) for γ-activated factor (gaf)-dependent cellular responses to stat1. low proportion of il-17a-and il-22-producing t cells, lower levels of intracellular il-17a and il-22 by t cells and impaired candidainduced secretion of il-17a and il-22 by leukocytes from cmc patients compared to that in healthy controls were found. the c.820c > t (r274w) mutation affecting the ccd and the c.1154c > t (t385m) mutation affecting the dbd of stat1 and resulted in gain-ofphosphorylation and gof. these mutant alleles enhanced the cellular responses to cytokines via stat1 signalling pathway. these data provide further insight into the mechanism of host defense against candida. heterozygous gain-of-function stat1 mutation is known as a major etiology of chronic mucocutaneus candidiasis. gof mutation affecting the stat1 coiled-coil domen (d165g) was initially discovered in 13-year boy with cmc. gof mutation of dna-binding domen (t385m) was found in the second our patient with cmc. both patients have early manifestation of recurrent or persistent infections of the skin, mucous membranes, and nails with candida albicans. they also have skin infections with dermatophytes. patient 1 presented from the first months of age with severe recurrent sinopulmonary infections. recurrent pneumonia and chronic bronchitis complicated by bronchiectasis, which resulted in cor pulmonale and congestive heart failure. patient 2 suffered from recurrent hsv infection, recurrent aphthous stomatitis, has several episodes of bacterial skin infections. he also has chronic bronchitis and several episodes of pneumonia, but does not have bronchiectasis. both boys developed esophageal stricture, patient 2 necessitating nissen fundoplication in the age of 5 years. the patients have mild autoimmune features: uveitis (p1) and alopecia (p2). immunological investigation revealed different impairment of immune system: more severe, similar to combined immunodeficiency in p1, which declines with age. the p2 does not have changes in lymphocyte number and immunoglobulin's, but impaired antibody production to pneumococcal antigens. western blotting performed with nuclear extracts of lymphocytes of both patients showed stronger stat1 phosphorylation after stimulation with cytokines ifnγ, ifnα, il-27. mononuclear blood cells from both patients released much smaller amounts of il-17a and il-22 than candida-exposed cells from healthy control. stat3 activation triggers transcription of interleukin (il)-17 which is crucial for mounting protective immune responses against fungi. several mutations affecting the stat3/il-17 pathway have been reported, resulting in selective susceptibility to fungal (candida) infection, a hallmark of chronic mucocutaneous candidiasis (cmc). in patients with autosomal-dominant (ad)-cmc we previously reported defective th17 responses and identified an underlying gain-of-function (gof) stat1 mutation leading to hyperphosphorylation of stat1. how this affects stat3 or leads to decreased il-17 remains to be determined. in patients with ad-cmc, we assessed how gof-stat1 mutations affect stat3 activation, dna-binding, gene expression, cytokine production and the effect of epigenetic modification. we show that stimulation of stat3 in the presence of gof-stat1 mutations leads to significantly reduced transcription of stat3-inducible genes (rorc/il-17/ il-22/il-10/c-fos/socs3/c-myc). this was not due to impaired stat3 phosphorylation, altered nuclear translocation nor sequestration of stat3 into stat1/stat3 heterodimers. dna binding to a stat-consensus binding site construct (hsie) was intact but binding to an endogenous stat3 dna target was impaired. the reduced stat3-dependent gene transcription could be normalized by inhibiting stat1 activation by fludarabine or enhancing acetylation with histone deacetylase (hdac) inhibitors trichostatin a or itf2357. silencing hdac1, hda2 and hdac3 indicated an important role for hdac1. impaired stat3-dependent gene transcription likely underlies decreased th-17 cytokine production, susceptibility to fungal infections and other pathology seen in ad-cmc patients and could be a new target for defining novel therapeutic approaches for this potentially lethal disease. autoimmune features have been long thought as association with immunodeficiency disorders, but are now viewed as a crucial component of some diseases attributed to the breakdown of self tolerance or defects of immune regulators. it had been previously established that a single gene defect of the foxp3 gene (foxp3 in humans) caused widespread autoimmunity in both humans and mice. the clinical syndromes observed in both scurfy mice and humans suffering from ipex are similar to those observed in experimental models in which treg are selectively depleted. in 2003, three groups demonstrated that these diseases were indeed the result of a regulatory cell deficiency. around one third of the patients with clinical manifestation closely resembling ipex syndrome, foxp3 is not mutated, these patients are referred to as ipex like. here we present a case; a female patient 13 years with multiple autoimmune manifestations; dm, coeliac disease and ulcerative colitis with marked decrease in the percent of cd4 + cd25 + foxp3+ cells. as she has a siblings suffering from dm; the whole family was investigated. the father and the mother had 0% cd4 + cd25 + foxp3+ cells. background: hids is an autosomal recessive disease, first recognized as a separated entity at 1984. in patients with hids, the activity of mevalonate kinase is reduced to 5-15% of normal levels. hids is caused by mutations in the mevalonate kinase gene (mvk), located on the long arm of chromosome 12 (12q24). it is manifested by cyclic attacks of fever initiated usually during first year of age. the frequency and severity of attacks tend to decrease later in life. materials and method: a retrospective analysis of medical history , clinical course and laboratory findings of two albanian children with periodic fever , diagnosed with hyper igd syndrome. results: case presentation 1: an 5-year-old boy admitted to the hospital because of periodic fever spikes, which occurred every 3-4 weeks and lasted 3-5 days, presented since the first year of life and coincided with the beginning of immunization. he had a tonsillectomy and adenoidectomy at the age of 3. the fever attacks were associated with chills, malaise, and abdominal pain without gastrointestinal signs. between attacks the patient was free of symptoms. from his family history, recurrent febrile episodes during childhood were reported to his father. physical examination showed normal findings, except for a cervical lymphadenopathy. laboratory: marked increase of erythrocyte sedimentation rate and crp. wbc-ranged from 5.710 to 12 000/mm 3. , high asto. serum igd was repeated several times and was always elevated (mean value: serum igd 122 iu/ml). the mutation v377i is found from the genetic examination done for gene mutations in chromosome 12 (12q24). he repeated attacks after initial treatment with corticosteroid ,than is suggested. the second case was a four-year-old girl hospitalized five times because of prolonged fever, and diagnosed as pneumonia, tonsillitis, acute otitis media and sinusitis, treated by antibiotics. her laboratory findings were not remarkable except for increased acute inflammatory responses. serum amyloid a (saa) 1100 μg/l (8 μg/l) and igd was extremely high 181.9 iu/ml. genetic examination for two mutations were negative, but reduced mevalonate kinase activity in white blood cells was demonstrated in more thorough investigations. treatment regime: colchicine conclusions: auto inflammatory syndromes always pose diagnostic and therapeutic challenges to the clinicians. the clinical description of the diversity of periodic fever syndromes is helpful in the assessment and management of these patients. although hids is predominantly identified in populations from northern european areas, it has to be considered in children with periodic fever. anastasiia bondarenko 1 ; liudmyla chernyshova 1 ; iryna sychova 2 1 shupik national medical academy of postgraduate education, kiev, ukraine. 2 dniepropetrovsk regional children's hospital, dniepropetrovsk, ukraine. background. aspergillus is an actual pathogen in chronic granulomatous disease responsible for about 20% of all infections. in 70-80% lungs are involved and in 5% -cns. case. we report a case of combined loci in 9-years old female patient with ar cgd. the child was born from iii pregnancy, ii delivery on 37 th week of gestation with body mass 2750g. she received bcg vaccination at 4 -th day of birth. at 3 months the local inflammation in site of bcg with regional lymphadenitis developed which was treated with isoniazid for 3 months. then bilateral purulent cervical lymphadenitis developed at 6, 9 and 11 months treated with wide spectrum antibiotics. culture from pus was negative. pcr for mycobacterium tuberculosis complex was negative. at18 months systemic infection without loci occurred with fever, lymphadenopathy, hepatosplenomegaly, loss of weight, progressive anemia, inflammatory changes in blood for almost 6 months. bacteriological cultures were negative. treatment with wide spectrum antibiotics was insufficient for 3 months. disseminated bcg infection was suspected and 4-compound amb treatment was started ex juvantibus with positive effect: the fever has stopped, the sizes of lymph nodes, liver and spleen have decreased, the weight of a body normalized. the child suffered from recurrent pyogenic infections, underwent disseminated salmonellosis. at the age of 5 years blood samples were tested at the laboratory of human genetics of infectious diseases, inserm (paris, france). an absence of p22 phox protein expression detected by western blot conferring a complete defect in cyba due to compound of geterozigous mutations in 16q24. at 6 years primary tuberculosis complex of right upper lobe (mbt -) was diagnosed. at the age of 7 during the unexplained fever multiple formations were identified in the liver, biopsy showed caseosis suspected the mycobacterial nature of lesions but mbt (-). at the age of 9 years because of shade in the left upper lobe and ineffective standard antibiotic treatment the tuberculosis again was suspected. due to ineffective antimycobacterial treatment for 8 months multi drug resistant tubercullosis was considered. anti-tb drugs ii line was appointed without clinical response. fever persisted. mri of brain revealed mass lesion in the left parietal lobe. because of suspected tumor the brain biopsy was done and the pus was obtained. microbiological studies revealed aspergillus fumigatus. at the same time subcutaneous tumor-like infiltrate 40 х 20 mm appeared on chest in a proection of lung lesions. the pus was obtained during thoracentesis. result of microbiological studies: aspergillus fumigatus. drainage of abscesses and intravenous voriconazolum led to dramatic clinical improvement and normalization of blood parameters. conclusion. features of our case is spread lesions of aspergillosis with relatively slow progression of infection. high incidence of tuberculosis in ukraine leads to a high suspicion regarding this infection. diagnosis of tb is mainly based on instrumental studies. radiological and histological differential diagnosis between tuberculosis and other infections with granulomas in cgd is difficult. high suspicion of tuberculosis led to late diagnosis of aspergillosis. great north children's hospital, newcastle upon tyne hospitals nhs foundation trust, and primary immunodeficiency group, institute of cellular medicine, newcastle university, newcastle upon tyne, uk even following the introduction of biologic disease modifying antirheumatic drugs (dmards), a small number of children suffering from severe, refractory autoimmune (ai), rheumatic and/or autoinflammatory disorders will not get into clinical remission (cr) and will potentially further suffer from multiple side-effects of combined and long-term immunosuppressive and anti-inflammatory therapies, in particular severe infections (marodi l, casanova jl. jaci 2010; abinun m. ped health 2010). whilst autologous t cell depleted hsct following the immunosuppressive conditioning regimen achieved complete clinical remission in majority of children with severe juvenile idiopathic arthritis (jia) (de kleer im et al. ann rheum dis 2004) , infection-related mortality remains significant (abinun m et al. mol immunol 2009) . therefore, following the success of allogeneic hsct in treating 6 children with immunodysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome (nademi z et al. bmt 2014) , we treated further 10 children with different severe ai (alps, autoimmune lymphoproliferative syndrome (n = 3); complex ai disorder (n = 1)), rheumatological (jsle, juvenile systemic lupus erythematosus (n = 1); jia (n = 2)) and autoinflammatory disorders (mkd, mevalonic kinase deficiency/ traps, tnf-receptor associated periodic fever syndrome (n = 1); eoc, early onset colitis (n = 2)). overall, 14 of the 16 children are alive (follow up 1-11 years), 12 in complete and 1 (complex ai disorder) in partial cr, original disease (alps) relapsed in 1, and 2 children died (1 each with alps and eoc). 2 children had significant, but transient acute (grade 3-4) and chronic (limited) graft vs. host disease (gvhd), 3 experienced multiple virus reactivation(s), and remarkably we saw significant secondary ai diseases post-hsct (transient nephritic syndrome (n = 2) and cytopaenias (n = 1); psoriasis, n = 1; and thyroid disorders (grave's thyrotoxicosis and hypothyroidism), n = 2). our data add to the positive experience and evidence acquired over the last 10-15 years (daikeler t et al. bmt 2009; snowden ja et al. bjh 2012) to propose the allogeneic hsct as a viable treatment option for the small group of children suffering from severe autoimmune disorders. the wiskott-aldrich syndrome (was) is an x-linked primary immune deficiency disorder characterized by thrombocytopenia, microthrombocytopenia, recurrent, mostly respiratory tract infections, eczema and increased risk of autoimmune disorders and malignancies. was is caused by mutations in the wasp gene which encodes wasp, a 502-amino acid protein. wasp plays a critical role in actin cytoskeleton organization, signalling and different functions of immune cells. we present here the results of genetic analysis of patients with was from eleven eastern and central european (ece) countries, turkey, iran and azerbaijan. clinical and laboratory information of 116 affected males and 63 carrier females from 106 was families were collected. the wasp gene was sequenced from genomic dna of patients with was, as well as their family members to identify carriers. in this large cohort, we identified 77 unique mutations including 23 novel sequence variants. the mutations were scattered throughout the wasp gene and included single base pair changes (20 missense and 14 nonsense mutations), 8 small insertions, 22 deletions, and 12 splice site defects. this study was financially supported by the tübi̇tak project 110s252 and bap project tda-2013-4075. background: chronic granulomatous disease (cgd) is a rare primary immunodeficiency disorder of phagocytes. resulting in impaired killing of bacteria and fungi. a mutation in one of the four genes encoding the components p22 phox , p47 phox , p67 phox and p40 phox of the leukocyte nadph oxidase leads to autosomal recessive (ar)-cgd. a mutation in the cybb gene encoding gp91 phox leads to x-linked recessive cgd. methods: we report here the results of genetically and functionally characterized 100 patients with cgd from 83 turkish families in turkey. results: most of the families (62%) have an ar genotype (%28 p22 phox , %20 p47 phox and %14 p67 phox ) and 38% have an x-linked genotype. patients with a22 0 , a67 0 and x91 0 phenotypes with oxidase null activity (dhr stimulation index of ≤1.5) were found in 73 patients. however, in p47 phox deficient cases and in 7 other ar cases with high residual oxidase activity (dhr stimulation index ≥3) were found in 27 patients. conclusions: residual oxidase activity is similarly lack in the x91 0 , a22 0 and a67 0 phenotype except 7 ar cases with missense mutation. in our cohort, the percentage of ar-cgd was different from european and usa registries (in comparison with %5, %5 and 30% of p22 phox , p67 phox and p47 phox deficient ar-cgd cases, respectively) with the higher percentage of patients with p22 phox (28%) and p67 phox -deficent (14%) phenotypes, and the lower percentage of patients with p47 phox -deficient (20%) phenotype. the basic difference in our results from those reported is the higher percentage of patients with ar-cgd (%62), which was lower than in the european and usa registries, probably because of the higher prevalence of consanguineous marriage in turkey. introduction: schnitzler syndrome is an autoinflammatory disorder of unknown etiology. at least some of its clinical presentation is mediated through an activation of inflammasome and release of il-1, as was repeatedly demonstrated by a prominent therapeutic effect of il-1 blockade. recent reports bring an evidence of an important role of mitochondria in inflammasome activation and in a pathogenesis of autoinflammatory diseases. we have therefore investigated mitochondrial function and structure in patients with schnitzler syndrome. materials and methods: activity and amount of oxidative phosphorylation complexes (oxphos) were analysed by spectrophotometry, histochemistry and imunoelectrophoretic methods in fibroblast cell lines derived from skin biopsies of three adult male patients with schnitzler syndrome. ultrastructure of mitochondria, mitochondrial network and reactive oxygen species (ros) were analysed by fluorescent and electron microscopy. results: the activities and amount of oxphos complexes i, iii and iv were decreased in patients with schnitzler syndrome. interindividual differences in the degree of impairment (from severe to moderate) in analyzed mitochondrial parameters were found. content of ros, previously suggested as main inducers of inflammasome, were not significantly increased in cells with schnitzler syndrome. we, however, did find consistent and prominent changes in mitochondrial structure of all three patients. disturbed mitochondrial network and mainly abnormal, partially swelling mitochondria with unusual and sparse cristae were characteristic for all patients. we did further notice marked accumulation of neutral lipids in all tested fibroblasts. conclusion: severe structural damage of mitochondria associated with milder functional changes represented a consistent feature found in all tested schnitzler syndrome patients. along with progress in basic and clinical immunology worldwide, the knowledge and activities in the field of primary immunodeficiencies (pids) have developed during last two decades. in 1997, a group of junior doctors and students joined seniors in this filed to establish iranian primary immunodeficiency registry (ipidr). several national and international research projects have been done so far which led to lots of publications, while improving the diagnosis of patients with pids, construction of iranian primary immunodeficiency association (ipia), and establishment of research center for immunodeficiencies were other activities which lead to better management of the patients. organizing annual meeting on clinical immunology and immunodeficiencies, celebrating pi week annually and active participation in the international congresses were all helped in to increase knowledge of physicians in the country. the overall activities in the field of pids led to an increased trend in recognition of more patients in the recent years, which was associated with decreased delay in diagnosis. based on recent report of the registry, published recently in the j clin immunol, more than 730 new patients with pid, in addition to previously 930 reported patients, were presented. predominantly antibody deficiencies were the most common form of disease, followed by combined immunodeficiencies, congenital defects of phagocytes, and other well-defined syndromes with immunodeficiency. the rapid progress in identification and registration of the patients with pids is important not only as of epidemiological aspect, but also as of timely diagnosis and appropriate treatment of the patients. the j project physician education and clinical research collaboration program was launched in 2004 in eastern and central europe (ece). in less than 10 years, it has achieved remarkable success. this project aims to increase knowledge in the field of primary immunodeficiency disorders (pid), and to improve the diagnosis and treatment of patients worldwide, particularly in countries with limited economic resources, which currently report fewer such patients than expected. in most ece countries, gene sequencing, which can provide a definitive diagnosis of pid, still remains unavailable. by contrast, such technology is used elsewhere to detect the more than 200 pid-causing genes that have been discovered in the last three decades. thus, pid awareness programs like the j project remain critically important, to improve diagnostic facilities and treatment and to promote clinical research collaboration. this paper highlights the achievements of the j project and the spread of its concepts and spirit to the countries of western asia. primary immunodeficiencis (pid ) are rare genetically determined diseases , occurring with an incidence of 10 per 100 000 inhabitants. it is heterogeneous group of disorders, from quite commonly found and usually asymptomatic iga deficiency (1 in 600), to a very rare diseases such as chediak -higashi (1 in 2 000 000 inhabitants). in 2005, within the framework of a government research project no. pbz-kbn-119/p05/ 2005 -" development , improvement and implementation of highly specialized diagnostic procedures for immune-mediated diseases", a network of cooperating national centers for diagnosis and treatment of pid, named polish working group on primary immunodeficiencies (pgr pno) was founded. as a result of joint efforts of the group as well as an implementation of three eu grants [euro-pid -nas qlrt -2001 -0274 ( 2001 -2004 , euro-policy -pid sp23 -ct-2005-006411 , euro-gene -scan (223, 293)] the number of centers actively working in the diagnosis and treatment of primary immunodeficiencies increased. up todate pgr pno includes 15 pediatric centers , and since 2012 -11 centers for adults. development and dissemination of new diagnostic and therapeutic standards contributed significantly to the increase in detection pid. with early diagnosis of the disease -the implementation of appropriate treatment, including gamma globulin replacement therapy together with quality of life has improved. in spite of all efforts recognition of pid in poland is very rare and currently is 1.44 to 100 000, what is almost 10 times smaller than in europe (www.esid.org). at the moment, a nationwide registry of children and adults with pid consists of 3 368 patients. in september 2013 pgr -pno summarized in the annual report the current status of the substitution therapy with intravenous and subcutaneous immunoglobulin therapy in children and adults with pid in poland. on the basis of available information, half of all patients (1 684 children and adults) were diagnosed to have antibody deficiency. these data are similar to the register of a european database esid , where the percentage of patients with pid with a predominance of antibody deficiency is more than 56% (www.esid.org) development and dissemination of new diagnostic and therapeutic standards as well as a national cooperation contribute significantly to the increased detection of pid. early diagnosis of the disease is followed by earlier implementation of appropriate treatment, including gammaglobulin replacement therapy together with improvement of quality of life. mutations of was were identified in 38 was boys and in 10 heterozygote mothers. frequently genetic damages occur in 1,2,7,10 exones and 6,8 intrones: deletions (11), splice-site mutation (9), missens (12), insertions (6). deletions and insertions lead to stop-codon in 7 cases. 31 nbs patients were homozygous for 657del5 mutation and oneheterozygote for 657del5 had 681delt x-cgd: deletions (10) and nonsens (6) mutations in different exons of cybb were observed most often. in 33 families was performed prenatal diagnosis: x-scid-4, higm1-4, xla-2, was-6, nbs-6, a-t-4, cgd-5, xlp-1, dnalig4-1. healthy children -31. recurrent severe complicated infections developed in 90% of pid patients. antibody deficiencies: bacterial infections -100%, enteroviral -17%, tuberculosis 2 cases, atypical mycobacteriosis -1 case. combined pid: bacterial infections -100%, fungal -40%, viral -37%, opportunistic (pneumocystis jiroveci) -48%, mycobacterial -70% (24 -complication of bcg vaccination, 5 -tuberculosis or atypical mycobacteriosis). cgd: bacterial -62% (staph.aureus, e.coli, b.cepatia, salmonella spp., klebsiella spp.), mycobacterial -75% (85% bcg origin, 15% -tuberculosis), aspergilosis -30% immune disregulation syndromes (xlp1 [10] and alps [41]): bacterial infections -20%, viral -8% (ebv). autoimmune violations were observed in 56% of all pid cases: 5% of combined pid, 35% of antibody deficiencies, 65% of other well defined syndromes, 100% of autoimmune syndromes. cytopenias developed in 68%, vasculitisin 15%, ulcer colitisin 14%, arthritisin 11%. oncology diseases developed in 4% of patients: mainly in nbs, a-t, was and cvid: t-and b-leukemia's, lymphomas, solid cancer. the study of primary immunodeficiency is a unique model for studying the molecular basis of immunity. question about intravital pid verification is still relevant. a regional center of clinical immunology was established in 1986 at the regional children's clinical hospital №1, yekaterinburg. the regional clinical hospital №1 joined the center in 2013. institute of immunology and physiology, ural branch of the russian academy of sciences carries scientific management of the center. the centre works closely with foreign counterparts in the international project j project and it has been one of the centers of jmf since 2013. over 28 years we formed a regional register of patients with primary immunodeficiency, comprising 309 patients: 224 children and 85 adults. analysis of the regional register allows to investigate the causes of deaths in patients with pid. infectious syndrome mortality -sepsis, generalized mycobacterial infection -22 patients, proliferative process -4 patients, dominate the mortality structure. creation of specialized immunological centers permit to raise the educational level of the medical staff, precisely identify nosological forms of pid among different groups of patients, to prevent the birth of children with pid, increase the length and quality of life such patients and take part in the collaboration with international experience in the pid. introduction: the aim of this retrospective study is to determine the frequency, and demographic, clinical and laboratory features of adult cvid patients referred to our clinic. materials and methods: we retrospectively evaluated 26 adult patients (9 female (%34,6), 17 male (%65,4); aged 18 to 67 years: median 27 years) who were diagnosed as cvid according to esid and pagid criteria during a 4 year period (january 2010-march 2014). results: the median current age of 26 patients was 27, and the median cvid diagnosis age was 22,5 years. the diagnostic delay in patients with cvid was 4,5 years (median). cvid patients presented lower levels of igm (12 patients, 46,1 %), iga (16 patients, 61,5 %) and igg (16 patients, 61,5 %). according to lymphocyte lmunophenotypes of cvid patients, cd4 (15 patients, 57,6 %), cd19 (13 patients, 50 %) and cd4/cd8 (13 patients, 50 %) values were observed the most lower ones. discussion: we found that both of the patients with bronchiectasis showed lower levels of immünoglobulins and lower imunophenotypes of b cell than the others that do not have bronchiectasis. in our patients cd4, cd19 and cd4/cd8 values have got enough priority to be mentioned about an immun deficiency. in conclusion, despite recent improvements in diagnostic tools, the diagnosis of mild or moderate cvid is often delayed. however, it seems that the diagnosis of cvid is delayed especially in adulthood on account of the fact that the lack of awareness of these illnesses among the medical professionals all over the world. primary immunodeficiency disease is important in turkey because of the high rate of consanguineous marriage. the lack of awereness about immunodeficiency can cause late-diagnosis and severe complications. the objective of this study was to assess pid awereness before and after clinical immunogy education among medical students. one hundred and thirty-two questionnaires 0 with 71 items (1) were distributed to seventh somestre medical students and 116 (88%) completed questionnaires were evaluated before (first) and after (second) their education about clinical immunology courses for 6 hours. questionnaire scores (qs) were detected as total correct answers. the mean of the first qs was 37.4 ± 3.7 and second qs was 42 ± 4.3 (p < 0.05). there was no statistically difference in gender (60 m and 56 f). of 69 questions, there were 39 related with pid directly. the correct responses rate less than 50% before education were 10 of 39 questions. all participants corrected their responses after education. the best improvement was detected in the responses of the clinical signs related with pid. it was remarkable that the participants have known the family history related with pid excellent before education. the majority of the participants (80%) believed that a lymphocyte count of 2500/mm3 was related to immunodeficiency. nbt and ch50 test were not found to be related with pid before education. it is also important to increase the awareness of pid among the physicians during their education in medical school and more comprehensive education in pid appears to be useful for medical students. chronic granulomatous disease (cgd) is a rare primary immunodeficiency with mutations in nadph oxidase enzyme complex which causes failure of phagocytic cells to produce superoxide and subsequent intracellular killing of microorganisms. we retrospectively analysed medical records of patients diagnosed with cgd in the last 35 years from immunological diagnostic centres from 9 central and eastern european countries (estonia, poland, belarus, ukraine, czech republic, slovakia, hungary, serbia and slovenia) and russia. genetic sequencing from patients' dna was performed in genetic centres in ljubljana, belarus and netherlands for mutations in known genes involved in cgd pathogenesis: cybb, cyba, ncf1, ncf2. we included 104 patients with cgd in our cohort, 7 were female. the mean age at presentation of the disease was 12 months and at diagnosis 3,9 years. lymphadenitis (43%), dermatitis (16%), enteritis (16%), pulmonary infections (13%), liver abscesses (4%) and septicaemia (6%) were the most common clinical presentation. complications of bcg vaccination (28%) were the most common presenting infection. in total 917,4 years of followup in our cohort, the patients suffered 834 different severe infectious episodes (0.9 per year). respiratory (25%), lymph node (25%) and gastrointestinal tract (18%) infections represented the most prevalent severe infections. we identified 87 different mutations out of 104 genes tested. in 78 patients we identified different mutations in cybb gene, 2 unrelated patients had the same mutation in cyba gene and in 5 patients had typical deletion in ncf1 gene. in our cohort we observed high incidence of bcg infections as a presenting symptom. apart from high bcg infections patients included in our study had similar frequencies of infections and infecting microorganisms as patients described in previous series. objectives: the aim of this study is to determine the carrier rate in healthy controls from central european and balkan region. methods: we screened more than 500 healthy subjects from 5 countries in the region. exon 2 and 10 was pcr amplified and subsequently sequenced with abi prism 310 genetic analyzer. results: heterozygous mutations were found in 4% of apparently healthy hungarians, 7% of slovenians, 8% of bosnians, 11% of serbians and in 16% of apparently healthy macedonians. mutations found in hungarian population were as follows: v726a (1), k695r (3). mutations found in slovenian population were: v726a (1), k695r (5) and e148q (1). mutations found in bosnian population were: v726a (1), k695r (6) and f756c (1). mutations found in serbian population were: e148q (6), k695r (5). mutations found in macedonian population were as follows: e148q (8), k695r (7) and m694v (1). conclusion: we found higher than expected carrier rate in screened populations, from 4% to 16%. it is interesting to note that more than half (60%) of detected carriers in all analyzed populations has k695r mutation. progress in the field of primary immunodeficiencies (pids) is reflected in national pid registries. data from slovenian pid registry were analyzed. patients' data were collected retrospectively before 2007 and prospectively afterward. patients were classified according to international classification and updated regularly. data of 193 patients with 45 different pids were analyzed. interestingly, complement deficiencies are the most common, accounting for 23% of all entries. second most common are antibody deficiencies with 19%, followed by well-defined syndromes (16%), immune dysregulation (13%), neutrophil defects (12%), combined deficiencies (9%), autoinflammatory disorders (6%) and defects of innate immunity (2%). prevalence of diagnosed pids in slovenia has changed in the last 5 years; less complement deficiencies and more antibody deficiencies were diagnosed in comparison to previous decades. the number of new pid cases has been gradually increasing, a more prominent increase has been noted in the last 10 years. the prevalence increased most for combined immunodeficiencies, cvid and autoinflammatory disorders. the spectrum of pid entities has also widened in the last decade. three patients with scid were diagnosed and successfully treated in the last three years (incidence -1:25.000 births). high prevalence of complement deficiencies reflects early implementation of good complement diagnostic facilities and awareness among infectologists. this group of patients was prospectively collected from 1987. combined immunodeficiencies, cvid and autoinflammatory syndromes were all probably underdiagnosed before due to lack of awareness among physicians. distribution of pid groups is more consistent with esid registry in the last five years. identification and successful treatment of scid patients in the last years is an important quality marker. bulgarian association for clinical immunology was set up in 2005 aiming to get together all specialists working in the field of clinical immunology. one of the important objectives of the association was to raise the public awareness and attract attention of specialists, national health system, government and other related societies in order to improve the diagnosis and access to treatment for children and adults with pid. efforts of immunologists led to the following results: 1. consensus on the diagnosis and treatment of the basic pid groups was created by the pid national working group that was established in 2010, and specific guidelines were disseminated as well. register for pid patients has been set up in bulgaria that allowed the collection of data on the incidence and prevalence of pid and the negative effect of these conditions on the population. 3. educational program to improve the qualification of the physicians and provide available resources to general practitioners and raise the public awareness were introduced. collaboration with patient's organizations was developed. 5. treatment of pid patients has been fully covered by the public health system since march 2013. all these steps made it possible to advance the diagnosis and management of pid in our country. pediatric pid patients care -single center experience g. petrova; p. perenovska; s. mihailova; e. naumova umhat "alexandrovska", sofia, bulgaria j-project in bulgaria started in 2005 and up to now we have elaborated programme with diagnostic criteria, well equipped laboratory, established some mutual connections with foreign colleagues, held regional meetings, conferences; created clinical standards for treatment and ensured an immunoglobulin treatment and replacement therapy. here are examples of some of the problems we face: 1. seven-years old boy with hypogamaglobulinaemia (normal number b-ly with abnormal function). ivig had some initial effect, but lately we noted very fast deceleration in the overall health status with possible need of lung transplantation. the case is posing a question what more could we do, could we have prevented this rapid worsening. 2. ten-month old girl with scid with severe bcg infection after first vaccination, referred relatively late to our center, but successfully transplanted. the case is posing a question about timing of bcg vaccination and of referral to specialized center. 3. nine-years old girl with unidentified immune deficiency, normal immunological follow up but clinical course as an immune deficiency with very favorable effect of ivig according the parents. the case is posing the question should we stop or should we continue ivig, despite failing to find immunological defect, based on the good clinical response. unfortunately pids are not very well recognized and sometimes the patients are referred late. sometimes poverty and lack of knowledge of patients leads to miscalculation and neglecting of their conditions by themselves, or refusal for specific tests for clarifying the diagnosis. background: although rare, primary immune deficiencies (pid) are manifested with high rate infections as well as with autoimmune and malignant disorders that are treated hardly and inefficiently. pid are not only immunological problem; they require close collaboration between immunologists, pediatricians, ent, lung and gut specialists, dermatologists, hematologists, oncologists, patients and administration. the aim of this study was to summarize the activity on registration and replacement therapy of pid patients in plovdiv region at the university hospital "st. george"-plovdiv for 1 year (03.2013-02.2014). methodology: children with pid of humoral immunity hospitalized at the clinic of pediatrics, and adults with hereditary angioedema (hae) were included in the study using immunological and other lab tests, clinical follow up and treatment: iv and sc ig for children with pid and c1 esterase inhibitor (ruconest, berinert) for hae patients. results: three national workshops and a national conference on pid were hosted and organized in plovdiv since 2005. well established university hospital immunological laboratory, detecting serum immunogloblins, blood lymphocyte populations and subpopulations and complement proteins; clinic of pediatric and genetic diseases and an information center for rare diseases and orphan drugs function in plovdiv. an expert center for diagnosis and treatment of pid was created at the university hospital. it is a team of two competent pediatricians, an immunologist and an allergist. the targets are hospitalized pid children, outpatient pid children and hae adults. the center introduced regular replacement therapy with iv and sc iv and c1 inhibitor, reimbursed by the national insurance. together with icrdod the experts provide education for patients and parents how to perform sc ig application as well as consultations of patients and relatives about pid.since march 2013 indicated reimbursed replacement therapy with iv ig -octagam, started regularly in 5 hospitalized pid children with bruton hypogammaglobulinemia, cvid, omenn syndrome or igg1 id. these patients, aged form 4 to 12 years, had 2 to 6 hospitalizations for one year. four outpatient children with omenn syndrome or igg1 subclass deficiency were subjected to sc ig -gammanorm, and 7 hae type 1 outpatients had good response for replacement c1 inhibitor therapy as follows: conestat alfa (ruconest)in 5 hae adults (150 iu/ml weekly), and berinert (20 u/kg b.w. weekley)in 2 hae adult patients. conclusions: the expert pid center in plovdiv university hospital provides competent diagnosis, therapy, education and consultations for pediatric and adult pid patients from plovdiv region. the recent introduction of reimbursed replacement therapy for pid patients (hospitalized or outpatients) allows regular immunological and clinical follow up of the diseases. background: intravenous immunoglobulins (ivigs) are scarce biological products used in a broad variety of disorders. tolerance to infusions is usually good but adverse events, including some serious ones, have been reported. methodology: a cohort study aimed for detection of adverse events that occur during and following intravenous immunoglobulin (ivig) infusions at cairo university children hospitals [patients were recruited at neonatal intensive care units (nicu), pediatric intensive care units (picu), general and specialized inpatient wards ] over a time period of six months, from april through september, 2013. the study included 62 transfusions for different disease conditions in 55 patients.three maltose-stabilized intravenous immunoglobulin products were administered to patients. assessments were done before, during and after the infusions. results: there were 22 symptoms and 26 laboratory changes of adverse events during ivig transfusions, with some patients experiencing more than one adverse reaction. adverse events were noted to occur most frequently within 1 to 6 h from onset of ivig infusion (n = 9, 39.1%). first hour after infusion onset was the most common timing for symptoms of adverse reactions (n = 5, 21.7%). patient characteristics of those with adverse reactions: adverse reactions occurred in 37.1 % of the infusions (n = 23) with the majority belonging to the 6-9 years old age group (n = 10, 43.5%), with variable diagnostic categories. ten patients observed during 14 infusions (60.9%) had one or more risk factors for complications, while 6 patients observed during 9 infusions (39.1%) had no risk factors. the commonest risk factor was administration of nephrotoxic drugs (n = 8, 34.7%), followed by presence of a suspected autoimmune disorder (n = 7, 30.4%) and preexisting renal insufficiency (n = 5, 21.7%). using regression analysis, the predicting variables for each complication were noted .for example ,fever and chills were related to infusion rate and dose whereas the predicting variables for pallor were infusion rate and presence of existing risk factors. conclusions: clinicians should be aware of the high need for special monitoring while infusing ivig to patients with primary immunodeficiency disorders, autoimmune hematological disorders and sepsis. certain diagnosis of a primary immunodeficiency disorders (pid) is most confirmedly performed by investigation of a gene defect, allowing genetic counseling and screening. molecular diagnosis helps both parents and index pid patient by carrier detection and pre-implantation testing for selecting appropriate reproductive decisions. furthermore, for confirmation of diagnosis and establishment of the inheritance pattern genetic analysis is necessary. this survey in all pid cohorts should be considered for long-term planning such as bone marrow transplantation of a pid infant at birth. the result of this testing also is important for screening of newborns and for those in specific family or ethnic groups. the prevalence of pids has been estimated to be more than 1/600 worldwide. based on the total population of iran reported in 2010 (76,424,000), the expected prevalence of pids in iran would be more than 127,300 individuals. however, because of the high rate of consanguineous marriages in iran and an increased risk for development of disorders with an autosomal recessive pattern of inheritance, this prediction is likely to be an underestimation. to date, 2247 clinically diagnosed patients of pids have been reported in iran, and a definite diagnosis, defined by mutation analysis, was made in 790 individuals. as a result, 1.76% of the expected pid patients have been identified, and among these, 35.2% have been diagnosed at a molecular level. the proportion of genetically definite diagnosis varied between 84.5 and 0% in the different disease categories. this wide spectrum might be due to unknown underlying genetic defects or modifying genes, especially in patients with predominantly antibody deficiency. on the other hand, the latter patients also had the lowest percentage of clinically diagnosed cases. croatia is a small country with 4 267 889 inhabitants . according to expected prevalence of pids we expected approximately four hundred patients with pid. for the past few years university hospital center zagreb is the reporting center for esid registry. it is also the national center for diagnosis and treatment of pids, including haematopoietic stem cell transplantation. current pid database is running manually, with the exception of patients reported to esid registry. most of them are of pediatric age, reffered to the hospital from all over the country. the pids patients are classified according to international classification ( iuis-international union of immunological societies ). there are 121 pids patients included in the database at the moment. the majority of them have antibody deficiency. combined immunodeficiency and well defined syndromes appear in equal distribution. other types of pids are reported in small numbers. although no consanguinity was reported, we noticed the geografic distribution of severe combined immunodeficiency patients (scid) mostly in 2 regions, istra and podravina. it can be explained with genetic isolation. the establishment of national online pid registry is in process. the aim is to improve the diagnosis od pid specially in adult patients, who are not included in present database, with exception of the patients diagnosed in childhood. primary immunodeficiency disorders are underdiagnosed in croatia, specially in adults. establishing national pid registry will improve the physicians awareness of pids, which is particularly important for adult patients. we expected the number of diagnosed pid patients will rise. this will give the opportunity to make progress in diagnosis and treatment, and the opportunity for further epidemiological and clinical studies. another 25 patients have well defined immunodeficiency syndromes from which: 11 are with di george anomaly; 2 with wiskott-aldrich syndrome; 4 with ataxia-teleangiectasia; 4 with hyper ige syndrome; 1 with schimke syndrome and 3 are with nijmegen breakage syndrome. in early nineties, two (2) of our patients had chronic mucocutaneus candidiasis. 3 patients are registered as x-linked scid and 1 with xlinked lymphoproliferative syndrome (xlp). 1 patient has chronic granulomatous disease; 3 have severe congenital neutropenia and 5 have hereditary angioedema. 2 patients are with whim syndrome. 6 patients are with anti-inflammatory disorders: 1 has hyper ig d syndrome (hids) and 5 have familial mediterranean fever. the j-project realization in the republic of kazakhstan allows to update the pid problem, to raise the availability of early diagnosis of pid, to improve the life quality of patients with pid by providing substitution therapy and as a result, the infant mortality and disability rate has been reduced. common variable immunodeficiency (cvid) is the most common primary immunodeficiency (pid) characterised by impaired immunglobulin production and immune dysregulation. chronic and recurrent infections and its results are typical manifestation of this disease. in addition there is a higher risk of autoimmune disorders, lymphoproliferative or granulomatous diseases and malignancies. successful management of cvid patients is based on prevention and consistent therapy of infections with sufficient immunoglobulin replacement and/or antibiotics, prevention and active screening of cvid related complications. in our study we analysed data of 41 patients gained from medical records. these data were also input into czech pid registry and pid registry organized by esid (european organisation for immunodeficiency). we aimed at the period before diagnosis-onset of the symptoms and their characters and the course of the disease-effect of therapy, occurence of the related complications. finally, we compared our data with similar performed studies. chronic and recurrent upper and lower respiratory infections were the most frequent first manifestation of our cvid patients, but developed chronic lung disease or autoimmune disorder as well. in all patients the intravenous or subcutaneous imunoglobulin replacement therapy, eventually combined therapy with antibiotic prophylaxis, was initiated. beside chronic lung disease the most common complications were autoimmunity disorders, especially autoimmune thyroiditis, evans syndrome, trombocytopenia (itp), autoimmune hemolytic anemia (aiha). on the contrary we revealed 2 patients with insuline dependent (type 1) diabetes mellitus and cvid. only few case reports have been published with such association. successful management of cvid patients is based on a prevention and a consistent treatment of infections with sufficient immunoglobulin replcement and/or antibiotic therapy, a prevention and an active screening of cvid related complications. such approach can significantly improve the prognosis of cvid patients and the quality of their life. laura zilinskaite 1 ; ieva bajoriuniene 1,2 ; raimundas sakalauskas 1 ; brigita sitkauskiene 1,2 1 department of pulmonology and immunology, lithuanian university of health sciences, kaunas, lithuania. background. primary immunodeficiency (pid) is considered to be a rare disease. despite that it is thought that six million people may be living with a pid worldwide. in the hospital of lithuanian university of health sciences (hluhs) patients with suspected immune disorders have been diagnosed and treated since 1998. we aimed to review the structure of pid diagnosed and treated in hluhs during the last five years. methods. data about patients with pid consulted in the hluhs was collected from the department of medical statistics. case histories of these patients were revised and patients' data was collected: onset of symptoms, type and duration of disorder, type of treatment. all patients with pid were divided into several groups according to the classification of international union of immunological societies primary immunodeficiency diseases classification committee (2011). results. there were 57 patients with pid diagnosed in the centre. antibody deficiency was diagnosed for 38 patients: 3 -bruton's disease, 14common variable immunodeficiency (cvid) and 21selective immunoglobulin (ig) a deficiency. complement deficiencies were diagnosed for 13 patients: 11 -c1 esterase inhibitor deficiency and 2 -c4 deficiency. another well-defined syndrome with immunodeficiency was found in six patients. the most prevalent symptom in patients with predominant igg deficiency was recurrent pneumonias which occurred at the age 11.9 ± 4.9 yrs. the mean time between the onset of symptoms and confirmation of the diagnosis was 14.6 ± 6.4 yrs. thirteen patients are on the replacement therapy with intravenous immunoglobulin. these patients had 2-6 infections/year before the treatment initiation and only 0-1 infections/year during the treatment. •pid center has successful sideproject as regional charitable public organization of invalids "society of patients with primary immunodeficiency diseases in st. petersburg" solovushka (nightingale) "". web site for patients (www.opidspb.ru) was opened due to mutual efforts of pid center staff and patients. pid center laboratory is based on spb pasteur institute central clinical diagnostics laboratory and laboratory of molecular immunology and seroepidemiology. main groups of tests perform for pid patients are: general clinical assays; flow cytometry (6-color assay on facs canto ii); humoral factors assays (ig levels, igg subclasses, post-vaccination igg levels, etc.); burst-test on flow cytometer; genetic analysis of btk, rag1, rag2,was, cybb genes. despite of relatively short story of spb pid center it has a variety of completely diagnosted and successfully cured cases of pid including agammaglobulinemia with b-cell in identical siblings, wiskott-aldrich syndrome, chronic granulomatous disease (cgd), etc. introduction: the primary immunodeficiency diseases are a group of disorders caused by basic defects in immune function that are intrinsic to, or inherent in, the cells and proteins of the immune system. there are more than 200 primary immunodeficiency diseases.. laboratory studies are necessary to determine the presence of a primary immunodeficiency diseases. the standard screening tests for antibody deficiency starts with measurement of immunoglobulin levels in the blood serum. these consist of igg, iga and igm levels. the results must be compared to agematched controls. additional studies used to evaluate patients with antibody deficiencies include measuring the different types of lymphocytes in the blood by marking those cells with molecules that can identify the different types. a commonly used test is called flow cytometry that can identify b-cells and t cells present in the circulation. methods: in 2008 in our laboratory of immunology is created the sector of examination for the pid and we measure the immunoglobulin levels with a beckman coulter immage immunochemistry system fully automated rate turbidometry and rate nephelometry method. we have determine the aged-matched levels of immunoglobulins in our laboratory. we have install also a new flow cytometer of beckman coulter company and we can determine the b cells by cd19 marker and t cell with cd3 , cd4 and cd8 marker. this examinations has been of great help in diagnosis of primary immunodeficiencies. results: in years 2010-2013 from the examination of children aged from 0-14 years old reported in our laboratory for immunological examination from the pediatric department of uhc mother teresa in tirana we have selected 41 cases with disorders regarding the primary immune deficiencies. we have 5 cases (3 cases reported in 2008-2009 and 2 new cases ) with nul b cell in cytoflorometry (b cd19 cell = 0 %). the level of immunoglobulins was indetectable for igg , iga and igm in the moment of diagnosis. they were boys and the age at diagnosis was 3 and 4 years and actually they are 4 and 6 years treated with ivig. we have classified them as bruton (xla). we have 12 case with low cd19 b-cell in circulation but only in 4 of them we have done the immunoglobulin level. the low level of cd19 b cell is accompanied with different kind of hypoglobulinemies: 1common variable immunodeficiency cvid, 1 with isolated iga deficiency, 1 with igg deficiency and 1 with both igg + igm deficiency. the immunoglobulins disorders : we have classified them according the antibody deficiencies (tab1) and we noted that the most frequent one is the deficiency of iga (12/19 or 63.2 % -from which 6 iga isolated, 5 accompanied with low igm and 1 with low igg ) with average age at diagnosis 5.09 years old. in children in the first years of live aged from 0.8 to 2 years old we found 8 case with transient hypogammaglobulinemia of infancy. according this survey we can report our 92 cases of primary immunodefiecies in uhc mother teresa of tirana with different classification conclusion: the finding are to be completed with other cases in albania and it is necessary to do the national register for pid in order to estimate exactly the prevalence of this disorders in our country. nyíregyháza-debrecen, hungary the stat3 mutation was confirmed in 2007 as the cause of autosomal dominant hyper-ige syndrome (ad-hies). the disease, which also mentioned in the literature as job's syndrome, is a rare primary immunodeficiency. this disease can be characterized by the following classic triad: recurrent purulent skin infections, cold abscessus which are formed in the ground of chronic ekcematoid dermatitis, pneumatocele, formation causing pneumonia and extremely high ige level. dental training interdependency as well as bone and connective tissue disorders frequently occur in the nonimmunological symptoms as multi-systemic disease. the stat 3 protein has an important role in the area of wound healing, immunity, tumor and neovascularization. we would like to show this in case of 3-years old kitti whom the perinatal medical history was eventless. there is a frequent hospitalization in kitti's case history since her infancy. due to the age of three months because of serious exsiccotoxicosis, 5 months old bilateral bronchopneumonia and pleurisy required icu care. bronchoscopy was made because of recurrent pneumonia, which excluded the bronchial malformation. lately rather otitis, mastoiditis and airway obstructive symptoms dominated the clinical picture. from serum immunoglobulins-igg is very low, whereas high levels of igewas indicated. on this basis, the diagnosis of job's syndrome was up, which is a negative stat3 mutations as the molecular genetic test performed demonstrated. the disease has a great clinical importance, since the risk of emergence of serious and often life-threatining complications are high. because of the prevention of disseminated infections, the early detection and the appropriate treatment are essential. in default of causal therapy, the treatment primarily concern to prevention of infections, or their aggressive antibiotic therapy. calcium and vitamin d and as well as histamine 1 on the case of itchy skin symptoms are reccurrended to use. laboratory of molecular immunogenetics, human genetics institute, cnrs and university montpellier 2, montpellier, france. we performed clinical, immunological and genetic studies of 12 hyper-ige syndrome (hies) patients from 4 hungarian, 2 lebanese, one russian, one polish, and one swedish families with autosomal dominant (ad) or sporadic forms of the disease to reveal cross-ethnicity of recurrent and novel mutations in the signal transducer and activator of transcription-3 gene (stat3). four patients from 3 hungarian families, and one russian, and one swedish patient carried the heterozygous r382w germline mutation at the dna-binding site of stat3. the recurrent v637m mutation affecting the src homology 2 (sh2) domain was detected in one lebanese and one polish family, and the v463del deletion located in the dna-binding domain was unveiled in another lebanese family. a novel h332y mutation affecting the dna-binding site of stat3 in three hungarian patients from a gypsy family was also found. the segregation of this mutation with hies, restriction fragment length polymorphism analysis of stat3 from patients and controls and the negligible production upon il-6 stimulation of monocyte chemotactic protein-1 by the patient's blood mononuclear cells suggested that the h332y mutation was disease-causing. these data suggest, that dominant negative mutations of the dna-binding and sh2 domains of stat3 cause ad and sporadic cases of hies in different ethnic groups with r382w as the predominant mutation found in 5 of the 9 families. functional and genetic data support that the novel h332y mutation may result in the loss of function of stat3 and leads to the hies phenotype. published in molecular immunology. 46:202-6. males with an expressed mutation in the sap (signaling lymphocyte activating molecule [slam]-associed protein) gene have an x-linked syndrome characterized by an increased vulnerability to infection with epstein-barr virus (ebv). we evaluated two related male patients with fatal infectious mononucleosis (fim) and mutation in the sap gene. sequence analysis revealed hemizygous g to a transition at nucleotide position 47 in exon 1 in one of the patients, and heterozygosity for this mutation in the genomic dna from his mother and maternal grandmother. this mutation resulted in asparagine instead of glycine in the sequence of the sap protein at amino acid position 16. to analyse the effect of this missense mutation on protein function cdna was generated by site-directed mutagenesis and cloned in pcmv-flag vector. we found that the mutant sap (sap/g16d) protein was defective in protein folding as manifested by the reduced half-life compared to that of wild type sap. furthermore, the sap/g16d protein was defective in binding to its philological ligands slam and 2b4. these results suggest that defects in protein folding and ligand binding collectively contribute to the loss of function of the sap protein in patients carrying g16d mutation. dedicator of cytokinesis 8 (dock8) deficiency is an innate error of adaptive immunity characterized by recurrent viral, bacterial and fungal infections, very high serum ige concentration and a progressive deterioration of t-and b cell-mediated immunity. traditional sanger sequencing may fail to identify mutations in dock8, due to overlapping large deletions in heterozygous patients. we studied the genetic and immunological features of two sisters (11 and 6 years of age) born to healthy hungarian parents. mutational analysis of genomic dna and cdna from the patients and parents by a combination of pcr and bidirectional targeted sequencing failed to identify the mutation. however, a multiple ligation-dependent probe amplification (mlpa) assay revealed two previously unknown large deletions, del1-14 exons and del8-18 exons, of dock8 in both patients. the children's mother was heterozygous for the del1-14 exons mutation, whereas the father carried the del8-18 exons deletion. immunoblot analysis showed an absence of dock8 protein from the peripheral blood lymphocytes of both patients. these data suggest that the new compound heterozygous del1-14 exons and del8-18 exons mutations result in a loss of dock8 protein function and a typical dock8 deficiency phenotype. our findings suggest that traditional sequencing technology may give misleading results in such cases and that mlpa may be indispensible for the definition of the large deletions frequently observed in patients with dock8 deficiency. we describe here a patient with invasive cryptococcus laurentii infection and the x-linked form of hyper-igm syndrome (x-higm). c.laurentii is an extremely rare human pathogen. this fungus was previously considered saprophytic and non-pathogenic to humans, but it has been isolated as the etiologic agent of skin infection, keratitis, endophthalmitis, lung abscess, peritonitis, meningitis, and fungemia. most affected individuals had a compromised immune system because of leukemia, cancer, diabetes mellitus, aids, or prematurity. repeated isolation of c.laurentii from the oropharynx of an immunocompromised patient has also been docuthe wiskott-aldrich syndrome (was) is an x-linked recessive immune deficiency disorder characterized by thrombocytopenia, small platelet size, eczema, recurrent infections, and increased risk of autoimmune disorders and malignancies. was is caused by mutations in the wasp gene which encodes wasp, a 502-amino acid protein. wasp plays a critical role in actin cytoskeleton organization and signalling, and functions of immune cells. we present here the results of genetic analysis of patients with was from eleven eastern and central european (ece) countries and turkey. clinical and haematological information of 87 affected males and 48 carrier females from 77 was families were collected. the wasp gene was sequenced from genomic dna of patients with was, as well as their family members to identify carriers. in this large cohort, we identified 62 unique mutations including 17 novel sequence variants. the mutations were scattered throughout the wasp gene and included single base pair changes (17 missense and 11 nonsense mutations), 7 small insertions, 18 deletions, and 9 splice site defects. genetic counselling and prenatal diagnosis were applied in four affected families. introduction: mhc-class ii deficiency is an autosomal recessively inherited combined immunodeficiency disorder characterized by less than 5% expression of hla-dr on b cells or monocytes. it is caused by mutation of genes (ciita, rfxank, rfx5, rfxap) regulating transcription factors which controls expression of mhc-class-ii molecules on cell surface. mhc-class ii molecules are expressed on thymic epithelial cells, antigen presenting cells (b lymphocytes, dendritic cells, monocytes and macrophages) and activated t cells. these molecules are of critical importance to immunity, cd4+ t cell development, antibody production, tolerance induction and inlammatory response. consequently, patients present with clinical findings related to combined immunodeficiency during infancy. material and methods: medical records of thirteen patients with mhc-class ii deficiency, followed-up in ankara university, medical school, division of pediatric immunology/allergy from 1999 to 2013, were evaluated retrospectively. findings: during study period, 6 male and 7 female patients were diagnosed with mhc-class ii deficiency. age of diagnosis were between 3 months and 4 years of age. consanguinity were present in eleven out of thirteen patients. most frequent clinical findings during initial diagnosis were failure to thrive, pneumonia and oral moniliasis. lymphopenia was absent in all of the patients, however, low serum igg level was present in all of them. except for the 15 yo female patient with a positive family history and whose hla-dr expression was 0,2%, hla-dr expression was 0% in the rest of the patients. eight patients underwent hematopoietic stem cell transplantation (hsct). two patients were lost soon after hsct due to complications and three patients died of opportunistic infections. four patients died of severe opportunistic infections without underwent hsct. results: mhc-class ii deficiency is a combined immunodeficiency and not considered a rare disease in our country. to date, only known treatment is hsct. since it has poor prognosis, hsct should be performed before development of chronic viral infections and sequelae related to infections in patients who have hla-matched sibling. fatih celmeli 1 ; giancarlo la marca 2 ; ines santisteban 3 ; michael s. hershfield 3 1 purine nucleoside phosphorylase deficiency (pnp deficiency) is a rare autosomal recessively inherited type of immunodeficiency. pnp deficiency constitutes about 1 to 2 percent of all combined immunodeficiencies. it is characterized by progressive combined immunodeficiency and neurologic findings which includes ataxia, developmental delay, and spasticity. the immunodeficiency is progressive, with normal immune functions at birth, but severe t cell deficiency with variable b cell functions presented by the age of 2 years . the only curative treatment is the hematopoietic stem cell transplantation (hsct). here, we present a 14 year-old girl with recurrent respiratory tract infections, short stature and spastic paraplegia. immunological, biochemically and genetics investigation revealed pnp deficiency with a117t mutation in pnp gene. case report: a 14 years-old girl was referred to our pediatric immunology clinic for recurrent sinopulmonary infections since 6 year of age. she was full term neonate and her parents were not consanguineous. she had a history of prolonged and resistant bronchopneumonia, and an attack of generalized chickenpox complicated with pneumonia. she had also severe zona infection resolved with ulceration in cornea, one year ago. she has been suffering from frequent infections like chronic sinusitis, oral moniliasis, recurrent pneumonia and sclerosing cholangitis. she has also nonprogressive cerebral palsy, spastic paraplegia, behavioral problems and limited motor and mental retardation. there was no family history of recurrent infections or immunological disorders. on her physical examination, there was failure to thrive, (her weight and height <3 rd p) oral moniliasis, bilateral crepitus ralls, and splenomegaly. she has also marked spasticity with brisk reflexes in lower extremities. laboratory tests revealed lymphopenia: hemoglobin 11 g/dl and platelets 158000/mm 3 wbc 4000/mm 3 , absolute lymphocyte 560/mm 3 . the laboratory results are shown in the table. lymphocyte proliferation is lower than normal limits in response to pha stimulation (wst1 assay). ppd response and hiv was negative. antihbs, and antihav were negative despite vaccination. uric acid level 1,8 mg/dl. direct coombs was negative, thyroid autoantibodies were within normal limits. genetic analysis revealed homozygous missense mutation (c.349g > a), which causes the a117t amino acid substitution in pnp gene, in exon 4, which has previously been reported. additionally, a homozygous g/a polymorphic site in ivs3 has been detected (c.285 + 10g (ivs3 + 10g)). discussion: pnp deficiency is caused by mutations in the pnp gene at 14q13.1. this gene encodes the protein purine nucleoside phosphorylase, one of the enzymes involved in the purine salvage pathway. adenosine deaminase (ada) deaminates adenosine to yield inosine, which is then converted to hypoxanthine by pnp. pnp also converts guanosine to guanine. a number of metabolites are elevated in the plasma and urine in pnp deficiency, including deoxyguanosine and deoxyinosine. there is an intracellular accumulation of their deoxy triphosphate compounds, particularly deoxyguanosine triphosphate (dgtp). the latter is toxic to t cells, a property similar to deoxyadenosine triphosphate in adenosine deaminase deficiency. in this report, we demonstrate the clinical characteristic of the patient with late diagnosis of pnp deficiency. pnp mutations likely lead to an intense alteration of the enzyme activity which in turn, cause severe and early onset of the clinical findings. however, in our case, the clinical onset of the disease is quite late (after 6 years) which can be explained by the residual activity of the pnp. in conclusion patients with pnp deficiency can be late onset. additionally, late diagnosis of this patient can cause severe comorbidity which limits the chance of bone marrow transplantation. di george syndrome, a disorder caused by a defect in chromosome 22 (22q11.2 deletion), results in the poor development of several body systems. clinical features include congenital heart defects, hypoparathyroidism and thymic hypoplasia or aplasia leading to t-cell immunodeficiency. the aim of our study is to screen and determine the incidence of di george syndrome within only one tube of blood in children with congenital heart anomalies in our population. children who were found to have a cardiac defect during routine visits in pediatric cardiology and neonatalogy departments were included into the study. cases with known genetic syndromes and newborns younger than 32 gestational weeks of age and small for gestational age (birth weight <2500 gr) were excluded. a total of 136 patients were included. there were 79 (58%) males and 57 (42%) females. age ranged between 0-80 months (10.2 ± 15.9 months). parental consanguinity was 17% (n = 23) in the study group. the majority of patients diagnosed after murmur was heard during the routine physical examination (n = 62, 46%). five patients (3%) diagnosed antenatally. remaining clinical signs on admission were as follows; respiratory distress (n = 33, 24%), tachycardia (n = 20, 15%) and central cyanosis (n = 16, 12%). echocardiagorafic examinations revealed ventricular septal defect (vsd) (n = 27), tetralogy of fallot (tof) (n = 17), vsd-asd (n = 13), aortic coarctation (n = 11), double outlet right venticle (dorv) (n = 10), transposition of the great arteries (n = 6), truncus arteriosus (n = 5) and pulmonary atresia (n = 6). pulmonary stenosis, endocardial cushion defects, total pulmonary venous return anomaly and hypoplastic left heart were the other defects. 22q11.2 deletion was ascertained in 7 (5.1%) patients; these patients were diagnosed to have tof (28.6%), truncus arteriosus (14.6%), dorv (14.3%), vsd (14.3%) and vsd-asd (14.3%). preliminary results of the study showed that the frequency of 22q11.2 deletion is 5% in patients with known cardiac defects. single tube of blood is enough for flow cytometric and genetic analyses. further studies involving higher number of patients is mandatory to give sufficient information about the exact incidence of the disease. di george syndrome -where do we stand now? małgorzata pac; małgorzata skomska; ewa bernatowska department of immunology, the children's memorial health institute, warsaw, poland di george syndrome (dgs) classically comprises t-cell deficiency (due to thymic hypoplasia), hypoparathyroidism, cardiac malformations,and facial abnormalities. deletions of the long arm of chromosome 22 at position q.11 are most commonly associated with dgs. syndrome is also found associated with other genetic abnormalities (10p deletions, char ge), certain teratogenic influences (retinoid acid, foetal alcoholic syndrome, maternal diabetes). the dgs phenotype is very heterogenous with variable expression of the different features including the immunodeficiency. the initial treatment emphasis is to control the hypoparathyroidism. correction of congenital heart defects (if present) is usually needed. the best treatment of the immune defects of dgs is still controversial. both hsct and transplant with fetal thymus are the option for complete dgs (cdgs). long term survival after hsct has been reported, though at a lower rate (41-48%) compared to survival after hsct for scid. survival in the subgroup receiving matched sibling donor transplants was better at over 60%. the use of post natal human thymus was pioneered by markert at duke university and has become established as the treatment of choice for cdgs, with the result of 43 out of 60 treated patients survived (72%). more recently this approach has also been used in london, at gosh. . under care of cmhi there are119 patients fulfilling esid criteria, 59 girls (49.6%) and 60 boys (50.4%), age 2/12 -23 y.o. in 70% of them 22q11 deletion in locus d22s75 was found. the vast majority children were diagnosed as partial dgs. none of them had significant hypogammaglobulinemia and no regular ivig therapy or antibiotic prophylaxis were required.. the mean number and percentage of cd3, cd8 and cd4 lymphocytes as well as lymphoproliferative answer to pha and cd3 in dgs patients were slightly diminished. in many improvement of cellular immunity was observed with age. about 86% presented with congenital heart disease, requiring surgery, while almost 50% had the symptoms of hypocalcemia and hypoparathyroidism, next 46% -speech and learning difficulties. one child was diagnosed as cdgs. scid is a rare, inherited condition, is caused by numerous molecular defects that lead to severe compromise in the number and function of t cells, b cells, and occasionally natural killer cells. seventeen patents with scid were registered during the period from 1994 till 2013 in the children's clinical university hospital. medical charts of these patients have been reviewed. there were 9 boys and 8 girls. positive family history was in 4 families. mean age at the onset of symptoms and scid diagnosis was 1.7 ± 1.23 and 4.03 ± 1.6 months, respectively. pneumonia (65%), candidacies (65%), bcg infection (47%), diarrhea (35%) were the most important infections. anemia and relative lymphopenia were in 76% cases , growth retardation, hypotrophy had 59% children . pathogens such as candida albicans (11), mycobacterium tuberculosis complex (8), cmv (3) and others have been identified. totally 15 patients died.two girls are alive (29 and 17 months post-transplant). autopsy was done in 8 patients. we saw different changes in thymus and lymphatic nodes. artemis deficiency (n = 1), t-b-scid (n = 4), t-b + scid (n = 5), γc deficiency (n = 1 mutation r224w in γ-chain of receptor for il-2), unspecified scid (n = 6) were detected. conclusion: generalized bcg infection had 47% of our scid patients. (incidence of tb is still high 36.2 / 100 000 population in latvia and newborns obligatory are vaccinated on the second to fifth day of life). due to possibility of absence for pid routine genetic identification in only few scid forms were identified precisely. children's hospital, university of freiburg, freiburg, germany. purpose: ipex (immunodysregulation, polyendocrinopathy, enteropathy, x-linked) is a rare x-linked recessive life-threatening disorder characterized by autoimmunity and early death. pulmonary complication related with ipex has not elucidated exactly. here, we report 4 i.e. patients, 3 of which died from severe pulmonary disease. methods: clinical data and laboratory findings included autoantibodies, immunoglobulin levels as well as number of t, b and nk cells were evaluated. foxp3 expression was performed by flow cytometry. genomic dna was isolated and all exons and exon-intron boundaries of the foxp3 gene were sequenced by sanger sequencing. results: patient i (pi) presented with nephrotic syndrome at 3 years of age and then developed autoimmune hepatitis without eczema, enteropathy or high ige and died at 9 years of age due to acute respiratory distress syndrome (ards). two cousins of pi had the same hypomorphic splice site mutation leading to normal foxp3 protein expression and suppressive capacity. however, they exhibited typical symptoms such as eczema, diabetes and enteropathy with eosinophilia at early age (pii, piii) and were transplanted in infancy. one of them had severe respiratory distress right after birth (piii). patient iv from another family presented with chronic diarrhea without autoimmune manifestations and died due to ards. conclusion: lung disease related to ipex syndrome has not been reported before and this entity could be a critical factor in disease outcome. severe combined immunodeficiencies (scids) are a group of primary immunodeficiencies that comprise a number of monogenic disorders characterized by a block in t cell differentiation with or without impairment of b cell and natural killer (nk) cells. without early diagnosis and treatment most children die in the first year of life. a lack or very low number of t-cell receptor excision circle (trec) detected by realtime quantitative polymerase chain reaction assay (qpcr) is consistent with t-cell lymphopenia and has repeatedly demonstrated clinical validity in population based newborn screening for scid. however, the impact of population screening will be less in communities with high consanguinity and family history of scid, in which targeted screening may be more appropriate. here, we screened 100 high risk neonates and infants (with one or more of the following: clinical presentation and/or family history suggestive of pid, failure to thrive otherwise unexplained, lymphopenia) at cairo university hospitals. their full history and clinical examination were recorded. immunoglobulin profile and immunophenotyping of peripheral lymphocytes were performed as confirmatory tests. sixteen classical scid cases were detected, as well as another 2 scid variants (omenn syndrome and major histocompatibility complex class ii deficiency) (totally 18.4% of all subjects screened). the rate of consanguinity in this group was 77.8%. secondary causes of low trecs, other than scid, in our series included: bacterial septicemia (4 preterm, 1full-term), prematurity (2 cases), one preterm with omphalocele and facial dysmorphism, one preterm with congenital adrenal hyperplasia, one full term with microvillus inclusion disease, and one full term with idiopathic tcell lymphopenia. this demonstrates that in populations with high consanguinity rates, as in egypt, targeted (non-population based) trecs assay may provide a more efficient screening strategy. case 1: a is a 4 years old female, 1 st kid of non consanguineous marriage presented with fever for 5 months. one week after the onset of the fever red patches appeared on the face, hands, abdomen, ll. mother sought medical advice and received antibiotics, antipyretics and oral steroids for about one month with no signs of improvements regarding fever. one week later the mother noticed pallor and sought medical advice and the baby was admitted to local hospital and received one bag of blood. and then she was referred to our hospital (zagazig university hospital) where she developed acute pallor again which needed recurrent blood transfusions. dark colored urine occurred in frequent attacks with abdominal enlargement and pain, and interestingly upto this time, no improvement regarding fever, bone pain or pallor. on examination she was underbuilt (all parameters are under 3rd centile), pallor, tinge of jaundice, generalized skin pigmentation, generalized lymphadenopathy, 2 nd degree clubbing and hsm investigations: cbc (pancytopenia with reticulocytopenia) -lft (increased ast with indirect hyperbilirubinemia)-kft (normal)-ldh (highly elevated 7238 u/l)-esr (105) -fibrinogen (0.336 gm/ l)serum triglycerides (315.7 mg/dl)-ebv-vcm igm positive and igg negative -c3 (1.3 normal) -rf and ana(-ve) -serum ferritin (32436 ng/ml) -cd 56 is low -bone marrow biobsy revealed dysplastic changes -l.n. biopsy revealed non specific inflammatory changes. treatment: hlh protocol 94 with no sct prognosis: patient passed initial phase with complete resolution and waiting for bmt case 2: m is 4 months old boy, 2 nd kid of non consanguineous marriage (the 1 st is healthy 3 years old female) presented with fever and difficult breathing since the age of 2 months. the fever was of gradual onset stationary course and not responding to treatment with antipyretics and antibiotics. one week later the mother noticed abdominal enlargement with red rash over the abdomen that was associated with pallor but there was no evidence of bleeding from any site, no change in the color of urine, no jaundice, no ecchymosis, no joint swelling then he is referred to our hospital (zagazig university hospital). this boy is delivered by nvd at term with no evidence of any problem either during pregnancy or delivery, he is exclusively breast fed and vaccinated as scheduled. examination: his weight was 6 kg, length 53 cm, and head circumference 35 cm all are average for age, he was pale with no jaundice or cyanosis he had hsm and other systemic examination was quite well. children with pid usually are admitting to the hospitals and intensive care units of infants' pathology regional children clinical hospital №1, but at a later date, in serious condition, after the development of clinical manifestations in the form of severe generalized infectious disease, or various complications including hematological. the screening technology approves children to be diagnosed in newborn period and to be observing by immunologist and hematologist for the next preventative therapy and bone marrow transplantation or hematopoietic stem cell transplantation. costs for the differential diagnosis and verification of the diagnosis before the manifestation and complications development of pid -51 245 usd. costs for the differential diagnosis and verification of diagnosis after manifestation and complications development of pid would be 115 208 usd in 2 months. economic loss prevention in pid -63 872 usd. economic loss prevention in t -lymphopenia -105 884 usd. we also should include into account the contribution to the state's economy, which will later be obtained, due to the presence of a healthy member of society. at is an autosomal recessive multisystem disorder characterized by progressive cerebellar ataxia, telangiectasia and increased susceptibility to infections and malignancies, particularly lymphomas and leukemia. laboratory immune investigations typically show decreased peripheral tcells, particularly of naïve t cells, with abnormal in vitro response to mitogens. most at patients have decreased serum iga and igg subclass concentrations. while about 10% of patients with at show raised serum igm concentrations during the course of the disease, it is unusual to find a high level of igm at onset. as cerebellar ataxia and oculocutaneous telangiectasia are not present at very young age, these patients are often erroneously diagnosed as hyper igm syndrome (higm). to prevent mistaking a-t patients for higm it is proposed to add dna repair disorders as a possible cause of higm. . diagnosis of at, suggested by elevated alfa-feto-protein and increased sensitivity of patients' cells to irradiation, can be confirmed by identifying a mutation in the atm gene.we report 3 female case of at that with diagnosis of hyper igm received ivig but later they had manifestation of ataxia in the course of their disease and then had telangiectasia of conjunctiva. first case was a 3 yrs girl that was suffered from itp and granulomatosis lesion of skin associated with hyper igm before at diagnosis. second case was a 6 years old girl with microcephaly, sever ftt , neurodevelopmental delay , abnormal faces and hypo and hyperpigmentation lesions and anemia. with respect to these manifestation and increased afp ,nijmegan breakage syndrome was suggested. third case was suffered from hyperigm before a t diagnosis for 2 years. we present a patient with a dock8 deficiency. mutations in the dedicator of cytokinesis 8 gene (dock8) cause a combined primary immunodeficiency syndrome that is characterized by elevated serum ige levels, depressed igm levels, eosinophilia, sinopulmonary infections, cutaneous viral infections, and lymphopenia. onset of the disease was observed at 1-month of age with severe eczema and recurrent respiratory infections (pneumonia, bronchitis, otitis). at 5 years of age neuroblastoma was diagnosed. from the age of 6 years he started with severe skin infection , subsequently recurrent mucocutaneous aspergillosis was established based on skin biopsy and bacteriological studies. immunological investigations revealed persistent leukocytosis, hypereosinophilia, low level of igm and increased ige up to 5 000 iu/ml. so hyper ige syndrome was diagnosed and genetically confirmed when a large deletion of the dock8-gene was identified. stem cell transplantation was performed in 2012 when the patient was 11 y.o. one year later progressive multifocal leukoencephalopathy secondary to infection by polyomavirus jc was diagnosed. but after immunosuppressive therapy with cyclosporine awas suspended our patient's condition improved: the load of polyomavirus jc on plasma showed a decrease; mri brain was essentially stable ; immunological tests showed an initial improvement of subpopulations and proliferative response to mitogens; donor chimerism 100% stable. özdemir öner 1 ; bozdoğan sıla 2 1 department of pediatrics, division of allergy/immunology, sakarya university, medical faculty, adapazarı, türkiye. background: bronchiolitis and infantile asthma are the most frequent causes for typical wheezing signs in infants. however, when a physician comes across patients with recurrent wheezing are resistant to β2-agonist and anti-cholinergic therapy, known as atypical wheezing cases; he should investigate for hypogammaglobulinemia in these patients. aim: here, three cases are reported to make pediatricians aware of hypogammaglobulinemia, which is one of the reasons causing recurrent and persistent wheezing attacks during infancy and beyond. case presentations: case 1: 24 month-old girl presented to us with complaining of coughing and persistent wheezing. she has been having wheezing and breathing difficulty for the last 2 months after she got upper respiratory tract infection. her symptoms persisted even though she was using religiously nebulized salbutamol + budesonid therapy. before this episode, she had had 9 other wheezing attacks in her past medical history beginning from 2 months of age. in her family history, her father has asthma. physical examination revealed her breathing difficulty. ronchi as well as rales were heard on the auscultation of her lungs. at the fourth day of admission, she was given ivig 500mg/kg/dose. later, her symptoms did improve and not recur for the last 5 months. laboratory findings showed normal routine biochemistry, complete blood count and sedimentation rate. chest x-ray showed normal findings. echography was normal. ph-metry for reflux investigation was normal. sweat test was normal. in the serological evaluation: low igg level for his age (358 mg/dl) was detected at two different times. igg subgroups, igm (156 mg/ dl), iga (34 mg/dl) and ige (68) levels were within normal. case 2: 8-month-old girl came to our outpatient clinic with complaints of coughing and wheezing. at 2 months of age, she had urinary and upper respiratory tract infections. despite antibiotic therapy, wheezing persisted for 2 months and wheezing severity increased and it did not respond to β2-agonist therapy. thereafter, she was admitted to the hospital for 7 days and symptoms resolved. however, she came back to hospital due to recurrence of her symptoms in 10 days. in her family history, grandmother and her cousins have asthma. physical examination showed breathing difficulty. ronchi as well as rales were heard on her lungs. although salbutamol, ipratropium, antibiotherapy (clarithromycin) and anti-reflux therapies were given, her symptoms did not improve for 2 weeks. at the 15th day of admission, she was given ivig 500mg/kg/dose. later, her respiratory system symptoms did not recur for the last several months. once she was evaluated for persistent wheezing attacks during admission, biochemistry, cbc, esr were normal. chest x-ray and echography were normal. in serological evaluation: low igg level for his age (304 mg/dl) was detected at two different times. igg subgroups, igm (106 mg/dl), iga (34 mg/dl) and ige (<5) levels were normal. case 3: 20 month-old boy was brought to us complaints of having frequent lower respiratory tract infections (bronchiolitis). he was experiencing recurrent wheezing attacks almost every other week for the last 6 months. in past medical history, he was diagnosed with trisomy 21 and hypothyroidism at the 3 months of age. he went thru an operation for atrio-venticular septal defect. physical exam revealed dyspnea, tachypnea and wheezing. crackles were heard on the chest auscultation. abdominal, cardio-vascular and the rest of the examination were normal. when he was evaluated for frequent wheezing attacks in our outpatient clinic, routine biochemistry, cbc and esr were normal. chest x-ray showed normal findings. in serological evaluation: low igg level for his age (300mg/dl) was detected twice. igg subgroups, igm, iga and ige levels were within normal. he was given ivig 400mg/kg/dose. for the last three months, he did not have any lower respiratory tract infection. conclusion: the awareness of immunodeficiency among pediatricians has been greatly improved. recurrent respiratory tract infections are major infections in these patients. thi is a relatively common condition associated with infant hypogammaglobulinemia. in patients with recurrent and/or persistent wheezing symptoms during infancy and beyond, especially resistant to therapy, hypogammaglobulinemia should be excluded from possible diagnoses. background: the acquisition of new food allergy after transplantation or transplant-acquired food allergy (tafa) is usually reported in adults and rarely in children. tafa is described mainly after liver, but also after small bowel/intestinal, lung and heart transplantations. in different studies, the male/female ratio is equal. literature data suggest that children with tafa typically present within the first year after surgery and they are typically allergic to multiple foods. aim: tafa is generally characterized with allergy to multiple foods and increased level of total and/or spesific ige. here, a patient although who had normal total. ige and specific ige test results, he developed reaction to skin prick test for cow's milk is presented and his clinical presentation will be discussed. case presentation: 15 month-old-boy came to our allergy clinic with complaints of vomiting after drinking cow's milk and skin rush on the area where contact ed with chocolate. in his past medical histroy, left lateral segment of liver (donor was his mother) was transplanted to him when he was at 5 months. the liver donor was not recorded as having a history of allergic disease. methylprednisolone and tacrolimus immunosuppression were used after the transplantation, and tacrolimus therapy was continued for prophylaxis of chronic rejection. when he was at 7 months, family fed the patient with cow's milk but 3 hours later he began to vomit. he vomited five times in two hours. then, he developed constipation. rectal irrigation was used. then oral intake stopped for two days. he was thought to be having food protein induced enterocolitis. his vomiting complaints repeated after intake of formula and baby food which include grain. so he fed with special formula including short-chain peptides and free aminoacids and his symptoms improved. past medical history: extrahepatic biliary atresia was diagnosed at 3 weeks age with conjugated hyperbilirubinemia (according to scintigraphy and biopsy results). family history: his father has penicillin allergy and his aunt has asthma. at our outpatient clinic: height and weight were within normal percentiles. physical examination reevaled normal examination findings. laboratory findings: wbc was 4.420/mm3, with 21% neutrophils, 6% eosinophils and 69% lymphocytes. his hemoglobin was 8.2 g/ dl , platelet count was 280.000/mm3. total ige : <5 and immunocap specific ige against milk, grain and other classsic foods was <0.35. skin prick test results: saline: 0x0mm, histamine 4x4mm, fresh cow's milk:2x2mm, other food allergens (peanut, egg, fish, soybean, wheat): 0x0mm. conclusion: our patient seemed to have cow's milk allergy related to liver transplantation. laboratory investigations and clinical presentation of the patient did not look like typical ige-mediated food allergy, which is expected in tafa. patient history: 33 years old male patient, the first pneumonia at the age of 18 followed by 3 times pneumonia attacks/year, otitis media and sinusitis. hospitalization due to respiratory insufficiency caused by bronchiolitis obliterans and diagnosed with hypogammaglobulinemia at the age of 27. no consanguinity. patient findings at diagnosis (02/2008) had hepatospenomegaly, bilaterally cervical, supraclavicular axillar lap, osteoporosis, bronchiolitis obliterans organizing pneumonia, hyper igm, lower igg, iga, ige, low b-cell. no response to tetanus toxoid. i̇sohemagglutinin antib was ¼. cd40,cd40l, aid, taci, baffr, icos gene mutation were negative. protein electrophoresis revealed polyclonal igm increase, immunofixation was no clonality. lymph node biopsy result was available paracortical expantion . cd3(+) t and cd20 were positive. b lymphocyte distribution were normal. malignancy ruled out. no giant germinal centers. evaluation of bone marrow aspiration/ biopsy were abnormal localization of megakaryocytes, dismegakaryopoiesis, blasts in normal range. lymphocyte ratio 10% mostly consisting of cd3+ cells, cd20+ cells were rare, plasma cell ratio was 2%, amyloid negative. progression of patient, igm level and spnenomegaly were increased and patient had respiratory failure. splenectomy could not be done due to respiratory failure. than patient was treated with rituximab (375mg/kg/week). after rituximab therapy, lymph nodes and splenomegaly were (5 cm), regressed and igm level decreased (from 4390 mg/dl to 2040 mg/dl), increased effort capacity. after 7 month after rituximab therapy splenomegaly and igm level were progressed. splenoctomy was performed. pathological evaluation of the spleen malignancy ruled out. current igm level is 6300 mg/dl. conclusion: the most convinient scenerio for this patient would be a csr defect of unknown etiology presented as cvid. the recent litarature revealed genetic defects of some molecules operating in dna repair pathways such as msh2, msh4, msh5, msh6, mlh1, rad50, rad 52, nbs1 leading to csr abnormality and impaired antibody maturation. cvid is characterized by hypogammaglobulinemia, recurrent respiratory and gastrointestinal bacterial infections. good's syndrome(gs) is a thymoma-related immunodeficiency and characterized by hypogammaglobulinemia, decreased b cell and variable deficiencies in cell-mediated immunity. case: a 47-year old male patient presented with a palpable anterior chest wall mass. in 2005, he was diagnosed with rheumatoid arthritis. laboratory showed hypogammaglobulinemia and all autoantibodies were negative. cd19 + , cd20 + , cd22 + cells were <1%. bone marrow(bm) examination demonstrated low cd20 + b-lymphocyte and increase in cd3 + t cells. tuberculin skin test was positive. t-cell proliferative response was normal. immunizations with h.influenzae type-b and tetanus toxoid revealed no response. anti-b titer was low. taci, btk and icos mutattions were negative. ultrasonography showed hepatosplenomegaly. mild edema, mononuclear cell infiltration (suggested the early stages of extrahepatic biliary obstruction) were detected on liver biopsy. in the medical history, he had reported chronic sinusitis, otitis and bronchitis dating back to 3rd decade of life. at age 38, he underwent surgery for thymoma. in follow-up, the computed tomography showed soft tissue mass on the anterior chest wall and pathology was thymoma. discussion: frequent respiratory infections with encapsulated pathogens beginning at the age of 30, lack of opportunistic pathogen infections, presence of hepatosplenomegaly and rheumatoid arthritis, bm examination findings and successful management of infections with ivig therapy all indicated a diagnosis of cvid. the coexistence of cvid and thymoma has been reported in the literature. introduction: common variable immunodeficiency (cvid) is a primary immunodeficiency disorder characterized by impaired b cell differentiation with defective immunoglobulin production. it has heterogeneous clinical manifestations including recurrent infections, chronic lung disease, autoimmune disorders, gastrointestinal disease, and susceptibility to lymphoma. patients with this disorder have evidence of immune dysregulation leading to autoimmunity. autoimmune cytopenias are a more common presenting disorder in children and may be the initial manifestation of the disease. we want to present a patient presenting with autoimmunue hemolytic anemia and finally diagnosed as cvid. case: a 15 years old, previously healthy female patient applied to emergency clinic with complaint of paleness, light headedness and yellow discoloration of her scleras. her history was not compatible with blood loss. she denied having melena, hemotochezia or hematuria. her mensturation history was also normal. her hemoglobin level was 7.2 gr/dl, reticulocyte count was % 4.17, complete blood count was otherwise normal. direct coombs was (+++). for the differential diagnosis of immune hemolytic anemia, viral serology, ana and anti-dsdna were studied, but the results were normal. igg, igm and iga levels were lower than normal normal for her age. other causes of hypogammaglobulinemia were excluded. blood group was arh (+), blood isohemagglutinin were anti a(-) and anti b (-/1+). lymphocyte subsets were also studided. as the patient has reduced immunoglobulin levels with normal lymphocyte subset analysis, she presented after puberty and other defined immunodeficiency states were excluded, she was diagnosed as cvid and monthly immunoglobulin replacement therapy was planned. she had aseptic meningitis after her first ivig transfusion. the ivig preparation was changed with another trade and she did not have any problem during the following treatments. it has been one year since she was diagnosed and she did not have any other medical problems. conclusion: the diagnosis of cvid requires decreased igg, igm and iga levels are also reduced but are less valuable for diagnosis. ige level is checked to exclude other disorders. igg subclass determinations are indicated if antibody titers are decreased but immunoglobulin levels are near normal. hypogammaglobulinemia secondary to other disorders should be excluded. autoimmune conditions can be the presenting signs/symptoms in cvid. autoimmune hematologic disorders may precede, present at the time of diagnosis or develope during the course of cvid in approximately one-half of the patients with autoimmune problems. selective immunoglobuline deficiency is an uncommon dysgamaglobulinemia, in which immunoglobuline levels except igm level are normal. it can be primary or secondary to cancer, autoimmune diseases, gastrointestinal system diseases and immunosuppressive therapy. patients can be asymptomatic or have recurrent infections, asthma, angioedema, autoimmune diseases, celiac disease and bronchiectasis. allergic diatheses are the second commonest presentation of selective igm deficiency. in this presentation, we report a case with asthma and angioedema who has selective immunoglobuline m deficiency. a 16 year old male patient who has been diagnosed with asthma for 12 years with a well controlled asthma for 2 years presented with labial angioedema. he had labial angioedema daily without antihistamines. he did not have any suspected food or drug allergy. he did not have family history of angioedema. physical examination was normal under antihistamine therapy. laboratory evaluation revealed a 4.3% percentage of eosinophils. absolute lymphocyte count was 2700, absolute neutrophil count was 7000 cells/mm 3 . immunoglobuline e value was 260 ng/ml, levels of immunoglobuline g and a were within normal limits for age. immunoglobulin m value was 28.7 mg/dl (73-448). anti a was 1/8, anti b was ½ positive, antihbs was above 1000 mlu/ml. lymphocyte subsets were normal. because of the continuous usage of antihistamines, prick tests could not be done. levels of d1 was17.8, d2 was12.4 ku a /l. thyroid functions, antitpo, c3, c4 and c1 esterase inhibitor values were normal. antinuclear antibodies and antitransglutaminase lga was negative. immunoglobuline m value of his father was normal, immunoglobulin m value of his brother was 53.50 mg/dl (88-322). selective immunoglobuline deficiency is a rarely seen dysgamaglobulinemia. it was reported in children with asthma, but it was not reported in children with angioedema. it can have value in the clinical evaluation of patients with angioedema. cukurova university, department of pediatric allergy and immunology, adana, turkey a patient who is at age of 11 years at present and who has been followed up at the our clinic with xla diagnosis presented with pain in his ankles and wrists and swelling of his right knee. he also was suffering from skin tightening of the lower extremities. his physical examination revealed arthitis of the right knee and sclerotic changes were detected in the skin. skin biopsy was performed and it revealed morphea (localized scleroderma). after the diagnosis of morphea and arthritis, ivig 2 gm/kg and nsaid were applied. following the treatment skin findings and arthritis resolved, however approximately two months later liver enzymes were detected to be high in his routine control. liver biopsy performed to clarify the aetiology of elevated liver enzymes was reported as autoimmune hepatitis. in addition to ivig 2 gm/kg, budenofalk 6mg/day was started. after 3 months of treatment, his liver enzymes normalized. currently he is being treated with ivig monthly and ursofalk daily. patients with xla typically present with recurrent bacterial infections and it might be associated with some autoimmune diseases. there are not any reports indicating an associaton of autoimmune hepatitis and scleroderma with xla. bruton agammaglobulinemia is an inherited immunodeficiency disease caused by mutations in the gene coding for bruton tyrosine kinase (btk). the median age at the diagnosis of the antibody deficiency is about 4.6 years in turkey. here, we report a case of bruton disease presenting with recurrent cervical abscess at two months old infant. a 2-months-old boy was firstly hospitalized for the treatment of the right cervical abscess at 25 days old. after the recurrence of swelling on the cervical area, the patient was referred to the admitted to hospital secondly. there was no consanguinity between parents and, his family history was unremarkable except four of the mother's cousins, died because of unknown etiology in infancy. on his physical examination, his weight, height andhead circumference were normal range by age. there were no visible tonsils. there was a palpable, mobile 1x1 cm mass on right upper cervical area. the molecular analysis of the causal gene for bruton's tyrosine kinase (btk gene) revealed the mutation in exon 19. this mutation g.67662delg (c.1750 + 57del) leads to the changes of amino acid order in the protein with the subsequent changes in activity of btk (at the level of dna: substitution of glutamic acid (p.glu507*) causes non-sense mutation leading to the formation of stop codon with premature end of dna transcription to cause stop codon. after initiating the intravenous immunoglobulin with antibiotics, the cervical mass was getting smaller in a short period, and had not observed again. neutropenia was improved within the 3 months. this case is an important example to diagnose bruton disease in early life. it demonstrates that maintaining a high level of clinical suspicion is essential for the diagnosis of bruton disease in a child with recurrent cervical masses. elif azarsiz; neslihan edeer karaca; guzide aksu; necil kutukculer ege university faculty of medicine, department of pediatric immunology, izmir, turkey transient hypogammaglobulinemia of infancy (thi) is characterized by recurrent infections and reduced serum immunoglobulin levels. typically, thi patients recover spontaneously, mostly within 30-40 months of age, but sometimes recovery may be delayed until 5-6 years. the use of intravenous immunoglobulin (ivig) as an alternative to antibiotic prophylaxis remains contraversial also in symptomatic patients. some authors believe that ivig therapy may cause a delay in the maturation of the humoral immune system because of the interference from passively transfered antibodies. the aim of this study was to investigate the effect of ivig replacement on recovery from immunodeficiency in these patients. 43 patients (65%) received ivig therapy while 23 patients (34.8%) showed spontaneous normalization without ivig. the percentages of patients who had more than six times the number of febrile infections in a year decreased from 91% to 21% in the group receiving ivig treatment. at admission, before being recruited to ivig therapy, serum immunoglobulin g (igg) levels and anti-hemophilus b (hib) antibody titers were found to be significantly low in cases who were selected for replacement. the percentages of patients who did not have protective levels of anti-hib, anti-rubella or anti-rubeola-igg were also significantly high in ivig cases. there was no statistically significant difference in the age at which igg levels normalized between both groups. patients in the ivig group and non-ivig group reached normal igg levels at the age of 42.9 ± 22.0 and 40.7 ± 19.8 months, respectively. in conclusion, ivig infusions do not cause a delay in the maturation of the immune system in thi patients. the very low and non-protective specific antibody responses against previously applied vaccines are important factors to consider when selecting patients for ivig therapy. zoltán ellenes-jakabffy 1 ; ibolya kovács 1 ; mihaela bătăneanţ 2 ; maria cucuruz 2 ; margit şerban 2 ; lászló maródi 3 1 department of pediatrics, clinical city hospital oradea, romania. objective: to study the amplitude of the chronic inflammatory phenomena: atopic and autoimmune diseases, as well as their associations in pediatric sigad patients and to study the sigad patients' family history (1 st degree relatives) for pids (primary immunodeficiencies). methods: retrospective analysis of the clinical and laboratory records of pediatric sigad patients diagnosed between 1998 and 2013 at the departments for pediatric immunology of the medical universities of debrecen (hungary), oradea and timişoara (romania). results: out of 148 patients, we found out 74 (50%) with atopic diseases, mostly with respiratory localizations (asthma and allergic rhinitis), 16 (10,81%) patients with autoimmune diseases (jia, psoriasis, celiac disease, thyroiditis etc.) and other 15 patients without clinical symptoms of autoimmunity but constantly elevated autoantibody levels. there were 7 patients with coexistent atopic and autoimmune diseases. regarding the family history, we identified 8 families with multiple cases of pids : 6 with multiple sigad, 1 with sigad and cvid, 1 with sigad and higms (hyper igm syndrome). conclusions: the chronic inflammatory phenomena are present in the majority of the studied sigad patients: symptomatic atopic diseases in 50%, symptomatic autoimmune diseases in 10,8%, that means a cumulative 61%. there are comorbid associations within the atopic and autoimmune disease groups and also between the two groups. sigad is the most common primary immunodeficiency. it's prevalence is 1/400-1/800. aim was to assess the prevalence of co-morbidity in patients with sigad in latvian pediatric population and the analysis of some immunological abnormalities in these patients. the study included 120 patients 4 -18 years old. medical charts have been reviewed. into account were taken the data, which were made at time of diagnosis. patients were divided into 4 groups: 1 st -patients with allergic disease, 2 nd -patients with autoimmune diseases, 3 rd -patients with infectious diseases, 4 th -asymptomatic patients or patients with sigad unrelated diseases. patients with multiple co-morbidities of various disease groups were not placed in any of these groups. each patient group was divided by age: 4 -6 years, 7 -13 years 14 -18 years. results.58.4% were boys and 41.6% girls. sigad in 53.9% of the cases were diagnosed before 7 years of age (inclusive). 85% of the patients had co-morbidities: allergic (30%), autoimmune (37.5%) or infectious diseases (17.5%). patients 4 -6 years old: children with infectious and autoimmune diseases have 1.3 times greater igg than healthy children or children with allergic diseases (p < 0,05); children with autoimmune diseases has 1.9 times more cd8 cells than children with allergic diseases (p < 0,05).; children with infectious diseases have 2.1 times lower absolute number of cd25 cells than children with autoimmune diseases (p < 0,05); patients 7 -13 years old: children with infectious diseases have 1.5 times more absolute number cd25 cells than children with allergic diseases (p < 0,05). patients 14 -18 years old: children with autoimmune disease have 1.6 times higher cd4/cd8 index than children with infectious diseases (p < 0,05) karakina m. l. 1,2,3 , tuzankina i. a. 1,2 , vlasova e. v. 2 1 introduction: antiepileptic drugs are known to cause immunosupression in some cases. levetiracetam is an anticonvulsant medication used to treat epilepsy in the posttraumatic seizures. we report a rare case of hypogammaglobulinemia and b cell aplasia associated with levetiracetam treatment. case report: a 45-year-old female was operated for pituitary tumor with transnasal surgery and required second operation for postoperative rhinorrhea. after operation, menengitis developed and antibiotic treatment was administrated. however, there was a poor response to this treatment after one month and craniotomy was performed due to the diagnosis of "shimic menengitis". her seizures occurred as a postoperative complication and levetiracetam was initiated. after the 9-month follow-up, the findings of menengitis could not be controlled with antibiotherapy. she was referred to our immunology department for chronic menengitis with fever, headache and high cerebrospinal white blood cell count. results: in her clinical evaluation, it was learned that she had previously healthy. laboratory examination showed that decreased levels of igg 778 mg/dl (normal: 913-1884) and iga 97 mg/dl (n: 139-378). peripheral blood flow cytometric analysis revealed the absence of b cells (cd19+ b cells; <1%). t cell subsets and natural killer cell numbers were normal. neutrophil function, chemotaxis, phagocytosis and oxidative burst activity were found to be normal. isohemaglutinin titer, levels of pneumococal and tetanus specific igg antibodies were also normal. antiepileptic drug was discontinued after epileptic seizure was controlled. b cells gradually increased three weeks later and returned to normal within two months (cd19+ b cells: 9.8%). conclusion: patients requiring levetiracetam should have serum immunoglobulins measured and lymphocyte subsets analysis performed if they experience recurrent or persistent infections. mustafa gulec 1 ; fevzi demirel 1 ; ugur musabak 1 ; ozgur kartal 1 ; sait yesillik 1 ; abdullah baysan 1 ; ergun ucar 2 ; osman sener 1 1 gulhane medical school, division of immunology allergic diseases, ankara, turkey. 2 gulhane medical school, department of chest diseases, ankara, turkey. introduction: common variable immune deficiency (cvid) may present with several clinical manifestations involved in different organs and tissues in adults. we present a case with a history of chronic cough for more than twenty years and further diagnosed as cvid. case: a 49-year-old male who works in a chemistry lab admitted to our clinic with a history of frequent upper respiratory tract infections for more than 20 years. he also had intermittent diarrhea symptoms and his respiratory symptoms have been worsened since 2009. he had been hospitalized due to pneumonia and empyema several times. he had undergone left lower lobectomy due to bronchiectasis in 2012. he had been admitted to intensive care unit due to worsening of his medical condition. he was further diagnosed with cvid and ivig treatment was initiated. he is currently under remission with monthly ivig treatment and without any respiratory or gastrointestinal symptoms. discussion and conclusion: cvid is a clinical disorder in which the humoral part of the immune system is affected. most frequent presenting symptoms belong to respiratory, gastrointestinal systems and skin. however, due to the organ specific physical examination and lack of awareness, the diagnosis is frequently overlooked. in our case, frequent upper respiratory infection, loss of weight, diarrhea, bronchiectasis and empyema with unknown etiology are the most informative clinical signs. medical history is the most important part of patient evaluation. immunoglobulin replacement therapy is a basic treatment in primary immunoglobulin deficiency disorders. immunoglobulin substitution can be given intravenously (ivig) or subcutaneously (scig) for patients with antibody deficiency. both of these treatments are effective in prevention and cure of infections, although differences in advers events profile and patients' quality of life can be seen. the authors describe here their experiences in switching patients from ivig treatment to scig and a few years observation of scig therapy of patients with antibody deficiencies. sirje velbri 1 , mirja varik 2 1 tallinn children's hospital, tallinn, estonia. 2 north-estonian regional hospital, tallinn, estonia. j clin immunol (2014) 34:696-747 antibody deficiencies are the most common group of primary immunodeficiencies. the main hallmark of antibody deficiencies are recurrent infections but the patients have also higher risk of autoimmune and allergic diseases. we analysed retrospectively the frequency and character of autoimmune diseases in 159 patients (98 children and 61 adult patients) with primary antibody deficiencies. there were ana-lysed 2 patients with xla, 24 patients with cvid, 65 patients with selective iga deficiency, 26 pa-tients with iga/igg subclass deficiency and 42 patients with isolated igg subclass deficiency. autoimmune diseases were found in 39 patients (24,5%) besides in 17 children (17%) and in 22 adult patients (36%). in two boys with xla there was not found autoimmune diseases but in patients with other forms of antibody deficiencies in 12-57% of cases. autoiimmune diseases were found more often in iga/igg subclass defi-ciency (57%) than in other forms of antibody deficiencies (0-25%). the spectrum of auto-immune diseases differed in adults and child-ren and in different forms of antibody de-ficiencies. immune thrombocytopenia was found in adult patients with cvid or igg subclass deficien-cy, autoimmune connective tissue disorders in iga and iga/igg subclass deficiency. in children there was found mainly thyroiditis, diabetes i type and juvenile arthritis. rostov state medical university. research institute of clinical immunology, rostov-on-don, russia. primary immunodeficiency (pi) is currently one of most important genetically determined immunological clinical pathology which is hard to manage . among different types of pis almost 60 % of cases occurs due to a deficiency in antibodies production. injections of immunoglobulin are current standard in the management of pi. however this treatment is expensive, often is hard for patients, and frequently has limited effectiveness. substitution of immune proteins frequently is inefficient for treatment of severe infectious conditions in pi patients. we investigated maturation, activation and differentiation of immune t-cells in the dynamics of ivig replacement therapy. we observed patients with with cvid (7) and xla (5 people) over a year of regular replacement therapy. we have found that recovery of the humoral component does not affect the maturation and the differentiation of t-cells, but can reestablish activation and regulatory properties. these changes are more evident among patients with cvid, which immuneregulatory and functional potential reestablish faster. obviously, the effects tlymphocytes increase the effectiveness of replacement therapy in patients with xla, cvid common variable immunodeficiency (cvid) is one of the most frequent symptomatic primary immunodeficiencies. the diagnosis is based on significantly decreased levels of immunoglobulins, with poor or absent response to vaccines and by excluding other defined causes of hypogammaglobulinaemia. as suboptimal antibody production is mainly due to b cell defects, therefore, we aimed to study lymphocyte subgroups of cvid patients and to compare the patterns with the clinical presentation in these patients. six adult patients with cvid diagnosis were studied. lymphocyte subpopulations were determined by flow cytometry. for b-cells subgroups cd3, cd27, igm and igd reagents w e r e u s e d . a l l l y m p h o c y t e s w e r e g a t e d f o r f i n d i n g cd19 + cd27 + memory b cells and from this population switched (cd19 + cd27 + igd -igm -) and non-switched (cd19 + cd27 + igd + igm + ) memory b cells were counted. all patients had normal levels of total lymphocyte count and absolute counts of cd4 + , cd8 + and cd16 + /cd56 + cells were also normal in all patients. only one patient showed low levels of cd19 + cells levels. according to paris classification scheme the patients could divided into two subgroups: mb0 and mb1. although almost all our cvid patients had normal number of total b cells, most of them showed reduced number of memory b cells and/or switched memory b cells. all of our patients had very low or absent level of class-switched memory b cells, therefore can be possible associated with a higher risk of granulomatous disease and splenomegaly. detailed investigation of b-cell phenotypes can better characterise cvid patients and can provide more information about possible clinical outcome. background: common variable immunodeficiency (cvid) is one of the most frequent symptomatic primary immunodeficiencies, often related to spectrum of infectious and autoimmune diseases. in cvid patients wide spectrum of gastrointestinal disorders, including infections, are frequently seen. inflammatory bowel disease, helicobacter pylori infection, giardia lamblia infection, campylobacter or salmonella infection have been reported. however, abnormal liver function test and liver disease are found in approximately 10% of cvid patients. case history: a 47-year old female patient was admitted to infectious disease department due to recurrent pneumonia and purulent rhinosinusitis. blood analysis confirmed panhypogammaglobulinaemia with impaired responses to vaccinations, elevated liver function tests and anti-hcv positivity, interpreted as old and passed infection. due to cvid intravenous immunoglobulin substitution was started. however, liver function tests remained elevated and with hcv-rna analysis high hepatitis c viral load was detected. chronic hepatitis c virus infection was diagnosed and treatment with peginterferon α-2a and ribavirin was started. conclusion: our case emphasizes the need for hcv-rna and hbv-dna analysis in patients with hypogammaglobulinaemia, as the serological detection is impaired and prognosis for chronic hepatitis in immunodeficiency patients is poor. outpaitent clinic of clinical immunology and allergology, east tallinn central hospital, tallinn, estonia background: primary antibody deficiencies are the most frequent primary immunodeficiencies. recurrent respiratory tract infections may result in permanent lung damage in 20-40% of patients, most commonly presenting with the development of bronchiectasis. we aimed to evaluate the lung function and radiographic pulmonary changes in our patients with primary antibody deficiency. material and methods: we reviewed the records of adult patients with a confirmed diagnosis of primary antibody deficiency at our clinic. 10 patients were included in this analysis in whom ct scan was performed during the last 24 months, comprising 8 patients with cvid and two with igg subclass deficiency (median age 33 years; range 19-68 years; 60% females). all patients were on regular immunoglobulin replacement and one of the patients was on prophylactic antibiotic at the time of analysis. mean trough levels of igg were calculated based on the results measured during the last 12 months prior to ct scan. the spirometry was performed according to published protocols. results: all patients demonstrated normal spirometry data based on fev1 and fvc. two patients had slightly lower mmef rates, however, the changes were not associated with higher rate of infection nor changes in ct scan. none of our patients had bronchiectasis or atelectasis. among parenchymal changes fibrotic lines were most frequently detected. in two patients ground glass due to fibrosis was noted. mean immunoglobulin trough levels in our patients were between 4.9-8.6g/l, with the median of mean trough levels of all our patients 6.9g/l. when comparing the trough levels to lung function and ct scan results, no significant associations were seen. conclusion: no remarkable changes in lung function or chest ct scan in our patients with primary antibody deficiency were noted. as regular immunoglobulin replacement therapy could have prevented the development of permanent lung damage. rationale: patients with primary immunodeficiencies (pi) (n = 83) were treated subcutaneously (sc) with immunoglobulin g (ig) preceded by recombinant human hyaluronidase (ighy) at 3 or 4 week intervals based on their previous intravenous ig (igiv) dose. we report data for a subset of 59 patients aged ≥18 years from the final efficacy, safety and tolerability data of a pivotal phase 3 trial of ighy. methods: patients received igiv for 3 months at prestudy doses and frequencies. subsequently, ig 10% was administered sc, at 108% of the weekly equivalent of the iv dose, following rhuph20 infused through the same sc needle at a dose of 75 u/g igg. after a ramp up from a 1-to a 3-or 4-week dose interval, 59 patients received ighy every 3 4 weeks for 12 months. the primary efficacy endpoint was the mean rate of validated acute serious bacterial infections (sbis) per patient-year during the efficacy period. results: fifty-nine patients received 985 ighy infusions; 97.8% were completed without administration changes due to tolerability concerns or adverse events (aes). median infusion sites/month was 1.13. the temporally associated systemic ae rate was 0.20/infusion (ighy) vs. 0.33/infusion (igiv). the local adverse drug reaction rate was 0.286/infusion. the annual sbi rate was 0.00 and 3.10/patientyear for all infections. conclusion: in adults with pi, ighy was effective in preventing infections. the majority of patients received full 3-to 4-weekly doses of ig using a single sc site with good local and systemic tolerability. rationale: in a pivotal phase 3 trial of facilitated-subcutaneous (sc) infusion of human immunoglobulin g (igg), 10%, and recombinant human hyaluronidase (rhuph20) (ighy) in patients with pi, rhuph20 permitted most patients to have a single-site infusion (every 3-4-week igg dosing) with bioavailability and infusion rates comparable to intravenously administered igg (igiv). we report the final analysis of the long-term extension of the initial phase 3 study, with a duration of up to 3 years of treatment with ighy plus additional follow-up. methods: sixty-six patients who completed the initial phase 3 study enrolled in the extension study. patients continued their pre-study ighy dose/frequency every 3-4 weeks. after 3 months, some patients switched to 2-week dosing to evaluate effects of shorter ighy interval on trough igg levels. from the final analysis, tolerability and safety after up to 3 years of treatment were evaluated. the ighy part of the extension study was followed by a 24-48 week observation period during which patients received igg 10% administered iv, or sc weekly without rhuph20. results: in the extension study, 66 patients were treated with ighy and 14 discontinued prior to safety follow-up. following discontinuation of rhuph20, 48 patients switched to follow-up. no patients withdrew due to ighy-related reactions (adrs). no serious adrs related to ighy were reported. the maximum ighy exposure for the initial and extension studies combined was 3 years (total exposure = 187.69 patient-years; n = 2959 ighy infusions); during this time, there were no clinically observable long-term changes in the skin or sc tissue. the rate of temporally related systemic adverse events (aes), excluding infections, was 0.159/ infusion. the rate of all local aes was 0.103/infusion. of the 1600 ighy infusions administered in the extension study, 97.9% had no administration changes (rate reduction, interruption or discontinuation) due to tolerability concerns or adverse events. the annual rate of all infections under ighy treatment was 2.86/patient-year. reducing the dosing interval from 4 to 2 weeks (same monthly dose) resulted in a 13% increase in trough igg levels. thirteen patients had at least 1 non-neutralizing anti-rhuph20 antibody titer of ≥1:160 with no associated aes; no patients had neutralizing anti-rhuph20 antibodies. conclusions: in the extension study, ighy was well tolerated and effective, with no serious adrs for treatment periods up to 2 years. over a maximum 3-year ighy exposure (for an individual patient) in the initial phase 3 and extension studies combined, no long-term changes in skin or sc tissue were observed. the rates of infections and adverse reactions were stable or decreased over the course of the two studies, suggesting no increased risk with continued exposure to ighy. rationale: we report interim analysis of safety, tolerability and pharmacokinetics (pk) of igsc 20% in patients with primary immunodeficiencies (pi) aged ≥2 years in europe. methods: epoch 1: igsc 16% or intravenous ig 10% (igiv) administered at pre-study doses every 3 months. epoch 2: igsc 20% administered 1 time per week for 12 months at epoch 1 doses. serum igg trough levels are maintained at >5 g/l. the primary endpoint is validated acute serious bacterial infection (sbi) rate. results: at the interim analysis in october 2013, 49 patients started the study. during igsc 20% treatment (n = 48), 1 acute sbi episode (pneumonia, moderate in severity) was reported. the infection rate per patientyear was 4.3 (igsc 20%). there were no serious adverse events considered related to any treatment. the rate of local adverse drug reactions (adrs) was 0.055/infusion and all were mild in severity; no severe systemic adrs were reported with igsc 20%. of 1812 igsc 20% infusions, only 0.4% required slowing or interrupting the administration rate. mean serum igg trough levels were 9.2 g/l (igsc 20%, n = 46), 9.1 g/l (igiv 3-week interval, n = 4) and 7.7 g/l (ivig 4-week interval, n = 25). conclusion: igsc 20% provided an effective and well-tolerated therapy, with no dose adjustments needed from pre-study ig dose. this study is ongoing to confirm results over 12 months. it is well known, that intravenous immunoglobulin (ivig) is the main therapeutic modality in b-cell primary immunodeficiencies (pid), it decreases mortality and morbidity in these patients dramatically. yet, it is also well known that all ivig products are very expensive, especially considering life-time use and almost normal life expectancy in these patients, if treated correctly. irregular treatment and problems with insurance/state coverage of ivig in some countries stems from this. goal: the goal of our study was to compare medical and other costs, related to the disease in patients with humoral pids with or without ivig treatment. patients: 21 patient with b-cell deficiencies (73% x-linked agammaglobulinemia, all genetically confirmed, 27% common variable immunodeficiency). the age of patients varied from 2 to 16 years. methods: we analyzed medical and other disease-related costs during 2 years preceding the diagnosis (without ivig therapy) retrospectively and during 2 years on regular ivig therapy. the costs included those incurred by the state (hospitalization, home visits, emergency calls) and by the parents (costs of drugs, private consults, etc). the state costs caused by parents missing work were also considered. for standardization purpose for calculation we used the prices for the end of 2013. results: the patients analyzed fell into 3 different categories: 1. the 1st group -(76% of patients studied) -patients with several severe infections before therapy, some chronic conditions as a result of those. age of diagnosis varied from 2 years to 16 years (with average time to diagnosis 5 years). in this group costs of ivig treatment were 2,5 times higher than before the diagnosis (fig. 1 ). 2 nd group(10% of all). patients with late diagnosis (average time to diagnosis 7 years), who had multiple severe infections before the ivig treatment and acquired serious chronic lung complications due to it. in this group the costs before the treatment were higher, than on ivig treatment. 3. very early diagnosis -within the first year of life (mostly because of preceding family history) (14%). the comparative analysis was not possible, but it was noted that these patients had no history of serious infections before of while on ivig therapy. the only additional costs, besides ivig, were related to bad venous access, requiring occasionally 1 day in-patient hospitalization for ivig infusion. as expected, in all 3 groups, the number of infectious episodes, the number of hospitalizations (fig. 2) and doctor visits (fig. 3) after beginning of regular ivig treatment dropped dramatically. we also followed 2 patients with xla, who did not receive ivig therapy because of social aspects. both patients died from severe infections. we evaluated this fact in economical prospective: in russia one worker, who works continuously and retires at 60 years of age brings about 130 mln roubles into the state budget (when costs for schooling and routine medical care are subtracted). if one supposes that an xla patient have been diagnosed very early in life, did not form complications prior to therapy and was on regular ivig therapy for life, this sum equals to 160 years on ivig. conclusions: regular ivig therapy not only leads to reduction of infectious episodes, hospitalizations, and as a result improved quality of life. in some cases it even brings down the disease-related costs, incurred by the medical system and the family, and is economically advantageous for the state. introduction: replacement of immunoglobulins is a standard therapy for patients with primary immunodeficiency disease (pidd) characterized by primary antibody deficiency (pad). this poster represents our clinical experiences of initiation of home-based treatment with subcutaneous immunoglobulin (scig) with the patients diagnosed with primary variable immunodeficiency (cvid). case report: the patient (age 30) has been treated at our immuno-allergy outpatient clinic since 2005 with the diagnosis of hypogammaglobulinemia (igg, iga, igm) with normal b cell count, withsusp. cvid. with the repeated administration of intramuscular and intravenous immunoglobulins (ivig, imig) repeatedly occurred serious adverse reactions, which resulted in discontinuation of the replacement therapy. in february 2012 the health condition of the patient worsened due to recurrent bacterial respiratory infections. there was a progressive decrease of serum concentrations of immunoglobulins (igg 2,1 g/l, igm 0,25 g/l, iga 0,23 g/l). the patient was admitted to the intensive care unit of the 1 st internal department, university hospital bratislava, for a subcutaneous immunoglobulin replacement trial. despite serious adverse reactions with previous administration of several types of immunoglobulins, there have not occurred any clinically relevant side effects. conclusion: compared with im or iv formulations and administration, for selected patients, scig is better tolerated, clinically efficacious, safe, and appreciated by the patients. background: common variable immunodeficiency (cvid) is primary immunodeficiency (pid) classically viewed as antibody deficit. although, cvid is considerd to be a humoral immunodeficiency, approximately 40% of cvid patients have low t-cell counts or abnormal tcell function. despite adequate immunoglobulin replacement patient morbidity and mortality is variable and a number of complications are not those typically seen in pure antibody pid e.g. xla. so, t-cell rather than b-cell phenotype could determine outcome in patients with cvid. many patients with cvid have clinical history suggestive of allergic respiratory disease, but prevalence of asthma and role of atopy have not been well established. apart from recurrent infections and their sequelae, cvid patients suffer from other disease-related complications in up to 45% of the cases. about 25% have onset before the age of 15 years. aims: 1) to present one more case of tadolescent with cvid and allergic asthma, 2) emphasise ultimate need of collaborative network of primary immunodeficiency centers. case report: parents of 12,5 y boy were sure that "something was wrong" with their son and were seaking for problem solution for many years. since age 3, child had frequent respiratory infections. adenoidectomy and tonsillectomy were performed at age 6. sinuitis was diagnosed several times. boy was complaining of fatique for a long time. last several years, his main problems were fever (max 41 c) usually lasting 5 days till 2 weeks, accompanying running nose, coughing, conjunctival problems; intermittently headache (lasting for a few hours till all day). oral aphtae were present almost every two weeks. he was incompletely vaccinated (bcg, and once diteper). morbilli and varicellae infections passed without complications. in jan 2012 he was diagnosed as allergic asthma in sarajevo (allergy to pollen, soya, nuts and antibiotics ("ceclor" and "pancef"). in february 2012 was admitted at children's hospital sarajevo for suspected primary mmunodeficiency. he had slightly lower levels of igg and iga (twice measured), normal ige and decreased number of t helper ly. due to suspected pid, boy was checked up in two nearest regional pid centers : hypogammaglobulinemia was confirmed (igg 4,7, iga 0,24, igm 0,46, as well as deficiency of igg1, igg2 and igg3). flow cytometry showed slightly raised concentration of lymphocites b (cd8), slightly raised number of nondifferentiated b cells. cvid was suspected but not proved. in july 2012 child visited center for primary immunodeficiencies in munchen, germany, were he was diagnosed ad probable cvid on the basis of hypogammaglobulinemia, lower levels od switch memory b cells, normal number of t-cells, positive antibodies to vaccinations and overcome infection (morbilli, varicellae). allergic asthma was additionaly confirmed in specialised pediatric pulmology hospital in germany (abnormal spirometry, normal ige, positive skin prick test, abnormal fractional exaled nitric oxid test, incipient brochiectasies due to asthma confirmed by high -resolution lung ct scan). low human immunoglobulin replacement was started (200 mg/kgbw) as well as antiasthmatic therapy (inhalatory steroids, antihistaminics). excellent therapeutical response were achieved : after one 1,5 y follow up, we can confirm patient has excellent general condition, no subjective symptoms, no tiredness, no severe infections. conclusion: diagnosing cvid is challenging task and quite often could be "per aspera ad astra". there is ultimate need of collaborative work of primary immunodeficiency network aimed of diagnosing patients on time. cvid patients with history suggestive of allergic asthma, are negativne on traditional tests, additional test designed to identify allergic asthma might be conducted. common variable immune deficiency (cvid) is a heterogeneous syndrome characterized by hypogammaglobulinemia, recurrent infections, immune disregularity (autoimmunity, autoinflamation) and propensity to malignancies. in the us report, 8.2% of cvid patients had a lymphoid malignancy, and cancers of other sorts developed in 7% of patients. it is not clear why cvid patients have higher risk of malignancy but chronic antigenic stimulation, chronic inflammation and increased chromosomal radiosensitivity may be the cause. cvid patients with higher igm level, reduced or absent b cell numbers, cd4 t cells lower than 200 and pli phenotype have higher prevalence of malignancy. allergy and clinical immunology department of rasool e akram hospital has registered 47 cvid patients. mean age of the onset of cvid symptoms was 11.2 years. mean diagnosis age was 20.2 years with mean diagnostic delay of 9 years. mean follow up time was 7 years (minimum 0.5-maximum 23 y). malignancy occurred in the follow up of 6 patients (13%). one patient had two different malignancies (breast cancer and gi adenocarcinoma). malignancy risk per case was 15%. hodgkin's lymphoma was the most common type (43% of cancers common variable immune deficiency is a heterogeneous syndrome characterized by hypogammaglobulinemia , recurrent infections , auto immunity and auto-inflammation . more than 25 % of cvid patients have auto immune complications and among them, auto immune cytopenia is the most common. cvid patients with higher igm levelhigher 21 low b cellslower t reg levelslower cd4/cd8 ratio and lower class switched memory b cells have higher prevalence of autoimmunity. allergy and clinical immunology department of this hospital has registered 47 cvid patients. mean age of onset of cvid symptoms was 11.2 years. mean diagnosis age was 20.2 years with mean diagnostic delay of 9 years. mean follow up time was 7 years (minimum 0.5 maximum 23 y). autoimmunity was detected in 23 cases (49 %) and 12 cases (26 %) had more than one autoimmunity. autoimmunity was the first symptom of cvid in 15 percent of cases. autoimmune disorders should be considered in the follow up of cvid patients. igg is major immunoglobulin and classified subgroups as igg1, igg2, igg3 and igg4. igg1 and igg3 subclasses are rich in antibodies aganist proteins such as the toxins produced by the diphtheria and tetanus, as well as antibodies aganist viral proteins. recurrent ear infections, sinusitis, bronchitis and pneumonia are common in ig g subclass deficiency. ig g1 is the major subclass of ig g. igg1 subclass deficiency is very rare. chronic eosinophilic pneumonia is one of the eosinophilic lung disease and is seen rarely. in the presence of peripheral eosinophilia and radiological pulmonary infiltrates diseases suspected. when increase in the number of eosinophils in bronchoalveolar lavage fluids and/or presence of eosinophils in lung tissue diagnosis is confirmed. according to different recording systems chronic eosinophilic pneumonia is %0-2 of the interstitial lung disase. there is not any criteria for diagnosis but also diagnoses is confirmed with suspected findings. symptoms inludes that: 1)in the presence of respiratory symptoms for two weeks long 2)eosinophilia at alveolar lavage and\or peripheral blood ( bal fluid cytological examination > %25, blood >1500/mm 3 ) 3)radiological imaging of the lung peripheral infiltration 4)exclusion of the other causes of eosinophlic lung disease there is eosinophilia over 1000/mm 3 nearly all patients. one of third or half of patients have diagnosed as asthma. disease begin with systemic symptoms such as night sweats, weight loss, anorexia and pulmoner symptoms such as cough, shortness of breath, wheezing. patients have restrictive or obstructive findings in pft. one of third patients, especially with history of asthma, have obstruction in pft. i̇n the pathologic biopsy findings include; thickening of alveolar walls and accumolation of eosinophils and lymphocytes. long time used corticosteroids treatment is recomended. relaps is common when treatment is discontinued. we present the patient who has ig g1 deficiency, chronic eosinophlia and %42 eosinophils in bronchoalveolar lavage fluid. the patient improved long time used oral steroid then inhaled steroids. this was presented in terms of clinical association. case: a 12 years old female patient who were followed due to asthma in the other center for two years, although use of combination inhaled fluticasone and salmeterol, patient was admitted with cough and sputum production. in thorax ct there were, bronchiectasis at right lower lobe, pneumonic consolidation in the right lower lobe and ground glass opacities. we detected as igg 924 mg/dl (835-2094mg/dl), iga 157 mg/dl ( 67-433 mg/dl), igm180 mg/dl (47-484 mg/dl), ige 108. 6 mg/dl, igg1 4896 mg/dl (5990-15600 mg/dl), igg2 2354 mg/dl (1110-5150mg/dl), igg3 1024 mg/dl( 290-2000mg/dl), igg4 616 mg/dl (40-1600 mg/dl). because eosinophilia (717 cells) and symptoms continued, bronchoscopy was performed. left main bronchus was normal, right bronchus were seen dilated. purulent secretion was aspirated on rigt bronchus with flexible bronchoscopy ..in cytological examination % 42 eosinophils was detected in bal. bronchoalveolar lavage cultures was negative. the patient was diagnosed chronic eosinophilic pneumonia and 1 mg/ kg oral steroid was began. ivig was given up to patient; because of frequently recurrent sinopulmonary infection and patient had igg1 subclass deficiency. in the third month of oral steroid therapy physical examination findings and pft were improved and started inhaled steroid. conclusion: immunodeficiencies often can be seen alone. although, the pathogenesis of immundeficiencies with lung disease is not well understood and associated with interstitial lung disease. therefore, investigations must be include bronchoscopy and bronchoalveolar lavage. scid is a congenital heterogeneous group of diseases characterized by severe impairment of t,b, nk cell development and function. the hematopoietic stem cell transplanted 93 patients with scid in the time period from 1994 to 2014 were evaluated. male/female ratio is 59/34. median follow-up time is 5 years (6 months-20 years). parental consanguinity ratio was 81,7%. mean age of onset of the symptoms was 3 ± 2,7 months. most common clinical findings on admission were; pneumonia (75,3%), moniliasis (62,4%), diarrhea (39,8%), dermatitis (19.4%). classification according to t, b, and nk cell counts; (figure 1 ). molecular genetic defects were determined in 60 patients (figure 2 ) are given. there is no difference between age groups according to occurrence of acute and chronic gvhd. death ratio increases with the increasing age. acute and chronic gvhd and number of deaths were significantly higher in peripheral stem cell transplanted patients. there is no statistically significant difference in occurrence of acute/chronic gvhd between b-and b + scid, who had been hla idantically transplanted from family donor. both groups have similar ratios of ivig treatment need after hsct. there is no statistically significant difference in occurrence of acute/ chronic gvhd between nk-and nk + scid, who had been hla idantically transplanted from family donor. both groups have similar ratios of ivig treatment need after hsct. death ratio was similar in groups. bcg dissemination in bcg vaccinated patients is significantly higher in the nk + group. the rate of complications due to severe infections is high and increases with age in patients with scid. hsct is curative, should be considered as early as possible. trec analysis for neonatal screening will give chance for early diagnosis and treatment of the patients. the hyper-ige syndromes are rare pids and are characterized by atopic dermatitis, skin abscesses recurrent pneumonias, elevated serum ige levels and sometimes mucocutaneous candidiasis. we have been evaluating and following up a group of patients with features suggestive for autosomal recessive hyper ige syndrome (ar-hies). homozygous dock8 mutations were identified in several of these patients. however, two siblings from a consanguineous family in this group of patients showed homozygous block in chromosome 20p1.2 in homozygosity mapping which includes stk4 gene. sanger sequencing was performed for stk4 deficiency and showed a novel c.57-60delgata mutation in stk4 gene causing a premature stop codon. clinical manifestations of stk4 deficiency, also known as a macrophage stimulating 1 (mst1) deficiency, stk4 deficiency comprise recurrent and severe viral skin infections including molluscum contagiosum and warts, fungal and bacterial infections and autoimmunity which are also the features of ar-hies. our patients's main features include autoimmune cytopenias (aiha and itp), cutaneous viral (molluscum contagiosum and mild perioral herpetic lesion), mild atopic dermatitis, seborrheic dermatitis, lymphopenia (particularly cd4 lymphopenia), and intermittent mild neutropenia. serum ige level was mildly high in these patients. our results indicates that patients that show clinical phenotype of ar-hies needs to be also evaluated for stk4 deficiency. determination of the underlying defect and reporting the patients are required for the description of the phenotypic spectrum of pids and the frequency of these diseases as well as for genetic counselling. mendelian susceptibility to mycobacterial disease (msmd) is a rare entity. patients with msmd have susceptibility to salmonella and some other intracellular microorganisms in addition to weakly pathogenic mycobacterial species. il-12rβ1 deficiency, most common form of msmd, is caused by mutations in the il12rb gene. 37 patients who have symptoms suggestive for msmd or history of sibling death due to bcgosis were evaluated. the expression of the il-12rb1 was detected in patients and family members by monoclonal antibodies on the lymphocyte surface by flow cytometry after the lymphocytes were stimulated in vitro with pha. mutation analyses was done by sanger sequencing. all index cases were presented either with bcg or salmonella infection. two patients, though they were bcg vaccinated had no clinical symptom, six presented with the symptoms of salmonella infections, two developed leukocytoclastic vasculitis, candidiasis was the accompanying feature in seven. recurrent leishmaniasis that necessitated subcutaneous interferon-γ and prophylactic amphotericine b therapy was present in a patient. in all patients the percentage of lymphocytes with il12rβ1 expression was found to be less than %1. prophylactic antimycrobial treatment and in severe and resistant infectious episodes if-γ therapy, should be given. many patients have associated mucocutaneous candidiasis. the prognosis is good unless the patient admits at the later stages of bcg infection. the results of our patients showed that the analysis of the surface expression of il12rβ1 on activated lymphocytes is an effective diagnostic method which can also be used in screening of the patients with probable msmd. ataxia-telangiectasia (a-t) results from the loss of ataxia-telangiectasia mutated gene (atm) function and is a heterogeneous, multisystemic disease and characterized by accelerated telomere loss, genomic instability, progressive neurological degeneration, immunodeficiency, premature ageing and increased neoplasia incidence. even in classic a-t with ataxia and telangiectasia, the onset of clinical symptoms and the rate of progression are variable. here, we report two siblings was diagnosed as a-t with severe and early hypogamaglobulinemia, decreased cd19, increased cd45ro, no ataxia and telangiectasia, purulent otitis and hemophagocytosis that harbor a rare frameshift mutation of atm gene. özdemir öner 1 ; bozdoğan sıla 2 1 department of pediatrics, division of allergy/immunology, sakarya university, medical faculty, adapazarı, türkiye. 2 department of pediatrics, sakarya university, medical faculty, adapazarı, türkiye. background: the acquisition of new food allergy after transplantation or transplant-acquired food allergy (tafa) is usually reported in adults and rarely in children. tafa is described mainly after liver, but also after small bowel/intestinal, lung and heart transplantations. in different studies, the male/female ratio is equal. literature data suggest that children with tafa typically present within the first year after surgery and they are typically allergic to multiple foods. aim: tafa is generally characterized with allergy to multiple foods and increased level of total and/or spesific ige. here, a patient although who had normal total. ige and specific ige test results, he developed reaction to skin prick test for cow's milk is presented and his clinical presentation will be discussed. case presentation: 15 month-old-boy came to our allergy clinic with complaints of vomiting after drinking cow's milk and skin rush on the area where contact ed with chocolate. in his past medical histroy, left lateral segment of liver (donor was his mother) was transplanted to him when he was at 5 months. the liver donor was not recorded as having a history of allergic disease. methylprednisolone and tacrolimus immunosuppression were used after the transplantation, and tacrolimus therapy was continued for prophylaxis of chronic rejection. when he was at 7 months, family fed the patient with cow's milk but 3 hours later he began to vomit. he vomited five times in two hours. then, he developed constipation. rectal irrigation was used. then oral intake stopped for two days. he was thought to be having food protein induced enterocolitis. his vomiting complaints repeated after intake of formula and baby food which include grain. so he fed with special formula including short-chain peptides and free aminoacids and his symptoms improved. past medical history: extrahepatic biliary atresia was diagnosed at 3 weeks age with conjugated hyperbilirubinemia (according to scintigraphy and biopsy results). family history: his father has penicillin allergy and his aunt has asthma. at our outpatient clinic: height and weight were within normal percentiles. physical examination reevaled normal examination findings. laboratory findings: wbc was 4.420/mm 3 , with 21% neutrophils, 6% eosinophils and 69% lymphocytes. his hemoglobin was 8.2 g/ dl, platelet count was 280.000/mm 3 . background: except for antibody deficiency and complement defects, hematopoietic stem cell transplantation (hsct) is the single best curative treatment defined for primary immunodeficiency (pid) so far. in the current study, we aimed to assess the role of pid type, donor type and clinical status on hsct success rates. materials and methods: we retrospectively reviewed the records of a total of 91 hscts procedures performed in 68 patients diagnosed with pid between 1997 and 2013, in ankara university pediatric allergy and immunology department. results: of the 68 patients, 40 had severe combined immunodeficiency (scid) and 28 had non-scid. the survival rates following hsct, in both scid and non-scid patients were 75%. when classified according to the source of donor, patients who had a hla-matched sibling donor (msd) in the scid group had 90.9% survival rate post transplantation, those who had a matched related donor (mrd) had 85.7% and those who received a haploidentical donor had 63.6% survival rates. in the non-scid group there were 3 patients with haploidentical transplants (2 omenn syndrome and 1 mhc class ii deficiency) and all patients died. we assessed several potential risk factors associated with survival in scid patients. patients diagnosed over 6 months of age with a pre-existing pulmonary infection, requiring intensive care and/or mechanical ventilation had significantly lower survival rates. conclusion: hsct is the best curative treatment for pid. our results demonstrated that hsct performed from matched family donors or even haploidentical parents is a lifesaving treatment in various types of pid's, especially in scid. introduction: in case of donor availability allogeneic hematopoietic stem cell transplantation (hsct) can be regarded as a definitive therapy for a variety of primary immunodeficiency syndromes (pids), including severe combined immunodeficiency (scid) and non-scid pids. study period: we retrospectively reviewed the hospital records of 32 consecutive children with pid, who had 41 allogeneic hsct in the last 22 years, between january 1991 and january 2014. our median follow-up time is 2,1 years (2 months -22,1 years) patients and methods: the median age of children at hsct was 10,5 months (3,5 months-18 years). 23 boys/9 girls diagnoses were based on anamnestic data, clinical findings, and immunological and genetic analysis. conditioning regimens included busulphan + cyclophosphamide, busulphan + cyclophosphamide + atg, fludarabine + melphalan or fludarabine + anti-cd52 in b-, t-, nk-scid cases conditioning was not used. indications for transplantation: patients' diagnoses were severe combined immunodeficiency (n = 16), wiskott-aldrich syndrome (was, n = 6), chronic granulomatous disease (cgd, n = 3), xlinked lymphoproliferative disease (xlp, n = 3), whim-syndrome (n = 1), hyper igm syndrome (cd40 ligand deficiency, n = 1), leukocyte adhesion deficiency (lad, n = 1), dock8 mutation (n = 1) transplantations: 41 hscts for 32 children were performed. 6 patients were retransplanted (4 pt once, 1 pt twice, 1 pt 3 times), because of rejections. at the first hscts in 5/32 cases sibling bone marrow, 1/32 sibling peripheral blood, 1/32 sibling cord blood, 4/32 unrelated cord blood, 9/32 unrelated bone marrow, 2/32 unrelated peripheral blood, 7/32 haploidentical donors were used median cd34 count was 2,52x10 6 /kg (0,13x10 6 -26,95x10 6 /kg) patients engrafted on median 27 ± 8 day (anc > 0,5 g/l) acute gvhd occured in 17/32 cases ( pathomorphological findes: distortion of the structure of lymph nodes due to enlargement of paracortical zones and follicules was found in all patients. presumed atypical cells (makrolymphoblastes and berezovsky-sternberg-reed cells), were detected in 2 patients overwise during follow-up for 2-5 years in these patients revealed no specific infectious and malignansy. accumulation of proliferating dn cells (a lot of mitoses) was characteristic fiture in lymph nodes and spleen of alps patients. setting of plasma cells in follicular zones in the lymph nodes was reveald in 5 cases, and eosinophilesin 2, pronounced immunoblast proliferation in 2, sinus histiocytosisin 3. multiple hyperplastic follicles, extended sinuses with many phagocytizing macrophages, lymphocytic infiltration was revealed by histological examination of the spleen in all cases. immunohistochemistry findes: t-and b-cells proliferation (ki-67 expression), atypical location of lymphocyte populations, settings of plasma cells in all lymph node zones. in paracortical zones was found cd5+, cd7+, cd3 + cd4-, cd3 + cd8-cells in 5 patients, cd38+, cd68+ cells in 1 patient; and in restricted follicular zones -cd19+, cd22+, hla-dr + cells in 4 patients, cd3+, cd15+ and cd68+ in 2 patients. the presentation of a case of kimura disease in a patient with was. the was was diagnosed in 1 year 2months, based on infectious syndrome, atopic dermatitis, hematological syndrome -skin hemorrhages and thrombocytopenia -25*10 9 /l. after he performed often sars. regularly received ivig at 400mg/kg, antibiotic therapy with a positive effect -infectious syndrome stopped, controlled hemorrhagic syndrome. since 2010 there was a herpes virus infection with frequent exacerbations on the face. patient received acyclovir, valacyclovir, famvir with a temporary effect. in 08.2011 on the left side of the face -periorbital region, eyelids, malar region appeared the site of soft tissue hyperproliferation 10 -12 cm in diameter with a thickness of 3-4 cm, with moderate moist, painful on palpation, the left eye was closed, the lid margin ciliated dramatically thickened, deformed. in october-november 2011 was admitted in diagnostic department, massive antibiotic, antimycotic, antiviral therapy, ivig therapy. the histological study showed the angiolimphoid hyperplasia with eosinophilia without immunomorphological signs of malignant tumor growth. based on histological, immunohistochemical studies of the skin, these clinical and laboratory findings, diagnosis: mass lesion of the left face -kimura disease. patient helded 6 courses of chemotherapy with positive dynamics: mass lesion decreased, erosive surfaces disappeared and now receives romiplostimum, acyclovir, biseptolum, ivig 0.5 g/kg 1 time per month. under the observation there is a family k., which is unique in the deletion variants of the same region 22q.11.2 and the presence of an adult patient with the di george syndrome. clinical manifestations of the syndrome in family members is differ. methods: clinical, laboratory, instrumental and genetic ( dna was isolated using a kit «qiaamp dna mini kit» and dna from dried blood spots was isolated using a commercial kit "dna-sorb-b». mlpaanalysis set salsa mlpa probemix p250-b2 digeorge, genetic analyzer applied biosystems 3500). mother (34 years), had few incidents of pneumonia before 10 years. she was diagnosed ullrich-turner syndrome at age 10 years. all childbirths by caesarean section. she has not any laboratory and instrumental findings of immunodeficiency, endocrinopathy, cardiac and thyroid abnormalities. she has deletion in the starting area of the di george region (lcr22-a), including genes cltcl1, hira, cdc45, cldn5, gp1bb, tbx1, txnrd2, dgcr8. father (36 years), healthy, current smoker. the dna microstructural violations in di george region haven't been identified. son was from i pregnancy, heart defect was set prenatally, marked growth retardation. childbirth at 38 weeks. he had unstable reduced cell parameters and hypogammaglobulinemia, the size reduction of the thymus and congenital heart disease: truncus arteriosus, dc 2b stage by lang. after 42 days of life operated for congenital heart disease. during the surgical intervention the thymus wasn't found in a typical place. the postoperative period was complicated by sepsis, heart and respiratory failure. received ivig, antibiotic and antifungal therapy. he suffered from sars, severe course, the degree of respiratory failure 2 (8 months) -death. in mlpa-dna defined microdeletion disorders starting region di george (lcr22-a) -from the 4-year-old dry blood sample (postmortem study a 3 year old boy with bleeding, eczema and recurrent pyogenic infection was admitted to our department. the child who received treatment in hematology accidentally a few times, but has not had the effect of treatment . laboratory parameters -cd3-cells-59% (5550), cd4-cells -22% (1221), cd8-cells -37% (2053), cd19-cells -29% (2728), cd16 / 56-cells -15% (1411), cd4 / cd8-0.59, hla-dr-22, nbt-73. igg-14.6 q /l, iga-3.3 q / l, igm -4.3 q / l, ige-328 u / ml. hb-40 g / l, erythrocytes-1.75x 10 12 / l, leukosytes -15.6x10 9 / l, neutrophils-31%, lymphocytes -56% (9408), eosinophils-2%, monocytes-2%, metamielosytes:-9%, plateletsrare was observed.normal bone marrow cells, the separation of platelets from meqakariosyt has become weak. the advantage of erythroid unordered have been observed. bone marrow puncture the mielokariosytes -87.0 x10 9 / l, meqakariosytes -0.01x10 9 / l, blasts-1.2%, myelosytes -7.4%, metamyelosytes -9.2%, seqments -14.2% neutrophils-% 51.6, eosinophils-1.4%, lymphocytes-5.8%, monocytes-0.4% erythroblasts-2.0%, pronormoblasts-2.8%, normoblasts-40.0% meqakariosytes -0.4%, plasmatic cells-0.2%.his younger brother with eczema, pyogenic infections (furunculosis) was admitted to our department. cd3-38% (1761) ataxia telangiectasia (a-t), is a genetic disorder caused by the homozygous mutation of the atm gene and, frequently associates with variable degrees of cellular and humoral immunodeficiency. however, the immune defects in patients with a-t are not well characterized. to our knowledge, there is no work on major lymphocyte subpopulations and recent thymic emigrants of a-t patients comparing to age-matched healthy controls. according to esid criteria, 17 patients diagnosed as a-t and 12 agematched healthy children were assigned to the study. both patients and healthy controls were grouped as 0-5, 6-10, 7-15 years and older than 16 years. using flow cytometer, major lymphocyte subpopulations and cd4 + cd45ra + cd31+ recent thymic emigrants (rte) were determined as per cent and absolute cell numbers and compared. no significant differences regarding all lymphocyte subpopulations were observed between age groups of a-t. comparing to the healthy controls, there were a decrease (in t cells, effector memory t4 cells, b cells, naïve b cells, switched b cells and rte) and there were an increase (in active t8 cells, naïve t4 cells and nonswitched b cells) in the absolute number and percent of some cell populations in the a-t group. findings of this study showed effector functions in some cell lymphocyte populations were decreased and could be thought that bone marrow of patients should be tried to increase the cells numbers. however, the study has an important limitation about patient and healthy population. ataxia-telangiectasia (a-t) results from the loss of ataxia-telangiectasia mutated gene (atm) function and is a heterogeneous, multisystemic disease and characterized by accelerated telomere loss, genomic instability, progressive neurological degeneration, immunodeficiency, premature ageing and increased neoplasia incidence. even in classic a-t with ataxia and telangiectasia, the onset of clinical symptoms and the rate of progression are variable. here, we report two siblings was diagnosed as a-t with severe and early hypogamaglobulinemia, decreased cd19, increased cd45ro, no ataxia and telangiectasia, purulent otitis and hemophagocytosis that harbor a rare frameshift mutation of atm gene. nijmegen breakage syndrome(nbs) is a rare autosomal recessive disease usually presenting at birth with microcephaly. here we present a case of nbs diagnosed at the age of twenty seven who admitted to our outpatient clinic with malaise, loss of appetite, weight loss and dyspnea on effort . she was hospitalized in another centre 2 years before because of pneumonia, pancytopenia, generalized lymphadenopathy, hepatosplenomegaly and hypogammaglobulinemia (very low igg and iga) where she couldn't have any definite diagnosis. her past medical history was remarkable for primary amenorrhea and basal cell carcinoma excision from preaericular region when she was 23. she had no severe infection history before 25 except frequent upper respiratory tract infections. her parents were consanginous and she had a brother died at 6 months of age. microcephaly together with short stature, multiple palpable lymphadenopathies , splenomegaly and absent secondary sexual characteristics were prominent features in physical examination. direct fluorescence sequencing of the nbn gene showed a homozygous mutation in exon 6 (c.657_661delacaaa) which confirmed the diagnosis of nbs in our patient. nbs is mostly diagnosed in childhood. the delay in diagnosis was partly due to the lack of severe infections in her past medical history until she was 25. another factor that lead to the delay is that it is an unknown disease among physicians in turkey. the longest known survival is 53 years in a patient who had no clinical features of nbs other than primary amenorrhea. as a result, our patient is one of the oldest patients reported in the literature presenting nearly all of the classical features of the disease and carrying the most common pathologic mutation. wiscott-aldrich syndrome (was) is a rare x-linked recessive immunodeficiency disorder characterized by thrombocytopenia, small platelets, eczama, recurrent infections and an increased risk of autoimunity and malignancy. the gene responsible for was is located in chromosome xp11.22-p11-23, which consists of 12 exons, and encodes a 502-amino acid protein. in this study, we aimed to screen was gene mutations and analyze the effects of determined mutations in 2 boys with non-classical was phenotype. ivs10 + 1g > a gene alteration in intron 10 of was gene was identified in case 1, previously. so, rna isolation and then cdna synthesized was carried out. in case 2, after amplification of 12 exons of was gene by pcr, the amplicons were sequenced. in this patient, g > c alteration was detected in the first base of intron 9. afterwards, cdna synthesized for detecting the splicing effect. based on the gel image results, cdna found to be 400 base pairs smaller than the normal in case 1. in case 2, g > c alteration was detected in the first base of intron 9 and then we determined multiple splicing products. two different splicing mutations (ivs10 + 1g > a and ivs9 + 1g > c) were detected in two cases with non-classical phenotype. ivs9 + 1g > a splicing mutation was stated in the literature, previously, but ivs9 + 1g > c mutation was first time identified in this study. whim is rare (<1 / 1 000 000), heterozygous, autosomal dominantly inherited pid, caused by mutations in the gene encoding for the chemokine receptor cxcr4, mapped on 2q21 locus. the altered cxcr4/ cxcl12 interaction impairs cellular homeostasis and trafficking, resulting in immunological dysfunctions with abnormal retention of mature neutrophils in the bone marrow (myelokathexis) and consecutive severe neutropenia, variable degree of lymphopenia and hypogammaglobulinemia. whim patients suffer from recurrent bacterial infections since early childhood and later on manifest a specific susceptibility to hpv infections with developing widespread warts. because of rarity of the disease, heterogeneity in clinical presentation and usually incomplete phenotype, the diagnosis is often delayed and whim syndrome is not suspected. a 13 year old boy who is suffering from recurrent bacterial infections, often complicated with bronchopneumonia, with severe neutropenia, but also, with lower level of lymphocytes and still, normal serum immunoglobulin level is presented. he has no developed warts; neither his parents nor relatives have warts. at the age of 10 years was unveiled the diseasecausing mutation in the cxcr4 gene (c.1000c > t; p.r334x; heterozygote). severe congenital neutropenia accompanied with lymphopenia and findings of mature neutrophils in bone marrow, might be an easy approach for getting closer to the clinical diagnosis of whim syndrome. early identification is important for clinical and therapeutic management, allowing a more comprehensive follow-up and administration of appropriate therapy. we report two cases of congenital heart disease (tetralogia fallot and interruption aortic arch) with confirmed microdeletion chromosome 22q11.2 by karyotype and fluorescence in situ hybridization analysis (fish). children underwent surgical correction of congenital heart defects with good postoperative outcome, although were complex. the phenotype of these patients can be extremely variable, frequently leading to clinical confusion, diagnostic delay, excess morbidity, early mortality. identification of these patients is essential for their adequate management and genetic counseling. a multidisciplinary approach is fundamental to ensure that the patient will be able to attain his or her maximal potential. the underlying cause of the juvenile periodentitis is not well understood but is now thought to be related to an abnormal immune system and to invading bacteria in the cementum of the teeth. instead painful fissures and recurrent pyogenic infections of the skin seem to be the most common medical complications. however, a number of pls patients with abscesses or pseudotumors of the liver have been described. there have been reports of pls patients with other stigmata such as growth retardation, non-symptomatic intracranial calcifications and mental retardation furthermore, coinheritance of pls and albinism type 1 has been reported. case presentation: a 13 yr old girl admitted to our hospital with chief compliant of skin lesions since early months of birth. in the past medical history; she had skin abscess and failure to thrive. on admission she had erythematous, shiny skin with generalized dry scaly predominantly palmoplantar hyperkeratosis and loss of teeth except four or five molar teeth. she informed that she had malformed teeth since childhood which fell off one by one. she had also poor oral hygiene non-pitting edema on lower extremities. no hepatosplenomegaly detected. family history was negative. she investigated for probable immune-deficiency with regard to skin lesions, history of skin abscess and ftt the lab finding are as following: cbc diff = normal ,crp = neg, esr = 6, ast = 31, alt = 15, alph = 1656, alb = 3.8, total pr = 6.2 , urea = 16, cr = 0.4 igg = 1620, igm = 230, iga = 43, ige = 10.8, cd19 = 17%, cd 8 = 20%, cd3 = 55%, cd4 = 34%, cd8 = 20%, cd 56 = 15% conclusion: according to nearly normal lab values and presenting signs such as: generalized pyogenic periodentitis, palmoplantar hyper keratosis and negative family history were attributed to a very rare autosomal recessive disorder with ectodermal dysplasia known as papillon-lefevresyndrome manifesting with palmoplantar hyperkeratosis and severe early onset of destructive periodontal leading to pre-mature loss of both primary and permanent dentitions. nijmegen breakage syndrome (nbs) is a rare autosomal recessive syndrome of chromosomal instability mainly characterized by microcephaly at birth, combined immunodeficiency and predisposition to malignancies. the disease is caused by mutations in the nbs1 gene, which encodes nibrin, a component of the hmre11-rad50-p95 complex involved in cellular response to dna doublestrand breaks. the aim of the present case report was to discuss two siblings with immunologically and clinically different phenotypes of disease presentation and to make an attempt to explain possible genotype-phenotype relation. the two patients and their non-consanguineous parents were clinically, laboratory and genetically investigated. for mother and father we observed no clinical presentation and no immunological abnormalities. sibling no.1 (6 years old boy) had no history of recurrent infections and no deviation in immunological tests. sibling no.2. (6 month old girl) had recurrent infections since birth and iga, igg2 and igg4 deficiency as well as t-and b-cells deficiency. cytogenetic analysis revealed variable percent of spontaneous chromosomal instability which was more severe (in 50% of chromosomes analyzed) in sibling no.2. additionally we sequenced bi-directionally (26 amplicons) the dna samples from all family members to survey the germline genetic variation in the nbs1gene. the 657del5 (exon 6) was detected in both siblings in homozygous and in both parents in heterozygous feature. in order to explain different clinical and immunological presentation of two siblings the rest of the nbs exones were analyzed for genetic heterogeneity. no additional changes were observed. in conclusion patients with the same nbs1 genotype may show different phenotypes. other gene/epigenetic factors seem to play a role in phenotype modulation. omenn syndrome [mendelian inheritance (omim 603554)] is an autosomal recessive form characterized by the presence of fatal generalized severe erythroderma, lymphoadenopathy, eosinophilia and profound immunodeficiency. objective: we studied clinical and immunological presentation of the disease manifestation and frequency c.368-369delaa (p.k86vfs118) in rag1 gene among eastern slavs population. results: we collected clinical and immunological data of 9 patients (1 from belarus, 5 -ukraine, 3 -russia) 6 females, 3 males. age of omenn syndrome manifestation varied from 1 st day of life to 1 yr 1 month. age of diagnosis -20 days to 1 year 10 months. 8 in 9 patients had classical immunological phenotype t(+/-)b-nk+, 1 pt had tlowb + nk + with cd3 + tcrgd + expansion. 6 in 9 pts had mutation in rag1 gene, 4 in 6 had c.368-369delaa (p.k86vfs118) in one or two alleles. at present moment 4 in 9 pts are alive, 3 were transplanted, 1 pt is prepared to bmt. conclusion: this study demonstrates that the most popular genetic abnormalities in eastern slavs children with omenn syndrome is c.368-369delaa (p.k86vfs118) in rag1 gene. this information may be useful for rapid diagnostic of omenn syndrome in laboratories used sscp (single strand conformation polymorphism) before sequencing. under examination the patient particularly bright phenotype attracted attention: microcephaly, "birdlike" facial features (sloping forehead, nape, hypoplasia of brow ridges, broad nasal bridge and protruding midface, hypoplasia of the mandible). in addition, besides specific anomaly of the facial bones we noted: big ears, sparse hair and clinodactyly of the fifth fingers. clinical and immunological characteristics: the feature of the case is pancytopenia syndrome we have diagnosed at the early stages of observation and which is continued throughout the period of observation. erc -2.9-3.6 x 10 12 /l hb -71-105 g/l leuk -0.5-3.0 x 10 9 /l neu -10-26 % (300-780 cells/mcl) plt -40-60 x 10 9 /l data of immunological examination: iga -0,06 g / l igm -0,6 g / l igg -0,75 g / l (other results of immunological examination are without features) the deep insufficiency of antibody production in our patient was the cause of serious, recurrent, and subsequently chronic bacterial sinopulmonary infections after 3 years old. the results of clinical laboratory and immunological examination without significant features: erc -4,0-5,4 x 10 12 /l hb -121-133 g/l leuk -11,5-6,2 x 10 9 /l neu -40-70% (2640-11680 cells/ mcl) plt -512 x 10 9 /l iga -0,85 g/l -1.45 g / l igm -1,33 g/l -0.66 g / l igg -11,3 g/l -11.8 g / l (other results of immunological examination are without features) the x-linked chronic granulomatous disease (cgd) is a primary phagocytic cell deficiency characterized by severe bacterial and fungal infections of various organs. we report of a 19 years of a male patient with xlinked cgd who presented with recurrent hepatic abscesses as the sole manifestation of the disease. phagocytic and bactericidal activities of granulocytes were studied by using microbiological assays. generation of superoxide anion by blood granulocytes was measured by the ferricytochrome c reduction test. cgd is an immunodeficiency caused by mutations in genes encoding subunits of the nadph oxidase complex. normally, assembly of the nadph oxidase complex in phagosomes of phagocytic cells leads to a "respiratory burst" essential for the clearance of microorganisms. cgd patients lack this mechanism, which results in life-threatening infections and granuloma formations. the leading cause of death are pneumonia and pulmonary abscess, septicemia and brain abscess. in neurogical manifestations various pathogens have been involved including aspergillus spp., s. prolificans, a. infectoria, salmonella and staphylococcus spp. there are only some several reports on fungal brain and spinal cord infection, aspergillus abscess resembling brain tumor, meningitis due to streptococcus and candida spp. in the past 20 years we treated 7 children with cgd. we present the infectological challenge of an x-linked cgd patient with brain abscess. in spite of our effort we were unable to identify its causative pathogen. empiric therapy sometimes resembles polypragmasia in cgd. to decrease mortality and morbidity from fungal infections in cgd the prophylactic use of itraconazole or voriconazole is widely recommended. a relatively new azole, posaconazole is active in pulmonary and cerebral fungal manifestations , indeed may be effective against fungi with inherent resistance to ampb or voriconazole. there was no etiological diagnosis in our case that did not respond to conventional antifungal and antibacterial treatment. based upon the findings and literature data we presume the causative agent might be some kind of moulds. we suppose the use of echinocandin and posaconazole as salvage ("prophylactic") therapy has resulted significant regression of the brain abscess. the diagnosis of chronic granulomatous disease (cgd) was verified in 7 children during the past few years in the department of clinical immunology of regional children's hospital of chelyabinsk (russia). in our opinionin 3 casesthe diagnosis was establishedearly enough. michael w. transferred to the neonatal pathology unit of our clinic at the age of 5 days with vesiculopustules. take into consideration our own experience of observing children with this form of primary immunodeficiency in previous years, the child was conducted immunological examination, in particular, the test of restoration nitroblue tetrazolium by superoxide anionformed when oxygen explodes in leukocyte (nitro blau tetrasolium -nbt-test). this decision is caused by the fact that previously observed children with chronic granulomatous disease, had vesiculopustules in 90 % at birth. the survey has revealed a complete lack of production of reactive oxygen species by neutrophils in the evaluation of nbt-test, which allowed us to suggest the diagnosis of cgd. the baby was banned vaccination against tuberculosis, and after reliever vesiculopustules the boy was discharged home. however, at the age of 1 month the baby suffered from glandular abscess and right-segmental pneumonia. in cbc there is expressed anemia (hb 60 g/l, erythrocytes -1.98 x 10 12 /l), leukocytosis (22x 10 9 /l), accelerated esr (36-55 mm/h). at the age of 3 months in the university of debrecen, medical and health science center debrecen, hungary, molecular genetic studywas conducted by prof. dr. laszlo marodi. it was revealed a mutation in c. 374 g > a in exon of 6 gene cybb (encoding subunit gp91-phox), after that the diagnosis of cgd was finally verified. it was assigned a basic preventive antimicrobial therapy by trimethoprimsulfamethoxazole, and itraconazoleto prevent fungal infections. despite this since 6 months the child has repeatedly and consistently suffered from bilateral groin lymphadenitis during the year, acute hematogenous osteomyelitis of the left ulna, 2 pneumonias, purulent mesadenitis, endoperitonitis. the fact that the child aged 8 months was diagnosed sepsisis evidence of the severity of infectious complications. clinical and biochemical blood tests were distinguished by consistently high levels of esr and the presence of leukocytosis, and also severe anemia. now the child is 2 years 11 months, he is undergoing treatment at the department of clinical immunology, russian children's clinical hospital (moscow) after bone marrow transplantation. case history № 2, 3 christina n. from the early history we know that the girl's newborn period was uneventful. she was vaccinated against tuberculosis in the nursing home (no reaction). it was noted the formation of abscesses after vaccination against whooping cough, diphtheria and tetanus in 3 and 4.5 months. in 2 years 8 months she suffered from mezootit. for the first time the girl came under our observation in the children's hospital in chelyabinsk (russia) at the age of 3 years with right segmental pneumonia, complicated by the destruction. after further examination it was diagnosed tuberculosis of intrathoracic lymph nodes to the right with upper lobe bronchopulmonary defeat. to take into consideration given above, it was conducted immunological test that revealed a complete lack of production of reactive oxygen species by neutrophils in the evaluation of nbt-test. on the basis of the history, clinical manifestations and results of immunological examination chronic granulomatous disease was diagnosed. after fourmonth period treatment a recovery came from tuberculosis and child was transferred to outpatient monitoring. in 2 years in this family a second daughter was born -arina n. taking into account revealed immunodeficiency of her sister, the child was not vaccinated against tuberculosis on our recommendations. a six-month-old child was conducted immunological examination, which, like her older sister, also shows a complete lack of production of reactive oxygen species by neutrophils in the evaluation of nbt-test. during the molecular genetic studies of both sisters it was identified identical mutation c. 73-74 del gt gene ncf1 (encoding subunit p47phox). now the older girl is 8 and her sister is 3 years old. observing them in the dynamics neither of them do not show any life-threatening infections. thus, the recovery test of nitrobluetetrazolium by superoxide anion formed when oxygen explodes in leukocyte (nitro blau tetrasolium -nbt-test) is a fairly reliable method of early diagnosis of chronic granulomatous disease. chronic granolomatous disease (cgd) is due to defective phagocyte superoxide production leading to impaired microbial killing. it is comprised of a group of five genotypes with a common phenotype, chracterized by recurrent severe bacterial and fungal infections and tissue granuloma formation. patients with cgd often present with pneumonia, liver abscess, skin infections, lymphadenitis, osteomyelitis. a five month old boy was referred to pediatric infection unit by lymphadenitis. on the medical history, he had taken antibiotic therapy for lymphadenopathy when he was 2 months old. the abnormal eye movement was noticed by the family and on the eye examination peripheric chorioretinal hypopigmented lesions were determined when he was 4 months old. i̇n his labratory examination, viral serology were negative. his parents were not consanguineous. his mother's brother had died at 3 years old. he had history of skin infections and osteomyelitis. his grandmother had been diagnosed as having tuberculosis lymphadenitis one year ago. on his physical examination, his growth was normal, patological findings were horizontal nistagmus, 2/6 degree systolic murmur, fistulized lymph tissue on the left submandibuler region. on the laboratory findings; he had anemia and neutropenia. immunglobulin levels and lymphocyte subtypes were normal. his respiratory burst activity was very low. chronic granulamatous disease was thougth in the patient by the clinical and laboratory findings. i̇t was detected gp91phox mutation in the genetical analysis. he was diagnosed as having x-linked cgd. antibiotic prophylaxis (tmpsmx, flucanazol) and interferon gamma were started. he is 14 months old now and he is on the list of match unrelated donor screening. patiens who has history of lympadenitis, skin infections and chorioretinal findings should be evaulated for the x-cgd. leucocyte adhesion deficiencies (lads) are rare autosomal recessive inherited disorders. three different forms of lads have been described so far. in lad-i, the most common leucocyte adhesion deficiency, the function of β2 integrin cd18 is lost. . while one of the first signs of the disease consist in delayed separation of the umblical cord, severe infections already start early during infancy. another feature of lad-i includes impaired wound healing. therefore, mortality during infancy is high. a fifty day old boy was referred to the hospital due to diarrhea and leukocytosis. the patient was delivered following an uncomplicated full term pregnancy. the parents were first degree cousins. father's brother and mother's uncle had died during infancy period. his umblical cord had separated on the 19th day. patient had applied because of diarrhea by starting in the first days of life and leukocytosis was detected (61.000/ mm 3 ). on the physical examination, his body weight was 5200 gr (10th to 25th percentile) and his height was 56cm (10th percentile). there was a granuloma on the umbliculus. other systems were normal. in the laboratory examination, leukocyte count was high, immunoglobulins, respiratory burst activity, gaita analysing and culture were normal. in the lymphocyte subtype analysing, cd19+ b cells ratio was mildly low. cd18, cd11a, cd11b, cd11c levels were found to be very low. in the genetic analysing, it was detected two deleterious mutations in the itgb2 gene. patient had been diagnosed as lad-1. he treated by antibiotics and then started prophylactic antibiotherapy. family screened for tissue match. his older sister was found full matched. he was referred to transplantation unit. it was applied bone marrow transplantation when he was 3 months old. presence of delayed umblical cord separation and leucocytosis should be considered in the diagnosis of lad but these patients might have different symptoms such as diarrea. mendelian susceptibility to mycobacterial disease (msmd) is a rare entity. patients with msmd have susceptibility to salmonella and some other intracellular microorganisms in addition to weakly pathogenic mycobacterial species. il-12rβ1 deficiency, most common form of msmd, is caused by mutations in the il12rb gene. 37 patients who have symptoms suggestive for msmd or history of sibling death due to bcgosis were evaluated. the expression of the il-12rb1 was detected in patients and family members by monoclonal antibodies on the lymphocyte surface by flow cytometry after the lymphocytes were stimulated in vitro with pha. mutation analyses was done by sanger sequencing. all index cases were presented either with bcg or salmonella infection. two patients, though they were bcg vaccinated had no clinical symptom, six presented with the symptoms of salmonella infections, two developed leukocytoclastic vasculitis, candidiasis was the accompanying feature in seven. recurrent leishmaniasis that necessitated subcutaneous interferon-γ and prophylactic amphotericine b therapy was present in a patient. in all patients the percentage of lymphocytes with il12rβ1 expression was found to be less than %1. prophylactic antimycrobial treatment and in severe and resistant infectious episodes if-γ therapy, should be given. many patients have associated mucocutaneous candidiasis. the prognosis is good unless the patient admits at the later stages of bcg infection. the results of our patients showed that the analysis of the surface expression of il12rβ1 on activated lymphocytes is an effective diagnostic method which can also be used in screening of the patients with probable msmd. introduction: mendelian susceptibility to mycobacterial disease (msmd, online mendelian inheritance inman 209950) is a rare immunodeficiency characterized by predisposition to infections caused by weakly virulent mycobacteria, such as mycobacterium bovis bacille calmette-gue´rin (bcg), environmental nontuberculous mycobacteria (ntm), and salmonella strains in otherwise healthy individuals. il-12rb1 deficiency is the most common form of msmd and is characterized by childhoodonset mycobacteriosis with frequent recurrence. it has been found that patients with il-12rb1 deficiencies are also prone to developing infections with nontyphoidal salmonella species with bacteremia and lymphadenopathy. here we present a girl with recurrent cutaneous leukocytoclastic vasculitis (clv) with salmonella enteritidis due to il-12rb1 deficiency. case report: a four year old girl that had been diagnosed serologically with recurrent salmonella infections, associated with lymphadenopathy and skin eruption was admitted as having henoch-schönlein purpura. she had been vaccinated with bcg and developed left axillary lymphadenitis which spontaneously drained and had recurrent oral monilia plaque. edema and purpuric eruptions were present on the upper and lower extremities and the abdomen.multiple mobile, painful,enlarged submandibular lymph nodes of about 2x2 cm in diameter were palpable. skin biopsy showed a dense inflammatory site with eosinophils, neutrophils and fibrin in the upper dermis and dermal vessel wall,compatible with leukocytoclastic vasculitis. serological studies to assess diagnostic markers for vasculitis and infectious agents were all negative. immune work-up were unremarkable other than hypergammaglobulinemia. salmonella enteritidis was identified in blood culture. she responded dramatically to ceftriaxone treatment within a few days and lesions cleared completely. extended immunological and molecular genetic examination of the patient was carried out for il-12/ifn-γ pathway defects. on the facs analysis of t cells for cell surface expression of the cytokine receptor chains, she did not express any il-12rβ1. discussion: in thepresent report, we describe a child with clv with salmonella enteritidis due to il-12rb1 defi-ciency.in a large cohort of patients with il-12rb1 deficiency, ntm, m. tuberculosis, disseminated bcg infection after inoculation with the vaccine, and salmonella infection have been described. sporadic cases with other infectious agents have also been reported. salmonella infections reported in these patients were due to extraintestinal, or septicemic, recurring infections caused by nontyphoidal salmonella species. only two il-12rb1-deficient patients have been identified with vasculitis due to salmonella strains; both came from turkey, where consanguineous marriages are common. kutukculer and colleagues reported the first case of s. enteritidis-associated clv. sanal and colleagues reported a clv case associated with group d salmonella infection. leukocytoclastic vasculitis is an immune complex mediated disease predominantly involving small vessels of the skin and can be associated with drugs or can be found as a component of other disease, such as infections, connective tissue disorders, and malignancies. infectious agents can cause vasculitis directly or clinically mimic primary vasculitis .multiple infectious agents have been suspected as triggering or contributory factors in the vasculitic process . several factors contribute to the primary vasculitis related to infections: a type 3 or immune-complex reaction, cell-mediated hypersensitivity, abnormal immune regulation, and direct endothelial cell invasion by infectious agents. in our case, extensive evaluation was performed to determine the underlying vasculitis process. clinical and laboratory examinations revealed no association between vasculitis and other infections or an underlying connective tissue disease or medication. she responded well to ceftriaxone treatment, and clinical manifestations gradually resolved within a few days, providing strong evidence that improvement of the vasculitic lesions was due to elimination of the salmonella with antibiotics conclusion: our patient has one of the exceptional forms of il-12rb1 deficiency, with recurrent clv due to salmonella enteritidis. although common presentations for salmonella infection in individuals with il-12rb1 deficiency are lymphadenopathy and bacteremia, it can be present clinically as clv. some infections such as salmonella may be responsible for different types of vasculitis even though they are not common . in this respect, clinicians should be aware of possible infectious causes of vasculitis, and children presenting with unusual recurrent infections caused by non typhoidal salmonella, bcg, or ntm should be investigated for ifn-γ ⁄ il-12 pathway defects. gülez n.; genel f. dr. behcet uz children hospital allergy-immunology department, izmir, turkiye the complement system is an important part of the innate immune defense and also plays a major role in shaping the adaptive immune response. these functions are required for a good defense against infections, especially bacteria. the c8 deficiency is a rare disease that is associated with recurrent neisserial infections, especially meningits caused by n. meningitidis. the patient, a seven years old girl was admitted to hospital with high fever and diffuse, purple-coloured skin lesions. her symptoms gave the diagnosise meningococcal meningitis. she had also earlier been diagnosed with the same disease when she was 5 years old. a sister to the patient had died from meningitis at 3 years of age. she has also one older and one younger sister. there is no consanguinity between her parents. the laboratory analyses of the classical pathway measured as complement hemolytic activity (ch50) and c8 concentration revealed no activity and absence of c8, respectively. analysis of serum from her younger sister showed the same results, while her older sister's ch50 and c8 levels were found normal. thus, our patient and her younger sister were diagnosed with hereditary c8 deficiency. the genetic analyses have not been completed yet. we here report the third and fourth cases of c8 deficiency in turkish patients. interferon-g receptor-1 (ifngr1) deficiency is caused by mutations in ifn-γ receptor-1 gene and is characterized mainly by susceptibility to mycobacterial disease. we report a boy with complete recessive ifngr1 deficiency, afflicted by recurrent mycobacterial diseases with m. bovis, m tuberculosis, m. avium intracellulare and m.fortuitum. genetic analysis showed a homozygous mutation (106inst) in ifngr1 gene leading to complete ifngr1 deficiency. in addition, he had atypical mycobacterial skin lesions caused by m.avium intracellulare and he developed scrotal and lower limb lymphedema secondary to compression of large and fixed inguinal lymphadenopathies. to our knowledge, the patient is the first case with interleukin-12/interferon − γ pathway defect and severe lymphedema. defects in the il-12/ ifn − γ pathway must be considered in patients with disseminated or recurrent mycobacterial infections and in patients with severe viral infections, especially in countries where bcg vaccination is part of the national health programme. it must be kept in mind that these patients may develop granuloma-like skin lesions and severe lymphedema. hsct must be applied at the earliest time before developing organ damages. t lymphocyte/nk cells. restricted defective molecules in the circuit and recently discovered cybb responsible for autophagocytic vacuole and proteolysis have been identified in around 60% of patients with the mendelian susceptibility to the mycobacterial disease (msmd) phenotype. primary defects in oxidase activity in chronic granulomatous disease (cgd) lead to severe, life-threatening infections. the role of phagocytic respiratory burst in host defense against mycobacterium tuberculosis was controversial. previous studied showed that the critical role at reactive oxidants is to serve as intracellular signals for activation of microbicidal enzymes, rather than excretions a microbicidal effect perse.the role of phagocytic respiratory burst in host defense against m. tb is further supported by recent studies discovered immunological defects secondarily affecting phagocyte respiratory burst function and resulting in primary immunodeficiencies with varied phenotypes, including susceptibilities to pyogenic or mycobacterial infections. the patients with severe pid's like scid have broader diverse infections susceptibility and mycobacterial infections as well, however, common variable immunodeficiency (cvid) mostly characterized by a deficiency of immunoglobulins and recurrent sinopulmonary infections. method: we overview the clinical rate of mycobacterial disease in our pid cases and evaluate the complex cases. results: two hundred pid cases were evaluate between 1996-2013 in our clinic, among 5% of them which diagnosed as msmd nearly all presented with mycobacterial infection.8% diagnosed as cgd and interestingly 60% of them have been experienced mycobacterial disease sometimes in their life, as disseminated bcg or late onset complications of bcg including osteomyelitis or mtb once or more than one episode through their life. also we have presented a cvid patient with disseminated tb and granulomatouse hepatitis, tb arthritis , peritonitis and a patient with lad and nontubercolouse mycobacterial infectiouse abcesses of her skin. conclusion: pid cases like cgd, msmd or cvid which are living in area's with high prevalence of mycobacterial infection could have quiet different presentations and the study of these complex cases has provided essential insights into the functioning of the immune system. despite the conventional view we have confirmed that the generation of rois by phagocytic respiratory burst may play a role in the defense of the host against m. tuberculosis by clinical evidence. goran ristic, srdjan pasic, bojana slavkovic division of clinical immunology, mother and child health care institute of serbia, belgrade chronic granulomatous disease (cgd) is a rare disease caused by mutation in any of the five components of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase in phagocytes, resulting in recurrent, life-threatening bacterial and fungal infections of the affected individuals. our proband male patient presented at age of 2 years with bilateral pneumonia and positive serology for aspergillus sp. the phorbol myristate acetate (pma) stimulated nitroblue tetrazolium (nbt) test showed no reduction (0%) in our patient and partial reduction (58%) in his mother. analysis of the cybb gene showed a deletion of nucleotide g (c.1237delg) exon 10 causing frame shift mutation and early termination of translation (p.val413serfs). this mutation was not previously described. at the moment of diagnosis, his mother was already pregnant, 19 th week of gestation. fetal ultrasound showed that she was carrying a male fetus. the fetal blood sample, obtained by the percutaneous umbilical cord blood sampling, showed male karyotype and the pma stimulated nbt test showed 100% reduction. there were no complication during pregnancy or delivery and a healthy boy was born. the proband patient underwent allogenic hsct, his sister was the identical sibling donor. in the cases where the family-specific mutations are unknown, partial or complete gene deletions can be recognized by multiplex ligase-dependent probe amplification (mlpa) or array comparative genomic hybridization ( features of the patients with ar-hies dock-8 deficient and non-dock-8 deficient group are given in the table 1 . all patients with dock-8 deficiency presented with cutaneous viral infections or early onset and severe atopic dermatitis. many have also food allergy and/or asthma. neurological complications and malignancy were seen in 20% and 27% respectively. sixty seven percent of patients had low t; 83 %, low cd4 levels; 100%, high ige. the latter features were shared between patients with or without dock-8 deficiency, except atopic dermatitis which was mild when present in patients without dock-8 deficiency and ige levels were only mildly high or normal. we identified stk4 and coronin 1a deficiency in two siblings each among the 11 patients who showed overlapping features with ar-hies and do not have dock-8 deficiency. our results showed that patients with dock8 deficiency have early onset and more remarkable eczema, food allergy, asthma, more marked eosinophilia, higher ige, low igm levels and development of malignancy. these features may be helpful in differentiation dock8 patients from patients without dock-8 deficiency. it seems routine lymphocyte subset study results are not helpful for this differentiation. alişan yıldıran 1 , stephan borte 2 murat elli 1 , tunç fışgın 1 1 ondokuz mayıs university, school of medicine, samsun-turkiye 2 immunodeficiency center leipzig (idcl) at klinikum st. georg ggmbh, leipzig-germany hsct might be curative for some pids. our immunology and transplantation center was newly established. we retrospectively reviewed all children with pid who diagnosed and received hsct at ondokuz mayıs university or somewhere between june 2010 and december 2013. twenty-two patients were identified. four of them were referred to us for hsct from other centers. the median age was 6 months (1 month-10 yr) at hsct. patients' diagnoses were scid (n = 11), chs (n = 2), leukocyte adhesion deficiency (n=2), mhc class ii deficiency (n=2), chronic granulomatous syndrome (n = 2), hlh (n = 1), was (n = 1) and omenn's syndrome (n = 1). seven patients received hla-matched related hsct; twelve haploidentical hsct and two matched unrelated hsct. one scid patient died just after her diagnosis. two patients developed bcgosis secondary to reactivation of pretransplant vaccination. one of them died due to hemophagocytic bone marrow aplasia, and the other has recovered. five patients had graft failure; two of them received no conditioning regimens because of general health status and the other because of cmv infection. at a median follow up of 6 months (range 0-64), 15 patients are alive, with overall survival of 68 %. we conclude that; our clinic undertakes an important duty in our region for pid patients. also, different pid's could be seen in our region. nerological complication 20 1 patient (epilepsy, brain infarct) outcome 3 died (one with post -bmt comp., 2 with malignancy all alive department of pediatrics and adolescent medicine 1 department of allergology, rheumatology and clinical immunology, university children's hospital, university medical centre, ljubljana, slovenia. developed.there is no severe life-threatening complications development. we describe here a patient with invasive cryptococcus laurentii infection and the x-linked form of hyper-igm syndrome (x-higm). c.laurentii is an extremely rare human pathogen. this fungus was previously considered saprophytic and non-pathogenic to humans, but it has been isolated as the etiologic agent of skin infection, keratitis, endophthalmitis, lung abscess, peritonitis, meningitis, and fungemia. most affected individuals had a compromised immune system because of leukemia, cancer, diabetes mellitus, aids, or prematurity. repeated isolation of c.laurentii from the oropharynx of an immunocompromised patient has also been documented. invasive c.laurentii infection has not been reported in patients with any form of primary immunodeficiency disorder emphasizing the true rarity of disease due to this fungus. two groups have recently reported that dectin-1 deficiency due to the mutation of clec7a or premature termination of the dectin-1 signal transduction molecule card9 may predispose patients to chronic mucocutaneous candidiasis (cmc). we studied the frequency of clec7a mutation in 76 healthy individuals, 10 patients with the hyper-ige syndrome (hies), and 8 patients with autoimmune polyendocrine syndrome type 1 (aps-1), all aged between 2 to 60 years. genomic dna was isolated from peripheral blood. monocytes and monocyte-derived dendritic cells (mddcs) were used to study the phenotypic expression of dectin-1. clec7a gene was sequenced with the big dye terminator cycle sequencing kit. mononuclear cells (mcs) were isolated from heparin-treated venous blood. mdccs were obtained by culturing monocytes isolated by immunomagnetic cell separation assay. receptor expression was assessed by flow cytometry. secretion of il-17a by mcs stimulated with killed c. albicans blastoconidia was assessed by elisa. we report here on healthy individuals with homozygous (one 38year-old man) or heterozygous (32 men and 44 women) tyr238x mutation in the dectin-1 gene but no signs of cmc. dectin-1 levels on monocytes and mddcs were negligible in the homozygous man and the heterozygous individuals displayed intermediate levels of dectin-1, between those of the homozygous man and the wild-type controls. markedly lower levels of il-17a production were observed in the cells of the man with the homozygous mutation than in the control cells. levels of production of this cytokine were intermediate in heterozygotes. the frequency of tyr238x heterozygous individuals was 21% among healthy donors and 10 % in patients with hies and 20 % in patients with aps1. importantly, the 10 year-old hies girl with heterozygous tyr238x mutations has never had mucocutaneous candidiasis. we suggest that dectin-1 receptor-mediated immunity is redundant for host defense against cmc, possibly due to the involvement of multiple lectin receptors in the recognition and uptake of candida. chronic mucocutaneous candidiasis (cmc) is a heterogenous group with recurrent chronic candida infections spesifically involving nails, skin and oropharynx. several immunodeficiencies as dock-8 deficiency, severe combined immune deficiency, autoimmune polyglandular syndrome type 1 (apeced), il-12rβ1 and il-12p40 deficiencies, card 9, stat-3 and stat-1 can cause cmc. we report here a 3 years old boy, born to consaginous parents with onicomycosis, moniliasis, recurrent pneumonia, recurrent herpetic lesions, autoimmune thyroiditis and trombocytopenia. last admission was due to generalized tonic-clonic convulsion and mycotic aneurism on middle cerebral artery was detected. flow cytometry revealed cd4 lymphopenia, immunoglobulin values were in normal range. polymorphism on exon 6 for aire gene (t/c heterozygot g227g) and heterozygot stat1 mutation (c.501 > a; q167h) were detected. various stat1 gof mutations (affecting the coiled-coin domain or the dna-binding domain) have been systematically associated with susceptibility to cmc. autoimmune manifestations associated with stat-1 mutations have been attributed to increased type 1 interferon. the aneurism formation is not elucidated whether it's due to candida infection or vascular damage directly affected by stat-1 mutation. purpose: chronic granulomatous disease (cgd) is a rare genetic disease of phagocytic system. affected patients commonly present with bacterial infections associated with pneumonia, abscesses and lymphadenitis. in this study, we investigated the clinical and laboratory findings of our cgd patients. materials and methods: the demographic data (age at diagnosis, initial presenting symptoms, family history, follow-up period), mutation analysis, therapy options, complications, radiological findings and prognosis were evaluated retrospectively. results: among 9 cgd patients, autosomal recessive form was detected in 4 of them. the age at onset was statistically lower in x' linked cgd patients than ar form (11.2 ± 7.6 mo vs 43.5 ± 21.5 mo; p = 0.001).respiratory tract infections (sinusitis, otitis, pneumonia) and recurrent abscesses were more commonly seen at onset. microbiological culture revealed a. fumigates from lung biopsy in one patient and s.marcescens from blood specimen in other ones. bcgitis was observed in one patient and five patients received anti-tb therapy. non-infectious complications were granulomatous uveitis, recurrent pericardial effusion, skin granuloma, noduler formation in lung and brain area. conclusion: due to high rate of consanguinity, autosomal recessive inheritance was observed highly in our patient cohort. since, patients with cgd are susceptible to tuberculosis and bcg complications; initiation of tuberculosis prophylaxis is advisable in countries where bcg is still administrated at birth. key words: chronic granulomatous disease, consanguinity, bcg. acquired immunodeficiency research center, isfahan university of medical sciences, isfahan, iranbackground: defects of the immune system in primary immunodeficient diseases (pids) predispose individuals to recurrent infections. complex genetic components for susceptibility to mycobacterial disease have been suggested. natural human immunity to the mycobacteria group, including mycobacterium tuberculosis(mtb), bacille calmette-guérin (bcg) or nontuberculous mycobacteria (ntm) relies on the functional il-12/23-ifn-γ integrity of macrophages (monocyte/dendritic cell) connecting to key: cord-017420-tjwxec77 authors: stephens, r. scott title: neutropenic fever in the intensive care unit date: 2019-07-09 journal: oncologic critical care doi: 10.1007/978-3-319-74588-6_118 sha: doc_id: 17420 cord_uid: tjwxec77 neutropenic fever is a common and potentially life-threatening condition in patients treated for cancer. rapid initiation of appropriate antimicrobial therapy is necessary to decrease the risk of mortality. most infections are due to gram-positive organisms, but the mortality rate is higher for gram-negative infections. multidrug-resistant organisms are an emerging threat to neutropenic patients. increasing data suggest that the pathophysiology of neutropenic fever and neutropenic sepsis is substantially different from non-neutropenic fever and sepsis. additional research is needed to both further elucidate the pathogenesis of neutropenic fever and to develop additional effective antimicrobials. neutropenic fever is one of the defining conditions of oncologic critical care. neutropenic fever is defined as a single temperature higher than 38.3 c or a sustained temperature greater than 38.0 c for more than 1 h, in the presence of an absolute neutrophil count [anc]) less than 1500 cells/mm 3 , though many centers and guidelines use an anc cutoff of less than 500 cells/mm 3 [28, 33] . exceedingly common in patients receiving cytotoxic chemotherapy, neutropenic fever is a medical emergency which requires urgent initiation of broad-spectrum antibiotics. any oncologic patient receiving myelosuppressive chemotherapy is at risk of developing neutropenia and opportunistic infections, but profound neutropenia with life-threatening infectious complications is most commonly seen in patients with hematologic malignancies. this can occur during aplasia and before engraftment in patients receiving hematopoietic stem cell transplant (hsct) or from disease-related or treatment-induced cytopenias in other hematologic malignancy patients. while there are many potential causes of fevers in neutropenic hosts, both infectious and noninfectious, fevers must be presumed to be infectious in origin. potential infectious agents and sources of infection are legion and include viruses, bacteria, and fungus. viral pathogens include respiratory viruses (e.g., respiratory syncytial virus, rhinovirus, adenovirus, coronavirus, influenza, parainfluenza), reactivated or de novo herpes viruses (e.g., herpes simplex, cytomegalovirus, epstein-barr virus), and many other potential candidates. bacterial infections may arise from gut translocation, oral mucosal translocation, infection of indwelling vascular catheters, skin and soft tissue infections, pneumonias, and urinary sources. fungal infections typically arise from gut translocation, fungal pneumonias (e.g., aspergillosis), and vascular catheters. drug fevers or "tumor fevers" are the most common example of noninfectious fevers, but these are diagnoses of exclusion. neutropenic fever occurs in up to 50% of patients with solid tumors receiving cytotoxic chemotherapy and in more than 80% of patients receiving chemotherapy for hematologic malignancies or undergoing hsct [28] . in 2012, more than 90,000 adults were hospitalized for cancer-related neutropenia, with a total cost of $2.3 billion [75] . in-hospital mortality for all patients admitted with neutropenic fever is nearly 10%; this increases to a hospital mortality rate of more than 15% for patients with leukemia admitted for neutropenic fever [37] . bacteremia is documented in up to 25% of neutropenic fever patients [28] . whereas in the past gram-negative organisms were commonly cultured, gram-positive organisms, including staphylococci, enterococci, and streptococci, are currently the most commonly isolated bacteria [28, 50] (table 1) . this shift is presumably due to the increased use of long-term indwelling vascular catheters and may also be affected by increased use of prophylaxis against gram[50, 59] . though grampositive infections are more common, gramnegative infections confer a higher risk of mortality [77] . fungal infections, particularly candida and aspergillus, are also frequent, especially in patients with prolonged or profound neutropenia [33] . respiratory viruses can be isolated in approximately 20% of patients [34] . despite best efforts, no causative organism can be identified in about 50% of cases of neutropenic fever [28, 33] . sepsis has been most recently defined as "lifethreatening organ dysfunction caused by a dysregulated host response to infection" [70] . a premium is placed on identification of organ dysfunction using either the sequential organ failure assessment (sofa) score or the quick sofa (qsofa) score; the presence of organ dysfunction in the setting of a suspected or proven infection is sufficient to diagnose sepsis (tables 2 and 3 ). septic shock is defined by the need for vasopressors to maintain a mean arterial pressure (map) !65 mmhg or a serum lactate >2 mmol/l despite adequate volume resuscitation. there is no specific consensus definition for neutropenic sepsis, other than sepsis occurring in the presence of neutropenia. any evidence of organ dysfunction, including an elevated lactate, in the presence of neutropenia should be treated as a potential indicator of sepsis. there are few reliable data on the incidence of neutropenic sepsis or neutropenic septic shock. it has been estimated that 50% of patients with neutropenic fever will develop sepsis, and up to 10% of patients with neutropenic fever will progress to septic shock [33] . among neutropenic allogeneic hsct patients, approximately 10% will develop severe sepsis during the engraftment period [38] . cytotoxic chemotherapy or cytotoxic radiation, whether given as an antitumor agent or conditioning regimen for hsct, induces neutropenia by injuring or destroying hematopoietic precursor cells within the bone marrow as well as injuring the bone marrow structure itself [44] . circulating [43] , so neutropenia develops rapidly after myelosuppressive therapy. depending on the chemotherapy employed, circulating neutrophils and other white blood cells may also be damaged or destroyed, causing a more rapid leukopenia than regimens which only affect the marrow. some agents will injure progenitor cells but not primitive stem cells (which re-populate the progenitor cells), whereas other agents will injure all cells in the marrow [44] . in the normal patient with a localized infection, neutrophils are rapidly recruited at the onset of focal infection and are essential to microbial killing and infection control [40, 45] . neutropenic fever and neutropenic sepsis have historically been thought of as variants of non-neutropenic fever and sepsis, just without an intact immune system, akin to a "fire without firefighters." however, increasing data suggest that the pathophysiology of neutropenic patients is much more complicated than simply an uncontrolled infection [36] . in addition to their key role in the response to localized infections (e.g., pneumonia), in sepsis neutrophils are rapidly recruited to organs (lungs, kidneys, liver) and are thought to contribute to tissue damage and organ dysfunction [45] . neutrophils also contribute to resolution of injury and tissue repair [36] , as well as regulation of the adaptive immune response [40, 76] . thus neutropenic patients are not only deficient in the immediate response to infection but also have an altered physiology regarding the development of systemic inflammation and sepsis and in the regulation of the immune response to injury and tissue repair. it is increasingly unclear whether neutropenic sepsis (and the organ damage sequelae) is the same disease as non-neutropenic sepsis. because cytotoxic chemotherapy is not selective, neutropenic patients are also lymphopenic, thrombocytopenic, and anemic. lymphocytes, including t cells, b cells, and natural killer (nk) cells, have protean roles in the immune system and a broad effect on the response to infection and septic physiology [20, 43] . these cells are also drastically reduced after cytotoxic chemotherapy, and lymphopenia has been associated with an increased risk of death [67, 71] . adding to the complexity, the kinetics of count recovery and engraftment vary, and reconstitution of all cell lines does not happen simultaneously [11] . though the total effect of lymphopenia on the pathophysiology of neutropenic fever and infection remains to be fully determined, patients with neutropenic sepsis have different cytokine profiles than patients with non-neutropenic sepsis: neutropenic patients have higher levels of il-6, il-8, and g-csf than non-neutropenic patients [60] . monocytes are also dysfunctional in neutropenic sepsis, with evidence not only of deactivation of monocytes in peripheral blood but also of deactivation of differentiated pulmonary macrophages in patients with neutropenic sepsis [48, 49] . the implications of pancytopenia for the response to infection extend beyond white blood cells. most neutropenic patients are thrombocytopenic, and platelets are increasingly recognized to play an important role in the immune response [23, 74] . thrombocytopenia is associated with poor outcomes in critical illness, including in sepsis [18] . in addition to secreting mediators and regulators of inflammation, platelets interact with neutrophils and monocytes and play a vital role in the defense against bacterial, viral, and fungal infections via the formation of neutrophil extracellular traps (nets) [16, 21, 23] . platelets also play roles in the development and resolution of organ failure in inflammatory states, including sepsis. in particular, thrombocytopenia has been demonstrated to potentiate lung injury [46, 86] , and platelets may play a role in the pathophysiology of acute kidney injury [23] . while the role of thrombocytopenia in infection remains incompletely elucidated, it seems clear that [70] thrombocytopenia is an important factor in the pathophysiology of neutropenic infection. other organ systems with relevance to neutropenic infections are also affected by cytotoxic chemotherapy. the most important of these is injury to the mucosal barrier of the intestinal tract [13, 14, 78] . disruption of this barrier, which can occur throughout the gastrointestinal (gi) tract, creates portals through which enteric pathogens, including bacteria and yeast, can enter the bloodstream. this is an important source of gram-positive, gram-negative, and fungal infections in neutropenic patients. the respiratory system is also affected in neutropenia. not only are pulmonary macrophages known to be qualitatively dysfunctional in neutropenic sepsis [48, 49] , but quantitative cell counts of alveolar macrophages, lymphocytes, and neutrophils are decreased during neutropenia [19] . neutropenia also appears to adversely affect lung repair after injury [12] . taken together, these data strongly support the hypothesis that neutropenic sepsis has a significantly different pathophysiology than non-neutropenic sepsis. as defined above, the diagnosis of neutropenic fever requires a single temperature higher than 38.3 c or a sustained temperature greater than 38.0 c for more than 1 h in the presence of neutropenia [28, 33] . while some patients are asymptomatic in the presence of neutropenic fever, many describe non-specific symptoms (e.g., cough, anorexia, nausea, fatigue, dizziness, myalgias, confusion, behavioral changes). patients may also present with respiratory symptoms (cough, shortness of breath, sinus pain or drainage) or abdominal symptoms such as pain or diarrhea. fewer than half of patients will feel feverish, shiver, or have rigors [17] . presentation to the hospital can be delayed, with one study suggesting a mean delay in presentation of 11 h and nearly 40% of patients delaying presentation for more than 12 h [17] . though neutropenic patients have higher fevers than non-neutropenic patients, there is no association between peak temperature and mortality. hypothermia during neutropenic sepsis is associated with worse outcomes [84] . neutropenic patients with septic shock tend to have more frequently positive blood cultures, more fungal infections, more multidrug-resistant bacterial infections, and higher mortality rates than immunocompetent patients. compared to non-neutropenic patients, patients with neutropenic sepsis have higher rates of shock and are at higher risk to sustain acute kidney injury [60] . early recognition of neutropenic fever is essential. regardless of whether neutropenic patients are hospitalized or not, frequent temperature checks are essential to detect fever; outpatients must particularly be educated on the importance of monitoring temperature. similarly, interventions that might mask fevers (e.g., antipyretics such as acetaminophen) should generally be avoided in neutropenic patients, and clinicians should be aware of other interventions (e.g., steroids, continuous renal replacement therapy) which may suppress fevers. once a fever is detected, blood cultures should be obtained without delay, and antibiotics initiated as quickly as possible. diagnostic measures must not interfere with the timely administration of antibiotics. a comprehensive physical exam should be performed and may uncover potential sources of infection (e.g., mouth sores, skin lesions, pulmonary findings, abdominal tenderness or pain). blood cultures are essential to the evaluation of neutropenic patients with fever. a minimum of two sets of blood cultures should be drawn upon presentation. current recommendations suggest obtaining two sets of cultures, including both peripheral blood cultures and cultures from a central venous catheter, if present [28, 33] . additional laboratory studies at presentation should include complete blood count with differential, electrolytes, and markers of renal and hepatic function (creatinine, blood urea nitrogen, transaminases). depending on the clinical scenario, strong consideration should be given to obtaining an arterial blood gas, coagulation studies, and lactate level [33] . lactate is of particular potential interest, as an elevated level may help detect early evidence of sepsis-induced malperfusion [33, 70] . other microbiologic studies can be targeted toward patient-specific indicators. for example, urinary symptoms or an abnormal urinalysis should prompt urine cultures. diarrhea, especially in a patient treated with antibiotics, should prompt evaluation for clostridium difficile colitis. fungal markers such as galactomannan or beta-d-glucan may be useful in some patients [33] . respiratory symptoms or abnormalities on chest imaging should be evaluated with testing for respiratory viruses and sputum culture [28] . symptom-guided imaging studies comprise an important part of the evaluation of neutropenic fever. chest computed tomography (ct) scanning should be performed in patients with respiratory symptoms, and potentially asymptomatic patients with cryptic fevers [33] . plain chest radiographs are of limited utility in this population and should not be routinely obtained in lieu of ct scans [87] . sinus, head, and abdominal imaging should be performed as indicated [28, 33] . the use of nuclear medicine techniques such as fdg-pet/ ct to identify foci of infections in febrile neutropenia has been described, but the utility of these techniques has not yet been proven, and remains impractical for current clinical use [80] . as noted above, respiratory symptoms and/or the presence of abnormalities on chest imaging should prompt evaluation for a respiratory infection. in most cases, this can be done noninvasively [6, 8, 10] . bronchoscopy may be indicated in some patients, but the benefits of potential diagnosis must be weighed against the risk of requiring endotracheal intubation during bronchoscopy. abdominal symptoms (pain, diarrhea) should lead to consideration of neutropenic enterocolitis, also known as typhlitis, which is an incompletely understood condition of ileocolonic inflammation which can lead to intestinal necrosis and perforation and is associated with a high mortality rate [63, 64] . diagnosis requires the presence of neutropenia, bowel wall thickening >4 mm over a >30 mm longitudinal distance, fever >38 c, and abdominal pain [31, 51, 63] . specific diagnosis is elusive in many patients, and diagnostic steps should be repeated if no source is found and fevers persist for 72-96 h. repeat blood cultures and repeat or expanded imaging studies may provide additional diagnostic information. risk stratification is an essential part of diagnosis, as it informs immediate management. the multinational association of supportive care in cancer (mascc) score (table 4 ) [35] was developed to predict which patients with neutropenic fever may be safely treated as outpatients. a score !21 identifies a standard risk patient, whereas a score <21 indicates a high-risk patient. additional criteria for outpatient treatment have been enumerated by heinz et al. [33] ; these focus on signs of clinical and social stability, with particular emphasis placed on expected good adherence to oral medications, adequate social support (the patient does not live alone), and the ability to present to the hospital within 60 min. it should be noted that the mascc score has limited utility in predicting either the risk of critical illness or icu outcomes. other factors which influence risk stratification include the depth and duration of neutropenia, with an anc 100 cells/mm 3 and >7 days duration, respectively, being markers of a high-risk patient [28] . accordingly, neutropenic modified from [35] patients with a hematologic malignancy or status post hsct are almost always higher risk than patients who become neutropenic during the course of cyclic treatment for a solid malignancy. comorbid conditions should also be integrated into any clinical risk assessment. pharmacologic prophylaxis in neutropenic patients before neutropenic fever develops due to the high risk of infection in neutropenia, many centers provide routine antimicrobial prophylaxis to patients deemed to be at high risk for infection and who are expected to have prolonged periods of profound neutropenia (e.g., anc 500 cells/mm 3 for >7 days). the rationale for antibacterial prophylaxis is to reduce the risk of gram-negative infections and streptococcal infection from oral mucositis. in high-risk patients, current guidelines suggest antibacterial prophylaxis with a fluoroquinolone such as ciprofloxacin or levofloxacin, with the latter preferred if severe mucositis is anticipated [29, 30] . antifungal prophylaxis against yeast is recommended in highrisk patients (hsct, chemotherapy for leukemia) with fluconazole, itraconazole, voriconazole, posaconazole, or an echinocandin (caspofungin or micafungin). anti-mold (aspergillus) prophylaxis (with voriconazole or posaconazole) is recommended in patients undergoing chemotherapy for leukemia or patients with anticipated very prolonged neutropenia or prior invasive mold infection [28] . antiviral prophylaxis acyclovir or valacyclovir is recommended for patients who are seropositive for herpes simplex virus (hsv) and for varicella zoster virus (vzv)-seropositive hsct patients. finally, leukemia and hsct patients should receive prophylaxis against pneumocystis jirovecii pneumonia. these prophylactic antimicrobials may lessen, but do not eliminate the risk infections while neutropenic; thus, patients and caregivers must remain vigilant for the development of fever. moreover, attention must be paid to the prophylactic regimen, as it will affect the choice of empiric antibiotics for neutropenic fever. neutropenic fever is a medical emergency, and appropriate empiric antibiotics must be started without delay: within 60 min of presentation [28, 33, 57] . some data suggest that even delays in antibiotic administration beyond 30 min are associated with increased mortality [62] . as noted above, it is desirable to obtain blood cultures if possible before antibiotic initiation, so long as this does not delay antibiotic administration. no other diagnostic maneuvers should be attempted before antibiotic initiation. empiric antibiotics must cover the common organisms discussed above (pseudomonas aeruginosa, staphylococcus aureus, streptococcal species) and should also be tailored according to prior patient-specific culture data and institutional epidemiology [28, 33] . appropriate empiric antibiotics include an antipseudomonal penicillin (e.g., piperacillin/ tazobactam), or antipseudomonal cephalosporin (e.g., cefepime), or a carbapenem (imipenem, meropenem) [28, 33] . some data suggest improved outcomes with prolonged antibiotic infusion times [58] though these data require confirmation. fluoroquinolones, which are frequently used as prophylactic therapy in neutropenia, should not be used as empiric monotherapy in neutropenic fever due to the possibility of resistance. though gram-positive organisms are common causes of neutropenic fever, vancomycin is not routinely indicated, but should be added in the presence of suspected catheter-related infection, soft tissue infection, oral mucositis, radiographically proven pneumonia, known colonization with resistant gram-positive organisms (e.g., methicillin-resistant staphylococcus aureus, penicillin-resistant streptococcus), or hemodynamic instability [28, 33, 57] . the use of combination antibiotic regimens (defined as dual gram-negative coverage with an antipseudomonal beta-lactam and an aminoglycoside) in neutropenic fever is controversial. though some studies have suggested a mortality benefit to combination antibiotics [39] , this has not been a consistent finding, and 2 metaanalyses have shown no benefit to the addition of an aminoglycoside to a beta-lactam and a higher risk of renal failure with combination therapy [55, 56] . accordingly, current guidelines for the management of neutropenic fever and sepsis recommend monotherapy with an antipseudomonal beta-lactam unless otherwise dictated by circumstances such as patient allergies, the presence of resistant organisms, or refractory hemodynamic instability [28, 57, 61] . it should be noted, however, that this recommendation does not preclude the use of vancomycin or antifungals; it is only directed at combination therapy targeted against gram-negative bacteria. even with appropriate antibiotics, fever in neutropenic patients typically persists for a median of 5 days; thus, ongoing fevers, unless accompanied by clinical instability, should not necessarily be viewed as evidence of failure of antibiotic therapy [73] . in patients with persistent or recurrent fevers after 3-5 days, a modification of antibiotic regimen is reasonable, especially if guided by new or changing clinical data. earlier antibiotic escalation is necessary in some patients, most commonly in patients with hemodynamic instability. in the face of hemodynamic instability, vancomycin should be added if not already part of the regimen. additionally, antipseudomonal cephalosporins or penicillins should be escalated to a carbapenem (e.g., meropenem), and strong consideration should be given to the addition of an aminoglycoside, fluoroquinolone, or aztreonam [28, 33, 57, 61] . drug-resistant and multidrug-resistant (mdr) organisms are an increasing problem in neutropenic infections [26, 29, 50] . vancomycin-resistant enterococcus (vre) bacteremia affects a substantial portion (10-35%) of patients during induction therapy for leukemia or after hsct and is associated with significantly worse outcomes [3, 53, 85] . early treatment with agents active against vre such as linezolid or daptomycin may improve outcomes. predictive models are being developed to assist with early identification of patients who might benefit from early initiation of antibiotics active against vre [83] . similarly, mdr gram-negative infections, particularly carbapenem-resistant enterobacteriaceae, are associated with high mortality rates, especially among allogeneic hsct patients [66] . successful treatment of these infections requires early use of multidrug antibiotic regimens, typically including aminoglycosides, carbapenems, and polymyxins. the use of surveillance rectal cultures, performed pre-transplant and then weekly after hsct, to identify patients with mdr infections and allow immediate initiation of antibiotic therapy targeted against mdr organisms may result in better outcomes [26] . fungal pathogens are a constant threat in neutropenic patients, and consideration must be given to the use of antifungal agents in all patients with neutropenic fever. in general, antifungal therapy should be initiated in the setting of persistent fever after 5-7 days of appropriate antibacterials [28, 33] . appropriate antifungals should have activity against molds, especially aspergillosis; examples include liposomal amphotericin, caspofungin, and voriconazole, though the data are more robust in favor of the former two [33, 81, 82] . fluconazole should not be used as empiric therapy. an increasing number of neutropenic patients are on antifungal prophylaxis with voriconazole prior to developing fever; the utility of changing antifungal agents upon fever development in this setting is unclear. antifungals should be strongly considered as early therapy in all patients who are hemodynamically unstable [28, 33] . with the advent of multidrug-resistant organisms, antibiotic stewardship is increasingly important, even in neutropenic patients. the optimum duration of antimicrobial therapy in the neutropenic patient and whether antimicrobials may be safely de-escalated in the face of clinical stability are ongoing areas of investigation. one randomized controlled trial suggests that empiric antibiotics may be safely discontinued after a patient has defervesced and remained afebrile for 72 h, regardless of whether neutrophil recovery has occurred [2] . another recent paper suggested that it is safe to withhold antibacterial therapy in children with neutropenic fever in whom infection with a respiratory virus has been proven [65] . neither of these tactics has become standard of care, but both highlight the increasingly realized importance of antibiotic stewardship, even in neutropenic patients. hematopoietic growth factors may be considered in select cases of neutropenic fever, as they have been shown to shorten the duration of neutropenia, but do not impact mortality [22] . according, due to a lack of proven mortality benefit, current guidelines recommend against the routine use of hematopoietic growth factors in neutropenic fever or neutropenic sepsis [29, 72] . granulocyte transfusions have been used to support patients with neutropenia, both to prevent infections and to help treat established infections. very few studies have been performed to evaluate this intervention, and those studies that are available are small. two recent cochrane metaanalyses have examined the use of granulocyte transfusions in neutropenic patients to prevent and treat infections, respectively. both concluded that there was insufficient evidence to determine whether granulocyte transfusions conferred any mortality benefit [24, 25] . early and appropriate administration of antimicrobials is essential to preventing death from neutropenic fever and neutropenic sepsis. good outcomes also depend on successfully managing the hemodynamic derangements and organ failure of sepsis. the intensive care unit (icu) is the bestsuited location to care for neutropenic patients with sepsis and septic shock, and earlier icu admission has been associated with improved survival rates [7, 9] . recent guidelines have been published for the management of sepsis and septic shock [61] ; these guidelines are also applicable to the management of neutropenic sepsis. key points include initial resuscitation with at least 30 ml/kg of intravenous crystalloid with additional fluid resuscitation as indicated and using norepinephrine as a first-line vasopressor to target a mean arterial pressure !65 mm hg. either vasopressin or epinephrine may be added if the response to norepinephrine is inadequate. there may be benefit to using balanced crystalloid solutions such as lactated ringer's or plasmalyte rather than normal saline [68, 69] . source control should be obtained if possible. though occasionally an abscess may be present and feasible to drain, most commonly, an indwelling central venous catheter is the only addressable source of infection. in the hemodynamically unstable patient with a suspected catheter infection, early catheter removal is associated with improved survival [39] ; accordingly, infected or potentially infected catheters should be removed without delay. patients with neutropenia and sepsis are at high risk of developing multi-organ failure, particularly the acute respiratory distress syndrome (ards) [4, 5] . the use of noninvasive ventilation in immunosuppressed patients with hypoxemic respiratory failure is increasingly controversial, and heated humidified high-flow oxygen may be a better option [5, 15, 27, 41, 42, 52] . once patients are intubated, low tidal volume ventilation should be used to maximize lung protection and minimize ventilator-induced lung injury [1] . adjuncts such as neuromuscular blockade and prone positioning should be used in patients with moderate to severe ards (pao2: fio2 < 150) [32, 54] . mortality for patients who develop ards in this context remains high, but outcomes are improving. attention to best practices for mechanical ventilation is essential. an algorithm for the empiric management of neutropenic fever can be found in fig. 1 . the risk of neutropenic fever and neutropenic sepsis resolves once the bone marrow recovers and neutrophil counts return to normal. if treated appropriately, neutropenic fever is a common, predictable, and manageable complication of cytotoxic therapy or hsct. neutropenic sepsis continues to confer a poor prognosis, with recent data suggesting an approximate 46% mortality rate in patients with hematologic malignancies who develop septic shock [7, 39, 47] . predictors of mortality include sepsis after allogeneic hsct, the presence of graft vs host disease, respiratory failure requiring mechanical ventilation, positive blood cultures, cardiac failure, renal failure, and hepatic failure [38, 47] . younger age (<70 years) and the presence of neutropenic enterocolitis are associated with improved survival [47] . encouragingly, survival in neutropenic sepsis and septic shock appears to be improving but is still worse than in non-neutropenic septic patients [9, 10, 39] . the emergence of multidrug-resistant organisms is a major concern, and there is an urgent need for novel antibiotics to address this threat. along these lines, additional work needs to be done to identify patients in whom antibiotic therapy can be safely de-escalated. the 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institute of allergy and infectious diseases mycoses study group caspofungin versus liposomal amphotericin b for empirical antifungal therapy in patients with persistent fever and neutropenia prediction of bloodstream infection due to vancomycin-resistant enterococcus in patients undergoing leukemia induction or hematopoietic stem-cell transplantation association between early peak temperature and mortality in neutropenic sepsis colonization, bloodstream infection, and mortality caused by vancomycin-resistant enterococcus early after allogeneic hematopoietic stem cell transplant platelets in the pathogenesis of acute respiratory distress syndrome the utility of routine chest radiography in the initial evaluation of adult patients with febrile neutropenia patients undergoing hsct key: cord-001120-fxd533b4 authors: everitt, aaron r.; clare, simon; mcdonald, jacqueline u.; kane, leanne; harcourt, katherine; ahras, malika; lall, amar; hale, christine; rodgers, angela; young, douglas b.; haque, ashraful; billker, oliver; tregoning, john s.; dougan, gordon; kellam, paul title: defining the range of pathogens susceptible to ifitm3 restriction using a knockout mouse model date: 2013-11-21 journal: plos one doi: 10.1371/journal.pone.0080723 sha: doc_id: 1120 cord_uid: fxd533b4 the interferon-inducible transmembrane (ifitm) family of proteins has been shown to restrict a broad range of viruses in vitro and in vivo by halting progress through the late endosomal pathway. further, single nucleotide polymorphisms (snps) in its sequence have been linked with risk of developing severe influenza virus infections in humans. the number of viruses restricted by this host protein has continued to grow since it was first demonstrated as playing an antiviral role; all of which enter cells via the endosomal pathway. we therefore sought to test the limits of antimicrobial restriction by ifitm3 using a knockout mouse model. we showed that ifitm3 does not impact on the restriction or pathogenesis of bacterial (salmonella typhimurium, citrobacter rodentium, mycobacterium tuberculosis) or protozoan (plasmodium berghei) pathogens, despite in vitro evidence. however, ifitm3 is capable of restricting respiratory syncytial virus (rsv) in vivo either through directly restricting rsv cell infection, or by exerting a previously uncharacterised function controlling disease pathogenesis. this represents the first demonstration of a virus that enters directly through the plasma membrane, without the need for the endosomal pathway, being restricted by the ifitm family; therefore further defining the role of these antiviral proteins. intrinsic cellular defense molecules are able to detect and restrict invading pathogens at the level of the infected cell and constitute an initial repertoire of proteins that prevent infection. such intrinsic defenses have the ability to detect the pathogen, and either directly block a component of the pathogen's life cycles and / or signal to the innate and adaptive immune system to further control the infection. in certain cases, these intrinsic restriction factors recognise non-self pathogenassocated molecular patterns, such as lipids, proteins and nucleic acids, from a broad range of pathogens through pathogen recognition receptors. this ability allows the host to detect bacterial, viral, fungal and protozoan pathogens [1] . for example, toll-like receptor 4 alone is able to detect gram-negative bacteria, fungi, trypanosomes and surface proteins on several viruses [2] . alternatively, other restriction factors, particularly those that target viruses, appear to have a more reduced range of pathogens that they can block, as is the case for many interferon-stimulated genes (isgs) [3] . however, restriction factors that work in defined cellular locations against a common pathogen feature of infection may also have broad anti-influenza properties. we and others have found that the isg interferon-inducible transmembrane 3 (ifitm3), initially defined as playing a developmental role in germ cell homing [4] , has a profound role in the restriction of viruses entering the cell through the acid endosomal pathway [5, 6] , including influenza and dengue viruses [7] . since the discovery of ifitm3's antiviral role, the number of viruses restricted by the ifitm family has expanded considerably [5, [7] [8] [9] [10] [11] [12] [13] [14] . this has led to the generation of hypotheses about how the ifitm family achieves restriction; namely through preventing the fusion of viral and cellular membranes [15, 16] . recently, the role of ifitm3 has been expanded by the discovery of nonenveloped reoviruses' restriction [9] . this has important implications, as nonenveloped viruses do not rely on membrane fusion to gain release from late endosomes. instead, it is hypothesised that these viruses may physically disrupt the endosomal membrane through their surface proteins [17, 18] . this therefore potentially broadens the actions of ifitm3 beyond enveloped viruses and may also include other non-viral pathogens. the role of ifitm3 in vivo shows it is crucial restriction factor for preventing the onset of severe influenza viral infections in a knockout mouse model [6] . further, the overrepresentation of a single nucleotide polymorphism (snp), rs12252 c allele in the human ifitm3 gene in two cohorts of patients hospitalised with influenza virus during the 2009 h1n1 pandemic shows that the rs12252 cc genotype confers an 4-5 fold increased risk developing a severe influenza virus infection [6, 19] . here we sought therefore to expand and define the role of ifitm3 in pathogen restriction by assessing the susceptibility of ifitm3-deficient (ifitm3 -/-) mice to bacteria (salmonella typhimurium, citrobacter rodentium, mycobacterium tuberculosis), a parasite (plasmodium berghei) and a virus (respiratory syncytial virus, rsv) to determine the specificity of this crucial antimicrobial protein. we show that ifitm3 is specifically an antiviral protein; yielding no significant phenotype in mice when challenged with bacteria and protozoa, despite studies implicating the ifitm family in restriction of these pathogens [20, 21] . we also show a novel role for ifitm3 in vivo in restriction of rsv: a virus that does not enter cells through the endosomal pathway, adding further to the role of ifitm3 as a central antiviral restriction factor that targets cellular entry. background-matched 8-10 week old wild type, ifitm3 -/-(wellcome trust sanger institute [22] ) and ifngr -/-mice (jackson laboratories), all of which were >95% c57bl/6, were maintained in accordance with uk home office regulations, uk animals scientific procedures act 1986 under the project license ppl 80/2596. animals were supplied with food and water ad libitum and were monitored daily for signs of illness. ifitm3 -/-mice were phenotyped through pipelines at the wellcome trust sanger institute as described previously [23, 24] . immunohistochemistry 5-µm sections of paraffin-embedded tissue were incubated with anti-ifitm3 antibody (abcam), which was subsequently bound to a secondary horse radish peroxidase-conjugated antirabbit antibody (dako). sections were counterstained with hematoxylin (sigma-aldrich) and were assessed for expression by microscopy. groups of 8 ifitm3 -/-and 8 c57bl/6j mice were challenged intravenously with 5 x 10 5 colony forming units (cfu) salmonella typhimurium m525 containing tetc, (fragment c of tetanus toxin, to act as an antigen for subsequent antibody quantification), and followed for 28 days. on day 14 postinfection (pi), four from each group of mice are culled and organs (spleen, liver and caecum) removed. a small piece of the spleen and liver was fixed in 4% formalin and then later processed to paraffin blocks as a biobank of infected tissue for histological study if interesting. the rest of the organs were weighed then homogenized, serially diluted and plated to determine viable bacterial load. at day 28pi, the remaining four mice are culled following a terminal bleed and the organs removed and processed as above. the blood was allowed to clot then centrifuged when the serum was removed and used to detect tetc antigen specific antibodies by enzyme-linked immuosorbent assay (elisa). mice were weighed and monitored daily for signs of clinical illness. groups of 8 ifitm3 -/-and 8 c57bl6j mice were orally infected by gavage with 1 x 10 9 cfu of and followed for 28 days. every 2-3 days faeces from infected mice are collected. these were then weighed and homogenized in 1 ml per 100 mg of faeces. this was then serially diluted and plated to determine viable bacterial load. on day 14pi four mice per group were culled and organs (spleen, liver, caecal contents, caecum, 6cm of colon) removed. a small piece of the distal colon was fixed in 4% formalin and processed to paraffin blocks as a biobank of infected tissue for histological analysis. the rest of the organs were weighed then homogenized, serially diluted and plated to determine viable bacterial load. on day 28pi the remaining four mice were culled and the above is repeated. mice were weighed and monitored daily for signs of clinical illness. mice were infected by low-dose aerosol exposure with h37rv m. tuberculosis using a glas-col (terre haute, in) aerosol generator calibrated to deliver approximately 100 bacteria into the lungs. bacterial counts in the lungs at each time point of the study were determined by plating serial dilutions of individual lung homogenates on duplicate plates of middlebrook 7h11 agar containing oadc enrichment. colony-forming units were counted after 3-4 weeks incubation at 37 °c. a transgenic plasmodium berghei anka reporter line, pbgfp-luc con (rmgm-28), that constitutively expresses a fusion protein of gfp and firefly luciferase [25] , was maintained by passage in balb/c mice. to induce experimental cerebral malaria (ecm), infected blood containing 5 x 10 5 infected red blood cells was injected intraperitoneally. from day six pi mice were monitored twice daily and scored for clinical signs of neurologic disease using a ten parameter murine coma and behaviour scale adapted from that of carroll et al. [26] . mice classified as having ecm were killed by cervical dislocation. on days 2 and 3 pi parasite growth was monitored by luciferase measurements in 3 μl of blood, which were collected from a tail vein into citrate-dextrose acd freezing buffer (sigma) and stored at -80°c until analysis using the promega bright-glo luciferase assay system with a berthold orion ii microplate luminometer. parasitemia was also monitored using giemsa-stained thin blood smears. plasma samples from p. berghei infected mice were analysed for cytokines using a cytometric bead array inflammation kit (bd biosciences) according to manufacturer's instructions. samples were acquired on a bd facs aria ii, and data analysed using bd fcap array software. rsv strain a2 (from prof p. openshaw, imperial college london) was grown in hep-2 cells and viral titer determined by plaque assay. mice were infected intranasally (i.n.) with 5 x 10 5 plaque forming units (pfu) under isoflurane anesthesia. weight was measured daily to monitor disease severity. to collect bronchoalveolar lavage (bal) fluid, the lungs of each mouse were inflated five times with 1 ml of pbs and bal fluid kept on ice; 100 μl was centrifuged onto glass slides and stained with hematoxylin and eosin for cell differentiation. the remainder was centrifuged, the supernatant retained at −80°c, and the pellet resuspended in rpmi medium with 10% fetal calf serum. lungs were removed, the smaller lobe was snap frozen in liquid nitrogen for rna extraction and the remainder was homogenized by passage through 100-μm cell strainers (falcon). red blood cells in the lung sample were lysed in ammonium chloride buffer, and the remaining cells resuspended in rpmi medium with 10% fetal calf serum. viable cell numbers were determined by trypan blue exclusion and lung cells types were differentiated by flow cytometry on a bd facs aria ii using antibodies from bd and ebioscience. rsv viral load was measured by quantitative rt-pcr for the rsv l gene using primers and probes previously described [27] , with l gene copy number determined using a rsv l gene standard and presented relative to μg lung rna. lungs were homogenised with a rotor-stator homogeniser, centrifuged and the supernatant collected for cytokine analyses. cytokines in lung homogenate and bal fluid were quantified using duosets from r&d systems. all results are expressed as mean +/-s.d.; statistical significance was calculated by analysis of variance (anova) followed by bonferroni's multiple comparison test when there were more than two groups and student's t-tests for the comparison of two groups. non-normally distributed data were assessed by mann-whitney u test. results regarding mouse survival were analysed by a log-rank (mantel-cox) test. all data regarding mouse phenotyping, including how individual traits were statistically analysed can be found at the wellcome trust sanger institute's mouse resources portal (http:// www.sanger.ac.uk/mouseportal/) all calculations were performed using graphpad prism 5.0 software, and results were considered significant at p < 0.05. many genes are embryonically lethal or lead to no overt phenotype when knocked out in mice. indeed, ifitm3 -/-mice have no discernable phenotype [22] . to examine in greater detail, uninfected mice were assessed against a panel of phenotypic assays [24] , incorporating a robust set of adult traits that are capable of detecting phenotypic variations. we observed no statistically significant differences across all of our key phenotyping categories, including those assessing immune functions compared to wild type control mice ( table 1) . each of the categories listed in table 1 can be further subdivided into a number of additional categories, again all being wild type in phenotype. the complete list of phenotyping can be accessed through the wellcome trust sanger institute's mouse resources portal (http://www.sanger.ac.uk/mouseportal/). we assessed the tissue distribution of ifitm3 by immunohistochemistry of wild type compared to ifitm3 -/-mice, including lymph node, lung, spleen, liver and intestine. the expression of ifitm3 was absent in all ifitm3 -/-mouse organs, as expected, but was highly expressed in all wild type organs ( figure 1 ). in wild type mice, the expression pattern of ifitm3 was noteworthy. the spleen and lymph nodes indicated that ifitm3 was predominantly expressed in the red pulp, but was absent from the white pulp. similarly, intestinal staining revealed ifitm3 expression to be high in the lamina propria, but not on the villus epithelium. conversely, lung and liver showed ubiquitous expression of ifitm3 throughout the tissues, with protein present in respiratory epithelial cells and hepatocytes, respectively. as the first indication of the crucial role of ifitm3 only appeared upon infection with influenza [6] and the tissue distribution suggests ifitm3 is important in multiple organ systems, we challenged the ifitm3 -/-mice with a number of different pathogens. wild type and ifitm3 -/-mice were intravenously dosed with 1 × 10 6 cfu of s. typhimurium m525 bacteria and observed for 28 days pi for signs of morbidity and weight loss (figure 2a ). all mice survived the challenge and gained weight over the time course of the study. ifitm3 -/-mice appeared to gain proportional weight at a slower rate, however, this was due to these mice being on average 5g heavier at the start of challenge and based on lack of bacterial counts and pathology, is unlikely to be due to infection. on day 28 pi, anti-s. typhimurium antibody titres were determined from the sera of wild type and ifitm3 -/-mice. this indicated that both genotypes of mice produced statistically similar antibody profiles ( figure 2b) . further, the bacterial load was determined in the spleen, liver and faecal contents ( figure 2c-e) . similarly, bacterial counts revealed no significant differences between wild type and ifitm3 -/-mice; together showing that ifitm3 does not play a role in resistance or susceptibility to salmonella infection. wild type and ifitm3 -/-mice were orally gavaged with 1 × 10 9 cfu of c. rodentium bacteria and monitored for 28 days pi for signs of morbidity. weight loss profiles showed that neither wild type nor ifitm3 -/-mice had any overt signs of illness over the course of infection ( figure 3a ). bacteria shed in the faeces of these mice also revealed no significant differences between the genotypes, with clearance of infection achieved by day 25 pi in ifitm3 -/-mice ( figure 3b ). mice were sacrificed on days 14 and 28 pi to determine any differences in the bacterial burden between wild type and ifitm3 -/-mice. counts in the caecum (total, caeceal patch and contents) and colon showed no significant differences in bacterial colonisation and clearance ( figure 3c-f) . similarly, analysis of the liver and spleen revealed no instances of bacteraemia in either wild type or ifitm3 -/-mice ( figure 3g,h) . taken together, these data suggest ifitm3 does not impact on c. rodentium infection or pathogenesis. wild type and ifitm3 -/-mice were aerogenically infected with an aerosolised dose of approximately 100 cfu of h37rv m. tuberculosis bacteria and monitored for signs of morbidity for the following 28 days. to determine whether ifitm3 was involved in the control of the bacterial infection, mice were sacrificed on days 0, 7, 14 and 28 pi to calculate the bacterial burden in the lungs. there were however, no significant differences between wild type and ifitm3 -/-mice (figure 4) , with bacterial growth kinetics indicating that ifitm3 does not effect on m. tuberculosis infection and pathogenesis. mice were intraperitoneally injected with 5 × 10 5 red blood cells infected with a p. berghei anka reporter line, pbgfp-luc con (rmgm-28), that constitutively expresses a fusion protein of gfp and firefly luciferase [25] . interferon gamma (ifnγ) receptor knockout mice (ifngr -/-) mice were included to act as control, as these mice do not succumb to lethal episodes of ecm. the experimental challenge revealed there to be no significant difference in phenotype seen in ifitm3 -/-mice compared with wild type littermate controls, with both showing susceptibility to ecm ( figure 5a ). the ~50% survival of wild type mice falls within acceptable boundaries owing to inherent inefficiencies in the delivery of parasites into the mice [28] . in contrast, ifngr -/-mice infected in parallel were fully protected from infection. analysis of parasite burden revealed that all mice were infected with p. berghei ( figure 5b ), but with no significant differences at day three pi. additionally, levels of the inflammatory cytokines ifnγ, tumor necrosis factor alpha (tnfα) and monocyte chemotactic protein-1 (mcp-1) were analysed by cytometric bead array, again no significant differences between wild type and ifitm3 -/-mice were observed ( figure 5c ). wild type and ifitm3 -/-mice were intranasally infected with 5 × 10 5 pfu of rsv-a (a2 strain) and were monitored daily for weight loss for seven days pi. cohorts of mice were sacrificed on days four and seven pi to quantify viral burden and immunological changes over the course of the challenge. ifitm3 -/-mice showed highly significant weight loss on days six and seven pi compared to wild type littermates (p < 0.01) ( figure 6a ). furthermore, ifitm3 -/-mice showed a significantly higher peak in viral load on day four pi (p < 0.05), which remained higher than wild type mice at seven days pi ( figure 6b ). bronchoalveolar lavage (bal) was performed at days four and seven pi and lungs harvested at day seven for cell and cytokine measurements. cellular infiltrate was quantified over the course of infection, which showed a significant increase in total cells resident in the lungs on day seven pi in ifitm3 -/-mice (p < 0.05, figure 6c ) and a similarly significant increase in total cellular infiltrate in the bal fluid on day four pi (p < 0.01, figure 6d ). flow cytometry revealed an increase in all cellular subpopulations in ifitm3 -/-mice relative to wild type littermates on day seven pi. in particular, numbers of total cd3+ t-cells (p < 0.05) in the lungs ( figure 6e) (p < 0.01, figure 6f ). analysis of inflammatory cytokines, including ifnγ, il-6 and il-1β revealed differences in their levels between genotypes of mice in the lungs and bal fluid on day seven pi ( figure 6g ,h), with significantly higher levels of ifnγ (lung: p < 0.05, bal: p < 0.05) and il-1β (p < 0.05) in ifitm3 -/-mice relative to wild type controls. the role of ifitm3 in restricting virus infections, where the virus enters the cell through the acidic endosomal pathway, is well established [5, 15, 29] . however, ifitm3's role in other infections or the effect in removing ifitm3 in vivo in the absence of infection is not well understood. here we show ifitm3 is expressed in many different murine tissues and cell types and does affect the response to rsv infection in mice. ifitm3 does not contribute to the infection phenotype of citrobacter, salmonella or mycobacterium bacterial infections. these bacterial species encompass a range of physiological and pathogenic niches. salmonella enterica serovar typhimurium (s. typhimurium) is an intracellular bacteria that enters cells through phagocytosis or by a bacterial-triggered entry mechanism and replicates within endosomal-like structures known as salmonella-containing vacuoles [30] . in contrast, citrobacter rodentium (c. rodentium) is a noninvasive, gram-negative bacterium used to model enteropathogenic and enterohaemorrhagic e. coli infections of the gut [31, 32] , and mycobacterium tuberculosis (m. tuberculosis) is an intracellular respiratory bacterium that replicates primarily within macrophages and dendritic cells, before forming latent granulomas in the infected organs [33] , and is the causative agent of tuberculosis (tb). we found no evidence for control of m. tuberculosis bacterial growth in murine lungs, despite the fact that the pathogen triggers a type i ifn response [34] , which subsequently up-regulates ifitm3 expression. further, a recent study implicated a snp (rs3888188) in the promoter of ifitm3 with susceptibility to tb [21] , wherein the minority rs3888188-g allele was significantly overrepresented in patients with tb compared to healthy controls in a han chinese population. we found no evidence in our murine model that the ablation of ifitm3 expression impacted on m. tuberculosis infection. however, it should be noted that we only assayed for initial infection and colonisation of the lungs and that the long term dormancy characteristic of human tb is not observed in the mouse model. furthermore, these differences may have arisen due to our challenge strain differing from that used in the human study; therefore inducing different gene expression signatures. further, we found that ifitm3 does not impact on the development of ecm in plasmodium berghei anka infection, although it is well established that malarial parasites elicit strong type i and type ii ifn responses in their hosts, which have been shown to impact on the severity of infection [35, 36] , with ifnα and ifnγ contributing to lethality in murine models. furthermore, eight snps in the ifn receptor, ifnar1, have been associated with the development of cerebral malaria in children; a finding that is corroborated in ifnar -/-mice, which also do not develop cerebral malaria [37] . ifitm3, along with several other isgs, are significantly up-regulated in patients that have become infected with p. falciparum [20] . it was shown that deletion of several of these isgs, including tbk1 and the double knockout of irf3 and irf7 prevented mice from developing lethal ecm [20, 36] . our work shows ifitm3 is not an isg involved in the pathogenesis of experimental cerebral malaria. however, we saw that ifitm3 was important in the control of rsv infection, leading to more severe disease in ifitm3 -/-mice, as assessed by weight loss, viral load and a dysregulated immune response. although these trends were seen with influenza virus infection of ifitm3 -/-mice, the phenotype seen in the rsv challenge is not as striking [6, 38] . this may be due to the response to the different viruses and the genetic background of the mice (c57bl/6 taconic), which we [39] and others [40] have shown is influential in rsv disease severity. rsv is one of the commonest respiratory pathogens in children that necessitates hospitalisation [41] ; accounting for three times more admissions to hospital than influenza viruses [42] . rsv, like influenza virus, is an enveloped virus that initially causes a mild upper respiratory tract infection that can develop into bronchiolitis and cause acute respiratory distress. our discovery that the lack of ifitm3 can alter the pathogenesis of rsv infection suggests ifitm3 either directly restricts rsv cell infection in vivo, or exerts a hitherto uncharacterised function controlling virus infection in vivo is novel and supports associations seen in the mouse model [43, 44] , in airway epithelial cultures [45] and in blood from hospitalised infants [46] . strikingly, rsv infects cells through the plasma membrane and does not require the endosomal pathway. rsv enters airway epithelial cells via f protein binding of nucleolin, which is situated in cholesterol rich microdomains / lipid rafts [47, 48] . rsv is proposed to bind to nucleolin via its f protein, which initiates hemifusion of the rsv envelope with the cell membrane [48] ; thus delivering the viral content directly into the cytoplasm without the need for endosomes. recently, li and colleagues [49] suggested that the ifitm family of proteins are capable of restricting viral hemifusion and the formation of syncytia by reducing membrane fluidity [15, 16] . ifitm3, however, is primarily distributed intracellularly on endosomal membranes [50] . of the ifitm family members, ifitm1 is primarily localised to the cell surface [15] : the site of rsv-cell fusion. therefore ifitm1, which is functional in the ifitm3 -/-mice [6] , may provide the strongest block to rsv infection. previous studies have shown a degree of overlap of function between ifitm1, -2 and -3, with certain ifitms having specificity for restricting particular viruses [7, 8] . importantly, ifitm1, -2 and -3 interact and may function co-operatively [15] , possibly with ifitm3 potentiating an ifitm1 restriction of rsv. indeed, ifitm1 has been shown to be up-regulated during rsv infection [51] . with the recent recognition of ifitm3-mediated restriction of nonenveloped reoviruses, the pleotropic effect of ifitm3 on diverse virus infections, or the co-operative role of all antiviral ifitm proteins as a layered defense to different virus infections, remains to be determined. mice were intraperitoneally injected with red blood cells containing p. berghei anka and were monitored for survival for 12 days pi. n = 9 for wild type, n = 2 for each of ifitm3 -/and ifngr -/-mutants (a). parasite biomass was monitored by measuring the activity of a luciferase reporter gene constitutively expressed by the parasite and expressed as relative light units (rlu) (b), and cytokine dysregulation was measured from the sera on day three pi by cytometric bead assay (c). ■: wild type, □: ifitm3 -/-, ○: ifngr -/-. results show means ± s.d. (n > 2). pattern recognition receptors and inflammation pathogen recognition and innate immunity the broad-spectrum antiviral functions of ifit and ifitm proteins ifitm/mil/fragilis family proteins ifitm1 and ifitm3 play distinct roles in mouse primordial germ cell homing and repulsion ifitm3 inhibits influenza a virus infection by preventing cytosolic entry ifitm3 restricts the morbidity and mortality associated with influenza the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus interferon inducible transmembrane protein 3 (ifitm3) restricts reovirus cell entry interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections a diverse range of gene products are effectors of the type i interferon antiviral response ifitm-2 and ifitm-3 but not ifitm-1 restrict rift valley fever ifitm1 is a tight junction protein that inhibits hepatitis c virus entry the cd225 domain of ifitm3 is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry strategy for nonenveloped virus entry: a hydrophobic conformer of the reovirus membrane penetration protein î¼1 mediates membrane disruption adenovirus protein vi mediates membrane disruption following capsid disassembly interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with severe influenza in chinese individuals innate immune recognition of an at-rich stem-loop dna motif in the plasmodium falciparum a functional promoter polymorphism of <italic>ifitm3</italic> is associated with susceptibility to pediatric tuberculosis in han chinese normal germ line establishment in mice carrying a deletion of the ifitm/ fragilis gene family cluster mouse large-scale phenotyping initiatives: overview of the european mouse disease clinic (eumodic) and of the wellcome trust sanger institute mouse genetics project genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many murine malaria parasite sequestration: cd36 is the major receptor, but cerebral pathology is unlinked to sequestration a rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria preexposure to cpg protects against the delayed effects of neonatal respiratory syncytial virus infection plasmodium berghei anka (pba) infection of c57bl/6j mice: a model of severe malaria interferon-inducible transmembrane proteins of the innate immune response act as membrane organizers by influencing clathrin and v-atpase localization and function salmonella enterica: a surprisingly well adapted intracellular lifestyle citrobacter rodentium of mice and man enhanced susceptibility to citrobacter rodentium infection in microrna-155-deficient mice immunology of tuberculosis mycobacterium tuberculosis triggers host type i ifn signaling to regulate il-1beta production in human macrophages cytokines: accelerators and brakes in the pathogenesis of cerebral malaria type i interferons suppress cd4(+) t-cell-dependent parasite control during blood-stage plasmodium infection ifnar1 controls progression to cerebral malaria in children and cd8+ t cell brain pathology in plasmodium berghei-infected mice ifitm3 limits the severity of acute influenza in mice genetic susceptibility to the delayed sequelae of neonatal respiratory syncytial virus infection is mhc dependent genetic susceptibility to respiratory syncytial virus infection in inbred mice respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology the burden of respiratory syncytial virus infection in young host transcription profiles upon primary respiratory syncytial virus systemic signature of the lung response to respiratory syncytial virus plasticity and virus specificity of the airway epithelial cell immune response during respiratory virus global gene expression profiling in infants with acute respiratory syncytial virus broncholitis demonstrates systemic activation of interferon signaling networks cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells advances in understanding respiratory syncytial virus infection in airway epithelial cells and consequential effects on the immune response ifitm proteins restrict viral membrane hemifusion the n-terminal region of ifitm3 modulates its antiviral activity by regulating ifitm3 cellular localization a systemsbased approach to analyse the host response in murine lung macrophages challenged with respiratory syncytial virus we would like to thank c. brandt for maintaining mouse colony health and wellbeing. key: cord-022393-s26d54ew authors: e. newcomer, christian; g. fox, james title: zoonoses and other human health hazards date: 2007-09-02 journal: the mouse in biomedical research doi: 10.1016/b978-012369454-6/50054-6 sha: doc_id: 22393 cord_uid: s26d54ew zoonoses refers to the infectious diseases and infestations that are transmissible directly from an animal host to humans. the biomedical literature contains numerous reports of zoonotic diseases and parasitic infestations from laboratory mice and their wild counterparts. the extended maintenance of the laboratory mouse over a number of generations under controlled and increasingly sophisticated laboratory animal housing conditions with veterinary oversight and effective infection control measures has markedly reduced the likelihood that zoonotic agents would be encountered in a modem animal care and use environment. wild caught mice that are maintained in naturalistic housing environments in the laboratory, laboratory mice that have contact with wild or feral mice, and mice kept as pets in the home environment are examples of animal management conditions that would be conducive to the expression and transmission of zoonotic diseases and other mouse-associated hazards. in addition to the zoonoses, mice are capable of inflicting bites on inadequately trained personnel and are a rich source of allergens for a substantial number of persons predisposed to develop mouse-associated allergic sensitivities. this chapter discusses the mouse-associated zoonotic diseases and other health hazards and explains the strategies that are helpful for reducing or eliminating the risk of personnel exposure to these conditions. zoonoses is derived from the greek words zoon (meaning animals), and noses (meaning disease), and refers to the infectious diseases and infestations that are transmissible directly from an animal host to humans. the biomedical literature contains numerous reports of zoonotic diseases and parasitic infestations from laboratory mice and their wild counterparts. the extended maintenance of the laboratory mouse over a number of generations under controlled and increasingly sophisticated laboratory animal housing conditions with veterinary oversight and effective infection control measures has markedly reduced the likelihood that zoonotic agents would be encountered in a modem animal care and use environment. however, when these essential animal program quality measures fail or are not incorporated into the animal facility operations, zoonotic pathogens may be unwittingly introduced and perpetuated, placing personnel at increased risk of exposure. wild caught mice that are maintained in naturalistic housing environments in the laboratory, laboratory mice that have contact with wild or feral mice, and mice kept as pets in the home environment are examples of animal management conditions that would be conducive to the expression and transmission of zoonotic diseases and other mouse-associated implications in the new world serocomplex group are present among the wild rodents endemic to the united states such as neotoma spp. and peromyscus spp. buchmeier et al. 2001; fulhorst et al. 2002) . research animal programs that import wild rodents for laboratory studies should stay abreast of the developments on the identification and host-range characteristics of the new world arenaviruses of emerging importance. this section will focus only on lymphocytic choriomeningitis (lcm), a naturally occurring viral zoonosis of the laboratory mouse. many published reports of human lcm infection are associated with laboratory animal and pet contact, particularly mice and hamsters, and these studies now span many decades (armstrong and lillie 1934; bowen et al. 1975; dykewicz et al. 1992; jahrling and peters 1992; lehmann-grube et al. 1979; rousseau et al. 1997) . there seems to be a resurgent awareness among physicians that lcm should be sought as an etiology in human neurological disease and in pediatric congenital brain disorders (barton and hyndman 2000; barton et al. 1995 barton et al. , 2002 romero and newland 2003) . lcmv is widely distributed among the wild mouse population throughout most of the world presenting a zoonotic hazard (childs et al. 1992; childs and peters 1993; morita et al. 1996; smith et al. 1993 ). surveys conducted within the urban environment of baltimore, maryland, reported that 9% of house mice and 4.7% of persons tested had measurable lcmv antibody titers (childs et al. 1991 (childs et al. , 1992 . a similar serological survey conducted in spain across urban and rural ecological settings also found a 9% prevalence in mice and a 1.7% prevalence among persons by immunofluorescence assay (lledo et al. 2003) . the recent serological detection of lcmv in five mice on the treeless, sub-antarctic, macquarie island of australia, indicates the extent of distribution of this agent to the remotest areas of our planet (moro et al. 2003) . the apparent ease with which lcmv is transmitted to humans also occurs in a variety of other laboratory animal species; hamsters, guinea pigs, swine, dogs, and nonhuman primates, especially callitrichids, which readily sustain natural infections. in the case of the callitrichids, there have been numerous reports of epizootic infectious hepatitis (callitrichid hepatitis) due to lcmv, with a high mortality rate in zoological parks in both the united states and england over the past two decades (lucke and bennett 1982; montali et al. 1989; stephensen et al. 1990 stephensen et al. , 1991 stephensen et al. , 1995 . rodent (mouse) infestations of these zoos and/or the supplementation of the diets of tamarins and marmosets with suckling mice, a common practice (richter et al. 1984) , are potentially rich sources for lcmv. in the research animal facility environment, the laboratory mouse continues to merit attention as the species of primary concern as a reservoir for cases of human lcm (dykewicz et al. 1992 ). in the laboratory mouse, and to a lesser degree the hamster, breeding colonies can become endemically infected when the virus is transmitted to pups in utero or early in the neonatal period, producing a tolerant subclinical infection characterized by chronic viremia and viruria. when infected, athymic and other immunodeficient mouse strains may be predisposed to harboring silent, persistent infections and present a higher risk to personnel (dykewicz et al. 1992) . under some circumstances lcmv also produces a pantropic infection and may be copiously present in blood, cerebrospinal fluid, urine, nasopharyngeal secretions, feces, and tissues of infected natural hosts and possibly humans. bedding material and other fomites contaminated by lcmv-infected animals can also be important sources of infection for humans, as demonstrated in a recent outbreak among laboratory animal technicians and on many previous occasions (biggar et al. 1975; dykewicz et al. 1992) . the experimental passage of tumors and cell lines contaminated with lcmv has long been recognized (haas and stewart 1956) and represents one of the biggest threats for the introduction of lcmv into animal facilities at the present time (bhatt et al. 1986; dykewicz et al. 1992; nicklas et al. 1993) . reported that 17 of 63 rodent transplantable tumors screened were positive for lcmv and identified contamination in 4 of 14 hamster tumors and 2 of 81 mouse tumors that had been propagated in animals (nicklas et al. 1993) . the growth of lcmv in insect cell lines has also been demonstrated (rahacek 1965) , and the article by hotchin summarized the work of others indicating that numerous experimentally infected, bloodsucking ectoparasites are capable of transmitting the disease to laboratory rodents (hotchin 1971) . lcm virus also has been recovered from cockroaches (armstrong and lillie 1934) . the diagnosis and control of lcmv infection in mouse colonies has been reviewed in chapter 7 of this volume. tumor and cell-line screening before animal passage, the control of wild rodent infestations in areas where animals are housed or used, and the early detection of colony infections through sound colony health surveillance practices are of critical importance to the prevention of infection in mouse colonies. once established in mouse breeding colonies, the high viral load characteristically shed by mice infected congenitally or neonatally represents a very serious hazard to personnel. most of the cases of human infection, whether involving exposure in the home, agricultural, or laboratory setting, have involved contact with live mice and their excreta or mouse carcasses (dykewicz et al. 1992; havens 1948; morbidity and mortality weekly report 1984) . several authors have emphasized the association between the actual handling of infected mice and the contraction of the disease by humans (dykewicz et al. 1992; havens 1948; smithard and macrae 1951) . several cases of human infection have suggested the possibility that infected rodent tissues can serve as a source of infection for laboratory personnel (baum et al. 1966; dykewicz et al. 1992; tobin 1968 ). humans may be infected by inhalation or by the contamination of mucous membranes or broken skin with infectious tissues or fluids from infected animals. the transmission of lcmv by the bite of an infected mouse can also occur (scheid et al. 1964) . also, hotchin reported the findings of other researchers that lcmv was transmissible experimentally through the intact skin of the guinea pig, but this finding has not been reported in humans (hotchin 1971) . airborne transmission is well documented and plays a very important role in human infections, especially through the ready dispersion and inhalation of viral-contaminated dust from the animal cage or room (biggar et al. 1975; hinman et al. 1975) . following an incubation period of 1 to 3 weeks, humans may experience asymptomatic or a mild febrile disease ranging to a serious flu-like illness characterized by anorexia, malaise, diffuse myalgia and arthralgia, fever, chills, vomiting, headache, stiff neck, and photophobia. some patients enter a second stage of the disease several days after the resolution of early mild symptoms, developing meningoencephalitis and exhibiting additional signs such as drowsiness, confusion, sensory disturbances, and motor abnormalities. patients can also develop more serious nonneurological manifestations of the disease such as maculopapular rash, lymphadenopathy, parotitis, orchitis, arthritis, and epicarditis (peters 1997 ). central nervous system involvement has resulted in death in several cases. infections during pregnancy pose a risk of infection for the human fetus (wright et al. 1997). wright et al. reported 26 cases in human infants, with lcmv confirmed serologically over a two-year period in a major u.s. medical center (wright et al. 1997) . these infants presented with ocular abnormalities, macrocephaly, and hydrocephalus with microcephaly. fifty percent of the mothers reported having had illnesses compatible with lcmv infection, and over half reported exposures to rodents during their pregnancies. the diagnosis of lcm infection in humans is currently made by serological testing using either the immunofluorescent antibody (ifa) test or the enzyme-linked immunosorbent assay (elisa) (barton et al. 2002) . both of these tests are available through the centers for disease control and prevention and are superior to the complement fixation test that is widely available commercially. although there are no proven effective antiviral therapies for lcm infection, intravenous ribavirin therapy reduces mortality in patients infected with lassa fever virus (a member of the old world arenavirus serocomplex) and may be of some benefit in patients with severe lcmv infections (andrei and de clercq 1993; mccormick et al. 1986 ). this disease can be prevented in the laboratory through periodic serological surveillance using elisa and ifa tests of newly introduced animals with inadequate disease profiles and of resident animal colonies at risk. thorough screening of all tumors and cell lines intended for animal passage using the highly sensitive mouse antibody production assay or newer pcr-based laboratory tests for the presence of lcmv is another crucial element in the program to prevent the introduction of lcmv into established animal colonies (besselsen et al. 2003 and chapter 7 of this volume). sound animal facility sanitation practices and the use of microbarrier caging systems with proper infection-control techniques should prevent or suppress the spread of lcmv if present in the environment. the elimination of wild rodent infestations in animal facilities is very important to prevent the introduction of lcmv into the animal facility environment. also, facilities with wild rodent infestations may encounter the relatively common, free-living, bloodsucking mite of the rodent, ornithonyssus bacoti, in abundance (personal communication). although the natural lcmv transmission to humans from bloodsucking ectoparasites is unproven, the control of potential ectoparasitic vectors of this type would be a prudent measure. rabies is an acute, almost invariably fatal disease that occurs worldwide with the exception of a few countries, generally island nations, and other regions that have excluded the disease through animal importation and control programs and the aid of geographic barriers. neither the laboratory mouse nor other small wild rodent hosts appear to be important as reservoirs of natural rabies infection. hence, the principal reason for our discussion of rabies as a zoonotic disease of the laboratory mouse is to provide information that should quickly allay the fears of uninformed research and animal facility staff who suffer the bite of a mouse. on the other hand, the experimental use of mice in the study and characterization of rabies virus and in rabies vaccine development is an important component of some animal care and use programs that deserves special attention by the institution during all phases of research planning and implementation. there are no known cases of human rabies from rodents in the united states (2002). the incidence in larger wild rodent species within the united states has increased in recent years, however. during the interval 1971-1984, a total of 97 cases of rabies in rodents were recorded, but from 1995 to 2000 approximately 52 cases were reported in large rodents annually (2002) . the rodents involved were woodchucks and beavers, both species presumably large enough to survive the chance encounter and attack by a wild rabid carnivore such as the raccoon, skunk, fox, or feral cat. earlier literature on the federal republic of germany summarized findings from 1961 to 1967, which indicated that three mice, one rat (species not given), nine norway rats, and three muskrats were infected with rabies and had bitten humans (scholz and weinhold 1969) . despite rare reports of this nature, rodents are not a proven source of rabies transmission to humans (johnson 1989 ). all mammals are generally regarded as susceptible to rabies. in humans, the course of the disease proceeds through several phases: incubation, prodromal, acute neurological, coma, and rarely, recovery (johnson 1989) . the incubation period varies from 9 days to over 8 months. during the prodromal stage lasting 2 to 4 days, patients experience a period of apprehension and develop headache, malaise, and fever. an abnormal, indefinite sensation at the site of a prior animal bite wound is the first specific symptom. patients also may develop intermittent periods of excitation, nervousness, or anxiety interspersed with quiet periods when the mental state appears normal. further progression of the disease involves paresis or paralysis, inability to swallow, and the related hydrophobia, delirium, convulsions, and coma. rabies produces an almost invariably fatal acute viral encephalomyelitis, with death due to respiratory paralysis. adult mice used experimentally in rabies studies usually exhibit clinical signs between 5 and 15 days following inoculation and die within 5 days of the onset of clinical signs coinciding with the period of viral shedding. clinical signs in the mouse consist of muscular tremors, incoordination, excitation, or paralysis. certain rabies virus isolates from skunks produce a spastic paralysis in adult mice followed by recovery in a high percentage of infected mice. also, infant mice inoculated with certain strains of street rabies virus are capable of full recovery (johnson 1989 ). personnel working with mice experimentally infected with rabies virus should adhere to the well-established and detailed procedures that have been described in other sources for animal inoculation, husbandry, and tissue harvest procedures (johnson 1989) . vaccination of personnel involved in rabies studies with laboratory animals also is clearly indicated, regardless of the animal species involved. four other viruses that produce natural infections in the mouse have been implicated previously or are known to be infectious for humans. these include hantavirus, sendai virus, reovirus 3, and mouse hepatitis virus. for none of these viral agents is there documented evidence of zoonotic transmission of the agent from naturally infected laboratory mice to personnel in the laboratory. hantaviruses are zoonotic viruses comprising at least 22 species that are maintained among natural rodent reservoirs, despite the presence of neutralizing antibody in the rodent host (elliott et al. 2000; meyer and schmaljohn 2000) . approximately half of the hantaviruses are known human pathogens producing virus-specific patterns of disease that include hantavirus pulmonary syndrome, hemorrhagic fever with renal syndrome, and its benign form, nephropathia endemica (mills and childs 1998) . although mus musculus can exhibit the pattern of persistent hantavirus infection in the presence of neutralizing antibody when induced experimentally (araki et al. 2003) , this does not occur in natural infections of the mouse (meyer and schmaljohn 2000) . this is the likely reason that the wild mouse apparently is not important as a natural reservoir for the hantaviruses. this interpretation is supported by serological studies of wild mus musculus that detected either no or a very low incidence of serological evidence to hantavirus exposure even when other wild rodent species in the vicinity had a high level of endemic infection (kantakamalakul et al. 2003; meyer and schmaljohn 2000; pacsa et al. 2002; zuo et al. 2004) . mus musculus is used in the laboratory as an animal model to study various aspects of hantavirus infection ranging from vaccine development (choi et al. 2003) , viral pathogenicity (ebihara et al. 2000; kim and mckee 1985) , and immunological aspects of persistence (araki et al. 2003) , and it is primarily in this context that hantavirus deserves mention as a zoonotic infection in the mouse. the control of wild rodent infestations in animal facilities, quarantine and testing of wild caught rodent species during importation, and proper observance of animal biosafety guidelines for hantavirus-infected animals would be expected to virtually eliminate the risk of this zoonosis in laboratory maintained mice. mouse hepatitis virus (mhv) remains a prevalent infection in many mouse colonies where it potentially impacts colony health and disrupts experimental studies (see chapter 6). earlier studies have demonstrated that human sera contained complement-fixing and neutralizing antibodies to mhv (hartley et al. 1964) . later studies suggested that this was most likely due to cross-reactive antibodies from human cold virus infections (bradburne 1970; mcintosh et al. 1967 ). mouse hepatitis virus and the two prototype human cold viruses (oc43 and 229e) are members of the antigenic group 2 coronaviruses, and it is now known that members of this group share four, and in some cases five, structural genes that could account for this crossreactivity (navas-martin and weiss 2003). the coronaviruses have a very narrow host range and generally replicate only in the cells of the host species (navas-martin and weiss 2003). however, under unique laboratory conditions involving persistent cell culture infection, the use of mixed cell cultures of murine and of a nonpermissive species, or the use of cells possessing modified receptors, mhv has been adapted to grow in human, nonhuman primate, and hamster cells. also, the many strains of mhv in combination with use of targeted rna recombinant system have also been very useful for experimental study of the molecular basis of coronavirus pathogenicity (masters 1999) . application of the targeted rna recombinant system to mhv for exploring of emerging coronavirus infections such as sars may delineate the molecular basis for expanding of host range. these studies may warrant the reader's future attention, but at the present, mhv can be reasonably dismissed as a zoonotic infection. sendai virus was once a prevalent agent in mouse colonies but has become a rarity in most institutions. this is due to its ease of eradication through the use of temporary cessation of breeding to eliminate a naive population that is susceptible to infection and through the use of caging systems that prevent transmission (see chapter 11). sendai virus was originally isolated and described in the 1950s during the investigation of cases of human respiratory illness (gerngoss 1957; kuroya et al. 1953a kuroya et al. , 1953b sano et al. 1953; zhandoff et al. 1957 ). in the original report involving the isolation of sendai virus from japanese newborn human infants suffering from fatal pneumonitis, lung suspensions from the newborns were inoculated intranasally into mice, producing lung consolidation and death in several cases (kuroya et al. 1953b) . in later studies, sendai virus isolates were reported to produce disease in human volunteers (kuroya et al. 1953a; yamada 1956) , and reports from many countries indicated that serological evidence of sendai virus infection was associated with outbreaks of human respiratory illness (demeio and walker 1958; gardner 1957; jensen et al. 1955). tennant et al. demonstrated that personnel working with laboratory animals had antibody titers to sendai virus, but personnel with no known exposure to laboratory animals also had significant titers to the agent (tennant et al. 1967) . recombinant sendai virus is widely used for gene transfer experiments, and these vectors can readily infect human airway epithelium and a variety of other human tissues under experimental conditions (nagai 1999; pinkenburg et al. 2004) . although these recent studies have clearly demonstrated that sendai virus is capable of infecting human tissues, the initial evidence for its role as a human pathogen remains doubtful. the mice used for the early isolations of the agent may have already been endemically infected with sendai and served as the source, or it may be that other serologically cross-reactive parainfluenza viruses, which were not characterized during this era, were responsible for producing false positive reactions to sendai virus (ishida and homma 1978) . reoviruses are generally regarded as the cause of childhood infections producing asymptomatic or very mild disease, and there are few reports linking these infections with a particular disease (tsai 2000) . reovirus 3 was originally isolated from the feces of a clinically ill child (stanley et al. 1953) , and it continues to receive attention as a possible etiology for neonatal hepatitis and extrahepatic biliary atresia in infants (richardson et al. 1994; steele et al. 1995) . reovirus 3 is highly infectious for infant laboratory mice and still receives some attention in the health-screening programs for the mouse and laboratory rodent species. jacoby and lindsey (1997) reported that mouse colonies in the united states continue to have a 5 to 20% prevalence of reovirus 3 infection. although there are no confirmed reports of reovirus 3 transmission from mice (or other laboratory animal species) to humans, the laboratory mouse should be considered a possible source for this infectious agent for humans and other susceptible species. the rickettsiae are fastidious, small pleomorphic coccobacilliary organisms maintained in nature in a cycle of infection involving mammalian hosts and arthropod vectors as reservoirs (saah 2000) . in most rickettsial infections, humans serve only as an incidental host and do not contribute to the propagation of the organism in nature. such is the case for rickettsialpox, a nonfatal, self-limiting zoonotic disease caused by rickettsia akari, which is perpetuated in a cycle of infection involving mus musculus as the primary host and a mite vector (liponyssoides sanguineus). isolation of the organisms from rats (rattus) and voles (microtus) has also been reported. the first cases of human rickettsialpox were described in patients in new york city (huebner et al. 1946a (huebner et al. , 1946b , and outbreaks of the disease have generally remained clustered within large urban areas of the united states and in rural north carolina, utah, south africa, korea, and the former soviet union . according to koss et al. approximately 800 cases of rickettsialpox have been reported in the literature, with nearly 500 of these within the three years following the original description of the disease . prior to the report by koss et al. the largest case study included 13 patients accumulated over a 10-year period. the recent report by koss et al., however, included 18 patients in new york city reporting over only a 20-month period in the wake of the september 11, 2001 attacks, suggesting that perhaps the heightened sensitivities to possible bioterrorism events stimulated an upsurge in the reporting of cases as a byproduct of increased patient concerns . authors have commented on a variety of other social and demographic factors that may also be contributing to the noticeable increase in the incidence of rickettsialpox and murine typhus in the urban environment (comer et al. 1999; paddock et al. 2003) . there are no reported cases of rickettsialpox in personnel related to exposure to naturally infected laboratory mice. the mite vector l. sanguineus has not been reported in laboratory mouse colonies either historically or contemporarily. however, the tropical rat mite (ornithonyssus bacoti) can be experimentally infected with r. akari but has not been shown to play a role in the natural cycle of infection. in the authors' experience of ornithonyssus bacoti infestations of laboratory mouse or rat colonies are still seen with some frequency in facilities that have resident wild or feral mouse populations and should be addressed in the institution's pest control and infection control programs. rickettsialpox has an incubation period of 7 to 21 days following the bite of the infected mite (saah 2000 mild to severe with an abrupt onset, and it typically presents with a classic triad of an eschar, fever, and a papulovesicular rash. the papule develops at the site of the bite and later ulcerates and progresses to an eschar, 0.5 to 3 cm in diameter, as r. akari proliferates locally in the skin and vasculitis develops saah 2000) . the rash begins with firm, generally nonpruritic, erythematous papules, 2 to 10 mm in diameter, that develop into vesicles and heal by crusting. in addition to fever, patients may experience chills and headache, and less commonly, backache, myalgia, and photophobia. the disease is mild and self-limiting within 6 to 10 days, and serious complications or death have rarely been reported (saah 2000) . patients typically respond quickly after the initiation of antirickettsial therapy with tetracycline, doxycycline, or other appropriate agent saah 2000) . however, following resolution of the infection, headache and lassitude can persist for 1 to 2 weeks. the reader should refer to koss et al. for information on other rash-producing or eschar-related diseases that should be considered in the differential diagnosis to rickettsialpox . rickettsia akari are diagnosed by complement fixation tests or the more sensitive indirect immunofluorescent antibody test. serum antibody to r. akari generally takes 2 weeks to develop, and paired sera are needed to confirm a four-fold rise in antibody titer (saah 2000) . during the acute phase of the infection, immunohistochemistry or pcr analysis can be used for a rapid diagnosis on biopsy material obtained from the papulovesicular rash or eschar paddock et al. 2003) . laboratory mice infected with r. akari develop fatal pneumonia with intranasal inoculation and severe illness and death with intraperitoneal inoculation. mice develop anorexia, depression, and dyspnea. peritonitis, splenomegaly, and lymphadenitis are found upon necropsy examination. subcutaneous inoculation produces an active infection for 1 month, with organisms being recovered from the spleen but not the feces or urine (bell 1970) . the control and eradicaton of r. akari infections depend on the prevention of wild mice and the mite vector from entering laboratory animal facilities and human dwellings. murine typhus is a rickettsial disease caused by rickettsia typhi (previously r. mooseri) that occurs worldwide with epidemics or with high prevalence in particular geographic areas (dumler and walker 2000) . in the united states, most human cases of the disease are concentrated in texas and southern california. the disease is now predominantly associated with the rat as the primary host species for the oriental rat flea (xenopsylla cheopis), which serves as the principal ectoparasitic vector transmitting the disease to humans. however, the mouse can also serve as a host for this flea, as well as for the northern rat flea (nasopsyllus fasciatus) and the mouse flea (leptosylla segnis), which also bite man and can be involved in the transmission of r. typhi (flynn 1973; pratt and wiseman 1962; yunker 1964 ). an early report in the literature indicated that x. cheopis was easily established in an animal facility inhabiting rooms used for housing laboratory mice (yunker 1964) . it is now also known that the cat and opossum and the cat flea (ctenocephalides felis) can be involved in sustaining the cycle in some geographic localities (azad et al. 1997) . clark and will (1994) reported on use of the laboratory mouse as an experimental host for rearing x. cheopis, but there have been no reports of natural infestations of mouse colonies with any of the flea vectors of r. typhi for several decades. also, r. typhi has not been isolated from natural infections in laboratory mice. murine typhus is a more serious disease than rickettsialpox and presents with fever, headache, chills, nausea, and vomiting. splenomegaly, hepatomegaly, central nervous system involvement, and multiorgan failure can occur as severe and potentially fatal complications. a skin rash, which is typically maculopapular in the case of murine typhus, occurs much less commonly than in rickettsialpox, and its absence should not dissuade the clinician from making a diagnosis of murine typhus and from promptly instituting therapy due to the potential severity of the disease (dumler and walker 2000) . the methods used for the laboratory diagnosis and treatment of the disease in humans and the principles of preventing the introduction of r. typhi into laboratory animal colonies are similar to those for r. akari. leptospirosis microorganisms were discovered in 1914 when they were isolated from jaundiced patients (inada et al. 1916) ; after further study they were named in 1917 (noguchi 1918) . leptospirosis is solely a zoonotic disease of livestock, pet and stray dogs, and wildlife, including wild rodents. rodent reservoir hosts of leptospirosis include, in addition to rats, mice, field moles, hedgehogs, gerbils, squirrels, rabbits, and hamsters (fox and lipman 1991; torten 1979) . human to human transmission is extremely rare. leptospira interrogans (comprising more than 200 serovars) have been isolated worldwide (tappero et al. 2000) . l. interrogans contains 23 serogroups with strains pathogenic for amphibians, reptiles, and mammals including humans. serovars australis, ballum, bataviae, hardjo, grippotyphosa, icterohemorrgagiae, javanica, and pomona are associated with rodent infections. leptospira serovars, including l. australis, bataviae, grippotyphosa, hebdomidis, icterohaemorrhagiae, pomona, and pyrogenes, are found in the house mouse (torten 1979) . leptospira ballum has also been reported from mice and is e. newcomer and james g. fox most commonly associated with zoonotic outbreaks (borst et al. 1948; friedmann et al. 1973; stoenner and maclean 1958) . although particular serovars usually have distinct host species, most serovars can be carried by several hosts. leptospira are well adapted to a variety of mammals, particularly wild animals and rodents. in the chronic form, the organism chronically infects the host and is shed in the urine inconspicuously for long periods of time. rodents are the only major animal species that can shed leptospires throughout their lifespan without clinical manifestations (fox and lipman 1991; torten 1979) . active shedding of leptospires by rodents can go unrecognized until personnel handling the animals become clinically infected or are infected by exposure to water or food contaminated by urine. rats and mice are common animal hosts for serotype, l. ballum, although it has been found in other wildlife as well. water can often be contaminated with infected rodent urine. the infection can persist unnoticed in laboratory rodents, though their carrier rates for laboratory-maintained rodents in the united states are unknown, but it is probably low. the organism is not routinely screened on health surveillance protocols for mouse colonies; however, there was a report of leptospirosis in 1984 in a research colony of mice in the united states being housed in a large research institution (alexander 1984) . l. icterohaemorrhagiaes antibody when compared to children living in the detroit suburbs. therefore, children living in rodent-infested tenements may be at increased risk of infection (demers et al. 1983) . in europe and more recently in north america, rodents including house mice have provided a source of leptospira infection for swine and by extension could also infect personnel working in swine production units (galton 1966; smith et al. 1992) . leptospira interrogans serovar bratislava is commonly reported in these mice. the disease may vary from unapparent infection to severe infection and death. a self-limited systemic illness is seen in approximately 90% of infected humans. the incubation period is usually 5 to 14 days. individuals infected with leptospira experience a biphasic disease (faine 1991; sanger and thiermann 1988; stoenner and maclean 1958) . they become suddenly ill with weakness, headache, myalgia, malaise, chills, and fever and usually exhibit leukocytosis. during the second phase of the disease, conjunctival suffusion and a rash may occur. upon examination, renal, hepatic, pulmonary, and gastrointestinal findings may be abnormal. intravenous penicillin is the drug of choice in treating early-onset and late-stage leptospirosis infection (faine 1991; taber and feigin 1979; watt et al. 1988 ). ampicillin and doxycycline also have been effective in treating people with mild to moderate forms of leptospirosis. because leptospirosis in humans is often difficult to diagnose, the low incidence of reported infection in humans may be misleading. from 1974 to 1979, only 498 cases were reported, for an incidence of 0.05 per 100,000 people per year (sanger and thiermann 1988) . leptospirosis was removed from the reportable disease category in the united states in 1995 because of the small number of cases reported. outbreaks have been documented in the united states from personnel working with laboratory mice (barkin et al. 1974; stoenner and maclean 1958) . in one study, 8 of 58 employees handling the infected laboratory mice (80% of breeding females were excreting l. ballum in their urine) contracted leptospirosis (stoenner and maclean 1958) . infection with leptospira most frequently results from handling infected animals (contaminating the hands with urine) or from aerosol exposure during cage cleaning (barkin et al. 1974; friedmann et al. 1973; stoenner and maclean 1958) . skin abrasions or exposure to mucous membranes may serve as the portal of entry. all secretions and excretions from infected animals should be considered infective. in one instance, a father apparently was infected after his daughter used his toothbrush to clean a contaminated pet mouse cage (boak et al. 1960) . rodent bites can also transmit the disease (looke 1986 ). in detroit, children from the inner city had a significantly higher because of the variability in clinical symptoms and the lack of pathognomonic pathologic findings in humans and animals, serologic diagnosis or actual isolation of leptospires is imperative (faine 1991) . as an aid to diagnosis, leptospires can sometimes be observed by examination or direct staining of body fluids or fresh tissue suspensions (sulzer et al. 1968 ). the definitive diagnosis in humans or animals is made by culturing the organisms from tissue or fluid samples, or by animal inoculation (particularly in 3-to 4-week-old hamsters) and subsequent culture and isolation. culture media with long-chain fatty acids with 1% bovine serum albumin are routinely used as a detoxicant (faine 1991) . serologic assessment is accomplished by indirect hemagglutination, agglutination analysis, complement fixation, microscopic agglutination, and fluorescent antibody techniques (faine 1991) . the serologic test most frequently used is the modified microtiter agglutination test. titers of 1:100 or greater are considered significant. molecular techniques including pcr and randomly amplified polymorphic dna fingerprinting are used for identification of serovars (tappero et al. 2000) . in mouse colonies infected with l. ballum, antibodies against l. ballum were detected in sera of mice of all ages, but 26. zoonoses and other human health hazards 727 leptospires could be recovered only from mature mice. progeny of seropositive females had detectable serum antibodies at 51 days of age but not at 65 days. it was also reported that progeny of seropositive female mice, which possessed antibody at birth and acquired additional antibody from colostrums, remained free of leptospires if isolated from their mothers at 21 days of age, despite exposure during the nursing period (stoenner 1957) . studies in mice experimentally infected with l. grippotyphosa demonstrated that maternal antibodies, whether passed through milk or placental transfer, conferred protection of long duration against the carrier state and shedding of leptospires. thus, serologically positive immune mothers do not transmit the disease to their offspring. however, mice born to nonimmune mothers, if infected at 1 day postpartum, become carriers with no trace of antibodies. thus a population of carrier pregnant mice without antibody could serve as a precipitator in outbreaks among susceptible mouse populations (birnbaum et al. 1972) . field surveys have supported this data in that a significant percentage of carrier mice do not have antibodies. this led to the diagnostic approach, which specifies that both serologic and isolation methods must be utilized to determine the rate of leptospiral infection in rodents (galton et al. 1962) . leptospira ballum is frequently found in the common house mouse (m. musculus) (brown and gorman 1960; yager et al. 1953) . therefore, eradication of infected colonies, use of surgically derived and barrier-maintained mice or of conventional laboratory mice free of leptospira infection, coupled with the prevention of ingress of wild rodents, should effectively preclude introducing of the organism into research and commercial laboratories (loosli 1967) . leptospira ballum has been eliminated from a mouse colony by administration of feed containing 1000 gm chlorotetracycline hydrochloride per ton for 10 days. after 7 days of antibiotic therapy, mice were transferred to clean containers and administered clean water, both having been sterilized by steam. mouse traps and rodenticides were used to destroy escaped mice and to prevent reintroduction of l. ballum by the common house mouse . commercial animal colonies maintained in research vivarium today are not routinely screened for leptospirosis, assuming that the organism has been effectively eliminated from commercial and research-maintained mice. rat bite fever can be caused by either of two microorganisms: streptobacillus moniliformis or spirillum minus. streptobacillus moniliformis causes the diseases designated as streptobacillary fever, streptobacillary rat bite fever, or streptobacillosis. haverhill fever and epidemic arthritic erythemia are diseases associated with ingestion of water, food, or raw milk contaminated with str. moniliformis. sodoku, derived from the japanese words for rat (so) and poison (doku), spirilosis, and spirillary rat bite fever are caused by another bacterium, spirillum minus. the bite of an infected rat is the usual source of infection. in some cases, other animal bites, including mice, gerbils, squirrels, weasels, ferrets, dogs, and cats, or rare traumatic injuries unassociated with animal contact, cause the infection. in a retrospective analysis coveting three decades of 45 s. moniliformis isolates (91% from humans) from the department of public health in berkeley, california, 50% of the isolates were from children < 9 years of age (graves and janda 2001) . in 75% of the human infections where a diagnosis was made, rat bite fever (rbf) was suspected; 83% of those suspected cases involved either known rat bite or exposure to rodents. two cases of rbf were attributed to exposurem in one case a squirrel, and in the second a mouse (graves and janda 2001). interestingly, > 9% of the cases could not be attributed to a rat bite or scratch, indicating that close contact with infected rodents can be sufficient to become infected (graves and janda 2001) . other reports have indicated that the disease can occur in individuals who have no history of rat bites, but reside or work in rat-infested areas or have pet rats with whom they have close contact (fordham et al. 1992; holroyd et al. 1988; rumley et al. 1987) . rat scratches can also be the source of the organism (edwards and finch 1986; shanson et al. 1985) . exposure to cats and dogs that prey on wild rodents may also be the source of the organisms. these organisms are present in the oral cavity and upper respiratory passages of asymptomatic rodents, usually rats (wilkins et al. 1988 ). mice can be infected with resulting morbidity and mortality due to arthritis and pneumonia. in one study, streptobacillus moniliformis was isolated as the predominant microorganism from the upper trachea of laboratory rats (paegle et al. 1976) . presumably the incidence of str. moniliformis is now lower in high-quality, commercially reared specific pathogen-free rats. surveys in wild mice indicate 0 to 25% infection with spirillum minus (hull 1955) . (2004). streptobacillus moniliformis incubation varies from a few hours to 2 to 10 days, whereas spirillum minus incubation ranges from 1 to 6 weeks (table 26-1) . fever is present in either form. inflammation associated with the bite and lymphadenopathy are frequently accompanied by headache, general malaise, myalgia, and chills (arkless 1970; cole et al. 1969; gilbert et al. 1971; mcgill et al. 1966) . the discrete macular rash that often appears on the extremities may generalize into pustular or petechial sequelae. arthritis occurs in 50% of all cases of s. moniliformis but is less common in spirillum minus. streptobacillus moniliformis may be cultured from serous to purulent effusion, which is recovered from affected larger joints. a total of 18 cases of endocarditis due to s. moniliformis were reported from 1915 to 2000 (shvartsblat et al. 2004 ). death has occurred in cases of s. moniliformis involving preexistent valvular disease or as a result of endocarditits in a previously healthy individual. infants can also die of the infection (sens et al. 1989) . if antibiotic treatmentmusually penicillin at doses of 400,000 to 600,000 daily for 7 daysmis not instituted early, complications such as pneumonia, hepatitis, pyelonephritis, enteritis, and endocarditis may develop (richter 1954 ). if endocarditis is present, the penicillin should be given parenterally at doses of 15 to 20 million units daily for 4 for 6 weeks. streptomycin and tetracyclines are also effective antibiotics for those individuals with penicillin-associated allergies. addition of streptomycin to standard therapy is also advised in cases where isolates of sir.. moniliformis are cell wall deficient (rupp 1992) . spirillum minus does not grow in vitro and requires inoculation of culture specimens into laboratory animals, with subsequent identification of the bacteria by dark-field microscopy. streptobacillus moniliformis grows slowly on artificial media but only in the presence of 15% blood and sera, usually 10% to 20% rabbit or horse serum incubated at reduced partial pressures of oxygen. sodium polyanethol sulfonate sometimes found in blood-based media because of its properties as a bacterial growth promoter should not be used due to its inhibitory effects on sir. moniliformis (lambe et al. 1973; shanson et al. 1985) . growth on agar consists of 1 to 2 mm gray, glistening colonies. the api zym diagnostic system can be used for rapid biochemical analysis and diagnosis. a pcr-based assay has also been described to diagnose sir. moniliformis (berger et al. 2001) . the genus salmonella are gram-negative bacteria with approximately 2000 serotypes. nontyphoidal salmonellosis is caused by any of these serotypes. other than salmonella typhi, the causative agent of typhoid fever, salmonellosis occurs worldwide and is important in humans and animals. s. typhi and salmonella choleraesuis have only one serotype, whereas the remaining 2000 serotypes are within the species salmonella enteritidis. references to the salmonella enteritidis serotypes are abbreviated such that "enteritidis" is dropped; for example, s. enteritidis serotype typhimurium is called salmonella typhimurium. salmonella typhimurium is the serotype most commonly associated with disease in both animals and humans. other serotypes most commonly reported from humans and animals are salmonella heidelberg, salmonella agona, salmonella montevideo, and salmonella newport. salmonellae are pathogenic to a variety of animals. salmonella are ubiquitous in nature and are routinely found in water or food contaminated with animal or human excreta. fecal-oral transmission is the primary mode for spreading infection from animal to animal or to humans. transmission is enhanced by crowding and poor sanitation. during the early 1900s, rodenticides containing live cultures of s. enteritidis were distributed on a large-scale basis by commercial and public health organizations in an attempt to eliminate feral rats. these cultures were known as "rat viruses" and were widely used in europe, england, and the united states as "rat poisons" (weisbroth 1979). however, enthusiasm for their use waned when it was discovered that the spread of the organisms couldn't be limited; predictably, the baiting program was implicated in several epidemics among exposed human populations (weisbroth 1979) . surprisingly, as late as the 1950s in england, s. enteritidis (serovar danzy) was isolated from adults living four miles apart. the source of infection was traced to contaminated cakes from a local bakery. mice that had acquired the (brown and parker 1957) . as with other fecal-oral transmitted diseases, control depends on eliminating contact with feces, food, or water contaminated with salmonella or animal reservoirs excreting the organism. salmonella survive for months in feces and are readily cultured from sediments in ponds and streams previously contaminated with sewage or animal feces. fat and moisture in food promote survival of salmonella. pasteurization of milk and proper cooking of food (56~ for 10 to 20 minutes) effectively destroys salmonella. municipal water supplies should be routinely monitored for coliform contamination (pavia and tauxe 1991) . clinical signs of salmonellosis in humans include acute sudden gastroenteritis, abdominal pain, diarrhea, nausea, and fever. diarrhea and anorexia may persist for several days. organisms invading the intestine may create septicemia without severe intestinal involvement; most clinical signs are attributed to hematogenous spread of the organisms. as with other microbial infections, the severity of the disease relates to the organism's serotype, the number of bacteria ingested, and the host's susceptibility. in experimental studies with volunteers, several serovars induced a spectrum of clinical disease ranging from brief enteritis to serious debilitation. incubation varied from 7 to 72 hours. cases of asymptomatic carriers, persisting for several weeks, were common (hull 1955) . salmonella are flagellated, nonsporulating, aerobic gramnegative bacilli that can be readily isolated from feces on selective media designed to suppress bacterial growth of other enteric bacteria. salmonella serotyping requires antigenic analysis (fox 1991) . salmonella gastroenteritis is usually mild and self-limiting. with careful management of fluid and electrolyte balance, antimicrobial therapy is not necessary. in humans, antimicrobial therapy may prolong rather than shorten the period that salmonella are shed in the feces (nelson et al. 1980; pavia and tauxe 1991) . in one double-blind placebo study of infants, oral antibiotics did not significantly affect the duration of salmonella carriage. bacteriologic relapse after antibiotic treatment occurred in 53% of the patients, and 33% of these suffered a recurrence of diarrhea, whereas none of the placebo group relapsed (nelson et al. 1980 ). tick-borne relapsing fever occurs primarily in foci in the western part of the united states, as well as other parts of the world. the disease is caused by at least 15 borrelia species and is transmitted to humans from a variety of rodents (chipmunks, squirrels, rats, mice, prairie dogs, hedgehogs) via soft ticks of the genus ornithodorus. of recent interest are the increasingly recognized enterohepatic helicobacter spp., which cause both hepatic and intestinal disease in mice (whary and fox 2004) . one of these, h. bilis, isolated routinely from mice, has been found using pcr-based assays in bile and gallbladder of chilean patients with chronic cholecystitis and in biliary cancers in japanese patients (fox et al. 1998; matsukura et al. 2002) . whether these new helicobacters will be linked to zoonotic transmission from wild or laboratory rodents will require further studies. pathogenic staphylococcus aureus of human phage type can cause clinical disease in mice and rats. this organism has been (davies and shewell 1964) 1 lab worker % not determined, alopecia, increased scaling on head (booth 1952) and back, 10 mice 1 bacteriologist 60 of 400, crusted or crustless plaques, circular with (cetin et al. 1965 ) prominent periphery; general alopecia; mortality in some mice 1 technician 20% colony with alopecia and scaly skin (dolan et al. 1958 ) 1 technician alopecia with crusting an erythema (povar 1965) introduced into spf barrier-maintained mouse colonies and spf rats and guinea pigs; the same phage type was isolated from their animal caretakers (davey 1962; shults et al. 1973) . colonization by normal s. aureus strains in the nasopharyngeal area of humans presumably minimizes the zoonotic potential of animal-originated s. aureus. as reviewed comprehensively in blank, reports of ringworm (favus) in the mouse began to appear in the european literature in the mid-nineteenth century and in the north american literature during the early twentieth century (blank 1957) . several of the early authors noted the similarities between the lesions of favus in the mouse and in humans. quincke, who is generally credited with isolating the causative agent which he named 3~-pilz (now trichophyton mentagrophytes), suggested that the infection in the mouse was also a source of infection of the cat, and thereby, of humans. earlier reports of murine ringworm referred to the causative agent as t. quinckeanum, but the successful crossing of t. quinckeanum with the perfect state of t. mentagrophytes, arthroderma behamiae, indicates that t. quinckeanum is not a distinct species (ajello et al. 1968) . a later study of the two varieties, t. mentagrophytes var. mentagrophytes and t. m. var. quinckeanum, noted that the conidia from both produced two morphological variants on cultivation (granular and fluffy), and these variants were a. behamiae type + and pathogenic (hejtmanek and hejtmankova 1989) . in addition to t. mentagrophytes, epidermophyton floccosum, mircrosporum gallinae, m. gypseum, m. canis, t. erinacai, t. schoenleini, and t. (keratinomyces) ajello have been reported as zoophilic dermatophytes that can infect mice and cause ringworm in humans (dvorak 1964; krempl-lamprecht and bosse 1964; marples 1967; papini et al. 1997; refai and ali 1970) . the dermatophytes are distributed worldwide and can involve a variety of small animal host species in addition to the mouse. chmel et al. (1975) conducted field studies in a wooded farm setting in czechoslovakia and detected an overall prevalence rate of 4.4% (57 positive of 1288) for t. mentagrophytes infection in 6 of 13 species sampled; the prevalence in mus musculus was 3.4%, with mice comprising 15.8% of the infections detected. of the species that harbored the infection, all frequented the barn or granary area; the seasonal incidence was highest during the winter months when the rodent carriers were more likely to seek harborage indoors. chmel et al. (1975) also analyzed patient data and demonstrated that t. mentagrophytes was the predominant isolate from those who did agricultural work, while t. verrucosum was the main isolate from individuals who worked with farm animals. also, human t. mentagrophytes infections were most common on the hands, wrist, forearm, face, and neck, unprotected skin sites readily contaminated by fodder, litter, or other materials while working in the barns. ringworm infections associated with the handling of bags of grain in which mice had been living have also been reported (alteras 1965; blank 1957) . ringworm infection in laboratory mice is often asymptomatic, remaining unrecognized until laboratory personnel become infected. early reports in the literature indicated that the prevalence of t. mentagrophytes was 80 to 90% among some laboratory mouse stocks (davies and shewell 1964; dolan et al. 1958 ). however, these reports predated the era of modem laboratory animal colony management marked by the commercial availability of cesarean-derived, barrier-maintained rodents. moreover, the modem production practices that have been universally adopted by the industry for decades and the use of microbarrier cages with appropriate technique have further reduced the opportunity for ringworm to become a significant problem in contemporary colonies. in recognition of this fact, none of the major commercial vendors in the united states survey their colonies for dermatophyte infections as part of routine health monitoring. sporadic cases of ringworm infections in rodents have been reported in the past three decades (hironaga et al. 1981; mizoguchi et al. 1986; papini et al. 1997) . programs involved in importing mice from sources that fail to meet contemporary rodent production and husbandry practices should consider screening mice for dermatophytes during the quarantine period. the ease of transmission of dermatophytes from animals to humans is well known and is a significant health hazard. laboratory mice, as well as other laboratory animal species, can harbor dermatophyte infection, with few or no visible skin lesions transmitting the infection to unsuspecting personnel (dolan et al. 1958) . transmission can occur through direct contact with the infected animal or through indirect contact with animal bedding or other materials in the environment of the contaminated animal room. rigorous facility and equipment sanitation has long been recognized as an essential element of an effective control program and should be undertaken in conjunction with efforts to treat valuable animals or to repopulate previously contaminated areas of a facility (davies and shewell 1964; dolan et al. 1958; mizoguchi et al. 1986 ). the importance of barrier protections by donning appropriate clothing, using gloves and other personal protective equipment, and modifying work practices to minimize skin exposure to dermatophytes has also been acknowledged for the prevention of transmission (dolan et al. 1958) . when prevention methods fail, allowing the introduction of dermatophyte infection into a mouse colony, and when transmission to humans occurs, clinical cases of dermatophytosis routinely respond well either to topical or systemic antifungal therapy. *found in laboratory animals that cause allergic dermatitis or from which zoonotic agents have been recovered in nature (see yunker 1964) . **wee, western equine encephalitis. +sle, st. louis equine encephalitis. ++rmse rocky mountain spotted fever. dermatophytosis or ringworm in humans can be asymptomatic and minor, often self-limiting and drawing little attention from the affected individual. the infection usually causes an expanding, scaly and erythematous inflammatory plaque on the skin that occasionally contains fissures or vesicles when severely eczematous. on the trunk and extremities, the lesion may consist of one or more circular lesions with a central clearing and sharply defined margins, forming a ring, and hence the name "ringworm" (fig. 26-1) (merlin et al. 1994) . other dermatophytes are named according to the sites of involvement on the body (e.g., tinea pedis for foot infections, tinea capitis for scalp infections). the dermatophyte infections of humans associated with direct or indirect contact from mice usually involve the body or extremities, especially the arms and hands. zoophilic t. mentagrophytes infection usually produces a highly inflammatory lesion and often undergoes rapid resolution. however, it can also produce furunculosis--deep infection of the hair follicles or widespread tinea corporism which is also seen in infections of e. f l o c c o s u m . in a laboratory-acquired infection with t. (keratinomyces) ajelloi, mice were the source of infection for a laboratory technican who developed small, grayish-white, scaly lesions on both hands. hand lesions yielded the organism, as did 2 of 250 apparently health mice (refai and ali 1970) . a. tapeworms a. reservoir and incidence although this parasite occurs in the mouse intestine, it is more commonly associated with rats and is especially common in wild norway (rattus norvegicus) and black (rattus rattus) rats throughout the world (faust and russell 1970; stone and manwell 1966; wardle and mcleod 1952 (voge and heyneman 1957) . larval development in tribolium sp. at 30~ requires 8 days. therefore, humans become infected only through ingestion of infected insects, such as flour beetles, which may contaminate rodent food or cereal marketed for human consumption. c. clinical signs the infection in humans is usually asymptomatic, but in moderate to heavy infections it may cause headaches, dizziness, abdominal discomfort, and diarrhea. the greatest length of an adult parasite removed from a patient was 1 meter. usually, adult parasites are 20 to 50 cm long and as much as 4 mm wide (markell et al. 1999 ). a. reservoir and incidence the dwarf tapeworm is a common parasite of both the wild house mouse and the laboratory mouse. as indicated earlier in the text, in most well-managed mouse colonies, r. nana incidence is low compared to earlier reports of its high incidence in rodent colonies (wescott 1982) . the estimate that 20 million humans in the world are infected was made many years ago but probably is an underestimate (markell et al. 1999) . surveys conducted in central europe report that this tapeworm in humans is more prevalent in warm than in temperate regions. an incidence of 10% has been noted in some south american countries (jelliffe and stanfield 1978) . it is most commonly diagnosed in children. diagnosis is made by observing characteristic eggs in the feces. b. mode of transmission r. nana is unique among tapeworms in that the adult worm develops after the egg is ingested. the hooked oncosphere then invades the intestinal mucosa and develops into a cysticeroid larva. rodentolepsis nana eggs can contaminate hands, be trapped on particulate matter, or be aerosolized, and then accidentally ingested. since no intermediate host is required, the eggs are readily infective for the reciprocal hosts (faust and russell 1970) . precautions against infection include strict personal hygiene, appropriate laboratory uniforms, and use of disposable gloves and face masks when handling contaminated bedding and feces. c. clinical signs the clinical picture of r. nana infection is quite cosmopolitan. in well-nourished persons, essentially no symptoms occur; the infection is noted when the proglottids or ova are seen in the stool. in other persons, the symptoms include headaches, dizziness, anorexia, inanition, pruritis of the nose and anus, periodic diarrhea, and abdominal distress. a tapeworm identified as r. nana was found in a tumor removed from the chest wall (jelliffe and stanfield 1978) . the diagnosis is based on identification of the characteristic eggs or proglottids in the stool. d. treatment praziquantfel, given orally in a single dose of 25 mg/kg body weight is the drug of choice. alternatively, niclosamide is given daily for 5 days because of the tissue phase of the parasite. the dose is 2 gm for adults and 1.5 gm for children > 34 kg, and 1.0 gm for children between 11 and 34 kg (markell et al. 1999) . recently, a parasite known to naturally colonize mice, r. microstoma, has been identified in the feces of humans living in the northwest of western australia (macnish et al. 2003) . although r. nana was the most common enteric parasite based on microscopic examination of feces, r. microstoma was identified as a mixed infection in 4 of 11 individuals by using a molecular-based assay consisting of restriction fragment length polymorphism of tapeworm dna as well as a sequencing of the pcr product of the internal transcribed spacer 1 region of ribosomal dna (macnish et al. 2002) . given that r. microstoma requires an intermediate host, tribolium confusum for its life cycle, it is understandable why it was not as common as r. nana in this study. however, given the morphological similarities of the eggs of r. nana and r. microstoma, the true prevalence of r. microstoma in humans won't be known until molecular techniques to differentiate the two species are utilized in future studies. syphacia obvelata is an ubiquitous parasite in both wild and laboratory mice. although parasitology texts report that syphacia is infectious to humans, this citation originates from a publication in 1919, in which two s. obvelata adult worms and eggs reportedly were found in the formalin preserved feces of a filipino child whose entire family of five was infected with h. nana (riley 1919) . no mention is made of the method of collecting the feces, nor is it known whether the feces could have been contaminated with murine feces or with the parasite and/or eggs. the only other report is an unpublished finding of s. muris eggs in the feces of two children and two rhesus monkeys, cited in a personal letter from dr. e. e. faust of tulane university, dated january 6, 1965 (stone and manwell 1966) . both of these cases may therefore be examples of spurious parasitism, but definitive information for that conclusion is lacking. regardless, no published information indicates that laboratory personnel have been infected by working with syphacia-infected mice. contamination of food or utensils or accidental ingestion of syphacia ova (e.g., via contaminated hands) could result in infection of humans. people working with infected mice probably ingest ova occasionally, but there is no evidence that this exposure results in active infection. because syphacia infection in humans has not been described, clinical signs have not been noted. there are striking differences in size between specimens of female s. obvelata and those of enterobius vermicularis, the pinworm, in humans (markell and voge 1965) . syphacia is 3.5 to 5.8 mm long, whereas the enterobius sp. female reaches a length of 8 to 13 mm. the male syphacia sp. measures 1.1 to 1.5 mm compared to 2.5 mm for enterobius. the size difference between the eggs of the two species is also marked: syphacia eggs are more than twice as long (125 gm versus 52/am as those of enterobius). it is unlikely therefore that syphacia sp. would be misdiagnosed as enterobius sp., assuming, of course, that the observer was aware of the size difference and measured the eggs. although many species of mites are found on laboratory mice, only ornithonyssus bacoti, the tropical rat mite, and liponysoides sanguineus, the house mouse mite, are vectors of human disease. ornithonyssus bacoti is seen in laboratory mice (fox 1982) ; l. sanguineus has been identified only on wild mice. bites from these mites, as well as from another mouse mite, haemalaelaps casalis, are responsible for allergic dermatitis, or local inflammation, in humans. ornithonyssus bacoti can be found on many rodents; the brown norway rat and the black roof rat are probably the primary host species (beaver and jung 1985) . since the time of the first report of human ornithonyssus bacoti-associated dermatitis in australia in 1913, and a 1923 report in humans in the united states, many other cases have continued to be described throughout the world (see table 26 -4) (charlesworth and clegern 1977; chung et al. 1998; dove and shelmire 1931; dowlati and maguire 1970; engel et al. 1998; fox 1982; haggard 1955; hetherington et al. 1971; riley 1940; theis et al. 1981; wainschel 1971; weber 1940) . ornithonyssus bacoti is an obligate bloodsucking parasite, usually tan but red when engorged with blood. both the male and female feed on a rodent as their preferred host. the female is 700 ~tm to 1 mm in length; the male is smaller (fig. 26-2) . eggs are laid in bedding or wall crevices by the female, which survives for about 70 days and feeds about every two days during this period. the mite has five developmental stages: adult, egg, nonfeeding larva, bloodsucking protonymph, and nonfeeding deutonymph. after feeding, the adults and protonymphs leave their host and seek refuge in cracks and crevices. the life cycle from adult to egg requires 7 to 16 days at room temperature. unfed protonymphs have survived for 43 days (brettman et al. 1981) . the mite often gains access to the premises on wild rodents and lives in crevices. if wild rodents are not readily available or are captured, the mite will seek blood elsewhere, either from the wild or laboratory rodent (if in an animal research facility) and/or humans. in some infestations, the rodent shows no clinical signs. however, in more chronic cases, dermatitis and anemia may develop. historically, this mite has been a troublesome parasite in certain laboratory animals, especially rats, mice, and hamsters (fox 1982; keefe et al. 1964 ). a. clinical signs tropical rat mites produce painful, pruritic lesions in humans. examination of patients often discloses papular lesions on the wrists, arms, abdomen, and chest. raised erythematous papules and nodules several millimeters to greater than 1 cm in size occur singly or in linear configuration ( fig. 26-3) . epidemiologically, cases usually occur in clusters that involve a common source of exposure to the mite. experimentally, cases have been shown to be a vector of pathogens. in the laboratory, mite transmission of various rickettsial species, pasteurella tularensis, and coxsackie virus between different laboratory animals has been shown (hopla 1951; petrov 1971; philip and hughes 1948; schwab et al. 1952) . affected individuals may be treated with topical lindane or treated symptomatically, given that the mite does not reside on humans for any extended periods. papular dermatitis will regress after a period of 7 to 10 days post-therapy. recurrence of ornithonyssus bacoti infestations is common unless the premises have been treated with an appropriate insecticide, and any feral rodents eradicated (engel et al. 1998 ). fleas are seldom found in laboratory mice but are common parasites of feral rodents. the oriental rat flea, xenopsylla cheopis, and another flea, nasopsyllus fasciatus, both naturally infest mice and rats; they are vectors for murine typhus. apparently, x. cheopis is easily established in animal facilities. at a midwestern u.s. university, it inhabited a room housing laboratory mice where, on two separate occasions, the flea caused distress by biting students (yunker 1964) . leptopsylla segnis, the mouse flea, bites humans and is a vector for plague and typhus, serious diseases in humans. also, l. segnis can serve as an intermediate host for the rodent tapeworms r. nana and r. diminuta, which can infect people. the flea's bite can also be irritating and cause allergic dermatitis. epidemiological perspective on the transmission of infectious diseases, principally rabies. the bite of the rat is far more powerful and more likely to be disfiguring than that of the mouse, and rat bites are known to be associated with the transmission of bacterial zoonoses such as rat bite fever and leptospirosis (see elsewhere in this chapter). one should assume that the mouse is also capable of transmitting these agents via bite. rabies transmission from small rodents in the wild occurs but is exceedingly rare (gdalevich et al. 2000) ; therefore, rabies is of concern only if experimental studies with the virus are being conducted in mice. mice can also transmit hantavirus infection and lymphocytic choriomeningitis virus infection via bites. anecdotally, most animal care and use programs report that rodent bites among personnel are a reasonably common occurrence that often are unreported to an institution's occupational medical service, despite the fact that bites inflict pain, produce anxiety, and may have significant health consequences. in addition to the hazard of zoonotic disease or local wound infection with pyogenic or toxin producing bacteria such as clostridium tetani, staphylococcus spp., streptococcus spp., escherichia coli, and bacillus subtilis, rodent bites, including those of the mouse, can induce a severe local allergic reaction or anaphylaxis in individuals previously sensitized to allergen (hesford et al. 1995; teasdale et al. 1983; thewes et al. 1999) . thus, bite wounds from mice should be immediately cleaned thoroughly and reported to the institutional occupational medical service to permit evaluation of the person's tetanus immunization status and need for additional local wound or other medical care. the need for additional training of bitten persons in animal handling may also be indicated. authoritative information on the incidence and impact of animal bites in the general population over the past several decades is scant, and reliable data on the incidence of mouse bites among personnel who work in laboratory animal facilities or among the general populace is lacking. there have been occasional studies on the occurrence and clinical characteristics of rat bites within urban populations, including a recent investigation of 622 bites over the period 1974-1996 that associated this phenomenon with urban blight, poverty, and unemployed populations (hirschhorn and hodge 1999) . traditionally, animal bites have received attention from the clinical perspective of wound management and complications and from the allergic skin and respiratory reactions to laboratory mice are very common in laboratory animal caretakers and technicians who work with these animals. a survey by lutsky (1987) demonstrated that three-fourths of all institutions with laboratory animals had animal care personnel with allergic symptoms. the prevalence of symptoms of laboratory animal-associated allergy (laa) among personnel working with laboratory animals has been estimated as between 10 and 46% in numerous recent studies, and among these individuals, approximately 10% are estimated to eventually proceed to the development of asthma (chan-yeung and malo 1994; eggleston and wood 1992; hollander et al. 1996; hunskaar and fosse 1990; knysak 1989; renstrom et al. 1994) . furthermore, other sources have suggested that among atopic individuals with preexisting allergic disease, up to 73% of persons exposed to lab animal allergens may eventually develop laa (committee on occupational health and safety in research animal facilities/national research council allergens 1997). the population at risk for work-related exposure to rodents was estimated at 90,000 (newill et al. 1986 ); this population has likely grown in the intervening years to the expanding populations of genetically modified mice that are used in contemporary biomedical research programs and require care. moreover, a recent study would seem to suggest that the risk of exposure to mouse allergens is not confined to those working in the laboratory animal facility environment. data analyzed from the first national survey of lead and allergens in housing in the united states demonstrated that 82% of homes of diverse types and income levels across geographic locations had evidence of mouse allergen; 57% had detectable levels on the kitchen floors specifically; and 22% had allergen concentrations greater that 1.6 ~tg/g of dust collected, a level previously correlated with the significantly increased rate of sensitization to mouse allergen (cohn et al. 2004) . the large number of staff at risk of exposure in the workplace or already presensitized, in combination with the substantial added costs to employers for the medical management, operational disruptions, and retraining efforts related to employees who develop laa and later proceed to asthma, should provide the impetus for many institutions to pay grater attention to this element of the occupational health and safety program (schweitzer et al. 2003) . the major allergen of the laboratory mouse is the mus m 1 protein, a member of the mouse major urinary proteins encoded by a multigene family consisting of approximately 35 genes (clark et al. 1984a (clark et al. , 1984b . the earlier literature on the subject of mouse allergy referred to the mouse urinary proteins (mups), whereas recent literature cites the specific protein (mus m 1) that is now known to be the primary offending allergen in the mup multigene family. the mus m 1 protein is in the lipocalin family of proteins that are produced in the saliva and liver and are excreted in the urine at levels 100 times higher than are present in mouse serum. lipocalins serve to bind small hydrophobic molecules and function biologically to transport vitamins, small volatile odorants, and pheromones conferring the characteristic odor to mouse urine (cavaggioni et al. 1999; flower et al. 1993; konieczny et al. 1997; santa et al. 1998; virtanen et al. 1999) . several studies have indicated that production of mus m 1 is under hormonal control and that the urine of male mice contains four-fold higher levels than the urine of female mice (hastie et al. 1979; lorusso et al. 1986; price and longbottom 1987) . in addition to being present in the saliva and urine, mus m 1 in the serum becomes incorporated into the pelt, conferring the allergenic property to mouse dander. the main allergens of many furred animals are structurally similar proteins within the lipocalin family, including those of the cow (bos d 2), horse (equ c 1), dog (can f 1), and rat (rat n 1) (virtanen et al. 1999) . the mus m 1 and rat n 1 lipocalin allergens, to which 90% of mouse and rat allergic individuals react, respectively, are closely related, sharing a 66% homology (clark et al. 1984a) . some have proposed that personnel exposed to laboratory animal allergens can be categorized into four basic risk groups based on their history of allergic disease and sensitization to animal proteins (committee on occupational health and safety in research animal facilities/national research council allergens 1997). these risk groups are (1) normal individuals, (2) atopic individuals with preexisting allergic disease, (3) asymptomatic individuals with ige antibodies to allergic animal proteins, and (4) symptomatic individuals with clinical symptoms upon exposure to animal allergens. individuals in the normal risk group do not have a history of allergic disease, and 90% will never develop symptoms of laa. if laa develops in individuals in the normal risk group, it usually appears during the first three years of exposure. however, infrequently individuals in this risk group who have remained free of laa for 10 or more years of exposure have developed a delayed onset of the condition (department of health and human services, national institute of occupational safety and health 1997). atopic individuals have a genetic predisposition for an exaggerated tendency to mount ige responses to common environmental allergens. atopic individuals have higher total levels of ige in the circulation and higher blood eosinophil counts compared to normal individuals, possibly as a result of the activation of cytokines involved in ige isotype switching, eosinophil survival, and mast cell proliferation (janeway et al. 2001 ). among atopic individuals, up to 73% of those exposed to allergenic animal proteins eventually develop symptoms (agrup et al. 1986) . in asymptomatic individuals with elevated circulating ige antibodies to animal allergens, up to 100% are at risk of developing allergic symptoms. of the individuals in risk groups that are already symptomatic for laa, approximately 33% will develop chest symptoms and 10% are likely to develop occupational asthma and face the prospect that continued exposure will result in permanent impairment. allergic rhinitis, allergic conjunctivitis, and contact urticaria are the most common disorders seen in laa (committee on occupational health and safety in research animal facilities/ national research council allergens 1997). clinically, allergic rhinitis and conjunctivitis present with the symptoms of sneezing, clear nasal discharge, nasal congestion, itchiness, and watery eyes. contact urticaria presenting as raised, circumscribed, erythematous lesions may also be present in laa patients who report an intense itchiness to the skin in the area of contact with the allergen. figs. 26-4 and 26-5 (fox and brayton 1982) illustrate the typical wheal and flare reaction on the skin provoked in an individual who had developed hypersensitivity to mouse urine over a period of several years and who was exposed by having a mouse with urine-contaminated feet walk over his arm (ohman 1978) . one large survey of laboratory animal workers summarized in the niosh alert (department of health and human services, national institute of occupational safety and health 1997) reported that of 5641 animal workers from 137 animal facilities, 23% developed allergic symptoms related to laboratory animals. of the workers reporting symptoms, 82% had nasal or eye symptoms, 46% had skin complaints, and 33% had asthma. patients who develop asthma as a more serious complication of laa manifest symptoms of wheezing, intermittent dyspnea or shortness of breath, cough, often nocturnal or in the early morning, and tightness of the chest. the key clinical sign in these patients is wheezing on auscultation, and physiological abnormalities include airflow obstruction, which may vary over time, bronchodilator responsiveness, and increased airway responsiveness (airway hyperreactivity) (tang et al. 2003) . though quite rare, generalized anaphylactic reactions that are potentially life threatening can occur in individuals highly sensitized to animal allergens. anaphylaxis may manifest as diffuse itching, hives, and swelling of the face, lips, and tongue. in some individuals, breathing becomes difficult owing to laryngeal edema, and others develop asthma and wheezing. laboratory animal-associated allergy is an example of the type i, ige antibody-mediated, immune reaction, and the reader should refer to other sources for a detailed discussion of the molecular mechanisms involved in developing this reaction (janeway et al. 2001 ). in the case of animal allergens, the usual route of initial exposure is airborne, although bite exposures (saliva) and direct contact with the skin can also become important in later clinical symptoms. in the type i reaction, upon exposure to antigen, which is often a protein or glycoprotein, the allergen is taken up and processed by cells of the innate and adaptive immune systems and by dendritic cells located in the mucosal-associated lymphoid cells, gut-associated lymphoid cells, and/or the dermis. the cytokine profiles of these cells favor the development of na'fve cd4 t cells into th2 cells that induce b cells to produce ige specific for the allergen. once the ige response is initiated, it can be further enhanced by basophils, mast cells, and eosinophils that also drive allergen-specific ige production (janeway et al. 2001) . ige is normally found only in low levels in the circulation because it binds to tissue mast cells and circulating basophils. in the sensitized individual, restimulation with the sensitizing allergen results in allergen binding to ige and the release of histamine and other chemical mediators from the mast cells and basophils. these mediators produce the array of clinical signs and symptoms that are characteristic of the allergy: itchiness, nasal congestion, sneezing, nasal and ocular drainage, coughing, wheezing, and shortness of breath. to establish the diagnosis of laa related to mouse exposure, the physician should begin by considering the strength of the history, physical examination findings, the temporal relationship between the patient symptoms and the environmental exposure to mice, and possibilities of alternative explanations for the patient's problems such as exposure to other potential allergens in the workplace or allergens of a nonoccupational nature. the development of clinical symptoms concomitant with or following exposure to an environment containing mice or mus m 1 laden mouse products should help in narrowing the number of allergens tested. the patient's family history of allergy is also very important to consider because atopy is a proven risk factor in developing laa (botham et al. 1995; meijer et al. 2002; venables et al. 1988 ). physical examination of the patient and clinical monitoring for the progression of allergic disease incorporate a number of approaches. pulmonary function tests such as bronchial hyperresponsiveness (in response to pharmacologic challenge with methacholine and not the specific allergen) and the forced expiratory volume in one second (fev1) are commonly used to evaluate the degree of airway impairment and the response to bronchodilators, glucocorticoids, and other therapeutic agents. radiographs may also be useful in patients with pulmonary involvement. routine laboratory tests may also aid in the characterization and management of the patient's condition, such as complete blood count and nasal smears for eosinophilia which is common in allergic individuals but also can be seen in those with perennial nonallergic rhinitis (dykewicz et al. 1998) . measurement of total serum ige has little value to the physician as an aid in distinguishing whether a particular patient has allergic disease, but it may offer some potential for the identifying of populations at risk for developing of laa as indicated in both prospective and cross-sectional studies of laboratory animal workers (hollander et al. 1996; renstrom et al. 1995) . use of the radioallergosorbent test (rast) for the detection of human ige antibodies of defined allergen specificity is also available for patient evaluation. however, the quality of the laboratory performing the in vitro rast assays, the specificity of the allergens used, and the potential for cross-reactivity are important considerations in adopting the rast as a diagnostic tool (hamilton 2003) . even when properly conducted, in vitro tests usually fail to detect a modest number of skin testpositive individuals, and on a per-test basis, skin tests have lower time and reagent costs (hamilton 2003) . clinicians agree that when properly performed, prick-puncture skin tests are generally considered the most convenient and least expensive screening method for detecting allergic reactions in most patients (demoly et al. 2003) . the valid interpretation of these tests relies on standardized allergens and methods, and negative prick-puncture tests may be confirmed by more sensitive intradermal techniques. even after falsepositive and false-negative tests have been eliminated, the proper interpretation of results requires thorough knowledge of the patient's history and physical findings. a positive skin test alone does not confirm a definite clinical sensitivity to an allergen in the asymptomatic patient but possibly predicts the onset of allergic symptoms. a positive skin test in conjunction with a history suggestive of clinical sensitivity strongly indicates the allergen as the cause of the disease (horak 1985) . strong positive skin tests along with a suggestive clinical history also correlate well with results of bronchial or nasal challenges with the antigen. the animal facility conditions and practices that contribute to mouse-associated laa as a serious and prevalent workplace hazard have received considerable study over the past several decades, enabling effective strategies for achieving control of exposures in most research animal care and use settings. in summary, these strategies involve exposure reduction through source reduction, containment of hazard through the use of modern equipment and engineering controls, and barrier protection with personal protective equipment. the mus m 1 allergen load in the environment is markedly increased when male mice are used in studies due to the fact that they excrete 4-fold higher levels of allergen in the urine than do female mice (lorusso et al. 1986 ). therefore, sources have recommended, that whenever scientifically possible, use of only female mice would be a means of reducing allergen load in the environment and minimizing the exposure of personnel (department of health and human services, national institute of occupational safety and health 1997; renstrom et al. 2001 ). furthermore renstrom et al. (2001) reported a three-fold higher rate of allergic sensitization in technicians who worked with male rodents. although this approach may be useful in a few studies, this method of source reduction would appear to have only very limited applicability across the broad scope of contemporary studies using mouse models. source reduction of mouse allergen is also achieved through reduction of animal density within an animal room (the number of animals per room volume) and through use of frequent, effective facility sanitation practices (department of health and human services, national institute of occupational safety and health 1997). the risk of exposure to mouse allergen varies by the type of animal-related activities conducted by personnel and by the type of animal housing systems and equipment containment devices employed in the use and maintenance of laboratory mice (gordon et al. 1997 (gordon et al. , 2001 schweitzer et al. 2003; thulin et al. 2002) . many studies have examined the different caging systems used for mouse housing, and the ability of each cage system design to reduce environmental allergen is well known (gordon et al. 2001; schweitzer et al. 2003) . the application of just a simple filter sheet top or fitted filter bonnet to an open cage is effective in reducing ambient allergen levels (reeb-whitaker et al. 1999) . however, studies indicate the clear superiority of individually ventilated caging (ivc) systems run under negative pressure for the purpose of controlling room allergen levels (gordon et al. 2001; reeb-whitaker et al. 1999; schweitzer et al. 2003) . gordon et al. (2001) suggested that the use of efficient negative ivc in combination with other engineering controls for allergen containment during procedures and waste processing would potentially produce a virtually allergen-free work environment. when negative pressure ivc housing is not available, the placement of cages in a hepafiltered, ventilated cabinet is effective at reducing room allergen loads (thulin et al. 2002) . gordon et al. (1997) reported that individuals who have direct contact with mice (animal technicians) have the highest exposure, followed by those who have intermittent contact with anesthetized animals or mouse tissues (scientists and necropsy technicians), followed by those with indirect contact (office workers or histology technicians). the specific animal husbandry activities that are known to result in high exposure of personnel to mouse allergen are cage-changing activities, including animal transfer, stacking dirty cages, and manual emptying of cages; handling animals directly (particularly males); and room sweeping (gordon et al. 1997) . for each of these activities, use of improved containment equipment and changes in equipment handling procedures are effective in the controlling the allergen hazard and should be encouraged. for example, use of ventilated cabinets or biological safety cabinets during cage changing and animal handling is effective in conjunction with the use of microisolation cages (gordon et al. 2001; schweitzer et al. 2003; thulin et al. 2002) . containment equipment has also been designed for the capture of airborne allergens generated when the bottom of one dirty cage is placed into the opening of another to stack the cages for transport to the cage wash area or when dirty bedding is removed prior to cage washing (gordon et al. 1997; kacergis et al. 1996) . room cleaning with a vacuum equipped with hepa filtration followed by mopping with a damp mop also aids in reducing the environmental allergen load and personnel exposure (kacergis et al. 1996) . use of personal protective equipment and dedicated work clothing for personnel involved in high-exposure activities is an important asset in reducing allergen exposure. it is important for the work clothing to remain at work, as evidenced by the finding that children of laboratory animal workers had a higher incidence of clinical signs during provocative testing, positive skin tests, and ige specific to laboratory rodents than did the children of parents who worked in other occupations (krakowiak et al. 1999) . full sleeve protection and gloves should be worn to prevent the urticarial reactions in persons who are highly sensitive to the mus m 1 allergen. personnel should also be provided with respiratory protection and eye or face protection when warranted. either filtering facepiece particulate respirators (n95 equivalent) or powered air purified respirators are effective in reducing exposure and alleviating clinical symptoms (schweitzer et al. 2003; thulin et al. 2002) . special attention must be paid to the selection and fitting of n95 filtering facepiece particulate respirators to ensure proper function (morbidity and mortality weekly report 1998). when the elimination of mouse allergen exposure in the workplace is not achieved through the use of engineering controls, work practices, and personal protective equipment, allergic reactions in persons sensitive to mus m 1 can be managed with pharmacological agents that have a long history of use for this condition. these include antihistamines, topical ~-adrenergic agents (bronchodilators), cromolyn sodium as a nasal spray, and intranasal potent glucocorticoids (austen 2004) . prophylaxis in patients with mild symptoms is often provided by topical cromolyn sodium on a continuous basis, supplemented with the intermittent use of antihistamines often at bedtime. the selection of the antihistamine has been an area of considerable discussion, and the reader should refer to casale et al. (2003) for further insights into this matter. in more serious cases, potent topical glucocorticoids may be necessary for alleviating clinical signs. immunotherapy, or hyposensitization, is typically reserved for patients who are unable or unwilling to escape the allergen provoking the response. although allergy to the dog or the cat can be ameliorated by immunotherapy (norman 1998 (norman , 2004 , the infrequent reports in the literature of immunotherapy for allergy to mice and other small laboratory animals have failed to establish the usefulness of this approach for the control of allergy to these species (sorrell and gottesman 1957; wahn and siraganian 1980) . the progress made in the past two decades in the evolution of health care systems responsible for the monitoring, control, and elimination of infectious diseases in laboratory mice as well as the advancements in the facilities, equipment, and techniques used to maintain mice in contemporary animal care and use programs, has vastly reduced the likelihood that personnel will encounter zoonotic diseases or other health hazards in the laboratory under most circumstances. continued program success in the control of mouse-associated hazards relies on the use of well-designed and maintained animal facilities, exclusion of wild rodents and other vermin, and quality control in animal care and veterinary care practices. in unique experimental situations that place personnel at a high risk of exposure to mouse-associated hazards, institutional review should ensure that procedures are carefully planned and conducted using personal protective equipment for worker safety. allergy to laboratory animals in laboratory technicians and animal keepers the relationship of trichophyton quinckeanum to trichophyton mentagrophytes alteras, i. 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respiratory infections and infectious mononucleosis exposure to leptospira icterohaemorrhagiae in inner-city and suburban children: a serologic comparison department of health and human services. national institute of occupational safety and health ringworm epizootics in laboratory mice and rats: experimental and accidental transmission of infection the tropical rat mite, liponyssus bacoti hirst 1914: the cause of a skin eruption of man, and a possible vector of endemic typhus fever rat mite dermatitis: a family affair rickettsia typhi (murine typhus) geophites zoophilic and anthropohilic dermatophytes: a review lymphocytic choriomeningitis outbreak associated with nude mice in a research institute diagnosis and management of rhinitis: complete guidelines of the joint task force on practice parameters in allergy pathogenicity of hantaan virus in newborn mice: genetic reassortant study demonstrating that a single amino acid change in glycoprotein g1 is related to virulence characterisation and antibiotic susceptibilities of streptobacillus moniliformis management of allergies to animals family bunyaviridae tropical rat mite dermatitis: case report and review leptospirosis craig and faust's clinical parasitology structure and sequence relationships in the lipocalins and related proteins parasites of laboratory animals rat bite fever without the bite man's worst friend (the rat) outbreak of tropical rat mite dermatitis in laboratory personnel zoonotic diseases. campylobacter infections and salmonellosis. semin zoonoses and other human health hazards hepatic helicobacter species identified in bile and gallbladder tissue from chileans with chronic cholecystitis infections transmitted from large and small laboratory animals leptospirosis ballum contracted from pet mice bear canyon virus: an arenavirus naturally associated with the california mouse (peromyscus californicus) leptospiral serotype distribution lists according to host and geographic area serological evidence of infection with sendai virus in england rabies in israel: decades of prevention and a human case peculiarities of the 1956 influenza outbreak in vladisvostok due to d virus rat-bite fever elimination of mouse allergens in the working environment: assessment of individually ventilated cage systems and ventilated cabinets in the containment of mouse allergens measurement of exposure to mouse urinary proteins in an epidemiological study rat-bite fever (streptobacillus moniliformis): a potential emerging disease lymphocytic choriomeningitis virus in mouse neoplasms rat mite dermatitis in children laboratory tests for allergic and immunodeficiency diseases antibodies to mouse hepatitis viruses in human sera multiple genes coding for the androgen-regulated major urinary proteins of the mouse lymphocytic choriomeningitis virus teleomorphs and mating types in trichophyton mentagrophytes complex anaphylaxis after laboratory rat bite: an occupational hazard rat mite dermatitis outbreak of lymphocytic choriomeningitis virus infections in medical center personnel trichophyton mentagrophytes skin infections in laboratory animals as a cause of zoonosis identification of risk factors in rat bite incidents involving humans cat and dog allergy and total ige as risk factors of laboratory animal allergy streptobacillus moniliformis polyarthritis mimicking rheumatoid arthritis: an urban case of rat bite fever experimental transmission of tularemia by the tropical rat mite manifestation of allergic rhinitis in latent-sensitized patients. a prospective study the contamination of laboratory animals with lymphocytic choriomeningitis virus rickettsialpox--a newly recognized rickettsial disease. v. recovery of rickettsia akari from a house mouse (mus musculus) rickettsialpoxma newyly recognized rickettsial disease. iv. isolation of a rickettsia apparently identical with the causative agent of rickettsialpox from allerdermanyssus sanguineus, a rodent mite rickettsialpox--a newly recognized rickettsial disease. i. isolation of the etiological agent diseases transmitted from animals to man allergy to laboratory mice and rats: a review of the pathophysiology, epidemiology and clinical aspects the etiology, mode of infection and specific therapy of weil's disease sendai virus health care for research animals is essential and affordable lymphocytic choriomeningitis virus. a neglected pathogen of man allergy and hypersensitivity diseases of children in the subtropics and tropics serologic evidence of american experience with newborn pneumonitis virus rabies virus air quality in an animal facility: particulates, ammonia, and volatile organic compounds prevalence of rabies virus and hantaan virus infections in commensal rodents and shrews trapped in bangkok ornithonyssus bacoti (hirst) infestation in mouse and hamster colonies pathogenesis of hantaan virus infection in suckling mice: clinical, virologic, and serologic observations animal aeroallergens the major dog allergens, can f 1 and can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms increased detection of rickettsialpox in a new york city hospital following the anthrax outbreak of 2001: use of immunohistochemistry for the rapid confirmation of cases in an era of bioterrorism allergy to laboratory animals in children of parents occupationally exposed to mice, rats and hamsters epidermophyton floccosum (harz) langeron and milochevitsch als spontanin fektion bei mausen newborn virus pneumonitis (type sendai) ii. the isolation of a new virus newborn virus pneumonitis (type sendai) ii. the isolation of a new virus possessing hemagglutinin activity streptobacillus moniliformis isolated from a case of haverhill fever: biochemical characterization and inhibitory effect of sodium polyanethol sulfonate lymphocytic choriomeningitis virus infection in a province of spain: analysis of sera from the general population and wild rodents weil's syndrome in a zoologist zoonoses in common laboratory animals immunologic and biochemical properties of the major mouse urinary allergen (mus m 1) an outbreak of hepatitis in marmosets in a zoological collection a worldwide survey of management practices in laboratory animal allergy trichophyton mentagrophytes in mice: infections of humans and incidence amongst laboratory animals a molecular phylogeny of nuclear and mitochondrial sequences in hymenolepis nana (cestoda) supports the existence of a cryptic species detection of the rodent tapeworm rodentolepis (=hymenolepis) microstoma in humans. a new zoonosis? medical parisitology non-domestic animals in new zealand and in rarotonga as a reservoir of the agents of ringworm reverse genetics of the largest rna viruses association between helicobacter bilis in bile and biliary tract malignancies: h. bilis in bile from japanese and thai patients with benign and malignant diseases in the biliary tract lassa fever. effective therapy with ribavirin rat-bite fever due to streptobacillus moniliformis growth in sucklingmouse brain of "ibv-like" viruses from patients with upper respiratory tract disease detection of workers sensitised to high molecular weight allergens: a diagnostic study in laboratory animal workers infectious and parasitic disease persistent hantavirus infections: characteristics and mechanisms ecologic studies of rodent reservoirs: their relevance for human health a new transmissible viral hepatitis of marmosets and tamarins epidemiologic notes and report: lymphocytic choriomeningitis virus-georgia mmwr seroepidemiological survey of lymphocytic choriomeningitis virus in wild house mice in china with particular reference to their subspecies pathogens of house mice on arid boullanger island and subantarctic macquarie island paramyxovirus replication and pathogenesis. reverse genetics transforms understanding sars: lessons learned from other coronaviruses treatment of salmonella gastroenteritis with ampicillin, amoxicillin, or placebo preemployment screening for allergy to laboratory animals: epidemiologic evaluation of its potential usefulness contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses morphological characteristics and nomenclature of leptospira (spirochaeta) icterohemorrhagiae (inada and ido) immunotherapy: past and present immunotherapy: 1999-2004 allergy in man caused by exposure to mammals hantavirus-specific antibodies in rodents and humans living in kuwait rickettsialpox in new york city: a persistent urban zoonosis microbial flora of the larynx, trachea, and large intestine of the rat after long-term inhalation of 100 per cent oxygen survey of dermatophytes isolated from the coats of laboratory animals in italy nontyphoidal. in bacterial infections of humans arenaviruses on the role of the mite ornithonyssus bacoti hirst as a reservoir and vector of the agent of tularemia the tropical rat mite, liponyssus bacoti, as an experimental vector of rickettsial pox recombinant sendai virus for efficient gene transfer to human airway epithelium ringworm (trichophyton mentagrophytes) infection in a colony of albino norway rats fleas of public health importance and their control allergy to mice. i. identification of two major mouse allergens (ag 1 and ag 3) and investigation of their possible origin cultivation of different viruses in tick tissue culture control strategies for aeroallergens in an animal facility laboratory acquired infection with keratinomyces ajelloi working with male rodents may increase risk of allergy to laboratory animals allergic sensitization is associated with increased bronchial responsiveness: a prospective study of allergy to laboratory animals prospective study of laboratory-animal allergy: factors predisposing to sensitization and development of allergic symptoms reovirus serotype 3 infection in infants with extrahepatic biliary atresia or neonatal hepatitis incidence of rat bites and rat bite fever in baltimore primates. in laboratory animal medicine a mouse oxyurid, syphacia obvelata, as a parasite of man rat mite dermatitis in minnesota viral meningitis and encephalitis: traditional and emerging viral agents lymphocytic choriomeningitis virus in southern france: four case reports and a review of the literature rat-bite fever as a cause of septic arthritis: a diagnostic dilemma streptobacillus moniliformis endocarditis: case report and review rickettsia akari newborn virus pneumonitis (type sendai) i. report: clinical observation of a new virus pneumonitis of the newborn a bovine dander allergen, comparative modeling, and similarities and differences in folding with related proteins distribution of the virus lymphocytic choriomeningitis virus in west germany the tropical rat mite (liponyssus bacoti) as an experimental vector of coxsackie virus reducing exposure to laboratory animal allergens fatal streptobacillus moniliformis infection in a two-month-old infant comparison of media with and without 'panmede' for the isolation of streptobacillus moniliformis from blood cultures and observations on the inhibitory effect of sodium polyanethol sulphonate staphylococcal botryomycosis in a specific-pathogen-free mouse colony fatal rat bite fever in a pet shop employee a serologic survey for viruses and mycoplasma pulmonis among wild house mice (mus domesticus) in southeastern australia a survey of house mice from iowa swine farms for infection with leptospira interrogans serovar bratislava lcm: associated human and mouse infections mouse allergy: case report studies on the pathogenesis of a hitherto undescribed virus (hepato-encephalomyelitis) producing unusual symptoms in suckling mice reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease isolation of an arenavirus from a marmoset with callitrichid hepatitis and its serologic association with disease identification, using sera from exposed animals, of putative viral antigens in livers of primates with callitrichid hepatitis cdna sequence analysis confirms that the etiologic agent of callitrichid hepatitis is lymphocytic choriomeningitis virus the laboratory diagnosis of leptospirosis leptospirosis (ballum) contracted from swiss albino mice elimination of leptospira ballum from a colony of swiss albino mice by use of chlortetracycline hydrochloride potential helminth infections in humans from pet or laboratory mice and hamsters comparison of diagnostic technics for the detection of leptospirosis in rats spirochetal infections in middleton's allergy: principles and practice anaphylaxis after bites by rodents incidence of murine virus antibody in humans in contact with experimental animals tropical rat mite dermatitis. report of six cases and review of mite infestations anaphylactic reaction after a mouse bite in a 9-year-old girl reduction of exposure to laboratory animal allergens in a research laboratory viruses transmissible from laboratory animals to man leptospirosis. in crc handbook series in zoonoses orthoreoviruses. in principles and practices of infectious diseases laboratory animal allergy in a pharmaceutical company important animal allergens are lipocalin proteins: why are they allergenic? development of hymenolepis nana and hymenolepis diminuta (cestoda: hymenolepididae) in the intermediate host tribolium confusum efficacy and specificity of immunotherapy with laboratory animal allergen extracts rat mite bite the zoology of tapeworms placebo-controlled trial of intravenous penicillin for severe and late leptospirosis bacterial and mycotic diseases helminths natural and experimental helicobacter infections rat-bite fever in a gerbil breeder congenital lymphocytic choriomeningitis virus syndrome: a disease that mimics congenital toxoplasmosis or cytomegalovirus infection natural occurrence of leptospira ballum in rural house mice and in an opossum on the biological character of hvj akitsugu strain and its pathogenicity in human beings as revealed after experimental inoculation of it in volunteers infections of laboratory animals potentially dangerous to man: ectoparasites and other arthropods, with emphasis on mites influenza d in early infancy key: cord-021555-rrverrsj authors: delano, margaret l.; mischler, scott a.; underwood, wendy j. title: biology and diseases of ruminants: sheep, goats, and cattle date: 2007-09-02 journal: laboratory animal medicine doi: 10.1016/b978-012263951-7/50017-x sha: doc_id: 21555 cord_uid: rrverrsj nan since the first edition of this book, the use of ruminants as research subjects has changed dramatically. formerly, large animals were primarily used for agricultural research or as models of human diseases. over the past decade, ruminants have continued in their traditional agricultural research role but are now extensively used for studies in molecular biology, genetic engi-british stock with egyptian and indian goats. this breed is relatively heat tolerant and produces milk with the highest butterfat (about 4-5%). fiber breeds include the angora and the cashmere. the angora, the source of mohair, originated in turkey. the cashmere breed is found primarily in mountainous areas of central asia. the la mancha, a newer breed of dairy goat first registered in the united states in 1958, has rudimentary ears that are a genetically dominant distinguishing characteristic of the breed. the meat breeds include the boer, sapel, ma tou, kambling, and pygmy. the pygmy goat is small and is sometimes used for both meat and milk. the mubend of uganda and the red sokoto of west africa produce quality skins for fine leather (smith and sherman, 1994) . most breeds of cattle are classified as "dairy" or "beef"; a few breeds are considered "dual-purpose." common dairy breeds in the united states include holstein-friesian, brown swiss, jersey, ayrshire, guernsey, and milking shorthorn. holsteins have the largest body size, whereas jerseys have the smallest. of breeds in temperate regions, jerseys have been considered to be the most heat tolerant, but holsteins have been found to adapt to warmer climates. there are many beef breeds. the more common in the united states include angus (also called aberdeen-angus), hereford (both polled and horned), and simmental (briggs and briggs, 1980; schmidt et al., 1988) . breeds indigenous to other continents, such as the cape buffalo, have been found to have unique innate immune characteristics that protect them from endemic trypanosomiasis (muranjan et al., 1997) . more detailed information regarding these and other ruminant breeds is available in briggs and briggs (1980) . "rare" or "minor" breeds of sheep, goats, and cattle are studied for their genetic and production characteristics. discussions of these and efforts at conservation are described in detail elsewhere (national research council, 1993) . several terms are unique to ruminants. in relation to sheep, a ewe is the female, and a ram is the adult intact male. a lamb is the young animal, and ram lamb and ewe lamb are commonly used terms. a wether is a castrated male. the birthing process is referred to as lambing. with respect to goats, a doe or nanny is the female. a buck or billy is the adult intact male. a kid or goatling is a young goat. a young male may be referred to as a buckling, and a young female may be referred to as a doeling. a castrated male in this species is also called a wether. the birthing process is called kidding. with respect to cattle, an adult female is a cow, and an adult male is a bull. a calf is a young animal. a heifer is a female who has not had her first calf. a steer is a castrated male. calving refers to the act of giving birth. ruminants have been used as research models since the inception of the land grant college system, first in production agriculture and now also in basic and applied studies for the anatomic and physiologic sciences and in biomedical research for a variety of purposes. healthy, normal young ruminants serve as models of cardiac transplantation and as preclinical models for evaluation of cardiac assist or prosthetic devices, such as vascular stents and cardiac valves (salerno et al., 1998) . for many years, ruminants have been useful research subjects for reproductive research, such as research on embryo transfer, artificial insemination, and control of the reproductive cycle (wall et al., 1997) . several important milestones in gene transfer, cloning, nuclear transfer, and genetic engineering techniques have been developed or demonstrated using these species (ebert et al., 1994; schnieke, 1997; cibelli et al., 1998a,b) (see fig. 1 ). one of many proposed uses of genetically engineered ruminants is the production of proteins that will be secreted in the milk and later isolated (ebert et al, 1994; memon and ebert, 1992) . healthy sheep and goats are also often used for antibody production (hanly et al., 1995) . genome mapping developed rapidly during the 1990s; extensive information is available and is increasing for sheep and cattle (broad et al., 1998; womack, 1998) . sheep are often selected for studying areas such as ruminant physiology and nutrition. these animals provide obvious bene-fits over the use of cattle in research from the standpoint of size, ease of handling, cost of maintenance, and docile behavior. sheep are also widely used models for basic and applied fetal and reproductive research (buttar, 1997; rees et al., 1998; ross and nijland, 1998) . the species is used for investigating circadian rhythms related to day length (lehman et al., 1997) , and the interaction between olfactory cues and behavior (kendrick et al., 1997) . the number and diversity of natural-and induceddisease research models in sheep are great and increasing. natural models include congenital hyperbilirubinemia/hepatic organic anion excretory defect (dubin-johnson syndrome) in the corriedale breed, congenital hyperbilirubinemia/hepatic organic anion uptake defect (gilbert syndrome) in the southdown breed, glucose-6-phosphate dehydrogenase deficiency in the dorset breed, gm~ gangliosidosis in the suffolk breed, and pulmonary adenomatosis (jaagsiekte) in many breeds (hegreberg, 198 l a) . induced models include arteriosclerosis, hemorrhagic shock, copper poisoning (wilson's disease), and metabolic toxocosis (hegreberg, 198 lb) . goats are used in a wide variety of agricultural and biomedical disciplines such as immunology, mastitis, nutrition, and parasitology research. vascular researchers select the goat because of the large, readily accessible jugular veins. goats with inherited caprine myotonia congenita ("fainting goats") have been used as a model for human myotonia congenita (thomsen's disease) (kuhn, 1993) . a line of inbred nubians serves as models for the genetic disease [3-mannosidosis and prenatal therapeutic cell transplantation strategies (lovell et al., 1997) . (these disorders are discussed in more detail in section iii,b,1.) goats are used as a model for osteoporosis research (welch et al., 1996) . cattle are often used as a source of ruminal fluid for research, teaching, or treatment of other cattle, by placing a permanent fistula in the left abdominal wall to allow sampling of ruminal fluid (dougherty, 1981) . cattle also serve as models of many infectious diseases, including zoonoses, and several inherited metabolic diseases. this species is useful for the basic and comparative research on the pathogenesis and immunology of inherited and infectious diseases. bovine trichomoniasis, caused by tritrichomonas (trichomonas)fetus, has been identified as a useful model for the human infection by trichomonas vaginalis (corbeil, 1995) . inherited cardiomyopathies have been found in the holstein-friesian, simmental-red holstein, black spotted friesian, and polled hereford with woolly coat (weil et al., 1997) . lipofuscinosis has been identified in ayrshires and friesians, and glycogenesis in shorthorns and brahmans. metabolic diseases such as hereditary orotic aciduria and hereditary zinc deficiency have been characterized in holstein-friesian or friesian cattle. holstein cattle also serve as a model for leukocyte adhesion deficiency syndrome (afip, 1995) . common breeds of normal, healthy ruminants are usually readily available, although seasonality may play a role, as noted below. agricultural sources and reputable farms may be located through land-grant universities or agricultural schools, cooperative extension and 4-h networks, regional ruminant breeders' associations, and farm bureaus. commercial sources of purposebred animals are found in technical publications and annual listings of research animal vendors. breeds carrying genetic traits of interest, either as animal models or as valuable production characteristics, may be located through literature or internet searches, animal science societies, breed or livestock conservation associations, and information resources such as the armed forces institute of pathology. organizations such as the institute for laboratory animal research (ilar), national center for research resources (ncrr), or the animal welfare information center (awic) may also serve as information sources about the animals needed. purpose-bred research sheep and goats are available from commercial vendors and are usually maintained in registered facilities under federal standards that are also acceptable to research animal accrediting agencies. these commercial animals are frequently described as specific pathogen-free (spf) and housed as biosecure or closed flocks. animal health programs are in place, and health reports or other quality assurance reports are usually available on request. agricultural sources of either small ruminant may be acceptable, but specific research needs may not have been addressed or may not be understood. lambs, kids, and milking goats may be difficult to locate in fall and winter months because most breeds of sheep and goats are seasonal breeders. management practices exist, however, to extend the breeding and milking seasons. most cattle used as animal models in research in the united states are from one of the dairy breeds, usually holstein, because this breed is now the most common. purpose-bred, specific pathogen-free research cattle are not typically available. because of selection and the management of dairy production units, calves and young stock are available year-round. availability of young beef cattle is more seasonal, according to production cycles typically followed by that industry. auction barns or sales are not appropriate sources for research ruminants. many of these animals are culls and will be poor-quality research subjects. they may be in poor body condition and stressed, may be sources of disease, and may contaminate other healthy animals, as well as the research facility. selection of the suppliers should be made only after research needs have been carefully considered. consistently working with and buying directly from as few sources as possible are best. certain types of research (i.e., agricultural nutrition studies) may better be served by selecting animals from local agricultural suppliers rather than commercial vendors located in a different geographical area. the selection of sources for research ruminants includes scrutiny of flock or herd record keeping; health monitoring, vaccination, and preventive medicine programs (including hoof care); production standards and management practices consistent with the industry; management of the breeding flock or herd; sanitation and waste handling programs; vermin and insect control measures (especially for flies and other flying insects); rearing programs for and condition of young stock; the location, health, and condition of the other animals on the premises; intensity of housing; and animal housing facilities. preliminary and periodic visits to the source farms should be conducted. it is important to establish a good relationship with the local attending large-animal veterinarians, who will be valuable resources for current approved therapies and practices. they may need to be oriented on the specific requirements of animal research. creative ways can be used to initiate and foster a good working relationship between the agricultural supplier and the research facility. supplying the vaccines or dewormers required for flock health programs, providing services such as quarterly serological testing or fecal examinations for the herd or flock, and paying a premium (rather than market price) for animals that meet the quality criteria established for the research animals are often helpful. a set of testing standards can be developed based on one high-quality supplier, and then flocks or herds can be "qualified" based on those standards. qualifying entails evaluations utilizing the facility and management aspects mentioned above and testing either a percentage of the herd or flock or the entire herd or flock for a number of infectious agents. the testing regimen itself should be carefully developed and evaluated. once qualified, each source farm should be reevaluated periodically to maintain its status. slaughter checks may be appropriate; otherwise necropsy of sentinel animals may be required. selected animals undergoing screening tests should be quarantined from the rest of flock or herd while awaiting test results. vaccination and deworming regimens can be instituted during these quarantine periods. a second quarantine should occur when animals arrive at the research facility. the animal screening process also depends on the origin of the animal (state, country) and the scientific program. federal and state regulations must be followed. socialization of the animals at the source facility should also be considered in terms of ease of handling and safety for personnel in the confinement of the research lab, barn, or farm. for example, frequently handled calves will be easier to manage, and adult dairy goats that have been acclimated to human contact are preferable. several texts provide information on industry standards for flock and herd management and preventive medicine strategies that can provide helpful orientation to those unfamiliar with these aspects. these references also provide information regarding vaccination products licensed for use in ruminants and typical herd and flock vaccination parasite control schedules ("current veterinary therapy," 1986 , 1999 "council report," 1994; "large animal internal medicine," 1996; smith and sherman, 1994) when designing a vaccination program during qualification of a source or at the research facility, it is important to evaluate the local disease incidence and the potential for exposure. vaccination programs should be conducted with an awareness of duration of passive immunity and stresses in ruminants' lives (e.g., weaning, grouping, management changes, and shipping) that may impair immunity or increase susceptibility to infectious diseases. it is also prudent to evaluate the cost-effectiveness of vaccination; labor and vaccine expenses may be much higher than the potential animal morbidity or mortality for diseases in a particular locality. not all of the vaccines mentioned subsequently will be necessary in all herds or flocks. vaccination needs for research animals will also depend on the local disease history, intent of the research, the age of the animals needed for research, and the length of time the animals will be housed. typical health screening programs for sheep include q fever (coxiella burnetii); contagious ecthyma; caseous lymphadenitis (corynebacterium pseudotuberculosis); johne's disease (mycobacterium paratuberculosis); ovine progressive pneumonia; internal parasitism such as nasal bots, lungworms, and intes-tinal worms; and external parasitism such as sheep keds. each supplier should be queried about vaccination programs for bluetongue, brucella ovis, campylobacter spp., chlamydia (enzootic abortion of ewes), clostridial diseases, pneumonia complex (parainfluenza 3, pasteurella haemolytica, and p. multocida), ovine ecthyma, rabies, dichelobacter (bacteroides) nodosus, arcanobacterium pseudotuberculosis, bacillus anthracis, and fusobacterium necrophorum. because of the limited number of biologics approved for small ruminants, products licensed for cattle have been used with success in sheep, and some licensed for sheep are used in goats ("council report," javma, 1994) . in some cases, approved feed additives, such as coccidiostats, are fed to sheep. the basic screening profile for goats should include q fever (coxiella burnettii), caprine arthritis encephalitis (cae), brucellosis, tuberculosis, and johne's disease (mycobacterium paratuberculosis) . goats may also be tested for caseous lymphadenitis, contagious ecthyma, or mycoplasma as needed. herd vaccination programs may include immunizations against tetanus and other clostridial diseases, chlamydia, campylobacter, contagious ecthyma, caseous lymphadenitis, corynebacterium pseudotuberculosis, and escherichia coli. cattle herds should be screened for johne's disease, brucellosis, tuberculosis, respiratory diseases, internal and external parasitism, and foot conditions such as hairy heel warts and foot rot. determination of the status of the herd with respect to bovine leukemia virus (blv) may be worthwhile. herd programs may include essential or highly recommended vaccines against bovine viral diarrhea virus (bvdv), infectious bovine rhinotracheitis virus (ibrv), bovine respiratory syncytial virus (brsv), parainfluenza 3 (pi-3), leptospira pomona, tritrichomonas fetus, rotavirus, coronavirus, campylobacter (vibrio) , pasteurella haemolytica and p. multocida, and brucella abortus. other vaccination programs, dependent on herd status, endemic diseases, or geographic location, may include immunizations against the clostridial diseases, moraxella bovis (pinkeye), fusobacterium necrophorum (foot rot), staphylococcus aureus (mastitis), haemophilus somnus, rabies, tetanus, bacillus anthracis, enterotoxigenic e. coll anaplasma, and other leptospira species. some products considered to have limited efficacy include vaccines against salmonella dublin and s. typhimurium. some autogenous vaccines may be more effective than the commercially available products--for example, the bovine papillomavirus (warts) vaccines. rearing programs for dairy calves differ from those for the smaller ruminants, including the withdrawal of calves from their dams immediately or by 24 hours after birth. in the cattle industry, antibiotics, ionophores (antibiotics that control selected populations of ruminal organisms), coccidiostats, probiotics, and other approved additives may be part of the milk replacers, grain and concentrate formulations, and/or creep feeding regimens. use varies by the segment of the industry, and regulations vary by country. subcutaneous hormonal implants (such as estradiol benzoate and progesterone combined, zeranol, or 17[~-estradiol) are administered, especially to beef calves destined for market rather than breeding, to promote growth. transportation of the animals from the source to the research facility must be carefully planned, and all applicable livestock travel regulations followed. it is best to have the animals transported in vehicles regularly utilized by the source farm. if commercial haulers are used, then disinfecting trucks, trailers, and associated equipment, such as ramps and chutes, beforehand is particularly important. the loading, footing, and distribution of the animals in the trailers and trucks, as well as environmental conditions during shipping, are important to consider to minimize stress and injury to the animals. sufficient time for acclimation to the facility, pens, handlers, feed, and water must be allowed once at the destination ("livestock handling and transport," 1998). recent publications address many general considerations as well as specifics about the facilities, husbandry, space requirements, and standard practices for research and production ruminants. institutions, private entities, researchers, and facility staff must also be aware of the recent adoption by the u.s. department of agriculture (usda) of specific guidelines for regulation of farm animals, such as ruminants, that are used in biomedical and other nonagricultural research. the usda animal care policy 29 notes that the "guide for the care and use of agricultural animals in agricultural research and teaching" and the "guide for the care and use of laboratory animals" provide additional information to supplement the existing animal welfare act regulations (cfr, 1985; fass, 1999; hays et al., 1998; nrc, 1996a; usda, 2000) . in all cases, stress should be considered and minimized in the husbandry and handling of ruminants. animals need to be provided adequate time to adapt to new surroundings. stress decreases feed intake, and the resulting energy, vitamin, and mineral deficiencies will affect the growth and development in younger animals. reproductive soundness and rumen function are affected by transport and similar stresses. standard practices such as weaning, castration, dehorning, vaccinations, deworming and treatments for external parasites, shipping and the associated feed and water deprivation, introduction to a new housing environment and new personnel, and intercurrent disease are all stressors (houpt, 1998) . animals should be acclimated to the use of halters and leads, temporary restraint devices, and other handling equipment associated with the research program. personnel in the research facility who are unfamiliar with ruminants should be trained in appropriate handling techniques. ap-preciation for ruminant behaviors has grown in recent years, and refined ruminant handling techniques have been published (houpt, 1998; grandin, 1998) . when ruminants are confinement-housed, care should be taken to provide adequate but draft-free ventilation. ammonia buildup and other waste gases may induce respiratory problems. in cold weather, if the ceiling, walls, or water pipes condense water, then the ventilation should be increased even at the expense of lower temperatures. even adult goats and younger cattle are quite comfortable in cold, even subfreezing temperatures, if provided with adequate amounts of dry dust-free bedding and draft protection. sheep, because of their wool, are remarkably tolerant to both hot and cold extremes. newborn lambs and recently shorn adults are susceptible to hypothermia, hyperthermia, and sunburn. therefore, in outside housing areas, sheep should be provided with shelters to minimize exposure to sun and inclement weather. animals housed under intensive confinement should be kept clean, and excreta should be removed from the pens or enclosures daily. feed and water equipment should be maintained in sound, clean condition and should be constructed to prevent fecal contamination. waterers should not create a muddy environment in paddocks or pens. there should be sufficient continuous-access waterers placed around the area to prevent competition or fighting. feeders should be constructed to conform to species size and feeding characteristics and to prevent entrapment of head and limbs. pens, other enclosures, passageways, chutes, and floors must be very sturdy to withstand such factors as the frequent cleaning; the strength, weight, and curiosity of all ages of animals; and the investigative and climbing behaviors of goats. chain-link fences are dangerous because goats (as well as some breeds and ages of sheep) are curious and tend to stand on their hind legs against fencing or walls. forelimbs may be caught easily in the mesh. floors in any areas where animals will be housed, led, or herded must ensure secure footing at all times to prevent slipping injuries. all ruminants are social and herding animals. therefore, they should be housed in groups or at least within eyesight and hearing of other animals. singly housed animals should have regular human contact. environmental enrichment should be governed by the experimental protocol or standard operating procedures, and durable play objects should be supplied to those animals that are housed in confinement. calves, in particular, that must be singly housed or that have been recently weaned, need play objects (morrow-tesch, 1997) . because sheep and goats are sensitive to changes in light cycle (especially reproductive parameters), photoperiod must be taken into account. normally, sheep and goats should be maintained on a cycle comparable to natural conditions. light intensity should be maintained at about 220 lux (ilar, 1996; fass, 1999) . light cycles can be manipulated for experimental reasons. the development of the digestive system and the unique function of the rumen are among the most notable comparative anatomic and physiologic characteristics of ruminants. there is a three-compartment forestomach (rumen, reticulum, and omasum) and a true stomach (abomasum). the mature rumen functions as an anaerobic fermentation chamber in which the enzymes, such as cellulase, of the resident bacteria allow the animals to prosper as herbivores. digestion is also aided by other microorganisms, such as protozoa (105-106/ml) and bacteria (109-101~ that contribute to rumen fermentation. the result is the production of volatile fatty acids (acetic, propionic, and butyric) . unlike in the monogastrics, fermentative digestion and volatile fatty acid absorption also occur in the large intestines. the main sources of energy for ruminants are volatile fatty acids (vfas) rather than glucose. glucose is formed from propionic acid (or from amino acids) for metabolism in the central nervous system (cns), uterus, and mammary glands. plasma glucose in ruminants is much lower than and is regulated differently from that in nonruminants. the rumen microorganisms also synthesize vitamins, such as b and k, and provide protein that is used by the animals' systems. large amounts of fermentation gases such as co2 and methane, and small amounts of nitrogen, are naturally eructed (hecker, 1983; schimdt et al., 1988) . intestinal immunoglobulin absorption by pinocytosis in the neonates is crucial to the success of passive transfer. this transfer mechanism is functional for approximately the first 36 hr after birth. neonatal ruminants are immunocompetent, however, and this condition is used to advantage for vaccinations against some common diseases of the neonatal and later juvenile periods, such as infectious bovine rhinotracheitis (ibr) vaccine (using modified live virus vaccines) to calves when their dams' colostrum is lacking antibody against this virus. unlike hepatic lipogenesis in humans, lipogenesis in sheep primarily occurs in adipose tissue and the mamrnary gland (hecker, 1983) . in addition to normal lymph node chains, and as in other ruminants, sheep have small red "nodes" associated with blood vessels. inadvertently named hemal "lymph nodes," they contain numerous red blood cells. sheep have a relatively large pituitary gland, and accessory adrenal medullary tissue may be interspersed throughout the abdominal cavity. three major ovine histocompatability classes have been identified and designated as ovar (ovis aries) classes i, ii, and iii (franz-werner et al., 1996) . bovines are recognized as having several unique aspects involving their immune systems. the bovine lymphocyte antigen (bola) system ranks after the hu-man (hla) and murine (h-2) systems in terms of depth of knowledge (lewin, 1996) . cattle are considered free of autoimmune diseases (schook and lamont, 1996) . the complexity of the immunobiology of the bovine mammary gland is being studied extensively because mastitis is the most prevalent disease in the dairy industry. several innate immune mechanisms and cellular defenses, and their variation throughout lactation, have been described (sordillo et al., 1997) . hematology and clinical reference texts are available for the ruminant species and include overviews of normal values for age, sex, and breed-specific ranges, as well as discussions regarding the influences on the hemogram of many management, nutritional, geographic, metabolic, physiologic (including lactation), medication, and iatrogenic variables (duncan and prasse, 1986; jain, 1986; kaneko et al., 1997) . these references should be consulted when preparing to include blood collection data in research protocols and when reviewing hematologic findings. in addition, most veterinary diagnostic laboratories have also developed databases for normal ranges for hematologic and clinical chemistry values based on subjects from their service areas, and these may be useful as local and breed references. appropriate control groups must be incorporated into each research plan, however, to establish the normal values (see table i ) for the particular locale, diagnostic facilities, breed, age, sex, and research circumstances. normal hematologic and clinical biochemistry data are presented in tables ii and iii. some general statements apply to most ruminants. most ruminants have fewer neutrophils than lymphocytes. the blood urea nitrogen (bun) values cannot be used as an indicator of renal function because of the metabolism of urea nitrogen by rumen microflora. because of the large volume of rumen water, ruminants can generally go several days without drinking before significant dehydration occurs. erythrocytes may become more fragile during rehydration, resulting in some degree of hemolysis and hemoglobinuria. severe dehydration can occur quickly, however, in animals that are ill. urine ph is generally alkaline in adult ruminants. ruminant erythrocytes are smaller than those in other mammals, and hematocrits tend to be overestimated unless blood samples are centrifuged for longer amounts of time for packing of the cell pellet. increased red-cell fragility is also associated with the smaller erythrocyte. rouleau formation does not occur in cattle but does to a limited extent in sheep and goats. in addition to fetal hemoglobin, sheep are reported to have at least six different hemoglobins (hecker, 1983) . blood coagulation in sheep is similar to that in humans. 2(di 0/3, dc 0/1, dp 3/3) = 20 2(di 0/3, dc 0/1, dp 3/3) = 20 2(di 0/3, dc 0/1, dp 3/3) = 20 permanent dental formula 2(10/3, c 0/1, m 3/3) = 32 2(10/3, c 0/1, m 3/3) = 32 2(10/3, c 0/1, m 3/3) = 32 avital sign data for goats are from "large animal internal medicine" (1996) . sheep weight data represent weights of feeder lamb and adult dry ewe (federation of animal science societies [fass], 1998) . goat weight data are for a large-breed male goat. cattle weight data represent weights of female holstein or guernsey dairy cattle (fass, 1998) . life span data for sheep and cattle are from brooks et al. (1984) . erythrocytes in pygmy and toggenburg goats tend to be more fragile than erythrocytes from other goat breeds. normal caprine erythrocytes lack central pallor because they are fiat and lack biconcavity. normal caprine erythrocytes may exhibit poikilocytosis. at least five blood groups have been reported in goats: b, c, m, r-o, and x. because transfusion reaction rates may be as high as 2-3%, cross-matching is advisable although not always practical (smith and sherman, 1994) . blood loss of up to 25% of the red cell mass at a single time point can be tolerated by healthy goats. blood may safely be obtained in volumes of 10 ml/kg body weight and given in volumes of 10-20 ml/kg. in general, aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) are not liverspecific in goats, and alanine aminotransferase (alt; formally serum glutamic-pyruvic transaminase, or sgpt) cannot be used to evaluate hepatic disease in goats. ~,-glutamyltransferase (ggt) and alkaline phosphatase (ap) are associated with biliary stasis, and elevations in ggt are generally associated with hepatic damage. the nutritional needs of ruminants vary considerably according to the species, breed type, different phases of development, the use of the animals, location, and different stresses in their lives. for example, mineral requirements and other nutritional requirements vary even among breeds of cattle. several references are available that describe the varying requirements and are useful for determining the requirements of ruminants consistent with the parameters noted above and the type of feeds available (jurgens, 1988; "large animal clinical nutrition," 1991; nrc, 1981 nrc, , 1989 nrc, , 1993 nrc, , 1996b "large animal internal medicine," 1996) . preformulated commercial feeds, concentrates, and supplements are available specifically for the different species of ruminants. some of these provide complete energy and protein requirements or may be used as supplements for what cannot be provided entirely by pasture, forage, hay, or silage. concentrate mixtures contain salt, minerals, and other elements. concentrates should contain a protein source such as soybean meal, cottonseed meal, or linseed meal. computer programs are also readily available for those who may need to formulate and balance rations. the palatability of feeds should be taken into account. mineral deficiencies and supplementation have been shown to influence several physiologic parameters such as immune function. introduction of young stock should include continuation of the feeding program of the source or gradual transition to appropriate feed for the animals available in the region of the research facility (nrc, 1996) . good-quality pasture can support ruminants under certain circumstances. lush spring pastures, especially pastures containing alfalfa, can induce bloat, diarrhea, grass tetany, or nitrate poisoning. ruminants not acclimated to lush pasture should be fed good-quality hay and slowly introduced to pasture environments. when ruminants have access to pasture, it is important to be aware of different eating habits. sheep and cattle are grazers. goats are browsers and will readily eat grasses, as well as seeds, nuts, fruit, and woody-stemmed plants. goats, however, can also be selective eaters and will only eat the leafy, more nutritious parts of the plant. therefore, goats have a tendency to "waste" hay. other eating habits should also be considered. finely ground concentrates are not tolerated well by goats; pelleted concentrates are preferred because the goat will pick out large particles in mixes. generally, goats do not prefer "sweet" feeds that contain molasses and do not need supplemental concentrates if a good-quality pasture or hay is fed. when given access to a salt block, goats generally are self-regulating. grass-fed goats and lactating goats may need supplementation with calcium and phosphorus, whereas alfalfa-fed goats do not (bretzlaff et al., 1991) . horse and sheep feeds may be fed to goats provided that the feed does not contain much molasses (bretzlaff et al., 1991) . the copper content of horse feed is not excessive for goats, as it is for sheep. pelleted horse feeds with 25-28% fiber and 12-14% protein are good goat rations. goats will consume 5-8% of body weight in dry-matter intake (whereas cattle will usually consume only 4% of body weight). goats enjoy human contact, and small alfalfa cubes make tasty treats for the goat. rations that have excessive calcium-phosphorus ratios or elevated magnesium levels may induce urinary calculi in male ruminants. these may also occur when forage grasses are high in silicates and oxalates. to increase ovulation rate in does, some producers "flush" females by feeding 0.5-1 lb concentrate per head per day for several weeks before and after the initiation of the breeding season. thin pregnant dairy goats should be fed 1 lb concentrate per 2) 4.2-9.1 (6.5) 5.6-6.5 potassium (k; mmol/l) hp 3.9-5.4 (4.8) 3.5-6.7 (4.3 ___ 0.5) 3.9-5.8 (4. adata presented as ranges with mean and standard deviation in parentheses, s, serum; p, plasma; hp, heparinized plasma. clinical biochemistry data from kaneko et al. (1997) . day, with the amount increasing to 1.5 lb per head per day during the last 6 weeks of gestation. forage should be fed ad libitum during this time. all newborn ruminants must receive passive immunity from colostrum, the first postpartum milk of a dam that contains concentrated protective maternal antibodies (most as igg1), functional leukocytes, cytokines, vitamins, minerals, and protein. colostrum also has laxative properties. trypsin inhibitors in the colostrum allow the passage of intact antibody molecules, by pinocytosis, through the neonate's gut wall and into the bloodstream during the first few days after birth. the quality of the colostrum is directly related to herd or flock management, vaccination programs, and the dam's overall condition and nutrition throughout gestation and at the time of parturition. ensuring effective colostrum transfer is also dependent on the timing and amount taken by the neonate. most neonatal ruminants can suckle well within 3 hr of birth. those that do so have been shown to have significantly less diarrhea (naylor, 1996) . neonates weakened by dystocia or hypothermia, for example, should be hand-fed or tube-fed colostrum. if necessary, the dam should be hand-milked and the newborn fed colostrum (for example, 20-40 ml for kids) every 2-4 hr for the first 1-2 days. in typical management situations, dairy calves either are separated from their dams immediately after birth and bottle-fed colostrum, or they remain with their dams for only about 24 hr and suckle fresh colostrum during this time. dairy producers then refrigerate and/or freeze the colostrum that cannot be consumed by the calf during that time and then feed this diluted 50:50 with warm water 3 times a day to the calves during the next 2-3 days. extra frozen colostrum for emergencies may be obtained from dairy farmers; it is advantageous to obtain colostrum from well-managed herds and from the multiparous cows in the herd (not heifers) in the same geographic locale. holstein calves, for example, should receive a minimum of 3-5 liters within 12 hr of birth and then be fed about 10-15% of body weight in colostrum by 24 hr of age. after 3 days, calves are then placed on milk replacers. although young ruminants generally do well receiving their dams' milk, commercially available milk replacers are available and should generally be prepared and fed according to the manufacturer's recommendations. containers used to prepare and feed these replacers should be sanitized daily. the fat content of both calf and lamb milk replacers is excessive; however, calf milk replacers can be used for kids if care is taken not to overfeed. young ruminants can be offered good-quality hay (such as second cutting) to nibble on by 1 week of age. calves may be provided with calf starter, a commercially available concentrate with appropriate levels of energy and protein, fed according to the manufacturer's recommendations at 2-3 weeks of age. they can be weaned off milk replacer by 4-7 weeks of age. young ruminants (4-12 months of age) need good-quality forage as well as grain and concentrate supplementation to promote development of the rumen. in farm management situations, forage can be silage, pasture, and hay. in a confinement situation like a research unit, good-quality hay, such as second cutting, is desirable. animals should not be overfed and should be offered a mineral mix free-choice. in contrast to dairy calves, beef calves remain with their mother cows until weaning at 7 months of age. calves tend to suckle many times per day. as they mature, calves are creepfed, with the energy and protein content of the ration determined by the milk production of the dams and by the available forage, such as pasture. several useful references addressing ruminant reproduction in detail are available ("current veterinary therapy: food animal practice," 1986 practice," , 1993 practice," , 1999 "large animal internal medicine," 1996; "current therapy in large animal theriogenology," 1997; hafez, 1987) . sheep are seasonally polyestrous; most breeds will express estrus in the fall (northern hemisphere) and subsequently lamb in the spring. some breeds of sheep may cycle in both the fall and the spring. between seasonal periods of receptivity, the females undergo a long period of sexual quiescence called anestrus. in a research environment, ewes can be artificially stimulated to progress from anestrous to estrous cyclicity by maintaining the females in 8 hr of light and 16 hr of dark for 8-10 weeks. puberty is reached at about 7-8 months (or earlier) in both rams and ewes; rams will typically reach puberty before their female counterparts. ewes will display signs of estrus for about 24-30 hr and will ovulate spontaneously at the end of estrus. the estrous cycle length is 14-19 days, with an average of about 17 days. following breeding, the average length of gestation is 147-150 days. slightly longer gestations are observed in animals carrying single lambs (singlets), in animals carrying rams, and in certain breeds such as those derived from merinos. prolificacy, or the number of lambs produced per gestation, tends to be dependent on the maturity of the dam (older dams tend to have multiple lambs) and on breed characteristics (some fine-wool breeds have fewer multiple births). the finn and dorset breeds are especially prolific. lambs vary in size at birth from about 3-4 lb up to 25 lb. factors that affect birthweight include parental size, number of lambs in the litter (fewer lambs or singlets tend to be larger), age of the ewe (younger ewes have smaller lambs), lamb gender (males tend to be heavier), nutrition, and season or temperature (spring lambs tend to be larger than fall lambs). goats are seasonally polyestrous in temperate regions, so that young are born in favorable times of the year. they are shortday breeders, in that estrus (heat) is brought about by the decreasing light of shorter days. in temperate climates of the northern hemisphere, goats are normally anestrous during the summer and begin cycling in the fall. the actual length of the sexual cycle depends on day length, breed, and nutrition. most dairy goats cycle between august and february or march. nubians often have extended breeding cycles, and the sexual season of some breeds, including the alpine, can be extended by artificial means. the caprine gestation length averages 150 days with a variation of 145-155 days. does bear singletons, twins, and triplets, with slightly shorter gestation when the doe is carrying triplets. cows are polyestrous. domestication of cattle has included selection against seasonality of the breeding season, particularly in dairy breeds but to some extent also in the beef breeds. in spite of this, cattle have been found to be still sensitive, in varying manifestations, to photoperiodicity. reproductive physiology in cattle is influenced by many factors. the reproductive programs in source herds and at well-managed facilities will be production-related. extensive coverage of both physiologic basics and specific industry-related criteriamfor retention of a cow as a breeder, for examplenare addressed in detail in texts and references oriented toward herd and production management ("current veterinary therapy," 1986). gestation in cattle is approximately 280 days, with a range of 270-292 days. the length of gestation in cattle is influenced by fetal sex; fetal numbers; age and parity of the cow; breed; genotype of cow, bull, or fetus; nutrition; and local environmental factors. as noted, these factors are also important in sheep and goats. cows usually bear single calves, although twin births do occur. when twins are combinations of male and female calves, the female should be evaluated for freemartinism. ovine estrus detection is usually accomplished by the ram. nonetheless, because artificial insemination is achievable in ewes, clinical signs of estrus are important. typically, ewes in heat will show a mild enlargement of the vulva, with slight increases of mucus secretion. ewes may isolate from the flock and appear anxious. it is often better and clearly more reliable to employ the help of a sterile ram to mark females when they are in standing heat. two mating systems commonly employed include hand mating and group mating. with hand mating, ewes are placed either singly or in small groups with the ram of choice. ewes are removed as serviced. group mating involves placement of a mature ram with approximately 50-60 ewes for the entire 6-week breeding season. in either mating system, it is best to attach a marking harness to the male so that individual ewes can be identified as serviced. this is important so that parturition dates can be calculated. an easy, natural way to estimate pregnancy is by placing sterile teaser rams with the ewes at the end of the breeding season. any animal marked by the ram probably has not conceived. ultrasound scanners are also used for pregnancy detection. the ultrasound transducer is placed against the right abdomen; presence of a fetus is indicated on the machine. claims of 98% accuracy at 6 weeks postbreeding have been made, although accuracy is generally best beyond 60 days of gestation. interrectal doppler ultrasound probes detect fetal pulses. fetal heart rate is in the range of 130-160 beats per minute, whereas maternal heart rates tend to be 90-110 beats per minute. accuracy is best beyond 60 days of pregnancy. rectal-abdominal palpation is an inexpensive alternative. a plastic probe is introduced intrarectally into the ewe, which is restrained on her back in a cradle. the plastic probe is then manipulated toward the abdomen while palpating for the fetus with the opposite hand. the age of the doe when she first expresses heat varies with breed. some does will express signs of heat between 3 and 4 months old. however, does should be 7-10 months old or at least 80-90 lb in weight before being bred. the caprine estrous cycle lasts 18-24 days. the duration of estrus is 24-96 hr but averages about 40 hr. the estrous cycle can be more erratic in the beginning than in the end of the breeding season (smith, season (with winter delaying), and the level of nutrition (with higher levels hastening puberty). in some cases, the presence of mature cycling cows influences heifer puberty. with adequate nutrition, dairy breeds will reach puberty at 10-12 months and beef breeds at 11-15 months, and estrous cycles will occur regularly after the pubertal (first) estrus, maturing heifers will often have one or more ovulations before showing overt signs of estrus. only one follicle usually ovulates per estrous cycle (hafez, 1987) estrus, or standing heat, in cattle averages 12-16 hr in length, with a range of 6-24 hr ("large animal internal medicine," 1996) . detection of standing heat is important because it is closely related to the time of ovulation. ovulation occurs approximately 25-32 hr after estrus. detection of estrus is usually accomplished by visual observation of vaginal mucous discharge, mounting behavior by other females (i.e., the cow standing to be mounted is the individual in estrus), and receptivity to a bull (willingness to stand). successful visual detection of standing heat is dependent on observation skills of handlers, knowledge of the herd, stresses (e.g., detection decreased in bos taurus during heat stress), barn and yard surfaces (estrus detected better on dirt than on concrete), and maintaining a consistent observation schedule. teaser animals outfitted with marking devices are also used. other methods of detecting estrus include monitoring progesterone levels; glass slide and other evaluations of cervical mucus; change in vaginal ph; and body temperature changes (hafez, 1987) . estrous cycles are usually 21 days in length, with a range of 17-25 days. it is recommended that a heifer deliver her first calf by 2 years of age. after successful conception, progesterone levels in the cow remain elevated for most of the pregnancy, as the result of the 1997). "standing heat" is usually 12-24 hr but can be as short 9 corpus luteum of pregnancy, and they decline only during the as a few hours. signs of estrus in goats include uneasiness, tail switching or "flagging," redness and swelling of the vulva, clear vaginal discharge that becomes white by the end of estrus, vocalization such as continuous bleating, and occasionally riding and standing with other does. a doe that is not in heat will not stand to back pressure or for attempts to hold her tail. does can be induced to show signs of heat by buck exposure and will ovulate within 7-10 days after introduction of the buck. goats ovulate during the later part of the estrous cycle, most between 24-36 hr after the onset of estrus. nevertheless, goats should be mated once signs of estrus are recognized and every 12 hr until the end of estrus. most goats kid only once a year, although some goats near the equator may kid twice. once bred successfully, a goat will only rarely show signs of heat again. in fact, the first sign of pregnancy is usually a failure to return to heat, so animals should be carefully watched. pregnancy can be affirmed by a variety of means. goats will generally decrease milk production with pregnancy and should have at least a 6-to 8-week dry period for the udder to fully involute and prepare for the next milking period. in cattle, age of first estrus is dependent on the breed, the final month. conceptus implantation occurs beginning at about day 17. if the pregnancy fails before this time, the cow will begin to cycle again between days 18-24, but if the pregnancy ends after day 17, there may be a delayed return to estrus. realtime ultrasonography can be used to determine pregnancy as early as 9 days after insemination, with embyros seen by days 26-29. fetal gender can also be determined by experienced personnel by this method by about day 55. detection of pregnancy can be successful by 25-40 days after conception by observation of failure to return to estrus or by palpation per rectum (detecting fetal membrane slip by days 30-35 and/or amniotic vesicle by days 28-35). palpation of the fetus is possible by day 65 and placentomes by approximately days 100-110. palpation later in presumed pregnancy will provide information based on differences in size of the two uterine horns, changes in the uterine wall, and fremitus in the miduterine artery. pregnancy can also be determined with reasonable success rates by determining if progesterone levels are elevated at days 20-24 after insemination. levels of bovine pregnancy-specific protein b may also be measured; this is produced by trophoblastic cells and is detectable by days 15-24 and elevated throughout pregnancy. placentation in sheep, goats, and cattle is epitheliochorial and 10 ft. evaluation of a cow's udder prior to breeding and especotyledonary, in contrast to the diffuse or microcotyledonary cially as parturition approaches is important in order to assure placentas of horses and pigs. the placentomes, the infolded adequate nutrition and success of passive transfer by the functional units of the placenta, are formed as the result of fu-neonate. if the udder is edematous or if mastitis is present, for sion of the villi of the fetal cotyledons projecting into the crypts example, an alternate source of colostrum (such as frozen reof the maternal caruncles (specialized projections of uterine " serves) must made be available. poor udder conformation may mucosa). caruncles of sheep and goats are concave in shape, whereas those of cows are convex. the placentomes are distributed between the pregnant and nonpregant horns of the uterus in sheep, and there are 90-100. in cattle, although the placentomes initially develop around the fetus, they will eventually be distributed to the limit of the chorioallantoic membrane even in the nongravid horn. the placentomes in the nongravid horn will be smaller than in the gravid horn. the total number will be 70-120. the best birthing preparation for all dams is to ensure a proper plane of nutrition (not overnutrition) and adequate exercise. if possible, the dam should be confined to a birthing pasture or sanitized maternity pen a few days prior to parturition. the birthing environment will be very important in the overall health of the dam and offspring; stress minimization and a clean environment will benefit the immune health of both in the short and long term. outdoor parturition in a small birthing pasture has advantages. there is less stress and less intensity of pathogens. indoor maternity pens should be clean, dry, warm, well bedded, well ventilated but draft-free, and well lighted. adequate space per pen minimizes losses of neonates from being stepped and sat on by the dam. management of these pens, especially if concentrated in an area, is important to minimize pathogens to which dam and young are exposed. water troughs or buckets should be elevated or placed outside the pen, because lambs and kids have a tendency to fall or be pushed into them. soiled bedding should be removed from the birthing pen between dams, the area sanitized and allowed to dry, and fresh bedding installed for the next occupant. moving the female immediately before or during parturition may delay the birthing process. in goats, furthermore, in utero death may occur if parturition is unduly delayed. dams should be monitored closely during parturition for dystocias; these may result in loss of young or in young severely weakened from the prolonged birthing process. prior to parturition, ewes should be sheared or crutched. crutching refers to removing wool around the perineal and mammary areas; this minimizes fetal contamination during the birth process. foot trimming can be done at this time as well. the tail and perineal area of the doe should be clipped and cleaned to improve postbirth sanitation. in general, the pregnant doe needs a 14 ft 2 (1.2 m x 1.2 m) area for the birthing process, and area needs to be increased after birthing to allow spacing for kids. each cow should have a minimum pen area of 10 ft x also be problematic; contingency plans should be made to ensure adequate support for the young if they cannot suckle from those udders. inexperienced heifers may react indifferently or aggressively to their offspring and should be monitored more closely than older, multiparous cows with uneventful calving histories. ewes approaching parturition generally isolate themselves from the flock, become restless, stamp their feet, blat, and periodically turn and look at their abdomen. the pelvic region will appear relaxed, and milk will be present in the udder. once hard labor contractions begin, lambs will usually be born quickly. animals that do not appear to be progressing correctly should be examined for dystocia. most cases of fetal malpresentation or malpositioning can be corrected via vagino-uterine manipulation. occasionally cesarean sections will be necessary. sanitation, cleanliness, and adequate lubrication are of utmost importance when performing obstetrical procedures. for about a week before parturition, rectal temperature of the doe will be above normal, or about 103~ depending on environmental temperatures. approximately 24 hr prior to birth, rectal temperature will fall to slightly below normal. many large dairy-goat facilities attempt to control the onset of parturition in order to assist birthing. the drug of choice to induce parturition in the goat is prostaglandin f2~ (pgf2~) (ott, 1982) . on day 144 of gestation, goats given pgf2~ (2.5-5 mg) will deliver kids within 28-57 hr. most goats prefer to kid alone and do so unaided. human interaction can actually interfere with normal birthing, especially in young or nervous does. some does may reject kids if extensive human interference occurs. does nearing parturition have an obviously swollen udder and a red, swollen vulva. pelvic ligaments at the base of tail relax. the doe may circle to make a bed, get up and down, look at her tail or sides, push other goats away, and bleat softly. signs of impending parturition include restlessness; vocalization (bleating softly); uneasiness, including getting up and down, pawing, and bedding; and a mucous discharge, leading to a moist tail. eight to 12 hr prior to parturition, the cervix will dilate and the cervical mucous plug will be evident as a tan, smeared substance on the tail and perineum of the dam. kids should present within 1-6 hr in either anterior or posterior position. a posterior presentation can be recognized by the presence of upward-pointing feet. most does will rest between fetuses and are best left alone. however, if labor is prolonged more than 1 hr, a vaginal exam is indicated. if the pregnant goat is housed with other goats, then herdmates will express great interest in the dam. unless moved prior to parturition, it is best to leave the dam with the group until after parturition, because removal may delay parturition. goats are not prone to retained placenta. normal kids will be quite active and will quickly attempt to stand and nurse. weak kids should be towel-dried, warmed (via heat lamp, heat pad, or warm water bottle), and assisted to nurse or fed colostrum. the goat is one of the few ungulate species that will exhibit "false pregnancy," or pseudopregnancy. this is a fairly common condition. does may have characteristically distended abdomens and may develop hydrometra and "deliver" large volumes of cloudy fluid at expected due dates. subsequent pregnancies can be normal. goats should be tested for pregnancy by 40 days of age. veterinary use of prostaglandins has been successful in treating this condition. as in other species, parturition in cattle results from a combination of hormonal changes associated with the maturity of the fetus, notably acth (adrenocorticotropic hormone) and subsequent increases in fetal corticosteriods within 2 days of birth. administration of acth to a fetus, or administration to the dam, results in premature birth. pregnancy is extended if fetal pituitary or adrenal glands are removed surgically. the fetal cortisol probably affects placental steroid production, accounting for sharp increases in the estrogens and estrogen precursors. coincident with this, maternal progesterone levels fall. the rising levels of estrogen cause release of maternal pgf2~ and induction of oxytocin receptors. most cows will separate themselves from the rest of the herd. a cow will lift her tail and arch her back when she is within a few hours of delivering the calf, and most cows are recumbent when delivering the calf. typically, the whole birthing process takes about 100 min. the length of labor of cows carrying larger calves also will be longer. nervous heifers will take longer to deliver, and if they are disturbed, their labor may cease. all postparturient animals should be monitored for successful passage of these fetal membranes within 12 hr of birth. veterinary intervention is required if not. cows occasionally eat placentas, which may subsequently obstruct rumen outflow and require surgical correction. for cattle, it is now recommended practice to remove membranes that have passed, in order to prevent ingestion. following lambing, it is critical that the newborns be "processed" so that they will have greatest survival chances. in a well-managed flock, many lambs and ewes will not need much assistance. when assistance is given, the newborn lamb's nose and mouth should be wiped free of secretions; gently swinging the lambs, head down, aids in removal of these fluids. the lamb should be dried off and stimulated through rubbing to aid its breathing. the lamb's navel should be dipped in an iodine solution to prevent subsequent navel infections. and the lamb should be identified by the application of an ear tag or ear notch. it is extremely important that the lamb be supplied with highquality colostrum within the first 12 hr of birth. lambs that are not nursing on their own should be tube-fed with colostrum that has been collected and saved previously (i.e., frozen in ice cube trays) or collected from the mother after parturition. passive transfer can be assessed by measuring serum y-glutamyltransferase (ggt) levels (tessman et al., 1997) . after the first few days, colostrum changes over to milk. nursing lambs will ingest increasing amounts of milk as they grow. if the ewe cannot produce sufficient milk, the lamb should be "grafted" onto another ewe or fed artificially with a baby bottle. powdered milk replacers are commercially available; the content of ewe milk is much different from that of cow's milk; thus lamb milk replacer should specifically be used. one report notes that 50-70% of lamb deaths occur during the first week of life and up to 90% occur within the first month. good management of ewes during gestation, care of the lamb at parturition, application of an appropriate vaccination program, and observation and intervention within the first several weeks of a lamb's life will minimize losses (ross, 1989) . immediately after birth, the placenta and any birthing materials should be removed from the doe's pen. kids do not usually need assistance. if kids are to be raised by the dam, they can be left alone; otherwise, kids should be towel-dried and removed from the dam. kids are cold-sensitive and may require a heat lamp or other source of added warmth in cold weather. navel cords should be dipped in tincture of iodine, and kids should be dehorned and castrated within the first several days of life. to control caprine arthritis encephalitis (cae), kids should be immediately removed from the dam and hand-fed heattreated colostrum. colostrum should be heat-treated for 1 hr at 131 ~ e the first feeding can be up to 125 ml of colostrum. kids should receive a total of 250 ml colostrum within the first 36-48 hr of birth. after day 3, kids can be placed on milk replacer. milk replacers should contain 16-24% fat and 20-28% milkbased protein. by 14 days of age, kids should be consuming approximately 1.1-1.4 liters of milk per day. kids should be introduced to forages as soon as possible and may be weaned by 6-10 weeks or 18-25 lb body weight. milk that is fed can be reduced by 4 weeks of age by decreasing either the volume fed or the number of feedings. as with other dams, a cow is usually very attentive to her newborn calf, cleaning and softly vocalizing to the neonate. calves typically are standing by 1 hr after birth and are suckling within 3 hr. as noted previously, dairy calves may be removed from the cow even before suckling, and the colostrum milked from the dam and given to the calf. assistance may be required for nervous heifers, after dystocias and in extreme circumstances such as severe cold. cleaning the newborn's nose and mouth, rubbing down the neonate, assuring that the calf does not get chilled, and assuring that it receives adequate colostrum are all important under any of these circumstances. a stressed calf's umbilical may be treated with an iodine or chlorhexidine solution, although some authors note no benefit of navel treatment, specifying that successful transfer of passive immunity and sound sanitary management of birthing area are the most crucial factors in preventing omphalitis (navel ill) (house, 1996; kersting, 1997; kasari and roussel, 1999) . because newborn calves can be deficient in vitamin a and iron, these may be injected to improve disease resistance (wikse and baker, 1996) . in cases in which the dams' colostrum is known to be deficient in antibodies against common diseases, vaccinations may be administered at 1 day old and followed with boosters at regular intervals. dehorning is performed when horn buds appear. castration is performed between 2 and 9 weeks of age or later. sexing the young in any of the ruminant species is straightforward. the vulva of the female young is located just ventral to the anus. the genitalia of the male include a penis, located along the ventral midline, and a scrotum, located in the inguinal region. the phenomenon of the freemartin, a genetic female born as a twin to a male, is the result of anastomoses between placental circulations of the twin fetuses; the mixing of bloodforming cells and germ cells results in the xx/xy chimeras. this occurs in 85-90% of phenotypic bovine females born as co-twins with males. the female will often have abnormal vulva and clitoris, and the vagina will be a blind end because of the lack of a cervix. sometimes singleton freemartins are born if the male fetus is lost after 30 days' gestation. multiple births are selected for and are common in sheep; the freemartin phenomenon is regarded as rare. twinning is common in goats, and freemartinism occurs in about 6% of male-female pairs of twins. intersexes are seen in some goat breeds and when polled goats are mated. proof is usually based on evidence of abnormal genital development and reports of abnormal sexual behavior. prior to weaning, it must be established that lambs can nutritionally survive without mother's milk. thus, grain, and later roughage, should be offered to lambs well in advance of the day of weaning so that they can adjust to the feedstuff. to prevent the ewes from ingesting the lamb ration, a "creep" should be set up by building an area adjacent to the ewe-lamb pen and devising a slatted entry for the lambs to enter but not the ewes. therefore, the lambs will be accustomed to the new ration through this creep-feeding process. if lambs and ewes will be pastured later in the spring, it is still beneficial to creep-feed lambs until pasture growth is adequate enough to fulfill the requirements of the growing lambs. lambs that are consuming 1.5-2 lb of creep feed per day may be weaned. depending on the individual program, lambs may be weaned as early as 4 weeks of age, although 6-8 weeks of age is more common. if ewes are of a breed that will cycle twice a year, and if it is expected that they will be rebred, then the lambs must be weaned as early as possible so that lactational anestrus will resolve and ewes will recycle. another factor is the cost of lactation rations for the ewes; if lamb grain is more economical than ewe grain, then lambs should be weaned. about 4-5 days prior to weaning, feeding of the lactation ration to the ewes should be discontinued, and only roughage fed. at weaning, the lambs should be removed in the creep, and the ewes removed to an area that is not within sight (and preferably sound) of the lambs. the ewes should be monitored for postweaning mastitis and treated as necessary. ewes that have physical or disease problems or that have not been productive at lambing or feeding their lambs should be culled. the lambs should be monitored to assure that they continue to gain weight and are eating the new ration. kids should be introduced to forages within the first week of life because the natural curiosity of these animals will cause them to investigate sources of feed. kids can be weaned by 6-10 weeks or 18-25 lb. hand-fed milk should be reduced by 4 weeks of age by reducing the volume fed or by decreasing the number of feedings. dairy calves are now usually removed from their dams immediately after birth. it is less common now to allow the calves to remain with their dams for about 24 hr and suckle fresh colostrum during this time, because their intake will be inadequate. dairy producers refrigerate and/or freeze the colostrum produced during the first 24 hr and feed this, diluted 50:50 with warm water, twice a day to the calves during the next 2-3 days. holstein calves, for example, should receive a minimum of 3-5 liters within 12 hr of birth and then be fed about 10-15% of body weight in colostrum by 24 hr of age. after 3 days, calves are then placed on milk replacers, preformulated powders reconstituted with water that provide complete nutrition. milk replacers are commercially available and should be fed according to manufacturer's recommendations vaccination programs for calves vary with the preventive medicine program for the overall herd. passive immunity provided by colostrum from cows on sound management programs will last until a calf is about 6-7 months old; normally vaccinations are not necessary and are contraindicated during those first 6 months. the duration of passive immunity varies considerably among calves, however; some producers choose to begin vaccinating calves at 1-2 months of age and continue with monthly booster immunizations until the animals are 7 months old, when passive immunity is no longer a possibility. artificial insemination (ai) in sheep is more difficult than in cattle because sheep are smaller and cannot be reproductively manipulated via the rectum and because the cervix of sheep is more difficult to traverse with the insemination pipette. breeding animals artificially with fresh semen produces pregnancy rates averaging 50% (not unlike that of cattle); artificial insemination with frozen semen is less successful. several artificial insemination techniques have been used. laparoscopic ai involves the surgical instillation of semen into the uterus through a small abdominal opening. the procedure is successful but is technically involved and costly. cervical ai involves the transvaginal introduction of semen into the cervix. a modification of this technique (transcervical ai) allows for penetration through the cervix into the uterus. this method (called the guelph system for transcervical ai) leads to successful penetration into the uterus in up to 75% of ewes when performed by an experienced inseminator. artificial insemination is now an integral part of dairy herding; natural insemination as a management practice is relatively rare. technicians performing the ai technique are available through commercial enterprises. dairy production employees are also trained. information regarding the management of the donors and recipients, the storage and handling of the semen, and the skills and record keeping required is covered extensively elsewhere (nebel, 1997) . because sheep are hormonally similar to other ruminants, estrous synchronization techniques are comparable. progesterone suppresses follicle-stimulating hormone (fsh) secretion, preventing animals from developing follicles and exhibiting estrus. artificial or natural progesterone can be administered in the feed, through parenteral injection, subcuticular implants, and vaginal pessaries. the progesterone is withdrawn in about 12-14 days, after which the fsh secretion will initiate the process of follicle development (trower, 1993) . estrus usually will occur in 36-60 hr (average is 48 hr). a natural method of synchronization, often applied to promote flock breeding within a short period of time (and thus parturition will be within a narrow window as well), is the introduction of sterile rams with the ewes before the beginning of the normal fall mating period. pheromones released from males naturally stimulate the females to cycle and to synchronize their heats. it should be noted that introduction of a male during late anestrus will often stimulate ovulation in about 6 days; however, this cycle will generally be without clinical signs of estrus (silent heat). vasectomy of rams is one method of producing sterile "teaser rams." introduction of the buck to a group of does will induce ovulation and may even synchronize does. does that are kept separate from the buck will show signs of estrus, will ovulate within 6-10 days, and will have normal pregnancies when introduced to a buck. bucks with horns and intact scent glands are better able to induce ovulation than dehorned bucks, whose scent glands often been removed. control of breeding in the goat has been studied mostly in dairy breeds in order to produce milk throughout the year and to reduce kidding labor. goats in the luteal phase of the estrous cycle, days 4-16, are sensitive to pgf2~ (2.5-5 mg im) and will show estrus in 36-60 hr postinjection (bretzlaff, 1997) . dosing cycling animals twice 11 days apart will synchronize goats, and artificial insemination using this method has resulted in 40-60% conception rates (bretzlaff, 1997; greyling and van niekerk, 1986) . programs for timed breeding have been described and involve administering progestogens (bretzlaff, 1997) . vaginal pessaries of fluorogestone acetate left in place for 21 days in the doe followed by an injection of pregnant mare serum gonadotropin (pmsg) at the time of pessary removal have proven successful. also, when primed by pgf2~, an 11day regimen of fluorogestone acetate with pmsg given on day 9 has been successful. synchronization of cattle estrous cycles and superovulation are used as management techniques in certain commercial cattle and dairy production settings where estrus synchronization or embryo transfer is advantageous to production and management. the methodology is also used in the research setting for coordinating donors and recipients of embryos or other genetically manipulated tissues for implantation. the options and dosing regimens are described in detail in veterinary clinical texts (wenzel, 1997; vanderboom et al., 1997) . in synchronization, the principle is lysis of the existing corpus luteum. the more common practices involve the use of products approved for use in cattle such as pgf2~, one of its analogs, or products containing estradiol valerate. progestogens are also used in conjunction with estradiol valerate. other approaches, involving management techniques combined with pharmacologic interventions, are considered less successful. superovulation regimens involve injections of fsh either alone or with pgf2~ at timed internals. estrus is expected 48 hr after the final injection, and two inseminations are performed at 12 hr intervals after estrus detection. preparation of recipients involves injection of pgf2~ or progestogens with gonadotropins such as pmsg. for greatest success as management tools, these must be combined with a consistent program that provides appropriate nutrition for all cattle involved. synchronization of animals is also influenced by several other factors, however, such as time in the cycle when hormones are administered, response by each individual animal, whether the cow is a dairy or beef animal, parity and maturity of the cows, success of heat detection after the luteolysis, and accurate record keeping. embryo transfer involves the removal of multiple embryos from a superovulated embryo donor and transferring them to synchronized recipients. this method maximizes the genetic potential of the donor animal. the donor animal is hormonally superovulated and inseminated. in sheep, about 1 week after breeding, the embryos are surgically removed from the donor's uterus. in cattle, the procedure is nonsurgical. about 75% of expected embryos (determined by counting corpora lutea) can be recovered; successful recovery is affected by factors such as age of the donor, reproductive health, and experience of the surgeon or technician. furthermore, not all collected embryos are of transferable quality. recipients are hormonally synchronized with the donor animals. on the day of embryo collection, transferable embryos are implanted into the uterus of the recipient; laparoscopy has been used in the past and is now being replaced by nonsurgical methods. pregnancy rates average about 70%. if recipients are not available, embryos, like sperm, can be frozen and kept for later transfer. embryo transfer is commonly practiced in cattle as a herd improvement technique and as a research technique for engineered embyros. disease screening programs for all animals involved are important because several pathogens can be transmitted directly or indirectly, such as bovine viral diarrhea virus, bluetongue virus, infectious bovine rhinotracheitis virus, and mycoplasmal species. in sheep flocks and goat herds, as noted, male young are usually castrated by 1 month of age. the elastrator method is the more popular for animals less than 1 week of age. other methods include the emasculatome (crushing) and surgical removal ("knife method"). the distress associated with castration and tail docking in lambs is the subject of debate and has been researched recently (kent et al., 1995) . as noted, male calves are usually castrated as early as possible and no later than 3 month of age. in some production situations, however, where maximum hormone responsive muscle development and grouping animals together for procedures dictate scheduling, the procedure may be performed on older males. open and closed techniques are used, depending on the age of animals and on veterinary or farm practice. breeding and vasectomized rams and bucks are usually maintained by medium to large production farms. smaller farms often borrow breeding males. breeding males are typically selected by production record, pedigree, and/or breed. vasectomized males are often retired breeders and should be tattooed or identified clearly to avoid any wasted breeding time. the vasectomy technique for both species is comparable (smith and sherman, 1994) . rams may be housed together for most of the year, whereas bucks are penned separately. because ewes will exhibit only a limited number of estrous cycles before becoming reproductively quiescent, it is critical that the male be capable of successfully breeding the female in an expeditious manner. any defects in the external genitalia, reproductive diseases, or musculoskeletal abnormalities may prevent successful copulatory behaviors. furthermore, it is impor-tant to know the semen quality of the ram as one indicator of fertility. semen can be collected via electroejaculation or by use of a teaser mount. once semen is collected, it should be handled carefully and kept warm to prevent sperm death, leading to improper conclusions about the male. typically, the characteristics usually evaluated as a determinate of sperm quality are volume (normal between 0.7 and 2.0 ml); motility (% of sperm moving in a forward wave; high quality is associated with motility of approximately 90%); concentration (sperm count per unit of volume as measured by a hemocytometer; high-quality semen should contain 1.8 x 109 sperm per ml); morphology (live versus dead cells, as determined by special stains and the percentage of abnormal-appearing sperm; neither the abnormalities nor the dead sperm should exceed 10% in high-quality semen). the extensive use of artificial insemination in the dairy cattle industry has minimized the use of bulls on many farms, although a farm may maintain a few bulls for heat detection and for "cleanup" breeding. breeding bulls are maintained in beef production establishments. breeding bulls must be part of the herd vaccination program, with special attention to appropriate timing of immunizations for the commonly transmitted venereal diseases campylobacteriosis and trichomoniasis. tail docking is a relatively recent development in dairy herd management and is practiced in the belief that it will minimize bacterial contamination of the udder and therefore the milk. tails are typically docked to about 10 inches in length. the practice is more popular in certain regions in the united states. to date, there is no published study indicating that this technique provides any distinctive advantage over keeping the tail switch hair clipped short. healthy ruminants have good appetites, chew cud, are alert and curious, have healthy intact coats, move without hindrance, and have clear, bright, clean eyes and cool dry noses. even adult animals, when provided sufficient space, will play. sheep and goats have tidy "pelleted" dark green feces. cattle have pasty, moist, dark green-brown feces. ruminants normally vocalize, and handlers will learn to recognize normal communication among the group or directed at caregivers in contrast to that when animals are stressed. excessive, strained vocalizations are often a sign of stress in cattle. "bruxism," or grinding of the teeth by a ruminant, is usually associated with discomfort or pain. other signs of discomfort, stress, or illness include decreased time spent eating and cud chewing, restlessness, prolonged recumbency with outstretched neck and head, and hunched back when standing. unhealthy ruminants may be thin, may arch their backs or favor a limb, or may have external lumps or swollen joints, an unusual abdominal profile, or rough or dull coats. all ruminants are herd animals to some extent and social individuals; therefore, every effort should be made to allow contact among animals, in terms either of direct contact or of sound, smell, or sight. human contact and handling should be initiated promptly and maintained regularly and consistently throughout the animal's stay in the research facilities. animals should be provided sufficient time to acclimate to handlers and research staff. cattle and sheep can hear at higher frequencies than humans can and may react to sounds not perceived by handlers. knowledge of the peculiarities of sheep behavior will increase the ease of handling and decrease stress-related effects in research. generally, fine-wooled breeds, such as rambouillet, are the most gregarious and are best handled in groups. the meat, or "downs," breeds tend to be less gregarious, and the long-wooled breeds tend to be solitary (ross, 1989; asia, 1996) . nonetheless, movement of animals is simplified by proper facility design. sheep have a wide-angle visual field and are easily scared by activities that are taking place behind them. sheep should be moved slowly and gently. to capture individuals within a flock, it is best to confine the flock to a smaller space and use a shepherd's crook or to gently catch the animal in front of the neck/thorax. grabbing the wool can injure the animals, as well as damage the wool and the underlying tissues. sheep move best in chutes that have solid walls, and individual animals will generally follow a lead animal. any escape route will be challenged and, if successfully breached, will disrupt the entire flock movement. sheep movement is also disrupted by contrasts such as light and shadows that impinge on a chute or corral. finally, like most animals, sheep have a flight zone (minimum zone of comfort), the penetration of which will result in sheep scattering. this minimal flight distance can be modified by increasing handling of the animals and working at the edge of the zone, but it should always be considered when working with animals in chutes, pens, or other confined areas. goats exhibit behavioral characteristics that make them quite distinct from other ruminants. their browsing activity makes them quite orally investigative. goats will readily nibble or chew just about anything they come in contact with, so researchers should keep all paperwork and equipment out of reach. a herd of goats will readily chew through wood gates and fencing, especially when confined in areas without alternatives for chewing behavior. goats are also inquisitive, restless, agile jumpers and climbers, and quite mischievous. if maintained in paddocks, strong high fences are essential, as are adequate spaces for exercise or boulders or rock piles for hoof maintenance and recreational climbing. goats are more tolerant of isolation and are more easily acclimated to human contact than sheep are, but goats will confront unfamiliar intruders and make sneezing noises. goats with horns will use them to advantage, and horns may also become entangled in fencing. although less strongly affected by flock behavior, goats are social animals. most goats raised in close human contact are personable and cooperative and can easily be taught to stand for various procedures, including blood collection. an understanding of breed behaviors, sources of stress in cattle, play behaviors, calf behaviors, and dominance determinants will contribute to prevention of injuries to handlers and better health and welfare of the animals. ruminants of all ages, especially cattle of all ages, should be handled with an appreciation of the serious injury to human handlers that may result (houpt, 1998) . cattle have a wide visual field, as sheep do, and a flight zone that varies in size, according to previous handling experiences (gentle handling and animal tameness make the flight zone smaller) and the circumstances of the moment (grandin, 1993) . groups of cattle are moved effectively around a facility by utilizing chute systems, with sequences of gates, that minimize chances of animals turning around. dairy cattle have been bred and selected over centuries for their docile, tractable characters and production characteristics. in contrast, beef breeds have not been selected for docility and are generally more difficult to handle and restrain. beef breeds, such as angus, are known for their independent natures and protective maternal instincts. all cattle respond well to feed as a reward for desired behavior. healthy cattle typically are very curious and watchful and are alert to sounds and smells. when not grazing or eating, they hold their heads up. when sleeping, the head and neck may be tucked back. because of ruminant digestive and metabolic needs, much of the day is spent eating or cud chewing. occasionally, adult cows sit upright like dogs. cattle maintained inside tend to be more docile. in addition to forced isolation from other cattle, sources of stress include rough attitudes of handlers and unfamiliar visual patterns, routines, or environments. these stressors may exacerbate signs of systemic illnesses. calves are known for non-nutritive suckling, bar licking, and tongue rolling. non-nutritive suckling behavior is greater in hungry calves and also right after a milk meal. it is best to provide nipples and other clean noninjurious materials for the animals to suck. non-nutritive suckling can be detrimental in group-housed calves because it can result in disease transmission and hair ball formation. environmental enrichment devices have been developed to cope with this behavior. the behavior diminishes as the animals are weaned onto solid food (morrow-tesch, 1997) . play activity and vocalizations of calves mimic adult dominance behaviors. play activity by young adult cattle is more common in males, can be quite rough, and is often triggered by a change in the environment. dominance behaviors are dependent on direct physical contact among the cattle, and dominance hierarchies are established within a herd. horns, age, and weight have been reported to be the most important determi-nants. aggressive behaviors in cattle may be triggered by newly introduced animals or unfamiliar visual patterns and by feeding when animals are very hungry. aggression is more common among intact adult males. this section focuses primarily on the more common diseases affecting sheep, goats, and cattle in the united states and elsewhere in north america and those that are reportable. for detailed information not included in this limited overview and for diseases of importance internationally, the authors recommend several excellent comprehensive and focused veterinary clinical texts and periodicals that address ruminant diseases, preventive medicine, and individual and flock or herd management. these are listed under "major references" in the reference list at the end of this chapter. recommendations for current drug therapies, both approved and off-label use in ruminants, including withholding prior to slaughter, formularies, and related information can be found in the references noted above and in formularies (hawk and leary, 1995; plumb, 1999) . in addition, the food animal residue avoidance databank (farad), accessible on the internet <http://www.farad.org>, should be used as a resource. farad is a food safety project of the u.s. department of agriculture and is an information resource to prevent drug and pesticide residues in food animals and animal products. food; may be anorexic, weak, unthrifty and depressed; and may salivate excessively. diagnosis is made based on clinical signs and is confirmed by culture. epizootiology and transmission. the organism penetrates wounds of the skin, mouth, nose, gastrointestinal tract, testicles, and mammary gland. rough feed material and foreign bodies may play a role in causing abrasions. actino bacillus lignieresii then enters into deeper tissues, where it causes chronic inflammation and abscess formation. lymphatic spread may occur, leading to abscessation of lymph nodes or infection of other organs. necropsy findings. purulent discharges of white-green exudate drain from the tracts that often extend from the area of colonization to the skin surface. exudates will also contain characteristic small white-gray (sulfurlike) granules. the pus is usually nonodorous. differential diagnosis. contagious ecthyma and caseous lymphadenitis are the primary differentials. diseases or injuries causing oral pain and discomfort, such as dental infections, foreign bodies, and trauma, should be considered. treatment. animals should be fed softer feeds. antibiotics such as sulfonamides, tetracyclines, and ampicillin are effective, although high doses and long durations of therapy are required. penicillin is not effective. weekly systemic administration of sodium iodide for several weeks is not as effective as antibiotic therapy. surgical excision and drainage are not recommended. etiology. actinobacillus lignieresii is an aerobic, nonmotile, non-spore-forming, gram-negative rod that is widespread in soil and manure and is found as normal flora of the respiratory, gastrointestinal, and reproductive tracts of ruminants. in sheep and cattle, a. lignieresii causes sporadic, noncontagious, and potentially chronic disease characterized by diffuse abscess and granuloma formation in tissues of the head and occasionally other body organs. this disease, called wooden tongue, has not been documented in goats. clinical signs. skin lesions are common. tongue lesions are more common in cattle than in sheep. lip lesions are more common in sheep. soft-tissue or lymph node swelling accompanied by draining tracts is observed in the head and neck regions, as well as other areas. animals may have difficulty prehending prevention and control. because the organism enters through tissue wounds, especially those associated with oral trauma, feedstuffs should be closely monitored for coarse material and foreign bodies. b. arcanobacterium infection (formerly actinomycosis, or "lumpy jaw") etiology. arcanobacterium (formerly known as actinomyces or corynebacterium) pyogenes and a. bovis are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. arcanobacterium bovis is a normal part of the ruminant oral microflora and is the organism associated with "lumpy jaw" in cattle; this syndrome is rarely seen in sheep and goats. this organism has also been associated with pharyngitis and mastitis in cattle. clinical signs and diagnosis. arcanobacterium bovis causes mandibular lesions primarily. the mass will be firm, nonpainful, and immovable. draining tracts may develop over time. if teeth roots become involved, painful eating and weight loss are evident. radiographic studies are helpful for determining fistulas. diagnosis is based on clinical signs, and culture is required to confirm arcanobacterium. the prognosis is poor for lumpy jaw. epizootiology and transmission. these organisms are normal flora of the gastrointestinal tracts of ruminants and gain entrance into the tissues through abrasions and penetrating wounds. necropsy. draining lesions with sulfurlike granules (as with actinobacillosis) are frequently observed. ious degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, hematuria, and so on. severe sequelae may include septicemia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, and endocarditis. research complications. young stock affected by omphalophlebitis may be inappropriate subjects because of growth setbacks and physiologic stresses from the infection. affected adult animals will not thrive and, even with therapy, may not be appropriate research subjects. pathogenesis. arcanobacterium pyogenes is known to produce an exotoxin, which may be involved in the pathogenesis. differential diagnosis. actinobacillus lignieresii and caseous lymphadenitis are important differentials for draining tracts. a major differential for omphalophlebitis is an umbilical hernia, which will typically not be painful or infected. there are many differentials for septic joints and polyarthritis: chlamydia spp., mycoplasma spp., streptococci, coliforms, erysipelothrix rhusiopathiae, fusobacterium necrophorum, and salmonella spp. tumors, trauma to the affected area, such as the mandible, and dental disease or oral foreign body should also be considered. prevention and control. arcanobacterium bovis lesions can be prevented or minimized by feeds without coarse or sharp materials. treatment. penicillin or derivatives such as ampicillin or amoxicillin are treatments of choice. sodium iodides (intravenous) and potassium iodides (orally) have been utilized also. extended antibiotic therapy may be necessary. surgical excision is an option. in addition to medications noted above, isoniazid is somewhat effective for a. bovis infections in nonpregnant cattle. research complications. the possibility of long-term infection and long therapy are factors that will diminish the value of affected research animals. omphalophlebitis, omphaloarteritis, omphalitis, and navel ill are terms referring to infection of the umbilicus in young animals. arcanobacterium pyogenes is the most common organism causing omphalophlebitis, an acute localized inflammation and infection of the external umbilicus. most cases occur within the first 3 months of age, and animals are presented with a painful enlargement of the umbilicus. animals may exhibit varetiology. bacillus anthracis is a nonmotile, capsulated, sporeforming, aerobic, gram-positive bacillus that is found in alkaline soil, contaminated feeds (such as bonemeal), and water. common names for the disease anthrax include woolsorters' disease, splenic fever, charbon, and milzbrand. clinical signs and diagnosis. anthrax is a sporadic but very serious infectious disease of cattle, sheep, and goats characterized by septicemia, hyperthermia, anorexia, depression, listlessness, depression, and tremors. subacute and chronic cases may occur also and are characterized by swelling around the shoulders, ventral neck, and thorax. the incubation period is 1 day to 2 weeks. bloody secretions such as hematuria and bloody diarrhea often occur. abortion and blood-tinged milk may also be noted. the disease is usually fatal, especially in sheep and goats, after 1-3 days. death is the result of shock, renal failure, and anoxia. diagnosis is based on the clinical signs of peracute deaths and hemorrhage. stained blood smears may show short, single to chained bacilli. blood may be collected from a superficial vein and submitted for culture. epizootiology and transmission. cattle and sheep tend to be affected more commonly than goats, because of grazing habits. older animals are more vulnerable than younger, and bulls are more vulnerable than cows. although the disease occurs worldwide, and even in cold climates, most cases in the united states occur in the central and western states, and outbreaks usually occur as the result of spore release after abrupt climatic changes such as heavy rainfall after droughts or during warmer, dryer months. spores survive very well in the environment. the anthrax organisms (primarily spores) are generally ingested, sporulate, and replicate in the local tissues. abrasive forages may play a role in infection. transmission via insect bites or through skin abrasions rarely occurs. necropsy. necropsies should not be done around animal pens or pastures, and definitive diagnoses may be made without opening the animals. incomplete rigor mortis, rapid putrefaction, and dark, uncoagulated blood exuding from all body orifices are common findings. blood collected carefully and promptly from peripheral veins of freshly dead animals can be used diagnostically. splenomegaly, cyanosis, epicardial and subcutaneous hemorrhages, and lymphadenopathy are characterisitic of the disease. pathogenesis. the rapidly multiplying organisms enter the lymphatics and bloodstream and result in a severe septicemia and neurotoxicosis. encapsulation protects the organisms from phagocytosis. liberated toxins cause local edema. differential diagnosis. although anthrax should always be considered when an animal healthy the previous day dies acutely, other causes of acute death in ruminants should be considered, e.g., bloat, poisoning, enterotoxemia, malignant edema, blackleg, and black disease. prevention and control. outbreaks must bereported to state officials. anthrax is of particular concern as a bioterrorism agent. any vaccination programs should also be reviewed with regulatory personnel. herds in endemic areas and along waterways are usually vaccinated routinely with the sterne-strain spore vaccine (virulent, nonencapsulated, live). careful hygiene and quarantine practices are crucial during outbreaks. dead animals and contaminated materials should be incinerated or buried deeply. biting insects should be controlled. the disease is zoonotic and a serious public health risk. treatment. treatment of animals in early stages with penicillin and anthrax antitoxin (hyperimmune serum, if available) may be helpful. amoxicillin, erythromycin, oxytetracycline, gentamicin, and fluoroquinolones are also good therapeutic agents. during epidemics, animals should be vaccinated with the sterne vaccine. research complications. natural and experimental anthrax infections are a risk to research personnel; the pathogen may be present in many body fluids and can penetrate intact skin. the organism sporulates when exposed to air, and spores may be inhaled during postmortem examinations. etiology. brucella is a nonmotile, non-spore-forming, nonencapsulated, gram-negative coccobacillus. brucella abortus is one of several brucella species that infects domestic animals but cross-species infections occur rarely. brucella abortus or b. melitensis may cause brucellosis in sheep, cattle, and goats. brucella melitensis (biovar 1, 2, or 3) is the primary cause of sheep disease (garin-bastuji et al., 1998) . brucella ovis is more commonly associated with ovine epididymitis or orchitis than abortion. in the united states, clusters of brucellosis are still found in western areas contiguous to yellowstone national park. bang's disease is the common name given to the disease in ruminants. clinical signs and diagnosis. brucella melitensis in the adult ewe is generally asymptomatic and self-limiting within about 3 months. however, because the organism may enter and cause necrosis of the chorionic villi and fetal organs, abortion or stillbirths may occur. abortion usually occurs in the third trimester, after which the ewe will appear to recover. it has been reported that up to 20% of infected ewes may abort more than once. rams will also be infected and may develop orchitis or pneumonia. the disease caused by b. ovis is manifested by clinical or subclinical infection of the epididymis, leading to epididymal enlargement and testicular atrophy. brucella ovis causes decreased fertility. brucella melitensis is the more common cause of brucellosis in goats. brucella abortus has been shown to infect goats in natural and experimental infections, and b. ovis has also been shown to infect goats experimentally. does infected with b. melitensis will also abort during the third trimester. infections with b. abortus in cattle produce few clinical signs. there may be a brief septicemia during which organisms are phagocytosed by neutrophils and fixed macrophages in lymph nodes. in cows, the organism localizes in supramammary lymph nodes and udders and in the endometrium and placenta of pregnant cows. infection may cause abortions after the fifth month, with resulting retained placentas. permanent infection of the udder is common and results in shedding of organisms in milk. in bulls, the organism may cause unilateral orchitis and epidydimitis and involvement of the secondary sex organs. organisms may be in the semen. in infected herds, lameness may also be a clinical sign. diagnosis of brucellosis can be made by bacterial isolation of the brucella organism from necropsy samples (especially the fetal stomach contents), as well as by supportive serological evidence. many serological tests are available, such as the tube and plate agglutination tests, the card or rose bengal test, the rivanol precipitation test, complement fixation, enzyme-linked immunosorbent assay (elisa), polymerase chain reaction (pcr), and others. test selection is often dependent on state requirements in the united states. epizootiology and transmission. the primary route of transmission of b. abortus is ingestion of the organism from infected tissues and fluids (milk, vaginal and uterine discharges) during and for a few weeks after abortion or parturition; contaminated semen is considered to be a minor source of infection. exposure to the organism may occur via the gastrointestinal tract (contaminated feed or water), the respiratory tract (droplet infection), or the reproductive tract (contaminated semen) and through other mucous membranes such as the conjunctiva. brucella ovis is transmitted in the semen, as well as orally or nasally through contaminated feed and bedding. necropsy findings. a sheep fetus aborted due to brucella will exhibit generalized edema. the liver and spleen will be swollen, and serosal surfaces will be covered with petecchial hemorrhages. peritoneal and pleural cavities often contain serofibrinous exudates. the placenta will be leathery. pathogenesis. ruminants are considered especially susceptible to brucella infection, because of higher levels of erythritol (a sugar alcohol), which is a growth stimulant for the organism. brucella utilizes erythritol preferentially over glucose as an energy source. placentas and male genitalia also contain high levels of erythritol. brucella organisms also evade lysis when phagocytosed by macrophages and neutrophils and survive intracellularly in phagosomes. abortion is the result of placentitis, typically during the third trimester of gestation. brucella ovis enters the host through the mucous membranes, then passes into the lymphatics, causes hyperplasia of reticuloendothelial cells, and is spread to various organs via the blood. the organism localizes in the epididymides, the seminal vesicles, the bulbourethral glands, and the ampullae. orchitis may be a sequelae of the disease. epididymitis can be diagnosed by identifying gross lesions by palpation of the epididymides, by serological evidence of antibodies to b. ovis, and by semen cultures. differential diagnosis. differential diagnoses include all other abortion-causing diseases. many other agents, such as actinobacillus spp., arcanobacterium (actinomyces) pyogenes, eschericia coli, pseudomonas spp., proteus mirabilis, chlamydia, mycoplasma, and others may be associated with ovine epididymitis and orchitis. a clinically and pathologically similar agent, actinobacillus seminis, has been isolated from virgin rams. this organism has morphological and staining characteristics similar to those of b. ovis and complicates the diagnosis (genetzky, 1995) . prevention and control. the rev 1 vaccine has been recommended for vaccination of ewe lambs in endemic areas, but this vaccine is not used in the united states. separating young rams from potentially infected older males, sanitizing facilities, and vaccinating them with b. ovis bacterin can prevent the disease. over the past 20 years, aggressive federal and state regulatory and cattle herd health programs in the united states have provided control and prevention mechanisms for this pathogen through a combination of serological monitoring of herds, slaughter of diseased animals, herd management, vaccination programs, and monitoring of transported animals. most states are considered brucellosis-free in the cattle populations; thus, procurement of ruminants that have been exposed to this infectious agent will be unlikely. cattle vaccination programs can be very successful when conducted on a herd basis to reduce likelihood of exposure. strain 19 and the recently validated attentuated strain rb51 are live vaccines and can be used in healthy heifer calves 4-12 months old. vaccination for older animals may be done under certain circumstances. vaccination of bull calves is not recommended, because of low likelihood of spread through semen and possibility of vaccination-induced orchitis. the strain 19 vaccine induces long-term cell-mediated immunity, protects a herd from abortions, and protects the majority of a herd from reactors during a screening and culling program. the vaccine will not, however, protect the animals from becoming infected with b. abortus. strain 19 vaccine induces an antibody response in cattle. the rb51 vaccine does not result in antibody titers and therefore is advantageous because infection with brucella can be determined serologically. the rb51 vaccine has been designated as the official calfhood bovine brucellosis vaccine in the united states by the u.s. department of agriculture's animal and plant health inspection service (aphis) (stevens et al., 1997) . brucella vaccine should be administered to unstressed, healthy cattle, with attention to particular side effects of the vaccination material and to prevention of compounding stresses associated with weaning, regrouping, other management changes, and shipping. the rb51 is regarded as less pathogenic and abortigenic in cattle. clinical signs and diagnosis. ovine vibriosis is a contagious disease that causes abortion, stillbirths, and weak lambs. the organism inhabits the intestines and gallbladder in subclinical carriers. abortion generally occurs in the last trimester, and abortion storms may occur as more susceptible animals, such as maiden ewes, become exposed to the infectious tissues. it is reported that 20-25% of the flock may become infected and up to 5% of the ewes will die (jensen and swift, 1982) . some lambs may be born alive but will be weak, and dams will not be able to produce milk. diagnosis is achieved by microscopic identification or isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharges. tentative identification of the organism can be made by observing curved ("gull-wing") rods in giemsa-stained or ziehl-neelsen-stained smears from fetal stomach contents, placentomes, or maternal uterine fluids. epizootiology and transmission. campylobacteriosis occurs worldwide. campylobacter spp., such as c. jejuni, normally inhabit ovine gastrointestinal tracts. transmission of the disease occurs through the gastrointestinal tract, followed by shedding, especially associated with aborted tissues and fluids. in abortion storms, considerable contamination of the environment will occur due to placenta, fetuses, and uterine fluids. ewes may have active campylobacter organisms in uterine discharges for several months after abortion. the bacteria will also be shed in feces, and feed and water contamination serve as another source. there is no venereal transmission in the ovine. necropsy. aborted fetuses will be edematous, with accumulation of serosanguinous fluids within the subcutis and muscle tissue fascia. the liver may contain 2-3 cm pale foci. placental tissues will be thickened and edematous and will contain serous fluids similar to those of the fetus. the placental cotyledons may appear gray. pathogenesis. the organism enters the bloodstream and causes a short-term bacteremia (1-2 weeks) prior to the localizing of the bacteria in the chorionic epithelial cells and finally passing into the fetus. should be considered in late gestation ovine abortions. a bacterin is available to prevent the disease. carrier states have been cleared by treating with a combination of antibiotics, including penicillin and oral chlortetra-cycline. aborting ewes should be isolated immediately from the rest of the flock. after an outbreak, ewes will develop immunity lasting 2-3 years. treatment. infected animals should be isolated and provided with supportive therapy. prompt decontamination of the area and disposal of the aborted tissues and discharges are important. research complications. losses from abortion may be considerable. campylobacter ssp. are zoonotic agents, and c. fetus subsp, intestinalis may be the cause of "shepherd's scours." ii. clinical signs and diagnosis. preliminary signs of a problem in the herd will be a high percentage of cows returning to estrus after breeding and temporary infertility. this will be particularly apparent in virgin heifers that may return to estrus by 40 days after breeding. long interestrous intervals also serve an indication of a problem. spontaneous abortions will occur in some cases, typically during the fourth to eighth months of gestation. severe endometritis may lead to salpingitis and permanent infertility. demonstration or isolation of the organism, a curved rod with corkscrew motility, is the basis for diagnosis. the vaginal mucous agglutination test is used to survey herds for campylobacteriosis. serology will not be worthwhile, because the infection does not trigger a sufficient antibody response. culture from breeding animals may be difficult because campylobacter will be overgrown by faster-growing species also present in the specimens. epizootiology and transmission. the bacteria is an obligate, ubiquitous organism of the genital tract. transmission is from infected bulls to heifers. older cows develop effective immunity. necropsy findings. necrotizing placentitis, dehydration, and fibrinous serositis will be found grossly. in addition, bronchopneumonia and hepatitis will be seen histologically. pathogenesis. campylobacter organisms grow readily in the genital tract, and infection is established within days of exposure. the resulting endometritis prevents conception or causes embyronic death. differential diagnosis. the primary differential diagnosis for campylobacteriosis is trichomoniasis. other venereal diseases should be considered when infertility problems are noted in a herd. these include brucellosis, mycoplasmosis, ureaplasmosis, infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (ibr-ipv), and bovine virus diarrhea (bvd). leptospirosis should also be considered. in addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. prevention and control. killed bacterin vaccines are available, either as oil adjuvant or as aluminum hydroxide adsorbed. the former is preferred because of duration of immunity but causes granulomas. that vaccine also has specific recommendations regarding administration several months before the breeding season. the latter product is administered closer to the breeding season, and the duration of immunity is not as prolonged. in both cases, boosters should be given after the initial immunization and as part of the regular prebreeding regimen. only one bacterin product is approved for use in bulls. many combination vaccine products contain only the aluminum hydroxide adsorbed product. artificial insemination (ai) is particularly useful at controlling the disease, but bulls used for ai must be part of a screening program for this and other venereal diseases such as trichomoniasis. treatment. cows will usually recover from the infection, and treatment with antibiotics such as penicillin, administered as an intrauterine infusion, improve the chances of returning to breeding condition. etiology. the most common caprine bacterial skin infection is caused by staphylococcus intermedius or s. aureus and is known as staphylococcal dermatitis (smith and sherman, 1994) . the staphylococcus organisms are cocci and are categorized as primary pathogens or ubiquitous skin commensals of humans and animals. staphylococcus aureus and s. intermedius are classified as primary pathogens and produce coagulase, a virulence factor. clinical signs and diagnosis. small pustular lesions, caused by bacterial infection and inflammation of the hair follicle, occur around the teats and perineum. occasionally, the infection may involve the flanks, underbelly, axilla, inner thigh, and neck. staphylococcal dermatitis may occur secondary to other skin lesions. diagnosis is based on lesions. culture will distinguish s. aureus. pathogenesis. simple boredom may cause rubbing, followed by staphylococcal infection of damaged epidermis. differential diagnosis. the presence of scabs makes contagious ecthyma a differential diagnosis, along with fungal skin infections and nutritional causes of skin disease. treatment. severe infections should be treated with antibiotics based on culture and sensitivity. severe lesions and lesions localized to the underbelly, thighs, and udder benefit by periodic cleaning with an iodophor shampoo and spraying with an antibiotic and an astringent (smith and sherman, 1994) . h. clostridial diseases i. clostridium perfringens type c infection (enterotoxemia and struck) etiology. clostridium perfringens is an anaerobic, grampositive, nonmotile, spore-forming bacterium that lives in the soil, in contaminated feed, and in gastrointestinal tracts of ruminants. the bacteria is categorized by toxin production. toxins include alpha (hemolytic), beta (necrotizing), delta (cytotoxic and hemoltyic), epsilon, and iota. types of c. perfingens are a, b, c, d, and e. this is a common and economically significant disease of sheep, goats, and cattle. clinical signs and diagnosis. the beta toxin associated with overgrowth of this bacterium results in a fatal hemorrhagic enterocolitis within the first 72 hr of a young ruminant's life. many animals may be found dead, with no clinical presentation. affected animals are acutely anemic, dehydrated, anorexic, restless, and depressed and may display tremors or convulsions as well as abdominal pain. feces may range from loose gray-brown to dark red and malodorous. morbidity and mortality may be nearly 100%. a similar noncontagious but acutely fatal form of enterotoxemia in adult sheep, called struck, occurs in yearlings and adults. struck is rare in the united states. the disease is also caused by the beta toxin of c. perfringens type c and is often associated with rapid dietary changes or shearing stresses in sheep. although affected animals are usually found dead, clinical signs include uneasiness, depression, and convulsions. mortality is usually less than 15%. diagnosis is usually based on necropsy findings, although confirmation can be made by culture of the organism. identification of the beta toxin in intestinal contents may be difficult because of instability of the toxin. necropsy findings. necropsy findings include a milk-filled abomasum, and hemorrhage in the distal small intestine and throughout the large intestine. petechial hemorrhages of the serosal surfaces of many organs, especially the thymus, heart, and gastrointestinal tract, will be visible. hydropericardium, hydroperitoneum, and hemorrhagic mesenteric lymph nodes will also be present. pulmonary and brain edema may also be seen. histologically, the gram-positive c. perfringens organisms may be visible in excess numbers along the mucosal surface of the swollen, congested, necrotic intestines. in cases of struck, necropsy findings include congestion and erosions of the mucosa of the gastrointestinal tract, serosal hemorrhages, and serous peritoneal and pericardial fluids. in late stages of the disease and especially if prompt necropsy is not performed, the organism will infiltrate the muscle fascial layers and produce serohemorrhagic and gaseous infiltration of perimysial and epimysial spaces. pathogenesis. hemorrhagic enterotoxemia is an acute, sporadic disease caused by the beta toxin of clostridium perfringens type c. neonates ingest the organism, which then proliferates and attaches to the gastrointestinal microvilli and elaborates primarily the beta toxins. the trypsin inhibitors present in colostrum prevent inactivation of the beta toxin. the toxins injure intestinal epithelial cells and then enter the blood, leading to acute toxemia. the intestinal injury may result in diarrhea, with small amounts of hemorrhage. associated electrolyte and water loss result in dehydration, acidosis, and shock. differential diagnosis. differential diagnoses include other clostridial diseases such as blackleg and black disease, as well as coccidiosis, salmonellosis, anthrax, and acute poisoning. clinical signs in chronic cases in older animals, such as adult goats, include soft stools, weight loss, anorexia, depression, and severe diarrhea, sometimes with mucus and blood. mature affected sheep may be blind and anorectic and may head-press. necropsyfindings. necropsy findings are similar to those seen with c. perfringens type c. additionally, extremely necrotic, soft kidneys ("pulpy kidneys") are usually observed immediately following death. (this phenomenon is in contrast to what is normally associated with later stages of postmortem autolysis.) focal encephalomalacia, and petechial hemorrhages on serosal surfaces of the brain, diaphragm, gastrointestinal tract, and heart are common findings. diagnosis can be made from the typical clinical signs and necropsy findings as well as the observation of glucose in the urine at necropsy. shock, probably through vascular damage. the noncontagious, peracute form of enterotoxemia occurs in suckling, fast-growing animals, either nursing from their dams or on high-protein, high-energy concentrates. the largest, fastest-growing animals generally are predisposed to this condition; for example, lambs, fat ewe lambs, and usually singleton lambs tend to be most susceptible. the hyperglycemia and glucosuria seen in acute cases are due to epsilon toxin effects on liver glycogen metabolism. should be administered to the pregnant animals prior to parturition. an alternative includes administration of an antitoxin to the newborn lambs. the disease may become endemic once it is on the premises. treatment. treatment is difficult and usually unsuccessful. antitoxin may be useful in milder cases, and the antitoxin and toxoid can also be administered during an outbreak. differential diagnosis. tetanus, enterotoxigenic e. coli, botulism, polioencephalomalacia, grain overload, and listeriosis are differentials. prevention and control. vaccination prevents the disease. maternal antibodies last approximately 5 weeks postpartum; thus young animals should be vaccinated at about this time. feeding regimens to young, fast-growing animals and feeding of concentrates to adults should be evaluated carefully. research complications. this disease can be costly in losses of neonates and younger animals. treatment. treatment consists of support (fluids, warmth), antitoxin administration, oral antibiotics, and diet adjustment. toxin that is proteolytically activated by trypsin. this disease caused by c. perfringens tends to be associated with sheep and is of less importance in goats and cattle. clinical signs. the peracute condition in younger animals is characterized by sudden deaths, which are occasionally preceded by neurological signs such as incoordination, opisthotonus, and convulsions. because the disease progresses so rapidly to death (within 1-2 hr), clinical signs are rarely observed. hypersalivation, rapid respirations, hyperthermia, convulsions, and opisthotonus have been noted. in acute cases, hyperglycemia and glucosuria are considered almost pathognomonic. etiology. clostridium tetani is a strictly anaerobic, motile, spore-forming, gram-positive rod that persists in soils and manure and within the gastrointestinal tract. at least 10 serotypes of c. tetani exist. clinical signs. infection by c. tetani is characterized by a sporadic, acute, and fatal neuropathy. after an incubation period of 4 days to 3 weeks, the animal exhibits bloat; muscular spasticity; prolapse of the third eyelid; rigidity and extension of the limbs, leading to a stiff gate; an inability to chew; and hyperthermia. erect or drooped ears, retracted lips, drooling, hypersensitivity to external stimuli, and a "sawhorse" stance are frequent signs. the animal may convulse. death occurs within 3-10 days, and mortality is nearly 100%, primarily from respiratory failure. diagnosis is based on clinical signs. musclerelated serum enzymes such as aspartate aminotransferase (ast), creatinine kinase (ck), and lactate dehydrogenase (ldh) might be elevated. (jensen and swift, 1982) . serum cortisol may also be elevated, and stress hyperglycemia may be evident. permanent lameness may result in survivors. contaminant and is often found as part of the gut microflora of herbivores. the organisms sporulate and persist in the environment. all species of livestock are susceptible, but sheep and goats are more susceptible than cattle. individual cases may occur, or herd outbreaks may follow castration, tail docking, ear tagging, or dehorning. mouth wounds may also be sites of entry. pathogenesis. tetanus, or lockjaw, is caused by the toxins of c. tetani. all serovars produce the same exotoxin, which is a multiunit protein composed of tetanospasmin, which is neurotoxic, and tetanolysin, which is hemolytic. a nonspasmogenic toxin is also produced. contamination of wounds results in anaerobic proliferation of the bacterium and liberation of the tetanospasmin, which diffuses through motor neurons in a retrograde direction to the spinal cord. the toxin inhibits the release of glycine and y-aminobutyric acid from renshaw cells; this resuits in hypertonia and muscular spasms. proliferation of c. tetani in the gut of affected animals may also serve as a source and may produce clinical signs. the uterus is the most common site of infection in postparturient dairy cattle with retained placentas. differential diagnoses. early in the course of the infection, differential diagnoses include bloat, rabies, hypomagnesemic tetany, polioencephalomalacia, white muscle disease, enterotoxemia in lambs, and lead poisoning. polyarthritis of cattle is a differential for the gait changes in that species. necropsy findings. findings are nonspecific except for the inflammatory reaction associated with the wound. because of the low number of organisms necessary to cause neurotoxicosis, isolation of c. tetani from the wound may be difficult. administering tetanus antitoxin (e.g., at least 500 iu in an adult sheep or goat); vaccinating with tetanus toxoid; administering of antibiotics (penicillin, both parenterally [potassium penicillin intravenously and procaine penicillin intramuscularly] and flushed into the cleaned wound), a sedative or tranquilizer (e.g., acepromazine or chlorpromazine) and a muscle relaxant; and keeping the animal in a dark, quiet environment. supportive fluids and glucose must be administered until the animal is capable of feeding. if the animal survives, revaccination should be done 14 days after the previous dose. prevention and control like other ubiquitous clostridial diseases, tetanus is impossible to eradicate. the disease can be controlled and prevented by following good sanitation measures, aseptic surgical procedures, and vaccination programs. tetanus toxoid vaccine is available and very effective for stimulating long-term immunity. tetanus antitoxin can be administered (200 iu in lambs) as a preventive or in the face of disease as an adjunct to therapy. both the toxoid and the antitoxin can be administered to an animal at the same time, but they should not be mixed in the syringe, and each should be administered at different sites, with a second toxoid dose administered 4 weeks later. animals should be vaccinated 2 or 3 times during the first year of life. does and ewes should receive booster vaccinations within 2 months of parturition to ensure colostral antibodies. research complications. unprotected, younger ruminants may be affected following routine flock or herd management procedures. contaminated or inadequately managed open wounds or lesions in older animals may provide anaerobic incubation sites. etiology. clostridium novyi, an anaerobic, motile, sporeforming, gram-positive bacteria, is the agent of bighead and black disease. clostridium novyi type d (c. hemolyticum) is the cause of bacillary hemoglobinuria, or "red water." clostridium chauvoei is the causative agent of blackleg. clinical signs. bighead is a disease of rams characterized by edema of the head and neck. the edema may migrate to ventral regions such as the throat. additional clinical signs include swelling of the eyelids and nostrils. most animals will die within 48-72 hours. black disease, or infectious necrotic hepatitis, is a peracute, fatal disease associated with c. novyi. it is more common in cattle and sheep but may be seen in goats. the clinical course is 1-2 days in cattle and slightly shorter in sheep. otherwise healthy-appearing adult animals are often affected. clinical signs are rarely seen, because of the peracute nature of the disease. occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency are observed prior to death. bacillary hemoglobinuria is an acute disease seen primarily in cattle and characterized by fever and anorexia, in addition to the hemoglobinemia and hemoglobinuria indicated by the name. animals that survive a few days will develop icterus. mortality may be high. blackleg, a disease similar to bighead, causes necrosis and emphysema of muscle masses, serohemorrhagic fluid accumulation around the infected area, and edema (jackson et al., 1995) . blackleg is more common in cattle than in sheep. the incubation period is 2-5 days and is followed by hyperthermia, muscular stiffness and pain, anorexia, and gangrenous myositis. the clinical course is short, 24-48 hr, and untreated animals invariably die. blackleg in cattle can be associated with subcutaneous edema or crepitation; these do not usually occur in sheep. most lesions are associated with muscles of the face, neck, perineum, thigh, and back. epizootiology and transmission. bighead is caused by the toxins of c. novyi, which enters through wounds often associated with horn injuries during fighting. the c. novyi type b organisms produce alpha and beta toxins, and the alpha toxins are mostly responsible for toxemia, tissue necrosis, and subsequent death. clostridium novyi type d is endemic in the western united states. it is hypothesized that the c. chauvoei organisms enter through the gastrointestinal tract. black disease and bacillary hemoglobinuria are associated with concurrent liver disease, often associated with fasciola infections (liver flukes); it is sometimes seen as a sequela to liver biopsies. the diseases are more common in summer months, and fecal contamination of pastures, flooding, and infected carcasses are sources of the organism. birds and wild animals may be vectors of the pathogen. ingested spores are believed to develop in hepatic tissue damaged and anoxic from the fluke migrations. necropsy. diagnosis of black disease is usually based on postmortem lesions. subcutaneous vessels will be engorged with blood, resulting in dried skin with a dark appearance. carcasses putrefy quickly. in addition, hepatomegaly and endocardial hemorrhages are common, and hepatic damage from flukes may be so severe that diagnosis is difficult. blood coagulates slowly in affected animals. pathogenesis. the propagation of the clostridial organisms is self-promoted by the damage caused by the toxins and the increased local anaerobic environment created. clostridium novyi proliferates in the soft tissues of the head and neck, and the resultant clostridial toxin causes increased capillary permeability and the liberation of serous fluids into the tissues. mixed infections with related clostridial organisms may lead to increasing hemorrhage and necrosis in the affected tissues. diagnosis is based on clinical signs. in black disease and bacillary hemoglobinuria disease, the ingested clostridial spores are absorbed, enter the liver, and cause hepatic necrosis. associated toxemia causes subcutaneous vascular dilatation; increased pericardial, pleural, and peritoneal fluid; and endocardial hemorrhages. the toxins produced by c. novyi, identified as beta, eta, and theta, and each having enzymatic or lytic properties or both, also contribute to the hemolytic disease. clostridium chauvoei spores proliferate in traumatized muscle areas damaged by transportation, rough handling, or injury. differential diagnosis. differential diagnoses include other clostridial diseases as well as photosensitization. hemolytic diseases such as babesiosis, leptospirosis, and hemobartonellosis should be included as differentials. treatment. for c. chauvoei infection (blackleg), early treatment with penicillin or tetracycline may be helpful. treatment for black disease is not rewarding even if the animal is found before death. carcasses from bacillary hemoglobinuria losses should be burned, buried deeply, or removed from the premises. prevention and control. vaccinating animals with multivalent clostridial vaccines can prevent these diseases. subcutaneous administration of vaccine material is recommended over intramuscular. vaccinations may be useful in an outbreak. careful handling of ruminants during shipping and transfers will contribute to fewer muscular injuries. for bighead, mature rams penned together should be monitored for lesions, especially during breeding season. control of fascioliasis is very important in prevention and control of black disease and in the optimal timing of vaccinations. etiology. clostridium septicum is the species usually associated with malignant edema, but mixed infections involving other clostridial species such as c. chauvoei, c. novyi, c. sordellii, and c. perfringens may occur. clostridium spp. are motile (c. chauvoei, c. septicum) or nonmotile, anaerobic, spore-forming, gram-positive rods. clinicial signs. malignant edema, or gas gangrene, is an acute and often fatal bacterial disease caused by clostridium spp. the incubation period is approximately 2-4 days. the affected area will be warm and will contain gaseous accumulations that can be palpated as crepitation of the subcutaneous tissue around the infected area. regional lymphadenopathy and fever may occur. the animal becomes anorexic, severely depressed, and possibly hyperthermic. edema and crepitation may be noted around the wound; death occurs within 12 hr to 2 days. epizootiology and transmission. the organisms are ubiquitous in the environment and may survive in the soil for years. the disease is especially prevalent in animals that have had recent wounds such as those that have undergone castration, docking, ear notching, shearing, or dystocia. necropsy findings. the tissue necrosis and hemorrhagic serous fluid accumulations resemble those of other clostridial diseases. pathogenesis. in most cases, the clostridial organisms cause a spreading infection through the fascial planes around the area of the injury; vegetative organisms then produce potent exotoxins, which result in necrosis (alpha toxin) and/or hemolysis (beta toxin). furthermore, the toxins enter the bloodstream and central nervous system, resulting in systemic collapse and high mortality. necropsy. spreading, crepitant lesions around wounds are suggestive of malignant edema. affected tissues are inflamed and necrotic. gas and serosanguineous fluids with foul odors infiltrate the tissue planes. large rod-shaped bacteria may be observed on histopathology; confirmation is made through culture and identification. intramuscular inoculation of guinea pigs causes a necrotizing myositis and death. organisms can be cultured from guinea pig tissues. treatment. infected animals can be treated with large doses of penicillin and fenestration of the wound is recommended. prevention and control. proper preparation of surgical sites, correct sanitation of instruments and the housing environment, and attention to postoperative wounds will help prevent this disease. multivalent clostridial vaccines are available. research complications. morbidity or loss of animals from lack of or unsuccessful vaccination and from contaminated surgical sites or wounds may be consequences of this disease. etiology. escherichia coli is a motile, aerobic, gram-negative, non-spore-forming coccobacillus commonly found in the environment and gastrointestinal tracts of ruminants. escherichia coli organisms have three areas of surface antigenic complexes (o, somatic; k, envelope or pili; and h, flagellar), which are used to "group" or classify the serotypes. colibacillosis is the common term for infections in younger animals caused by this bacteria. clinical signs. presentation of e. coli infections vary with the animal's age and the type of e. coli involved. enterotoxigenic e. coli infection causes gastroenteritis and/or septicemia in lambs and calves. colibacillosis generally develops within the first 72 hr of life when newborn animals are exposed to the organism. the enteric infection causes a semifluid, yellow to gray diarrhea. occasionally blood streaking of the feces may be observed. the animal may demonstrate abdominal pain, evidenced by arching of the back and extension of the tail, classically described as "tucked up." hyperthermia is rare. severe acidosis, depression, and recumbancy ensue, and mortality may be as high as 75%. the septicemic form generally occurs between 2 and 6 weeks of age. animals display an elevated body temperature and show signs suggestive of nervous system involvement such as incoordination, head pressing, circling, and the appearance of blindness. opisthotonos, depression, and death follow. occasionally, swollen, painful joints may be observed with septicemic colibacillosis. blood cultures may be helpful in identifying the septicemic form. in ruminants, e. coli is is a less common cause of cystitis and pyelonephritis. the cystitis is characterized by dysuria and pollakiuria; gross hematuria and pyuria may be present. the infection may or may not be restricted to the bladder; in the later presentation, and in cases of pyelonephritis, a cow will be acutely depressed, have a fever and ruminal stasis, and be anorexic. in chronic cases, animals will be polyuric and undergo weight loss. escherichia coli may also cause in utero disease in cattle, resulting in abortion or weakened offspring. epizootiology and transmission. escherichia coli is one of the most common gram-negative pathogens isolated from ruminant neonates. zeman et al. (1989) classify e. coli infections into four groups: enterotoxigenic, enterohemorrhagic, enteropathogenic, and enteroinvasive. enterotoxigenic e. coli (etec) attach to the enterocytes via pili, produce enterotoxins, and are the primary cause of colibacillosis in animals and humans. fimbrial (pili) antigens associated with ovine disease include k99 and f41. enterohemorrhagic e. coli (ehec) attach and efface the microviuus, produce verotoxins, and occasionally cause disease in humans and animals. enteropathogenic e. coli (epec) colonize and efface the microvillus but do not produce verotoxins. epec are associated with disease in humans and rabbits and cause a secretory diarrhea. enteroinvasive e. coli (eiec) invade the enterocytes of humans and cause a shigella-like disease. overcrowding and poor sanitation contribute significantly to the development of this disease in young animals. the organism will be endemic in a contaminated environment and present on dams' udders. the bacteria rapidly proliferate in the neonates' small intestines. the bacteria and associated toxins cause a secretory diarrhea, resulting in the loss of water and electrolytes. if the bacteria infiltrate the intestinal barrier and enter the blood, septicemia results. diagnosis of the enteric form can be made by observation of clinical signs, including diarrhea and staining of the tail and wool. necropsy findings. swollen, yellow to gray, fluid-filled small and large intestines, swollen and hemorrhagic mesenteric lymph nodes, and generalized tissue dehydration are common. septicemic lambs may have serofibrinous fluid in the peritoneal, thoracic, and pericardial cavities; enlarged joints containing fibrinopurulent exudates; and congested and inflamed meninges. isolation and serotyping of e. coli confirm the diagnosis. elisa and latex agglutination tests are available diagnostic tools. differential diagnosis. differential diagnoses include the enterotoxemias caused by c. perfringens type a, b, or c; campylobacter jejuni; coccidia, rotavirus, coronavirus, salmonella, and cryptosporidia. other contributing causes of abomasal tympany in young ruminants, such as dietary changes, copper deficiency, excessive intervals between feedings of milk replacer, or feeding large volumes should be considered. prevention and control. the best preventive measures are maintenance of proper housing conditions, limiting overcrowding, and frequently sanitizing lambing areas. attention to colostrum feeding techniques and colostral quality are important means of preventing disease. treatment must include intravenous fluid hydration and reestablishment of acid-base and electrolyte abnormalities. treatment. antibiotics such as trimethoprim-sulfadiazine, enrofloxacin, cephalothin, amikacin, and apramycin may be helpful; oral antibiotics are not recommended. vaccines are available for prevention of colibacillosis in cattle. etiology. corynebacterium pseudotuberculosis (previously c. ovis) are nonmotile, non-spore-forming, aerobic, short and curved, gram-positive coccobacilli. caseous lymphadenitis (cla) is such a common, chronic contagious disease of sheep and goats that any presentation of abscessing and draining lymph nodes should be presumed to be this disease until proven otherwise. the disease has been reported occasionally in cattle. clinical signs and diagnosis. abscessation of superficial lymph nodes, such as the superficial cervical, retropharyngeal, subiliacs (prefemoral), mammary, superficial inguinals, and popliteal nodes, and of deep nodes, such as mediastinal and mesenteric lymph nodes, is typical. radiographs may be helpful in identifying affected central nodes. peripheral lymph nodes may erode and drain caseous, "cheesy," yellow-green-tan secretions. the incubation period may be weeks to months. over time, an infected animal may become exercise-intolerant, anorexic, and debilitated. fever, increased respiratory rates, and pneumonia may also be common signs. exotoxin-induced hemolytic crises may occur occasionally. morbidity up to 15% is common, and morbid animals will often eventually succumb to the disease. diagnosis is based on clinical lesions; elisa serological testing is also available. smears of the exudate or lymph nodes aspirates can be gram-stained. lymph node aspirates may also be sent for culturing. epizootiology and transmission. the organism can survive for 6 months or more in the environment and enters via skin wounds, shearing, fighting, castration, and docking. ingestion and aerosolization (leading to pulmonary abscesses) have been reported as alternative routes of entry. necropsy findings. disseminated superficial abscesses as well as lesions of the mediastinal and mesenteric lymph nodes will be identified. cut surfaces of the affected lymph nodes may appear lamellated. lungs, liver, spleen, and kidneys may also be affected. cranioventral lung consolidation with hemorrhage, fibrin, and edema are seen histologically. pathogenesis. corynebacterium pseudotuberculosis produces an exotoxin (phospholipase d) that damages endothelial and blood cell membranes. this process enhances the organisms' ability to withstand phagocytosis. the infection spreads through the lymphatics to local lymph nodes. the necrotic lymph nodes seed local capillaries and hematogenously and lymphatically spread the organisms to other areas, especially the lungs. differential diagnosis. differentials include pathogens causing lymphadenopathy and abscessation. treatment. antibiotic therapy is not usually helpful. abscesses can be surgically lanced and flushed with iodinecontaining and/or hydrogen peroxide solutions. abscessing lymph nodes can be removed entirely from valuable animals. during warmer months, an insect repellent should be applied to and around healing lesions. all materials used to treat animals should be disposed of properly. because of the contagious nature of the disease, animals with draining and lanced lesions should be isolated from cla-negative animals at least until healed. commercial vaccines are available (piontkowski and shivvers, 1998) . minimizing contamination of the environment, using proper sanitation methods for facilities and instruments, segregating affected animals, and taking precautions to prevent injuries are all important. research complications. this pathogen is a risk for animals undergoing routine management procedures or invasive research procedures, because of its persistence in the environment, its long clinical incubation period, and its poor response to antibiotics. etiology. corynebacterium renale, c. cystitidis, and c. pilosum are sometimes referred to as the c. renale group. these are piliated and nonmotile gram-positive rods and are distinguished biochemically. corynebacterium renale causes pyelonephritis in cattle, and c. pilosum and c. cystitidis cause posthitis, also known as pizzle rot or sheath rot, in sheep and goats. in many references, all these clinical presentations are attributed to c. renale. clinical signs and diagnosis. acute pyelonephritis is characterized by fever, anorexia, polyuria, hematuria, pyuria, and arched back posture. untreated infections usually become chronic, with weight loss, anorexia, and loss of production in dairy animals. relapses are common, and some infections are severe and fatal. diagnosis of pyelonephritis is based on urinalysis (proteinuria and hematuria) and rectal or vaginal palpation (assessing ureteral enlargement). urine culturing may not be productive. in chronic cases, e. coli and other gram-negatives may be present. posthitis and vulvovaginitis are characteriazed by ulcers, crusting, swelling and pain. the area may have a distinct malodor. necrosis and scarring may be sequelae of more severe infections. fly-strike may also be a complication. diagnosis is based on clinical signs and on investigation of feeding regimens. epizootiology and transmission. ascending urinary tract infections with cystitis, ureteritis, and pyelonephritis are widespread problems, but incidence is relatively low. the vaginitis and posthitis contribute to the venereal transmission, but indirect transmission is possible because the organisms are stable in the environment and present on the wool or scabs shed from affected animals. posthitis occurs in intact and castrated sheep and goats. necropsy findings. pyelonephritis, multifocal kidney abscessation, dilated and thickened ureters, cystitis, and purulent exudate in many sections of the urinary tract are common finding at gross necropsy. of bovine genitourinary tracts. the pilus mediates colonization. conditions such as trauma, urinary tract obstruction, and anatomic anomalies may predispose to infection. in addition, more basic ph urine levels may block some immune defenses. infections ascend through the urinary tract. the bacteria are urease-positive when tested in vitro, and the ammonia produced in vivo during an infection damages mucosal linings, with subsequent inflammation. corynebacterium cystitidis and c. pilosum are normally found around the prepuce of sheep and goats. high-protein diets, resulting in higher urea excretion and more basic urine, are contributing factors. posthitis and vulvovaginitis may develop within a week of change to the more concentrated or richer diet, such as pasture or the addition of high-protein forage. the ammonia produced irritates the preputial and vulvar skin, increasing the vulnerability to infection. differential diagnosis. urolithiasis is a primary consideration for these diseases. contagious ecthyma should be considered for the crusting that is seen with posthitis and vulvovaginitis, although the lesions of contagious ecthyma are more likely to develop around the mouth. ovine viral ulcerative dermatosis is also a differential for the lesions of posthitis and vulvovaginitis. prevention and treatment. because high-protein feed is often associated with posthitis and vulvovaginitis, feeding prac-tices must be reconsidered. clipping long wool and hair also is helpful. treatment. long-term (3 weeks) penicillin treatment is effective for pyelonephritis. reduction of dietary protein, clipping and cleaning skin lesions, treating for or preventing fly-strike, and topical antibacterial treatments are effective for posthitis and vulvovaginitis; systemic therapy may be necessary for severe cases. surgical debridement or correction of scarring may also be indicated in severe cases. etiology. erysipelothrix rhusiopathiae is a nonmotile, nonspore-forming, gram-positive rod that resides in alkaline soils. clinical signs. erysipelothrix causes sporadic but chronic polyarthritis in lambs less than 3 months of age. in older goats, erysipelas has been associated with joint infections. epizootiology and transmission. the disease may follow wound inoculation associated with castration, docking, or improper disinfection of the umbilicus. following wound contamination and a 1-to 5-day incubation period, the lamb exhibits a fever and stiffness and lameness in one or more limbs. joints, especially the stifle, hock, elbow, and carpus, are tender but not greatly enlarged. necropsy findings. thickened articular capsules, mild increases in normal-appearing joint fluid and erosions of the articular cartilage are usually found. the joint capsule is infiltrated with mononuclear cells, but bacteria are difficult to find. diagnosis is based on clinical signs of polyarthritis, and confirmation is made by culturing the organism from the joints. differential diagnosis. differential diagnoses include polyarthritis caused by chlamydia or other bacteria and stiffness caused by white muscle disease. other bacteria causing septic joints include areanobacterium pyogenes and fusobacterium necrophorum. caprine arthritis encephalitis (cae) should also be considered. prevention and control. proper sanitation and prevention of wound contamination are important in preventing the infection in lambs. screening of goat herds for cae is recommended. therapy. erysipelas is sensitive to penicillin antibiotic m. etiology. dermatophilus congolensis is an aerobic, grampositive, filamentous bacterium with branching hyphae. dermatophilosis is a chronic bacterial skin disease characterized by crustiness and exudates accumulating at the base of the hair or wool fibers (scanlan et al., 1984) . clinical signs. animals will be painful but will not be pruritic. two forms of the disease exist in sheep: mycotic dermatitis (also known as lumpy wool) and strawberry foot rot. mycotic dermatitis is characterized by crusts and wool matting, with exudates over the back and sides of adult animals and about the face of lambs. strawberry foot rot is rare in the united states but is characterized by crusts and inflammation between the carpi and/or tarsi and the coronary bands. animals will be lame. in goats and cattle, similar clinical signs of crusty, suppurative dermatitis are seen; the disease is often referred to as cutaneous streptothricosis in these species. lesions in younger goats are seen along the tips of the ears and under the tail. diagnosis is based on clinical signs as well as the typical microscopic appearance on stained skin scrapings, cultures, and serology. epizootiology and transmission. the disease occurs worldwide, and the dermatophilus organism is believed to be a saprophyte. transmission occurs by direct or indirect contact and is aggravated by prolonged wet wool or hair associated with inclement weather. biting insects may aid in transmission. necropsy findings. lymphadenopathy as well as liver and splenic changes may be observed. histopathologically, superficial epidermal layers are necrotic and crusted with serum, white blood cells, and wool or hair. dermal layers are hyperemic and edematous and may be infiltrated with mononuclear cells. pathogenesis. lesions typically begin around the muzzle and hooves and the dorsal midline. prevention and control. potash alum and aluminum sulfate have been used as wool dusts in sheep to prevent dermatophilosis. minimizing moist conditions is helpful in controlling and preventing the disease. in addition, controlling external parasites or other factors that cause skin lesions is important. lesions will resolve during dry periods. treatment. animals can be treated with antibiotics such as penicillin and oxytetracycline. treating the animals with povidone-iodine shampoos or chlorhexidine solutions is also useful in clearing the disease. n. etiology. two bacteria, dichelobacter (bacteroides) nodosus and fusobacterium necrophorum, work synergistically in caus-ing contagious foot rot in sheep and goats. other organisms may be involved as secondary invaders. both dichelobacter and fusobacterium are nonmotile, non-spore-forming, anaerobic, gram-negative bacilli. foot rot is a contagious, acute or chronic dermatitis involving the hoof and underlying tissues (bulgin, 1986) . it is the leading cause of lameness in sheep. at least 20 serotypes of dichelobacter are known. arcanobacterium pyogenes may also contribute to the pathogenicity or to foot abscesses in goats. foot scald, an interdigital dermatitis, is caused primarily by d. nodosus alone. clinical signs. varying degrees of lameness are observed in all ages of animals within 2-3 weeks of exposure to the organisms. severely infected animals will show generalized signs of weight loss, decreased productivity, and anorexia associated with an inability to move. the interdigital skin and hooves will be moist, with a distinct necrotic odor. morbidity may reach 70% in susceptible animals. diagnosis is based on clinical signs. smears and cultures confirm the definitive agents. clinical signs of the milder disease, foot scald, include mild lameness, redness and swelling, and little to no odor. epizootiology and transmission. fusobacterium necrophorum is ubiquitous in soil and manure, in the gastrointestinal tract, and on the skin and hooves of domestic animals. in contrast, dichelobacter contaminates the soil and manure but rarely remains in the environment for more than about 2 weeks. some animals may be chronic carriers. overcrowded, warm, and moist environments are key elements in transmission. outbreaks are likely in the spring season. shipping trailers and contaminated pens or yards should be considered also as likely sources of the bacteria. pathogenesis. both organisms are transmitted to the susceptible animal by direct or indirect contact. the organisms enter the hoof through injuries or through sites where strongyloides papillosus larvae have penetrated. fusobacterium necrophorum initiates the colonization and is followed by d. nodosus. the latter attaches and releases proteases; these cause necrosis of the epidermal layers and separation of the hoof from the underlying dermis. the pathogenicity of the serotypes of d. nodosus is correlated with the production of these proteases and numbers of pili. additionally, f. necrophorum causes a severe, damaging inflammatory reaction. differential diagnosis. foot abscesses, tetanus, selenium/ vitamin e deficiencies, copper deficiency, strawberry foot rot, bluetongue virus infection (manifested with myopathy and coronitis), and trauma are among the many differentials that must be considered. treatment. affected animals are best treated by manually trimming the necrotic debris from the hooves, followed by application of local antibiotics and foot wraps. systemic antibiotics such as penicillin, oxytetracycline, and erythromycin may be used. goats have improved dramatically when given a single dose of penicillin (40,000 u/kg) (smith and sherman, 1994) . footbaths containing 10% zinc sulfate, 20% copper sulfate, or 10% formalin (not legal in all states) can be used for treatment as well as for prevention of the disease. affected animals should be separated from the flock. vaccination has been shown to be effective as part of the treatment regimen. some breeds of sheep and some breeds and lines of goats are resistant to infection. individual sheep may recover without treatment or are resistant to infection. epizootiology and transmission. cases may be sporadic, or epizootics may occur. bos taurus dairy breeds and animals with wide interdigital spaces are more commonly affected. the factors here are comparable to those present in foot rot of smaller ruminants. necropsy findings. findings at necropsy include dermatitis and necrosis of the skin and subcutaneous tissues. although necropsy would rarely be performed, secondary osteomyelitis may be noted in severe cases by sectioning limbs. prevention and control. prevention and control programs involve scrutiny of herd and flock management; quarantine of incoming animals; vaccination; segregation of affected animals; careful and regular hoof trimming; discarding trimmings from known or suspected infected hooves; maintaining animals in good body condition; avoiding muddy pens and holding areas; and culling individuals with chronic and nonresponsive infections. dichelobacter nodosus bacterins are commercially available; cross protection between serotypes varies. biannual vaccinination in wet areas may be essential. some breeds may develop vaccination site lumps. footbaths of 10% zinc sulfate, 10% formalin (where allowed by state regulations), or 10% copper sulfate are also considered very effective preventive measures. goats are less sensitive than sheep to the copper in the footbaths. treating and controlling foot rot is costly in terms of time, initial handling and treatments and their follow-up, housing space, and medications. etiology. interdigital necrobacillosis of cattle is caused by the synergistic infection of traumatized interdigital tissues by fusobacterium necrophorum and bacteroides melaninogenicus. like f. necrophorum, b. melaninogenicus is a nonmotile, anaerobic, gram-negative bacterium. dichelobacter nodosus, the agent of interdigital dermatitis, may be present in some cases. this is a common cause of lameness in cattle. clinical signs. clinical signs include mild to moderate lameness of sudden onset. hindlimbs are more commonly affected, and cattle will often flex the pastern and bear weight only on the toe. the interdigital space will be swollen, as will be the coronet and bulb areas. characteristic malodors will be noted, but there will be little purulent discharge. in more severe cases, animals will have elevated body temperature and loss of appetite. the les~ons progress to fissures with necrosis until healing occurs. the diagnosis is by the odor and appearance. anaerobic culturing confirms the organisms involved. pathogenesis. the bacteria enter through the skin of the interdigital area after trauma to the interdigital skin, from hardened mud, or from softening of the skin due to, for example, constant wet conditions in pens. colonization leads to cellulitis. in addition, f. necrophorum releases a leukocidal exotoxin that reduces phagocytosis and causes the necrosis, whereas the tissues and tendons are damaged by the proteases and collagenases produced by b. melaninogenicus. zinc deficiency may play a role in the pathogenesis in some situations. differential diagnoses. the most common differentials for sudden lameness include hairy heel warts and subsolar abcesses. bluetongue virus should also be considered. grain engorgement and secondary infection from cracks caused by selenium toxicosis should also be considered. the exotic footand-mouth disease virus would be considered in areas where that pathogen is found. prevention and control. as with foot rot in smaller ruminants, management of the area and herd are important. paddocks and pens should be kept dry, well drained, and free of material that will damage feet. footbaths and chlortetracycline in the feed have been shown to control incidence. affected animals should be segregated during treatment. chronically affected or severely lame animals should be culled. new cattle should be quarantined and evaluated. ing within a week include cleaning the feet and trimming necrotic tissue; parenteral antimicrobials, such as oxytetracycline or procaine penicillin, or sulfonomethazine in the drinking water or tetracyclines in feed; and footbaths (such as 10% zinc sulfate, 2.5% formalin, or 5% copper sulfate) twice a day. in severe cases, more aggressive therapy such as bandaging the feet or wiring the digits together may be needed. animals can recover without treatment but will be lame for several weeks. acquired immunity is reported to be poor. research complications are comparable to those noted for foot rot in smaller ruminants. fusobacterium necrophorum is also associated with foot abscesses, the infection of the deeper structures of the foot, in sheep and goats. only one claw of the affected hoof may be involved. the animals will be three-legged lame, and the affected hoof will be hot. pockets of purulent material may be in the heel or toe. etiology. bacteria such as fusobacterium spp., bacteroides spp., and dichelobacter nodosus have been isolated from bovine heel lesions. spirochete-like organisms have also been shown in the lesions of cows with papillomatous digital dermatitis (pdd), in the united states and europe; these have culturing requirements similar to those of treponema species. treatment. antibiotic and antiseptic regimens have been used successfully for this problem. antibiotics include parenteral cephalosporins and pencillins, as well as topical tetracyclines with bandaging. antiseptic or antibiotic solutions in footbaths include tetracyclines, zinc sulfate, lincomycin, spectinomycin, copper sulfate, and formalin. the footbaths must be well maintained, minimizing contamination by feces and other materials. tandem arrangements, such as the cleaning footbaths and then the medicated footbaths, and preventing dilution from precipitation are useful. other treatments such as surgical debridement, cryotherapy, and caustic topical solutions have been successful. research complications. infectious, contagious ppd is one of the major causes of lameness among heifers and dairy cattle and is a costly problem to treat. the outbreaks are generally worse in younger animals in chronically infected herds. the immune response is not well understood, and it may be temporary in older animals. clinical signs. all lesions occur on the haired, digital skin. one or all feet may be affected. most lesions occur on the plantar surface of the hindfoot (near the heel bulbs and/or extending from the interdigital space), but the palmar and dorsal aspect of the interdigital spaces may also be involved. progression of lesions, typically over 2-3 weeks, includes erect hairs, loss of hair, and thickening skin. moist plaques begin as red and remain red or turn gray or black. exudate or blood may be present on the plaque. plaques enlarge and "hairs" protrude from the roughened surface. lesioned areas are painful when touched. the lesions may or may not be malodorous. epizootiology and transmission. facility conditions and herd management are considered contributing factors. the following have been examined as contributing factors: nutrition, particularly zinc deficiency; poorly drained, low-oxygen, organic material underfoot; poor ventilation; rough flooring; damp and dirty bedding areas; and overcrowding. these interdigital lesions occur commonly in young stock and in dairy facilities throughout the world. the disease is seen only in cattle. pathogenesis. the organisms noted above, combined with poor facility and herd management, are critical in the pathogenesis. differential diagnosis. differentials for lameness will include sole abscesses, laminitis, and trauma. prevention and control. each facility and management condition noted above should be addressed in conjunction with appropriate antibiotic and/or antiseptic treatment regimens. all equipment used for hoof trimming must be cleaned and disinfected after every use. trucks and trailers should also be sanitized between groups of animals. etiology. haemophilus somnus is a pleomorphic, nonencapsulated, gram-negative bacterium. diseases caused by this organism include thromboembolic meningoencephalitis (teme), septicemia, arthritis, and reproductive failures due to genital tract infections in males and females. haemophilus somnus is a also major contributor to the bovine respiratory disease complex. haemophilus spp. have been associated with respiratory disease in sheep and goats. clinical signs. the neurologic presentation may be preceded by 1-2 weeks of dry, harsh coughing. neurologic signs include depression, ataxia, falling, conscious proprioceptive deficits; signs such as head tilt from otitis interna or otitis media, opisthotonus, and convulsions may be seen as the brain stem is affected. high fever, extreme morbidity, and death within 36 hr may occur. respiratory tract infections are usually part of the complex with infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza 3, mycoplasma, and pasteurella, and the synergism among these contributes to the signs of bovine respiratory disease complex (brdc). in acute neurologic as well as chronic pneumonic infections, polyarthritis may develop. abortion, vulvitis, vaginitis, endometritis, placentitis, and failure to conceive are manifestations of reproductive tract disease. in all cases, asymptomatic infections may also occur. diagnosis based on culture findings is difficult because h. somnus is part of the normal nasopharyngeal flora. paired serum samples are recommended; single titers in some animals seem to be high because of passive immunity, previous vaccination, or previous exposure. in cases of abortion, other causes should be eliminated from consideration. because the organism is considered part of the normal flora of cattle and can be isolated from numerous tissues, the distinction between the normal flora and the status of chronic carrier is not clear. outbreaks are associated with younger cattle in feedlots in western united states, but stresses of travel and coinfection with other respiratory pathogens are involved in some cases. adult cattle have also been affected. vaccination for viral respiratory pathogens may increase susceptibility. transmission is by respiratory and genital tract secretions. the organism does not persist in the environment. times of stress to the cattle is worthwhile. killed whole-cell bacterins are commercially available; these have been shown to be effective in controlling the respiratory disease presentation. control of other clinical aspects of the h. somnus disease by these bacterins has not been well described. treatment. rapid treatment at the first signs of neurologic disease is important in an outbreak. haemophilus somnus is susceptible to several antibiotics, such as oxytetracycline and penicillin, and these are often used in sequence until the cattle are recovered. necropsy findings. pathognomonic central nervous system lesions include multifocal red-brown foci of necrosis and inflammation on and within the brain and the meninges. many thrombi with bacterial colonies will be seen in these affected areas. ocular lesions may also be seen, including conjunctivitis, retinal hemorrhages, and edema. usually animals with neurological disease will not have respiratory tract lesions. the respiratory tract lesions include bronchopneumonia and suppurative pleuritis. when combined with pasteurella infection, the pathology becomes more severe. aborted fetuses will not show lesions, but necrotizing placentitis will be evident histologically. pathogenesis. inhalation of contaminated respiratory secretions from carrier animals is the primary means of transmission. the anatomical location of bacterial residence within the carriers has not been identified. after gaining access by way of the respiratory tract, the bacteria proliferate, and a bacteremia develops. the bacteria are phagocytosed by neutrophils but are not killed. the thrombosis formation is due to the adherence by the nonphagocytosed organisms to vascular endothelial cells, degeneration and desquamation of these cells, and exposure of subendothelial collagen, with subsequent initiation of the intrinsic coagulation pathway. antigen-antibody complex formation, resulting in vasculitis, is also correlated with high levels of agglutinating antibodies. other pathogens associated with neurological disease and respiratory disease such as pasteurella hemolytica, p. multocida, and p. aeruginosa. in smaller ruminants, corynebacterium pseudotuberculosis should be considered. prevention and control. stressed animals or those exposed to known carriers can be treated prophylactically with tetracycline administered parenterally or orally (in the feed or water). the late-stage polyarthritis is resistant to antibiotic therapy, because of failure of the antibiotic to reach the site of infection. planning vaccination programs carefully will decrease chances of outbreaks. for example, avoiding vaccinating animals for infectious bovine rhinotrachetitis and bovine viral diarrhea during clinical signs. leptospirosis is a contagious but uncommon disease in sheep and goats. the disease may cause abortion, anemia, hemoglobinuria, and icterus and is often associated with a concurrent fever. after a 4-to 10-day incubation period, the organism enters the bloodstream and causes bacteremia, fever, and red-cell hemolysis. leptospiremia may last up to 7 days. immune stimulation is apparently rapid, and antibodies are detectable at the end of the first week of infection; crossserovar protection does not occur. during active bacteremia, hemolysis may result in hemoglobin levels of 50% below normal. hyperthermia, hemoglobinuria, icterus, and anemia may be observed during this phase, and ewes in late gestation may abort. abortion usually occurs only once. mortality rates of above 50% have been reported in infected ewes and lambs (jensen and swift, 1982) . subclinical infection is more common in nonpregnant and nonlactating animals. sheep infected with leptospirosis may display a hemolytic crisis associated with igm acting as a cold-reacting hemagglutinin. acute and chronic infections in cattle are more common than infections in sheep and goats. acute forms in cattle display signs similar to those in sheep. acute infection in calves may progress to meningitis and death. lactating cows will have severe drops in production. chronic cases may lead to abortion, with retained placenta, and weakened calves or animals that carry the infection. infertility may also be a sequela. epizootiology and transmission. leptospires are a large genus, and leptospirosis is a complicated disease to prevent, treat, and control. the organism survives well in the environment, especially in moist, warm, stagnant water. cattle, swine, and other domestic and wild animals are potential carriers of serovars common to particular regions. wild animals often serve as maintenance hosts, but domestic livestock may be reservoirs also. organisms are shed in urine, in uterine discharges, and through milk. animals become carriers when they are infected with a host-adapted serovar; sporadic clinical disease is more commonly associated with exposure to a non-hostadapted serovar (heath and johnson, 1994) . infection may occur via oral ingestion of contaminated feed and water, via placental fluids, or through the mucous membranes of the susceptible animal. placental or venereal transmission may occur. as the organisms are cleared from the bloodstream, they chronically infect the renal convoluted tubules and the reproductive tract (and occasionally the cerebrospinal fluid or vitreous humor). chronically infected animals may shed the organism in the urine for 60 days or longer. necropsy. diagnosis is confirmed by identification of leptospires in fetal tissues. the leptospires are visible in silver-or fluorescent antibody-stained sections of liver or kidney. leptospires may also be seen under dark-field or phase-contrast microscopy of fetal stomach contents. fetal and maternal serology, and diagnostic tests such as the microscopic agglutination test, are useful; interpretation is complicated because of cross reaction of antibodies to many serovars. differential diagnosis. more than one serovar may cause infection in one animal, and each serovar should be considered as a separate pathogen. because of the associated anemia, differential diagnoses should include copper toxicity and parasites, in addition to other abortifacient diseases. prevention and control. polyvalent vaccines, tailored to common serovars regionally, are available and effective for preventing leptospirosis in cattle. immunity is serovar specific. because serological titers tend to diminish rapidly (40-50 days in sheep [jensen and swift, 1982] ), frequent vaccination may be necessary. other prevention measures such as species-specific housing, control of wild rodents, and proper sanitation should be instituted. treatment. antibiotic treatment is aimed at treating ill animals and trying to clear the carrier state. treatment methods for acute leptospirosis include oxytetracycline for 3-6 days. addition of oxytetracycline or chlortetracycline to the feed for 1 week may be helpful. these antibiotics are considered best for removal of the carrier state of some serovars. vaccination and antibiotic therapy can be combined in an outbreak. research complications. leptospirosis is zoonotic and may be associated with flulike symptoms, meningitis, or hepatorenal failure in humans. etiology. listeria monocytogenes is a pleomorphic, motile, non-spore-forming, [3-hemolytic, gram-positive bacillus that inhabits the soil for long periods of time and has been often found in fermented feedstuffs such as spoiled silage. of the 16 known serovars, several produce clinical signs in ruminants. listeria ivanovii (associated with abortions in sheep) is serovar 5. clinical signs. listeriosis is an acute, sporadic, noncontagious disease associated with neurological signs or abortions in sheep and other ruminants. the overall case rate is low. the disease may present as an isolated case or with multiple animals affected. three forms of disease are described: encephalitis, placentitis with abortion, and septicemia with hepatitis and pneumonia. the encephalitic form is most common in sheep; septicemic forms may occur in neonatal lambs (scarratt, 1987) . clinically, the encephalitic form begins with depression, anorexia, and mild hyperthermia after an incubation period of 2-3 weeks. as the disease progresses, animals exhibit nasal discharges and conjunctivitis and begin to walk in circles, as if disoriented. facial paralytic lesions, including drooping of an ear or eyelid, dilation of a nostril, or strabismus occur unilaterally on the affected side as the result of dysfunction of some or all the cranial nerves v-xii. the neck will by flexed away from the affected side. facial muscle twitching, protrusion of the tongue, dysphagia, hypersalivation, and nasal discharges may be noted. the hypersalivation may lead to metabolic acidosis in advanced cases in cattle. anorexia, prostration, coma, and death follow. the placental form usually results in last-trimester abortions in ewes and does, which typically survive this form of the disease. the affected females may be asymptomatic or may show severe clinical signs such as fever and depression, with subsequent retained placenta or endometritis. abortion usually occurs within 2 weeks of listeria infection. in cattle, abortion occurs during the last 2 months of gestation and has been induced experimentally 6-8 days after exposure. cows present with the range of clinical signs seen in smaller-ruminant dams. there is no long-term effect on the fertility of affected dams. epizootiology and transmission. the organism is transmitted by oral ingestion of contaminated feeds and water or possibly by inhalation. by the oral route, the organism enters through breaks in the oral cavity and ascends to the brain stem by way of nerves. when severe outbreaks occur, feedstuffs should be assessed for spoilage. listeria organisms can be shed by asymptomatic carriers, especially at the end of pregnancy and at lambing. diagnosis and necropsy findings. diagnosis is usually made from clinical signs. culture confirms the diagnosis (cold enrichment at 20~ is preferable but not essential for isolation). impression smears will show the pleomorphic gram-positive characterisitics of the pathogen. tissue fluorescent antibody techniques may also be utilized. gross lesions are not observed with the encephalitic form. microscopic lesions include thrombosis, neutrophilic or mononuclear foci in areas of inflammation, and neuritis. the pons, medulla, and anterior spinal cord are primarily affected in the encephalitic form. microabscesses of the midbrain are characteristic of listeria encephalitis in sheep. aborted fetuses that are intact may show fibrinous polyserositis, with excessive serous fluids; small, necrotic foci of the liver; and small abomasal erosions. necrotic lesions of the fetal spleen and lungs may also be seen. in goats, listeria-induced neurological lesions occur only in the brain stem. placentitis, focal bronchopneumonia, hepatitis, splenitis, and nephritis may be seen with other forms. pathogenesis. with the encephalitic form, the organism penetrates mucosal abrasions and enters the trigeminal or hypoglossal nerves. the listeria organisms then migrate along the nerves and associated lymphatics to the brain stem (medulla and pons). in the septicemic form, the organism penetrates tissues of the gastrointestinal tract and enters the bloodstream, to be distributed to the liver, spleen, lungs, kidneys, and placenta. after infection, organisms are shed in all body secretions (infected milk is an important risk factor for zoonosis). a toxin produced by listeria monocytogenes is correlated with pathogenicity, but the mechanism of the pathogenesis of this molecule has not been elucidated. differential diagnoses. rabies, bacterial meningitis, brain abscess, lead toxicity, and otitis media must be considered as differentials. in sheep, the differentials include organisms that cause abortion, and neurological signs, such as enterotoxemia due to clostridium perfringens type d. in goats, the major differentials include caprine arthritis encephalitis viral infection and chlamydial and mycoplasmal infections. in both species, scrapie is a differential. in cattle, aberrant parasite migration or hemophilus somnus infection must also be considered. prevention and control. affected dams should be segregated and treated. other animals in the group may be treated with oxytetracycline as needed. aborted tissues should be removed immediately. proper storage of fermented feeds minimizes this source of contamination. when silage spoils, the ph increases, producing a suitable growth environment for the organism. commercial vaccines are not available in the united states. treatment. affected animals can be treated aggressively with penicillin, ampicillin, oxytetracycline, or erythromycin. exceptionally high levels of penicillin are required for treating affected cattle. severely affected animals should receive appropriate fluid support and other nursing care. treatment is less successful, and mortality is especially high in sheep. recovered animals tend to resist reinfection. research complications. in addition to the loss of fetal animals, stress to the dams, and risks to other animals, any aborted tissue by a ruminant should be regarded as a potential zoonotic risk. listeria can cause mild to severe flulike symptoms in humans and may be a particular risk for pregnant women and for older or immune-compromised individuals. listeriosis in humans is a reportable disease. etiology. lyme disease is caused by the spirochete borrelia burgdorferi. clinical signs and diagnosis. reports in ruminants indicate seroconversion to b. burgdorferi, but there are few definitive correlations to the arthritis that is present. diagnosis requires culturing from the affected joints and diagnostic elimination of other causes of lameness and arthritis. epizootiology and transmission. the organism is present throughout much of the northern hemisphere and has been reported in many mammals and also in birds. ticks of the ixodes ricinus complex are the major vectors of the spirochete and must be attached for 24 hr for successful transmission. pathogenesis. the ixodes ticks have three life stages: larval, nymphal, and adult. feeding occurs once during each stage, and wild animals are the source of blood meals. the larval stages feed from rodents, such as the white-footed deer mouse, peromyscus leucopus, from which they acquire the spirochete. the nymphal stage is that which usually infects other animals. the adult ticks are usually found on deer. differential diagnosis. seroconversion to b. burgdorferi does not necessarily confirm the cause of arthritis. other causes of arthritis and lameness in ruminants include trauma, caprine arthritis encephalitis virus, mycoplasma spp., chlamydia psittaci, erysipelothrix spp., arcanobacterium pyogenes, brucella spp., and rickets. prevention and control. control of the tick vector is the most important factor in preventing the possibility of exposure or disease. treatment. antibiotic therapy, with tetracycline, penicillin, amoxicillin, and cephalosporins, is used for diagnosed or suspected lyme arthritis. research complications. lyme disease is zoonotic, and the lxodes ticks transmit the disease to humans. v. mastitis i. ovine mastitis mastitis in ewes may be acute, subclinical, or chronic. acute mastitis often results in anorexia, fever, abnormal milk, and swelling of the mammary gland. pasteurella haemolytica is the most common cause of acute mastitis. additional isolates may include, in order of prevalence, staphylococcus aureus, actinomyces (corynebacterium) spp., and histophilus ovis. escherichia coli and pseudomonas aeruginosa have also been found to cause acute mastitis. as many as six serotypes of pasteurella haemolytica have been isolated from the mammary glands of mastitic ewes. furthermore, intramammary inoculation of these organisms isolated from ovine and bovine pulmonary lesions has resulted in clinical mastitis in ewes (watkins.and jones, 1992) . subclinical mastitis is detected only indirectly, by counting somatic cells. the most common isolate from ewes with subclinical mastitis is coagulase-negative staphylococci. other isolates include actinomyces bovis, streptococcus uberis, s. dysgalactiae, micrococcus spp., bacillus spp., and fecal streptococci. most of these organisms are commonly found in the environment. diffuse chronic mastitis, or hardbag, results from interstitial accumulations of lymphocytes in the udder. both glands are usually affected, but no inflammation is present. serological evidence suggests that diffuse chronic mastitis is caused by the retrovirus that causes ovine progressive pneumonia (opp or maedi/visna virus). other bacterial agents or mycoplasma have not usually been isolated from udders with this type of mastitis. acute mastitis occurs in approximately 5% of lactating ewes annually, and it usually occurs either soon after lambing or when lambs are 3-4 months old (lasgard and vaabenoe, 1993) . subclinical mastitis occurs in 4-50% of lactating ewes (kirk and glenn, 1996) . subclinical mastitis is more common in ewes from high-milk-producing breeds. skin or teat lesions and dermatitis increase the prevalence of disease. acute mastitis can be diagnosed in ewes with associated systemic signs of disease by physical examination of the udder and inspection of the milk. subclinical mastitis is often suggested by somatic cell counts elevated above 1 x 106 cells/ml. when high somatic cell counts are identified, subclinical mastitis can be diagnosed by milk culture. the california mastitis test may also be helpful as an indicator of mastitis. manual palpation of a hard, indurated udder as well as serological testing for the maedi/visna virus is helpful in confirming the diagnosis of diffuse chronic mastitis. treatment for acute bacterial mastitis should include aggressive application of broad-spectrum antibiotics (intramammary and systemic) and supportive therapy such as fluids and anti-inflammatory drugs. it is may be helpful to milk out the infected ud-der frequently; oxytocin injections preceding milking will improve gland evacuation. because somatic cell counting is often not routinely performed, treatment of subclinical mastitis is seldom done. there is currently no treatment available for diffuse chronic mastitis. ii. caprine mastitis lactating goats are subject to inflammation of mammary gland, or mastitis. the primary causative organisms are staphylococcus epidermidis and other coagulasenegative staphylococcus spp. clinical signs of mastitis include abnormal coloration or composition of milk, mammary gland redness, heat and pain, enlargement of the mammary gland, discoloration of the mammary gland, and systemic signs of septicemia. large abscesses may be present in the affected gland. staphylococcus aureus is also associated with caprine mastitis, and toxemia may be part of the clinical picture. this organism produces a necrotizing alpha toxin that can result in gangrenous mastitis. caprine mastitis may be clinical or subclinical, and the first indication of mastitis may be weak, depressed, or thin kids. diagnosis is based on careful culture of mastitic milk. treatment includes frequent stripping, intramammary antibiotics, and nonsteroidal anti-inflammatory drugs. oxytocin (5-10 u) may help milk letdown for frequent strippings. bovine mastitis products can be used in the goat; however, care should be taken not to insert the mastitis tube tip fully, because damage to the protective keratin layer lining the teat canal may occur. in severe acute systemic cases, steroids, fluids, and systemic antibiotics may be necessary. other less common causes of mastitis in goats include streptococcus spp. (s. agalactiae, s. dysgalactiae, s. uberis, and zooepidemicus). gram-negative causes of caprine mastitis include escherichia coli, klebsiella pneumoniae, pasteurella spp., pseudomonas, and proteus mirabilis. corynebacterium pseudotuberculosis can cause mammary gland abscessation, whereas mycoplasma mycoides may cause agalactia and systemic disease. "hard udder" can be caused by caprine arthritis encephalitis virus (caev). brucellosis and listeriosis can cause a subclinical interstitial mastitis (smith and sherman, 1994) . iii. bovine mastitis mastitis is the disease of greatest economic importance for the dairy cattle industry. the majority of the impact will be on the production and overall health of the cows, but low-incidence herds also diminish the risk of calves' ingesting or being exposed to pathogens. the most common bovine mastitis pathogens include staphylococcus aureus and streptococcus agalactiae, s. dysgalactiae, and s. uberis; coliform agents such as escherichia coli, enterobacter aerogenes, serratia marcescens, and klebsiella pneumoniae; mycoplasmal species such as mycoplasma bovis, m. bovigenitalium, m. californicum, m. canadensis, and m. alkalescens; and salmonella spp. such as s. typhimurium, s. newport, s. enteritidis, s. dublin, and s. muenster. many of these agents such as staphylococcus spp., salmonella spp., and the coliforms can cause both acute and chronic mastitis, as well as severe systemic disease, including fever and anorexia. these must be regarded as herd and environmental pathogens in terms of treatment and prevention. the pathogenesis of staphylococcal infections is comparable to that in goats. staphylococcus agalactiae can be cleared from udders because it does not invade other tissues, is an obligate resident of the glands, and is susceptible to penicillin. in contrast, s. uberis and s. dysgalactiae are environmental organisms and can be highly resistant to pencillin. mycoplasma bovis is the more common of the mycoplasmal pathogens and can cause severe infections. transmission of the mycoplasmas is not well defined but may be related to their presence in other organ systems. treatments for mycoplasmal mastitis are not successful; culling is recommended. there are many interrelated factors associated with prevention and control of mastitis in a herd, including herd health and dry cow management, order of animals milked, milking procedures, milking equipment, condition of the teats, and the condition of the environment. management of the overall herd includes aspects such as vaccination programs, nutrition, isolation of incoming animals, and quarantine and treatment of or culling diseased individuals. culturing or testing newly freshened cows and monitoring the bulk milk tank serve as indicators of subclinical mastitis. herd management will diminish teat lesions. bacterin vaccines are available for preventing and controlling coliform mastitis and s. aureus mastitis. at the time of dry-off, all cows must be treated by intramammary route. some infections can be successfully cleared during this time. younger, disease-free animals should be milked first; any animals with diagnosed problems should be milked after the rest of the herd and/or segregated during treatment. milkers' hands easily serve as a means of pathogen transmission, and wearing rubber gloves is recommended. teat and udder cleaning practices include washing and drying with single-service paper or cloth towels or pre-and postmilking dipping. milking equipment must be maintained to provide proper vacuum levels and pumping rates, and liners should be the appropriate size. facilities that provide clean and dry areas for the animals to rest, feed, and move will diminish teat injuries and reduce exposures to mastitis pathogens. in that regard, inorganic bedding such as clean sand harbors few pathogens in contrast to shavings and sawdust. w. etiology. moraxella bovis, a gram-negative coccobacillus, is the most common cause of infectious bovine keratoconjunctivitis (ibk) in cattle. this organism is not a cause of keratoconjunctivitis in sheep and goats. the disease includes conjunctivitis and ulcerative keratitis. the pathogenic m. bovis strain is piliated, and at least seven serotypes exist. clinical signs. lacrimation, photophobia, and blepharospasm are seen initially. conjunctival injection and chemosis develop within a day of exposure, and then keratitis with corneal edema and ulcers. anterior uveitis may be a sequela within a few days, and thicker mucopurulent ocular discharge may be seen. corneal vascularization begins by 10 days after onset. reepithelialization of the corneal ulcers occurs by 2-3 weeks after onset. diagnosis is usually based on clinical signs, but culturing is helpful and fluoroscein staining is useful for demonstrating corneal ulceration. epizootiology and transmission. the disease is more severe in younger cattle. the clinical signs of ibk tend to be more severe in cattle that are also infected with infectious bovine rhinotracheitis (ibr) virus or those that have been vaccinated recently with modified live ibr vaccine. the bacteria are shed in nasal secretions and cattle with no clinical symptoms may be carriers. transmission is by fomites, flies, aerosols, and direct contact. incidence in winter months is very low. nonhemolytic strains are associated with the winter epidemics, and hemolytic strains are associated with summer epidemics. necropsy findings. necropsy is not typically performed on these cases. corneal edema, ulceration, hypopyon, and uveitis would be noted, depending on the stage of infection. pathogenesis. the pili ofm. bovis bind to receptors of corneal epithelium. the virulent strains of the bacteria then release the enzymes that damage the corneal epithelial cells. other factors contributing to infection include ultraviolet light and trauma from dust and plant materials. differential diagnoses. infectious bovine rhinotrachetitis virus causes conjunctivitis, but the central corneal ulceration that is characteristic of ibk is not seen with m. bovis infections. mycoplasma, listeria, branhamella (neisseria) , and adenovirus may be cultured from affected bovine eyes but none has been shown to produce the corneal lesions when inoculated into susceptible animals. prevention and control. cattle should not be immunized intranasally with modified live infectious bovine rhinotracheitis vaccine during ibk outbreaks; this will likely exacerbate the infection. new animals should be quarantined and treated prophylactically before introduction to herds. the available vaccines, containing. m. bovis pili or killed m. bovis, help decrease incidence and severity of disease; these preparations are not completely effective, because the m. bovis strain may not be homologous to that used for the vaccine preparation. other preventive measures include 10% permethrin-impregnated bilateral ear tags, pour-on avermectins, or dust bags or face rubbers containing insecticide (such as 5% coumaphos) to control flies throughout the season and premises; mowing of high pasture grass to minimize ocular trauma; provision of shade; control of dust and sources of other mechanical trauma; and segregation of animals by age. treatment. cattle can recover without treatment, but younger animals should be treated as soon as the infection is detected. antibiotic treatments include topical, subconjunctival administration and intramuscular dosing. several standard topical antibiotics have been shown to be effective, including oxytetracycline, gentamicin, and triple antibiotic combinations. these should be administered twice per day. subconjunctival injections of antibiotics, such as penicillin g, provide higher corneal levels of drug; these are typically administered only once or twice in severe cases. intramuscular doses of long-acting oxytetracycline, given on alternate days, are effective in larger herds, and 2 doses 72 hr apart eliminate carriers. third-eyelid flaps, temporary tarsorrhaphy, or eye patches may be useful in certain cases. epizootiology and transmission. although m. bovis can be killed by sunlight, it otherwise survives a long time in the environment and in cattle feces. animals acquire the infection from the environment or from other animals via aerosols, from contaminated feed and water, and from secretions such as milk, semen, genital discharges, urine, and feces. clinically normal animals may serve as carriers. the bacilli stimulate an initial neutrophilic tissue response. neutrophils become necrotic and are phagocytosed by macrophages, forming giant epithelioid cells called langhans' giant cells. an outer lymphocytic zone is formed, and fibrotic encapsulation creates the classical caseous nodules. vascular erosion and hematogenous migration of the organisms may lead to lesions throughout the body. necropsy findings. yellow primary tubercles (granulomas) with central areas of caseous necrosis and calcification are present in the lungs. caseous nodules are also associated with gastrointestinal organs and mesenteric lymph nodes. research complications. this pathogen does present a complication due to the carrier status of some animals, the likelihood of herd outbreaks, the severity of disease in younger animals, and the morbidity, possible progression to uveitis, and time and treatment costs associated with infections. the overall condition of the cattle will be affected for several weeks, and permanent visual impairment or loss, as well as ocular disfigurement, may occur. mycobacterium bovis infection (tuberculosis) etiology. mycobacteria are aerobic, nonmotile, non-sporeforming, acid-fast pleomorphic bacteria. most cases of tuberculosis in sheep are related to mycobacterium bovis or m. avium. cases in goats have been attributed to m. bovis, m. avium, or m. tuberculosis. mycobacterium bovis, or the bovine tubercle bacillus, is the cause in cattle but has been isolated from many domestic and wild mammals. other agents of mammalian tuberculosis include m. microti and m. africanum. clinical signs. tuberculosis is a sporadic, chronic, contagious disease of ruminants and is zoonotic. the infection is often asymptomatic later in the illness, and it may be diagnosed only at necropsy. the respiratory system (m. bovis) or the digestive system (m. avium) is the primary site of infection; other tissues such as mammary tissue and reproductive tract may be infrequently involved. locations of the characteristic tubercles will determine whether clinical signs are seen. respiratory signs may include dyspnea, coughing, and pneumonia. digestive tract signs include diarrhea, bloat, or constipation; diarrhea is most common. lymphadenopathy occurs in advanced cases. fever and generalized disease may be seen after calving. infected goats lose weight and develop a persistent cough. prevention and control. significant progress has been made in eradication programs in the united states during the past several decades, but during the 1990s, infected animals continued to be found in domestic cattle herds and particularly in captive deer herds in hunting preserves. the intradermal tuberculin test, using purified protein derivative (ppd), is usually used as a diagnostic indicator in live animals. this test should be performed annually on bovine and caprine dairy herds (and bison herds); the official tests are the caudal fold, comparative cervical, and single cervical tests. notification to state officials is required following identification of intradermal-positive animals. great care must be exercised in any handling of tissue or necropsies of reactors, and state animal health officials should be consulted regarding disposal of materials and cleaning of premises following depopulation of positive animals. no treatment is recommended, and treatment is usually not allowed, because of the zoonotic potential, chronicity of the disease, and the treatment costs. slaughter is preferred, to prevent potential transmission to humans. paratuberculosis, or johne 's disease (mycobacterium paratube rculo sis) etiology. mycobacterium paratuberculosis, the causative agent of johne's disease, is a fastidious, non-spore-forming, acid-fast, gram-positive rod. the organism is actually a subspecies of m. avium, but m. paratuberculosis does not produce the siderophore mycobactin (an iron-binding molecule) of m. avium. clinical signs and diagnosis. johne's disease is a chronic, contagious, granulomatous disease of adult ruminants and is characterized by unthriftiness, weight loss, and intermittent diarrhea. in sheep and goats, chronic wasting is usually seen, occasionally with pasty feces or diarrhea. in cattle, chronic diarrhea and rapid weight loss are the most common clinical signs of the disease. usually older adult animals are infected, but over time in an infected herd, younger animals will become infected when sufficient doses of organisms are ingested. although clinical signs are nonspecific, johne's disease should be considered if the affected diarrheic animals have a good appetite and are on a good anthelmintic program. the disease is diagnosed based on clinical signs and laboratory analyses, although none of the tests is more than 50% sensitive. in addition, the sensitivity of the serological tests differs between species. the standard is the fecal culture that takes 8-12 weeks. theenzyme-linked immunosorbent assay (elisa) is now considered the most reliable serological test, but false negatives do occur. other serological tests such as agar gel immunodiffusion (agid) and complement fixation are useful. herd screening may be done using the agid or elisa serological tests. identification of the organism on culture, or the presence of acid-fast organisms on mucosal or mesenteric lymph node smears or from rectal biopsies, helps confirm the diagnosis. some animals serologically negative for johne's disease, however, have been found to be positive on fecal culture. commercial agid tests approved for use in cattle may be useful in diagnosing johne's disease in sheep (dubash et al., 1996) . serological tests cross-react with other species of mycobacterium, especially m. avium. epizootiology and transmission. the organism is prevalent in the environment and is transmitted to young animals by direct or indirect contact. although vertical transmission has been reported, the organism more commonly enters the gastrointestinal tract and penetrates the mucosa of the distal small intestine, primarily the ileum. chronic carriers may intermittently shed the organisms. parasite that grows only in macrophages of infected animals. nursing infected dams are a primary source of infection of neonates. if the organism is not cleared, it proliferates slowly in the tissue, leading to inflammatory reactions that progress through neutrophilic to mononuclear stages. the organism may penetrate the lymphatics and proliferate in mesenteric lymph nodes. after an incubation period of a year or more, some of the carriers will progress to clinical disease manifested by fibrotic and hyperplastic changes in the ileum, leading to the classic thickening in the region. gut changes result in intermittent diarrhea, with subsequent dehydration, electrolyte imbalances, and malnutrition, although this clinical sign is more common in cattle than in sheep or goats. necropsy and diagnosis. the ileum from infected cattle is grossly thickened; this is not seen in sheep and goats. ileal and ileocecal lymph nodes provide the best samples for histology and acid-fast staining. differential diagnosis. diseases causing chronic wasting and poor coat and body condition of all ruminants should be considered. these include chronic salmonellosis, peritonitis, severe parasitism, winter dysentery, and pyelonephritis. deer can be infected, and the lesions can be confused with those of tuberculosis. prevention and control. prevention is the most effective method to manage this pathogen. efforts should be focused on eliminating the disease through test and slaughter. neonates should not be reared by infected dams. some states have johne's disease eradication programs. facilities and pastures where animals testing positive for johne' disease were maintained should be thoroughly cleaned and kept vacant for a year after culling. other considerations. mycobacterium paratuberculosis is being investigated as a factor in the development of crohn's disease in humans. etiology. the most common organism causing infection of the umbilicus is arcanobacterium (formerly actinomyces, corynebacterium) pyogenes; other bacteria may be present. arcanobacterium spp. are anaerobic, nonmotile, non-sporeforming, gram-positive, pleomorphic rods to coccobacilli. other environmental contaminants are also associated with this disease, such as escherichia coli, enterococcus spp., proteus, streptococcus spp., and staplylococcus spp. clinical signs and diagnosis. navel ill is an acute localized inflammation and infection of the external umbilicus. animals present with fever and painful enlargement of the umbilicus. animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, and hematuria. other common severe sequelae include septicemia, pneumonia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, uveitis, endocarditis, and diarrhea. neonates, and most cases occur within the first 3 months of age. cleanliness of the birthing and housing environment and successful transfer of passive immunity are important factors in the occurrence of the disease. dystocia resulting in weak neonates can be a factor predisposing to the development of the disease. navel ill is diagnosed by typical clinical signs. the presence of microabscesses and palpation of the umbilical area for firm intra-abdominal structures extending from the umbilicus are abnormal. assessment of colostral immunoglobulin transfer may contribute to determination of the prognosis. navel ill should always be considered for young ruminants with fever of unknown origin during the first week of life and for slightly older lambs, kids, or calves that are not thriving. arthrocentesis of affected joints and culture of the fluid for identification of the pathogen are also diagnostic options and essential for effective antimicrobial selection. differential diagnosis. the major differential is an umbilical hernia, which will typically not be painful or infected and can often be reduced. mycoplasmal arthritis is a differential in kids. in the past, erysipelothrix rhusopathiae was a common navel ill pathogen in sheep. treatment. omphalitis can be treated with a 10 to 14 day course of broad-spectrum antibiotics such as ampicillin, amoxicillin, penicillin, ceftiofur, florfenicol, and erythromycin. if an isolated abscess is palpable, it should be surgically opened and repeatedly flushed with iodine solutions. surgical reduction of the infected umbilicus is indicated if intra-abdominal structures are involved. the prognosis for recovery is good if systemic involvement has not occurred. prevention and control. the disease is best prevented and controlled by providing clean birthing environments, ensuring adequate colostral immunity, thoroughly dipping the umbilicus of newborns in tincture of iodine or strong iodine solution (lugol's), monitoring for dystocias, and maintaining young growing animals in noncontaminated environments. may invade the bloodstream, causing disseminated septicemia. clinically, the lambs may exhibit nasal discharge of mucopurulent to hemorrhagic exudate, hyperthermia, coughing, dyspnea, anorexia, and depression. with the respiratory form, auscultation of the thorax suggests dullness and consolidation of anteroventral lobes; this will be confirmed by radiographs. the disease is diagnosed by clinical signs, blood cultures from septicemic animals, blood smears showing bipolar organisms, and history of predisposing stressors. in cultures, p. hemolytica is distinguished from p. multocida by hemolysis on blood agar; only p. multocida produces indole. epizootiology and transmission. the organism is ubiquitous in the environment and in the respiratory tracts of these animals. younger ruminants, between 2 and 12 months of age, are especially prone to infection during times of stress, such as weaning, transportation, dietary changes, weather changes, and overcrowding. the pneumonic form appears as a complex associated with concurrent infections such as parainfluenza 3, adenovirus type 6, respiratory syncytial virus, mycoplasmas, chlamydia, pasteurella multocida and bordetella parapertussis (martin, 1996; brogden et al., 1998) . the organism is transmitted between animals by direct and indirect contact, through inhalation or ingestion. necropsy findings. necropsy lesions include areas of necrosis and hemorrhage in the small intestines and multifocal 1 mm lesions distributed on the surfaces of the lungs and liver. with the pneumonic form, serofibrinous exudates fill the alveoli; ventral lung lobes are consolidated and are congested and purple-gray in color. fibrinous pleuritis, pericarditis, and hematogenously induced arthritis also may be evident.. the disease can be costly to treat, and the toll taken on young animals due to the consequences of systemic infection may detract from their research value. etiology. pasteurella hemolytica and p. multocida are aerobic, nonmotile, non-spore-forming, bipolar, gram-negative rods. biotype a serotypes are associated with pneumonia and septicemia in all ruminants (ellis, 1984) . serotype 1 of p. hemolytica is considered a major cause of pulmonary lesions of bovine bronchopneumonia and fibrinous bronchopneumonia. clinical signs. pasteurellosis is an acute bacterial disease characterized by bronchopneumonia, septicemia, and sudden death. the organism invades the mucosa of the gastrointestinal tract or respiratory tract and causes localized areas of necrosis, hemorrhage, and thrombosis. the lungs and liver are frequent areas of formation of microabscesses. acute rhinitis or pharyngitis often precedes the respiratory form. the organism also pathogenesis. a leukotoxin is considered to be a key factor in the pathogenesis of the p. hemolytica infection. macrophages and neutrophils are lysed by the toxin as they arrive at the lung, and the enzymes released by the neutrophils cause additional damage to the tissue. treatment. treatment may include the use of antibiotics such as penicillin, ampicillin, tylosin, sulfonamides, or oxytetracycline. newer antibiotics, such as ceftiofur, tilmicosin, spectinomycin, and florfenicol, are very effective and approved for use in cattle. in outbreaks, cultures from fresh necropsies are helpful for determining sensitivities useful for the remaining group. prevention and control. the incidence of disease can be decreased by minimizing the degree of stress; by improving management, such as nutrition and control of parasitism; and, in cattle and sheep, by vaccinating for viral respiratory infections such as parainfluenza. early pasteurella hemolytica bacterin vaccines for use in cattle are not considered effective, but newer products based on immunizing against the leukotoxin and some bacterial capsule surface antigens are effective. pasteurella multocida bacterins and live streptomycin-dependent mutant vaccines are available. in young animals, passive immunity is protective. preventive measures also include maintaining good ventilation in enclosures and barns. new animals to the flock or herds should be quarantined for at least 2 weeks before introduction. etiology. salmonella typhimurium is a motile, aerobic to facultatively anaerobic, non-spore-forming, gram-negative bacillus and is the organism associated with enteric disease and some abortions in ruminants. it is a common inhabitant of the gastrointestinal tract of ruminants. current nomenclature categorizes s. typhimurium as a serovar within the species s. enteritidis (the other two species are s. typhi and s. choleraesuis). salmonella typhimurium, s. dublin, and s. newport are the common species seen in bovine cases. salmonella typhimurium, s. dublin, s. anatum, and s. montevideo are seen in ovine and caprine cases, although a host-adapted species has not been identified in the goat. ovine abortions due to various salmonella species are not reported in the united states but are enzootic in other countries. salmonella serotypes have been associated with aborted fetuses in all ruminant species. clinical signs and diagnosis. salmonellosis causes acute gastroenteritis, dysentery, and septicemia (anderson and blanchard, 1989) . clinically, the animals become anorexic and hyperthermic. diarrhea or dysentery develops; feces may contain mucus and/or blood and have a putrid odor. animals become severely depressed and weak, losing a high percentage of their body weight. animals may die in 1-5 days because of dehydration associated with dysenteric fluid loss, septicemia, shock, and acidosis. morbidity may be 25%, and mortality may be high. septicemia may result in subsequent meningitis, polyarthritis, and pneumonia. chronically infected animals may have intermittent diarrhea. in goats, salmonellosis may be recognized as diarrhea and septicemia in neonates, as enteritis in preweaned kids and mature goats, and, rarely, as abortion. adult cases may be sporadic, with intermittent bouts of diarrhea, subacute or even chronic. morbidity and mortality will be highest in neonates, and some may simply be found dead. the older animals generally tend to fare better during the disease. abdominal distension with profuse yellow feces is common. kids become severely depressed, anorexic, febrile (with temperatures as high as 106~176 dehydrated, acidotic, recumbent, and comatose. salmonella abortions may occur throughout gestation. there may not be any other clinical signs, or abortion may be seen with diarrhea, fever, and vulvar discharges. hemorrhage, placental necrosis, and edema will be present. metritis and placental retention may occur. some mortality of dams may occur. diagnosis is based on clinical signs and can be confirmed by culturing fresh feces or at necropsy. because of intermittent shedding of organisms, culture may be difficult; repeated cultures are recommended. leukopenia and a degenerative shift to the left are not uncommon hematological findings. epizootiology and transmission. stresses associated with recent shipping, overcrowding, and inclement weather may predispose the animal to enteric infection. birds and rodents may be natural reservoirs of salmonella in external housing environments. transmission is fecal-oral. after ingestion, the organisms may proliferate throughout the gastrointestinal tract and may penetrate the mucosa of the intestines, invade the peyer's patches and lymphatics, and migrate to the spleen, liver, and other organs. animals that survive may become chronic carriers and shedders of the organisms, and this has been demonstrated experimentally (arora, 1983) . fecal-oral transmission is also associated with salmonella abortion; veneral transmission has not been reported. necropsy findings and diagnosis. animals will have noticeable perineal staining. intestines (particularly the ileum, cecum, and colon) may contain mucoid feces with or without hemorrhages. petechial hemorrhages and areas of necrosis may be noticed on the surface of the liver, heart, and mesenteric lymph nodes. the wall of the intestines, gallbladder, and mesenteric lymph nodes will be edematous, and a pseudodiphtheritic membrane lining the distal small intestines and colon may be observed. this membrane is not normally seen in the goat (smith and sherman, 1994) . splenomegaly may be present. aborted fetuses will often be autolysed. placentitis, placental necrosis, and hemorrhage are commonly seen. serologic evidence of recent infection can be demonstrated in the dam. salmonella can be isolated from the aborted tissues. pathogenesis. after ingestion, the organism proliferates in the intestine. damage to the intestines and the resulting diarrhea are due to the bacterial production of cytoxin and endotoxin. although the salmonella organisms will be taken up by phagocytic cells involved in the inflammatory response, they survive and multiply further. septicemia is a common sequela, with the bacteria localizing throughout the body. in latently infected animals, it is often shed from the gallbladder and mesenteric lymph nodes. younger animals may be susceptible because of immature immunity and intestinal flora and higher intestinal ph. carriers may develop clinical disease when stressed. differential diagnoses. in young animals, differentials include other enteropathogens: escherichia coli, rotavirus and coronavirus, clostridia, cryptosporidia, and other coccidial forms. these pathogens may also be present in the affected animals. differentials in adults include bovine viral diarrheas and winter dysentery in cattle and parasitemia and enterotoxemia in all ruminants. prevention and control. affected animals should be isolated during herd outbreaks. samples for culture should include herdmates, water and feed sources, recently arrived livestock (other species), and area wildlife, including birds and rodents. repeated cultures, culling of animals, intensive cleaning, and disinfection of facilities are all important during outbreaks. the bacteria survive for about a week in moist cow manure. vaccination using the commercially available killed bacterin or autologous bacterins may be useful in outbreaks involving pregnant cattle, although the j-5 bacterin is now considered better. treatment. nursing care includes rehydration and correction of acid-base abnormalities. antibiotic therapy may be useful in cases with septicemia, but it is controversial because it may induce carrier animals. gentamicin, trimethoprim-sulfadiazine, ampicillin, enrofloxacin, and amikacin antibiotics may be successful. negative, rod-shaped bacterium. type a is more virulent than type b. clinical signs. although tularemia is a disease of livestock, pets, and wild animals, sheep are most commonly affected. the disease is characterized by hyperthermia, muscular stiffness, and lymphadenopathy. infected animals move stiffly, are depressed, and are hyperthermic. anemia and diarrhea may develop, and infected lymph nodes enlarge and may ulcerate. mortality may reach 40%. animals that recover will have immunity of long duration. epizootiology and transmission. the disease is most commonly transmitted by ticks or biting flies. the wood tick, dermacentor andersoni, is an important vector in transmitting the disease in the western united states, and, as natural hosts, wild rodents and rabbits tend to be reservoirs of the pathogen. research complications. salmonellosis is zoonotic, and some serotypes of the organism have caused fatalities even in immunocompetent humans. attempts should be made to identify and cull carrier animals. pathogenesis. the organisms, entering the tick bite wound, move via lymphatics to lymph nodes and subsequently to the bloodstream, where they cause septicemia. the organisms can also be transmitted orally through contaminated water. etiology. spirochete-like organisms are associated with this disease; it is now recognized that the agent is not a chlamydial organism. the disease has been reported only in the foothills bordering the central valley of california. necropsy findings. ticks may also be present on the carcasses. suppurative, necrotic lymph nodes are typical. lungs will be congested and edematous. diagnosis is confirmed by prompt culturing of the organism from lymph nodes, spleen, or liver where granulomatous lesions form; p. tularensis does not survive for long periods in carcasses. serological findings may also be helpful. clinical signs. cows that become infected with the causative agent before 6 months of gestation abort or give birth to weak calves without any clinical sign of infection. cows infected after 6 months of gestation give birth to normal calves. affected cows rarely abort in subsequent pregnancies. the tick vector is ornithonecropsy. fetuses show several pathological changes, including enlargement of the cervical lymph nodes, spleen, and liver. the calf's thymus will be small, and histologically there will be losses of thymic cortical lymphocytes. histologic changes in lymph nodes and spleen include vasculitis, necrosis, and histiocytosis. treatment. chlortetracycline treatment has been effective in controlling this disease. etiology. tularemia is caused by pasteurella (francisella) tularensis a nonmotile, non-spore-forming, aerobic, gram-control and prevention. eliminating the tick vectors can prevent tularemia. animals should be provided with fresh water frequently. the organism can survive in freezing conditions and in water and mud for long periods of time. caretakers, veterinarians, and researchers should take special precautions before handling the tissues of infected sheep, because this is a method of zoonotic spread. research complications. the disease is zoonotic, and transmission to people may result from tick bites or from handling contaminated tissues. although not a major disease of concern in sheep, researchers using potentially infected animals from western range states of the united states should be aware of it. the organism is antigenically related to brucella spp. etiology. yersiniosis is caused by infections with yersinia enterocolitica, a gram-negative, aerobic, and facultative anaerobe of the family enterobacteriaceae. there are 50 serotypes reported for y. enterocolitica. yersinia pseudotuberculosis infections have also been seen in ruminants. enteric infections predominate in the diseases caused by these bacteria. clinical signs and diagnosis. clinical disease may be seen rarely in many groups of ruminants. goats of 1-6 months old suffer from the enteric form of the disease, which is characterized by sudden death or the acute onset of watery diarrhea lasting 1 or more days. spontaneous abortions and weak neonates are also clinical manifestations of infection. lactating does may have mastitis that becomes chronically hemorrhagic. bacteremia results in internal abscesses, abortion, and acute deaths. yersinia pseudotuberculosis has been associated with laboratory goat epizootics (obwolo, 1976) . diarrhea in pastured sheep, stressed by other factors, has also been reported. diagnosis is based on culture and serology. epizootiology and transmission. the bacteria are carried by wild birds and rodents, and transmission is by ingestion of contaminated feed and water. research complications. yersinia is zoonotic. prevention and control. control measure are not well defined, because the epidemiology of the disease is poorly understood (smith and sherman, 1994) . tissues from affected goats must be handled and disposed of properly. areas housing affected goats must be thoroughly sanitized. treatment. in case of an abortion storm, treatment of goats with tetracycline has been useful. other broad-spectrum antibiotics may also be useful. clinical signs. contagious caprine pleuropneumonia is characterized by severe dyspnea, nasal discharge, cough, and fever (mcmartin et al., 1980) . infections with other mycoplasma species also have similar clinical signs. septicemia without respiratory involvement may also be a presentation. epizootiology and transmission. this disease is highly contagious, with high morbidity and mortality. transmission is by aerosols. mycoplasma mycoides subsp, mycoides has become a serious cause of morbidity and mortality of goat kids in the united states. necropsy. large amounts of pale straw-colored fluid and fibrinous pneumonia and pleurisy are typical. some lung consolidation may be present. meningitis, fibrinous pericarditis, and fibrinopurulent arthritis may also be found. diagnosis is usually made at necropsy by culture of the organism from lungs and other internal organs. differential dagnosis. in the united states, the principal differential for m. mycoides subsp, mycoides is caprine arthritis encephalitis. treatment. tylosin and oxytetracycline are effective. some infections are slow to resolve. prevention and control. vaccines are available in some areas. infected herds are quarantined. new goats should be quarantined before introduction to the herd. research complications. the worldwide distribution of the f38 biotype, as well as the aerosol transmission and high mor-bidity and mortality characteristics of mycoplasmal infectious, make these infections economically important diseases. considerable attention is presently given to this genus as a source of morbidity and mortality in goats. iv. mycoplasma conjunctivae (mycoplasmal keratoconjunctivitis) etiology. mycoplasma conjunctivae causes infectious conjunctivitis, or pinkeye, in sheep and goats with associated hyperemia, edema, lacrimation, and corneal lesions. mycoplasma mycoides subsp, mycoides, m. agalactiae, m. arginini, and acholeplasma oculusi have also been associated with keratoconjunctivitis in these species. respiratory disease and other infections, such as mastitis, may also be observed. clinical signs and diagnosis. all ages of animals may be affected. initially, lacrimation, conjunctival vessel injection, and then keratitis and neovascularization are seen. sometimes uveitis is evident. although the presentation is usually unilateral, bilateral involvement is possible. recurring infections are common. culturing provides the better diagnostic information, and cultures will be positive even after clinical signs have diminished. ily between animals by direct contact. animals can become reinfected, and carrier animals may be a factor in outbreaks. necropsy. it is unlikely that animals would die or be euthanized and undergo necropsy for this problem. conjunctival scrapings would include neutrophils during earlier stages and lymphocytes during later stages. epithelial cell cytoplasm should be examined for organisms. differential diagnosis. the primary differential in sheep and goats is chlamydia, as well as branhamella, rickettsia (colesiota) conjunctivae, and infectious bovine rhinotracheitis in goats only. it is important to consider these differentials if arthritis, pneumonia, or mastitis is present in the group or the individual. treatment. animals do recover spontaneously within about 10 weeks. tetracycline ointments and powders are also used. third-eyelid flaps may be necessary if corneal ulceration develops. prevention and control. new animals should be quarantined and, if necessary treated, before introduction to the flock or herd. etiology. eperythrozoonosis is a rare, sporadic, noncontagious, blood-borne disease in ruminants worldwide caused by the rickettsial agent eperythrozoon. host-specific species of importance are e. ovis, the causative species in sheep and goats, and e. wenyoni, e. tegnodes, and e. tuomii, the causative agents in cattle. although the disease is of minor importance, it can cause severe anemia and debilitation in affected animals. haemobartonella bovis is also rare, and is usually found only in association with other rickettsial diseases. clinical signs and diagnosis. the disease is more severe in sheep. following an incubation period of 1-3 weeks, infected animals exhibit episodic hyperthermia, weakness, and anemia. losses may be greater in younger lambs. cattle are usually latently infected but may have swollen and tender teats and legs. fever, anemia, and depression will be present if the cattle are stressed by another systemic disease. diagnosis is based on clinical evidence of anemia and is confirmed by observing the rickettsiae on the surface of red blood cells in a blood smear. epizootiology and transmission. the rickettsial organisms are transmitted typically to young sheep by biting insects, ticks, contaminated needles or blood-contaminated surgical instruments. necropsyfindings. necropsy findings include splenic enlargement and tissue icterus. has resulted in transient hyperthermia, mild respiratory disease, and mastitis. abortions, stillbirths, and births of weak lambs are also seen. epizootiology and transmission. coxiella burnetii is extremely resistant to environmental changes as well as to disinfectants; persistence in the environment for a year or longer is possible. the organism is associated with either a free-living or an arthropod-borne cycle. coxiella burnetii is found in a variety of tick species, such as ixodid or argasid, where it replicates and is excreted in the feces. once introduced into a mammal, coxiella may be maintained without a tick intermediate. the organism is especially concentrated in placental tissues, replicates in trophoblasts, and will be in reproductive fluids. additionally, the organism is shed in milk, urine, feces, and oronasal secretions. necropsy findings. no specific lesion will be seen in aborted or stillborn fetuses, but necrotizing placentitis will be a finding in cases of abortion. the placenta will contain white chalky plaques and a red-brown exudate. the disease can be diagnosed by identifying the rickettsial organisms in smears of placental secretions. the organism has been found in the placentas of clinically normal animals. the organism stains red with modified ziehl-neelsen and macchiavello stains and purple with giemsa stain. pathogenesis. the organism invades and destroys red blood cells. it is believed that intravascular hemolysis and erythrophagocytosis contribute to the macrocytic anemia. as with other red blood cell parasites, splenectomy aggravates the disease. differential diagnosis. because of the organisms' similarity to chlamydia, confirmation must be made by culture techniques, immunofluorescent procedures, elisa, and complement fixation tests. differential diagnosis. clontridium novyi type d, babesiosis, and leptospirosis are the primary differentials. prevention and control. following strict sanitation practices for surgical procedures and controlling external parasites prevent the disease. treatment. treatment is not usually recommended, but oxytetracycline has been used. sheep will develop immunity if supported nutritionally during the disease. research complications. splenectomized animals are the experimental models used to study these diseases. ii. q fever, or query fever (coxiella burnetii) etiology. coxiella burnetii is a small, gram-negative, obligate intracellular rickettsial organism that causes query fever and is regarded as a major cause of late abortion in sheep. clinical signs. infection of ruminants with c. burnetii is usually asymptomatic. experimental inoculation in other mammals treatment. coxiella can be treated with oxytetracyclines. a vaccine is not commercially available. prevention and control. any aborting animals should be segregated from other animals, and other pregnant animals should be treated prophylactically with tetracycline. serologic screening of ruminant sources should be performed routinely. barrier housing, a review of ventilation exhaust, and defined handling procedures are often required. all placentas and all aborted tissues should be handled and disposed of carefully. q fever has been reported in many mammalian species, including cats. research complications. coxiella burnetii-free animals are particularly important in studies involving fetuses and placentation. because of its zoonotic potential, c. burnetii presents a unique problem in the animal research facility environment. a single organism has been shown to cause disease. some of the greatest concerns are the risk to immunocompromised individuals, pregnant women, and other animals, and the presence of carrier animals or those that may shed the organism in placentas, for example. etiology. the ruminant adenoviruses are dna viruses that cause respiratory and reproductive tract diseases. nine antigenic types of the bovine adenovirus have been identified, with type 3 associated with respiratory disease. two of the ovine and two of the caprine antigenic types have been identified. clinical signs. signs of infection range from subclinical to severe, including pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, weak calf syndrome, and abortion. respiratory tract and intestinal tract diseases may be concurrent. infections caused by this virus are often found associated with other viral and bacterial infections. epizootiology and transmission. the virus is believed to be widespread, but prevalence and characteristics of infection have not been characterized. transmission of adenoviruses in other species (e.g., canine) is by aerosols or fecal-oral routes. necropsy findings. lesions found after experimental infections include atelectasis, edema, and consolidation of the lungs. etiology. the bluetongue virus is an rna virus in the orbivirus genus and reoviridae family. five serotypes (2, 10, 11, 13, and 17) have been identified in the united states, where it is seen mostly in western states. bluetongue is an acute arthropodborne viral disease of ruminants, characterized by stomatitis, depression, coronary band lesions, and congenital abnormalities (bulgin, 1986) . clinical signs and diagnosis. sheep are the most likely to show clinical signs. clinical disease is less common in goats and cattle. early in the infection, animals will spike a fever and will develop hyperemia and congestion of tissues of the mouth, lips, and ears. the virus name, bluetongue, is associated with the typical cyanotic membranes. the fever may subside, but tissue lesions erode, causing ulcers. increased salivary discharges and anorexia are often related to ulcers of the dental pad, lips, gums, and tongue, although salivation and lacrimation may precede apparent ulceration. chorioretinitis and conjunctivitis are also common signs in cattle and sheep. lameness may be observed associated with coronitis and is evident in the rear legs. skin lesions such as drying and cracking of the nose, alopecia, and mammary glands are also observed. secondary bacterial pneumonia may also occur. animals may also develop severe diarrhea and become recumbent. sudden deaths due to cardiomyopathy may occur at any time during the disease. hematologically, animals will be leukopenic. the course of the disease is about 2 weeks, and mortality may reach 80%. if animals are pregnant, the virus crosses the placenta and causes central nervous system lesions. abortions may occur at any stage of gestation in cattle. prolonged gestation may result from cerebellar hypoplasia and lack of normal sequence to induce parturition. cerebellar hypoplasia will also be present in young born of the infected dams, as well as hydrocephalus, cataracts, gingival hyperplasia, or arthrogryposis. diagnosis is suspected with the characteristic clinical signs and exposure to viral vectors. virus isolation is the best diagnostic approach if blood is collected during the febrile stage of the disease or brains from aborted fetuses. fluorescent antibody tests, elisa, virus neutralization tests, pcr, and agar gel immunodiffusion (agid) tests are also used to confirm the diagnosis. necropsy findings. at necropsy, erosive lesions may be observed around the mouth, tongue, palate, esophagus, and pillars of the rumen. ulceration or hyperemia of the coronary bands may also be seen. many of the internal organs will contain petechial and ecchymotic hemorrhages of the surfaces, and hemorrhage may be seen at the base of the pulmonary artery. pathogenesis. the virus multiplies in the hemocoel and salivary glands of the fly and is excreted in transmissible form in the insect's saliva. after entering the host, the virus causes prolonged viremia. the incubation period is 6-14 days. the virus migrates to and attacks the vascular endothelium. the resulting vasculitis accounts for the lesions of the skin, mouth, tongue, esophagus, and rumen and the edema often found in many tissues. ballooning degeneration of affected tissues, followed by necrosis and ulceration, occurs. the effects on fetuses appear to be due to generalized infections of developing organs. differential diagnosis. differentials include other infectious vesicular diseases such as foot-and-mouth disease, contagious ecthyma, bovine viral diarrhea virus-mucosal disease, infectious bovine rhinotracheitis, bovine papular stomatitis, and malignant catarrhal fever. rinderpest is a differential in countries where it is endemic. photosensitization should be considered. foot rot is a differential for the lameness and coronitis. differentials for the manifestations such as arthrogryposis include border disease virus and genetic predispositions of some breeds such as charolais cattle and merino sheep. prevention and control. cellular and humoral immunity are necessary for protection from infection. the bluetongue virus is insidious because the genome is capable of reassortment, and some vaccines will not have the antigenic components represented in the local infection. in addition, there is little to no cross protection between strains. modified live vaccines are available in some parts of the united states but should not be used in pregnant animals. vaccinating lambs and rams in an outbreak is worthwhile, for example, but vaccinating lategestation ewes may cause birth defects or abortions. congenital defects are more common from vaccine use than from naturally occurring infection. minimizing exposure to the vector in endemic areas will decrease the incidence of the disease. treatment. supportive care and nursing care are helpful, including gruels or softer feeds, easily accessed water, and shaded resting places. nonsteroidal anti-inflammatory drugs are often administered. for the cases of secondary bacterial pneumonia and some cases of bluetongue conjunctivitis, antibiotics may be administered. research complications. this is a reportable disease because clinical signs resemble foot-and-mouth disease and other exotic vesicular diseases. etiology. bovine lymphosarcoma refers to lymphoproliferative diseases in young cattle that are not associated with bovine leukemia virus (blv) infection, and those in older cattle that are associated with b lv. b lv is a b lymphocyte-associated retrovirus (johnson and kaneene, 1993a,b,c) . clinical signs. forms of bovine lymphosarcoma that are not associated with blv infection are calf, or juvenile; thymic, or adolescent (animals 6 months to 2 years old); and cutaneous (any age). the calf form is rare and characterized by generalized lymphadenopathy. onset may be sudden, and the disease is usually fatal within a few weeks. signs include lymphadenopathy, anemia, weight loss, and weakness. some animals may be paralyzed because of spinal cord compression from subperiosteal infiltration of neoplastic cells. the adolescent form is also rare, the course rapid, and the prognosis poor. the disease is seen most often in beef breeds such as hereford cattle and is characterized by space-occupying masses in the neck or thorax. these masses are also often present in the brisket. secondary effects of the masses are loss of condition, dysphagia, rumen tympany, and fatal bloat. the cutaneous presentation has a longer course and may wax and wane. the masses are found at the anus, vulva, escutcheon, shoulder, and flank; they are painful when palpated, raised, and often ulcerated. the animals are anemic, and neoplastic involvement may affect cardiac function. generalized or limited lymphadenopathy may be apparent. only the adult, or enzootic, form of bovine lymphosarcoma is associated with blv infection. many animals do not develop any malignancies or clinical signs of infection and simply remain permanently infected. some cows manifest disease only during the periparturient period. malignant lymphoma is the more common, whereas leukosis, due to b-lymphocyte proliferation, is rare. clinical signs are loss of condition and a drop in production of dairy cattle, anorexia, diarrhea, ataxia, paresis, and other signs dependent on the location of the neoplastic tissue. tumors are associated with lymphoid tissues. common sites also include the abomasum, spinal canal, and uterus. cardiac tumors develop at the right atrial or left ventricular myocardium, and associated beat and rate abnormalities may be auscultated. the common ocular manifestation of the disease is exophthalmos due to retrobulbar masses. many internal organs may be involved, and tumors may be palpable per rectum. secondary infections will be due to immunosuppression and the weakened state of the animal. sheep have acquired blv infection naturally and have been used as experimental models; in both situations, this species is susceptible to tumor and leukemia development. goats seroconvert but do not develop the clinical syndromes. diagnosis is based on the animal's age, clinical signs, serology, hematology findings according to the form, aspirates or biopsies of masses, and necropsy findings. kits are available for running agid, for which the blv antigens gp-51 and gp-24 are used; antibodies may be detected within weeks after exposure and may also help in predicting disease in clinically normal cattle. elisa and pcr diagnostic aids will also be helpful. worldwide. it is estimated that at least 50% of the cattle in the united states are infected with blv. as few as 1% of these animals develop lymphosarcoma, but the adult form of the disease described here is the most common bovine neoplastic disease in the united states. larger herds tend to have higher rates. genetic predisposition may be involved; in addition to the presence of blv, the type of bovine lymphocyte antigen (bola) may be correlated to resistance or susceptibility and to the course of the disease. transmission is believed to be by inhalation of blv in secretions; in colostrum; horizontally by contaminated equipment not sanitized between cattle; and by rectum (e.g., mucosal irritation during per-rectum exams or procedures). natural-service bulls may transmit the infection to cows. cows infected with blv may transmit the infection to their calves in utero. tabanid and other flies also serve as vectors, but these represent a minor means of transmission. necropsy findings. neoplastic infiltration of many organs and tissues are found in the calf form and the cutaneous forms. tumors may be local or widely distributed in the enzootic form. definitive diagnosis of neoplastic tissue specimens is by histology. pathogenesis. as with other retroviruses, the blv integrates viral dna into host target cell dna by means of the reverse transcriptase enzyme, creating a provirus. epizootiology and transmission. the virus is reported to be widespread. occurrence is often seasonal, and biting insects may be vectors. transmission with successful infection requires deep penetration of the skin. transmission may be by contaminated milkers' hands, contaminated equipment, and other fomites. differential diagnosis. differential diagnoses include other diseases that cause lesions on teats such as pseudocowpox, papillomatosis, and vesicular stomatitis. other vesicular diseases may be considered, but other more severe clinical signs might be associated with those. there is no vaccine for this disease. development and maintenance of a blv-free herd, or controlling infection within a herd, requires financial and programmatic commitments: blv-positive and blv-negative animals maintained separately; serologic testing (such as at least every 6 months) and separating positive animals; and washing and then disinfecting instruments, needles (or using sterile singleuse products), and equipment for ear tagging and dehorning and other such equipment between animals. a fresh rectal exam sleeve and lubricant should be used for each animal examined. otherwise serologically positive cows may have undetectable antibodies during the periparturient period. embryo transfer recipients should be negative, and the virus will not be transferred by the embryonic stage. calves should be fed colostrum from serologically negative cows. treatment. treatment regimens of corticosteroids and cancer chemotherapeutic agents provide only short-term improvement. in cases where ova, embryos, or semen need to be collected, supportive care for the affected animals is essential. research complications. the united states and several countries, some in europe, have official programs for eradication of enzootic bovine leukosis. prevention and control. established milking hygiene practices are important control measures: having milkers wash their hands with germicidal solutions or wear gloves, cleaning equipment between animals, and separating affected animals. treatment. there is no treatment, and affected animals should be separated from the herd and milked last. lesions can be cleaned and treated with topical antibacterials. etiology. the bovine viral diarrhea virus (bvdv) is a pestivirus of the flaviviridae family. the flaviviridae include hog cholera virus and border disease virus of sheep. the virus contains a single strand of positive-sense rna. a broad range of disease and immune effects is produced by b vdv only in cattle. in addition, this virus is important in the etiology of bovine pneumonias. bovine viral diarrhea/mucosal disease (bvd/md) is one of the most important viral diseases and one of the most complex diseases of cattle. strains of bvdv are characterized as cytopathic (cp) and noncytopathic (ncp), based on cell-culture growth characteristics. the virus has also been categorized as type 1 and type 2 isolates. heterologous strains exist that may confound even sound vaccination programs. etiology. bovine herpesvirus 2 causes bovine herpes mammillitis, a widespread disease characterized by teat and udder lesions, as well as oral and skin lesions. clinical signs and diagnosis. lesions begin suddenly with teat swelling; the tissue will be edematous and tender when touched. the udder lesions may extend to the perineum. the lesions progress to vesicles, then to ulcers; these may take 10 weeks to heal. lesions rarely may also develop focally around the mouth and generally on the skin of the udder. secondary mastitis may occur, because of bacteria associated with the scabs. diagnosis is by clinical signs and serologically. clinical signs and diagnosis. signs of bvdv infections may be subclinical but also include abortions, congenital abnormalities, reduced fertility, persistent infection (pi) with gradual debilitation, and acute and fatal disease. the presence of antibodies, whether from passive transfer or immunizations, does not necessarily guarantee protection from the various forms of the disease. an acute form of the disease, caused by type 2 bvdv, occurs in cattle without sufficient immunity. after an incubation period of 5-7 days, clinical signs include fever, anorexia, oculonasal discharge, oral erosions (including on the hard palate), diarrhea, and decreased milk production. the disease course may be shorter with hemorrhagic syndrome and death within 2 days. clinical signs of b vdv in calves also include severe enteritis and pneumonia. when susceptible cows are infected in utero from gestational be found extending throughout the gastrointestinal tract to the days 50-100, or gestational cows are vaccinated with a modi-cecum. the respiratory tract lesions will often be complicated fled live vaccine, abortion or stillbirth result. congenital defects caused by bvdv during gestational days 90-170 include impaired immunity (thymic atrophy), cerebellar hypoplasia, ocular defects, alopecia or hypotrichosis, dysmyelinogenesis, hydranencephaly, hydrocephalus, and intrauterine growth retardation. typical signs of cerebellar dysfunction will be evident in calves, such as wide-based stance, weakness, opisthotonus, hyperflexion, hypermetria, nystagmus, or strabismus. some severely affected calves will not be able to stand. ophthalmic effects include retinal degeneration and microphthalmia. fetuses can also be infected in utero, normal at birth, immunotolerant to the virus, and persistently infected (pi). the term mucosal disease is commonly associated with this form of the infection. many pi animals do not survive to maturity, however, and many have weakened immune systems. the pi animals are important because they shed virus and will probably show the clinical signs of mucosal disease (md) caused by a cp b vdv strain derived from an ncp b vdv strain. these md clinical signs include fever, anorexia, and profuse diarrhea that may include blood and fibrin casts, and oral and pharyngeal erosions, as well as erosion at the interdigital spaces and on the teats and vulva. many other associated clinical signs include anemia, bloat, lameness, or corneal opacities and discharges. secondary effects of hemorrhage and dehydration also contribute to the morbidity and mortality. animals that do not succumb to the disease will be chronically unthrifty, debilitated, and infection-prone. diagnosis in affected calves is based on herd health history, clinical signs, and antibodies to b vdv in precolostral serum. viral culturing from blood may be useful. in older animals, oral lesions, serology, detection of viral antigen, and virus isolation contribute to the diagnosis. leukopenia, and especially lymphopenia, are seen. serology must be interpreted with the awareness of the possibility of pi immunotolerant animals. vaccination against the disease carries its own set of side effects and potential problems, especially when using modified live vaccines, whether against cp or ncp strains. the condition of the animals is also a variable. epizootiology and transmission. bvdv is present throughout the world. transmission occurs easily by direct contact between cattle, from feed contaminated with secretions or feces, and by aborted fetuses and placentas. pi females transmit the virus to their fetuses. semen also is a source of virus. necropsy findings. in affected calves, histopathologic findings include necrosis of external germinal cells, focal hemorrhages, and folial edema. later in the disease, large cavities develop in the cerebellum, and atrophy of the cerebellar folia and thin neuropil are evident. older calves may have areas of intestinal necrosis. in cases where oral erosions occur, erosions will by secondary bacterial pneumonia. when the hemorrhagic syndrome develops, petechiation and mucosal bleeding will be present. pathogenesis. the cp and ncp strains are thought to be related mutations of the bvdv; the cp short-lived isolates are believed to arise from the ncp strains. the ncp strains are those present in the pi animals, and the strains are maintained in cattle populations. cp and ncp isolates vary in virulence, and classification of these types is based on viral surface proteins. considerable antigenic variation also exists between strains and types. other viral infections, such as bovine respiratory syncytial virus and infectious bovine rhinotracheitis, may also be present in the same animals. the pathology caused by b vdv is due to its ability to infect epithelial cells and impair the functioning of immune cell populations through out the bovine system. in type 2 bvdv hemorrhagic syndrome, death results from viral-induced thrombocytopenia. in fetuses, the virus infects developing germinal cells of the cerebellum. the purkinje's cells in the granular layer are killed, and necrosis and inflammation follow. the immune effects are the result of the virus's interfering with neutrophil and macrophage functions and of lymphocyte blastogenesis. all of these predispose the affected animals to bacterial infections with pasteurella haemolytica. b vdv damages dividing cells in fetal organ systems, resulting in abortions and congenital effects. differential diagnosis. many differentials must be considered for the clinical manifestations of b vdv infections. differentials for enteritis of calves include viral infections, cryptosporidia, escherichia coli, salmonella, and coccidia. salmonella, winter dysentery, johne's disease, intestinal parasites, malignant catarrhal fever (mcf), and copper deficiency are differentials for the diarrhea seen in the disease in adult animals. respiratory tract pathogens such as bovine respiratory syncytial virus, pasteurella, haemophilus, and mycoplasma must be considered for the respiratory tract manifestations. oral lesions are also produced by mcf, vesicular stomatitis, bluetongue, and papular stomatitis. infectious bovine herpesvirus 1, leptospirosis, brucellosis, trichomoniasis, and mycosis should be considered in cases of abortion. prevention and control. combined with sound management in a typical cattle herd, vaccination is the best way to prevent b vdv and should be integrated into the herd health program, timed appropriately preceding breeding, gestation, or stressful events. vaccine preparations for b vdv are modified live virus (mlv) or killed virus. each has advantages and disadvantages. the former induces rapid immunity (within 1 week) after a single dose, provides longer duration of immunity against sev-eral strains, and induces serum neutralizing antibodies. mlv vaccines are not recommended for use in pregnant cattle, may induce mucosal disease, and may be immunosuppressive at the time of vaccination. the immunosuppression is detrimental if cattle are concurrently exposed to field-strain virus because it will facilitate infection and possible clinical disease. the mlv strains may cross the placenta, resulting in fetal infections. the killed vaccines are safer in pregnant animals but require booster doses after the initial immunization, may need to be given 2-3 times per year, and do not induce cell-mediated immunity. passive immunity may protect most calves for up to 6-8 months of age. subsequent vaccination with mlv may provide lifelong immunity, but this is not guaranteed. annual boosters are recommended to protect against vaccine breaks. the virus persists in the environment for 2 weeks and is susceptible to the disfectants chlorhexidine, hypochlorite, iodophors, and aldehydes. maintenance of a closed herd to prevent any possibility of the introduction of the virus is difficult. isolation of new animals, avoidance of the purchase of pregnant cows, scrutiny of records from source farms, use of semen tested bulls, minimization of stress, testing of embryo-recipient cows, and maintainenance of populations of ruminants (smaller or wild species) separately on the premises will minimize viral exposure. other management strategies may require a program for testing and culling pi cattle. this can be expensive but may be a worthwhile investment to remove the virus shedders from a herd. no specific treatment is available. supportive care and treatment with antibiotics to prevent secondary infection are recommended. animals that survive the infection should be evaluated a month after recovery to determine their status as pi or virus-free. etiology. cache valley virus (cvv), of the arbovirus genus of the bunyaviridae family, is a cause of congenital defects in lambs. cvv infection in fetal and newborn lambs include arthrogryposis, microencephaly, hydranencephaly, porencephaly, cerebellar hypoplasia, and micromyelia. stillbirths and mummified fetuses are seen. lambs will be born weak and will act abnormally. diagnosis is by evidence of seroconversion in precolostral blood samples or fetal fluids, as the result of in utero infection. western united states, although it has been isolated in a few midwestern states. although considered a disease of sheep, virus has been isolated from cattle and from wild ruminants and antibodies found in white-tailed deer. transmission is by arthropods during the first trimester of pregnancy. etiology. caprine arthritis encephalitis virus (caev) occurs worldwide, with a high prevalence in the united states. caprine arthritis encephalitis (cae) is considered the most important viral disease of goats. the caev is in the lentivirus genus of the retroviridae family. it causes chronic arthritis in adults and encephalitis in young. caev is in the same viral genus as the ovine progressive pneumonia virus (oppv). clinical signs and diagnosis. the most common presentation in goats is an insidious, progressive arthritis in animals 6 months of age and older. animals become stiff, have difficulty getting up, and may be clinically lame in one or both forelimbs. carpal joints are so swollen and painful that the animal prefers to eat, drink, and walk on its "knees." in dairy goats, milk production decreases, and udders may become firmer. this retrovirus also causes neurological clinical signs in young kids 2-6 months old. kids may be bright and alert, afebrile, and able to eat normally even when recumbent. some kids may initially show unilateral weakness in a rear limb, which progresses to hemiplegia or tetraplegia. mild to severe lower motor neuron deficits may be noted, but spinal reflexes are intact. clinical signs may also include head tilt, blindness, ataxia, and facial nerve paralysis. older animals in the group may experience interstitial pneumonia or chronic arthritis. the pneumonia is similar to the pneumonia in sheep caused by oppv; the course is gradual but progressive, and animals will eventually lose weight and have respiratory distress. some animals in a herd may not develop any clinical signs. diagnosis is based on clinical signs, postmortem lesions, and positive serology for viral antibodies to caev. an agar gel immunodiffusion (agid) test identifies antibodies to the virus and is used for diagnosis. kids acquire an anti-caev antibody in colostrum, and this passive immunity may be interpreted as indicative of infection with the virus. the antibody does not prevent viral transmission. ep&ootiology and transmission. the virus is prevalent in most industrialized countries. the common means of transmission, from adults to kids, is in the colostrum and milk in spite of the presence of anti-caev antibody in the colostrum. transmission may occur among adult goats by contact. intrauterine transmission is believed to be rare. transmission to sheep has occurred only experimentally; there is no documented case of natural transmission. necropsy findings. necropsy and histopathology reveal a striking synovial hyperplasia of the joints with infiltrates of lymphocytes, macrophages, and plasma cells. other histologic lesions include demyelination in the brain and spinal cord, with multifocal invasion of lymphocytes, macrophages, and plasma cells. in severe cases of mastitis, the udder may appear to be composed of lymphoid tissue. tem, resulting in the formation of non-neutralizing antibody to viral core proteins and envelope proteins. immune complex formation in synovial, mammary gland, and neurological tissue is thought to result in the clinical changes observed. most commonly, the carpal joint is affected, followed by the stifle, hock, and hip. the infection is lifelong. differential diagnosis. the differential diagnosis for the neurologic form of caev should include copper deficiency, enzootic pneumonia, white muscle disease, listeriosis, and spinal cord disease or injury. the differential diagnosis for caev arthritis should include chlamydia and mycoplasma. prevention and control. herds can be screened for cae by testing serologically, using an agid or an enzyme-linked immunosorbent assay (elisa) test. the elisa is purported to be more sensitive, whereas the agid is more specific. individual animals show great variation in development of antibody. because cae is highly prevalent in the united states, and because seronegative animals can shed organisms in the milk, retesting herds at least annually may be necessary. recently, an immunoprecipitation test for cae has been developed that has high sensitivity and specificity. control measures include management practices such as test and cull, prevention of milk transmission, and isolation of affected animals. parturition must be monitored, and kids must be removed immediately and fed heat-treated colostrum (56~ for 1 hr). caev-negative goats should be separated from caevpositive goats. treatment. there is no treatment for caev. is also referred to as bovine herpesvirus 1 (bhv-1) and is an alphaherpesvirus. ibrv causes or contributes to several bovine syndromes, including respiratory and reproductive tract diseases. it is one of the primary pathogens in the bovine respiratory disease complex. strains include bhv-i.1 (associated with respiratory disease), bhv 1.2 (associated with respiratory and genital diseases), and bhv 1.4 (associated with neurological diseases), which has been reclassified as bovine herpesvirus 5. clude conjunctivitis, rhinotracheitis, pustular vulvovaginitis, balanoposthitis, abortion, encephalomyelitis, and mastitis. the respiratory form is known as infectious bovine rhinotracheitis, and clinical signs may range from mild to severe, the latter particularly when there are additional respiratory viral infections or secondary bacterial infections. the mortality rate in more mature cattle is low, however, unless there is secondary bacterial pneumonia. fever, anorexia, restlessness, hyperemia of the muzzle, gray pustules on the muzzle (that later form plaques), nasal discharge (that may progress from serous to mucopurulent), hyperpnea, coughing, salivation, conjunctivitis with excessive epiphora, and decreased production in dairy animals are typical signs. open-mouth breathing may be seen if the larynx or nasopharygneal areas are blocked by mucopurulent discharges. neonatal calves may develop respiratory as well as general systemic disease. in these cases, in addition to the symptoms already noted, the soft palate may become necrotic, and gastrointestinal tract ulceration occurs. young calves are most susceptible to the encephalitic form; signs include dull attitude, head pressing, vocalizations, nystagmus, head tilt, blindness, convulsions, and coma, as well as some signs, such as discharges, seen with respiratory tract presentations. this form is usually fatal within 5 days. abortion may occur simultaneously with the conjunctival or respiratory tract diseases, when the respiratory infection appears to be mild, or may be delayed by as much as 3 months after the respiratory tract disease signs. infectious pustular vulvovaginitis is most commonly seen in dairy cows, and clinical signs may be mild and not noticed. otherwise, signs are fever, depression, anorexia, swelling of the vulvar labia, vulvar discharge, and vestibular mucosa reddened by pustules. the cow will often carry her tail elevated away from these lesions. these soon coalesce, and a fibrous membrane covers the ulcerated area. if uncomplicated, the infection lasts about 4-5 days, and lesions heal in 2 weeks. younger infected bulls may develop balanoposthitis with edema, swelling, and pain such that the animals will not service cows. epizootiology and transmission. ibrv is widely distributed throughout the world, and adult animals are the reservoirs of infection. the disease is more common in intensive calf-rearing situations and in grouped or stressed cattle. transmission is primarily by secretions, such as nasal, during and after clinical signs of disease. modified live vaccines are capable of causing latent infections. necropsy findings. fibrinonecrotic rhinotracheitis is considered pathognomic for ibrv respiratory tract infections. there will be adherent necrotic lesions in the respiratory, ocular, and reproductive mucosa. when there are secondary bacterial infections, such as pasteurella bronchopneumonia, findings will include congested tracheal mucosa and petechial and ecchymotic hemorrhages in that tissue. lesions from the encephalitic form include lymphocytic meningoencephalitis and will be found throughout the gray matter (neuronal degeneration, perivascular cuffing) and white matter (myelitis, demyelination). intranuclear inclusion bodies are not a common finding with this herpesvirus. pathogenesis. in the encephalitic form, the virus first grows in nasal mucosa and produces plaques. these resolve within 11 days, and the encephalitis develops after the virus spreads centripetally to the brain stem by the trigeminal nerve dendrites. latent infections are also established in neural tissue. differential diagnosis. the severe oral erosions seen with bvdv infections are rare with infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (ibr-ipv). the conjunctivitis of ibr may initially be mistaken for that of a moraxella bovis (pinkeye) infection; the ibr will be peripheral, and there will not be corneal ulceration. bovine viral diarrhea virus and ibrv are the most common viral causes of bovine abortion. differentials for balanoposthitis include trauma from service. vated, attenuated, modified live, and genetically altered preparations. some are in combination with parainfluenza 3 (pi-3) virus. the mlv preparations are administered intranasally; these are advantageous in calves for inducing mucosal immunity even when serologic passive immunity is already present and adequate. some newer vaccines, with gene deletion, allow for serologic differentiation between antibody responses from infection or immunization. bulls with the venereal form of the infection will transmit the virus in semen; intranasal vaccine may be used to provide some immunity. treatment. uncomplicated mild infections will resolve over a few weeks; palliative treatments, such as cleaning ocular discharges and supplying softened food, are helpful in recovery. antibiotics are usually administered because of the high likelihood of secondary bacterial pneumonia. the encephalitic animals may need to be treated with anticonvulsants. etiology. parainfluenza 3, an rna virus of the family paramyxoviridae, causes mild respiratory disease of ruminants when it is the sole pathogen. the viral infection often predisposes the respiratory system to severe disease associated with concurrent viral or bacterial pathogens. viral strains are reported to vary in virulence. serotypes seen in the smaller ruminants are distinct from those isolated from cattle. clinical signs and diagnosis. infections ranging from asymptomatic to mild signs of upper respiratory tract disease are associated with this virus by itself; infections are almost never fatal. clinical signs include ocular and nasal discharges, cough, fever, and increased respiratory rate and breath sounds. in pregnant animals, exposure to pi-3 can result in abortions. clinical signs become apparent or more severe when additional viral pathogens are present, such as bovine viral diarrhea virus, or a secondary bacterial infection, such as pasteurella haemolytica infection, is involved. greater morbidity and mortality will be sequelae of the bacterial infections. viral isolation or direct immunofluorescence antibody (ifa) from nasal swabs can be used for definitive diagnosis. presently it is assumed that the virus is widespread in goats, but firm evidence is lacking. for an infection of pi-3 only, findings will be negligible. some congestion of respiratory mucosa, swelling of respiratory tract-associated lymph nodes, and mild pneumonitis may be noted grossly and histologically. intranuclear and intracytoplasmic inclusion bodies may be present in the mucosal epithelial cells. findings will be similar but not as severe as those caused by bovine respiratory syncytial virus. immunohistochemistry may also be used. pathogenesis. pi-3 infects the epithelial mucosa of the respiratory tract; however, the disease is often asymptomatic when uncomplicated. differential diagnosis. differentials, particularly in cattle, include infections with other respiratory tract viruses of ruminants: ibrv, bvdv, bovine respiratory syncytial virus, and type 3 bovine adenovirus. prevention and control. immunization, management, and nutrition are important for this respiratory pathogen, as for others. in cattle, modified live vaccines for intramuscular (im), subcutaneous (sc), or intranasal (in) administration are available. the im and sc routes provide immune protection within 1 week after administration but will not provide protection in the presence of passively acquired antibodies. it is contraindicated for pregnant animals because it will cause abortion. the in route immunizes in the presence of passively acquired antibodies, provides immunity within 3 days of administration, and stimulates the production of interferon. other vaccine formulations, about which less information is reported, include inactivated or chemically altered live-virus preparations; both are administered im, and followup immunizations are needed within 4 weeks. booster vaccinations are recommended for all preparations within 2-6 months after the initial immunization. all presently marketed vaccine products come in combination with other bovine respiratory viruses as multivaccine products. the humoral immunity protects against pi-3 abortions. there is no approved pi-3 vaccine for sheep and goats. the use of the cattle formulation in these smaller ruminants is not recommended. sound management of housing, sanitation, nutrition, and preventive medicine programs are all equally important components in prevention and control. treatment. uncomplicated disease is not treated. etiology. the respiratory syncytial viruses are pneumoviruses of the paramyxoviridae family and are common causes of severe disease in ruminants, especially calves and yearling cattle. two serotypes of the bovine respiratory syncytial virus (brsv) have been described for cattle; these may be similar or identical to the virus seen in sheep and goats. clinicalfindings and diagnosis. infections may be subclinical or develop into severe illness. clinical signs include fever, hyperpnea, spontaneous or easily induced cough, nasal discharge, and conjunctivitis. interstitial pneumonia usually develops, and harsh respiratory sounds are evident on auscultation. development of emphysema indicates a poor prognosis, and death may occur in the severe cases of the viral infection. secondary bacterial pneumonia, especially with pasteurella haemolytica, with morbidity and mortality, is also a common sequela. abortions have been assciated with brsv outbreaks. diagnosis is based on virus isolation and serology (acute and convalescent). nasal swabs for virus isolation should be taken when animals have fever and before onset of respiratory disease. prevention and control. vaccination should be part of the standard health program, and all animals should be vaccinated regularly. vaccinations should be administered within 1-2 months of stressful events, such as weaning, shipping, and introduction to new surroundings. currently available vaccines include an inactivated preparation and a modified live virus preparation administered intramuscularly or subcutaneously; immunity develops well in yearling animals, and colostral antibodies develop when cows are vaccinated during late gestation. passive immunity from colostrum provides at least partial protection to calves in herds where disease is prevalent. but this immunity suppresses the mucosal iga response and serum antibody responses. the basis for successful immune protection is the mucosal memory iga, but this is difficult to achieve with present vaccine formulations. the virus is easily inactivated in the environment. preventive measures in preweaning animals should include preconditioning to minimize weaning stress. treatment. recovery can be spontaneous; however, antibiotics and supportive therapy are useful to prevent or control secondary bacterial pneumonia. in severe cases, antihistamines and corticosteriods may also be necessary. use of vaccine during natural infection is not productive and may result in severe disease. etiology. ulcerative dermatosis is a contagious disease of sheep only. it is caused by a poxvirus similar to but distinct from the causative agent of contagious ecthyma ("current veterinary therapy," 1993). epizootiology and transmission. these viruses are considered ubiquitous in domestic cattle and are transmitted by aerosols. teroventral lung lobes. edema and emphysema are present. as the name indicates, syncytia, which may have inclusions, form in areas of the lungs infected with the virus. necrotizing bronchiolitis, bronchiolitis obliterans, and hyaline membrane formation will be evident microscopically. crusts associated with the skin and mucous membranes of the genitalia, face, and feet (bulgin, 1986) . genital lesions are much more common than the facial or coronal lesions. discomfort may be associated with the lesions. paraphimosis occasionally occurs. these lesions are painful; during breeding season, animals will avoid coitus. morbidity is low to moderate, and mortality negligible if the flock is otherwise healthy. diagnosis is based on clinical signs. pathogenesis. the severe form of the disease, which often follows a mild preliminary infection, is thought to be caused by immune-mediated factors during the process of infection in the lung. virulence may vary greatly among viral strains. united states, ulcerative dermatosis is transmitted through direct contact with abraded skin of the prepuce, vulva, face, and feet. necropsy findings. necropsy would rarely be necessary to diagnose an outbreak in a healthy flock. findings will be similar to those described for contagious ecthyma. when no contact with cattle has occurred. persistently infected animals, such as lambs, are shedding reservoirs of the virus in urine, feces, and saliva throughout their lives. pathogenesis. following an incubation period of 2-5 days, the virus replicates in the epidermal cells and leads to necrosis and pustule formation. pustules rapidly break, forming weeping ulcers. the ulcers scab over and eventually form a fibrotic scar. the disease usually resolves in 2-6 weeks. rarely, the disease will persist for many months to more than a year. differential diagnosis. the main differential is contagious ecthyma, which is grossly and histopathologically associated with epithelial hyperplasia. this is also a feature of ulcerative dermatosis. imals, especially males, should not be used for breeding. treatment. affected animals should be separated from the rest of the flock. treatment is supportive, including antiseptic ointments and astringents. research complications. breeding and maintenance of the flocks' condition, because of the pain associated with eating, will be compromised during an outbreak. etiology. border disease, also known as hairy shaker disease (or "fuzzies" in the southwestern united states), is a disease of sheep caused by a virus closely related to the bovine viral diarrhea virus (bvdv), a pestivirus of the togaviridae family. goats are also affected. the virus causes few pathogenic effects in cattle. clinical signs and diagnosis. border disease in ewes causes early embryonic death, abortion of macerated or mummified fetuses, or birth of lambs with developmental abnormalities. lambs infected in utero that survive until parturition may be born weak and often exhibit a number of congenital defects such as tremor, hirsutism (sometimes darkly pigmented over the shoulders and head), hypothyroidism, central nervous system defects, and joint abnormalities, including arthrogryposis. later, survivors may be more susceptible to diseases and may develop persistent, sometimes fatal, diarrhea. the virus infection produces similar clinical manifestations in goats, except that the hair changes are not seen. diagnosis includes the typical signs described above, as well as serological evidence of viral infection. virus isolation confirms the diagnosis. wide, and reports of disease are sporadic. disease has occurred necropsy findings. lesions include placentitis, and characteristic joint and hair-coat changes in the fetus. histologically, axonal swelling, neuronal vacuolation, dysmyelination, and focal microgliosis are observed in central nervous system structures. pathogenesis. the virus entering the ewe via the gastrointestinal or respiratory tracts penetrates the mucous membranes and causes maternal and fetal viremia. infection during the first 45 days of gestation causes embryonic death. in lambs infected between 45 and 80 days, the virus activates follicular development, diminishes the myelination of neurons, and causes dysfunction of the thyroid gland. infection after 80 days of gestation results in lambs that are born persistently infected. infected lambs have high perinatal mortality; survivors have diminished signs over time but, as noted, continue to shed the virus. prevention and control. border disease can be prevented by vaccinating breeding ewes with killed-bvdv vaccine. congenitally affected lambs should be maintained separately and disposed of as soon as humanely possible. new animals to the flock should be screened serologically. if cattle are housed nearby, vaccination programs for bvdv should be maintained. treatment. there is no treatment other than supportive care for affected animals. etiology. contagious ecthyma, also known as contagious pustular dermatitis, sore mouth, or off, is an acute dermatitis of sheep and goats caused by a parapoxvirus. this disease occurs worldwide and is zoonotic. naturally occurring disease has also been reported in other species such as musk ox and reindeer. other parapoxviruses infect the mucous membranes and skin of cattle, causing the diseases bovine pustular dermatitis and pseudocowpox. clinical signs and diagnosis. the disease is characterized by the presence of papules, vesicles, or pustules and subsequently scabs of the skin of the face, genitals of both sexes, and coronary bands of the feet. lesions develop most frequently at mucocutaneous junctions and are found most commonly at the commissures of the mouth. off is usually found in young animals less than 1 year of age. younger lambs and kids will have difficulty nursing and become weak. lesions may also develop on udders of nursing dams, which may resist suckling by offspring to nurse, leading to secondary mastitis. the scabs may appear nodular and raised above the surface of the surrounding skin. morbidity in a susceptible group of animals may exceed 90%. mortality is low, but the course of the disease may last up to 6 weeks. diagnosis is based on characteristic lesions. biopsies may reveal eosinophilic cytoplasmic inclusions and proliferative lesions under the skin. electron microscopy will reveal the virus itself. disease is confirmed by virus isolation. epizootiology and transmission. all ages of sheep and goats are susceptible. seasonal occurrences immediately after lambing and after entry into a feedlot are common; stress likely plays a role in susceptibility to this viral disease. older animals develop immunity that usually prevents reinfection for at least 1 or more years. resistant animals may be present in some flocks or herds. the virus is very resistant to environmental conditions and may contaminate small-ruminant facilities, pens, feedlots, and the like for many years as the result of scabs that have been shed from infected animals. transmission occurs through superficial lesions such as punctures from grass awns, scrapes, shearing, and other common injuries. necropsy findings. necropsy findings include ballooning degeneration of epidermal and dermal layers, edema, granulomatous inflammation, vesiculation, and cellular hyperplasia. secondary bacterial infection may also be evident. pathogenesis. the virus is typical of the poxviridae, resembling sheep poxvirus (not found in the united states) and vaccinia virus and replicating in the cytoplasm of epithelial cells. following an incubation period of 2-14 days, papules and vesicles develop around the margins of the lips, nostrils, eyelids, gums, tongue, or teats; skin of the genitalia; or coronary band of the feet. the vesicles form pustules that rupture and finally scab over. virus should be considered in both sheep and goats. an important differential in goats is staphylococcal dermatitis. prevention and control. individuals handling infected animals should be advised of precautions beforehand, should wear gloves, and should separate work clothing and other personal protective equipment. clippers, ear tagging devices, and other similar equipment should always be cleaned and disinfected after each use. colostral antibodies may not be protective. vaccinating lambs and kids with commercial vaccine best prevents the disease. dried scabs from previous outbreaks may also be used by rubbing the material into scarified skin on the inner thigh or axilla. animals newly introduced to infected premises should be vaccinated upon arrival. precautions must be taken when vaccinating animals, because the vaccine may induce orf in the animal handlers; it is not recommended to vaccinate animals in flocks already free of the disease. affected dairy goats should be milked last, using disposable towels for cleaning teat ends. treatment. affected animals should be isolated and provided supportive care, especially tube feeding for young animals whose mouths are too sore to nurse. treatment should also address secondary bacterial infections of the orf lesions, including systemic antibiotics for more severe infections. treatment for myiasis may also be necessary. the viral infection is self-limiting, with recovery in about 4 weeks. research complications. carrier animals may be a factor in flock or herd outbreaks. contagious ecthyma is a zoonotic disease, and human-to-human transmission can also occur. the virus typically enters through abrasions on the hands and results in a large (several centimeters) nodule that is described as being extremely painful and lasting for as many as 6 weeks. lesions heal without scarring. etiology. foot-and-mouth disease (fmd) is caused by the foot-and-mouth disease virus, a picornavirus in the aphthovirus genus. the disease is also referred to as aftosa or aphthous fever. seven immunologically distinct types of the virus have been identified, with 60 subtypes within those 7. epidemics of the disease have occurred worldwide. north and central america have been free of the virus since the mid-1950s. this is a reportable disease in the united states; clinical signs are very similar to other vesicular diseases. cattle (and swine) are primarily affected, but disease can occur in sheep and is usually subclinical in goats. clinical signs and diagnosis. in addition to vesicle formation around and in the mouth, hooves, and teats, fever, anorexia, weakness, and salivation occur. vesicles may be as large as 10 cm, rupture after 2 days, and subsequently erode. secondary bacterial infections often occur at the erosions. anorexia is likely due to the pain associated with the oral lesions. high morbidity and low mortality, except for the high mortality in young cattle, are typical. diagnosis must be based on elisa, virus neutralization, fluorescent antibody tests, and complement fixation. epizootiology and transmission. domestic and wild ruminants and several other species, such as swine, rats, bears, and llamas are hosts. asymptomatic goats can serve as virus reservoirs for more susceptible cohoused species such as cattle. greater mortality occurs in younger animals. the united states, great britain, canada, japan, new zealand, and australia are fmd-free, whereas the disease is endemic in most of south america, parts of europe, and throughout asia and africa. the virus is very contagious and is spread primarily by the inhalation of aerosols, which can be carried over long distances. transmission may also occur by fomites, such as shoes, clothing, and equipment. human hands, soiled bedding, and animal products such as frozen or partially cooked meat and meat products, hides, semen, and pasteurized milk also serve as sources of virus. necropsy findings. vesicles, erosions, and ulcers are present in the oral cavity as well as on the rumen pillars and mammary alveolar epithelium. myocardial and skeletal muscle degeneration (zenker's) is most common (and accounts for the greater mortality) in younger animals. histological findings include lack of inclusion bodies. vesicular lesions include intracellular and extracellular edema, cellular degeneration, and separation of the basal epithelium. replicates in the pharynx and digestive tract in the cells of the stratum spinosum, and viremia and spread of virus to many tissues occur before clinical signs develop. virus shedding begins about 24 hr before clinical signs are apparent. vesicles result from the separation of the superficial epithelium from the basal epithelium. fluid fills the basal epithelium, and erosions develop when the epithelium sloughs. persistent infection also occurs, and virus can be found for months or years in the pharnyx; the mechanisms for the persistence are not known. differential diagnosis. vesicular stomatitis is the principal differential. other differentials include contagious ecthyma (orf), rinderpest, bluetongue, malignant catarrhal fever, bovine papular stomatitis, bovine herpes mammillitis, and infectious bovine rhinotracheitis virus infection. products from endemic areas is regulated. quarantine and slaughter are practiced in outbreaks in endemic areas. quarantine and vaccination are also used in endemic areas, but vaccines must be type-specific and repeated 2 or 3 times per year to be effective and will provide only partial protection. autogenous vaccines are best in an outbreak. passive immunity protects calves for up to 5 months after birth. the virus is inactivated by extremes of ph, sunlight, high temperatures, sodium hydroxide, sodium carbonate, and acetic acid. treatment. nursing care and antibiotic therapy to minimize secondary reactions help with recovery. humoral immunity is considered the more important immune mechanism, with cellmediated immunity of less importance. research complications. rare cases in humans have been reported. importation into the united states of animal products from endemic areas is prohibited. etiology. malignant catarrhal fever (mcf) is a severe disease primarily of cattle. the agents of mcf are viruses of the gammaherpesvirinae subfamily. alcelaphine herpesvirus 1 and 2 and ovine herpesvirus 2 are known strains. the alcelaphine strains are seen in africa. the ovine strain is seen in north america. the alcelaphine and ovine strains differ in incubation times and duration of illness. disease may occur sporadically or as outbreaks. clinical signs and diagnosis. signs range from subclinical to recrudescing latent infections to the lethal disease seen in susceptible species, such as cattle. sudden death may also occur in cattle. presentations of the disease may be categorized as alimentary, encephalitis, or skin forms; all three may occur in an animal. corneal edema starting at the limbus and progressing centripetally is a nearly pathognomonic sign; photophobia, severe keratoconjunctivitis, and ocular involvement may follow. other signs include prolonged fever, oral mucosal erosions, salivation, lacrimation, purulent nasal discharge, encephalitis, and pronounced lymphadenopathy. as the disease progresses, cattle may shed horns and hooves. in north america, cattle will also have severe diarrhea. the course of the disease may extend to 1 week. recovery is usually prolonged, and some permanent debilitation may occur. the disease is fatal in severely affected individuals. history of exposure, as well as the clinical signs and lesions, contributes to the diagnosis. serology, pcr-based assays, viral isolation, and cell-culture assays, such as cytopathic effects on thyroid cell cultures, are also used. because of the susceptibility of rabbits, inoculation of this species may be used. in less severe outbreaks or individual animal disease, definitive diagnosis may never be made. necropsy. gross findings at necropsy include necrotic and ulcerated nasal and oral mucosa; thickened, edematous, ulcerated, and hemorrhagic areas of the intestinal tract; swollen, friable, and hemorrhagic lymph nodes and other lymphatic tissues; and erosion of affected mucosal surfaces. lymph nodes should be submitted for histological examination. histological findings include nonsuppurative vasculitis and encephalitis; large numbers of lymphocytes and lymphoblasts will be present without evidence of virus. pathogenesis. the incubation period may be up to 3 months. vascular endothelium and all epithelial surfaces will be affected. the virus is believed to cause proliferation of cytotoxic t lymphocytes with natural killer cell activities, and the resulting lesions are due to an autoimmune type of phenomenon. differential diagnoses. the differentials for this disease are bovine viral diarrhea/mucosal disease, bovine respiratory disease complex, infectious bovine rhinotracheitis, bluetongue, vesicular stomatitis, and foot-and-mouth disease. causes of encephalitis, such as bovine spongiform encephalopathy and rabies, should be considered. in africa, rinderpest is also a differential. other differentials are arsenic toxicity and chlorinated naphthalene toxicity. in north america, sheep, as well as cattle that have been either exposed or that have survived the disease, are reservoirs for outbreaks in other cattle. if there is concern regarding presence of the virus, animals should be screened serologically; once an animal has been infected, it remains infected indefinitely. lambs can be free of the infection if removed from the flock at weaning. the virus is very fragile outside of host's cells and will not survive in the environment for more than a few hours. lobes; and hematological findings indicate anemia and leukocytosis. the rare neurological signs include flexion of fetlock and pastern joints, tremors of facial muscles, progressive paresis and paralysis, depression, and prostration. death occurs in weeks to months. the disease can be serologically diagnosed with agar gel immunodiffusion (agid) tests, virus isolation, serum neutralization, complement fixation, and enzyme-linked immunosorbent assay (elisa) tests. sixty-eight percent of sheep in some states have been infected with the virus (radostits et al., 1994) . it is transmitted horizontally via inhalation of aerosolized virus particles and vertically between the infected dam and fetus. in addition, transmission through the milk or colostrum is considered common (knowles, 1997) . necropsy findings. lesions are observed in lungs, mammary glands, joints, and the brain. pulmonary adhesions, ventral lung lobe consolidation, bronchial lymph node enlargement, mastitis, and degenerative arthritis are visualized grossly. meningeal edema, thickening of the choroid plexus, and foci of leukoencephalomalacia are seen in the central nervous system (cns). histologically, interalveolar septal thickening, lymphoid hyperplasia, histiocyte and fibrocyte proliferation, and squamous epithelial changes are seen in the lungs. meningitis, lymphoid hyperplasia, demyelination, and glial fibrosis are seen in the cns. affected and any exposed animals should be isolated from healthy animals. there is no specific treatment for mcf; supportive treatment may improve recovery rates. corticosteroids may be useful. etiology. an rna virus in the lentivirus group of the retroviridae family causes ovine progressive pneumonia (opp), or maedi/visna. maedi refers to the progressive pneumonia presentation of the disease; visna refers to the central nervous system disease, which is reported predominantly in iceland. visna has been reported in goats but may have been due to caprine arthritis encephalitis infection. clinical signs and diagnosis. opp is a viral disease of adult sheep characterized by weakness, unthriftiness, weight loss, and pneumonia (pepin et al., 1998; de la concha bermejillo, 1997) . clinically, animals exhibit signs of progressive pulmonary disease after an extremely long incubation period of up to 2 years. respiratory rate and dyspnea gradually increase as the disease progresses. the animal continues to eat throughout the disease; however, animals progressively lose weight and become weak. additionally, mastitis is a common clinical feature. thoracic auscultation reveals consolidation of ventral lung pathogenesis. the virus has a predilection for the lungs, mediastinal lymph nodes, udder, spleen, joints, and rarely the brain. after initial infection, the virus integrates into the dna of mature monocytes and persists as a provirus. later in the animal's life, infected monocytes mature as lung (and other tissue) macrophages and establish active infection. the virus induces lymphoproliferative disease, histiocyte and fibrocyte proliferation in the alveolar septa, and squamous metaplasia. pulmonary alveolar and vascular changes impinge on oxygen and carbon dioxide exchange and lead to serious hypoxia and pulmonary hypertension. secondary bacterial pneumonia may contribute to the animal's death. pulmonary adenomatosis is the differ-prevention and control. isolating or removing infected animals can prevent the disease. facilities and equipment should also be disinfected. ii. proliferative stomatitis (bovine papular stomatitis) etiology. a parapoxvirus is the causative agent of bovine papular stomatitis. this virus is considered to be closely related to the parapoxvirus that causes contagious ecthyma and pseudocowpox. it is also a zoonotic disease. the disease is not considered of major consequence, but high morbidity and mortality may be seen in severe outbreaks. in addition, lesions are comparable in appearance to those seen with vesicular stomatitis, bovine viral diarrhea virus, and foot-and-mouth disease. the disease occurs worldwide. clinical signs and diagnosis. raised red papules or erosions or shallow ulcers on the muzzle, nose, oral mucosa (including the hard palate), esophagus, and rumen of younger cattle are the most common findings. in some outbreaks, the papules will be associated with ulcerative esophagitis, salivation, diarrhea, and subsequent weight loss. lesions persist or may come and go over a span of several months. morbidity among herds may be 100%. mortalities are rare. bovine papular stomatitis is associated with "rat tail" in feedlot cattle. animals continue to eat and usually do not show a fever. no lesion is seen on the feet. the infection may also be asymptomatic. diagnosis is based on clinical signs, histological findings, and viral isolation. epizootiology and transmission. cattle less than 1 year of age are most commonly affected, and disease is rare in older cattle. transmission is by animal-to-animal contact. necropsy findings. raised papules may be found around the muzzle and mouth and involve the mucosa of the esophagus and rumen. histologically, epithelial cells will show hydropic degeneration and hyperplasia of the lamina propria. eosinophilic inclusions will be in the cytoplasm of infected epithelial cells. pathogenesis. following exposure to the virus, erythematous macules most commonly appear on the nares, followed by the mouth. these become raised papules within a day, regressing after days to weeks; the lesions that remain will be persistent yellow, red, or brown spots. some infections may recur or persist, with animals showing lesions intermittently or continuously over several months. differential diagnosis. pseudocowpox, vesicular stomatitis, foot-and-mouth disease, and bovine viral diarrhea virus infection are the differentials for this disease. the differential for the "rat tail" clinical sign is sarcocystis infection. there is no vaccine available for bovine papular stomatitis. because of the similarity of this virus to the parapoxvirus of contagious ecthyma, it is important to be aware of the persistence in the environment and susceptibility of younger cattle. vaccination using the local strain, and the skin scarification technique for off, have been protective. handlers should wear gloves and protective clothing. treatment. cattle usually will not require extensive nursing care, but lesions with secondary bacterial infections should be treated with antibiotics. their hands at sites of contact with lesions of cattle. iii. pseudocowpox etiology. pseudocowpox is a worldwide cattle disease caused by a parapoxvirus related to the causative agents of contagious ecthyma and bovine papular stomatitis (see sections iii,a,2,m and iii,a,2,q,ii). lesions are confined to the teats. this is also a zoonotic disease. clinical signs and diagnosis. minor lesions are usually confined to the teats. these are distinctive because of the ring-or horseshoe-shaped scab that develops after 10 days. additional lesions sometimes develop on the udder, the medial aspect of the thighs, and the scrotum. the teat lesions may predispose to mastitis. etiology. pulmonary adenomatosis is a rare but progressive wasting disease of sheep, with worldwide distribution. pulmonary adenomatosis is caused by a type d retrovirus antigenically related to the mason-pfizer monkey virus. jaagsiekte was the designation when the disease was described originally in south africa. progressive respiratory signs such as dyspnea, rapid respiration, and wasting. the disease is diagnosed by these chronic clinical signs and histology. epizootiology and transmission. the disease is transmitted by aerosols. body fluids of viremic animals, such as milk, blood, saliva, tears, semen, and bronchial secretions, will contain the virus or cells carrying the virus. necropsy. the adenomas and adenocarcinomas will be small firm lesions distributed throughout the lungs. the adenocarcinomas metastasize to regional lymph nodes. pathogenesis. as with ovine progressive pneumonia (opp), the incubation period is up to 2 years long. adenocarcinomatous lesions arising from type ii alveolar epithelial cells may be discrete or confluent and involve all lung lobes. with or is a differential diagnosis for opp. etiology. cutaneous papillomatosis is a very common disease in cattle and is much less common among sheep and goats. the disease is a viral-induced proliferation of the epithelium of the neck, face, back, and legs. these tumors are caused by a papillomavirus (dna virus) of the papovaviridae family, and the viruses are host-specific and often body site-specific. most are benign, although some forms in cattle and one form in goats can become malignant. in cattle, the site specificity of the papillomavirus strains are particularly well recognized. designations of the currently recognized bovine papillomavirus (bpv) types are bpv-1 through bpv-5. clinical signs and diagnosis. the papillomas may last up to 12 months and are seen more frequently in younger animals. lesions have typical wart appearances and may be single or multiple, small (1 mm) or very large (500 mm). the infections will generally be benign, but pain will be evident when warts develop on occlusal surfaces or within the gastrointestinal tract. in addition, when infections are severe, weight loss may occur. when warts occur on teats, secondary mastitis may develop. in cattle, bpv-1 and bpv-2 cause fibropapillomas on teats and penises or on head, neck, and dewlap, respectively. bpv-3 causes flat warts that occur in all body locations, b pv-4 causes warts in the gastrointestinal tract, and b pv-5 causes small white warts (called rice-grain warts) on teats. warts caused by bpv-3 and bpv-5 do not regress spontaneously. prognosis in cattle is poor only when papillomatosis involves more than 20% of the body surface. in sheep, warts are the verrucous type. the disease is of little consequence unless the warts develop in an area that causes dis-comfort or incapacitation such as between the digits, on the lips, or over the joints. in adult sheep, warts may transform to squamous cell carcinoma. in goats, the disease is rare, and the warts are also of the verrucous type and occasionally may develop into squamous cell carcinoma. warts on goat udders tend to be persistent. diagnosis is made by observing the typical proliferative lesions. epizootiology and transmission. older animals are less sensitive to papillomatosis than young animals, although immunosupressed animals of any age may develop warts as the result of harbored latent infections. the virus is transmitted by direct and indirect (fomite) contact, entering through surface wounds and sites such as tattoos. pathogenesis. the incubation period ranges from 1 to 6 months. the virus induces epidermal and fibrous tissue proliferation, often described as cauliflower-like skin tumors. the disease is generally self-limiting. differential diagnosis. in sheep and goats, differentials include contagious ecthyma, ulcerative dermatosis, strawberry foot rot, and sheep and goat pox. for cattle) or autogenous vaccines must be used with a recognition that papovavirus strains are host-specific and that immunity from infection or vaccination is viral-type-specific. autogenous vaccines are generally considered more effective. some vaccine preparations are effective at prevention but not treatment of outbreaks. viricidal products are recommended for disinfection of contaminated environments. minimizing cutaneous injuries and sanitizing equipment (tattoo devices, dehorners, ear taggers, etc.) in a virucidal solution between uses are also recommended preventive and control measures. halters, brushes, and other items may also be sources of virus. treatment. warts will often spontaneously resolve as immunity develops. in severe cases or with flockwide or herdwide problems, affected animals should be isolated from nonaffected animals, and premises disinfected. warts can be surgically excised and autogenous vaccines can be made and administered to help prevent disease spread. cryosurgery with liquid nitrogen or dry ice has also proven to be successful for wart removal. topical agents such as podophyllin (various formulations) and dimethyl sulfoxide may be applied to individual lesions once daily until regression. etiology. pseudorabies is an acute encephalitic disease caused by a neurotropic alphaherpesvirus, the porcine herpesvirus 1. one serotype is recognized, but strain differences exist. the disease has worldwide distribution. it is a primarily a clinical dis-ease of cattle, with less frequent reports (but no less severe clinical manifestations) in sheep and goats. during the rapid course of this usually fatal disease. at the site of virus inoculation or in other locations, abrasions, swelling, intense pruritus, and alopecia are seen. pruritus will not be asymmetric. animals will also become hyperthermic and will vocalize frantically. other neurological signs range from hoof stamping, kicking at the pruritic area, salivation, tongue chewing, head pressing and circling, to paresthesia or hyperesthesia, ataxia, and conscious proprioceptive deficits. nystagmus and strabismus are also seen. animals will be fearful or depressed, and aggression is sometimes seen. recumbency and coma precede death. diagnostic evidence includes clinical findings; virus isolation from nasal or pharyngeal secretions or postmortem tissues; and histological findings at necropsy. serology of affected animals is not productive, because of the rapid course. if swine are housed nearby, or if swine were transported in the same vehicles as affected animals, serological evaluations are worthwhile from those animals. epizootiology and transmission. swine are the primary hosts for pseudorabies virus, but they are usually asymptomatic and serve as reservoirs for the virus. the infection can remain latent in the trigeminal ganglion of pigs and recrudesce during stressful conditions. other animals are dead-end hosts. the unprotected virus will survive only a few weeks in the environment but may remain viable in meat (including carcasses) or saliva and will survive outside the host, in favorable conditions, in the summer for several weeks and the winter for several months. transmission is by oral, intranasal, intradermal, or subcutaneous introduction of the virus. when the virus is inhaled, the clinical signs of pruritus are less likely to be seen. transmission can also be by inadvertent exposure (e.g., contaminated syringes) of ruminants to the modified live vaccines developed for use in swine. spread between infected ruminants is a less likely means of transmission, because of the relatively short period of virus shedding. transport vehicles used for swine may also be sources of the virus. raccoons are believed to be vectors of the virus. horses are resistant to infection. there is no pathognomonic gross lesion. definitive histologic findings include severe, focal, nonsuppurative encephalitis and myelitis. eosinophilic intranuclear inclusion bodies (cowdry type a) may be present in some affected neurons. methods such as immunofluorescence and immunoperoxidase staining can be used to show presence of the porcine herpesvirus 1. pathogenesis. the incubation period is 90-156 hr and duration of the illness is 8-72 hr. the longest duration is seen in animals with pruritus around the head. differential diagnoses. differentials for the neurologic signs of pseudorabies infection include rabies, polioencephalomalacia, salt poisoning, meningitis, lead poisoning, hypomagnesemia, and enterotoxemia. those for the intense pruritus include psoroptic mange and scrapie in sheep, sarcoptic mange, and pediculosis. prevention and control. pseudorabies is a reportable disease in the united states, where a nationwide eradication program exists; states are rated regarding status. effective disinfectants include sodium hypochlorite (10% solution), formalin, peracetic acid, tamed iodines, and quaternary ammonium compounds. five minutes of contact time is required, and then surfaces must be rinsed. other disinfectant methods for viral killing include 6 hr of formaldehyde fumigation, or 360 min of ultraviolet light. transport vehicles should be cleaned and disinfected between species. serological screening for pseudorabies of swine housed near ruminants is essential. there is no treatment, and most affected ani-research complications. swine housed close to research ruminants should be serologically screened prior to purchase, and all transport vehicles should be cleaned and disinfected between loads of large animals. humans have been reported to seroconvert. the porcine herpesvirus 1 shares antigens with the infectious bovine rhinotracheitis virus. etiology. rabies is a sporadic but fatal, acute viral disease affecting the central nervous system. the rabies virus is a neurotropic rna virus of the lyssavirus genus and the rhabdoviridae family. sheep, goats, and cattle are susceptible. the zoonotic potential of this virus must be kept in mind at all times when handling moribund animals with neurological signs characteristic of the disease. rabies is endemic in many areas of the world and within areas of the unites states. this is a reportable disease in north america. clinical findings and diagnosis. animals generally progress through three phases: prodromal, excitatory, and paralytic. many signs in the different species during these stages are nonspecific, and forms of the disease are also referred to as dumb or furious. during the short prodromal phase, animals are hyperthermic and apprehensive. animals progress to the excitatory phase, during which they refuse to eat or drink and are active and aggressive. repeated vocalizations, tenesmus, sexual excitement, and salivation occur during this phase. the final paralytic stage, with recumbency and death, occurs over several hours to days. this paralytic stage is common in cattle, and animals may simply be found dead. the clinical course is usually 1-4 days. diagnosis is based on clinical signs, with a progressive and fatal course. confirmation presently is made with the fluorescent antibody technique on brain tissue. epizootiology and transmission. the rabies virus is transmitted via a bite wound inflicted by a rabid animal. cats, dogs, raccoons, skunks, foxes, wild canids, and bats are the common disease vectors in north america. virus is also transmitted in milk and aerosols. necropsy findings. few lesions are seen at necropsy. many secondary lesions from manic behaviors during the course of disease may be evident. histological findings will include nonsuppurative encephalitis. negri bodies in the cytoplasm of neurons of the hippocampus and in purkinje's cells are pathognomonic histologic findings. pathogenesis. after exposure, the incubation period is variable, from 2 weeks to several months, depending on the distance that the virus has to travel to reach the central nervous system. the rabies virus proliferates locally, gains access to neurons by attaching to acetylcholine receptors, via a viral surface glycoprotein, migrates along sensory nerves to the spinal cord and brain, and then descends via cranial nerves (trigeminal, facial, olfactory, glossopharyngeal) to oral and nasal cavity structures (i.e., salivary glands). the fatal outcome is currently believed to be multifactorial, related to anorexia, respiratory paralysis, and effects on the pituitary. differential diagnosis. rabies should be included on the differential list when clinical signs of neurologic disease are evident. other differentials for ruminants include herpesvirus encephalitis, thromboemobolic meningoencephalitis, nervous ketosis, grass tetany, and nervous cocciodiosis. prevention and control. vaccines approved for use cattle and sheep are commercially available and contain inactivated virus; there is not one available in the united states for goats. ruminants in endemic areas, such as the east coast of the united states, should be routinely vaccinated. any animals housed outside that may be exposed to rabid animals should be vaccinated. vaccination programs generally begin at 3 months of age, with a booster at 1 year of age and then annual or triennial boosters. awareness of the current rabies case reports for the region and wildlife reservoirs, however, is important. monitoring for and exclusion of wildlife from large-animal facilities are worthwhile preventive measures. the virus is fragile and unstable outside of a host animal. research complications. aerosolized virus is infective. personal protective equipment, including gloves, face mask, and eye shields, must be worn by individuals handling animals that are manifesting neurological disease signs. bovine spongiform encephalopathy, a transmissible spongiform encephalopathy (tse), is not known to occur in the united states, where since 1989 it has been listed as a reportable disease. the profound impact of this disease on the cattle industry in great britain during the past two decades is well known. the disease may be caused by a scrapielike (prion) agent. it is believed that the source of infection for cattle was feedstuff derived from sheep meat and bonemeal that had been inadequately treated during processing. the incubation period of years, the lack of detectable host immune response, the debilitating and progressive neurological illness, and the pathology localized to the central nervous system are characteristics of the disease, and are is comparable to the characteristics of other tse diseases such as scrapie, which affects sheep and goats. in addition, the infectious agent is extremely resistant to dessication and disinfectants. confirmation of disease is by histological examination of brain tissue collected at necropsy; the vacuolation that occurs during the disease will be symmetrical and in the gray matter of the brain stem. molecular biology techniques, such as western blots and immunohistochemistry, may also be used to identify the presence of the prion protein. differentials include many infectious or toxic agents that affect the bovine nervous and musculoskeletal systems, such as rabies, listeriosis, and lead poisoning. metabolic disorders such as ketosis, milk fever, and grass tetany are also differentials. there is no vaccine or treatment. prevention focuses on import regulations and not feeding ruminant protein to ruminants; recent usda regulations prohibit feeding any mammalian proteins to ruminants. etiology. scrapie is a sporadic, slow, neurodegenerative disease caused by a prion. scrapie is a reportable disease. it is much more common in sheep than in goats. the disease is similar to transmissible mink encephalopathy, kuru, creutzfeldt-jakob disease, and bovine spongiform encephalopathy (mad cow disease). prions are nonantigenic, replicating protein agents. clinical signs and diagnosis. during early clinical stages, animals are excitable and hard to control. tremors of head and neck muscles, as well as uncoordinated movements and unusual "bunny-hopping" gaits are observed. in advanced stages of the disease, animals experience severe pruritus and will self-mutilate while rubbing on fences, trees, and other objects. blindness and abortion may also be seen. morbidity may reach 50% within a flock. most animals invariably die within 4-6 weeks; some animals may survive 6 months. in goats, the disease is also fatal. pruritus is generally less severe but may be localized. a wide range of clinical signs have also been noted in goats, including listlessness, stiffness or restlessness, or behavioral changes such as irritability, hunched posture, twitching, and erect tail and ears. as with sheep, the disease gradually progresses to anorexia and debilitation. diagnosis can be made by clinical signs and histopathological lesions. a newer diagnostic test in live animals is based on sampling from the third eyelid. tests for genetic resistance or susceptibility require a tube of edta blood and are reasonably priced. epizootiology and transmission. the suffolk breed of sheep tends to be especially susceptible. scrapie has also been reported in several other breeds, including cheviot, dorset, hampshire, corriedale, shropshire, merino, and rambouillet. it is believed that there is hereditary susceptibility in these breeds. targhees tend to be resistant. genomic research indicates there are two chromosomsal sites governing this trait; these sites are referred to codons 171 (q, r, or h genes can be present) and 136 (a or v genes can be present). of the five genes, r genes appear to confer immunity to clinical scrapie in suffolks in the united states. affected suffolks in the united states that have been tested have been aa qq. the disease is also enzootic is many other countries. the disease tends to affect newborns and young animals; however, because the incubation period tends to range from 2 to 5 years, adult animals display signs of the disease. scrapie is transmitted horizontally by direct or indirect contact; nasal secretions or placentas serve as sources of the infectious agent. vertical transmission is questioned, and transplacental transmission is considered unlikely. necropsy findings. at necropsy, no gross lesion is observed. histopathologically, neuronal vacuolization, astrogliosis, and spongiform degeneration are visualized in the brain stem, the spinal cord, and especially the thalamus. inflammatory lesions are not seen. pathogenesis. replication of the prions probably occurs first in lymphoid tissues throughout the host's body and then progresses to neural tissue. differential diagnosis. in sheep and goats, depending on the speed of onset, differentials for the pruritus include ectoparasites, pseudorabies, and photosensitization. prevention and control. if the disease diagnosed in a flock, quarantine and slaughter, followed by strict sanitation, are usually required. the u.s. department of agriculture has approved the use of 2% sodium hydroxide as the only disinfectant for sanitation of scrapie-infected premises. prions are highly resistant to physicochemical means of disinfection. artificial insemination or embryo transfer has been shown to decrease the spread of scrapie (linnabary et al., 1991) . research complications. as noted, this is a reportable disease. stringent regulations exist in the united states regarding importation of small ruminants from scrapie-infected countries. etiology. vesicular stomatitis (vs) is caused by the vesicular stomatitis virus (vsv), a member of the rhabdoviridae. three serotypes are recognized: new jersey, indiana, and isfahan. the new jersey and indiana strains cause sporadic disease in cattle in the united states. the disease is rare in sheep. clinical signs and diagnosis. adult cattle are most likely to develop vs. fever and development of vesicles on the oral mucous membranes are the initial clinical signs. lesions on the teats and interdigital spaces also develop. the vesicles progress quickly to ulcers and erosions. the animal's tongue may be severely involved. anorexia and salivation are common. weight loss and decreased milk production are noticeable. morbidity will be high in an outbreak, but mortality will be low to nonexistent. diagnostic work should be initiated as soon as possible to distinguish this from foot-and-mouth disease. diagnosis is based on analysis of fluid, serum, or membranes associated with the vesicles. virus isolation, enzyme-linked immunosorbent assay (elisa), competitive elisa (celisa), complement fixation, and serum neutralization are used for diagnosis. epizootiology and transmission. this disease occurs in several other mammalian species, including swine, horses, and wild ruminants. vsv is an enveloped virus and survives well in different environmental conditions, including in soil, extremes of ph, and low temperatures. outbreaks of vs occur sporadically in the united states, but it is not understood how or in what species the virus survives between these outbreaks. incidence of disease decreases during colder seasons. equipment, such as milking machines, contaminated by secretions is a mechanical vector, as are human hands. transmission may also be from contaminated water and feed. transmission is also believed to occur by insects (blackflies, sand flies, and culicoides) that may simply be mechanical vectors. it is believed that carrier animals do not occur in this disease. necropsy. it is rare for animals to be necropsied as the result of this disease. typical vesicular lesion histology is seen, with ballooning degeneration and edema. there is no inclusion body formation. pathogenesis. lesions often begin within 24 hr after exposure. the virus invades oral epithelium. injuries or trauma in any area typically affected, such as mouth, teats, or interdigital areas, will increase the likelihood of lesions developing there. animals will develop a long-term immunity; this immunity can be overwhelmed, however, by a large dose of the virus. differential diagnosis. foot-and-mouth disease lesions are identical to vs lesions. other differentials in cattle include bovine viral diarrhea, malignant catarrhal fever, contagious ecthyma, photosensitization, trauma, and caustic agents. prevention and control. quarantine and restrictions on shipping infected animals or animals from the premises housing affected animals are required in an outbreak. vaccines are available for use in outbreaks and have decreased the severity of lesions. phenolics, quaternaries, and halogens are effective for inactivating and disinfecting equipment and facilities. treatment. affected animals should be segregated from the rest of the herd and provided with separate water and softened feed. these animals should be cared for after unaffected animals. any feed or water contaminated by these animals should not be used for other animals; contaminated equipment should be disinfected. topical or systemic antibiotics control secondary bacterial infections. cases of mastitis secondary to teat lesions must be treated as necessary. any abrasive materials that could cause further trauma to the animals should be removed. research complications. animals developing vesicular lesions must be reported promptly to eliminate the possibility of an outbreak of foot-and-mouth disease. personal protective equipment, especially gloves, should be worn when handling any animals with vesicular lesions. vsv causes a flulike illness in humans. x. viral diarrhea diseases i. ovine. rotavirus, of the family reoviridae, induces an acute, transient diarrhea in lambs within the first few weeks of life. four antigenic groups (a-d) have been identified by differences in capsid antigens vp3 and vp7. primarily group a, but also groups b and c, have been isolated from sheep. the disease is characterized by yellow, semifluid to watery diarrhea occurring 1-4 days after infection. the disease can progress to dehydration, anorexia and weight loss, acidosis, depression, and occasionally death. the virus is ingested with contaminated feed and water and selectively infects and destroys the enterocytes at the tips of the small intestinal villi. the villi are replaced with immature cells that lack sufficient digestive enzymes; osmotic diarrhea results. virus may remain in the environment for several months. the disease is diagnosed by virus isolation, electron microscopy of feces, fecal fluorescent antibody, fecal elisa tests (marketed tests generally detect group a rotavirus), and fecal latex agglutination tests. rotavirus diarrhea is treated by supportive therapy, including maintaining hydration, electrolyte, and acid-base balance. a rotavirus vaccine is available for cattle; because of cross-species immunity, oral administration of high-quality bovine colostrum from vaccinated cows to infected sheep may be helpful ("current veterinary therapy," 1993). coronavirus, of the family coronaviridae, produces a more severe, long-lasting disease when compared with rotavirus. clinical signs are similar to above, although the incubation period tends to be shorter (20-36 hr), and animals exhibit less anorexia than those with rotavirus. additionally, mild respiratory disease may be noted (janke, 1989) . like rotavirus, coronavirus also destroys enterocytes of the villus tips. the virus can be visualized with electron microscopy. treatment is supportive; close consideration of hydration and acid-base status is essential. bovine vaccines are available. ii. caprine. rotavirus, coronavirus, and adenoviruses affect neonatal goats; however, little has been documented on the pathology and significance of these agents in this age group. it appears that bacteria play a more important role in neonatal kid diarrheal diseases then in neonatal calf diarrheas. iii. bovine. rotaviruses, coronaviruses, parvoviruses, and bovine viral diarrhea virus (bvdv) are associated with diarrheal disease in calves. each pathogen multiplies within and destroys the intestinal epithelial cells, resulting in villous atrophy and clinical signs of diarrhea (soft to watery feces), dehydration, and abdominal pain. these viral infections may be complicated by parasitic infections (e.g., cryptosporidium, eimeria) or bacterial infections (e.g., escherichia coli, salmonella, campylobacter). treatment is aimed at correcting dehydration, electrolyte imbalances, and acidosis; cessation of milk replacers and administration of fluid therapy intravenously and by stomach tube may be necessary, depending on the presence of suckle reflex and the condition of the animals. diagnosis is by immunoassays available for some viruses, viral culture, exclusion or identification of presence of other pathogens (by culture or fecal exams), and microscopic examination of necropsy specimens. prevention focuses on calves suckling good-quality colostrum; other recommendations for calf care are in section ii,b,5. combination vaccine products are available for immunizing dams against rotavirus, coronavirus, and enterotoxigenic e. coli. additional supportive care for calves includes providing calves with sufficient energy and vitamins until milk intake can resume. rotaviruses of serogroup a are the most common type in neonatal calves; 4-to 14-day old calves are typically affected, but younger and older animals may also be affected. the small intestine is the site of infection. antirotavirus antibody is present in colostrum, and onset of rotavirus diarrhea coincides with the decline of this local protection. transmission is likely from other affected calves and asymptomatic adult carriers. the diarrhea is typically a distinctive yellow. colitis with tenesmus, mucus, and blood may be seen. this virus may be zoonotic. coronaviruses are commonly associated with disease in calves during the first month of life, and they infect small-and large-intestinal epithelial cells. the virus infection may extend to mild pneumonia. transmission is by infected calves and also by asymptomatic adult cattle, including dams excreting virus at the time of parturition. calves that appear to have recovered continue to shed virus for several weeks. parvovirus infections are usually associated with neonatal calves. b vdv infections also are seen in neonates and also affect many systems and produce other clinical signs and syndromes that are described in section iii,a,2,e. iv. winter dysentery. winter dysentery is an acute, winterseasonal, epizootic diarrheal disease of adult cattle, although it has been reported in 4-month-old calves. the etiology has not yet been defined, but a viral pathogen is suspected. coronavirus-like viral particles have been isolated from cattle feces, either the same as or similar to the coronavirus of calf diarrhea. outbreaks typically last a few weeks, and first-lactation or younger cattle are affected first, with waves of illness moving through a herd. individual cows are ill for only a few days. the incubation period is estimated at 2-8 days. the outbreaks of disease are often seen in herds throughout the local area. clinical signs include explosive diarrhea, anorexia, depression, and decreased production. the diarrhea has a distinctive musty, sweet odor and is light brown and bubbly, but some blood streaks or clots may be mixed in with the feces. animals will become dehydrated quickly but are thirsty. respiratory symptoms such as nasolacrimal discharges and coughing may develop. recovery is generally spontaneous. mortalities are rare. diagnosis is based on characteristic patterns of clinical signs, and elimination of diarrheas caused by parasites such as coccidia, bacterial organisms such as salmonella or mycobacterium paratuberculosis, and viruses such as b vdv. pathology is present in the colonic mucosa, and necrosis is present in the crypts. etiology. chlamydia psittaci is a nonmotile, obligate, intracytoplasmic, gram-negative bacterium. clinical signs. enzootic abortion in sheep and goats is a contagious disease characterized by hyperthermia and late abortion or by birth of stillborn or weak lambs or kids (rodolakis et al., 1998) . the only presenting clinical sign may be serosanguineous vulvar discharges. other animals may present with arthritis or pneumonia. infection of animals prior to about 120 days of gestation results in abortion, stillbirths, or birth of weak lambs. infection after 120 days results in potentially normal births, but the dams or offspring may be latently infected. latently infected animals that were infected during their dry period may abort during the next pregnancy. ewes or does generally only abort once, and thus recovered animals will be immune to future infections. and specific antigens associated with the cell surface. the group antigen is common among all chlamydia; the specific antigen is common to related subgroups. two subgroups are recognized, one that causes eae and one that causes polyarthritis and conjunctivitis. the disease is transmitted by direct contact with infectious secretions such as placental, fetal, and uterine fluids or by indirect contact with contaminated feed and water. necropsy. placental lesions include intercotyledonary plaques and necrosis and cotyledonary hemorrhages. histopathological evidence of leukocytic infiltration, edema, and necrosis is found throughout the placentome. fetal lesions include giant-cell accumulation in mesenteric lymph nodes and lymphohistiocytic proliferations around the blood vessels within the liver. diagnosis is based on clinical signs and laboratory (serological or histopathological) identification of the organism. impression smears in placental tissues stained with giemsa, gimenez, or modified ziehl-neelsen can provide preliminary indications of the causative agent. immunofluorescence, enzyme-linked immunosorbent assay (elisa), and polymerase chain reaction (pcr) methods also aid in diagnosis. differential diagnosis. q fever will be the major differential for late-term abortion and necrotizing placentitis. campylobacter and toxoplasma should also be considered for late-term abortion. treatment. animals may respond to treatment with oxytetracycline. abortions are prevented through administration of a commercial vaccine, but the vaccine will not eliminate infections. this is a sheep vaccine and should be administered before breeding and annually to at least the young females entering the breeding herd or flock. research complications. in addition to losses or compromise of research animals, pregnant women should not handle aborted tissues. etiology. chlamydia psittaci is a nonmotile, obligate intracellular, gram-negative bacterium. chlamydial polyarthritis is an acute, contagious disease characterized by fever, lameness (bulgin, 1986) , and conjunctivitis (see section iii,a,3,c) in growing and nursing lambs. clinical signs. clinically, animals will appear lame on one or all legs and in major joints, including the scapulohumeral, humeroradioulnar, coxofemoral, femorotibial, and tibiotarsal joints. lambs may be anorexic and febrile. animals frequently also exhibit concurrent conjunctivitis. the disease usually resolves in approximately 4 weeks. joint inflammation usually resolves without causing chronic articular changes. epizootiology and transmission. the disease is transmitted to susceptible animals by direct contact as well as by contaminated feed and water. the organism penetrates the gastrointestinal tract and migrates to joints and synovial membranes as well as to the conjunctiva. the organism causes acute inflammation and associated fibrinopurulent exudates. necropsy findings. lesions are found in joints, tendon sheaths, conjunctiva, and lungs. pathological sites will be edematous and hyperemic, with fibrinous exudates but without articular changes. lesions will be infiltrated with mononuclear cells. lung lesions include atelectasis and alveolar inspissation. diagnosis is based on clinical signs. synovial taps and subsequent smears may allow the identification of chlamydial inclusion bodies. treatment. animals respond to treatment with parenteral oxytetracycline. etiology. chlamydia psittaci, a nonmotile, obligate intracellular, gram-negative bacterium, is the most common cause of infectious keratoconjunctivitis in sheep. chlamydia and mycoplasma are considered to be the most common causes of this disease in goats. chlamydial conjunctivitis is not a disease of cattle. clinical signs. infectious keratoconjunctivitis is an acute, contagious disease characterized in earlier stages by conjunctival hyperemia, epiphora, and edema and in later stages by, corneal edema, ulceration, and opacity. perforation may result from the ulceration. animals will be photophobic. in less severe cases, corneal healing associated with fibrosis and neovascularization occurs in 3-4 days. lymphoid tissues associated with the conjunctiva and nictitating membrane may enlarge and prolapse the eyelids. morbidity may reach 80-90%. bilateral and symmetrical infections characterize most outbreaks. relapses may occur. other concurrent systemic infections may be seen, such as polyarthritis or abortion in sheep and polyarthritis, mastitis, and uterine infections in goats. epizootiology and transmission. direct contact, and mechanical vectors such as flies easily spread the organism. necropsy. if the chlamydial or mycoplasmal agents are suspected, diagnostic laboratories should be contacted for recommendations regarding sampling. conjunctival smears are also useful. pathogenesis. the pathogen penetrates the conjunctival epithelium and replicates in the cytoplasm by forming initial and elementary bodies. the infection moves from cell to cell and causes an acute inflammation and resultant purulent exudate. the chlamydial organism may penetrate the bloodstream and migrate to the opposite eye or joints, leading to arthritis. diagnosis is suggested by the clinical signs. cytoplasmic inclusions observed on conjunctival scrapings and immunofluorescent techniques help confirm the diagnosis. differential diagnosis. nonchlamydial keratoconjunctivitis also occurs in sheep and goats. the primary agents involved include mycoplasma conjunctiva, m. agalactiae in goats, and branhamella (neisseria) ovis. a less common differential for sheep and cattle is listeria monocytogenes. other differentials include eye worms, trauma, and foreign bodies such as windblown materials (pollen, dust) and poor-quality hay; these latter irritants and stress may predispose the animals' eyes to the infectious agents. should be minimized whenever possible. quarantine of new animals and treatment, if necessary, before introduction into the flock or herd are important measures. shade should be provided for all animals. treatment. the infections can be self-limiting in 2-3 weeks without treatment. treatment consists of topical application of tetracycline ophthalmic ointments. systemic or oral oxytetracycline treatments have been used with the topical treatment. atropine may be added to the treatment regimen when uveitis is present. shade should be provided. a. protozoa i. anaplasmosis etiology. anaplasmosis is an infectious, hemolytic, noncontagious, transmissible disease of cattle caused by the protozoan anaplasma marginale. anaplasma is a member of the anaplasmatacae family within the order rickettsiales. in sheep and goats, the disease is caused by a. ovis and is an uncommon cause of hemolytic disease. anaplasmosis has not been reported in goats in the united states. some controversy exists regarding the classification. most recently it is classified as a protozoal disease because of similarities to babesiosis. it has also been classified as a rickettsial pathogen. this summary addresses the disease in cattle with limited reference to a. ovis infections, but there are many similarities to the disease in cattle. clinical signs and diagnosis. acute anemia is the predominant sign in anaplasmosis, and fever coincides with parasitemia. weakness, pallor, lethargy, dehydration, and anorexia are the result of the anemia. four disease stagesnincubation, developmental, convalescent, and carriermare recognized. the incubation stage may be long, 3-8 weeks, and is characterized by a rise in body temperature as the infection moves to the next stage. most clinical signs occur during the 4-to 9-day developmental stage, with hemolytic anemia being common. death is most likely to occur at this stage or at the beginning of the convalescent stage. death may also occur from anoxia, because of the animal's inability to handle any exertion or stress, especially if treatment is initiated when severe anemia exists. reticulocytosis characterizes the convalescent stage, which may continue for many weeks. morbidity is high, and mortality is low. the carrier stage is defined as the time in the convalescent stage when the animal host becomes a reservoir of the disease, and anaplasma organisms and any parasitemia are not discernible. common serologic tests are the complement fixation test and the rapid card test. these become positive after the incubation phase and do not distinguish between the later three stages of disease. definitive diagnosis is made by clinical and necropsy findings. staining of thin blood smears with wright's or giemsa stain allows detection of basophilic, spherical a. marginale bodies near the red blood cell peripheries. evidence will most likely be found before a hemolytic episode. a negative finding should not eliminate the pathogen from consideration. epizootiology and transmission. the disease is common in cattle in the southern and western united states. anaplasma organisms are spread biologically or mechanically. mechanical transmission occurs when infected red blood cells are passed from one host to another on the mouthparts of seasonal biting flies. sometimes mosquitoes or instruments such as dehorners or hypodermic needles may facilitate transfer of infected red cells from one animal to another. biological transmission occurs when the tick stage of the organism is passed by dermacentor andersoni and d. occidentalis ticks. the carrier stage covers the time when discernible anaplasma organisms can be found on host blood smears. recovered animals serve as immune carriers and disease reservoirs. necropsy. pale tissues and watery, thin blood are typical findings. splenomegaly, hepatomegaly, and gallbladder distension are common findings. pathogenesis. the parasites infect the host's red blood cells, and acute hemolysis occurs during the parasites' developmental stage. the four stages of the parasite's life cycle are described above because these are closely linked to the clinical stages. differential diagnosis. the clinical disease closely resembles the protozoal disease babesiosis. whole organism) programs are not entirely effective, and vaccine should not be administered to pregnant cows. neonatal isoerythrolysis may occur because of the antierythrocyte antibodies stimulated by one vaccine product. vaccinated animals can still become infected and become carriers. the cattle vaccine has shown no efficacy in smaller ruminants, and there is no a. ovis vaccine. identifying carriers serologically and treating with tetracycline during and/or after vector seasons may be an option. removing carriers to a separate herd is also an approach. interstate movement of infected animals is regulated. treatment. oxytetracycline, administered once, helps reduce the severity of the infection during the developmental stage. other tetracycline treatment programs have been described to help control carriers. ii. babesiosis (red water, texas cattle fever, cattle tick fever) etiology. babesia bovis and ba. bigemina are protozoa that cause subclinical infections or disease in cattle. these are intraerythrocytic parasites. babesia bovis is regarded as the more virulent of the two organisms. this disease is not seen in the smaller ruminants in the united states. clinical signs and diagnosis. the more common presentation is liver and kidney failure due to hemolysis with icterus, hemoglobinuria, and fever. hemoglobinuria indicates a poor prognosis. acute encephalitis is a less common presentation and begins acutely with fever, ataxia, depression, deficits in conscious proprioception, mania, convulsions, and coma. the encephalitic form generally also has a poor prognosis. sudden death may occur. thin blood smears stained with giemsa will show babesia trophozoites at some stages of the disease, but lack of these cannot be interpreted as a negative. the trophozoites occur in a variety of shapes, such as piriform, round, or rod. complement fixation, immunofluorescent antibody, and enzyme immunoassay are the most favored of the available serologic tests. babesiosis is present on several continents, including the americas. in addition to domestic cattle, some wild ruminants, such as white-tailed deer and american buffalo, are also susceptible. bos indicus breeds have resistance to the disease and the tick vectors. innate resistance factors have been found in all calves. if infected, these animals will not show many signs of disease during the first year of life and will become carriers. stress can cause disease development. prevention and control. offspring of immune carriers resist infection up to 6 months of age because of passive immunity. vector control and attention to hygiene are essential, such as between-animal rinsing in disinfectant of mechanical vectors such as dehorners. there is no entirely effective means, however, to prevent and control the disease. vaccination (killed necropsy findings. signs of acute hemolytic crisis are the most common findings, including hepatomegaly, splenomegaly, dark and distended gallbladder, pale tissues, thin blood, scattered hemorrhages, and petechiation. animals dying after a longer course of disease will be emaciated and icteric, with thin blood, pale kidneys, and enlarged liver. pathogenesis. the protozoon is transmitted by the cattle fever ticks boophilus annulatus, b. microplus, and b. decoloratus; these one-host ticks acquire the protozoon from infected animals. it is passed transovarially, and both nymph and adult ticks may transmit to other cattle. only b. ovis is transmitted by the larval stage. clinical signs develop about 2 weeks after tick infestations or mechanical transmission but may develop sooner with the mechanical transmission. hemolysis is due to intracellular reproduction of the parasites and occurs intra-and extravascularly. in addition to the release of merozoites, proteolytic enzymes are also released, and these contribute to the clinical metabolic acidosis and anoxia. the development of the encephalitis form is believed to be the result of direct invasion of the central nervous system, disseminated intravascular coagulation, capillary thrombosis by the parasites and infarction, and/or tissue anoxia. differential diagnosis. in addition to anaplasmosis, other differentials for the hemolytic form of the disease are leptospirosis, chronic copper toxicity, and bacillary hemoglobinuria. several differentials in the united states for the encephalitic presentation include rabies, nervous system coccidiosis, polioencephalomalacia, lead poisoning, infectious bovine rhinotracheitis, salt poisoning, and chlorinated hydrocarbon toxicity. prevention and control. control or eradication of ticks and cleaning of equipment to prevent mechanical transmission, as noted in section iii,a,3,a,i, are important preventive measures. some vaccination approaches have been effective, but a commercial product is not available. treatment. supportive care is indicated, including blood transfusions, fluids, and antibiotics. medications such as diminazene diaceturate, phenamidine diisethionate, imidocarb diprionate, or amicarbalide diisethionate are most commonly used. treatment outcomes will be either elimination of the parasite or development of a chronic carrier state immune to further disease. research complications. this is a reportable disease in the united states. iii. coccidiosis etiology. coccidiosis is an important acute and chronic protozoal disease of ruminants. in young ruminants, it is characterized primarily by hemorrhagic diarrhea. adult ruminants may carry and shed the protozoa, but they rarely display clinical signs. intensive rearing and housing conditions and stress increase the severity of the disease in all age groups. coccidia are protozoal organisms of the phylum apicomplexa, members of which are obligatory intracellular parasites. there are at least 11 reported species of coccidia in sheep, of which several are considered pathogenic: eimeria ashata, e. crandallis, and e. ovinoidalis (schillhorn van veen, 1986). at least 9 species of eimeria have been recognized in the goat (foreyt, 1990) . eimeria ninakohlyakimovae, e. arloingi, and e. christenseni are regarded as the most pathogenic. eimeria bovis and e. zuernii (highly pathogenic), and e. auburnensis and e. alabamensis (moderately pathogenic), are among the 13 species known to infect cattle. eimeria zuernii is more commonly seen in older cattle and is the agent of "winter coccidiosis." clinical signs and diagnosis. hemorrhagic diarrhea develops 10 days to 3 weeks after infection. fecal staining of the tail and perineum will be present. animals will frequently display tenesmus; rectal prolapses may also develop. anorexia, weight loss, dehydration, anemia, fever (infrequently), depression, and weakness may also be seen in all ruminants. the diarrhea is watery and malodorous and will contain variable amounts of blood and fibrinous, necrotic tissues. the intestinal hemorrhage may subsequently lead to anemia and hypoproteinemia. depending on the predilection of the coccidial species for small and/or large intestines, malabsorption of nutrients or water may occur, and electrolyte imbalances may be severe. concurrent disease with other enteropathogens may also be part of the clinical picture. in sheep, secondary bacterial infection with organisms such as fusobacterium necrophorum may ensue. young goats may die peracutely or suffer severe anemia from blood loss into the bowel. older goats may lose the pelleted form of feces. cattle may have explosive diarrhea and develop anal paralysis. the disease is usually diagnosed by history and clinical signs. numerous oocysts will frequently be observed in fresh fecal flotation (salt or sugar solution) samples as the diarrhea begins. laboratory results are usually reported as number of oocysts per gram of feces. coccidia seen on routine fecal evaluations reflect shedding, possibly of nonpathogenic species, without necessarily being indicative of impending or resolving mild disease. epkzootiology and transmission. as noted, coccidiosis is a common disease in young ruminants. in goats, young animals aged 3 weeks to 5 months are primarily affected, but isolated outbreaks in adults may occur after stressful conditions such as transportation or diet changes. coccidia are host-specific and also host cell-specific. the disease is transmitted via ingestion of sporulated oocysts. coccidial oocysts remain viable for long periods of time when in moist, shady conditions. necropsy. necropsies provide information on specific locations and severity of lesions that correlate with the species involved. ileitis, typhlitis, and colitis with associated necrosis and hemorrhage will be observed. mucosal scrapings will frequently yield oocysts. various coccidial stages associated with schizogony or gametogony may be observed in histopathological sections of the intestines. fibrin and cellular infiltrates will be found in the lamina propria. pathogenesis. this parasite has a complex life cycle in which sexual and asexual reproduction occurs in gastrointestinal enterocytes (speer, 1996) . the severity of the disease is correlated primarily with the number of ingested oocysts. specifics of life cycles vary with the species, and those characteristics contribute to the pathogenicity. in most cases, the disease is well established by the time clinical signs are seen. oocysts must undergo sporulation over a 3-to 10-day period in the environment. after ingestion of the sporulated oocysts, sporozoites are released and penetrate the intestinal mucosa and form schizonts. schizonts initially undergo replication by fission to form merozoites and eventually undergo sexual reproduction, forming new oocysts. the organisms cause edema and hyperemia; penetration into the lamina propria may lead to necrosis of capillaries and hemorrhage. differential diagnosis. differential diagnoses include the many enteropathogens associated with acute diarrhea in young ruminants: cryptosporidia, colibacilli, salmonella, enterotoxins, yersinia, viruses, and other intestinal parasites such as helminths. in cattle, for example, bovine viral diarrhea virus and helminthiasis caused by ostergia must be considered. management factors, such as dietary-induced diarrheas, are also differentials. in older animals, differentials in addition to stress are malnutrition, grain engorgement, and other intestinal parasitisms. prevention and control. good management practices will help prevent the disease. oocysts are resistant to disinfectants but are susceptible to dry or freezing conditions. proper sanitation of animal housing and minimizing overcrowding are essential. coccidiostats added to the feed and water are helpful in preventing the disease in areas of high exposure. treatment. affected animals should be isolated. on an individual basis, treatment should also include provision of a dry, warm environment, fluids, electrolytes (orally or intravenously), antibiotics (to prevent bacterial invasion and septicemia), and administration of coccidiostats. coccidiostats are preferred to coccidiocidals because the former allow immunity to develop. although many coccidial infections tend to be self-limiting, sulfonamides and amprolium may be used to aid in the treatment of disease. other anticoccidial drugs include decoquinate, lasalocid, and monensin; labels should be checked for specific approval in a species or specific indications. animals treated with amprolium should be monitored for development of secondary polioencephalomalacia. pen mates of affected animals should be considered exposed and should be treated to control early stages of infection. mechanisms of immunity have not been well defined but appear to be correlated with the particular coccidial species and their characteristics (for example, the extent of intracellular penetration). immunity may result when low numbers are ingested and there is only mild disease. immunity also may develop after more severe infections. iv. cryptosporidiosis etiology. cryptosporidium organisms are a very common cause of diarrhea in young ruminants. four cryptosporidium species have been described in vertebrates: c. baileyi and c. meleagridis in birds and c. parvum and c. muris in mammals. cryptosporidium parvum is the species affecting sheep (rings and rings, 1996) . debate continues regarding whether there are definite host-specific variants. clinical signs and diagnosis. cryptosporidiosis is characterized by protracted, watery diarrhea and debilitation. the diarrhea may last only 6-10 days or may be persistent and fatal. the diarrhea is watery and yellow, and blood, mucus, bile, and undigested milk may also be present. infected animals will display tenesmus, anorexia and weight loss, dehydration, and depression. in relapsing cases, animals become cachectic. overall, morbidity will be high, and mortality variable. mucosal scrapings or fixed stained tissue sections may be useful in diagnosis. the disease is also diagnosed by detecting the oocysts in iodine-stained feces or in tissues stained with periodic acid-schiff stain or methenamine silver. cryptosporidium also stains red on acid-fast stains such as kinyoun or ziehl-neelsen. fecal flotations should be performed without sugar solutions or with sugar solutions at specific gravity of 1.27 (foryet, 1990) . fecal immunofluorescent antibody (ifa) techniques have also been described. epizootiology and transmission. younger ruminants are commonly affected: lambs, kids (especially kids between the ages of 5 and 10 days old), and calves less than 30 days old. like other coccidians, cryptosporidium is transmitted via the fecal-oral route. in addition to local contamination, water supplies have also been sources of the infecting oocysts. the oocysts are extremely resistant to desiccation in the environment and may survive in the soil and manure for many months. necropsy findings. the lesions caused by cryptosporidium are nonspecific. animals will be emaciated. moderate enteritis and hyperplasia of the crypt epithelial cells with villous atrophy as well as villous fusion, primarily in the lower small intestines, will be present. cecal and colonic mucosae may sometimes be involved. gastrointestinal smears may be made at necropsy and stained as described above. pathogenesis. although cryptosporidium infections are clinically similar to eimeria infections (moore, 1989) , cryptosporidium, in contrast to eimeria, invades just under the surface but does not invade the cytoplasm of enterocytes. there is no intermediate host. the oocysts are half the size of eimeria oocysts and are shed sporulated; they are, therefore, immediately infective. within 2-7 days of exposure, diarrhea and oocyst shedding occur. the diarrhea is the result of malabsorption and, in younger animals, intraluminal milk fermentation. autoinfection within the lumen of the intestines may also occur and result in persistent infections. in addition, several other pathogens may be involved, such as concurrent coronavirus and rotavirus infections in calves. environmental stressors such as cold weather increase mortality. intensive housing arrangements increase morbidity and mortality. differential diagnosis. other causes of diarrhea in younger ruminants include rotavirus, coronavirus, and other enteric viral infections; enterotoxigenic escherichia coli; clostridium; other coccidial pathogens; and dietary causes (inappropriate use of milk replacers). in addition, these other agents may also be causing illness in the affected animals and may complicate the diagnosis and the treatment picture. eimeria is more likely to cause diarrhea in calves and lambs at 3-4 weeks of age. giardia organisms may be seen in fecal preparations from young ruminants but are not considered to play a significant role in enteric disease. blood. animals exhibit fever, dehydration, and depression. chronic cases may result in a "poor doer" syndrome with weight loss and unthriftiness. giardia can be diagnosed by identifying the motile piriform trophozoites in fresh fecal mounts. oval cysts can be floated with zinc sulfate solution (33%). standard solutions tend to be too hyperosmotic and to distort the cysts. newer enzyme-linked immunosorbent assay (elisa) and ifa tests are sensitive and specific. epizootiology and transmission. giardia infection may occur at any age, but young animals are predisposed. chronic oocyst shedding is common. transmission of the cyst stage is fecaloral. wild animals may serve as reservoirs. necropsy findings. gross lesions may not be evident. villous atrophy and cuboidal enterocytes may be evident histologically. prevention and control. precautions should be taken when handling infected animals. affected animals must be removed and isolated as soon as possible. animal housing areas should be disinfected with undiluted commercial bleach or 5% ammonia. formalin (10%) fumigation has proven successful (foryet, 1990) . after being cleaned, areas should be allowed to dry thoroughly and should remain unpopulated for a period of time. because enteric disease often is multifactorial, other pathogens should also be considered, and management and husbandry should be examined. no known drug treatment is available. the disease is generally self-limiting, so symptomatic, supportive therapy aimed at rehydrating, correcting electrolyte and acid-base balance, and providing energy is often effective. supplementation with vitamin a may be helpful. age resistance begins to develop when the animals are about 1 month old. research complications. cryptosporidiosis is a zoonotic disease. it is easily spread from calves to humans, for example, even as the result of simply handling clothing soiled by calf diarrhea. adult immunocompetent humans are reported to experience watery diarrhea, cramping, flatulence, and headache. the disease can be life-threatening in immunocompromised individuals. v. giardiasis etiology. giardia lamblia (also called g. intestinalis and g. duodenalis) is a flagellate protozoon. giardiasis is a worldwide protozoal-induced diarrheal disease of mammals and some birds (kirkpatrick, 1989 ), but it not considered to be a significant pathogen in ruminants. clinical signs and diagnosis. diarrhea may be continuous or intermittent, is pasty to watery, is yellow, and may contain pathogenesis. following ingestion, each giardia cyst releases four trophozoites, which attach to the enterocytes of the duodenum and proximal jejunum and subsequently divide by binary fission or encyst. the organism causes little intestinal pathology, and the cause of diarrhea is unknown but is thought to be related to disruption of digestive enzyme function, leading to malabsorption. disturbances in intestinal motility may also occur (rings and rings, 1996) . prevention and control. intensive housing and warm environments should be minimized. cysts can survive in the environment for long periods of time but are susceptible to desiccation. effective disinfectants include quaternary ammonium compounds, bleach-water solution (1:16 or 1:32), steam, or boiling water. after cleaning, areas should be left empty and allowed to dry completely. treatment. giardia has been successfully treated with oral metronidazole. benzimidazole anthelmintics are also effective, but these are not approved for use in animals for this purpose. should be taken when handling infected animals. etiology. neosporosis is a common, worldwide cause of bovine abortion caused by the protozoal species neospora caninum. abortions have also been reported in sheep and goats. neonatal disease is seen in lambs, kids, and calves. until 1988, these infections were misdiagnosed as caused by toxoplasma gondii. some similarities exist between the life cycles and pathogeneses of both organisms. clinical signs and diagnosis. abortion is the only clinical sign seen in adult cattle and occurs sporadically, endemically, or as abortion storms. bovine abortions occur between the third and seventh month of gestation; fetal age at abortion correlates with the parity of the dam as well as with pattern of abortion in the herd. although cows that abort tend to be culled after the first or second abortion, repeated n. caninum-caused abortions will occur progressively later in gestation (up to about 6 months) and within a shorter time frame in the same cow (thurmond and hietala, 1997) . although infections in adults are asymptomatic other than the abortions, decreased milk production has been noted in congenitally infected cows. many neospora-infected calves will be born asymptomatic. weakness will be evident in some infected calves, but this resolves. rare clinical signs include exophthalmos or asymmetric eyes, weight loss, ataxia, hyperflexion or hyperextension of all limbs, decreased patellar reflexes, and loss of conscious proprioception. some fetal deaths will occur, and resorption, mummification, autolysis, or stillbirth will follow. immunohistochemistry and histopathology of fetal tissue are the most efficient and reliable means of establishing a postmortem diagnosis. serology (ifa and elisa) is useful, including precolostral levels in weak neonates, but this indicates only exposure. titers of dams will not be elevated at the time of abortion; fetal serology is influenced by the stage of gestation and course of infection. earlier and rapid infections are less likely to yield antibodies against neospora. none of the currently available tests is predictive of disease. epizootiology and transmission. the parasite is now acknowledged to be widespread in dairy and cattle herds. the life cycle of n. caninum is complex, and many aspects remain to be clarified. the definitive host is the dog (mcallister et al., 1998) . placental or aborted tissues are the most likely sources of infection for the definitive host and play a minor role in transmission to the intermediate hosts. the many intermediate hosts include ruminants, deer, and horses. transplacental transmission is the major mode of transmission in dairy cattle and is the means by which a herd's infection is perpetuated. a less significant mode of transmission is by ingestion of oocysts, which sporulate in the environment or in the intermediate host's body. reactivation in a chronically infected animal's body is the result of rupture of tissue cysts in neural tissue. seropositive immunity does not protect a cow from future abortions. many seropositive cows and calves will never abort or show clinical signs, respectively. some immunological cross-reactivity may exist among neospora, cryptosporidia, and coccidium. necropsy findings. aborted fetuses will usually be autolysed. in those from which tissue can be recovered, tissue cysts are most commonly found in the brain. spinal cord is also useful. histological lesions include mild to moderate gliosis, nonsuppurative encephalitis, and perivascular infiltration by mixed mononuclear cells. pathogenesis. as with toxoplasma, cell death is the result of intracellular multiplication of neospora tachyzoites. neospora undergoes sexual replication in the dog's intestinal tract, and oocysts are shed in the feces. the intermediate hosts develop nonclinical systemic infections, with tachyzoites in several organs, and parasites then localize and become encysted in particular tissues, especially the brain. infections of this type are latent and lifelong. except when immunocompromised, most cattle do not usually develop clinical signs and do not have fetal loss. fetuses become infected, leading to fetal death, mid-gestation abortions, or live calves with latent infections or congenital brain disease. it usually takes 2-4 weeks for a fetus to die and to be expelled. many aspects of the role of the maternal immune response and pregnancy-associated immunodeficiency in the patterns of neospora abortions remain to be elucidated. differential diagnosis. even when there is a herd history of confirmed neospora abortions, leptospirosis, bovine viral diarrhea virus (bvdv), infectious bovine rhinotracheitis virus (ibrv), salmonellosis, and campylobacteriosis should be considered. bvdv in particular should be considered for abortion storms. differentials for weak calves are b vdv, perinatal hypoxia following dystocia (immediate postpartum time), bluetongue virus, toxoplasma, exposure to teratogens, or congenital defects. prevention and control. the primary preventive measure is preventing contact with contaminated feces. oocysts will not survive dry environments or extremes of temperature. dog populations should be controlled, and dogs and other canids should not have access to placentas or aborted fetuses. dogs should also be restricted from feed bunks and other feed storage areas. preventive culling is not economically practical for most producers. a vaccine recently became available. if embryo transfer is practiced, recipients should be screened serologically before use. laxis. there is no known treatment or immunoprophyclinical signs and diagnosis. clinical signs of sarcocystosis infection are seen in cattle during the stage when the parasite encysts in soft tissues. often the infections are asymptomatic. fever, anemia, ataxia, symmetric lameness, tremors, tail-switch hair loss, excessive salivation, diarrhea, and weight loss are clinical signs. abortions in cattle occur during the second trimester and in smaller ruminants 28 days after ingestion of the sporulated oocysts. definitive diagnosis is based on finding merozoites and meronts in neural tissue lesions. clinical hematology results include decreased hematocrit, decreased serum protein, and prolonged prothrombin times. sarcocystis-specific igg will increase dramatically by 5-6 weeks after infection. there is no cross-reaction between sarcocystis and toxoplasma. epizootiology and transmission. infection rates among cattle in the united states are estimated to be very high. transmission is by ingestion of feed and water contaminated by feces of the definitive hosts. dogs are the definitive hosts for the species that infect the smaller ruminants. cats, dogs, and primates (including humans when s. hominis is involved) are the definitive hosts for the species that infect cattle. necropsy. aborted fetuses may be autolysed. lesions in neural tissues, including meningoencephalomyelitis, focal malacia, perivascular cuffing, neuronal degeneration, and gliosis, are most marked in the cerebellum and midbrain. lesions may be found in other tissues, such as lymphadenopathy, and hemorrhages may be found in muscles and on serous surfaces. cysts in cardiac and skeletal muscles are common incidental findings during necropsies. pathogenesis. ingestion of muscle flesh from an infected ruminant results in sarcocystis cysts' being broken down in the carnivore's digestive system, release of bradyzoites, infection of intestinal mucosal cells by the bradyzoites, differentiation into sexual stages, fusion of the male and female gametes to form oocysts, and shedding as sporocysts by the definitive hosts. the sporocysts are eaten by the ruminant and penetrate the bowel walls; several stages of development occur in endothelial cells of arteries. merozoites are the form that enters soft tissues, such as muscle, and subsequently encysts. prevention and control. feed supplies of ruminants must be protected from fecal contamination by domestic and wild carnivores. these animals should be controlled and must also not have access to carcasses. in larger production situations, monensin may be fed as a prophylactic measure. treatment. monensin fed during incubation is prophylactic, but the efficacy in clinically affected cattle is not known. etiology. toxoplasmosis is caused by the obligate intracellular protozoon toxoplasma gondii, a coccidial parasite of the family eimeridae. cats are the only definitive hosts, and several warm-blooded animals, including ruminants, have been shown to be intermediate hosts. the disease is a major cause of abortion in sheep and goats and less common in cattle. clinical signs and diagnosis. clinical signs depend on the organ or tissue parasitized. toxoplasmosis is typically associated with placentitis, abortion, stillbirths, or birth of weak young (underwood and rook, 1992; buxton, 1998) . it has also been shown to cause pneumonia and nonsuppurative encephalitis. the enteritis at the early stage of infection may be fatal in some hosts. hydrocephalus does not occur in animals as it does in human fetal toxoplasma infections. rare clinical presentations in ruminants include retinitis and chorioretinitis; these are usually asymptomatic. infection of the ewe during the first trimester usually leads to fetal resorption, during the second trimester leads to abortion, and during the third trimester leads to birth of weak to normal lambs with subsequent high perinatal mortality. congenitally infected lambs may display encephalitic signs of circling, incoordination, muscular paresis, and prostration. in sheep, weak young will develop normally if they survive the first week after birth. infected adult sheep show no systemic illness. infected adult goats, however, may die. diagnosis may be difficult, and biological, serological, and histological methods are helpful. serological tests are the most readily available. complement fixation and the sabin-feldman antibody test may assist in diagnosis. antibodies found in fetuses are indicative of congenital infection and are typically detectable 35 days after infection; fetal thoracic fluid is especially useful in demonstrating serological evidence of exposure. biological methods, such as tissue culture or inoculation of mice with maternal body fluids, or with postmortem or necropsy tissues, are more time-consuming and expensive. epizootiology and transmission. this protozoon is considered ubiquitous. fifty percent (50%) of adult western sheep and 20% of feedlot lambs have positive hemagglutination titers (1:64 or higher) (jensen and swift, 1982) . transmission among the definitive host is by ingestion of tissue cysts. necropsy findings. at necropsy, placental cotyledons contain multiple small white areas that are sites of necrosis, edema, and calcification. fetal brains may show nonspecific lesions such as coagulative necrosis, nonsuppurative encephalomyelitis, pneumonia, myocarditis, and hepatitis. histologically, granulomas with toxoplasma organisms may be seen in the retina, myocardium, liver, kidney, brain, and other tissues. impression smears of these tissues, stained appropriately (e.g., with giemsa), provide a rapid means of diagnosis. identification of the organism in tissue sections (especially of the heart and the brain) also confirms the findings. toxoplasma gondii is crescent-shaped, with a clearly visible nuclei, and will be found within macrophages. pathogenesis. the protozoon has three infectious stages: the tachyzoite, the bradyzoite, and the sporozoite within the oocyst. the definitive hosts, felids, become infected by ingesting cyst stages in mammalian tissues, by ingesting oocysts in feces, and by transplacental transfer. ingested zoites invade epithelial cells and eventually undergo sexual reproduction, resulting in new oocysts, which the cats will shed in the feces. cats rarely show clinical signs of infection. one cat can shed millions of oocysts in 1 gm of feces, but the asymptomatic shedding takes place for only a few weeks in its life. oocysts sporulate in cat feces after 1 day. ruminants are intermediate hosts of toxoplasmosis and become infected by ingesting sporulated oocyst-contaminated water or feed. as in the definitive host, the ingested sporozoite invades epithelial cells within the intestine but also further invades the bloodstream and is transported throughout the host. the organism migrates to tissues such as the brain, liver, muscles, and placenta. placental infection develops about 14 days after ingestion of the oocysts. the damage caused by an infection is due to multiplication within cells. toxoplasma does not produce any toxin. campylobacter, chlamydia, and q fever. prevention and control. feline populations on source farms should be controlled. eliminating contamination of feed and water with cat feces is the best preventive measure. sporulated oocysts can survive in soil and other places for long periods of time and are resistant to desiccation and freezing. vaccines for abortion prevention in sheep are available in new zealand and europe. treatment. toxoplasmosis treatment is ineffective, although feeding monensin during pregnancy may be helpful (underwood and rook, 1992) . (monensin is not approved for this use in the unites states.) weak lambs that survive the first week after birth will mature normally and will not deliver toxoplasmainfected young. research complications. because toxoplasmosis is zoonotic, precautions must be taken when handling tissues from any abortions or neurological cases. infections in immunocompromised humans have been fatal. etiology. trichomoniasis is an insidious venereal disease of cattle caused by tritrichomonas (also referred to as trichomonas) fetus, a large, pear-shaped, flagellated protozoon. the organism is an obligate parasite of the reproductive tract, and it requires a microaerophilic environment to establish chronic infections. in the united states, it is now primarily a disease seen in western beef herds. there are many similarities between trichomoniasis and campylobacteriosis; both diseases cause herd infertility problems. clinical signs and diagnosis. clinical signs include infertility manifested by high nonpregnancy rates as well as periodic py-ometras and abortions during the first half of gestation. often the problem is not recognized until herd pregnancy checks indicate many "open," delayed-estrus, late-bred cows, or cows with postcoital pyometras. the abortion rate varies from 5% to 30%, and placentas will be expelled or retained. tritrichomonas fetus also causes mild salpingitis but this does not result in permanent damage. other than these manifestations, infection with t. fetus causes no systemic signs. diagnosis is based on patterns of infertility and pyometras. for example, pyometras in postcoital heifers or cows are suggestive of this pathogen. diagnostic methods include identifying or culturing the trichomonads from preputial smegma, cervicovaginal mucus, uterine exudates, placental fluids, or abomasal contents of aborted fetuses. other nonpathogenic protozoa from fecal contamination may be present in the sample. the trichomonad has three anterior flagellae, one posterior flagella, and an undulating membrane; it travels in fluids with a characteristic jerky movement. culturing must be done on specific media, such as diamond's or modified pastridge. real exposure from breeding bulls or cows or, in some cases, contaminated breeding equipment. necropsy findings. nonspecific lesions, such as pyogranulomatous bronchopneumonia of fetuses and placentitis, may be seen in aborted material; some cases will have no gross lesions. histologically, trichomonads may be visible in the fetal lung lesions and the placenta; those tissues are also the most useful for culturing. pathogenesis. tritrichomonas fetus colonizes the female reproductive tract, and subsequent clinical manifestations may be related to the size of the initial infecting dose. tritrichomonas fetus does not interfere with conception. embryonic death occurs within the first 2 months of infection. affected cows will clear the infection over a span of months and maintain immunity for about 6 months. infections in younger bulls are transient; apparently organisms are cleared by the bulls' immune systems and are dependent on exposure to infected females. older bulls become chronic carriers, probably because of the ability of t. fetus to colonize deeper epithelial crypts of the prepuce and penis. differential diagnosis. campylobacteriosis is the other primary differential for reduced reproductive efficiency of a herd. other venereal diseases should be considered when infertility problems are noted in a herd: brucellosis, mycoplasmosis, ureaplasmosis, and infectious pustular vulvovaginitis. in addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. heifers, cows, and breeding bulls are vaccinated subcutaneously twice at 2 to 4 week intervals, with the booster dose administered 4 weeks before breeding season starts. similar timing is recommended for administration of the annual booster; a long, anamnestic response does not occur. bulls used for artificial insemination (ai) are screened routinely for t. fetus (and campylobacter) . ai reduces but does not eliminate the disease. the use of younger, vaccinated bulls is recommcmded in all circumstances. new animals should be tested before introduction to the herd. control measures also include culling affected cows or else removing them from the breeding herd for 3 months to rest and clear the infection. culling chronically infected bulls is strongly recommended. treatment. imidazole compounds have been effective, but the use of these is not permitted in food animals in the united states. therapeutic immunizations are worthwhile when a positive diagnosis has been made. these will not curtail fetal losses but will shorten the convalescence of the affected cows and improve immunity of breeding bulls. research complications. trichomoniasis should be considered whenever natural service is used and fertility problems are encountered. nematodes are important ruminant pathogens that cause acute, chronic, subclinical, and clinical disease in adults and adolescents. the major helminths may cause gastroenteritis associated with intestinal hemorrhage and malnutrition. nematodiasis is associated with grazing exposure to infective larvae; animals procured for research may have had exposure to these helminths. mixed infections of these parasites are common. generally, older animals develop resistance to some of the species; thus, animals between about 2 months and 2 years of age are most susceptible to infection. because of the parasites' effects on the animals' physiology, infection in these younger animals is a major contributor to a cycle of poor nutrition and digestion, compromised immune responses, and impaired growth and development. diagnosis is primarily based on fecal flotation techniques; however, because many of these nematodes have similar-appearing ova, hatching the ova and identifying the larvae are often required (baermann technique). a number of anthelmintics can be used to interrupt nematode life cycles. see zajac and moore (1993) and pugh et al. (1998) for comprehensive reviews of treatment and control of nematodiasis. i. haemonchus contortus, h. placei (barber's pole worm, large stomach worm) . haemonchus contortus is the most important internal parasite of sheep and goats, and the brief description here focuses on the disease in the smaller ruminants. haemonchus contortus and h. placei infections do occur in younger cattle and are similar to the disease in sheep. haemonchus is extremely pathogenic, and the adults feed by sucking blood from the mucosa of the abomasum. severe anemia may lead to death. weight loss, decreased milk production, poor wool growth, and intermandibular and cervical edema due to hypoproteinemia ("bottle jaw") are also common clinical signs. diarrhea is not seen in all cases but may sometimes be severe or chronic. the life cycle is direct. under optimal conditions, a complete life cycle, from ingestion of larvae to eggs passed in the feces, occurs in 3 weeks. embryonated eggs may develop into infective larvae within a week. hypobiotic (arrested) larvae may exist for several months in animal tissues, serving as a reservoir for future pasture contamination. periparturient increases in egg shedding by ewes contribute to large numbers of eggs spread on spring pastures ("spring rise"). resistance to common anthelmintics has developed; currently ivermectin or benzimidazole products are used, with a minimum of 2 dosings given 2-3 weeks apart. levamisole is also used. in severe cases, animals may benefit from blood transfusions and iron supplementation. because animals may easily acquire infective larvae from ingestion of contaminated feed and from contaminated pastures, general facility sanitation and pasture management and rotation are important preventive and control measures. haemonchus contortus is susceptible to destruction by freezing temperatures and dry conditions. ii. ostertagia (teladorsagia) circumcincta (medium stomach worm). ostertagia circumcincta is also highly pathogenic for sheep and goats and, like haemonchus, attaches to the abomasal mucosa and ingests blood. the life cycle is comparable to that of haemonchus, including the phenomenon of hypobiosis. larvae are especially resistant to cool temperatures, however, and will overwinter on pastures. larvae-induced hyperplasia of abomasal epithelial glands results in a change of gastric ph from about 2.0 to near 7.0, leading to decreased digestive enzyme activity and malnutrition. clinical syndromes are categorized as type 1 or type 2. the former type is associated with infections acquired in fall or spring and is seen in younger animals. the latter type is associated with emergence of the arrested larvae during spring or fall. clinical signs include anemia, weight loss, decreased milk production, and unthriftiness. diarrhea is usually seen in type 1 only; the symptoms of type 2 are comparable to those of haemonchus infections. anthelmintic drug therapy is comparable to that for haemonchus, and drug resistance is also a problem with ostertagia. iii. ostertagia ostertagi (cattle stomach worm). ostertagia ostertagi is the most pathogenic and most costly of the cattle nematodes. ostertagia leptospicularis and o. bisonis also cause disease. the life cycle is direct, and egg shedding by the cattle may occur within 3-4 weeks of ingestion of infective larvae. hypobiosis is also a characteristic of o. ostertagi. in the initial steps of infection, the normal processes of the abomasum are profoundly disrupted and cells are destroyed as the larvae develop within and emerge from the glands. moroccan leather appearance is the term to describe the result of cellular hyperplasia and loss of cell differentiation. cycles of infection and morbidity depend on geographic location, climate, and production cycles. type 1 cattle ostertagiasis is associated with ingestion of large numbers of infective larvae, occurs in animals less than 2 years old, and causes diarrhea and anorexia. type 2 ostertagiasis occurs in cattle 2-4 years old and older adults, is the result of the emergence and development of hypobiotic larvae, and in addition to signs seen with type 1, hypoproteinemia with development of submandibular edema, fever, and anemia is a clinical sign. treatment options include ivermectin, fenbendazole, and levamisole; all are effective against the arrested larvae. ostertagia is susceptible to desiccation but is resistant to freezing. iv. trichostrongylus vitrinus, t. axei, t. colubriformis (hair worms) . trichostrongylus species favor cooler conditions, and some larvae may overwinter. although the different species may affect different segments of the gastrointestinal tract, the nematode attaches to the mucosa and affects secretion and/or absorption. trichostrongylus vitrinus and t. colubriformis infect the small intestine of sheep and goats. trichostrongylus axei infects the abomasum of cattle, sheep, and goats and causes increases in abomasal ph similar to those seen with ostertagia. mucosal hyperplasia is not seen. the prepatent period is about 3 weeks. affected animals display unthriftiness, anorexia, decreased milk production, weight loss, diarrhea, and dehydration. these worms show intermediate resistance to freezing temperatures and dry conditions. v. nematodirus spathiger, n. battus (thread-necked worms vii. strongyloides papillosus. strongyloides papillosus is a small-intestinal parasite of sheep and cattle. strongyloides has a different life cycle from that of many nematodes. the eggs, expelled in the feces, are larvated, and when they hatch, they form both free-living males and females or parasitic females only. the parasitic females may enter the gastrointestinal tract through oral ingestion, such as in milk during nursing, or through direct penetration of the skin. penetrating larvae enter the bloodstream and are transported to the lungs, where they penetrate the alveoli, are coughed up, and then swallowed to ultimately enter the gastrointestinal tract. adult females may reproduce in the small intestines by parthenogenesis. clinical signs associated with strongyloides include weight loss, diarrhea, unthriftiness, and dermatitis in cases where large numbers migrate through the skin. the current broad-spectrum anthelmintics are effective against strongyloides. strongyloides, bunostomum infection may involve oral ingestion or direct penetration of the skin (followed by tracheal migration and swallowing). the larvae mature in the small intestines and suck blood. larvae are susceptible to desiccation and freezing. heavy infection with bunostomum may result in anemia, diarrhea, intestinal hemorrhage, edema, and weight loss. ix. oesophagostomum columbianum, o. venulosum (nodule worms) . oesophagostomum spp. primarily infect the large intestine and occasionally the distal small intestine, causing nodule worm disease, or simply gut. oesophagostomum columbianum and o. venulosum infect sheep and cattle. these nematodes may affect sheep from 3 months to 2 years of age, and the prepatent period is about 6 weeks. larvae are highly sensitive to freezing and desiccation and rarely overwinter. larvae penetrate the large-intestinal mucosa but occasionally move into the deeper areas of the intestinal wall near the serosa. the resultant inflammatory reaction may lead to the formation of a caseous nodule that may mineralize over time. intestinal lesions may accelerate peristalsis, leading to diarrhea, or may inhibit peristalsis (later stages), resulting in constipation. clinical signs include weakness, unthriftiness, alternating episodes of diarrhea and constipation, and severe weight loss. nodular lesions are typical at necropsy. x. chabertia ovis (large-mouth bowel worm). chabertia ovis is a minor colon parasite of sheep, goats, and cattle and is seen primarily in sheep. signs of infection are not usually seen in cattle. prepatent periods are up to 50 days. heavy infection, which may result from as few as 100 worms located at the proximal end of the colon, may lead to hemorrhagic mucoid diarrhea, weight loss, weakness, colitis, and mild anemia. xi. trichuris (whipworms). trichuris spp. are mildly pathogenic nematodes and are usually attached to the cecal mucosa. trichuris has a rather long prepatent period, extending from 1 to 3 months. the oval eggs are double-operculated and survive well in pasture environmental extremes. the adult worms also have a characterisitic morphology, with one thicker end appearing as a whip handle. the nematodes cause a minor cecitis and will feed on blood. clinical infection is rare and results in diarrhea with mucus and blood. treatment and prevention methods are similar to those for other nematodes. xii. dictyocaulus (lungworms). dictyocaulus spp., or lungworms, are nematodes that cause varying clinical signs in ruminants. in sheep, dictyocaulus filaria, protostrongylus rufescens, and muellerius capillaris cause disease; dictyocaulus is the most pathogenic. goats are infected by the same species as sheep, but infections are uncommon. dictyocaulus viviparus is the only lungworm found in cattle, causing "fog fever." infections with these parasites in the united states tend to be associated with cooler, moister climates. lungworms induce a severe parasitic bronchitis (known as husk, or verminous pneumonia) in sheep between approximately 2 and 18 months of age. sheep infected with any of the lungworm species may display coughing, dyspnea, nasal discharge, weight loss, unthriftiness, and occasionally fever. coughing and dyspnea are symptoms in goats. diagnosis is suggested by persistent coughing and nasal discharge and is confirmed by identifying larvae in the feces or adults in pathological samples. the baermann technique, involving prompt examination of room-temperature feces, is usually used; zinc sulfate flotation is also used. dictyocaulus has a direct life cycle. the adult worms reside in the large bronchi. dictyocaulus produces embryonated eggs that are coughed up and swallowed; the eggs then hatch in the intestines, and larvae are expelled in the feces. the expelled larvae are infectious in about 7-10 days and, after ingestion, penetrate the intestinal mucosa and move through the lymphatics and blood into the lungs, where they develop into adults in about 5 weeks. dictyocaulus filaria causes an especially severe bronchitis in sheep. protostrongylus inhabits smaller bronchioles. muellerius is of minor pathogenicity. protostrongylus and muellerius require the snail or slug as an intermediate host. infection occurs through ingestion of infected snails; infections are less likely than those caused by the direct ingestion of dictyocaulus larvae. immunity wanes over a year. viral and bacterial respiratory tract infections may be associated with the parasitic infection. more severe illness is seen after infections with cooperia and ostertagia, because of a synergism between the nematodes even if the cattle are not currently infected with those parasites. hypobiosis (arrested development of immature worms in lung tissue) is associated with dictyocaulus infections; cattle will be silent carriers, showing no clinical signs and serving as a means for the infection to survive over winter or a dry season. pastures can be heavily contaminated during the next grazing season. necropsy lesions include bronchiolitis and bronchitis, atelectasis, and hyperplasia of peribronchiolar lymphoid tissue. nematodes frequently reside in the bronchi of the diaphragmatic lung lobes and are frequently enmeshed with frothy exudate. prevention and control of the disease involve appropriate pasture management. elimination of intermediate hosts is important in sheep and goat pastures. in a laboratory setting, animals may be procured that are already harboring the disease. infected animals can be treated with anthelmintics such as ivermectin or levamisole. muellerius tends to be resistant to levamisole. there is no anthelmintic currently approved for goats, but fenbendazole, administered 2 weeks apart, has been effective for all three tapeworms are rarely of clinical or economic importance. in younger animals, heavy infections result in potbellies, constipation or mild diarrhea, poor growth, rough coat, and anemia. moniezia expansa, and less commonly moniezia benedini, inhabit the small intestines of grazing ruminants. moniezia expansa has the widest distribution of the tapeworm species in north america. soil mites (galumna spp. and oribatula spp.) contribute to the life cycle as intermediate hosts, a period that lasts up to 16 weeks. cysticercoids released from the mites are grazed, pass into the small intestines, and mature. no clinical or pathological sign is usually observed with moniezia infection; diagnosis is made by observing the characteristic triangularshaped eggs in fecal flotation examinations. infection is treated with cestocides. thysanosoma actinoides, or the fringed tapeworm, is a cestode that resides in the duodenum, bile duct, and pancreatic duct of sheep and cattle raised primarily west of the mississippi river in the united states. thysanosoma is of the family anoplocephalidae. the life cycle is indirect, and the intermediate host is the psocid louse. larval forms, or cysticercoids, are ingested by grazing animals, and the prepatent period is several months. typically, no clinical signs are observed with thysanosoma infection; nonetheless, liver damage, resulting in liver condemnation at slaughter, occurs. necropsy lesions include bile and/or ductal hyperplasia and fibrosis. thysanosoma is diagnosed premortem by identifying the gravid segments in the feces. ii. abdominal or visceral cysticercosis. abdominal or visceral cysticercosis is an occasional finding at slaughter. the socalled bladder worms typically affect the liver or peritoneal cavity and are the larval form of taenia hydatigena, the common tapeworm of the dog family. taenia hydatigena resides in the small intestines of canids, and its gravid segments, oncospheres, contaminate feed and water sources. after ingestion, the larvae penetrate the intestinal mucosa, are transported via the bloodstream to the liver, and cause migration tracts throughout the liver parenchyma. the larvae may leave the liver and migrate into the peritoneal cavity, where they attach and develop over the next 1-9 months into small fluid-filled bladders. the life cycle is completed only after these bladders are ingested by a carnivore, thus completing the maturation of the adult tapeworms. although larval migration may cause nonspecific signs such as anorexia, hyperthermia, and weight loss, affected animals are usually asymptomatic. at necropsy, the bladder worms will be observed attached to the peritoneal or organ surfaces. migration tracts may result in fibrosis and inflammation. diagnosis is usually made at necropsy. because of the migration through the liver, fasciola hepatica is a differential diagnosis. minimizing exposure to canine feces-contaminated feeds and water effectively interrupts the life cycle. research animals may have been exposed prior to purchase. echinococcosis, like cysticercosis, is an occasional finding at slaughter or necropsy. the hydatid cyst is the larval intermediate of the adult tapeworm echinococcus granulosus, which resides in the small intestines of dogs and wild canids. embryonated ova are expelled in the feces of the primary host and are ingested by herbivores, swine, and potentially humans. the eggs hatch in the gastrointestinal tract, and the oncospheres penetrate the mucosal lining, enter the bloodstream, and are transported to various organs such as the liver and lungs. the cystic structure develops and potentially ruptures, forming new cystic structures. clinically, echinococcosis presents minimal clinical signs; unthriftiness or pneumonic lesions may be associated with infected organs. cysts are typically observed at necropsy. prevention should be aimed at decreasing fecal contamination of feed and water by canids. additionally, tapeworm-infected dogs can be treated with standard tapeworm therapies. treatment of infected ruminants is uncommon. iv. gid. coenuris cerebralis, the larval form of the canid tapeworm taenia (multiceps) multiceps, is the causative agent of the rare condition called gid. the disease occurs in ruminants as well as many other mammalian species. the larval parasite, ingested from fecal-contaminated food and water, invades the brain and spinal cord and develops as a bladder worm that causes pressure necrosis of the nervous tissues. the resultant signs of hyperesthesia, meningitis, paresis, paralysis, ataxia, and convulsions are observed. diagnosis is usually made at necropsy. eliminating transfer from the canid hosts prevents the disease. the cercariae leave the intermediate host, swim to grassy vegetation, lose their tail, and become a cystlike metacercaria. the metacercariae may remain in a dormant stage on the grass for 6 months or longer until ingested by a ruminant. the ingested metacercariae penetrate the small-intestinal wall and migrate through the abdominal cavity to the liver. there they locate in a bile duct, mature, and remain for up to 4 years. acute liver fluke disease is related to the damage caused by the migration of immature flukes. migratory flukes may lead to liver inflammation, hemorrhage, necrosis, and fibrosis. fascioloides magna infections in sheep and goats can be fatal as the result of just one fluke tunneling through hepatic tissue. in cattle, infections are often asymptomatic because of the host's encapsulation of the parasite. liver fluke damage may predispose to invasion by anaerobic clostridium species such as c. novyi that could lead to fatal black disease or bacillary hemoglobinuria. chronic disease may result from fluke-induced physical damage to the bile ducts and cholangiohepatitis. blood loss into the bile may lead to anemia and hypoproteinemia. liver damage also is evidenced by increases in liver enzymes such as y-glutamyl transpeptidase (ggt). persistent eosinophilia is also seen with liver fluke disease. other clinical signs of liver fluke disease include anorexia, weight loss, unthriftiness, edema, and ascites. at necropsy, livers will be pale and friable and may have distinct migration tunnels along the serosal surfaces. bile ducts will be enlarged, and areas of fibrosis will be evident. diagnosis can be made from clinical signs and postmortem mites cause a chronic dermatitis. the principal symptom of these infections is intense pruritus. in addition, papules, crusts, alopecia, and secondary dermatitis are seen. anemia, disruption of reproductive cycles, and increased susceptibility to other diseases may also occur. mites are rare in ruminants in the united states, but infections of sarcoptes and psorergates mange must be reported to animal health officials. ruminants in poorly managed facilities are generally the most susceptible to infection, and infections are more frequent during winter months. diagnosis is based on signs, examination of skin scrapings, and response to therapy. no effective treatment for demodectic mange in large animals has been found. the differential for mite infestations is pediculosis. several genera of mites may affect sheep. these have been eradicated from flocks in the united states or are very rare and include psoroptes ovis (common scabies), sarcoptes scabiei (head scabies, barn itch), psorergates ovis (sheep itch mite), chorioptes ovis (foot scabies, tail mange), and demodex ovis (follicular mange). goats can also be infected by sarcoptic, chorioptic, and psoroptic mange. the scabies mite sarcoptes rupicaprae invades epidermal tissue and causes focal pruritic areas around the head and neck. the chorioptic mite, either chorioptes bovis or c. caprae, does not invade epidermal tissue but rather feeds on dead skin tissue. the chorioptic mite prefers distal limbs, the udder, and the scrotum and can be a significant cause of pruri-tus. the psoroptic mite psoroptes cuniculi commonly occurs in the ear canal and causes head shaking and scratching. repeated treatments of lime sulfur, amitraz, or ivermectin may be effective (smith and sherman, 1994) . goats are also susceptible to demodectic mange caused by demodex caprae. adult mites invade hair follicles and sebaceous glands. pustules may develop with secondary bacterial infection. psoroptes bovis continues to be present in cattle in the united states, although it has been eradicated from sheep. chorioptes bovis typically infects lower hindlimbs, perineum, tail, and scrotum but can become generalized. the sarcoptic mange mite s. scabei can survive off the host, so fomite transmission is a factor. the mange usually begins around the head but then spreads. this parasite can be transmitted to humans. demodex bovis infects cattle; nodules on the face and neck are typical. demodex bovis infections may resolve without treatment. lindane, coumaphos, malathion, and lime sulfur are used to treat psoroptes and psorergates. ivermectin is effective against sarcoptes and is approved for use in cattle. lice that infect ruminants are of the orders mallophaga, biting or chewing lice, and anoplura, sucking lice. these are wingless insects. members of the mallophaga are colored yellow to red; members of the anoplura are blue gray. lice produce a seasonal (winter-to-spring), chronic dermatitis. in sheep, biting lice include damalinia (bovicola) ovis (sheep body louse). sucking lice that infect sheep include linognathus ovillus (blue body louse) and l. pedalis (sheep foot louse). in goats, biting lice infection are caused by d. caprae (goat biting louse), d. limbatus (angora goat biting louse), and d. crassipes. suckir/g louse infections in goats are caused by l. stenopis and l. africanus. damalinia bovis is the cattle biting louse. sucking lice include l. vituli, solenopotes capillatus, haematopinus eurysternus, and h. quadripertusus. pruritus is the most common sign and often results in alopecia and excoriation. the host's rubbing and grooming may not correlate with the extent of infestation. hairballs can result from overgrooming in cattle. in severe cases, the organisms can lead to anemia, weight loss, and damaged wool in sheep and damaged pelts in other ruminants. young animals with severe infestations of sucking lice may become anemic or even die. pregnant animals with heavy infestations may abort. in sheep infected with the foot louse, lameness may result. lice are generally species-specific. those infecting ruminants are usually smaller than 5 mm. goats may serve as a source of infection for sheep by harboring damalinia ovis. transmission is primarily by direct contact between animals. transmission can also occur by attachment to flies or by fomites. some animals are identified as carriers and seem to be particularly susceptible to infestations. biting or chewing lice inhabit the host's face, lower legs, and flanks and feed on epidermal debris and sebaceous secretions. sucking lice inhabit the host's neck, back, and body region and feed on blood. lice eggs or nits are attached to hairs near the skin. three nymphal stages, or instars, occur between egg and adult, and the growth cycle takes about 1 month for all species. lice cannot survive for more than a few days off the host. all ruminant mite infestations are differentials for the clinical signs seen with pediculosis. animals that are carriers should be culled, because these individuals may perpetuate the infection in the group. lice are effectively treated with a variety of insecticides, including coumaphos, dichlorvos, crotoxyphos, avermectin, and pyrethroids. label directions should be read and adhered to, including withdrawal times. products should not be used on female dairy animals. treatments must be repeated at least twice at intervals appropriate for nit hatches (about every 16 days) because nits will not be killed. fall treatments are useful in managing the infections. systemic treatments in cattle are contraindicated when there may be concurrent larvae of cattle grubs (hypoderma lineatum and h. bovis). back rubbers with insecticides, capitalizing on self-treatment, are useful for cattle. sustained-release insecticide-containing ear tags are approved for use in cattle. etiology. ruminants are susceptible to many species of ixodidae (hard-shell ticks) and argasidae (softshell ticks). many diseases, including anaplasmosis, babesiosis, and q fever are transmitted by ticks. clinical signs and diagnosis. tick infestations are associated with decreased productivity, loss of blood and blood proteins, transmission of diseases, debilitation, and even death. feeding sites on the host vary with the tick species. ticks are associated with an acute paralytic syndrome called tick paralysis. this disease is characterized by ascending paralysis and may lead to death if the tick is not removed before the paralysis reaches the respiratory muscles. diagnosis is based on identification of the species. epizootiology and transmission. ticks are not as host-specific as lice. ticks are classified as one-host, two-host, or three-host; this refers to whether they drop off the host between larval and nymphal stages to molt. pathogenesis of tick infestations. patterns of feeding on the host differ between argasidae and ixodidae. the former feed repeatedly, whereas the latter feed once during each life stage. pathogenesis of tick paralysis. following a tick-feeding period of 4-6 days, the tick salivary toxin travels hematogenously to the myoneural junctions and spinal cord and inhibits nerve transmission. removal of the ticks reverses the syndrome unless paralysis has migrated anteriorly to the respiratory centers of the medulla. in these cases, death due to respiratory failure occurs. insecticides. ticks can be treated using systemic or topical h. other parasites i. nasal bots (nasal myiasis, head grubs). nasal myiasis causes a chronic rhinitis and sinusitis. the disease is caused by the larval forms of the botfly oestrus ovis. the botfly deposits eggs around the nostrils of sheep. the ova hatch, and the larvae migrate throughout the nasal cavity and sinuses, feeding on mucus and debris. in 2-10 months, the larvae complete their growing phase, migrate back to the nasal cavity, and are sneezed out. the mature larvae penetrate the soil and pupate for 1-1.5 months and emerge as botflies. clinically, early in the disease course, animals display unique behaviors such as stamping, snorting, sneezing, and rubbing their noses against each other or objects. hypersensitivity to the larvae occurs (dorchies et al., 1998) . later, mucopurulent nasal discharges associated with the larval-induced inflammation of mucosal linings will be observed. at necropsy, larvae will be observed in the nasal cavity or sinuses. mild inflammatory reactions, mucosal thickening, and exudates will accompany the larvae. the disease is diagnosed by observing the behaviors or identifying organisms at necropsy. up to 80% of a flock will potentially be infected; treatment should be employed on the rest of the flock. ivermectins and other insecticides will eliminate the larvae; but treatment should be done in the early fall, when larvae are small. fly repellents may be helpful at preventing additional infections. ii. screwworm flies. cochliomyia hominivorax (callitroga americana) is the the screwworm that causes occasional disease in the southwestern united states along the mexico border. eradication programs have been pursued, and the disease is reportable. large greenish flies lay large numbers of white eggs as shinglelike layers at the edges of open wounds (including docking and castration sites), soiled skin, or abrasions. eggs hatch within 24 hr. larvae are obligate parasites of living tissue, and the cycle is perpetuated because the increasingly large wound continues to be attractive to the next generation of flies. larvae eventually drop off, pupate best in hot climates, and hatch in 3 weeks. large cavities in parasitized tissue are formed, and lesions are characterized by malodor, large volumes of brown exudate, and necrosis. single animals or entire herds may be affected. treatment is intensive, with dressings and larvicidal applications. if there is no intervention, the host succumbs to secondary infections and fluid loss. effective current control regimens include subcutaneous injection of ivermectin and programs that release sterile male flies. iii. sheep keds ("sheep ticks"). in sheep and goats, sheep keds produce a chronic irritation and dermatitis with associated pruritus. the disease is caused by melophagus ovinus, which is a fiat, brown, blood-sucking, wingless fly; the term sheep tick is incorrectly used. the adult fly lives entirely on the skin of sheep. females mate and produce 10-15 larvae following a gestation of about 10-12 days. the larvae attach to the wool or hair and then pupate for about 3 weeks. the adult female feeds on blood and lives for 4-5 months; the life cycle is completed in about 5-6 weeks. infection is highest in fall and winter. pruritus develops around the neck, sides, abdomen, and rump. in severe cases, anemia may occur. keds can transmit bluetongue virus. keds are diagnosed by gross or microscopic identification. ivermectin or other insecticides are useful treatment agents. portant, other immune mechanisms are not well understood. immunity may not be of long duration. recovery is enhanced by correcting nutritional deficiencies and improving housing and ventilation problems. a number of topical treatments, such as 2-5% lime-sulfur solution, 3% captan, iodophors, thiabendazole, and 0.5% sodium hypochlorite, can be used. in severe cases, systemic therapy with griseofulvin may be successful. prevention and control. the animals' environment and overall physical condition should be reassessed with particular attention to ventilation, crowding, sanitation, and nutrition. pens should be thoroughly cleaned and disinfected. research complications. ringworm is a zoonotic disease. etiology. dermatophytosis, or infection of the keratinized layers of skin, is caused mostly by species of the genera trichophyton and microsporum. the primary causes in sheep are t. mentagrophytes and t. verrucosum. in goats, the agents are t. mentagrophytes, m. canis, m. gypseum, t. verrucosum, t. schoenleinii, and epidermophyton floccosum. in cattle, t. verrucosum is the primary causative agent. dermatophytosis is a common fungal infection of the epidermis of cattle and is less common in sheep and goats. clinical signs and diagnosis. multiple, gray, crusty, circumscribed, hyperkeratotic lesions are characteristic of infection. lesions will vary in size. in all ruminants, lesions will be around the head, neck, and ears. in goats and cattle, lesions will extend down the neck, and in cattle, lesions develop particularly around the eyes and on the thorax. cattle lesions are unique in the marked crustiness, which progressively appears wartlike. hair shafts become brittle and break off. intense pruritus is often associated with the alopecic lesions. the disease can be diagnosed by microscopic identification of hyphae and conidia on the hairs following skin scraping and 20% potassium hydroxide digestion. dermatophyte test media (dtm) cultures are the most reliable means to diagnose the fungus. broken hairs from the periphery of the lesion are the best sources of the fungus. epizootiology and transmission. younger animals are more susceptible, and factors such as crowding, indoor housing, warm and humid conditions, and poor nutrition are also important. transmission is by direct contact or by contact with contaminated fomites, such as equipment, fencing, or feed bunks. pathogenesis. incubation can be as long as 6 weeks. the organisms invade and multiply in hair shafts. treatment. spontaneous recovery occurs in all species in 1-4 months. although cell-mediated immunity is considered iminverted eyelids are a common inherited disorder of lambs and kids of most breeds. generally, the lower eyelid is affected and turns inward, causing various degrees of trauma to the conjunctiva and cornea. young animals will display tearing, blepharospasm, and photophobia initially. if the disorder is left uncorrected, corneal ulcers, perforating ulcers, uveitis, and blindness may occur. placing a suture or a surgical staple in the lower eyelid and the cheek, effectively anchoring the lid in an everted position, successfully treats the condition. the procedure likely results in the formation of some degree of scar tissue within the lower lid, because when the suture eventually is removed, the condition rarely returns. other treatments include the injection of a "bleb" of penicillin in the lid, regular manual correction over a 2-day period early in the animal's life, and application of ophthalmic ointments, powders, and solutions. boric acid or 10% argyrol solutions have been used as treatments. because of the genetic predisposition, prevention of the condition requires removal of maternal or paternal carriers. [3-mannosidosis is an autosomal recessive lysosomal storage disease of goats. the disease affects kids of the nubian breed and is identified by intention tremors and difficulty or inability of newborns to stand. cells of affected animals are vacuolated because of a lack of lysosomal hydroxylase, which results in accumulation of oligosaccharides. newborn kids are unable to rise, and they have characteristic flexion of the carpal joint and hyperextension of the pastern joint. kids are born deaf and with musculoskeletal deformities such as domed skull, small narrow muzzle, small palpebral fissures, enophthalmos, and depressed nasal bridge (smith and sherman, 1994) . carrier adults can be identified by plasma measurements of [3-mannosidase activity. caprine congenital myotonia is an inherited autosomal dominant disease that affects voluntary striated skeletal muscles. goats with this disease are commonly known as fainting goats. "fainting" is actually transient spasms of skeletal musculature brought about by visual, tactile, or auditory stimuli (smith and sherman, 1994) . muscle fiber membranes appear to have fewer chloride channels than normal, resulting in decreased chloride conduction across the membrane, with subsequent increased membrane excitability and repetitive firing (smith and sherman, 1994) . contractions of skeletal muscle are sustained for up to 1 min. kids exhibit the condition by 6 weeks of age, and males appear to exhibit more severe clinical signs than females (smith and sherman, 1994) . electromyographic studies produce an audible "dive-bomber" sound characteristic of hyperexcitable cell membranes (smith and sherman, 1994) . i. congenital erythropoietic porphyria. congenital erythropoietic porphyria (cep) is an autosomal recessive disease of cattle seen primarily in holsteins, herefords, and shorthorns. the disease also occurs in limousin cattle, humans, and some other species. in the homozygous recessive animal, symptoms of the disease may vary from mild to severe and occur at different times of the year and in different ages of animals. a reddish brown discoloration of teeth and bones is a characteristic of the disease, as is discolored urine, general weakness and failure to thrive, photosensitization, and photophobia. bones are more fragile compared with bones of normal animals. a regenerative anemia occurs as the result of the shortened life span of erythrocytes, due to accumulations of porphyrins. the genetic defect is associated with low activity of an essential enzyme, uroporphyrinogen iii synthase, in the porphyrin-heme synthesis pathway in erythrocytic tissue. the ranges in the presentation of the disease are believed to be related to varying cycles of porphyrin synthesis. porphyrins are excreted in varying amounts in the urine and the discoloration fluoresces under a wood's lamp. diagnosis is based on these clinical and visible signs of porphyria; skin biopsy provides definitive diagnosis. heterozygotes may have milder symptoms. many other genetic defects, in all major organ systems, have been described in numerous breeds of cattle and are described in detail elsewhere ("large animal internal medicine," 1996) . in many cases, the genetic basis has been clarified, and associated defects also noted. many defects are reported in particular breeds, but as crossbreeding increases and new breeds are developed, these traits are appearing in these animals. the bovine genome continues to be further characterized, and more linkage maps and gene locations are forthcoming (womack, 1998) . some bovine genetic defects are also regarded as models of genetic disease, such as leukocyte adhesion deficiency of holstein cattle. some of the more commonly reported defects include syndactyly in holsteins and other breeds and polydactyly in simmentals; lysosomal storage diseases such as a-mannosidosis in some beef breeds; enzyme deficiencies such as citrullinemia in holsteins; and progressive degenerative myeloencephalopathy ("weaver") in brown swiss. ii. goiter of sheep. a defect in the synthesis of thyroid hormone has been identified in merino sheep (radostits et al., 1994) . lambs born with the defect have enlargement of the thyroid gland, a silky appearance to the wool, and a high degree of mortality. edema, bowing of the legs, and facial abnormalities have also been noted in animals with this disorder. immaturity of the lungs at birth causes neonatal respiratory distress and resuits in dyspnea and respiratory failure. spider lamb syndrome is an inherited, often lethal, musculoskeletal disorder primarily occurring in suffolk and hampshire breeds. severely affected lambs die shortly after birth. animals that survive the perinatal period develop angular limb deformities, scoliosis, and facial deformities. with time, affected animals become debilitated, exhibit joint pain, and develop neurological problems associated with the spinal abnormalities. radiologically, secondary ossification centers--especially the physis, subchondral areas, and cuboidal bonesmare affected. abnormal endochondral ossification leads to excess cartilage formation, notably apparent in the elbows. lambs will typically display abnormally long limbs, medial deviation of the carpus and tarsus, flattening of the sternum, scoliosis/kyphosis of the vertebrae, and a rounded nose. muscle atrophy is common. diagnosis can be based on typical clinical signs, which are similar to those seen with marfan syndrome in humans (rook et al., 1986) . long-term survival is rare; treatment is unsuccessful. i. abomasal and duodenal ulcers. abomasal and duodenal ulcers occur more frequently in calves and adult cattle than in sheep and goats. like rumenitis, abomasal and duodenal ulcers may be associated with lactic acidosis. concurrent disease, such as salmonellosis, bluetongue, or overuse of anti-inflammatory drugs, or recent shipping or environmental stresses may also lead to ulcer formation. copper deficiency, dietary changes, mycotic infections, clostridium perfringens abomasitis, and abomasal bezoars are associated with this disease in calves. in older adult cattle, abomasal lymphosarcoma may be the underlying condition. gastric acid hypersecretion in conjunction with insufficient gastric mucous secretion will physically destroy the gastric epithelium. deep ulceration may cause serious hemorrhage and/or perforation with peritonitis. chronic hemorrhage may lead to anemia. although ulcers are often asymptomatic in calves, perforation with peritonitis is more common than hemorrhage. dark feces or melena and abdominal pain may be observed. arched back, restlessness, kicking at the abdomen, bruxism, and anorexia are common signs of abdominal pain. fecal occult blood is as an easy diagnostic test. treatment includes gastrointestinal protectants and histamine antagonists. anemia may be symptomatically treated with parenteral iron injections and anabolic steroids. preventive measures in cattle herds include ensuring optimal passive immunity for calves, minimizing stress to calves, and striving for a herd free of bovine leukosis virus. ii. abomasal emptying defect. abomasal emptying defect of sheep is a sporadic syndrome associated with abomasal distension and weight loss. suffolks tend to be especially predisposed, although the disease has been diagnosed in hampshires, columbias, and corriedales. the mechanism of the disease is unknown. affected animals will exhibit a gradual weight loss with a history of normal appetites. feces will continue to be normal. ventral abdominal distension associated with abomasal accumulation of feedstuffs will be apparent in many of the animals. diagnosis is primarily based on history and clinical signs. elevations in rumen chloride concentrations (> 15 meq/liter) are commonly found. radiography or ultrasonography may be helpful at identifying the distended abomasum. abomasal emptying defect is usually eventually fatal. medical treatment with metoclopramide and mineral oil may be helpful in early disease. iii. abomasal displacement. displaced abomasum (da) is a sporadic disorder usually associated with multiparous 4-to 7year-old dairy cows in early lactation, but the condition can occur even in young calves. displacement to the right (rda) may be further complicated by torsion (rta), a surgical emergency. left displacement (lda) is more common than rda. clinical signs include anorexia, lack of cud chewing, decreased frequency of ruminal contractions, shallow respirations, increased heart rate, treading, and decreased milk production. diagnosis is based on characteristic areas of tympanic resonance during auscultation-percussion of the lateral to lateral-ventral abdomen ("pings"), ruminal displacement palpated per rectum, and clinical signs. cow-side clinical chemistry findings include hypoglycemia and ketonuria; more extensive evaluations will often indicate moderate to severe electrolyte and acid-base abnormalities. da occurs because of gas accumulation within the viscus, and the abomasum "floats" up from its normal ventral location to the lateral abdominal wall. no exact cause of da has been identified, but it is commonly associated with stress; high levels of concentrate in the diet, leading to forestomach atony; and many disorders, including lack of regular exercise, mastitis, hypocalcemia, retained placenta, metritis, or twins. factors such as body size and conformation indicate the possibility of genetic predisposition. treatments include surgical and nonsurgical techniques for lda; the former has a better chance of per-manent correction. emergency surgery is necessary for rta; the disorder is fatal within 72 hr. recurrence is rare after surgical correction. electrolyte and acid-base imbalances are likely in severe cases and especially with rta. prevention includes reducing stress, taking greater care in the introduction and feeding of concentrates, and reducing incidence of predisposing diseases noted above (rohrbach et al., 1999) . fat cow syndrome is seen in peri-or postparturient overconditioned or obese multiparous dairy cows. factors in the development of the condition include negative energy balance related to the normal decreased dry matter intake as parturition approaches; hormonal changes associated with parturition; and concurrent diseases of parturition that decrease feed intake and increase energy needs. the possible concurrent diseases include metritis, retained fetal membranes, mastitis, parturient paresis, and displaced abomasum. signs are nonspecific and include depression, anorexia, and weakness. prognosis is usually guarded. diagnosis is based on herd management, the animal's condition, ketonuria, and clinical signs. in prepartum cattle and in lactating cows, blood levels of nonesterified fatty acids (nefa) greater than 1000 ~teq/liter and 325-400 ~teq/liter, respectively, are abnormal (gerloff and herdt, 1999) . triglyceride analysis of liver biposy specimens are useful. in affected cows, body fat is mobilized, in the form of nefa in response to the energy demands. hepatic lipidosis occurs rapidly as the nefa are converted into hepatic triglycerides. the ability of the liver to extract the albumin-bound nefa from the blood is better than that of other tissues that need and can also use nefa as an energy source. treatment for any concurrent diseases must be pursued aggressively, as well as measures to increase and stabilize blood glucose, decrease nefa production, and increase forestomach digestion to improve production of normally metabolized volatile fatty acids. therapeutic measures include intravenous glucose drips, insulin (nph or lente) injections every 12 hr, and transfaunation of ruminal fluid from a normal cow. prevention includes minimizing stress to lategestation cows. dry and lactating cows should be maintained separately; their energy, protein, and dry matter requirements are very different. cows with prolonged lactation or delayed breeding should be managed to prevent weight gain. i. bloat. bloat or tympanites refers to an excessive accumulation of gas in the rumen. the condition most frequently occurs in animals that have been recently fed abundant quantities of succulent forages or grains. bloat is classified into two broad categories: frothy bloat and free-gas bloat. frothy bloat is associated with ingestion of feeds that produce a stable froth that is not easily expelled from the rumen. fermentation gases such as co2, ch4, and minor gases such as n2, 02, h2, and h2s incorporate into the froth, overdistend the rumen, and eventu-ally compromise respiration by limiting diaphragm movement. the froth is often derived from a combination of salivary mucoproteins, protozoal or bacterial proteins, and proteins, pectins, saponins, or hemicellulose associated with ingested leaves or grain. typical foodstuffs that cause frothy bloat include green legumes, leguminous hay (alfalfa, clover), or grain (especially barley, corn, and soybean meal). free-gas bloat is less related to feeds ingested; rather, it is caused by rumen atony or by physical or pathological problems that prevent normal gas eructation. some examples of causes of free-gas bloat are esophageal obstructions (foreign bodies, tumors, abscesses, and enlarged cervical or thoracic lymph nodes), vagal nerve paralysis or injury, and central nervous system conditions that affect eructation reflexes. clinically, the animal will exhibit rumen distension, and tympany will be observed in the left paralumbar fossa. additional signs may include colic-like pain of the abdomen and dyspnea. passage of a stomach tube helps to differentiate between free-gas bloat and frothy bloat; and with free-gas bloat, expulsion of gas through the stomach tube aids in treatment of the disorder. once rumen distension is alleviated with free-gas bloat, the underlying cause must be investigated to prevent recurrence. frothy bloat is more difficult to treat, because the foam blocks the stomach tube. addition of mineral oil, household detergents, or antifermentative compounds via the tube may help break down the surface tension, allowing the gas to be expelled. in acute, life-threatening cases of bloat, treatment should be aimed at alleviating rumen distension by placing a trocar or surgical rumenotomy into the rumen via the paralumbar fossa. limiting the consumption of feedstuffs prone to induce bloat can prevent the disease. additionally, poloxalene or monensin will decrease the incidence of frothy bloat. ii. lactic acidosis. lactic acidosis, or rumen acidosis, is an acute metabolic disease caused by engorgement of grains or other highly fermentable carbohydrate sources. the disease is most frequently related to a rapid change in diet from one containing high roughage to one containing excessive carbohydrates. diet components that predispose to acidosis include common feed grains; feedstuffs such as sugar beets, molasses, and potatoes; by-products such as brewer's grains; and bakery products. biochemically, ingestion of large amounts of the carbohydrate-rich diet causes the normally gram-negative rumen bacterial populations to shift to gram-positive streptococcus and lactobacillus species. the gram-positive organisms efficiently convert the starches to lactic acid. the lactic acid acidifies the rumen contents, leading to rumen mucosal inflammation, and increases the osmolality of rumen fluids, leading to sequestration of fluids and osmotic attraction of plasma and tissue fluid to the rumen. lactic acid-induced rumenitis predisposes the animal to ulcers, to liver abscesses from "absorbed" bacterial pathogens, to laminitis from absorbed toxins, and to polioencephalomalacia from the inability of the new rumen bacterial populations to produce sufficient thiamine needed to maintain normal nervous system function. clinically, animals will become anorexic, depressed, and weak within 1-3 days after the initial insult. incoordination, ataxia, dehydration, hemoconcentration, rapid pulse and respiration, diarrhea, abdominal pain, and lameness will also be noted 9 rumen distension and an acetone-like odor to the breath, milk, or urine may also be observed. diagnosis is based on history and clinical signs. blood, urine, or milk ketones can be detected (moore and ishler, 1997) . additionally, rumen ph, which is normally above 6.0, will drop to less than 5.0 and in severe cases may achieve levels as low as 3.8. similarly, urine ph will become acidic, blood ph will drop below 7.4, and hematocrit will appear to increase due to the relative hemoconcentration. necropsy findings will be determined by secondary conditions. the primary lactic acidosis will cause swelling and necrosis of rumen papillae and abomasal hemorrhages and ulcers. treatment must be applied early in the syndrome. in early hours of severe carbohydrate engorgement, rumenotomy and evacuation of the contents are appropriate. the 9 t 9 patient should be given mineral oil and antlfermentatlves to prevent the continued conversion of starches to acids and the absorption of metabolic products. bicarbonate or other antacids like magnesium carbonate or magnesium hydroxide introduced into the rumen will aid in adjusting rumen ph. furthermore, animals can be given oral tetracycline or penicillin, which will decrease the gram-positive bacterial population. iii. rumen parakeratosis. parakeratosis is a degenerative condition of the rumen mucosa that leads to keratinization of the papillary epithelium 9 excessive and continuous feeding of diets low in roughage causes the mucosal changes 9 generally, this condition is seen in feedlot lambs and steers that are fed an all-grain diet. clinically, animals may exhibit only poor rates of gain, due to changes in the absorptive capacity of the injured mucosa. at necropsy, papillae will be thickened and rough. they will frequently be dark in color, and multiple papillae will clump together. abscessation may be observed. histopathologically, papilla surfaces will have hyperkeratinization of the squamous epithelium. chronic laminitis may be observed. however, diagnosis of parakeratosis is generally made at necropsy. feeding adequate roughage, such as stemmy hay, will prevent the disease. antibiotics may be administered to prevent secondary liver abscess formation. iv. rumenitis. rumenitis is an acute or chronic inflammation of the rumen, which occurs most commonly as a sequela to lactic acidosis 9 in addition to concentrate feeding, inadequate roughage in the diet is also associated with this disorder 9 rumenitis may occur with contagious ecthyma infection or following ingestion of poisons or other irritants. because rumenitis is often associated with lactic acidosis, it tends to occur in feedlot animals. the inflamed ruminal epithelium becomes necrotic and sloughs, creating ulcers. endogenous rumen bacteria such as fusobacterium necrophorum may invade the ulcers, penetrate the circulatory system, and induce abscesses of the liver. clinically, the animals will appear depressed and anorexic. rumen motility will be decreased, and animals will lose weight. the disease may resolve in a week to 10 days; mortality may reach 20%. necropsy lesions include rumen inflammation and ulcers in the anteroventral sac. granulation tissue and scarring may be observed following healing. rumenitis is not typically diagnosed clinically; thus, specific treatment is not commonly done. the disease can be prevented by minimizing the incidence of lactic acidosis. etiology. traumatic reticulitis-reticuloperitonitis is a disease of cattle related to their exploratory tendencies and ingestion of many different, nonvegetative materials. the disease is rarely seen in smaller ruminants. clinical signs. clinical signs range from asymptomatic to severe, depending on the penetration and damage by the foreign object after settling in the animal's forestomach. many signs during the early, acute stages will be nonspecific, ranging from arched back, listlessness, anorexia, fever, decrease in production, ketosis, regurgitation, decrease or cessation of ruminal contractions, bloat, tachypnea, tachycardia, and grunts when urinating, defecating, or being forced to move. the prognosis is poor when peritonitis becomes diffuse. sudden death can occur if the heart, coronary vessels, or other large vessels are punctured by the migrating object. epizootiology and transmission. this is a noncontagious disease. the occurrence is directly related to sharp or metallic indigestible items in the feed or environment that the cattle mouth and swallow. necropsy findings. in severe cases, necropsy findings include extensive inflammation throughout the cranial abdomen, malodorous peritoneal fluid accumulations, and lesions at the reticular sites of migration of the foreign objects. cardiac puncture will be present in those animals succumbing to sudden death. pathogenesis. consumed objects initially settle in the rumen but are dumped into the reticulum during the digestive process, and normal contraction may eventually lead to puncture of the reticular wall. this sets off a localized inflammation or a localized or more generalized peritonitis. the inflammation may also temporarily or permanently affect innervation of local tissues and organs. further damage may result from migration and penetration of the diaphragm, pericardium, and heart. diagnosis is based on clinical signs, knowledge of herd management techniques in terms of placement of forestomach magnets, and reflection of acute or chronic infection on the hemogram. radiographs and abdominocentesis may be useful. differential diagnosis. differentials include abomasal ulcers, hepatic ulcers, neoplasia (such as lymphosarcoma, usually in older animals, or intestinal carcinoma), laminitis, and cor pulmonale. infectious diseases that are differentials include systemic leptospirosis and internal parasitism. diseases causing sudden death may need to be considered. prevention and control. this problem can be prevented entirely by elimination of sharp objects in cattle feed and in the housing and pasture environments. adequately sized magnets placed in feed handling equipment and forestomach magnets (placed per os with a bailing gun in young stock at 6-8 months of age) are also significant prevention measures. treatment. provision of a forestomach magnet, confinement, and nursing care, including antibiotics, are the initial treatments. in severe cases, rumenotomy may be considered. etiology. pregnancy toxemia is a primary metabolic disease of ewes and does in advanced pregnancy. beef heifers are susceptible to protein energy malnutrition (pem) syndrome, which is also referred to as pregnancy toxemia. clinical signs. in sheep, this disease is characterized by hypoglycemia, ketonemia, ketonuria, weakness, and blindness. hypoglycemic and ketotic ewes begin to wander aimlessly and to move away from the flock. they become anorexic and act uncoordinated, frequently leaning against objects. advanced signs may include blindness, muscle tremors, teeth grinding, convulsions, and coma. body temperature, heart rate, respiratory rate, and rumen motility continue normally. up to 80% of infected ewes may die from the disease. the course of the disease may last up to a week. in goats, the disease usually occurs in the last 6 weeks of gestation, especially in does carrying triplets. pregnancy toxemia should be considered with any goat showing signs of illness in late gestation. the doe may separate herself from the herd, stagger, or circle and may appear blind. appetite is poor, and tremors may be evident. a rapid metabolic acidosis results in subsequent recumbency. urinalysis will readily reveal ketonuria. if fetal death occurs, acute toxemia and death of the doe may result. in beef heifers, weight loss and thin body condition, weakness and inability to stand, and depression are clinical signs. some cows develop diarrhea. because the catabolic state is often so advanced, most affected heifers die even if treated. pregnancy toxemia is diagnosed by evidence of typical clinical signs. sodium nitroprusside tablets or ketosis dipsticks may be used to identify ketones in the urine or plasma of ewes and does. blood glucose levels found to be below 25 mg/dl and ketonuria are good diagnostic indicators. in cattle, ketonuria is not a typical finding; hypocalcemia and anemia may be present. that are obese or bearing twins or triplets. the disease develops during the last 6 weeks of pregnancy. pem most frequently occurs in heifers during the final trimester of pregnancy. necropsy findings. at necropsy, affected ewes will often have multiple fetuses, which may have died and decomposed. the liver will be enlarged, yellow, and friable, with fatty degeneration. the adrenal gland may also be enlarged. in cattle, heifers will be very thin, and in addition to a fatty liver, signs of concurrent diseases may be present. pathogenesis. rapid fetal growth, a decline in maternal nutrition, and a voluntary decrease in food intake in overfat ewes result in an inadequate supply of glucose needed for both maternal and fetal tissues. the ewe develops a severe hypoglycemia in early stages of the disease. the ruminant absorbs little dietary glucose; rather, it produces and absorbs volatile fatty acids (acetic, propionic, and butyric acids) from consumed feedstuffs. propionic acid is absorbed and selectively converted to glucose through gluconeogenesis. when the animal is in a state of negative energy balance, it hydrolyzes fats to glycerol and fatty acids. glycerol is converted to glucose while the fatty acids are metabolized for energy. the oxidation of fatty acids in the face of declining oxaloacetate levels (required for normal krebs cycle function) results in the formation of ketone bodies (acetone, acetoacetic acid, and [3-hydroxybutyric acid), thus causing the condition ketoacidosis. heifer cattle have high energy requirements for completing normal body growth and supporting a pregnancy. additional energy requirements are needed during pregnancy for winter conditions and during concurrent diseases. marginal diets and poor-quality forage will place the cows in a negative energy balance. differential diagnosis. hypocalcemia is a common differential diagnosis. in cattle, differentials include chronic or untreated diseases such as johne's disease, lymphosarcoma, parasitism, and chronic respiratory diseases. prevention and control. pregnancy toxemia can be prevented by providing adequate nutrition during late gestation and by maintaining animals in appropriate nonfat condition during pregnancy. in late pregnancy, the dietary energy and protein should be increased 1.5-2 times the maintenance level. pem can be prevented by maintaining appropriate body condition earlier in pregnancy and supplying good-quality forage for the last trimester. treatment. in sheep, because the morbidity may be as high as 20%, treatment should be directed at the flock rather than the in-dividual. treating the individual is usually unsuccessful. oral administration of 200 ml of propylene glycol or 50% glucose twice a day, anabolic steroids, and high doses of adrenocorticosteroids may be helpful. if ewes are still responsive and not severely acidotic or in renal failure, cesarean section may be successful by rapidly removing the fetus, which is the dietary drain for the ewe. in goats, pregnancy toxemia is best treated by removal of the fetuses either by cesarean section or induction of parturition. parturition can be induced in does by either dexamethasone (10 mg) or pgf2a (10 ~tg). in addition, goats may be treated with 10% dextrose (100 to 200 ml iv) or propylene glycol (60 ml per os 2 or 3 times a day). adjunctive therapy includes normalizing acid base and hydration status, administration of vitamin b 12 and transfaunation. heifers may be force-fed alfalfa gruels, given propylene glycol per os, placed on iv 50% glucose drips, and treated for concurrent disease. research complications. in research requiring pregnant ewes in late stages of gestation, for example, this disease should be considered if the animals are likely to bear twins and will be transported or stressed in other ways during that time. f hypocalcemia (parturient paresis, milk fever) etiology. hypocalcemia is an acute metabolic disease of ruminants that requires emergency treatment; the presentation is slightly different in ewes, does, and cows. clinical signs and diagnosis. in sheep, the disease is seen in ewes during the last 6 weeks of pregnancy and is characterized by muscle tetany, incoordination, paralysis, and finally coma. as calcium levels drop, ewes begin to show early signs such as stiffness and incoordination of movements, especially in the hindlimbs. later, muscular tremors, muscular weakness, and recumbency will ensue. animals will frequently be found breathing rapidly despite a normal body temperature. morbidity may approach 30%, and mortality may reach as high as 90% in untreated animals. affected does become bloated, weak, unsteady, and eventually recumbent. cows are affected within 24-48 hr before or after parturition. cows initially are weak and show evidence of muscle tremors, then deteriorate to sternal recumbency, with the head usually tucked to the abdomen, and an inability to stand. tachycardia, dilated pupils, anorexia, hypothermia, depression, ruminal stasis, bloat, uterine inertia, and loss of anal tone are also seen at this stage. the terminal stage of disease is a rapid progression from coma to death. heart rates will be high, but pulse may not be detectable. hypocalcemia is diagnosed based on the pregnancy stage of the female and on clinical signs. it is later confirmed by laboratory findings of low serum calcium. with hypocalcemia in ewes, the plasma concentrations of calcium drop from normal values of 8-12 mg/dl to values of 3-6 mg/dl. in cattle, plasma levels below 7.5 mg/dl are hypocalcemic; at the terminal stages levels may be 2 mg/dl. ewes during the last 6 weeks of pregnancy or during the first few weeks of lactation. the disease is not as common in the dairy goat as in the dairy cow. high-producing, older, multiparous dairy cows are the most susceptible, and the jersey breed is considered susceptible. cows that have survived one episode are prone to recurrence. in addition, dry cows must be managed carefully regarding limiting dietary calcium. the disease is not common in beef cattle unless there is an overall poor nutrition program. ing at necropsy. there is no pathognomonic or typical find-pathogenesis. during the periparturient period, calcium requirements for fetal skeletal growth exceed calcium absorbed from the diet and from bone metabolism. additionally, dietary calcium intake is thought to be compromised because, in advanced pregnancy, animals may not be able to eat enough to sustain adequate nutrient levels, and intestinal absorption capabilities do not respond as quickly as needed. after parturition, calcium needs increase dramatically because of calcium levels in colostrum and milk. recent information suggests that legume and grass forages, high in potassium and low in magnesium, create a slight physiological alkalosis (at least in cattle), which antagonizes normal calcium regulation (rings et al., 1997) . thus, bone resorption, renal resorption, and gastrointestinal absorption of calcium are less than maximal. prevention and control. maintaining appropriate nutrition during the last trimester is helpful in preventing the disease. in cows and does, for example, limiting calcium intake by removing alfalfa from the diet is helpful. treatment. hypocalcemia must be treated quickly based on clinical signs; pretreatment blood samples can be saved for later confirmation. twenty percent calcium borogluconate solution should be administered by slow intravenous infusion. response will often be rapid, with the resolution of the animal's dull mentation. less severely affected animals will often try to stand in a short time. relapses are common, however, in sheep and cattle. hypermagnesemia and hypophosphatemia often coincide with hypocalcemia. these imbalances should be considered when animals appear to be unresponsive to treatment. hypocalcemia in the goat can be treated with 50-100 ml of calcium borogluconate. heart rate should be monitored closely throughout calcium administration. if an irregular or rapid heart rate is detected, then calcium treatment should be slowed or discontinued. calcium gels and boluses are also available for treatment (rings et al, 1997) . prognosis is generally good if the animal is treated early in the disease, but the prognosis will often be poor when treatment is initiated in later stages of the disease. etiology. urolithiasis is a metabolic disease of intact and castrated male sheep, goats, and cattle that is characterized by the formation of bladder and urethral crystals, urethral blockage, and anuria (murray, 1985) . the disease occurs rarely in female ruminants. clinical signs and diagnosis. affected animals will vocalize and begin to show signs of uneasiness, such as treading, straining postures, arched backs, raised tails, and squatting while attempting to urinate. these postures may be mistaken for tenesmus. male cattle may develop swelling along the ventral perineal area. affected animals will not stay with the herd or flock. small amounts of urine may be discharged, and crystal deposits may be visible attached to the preputial hairs. additionally, in smaller ruminants, the filiform urethral appendage (pizzle) often becomes dark purple to black in color. the pulsing pelvic urethra may be detected by manual or digital rectal palpation, and bladder distention may be noticeable in cattle by the same means. as the disease progresses to complete urethral blockage, the animal will become anorexic and show signs of abdominal pain, such as kicking at the belly. the abdomen will swell as the bladder enlarges, and rupture can occur within 36 hr after development of clinical signs. bladder or urethral rupture may cause a short-lived period of apparent pain relief; subsequent development of uremia will eventually lead to death. the disease may progress over a period of 1-2 weeks, and the mortality is high unless the blockages are reversed. diagnosis is made by the typical clinical signs. abdominal taps may yield urine. calculi are usually composed of calcium phosphate or ammonium phosphate matrices. clinical disease is usually seen in growing intact or castrated males. the disease may be sporadic or there may be clusters of cases in the flock or herd. necropsy findings. necropsy findings include urine in the abdomen with or without bladder or urethral rupture. renal hydronephrosis may be evident. calculi or struvite crystal sediment will be observed in the bladder and urethra. histologically, trauma to the urethra and ureters will be present. etary, anatomical, hormonal, and environmental factors. male sheep and goats have a urethral process that predisposes them to entrapment of calculi. in cattle, the urethra narrows at the sigmoid flexure, and calculi lodge there most frequently. additionally, the removal of testosterone by early castration is thought to result in hypoplasia of the urethra and penis. this physical reduction in the size of the excretory tube may predispose to the precipitation of and blockage by the struvite minerals. grains fed to growing animals tend to be high in phosphorus and magnesium content. these calculogenic diets lead to the formation of struvite (magnesium ammonium phosphate) crystals. other minerals associated with urolithiasis include silica (range grasses), carbonates (some grasses and clover pastures), calcium (exclusively alfalfa hay), and oxalates (fescue grasses). differential diagnosis. grain engorgement colic, gastrointestinal blockage, and causes of tenemus, such as enteritis or trauma, are differentials. trauma to the urethral process should be considered. urinary tract infections are uncommon in ruminants. prevention and control. one case often is indicative of a potential problem in the group. urolithiasis can be minimized by monitoring the calcium:phosphorus ratio in the diet. the normal ratio should be 2:1. additionally, increasing the amount of dietary roughage will help balance the mineral intake. increasing the amount of salt (sodium chloride, 2-4%) in the diet to increase water consumption, or adding ammonium chloride to the diet, at 10 gm/head/day or 2% of the ration, to acidify the urine, will aid in the prevention of this disease. palatability of and accessibility to water should be assessed as well as functioning of automatic watering equipment. treatment. treatment is primarily surgical (van metre et al. 1996) . initially, amputation of the filiform urethral appendage may alleviate the disease since urethral blockage often begins here. as the disease progresses, urethral blockage in the sigmoid flexure as well as throughout the urethra may occur. in more advanced stages, perineal urethrostomy may yield good results. the prognosis is poor when the condition becomes chronic, reoccurs, or surgery is required. research complications. young castrated and intact male ruminants used in the laboratory setting will be the susceptible age group for this disorder. rickets is a disease of young, growing animals but rarely occurs in goats. it is a metabolic disease characterized by a failure of bone matrix mineralization at the epiphysis of long bones due to lack of phosphorus. the condition can occur as an absolute deficiency in vitamin d2, an inadequate dietary supply of phosphorus, or a long-term dietary imbalance of calcium and phosphorus. the syndrome must be differentiated from epiphisitis (unequal growth of the epiphyses of long bones in young, rapidly growing kids fed diets with excess calcium). clinical signs include poor growth, enlarged costochondral junctions, narrow chests, painful joints, and reluctance to move. spontaneous fractures of long bones may occur. animals will recover when dietary phosphorus is provided and if joint damage is not severe. a. copper deficiency (enzootic ataxia, swayback) etiology. chronic copper deficiency in pregnant ewes and does may produce a metabolic disorder in their lambs and kids called enzootic ataxia. in goats, this deficiency also causes swayback in the fetuses. clinical signs and diagnosis. this disease results in a progressive hindlimb ataxia and apparent blindness in lambs up to about 3 months of age. additionally, because copper is essential for osteogenesis, hematopoiesis, myelination, and pigmentation of wool and hair, ewes may appear unthrifty, may be anemic, and may have poor, depigmented wool with a decrease in wool crimp. affected kids are born weak, tremble, and have a characteristic concavity to the spinal cord, leading to the name swayback. when the deficiency occurs later during gestation, demyelination is limited to the spinal cord and brain stem. kids are born normally but develop a progressive ataxia, leading to paralysis, muscle atrophy, and depressed spinal reflexes with lower motor neuron signs. diagnosis is based on low copper levels found in feedstuffs and tissues at necropsy. diagnosis is based on clinical signs, feed analysis, and pathological findings. epizootiology and transmission. enzootic ataxia is rarely seen in western states; most north american diets have sufficient copper levels to prevent this disease. copper antagonists in the feed or forage at sufficient levels, such as molybdenum, sulfate, and cadmium, however, may predispose to copper deficiencies. pathogenesis. the maternal copper deficiency leads to a disturbance early in the embryonic development of myelination in the central nervous system and the spinal cord. copper is part of the cytochrome oxidase system and other enzyme complexes and is important in myelination, osteogenesis, hematopoiesis (iron absorption and hemoglobin formation), immune system development, and maintenance and normal growth (smith and sherman, 1994) . differential diagnosis. the differential diagnosis for newborns includes [3-mannosidosis, hypoglycemia, and hypothermia. for older animals the differential should include caprine arthritis encephalitis (goats), enzootic muscular dystrophy, listeriosis, spinal trauma or abscessation, and cerebrospinal nematodiasis. prevention and control. copper deficiency can be prevented by providing balanced nutrition for pregnant animals. necropsy findings. gross encephalomalacia has been noted. histopathologically, white matter of the brain and spinal cord displays gelatinization and cavitation. extensive nerve demyelination and necrosis are evident. postmortem lesions include extensive demyelination and neuronal degeneration. treatment. because the condition is developmental, supplemental copper may improve clinical signs but not eliminate them. necropsy findings. common findings at necropsy include icterus; a soft, dark, friable, enlarged spleen; an enlarged, yellow-brown friable liver; and "gun-barrel" black kidneys. hemoglobin-stained urine will be visible in the bladder. copper accumulations in the liver reaching 1000-3000 ppm are toxic. pathogenesis. hemolysis occurs when sufficient amounts of copper are ingested or released suddenly from the liver and is believed to be due direct interaction of the copper with red-cell surface molecules. stresses such as transportation, lactation, and poor nutrition or exercise may precipitate the hemolysis. etiology: acute or chronic copper ingestion or liver injury often causes a severe, acute hemolytic anemia in weanling to adult sheep and in calves and adult dairy cattle. growing lambs may be the most susceptible. copper toxicosis is rare in goats. differential diagnosis. other causes of hemolytic disease include babesiosis, trypanosomiasis, and plant poisonings such as kale. arsenic ingestion, organophosphate toxicity, and cyanide or nitrate poisoning should also be considered as the source of poisoning. urethral obstruction and gastrointestinal emergencies should be considered for the abdominal pain. clinical signs and diagnosis. the clinical course in sheep can be as short as 1-4 days, and mortality may reach 75%. hemolysis, anemia, hemoglobinuria, and icterus characterize the acute hemolytic crisis, associated with copper released from the overloaded liver. some clinical signs are related to direct irritation to the gastrointestinal tract mucosa. weakness, vomiting, abdominal pain, bruxism, diarrhea, respiratory difficulty, and circulatory collapse are followed by recumbency and death. hepatic biopsy is currently considered the best diagnostic approach; serum or plasma levels of copper and hepatic enzymes such as aspartate aminotransferase (ast) and y-glutamyltransferase (ggt) may provide some information, but it is generally believed that these will not accurately reflect total copper load or hepatic damage. and goats is the range of 20-100 mg/kg, and for cattle it is 220-880 mg/kg. chronic poisoning in sheep may occur when 3.5 mg/kg is ingested. copper-containing pesticides, soil additives, therapeutics, and improperly formulated feeds may potentially lead to copper toxicity. phytogenous sources include certain pastures such as subterranean clover. feed low in molybdenum, zinc, or calcium may lead to increased uptake of copper from properly balanced rations. a common cause of the disease in sheep is feeding concentrates balanced for cattle; cattle feeds and mineral blocks contain much higher quantities of copper than are required for sheep. chronic ingestion of these feedstuffs leads to copper accumulation and toxicity. copper toxicosis has been reported in calves given regular oral or parenteral copper supplements, and in adult dairy cattle given copper supplements to compensate for copper-deficient pasture. pregnant dairy cattle may be more susceptible to copper toxicity. rare sources of copper ingestion may include copper sulfate footbaths. control and prevention. the disease is prevented by carefully monitoring copper access in sheep and copper supplementation in cattle. sheep and goats should not be fed feedstuffs formulated for cattle, and dairy calf milk replacer should not be used for lambs and kids. molybdenum may be administered to animals considered at high risk. molybdenum-deficient pastures may be treated with molybdenum superphosphate. herd copper supplementation should be undertaken with the knowledge of existing hepatic copper levels, and existing copper and molybdenum levels, in the feedstuffs. treatment. oral treatment for sheep consists of ammonium or sodium molybdenate (50-100 mg/day), and sodium thiosulfate (0.5-1.0 mg/day) for 3 weeks aids in excretion of copper. oral d-penicillamine daily for 6 days (50 mg/kg) has also been shown to increase copper excretion in sheep. ammonium molybdenate has been administered intravenously to goats at 1.7 mg/kg for 3 treatments on alternate days. cattle have been treated orally with sodium molybdenate (3 gm/day) or sodium thiosulfate (5 gm/day). treatment for anemia and nephrosis may be necessary in severe cases. merino crosses and the british breeds, may be more susceptible to copper toxicosis caused by phytogenous sources. (nutritional muscular dystrophy, nutritional myodegeneration, white muscle disease, stiff lamb disease) etiology. white muscle disease, also known as stiff lamb disease, is a nutritional muscular dystrophy caused by a deficiency of selenium or vitamin e. clinical signs and diagnosis. clinically two forms of the disease have been identified: cardiac and skeletal. the cardiac form occurs most commonly in neonates. in these, respiratory difficulty will be a manifestation of damage to cardiac, diaphragmatic, and intercostal muscles. young will be able to nurse when assisted. in slightly older animals, the disease is characterized by locomotor disturbances and/or circulatory failure. clinically, animals may display paresis, stiffness or inability to stand, rapid but weak pulse, and acute death. mortality may reach 70% (jensen and swift, 1982) . paresis and sudden death in neonates with associated pathological signs are frequently diagnostic. with the skeletal form, affected animals are stiff and reluctant to move, and muscles of affected animals are painful. young will be reluctant to get up but will readily nurse when assisted. peracute to acute myocardial degeneration may occur in the cardiac form, and animals may simply be found dead. serum selenium levels are usually below 50 ppb (normal is 158-160 ppb) (nelson, 1983) . diagnosis may also include determination of antemortem whole blood levels of selenium and plasma levels of vitamin e. glutathione peroxidase levels in red blood cells can be measured as an indirect test. clinical biochemistry findings of significant elevations of aspartate aminotransferase (ast) in creatinine kinase (ck) are also supportive of the diagnosis. epizootiology and transmission. selenium deficiency has been associated with formulated diets deficient in selenium, forages grown on selenium-deficient soils in certain geographic regions, and forages such as alfalfa and clover that have an inability to efficiently extract available selenium from the soils. rumen bacterial reduction of selenium compounds to unavailable elemental selenium may also contribute to the disease. necropsy findings. necropsy lesions include petechial hemorrhages and muscle edema. hallmarks are pale white streaking of affected skeletal and cardiac muscle. these are due to coagulation necrosis. pale striated muscles of the limb, diaphragm, and tongue are also seen. antioxidants that protect lipid membranes from oxidative destruction. selenium is a cofactor for glutathione peroxidase, which converts hydrogen peroxide to water and other nontoxic compounds. lack of one or both results in loss of membrane integrity. differential diagnosis. in neonatal ruminants presenting with respiratory and cardiac dysfunction, differentials include congenital cardiac anomalies. differentials generally for weak neonates or sudden or peracute neonatal deaths should include septicemia, pneumonia, toxicity, diarrhea, and dehydration. prevention and control. awareness of regional selenium deficiencies is important. control involves providing good-quality roughage, vitamin e and selenium supplementation, and parenteral injections prior to parturition and weaning. treatment. affected animals may be treated by administering vitamin e or selenium injections. administering vitamin e or selenium to ewes in late pregnancy can prevent white muscle disease (kott et al., 1998) . the label dose for selenium is 2.5-3 mg/45 kg of body weight. combination products are available and can be used in goats at the sheep dose (smith and sherman, 1994) . proper mineral balance in the diet is critical. selenium toxicity occurs most frequently as the result of excessive dosing to prevent or correct selenium deficiency or as the result of ingestion of selenium-converting plants. the main preventive measure for the former is the use of the appropriate product for the species. secondarily, the concentration of the available product should be double-checked. in the united states, ruminants in the midwest and western areas may be subject to selenium toxicity when pastured in areas containing selenium-converting plants. signs of overdosing include weakness, dyspnea, bloating, and diarrhea. shock, paresis, and death may occur. initial clinical signs of excessive selenium intake from plants are observed in the distal limb, with cracked hoof walls and subsequent infection and irregular hoof growth. etiology. polioencephalomalacia (pem) is a noninfectious, noncontagious disease characterized by neurological signs. growing and adult ruminants on high-concentrate diets are typically affected. animals exposed to toxic plants or moldy feed containing thiaminases, feed high in sulfates, or unusually high doses of some medications are also at risk. clinical signs and diagnosis. an early sign may be mild diarrhea. acute clinical signs include bruxism, hyperesthesia, involuntary muscle contractions, depression, partial or complete opisthotonus, nystagmus, dorsomedial strabismus, seizures, and death. in subacute cases of the disease, animals may appear to walk aimlessly as if blind or may display head-pressing postures. hypersalivation may be present, but body temperatures and ocular reflexes are normal. morbidity and mortality may be high, especially in younger animals. diagnosis is suggestive from clinical signs and from response to intensive parental thiamine hydrochloride. epizootiology and transmission. pem is caused by a thiamin deficiency. the disease tends to be seen more frequently in cattle and sheep feedlots where the concentrates fed are high in fermentable carbohydrates. pastured animals are also vulnerable if grain is feed. thiaminase-containing plants, such as bracken fern, are often unpalatable so will less likely be a contributing factor. recent studies have also indicated that high levels of sulfate in the diet, such as in the fermentable, low-fiber concentrates, may play an important role. medications such as as amprolium, levamisole, and thiabendazole have thiaminantagonizing activity when given in excessive doses. sherman, 1994) . vitamin a deficiencies associated with hyperkeratosis have been reported, as well as vitamin e-responsive and selenium-responsive dermatitis. necropsy signs. cerebral lesions characterized by softening and discoloration are grossly observed in the gray matter. microscopically, neurons will exhibit edema, chromatolysis, and shrinkage. gliosis and cerebral capillary proliferation may be observed. a lack of thiamin results in inappropriate carbohydrate metabolism and accumulation of pyruvate and other intermediaries that lead to cerebral edema and neuronal degeneration. differential diagnosis. several important differentials include acute lead poisoning, nitrofuran toxicity, hypomagnesemia, vitamin a deficiency, listeriosis, pregnancy toxemia, infectious thromboembolic meningoencephalitis, and type d clostridial enterotoxemia. prevention and control. the disease can be prevented by monitoring the diet and by providing adequate roughage necessary to prevent overgrowth of thiaminase-producing ruminal flora and to maximize ruminal production of b vitamins. if excess sulfur is the primary factor, immediate removal of the source is critical. neonatal ruminants are born without immunoglobulins and must receive colostrum by 24 hr after birth. the morbidity and mortality associated with failure of or inadequate passive transfer, such as enteric and respiratory illnesses, can be severe. measures to assure passive immunity for neonatal ruminants are covered in section ii,b,5, and clinical signs of illness associated with lack of immunity are addressed in the discussions of bacterial diseases (e.g., escherichia coli infections) and, of viral diseases (e.g., diarrheas) in section iii,a,1 and iii,a,2. generally, transfer of less than 600 mg/dl of immunoglobulins in the serum is classified as failure of transfer, 600-1600 mg/dl is partial, and above 1600 mg/dl is complete transfer. methods to determine success of transfer should be performed within a week of birth and include single radial immunodiffusion (quantitates immunogloblin classes); zinc sulfate turbidity (semiquantitative); sodium sulfite precipitation (semiquantitative); glutaraldehyde coagulation (coagulates above specific level); and, y-glutamyltransferase (assays enzyme in high concentration in colostrum and absorbed simultaneously with colostrum). treatment. early aggressive treatment is essential to save animals. the disease is treated by frequent parenteral administration of thiamine hydrochloride, the first dose being administered intravenously. dexamethasone, b vitamins, and diazepam may also be required. treatment is less successful when sulfur plays a prominent role in the etiology. research complications. this disease is preventable. although the disease is less likely to occur in smaller groups of confined ruminants, the risks of feeding concentrates or moldy feed, for example, with minimal good-quality roughage, should be kept in mind. vitamin d toxicity can result either from iatrogenic overadministration or ingestion of the plant trisetum flavescens. serum calcium levels may be high enough that blood in edta tubes will clot. laminitis is common in ruminants and can be caused by sudden changes in diet, excess dietary energy, and grain overload (or overeating). laminitis is also associated with mastitis and metritis. facility conditions, such as concrete flooring, poor manure management, and inadequate resting areas may also contribute to the pathogenesis of the disease. the complete pathogenesis of laminitis is poorly understood; however, it is thought that changes in the diet cause changes in rumen microbial populations, resulting in acidosis and endotoxemia. dramatic changes in the vascular endothelium result in chronic inflammation of the sensitive laminae of the hoof, separation of corium and hoof wall, and rotation of the third phalanx. affected animals may be reluctant to get up or walk, will shift their weight frequently, and will grind teeth or walk on carpi. chronically, the hoof wall takes on a "slipper" appearance. treatment consists of identifying the underlying cause, administering antiinflammatories (phenylbutazone, flunixin meglumin), feeding good-quality forages only, and regular foot trimming. in goats, nutritional deficiencies often manifest as a generalized poor coat that is dry, scaly, thin, and erectile. zincresponsive dermatitis has been reported in goats (smith and otherwise normal, well-managed lambs, kids, and calves can develop loose, pasty feces due to a nutritional imbalance caused by overfeeding and/or improper mixing of milk replacers. only milk replacer formulated for the particular species should be used. once nutritional imbalances are corrected, the feces readily return to normal. sudden changes in diet can also result in loose feces. photosensitization is an acute dermatitis associated with an interaction between photosensitive chemicals and sunlight. the photosensitive chemicals are usually ingested, but in some cases exposure may be by contact. animals with a lack of pigment are more susceptible to the disease. three types of photosensitization occur: primary; secondary, or hepatogenous; and aberrant. primary photosensitization is related to uncommon plant pigments or to drugs such as phenothiazine, sulfonamides, or tetracyclines. secondary photosensitization is more common in large animals and is specifically related to the plant pigment phylloerythrin. phylloerythrin, a porphyrin compound, is a degradation product of chlorophyll released by rumen microbial digestion. liver disease or injury, which prevents normal conjugation of phylloerythrin and excretion through the biliary system, predisposes to photosensitization. the only example of aberrant photosensitization is congenital porphyria of cattle (see section iii,b,1). pathologically, the photosensitive chemical is deposited in the skin and is activated by absorbed sunlight. the activated pigments transfer their energy to local proteins and amino acids, which, in the presence of oxygen, are converted to vasoactive substances. the vasoactive substances increase the permeability of capillaries, leading to fluid and plasma protein losses and eventually to local tissue necrosis. photosensitization can occur within hours to days after sun exposure and produces lesions of the face, vulva, and coronary bands; lesions are most likely to occur on white-haired areas. initially, edema of the lips, corneas, eyelids, nasal planum, face, vulva, or coronary bands occurs. the facial edema, nostril constriction, and swollen lips potentially lead to difficulty in breathing. with secondary photosensitization, icterus is also common. necrosis and gangrene may occur. diagnosis is based on clinical lesions and exposure to the photosensitive chemi-cals and sunlight. treatment is symptomatic. the prognosis for hepatogenous type may be guarded if hepatic disease is severe. from excessive straining associated with dysuria from the pressure of the fetuses and/or abdominal contents on the bladder. if the prolapse obstructs subsequent urination, rupture of the bladder may occur. the vaginal prolapse can be reduced and repaired if discovered early, and techniques in small and large ruminants are comparable. the animal should be restrained, and the prolapsed tissue should be cleansed with disinfectants. best done under epidural anesthesia, the vagina is replaced into the pelvic canal and the vulvar or vestibular opening is sutured closed (buhner suture). alternatively, a commercial device called a bearing retainer (or truss) can be placed into the reduced vagina and tied to the wool, thereby holding the vagina in proper orientation without interfering with subsequent lambing. vaginal prolapses may have a hereditary basis in ewes and cows and may prolapse the following year. these animals should be culled. vaginal prolapses may occur in nonpregnant animals that graze estrogenic plants or as a sequela to docking the tail too close to the body (ross, 1989) . uterine prolapses occur sporadically in postpartum ewes and cattle. the gravid horn invaginates after delivery and protrudes from the vulva. the cause is unknown, but excessive traction utilized to correct dystocia or retained placenta, uterine atony, hypocalcemia, and overconditioning or lack of exercise have been implicated. in cattle, the uterine prolapses usually develop within 1 week of calving, are more common in dairy cows than in beef cows, and are often associated with dystocia or hypocalcemia. cows may also have concurrent parturient paresis. initially, the tissue will appear normal, but edema and environmental contamination or injuries of the tissue develop quickly. clinical signs will include increased pulse and respiratory rates, straining, restlessness, and anorexia. if identified early, the uterus can be replaced as for vaginal prolapses. electrolyte imbalances should be corrected if present. additional supportive therapy, including the use of antibiotics should always be considered. tetanus prophylaxis should be included. oxytocin should be administered to induce uterine reduction. vaginal closures are less successful at retaining uterine prolapses. preventive and control measures include regular exercise for breeding animals, and management of prepartum nutrition and body condition. vaginal and uterine prolapses occur in ewes, does, and cows. the conditions are not common in does. vaginal prolapses usually occur during late gestation and may be related to relaxation of the pelvic ligaments in response to hormone levels. in sheep, these are most common in overconditioned ewes that are also carrying twins or triplets. overconsumption of roughages, which distends the rumen, and lack of exercise leading to intraabdominal fat may predispose an animal to vaginal prolapse by increasing intra-abdominal pressure. the condition may result f rectal prolapse rectal prolapse is common in growing, weaned lambs and in cattle from 6 months to 2 years old. the physical eversion of the rectum through the anal sphincter is usually secondary to other diseases or management-related circumstances. rectal prolapses may occur secondary to gastrointestinal infection or inflammation, especially when the colon is involved. diseases that cause tenesmus, such as coccidiosis, salmonellosis, and intestinal worms, may result in prolapse. urolithiasis may result in prolapses as the animal strains to urinate. any form of cystitis or urethritis, vaginal irritation, or vaginal prolapse and some forms of hepatic disease may lead to rectal prolapse. abdominal enlargement related to advanced stages of pregnancy, excessive rumen filling or bloat, and overconditioning may cause prolapse. finally, excessive coughing during respiratory tract infections, improper tail docking (too short), growth implants, prolonged recumbency, or overcrowded housing with animal piling may lead to prolapses. diagnosis is based on clinical signs. early prolapses may be corrected by holding the animal with the head down, while a colleague places a pursestring suture around the anus. the mucosa and underlying tissue of prolapses that have been present for longer periods of time will often become necrotic, dry, friable, and devitalized and will require surgical amputation or the placement of prolapse rings to remove the tissue. rectal prolapse may also be accompanied by intestinal intussusceptions that will further complicate the treatment and increase mortality. occasionally, acute rectal prolapse with evisceration will result in shock and prompt death of the animal. prognosis depends on the cause and extent of the prolapse as well as the timeliness of intervention. in all cases of treatment, determination and elimination of the underlying cause are essential. gastrointestinal accumulations or obstructions of hair (and/ or sometimes very coarse roughage, forming bezoars) occur in cattle and sheep. cattle that are maintained on a low-roughage diet, that lick their coats frequently, that have long hair coats from outdoor housing, or that have heavy lice or mite infestations and associated pruritus will often develop bezoars. in addition, younger calves with abomasal ulcers have been found to be more likely to have abomasal tric. hobezoars as well. clinical signs may be mild or severe according to size, number, and location. ruminal trichobezoars rarely result in clinical signs. obstruction will be accompanied by signs of pain, development of bloat, and decreased fecal production. serum profiles will show hypochloridemia; other imbalances depend on the duration of the problem. diagnosis is also based on abdominal auscultation, rectal palpation, and ultrasound (useful in calves and smaller ruminants). treatment is surgical, such as paracostal laparotomy (for abomasal), paralumbar celiotomy with manual breakdown, or enterotomy. supportive care should be administered as necessary to correct electrolyte imbalances and to prevent inflammation and sepsis. prognosis is generally good if the condition is diagnosed and treated before dehydration and imbalances become severe and peritonitis develops. prevention includes providing good-quality roughage and treating lice and mange infestations. wounds may be sustained from poorly constructed pens or fences, or from skirmishes among animals. predators will usu-ally be sources of bite wounds. standard veterinary wound assessment and care are essential for wounds or bites. tetanus antitoxin may be indicated. use of approved antibiotics may be appropriate. the lesion should be cleaned with disinfectants and repaired with primary closure if it is clean and uncontaminated. thorough cleaning, regular monitoring, and healing by second intention are recommended for older wounds. abscesses may also occur in the soft tissues of the hooves (sole abscesses; see section iii,c,3) because of entrapped foreign bodies or hoof cracks that fill with dirt. preventive measures include improvement of housing facilities, pens, and pastures; monitoring hierarchies among animals penned together; and implementing predator control measures, such as sound fencing, flock guard dogs, or donkeys, in pasture situations. acute anaphylatic reactions in sheep, goats, and cattle are often clinically referable to the respiratory system. anaphylactic vaccine reactions cause acute lung edema; lungs are the primary site of lesions if collapse and death are sequelae. the animals will also be anxious and shivering and will become hyperthermic. salivation, diarrhea, and bloat also occur. immediate therapy must include epinephrine by intravenous infusion at (1 ml of 1:1000 per 50 kg of body weight for goats and 1:10,000 (0.1 mg/ml) or 0.01 mg/kg (about 5 ml) for adult cows.) furosemide (5 mg/kg) may be beneficial to reduce edema. prognosis is usually guarded. recovery can occur within 2 hr. in a research environment, catheter sites or experimental surgeries may be sources of iatrogenic infection. traumatic injuries to peripheral nerves can cause acute lameness. improper administration of therapeutics can easily cause this type of lameness. injections given in gluteals or between the semimembranosus and semitendinosus can cause irritation to the sciatic nerve and subsequent lameness. contraction of the quadriceps results in the limb being pulled forward. injections in the caudal thigh can damage the peroneal nerve and cause knuckling at the fetlock. traumatic injury to the radial nerve can result in a "dropped elbow" (nelson, 1983) . husbandry procedures such as tail docking, castration, dehorning, dosing with a bailing gun, and shearing may result in superficial lesions, dermal infections, or cases of tetanus. bailing-gun injuries to the pharynx may lead to cellulitis with coughing, decreased appetite, and sensitivity to palpation. standard veterinary assessment and care are essential for these cases. local and systemic antibiotics with supportive care may be indicated. swelling around peripheral nerves caused by inoculations may be reduced by diuretics and anti-inflammato-ries. mild cases of peripheral nerve damage may recover in 7-14 days. personnel training, including review of relevant anatomy, preprocedure preparation, appropriate technique, careful surgical site preparation, rigorous instrument sanitation, and sterile technique will minimize the incidence of potential complications from surgical procedures. albumin values and foaming urine. the proteinuria also distinguishes amyloidosis (and glomerulonephritis) from other causes of weight loss and diarrhea in cattle such as johne's disease, parasitism, copper deficiency, salmonellosis, and bovine viral diarrhea virus infection. prognosis is poor, and no treatment is reported. neoplasia and tumors are relatively rare in ruminants. lymphosarcoma/leukemia in sheep has been shown to result from infection by a virus related (or identical) to the bovine leukemia virus. pulmonary carcinoma (pulmonary adenomatosis) and hepatic tumors are found in sheep. virus-induced papillomatosis (warts), discussed in section iii,a,2,s, and squamous cell carcinomas have also been reported in sheep. in goats, thymoma is one of the two most common neoplasias reported, although no distinct clinical syndrome has been described. cutaneous papillomas are the most common skin and udder tumor of goats, and although outbreaks involve multiple animals, no wart virus has been identified. persistent udder papillomas may progress to squamous cell carcinoma. lymphosarcoma is reported rarely in goats. although adrenocortical adenomas have been reported frequently and almost exclusively in older wethers, no clinical condition has been described. lymphosarcoma of various organ systems and "cancer eye" (bovine ocular squamous cell carcinoma, or oscc) are the most commonly reported cancers in cattle. lymphosarcoma is described in section iii,a,2,c. lack of periocular pigmentation and the amount and intensity of exposure to solar ultraviolet light are considered important factors in oscc. genetic factors may also play a role. many cases occur in herefords. this is a disease of older cattle; no case has been reported in animals less than 4 years of age. the cancer metastasizes through the lymph system to major organs. treatment in either lymphosarcoma or oscc is recommended only as a palliative measure. the extent of ocular neoplastic involvement is a significant criterion for carcass condemnation. papillomatosis (warts) are common in cattle (see section iii,a,2,s). dental wear is seen most commonly in sheep. as sheep age, excessive dental wear may lead to an inability to properly masticate feed, manifesting as weight loss and unthriftiness. several factors predisposing to dental wear should be considered. the diet should be properly balanced for minerals, especially calcium and phosphorus, because primary or secondary calcium deficiency during teeth development results in softening of the enamel and dentin. dietary contamination with silica (i.e., hays and grains harvested in sandy regions) will lead to mechanical wear on the teeth. likewise, animals grazing or being fed in sandy environments will have excessive tooth wear. sheep older than about 5 years of age are especially prone to tooth wear and should be checked frequently, especially if signs of weight loss or malnutrition are evident. managing the content and consistency of the diets can best prevent the disease. of the ruminants, cows are the most frequently affected by subsolar absesses. dirt becomes packed into cracks in the horny layer of the sole of the hoof, and contamination eventually extends into the sensitive areas of the hoof, with lameness and infection resulting. animals maintained in very soiled or muddy conditions, combined with poor hoof care, are more likely affected. fusobacterium necrophorum is often the pathogen involved. separation of the animal, supportive care, surgical drainage, and antibiotic treatment are indicated. amyloidosis amyloidosis in adult cattle is due to accumulations of amyloid protein in the kidney, liver, adrenal glands, and gastrointestinal tract. the disease has been classified as aa type, or associated with chronic inflammatory disease, although other unknown factors are believed to be involved in some cases. clinical signs include chronic diarrhea, weight loss, decreased production, nonpainful renomegaly, and generalized edema. the loss of protein in the urine contributes to abnormal plasma advances in sheep and goat medicine animals and animal products, subchapter a, animal welfare formulary for laboratory animals domestic animal behavior for veterinarians and animal scientists schlam's veterinary hematology diseases of sheep animal feeding and nutrition guide for the care and use of 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sheep ovine listeric encephalitis coccidiosis in ruminants principles of dairy science human factor ix transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts the major histocompatibility complex region of domestic animal species clinical reproductive anatomy and physiology of the doe immunobiology of the mammary gland coccidiosis brucella abortus strain rb51: a new brucellosis vaccine for cattle use of age and serum gamma-glutamyltransferase activity to assess passive transfer status in lambs effect of congenitally acquired neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle artificial control of breeding in ewes toxoplasmosis infection in sheep bovine reproductive biotechnology transgenic dairy cattle. genetic engineering on a large scale the effect of intra-mammary inoculation of lactating ewes with pasteurella haemolytica isolates from different sources bovine surgery and lameness reduction of myocardial myoglobin in bovine dilated cardiomyopathy intraosseous infusion of prostaglandin e2 prevents disuse-induced bone loss in the tibia estrous cycle synchronization the bronchopneumonias (respiratory disease complex of cattle, sheep, and goats) the cattle gene map treatment and control of gastrointestinal nematodes in sheep diagnosis, treatment, and management of enteric colibacillosis key: cord-018651-phb44k66 authors: hammoud, dima a. title: neuroimaging date: 2017-05-26 journal: imaging infections doi: 10.1007/978-3-319-54592-9_8 sha: doc_id: 18651 cord_uid: phb44k66 imaging of infection in the cns has been handled using cross-sectional imaging for more than two decades now resulting in a large array of descriptive diagnostic criteria, capable, in most circumstances of narrowing the differential diagnosis, detecting life-threatening complications and establishing baseline for assessment of treatment response. limitations however exist, and in many circumstances, both cross-sectional imaging and nonspecific molecular imaging, such as (18)f-fdg, fail to establish a diagnosis. the availability of pathogen-specific imaging agents/ligands would have a great effect on the management of patients with cns infection. besides early diagnosis, avoidance of diagnostic brain biopsies can have significant effect on the mortality and morbidity of patients. to infection such as activation of microglia or other components of the immune system. among those approaches, direct targeting of the pathogen is probably of most clinical significance and would be most interesting to translate. targeting the pathogen is limited by lack of pathogen-specific imaging ligands. another limitation inherent to the cns is the presence of the blood-brain barrier limiting brain access to few molecules with special characteristics (e.g., small size, appropriate lipophilicity). even though the bbb can be disrupted in infectious processes, this is not always the case. in fact, with indolent less aggressive pathogens, the bbb can remain intact or minimally breached. in theory, directly imaging the infectious agent can be performed using ligands incorporated only by the pathogen but not by eukaryotic cells or through targeting of pathogen enzymes with labeled substrates. one example of the latter approach is imaging herpetic (hsv-1) encephalitis using 18 f-fhpg, a fluorinated derivative of ganciclovir, which is selectively phosphorylated by the hsv thymidine kinase (tk) enzyme and subsequently trapped within infected cells [1] . selective uptake of the ligand is then visualized using pet imaging of rats that received intranasal injection of hsv-1 compared to sham animals. this approach however is limited to pathogens that possess the tk enzyme, but more importantly by the inability of tk substrates such as fhpg, fhbg, and fiau to cross an intact bbb. even when the bbb is disrupted, the substrate can "leak" resulting in nonspecific accumulation. a well-designed experiment by buursma et al. used a freezing injury model causing sterile disruption of the bbb; even though 18 f-fhpg accumulated initially, it washed out quickly suggesting that the fhpg uptake in actual infections is due to irreversible trapping of the ligand in phosphorylated form within the infected cells [1] . , depends on its selective uptake by bacteria, in this case, the enterobacteriaceae family of gram-negative bacteria. sorbitol is not taken by eukaryotic cells and was found to be readily incorporated in gram-negative but not gram-positive bacterial cells [2] . 18 f-fds was previously synthesized through chemical reduction of the widely available ligand, 18 f-2-fluoro-deoxy-d-glucose ( 18 f-fdg) [3] . even though originally designed to image brain tumors [3] , a significantly higher uptake of 18 f-fds was seen in the e. coli-infected brain versus brain tumor or normal brain, suggesting specific uptake and retention by the bacteria (figure 8 .2) [2] . this is rendered even more sensitive by the demonstrated lack of background uptake in the normal brain. even though fds can only detect infections due to the enterobacteriaceae family, it can be greatly used in the right clinical setting, providing localization of the infection, differentiation of active infection from host inflammatory reaction, and evaluation of response to treatment. development of pathogen-specific ligands with wider detection range and low background uptake is the ultimate goal. much more commonly seen than direct pathogen detection in the brain is molecular imaging using genetically modified pathogens that are traceable through a reporter gene mechanism. a common approach is the use of recombinant viruses engineered to express a luciferase enzyme which allows performing longitudinal imaging, accurately determining the site(s) of infection, describing the temporal systemic dissemination of the virus, and eventually quantifying viral titers in various organs. a major limitation however is the inherent potential for attenuation of viral replication and/or loss of virulence of the pathogen [4] [5] [6] [7] [8] . both latter factors thus need to be carefully evaluated prior to starting imaging experiments. early examples coli-infected brain (top row), normal brain (middle row), and brain tumor tissues (lower row). uptake of the ligand is only noted in association with gram-negative infection suggesting specific uptake by the pathogen. adapted from weinstein and ordonez et al. [2] included developing recombinant viruses that expressed various luciferase reporter proteins such as hsv-1 [6] , vaccinia virus [9] , and the mouse hepatitis virus (mhv), a member of the coronavirus family [10] . an interesting finding in the paper by raaben et al. is the detection of the protective role of type i interferon in mhv infection, with widespread viral dissemination, elucidated through bioluminescence, seen in mice lacking a functional type i interferon response and reversal of the process through the administration of type i interferons to the mice [10] . this is an example of molecular imaging providing insight into potential therapeutic applications. more recent examples include the use of recombinant murine gammaherpesvirus 68 (mhv-68) expressing the firefly luciferase (fluc) to monitor virus progression after cns infection [11] and of recombinant dengue virus, for realtime evaluation of replication kinetics in the brain of infected mice [12] . western equine encephalitis virus (weev; alphavirus), a mosquito-borne virus that can cause severe encephalitis in humans, was also evaluated in a recombinant form (expressing fluc) in intranasally infected mice [13] . the genetically engineered reporter gene approach however is not limited to viruses and has been used with other pathogens such as parasites. a genetically modified and fully virulent african il1392 trypanosoma vivax strain that stably expresses fluc was monitored in vivo in the brain of infected immunocompetent mice. light emitted by the brain increased slightly up till day 20 post-infection (<400-fold the background) then rose substantially by day 25, reaching up to 2000fold the background, thus providing evidence of parenchymal infiltration [14] . other examples of reporter gene use include imaging of toxoplasma gondii encephalitis with spatiotemporal demonstration of recrudescence of the infection from the cns of immunocompromised mice [15] . virulent recombinant pathogens can also be used to assess responsiveness to treatment. for example, a streptococcus pneumoniae strain modified by the integration of a luminescent lux operon was used to determine responsiveness of the infection to daptomycin, an alternative antibiotic, eventually found to be as effective as vancomycin [16] . similarly, the use of luciferase-expressing trypanosoma brucei brucei allowed the identification of pathogen entry into the brain (using multiphoton microscopy) in early stages and determination of their responsiveness to various drugs (bioluminescent parasites were permanently eliminated by treatment with melarsoprol and db829 but not with diminazene). the interesting finding in this paper is that the investigators did not have to wait for the clinical development of the encephalitic stage of disease which could take as late as 180 days after the initial stage of the infection. instead, by using serial noninvasive bioluminescence imaging, they reduced the time needed to assess drug efficacy by two-thirds [17] . an alternative model to reporter gene-expressing pathogens is to genetically modify the animal model, usually mice. for example, luker et al. developed a transgenic reporter mouse that uses the promoter from hsv-1 thymidine kinase to control expression of fluc [6] . exposure to hsv-1 (three different strains tested) would then activate the expression of fluc with bioluminescence increasing in proportion to increasing input titers of the virus. this study however did not detect brain involvement with hsv-1. more complex models such as the transgenic reporter mouse strain that expresses fluc under the regulatory control of a concatenated gal4 promoter [18] and the transgenic reporter mouse in which luciferase expression is driven by the nuclear factor b (nf-b)-dependent portion of the human immunodeficiency virus-1 long terminal repeat (hiv-1 ltr) [19] allowed the visualization of brain luciferase expression in response to adenovirus infection [18] and lps intraperitoneal injection [19] , respectively. a particularly interesting example combines the two approaches (reporter gene and reporter mouse), using streptococcus pneumoniae strain engineered for bioluminescence (lux) in a transgenic mouse model containing an inducible fluc reporter gene under transcriptional control of the mouse glial fibrillary acidic protein (gfap) promoter. the two reporters were then separated based on the different spectra of light emission and substrate requirements for lux and fluc, using a highly sensitive in vivo imaging system. this approach allowed the monitoring of disease progression and response to treatment on one hand while documenting brain damage at the same time [20] . an indirect way of imaging the sequelae of intracranial infection is by imaging the host reaction. perhaps the most commonly used approach in this setting is imaging infection-associated neuroinflammation. this can be achieved through targeting of the translocator protein (tspo), an outer mitochondrial membrane receptor that gets upregulated in activated microglia, astrocytes, and blood-derived macrophages in response to cns central nervous system injury. the classical ligand for tspo has always been 11 c-pk11195; however a multitude of newer (second-generation) ligands designed to overcome the limitations of pk11195 have been developed over the last couple of decades [21] . using two of the second-generation ligands, 11 c-dpa-713 and 18 f-dpa-714, along with the prototype ligand 11 c-pk11195 [22] , neuroinflammation in the setting of hsv encephalitis could be imaged, with 11 c-dpa713 showing the lowest nonspecific uptake, suggesting it may be the most suitable for visualizing mild inflammation. other pathogens in which neuroinflammation was visualized using tspo ligands include simian immunodeficiency virus (siv) in macaques [21] and taenia solium (neurocysticercosis) in patients [23] . in the latter study, perilesional edema surrounding calcified lesions was visualized using 11 c-pbr28 suggesting resurgence of inflammation despite the chronicity of the infectious process. major limitations of tspo imaging however exist. the main limitation is its inability to specifically visualize infection with ligand accumulation readily seen with sterile inflammation such as that induced by injection of quinolinic acid in the rat striatum [24] (figure. 8.3 ). another main limitation in human translation of tspo imaging is a recently described polymorphism (rs6971) located in exon 4 of the tspo gene which results in nonconservative amino acid substitution at position 147 from alanine to threonine (ala147thr) in the fifth transmembrane domain of the tspo protein [25] . this polymorphism results in three binding levels: high, medium, and low binding. to account for the variability, in most cases, genotyping of subjects prior to quantitative assessments of tspo upregulation using pet is necessary. another problem with tspo imaging in non-focal brain infections such as neuro-hiv is the lack of a reference region due to diffuse involvement of the brain. analysis of the data in such cases requires labor-intensive arterial blood sampling and analysis of metabolites. recent description of tspo ligands that are not sensitive to polymorphism is encouraging [26] . other currently evaluated neuroinflammation imaging targets include the cannabinoid receptors cb1 and cb2 which together constitute the endocannabinoid system [27] . multiple ligands targeting cb2 are being developed [28] [29] [30] with encouraging animal applications [29] . targeting leukocytes and platelets changes associated with different infectious processes has also been attempted. one example is the use of single-chain antibodies that recognize the ligand-induced binding sites (libs) of gpiib/iiia in activated platelets [31] . gpiib/iiia becomes exposed upon activation and therefore offers the opportunity to target activated platelets [32] . by conjugating the libs antibody to iron oxide nanoparticles, they used it in an animal model of cerebral malaria and were able to detect abnormal vascular platelet aggregation in vivo. this approach allowed imaging of the infection before pathology could be visualized by conventional in vivo mri [31] . as an offshoot of optical imaging, two-photon microscopy has further opened the doors to better understanding of pathogen-host interaction in the brain. up till now a handful of studies have used this technology in the evaluation of infectious processes; however the available literature points toward a big role for this technique in better understanding the role of specific immune cells such as cd+ lymphocytes and antigen-presenting cells (apcs) in the pathophysiology of cns infection. the potential applications are limitless. for example, in the brains of mice chronically infected with toxoplasma gondii, two-photon microscopy showed that antigen (ag)-specific cd8 t cells were recruited and persisted in the presence of ongoing ag recognition. the dynamics of the interactions between t cells and the parasite could then be visualized real time: while cd8 t cells made transient contacts with granuloma-like structures containing parasites and with individual apcs, including some that did not contain parasites, they ignored intact ag-bearing cysts, astrocytes, and neurons, including infected neurons [33] . in another example, two-photon imaging was used in mice infected with different strains of malaria, showing that cd8+ t cells tended to accumulate mainly in the perivascular spaces of experimental cerebral malaria (ecm) mice. this however was also seen with strains that did not cause ecm, indicating that the presence of perivascular t cells per se is insufficient to cause ecm [34] . what differentiated the strains however were dynamic time-lapse movies that showed distinct differences in t-cell motility rather than localization, with the arrest of t cells in the perivascular spaces correlating with the pathogenic potential [34] . this dynamic real-time demonstration of host-pathogen (immune system) interactions within living tissue will surely facilitate and improve our understanding of immune responses to persistent pathogens in the brain. imaging of infection in the cns has been handled using cross-sectional imaging for more than two decades now resulting in a large array of descriptive diagnostic criteria, capable, in most circumstances of narrowing the differential diagnosis, detecting life-threatening complications and establishing baseline for assessment of treatment response. limitations however exist, and in many circumstances, both cross-sectional imaging and nonspecific molecular imaging, such as 18 f-fdg, fail to establish a diagnosis. the availability of pathogen-specific imaging agents/ ligands would have a great effect on the management of patients with cns infection. besides early diagnosis, avoidance of diagnostic brain biopsies can have significant effect on the mortality and morbidity of patients. 18f]fhpg positron emission tomography for detection of herpes simplex virus (hsv) in experimental hsv encephalitis imaging enterobacteriaceae infection in vivo with 18f-fluorodeoxysorbitol positron emission tomography the synthesis of 18f-fds and its potential application in molecular imaging luciferase imaging of a neurotropic viral infection in intact animals persistent gammaherpesvirus replication and dynamic interaction with the host in vivo noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice in vitro and in vivo characterization of a dual-function green fluorescent protein--hsv1-thymidine kinase reporter gene driven by the human elongation factor 1 alpha promoter application of bioluminescence imaging to the prediction of lethality in vaccinia virus-infected mice bioluminescence imaging of vaccinia virus: effects of interferon on viral replication and spread non-invasive imaging of mouse hepatitis coronavirus infection reveals determinants of viral replication and spread in vivo persistent infection of a gammaherpesvirus in the central nervous system noninvasive bioluminescence imaging of dengue virus infection in the brain of a129 mice bioluminescent imaging and histopathologic characterization of weev neuroinvasion in outbred cd-1 mice non-invasive in vivo study of the trypanosoma vivax infectious process consolidates the brain commitment in late infections localized recrudescence of toxoplasma infections in the central nervous system of immunocompromised mice assessed by in vivo bioluminescence imaging. microbes infect daptomycin in experimental murine pneumococcal meningitis in vivo imaging of trypanosome-brain interactions and development of a rapid screening test for drugs against cns stage trypanosomiasis generation of a highly inducible gal4-->fluc universal reporter mouse for in vivo bioluminescence imaging bioluminescent detection of endotoxin effects on hiv-1 ltr-driven transcription in vivo reduction of astrogliosis by early treatment of pneumococcal meningitis measured by simultaneous imaging, in vivo, of the pathogen and host response longitudinal in vivo positron emission tomography imaging of infected and activated brain macrophages in a macaque model of human immunodeficiency virus encephalitis correlates with central and peripheral markers of encephalitis and areas of synaptic degeneration dpa-714 as new pet tracers for tspo: a comparison with [11c]-(r)-pk11195 in a rat model of herpes encephalitis pet reveals inflammation around calcified taenia solium granulomas with perilesional edema lack of neuroinflammation in the hiv-1 transgenic rat: an [(18)f]-dpa714 pet imaging study an 18-kda translocator protein (tspo) polymorphism explains differences in binding affinity of the pet radioligand pbr28 synthesis and evaluation of translocator 18 kda protein (tspo) positron emission tomography (pet) radioligands with low binding sensitivity to human single nucleotide polymorphism rs6971 the emerging role of the cannabinoid receptor family in peripheral and neuro-immune interactions whole-body biodistribution and radiation dosimetry of the cannabinoid type 2 receptor ligand [11c]-ne40 in healthy subjects cannabinoid cb2 receptors in a mouse model of abeta amyloidosis: immunohistochemical analysis and suitability as a pet biomarker of neuroinflammation discovery of a high affinity and selective pyridine analog as a potential positron emission tomography imaging agent for cannabinoid type 2 receptor a contrast agent recognizing activated platelets reveals murine cerebral malaria pathology undetectable by conventional mri conformation-specific blockade of the integrin gpiib/iiia: a novel antiplatelet strategy that selectively targets activated platelets dynamic imaging of t cell-parasite interactions in the brains of mice chronically infected with toxoplasma gondii perivascular arrest of cd8+ t cells is a signature of experimental cerebral malaria key: cord-009967-fyqc5bat authors: gleckman, richard; gantz, nelson m. title: cost‐effective antibiotic prescribing date: 2012-01-24 journal: pharmacotherapy doi: 10.1002/j.1875-9114.1983.tb03264.x sha: doc_id: 9967 cord_uid: fyqc5bat antibiotics are often misused, resulting in a high frequency of adverse effects, emergence of drug‐resistant organisms, and excessive costs. the high cost of antibiotics is currently receiving the greatest attention. considerable cost savings can be achieved by appropriate prescribing of antibiotics for patients receiving these drugs prophylactically as well as for those with established infections. this article cites specific examples of how cost‐effective antibiotic prescribing practices can realize substantial cost savings without any diminished quality in patient care. therapy of established disease appropriate indications use of "therapeutic equivalents" single agent therapy oral versus parenteral route treatment duration hidden costs self or family administration no class of drugs in the hospital formulary is currently receiving closer scrutiny than the antibiotics. these agents are being subjected to intensive examination because of increased pharmaceutical lobbying efforts, concern over the emergence of drugresistant organisms, and the continuous introduction of new compounds. undoubtedly, however, the keen interest in antibiotics primarily stems from the fact that, as a class of drugs, they account for the single largest component of the pharmacy budget. in this age of cost-containment antibiotics will continue to remain in the limelight, particularly for those pharmacists and hospital administrators dedicated to holding the fiscal line. we shall identify antibiotic prescribing practices, both prophylactic and therapeutic, that can be changed to permit cost reductions. we will also describe innovative approaches that are being implemented to counteract the spiraling costs of health delivery. it is often unappreciated that between one-fourth and one-half of all antibiotics prescribed within a hospital are dispensed for prophylaxis. most of these agents are administered to surgical patients to prevent postoperative infections. it thus becomes apparent that if guidelines for perioperative antibiotic use are complied with, significant cost savings can be realized. postoperative wound infections are not only unsightly, but they contribute to morbidity and, most importantly, constitute a threat to the patient's life. these postoperative infections can cause extensive focal infection, bacteremia, hematogenous dissemination, septic shock and multiple organ failure. in addition, postoperative wound infections significantly increase the expense of hospitalization. ' prophylactic antibiotics are indicated exclusively for selective clean and clean-contaminated surgical procedures. clean procedures are those in which neither the respiratory, alimentary, genitourinary, or oropharyngeal cavities are entered and there is no break in technique. clean-contaminated procedures are those in which these cavities are entered without unusual contamination. antibiotic prophylaxis is indicated for a "clean" procedure when a prosthesis is being implanted, or the risk exists for a catastrophic infection. antibiotic prophylaxis is indicated for a "clean-contaminated" procedure when the incidence and consequences of infection are great, and the responsible organisms are predictable and susceptible to antibiotics. infections that are associated with frankly contaminated and dirty procedures merit antibiotics, but here the indication is not prophylaxis but definitive therapy of an established infection. a number of factors have been identified that contribute to postoperative wound infections. surgical factors have included the duration of the operation, the extent of local contamination, and the presence of hematomas, debris and foreign bodies.* host factors that predispose to wound infections include age greater than 60 years, malnutrition, obesity, diabetes mellitus, malignant diseases and the presence of remote infe~tion.~ concerns about prophylactic antibiotics have focused on four issues: drug expense, adverse drug reactions, alterations in the patient's indigenous microflora with the risk for superinfection, and the emergence of drug-resistant organisms that pose a threat to other patients exposed to the hospital flora. these concerns can only be allayed by the intelligent use of perioperative antibiotics in well defined indications. preferred prophylactic antibiotics should be nontoxic, inexpensive, and possess activity against the major pathogens likely to be encountered in the operative area. however, the antibiotics need not be active against every bacterial species present in the operative area. a very limited role exists for the second generation agent cefoxitin, and no indication exists for the third generation cephalosporins cefotaxime, moxalactam, and cefoperazone for perioperative pro phyla xi^.^ these more expensive agents have not been found to be more effective than less expensive agents, and their unrestrained use could encourage the emergence of drug-resistant organisms. effective antibiotic prophylaxis requires attainment of significant tissue concentrations during the "critical period", the period of the early inflammatory response to bacterial contamination. as a general rule adequate tissue concentrations of an antibiotic during the critical period can be obtained by a single dose of the drug administered shortly before the operation, and additional doses dispensed either earlier or later are usually unnecessary. the most common error in surgical prophylaxis appears to be excessive duration of admini~tration.~ no study indicates value to extending prophylaxis beyond 48 hours. limiting perioperative antibiotic prescribing to the first 48 hours would reduce drug-related adverse reactions, the rate of emergence of resistance microorganisms, and the cost of medications. table 1 , 2, and 3 outline those clean and cleancontaminated surgical procedures that merit perioperative prophylactic antibiotics. the tables describe the indication for prophylaxis and also provide a recommended antibiotic program. among the first generation cephalosporins cefazolin is probably the preferred agent for prophylaxis. this drug produces the highest and most sustained serum concentrations, can be given as infrequently as every 8 hours, and when administered according to a three times daily schedule, is the least expensive cephalosporin. when cefaxolin is prescribed for prophylaxis 1 gram of the drug should be administered i.m. on call to the operating room or i.v. at anesthesia induction. prophylactic antibiotics are also indicated for patients with congenital valvular disease, acquired valvular heart disease or prosthetic valves who are to be subjected to dental, urinary tract, biliary tract or lower intestinal tract instrumentation or surgery. the prophylaxis is designed to prevent bacterial endocarditis. detailed recommendations have been previously published to assist the physician in managing these patients6 the suggestion has also been made that patients with orthopedic implants be considered candidates for prophylactic antibiotics when they are exposed to procedures that could result in bacteremia. the implant could serve as a locus minoris resistentiae, and a deep wound infection would ensue. this suggestion has, however, neither been confirmed nor refuted by properly executed prospective controlled studies. table 4 lists those operative procedures where prophylactic antibiotic administration has been a common practice despite the lack of controlled studies and scientific justification. we feel that perioperative antibiotics should not be routinely prescribed for these procedures until properly performed clinical studies document their value. major cost savings and a reduction of adverse effects can be attained by appropriate antibiotic usage. a number of investigations have confirmed that at least 25 to 50% of prescribed antibiotics are not indicated. approximately one quarter to one half billion dollars could be saved annually in the united gleckman and gantz 24 1 one day is clamped ahigh risk patients for cesarean section prophylaxis include women from the lower socioeconomic status, obese women and patients who have internal fetal monitoring. some experts also recommend prophylaxis for those women with prolonged labor, membranes ruptured more than 6 hours or women subjected to multiple vaginal examinations. states, without compromising patient care by more appropriate antibiotic pre~cribing.'-~ one example of the problem of antibiotic misuse is the practice of prescribing antibiotics for those disorders in which studies have failed to demonstrate any benefit from drug therapy. no justification exists to treat nonpregnant women or elderly chronically catheterized patients with asymptomatic bacteriuria.'o similarly, antibiotics appear to be of no proven value when administered to patients with acute symptomatic bronchitis or acute symptomatic exacerbations of chronic bronchitis. acute bronchitis, an inflammatory disorder of the trachea and bronchi, occurs predominantly in the winter and is often preceded by an upper respiratory infection. invariably a self-limited disease, acute bronchitis is caused most frequently by viruses. antibiotic treatment is not indicated for this inflammatory process. acute exacerbations of chronic bronchitis, consisting of a change in the color, consistency and amount of sputum as well as increasing cough and dyspnea, have been ascribed to viruses (rhinovirus, coronavi rus, influenza) , mycoplasma pneumoniae and bacteria, including haemophilis influenzae, streptococcus pneumoniae, gram-negative bacilli and "normal respiratory flora." antibiotics have been prescribed to shorten the duration of the exacerbation, prevent respiratory failure or forestall progressive pulmonary deterioration that often occurs in patients with chronic bronchitis. no evidence has emerged that antibiotics can accomplish any of these desired goals. in fact, nicotra and associates could not document any beneficial effect by the addition of antibiotics to the conventional modes of therapy in patients with acute exacerbations of chronic bronchitis requiring hospitalization.'* it has become ritual to prescribe antibiotics to noncompromised patients with localized cutaneous abscesses. for patients with normal host defenses drainage is adequate therapy, and antibiotics are not indicated. i 3 in addition to the problem of prescribing antibiotics for disorders in which no benefit has been established, physicians often equate "best" treatment with the newest, invariably more expensive, antibiotic. penicillin g remains the drug of choice for pneumococcal pneumonia, community-acquired aspiration pneumonia, dental infections, streptococcal pharyngitis and syphilis. although the more expensive cephalosporins possess an expanded spectrum of activity, cure rates are not improved by prescribing these antibiotics instead of penicillin. only when controlled studies show enhanced efficacy or reduced toxicity should the newer, more expensive agent be prescribed. similarly, the inexpensive sulfonamides remain highly effective agents to treat communityacquired symptomatic bacterial cystitis in women.14 in the absence of a history of an allergic reaction to sulfonamide, there is no reason to prescribe the more expensive compounds, such as cefaclor, cephalexin, cefadroxil, nitrofuratoin or trimethoprimsulfame-thoxazole, when treating a woman with symptomatic, community-associated bacterial cystitis. generic gentamicin is available at a cost that is approximately one-third that of the other aminoglycosides tobramycin, netilmicin, or amikacin. for hospitals, particularly community hospitals, where gentamicin-resistant gram negative bacilli are uncommon pathogens, generic gentamicin can be selected as the preferred aminoglycoside. in other hospitals, generic gentamicin can be substituted for the other aminoglycosides as soon as results of antibiotic susceptibility tests permit. for patients with suspected or established infections caused by pseudornonas aeruginosa, tobramycin should be selected while awaiting the results of antibiotic susceptibility tests. tobramycin is also preferred by some experts for treating patients with renal insufficiency who have infections requiring an aminoglycoside; the rationale 243 is based on a lower frequency of nephrotoxicity, as measured by serum creatinine values. amikacin should be selected to treat infections caused by proteus vulgaris or providencia stuarfii while awaiting susceptibility reports. for patients with nosocomial gram negative infections requiring an aminoglycoside, tobramycin or amikacin should be selected initially if gentamicin resistant strains are prevalent in the hospital. as soon as the results of antibiotic susceptibility tests are available, gentamicin can be substituted for tobramycin or amikacin. with the above exceptions, gentamicin can be selected initially or after the results of susceptibility tests are known; this strategy will result in considerable cost savings to patients. cephalosporins account for up to one third of total pharmacy drug expenditures. the first generation cephalosporins cephalothin, cephapirin and cefazolin have essentially the same spectrum of activity. pharmacokinetically, cephalothin and cephapirin are interchangeable, but use of cephapirin can result in substantial savings. because of its more favorable pharmacokinetic profile, cefazolin can be given in doses that are one third those of cephalothin or cephapirin, with comparable effectiveness. cefazotin also requires less frequent dosing per day than cephalothin (four versus six times). since cefazolin can be given intramuscularly, intravenous administration costs are saved. cefazolin also is subject to competitive pricing since it is sold by two drug companies. with desirable pharmacokinetic properties as well as the ability to purchase the drug on bid, cefazolin is probably the first generation cephalosporin of choice. a common error in antibiotic prescribing is the failure to modify therapy when the results of antimicrobial susceptibility tests are available. an example of this error is the severely ill patient presenting with high fever, sweats, chills, hypotension and no obvious source of infection. broad spectrum therapy with two antibiotics is usually prescribed pending the results of cultures and antimicrobial susceptibility tests. initial therapy should not dictate later therapy, however. when the infecting organism is known and the susceptibility report is available, it is often possible to discontinue the initial broad spectrum combination drugs and prescribe a single, less expensive and potentially less toxic agent. while multiple drugs may be preferred therapy to treat selected infections such as fneumocysfis carinii pneumonia, malaria, toxoplasmosis, tuberculosis, enterococcal endocarditis, infection caused by psuedomonas aeruginosa or gram negative bacteremia in an immunosuppressed host, a single pathogen usually requires therapy with only one antibiotic.15 prescribing more than one drug to the patient is not only more expensive, but this practice often entails greater risk for untoward events. the use of a combination of antibiotics has been the conventional initial therapy for patients with intraabdominal and pelvic sepsis. until the results of cultures and susceptibility tests are available, usually an aminoglycoside with clindamycin, metronidazole, chloramphenicol or one of the extended spectrum pencillins (carbenicillin or ticarcillin) is prescribed to assure activity for the anaerobic components, particularly bacferoides fragilis, as well as the facultative gram-negative rods contributing to these infections. 16 recently, cefoxitin therapy has been compared with the combination of clindamycin plus amikacin for the treatment of mixed aerobicianaerobic infections." a prospective randomized trial of 70 patients given these therapies revealed no difference in therapeutic efficacy or incidence of toxicity. however, the cost of cefoxitin therapy was significantly less than the cost of the drug omb bin at ion.'^ these results were confirmed in a subsequent study of 90 patients given either cefoxitin or a combination of clindamycin and an aminoglycoside for the treatment of polymicrobial pelvic and abdominal infections.18therefore, it appears that cefoxitin alone may well be as safe and therapeutically effective as the standard combination treatments when it is administered to selected patients with community-acquired mixed anaerobidaerobic infections that result from appendicitis, diverticulitis, bowel trauma, pelvic inflammatory disease or endometritis. however, it would be preferable to add an aminoglycoside to cefoxitin for those patients with intraabdominal or pelvic infections who have received antibiotics within the preceding weeks or who have experienced a nosocomial abdominal or pelvic infection. considerable cost savings can be achieved by changing from parenteral to oral antibiotic administration and by replacing hospitalization with carefully supervised home treatments. oral therapy eliminates the cost of intravenous solutions and sets and the personnel time involved in preparation and infusion. recent studies have confirmed the efficacy and reduced expense of oral antibiotics prescribed for selected children with osteomyelitis and septic arthritis.1"22 in 1973, it was reported that favorable results ensued when oral antibiotic therapy was prescribed for hospitalized patients with serious infectionsz3 fourteen patients with osteomyelitis were treated successfully with oral cephalexin after they had received a short course of parenteral cephaloridine. however, it was not until 1978 when tetzlaff and associates reported on 35 children with osteomyelitis and septic arthritis, who had been treated with a brief initial course of intravenous therapy followed by oral antibiotics, that major attention was directed to this novel cost saving approach.20 the children with acute osteomyelitis and septic arthritis were hospitalized for the entire course of therapy. parenteral therapy was given initially for about one week, and volume 3, number 4, july~august 1983 then oral antibiotics were administered for a mean of approximately 3 weeks. drug absorption was monitored by measuring serum antibiotic concentrations and by determining serum bacterial activity. the oral antibiotics were well tolerated and all infections except one responded in 1979, prober and associates reported on their experience treating 63 children with serious staphylococcal infections (predominantly osteomyelitis). the children were treated with a short course of parenteral therapy followed by oral administration of antibiotic~.~~ once the children became asymptomatic they were discharged from the hospital for supervised oral therapy at home. the children were seen once or twice weekly in an outpatient setting; serum bactericidal levels of the antibiotics were monitored. this report and subsequent studies appear to indicate that with careful monitoring oral antibiotic therapy can be as effective as the standard prolonged intravenous therapy for specific skeletal infections in 26 oral therapy is cost effective, particularly when given at home, and this form of treatment is not associated with the inherent risks of intravenous infusion, namely, chemical phlebitis and bacteremia. home oral therapy permits increased patient comfort for the child. however, oral therapy necessitates careful sequential clinical monitoring; demonstration of therapeutic serum bactericidal antibiotic concentrations is since follow-up evaluations beyond 2 years on children who have received oral therapy have not been reported, prolonged vigilance will be required to detect recrudescent disease; relapses have been detected more than 10 years after the first attack of osteomyelitis. 26 hospitalization and parenteral antibiotic therapy has been considered the conventional treatment program for women with acute symptomatic community-acquired bacterial pyelonephritis. however, this infection can be treated in an outpatient setting when diagnosis is secure, the patient does not appear "toxic", the patient can tolerate oral medication, clinical and laboratory "follow-up" can be obtained, and the patient has not recently received antibiotics or been subjected to instrumentation. trimethoprimsulfamethoxazole possesses a spectrum of activity that encompasses most organisms that cause community-oriented bacterial pyelonephritis in women, and this drug has been successfully used for the outpatient treatment of symptomatic pyelonephri-ti^.^^ metronidazole administered orally is well absorbed even in the presence of food. consequently, the serum concentrations of metronidazole that are achieved are similar after either oral or intravenous administration of the drug.28 patients who are responding to parenteral metronidazole therapy can be successfully switched to oral metronidazole when the clinical situation dictates, and this results in substantial savings. if a patient is unable to swallow tablets, the drug can be given as a liquid preparation that can be formulated by the hospital pharmacist. duration of drug therapy contributes to antibiotic costs. virtually all recommendations as to how long drug therapy should continue are empiric, even for common disorders such as pneumococcal pneumonia and streptococcal c e l l~l i t i s .~~~ 30 since limited information is available on the precise duration of drug therapy, patients are probably treated for unnecessarily long periods. as new data emerge, however, we learn that antibiotic therapy can often be shortened, thereby resulting in cost savings and diminished toxicity. single dose therapy has emerged as the preferred treatment tactic for acute, symptomatic bacterial cystitis in young women.i4 when compared to the conventional 7-1 0 day course of treatment single dose therapy is less expensive, safer, equally effective, and associated with better compliance. single dose treatment of women with bacterial cystitis has not resulted in bacteremias, hospitalization or death.3' single dose treatment should be limited to women who are not pregnant, have neither renal insufficiency nor structural abnormalities of the urinary tract, and are able to provide post-treatment cultures. the following antimicrobial agents are safe and effective single dose therapy: sulfisoxazole 1 g; trimethoprim 80 mg; trimethoprim-sulfamethoxazole 2 regularstrength tablets and amoxicillin 3 g."5 no explanation exists for why cephalosporins have consistently failed as single dose therapy.i4 a single oral dose of 2 grams of metronidazole (eight 250 mg or four 500 mg tablets) is less expensive, as effective, and as well tolerated as the conventional 7-1 0 day course of therapy to treat vaginitis caused by trichomonas ~a g i n a l i s .~~ studies have also documented the efficacy of shorter treatment courses employing regimens of ampicillin, tetracycline or erythromycin to treat disseminated gonococcal i n f e~t i o n .~~ formerly, disseminated gonococcal infections were treated for a minimum of 2 weeks by the intravenous route exclusively. adults with disseminated gonococcal infection can be effectively treated with a one week program consisting initially of 2 million units of penicillin g administered every 4 hours followed by oral ampicillin or amoxicillin prescribed as 500 mg four times daily.34,35 hospitalization is usually recommended to establish the diagnosis of disseminated gonococcal disease since misdiagnosis occurs not infrequently with this disorder.33 selected patients can complete the oral regimens in an outpatient setting or, alternatively, they can be treated entirely without ho~pitalization.~~ acceptable oral regimens consist of giving amoxicillin (500 mg four times daily), tetracycline (500 mg four times daily), or erythromycin (500 mg four times daily) for at least 7 days. 35 the following requirements should be met before home treatment is recommended: the diagnosis should be well established; the patient should be considered compliant; complications, such as purulent joint effusions, must be cost-effective anti b iotlc prescribing gleckman and ganfz 245 absent; and the patient must be able to return for follow-up observations. the standard treatment for pulmonary tuberculosis consists of the combination of isoniazid (inh)ethambutol, inh-rifampin, or rifampin-ethambutol prescribed for 18-24 months. prolonged chemotherapy is expensive and is associated with compliance and toxicity problems. studies have confirmed that specific regimens given for 6 to 12 months to adults with uncomplicated pulmonary tuberculosis are as effective as more prolonged therapy and have the advantage of being less expensive and well tolerated.36s 37 short course treatment regimens for adults consist of administering inh (300 mg) and rifampin (600 mg) daily for 6 to 12 months. if the patient has had previous antituberculosis therapy or has emigrated from an area such as asia or africa, where high levels of initial drug resistance exist, then the inh-rifampin should be supplemented with streptomycin (12-25 mg/kg), pyrazinamide (30 mg/kg), or ethambutol ( 1 5 2 5 mg/kg) daily for the first 2 months, pending the results of susceptibility tests. if resistance to inh or rifampin is found by susceptibility tests, then short course chemotherapy is not indicated. a regimen should be selected using two or three drugs to which the organisms are susceptible, and it should be given for a period of 18 to 24 months.38 for the abbreviated treatment to be successful, patient compliance is critical. patients should be seen monthly, pill counts should be performed, urines should be tested for the presence of inh and rifampin, and bacteriologic examinations of sputum must be done. treatment should be continued until at least 6 months have elapsed from the time of conversion of the sputum culture from positive to negative. for most patients, the total duration of therapy will be 9 months. patients should be followed closely for 1 year after completing the short course regimen in order to detect relapse. for noncompliant patients, after an initial phase of daily inhrifampin treatment administered for 1 to 2 months, therapy can be continued twice-weekly with inh (15 mg/kg) and rifampin (600 mg) for 7 to 10 months. 38 the medications must be administered under supervision, and patients who receive intermittent rifampin should be monitored for the development of thrombocytopenia and a "flu syndrome." these abbreviated treatments cannot be recommended for children, for patients harboring drug-resistant organisms, for patients with extra-pulmonary tuberculosis, for patients with unique predisposing concomitant disease, such as silicosis or diabetes, or patients who have experienced previous drug failure or microbiological relapse. conventional therapy for endocarditis caused by penicillin-susceptible streptococci, defined as those strains with a minimum inhibitory concentration of d 0.1 pg/ml, consist of either 12 million units of penicillin per day administered intravenously alone for four weeks or 12 million units of parenteral penicillin for four weeks and concomitant streptomycin (1 g/day) during the initial two weeks. a regimen consisting of penicillin and streptomycin prescribed for only two weeks appears to be as safe and as effective as these four week regimen^.^^.^^ no data are available showing that the relapse rate after the two week treatment course exceeds that of the conventional 4-6 week treatment program. the patients are treated in the hospital and receive procaine penicillin (1.2 million units every six hours) and streptomycin (500 mg every 12 hours) intramuscularly for 2 weeks. short term therapy is not, however, recommended for patients who have had symptoms that exceed three months or have infection involving a prosthetic valve.39 preexisting vestibular disease, the presence of complications, (mycotic aneurysm, shock, cerebritis), abnormal renal function, or the identification of streptococcal endocarditis caused by resistant (mic > 0.1 pg/ml) or nutritionally dependent strains precludes short course therapy. the principal disadvantages of the two week regimen are the frequent intramuscular injections required and the risk of streptomycin-induced otoxicity. the total cost of antibiotic therapy consists of a number of components, only one of which is the drug price. "hidden" costsadministration sets and supplies, tests for laboratory monitoring, pharmacy preparation time and nursing timeare usually omitted in cost analyses. these ancillary costs can account for approximately one-half of the total expense of antibiotic the rap^.^' potentially less toxic antibiotics that require minimal laboratory monitoring for evidence of adverse reactions can decrease drug costs. for example, laboratory monitoring of renal function and aminoglycoside serum concentrations can contribute as much as one-third of the total antibiotic costs when aminoglycoside antibiotics are pre-s~ribed.~' an antibiotic such as cefazolin that possesses desirable pharmacokinetic properties (i.e., infrequent dosing, reduced dosage and diminished nephrotoxicity when compared with other first generation cephalosporins) and can be prescribed by the intramuscular route can decrease these "hidden" costs considerably. for example, by substituting intramuscular cefazolin for an intravenous betalactam resistant penicillin, a savings of $78 per day could occur.42 when studies demonstrate that different antibiotics provide equivalent therapeutic eff icacy and safety, these "hidden" costs should be considered when antibiotic recommendations are being offered. prolonged administration of intravenous antibiotics, i.e., therapy that exceeds 4 weeks in duration, has emerged as the preferred treatment course for patients with osteomyelitis, infective endocarditis and systemic fungal infections. traditionally, these patients have remained in the hospital for the dura-246 pharmacotherapy volume 3, number 4, july/august 1983 tion of treatment even though one to two weeks after the onset of therapy many patients feel well and are anxious to return home to complete their treatment. one innovative approach to the treatment of these serious infections has focused on the use of selfadministration of intravenous antibiotics in the the cost of home parenteral antibiotic therapy is about one-fourth to one-third of the inhospital the concept of home intravenous therapy is not new. successful home intravenous programs have included patients with hemophilia who receive clotting factors, patients with parenteral alimentation, and patients sustained by chronic hemodialysis. in 1974, rucker and harrison were the first investigators to report on the use of intravenously administered antibiotics given in the home.43 sixty-two children with cystic fibrosis were treated at home with either intravenous gentamicin or colistimethate for the management of pseudomonas-related pulmonary infections. in that study, 127 infectious episodes were treated at home, resulting in a 68% reduction in the need for hospitalization. the patients were seen once weekly, and no major complications were noted.43 subsequent reports on parenteral administration of antibiotics at home appeared in 1 978,455 46 (table 5) and to date 363 patients have been treated at five centers in the united states and almost half of the patients (150) were treated at home in a program developed at the fairfax hospital, a large community-teaching hospital in the washington, d.c. area. 44 patients selected for home treatment are considered to have responded satisfactorily to the intravenous program initiated in the hospital and require only a more extended course of intravenous antibiotics. home intravenous programs are coordinated by a team consisting of infectious disease specialists, pharmacists and nurses skilled in performing intravenous infusion. one to two days prior to discharge the patients and a family member are taught the techniques necessary to care for an i.v. cannula. the cannula is changed twice a week at home by a visiting nurse or in the hospital's outpatient department. the duration of home i.v. therapy averaged two to three weeks. in each of the published series, patients with osteomyelitis and septic arthritis have predominated, but patients with other infections have also been successfully managed (table 5) . a vast array of antibiotics have been used, and the solutions, which have been prepared in the hospital pharmacy, are kept refrigerated at home. antibiotics with long half-lives, such as cefazolin, are preferred since they permit dosing every six or eight hours. patients may be given a four or five day supply of antibiotic, depending on the stability of the drug, and they are instructed to return to the outpatient department once or twice weekly to have their progress evaluated. all studies have monitored patients for cornplications of the initial infection, compliance, adverse effects, including antibiotic toxicities and i.v. compli-cations, and superinfection. to date, all programs have confirmed the safety and efficacy of this form of therapy. long-term follow-up is not available in all of the studies, but short term efficacy data parallel the experience of in-hospital care.44 patients have been able to return to work and to school. home antibiotic programs require compliant patients, appropriate close monitoring, and the 24 houra-day availability of a hospital team consisting of a pharmacist, i.v. nurse and physician. successful programs also require that insurance carriers reimburse patients for these out-of-hospital extended charges. to date, medicare has not paid for outpatient antibiotic therapy, and some insurance carriers will reimburse policy holders for only 80% of the charges. it appears that for selected patients substantial cost savings can be realized with this novel approach to prolonged antibiotic the rap^.^' success of home intravenous antibiotic programs mandates careful selection of patients. those selected must be well enough to go home (except for the need for intravenous therapy), be compliant, and be proficient or have a family member trained in the administration and aseptic care of an i.v. cannula. various strategies have been advocated to reduce antibiotic misuse. approaches have included physician education, omission from formulary, restriction of selected antibiotics by pharmacists or infectious disease specialists, peer audits of prescribing practices, and surveillance of drug use by clinical pharm a c i s t~.~~ an additional strategy for improving antibiotic prescribing practices consists of providing more readily accessible information on antibiotics to clinic i a n~.~~ unfortunately, these efforts have often had limited success.54 in a study by jones et al, hospital staff did not improve their usage of antibiotics after an intensive educational program.54 a greater impact on unjustified antibiotic usage has been reported in studies employing direct control measures. substantial savings resulted when usage of selected antibiotics required either an infectious disease consultation or written justification by the 56 mcgowan and finland demonstrated that by removing an antibiotic from the restricted list, there was a marked increase in usage of that agent5' in a study by durbin and associates, physicians were required to indicate the rationale for antibiotic usage.58 depending on the category selected, drugs were discontinued after 2 days for prophylaxis, after 3 days for empirical therapy, and after 7 days for a therapeutic indication. a new prescription form had to be completed for the drug to be reordered. this program resulted in a 50% reduction in the mean duration of antibiotic prophylaxis. there was, however, little impact on antimicrobial use on the medical service with the prescription system. another approach that could reduce indiscriminate antibiotic usage is for hospitals to develop their own antibiotic guidelines similar to those developed the guidelines should be developed by a multidisciplinary committee composed of physicians who prescribe antibiotics as well as representatives from infectious diseases, pharmacy, and hospital administration.60 there should be agreements as to what constitutes appropriate and unacceptable antibiotic usage. once antimicrobial surveillance data to monitor compliance are accumulated, corrective action will require peer pressure from chiefs of services and strong administrative support. postoperative wound infection: a controlled study of the increased duration of hospital stay and direct costs of hospitalization postoperative wound infections: the influence of ultraviolet irradiation on the operating room and of various other factors surgical wound infection occurrence in clean operations the third-generation cephalosporins: a plea for restraint use of antimicrobial drugs in general hospitals: patterns of prophylaxis prevention of bacterial endocarditis use of antibiotics: a brief exposition of the problem and some tentative solutions this is medical progress? trends and consequences of antibiotic use in the united states a study of antimicrobial misuse in a university hospital the controversy of treatment of asymptomatic bacteriuria in nonpregnant women-resolved role of infection in chronic bronchitis antibiotic therapy of acute exacerbations of chronic bronchitis. a controlled study using tetracycline cutaneous abscesses clinical management of urinary tract infection antibiotic combinations: the clinical relevance of synergy and antagonism prospective, randomized, comparative study of clindamycin, chloramphenicol, and ticarcillin, each in combination with gentamicin, in therapy for intraabdominal and female genital tract sepsis cost comparison of two antimicrobial regimens for treating mixed aerobic-anaerobic infections a prospective randomized controlled trial of cefoxitin versus clindamycin-aminoglycoside in mixed anaerobic-aerobic infections clindamycin treatment of osteomyelitis and septic arthritis in children oral antibiotic therapy for skeletal infections of children. ii. therapy of osteomyelitis and suppurative arthritis high-dose dicloxacillin treatment of acute staphylococcal osteomyelitis in children long-term follow-up of ambulatory management of osteomyelitis success with cephalordine-cephalexin therapy use of the serum bactericidal titer to assess the adequacy of oral antibiotic therapy in the treatment of acute hematogenous osteomyelitis marks mi et at. oral antibiotic therapy of skeletal infections in children oral antibiotic therapy for bone and joint infections oral versus parenteral therapy of pyelonephritis metronidazole: an update on its expanding role in clinical medicine for how long should antimicrobial therapy continue (editorial) current practices in antimicrobial dosing efficacy of single-dose and conventional amoxicillin in urinary-tract infections localized by the antibody coated bacteria technique treatment of trichomonas vaginalis infections treatment of disseminated gonococcal infections gonococcal tenosynovitis-dermatitis and septic arthritis sexually transmitted diseases treatment guidelines short-course chemotherapy for tuberculosis with largely twice-weekly isoniazid-rifampin british thoracic association. a controlled trial of six months chemotherapy in pulmonary tuberculosis. second report: results during the 24 months after the end of chemotherapy guidelines for short-course tuberculosis chemotherapy antimicrobial therapy for penicillin-sensitive streptococcal infective endocarditis: two-week regimens outpatient intravenous medications in the management of cystic fibrosis intravenous antibiotic therapy in an outpatient setting intravenous antibiotic therapy at home feasibility of outpatient self-administration of parenteral antibiotics outpatient intravenous antibiotics experience with 65 patients intravenous antibiotic therapy at home training patients to administer intravenous antibiotics at home self-administration of intravenous antibiotics: an efficient, cost-effective home care program. cma j 51. frame pt. outpatient intravenous antibiotic therapy (editorial) evaluation of antibiotic usage: a comprehensive look at alternative approaches antimicrobial misuse, antibiotic policies and information resources the effect of an educational program upon hospital antibiotic use the antibiotic utilization committee: an effective tool in the implementation of drug utilization review that monitors the medical justification and cost of antibiotic use infection and antibiotic usage at boston city hospital: changes in prevalence during the decade 1964-1973 usage of antibiotics in a general hospital: effect of requiring justification improved antibiotic usage following introduction of a novel prescription system successful two-week treatment schedule for penicillin-susceptible streptococcus viridans endocarditis guidelines for peer review, veterans administration ad hoc interdisciplinary advisory committee on antimicrobial use, audits of antimicrobial usage the influence of dose frequency 1241-5 key: cord-023698-wvk200j0 authors: hammerschlag, margaret r.; kohlhoff, stephan a.; gaydos, charlotte a. title: chlamydia pneumoniae date: 2014-10-31 journal: mandell, douglas, and bennett's principles and practice of infectious diseases doi: 10.1016/b978-1-4557-4801-3.00184-3 sha: doc_id: 23698 cord_uid: wvk200j0 nan the first isolates of c. pneumoniae were serendipitously obtained during trachoma studies in the 1960s. after the recovery of a similar isolate from the respiratory tract of a college student with pneumonia in seattle, grayston and colleagues 8 applied the designation twar after their first two isolates, tw-183 and ar-39. only one serotype of c. pneumoniae has been identified so far. studies have found a high degree of genetic relatedness (greater than 98%) among human c. pneumoniae isolates tested. 4, 9 microbiology chlamydiae have a gram-negative envelope without detectable peptidoglycan; however, recent genomic analysis has revealed that both c. trachomatis and c. pneumoniae encode for proteins that form a nearly complete pathway for synthesis of peptidoglycan, including penicillinbinding proteins. 10 chlamydiae also share a group-specific lipopolysaccharide antigen and use host adenosine triphosphate (atp) for the synthesis of chlamydial protein. 10 although chlamydiae are auxotrophic for three of four nucleoside triphosphates, they do encode functional glucose-catabolizing enzymes, which can be used for generating atp. 10 as with peptidoglycan synthesis, for some reason, these genes are turned off, which may be related to their adaptation to the intracellular environment. all chlamydiae also encode an abundant protein called the major outer membrane protein (momp or ompa) that is surface exposed in c. trachomatis and c. psittaci but apparently not in c. pneumoniae. 10 the momp is the major determinant of the serologic classification of c. trachomatis and c. psittaci isolates. chlamydiae are susceptible to antibiotics that interfere with dna and protein synthesis, including tetracyclines, macrolides, and quinolones. c. pneumoniae lacks a tryptophan recovery or biosynthesis pathway and is resistant to sulfonamides and trimethoprim. 4 chlamydiae have a unique developmental cycle with morphologically distinct infectious and reproductive forms: the elementary body (eb) and reticulate body (rb; fig. 184-1 ). after infection, the infectious ebs, which are 200 to 400 nm in diameter, attach to the host cell by a process of electrostatic binding and are taken into the cell by endocytosis that does not depend on the microtubule system. • obligate intracellular bacterium, must be grown in tissue culture • capable of causing persistent infection, often subclinical • worldwide distribution, infects many animals as well as humans • primarily a respiratory pathogen in humans, causing community-acquired pneumonia • can cause epidemics in enclosed populations: military bases, schools, nursing homes • chlamydia pneumoniae causes pneumonia; clinically it cannot be differentiated from other causes of atypical pneumonia, especially mycoplasma pneumoniae. • the most accurate method of diagnosis is identification of the organism in respiratory samples by culture or nucleic acid amplification test (naat). • serology is of limited value, requires paired sera, and many patients who are positive by culture or naat will be seronegative. • c. pneumoniae is susceptible to macrolides, quinolones, and tetracyclines. data on efficacy are limited, including optimal dose and duration of therapy. • ten-to 14-day courses of erythromycin, clarithromycin, doxycycline, levofloxacin, or moxifloxacin or 5 days of azithromycin are clinically effective and result in approximately 80% microbiologic eradication. aberrant inclusions, on average 4 µm in diameter, containing about 60 abs that were similar in size to normal rbs but appeared electron dense and no longer retained a smooth spherical shape. these dense abs retained the characteristic chlamydial outer membrane structure, with very little periplasmic space, and the membranes more tightly bound to the chlamydial body, similar to normal rbs. no ebs were observed in these inclusions. these findings show that the developmental cycle of c. pneumoniae can combine the typical development forms with the persistent phase in tissue culture. another possible mechanism of chlamydial persistence could be through a direct effect on the host cell, possibly through an effect on apoptosis, which is an important regulator of cell growth and tissue development. apoptosis is a genetically programmed, tightly controlled process, unlike necrosis, which involves nonspecific inflammation and tissue damage and intracellular enzymes, condensation of nucleus, and cytoplasm and fragmentation. many microbial pathogens, including chlamydiae, have been found to modulate cellular apoptosis to survive and multiply. chlamydia spp. have been shown to both induce and inhibit host cell apoptosis, depending on the stage of the chlamydial developmental cycle. 13 chlamydiae protect infected cells against apoptosis as a result of external stimuli during early stages of infection and may induce apoptosis of the host cell during later stages of the life cycle. thus, chlamydiae may protect infected cells against cytotoxic mechanisms of the immune system, and the apoptosis observed at the end of the infection cycle may contribute to the inflammatory response because apoptotic cells secrete proinflammatory cytokines and facilitate the release of the organism from the infected cells. studies with ifn-γ-treated cultures have reported that cells infected with c. trachomatis and c. pneumoniae resist apoptosis as the result of external ligands, via inhibition of caspace activation. data from studies with the long-term continuously infected cell model showed marked differences in the effect of c. pneumoniae on apoptosis in acute and chronically infected a549 cells. 13 acute c. pneumoniae infection induced apoptotic changes in a549 cells within the first 24 and 48 hours after infection. induction of apoptosis in acute infection may facilitate release of c. pneumoniae from the host cell. chronic c. pneumoniae infection inhibited apoptotic changes within the first 24 hours and up to 7 days. these results suggest that inhibition of apoptosis may help to protect the organism when it is in the intracellular, persistent state. although numerous methods can be used to detect c. pneumoniae in clinical samples, in practice, detection is very difficult, primarily because of the lack of standardized well-validated methods. determination of whether c. pneumoniae infection is an acute primary infection or reinfection, a chronic persistent stage, or a past infection is also very difficult. cell culture, immunohistochemistry (ihc), and nucleic acid amplification tests (naats) detect living bacteria, antigen, and nucleic acid, respectively. these techniques are primarily used in research settings or require experienced specialized laboratories. in clinical settings, routine diagnosis of c. pneumoniae infection has been based on results of serologic testing to identify anti-c. pneumoniae immunoglobulin g (igg), iga, and igm antibodies. this approach is problematic for a number of reasons subsequently outlined in detail. recently, a new polymerase chain reaction (pcr) assay for the detection of c. pneumoniae became commercially available and was cleared by the u.s. food and drug administration (fda) in july 2012 (discussed later). 14 future use of this assay will provide new diagnostic capability. c. pneumoniae, as an obligate intracellular parasite, can be isolated by means of cell culture, but the organism is fastidious and slow growing. c. pneumoniae will grow, although not as readily, in cell lines that are usually susceptible to c. trachomatis, such as mccoy and hela cells. growth has been observed to be somewhat easier in hl (human line) and hep-2 cells. 15, 16 c. pneumoniae has been isolated from the respiratory tract (nasopharyngeal and throat cultures, bronchoalveolar lavage fluids) and ebs are sporelike; they are metabolically inactive but stable in the extracellular environment. within the host cell, the eb remains within a membrane-lined phagosome, with inhibition of phagosomallysosomal fusion. the inclusion membrane is devoid of host cell markers, but lipid markers traffic to the inclusion, which suggests a functional interaction with the golgi apparatus. chlamydiae appear to circumvent the host endocytic pathway, inhabiting a nonacidic vacuole that is dissociated from late endosomes and lysosomes. ebs then differentiate into rbs that undergo binary fission. after approximately 36 hours, the rbs differentiate back into ebs. despite the accumulation of 500 to 1000 infectious ebs in the inclusion, host cell function is minimally disrupted. at about 48 hours, release may occur via cytolysis or a process of exocytosis or extrusion of the whole inclusion, leaving the host cell intact. this strategy is very successful and enables the organism to cause essentially silent chronic infection. a number of in vitro studies have challenged this biphasic paradigm. chlamydiae may enter a persistent state in vitro after treatment with certain cytokines, such as interferon-γ (ifn-γ); treatment with antibiotics, specifically penicillin; restriction of certain nutrients, including iron, glucose, and amino acids; infection in monocytes; and heat shock. 4, 11 while in the persistent state, metabolic activity is reduced, and the organism is often refractory to antibiotic treatment. these different systems produce similar growth characteristics, including loss of infectivity and development of small inclusions that contain fewer ebs and rbs and ultrastuctural findings, specifically, morphologically abnormal rbs, which suggests that they are somehow altered during their otherwise normal development. these abnormal rbs are often called aberrant bodies (abs). restriction of certain nutrients has also been shown to induce persistence in chlamydiae. ultrastructural analysis of ifn-γ-treated c. pneumoniae also reveals atypical inclusions that contain large reticulate-like abs with no evidence of redifferentiation into ebs. another model of persistent c. pneumoniae infection is long-term continuous infection. in contrast to the previously described models, continuous cultures become spontaneously persistent when both chlamydiae and host cells multiply freely in the absence of stress. c. pneumoniae infection was maintained in hep-2 and a549 cells for more than 4 years without centrifugation, addition of cycloheximide, or ifn-γ. 12 infection levels in these infected cells were high (70% to 80%). ultrastructural studies revealed three types of inclusions in these cells. approximately 90% were typical large inclusions that ranged approximately from 5 to 12 µm in diameter. the second type (altered inclusions) contained both normal ebs and rbs, but in considerably lower numbers than typical inclusions, and pleomorphic abs, which were up to four to five times the size of normal rbs (2.5 µm in diameter); their cytoplasm was homogeneous. the third type of inclusion was small multiple in-house naat (such as pcr) methodologies have been published, but the literature has been confounded by lack of standardization and validation. in 2000, a centers for disease control and prevention (cdc) workshop suggested a few assays that were considered to be "validated" enough to be used for research. 18 some of these included early developed and validated ones. [20] [21] [22] [23] [24] these have been improved, and others have been developed since then. 25, 26, 27, 28 the advantages of these naat or pcr assays are their sensitivity, decreased possibility of contamination, and ability to quantify dna. nearly a decade later, however, many of these tests turned out to be highly prone to false-positive results. 29, 30 until recently, not a single naat for the detection of c. pneumoniae was commercially available or listed in the in vitro diagnostic (ivd) database of the fda (www.accessdata.fda.gov/ scripts/cdrh/cfdocs/cfivd/index.cfm). numerous in-house pcr-based tests still are performed, but these assays range from those that are well validated to those that are not validated at all. although naats offer the promise of exquisite sensitivity, theoretically allowing detection of a single organism in a clinical sample, both false-negative and -positive results can and do occur because of a large number of technical issues that were summarized recently. 31 currently, real-time pcr (rt-pcr) technology for the detection of c. pneumoniae should be used. 32, 33 rt-pcr offers significant advantages over conventional pcr in its rapidity, the ease with which it can be automated, the potential decreased risk of carryover contamination, and the potential provision of a quantitative result. until recently, there have been no commercially available naat assays. abbott laboratories (abbott park, il) developed a researchuse-only pcr assay that was used in a multicenter study comparing pcr results by using in-house pcrs from five different laboratories. the assay performed very well, but it was never taken to a clinical trial. 34 becton dickinson (franklin lakes, nj) performed a clinical trial for a strand displacement assay, but it was not cleared by the fda. biofire technologies (formerly idaho technologies; salt lake city, ut) developed a filmarray assay for the detection of 17 viruses, which is fda cleared. 35, 36 the filmarray system now includes assays on the same platform for some of the atypical agents of pneumonia, including c. pneumoniae, mycoplasma pneumoniae, and bordetella pertussis. this assay received fda clearance in july 2012. the filmarray system combines nucleic acid extraction, nested pcr, detection, and data analysis in a single-use pouch. 14 the automated system is simple to perform, and results are ready in 1 hour. this single test platform enables the detection of numerous viral and bacterial respiratory pathogens in a single test. water is added to hydrate the lyophilized reagents, and the respiratory specimen is added. the pouch is loaded into the filmarray instrument, and the remainder of the test is completely automated. after extraction of nucleic acid, a nested pcr reaction is performed within the pouch in an entirely closed system. the first-step pcr is a multiplexed reaction containing primers for all of the viral and bacterial targets; the amplicons from the first pcr are then diluted, and a second round of pcr reactions is performed in a multiwell array, each well containing a single primer set targeting a specific pathogen. both amplification and melt curve analysis allow the filmarray software to generate a result for each target. the system is very robust, detecting a low concentration of pathogen in the presence of a high concentration of a second pathogen, with results available in 1 hour. the respiratory panel detects adenoviruses, bocaviruses, coronaviruses, influenza a and b, influenza a subtypes (novel h12009, h1, h3), metapneumovirus, parainfluenza viruses 1 to 4, respiratory syncytial virus, and rhinoviruses, as well as b. pertussis, c. pneumoniae, and m. pneumoniae. 14, 35 specimens for research pcr testing include nasopharyngeal swabs, secretions from the respiratory tract including sputum and bronchoalveolar lavage fluid (bal), tissue, and peripheral blood mononuclear cells. swabs should be sent in tubes without transport medium. sputum, bal, and tissue should also be collected in a sterile device without transport medium. if necessary, sputum can be diluted by homogenizing it with tris-edta (tris-[hydroxymethyl] aminomethane-ethylenediaminetetraacetic acid) buffer. tissue has to be homogenized before dna extraction. if dna extraction for pcr tissue biopsies, including lung and adenoids. the organism can also be isolated from sputum, but sputum can be toxic to cell culture and often is contaminated by overgrowing fungi or bacteria. if nasopharyngeal or pharyngeal swab specimens are collected, use of aluminum or plastic-shafted dacron tip swabs is mandatory because calcium alginate on cotton tips and those with wooden shafts may inhibit the growth of the organism in tissue culture and may be toxic to cells. specimens for culture must be stored in a suitable transport medium optimized for chlamydiae. a suitable medium is sucrose-phosphate glutamic (spg) buffer with antibiotics and fetal calf serum, but readyto-use media are also commercially available. specimens that can be processed within 24 hours should be kept refrigerated at 4° c and shipped on wet ice. samples that cannot be processed within 24 hours should be held at 4° c before freezing at −70° c because more rapid freezing decreases the titer of viable organisms. specimens need to be treated with sterile glass beads or sonication to disrupt cells and then centrifuged onto the cell monolayers to facilitate absorption. cell cultures are incubated at 37° c with 5% carbon dioxide for at least 72 hours per passage. culture confirmation is assessed by staining inclusion bodies, using a chlamydia genusspecific fluorescent antibody and epifluorescence microscopy ( fig. 184-2 ). more than one subculture may be necessary for isolation; thus, culture is not a straightforward attempt to diagnose the microorganism in a timely fashion. because the organism has been difficult to grow and because of the lack of a commercially available other diagnostic assay, most original associations with respiratory diseases have been use of serology with the microimmunofluorescence (mif) test. patients who have igg autoantibodies against igm may cross react with anti-c. pneumoniae igm antibody. 17 c. pneumoniae has also been detected in tissue sections or cells with monoclonal antibodies labeled with a peroxidase (ihc) or fluorescent (immunofluorescent) marker. antigen detection testing, in general, allows preservation of tissue morphology. on the other hand, interpretation of the staining pattern to distinguish the organism from background or nonspecific staining is subjective and influenced by a number of technical issues. 18 in complex biologic samples, ihc gives rise to cross-reactions between antitarget antibodies and nontarget proteins that produce nonspecific signals (e.g., immunoreactivity for c. pneumoniae was frequently present in atheroma and nonatheroma sections of vessel walls). the sites with positive results with c. pneumoniae ihc assays precisely matched the sites with autofluorescent ceroid deposits. 19 the interpretation of ihc staining must be performed with the utmost caution. summary, serology seems not only to be insufficient for diagnosis of c. pneumoniae respiratory tract infection but also to be an inadequate methodology to study associations between c. pneumoniae and other diseases. it is important to know that new environmental chlamydia spp. are being steadily described. ample evidence exists for a huge diversity and wide distribution of chlamydiae in nature, and humans are exposed to that diversity of species. as an example, the recovery of a novel environmental chlamydia strain from activated sludge with cocultivation with an acanthamoeba sp. was reported; it was shown to also invade mammalian cells. 41 these new environmental chlamydiae (i.e., simkania, waddlia, and parachlamydia) may interfere with serologic testing for traditional chlamydiaceae (chlamydia). 42, 43 in summary, c. pneumoniae serology is most problematic in terms of defining specificity, reproducibility, and titer in a given clinical picture or disease, even if prospectively defined. the mode of transmission of c. pneumoniae remains uncertain but probably occurs through infected respiratory secretions. acquisition of infection via droplet aerosol was described during a laboratory accident. 44 c. pneumoniae can remain viable on formica countertops for 30 hours and can survive small-particle aerosolization. 45 spread within families and enclosed populations, including military recruits, prisons, and nursing homes, has been described. 33, [46] [47] [48] several serologic surveys have documented rising chlamydial antibody prevalence rates, beginning in school-aged children and reaching 30% to 45% by adolescence. 8 seroprevalence antibody, as determined with the mif method, can exceed 80% in some adult populations. 49, 50 the proportion of community-acquired pneumonia (cap) in children and adults associated with c. pneumoniae infection has ranged from 0% to more than 44%, varying with geographic location, the age group examined, and the diagnostic methods used (table 184-1) . 51 the proportion of cap attributable to c. pneumoniae appears to be significantly lower in studies published after 2000. whether this is secondary to the methods used (most of the recent studies have used rt-pcr) or possible cycling, as is seen with m. pneumoniae, is unknown. four studies published after 2010, from diverse geographic areas (europe, africa, and thailand), that used rt-pcr found prevalences of c. pneumoniae infection ranging from 0% to 3.8%. [52] [53] [54] [55] this was compared with 11.4% in a chinese study that used mif serology; 30% of the patients only had a single serum sample (see table 184 -1). 56 early studies that relied on serology suggested that infection in children younger than 5 years was rare; however, subsequent studies with culture or pcr assay have found the prevalence rate of infection in children beyond early infancy to be similar to that found in adults. 51 approximately 50% or is performed within 24 hours, storage at 4° c is sufficient; otherwise, specimens should be kept at least at −20° c. several types of serologic assays are currently commercially available for the detection of antibodies to c. pneumoniae. however, none are currently approved by the fda for this indication. the test used most frequently and recommended by the cdc remains the mif assay. 18 tests based on an enzyme-linked immunosorbent assay (elisa) format are particularly easy to perform and do not need sophisticated laboratory equipment, which makes them the preferentially offered diagnostic chlamydial tool for laboratories. c. pneumoniae, however, is an intracellular pathogen, and the poor correlation between direct detection (e.g., with culture or naat) and serologic results is not surprising. besides specificity issues, it is not at all clear which classes and titers of antibodies might represent acute first infection or reinfection, chronic, persistent, or past c. pneumoniae infection. 18 this is true for complement fixation tests (measurement of antibodies against chlamydial lipopolysaccharide-therefore not specific for c. pneumoniae), elisa-based tests (purified c. pneumoniae ebs or recombinant antigens detected; specificity unclear), and also the gold-standard mif test (formalinized c. pneumoniae ebs fixed onto glass slides). a serologic test can only be as specific as the antigen used. crossreactivity between c. pneumoniae and other chlamydia species has been shown with the mif test. factors such as strain type, purity, and concentration of antigen used, and the assay procedure itself, might contribute to the fact that the mif is less specific for c. pneumoniae than thought 20 years ago. data also show significant problems with subjective interpretation and intralaboratory and interlaboratory reproducibility. 37 the problems in context with c. pneumoniae serology have been discussed in detail in two recently published review articles. 38, 39 for an example of the complexity of this issue, consider that two multicenter pneumonia treatment studies in children showed that although 7% to 13% of the patients in the study had positive culture results and 7% to 18% met the serologic criteria with the mif test for acute infection, they were not the same patients. only 1% to 3% of the patients with positive culture results met the serologic criteria, and approximately 70% with positive culture results for c. pneumoniae were seronegative. 40 benitez and co-workers 33 reported similar data in adults from an investigation of a c. pneumoniae outbreak in a prison. mif serology (igg and igm) had only had a positive predictive value of 30% compared with rt-pcr. another problem with serologic diagnosis of c. pneumoniae infection is that the mif method used to detect serum antibodies is not standardized; recent studies have shown substantial interlaboratory variation in the performance of these tests. 37 in has been selected in vitro after passage of c. pneumoniae in subinhibitory concentrations of moxifloxacin. 66 these isolates were found to have a point mutation in the gyra gene. studies with long-term continuously infected cells suggest that c. pneumoniae may be refractory to antibiotics when in the persistent state. 67 on the basis of these few data, the following regimens can be used for respiratory infection from c. pneumoniae: in adults, doxycycline, 100 mg orally twice daily for 14 to 21 days; tetracycline, 250 mg orally four times daily for 14 to 21 days; azithromycin, 500 mg once a day followed by 250 mg/day for 4 days; clarithromycin, 500 mg orally twice a day for 10 days; levofloxacin, 500 mg, intravenously or orally once a day for 7 to 14 days; or moxifloxacin, 400 mg orally once a day for 10 days. for children, erythromycin suspension, 50 mg/kg per day for 10 to 14 days; clarithromycin suspension, 15 mg/kg per day for 10 days; or azithromycin suspension, 10 mg/kg once on the first day, followed by 5 mg/kg once daily for 4 days. some patients may need retreatment. one of the distinguishing characteristics of chlamydiae is the ability to cause persistent, often subclinical, infections. from a clinical standpoint, chlamydiae may be the persistent infection par excellence, capable of persisting in the host for months to years, often without causing obvious illness. from a microbiologic standpoint, persistence also refers to long-term intracellular infection that can be detected with antigen, microscopy, or nucleic acid-based amplification methods. chronic persistent infection with c. pneumoniae has been implicated in the pathogenesis of several chronic diseases, initially not thought to be infectious, including asthma, arthritis, and atherosclerosis. however, studies of the association of c. pneumoniae and these disorders have been hampered with difficulty in diagnosis of chronic persistent infection with the organism, which, in turn, makes determination of the efficacy of interventions difficult, especially with antibiotics. infection with c. pneumoniae has been linked to asthma by a large number of epidemiologic and clinical studies. the controversy about definition of infection and diagnostic tests contributes to the difficulty in interpretation and comparison of studies. the field is further complicated by differences in study populations in regard to asthma phenotype and the presence of acute symptoms. table 184 -3 summarizes selected studies that have examined the association of c. pneumoniae infection and asthma. the wide range of positivity illustrates the sometimes contradictory findings regarding an association between c. pneumoniae and asthma, some of which may be explained by differences in populations and diagnostic methods. in 1991, hahn and colleagues 68 reported an association between serologic evidence of acute c. pneumoniae infection and adult-onset more children with culture-documented c. pneumoniae respiratory infection (pneumonia and asthma) show seronegativity with the mif. 51 prolonged respiratory infection, documented with culture, that lasts from several weeks to several years after acute infection has been reported. 57 coinfections with other organisms, specifically, streptococcus pneumoniae and m. pneumoniae, may occur frequently. 51, 58 clinically, these patients cannot be differentiated from those infected with a single organism. in these cases, c. pneumoniae may not be the primary cause of the pneumonia but might disrupt the normal clearance mechanisms and enable other pathogens to invade. this may have been the case in an outbreak of pneumonia and fatal pneumococcal meningitis among u.s. army trainees. 48 six of 12 trainees with pneumonia were infected with c. pneumoniae, which suggested a simultaneous outbreak of both infections. asymptomatic respiratory infection may occur in 2% to 5% of adults and children. 49, 58 the role asymptomatic carriage plays in the epidemiology of c. pneumoniae is not known. acute respiratory infection with c. pneumoniae does not appear to vary by season, but no systematic surveillance for c. pneumoniae infection exists in the united states. most respiratory infections from c. pneumoniae are probably mild or asymptomatic. initial reports emphasized mild atypical pneumonia clinically resembling that associated with m. pneumoniae. 8 subsequent studies have found that pneumonia associated with c. pneumoniae has been clinically indistinguishable from other pneumonias. 51,59 c. pneumoniae has been associated with severe illness and even death, although the role of preexisting chronic conditions as contributing factors in many of these patients is difficult to assess. c. pneumoniae can be a serious pathogen even in the absence of underlying disease. c. pneumoniae was isolated from the respiratory tract and the pleural fluid of a previously healthy adolescent boy with severe pneumonia complicated by respiratory failure and pleural effusions. 60 the role of host factors remains to be determined. although c. pneumoniae has been detected in bronchoalveolar lavage fluid from 10% of a group of patients with acquired immunodeficiency syndrome and pneumonia, its clinical role in these patients is uncertain because most were coinfected with other well-recognized pathogens such as pneumocystis jirovecii and mycobacterium tuberculosis. 61 gaydos and colleagues 62 identified c. pneumoniae infection with pcr assay in 11% of a group of immunocompromised adults with human immunodeficiency infection, malignant neoplasms, and other immune disorders, including systemic lupus erythematosus, sarcoidosis, and common variable immunodeficiency. c. pneumoniae was responsible for 6 of 31 episodes (19%) of acute chest syndrome in children with sickle cell disease in new york. 63 c. pneumoniae infection in these patients appeared to be associated with more severe hypoxia than was infection with m. pneumoniae. the relationship of c. pneumoniae and upper respiratory infections, including pharyngitis, sinusitis, and otitis media, is less clear. data on treatment of c. pneumoniae respiratory infection are limited. c. pneumoniae is susceptible to antibiotics that affect dna and protein synthesis, including macrolides; azalides, specifically azithromycin; tetracyclines; and quinolones (table 184-2) . however, in vitro activity may not always predict in vivo efficacy. most published pneumonia treatment studies have used serology alone for diagnosis of c. pneumoniae infection, which is at best a clinical end point. results of several multicenter treatment studies that used culture showed 70% to 86% efficacy of treatment with erythromycin, clarithromycin, azithromycin, levofloxacin, and moxifloxacin in eradicating c. pneumoniae from the nasopharynx of children and adults with cap. 40 most patients had clinical improvement despite persistence of the organism. persistence did not appear to be the result of the development of antibiotic resistance because the minimum inhibitory concentrations (mics) of the isolates obtained after treatment did not change. antibiotic resistance is unusual in chlamydiae. 40, 64 investigators were unable to select for macrolide resistance after passage of c. pneumoniae in subinhibitory concentrations of azithromycin. 65 in contrast, resistance to quinolones wheezing and anti-c. pneumoniae ige in children infected with c. pneumoniae was shown, which suggests a th2 response to the bacterium in patients with asthma. 76 the role of stress and host genetics in delayed or suboptimal th1 response to chlamydial infection and development of complications in certain individuals, and the role of specific c. pneumoniae antigens eliciting harmful immune responses in patients with asthma, is currently unclear. if infection with c. pneumoniae contributes to inflammation in patients with allergic asthma, diagnosis and treatment of these infections is important. interactions may also exist between c. pneumoniae infection and asthma drugs. treatment of asthma exacerbations frequently includes systemic steroids, which have been shown to enhance the in vitro infectivity of c. pneumoniae 77 ; this was reflected in significant increases of inclusions but did not affect the in vitro activities of azithromycin, erythromycin, and doxycycline against c. pneumoniae. 77 several studies have addressed the question of whether antibiotic treatment of c. pneumoniae infection in patients with asthma leads to improvement in disease activity. study design has been complicated by the fact that macrolides, quinolones, and tetracyclines all have immunomodulatory activity independent of their antimicrobial activity. 78, 79 any positive treatment outcomes may therefore be the result of antichlamydial or immunomodulatory effects, or a combination of the two. several uncontrolled studies showed beneficial effects of antibiotics on patients with asthma with proven or presumed c. pneumoniae infection. 59, 80 subsequent placebo-controlled trials attempted to confirm the benefits suggested by these preliminary studies. a placebo-controlled 6-week trial of roxithromycin in patients with asthma who were seropositive for c. pneumoniae showed significantly higher morning peak expiratory flow in the treatment group at the end of treatment but not at subsequent time points. 81 in the absence of clear evidence that asthma and asthmatic bronchitis in the united states. studies that have shown an association or lack of association between the presence of antibodies (igg, igm, and iga) and higher antibody titers (igg) against c. pneumoniae with asthma have been reported since then in a variety of populations. 69 studies that used direct detection methods (culture or pcr) were more consistent in establishing a role of c. pneumoniae in exacerbations of asthma (see table 184 -2). in patients with stable asthma symptoms, evidence for infection with c. pneumoniae of up to 22% with pcr, alone or in combination with m. pneumoniae, may suggest chronic infection. 69 the clinical implications of c. pneumoniae infection in patients with asthma who have no acute symptoms are not clear; the obvious concern is that the persistent presence of the pathogen may lead to ongoing inflammation and thus contribute to severity and progression of asthma. 70 currently, minimal data exist to examine the immunologic basis for the association between c. pneumoniae and asthma pathology. persistent infection with c. pneumoniae, which has been shown in patients with asthma, might be the result of an insufficient th1 response in these patients, which is critical for clearance of the intracellular bacterium. 59, 71 in analogy to the correlation of abnormal host immune response to c. trachomatis infection and tissue sequelae, a similar relationship may conceivably exist between respiratory infection with c. pneumoniae and asthma pathology. 72 abnormal cellular immune responses to respiratory infections with c. pneumoniae in patients with asthma may in part be related to genetic variation in immune mediator genes. 73 genetic variation of toll-like receptor 2 is under investigation as a major factor in the development of asthma and may be related to susceptibility to c. pneumoniae. in toll-like receptor 2 −/− mice, decreased ifn-γ and adaptive cell responses led to poor control of respiratory infection with c. muridarum and prolonged inflammation. 74 differences in c. pneumoniae igg antibody responses were seen in children with asthma, depending on variant mannose-binding lectin alleles. 75 an association between mice. 88 in vitro studies have shown that c. pneumoniae can infect and replicate within monocytes, macrophages, and vascular endothelial and smooth muscle cells and that all are important components of atherosclerotic plaque. 86, 89 in vitro infection also results in oxidation of cellular low-density lipoprotein; the production of proinflammatory cytokines involved in atherogenesis, including tumor necrosis factor-α, interleukin-6 and -1β, and inf-α; and the transendothelial migration of neutrophils and monocytes. 86, 89 c. pneumoniae can induce human macrophage foam cell formation in vitro, a key event in early atheroma development. 90 however, this may not be specific because the key component appears to be the chlamydial lipopolysaccharide, which is conserved in all chlamydial species, including c. trachomatis. however, no single serologic, pcr, or ihs assay has been used consistently across all studies, and these assays are not standardized. in 2002, boman and hammerschlag 38 reviewed 14 seroepidemiologic studies published from 1992 to 2000 and found a great deal of heterogeneity among these studies in terms of the serologic tests used and the criteria for seropositivity. in some studies, an igg or iga titer of 1 : 64 or more was used as an indicator of chronic infection; in others, the same criteria were used as indicators of past infection. nine of these studies used the mif assay; all were in-house tests. the antigen used was only specified in four of the mif assays. the remaining studies used a variety of other methods, including genus-specific enzyme immunoassays and whole-cell immunofluorescence. as stated previously, mif assays are not standardized and are subject to significant operator variation. 37 background seropositivity rates in the general population often exceed 70%, which can also make demonstration of an association between the presence of c. pneumoniae antibodies and cad difficult. earlier case-control studies that showed an association were generally small and based on single serum samples, which does not take into account that antibody titers fluctuate over time. a metaanalysis of 12 studies only found combined odds ratios of 1.15 and 1.13 for igg and iga antibodies, respectively. 91 boman and hammerschlag 38 also analyzed 43 studies, published from 1992 through 2000, that examined 2679 samples of atheromatous tissue for the presence of c. pneumoniae with culture, electron microscopy, pcr, and ihs. the overall rates of detection of c. pneumoniae ranged from 0% to 100%, with 49.7% being positive by at least one method. however, when specimens were analyzed with more than one method, the prevalence rate of specimens positive by at least two methods (usually ihs and pcr) was only 15.14%. as with the serologic studies, major variation was found in the methods, including the antibodies and techniques for ihs and pcr. ihs has also been found to have problems with interpretation and reproducibility. studies from hoymans and colleagues 19 reported that ceroid, an insoluble lipid present in plaque, could cause nonspecific reactions with ihs. the extent of interlaboratory variation with performance of pcr was shown by apfalter and colleagues, 29 who sent a panel of 15 homogenized clinical atheroma specimens (carotid and coronary) and control specimens to nine laboratories in europe and the united states for detection with pcr. the positivity rate in the clinical specimens ranged from 0% to 60%, and three laboratories identified c. pneumoniae in negative control specimens. the concordance between the assays was only 25% for one specimen. subsequently, apfalter and colleagues 31 showed that contamination was practically impossible to avoid with nested pcr assays. ieven and hoymans 39 published an analysis of studies reported since 2000, many of which used rt-pcr and were found to be predominantly negative. rt-pcr is much less subject to contamination from amplicon carryover. ieven and hoymans 39 also noted that in studies where serology was done in addition to pcr, no correlation was found between presence of c. pneumoniae in the atheroma tissue and presence of anti-c. pneumoniae antibodies in individual patients. with extrapolation from the observation that c. pneumoniae can disseminate systemically in mice after intranasal inoculation, 85 the presence of c. pneumoniae in peripheral blood mononuclear cells (pbmcs) has been suggested to act as a surrogate marker for infection with c. pneumoniae in individuals with cardiovascular and other diseases. 38, 39 more than 20 studies that examined the presence of c. pneumoniae dna in pbmcs have been published, and as seen with studies of vascular tissue, the reported prevalence rate of c. pneumoniae dna patients with asthma in this study had persistent c. pneumoniae infection, one could conclude that the treatment effect was the result of the anti-inflammatory action of roxithromycin, which disappeared after stopping the drug. a double-blind, randomized, placebo-controlled study of telithromycin in patients with acute exacerbations of asthma found reduction of asthma symptoms among those treated with the active drug; however, the study could not adequately assess the effect of infection because only one of 278 enrolled patients was positive for c. pneumoniae with pcr assay of upper airway samples. 82 similarly, in a randomized, controlled study of clarithromycin, the number of asthma patients who were pcr-positive for either c. pneumoniae or m. pneumoniae was only 12 and therefore was underpowered to test the effect of clarithromycin on asthma outcome variables. 83 a randomized controlled trial of minocycline in patients with allergic asthma showed improved asthma symptoms and reduced total serum ige, a beneficial effect that did not appear to be the result of a respiratory infection with c. pneumoniae; seropositivity for c. pneumoniae was not significantly different between patients and control subjects, and no patient had positive nasopharyngeal cultures for c. pneumoniae. 84 comparing studies of antibiotic treatment of patients with asthma is complicated by the use of different criteria of c. pneumoniae infection status (culture, pcr, serology, or a combination of these tests), use of nonstandard methods, and the unclear definition of chronic infection. most studies have been underpowered to show effects of infections status. in conclusion, although diagnosis and treatment of c. pneumoniae infections in patients with asthma with signs and symptoms of an airway infection are recommended, the benefit of using antibiotics with activity against atypical bacteria in patients with asthma without laboratory evidence of infection remains controversial. persistent c. pneumoniae infection has also been implicated in the pathogenesis of several chronic diseases, initially not thought to be infectious, including atherosclerosis, multiple sclerosis (ms), temporal arteritis, stroke, alzheimer's disease, lung cancer, and macular degeneration. 69 studies in mice have shown that c. pneumoniae disseminates to the spleen and other organs after respiratory infection via macrophages. 85 however, this effect has not been conclusively shown to occur in humans. in addition, studies of the association of c. pneumoniae and these disorders have been hampered by difficulty in diagnosis of chronic persistent infection with the organism; no validated serologic or other surrogate markers exist for chronic c. pneumoniae infection. 18 the high prevalence of chlamydial infections and transient immunity after infection makes differentiation of persistent infection from reinfection or even past infection difficult. this, in turn, makes determination of the efficacy of any therapeutic intervention difficult. conventional risk factors, including cigarette smoking, hypertension, and high serum lipid levels, do not fully explain the incidence, prevalence, and distribution of coronary artery disease (cad). inflammation of the vessel wall plays an essential role in the initiation and progression of atherosclerosis, erosion, fissure, and eventual rupture of the atheromatous plaques. 86 various markers of systemic inflammation, including c-reactive protein, have been found to predict future cardiovascular events, including nonfatal and fatal myocardial infarction and stroke. although inflammation is present, the exact cause is still not known. infectious agents, including cytomegalovirus, human herpesviruses, enteroviruses, helicobacter pylori, bacteria involved with periodontal disease, and c. pneumoniae have also been investigated as possible causes for this inflammation. the first report that suggested a possible association between c. pneumoniae infection and cad came from a case-control study from finland published in 1988, showing that patients with proven cad were significantly more likely to have antibodies to c. pneumoniae than control subjects selected at random. 87 this report was quickly followed by additional seroepidemiologic studies and studies that identified c. pneumoniae in atheroma with various methods, including culture, immunohistochemical staining (ihs), and pcr. 38, 39 animal studies have shown that c. pneumoniae can either induce or enhance the development of atherosclerosis in 400 mg/day per month for 2 years. the duration of follow-up ranged from 3 months to 2 years. the results of 2 of 6 of the earlier small studies (≤150 patients in each arm) favored antibiotic, but all of the remaining 5 large studies favored placebo for all end points, including total mortality; subsequent myocardial events, including infarction; and unstable angina. also, no relationship was found between outcome and c. pneumoniae serologic status. a similar analysis with similar results was published by baker and couch in 2007. 97 a number of possible reasons have been proposed for the failure to show a positive effect of antibiotic treatment, including populations studied, trial design, and duration of treatment. given lack of a reliable marker for endovascular c. pneumoniae infection and the largely negative results in recent organism detection studies, additional studies are unlikely to show any benefit of long-term antibiotic treatment in reducing mortality or cardiovascular events in patients with cad. during the past 60 years, 20 different bacteria and viruses have been proposed to be associated with ms. the results were often inconsistent. the possible association of c. pneumoniae and ms was first described in a case study from researchers at vanderbilt university medical center (vumc); this study was then followed by a series of patients from vumc in which the researchers claimed that they identified the organism with culture and pcr. 98 the hypothesis of how c. pneumoniae might cause ms was not clear. the results of subsequent studies from a number of other groups were conflicting, some finding c. pneumoniae dna in approximately 30% to more than 80% of cerebrospinal fluid (csf) specimens from patients with ms and in approximately 20% of csf specimens from patients with other neurologic diseases, and others finding none in csf and brain tissue with culture and pcr. 99, 100 in an effort to deal with the issue of laboratory-to-laboratory differences in methods used to detect c. pneumoniae in studies of ms, prospectively collected csf specimens from patients with ms and other neurologic diseases were sent to laboratories at vumc, johns hopkins university (jhu), and the university of umea (uu) in sweden and subsequently also to the cdc. 101 thirty specimens from patients with ms and 22 control specimens were tested; none was positive with pcr at jhu, uu, and the cdc, but 73% of the csf specimens from patients with ms and 23% of the control specimens were positive with pcr at vumc. reasons for these discrepant results were discussed and included poor sensitivities of the assays used by jhu, uu, and the cdc or specificity problems with the assays used by vumc. the primer sets used by vumc in the multicenter study were analyzed at the cdc and were found to have high sequence similarity to human dna, as determined with a blast (basic local alignment search tool), suggesting that they were not specific for c. pneumoniae. 102 in pbmcs has also varied significantly, from 0% to 59% of patients with cad and 0% to 46% of healthy blood donors. 92 kohlhepp and others 93 examined pbmcs from more than 300 blood donors, either younger than 20 years or older than 60 years. the samples were divided and sent to two different laboratories, where they were tested for c. pneumoniae dna with rt, touchdown, and nested pcr assays. only two samples from the younger-than-20-year-old group were positive in one of the laboratories but negative in the second. none of the samples for the more-than-60-year-old group was positive in either laboratory. this study showed that two different laboratories, with different extraction methods and rt-pcr targets, did not detect c. pneumoniae dna in both cohorts of patients, but evidence of interlaboratory discrepancy was found with two specimens. more recently, west and co-workers 94 examined pbmcs from 86 patients with angiogramdocumented coronary artery disease and 91 age and gender control subjects for the presence of c. pneumoniae dna by using two different rt-pcr assays that used different genetic targets. no c. pneumoniae dna was detected in any of the specimens, including serial specimens from a subset of patients followed for 8 months. the background prevalence of anti-c. pneumoniae igg of greater than or equal to 1 : 16 was 74% in case and control subjects. results of the initial seroepidemiologic and organism detection studies led to several preliminary studies that investigated the efficacy of antibiotic treatment directed at c. pneumoniae for the prevention of secondary cardiac events. the results of these preliminary studies suggested an effect but were underpowered and raised questions about the antibiotic regimens used and methods of identification of patients with c. pneumoniae infection. the major assumption of many of the seroepidemiologic studies of the association of c. pneumoniae and atherosclerosis and other chronic conditions is that the presence of anti-c. pneumoniae antibody implies the presence of the organism somewhere in the body. however, earlier studies of patients with respiratory infection often found a poor correlation between serology and isolation of the organism from the respiratory tract. 51 gupta and others 95 randomized 60 men with prior myocardial infarction and who were seropositive with mif (igg ≥ 8) to receive either azithromycin 500 mg/day for 3 or 6 days or placebo. they found that the patients who received azithromycin showed a decrease in mif igg titers and had a lower risk of a secondary adverse cardiac event than the patients who received placebo. the antibiotic regimen used by gupta and colleagues 95 was never studied for treatment of c. pneumoniae infections. a meta-analysis of 11 randomized trials, which enrolled a total of 19,217 patients, was published in 2005. 96 seven of these trials used azithromycin; length of treatment ranged from 500 mg/day for 3 to 6 days to 500 to 600 mg/wk for 6 weeks to 1 year. three studies used roxithromycin for 30 days to 6 weeks; one used clarithromycin, 500 mg/day for 85 days; and one used gatifloxacin the complete reference list is available online at expert consult. emended description of the order chlamydiales, proposal of parachlamydiaceae fam. nov. and simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family chlamydiaceae, including a new genus and five new species, and standards for identification of organisms international committee on systematics of prokaryotes. subcommittee on the taxonomy of chlamydiae. minutes of the closed meeting parachlamydiaceae: potential emerging pathogens chlamydia pneumoniae: modern insights into an ancient pathogen unity in variety-the pan-genome of the chlamydiae a new respiratory tract pathogen: chlamydia pneumoniae strain twar comparative genomes of chlamydia pneumoniae and c. trachomatis genome sequencing and our understanding of chlamydiae chlamydia persistence-a tool to dissect chlamydia-host interactions an automated nested multiplex pcr system for multipathogen detection: development and application to respiratory tract infection use of hep-2 cells for improved isolation and passage of chlamydia pneumoniae standardizing chlamydia pneumoniae assays: recommendations from the centers for disease control and prevention (usa) and the laboratory centre for disease control (canada) immunohistostaining assays for detection of chlamydia pneumoniae in atherosclerotic arteries indicate cross-reactions with nonchlamydial plaque constituents comparison of a new quantitative ompa-based 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from individuals younger than 20 years and older than 60 years detection of circulating chlamydophila pneumoniae in patients with coronary artery disease and healthy control subjects elevated chlamydia pneumoniae antibodies, cardiovascular events, and azithromycin in male survivors of myocardial infarction effects of antibiotic therapy on outcomes of patients with coronary artery disease. a meta-analysis of randomized controlled trials azithromycin for secondary prevention of coronary artery disease: a meta-analysis multiple sclerosis associated with chlamydia pneumoniae infection of the cns infectious agents and multiple sclerosis: are chlamydia pneumoniae and human herpes virus 6 involved? chlamydia pneumoniae and multiple sclerosis: fact or fiction is chlamydia pneumoniae found in spinal fluid samples from multiple sclerosis patients? conflicting results is chlamydia pneumoniae present in the cerebral spinal fluid of multiple sclerosis patients? key: cord-002222-rgqwm3vb authors: olarte-castillo, ximena a.; hofer, heribert; goller, katja v.; martella, vito; moehlman, patricia d.; east, marion l. title: divergent sapovirus strains and infection prevalence in wild carnivores in the serengeti ecosystem: a long-term study date: 2016-09-23 journal: plos one doi: 10.1371/journal.pone.0163548 sha: doc_id: 2222 cord_uid: rgqwm3vb the genus sapovirus, in the family caliciviridae, includes enteric viruses of humans and domestic animals. information on sapovirus infection of wildlife is limited and is currently lacking for any free-ranging wildlife species in africa. by screening a large number of predominantly fecal samples (n = 631) obtained from five carnivore species in the serengeti ecosystem, east africa, sapovirus rna was detected in the spotted hyena (crocuta crocuta, family hyaenidae), african lion (panthera leo, family felidae), and bat-eared fox (otocyon megalotis, family canidae), but not in golden or silver-backed jackals (canis aureus and c. mesomelas, respectively, family canidae). a phylogenetic analysis based on partial rna-dependent rna polymerase (rdrp) gene sequences placed the sapovirus strains from african carnivores in a monophyletic group. within this monophyletic group, sapovirus strains from spotted hyenas formed one independent sub-group, and those from bat-eared fox and african lion a second sub-group. the percentage nucleotide similarity between sapoviruses from african carnivores and those from other species was low (< 70.4%). long-term monitoring of sapovirus in a population of individually known spotted hyenas from 2001 to 2012 revealed: i) a relatively high overall infection prevalence (34.8%); ii) the circulation of several genetically diverse variants; iii) large fluctuations in infection prevalence across years, indicative of outbreaks; iv) no significant difference in the likelihood of infection between animals in different age categories. the likelihood of sapovirus infection decreased with increasing hyena group size, suggesting an encounter reduction effect, but was independent of socially mediated ano-genital contact, or the extent of the area over which an individual roamed. environmental contamination might be an important route for fecal-oral transmission of sapovirus, for example when spotted hyenas sniff virus infected feces or ingest water contaminated with virus infected feces. if so, individuals with a limited range may be less likely to encounter virus infected feces than those with an extensive range. in the serengeti np, adult and subadult hyenas (i.e. those !12 months of age) not only range throughout the approximately 56 km 2 of their clan's territory, but also undertake long distance foraging trips (of approximately 140 km distance round-trip) from their clan territory [46, 47] . the ranges of cubs (<12 months of age) are by comparison extremely limited, being restricted to the communal den area within the clan territory [47] . if the extent of an animal's range determines its chance of encountering virus infected feces, then all else being equal, cubs should be less often infected with sapovirus than older animals. finally, sapovirus transmission might depend on basic population parameters such as population density, principally represented by clan (group) size. if the chance of transmission increases with animal density, then individuals living in larger clans should be more likely to be infected than those in smaller clans. however, if an encounter reduction effect operates [48, 49] , then we expect the chance of susceptible individuals encountering an infected animal to decline with clan size. in humans, sapovirus infection is currently thought to provide immunological protection, at least to antigenically homologous sapoviruses, although specific immunological responses are still unknown [16] . currently nothing is known about the immunological responses of spotted hyenas to sapovirus infection, or the length of immunological protection following sapovirus infection. even so, if sapovirus infection induces long-term immunity against reinfection regardless of strain-type, we would expect cubs (i.e. naïve animals) to be more prone to infection than adults, as is the case for coronavirus infection in this species [35] . however, if sapovirus infection provides only short-term immunity, we would expect re-infections among animals of all ages. if immune responses are strain-specific, re-infection would also be expected in animals of all ages, following the appearance of a divergent strain. this study aims to advance knowledge of sapovirus infection in wild carnivore communities in africa. we report the identification of sapoviruses in wild carnivores in africa and investigate the genetic diversity of strains infecting sympatric carnivore species in the serengeti ecosystem. we assess temporal changes in sapovirus infection in a large population of individually known spotted hyenas during a period spanning more than a decade and investigate whether sapovirus infection provides long-term immunity against future infection. furthermore, we test three mechanisms likely to affect the fecal-oral spread of sapovirus infection in spotted hyenas. this study was conducted in the serengeti np, from february 2001 to march 2012. fresh fecal samples (n = 514) were collected shortly after deposition from individually known spotted hyenas including 146 samples from adults (females n = 93, males n = 53), 41 samples from subadults (females n = 20, males n = 21) and 327 samples from cubs (females n = 152, males n = 175) from three large clans (denoted in fig 2 as i, p, and m). fecal samples were also collected from other carnivores in the serengeti np (african lion, panthera leo, n = 9; bat-eared fox, otocyon megalotis, n = 9; silver-backed jackal, canis mesomelas, n = 74 samples; golden jackal, canis aureus, n = 25). following collection, feces were thoroughly mixed and divided in aliquots. tissue samples (5 intestine, 2 liver, 9 lung, 10 lymph node, 12 spleen, 10 blood, 1 muscle, 1 saliva) were also collected opportunistically from dead spotted hyenas which were mostly killed by lions or when hit by motor vehicles [40] and hence were not necessarily members of study clans, and from two other carnivore species (bat-eared fox: 2 intestines; silver-backed jackal: 1 intestine, 2 liver, 3 lung). both fecal and tissue samples were stored and transported frozen at -80°c, or were preserved in rnalater (sigma-aldrich inc., st. louis, mo, usa), stored initially at -10°c, and finally stored at -80°c until analyses [35, 36] . currently, porcine enteric calicivirus (pec) cowden strain [50, 51] is the only known sapovirus that can be cultured. hence, viral detection and initial characterization involves mostly molecular methods based on sequence data of the well-conserved rna-dependent rna polymerase (rdrp) and the variable structural vp1 genes [16, 21] . in this study, sapovirus rna was detected by targeting the highly conserved rdrp gene. total rna was extracted from 200μl of 10% (wt/vol) fecal suspension in depc-treated water using the qiaamp minelute virus spin kit (qiagen, hilden, germany), according to manufacturer's instructions. sapovirus rna was detected with the broadly reactive primer pair p289 and p290 [52] targeting highly conserved motifs in the rdrp protein of caliciviruses. based on the sequences initially generated, nested primers were designed, cali2f (5'-cag tga cag cca cat cct tg-3') and cali2r (5'-agc act gca gca gca aag ta-3'), targeting the rdrp gene. rt-pcr was performed using superscript tm iii one-step rt-pcr system with platinum 1 taq dna polymerase (invitrogen, karlsruhe, germany) following the user manual's instructions in a total reaction volume of 25μl. amplicons of the expected size were purified using the qiagen pcr purification kit (qiagen, hilden, germany). in order to avoid rnases, all surfaces were cleansed with rnase away (molecular bioproducts, san diego, ca, usa). the purified products were sequenced bidirectional using the big dye terminator cycle sequencing kit 1.1 (applied biosystems [abi], darmstadt, germany) following the manufacturer's instructions. a 3130 genetic analyzer (abi) was used for the sequencing. subsequently, sequences were assembled in geneious v 9.0.2 (biomatters ltd, auckland, new zealand) or bioedit 7.0.9.0 [53] . samples that could not be sequenced were considered positive when bands of the expected size were present with both primer pairs. for these samples the rt-pcrs were run in duplicate to ensure that the results were reliable. to obtain a longer segment of the rdrp gene, the primer 90r (5'-rcc ctc cat ytc aaa cac ta-3') was used together with the primer calir2.. genbank accession numbers for 20 sequences identified by this study are designated kt777545-kt777564. these accession number are included in our phylogenetic tree (fig 2) , together with the host species, the year in which the variant was collected and for spotted hyenas also clan membership, denoted as i,m,p if know or z if not known. all partial rdrp genes sequences (210 nucleotides, 70 amino-acids) presented the characteristic caliciviral glpsg motif. one sample from a spotted hyena in 2011 was sequenced for a longer fragment of the rdrp gene (700 nucleotides, 233 amino-acids, accession number kt777560) which presented both the glpsg motif and the ygdd motif. sapovirus sequences obtained in this study for the partial rdrp gene together with others retrieved from genbank were aligned using the muscle algorithm [54] in geneious v 9.0.6 (biomatters ltd, auckland, new zealand). at least one reference sequence of each of the five genogroups of sapovirus (gi-gv) was included in the analysis (gi, n = 5, accession numbers ay237422, ay694184, dq366345, u95644, u73124, gii, n = 3, ay646855, ay237420, ay603425, giii, n = 3, fj715800, af182760, fj387164, giv, n = 1, dq058829, gv, n = 1, ay646856). additional sapovirus sequences from domestic dog (jn387135, jn387134), california sea lion (jn420370), mink (ay144337) and bats (jn899072, jn899074, jn899075) were included. viruses from other genera in the caliciviridae family known to infect carnivores were also included, such as feline calicivirus (af098931-32) and canine calicivirus (af053720, ab070225) from the vesivirus genus, and a norovirus reported from a captive african lion (genus norovirus, ef450827). average nucleotide and amino-acid similarities were calculated using discovery studio visualizer 4.0 (accelrys software inc, san diego, usa). phylogenetic relationships were reconstructed using maximum likelihood (ml) and bayesian markov chain monte carlo (mcmc) phylogenetic inferences. the ml analysis was performed in paup ã 4.0b10 [55] using 1,000 bootstrap replicates to estimate the statistical support of the branches. the bayesian analysis was carried out using mrbayes version 3.1 [56, 57] . the mcmc search was set to 10,000,000 iterations, with trees sampled every 1,000 th iteration. the nucleotide substitution model used in the ml analysis was obtained using modeltest 3.7 [58] and for the bayesian analysis using mrmodeltest 2.3 [59] . for both cases the akaike information criterion (aic) was used to select the best-fitting model. to determine factors influencing the likelihood of sapovirus infection and changes in long-term infection prevalence we screened feces from individually recognized spotted hyenas in three study clans (i,m,p). age was estimated when individuals were first sighted as cubs, to an accuracy of ± 7 days [60] using pelage characteristics, whether their ears were flattened or upright, and their coordination during locomotion [61, 62] . we classified animals as cubs when less than 12 months of age, as subadults when between 12 and less than 24 months of age, and as adults when ! 24 months of age [63] . sex was determined by the dimorphic glans morphology of the erect phallus [64] . total clan size comprised all adults, subadults and cubs of both sexes. access to food resources in clan territories is determined by social status: all immigrant males are socially subordinate to female clan members and their offspring at food resources in the clan territory [65] . we determined the rank of adults in separate female and breeding male linear dominance hierarchies using the outcome of submissive responses in dyadic interactions within each sex, as detailed in [43, 60, 63] . to compare individual ranks across clans of different sizes, we used standardized ranks. we calculated the standardized rank of each individual within its clan on the date it was sampled using the method described by [66] . this method assigns standardized ranks between -1 (held by the animal with the lowest rank) and +1 (held by the animal with the highest rank) [60, 63] . adult females with standardized ranks higher or equal to the median standardized rank of 0 were classified as holding high social status, those with standardized ranks below 0 as low social status [43] . cubs and subadults were assigned the social status of their mother [60] . all immigrant males held a social status below adult clan females [63] . if sapovirus infection depends on intra-specific contact rates, we would expect the dynamics of social interactions within each clan to determine exposure to pathogens. for this purpose we constructed an index of social (ano-genital during greeting ceremonies) contact rates in spotted hyenas as follows. we combined social status and sex in that high ranking females and their offspring were given a high score (for contact rate), low ranking females and their offspring were given a medium score, and immigrant and reproductively active natal males were given a low score. in order to assess whether the range of an animal, the size of the area over which an individual typically roams, determines the chance of exposure to pathogens, we classified adults and subadults of both sexes with an extensive geographical foraging range as 'roaming' , because they range both within their clan territory (~55-75 km 2 ) and undertake long distance foraging trips outside the clan territory [46, 47] . cubs were classified as 'den-bound' , i.e., with a small range restricted to the vicinity of the communal den inside the clan territory. for the purpose of considering the effect of basic population parameters such as population density on incidence of infection we used total clan size on the date each animal was sampled. to investigate whether sapovirus infection provided immunity against re-infections we genetically screened feces from 91 individually known spotted hyenas from which fecal samples were obtained on at least two different dates. of these, 76 individuals were screened on two different dates, 10 individuals on three different dates and 5 individuals on four different dates. using these screening results we calculated the average interval duration between two successive sampling dates. we used nonparametric models, including the mann-whitney u-test and the kruskal-wallis test, to compare medians [67] and the kaplan-meier survivorship and the logrank test in survival analyses to compare the survivorship curves of intervals between different combinations of incidences of infection [68] . to investigate differences in the prevalence of sapovirus in the spotted hyena population studied between 2001 and 2012 we first tested for differences in the prevalence of infection across years, using a log-likelihood ratio-test. for this test we only considered years with a sample size of at least 20 individuals, thus years 2001, 2002 and 2012 were excluded where sample sizes were 17, 11 and 5, respectively. we also checked for possible differences between age categories, using the same statistical test. these analyses were run in systat version 13 (systat software inc., richmond, va, usa). we then ran models to assess which of three possible mechanisms influenced the likelihood of sapovirus infection in our study population. for this purpose we used binary logistic regression models [69] , with predictor variables contact rate, lifetime range and clan size, and ran these as mixed models with animal identity as a random variable to account for the fact that some individuals contributed more than one tissue or fecal sample to the data set. if a genetic screening result was available for more than one organ or fecal sample for an individual on the same sampling date, only one result was included in the dataset for the prevalence models; if we obtained both a positive and negative result from an animal on the same day, the positive result was selected. this applied to 7 individuals where we had two fecal samples from the same day, and to 7 individuals from which altogether 16 tissue samples were examined. we included data from all individuals sampled during the years which could either be classified as outbreak or non-outbreak years (see results). models were run with the glmer function of package lme 4 version 1.1-8 in in r (r development core team, v. 3.1.1). we used log-likelihood ratio tests and information criteria (aic and schwartz's [bic s ] and raftery's bayesian information criterion [bic r ]) to check whether the final model was superior to an intercept-only or a reduced model. models were considered similar if differences in aic were less than 2.5 and preferable if the difference exceeded 6.0 [70] ; similar if differences in bic r were less than 2.0, a positive degree of preference if values of bic r varied between 2.01 and 6.0 and a strong degree of preference if values of bic r differed by more than 6 (a. raftery in [71] , p73). as the evaluation of our models with all information criteria produced similar conclusions, we report only aic values. the significance of each predictor variable was assessed in the following way. we calculated the marginal contribution of each parameter to the full model by subtracting from the full model the log-likelihood ratio of a second model with each variable removed and testing the difference against a chi-square distribution with the appropriate degrees of freedoms (see discussions in [69, 71] ). in order to illustrate the effect of clan size on the chance of infection, we proceeded as follows. we calculated "covariate adjusted estimates" of the logits for each record over the observed range of values by adjusting them to the median of the remaining covariates (contact rate, lifetime range) of their log-odds (logit) for being infected ( [69] , p80), and then converted the resulting estimates into probabilities using the logistic equation. this permitted us to show the effect of clan size on the likelihood of infection whilst controlling for the covariates contact rate and lifetime range at their middle values. the significance threshold for all tests was fixed at 5% and all tests were two-tailed. the data used for the statistical analyses is contained in s1 table. ethics statement the study was approved by the tanzanian commission of science and technology (cost-ech) and the tanzania wildlife research institute (tawiri). permission to work in the serengeti national park was granted by the tanzanian national parks authority (tanapa). the work was also approved by the internal ethics committee of the leibniz institute for zoo and wildlife research (izw), approval no. 2011-04-03. screening targeting the conserved rdrp gene revealed sapovirus rna in feces from spotted hyena (33.3%, 171/514 samples), african lion (33.3%, 3/9 samples) and bat-eared fox (22.2%, 2/9 samples). no sapovirus rna was found in fecal samples from golden (0/25) or silverbacked jackals (0/74). sapovirus rna was found in tissue samples collected opportunistically from dead spotted hyenas (spleen, 6/12 samples, liver 1/2 samples, lymph node 2/10 samples), but not in intestine (0/5 samples) or lung samples (0/9 samples). animals with positive spleen samples were negative for sapovirus rna in their other available tissues (2 lymph nodes; 1 liver; 1 lung). a total of 20 partial rdrp gene sequences (16 from spotted hyenas, 3 from african lions and 1 from bat-eared foxes) were obtained and used for the phylogenetic analysis, together with publically available sequence data from 25 representatives of all sapovirus genogroups, divergent unclassified sapoviruses, and other genera in the caliciviridae family, including norovirus and vesivirus. the sapovirus strains from wild carnivore species in the serengeti ecosystem were placed together in one independent monophyletic cluster (fig 2) , and separately from all recognized sapovirus genogroups (gi to gv) and other unclassified sapoviruses. nucleotide sequence comparison between strains within the african wild carnivore group and other sapoviruses revealed low nucleotide similarity, ranging from 70.4% ± 1.4 with two domestic dog strains to 56.2% ± 1.5 with sequences from genogroup gii strains. at the amino acid level the highest similarity was with strains within genogroup giv (84.9% ± 1.5) and the lowest with strains from one bat species in asia (74.8% ± 0.6). nucleotide sequence comparison with members of other genera in the calicivirus family known to infect carnivores also showed low similarity values (feline and canine calicivirus; 60.3% ± 1.7 and 59.3% ± 2.1, respectively, norovirus from a captive african lion; 56.1% ± 0.9). within the group of african wild carnivore strains, the sapovirus strains from spotted hyenas grouped together and separately from those obtained from african lions and bat-eared foxes (fig 2) . one strain from a member of the p clan (all from members of the p study clan), all the clusters contained variants obtained from members of at least two of the three study clans. notably, one variant obtained from a bat-eared fox in 2011 was placed separately from those obtained from spotted hyenas in that same year, but close to the three variants obtained in 2010 from african lions. comparison of nucleotide sequences revealed that the average similarity between all strains from spotted hyenas (96.1% ± 3.8) was lower than between strains from african lions and bateared foxes (99.1% ± 0.3). the percentage similarity from the variant in 2007 placed separately from the other spotted hyena variants was lower (87.4% ± 0.6) than that of all the other spotted hyena variants that grouped more closely (97.4% ± 1.6). however, comparison at the amino acid level revealed that all strains from spotted hyenas were identical. the average similarity of the sequences from african lions and bat-eared foxes was 99.2% ± 0.9 (with differences at two amino acid positions). the average nucleotide and amino acid similarity between the spotted hyena strains and those from african lions and bat-eared foxes was 85.1% ± 1.0 and 99.6% ± 0.7, respectively. overall, sapovirus infection prevalence (combining results from fecal and tissue samples) in spotted hyenas was 34.8% (180/517 samples, table 1 ). infection prevalence between 2001 and 2012 (fig 3) fluctuated substantially between years (log likelihood ratio = 69.157, df = 11, p < 0.00001). we considered infection prevalence in any given year equal to or above 40% as indicative of an outbreak of sapovirus infection in that year. by this definition, 2003, 2004, 2006, 2007, 2010 were considered 'outbreak years' , and 2005, 2008, 2009, 2011 were considered 'non-outbreak years' in which infection prevalence was below 40%. of the 16 partial rdrp gene sequences obtained from spotted hyenas, 11 of these were from outbreak years, three were from non-outbreak years and two were from years that could not be classified (2002). in the phylogenetic tree strains from non-outbreak years clustered with those from outbreak years (fig 2) . to determine whether the prevalence of sapovirus infection was affected by age, we screened feces from animals in different age categories (i.e., cubs, subadults and adults). we found no significant differences in infection prevalence between different age categories across all years (chi square test, likelihood ratio = 2.045, df = 2, p = 0.36), in non-outbreak years (likelihood ratio = 3.860, df = 2, p = 0.15), or outbreak years (likelihood ratio = 3.331, df = 2, p = 0.19). we screened for sapovirus rna in feces obtained from 91 individuals on two separate occasions (s2 table) . of these, 15 individuals were sampled on at least three separate occasions, and from five of these animals on a fourth occasion ( table 2) . results revealed 32 transitions of an individual from sapovirus rna negative to positive and 15 transitions from positive to negative. in many cases the infection status did not change between sampling dates, for both initially negative (negative to negative, 53 cases) and initially positive individuals (positive to positive, 11 cases). we found no cases of transitions from positive to negative to positive ( table 2) . transition intervals were similar between first and second, second and third, and third and fourth sampling date (kruskal-wallis test, h = 2.567, df = 2, p = 0.28). we therefore analyzed the relationship between the duration of time intervals between successive samples and changes in infection status without regard to the number sampling repeats per individual. the duration significantly varied between different categories of changes of infection status (survival analysis, log-rank test, log-likelihood ratio = 10.114, df = 3, p = 0.018). similar intervals were observed for changes in infection status from negative to positive (mean: 466.6 days, 95% c.i. 285.5-647.6 days, median 158 days, n = 32) and from positive to negative (mean: 602.8 days, 95% c.i. 185.8-1019.8 days, median 180 days, n = 15). the shortest and longest intervals were observed when there was no change in status: negative to negative (mean: 241.1 days, 95% c.i. 132.5-349.7 days, median 82 days, n = 53) and positive to positive (mean: 769.0 days, 95% c.i. 412.5-1125.5 days, median 715 days, n = 11), respectively. we used a mixed-effects binary logistic regression model to test factors influencing the likelihood of infection in spotted hyenas (log-likelihood ratio = 16.717, df = 4, p = 0.0022, n = 484 samples from 380 individuals with complete information). the results revealed that infection was not significantly altered by either contact rates or the extent of an individual's range ( table 3 ). the likelihood of infection significantly declined as clan size increased: with every additional individual in the clan, clan members were 1.02 times less likely to be infected with sapovirus ( table 3) . as the actual clan sizes ranged from 65 to 145 individuals, this implied a more than two-fold change in the likelihood of infection across the observed range of clan sizes (fig 4) . correspondingly, median clan sizes were significantly lower during outbreak (median = 77, mean = 83.6, with 95% c.i.: 81.4-85.9) than non-outbreak (median = 89, table 2 . rt-pcr fecal screening results for known spotted hyenas sampled at least three dates. this is the first report of sapovirus infection in wildlife species in africa. our results extend the host species range for this genus to include the spotted hyena, african lion and bat-eared fox. prior to our study, sapovirus infection in carnivores worldwide was not known from any species belonging to the felidae (including the domestic cat) [72] , or hyaenidae, but was reported only for species in the families otariidae (californian sea lion) [23] , mustelidae (mink) [25] and canidae (domestic dog) [26] . our phylogenetic analysis based on partial rdrp gene sequences revealed that sapovirus strains from wild carnivores in the serengeti ecosystem formed a monophyletic group that was distinct from other sapovirus strains worldwide, including strains from the three previously identified carnivore hosts (fig 2) . strains from spotted hyena formed a separate sub-group from those obtained from african lions and bat-eared foxes, even within the same sampling year (fig 2) , suggesting that strains circulating in the spotted hyena population are distinct from those in the african lion and bat-eared fox populations. evidence for a degree of speciesspecificity in host range is apparent in other viruses of carnivores in the serengeti ecosystem. genetically distinct alphacoronavirus variants infect spotted hyenas and sympatric silverbacked jackal during the same year [35] , genetically distinct strains of kobuvirus infect domestic dogs and wild carnivores [36] , and during the 1993/1994 canine distemper epidemic in the serengeti np, genetically distinct strains circulated in non-canids (african lion and spotted hyena) and canids (domestic dog and bat-eared fox) [73] . more extensive characterization of sapovirus strains infecting carnivore species in the serengeti ecosystem would clarify their host range and help identify which species in the large carnivore guild are infected with sapovirus. currently it is not known whether or not domestic dogs and domestic cats in africa are infected with sapovirus. our results support the conclusion of previous studies, which emphasize the importance of long-term monitoring when documenting the genetic diversity of sapovirus strains [20, 28, 31] . clearly we would have detected far less genetic diversity in our partial rdrp gene sequence data had our sampling of spotted hyenas been limited to a time frame of one or two years (fig 2) , and particularly if sampling was (by chance) only undertaken during non-outbreak years when infection prevalence was low (fig 3) . samples obtained during outbreak years revealed considerable genetic diversity; for example from 2006 to 2007 we obtained sequence data from five different variants, including the distinct 2007 variant (kt777556) which was the least similar to all others from this host species. as we were not able to sequence data from all rt-pcr positive samples, we cannot exclude the possibility that the genetic diversity among spotted hyena strains was higher than our results indicate. even so, in line with a previous study [74] , our result show that sequence data from the non-structural rdrp gene yields useful information on the genetic diversity of circulating sapovirus strains. some outbreaks of sapovirus infection in humans can be linked to the emergence of specific genotypes [18, 75, 76] , suggesting that herd immunity against prevailing genotypes may be evaded by the emergence of genetically novel strains. our long-term monitoring of sapovirus infection in spotted hyenas revealed significant changes in yearly prevalence during the study (fig 3) and the occurrence of three outbreaks of infection. the highest infection prevalence (above 72.4%) occurred during an outbreak from 2002 to 2004, whereas infection prevalence in two later outbreaks (from 2006 to 2007 and in 2010) was considerably lower. presumably the 2002/2004 sapovirus outbreak induced herd immunity to the genetic strains that circulated in spotted hyenas during this period, but our phylogenetic analysis (fig 2) did not reveal the emergence of genetically distinct strains in response to this. even so, our partial sapovirus rdrp gene sequence data are insufficient to draw strong conclusions. for this, a more extensive genetic investigation is needed, particularly using the vp1 gene used to place sapoviruses in genogroups [16, 21] , that may reflect the antigenic relationships between sapovirus [77] . overall sapovirus infection prevalence in spotted hyenas (34.8%) in the serengeti np was several magnitudes higher than the prevalence reported for the domestic dog (< 2%) [26, 72] and the bat h. pomona (1.6%) [28] . moreover, our long-term monitoring reveals that infection prevalence in spotted hyenas was typically high, being above 20% in most years (fig 3) . there has been much discussion about the effect of human age on sapovirus infection (reviewed by [16] ), mostly based on studies on individuals with gastrointestinal infections. however, there is growing evidence from research on humans with and without clinical symptoms which demonstrates sapovirus infection across a wide range of ages [76] , including elderly people [78] . our results on sapovirus infection across different age categories indicate that the likelihood of infections in spotted hyenas was not significantly influenced by age ( table 1) . the long-term perspective of our study allowed us to assess the sapovirus infection status of several individually known spotted hyenas on different sampling dates. several animals transitioned from positive to negative, and we interpret this to indicate that they successfully cleared the virus following infection. if, following an initial infection, spotted hyenas gained long-term immunity against further infection, infection prevalence should decline with age. as prevalence amongst adults reached almost 50% during outbreak years, our results do not provide strong evidence for long-term immunity in this species (table 1) . even so, we found no individual that changed from rt-pcr positive to negative to positive (table 2) which would have provided direct evidence of re-infection. during outbreaks of sapovirus in humans and pigs, cases of re-infection with sapovirus belonging to different genogroups have been reported in both species, suggesting genogroup-specific immunity for sapoviruses [74, 79, 80] . more extensive investigation of the genetic diversity across strains circulating in our spotted hyena population is needed to determine whether sapovirus strains induce (1) short-term immunity, which would permit re-infection with strains from the same sapovirus genogroup, (2) genogroup-specific immunity, in which re-infection would involve strains from different genogroups, or (3) possibly a complex interplay between the two, as hypothesized for the genetically and antigenically diverse norovirus which is closely related to the sapovirus genus [81, 82] . interestingly, infection status depended on the length of time between repeated samples. animals that were negative on two separate dates (table 2 ) had the shortest median period between sampling dates (82 days), those that changed from negative to positive (158 days) and from positive to negative (180 days) had an intermediate median number of days, whereas animals that were positive on both sampling days (715 days) had the longest median period. taken together, these results suggest that when a spotted hyena is infected, infection is cleared, and reinfection is unlikely within a period of several months, which is consistent with the idea that exposure to sapovirus does not provide long-term immunity against further infection. in humans, sapovirus shedding often subsided 14 days after the onset of illness [83] , but can persist for up to 38 days [74] . hence, we speculate that the spotted hyenas which were positive on two sampling dates several months apart were animals that were re-infected rather than individuals with persistent long-term infections. however, currently we cannot exclude the possibility that there may be spotted hyenas that shed sapoviruses for periods spanning several months. spotted hyena social and ranging behavior has been shown to structure transmission routes and the likelihood of infection by a broad range of pathogens [35, [41] [42] [43] [44] ]. yet when we tested whether these two factors influenced sapovirus infection in this species, neither the predicted effect of contact rates based on known patterns of greeting ceremonies, nor the extent of an individual's range significantly influenced the likelihood of infection in our study population (table 3) . clan size was a significant factor, but contrary to our expectation, the likelihood of infection declined with increasing clan size. this phenomenon is known in the behavioral literature as the attack-abatement effect [84] or as the encounter-reduction effect [48, 85, 86] . in multi-species host assemblages the same phenomenon in the ecological literature is termed a 'dilution' effect. at least five non-exclusive mechanisms have been identified [49] that can cause a reduction in infection incidence as the number of host species increases, but not all of them are relevant to intraspecific sapovirus infections in spotted hyenas. we have no evidence that sapovirus infection increases the death rate of infected individuals (mechanism 1), as no obvious clinical signs are associated with sapovirus infection in most spotted hyenas. although we lack a precise measure of the recovery rate (mechanism 2), the general absence of obvious clinical signs of sapovirus infection suggest that the recovery rate is already very high, so that a change in this factor is likely to be modest. a decrease in the density of susceptible individuals (mechanism 3) is unlikely unless this results from a substantial increase in the proportion of clan members immune to sapovirus infection, even if immunity is relatively transient. a substantial increase in the prevalence of immune clan members would result in far fewer sapovirus susceptible individuals. moreover, such an increase in herd immunity would probably also lead to a decrease in the prevalence of sapovirus excreting clan members, thereby reducing the probability of: (1) sapovirus transmission per encounter (mechanism 4) between clan members and (2) the encounter rate between susceptible and infectious individuals (mechanism 5) in a clan. further research and more detailed modeling of the interplay between clan size and the prevalence of clan members in different sapovirus infection states (susceptible, infected/excreting virus, and immune) is required to test this idea. in the context of our study, mechanism 5 (encounter rate between susceptible and infectious individuals) is similar to mechanism 3 (decrease in the density of susceptible individuals) because a reduced density of susceptible clan members caused by a rise in the prevalence of transient immunity would also curb the number of infected animals in a clan and may prevent their number growing in proportion to increasing clan size, and possibly holding them at or below a fixed number (threshold). an increase in the risk of internal pathogen infection with a decrease in a group size component has been reported by other studies [3, 42] . as sapoviruses cannot be cultured, with the exception of the strain pec cowden [50, 51] , knowledge of viral tropism and the receptor use for entry to host cells is limited. moreover, sapovirus typically infects intestinal tissue and to our knowledge is not known to infect other tissues, as illustrated by the absence of viral rna in any extra-intestinal tissues in gnotobiotic pigs inoculated with pec cowden [87] . however, in a few dead spotted hyenas (typically road casualties) from which we opportunistically obtained tissue samples, we detected sapovirus rna most often in the spleen and occasionally also in lymph nodes, intestines and the liver. to our knowledge this is the first report of sapovirus rna in extra-intestinal tissues following natural infections. the possibility that sapoviruses disseminate to extra-intestinal tissues may be of clinical importance [88] . notably, in asymptomatic mice shedding murine norovirus in feces, viral rna was also found in several extra-intestinal organs, including the liver, spleen and lymph nodes [89] . studies on caliciviruses infecting wild carnivores have focused on feline calicivirus (fcv, genus vesivirus, family caliciviridae). for example, serological surveys documented that african lions [90] and spotted hyenas in the serengeti ecosystem were exposed to fcv [91] with a high prevalence. our phylogenetic analysis shows that the variants we report from wild carnivores in africa are distinct from both fcv and canine calicivirus (fig 2) . a norovirus (genus norovirus, family caliciviridae), genetically related to human noroviruses was reported from a captive african lion [92] and subsequently identified in domestic dogs and domestic cats [93, 94] . combining these findings with our results suggests that the spotted hyena and african lion can be infected by viruses belonging to three different genera of the family caliciviridae. we provide the first report of sapovirus infection in wild carnivore species in africa, including the spotted hyena, african lion and bat-eared fox. long-term monitoring revealed considerable genetic diversity of variants from these species which were phylogenetically distinct from previously reported sapovirus strains from other geographical areas worldwide. sapovirus prevalence in the spotted hyenas varied between years and was not influenced by age. an individual's likelihood of infection significantly declined with increasing group size, consistent with an encounter reduction effect. our results reinforce the importance of long-term studies in 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genotype diversity and prevalence of infection in wild carnivores in the serengeti national park molecular characterization of canine kobuvirus in wild carnivores and the domestic dog in africa population dynamics, population size and the commuting system of serengeti spotted hyenas. sinclair are and arcese p, editors. serengeti. ii. dynamics, management, and conservation of an ecosystem crocuta crocuta spotted hyena canine distemper virus infection in serengeti spotted hyenas regular exposure to rabies virus and lack of symptomatic disease in serengeti spotted hyenas a hepatozoon species genetically distinct from h. canis infecting spotted heynas in the serengeti ecosystem factors influencing dypylidium sp. infection in a free-ranging social carnivore, the spotted hyaena (crocuta crocuta) does lactation lead to resource allocation trade-offs in the spotted hyaena? the impact of a pathogenic bacterium on a social carnivore population the erect 'penis' is a flag of submission in a female-dominated society: greetings in serengeti spotted hyenas the commuting system of serengeti spotted hyaenas: how a predator copes with migratory prey. ii. intrusion pressure and commuters space use the commuting system of serengeti spotted hyaenas: how a predator copes with migratory prey. iii. attendance and maternal care animal grouping for protection from parasites: selfish herd and encounter-dilution effects effects of species diversity on disease risk serial propagation of porcine enteric calicivirus-like virus in primary porcine kidney cell cultures serial propagation of porcine enteric calicivirus in a continuous cell line. effect of medium supplementation with intestinal contents or enzymes design and evaluation of a primer pair that detects both norwalk and sapporo-like caliciviruses by rt-pcr bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt muscle: a multiple sequence alignment method with reduced time and space 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sapovirus infection in pig litters raised under experimental conditions norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations genetic and antigenic diversity among noroviruses natural history of human calicivirus infection: a prospective cohort study attack abatement: a model for group protection by combined avoidance and dilution parasitism and group size in social animals: a meta-analysis parasites and the behavior of animals comparative pathogenesis of tissue cultureadapted and wild-type cowden porcine enteric calicivirus (pec) in gnotobiotic pigs and induction of diarrhea by intravenous inoculation of wild-type pec the mucosal lesion of the proximal small intestine in acute infectious nonbacterial gastroenteritis desk encyclopedia of human and medical virology prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging lions in east africa antibodies to canine and feline viruses in spotted hyenas (crocuta crocuta) in the masai mara national reserve norovirus in captive lion cub (panthera leo). emerg infect dis detection and molecular characterization of a canine norovirus discovery and genomic characterization of noroviruses from a gastroenteritis outbreak in domestic cats in the us key: cord-022501-9wnmdvg5 authors: nan title: p1460 – p1884 date: 2015-12-28 journal: clin microbiol infect doi: 10.1111/j.1470-9465.2006.12_4_1431.x sha: doc_id: 22501 cord_uid: 9wnmdvg5 nan resistance rates (rr) over a period of 5 years were generally better than those reported for a total of 40 icus. the mean mrsa rr was 22.1%, for all the sari icus, whereas it was only 2.7% in the study icu. by the end of 2003 duration of treatment for pneumonia had been reduced to 5-7 days and written guidelines on empiric antibiotic treatment and prophylaxis were revised with respect to the resistance situation of the study icu. the significant decrease between 2000 and 2004 in total antimicrobial ad from 1,099 to 607 in the study icu resulted mainly from the reduced consumption of 2nd generation cephalosporins, carbapenems and imidazoles. ni did not change significantly over time. compared to the year 2000, the costs for antibiotics were halved from €51,102 to 22,324, which corresponds to €18.7/pd and €6.6/pd, respectively. the percentage of antibiotics in the total icu budget for pharmaceuticals decreased from 14.6% to 10.4%. conclusion: surveillance and feedback of antibiotic use and resistance can serve as a valuable quality control instrument and can have an impact on antibiotic treatment. from 2000 to 2004, antibiotic use was reduced by 45% and costs for antibiotics/pd were cut by two third in the icu study without any increase in device associated nosocomial infection rates. the resistance situation was generally better than in all sari icus, but showed heavy fluctuations. similar illness burden but different antibiotic prescription to children: a population-based study k. hedin, m. andre, a. håkansson, n. rodhe, s. mö lstad, c. petersson (växjö, falun, malmö, linköping, se) objectives: respiratory tract infections are the most common reason for antibiotic prescription in sweden as in other countries. the prescription rates vary markedly in different countries, counties and municipalities. the reasons for these variations in prescription rates are not obvious. the aim of the study was to find possible explanations for different antibiotic prescription rates in children. therefore a prospective population based log book study was conducted in four municipalities which, according to official statistics, had high and three municipalities which had low antibiotic prescription rates. methods: during one month, parents recorded all infectious symptoms, physician consultations and antibiotic treatments, from 848 18-month-old children in a log book. the children's parents also answered a questionnaire about socioeconomic factors and concern about infectious illness. results: antibiotics were prescribed to 11.6% of the children in the high prescription area and 4.7% in the low prescription area (crude or 2.67 (95% ). after multiple logistic regression analyses taking account of socioeconomic factors, concern about infectious illness, number of symptom days and physician consultations, differences in antibiotic prescription rates remained (adjusted or 2.61 (95%ci 1.14-5.98)). the variable that impacted most on antibiotic prescription rates although it was not relevant to the geographical differences was a high level of concern about infectious illness in the family. conclusion: the differences in antibiotic prescription rates could not be explained by socioeconomic factors, concern about infectious illness, number of symptom days and physician consultations. the differences may be attributable to different prescription customs, in which case physicianś prescription patterns are not always rational. decreasing outpatient antibiotic prescribing in germany, 1995 germany, -2004 , does not include newer macrolides, fluoroquinolones and extendedspectrum beta-lactams w.v. kern, k. de with, k. nink, h. schrö der (freiburg, bonn, de) objective: the esac (european surveillance of antibiotic consumption, www.ua.ac.be/esac) project has shown that outpatient antibiotic prescribing in germany has been comparatively low among european countries. we assessed trends over time and regional variation of outpatient antibiotic use in germany, and wondered if the observable decreasing trend included all drug classes to a similar extent. methods: prescription data (compulsory health insurance covering >90% of the population, sample of 0.4% until the year 2000, all prescriptions thereafter) were analysed using the atc/who methodology and current ddd definitions. we specifically defined the following drug groups: ''basic'' penicillins (bpens, oral penicillin or aminopenicillins), extended-spectrum betalactams (esbls, oral cephalosporins, staphylococcal penicillins, aminopenicillin/betalactamse inhibitor combinations, parenteral cephalosporins and broadspectrum betalactams), newer macrolides (nmls, roxithromycin, clarithromycin, azithromycin) versus older macrolides (omls). quinolones (fqs), folate synthesis inhibitors (t/ss) and tetracyclines (tets) were also assessed. data were expressed in yearly ddd/1000 persons covered by the insurance (ddd/1000). findings: outpatient prescribing in 1995 was 6140 ddd/1000 (corresponding to 16.8 did = ddd/1000 and day) and decreased to 5430 ddd/1000 in the year 2000 and to 4672 ddd/1000 in 2004. the decreasing trend over the last 4 years was observed in all regions. the decrease was most significant for omls ()55%), t/ss ()48%), tets ()36%), and bpens ()13%) while there was no decreasing use of esbls (±0%) and increases in the rate of prescribing nmls (+13%) and fqs (+43%). tets and bpens, however remained the most prescribed antibiotics in 2004. regional variations in 2004 remained large for bpens (>3-fold) with very low prescribing rates in the eastern region, but were small for t/ss, nmls and fqs (<2-fold). conclusions: over a decade we observed a 24% decreasing outpatient antibiotic prescribing that included relevant antibiotic drug classes except esbls, nmls and fqs. the relative increase was most significant for fqs. severe community-acquired pneumonia admitted to the intensive care unit: impact of antibiotic therapy delay on hospital mortality antibiotic therapy were enrolled in the study. pts were divided in 4 groups according to time to treatment (<2 h gi, [2] [3] [4] [4] [5] [6] [7] [8] . baseline severity scores (apache ii, sofa, psi, curb-65), microbiological documentation, and hospital outcome were compared for all groups. results: 152 pts were included in the study. microbiological documentation was achieved in 80% of all pts, positive blood cultures in 54/152 (35.5%), s. pneumoniae in 64/152 (42%). mean age was 66 ± 13, apache ii 25.8 ± 7.6, sofa 8.8 ± 3.7, psi 158 ± 40, curb-65 3.2 ± 1, mechanical ventilation in 67.7% and vasopressors use in 63.1%. overall icu and hospital all-cause mortality were 27.5% and 35.5%, respectively. baseline severity scores were comparable in all 4 groups and their respective hospital mortality is provided in table 1 . conclusions: in severe cap, treated with a combination therapy, time to treatment seems to have an impact on hospital all-cause mortality. based on our results, antibiotic treatment should be initiated within the first 2 hours after hospital admission. (period ii) -cefoperazone/ sulbactam (8 g a day) as monotherapy were used as the empirical antibacterial therapy of vap. the rotation from cefepime to cefoperazone/sulbactam was performed due to our previous study demonstrated high frequency of esbl producers among enterobacteriaceae. the samples from the lower respiratory tract were obtained by mini-bal. the sensitivity of microorganisms to the antibiotics studied (ceftazidime, cefepime, cefoperazone/sulbactam and carbapenems) was determined by the disk diffusion method. results: the main pathogens of vap were s. aureus (20%), p. aeruginosa (19%), enterobacteriaceae (23%) and this structure did not changed during both periods. the antibiotic sensitivity of p. aeruginosa and enterobacteriaceae (k. pneumoniae and e. coli), was studied separately. a high level of resistance of enterobacteriaceae to cefepime can be explained by the strains prevailing in the given icu, which produced extended spectrum beta-lactamases (ctx-m). the resistance of enterobacteriaceae to cefepime was 57.5% in i period and 80.5% in ii period, to ceftazidime -90.0 and 100%, meropenem -0 and 0%, imipenem -5.1 and 2.1%, cefoperazone/sulbactam -10.9 and 20.8%, respectively. a change of cefepime for cefoperazone/sulbactam was not followed by any decrease of enterobacteriaceae resistance level to cefepime during ii period. the resistance level of p. aeruginosa to cefepime was 20.5% in i period and 24.1% in ii period, to ceftazidime -22. 5 and 33.0%, meropenem -44.7 and 39.5%, imipenem -50.1 and 39.5%, cefoperazone/ sulbactam -16.9 and 12.5%, respectively. conclusion: the exclusion of cefepime for 9 months didn't improved the sensitivity of enterobacteriaceae to this medication. the level of resistance of p. aeruginosa and enterobacteriaceae to cefoperazone/sulbactam did not increased despite a wide use of this antibiotic during 9 months. antibiotic consumption in german acute care hospitals m. steib-bauert, k. de with, e. meyer, p. straach, w.v. kern (freiburg, frankfurt, de) objective: outpatient antibiotic use in germany differs substantially between eastern and southern parts of the country (relatively low use) and western part (relatively high use). there is no nationwide estimate of hospital antibiotic use and its geographic variation if any. the aim of the present study was to provide an estimate of recent hospital antibiotic use density in germany and to identify basic unit/hospital characteristics associated with excess use. methods: data on hospital consumption of systemic antibiotics in anatomical therapeutic chemical (atc) class j01 were obtained from a convenience sample of 145 acute care hospitals in germany that participated in an ims survey in the year 2003 and had complete data (dispensed drugs and patient-days per year) for at least one non-paediatric, non-psychiatric department or ward. a total of 275 non-icu surgical departments/wards, 229 non-icu non-surgical (general medicine, oncologyhaematology, neurology/stroke) departments/wards, and 184 icus covering >16 million patient-days were analysed. data were expressed in ddd (who/atc definition version 2001) or ''prescribed/recommended daily doses'' (pdd, better reflecting [high] dosages given to hospitalized patients) per 100 patient days (ddd/100 and pdd/100). findings: the weighted mean over all departments/wards incl. icus was 49.8 ddd/100 (31.4 pdd/100). as expected, icu antibiotic use density was much higher than use in non-icu areas, and use in haematology-oncology was higher than in other non-surgical departments/wards. in univariate analyses, bed-size category and university affiliation (icus, surgical wards), region (icu, surgical and non-surgical wards) and haematology-oncology as specialty (non-surgical wards) were associated with use density, but these associations were only partly confirmed in multivariate logistic regression analyses of factors associated with excess ( ‡75%) use density which showed university affiliation and haematology-oncology to be independently associated with high use. conclusions: based on this hospital sample, antibiotic use in german hospitals shows little, non-significant regional variation and appears to be similar to what has been described from other european countries. adjustment of the data at least for university affiliation and haematology-oncology is important in comparative analyses of hospital antibiotic consumption. impact of formulary change in medical intensive care unit on outcome of infection and antimicrobial resistance sought to evaluate a formulary change and impact it has on infection and resistant. methods: prospectively, all patients in a 20-bed icu were followed for a period of 4 months in phase i (390 patients per 2379 patient days) and to collect baseline data after a decrease in the use of piperacillin-tazobactam (pt) when substituted by cefepime for a period of 4 months in phase ii (383 patients per 2260 patient days). results: total infections in phase i vs. phase ii were lower respiratory tract (lrti) 214 patients (55%) vs. 203 patients (53%); urinary tract infection (uti) 94 patients (24%) vs. 96 patients (25%); and sepsis of undetermined aetiology 70 patients (18%) vs. 65 patients (17%), respectively. there were no significant differences in death (22% vs. 19%) , cure or improvement of infection (53% vs. 56%), readmission to the unit (3.5% vs. 3.2%), hospital risk of death (29.8% vs. 30.2%), mean length of icu stay (6.1 days vs. 5.9 days), or rates of nosocomial infection (6.3% vs. 5.1% for lrti; 4.0% vs. 4.2% for uti; 0.8% vs. 0.0% for soft tissue infection; 0.8% vs. 1.0% for bacteremia; 2.1 vs. 1.0 per 1000 patient days for intravenous catheter infection) in phase i and ii respectively. the cost of antimicrobial acquisition in phase i and ii were $548 and $433 per patient respectively (p < 0.001). the mean antimicrobial treatment costs per patient for pt were $145 vs. $100 and cefepime were $80 vs. $105 in phase i and ii respectively (p < 0.01). the in vitro susceptibility and rate of infection and colonization with escherichia coli were unchanged in both study periods. there were 68 vs. 39 staphylococcus aureus (p < 0.001); of these 94percnt vs. 87percnt were methicillinresistant s. aureus and 11 vs. 9 enterococcus faecium (82% vs. 77% vancomycin-resistant enterococci) in phase i and ii respectively. there were 73% vs. 31% pseudomonas aeruginosa and 70% vs. 4% klebsiella pneumoniae extended spectrum beta lactamases in phase i and ii respectively. conclusion: the implementation of formulary substitution of pt to cefepime in the medical icu had resulted in a decrease in the use of pt. in addition, there were decreased costs and less s. aureus infections without adversely affecting the outcome of infection or antimicrobial resistance. intravenous antibiotic use in scottish hospitals; evaluation of the glasgow antimicrobial audit tool r.a. seaton, d. nathwani, p. burton, e. douglas (glasgow, dundee, uk) introduction: there are few data on antibiotic prescribing within scottish hospitals and a coordinated multisite point prevalence survey had not been performed before. there is concern that antimicrobials are overused in hospitals. methods: antibiotic use in acute medical and surgical units in 10 scottish hospitals across 5 trusts, was investigated using a point prevalence survey. data were collected by pharmacists. appropriateness of the iv route of administration was determined by review of data by an infectious diseases physician (idp) and compared with a specifically designed computerised algorithm. the idp also judged the appropriateness of the chosen iv agent against local guidelines. 3826 patients from 10 hospitals in 5 regions were surveyed on a single day. 1079 (28.3%) were receiving an antibiotic, 381 (35.3%) intravenously. 197 receiving oral antibiotics had received an iv previously. median duration of iv therapy was 4 days (iqr 2-7 days) and time from iv to oral switch was 3.5 (2) (3) (4) (5) (6) . the idp judged appropriate iv route in 84% patients compared with 84.8% by the algorithm. the sensitivity of the algorithm was 93.4% and specificity 60.7%. the positive predictive value was 92.6% and the negative predictive value was 63.8%. the idp judged iv agents to have been chosen and administered appropriately in 80%. most frequently prescribed iv agents were 3rd generation cephalosporins (3gc) (28.3%), co-amoxiclav (20.2%), metronidazole (19.2%), glycopeptides (18.6%). significant regional differences were seen for most antibiotic groups including 3gcs (49.2% (site 3) vs 24.4% (sites 1, 2, 4, 5) , p < 0.001) and glycopeptides [31.8% (site 1) vs 9.3% (site 2, 3, 4, 5) , p < 0.001]. it is possible to coordinate, collect and compare data from scottish hospitals. the gaat gives a good estimate of the appropriateness of iv therapy. significant differences in prescribing patterns between similar patient groups across different hospital sites were demonstrated. such data may usefully inform local and national audit and support prescribing initiatives. associations between continuous variables were tested in univariate analysis with the spearman correlation test (r). multiple linear regression analysis was performed in a backward stepwise approach. results: the median rate of total hospital glycopeptides use was 4.11 (range 0.21 to 27.22) ddds per 1,000 pd with higher consumption in large public hospitals. consumption was higher in intensive care areas (median 46.51; range 7.19 to 134) than in surgery areas (median 4.5; range 0.17 to 24.76 ) and in medicine (median 4.26 ; range 0 to 41). glycopeptides use correlated with number of central line per 1,000 pd (r: 0.44; p: 0.03) and with size of the various areas in the hospital (for intensive care, r: 0.50; for medicine areas, r: 0.33 and for surgery areas, r: 0.42; p < 0.05). median incidence of mrsa was 0.87 per 1,000 pd. incidence of mrsa explained a small proportion of the variation in hospital glycopeptides consumption (r2: 0.13). in a multivariate linear regression model, incidence of mrsa and number of beds in surgery areas were independent predictors of total glycopeptides use in the hospital (r2 adjusted: 0.39). after controlling for these factors, number of central-line per 1,000 pd was no more associated with glycopeptides use. conclusion: in our hospitals, total glycopeptides use was not heavily determined by incidence of mrsa. although glycopeptides use in surgery areas was not the highest, the total number of surgery beds in the hospital explained a large variation of the total hospital glycopeptides use. therefore we had to take it into account to interpret these consumption and to decide further evaluation. antibiotic management of acute lower respiratory tract infections among dutch elderly patients in primary care j. bont, c. birkhoff, t. verheij, e. hak on behalf of esprit objectives: acute lower respiratory tract infection (lrti) can cause various complications leading to morbidity as well as mortality notably among elderly patients. antibiotic treatment of lrti is common, despite dutch clinical guidelines recommending antibiotics only in case of pneumonia or high risk of serious complications. we assessed the course of illness and outcome of pneumonia, acute bronchitis and exacerbations of copd or asthma among dutch elderly patients in primary care and assessed whether gps were inclined to prescribe antibiotics more readily to patients with potential risk factors for complications in acute bronchitis or exacerbations of copd/ asthma. methods: we retrospectively analysed medical data from 3,166 episodes of lrti among patients ‡65 years of age presenting in primary care to describe the course of illness and outcome. the relation between prescriptions of antibiotics and patients with risk factors for a complicated course was assessed by means of multivariate logistic regression. risk factors for a complicated course included heart failure, history of myocardial infarction, angina pectoris, diabetes, history of stroke, dementia, malignancy, and history of pneumonia or hospitalisation in preceding year. results: one or more complications arose in 17% of episodes of lrti. among these, 8% suffered from pulmonary complications, 5% had cardiovascular complications (heart failure, myocardial infarction etc.), 5% had a protracted course and 0.6% had a diabetes event. in 6.9% of the patients complications led to hospital admission and in 2.4% lrti were fatal. antibiotics were more readily prescribed to patients aged ‡90 years, when heart failure was present and in patients with diabetes. no significant association was observed in patients with other co-morbid conditions. patients diagnosed with an exacerbation of copd or acute bronchitis with a history of pneumonia or hospitalisation in the preceding year were not more likely to receive antibiotics. conclusions: a considerable part of elderly patients with a lrti suffers from a severely complicated course in primary care. although gps are inclined to prescribe more readily antibiotics in the very old and those with heart failure or diabetes, other potential risk factors are not taken into account. objectives: in this study it was aimed to analyse the infectious diseases (id) trainees' night/weekend shift consultation process in terms of patient and consultant characteristics, types of recommendations, and compliance with recommendations. methods: all consultations performed by id trainees in night shift and at the weekends between june 10th-august 10th 2004 were analysed in terms of consultation type [treatment continuation (tc), consultation for surgical antibiotic prophylaxis (pa), and consultation with or without a request of a specific antibiotic (others)]. appropriateness of recommendations was assessed the day after the consultation by infectious diseases specialists (ids). adherence to recommendations was assessed 3 days after the consultation by idss. recommendations including antibiotics were considered appropriate, if they were appropriate according to national and international guidelines. recommendations were considered complied, if they were done in up to 72 hours after the consultation (except the consultations in the emergency medicine and the consultations in which antibiotics were started by the counselling idss). results: of 436 consultations 79 was for tc, 74 was for pa and 290 was for others. the clinic where id consultations were requested mostly was general surgery clinic (152/436, 34.9%) . in 2% of all consultations trainees consulted the specialists. overall 146 consultations (74 for sp, 69 for a clinical infectious disease diagnosed clinically, 9 for an infectious disease diagnosed microbiologically) were for requesting spesific antibiotic(s). pa were approved in 68 of 74 consultations. antibiotic was not recommended in 14 of 290 other consultations. in six of 74 consultations for pa antibiotic was changed for a clinically diagnosed infectious disease. in one of 79 consultations for tc antibiotic was changed due to lack of response to the given antibiotic, in others tc was approved. inappropriate antibiotic recommendation rate was 2.4% (8/32, 4 inappropriate choice, 3 inappropriate dosage, one antibiotic unnecessary). overall compliance to id recommendations was 76.1% (476/608). rate of compliance to antibiotic recommendations was evaluated in 240 consultations and was found 98.2% (277/282) and was higher than compliance to other (microbiology etc.) recommendations (60.8%, 199/327, chi square p < 0.05). conclusion: methodologies to improve the compliance to nontreatment based recommendations and optimizing antibiotic selection is necessary. study of the influence of online practice guidelines on the appropriateness of antibiotic prescribing in a university-affiliated psychiatric hospital j.f. westphal, c. nonnenmacher, d. gregoire, m. hittinger, c. oulerich, f. jehl (brumath, strasbourg, fr) background: problems with the dissemination of guidelines are frequently cited as a major reason for failure to impact practice. reviews of the effectiveness of various methods of guideline dissemination show that the most predictable impact is achieved when the guideline is made accessible through computer-based reminders that are integrated into the clinician's workflow. we report a time-series prospective investigation aimed at comparing the appropriateness of antibiotic (ab) orders for pneumonia at the treatment initiation level after vs. before having embedded our current ab guidelines for pneumonia in the computerized physician drug-order entry system of our teaching psychiatric hospital comprising 410 adult beds. methods: in total, 160 consecutive ab orders for pneumonia were evaluated by the pharmacy department, including 80 orders just before and 80 orders just after implementation of online ab guidelines. appropriateness of ab orders relative to the guidelines was assessed according to 3 criteria: (1) the choice of ab with respect to the mode of acquisition (community-or hospital-acquired) of pneumonia or the presence of clinical risk factors for involvement of gramnegative bacilli, (2) the daily dosage, (3) the planned duration of treatment. data were extracted from the computerized infection declaration system that recorded all ab-requiring infections in our hospital. results: the number of ab orders with at least 1 criterion of inappropriateness tended to decrease, yet not significantly (p = 0.11), after vs. before implementation of online guidelines: 30/80 (36.5%) and 40/80 (50.0%), respectively. the number of criteria of inappropriateness relative to all ab orders for pneumonia was significantly lower in the post-implementation period: 43.8% vs. 65.0% before implementation (difference 21.2%, 95% ci 6. 1-36.3, p < 0.01) , with a trend to a decreased number of orders containing more than 1 criterion of inappropriateness. analyzed separately, the numbers of inappropriate orders for the choice of the ab, or the daily dosage, or the planned duration of treatment decreased, yet not significantly (p > 0.1 for each criterion), in the post-vs. preimplementation period : 11 vs. 18, 12 vs.15, 12 vs.19, respectively. conclusion: in this study, the moderate impact on ab prescribing practices of online guidelines available at the time of drug order shows that additional types of intervention are needed to improve further the quality of ab prescribing. material: the pilot hospitals had a median capacity of 654 (range, 154 to 1597) beds; their regional distribution was representative of population size; 18 were general hospitals, 8 teaching hospitals and 10 general hospitals with teaching beds. results: ams were internists (28), microbiologists (13) and pharmacists (13). amts included a mean of 10 members who met every 6 weeks on average. all hospitals irrespective of size or affiliation had undertaken a wide range of antibiotic management interventions in 2003, which increased in 2004; these included (in 2003 and 2004, respectively) : major review of formulary (in 10 and 23 hospitals), development of clinical guidelines (69 and 215 topics), restricted access to selected antibiotics (carbapenems, glycopeptides, quinolones, new drugs; in 25 and 33 hospitals). in 2003, antibiotic consumption databases were established in 35 hospitals and antibacterial susceptibility databases in 31 hospitals. in 2004, cross-analysis of these databases was performed in 28 hospitals. in 2004, prescribing assistance, antibiotic stop orders, treatment streamlining and iv/po therapy switch were implemented in 32, 21, 24 and 24 hospitals, respectively. in 2004, 26 hospitals reported a better use of target antibiotics, 15 hospitals a decrease in consumption of restricted antibiotics, 5 hospitals a decrease of total antibiotic consumption, 2 hospitals a decrease in high consumer departments. conclusion: all hospitals participating in the amt pilot scheme have developed multiple antibiotic policy interventions and established monitoring and guidance of antibiotic prescription. preliminary data from some hospitals indicated success in meeting self-defined targets of appropriate use and reducing the consumption of selected antimicrobial agents. more systematic evaluation using standard quantitative and qualitative indicators is planned. antibiotic prescribing practices at two linked london teaching hospitals p1476 comparison of different antibiotic consumption measurement methods in large multidisciplinary hospital e. pujate, i. apine, u. dumpis (riga, lv) objectives: antibiotic selection pressure is determined by the total amount of antibiotics, number and density of patients treated with antibiotics in the particular geographical area. several antibiotic consumption detection methods should be combined in the hospital setting. our objective was to evaluate efficacy of different approaches in large multidisciplinary hospital. methods: point prevalence studies were repeated annually at [2002] [2003] [2004] in stradins university hospital (1000 beds) in latvia. all patients receiving antibiotics on the day of the survey were identified and their medical records were reviewed. data on antibiotics, dose and route of administration were collected. in addition, annual data on antibiotics dispensed to the departments were collected from pharmacy. total used grams for each antibiotic were expressed into defined daily doses (ddd-who). bed days (bd) and admission days (ad) were used as denominators. results: table 1 total use of antibiotics in stradins university hospital 2002 hospital -2004 the most commonly used antibiotic groups in the pharmacy study were 1st generation cephalosporins (13.35 ddd/100 bd in 2002 , 11.6 in 2003 , 10.8 in 2004 ) and penicillin's with extended spectrum (11.20, 11.98, 13.15 ) followed by fluoroquinolones (6.26, 8.13, 8.69 ) and metronidazole (4.42, 4.59, 5.61 ). there was no significant difference between distribution of different antibiotics from prevalence and pharmacy studies if calculated in ddds. in contrast, distribution of antibiotics calculated per patient in the prevalence study was quite different; 1st generation cephalosporins (8. 71%, 8.63%, 5.84% in 2002, 2003, 2004 respectively) and fluoroquinolones (3.05%, 6.69%, 5.63%) with smaller proportion of extended spectrum penicillins (4.36%, 3.88%, 3.93%) and metronidazole (4.03%, 3.56%, 4.68%). conclusions: there were no differences in the distribution of antibiotics calculated in ddds per bed days and admissions. distribution of antibiotics in annual pharmacy studies and point prevalence studies if calculated in ddds were also similar. in contrast, the prevalence data expressed as a proportion of patients with selected antibiotics showed quite different distribution. studies using only ddds may overestimate use of certain antibiotic groups in our setting where who ddds are significantly different from actual pdds used. a study of prescribing patterns and errors of antibiotics in a saudi hospital m. al-jamal, m. al-barrak (riyadh, sa) background: the term ''prescribing patterns'' has been used extensively in studies to describe different aspects of the prescribing process. antibiotics as well as other drugs are prescribed for the purpose of achieving definite therapeutic outcomes that improve a patient's quality of life while minimizing risk. in the clinical literature, the incidence of antibiotics prescribing errors ranges between 0.5% and 18.8%. objective: in this study we will address antibiotics prescribing patterns and the incidence of prescribing errors in a tertiary hospital and the potential relationship between them. methods: a prospective study of all prescriptions in a 3-month period (june to august 2003) in a tertiary hospital has been analysed. the hospital provides both primary and secondary levels of care. criteria used include frequency of selected prescribed drugs, average number of items per prescription, compliance to the hospital formulary, frequency of prescriptions for antibiotics, generic prescribing and diagnosis. the prescribing patterns and the incidence of prescribing errors were performed. results: total number of prescriptions for the 3-month study was 24,404. emergency room (er) and primary care have the highest number of prescriptions (37.1%). the average number of items per prescription is 2.1. the most prescribed drugs by primary care (25.3% errors), emergency are antibiotics (28.2%), medicine (3.7), ophthalmology (22.3), gynaecology (7.8), and paediatrics (17.8). the prescription errors were 13.6% in primary care and 22.3% in emergency department. discussions and conclusions: over 24000 prescriptions were included in this study. the incidence of prescribing errors was 18.8% the average number of items per prescription was 2.1. total prescription errors are also related to frequency of prescribing antibiotics. there was a relation between prescribing of antibiotics and prescribing of trade names (p < 0.01), and compliance to the hospital formulary (p < 0.001). several factors influence prescribing patterns and variations in prescribing rates has been identified. these include general physician behavior, differences in morbidity and mortality patterns, social perception toward illness, and physician clinical skills, experience and qualification, as well as physician continuing education and training. special antibiotic prescribing guidelines and restrictions should target primary care and emergency department physicians. effect of a policy for restriction of selected classes of antibiotics on antimicrobial drug cost and resistance of the non-restricted antibiotics. the logistic regression model we performed showed that the new policy had an independent positive effect on the in vitro antimicrobial susceptibility of pseudomonas aeruginosa (p = 0.051) but not of acinetobacter baumannii and escherichia coli isolates. conclusion: our data suggest that there are considerable limitations of the programs aiming to reduce the consumption of restricted antibiotics through the approval of their use by specialists, at least in a proportion of settings. education programs that aim to involve the medical staff directly responsible for the care of patients in voluntary decisions regarding the appropriate use of antimicrobial agents may have more profound and sustainable success, and thus, deserve to be studied. estimating hospital versus ambulatory care consumption of antibiotics in southwestern germany k. de with, m. steib-bauert, h. schrö der, k. nink, w.v. kern (freiburg, bonn, de) objective: preliminary data from the esac (european surveillance of antibiotic consumption, www.ua.ac.be/esac) project indicated that the proportion of hospital care (hc) antibiotic use on total antibiotic use in several european countries ranges between 5 and 20%. only few countries, however, have so far been able to report representative countrywide information on both hc and ambulatory care (hc) antibiotic consumption. we estimated ac versus hc consumption of antibiotics for one of the 16 german federal states located in the southwestern part of the country with a 10.5 million population. methods: data on hc consumption (atc class j01) were obtained from a convenience sample of acute care general hospitals (n = 42), extrapolated to state-wide consumption (using official statistics for the total state-wide 152 general plus 77 special non-psychiatric/non-paediatric/non-radiotherapy hospitals), expressed in defined daily doses per 1000 inhabitants and day (did), and finally compared to ambulatory care antibiotic use density in the same region and period of time (years 2001 and 2002) . findings: the estimated state-wide hc consumption of antibiotics was 2.1 did (95% confidence interval, 1.9 to 2.2 did) in both years. state-wide antibiotic consumption in the ac setting during the same time was 12 did (~85% of total consumption). ac consumption of fluoroquinolones (1.1-1.2 did, 79%) and macrolides/clindamycin (1.8 did, 95%) made up a major proportion of total use of that drug classes. conclusions: hospital antibiotic use in the southwestern part of germany can be estimated to contribute~15% to overall antibiotic consumption in the general population. antibiotic use profile and temporal trends during a 5-year period at a greek university hospital: implications for antibiotic policy changes e.i. kritsotakis, p. assithianakis, p. kanellos, n. tzagarakis, m.c. ioannides, a. gikas (heraklion, gr) objectives: to investigate the profile and temporal trends of inpatient antimicrobial use over a 5-year period at the university hospital of heraklion crete, greece. further, to examine the way in which frequency of data collection and stratification by different patient-care areas provides guidance to antibiotic policy changes. methods: retrospective monitoring of antimicrobial consumption was carried out according to the who anatomic therapeutic chemical classification (atc) and defined daily dose (ddd) measurement methodology. pharmacy records were used to obtain aggregate data of drug deliveries to individual wards. results were expressed as usage density rates in ddds per 100 bed-days (ddd/100bd). linear regression was used in order to assess the statistical significance of a temporal trend in usage densities. results: during 1998 -2002 , hospital-wide antimicrobial use (atc group j01) significantly increased by 22%, from 86.97 to 106.24 ddd/100bd. the annual average increase rate was 4.8 ddd/100bd. stratification by clinical service demonstrated differences in the intensity and profile of class use, as well as varying temporal trends (figures 1, 2) . pooled usage rates in ddd/100bd, overall percentage increases and annual average increase rates were respectively 109. 97, 35.6%, 8.1 for medical wards; 98.21, 48.7%, 9.1 for icu's; and 74.46, 34.3%, 5.7 for haemato-oncology wards. surgical wards had a fairly constant usage rate (98.36). a shift towards the newer broad-spectrum antibiotics to the detriment of the older penicillins and cephalosporins was noted in all hospital areas. conclusion: surveillance of aggregate data on the consumption of antimicrobials using the atc/ddd system provided a clear picture of the profile of hospital usage. monthly data over a sufficient surveillance period allowed the assessment of temporal trends. stratification of usage rates by clinical service allowed areas of concern to be specified. thus, surveillance of monthly antimicrobial consumption rates stratified by patientcare area can provide a simple, rapid and efficient tool for triggering antibiotic policy changes in the hospital and targeting more detailed quality-of-use audits. appropriate use of aminoglycosides: the impact of an antibiotic control team c. rioux, p. lesprit, j.r. zahar, a. hulin, a. bernier-combes, c. brun-buisson, e. girou (créteil, paris, fr) objectives: many factors are involved in the appropriate use of aminoglycosides (ag), such as modalities of administration, serum monitoring and duration of treatment. we assessed prospectively the risk factors and the impact of an antibiotic control team on the appropriateness of ag prescriptions. methods: in a setting of a restricted delivery system of ag in our hospital, we first performed an observational audit (oa) to assess the appropriateness of prescriptions including justification of prescribing, adequacy of drug choice, adequacy of administration modalities, modalities of serum monitoring and duration of treatment. after implementation of specific guidelines hospital wide, we then performed an interventional audit (ia) where an antibiotic control team could interfere when ag prescriptions were considered inappropriate. appropriateness of ag prescriptions between the 2 audits was then compared. results: 200 prescriptions were analysed. during the ia, 32% of prescriptions were modified by the control team. as compared to the oa, prescriptions in the ia were significantly more appropriate with regard to treatment duration (73 vs 55%, p = 0.009) and serum monitoring (61 vs 40%, p = 0.05). median treatment duration was shorter in the ia (4 d) than in the oa (6 d) (p < 0.0001). a logistic regression model showed that risk factors for appropriate treatment duration were (adjusted or, 95% ci, p value): hospitalization in intensive care unit (4.39, 1.57-12.2, 0.005) , polymicrobial infection (4.08, 1.38-12.08, 0.01) and antibiotic control team intervention (2.41, 1.23-4.72, 0.01) . table: conclusions: despite a restricted delivery system, ag use was frequently associated with excessive treatment duration and errors in monitoring modalities. reinforcing practice guidelines through direct counselling improved appropriateness of prescriptions. hospital antibiotic consumption in southern and eastern mediterranean countries: preliminary results from the armed project p. zarb, m.a. borg, h. goossens, m. ferech for the armed participants introduction: armed is an international research project investigating antimicrobial resistance and consumption in 7 southern and eastern mediterranean countries through the collection of comparable and validated antimicrobial resistance data as well as information about antibiotic consumption patterns and infection control initiatives. objectives: the consumption part of the study aims to collect data on antimicrobial use within participating hospitals in the region, which information is currently unavailable. methods: data collection is planned over a 24-month period using anatomical therapeutic chemical (atc) classification, a validated methodology adopted by the european surveillance of antimicrobial consumption (esac -www.ua.ac.be/esac). 23 hospitals are participating: cyprus (5 hospitals); egypt (7); jordan (1); malta (1); tunisia (3); turkey (6). results are expressed in ddd/1000 bed-days. results: data from 2004, the first year of data collection, indicates that turkish hospitals seem to show the lowest overall consumption [230-480 ddd/1000bed days], whilst the cypriot hospitals show highest values [2900-7500 ddd/ 1000bed days]. the most common antibiotics used are the beta-lactams, especially the penicillins although in jordan and turkey cephalosporin consumption is very close to the penicillins. broad-spectrum penicillins [j01ca] are the mostly utilised penicillins in cyprus, jordan and tunisia whereas in malta and turkey the combination penicillins [j01cr] are the most widely used. there is more variability where cephalosporin consumption is concerned. cyprus utilises mainly first generation, jordan and malta the second generation. in egypt, tunisia and turkey there is significant variability between hospitals; nevertheless use of third generation cephalosporins appears to be significant. conclusion: a significant variability was evident between countries. this is likely to be multifactorial depending on the antibiotics licensed in a country, the national and/or hospital formulary, the type of hospital as well as any antibiotic donations that are relevant in some of the study hospitals. nevertheless, the preliminary results suggest that trends within hospitals of the same country tend to be similar. furthermore, the region as a whole seems to utilise a considerable quantity of broad-spectrum antimicrobials. this can be a factor in the high prevalence of resistance already documented in the study. russian pharmacoepidemiology study of the antibiotic prescription during pregnancy results: mean age of the patients was 25.6 ± 6.0 (min -14, max -52) years, mean gestational ages at admission to hospital was 27. 0 ± 10.8 (3 to 42) weeks. most often (77.5%) infection was community acquired and 2.1% -nosocomial, in 20% patients there was not to estimate origin of the infection. the most prevalent infections during pregnancy in russia was urinary tract infections -39.9%, std -19.3%, candidiasis -17.4%, rti -6.6% therefore the most interest was analysing the antibiotic prescription for uti in pregnancy (table) . in 28% cases were used topical (intravaginal) antimicrobial administration. most often of topically administrated antimicrobials (19.02% of all prescriptions) were prescribed combined drugs included antibacterials and amtimycotics. in 80.98% cases antimicrobials were prescribed systemically. mostly prescribed antimicrobials were beta-lactams (16.2% for outpatients and 57.7% for inpatients), ampicillin was prescribed more often (4.6% for outpatients and 31.5% for inpatients). amoxicillin + clavulanic acid was prescribed in 6.4% of outpatients and 5.6% inpatients pregnant women with uti. cephalosporins were prescribed in 5.2% and 20.6% for outpatient and inpatient uti (mainly iii-and ist generations). mitroimidazoles -1.8-7.4% (in general metronidasole), nitro-furanes -8. [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] .0%, aminolglycosides -4.6-3.8% were prescribed quite often but unjustified. other antimicrobials (fluoroquinolones, doxicicline, antiviral drugs, antifungals) were prescribed relatively rarely. despite the fact that most prescribed drugs were class b by fda, 5.8% all antimicrobials prescribed to pregnancy were class c, 0.8% class d and 9.1% were unclassified. conclusions: most often prescribed antimicrobials for uti (the most prevalent infections during pregnancy in russia) are betalactams and combined topical antibacterials. in 15.7% cases were prescribed antimicrobials of class c, d or unclassified by fda. in 25% outpatient and 53.6% inpatient were used antibiotics with low in vitro activity for uropathogens. objectives: to study the dynamics of the antibiotic usage in children from orphanages located in different russian cities as the result of interventions with the increased use of the most active antimicrobials and restrictions on use of the least active. methods: the study was performed in 12 orphanages (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) from 5 cities of european russia (moscow, saint-petersburg, smolensk, karachev, bryansk) . use of antimicrobials during the previous 12 months was analysed upon reviews of medical records of 743 children <7 years in 2003. appropriate recommendations on predominant use of selected beta-lactams (e.g. amoxicillin/clavulanate -amc) with restriction of antimicrobials of other classes (e.g. co-trimoxazole -sxt) were made where applicable on the basis of the expert analysis of antibiotic usage and pneumococcal nasopharyngeal resistance rates. repeated antibiotic usage analysis was performed 7 months later in 2004 upon reviews of medical records of 752 children <7 years. results: total usage of antimicrobials increased 1. increase of resistance to pen and aminopenicillins. enhanced use of cephalosporins led to increase of resistance to these drugs. in spite of recommendations to restrict usage of am/ox, aminoglycosides and sxt, the analysis showed that these antimicrobials still accounted for 13.2%, 7.8% and 7.5% of all prescriptions, respectively, thus dictating the need for further enforcement measures. antibiotic consumption in ambulatory care in latvia, 2004 s. berzina, m. ferech, g. ozolins, h. goosens (riga, lv; antwerp, be) objectives: to collect data on antibiotic consumption in ambulatory care (ac) in latvia according to the esac data collection protocol. esac (european surveillance of antimicrobial consumption, granted by dg sanco of the ec) is an international network of national surveillance systems, aiming to collect reliable and comparable data on antibiotic consumption in europe. methods: the data on ac antibiotic consumption for 2004 have been collected using atc/ddd classification (who, version 2005) and expressed in defined daily doses per 1000 inhabitants per day (did). data were obtained from the state medicinal agencybased on the reports of the wholesalers for ac. results: the overall use of antibiotics in ac was 11.7 did in 2004, which positions latvia to countries with comparatively low antibiotic consumption in europe. the mostly used class of antibiotics in ac were penicillins with extended spectrum (mainly amoxicillin) -3.97 did (33.9%). other frequently used antibiotics were tetracyclines (mainly doxycycline), representing 2.41 did (20.6%), combinations of penicillins/with betalactamase inhibitors (essentially co-amoxiclav) -1.18 did (10.1%), macrolides (mainly clarithromycin) 0.85 did (7.26%), fluoroquinolones (essentially ciprofloxacin) -0.84 did (7.17%) and combinations of sulphonamides and trimethroprim, incl.derivatives -1.02 did (8.71%). the most frequently used antibiotics in ac in latvia, in 2004, were amoxicillin (3.97 did) , doxycycline (2.41 did) , and co-amoxiclav (1.18 did) . conclusions: valid data on outpatient antibiotic use in latvia has been for the first time collected and delivered to european surveillance of antimicrobial consumption. this allows international comparison of the pattern of antibiotic consumption in latvia with other european countries. trends in glycopeptide antibiotics consumption over a 6-year period in a general hospital, athens, greece introduction: glycopeptide use is under restriction in hellenic hospitals since late '80s. the aim of our study was to record trends in their consumption over the last 6 years in our hospital (''a. fleming'' general hospital -300 beds) and to correlate these data with the numbers of important gram (+) strains isolated in our hospital during the same time period. methods: we measured glycopeptide use for the period 1999-2004 by using data from the pharmacy computer. consumption was expressed as ddds/1000 patient days (abc calc 3.0). furthermore we correlated these data with data from the microbiology department concerning numbers of mrsa, mrse and enterococci isolated during the same period. results: glycopeptide consumption was 1.3, 6.1, 6.3, 7.9, 4.5 and 13 ddds/1000 patient days for the years 1999, 2000, 2001, 2002, 2003, 2004 (900% increase) . at the same time the cumulative number of mrsa, mrse and enterococci isolated were 175, 224, 207, 415, 338, 479 respectively (170% increase) . when both types of data were put on the same graph, glycopeptide consumption correlated well with the number of important gram(+) strains isolated (figure). furthermore vre percentage among enterococci was 0, 0, 3.0, 0.6, 3.5, 1 for the study years respectively. it is worth noting that 87% of our mrsa strains were sensitive to rifampin, 83% to clindamycin, 74% to cotrimoxazole, 80% to clindamycin + rifampin and 74% to cotrimoxazole + rifampin. linezolid has not been introduced in our hospital yet. conclusions: (a) despite the restriction policy, a tremendous increase in glycopeptide use was recorded in our hospital during the study period and this correlated to the number of the important gram(+) strains isolated; (b) nevertheless, vre is not a significant problem for our hospital yet; and c. the huge increase in glycopeptide use could be avoided at least in part, since other, older and simpler antibiotics could substitute for glycopeptides in many cases. an audit of linezolid use in a university teaching hospital, galway, ireland objective: to audit linezolid use over a six-month period among the in-patient population of a 504-bed teaching hospital that includes most medical and surgical specialties with the exception of nephrology, rheumatology and orthopaedics. methods: a prospective audit was carried out of the prescribing of linezolid to in-patients from october 2004 to april 2005. the ward pharmacist recorded the details of all patients who were prescribed linezolid. a chart review was performed to assess the profile of patients prescribed linezolid, clinical and microbiological indications for treatment, adherence to treatment guidelines and documented adverse events. results: over the 6-month period 53 courses of linezolid were prescribed. fifty two percent of the patients for whom linezolid was prescribed were from surgical specialties; half of these patients were under the care of one surgeon. pneumonia was the clinical indication for use in 32% of cases and soft tissue infection in 36% of cases. the microbiological indication was clear in 64% of cases where mrsa or vre had been isolated. in 36% of cases therapy was either (1) empiric with no significant organisms isolated prior to prescription of linezolid or (2) therapy was directed against an organism that could have been treated with an alternative agent. duration of treatment exceeded 10 to 14 days in 40% of courses. an adverse event was recorded in the case of only one course of linezolid. conclusion: in more than a third of cases linezolid use was prescribed without clear justification. avoidable use of linezolid is associated with increased costs and risks of acquired resistance. participants prior to on an oral interview during which the interviewer filled in the answers. results: a total of 62 cm specialists completed the inquiry. this represents approximately 25% of the national quorum. mean age was 48 years (32-63). 22 of the 62 interviewed cm specialists worked full-time with more full-time employment in hospital labs (hl). next to routine microbiology, other activities performed by cm specialists are mainly the other domains of clinical biology, hospital hygiene and to a lesser extent quality control and lab management. almost two thirds of the interviewed cm specialists believes that their training hasn't prepared them properly for the tasks they are performing now. most desired changes include more emphasis on the clinical aspect of infectious diseases and on antibiotic treatment counselling. cm does not exist as a separate speciality in belgium but is included in the 'clinical biology' speciality training. the majority of the respondents thinks that cm should become a sub-speciality (still part of clinical biology) but with a specific minimal training that needs to be defined. the majority of the cm specialists also believes that cm can share lab infrastructure with other disciplines and that the essential aspect of cm lies predominantly in the medical expertise. conclusion: cm training should put more emphasis on the clinical aspect of infectious diseases and on antibiotic treatment counselling. the majority of the respondents feels that cm should become a sub-specialty (still part of clinical biology), with a well defined training curriculum. objectives: since more than 10 years, all infectious disease consultations have been recorded in a computerized database (epi info 6.04d, cdc). here we report on 1163 consultations of a fellow, conducted during 1 year, compared with 5708 consultations conducted by two veteran board-certified infectious disease consultants during the same period. methods: we analysed computerized consultation records, including demographic details of patients; referring department; initiative for, route and purpose of the consultation; and recommendations; and compared between the different consultants. results: a larger percentage of veterans' compared to the fellow's consultations, were requested by attending physicians (72% vs. 50%, p < 0.001), while follow-up (15% vs. 23%, p < 0.001), laboratory results (11% vs. 23%, p < 0.001) or prescription for a restricted antimicrobial agent (1.3% vs. 3 .3%, p < 0.001) were more prevalent in fellow consultations. the fellow had a higher rate of additional consultations (in which the patient was seen more than once) (40% vs. 30%, p < 0.001), and performed more bedside consultations (68% vs. 47%, p < 0.001) or consultation by curb side discussion (25% vs. 13.2%, p < 0.001), and less consultations by telephone (6.6% vs. 40.1%, p < 0.001). diagnosis and prophylaxis were more often the purposes of the veterans' consultations (44.8% vs. 36.1%, p < 0.001, 3.9% vs. 2.1%, p < 0.01, respectively), and they also offered new diagnoses more frequently (p < 0.005). the veteran consultants more often conducted consultation for communityacquired infections (53% vs. 44%, p < 0.001), and more often started antibiotic treatment (19% vs. 8 .9%, p < 0.001). conclusions: significant differences were detected between consultations conducted during the first year of a fellow compared to those of veteran infectious disease consultants. these differences reflect the changing demands and activities in the consultant's work as experience and knowledge accumulate. periodic analysis of computerized data of consultations facilitates supervision as well as direction of consultants' work, addressing issues such as antibiotic use and patterns of microbial resistance. bridging the gap between health care and public health; capacity building in infectious disease control objectives: in recent years, the european union (eu) has developed and supported many activities in the field of communicable diseases. these activities not only concern surveillance networks of specific infectious diseases (e.g. enter-net for salmonella and escherichia coli infections, ewgli for legionella infections, eiss for influenza infections), but also eu training programmes like epiet (european programme for intervention epidemiology training) and a eu communicable disease bulletin. even more recent are the eu's initiative bichat to improve preparedness and response to bioterrorism, and the development of a eu cdc. moreover, a major part of the new programme of community action in the field of public health (ph) (2003) (2004) (2005) (2006) (2007) (2008) concerns id, with not only a commitment to improve information and information exchange, but particularly to strengthen the international rapid response capacity.all this, to illustrate the importance of idc on the eu agenda. methods: the european public health association (eupha) is an umbrella organisation for ph associations in eu. in 2003 eupha has created an eupha section idc bringing together eupha members with expertise in this field and representatives of the various above mentioned eu initiatives in order to: promote and strengthen research in the field of idc; provide a platform for the exchange of information, experience and research in idc; bring together researchers, ph practitioners and policymakers active in idc; encourage joint activities in idc; and improve idc training. results: by now the section has 307 members from 50 different countries. as of 2005 the section is represented in the ecdc advisory forum. different section activities: organising 8 workshops, a breakfast meeting and a pre-conference meeting on timely idc topics during eupha conference. objectives: to establish a cross-border dutch-german network (www.mrsa-net.org) providing a user-friendly knowledge centre for hospitals, public health authorities, gps, nursing homes and laboratories. primary purpose is to aid in the reduction of mrsa-rates and limit the cross-border transmission of mrsa. guidelines and their implementation play a significant role in reaching these aims. cross-border (ca-) mrsa guidelines will be redesigned according to international standards and socio-cultural differences between the nations. methods: based on quality standards for safety and healthcare documentation used in high risk chemical organizations, a framework for a systematic content analysis of national mrsaguidelines was developed. national guidelines were analysed on the basis of this framework. results: a content analysis of the current national mrsaguidelines showed five dominating mrsa-perspectives: rule-, expert-, risk-, demand-and community-driven. german guidelines are mainly dominated by the rule-and expertdriven perspectives (guidelines are literally derived from law and follow the infection transmission route), in contrast to the dutch which focus on the demand of the user and the community (addressed to public health and acceptability of guidelines by users). conclusion: the analysis showed that the fact that there are different guideline-perspectives results in an enormous, confusing set of guidelines. the management and use of guidelines becomes uncontrollable and leads to an illusory organisation where healthcare workers don't act in accordance with the guidelines and start applying their own insights. this might lead to cost-increasing and contrasting situations. to implement guidelines successfully in a cross-border situation, a cultural and technical synchronisation alongside an integrated approach of the different perspectives of guidelines is necessary, inline with the current disease management models. further research about the redesign and the evaluation of those guidelines in practice will help achieving this. r. tsiklauri (tbilisi, ge) background: animal bites are a common but under recognized public health problem. it has been estimated that there are 8-10 000 bites each year in georgia, and based on an average visit and post-exposure treatments cost at list $120 000 per year. despite the frequency and expense of these injuries, there is little information about the incidence of animal bites because of a lack systematic reporting and a lack of measurement of the quality and completeness of reported data. objectives: to investigate animal bites and rabies reported cases, revealed unreported cases, analyse and based on study results find more effective epidemiological measures of animal bites and deaths (due to rabies) prevention in georgia. methods: the capture-recapture method was used, along with log-linear modelling. for sources were used to identify victims: policlinic/ambulatory reports, hospital reports, animal control reports and victim reports. results: in 1980-2001 years 150 700 dog and other animal bites were reported. the capture-recapture method estimated that there were 146 200 unreported bites. during these period 118 deaths due to rabies was registered in georgia and 67 (57%) cases among them have been registered during the last 6 years. the reasons of fatal cases were untreated (47%), uncompleted treated (34%) and late began post-exposure treated (19%) cases of bites (mostly dog bites). about 56% of bitted persons did not know about rabies and it's prevention measures. about 32% had incorrect information about prevention and only 12% of them knew epidemiological and clinical aspects of disease. about 16% of physicians who were responsible on quality post-exposure treatment had not an adequate knowledge. conclusion: dog and other animal bites are common but preventable injuries. to improve surveillance and prevention of rabies in georgia, the focus should be on educating the general public about the serious consequences of animal bite injuries and developing the animal's vaccination strategy. pharmacoeconomics and electronic resources p1493 the expected economic burden of methicillinresistant staphylococcus aureus in complicated skin and skin structure infections: a modelling approach a. kuznik, r. mallick, d. weber (collegeville, chapel hill, us) objective: to model the expected rate of clinical failure of initial empiric therapy and economic burden likely to be associated with the increasing prevalence of methicillinresistant staphylococcus aureus (mrsa) in patients hospitalised with complicated skin and skin structure infections (csssi) in the united states. methods: using published data on (1) the prevalence of mrsa and other bacterial pathogens causing csssi in the us, (2) the in-vitro susceptibility rates of commonly used regimens in csssi in the us in relation to the most pervasive pathogens identified above, and (3) estimated costs of failure of initial, empiric treatment from a recent study of a large us multi-hospital database, we developed a model to predict the expected clinical and economic impact of increasing prevalence of mrsa. specifically, clinical failure of 5 of the more commonly used initial regimens in csssi was modeled in terms of their in-vitro susceptibility rates with respect to mrsa, weighted by mrsa prevalence. varying the rate of mrsa further yielded projected clinical failure rates and costs attributable to increasing levels of methicillin resistance over time. results: given current 55% prevalence of s. aureus pathogens in csssi, half of them methicillin-resistant (base case mrsa = 27%), the model projected an overall clinical failure rate of 35.9% for 5 of the more commonly used initial regimens, with an expected overall treatment cost (in us dollars) of $5,492 per patient (range, $4,566-5,633) . if none of the s. aureus pathogens were resistant (mrsa = 0%), clinical failure rate was projected to be 18.4% and treatment cost to be $4,869 per patient. the differences in the two scenarios translated to an expected clinical failure rate of 17.5%, an incremental cost of $623 per patient, and for the 800,000 patients hospitalised for csssi annually in the us, an expected health care system burden of $498 million attributable to mrsa. under a "worst-case" scenario in which mrsa was the only causative pathogen (mrsa = 100%) in csssi, clinical failure rate was projected to be 79.3%, and treatment cost per patient was expected to be $7,035. conclusions: going beyond existing estimates, our model generated a substantial expected clinical failure rate and economic impact attributable to current mrsa levels, as well as simulations of the expected impact of increasing mrsa prevalence over time, varying levels of mrsa across regions and choice of initial empiric regimens. treatment of complicated skin and skin structure infections in the us: expected cost differences between tigecycline and vancomycin/aztreonam r. mallick, a. kuznik, d. weber (collegeville, chapel hill, us) objective: to compare tigecycline and vancomycin/aztreonam in terms of treatment-related costs for patients hospitalised in the united states with complicated skin and skin structure infections (csssi). methods: we conducted a retrospective analysis of pooled data from us centres in two randomized, double-blind clinical studies comparing tigecycline and vancomycin/aztreonam in the treatment of csssi. using regression analysis, we estimated the effect of tigecycline treatment on hospital length of stay (los), controlling for other significant predictors. using published estimates of daily hospitalisation cost of csssi in the us from a multi-hospital audit, we then translated the estimated impact on los into economic terms. this analysis was repeated for the subgroup of patients in which the primary pathogen was methicillin-resistant staphylococcus aureus (mrsa). clinical efficacy (tigecycline 81%, vancomycin/aztreonam 83.1%; p = 0.376) was similar across treatments and was not included as a model parameter. results: our retrospective analysis of the pooled clinical data from us centres found that tigecycline was associated with a shorter los [)1.85 days (p = 0.015)] compared with the combination of vancomycin/aztreonam in the treatment of patients with csssi. at a mean daily hospitalisation cost (in us $) of $794, excluding antibiotic costs, this translated into expected medical cost savings of $1,469 per patient for tigecycline compared with vancomycin/aztreonam. in the mrsa subgroup, comprising 26% of the clinical study sample, tigecycline was associated with a greater reduction in los [)2.82 days (p = 0.011)] compared with vancomycin/ aztreonam, translating to expected medical cost savings of $2,239 per patient treated with tigecycline. these expected medical cost savings more than offset the higher average daily drug acquisition costs of tigecycline ($118/day) relative to the vancomycin/aztreonam combination ($110/day). conclusion: in a retrospective analysis of pooled clinical data of patients with csssi treated at us centres, tigecycline was associated with a significantly reduced length of hospital stay relative to vancomycin/aztreonam; this translated into substantial cost savings, especially in the subset of csssi patients with mrsa. the economic impact of linezolid in the treatment of skin and soft tissue mrsa infections in italy m. eandi, p. dale, s. sorensen, t. baker, m. procaccini, s. duttagupta (turin, it; london, uk; bethesda, us; rome, it; new york, us) objective: linezolid has been shown to be highly effective against infections caused by methicillin-resistant staphylococcus aureus (mrsa) in patients with complicated skin and soft tissue infections (cssti). the objective of this study was to evaluate the clinical and economic consequences of using linezolid for the empiric treatment of cssti from the italian hospital perspective. methods: a decision-analytic model was developed to calculate the clinical and cost outcomes of empiric treatment of hospitalized patients with cssti in italy prescribed linezolid, vancomycin or teicoplanin. efficacy data were derived from clinical trials. costs from published sources were applied to tests, adverse events, and days of intravenous and oral (linezolid only) treatment and hospitalization by ward type (general, intensive-care). resource use and utilization patterns were obtained from a combination of clinical trial data and expert opinion. outcomes included total costs per patient, cost per cure and cost per death avoided. uncertainty surrounding the ce ratio was tested using one-way sensitivity analysis. results: starting empiric treatment with linezolid resulted in 98.4% of patients cured from mrsa compared to 98.0% with vancomycin. the average cost per patient treated with linezolid was €6,305 versus €6,228 for patients treated with vancomycin. this resulted in a cost per cure of €22,404. in a separate analysis more patients were cured using linezolid (97.6%) compared to teicoplanin (94.7%). the average total cost per episode was €6,401 for linezolid treated patients versus €5,897 for teicoplanin treated patients, resulting in a cost per cure of €17,100. the most sensitive parameters included hospital los and mrsa resistance rate. conclusions: in the treatment of cssti due to suspected mrsa in italy, the empiric use of linezolid is cost-effective when compared to vancomycin and teicoplanin p1496 outpatient and home parenteral antimicrobial therapy for the treatment of cellulitis: evaluation of efficacy and cost h. ziglam, r. tilley, c. wootton, j. morrison, d. nathwani (dundee, uk) objective: outpatient and home parenteral antibiotic therapy (ohpat) programmes are effective, well tolerated and economically advantageous in carefully selected patient populations. skin and soft tissue infections represent a high burden disease which in amenable to treatment by ophat programmes. we retrospectively analysed our outcomes registry to evaluate the clinical and health economic impact of treating cellulitis in this setting. methods: we have reviewed 465 patients with cellulitis and erysipelas who were treated with ohpat. each patient treatment has a full integrated care pathway (icp). the icp documents the microbiological outcome, drug and vascular access complication rates, impact on drug costs and in-patient bed days on the 465 number of patients treated from april 1998 to march 2005 are presented here. we also reviewed using the smr1o inpatient discharge diagnosis data from the information statistics division scotland (isd) and the dundee infectious diseases units (didu) outcomes registry database. the key diagnosis (icd 10 codes) groups considered were cellulitis (lo30, 31, 32, 33, 38, 39) and erysipelas (a46x) over eight consecutive years (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) . results: the patients received intravenous antibiotic therapy for a mean duration of 5.64 days. the two primary agents administered were once-daily ceftriaxone in 84%of patients and teicoplanin in 9.8% of patients. of the 465 patients, 431 (92.6%) were cured or improved; 5 worsened and required surgery. tinea pedis was found in 31% of patients treated for cellulitis. economic benefits were realized despite use of more expensive agents. data from the dundee outcomes registry revealed a mean reduction in length of hospitalization from 5.8 days (1996/ 1997) to3.2 in 2003-2004-a reduction of 48% compared to scottish data from isd which did not show any changes in length of hospitalization between year 1996/1997 (5.33 days) and year 2003-2004 (5.92 days) . conclusions: we have found that ohpat is clinically effective and can be administered safely and successfully in an outpatient setting. the majority of complications were minor, and 93% of patients were cured. tinea pedis and were found to be significant risk factors for acute cellulitis and indicate that improved awareness and management of toe web intertrigo might reduce the incidence of cellulitis. this analysis also supports the premise that an adult ohpat programme can substantially reduce healthcare resource use in the european healthcare setting. cost-effectiveness analysis of intravenous moxifloxacin compared to levofloxacin in hospitalised elderly patients with communityacquired pneumonia objective: to evaluate the cost-effectiveness of moxifloxacin compared to levofloxacin in hospitalised patients aged ‡65 with community acquired pneumonia (cap). methods: a randomised double-blind parallel group study was conducted in 47 us hospitals. patients had radiological evidence of bacterial pneumonia confirmed by at least 2 other signs, were aged ‡65 years and were managed as inpatients on initiation of treatment. patients initially received moxifloxacin 400 mg iv o.d. or levofloxacin 500 mg iv o.d., and once stabilised were switched to oral therapy with the same agent. the effectiveness endpoint for the economic analysis was the percentage of patients successfully treated, defined as patients with marked improvement, resolution or clinical cure at test of cure visit after 7-14 days of therapy who did not experience a serious cardiac adverse event. total costs were estimated from the perspective of the treating hospital and included antibiotic drugs, hospital stay, hospital re-admission within 28 days and cost of managing treatment failures. results: 394 patients were included in this analysis, 195 randomised to moxifloxacin and 199 to levofloxacin. 82% (95% ci: 77-88%) of moxifloxacin and 75% (69-81%) of levofloxacin treated patients were successfully treated (resolution, clinical cure at toc and no serious drug related aes). in the moxifloxacin group patients reported a mean of 3.5 days of iv antibiotic treatment and 5.7 days inpatient stay with 3.4 days iv antibiotic treatment and 6.1 days inpatient stay in the levofloxacin group. mean per patient drug cost was $257 in the moxifloxacin group, $222 in the levofloxacin group. mean total cost was $8,339 in the moxifloxacin group and $8,354 in the levofloxacin group. findings were consistent across a range of patient subgroups. costs were sensitive to length of hospital stay. conclusions: patients in the moxifloxacin group had higher rates of successful treatment at slightly lower average costs than the levofloxacin group. this confirms the results of the target study where moxifloxacin showed superior clinical efficacy in comparison to co-amoxiclav with or without clarithromycin in hospitalised cap patients. antibiotic costs were slightly higher in the moxifloxacin group than the levofloxacin group but total costs were slightly lower, due to reduced hospital stay. economic impact of invasive fungal infections in icu patients in a tertiary care hospital in switzerland a. imhof, w. zingg, r. laffer, c. ruef (zurich, ch) objectives: invasive fungal infections (ifi) cause significant morbidity and mortality. the management of invasive fungal infections is currently undergoing important changes due to the availability of new therapeutic agents with improved safety profiles but the acquisition costs of these new agents are high. we evaluated the average overall cost of management (microbiological diagnosis and treatment) of invasive fungal infection in critically ill patients at a large university hospital. methods: a retrospective (2003) (2004) , pairwise-matched cohort study was performed on 4 surgical icus and one medical icu at our university hospital. icu patients with documented ifi (n = 23) were matched with control subjects (n = 46) on the basis of disease severity, sex and age (±5 years). clinical outcome was principally evaluated by in-hospital mortality. the economic impact of microbiological studies and antibiotic treatment was assessed. calculations were based on the period between admission and diagnosis of ifi in cases and the duration of hospital stay in controls, respectively. results: the median length of hospital and icu stay differed significantly between cases and controls (43 vs 14 days, 19 vs 3 days, p < 0.001, respectively). ifi occurred after a median hospital stay of 22 (range 2-95) days. the mortality rate for patients with ifi and matched control subjects were 30.4% and 10.9%, respectively (p = 0.04). there was no significant difference between cases and controls for charlson index, mccabe and saps ii score. median number of antibiotic treatment courses was 3 for cases and 1 for controls (p = 0.003), with a median duration of therapy of 6 days vs 1 day (p < 0.0001), respectively. microbiological studies (mis) were conducted 78 times/100 patient-days (pd) in cases and 17 times/100 pd in the control group (p = 0.03). the most frequent samples were bloodcultures in both groups. swabs were ordered significantly more frequently in cases (median 25 (range: 1-150) vs. 2 (0-23); p = 0.04). the cost for mis was 13'728 euro/100 pd in cases vs. 3742 euro/100 pd in controls, and the costs for antifungal therapy 4'373 euro/100 pd vs. 99 euro/100 pd, respectively. conclusions: ifi is associated with excess length of icu and hospital stay, increased use of antibiotics and microbiological diagnostics. the microbiological studies have a significant economic impact on the treatment of ifi. cost-effectiveness of voriconazole to amphotericin b deoxycholate in early and late treatment of invasive aspergillosis r. greene, j. mauskopf, c. roberts, t. zyczynski, h. schlamm (boston, research triangle park, new york, us) objective: we estimate the cost-effectiveness of alternative initial drug treatments of invasive pulmonary aspergillosis (ipa) in suspected earlier and later lung involvement, based on the presence or absence of the halo sign on thoracic computed tomography (ct). methods: we constructed a decision analysis model comparing 12-week treatment outcomes for a subset of patients enrolled in a clinical trial of initial treatment of ipa with amphotericin b dexoycholate (ambd) vs voriconazole (vor). patients included those with suspected lung involvement who underwent a baseline thoracic ct. the subset was subdivided into two groups based on the presence or absence of a characteristic ct halo sign, a perimeter of ground glass ct opacity surrounding a solid lung nodule ‡1 cm diameter, known as an early indicator of ipa. healthcare resource use and survival data were obtained directly from the clinical trial. us unit costs for drugs and health care services were applied from standard data sources. cost and survival at 12-weeks were estimated for those with and without a halo sign at baseline. incremental cost-effectiveness ratios comparing vor to ambd were calculated for both patient subgroups. sensitivity of results to uncertainty in health care use and cost estimates was tested. results: patients in the halo subgroup had better survival than those in the no-halo subgroup (70.6% vs 54.4%), with lower total treatment cost ($44,352 vs $47,077) . survival was higher for vor than for ambd in both patient subgroups (halo: 75.3% vs 65.2%; no-halo: 68.3% vs 39.5%). in the halo subgroup, total costs were lower for those treated with vor than for those treated with ambd ($40,380 vs $48,985) . in the no-halo subgroup, total cost per patient was slightly higher for those treated with vor ($48,133 vs $45,938) . accounting for the difference in survival, the incremental cost-effectiveness ratio for vor compared to ambd was $7,625 per additional 12-week survivor in this subgroup. conclusions: earlier identification and treatment of ipa appears to result in better survival and potentially lower costs than later treatment. initial treatment of ipa with vor improves survival in patients with early or late disease compared with ambd, is cost saving in the halo sub-group, and is cost-effective in the no-halo subgroup, within the constraints of our analysis. objective: to describe and compare the nursing labor time required for preparation and administration of liposomal amphotericin b (l-amb), amphotericin b deoxycholate (ambd), and voriconazole (vor). methods: activities associated with nurse preparation and administration of the three study drugs were timed by trained observers at five hospitals (one in italy, three in france, and one in the united kingdom). target tasks were classified as those likely to be affected by the difference between the drugs and excluded those tasks likely to differ because of site-specific factors (e.g., travel time to a patient room in different hospitals). target tasks included: obtain supplies and medications; prepare medications; educate patient; administer medications; monitor for adverse events; and prepare follow-up medications. the mean times for administration of a single day of study drug were summarised and compared, accounting for a single daily dose of l-amb and ambd and 2 daily doses of vor iv or oral. results: sixty-nine patients were observed receiving 256 doses of study medications at the five hospitals. time of administration in minutes per day was 20, 16, 14, and 3 for l-amb, ambd, vor iv, and vor oral, respectively. administration time was significantly lower for vor iv compared with l-amb (p < 0.05) and for vor oral compared to all iv regimens (p < 0.05). the task of preparation of medications required the most time for iv formulations, and was longer in the l-amb group than the others (l-amb: 12 mins vs ambd: 7 mins; vor iv: 9 mins). ambd required more time for patient monitoring and administration of followup drugs than other formulations (ambd: 7 mins vs l-amb: 4 mins; vor iv: 2 mins). conclusion: vor iv required significantly less time to prepare and administer on a daily basis compared to l-amb. measurements of iv antifungal versus oral vor administration suggest the opportunity to save 10-17 minutes per day by switching to oral therapy when possible. need of cost-effectiveness investigation focused on diagnosis, management and prevention of osteopenia and osteoporosis in the setting of hiv disease treated with haart: when to act, how to act, which patients are the first target of intervention r. manfredi, l. calza, f. chiodo (bologna, it) background: osteopenia/osteoporosis are emerging untoward effects of hiv infection/haart. the pathogenesis is multifactorial, involving all classes of anti-hiv drugs, although protease inhibitor use, overall haart duration, and the male sex, seem related to a greater risk.epidemiologicalclinical data. in an ongoing study at our centre where >1000 hiv-infected patients (p) are followed, bone mineral density was assessed in lumbar spine/femural head by a dual energy xray absorptiometry (dexa) exam to estimate the prevalence of osteopenia/osteoporosis. in a screening of~100 p, the frequency of osteopenia and osteoporosis (based on lumbar t-score) was 38% and~10%, respectively. an increased risk was found in p treated with protease inhibitors versus those receiving nonnucleoside reverse transcriptase inhibitors or triple nucleoside/ nucleotide combinations. discussion and future insights: prospective studies of extensive p samples are needed, to elucidate the epidemiology, pathogenesis, clinical issues and evolution of hiv-associated bone metabolism anomalies. when planning strategies for their early diagnosis, prevention and management also cost-effectiveness issues should be considered, since no pharmacoeconomic data still exist in this setting. although severe consequences (e.g. pathological fractures, prosthetic implants) are expected to be infrequent their consequences in terms of length and intensity of hospitalization, related costs, and especially severe consequences on the p's quality of life, play a notable role. anyway, the most reliable diagnostic procedure (dexa) has affordable costs (around eur 43.40 for a total-body scan which also offers a body composition assessment), as well as the first-line drugs for osteopenia, e.g. supplementation with calcium (eur 5-6.5/month), and vitamin d (eur 7/month). these costs cannot be compared with the costs of a standard care of an asymptomatic haart-treated p (eur 471 to 774/month) and the immunological, virologic, laboratory and clinical controls made at least quarterly. like postmenopausal osteopenia/osteoporosis (burdened by a greater risk of bone mass anomalies) also hiv disease should be investigated from multiple cost-effectiveness points of view to establish which p are the preferred candidates for a dexa screening when this examination is more useful during hiv disease course and therapy, when the exam should be repeated and when and how to intervene pharmacologically to prevent serious and potentially invalidating complications. a comparative study on the cost of new drugs in different therapeutic categories m. falagas, k. fragoulis, g. zouglakis, i. karydis (athens, gr) objectives: drug treatment is becoming more expensive due to the increased cost for the introduction of new drugs and there seems to be an uneven distribution of medication cost for different therapeutic categories. we hypothesized that the cost of new antimicrobial agents may differ from that of other therapeutic categories and this may play a role in the stagnation of development of new antibiotics. methods: we performed a pharmaco-economical comparative analysis of the drug cost of treatment for new agents introduced in the united states drug market in various therapeutic categories. we calculated the drug cost [in us dollars (usd)] of a 10-day treatment of all new drugs approved by the fda during the period between january 1997 and july 2003, according to the 2004 red book pharmacy's fundamental reference. results: new anti-neoplastic agents were found to be the most expensive drugs in comparison to all other therapeutic categories with a median 10-day drug-treatment cost of 848 usd compared to the median 10-day drug-treatment costs of all other categories ranging from 29 to 301 usd (table) . on the other hand, new antimicrobial drugs were found to be much less expensive with a median 10-day drug-treatment cost of 137 and 85 usd for all anti-microbial agents and for anti-microbial agents excluding anti-hiv medications, respectively. conclusion: the drug-treatment cost of new medications varies considerably by different therapeutic categories. this fact may influence industry decisions regarding the development of new drugs and may play a role in the shortage of new anti-microbial agents in the fight against the serious problem of anti-microbial resistance. usage and expenditure of 4f-quinolones in a tertiary hospital in 2004 t.a. peppas, k. malengou, n. zachos, d. voutsinas, o. kosmopoulou, n. galanakis (piraeus, athens, gr) objective: our aim was to assess 4-f-quinolone (4fq) usage, distribution and expenditure over 2 years. objective: to analyse antiobiotic utilization in croatia using anatomical-therapeutic-chemical (atc) drug classification system and number of defined daily doses (ddd). methods: data on the number of packages and purchase price were collected for each individual drug. these data were used to calculate the number of defined daily doses (ddd) and ddd per 1000 inhabitants per day (ddd/tid). data obtained from 80% of pharmacies and 65% of hospitals were extrapolated to the total number of pharmacies and hospitals in croatia. drug utilization 90% (du90%) segment was used as a prescribing quality indicator. results: in 2004, the overall utilization of antibiotics in croatia amounted to 23.35ddd/tid. according to drug groups, penicillins (j01c) showed highest utilization (12.24 ddd/tid), predominated by the subgroup of penicillin combinations (including beta-lactamase inhibitors, j01cr) with6.86 ddd/ tid, within which the combination of amoxicillin + clavulanic acid accounted for99.9% with 6.85ddd/tid. broad-spectrum penicillins (j01ca) accounted for 43.2%(5.28 ddd/tid) of total penicillin utilization, with a 97.7% predominance of amoxicillin (5.16 ddd/tid). cephalosporins (j01d) ranked second with 4.00ddd/tid, followed by macrolides and lincosamides (j01f) with 2.41 ddd/tid, with an 86.84%predominance of macrolides (j01fa) with 2.09ddd/tid. among the latter, azithromycin showed highest utilization with 1.44 ddd/tid, accounting for 68.66% of total macrolide utilization. tetracyclines (j01a) ranked fourth with 2.02ddd/tid, accounting for 8.65% of overall antibiotic utilization, followed by quinolones with 1.65 ddd/tid, other antimicrobials with 0.91 ddd/tid, and aminoglycosides with 0.16ddd/tid. sulfonamides (j01e) accounted for a negligible proportion of overall utilization. du 90% segment included 9 of 43 antibiotics registered in croatia, with amoxicillin + clavulanic acid as the leading one, followed by cephalexin with 1.83, cefuroxime with 1.53, azithromycin and norfloxacin with 1.3each, nitrofurantoin with 0.56 and clarithromycin with 0.53ddd/tid. hospital utilization accounted for 7.1% of overall antibiotic utilization expressed in ddd/tid and 22.7% of the respective financial cost, predominated by aminoglycosides (j01g) with 72% and 94.2%, and lowest proportion of tetracyclines (j01a) with 4.3%, and 2.4%, respectively. conclusion: the utilization of antibiotics in croatia is among the highest in europe, mostly due to overuse of amoxicillin + clavulanic acid, which has no rational ground in professional guidelines. objective: the objective of the research was to analyse antibiotics prescripsion behavior by family doctors and specialists treatments prior to and after the introduction of the health care reform in poland. materials and methods: prescriptions from the first six months of 1999 and 2005 were compared. the data was collected from two randomly chosen pharmacies in the city of zabrze that supply the citizens of the silesian agglomeration from various social backgrounds. taking into account the value of a single antibiotics package and the price a patient has to pay for it an average price of medications prescribed by family and specialist doctors was calculated. results: a total of 30793 prescriptions were analysed out of which 24834 dated from 1999 and 5959 from 2005.in the first half-year of 1999 the percentage of prescriptions for antibiotics reached 7.76% on average, and in the year 2005 -the average was 11.6%. in the first half-year of 1999 family doctors mostly prescribed: penicyllins (43%), makrolids (26%), cephalosporins (16%), tetracyclins (11%), chinolons (3%). in the same period spcialist doctors prescribed: penicyllins (41%), cephalosporins (17%), makrolids (15%), tetracyclins (13%), lincozamids (9%), chinolons (4%). in the first half-year of 2005 family doctors most often prescribed penicyllins -(44.8%), makrolids -(27.1%), cephalosporins -(12.5%), tetracyclins -(9.4%) and lincozamidsbased (3.1%) treatments specialist doctors, on the other hand, prescribed penicllins (41.7%), makrolids (17.9%), cephalosporins (17.7%), tetracyclins (12.1%), lincozamids (5.2%) and chinolons (3%). the average prices of the prescribed medications in the years 1999 and 2005 were, respectively: for family doctors-eu 3.08 and 4.01, for specialist doctors eu 4.44 and 3.87. conclusions: there has been a considerable increase in the percentage of prescriptions for antibiotics from 7.45% (in 1999) to 11.3% (in 2005). the tendency towards prescribing antibiotics in the specific groups of doctors has not changed significantly. in both years prescriptions for antibiotics were in line with the recommendations. also, prices of medications prescribed by family doctors have risen. internet guide on antimicrobial resistance a. sosa, f. traub, s. valovic, p. chea (boston, us) objectives: (1) to organize the plethora of information available, providing clinicians the tools to easily access available online resources that include academic institutions, professional societies as well as sites maintained by private individuals; (2) to inform clinicians of new advances in the epidemiology, diagnosis, treatment, and prevention of most common infections; (3) to inform on subjects such as clinical trials in antimicrobial resistance, information about specific pathogens and their infections, genomic resources, culture collections, electronic images of pathogens and antimicrobial agents, antimicrobial resistance lecture and teaching materials, environmental health and safety information, and a listing of websites of infectious disease and clinical microbiology professional societies. methods: we defined four inclusion criteria after extensive consulting with apua staff and scientific advisory members: (1) recognized/reputable source; (2) high quality of information presented; (3) potential usefulness to medical professionals and the general public; and (4) ease of navigation. ideal parameters were determined for the guide's scope and the appropriate sources identified online were subsequently reviewed. a set of broad categories was established to organize the topics and the online resources. sites reviewed included those maintained by the federal government, academic institutions, nonprofit organizations, and commercial entities. some personal websites were included because of their quality and their association with academic institutions. this review is intended as an introduction to amr websites. results: with use of popular search engines, such as google, yahoo!, and altavista, we initially identified a great number of websites. using broad search terms, such as "antibiotic resistance," we identified 1,310,000 web addresses. the term "antimicrobial resistance" generated 578,000 hits, and the term "drug resistance" generated 6,190,000 hits. conclusions: websites found were classified following a systematic topic structure. each website listed describes: (1) full citation of the resource: author/editor and title of website; (2) date of publication or last revision; (3) methods and results: the portal is free to use but requires registration and has more than 1850 registered users as of november 2005. of these, 20% are in-training positions, 28% hold a faculty position in infectious diseases (id), microbiology or hemato-oncology, 24% are specialists in a non-university, but a teaching setting. the remaining are a mixed group of primary physicians, other speciality doctors and pharmaceutical company workers. running costs of the portal are partially covered by educational grants from pharmaceutical sponsors who have no role in organization of the site, but their names are acknowledged. articles chosen by the two faculty members, one id physician and one haematologist, are sent to registered users by daily e-mail postings. these are selected from toc alerts of various core clinical journals and well known educational web sites (e.g. cdc, who, medscape). a short turkish summary is provided and the reader is referred to the abstract and if available, to the free full text of original article with a link. other materials included guidelines, free slide sets, study protocols and updates from the group, cme activities and meeting announcements. registrants may also use the site for expert opinion. during the trial period, the site has been visited 30740 times with 282239 hits. approximately 5.5 gb material was downloaded. the frequency of readings are related with the time (highest between 9.00-11.00 am during weekdays and lowest during weekends), the type of documents (i.e. educational materials and guidelines), popularity of the news (e.g. peaked during an epidemic of avian influenza when related news and articles announced). the pages are most frequently visited by id specialists followed by clinical microbiology, haematology and pharmaceutical company workers (33%, 19%, 9% and 8% respectively). conclusion: timely published medical data have high attraction rates among physicians. our results also indicated that, a web page gets ''old'' after about a month of publishing, emphasizing the importance of well-timed announcements of the portal material. neli and nric survey: information needs of infectious disease professionals p. kostkova, s. d'souza, g. madle, j. mani-saada, s. wiseman, a. roy, j. weinberg (london, uk) healthcare professionals are increasingly facing the problem of information overflow. it is getting impossible to keep up-to-date with the latest research findings, care guidelines and pathways, government strategies and national and local policies. internet enables an instant information dissemination enabling access to the latest results at any time as well as informal knowledge exchange by using chat rooms and discussion forums. however, it is getting increasingly difficult for busy professionals to find reliable quality-assured information on the internet when they need it. national internet libraries in the uk are addressing this problem: the umbrella portal national electronic library of infection neli (http://www.neli.org.uk) providing a single access portal to quality-assured information on treatment, diagnosis, prevention and management of infection diseases, and the national resource for infection control nric (http:// www.nric.org.uk) -a single-stop shop for policies, guidelines and research around infection control, hosted by neli. to better meet the information needs of these internet portals, accessed by 2000 unique users per month, we are conducting an information needs study to explore clinical questions, user needs and disease priorities of users seeking answers on neli and nric. a pilot qualitative online questionnaire-based study revealed that our users come from the variety of professionals: clinical scientist, consultant, registrar, psychotherapist, lecturer, gp, medical librarian, information scientist, health protection. these have questions mainly around hiv, tinea, molluscum contagio, meningitis, cold, mrsa, lyme, toxoplasma, chicken pox, influenza, diarrhoea and vomiting, rash, staph. aureus, traveller infections antibiotics resistance, malaria, mmr, meningitis, viral myocarditis, anthrax, smallpox, and tb. this is in line with our quantitative weblog-based evaluation of the most commonly access topics on neli by nhs-based users: antimicrobial resistance and hae (10.27%), tb (9.54%), meningitis (9.47%), hiv (8.95%), chlamydia (6.31%), e. coli (5.54%), staph. aureus (5.26%), adenovirus (4.84%), blood borne infections (4.44%). the results of the ongoing analysis of google search keywords that brought users to neli and nric will be discussed. further results identifying the needs specific to the infection disease professions will be discussed in relation to differences in the national variations in information needs and priorities. training in infection s. d'souza, p. kostkova, f. cooke, a. holmes (london, uk) specialists today require prompt access to quality information in order to work effectively. the diversity of specialist interests in the field of infection has led to the formation of a large number of professional and scientific societies. these play an increasingly important role in ensuring that the trainee is effectively supported, not only during the period of training, but also in longer-term personal development. details of relevant societies, conferences, grants etc are, on most society websites, confined to those for either that society or others in that specialty only, and knowledge of the numerous places in which to look for this information is necessary to find out the latest information. training in infection (tii -www.trainingininfection.org.uk) is an online resource, primarily aimed at infection specialist trainees but useful throughout the career path, which brings together this information into one central access point, so that users from all infection specialties can find the appropriate information for their specialty quickly and easily. it identifies and links to the key relevant resources covering a broad range of infection related disciplines in a dynamic database structure. information on societies, conferences, grants, journals, textbooks and more are available on the site, and have been put together to create a one-stop infection training portal. online discussion forums to be implemented will allow trainees' to share ideas and make the most of their combined expertise, and users will be able to receive alerts on new information in their specialty as well as be reminded of conference deadlines, journal submission deadlines etc. the ability to discuss regional issues online within specialties also aims to promote greater local and international collaboration. training in infection is endorsed by the national electronic library of infection (neli -www.neli.org.uk), an established digital library bringing together the best available online evidence-based resources on the investigation, treatment, prevention and control of infectious diseases. research designs and statistical methods in medical abstracts m. kompoti, m. matsagoura, a. koutsovasilis, a. koutsovasili, s. drimis (athens, gr) statistical methods used in biomedical research articles are being increasingly scrutinized in medical journals. however, no such strict policy is generally applied in abstracts presented in medical congresses. objective: this study aimed at assessing the frequency of research designs and statistical methods reported in abstracts presented in two successive years of the european congress of clinical microbiology and infectious diseases (eccmid). material and methods: we reviewed all abstracts included in the abstract book of the 14th eccmid (prague 2004) (pg) and the 15th eccmid (copenhagen 2005) (cp). all abstracts of original research studies but no abstracts of lectures were included in our study. two independent investigators read all abstracts and extracted information concerning origin, type (clinical, laboratory, animal model), research design, sample size and statistical methods used in the study. data analysis was performed with logistic regression and pearson's chi-square test for categorical variables and student's t-test for continuous variables. statistical significance level was set at p < 0.05. results: a total of 4178 abstracts were included in the analysis according to eligibility criteria (2110 from pg and 2068 from cp). laboratory studies prevailed (61%) followed by clinical studies (35%) and experimental studies with animal models (4%). the majority (79.3%) of the studies were observational (retrospective, prospective, cross-sectional) of which 6.3% concerned diagnostic accuracy testing of laboratory methods and 2.3% were pharmacological studies, 3.8% were randomized controlled trials. statistical evaluation was clearly described in 26.5% of abstracts (26.1% in pg and 19.8% in cp, p < 0.001), while the rest of abstracts included only descriptive statistics or no statistics at all. the proportion of statistical methods reporting varied according to the type of the study (animal model studies 49.4%, clinical studies 38.9% and laboratory studies 12.0%, p < 0.001). multicentre research studies reported statistics more frequently than single-center studies (26.5% vs. 21.5%, respectively, p = 0.005). conclusions: statistical analysis is an inseparable part of original research. research design as well as the implemented statistical methods should always be reported in an adequate manner, thus improving the scientific quality of abstracts. antimicrobial pk/pd p1512 nxl103-oral streptogramin: a phase i, doubleblind, single escalating oral dose study to evaluate safety, tolerability and pharmacokinetics in healthy adult male volunteers m. rangaraju, j. rey, j. hodgson (romainville, vitry sur seine, fr) background: nxl 103 (formerly xrp 2868) is a novel semisynthetic oral streptogramin that consists of a 30/70 (w/w ratio) association of a pristinamycin ia (pi) derivative and a pristinamycin iib (pii) derivative. nxl 103 is being developed for the treatment of respiratory tract and skin and skin structure infections. methods: 60 healthy male subjects were enrolled in this study. 10 subjects in each of 6 cohorts (125 mg, 250 mg, 500 mg, 1000 mg, 1500 mg and 2000 mg) received either nxl103 (8) or placebo (2) . an additional cohort of 10 subjects received a single dose of 500 mg nxl103 in fasting and fed conditions. blood and urine samples for pk analysis were collected at multiple time points. safety was assessed via adverse events, physical examination, clinical laboratory data, ecg and cardiac monitoring. results: nxl103 administered as 125 mg capsules at single doses from 125 mg to 2000 mg was well tolerated and safe. there was no serious or severe adverse event, no dosedependency in the number of aes or their severity, no significant variation in blood pressure or heart rate, no abnormality on ecg recording, and no clinically significant changes compared to baseline for laboratory parameters. both components were rapidly absorbed; pi being slightly more rapidly absorbed than pii. the cmax and auc (0-t) increased approximately in proportion with dose. the proportion of pi and pii components estimated on mean exposure values was approximately comparable to that administered (30/70), indicating that the relative bioavailabilities of pi and pii are simila. elimination half-life ranged from between 2 to 3 hours for pi to 4 to 6 hours for pii. food increased the bioavailability of pi and pii by approximately 20%. conclusions: nxl103 is safe, well tolerated and exhibits predictable pk properties in healthy volunteers in doses up to 2000 mg administered as a single dose. correlation of vancomycin and daptomycin susceptibility in staphylococcus aureus in reference to accessory gene regulator polymorphism and function w. rose, m. rybak, b. tsuji, g. kaatz, g. sakoulas (detroit, new york, us) objective: polymorphism at the accessory gene regulator (agr) locus in s. aureus (sa) defines 4 groups (i-iv). agr group ii sa have been associated with glycopeptide treatment failure in patients. sa with loss of agr function appear to have a higher tendency to become vancomycin (v) resistant. it is unknown whether this association only pertains to glycopeptides. we examined the effect of varying v and daptomycin (d) against agr+ and agr null pairs in an in vitro pharmacodynamic model (ivpm). methods: agr group i and ii wild-type prototype and knockout (tetm::agr) pairs were evaluated. mic values were determined according to clinical laboratory standards institute. ivpm glass and hollow fibre models were used to simulate dosages and auc/mic exposures for v ranging from 62.5 mg-1 g q 12 h fauc/mic range 31-665 mg/l*h, and d 0.75 mg/kg-6 mg/ kg/day fauc/mic range 37.5-346. the dosage regimen and auc/mic breakpoints that produced resistance was then evaluated in the hollow fibre ivpm. all ivpm simulations were performed in duplicate over 72 h. resistance was evaluated using 3 and 6 x mic screening plates at 0, 8, 24, 48, 56 and 72 h. results: pre-exposure mic values for agr i± and agr ii± were 0.75/1 and 1 for v and 0.25 lg/ml for d respectively. vintermediate resistance (mic = 8 mg/l) was detected in both agr i and ii null strains at a simulated v dosage of 62.5 mg q 12h (auc/mic 31), representing an mic increase of 8-10 fold. this breakpoint for resistance was verified in the hollow fibre model. although significant regrowth was noted with suboptimal dosing of d, no resistance was detected on d screening plates for any daptomycin regimen evaluated. conclusions: exposure of sa to v approximating 1/6 of optimal serum concentrations resulted in the development of heteroresistance in the agr null group i and ii. loss of agr function did not correlate with the development of d resistance despite suboptimal simulations of d exposures. these results implicate loss of agr function important to the development of glycopeptide resistance but not to loss of susceptibility to d. teicoplanin efficiently penetrates into the rabbit infected vitreous but may enhance expression of virulence factors at sub-inhibitory concentrations e. forestier, f. jehl, c. gallion, r. andres, s. bronner, l. leininger, g. prévost (strasbourg, fr) objectives: fluoroquinolones are the antibiotics that most efficiently penetrate inside vitreous. however, alternative treatments for endophtalmitis may be required in some cases for example resistant bacteria. we used a rabbit experimental model of endophtalmitis to evaluate the penetration of teicoplanin in different conditions. the influence of subinhibitory concentrations of teicoplanin was also evaluated on the expression of s. aureus virulence factors. methods: new zealand rabbits (>3 kgs) received one or repeated doses of intra-venous (iv) teicoplanin (60 mg/kg) every 12 hours for 3 days plus one dose a day for 2 more days. another group of rabbits was infected by 1000 cfu of a methicillin resistant s. aureus ibs 3284 (cmi = 1.5 mg/l) producing enterotoxin a, panton-valentine leucocidin and luke-lukd. they were administrated 24-36 hours later with 60 mg/kg teicoplanin, as a single dose or as to reach the steady state. vitreous (200 ll) was sampled before new injections of teicoplanin or at indicated time as well as blood before or 15 min after teicoplanin injection. teicoplanin concentrations were measured by hplc. bacterial counts were recorded and expression of virulence factors was semi-quantified by dedicated competitive rt-pcr tests. results: in safe eyes, teicoplanin penetration remains moderate reaching about 4 mg/l within about 6-8 h after one iv injection. the half-life of teicoplanin in the rabbit vitreous is about 25 hours. after 5 days of repeated injections, intra-ocular concentration stabilises around 4 mg/l while residual blood concentrations were comprised between 30-40 mg/l. in infected eyes, teicoplanin, when repeatedly administrated after the beginning of clinical signs, i.e. 24 h postinfection = 106 cfu/ml, reaches intra-vitreal concentrations of 9.3 ± 2.3 mg/l 40 h post-infection, and increases to 14.3 ± 5.4 mg/l 84 h post-infection and 12 h after a fourth injection. however, at sub-inhibitory concentrations (~2 mg/l), it may be responsible for a significant increase of agr, gammahemolysin hlga, luked and panton-valentine leucocidin luk-pv expressions with ratio ranging from 15 to 100 folds. conclusion: these preliminary results strongly suggest that teicoplanin iv administration constitutes an interesting alternative therapy for endophtalmitis provided high intraocular concentrations are rapidly obtained. investigations now concern optimisation of teicoplanin dosage regimen. pharmacokinetics of temocillin in intensive care patients and monte carlo simulations to evaluate susceptible breakpoints j.w. mouton, r. dejongh, v. basma, p. tulkens, s. carryn (nijmegen, nl; genk, brussels, be) background: temocillin (tmo) is a narrow spectrum penicillin with good activity against gram negative micro-organisms including esbl and ampc producers. little pharmacokinetic data are available however. we performed a pharmacokinetic study in 6 icu patients receiving tmo 2g q12h. parameter estimates were used to predict concentrations during continuous infusion (coinf) and compared with data obtained from 6 other icu patients receiving coinf to validate the model. the model was then used to perform monte carlo simulations (mcs) to determine probabilities of target attainment (ptas) for pharmacodynamic indices (pdi) in order to evaluate and suggest clinical breakpoints. methods: blood samples were taken from icu patients prior to (t = 0) and after (t = 1, 2, 3, 6, 12 h) a 30 m infusion of 2 g tmo (n = 6) or after 48 h during coinf with 4 g/24 h (n = 6), and then cooled, centrifuged and stored at )70°c until analysis by hplc. protein binding was determined using an ultrafiltration method. results were used to estimate population pharmacokinetic parameters by winnonmix including the covariance matrix. miclab233 was used to perform simulations for coinf as well as to perform mcs (10000 cycles) and obtain ptas for the unbound fraction including 95% confidence intervals (ci) for the target concentrations. ft>mic was chosen as the pdi because of the pharmacodynamic properties of tmo. results: protein binding was 75%. a one-compartment model best fitted to the data, with estimates (se) of vc = 14.3 (0.87) l and k10 = 0.172 (0.059) 1/h corresponding to a mean half-life of 4.03 h. using these estimates, the predicted unbound concentration during coinf was 16.9 mg/l, while the mean concentration in the 6 other patients was 17.03 mg/l, a bias of less then 1%. the breakpoint mic for a mean ft>mic of 50% was 16 mg/l. however, mcs -taking the variation in the population into account -indicated that 100% ptas of a 2g q12h dose were obtained at 4, 4, and 2 mg/l for 40, 50 and 60% ft>mic, respectively. the 95% ci at 50% ft>mic indicated a clinical breakpoint of 8 mg/l. the 95% ci was relatively large, as expected from data obtained in patients rather than volunteers. conclusion: the population pharmacokinetic estimates from 6 icu patients were very well in agreement with the validation study, with a bias of <1%. the mcs indicate a susceptible breakpoint for temocillin of £8 mg/l provided an administration of 2 g q12h is used. tissue penetration and pharmacokinetics of moxifloxacin in diabetic foot infections:an interim analysis j. majcher-peszynska, k. karrasch, m. saß, r. mundkowski, a. gussmann, p. kujath, b. ruf, w. schareck, h. koch, b. drewelow (rostock, bad saarow, lubeck, leipzig, beeskow, de) objectives: with its broad spectrum of activity against grampositive, gramnegative and anaerobic organisms moxifloxacin covers the pathogens of the mainly polymicrobial infections associated with the diabetic foot. inflammatory and fibrotic processes in diabetic foot infections (dfi) contribute to impaired tissue penetration of antibiotics. in addition, diabetic patients represent a pharmacological risk population, physiological changes in diabetic patients may alter the pharmacokinetics of antibiotics. the study was designed to investigate the penetration of moxifloxacin into perinecrotic tissue in 60 patients with dfi and the pharmacokinetic properties of moxifloxacin in diabetic patients. methods: the interim analysis of this open, multicentre study included 30 adult, hospitalized male and female patients (mean age: 69.8 years) with type 2 diabetes mellitus and dfi. the pharmacokinetic parameters of moxifloxacin and penetration into dfi tissue at steady state (day 4 to 8) following once daily administration of 400 mg iv or po were evaluated. correlations between penetration of moxifloxacin and clinical and laboratory parameters were examinated. results: in all 30 patients the moxifloxacin concentrations measured in infected diabetic foot wounds 3 hours after administration exceeded the in vitro mic90 values of susceptible staphylococci (0.125 mg/l). the moxifloxacin concentrations achieved in dfi tissue correlated more strongly with the auc0-24 (r = 0.659; p < 0.01) than with the corresponding plasma concentrations (r = 0.492, p < 0.01), but not with the extent of the systemic inflammation and the blood glucose level. taking into account the predictive pk/pd parameters for moxifloxacin (based on an in vitro mic90 value of 0.125 mg/l for staphylococcus aureus) a therapeutic success can be expected (auc24/mic: 204.3; cmax/mic: 22.9). significant differences between the routes of administration (iv vs po) were only observed for tmax (p < 0.01) and t1/2 (p < 0.05), but not for other clinically relevant parameters (auc0-24, cmax, moxifloxacin tissue concentration). this allows sequential therapy i.v./p.o. in this indication. conclusion: based on adequate plasma concentrations in diabetic patients, the sufficient penetration into dfi tissue and the possibility of a sequential therapy, moxifloxacin representsfrom a pharmacological point of view -a valuable therapeutic option in the treatment of diabetic foot infections caused by susceptible organisms. fluoquinolones effects on patient lymphocytes during prolonged treatments e. bertazzoni minelli, a. benini, d. doria, p. franceschetti, m.e. fracass (verona, it) fluoroquinolones (fq), widely used in clinical practice, are well tolerated. the most common adverse reactions are those affecting gastrointestinal tract, phototoxicity and allergy. the aim of the study is to evaluate the possible cellular damage in lymphocytes of patients treated with different fqs according to pharmacokinetic data. blood samples obtained from thirty-six patients treated with ciprofloxacin (cpx, 14 pts), levofloxacin (lvx, 15 pts.) and moxifloxacin (mfx, 7 pts) at different doses were analysed. patients treated with cpx and lvx were in therapy with other drugs (diuretics, cardiovascular drugs, omeprazole, antiinflammatory drugs, etc.). mxf treated patients were not in therapy with other drugs. samples were collected at time 0 (before fqs administration) and after 3 and 9 days of treatment. serum levels of fqs were determined with microbiological method and hplc. comet test was performed on lymphocytes, to evaluate dna damage. gsh levels were determined as efficiency marker in metabolic process of detoxification. cpx showed good serum concentrations; its levels increases proportionally with administered doses (from 250 to 1000 mg). lvx concentrations resulted in good inhibitory levels after 3 treatments (500 mg) both per os and i.v. patients orally treated with 250 mg showed similar serum levels (from 0.8 to 2.1 mg/l). mfx levels were between 3.6 and 1.6 mg/l after 3 and 9 days. repeated cpx administration induced a dose-dependent increase in all dna damage parameters, with statistical differences after 9 treatments. mfx (400 mg) and lvx administration didn't induce dna damage after 3 and 9 days. intracellular levels of gsh were similar in all treated groups, even if cpx treated patients showed the lowest concentrations. no statistical correlations were found between all parameters studied. these data indicate that cpx induce dna damage in lymphocytes in combination with a reduced efficiency in detoxification system. this effect does not seems to depend on high intersubject variability for fqs administered doses, co-administration of other drugs, different ages of patients and low samples numbers. effects of single fqs molecules seems to be structurespecific and selective. objectives: ertapenem is a carbapenem commonly used to treat intra-abdominal infections. the antibacterial spectrum includes the major causative pathogens. clinical trials proved excellent clinical and microbiological efficacy in peritonitis. on the other hand in inflammatory pancreatic diseases sufficient antibiotic concentration in the inflammatory tissue is vital for the outcome of the disease. we therefore investigated ertapenem concentrations in pancreatic tissue and juice in comparison to the plasma levels measured at the same time. methods: in a prospective clinical trial ertapenem was given in a dosage of 1 g i.v. 30 minutes prior to operation in patients (18-70 years) suffering from chronic pancreatitis or pancreas carcinoma undergoing pancreas resection. blood samples were collected every 60 minutes during the operation. moreover we collected pancreatic tissue and pancreatic juice shortly before resection and shortly before finalisation of the anastomosis. the samples of ertapenem (blood, juice, tissue) were determined by hplc. results: in 13 patients (5 female, 8 male, mean age 54.8 ± 11.2 years) ertapenem blood concentrations were determined and demonstrated intraoperatively high concentration (20 ± 8 mg/l) above mic90 values for major expected pathogens. concomitantly in 10 of these patients ertapenem concentration was determined also in pancreas tissue and pancreas secretion (in further 3 patients in pancreas secretion only). in 7/10 patients sufficiently high ertapenem levels were detected in pancreatic tissue. in 3 patients with chronic pancreatitis no accumulation was seen. mean pancreas tissue concentration was 0.8 ± 0.4 lg/g tissue. 5 of 6 patients with pancreas carcinoma had increased ertapenem levels in pancreas secretion but only 2 of 6 patients with chronic pancreatitis. conclusion: in patients with pancreas carcinoma, ertapenem levels were measured in pancreatic tissue as well as in pancreatic secretion and penetration seems to be similar to imipenem. due to chronic inflammation and possibly altered microcirculation only in one half to one third of chronic pancreatitis patients ertapenem levels were detected. bacterial strain-independent pharmacodynamics of linezolid/doxycycline combinations with staphylococcus aureus: 5-day simulations using an in vitro dynamic model m. smirnova, i. alferova, i. lubenko, y. portnoy, s. zinner, a. firsov (moscow, ru; cambridge, us) objective: to delineate the possible advantages of linezolid (l)/doxycycline (d) combinations over monotherapy, the pharmacodynamics of l, d and l+d were studied with s. aureus. methods: s. aureus atcc 43300 and a clinical isolate s. aureus 479 were exposed to twice-daily l (half-life 6 h) and once-daily d (half-life 15 h), alone and in combination (1:3 ratio based on 24-h auc/mics), for five consecutive days. to provide simultaneous mono-exponential elimination of l and d with different half-lives, a previously described dynamic model was modified according to blaser and zinner. the mics of l were 1.56 and 1.56 mg/l and mics of d were 0.1 and 0.05 mg/l for s. aureus atcc 43300 and s. aureus 479, respectively. nine dosing regimens were simulated with each organism exposed to different auc/mics (in hours): l30, l60 and l200; d90, d180 and d520; l30 + d90, l60 + d180 and l200 + d520. the cumulative antimicrobial effect was expressed by its intensity (ie) measured from the start of treatment to the time after the last antibiotic dose when numbers of antibiotic-exposed bacteria reached at least 108 cfu/ml. emergence of resistance was monitored daily by quantitating surviving organisms on agar plates containing 2x and 4xmic of l or d. results: with both s. aureus atcc 43300 and s. aureus 479 exposed to l or d, ie increased with increasing simulated auc/ mic ratios, although significantly higher ies were produced with l30, l60 and l200 treatments relative to d90, d180 and d520 treatments. each of the combined treatments, i.e., l30 + d90, l60 + d180 and l200 + d520, produced much greater ies than the sum of l and d ies observed in the respective mono-treatments with both s. aureus strains. based on population data, a pronounced selection of s. aureus resistant to d occurred in all mono-treatments with d. it was also observed with l30 + d90 and, to a lesser extent, with l60 + d180 but not with l200 + d520. no resistance to l was observed with l mono-or combination treatments. conclusions: these data predict a synergistic interaction of l with d against s. aureus. anti-staphylococcal effects of telavancin in an in vitro dynamic model: impact of different half-lives and initial concentrations that simulates normal (nek) and impaired elimination kinetics (iek). materials and methods: a glycopeptide intermediately susceptible strain of s. aureus (gisa) mu-50 with a telavancin mic of 0.5 mg/l was selected for the study. with both nek and iek simulations at a starting inoculum of 6 log cfu/ml, gisa mu-50 was exposed to different ratios of the peak concentration (cmax) to the mic of telavancin (as a single dose), i.e., 2.6, 7.8 and 14. based on time-kill data, the intensity of the antimicrobial effect (ie -the area between control growth and time-kill curves) was determined from time zero to the time when the effect no longer could be detected, i.e. the time after the last dosing at which the number of antibiotic-exposed bacteria reached 7 log cfu/ml. results: in each treatment, bacterial regrowth followed gradual reduction in the starting inoculum during the first 12 h (similar in nek and iek simulations) that led to significantly lower minimal numbers of surviving organisms in iek simulations compared to nek simulations. despite similar rates of initial killing, times to regrowth were much longer in iek than nek simulations. at a given cmax/mic ratio, the ies observed in iek were greater than in nek simulations (figure). conclusions: these findings demonstrate pharmacokineticdependent pharmacodynamics of telavancin with staphylococci. pharmacokinetics of amoxicillin in pregnant women with pre-term premature rupture of the membranes objectives: amoxicillin is widely used during pregnancy, in particular to treat group b streptococcus. insufficient knowledge on the pharmacokinetics just before and during delivery, could pose patients with preterm premature rupture of the membranes (pprom) at serious risk for under dosing. we investigated the pharmacokinetics in patients with pprom in this critical situation. methods: seven healthy women at 34-37 weeks of gestation were included. they received 1 g (first dose 2 g) amoxicillin for pprom according to local guidelines. from each patient 14-28 blood samples were taken. antibiotic serum concentrations were determined by a validated hplc method. pharmacokinetic parameters were estimated by population pk modeling using nonmem. to discriminate between various models the minimum value of the objective function (mvof) was used. a reduction of >10 in mvof was considered significant. results: a three-compartment pharmacokinetic model best described the time course of amoxicillin. the clearance and volume of distribution of the central compartment (vc) were estimated at respectively 23 ± 1.8 l/h and 6 ± 0.8 l (mean ± se). estimates of the parameters and model discrimination improved when we assumed the size of the third compartment to be equal to the first compartment. the residual error was found to be proportional to the serum concentrations. most of the inter-individual variability could be explained by variation of clearance. the mean volume of distribution at steady state (vss) and terminal half-life were 23.9 l and 1.34 h respectively. estimated values of elimination and distribution rate constants were: k10 = 3.9 h-1, k12 = 1.7 h-1, k21 = 0.8 h-1, k13 = 8.8 h-1 and k31 = 8.8 h-1. as was to be expected due to the small population size, no significant relationship was observed between the individual posthoc estimates for clearance and patient characteristics. conclusion: here we describe the pharmacokinetics of amoxicillin in pregnant women with pprom. it was found that the pharmacokinetics clearly differs from that in nonpregnant individuals. clearance and vss were significantly higher and the terminal half-life was shorter. furthermore, a 3-compartment model was found to describe the data better than a 2-compartment model. it is an intriguing question whether this 3rd compartment is a unique feature associated with pregnancy. these data offer a theoretical basis to make proper dose-adjustments in a particular patient group in a critical condition. penetration of piperacillin and tazobactam in severe acute pancreatitis objectives: acute necrotizing pancreatitis is still related to an extremely high mortality rate, based on local infectious complications, particularly in necrotizing areas. limited penetration of antimicrobial drugs in these areas is considered to be a major cause for failure of therapy of severe infections. combinations of beta-lactamase inhibitors (bli) and beta-lactam antibiotics like broad-spectrum penicillines (bsp) have antibacterial activity against most of the common pathogens in severe necrotizing pancreatitis. co-administration leads to an increase of antibacterial activity due to an inhibition of betalactamases. on that score, the penetration of co-administrated pip and bli into inflamed or necrotic pancreatic tissue has not been investigated yet. methods: adressing the penetration capability of bsp and bli a clinical trial was designed to investigate the penetration of piperacillin (pip) and tazobactam (taz) in patients with severe necrotizing pancreatitis undergoing pancreas surgery. samples (n = 21) were taken from plasma (pl), necrotic areas of pancreatic tissue (pn), peripancreatic fatty tissue (pft) and bursa secretion (bs) following intravenous administration of 4.0 g pip and 0.5 g taz. concentrations of pip/ taz were determined by hplc/ uv. results: mean plasma concentrations at 1.5 h after application were 56.4 ± 29.5 mg/l (pip) and 16.9 ± 15.9 mg/l (taz). exceeded in pl and bs, nearly reached in pn but not in pft. the concentration of pip in combination with taz exceeded or reached the mic90 in pl, pn and bs against e. coli, klebs. spp., enterobacter, proteus spp. and clostr. spp., in pl and bs even against pseudomonas and bacteroides. conclusion: given in combination both -pip and taz -have been demonstrated to reach rapidly effective inhibitory concentrations in inflamed and necrotic compartments of pancreatic and peripancreatic tissue. co-administration of piperacillin and tazobactam may have a potential clinical benefit in prevention and treatment of local infectious complications of severe necrotizing pancreatitis. pk/pd challenges of in vitro time-kill curves -a new modelling approach s. schmidt, o. burkhardt, w. treyaprasert, h. derendorf (gainesville, us; bangkok, th) objective: in vitro pk/pd models, based on time-kill curve data, have become a powerful tool to predict the in vivo situation. up to date, several modelling approaches have been undertaken to develop suitable pk/pd models that fit in vitro data sufficiently well. widely used simple sigmoid emax models meet these criteria only partly. a further approach was undertaken to address the weak points of currently used models and applied to model the effects of ceftriaxone against escherichia coli. methods: constant concentration time-kill curves were performed in mueller-hinton broth (mhb, difco) with and without bovine serum albumin (bsa) 40 g/l. using concentrations of ceftriaxone, ranging from 0.25 mic to 16 mic, the change in number of bacteria (cfu/ml) versus time was linked to its effect. escherichia coli atcc 25922 was employed as the test organism. samples were taken at 0, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 hours. the data were modelled simultaneously, using a modified sigmoid emax model and the software scientist ò for windows tm . results: a differential equation, characterized by growth rate constant (k0) times the starting number (n) of bacteria represent the simplest case. barging from log-growth phase to stationary phase can be described by an additional nmax term. however, bacteria do not necessarily start growing in the loggrowth phase. this delay in onset of growth can be modeled by an exponential term, characterized by a factor beta (b) and time (t). to describe the overall change in number of bacteria not only growth but also concentration (c) dependent kill has to be taken into account. from certain drug specific concentrations on, a maximum effect is reached, described by the maximum kill rate constant kmax. however, it may be necessary to model a delay in the onset of kill with an additional exponential term, characterized by a factor alpha (a) and time (t). finally, a hill factor/shape factor (h) is necessary to smooth the predicting curves out. as shown in figure 1 this new model meets the in vitro time-kill curve data sufficiently well. the final equation including all parameters described above is: conclusion: the proposed model was able to describe the observed data much better than a simple emax-model. incorporating two additional terms into the model, the in vitro situation could be described much better, taking the delay in onset of growth and kill into account. objectives of this study were (1) to describe the pharmacodynamic (pd) profiles of bpr 500 mg iv q8h as a 2hr infusion & 500 mg iv q12h as a 1-hr infusion; (2) to determine the overall probability of target attainment (pta) by weighting for the expected distributions (dis) of renal function (rfx) in the populations (pop) of interests; (3) to determine the organism-specific pta against the pathogens encountered in phase ii trials. methods: 150 subjects (total 2443 samples) were studied (phase i/ii subjects). samples were analysed using bignpod. to assess the impact of differing degrees of rfx impairment on pta, crcl (crcl-cockcroft & gault method) was employed as a covariate in the pop pk analysis. monte carlo simulation (mcs) (9999 subjects) was performed with adapt ii. overall pta was calculated for 30-60% ft>mic. weighting for the expected dis of rfx in the pop of interests was accomplished by using the dis of crcl observed in 2 previous registration studies of the same indications (cssti and np). dis of mics for pathogens was supplied by sponsor. results: in the pop pk analysis, the pop mean (sd) values for volume, clslope, clintercept, kcp and kpc were:7.65 (3.89) l, 0.51 (0.32) l/hr, 2.35 (1.08), 3.05 (5.14) hr-1 and 1.10 (0.95) hr-1, respectively. the obs-pred plot was obs = 1.003 x pred + 0.627; r2 = 0.947 after the bayesian step. in the mcs analysis of bpr 500 mg iv q12 h, the pta of achieving 30% ft>mic & 50% ft>mic exceeded 90% for mics values £2 mg/l & £0.5 mg/l, respectively. for bpr 500 mg iv q8h, the pta of achieving 60% ft>mic exceeded 90% for mics values £2 mg/l. in the organism-specific analysis, the pta of a static effect (30% ft>mic) exceeded 90% for both mssa & mrsa for bpr 500 mg iv q12h. bpr 500 mg iv q8h provided a >90% pta of a cidal effect (50% ft>mic) for both mssa & mrsa. for gnb, the pta of bpr 500 mg iv q8h in achieving a cidal effect (60% ft>mic) exceeded 90% for non-ampc-bearing gnb. for ampc-bearing gnb, the pta of achieving a cidal effect was 84.8%. conclusions: an extensive evaluation of the pd of bpr was performed to estimate the overall activity of bpr against target pathogens. these findings need to be validated in the clinical trial arena. investigation of different levofloxacin regimens in patients with acute complicated urinary tract infections p. tenke, r. benko (budapest, szeged, hu) objective: in the present study we aimed to find out if a continuous or an intermittent levofloxacin (1 · 500 mg) treatment is more advantageous for patients with acute complicated urinary tract infection (uti) caused by urinary obstruction. we investigated if levofloxacin adsorbs to the surface of the foreign body, which was inserted with the aim of temporary resolution of ureteral obstruction. preventive effect of levofloxacin on bacterial biofilm formation and incrustation was also evaluated. methods: we enrolled and randomised 24 patients who had acute uti caused by urinary obstruction. obstruction was resolved with double j stent (djs) insertion or percutaneous nephrostomy (pcn) and meanwhile, antibiotic treatment was started in all patients. 12 patients (group 1) were on antibiotics till the day of definitive curative operation when all foreign bodies were removed. in the other 50% of the patients (group 2) the antibiotic therapy was stopped 7 days after the djs or pcn insertion. short term antibiotic course -which is advisable for prevention of uti before invasive endoscopic treatmentwas administered in both groups from the day of the operation (after djs or pcn removal) and it was continued until the removal of all possible urinary foreign bodies used during the operation. in both groups of patients we recorded and evaluated early and late clinical and microbiological recovery. retrieved stents were sectioned for further laboratory examinations. adsorbed levofloxacin in the conditioning film layer and on the stent surface was detected by hplc. rasterelectron microscopy (rem) was used to examine biofilm formation and encrustation. results: we did not find any significant differences between the two groups of patients, neither in clinical (presence of fever, back pain, flank pain, leukocyte count) nor in microbiological recovery. statistical analysis showed that significantly greater amount of levofloxacin adsorbed to the conditioning film than to the stent surface in both groups of patients (0.72 ± 0.83 vs. 0.20 ± 0.34 in group 1 and 0.24 ± 0.28 vs. 0.1 ± 0.15 in group 2). no viable, adherent bacteria were recovered by sonication and culture in any of the patients, and no biofilms or encrustation were seen under rem either. conclusion: our data prove the hypotheses that continuous antibiotic treatment does not have any clinical or microbiological advantages in patients with indwelling ureteral stents compared to intermittent therapy. objective: the prophylaxis of bacterial infections during cardiac surgery is widely used in clinical practice. staphylococcus aureus, staphylococcus epidermidis and enterococcus spp are the pathogens most frequently involved in infective complications of cardio-pulmonary bypass (cpb) surgery. it is generally agreed that the success of prophylaxis is dependent on the ability to reach and maintain free antibiotic concentration in tissues higher than the mics for the most common pathogens. so we estimated the tissue concentrations of linezolid into sternal bone of patients undergoing cpb surgery. methods: six patients undergoing routine cpb surgery were given 600 mg linezolid as a 30 min iv infusion along with conventional prophylaxis of 1.5 g of cefuroxime immediately before surgery. two hours after the end of infusion blood samples and sternal bone tissues were collected. the local medical research ethics committee approved this study and all patients gave written informed consent. samples were assayed for the presence of linezolid by a high-performance liquid chromatography (hplc) method. results: following a 600 mg infusion of linezolid, mean serum concentration for the six patients were 8.48 mg/l (range 7.3-12.36 mg/l) 2 hours after the end of infusion. the concentration of linezolid into sternal bone was 4.35 mg/l (range 3.9-5.2 mg/ l) 2 hours after the end of infusion. the penetration of linezolid into sternal bone was 52.2%. conclusion: the penetration of linezolid into bone was 52.2% of the simultaneous blood levels. in all bone samples the concentration of linezolid exceeded the mic for susceptible pathogens (<4 mg/l). although these data have been obtained from healthy, well-perfused bone the values suggest that linezolid may be a useful agent in the management of multidrug-resistant gram-positive bone infections. the antibacterial effect of daptomycin, teicoplanin and vancomycin against s. aureus studied in an in vitro pharmacokinetic model of infection a. noel, k. bowker, a. macgowan (bristol, uk) objectives: daptomycin (dap) is the first cyclic lipopeptide antibiotic approved for parenteral use in gram-positive infection. as yet, no comparative pharmacodynamic studies have been performed using dap and the two most common iv therapies teicoplanin (tei) and vancomycin (van). we used a pharmacokinetic (pk) model to study the antibacterial effect (abe) of these agents against two typical mrsa strains (ukemrsa15 & 16) and a hetero vancomycin intermediate mrsa (hvisa). methods: an in vitro dilutional model was used to simulate the free drug concentration over 48 h associated with doses of -dap 6 mg/kg 24 hrly (cmax 6.4 mg/l, t 1/2 8 h); tei 400 mg 24 hrly (cmax 4.5 mg/l, t1/2 24 h); van 1 g 12 hrly (cmax 13.5 mg/l, t1/2 6 h). an inoculum of 10 6 cfu/ml was used and the experiments performed in triplicate. abe was assessed by area-under-the-bacterial-kill-curve 0-24 h (aubkc24) and logcfu/ml.h objectives: linezolid, the first oxazolidinone, is active against methicillin resistant staphylococcus aureus and has been effective in a variety of acute infections. however long-term administration, although desirable in bone infections caused by resistant gram-positive organisms, is hampered by the occurrence of anaemia and thrombocytopenia. administration of vitamin b6 has been reported to prevent myelosuppression. methods: patients attending the infectious disease clinic with bone infections caused by resistant gram-positive bacteria and treated with linezolid (600 mg b.d. orally), received vitamin b6 (125 mg o.d. orally) for the period of administration of linezolid. full blood counts were followed-up weekly. linezolid treatment was discontinued if haemoglobin declined below 9 mg/dl or platelets below 120000/ll. data from sixteen patients with osteomyelitis and 8 with prosthetic joint infections were evaluated. comparisons were performed with 20 matched historical controls receiving linezolid without b6 by kaplan-meyer curves with the log-rank test. results: the median follow-up of patients receiving b6 was 3.5 weeks and of controls 4 weeks. in the b6 group 63% of the patients discontinued while in the control group 44% of the patients discontinued treatment because of side effects (p ns). 47% of patients receiving b6 discontinued due to thrombocytopenia and 20% due to anaemia. respective percentages in the control group were 21% and 38% (p ns for all comparisons). mean time to the occurrence of thrombocytopenia was 4 weeks in the patients who received b6 and 3.25 weeks in the control patients. respective times to occurrence of anaemia were 3.4 and 3.85 weeks. all cases of myelosuppression were reversible. conclusions: administration of b6 failed to prevent or delay both thrombocytopenia and anaemia in patients receiving linezolid. other methods should be investigated to facilitate longer administration of linezolid in this group of patients. therapeutic drug monitoring of colistin -a 7-year review from a uk clinical antibiotic assay service k. bowker, a. noel, s. tomaselli, a. macgowan (bristol, uk) objectives: over the last 5 years there has been increased use of colistin (col). however, little clinical data is available on the therapeutic levels of colistin. monitoring of col is useful in terms of therapeutic levels and avoidance of toxicity for patients with cystic fibrosis and complicated gram-negative infections. previous in vitro data has shown that col is bactericidal at col concentration is ‡3 mg/l. here we assessed col data collected from our antibiotic assay service from the last seven years in order to establish if such levels were obtained. methods: col levels were determined by bioassay. data was retrospectively collected from the hospital information management system. data was assessed collectively and stratified by known cystic fibrosis patients, sex and age. objectives: ßlactams like ceftriaxone (cfx) and quinolones such as moxifloxacin (mox) are widely used to treat pneumococcal infection. we studied the antibacterial effect of (abe) after the first dose exposure to free drug serum concentrations of iv cfx 2 g and po mox 400 mg against s. pneumoniae strains with typical mics at low and high inocula. methods: a hollow fibre in vitro model was used to simulate free drug concentrations over 24 h of cfx (2 g 24 h iv, cmax 26 mg/l, auc 56 mg/laeh, t1/2 6 h) and mox (400 mg, 24po, cmax2.0 mg/l, auc20 mg/laeh, t1/2 10.5 h). the cfx mic was 0.06 mg/l (t>mic cfx 100%) and mox mic 0.06 mg/l (auc/ mic 334). initial abe was measured by the slope of the log viable count 0-5 h and total abe over the dose interval (24 h) by log reduction in viable count at 24 h (d24) and the area-underthe-bacterial-kill-curve (aubkc24). inocula of 106 cfu/ml and 108 cfu/ml were used. results: the initial and total abe at low and high inocula were: given the pk/pd indices modelled both drugs showed a maximal effect. clearance from the model occurred at 12 h (106 inoculum) and 24 h (108 inoculum). there were no significant differences in speed or extent of abes comparing cfx and mox. conclusion: the abes of iv cfx and po mox against s. pneumoniae is similar in the first 24 hrs of drug exposure. emergence of resistance in e. coli and ent. cloacae after exposure to ceftriaxone or ertapenem in an in vitro model of infection k. bowker, a. noel, a. macgowan (bristol, uk) objectives: emergence of resistance (eor) is an emergent factor in therapeutic choice. we studied eor to ceftriaxone (cfx) and ertapenem (ert) in e. coli (ec) and ent. cloacae (entclo), a more challenging species within inducible blactamases. methods: an in vitro dilutional model was used to simulate free drug concentrations associated with 1 g 24 hrly cfx (cmax 22 mg/l, t½ 8 h) and ert (cmax 13 mg/l, t½ 5 h) over 72 h. two inocula 106 and 108 cfu/ml were used and eor assessed by population-analysis-profiles (pap). the area under the pap (auc-pap) was used to measure eor. ert mics were 0.018 mg/l ec and 0.25 mg/l entclo, cfx mic 0.14 mg/l ec and 0.5 mg/l entclo. experiments were performed in triplicate and mean values presented. results: observations at 72 h were similar to those at 48 h, hence data to 48 h is given. at 106 and 108 cfu/ml, ec viable counts were reduced by 2 fi 4 logs, there was no eor. against entclo inoculum 106 and 108, cfx resulted in an initial 0-2 log drop, then regrowth, ert produced a >4 log reduction. eor as measured by the mean auc-pap (n = 3) with entclo is shown below: dosing with cfx resulted in eor to cfx and ert at high and low inoculum. dosing with ert resulted in no eor at 106 inoculum, at 108 resistance emerged to both cfx and ert. conclusions: eor depends on species (entclo > ec); duration of exposure (long > short) and agent (cfx > ert). ert appears to induce less eor both to itself and cfx than cfx does to itself and ert. however, initial use of cfx may reduce the effectiveness of ert. comparative serum activity of telithromycin, azithromycin, and amoxicillin/clavulanate against aerobic and anaerobic respiratory pathogens objectives: the purpose of this investigation was to study the clinical potential of telithromycin, a new ketolide antibiotic, for the treatment of mixed aerobic/anaerobic respiratory infections. in this study, we compared the pharmacodynamics (duration of inhibition/killing) of telithromycin (tel) to azithromycin (azi) and amoxicillin/clavulanate (a/c) against aerobic and anaerobic pathogens associated with mixed respiratory infections. methods: following written informed consent, ten healthy adult subjects (ages, 20-47 yrs) received single doses of tel (800 mg), azi (500 mg), and a/c (875 mg) one week apart following a 12-h fast. venous blood samples were obtained at 2, 6, 12, and 24 h after the dose and stored at )70°c. inhibitory and bactericidal titre s were determined by microdilution (s. pneumoniae & h. influenzae) and agar dilution (peptostrep. magnus, peptostrep. micros, prev. bivia, & prev. melaninogenica) procedures following clinical & laboratory standards institute methodology. bactericidal titres in serum endpoint was determined as the highest dilution of serum yielding 99.9% killing. the median titres at each time point were calculated and the duration of activity was used for comparison of these agents. conclusions: in this ex-vivo study, we found that tel can provide prolonged (50% of the dosing interval) inhibitory activity in serum against macrolide-resistant strains of s. pneumoniae, bl pos. and neg. strains of h. influenzae, and common respiratory anaerobic pathogens. these findings suggest that tel could have clinical utility in the treatment of community-acquired mixed aerobic-anaerobic respiratory tract infections, including sinusitis, bronchitis, and pneumonia. objectives: increasing resistance in isolates of e. coli (12%) and p. aeruginosa (40%) to fluoroquinolones (fq) is a concern since these antibiotics are commonly used in the treatment of complicated urinary tract infections (utis). currently, no interpretive standards exist for ''susceptible'' isolates in urine for the newer fq. the purpose of this investigation was to evaluate the activity of high-dose levofloxacin against fqresistant urinary pathogens. methods: in this study, we determined the serum and urine levels of high dose (750 mg) levofloxacin (l) as well as its bactericidal activity in urine (uba) against l-resistant isolates of e. coli (mics = 8 to 64 lg/ml) and p. aeruginosa (mics = 8 to 64 lg/ml). following written informed consent, blood and urine samples were collected from 10 healthy adult (ages, 20-47 y/o) fasting subjects (5 m and 5 f) prior to and at 1.5, 4, 8, 12 , and 24 hours after a single 750 mg dose of l. serum and urine concentrations were measured by a validated hplc assay (0.9-2.1% cv). the testing methodology for uba was similar to the microdilution assay used for serum bactericidal testing (clsi) with the exception that antibiotic-free urine was used to dilute these samples. the median titre (1:2-1:32) at each time point for the 10 subjects was used to determine uba. results: the mean serum pharmacokinetic parameters were similar to previously published values: cmax = 9.2 lg/ml, auc = 95 mgaeh/l, and t1/2 = 7.6 h. mean urine concentrations ranged from 475 lg/ml (4 h) to 186 lg/ml (24 h). uba (titres >1:2) was maintained for at least 12 hours in all subjects for e. coli isolates with mics = 8, 16, and 32 lg/ ml. for the e. coli strain with a mic = 64 lg/ml, 8 subjects exhibited uba at 8 h but only 2 subjects exhibited uba at 12 h. similar results were observed against the p. aeruginosa isolates. conclusions: the results from this ex-vivo pharmacodynamic study in healthy volunteers found that 750 mg of l provides prolonged (at least half the dosing interval) uba against l-resistant strains of e. coli and p. aeruginosa up to 32 lg/ml. this suggests that a separate urinary susceptibility breakpoint is indicated for urine isolates treated with 750 mg doses of l. objectives: exposure of methicillin-resistant s. aureus (mrsa) to acid ph restores its susceptibility to beta-lactams (sabath and al., aac, 1972) . in macrophages, s. aureus is mainly confined within phagolysosomes where the ph is acidic. we showed that meropenem (mem) displays similar intracellular activity against mrsa atcc 33591 and mssa atcc 25923 in macrophages. in the present study, we have investigated the intraphagocytic activity of mem and cloxacillin (clx) against 3 mrsa clinical isolates (including one visa strain), in comparison with the reference mrsa atcc 33591 and mssa atcc 25923 strains. methods: mic's were determined in mhb (plus nacl 2%) by micro-dilution method. meca expression was examined at neutral and acidic ph by a semi-quantitative rt-pcr (16 s rrna as housekeeping gene). intracellular activity was assessed in human thp-1 macrophages exposed to extracellular concentrations equivalent to human cmax (total drug; mem: 50 mg/l; clx: 8 mg/l) by examining the decrease in cellassociated cfu after 24 h from the original, post-phagocytosis inoculum (controls [no antibiotic]; approx. 1 log cfu increase). results: the table shows the mics (in broth) at neutral and acid ph and the intracellular activity for the 5 strains studied. in atcc 33591, meca expression was similar for bacteria maintained in broth at ph 7. conclusions: the intracellular environment markedly enhances the activity of beta-lactams against mrsa, probably through exposure to acid ph, although the latter does not affect meca expression. comparative activity of dalbavancin against european gram-positive isolates i. morrissey, c. dencer, j.w.t. dallow, j. childers, a. brook, j. cowan (london, uk) objectives: dalbavancin (dal) is a new semisynthetic lipoglycopeptide with a half-life of 8.5 days, enabling onceweekly dosing. this study compared the activity of dal with other agents against gram-positive isolates from europe. methods: isolates from belgium, the czech republic, denmark, finland, france, germany, hungary, italy, the netherlands, poland, spain, sweden and the uk were included. the clsi broth microdilution method was used to determine mic using dried microtitre plates. the following antimicrobial agents were evaluated: dal, vancomycin (van), teicoplanin (tei), daptomycin (dap), linezolid (lzd), dalfopristin/quinupristin (syn), erythromycin (ery), levofloxacin (lev) and tetracycline (tet). results: selected data are shown in the objectives: dalbavancin (dal) is a next generation lipoglycopeptide antibiotic in development for the treatment of complicated skin and skin structure infections (csssi). a population pharmacokinetic (pk) analysis was performed to estimate patient parameters and to determine significant covariates. incorporating the pk model, pharmacodynamic (pd) parameters were simulated to support the effectiveness of a weekly dosage. methods: the pk analysis included 1668 dal concentrations from 532 patients across 3 clinical trials. most patients received 1000 mg on day 1 and 500 mg on day 8. possible covariates examined included demography and concomitant medications, including medications that are considered inhibitors, inducers, and substrates of cytochrome p450 enzymes. the pk-pd analysis employed monte carlo simulations of time-dependent and concentration-dependent parameters. distributions of mics were obtained directly from clinical studies, and were also simulated to explore the effect of higher mics. results: dal pk fit a 2-compartment model with interpatient variability (ipv) on all parameters. the typical value and ipv (cv%) of clearance (cl) was 0.0571 l/h (18.0%), influenced by body surface area (bsa) and creatinine clearance (clcr). volume of distribution (v1) was 4.15 l (24.5%) and influenced by bsa. the inter-compartmental clearance and peripheral volume were 0.476 l/h and 11.4 l, respectively. free drug concentrations were simulated using a dal protein binding of 93%. for a weekly 2-dose regimen, free dal remained above 1 mg/l for the majority (>90%) of patients for more than 14 days. using previously described area under the curve (auc)/mic targets for staphylococcus aureus, a proposed mic of at least 0.5 mg/l was associated with a greater than 90% probability of target attainment. conclusions: dal pk were predictable, demonstrating low ipv. bsa and clcr were the only sources of variability, but described less than 25% of the ipv. pd simulations support the use of dalbavancin in a weekly regimen. objectives: methicillin-resistant staphylococcus joint infection due to peri-operative contamination is a complication after arthroplasty. the objective of this study was to assess the distribution of radioactivity in bone and related structures using quantitative autoradiography after administration of [14c]dalbavancin in rabbits. methods: new zealand white male rabbits were given a single intravenous (iv) bolus dose of 20 mg/kg [14c]-dalbavancin (n = 18) or control vehicle (n = 3). plasma, cerebrospinal fluid (csf), bone, bone marrow, and nucleus pulposus were collected at 12, 24, 72, 120, 168 and 336 h post-dose by necropsy, homogenized, combusted, and analysed for total drug-derived radioactivity using liquid scintillation counting (lsc). in addition, the left hindlimb from 1 rabbit/time point was flash-frozen and cryosectioned for quantitative autoradioluminography. results: [14c]-dalbavancin-derived radioactivity was rapidly and widely distributed into bone, bone marrow and, to a lesser extent, in csf and nucleus pulposus. autoradioluminography data indicated that concentration of radioactivity was highest in bone marrow, whole blood, articulate cartilage, ligament, epiphyseal plate, periostium, and meniscus. at 336 h postdose, [14c]-dalbavancin-derived radioactivity was measurable in all tissues, and remained at relatively high concentrations in bone marrow (30.27 lg equiv/g), epiphyseal plate (8.42 lg equiv/g), periostium (5.18 lg equiv/g), and articular cartilage (4.84 lg equiv/g). in homogenized bone using lsc, mean concentration after 336 hours was 1.96 lg equiv/g. conclusion: [14c]-dalbavancin-derived radioactivity rapidly penetrated knee joint tissues and persisted at relatively high concentrations for at least 336 h after a single iv dose in rabbits. objectives: telavancin (tlv), a bactericidal lipoglycopeptide with multiple mechanisms of action, is in phase 3 trials for the treatment of hospital-acquired pneumonia (hap) with a focus on infections due to methicillin-resistant s. aureus. tlv is primarily eliminated by the kidneys and requires dosage adjustment for renal dysfunction. tlv is highly protein bound (93%) in healthy subjects which would suggest that it would not be removed by dialysis, but its small volume of distribution (0.15 l/kg) means that it may be removed by cvvh. cvvh is widely used in the management of critically ill patients. the objective of this study was to determine cvvh telavancin transmembrane clearance (cl) with 2 commonly used hemofilters (an69, polysulfone) at conventional ultrafiltrate flow rates. methods: tlv cl was assessed in our in vitro cvvh model using citrate anticoagulated bovine blood and b. braun diapact machine. experiments were run using an69 (m100, gambro) and polysulfone (f160nr, fresenius) hemofilters. ultrafiltrate (uf) flows were 1, 2, 3 and 6 l/hr with sufficient blood flows [(qb) 200-350 ml/min] to maintain uf rates. blood samples were collected from the pre-filter line and uf samples from the post-filter uf port. concentrations of tlv in plasma and uf samples were assayed using validated lc-ms/ms methods. tlv cl was determined using the following formula:cl = (uf flow rate) [tlv]uf/[tlv]arterial. cl differences between the filter types were compared using a two-tailed, unpaired t-test. conclusion: tlv is substantially cleared by cvvh and cl increases significantly with increasing uf rate. cl did not differ by hemofilter type. cvvh cl at higher uf flows exceeds the total cl reported in patients with normal renal function. tlv likely will require dose adjustments in patients receiving cvvh. objectives: telavancin (tlv), a bactericidal lipoglycopeptide with multiple mechanisms of action, is in phase 3 trials for the treatment of hospital-acquired pneumonia (hap) with a focus on infections due to methicillin-resistant s. aureus. tlv is primarily eliminated by the kidneys and requires dosage adjustment for renal dysfunction. tlv is highly protein bound (93%) in healthy subjects which would suggest that it would not be removed by dialysis, but its small volume of distribution (0.15 l/kg) means that it may be removed by cvvhd. cvvhd is used in the management of critically ill patients. the objective of this study was to determine cvvhd tlv transmembrane clearance (cl) with 2 commonly used hemodialyzers at conventional cvvhd dialysate flow rates. methods: tlv cl was assessed in our in vitro cvvhd model using citrate anticoagulated bovine blood and b. braun diapact machine. experiments were run 5 times using an69 (m100, gambro) and polysulfone (f160nr, fresenius) hemodialyzers. dialysate flows were 1, 2, 3 and 6 l/hr with sufficient blood flows [(qb) 200-350 ml/min] to maintain appropriate transmembrane pressures. blood samples were collected from the pre-filter port (a) and post-filter port (v), and spent dialysate samples (d) from the post-filter d port. plasma tlv concentrations (arterial and venous) and dialysate samples were assayed using validated lc-ms/ms methods and tlv cl was determined using the following formula: cl = (d flow rate) [tlv]d/(([tlv]arterial+[tlv]venous)/2). dialytic cl between filter types was compared using a two-tailed, unpaired t-test. conclusion: tlv is effectively cleared by cvvhd. the higher permeability polysulfone dialyzer was associated with significantly increased cl vs. the an69 dialyzer as dialysate flow increased. the degree of tlv cl seen with cvvhd suggests that dose adjustments will be necessary in patients receiving cvvhd. objective: it is still a subject of controversy that only free, unbound drug is responsible for antibacterial activity of antibiotics. to provide further proof, that only free drug contributes to antimicrobial efficacy a comparative, doseranging time-kill curve study was performed. to exclude influence factors resulting from different mechanisms of action this was done within the antibiotic class of carbapenems, using compounds with different serum protein binding. methods: constant concentration time-kill curves were performed in 50% serum for the slightly serum protein bound mer-openem (~2%) and imipenem (10%) as well as for the highly serum protein bound ertapenem (95%) and faro-penem (95-96%), ranging from 1 · mic to 16 · mic. the change in number of bacteria (cfu/ml) versus time was linked to their effect. escherichia coli atcc 25922, klebsiella pneumoniae bay 63, staphylococcus aureus bay 133 and streptococcus pneumoniae atcc 6303 were used as the test organisms. samples were taken at 0, 0.5, 1 2, 4, 6 and 8 hours. the data were modelled simultaneously using the software scientist ò for windows tm and a modified sigmoid emax model characterized by growth rate constant (k0), maximum kill rate (kmax), and concentration at half maximum effect (ec50). results: for all four bacterial strains investigated, there were dramatic increases (400-700%) in ec50 for the highly se-rum protein bound carbapenems (ertapenem, faropenem) in the presence of serum proteins (fig. 1) . for both substances no significant differences in k0 and kmax were determined. in contrast, imipenem and meropenem showed only minor differences in ec50 in the presence and the absence of 50% serum. conclusion: only free, unbound drug is responsible for the antimicrobial activity. analysis of these time-kill curves clearly showed that the antibacterial efficacy was significantly decreased in the presence of 50% serum for the highly bound ertapenem and faropenem while being unaltered for the slightly bound meropenem and imipenem. objective: numerous in vitro experiments have shown that protein binding (pb) is an important factor for antimicrobial activity, especially for highly bound antibiotics. however, the experimental conditions that simulate the in vivo situation best are still subject of controversy. therefore, an in vitro microdialysis experiment was performed that evaluates various influence factors on the pb of the highly bound betalactams ceftriaxone (pb 95-98%). methods: a comparative, dose-ranging in vitro microdialysis study was conducted to determine free, unbound ceftriaxone concentrations in lactated ringer's solution and todd hewitt broth (thb) both with and without bovine serum albumin (bsa; sigma, st. louis) 40 g/l and human plasma at 37°c. furthermore, in vitro constant concentration time-kill curves were performed, using escherichia coli atcc 25922, streptococcus pneumoniae atcc 6303 and streptococcus pneumoniae atcc 49619 as the test organisms. the data was analysed using an appropriate pk/pd model, characterized by growth rate constant (k0), maximum kill rate (kmax), and concentration at half maximum effect (ec50) and correlated to free ceftriaxone thb concentrations determined by hplc-uv. results: there were only minor differences in both unbound drug concentrations and anti-infective activity when bsa 40 g/ l was added to either lactated ringer's (pb 0% and 12.5±2.5%, with and without bsa respectively) or thb (pb 27% and 25.5±2.5%, with and without bsa respectively). no significant changes in k0, kmax and ec50 were observed. however, using human plasma, unbound concentrations (pb 60%)88%) were altered dramati-cally. conclusion: only free, unbound drug is responsible for the antimicrobial activity. however, one cannot rely on that binding to commercially purchased bsa is consistent with reported protein binding values. unbound concentrations should be measured under the respective experimental conditions to be able to correctly interpret the experimental results. in vitro postantibiotic effect of faropenem on penicillin-resistant streptococcus pneumoniae and beta-lactamase-producing haemophilus influenzae c.l. young, i.a. critchley, u.a. ochsner, n. janjic (louisville, us) objectives: faropenem (far) is an oral penem with potent activity against respiratory pathogens such as penicillin (pn)resistant streptococcus pneumoniae (sp) and beta-lactamase (bla)-producing haemophilus infleunzae (hi). the postantibiotic effect (pae) is a pharmacodynamic (pd) parameter that monitors suppression of bacterial growth following short exposure and removal of the drug. paes are clinically important for agents such as far with short half-lives (1 h) . the aim of the study was to determine the pae of far on resistant phenotypes of sp and hi. methods: nine clinical isolates of sp, 3 pn-s, 3 pn-i, and 3 pn-r and six clinical isolates of hi, 3 bla-negative and 3 blapositive were tested in pae studies. paes were determined in cation-adjusted mueller-hinton broth with 3-5% lysed horse blood for sp and haemophilus test medium for hi. exponential cultures (106 cfu/ml) were exposed to far at 1, 4 and 10 x mic. far was removed by serial washing (10,000-fold dilution) prior to transfer to fresh media. control cultures were treated in the same way. bacteria were incubated with shaking and viable cfus determined at 1, 4, 6, 8, 16 and 20 h. counts of log10 cfu were plotted against time and pae defined as the difference is the time required for count in test culture and control (untreated culture) to increase 1 log10 above the count observed immediately after removal. results: significant paes of >0.5 h were observed for all strains of sp at 4 and 10 x mic. however, the pae was more prolonged on the pn-r strains with mean paes of 3.9 h at 4 and 10 x mic. among hi, little or no pae was observed on bla-negative strains but a significant pae was observed on the bla-positive isolates (mean paes of 5.9 h and 8.9 h at 4 and 10 x mic respectively). conclusions: far demonstrates a prolonged pae on key resistant phenotypes of sp (pn-r) and hi (bla-positive) compared with susceptible strains. the observation of pae in bla-positive hi is unique in the class of beta-lactams. far exhibits in vitro pd properties that may contribute to its clinical efficacy against pn-r sp and bla-positive hi. telavancin is more efficacious than vancomycin in a murine model of bacteraemic peritonitis induced by methicillin-resistant staphylococcus aureus s. hegde, n. reyes, b. benton, r. skinner (south san francisco, us) objective: telavancin (tlv) is a novel lipoglycopeptide that operates through multiple mechanisms to produce potent and rapid bactericidal activity against clinically relevant grampositive bacteria including methicillin-resistant staphylococcus aureus (mrsa). the present studies evaluated the in vivo efficacy of tlv vs vancomycin (van) in a model of mrsa induced peritonitis in neutropenic mice. methods: female nsa immunocompromised mice were inoculated intraperitoneally with atcc mrsa 33591 and treated, beginning at 4 h post-infection, with 2 subcutaneous doses (q 12h) of vehicle (veh) or test compound. mouse pharmacokinetic data were generated and used to choose doses of tlv (40 mg/kg) and van (110 mg/kg) in order to equate clinical exposures (auc's (free drug) of 44 and 132 lg.hr/ml, respectively). in survival studies, deaths were recorded for 14days post-infection and survival curves were compared using log-rank test. in bacterial titer determination studies, designated groups of control and drug-treated surviving animals were humanely euthanized at various times post-treatment and their blood and spleen were harvested to determine bacterial titers. results: mics of tlv and van were 0.5 and 1.0 lg/ml, respectively. mortality was 100% in animals treated with veh or van. mortality was 7% in tlv-treated animals (p < 0.05 vs veh and van). the pre-treatment bacterial titres were 4.3 log cfu/ml and 8.7 log cfu/g in the blood and spleen, respectively. analysis of the time kill curves for both blood and spleen revealed that tlv exhibited significantly greater killing activity than van (p < 0.05, two-way anova). at 6 hrs after the first dose, the titers in the blood were reduced to a greater extent by tlv ()2.5 log cfu/ml) compared to van ()1.2 log cfu/ml). at 6 hrs after the second dose, the splenic titers were reduced to a greater extent by tlv ()3.8 log cfu/g) when compared to van ()1.4 log cfu/g). conclusions: the data described here demonstrate that tlv's in vivo bactericidal activity is superior to that of van against mrsa and results in successful infection resolution and, consequently, improved survival in the murine peritonitis model. proper use of carbapenems for blood-derived clinical isolates of pseudomonas aeruginosa y. kobayashi (tokyo, jp) methods: regimens of carbapenems were given to 1500 healthy adult subjects. changes in their blood concentrations of carbapenems were compared by using pharmacokinetic parameters (two-compartment model analysis) of meropenem (mepm), imipenem (ipm), and panipenem (papm) and by applying the lognormal distribution to the probability distribution of distribution volume and plasma half-life with monte carlo simulation (mcs). based on the data on distributions of the minimal inhibitory concentrations (mic) of various carbapenems for 77 blood-derived clinical isolates of pseudomonas aeruginosa isolated/identified at keio university hospital between october 1997 and october 2004 (mic90: mepm 8 mcg/ml, ipm 16 mcg/ml, papm 32 mcg/ml), the mics in the 1500 subjects were obtained with mcs. from the changes in blood concentrations and mics in the subjects, the probability of achieving t>mic was calculated for each carbapenem regimen, using the formula reported by kuti et al.: %t>mic = ln (dose/vd*mic)*(t1/2/0.693)*(100/di) <<ln: natural logarithm, dose: 1 dose (mg), vd: distribution volume (l), t1/2: plasma half-life (h), di: dosing interval (h)>>. based on craig's data, the maximum bactericidal effect on gram negative bacilli is attained when %t>mic is approximately 50%. we focused on this information and analysed our data. results: the probability of achieving t>mic50% was 43.2% for mepm 500 mg bid, followed by 4.2% for ipm 500 mg bid and 3.6% for papm 500 mg bid. when the dose was increased from 500 mg to 1000 mg, it was 60.7% for mepm 1000 mg bid, followed by 10.3% for ipm 1000 mg bid and 9.9% for papm 1000 mg bid. when the dose remained at 500 mg and the dosing frequency was increased to three times daily, it was 76.5% for mepm 500 mg tid, followed by 43.0% for ipm 500 mg tid and 18.8% for papm 500 mg tid. regarding mepm, it was 85.9% for 500 mg gid? and 86.7% for 1000 mg tid showing higher probabilities. discussion: in severe sepsis caused by pseudomonas aeruginosa, remarkably higher t>mic50% was achieved with carbapenems at 500 mg tid, although the daily dose (1500 mg) was lower, compared to 1000 mg bid. carbapenems with a low mic distribution, i.e. a superior antibacterial activity, showed higher probability of achieving t>mic. therefore, the optimal treatment for such sepsis is mepm 500 mg tid. mepm 500 mg qid appeared to provide comparable therapeutic effects with those at 100 mg tid, the usual dose in foreign countries. penetration of moxifloxacin into normal and infected subcutaneous tissue in patients with spinal cord injury measured by microdialysis background: skin breakdowns, also termed decubitus ulcers or pressure sores, are a major complication associated with spinal cord injury, resulting in infection and tissue death. moxifloxacin (mfx) is approved for the treatment of sssi. our objective was to construct a population pk model for mfx disposition in plasma, normal and infected subcutaneous tissue in spinal cord injured patients with infected decubitus ulcer. methods: 4 patients receiving 400 mg mfx orally daily were enrolled in this study. blood, saliva and interstitial tissue fluid samples (microdialysis in normal and infected tissue) were collected over a time period of 8 hrs. mfx concentrations were measured by a validated hplc. concentration-time data obtained in the present study were pooled with previously published mfx data (n = 12). population pk modelling was performed with nonmem. results: the concentrations of mfx achieved in plasma, saliva, normal subcutaneous tissues and infected decubitus ulcers showed parallel profiles versus time. the pk was best described by a 2-compartment model with a link to interstitial tissue fluid. the population pk parameters were as follows (given as estimate with percent interindividual variability in parentheses): cl 10.2 l/h (3%); central vd 83.2 l (15%); intercompartmental cl 31.9 l/h (16%); peripheral vd 92.2 l (17%); and elimination rate constant for interstitial tissue fluid 1.57 h)1 (17%). with a conservative mic90 of 0.25 mg/l, the peak/mic ratios were higher than 10 and the auc24/mic ratios were higher than 100 for plasma, saliva and interstitial tissue fluids. conclusions: this study showed the good diffusion of mfx into subcutaneous tissue in spinal cord injured patients with decubitus ulcers. the interstitial tissue fluids reached bactericidal levels for common bacteria found in infected skin lesions. objective: investigations of pharmacodynamic parameters such as postantibiotic effect and postantibiotic subminimum inhibitory concentration effect have been employed for design of dosing schedules of antimicrobial agents. in this study we compared postantibiotic effect and postantibiotic subminimum inhibitory concentration effect of ciprofloxacin, levofloxacin, and moxifloxacin for clinical isolates of methicillin susceptible staphylococcus aureus, methicillin resistant staphylococcus aureus and pseudomonas aureginosa. methods: the following strains were tested in this study: methicilline-susceptible staphylococcus aureus (n:4), methicilline resistant -staphylococcus aureus (n:2) and pseudomonas aeruginosa (n:2). the pae was determined by viable plate count method using mueller hinton broth. tubes containing 5 ml of broth and the antibiotic to be tested at 1, 2, 4 and 8x the mic were inoculated with approximately 5 · 106 cfu/ml. growth controls with an inoculum but not antibiotic were included with each experiment. result: postantibiotic effects of ciprofloxacin, levofloxacin and moxifloxacin increased with increasing concentration of the drug. the longest postantibiotic effect was observed for moxifloxacin. moxifloxacin showed no postantibiotic effect one p. aureginosa at all concentration and had no post antibiotic effect to another p. aureginosa at x2mic and mic. in our study the longest postantibiotic subminimum inhibitory concentration effect against mssa was determined with moxifloxacin. similarly the moxifloxacin induced the longest effect against mrsa. however, this time frame was shorter than that of mssa. conclusions: all three antibiotics, showed for longer postantibiotic subminimum inhibitory concentration effect in all submic concentrations, immeasurable within the study period i.e. 24 hours. lack of horizontal transmission of fluoroquinolone resistance between s. mitis and s. pneumoniae objectives: fluoroquinolone (fq) resistance can arise in s. pneumoniae through acquisition of dna from s. mitis and subsequent homologous recombination. the frequency at which this occurs is unknown, and while likely a rare event, increases in fq resistance among s. mitis may increase the rate at which horizontal transmission occurs. we sought to determine the frequency at which fq resistance could be transferred from s. mitis to s. pneumoniae or from s. pneumoniae to s. mitis. methods: s. mitis (either fq^r,tetracycline[tet^s], fq^r,penicillin[pen^s], or fq^s,pen^r) and s. pneumoniae (either fq^s,tet^r, fq^s,pen^r or fq^r,pen^s) were grown in co-culture using a pharmacodynamic model in the presence of either moxifloxacin (mxf) or levofloxacin (lfx) at salivary drug concentrations. after incubation, aliquots were plated onto either tet or pen containing sba plates to select for the recipient strains. fq susceptibility was performed using microbroth dilution. the entire parc and gyra genes were amplified and sequenced to determine if horizontal transmission occurred. results: in initial experiments tet was used as the selective agent. however tet resistance was transferred and therefore pen^r was used as a selective marker. an increase in the lfx mic in was observed in 3 s. pneumoniae and 1 s. mitis strain. sequencing of the parc gene revealed the selection of 2 ser79phe and 1 ser79tyr mutations in s. pneumoniae and ser79phe in s. mitis consistent with fq resistance. sequencing of the entire gene failed to uncover evidence of horizontal transmission. no mutations were detected in gyra. selection of 1st step parc clinical microbiology and infection, volume 12, supplement 4, 2006 mutations occurred only after exposure to lfx. mxf eradicated both s. mitis and s. pneumoniae and failed to either select for resistance or support horizontal transmission. conclusions: although 1st step parc mutations were selected in 4 strains (3 s. pneumoniae, 1 s. mitis), we failed to find evidence of horizontal transmission between s. pneumoniae and s. mitis under our laboratory conditions. the phenomenon of horizontal transfer resulting in fq resistance has been described, however, based on our results, we must speculate that it is an extremely rare event and not likely to be a major driver of fq resistance. of interest, the parc mutations were selected only under the selective pressure of lfx. mxf completely eradicated both s. pneumoniae and s. mitis and did not select for the development of fq^r mutations. objectives: the aim of the present study was to assess the killing activity of ertapenem (ert) and metronidazole (mtr) against four selected bacteroides fragilis strains with different mic values in an in vitro pharmacokinetic/pharmacodynamic (pk/pd) model. since anaerobes are often present in mixed infections, kill kinetics were also established for mixed inocula employing the b. fragilis strains together with four selected escherichia coli strains. the killing activity was analysed for kinetic concentrations of the antimicrobial agents simulating human serum kinetics. methods: a pk/pd in vitro model was established by adding appropriate amounts of broth every half hour. at the same time intervals samples were obtained and plated. after incubation colony forming units were counted. human serum concentrations were simulated with cmax = 100 mg/l and t1/2 of 5 hours for ert and cmax = 14.0 mg/l and t1/2 of 7 hours for mtr. mann trend test was used for statistical analysis. results: as to be expected the e. coli strains were not killed by mtr both in pure as well as in mixed cultures whereas the susceptible e. coli strains were effectively killed by ert. in pure cultures the b. fragilis strains were effectively killed by mtr and the growth of the susceptible b. fragilis strains was reduced by ert by about two to four logs. however, in some mixed cultures the killing activity of mtr against the b. fragilis strains was significantly reduced. conclusion: the in part moderate in vitro activity of ert against the b. fragilis strains and the reduced activity of metronidazole in mixed cultures against the b. fragilis strains may explain some of the difficulties in treating mixed aerobic/ anaerobic infections. penetration of ciprofloxacin into human cerebrospinal fluid and brain tissue a. tsona, s. metallidis, e. koumentaki, j. nikolaidis, p. kollaras, g. lazaraki, p. nikolaidis (thessaloniki, gr) objectives: the aim of the present study was to determine the penetration of ciprofloxacin into cerebrospinal fluid (csf) and brain tissue of humans. methods: a total of 10 patients undergoing brain tumor excision were evaluated. the patients received a single intravenous dose of 400 mg ciprofloxacin. samples of blood, cerebrospinal fluid and brain (brain-adjacent tumour tissue) were collected during surgery 2 h after drug administration. ciprofloxacin concentrations in serum, cerebrospinal fluid and brain homogenate were analysed by means of a validated hplc method. results: ciprofloxacin concentrations in plasma (mcg/ml), cerebrospinal fluid (mcg/ml) and tissue homogenate (mcg/g), respectively, after 2 h ranged 0.87-2.67 mcg/ml, 0.07-0.37 mcg/ ml and 0.65-4.02 mcg/g. csf-to-serum ratio ranged between 0.06 and 0.14. tissue-to-serum ratio ranged between 0.41 and 4.25. mean (±s.d.) csf/serum concentration ratios and brain tissue/serum concentration ratios were respectively 0.10 ± 0.03 and 1.70 ± 1.17. conclusion: these findings suggest that valuable informations on brain tissue penetration can be obtained only from brain material. data from csf penetration cannot be extrapolated to the brain since the blood: sf barrier differs from the blood:brain barrier. concentrations of ciprofloxacin in cerebrospinal fluid were lower than those in serum, in contrast to the brain tissue concentrations that exceeded serum concentrations. the achieved concentrations in brain tissue were generally above the mic90 of common pathogens in central nervous system infections (h. influenze, n. meningitidis, s. pneumoniae, l. monocytogenes, escherichia coli, aerobic gram-negative bacilli, group b streptococci, mssa). cerebrospinal fluid concentrations exceed the mics of neisseria meningitidis and most gram-negative aerobic bacilli. our findings suggests that ciprofloxacin may be an acceptable alternative for the treatment of meningitis due to susceptible gram-negative aerobic organisms and for the treatment of brain abscesses. objective: to model the performance of imipenem (imi), meropenem (mem), and ertapenem (etm) against esbl producing e. coli and klebsiella spp in order to identify possible pd differences among compounds. methods: minimal inhibitory concentrations (mics) were generated for 133 randomly selected esbl producing isolates of ec (n = 29) and kl (n = 104) collected during 2004 from brazilian hospitals as part of the mystic program. mic testing for imi, mem, etm, ceftazidime (ctz), and cefotaxime (ctx) were done by e-test methodology. esbls were confirmed via ctz/clavulanate and ctx/clavulanate e-test. pd exposure, measured as percent time above the mic for free drug (ft>mic), was modelled via a 5000 subject monte carlo simulation for the following 30-minute infusions:imi 1 gram every 8 hours, mem 1 gram every 8 hours, and etm 1 gram every 24 hours, using pharmacokinetics from healthy volunteers. the bactericidal cumulative fraction of response (cfr) was calculated for each regimen against the populations of ec, kl, and against all esbl isolates together. bactericidal cfr was defined as 40% ft>mic for all agents. results are reported as cfr (95% confidence interval). results: isolates were 100% susceptible (s) to imi and mem (mic range 0.125-1.5 and 0.023-4 mg/l, respectively), and 97% s to etm (mic range 0.008-32 mg/l conclusions: these findings support other data that although etm is likely to be an effective empiric agent against most esbl producing ec and kl, its ability to achieve high bactericidal pd exposure will be dependent on the presence of less susceptible organisms in the population. imi and mem should remain first line for esbl infections. objectives: this study analyses eradication and resistance selection in streptococcus pneumoniae with moxifloxacin, levofloxacin and azithromycin, using a parental serotype 3 infecting strain (a) and subsequent resistant step-mutants (isolates b, c and d) selected in vivo in a patient with pneumonia. methods: moxifloxacin, levofloxacin and azithromycin mics were 1, 2 and 0.5 lg/ml for the parental strain, 4, 16, and 4 lg/ ml for isolate b, and 4, 16 and >128 lg/ml for isolates c and d, respectively. a pharmacokinetic computerized device was used to simulate serum and epithelial lining fluid (elf) concentrations. initial inocula was approx. 108 cfu/ml. population analysis profiles were performed using plates with increasing antimicrobial concentrations on a mic basis. results: in serum, moxifloxacin eradicated the parental isolate (isolate a), with an auc0-24 h/mic value of 39.6. serum auc0-24 h/mic values of 26.5 and 5.5 for levofloxacin and azithromycin, respectively, were not able to eradicate isolate a. in elf, moxifloxacin showed a bactericidal pattern against all isolates with a minority (approx. 100 cfu/ml) of the survival population (isolates b, c and d) growing in plates with moxifloxacin concentrations higher than those obtained in elf. levofloxacin and azithromycin showed a bactericidal pattern only against isolate a, with the whole population of isolates b, c and d growing in plates with levofloxacin concentrations higher (16-64 lg/ml) than those obtained in elf, and in plates with azithromycin concentrations as high as 2048 lg/ml (for isolates c and d). in elf, moxifloxacin auc0-24 h/mic values were 201.0 for isolate a, and 50.3 for isolates b, c and d. levofloxacin auc0-24 h/mic values were 94.0 for isolate a, and 11.8 for isolates b, c and d. azithromycin auc0-24 h/mic values were 8.0 for isolate a; 10.0 for isolate b; and 0 for isolates c and d. conclusion: if prevention of resistance depends more on the eradication of possible emerging mutants in pulmonary tissues than of the parental susceptible strain, moxifloxacin concentrations in elf may provide advantages over previous quinolones and macrolides in preventing clinical failures. objectives: to explore how antimicrobial pressure influences the evolution of streptococcus pneumoniae populations sharing the same ecological niche. methods: an in vitro computerized pharmacodynamic model simulating physiological concentrations obtained over 24 h after 500 mg o.d levofloxacin, 750 mg b.i.d ciprofloxacin, and 500 mg o.d azithromycin was used to investigate its effect on a mixed culture of five s. pneumoniae serotypes (s) as an approach to ecology of population dynamics. resistance patterns were: s12 was susceptible to study drugs, s31 was low-level macrolideresistant (efflux phenotype), s11 was high-level macrolideresistant (erm genotype), s9v was low-level quinoloneresistant, and s3 was high-level quinolone-resistant. initial mixed inocula (time 0) included similar percentages of each serotype. results: mean colony counts in antibiotic-free plates (whole pneumococcal population) increased (from 0 to 24 h) from log10 6.9 to 8.8 in drug-free simulations (control), from log10 6.8 to 7.9 in levofloxacin simulations, from log10 6.8 to 8.6 in ciprofloxacin simulations, and from log10 7.1 to 8.8 in azithromycin simulations. at 24 h of control drug-free experiments, dominant strains were s9v (57.4%) and s12 (41.8%) with marginal populations of s31, s3 and s11. azithromycin selected in a much higher extent the strain with low-level resistance to macrolides (s31) than the strain with high-level resistance (s11) (accounting for 99.9% vs. 0.1% of total population at 24 h). ciprofloxacin selected in a higher extent low-level (s9v) than high-level (s3) quinolone resistance (72.4% vs. 27.6%). levofloxacin decreased the proportion of the predominant s9v in controls to 22.2% (an intermediateresistant strain with mic = 4 lg/ml), and unmasked the highlevel resistant strain (mic = 32 lg/ml) up to 77.8%. conclusion: strain distribution in antibiotic-free environment depends on bacterial fitness in mono-and multi-strain niches. the selective pressure of antimicrobial regimens eradicate some populations and unmask minor populations, thus redistributing the whole population. selective potential only for resistance phenotypes with very low prevalence (as high-level quinolone resistance) in the community should be preferred to that selecting more prevalent resistance phenotypes. re-evaluation of the role of broad-spectrum cephalosporins against staphylococci applying contemporary in vitro results and pharmacokinetic-pharmacodynamic principals h. sader, s.m. bhavnani, p.g. ambrose, r. jones (north liberty, us) objectives: to re-evaluate the current in vitro activity and to assess the pk-pd target attainment of cefepime (cpm), ceftriaxone (cro) and ceftazidime (caz) against staphylococcus spp. methods: the potency of cpm, cro and caz against staphylococci was accessed through the sentry antimicrobial surveillance program database, worldwide. during the 1998-2004 period 41,883 s. aureus (sa; 63% oxacillin [oxa]-susceptible [s]) and 14,349 coagulase-negative staphylococci (cons; 22% oxa-s) were s tested against cpm, cro, caz and numerous comparators by clsi broth microdilution methods. using volunteer pk data and a linear intermittent intravenous infusion model, and an animal-derived pk-pd target of 25% time above mic, expected probabilities of target attainment (pta) for cephems were evaluated using monte carlo simulation. pta were determined for the following dosing regimens: cpm 1gm q12 and q8 hours, caz 1 gm q8 hours and cro 1 gm q24 hours, each representing the most common dosing patterns applied clinically. cephem susceptibility (%s) was calculated based on the current clsi (2006) breakpoints (bkps) and also on bkps derived from a pta >90%. results: against oxa-s sa, mic50/90 values were (in mg/l): 2/4 for cpm, 4/4 for cro and 8/16 for caz, respectively; and against oxa-s cons mic50/90 values were (in mg/l) 0.5/2 for cpm, 2/4 for cro, and 4/8 for caz, respectively. the calculated %s of these cephems are summarized in the table: twenty year-old clsi bkps would rank the tested agents cpm ‡ cro > caz and by pk-pd pta cpm ‡ caz > cro. cpm has a potency advantage over caz (4-to 8-fold) and superiority at the usual dosing over cro (22.7-66.1%) for oxa-s staphylococci. caz pk overcomes by-weight activity disadvantages, while a low proportion (<5%) of active freedrug penalizes cro in the pta calculations. pta remained at >90% to a bkp of 16 mg/l for cpm (1 gm q8) and caz and to a bkp of 2 mg/l for cro. conclusions: regardless of applied bkp (clsi or pk-pd), cpm has the widest and more potent anti-staphylococcal activity among commonly used ''third-or fourth-generation'' cephems. when used at doses ‡ 3 gm/day, cpm assures maximal coverage of oxa-s staphylococci whether using existing (clsi) or modified (pk/pd) bkps. cro should be used with caution. methods: the mic for all strains were determined by serial two-fold macrodilutions. an in vitro kinetic model was used to investigate the antibacterial efficacy of constant drug concentrations during 6 hours. the selection of the doses of azithromycin tested in each bacterial strains was based on their mic values. bacterial counts were determined on appropriate agar plates using an adapted drop-plate method. twelve different pk/pd models were fitted and compared to the time-kill data by using non-linear regression. results: a simple pk-pd model was not sufficient to describe the pharmacodynamic effects for the four bacterial strains. appropriate models that gave good curve fits included a saturation term for the number of bacteria (nmax), delay terms (1-e-zt) for the initial bacterial growth phase and/or the onset of anti-infective activity as well as a hill factor (h) to capture the steepness of the concentration-response relationship. azithromycin had high potency against s. pneumoniae strains and m. catarrhalis while the potency of azithromycin against h. influenzae was poor. conclusions: the developed pk/pd models are suitable for describing the pharmacodynamics of azithromycin. applications of these pk-pd models will eventually provide a tool for rational antibiotic dosing decisions. objectives: optimal antimicrobial dosage regimens aim to achieve successful clinical outcomes without drug toxicity or emergence of bacterial resistance. for concentration dependent antibiotics, such as the fluoroquinolones, in humans a cmax:mic ratio of >10 is considered more important for efficacy and reduced selection of resistance than prolonged antibiotic concentrations just above the mic. fluoroquinolone resistance in zoonotic bacteria is a matter of public health concern, and fluoroquinolone treatment of poultry can rapidly select for bacteria with reduced fluoroquinolone susceptibility. in this study we compared basic pharmacokinetic parameters for the recommended dose of baytril (enrofloxacin) 10% oral solution in poultry to 2.5x this dose for birds dosed by continuous water (standard) compared to pulsed water treatments and dosing by gavage.methods. for the pulsed versus continuous water treatments, groups of chickens received baytril 10% oral solution at 50 (recommended) or 125 ppm continuously in the water or at 10 (recommended) or 25 mg/kg pulsed in the water. for each group, three birds were killed at 0, 2, 4, 6, 8, 10 and 24 hours after start of antibiotic treatment and caecal contents, liver, lung and sera were taken and the concentration of fluoroquinolone determined by fluorescence hplc. for gavage treatment, dosing was at 10 and 25 mg/kg by crop intubation and four birds were killed in each group at 2, 6 and 24 hours after gavage; caecal contents, liver and sera were taken and analysed as above. basic pharmacokinetic parameters were determined using pk solutions software. results: the mean fluoroquinolone cmax in caecal contents (and sera) for gavage, pulsed water and continuous water treatments respectively was 78.01 (1.81), 53.19 (1.17) and 24.73 (0.73) mg/ml after the recommended dose and 115.86 (3.86), 115.63 (2.74) and 68.02 (1.17) mg/ml after 2.5x the recommended dose. cmax of antibiotic in liver and lung was increased by the modified regimens in similar proportions to above. both pulsed water and gavage treatment not only resulted in higher cmax values, but also a faster rate of fluoroquinolone clearance than continuous water treatment ( figure 1 ). dosing by gavage is not practical for thousands of chickens. however, pulsed dosing at 2.5x the recommended dose can increase cmax values about fourfold and so could improve efficacy and reduce selection of resistance, compared to the current recommended treatment regime. objectives: nephrotoxicity is the major concern arising with the use of intravenous colistimethate sodium. methods: a prospective cohort study was performed at ''henry dunant'' hospital, a 450-bed tertiary care center in athens, greece. patients who received intravenous colistin for at least 7 days for the treatment of multidrug resistant gram-negative bacterial infections were included in the study. the development of nephrotoxicity through evaluations of serum creatinine, blood urea, serum electrolytes, urinalysis, and creatinine and sodium in 24-hour urine collection during intravenous colistin therapy was the primary end point of the study. results: twenty-six patients were included in the study, 21 of whom received colistimethate sodium (cms) for at least 7 days and were evaluated further. the mean (± sd)/median daily dose, cumulative dose, and duration of treatment of intravenous cms was 5.5 (± 1.9)/6 million iu, 90.2 (±52.0)/72 million iu, and 17.7 (±11.7)/15 days (range 7-54 days), respectively. three of the 21 evaluable patients (14.3%) developed nephrotoxicity during the intravenous treatment with cms. the cumulative dose of the administered cms was statistically correlated with the difference between the end and start of cms treatment values of serum creatinine (r = 0.6, p = 0.004 by spearman's test). a statistically but not clinically significant decrease of the mean baseline serum sodium concentration was observed between start and end of treatment [mean 144.2 (±6.9) to 142.1 (±6.1) mmol/l, p = 0.04]. no other toxic events were noted during the intravenous administration of colistimethate sodium. conclusion: although this is an evaluation of a small number of patients, our prospective study shows that nephrotoxicity was not commonly observed in this group of patients who received intravenous colistimethate sodium. however, caution should be taken to avoid the prolonged administration of the antibiotic. objectives: the objective of the present work is quantitative structure-activity relationship (qsar) analysis of antimicrobial activity of the 4-thiazolidone derivatives and consequent computational design of new antimicrobials. methods: for the achievement of the formulated objectives the qsar investigation has been carried out using computational chemistry approach based on simplex representation of molecular structure (sirms). on the framework of sirms it is possible to develop the molecular design of the new effective antimicrobials. results: systematic researches of relationship between antimicrobial activity (staphylococcus aureus -methicillin-sensitive (mssa) strain, pseudomonas aeruginosa -r and s strain, klebsiella pneumoniae, candida albicans s and ñ itrobacter freundii) and a structure of about one hundred fifty compounds (4-thiazolidone derivatives and analogs). the elucidation of structure-activity relations allows predicting biological properties of such compounds, to execute their direct synthesis and to receive the indispensable information for research of mechanisms of their biological effect. completely adequate statistical partial least squares models (r2 = 0.841-0.990, q2 = 0.600-0.814) have been obtained for all of the studied cultures. on the base of the first ones the molecular fragments both promoting and interfering the given antimicrobial activity have been determined. they give a possibility to realize the computer high throughput screening and molecular design of active compounds. the results of prognosis are verifying by the experimental investigations. also the influence of heterocycle system evolution on antimicrobial activity has been revealed. conclusion: qsar analysis of antimicrobial activity of 4-thiazolidone derivatives allows us to discover that the presence of naphthalene-substituted fragment (independently on its location in molecule) has distinctly negative influence on antimicrobial action. the requirements to molecular design have been formulated. for example, high active compounds must include 3-indolyl fragment. computational design of the new antimicrobials based on the substituted crown ethers activity of the row of substituted crown ethers and consequent molecular design of new antimicrobials. methods: the well-known hierarchic system of qsar models based on simplex representation of molecular structure has been used for the solution of the formulated problem, within the framework of which one it is possible to develop the molecular design of the new effective antimicrobial agents. results: we tried to conduct systematic researches of relationship between antimicrobial activity (planococcus citreus, streptococcus lactis, micrococcus lysodeiktious, staphylococcus aureus, streptococcus faecalis, bacillus subtillum) about two hundred fifty crowns ethers including aromatic, cyclic and heterocyclic etc. fragments and a structure of these molecules, in particular -macro cycle size, it dentacy, lipophily, nature of the substituents, and other factors. the elucidation of similar relations allows predicting biological properties of crown compounds, to execute their direct synthesis and to receive the indispensable information for research of mechanisms of biological effect of such kind of compounds. completely adequate qsar models (r2 = 0.841-0.995, q2 = 0.660-0.954) have been obtained using partial least squares method for all of the studied cultures. on the base of the first ones the molecular fragments with positive or negative influence on the explored properties have been determined. they give a possibility to realize the virtual screening and molecular design of compounds with the high level of target activity. the results of prognosis are verifying by the experimental investigations. conclusion: qsar analysis of antimicrobial activity of crown ethers allows us to suppose the presence of two different mechanisms of their antimicrobial action. it is discovered that the presence of diphenyloxide and tert-butyl fragments promotes; diphenyl-sulphide and diamino-biphenyl -prevents the antimicrobial action. it is shown that the hexadenthal crown ethers containing aromatic fragments with a tert-butyl group are the most perspective antimicrobials. objectives: methionyl trna synthetase (mrs) catalyses the covalent attachment of methionine to its cognate trna. rep8839 is a synthetic inhibitor of mrs with potent antibacterial activity against staphylococcus aureus including clinically-relevant resistant strains (mic90 equals 0.06 to 0.5 lg/ml). we determined the biochemical potency and mechanism of action of rep8839 and related compounds with respect to s. aureus mrs enzymatic activity. we also evaluated the enzyme kinetic properties of mutated forms of s. aureus mrs. methods: the mets gene from s. aureus was expressed in e. coli and mrs was purified to near homogeneity by ammonium sulfate fractionation and anion exchange chromatography. aminoacylation of trnamet was measured using scintillation proximity assays (spa). the kinetics of the atp:ppi exchange were determined using thin layer chromatography (tlc). mutants of s. aureus mrs were selected by serial passage and spontaneous resistance in the presence of rep8839. results: rep8839 exhibited strong inhibition of s. aureus mrs in the aminoacylation reaction, having an ic50 limited by the enzyme concentration. in order to estimate the true inhibition constant (ki), we utilized an atp:ppi exchange assay. rep8839 showed potent inhibition of s. aureus mrs, with a ki of 10 pm. related inhibitors were analysed, and a correlation was observed between the ki for mrs and the mic for s. aureus. rep8839 was found to be competitive with methionine binding, but uncompetitive with atp binding (i.e., increasing the atp concentration resulted in tighter binding of rep8839). the majority of analogs exhibited comparable mechanism of action; altered mechanism of action was observed with a subset of analogs. mutated s. aureus mrs variants (derived from strains with elevated mics) showed substantially weaker binding by rep8839. all of the mutated enzymes exhibited impaired trna aminoacylation activity, with defects ranging from reduced turnover rates to weaker affinities for one or more substrates. conclusions: rep8839 is a potent inhibitor of s. aureus mrs. enzymatic potency of this class of inhibitors correlates with microbiological potency. mutations that confer resistance to rep8839 result in functionally impaired mrs, encompassing a wide variety of enzymatic phenotypes. we report here the antibacterial and antifungal activity of 8 newly synthesized and physico-chemically characterised thioureides of 2-(4-chlorophenoxy)-benzoic acid. the new compounds were prepared in three stage. firstly, the 2-(4-chlorophenoxymethyl)-benzoic acid was prepared by treating the phtalide with p-chlorophenol potassium salt in xylene. the second stage was the synthesis of 2-(4-chlorophenoxymethyl)benzoyl chloride by treating the corresponding acid with thionyl chloride using anhydrous 1,2-dichloroethane as solvent, followed in the third stage, by the treatment of the above-mentioned chloride with ammonium thiocyanate. the 2-(4-chlorophenoxymethyl)-benzoyl isothiocyanate resulted after refluxing the reaction mixture in dry acetone. the new compounds were prepared by refluxing the isothiocyanate with primary aromatic amines in dry acetone. the obtained compounds have been characterized by their physical properties and their chemical structures were confirmed using the spectral analysis. the aim of this study was also to evaluate the in vitro antimicrobial activity of the new compounds. the in vitro antimicrobial testing was performed by binary microdilution method, in 96 multi-well plates, in order to establish the minimal inhibitory concentration (mic), against gram-positive (listeria (l.) monocytogenes, staphylococcus (s.) aureus, bacillus (b.) subtilis), gram-negative (psedomonas (p.) aeruginosa, escherichia (e.) coli, salmonella (s.) enteritidis), as well as candida sp., using both reference and clinical, multidrug resistant strains. our results showed that the tested compounds exhibited a specific antimicrobial activity, depending on the nature of the substituents and their position on the benzene ring, both concerning the microbial spectrum and the mic value. the mics values widely ranged between 1024 mcg/ml and 32 mcg/ml. the most active proved to be n-[2-(4-chlorophenoxymethyl)-benzoyl]-n'-(2,6-dichloro-phenyl)-thiourea and n-[2-(4-chlorophenoxymethyl)-benzoyl]-n'-(4-bromo-phenyl)-thiourea, showing a large spectrum of antimicrobial activity against enterobacterial strains (e. coli and s. enteritidis), l. monocytogenes, s. aureus and candida sp. all the tested compounds were highly active against s. aureus (mic = 32 mcg/ml). four of the tested compounds exhibited antifungal activity (mic = 256-32 mcg/ml), and p. aeruginosa as well as b. subtilis were resistant to all tested compounds. in vitro antimicrobial activities of novel dianthraquinones produced by a marine streptomyces sp. against clinical staphylococcus aureus and enterococcus faecium isolates k.l. laplante, k. lor, a. socha, d.c. rowley (providence, north kingston, us) objectives: the escalation of antibiotic resistance among grampositive pathogens presents increasing treatment challenges and requires the development of new therapeutic agents. recently we discovered a new class of dianthraquinone antibiotics produced by a marine streptomycete. the inhibitory and bactericidal activity of four dianthraquinone secondary metabolites and four semi-synthetic derivatives were measured against clinical strains of vancomycin resistant e. faecium (vre), methicillin susceptible and methicillin resistant s. aureus (mssa and mrsa, respectively). two compounds, daq550a and daq552, were tested against an expanded panel of pathogens. methods: thirty-two clinical strains of vre (n = 10), mssa (n = 12) and mrsa (n = 12) were obtained from patients at the veterans affairs medical center in providence, ri. mic's were performed using methodologies described by clsi. control isolates were atcc52923 and atcc29213. the bactericidal activity of each antimicrobial agent was evaluated with time-kill experiments using 6 randomly selected mssa (n = 2), mrsa (n = 2), and vre (n = 2) isolates tested at 4 times the respective mic. conclusions: the potent activities and unusual structures of the dianthraquinones tested here suggest that these may provide a new molecular scaffold for the development of novel antimicrobial agents. more biological testing is warranted to more fully explore the clinical potential of these antibiotics. efficacy of the novel antimicrobial peptide plectasin to staphylococci objective: the purpose of the investigation was to investigate the in vitro efficacy and kill kinetics of plectasin against staphylococcus aureus. plectasin is a newly discovered defensintype antimicrobial peptide found in the fungus pseudoplectania nigrella which showed activity against several gram-positive bacteria including drug resistant strains (mygind ph. et al. plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. nature 2005; 437:975-980). methods: all experiments were determined according to clsi/ nccls guidelines. bactericidal activity was characterized by time kill experiments at 2 and 10 times the mic. staphylococcus aureus (s. aureus) atcc29213 were used as the test organism and vancomycin was used for comparison. the kill kinetics and post antibiotic effect (pae) were evaluated by cfu determination. inoculum sizes ranging from 10e4 to 10e7 cells were used to test the inoculum effect. 10e10 cells were employed for determination of mutant prevention concentration (mpc) and the frequency of spontaneous resistance. results: plectasin is bactericidal as evidenced by kill kinetics showing a 2.1 log reduction in cfu/ml after 1 hour of incubation and a reduction of 3.1 log cfu/ml after 2 hours. this is superior compared to the activity of vancomycin. no inoculum effect was observed in the employed range of cells. the observed pae had a duration of 2 hours and 42 minutes. no spontaneously resistance mutation was observed among 10e10 cells of staphylococci and the mpc were determined to be 8 times mic. conclusions: plectasin is a novel antimicrobial peptide that shows potent antimicrobial activity against gram-positive bacteria including drug-resistant organisms. the potent, excellent bactericidal activity in vitro, lack of cross-resistance to clinical used antibiotics, low spontaneously resistance mutation frequency and good pae properties, suggest that plectasin may have potential as a therapeutic agent against staphylococci. in vitro antimicrobial activity of the novel polymeric guanidine akacid plus ò c. kratzer, s. tobudic, w. graninger, a. buxbaum, a. georgopoulos (vienna, at) objectives: cationic antimicrobials are widely used for disinfection within clinical settings. in the present study the bactericidal and fungicidal activity of akacid plus ò , a novel polymeric compound of the cationic family of disinfectants, was evaluated against quality control strains of staphylococcus aureus, enterococcus hirae, escherichia coli, pseudomonas aeruginosa, candida albicans and aspergillus niger in comparison to chlorhexidine digluconate. methods: the in vitro activity of akacid plus ò and chlorhexidine was determined by quantitative suspensions tests according to the european committee for standardization at concentrations of 0.01-0.5% against bacterial strains and c. albicans and at concentrations of 0.5-4% against a. niger after exposure for 5, 15 and 60 min in the presence and absence of 0.3% bovine albumin and dilution in distilled and hard water. results: in the basic quantitative suspension test akacid plus ò destroyed all bacterial pathogens at a concentration of ‡0.1% in £5 min contact time. chlorhexidine was also highly active against s. aureus, e. coli and p. aeruginosa, but failed to eliminate e. hirae within 5 min. under high organic burden, the bactericidal activity of both disinfectants was slightly reduced. akacid plus ò showed fungicidal activity against c. albicans within 15-60 min and eliminated a. niger at a concentration of ‡1% in 5 min contact time. chlorhexidine was fungicidal against c. albicans, but did not achieve biocidal activity against a. niger. conclusion: the novel polymeric guanidine akacid plus ò when compared to chlorhexidine digluconate showed similar bactericidal activity against s. aureus, e. coli and p. aeruginosa and superior biocidal activity against e. hirae and a. niger. investigation of emergence of bacterial resistance to the novel antibacterial photodynamic agent xf-42 are novel, light activated antibacterial agents (1) active against gram-positive bacteria, which have greater potency than antibiotics. the emergence of resistance to xf-42 has been investigated. methods: 0.382 mg/l of xf-42 was added to 108 cells/ml of mrsa. after 5 minutes incubation in the dark the unbound xf-42 was removed and the culture illuminated with 13.7 j/cm 2 of light at 422 nm and cfu analysis undertaken to determine the number of viable cells remaining. 5 surviving clones of the treatment were cultured and subjected to further treatment. 10 cycles were undertaken to determine whether the number of surviving cells increased, suggesting resistance build up to xf-42. results: the survival of methicillin-resistant staphylococcus aureus (mrsa) (atcc baa-44) is expressed as log n0/n, where n0 and n are the cfu of untreated and treated suspensions, respectively. the results demonstrate that no detectable resistance build up to the activity of xf-42 was seen after 10 successive treatments. a low propensity for emergence of resistance is a valuable attribute for new anti-bacterial agents. xf-42 might be effectively employed in the clinical setting for prophylactic use to decolonise skin and nares and therapeutic use to treat infected wounds/ulcers. objectives: the xf drugs are novel, light activated antibacterial agents (1) active against gram-positive bacteria which have superior potency to antibiotics but possess a low propensity to induce resistant bacterial strain emergence. a novel ex-vivo porcine skin model has been developed to test the antibacterial activity of xf-73 on the surface of skin. methods: 10x7 cells of methicillin-resistant staphylococcus aureus (mrsa) were inoculated onto a 1.32 cm 2 area of ex-vivo porcine skin samples, immobilised in agar. after drying, solutions of xf-73 were applied and after 60 minutes, the samples were illuminated for 15 minutes with blue light (422 nm) with various total light doses using a lumacare tm lc-122m lamp. cfu analysis were undertaken to determine the number of viable cells remaining after treatment. controls of drug alone and light alone were included. results: using 7.64 mg/l of xf-73, cfu analysis demonstrated that at a total light dose of 5 j/cm 2 , there was~90% kill of bacteria. at 10 j/cm 2 , there was 99.9% kill of bacteria, and 99.99% at 20 j/cm 2 and 40 j/cm 2 . at a total light dose of 20 j/ cm 2 , it was found that there was a <99% kill by xf-73 at concentrations of 0.76, 1.53 and 3.8 mg/l. at a concentration of 7.64 mg/l, there was a >99.9% kill. this kill did not significantly increase at 22.93 and 76.4 mg/l. conclusions: the results demonstrate that xf-73 has exceptional activity at low concentrations against mrsa on the surface of porcine skin. xf-73 and light are non-toxic to skin at therapeutic concentrations. work is in progress to clinically evaluate the effectiveness of this compound in eradicating staphylococcal nasal carriage. objectives: the rise of epidemic methicillin-resistant staphylococcus aureus (emrsa) and the emergence of mupirocin resistance means that it is essential to develop new therapies that cannot be readily overcome by microorganisms. the xf series of novel light activated antibacterial agents (1) active against gram-positive bacteria addresses this issue and have superior levels of activity to antibiotics but with less likelihood of resistance emergence. the antibacterial activity of the xf drugs against emrsa has been investigated. methods: mic and mbc assays were used to investigate the antibacterial activity of xf-73, a novel antimicrobial photodynamic agent against a range of staphylococcus aureus strains. a concentration range of 2-0.001 mg/l was investigated. 15 minutes of 420 nm light activation (13 j/cm 2 ) was applied. light alone had no effect. results: conclusions: the results demonstrate that xf-73 has exceptionally low mic and mbc values against all of the s. aureus strains tested. the results also demonstrate that xf-73 is equally effective against mrsa and methicillin-sensitive staphylococcus aureus (mssa) indicating its mode of action is independent of antibiotic resistance. xf-73 may therefore be useful in prevention and treatment of emrsa. xf-73 is non-toxic to skin at prophylactic/therapeutic concentrations and has potential for the treatment of skin sepsis and the eradication of nasal and skin mrsa carriage. work is in progress to evaluate the effectiveness of this compound in eradicating staphylococcal nasal carriage. objective: nxl101 is a novel antibacterial currently in preclinical development. the mechanism of action is directed against topoisomerase, and the spectrum of activity is exclusively against gram positive organisms. the goal of the study was to characterise the activity and time/kill kinetics against common aerobic cocci in comparison to currently marketed molecules: linezolid (lin), vancomycin (van), quinupristin/dalfopristin (q/d) and moxifloxacin (mox). methods: (i) in vitro susceptibility tests: the strains used were from the culture collection of novexel and were of clinical origin. mics were determined by an agar dilution technique. mueller hinton agar medium was used, supplemented with 5% horse blood for group a streptococci (gas), group b streptococci and s. pneumoniae. overnight cultures were diluted to obtain the final inoculum of 10 4 cfu/spot. the mic was the lowest concentration which inhibited all visual growth (3 or less colonies were ignored). (ii) time/kill kinetics: experiments were performed against strains of s. aureus (n = 8) and s. pneumoniae (n = 4) in 20 ml volumes of appropriate growth medium with initial inoculum of around 10 6 cfu/ml of logarithmically growing culture. timed samples over a 24 hour period were enumerated using a spiral plating method. nxl101 was compared to linezolid and vancomycin and the concentrations tested were 4, 8 and 16-fold the mic90 for both species. results: (i) the mic90s of nxl101 versus comparators are shown in the table. (ii) time kill experiments showed that nxl101 was bactericidal against s. aureus, including methicillin resistant strains (>3log10 reduction within 6-8 hours) compared to a slowly bactericidal effect for vancomycin (24 hours). nxl101 and vancomycin were both bactericidal against s. pneumoniae within 6-8 hours. linezolid was bacteriostatic against all strains tested. conclusion: nxl101 exhibits bactericidal activity against common gram positive cocci, including strains which exhibit resistance to methicillin, vancomycin and fluoroquinolones. nxl101 warrants further investigation. objectives: the aim of this study was to identify bacterial proteins as targets of the endogenous antiseptic n-chlorotaurine (nct), which is a promising microbicidal agent for topical treatment of infections. in addition, a combination of nct with ammonium chloride which enhances the microbicidal activity significantly was investigated. methods: escherichia coli and staphylococcus aureus were treated with nct and nct plus ammonium chloride for different incubation times between 1 and 30 min -a period where killing takes place. to find out protein changes, 2d-page of bacterial proteins followed by mass spectrometry was performed. results: incubation in 1% nct revealed a change of the charge and a separation of numerous proteins into a series of spots with a different isoelectric point. moreover, in e. coli heat shock protein 60 appeared, while ribosome releasing factor, d-ribose periplasmic binding protein, and malonyl-coa transacylase spots decreased. in s. aureus, enolase and a translation elongation factor decreased. these changes appeared more rapidly in the presence of ammonium chloride, which can be explained by formation of the more lipophilic and microbicidal monochloramine. molecular mechanisms of attack comprised mainly oxidation of thio and amino groups as confirmed with model peptides. conclusion: these results fit very well to previous preclinical and clinical findings. they indicate both surface attack and penetration of oxidation capacity into the bacteria and destruction of essential proteins by nct and nct plus ammonium chloride, respectively. objectives: ceftobiprole is a new extended-spectrum cephalosporin with activity against methicillin-susceptible and methicillin-resistant staphylococci, as well as against most enterobacteriaceae. in this study the anti-staphylococcal activity of ceftobiprole is reported from a set of isolates from a recent clinical trial. methods: consecutive clinical isolates of staphylococci from 340 patients enrolled in a multicentre clinical trial involving complicated skin infections were examined for their susceptibility to ceftobiprole and selected anti-gram-positive agents. mics were determined using clsi methodology. results: among these isolates, 525 staphylococcus aureus and 84 coagulase-negative staphylococci (cons) were identified. the percentages of methicillin-resistant strains were 43% for s. aureus and 50% for cons. all strains (except one cons with a linezolid mic of 8 mg/l) were susceptible to vancomycin and linezolid, with mics <2 mg/l.against methicillin-susceptible s. aureus, ceftobiprole mic50 and mic90 values were 0.25 and 0.5 mg/l, respectively, and against methicillin-resistant s. aureus, ceftobiprole mic50 and mic90 values were 0.5 and 2 mg/l, respectively. ceftobiprole mics ranged from £0.06 to 1 mg/l against methicillin-susceptible-cons (ms-cons) and methods: consecutive, non-duplicate bacterial isolates (10,068 strains) acquired from patients with bloodstream, respiratory, and skin and skin structure infections both nosocomial and community acquired were submitted from >70 medical centres in europe, the americas and the asia-pacific region. all isolates were tested using clsi/nccls broth microdilution methods against grn, the currently marketed fluoroquinolones (fq) including cipro, levofloxacin (levo), gatifloxacin (gati) and representative comparator agents. oxa-and cipro-s and -r subsets were included. a grn-s breakpoint of £0.12 mg/l was applied for comparative purposes only and was based upon the mic population distributions of strains that included quinoloneresistance determining region (qrdr) mutations. results: potency for grn and comparator fqs tested against sa: (see table) . key resistance patterns (%) among this sa collection included oxa (40.3), cipro (36.4), erythromycin (47.5), clindamycin (13.4), tetracycline (9.7), and trimethoprim/ sulfamethoxazole (4.8%); gram-positive-targeted comparator including vancomycin, linezolid, daptomycin and quinupristin/dalfopristin all remained >99% s. compared with currently marketed fqs when tested against all sa, grn was 2-to 16-fold more active (mic50, £0.03 vs. 0.06 or 0.5 mg/ l). against both oxa-s and -r sa, grn displayed markedly enhanced potency compared with cipro and levo ( ‡4-fold), and gati (2-to 4-fold). among cipro-r isolates, grn also maintained ‡4-fold greater potency (mic50, 1 vs. ‡4 mg/l) although overall s for all fqs was 0-1%. compared to the fq agents tested against sa, grn was the most potent agent and maintained the broadest coverage against oxa-and cipro-r strains even when applying a very conservative epidemiologic breakpoint. when a fq is indicated for staphylococcal coverage, this des-f(6) quinolone may represent a superior alternative among fq class agents, while minimizing selection of resistance. objective: to assess the garenoxacin (grn) potency against a vast number of international respiratory tract infection (rti) pathogens, especially versus phenotypic (high mic) or genotypic (sequence change) qrdr mutants. a total of 40,423 isolates from 6 continents were analysed (1999) (2000) (2001) (2002) (2003) (2004) (2005) table) conclusions: grn maintains clinically usable activity (mic, £1 mg/l) against important community-acquired rti pathogens having r to presently marketed fluoroquinolones and against those isolates with documented qrdr mutations. continued development of this novel des-f(6) quinolone agent appears desirable. in vitro activity of garenoxacin tested against ciprofloxacin-susceptible and -resistant enterobacteriaceae and acinetobacter spp. strains collected worldwide by the sentry antimicrobial surveillance program (2004) (2005) h. sader, t. fritsche, p. strabala, r. jones (north liberty, us) objective: to evaluate the contemporary activity of garenoxacin (grn) against ciprofloxacin (cipro)-susceptible (s) and cipro-resistant (r) enterobacteriaceae (ent) and acinetobacter spp. (asp). unlike recently marketed fluoroquinolones (fq), grn, a des-f(6) quinolone lacks the c-6 fluorine. methods: a total of 9,017 isolates (8,247 ent and 770 asp) were consecutively collected from > 70 medical centres from bloodstream, respiratory, urinary and skin and soft tissue infections and tested by reference broth microdilution methods according to clsi/nccls methods and interpretative criteria. a grn s breakpoint of £1 mg/l was applied for comparison purposes only. results: the results of the major organism groups tested: (see table) . grn showed excellent activity against this large collection of ent (mic50, 0.12 mg/l) and 82.7% of isolates were inhibited at £1 mg/l. objectives: garenoxacin (grn) is a novel, broad-spectrum des-f(6)-quinolone with activity against gram-negative and grampositive aerobes and anaerobes including quinolone-resistant staphylococcus aureus. the objective of this analysis was to compare the microbiologic efficacy of grn to that of comparators against common pathogens involved in complicated skin and skin structure infections (csssi). methods: two multinational, double-blind, randomized studies were conducted. in the first study, subjects received grn (600 mg iv to po qd) or piperacillin/tazobactam (3.375 g iv q6h) with transition to po amoxicillin/clavulanate (500 mg po q8h). in the second study, subjects received grn (600 mg po qd) or ciprofloxacin/metronidazole (500 mg q12h/500 mg q8h). all antimicrobials were administered for 7 to 14 days. subjects were adults ( ‡18 y) newly hospitalized or ambulatory outpatients with evidence of csssi who did not have underlying osteomyelitis. microbiologic efficacy was determined 5 to18 days post-therapy. results: a total of 567 subjects were microbiologically evaluable (grn, n = 283; comparators, n = 284). the disease diagnosis was similar between grn and comparators and included infected pressure sore (5% vs 3%), infected diabetic foot ulcer (18% vs 19%), major abscess (66% vs 67%), or postsurgical wound infection (11% vs 12%). the majority of common skin pathogens were eradicated by grn background: acute bacterial sinusitis (abs) is a common infection world-wide, with many patients having an associated an allergic component/history. however the role of antibacterials in these patients (pts) has not been examined. as some fluoroquinolones (fq) have an in-vitro immunomodulatory effect (ie) the clinical efficacy of gem was compared other agents in abs pts with or without allergic rhinitis (ar). methods: 5 phase 3 clinical trials were pooled and pts where allergic rhinitis was identified (584 pts) were compared with pts not reporting ar (1834 pts). clinical response (success or failure) at end of therapy (eot) & at follow-up (fu, approx. 1-3 weeks after treatment) was studied. comparators (cmp) were cefuroxime (cef) and trovafloxacin (tro). results: % success based on clinical outcome at eot and fu for ar and non ar pts are shown in the table. for all treatments eot success was high for the non ar pts, but at fu this was reduced, especially with both fqs. in contrast, gem retained a high clinical success rate in pts with ar unlike cef or tro. conclusion: gem has been shown to be very efficacious in a sub group of problematic abs pts. this advantage may be due to the high antibacterial activity of gem vs key abs pathogens and/or a stimulatory ie. both being important with pts having decreased local immune defences. these data also show that not all fluoroquinolones have immuno-stimulatory properties. garenoxacin efficacy against multidrug-resistant streptococcus pneumoniae: retrospective analysis of community-acquired pneumoniae isolates obtained from nine phase ii and iii clinical studies (1999) (2000) (2001) (2002) (2003) t. black, h. waskin, r. hare (kenilworth, us) objective: garenoxacin (grn) is a novel, des-f(6)-quinolone with excellent activity against s. pneumoniae, one of the most common pathogens causing community-acquired pneumoniae (cap). the incidence of infections caused by antibiotic-resistant isolates of streptococcus pneumoniae is on the increase, therefore information regarding the activity of new anti-infective drugs against populations of s. pneumoniae that are multi-drug resistant (mdr) is critical. mdr s. pneumoniae (mdrsp) includes isolates previously known as prsp (penicillinresistant s. pneumoniae), as well as strains resistant to two or more of the following antibiotics: second-generation cephalosporins, macrolides, tetracyclines, and trimethoprim/ sulfamethoxazole. methods: pretreatment sputum and blood isolates collected worldwide during grn phase 2/3 clinical cap trials (1999) (2000) (2001) (2002) (2003) were retrospectively analysed for the mdrsp phenotype. of the 352 s. pneumoniae isolates originally identified, 208 from 180 subjects were subjected to secondary mdr susceptibility testing by central laboratories. confirmed mdrsp isolates were matched to individual subjects to assess clinical and microbiological outcomes for mdrsp-infections treated with grn. results: expanded susceptibility testing identified 53/208 mdrsp isolates from 44 unique subjects. the lowest mic50 and mic90 values for mdrsp isolates tested against a panel of representative drugs were observed for grn (table 1 ; 0.03 lg/ ml and 0.06 lg/ml, respectively). the incidence of resistance to the five classes of drugs was 11%, 12%, 21%, 19% and 18% for penicillin, 2nd generation cep., macrolides, tetracycline and tri/sulf, respectively. no isolates were resistant to grn using a proposed susceptibility breakpoint value of £1 lg/ml. thirtyfive percent, 28%, 15%, 9% and 13% of isolates were resistant to 1, 2, 3, 4 and 5 drug classes, respectively. the worldwide incidence of mdrsp was 18% with an equivalent geographic distribution of 19%, 21% and 16% among north america, europe and the rest of world. overall, grn provided clinical and bacteriological success for 32/35 (91%) cap evaluable subjects with mdr infection, which was similar to clinical success for evaluable subjects with non-mdrsp cap infections 151/165 (92%). conclusions: these data demonstrate the ability of grn to successfully eradicate mdrsp associated with cap. . per cent success is shown in the table (ab, antibiotics, copd, chronic bronchitis and obstructive lung disease, hd, heart disease). results: although gemifloxacin showed lower % success than comparator against cap patients with no defined risk factor, gemifloxacin was considerably more successful than comparator against patients associated with risk factors, especially diabetic patients where comparator success was low. this advantage was often more prominent at fu than at eot. patients with other comorbidities such as renal failure or malignancy were not recruited in sufficient number for analysis. conclusions: these data support the use of gemifloxacin in the treatment of cap, especially where the patient has recognised idsa risk factors. microbiologic efficacy of garenoxacin vs. comparators against common pathogens associated with community-acquired pneumonia objectives: garenoxacin (grn) a novel, broad-spectrum des-f(6)-quinolone is active against many clinically important respiratory pathogens including penicillin-resistant strains of streptococcus pneumoniae. grn has dual sites of inhibition (dna gyrase and topoisomerase iv) and may be less likely to promote resistance. the objective of this analysis was to compare the microbiologic efficacy of grn to that of comparators against common pathogens involved in community-acquired pneumonia (cap). methods: two multinational, double-blind, randomized studies were conducted. in the first study, subjects received grn (400 mg po qd for 5 d) or amoxicillin/clavulanate (a/c; 500 mg po q8h for 7-10 d). in the second study, subjects received grn (400 mg po qd for 7-10 d) or levofloxacin (lev; 500 mg po qd for 7-10 d). adults (18 years of age or older) were enrolled with clinical and radiologic evidence of cap [new infiltrate(s) on chest radiograph and fever, leukocytosis, cough, chest pain, auscultatory findings, or sputum production]. the majority of subjects were fine class i/ii in both studies. bacteriologic eradication was assessed 5 to18 days post therapy. results: a total of 377 treated subjects had pretreatment pathogens (grn, n = 179; comparators, n = 198) . the overall eradication rate in all treated subjects was 91% (129/142) for grn and 85% (123/145) for the comparators. eradication rates for s pneumoniae were 92% (24/26) for garenoxacin and 96% (49/ 51) for the comparators. eradication of s pneumoniae was 100% and 89% for a/c and lev, respectively. in strains with reduced susceptibility to penicillin eradication rates were 86% (6/7) vs 67% (2/3) in favour of grn. eradication rates for h. influenzae were 89% (40/45) and 75% (30/40) for grn and comparators, respectively. lev eradicated 71% of h. influenzae isolates and a/c eradicated 77% of the strains isolated. there were very few isolates (4) of moraxella catarrhalis in the 2 studies. in 1 study grn was 100% effective against 3 strains of m. catarrhalis and in the other a/c was 100% effective against the 1 strain isolated. grn eradicated 95% (19/20) of the staphylococcus aureus isolates vs 100% (11/11) for the comparators. conclusions: grn was highly active against pathogens commonly associated with cap including drug-resistant strains of s pneumoniae and represents an effective therapeutic option for this patient population. objectives: garenoxacin (grn) a novel, broad-spectrum des-f(6)-quinolone is active against many clinically important respiratory pathogens including penicillin-resistant strains of streptococcus pneumoniae. there is a growing problem of resistance in strains of s pneumoniae, with multi-drug-resistant s pneumoniae (mdrsp) becoming increasingly more common. the objective of this study was to evaluate the clinical and microbiologic efficacy of grn in the treatment of communityacquired pneumonia (cap) caused by mdrsp. methods: this was a multinational, open-label, noncomparative study. subjects were adults ( ‡18 and <75 y) with clinical (clinical signs, sputum production), radiologic (new infiltrates on chest radiograph), or microbiologic (predominance of gram-positive cocci in pairs on sputum gram-stain or a positive blood culture for s. pneumoniae) evidence of cap caused by s. pneumoniae. subjects received grn 400 mg po qd or grn 400 mg iv with transition to 400 mg po qd for 7 to 14 days. clinical and microbiologic responses were determined at a test-of-cure visit 7 to 14 days posttherapy. results: a total of 121 subjects were enrolled. of these, 47 (17 po only, 30 iv to po) were clinically and microbiologically evaluable. clinical and microbiologic success rates were 91% (43/47) and 89% (42/47), respectively. clinical success rates were 94% (16/17) and 90% (27/30) for po and iv to po, respectively. documented s. pneumoniae bacteremia was present in 28% (n = 13) of subjects with a clinical success rate of 92%. among evaluable subjects, resistance rates for s. pneumoniae were penicillin 13%, second-generation cephalosporin 17%, macrolides 21%, tetracyclines 21%, and trimethoprim/ sulfamethoxazole 17%. twelve evaluable subjects had pneumonia caused by mdrsp. clinical success rate was 92% (11/12) in subjects with mdrsp and 91% (32/35) in non-mdrsp subjects. clinical success of grn for strains resistant to 2, 3, 4, or 5 antimicrobial drug classes, were 100% (5/5), 100% (1/1), 100% (2/2), and 75% (3/4), respectively. microbiologic success was 83% (10/12) and 91% (32/35) for mdrsp and non-mdrsp (susceptible or resistant to 1 class) strains, respectively. grn was generally well tolerated with drug-related adverse events (ae) reported in 14% (8/58; po) and 21% (13/63; iv to po) of subjects. conclusions: grn (po or iv to po) is an effective treatment for cap caused by mdrsp and non-mdrsp. grn is well tolerated. in vitro bactericidal activity of daptomycin against staphylococcus aureus and enterococcus spp.: comparison with vancomycin, teicoplanin and linezolid h. drugeon, m. juvin (nantes, fr) objectives: the aim of this study was to evaluate the bactericidal activity (by killing kinetics) of daptomycin (dap) against staphylococcus aureus (sa) clinical isolates with different teicoplanin mics and against enterococcus faecalis (efl) and e. faecium (efm) with different mechanisms of glycopeptide resistance. dap has been compared with teicoplanin (tei), vancomycin (van) and linezolid (lin). methods: 6 sa strains (2 mssa and 4 mrsa) with tei mic distributed from 0.5 to 8 mg/l, 6 enterococcus (3 efl and 3 efm) with glycopeptide phenotypes [s, r-vana, r-vanb] were studied using a killing curve method. 7 antibiotic concentrations were used from 1 mg/l to 64 mg/l in two fold dilutions. surviving bacteria were counted at t0, t30', t1, t3, t6, t12 and t24 hours using agar plates with inhibitors to prevent antibiotic carry-over. antibiotics tested were daptomycin (dap), teicoplanin (tei), vancomycin (van) and linezolid (lin). results: all the sa isolates were susceptible to dap (mic = 0.25-1 mg/l), to lin (mic = 1-2 mg/l), to van (mic = 1-2 mg/l) regardless of susceptibility to methicillin. dap showed the same strong concentration dependent bactericidal activity with mssa and mrsa: at t30' bactericidal activity (ba) (decrease of 3 log10 cfu/ml) was observed with 8-16 mg/l of dap; at t3 hours, 1-4 mg/l of dap was sufficient and at t6 hours, ba was obtained with 1 mg/l of dap. the other antibiotics showed a time dependent bactericidal activity but ba was observed only with long exposure ( ‡12 hours) and with high concentrations. all the enterococcus isolates were susceptible to dap (mic = 1-2 mg/ l) and to lin (mic = 1-2 mg/l) regardless of the resistance to glycopeptides. ba of dap was also concentration dependent. ba was obtained with 4-8 mg/l after 6 hours of contact and with 1 mg/l after 12 hours of contact for efl. ba was observed with 8-16 mg/l after 6 hours of contact and with 1-2 mg/l after 24 hours of contact for efm. the other antibiotics had a time dependant activity but didn't show bactericidal activity with concentrations 32 mg/l. conclusion: the bactericidal activity of daptomycin was very strong, concentration dependent, and not influenced by the level or mechanism of glycopeptide resistance the bactericidal activity of linezolid was time dependent and observed only with the highest concentration and the bactericidal activity of vancomycin and teicoplanin was time dependent but was influenced by the mechanism of glycopeptide resistance. objectives: telavancin (tlv) is a bactericidal lipoglycopeptide with multiple mechanisms of action that is in phase 3 clinical trials for the treatment of complicated skin and skin structure infections and hospital-acquired pneumonia with a focus on infections due to methicillin-resistant staphylococcus aureus (mrsa). this study evaluated and compared the antibacterial activity of tlv with that of other antibacterial agents against recent gram-positive clinical isolates from germany. methods: a total of 363 aerobic gram-positive bacterial strains recently collected were included. antibiotics tested were tlv, vancomycin (van), teicoplanin, penicillin, oxacillin, ampicillin, cefuroxime, ceftriaxone, daptomycin (dap), linezolid (lzd), quinupristin-dalfopristin, clindamycin, ciprofloxacin, levofloxacin, gentamicin, streptomycin, erythromycin, telithromycin, co-trimoxazole and tetracycline. mics were determined by the broth microdilution procedure according to the guidelines of the clsi. results: tlv exhibited potent activity against all grampositive bacteria including resistant isolates such as mrsa, van-resistant enterococci, pneumococci (including multiple resistant strains with various antibiotic resistance phenotypes) and other streptococcal species. tlv showed excellent in vitro activity against the species irrespective of the antibiotic phenotype tested. for methicillin-susceptible s. aureus (mssa, n = 33) and mrsa (n = 42) mic90 of tlv for both phenotypes were 0.5 mg/l. for coagulase-negative staphylococci (n = 72, incl. msse, mrse, mssh, mrsh and others) mic90s were 0.5 or 1 mg/l. mic90s of tlv for enterococcus faecalis (n = 30) and e. faecium (n = 32) were 1 and 2 mg/l, respectively. for van-resistant strains of e. faecalis (n = 2) or e. faecium (n = 7) mics for tlv ranged from 0.12 to 4 mg/l. against streptococcus pneumoniae (n = 60) tlv mics ranged from £0.001 to 0.03 mg/l. all streptococcus pyogenes, streptococcus agalactiae and all viridans group streptococci (n = 94) had mics of £0.125 mg/l. conclusion: based on mic90, tlv was more potent than van, dap or lzd against staphylococci, streptococci and e. faecalis. it was superior to dap and lzd against e. faecium and at least as active as dap or lzd against most van-resistant enterococci. tlv appears to be a promising new antimicrobial agent for the treatment of infections caused by gram-positive organisms including multiply resistant isolates. the extent of protein binding (pb) of dap is still under investigation and data available so far indicate pb of either 92% or 63%. therefore we tested two fscs: 22.0 (corresponding to 63% pb) and 4.8 (corresponding to 92% pb). the activity of dap was determined in mueller-hinton broth supplemented with 50 mg/l calcium. viability counts were performed at 0.25, 0.5, 1, 3, 6, 12 and 24 h. one methicillin-susceptible staphylococcus aureus (mssa), two methicillin-resistant s. aureus (mrsa), one vancomycin-susceptible (van-s) and one van-resistant (van-r) enterococcus faecalis, one van-s and one van-r enterococcus faecium were tested. bactericidal activity was defined as >99.9% killing during incubation. results: dap was bactericidal at concentrations of 60.0 mg/l and 22.0 mg/l in all seven strains. the concentration of 4.8 mg/l was bactericidal against the two mrsa and against the van-s e. faecium. in the other four strains the maximum reduction of initial inoculum ranged from 1.52 to 2.53 log10 cfu/ml. in six strains a bactericidal effect at 60.0 mg/l and 22.0 mg/l of dap, respectively, occurred between 15 minutes and 3 h and after 6 h in the van-s e. faecalis. van at 40.0 mg/l or 18.0 mg/l was bactericidal in only two strains after 24 h (1 mssa, 1 mrsa). against the other five strains, van was bacteriostatic with maximum reduction of initial inoculum between 1.91 and 2.78 log10 cfu/ml at 40 mg/l after 24 h, respectively. both tpl and lzd were consistently bacteriostatic against the test strains. conclusion: dap at psc of 60.0 mg/l as well as at fsc of 22.0 mg/l showed a pronounced bactericidal effect within 3 h in 6/7 strains. van was bactericidal in only 2/7 strains after 24 h. compared to van bacterial killing by dap was very rapid. tpl and lzd were bacteriostatic only. the effect of human serum on the bactericidal activity of daptomycin and comparators against staphylococcus aureus and enterococcus spp. background: daptomycin is a new cyclic lipopeptide antibiotic that shows rapid bactericidal activity and has high protein binding when assessed by standard methodology. this study investigated the bactericidal activity of daptomycin and the effect of protein binding by the addition of 50% human serum (hs). methods: exponentially-growing methicillin-susceptible andresistant s. aureus (mssa, mrsa) and vancomycin-susceptible enterococcus faecium (vse) and -resistant enterococcus faecium (vre) (ca. 106 cfu/ml) were exposed to daptomycin (dap), vancomycin (van), teicoplanin (tei), piperacillin-tazobactam (ptz) or linezolid (lzd) at peak (p) and trough (t) serum concentrations in mueller hinton broth supplemented with ca2+ to 50 mg/l with or without hs. viable count was determined at 0.25, 0.5, 3, 6 & 24 h. plots were made of log reduction in viable count over time and the area-under-thecurve measured to calculate bactericidal indices (bis) from these plots (j antimicrob chemother 1997, 39: 713-717). results: daptomycin reduced viable count of mssa & mrsa by approx. 5 logs or more within 0.25 h and vse or vre within 3 h at p. other agents either did not achieve this or required 24 h to do so (not shown). bi data are shown below (>represents kill beyond the limit of detection). hs had little effect on dap kill, except against the vre at t. nevertheless, dap at t against vre was more bactericidal than any other antibacterial except dap at p. conclusions: dap was the most bactericidal agent tested as measured either by bi or rate of kill. dap at p reduced mssa and mrsa to below detection within 15 min. the effect of hs was minimal which suggests that protein binding is either weak or highly reversible. these data support the use of dap in the treatment of infections caused by these organisms. daptomycin activity against multi-resistant staphylococcus haemolyticus bloodstream isolates from severe infections objectives: daptomycin, a new cyclic lipopeptide with activity against multidrug-resistant gram-positive pathogens including mrsa, is approved for use in cssst infections (us-fda) and is being reviewed by emea for approval in eu member countries. the rapid bactericidal activity of daptomycin, due to its unique mechanism of action, makes it an attractive antibiotic for serious gram-positive infections. the study was performed: (i) to evaluate the activity of daptomycin and other drugs against 50 multi-resistant clinically relevant staphylococcus haemolyticus (mrsh), isolated from bloodstream infections in various hospitals in italy (ii) to determine epidemiologic and genetic correlation among strains, and (iii) to characterize the sccmec dna of these strains. methods: the mrsh strains were tested against a panel of antimicrobial agents, by broth microdilution method performed according to clsi (clinical laboratory standards institute) guidelines, including supplementation of 50 mg/l calcium for daptomycin. moreover, phenotypic tests and antibiotic susceptibility profiling were carried out and the results compared with molecular typing analysis by using smai-pfge fingerprints and pcr to characterize the mec-complex. results: all isolates were resistant to erythromycin, gentamicin, ciprofloxacin, 16 strains showed reduced susceptibility to vancomycin (mics 2 mg/l), 24 strains were resistant to cotrimoxazole, 16 strains to clindamycin, 6 strains to chloramphenicol and 3 strains to tetracycline. almost all isolates were inhibited by £1 mg/l of daptomycin, and only four strains exhibited a mic value of 2 mg/l. pfge analyses showed the existence of at least two multi-resistant s. haemolyticus clones widespread in different hospitals. methicillin-resistance was correlated to the presence of the meca and preliminary results regarding the genetic element carrying the gene, showed an organization of the mec-complex of class a and class c. conclusions: our results suggest that daptomycin has excellent activity against multiresistant mr s. haemolyticus isolates, which represent a serious threat in catheter-related bloodstream infections. furthermore, the emergence of s. haemolyticus exhibiting reduced susceptibility to vancomycin is of particular concern, probably due to the common use of vancomycin as initial therapy for such infections. moreover, the use of additional molecular techniques to fingerprint isolates makes this study of clinically important cons more accurate. objectives: ceftobiprole is a new cephalosporin with a broad spectrum of action including methicillin-resistant staphylococci (mrs) as well as many other gram-positive and gram-negative pathogenic bacteria. this study investigates the structural basis for the good activity against mrs. methods: the primary beta-lactam resistance determinant of mrs, penicillin-binding protein pbp 2' (or 2a) has been cloned and expressed as a soluble form in which the amino-terminal residues forming a membrane-anchor have been deleted. the soluble form has been crystallized and the structure of the complex formed after soaking crystals in a solution containing ceftobiprole has been determined at 2.8 angstrom resolution. additional data on the structure of the ceftobiprole-pbp2' complex formed in solution has been obtained using spectroscopic methods such as uv-circular dichroism. results: ceftobiprole reacts rapidly with pbp2' to form a stable acyl-enzyme complex. the ceftobiprole moiety is positioned deep within the active site of the acyl-enzyme complex formed with pbp2', where it forms several hydrogen bonds and hydrophobic interactions. in particular, the 7-aminothiadiazolylhyroxyiminoacetyl side chain of ceftobiprole sits more deeply within the side-chain binding pocket of pbp 2' than does the 7-acylamino side chain of nitrocefin in the previously determined complex structure. the additional interactions probably add to the enhanced stability of the acyl-enzyme complex formed with ceftobiprole, compared to complexes formed with other betalactams that are inactive against mrs. significant structural rearrangements between apo-enzyme and acyl-enzyme are evident in the crystal structure and in solution. conclusion: ceftobiprole readily forms a stable inhibitory acylenzyme complex with the pbp 2', the beta-lactam resistance determinant of mrs. this, together with potent inhibition of the normal complement of beta-lactam sensitive penicillin-binding proteins, accounts for its excellent activity against staphylococci and probably accounts for the low rates of resistance development observed in experimental conditions. incidence of staphylococcus aureus with reduced susceptibility to glycopeptides in a french hospital (november 2004 -april 2005 c. morate, a. charron, c. bebear, j. maugein (bordeaux, fr) staphylococcus aureus are a major cause of nosocomial infections around the world. glycopeptides remain the drug of choice for severe infections caused by mrsa. however, after the emergence of vancomycin resistance in enterococcus and in the coagulase negative staphylococcus, strains of staphylococcus aureus with reduced susceptibility to glycopeptides (gisa) have been reported in different countries like japan, france, spain, the uk and the united states. the aim of our study was to determine the proportion of vancomycin resistance in clinical s. aureus isolates in a french university hospital, between november 2004 and april 2005, then we wanted to define if there was an epidemic clone and study the clinical impact of these gisa strains. the protocol of detection was, first, a screening test on bhi agar containing 4 mg/l of teicoplanin, then, the vancomycin and teicoplanin mics were determined by the method of etest with an inoculum of 2.0 mcf on the selected strains. finally, the isolates with mic of the teicoplanin ‡8 mg/ l and mic of the vancomycin ‡4 mg/l or mic of the teicoplanin ‡12 mg/l and mic of the vancomycin £4 mg/l were studied on population analysis. after that, pulsed-field gel electrophoresis (pfge) was performed on the different isolates and the pulsotypes were compared. from november 2004 to april 2005, 468 s. aureus isolates were collected from 331 patients and screened for glycopeptide resistance on an initial agar screening test containing 4 mg/l of teicoplanin. the teicoplanin mic was >4 mg/l for 65 isolates (13.9%) from 59 patients and these strains were selected for the determination of the mics by ''macromethod'' etest. by this technique, 39 strains were selected and studied by population analysis. all the profiles were compared to the reference strain mu3 profile. this procedure detected 5 isolates (from 5 patients) with heterogeneous reduced susceptibility to glycopeptides (hgisa). so the incidence of staphylococcus aureus with reduced susceptibility to glycopeptides in our hospital was found to be 1.1%. four strains were resistant to methicillin and 3 were also resistant to gentamicin. the diversity of the strains was confirmed by pfge: there was not an epidemic clone in the hospital. the clinical history showed that 4 patients had received a prior treatment with vancomycin, and that 3 patients had a failure in treatment: 2 of them had cystic fibrosis. objectives: enterococcus faecalis was the most prevalent organism (72.5%) involved in enterococcal infections at tehran hospitals followed by e. faecium (22.5%). due to widespread expansion of aminoglycoside modifiying enzymes (agmes) genes, the rate of resistance to high level concentration of aminoglycosides has increased in these years. the rate of high level gentamicin resistant isolates of enterococci (hlgr) is high in iran (46%). the aim of this study was to determine the genes encoding resistance to aminoglycosides among enterococci in iran. methods: disks containing 120 lg gentamicin were used to detect hlgr isolates. primers specific for aac (6') aph (2") and aph (3') iiia genes were used in pcr to possibly detect acetyltransferases and phosphotransferas, the common agmes among 113 isolates of enetrococci. theses isolates were resistance to different concentration of gentamicin. results: a 222 bp region of the aac (6')-aph (2") gene was amplified by pcr in 95% hlgr isolates as well as in 40% of low level getamicin resistant isolates (llgr). moreover the gene aph (3') iiia was detected in 92.5% and 72% of isolates of hlgr and llgr respectively. differences between isolates of e. faecalis and e. faecium were found in term of prevalence of aph (3') iiia gene. conclusion: the bifunctional enzyme aac (6')-aph (2") is the main cause of resistance to high concentration of aminoglycosides in our collection of enterococci. this enzyme confers resistance to all clinically useful aminoglycosides with the exception of streptomycin. in the absence of aac (6')-aph (2"), gentamicin could be used in combination therapy. prevalence and genetic analysis of methicillinresistant staphylococcus aureus expressing highlevel and low-level mupirocin resistance m. kural, t. us, y. akgun (eskisehir, tr) objectives: to investigate the genetic location of mupa gene which encoded mupirocin resistance and characterize mupirocin-resistant methicillin resistant staphylococcus aureus (mrsa) isolated from patients in a turkish university hospital by polymerase chain reaction (pcr) and plasmid analysis. methods: methicillin and mupirocin resistance were detected by disk diffusion (oxoid, uk). the etest (ab biodisk, sweden) was performed to determine mupirocin minimum inhibitory concentrations (mics). the presence of mupa and meca were detected by pcr using specific primers. plasmid analysis were used to study the genetic location of mupa gene. results: a total of 595 (44.9%) mrsa strains were identified by disk diffusion in 1324 s. aureus. of the 595 clinical isolates 394 (66.2%) were from wound, 91 (15.3%) from blood, 41 (6.9%) from catheter, 36 (6.1%) from lower respirator tract (bronchoalveolar lavage, pleural fluid and transtracheal aspirates), 13 (2.2%) from sputum, 12 (2.0%) from urine and 8 (1.3%) from other (serebrospinal fluid, parasynthesis fluid, peritoneal fluid, and bone marrow) clinical samples. among the mrsa isolates, mupirocin resistance was detected in 35 (5.9%) strains with disk diffusion and etest. of the 35 mupirosin-resistant isolates 23 (3.9%) expressed high-level (muh) and 12 (2%) expressed lowlevel (mul) mupirocin resistance. all isolates were vancomycin, teicoplannin susceptible and chloramphenicol resistant with disk diffusion. isolates with high-level and low level mupirocin resistance due to the mupa gene were also detected with pcr. plasmids were detected in all of the 35 isolates. however only the muh isolates contained a 38 kb plasmid that encoded highlevel resistance. all of the isolates contained a 4.4 kb plasmid and resistant to chloramphenicol. conclusion: our results indicated that the mrsa clones detected in the hospital had acquired a high-level mupirocin resistant plasmid. the past observations and recent studies suggested that the numbers of such strians have increased following extensive topical use of mupirocin. the usage of mupirocin in our hospital has not yet been systematically implemented. it is frequently prescribed for the treatment of staphylococcal skin infections and less to eliminate nasal carriage of mrsa. in our hospital we should be aware of the possible emergence and increase of mupirocin highly resistant mrsa strains in the future so that we should be considered when using mupirocin to control the spread of mrsa in hospital. emergence and spread of acquired fusidic acid resistance in staphylococcus aureus objectives: a major route to fusidic acid resistance (fusr) in s. aureus involves acquisition of fusb, a resistance determinant first clinical microbiology and infection, volume 12, supplement 4, 2006 identified on plasmid pub101. here we show that (i) the two currently-circulating major clones of fusr s. aureus identified to date have acquired fusb from pub101 (or from the same ancestral source as pub101), and (ii) that the pub101-encoded fusb is only one of at least three lineages of this protein that appear to have evolved since recruitment of the original, ancestral fusb to the staphylococci. methods: plasmid purification, dna sequencing, pcr amplification, and cloning in s. aureus rn4220 using shuttlevector pcu1, were all performed using established methods. antibiotic susceptibility testing was performed by agar dilution. results: the epidemic european fusidic acid-resistant impetigo clone (eefic) and community-acquired mrsa strain st80 have been shown to carry chromosomal and plasmid-encoded fusb, respectively. dna sequencing of fusb and its surrounding regions in these backgrounds revealed that they are identical to sequences on pub101. however, acquired fusr does not always result from acquisition of the prototypical fusb gene. a gene encoding a fusb homologue was recently identified during sequencing of s. aureus strain mssa476, and we identified an additional homologue encoded in the genome of s. saprophyticus strain atcc15305. the products of these genes exhibit~40% homology to fusb and to each other. cloning of pcr amplicons corresponding to these genes and their upstream expression signals into s. aureus established that they both confer resistance to fus. since these functional homologues are more closely related to each other than to those from other gram-positive organisms, it is highly likely that they evolved from an ancestral fusb after its recruitment to the staphylococci. conclusions: the three members of the staphylococcal family of fusb proteins appear to have evolved from the same ancestral protein, which, based on the low level of sequence homology between fusb genes at the nucleotide level, clearly occurred well before the introduction of fus into the clinic. of the three, the fusb protein encoded by pub101 is by far the most successful, and this gene/plasmid represents the source of (or shares a source with) the major fusr strain lineages. telithromycin activity is reduced by efflux in streptococcus pneumoniae c. benvenuti, r. koncan, g. bahar, a. mazzariol, g. cornaglia (verona, it; ankara, tr) objectives: telithromycin shows an excellent activity against m-type erythromycin-resistant streptococcus pneumoniae, thus is commonly regarded as being capable of overcoming the efflux resistance mechanism. nevertheless, telithromycin mic values in those strains appear to be distinctly higher than in the erythromycin-susceptible ones. the possibility of telithromycin acting as an actual efflux substrate, as it was already demonstrated in streptococcus pyogenes, seemed worth investigating. methods: telithromycin mic distribution was analysed in a collection of 423 italian s. pneumoniae strains originating from multi-centre studies (2000) (2001) (2002) (2003) . the effect of an efflux mechanism was investigated using [3h]-telithromycin. results: telithromycin mic ranges were £0.002-0.12 mg/l (mic50 0.008 mg/l and mic90 0.03 mg/l) in erythromycinsusceptible strains (lacking both mef and erm genes) and 0.016-1 mg/l (mic50 0.25 mg/l and mic90 0.5 mg/l) in strains endowed with the m phenotype. a distinct telithromycin efflux was detected in the strains expressing the mef gene, but not in those expressing the erm(b) gene, nor in the susceptible strains lacking mef or erm genes. efflux reversibility by addition of an inhibiting compound (sodium arsenate) was demonstrated. an msr-like sequence was also found in all strains effluxing telithromycin, but not in the others. conclusions: this is the first time that telithromycin has been shown to be effluxed by s. pyogenes isolates. that the efflux is related to the presence of both the mef and the msr-like genes is clearly demonstrated, but -owing to the increasingly evident complexity of s. pneumoniae efflux systems -other genes might also contribute to the efflux. an unusual phenotype of enterococcus faecalis in greece expressing low-level resistance to clindamycin and dalfopristin but susceptibility to quinupristin-dalfopristin m. maniati, f. kontos, p. liakos, e. petinaki, i. spiliopoulou, a. maniatis (larissa, patras, gr) objectives: to investigate the resistance mechanism of a new described phenotype among enterococcus faecalis expressing lowlevel resistance to clindamycin and dalfopristin but susceptibility to quinupristin-dalfopristin (q-d). methods: in greece, during 2005, three enterococcus faecalis isolates, expressing this unusual phenotype, were recovered from urine samples. the isolates were studied by pcr for the lsa-gene and by pfge. nucleotide sequencing analysis of lsa and 309 bp of the upstream region was performed. the isolates were also tested by rt-pcr for the expression of the lsa-gene. results: the isolates belonged to three distinct clones and carried the lsa-gene. no stop codons were found in any strain, while some point mutations in the lsa-gene were detected. comparing the lsa mrna production of these unusual strains with that obtained from fully q-d resistant ones no quantitative differences were found. conclusions: the findings of the present study clearly show that the resistance mechanism of quinupristin-dalfopristin is not only correlated with the presence and the expression of the lsagene. some mutations detected in the lsa gene probably are responsible for the production of an lsa protein with decreased activity, resulting to the q-d susceptibility. the presence of erm tr gene is responsible for the macrolide-resistance of streptococcus agalactiae objectives: to investigate the mechanism of resistance to macrolides in strains of streptococcus agalactiae in the area of thessalia, greece during the period 2000-2005. methods: the subject of this study were 147 strains of s. agalactiae which were collected from clinical specimens (90% vaginal swabs) from pregnant and non pregnant women. the strains were identified by gram stain, the lancefield b antigen, and by api strep system (biomerieux, france). susceptibility to macrolides, lincosamides and streptogrammines b was studied by the disk diffusion method. the mics were also measured by the use of e-test. the differentiation between m and mlsb inducible type was tested by the double disk synergy test (ddst). the detection of the genes mef a, erm tr, and erm b was performed by polymerase chain reaction (pcr). the clonality of the resistant strains was studied by pulse-field gel electrophoresis. results: of the 147 strains, 15 were resistant to erythromycin, lincosamid and streptogrammines b. none was found to be resistant to erythromycin only (m-phenopype). 60% of the strains were mlsb constitutive phenotype, while 40% were mlsb inducible. all strains were found to carry the erm tr gene. only one strain was found to carry both erm tr and erm b genes. pfge analysis revealed the emergence of multiple resistant clones. conclusions: the resistance of s. agalactiae to mlsb antibiotics is related with the presence of erm tr gene in central greece. emergence of novel clindamycin resistance phenotype among invasive streptococcus pyogenes isolates in sweden a. jasir, b. luca, c. schalen (lund, se) objectives: in some recent throat group a streptococci (gas) isolates from our diagnostic laboratory total resistance to clindamycin but susceptibility to erythromycin and other 14-as well as 15-membered macrolides was found. the isolates were susceptible to 16-membered macrolides and streptogramin b. these atypical strains thus did not agree with previously known mls resistance phenotypes. the main objective was to characterize theses resistance phenotype and genotypes. method and results: the isolates were examined for resistance genes by pcr. out of 14 strains one harboured an erma gene. the gene was sequenced and showed a mutation in regulatory part and was localized on a transposons. all other strains were negative for any erm genes and were also tested for 23s rrna mutations with negative outcome. strains were t-and emm typed and showed to belong to different types. conclusions: gas account for common human infections such as acute pharyngotonsillitis and impetigo, which untreated may be followed by the nonsuppurative complications rheumatic fever and acute poststreptococcal glomerulonephritis gas may also give rise to invasive, often life-threatening acute disease, such as scarlatina, erysipelas, endometritis, necrotising fasciitis and sepsis, often accompanied by toxic shock. without known exceptions, gas are fully susceptible to betalactams, which are first-choice drugs for treatment. in cases of allergy or intolerance to penicillins, macrolides are most used, and possibly as a consequence, a significant resistance development to these agents has evolved in many parts of the world. though the role of clindamycin for treatment of streptococcal disease is more limited this drug was shown to be particularly effective in eradicating streptococci after penicillin treatment failure of pharyngotonsillitis. clindamycin, often as a supplement to betalactams, also may have a life-saving effect in the treatment of fulminant streptococcal infections. due to its important role in the treatment of invasive streptococcal disease, resistance development to clindamycin in gas is considered highly undesirable. the alarming finding of a possibly new phenotype of selective clindamycin resistance in gas will motivate a thorough analysis of the phenotype as well as identification of its resistance determinants. a. al-lahham, m. van der linden, r.r. reinert (aachen, de) objectives: telithromycin is a novel ketolide antibiotic with significant in-vitro activity against streptococcus pneumoniae. the aim of this study is to characterize the resistance mechanisms of clinical isolates of s. pneumoniae with reduced susceptibility to telithromycin (>1 mg/l) and to perform the time-kill kinetics with telithromycin. methods: determination of mics was performed by the microbroth dilution method according to the clsi and the serotyping by the neufeld quellung reaction. multilocus sequence typing, sequencing of the 23s rrna, sequencing of genes encoding ribosomal proteins (l4 and l22), and ermb were performed according to standard methods. four isolates were selected for time-kill, two of which with a telithromycin mic 2 mg/l and two strains with a telithromycin mic of 8 mg/l. results: in two nation-wide studies and one european surveillance study (n = 6604) performed at the national reference center for streptococci (nrcs) in germany, reduced susceptibility to telithromycin (>1 mg/l) was detected in 17 isolates (0.3%). mic50/mic90 (mg/l) of the strains to other antibiotics were as follows: telithromycin 2/8, penicillin g 2/2, cefuroxime 8/8, erythromycin a >32/>32, clindamycin >32/>32, tetracycline 32/32, and gatifloxacin 0.25/0.25. two major serotypes were observed, serotype 14 (58.8%) and serotype 19a (29.4%). all isolates possess the cmlsb phenotype (ermb positive). the isolates showed a wide range of combinations of resistance determinants including multiple alterations in the 23s rrna (a138g, c150t, a260g, a1745t, and c2216t), a s20n alteration in the ribosomal protein l4 (n = 9), and a n100s alteration in the erm(b) gene (n = 14). the predominant clone was serotype 14 sequence type 143 (8 of 17 isolates), which was seen in france (n = 7) and germany (n = 1). telithromycin-resistance has also spread to the spain23f-1 clone (st 81; n = 1) and its serotype 19a variant. in vitro time-kill showed a minimal kill from 0-8 hours and then regrowth. bactericidal activity was achieved only with 8 times the mic in all strains. conclusions: although the incidence of telithromycin resistance remains rare world-wide, the spread of telithromycin resistance to multi-drug resistance clones with world-wide distribution is worrisome. gbs obtained from non-pregnant women. the erythromycin resistant-gbs were identified, phenotypically analysed, screened by pcr for mre(a) gene and for erythromycin resistance genes: erm(b), erm(tr), mef(a) and mef(e), and serotyped with type specific antisera for serotypes ia, ib, ii, iii, iv, and v. results: among the total of gbs, 161 (28.65%) were erythromycin-resistant: 86 (24.29%) erythromycin-resistant gbs were isolated from vaginal swabs of pregnant women and 75 (36.06%) from non-pregnant women. the frequency of serotypes in 151 erythromycin-resistant gbs tested, the distribu-tion of their resistance genes and the distribution of serotypes among the different genotypes are illustrated in the table. nt, nontypeable, the mre(a) gene was found in all the gbs strains tested. mics of erythromycin in erythromycin-resistant gbs were: mic50 and mic90, >128 mg/l; range, 4 to >128 mg/l for gbs harbouring erm(b) and erm(b)+erm(tr) and mic50 and mic90, 8 mg/l and >128 mg/l, respectively; range, 0.5 to >128 mg/l for gbs harbouring erm(tr). conclusion: erm(b) was the erythromycin-resistant gene most prevalent among the gbs isolates and these isolates showed the highest mics of erythromycin. the commonest serotypes among erythromycin-resistant gbs isolated were iii, ii and i, and showed genotypic variability harbouring either of the two most prevalent genes, erm(b) or/and erm(tr). methods: we studied the rates of resistance to tetracycline and minocycline among 161 erythromycin-resistant gbs strains isolated at the university hospital lozano blesa of zaragoza, spain. isolates were subsequently phenotypically analysed by means of the disk diffusion method and screened by pcr for erythromycin and tetracycline resistance genes [erm(b), erm(tr), mef(a/e), tet(m) and tet(o)]. the susceptibility to erythromycin, josamycin, tetracycline and minocycline was tested by the agar dilution method according to the nccls. the strains were serotyped with type specific antisera for serotypes ia, ib, ii, iii, iv, and v. results: among the total of 161 isolates of macrolide-resistant gbs collected from may 2002 to april 2004 in our hospital (28.6% of the total sgb isolated), 124 (78.85%) were tetracyclineresistant. the distribution of tet(m) and tet(o) among the erythromycin-resistant gbs harbouring erm(b) was (66.1% and 54.2%, repectively) and harbouring erm(tr) was (63.8% and 34.5%). the distribution of tetracycline resistance genes and serotypes among the different genotypes in 121 gbs are illustrated in the table.*nt: non typable isolates carrying tet(m) or tet(m)+tet(o) presented the following mics: tetracycline (mic90 64, mic50 32, range 16-128 mg/l); minocycline (mic90 32, mic50 16, range 4-32 mg/l). isolates carrying tet(o) presented the following mics: tetracycline (mic90 64, mic50 32, range 32-64 mg/l); minocycline (mic90 16, mic50 16, range 4-32 mg/l). conclusion: the majority (61.1%) of tetracycline-resistant gbs harboured tet(m) alone or in combination with tet(o). the most prevalent serotypes among the total of tetracycline-resistant gbs was the serotype iii (38%) and the serotype ii (28%). serotype iii was more prevalent among the gbs harbouring the tet(m) gene and serotype ii was more prevalent among the gbs harbouring the tet(o) gene. objectives: to know the prevalence of resistance to macrolides in viridans streptococci, its mechanism and the genetic elements which are involved. methods: we studied 89 viridans streptococcus pharyngeal isolates from different patients. mics for macrolides were determined by the agar dilution method. the presence of mef and erma, ermb and ermtr genes, and the presence of mega (macrolide efflux genetic assembly) or tn1207 in resistant, mef + isolates was determined by pcr with specific primers. the similarity to mef genes first described in pneumococci (mefe) and streptococcus pyogenes (mefa) was determined by sequencing. results: 48 viridans streptococci isolates were resistant to macrolides (53.9%). 42 out of the 48 resistant isolates harboured mef genes (87.5%), one harboured ermb (2.1%), and 5 isolates harboured both mef and ermb genes (10.4%). no isolates harboured erma or ermtr genes. we studied genetics elements which harbour mef genes in other streptococci, in 15 mef (+) isolates. we found mega insertion element in 14 of 15 isolates (93.3%), all of them harbouring mefe. the only isolate in which we found mefa, did not harbour mega, but tn1207. conclusions: m phenotype is frequent in viridans streptococci, and all of them harbour mef genes. most mlsb phenotype viridans streptococci do not harbour erm genes alone; most of them combine erm and mef genes. most isolates contained the mef sequence corresponding to mefe, and the genetic element (mega) usually described in pneumococci as harbouring this gene. one isolate contained the sequence corresponding to mefa, and the genetic element usually described in s. pyogenes (tn1207). the increasing presence of macrolide-resistant pneumococci harbouring mega element might be related with its wide presence in viridans streptococci. the acquisition of mega by pneumococci from viridans streptococci through transformation is being studied. objectives: the principal mechanism of macrolide resistance in streptococcus pneumoniae in italy is target site modification mediated by erm(b). the erm(a) gene is common in streptococcus pyogenes but rare in s. pneumoniae, even if recent studies have demonstrated an increased detection of this resistance determinant. recently, a clinical s. pneumoniae isolate carrying erm(a) has been obtained from a patient with meningitis in italy. the aim of this study is the molecular characterization of this isolate. methods: antimicrobial susceptibility tests were determined by etest. the presence of erythromycin resistance determinants was detected by pcr assays. genotyping was performed by pfge and mlst. the flanking regions of erm(a) were analysed by sequencing a fragment amplified by inverse pcr. transformation and conjugation experiments were carried out. transformants were analysed by pfge and hybridization with an erm(a) probe. results: the isolate belonging to serotype (st) 11a, was resistant to erythromycin, inducibly resistant to clindamycin and susceptible to penicillin and tetracycline. by pcr, the only macrolide resistance determinant detected was erm(a). pfge analysis revealed the genetic correlation of this strain with other st 11a s. pneumoniae italian isolates. mlst confirmed this data since the isolate belonged to st2003, which is a single-allele variant of st62, the most common st among italian st 11a isolates. a 5200 bp dna fragment, obtained by inverse pcr and containing the erm(a) gene, was sequenced. this fragment contains an orf upstream erm(a), the gene erm(a) identical to that described in s. pyogenes and 3 orfs, downstream erm(a), one homologous to a hypothetical kinase and the other two to transposases of other gram-positive species. in transformation experiments the gene erm(a) was transferred to an erythromycin susceptible recipient. hybridization analysis of one transformant revealed that the size of the transferred dna fragment was approximately 50 kb. no transconjugant was obtained in mating experiments. conclusions: this is the first italian report of an s. pneumoniae isolate carrying erm(a). erm(a) appears to be contained in a genetic element that includes two transposases, although the gene is not transferable by conjugation. objective: treatment with the first oxazolidinone antibiotic, linezolid, of infections caused by staphylococci has proved effective in most cases. in the present study we present the first three cases of linezolid resistant staphylococci in our hospital. methods: we examined three coagulase negative staphylococcus strains isolated from blood cultures (bactec, becton dickinson). identification and susceptibility testing were performed by the vitek ii automated system (biomerieux) and the results were confirmed by the api system (biomerieux) and e-test (ab biodisk, solna, sweden), according to nccls guidelines. results: three linezolid resistant staphylococci were isolated from blood cultures. identification showed that all three isolates were staphylococcus cohnii subsp. urealitycum and the mic values were 12 lg/ml (n = 1) and 24 lg/ml (n = 2) which are much higher than the value of 4 lg/ml that characterizes sensitive strains. the isolates derived from three patients in different wards of our hospital. the first two isolates were recovered from two icu patients in april and august 2005 and the last staphylococcus cohnii was isolated from a patient in the neurosurgery ward, who is still hospitalized. all patients received prolonged treatment with linezolid. conclusions: although six linezolid resistant clinical isolates of s. aureus were previously reported in the literature, these three isolates are the first coagulase negative staphylococcus isolates resistant to linezolid. it is imperative to screen for resistance to linezolid all staphylococci and take the necessary precausions in order to prevent the spread of a linezolid resistant strain in other wards of our hospital. correlation between mic and number of mutated 23s rrna genes in oxazolidinone-resistant staphylococcus aureus objectives: to determine the number of mutated 23s rdna alleles present in clinical and laboratory-generated linezolidresistant staphylococcus aureus isolates. methods: 36 linezolid-resistant isolates were tested, 9 of them clinical isolates (mics 16-32 mg/l) and 27 mutants selected in-vitro (mics 8-64 mg/l). the mutants were raised by repeated passage on increasing linezolid concentrations and their parentage was verified by pfge. mics were determined by agar dilution. genomic dna was digested with ecori and hybridized with a 420 bp probe corresponding to domain v of the genes encoding 23s rrna, to determine gene copy number. pyrosequencing was used to quantify the proportions of wildtype and mutated alleles present; assays were designed to detect the presence of 7 mutations conferring oxazolidinone resistance. pyrosequencing and hybridization data were combined to determine the number of mutated alleles present. results: resistance selected in-vitro proved less stable than that in the clinical isolates. pyrosequencing showed that all 9 clinical isolates had the g2576t mutation, 16 of the in-vitro selected mutants had g2576t, 7 had t2504c, 2 had t2500a, 1 had a2503g and 1 had g2505a. the 23s rdna copy number in the oxazolidinone-resistant clinical isolates varied from 4-6, and from 5-9 in the laboratory-generated mutants; 14/27 laboratoryselected mutants had changes in copy number, compared with their parent strains, and 3 had changes in fragment size, but not number. the number of mutated copies in lin-resistant isolates ranged from 1-5 in laboratory-selected mutants and from 2-4 in clinical isolates. an increasing number of mutated genes correlated with increasing linezolid mic. conclusions: in combination, pyrosequencing and hybridization successfully determined the number of mutated 23s rdna alleles. exposure to linezolid selected changes in 23s rrna gene copy number as well as sequence in 50% of in-vitro selected mutants. there was a positive correlation between both the number and proportion of mutated 23s rdna copies and mic, previously unproven for staphylococci. objectives: linezolid (lzd) is an important antibiotic for the treatment of enterococcal infections, especially when the corresponding strain possesses multiresistance including resistance to vancomycin (van). we report the emergence lzd resistance in clonally related van-susceptible and vanresistant enterococcus faecium isolates originated from an icu patient only 12 days after initiation of linezolid therapy. patient and methods: van-resistant e. faecium was repeatedly isolated from intraabdominal cultures of a 76-year-old female icu-patient with infected necrotizing pancreatitis after pancreaticoduodenectomy (whipplés procedure). antibiotic susceptibility testing of the bacteria was performed by e-tests; vana gene were detected by pcr. the possible lzd resistance mechanism (mutation in the 23s rdna of one or more of the six 23s rrna alleles of e. faecium) was examined by a pcr-based method. molecular typing of the strains was performed by smai macrorestiction analysis. results: van-resistant but lzd-sensitive e. faecium (vrlse) were initially detected in intraabdominal cultures, however, already twelve days after initation of lzd therapy, van-and lzd-resistant e. faecium (vrlre) strains were detected. resistance to lzd was confirmed: mics ranged from 16 to 32 mg/l. all e. faecium isolates showed identical or closely related pfge patterns. throughout the icu period, van-and lzd-susceptible e. faecium (vslse) strains were repeatedly detected in the same specimens from which the vrlse and vrlre were isolated. additionally, van-susceptible e. faecium isolates with resistance to lzd (vslre) were detected. mutations in the 23s rdna of three out of six alleles led to lzd resistance in the e. faecium isolates examined. two weeks after termination of the lzd therapy, no lzd-resistant strain could be detected in follow-up swabs. conclusions: resistance to lzd in e. faecium can occur already shortly after the initiation of lzd therapy. assessment of antibiotic susceptibilities of all isolates at the start of therapy and regularly during the therapy is advisable, especially during therapy of severe infections. the epidemiological and clinical repercussions of resistance to lzd among enterococci cannot be predicted at this time. attention to proper dosing and prompt removal of infected devices, when feasible, could limit occurrence and spread of lzd-resistant e. faecium. objectives: to investigate the mechanisms of resistance to tetracycline in shigella spp. methods: one hundred and eleven tetracycline-resistant shigella spp strains (67 s. sonnei, 44 s. flexneri), 6 were isolated as a cause of enteritis in our geographical area and the remaining recovered from patients with traveller's diarrhoea. antimicrobial susceptibility to tetracycline was determined by the kirby-bauer method. presence of teta, tetb and tetg genes was established by pcr. sequencing of amplified products were used to corroborate the reliability of the pcr results. maentel haenszel test was used to establish the statistical significance. results: the statistical analysis showed that the teta gene was more frequent in s. sonnei (p < 0.05), while tetb was more usual in s. flexneri (p < 0.0005). although without statistical significance (p:0.07), presence of non-determined mechanisms of tetracycline-resistance seems to be more frequent among s. sonnei. conclusions: species-specific differences in the distribuition of the teta and tetb genes has been shown. moreover 22.5% of the analysed strains did not show any of the analysed determinants of tetracycline-resistance. the concomitant presence of more than one of the analysed genes is a rare event. distribution and genetic determinants of tetracycline resistance in laribacter hongkongensis isolates from humans and fish objectives: to study the distribution of tetracycline resistance and to clone and characterize a tetracycline resistance determinant in laribacter hongkongensis, a recently discovered bacterial genus and species associated with communityacquired gastroenteritis. methods: twenty-four l. hongkongensis strains isolated from patients with community-acquired gastroenteritis and 24 l. hongkongensis strains isolated from freshwater fish in hong kong were used in this study. genetic determinants for tetracycline resistance were looked for by screening a genomic dna library of l. hongkongensis. the prevalence of teta gene in other strains of l. hongkongensis was studied by pcr using laboratory-designed primers. the presence of the tetracycline resistance determinants in plasmid was examined by southern blot analysis. results: among 24 human and 24 fish isolates tested, 3 human and 1 fish isolates were tetracycline-resistant. a 3566-bp gene cluster, which consists of 2 putative transposases, a tetr and a teta gene, was cloned by inserting restriction fragments of genomic dna from a resistant strain, hlhk5, into pbk-cmv. the 1266-bp teta and 636-bp tetr genes shared significant nucleotide sequence homology with known teta and tetr genes. while the flanking regions and 3' end of the teta were identical to the corresponding regions of a tetc island in chlamydia suis, the teta was almost identical to that of transposon tn1721 and plasmids found in many gram-negative bacteria, suggesting that illegitimate recombination may have occurred to produce the present tetracycline resistant determinant. southern hybridization suggested that the teta gene of hlhk5 was plasmid-encoded. the tetracycline resistance in l. hongkongensis was associated with teta. pcr amplification of the teta gene in the 48 isolates of l. hongkongensis, including hlhk5, showed the presence of teta in all the four tetracycline resistant isolates but none of the tetracycline susceptible ones. in contrast to strain hlhk5, the teta of two strains were identical to that of tn1721, while that of the other strain was more closely related to other gram-negative bacteria plasmids. conclusion: our results indicate that horizontal transfer of genes, especially through tn1721 and related plasmids, between l. hongkongensis and other gram-negative bacteria is probably a frequent event and is an important mechanism for acquisition and dissemination of tetracycline resistance in l. hongkongensis. succesful treatment of infective endocarditis with linezolid t. hryniewiecki, u. lopaciuk, j. stepinska (warsaw, pl) objectives: there is an increasing proportion of resistant strains causing infective endocarditis in recent years. it has changed the approach to choice of antibiotic therapy. linezolid (zyvoxid ò ) is a new bacteriostatic antibiotic with a wide spectrum of activity against gram-positive organisms and with good efficacy in experimental animal models of endocarditis. unfortunately clinical experience with linezolid in the treatment of endocarditis is limited. the aim of the study was to observe efficacy of linezolid in the treatment of infective endocarditis. methods: the study group consisted of 8 patients hospitalised in institute of cardiology in warsaw (2003 warsaw ( -2005 due to clinically resistant infective endocarditis. the diagnosis of endocarditis was established according to the duke criteria by clinical examination, echocardiography, laboratory investigations and positive blood cultures with vancomycin mic estimation (in 5 pts). all patients were treated surgically (valve replacement, artificial material removal) in conjunction with different conventional antibiotics and afterwards with 600 mg of linezolid every 12 hours intravenously. results: infective endocarditis was diagnosed as caused by mrcns in 2 pts, mssa in 1 pt, enterococcus faecalis in 1 pt and staphylococcus epidermidis mr in 1 pt. vancomycin mic vary from 2 to 4 mg/l. in 3 pts culture-negative endocarditis was diagnosed. all patients were treated with linezolid intravenously 2 to 4 weeks (average 3,1). clinical response and eradication of bacteremia were achieved in all patients. leukopenia nad thrombocytopenia as an adverse reaction occurred in 1 patient. conclusions 1. linezolid is effective in patients with grampositive endocarditis. 2. linezolid could be also effective in some patients with culture-negative endocarditis. 3. linezolid may provide an alternative in the treatment of infective endocarditis due to multi-resistant bacteria, in patients with resistant course or with adverse reaction to conventional antibiotics. objectives: to evaluate the safety and efficacy of lzd in a chinese population. methods: this randomized, double-blind, multi-centre study was conducted in china. after obtaining written informed consent, patients from 18 to 75 years of age with pneumonia (pneu) or skin and soft tissue infection (ssti) known or suspected to be caused by a gram-positive pathogen were randomized 1:1 to receive either lzd, 600 mg, or vancomycin (van), 1 g, each given iv q12h. patients were to be treated for 7 to 21 days, and outcomes were assessed at end-of-treatment (eot) and follow-up (f-u) visits. results: one hundred forty-two patients were enrolled and received study medication, 80 with pneu and 62 with ssti. clinical assessments (effective = ''cured'' plus ''marked improvement'') for patients in the fully evaluable population are summarized in the table. the most frequently isolated pathogen was staphylococcus aureus: all isolates were susceptible to both study drugs. the eradication rates for all pathogens in evaluable patients at the f-u evaluation were 54/58 (93.1%) in lzd-treated patients and 42/56 (75.0%) in van-treated patients (p = 0.0072). all patients receiving study drug were evaluated for safety. drug-related adverse events (aes) were reported in 18 (25.4%) lzd-treated and 12 (16.9%) van-treated patients. the most commonly reported drug-related aes in lzd-treated patients were mild abnormalities in liver function tests and leucopenia (4.2% each); rash (7.0%) was the most commonly reported ae in van-treated patients. seven (9.9%) lzd-treated and 10 (14.1%) van-treated patients discontinued study drug because of an ae. conclusions: linezolid is an effective drug for the treatment of infections caused by gram-positive pathogens and is welltolerated. eradication in one patient, by rifamycinlinezolid, of a methicillin-resistant staphylococcus aureus producing panton-valentine leukocidin, responsible for 7 relapses over 18 months, and decolonisation of her family by mupirocin objective: we report the case of the mother who experienced 7 relapses over the period. methods: pvl-mrsa were isolated on routine and mrsa agars (biorad). antibiotypes were studied by disk diffusion method. genetics and pulsotypes were studied by the french centre national de référence des staphylocoques in lyon (cnr). results: mrs kym had her 4th child on october 8, 2003 in the hospital of orléans. she was healthy and presented no risk factor for delivery. three weeks later she was addressed for surgical treatment of an abscess on buttocks. cultures yielded the special antibiotype: methicillin-r, kanamycin-r and tobramycin-s, of the pvl-mrsa currently spreading across europe and maghreb. the cnr found the luks-pv and lukf-pv-genes.through march 2005, she relapsed 7 times and was treated by pyostacin for a total of 32 weeks. two of her children were addressed for abscesses (1 buttocks, 1 thumb) yielding the same bacteria. in march 2005, mrs kym was addressed to the infectious diseases ward because of nasal furonculosis. samples yielded a pvl-mrsa with mls-b phenotype. treatment by rifamycin-linezolid 14 d was initiated. the whole family was screened. the father and the boy, 9, who had the infected thumb 17 months earlier, were carriers. the girl, 12, who had an abscess on buttocks 8 months earlier was not. in april the whole family accepted an attempt for decolonisation by 2% nasal mupirocin 2 or 3/d for 5 d. cure and decolonisation were confirmed by nares and cutaneous folds samples in may and june. they missed an additional appointment in the beginning of term, but a phone call to the social worker confirmed none of them relapsed. the cnr studied 7 strains (3 from mrs kym 2003 , 2005 , 2 from children abscesses in 2003 and 2004, 2 from boy's and father's nares 2005), and confirmed that they were all identical along the period and across the family. conclusion: short treatment with linezolid-rifamycin in the relapsing case associated with familial decolonisation by nasal mupirocin was an effective strategy to stop a time-prolonged familial outbreak of pvl-mrsa infection. multiple brain abscesses and purulent meningitis by listeria monocytogenes in an otherwise healthy man. favourable linezolid response, hampered by a suspected early drug myelotoxicity introduction: l. monocytogenes cns infection in immunocompetent adults remains rare. meningitis is the most common cns manifestation, with brain abscesses being <1% of overall episodes. anecdotal episodes of cns l. monocytogenes infection were reported from immunocompetent patients where both diagnosis and treatment may be hampered by low clinical suspicion and a frequent non-specific presentation. in a 15-yearlong survey conducted in dallas (us), only 4 cases of nonneonatal l. monocytogenes meningitis were found (estimated incidence rate: 0.3%). case report: a 50-year-old male with a negligible history and no obvious exposure to l. monocytogenes was hospitalized owing to dizziness. a brain ct scan showed a small, late ischemic lesion. a few days later hyperpyrexia, headache, vomiting and altered mentation occurred. the csf study detected an elevated albumin content (217 mg/dl), low glucose (4 mg/dl) and a wbc count of 256 cells/ll (60% neutrophils) so that ceftriaxone-chloramphenicol were immediately started. clinical-neurological conditions deteriorated while l. monocytogenes was cultured from the csf so that treatment was changed towards high-dose ampicillin-gentamicin. the persistence of severe clinical-neurological conditions and altered csf assay prompted the introduction of rifampicin-cotrimoxazole after 10 days, but 8 days later other focal neurological deficits appeared and a mri showed small, hyperintense focal lesions involving the medulla oblongata, interpreted as multiple abscesses. the introduction of linezolidmeropenem, despite anemia (requiring 2 rbc transfusions after 7-14 days) led to a progressive clinical-csf improvement. our patient recovered completely and a control mri carried out 1 month after discharge confimed the complete disappearance of the multiple brain listeria abscesses. discussion: the l. monocytogenes meningitis and multiple subtentorial abscesses (including rare localizations at cerebellum, bulb, and pons varolii), had an evolving cumbersome presentation. despite the in vitro activity of a broad spectrum of agents, multiple therapeutic changes became necessary, until the last linezolid-meropenem combination, which was proved very effective, although it was affected by relapsing anemia probably attributable to linezolid. linezolid, due to its elevated csf-brain penetration, and its activity against a broad spectrum of cns pathogens (including the intracellular l. monocytogenes), is expected to become a key antimicrobial compound, waiting for rct. discrepancy between favourable in vitro microbiological data and a severe clinical course of a staphylococcal knee and soft tissue infection responsive to oxazolidinone linezolid only after failure of all other therapeutic attempts introduction: to offer therapeutic alternatives for the emerging, multiresistant, serious gram-positive infections, novel molecules (quinupristin/dalfopristin, linezolid, daptomycin) were introduced and are made available when multiresistant gram-positive cocci are documented as no more susceptible to all available drugs including glycopeptides. however, inezolid encompasses unique tissue penetration and diffusion features (regarding soft tissues, lungs, joints and central nervous system) which make this last drug extremely promising in all circumstances where the penetration rate into infectious foci becomes critical. clinical experience: a very intriguing case report of a severe, staphylococcal knee arthtiris associated to an extensive local cellulitis/fasciitis and haematogenous dissemination occurring after a surgical curettage was characterized by a complete lack of response to a prolonged vancomycin/teicoplanin plus rifampicin therapy based on the apparently favourable in vitro sensitivity assays of methicllin-resistant staphylococci, but rapidly responded to i.v. (followed by oral) linezolid administration. the complete lack of clinical activity of a 2-week glycopeptiderifampicin administration cannot be explained by the in vitro measured mic90 values of isolated pathogens which showed complete sensitivity of staphylococcus aureus against vancomycina/teicoplanin and rifampicin and susceptibility of a concurrent hematogenous s. epidermidis strain to glycopeptidesrifampicin. since an abscess formation and an underlying osteomyelitis were carefully excluded by adequate instrumental examinations, from a theoretical point of view the active glycopeptide-rifampicin molecules should have been provided appropriate cure. on the other hand, from a strictly clinical issue, only a 2-week administration of i.v. linezolid followed by one more week of oral linezolid allowed to obtain a complete clinical-bacteriological cure and a complete function recovery without any sequelae after a 1.5-year follow-up. conclusions: when the management of severe, multiresistant gram-positive infections is of concern, the in vitro activity of single drugs and therapeutic classes should be carefully evaluated in relation with the expected penetration and diffusion rates of these drugs into the relevant organs and tissues involved by the ongoing infectious localizations. otherwise, apparently unexplained failures may occur also when in vitro studies point out a complete activity of the tested compounds. epidemiology of resistance to antibiotics -ii p1610 contemporary prevalence of bro betalactamases in m. catarrhalis: report from the sentry antimicrobial surveillance program (usa; 1997 l. deshpande, h. sader, r. jones (north liberty, us) objectives: to evaluate the prevalence of bro-1 and bro-2 among b-lactamase (bl)-producing m. catarrhalis (mcat) in the usa. although the bl-mediated penicillin (pen) resistance (r) in mcat has been stable at 95%, the bro-1 and -2 occurrence has not been determined in usa isolates since 1998. bro-2 rates have been reported at <15 (1980s), 4.8 (1984-94), 2.1 (1994-95) and 3.1% (1997-98) . methods: community-acquired mcat isolates (sentry program 1997 were tested by clsi broth microdilution methods including: 7,860 worldwide and 3,671 in north america (na). bro-1 and -2 was detected by pcr methods (levy and walker; 2004), compared to epidemiologic tests, and mic values. 100 b-lactamase-positive (bl+) mcat samples per year from usa (39 sites) and canada (ca; 7 sites) were tested for the odd-numbered years. results: the bro-2 rate was 4, 4, 3, and 3% for 1997, 1999, 2001 and 2003, respectively; rates in ca (8 isolates) > usa (6). several agents remained active: amoxicillin/clavulanate (mic90, £0.25 mg/l), ceftriaxone (ctri; 0.5), cefuroxime (2), erythromycin (£0.25-0.5), levofloxacin (£0.03-0.06), tetracyclines (2) and trimethoprim/sulfamethoxazole (tmp/ smx; £0.5/9.5). pen mic distribution was tri-modal (£0.03, 1-2, >4 mg/l) and ctri bi-modal (0.016, 0.5), yet bro-1 and -2 mic/zone distributions overlap (best discriminated by methicillin (mean zone, 10.6 vs. 19.4 mm) and pen (13.9 vs. 24.1) disks). possible bro-2 epidemic clusters could not be excluded due to a very common ribotype in 3 centres (ca, 2 sites; usa, 1). conclusions: this bro-1 and -2 enzymes na prevalence update in mcat isolates (1997) (1998) (1999) (2000) (2001) (2002) (2003) shows stability at 96-97% and 3-4%, respectively. phenotypic tests (zones or mics) cannot easily distinguish between these b-lactamase types, necessitating the use of molecular applications. objective: although resistance to penicillin in beta haemolytic streptococci has not been reported yet, increasing resistance rates for alternative drugs, such as erythromycin, clindamycin or tetracycline is an emerging concern which brings the necessity to carefully monitor penicillin susceptibility. materials and methods: in order to detect any changes in penicillin mics, we performed antimicrobial susceptibility testing for all isolated beta haemolytic streptococci in our hospital between january 2000 and november 2005. identification to serogroup level was done using a commercial latex agglutination kit (avipath strep, omega diagnostics ltd., scotland, united kingdom). results: a total of 1890 isolates were identified, distribution of groups for serogroup a, b, c and g were 75.6%, 18.9%, 2.8% and 2.7%, correspondingly. penicillin susceptibility was determined using etest (ab biodisk solna, sweden) strips according to manufacturers' instructions. when results are evaluated in 2year periods, mic90 increased from 0.012 to 0.016 mg/ml for group a, from 0.064 to 0.094 mg/ml for group b, from 0.012 to 0.064 mg/ml for group c and from 0.016 to 0.023 mg/ml for group g (table) . conclusions: even though highest mic90 values were to be found in group b (0.094 mg/ml), our results indicate the steady increase in penicillin mic for all serogroups. three group a and six group b isolates with penicillin mic of 0.125 mg/ml, reaching susceptibility breakpoint concentration according to clsi, and also highly elevated mic90 concentrations for group b streptococci may be messengers of possible forthcoming resistant strains. objective: to study trends in macrolide resistance rates among s. pneumoniae isolated from children aged 3 to 40 months attending day-care centres in france following implementation of prudent antibiotic use campaigns (alpes maritimes 2001 , france 2003 and pneumococcal conjugate vaccine (pcv) (2003) . method: nasopharyngeal aspirates were obtained from a random 2-stage cluster sample of children attending day-care centres in the nord (n) and alpes maritimes (am) areas during 3 consecutive surveys between january and march 1999 march , 2002 march and 2004 . susceptibility to erythromycin and clindamycin and resistance phenotype were analysed by disk diffusion method. serotypes were determined using the quelling reaction. pneumococcal immunization status and antibiotic prescriptions over the 3 previous months were recorded. results: sp was isolated from 278/548, 289/534 and 269/567 children in 1999, 2002 and 2004 , respectively (p < 10 )3 ). resistance to macrolides declined overall from 77.0% to 64.3% of strains between 1999 and 2004 (p < 10 )2 ). among erythromycin-resistant (e-r) isolates, percentage of erm-b phenotype increased from 81.0% to 90.2% (p = 0.001). while the proportion of penicillin non-susceptible strains declined from 33.6% to 24.7% of sp isolates (p < 10 )3 ), erythromycin resistance remained stable among these strains at 92.0%. overall proportion of treated children fell from 64.3% to 48.7% (p < 10 )3 ) between 1999 and 2004; in am this reduction was observed in 2002 (16.0%; p < 10 )3 ), while in n it occurred in 2004 (24%; p < 10 )3 ) and the percentage of macrolides among prescriptions fell from 13.0% to 9.0% (v 2 for trend: p = 0.06). serotype distribution showed most e-r isolates were 6b, 14, 19f and 23f. a 50% reduction in serotype 23f was observed in am in 2002 and in n in 2004. immunisation with pcv concerned at least 33.5% of children in 2004. conclusion: macrolide resistance has followed a parallel decline with penicillin resistance as a result of antibioticprescription reducing campaigns and pneumococcal immunization against the most prevalent macrolide-resistant serotypes. objective: to evaluate the prevalence of resistance of invasive strains of s. pneumoniae to erythromycin after decline in macrolide consumption. methods: the number of packages of antibiotics was obtained from the institute of public health of slovenia. for the period 1999-2004 the data on outpatient antibiotic consumption were collected using the atc/ddd classification (who version 2005) and the results were expressed in ddd/1000 inhabitants per day (did). all invasive strains of s. pneumoniae isolated from sterile body fluids in all slovenian hospitals were included in the study. susceptibility testing was performed using nccls approved disk diffusion test. results: during 1999-2004 the total use of antibacterials in slovenia decreased for 17.4% from 20.23 did to 16.71 did. the consumption of macrolides which constituted 17.1-19.4% of total use of antibacterials decreased for 21.3% (3.81 to 3.0 did). short-acting (erythromycin, miocamycin), intermediate-acting (midecamycin, roxithromycin, clarithromycin), and long-acting (azithromycin) decreased for 33.4%, 25.8% and 13% respectively. in all years the use of intermediate-acting macrolides was the most prescribed subclass of macrolides corresponding for 1.50-2.21 did, followed by long-acting (1.25-1.47 did) and short-acting (0.07-0.12 did). the resistance of s. pneumoniae strains to erythromycin increased from 5.4% (6/110) to 11.2% (19/170); in children from 2.9% (1/ 34) to 18.1% (6/33) and in adults from 6.5% (5/76) to 9.5% (13/137) respectively. rates of the isolates resistant to erythromycin and at least 2 of the following agents: penicillin, tetracycline, tmp/smx, chloramphenicol increased from 0.9% (1/110) to 7.7% (13/169); in children from 0% (0/ 34) to 15.1% (5/33) and in adults from 1.3% (1/76) to 5.8% (8/ 137) respectively. conclusion: despite a reduction of macrolide consumption in outpatients the resistance of invasive strains of s. pneumoniae was increasing during the observation period especially in children. multiple drug resistance explains best the changes in s. pneumoniae resistance in a ten-year surveillance study in belgium objective: belgium is located between countries with very high and very low antibiotic resistance rates. modeling how resistance changes over time and place in belgium provides insights into correlates of s. pneumoniae resistance at the population level. methods: surveillance data consists of 14,488 s. pneumoniae invasive isolates from 1994-2004, identified by postal code as well as clinical and demographic information. antimicrobial consumption (ims health services) is expressed in defined daily doses (ddd) per 1000 inhabitants per day. changes in resistance by month and postal code were evaluated using mixed effects models for repeated measures, using mathematical models of transmission for the curve shape, and taking into account seasonality. resistance to penicillin, erythromycin, tetracycline, and ofloxacin was considered in the analysis. results: resistance to penicillins, macrolides and tetracyclines peaked in the year 2000, and their levels in 2004 were 11.6%, 35.6% and 28.7% respectively. the shape of the curves is similar for most of the antibiotics studied, with a steep rise from 1994 to 2000 and a plateau thereafter. resistance to two or more antibiotic classes corresponded to 72% of all resistant isolates and in a multivariate model explains most of the variability through time and place of the antibiotics studied. resistance to only one antibiotic (any) decreased from 16.3% in 1994 to 11.7% in 2004, while resistance to two or more increased 2.5 times (95% ci 1.95-3.12, p < 0.0001) from 14.1% in 1994 to 28.8% of all isolates 10 years later. more than nine out of ten isolates that were macrolide or tetracycline resistant were also multiply resistant (mr). mr increases 0.57% for each ddd of overall cumulative antimicrobial consumption, and out of all antibiotic classes, macrolides and broad-spectrum penicillins are most associated with resistance. conclusion: resistance to two or more antibiotics is the most important factor in understanding the changes over time for all studied antibiotic classes in belgium. the cumulative impact of antimicrobial exposure of separate antibiotic classes at the population level facilitates the survival and transmission of any isolate that is resistant to two or more antibiotic classes. methods: the isolates were identified by biochemical tests and specific serotyping. antimicrobial susceptibility to ampicilllin (amp), amoxicillin plus clavulanic acid (auc), cloramphenicol (cm), gentamicin (gm), cotrimoxazole (sxt), nalidíxic acid (nal) and tetracycline (tc) were established by the method of kirby bauer. the presence of beta-lactamases encoding genes (tem, carb, oxa2-like) as well as the teta, tetb, tetc, tetg, cmla and flor genes, and integrons type 1 was established by pcr, while the presence of plasmid-mediated dhfr was determined by pcr-rflp and the cat activity by a colorimetric assay. results: seven different resistance patterns were identified: i. susceptible (38 strains); ii. amp, sxt, gm, a/c (37); iii. amp, tc, cm (2); iv. amp, tc, sxt, cm (2); v. amp, a/c (4); vi. amp, sxt, a/c (1); vii (1). -sxt. no isolate resistant to nalidixic acid was detected. resistance to beta-lactam agents was due to the presence of beta-lactamases type tem-like (pattern v), carb-2 (iii) and tem-like plus oxa-30 (ii, v, vi). meanwhile resistance to cloranphenicol and tetracycline was associated to cat activity (iii, iv) and flor (iii), and tetb (iv) and tetg (iii) respectively. no mechanism of cotrimoxazole resistance was detected in the isolates of the patterns ii, vi and vii, while dfra1 was detected in the isolates of the group iv. resistance to gm was associated to the presence of the gene aadb, detected in the analysis of integrons type 1. type 1 integrons were detected in isolates belonging toi the pattern ii (750 bp -aadb; 2000 bp -oxa30, aada1), iii (900 bp -carb2, 1100 -aada1), iv (1500 bp -dfra1, aada1), v and vi (2000 bp -oxa30, aada1). conclusions: a great diversity of resistance mechanisms has been detected. those mechanisms might spread among microorganisms resulting in a serious health problem due to the limited number of antibiotic treatments available in the area. small outbreaks of veb-1 esbl producing acinetobacter baumannii in belgian nursing homes and hospitals through cross-border transfer of patients from northern france methods: from 01/04 to 03/05, all belgian acute hospitals were invited to report cases of nosocomial infections/colonisations due to mdr ab isolates presenting a resistance profile similar to the french epidemic strain (resistance to all agents except carbapenems and colistin) and to send such isolates to the reference laboratory for phenotypic confirmation and for genotypic characterization (pcr of veb-1 and class 1 integron, pfge typing). guidelines for detection of the epidemic strain, screening for carriage in patients transferred from hospitals or nursing homes (nh) close to the french border as well as infection control measures were sent to all hospitals. results: overall 73 ab strains from 30 hospitals were sent to the reference laboratory. only, 26 of these fulfilled the phenotypic resistance patterns and 6 were definitely confirmed as veb-1 ab and had a pfge pattern identical to the french epidemic clone. two mini-outbreak clusters (each involving 3 cases) were documented in hospitals from two cities (tournai and chimay) closed to the french border. two patients died from their infection. in the first outbreak, all 3 patients were residents who lived in the same nh. two of them were french citizens who had been hospitalised in different acute care hospitals in the north of france within the last year. in the second outbreak, the index case had also been previously hospitalised in a french hospital. secondary transmission to two other hospitalised patients occurred in this outbreak. conclusion: despite the large extension of the veb-1 ab outbreak in france no similar problem occurred in belgium. however, this national alert allowed to detect two small outbreaks in belgian institutions located close to the french border. in both outbreaks the epidemic strain was imported from france through patient circuits. this study illustrates that transfers between acute care hospitals and nh may explain cross-border spread of multi-resistant epidemic strains. types of extended-spectrum beta-lactamases in salmonella spp. and decreased susceptibility to fluoroquinolones objectives: the aim of this study was to determine the rate of esbl production in clinical isolates of salmonella spp. and to detect decreased susceptibility to fluoroquinolones in esbl positive isolates in turkey. methods: a total of 620 salmonella spp. isolated from clinical samples from thirteen centres between 2000 and 2002 were included in the study. in vitro susceptibility to ampicillin, amoxicillin/clavulanic acid, cefotaxime, gentamicin, chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole and ciprofloxacin were determined using the agar dilution method on mueller-hinton agar following the clinical and laboratory standards institute (clsi) guidelines. decreased susceptibility to ciprofloxacin was defined as an mic of 0.125-1 mg/l. salmonella isolates were screened for esbl production by double disk synergy method using amoxicillin/clavulanic acid, cefotaxime and ceftazidime disks. types of esbl enzymes were analysed by pcr for tem, ctx-m, shv and per-1 genes. results: in salmonella spp. the highest level of resistance was observed against ampicillin (41.3%) followed by chloramphenicol (36.6%), tetracycline (34.2%) amoxicillin/ clavulanic acid (32.4%), trimethoprim/sulfamethoxazole (2.3%), gentamicin (1.6%), and cefotaxime (0.8%). ciprofloxacin resistance was observed in one isolate (0.2%). among 620 salmonella isolates, 6 (0.97%) were shown to produce esbl by double disk synergy testing. these isolates were salmonella typhimurium (n = 2), serogroup c1 (n = 2) and salmonella enteritidis (n = 2). three isolates were from fecal samples two were from urine and one was from blood. one of the esbl producing isolates were susceptible to cefotaxime in vitro. two isolates showed decreased susceptibility to ciprofloxacin. all the esbl producers were resistant to ampicillin, amoxicillin/ clavulanic acid, chloramphenicol and harbored ctx-m type enzymes. in three isolates a tem-type enzyme was also present. conclusion: albeit being rare, esbl production is an important resistance factor among salmonella spp. in order to prevent treatment failures, decreased susceptibility to fluoroquinolones should be investigated routinely in invasive isolates as well as esbl production. incidence of faecal carriage of esbl-producing enterobacteriaceae in hospital and community patients during two non-outbreak periods of time the identities of the esbl-producing isolates recovered during 2002 were: e. coli (n = 98), k. pneumoniae (n = 2), p. vulgaris (n = 2) and e. cloacae (n = 1), and isolates recovered during 2004 were: e. coli (n = 340), k. pneumoniae (n = 3), k. oxytoca (n = 1), p. vulgaris (n = 1), p. mirabilis (n = 2), e. cloacae (n = 1) and e. aerogenes (n = 1). conclusions: a dramatic, significant increase in the frequency of faecal carriage of esbl-producing isolates was demonstrated in 2004 among hospitalized (8.1%) and ambulatory patients (7.2%).the results revealed that the prevalence of faecal carriage among ambulatory patients and hospitalized patients was not significantly different in both periods of time. outpatients came from the community carrying enterobacteria harbouring esbl in the intestinal tract, suggesting that the community could be a reservoir for these microorganisms and enzymes. methods: a total of 71 k. pneumoniae, 3 k. oxytoca, 11 e. coli, 4 c. freundii, 3 s. marcescens, and 3 e. cloacae from 6 university hospitals, isolated from blood, wound, urine, sputum and other clinically significant specimens were proven to produce esbls. antimicrobial susceptibility was determined according to clsi, 2002 ; conjugation on a solid medium was performed; isoelectric focusing was followed by bioassay; pcr with beta-lactamase group-specific oligonucleotides was applied, followed by nucleotide sequencing; rapd with eric-1a and eric 2 primers was performed. results: mic of ceftazidime varied from 4 to >512 mg/l, mic of cefotaxime -2-512 mg/l; the addition of sulbactam 1: 1 reduced mic > 3-fold. transconjugants exhibited resistance both to extended-spectrum cephalosporins and aminoglycosides in 37 of 39 strains. according to their pi, two clusters of betalactamase producers could be described: first one -esbls focussed at pi 8.2, and the second -pi at 6.3. results from pcr confirmed the presence of two groups esbls: tem and shv. sequencing of representative strains showed the presence of shv-12 in two participating hospitals and of shv-5 in only one strain e. cloacae, while tem-3 like enzyme was found in 2 centres and had a clonal dissemination. objectives: during treatment with selective decontamination of the digestive tract (sdd), four strains of multidrug-resistant (mdr) gram-negative bacteria (three escherichia coli strains and one klebsiella pneumoniae) were isolated at the intensive care unit (icu) in the academic medical center (amc) in amsterdam. these isolates were extended spectrum betalactamase (esbl) positive. we investigated whether this was due to interspecies transfer of resistance genes. methods: the strains were typed by amplified fragment length polymorphism analysis. the plasmids from these strains were characterized by restriction fragment length polymorphism. resistance genes of the mdr-strains were characterized by pcr and sequence analysis. results: aflp analysis confirmed that the three mdr e. coli isolates represented three different strains. the mdr-strains were shown to harbour the same plasmid with identical extended-spectrum â-lactamase (esbl) genes; ctx-m-15 and shv-5. conclusions: identification of the emergence of such mdr gram-negative bacteria and recognition of resistance plasmid transfer during sdd treatment is crucial for optimal application of this regimen in icus. the use of the third generation cephalosporins in sdd may associate with emergence and increase in the prevalence of esbls. therefore, for optimal screening of resistance to cephalosporins in icus, the screening for esbls should be included. objective: carbapenems are the drugs of choice for the treatment of serious infections caused by esbl-producing enterobacteriaceae and the emergence of carbapenem resistance is rarely documented. we investigated pairs of carbapenem-susceptible and resistant k. pneumoniae isolates from three patients, collected before and after therapy with carbapenems. methods: pre-and post-therapy pairs of esbl-producing k. pneumoniae isolates were from three patients with urinary catheter-associated infections who were treated with ertapenem (erp, 2 cases) or meropenem (mem, one case) in a district general hospital with a low incidence of esbl-producing organisms (0.43/1000 bed days), and meropenem use of 1160 ddd/year. isolates were compared by pfge of xbai-digested genomic dna. mics were determined and interpreted by british society for antimicrobial chemotherapy methodology. blactx-m alleles were sought by multiplex pcr. outer membrane proteins (omps) were extracted, and analysed by sds-page. results: the three patients relapsed following erp or mem therapy, and the post-therapy isolates from repeat urine samples were resistant (table), with mics erp>mem>ipm. all six isolates from the three patients belonged to the same pfge strain, but transmission of the resistant variants is unlikely as the patients were geographically and temporally unrelated and separate selection of resistance in individual patients seems more likely. all isolates had a group 1 ctx-m esbl; the resistant isolate in each pair had lost a major omp, consistent with a porin, compared with its susceptible 'parent'. all three patients were successfully treated with amikacin. the emergence of carbapenem resistance in ctx-m-producing k. pneumoniae following therapy severely limits treatment options. whilst unusual in general, such selection has occurred repeatedly with this strain. wide geographic spread of diverse acquired ampc beta-lactamases in escherichia coli and klebsiella spp. in the uk and ireland objective: to determine the distribution of genes encoding acquired ampc beta-lactamases in cephalosporin-resistant isolates of e. coli and klebsiella spp. submitted to the uk national reference laboratory. methods: mics were determined by agar dilution and interpreted according to breakpoints of the british society for antimicrobial chemotherapy. isolates of e. coli or klebsiella spp. resistant to cefotaxime and ceftazidime, irrespective of addition of clavulanic acid, were inferred to have possible ampcmediated resistance. genes encoding six phylogenetic groups of acquired ampc enzymes were sought with a multiplex pcr assay (perez-perez & hanson. j clin microbiol 2002; 40:2153-62) . selected isolates were compared by pfge, and selected blaampc amplicons were sequenced. results: 47 e. coli isolates and 8 klebsiella spp. from separate patients yielded pcr amplicons indicating the presence of genes encoding acquired ampc enzymes. forty of these e. coli isolates (from 20 hospitals) produced cit-type enzymes, 6 (from 3 irish hospitals) produced acc types, and 1 a dha type. the klebsiella spp. produced acc (5 isolates from 2 irish hospitals), fox (2 isolates from 2 welsh hospitals) or dha (1 irish isolate) enzymes. genes encoding ebc-/ent-and moxtype enzymes were not detected. twelve e. coli isolates from one hospital all produced a cit-type enzyme; these 12 isolates belonged to an epidemic uk strain, designated strain a; 8 isolates also contained blactx-m-15 linked to an upstream copy of is26, as is characteristic of strain a; 4 isolates lacked blactx-m-15. sequencing of a representative blaampc amplicon indicated production of a novel cmy-2 variant in these isolates. conclusions: diverse acquired ampc enzymes are present in e. coli and klebsiella spp. in the uk and ireland, with cit-types the most common, and acc types linked to ireland. the broad resistance profiles of ampc enzymes compromises patient management. hence, the acquisition of a cmy-2-like enzyme by epidemic e. coli strain a suggests that acquired ampc enzymes are poised to become an important public health issue in the uk. objective: to characterize the spectrum of activity and potency of dor (formerly s-4661) and comparator agents against contemporary wild-type bacterial isolates from medical centres in europe and the middle east in 2004. dor is a novel parenteral 1-b-methyl carbapenem in late stage clinical development whose molecular structure confers stability to b-lactamases and resistance (r) to renal dehydropeptidases. methods: the collection included 6480 non-duplicate, consecutive clinical isolates from patients in 24 medical centres in europe (21), turkey (2) and israel (1) that were submitted to the dor surveillance program (2004) for identification confirmation and susceptibility (s) testing. mic values for >30 antimicrobials were determined using nccls broth microdilution methods (2003) . a tentative dor susceptible (s) breakpoint of £4 mg/l (£0.25 mg/l for s. pneumoniae) was used for comparative purposes; clsi (2005) criteria were used for other tested agents. results: antimicrobial activities of dor and other carbapenems vs. selected isolates. dor consistently displayed activity against staphylococci and streptococci (mic90, 0.06 and 0.5 mg/l) most similar to that of imipenem, and against e. coli and klebsiella spp. (mic90, 0.06 and 0.5 mg/l, respectively, including 8.7 and 26.9% of strains that met esbl screening criteria), most similar to that of meropenem. enterobacter spp. isolates, including 35.8% that were ceftazidime-r (indicative of ampc production), were also highly s to dor and other carbapenems (0.5 to 1.4% r). dor also provided slightly enhanced coverage against p. aeruginosa (82.7% s) and acinetobacter spp. (54.4% s) compared to other carbapenems. carbapenem r among these latter strains is, however, a particularly worrisome development. conclusions: dor is a new carbapenem with a competitive profile that incorporates both potent gram-negative and grampositive activity, with enhanced activity against the commonly occurring non-fermentative gram-negative bacilli. carbapenems are assuming a greater therapeutic role in many nations as multi-drug resistance (including emergence of ambler class a, c and d b-lactamases) spreads, necessitating their accelerated development. phenotypic and genetic characterisations of enterococcal isolates in tehran sewage, with emphasis on detection of vana and vanb genes objectives: enterococci are members of the normal gut flora of animals and humans and are thus released into the environment directly or via sewage outlets, where they can survive for long time periods. during the last decade the concern has been focused on enterococci that are resistant to the glycopeptide antibiotic vancomycin [vancomycin-resistant enterococci (vre)]. the aim of the study was to detect and to analyse the biochemical diversity of the entrococci strains in tehran sewage and to determine the genetic characterization of vre. methods: a total of 410 isolates of enterococci were selected on me agar medium. all of the isolates were identified at the species level by the common biochemical tests. drug susceptibility test of isolates was done by disk diffusion method with 6 antibiotics vancomycin, erythromycin, gentamicin, tetracycline, chloramphenicol and ciprofloxacin. the mic was also done by macrobroth dilution assay. analysis of the plasmid profiles and the pcr tests for vana and vanb genes were done. methods: we studied 153 vre isolates collected in the north and center of portugal (1996 portugal ( -2004 from: (i) 81 clinical isolates from 3 hospitals in different cities, (ii) 4 faecal samples from healthy volunteers, (iii) 2 river water samples, (iv) 6 samples collected downstream of hospital sewage water, (v) 2 samples from urban sewage water, (vi) 4 swine faeces (vii) 27 poultry food samples for human consumption. identification and characterization of vancomycin resistant genes vana, vanb, vanc1 and vanc2 were determined by a multiplex pcr. the backbone structure of tn1546 was characterized by a pcr overlapping assay (10 overlapping fragments), and further sequencing. conclusion: beta-lactamase production among hi strains has declined significantly since 1999 among children attending daycare centres as antibiotic prescriptions fell among this population. results: the mic distribution of am showed 4% of strains (n = 332) with a mic > 4 mg/l and 7% with a mic of >2 mg/l, indicating that resistance to am is still relatively rare and does increase as compared to nethmap2004. the lognormal distribution of both am and amc (1 strain r) extended to 1 mg/l but showed tailing to 4 mg/l. this may indicate hidden less susceptible strains but could equally well be explained by testing circumstances, since the left part of the mic distribution showed comparable tailing. all strains were susceptible to moxifloxacin, levofloxacin and cefotaxim. the lognormal distribution of sxt extended to 0.25 mg/l with 10% of strains showing higher values. doxycyclin resistance was less than 5%. most of the strains were resistant to clarithromycin and azithromycin with a mic10 >0.5 mg/l for both. conclusions: resistance of hi to common antimicrobials in the netherlands is still low and does not increase. objectives: s. pneumoniae (sp) and h. influenzae (hi) are the two most common pathogens associated with community-acquired pneumonia. changes in the prevalence of resistance or multidrug resistance (mdr) among these pathogens have important therapeutic ramifications. the global surveillance initiative is a longitudinal study that benchmarks antibacterial resistance among respiratory pathogens. methods: during 2005, 964 sp and 924 hi were isolated from patient specimens collected at hospital laboratories in france (fr), germany (ger), italy (it), spain (spa), and the united kingdom (uk). isolates were centrally tested by broth microdilution against lev, penicillin (pen; sp only), azithromycin (azi), erythromycin (ery), clindamycin (cli), ceftriaxone (ctx), cefuroxime (cfx), and trimethoprimsulfamethoxazole (tmp-smx) (nccls, 2005) . data were analysed according to pen resistance, mdr, and b-lactamase status. mdr was defined concurrent resistance to ‡2 of the following agents: ctx, cfx, ery, lev, pen, and tmp-smx. results: for sp, pen r was 1.8% in ger, 2.6% in the uk, 8.5% in it, 15.7% in spa, and 27.1% in fr. azi r was 13.0% in the uk, 22.1% in ger, 27.8% in spa, 44.3% in it, and 46 .3% in fr. overall, lev r was rare (£1%) and mic90s = 1 mg/l in all countries. 62.9% of isolates were susceptible to all of the drugs tested, the most common phenotype encountered. the prevalence (%) of mdr among sp ranged from 4.7 in uk to 32.3 in fr. resistance to pen, ery, cfx, and tmp-smx was the most prevalent mdr phenotype found in europe. overall 97.4% of mdr sp were susceptible to lev. for hi, b-lactamase rates varied by country from 4.5% in it to 21.9% in fr. based on mic90 lev and ctx were the most active agents tested against hi, regardless of b-lactamase status. conclusions: lev showed potent activity against sp and hi. for sp, lev activity was independent of resistance to pen or mdr phenotype. lev maintained consistent activity against sp based on mic90, regardless of country studied. antimicrobial surveillance data from studies such as the global offer guidance to physicians for empiric prescribing. sxt was obtained (2001 sxt was obtained ( -2004 . conclusions: our results suggest that beta-lactamase production does not constitute a threat in hi therapy since values were almost constant. although with an unregulated fluctuation on arnblp percentages, it seems that this mechanism is gaining importance in relation to beta-lactamase production. thus, we conclude the need to be aware of arnblp, as these strains are difficult to detect using the nccls (2004) breakpoints. further molecular studies of the resistance genes responsible of this resistance mechanism are needed. resistance of beta-lactamase producer strains, to other antibiotics decreased during the period of study, due to the diminished use of these antibiotics. this study shows the importance of monitoring antibiotic resistance in hi in order to detect emerging mechanisms. antimicrobial susceptibility of respiratory haemophilus influenzae strains in northern greece k. koraki, p. karapavlidou, d. sofianou (thessaloniki, gr) objectives: to investigate the antimicrobial susceptibility of haemophilus influenzae, one of the most frequent bacterial pathogens of respiratory tract infections. treatment of these infections is most often empirical and considerable geographical resistance variation has been reported. methods: eighty h. influenzae strains were collected from respiratory tract specimens (sputum, bronchoalveolar lavages, endotracheal secretions) in a 4-year period (2000) (2001) (2002) (2003) (2004) . identification was made by colonial morphology, gram staining characteristics, x-and v-factor requirements and api nh (biomerieux, france). antibiotics were selected to reflect representative current treatment options and susceptibility was determined by kirby-bauer disc diffusion method on haemophilus test medium according to nccls guidelines. results: out of the 80 h. influenzae strains 58 were isolated from children and 22 from adults. 25% of isolates came from children admitted to the intensive care units and 18.7% from cystic fibrosis patients. a seasonal trend was reported for infections since 41.2% of isolates were collected during springtime and 25% during autumn months. overall ampicillin resistance was 13.3% and resistant strains were isolated exclusively from children. ampicillin resistance was doubled among cystic fibrosis patients (27.3%). all isolates were susceptible to amoxicillin/clavulanate, chloramphenicol, ciprofloxacin and imipenem. the rank order of cephalosporin activity was cefotaxime and ceftriaxone (100%) followed by cefuroxime and cefaclor (95.8% and 88.9% respectively). trimethoprim/ sulfomethoxazole was active against 95.2% of isolates while erythromycin was the least potent antimicrobial agent with 75% of isolates being susceptible to it. no multiresistant phenotypes were detected. conclusion: our results demonstrated that ampicillin resistance among h. influenzae in our area is still relatively low and overall antibacterial susceptibility rates are high. knowledge of antimicrobial resistance among these pathogens is imperative for physicians to choose the most appropriate therapeutic agent. results: 396 nosocomial gram-negative uropathogens were studied. most common uropathogens were p. aeruginosa (30.8%), e. coli (25.8%), k. pneumoniae (11.1%), followed by a. baumannii (7.3%), enterobacter spp. (5.8%), s. marcescens (4.5%), proteus spp. (3.8%) and other gram-negative rods (10.9%). resistance rates (i+r, %) among p. aeruginosa were: gentamicin -87%, levofloxacin -84%, ciprofloxacin -81%, cefoperazone -79%, cefoperazone/sulbactam -71%, cefepime -62%, piperacillin -55%, amikacin -48%, ceftazidime -46%, imipenem -45%, meropenem -43%, piperacillin/tazobactam -43%, polymyxin b -7%. resistance rates (i+r, %) among e. coli were: piperacillin -75%, ticarcillin/clavulanic acid -69%, amoxicillin/clavulanic acid -62%, ciprofloxacin -54%, gentamicin -54%, moxifloxacin -54%, levofloxacin -53%, cefoperazone -48%, ceftriaxone -45%, cefepime -40%, ceftazidime -32%, cefoperazone/sulbactam -19%, piperacillin/tazobactam -18%, amikacin -17%, all strains were susceptible to ertapenem, imipenem, meropenem. resistance rates (i+r, %) among k. pneumoniae were following: piperacillin -82%, cefoperazone -75%, ceftriaxone -71%, gentamicin -71%, amoxicillin/clavulanic acid -66%, cefepime -64%, ceftazidime -57%, ciprofloxacin -53%, piperacillin/ tazobactam -46%, moxifloxacin -43%, cefoperazone/sulbactam -43%, levofloxacin -41%, amikacin -27%, ertapenem -7%, imipenem and meropenem were active against all isolates. conclusion: p. aeruginosa, e. coli and k. pneumoniae are the main gram-negative uropathogens in russian icus patients. imipenem, meropenem, ertapenem showed prominent activity against e. coli and k. pneumoniae. cefoperazone/sulbactam, piperacillin/tazobactam, amikacin exhibited considerable activity versus e. coli, while k. pneumoniae were more resistant to them. p. aeruginosa were highly resistant to all tested antimicrobials except polymyxin b, thus leaving virtually no choices for therapy in terms of acceptable patient safety. results: overall 69 gram-negative anaerobic bacteria from 41 patients were studied. isolation sites were represented by intraabdominal -25 (60.9%), soft tissue -7 (17.1%), prostate fluid -5 (12.2%), bone -3 (7.4%), and dental -1 (2.4%) infections. susceptibility of 31 (44.9%) prevotella spp., 23 (33.3%) bacteroides spp. (predominantly b. fragilis group -20 strains), 7 (10.2%) fusobacterium spp., 4 (5.8%) porphyromonas spp., and 4 (5.8%) veillonella spp. to ampicillin, clindamycin, metronidazole, imipenem, ertapenem, amoxicillin/clavulanic acid and cefoperazone/sulbactam was determined. all species were susceptible to carbapenems. in prevotella spp. there were 64% and 3% strains resistant to ampicillin and clindamycin and 4% of strains with intermediate resistance to metronidazole. among bacteroides spp. 92% of strains were resistant to ampicillin and 22% to clindamycin. no resistance to metronidazole was detected in bacteroides spp. objectives: the objectives of this study were to: analyse our current blood culture practice; describe the frequency of occurrence and antimicrobial susceptibility of bloodstream infections (bsi) isolates; determine the contamination rate. methods: we performed a prospective survey of all positive blood cultures received in the department of microbiology of tartu university hospital (938 beds) in 2004. blood culture system used was bactec 9120. duplicates within one week were excluded. isolates were identified using conventional microbiology methods and susceptibility tests were those recommended by nccls. to determine extended spectrum beta-lactamase (esbl) producers an e-test with cefepime and cefepime combined with clavulanic acid was used. nosocomial infections were defined according to cdc criteria. results: during study period 7230 blood culture bottles were received, comprising 2840 blood culture sets (10.4 sets per 1000 patient-days). these resulted in 427 (17.2%) positive blood cultures, 127 (29.7%) were considered contaminants and contamination rate was 5.1%. a total of 273 bsi episodes involving 246 patients were identified and 161 (59%) of these were nosocomial. the incidence of nosocomial bsi (n-bsi) and community-acquired bsi (ca-bsi) was 0.6 and 0.4 per 1000 patient-days, respectively. polymicrobial bsi was detected in 25 patients. among n-bsi dominated coagulase-negative staphylococci (49/30.4%), staphylococcus aureus (19/11.8%), klebsiella spp. (21/13%), and escherichia coli (16/9.9%). the most frequent pathogens of ca-bsi were e. coli (31/27.7%), s. aureus (14/12.5%), haemophilus influenzae (11/9.8%), and streptococcus pneumoniae (10/8.9%). susceptibility to oxacillin of s. aureus and cons was 100% and 21.4%, respectively. all s. pneumoniae isolates were susceptible to penicillin. 97.8% of e. coli strains were susceptible to ciprofloxacin, 67.4% to ampicillin, and 100% to gentamicin. susceptibility of klebsiella spp. to both ciprofloxacin and gentamicin was 96.7%, and to ampicillin 6.7%. 14.3% of klebsiella spp. and none of e. coli isolates were esbl-producers. the susceptibility patterns of n-bsi and ca-bsi pathogens were similar to each other. conclusion: compared to west and north european countries our number of blood culture sets per 1000 patient-days is low. this may explain the relatively low incidence of bsi. the interventions to reduce contamination rate need to be implemented. the susceptibility among bsi isolates was high. recent outbreaks of c. difficile associated diarrhoea (cdad) reported in north america, united kingdom and the netherlands have emphasized the importance for an ongoing surveillance of cdad. the aims of the present study was to determine the epidemiology of cdad over the past 5 years and the rate of nosocomial transmission in our acute care hospital (750-beds). materials and methods: all the cases of cdad diagnosed between january 1st 2000 and december 31st 2004 were retrospectively reviewed. a cdad case was defined as diarrhoea in hospitalised patients with a positive result for c. difficile cytotoxin or with a positive toxigenic culture. cdad was considered as severe if patient fulfilled at least 2 of the following 3 criteria: fever > 38.5c abdominal pain or leukocyte count >10,000/mm 3 or if the patient had an endoscopically proven pseudomembranous colitis or complications (toxic megacolon, perforation…). cdad was considered as community acquired if the diarrhoea occurred in patients within 72 h after admission and if the patient had no history of hospitalisation in the previous 2 months, otherwise cdad was considered as nosocomial. all the strains were serogrouped and characterized by toxinotyping and pcr-ribotyping. detection of toxin a, toxin b and binary toxin was performed by pcr. results: 151 cases of cdad were diagnosed: 147 clinical charts could be reviewed and 131 strains were studied. global incidence of cdad was 1.1 per thousand discharges with higher rates in 2003 and 2004. diarrhoea was community acquired in 19% of patients. for patients with nosocomial cdad, transmission of the strain from patient to patient (i.e. strain with the same serogroup and pcr-ribotype than the strain from another patient hospitalised in the same ward in the previous 2 months) was demonstrated in 10.1% of cases. binary toxin was positive in 11% of strains. binary toxin was associated to a more severe diarrhoea (p < 0.01) and to a higher case fatality (p < 0.01). a specific clone accounted for 25% of all the strains (serogroup h, pcr-ribotype ''026'') but this clone was found both in nosocomial or community cases. three strains belonged to toxinotype iii but further investigations are needed to know whether these strains correspond to the hypervirulent strains involved in recent outbreaks. conclusion: incidence of cdad is low in our hospital and cross infection is limited. these results also suggest that strains with binary toxin might be more virulent. the development and application of a new exact typing method for clostridium difficile: multilocus variable number of tandem repeat analysis objectives: to study the epidemiology of clostridium difficile, a typing method with a higher discriminatory power, typeability and reproducibility than currently available methods is required. multi-locus variable number of tandem repeat analysis (mlva) is a new candidate technique, that has already been tested successfully on a number of bacterial and fungal species. using the whole genomic sequence, we developed mlva for c. difficile and compared the method to standardized pcr-ribotyping. additionally, mlva was tested on a collection of the new emerging hypervirulent pcr-ribotype 027 strains. methods: short tandem repeat loci (3 to 9 bp) were identified using tandem repeat finder v3.21 on the genome of c. difficile strain 630. amplification of the repeats was performed using a single pcr-protocol. pcr-fragments were analysed using multicoloured capillary electrophoresis on an abi3100, with a rox500-marker as internal marker for each sample. the number of repeats per fragment was subsequently determined.the discriminatory power of the mlva was tested on 23 reference strains representing 11 serogroups and 12 toxinotypes. the ability to subtype specific pcr-ribotypes was investigated with 7 subtypes of pcr-ribotype 001 (rep-pcr types 1-7), 6 tcda-/tcdb+ strains of pcr-ribotype 017, and 11 strains belonging to pcr-ribotype 027. of these 11 type 027 strains, 9 were isolated from 3 outbreaks and 2 from endemic cases. results: a total of 7 regions with short tandem repeats were identified. mlva discriminated all 23 reference strains and the 7 known reference strains of pcr-ribotype 001 (rep-pcr 1-7). two mlva-types were recognized among 6 tcda-/tcdb+ strains; the differences were present in only one of the 7 repeat-regions. of 11 pcr-ribotype 027 strains, 9 outbreak-related strains were identical to each other. interestingly, two endemic type 027 strains differed from the other strains in 2 of the 7 regions. conclusion: mlva is a highly discriminatory genotyping method for c. difficile and is capable to subtype various crribotypes. mlva is also an important new tool to study the epidemiology of the emerging pcr-ribotype 027 strains. comparative study of clostridium difficile diarrhoea in elderly patients treated with moxifloxacin versus amoxycillin for lower respiratory tract infections l. mooney, m. wilcox (leeds, uk) fourth generation fluoroquinolones such as moxifloxacin have improved anti-anaerobic activity. consequently, these new agents could induce c. difficile infection (cdi) by inhibition of 'protective' anaerobic flora. recent reports have suggested such an association. however, further studies are warranted to determine the risk of cdi in elderly in-patients treated with these agents, and notably where exposure to cd is measured/ controlled. methods: we prospectively investigated the propensity of moxifloxacin (mox) or amoxycillin/macrolide (aml/mac) to induce cdi when used to treat lower respiratory tract infections (lrtis) in elderly in-patients, using a 4-ward, crossover design (18 months total). patients prescribed mox or aml/mac were monitored for gastrointestinal symptoms. diarrhoea was assessed as due to cd, viral or other cause. relevant clinical data were collected. concurrent epidemiological surveillance was also performed to determine environmental exposure to cd. results: 128 patients were studied, 24 receiving mox and 104 had aml/mac. univariate analysis indicated that there was no significant difference between mox and aml/mac patients in gender, age (83.6 vs 86.6 mean years, respectively), or duration of hospitalisation (total, prior to and post diarrhoea). duration of antibiotic therapy did not differ significantly between mox and comparator patients (either total days or days before diarrhoea onset). there was a significant association between mox and overall risk of diarrhoea. however, there was no significance between mox treatment and cd, viral or other cause of diarrhoea. risk factor analysis to inform on possible confounders was performed. initial epidemiological survey results indicate that there was no change in environmental exposure levels to cd on each hospital ward. molecular typing of all clinical and environmental isolates of cd is ongoing. conclusions: although recent reports have highlighted a risk of cdi associated with fluoroquinolones (and increased age), none have specifically studied hospitalised elderly populations prospectively and controlled for exposure to cd. diarrhoea occurs relatively frequently after antibiotic therapy in the elderly. mox was associated with an increased rate of diarrhoeal symptoms, but causes other than cdi explained this association. mox treatment was not significantly associated with cdi when compared with amox/mac treatment for lrti in elderly in-patients. prevalence and association of macrolidelincosamide-streptogramin b resistance with resistance to moxifloxacin in clostridium difficile strains isolated from symptomatic adults and children hospitalised in two university hospitals in warsaw h. pituch, d. wultanska, g. nurzynska, p. obuch-woszczatynski, f. meisel-mikolajczyk, m. luczak (warsaw, pl) objectives: clostridium difficile is the main aetiological agent of nosocomial diarhoea. clindamycin, penicillins, and cephalosporins have been associated with cdad. however, several case reports of fluoroquinolone-associated c. difficile diarrhea have been published. c. difficile strains usually exhibits susceptibility to metronidazole, and vancomycin. we describe prevalence and association of macrolide-lincosamide-streptogramin b (mlsb) type resistance with resistance to moxifloxacin of c. difficile strains isolated from adults and children. methods: eighty-three c. difficile strains recovered from adults and children hospitalised in two university hospitals were investigated (hospital 1: adults n = 38, and children n = 30; hospital 2: adults n = 15). toxin types were determined by commercial test for toxin a and cytotoxicity test for toxin b. tcda, tcdb were detected by pcr. mics of erythromycin, clindamycin, moxifloxacin, vancomycin and metronidazole were determined by e-test (ab biodisk, sweden). the ermb gene was detected by pcr. results: sixty-seven (81%) c. difficile strains were toxigenic. among these, 44 were a+b+, and 23 were a-b+. all 83 strains were susceptible to vancomycin and metronidazole. high level resistance to erythromycin, clindamycin and moxifloxacin was found in 46%, 42%, 27% of the tested strains, respectively. twenty-one c. difficile strains harboured high level resistance to erythromycin, clindamycin and moxifloxacin, simultaneously. among these, all were a-b+ and were isolated from adults, only. twenty-one of the macrolide-lincosamide-streptogramin b (mlsb)-resistant a-b+ strains carried the erythromycin resistance methylase gene (ermb). conclusion: resistance against clindamycin, erythromycin and moxifloxacin among polish a-b+ c. difficile strains was very frequent. fluoroquinolone resistance is associated with resistance to mlsb antimicrobials. we suggest that increasing use of fluoroquinolones is selective pressure for clonal dissemination of a-b+ c. difficile strains. fluoroquinolones use is a strong risk factor for cdad in our hospitals. acknowledgement: this work was supported by the ministry of scientific research and information technology, grant no. 2 p05d 074 27. national surveillance to the incidence of clostridium difficile-associated diarrhoea in the netherlands s. paltansing, r. guseinova, r. van den berg, c. visser, e. van der vorm, e.j. kuijper (leiden, amsterdam, nl) objectives: the recent outbreaks of clostridium difficileassociated diarrhoea (cdad) due to the new emerging pcrribotype 027, toxinotype iii strains has renewed the interest of cdad as an important nosocomial infection. to determine the incidence of cdad in the netherlands, we conducted a prospective surveillance study in 13 hospitals in the netherlands. clinical microbiology and infection, volume 12, supplement 4, 2006 methods: from may 1st to july 1st of 2005, 13 participating hospitals registered all patients diagnosed with cdad. a standardized questionnaire was devised to obtain patient information. faeces samples or isolated strains were sent to the reference laboratory at the lumc for culture and the presence of genes for toxins a and b (tcda and tcdb). pcrribotyping was performed according to the method of bidet and toxinotyping as described by rupnik et al. results: routine methods to diagnose cdad in 13 laboratories included combinations of cytotoxicity tests (59%), enzymeimmunoassays (56%) and culture of toxinogenic strains (31%). in total, 91 patients with cdad were reported. the overall incidence (median) of cdad was 17 for 10,000 patient admissions and varied from 1 to 75. of 91 patients with cdad, 41% was community acquired. the median age of 54 patients with nosocomial acquired cdad was 59 years. of 54 patients with cdad, 7 (13.9%) died during the study period. at least 41 different pcr-ribotypes could be recognized among 91 strains. type 027 was identified in 9 patients from 1 hospital. toxinotyping revealed the presence of at least 7 different types. of 91 strains, 87% were tcda+/tcdb+, 10% tcda-/tcdb-and 3% tcda-/tcdb+. conclusions: the incidence of cdad in the netherlands is lower than reported in usa and canada, but varied considerably per hospital. the new emerging type 027 was found in 9 patients from 1 hospital with a high incidence of cdad (39 per 10,000 admissions). outbreak of clostridium difficile pcr-ribotype 027 toxinotype iii in harderwijk, the netherlands objectives: since 2002, several epidemics of clostridium difficileassociated diarrhoea (cdad) caused by c. difficile pcr-ribotype 027 toxinotype iii have occurred in usa, canada, and the uk. in april 2005, the first outbreak encompassing 45 patients was observed in a medium large hospital of 350 beds in the netherlands. the isolated 027 strain was completely resistant to erythromycin and ciprofloxacin. the patient characteristics, predisposing factors and outcome of cdad were studied. methods: a case-control study was performed in 45 patients and 90 at random selected controls without diarrhoea who stayed at the same department as the patients when the diagnosis of cdad was made. standardized questionnaires were designed to collect data from the patient records and all surviving patients were interviewed 3 months after the diagnosis. faeces samples were cultured for the presence of c. difficile and isolates were typed. results: the incidence of cdad increased from 4 per 10,000 patient admissions in 2004 to 81.5 per 10,000 admissions in 2005. between april and september 2005, 45 patients with cdad due to type 027 were identified. of 45 patients, 9 (20%) died of which 3 (7%) as a direct result of cdad. eleven (24%) patients experienced one or more relapses. the average age of the cases was 72 yrs, 42.2% of the patients was male. in a multivariate analysis, antibiotic use (or 15.3, p < 0.001), duration of hospital stay (cases 31 days, controls 13 days; p < 0.001) and tube feeding (or 3.5, p = 0.03) were found to be significantly associated with cdad. in particular, the use of ciprofloxacin (or 11.8, p < 0.001) and cephalosporins (or 5.8, p < 0.001) were associated. no association was found between the use of protonpump inhibitors and the risk of cdad. the use of erytromycin was significantly higher in cases (11.1%) than in controls (2.2%) in a univariate analysis (p < 0.041), but this relation was not significant in a multivariate analysis. conclusion: antibiotic use (especially ciprofloxacin and cephalosporins), duration of hospital stay and tube feeding were significantly associated with cdad caused by c. difficile type 027, toxinotype iii in the netherlands. we could not confirm the previously described relation between use of protonpump inhibitors and risk of cdad. clostridium difficile pcr ribotype 027, toxinotype iii in the netherlands objectives & methods: shortly after the reports in june 2005 of clostridium difficile pcr ribotype 027, toxinotype iii in england, this more virulent type was also detected in the netherlands. in response, the dutch centre for infectious disease control has undertaken measures to monitor and control the outbreak. c. difficile 027 guidelines for infection control and treatment were formulated, separately for hospitals and nursing homes. the leiden university medical centre serves as a reference centre for diagnostics and typing of c. difficile. laboratories are encouraged to send in samples for typing in case of a clear rise in the incidence in c. difficile, rapid spread, or several clinically suspect cases.organisation-based surveillance was set up: questionnaires are sent monthly to institutions with c. difficile associated diarrhoea (cdad) outbreaks to obtain information on incidence, c. difficile testing strategies, antibiotics use and control measures taken.measures taken in hospitals dealing with an outbreak of type 027 include: treatment of cdad with vancomycin in stead of metronidazole, emphasis on frequent and thorough cleaning and disinfection, isolation of all patients with diarrhoea until tested negative for c. difficile toxin, cohort isolation of cdadcases if individual isolation capacity is exceeded and strong restriction of certain antibiotics, including fluorochinolones. results: until november 1st, 2005, 224 samples from 23 institutions have been sent in for typing, resulting in 74 type 027 positives. epidemic spread of type 027 has been detected in 7 hospitals and one nursing home. furthermore, in retrospective studies in four hospitals isolated cases of type 027 were detected. it became clear that in one region with three hospitals, the cdad incidence had already risen in 2002, 2003 and 2004, respectively . unfortunately, no samples from that period were available for typing. in the hospitals with epidemic spread of type 027, a wide range in the monthly incidence of cdad was observed, from 50 to 114 per 10,000 admissions during the outbreaks. the incidence in the pre-epidemic period varied from 3 to 7 (see figure) . conclusions: the outbreaks in hospitals are difficult to control: most hospitals continue to have new cases for a long period, although the incidence is decreasing in several hospitals. fortunately, once a c. difficile 027 outbreak in a hospital is recognised, spread to other hospitals has not been observed. objectives: c. difficile is a major cause of antibiotic associated diarrhoea (aad) and colitis (c). the aim of this study was to determine the incidence of these infections in our hospital (450 beds), during a period of 6 months (march-october 2005) . methods: a number of 182 liquid stools from equal adult patients (mean age 61 y, m:110, f: 72) receiving broad spectrum antibiotics (especially cephalosporins) were plated in ccfa (oxoid) and anaerobic brucella agar (ba), after alcohol shock procedure. if the culture was positive, an immunochromatographic test was performed for toxin a (colorpactm toxin a, bd, usa). if the last test was negative, a rapid enzyme immunoassay was performed for toxins a+b (immunocard, meridian bioscience inc. cincinatti, ohio). results: c. difficile was isolated in 23/182 (12.6%) samples. seventeen men (pathological -p, pneumological -pn, surgical -s, urologic -u, outpatients -o, 7, 6, 2, 1, 1 respectivly) and 6 women (p:4, pn:1, o:1) harbored c. difficile in their intestin. twelve out of 23 strains (52%) produced toxin a, while the remaining (48%) produced toxin b. eleven patients had severe diarrhoea (7-10 days). one patient got endoscopic examination, which confirmed colitis findings. the two outpatients received oral cefuroxime in the preceding week of the positive culture. conlusions: (1) the incidence of c. difficile infections in this study is among these reported in international bibliography (12.6%). (2) since toxigenic b c. difficile strains were demonstrated in half cases, the use of the tests detecting both toxins a and b by clinical laboratories is recommended.(3) molecular technics application (e.g. pfge and ribotyping) will offer a better knowledge of c. difficile spread in our hospital. assay of the cytotoxicity of stool samples to cells in tissue culture is commonly considered the 'gold standard' for detection of c. difficile toxin. however the method is slow and therefore its use can result in delayed patient treatment and implementation of infection control measures. we undertook a comparison of two microtitre plate-based elisa kits (techlab c. difficile tox a/b ii and meridian premier toxins a & b) and three rapid immunoassay card kits (remel xpect clostridium difficile toxin a/b, meridian immunocard toxins a & b and techlab tox a/b quik chek) with an in-house cytotoxin assay. all samples tested had been referred for routine microbiological examination. toxin tests were done on unformed samples from adult hospital in-patients and bone marrow transplant recipients and on samples where c. difficile toxin testing was requested by the referring clinician. all kits were used according to manufacturers' instructions. three hundred and thirty three specimens were tested using all five kits and cytotoxin assay. sensitivities and specificities were calculated both (a) assuming the cytotoxin assay to be the 'gold standard' (universally correct) test and (b) taking a concensus view that any sample with at least two tests positive is truly positive. data are shown in the table below. overall, the microtitre plate-based elisa kits were more sensitive than the rapid immunoassay card kits. the cytotoxin assay was negative for seven samples that were positive by at least two other tests. thus the plate-based elisa kits were also more sensitive (but less specific) than the cytotoxin assay if consensus data was used to judge true positivity. we conclude that some immunoassay kits offer an acceptable alternative to cytotoxin assays for the detection of c. difficile toxin, allowing more rapid diagnosis. location of the enterotoxin gene in strains of clostridium perfringens associated with gastroenteritis objectives: clostridium perfringens type a is a common cause of food poisoning and is also associated with non-food borne gastroenteritis including antibiotic associated, infectious and sporadic diarrhoea. the disease symptoms are due to an enterotoxin produced when the organism sporulates in the human small intestine. the c. perfringens entertoxin gene (cpe) has been shown to be located either on the chromosome or on one of two large plasmids and it is generally accepted that c. perfringens strains associated with food poisoning have a chromosomal cpe gene whilst strains isolated from non-food borne diarrhoea have a plasmid encoded cpe gene. spores from strains possessing a chromosomal cpe gene have been found to be far more heat resistant than spores from strains with a plasmid encoded cpe gene. heat resistant spores are more able to survive the cooking process and go on to cause food poisoning, thus explaining why most food poisoning strains have been found to have chromosomally located cpe genes. the purpose of this study was to determine the location of the cpe gene in a range of c. perfringens strains from the uk, including those from both food borne and non-food borne illness. method: a multiplex pcr assay described by miyamoto et al., (2004) was used to determine the location of the cpe gene in 107 strains of c. perfringens isolates associated with food borne illness and 16 strains associated with non-food borne illness. results: by multiplex pcr assay 35% of c. perfringens strains associated with food borne outbreaks in the uk were found to have a plasmid encoded cpe gene. these findings have not been described before. all strains associated with non-food borne illness had the cpe gene located on one of two plasmids, as anticipated. conclusions: a significant number of food borne outbreaks of c. perfringens food poisoning were found to be caused by strains of c. perfringens carrying a plasmid encoded cpe gene. since strains of c. perfringens with a chromosomal cpe and plasmid cpe genes have different physiological characteristics this may have a profound impact on their mode of transmission. references miyamoto, k., wen, q. and mcclane, b. a. (2004) multiplex pcr genotyping assay that distinguishes between isolates of clostridium perfringens type a carrying a chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an is1470-like sequence, or a plasmid cpe locus with an is1151 sequence. journal of clinical microbiology 42, 1552-1558. novel multiplex-pcr method for simultaneous detection of clostridium difficile toxin a and toxin b and the binary toxin (cdta/cdtb) genes applied on a danish cohort k.e.p. olsen, s. persson (copenhagen, dk) objectives: a new multiplex pcr method was developed for the detection of the clostridium difficile toxin genes: tcda, tcdb, cdta and cdtb. this method was applied on 210 clostridium difficile strains isolated from 175 danish hospitalised patients with diarrhoea in the period from april to october 2005, in order to investigate the present toxin profiles and their correlation to sex and age. method: a 5-gene multiplex pcr method was developed for the simultaneous amplification of the four clostridium difficile toxin genes tcda, tcdb, cdta, cdtb and 16s rdna as an internal positive control. template dna was prepared from plate grown bacterial colonies by a simple boiling procedure, and amplicons were visualized by standard gel electrophoresis. results: three different toxin profiles were detected in the danish cohort: 43 tcda+, tcdb+, cdta+/cdtb+; 107 tcda+, tcdb+, cdta-/cdtb-and 24 non-toxigenic tcda-, tcdb-, cdta-/ cdtb-. the prevalence of the binary toxin genes in this study was 25% of the clinical isolates.more than half of the strains (62%) were isolated from the elderly part of the population (>59 years), and 74% of these strains displayed the tcda+, tcdb+, cdta+/cdtb+ profile. of the non-toxigenic strains, 83% of the patients were females. one fourth of the strains isolated from children under 2 years of age were non-toxigenic. in four patients, two different toxin profiles were obtained from independent faecal samples. conclusion: this method offers a one-step, rapid and specific identification of clostridium difficile toxin genes. this specific toxin profiling allows an evaluation of the pathogenic potential of the isolated clostridium difficile and surveillance of emerging toxin profiles. further studies of the isolated toxigenic clostridium difficile strains will include gene deletion analyses of the tcda and the tcdc (toxin regulating gene) which independently have been observed to cause enhanced pathogenicity. prevalence of clostridium difficile-associated diarrhoea in hospitalised patients with nosocomial diarrhoea in university of medical sciences hospitals, tehran, iran objectives: this study was aimed at determining the prevalence of clostridium difficile associated diarrhoea in hospitalized patients with nosocomial diarrhoea at three university hospitals in tehran from december 2002 to august 2004. methods: during the study period, the stool samples of 1500 hospitalized patients with nosocomial diarrhoea were cultured and tested by stool cytotoxin assay, toxigenic culture and also 650 of the samples were examined by enzyme immunoassay. results: in 159 (10.9%) of samples c. difficile grew and 108 stool samples (prevalence: 7.2%) were toxin positive by stool cytotoxin assay, enzyme immunoassay or toxigenic culture. there were no significant relationships between c. difficileassociated diarrhoea and sex and age of patients. the results of the present study showed that among requested samples the highest percentage of c. difficileassociated diarrhoea was observed from the transplantation department (13.9%), followed by icu and paediatric section. objectives: the prevalence of toxigenic clostridium difficile (c. difficile) has been reported about 70-80% in korea. toxin a(-)/ toxin b(+) variant c. difficile strain is also important in nosocomial c. difficile infection. however, characterization of clostridial toxin (toxin a, toxin b) had not been studied. methods: we used pcr for toxin a and toxin b genes in 260 c. difficile isolates from patients admitted in three tertiary hospitals during january to december, 2004. primers for toxin a genes were nk1-nk2, nk2-nk3 and nk9-nk11 and toxin b gene was nk104-nk105. results: toxin a and toxin b positive rates using nk1-nk2, nk3-nk2 and nk9-nk11 were concordant and ranged from 62.1% to 90.9% in 3 hospitals. the proportions of non-toxigenic strains were 10-40%. however, we could differentiate toxin a(-)/toxin b(+) variants using nk9-nk 11 primers. the proportion of toxin a(-)/toxin b(+) c. difficile variants were 43.0%, 9.7% and 36.4% in 3 hospitals respectively. objective: administration of antibiotic drugs has long been known to cause alterations in the gut ecosystem. in some patients, these alterations may create a niche that allows the overgrowth of some pathogens such as clostridium difficile, the main causative agent in nosocomial infectious diarrhoea. a predictive tool to assess the risk of development of clostridium difficile, would be of utmost clinical relevance. it remains to be determined whether specific patterns in pre-existing gut microbiota can predict the risk of onset of clostridium difficile, upon initiation of antibiotic treatment. using samples from 87 subjects enrolled in a previously published clinical study on antibiotic-associated diarrhoea (aad), we investigated the potential relationship between their dominant faecal microbiota and the subsequent development of clostridium difficile when subjects received antibiotics. methods: temporal temperature gradient gel electrophoresis (ttge) was used to assess dominant species distribution in gut microbiota. each electrophoregram was digitised from the migration distances and a regression model [partial least square-discriminant analysis (pls)] was built to investigate the correlation between pre-treatment dominant faecal microbiota and the acquisition of clostridium difficile during antimicrobial chemotherapy. results: this pls model could explain 46% of the subsequent onset of clostridium difficile. this result supports the concept of ''permissive'' flora with preliminary data focusing on clostridium coccoides-phylogenetic group. conclusion: to our knowledge it is the first time that dominant faecal microbiota is found to heighten susceptibility to the subsequent onset of clostridium difficile upon initiation of antibiotic treatment. these findings insinuate that strategies reinforcing the control of dominant faecal microbiota at homeostasis would be of clinical relevance. this study has been partially financed by biocodex laboratories. objectives: selective therapy of c. difficile diarrhea (cdd) requires the reduction of pathogen counts in the colon, but spare the normal flora. to determine if par 101 is selective for cdd, serial stool samples were collected at study entry, at day 10, and weekly x5 during the conduct of a phase 2a study of cdd treatment. methods: patients (n = 32) were randomized to receive 50, 100 or 200 mg twice daily of par 101 for 10 days. no prior therapy was given to 24 patients; 8 receive 1 or 2 doses of standard therapy. as treatment controls, 7 additional patients were treated with vancomycin 125 mg qid for 10 days. five well persons donated stools as normal flora controls. fresh stool samples were cultured 10-2, 4, 6, 8 for c. difficile vegetative and spore forms; faecal filtrates were tested for cytotoxin b by cell assay. strains were characterized by tcda/b, ermb, cdta/b pcr and by ribotyping. at study entry and day 10, aerobic and anaerobic faecal flora cultures, diluted 10-3, 5, 7, 9, were examined for major floral shifts. since bacteroides group organisms are ubiquitously present and cultivable, this genera was selected as a indicator of the integrity of the microbial flora. results: at study entry, mean log 10 cfu + sd vegetative counts of c. difficile (all par 101 patients) were 6.8 + 3.6, range 2-10.95; at day 10, with the exception of one patient receiving 50 mg, all other patients had c. difficile quantitative counts reduced < 2 log10/gm faeces. vancomycin was similarly effective. at study entry, bacteroides group counts were < 3, 3-8, & 8.5-10 log10 cfu/gm in~1/3 each of patients. all normal stools showed complex, multi-genera in high counts, with 4-5 bacteroides group species > 11 log10 cfu/g. mean + sd of log10 cfu of bacteroides group counts/g feces wet weight at study entry and day 10 for 50 mg/day (n = 10) were 6.6 + 2.8/ 8.2 + 2.6 (p = 0.11, wilcoxon matched pairs signed-ranks test, 2 tailed); for 100 mg/day (n = 8) were 6.6 + 2.8/6.3 + 2.5 (p = 0.44); for 200 mg/day (n = 11) were 7.0 + 2.9/7.3 + 3.1 (p = 0.56); and for vancomycin (n = 7) 7.4 + 2.7/3.6 + 1.9 (p = 0.03). conclusion: patients with cdd have variably impaired normal flora. par 101 was effective in all dosages in eradicating c. difficile. a dose-dependent reduction in bacteroides counts was not observed. vancomycin significantly reduces bacteroides counts during cdd treatment. par 101 is effective against c. difficile in-vivo, and is relatively sparing of the normal flora. results: the three rt pcr assays were able to detect all enterovirus strains in cell culture supernatants. however the detection limit of the mgb rt pcr was 1 to 2 log10 more sensitive in 14 out of 16 dilutions assays of vc supernatants compared to the rab and ver rt pcr. all ver and mgb negative csf were vc negative. thirty-two csf specimens from 68 patients suspected of viral meningitis were positive by all rt pcr (47.1%), whereas only 8 were found positive by vc (11.8%). the rab rt pcr failed to detect 4 csf confirmed positive by vc (1 echo 6 and 3 non typable ev). among samples positive by rt pcr, sensitivity of ver, mgb and rab was respectively 100%, 100% and 71.9%. conclusion: in our laboratory, mgb rt pcr has a good correlation with ver rt pcr whereas rab rt pcr is less sensitive especially for the detection of echovirus 6. the mgb rt pcr seems to be the most sensitive of the 3 rt pcr. further studies, including more ev strains should help to precise the sensitivity of this assay. a. dalwai, s. ahmad, e. hussein, a. pacsa, w. al-nakib (kuwait, kw) objectives: enteroviruses generally share tissue tropism and present with overlapping disease spectrum, however certain enteroviruses may be over represented in certain diseases than others. coxsackievirus a7 though has been reported to cause several diseases such as febrile illness, herpangina, aseptic meningitis and acute flaccid paralysis, the frequency was very low. the study aimed to determine the prevalent enteroviruses causing non-specific febrile illness, aseptic meningitis, encephalitis, neonatal disease and myositis, in kuwait. it also aimed to study the association between a certain enterovirus and a particular disease and its severity. methods: diagnosis of enteroviral infection was based on detection of enteroviral rna by semi-nested rt-pcr of a portion of the 5'utr of the enteroviral genome followed by southern hybridization with an enterovirus specific probe to confirm the results. the enterovirus was genotyped by sequencing of the 5'utr, the vp4 and a portion of the vp2 encoding regions, and the sequence was analysed by blast analysis, clustalw alignment and phylip phylogenetic analysis package. results: enteroviruses were the only etiological agents detected in 24% (217) of 920 disease cases investigated. coxsackievirus a7 was identified to be the second most predominant enterovirus (24%; 44 of 187 cases genotyped) associated with disease, after only echovirus 9 (39%; 70/187). although identified in all the diseases investigated, coxsackievirus a7 occurred less frequently in cns disease cases (17%; 25/147) than in febrile illness cases (43%; 15/34). in a preliminary study, it was also predominantly detected in 66% (4/6) of myositis cases. the 5'utr of this virus showed 96% homology with that of coxsackievirus a7 prototype strain (parker strain) whereas the vp4 and the adjoining region showed greater homology to human enterovirus b genotype sequence. conclusions: coxsackievirus a7 was determined to be an emerging enterovirus associated with different diseases in kuwait. it was frequently represented in mild febrile illness and myositis cases than in cns disease suggesting that the isolate might be less neurovirulent. molecular analysis suggests that the isolate might have emerged due to recombination between coding and non-coding segments of coxsackievirus a7 and human enterovirus b group genomes. acknowledgement: supported by research administration project grants mi 04/01, ym 03/02 and college of graduate studies, kuwait university. the new proposed enterovirus type 75 is causing meningitis in spain introduction: several new proposed enteroviruses (ev) have been recently described, including the named ev75 [1] . a total of 8 isolates of this serotype were identified from 1974 to 2000 in america, africa or asia associated mainly with acute flaccid paralysis or unspecified disease. objective: to determine if this new serotype circulates in spain and what type of disease produces. methods: a total of 300 ev isolates coming in 2005 to the spanish enterovirus reference laboratory were studied both by micro neutralization assays and by typing pcr [2] . in the isolates in which ev75 was suspected by the mentioned methods complete vp1 gene was amplified and sequenced with specific designed primers. results: four isolates from two different regions of spain were identified as ev75 (more than 80% of homology with the published sequences). three of them corresponded to aseptic meningitis in children and were isolated from csf. discussion: the present work demonstrates that this new proposed virus circulates also by europe and is associated to aseptic meningitis. till the moment it seems that is represented in a minor proportion (4/300 studied), however the possibility of spreading of this viral infection should be considered, as evs may behave in that way, as previously have been demonstrated [3] . objectives: rotavirus is the most important cause of severe gastroenteritis in infants and young children through the world and is responsible of 500,000 deaths annually, mostly in developing countries. therefore, development of rotavirus vaccine is a high priority. rotavirus strains with g1 types account for the majority of the diarrhoea episodes. recently, a monovalent g1 attenuated rotavirus vaccine was licensed in mexico. in view of a hypothetical introduction of such vaccine in europe, we investigated the variability over time of vp7 antigenic genes of g1 rotavirus strains in our area. methods: fifty strains were selected from a total of 780 g1 strains obtained from children of less than 5 years of age hospitalised with acute gastroenteritis at the ''g. di cristina'' children's hospital of palermo in the period 1986-2004. the selected strains were genotyped by rt-pcr and 35 of them were submitted to vp7 gene sequence analysis. results: all but one of the 50 strains were genotyped as g1p(8). the vp7 sequences of 35 of them were distributed into lineages i and ii. lineage i included 14 strains from 7 different years in the range 1986-2004. lineage ii included 21 strains from 9 different years in the range 1989-2004. the degree of similarity among the nucleotide sequences of italian strains in each lineage were comprised between 94% and 100%. an alignment of the deduced amino acid sequences showed major lineage specific amino acid changes in the variable antigenic regions with respect to the reference wa strain. conclusions: sequence analysis indicated that in palermo there was co-circulation of g1 strains belonging to two different lineages. overall, the g1 strains showed a high degree of similarity inside each lineage and shared specific amino acid modifications. the antigenic differences between circulating strains might permit them to escape neutralization and persist in the infantile population. our results suggest that rotavirus strains belonging to the two g1 lineages should be both included in a rotavirus vaccine preparation. epidemic spread of recombinant noroviruses with four capsid types in hungary objectives: noroviruses (''winter vomiting diseases'') are the predominant etiological agent in hungary and common pathogen worldwide in outbreaks of gastro-enteritis in humans. noroviruses are genetically diverse group of viruses with multiple genogroups (gg) and genotypes. more recently, naturally occurring recombinant noroviruses were identified. these viruses had a distinct polymerase gene sequence (orf1, designated ggiib/hilversum) and were disseminated through waterborne and food-borne transmission in europe. our aim was to characterize these emerging recombinant noroviruses causing outbreaks of gastro-enteritis in hungary. methods: stool and rna samples -from norovirus outbreaks between january 2001 and may 2005 -containing ''ggiib/ hilversum polymerase'' (ggiib-pol) were selected for analysis of the viral capsid region (orf2) by reverse transcriptionpolymerase chain reaction (rt-pcr) followed by sequence-and phylogenetic analysis. results: forty (12.6%) of 318 confirmed norovirus outbreaks were caused by the new-variant lineage with the ggiib-pol. viral capsid region was successfully characterized in 24 ggiibpol outbreaks. four different recombinants were detected with capsids of hu/nlv/ggii-3/mexico/1989 (n = 10, 41.7%), hu/ nlv/ggii-2/snow mountain/1976 (n = 9, 37.5%), hu/nlv-1/ggii/hawaii/1971 (n = 4, 16.6%) and hu/nlv/ggii-4/ lordsdale/1993 (n = 1, 4.2%). interestingly, outbreaks caused by recombinant ggiib-pol strains mostly associated with outbreaks among children (47.5%) and had non-winter seasonality. conclusions: epidemic spread of emerging multiple recombinant norovirus strain ggiib-pol were detected in hungary that became the second most common norovirus variants -next to the endemic ggii-4/lordsdale virus -causing epidemics of gastroenteritis in the last 4.5 years. the respiratory infections are the most common diseases in the world being the origin of a great morbidity and mortality especially in infants and elderly. (1) human metapneumovirus (hmpv) was first described in dutch children with acute respiratory tract infections (artis) in june 2001. (2) very limited studies data are available from tropical and developing countries. we sought to determine the role of hmpv in upper and lower respiratory tract infections in cuban patients and correlated the presence of virus with clinical characteristics of the disease. between 1 october 2002 to 30 september 2003 clinical samples received from the national surveillance program of artis at the national reference laboratory of respiratory viruses, for virological study, were used to detect hmpv by rt-nested pcr, amplifying a conserved fragment of 170 nucleotides in the polymerase gene. we found rna hmpv in 9.6% of samples from the patients with artis. 44.4% of individuals who tested positive for hmpv were under 12 months of age. patients with evidence of hmpv had symptoms consistent with either upper or lower respiratory tract disease or both. 66.6% of hmpv positive individuals were detected during august-october (table1). the results of this preliminary study shows that hmpv is present among cuban patients with arti. constitute the first report of the frequency of hmpv infection in a non-preselected group of cuban patients with ages ranged from 3 months to 60 years old. it should be noted that this is the first report of hmpv infection in central america and in the caribbean region, further confirming the worldwide distribution of the virus (3) (4) (5) . detection of human metapneumovirus in paediatric nasopharyngeal aspirates by a taqman minor groove binder probe assay: a one-year prospective study in belgium w. verstrepen, p. bruynseels, a. de smet, a. mertens (antwerp, be) objective: human metapneumovirus (hmpv) has a relative high incidence in acute respiratory infections in children but is difficult to isolate in culture. the aim of the study was to decrease the number of undiagnosed viral respiratory infections in our hospital by means of a taqman minor groove binder (mgb) probe assay. methods: from october 2004 to september 2005 a total of 1387 nasopharyngeal aspirates from children presenting at our paediatric facility were analysed. rna extracts from specimens negative for rsv, parainfluenzavirus and influenzavirus with an (in) direct immunofluorescence assay (ifa) were subjected to a taqman mgb probe assay in parallel with a previously published taqman assay. results: of the 1387 specimens, 371 (27%) were positive by ifa for either rsv (226), parainfluenzavirus (47), influenzavirus a (83) or influenzavirus b (15). hmpv was detected in 83 (8.4%) of the remaining 988 specimens subjected to the newly developed pcr. of the patients with a positive hmpv assay, 46/52 (88.5%) presented with respiratory symptoms. 72% of the positive specimens were from children less than 2 year as compared to only 6% from children older than 5 years. viral load was highest in children less than 1 year. a prominent seasonal variation was noted since more than half of the positive specimens occurred during the months march and april. there was no significant difference in the proportion nor viral load of positive specimens from ambulatory patients, patients admitted to a general ward or patients requiring intensive care. as compared to the published taqman assay, diagnostic sensitivity and specificity were 97.6% and 99.9% respectively, whereas ppv and npv were 98.8% and 99.8%. method comparison (nccls guideline ep-9a) failed to demonstrate a significant difference between both assays when the threshold cycle (ct) was between 22 and 41. strongly positive specimens (ct < 22) were associated with a lower ct using the published taqman assay. however, the new taqman mgb probe assay appeared to be more sensitive for weakly positive specimens (ct > 41). conclusion: the number of viral respiratory infections confirmed in our hospital was substantially increased by means of the hmpv taqman mgb probe assay. the new assay is a reliable alternative to the previously published taqman assay for detection of hmpv in nasopharyngeal aspirates. nucleic acid sequence based amplification and molecular beacon detection for the real-time identification of respiratory syncytial virus in paediatric respiratory specimens r. manji, f. zhang, c. ginocchio (lake success, us) background: respiratory syncytial virus (rsv) is the leading cause of lower respiratory tract infection in infants and young children, with bronchiolitis and pneumonia being the major clinical manifestations. the rapid diagnosis of rsv infections is of central importance for individual patient management (rational use of antibiotics and antiviral agents), hospital infection control and monitoring epidemiological disease patterns. this study included a technical validation and a retrospective clinical evaluation of a real time nasba assay for the detection of rsv a and rsv b in paediatric respiratory samples. methods: samples tested included: dilution panels of in vitro transcribed rna, local rsv isolates, isolates of common respiratory pathogens, and frozen respiratory specimens (nasopharyngeal aspirates, washes or swabs) from 231 children (age range: 5 d to 2 yr) who were evaluated in the paediatric emergency department for respiratory disease. nucleic acid (na) isolation, amplification and detection were performed using the nuclisens easyq basic kit and nuclisens easyq rsv a+b reagents (biomérieux). specimen nas and a rsv specific internal rna control (ic) were co-extracted using nuclisens magnetic extraction reagents and the nuclisens minimag instrument (biomérieux) and co-amplified using a single rsv specific primer pair. included in the reaction were a rsv specific molecular beacon (5'-fam) and an ic specific molecular beacon (5'-rox). target amplification and continuous monitoring of emitted fluorescence were performed using a nuclisens easyq analyzer (biomérieux). results were compared to direct immunofluorescence (dfa) and/or viral culture using r-mix cells (diagnostic hybrids, oh). results: the limit of detection for rsv was 2 rna copies/rxn and the 100% detection rate was 5 copies/rxn. the assay was 100% specific for rsv with no cross reactivity to other respiratory pathogens. the nasba assay detected 10% more positive specimens than dfa and 44% more positive samples than vc. the npvs of the assays were: nasba 94.6%, dfa 90.8% and vc 78.0%. the nuclisens easyq rsv assay demonstrated superior sensitivity to both dfa and viral culture for the detection of rsv a and b from respiratory specimens. the assay was easy to use, required minimal hands on time (1 hr) and a faster time to results as compared to rapid culture (4 hr vs. 24-72 hr). respiratory syncytial virus (rsv) is a major cause of acute lower respiratory tract infection in infants and young children. it has previously been shown that hrsv isolates can be divided into two antigens groups a and b. the g protein is the most divergent both between and within the two subgroups and appears to accumulate amino acid changes with time, suggesting evolution under selective pressure. our knowledge of the molecular epidemiology of rsv has so far been based mainly on studies done in the developed world with e temperate climate. very limited epidemiological data are available from tropical and developing countries, where rsv infections may follow a different pattern. in this report we examine the molecular epidemiology and evolutionary pattern of the g protein of both subgroups a and b rsv through consecutive epidemics in cuba. sixthly four nasopharyngeal swabs were collected from children under 1 years of age with respiratory disease to different hospitals in cuba between 1994 and 2000, to examine the molecular epidemiology and evolutionary pattern of the g protein of rsv. all samples collected from 1994 to 1999 were rsv subgroup a; however both subgroups co-circulate during 2000. the cuban isolated from 1994 to 1996 showed a great homogeneity between them and were resemble to an ancient strain (long) with only five nucleotide differences, this also occur in 1998 and 2000 with two strain. furthermore was detected different size of g protein (297 or 298 for rsv a and 295 for rsv b) due to change in stop codon used he genetic homogeneity of the cuban isolates (1994) (1995) (1996) and their resemble to an ancient strain such as long was an unusual finding in our country. in both subgroups was observed the predominance of strains with almost similar sequences. phylogenetic analysis for subgroup a strains showed that strains were cluster in different genotypes with virus isolated in different geographic regions. both subgroups co-circulated during 2000 and clustered whit south african strains that circulate during at the same time. point mutations in respiratory syncytial virus detected by lightcycler pcr and melting curve analysis u. germer, l. nielsen, k. boye, h. westh (copenhagen, dk) objective: the objective was to analyse rsv real-time pcrpositive isolates from clinical samples, which appeared to belong to three different groups according to melting temperature (tm) of the amplicons. the analysis was done according to genotypic and phenotypic difference and related to geographical distribution. materials and methods: 322 nasopharyngeal aspirates were collected from children with respiratory distress in the city of copenhagen. viral rna extracted using the magnapure lc automated extraction system was amplified in a real-time rt-pcr previously described (1) . five samples from each of the three groups with different tm's were selected for bidirectional dna sequencing using the rsv primers. sequences were analysed using chromas lite version 2.3. results: a total of 322 clinical samples were analysed. 170 (53%) of the 322 samples were positive and 152 (47%) were negative for rsv. three distinctive groups with different tm's could be identified from the melting curve analysis. group 1 (n = 91) had a tm with a median of 60.9°c, group 2 (n = 59) and 3 (n = 20) had lower tm's with a median tm of 58.7°c and 56.8°c respectively. sequence analysis of amplicons showed that the difference in tm was due to differences in genotype between the three groups. genotype 1 and 3 were closely related, differing only in two nucleic acids in position 12525 (c to t) and 12564 (a to t). both were silent mutations. only position 12564 is targeted by the probe. genotype 1 and 3 were both blasted to a complete genome sequence of respiratory syncytial virus subgroup a (genbank rsu39661) with the highest identity score for genotype 1. genotype 2 sequences were blasted to human respiratory syncytial virus mutant cp52 subgroup b (genbank af013255). geographical analysis showed a higher prevalence of the mutant strain (genotype 3) in the northern areas of the greater copenhagen area compared to central, southern and western areas (p = 0.01). conclusion: we found three genotypes of rsv according to the tm of the pcr product. two of the genotypes were closely related with only two point mutations and the same phenotype. genotype 3 was mainly found in clinical isolates from the northern part of copenhagen, suggesting a local epidemic spread. objectives: biomerieux is developing a real-time nasba assay to detect influenza a and b rna in different kind of respiratory clinical samples, by using the nuclisens ò easyq basic kit in combination with specific primers and molecular beacons. methods: 89 nasal/throat swabs in transport medium from hospitalised children (0-16 yrs from edouard herriot hospital, lyon, france) were used for this evaluation. influenza rna is isolated using the nuclisens ò minimag extraction. an internal control is added to the sample prior to nucleic acid extraction. the assay is designed to detect in a single tube, using a three-label approach, the internal control and both influenza a and influenza b rna. amplification reactions were performed in a nuclisens ò easyq analyser allowing real-time detection. the results of the clinical samples were compared to cell culture results. results: among 89 swabs tested, real-time nasba detected 10 (11.2%) samples for influenza a and 2 (2.2%) samples for influenza b. comparatively, by cell culture only 5 (5.6%) samples were identified as influenza a and non as influenza b. interestingly, 1 influenza a positive sample identified by cell culture was found negative in real-time nasba. conclusions: the data showed that nuclisens ò easyq influenza a/b assay detected 50% more influenza a virus than cell culture method. moreover, real-time nasba detected 2 influenza positive samples, which were not detected by cell culture. with this assay a qualitative detection of influenza a and influenza b viruses in a single reaction can be done within 3 hours. it provides a valuable alternative to cell culture method for the clinical management of patients with influenza infections. results: 299 patients have developed mumps meningitis and 17 patients were diagnosed with mumps meningoencephalitis. age limits were from 3 to 17 years and sex ratio m/f was 7/ 4.clinical manifestations involved fever (95%), stiff neck (92%), nausea and vomiting (89%), headaches (86%), photophobia (73%) and neurological manifestations such as: equilibrium disorders and drowsiness (6%), convulsions (0.17%), cerebellum syndrome (0.12%). meningeal symptoms have occurred shortly after parotiditis in 94% of cases and before parotiditis in 3% of cases; the other cases have evolved without parotid swelling. other localizations of the mumps infection were: parotiditis (97%), pancreatitis (61%), submaxillitis (40%) and orchitis (4%). lumbar puncture yields csf containing between 130 and 2830 wbc/mm3. the predominating cells were usually lymphocytes, but 12% of the patients have polymorphonuclear leukocyte predominance at the first puncture. protein levels are normal to mildly elevated in all cases and hypoglycorrachia was founded in 10% of the patients. therapy for mumps meningitis was symptom-based (analgesics and antipyretics) in 67% of cases and glucocorticoid therapy in 33% of cases. conclusions: (1) neurological involvement in mumps occurred in 13.32% of cases; (2) men are afflicted two times more often as women, but the age distribution is the same as for uncomplicated mumps; (3) mumps meningitis was the only localization of the mumps infection in 3% of cases. mumps is acute generalized infection occurs primarily in schoolaged children and adolescents. most prominent manifestation of mumps is swelling and tenderness of salivary glands especially parotid gland. uveitis is a rare manifestation of mumps. here we present a mumps case complicating with uveitis. 26 years old paediatric nurse was admitted to our emergency department because of headache and malaise. on physical examination bilateral parotid enlargement was noticed. opthalmology consulatation revealed anterior uveitis. local prednisolon and cyclopentholate treatment were prescribed. lumbar puncture revealed lymphocytic pleocytosis without hypoglychorachea and elevated protein levels. mumps igm was found positive. differential diagnosis made with other viral infections and sarcoidosis. her headache diminished day after the hospitalisation. uveitis responded very well to local therapy and patient got well in 2 weeks. clinical and epidemiological aspects of a measles epidemic, bucharest, romania results: there were 108 cases; sex ratio m/f:58/50. the mainly affected age group is under 13 months (38.9%) followed by 13 months-2 years (23.15%), 2-7 years (22.22%), > 16 years (10.18%) and 7-16 years (5.55%). 70.37% cases were hospitalacquired (mostly in paediatric clinics), 3.7% were communityacquired; in 25.9% cases the source was unknown. the most common clinical features were fever (100%), rash (100%), conjunctival hiperemia (78.7%), cough (70.37%), micropoliadenopatia (64.8%), diarrhoea (39.81%). pulmonary complications were described in 62.3% of cases; 22.38% of them were bacterial pneumonia, 77.62% were viral pneumonia. in 20.37% of cases we diagnosed acute stomatitis, in 12.96% bacterial conjunctivitis; in 14.81% of cases -otitis; in 1.85% of cases -pharingitis, and in one case (0.92%) -urinary tract infection. 7.4% of the patients were previously diagnosed and treated for pulmonary tb. all cases were confirmed serologically through detection of specific igm antibodies. 25 patients (23.15%) had severe clinical forms of measels. the evolution was good in all cases. conclusion: 1. this year in the south-east part of the country, evolves a measels epidemic with different features comparing to the previous one (1997) (1998) we investigated the recombinant proteins np and hn to develop new antigen with useful properties for applied in elisa test systems. methods: significant antigenic epitopes of nucleoprotein (np) and haemagglutinin (hn) measles virus strain edmonston were generated by computer analysis. using standard geneengineering techniques was evaluated two fusion peptides np and hn consist from only linear t-cell antigenic determinants. the virus-neutralization activity of hyperimmune serum on recombinant proteins was determined by plaque reduction neutralization test (prn). the level of specific igg in serum to genotypes a, d4, and d6 of measles virus was determined by enzyme-linked immunosorbent assay (elisa). we used recombinant proteins np and hn as antigens for elisa. results: hyperimmune serum was collected from mice after immunization by np and hn recombinant proteins. the level of neutralize activity was measured in the prn assay with strain edmonston. the titre reached up to 1:13.5 and 1:22.9 for np and hn recombinant proteins, respectively. interestingly that, hyperimmune serum on recombinant protein np in elisa reacted both with np (titre 1:6400) and with hn (titre 1:3200), and in turn serum on recombinant protein hn reacted only with hn (titre 1:12800). the estimation immunological properties of proteins with use of the panel of serum (50 samples) collected from patients. the diagnosis of measles infection was confirmed in laboratory (by rt-pcr). the nucleotide sequences of rt-pcr products used for genotyping of mv. selective interaction of antibodies in elisa with recombinant proteins in relation to various genotypes is revealed. the interaction with genotypes a and d6 was expressed with high level of correlation whereas with genotype d4 any serum did not react authentically (as the control was used recombinant protein n of sars virus). conclusion: we have shown that neutralize antibodies formed hot only on superficial proteins such as hn, f and sh but also on core proteins such as np. our data demonstrate that the recombinant proteins np and hn could be a cost-effective alternative to current whole virus based elisas for surveillance for immunity to measles and could more efficient in detecting susceptibility to measles in relation to genotypes a and d6. episode 1: a pregnant woman with thirty-eight week of gestation was hospitalized in obstetrics clinic with the complaints of fever, malaise, and severe vaginal bleeding. on admission, white blood cell count was 6700/mm 3 , haemoglobin was 11.5 g/l, platelet count was 8 000/mm 3 . the level of ast was 591 iu, alt 163 iu, lactate dehydrogenase 1079 iu, and creatinin phosphokinase 2132 iu. the baby was delivered by cesarean section. in serum cchf igm was positive by elisa, and per oral ribavirin was administered after delivery. at the first day of delivery, the clinical and laboratory of findings of the baby were found to be normal. however, on his 5th day, he died because of massive bleeding. his cchf igm was found to be negative. episode 2: a pregnant woman with 19 week of gestation was admitted to the hospital. her complaints were fever, malaise, headache, myalgia, nausea, vomiting, diarrhoea, and subconjuntival bleeding. in her laboratory investigation, white blood cell count was 3400/mm 3 , haemoglobin level was 10.4 g/l and platelet count was 53000/mm 3 . the level of ast was 813 iu, alt 539 iu, and ldh 741 iu. in her serological analysis cchf igm and cchf virus -pcr was found to be positive. at the twenty six week of gestation in obstetric ultrasound, fetal intraabdominal fluid was visualized and amniocentesis was performed. in serological analysis of amniotic fluid cchfv-pcr was found to be negative. intraabdominal fluid had increased and scrotal edema was visualized at thirty eighth weeks of the gestation. after her vaginal delivery, baby was severely ill and was operated with the diagnosis of necrotizing enterocolitis. his laboratory findings were normal except high white blood cell count. on his fifth day, thrombocytopenia occurred and he died because of massive bleeding. his cchf igm and pcr were negative. conclusion: to our knowledge, these are the first episodes of intrauterine cchf infection. these episodes show that cchfv can transmit through placenta. obstetricians in endemic countries should consider cchf infection among the patients with massive bleeding and thrombocytopenia. objective: to detect the asymptomatic crimean congo hemorrhagic fever virus (cchfv) infections in an endemic area, and calculate the attack and the infection rate. methods: the study was performed in a cchf endemic region. the household members of the index cases were screened for cchfv igg and igm by elisa. the data related to risk exposure were obtained by a structured form. results: eleven index cases were admitted to the clinic, 45 household members of these cases were screened. all the index patients had positive igm or pcr for cchfv. among the household members, three individuals had igg positivity (%), and only one patient had igm positivity. none of the screened individuals had symptoms. the mean age was 28 (sd 17), and 52% of the subjects were female. tick bite was detected a risk factor (p = 0.040) for cchv infection, whereas patient care and contact with body fluids of the patients were not (p > 0.05). eighteen patients had the history of tick bite, and 8 became infected (44%), and five (27%) became ill. among the infected eight individuals, five became ill (63%). conclusion: although we consider that some of the patients do not notice tick bite, we can still suggest that the infection rate of the virus is rather high compared to similar diseases. tick bite is the major risk factor, in comparison to exposure to blood and body fluids of the infected cases. results: 457 children were included in our study. distribution according sex was: 57.6% female and 42.4% male. median of age was 13 years (iqr = 6). during the follow-up study we recorded 2 years when the number of cases increased. the distribution of cases among the study was: 8.4% in 2000, 33.9% in 2001, 20.9% in 2002, 6.6% in 2003, 7.0% in 2004 and 23.1% in 2005 . the proportion of paediatric patients also varied from; 11.9% in 2000, 9.6% in 2001, 12.4% in 2002, 9.7% in 003, 7.8% in 2004 and 4.4% in 2005 . in panama city we recorded 65.3% of the infants. we detected an increase in the number of patients in the rain season, from may till november. the mean of days between the onset of symptoms and the first blood sample was 6.3 days (ds: 6.7) a second sample was obtained in 23.6% of our infants with an average time of 10.1 days (ds: 7.4). the frequency of classical symptoms related to dengue virus infection was: fever (95.2%), severe headache (74.2%), chill (65.9%) rash (63.5%), myalgia (51.9%), retro-orbital pain (51.6%), arthralgia (43.3%), gastrointestinal symptoms (37.4%), inflamed pharynx (26.7%), cough (26.5%), mild respiratory symptoms (18.6%) and diarrhoea (10.7%). in our infants the symptoms which were detected first were; fever, severe headache, chill, myalgia, retroorbital pain, arthralgia, mild respiratory symptoms, cough and inflamed pharynx. we did not observed differences on clinical features between girls and boys. however, we detected detected significative differences among symptoms when we compared infants who were £5 years old with those who were older (p < 0.005). four of our patients died because of dengue hemorrhagic fever. conclusion: dengue is endemic in panama as in most tropical countries and is one of the world s major emerging infectious disease. more data about this illness are needed to elaborate sanitary programmes which contribute to control this infection. diagnosis of dengue infection by enzyme-linked immunosorbent assay and reverse transcriptionpolymerase chain reaction from oral specimens dengue. salivary elisa has been shown by various investigators to be useful for dengue diagnosis. we sought to perform a pilot evaluation of diagnostic value of elisa and pcr of oral brushes and saliva for dengue diagnosis in adults. methods: adults with acute fever and suspected of dengue infection admitted to our university hospital were enrolled. dengue diagnosis was made by standard elisa using serum or plasma. patients with negative elisa served as controls. buccal mucosal cells were collected for rt-pcr and saliva for both rt-pcr and elisa at least twice, 7-14 days apart. our elisa criteria for saliva were single igm > 20 units or single igg > 60 units or 2-fold increase in igg titre with the second titre >40 units for secondary dengue infection. criteria for primary dengue infection were the same as secondary infection plus igm:igg ratio of over 1.8. results: 23 cases and 16 controls were enrolled. our country is endemic for dengue and thus there was no primary dengue adult case in this study. as the study was performed in hospitalized patients, most of the first samples were collected one day before or on the day of defervescence. the specificities of either methods and the sensitivity of elisa method for saliva were 100%. sensitivities were approximately 32-33% for rt-pcr using buccal cells or saliva specimens. however, a combination of rt-pcr results for both types of oral specimens gave a sensitivity of 52%. the results are summarised in the table. conclusions: collection of oral specimens is less invasive and may be more acceptable in certain situations. a single, acute specimen is adequate for diagnosis by rt-pcr. our specimens, however, were collected late in the course of illness which affected the sensitivity of rt-pcr's. earlier specimens may give a better yield. a study in paediatric patients is needed to assess the value of these methods for primary dengue infection. objective: the aim of this study was to assess the proportion of seropositives against hantaviruses among healthy blood donors. methods: 1607 volunteer donors were recruited by the institute of transfusion medicine, representing the demographic situation in the tyrol regarding gender and residence. sera were tested for igg with a commercially available elisa. positive samples were confirmed by a commercially available dot blot which was also used for identification of the serovar. setting: the study area comprises north tyrol (austria, north of the main ridge of the alps), south tyrol (italy) and east tyrol (austria, both south of the main ridge of the alps). south tyrol belongs to the catchment area of the etsch river, which drains into the adriatic, while north-and east tyrol are part of the catchment area of the danube, which drains to the black sea. results: none of 617 samples from the italian part of the study area yielded a positive result, wherein 7 of 988 donors of the austrian part turned out to be seropositive. two patients were positive for hantaan, 3 patients were positive for puumala, one patient was positive for dobrava and one patient had antibodies against hantaan and dobrava. only one of those patients reported extensive travelling abroad. conclusions: evidence was found for the occurrence of hantaviruses in the austrian part of the region covering the catchment area of the danube, but not in the italian part of the study area covering the catchment area of the etsch river. seropositivity to hantaviruses differs by hydrogeographic areas. objectives: canine coronavirus (ccov) is an enveloped, singlestranded rna virus, belonging to group i coronaviruses within the family coronaviridae. two different ccov genotypes have been recognised, that are designated ccovs type i and type ii on the basis of their genetic relatedness to feline coronaviruses (fcovs) type i and type ii, respectively. ccov is usually responsible for mild, self-limiting infections restricted to the enteric tract. we report the molecular characterisation of a pantropic variant of ccov that caused fatal disease in pups. methods: ccov type ii strain cb/05 was isolated from an outbreak of fatal disease affecting seven dogs housed in a pet shop in apulia region, italy and characterised by fever, lethargy, inappetance, vomiting, haemorrhagic diarrhoea, neurological signs, and severe lesions in the parenchymatous organs. in all tissues, ccov antigen was detected by immunoistochemistry and ccov type ii rna was identified by genotype-specific realtime rt-pcr. the 3' end of the genome of strain cb/05 was determined by amplification of seven partially overlapping fragments. the pcr-amplified products were subjected to direct sequencing and the obtained nucleotide (nt) sequences were assembled and analysed using the bioedit software package and the ncbi's and embl's analysis tools. genbank accession number dq112226 was assigned to the sequenced 8.7-kb fragment. the inferred amino acid sequences (aa) were compared to the analogous proteins available in the online databases. results: the structural proteins s, e, m, n of strain cb/05 displayed a high degree of aa identity to the cognate orfs of ccov type ii, although the s protein showed the highest identity to type ii fcovs. while the nonstructural protein (nsp) 3a had the same length of known ccovs, the nsp3b was 49-aa shorter than expected due to the presence of a 38-nt deletion at position 4704 and to a frame shift in the sequence downstream the deletion that introduced an early stop codon. conclusions: association of strain cb/05 to a severe, fatal disease of dogs, together with virus isolation from organs with remarkable lesions, strongly suggests that this virus has changed the tropism, acquiring the ability to spread from the enteric tract to the internal organs. by sequence analysis of the viral genome, the only striking change was the truncated form of nsp3b, but the role of the deletion in the orf3b in determining the patho-biological change deserves more in-depth investigation. objectives: to perform a surveillance study for sars coronavirus (sars-cov)-like virus in non-caged wild animals from the wild of hong kong special administrative region (hksar). methods: from summer 2004 to spring 2005, 127 bats, 60 rodents and 20 monkeys from 11 locations in hksar were captured. nasopharyngeal and anal swabs and blood samples were collected and tested for sars-cov-like virus rna by rt-pcr using conserved primers targeted to a 440-bp fragment of the rna-dependent rna polymerase (pol) gene. the complete genome of the sars-cov-like virus from bats (bat-sars-cov) was sequenced using rna extracted from three anal swabs of three bats as template. phylogenetic tree construction was performed using neighbor-joining method with growtree using jukes-cantor correction. prediction of signal peptides and cleavage sites was performed using signalp, transmembrane domains using tmpred and tmhmm, potential n-glycosylation sites using scanprosite and protein family analysis using pfam and interproscan. antibodies were detected using a recombinant bat-sars-cov nucleocapsid protein enzyme immunoassay and neutralization assay for human sars-cov. results: we identified a coronavirus closely related to sars-cov (bat-sars-cov) from 23 (39%) of 59 anal swabs of wild chinese horseshoe bats by rt-pcr. sequencing and analysis of three bat-sars-cov genomes from samples collected at different dates showed that bat-sars-cov is closely related to sars-cov from humans and civets. phylogenetic analysis showed that bat-sars-cov formed a distinct cluster with sars-cov as group 2b coronaviruses, distantly related to known group 2 coronaviruses. most differences between the bat-sars-cov and sars-cov genomes were observed in the spike gene, orf 3 and orf 8, which are the regions where most variations were also observed between human and civet sars-cov genomes. in addition, the presence of a 29-bp insertion in orf 8 of bat-sars-cov genome, not in most human sars-cov genomes, suggests that it has a common ancestor with civet sars-cov. antibody against recombinant bat-sars-cov nucleocapsid protein was detected in 84% of chinese horseshoe bats using an enzyme immunoassay. neutralizing antibody to human sars-cov was also detected in those with lower viral loads. conclusion: our data support the existence of sars-cov-like virus in chinese horseshoe bats in hksar. noroviruses are genetically heterogeneous and form at least 27 genotypes within 5 genogroups, gi, gii, giii, giv, and gv, based on the capsid genes. human novs cause an estimated 23 million cases of illness annually in the united states alone and >90% of nonbacterial epidemic gastroenteritis worldwide. porcine calicivirus have been found to be genetically similar to human gii novs or to sapoviruses but calicivirus rna has been detected at low frequency by rt-pcr in adults or fattening pigs. the close genetic relationships between human and porcine novs raise public health concerns regarding their potential for zoonotic transmission and as a potential source of new epidemic human strains. methods: a total of 46 faecal samples of nursing and weaning piglets with enteritis were collected during 2003-2005 in porcine herds in italy. an additional 44 samples were include in the analysis, that had been collected during a rotavirus (rv) surveillance study in 2003-2005, all which tested positive to rv by electron microscopy and by rt-pcr. viral rna was extracted by the guanidine thiocyanate/glass milk method to eliminate enzyme inhibitors. primer pair con2-con3, targeted to the vp4 outer capsid protein, was used for rv detection. a degenerated version of primer pair 289/290 was used for nv detection, that targets a conserved region in the rnapolymerase. results: nov rna was detected in 20/46 of the screened samples, while rvs were detected in 24/46 samples. mixed infections nov+rv were found in 10 samples. screening of the 44 rv positive samples allowed detection of 15 mixed infections with novs. conclusions: in previous investigations novs were detected in 4 of 1,017 normal slaughtered pigs in japan, in 2 of 100 pooled pig faecal samples of 3-to 9-month-old fattening pigs in the netherlands and in 6 out of 274 healthy adult and finisher pigs in the united states. interestingly, in this study a high rate of positivity to novs (35/90) was found in nursing and weaning piglets with diarrhoea, a finding that may suggest a higher frequency of infection by nov in young pigs or an association between nov infection and occurrence of enteric disease. altogether, these findings demonstrate that novs are common in porcine herds in italy and provide new insights into the ecology of novs. detection of calicivirus genome in calves using ni/e3 primers m. mahzounieh, t. zahraeisalehi, e. moghtadaei khorasgani (shahrekord, tehran, ir) caliciviruses may cause a wide spectrum of disease in animals and are important etiological agent of viral gastroenteritis in humans. members of the family caliciviridae are small nonenveloped viruses 27 to 35 nm in diameter. they possess a single stranded poly adenylated rna genome. caliciviruses have been isolated from mink, dog, cattle and non-human primates. "norwalk-like viruses" (nlvs) are the most common cause of acute non-bacterial gastroenteritis in humans. cattle may be a reservoir of nlvs although never bovine nlvs have been found in humans. in this study, we try to detect enteric caliciviruses genome from 50 faecal samples of 6 dairy cattle herds in shahrekord area using reverse transcriptase polymerase chain reaction (rt-pcr) assays specific for nlvs found in humans. the primers used for pcr amplification were ni and e3, which amplify a 113-bp product for the detection of both genogroups i and ii srsv rna in fecal material. our results showed that nine specimens (18%) were positive. these findings suggest that calicivirus infection is endemic in dairy herds in shahrekord, iran and may be have an important role in calf diarrhea. objectives: reoviruses are non-enveloped, 10-segmented dsrna viruses. in humans and mammalians three distinct serotypes exist, whose prototypes are strains lang (t1), jones (t2) and dearing (t3). although reoviruses have been isolated both from the enteric and respiratory tract, no diseases has been clearly associated to reovirus infection in humans. the potential association with extra-hepatic biliary atresia, myocarditis, and, above all, neurological and cutaneous diseases require further investigations. reoviruses are ubiquitous and scarcely speciesspecific. reovirus identification is usually based on electronic microscopy or gel electrophoresis and reovirus incidence seems to be very low in humans and most mammalians. in this study, we investigated the presence of reoviruses in dogs by means of molecular methods. methods: one hundred ninety-two rectal samples from dogs with diarrhoea, 12 ocular swabs, 19 nasal swabs and 27 oropharyngeal swabs from dogs with ocular/nasal discharge were subjected to an rt-pcr assay targeting a conserved region of viral genome segment l1 (primers l1-rv5/l1-rv6). positive samples were characterised by polyacrylamide gel electrophoresis (page), serotype-specific rt-pcr assays targeting segment s1 and sequence analysis. to increase the sensitivity, a nested pcr using primers l1-rv7/l1-rv8 was performed on samples tested rt-pcr negative. results: only 4 faecal swabs (2.1%) were found positive (rt-pcr product of 416 bp). by using a serotype-specific rt-pcr assay and/or sequence analysis, two strains was characterised as type 3 and the other ones as type 1. page of viral dsrna confirmed the genetic characterisation. unexpectedly, in secondround pcr 50 faecal samples (26%), 9 ocular swabs and 10 nasal swabs yielded a 344 bp product, while no oropharyngeal swab was positive. conclusions: these data suggest a wider distribution of reoviruses in dogs than previously thought, even if most reovirus infections were detected only by nested pcr. the ability of reoviruses to induce disease in dogs, alone or in synergism with other pathogens, is still unclear, since attempts to reproduce a specific disease in germ-free dogs have given contradictory results. due to their poor species-specificity, reoviruses may be easily transmitted from animals to humans (and vice-versa). further studies are required to understand reovirus ecology and their potential zoonotic impact. objectives: parvovirus b19 is a member of a family parvoviridae. on the basis of genetic distances and evolutionary relationships, human parvoviruses are divided into three genotypes: genotype i corresponding to b19-related isolates, genotype ii to lali-related isolates, and genotype iii to v9-related isolates. parvovirus b19 causes a common exanthematous disease in childhood or adult age, arthropathy, hydrops fetalis, various haematological disorders and myocarditis. up to now, we have had no data of the prevalence of b19 virus in slovenian population. consequently, we also lack information on the genotypes of parvovirus b19 that are involved in the patients who suffer from the infection. methods: to gather information of the genetic variants of b19 virus present in slovenia, we extracted dna from 48 serum samples that were sent for serologic diagnostic of parvovirus b19 infection and were positive for specific igm in the period from january 1997 to june 2005. nearly half of all 48 patients were children and young adults up to 25 years. the ns1 region of parvovirus b19 was amplified by the nested pcr (primers pb19f1, r1 and pb19f2, r2). all pcr products were directly sequenced. the results of our study show that dna of parvovirus b19 was present in all 48 samples that tested positive for specific igm antibodies. after the first round of pcr reaction, 17 samples were positive, and after the second reaction, all 48 samples were positive. altogether 12 unique genotype variants of parvovirus b19 were identified and all were clustered in the genotype i group of b19-related isolates. most of the distinct 12 genetic variants differed in 1% to 2% from the sequences deposited in gene bank. the majority of sequences obtained from the b19 virus epidemic in 1998 represents a single variant of genotype i with the gene bank acc. no. aj781038. we also found that different genetic variants of parvovirus b19 were circulating in 2002 and were 100% or 99% identical to the genotype i variant with the gene bank acc. no. z68146. in our study, we were not able to identify any variants of other rare genotypes (lali or v9). conclusion: parvovirus b19 dna was successfully amplified from all igm positive serum samples of the patients. the genotype i of parvovirus b19 is dominating in infections with parvovirus b19 in slovenia. objectives: great britain has been free of animal brucellosis since 1985 (european commission decision 93/52). the main source of infection for uk residents is through contact with infected material in foreign countries. the objective of this study is to type human samples received in the uk since 1999 using variable number tandem repeats (vntr) molecular typing to confirm results obtained by classical typing and relate these results to the suspected source of infection. methods: classical typing is traditionally used and is based on the phenotypic attributes of each strain and biovar. vntr typing is a recently developed molecular method, which is based on short repeats contained in the dna that can be amplified to give a banding pattern specific to each strain. results: results found using both methods are consistent. the results show geographical differences, consistent with observations of strain genotype distribution found in animal brucellosis. conclusion: patient history has been gathered where possible giving information on recent travels. along with results found by classical typing and confirmed by vntr typing we can draw a picture of the sources of infection. these results illustrate the potential of vntr typing as a tool to aid conventional approaches to epidemiological traceback that, in the presence of a suitably comprehensive database of strain genotypes, could help identify the source of an infection. is711-fingerprinting of brucella isolates from humans e.stubberfield (surrey, uk) objective: brucellosis is a zoonotic disease usually associated with cattle, sheep, goats and pigs. human infection has been attributed to b. melitensis, b. abortus, b. suis, b. canis and b. maris. although the uk is officially bucellosis free there are a number of human cases due to travel and occupation that are submitted to our laboratory for diagnosis. definitive diagnosis of brucella is by bacteriological culture and microbial tests (classical typing), however these require skilled personnel and the results can be subjective. there are a number of molecular tests that have been developed to assist with diagnosis more rapidly and in some cases to strain level less subjectively. is711-fingerpinting is a molecular technique that has proved useful for the identification of brucella isolates to species and in some cases stain level. is711-fingerprinting relies on the variable number and location of the is711 mobile genetic element found in all brucella isolates. method: 73 brucella isolates from humans have been tested. genomic dna was extracted, digested using restriction endonuclease ecor1, and electrophoresed. southern blotting was performed, hybridising with a dig-labelled is711 probe. results: the number of brucella is711 copies range from 5 to more than 20. brucella melitensis remains the most commonly acquired brucella species of travellers, while occupational infections have included b. abortus isolated from cattle farmers and b. suis associated with pig butchers. two marine brucella strains have been isolated originating from an occupational perspective (a laboratory worker) and a natural setting from an unknown source. 7 unusual patterns have been observed, 5 of which are unique. one of the new patterns has been observed only in isolates originating in east african countries. conclusion: although the diagnosis of brucella to species and strain level is not essential for the treatment of human brucellosis, it is useful for epidemiological studies. is711fingerprinting is able to identify the three biovars of b. melitensis, many other techniques do not offer this capability, because of this it may be a useful test in epidemiological studies. this method remains an important diagnostic tool for brucella identification. rapid diagnosis of brucellar epididymo-orchitis by real-time pcr assay in urine samples objectives: to study the diagnostic yield of a real-time pcr assay in urine samples for the rapid diagnosis of brucellar epididymo-orchitis, in comparison with conventional microbiological techniques. methods: ten consecutive patients with brucellar epididymoorchitis were included in the study. the diagnosis of brucellosis was established according to one of the following criteria: first, isolation of brucella spp in blood or any other body fluid or tissue sample or, second, the presence of a compatible clinical picture together with the demonstration of specific antibodies at significant titers or seroconversion. epididymo-orchitis was diagnosed in patients with scrotal enlargement, swelling and pain not due to other causes.for dna amplification we used a sybr green i lightcycler-based real-time pcr assay. the assay amplifies a 223 bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein (bcsp31). the pair of 21 nucleotide primers b4 (5' tgg ctc ggt tgc caa tat caa 3') and b5 (5' cgc gct tgc ctt tca ggt ctg 3) were used in the amplification process. after dna amplication, we performed melting curve analysis to verify the specificity of the pcr products. in order to study the specificity of the technique, all the samples from the patients with brucellosis were paired with an equal number of samples from controls with urinary tract infection. (e. coli four cases, k. pneumoniae two cases, p mirabilis two cases, and c. freundii and p. aeruginosa one of each). results: the mean age was 38.4 years (range 21-69). the duration of the symptoms prior to diagnosis was 30.2 ± 16.2 days (range: 5-51). b. melitensis was isolated from blood cultures in nine cases (90%). wright's seroagglutination was negative or inconclusive in 30% of cases. brucella was isolated from urine in only one case whereas real-time pcr assay in urine was positive in nine (90%) cases and the results were available in four hours, whereas the mean time to availability of the final blood culture results was 5.8 days (range 4.5-7 days). real-time pcr was negative for all the control samples from patients with urinary tract infections. conclusion: sybr green i lightcycler-based real-time pcr assay in urine samples is highly sensitive and specific, easy to perform and could provide the clinician with the results in under five hours. the technique could be a practical and useful tool for the rapid diagnosis of genitourinary complications of human brucellosis. objectives: although the united kingdom remains brucellosisfree, there are more than 500,000 new cases of human brucellosis reported each year according to the world health organisation. uk residents returning from worldwide travel may have encountered exposure through contact with infected animals and animal products such as dairy produce and meat. phenotypic characterisation or classical methods remain the definitive diagnosis though require skilled personnel and have their limitations. the increasing range of molecular techniques can aid the rapid detection and characterisation of brucella species and their biovars and may have significance in epidemiological studies. methods: a study of brucella reference and field strains of mainly human isolates from different geographic locations were analysed for diversity of their genes encoding the outer membrane proteins (omps) 25, 2a and 2b. pcr products of the three genes digested with seven restriction enzymes were analysed for polymorphisms. results: a re-occurring unique pattern profile seen only in human isolates was observed originating in some european countries and beyond. a growing database of strain types giving a recent overview of brucella infection of humans of many countries. conclusions: molecular typing methods may have an advantage over classical typing concerning brucella melitensis, the most common brucella infection of humans. the characterisation of human brucella isolates may be useful in epidemiological studies for a variety of purposes. objectives: a study to demonstrate the rapid detection and speciation of campylobacter jejuni and of campylobacter coli isolates directly from enrichment broth using a taqman ò assay. single nucleotide polymorphism analysis of mapa positive strains was used for rapid identification of c. jejuni clonal complexes. methods: thermotolerant campylobacter species were initially confirmed by culture according to the modified draft iso 17995 method, where water samples were filtered through 0.2 mm pore size nylon membrane. the filters were transferred to selective enrichment in preston broth to improve their recovery and therefore detection of any campylobacter cells present. dna was extracted directly from the enrichment broth culture for real-time detection of c. jejuni and c. coli using the taqman ò . samples, which were map a positive were, further characterise by single nucleotide polymorphism profiling for rapid recognition of c. jejuni clonal complexes. results: environmental samples, which were confirmed by culture were also map a positive by taqman ò . snp profiling of mapa positive isolates identified clonal complexes, which are predominantly contained in isolates of human disease and chicken. conclusions: this study has demonstrated the feasibility of rapid detection and identification of c. jejuni and c. coli following short enrichment incubation using a taqman ò assay. a rapid turnaround time of between 3-4 h per batch of 96 samples was achieved. snp profiling offers important epidemiological grouping at strain level, enabling accurate and phylogenetically valid strain identification for c. jejuni, which may have important host associations for tracing sources of infection and consequently improve public health responses. objectives: campylobacter jejuni and c. coli are recognized as the most common causes of acute bacterial gastroenteritis in humans, c. jejuni being the predominant species in most developed countries. the hippurate hydrolysis test is widely used to differentiate c. jejuni from other campylobacter species. about 10% of c. jejuni isolates fail to hydrolyze hippurate under laboratory conditions. molecular methods represent an alternative to the phenotype-based methods. we tested two multiplex pcr assays for species identification of human campylobacter strains and compared the results with the hippurate hydrolysis test. methods: campylobacter strains isolated from patients were tested for hippurate hydrolysis with rosco diagnostic tablets. 50 hippurate-negative and 15 hippurate-positive strains were selected for two multiplex pcr assays. one pcr-method was based on distinctive ceue-genes of c. jejuni and c. coli, the other pcr-method detected genes from five major clinically relevant campylobacter species: hipo from c. jejuni, glya from c. coli, c. lari and c. upsaliensis, sapb2 from c. fetus subsp. fetus, and 23s rrna gene from campylobacter spp. as an internal validation control. results: the c. jejuni hipo gene was detected in all of the 15 hippurate-positive strains and 10 of the 50 hippurate-negative strains. the c. coli glya was detected in 39 of the hippuratenegative strains. in one hippurate-negative strain, sapb2 from c. fetus subsp. fetus was detected. species-specific genes were detected in 53 of the 65 strains with the ceue-based pcr assay. c. jejuni ceue was detected in 12 hippurate-positive and 5 hippurate-negative strains. c. coli ceue was detected in 36 hippurate-negative strains. conclusion: all hippurate-positive strains were identified as c. jejuni. of the hippurate-negative strains, 78% were identified as c. coli, whereas 20% were identified as c. jejuni and one strain as c. fetus subsp. fetus. the results of the two pcr assays were concordant, although some strains could not be identified with the ceue-based pcr assay. the results suggest that molecular species identification should be performed on hippuratenegative strains after the hippurate hydrolysis test for accurate species identification. multiplex-pcr is quick and easy to perform. using the pcr assay that simultaneously detects five campylobacter species also diminishes the need for further phenotypic testing. phenotypic typing of cryptosporidium species isolated from children in kuwait: a role in unique transmission j. iqbal, p. hira (safat, kw) background: cryptosporidiosis is recognized worldwide as a significant cause of diarrhoeal diseases in both adults and children especially in children less than 2 years of age. objective: cryptosporidium spp. isolated from young children in kuwait were characterized at the molecular level to understand the transmission of infection. the study was approved by the ethical committee, faculty of medicine, kuwait. methodology: over a period of 2 years, faecal specimens from 97 kuwaiti children with persistent diarrhoea found to be positive for cryptosporidium spp. by microscopy were genotyped and sub-typed with a small subunit rrna-based pcr-restriction fragment length polymorphism analysis. informed consent was taken from all individuals included in the study. results: the median age of infected children was 4.9 years, and the majority of the infections (>70%) occurred during the cooler months january-april, indicating a marked seasonal variation. more than 85% of the children with cryptosporidiosis had only cryptosporidium infection. socio-demographic information did not reveal any particular mode of transmission of infection. genotyping of the organisms isolated showed that ninety-two (95%) of the children had c. parvum, 4 (4%) had c. hominis, and 1 (1%) had both c. parvum and c. hominis. altogether, 9 subtypes of c. parvum and c. hominis were observed. objectives: the intracellular respiratory pathogen chlamydophila pneumoniae (cp) might be involved in the pathogenesis of atherosclerosis. several studies have demonstrated a serological association between cp and cardiovascular disease and dna from the bacteria has been found in various atheromatous vessels. after infection in the respiratory tract, cp is believed to be disseminated systemically within alveolar macrophages. the prevalence of cp within peripheral blood mononuclear cells (pbmc) has in some studies been shown to be higher in patients suffering from cardiovascular disease than in control patients. we investigated the presence of cp dna in aortic heart valves and pmbc in 55 patients (37 men; 18 women; mean age 69 years) undergoing aortic valve replacement because of aortic stenosis. also, the presence of cp mrna was investigated in the sclerotic aortic heart valves as a marker of viable bacteria. methods: dna was extracted from aortic valve biopsies and pbmc using the qiaamp dna mini kit (qiagen). mrna and dna were extracted from another piece of the same biopsy using trizol (invitrogen). real-time pcr directed against the chlamydia momp gene was used to detect cp-specific dna and mrna. patient sera were tested for cp-specific igm, igg and iga antibodies by the microimmunofluorescence technique. results: cp dna was found in aortic heart valves from 20% (11/55) of the patients and in pbmc from 5% (3/55) of the patients. in one patient cp dna was found in both pbmc and heart valve. no patient had cp-specific igm antibodies. in patients that were pcr-positive for cp dna in the aortic heart valves, 73% had igg ‡1:64 and 45% had iga ‡1:32. in patients that were pcr-negative in the aortic heart valves, 59% had igg ‡1:64 and 23% had iga ‡1:32. cp-specific mrna in aortic heart valves will be presented on the poster. conclusion: cp-specific dna was found in sclerotic aortic heart valves from 20% of patients undergoing aortic valve replacement. this confirms previous investigations supporting a role for cp in the pathogenesis of aortic valve sclerosis. the prevalence of cp in pbmc was 5% which is comparable to that reported in healthy blood donors and lower than that recorded in patients suffering from other cardiovascular diseases. if the bacteria are involved in the pathogenesis of aortic sclerosis they have likely been spread to the aortic valve long before the patient is in need of surgery because of the stenotic valve. introduction: diarrhoea is one of the most common causes of morbidity and mortality among young children in developing countries. diarrheagenic e. coli strains include several emerging pathogens of worldwide public health. six important categories are entero-aggrigative e. coli (eaec), entropathogenic e. coli (epec), enterotoxigenic e. coli (etec), enterohemorrahgic e. coli (ehec), entroinvasive (eiec) and shigatoxin-producing e. coli (stec). this study investigated the role of different diarrheagenic e. coli in iranian children with acute diarrhoea by molecular methods and the antibiotic susceptibility of isolated strains. methods: from april 2003 to january 2005, one thousand eighty five children with acute diarrhoea in tehran hospitals in were enrolled in the study. the fecal samples were cultured on macconkey for conventional bacterial pathogen and sorbitol macconkey agar for non sorbitol fermenting phenotype, than they were incubated in 37ordm;c. the primary stool cultures were subjected to six different pcr reactions targeting stx1 and stx2 gene, heat-labile enterotoxin (lt) producing gene, heatstable enterotoxin (st) producing gene, eae gene and pcvd432 plasmid. the kirby -bauer disc diffusion method was used for antibiogram of 110 isolated strains from different diarrheagenic e. coli by 12 different antibiotics. results: two hundred seventy one diarrheagenic e. coli strains were detected. stec was the most prevalence with 125 (46.1%). the frequency of other strains was 28.7%, 16.3%and 8.8% for etec, eaec and epec, respectively. out of 120 stec isolated 86 strains (% 68.8) had stx1 or stx2 gene, and 4 strains had stx1 and stx2 gene. the eae gene was found in 13 (10.4) stec strain. out of 110 tested strains, 102(92.5%) were resistance to ampicllin and cefalotin, and 99 (90%) to streptomycin. conclusion: in this study stec was the most frequent associated with diarrhoea. the strong association between use of antibiotics and colonization with antibiotic resistant e. coli, suggest a major role for selection of resistant strains while using antibiotics. the existence of other unknown intestinal adherence factors has been suggested by the isolation of stec strains that lack the eae gene but are still associated with bloody diarrhoea or hemolytic ureamic syndrome (hus). since there is no specific treatment, there is an urgent need for effective preventive measures based on detailed understanding of the epidemiology of stec infections. identification of shiga toxin-producing escherichia coli in raw beef using dna hybridization with digoxigenin-labelled probes and multiplex pcrs m. weiner, j. osek (pulawy, pl) shiga toxin-producing escherichia coli (stec) is an important cause of bloody diarrhoea, haemorrhagic colitis, haemolytic uremic syndrome and thrombotic thrombocytopenic purpura. transmission of stec occurs through consumption of contaminated food, especially meat, dairy products and water. objectives: to develop a three-steps procedure based on two multiplex pcrs and dna hybridization with digoxigeninlabelled probes for identification of stec in raw beef. methods: beef samples inoculated with different number of e. coli o157:h7 cells were incubated in tsb medium at 37°c for 6 h. the cultures were then transferred to tsb with mitomycin c and incubated for another 18 h. the resulted cultures were used as a source of dna template. the mpcr-1 was established to identify shiga toxins genes (conserved sequence). the positive culture samples were subjected to dna hybridization with dig-labelled probes as follows: the culture was diluted and inoculated onto agar plates supplemented with tergitol ò and incubated at 37°c for 18 h. then, the nylon membranes were put on agar plates, carefully removed and incubated in denaturation, neutralisation and equilibration solutions following incubation with the stx-specific dig-labelled probes, anti-dig conjugates and finally developed with enzyme substrates (bcip and nbt). dark spots visible on the membranes were compared with the respective bacterial colonies on the original agar plates. the corresponding bacterial colonies were isolated and characterized using the mpcr-2 test which allows amplification of stx1 (shiga toxin type 1), stx2 (shiga toxin type 2), rfbo157 (e. coli o157) and flich7 gene (h7 antigen). an internal control of amplification (e. coli 16s rrna gene) was also included in both mpcr tests. results: the first mpcr resulted in two amplification products: 230 bp for stx and 401 bp for 16s rrna genes. the positive meat samples were further tested with dna probes and positive colonies were then characterized with the second test (mpcr-2), generating the amplicons either of 348 bp (stx1), 584 bp (stx2), 420 bp (rfbo157), 247 bp (flich7) or 798 bp (16s rrna). the specificity of this procedure was confirmed by testing e. coli o157:h7, o157:h-and non-stec bacteria. the sensitivity of the method was estimated as 4 cfu/g of meat. conclusion: the obtained results demonstrated the high specificity of the procedure developed and the possibility of using it for identification of shiga toxin-producing e. coli in raw beef. correlation between virulence pattern, phylogenetic group and extended spectrum betalactamases genes in escherichia coli strains isolated from blood cultures m. damian, c. usein, d. tatu-chitoiu, s. ciontea, d. jardan, a. palade (bucharest, ro) e. coli, heterogeneous species consisting of commensal and pathogenic strains, is causing a broad spectrum of human diseases, including extra intestinal and enteric infections.the strains isolated from invasive infections were documented to be carriers of a large number of genetic structures coding for virulence, as well as for resistance to antimicrobial agents. the aim of this study was to evaluate the virulence of strains in comparison with the presence of esbl genes and their distribution among the different phylogenetic groups. a total of 51 e. coli strains, isolated from blood cultures, in hospitalised patients, adults and children, were screened for virulence factors-encoding genes (pap, sfa/foc, afa, hly, cnf, aer and fimh), for genes encoding resistance to extended spectrum betalactam antibiotics (bla shv and bla tem genes) and the appurtenance to one of the main four phylogenetic group based on presence or absence of markers chua, yjaa and tspe4.c2 three strains, negative for all virulence genes, were included in the phylogenetic group a. ten strains, which were positive for five or six virulence genes, were identified as b2 group. no matter the phylogenetic grouping, the remaining strains possessed at least one virulence gene. no strain was pcr positive for all seven virulence genes targeted. among the 16 strains which were positive in the double disk test, 12 strains exhibited both bla shv and bla tem genes and 4 strains only bla tem gene. restriction with pst i and dde i and sequencing of the amplicons were performed in order to identify the type of esbl gene expression product. taking into account the link between phylogenetic group and virulence, we obtained a good correlation for the bacteremic e. coli strains analysed, but there was no relationship with the production of esbls. isolation of shiga-toxin producing escherichia coli from meat samples, phenotypic and genotypic characterisation of isolated strains f. baghbani-arani, f. jafari, m.r. zali, s. salmanzadeh-ahrabi (tehran, ir) objective: shiga-toxin producing escherichia coli (stec) is an emerging foodborne pathogen of worldwide public health importance. this bacterium has been reported as an etiological agent of many outbreaks and sporadic cases. definition of the diversity and antimicrobial susceptibility of (stec) may be helpful in the management of sporadic cases and outbreaks. studies in different countries show that food items maybe contaminated by this pathogen. the present study was carried out to determine the frequency of contamination of meat samples by stec collected in tehran as well as defining genotypes, serotypes, antibiogram susceptibility patterns and molecular diversity of isolated bacteria. methods: from july 2004 to june 2005, 250 beef samples were collected from different part of tehran. a 25 grams of each samples was enriched in ec broth and subculture on mac-conkey agar. dna was extracted from a loop full of bacteria taken from primary first streaking area of mac-conkey agar and was subjected to three different pcr reactions targeting stx1, stx2 and eae genes. as much as colonies required were tested for finding the colony responsible for positive results in the first pcr. antibiogram susceptibility patterns of isolated strains were determined by standard disk diffusing method. the 17 antimicrobial agents were used at this study. all isolates were serotyped by slide agglutination test using standard antisera (mast groups) subtyping of strains was done with rapd-pcr by 1283primer. results: among 250 samples, 47 (19%) samples were positive and their genotypes were as follow: 1(0.4%) stx1+, stx2-, eae-, 35(14%) stx1-, stx2+, eae-, 6(2.4%) stx1-, stx2+ and eae+.4 (1.6%) stx1+, stx2+, eae-, 1(0.4%) stx1+, stx2+, eae+. among these positive samples 30 strains were isolated. according to the antibiotic susceptibility tests, all isolates were resistance to erythromycin (e) and oleandomycin (ol), and were sensitive to imipenem (i); gentamicin (g) norofloxazin (nx) enterofloxazin (ex) ciprofloxazin (cf) and ceftazidim (ca). in otyping and htyping the most frequency were o112ac and h2 serotypes. analysis of isolates by rapd-pcr yielded 17different patterns. conclusion: our results show that contamination of meat samples by stec is a life-threatening health problem. combinational analysis of antibiogram susceptibility patterns and serotypes with rapd-pcr patterns can aid to survey the characteristics of stec strains. factors affecting the conjugative transfer of plasmid pip501 in enterococcus faecalis a.m. al-qurashi (dammam, sa) objectives: factors which are known to influence plasmid transfer were studies using the conjugative plasmid pi501, which encodes erythromycin resistance, in enterococcus faecalis. methods: the donors strains streptococcus a agalactiae v666 (group b) is resistant to in rifampicin and fusidic acid, non hemolytic and b-lactamase-negative. it contains the broad host range plasmid pi501, which confers resistance to erythromycin and chloramphenicol. the recipient is enterococcus faecalis strain jh2-2 group d. results: transfer of pip501 occured on a agar, on filters and in broth cultures at relatively high densities (106-108 bacteria/ml). transfer frequency was largely unaffected over a wide range of temperatures (23-42°c). the ph of the medium, in the range ph 5-9 had little effect on the transfer frequency. log phase cultures and donor: recipient ratios of 1:1000-100:1 were required for optimal for plasmid transfer. conclusion: factors which modified the transfer efficiency of the conjugative plasmid pip501 were mating media, solid or liquid environment, mating time, mating temperature, selection temperature, growth temperature of donor and recipient of prior to mating, ph culture age, and donor/recipient cells ratio, to obtain a better understanding of this plasmid and its transfer process will help understand what role they may have in the dissemination resistance among streptococcal and enterococcal populations. enterococcus faecium blood-culture isolates collected during a five-year period h. billströ m, å . sullivan, b. lund (stockholm, se) background: enterococcus faecalis and enterococcus faecium have during the last years become a significant nosocomial problem. this could be due to the enterococcus hardy nature combined with intrinsic and acquired antibiotic resistance. since most individuals harbour enterococci in their normal intestinal microflora there has been a discussion regarding the origin of these isolates. during the last ten years the isolation ratio between e. faecalis and e. faecium have shifted from 10:1 to 3:1. this could be because of increasing antibiotic resistance among infectious e. faecium isolates compared to infectious e. faecalis ones. it is possible that this increase also depends upon different virulence genes such as enterococcal surface protein (esp), hyaluronidase variant gene (hylefm) and e. faecalis antigen a variant (efafm). objectives: the objectives in this study were to determine the presence and frequencies of seven different enterococcal virulence genes in infectious isolates. further objectives were to see if the number of virulence genes in these isolates vary or increase over time. methods: a total of 257 strains isolated from bacteraemia patients during year 2000-2005 at the karolinska university hospital, huddinge were used. all isolates were screened for seven different virulence genes using a multiplex pcr. these seven virulence genes were aggregation substance (asa), cytolysin (cyt), collagen binding protein (ace), e. faecalis antigen a variant (efafm), enterococcal surface protein (esp), gelatinase (gel) and hyaluronidase (hyl). results: according to the results about half of all isolates were esp-positive. the prevalence of the other virulence genes asa, efafm, gel and hyl were detected, but in low frequencies (<5%). conclusion: it seems like the esp gene is the most dominant virulence gene in e. faecium isolates. the occurrence of virulence traits in these isolates further indicates that the potential to cause infection is potentiated among this enterococcal population the data from this investigation supports the hypotheses that enterococci causing infection in hospitalized patients are probably of nosocomial origin rather than endogenous. objectives: the ability of l. monocytogenes to tolerate alkaline stress is of particular importance, as this pathogen is often exposed to such stress in food processing environments cleaned with alkaline detergents or in the mildly alkaline ph values which prevail within engulfing phagolysosomes. this study aims to investigate the alkaline tolerance response (altr) in listeria monocytogenes 10403s using dna microarray technology. knowledge of the alkaline-induced stress response will be useful in understanding how this pathogen tolerates alkaline stress. methods: transcription profiling of l. monocytogenes 10403s was carried out at 15, 30 and 60 min at high ph in order to capture an early, an intermediate and a prolonged expression response to alkaline stress using oligo arrays from the pathogen functional genomic resource centre. to verify the microarray results the regulation of some ph stress response genes were confirmed by real time quantitative polymerase chain reaction (rt-pcr). results: about 1584 genes were upregulated and 1754 genes (of 6347 open reading frames represented on the arrays) were down regulated at least 1.5 fold upon alkaline shock. many of the repressed genes encode enzymes that are involved in the biosynthesis of amino acids, nucleotides and coenzymes, indicating a metabolic adjustment of the cells to the high ph. notably, the strongest alkaline-inducible genes were involved in the membrane transport systems. conclusion: the analysis of the data revealed that cells sense and respond to alkaline stress with an extensive program of changes in gene expression. interestingly, there is a strong correlation between the altr and virulence gene expression. comparison to various microarray data already in the literature revealed similarity between the response to alkaline stress and the transcriptional response to stresses such as osmotic shock. engineering improved listerial stress tolerance "with a twist"! r. sleator, c. hill (cork, ie) objectives: to engineer listeria monocytogenes strains with a significantly improved ability to tolerate stresses encountered in the external environment and during gastrointestinal transit, thus, improving listeria's efficacy as a potential vaccine and drug delivery platform. methods and results: using a directed evolution approach, based on a random mutagenesis strategy involving the e. coli xl1-red mutator strain, we generated a mutant variant of the listerial betl gene (designated betl*), encoding a secondary betaine uptake system. the mutant betl* promotes a dramatic increase in resistance to a number of biologically relevant stresses when expressed in a variety of different surrogate hosts. using a luciferase (lux) reporter system in combination with the ivis imager system (xenogen corporation, alameda, ca), we tracked betl* expression, in real time, both in vitro under various environmental stresses and in vivo in animal models of infection. in each case strains expressing betl* demonstrated a marked improvement over those expressing wild type betl, both in terms of gene expression and bacterial growth. sequence analysis of the mutated gene revealed a single nucleotide deletion in the spacer region between the -10 and )35 promoter elements upstream of the betl coding region. this deletion presumably introduces a conformational 'twist' in the putative promoter, thereby increasing its transcriptional output. furthermore, the betl* mutation appears to counter the heretofore unreported 'twisted' cell morphology observed using scanning electron microscopy of l. monocytogenes grown at elevated osmolarities. conclusions: it is possible to selectively improve genes required for bacterial stress survival both inside and outside the host. such mutated genes systems may ultimately be used for the construction of more physiologically robust bacterial based vaccine and drug delivery platforms. a.r. samarbaf-zadeh, s. tajbakhsh, s.m. moosavian (ahwaz, ir) introduction: peptic ulceration following infection of stomach with h. pylori is a common disease. accurate and rapid detection of the bacteria can lead to implementation of appropriate treatment and recovery. this research was undertaken to evaluate the sensivity and specificity of fluorescent in-situ hybridization (fish) in the detection of h. pylori in patients who were suffering from dyspepsia. methods: for this purpose, one hundred gastric biopsy samples taken from antrum and corpus of stomach by endoscopy were tested by fish and compared with conventional culture method complemented with biochemical tests. results: fish detected h. pylori in 48 clinical samples while conventional method detected 42 samples. the sensivity and specificity of fish for detection of h. pylori were calculated as 98% and 100% respectively. conclusion: the findings of this study suggest that fish is a highly suitable and rapid method for diagnosis of h. pylori, especially when the samples are taken from the antrum and the corpus of the stomach this technique potentially can be applied routinely for detection of this bacterium in clinical samples. objective: numerous studies have demonstrated that h. pylori is ubiquitous; approximately 50% of the world's population is infected with the organism. gastroduodenal diseases associated with h. pylori infection are manifested principally in adults. however, it's usually during chilhood that the infection is acquired, and it is possibile that mucosal and humoral responses at this time may determine, at least in part, the course of the natural infection. our study will describe the prevalence of the h. pylori oral carriage in children resident in bari, south of italy, using the pcr method. methods: the evaluation was performed in 404 children, with ages ranging from 6 to 11 years, from primary school district of local health unit of bari, italy (ausl ba/4). the school and the class have been selected using the cluster sampling method. a standardized questionnaire was used to verify socio-economic standard, hygiene and history of previous gastrointestinal disorder. a standard full-mouth examination was made to detect periodontal diseases, then dental plaque and saliva collected from children were placed in pbs and transported in laboratory. h. pylori infection status was checked by pcr method. dna was extracted from oral samples by the boiling method and evaluated for the presence of h. pylori caga and urea genes using commercial kit (ab analitica, padova). results: a total of 404 children (212 females and 192 males) partecipated to the study. the presence of gene coding for caga was found in 57 children (14%), but gene urea was detected only in 22 (5%). the bacteria was detected in saliva, supragingival and subgingival plaque, suggested that these sites may be considered reservoirs for h. pylori in ureasi-positive patients. there was statistically significant relationship between who didn't wash their hands frequently and the presence of urea gene (o.r. 1.71). conclusions: current knowledge implies that acquisition of h. pylori seems to occur predominantly in childhood and that once acquired the infection persists life-long in most infected subjects. it has been reported at a worldwide level that h. pylori infection prevalence in children varies between 10% and 80% and increases with low socio-economic and educational levels and age. the results of this study suggest that oral carriage of h. pylori may play a role in the transmission of infection and that the hand may be instrumental in transmission. the role of helicobacter pylori in otitis media with effusion t. yilmaz, m. ceylan, y. akyon, o. ozcakir, b. gursel (ankara, tr) objectives: otitis media with effusion (ome) is such a common disease of childhood and its pathogenesis still remains unsettled. pepsinogen and pepsin has been shown in the middle ear fluid of patients with ome, indicating that gastric juice could reach as far as middle ear. if gastric juice could enter the middle ear, helicobacter pylori, a common inhabitant of gastric juice and mucosa, would also be expected to be found in the middle ear of patients with ome. the objective of this study was to evaluate possible role of helicobacter pylori in pathogenesis of otitis media with effusion. methods: the study group consisted of 22 children who are to undergo bilateral ventilation tube insertion, adenoidectomy, tonsillectomy with a diagnosis of ome, adenoid hypertrophy and chronic tonsillitis. the control group consisted of 20 children who are to undergo adenoidectomy, tonsillectomy with a diagnosis of adenoid hypertrophy and chronic tonsillitis. for the study group, middle ear fluid was aspirated and a small biopsy was taken from the promontorium mucosa. for the control group, myringotomy was done and a small biopsy was taken from the promontorium mucosa. for both groups, 5 mm deep tissue specimens were obtained from tonsil and adenoid. for all the specimens taken from the patients, culture and a nested-pcr were performed to show helicobacter pylori. results: middle ear fluid culture was positive for h. pylori in 2 patients and mucosa culture was positive in 1 patient only. in the control group middle ear mucosa cultures were always negative. when culture and pcr results were combined together; the middle ear was positive for h. pylori in 10 patients in the study group and in 2 patients in the control group. this difference was statistically significant. h. pylori presence in the tonsillar and adenoid tissues by culture and pcr was also significantly more frequent in the study group compared to the control group. conclusion: this study is the first to grow h. pylori in the middle ear in ome. significantly increased colonization by h. pylori of the middle ear, tonsillar and adenoid tissue in patients with ome indicates that the bacteria reaching the middle ear through gastroesophageal reflux might be involved in the pathogenesis of ome. for ome cases resistant to medical treatment it may meaningful to evaluate the patient for gastroesophageal reflux and h. pylori. distribution of the serine-aspartate repeat protein-encoding sdr genes among nasal carriage and invasive staphylococcus aureus strains objectives: this study was designed to examine the distribution of the sdr genes among nasal carriage and invasive staphylococcus aureus strains as well as methicillinsensitive s. aureus (mssa) and methicillin-resistant s aureus (mrsa). methods: the presence or absence of the sdr genes using dna from 497 s. aureus strains was determined by a novel triplex pcr procedure. the two-tailed fisher's exact test was used to analyse the distribution of the sdr genes among s. aureus strains originating from different hosts. p values less than 0.05 were considered a statistically significant difference. results: the sdr locus was found in all 497 investigated s. aureus strains although in 29 strains it contained only the sdrc gene (sdrd -sdre-). the sdrc + sdrd -sdre-gene profile was exclusive to mssa strains (fisher's exact test; p = 0.0005) and was not found in the strains collected from bone infections (p = 0.0019). we also found a strong association between the presence of the sdrd gene and mrsa strains (p < 0.0001). conclusion: our findings suggest that mssa strains with the newly uncovered sdrc + sdrd -sdre-gene profile have a substantially decreased potential to establish bone infection. sequencing of luks-pv and lukf-pv in methicillin-sensitive and methicillin-resistant staphylococcus aureus of diverse genetic backgrounds in a swedish county c. berglund, b. sö derquist (ö rebro, se) objectives: community-aquired methicillin-resistant staphylococcus aureus (ca-mrsa) have been reported to carry the loci for panton-valentine leukocidin (pvl) in high frequency. the aim of this study was to describe variations within the pvl genes (luks-pv and lukf-pv) in methicillinsensitive and methicillin-resistant s. aureus of diverse genetic backgrounds. methods: twelve pvl-positive s. aureus were characterised by multilocus sequence typing (mlst) and mrsa also by staphylococcal cassette chromosome mec (sccmec) typing. ten of these were isolated between 1990-2005 in ö rebro county, sweden. oligonucleotide primers were designed to yield a product size of~2500 bp including luks-pv and lukf-pv and flanking regions by pcr amplification. cyclic sequencing was performed with several sets of primers to overlap the sequences on both strands and was separated on abi prism ò 3100 genetic analyzer (applied biosystems). the nucleotide sequences were analysed using abi prism ò autoassembler tm dna sequence assembly 1.4.0 software and compared using bioedit 7.0.1. results: analysis with mlst differentiated the pvl-positive ca-mrsa into six different sequence types (st8, 36, 80, 152, 154 and 256) with either sccmec type iv, iv c, v or unknown types. six additional sts (st5, 22, 25, 30, 88 and new) were detected among the pvl-positive methicillin-sensitive s. aureus. sequencing luks-pv and lukf-pv revealed eight point mutations among these isolates with twelve different origins. five substitutions had occurred in luks-pv and three in lukf-pv. only one substitution was nonsynonymous (histidine fi arginine). conclusion: the pvl-genes were well conserved despite the different genetic origins of the isolates analysed. the pvl is an extracellular product and the genes are not subject to any selective forces and thereby diversify very slowly. additional nonsynonymous mutations might result in a non-functional toxin. the first case of staphylococcus pseudintermedius in humans isolated from an icd lead l. van hoovels, a. vankeerberghen, k. van vaerenbergh, a. boel, h. de beenhouwer (aalst, be) introduction: staphylococcus pseudintermedius is recently described as a new coagulase-positive species from animals (devriese et al., 2005) . the pathogenic significance of this novel species remains unclear and to our knowledge no human infection due to s. pseudintermedius has been reported to date. here, we present the first isolation of s. pseudintermedius in humans with important clinical significance. patient and methods: a 60-year old male patient was referred to our centre for an ischemic cardiomyopathy and ventricle tachycardia for which he recieved an implantable cardioverterdefibrillator (icd) in january 2004. in august 2005 he presented with complaints of migration of the icd device. clinical examination revealed perforation of the icd pocket. infection was suspected and confirmed by the presence of pus in the pocket. the infected icd was completely removed and several samples (ventricular lead, pus and a tissue sample from the pocket) were sent for culture.bacteria obtained by routine culture were further characterised by phenotypical identification, pastorex ò staph-plus (biorad), api staph ò (biomérieux) and phoenix ò (bd). for molecular analysis, pcrs were performed targeting the nuclease (nuc) and coagulase (coag) genes of s. aureus. additionally, sequencing of the 16s rrna gene was performed and further analysed using blast. results: staphylococci with identical phenotypical appearance were isolated from 2 of the 3 icd samples (lead and pus). colonies were beta-hemolytic on sheep blood agar, dnase and coagulase positive but clumping factor, mannitol and pastorex ò negative. biochemical identification by api staph ò and phoenix ò gave a presumptive identification of s. aureus with a confidence value of respectively 88,5% and 97%.the pcrs for the nuc and coag genes were both negative. 16s rrna gene sequencing resulted in the identification of s. pseudintermedius based on a 100% sequence similarity with a previous reported sequence by devriese et al. conclusion: this case report describes the first identification of s. pseudintermedius as a significant pathogen in human. growth characteristics and commercial identification systems misidentify the organism as s. aureus. when confronted with an inconsistent phenotypical identification pattern, clinical labs should consider the use of 16s rrna gene sequencing for final confirmation. characterisation of staphylococcus aureus isolates recovered from dairy sheep farms (agr group, adherence, slime, resistance to antibiotics) e. vautor, m. sabah, g. mancini, m. pepin, h. carsenti-dellamonica (sophia-antipolis, nice, fr) objectives: the purpose of this study was to investigate 46 staphylococcus aureus natural isolates associated with dairy sheep mastitis for epidemiological key features (agr group, adherence, slime production and antibiotics resistance). methods: the s. aureus isolates (n = 46) were recovered from a field study in the southeast of france in 2001-2004 (28 from subclinical mastitis, 10 from clinical mastitis, 8 from the environment of the dairy sheep farm). a total of thirteen dairy sheep farms, producing cheeses manufactured with raw ewe's milk, were involved. the agr group were determined by multiplex and real-time pcr. the evaluation of adherence and slime production were assessed with methods previously described by christensen et al. (1982) . the susceptibility patterns to 11 antibiotics were determined using the discdiffusion method on mueller-hinton agar plates. oxacillin susceptibility testing was performed on all the isolates. the 10 others antibiotics susceptibility was only studied on the 28 isolates recovered from subclinical mastitis as they represent the major source of cheese contamination. results: 80% (37/46) of the isolates belonged to agr group 3, regardless of clinical findings. 39% (18/46) were adherent, strongly adherent or with maximal adherence (biofilm producers). 26% (12/46) were slime producers (moderate or strong producers). all the isolates (n = 46), but seven, were susceptible to all the antibiotics tested. two isolates recovered from subclinical mastitis were resistant to oxacillin and partly resistant to ampicillin and penicillin-g. the five other isolates were found: partly resistant to erythromycin (n = 1), cefoperazone and penicillin-g (n = 1), erythromycin (n = 1), neomycin (n = 1) or resistant to enrofloxaxin and partly resistant to ampicillin and penicillin (n = 1). conclusions: s. aureus isolates recovered from sheep mastitis in the southeast of france are mainly related to agr group 3 suggesting a role for agr-regulated proteins in the persistence of this bacteria in the sheep udders. biofilm and slime production may also be an important aspect for intracellular survival of s. aureus which could promote the development of persistent intramammary infections. finally, ewe's milk does not appear to represent a source of resistant s. aureus and specially methicillin (oxacillin)-resistant s. aureus (mrsa) for human health. detection of virulence genes in staphylococus aureus isolates from dairy sheep, goats and cows mastitis, using single-dye dna microarray e. vautor, v. magnone, g. rios, m. pepin, p. barbry (sophia-antipolis, fr) objectives: staphylococcus aureus is a common cause of mastitis in dairy farms animals. although many putative virulence factors have been identified in s. aureus genomes (kuroda et al., 2001) , the differences in pathogenic potential between naturally occurring isolates remain largely unaddressed. the relative importance of host (tissue) factors versus bacterial virulence determinants in disease pathogenesis is not well known, but it is widely accepted that bacterial factors including toxins, cell wall-associated adhesions, and secreted exoproteins are involved in the process. in this study, we use a single-dye dna microarray assay to investigate the presence or absence of 196 putatives virulence genes in 75 s. aureus isolates recovered from cases of ovine, caprine and bovine mastitis. methods: mastitis s. aureus isolates: sheep (n = 25), goats (n= 22), cows (n = 20).dna microarray: the arrays were spotted with long oligonucleotides (65-mer) representing 192 known virulence genes and new candidates identified in mu 50 genome (a human strain) and other s. aureus genomes. each gene were spotted four time. dna extracted from the strains were labelled with fluorescent cy5 using the bioprime ò array cgh (invitrogen). control strains with known genetic and phenotypic characteristics were used to normalize the data. results: (i) the majority of the virulence gene was detected in all the isolates (e.g. coa, ica adbc operon, htra, hysa, nuc, sbi, sdre, ssp, feob, fnb, sib, spa). (ii) genes were not detected in the majority of the isolates (e.g. cna, edin, lukf-pv, sav1999,…). (iii) genes were not found in isolates, depending on the herd (e.g. aur or sav1038 absent in isolates from some dairy sheep farm), on the isolates whatever the species (i.g. bsap, caph, entk, eta, fnbb, hsds, lpl2, lukd, …) . but we found gene mainly related to species (e.g. agriii, sav2496,…) comprehensive results will be given in the poster. conclusions: the present study indicated that the prevalence of virulence genes among s. aureus isolates recovered from dairy farm species depends on the gene. these observations suggest a common occurrence of host-adapted (or tissueadapted) s. aureus strains in which particular virulence genes may play a significant role. when taken with complementary methods such as pcr or/and southern hybridisation, singledye dna microarrays may provide a powerful tool to type s. aureus strains for epidemiological and possibly pathogenesis studies. detection of dna sequences distinguishing two closely related genomes of staphylococcus aureus from subclinical versus gangrenous ewe mastitis strains n. chevalier, c. huard, r. thiery, e. vautor (sophia-antipolis, fr) objectives: staphylococcus aureus is a common cause of mastitis in dairy sheep. the severity of mastitis ranges from subclinical to gangrenous forms. subclinical mastitis is an inflammation that is not readily detected clinically whereas gangrenous form is an acute necrotizing mastitis. with the ain to find genetic markers or virulence factors that are only present in gangrenous strains a suppression substractive hybridisation (ssh) method was used in the present study to compare two strains of s. aureus respectively recovered from subclinical or gangrenous mastitis in the same dairy sheep herd. methods: 42 ewes were held in the investigated farm. the subclinical strain was recovered in january 2002 from the milk of 6 ewes. the gangrenous strain was recovered in december 2002 from an primipare dairy sheep that subsequently died from this acute mastitis. dna extracted from the strains were first compared by pulsed field gel electrophoresis (pfge). then, ssh was performed by using dna from the subclinical strain (driver), as described in a commercial kit (clontech pcr-select bacterial genome substraction kit). results: using pfge, four band differences were found between the two strains. two dna fragments, presumably specific from the gangrenous strain were detected by ssh and sequenced: (i) a 262 bp (98% of homology with the sulfide quinone reductase contained in orf 11 pathogenicity island of the mrsa 252 strain) (ii) 280 bp (98% homology with a gene coding a bacteriophage holine contained in the s. aureus n315 genome). control pcr tests using primers designed from these specific gene candidates confirmed that they were only present in the s. aureus gangrenous strain. conclusions: according to tenover et al. (1995) , a 4 band difference using pfge indicates that the strains may possibly be related genetically. although genes classically involved in the virulence of s. aureus were not detected in the present study, two putative virulence factors were detected. the sulfide quinone reductase allows s. aureus to growth on sulfide (found in animal manure). the holine protein breaks the internal membrane of s. aureus to release daughter phages suggesting that a mechanism of horizontal gene transfer could have been mediated by bacteriophages and could explain the acquisition of virulence factors. antimicrobial clinical trials p1702 outpatient treatment of acute pyelonephritis in pregnancy after 24 weeks. a randomised controlled trial z. ahmadinejad, s. hantooshzadeh (tehran, ir) objectives: the purpose of this study was to compare the safety and efficacy of outpatient and inpatient treatment of acute pyelonephritis in pregnancy. methods: this was a randomized controlled, clinical trial. one hundred twenty eight gravidas past 24 weeks' gestation admitted in imam khomeini hospital, tehran & sahid dr bahonar hospital, kerman, divided by random blocks to outpatient or inpatient therapy, received two 1-g doses of intramuscular ceftriaxone at 24-hour intervals while hospitalized, then were discharged and reevaluated within 48-72 hours or remained hospitalized until afebrile for 48 hours. all patients completed a 14-day course of oral cephalexin. we performed urine cultures on admission and 10-14 days after therapy. results: the two groups were similar with respect to age, parity, temperature, estimated gestational age, initial white blood cell count, and incidence of bacteremia. there were not any significant differences between two groups about the clinical improvement after 48-72 hours, bacteriuria 10-14 days after treatment, relapse of pyelonephritis, requirement to change in antibiotic, date of pregnancy at delivery and preterm labor. the relapse of bacteriuria and preterm labor in inpatients were significantly more than outpatients (pv = 0.0077 and 0.030 respectively). the birth weight of neonate in outpatients were significantly more than inpatients (pv = 0.013). conclusion: outpatient antibiotic therapy is effective and safe in selected pregnant women with pyelonephritis. however in this study, the neonatal outcomes were better in outpatients and the maternal outcomes in inpatients. experience with daptomycin in patients with renal insufficiency investigators collected demographic, disease state, clinical and microbiological data; outcomes were defined using standard definitions. patients nonevaluable for outcome were excluded. core data were divided and data on cohorts of pts with a creatinine clearance (crcl) ‡30 or <30 ml/min were examined. results: of the 1160 pts enrolled, 770 (66%) had evaluable pt outcomes and either crcl ‡30 ml/min (nml, n = 598) or crcl <30 ml/min not yet requiring renal replacement therapy (ri, n = 172). the distribution of males and females was equal in both groups. ri pts were older (45% ‡66 yrs vs 29%, p < 0.01). the groups did not differ in the percent coming from the community setting prior to starting dap (nml 46%, ri 49%). nml had more frequent history of fractures/orthopaedic procedures (11 vs 4%, p < 0.01) and haematological cancers (6 vs 1%, p < 0.02) while ri had higher rates of any renal disease (4 vs 17%, p < 0.01), chf (6 vs 12%, p < 0.05) and other immunologic/ inflammatory disease (2 vs 11%, p < 0.01). ri had higher rates of skin infections (60 vs 69%, p < 0.05) and endocarditis (3 vs 6%, p < 0.05). infections that were frequently reported for nml and ri were bacteremia, non-catheter-related (10 vs 10%), bacteremia, catheter-related (10 vs 4%), osteomyelitis (14 vs 7%), and foreign body-orthopaedic (6 vs 5%), all p > 0.05. methicillin-resistant staphylococcus aureus was the most common pathogen; nml 41%, ri 38%. ri had higher rates of coagulase-negative staphylococci (9 vs 15%, p < 0.02) and viridans streptococci (0.3 vs 1.7%, p < 0.05). there was no difference in the percentage receiving antibiotics prior to dap; nml 75%, ri 68%. the mean dap dose and duration were similar; nml 4.7 mg/kg for 18 d, ri 4.6 mg/kg for 16 d. the most frequent dose was 4 mg/kg; nml 58%, ri 39%. ri initial dap dosing was more frequent than recommended (q 48 h) in 76%. the mean time to clinical response was similar; nml 5.2 d, ri 5.4 d. more pts in nml received concomitant antibiotics with dap; 53 vs 35%, p < 0.01). the clinical success (cure and improved) rates were; nml 95%, ri 96%. conclusion: dap shows favourable clinical success rates in pts regardless of the presence of renal insufficiency. in vitro activity of second line antibiotics against helicobacter pylori infection objective: the aim of our study was to determine the in vitro activity of levofloxacin, ciprofloxacin and rifampicin in clinical strains of h. pylori. material and methods: 40 isolates of h. pylori from biopsies of dyspeptic patients were obtained following standard methodology. in vitro activity of metronidazole, clarithromycin, levofloxacin, ciprofloxacin and rifampicin was determined by e-test using 5% sheep blood agar and incubated at 37ordm;c during 3-5 days in a co2 atmosphere. mic was determined as the point of complete inhibition of growth. breakpoint of the nccls for other microorganisms were considered for fluorquinolones: resistant if mic > 4 mg/ l. for rifampicin we considered the strain susceptible if mic <32 mg/ l, as same studies reported. results: 42.5% of the strains were resistant to metronidazole and 50% to clarithromycin. mic50, mic90 and range (mg/l) was: 0.064, 0.125 and 0.012 fi 32 for levofloxacin, 0.064, 0.25 and 0.006 fi 32 for ciprofloxacin and 0.75, 1.5, and <0.002-4 for rifampicin. all the strains were susceptible to rifampicin and only 2% of them were resistant to fluorquinolones. conclusions: the fluorquinolones tested and rifampicin showed an excellent in vitro activity against h. pylori, despite the high resistance rate to metronidazole and clarithromycin. however, in vitro susceptibility test should be done before the use in clinical practice. vibrio antibodies in serum and breast milk samples of parturient women in calabar, nigeria objectives: serum and breast milk samples from 335 parturient women and serum from non-parturient controls were analysed for prevalence and titres of vibrio antibodies. methods: v. cholerae agglutinins and vibriocidal antibodies in serum samples were analysed by direct agglutination and immune bacteriolysis techniques respectively, using 96 well microtitre plates. the protective value of breast milk was evaluated by haemagglutination inhibition and rabbit intestinal mucosal attachment of v. cholerae cells. results: vibrio agglutinins were detected in serum samples of 191(57.0%) parturient and 78 (33.9%) non-parturient subjects (p < 0.05). high prevalence rates of 68.9% and 39.1% occurred among parturient and control subjects of 21-25 years of age respectively. at 1:160 cut off titre to evaluate vibrio cholerae specific bacteriocidal antibodies, activity was detected in samples of 31 (57.1%) and 9 (56.3%) parturients and controls respectively aged 26-30 years. breast milk from 67 (20.0%) parturients contained vibrio agglutinins with titres ranging between 1:20 and 1:320, while milk samples from 32 subjects showed haemagglutination inhibition (hi) activity titres of p 1:40. of the 19 hi positive milk samples 17 (89.5%) showed inhibition of v. cholerae adherence to rabbit intestinal mucosa at titres p 1:80, and 53-92% reductions in cell attachment. conclusion: our study confirms that parturient women in calabar may benefit from significant serum titres of v. cholerae antibodies and provide immune protection for their babies through breast milk secretions. moxifloxacin vs clarithromycin for treatment of community-acquired pneumonia associated with common respiratory pathogens: a pooled analysis objectives: streptococcus pneumoniae and haemophilus influenzae are pathogens commonly associated with community-acquired pneumonia (cap). this study compared the clinical and bacteriologic efficacy of moxifloxacin (mxf) to clarithromycin (clar) in cap patients with these pathogens. patients and methods: data were pooled from three doubleblind, multicenter, phase iii trials comparing oral mxf 400 mg qd to clar 500 mg bid for 10 days. all patients included had mild-to-moderate cap. clinical and bacteriologic success rates were identified for s. pneumoniae and h. influenzae isolated from these studies. data for the efficacy-valid population was recorded at the test-of-cure (toc) visit (7-35 days post-therapy). results: 1437 patients were entered, of which 356 were microbiologically evaluable. infection with s. pneumoniae and/or h. influenzae was documented in 149 (42%) of patients (5 mxf and 2 clar patients had mixed infection). within this cohort, the two treatment groups were well balanced based on demographic/baseline medical characteristics (55% male, mean age 50 yrs, 41% smokers, 8% recent antimicrobial therapy). clinical success and bacteriologic eradication rates (one response per patient) at toc are presented in the table. conclusions: in cap associated with s. pneumoniae and h. influenzae there was a trend towards greater bacterial eradication for mxf vs clar. clinical success rates were significantly higher for mxf monotherapy vs clar. variability of creatinine clearance measurements in inpatients with community-acquired pneumonia r. grossman, s. choudhri, d. haverstock (mississauga, ca; west haven, us) objectives: moxifloxacin, levofloxacin and gatifloxacin have been recommended as empiric therapies for patients with community-acquired pneumonia (cap). levofloxacin and gatifloxacin require dose-adjustment for renal insufficiency while no dose adjustment is required for moxifloxacin. this study was designed to determine the frequency and underlying variability of renal insufficiency in patients with cap. methods: a pooled analysis of data from 2211 patients with mild to moderate or severe cap entered into one of six randomized, controlled clinical trials was undertaken. renal function (calculated creatinine clearance; crcl) was assessed in each patient prior to treatment with mxf and then again during and post-treatment. results: baseline crcl levels in this pooled population of patients with cap were: <25 ml/min in 47 (2.1%) of patients, 25-49.9 ml/min in 436 (19.7%) and ‡50 ml/min in 1728 (78.2%) patients. after the pre-treatment crcl measurement 88 patients (4%) were lost to follow-up, so there was no during or post treatment value. in patients with cap the crcl improved from baseline in many patients during or post-treatment, while some patients experienced a worsening of renal function (see table) . conclusions: renal function (crcl) is highly variable in cap patients with baseline evidence of renal insufficiency. renal function should be monitored closely to permit appropriate dose adjustments if levofloxacin or gatifloxacin is used in this patient population. moxifloxacin may be a better empiric choice in this setting as it does not require dose adjustment in patients with renal insufficiency or renal failure. a prospective, controlled, randomised, nonblind, comparative study of the efficacy and safety of high-dose single daily ceftriaxone plus ciprofloxacin versus thrice-daily ceftazidime plus amikacin in the empirical therapy of febrile neutropenic patients objective: empirical antibiotic treatment for febrile neutropenia is well established. the best regimen is still controversial. the purpose of this study was to evaluate the efficacy, safety and cost of high-dose single daily ceftriaxone plus ciprofloxacin versus thrice daily ceftazidime plus amikacin in neutropenic febrile patients. patients and methods: ninety-five patients with febrile neutropenia were included in a prospective, controlled, randomized, non-blind, comparative study. patients were randomly assigned to either treatment group (63 in the ceftriaxone/ciprofloxacin group and 32 in the ceftazidime/ amikacin group) and evaluated as successes or failures according to defined criteria. daily assessments were made on all patients all adverse events were record. results: the overall incidence of documented infections was 45.9%: 24/47 (51.1%) in the ceftriaxone/ciprofloxacin group and 10/27 (37%) in the ceftazidime/amikacin group. there was significant difference in clinical efficacy between groups (p = 0.011) at the end of therapy. ceftriaxone/ciprofloxacin group had an overall incidence of resolution and improvement of 95,7% in comparison to the 75% of the ceftazidime/amikacin group. thirty-nine organisms were isolated, 26 (66.67%) gramnegative and 13 (33,33%) gram-positive. there was low incidence of adverse events in both groups. conclusion: the combination of high dose single daily ceftriaxone plus ciprofloxacin was more effective than the standard combination of thrice daily ceftazidime plus amikacn with no significant adverse events in either group. objective: in past studies of azithromycin in children, a posttreatment (pt) benefit was observed at day 28. in 5 recent phase 3 trials in adults, single-dose zmax was at least as effective as standard comparators for treatment of respiratory tract infections (rtis), including cap. our objective is to demonstrate a pt benefit in this adult population. methods: post-hoc analyses, including respiratory adverse event burden (raeb), were conducted on the all treated population (n = 2596; 1292 az-m, 1304 comparators) in the 5 phase 3 studies. the raeb is the sum of duration, in days, of all respiratory adverse events, divided by total number of observation days of all patients, normalized to 1 year. the overall and per study day raeb were calculated for zmax and the pooled comparators for the 5 studies combined. results: raeb, in days/patient year, was 15.5 for az-m patients vs 20.2 for comparator patients (p = 0.08). the difference in raeb consistently and progressively favoured zmax, beginning at day 11 and achieving statistical significance between days 29 and 35, when the upper limits of the 95% cis around the differences were below zero (figure). faropenem medoxomil (fm) is an oral penem with potent activity against streptococcus pneumoniae and haemophilus influenzae. this integrated analysis was conducted to summarize the efficacy of 10 days of 300 mg bid of fm compared with other beta lactams in the management of community acquired pneumonia (cap). methods: efficacy was determined in three multicenter randomized double-blind controlled trials (rct) and a single uncontrolled study of faropenem medoxomil. comparators were 14 days of cefpodoxime (c), 10 days of amoxicillinclavulanate (ac), or 10 days of amoxicillin (a). the analysis allowed examination of treatment effects by age, race, gender and study site subgroups. results: a total of 2267 subjects were studied. studies 1 and 4 were conducted in n. america, studies 2 and 3 in europe, latin america, israel, and s. africa. n. american vs. other studies included subjects at least 12 (vs. at least 18) years of age and only out patient (vs. outpatient and hospitalized) subjects. the clinical responses for fm in both per protocol and intention-to-treat populations were non-inferior to comparator for each study and for the three trials combined. no differences were found in treatment effect by age, race, gender, or country. recovery of an etiologic agent from initial respiratory or blood culture varied between 9.4 and 17.5% of cases in the 4 studies for a total of 236 microbiologically evaluable subjects. s. pneumoniae was eradicated or presumed eradicated in 82/90 (91.1%) and 49/52 (94.2%), h. influenzae in 49/61 (80.3%) and 30/32 (93.8%), s. aureus in 13/14 (93%) and 8/10 (80%), h. parainfluenzae in 6/8 (75%) and 0/2 (0%), and m. catarrhalis in 6/7 (85.7%) and 4/4(100%) fm and comparator recipients, respectively. clinical response for s. pneumoniae bacteremic patients was 18/21 (85.7%) for fm. conclusions: fm efficacy was consistent across studies, within subgroups, and non-inferior to comparators. it is efficacious against the most common bacterial pathogens and in the most severe form (bacteremic) disease. fm is a good option for the treatment of cap. propionibacterium acnes strains isolated from acne vulgaris and severe infections c. oprica, c.e. nord (stockholm, se) propionibacterium acnes is a member of the resident flora of the skin and is an important factor involved in inflammatory reactions in acne patients. during the last years the prevalence of different severe infections due to p. acnes has increased. objectives: 1) to detect the prevalence of resistant p. acnes strains isolated from acne patients in stockholm and different severe infections in europe; 2) to identify the mechanisms of resistance and the genetic diversity among resistant strains. methods: p. acnes strains isolated from acne vulgaris and severe infections were tested against clindamycin, erythromycin, linezolid and tetracycline and pulsed-field gel electrophoresis was used for further characterization. pcr and sequencing of the genes encoding domain v of 23s rrna for clindamycin and erythromycin resistant strains and 16s rrna for tetracycline resistant strains were performed. results: i) antibiotic-resistant strains were more often isolated from antibiotic treated patients with moderate to severe acne area than from non-antibiotic treated acne patients. an individual might harbor different pulsotypes of p. acnes with various degrees of resistance. ii) among the 304 clinical isolates from 13 european countries were found resistant strains to tetracycline, clindamycin, and erythromycin. overall, in the southern europe a higher prevalence of erythromycin-resistant strains was noticed and in southern and eastern europe a higher prevalence of resistance to clindamycin. it was noticed a high genomic diversity and the geographical spread of some clones in related areas but also in geographically distant countries. most clindamycin or erythromycin resistant p. acnes isolates, were found to be members of a single clone that has spread in different geographically countries. iii) p. acnes clindamycin and erythromycin resistant strains carrying one of the described mutations within the 23s rrna were predominantly isolated from swedish acne patients compared to strains from other infections. forty-four per cent of tetracycline resistant strains were found to carry a mutation in the 16s rrna. these strains were isolated from swedish acne patients, were highly resistant and were clustered in one pulsotype. conclusion: surveillance of both the prevalence of resistant p. acnes strains and associated resistance mechanisms is important due to the rapid variation in resistance patterns, both in acne patients and other severe infections. antimicrobial activity of unisepta quick and deconex solarsept on the surface contamination and dental instrument in dental clinics in iran f. shahcheraghi (tehran, ir) objectives: quaternary ammonium compounds (qacs) are amphoteric surfactants that are widely used for the control of bacterial growth in clinical and industrial invironment.unisepta quick and deconex solarsept are new generation of qacs is widely used as adjuncts in iran to hygine in dental clinics.the aim of present study was to investigate clinical efficiency of these substances on the surface and instruments in dental clinics. material and methods: the following bacteria and fungi on the base of aoac standard were used.pseudomonas aeruginosa (atcc 27853), staphylococcus aureus (atcc 25923) bacillus. subtilis atcc (6051), mycobacterium bovis atcc (35443) and wild types of trichophyton mentagraphit, p. aeruginosa and salmonella typhimorium (a common fungi in iran). a stock solution of deconex solarsept (borer chemie) and unisepta quick (micro 10 unident) was prepared as recommended by the manufacturer. the concentration of bacterial suspention was 0.5 macfarland and the results were reported on the base of decreasing in (cfu) colony forming unit from 107 to 102. results: the results shows that both of these disinfectants have bactericidal and fungicidal activity on the standard p. aeruginosa, s. aureus, s. typhimurium and trichophyton mentagraphit, the number of bacteria decreased significantly (p < 0.05), but no significant difference was seen with b. subtilis, wild type of p. aeruginosa and m. bovis. conclusion: the results confirm that these qacs are not able to sterilize or disinfect medical and dental instruments, and they can not be used lonely, and it must be used with the other methods for sterilization of surface and dental instruments. macrolide as long-term treatment in patients with bronchiectasis colonised by p. aeruginosa background: a certain efficacy of macrolide against p. aeruginosa has been described in vitro, mainly through mechanisms such disruption of quorum sensing and suppression of inflammation. aim: to evaluate the efficacy of macrolide in patients with bronchiectasis colonised by p. aeruginosa. methods: the study prospectively included patients with bronchiectasis and p. aeruginosa isolated in sputum in stable state. all subjects received either azithromycin 250 mg · 3 days/week or clarithromycin 500 mg daily on long term and completed daily diary cards for symptoms and pef values until the end of therapy. follow-up period was 1 year. results: 26 patients with bronchiectasis and p. aeruginosa evidence in sputum were included (17 men, mean age 55.0 ± 12.1 yrs.). 12 patients received azithromycin and 14 patients clarithromycin, with a mean duration of 4.5 ± 1.7 months. five (19.2%) patients discontinued treatment after less than 2 weeks because of adverse events. at the end of therapy, 12 (46.1%) patients showed no evidence of p. aeruginosa in sputum while 9 (34.6%) patients still had ps. aeruginosa in sputum. an improvement in the following parameters could be observed in all patients: sputum volume (75.5 ml/day before therapy versus 31.6 ml/day after therapy, p = 0.04); pef (311.2±77.1 l/min before therapy versus 476.2 ± 30.2 l/min after therapy, p = 0.03); number of exacerbations/year (2.8 in the previous year versus 1.7 in the follow-up year, p = 0.01). conclusion: the study shows that macrolide may be an effective therapy in patients with bronchiectasis colonised by p. aeruginosa. independently of the microbial eradication, an improvement of the clinical symptoms and a reduction of exacerbations were observed in all patients. fungal pathogens from haematoncology patients and their susceptibility to new and old antifungal drugs the expanding population of immunocompromised hosts has been infected with many established and emerging opportunistic fungi. most pathogens can be treated empirically whereas for an increasing number of species proper treatment starts once the mic becomes available. though invasive aspergillosis remains the principle life threatening complication in the haematoncology patients (hop) other pathogens cannot be ignored as selection and resistance during prophylaxis increases the risk of treatment failure.in order to understand the frequency of rare fungal pathogens, selection and emergence of resistance in our trust all fungi from hop were identified using standard mycological techniques and the mics to amphotericin b (amb), flucytosine (5fc), fluconazole (fcz), itraconazole (itz), voriconazole (vcz) and caspofungin (cfg) were determined using the nccls method. 513 specimens were processed, 53% respiratory, 17.5% blood, 12.8% oral, 8.4% other sterile (bile, csf, drains, lines and tissue biopsies) and 8.3% nonsterile sites. yeasts accounted for 85% and filamentous fungi (ff) for 15%, representing 12 candida sp, 7 other types of yeast, 6 aspergillus sp and 8 other types of ff. c. albicans represented 35.7%, c. glabrata 29.3%, c. krusei 8.6%, a. fumigatus 10% and other aspergillus sp 2% of all isolates. the mic 90s for all isolates were amb 0.5, 5fc 2, fcz >64, itz 1, vcz 1 and cfg 0.06 mg/ l. with the exception of acremonium sp, a. versicolor, a. terreus and scedosporium apiospermum all isolates including the 15 isolates of c. lusitaniae were sensitive to amb. most but not all ff and only one isolate of c. albicans from the yeasts were resistant to 5fc. all ff, rhodotorula sp, c. albicans 3%, c. glabrata 53% and c. krusei 41% were resistant to fcz. only absidia corymbifera, acremonium sp 25%, c. albicans 1%, c glabrata 33% and saccharomyces cerevisiae 4% were resistant to itz. for vcz a. corymbifera, acremonium sp 25%, c. albicans 2%, c. glabrata 31%, c. krusei 2%, c. tropicalis 10%, rhodotorula sp 66.6% and p. aecilomyces variotii 50% had an mic ‡2 mg/l. with cfg the effective concentration was ‡0.5 mg/l for a. corymbifera, fusarium solani, geotrichum capitatum, sporobolomyces salmonicolor, acremonium sp 50% and c. parapsilosis 25%.the data show that hop are exposed to many different fungal pathogens some of which are resistant to the old and the new antifungals and that amb is still the drug with the broader spectrum and less developed resistance for both yeasts and ff. faropenem medoxomil in the treatment of acute bacterial sinusitis: an integrated analysis s. kowalsky, r. tosiello, r. echols (milford, us) background: faropenem medoxomil (fm) is an orally absorbed, synthetic, penem antibacterial agent with in vitro activity against community-acquired respiratory pathogens. methods: the efficacy of fm in subjects with acute bacterial sinusitis (abs) was evaluated in 3 phase iii trials; 100288, 10186, and 100287. study 100288 was conducted in n. america, study 10186 was conducted in europe and israel and study 100287 was conducted in the us and argentina. 100288 and 10186 were prospective, randomized, double-blind, active comparator trials and 100287 was an open-label ''sinus tap'' trial. the dose of fm was 300 mg bid in all 3 studies. the comparator in 100288 and 10186 was cefuroxime axetil (cfx) 250 mg bid. the duration of fm treatment in 100288 was 7 days and 10 days vs cfx for 10 days. in 10186, fm or cfx were given for 7 days. in 100287, fm was administered for 7 days. the primary efficacy variable in all 3 studies was clinical response at the test-of-cure (toc). microbiologic response at the toc was a secondary efficacy variable in 10186 (sinus puncture and endoscopic collection) and 100287 (sinus puncture and aspiration). non-inferiority was defined as the difference in cure rates (fm minus comparator) where the lower boundary of the 95% ci was greater than -10%. results: the cure rates at the toc are shown in the table for the valid per protocol (vpp) and the intent-to-treat (itt) populations. the frequency of isolation of key pathogens and the rate of eradication in samples obtained by endoscopicallyguided swab and in samples obtained by tap were consistent across studies. the eradication rates for s. pneumoniae, h. influenzae, and m. catarrhalis were 94.8% vs. 96.3% (fm 7/ 10 d vs. cfx 7/10 d), 82.4% vs. 90.5% (fm vs. cfx) and 96.2% vs. 83.3% (fm vs. cfx), respectively. conclusions: fm 300 mg bid x 7 days was shown to be noninferior to cfx in clinical efficacy in two prospective, doubleblind, comparative trials. a third, open-label trial, demonstrated similar efficacy in microbiologically documented abs caused by key pathogens. longer (10 d treatment) with fm provided no additional efficacy. faropenem medoxomil in the treatment of acute exacerbation of chronic bronchitis: an integrated analysis s. kowalsky, r. tosiello, r. echols (milford, us) background: faropenem medoxomil (fm) is an orally absorbed, synthetic, penem antibacterial with in vitro activity against community-acquired respiratory pathogens. methods: the efficacy of fm in subjects with acute exacerbation of chronic bronchitis (aecb) was evaluated in 2 phase iii trials. study 10187 was conducted in europe, israel, mexico, and south africa. study 100291 was conducted in the us and argentina. both were prospective, randomized, double-blind, active comparator trials. the dose of fm was 300 mg bid for 5 days in both studies. the comparators were clarithromycin (clr) 500 mg bid for 7 days and azithromycin (azi) qd for 5 days (500 mg on day 1 and 250 mg on . the primary efficacy variable was clinical response at the test-of-cure (toc). microbiologic response at the toc, in subjects with a baseline pathogen was a secondary variable. non-inferiority was defined as the difference in cure rate (fm minus comparator) where the lower boundary of the 95% ci was greater than )10%. results: the cure rates are shown below for the valid per protocol (vpp), intent-to-treat (itt) and modified itt populations (all itt subjects who met inclusion/exclusion criteria). in both the individual studies and the pooled analyses, for all populations, treatment with fm was not less effective than either comparator. 20% of treated subjects in 10187 and 28% of subjects in 100291 were evaluable for microbiological response. in 10187, the eradication rates for the microbiologically evaluable population was higher in the clr group (77.1%) compared with the fm group (68.6%) (95% ci )23. 4, 6.3) . in contrast, the eradication rate in 100291 was similar in the fm (80.0%) and azi (78.9%) groups (95% ci -9.7, 11.8). when the data were pooled across studies, the response rates were similar with fm (75.8%) and combined comparator (78.2%) groups (95% ci -11.0, 6.3). the combined eradication/presumed eradication rates in the pooled fm and comparator groups were 81.8% vs. 86.6%, respectively for s. pneumoniae and 76.0% vs. 77.6%, respectively, for h. influenzae. conclusions: fm was shown to be non-inferior to either azi or clr in clinical efficacy in two adequate and well-controlled trials. pooled analysis further strengthened the clinical noninferiority conclusion. the difference in eradication rates observed in study 10187 (clr) was not supported by study 100291 (azi). an integrated safety analysis of faropenem medoxomil: results of 5,023 subjects from phase ii/iii clinical trials r. echols, r. tosiello (milford, us) objective: to evaluate the safety profile of faropenem medoxomil (fm), a novel oral penem antibiotic. methods: 5,023 subjects from 3 phase ii and 14 phase iii clinical trials received fm, 300 mg bid for 3-10 days for treatment of acute bacterial infections. randomized controlled trials (rcts) included 4,223 fm and 3795 comparator treated subjects. analyses were conducted to identify possible disparate adverse event (ae) reporting based on type of infection, subject age (12-17, 18-45, 4664, 65-75, >75) and gender, duration of treatment (3/5 d v. 7/10 d), geography (na, eu, row), study design (open label v rct), relationship to treatment. comparisons were made to control treatment based on antibiotic class (b-lactam v. other), and individual antibiotic treatments. results: fm compared favourably to penicillins, cephalosporins and macrolides. fm was better tolerated than tmp/smx and co-amoxiclav. open labeled trials had higher aes reported v. rcts. aes reporting na = row > eu except serious aes and deaths where row = eu>na. aes for fm 3/5 d = fm 7/10 d. underlying infection did influence ae reporting. female gender had higher ae reporting than male gender. fm was tolerated equally well across age ranges, although deaths and saes were more common in >75 age group. common aes (>1%/related from rcts) were diarrhoea, nausea, fungal vaginosis and headache and were generally less frequent with fm than control rx. no evidence of neuro or cardio toxicity was identified. laboratory tests identified no hepatic, renal or hematopoietic signals. conclusion: faropenem medoxomil, a novel oral penem antibiotic, has the safety profile expected of a b-lactam but is better tolerated than co-amoxiclav with approximately one-third the gi side effects. the efficacy of non-surgical and systemic antibiotic treatment regimens in smoking and non-smoking patients e. pähkla, k. lõ ivukene, p. naaber, m. saag (tartu, ee) periodontitis is a chronic infectious disease, which leads to the destruction of periodontal ligament fibres and alveolar bone until tooth loss. the objective of this study was to compare the longitudinal effect of combination of non-surgical periodontal therapy with systemic antibiotics in smoking (s) and nonsmoking (ns) patients. methods: there were total of 28 patients with severe generalized chronic periodontitis involved in this study (14 s, 14 ns), who did not respond well to previous mechanical periodontal treatment. the clinical examination included recordings of visible plaque index (vpi), modified gingival index (mgi), bleeding on probing (bop) and suppuration after probing (sup), probing pocket depths (pd) and clinical attachment levels (cal). the non-surgical periodontal therapy was performed within 4 weeks. clinical parameters were recorded at baseline, 2-3 weeks after the first mechanical treatment and 14 months after combined treatment, during a regular check-up visit. as the patients did not respond to the conventional periodontal therapy, the microbiological analyses were taken and a combination of systemic amoxicillin 500 mg · 3 and metronidazole 250 mg · 2 for 7 days, was prescribed. results: the results suggested that the combined systemic antibiotic therapy is effective in case of severe generalized chronic periodontitis, as vpi, bop, sup, cal, and mgi improved significantly after the treatment. in the ns group all parameters, except cal, improved significantly after the treatment. the s showed markedly smaller reduction in sup, mgi, and cal.after instrumentation, no periodontal pathogens were isolated in 11 (39%) patients, while 17 patients (61%) were infected with one to three different pathogens. among the pathogens, prevotella intermedia/nigrescens (10 patients) and actinobacillus actinomycetemcomitans (8 patients) were dominating. the total level of microbial load (log10 cfu/ml) as well as the spectrum of pathogens in s and ns patients remained similar. conclusions: despite of positive treatment effect in general, there were insignificant improvements in any clinical parameters in the smoking group. smoking has adverse effect on periodontal therapy; therefore the dentist should cooperate with patients in counselling of smoking cessation to achieve better results in the treatment of periodontitis. objectives: laminin (ln), which is a large multidomain glycoprotein of the extra cellular matrix, has attracted much attention because of its importance in many cellular functions, including induction of cell adhesion, growth promotion and mediation of cell communication. the target of this study was to find out whether there is any relation between the levels of serum ln and the inflammatory activity of a microbial infection. patients/methods: from june to october 2005, 48 immunocompetent adults, with confirmed bacterial infection were admitted to our hospital (17 with pneumonia, 24 with pyelonephritis and 8 with cholecystitis) (group 1). at the same time 50 hospitalised patients for non-infectious causes (stroke, gastrointestinal bleeding, anaemia) were also studied (group 2). the levels of serum ln and crp were measured on the day of admission in both groups. the levels of ln were measured using an enzyme immunoassay kit (takara laminin eia kit) and 100 healthy volunteers were used to determine its normal limits (130-520 ng/ml). plasma crp concentration was assessed by immunoturbidometric method (using randox, uk kits). normal values were considered those below 10. results: the mean serum ln levels of patients of group 1 were 800.90 ± 249.29 (much higher that the normal limits), while the mean crp value was 117.53 ± 87.78. the mean corresponding values in group 2 were 475.79 ± 239.25 for ln (within normal limits) and 24.15 ± 32.77 for crp. there is a statistically significant difference between the mean ln levels of the two groups (p < 0.001). additionally, there is a statistically significant correlation between the levels of ln and crp (a well studied serum inflammatory marker) in patients with bacterial infection (group 1) (pearson correlation coefficient r = 0.565, p = 0.01). conclusions: the definition of the ln levels could constitute a new reliable, simple, direct serum marker for the confirmation of an active bacterial infection. additionally, as the crp levels are above normal in group 2 too (patients without infection) while ln lies within normal limits, maybe ln is even more specific than crp. more studies are required in the future, with more patients included, in order to confirm the outcome of this study. performance and clinical significance of a direct tube coagulase test using serum separator tubes for rapid identification of staphylococcus aureus from blood culture broth d. kwa, t. schü lin-casonato, p. sturm (nijmegen, nl) objective: blood cultures are important in the diagnosis of serious infections. early administration of effective antibiotics is associated with improved patient outcome. the performance of the direct tube coagulase (dtc) using serum separator tubes (ssts) for rapid identification of s. aureus from blood culture broth (bcb) was investigated. the clinical significance of rapid identification was assessed. methods: consecutive blood cultures with gram-positive cocci in clusters were tested. bcb was collected in ssts using a subculture-venting unit. after centrifugation, the supernatant was discarded and 1 ml rabbit plasma was added to the remaining pellet of bacteria. coagulation was evaluated after 2 and 4 hours incubation at 37°c, and after overnight incubation at room temperature. in parallel, a direct tube coagulase test was performed using a 1:10 saline dilution of bcb as described previously. isolates were identified by standard microbiology procedures. clinical significance was measured by comparison of antimicrobial prescription based on gram stain results, direct coagulase results, and culture results. results: over a 6-week period, 90 bcbs from 46 patients were tested. s. aureus was present in 22 bcbs. using the serum separator tube method and the saline dilution method, the sensitivity of the dtc after 2 hours incubation was 91% and 41%, and after 4 hours 100% and 88%, respectively. the specificity of both methods was 100%. rapid identification of s. aureus resulted in initiation (n = 1) or streamlining (n = 4) of antimicrobial therapy in 5 of 13 patients with s. aureus bacteremia. rapid identification of coagulase-negative staphylococci resulted in changes in antimicrobial therapy in 1 of 33 patients. conclusion: the dtc using ssts for bacterial enrichment is a very reliable, rapid, cheap and easy to perform method for identification of s. aureus from bcb. implementation of this test can improve antimicrobial therapy. evaluation of the results of the spanish seimc external quality control program for the diagnosis of enterococcus faecalis and klebsiella pneumoniae infections r. guna, j.l. pérez, n. orta, c. gimeno on behalf of seimc objectives: to evaluate the results obtained from four shipments of two different strains by the participants in the seimc external quality control program (eqcp). these controls were intended to analyse the percentages of correct species identification and the ability of the participants in detecting some special features of the control strains: vanb phenotype in the case of e. faecalis, and the production of extended spectrum betalactamase (esbl) in k. pneumoniae. methods: the same strain of each microorganism was sent in two different shipments. the vanb e. faecalis strain was sent both in a control of year 2002 as well as in other of 2004, while the esbl-producing k. pneumoniae was sent in 1999 and in 2005 to an average of 200 laboratories. the results obtained were compared with those of a reference laboratory that certified both the species identification and the resistance features. results: in the 2002 control, 92.6% of participants identified correctly e. faecalis, while 97.8% did it in 2004. as for the glycopeptide resistance pattern of the enterococcal strain, 39.9% and 53.3% of participants detected the vanb phenotype in 2002 and 2004, respectively. overall, the k. pneumoniae strain was correctly identified in both separate controls by most of the participants (97.1% and 98.6%, respectively). interestingly, the percentage of laboratories that detected the presence of the esbl in the k. pneumoniae strain sharply increased from 55.0% in 1999 to 89.4% in 2005. the overall percentages of correct species identification were high for the two microorganisms and for both control points. most important, the ability of the spanish clinical laboratories in detecting the special resistance features of these strains clearly improved along the study period. these data confirm the importance of implement a continuous surveillance of the diagnostic training in the clinical laboratory, as well as the possible positive intervention of the seimc external quality control program in such improvement, since the analysis of results is accompanied of updated reviews on the subject of each control. a. bonnet-pierroz, a. resenterra, o. péter (sion, ch) objective: to evaluate 2 new elisa ridascreen ò borrelia igg and igm for antibody response in patients with confirmed lyme borreliosis and to compare to the results of vidas lyme (igg-igm) and in-house immunoblots (b. garinii igg and igm for early cases or b. burgdorferi sensu stricto, b. afzelii, b. garinii, b. valaisiana igg for late cases). methods: elisa ridascreen ò borrelia igg and igm was used to screen sera from patients with clinically confirmed erythema migrans em (n = 30). patients with confirmed neuroborreliosis by intrathecal antibody synthesis (n = 45) were evaluated for igg antibodies to borrelia. sera from patients with acrodermatitis chronica atrophicans aca (n = 30) and sera and synovial fluids from patients with lyme arthritis (n = 30) were also evaluated for igg antibodies. patients with syphilis (n = 10) and infectious mononucleosis (n = 10) were screened for igg and igm antibodies to borrelia in order to estimate the specificity. conclusion: the elisa ridascreen ò borrelia igg and igm have shown a good sensitivity for the serological diagnosis of lyme borreliosis. the short evaluation for the specificity of the igg test revealed a good assay with few false positive reactions, whereas the igm assay was, as expected more prompt to give false positive results with sera from patients with infectious mononucleosis. so far any equivocal or positive tests should be confirmed by immunoblots. is it necessary to incubate the bact/alert blood culture bottles more than 3 days? objective: to assess the incubation time reduction of the aerobic and anaerobic bact/alert system bottles from 5 to 3 days. methods: from 1996 to 2004 we processed 94.303 blood culture sets and detected 9.432 (10%) positive blood cultures with clinical significance. we retrospectively examined the detection time of positive bottles and assessed the clinical significance of the bottles that were positive between the fourth and fifth day. results: out of 9432 positive blood cultures with clinical significance, 9238 (97.9%) were detected within the first 3 days of incubation. out of the 194 positive blood cultures detected between the fourth and fifth incubation days, 105 were recovered in concurrent cultures within the first 3 days. chart reviews were conducted from 78 patients with the remaining 89 isolates. only in 24 patients (0.3% positive blood cultures) changes in antimicrobial therapy based upon the positive blood culture results on day 4 to 5 were made, in the other patients the empirical treatment was adequate. the isolated microorganisms in those 24 patients were: 7 gram-positive cocci (3 staphylococcus spp. not s. aureus, 1 staphylococcus aureus, 2 streptococcus viridans and 1 streptococcus pyogenes), 5 anaerobes, 3 enterobacteriaceae, 2 pseudomonas aeruginosa, 2 campylobacter spp., 2 candida spp. 1 cryptococcus neoformans, 1 brucella spp. and 1 haemophilus influenzae. conclusions: incubation of bact/alert blood cultures bottles only for 3 days would have represented a detection loss of 0.3% of the clinically significant isolates, which led to antimicrobial therapy changes. although we keep employing a 5-day incubation for routine blood cultures, we could reduce the incubation time to 3 days depending on current instrument capacity. an enzyme immunoassay for anti-diphtheria antibodies: a practical alternative to the vero cell assay r. budd, e. harley, r. george, a. efstratiou, k. broughton, a. bradwell (birmingham, london, uk) introduction: in this extended study, results from an anti-diphtheria toxoid enzyme immunoassay (eia), specifically designed to detect higher affinity antibodies, were compared with those from a vero cell assay (vca). methods and results: 154 serum samples with antibody concentrations ranging from 0.008-8.000 iu/ml on the vca from the respiratory and systemic infectious laboratory (rsil) were assayed by eia (the binding site ltd, uk). a further 100 samples from rsil selected on the basis of being close to the protective level, were assayed to confirm the performance of the eia. the eia was calibrated, against the nibsc reference material 00/496 and the assay measuring range was 0.004-3.0 iu/ml results were compared using the who guidelines of 0.01-0.1 iu/ml as minimum protective level, and > 0.1 iu/ml as protective. relative agreement, sensitivity and specificity for the first 154 samples were: 91.6%, 98.8% and 83.3% respectively, for the second set of 100 samples performance was: 90.0%, 84.5% and 97.6%, and for the combined 254 samples results were: 91.3%, 92.9% and 89.4% respectively. roc analysis of the total 254 samples confirmed the highest sensitivity 92.9% and specificity 89.4% occurred at a cut-off of precisely 0.1 iu/ml for the elisa assay. conclusion: of the total 22 discrepant samples, 14 had vca and eia values < 0.149 iu/ml, therefore we suggest the possibility of establishing an equivocal zone for the interpretation of the eia results. if the test is part of a general immune status assessment a grey zone is not required. if undertaken to determine the requirement for immunization, the use of the equivocal zone is recommended. by applying these criteria in the eia, only one sample would have suggested inappropriate immunization, as indicated by a vca result > 0.149 iu/ml. because of the > 90% agreement between the two assays, significant advantages of cost and speed, ease of use and the potential for automation, the eia could therefore be considered as an alternative to the vca. evaluation of accuracy limits of countable colony-forming units on agar plates j. arbique, a. rendell, k. forward (halifax, ca) objectives: accurate colony counts are an essential component of many microbiology research projects and clinical laboratory processes. the suggested range of accuracy of colony-forming units (cfu) extends from 30 to 300 (standard methods for the examination of water and wastewater). this recommendation dates to 1907, and fails to adequately address the numerous sources of inter-and intra-variability. without more detailed analysis it is difficult to estimate the sample size and number of replicates necessary to ensure accurate results. the purpose of this study was to determine the validity of accuracy limits for quantifying cfus on agar plates. methods: escherichia coli (atcc 25922) and staphylococcus epidermidis (atcc 12228) were used to prepare series of four organism densities ranging from approximately 40-500 cfu, on three different days. on each day, each of the 4 densities for both organisms was plated on sba and viable organisms were counted following incubation. an average of the margins of error obtained over the 3 days of testing was used to determine the reproducibility of agar plate counts, and to estimate the optimum number of replicate plates (sample size) required for each organism at each concentration. results: margins of error for both organisms were greatest with suspensions yielding approximately 40 cfu, and lowest for suspensions yielding 300 and 500 cfu. nine replicate plates were required for a suspension of s. epidermidis yielding 40 cfu to achieve the same margin of error as obtained with 3 replicate plates at concentrations yielding 100-300 cfu. seven replicates plates were required for a suspension of e. coli yielding 40 and 100 cfu to achieve similar margins of error to those obtained with 4 replicate plates at concentrations yielding 300 cfu, and 3 replicate plates at concentrations yielding 500 cfu. conclusion: we found that the greater the concentration (300 and 500 cfu), the fewer replicate plates necessary to reliably estimate organism concentrations. the lower the organism density (40 cfu), the more plates necessary to reliably estimate cfus. contrary to the recommendations described in standard methods for the examination of water and wastewater, cfu of 500 were reliably reproducible. for greatest accuracy, experiments should be conducted so as to assure that colony counts are in the range of 300-500. direct microscopy: a valuable instrument for diagnosis and prognosis of periodontal disease objective: to appreciate the composition of micro flora from periodontal pockets, using light microscopy and to compare it with clinical status. introduction: it is generally accepted that periodontal disease occurs when anaerobic gram-negative flora increase in number with the subsequent decrease of facultative anaerobe grampositive bacteria. in other words, the switch from gram-positive to gram-negative of sub-gingival flora has a pathologic significance and could be observed using direct microscopy. materials and methods: 30 specimens sampled with sterile paper points from periodontal pockets and 10 samples from clinical healthy persons were included in this study. each sample was diluted in 0.5 ml saline solution and, with a calibrated loop, was taken 10 ll aliquots in order to prepare a smear for microscopic examination and for inoculation on solid media (columbia with 5% sheep blood). the smears were gram stained and the culture plates were incubated in anaerobic conditions (72 h, 37°c) and in air (24 h, 37°c). results: in 95% of samples from patients with periodontal disease, easily notable, high number of gram-negative bacteria at direct microscopy, associated with abundant growth in anaerobic condition and poor growth in air. in 9 from 10 healthy patients, the gram-negative flora was almost absent and gram-positive bacteria were in high number, correlated with the absence of bacterial growth in anaerobiosis and some growth in air.the presence of treponema spp. at direct microscopy was associated with deep and bleeding periodontal pockets. after few days of proper therapy, the good clinical status was well correlated with an increasing number of gram-positive bacteria. conclusions: 1) using a diluted sample for microscopic examination, the value of the method increase, offering important information about the composition of sub-gingival flora.2) the good correlation between the clinical status and microscopic finding recommend it as an easy to use diagnostic method in dentistry. identification of species and glycopeptide resistance among enterococcal isolates by bd phoenix objectives: vancomycin resistant enterococci are emerging in europe necessitating their fast and accurate identification by the laboratory. there was an attempt to evaluate the performance of the bd phoenix automated microbiology system (bd diagnostic systems, sparks, md.) for the correct identification of species and glycopeptide resistance in comparison to the gold standard of diagnosis, pcr, using a large collection of clinical strains. methods: a total of 232 enterococcal isolates were tested by the bd phoenix sytem. these strains were isolated from 145 faecal, 55 urine, 18 pus, 5 blood and 9 samples from other body sites cultures. a multiplex pcr was applied using 8 different pairs of primers, specific for the identification of e. faecium, e. faecalis and the vana, vanb, vanc, vand, vang, vane glycopeptide resistance genotypes. susceptibility to the glycopeptides was also confirmed by the etest (ab biodisk, solna, sweden). results: according to the pcr, there were 101 e. faecium (including 85 vana-positive strains), 72 e. faecalis (including 1 vana-positive and 1 vanb-positive strains) and 58 e. cass/gall isolates. two strains were not identified and were excluded from the analysis. discrepant results between the multiplex pcr and the phoenix system were obtained for 23/232 isolates (10%) with similar rates amongst faecal (15/145, 10.4%) and the rest of the isolates (8/87, 9.4%). the most common discrepancies were the misidentification of 11 e. faecium vana strains and 7 e. faecalis strains as e. cass/gall by phoenix. two e. faecalis strains were incorrectly characterized as vancomycin resistant, two e. faecium strains were misidentified as e. hirae and e.cass/gall, respectively, and one e.cass/gall strain was reported as e. faecium resistant to both glycopeptides. thus, the sensitivity and specificity for the identification of e.cass/gall by phoenix were 98.3% (57/58 strains) and 88.4% (145/164 strains), respectively, while 12.8% of vana strains (11/86 strains) were not recognized by this system. conclusion: this study demonstrates that the new identification system, phoenix, similarly to other automated or manual systems, presents with problems regarding correct identifica-tion of enterococcal species and glycopeptide resistance. specifically, laboratories should be aware that clinically significant isolates identified as e.cass/gall should be confirmed by another method. an audit of sputum requisition practices p. lal, i. balakrishnan (london, uk) objectives: to analyse the indications and rationale for the processing of sputum specimens in a london teaching hospital. methods: sputum samples received from 01/12/2004 -28/02/ 2005 were included in this study. data were obtained from the patient requisition forms and the winpath systems and were analysed further as per the objectives. results: a total of 1309 specimens were received during this period. 1202 (92%) from hospital in-patients and 107 (8%) from general practitioners. out of the total of 1202 samples received from hospital-in patients 167 (13.9%) had > 25 epithelial cells/ lpf. no clinical details were mentioned in 258 (21.4%) and 388 (32.2%) were from patients already on antibiotics. repeat specimens within one week were sent in 340 (28.2%) cases and 375 (33%) had atypical serology also sent. out of the 1202 hospital-in patient samples 372 (30.9%) had a significant isolate and 488 (40.5%) had normal respiratory tract flora isolated. others were reported as: ''gross oral contamination'' [168 (13.9%)], ''no growth'' [29 (2.4%)] and ''no significant growth'' [37 (3.07%)]. there were a few specimens reported as ''inappropriate specimen -two days old'' [35 (2.9%)] and ''leaking'' or ''saliva only' ' [22 (1.8%) ]. out of a total of 107 samples received from gp patients 14 (13.1%) of samples had > 25 epithelial cells/lpf, 25 (23.3%) had no clinical details provided, 5 (4.6%) samples were sent while patients were on antibiotics and 9 (8.4%) samples were repeated within one week. only 4 (3.9%) had atypical serology also sent. conclusions: -less than one-third of specimens yielded a significant pathogen.-adequate clinical details were lacking in about one-fifth of specimens.-nearly one-third of specimens were repeated within one week, without a clear indication.-about 16% of specimens were of poor quality.-atypical serology was only performed in 3.9% of outpatients, as compared with 33% of in-patients.this audit brings forth the fact that the clinical indications for which sputa are being sent for culture need to be clearly defined and an educational campaign instituted amongst relevant healthcare professionals. sputum collection techniques need to be rigorously applied if good-quality specimens are to be obtained. indications for performing atypical serology need to be defined and reinforced, particularly in primary care. a new approach to laboratory diagnostic of infectious gastroenteritis -a follow-up objectives: in order to optimize use of laboratory facilities and ensure flexibility in relation to current epidemiology, a new approach to laboratory diagnosis of infectious gastroenteritis was applied: from an algorithm the decision of which organisms to test for was defined by the demographic, clinical and epidemiological information submitted to the laboratory on paper/electronic request forms. methods: from april 1, 2004 -june 30, 2005 , hospitals and general practitioners submitted a request form with the following information together with the stool sample (s): (1) acute or persistent diarrhoea (duration > 2 weeks); (2) bloody stools; (3) recent history of foreign travel; (4) > 2 patients within same epidemiological setting; and (5) nosocomial infection. provision of data is mandatory when submitting electronically. based on these data, analyses were performed according to an algorithm. examination for salmonella, shigella, yersinia, campylobacter, and clostridium difficile was done by culturing. verotoxin producing e. coli (vtec), enteropathogenic e. coli (epec), enterotoxigenic e. coli (etec) and enteroinvasive e. coli (eiec) were identified by pcr for virulence genes and serotyping. rota and adenovirus were detected by antigen tests and parasites by microscopy. results: in total we examined 8,628 samples from 4,088 patients. a pathogen was isolated in 19% of patients. in 673 cases (17%) clinical/epidemiological data were missing. • 1,205 patients had diarrhoea < 2 weeks: 13% with campylobacter, 5% with salmonella, 4% with etec, 3% with giardia, and 1% each with eiec and vtec • 1,502 patients had diarrhoea > 2 weeks: 3% with campylobacter, 2% with giardia, 1% each with epec and vtec • 740 patients had a history of foreign travel: 7% with campylobacter, 6% with etec, 4% with salmonella, and 4% with giardia • 214 patients had bloody stools: 18% with campylobacter, 3% with salmonella, and 2% with vtec • 896 patients were < 7 years: 4% with campylobacter, 3% with epec, 2% each with giardia, vtec and salmonella, and 1% with etec conclusions: campylobacter was the most common bacterial pathogens in all groups and rotavirus was the most common pathogen in children < 7 years. the new approach had a number of advantages: more relevant microbiological analysis, collection of data on defined patient groups, and flexibility regarding adaptation to current epidemiological knowledge. increasing use of electronic submission of request forms will optimize the approach used. objectives: small colony variants (scvs) are an emerging infectious disease problem, presenting as a naturally occurring, slow-growing subpopulation of staphylococcus aureus that are characterized by tiny colonies on solid media. studies on scvs recovered from patients with persistent infections are hampered due to their frequent unstable phenotype. in particular, scvs are not easily distinguishable from the normal phenotype in broth media and a reversion of scvs into the normal phenotype is not traceable. methods: a set of isogenic s. aureus isolates comprising the (i) normal and the (ii) scv phenotype (isogenic to the isolate with normal phenotype) recovered from clinical specimens, as well as (iii) corresponding mutants mimicking the scv phenotype (knock-out of hemb), and (iv) their complemented mutants were used to investigate the feasibility of fourier-transform infrared (ftir) spectroscopy to trace the expressed phenotype in broth media. the respective isolates cultured on solid media served as controls. in addition, all isolates were genotyped by pulsed-field gel electrophoresis and spa typing. results: using first-derivative infrared spectra to calculate spectral distances, hierarchical clustering based on spectral information in three different spectral ranges resulted in a dendrogram that showed a clear discrimination between both staphylococcal phenotypes. distinct clusters comprising the clinical and mutant scv phenotype on one hand and the normal phenotype (isolate with normal phenotype and complemented mutant) on the other hand were found. thus, scvs from different clonal lineages gave spectra that were more similar to one another than to their normal growth parent. ftir was also shown to be able to trace the switch of the phenotypes in broth when the medium was supplemented. conclusion: ftir spectroscopy allows a rapid, reproducible and clear discrimination of different phenotypes of s. aureus in fluid media for diagnostic and research purposes. in contrast to genotyping approaches, ftir staphylococcal fingerprinting is only reliable for typing purposes if the isolates exhibit the same phenotype. in future studies, this technique may also provide an approach for tracing the scv phenotype in infected tissues. objectives: triggering receptor expressed on myeloid cells-1 (trem-1) is a recently discovered cell surface molecule whose expression on phagocytes is up regulated by exposure to bacteria or fungi. a soluble form of trem-1 (strem-1) can be measured in various body fluids. we studied whether strem-1 in cerebrospinal fluid (csf) could serve as a biomarker for the presence and outcome in patients with bacterial meningitis. methods: in this retrospective study on diagnostic accuracy we used an elisa to determine levels of strem-1 in csf from 46 adults with bacterial meningitis, confirmed by csf culture, who participated in the prospective dutch meningitis cohort study; 8 patients with viral meningitis, confirmed by polymerase chain reaction of csf; and 9 healthy control subjects, who underwent lumbar puncture to exclude the diagnosis of subarachnoid haemorrhage. the mann-whitney u test and the chi-square test were used to identify differences between groups. a receiveroperating-characteristic curve (roc) was constructed to illustrate various cut-off csf levels of strem-1 in differentiating between the presence and absence of bacterial meningitis and diagnostic accuracy was quantified by 95% confidence intervals (95% ci). results: levels of strem-1 in csf were higher in patients with bacterial meningitis as compared to those with viral meningitis [median, 159 pg/ml (range, 0 to 988 pg/ml) versus 0.5 pg/ml (range, 0 to 48 pg/ml); p = 0.001] and controls [0 pg/ml (range, 0 to 36 pg/ml); p < 0.001; fig]. patients with viral meningitis and controls had similar csf strem-1 levels. the area under the roc curve for discriminating between patients with and without bacterial meningitis was 0.88 (95% ci, 0.80 to 0.96; p < 0.001). at a cut-off level of 40 pg/ml, strem-1 yielded a sensitivity of 0.80 (95% ci, 0.69 to 0.88) and a specificity of 0.94 (95% ci, 0.77 to 0.99). in patients with bacterial meningitis, csf strem-1 levels were associated with mortality [survivors versus nonsurvivors: median 99 pg/ml (range, 0 to 365 pg/ml) versus 214 pg/ml (range, 0 to 988 pg/ml); p = 0.02]. conclusions: measuring strem-1 in csf may be a valuable new approach to accurately diagnose bacterial meningitis and identify patients at high risk for adverse outcome. therefore, a prospective study on strem-1 as biomarker in bacterial meningitis is needed. systematic review of rapid diagnostic tests for enterohaemorrhagic e. coli i. abubakar, l. irvine, l. shepstone, s. schelenz, c. aldus, p. hunter (norwich, uk) objective: a variety of rapid tests for the detection of enterohaemorrhagic escherichia coli (ehec) have recently emerged. culture on sorbitol macconkey (smac) agar and biochemical identification, while easy to use and inexpensive, is slow and lacks sensitivity in the detection of non o157:h7 serotypes. this study sought to determine the accuracy of rapid serological or polymerase chain reaction (pcr) assays which have been evaluated for the detection of all ehec serotypes compared to culture. methods: a systematic review and meta-analysis of 146 articles, identified via searches of electronic databases, hand searching of selected journals, and through contact with experts and commercial test manufacturers. the majority of these needed to be excluded due to low quality or lack of accuracy data. sensitivity and specificity of each method was calculated using full biochemical identification as the reference standard. twenty-one studies met the inclusion criteria, of which 7 used pcr methods and 10 used serological assays and 4 were based on culture. a summary receiver operator curve (sroc) was constructed from these data and the area under the curve (auc) calculated (using the trapezium rule). results: serological tests had individual sensitivities ranging from 0.82 to 1.00 and specificities ranging from 0.67 to 1.00. pcr tests had individual sensitivities ranging from 0.94 to 1.00 and specificities ranging from 0.92 to 1.00. additional analysis comparing smac agar culture with toxin detection methods showed poor sensitivity compared to pcr and serological tests (ranging from 0.24 to 0.38) yet the specificity was very good (1.00 for all 4 studies considered). our results suggest that both molecular and serological tests may have a potential role in detecting ehec infection. whilst there is very little difference in the effectiveness of these techniques, both are faster and have improved sensitivity when compared to traditional culture methods. fast, reliable diagnosis could lead to more informed treatment choices and improved outbreak control measures. however, given the substantial extra cost of these assays, an assessment of economic feasibility is necessary prior to use in everyday practice. antibodies against bordetella pertussis detected by slow agglutination test and elisa in two agerelated groups of vaccinated people suspected of acute pertussis: a comparative study objective: the aim of presented study was to describe differences between results of two tests used for detection of antibodies against bordetella pertussis (slow agglutination and elisa) in two age-related groups of patients suffering from respiratory infection. each of the people has undergone vaccination against b. pertussis. methods: paired sera obtained from two age-related groups of patients [(1). age 0-6 years, n = 49; (2). age above 6 years, n = 455] suffering from acute respiratory infection were tested. the first group comprised the children who were vaccinated earlier than one year before testing; the second group was determined by longer interval between the vaccination and the testing. the criterion of positivity of the slow agglutination was based on quadruple increase/decrease of the titer of specific antibodies; the criterion of serodiagnosis of the illness was the same. each of the patients was tested by elisa iga,igg,igm (virotech) during the same period, positive results of each of class of immunoglobulins were evaluated as positive elisa. the differences of results obtained by the two tests were assessed inside and between the groups. results: there were found 12.2% (respective 16.9%) concordant positive and 36.7% (respective 19.6%) concordant negative results between the tests in the first (respective second) group. there were found the following discrepancies in the frame of non equal results: agglutination positive/elisa negative sera were present in 8.2% (respective 13.2%) persons and agglutination negative/elisa positive samples were present in 42.9% (respective 62.2%) persons. conclusion: (1) . the frequency of serologically confirmed infection based on results of slow agglutination is higher in the group of people older six; the interpretation of the results in the younger group is limited by the influence of actual vaccination. (2) . the elisa evaluated as described above shows extremely high frequency of positivity in both groups, thus, the usefulness for diagnostics of acute infection seems to be low. (3) . the study will be continued to asses relationships between the positive results detected by slow agglutination and the positive ones detected by elisa in separate classes of specific immunglobulins. accuracy of the microscan walkaway system to identify coagulase-negative staphylococci a. sáez, b. ruiz, l. martínez-martínez (santander, es) objective: to determine the reliability of the identification of coagulase-negative staphylococci (cons) with the microscan walkaway 96 (wa, dade behring) system at species level when a > = 90% probability is obtained, considering as a reference the results of molecular identification. methods: one hundred and sixty-eight isolates of cons from clinical samples (october 2003 -may 2005 for which the identification with the wa system was ‡ 90%, and 9 atcc type strains were evaluated. bacteria were identified with the wa system using pos combo 1s panels. absence of coagulase was determined with a latex assay (pastorex ò staph-plus, bio-rad). reference identification was established by sequencing of the 16s rrna; when identification with wa and 16s rrna disagree, definitive identification was defined after sequencing of the soda and tuf genes, as previously described (drancourt et al. jcm 2000; 38: 3623-30 and heikens el al. jcm 2005; 43: 2286-90) . for identification, the sequences of 16s rrna, soda and tuf were compared with those in genebank. homologies values above 97% were considered reliable. results: all 9 type strains were correctly identified by 16s rrna sequencing as named by the atcc. among the 168 clinical isolates, the molecular method identified the following species (number): s. hominis (39), s. haemolyticus (35), s. saprophyticus (30), s. epidermidis (27), s. lugdunensis (12), s. schleiferi (7), s. capitis (7), s. simulans (4), s. pasteuri (2), s. warneri (2), s. intermedius (2) and s. equorum (1). the wa system correctly identified 8 out of the 9 atcc strains. s. pasteuri is not included in the wa database, and the corresponding atcc strain was misidentified as s. warneri. one hundred and fiftyseven out of the 168 (93.4%) clinical isolates were correctly identified by the wa. five s. haemolyticus were identified by wa as s. auricularis (2), s. simulans (2) and s. warneri (1) . other errors corresponded to: two s. pasteuri misidentified as s. warneri, one s. epidermidis as s. hominis, one s. lugdunensis as s. schleiferi, one s. hominis as s. haemolyticus and one s. equorum as s. cohnii. all isolates of s. saprophyticus, s. schleiferi, s. capitis, s. simulans, s. warneri and s. intermedius were correctly identified by the wa system. conclusions: the microscan walkaway 96 is reliable to identify cons at species level when a probability of > = 90% is obtained. s. pasteuri should be incorporated to the wa database in order to improve its performance. objectives: the aim of this study was to analyse the results of proficiency testing obtained by polish microbiology laboratories participating in polmicro. haemophilus influenzae is an important pathogen causing a variety of community-acquired respiratory tract infections, acute otitis media and purulent meningitis. two mechanisms of ampicillin (amp) resistance in this organism are described. one is mediated by the production of beta-lactamases tem-1 and rob-1; these amp-resistant strains are termed beta-lactamase-producing, amp-resistant (blpar). the second mechanism involves development of altered penicillin-binding proteins (pbp) with decreased affinity to amp and other beta-lectam agents. strains with resistance mechanisms mediated by pbp alterations are termed beta-lactamase-nonproducing, amp-resistant (blnar) h. influenzae. methods: four hundred seventy eight laboratories participated in this part of the scheme. each participating laboratory received haemophilus influenzae (pm-63)-beta-lactamase negative, ampicillin-resistant strain (blnar). the laboratories were asked to provide identification to the species level and of the susceptibility results and interpretation. results: correct identification to the species level of this strain was reported by 454 laboratories (97.2%) of the 467 labs involved. thirteen laboratories reported the analysed strain as haemophilus parainfluenzae. three hundred ninety eight laboratories (88.1%) of 452 correctly detected the mechanism of resistance to beta-lactams. only three laboratories incorrectly reported the organism as beta-lactamase producer. the greatest dispersion of inhibition zone was observed in the susceptibility of h. influenzae to ampicillin, amoxicillin-clavulanic acid and clarithromycin. conclusions: over 90% of the laboratories correctly identified and interpreted beta-lactamase-nonproducing, amp-resistant (blnar) h. influenzae strain. purpose and methods.the architect syphilis tp assay is a chemiluminescent eia that employs three recombinant antigens of treponema pallidum on the solid phase and an anti-human igm and igg conjugate. we evaluated this assay in comparison with a conventional eia (diesse enzywell syphilis screen recombinant) on unselected routine serum samples and on repository specimens for whom the results for specific igg and igm and of the rapid plasma reagin (rpr) assay were already known. in both instances an immunoblot (ib: inno-liatm syphilis score, innogenetics) has been employed on discordant specimens as a confirmatory assay. the precision and robustness of the architect assay were also evaluated.results.on 1.165 routine samples (975 from volunteer blood donors and 190 from in and outpatients) the concordance between architect and eia was high (1.155 samples, or 99.1%; 7 positives, 1,148 negatives). one of the discordant, positive by architect and negative by eia, was confirmed by ib. the specificity of the architect assay was 99.4% (95% confidence limits: 98.9-99.8). the 177 repository samples assayed belonged to three groups: 1) 9 biological false positives from 6 subjects: all negative by architect; 2) 134 true positives, all positive by architect, with a significantly stronger signal (average s/co: 26.19 vs. 14.53) on the 23 igm positive samples, all of whom were also positive by rpr; 3) 34 samples positive by rpr and negative by eia igg: 32 of them were negative by architect as well and for igm, while two specimens were strongly positive by architect and positive also for specific igm and with three specific bands by inno-lia, suggesting a pattern of recent infection. the reproducibility of the architect assay was good, with cvs of 7.78%, 5.71% and 5.51% on 28 replicates over 4 weeks of the assay's negative and positive control and of an internal control; finally, the s/co distribution of negative specimens confirmed the robustness of the assay, with a mean of 0.09, a median of 0.06, 10 standard deviations between the mean and the cut-off value and the 99th percentile at a s/co value of 0.53.conclusion.the automated assay for anti-treponema pallidum antibodies on the architect system has an excellent sensitivity and a good specificity. the analytical performances, coupled with the elevated throughput and minimal samples handling, make this method a first-choice option for syphilis screening and diagnosis in medium and large volume laboratories. objective: quantitative urine culture is the gold standard for defining the diagnosis of urinary tract infection (uti), because it allows identification of the uropathogenic species. however, this method is time consuming and expensive. approximately, up to 70% of urine cultures are negative with high cost for unnecessary testing. thus, we have evaluated the usefulness of two automated analysers for uti screening to quickly identify the negative samples that can be prompt reported to the clinicians, improving in the quality of patient care and allowing the laboratory to direct more effort into positive samples. methods: 1.165 of midstream urine samples submitted for microbiological examination were analysed by conventional urine culture plates (mcconkey agar + trypticase soy agar + bile esculine azide), sysmex uf-100 (sysmex, japan) and coral uti screen (coral biotechnology, ca, usa) automated analysers. uti was defined positive as follows: one or two strains of bacteria with at least 10 5 ufc/ml for the culture plates, more than 5.000 bacteria/ll and more than 15 wbc/ll for the uf-100 and/or an rlu value grater than 2% of the calibrator value for the coral. when more than two strains of bacteria were found, the culture was classified as contamined. results: the diagnostic performance of sysmex uf-100 and coral uti screen are shown in table 1. the sensitivity (95.8%) and negative predictive value (96.4%) confirm that sysmex uf-100 and coral uti screen are an excellent screening for uti. after this evaluation, we decide the use of the sysmex uf-100 and coral uti screen on our routine workflow for uti screening. the results of both the analysers are sent to a software system (labfinity dasit, italy) connected to the lis. if the results are lower than the cut-off values, uti can be excluded and directly reported to the physician. positive results are submitted to microbiological culture and reported within 24 or 48 hours depending on negative or positive bacterial growth. in our experience, evaluated on further 2.955 samples, this means that 78% of samples are immediately reported within very few hours. of the 22% of positive samples, 222 (35%) were confirmed by culture and reported within 48 hours, 418 (65%) were not confirmed and reported within 24 hours. comparison of the blood and bone marrow culture positivity rates for the diagnosis of brucellosis objectives: brucellosis is a common disease, seen worldwide as well as in our country. the diagnosis of brucellosis is made with certainly when brucellae are recovered from blood, bone marrow. in our study, we aimed to compare the blood and bone marrow culture positivity rates in patient with brucellosis. methods: this study was performed in the infectious diseases and clinical microbiology department of ankara research and training hospital between 2002 and 2004. the diagnosis of brucellosis was made on the history, physical findings, serologic findings and the isolation of the organism. the number of patients with brucellosis included to the study was 102. blood and bone marrow samples were taken from all of the patients on admission and cultured by using the bactec 9050 system. results: blood culture positivity for brucellosis was 48% (49/ 102), while bone marrow culture positivity was 34% (35/102). the difference between those positivity rates was found to be statistically significant (p < 0.05). the isolation ratio from blood cultures among acute cases was 66% (40/61) while it was 31% (9/29) among subacute cases. brucella isolation from blood was not detected in 12 chronic cases. the isolation rates of the microorganism from bone marrow of acute, subacute and chronic cases were 45.9%, 20.7%, 0.9% respectively. among our patients, 32 had history of medical therapy for brucellosis before admission and 27 of them was treated inadequately. of those 27 cases, the organism was isolated in 7 (25%) from blood and in 9 (33%) from bone marrow.in the cases with high standard tube agglutination titers, the rate of positivity was also high both in blood and bone marrow cultures. however when compared with low standard tube agglutination titers, that difference was not statistically significant.the mean growing time for the positivity of cultures was 4.2 days for bone marrow and was 5.8 days for blood cultures. the difference between the mean growing times of two culture types was found statistically significant (t-test. p < 0.05). conclusion: premedication, subacute and especially chronic phases decrease the possibility of isolation of the microorganism from blood culture. therefore we suggest taking bone marrow culture only for these kinds of patients as it. is a traumatic process. serological findings in blood sera of patients with yersinia-triggered arthritis e. golkocheva, r. stoilov, h. najdenski (sofia, bg) objectives: immunoblot analysis of iga and igg antibody response of blood sera from patient with yersinia triggered reactive arthritis and with undifferentiated arthritis were made. patients and methods: serum samples were obtained from patients admitted to clinic of rheumatology at medical university, sofia, bulgaria with suspicion of yersinia triggered reactive arthritis, based on diagnostic criteria. a total of 20 blood serum samples were analysed by immunoblot analysis with specific antigens-yops (yersinia outermembrane proteins). when y. enterocolitica is cultivated at 37 o c under calcium restriction (0.2 mm ca 2+ ), large amounts of yops are secreted into medium. these proteins were separated by 2d-sdselectrophoresis. results: immunoblot analysis of iga and igg antibody response against yops in 20 blood sera from patients with arthralgias and polyarthropathies was carried out. yersinia enterocolitica, serotype o: 8, was used as source for yop. seven strong bands of the molecular weights 26 kda-yope, 33 kda-yopn, 36 kda-yopd, 41 kda -v-ag, 43 kda-yopb, 46 kda-yopm and 51 kda-yoph were visualized. for immunoblot assay the optimal concentration of antigen was established by analytical electrophoresis. of the 10 blood sera from the patients with yersinia triggered reactive arthritis igg antibodies were detected against yoph, yopm, yopb, yopd, yopn and yope. iga antibodies were established against yopm, yopb, yopd, yopn and yope. all 5 sera from the patients with other rheumatic diseases were negative for the presence of anti-yersinia iga antibodies and two of them were positive for igg against yopd. antibodies from two classes were not detected in 5 sera samples from healthy people. conclusions: yops are borne by the virulence plasmid, which mean that they are clearly associated with virulence properties of pathogenic strains. moreover, yops is not restricted to single serotype and this made them a specific antigen in diagnosis of different yersinia infections. conventional techniques such as culture and demonstration of serum agglutinins prove to be insufficient to demonstrate invasive or chronic yersiniosis in contrast with the determination of specific serum iga and igg antibodies by immunoblot analysis and antigen detection. the detection of anti-yops igg and iga antibodies by immunoblot can be used for diagnosis of yersinia triggered arthritis. acknowledgements: this work was sponsored by natoreintegration grant 981256. objectives: to evaluate the identification and susceptibility results by using suspensions obtained directly from positive blood cultures. methods: during the period between 1st august and 31st october 2005 we selected all positive cultures grown in bact/ alert ò sa and sn bottles (biomérieux) from gram-negative bacilli. only the first culture positive from each patient was included. we inoculated 5 ml fluid from a positive bottle into a serum separator tube (bd vacutainer systems, plymouth, united kingdom) and centrifuged at 1500 x g for 10 minutes and the supernatant was carefully aspirated. using a cotton swab the bacteria were removed from the top of the separator layer to be suspended in 0.45% saline solution to get 0.6 mcfarland. the suspension was processed according to standard inoculation procedure for gn and ast-n020 vitek ò 2 cards. positive bact/alert 3d bottles were also sub cultured and after an overnight incubation several colonies were used to make a 0.63 mcfarland suspension in 0.45% saline. the suspension was processed according to standard vitek ò 2 inoculation procedure for gn and ast-n020 cards. results: identification: a total 56 gram-negative bacillus from positive blood cultures were investigated. fifty (89.2%) strains were correctly identified to the species level, four (7.1%) strains were not identified and two (3.6%) strains were misidentified. antimicrobial susceptibility testing: in all, 1040 mics were determined for 52 isolated by both methods. the unidentified strains (4) were excluded. the overall mic agreement between direct and standard inoculation was 93.9%. all individual antimicrobial agents scored > 90%. the overall minor error rate was 2.8% (30 of 1040). the overall major error rate was 1.05% (11 of 1040). the overall very major error rate was 2.1% (22 of 1040). the highest rate of mic agreement was for amikacin, norfloxacin (100%), meropenem (98%), gentamicin and ofloxacin (96.1%). conclusion: the direct method from positive bact/ alert&#61650; cultures cannot totally replace the approved methods of identification and susceptibility but in some cases provides earlier information which allows a better patient management and also reduce cost in patient care. investigation of listeria monocytogenes "o" antibodies in maternal and cord sera with the agglutination test e. us, a.t. cengiz, o. gelisen (ankara, tr) objectives: listeria monocytogenes is a gram-positive food borne pathogen that is responsible for listeriosis, a human infection with a mortality rate of 30%, which could cause severe motherto-child infections. this serious pathogen in pregnancy could be treated if diagnosed, but there is no routine screening test for susceptibility to listeriosis during pregnancy. therefore, we investigate different l monocytogenes serotype o antibodies for diagnosis of listeriosis in 275 maternal sera with agglutination test. 69 of them had spontaneous abortion, premature labour or stillbirth (group i), while 206 had no obstetric patology (group ii) in their previous pregnancies. cord bloods were also obtained at the delivery and tested. methods: all sera were being tested against antigens with the o formulation of serotype 1/2c, 3b, 4ab, 4c and 4d. the antigens were prepared by the method of osebold, and larsen et all. the bacterial suspensions were trypsinized for 15 min at 37°c to prevent cross-reactions and contaminations. sera were diluted by doubling serially in saline followed by addition of an equal volume of antigen. a positive titre of greater than or equal to 1:320 was chosen as positive test result to maximize the sensitivity and specificity. results: 4.36% of cases have ingested raw milk and diary products, 1.81% ready-to-eat foods, and 5.81% developed nonspecific febrile illness (nfi) during their pregnancies. 20% of group i were found positive (28.5% developed nfi) while at group ii 22% had positive (26.6% developed nfi) agglutination titres to one ore more serotypes. all the cord blood sera of group i were found negative, whereas two in group ii (all 4ab) were positive, with the positive maternal sera of the same serotype. it's evaluated as transmission of the antibody from mother to foetus. at group i the frequent serotypes were 1/2c = 4ab, at group ii 4ab, 1/2c, respectively. the newborns showed no symptoms or signs of listerial foeto-maternal infection. conclusion: the women encountered the antigens of l monocytogenes in any period of their life time (most 21-25 years of age) and produce antibodies against this pathogen. there is a relationship between nfi and positive titres. if the disease is recognized, it is possible to treat the mother and allow the birth of a healthy infant. we propose the less time consuming and easy to perform agglutination test as a routine screening test for susceptibility to listeriosis during pregnancy to prevent bad pregnancy outcomes. objectives: to evaluate the performance of a real time pcr assay (with a fluorogenic target-specific probe), mrsa-idi (geneohm sciences) for mrsa detection directly from mucocutaneous swabs in hospitalized patients. methods: clinical swabs (1 to 14 samples with a median of 2.1 samples per patient) from nares (n = 330) and skin (n = 326) were prospectively collected for mrsa screening from 321 patients admitted to a 858-bed teaching hospital. swabs were inoculated onto selective mrsa agar (mrsa-id, biomérieux), into the buffer extraction solution for idi-mrsa pcr assay and into enrichment broth (bhi with 7.5% nacl). after 24h, bhi broths were subcultured onto mrsa-id agar. selective agars were incubated for 48 h at 35ordm;c and examinated daily. suspected colonies were identified by coagulase testing; oxacillin resistance was tested by cefoxitin disk diffusion according to clsi recommendations. the pcr assay was performed according to the manufacturer's instructions. pcr results were compared with phenotypic identification test results. in case of discordant results, the assay was repeated, but only results from first testing were considered for calculating test performance. results: mrsa was detected by culture in 74 specimen (11.3%) from 38 patients. the sensitivity and specificity of the pcr compared with culture was 82.4% and 96.9%, respectively. positive predictive value and negative predictive value were 77.2% and 97.7%, respectively. the sensitivity of pcr (92%) was higher on nasal swabs than on swabs from other sites (77.5%, p < 0.001). the pcr assay detected mrsa in 34 patients (89.5%). the pcr assay provided results in 2 to 12 versus 48 to 72 hours for conventional method. conclusion: in our hospital, the id-mrsa pcr assay detected 89.5% mrsa carriers in less than 12 hours when performed on multiple specimen. the assay appeared more sensitive in testing nasal swabs than other clinical specimens. prospective studies are needed to evaluate the impact of this assay for rapid implementation of infection control procedures and its global costs and benefits. the purpose of this study was to establish a rapid and sensitive real-time polymerase chain reaction (pcr) method for detection of methicillin-resistant staphylococcus aureus (mrsa) from blood culture bottle. as a result of over use of broad-spectrum antibiotics after the 1960s in whole the world, an outbreak of mrsa infection has been seen. severe nosocomial infections with mrsa such as bacteraemia and sepsis may lead to multiple organ failure and high mortality in the hospital. although standard method took at least 48 hours to identify mrsa by the blood culture method, the presence of meca and nuc genes which is specific for methicillin resistance and s. aureus was determined by real-time pcr method within only 2 hours after blood culture signal positivity. nineteen s. aureus and 33 coagulase negative staphylococci positive blood culture bottles were studied retrospectively for detection of s. aureus and methicillin resistance. staphylococci were identified with classical methods and mics of oxacillin were determined by etest (ab biodisk) on mueller-hinton agar supplemented with 2% nacl. real-time pcr was performed to all positive blood culture samples for s. aureus and methicillin resistance determination. nineteen (100%) s. aureus were determined correctly by real-time pcr method. forty-four methicillin resistant and 8 methicillin sensitive staphylococci were detected by etest. using the real-time pcr method, the meca gene was detected in 47 staphylococci except 3. when compared with etest and realtime pcr method gave sensitivity, specificity, and positive and negative predictive values of 100%, 63%, 94%, 100% for both positive and negative tests, respectively. agreements between two methods were high (94%); there were 3 discrepant results among the 52 strains were tested. detection of mrsa bacteraemia and methicillin resistance with real-time pcr definitely is useful for reducing mortality and morbidity of this type infection. in conclusion, this method, as many as sensitive and specific for detection of mrsa bacteraemia and clinically should be beneficial for prevention of unnecessary antibiotic use and determination of appropriate antibiotic treatments of mrsa infection. pcr detection of class b, c and d betalactamases in environmental and clinical aeromonas strains t. fosse, c. giraud-morin, f. la louze (nice, fr) objectives: aeromonas spp. strains are waterborne opportunistic pathogens. they are able to produce different types of beta-lactamases (class b, c and d). the determination of beta-lactamase content is not easy by phenotypic methods. we have developed a pcr tool to study diversity and distribution of class b, c and d beta-lactamases in a set of representative clinical and environmental aeromonas species. method: a total of 22 references, 20 environmental and 80 clinical strains were tested. identification was realized by conventional tests and gyrb sequence analysis. beta-lactam antibiotic susceptibility was determined by diffusion agar and micro broth dilution methods. three sets of specific primers were defined for the pcr amplification of the internal region of class b beta-lactamase (mei1 and mei2, 297 bp size), class c beta-lactamase (aercp1 and aercp2, 840 bp) and class d betalactamase (aerd1 and aerd2, 514 bp). all pcr products were sequenced. results: class d pcr was positive with most strains except a. trota, a ticarcillin susceptible species (3 strains) . class c pcr was positive with most cephalothin resistant strains (mic >16 mg/l; 61/67 strains, 91%) including a. hydrophila and a. caviae phenospecies. class b pcr was positive with most strains of a. hydrophila and a. veronii phenospecies (33/38; 87%) including three imipenem susceptible strains (mic < 5 mg/l). beta-lactamase type distribution was species related and was particularly useful to better characterize environmental species such as a. bestiarum, a. popoffii and a. allosaccharophila. partial beta-lactamase gene sequence analysis allowed phylogenic studies. some cephalosporinase gene from environmental species was probable progenitor of ampc plasmidic beta-lactamase. conclusion: pcr with specific primers was a good method to detect class b, c and d beta-lactamase in aeromonas species. beta-lactamase type distribution and sequence analysis phylogeny were largely species related and could be helpful for molecular diagnostic and taxonomic purpose. objectives: the aim of this study was to develop a convenient dna extraction method and to optimise a pcr reaction in order to detect enterotoxin b producing s. aureus strains directly from milk. methods: we applied a chemical extraction method of bacterial dna from milk samples artificially inoculated with s. aureus. a pcr based method was used for the detection of seb gene (coding for enterotoxin b) and nuc gene (coding for termonuclease). a protocol for the multiplex pcr was developed and optimized. the sensitivity of the reaction was checked by determining the minimum number of organismsaeml -1 , which can be detected in the multiplex pcr and in each single pcr reaction. amplification specificity of the seb gene was verified by amplicon digestion with restriction endonucleases. results: the specific bands for both genes in the multiplex pcr were detected in samples containing a dna quantity corresponding to 5000 organismsaeml )1 . in the same reaction, the amplicon for nuc gene was visible for as little as the dna concentration corresponding to 1000 organismsaeml )1 . the sensitivity of each single pcr reaction was similar with those of multiplex pcr reaction. conclusion: the applied dna extraction method allowed us to obtain a good quality dna and can be used for a direct milk extraction. multiplex pcr reaction is a simple, rapid and reliable method for detecting enterotoxin b producing s. aureus strains from milk. objective: to detect the resistance to fluoroquinolones in 30 acinetobacter baumannii strains by a pcr-rflp assay. methods: thirty a. baumannii clinical isolates were obtained from different specimens (bronchial aspirates, blood-cultures, catheters, etc.) . the mics (minimal inhibitory concentrations) for ofloxacin were determined by agar dilution following standard methodology.a pcr-rflp method using one primer pair for amplification of a 344 bp fragment related to gyra gene (which codifies subunity a of dna-gyrase) and using one restriction enzyme hinf i was developed to study the resistance to ofloxacin in the different a. baumannii strains. when an a. baumannii strain is resistant to fluoroquinolones, a mutation in the position ser 83 of the dna-gyrase has been detected, decreasing the affinity for the antimicrobial. agarosa gel was used to determine the dna pattern: 2 fragments of 289 bp and 55 bp when there is not mutation and 1 fragment of 344 bp when the ser 83 to leu 83 mutation is present. results: the relationship between the pcr-rflp pattern and the mic to ofloxacin is shown in the table 1. the results of pcr-rflp analysis of most strains were in agreement with the results of mic. one isolate was susceptible to ofloxacin by agar dilution (mic = 0.25 mg/l) whereas by pcr-rflp this isolate seems to be resistant because it presents the mutation in gyra gene. two isolates with intermediate mic (4 mg/l) showed mutation in gyra. the genotypic study by pcr-rflp proved that ofloxacin resistant a. baumannii strains showed a punctual mutation in gyra gene, in the same position inside the sequence of gene. evaluation of a rapid amplification-detection assay for the identification of vancomycinresistant enterococci j. fuller, l. turnbull, s. shokoples, b. lui, l. rosmus, r. rennie (edmonton, ca) objective: the routine identification of vancomycin-resistant enterococci (vre) in clinical laboratories often yields a lengthy turn-around-time that may impede infection control efforts, particularly in an outbreak situation. in search of an improved vre test, we evaluated the genotype ò enterococcus assay (hain lifescience, germany), which provides both species and van gene identification for vre, and compared the results to conventional methods. methods: forty clinical enterococcal strains isolated on vrescreen agar media were selected for study. lactococcus and pediococcus were used as negative controls. conventional testing involved basic culture and identification tests, e-test susceptibility testing for vancomycin and teichoplanin, and pcr for vana, b, and c genes. the genotype ò enterococcus assay involved multiplex dna amplification and reverse hybridization of amplified product on an immobilized dna strip-blot containing probes for e. faecium, e. faecalis, e. casseliflavus, e. gallinarum, vana, vanb, vanc1, and vanc2/3. the genotype ò enterococcus assay produced correct species and van gene identification for all 40 (100%) vre isolates, including 7 e. faecalis vanb, 12 e. faecium vana, 12 e. faecium vanb, 6 e. gallinarum vanc1, 1 e. gallinarum vana-vanc1, and 2 e. casseliflavus vanc2/3. the only minor discrepancy was an e. casseliflavus that hybridized very weakly with the vanc1 probe in addition to the expected vanc2/c3 probe. the costs per specimen were comparable for each test method. however, the genotype ò enterococcus assay could be completed within a normal working day in contrast to conventional testing, which required a minimum of two days from the point of isolation on the vancomycin-screen media. conclusion: from this preliminary evaluation, the genotype ò enterococcus amplification-detection assay provides vre species and van genotype identification in a rapid and costeffective manner, superior to conventional culture methods. although further study is required, this kit may have clinical utility during a vre outbreak. application of minimal sequence quality values prevents misidentification of blashv type in single bacterial isolates carrying different shv extended-spectrum beta-lactamase genes background: detection of extended spectrum beta-lactamase (esbl) genes by pcr and sequence analysis is the gold standard for detection of shv-type beta lactamases. usually, quality values of sequence analyses are not reported. during a study on esbl epidemiology, three strains for which the default sequence assembly showed an shv)2 or shv-5 gene, showed low quality values at certain positions in individual sequence traces. we investigated the reason for these lower values. methods: shv genes were amplified by pcr from three isolates (escherichia coli, enterobacter cloacae and pseudomonas aeruginosa). individual sequence traces were analysed with the computer programs phred and codon code. pcr products were ligated in vector pcr2.1 and transformed to e. coli. sequence analysis was performed on eight individual clones from each transformation. results: visual inspection of the low quality positions in the sequence traces showed signals for two different nucleotides at three positions in the shv sequence: a or t at position 92, a or g at position 402 and a or g at position 703. the polymorphisms at positions 92 and 703 lead to aminoacid substitutions, the four different combinations would give shv types 2, 2a, 5 or 12. the double signals suggested that two or more blashv alleles were amplified. pcr amplicons were cloned in e. coli, in the sequences of individual clones only two combinations of the three polymorphisms were present: a92g402a703 and t92a402g703. these two combinations correspond to shv-2 and shv-12, respectively. conclusions: (i) in isolates of three different species, two different shv genes were present: shv-2 and shv-12. (ii) genotypic detection with default sequence assembly parameters may lead to misidentification of the number and type of shv genes carried by a single strain. (iii) careful interpretation of sequence data of shv genes, including analysis of low quality positions, may further improve our understanding of the epidemiology and evolution of these esbl genes. antimicrobial susceptibilities and epidemiological analysis of salmonella typhimurium human isolates in slovakia by phage typing and pulsed-field gel electrophoresis v. majtán, l. majtánova, m. szabó ová (bratislava, sk) objectives: salmonella typhimurium is a common cause of salmonellosis among humans and animals in many countries. in the last few decades the incidence of multidrug-resistant s. typhimurium infections appears to pose a particular health risk. the objectives of this study were analysis by antibiotic susceptibility, phage typing and pulsed-field gel electrophoresis (pfge) of s. typhimurium human isolates. methods: a total of 145 strains isolated during 1997-september 2005 were analysed. the susceptibility of isolates to ten antibiotics was evaluated by a disk diffusion method. the phage types were identified according to anderson et al. (1977) in the national reference center for phage typing of salmonellae. pfge was used to resolve xbai macro restriction fragments from all strains. results: of human isolates 88 (60.7%) were resistant to more than two antibiotics. sixty-three of isolates (43.4%) showed a classic dt104 resistance profile to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline (acssut). among this resistance type 37.2% were dt104, 7.6% were dt120 and one strain was dt20a. isolates encompassed 18 phage types. the majority of isolates was found to be definitive phage type dt104, representing 62.8% of all isolates. other phage types were mainly dt120, dt41 and dt20a. nine pulsotypes and 18 subpulsotypes were obtained using xbai restriction enzyme, but pattern x1 with its subtypes predominated (86.9%). a major pulsotype x1 was represented by 51.7% of dt104 isolates and was also found among dt120 isolates. conclusion: results indicated the spread of different clones of the multidrug-resistant s. typhimurium in the slovakia, but with predominance of one clone represented mainly by dt104 isolates. the phage typing as well as pfge may offer an improved level of discrimination for the epidemiological investigation of s. typhimurium human strains. novel reverse hybridisation assay to identify ctx-m genotype in cephalosporin-resistant isolates from uk and india to validate the assay results by dna sequencing. methods: isolate collection 1: 110 enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in london and south-east england. these isolates were known to carry phylogenetic group 1 blactx-m, but precise genotypes had not been determined. isolate collection 2: 130 enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in aligarh, north india. resistance determinants had not been investigated previously. a novel multiplex pcr was used to amplify blactx-m. reverse hybridisation was carried out using biotinylated pcr amplicon and sequence-specific oligonucleotides designed to identify members of ctx-m phylogenetic group 1. hybridisation results were validated by dna sequencing for 20 representative isolates from each collection. results: 109/110 london and se england isolates known to carry group 1 blactx-m gave a consistent profile, corresponding to that for ctx-m-15 and ctx-m-28; 1/110 gave a profile corresponding to ctx-m-3 and ctx-m-22. 82/130 indian isolates had blactx-m genes, all of which belonged to group 1, and all these gave a hybridisation profile corresponding to ctx-m-15 or ctx-m-28. ctx-m-28 and ctx-m-22 are rare variants, suggesting that the enzymes present were more likely to be ctx-m-15 and ctx-m-3, and this was confirmed by dna sequencing. conclusions: this is the first reported application of this novel reverse hybridisation assay to the analysis of large numbers of cephalosporin-resistant enterobacteriaceae. results were validated by dna sequencing. the assay is cheap and convenient, enables reasonable throughput, provides results within one day and can be used in place of dna sequencing. we believe it will be valuable for monitoring the prevalence and genotypes of blactx-m genes in enterobacteriaceae. detection of mexa and mexx efflux genes in p. aeruginosa: correlation between qc-rt-pcr and real-time pcr objectives: efflux systems are rarely identified as such in clinical microbiology laboratories. yet, over expression of transporters such as mexab-oprm and mexxy-oprm are likely to cause antibiotic multi-and cross-resistance in pseudomonas aeruginosa, leading to potential clinical treatment failures because of their inducible character. we have previously developed and validated with reference strains a qc-rt-pcr method to quantify mexa and mexx expression levels (eccmid 2005 . in the present study, we have developed a real-time-pcr assay and present here the correlation between both methods using control strains and clinical isolates. methods: expression levels of mexa and mexx were measured by both techniques in (i) 4 reference strains expressing only one of these efflux mechanisms [mexa (2) or mexx (2) ]; and (ii) 8 clinical isolates, in comparison with the wild-type strain pao1 (basal mexa and mexx expression levels). results: real-time pcr showed an inter-day reproducibility of 95 ± 5.3% (triplicates of 10 strains). among the clinical strains, 5 over expressed mexa and 3 mexx. the table shows (i) the mean level of overexpression of mexa and mexx in comparison with the wild type strain pao1 (set at 1), as detected by real-time pcr for all strains; (ii) the ratio of these values to those observed by qc-rt-pcr for the corresponding transporters. conclusions: both qc-rt-pcr and real-time-pcr are potentially useful in clinical laboratories as sensitive and rapid diagnostic tools to quantify the expression level of mexa and mexx in p. aeruginosa. combined with phenotypic characterization, this approach may help in a better understanding of the resistance mechanisms and epidemiology of resistance in this difficult-to-treat nosocomial pathogen. molecular detection of penicillin resistance in streptococcus pneumomiae n.g. rizk, n.a. abo khadr, s.m. abdel salam, n.m. gamil, m. hassan (alexandria, eg) objectives: the aim of the study was to detect penicillin resistant streptococcus pneumoniae by using seminested polymerase chain reaction (pcr) and to compare it with minimum inhibitory concentration (mic) of penicillin g. methods: fifty clinical isolates of streptococcus pneumoniae where isolated from patients admitted to alexandria main university hospital in egypt and were recovered from sputum (36 strains), throat swabs (11 strains), and pleural effusion (3 strains) . two species-specific primers 1a-1 and 1a-2, which amplified 1043 bp region of the pbp1a penicillin-binding gene, were used for pneumococcal detection. two resistance primers, 1a-r1 and 1a-r2, were used to bind to altered areas of pbp1a gene which, together with the down stream primer 1a-2, amplify dna sequences of 224 bp and 569 bp from isolates with penicillin mic > 0. objective: lipopolysaccharide-binding protein (lbp) is an acute phase protein produced in the liver. the objective of our study was to evaluate lbp as a marker of severity and prognosis in patients with bacteraemia. methods: 42 adult patients with community-acquired bacteraemia were included in a prospective manner. daily blood sampling for lbp and interleukin-6 (il-6) was performed. the patients were classified according to the systemic inflammatory response syndrome (sirs) criteria. demographic data, co-morbidity, microbiological aaetiology, routine biochemical parameters, focus of infection, severity score and mortality on day 28 were recorded. lbp and il-6 levels were analysed on plasma samples with a chemiluminescent immunometric assay (immulite-1000 ò ). results: the median age was 71 yrs. the mortality rate on day 28 was 16.6%. 5 patients had bacteraemia without sirs, 17 patients had sepsis and 20 patients had severe sepsis. lbp concentrations are presented as medians and range: 32.2 lg/ml (28. 2-34.1) in patients without sirs, ) in patients with sepsis and 50.9 lg/ml (22.9-96.5) in patients with severe sepsis (p < 0.05). lbp levels correlated to levels of il-6 (rs 0.52), c-reactive protein (rs 0.68), leukocytes (rs 0.44) and neutrophils (rs 0.46) (p < 0.01). lbp did not predict the outcome of the patients with bacteraemia. conclusion: lbp levels increased with the severity of sepsis in patients with bacteraemia. lbp correlated to il-6, c-reactive protein, leukocytes and neutrophils. lbp did not predict the outcome of the patients in this small cohort. pyrosequencing of the gra6 gene to discriminate type i, ii and iii toxoplasma gondii in clinical samples b. edvinsson, b. evengård on behalf of the esgt objectives: infection with toxoplasma gondii in immunocompromised transplant recipients is rare but often fatal. to increase our knowledge about the significance of the genotype of the parasite during infection, methods suitable for routine use need to be developed. pyrosequencing is a rapid sequencing-bysynthesis method performed in real-time. it is developed for detection of short nucleotide polymorphisms (snps), and is suitable for molecular genotyping of microorganisms. we here present a pyrosequencing assay for rapid and reliable discrimination of toxoplasma gondii type i, ii and iii in clinical samples. methods: twenty-two isolates of t. gondii were used for pyrosequencing analysis of the gra6 gene. real-time pcr was performed using a lightcycler 2.0 instrument to amplify a 176 bp fragment of the gra6 gene. pyrosequencing analysis of two different snps contained within a 10 bp fragment of the amplified product was preformed to identify t. gondii type i, by detection of nucleotides g and a at these respective positions. type ii was g and g, and type iii was a and a. to test the assay in a clinical context, blood samples and lung tissue from an immunocompromised patient was analysed. results: the detection limit of the assay is 10 parasitic genomes in a sample. reproducibility (r) was calculated as r = nr/n (nr = the number of isolates assigned the same type on repeat testing and n = the number of isolates tested). r was determined using three independent runs, and was 1, suggesting clearly interpretable results with little variation. typeablility (t) of the assay was calculated as t = nt/n (nt = the number of typeable strains and n = the number of isolates tested). t was determined using three independent runs, each including four atypical isolates. t was 0.82, suggesting that the assay discriminates correctly between the three main genotypes of t. gondii, but does not detect atypical strains. analysis of the clinical samples revealed type ii t. gondii in blood samples and lung tissue. conclusion: when preceded by real-time pcr, pyrosequencing is a rapid process with a high reproducibility and throughput. this makes it a good candidate for routine use. the method does, however, not detect atypical or recombinant strains. more than one gene may have to be analysed for that purpose. acknowledgement: in particular, we want to thank marie-laure dardé and hervé pelloux for provision of the t. gondii isolates. virulence genes in escherichia coli isolates from calves in shahrekord area, iran shiga toxin-producing escherichia coli (stec) strains, also called verotoxin-producing e. coli (vtec) strains, represent the most important recently emerged group of food-borne pathogens around the world. members of this group are a major cause of gastroenteritis that may be complicated by hemorrhagic colitis (hc) or the hemolytic uremic syndrome (hus), which is the main cause of acute renal failure in children. domestic ruminants, mainly cattle, sheep, and goats, have been implicated as the principal reservoir. transmission occurs through consumption of undercooked meat, unpasteurized dairy products and vegetables, or water contaminated by feces of carriers because stec strains are found as part of the normal intestinal floras of the animals.we studied the prevalence of shiga toxinproducing escherichia coli (stec) in stool specimens of calves with diarrhoea or other gastrointestinal alterations from 10 dairy cattle farms of shahrekord city (central of iran). the virulence genes, stx1, stx2, eae, intimin hly, enterohemolysin, st, lt, were detected by multiplex pcr method. stec strains were detected in 7 (11.5%) of 61 e. coli from 180 cases investigated. stec o157 was isolated in 7 cases (11.5%), whereas non-o157 stec strains were isolated from 4 animals (6%). stec strains were the most frequently recovered enteropathogenic bacteria. pcr showed that 5 (8.2%) isolates carried st gene. none of isolates carried an ehxa, eae, and lt (labile toxin) genes. our results suggest that stec strains are a significant cause of calf infections in this area and confirm that, infections caused by stec non-o157 strains are more common than those caused by o157:h7 isolates. the high prevalence of stec strains (both o157 and non-o157 strains) also found in human patients by other investigators, and their association with serious complications, strongly supports the utilization of protocols for detection of all serotypes of stec in spanish clinical microbiology laboratories. objectives: shiga toxins are a-b holotoxin including one enzymatically active a subunit associated non-covalently to five identical receptor binding b subunits. each subunit can cause different signalling pathways in different cells. to assess the effect of each single subunit the specific clones for expressing the single subunit was designed. periplasmic expression yielded native ab5 holotoxin or b5 pentamer. methods: o157 was used as bacterial strain for pcr amplification of shiga toxin gene. each subunit was amplified by specific primers and the amplified genes were cloned in pbad expression vector. the expression of the cloned genes was induced and optimized by different concentration of arabinose. the expressed proteins was assessed on sds-page and detected by elisa and western blotting. the expressed recombinant ab5 holotoxin and b subunit were purified and assessed for its biological activity on cells. cell cytotoxicity was shown by the expressed (ab5) holotoxin. moreover inhibition was observed by b subunit and antibody against it. results: e. coli clones expressing recombinant shiga toxin a and recombinant shiga toxin b subunits were established to release the toxin to periplasmic space. expressed toxin was examined by sds-page to visualize two subunits. the whole structure of these expressed subunits was checked in native gel. active ab5 structure expressed in periplasmic space was extracted by polymyxine b. the biological activity of the constructed recombinant shiga toxin showed both vero cell cytotoxicity and inhibition of in vitro protein synthesis. conclusion: in this study it was shown that for b subunit assembly and secretion to periplasmic space as b5 pentamer homologous leader sequence is not needed. although for biological active holotoxin (ab5) secretion to periplasmic space the presence of homologous leader sequence of gene is essential. these subunits can be used for studying on cell cytotoxicity and also as a vector for antigen presentation in immunotherapeutic approaches. characterisation of gram-positive anaerobic cocci by biochemical tests and partial 16s rrna sequencing a. bryk, a. kanervo-nordstrom, m. hyvonen, e. kononen (helsinki, fi) objective: gram-positive anaerobic cocci, which are common findings in various infections, are difficult to identify in clinical microbiology laboratories, where identification is based only on few phenotypic tests. in recent years, this group of organisms (traditionally known as peptostreptococci) has encountered several taxonomic changes. the aim of the present study was to compare the characterization made by a selection of key phenotypic tests to that by partial sequencing of the 16s rrna gene. methods: fifty-nine clinical isolates sent to our laboratory as gram-positive anaerobic cocci were examined for their colony and cell morphologies and biochemically characterized using spot catalase and indole reaction, 8 enzyme reactions by individual diagnostic tablets (rosco), sodium polyanethol sulphate susceptibility, glucose fermentation, and determination of metabolic end products. in addition, commercial identification test kit (rapid id 32a) patterns were performed. the sequencing of the 16s rrna gene of the 59 clinical isolates and 4 reference strains comprised of about 470 bp, and the sequences obtained were compared to those in genbank database by using the multisequence advanced blast comparison software from the national center of biotechnology information. results: the biochemical characteristics of the isolates were consistent with those of peptostreptococcus anaerobius (n = 8), peptostreptococcus (micromonas) micros (n = 6), finegoldia magna (n = 25), peptoniphilus asaccharolyticus (n = 8), peptoniphilus sp. (n = 8) and anaerococcus sp. (n = 2), whereas 2 isolates remained as unidentified gram-positive anaerobic cocci. biochemical identification correlated with that obtained by partial 16s rrna sequencing in 56/59 (95%) isolates at genus level and in 40/59 (68%) isolates at species level. the agreement of the biochemical and sequence-based identification was 100% for p. micros and f. magna. of 8 isolates biochemically identified as p. asaccharolyticus, 2 isolates were identified as peptoniphilus harei and 6 remained as peptoniphilus sp. by sequencing. according to the sequence data, the 2 unidentified isolates were peptoniphilus ivorii. conclusion: most isolates from human infections proved to be f. magna. a relatively good agreement of identification was obtained using biochemical testing and partial 16s rrna sequencing. objectives: molecular methods for identification of infectious agents in patients with clinical infectious disease are increasingly being used. especially in cases where antibiotics have been given prior to sampling or when fastidious bacteria difficult to grow are the aaetiology of the infection. infectious arthritis is a serious disease where identification of the etiological agent is mandatory for optimal antibiotic treatment as well as indication of the primary focus if not the joint it self. methods: in the present prospective study, 227 synovial fluids taken from patients in elucidation of affected joints and sent to a clinical microbiological laboratory in the copenhagen area, denmark, were examined by conventional (culture, phenotypic tests) and molecular methods (pcr/sequencing of 23s ribosomal genes). conventional methods included gramstaining and microscopy, aerobic and anaerobic culture and identification. pcr/sequencing included dna extraction, pcr assay which produced a 700 bp fragment of 23s rdna, and sequencing of both dna strands of the amplicons. sequencing data were edited and a blast search in the ncbi database was done. results: overall a microorganism was identified in 24 of the 227 synovial fluids (10.6%). in 14 synovial fluids from nine patients bacteria were identified by either methods [staphylococcus aureus (n = 8), streptococcus pneumoniae (n = 3), streptococcus dysgalactiae (n = 2), citrobacter freundii (n = 1)]. six synovial fluids were only culture positive; in four of those six specimens coagulase negative staphylococci were isolated. in three of the 227 synovial fluids a microorganism was identified by 23s pcr only. in two synovial fluids 23s pcr identified only one microorganism, whereas culturing resulted in two isolates. conclusion: the present study indicates a significant contribution by molecular methods (pcr/sequencing of 23s ribosomal genes) in recognizing and identification of microorganisms from foci normally considered sterile like synovial fluids. continued suspicion of infected arthritis despite of negative cultures should result in use of molecular diagnostics. direct detection of cardiobacterium hominis by broad-range 16s rrna pcr and sequencing in the serum of a patient with infective endocarditis e. malli, d. klapsa, a. vasdeki, m. morava, m. pitsitaki, e. petinaki, a. maniatis (larissa, gr) objectives: to describe the detection of cardiobacterium hominis directly in the serum of a patient with infective endocarditis, by employment of broad-range 16s rrna pcr followed by sequencing. methods: a series of blood cultures were taken from the patient before starting empirical treatment. in addition, 10 ml whole blood was collected in rubber sealed pyrogen-free tubes for direct detection of bacterial dna. bacterial dna was detected by a broad range pcr reaction and sequencing process allowed identification of bacteria species. results: cardiobacterium hominis was identified as the causative agent of infective endocarditis, on two days after the serum collection. blood cultures, simultaneously obtained with the serum sample, remained negative after 5 days of routine incubation; however, after a prolonged incubation of twelve days a gram negative bacterium was isolated from the aerobic bottles, that was identified as c. hominis species, by the usual phenotypic studies (catalase, oxidase reaction, indole, nitrate, etc) which are time-consuming. conclusions: to our knowledge this is the first report of direct detection of c. hominis in the serum using molecular methods, emphasizing the need for the establishment of such methods especially for infections caused by fastidious organisms. identification of dangerous bacterial pathogens by 16s ribosomal rna gene sequence analysis w. ruppitsch, a. stoeger, a. indra, d. schmid, k. grif, c. schabereiter-gurtner, a. hirschl, f. allerberger (vienna, at) to assess the usefulness of partial 16s rrna sequence analysis for identification of dangerous bacterial pathogens, a total of 28 isolates comprising bacillus anthracis, brucella melitensis, biovars melitensis, suis, abortus and bovis, burkholderia mallei, burkholderia pseudomallei, francisella tularensis, yersinia pestis, and 9 genus-related and unrelated control strains were sequenced and analysed using the genbank database (blast 2.2.10, national institute of health, u.s.a), the microseq500 database (version 1.4.1 and v1.0, applied biosystems, foster city, u.s.a.) , the ribosomal database project-ii database (rdp-ii, release 9, update 26, michigan state university, u.s.a), and the ribosomal differentiation of medical microorganisms database (ridom, university of wuerzburg, germany). on genus level all isolates were identified using genbank, rdp-ii, and microseq v1.0. the older microseq 1.4.1 database identified 92% of the tested samples correctly on genus level. the ridom database did not include sequence data of the tested species even on genus level, the ridom database none (''there seems to be, at least currently, no close relative available''). genbank and rdp-ii identified all dangerous pathogens correctly. the microseq v 1.0 database identified four of the six species of dangerous pathogens. on species level none of the dangerous pathogens was correctly identified using microseq 1.4.1 or ridom. as previously noted by various other authors, the most important reason for failure of databases in identifying a bacterium is a lack of the 16 s rrna gene sequence of the particular bacterium in the database rather than misidentification because of poor sequence quality. one must also be aware that the following bacterial species or subspecies have the same 16s rrna gene sequence, which makes differentiation by sequence analysis impossible: b. anthracis and b. cereus, y. pestis and y. pseudotuberculosis, all brucella subspecies, and francisella tularensis ssp. holarctica and mediasiatica. in addition to 16s rrna gene analysis complementary methods are essential to discriminate between these bacteria on species or subspecies level. identification of nontuberculous mycobacteria by sequence analysis of the 16s ribosomal rna, the heat-shock protein 65 and the rna polymerase beta-subunit genes s. shin, j.h. yoon, e.c. kim (seoul, kr) objectives: the diagnosis of diseases caused by nontuberculous mycobacteria (ntm) is difficult because ntm are prevalent in the environment such as soil and water and because they have fastidious properties. in this study, we investigated the distribution pattern of ntm clinical isolates and the identification to the species level. methods: among the presumptive ntm clinical isolates, cultured in a third referral hospital from 21-jan-2003 to 20-jan-2004 in seoul, south korea, which were negative by probe hybridization method for mycobacterium tuberculosis complex, we selected those of more than 10 colonies or those cultured more than twice in a same patient. a total of 120 isolates were studied for the distribution of ntm including 97 isolates recruited for species identification by direct sequencing of 16s rrna, hsp65 and rpob gene segments. (2.5%) were also identified in the presumptive ntm isolates. the identification rate by sequencing of 16s rrna, rpob, and hsp65 were 65%, 82% and 87%, respectively. hsp65 or rpob gene was more efficient than 16s rrna in identification of ntm by sequencing. conclusions: some ntm are considered to be the causative organisms of clinical diseases even in the countries with intermediate burden of tuberculosis, so accurate identification method by direct sequencing can be adapted to clinical laboratories. evaluation of the genotype mtbdr assay for the simultaneous detection of resistance to rifampicin and isoniazid of mycobacterium tuberculosis clinical strains f. brossier, c. truffot-pernot, n. veziris, v. jarlier, w. sougakoff (paris, fr) objectives: the rapid determination of drug resistance in mycobacterium tuberculosis is an important challenge to ensure a rapid effective chemotherapy. the genotype mtbdr test is a commercially available dna strip assay enabling the molecular genetic identification of the m. tuberculosis complex and its resistance to rifampicin (rif-r) and isoniazid (inh-r) by detecting the most commonly found mutations in the genes rpob (asp516val, his526tyr, his526asp, ser531leu) and katg (ser315thr). here, we report the evaluation of the genotype mtbdr assay from a set of 106 clinical isolates of m. tuberculosis. methods: 106 clinical isolates were collected in france over a 2 years period (2003) (2004) and were included in the study: 77 were rif-r, 96 were inh-r (of which 73 were also rif-r) and 6 were susceptible to both drugs. the susceptibility tests were carried out by the standard proportion method. the mutations involved in rif-r and inh-r in rpob, katg, inha and his promoter region, were characterized by dna sequencing. results: the genotype mtbdr assay identified 100% of the 77 rif-r strains harbouring mutations in the rpob gene, of which 37 (48%) showed a ser531leu mutation and 14 (18%) a his526asp or tyr mutation. 61 of the 96 inh-r strains (63%) harboured a ser315thr mutation in katg, all identified by the genotype mtbdr assay. 58 of this 61 strains displayed a high level of inh-r. among the other inh-r strains, 3 showed a katg mutation at the level of the 315 regions, which was different from ser315thr (2 of which showing a low level of inh-r), and one harboured a deletion in katg (with a high level of inh-r). these 4 mutations were also detected by the strip. finally, among the 31 remaining inh-r strains not detected by the mtbdr assay, 13 were characterized by a mutation in position -15 of the promoter region for the maba-inha regulon (10 with a low level of inh-r), 7 by a ser94ala mutation in inha (all with a low level of resistance) and 11 by other mutations. conclusions: the mtbdr assay, which can readily be included in a routine laboratory workflow, identified 100% and 68% of the strains resistant to rif and inh, respectively. interestingly, 60 of the 68 inh-r strains showing a high level of resistance (88%), but only 5 of the 28 inh-r strains with a low level of resistance (18%), were detected by the mtbdr assay, indicating that complementary tests are necessary for detection of the m. tuberculosis strains having a low level of resistance to inh. variation in the streptococcal 16s rdna detected by pyrosequencing m. haanperä, p. huovinen, j. jalava (turku, fi) originally the aim of this study was to identify alpha-haemolytic streptococcal isolates to the species level by pyrosequencing the v1 and v2 regions of the 16s rdna and comparing the results to the sequences of type strains that have been determined earlier. however, the isolates could not be unambiguously identified due to sequence variations detected in the alpha-haemolytic isolates. materials and methods: invasive s. pneumoniae isolates (n = 17), alpha-haemolytic streptococcal blood culture isolates (n = 16) and alpha-haemolytic streptococcal isolates from the normal pharyngeal microbiota (n = 34) of six elderly persons were analysed by pyrosequencing the v1 and v2 regions. results: varying degree of genetic variation was found in different types of streptococcal isolates. in the pneumococcal isolates, no sequence variation was detected as all the isolates contained the sequence specific for s. pneumoniae in both regions. also the sequences of the alpha-haemolytic blood culture isolates were well in agreement with the sequences of the streptococcal type strains. however, most of these isolates could not be unambiguously identified, as they contained sequences belonging to different species in the v1 and v2 region. consequently, only five of the isolates could be unequivocally identified as s. gallolyticus (n = 2), s. anginosus (n = 1), s. mitis (n = 1) and s. sanguinis (n = 1). the commensal streptococci contained numerous sequences to which an identical type sequence could not be found. also sequences identical to type strains were found; but similarly to the blood culture isolates, the results enabled the identification of only four isolates: s. mitis (n = 2), s. parasanguinis (n = 1), and s. salivarius or s. vestibularis (n = 1). moreover, the pyrograms of three blood culture isolates and ten pharyngeal isolates indicated heterogeneous 16s rdna alleles. one such pyrogram of the v1 region is presented in the figure. interestingly, four of the eight different nonheterogeneous v1 and v2 sequence combinations of the blood culture isolates were also present among the pharyngeal isolates. the results of this study indicate that the variation in commensal streptococci is greater than that of the streptococcal type strains and pathogenic isolates. the presence of identical sequence combinations among the blood culture and pharyngeal isolates supports the assumption that potentially pathogenic isolates are present in the normal microbiota. evaluation of partial 16s rrna gene sequencing for identification of clinical isolates of nocardia species m. marín, m. sánchez, m. del rosal, e. cercenado, p. martín-rabadán, e. bouza (madrid, es) new species of nocardia are being described. conventional identification based on biochemical characteristics and pcrrestriction enzyme analysis is frequently unable to distinguish them. partial sequencing of 16s rrna gene has proven useful in the identification of bacteria. objective: to evaluate the utility of 5'end 16s rrna gene pcr and sequencing in the identification of clinical isolates of nocardia sp. compared with conventional methods and pcr-rflp of hsp65. methods: 48 clinical isolates of nocardia sp. were characterized by biochemical reactions and disk diffusion susceptibility testing. molecular identification was performed by hsp65 pcr-rflp and pcr of 5'end of 16s rrna gene followed by sequencing. the sequences obtained were compared with those included in genebank. only alignments with similarities higher than 99% were considered. a comparison of sequences of our nocardia isolates with those deposited in genebank and well characterized phenotypically was performed using clustal x 1.8 software. results: distribution of species after pcr-rflp of hsp65 was n. asteroides vi (22), n. farcinica (11), n. nova (6), n. asteroides i (4), n. otitidiscaviarum (4) and n. asteroides iv (1) . partial sequence analysis of 16s rrna revealed a great heterogeneity between the isolates of n. asteroides vi, as follows: n. cyriacigeorgica (15 isolates), n. abscessus (3 isolates) and n. carnea (1 isolate). for 3 isolates, no genebank sequence was found with more than 99% similarity. all n. farcinica isolates had the same sequence and showed 100% similarity with those deposited in genebank. n. nova, n. asteroides i and n. otitidiscaviarum also showed sequence heterogeneity. three n. nova isolates matched with the recently described n. veterana and 3 with n. nova. n. asteroides i isolates were identified as n. abscessus (1) and n. beijingensis (3) . all n. otitidiscaviarum were identified properly. the isolate of n. asteroides iv was identified as n. transvalensis. conclusions: sequencing of 5'end 16s rrna gene is a useful and rapid molecular tool for the identification of nocardia clinical isolates. this method could provide more accurate results than the conventional ones used routinely in our laboratory. sequence analysis of the 5'end 16s rrna has enabled us to recognize great diversity and new species among our nocardia isolates. several species would have gone unnoticed using non-sequencing-based methods. antibacterial susceptibility studies-iii p1765 anaerobic bacteraemia due to fusobacterium necrophorum and clostiridium cadaveris: a case report m. panopoulou, e. alepopoulou, e. chrisafidou, a. tsaroucha, c. simopoulos, s. kartali (alexandroupolis, gr) introduction: anaerobic bacteremia is uncommon accounting 0.5-9% of bacteremias and it is associated with a high mortality rate, which is strongly and independently associated with underlying liver disease. case report: a 62 year-old man presented to our hospital with a 2-day fever and rigor. he had a history of cancer of the extrahepatic biliary tree, which was found incidentally during an operation for the treatment of echinococcal cyst of the liver. physical examination reveals high fever (39 c) and tachycardia. blood tests showed the following results: hb: 9.9 gr/dl, wbc: 22.500 /ul, plt: 296.000 /ul, tprot: 5.9 each colony type subcultured to blood agar plates and incubated aerobically and anaerobically (aerotolerance test). after 24 hours of incubation the two organisms grew only in anaerobic conditions. they identified by the api 20a system (bio-merieux-france) as fusobacterium necrophorum and clostiridium cadaveris. the patient's treatment started with metronidazole, amikacin and ceftriaxone and followed by metronidazole and imipenem. he was discharged after 3 weeks in a good condition. conclusions: although anaerobic bacteremia is rare, there is value in performing separate anaerobic blood cultures. the early recognition of anaerobic bacteremia and administration of the appropriate antimicrobial therapy play a major role in preventing mortality especially in patients with underlying disease. fluoroquinolone resistance among enterobacteriaceae strains isolated from urinary tract infections v. skandami-epitropaki, p. fostira, a. tsiringa, a. xanthaki, k. zampitha, m. toutouza (athens, gr) objectives: to study the frequency and antibiotic susceptibility of quinolone resistant bacterial stains isolated from patients with community-aquired bacteriuria and compare it with urinary pathogens from hospitalized patients. methods: during a 12-month period (october 2004 -october 2005 a total of 772 bacterial strains were isolated out of 8369 urine samples submitted for culture in our hospital laboratory from the community and from hospitalized patients with urinary tract infection symptoms. cultures and bacterial identification were obtained by conventional methods. antibiotic susceptibility testing was done by kirby-bauer disk diffusion method according nccls criteria. results: of the 772 bactrial strains studied (escherichia coli 604, klebsiella pneumoniae 97, proteus mirabilis 71), 14.6% of them were found to be quinolone resistant. the percentage of quinolone resistance was 19.9% for hospitalized patients (hp) and 5.7% for community patients (cp). the quinolone resistance for e. coli was 11.3% (16.5% for hp and 4.3% for cp), for k. pneumoniae 24.7% (28.0% for hp and 6.7% for cp) and for p. mirabilis 29.6% (29.6% for hp, 29.4% for cp). susceptibility pattern of the 113 quinolone resistant isolates to other antimicrobial agents was for hospitalized patients and community patients respectively as following: for e. coli ampicillin (am) 7%-9.1%, amoxicillinclavulanate (amc) 45.6%-54.5%, piperacillin-tazobactam (tzp) 69.4%-81.8%, cefuroxime (cxm) 64.9%-72.7%, trimethoprimsulfamethoxazole (sxt) 15.8%-18.2%, ceftazidime (caz) 66.7%-72.7%, cefepime (fep) 66.7%-81.8%, gentamicin (gm) 80.7%-72.7%. for k. pneumoniae am 0%-0%, amc 25%-0%, tzp 68.4%-100%, cxm 37.5%-100%, sxt 15.8%-100%, caz 37.5%-100%, fep 41.7%-100%, gm 79.2%-100%. for p. mirabilis am 9.5%-20%, amc 23.8%-40%, tzp 90.5%-100%, cxm 25%-40%, sxt 4.8%-0%, caz 23.8%-40%, fep 100%-100%, gm 56.3%-60%. seven strains of k. pneumoniae (29.2%) were carbapenem resistant and metallo-beta lactamase producing. conclusions: high resistance rates to fluoroquinolones were observed in uropathogen bacteria isolated not only from hospitalized patients but also from patients with communityacquired urinary tract infections in greece. increasing resistance rates to the rest antibiotic agents make the treatment of urinary tract infections a very difficult problem. susceptibility of pseudomonas aeruginosa isolated from the mystic programme to the carbapenems: meropenem and imipenem p.j. turner (macclesfield, uk) objectives: the meropenem yearly susceptibility test information collection programme (mystic) was initiated in 1997 in order to track the susceptibility of organisms in centres that were prescribing meropenem. this poster seeks to examine the susceptibility of pseudomonas aeruginosa isolates over this period to the carbapenems; meropenem and imipenem and, in particular, records the susceptibility of imipenem-resistant isolates to meropenem and vice versa. methods: pseudomonas aeruginosa isolates were speciated by the methods in current use at the participating centres. minimum inhibitory concentrations of meropenem and imipenem were determined using reference methods described by clsi. results: a total of 15709 isolates of pseudomonas aeruginosa have been tested globally, of these 78.2% were susceptible to meropenem at the breakpoint of < 4 mg/l and 70.1% to imipenem. globally, susceptibility to the two carbapenems has remained stable over the period 1997-2005, however when imipenem-resistant isolates were examined (n = 4625) 32.7% proved to be susceptible to meropenem, conversely of the 3359 meropenem-resistant isolates only 7.8% proved to be susceptible to imipenem. a similar pattern was seen when isolates were separated into global regions:usa 425 imipenem-resistant isolates, 25.4% susceptible to meropenemusa 346 meropenemresults: bacteroides fragilis group (bafg) accounted for 53% of the isolates, fusobacterium spp. for 7%, other gram negative bacilli (ognb) for 11%, clostridia (clos) for 14%, nonsporeforming gram-positive bacilli (nsfgpb) for 7% and cocci for 8%. beta-lactamases (bl) were detected in 61% of isolates. most bl + strains belonged to bafg (98%) and ognb (70%). at nccls-recommended breakpoints, more than 95% of isolates were susceptible to tzp, mtz, chl and mem, 92% to amc but only 77%, 70%, 62% and 33% to fox, ctt, cli and pen respectively. no nccls-breakpoints for anaerobes are available for mxf, lzd and tig. mic 50 and mic 90 for mxf were 1 and 64 mg/l, for lzd 2 and 4 mg/l and for tig 0.5-8 mg/l. in comparison with similar surveys conducted in 1987 and 1993 -1994 susceptibility of bafg to clindamycin decreased from 83% in 1987 , to 66% in 1993 -1994 and 48% in 2004 in bafg 92% of b. fragilis and 78% of non-b. fragilis were susceptible to amc in this study; in 1993-1994 susceptibility in these groups was 95% and 89% and in 1987-97% and 94% respectively. all isolates, except 6 bafg and 1 clos, were susceptible to mem. 98% of the isolates were susceptible to chl. susceptibility to mtz remains stable and is high in all groups except nsfgpb where mtz is active on merely 35% of the isolates. conclusions: tzp, mem and mtz remain very potent antimicrobial agents in the treatment of anaerobic infections. although still rare, resistant organisms were detected to each of them. therefore susceptibility testing of anaerobic isolates is indicated in severe infections to confirm appropriateness of antimicrobial therapy. further monitoring of background susceptibility is necessary to guide empiric treatment. comparative in vitro activity of levofloxacin against escherichia coli isolated from acute pyelonephritis in france in 2005 c.j. soussy, c. lascols, c. dib-smahi and the multicenter group study. objectives: the objective of this study was to evaluate the in vitro activity of levofloxacin (lvx) comparatively to other antibiotics against escherichia coli strains isolated from acute pyelonephritis in women consulting emerging rooms by 23 french hospitals in 2005. methods: mics of lvx, ofloxacin (ofx), ciprofloxacin (cip), nalidixic acid (nal), amoxicillin-clavulanic acid (amc), ceftriaxone (cro), cefixime (cfm), amikacin (an), gentamicin (gm) and cotrimoxazole (sxt) were determined by agar dilution according to the eucast breakpoints approved by 2005 recommendations of the comité de l'antibiogramme de la société française de microbiologie. quality control was performed with e. coli strain atcc 25922. results: a total of 231 strains were collected. 46.3% of strains were isolated from urinary samples, 10.8 % from blood culture and 42.9% from the two specimens. mics 50/90 (mg/l), the range of mics and the percentage of susceptibility (%) are presented in the following table: concerning the fluoroquinolones, mics50/90 of lvx were one/two dilution lower than those of ofx and two/one dilution higher than those of cip. for the other antibiotics, a higher percentage of susceptibility was observed with cro and an, when a lower percentage of susceptibility was observed with amc and sxt. conclusions: levofloxacin exhibited good in vitro activity against e. coli strains isolated from acute pyelonephritis with 93.1% of susceptible strains. in vitro activity of double and triple combinations of colistin, imipenem, rifampicin and linezolid against epidemic strains of multidrug-resistant acinetobacter baumannii producing oxa carbapenamases d.w. wareham, d.c. bean (london, uk) objectives: a. baumannii has emerged as an important cause of nosocomial infection in critically ill patients worldwide. in the uk three strains in particular exhibiting multi-drug resistance and producing oxa carbapenamases have been responsible for ongoing outbreaks. treatment options for infection with these organisms are limited as only colistin and tigecycline retaining significant activity in vitro. animal models and in vitro studies using other multi-resistant strains suggest that drugs in combination with colistin may be effective. we assessed the activity of colistin in combinations including imipenem, rifampicin and linezolid against epidemic strains from a recent uk outbreak. methods: isolates of a. baumannii exhibiting resistance to carbapenems were recovered from patients at barts and the london nhs. isolates were referred to the health protection agency and confirmed as belonging to clones producing oxa carbapenemases. activities of polymyxin, imipenem, rifampicin and linezolid alone and in double and triple combinations were determined using standard chequerboard assays with increasing concentrations of drug 1 on the x axis, drug 2 on the y axis and drug three in multiple replicate plates. after incubation at 24 hours wells were examined for growth and mic's determined for each combination. synergy between agents was defined as a fixed inhibitory concentration index (fici) of < 0.5. results: the isolates tested belonged to the oxa-23 clone 1, oxa-23 clone 2 and the south east clone, as confired by the hpa. colistin was the most active agent alone with mics from 1-2 mg/l. imipenem mic's varied from 4-32 mg/l. the most active combinations were colistin plus rifampicin (fici = 0.38) and colistin, rifampicin and imipenem (fici = 0.39). synergy was not seen with colistin in combination with imipenem alone. linezolid in combination with colistin (fici = 0.37), or imipenem (fici = 0.38) was synergistic but at therapeutically unobtainable linezolid concentrations (64 mg/l). conclusion: multidrug resistant strains of a. baumannii from the uk producing oxa carbapenemases remain susceptible to polymyxin in vitro. polymyxin exerts its effect on the bacterial cell wall; theoretically assisting other antibiotics to reach their respective targets, and seems a logical choice for inclusion in combination therapy. we have shown that rifampicin is synergistic with polymyxin against these isolates in vitro and may be effective in treating severe a. baumannii infections in man. a comparative in vitro evaluation of resistance development after exposure to teicoplanin, vancomycin, linezolid and quinupristin/ dalfopristin in staphylococcus spp. and enterococcus spp. mssa, mrse, msse, e. faecium and e. faecalis strains was determined on agar plates containing each antibiotic at clsi resistance breakpoints and at peak blood concentrations. after incubation at 37°c for 48 h colonies were counted and compared to the inoculum to calculate frequency of mutation. colonies grown in plates containing antibiotics were sampled for determination of mic values. results: frequency of mutation was less than 10-9 for all the tested antibiotics at peak blood concentrations. same results were obtained when breakpoint concentrations for each drug were used. conclusion: this one-step in vitro study demonstrated the ability of teicoplanin, vancomycin, linezolid and quinupristin/ dalfopristin to prevent growth of resistant mutants of staphylococci and enterococci, thus suggesting no occurrence of mutational events leading to resistance when bacteria are exposed to blood concentrations of these drugs. in order to establish the development of resistance after in vitro serial exposure to the same antibiotics simulating different in vivo concentrations, further studies are needed and are now in progress (multi step induction of resistance). in vitro activity of antimicrobial agents against legionella obtained from hotel water systems in turkey objectives: the aim of this study was to evaluate the in vitro activity of colistin against endemic pan-resistant acinetobacter baumannii (including resistance to imipenem) isolated during a 4 year period in a university hospital. methods: 150 imipenem-resistant acinetobacter spp. isolates were collected between january 2001 and october 2004, from a variety of clinical specimens of different patients attending distinct wards in a university teaching hospital. isolates were identified by api32gn and by sequencing the 16s rrna gene. mics of colistin were determined by agar dilution method, according to nccls susceptible breakpoint (£ 2 mg/l). pfge (apai restriction enzyme) was performed. results: 141 of 150 a. baumannii isolates (94%) were susceptible to colistin. colistin resistance (mic ‡ 4 mg/l) was observed in 9 isolates (5 isolates with a mic of ‡ 16 mg/l and 4 isolates with a mic of 4 mg/l) recovered from different patients in distinct wards. among these imipenem-and colistin-resistant isolates, 3 distinct pfge patterns were identified (clones a, b, and c). resistance to almost all beta-lactams (including carbapenems) and variable susceptibility to aztreonam, amikacin and tobramycin was a common feature of clone a. isolates belonging to clone b showed resistance to imipenem, amoxicillin and its association with clavulanic acid (amc), ureidopenicillins and their associations; susceptibility to ceftazidime; and variable behaviour to meropenem, cefepime, cefpirome and aztreonam. the susceptibility profile to aminoglycosides was variable, differing from clone a in its susceptibility to netilmicin and minocycline. clone c was resistant to imipenem, amoxicillin, amc, piperacillin, piperacillin + tazobactam, ticarcillin and ticarcillin + clavulanic acid, but remained susceptible to meropenem, aztreonam, cefpirome, ceftazidime and cefepime. conclusion: only colistin, one of the few effective drugs available against multi-drug-resistant acinetobacter infections, showed in vitro activity against the majority of acinetobacter spp. strains isolated within the sampled hospital. the observed 6% a. baumannii resistance to the recently re-introduced colistin seems like the first chapter of a novel repeatedly told for several antibiotics. emergence of high-level gentamicin resistance in clinical enterococcal isolates of companion animals in portugal objectives: to characterize in vitro gentamicin susceptibility among enterococci causing infections in cats and dogs, in order to evaluate the impact of high-level gentamicin resistance in small animal therapeutics. methods: the samples were collected at the veterinary teaching hospital of the faculty of veterinary medicine and at veterinary private practices in the lisbon area. from january 1998 until november 2005, a total of 43 enterococci were isolated from dogs and cats with urinary tract infection (uti), otitis externa (oe) and pioderma. bbl crystal gram positive id system was used for identification at the species level. minimal inhibitory concentrations (mic) were determined by the microdilution method according to nccls (1) . the bifuntional enzyme gene that confers high-level gentamicin resistance (hlgr) was detected using pcr (2) . results: enterococcus faecalis was the predominant isolate (n = 35), followed in frequency by enterococcus faecium (n = 6). mic cumulative data analysis showed that mic50 values were 8 lg/ ml and mic90 1024 lg/ml. six (14%) hlgr clinical enterococcal isolates were detected, with mic ranges between 1024-2048 lg/ml. four of these enterococci were isolated from uti and 2 from oe. four of the phenotypically high-level gentamicin resistant isolates carried the aac(6')-ie-aph(2'')-ia gene. conclusions: the importance of enterococcal infection in small animal clinical samples has increased over the last years. mic cumulative data points out low-level gentamicin resistance among clinical enterococci isolates of veterinary origin and the emergence of high-level isolates, as previously detected (2). this fact compromises cell-wall active agents (such as ampicillin or vancomicin) and aminoglicoside in vivo synergy. the aac (6')-ieaph(2'')-ia gene carriage is of concern because its expression confers resistance also to tobramicin, netilmicin, amikacin and kanamicin. our findings are of critical importance, as they may have a direct impact in therapeutic decision in the management of companion animal's infections by enterococci. furthermore, transfer of resistance genes and resistance strains between animals and owners/caretakers by direct contact is a concerning probability. references: (1) results: interpretative criteria were used according to nccls 2002.during the study period penicillin resistant strains of s. pneumoniae was noted as follows: 67% in sputum or ta,19% in blood,12%in csf,and 75% in others,against cefuroxim resistant strains:15% in sputum or ta,10% in blood,4% in csf.regarding the susceptibility to ofx,penicillin resistant s. pneumoniae strains from sputum or ta revealed 97.9%.the penicillin resistant strains coming from sputum or ta showed resistance as follows; 48% to em and 78% to sxt,against strains isolated from others: 58% to em and 49% to sxt.no resistant strain to va was found. conclusion: the percentage of the penicillin resistant s. pneumoniae isolates from the lower respiratory tract, middle ear fluid, eye fluid and sinus was markedly higher than that of the isolates from blood and csf. the most efficient drugs against penicillin resistant pneumococci were cefuroxim and ofloxacin. these results from romania also underline the previous observations regarding the higher emerging rates of resistance in s. pneumoniae worldwide. penicillin resistance in streptococcus agalactiae objectives: streptococcus agalactiae has become recognized as a cause of serious illness in newborns, pregnant women, and adults with chronic medical conditions. heavy colonization of the genital tract with streptococcus agalactiae also increases the risk that a woman will deliver a preterm low-birthweight infant. early-onset infections (occurring at < 7 days of age) are associated with much lower fatality than when they were first described, and their incidence is finally decreasing as the use of preventive antibiotics during childbirth increases among women at risk. penicillin or ampicillin remains the drug of choice for intrapartum antibiotic prophylaxis for streptococcus agalactiae colonization in pregnant women. erythromycin and clindamycin are the drugs of choice for women with serious penicillin allergy who are colonized with streptococcus agalactiae. the objective of this study is to estimate the insorgence of penicillin resistance among streptococcus agalactiae. methods: all streptococcus agalactiae were tested against penicillin by agar dilution method according to clinical and laboratory standards institute 2005 (clsi); breakpoints for resistance were those recommended by the clsi. antimicrobial agents were obtained from their manufacture as laboratory grade powder. results and discussion: four hundred seven (407) clinical isolated were analysed during 2005. streptococcus agalactiae resulted resistant to penicillin in 1 case; and about 2% resulted borderlines.the present findings indicate a probable evolution in s. agalactiae toward penicillin resistance this finding suggest the need a continuous national and international surveillance programs to provide timely data on the evolution of incidence of penicillin resistance in this pathogen. ciprofloxacin susceptibility of the most common isolates at bacterial conuctivitis conclusion: according to the average numerals we concluded that all the isolated strains are highly susceptible at ciprofloxacin. its application in the conuctivial saccus is especially important in curing the conuctivial infections with resistent strains like pseudomonas aeruginosa. we successfully cure the bacteria chronic conuctivitis with the adequately used therapy according to antibiogram. antimicrobial resistance patterns of acinetobacter baumannii in clinical isolates g.t. tsilika, v.p. pliatsika, m.t. tsivitanidou, d.s. sofianou (thessaloniki, gr) objectives: a. baumannii is a nosocomial pathogen, commonly isolated from critically ill and immunocompromised patients. the aim of the present study was to evaluate the antimicrobial resistance of a. baumannii strains isolated in a tertiary care hospital througout a three-year period. methods: a total of 1311 a. baumannii strains were selected from january 2002 to december 2004.the specimens were obtained from inpatients hospitalized in intensive care unit (icu) and pediatric intensive care unit (picu) and other departments of our hospital.the identification and the antimicrobial susceptibility testing were performed using the vitek 2 automated system(biomerieux,france conclusions: the emergance and rapid spread of multidrug resistant a. baumannii isolates are of a great concern worldwide.imipenem was one of the most potent agents for treatment of those infections caused by multiresistant strains.the increasing prevalence of imipenem resistance limits therapeutic options and leads to outbreaks of carbapenems resistant strains. tigecycline in vivo studies objectives: antibacterial agents disrupt the ecological balance of the normal human microflora. disturbances may lead to the emergence of antibiotic resistance and/or to infections by potentially pathogenic bacteria. tigecycline, a member of a new class of antibiotics (glycylcyclines), has been shown to have a potent expanded broad-spectrum activity against most grampositive and gram-negative aerobic and anaerobic bacteria. the aim of the study was to investigate the ecological effects of tigecycline on the normal oropharyngeal and intestinal microflora in healthy subjects. methods: thirteen (13) white subjects (6 women, 7 men) aged 20 to 31 years, received 100 mg of tigecycline in the morning on day 1 as a 30-minute intravenous (iv) infusion, followed by 50mg doses of tigecycline given every 12 hours as a 30-minute infusion for 10 days. one (1) subject was withdrawn on day 2 because of an adverse event. serum, saliva, and faecal samples were collected before, during, and after administration for microbiologic cultivation and for assays of tigecycline. all new colonizing bacteria were tested for susceptibility (resistance >8 mg/l) during the investigation period. results: the serum concentrations on day 9, 12 hours after dosing, were 0.2 to 2.1 mg/l (mean value 0.4 mg/l, median value 0.2 mg/l, and sd 0.5 mg/l). the faecal concentrations on day 8 were 3.0 to 14.1 mg/kg (mean value 6.0 mg/kg, median value 5.6 mg/kg, and sd 2.9 mg/l). saliva concentrations were generally low, with highest mean value 0.18 mg/l, median value 0.22 mg/l, on day 10, 3 hours after dosing. a minor effect on the oropharyngeal microflora was observed. the numbers of enterococci and escherichia coli in the intestinal microflora were reduced at day 8, while other enterobacteria and yeasts increased. there was a marked reduction of lactobacilli and bifidobacteria but no impact on bacteroides. no clostridium difficile strains were isolated. two (2) klebsiella strains and 5 enterobacter strains resistant to tigecycline were found. conclusion: tigecycline had a minor effect on the oropharyngeal microflora. tigecycline's effect on the intestinal microflora was due to its spectrum of antibacterial activity and intestinal concentrations. objectives: to examine and report the use of tigecycline (wyeth) in the treatment of multidrug resistant acinetobacter (mdra) culture positive sepsis in 11 patients requiring mutiorgan support. methods: all patients were managed within the liver intensive care unit. physiological data was collected prospectively and entered onto a specialist database. patients received standard intensive care management; antibiotic and antifungal therapy administered as indicated by microbiological cultures. systemic inflammatory response (sirs) features initiated blood cultures (vascular lines and peripheral), drain fluid culture and broncoalveolar lavage (bal). screening swabs were undertaken weekly and samples sent for culture at laparotomy. mdra positive cultures from blood, bal, drain fluid or samples taken at laparotomy in the context of sirs resulted in the initiation of tigecycline treatment. results: 11 patients received tigecycline treatment for mdra infections. the underlying disease states were necrotizing pancreatitis (1), post hepatectomy (1), polytrauma (1), all with postive intra-abdominal cultures. acute and acute on chronic liver failure (4), mdra +ive broncho-alveolar lavage ± blood cultures and 4 post liver transplant patients (necrotising pancreatitis in one, 2 with recurrent small bowel perforation and 1 with retroperitoneal haemorrhage) all with positive blood cultures and in 3 positive intra-abdominal tissue/clot. mean time from admission to treatment for mdra was 25 days. mean duration of treatment was 10 days (range 4-15). mean apache ii score at initiation of therapy was 18 (range 13-26); 4/11 patients survived to intensive care discharge and 3/11 to hospital discharge. microbiological clearance of mdra was observed in 8/11 cases. in those who did not achieve microbiological clearance cause of death was intra-abdominal haemorrhage, recalcitrant organ failure with recurrent small bowel perforation and vasopressor resistant shock. in these patients one remained culture positive for intraabdominal sepsis despite full treatment (small bowel perforation x5). the drug was well tolerated with the only side effect being that of hypercalcaemia observed in 5/12 patients, mean corrected calcium 2.59 mmol/l, range 2.32-2.81. in all cases this resolved on drug discontinuation. conclusion: tigecycline appears to be an efficacious agent in the treatment of deep seated mdra infections. objectives: nausea (n) and vomiting (v) have been reported with tigecycline, a new glycylcycline with expanded broad spectrum activity. exposure-response relationships and patient covariates predictive of the first n and v occurrence were evaluated in patients with complicated intra-abdominal infections (ciai). methods: data from patients from 3 ciai trials (one phase 2 and two phase 3), receiving 100 mg loading dose and 50 mg every 12 hours, were pooled for analysis. n and v (definitely, possibly, or probably related to tigecycline) reported from the start of infusion until 24 hours after the last dose were included. individual exposure measures [auc0-12 and cmax] were calculated using a previously developed population pk model. logistic regression was used to evaluate predictors of first n and v occurrence. covariates included age, weight, sex, region of treatment, and baseline n and v. results: the dataset included 928 patients (218 with pk). mean (sd) age and weight were 46 (18) years and 73 (16) kg. 64% of patients were men and 24%, 37%, and 18% were enrolled in north america, europe, and latin america, respectively. baseline nausea or vomiting was reported in 47% and 35%. overall, n and v occurred in 18% and 13% of patients receiving tigecycline, however most (62%; 67%) of first n and v events were mild in nature. women had more n and v (23%; 17%) than men (15%; 11%). n and v were lower in europe (10%; 6%) than in other regions. auc0-12 and cmax were not predictive. the final nausea model included weight, sex, region, baseline nausea, and the interaction of weight/region as predictors of the first nausea occurrence (p = 0.671, 0.0006, 0.205, 0.033, & 0.023, respectively objective: because hospitalisation for community-acquired pneumonia (cap) is associated with substantial morbidity and health resource utilisation, we evaluated the predictors of prolonged hospital length of stay (los) and treatment duration. methods: we conducted a retrospective analysis of data from a double-blind, randomised, multicentre clinical study that compared the efficacy and safety of tigecycline with that of levofloxacin in the treatment of patients with cap requiring hospitalisation. patients were stratified by the fine pneumonia severity index and randomly assigned to receive tigecycline or levofloxacin via iv administration for at least 7 days. treatment duration and hospital discharge were based on physician assessment of signs and symptoms of infection and patient condition. we used cox proportional hazards modelling with stepwise selection to identify statistically significant predictors (p < 0.05) of treatment duration and hospital los. results: among 426 patients with cap in the clinical intent-totreat population with complete hospitalisation data, mean age was 49.8 years (range 17-92) and 22.8% of patients were aged ‡65 years. diabetes (11.5%), chronic obstructive pulmonary disease (7.8%), and congestive heart failure (7.0%) were leading co-morbidities. about 37.1% of patients were smokers and 5.6% were characterised by alcohol abuse. median fine pneumonia severity index score was 3; 21.1% of patients had a score >4. . there were no significant differences between the 2 groups in treatment duration or los. conclusions: tigecycline, a first-in-class glycylcycline, was associated with treatment duration and los similar to that of levofloxacin, adjusting for several identified risk factors. tigecycline effective in treating patients with intra-abdominal or skin/skin structure infections who have bacteraemia e.j. ellis-grosse, r. maroko (collegeville, us) objectives: the treatment of bacteraemia, which is a potentially fatal complication of infections originating at other body sites, is complicated by increasing resistance. tigecycline, a first-in-class glycylcycline, has an expanded spectrum of activity against gram-positive, gram-negative, anaerobic, and atypical bacteria including resistant strains. tigecycline is safe and effective in treating complicated skin and skin structure (csssi) and intraabdominal infections (ciai). this analysis examines tigecycline clinical trial experience in patients with ciai or csssi who had bacteraemia (presence of bacteria in blood) at baseline. objectives: treatment of complicated intra-abdominal infections (ciai) is challenging due to diverse bacteriology and bacterial resistance. the efficacy and safety of tigecycline (tgc), a first-in-class glycylcycline approved in mexico, brazil, peru, colombia and usa for treating ciai and complicated skin and skin structure infections, was compared with imipenem/cilastatin (imi/cis) in adult hospitalised patients with ciai in two double-blind, phase 3 multinational trials. this analysis evaluated tgc efficacy and safety in the european region of the integrated results of these two trials. methods: one study was conducted in 96 centres (17 countries) and the other study was conducted in 94 centres (27 countries). patients were stratified by disease severity (apache ii score £15 vs >15 but £30), and randomly assigned to iv tgc (100 mg loading, then 500 mg q12h) or iv imi/cis (500/500 mg q6h) for 5-14 days. clinical response at test-of-cure (toc, 12-44 days after therapy) for microbiological evaluable (me) and microbiological modified intent-to-treat (m-mitt) were co-primary efficacy endpoints where cure/failure responses were determined. safety was assessed by physical examination, laboratory results, and adverse event (ae) reporting. results: in the european analysis, 703 patients were mitt (received ‡1 dose), 556 m-mitt (283 tgc, 273 imi/cis) and 460 me (237 tgc, 223 imi/cis). treatment groups were balanced with respect to demographics. patients were mostly white (98.2%) men (58%) with a mean age of 50 years. for me, clinical cure rates at toc were 92.4% (219/237) for tgc vs 88.8% (198/ 223) for imi/cis (95% ci = )2. 2, 9.4 ; test for non-inferiority p < 0.0001). clinical cure rates for m-mitt were 87.3% (247/283) for tgc vs 83.5% (228/273) for imi/cis (95% ci = )2.5, 10.0; test for non-inferiority p < 0.0001). most commonly reported treatment emergent aes (teaes, mitt) for tgc and imi/cis were nausea (14.7% and 11.8%, p = 0.267) and vomiting (10.7% and 7.3%, p = 0.146). the imi/cis group had significantly higher teaes of fever (7.3% imi/cis vs 3.2% tgc, p = 0.017), hyperglycaemia (1.7% imi/cis vs 0 tgc, p = 0.031) and dyspnoea (2.8% imi/cis vs 0.3% tgc, p = 0.011) where tgc had significantly higher amylase increase (3.2% tgc vs 0.6% imi/cis, p = 0.011) and bun increase (2.3% tgc vs 0 imi/cis, p = 0.003). conclusions: similar to the overall integrated analysis of the two phase 3 trails, in the european analysis, tgc was safe and effective in the treatment of hospitalised patients with ciai in comparison with imi/cis. tigecycline is safe and effective in the treatment of complicated skin and skin structure infections: european experience of two double-blind phase 3 comparison studies with vancomycin/aztreonam r. maroko, n. dartois, d. sarkozy, j. goodrich, e.j. ellis-grosse on behalf of the tigecycline 300 and 305 study groups objectives: tigecycline (tgc) a first-in-class expanded spectrum glycylcycline, has been approved in mexico, brazil, peru, colombia and usa for treating complicated skin and skin structure infections (csssi) and complicated intraabdominal infections. two phase 3, randomised, double-blind studies were conducted in hospitalised men and women with csssi to determine tgc safety and efficacy compared with vancomycin/aztreonam (v/a). the objective of this analysis was to evaluate the efficacy and safety seen in the european population of the integrated analysis of these 2 phase 3 trials. methods: one study was conducted in 53 centres in 8 countries while the other study was conducted in 65 centres in 21 countries. patients were randomly assigned (1:1) to receive either tgc (100 mg, followed by 50 mg iv twice daily) or vancomycin (1 g iv twice daily) plus aztreonam (2 g iv twice daily) for up to 14 days. clinical response at test-of-cure (toc, 12-92 days after therapy) for clinically evaluable (ce) and clinical modified intent-to-treat (c-mitt) populations were coprimary efficacy endpoints in which cure/failure responses were determined. secondary objectives included determination of in vitro susceptibility to tgc of a range of bacteria that cause csssi and microbiological efficacy. safety was assessed by physical examination, laboratory results, and adverse event (ae) reporting. results: in the european analysis, 385 patients comprise mitt (received ‡1 dose of study drug), 326 comprised ce (167 tgc, 159 v/a/cis) and 376 comprised c-mitt (189 tgc, 187 v/a/ cis). treatment groups were balanced with respect to demographics. patients were mostly white (99.7%) men (63.6%) with a mean age of 50 years. in the european region, clinical responses to tgc and v/a at test-of-cure were similar: c-mitt, 84.7% (160/ 189) versus 86.6% (162/187), difference tgc-v/a was -2.0% (95% ci -9.5, 5.6). similar results were noted in the ce population with tgc curing 89.8% (150/167) and v/a curing 95.0% (151/ 159), difference tgc-v/a was -5.1% (95% ci -11.6, 1.2).most commonly reported treatment emergent aes (teaes, mitt) for tgc and v/a were nausea (17.3% and 3.2%, p < 0.001) and vomiting (6.1% and 1.6%, p = 0.032). the v/a group had significantly higher teaes of sgpt increase (6.9% v/a vs 2.0% tgc, p = 0.025) and rash (2.6% v/a vs 0 tgc, p = 0.028). conclusion: in the european analysis of the integrated phase 3 worldwide clinical studies, tgc monotherapy is as safe and efficacious as the combination of v/a in the treatment of patients with csssi. safety and tolerability of tigecycline r. maroko, n. dartois, g. rose, e.j. ellis-grosse (collegeville, us; paris, fr) objectives: tigecycline (tgc), a glycylcycline, is a first-in-class, extended, broad-spectrum iv antibiotic that has demonstrated clinical activity in patients with complicated intra-abdominal infections (ciai) and complicated skin and skin-structure infections (csssi). the safety of tigecycline was evaluated in four phase iii trials. methods: a total of 1415 hospitalized patients from these trials were pooled and evaluable for safety analysis. in the ciai trial, patients received tgc 50 mg q 12 hrs (following a 100-mg loading dose) or imipenem 500 mg and cilastin 500 mg q 6 hrs. those in the csssi study were treated with either tgc (same dose/schedule) or vancomycin 1 gm with or without aztreonam 2 gm q 12 hrs. results: the most frequently reported adverse events (aes) in both tgc-treated groups were nausea (n) and vomiting (v). the incidence of n was 29.5% while v was approximately 19.5%; these were generally mild to moderate in severity. infection-related serious aes were slightly more frequent with tgc versus comparators (6.7% vs 4.6%). discontinuations due to treatment-emergent aes (including n/v) occurred at similar rates with tgc and comparators (5.0% vs 4.7%). six patients (0.36%) treated with tgc presented with intestinal perforations and developed sepsis/septic shock compared with 2 (0.12%) for imipenem/cilastatin, with higher baseline apache ii scores in the tgc group; the relationship to treatment could not be determined. in the overall efficacy analysis, subjects with ''perforation of the intestines'' were balanced between the two groups, and overall efficacy was not statistically different. no clinically significant renal, hepatic, cardiac (qtc), bone marrow, or cns toxicities were noted with tgc. conclusion: tgc appears to be safe and tolerable for patients with ciai and csssi. n/v were generally mild to moderate in severity, self-limiting, and did not result in increased overall drug discontinuation. there did not appear to be clinically significant renal, hepatic, cardiac, bone marrow, or neurological toxicities related to tgc treatment. all-cause mortality rates did not statistically differ between those treated with tgc and the comparators. its demonstrated efficacy and favourable toxicity profile make tgc a good monotherapy option for selected serious infections. tigecycline as effective as imipenem/cilastatin in the treatment of complicated intra-abdominal infections: experience in india objective: due to diverse bacteriology and bacterial resistance, treatment of complicated intra-abdominal infections (ciai) is a challenge. in a double-blind, phase 3, multinational trial, the efficacy of tigecycline, a first-in-class glycylcycline, was compared with imipenem/cilastatin (imi/cis) in hospitalised patients with ciai. this subanalysis evaluated tigecycline safety and efficacy from 12 investigational sites in india. methods: patients were stratified by disease severity (apache ii score £15 vs >15 but <31), and randomly assigned to iv tigecycline (100 mg loading, 50 mg q12h) or iv imi/cis adjusted for body weight (500/500 mg q6h for ‡70 kg) for 5-14 days. clinical response at test-of-cure (toc, 12-44 days after therapy) for microbiological evaluable (me) and microbiological modified intent-to-treat (m-mitt) populations were co-primary efficacy endpoints where cure/failure responses were determined. safety evaluations included vital signs, laboratory tests and record of adverse events (aes). results: in india, 167 patients received at least 1 dose (mitt, 84 tigecycline, 83 imi/cis), 149 patients were clinically evaluable (ce), 85 were me, 120 were m-mitt. treatment groups were balanced with respect to demographic/baseline medical characteristics. primary diagnoses (mitt) were complicated appendicitis (31%), gastric/duodenal perforation (26%), perforation of intestine (18%), cholecystitis (12%), peritonitis (6%), and intraabdominal abscess (5%). cure rates at toc in me in india were 36/41 (87.8%) tigecycline and 40/44 (90.9%) imi/ cis, which are consistent with overall me results [80.6% (199/ 247) tigecycline vs 82.4% (210/255) imi/cis (95% ci = )8.4, 5.1; non-inferiority p < 0.001)]. in india m-mitt, cure rates at toc were 47/59 (79.7%) tigecycline and 54/61 (88.5%) imi/cis, similar to the overall m-mitt results [73.5% (227/309) tigecycline vs 78.2% (244/312) imi/cis (95% ci = )11.0, 2.5; non-inferiority p < 0.001)]. noninferiority of tigecycline among india patients could not be statistically demonstrated because of insufficient sample sizes, however, magnitude of response to study drugs in patients treated in india was comparable to that in overall patients. in india, treatment aes were similar with significantly higher incidence of dyspnoea in tigecycline (9.5%) vs imi/cis (0.0%), p = 0.007. conclusions: efficacy results in india are consistent with findings from the overall study and results at other centres, suggesting tigecycline is noninferior to comparator in treating ciai. nosocomial infection: control of environment, viral infections p1788 bacterial flora contamination of blood pressure cuffs in use on hospital wards n. walker, r. gupta, j. cheesbrough (preston, uk) blood pressure cuffs are a plausbile vehicle for the transmission of nosocomial infection between patients. despite this, few studies have examined the level of bacterial contamination and tested for the presence of common nosocomial pathogens on their surface. we swabbed 24 cuffs currently in use on hospital wards. using sterile gloves, a disposable template measuring 10 · 10 cms was placed onto the cuff and a moistened sterile swab was rubbed onto the defined area for 1 minute and then transported in 10 mls of buffer medium. from each sample, 0.5 mls of the buffer was plated onto 6 different media which included a non-selective agar medium for total viable count (tvc) and selective media for s. aureus, mrsa, c. difficile, coliforms and vancomycin resistant enterococci (vre.) bacterial growth was recovered from all 24 cuffs. pathogenic organisms were isolated from 14 cuffs (58%). mssa from 5, mrsa from 1 and c. difficile from 5. the remaining three cuffs grew more than one pathogenic organism; mssa + mrsa + c. difficile from one and mssa + c. difficile from 2 cuffs. colifroms and vre were not isolated from any of the cuffs. the range of total viable counts recovered per 100 cm 2 area of the cuff varied from 1000 > 20000 cfu and the cuffs with the highest counts tended to have more pathogens present. mssa and c. difficile were isolated from 33% of the cuffs sampled and mrsa from 8%. while the actual importance of this potential route of transmission for nosocomial pathogens remains unclear, it can not be dismissed. the impracticality of decontaminating blood pressure cuffs between patients suggests that single patient use cuffs or a barrier between cuff and skin would be a more viable option on a busy general ward. needlestick and sharp injuries of health care personnel in a newly founded tertiary hospital: a prospective study m. falagas, i. karydis, g. georgoulias, p. hatzopoulou, d. nikita, i. kostogiannou (athens, gr) objectives: needlestick and sharp injuries of health care workers are a major cause of anxiety and may expose susceptible employees to the risk of infectious diseases. however, the incidence of such injuries has not been examined in a newly founded hospital while preventive programmes are taking place. methods: we prospectively studied the needlestick and sharp injuries of employees in a newly founded tertiary hospital in athens, greece while a vaccination program against hepatitis b virus as well as educational activities for avoidance of injuries were taking place. serologic studies for hepatitis b and c virus as well as human immunodeficiency virus (hiv) were performed in all injured employees and the source patients (when known). results: sixty-eight needlestick, 8 sharp injuries, and 3 splashes were reported during the study period (01/10/2002 to 28/02/ 2005) in 71 nurses, 2 housekeepers, 3 technicians, and 3 ambulance workers. the overall incidence (percutaneous injuries and splashes) per 100full-time employment-years (100 fteys) was 2.4% whereas the incidence of percutaneous injuries alone per 100 fteys was 2.3%. a higher incidence of injuries was noted during the first than the second half of the study period (3.8% versus 1.8%, p = 0.003). no source patient was found positive for hepatitis c or hiv. the use of high-titre immunoglobulin after adjustment for the incidence of injuries was higher in the first than the second half of the study period (22.6% vs 3.8%, p = 0.05). conclusion: although we did not adjust for possible confounders, our data show that educational and vaccination preventive programs for needlestick and sharp injuries led to a statistically significant decrease in the incidence of such injuries and use of high-titre immunoglobulin. epidemiology of occupational needlestick and sharps injury among healthcare-workers in turkey s. hosoglu on behalf of the occupational infections study group, turkey background: health care workers (hcws) are frequently exposed to the danger of infectious agents through needle stick and sharps injury (nssi) in their occupational efforts. in turkey, the hepatitis b and c viruses cause an essential threat to the hcws because of their prevalence rate (2%-4% and 0.5%-1%, respectively). a cross-sectional countrywide survey study was performed on the epidemiology of nssi among hcws at 30 hospitals in 19 cities throughout the country. data relating to the epidemiology of nssis were collected using a standard questionnaire in 2004. results: totally 5048 hcws completed the questionnaire forms. nurses are the leading group (2016 persons) that joined into the study were followed by doctors (1452 persons) andlaboratory technicians (475). totally 2498 of them (49.5%) declared an occupational exposure or nssi in the last 12 months related their job. needle stick injury was reported in 1698 of them (33.6%), splash into the eye in 1132 (22.4%), sharp injury in 737 (14.6%), and the other injuries in 419 (7.0%). the hepatitis positivity was reported in 567 cases (2.27%) objectives: to assess the microbiological status of reprocessed single-use devices for interventional cardiology by testing bioburden, sterility and pyrogenic load. methods: a total amount of 154 electrophysiology non-lumen catheters (ep) were collected after the first clinical use on patient. 20 devices were contaminated with bacteria spiked human blood and underwent four different pre-sterilization protocols including chlorine, polyphenol, and enzymatic agents. treated samples were assayed by cultural quantitative methods (cqm) for bactericidal properties and electron microscopy (em) for biologic residuals. 73 ep were tested for sterility. by the repetition of simulated-use (bacteria spiked blood) and regeneration (enzymatic and chlorine treatment, gas plasma sterilization) we obtained 54, 39, 26, 28, 36, 22 samples respectively reprocessed 1, 2, 3, 4, 5, 6 times. devices were cultured for 28 days in trypticase soy broth. the pyrogenic status of 61 ep was monitored after clinical use, after decontamination-cleaning treatments and after complete reprocessing by lal test. results: high-resolution em and cqm confirmed the superior properties of chlorine releasing agent added to enzymatic detergent for devices treatment before sterilization. hypochlorous acid based protocols were more biocide (>3.1 log cfu reduction) than polyphenolic (3.2-1.9 log cfu reduction). sterility tests showed no positive sample to inoculated strain until the fourth cycle of reprocessing. catheters showed the growth of the inoculated strain, bacillus subtilis in 1/35 and 1/22 samples after five cycles and six cycles respectively. every reprocessed device was non-pyrogenic (<20 eu/catheter). in addition, tests conducted on in-vitro spiked catheters showed that pyrogenic loads of 200 eu/device were reduced to less than 11 eu/device. conclusions: reprocessing procedures following the adopted regeneration protocol were able to satisfy the fundamental microbiological requirements until five in-vitro reuses. sterility tests showed that devices' sterility was not guaranteed after five reuses. pre-sterilization treatments including enzymatic solutions and chlorine revealed high cleaning properties with effective bioburden reduction. storage intervals among reprocessing steps longer than 24 hours should be avoided in order to limit contamination and pyrogenic load. technical considerations suggest to consider the introduction of reprocessing procedure only in hospitals with a considerable workload. room disinfection in the hospital setting using akacid plus ò c. kratzer, s. tobudic, w. graninger, a. buxbaum, a. georgopoulos (vienna, at) objectives: akacid plus ò , a novel polymeric guanidine with broad antimicrobial activity also against multi-resistant bacterial strains, was used in the present study as room disinfectant. methods: disinfection of closed rooms experimentally contaminated with antibiotic-susceptible and multi-resistant staphylococcus aureus (mrsa), pseudomonas aeruginosa and escherichia coli was performed using akacid plus ò at concentrations of 0.1%, 0.25% and 0.5% for 100 minutes. bacterial suspensions were distributed on stainless steel plates and placed in a test and control room. recovery of the test bacteria was determinedbefore nebulizing, 60 and 100 minutes after the beginning and 4 hours after the end of room disinfection by a modified simple swab-rinse technique. for the detection of mrsa in isolation units, surface samples were collected by direct swab and enrichment culture. results: the swab-rinse method demonstrated a dose-and time-dependent effectiveness of akacid plus ò in eradicating s. aureus, e. coli and p. aeruginosa on stainless steel plates. nebulizing of 0.5% akacid plus was successful in eliminating all hospital pathogens in 340 min contact time, while mrsa was still detectable after use of 0.25% akacid plus ò . 0.1% akacid plus ò achieved a reduction >100 cfu of s. aureus and p. aeruginosa, but was only able to eradicate e. coli during the observation time. the results suggest that nebulized akacid plus ò at a concentration of 0.5% is a potent substance for eradication of pathogenic organisms in the hospital setting. study on the antiviral efficacy of citrofresh ò , a flavonoid based organic acid complex sanitizer z. nack (north-geelong, au) objective: determine the antiviral efficacy of this organic sanitizer against enveloped and non-enveloped viruses using a carrier based method. seeking registration for citrofresh ò in australia and in the eu as a hospital grade antiviral sanitizer. methods: the study was performed according to the american society of testing and materials (astm) designation (e 1053-85) recommended by the australian therapeutic goods administration (tga) to determine the efficacy of a disinfectant intended to use on inanimate, environmental surfaces. we tested citrofresh ò (diluted in standard hard water) in three different concentrations: 1%, 2% and 4% on adherent cell lines (pk-15, mrc-5, mdck, a549, l929) in four replicates against five different viruses including: porcine parvovirus (non-enveloped, high resistant against sanitizer); human rhinovirus-16 (non-enveloped, high resistant against sanitizer); human adenovirus-4 (non-enveloped, moderate resistant against sanitizer); human influenza type a (h3n2) virus (enveloped, moderate resistant against sanitizer); human herpes simplex virus type 1 (enveloped, low resistant against sanitizers). prior to the viral testings, acute toxicity assay was carried out to determine the adherent cells viability against citrofresh ò . results: cell lines exhibited >80% viability after exposure to all three concentration. herpes simplex type 1, human influenza type a and human adenovirus-4 exhibited the most significant viral log reduction of log10 4 to 5 at 4% concentration of citrofresh ò followed by the human rhinovirus-16 and porcine parvovirus log10 4 reduction at 4% concentration. the reduction of viable virus load was exhibited after 1 minute exposure time to citrofresh ò , which means no time-dependant activity. citrofresh ò clearly exhibited concentration and ph dependent viral load reduction activity against influenza type a and the human adenovirus -4 and human herpes simplex type 1 virus. the reduction in viral titre for porcine parvovirus and human rhinovirus-16 is probably ph dependent (the ph of 1% citrofresh ò is 6.5, 2% is 4.5 and 4% is 3.5). conclusion: our investigation shows that citrofresh ò is an effective disinfectant on environmental surfaces, eliminating enveloped and non-enveloped viruses and sufficient to achieve the minimum 4-log reduction with complete viral inactivation which is prerequisite for registration. rapid environmental recontamination of an intensive care unit after decontamination with hydrogen peroxide vapour objectives: to evaluate the effectiveness of hydrogen peroxide vapour (hpv) to reduce the levels of total bacterial and methicillin resistant staphylococcus aureus (mrsa) environmental contamination on an intensive care unit (icu), and to establish the rate of environmental recontamination. methods: the study took place on a 9 bed open plan icu. on each environmental screen 3 sites in each bed space (under the bed, the workstation and the monitor) were examined using broth enrichment for the detection of mrsa. in addition total bacterial counts were determined for under the bed and workstation using rodac plates. environmental screening was carried out monthly for the 3 months preceding the usage of hpv, increasing to weekly for the 4 weeks prior to usage. additional sampling was carried out immediately before patients were discharged from icu, following the subsequent terminal clean and then immediately after hpv use. after readmission of patients sampling was carried out at 24 h, 48 h and then weekly for a period of 8 weeks. patients were screened for mrsa on admission and then weekly. results: sampling of the environment prior to the usage of hpv revealed contamination of the environment with mrsa on 6/7 occasions, with mrsa colonised patients being present on only 3/7 occasions. after discharge of the patients and terminal cleaning of the environment, mrsa was isolated from 5 (13%) environmental sites. after the use of hpv, mrsa was not isolated from any environmental sites upon immediate sampling, but 24 h after patients were readmitted, including 2 patients known to be colonised with mrsa, mrsa was isolated from 5 sites. these sites were not clustered around the colonised patients but were widespread across the icu. in the 8 weeks post hpv usage mrsa has been isolated every week. the mean total bacterial counts prior to the use of hpv were 22.0/10 cm 2 underneath the beds and 3.8/10 cm 2 on the workstations, this was reduced after hpv to 0.1/10 cm 2 and 0.2/10 cm 2 respectively. after patients readmission the counts were 4.1/ 10 cm 2 underneath the beds and 1.2/10 cm 2 on the workstations after 48 h and returned to pre-hpv levels of 16.9/10 cm 2 and 4.8/10 cm 2 at each site respectively after 1 week. conclusion: hydrogen peroxide vapour is effective in eliminating bacteria from the environment. the rapid rate of recontamination of the environment suggests that the use of hpv is not an effective means of maintaining low levels of environmental contamination on an open plan icu. objectives: the nosocomial infections are more serious and dangerous than community acquired infections since they have high rate of morbidity and mortality as well as they increase the cost of therapy. recently many precautions have been taken to prevent these infections. one of these applications is that covering of the floor of the wards, clinics, intensive care units and operating rooms of the hospitals with vinyl flooring material, which is believed to be cleaned easily and effectively. in this study it was aimed to determine the duration of survive of the staphylococcus aureus, enterococcus feacalis, escherichia coli and pseudomonas aeruginosa, which were most common encountered as nosocomial infection agents, on the surface of flooring materials such as vinyl flooring, ceramic laminated wood and galvanized sheet at room temperature. methods: four kinds of flooring materials were prepared approximately in 4-6 cm 2 coupons and sterilized. separate bacterial suspensions equal to mc farland 1 turbidity were swapped to the surface of each flooring materials by sterile cotton swabs. all contaminated test materials were put in sterile petri dishes with cover and kept at room temperature without subjecting to the direct sunlight. on the third day, culture samples were taken from the surface of each material by sterile cotton swaps soaked with sterile saline and streaked on the blood agar surface. culturing procedure was repeated every other day until no growth detected. in case of three consequently, negative culture results obtained culturing was ended. results: overall results of the study were presented on table 1. conclusions: among the four flooring materials, galvanised sheet seemed to be the most unsuitable one for the bacteria to survive long period. in other words this material should be preferred as to laminated wood for covering benches and laboratory tables. as for the flooring of the floors the vinyl flooring material is better than ceramic. covering the complete cmv ie-1 and pp65 proteins.results: cmv seropositive transplant recipients had significantly hightened ie-1 and pp65 specific t cell frequencies compared to seronegative individuals. patients withevidence of cmv antigenemia or dnaemia could not be discriminated based on cmv-and donor-reactive t cells or serum creatinine. however, recipients of seropositive grafts with low ie1 response showed a tendency towards more frequent cmv infection. cmv disease was observed in only 3/64 individuals. 2 had no detectable ie1or pp65-t cell response, the third presented with a dominant pp65 response. interestingly, ie1-specific t cells correlated inversely with early post-tx donor-reactive t cell frequencies during weeks 1-2 post-tx. most importantly, ie1-specific t cell frequencies correlated inversely with serum creatinine at 6 and 12 months at several times post-tx. in patients without acute rejection, even pre-transplant ie-1 specific t cells correlated inversely with 6 and 12 months creatinine. conclusion: these data suggest subclinical control of cmv infection by ie-1 specific t cells and subsequently less graft injury by (cmv-induced) alloimmunity. universal precautions: knowledge, attitude and practice of healthcare workers regarding hiv, hepatitis b and c v. gupta, s. bhoi, a. goel, p. aggarwal (new delhi, in) objectives: increasing incidence of hiv, hepatitis b (hbv) and hepatitis c (hcv) in the patients expose the healthcare professionals of acquiring these infections during occupational exposure. we studied the knowledge, attitudes and practices of healthcare workers regarding hiv, hbv, hcv and the risk of occupational transmission of these diseases. methods: an interview survey was conducted among all the health care workers (hcw) using a standardised questionnaire comprising of 34 items in english and local language, as suitable, by an expert in the emergency ward of a tertiary care teaching hospital of a developing nation. data analysis (bivariate and multivariate analysis) was done using spss version 10. results: 170 (response rate: 80%) hcw participated in the study. the mean age was 33 ± 6 years, 54 were females. the study population comprised of 40% doctors, 23% nurses, 25% lab technicians and 12% support staff. respondents had adequate knowledge about causative (65%) usual transmission (63%), symptoms (69%) of aids but poor knowledge about hbv and hcv (43%, 41% and 30% respectively). inadequate knowledge was also revealed about the infectious bodyfluids (31%), disinfection of equipments (32%), pregnancy in hcw as a susceptibility factor (22%), post exposure prophylaxis (34%) and comparative infectivity of hiv and hepatitis (38%). 70% of hcw became anxious while treating these patients. poor compliance with universal precautions was noticed. high compliance was reported for wearing masks (72%) and wearing gloves (53%). doctors were more likely to suffer needlestick injury (p = 0.04) occupational exposures was found to be high (48%) with poor declaration rate (10%). guidelines adherence was influenced by profession (p < 0.001), availability or adequacy of protective equipments but not by work experience as hcw (p = 0.8). all of the respondents urged for an interactive information session. conclusions: results from this study reveal that there is a fair level of knowledge about hiv/aids but hepatitis b and c have not generated adequate concern among the hcw. incongruity between perceived knowledge and reported practice suggests that there is a need for an interactive awareness course about the universal precautions. the educational programmes need to consider attitudes in conjunction with empirical knowledge. objectives: the sero-prevalence of hepatitis a (hav) antibodies are known to be low in young adults in korea. recently, seventeen cases of hepatitis a have been reported in health-care workers (hcw) of icu in a university hospital from may 2005 to july 2005. we performed surveillance, and determined molecular identification of outbreaks. methods: 1. we checked the hav igm from all the patients of sicu with elevated ast/alt retrospectively and screened ast/alt level from all the nurses and the doctors in contact with suspicious index case. 2. when we determined the existence of outbreak, the molecular subtypes of hav from a blood of hcw were determined to provide the data for epidemiologic study. we determined the index case, a transmission route and the intervention for control an outbreak were planned. results: 1. seventeen hcw including 13 nurses and 4 doctors who are 22 to 32 years old, suffered from acute hav over 7 weeks period. 2. the possible transmission of hav was fecaloral route from the bed-ridden patients with diarrhea to the exposed hcw. 3. seventeen hcw were identified with a positive anti-hav igm. the eight hcw had a positive hav rna. analysis of the vp1-p2a region of each isolate showed genotype 1a in five strains and co-circulation of 1a and 1b in others. conclusions: the occurrence of hav outbreak highlights the importance of standard precaution in a hospital. the hav vaccination is considered in young aged-hcw. the genotype identification of blood would be useful for the epidemiologic study of suspicious hav outbreak in a hospital. management of a norovirus-associated gastroenteritis outbreak on two psychiatric wards a. buehling, u. arnold (magdeburg, de) objectives: we report a norovirus-associated outbreak of gastroenteritis on a closed psychiatric and a gerontopsychiatric wards from december 2004 to february 2005. during this time 46 patients and 25 healthcare workers (hcws) were affected. introduction and results of hygiene measures based on published guidelines on psychiatric wards are described. methods: effective and adapted measures had to be implemented to stop the outbreak and to prevent the spread of disease to other areas of our hospital. isolation or cohorting of the psychiatric patients was excluded for therapeutic reasons. regular hand disinfection in patient rooms was impossible because of the high risk of abuse. the following measures have been introduced: use of gowns, masks and gloves by hcws during care of infected patients-frequent hand disinfections with alcohol-based disinfectants by hcws using ''pocket bottles''; recommendation for all persons entering the station to use gowns, gloves and masks and to disinfect their hands frequently, distribution of handouts describing the measures; hand disinfection by all patients after using toilet, before and after taking meals (distribution of disinfectants by hcw); increased frequency of routine surface disinfection (3 times daily) instead of routine cleaning once daily; routine disinfection of door handles, handrails, wash-basins and -fittings and light switches 3-4 times a shift; avoidance of patient transfer via hospital; visitor restriction during outbreak time; daily evaluation of recommended measures and adaptation to the current situation; exclusion of affected staff from the ward until 48 h symptom free. results: the hygienic measures have been explained to the local hcws in daily meetings. they have been fully accepted only after a severe staff shortage in the fifth week of outbreak because of 3 new cases of gastroenteritis during hcws and 14 newly infected patients. because of the restrictive application of the adapted guidelines for these special wards the outbreak has been stopped within 4 further weeks. conclusion: in case of norovirus-based gastroenteritis outbreaks on closed psychiatric wards hygienic measures which are adapted to the concrete situation are necessary. especially in these cases the compliance with guidelines can be increased by daily meetings and daily evaluation of recommendations. staff shortage during the outbreak forced the strict compliance with the recommended measures. regional spread of antibiotic resistance methods: we performed surveillance of patients, healthcare stuff and icu environment and we registered the infections of ab during 3 periods of 21 days each one. the interval between 1st-2nd period was 6 months and 2nd-3rd period was 1 year. rectal, oropharyngeal swabs tracheal aspirates from patients, handswabs from stuff and samples from environment were taken weekly. the identification of ab was performed using vitek ii system the susceptibility was tested by kirby-bauer and mic methods and the <<dna fingerprints>>obtained by pulsed field gel electrophoresis (pfge). results: during the 1st 2nd and 3rd period, 19 patients (14 men, 5 women), 13 patients (7 men, 6 women) and 12 patients (8 men, 4 women) were hospitalized in icu respectively. ab was isolated in 74 from 250 samples (29.6%) at the 1st period, 59 from 336 (17.5%) at the 2nd and 62 from 260 (24%) at the 3rd period. totally ab was isolated in 195 from 846 specimens (23%) at the 1st 2nd and 3rd period among the patients carrying ab, 7/14 (50%), 7/10 (70%) and 5/8 (62%) were infected respectively. the infections observed during the study period were: sepsis (8), urinary tract infection (1), pneumonia (11), meningitis (1), thrombophlebitis (2) . all the isolated ab strains were multiresistant to antimicrobial agents. molecular analysis of 169 isolated strains by pfge distinguished the following types: a (64, subtypes a1-a7), b (10) at the 1st period a(6), c(7), d(22), e(2), f(1), g(1), h(2), i(1). j(1) at the 2nd period a(25), b (16), d(1), h(3), k(2). l(5) at the 3rd period. infections were caused mainly by a and d types while the same types were isolated from the environment and the hands of the icu stuff. conclusion: there was a high rate of colonization and infection of icu patients by multiresistant clones of ab. the persistence of clone a of a. baumannii and the appearance of b type at the 3rd period after its disappearance at the 2nd period despite the application hygiene measures, indicates the need for more strict reinforced infection control in icu. the transmission via the hands of stuff to patients has become the most important contributor factor in patient colonization and/or infection. objectives: the antibiotic resistance and its mechanism of group a streptococci (gas) varies according to nations or study period. we have investigated antibiotic resistance and mechanism of macrolide resistance for the strains isolated from korean children and compared to the previous (2002) results. methods: throat cultures were taken from 2351 elementary school children in jinju, korea from october to december, 2004 to isolate gas. antibiotic susceptibility test to erythromycin (em), clindamycin (cc), and tetracycline (tc) was performed by disk diffusion method. macrolide resistance phenotype and genotype as well as emm genotype were studied. results: isolation rate of gas was 14.0% (328/2351). resistance rates of em, cc, and tc were 9.8%, 8.8%, and 18.3% respectively, which were dramatically decreased from 51%, 34%, and 30% in 2002 at the same area. emm44/61 was prevalent (29%), while emm12 was the most common type (34%) in 2002. cmlsb, m, and imlsb were observed in 87.5%, 9.4%, and 3.1% respectively, compared to 64%, 34%, and 2% in 2002. the strains with cmlsb and imlsb had ermb gene and the ones with m phenotype were positive with mefa gene. conclusion: the resistance rates to em and cc were dramatically decreased compared to the past (2002). education to the public and physicians, decreased consumption of antibiotics, acquisition of immunity to the resistant strains, or change of prevalent emm types could be considered to explain the reason of decrease of antibiotic resistance. although antibiotic resistance rate was decreased, cmlsb type which has high mic was prevalent suggesting treatment failure for those children carrying these resistant strains in jinju, korea. analysis of skin and soft tissue infections in european medical centres: report from the sentry antimicrobial surveillance program (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) g. moet, p. strabala, h. sader, t. fritsche, r. jones (north liberty, us) objective: to analyse the skin and soft tissue infections (ssti) or wound infections in hospitalized patients in the sentry program for pathogen prevalence and resistance (r) variations in european (eu) medical centres for the years 1998 to 2005 (8 years). this program also included north america (na) and latin america (la) for the same years, except 2003. methods: 50 consecutively isolated pathogens/site were collected from each centre per year and varied in number of sites each year in eu: 1998 eu: (23), 1999 eu: (6), 2000 eu: (16), 2002 eu: (3), 2003 eu: (25), 2004 eu: (23), and 2005 . susceptibility testing was determined by clsi (formerly the nccls) broth microdilution methods and interpreted by current (2005) breakpoints. results: table of all years total of ssti pathogens. (see table) sa was the predominant pathogen in eu ranging from 35.7% of ssti isolates in 1998 to 47.5% in 2005. the top 5 most prevalent organisms accounted for 80.0% of isolates in all years with psa and ec ranking second and third, respectively with 22.5% combined, and enc and ent ranked fourth and fifth with 10.7% of the total isolates. compared to the americas, mrsa and vre isolation was at a lower occurrence rate in the eu; but between the rates of other monitored continents for ctz-r psa, cipro-r ec and ctz-r ent (ampc). vre increased in eu over the 8 year. conclusions: pathogen prevalence in ssti for eu has been consistent over the 8 monitored years although sa (with mrsa) appears to be increasing. eu is not a world leader in any key r marker compared to the americas. however, the r rates are evolving which suggests continued need for surveillance programs at regular intervals to detect mobile genetic r elements. objectives: carbapenems play an important role in the therapy of pseudomonas aeruginosa infections. the aim of our study was to characterize the molecular alteration responsible for changed susceptibility towards carbapenems in multiresistant p. aeruginosa strains from germany. methods: 19 multiresistant p. aeruginosa strains from 9 cystic fibrosis and 9 non cystic fibrosis patients were collected in 3 german hospitals in 2004. the strains showed reduced susceptibility (intermediate or resistant; din guidelines) to imipenem, piperacillin, ciprofloxacin and gentamicin. clonality was tested using pfge. a pcr screening for vim and imp was carried out. effluxpump overexpression was detected using an effluxpump inhibitor (epi) test. oprd and for strains with positive results in the epi test the effluxpump repressorgenes mexr and nfxb were sequenced. results: pfge patterns revealed no clonal relationship among the multiresistant strains. neither vim nor imp was detected. the geno-and phenotypes found are depicted in table 1. defective oprd genes caused by premature stopcodons or frameshifts were found in 17 strains. among those 11 had no mutations in mexr or nfxb and showed the highest mics found ranging from 8 to >32 and 4 to 32 mg/l of imipenem and meropenem, respectively. 3 additionally had defective mexr genes, but intact nfxb genes, 2 also had modifications in mexr and nfxb, and 1 showed only in nfxb additional alterations. for 2 strains no alterations in oprd but in mexr were proven. conclusions: the predominating mechanism of carbapenem resistance in multiresistant p. aeruginosa strains from germany was the loss of oprd. accessory overexpression of mexaboprm due to modifications in mexr did not result in significantly elevated mics of meropenem. moreover, the additional overexpression of mexcdoprj did not lower the mic of imipenem. in 2 strains with modifications only in mexr only elevated mics of imipenem indicate a reduced expression of oprd accompanied by overexpression of mexefoprn as conferred by nfxc-type mutants. objective: mart (study for monitoring antimicrobial resistance trends) is an ongoing global antimicrobial surveillance program focused on clinical isolates from intraabdominal infections (iai). the aim of this sub-analysis was to assess antimicrobial susceptibility patterns among gramnegative bacilli from 5 different regions of the world during 2004. methods: 6pa total of 81 major medical centres in north america, latin america, europe, middle east/africa, & asia/ pacific tested the in vitro activity of antimicrobial agents commonly used to treat iai against consecutive unique aerobic and facultative gram-negative bacilli from iai using microdilution techniques according to clsi guidelines & breakpoints. results: enterobacteriaceae were recovered from 4905 (88%) & non-enterobacteriaceae were recovered from 826 (15%) of the 5596 patients in the study worldwide, constituting 5317 (86%) & 839 (14%) of the 6156 total isolates, respectively. e. coli (n = 2979; 56%) and klebsiella spp. (n = 978; 18%) were the most commonly isolated enterobacteriaceae. pseudomonas spp. (n = 605; 72%) and acinetobacter spp. (n = 110; 13%) were the most commonly isolated non-enterobacteriaceae. isolates from asia/pacific and latin america were generally more resistant. 2267 (43%) of the enterobacteriaceae & 272 (32%) of the non-enterobacteriaceae were recovered <48 hours after hospitalization. the % susceptible isolates are reported below: conclusion: in this study, enterobacteriaceae were the predominant intraabdominal isolates recovered both <48 h and >48 h after hospitalization. carbapenems were overall the most active agents against enterobacteriaceae worldwide. resistance rates varied among geographic regions, with the asia/pacific and latin america regions generally having the most resistance. characterisation of streptogramin resistance genes among enterococcus faecium isolates from austrian animal husbandry a. eisner, g. gorkiewicz, g. feierl, f. dieber, e. marth, j. kö fer (graz, at) objectives: the streptogramin virginiamycin has been widely used as a growth promoter in animal husbandry in the european union but was banned in 1998 because of concerns about evolving cross-resistance to the streptogramin quinupristin-dalfopristin used in human medicine. the aim of the present study was to investigate the prevalence of streptogramin resistance genes of enterococcus faecium recovered from animal faecal specimens collected in southeast austria. methods: we analysed 300 e. faecium isolates of cattle (n = 100), pig (n = 100), and poultry (n = 100) for the presence of streptogramin resistance genes. we used selective enterococcal broth for isolation. species identification was done on basis of gram stain, catalase and pyrrolidonyl arylamidase activity, motility, lancefield group d antigen typing, and by the 20 strep apitest (biomérieux). detection of the resistance genes vat(e), vat(d), and erm(b) was done by pcr. results: the erm(b) gene encoding macrolide, lincosamine, and streptogramin b (mlsb) resistance was found in each 2 e. faecium isolates recovered from pig and cattle, none of the isolates from these animals carried genes coding for streptogramin a resistance. on the contrary, 14 e. faecium isolates from broiler specimens contained the vat(e) gene and one isolate contained the vat(d) gene. all of these isolates also contained the erm(b) gene. conclusion: our data indicate that the use of the meanwhile banned antimicrobial feed additive virginiamycin has created a reservoir of streptogramin-resistant e. faecium in southeast austrian poultry. characterisation of macrolide-resistant streptococcus pneumoniae isolates from russia objectives: streptococcus pneumoniae (spn) resistance to macrolide antibiotics continues to be of major concern. the aim of present study was to analyse phenotypic and genotypic characteristics of macrolide resistant spn isolates. methods: eighty one macrolide resistant spn isolates were collected in moscow, 2003 moscow, -2005 . the susceptibility testing was performed according to the clsi guidelines. macrolide resistance phenotypes were characterized by triple-disk diffusion test, using erythromycin, clindamycin and rokitamycin disks. detection of genes, coding resistance to macrolides, was done by rt-pcr. sequencing for qrdr mutations was performed on levofloxacin resistant spn isolates. selected isolates were analysed by mlst. results: by the triple-disk test, 21 isolates were assigned to the m phenotype, 20 of them were carrying mefa gene, and one was negative. twenty eight isolates were cmlsb-phenotype, 25 of them were carrying both ermb and mefa genes, in two isolates only ermb was detected, and one isolate was negative for both genes. imclsb phenotype was demonstrated by 29 isolates, both ermb and mefa genes were detected in 6 of them, and only ermb in 23. three isolates didn't demonstrated blunting of zone of inhibition around rokitamycin disk. associated resistance to penicillin g, tetracycline, chloramphenicol and co-trimoxazole was observed in 67.9%, 86.4%, 42.0% and 75.3% of spn isolates respectively. nine multidrug resistant isolates, harbouring both mefa and ermb genes, were subjected to mlst. among them one isolate was found to share the allelic profile st 81 (spain23f-1 clone), and four isolates were single-allele variants of st 81. in four isolates new allelic profiles were detected. three isolates were resistant to levofloxacin (mic ‡4 mg/l), in two of them with levofloxacin mic > 32 mg/l (st81 single-allele variants) e85k, s79f and i460v substitutions were detected in gyra, parc and pare, respectively. d83n and i460v substitutions were detected in parc and pare of one isolate with new allelic profile. conclusion: high prevalence of macrolide resistant spn, harboring both ermb and mefa genes is observed in moscow, macrolide resistance is associated with resistance to other groups of antibacterials. some multidrug resistant isolates are highly related to internationally disseminated multiresistant clone spain23f-1. strains with fluoroquinolone resistance in moscow were all single locus variants of the spain23f-1 clone. occurrence of tet(w) gene in a clostridium difficile clinical isolate p. mastrantonio, f. barbanti, p. spigaglia (rome, it) objectives: to investigate the presence of tet(w), a tetracycline resistance gene recently identified in anaerobic commensal bacteria from animals and humans, in c. difficile clinical isolates. methods: several c. difficile clinical isolates from different italian hospitals were analysed for the presence of a tet(w) gene by pcr assays. the primers used were designed on the tet(w) sequences available in genbank. pcr fragments obtained by these amplifications were sequenced. tet(w) dna flanking regions were also examined with a set of pcrs constructed on the sequence of the conjugative transposon tnb1230 of butyrivibrio fibrisolvens 1.230, that is the only element carrying a tet(w) partially characterized so far. tet(w) positive isolates were also examined for the tet(m) gene and for the presence of int and tndx, markers for the tn916 and the tn5397like elements, respectively. the tn916-like elements were further characterized by pcrs designed on enteroccus faecalis tn916 sequence. tetracycline mic values were determined by the e-test method. tetracycline resistance gene transfer was evaluated by filter mating experiments, using c. difficile p881r strain as recipient. results: a tet(w) gene was found in only one isolate, c. difficile cd5, also positive for the tet(m) gene. this isolate was resistant to tetracycline with a mic of 8 mg/l. sequence analysis of the tet(w) pcr fragment (about 1860 bp) showed that this gene had an identity of 99% with the genes found in clostridium spp strain k10, mitsuokella multacida and butyrivibrio fibrisolvens. no amplifications were obtained with the primers designed on tnb1230, indicating the presence of a different genetic support for tet(w) in c. difficile. tet(m) gene of c. difficile cd5 was carried by a tn916-like element that showed nucleotide sequence mutations in the region containing orf 17-20 compared to the element of e. faecalis. conjugative transfer of tet(w) was not observed, whereas the tet(m) gene was transferred to the recipient strain. c. difficile transconjugants were resistant to tetracycline with a mic of 8 mg/l. conclusion: the results obtained in this study demonstrate for the first time the presence of a tet(w) gene in a clinical isolate of c. difficile, providing further evidence of the spread of this resistance determinant among gastrointestinal bacteria. macrolide resistance determinants are prevalent and readily selected for in viridans group streptococci among healthy norwegian adults background: norway has a low prevalence of antimicrobialresistant bacteria including macrolide resistant (mr) respiratory tract pathogens. we have observed an increase in macrolide consumption in norway and there is a lack of knowledge on the reservoir of macrolide resistance determinants among viridans group of streptococci (vgs) in the pharyngeal flora. objectives: examine the occurrence, selection and persistence of macrolide resistance determinants in vgs pharyngeal flora in healthy norwegian adults before and after treatment with azithromycin. methods: throat samples were collected before (day 1), after treatment (day 7) and after 3 months (day 90) from 20 healthy volunteers. the samples were plated directly as a lawn on pdmii agar plates with 5% defibrinated blood with an erythromycin etest strip. photos were used as quantitative comparisons. up to 10 morphological different colonies with erythromycin etest mic ‡1lg/ml from each specimen were collected; day1 (n = 59), day 7 (n = 157) and day 90 (n = 76). in total 86 representatives mr, vgs-isolates were selected for further studies: (i) mics of erythromycin, tetracycline and penicillin were determined by etest. (ii) pcr's for erm(b), erm(tr), and mef(a/e), and subsequent sequence-typing of mef. species identification was performed by soda sequencing. results: a total of 17/20 persons carried a low number (<5) of mr vgs in day1 specimens, while 20/20 had a significant higher number (>100) of mr strains in day2 specimens. in day90 specimens, 20/20 carried a low number of mr, resembling day1. reduced susceptibility to penicillin was observed in 32/86 (34%) isolates. tetracycline resistance was found in 43/86 (57%), and mainly in erm(b)-positive strains. mef(a/e)-positive dominated day1 (53%) and erm(b) day2 specimens 52%. sequence typing revealed mef(e) (n = 41) and mef(a) (n = 3). soda sequence; s. mitis (n = 42), s. oralis (n = 9), s. parasanguinis (n = 12), s. salivarius (n = 22), and s. sanguinis (n = 1). conclusion: there is a pool of vgs carrying macrolide resistance determinants in the normal pharyngeal flora of healthy adults that are readily selected for during azithromycin exposure. the mef(e) and erm(b) were the most prevalent resistance genes and co-resistance to tetracycline was frequently observed, resembling the findings in norwegian clinical isolates of s. pneumoniae. these vgs may provide a pool of resistant bacteria that may transfer resistance determinants to more pathogenic organisms. relationships in genotype, phenotype, t type and pfge type among macrolide-resistant streptococcus pyogenes strains isolated in the czech republic v. jakubù , p. urbášková, l. straková (prague, cz) objectives: to determine relationships between phenotypic and genotypic methods among erythromycin-resistant s. pyogenes strains. methods: a total of 1331 clinical isolates of s. pyogenes resistant to erythromycin were collected in 39 microbiology laboratories during 2001-2003. erythromycin susceptibility was tested by the disk diffusion method. strains with an inhibition zone <21 mm around the erythromycin disk (15 lg) were sent to the national reference laboratory for antibiotics (nrl). presences of mlsb resistance genes (ermtr, ermb and mefa) were tested by pcr. t serotypes were determined in randomly selected representatives of each phenotype (n = 370). pfge type were determined in strains from year 2003 only (n = 68). results: the rate of the most prevalent phenotype (constitutive mlsb resistance) was 63%, 55% in the year 2001 and 2003, respectively. the major prevalent t types among the analysed strains were serotype t 28 (58%), t 12 (10%), t 4 (8%) and t b3264 (8%). gene ermb was the most frequent (54%). the results of pcr method was highly congruent with observed phenotype of resistance. pfge patterns of strains with constitutive mlsb resistance were highly identical. conclusion: m phenotypes, constitutive and inducible resistance to mlsb antibiotics were found and ermtr, ermb and mefa genes were detected among the analysed strains. the t serotype 28 was identified the mainly prevalent in our collection. the majority of strains harbouring t serotype 28 were constitutively resistant to macrolides. the study showed close relationships among genotypes, t types, specific resistotypes (phenotype) and pfge types. objectives: since recognition of transferable clindamycin and tetracycline resistance in bacteroides, we have undertaken a us national survey on the susceptibility of b. fragilis group to analyse emergence of resistance and trends, since these species are not routinely tested for susceptibility in hospital clinical laboratories. methods: agar dilution mics were determined for 5225 isolates from 1997-2004 for b. fragilis and related species from 9 geographically diverse centers in the us. antibiotics included 3 carbapenems, 3 b-lactam/b-lactamase inhibitors, 2 quinolones, a tetracycline, clindamycin, metronidazole, chloramphenicol, a glycylcycline and linezolid. isolate identity was confirmed by api 20a. results: analysis of resistance trends from 1997-2004 showed a decrease in geometric mean mic's (geomic) for imipenem (0.80 mcg/ml to 0.32 mcg/ml, p < 0.001) and meropenem (0.52 mcg/ml-0.42 mcg/ml, p = 0.003) for the bacteroides species. ertapenem geomic remained unchanged (0.99 mcg/ ml). for the b-lactamase inhibitors, piperacillin-tazobactam geomic declined from 2.56 mcg/ml to 2.22 mcg/ml (p < 0.001). ampicillin-sulbactam geomic did not change. few isolates were resistant to any carbapenem or b-lactamase inhibitor combination. clindamycin resistance increased, especially for b. fragilis, b. ovatus and b. thetaiotaomicron (all p < 0.001). among quinolones, resistance of bacteroides to moxifloxacin increased (geomic went from 2 mcg/ml to 3.5 mcg/ml, p < 0.001). b. fragilis remains the most sensitive bacteroides species to moxifloxacin, although approximately 45% of stains have mic's ‡to 4 mcg/ml in 2004. tigecycline susceptibility, tested over 5 years, did not change. the first confirmed metronidazole-resistant isolate (mic = 64 mcg/ml) obtained in the us was noted in 2002 but none were noted in 2003 or 2004. conclusion: improved susceptibility of bacteroides species to some carbapenems and the b-lactamase inhibitor combinations is unexplained but significant. clindamycin resistance continues to increase, especially for b. fragilis. moxifloxacin susceptibility for the non fragilis species shows that the majority of strains are resistant. the first metronidazole resistant isolate has been reported from the us. since resistance trends are associated with species, the differentiation within the species is of extreme importance, since it may impact the choice of antimicrobial agent for the treatment of infections caused by this group of anaerobes. observed duration of nasopharyngeal carriage of penicillin-resistant pneumococci: relations to age and serogroup p. geli, l. hö gberg, h. ringberg, e. melander, m. lipsitch, k. ekdahl (solna, malmö, lund, stockholm, se; boston, us) background and objectives: knowledge of how the duration of pneumococcal carriage varies with age and serogroup is essential to understanding how immunity to carriage arises throughout the course of life, and designing appropriate models for the effects of vaccination or other public health initiatives aiming to reduce the pneumococcal transmission in the community. using data from an ongoing swedish intervention project, the duration of nasopharyngeal carriage of penicillinresistant pneumococci (mic pcg >0.5 mg/l) stratified by both serogroup and age of the carrier were estimated. methods: the mean duration and corresponding 95% confidence interval was estimated by fitting a gamma distribution to the observed duration of carriage for each serogroup and age stratum. results: the mean duration of carriage for all cases was 37 days (95% ci 35-38). children below the age of 5 years carried prp for significantly longer periods (43 days, 95% ci 41-45) compared with older individuals (25 days, 95% ci 24-27). there were also differences within the group of cases below the age of 5 years, as the duration of carriage became significantly shorter for each year older the cases were. serogroup 9 and 14 were carried for significantly shorter periods compared with serogroup 6. serogroup 9 also had significantly shorter carriage duration compared with serogroups 19 and 23 for cases 0-4 years. for cases 5 years or older, no significant difference in carriage duration for different ages or serogroups could be noted. conclusions: even though the estimate does not cover any correction for the censored carriage duration and therefore not yield an estimate of the total length of carriage, the results highlight the importance to take both serogroup and age of the p1823 exploring the molecular basis for differences in phenotype of salmonella enteritidis typing phage n. delappe, d. morris, m. cormican (galway, ie) objectives: the salmonella enteritidis phage tying scheme of the laboratory of enteric pathogens, health protection agency, uk, is a widely used method for subtyping this important pathogen. the method is rapid and highly discriminatory. interpretation of results can be subjective and the typing phage which are central to the method have not been well characterised. complete sequence data is available for the salmonella typhimurium podovirus phage p22. methods: the 16 typing phage were propagated on s. enteritidis pt1b (pb406). phage were visualised by electron microscopy. phage dna was extracted and digested with hindiii. consensus pcr primers were designed based on sequences of p22 and other s. typhimurium phage. additional primers were designed based on the sequence of the s. enterititidis typing phage 3 (a siphovirus). amplification, sequencing and dna probe hybridisation of various phage genes were performed using standard techniques. results: on em the 16 typing phage comprise 6 podoviridae (phage 1, 8, 10, 14, 15 and 16) , 6 siphoviridae (phage 3, 5, 7, 11, 12 and 13) and 4 myoviridae (phage 2, 4, 6 and 9). digestion with hindiii subdivided each morphotype into 3 groups. the podoviridae contained genes homologous to p22 while the siphoviridae contained genes homologous to the sequenced s. enteritidis typing phage 3. some sequence variation was detected in podovirus and siphovirus genes however in some cases phage, which differ in their phenotype had no difference detected in hindiii digestion pattern or partial sequence. conclusions: the s. enteritidis typing phage set comprise 3 distinct phage morphotypes. in some instances distinct phage that contribute to differentiation between s. enteritidis phage types had no dna sequence variation detected. variations in phage typing reactions may in part be due to epigenetic difference in typing phage, e.g. due to methylation of phage dna. salmonella enteritidis typing phage biology could provide a model for developing approaches to phage therapy. tularemia is a zoonotic bacterial disease. the causative agent, francisella tularensis, is spread to humans by direct contact with infected rodents, inhalation, ingestion of contaminated water or by arthropod bites. in some endemic regions, outbreaks occur frequently, whereas nearby rural parts may be completely free. we presented two cases of tularemia in non endemic region of the turkey. case 1: a 46 year old female patient referred to tertiary hospital due to swollen on the neck for 2 months. before admission beta lactam antibiotics had been prescribed to her for tonsilopharyngitidis. but her complaints had been continued. so she admitted to our hospital. she had been suffered fever sore throat and neck pain. she had a palpable and painfull cervical lymphadenopathy which was not suppurated. leukocytosis and elevated c reactive protein were predominant. at screening there were not any lymphadenopathy detected elsewhere. she had been examined about cytomegalovirus epstein barr virus and brucellosis. they were negative. fine needle aspiration from neck was negative considered as malignancy. cultures were negative for routine bacteriologic examination. microagglutination test for tularemia was 1/320 positive. then we decided to treat her with gentamycin for 10 days. after treatment cervical lymphadenopathy became small. leukocyte count and c reactive protein levels were reach normal range. case 2: a 29 year old female patient referred to university hospital due to cervical lymphadenopathy and fever and sore throat. before admission beta lactam antibiotics were prescribed to her for 2 weeks. but no apparent benefits had been detected. there was a palpable and fistulated cervical lymphdenopathy. drainage was examined microscopically and cultured for bacteria, mycobacteria and fungi. on routine cultures no microorganisms were grown. fine needle aspiration was done. it was reported that suppurative granulamatous lympadenitis. so we were examined for tularemia, cat scratch disease. microaggltunation test for tularemia was 1/320 positive. then streptomycin had been given for 10 days and excision of lymphadenopathy had been done. no complications or recurrence occur. results: both patients were applied to us from non endemic and different regions of the turkey. they had no known insect bite history. both of them were diagnosed by serological tests. conclusions: in the differential diagnosis of tonsillopharyngitidis, tularemia also must be considered in the non endemic regions. tularaemia presenting with tonsillopharyngitis and cervical lymphadenitis: two case reports b. kandemir, i. erayman, m. bitirgen, e. turk aribas, a.c. inkaya, s. guler (konya, tr) tularemia is a zoonotic disease caused by francisella tularensis. francisella tularensis is transmitted to humans by direct contact or ingestion of infected animal tissues, through the bite of infected arthropods, by consumption of contaminated food or water, or from inhalation of aerosolized bacteria. in this report we describe two cases of oropharyngeal tularemia who presented with tonsillopharyngitis and cervical lymphadenitis. case i: a 43 years old woman with multiple cervical lymphadenitis has been admitted to our clinic. her complaints started 2 months ago with signs and symptoms of tonsillopharyngitis. she had received non specific treatment (ampicillin+sulbactam) and ten days later cervical lymph nodes appeared. the diagnosis was made serologically. the antimicrobial therapy (streptomycin 1 · 1 g im) was given for fourteen days. the patient recovered completely. case ii: a 16 years old girl with multiple cervical lymphadenitis was admitted to hospital. her complaints started 3 months ago with throat ache after which multiple cervical lymphadenitis appeared. she was admitted to our out patient clinics and diagnosed to have tularemia. anti-microbial therapy (streptomycin 1 · 1 g im+doxycyciline 2 · 100 mg) was given for four weeks but no clinical response was achieved. patient was admitted to the hospital and surgical drainage was performed. treatment against tularemia was prolonged. patient was finally recovered at the end of nine weeks of therapy. it can be concluded that early diagnosis and treatment of tularemia are important. some patients may benefit from surgical drainage and prolonged therapy. a case of nonclostridial crepitant cellulitis which is due to escherichia coli c. ayaz, m. ulug, m.k. celen, m.f. geyik, s. hosoglu (diyarbakir, tr) objectives: this condition is caused by gas forming bacteria that involve the skin, either or as an extension from deeper structures. the origin of infection is an abdominal wound, perianal disease, or operative incisions that have become secondarily infected. tracking of gas-forming organisms from deeper sites of infection may also present as crepitant cellulitis without a break in the skin. diabetics are more likely to acquire such infections, especially in the lower extremites. among the bacteria isolated are anaerobic organisms such as bacteriodes or anaerobic streptococci, or coliform bacteria, especially escherichia coli and klebsiella. because of this reason we reported a case of a nonclostridial crepitant cellulitis which is due to escherichia coli. case: a 40 year old man who was previously healthy, has come with fever, pain, oedema, erythema, crepitant and limitation of movement at the right lower extremity. in his history he had no complaint until 2 weeks ago. perianal abscess has developed at this time and it has drainged spontaneously 3 days later. than his complaints has comprised 3 day duration. on physical examination, the temperature was 38.9°c, pulse rate 116/ minute, respiratory rate 42/minute and blood pressure was 90/50 mmhg. laboratory evaluation showed a haemoglobin 8.1 g/dl, leucocyte count of 32700/mm 3 (neutrophils 92%). serum electrolytes, renal and liver function tests were within normal limits. c reactive protein was elevated up to 354 mg/dl, esr was 77 mm/h. escherichia coli was isolated from wound and blood cultures. he was treated initially with ampicilinsulbactam (6 g/day) and required attempt. even with optimal surgical and medical therapy, he dies at the third day of the treatment from septic shock. conclusion: the onset is generally gradual, and there is usually mild local pain and systemic toxicity, allowing clinical differentiation from the more fulminant clostridial myonecrosis. the surgical approach should be aggressive, but tailored specifically to the underlying cause of infection. antibiotic therapy is directed at a mixed aerobic-anaerobic flora, until culture reports are available. a case of iliopsoas abscess which is due to pseudomonas aeruginosa objectives: pyogenic psoas abscess, a rare but life-threatening infection, results from primary suppuration or is secondary to the spread of infection from an adjacent structure. primary iliopsoas abscess occurs probably as a result of hematogenous spread of an infectious process from an occult source in the body. primary iliopsoas abscess can occur in diabetus mellitus, intravenous drug abuse, aids, renal failure and immunosupression. ultrasound is diagnostic in only 60% of the cases. computed tomography should be done for definitive diagnosis and is considered the gold standard. stapylococcus aureus is the causative organism in patients with primary iliopsoas abscess, but pyogenic psoas abscess caused by pseudomonas aeruginosa is uncommon. because of this reason we reported this case. case: a previously well 67 year old woman presented with a month history of right loin to groin pain, limping or limitation of hip movement, fever and nausea. she was a diabetus mellitus patient for 7 years. on her physical examination, the temperature was 38.1°c, pulse rate 104/minute, respiratory rate 28/minute and blood pressure was 140/90 mmhg. examination of the respiratory system, cardiovascular system and abdomen were found to be normal. laboratory investigations revealed total leucocyte count of 17800/mm 3 (polymorphs 88%), c reactive protein was elevated up to 168 mg/dl, esr was 77 mm/h. serum electrolytes, renal and liver function tests were within normal limits, but serum glucose level was elevated to 351 mg/dl. her blood cultures were sterile, but abscess culture yielded pseudomonas aeruginosa which was taken during the surgery. she was treated imipenem (2 g/ day) + amicasin (1.5 g/day) and required surgical drainage. she was treated and followed up 21 days, and discharged at the end of the treatment. conclusion: in these patients treatment involves the use of appropriate antibiotics along with drainage of the abscess. an adequate knowledge of the causative organisms should guide the initial choice of antibiotics. depending on the results of the abscess fluid culture and sensitivity, adjustments should be made. percutaneous drainage or surgical drainage may be done in them. in conclusion early recognition, empiric antimicrobial coverage and aggressive drainage or debriment are indicated in these patients. cervical lymphadenitis in a diabetic woman f. ç okça, a. azap, s. gö çmen, h. erdi sanli, s. gü l (ankara, kirikkale, tr) objective: rhodococcus equi infections are commonly seen in immunocompromised patients. exposure to domestic animals, such as horses and pigs may play a role in some cases. two thirds of the r. equi infections in immunocompromised were reported in hiv infected patients, and the rest divided between transplant recipients, immunosupressive medications and other kinds of immunosupression. the clinical picture presents with pulmonary infection in 80% of patients. here, we report a rare case of cervical lymphadenitis in a diabetic women due to r. equi. case: a sixty-year-old diabetic woman was admitted with the complaints of fever, right cervical erythematous swelling with tenderness and warmth. on physical examination; inflammation beginning from the right submandibular region and descending to the upper chest was detected. a tender mass of 8 · 5 · cm. was palpated on the right cervical region. ampicillin/sulbactam 4 g/day was given emprically for a week with no improvement. the ct scan of the neck showed conglomerated lymphadenopathy extending from the submandibular area to the supraclaviculary region with 10.5 · 3 cm in size. the mass began to fluctuate and 500 cc abscess material was drained surgically. gram's stain of the purulent material showed polymorphonuclear leukocytes with pleomorphic gram positive coccobacilli. the cultures of the material grew r. equi. therapy was changed to teicoplanin and ciprofloxacin combination and surgical care of the wound with antiseptics was performed. after a month, intrevenous medical therapy was changed to oral route with roxythromycin and ciprofloxacin and was continued to 2 months with complete resolution. conclusion: increased awareness and improved laboratory techniques help for the early diagnosis of rhodococcal infections. timely diagnosis is important because the microorganism is usually resistant to penicillin g, oxacillin, ampicillin, carbenicillin and cefazolin. the use of at least one antibiotic with intracellular activity is necessary in the treatment of r. equi infections. empirical two drug regimens with erythromycin, rifampin and/or ciprofloxacin are recommended. objectives: to analysed the features of spondylodiscitis (sd), their clinical presentation, the commonest diagnostic methods and the kind of treatment applied according to the different groups of the study. methods: a retrospective and descriptive study taking place amongst the patients diagnosed as having sd from 1998 till 2003. in each case we studied the presence of underlying disease, primary infectious sources in the prior 6 months, the way symptoms started, location, diagnostic methods, treatment and evolution, comparing between different aetiologies. results: 67 patients with sd were studied.53 of them had pyogenic sd,(35 had spontaneous sd and 18 had an sd after spinal surgery) and 14 patients had tuberculous sd. 45 were men (16 to 84 years; mean 51.8). patients with postoperative sd were the youngest (mean 42.17 y, p = 0.001). underlying diseases were found in 75% of patients, mainly in postoperative sd (50% of cases) (p = 0.009). an episode of previous bacteremia or infectious source was found in 40% and 63% respectively of patients with spontaneous pyogenic sd, significantly higher than in surgical sd (6% had bacteremia and 11% other infectious source, p < 0.01). the most common presenting symptoms were back pain (98.5%) and neurological deficits (54%). frank fever occurred in 29% of cases, being more frequent in spontaneous sd (49%) than in postoperative sd (6%) or tuberculous sd (7%), p £ 0.002. leukocytosis was found only in 28% of patients. postoperative sd presented the lowest levels of esr (p = 0.008). s. aureus was the most frequent bacteria isolated (31%) in pyogenic spontaneous sd, as coagulase negative staphylococci was in surgical sd. lumbosacral localization was detected in 45% of spontaneous pyogenic sd and in 95% of postoperative sd. tuberculous sd predominate in dorsolumbar region. paravertebral abscess formation was observed in 39% of pyogenic sd and in 93% of tuberculous sd (p = 0.001). surgical treatment was required in 46.2% of tuberculous sd and in 9% of pyogenic sd (p = 0.005). outcome of patients with spontaneous sd was worse (sequelae in 62%), than in patients with surgical sd (38.2%) or tuberculous sd (14%) (p = 0.015). conclusions: 1) spontaneous sd was the most frequent and it occurred mainly in patients suffering from underlying diseases; 2) nearly all patients had pain but only in 1/3 of them was accompanied by fever; 3) the lumbar zone was the most frequent location; 4) the majority of patients had a complete resolution of their symptoms only with medical treatment. background: the ethiopathogenesis of cns abscess includes a broad spectrum of pathogens and predisposing conditions, so that a polymicrobial flora is a quite frequent event. capnocytophaga spp. includes fastidious gram-negative organisms, usually underestimated in the common clinical practice, and poorly tested in vitro for antimicrobial susceptibility. surprisingly, also agents usually active on gram-positive pathogens demonstrated some efficacy against capnocytophaga spp. (i.e. erythromycin, rifampin, tetracyclines, cotrimoxazole, chloramphenicol, and glycopeptides), which is usually responsible of anecdotal episodes of cns infection (meningitis, brain abscess, and subdural empyema). methods and results: the fourth case report of capnocytophaga spp. brain abscess is herewith reported. a probable origin from a recent cat bite and a mandibular granuloma is suspected. due to the lack of clinical and neuroradiological response to neurosurgical debridement and an association therapy including imipenem, amikacin, clindamycin and fluconazole, empiric administration of linezolid (1200 mg/day) was attempted, and a rapidly favorable clinical, microbiological, and neuroradiological response was achieved. notwithstanding the identification of capnocytophaga spp. as the sole microorganism yielded by purulent drainage of a cns abscess, patients with multiple risk factors and recent surgery are expected to suffer from a polymicrobial cns infection. due to its favourable cns penetration and its dual mode of administration (both i.v. and oral), linezolid may represent an alternative option in the event of cns diseases borne by numerous risk factors and a suspected polymicrobial origin, especially when a lack of response to first therapeutic attempts is of concern. in the management of a cns abscess where the role of microorganisms with an unpredictable sensitivity profile remains of concern, chemotherapy should be directed also against potentially multiresistant organisms. considering also the relevant limitations given by the often poor cns penetration, the activity of glycopeptide agents is limited, compared with that of linezolid. aetiologies and antimicrobial resistance profiles of purulent meningitis study carried out in a hospital of infectious diseases, algiers objectives: bacterial meningitis is a serious clinical and medicolegal consequences if management is incorrect. meningitis protocols have recently been published by the british infection society/meningitis research foundation and are widely disseminated in our institution. local guidelines are also available on the hospital intranet and in the emergency department and acute medical admissions wards. this study investigated the level of understanding about meningitis and knowledge of the guidelines in medical staff of different grades working in the emergency department and the acute medical admissions unit in a large teaching and emergency hospital. methods: 90 medical staff were interviewed faced to face and asked a series of questions on the management of meningitis. results were stored on a database and responses were analysed. results: general knowledge about meningitis was variable. although 96% knew that bacterial meningitis was a notifiable disease only 40% knew the procedure for informing the health protection agency and only 43% would notify viral meningitis. only 30% of responders were aware that guidelines could be viewed on the hospital intranet. only 41% correctly identified the indications and cautions for lumbar puncture. although the majority recognised the need for urgent administration of antibiotics 18% would omit antibiotics until further assessment and lumbar puncture results. only 30% were aware of the need to consider adding ampicillin to cover listeria in patients over 55 years of age and there was uncertainty about the management of patients with penicillin resistance. conclusions: although protocols and guidelines for meningitis have been produced and are easily accessible the majority of medical staff were uncertain how to access and utilise this information. the level of knowledge and expertise in managing meningitis amongst medical staff working in a and e and the acute medical unit was poor and there is a need for further education to improve patient management. guidelines are of no value if they are not disseminated to front-line medical staff. objectives: the aim of this study was to evaluate the prevalence of penicillin resistant and multi-drug resistant pneumococci isolates in streptococcus pneumoniae meningitis. methods: a retrospective study was carried out on 46 clinical records between january 2001 and october 2005. among the 46 csf samples the pneumococcal ethiology was confirmed by 55% positive cultures and 70% latex agglutination. antibiotic susceptibility testing was performed by disk diffusion method according to nccls standards. isolates of pneumococci with oxacillin zone sides of >20 mm are susceptible (mic < 0.06 microg/ml) to penicillin, while at those of <19 mm the mic has to be determined (by e -test). results: isolates from 12 patients (26%) were found with penicillin-resistance (prp) -of which 67% were multi-drug resistant-and 34 (84%) with penicillin susceptibility -of which 35% were resistant to other drugs. an abrupt onset of disease was found in 58% prp patients and 79% from non-prp ones. chest x ray pulmonary determinations were found in 50% prp patients and 35% non-prp ones. sixty-six per cent of prp patients and 29% of non-prp ones had a prior hospitalization. only 8% of non prp patients had a positive blood culture. antibiotic switch was made in 41% cases with prp isolates and 29% cases with non prp ones. the overall rate of mortality was 8%, with 16% for prp patients and 6% for non-prp ones. conclusions: non-prp isolates were the prevalent ethiology of s. pneumoniae meningitis. 35% of non -prp strains developed other drug resistance, and 67% prp strains were multi -drug resistant. prp meningites evolved more as a hospital-related pathology, with an abrupt onset, frequently associated with pulmonary determinations and higher mortality rate. background: although vaccination strategies have shifted the age distribution of meningitis to older age groups, few studies have specifically examined bacterial meningitis in the older adult. methods: from october 1998 to april 2002, we prospectively included 696 episodes of community-acquired bacterial meningitis, confirmed by culture of cerebrospinal fluid, which occurred in patients aged >16 years. we dichotomized the cohort with respect to age: patients aged ‡60 years were defined as older adults and patients aged 17-59 years as younger adults. predictors for an unfavourable outcome (defined as score 1-4 on the glasgow outcome scale) were determined by logistic regression. we tested for statistical interaction between age group and potential prognostic factors by adding multiplicative interaction terms to the model. the mann-whitney u test and the chi-square test were used to identify differences between groups. results: 257 of 696 episodes (27%) occurred in older adults and 439 episodes in younger adults (69%). streptococcus pneumoniae was the most common pathogen in older adults (69%). meningitis in younger adults was caused by neisseria meningitidis and s. pneumoniae in 50% and 40% of the episodes, respectively. older adults were more likely to present with the classic triad of bacterial meningitis (fever, neck stiffness and altered mental status) than younger adults (58% versus 36%; p < 0.001). the prognostic value of independent risk factors for unfavourable outcome was similar in both age groups. older adults had more complications during clinical course, resulting in a higher mortality rate than in younger adults (34% versus 13%; p < 0.001). sepsis was the most common cause of death in both age groups (30% in older adults versus 32% in younger adults; fig) . whereas older adults tended to die more often due to cardiorespiratory failure (25% versus 11%; p = 0.06), younger adults more often died due to brain herniation (23% versus 2%; p = 0.004). conclusions: bacterial meningitis in older adults is associated with high morbidity and mortality rates. elderly patients often present with classic symptoms and s. pneumoniae is the most common pathogen within this age group. whereas older adults often die due to cardiorespiratory failure, younger adults more often die due to brain herniation. incidence of serogroups and penicillin susceptibility in neisseria meningitidis isolates (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) objective: the aim of this study was to analyse the serogroup incidence and penicillin susceptibility in n. meningitidis before and after spanish epidemic outbreak in 1997. in this year the public health service decided a massive vaccination in our sanitary area (galicia, north-west of spain, 459.180 inhabitants) and in autum of 2000 the inclusion of vaccine against n. meningitidis serogroup c in vaccination programme. methods: retrospective study of all cases of meningococcal disease confirmed by culture and/or pcr in the health care area of santiago de compostela (galicia) from 1996 to 2004. results: in the period 1996-2004 we identified 151 meningococcal disease episodes by microbiologic diagnosis (56.3%, 41.7% and 2.0% to b, c and w135 respectively). in 1996, serogroup c were the 80% of the isolates. in 1997 and 1998 the serogroup incidence was almost the same (b = 45.7%, c = 48.6%). from 1999 an increase in b serogroup cases were detected, in 1999 (66.6%), in 2000 (88.9%) and in 2003 (76.9%). the most frecuent phenotype has been 2b p1:2.5 in c serogroup. during this period an increase in penicillin susceptibility was observed (in b serogroup 0% in 1996 and 44% in 2004 of the isolates were susceptible and in c serogroup 0% in 1996 and 50% in 2004). conclusions: the b serogroup is the most frecuent isolate during this period except in the years 1996 and 1997. the strain that cause the epidemic outbreak in 1997 (c:2b p1:2.5) was not isolated since 2000. in our health care area, c:2a serotype, was isolated for first time in 2000, and since then, is the unique serotype isolated in c serogroup. incidence rate in c serogroup has changed from 3.34/100.000 in 1997 to 1.09/100.000 in 2004. this decrease was caused by the drop of incidence rate on the youngest groups (<2 years and 2-4 years). the incidence rate in b serogroup during these years was modified from 1.48/100.000 in 1997 to 2.17/100.000 in 2004. a four-year retrospective analysis of infective endocarditis in a belgian university hospital n. de visscher, b. delaere, b. krug, y. glupczynski (yvoir, be) objectives: to establish the epidemiology of infective endocarditis (ie) and determine the prognostic factors for adverse outcome in patients admitted to a university hospital with a cardiovascular surgery department. methods: between 01/00 and 12/04, the clinical and laboratory features of all consecutive adult patients with a definite diagnosis of ie (duke criteria) were evaluated retrospectively by two infectious diseases physicians on the basis of clinical data charts and microbiological laboratory. results: 45 patients (24 men, 21 women) presented with a definite diagnosis of ie. mean age was 64, 6 yrs; 34 cases (76%) were native valve endocarditis (nve) and 11 (24%) were prosthetic valve endocarditis (pve); 30% of patients with nve had underlying valvular abnormalities (3 bicuspidies, 4 mitral prolapsus or regurgitation, 3 others). ten out of 11 cases of pve were late-onset episodes (>1 year after surgery). global mortality was 42% (18/45 pts), including 15 patients (33%) still under antibiotic therapy. a higher mortality rate was observed in pve [7/11; (64%)] than in nve [11/34; (32%)]. overall, 18 pts (42%) underwent surgery (mean: 18 days following admission). valvular replacement was contra-indicated in 11 pts because of critical status and/or major co-morbidities. the distribution of isolated pathogens was: streptococci: 20 cases (44%) including 4 cases of s. bovis, s. aureus: 14 cases (31%, including 2 mrsa), enterococci: 3 cases (7%), miscellaneous: 8 cases. the affected valves were: only aortic: 19 (43%), only mitral: 15 (33%), only tricuspidal: 1, aortic and mitral: 4, mitral and tricuspidal: 2, aortic, mitral and tricuspidal: 2. a high mortality rate was observed in s. aureus ie (9/14 [64%]), especially in the subgroup of patients with a pve (4/5 pts [80%] ). the mortality rate in patients with ie episodes caused by streptococci amounted 21% (5/24 pts). clinical microbiology and infection, volume 12, supplement 4, 2006 related, 20% (n = 40) had previous surgery and 9% (n = 19) were related to urinary or digestive tract procedures. only 2 patients had illegal substance abuse. the most frequent predisposing acquired cardiac condition for native valve endocarditis was degenerative valvular disease in 55% (71/ 129). twelve percentage (n = 24) had prior ie. the most frequent predisposing congenital cardiac condition was a bicuspid aortic valve in 5% (n = 11). in 16% (n = 32), no predisposing heart disease was discernible. causative microorganisms included: staphylococci in 43% (n = 87) with s. aureus in 31% (n = 62), cons in 12% (n = 25), streptococci in 26% (n = 52) with s. viridans in 12% (n = 25), s. bovis in 8% (n = 16), enterococci in 17% (n = 34) and other pathogens in 3% (n = 7). culture negative ie was reported in 11% (n = 23). both in community-acquired and nosocomial ie, s. aureus was the most frequent causative agent. twenty-three percentage (14/62) were methicillin-resistant s. aureus. s. viridans ie was mainly community-acquired while enterococcal ie was nearly equally distributed between community and nosocomial origin. conclusion: compared to older series, we observed a high proportion of nosocomial ie and of prosthetic valve ie. s. aureus and e. faecalis were the most prevalent causative microorganisms. enterococci were nearly equally distributed between community and nosocomial origin, suggesting that nosocomial enterococcemia should be added as a major criterion, as proposed before for s. aureus. the role of aminoglycosides in combination with a beta-lactam for the treatment of bacterial endocarditis: a meta-analysis of comparative trials m. falagas, d. matthaiou, p. papastamataki, i. bliziotis (athens, gr) objectives: the addition of an aminoglycoside to a beta-lactam for the treatment of patients with infective endocarditis has been supported mainly from data from laboratory and animal studies. we sought to review the evidence from the available comparative clinical trials regarding the role of aminoglycosides in combination with a beta-lactam for the treatment of bacterial endocarditis due to gram-positive cocci. methods: the studies for our meta-analysis were retrieved from searches of the pubmed database and from references of relevant articles. included studies were trials that provided comparative data regarding the effectiveness of the treatment and/or mortality in patients receiving monotherapy with a betalactam or beta-lactam/aminoglycoside combination therapy. two independent reviewers performed the literature search, study selection, and extraction of data from relevant studies published in english during the period 01/1975-08/2005. results: no clinical trial comparing beta-lactam monotherapy to beta-lactam/aminoglycoside combination therapy for the treatment of enterococcal endocarditis was found. we performed a meta-analysis of 5 available comparative trials (4 randomized controlled trials and 1 comparative prospective trial) that included 261 patients with bacterial endocarditis in native valves due to staphylococcus. aureus (4 studies) or streptococcus viridans (1 study). there was no statistically significant difference between the compared arms regarding mortality (or 0.59, ci 95% 0.21-1.66), treatment success (or = 1.25, ci 95% 0.49-3.05), treatment success without surgery (or = 1.66, , and relapse of endocarditis (or = 0.79, . nephrotoxicity was less common in the beta-lactam monotherapy arm compared to the beta-lactam/aminoglycoside combination therapy (or = 0.38, ci 95% 0.16-0.88, p = 0.024). conclusion: the limited evidence from the available prospective comparative studies does not offer support for the addition of an aminoglycoside to beta-lactam treatment of patients with endocarditis due to gram-positive cocci. a large multicenter randomized controlled trial may be necessary to reach a definitive conclusion on this issue. outpatient antimicrobial therapy for infective endocarditis. single-centre experience objectives: to evaluate the characteristics and outcome of infective endocarditis (ie) patients included in a outpatient antimicrobial therapy (opat) program. methods: from january 1997 to may 2005 all patients who received opat therapy for an ie were prospectively evaluated. inclusion in opat program require clinical stability and agreement of patients. active drug addiction was contraindicated for inclusion. antibiotic treatment was administered in bolus for once-daily antibiotics regimens. we used cadd-legacy tm plus (deltec, inc. st paul. usa) portable infusion system for either continuous or intermittentprogrammed bolus infusion. results: we included 65 patients, 51 male (78%), mean age 60 years old (sd: 19.3 years). the diagnostic of ie was definite in 45 cases (13 with pathologic diagnosis), 15 probable and 5 possible. mostly of the cases were community-acquired ie (83%). mitral valve ie was the most frequent anatomical site involved (46%), followed by aortic (32%). native-valve ie represent the majority of cases (55%), but 32% were prostheticvalve and 12% were pacemaker lead ie. viridans group streptococci was the most frequent isolate (31 patients, 48%) with 4 cases of s. bovis ie. eleven patients had s. aureus ie (17%). at the time of the diagnosis, 10 patients had valve rupture and 4 patients had periannular abscess. a total of 15 patients required some surgical intervention for the ie [9 valvular replacement (2 of them associated with aortic graft), 5 pacemaker extraction and 1 aortic graft]. the majority of the patients received outpatient monotherapy (65%). the most frequent antibiotic used was ceftriaxone (55% of the cases), followed by cloxacillin 20%, gentamycin 20%, vancomycin 14%, teicoplanin 14%, ampicillin 8% and other antibiotics in 14%. in 60% of the patients the vascular access was a perifericallyinserted venous central catheter and in 26% we used a portable infusion system. twelve patients (18%) had some complication during opat that require hospital readmission, of which 5 could return to opat program. three patients had a fatal outcome (deaths) during admission, not related to ie complications. the mean duration of opat was 18.9 days per patient, and globally supposed 1.230 days of hospital admission savings. conclusion: opat for ie can be a good therapeutic option for ie stable patients. this procedure can represent a considerable amount of hospital admissions savings, improving also patients' well-being, and must be take into account for the treatment of this disease. objectives: botulism, a neuroparalytic illness, is caused by toxin produced by clostridium botulinum. food born botulism, a potentially lethal neuroparalytic disease, is caused by ingestion of preformed toxin. clinical illness is characterised by cranial nerve paralysis, followed by descending flaccid muscle paralysis. in this article we report a case series including a family group of type e botulism after ingestion of an iranian traditional soup. methods: in january 2005, 11 patients of a family group developed clinical manifestations of botulism 1-2 hours following ingestion of a traditional soup. their main clinical presentations were severe weakness (90.9%-10 case) and lethargy (72.8%-8 case). other signs and symptoms were blurred vision, fixed and dilated pupils, diplopia, dry mouth and decreased gag reflex. based on clinical finding, all patients received 3 monovalent antitoxins (a, b, c). stool, gastric fluid and serum samples were sent for toxicological evaluation using the standard mouse bioassay. results: type e toxin was detected in the stool and serum sample of only one patient. all patients recovered and discharged one week after admission. conclusion: this study confirmed that prompt administration of antitoxin can prevent progression of disease based on clinical judgment and also may be life saving. in this case series study, we observed a short incubation period of 1-2 hours only in type e botulism. an outbreak of group g streptococcal pharyngitis among hospital personnel considered to be foodborne n. karabiber, a. gurbuz ertas, m. karahan, e. aykut arca, z.c. karahan, a. tekeli (ankara, tr) introduction: food-born outbreaks of streptococcal pharyngitis are relatively rarely reported,and while group a streptococci are the main causative agents, only a few epidemics caused by group g streptococci have been published. we describe here an outbreak of group g streptococcal pharyngitis occurred among the staff of a teaching hospital in ankara.the outbreak: an explosive outbreak of pharyngitis occured mainly among the staff working in certain departments (i.e. intensive care units, operation rooms) of tü rkiye yü ksek ihtisas teaching hospital, in 29 january 2004. methods: a total of 377 (111 and 266; 3 and 47 from catering firm personel) throat cultures were evaluated in 2 days,and 124 bhs strains were isolated, 65 and 59 on the first and the second days, respectively. presumptive identification by nbacitracin and trimethoprim/sulfametoxazole disk diffusion test showed that 121 strains were non-group a, 3 strains were group a streptococci. in definite grouping by streptococcus grouping kit (avipath-strep,omega),121 strains were found to be lancefield group g,3 strains were found to be group a streptococci (gas). one of the gas strains was isolated from a catering staff on the first day, the other two were isolated from two health care personnels on the second day. during the outbreak, 16 of 50 catering firm personel (32%) were found to positive for group g streptococci. all the bhs tested were found sensitive to penicillin g and erythromycin by agar disc diffusion method. conclusions: the configuration of the epidemic curve suggested a common source of exposure. since respiratory spread of streptococci in such a rapid fashion would be highly unlikely and that 16 of 121 positive throat culture were from the staff of the catering firm that provide all the food services for the hospital, and that most of them were working at the departments in which the outbreak occurred, we considered that the outbreak might be food-borne. prompt treatment with penicillin all the ill personnel and 9-day holiday coming consequently 30 january, terminated the outbreak. all the strains were cryopreserved for further typing studies.we are now typing these strains by pulsed field gel electophoresis (pfge) after digestion with smai restriction endonuclease. our initial results show that these strains are of the same origin. outbreak of acute gastroenteritis in an air force base in western greece e. jelastopulu, t. constantinidis, t. kolokotronis, d. venieri, g. komninou, c. bantias (patras, andravida, gr) objectives: on 20 september 2005, an operative training day at the air force base in western greece, soldiers and staff experienced an outbreak of acute gastroenteritis. the purpose of this study was to determine the causes of the outbreak and develop control measures. methods: following the assessment of descriptive epidemiology, a case-control analytic approach was utilized with 100 randomly selected cases and 66 controls. patients completed a questionnaire pertaining to the presence and severity of gastrointestinal disturbances, date and time of symptoms onset and consumption of food items served in the base on the implied training day. adequate questionnaire was administered to the controls. odds ratios were calculated and statistical significance was determined using x2 test. samples of food items were collected for bacteriological examination. results: the overall attack rate was at least 50% among the approximately 1050 attendees. the outbreak started abruptly in the late afternoon on 20 september, peaked at midnight and ended about 25 hours later. from the interviews and the analysis it was established that the lunch (beef, macaroni, tomato sauce and grated cheese) consumed several hours prior to onset of symptoms by affected military personnel was the likely source of the outbreak with a strong statistical association. there was only one subject who did not eat lunch. among the symptoms the most prominent were watery diarrhoea (96%) and abdominal pain (73%). relatively few indicated vomiting (8%) and nausea (7%). the mean incubation period was 9 h. in the bacteriological examination, staphylococcus aureus was detected in a sample of raw beef and in two samples of grated cheese (rest-cheese from lunch and an unopened package). conclusion: the short incubation period with abrupt onset, the symptomatology and the short, self-limiting nature of the illness, are suggestive of gastroenteritis caused by an enterotoxin-producing bacterium. s. aureus is considered to be the most likely cause. although mortality and longer-term morbidity are uncommon with food poisoning caused by enterotoxin-producing bacteria, this outbreak highlights its capacity to cause short term, moderately-severe illness in a young and healthy population. it underscores the need for proper food handling practices and reinforces the importance of appropriate microbiological specimen collection from cases, as well as the public health importance of timely notification of such outbreaks. occurrence, characterisation and antimicrobial resistance pattern of staphylococcus aureus strains isolated from dairy products in southern italy g. la salandra, e. goffredo, c. pedarra, m.c. nardella, a. parisi, a. dambrosio, n.c. quaglia, g.v. celano, g. normanno (foggia, valenzano, it) objectives: the ingestion of food contaminated by enterotoxins (ses) synthesized by staphylococcus aureus is responsible of one of the most common foodborne diseases (staphylococcal food poisoning-sfp). since s. aureus is often involved in cases of subclinical mastitis of ruminants, milk may results contaminated. infact, the dairy products are frequently related to cases of sfp, expecially in areas characterized by a high level of consumption of these products. consequently an active microbiological surveillance is needed in order to control the risk of sfp and to allow the improvement of the public health standards. s. aureus also show a large antimicrobial resistance pattern. in this work are reported the results of a survey conducted on the occurrence of s. aureus in dairy products from apulia region (southern italy). furthermore, the isolated strains were characterized in order to determine their ability in synthesizing ses and to evaluate their antimicrobial resistance pattern. methods: 250 samples of dairy products (milk, cheese, mozzarella cheese, ricotta cheeses) were analysed for the detection of s. aureus. the isolated strains were tested for the detection of ses, using the reverse passive latex agglutination test (sea to sed) and submitted to pcr to detect enta, entb, entc, entd and ente genes. furthermore, the strains were tested for susceptibility to ampicillin, tetracycline, gentamicin, eritromycin, enrofloxacin, co-trimoxazole, teicoplanin and vancomycin, by the agar diffusion method. results: out of 250 samples analysed, 36 (14.4%) resulted contaminated with s. aureus and, among these, 19 (52.7%) have been recognized as enterotoxigenic strains (10 samples of milk, 5 samples of mozzarella cheese, 3 samples of cheese from ovine milk and 1 sample of cheese). all the strains tested (one per each positive sample) showed antimicrobial resistance properties but none of these was resistant to teicoplanin and vancomicin. conclusions: the results obtained from this survey show that milk and dairy products from southern italy are frequently contaminated by enterotoxigenic strains of s. aureus and highlighted the need to implement strict hygienic control measures along the food chain in order to decrease the risk of spf. furthermore, the presence of antimicrobial-resistant strains of s. aureus in food may be considered a source of communityacquired infections, with the direct risk of transfer of the antimicrobial-resistance to intestinal human microflora. objectives: infection accounts for about one-third of cases of fever of unknown origin (fuo), which remains a major diagnostic challenge. recently, f-18-fluorodeoxyglucose (fdg) positron emission tomography (pet) has entered the field of clinical infectious diseases. fdg accumulates in tissues with a high rate of glycolysis, which is present in malignant cells and in all activated leukocytes. the aim of this prospective multi-centre study was to validate the use of fdg-pet as part of a structured diagnostic protocol in the general patient population with fuo. methods: from december 2003 to july 2005, 70 patients with fuo, defined according to the revised petersdorf criteria, were recruited from one university hospital and five community hospitals. a structured diagnostic protocol was used. fdg-pet was performed after certain obligatory laboratory tests, chest xray and abdominal ultrasound. the final clinical diagnosis was used for comparison with the fdg-pet results. results: a final diagnosis was established in 35 patients (50%): 12 infections, 5 malignancies, 16 non-infectious inflammatory disorders and 2 miscellaneous causes. of the total number of fdg-pet-scans, 33% were helpful. positive predictive value of fdg-pet was 70% and negative predictive value was 95%. fdg-pet was helpful in all patients diagnosed with an infection except for one case of pyelonephritis. contribution of fdg-pet to the final diagnosis did not differ significantly between the university hospital and the community hospitals. fdg-pet was not helpful in any of the patients with normal erythrocyte sedimentation rate (esr) and c-reactive protein (crp). conclusion: in addition to the apparent value of fdg-pet in diagnosing different infectious diseases as described in several case series, fdg-pet is a valuable imaging technique as part of a diagnostic protocol in the general patient population with fuo and a raised esr or crp. based on previous studies comparing gallium-67-citrate or labelled leukocyte scintigraphy and fdg-pet in patients with fuo and resulting from favourable characteristics of fdg-pet, conventional scintigraphic techniques may be replaced by fdg-pet in institutions where pet is available. emergence of clindamycin-resistant streptococcus pyogenes causing cellulitis epidemiology of viral respiratory infections a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus as a cause of community-acquired respiratory illness seroprevalence of human metapneumovirus in japan 1626-28. carriers into account when studying the dynamics of pneumococcal transmission and modelling the effect of pneumococcal vaccination in young children erythromycin-resistant streptococcus pneumoniae isolated in spain: serotypes, clones and mechanisms of resistance 14 (10%) and 23f (10%) that accounted for 73% of eryr strains. among eryr strains 109 (87%) had mlsb phenotype (73% constitutive and 14% inducible) and 16 (13%) had m phenotype. the genes detected in mlsb isolates were: ermb in 102 isolates, and ermb and mefe genes in 7 isolates. all 96 (88%) mlsb isolates with resistance to tet had tetm gene and 79 of them had int gene (related to tn916-like). seven positive ermb strains susceptible to tet had int gene spain23f-1, spain6b-2, sweden 15a-25 and st89-19f) accounted for 44% of these strains. capsular switching was observed in two clones, spain23f-1 (serotypes 19f and 19a) and sweden15a-25 (serotypes 23a, 23f suggesting the spread of tn916-like elements. the ermb positive strains, related to spain23f-1, spain6b-2, sweden 15a-25 and st89-19f clones, were more frequently isolated in adults us) objective: to characterize changes in the frequency of occurrence of bacterial pathogens responsible for pneumonia in hospitalized patients in europe for the years 1998-2004 and examine select antimicrobial susceptibilities (s) for predominant pathogens. the emergence of resistance (r) among pathogens responsible for pneumonia has resulted in changes to empiric therapy, with increasing reliance upon third-and fourthgeneration cephalosporins, beta-lactam/beta-lactamase inhibitor combinations, carbapenems and fluoroquinolones. methods: participating european medical centres (10-31/year) referred 50 consecutive, non-duplicate pathogens (8419 isolates) from lower respiratory tract sites determined to be significant by local criteria as the probable cause of pneumonia. all identified isolates were tested for s by the broth microdilution method 7 and 49.0%, respectively), increased significantly in 2001 (84.9% s), and returned to near-1998 levels in 2004. esblphenotypes (cro or caz or aztreonam mic ‡2 mg/l) remained essentially unchanged among ec between 1998 and 2004 (8.2% and 8.3%, respectively), whereas among ksp increases were more substantial (16.7% and 26.9%). metallobeta-lactamase-producing pa were identified during the study from italy vim-1) methods: four-hundred consecutive mrsa isolates were collected at 10 centres (max. 40 isolates per centre) as part of a multicentre study conducted throughout germany in 2004. isolates were collected from various sources, including colonization sites as well as infectious foci. only one isolate per patient was included and all isolates were spa-genotyped. cps were determined by slide agglutination with cp-specific antibodies (anti-t5-dt, anti-t8-conjugate, anti-336-repa). the serotypes were confirmed by immunodiffusion using lysostaphin-digested cell lysates. results: in the present study, we serotyped mrsa isolates collected most recently in a german multicentre study. all 400 mrsa isolates evaluated were one of the serotypes tested invasive pneumococcal disease in adults in north-rhine westphalia methods: surveillance for our current study focused on north-rhine westphalia, the largest federal state in germany (18 million inhabitants). 202 (52.2%) acute care hospitals27 microbiological laboratories serving these hospitals agreed to participate. we studied hospitalized patients older than 15 years of age. a case of ipd was identified by the isolation of s. pneumoniae from an otherwise normally sterile site. isolates were verified for species diagnosis by optochin testing and bile solubility, and for serotyping by the neufeld quellung reaction. mics of penicillin g, amoxicillin, cefotaxime, cefpodoxime, cefuroxime, clarithromycin us) objectives: carbapenems are the most reliably active betalactam antibiotics against enterobacteriaceae and are often the treatment of choice for infections caused by multi-drug resistant isolates. while carbapenem resistance has occasionally been reported in enterobacteriaceae, there are limited data on its frequency and distribution. methods: two large ongoing surveillance databases were searched for imipenem (imp) and ertapenem (etp) resistance in enterobacteriaceae: smart (study for monitoring antimicrobial resistance trends), a worldwide program focusing on community-and hospital-acquired intra-abdominal pathogens, and iss (icu surveillance survey), a us program focusing on icu isolates from any sterile body site. results: the overall frequencies of carbapenem-resistant enterobacteriaceae remained <2% in smart and <4% in iss throughout the periods of observation (see table). for 8% of esbl producing k. pneumoniae and e. coli were resistant to etp or imp and rates varied by geographic region. all isolates studied to date have exhibited multiple resistance mechanisms. conclusion: carbapenem resistance was uncommon among clinical isolates of enterobacteriaceae in these surveillance studies. its observed frequency varied by species and geographic region no significant seasonal variability in the prevalence of emergence of streptococcus b-haemolyticus strains in swabs was observed. conclusions: 1. seasonal fluctuations of pharyngeal discussion: with regard to high prevalences of giardiasis and enterobiasis it increase the prevalence that intestinal parasitic infections. it is suggested to decrease the rate of these parasitic infections in the region by strict programes that help to increase the knowledge of students, their parents and teachers about hygen. results: the study included 634 patients, with a mean age of 57.58 ± 19.2. 456 (71.9%) were women. the predisposing factors were: renal lithiasis patients (26.7%), prostatic adenoma (8.2%), vesical structure disease (4.3%), vesical functional disorder (6.6%), chronic kidney failure (4.3%). the underlying diseases included: diabetes mellitus (30.3%), immunosuppression (6.8%), previous urinary tract instrumentation (4.3%), permanent catheter (3.6%). the mean hospital stay was 10.93 ± 8.7 days. the mean duration of symptoms was 5.9 ± 8.2 days the absence of leukocyturia or mictional syndrome does not exclude the presence of complicated upper uti. 3) the high percentage of bacteriemia necessitates blood cultures, with e. coli being the most common pathogen. 4) the associated morbidity and mortality are important in association with sepsis or septic shock gr) objectives: to estimate the incidence of streptococci in community acquired urinary tract infections (uti) and also to carry out the in vitro antibiotic resistance of streptococci in urinary tract infections 3%) were enterococcus avium, 6 (2.0%) were enterococcus gallinarum and 38 (12.6%) were streptococci group b. the in vitro antibiotic resistance of enterococcus faecalis was: penicillin g 38.9%, ampicillin 2.4%, gentamicin500 29%, streptomycin2000 52.7%, nitrofurantoin 0%, ciprofloxacin 44.4%, tetracyclines 54.6%, vancomycin 4.3%, linezolid 0%. the in vitro antibiotic resistance of enterococcus faecium was: penicillin g 79%, ampicillin 69.8% tremolieres for the french aup study group background: short-course therapy for acute uncomplicated pyelonephritis (aup) is the newly suggested standard. talan et al. have demonstrated that oral ciprofloxacin (cip) for 7 days, was associated with greater cure rates than a 14-day trimethoprim-sulfamethxazole regimen. we assessed efficacy and tolerance of a 7-d. regimen of cip (study i), then of levofloxacin (lvx) in study ii results: of 171 (i) and 82 (ii) enrolled pts. 139 aged 31.7 ± 12.9 y., and 69, aged 3.58 ± 15.0 y. were retained for itt analysis; 16.5% and 16.0% had positive blood cultures. escherichia coli was the uropathogen in 99.2% and 100% of cases. finally 122 (i) and 60 (ii) were retained for per protocol (pp) analysis. at v4 bacterial eradication rates were 93.4% and 98.3 %. global cure rates were 89.0 and 92.8% at v4 and 77.3% and 75.0% at v5 with only less than 7% of lost to follow-up between v4 and v5 in both cases. side effects were observed in 12.8% and 27.6% of pts. who received 1 or more fq doses. conclusions: aup treatment with lvx 500 mg hiv-infected patients and drug addicts were excluded. antimicrobial susceptibilities of all s. pyogenes isolates were studied by microdilution method, and macrolide resistance phenotype by double disc test. macrolide resistance genes were detected by pcr. results: over the 10-year study period, there were 523 episodes of cellulitis. the infection was microbiologically 020). of note, all 4 cases of cellulitis due to clindamycin-resistant strains occurred during the last 3 years of the study. five (13%) patients presented with stss and died (1 due to an erythromycin-resistant strain). overall mortality (<30 days) was 33% this resistance might become a problem when treating s. pyogenes infections, especially stss cases. p1864 risk factors for community-acquired bacteraemic gram-negative cellulitis an administrative database was used. then we selected the patients with blood cultures obtained at the time of the cellulitis episode using the microbiology laboratory database. nosocomial cellulitis were excluded. a standardized data collection form was used to review the hospital records. in statistical analyses, student's t test was used for the comparison of mean values and chi square test and fisher's exact test for the comparison of categorical data (two tailed). results: of the 2251 patients with limb cellulitis identified in the study period, 301 patients had blood cultures and were selected for the analysis. bacteremia was detected in 59 of the 301 patients (19.6%), 15 of them due to gram-negative microorganisms hemorrhagic rash was present in 4.14% cases. koplick spot was found in 74.56% cases. measles was associated with streptococcal tonsillitis in 47.92% cases, with oral candidiasis in 20.22% cases and with pulmonary tuberculosis in 8.12% cases. severe forms of evolution were observed in complicated cases with: encephalitis (0.79%) or bronchopneumonia (5.89%), which required intensive care unit survey. we registered only one deceased, in a case of measles encephalitis in gipsy collectivities even it's very difficult, it's necessary to was performed. respiratory samples were tested routinely for twelve common respiratory pathogens. results: over the study period, of 587 samples processed, 40 -six cases were community-acquired and 23 (60%) patients had significant co-morbidities. cough was the predominant reported symptom. chest x-ray was performed in 31 cases, of which 28 showed abnormalities. bronchiolitis (22/ 39) was the commonest initial clinical diagnosis. the majority (69%) of patients received antibiotic therapy, but a convincing bacterial pathogen was isolated in less than half of these cases. thirty patients were admitted for management. more than one virus was identified from 14 patients, with rhinovirus being the predominant co-infection. overall, the average length of stay was 6.6 days. however, where hmpv was the sole pathogen identified, average length of stay was 3.1 days. conclusion: our data suggests that hmpv infections are more common in children with underlying co-morbidities. the rate of radiological imaging was higher than expected and perhaps is a reflection of the patient population or the degree of severity of illness. nosocomial acquisition occurred in 3 cases, which has implications for patient cohorting acknowledgements: the financial support for this study was provided by kuwait university research grant 03/03. evaluation of infection control practices in 31 haematopoietic stem-cell transplant facilities in german-speaking countries: variation of measures reflects lacking evidence s. wenzler-rö ttele, a. conrad, w.v. kern, h. bertz, f.d. daschner, m. dettenkofer (freiburg, de) objective: haematopoietic stem cell transplant (hsct) recipients are highly immunocompromised during pre-and postengraftment. thus, they are cared for in specialised facilities and versatile precautions are practised in order to prevent nosocomial infections. however, there is a lack of evidence whether these interventions are effective. furthermore, most of the measures are cost-intensive and restrict the patients' comfort. for evaluation of precautions, a survey was performed to assess the spectrum of measures commonly practised. methods: a questionnaire was compiled asking in detail for infection control measures differing according to allogeneic and autologous hsct recipients. the questionnaire was sent to 61 hsct facilities in germany, austria and switzerland. results: 31 questionnaires (51%) were filled in and sent back. among the 31 centres, 24 were university hospitals, and 4 teaching hospitals. the overall number of transplantations that were performed by the facilities varied considerably and ranged from 3 to 120/y for auto hscts and from 3 to 110/y for allo hscts. 80% of the institutions performing allo and auto hsct have implemented different precaution standards for each group. some measures regarding allo hsct were routinely adhered to in practically all institutions: accommodation in single rooms (96%), interdiction of plants and opening of windows (100% each) and protection from waterborne bacteria by use of terminal tap water filters (92%). 83% of hsct facilities perform their allo transplantations in hepa-filtered rooms and 54% are providing laminar air-flow for this population. there was a broad spectrum of different measures regarding barrier precautions: gowns when entering the room (required in 43% of centres for allo and 24% for auto hsct) and face masks (83% allo and 66% auto hsct). precautions to be followed by the patient varied among centres, e.g. specification of the face mask/respirator to be worn outside the isolation room (for allo hsct: 41% surgical mask, 5% ffp1, 36% ffp2 and 18% ffp3). conclusion: the broad variety of different preventive measures performed by the different facilities reflects lacking evidence for many infection control precautions that are commonly practised in the care of hsct recipients. this survey provides the basis for further studies within the onko-kiss project (hospital infection surveillance system for patients with haematologic/ oncologic malignancies). objectives: in this study it was our aim to evaluate the microbiological contamination of physiological serum flasks in use in medical day center for wound cleaning and to identify the isolated microorganisms. methods: we have collected 44 saline solutions from 22 health care centres localized in the health sub-region of coimbra. from each centre we have recovered 2 aleatory flasks in current use.the samples were transported at 4ordm;c and maintained at this temperature until its processing. saline solutions were seeded by the pour-plate technique in plate count agar and plates incubated at 28 and 37ordm;c for 48 h. the saline solutions were evenly spread over the surface of blood agar and sabouraud chloramphenicol agar (sab chl-d). the transfers of saline solutions flasks were also tested for microbiological contamination with a sterile cotton swab that was rubbed vigorously, over the transfer surface and directly applied on blood agar media. blood agar plates were incubated at 37°c for 48 h and sab chl-d plates were incubated at 28°c and 35°c and examined daily for a period of 30 days before declared as culture negative. microbial identification was firstly accomplished by employing conventional morphological and biochemical tests. when identification was not possible by these methods, 16s rrna gene sequence determination and phylogenetic analysis were used for bacterial strains and in the case of moulds we performed the amplification and sequencing of internal transcriber spacers region of 5.8s gene. results: from the 44 saline solutions analysed, 54.5% were contaminated. a total of 38 strains were isolated, 66% could be identified to species level using morphological and biochemical tests, the remaining 34% were identified by gene amplification and sequencing. about 69.6% of the identified strains were gram-positive cocci, the second dominant type of strain were gram-positive bacilli (13%), and the third dominant type of strains were gram-negative bacilli and moulds, both with 8.7%. the most frequent contaminants belong to human normal flora (64%), supporting the idea that the source of contamination of saline solutions analysed was human, in contrast with 36% of contamination due to the environment. conclusions: the contamination of the saline solutions is due to inadequate clinical practices. these results claim for more strict hygienic measures and for the replacement of big flasks by single use flasks with an incorporated overture used for wound irrigation. frequencies of cmv-ie specific memory t cells are inversely correlated with alloimmune memory and serum creatinine in kidney transplant patients p. nickel, g. bold, f. presber, c. schö nemann, j. pratschke, d. bitti, f. kern, h.-d. volk, p. reinke (berlin, de) background/aims: cytomegalovirus infection is a significant cause of morbidity in transplant patients and has been associated with allograft rejection. in this study frequencies of ifng-producing t cells following ex-vivo stimulation with protein-spanning peptide pools for cmv proteins pp65 and ie1 as well as donor-reactive t cell frequencies were serially determined during the first 6 months after renal transplantation (tx) to analyse the relation of cmv specific t cells, virus control and alloimmunity. patients: 64 kidney transplant recipients were included. immunosuppression generally consisted of anti-il-2r mab, calcineurin inhibitor, mmf and steroids. 2 presensitized patients received an induction by 2x low dose okt-3, anti-tnf mab, anti-cd20 mab and 5x plasmapheresis. 8 patients received fty-720, cyclosporine and steroids. methods: pbmcs from renal transplant recipients were analysed in a computer-assisted elispot assay before and at multiple times (mean 5) post-transplantationfor ifn-yproducing t cells following in-vitro stimulation for 24 hrs by irradiated donor cells and pools of overlapping peptides conclusions: although temporary r declines were seen among some european pneumonia pathogens, all showed increasing r to most class agents during the study period. the increase in esbl among enterobacteriaceae, and r among pa to most agents except polymyxin b, are especially worrisome. continued longitudinal comparisons of emerging pathogens and changing susceptibility profiles are critical elements in guiding empiric therapies and epidemiologic interventions. week, all participating hospital inpatients were swabbed on three anatomical sites: throat, nose and groin. we investigated the molecular epidemiology of the 46 mrsa isolates collected from 34 patients in 5 hospitals using the pfge method with the smai restriction enzyme. cluster analysis was carried out using bionumerics software. band-based similarity dice coefficients were used for dendrogram construction, which provides a quantitative assessment of strain similarity. samples were defined to belong to a cluster using a similarity coefficient of 75% or higher. pfge profiles were compared with the most similar strains from the harmony iums global mrsa database.results: 21 different restriction profiles were observed among the 46 mrsa isolates and 34 patients. isolates from the same patient but from different anatomical sites had similar pfge profiles. 7 clusters of mrsa strains could be identified with the two largest clusters containing 15 (44%) and 10 (29%) patients, respectively. strains from these 2 major clonal clusters occurred in 5 and 4 out of the 5 hospitals, respectively. isolates from the cluster with 15 patients were most similar to the well-known iberian clone: france a (10 strains), belgium e1 (2 strains), france b, france c and northern germany i (1 strain each). isolates from the next largest cluster of patients correlated with a group of strains previously found in finland and belgium: belgium e3 (2 strains), finland e1 (7 strains) and finland e7 (1 strain). the remaining strains were most closely related to belgium ec2 (3 strains), berlin iv (1 strain), southern germany ii (2 strains) and uk e15 (3 strains). conclusion: two major clonal clusters of mrsa strains were found to be dominant among hospitals inpatients in luxembourg. the molecular diversity of circulating strains was fairly diverse and profiles were very similar to previously described patterns in neighbouring countries and europe. further sequence-based genotyping is warranted to gain a better understanding of the clonal structure and elucidate transmission patterns. enterococci were identified by basic tests and by pcr amplification of ddl genes. susceptibility testing was performed using the icls broth microdilution method. resistance genes were detected by pcr, selected vana, vanb and vanc1 amplicons were sequenced. macrorestriction analysis (smai) resolved by pulsed-field gel electrophoresis (pfge) was performed. results: during the study 31 vre isolates with different phenotypes of resistance to glycopeptides were obtained from 3578 specimens. the prevalence of vre in the gastrointestinal tract was 3.5%. one e. faecalis (isolated from patient arrived from us) and 23 e. faecium isolates, harbouring vana genes, demonstrated mic's of vancomycin (van) and teicoplanin (tec) 256-1024 and 16-128 mg/l respectively. three e. faecium and four e. gallinarum isolates were vanb-positive, with van and tec mic's >32 and 0.25-1 mg/l respectively. all stains were susceptible to linezolide. among e. faecium isolates with vana genes one predominant pegf type was observed, differentiated in nine pegf sub-types. each of three other pegf types detected seemed to be unique. among six vana genes sequenced, four demonstrated similarity to vana gene from e. faecium (genebank af516335) and two to -vana gene from e. faecalis (genebank ay697425). in two sequenced vanb genes from in e. gallinarum nucleotide substitutions, resulting in seven new amino acid substitutions, were detected. conclusions: heterogeneity of glycopeptide-resistance genes, circulating in haematological centre, leads to the conclusion that their spread is not a local phenomenon. spread of vre is an emerging and, possibly, underestimated problem for russia. study of resistance and clonal relatedness of clinical isolates of stenotrophomonas maltophilia from a hospital in northern spain c. valderrey, e. sevillano, f. calvo, l. gallego (bilbao, es) objectives: the aim of this work was to study the antibiotic resistance and genetic relatedness among clinical isolates of s. maltophilia isolated from patients with tract respiratory infections. methods: the study included 36 s. maltophilia isolates obtained in a hospital from bilbao (northern spain) during 2005 (from january to october). susceptibility to antimicrobial agents was determined by the disk diffusion method following the nccls recommendations. the antibiotics tested were imipenem, meropenem, cefotaxime, ceftazidime, cefepime, aztreonam, amikacin, tobramicin, ciprofloxacin, ofloxacin and trimethroprim/ sulfamethoxazole. total dna was used as target for pcrfingerprinting experiments with primers rd1, eric2, ap3, m13 and rnar 1 and 2. to detect class 1 integrons, primers 3cs and 5cs were used in amplification experiments.results: resistance to antibiotics tested was the following: imipenem (100%), meropenem (67%), cefotaxime (92%), ceftazidime (28%), cefepime (64%), aztreonam (86%), amikacin (8%), tobramicin (14%), ciprofloxacin (25%), ofloxacin (25%) and trimethroprim/sulfamethoxazole (0%). pcr-fingerprinting technique was only useful when eric2 primer was used identifying 28 distinct genotypes. the other primers were not able to produce reliable band patterns. patients with several isolates maintained the same clone along time, although there are two patients from which two different genotypes have been isolated, and two clones that have been isolated from more than one patient. class 1 integrons were detected in 56% of isolates ranging in size from of 1500 to 300 bp (8 isolates bored combinations of two structures). conclusions: trimethroprim/sulfamethoxazole and amikacin showed the best activity against the isolates tested. for pcrfingerprinting experiments the best primer was eric2 which produced reliable and reproducible band patterns. there was a high clonal diversity since 28 different genotypes were identified among the 27 patients included in the study. many isolates bored class 1 integrons with sizes similar to those detected in other nonfermenters bacilli from the same environment.methods: -identification: the strains were identified by colonial morphology, haemolysis on blood agar plates, biochemistral and antigenic identification; antibiotic susceptibility testing: all the strains were tested by disk diffusion according to the national committee for clinical laboratory standard methods. mics were determinated by screening test or mic evaluation in solid media. results: our study concerns 252 bacterial strains isolated from january 2000 to june 2005. among the strains isolated, neisseria meningitidis represented the most number of cases with 43.7 %.these were distributed among all different age groups. serogroup a was the most predominant and represented 54.5% of total strains while groups b and c represented 15.5% and 14.5% respectively. streptococcus. pneumoniae represents the second causes of purulent meningitis with 32.5% while haemophilus. influenzae b is the third causative bacterial agent with 23.8%.this last agent is most predominant among infants less than 2 years of age in 80% of cases. neisseria. meningitis is susceptible to all types of antibiotics tested. however, haemophilus. influenzae b produced an inactivating enzyme (penicillinase) in 26.7% of cases. the resistance was associated to cotrimoxazole in 68.7% of cases. the results of mic done on streptococcus. pneumoniae show that 35.4% of strains has an intermediate resistance to penicillin and high level of resistance in 4.9%. the amoxycillin is active in 98.8% of the strains,in the opposite cefotaxim has an intermediate resistance in 6.1% and a high level of resistance in 2.4% of the strains. the resistance to penicillin was associated with resistance to erythromycin, cotrimoxazole or to both in some cases. conclusion: streptococcus. pneumoniae represents the second causative bacterial agent responsible of purulent meningitis and showed an increasing prevalence of resistance profiles to penicillin and cefotaxim in our hospital. this implicates an effective microbiological and epidemiological control.conclusion: as expected in a referral hospital with a cardiac surgery department, the prevalence of s. aureus ie was elevated as well as the attributable mortality rate. the high global mortality rate may be explained by the high frequency of severe co-morbidities and by the late referral of patients to hospital. our data suggest that there is room for improvement in the diagnosis and management of ie in a multidisciplinary collaborative approach. objective: to determine the clinical, epidemiological, diagnostic, and therapeutic characteristics of a series of 111 cases of prosthetic valve endocarditis. methods: we undertook a retrospective, descriptive study of 111 cases of prosthetic valve endocarditis obtained from a series of definite or probable 626 left sided infectious endocarditis from six second-or third-level andalusian hospitals from 1985 to 2005. results: of the 111 cases of prosthetic valve endocarditis, 96 (86.5%) were definite and 15 (13.5%) possible. the mean age was 58 ± 13 years, and they were more common in men (61%). late infection was more common than early involvement (74 vs. 37 cases). the aortic valve was involved in 54 cases (48%) and the mitral valve in 48 cases (43%. most (66%) of the valves were made of metal and prior handling had taken place in 26 cases (23%). clinical characteristics were fever 88%, constitutional syndrome 36%, murmur 41%, vascular events 37%, and immune phenomena 17%. complications included left ventricular failure 54%, kidney failure 22%, peripheral embolism 20%, cns embolisms 18% and heart block 9%. the etiology was as follows: in early prosthetic valve endocarditis the three most common pathogens were s. coagulase-negative (40%), s. aureus (22%) and enterococcus (11%). late prosthetic valve endocarditis involved s. viridans (30%), s. coagulase-negative (17%) and s. aureus (13%). transesophageal echocardiography alone in 14 cases (12%), and transthoracic followed by transesophageal echocardiography in 53 cases (48%). medical therapy was applied in 54 cases (48.6%) and surgery in 57 (51.3%). a cure was achieved in 71 cases (64%), the other 40 (36%) dying. of those who underwent surgery, 38.5% died and 31.4% of those who were treated medically died. the death rate from early prosthetic valve endocarditis was greater than that for late prosthetic valve endocarditis (54% vs. 29%). conclusions: 1) prosthetic valve endocarditis is a very serious infection which is still associated with an excessively high mortality, despite advances in diagnosis and treatment. 2) early prosthetic valve endocarditis has a worse prognosis than late prosthetic valve endocarditis, due to its distinguishing pathophysiological features. 3) the greater mortality seen in patients who underwent surgery is probably associated with the fact that they had more complications, such as perivalvular abscesses or persistent infection. outcome of infective endocarditis e.e. hill, s. vanderschueren, p. herijgers, m-c. herregods, p. claus, w.e. peetermans (leuven, be)objectives: despite progress in diagnosis and therapy, almost half of patients with infective endocarditis (ie) has at least one complication and overall mortality remains high. the aim of the present 5-year prospective observational study was to define predictors of outcome in patients with ie. methods: from june 2000 through december 2004, all first episodes of definite ie by the modified duke criteria, encountered in a single tertiary-care medical center, were registered and followed-up for 6 months. results: overall, 193 patients suffered 203 ie episodes. sixtyone percentage were males. the median age was 62 years (range 54-73). fifty-five percentage of episodes were referred from another hospital. at least one complication occurred in 79%. surgical intervention was performed in 63% and was mainly indicated because of congestive heart failure. the median time from diagnosis to surgery was 6 days (range 0-92). six-months mortality was 22% (n = 42). in bivariable analyses, factors associated with 6-months mortality were: age, female gender, causative microorganism, nidus of infection and therapeutic policy. six-months mortality was 18% for native valve ie and 31% for prosthetic valve ie; twenty-five% for nosocomial ie and 19% for community-acquired ie. six-months mortality rates for microorganisms were: staphylococci 33% (n = 26) [s. aureus 32% (n = 19) and cons 35% (n = 7)], enterococci 24% (n = 8), streptococci 8% (n = 4) and other microorganisms 14% (n = 4). the 6-months mortality for patients with a contraindication to surgery was 74% (n = 20), for patients conservatively treated without a contraindication 7% (n = 3) and for combined surgical-medical treatment 16% (n = 19). in multivariable logistic regression predictors of 6-months mortality were age (or, 1.05; 95% ci, 1.01-1.1; p = 0.03), causative microorganism (or, 0.74; 95% ci, 0.55-1; p = 0.049) and a contraindication to surgery (or, 32.26; 95% ci, 7.2-145; p < 0.001). conclusion: in the present prospective single centre study of 193 patients with definite ie, 6-months mortality rate was 0.22, and was especially high in patients with preestablished contraindications to surgery, in the elderly and in patients with staphylococcal ie. six-months mortality in patients with combined surgical-medical treatment versus exclusively medical therapy in patients without a contraindication to surgery was not statistically significant. staphylococcal and enterococcal ie had a worse prognosis compared to streptococcal ie. epidemiology and aetiology of infective endocarditis e.e. hill, p. claus, m-c. herregods, p. herijgers, s. vanderschueren, w.e. peetermans (leuven, be)objectives: the epidemiological features of infective endocarditis (ie) have changed. we report the results of a 5-year prospective observational study investigating trends in the epidemiology and etiology of ie. methods: from june 2000 through december 2004, we registered 203 definite ie episodes according to the modified duke criteria in 193 patients older than 16 years, hospitalized in a single tertiary-care center. results: sixty-one% of episodes involved males. the median age was 62 years (range 54-73).fifty-five percentage (n = 112) were referred from another hospital. forty-four percentage (n = 90) were nosocomial. thirty-four percentage (n = 70) involved prosthetic valves and 17% (n = 12) thereof were of early postoperative onset. the mitral valve was most frequently involved. exposure to ie risk factors during the previous 6 months was recorded in 67% (n = 136) of the episodes. twenty-four percentage (n = 49) were intravascular catheterobjective: to determine the eco-epidemiology of cryptosporidiosis in the health services executive -western area (formerly the western health board).concerns about the incidence of cryptosporidiosis in the western area prompted the department of public health to undertake further investigation of potential links between cryptosporidiosis and environment by focusing on farming activity and water supplies in the first instance. background: cryptosporidiosis was not notifiable in the republic of ireland prior to 2004, unless cited as a cause of gastroenteritis in a child less than two years old. as a result the incidence of cryptosporidiosis in the republic of ireland at the time was unknown. nationally it was estimated that up to 8% of cases of gastroenteritis in children less than two years old could be attributed to cryptosporidium. in the western area from 1999 to 2003 the proportion of cases of gastroenteritis in children less than two years old attributable to cryptosporidium ranged from 10.8% to 24.5%. this was cause for concern.many rural locations in the western area are served by voluntarily-operated water schemes. water quality from these schemes is often microbiologically unsatisfactory. the department of public health methods: initial research involved analysis of notification records for cases of cryptosporidiosis received from 1999 to 2003 inclusive. crude incidence rates for cryptosporidiosis in the western area were compared with crude incidence rates in england & wales, northern ireland, and scotland for the same time period. cases of cryptosporidiosis from the western area were geo-coded and mapped to visualize the geographic spread of cases, and are being contrasted with geographic data for farming activity, and also with available data on water supplies. the results of the initial phase of this research indicated the incidence of cryptosporidiosis in the western area may be cause for concern. the geographic spread of cases and potential links to farming practices and water supplies will be presented. objective: the evaluation of epidemiology and seasonal fluctactions of bacterial flora in pharyngeal swabs taken from family doctors' patients. material and methods: a total of 7807 of positive pharyngeal swabs ordered by primary care physicians from silesia were examined during the 2000-2005 period. the microbiological analysis was performed in silesian analytic laboratories. the intake of material, its transport and final identification complied with laboratory standards. results: the most common pathogens were, in order of prevalence: streptococcus viridans (57.4%), moraxella catarrhalis (45.2%), staphylococcus aureus along with mrsa (31.6%) and mrsa alone (1.6%), e. coli (18.8%), klebsiella pneumoniae (6.6%), streptococcus b. haemoliticus (4.7%). candida albicans was identified in 14.2% of positive specimens. considering seasonal fluctuation, the number of positive swabs in each month tended to gradually increase in spring with its culmination in may (10.4%). as for the most common pathogens streptococcus viridans and moraxella catarrhalis mirrored the general tendency and dominating in spring season (up to 11.7 and 12.1%, respectively) and having less stronger impact in automn (up to 9.3 and 9.4%). the frequency of isolation of the other pathogens revealed seasonal fluctuations confined to either spring, as in the case of klebsiella pneumoniae, escherichia coli and staphylococcus aureus strains (up to 17.7, 12.3 clinical microbiology and infection, volume 12, supplement 4, 2006 aim: the aim of this study was to identify the microorganisms isolated from corneal and conjuntival samples, isolated from patients attending the ophtalmology department of a spanish hospital. material and methods: a total of 69 corneal scrapes and 544 conjunctival swabs were obtained since october of 2002 to october of 2005 in an university hospital of madrid. samples were cultured into blood and chocolate agar plates and incubated at 35ordm;c in o 2 and co 2 atmospheres, respectively, for two days (conjunctival swabs) and fifteen days (corneal scrapes). identification and susceptibility tests were performed following standard methodology. results: thirty four (49.3%) out of 69 corneal samples and 96 (17.6%) out of 544 conjunctival swabs yielded positive cultures, respectively. results are summarized in the following table:conclusions : corneal scrapes yielded a higher number of positive cultures than conjunctival swabs. gram-positive microorganisms were more prevalent both from corneal scrapes and conjuntival swabs although the difference was more evident in corneal scrapes. s. aureus was the specie most prevalent in conjunctival samples meanwhile cns were the most prevalent in corneal scrapes. methods: vitreous fluid samples (n = 177) were obtained from 150 patients (94 male, 56 female) undergoing vitrectomy for endophthalmitis between january 2001 and october 2005. specimens of undiluted aqueous and vitreous fluid were cultured for aerobic, anaerobic bacteria and fungi by conventional methods. identification and antibiotic susceptibility were performed by the api system, vitek ii system (biomerieux) and the agar disk diffusion methods according to clsi recommendations. results: ninety one isolates were recovered from the samples. gram stain was positive in 133/177 (75.1%), while cultures were positive in 94/177 (53.2%) samples. gram-positive bacteria were the most common isolates (71/91, 78%), followed by gramnegative bacteria (11/91, 12%) and fungi (9/91, 10%). staphylococci coagulase-negative were isolated in 41/91 (45%). the next most common species isolated among gram-positive bacteria were s. aureus (6.6%), streptococcus spp (9.9%), propionibacterium acnes (9.9%), bacillus spp (3.3%), streptococcus. pneumoniae (1%) and enterococcus faecalis (1%). among gramnegative bacteria eight isolates were enterobacteriaceae, two were non fermenters and one was haemophilus inlfuenzae. two of the fungal isolates were candida albicans, one acremonium spp and six aspergillus fumigatus. polymicrobial growth was observed in six patients with two at least isolates. of staphylococci coagulase-negative 10/41 (24%) were resistant to methicillin. only one strain of staphylococcus aureus was methicillin resistant. all gram positive isolates were susceptible to vancomycin. all isolates were sensitive to amikacine and ceftazidime while resistance was observed in 9/177 (5%) isolates to fluoroquinolones. conclusion: a variety of microorganisms was isolated from the vitreous fluid of patients. the predominant isolates were grampositive bacteria, especially staphylococci coagulase-negative with low resistance rate to methicillin. so, therapy should be based on the isolation and identification of the infecting agent and the in vitro antibiotic susceptibility to the appropriate antibiotics. the prevalence of intestinal parasitic infection in the students of primary schools in nazloo region in urmia during [2004] [2005] k. hazrati tappeh (urmia, ir)background: intestinal parasitic infections are of the most important hygienic and economical problems of millions of people in all over the world, mostly from developing countries. understanding their epidemiological situation and relation to environmental and social factors is necessary for struggling with them in every society. this investigation was designed to study the prevalence of parasitic intestinal infections among primary school attending students in nazloo region of urmia district in 2004. materials and methods: 271 students were chosen randomly from 7 schools upon their population. having their questionnaires filled, two faecal samples were taken from each student and examined with direct wet mount and formalinether sedimentation technique. scotch tape was also applied in order to detect the enterobiasis and taeniasis. 271 students completed the test. all infected persons by e. vemicularis, h. nana were treated by mebandazole and giardia lamblia were treated by metronidazole. results: overall prevalence of parasitic protozoan infections was 29.5%. giardia lamblia was found in 28 cases (10.3%), entamoeba coli in 27 cases (10%) and blastocystis hominis in 2 cases legionella pneumophila as an occupational risk factor for inter-city bus drivers y. polat, ç . ergin, i. kaleli, a. pinar (denizli, ankara, tr)objectives: legionellaceae are ubiquitous aquatic microorganisms that usually isolated from evaporative condensers. various man-made sources such as cooling towers, whirlpools and spas are sources for legionella pneumophila. in hot climate, bus air-conditioning and aircirculating systems are possible sources for the organism. in this study, serologic status of bus drivers and their assistants for legionella infections as well as bus air-conditoner moisture exit samples for legionella species were investigated.methods: serum samples were collected from bus drivers (n = 63) and their assistants (n = 19). samples were tested for anti-legionella antibodies by indirect immunofluorescence technique. 1/320 dilution was accepted as a positive result for anti-legionella pneumophila antibodies. results were analysed according to risk factors based on hot/cold climate route (aegean and mediterranean parts of the turkey were accepted as hot climate region), immundeficiency, chronic diseases and work hours. according to serologic test results, air-conditioners of buses which has been driven by 1/10 dilution seropositive persons, were investigated. air-conditioner moisture exit samples were cultured on bcye-alpha agar supplied with bmpa. same samples were tested by pcr targeting a 108-bp fragment of the 5s rrna gene of legionella. results: anti-legionella pneumophila antibodies were positive in 12 (15.2%) bus-persons. bus drivers' seropositivity was higher than assistants (p < 0.05). in hot climate route, seropositivity was higher than cold climate route (p < 0.05). no positive pcr result was detected. coclusion: in conclusion, higher seropositivity rates in bus drivers were pointed out a newer occupational risk factor for legionellosis. although pcr positivity was not detected for bus air-conditioners, high seropositivity rates show that bus drivers have been somehow exposed to legionella. further legionellosis surveillance studies for bus drivers may help to understand legionella exposure during travel. objective: asymptomatic bacteriuria is an important risk factor contributing to pyelonephritis and renal disfunction in diabetic patients. in this study, the relationship between microalbuminuria and age, body mass index, duration of the disease, the level of glycohemoglobin, glycosuria and glomerular filtration rate is studied prospectively in diabetic patients who have asymptomatic bacteriuria. methods: a hundred and twenty-three type 2 diabetic outpatients who were admitted to baskent university konya medical and research center between january-october 2005 were included in the study. ages of the patients were within the range of 26-80 years. the diagnosis of asymptomatic bacteriuria was established according to the cdc criteria. concurrent samples for urinary culture, glomerular filtration rate, microalbuminuria and glycohemoglobin were obtained. results: twenty-two of 123(17.8%) patients had significant bacteriuria. of these patients 74% were female. although age, body mass index, creatinine clearence and presence of microalbuminuria were similar, there was a significant difference in glycohemoglobin levels, duration of diabetes and glycosuria between the two groups (p < 0.05). e. coli was the most common microorganism obtained from urinary samples. risk factors for asymptomatic bacteriuria were shown in the table. conclusion: the frequency of asymtomatic bacteriuria was found to be similar with the previous studies. high glycohemoglobin levels and long duration of diabetes were found to be the risk factors contributing to asymptomatic bacteriuria in type 2 diabetic patients. descriptive study of complicated pyelonephritis objective: the evaluation of prevalence and contributory factors associated with the development of urinary tract diseases among women with urinary incontinence. material and methods: 124 women aged from 35 to 65 years had their urine culture examination performed. the material was taken from the central stream of first catch urine and transported on uromedium. antibiogram was carried out with the use of becton-dicinson's discs. results: in 14 cases the urine culture tested positively which accounted for 11.3% of subject women. the most common pathogens of urinary tract were, in order of prevalence:e. coli-64.3%, staphylococcus aureus -14.3%,citrobacter diversus-7.13% and klebsiella pneumoniae-7.13%. candida albicans strains were isolated in one patient. e. coli had the highest sensitivity to norfloxacin -100% and cefuroxim -100%, amoxicillin with clavulonian acid -88.8%, ampicillin nitrofurantoin and trimethiprim -sulfamethoxazole -77.7% in each case, cefalothin -66.6%, tetracycline -55.5%, and amikacin -33.3% but only in 11.1% to amoxacillin. staphylococcus aureus proved sensitivity only to gentamicin (100%) and nitrofurantoin (100%). in the case of citrobacter diversus 100% sensivity to norfloxacin, nitrofurantoin, tetracycline, trimethoprim / sulfamethoxazole, ceftazidim and cefotaksym was confirmed.klebsiella pneumoniae also proved sensitivity to amoxicillin with clavulonian acid, cefuroksime, nitrofurantoin, norfloxacine, tetracyclin and trimethoprim / sulfamethoxazole. when considering the sensitivity of pathogens to antibiotics in the family practise setting of higher reliabilty are nitrofurantoina, norfloksacyna.after the administration of guided therapy complete release from symptoms was observed in 10 women (71 %).conclusions: women with urinary incontinence relatively seldom suffer from urinary tract infections. the most common pathogen among women with urinary incontinence was e. coli sensitive to floxacins and cephalosporins but with impaired reaction to amoxycillin. incidence and in vitro antibiotic resistance of streptococci in community-acquired urinary tract infections uncomplicated community-acquired urinary tract infections (ca-utis) and non-pregnant women in london hospital in kuwait over a period of two years. methods: eighty-six pregnant and 90 non-pregnant women with signs of ca-utis were enrolled in the study. the strains isolated from the patients who had significant bacteriuria were included in the microbiological analyses. the identification of the strains was performed using the api 20e system (biomerieux), while their susceptibility was determined by disk diffusion method. the interpretation of the results was realized according to nccls guidelines. quality control was performed using reference strain e. coli atcc 25922. oserotyping was carried out with polyvalent and monovalent antisera. hemolysin production was tested on human blood agar plates. possession of k1 antigen by e. coli was tested with agglutination by murine monoclonal antibodies to the group b meningococcal capsule. results: we found o serogroups o1, o2, o4, o6, o7, o18 and o75 among strains isolated from pregnant and non-pregnant women. hemolysin was presented in 35% and 40% respective. k1 antigen was presented in 40% of strains in studied groups.there are some statistically significant differences in antimicrobial resistance between both groups. amoxicillinclavulanate (amx-clv) resistance was higher among uti haemolytic isolates of e. coli in pregnant women (54%) then in non-pregnant women (38%). similar distinction in cefuroxime resistance was found -15% and 0. amikacin resistance was higher among uti isolates of e. coli in non-pregnant women (47%) then in pregnant women (23%).conclusions: there are no significant differences in expression of virulence factors of e. coli from pregnant and non-pregnant women with ca-utis in london hospital, kuwait. the resistance rates of e. coli from pregnant women to amx-clv and cefuroxime are significantly higher than in non-pregnant women. the penetration of telithromycin in gynaecological tissues and activity in cervicitis patients h. mikamo (gifu, jp)objectives: chlamydia trachomatis and neisseria gonorrhoeae are major causative organisms for sexual transmitted infections in japan. although several oral antimicrobial agents are active against c. trachomatis, few effective oral antimicrobial agents against n. gonorrhoeae exist in japan. two studies were conducted: a clinical pharmacology study examining penetration of telithromycin (tel), an oral ketolide antibiotic, in female genital organ tissues and a clinical study examining tel 600 mg once daily (qd) in cervicitis patients (pts chronic prostatitis (cp) is believed to be an infectious disease in most cases. both aerobic and anaerobic bacteria are involved in the polymicrobial microbiocenosis found in prostate specific specimens. coryneform bacteria form a remarkable part of this community, yet scarce knowledge exists about their clinical significance, species composition and antibiotic susceptibility.our aim was to compare the corynebacteria of the seminal fluid of cp patients and controls and to evaluate their antibiotic susceptibility.material and methods: semen samples from 66 controls and 49 cp patients (nih iiia or iv category) were analysed. corynebacterium seminale was identified by beta-glucuronidase activity, the rest of coryneforms using api coryne (biomerieux). e-test method was used for susceptibility testing.results: coryneforms were found from 78% cp patients and 84% controls (p > 0.05). twelve species and genera were found among 120 strains identified, the most frequent being c. seminale (in 60% cp patients and 58% controls). cp patients harboured significantly more arthrobacter sp. (17% vs 2%, p = 0.03) andcorynebacterium group g (14% vs 2%, p = 0.01), the latter association was especially eminent in case of patients with serious inflammation (>1 wbc/ml): 33% vs 2%, p = 0.0003. all tested 65 strains were susceptible to ampicillin-sulbactam, single strains were resistant to doxycycline (5%) and tmp/smx (5%), however, moderate resistance was common to doxycycline (29%). resistance to clindamycin (38%), benzylpenicillin (28%), nitrofurantoin (17%), erythromycin (16%) and norfloxacin (11%) was observed as well. half of cp-related corynebacterium group g strains showed resistance to nitrofurantoin and benzylpenicillin. in addition, they were often moderately resistant to clindamycin, erythromycin and, finally, norfloxacin frequently used to treat cp. conclusions: most of men have coryneforms in their semen, more than half harbour c. seminale. corynebacterium group g and arthrobacter sp are more frequently found in cp patients than the controls. in the treatment of cp of unknown etiology it is useful to take into consideration the susceptibility profile of corynebacterium group g. objective: to evaluate the role of cmv and listeria monocytogenes in abortion.methods: this descriptive prospective study was done on 450 women, 250 women with spontaneous abortion before 20th weeks of pregnancy as a case group and 200 healthy woman with full term delivery as a control group. serum samples were taken from all patients. elisa test was done for evaluation of cmv (igg and igm) and listeria antibodies in both groups. prevalence of seropositivity was determined. data were analysed by x2 and chi-square test.results: 450 seologic tests were done on samples. average age in case group was 25.6 ± 7.6 and in control group was 25.3 ± 6.5 years. in cases with abortion 89 (35.6%) and in control group 35 (17.5%) were seropositive for listeria monocytogenes. difference in seropositivity between 2 groups is statistically significant (p = 0.0001). cmv igg antibodies were positive in 235 (94%) of case group and in 150 (75%) of control group; the difference is significant statistically (p = 0.0001). cmv igm antibody was positive in 13 (5.2%) of case group and none in control group. difference is significant (p < 0.0001) there was no correlation number of previous abortion and seropositivity for listeria and cmv. conclusion: the present study showed an important role of listeria monocytogenes and cmv infection in abortion. serum and prostatic tissue concentrations of moxifloxacin (400 mg) after a single intravenous infusion in patients with benign prostatic hyperplasia undergoing transurethral resection of the prostate background: the spectrum of bacterial prostatitis comprises gram-negative, gram-positive and atypical pathogens. because of its broad spectrum of activity, moxifloxacin might be a suitable antibiotic for the treatment of bacterial prostatitis. aim: in this study the penetration of moxifloxacin into prostatic tissue after intravenous application of 400 mg as single dose was investigated.methods: in a prospective, multicentric study patients with benign prostatic hyperplasia received a single dose of moxifloxacin 400 mg in an 1 hour lasting infusion (250 ml) for perioperative prophylaxis before undergoing transurethral resection of the prostate (tur-p). serum concentrations were determined in all patients before infusion, at the end of infusion (time point 0), 0.5, 1 and 2 h after the end of infusion. patients were randomized for tissue sampling either 0, 0.5, 1 or 2 h after the end of infusion. at the beginning of tur-p approximately 1 g of tissue was sampled for analysis. concentrations of moxifloxacin in serum and tissue were determined by hplc. results: 39 patients were evaluated in the study. the concentrations (mean, sd, median, 25/75% quantile) are shown in the table. the prostatic tissue concentrations of moxifloxacin were approximately twice as high as in serum. at the end of infusion the tissue and serum concentrations were already equilibrated, because the tissue-serum ratios did not differ significantly from the end of infusion until 2 h after the end of infusion. after an intravenous infusion of 400 mg the serum and prostatic tissue concentrations of moxifloxacin were well above the mic values of the most important prostatic pathogens until 2 h after the end of infusion. therefore, moxifloxacin might be a good alternative for the treatment of bacterial prostatitis and/ or perioperative prophylaxis for tur-p. statistical significant differences were detected between patients with and without bgnc in the proportion of patients older than 65 years (81.3% vs 42.5%), the antecedent of recent animal bite (18.8% vs 0.7%), the presence of immunosuppression (12.5% vs 1.8%), the presence of haematological illness (12.5% vs 0.7%), and the degree of leukocytosis at admission (8989 ± 3736 vs 12690 ± 6028 cel/ll). conclusions: bgnc is frequently detected in our patients. age older than 65 years, the existence of immunosuppression, the existence of haematological illness, and the antecedent of animal bite are more frequent among patients with bgnc. patients with bgnc had a lower degree of leukocytosis at admission. these factors should be borne in mind to select empiric therapy for patients with cellulitis. is erysipelas-associated tinea pedis a site of streptococcal colonisation? objectives: tinea pedis is considered the most frequent portal of entry of erysipelas of legs (sel) but whether it is the site of streptococcal colonisation is unknown. methods: from june to october 2005 we prospectively searched for clinical tinea pedis in patients hospitalised in our infectious diseases ward for sel (acute and unilateral feature with fever were only retained). all patients had bacteriological samples on inter-digital spaces of both feet (sel side and contra lateral side).results: fifteen patients were included. all but one were treated by intra-venous penicillin-g followed by oral amoxicillin. on sel side: tinea pedis was found in 10/15 (67%) and, when present, streptococcal colonisation (c or g streptococcal groups) was found in 7/10 (70%), although streptococcal colonisation was never found (0/5) in its absence. on contra lateral sides : no streptococcal colonisation was found without tinea pedis, which was observed in 7/15, with streptococcal colonisation in 6/7. then there is a strength statistical association between streptococcal colonisation and tinea pedis, on sel side (p = 0.025) as well as on contra lateral side (p = 0.003). in one patient blood-cultures yielded with the same streptococcus than found in foot samples. discussion: streptococcal colonisation of tinea pedis is a common finding on both feet of patients hospitalised for sel. whether inter-digital colonisation is a primary stage of invasive disease remains unproved. in our experience, a strain of streptococcus that colonised inter-digital space was isolated in patient's blood, suggesting this hypothesis may be true in some cases. if confirmed, this concept could lead to a new strategy for secondary prophylaxis of recurrent sel by decontaminating streptococcal colonisation of tinea pedis. among ggs, 11 different emm types were found; stg6, stg480 and stg643 predominated. among gas, 8 types were found, emm81 predominated. one patient had the same ggs isolate in throat and skin. six patients had recurrent infections during the study; two of them with 3 disease episodes. of the 35 culture positive skin samples, 12 were taken from the erysipelas infection focus (42% positive for ggs) and 23 from another site (61% positive for ggs), e.g. wound, intertrigo, between toes or an unknown site.conclusion: a predominance of ggs was seen in the throat of erysipelas patients and their families whereas ggs was not present in control subjects. ggs, instead of gas, also seems to predominate in erysipelas skin lesions. several emm types were present in both groups and there was no clear predominance of a distinct type. the recurrent nature of erysipelas became evident also during this study. the evaluation of fournier's gangrene severity index score in 11 patients m. ulug, m.k. celen, m.f. geyik, c. ayaz, s. girgin (diyarbakir, tr)objectives: fournier's gangrene is synergistic necrotizing fasciitis of the perineum and abdominal wall along with the scrotum and penis in men and the vulva in women. it is rare but life-threatening process. in this study we identify effective factors in the survival of patients with fournier's gangrene and to determine the accuracy of the fournier's gangrene severity index score (fgsis). methods: we evaluate 11 patients with fournier's gangrene who were threated and follewed up from us between january 2005 and september 2005 in the department of general surgery prospectively.results: the results were evaluated in two groups: those who died (n: 3) and those who survived (n: 8). no statiscally significant difference was found between the age of the survivors and those who died. the admission and final laboratory parameters that correlated statiscally signinificant with outcome includes leucocyte count, hematocrit, urea, creatinine, lactate dehydrogenase, bicarbonate and albumin. sites of culture were skin/soft tissue (75, 74 and 92%), respiratory tract (3, 4, and 3%) , blood (4, 7 and 1%), urine (3, 11 and 0%) , and other (3, 2 and 2%) . 30-day mortality was 0% in this population. 50% of patients received antibiotic therapy alone, 5% surgery alone, 30% antibiotics + surgery, 3% other therapy, and 12% no treatment. the most common antimicrobial classes received were vancomycin (27%), beta-lactams (23), fluoroquinolones (11), and cotrimoxazole (10) with 14% of patients receiving multiple agents. median duration of antibiotic therapy was 12, 15, and 10 days, in the ca-mrsa, ha-mrsa and ca-mssa groups respectively. 47, 82, and 57% received adequate antimicrobial therapy (p < 0.001). hospital admission was required in 46, 57, and 17% of patients (p < 0.001). clinical success rates of initial therapy were 61, 63, and 84% (p < 001), and recurrences were more common in the ca-mrsa group, (18, 0, and 6%, p < 001). characteristics associated with outcome are listed in table 1 . in multivariate analysis, presence of mrsa and diabetes were predictive of clinical failure.conclusion: in the community setting, mrsa infections are associated with an adverse impact on outcome compared to mssa infections and patients with ca-mrsa are significantly less likely to receive adequate antibiotic therapy. microbiological analysis of root canals associated with periapical abscesses and the antimicrobial susceptibility of isolated bacteria s. ozbek, a. ozbek, m. koseoglu, s. evcil, a. erdogan, a. ayyildiz (erzurum, tr)objective: the periapical abscess is a collection of pus in the pulp or around the root of teeth. many odontogenic infections can be managed without antimicrobial therapy or bacteriologic investigation. however, when an acute bacterial infection has progressed or antimicrobial therapy might be of benefit to patients, antibiotics are prescribed. we aimed to identify microorganisms in root canals with periapical abscess and the antimicrobial susceptibility profile of them and to revise antimicrobial treatment protocols when antimicrobials is used empirically. methods: 30 patients with odontogenic infections included in this study. the microbiologic investigation was performed under strict aseptic conditions. a standardize routine of root canal therapy was instituted, and in each case a single root canal was sampled. in multirooted teeth only the largest canal was sampled to preserve the identity of a single endodontic/ microbiologic ecosystem. for microbial sampling, two sequential paper points were introduced into the full length of the canal, and kept in place for 1 min. one of the paper points was used for aerobic culture and the other one for anaerobic culture. to identify isolated bacteria, whole bacterial fatty acid profiles were evaluated by using microbial identification system. antimicrobial susceptibility results were obtained by disc diffusion test for aerobics, and e-test for anaerobics. results: totally 156 bacterial strains were isolated. 86 of them were aerobic and 70 of them were anaerobic. 18 or 60% of cultured specimens yielded mixed (aerobic and anaerobic) species. the most prevalent bacteria were staphylococcus spp. as aerobic, peptostreptococcus prevotii and streptococcus morbillorum as anaerobic. conclusion: beta-lactam antibiotics combined with beta-lactam inhibitor (amoxicillin-clavulanic acid) had a quite effect on gram (+) and (-) aerobics. when we take into consideration that beta-lactam antibiotics stimulate production of beta-lactamase, amoxicillin-clavulanic acid combination appears a good first step antimicrobial. clindamycin may be second alternative for that purpose. for anaerobics, cefoxitin and metronidazol had well effect. although imipenem and piperasilin-tazobactam are perfect, they should not be first step of therapy. due to the frequency of mixed infections, a combination of amoxicillinclavulanic acid and metronidazol or a combination of clindamycin and metronidazol considered to have well effect for mixed infections. clinical microbiology and infection, volume 12, supplement 4, 2006 study is to review the spectrum of p. multocida infections in our centre. methods: we studied the medical records of all patients who had positive cultures for p. multocida between 1993 and 2004. demographic, epidemiological, clinical and microbiological data including age, sex, animal exposure, site of infection, underlying diseases, type of therapy and outcome were evaluated. all isolates were identified by standard conventional microbiological methods. antibiotic susceptibility testing was performed by the disk diffusion method onto muller-hinton agar supplemented with 5% sheep blood and the mics of the antibiotics tested were determined by the e-test method. results: thirteen cases of p. multocida infections were diagnosed during this period. the male to female ratio was 10:3 and most patients (62%) were >70 years of age. respiratory tract infections were most commonly encountered (61.5%), followed by soft-tissue infections (30.8%) and septicemia (7.7%). underlying disease was present in 8 (61.5%) patients. among them, 4 presented a kind of malignancy. bullous pemphigoid, mitral valve stenosis, coronary disease, chronic obstructive pulmonary disease, and intracranial haemorrhage served also as predisponding factors. a traumatic animal exposure was reported in only 3 patients and non-traumatic in 2 cases. all isolates were susceptible to beta-lactams (penicillin, amoxicillin, amoxicillin/clavulanic acid, cefepime, cefuroxime, ceftriaxone, imipenem, and meropenem), quinolones (ciprofloxacin, norfloxacin, levofloxacin, and sparfloxacin), chloramphenicol, tetracycline, trimethoprim/sulfamethoxazole and 54% were intermediately resistant to aminoglycosides (gentamicin). appropriate antibiotic therapy was administered to all patients and a clinical response was observed in 10 (77%) of them. mortality rate was 23%. conclusions: pasteurella multocida must be considered as a possible etiology for a variety of infections, even without an obvious animal exposure. although this organism is susceptible to a large spectrum of antibiotics, a failure to treatment may be recorded especially in severe infections and in compromised patients. infections caused by nocardia cyriacigeorgici in zaragoza, spain: identification and antibiotic susceptibility c. villuendas, b. moles, v. rodriguez-nava, a. couble, f. laurent, m. revillo, p. boiron (zaragoza, es; lyon, fr)objectives: nocardia species known to date differ in their clinical presentation, antibiotic resistance patterns and geographic distribution. nocardia cyriacigeorgici is a recently described species.the aim of this study is to analyse the identification results, antimicrobial susceptibility together with the clinical data, of 13 n. cyriacigeorgici clinical isolates, recovered from 1998 to 2004 in our laboratory. methods: identification of nocardia spp. isolates was achieved in our laboratory on the basis of the following: visualization of the colony, gram stain and parcial acid-fast positivity by modified acid-fast staining, casein, xanthine and tyrosine hydrolysis, opacification of middlebrook 7h10 agar, production of arylsulphatase after 14 days incubation and antimicrobial susceptibility pattern.identification at species level was achieved by 16s rdna gene sequencing (laboratoire de mycologie. faculté de pharmacie. lyon. france)antimicrobial susceptibility tests included commercial broth microdilution (emiza 9 ef sensititre ò ) and gradient strip agar dilution (e-test ab biodisk ò ). interpretation of results was done according to nccls standard guidelines. in the six years of study, 129 isolates of nocardia spp. were recovered, 13 of them belonging to n. cyriacigeorgici species (10%). n. cyriacigeorgici represents the third species in frecuency in our serie, after n. abscessus and n. farcinica. the 13 strains were recovered from 13 patients, 12 from respiratory specimens and one from blood-culture.pneumonia was the most frequent clinical manifestation, being copd and previous corticosteroid therapy the most common predisposing conditions. all n. cyriacigeorgici isolates showed susceptibility to: amikacin, tobramycin, cefotaxime, imipenem, trimethoprimsulfamethoxazole and linezolid, and resistance to: amoxicillinclavulanic acid and ciprofloxacine. conclusion: n. cyriacigeorgici is not an infrequent cause of nocardiosis in our geographical area. the uniformity showed in the antimicrobial profile can be useful for its identification. in our hospital, patients with copd and receiving corticoid therapy is the most important group of risk for adquiring n. cyriacigeorgici infection. whit the technics available in our laboratory the isolates were identified as nocardia spp. and identification at species level was only possible by phylogenetic analysis using 16 rdna sequencing. high frequency of single-step resistance mutations in nocardia farcinica exposed to quinolones u.s. jensen, j.d. knudsen, k. schønning (hvidovre, dk)objectives: nocardia farcinica infections often require prolonged antibiotic therapy and perorally administered agents are desirable. isolates commonly display in vitro susceptibility to quinolones when tested by disc diffusion methodology. in the present study, we investigated the activity of three different quinolones (ciprofloxacin, levofloxacin and moxifloxacin) against n. farcinica and assessed the robustness of their activity by determining the frequency of single step resistant mutants when exposed to inhibitory concentrations of quinolones. methods: 10 isolates of n. farcinica were used in the study; correct identification to the species level was verified by 16s rdna sequencing. mics of ciprofloxacin, levofloxacin and moxifloxacin against n. farcinica as well as s. aureus atcc 25923 and e. coli ccug 17620 were determined by the agar dilution method using inocula of approximately 10.000 cfu and 48 h of incubation. single step mutation frequencies were determined by heavily inoculating selective agar plates containing quinolone at a concentration of 4x mic and counting resistant colonies after 5 days incubation. inoculum was quantified by seeding a dilution series of the inoculum employed on unselective plates and counting colonies after 48 h of incubation and frequencies were calculated by dividing the number of resistant colonies by the number of cfu present in inoculum. results: when mics were determined by agar dilution method all quinolones displayed roughly the same potency against n. farcinica isolates (mics between 0.25 and 4). as expected moxifloxacin were the most potent quinolone against s. aureus. however, all three quinolones selected for single step resistant mutants, the frequency of which was higher for ciprofloxacin (~10 )6 ) than for levofloxacin (10 )7 -10 )8 ), which again was higher than for moxifloxacin (10 )8 -10 )9 ). however, even for moxifloxacin the frequency against n. farcinica was comparable to the single step mutation frequency of ciprofloxacin against s. aureus (10 -9 ).conclusions: although quinolones may exhibit activity against n. farcinica, n. farcinica is capable of rapid development of resistance. therefore, quinolones should probably be avoided, at least as single agents, in the treatment of nocardia infections. correlation between clinico-laboratory findings and a positive igm elisa test for leptospira: a retrospective study e. mendrinou, p. goudas, a. regli (patra, gr) objective: to correlate a positive elisa test for igm antibodies against leptospira with the clinical and laboratory findings in patients with suspected leptospirosis. method: we retrospectively analysed the history, clinical course and laboratory findings in a total of 420 patients, with suspected leptospirosis. all patients fulfilled the criteria for clinical diagnosis of leptospirosis. from the 420 patients, 21 had to be transferred to the dialysis unit for haemodialysis and 6 patients had to be admitted to the intensive care unit (icu) due to severe pulmonary haemorrhage. serum samples from all patients were tested for igm antibodies against leptospira. results: from the total of 420 patients death occurred to only four, due to respiratory failure from severe pulmonary haemorrhage. the rest of the patients recovered completely. from the total of 420 patients 25 had a positive elisa igm test for leptospirosis (5.95%). however, from the 21 patients that were transferred to the dialysis unit, 8 had a positive leptospirosis test (25%) and from the six patients admitted to the icu, three had a positive test (50%). among other laboratory findings there was a stronger correlation between very low platelet levels (<30.000 mm3) and very high blood bilirubin levels (>20 mg/dl) with a positive test for leptospirosis. all 25 patients with a positive test had less than 30.000 platelets per mm3 and 18 had blood bilirubin over 20 mg/dl. the differential diagnosis of icterohemorrhagic fevers includes a vast number of pathogens, some of which are untraceable with the common laboratory methods. in our study, from the total of 420 patients, only 5.95% had a positive test for leptospirosis. in 92 of the rest 395 patients, many different pathogens were traced, most of them being several kinds of viruses (cmv, ebv), brucella and coxiella. in 303 of the patients no pathogen was traced. conclusions: taking into consideration the high sensitivity of the elisa test we conclude that: 1. icterohemorrhagic leptospirosis comprises only a small subtotal of icterohemorrhagic fevers; 2. there is a correlation between higher levels of bilirubin and/or very low platelet levels with leptospira infection; 3. there seems to be a correlation between leptospira infection and severity of icterohemorrhagic fevers. evaluation of continuous ambulatory peritoneal dialysis-related peritonitis attacks in ankara s. tekin koruk, m.a. yetkin, i. koruk, f.s. erdinc, s. sahan, n. tulek, m. duranay, a.p. demirö z (ankara, konya, samsun, tr) objectives: peritonitis is a common clinical problem that occurs in patients with end stage renal disease treated by peritoneal dialysis. the aims of this study were to assess demographic aspects, rates of peritonitis, causative organisms, clinical outcomes and treatment approach for continuous ambulatory peritoneal dialysis (capd) -related peritonitis cases. methods: seventy cases of peritonitis occurred in 55 patients treated in infectious diseases and clinical microbiology department between may 2003 and april 2004 were enrolled into this study. the mean age of the patients was 48.4 years (range 16-77 years). cloudiness of the peritoneal dialysis fluid and/or abdominal pain were considered suggestive of peritonitis and were confirmed by cell count and culture. baseline cell count, gram stain, and cultures were obtained, and repeated with periodic follow-up. results: the overall incidence of peritonitis was 2.5 ± 2.5 episodes/patient-year. in 52.7% of patients there were only one peritonitis attack, where as in 47.3% of them had two or more attacks. age, gender, education and profession of the patients have not been found as a risk factor in peritonitis attacks.the most common presenting symptoms of the patients were abdominal pain, cloudiness of the peritoneal dialysis fluid, nausea and vomiting. peritoneal dialysate fluid white blood cell count was 1773 ± 1224/mm3 in 70 episodes. cultures were positive in 51 (72.9%) peritonitis episodes; coagulase-negative staphylococci was the most common organism (%22.8), followed by staphylococcus aureus (%21.4), 19 episodes (%27.2) had negative culture results. there was a statistically significant decrease in serum crp and esr levels and at the end of the treatment when compared with the levels on admission.at the end of the study, 61 episodes of 70 peritonitis cases were treated with ip cefazolin and gentamicin protocol. seven of the patients did not respond initiate therapy and the therapy was converted to iv protocol. seven episodes were treated with iv antibiotics on admission for medical reasons (systemic infection and/or concurrent exit-side or tunnel infection). there were two deaths. two catheters were removed and the patients were transferred to haemodialysis programme. conclusion: despite all technical improvements during recent decades peritonitis is still the major complication of capd. for the accurate treatment of complications, causative organisms and their antimicrobial susceptibilities must be known. objective: viruses are a frequent cause of upper respiratory tract infections in children. among the respiratory viruses, influenza viruses are known to cause outbreaks globally. the present study was carried out to identify the influenza virus serotypes causing acute respiratory infection in children attending univesity hospital in konya in turkey. methods: thorat swabs were collected from 117 acute viral upper respiratory infection suspected children attending the out patient clinic of meram medical faculty hospital. two swabs were taken fron each chidren and one of the swabs was used for bacteriological cultures and if these were negative the other one was used for viral diagnosis. totally 100 bacteriological cultures negative swabs were investigated by real-time pcr for the presence of parainfluenza 1, 2 and 3, influenza a and b. results: one or more viral pathogens were detected in 24 children, with parainfluenza 1 9% being the most commonly identified virus. parainfluenza 2 in 7% and parainfluenza 3 in 5%, influenza a were identified in 5% and influenza b in 1%. from the specimens of 3 children more than one virus detected. conclusion: the influenza viruses cause morbidity and mortality among children and elderly. this study analysed the occurrence of influenza and paranfluenza respiratory ifections due to influenza and paranfluenza viruses. molecular methods used directly on clinical material have an important role in the rapid diagnosis and surveillance of influenza viruses and can be applied in clinical practice for correct diagnosis and administration of effective treatment. 1, 2, 5, 6, 7, 8 and 35 . the demographics, clinical presentations and laboratory findings of the 147 patients with serotype 3 were presented. results: the mean age was 4 y7 m, ranging from 5 months to 12 y2 m. seventy percents of children with serotype 3 infection clustered between october 2004 and january 2005. the mean duration of a positive culture result was 8.5 days. the mean duration of fever was 6.0 days, with 4 days before admission. forty (27%) children were treated as outpatients. the mean length of hospital stay was 5.4 days. the 3 most common diagnoses were exudative tonsillitis (25%), pneumonia or bronchopneumonia (24%) and pharyngoconjunctival fever (12%). the 3 most common symptoms and signs were fever (100%), cough (88%) and coryza (74%). neurologic complications were noted in 2 children. eighteen children had documented coinfection (including virus, bacteria and mycoplasma pneumoniae). leukopenia (wbc < 5000/microliter) was noted in two of 111 cases while leukocytosis (wbc > 15000/microliter) in 32 (29%). six (5.5%) of 110 cases had a normal serum c-reactive protein (crp) level (<10 mg/l), while 80% of 110 children had a serum crp greater than 40 mg/l. seventy (47.6%) of 147 children ever received antibiotics therapy. the outcomes were excellent in these cases. conclusion: recognizing that children with adenoviral serotype 3 infection may present with prolonged high fever, leukocytosis and elevated crp, which mimics bacterial infection, the clinician may not prescribe unnecessary antibiotics for these children. the infectious mononucleosis like syndrome (im) is an acute febrile disease of older children and young adults, and is characterized by lymphadenopathy, tonsillitis, splenomegaly, liver dysfunction and by the presence of peripheral lymphocytosis with >10% atypical lymphocytes. epstein -barr virus (ebv) is responsible for over 90% of the cases, cytomegalovirus (cmv) for 5%-7% and toxoplasma gondii <1%.herpes simplex, rubella and adenovirus are rare. the infection is usually characterized by mild symptoms. however in some cases the clinical manifestations may be rather atypical and severe. objective: to determine the prevalence of im like syndrome among patients in a children's hospital and its possible association with etiologic factors, age, major symptoms and atypical manifestations. material and methods: during a one-year period (january to december 2004) a total of 700 samples were examined in our laboratory. the study population was children between 1-15 years old, which either examined in the outpatient's clinic or hospitalized. all serum specimens were examined by1. indirect immunofluorescence for the presence of igg and igm antibodies against the viral capsid antigen (vca) ebv, 2.immuno chemistry luminescence for the detection of igg and igm antibodies to cmv and 3.eia for the detection of igg,igm abs of herpes simplex i and ii and toxoplasma gondii. results: of the 700 children examined 43 (6.1%) were found positive for igg and igm vca antibodies and 28(4%) showed positive specific igg and igm antibodies for cmv. these patients had one or more of the primary following symptoms: fever (87%), lymphadenopathy (80%), pharyngalgia (50%), cough (15%), skin eruption (9%). atypical manifestations as meningoencephalitis were found in two children one aged 20 months (caused by ebv) and the other of 7 years old (caused by hsv i) confirmed by pcr. the laboratory data showed positive serology for ebv and cmv infection, the existence of atypical lymphocyte (76%), ldh, asat and alat were moderately elevated (44 %) and crp increased (18%). conclusion: the frequency of im like syndrome in greece, though it's relatively low, it's not rare. the above results suggested that ebv, cmv, hsv should be considered in any young patient with im and acute neurological illness of uncertain etiology. objectives: enterovirus, parvovirus b19 and human herpes virus type 6 (hhv-6) are a common cause of infection in young infants. the objective of this study was to determine what portion of the infants who received a clinical diagnosis of febrile syndrome have a viral etiology by these three genera of viruses. methods: ninety-six patients were included in the study, all of them were admitted to the pediatric casualty of a tertiary care hospital, and all of them presented a febrile syndrome without a clear focus of infection (urinary tract, lung and meningeal infections were discarded). the assay was carried out in 96 blood samples by real-time pcr. dna was isolated from 200 ll of blood by semi-automated system magna pure lc total nucleic acid isolation kit (roche diagnostics, nederland bv). pcr was performed in a lightcycler instrument (roche molecular biochemicals) by a uniform cycling parameters: 10 min at 95°c for polymerase activation, and 50 cycles of 15 s at 95°c and 60 s at 60°c for amplification of the specific target sequence (5 utr gene for enterovirus, vp2 gene for parvovirus b19 and dna polymerase gene for hhv-6). pcr product formation was detected continuously by the use of taqman probes. results: a viral amplification was detected in 52 (54%) of the 96 patients included in this study. enterovirus was detected in 12 (12.5%) of the patients, parvovirus b19 in 10 (10.4%) and hhv-6 in 35 (36.4%). in five cases two viral amplifications were detected at the same time: 3 parvovirus b19/hhv-6 and 2 enterovirus/hhv-6. the mean age of the patients was 2 years old (range from 24 days to 14 years). in group of infants <6 months old (n = 27) there were 7 enterovirus and 6 hhv-6. in the infants from 7 months to 3 years old (n = 53) there were 3 enterovirus, 3 parvovirus and 24 hhv-6. in the last group of infants >3 years old (n = 16) there were 2 enterovirus and 7 parvovirus b19. conclusions: viral infections are an important cause of sepsis in infants admitted to hospital. enterovirus was the most frequent virus detected in infants <6 months, parvovirus b19 the most frequent in children >3 years old, and the hhv-6 was detected in all age groups. qualitative real-time pcr in blood is a rapid and sensitive method for diagnosis of enterovirus and parvovirus. however, is not the better method for diagnosis of hhv-6, a latent virus, in which this technique is not capable of distinguish between recent and acute infection. objectives: group a rotaviruses are a major cause of acute gastroenteritis in infant and young children worldwide. in this study, the molecular epidemiology and clinical features of rotavirus infection in iranian children was investigated. methods: between february 2004 to january 2005, thirty hundred and seventy two diarrhoea stools from children under 5-years-old with acute diarrhoea that attended the biggest paediatric hospital in tehran (iran), were analysed using elisa, electropherotyping and reverse transcriptionpolymerase chain reaction (rt-pcr). results: ninety-four samples (25.3%) were positive for the presence of rotavirus either by page, elisa, or both. according to page, the predominant electrophoretic pattern detected was the long profile 62 of 67 (92.5%) followed by the short electropherotype five of 67 (7.5%). out of the positive samples, 49 were further characterized by rt-pcr typing assay for identification of g types, resulting in 29 strains of g1 genotype while 20 samples could not be assigned a g type. all of g1 genotypes had a long rna electropherotype. among the patients with rotavirus infection, 23 (24.5%) required hospitalization. watery diarrhoea (92.6%), vomiting (73.4%) and fever (64.9%) were significantly more frequent in children suffering from rotavirus gastroenteritis. seven out of 94 rotavirus-positive patients had severe dehydration (p < 0.0001). rotavirus infection mostly affected children under 2 years of age with a peak incidence of 40% in children 1-2 years of age and it occurs year round with a seasonal pattern: more frequently during winter (46.2%). conclusion: this study revealed that rotavirus is an important etiological agent of acute gastroenteritis in tehran. we found that a major proportion of the specimens were untypeable. improved detection and characterization of incompletely typed strains will help to develop comprehensive strain information that may be required for tailoring effective rotavirus vaccines. serological study of prevalent rotaviruses in tehran e. habibi, s. ghorbani, a. jarollahei, z. habibi (tehran, zanjan, ir)objectives: rotaviruses are icosohedral and non-enveloped viruses that belong to reoviridae family which consist of three layers of protein surrounding 11 segments of dsrna. rotavirus is one of the most important agents of acute gastroenteritis in children. in this survey, the most prevalent serotypes in tehran and seasonal distribution in a year were detected. methods: in this study, a total number of 180 specimens of faecal samples of children and infants with acute gastroenteritis were collected from two children hospitals in tehran. the samples were tested by elisa procedure. serotyping investigation of iranian rotavirus isolates, using 7 serotypes monoclonal antibodies (g1-g2-g3-g4-g6-g8-g9) in elisa tests and immunosorbent electron microscopical studies using trapping and decoration techniques were performed. results: rotavirus type a infection was identified in 66 samples (37%). serotyping investigation in elisa tests proved that serotypes g1 and g4 were the most common serotypes circulating among infected children and infants in tehran. by electron microscopic studies the characteristic of rotavirus particles were observed in the faecal samples of infected children. the maximum incidence of infection was determined to occur among the cold months of the year. conclusion: it was approved that g1 and g4 serotypes are the main rotavirus serotypes present among children in tehran. it was detected that rotavirus diarrhoea was most prevalent among children of under 3 years of age. results: from 447 patients with varicella 19 presented neurological manifestations (sex ratio m/f:10/9). 18 had acut cerebellar ataxia and one had encephalitis. we estabilished the diagnosis on the basis of clinical aspects (including neurological examination), cerebrospinal fluid examination and electroencephalogram. the age interval was between 7 months and 30 years. most cases were diagnosed in children and teenager (17); one case toddlers, and 2 cases in adults. neurological manifestations appeared in most cases among 7 and 10 days after the onset of rash (15 cases). in the order of frequency: gait disorders (15), cerebellar ataxia (11), fever (7), vomiting (5), nistagmus (3), seizures and coma (1) . csf showed limphocytic pleiocytosis and elevated levels of protein (10 cases); in 9 cases csf had normal aspects. electroencephalogram had dominant theta wave with totally or partially suppression of alpha activity in all patients. all cases showed clinical and eeg improvement at the end of the treatment. conclusions: the most frequent neurological manifestation was cerebellar. the evolution was good under treatment, with no sequelae at 1 month of follow up. key: cord-023369-xwclh6ih authors: kim, faith; reichman, victoria; hooven, thomas a title: human herpesvirus-6 meningitis in a premature infant with fevers: a case and literature review date: 2020-04-18 journal: clin med insights case rep doi: 10.1177/1179547620912952 sha: doc_id: 23369 cord_uid: xwclh6ih human herpesvirus-6 (hhv-6) is a common virus that can cause nearly universal infection in infancy and early childhood. it typically manifests as an acute febrile illness. we describe a case of a premature infant with congenital hydrocephalus secondary to aqueductal stenosis with a ventriculoperitoneal shunt in place who developed intermittent fevers while she was admitted to the neonatal intensive care unit. she was ultimately diagnosed with acute hhv-6 meningitis. in addition to this report, we present a literature review regarding this virus’s potential modes of transmission and forms of clinical presentation in the neonatal period. human herpesvirus-6 (hhv-6) is a ubiquitous dna virus that causes near-universal infection in childhood. similar to other herpesviruses, it can establish latency in monocytes and macrophages and is capable of reactivation. 1 there are 2 expressed variants including hhv-6a and hhv-6b, the latter more prevalent and known to cause roseola infantum or sixth disease, which is associated with 10% to 20% of acute febrile seizures in young children. 2 infants and children between 6 months and 3 years of age acquire primary hhv-6 infection following the loss of protective maternal antibodies with seroprevalence of hhv-6 reaching more than 80% in children greater than 2 years of age; most have evidence of infection by 12 months of age. 2, 3 there is a wide clinical spectrum of hhv-6 infection ranging from asymptomatic disease to more serious disease including neonatal hepatitis, infectious mononucleosis-like syndrome, hemophagocytic syndrome, or viral myocarditis, particularly in the immunocompromised population. 4, 5 although infrequent, primary hhv-6 infection is associated with neurological disease including meningitis and encephalitis that can lead to longterm neurologic sequelae and poor outcomes. congenital hhv-6 infection, which is defined as presence of hhv-6 dna in an infant at birth, has been previously reported in approximately 1% of cord-blood samples by hall et al. 6 however, congenital infection could originate from multiple sources-one of which is by hereditary transmission of chromosomally integrated hhv-6 (cihhv-6), which has recently become recognized as an inherited condition with a unique mode of transmission. 7, 8 although the majority of hhv-6+ cases are due to latent cihhv-6, a significant subset of hhv-6+ neonates acquire active infection transplacentally, usually from mothers with cihhv-6 that has activated and produced replicating virions during pregnancy. 7, 8 the significance of this virus along with its potential neurologic predilection and reactivation capabilities remain unclear in the neonatal period. we report a case of a premature infant with congenital hydrocephalus and a ventriculoperitoneal (vp) shunt in place who presented with intermittent fevers ultimately found to have acute hhv-6 meningitis. a baby girl was born at 33 weeks' gestation via tertiary repeat cesarean section. she was born to a 27-year-old woman who presented unregistered to our institution in active preterm labor with vaginal bleeding concerning for abruption versus premature rupture of membranes. prenatally, the fetus was noted to have severe congenital hydrocephalus and bilateral cleft lip and palate diagnosed at 17 weeks' gestation at a previous institution. she had a fetal brain magnetic resonance imaging (mri) suggestive of aqueductal stenosis. the mother had scant prenatal care with her last visit at 20 weeks' gestation. her initial labs included a negative urine toxicology screen on admission; her routine prenatal serology tests ultimately returned negative. the baby was born with apgar scores of 6 and 8 at 1 and 5 minutes, respectively. her birth weight was 2.8 kg with a head circumference of 43.5 cm (>99th percentile). her exam was notable for macrocephaly with bulging fontanelles, large bilateral cleft lip/ palate, nasal encephalocele, and bilateral microphthalmia. her neurologic exam was notable for diffuse hypotonia but spontaneous movement of her extremities. during her admission to the neonatal intensive care unit (nicu), she underwent a vp shunt placement on day of life 2 and a gastrostomy tube placement on day of life 19 given her inability to feed by mouth with 2 clinical medicine insights: case reports her severe craniofacial anomalies. she was extubated to room air following each surgery with no difficulties and was tolerating full enteral feeds awaiting placement in a long-term care facility, formal adoption, and staged cleft repair at 3 months' corrected age. her medications included vitamin d and iron supplementation. in terms of a genetic work-up for her underlying anomalies, she had a negative microarray and fluorescence in situ hybridization (fish) analysis with no further genetic testing performed. on day of life 23, she had an axillary temperature of 38°c, pulse of 178 per minute and blood pressure of 73/35. her exam at the time of the first fever was notable for tachycardia, cool and clammy distal extremities, and overall fussiness but an otherwise unchanged neurological exam. her gastrostomy tube and vp shunt sites were not suggestive of a skin infection, and her respiratory status was stable. blood and urine cultures were drawn, a respiratory viral panel was negative, which tests for common respiratory viruses and bacteria via molecular identification at our institution (table 1) . her initial complete blood count (cbc) demonstrated a mild leukopenia with white blood cell (wbc) count 5.7 × 10 3 ul −1 , hemoglobin 8.5 g/dl, mean corpuscular volume 99.2 fl, and platelets of 417 × 10 3 ul −1 . manual differentiation showed 46% segmented neutrophils, 2% bands, and 45% lymphocytes. a repeat cbc on her fifth day of fever demonstrated leukocytosis with increased wbc count 21.9 × 10 3 ul −1 and elevated platelet count 584 × 10 3 ul −1 with 53% segmented neutrophils, 3% bands, and 33% lymphocytes on a manual differential. there were no atypical lymphocytes noted. her initial c-reactive protein (crp) was 28.3 mg/l, which peaked at 298.4 mg/l on her second day of fever. her basic metabolic panel was within normal limits. she had a chest radiograph that did not show a focal infiltrate. she was initiated on vancomycin and ceftazidime at meningitis treatment doses. she continued to have intermittent fevers as high as 39°c multiple times per day for the next 6 days and would defervesce each time with acetaminophen. with her fevers she would become tachycardic and diaphoretic, but in between fevers, her exam was at her baseline. after 48 hours of negative blood and urine cultures, her antibiotics were stopped. she had increased nasal congestion and secretions, so a repeat respiratory viral panel was performed on her second day of fever but returned negative. on her fifth day of fever, her exam was notable for increasing irritability and more persistent tachycardia with elevated inflammatory markers, so a repeat blood culture was drawn and a lumbar puncture was performed. she was also given her first transfusion of packed red blood cells. her lumbar puncture demonstrated 36 ul −1 total nucleated cells, 4000 ul −1 red blood cells with manual differentiation of 91% lymphocytes, 2% neutrophils, and 6% monocytes. gram stain was negative and culture showed no growth. her cerebrospinal fluid (csf) glucose was 39 mg/dl, while serum glucose was 95 mg/dl and the csf protein concentration was 363 mg/dl. a biofire filmarray meningitis/encephalitis csf multiplex polymerase chain reaction (pcr) panel returned positive for hhv-6 ( table 2) . antibiotics were not re-initiated. after 6 days, her fevers resolved, and she was presumed to have hhv-6 meningitis but was clinically improving thus antiviral therapy was not started. she was monitored closely for seizures given her intracranial abnormalities. she had a routine electroencephalogram (eeg) 2 weeks earlier that did not demonstrate any definite seizures. she remained at her neurological baseline once she recovered from her viral illness. she was noted to have a diffuse maculopapular rash on her face, neck, and chest about 48 hours after her fevers resolved. she was transferred on day of life 57 to a long-term rehabilitation facility. we describe a case of a premature infant with multiple congenital anomalies including congenital hydrocephalus status post placement of a vp shunt, bilateral cleft lip and palate, bilateral microphthalmia, and nasal encephalocele who was found to have acute hhv-6 meningitis in the setting of intermittent fevers during her nicu course. the diagnosis of hhv-6 central nervous system (cns) infection was supported by her clinical presentation and detection of viral dna 3 in her csf from a lumbar puncture. in terms of risk factors for both a bacterial and viral infection, she had a vp shunt and a newly placed gastrostomy tube and had been admitted to an nicu for more than 3 weeks in the midst of the winter season. in the setting of her fevers, which are defined as a temperature ⩾38°c in the neonatal population, etiologies considered included bacterial pneumonia, viral upper or lower respiratory tract infection, urinary tract infection, bacteremia, cellulitis in the setting of 2 indwelling devices, and meningitis given her intracranial anomalies with an indwelling device in place. she had laboratory evidence of inflammation with leukopenia initially and elevated inflammatory markers with 2 negative blood cultures, urine culture, respiratory viral panels, and csf culture that was drawn 72 hours after cessation of broad-spectrum antibiotics. her csf demonstrated elevated nucleated cells with a lymphocytic predominance, a low glucose, and elevated protein concentration outside the range of normal in a neonate; however, these numbers are difficult to interpret given her elevated red blood cell count. because she had a shunt, we presumed she was at higher risk for acquiring bacterial meningitis; however, a viral etiology fit more with her clinical picture of a self-limiting illness with an improved fever curve and resolution of symptoms without reinitiating antibiotic therapy. in the literature, there are only a few cases of hhv-6 meningitis or encephalitis reported in infants, and to our knowledge, this is the first reported case of cns infection in a premature infant. huang et al 9 reported 2 cases of hhv-6 meningitis in a 4-month-old boy and 7-month-old girl who had typical courses of roseola infantum with high fever for a few days followed by a skin rash eruption with evidence of csf pleocytosis, serum lymphocytosis, and serum serological evidence of igm anti-hhv-6. it is well-established that passively transferred maternal antibodies gradually decrease until the lowest level is reached around 4 to 7 months after birth, so these infants were in the expected time period to manifest a primary infection. 1 in addition, sugimoto et al 10 reported 2 neonatal cases of exanthema subitum caused by hhv-6 in a 27-day-old full-term boy and a 14-day-old full-term boy who each presented with high fevers followed by a classic skin rash. they both had igm antibodies in the acute phase and pcr detection of hhv-6 dna in the serum at high copy numbers suggestive of a primary infection despite presence of preexisting maternal antibodies, which the authors isolated from both mothers. although classic descriptions of primary hhv-6 infection typically occur after 6 months of age, authors have speculated that the level of passive maternal antibodies may not be uniformly protective as in these previous cases. in a prospective study evaluating for incidence of primary hhv-6 infection as the cause of acute febrile illnesses, infants who acquired their infection in the first few months of life had lower mean antibody titers than infants who did not have a primary infection. 11 providers have speculated that the level of viremia targeted to blood mononuclear cells likely causes symptomatic infection once the level of passive maternal antibodies has declined or if the viral load is particularly high. multiple modes of transmission of hhv-6 have been described in the literature including both vertical and horizontal transmissions, as well as a unique mechanism secondary to chromosomal integration of hhv-6 (cihhv-6), which can remain latent or undergo transplacental passage in utero. the virus can become activated in immunosuppressed individuals as well as during pregnancy, although the exact mechanism behind reactivation remains unknown. both in vivo and in vitro studies have shown that certain drugs including steroids like progesterone, anti-epileptics, antibiotics, and even nonsteroidal anti-inflammatory drugs can activate the virus. 12,13 viral dna has also been detectable in vaginal swabs with an incubation period of about 10 days suggesting that horizontal transmission from mothers to babies is possible. 14 maternal-fetal infection with hhv-6 has also been described and may be linked with a higher rate of fetal loss. in one study, hhv-6 antibodies were assayed in 30 mothers with spontaneous abortions in the first trimester, and the authors found that 10% of the cohort were positive for hhv-6 igm antibody, while the hhv-6 antigen was detected in the majority of abortive villous tissue suggesting that viral infection could predispose mothers to fetal loss. 15 similarly, a group from the united kingdom investigated occurrence of viral infection in fetal death and detected viral dna in 34% of tissue samples including detection of hhv-6 and hhv-7 in 5 samples. 16 another group analyzed over a thousand samples from multiple sites including tissue biopsies for detection of hhv-6 by pcr and identified a case of primary hhv-6a seroconversion occurring in a young pregnant woman with subsequent transmission to the fetus and unfortunately a spontaneous abortion at 24 weeks. 17 in general, hhv-6b is transmitted through contact with infected oral secretions with previous detection of this strain in the oropharynx of asymptomatic adults thus representing a major source of transmission to young children; however, there is no known information about how hhv-6a is spread. 2 moreover, congenital infections detected as hhv-6 dna in cord blood have been identified as another source of transmission similar to congenital cytomegalovirus (cmv) infections. 17 but in contrast to cmv, the majority of congenital hhv-6 infections are thought to be secondary to chromosomal integration of the virus into different human chromosomes within the whole genome, which is a proven phenomenon. 18 in a prospective study that examined the frequency and characteristics of cihhv-6, 86% of infants with congenital infections were primarily from cihhv-6, while the remaining 14% were secondary to transplacental infections who did not inherit cihhv-6. 18 infants with congenital infection due to cihhv6 had evidence of high viral loads in the cord blood and detection of hhv-6 dna in hair follicles in both the infants and at least one parent. 18 the transplacental transmission was primarily from cihhv-6 mothers, while only a small proportion of congenital hhv-6 infections resulted from the activation of hhv-6 from a mother with inherited cihhv-6. in other words, they found no evidence of transplacental infections except from mothers with cihhv-6 who suffered a reactivation of their integrated virus during pregnancy. 8, 18 importantly, the identification of cihhv-6 in an infant does not rule out active transplacental infection because the chances of cihhv-6+ infants acquiring a transplacental infection should be the same as in infants without evidence of cihhv-6. studies have attempted to investigate the congenital transmission of active hhv-6 virus prior to the discovery of cihhv-6. one study analyzed cord-blood specimens for evidence of congenital hhv-6 infection in 799 random samples that were originally collected to assess for congenital cmv infection. 19 only 2 samples were repeatedly positive for hhv-6-specific igm antibody identified by enzyme-linked immunosorbent assay (elisa) method but were negative for hhv-6 genomic dna by pcr testing representing active infection at approximately two-thirds the rate of congenital cmv infection in this same group. 19 the authors speculated that congenital transmission of hhv-6 similar to cmv is plausible yet rare, but given the retrospective nature of this analysis there were no information regarding the clinical presentations of both infants. 19 however, if both these infants were asymptomatic, then the presence of hhv-6 igm in their blood would be indicative of intrauterine transmission of either reactivation of hhv-6 and/ or activation of hhv-6 in mothers with cihhv-6 resulting in transmission of replication virions. because breast milk is another mode of transmission for cmv, another study was done to assess for hhv-6 in breastmilk. the authors evaluated 120 randomly selected human breast milk samples and tested them for hhv-6 dna by pcr. however, none of the 120 specimens had evidence of hhv-6, suggesting no transmission through breastmilk consumption. 20 although there have been cases of neonatal hhv-6 infection characterized by fever and classic rash despite evidence of maternal antibodies, our patient who was otherwise considered immunocompetent developed a presumed cns infection due to hhv-6 at an early age. as previously mentioned, congenital infections are mostly asymptomatic, but in our patient's case, the question of mode of transmission was challenging to decipher. however, 2 studies have demonstrated that 100% of active infections investigated were acquired from mothers with cihhv-6 that had activated during pregnancy suggesting that transmission of activated maternal cihhv-6 is the primary cause of non-inherited congenital infection. 8, 18 thus, there are 5 possibilities of congenital hhv-6 infection in an infant like our patient: 1. she had cihhv-6 but no active infection; however, these patients would be asymptomatic with evidence of hhv-6 dna but no igm antibodies. 2. she had cihhv-6 with active infection from a cihhv-6+ mother who reactivated the virus during pregnancy and subsequently transmitted the active virus, but it is impossible to differentiate active infection in a cihhv-6 patient using pcr methods alone. 3. she did not have cihhv-6 but had evidence of active infection from a cihhv-6+ mother who reactivated and transmitted the replicating hhv-6 virus transplacentally. 4. she acquired it transplacentally from a mother reinfected with hhv-6 or whose latent hhv-6 reactivated with no evidence of maternal cihhv-6. 5. she acquired it postnatally from another person in the nicu about 2 weeks prior to the onset of symptoms. we do not know the viral state of the maternal hhv-6 dna to determine whether reactivation or transcription of viral genes from an integrated genome were a possibility or if this was an acquired primary hhv-6 meningitis with a high enough viral load that overwhelmed her passive maternal antibodies. we hypothesize similarly to previous authors that she may have been exposed to a significant amount of viral replication leading to viremia with clinical symptoms, or potentially her maternal antibodies were low to begin with given her manifestation of cns disease. the role of hhv-6 cns disease continues to be an area of ongoing investigation particularly given its ranges of manifestations from febrile seizures to meningitis, encephalitis, and demyelinating disorders as reported previously in the literature. 3 currently, the route of hhv-6 entry into the cns is unknown in the literature despite its association with a wide kim et al 5 variety of neurologic disease from meningoencephalitis to multiple sclerosis in adults. 21 using autopsy specimens, harberts et al 22 demonstrated that hhv-6 potentially infects the cns via the olfactory pathway with highest prevalence of the virus in olfactory issues and the nasal cavity. we speculate that our patient may have been more susceptible to transmission of the virus to her cns given her significant craniofacial anomalies and nasal encephalocele. in one prospective study that assessed complications of primary hhv-6 infection, seizures were the principle complication accounting for approximately one-third of first-time febrile seizures among children less than 2 years old. 23 although investigators have been unable to successfully culture hhv-6 from csf, it is often detected in csf and other bodily fluids by pcr, as in our patient. there have been conflicting results in the literature regarding the frequency of hhv-6 pcr identification in csf with one study reporting positivity in up to 70% to 90% of children who had neurologic symptoms during their primary hhv-6 infection. 3, 24 another study examined csf samples from 245 pediatric patients who underwent evaluation for possible sepsis or neurologic symptoms and tested them for hhv-6 dna by pcr and found evidence of it in 3 patients who were all less than 2 months of age. 3 in 2 of the patients who were full term and less than 1 month old, they presented with fevers and had evidence of csf pleocytosis defined in this study as >22 leukocytes × 10 6 l −1 . both were diagnosed with aseptic meningitis at discharge and found to have hhv-6 dna in their csf samples when tested retrospectively. the last patient was a former 24-week baby boy who developed positive blood cultures for candida on day of life 10 but had no evidence of csf pleocytosis with negative bacterial, viral, and fungal cultures and also tested positive for hhv-6 dna, which was thought to be an incidental finding with no clinical implications. the authors concluded that the clinical features of the first 2 patients and the absence of other identified infectious agents were secondary to hhv-6 as a cause of aseptic meningitis. however, similar to our case in which the patient's csf results may have been from contamination by blood, the presence and magnitude of a pleocytosis are difficult to interpret with the number of red blood cells. the authors also commented that it was difficult to say with certainty that the hhv-6 dna isolated from the csf was not due to contamination from peripheral blood. 3 in addition, the assay used to detect hhv-6 dna pcr is an important consideration. our institution routinely uses the biofire diagnostics filmarray meningitis/encephalitis pcr (mep) panel, which provides a rapid pcr-based detection in the csf of various bacteria and viruses compared to conventional diagnostic methods of culture and pathogen-specific pcr testing. one retrospective study compared the real-world performance of mep panel compared to conventional testing in 138 csf samples and reported an overall agreement of 96% and a mean time to hour diagnosis of 3 hours compared to 13 hours, respectively; however, the authors did comment that the mep panel can potentially detect persistent, reactivated, or cihhv-6 that may not be causing active infection and must be interpreted with caution in the right clinical context. 25 finally, congenital hhv-6 may be compared to congenital cmv in the literature as they are closely related, and while it is true that most cihhv-6 congenital infections are asymptomatic, at least 28% of congenital infections are the same as congenital cmv with active virus transmitted to the fetus during pregnancy. 8, 26 cytomegalovirus is an abundant virus that is known to cause neurodevelopmental disability including sensorineural hearing loss and other developmental disabilities. congenital cmv infection results from primary infection, reinfection or reactivation of latent virus in the mother compared to congenital hhv-6 infection, which does not appear to be commonly due to reactivation or reinfection in the mother. caserta et al 26 performed a prospective double-blind controlled study comparing infants with congenital hhv-6 infection versus those without infection using a set of neurocognitive assessments to determine whether hhv-6 has an impact on early neurodevelopmental outcomes similar to cmv. infants with congenital hhv-6 infection were defined as having hhv-6 dna present in their cord-blood mononuclear cells, and chromosomal integration was confirmed by detecting dna in hair follicle specimens. major findings included significantly lower scores at 12 months of age on bayley-mental development index scores in the congenital infection group even after controlling for covariates potentially linking hhv-6 infection and neurologic disease; however, there were no specific clinical manifestations identified at birth such as hearing loss. 26 although there are limited longitudinal studies regarding the potential impact on neurodevelopmental impairment in this cohort that has yet to be confirmed, hall study brings to light this important question particularly if there are progressive, detrimental effects over time. consequently, the question remains regarding the clinical significance of this finding in the neonatal population given this virus's potential pathogenic role in the cns. hhv-6 is very common in young infants accounting for 20% of visits among 6-to 12-month-old infants presenting to the emergency department for febrile illnesses, but the frequency of mild or asymptomatic primary infection as well as its longterm impact remains undefined. 11 in hall et al's 11 study, of the 160 patients diagnosed with a primary infection, 13% were under 2 months of age with a mean duration illness of 6 days and a higher rate of hospitalization compared to age-matched healthy infants. thus, it is important for clinicians to maintain a high index of clinical suspicion for viral infections in this population as well as its complications prenatally and postnatally. in one cohort, pityrasis rosea, which is a rash often associated with both hhv-6 and hhv-7, was noted in 57% of pregnant women who ultimately miscarried with evidence of hhv-6 dna in fetal tissue in 3 out of 4 stillborns, suggestive of an association between hhv-6 and potential fetal death. 27 it is still unclear about the role congenital hhv-6 infections play in the perinatal and neonatal period particularly when it 6 clinical medicine insights: case reports comes to neurodevelopmental outcomes; however, there does not appear to be a severe congenital hhv-6 syndrome unlike congenital cmv, which often occurs in women who acquire primary infection early in pregnancy. hhv-6 infection occurs in essentially all children within the first few years, therefore preexisting immunity in the mother may be protective against such severe disease in most infants. increasing use of pcrbased pathogen detection panels in the neonatal population may lead to more frequent diagnoses of hhv-6 and improved understanding of its epidemiology and natural history. in summary, we present a case of a premature infant with multiple anomalies who acquired acute hhv-6 viral meningitis in the setting of intermittent high fevers, elevated inflammatory markers, and diagnostic testing from her csf that confirmed the diagnosis. given her hydrocephalus and vp shunt, neurology and neurosurgical subspecialists will continue to follow her as an outpatient. with her intracranial abnormalities, she remains high risk for seizures. she also failed her hearing tests multiple times throughout her course likely as a complication of her intracranial and craniofacial anomalies; her prematurity and comorbidities place her at high risk for significant neurodevelopmental impairment now possibly increased following her hhv-6 cns infection. it is important to consider hhv-6 infection as an etiology of fevers even during the neonatal period as timely diagnosis may prevent further unnecessary treatment with empiric antimicrobials and diagnostic evaluations. there are several limitations of this case report. it is based on a single case instead of a case series given the low prevalence of this infection in the premature population. the patient also has multiple underlying craniofacial abnormalities with a potentially underlying genetic diagnosis that has not yet been uncovered making it difficult to generalize her case to other infants, particularly if she is predisposed compared to others. finally, there was a lack of confirmation in hhv-6 detection in other bodily fluids or serum due to unavailable routine testing at our institution by either culture, serology, or pcr that could have strengthened this finding of an active infection based on her clinical presentation. laboratory and clinical aspects of human herpesvirus 6 infections update on human herpesvirus 6 biology, clinical features, and therapy human herpesviruses 6 and 7 and central nervous system infection in children clinical impact of primary infection with roseoloviruses review part 3: human herpesvirus-6 in multiple non-neurological diseases congenital infections with human herpesvirus 6 (hhv6) and human herpesvirus 7 (hhv7) transplacental human herpesvirus 6 (hhv-6) congenital infection caused by maternal chromosomally integrated virus sequence analysis of transplacentally acquired human herpesvirus 6 dna is consistent with transmission of a chromosomally integrated reactivated virus meningitis caused by human herpesvirus-6 human herpesvirus-6 infection in neonates: not protected by only humoral immunity human herpesvirus-6 infection in children: a prospective study of complications and reactivation chromosomal integration by human herpesviruses 6a and 6b possible progesterone-induced gestational activation of chromosomally integrated human herpesvirus 6b and transplacental transmission of activated human herpesvirus 6b hhv6 and hhv7: persistence and vertical transmission hhv-6 infection during pregnancy and spontaneous abortion viral infection in hydrops fetalis, spontaneous abortion and unexplained fetal death in utero detection of hhv-6 in over a thousand samples: new types of infection revealed by an analysis of positive results chromosomal integration of human herpesvirus 6 is the major mode of congenital human herpesvirus 6 infection serological evidence for congenital transmission of human herpesvirus 6 examination of human breast milk for evidence of human herpesvirus 6 by polymerase chain reaction human herpesvirus 6 and neuroinflammation human herpesvirus-6 entry into the central nervous system through the olfactory pathway association of human herpesvirus 6 infection of the central nervous system with recurrence of febrile convulsions human herpesvirus 6 dna in cerebrospinal fluid specimens from allogeneic bone marrow transplant patients: does it have clinical significance? potential clinical impact of the film array meningitis encephalitis panel in children with suspected central nervous system infections early developmental outcomes of children with congenital hhv-6 infection evidence of human herpesvirus-6 and -7 reactivation in miscarrying women with pityriasis rosea we are grateful to the family of our patient, the clinical microbiology laboratory at morgan stanley children's hospital of new york-presbyterian, and members of the neurosurgery team who consulted on this case. written informed consent from the current legal guardian was obtained and is maintained by the primary author. faith kim https://orcid.org/0000-0002-0344-0613 key: cord-017534-0ai8chbu authors: andersen, bjørg marit title: background information: isolation routines date: 2018-09-25 journal: prevention and control of infections in hospitals doi: 10.1007/978-3-319-99921-0_21 sha: doc_id: 17534 cord_uid: 0ai8chbu the isolation of patients with suspected or documented infections—to not spread to others—has been discussed for hundreds of years. guidelines are many, methods are different, attitudes show vide variations, routines and procedures are still changing, regulations by law may be absent, and some healthcare professionals may be afraid of adverse outcomes of isolation [1–44]. microbes that are spread in the environment, on the hands and equipment are invisible. the invisible agent does not call on attention before the infection; clinical disease, hospital infection or nosocomial infection is a factum that can be registered [23, 28, 29, 35–37]. how to stop the transmission is often “to believe and not believe” in infection control. like luster in sogn and glitre in akershus or in separate isolation buildings at the hospital, like epidemic buildings at ullevål hospital. despite academic controversy about "infection or not" from the late 1700s to the 1860s, the isolation treatment was used for presumably "infectious diseases" like typhoid, cholera, scarlet fever, diphtheria, smallpox, plague, etc. patients were isolated in hospital, at home or in own local epidemic houses in the country. chronic carriers of typhoid bacteria (salmonella typhi) were isolated alone at home or at the counties epidemic house by themselves (smitteborg, "infection castle" in skreia, norway, is still a local name). the epidemic houses had access control and were monitored by the healthcare board mayor. hygiene and infection control were very important since they had few curative agents. it was prepared vaccines and carried out partly or complete mass vaccinations against some infections. pulmonary tuberculosis was treated at sanatoriums in the mountains or at coast hospitals. the patient did not come home until he was free from infection. during outbreaks of typhoid fever, diphtheria or scarlet fever, the patients were isolated in separate sick barracks or at home. to be a carrier of an infectious disease could lead to work prohibition and isolation. schools were closed intermittently during outbreaks, and posters were put on houses with infectious disease as a warning. disinfection of houses, clothing and all of its contents was completed after the termination of the isolation period, using chlorine and sulphur smoke. carbolic acid and hydrochloric acid were also used where sulphur fumes treatment failed. bedding, clothing and fixtures that could not be washed or disinfected were often burned, with a financial compensation for this "outlay." patients who broke the isolation routines could be penalized and committed to further isolation. the society's need went first before each individual's needs [46] . during the 20 years from 1911 to 1930, among approximately 2.4 million inhabitants in norway, there were a total of 2,146,702 cases of acute laryngitis and bronchitis, 470,099 with acute gastroenteritis, 181,752 with "croup pneumonia," 89,100 cases of diphtheria, 75,000 with scarlet fever, 71,000 with rheumatic fever, 15,000 with typhoid and paratyphoid fever, 5300 with poliomyelitis and 3200 with dysentery! [46] knowledge of infection, sanitation and housing conditions (less overcrowded), clean water, good personal hygiene, vaccinations and nutritional status were factors mitigating epidemic outbreaks beginning of the twentieth century. this demonstrates that most infectious diseases can be prevented and controlled, even without antibiotics. in 1937, before the breakthrough of the penicillin, there were the most important precautions against infectious diseases in norway: [1] isolation of the source of infection, [2] disinfection, [3] vaccination, [4] quarantine, [5] food hygiene and [6] eradication of insects [46] . airborne infection was estimated as "the most important form of spreading infections" which may be correct even today [46, 47] . the last 125 years of isolation treatment carried out in norway have been in separate houses and buildings until ca. 1960 when most infectious diseases were integrated into appropriate wards for the underlying diseases. in 1970 there was still isolate buildings where all kinds of infections were taken care of and with good routines. today, this type of isolation is gone, with exception of some buildings in preparedness for strict isolation. in each third to fifth hospital bed, there is an infected patient, and each third patient is treated with resistance-driving antibiotics today [28, 29, 35, 48] . isolation units are therefore of great need. in norway with 5.5 million inhabitants, ca. 900,000 patients are treated annually in hospitals and about 50,000 receive hospital infections, resulting in at least 450,000 extra days in hospitals because of infection. hospital infections with staphylococcus aureus occur in 2% of the patients [28, 29] . isolation treatment is laborious and expensive (ca. 10,000 nkr or 1400 usd per day). out of the 13,700 somatic beds in norway in 2013, hospital infections charged ca. 20% of these, which would generate a need for ca. 2740 infection beds. however, the somatic bed capacity in norway is steadily being reduced, which creates several problems in the healthcare. there is an increased need of isolates for patients with infections, especially due to pulmonary tuberculosis, mrsa, vre, clostridium difficile (cd), multiresistant gram-negative bacteria and other "multidrug-resistant organisms" (mdro) [23, 24, 36, 37, [48] [49] [50] [51] . global incidence of entero-pathogenic bacteria and imported resistant agents increases, and there is an ongoing microbial spread via food, animals, fish, water, supplies and the environment [48, 51] . new knowledge of transmission, robustness and ability to survive in the environment may reinforce the methods for isolation. cd, originally defined as contact transmission, may also spread through the air [52] . cd problem increases, particularly in the united states where half a million patients are infected each year and 29,000 of them died in 2011 [53] . a special dangerous cd type (nap1) is detected [53] . the incidence of new respiratory viruses like human metapneumovirus, bocavirus, human coronavirus, etc. increases. for the first time in more than 100 years, a highly pathogenic virus as ebola has been introduced to norway and to other countries in europe. pandemic-actual viruses such as sars, mers, different types of bird flu, new outbreaks of ebola and other viral haemorrhagic fevers show the need for isolation and emergency preparedness [30] [31] [32] [33] [34] . children's diseases, who disappeared with vaccinations, like whooping cough, mumps, rubella and measles, are now increasing with outbreaks in areas with high vaccination rate in america and europe [1] . from 2014 to 2015, europe had more than 22,000 measles cases [54] . by hospitalization, these need airborne infection isolation units. noro-, rota-and sapoviruses and other types of virus gastroenteritis are highly infectious with vomiting and producing of aerosols, i.e. airborne infection. growing global outbreaks of norovirus may cause fast and widely spread of gastroenteritis among patients and personnel in health institutions and cause inactivity and stop of new admissions [55, 56] . the closure of wards or hospitals. outbreaks often end with the closure of the ward, as shown in one study, median in 14 days [16] . geriatric wards are usually closed during outbreaks. the most frequent cause is outbreaks of norovirus, 44%, p < 0.001 and influenza/parainfluenza, 38.5%, p < 0.001 [16] . intensive care units (icu) are often particularly vulnerable to infection. crosscontamination between such patients is normal [57] . in a 16-bed icu for adults, 430 patients were followed for 3950 patient days and cross-transmission was followed by genetic typing of the microbes [57] . a total of 40 episodes of cross-transmission was demonstrated and dominated by pseudomonas aeruginosa, other gram-negative bacilli and s aureus. cross-transmission was associated with understaffing, use of nasogastric tube, ventilator and other multiple patient-to-nurse contacts and immune-compromised patients [57] . patients treated at understaffed icus were three times more likely to be infected; immunosuppressed had four times greater risk, and bronchoscopy-treated patients led five times higher risk [57] . intensive units often have no isolates, and if they have, they are not so much used because of staff shortages. airborne infection isolation units will quickly be economic by more control over the spread of infection, fewer extra days in hospitals and fewer sick leave for personnel, as was demonstrated during the sars epidemic [33] . the larger outbreaks of infectious childhood diseases require more isolation for airborne infections [58] . the same goes for whooping cough which 10 years ago was an unknown phenomenon in norway and now in widespread child and adult disease [59] . imported infections are increasing, and hospital infections like wound infections, respiratory tract infections and antibiotic-associated diseases are also increasing. in the 1980s, the state board of health in norway recommended that the number of isolation beds should be 10% of the hospital's total bed numbers and the icu 25% [60] . at ullevål hospital, 5% of the beds were airborne infection isolates, and 3% contact isolates in 2008 [22, 28, 29 ]. in 2009, a european investigation was done as regards the number of "high-level isolation rooms" (hirs), i.e. airborne infection isolation units with negative pressure (not defined) with at least 6 air changes per hour and sluice (anteroom) [61] . this is a unit intended for high-risk infections like haemorrhagic viral infection, sars, the starting phase of pandemic influenza, xdr (extended drug-resistant) tuberculosis, anthrax, plague, biological terrorism, etc. [61] in all, 16 european network countries (eunid) with 211 hospitals and about 1790 isolation beds participated. many of these had fewer than 6 air changes per hour [61] . the total proportion of airborne infection isolates (hir) per country were calculated per million inhabitants. the largest proportion of these isolation units were detected in luxemburg, 31.5 per million; followed by finland, 28 [61] . most countries used hepa filter for air extraction from the isolates (9 out of 11). among 13 countries, 4 had localized airborne isolates (hir) to own buildings or wards, in 4 was hir in the same ward as other patient rooms, and in 5 both solutions were adopted [61] . a total of 342 hirs were equipped for intensive care, most in italy (245 beds) and in denmark (40 portable beds) [61] . after year 2000, hirs have been used in europe to approximately 10 imported haemorrhagic fever cases and 33 confirmed sars cases. in the united states, there are three "high-level isolation units" (hliu), placed at the same level as laboratory with biosafety level (bsl) 3 and 4, in separate buildings, with limited and controlled access, negative air pressure and special treatment of sewage and garbage [62] . isolates rely on "evidence-based design," i.e. knowledge of the construction, design, materials, equipment, furnishing, standards, etc., which are considered as core elements of the infection prevention [63] . solid evidence of the spread of infection is used to control good environmental measures, disinfection and proper use of the isolates [64] [65] [66] . infection isolation involves admittance of the patient to a separate room with disinfection/bathroom, sluice or anteroom (see fig. 21 .1). it is always an advantage with some negative pressure of infection isolates-even for contact infection-to avoid spread of infections outside the isolate. a contact isolate has sluice, patient room and disinfection/bathroom, about. 35 m 2 . the sluice should be at least 6 m 2 with room for bed or stretcher and wash basin. it shall have a defined clean side, space for clean clothes and unused ppe and a dirty side, for infectious waste and infectious waste bags or containers. patient room should be at least 20 m 2 , with the head end against the wall and about 2 m free zone on both long sides and from the foot end, to avoid too close contact with the patient. disinfection room, combined with bathroom, 8-10 m 2 , contains shower, toilet and wash basin, with plenty of space to be able to help the patient as needed. it should also be equipped with decontaminator, preferably a well-sealed, throughput decontaminator from disinfection room to the sluice. in throughput decontaminators (or autoclaves), infected equipment can be put in from the disinfection room and removed decontaminated in the sluice. all throughput systems should be assured with complete sealing against air leakage. it should only be opened from one side at the time, i.e. interlock. avoid throughput cabinets between the sluice and disinfection room. all isolation unit surfaces must be smooth, robust and withstand disinfectants, both liquid and dry gaseous form. avoid tiles, gaps and joints where dirt can form the basis for growth and biofilms of microbes which may prevent effect of disinfection. there should be at least eight air changes per hour (> six air changes [61] ), which is important to remove the infectious agent in the room air. incoming air must enter on the wall or ceiling; air extraction shall go out by the floor (15 cm above), preferably in the disinfection room. the isolate should have separate ventilation system so korridor ute dekont. that the infection does not come across to other patients by setbacks in joint ventilation ducts. intake air should be a certain distance from the air outlet. hepa filters should be set on air outlet, and procedures for maintenance and control should be established. airborne infection isolate is provided and formed as for a contact isolate. the isolate should be organized for direct access from outside. in addition, the controlled and defined negative pressure is graded down from the sluice to the disinfection-bathroom compartment [22] . pressure is usually, −16 to −25 pascal relative to the corridor and the adjacent rooms. in the sluice there is 6-10 pascal, in the patient room −16 to −25 pascal and disinfection room −25 pascal. the air will then be drawn from the cleanest room (sluice) to the dirtiest room (disinfection room), if the doors are closed. the most stable, controlled negative pressure may be made by doors opened outwards from the patient room to the sluice and further from the sluice to the corridor (see fig. 21 .1). the vacuum will suck the door even tighter to the door frame to hinder air leakage. interlocked doors should be used so that only one door at a time may be opened into the patient room. the stability is dependent on the number of air changes per hour. the minimum and most stabile is 6 air changes per hour (6) (7) (8) (9) (10) (11) (12) [19, 22, 61] . each air exchange reduces the pathogenic agents in the air substantially. all air expelled exits at the floor (15 cm above) and is gathered into an outgoing ventilation channel from the disinfection compartment. avoid "shortcuts" of the air circulation. as for contact isolates, airborne infection isolates should have separate ventilation systems with no setbacks in ventilation ducts; external intake of air over the roof should be well away from the external air outlet over the roof, and all air from the unit is hepa filtrated. procedures for maintenance, disinfection and control of filters and ducts should be established. cohort isolation of several patients with the same infectious agent may be appropriate. it is a good initiative during pandemics. the sars epidemic released a rapid learning process where the chinese built-within a week-a dedicated sars hospital for 1000 patients in beijing [33] . in a study from brazil, all patients with proven mdros (multidrug-resistant organisms) were transferred to a 34-bed unit for isolation treatment by a trained personnel, where personnel, patients and family were weekly taught about how to handle mdro's by a special team [67] . after the intervention, the mdro rate was significantly reduced, specially by the reduction of vre. isolated patients had at least the same good treatment quality as not isolated patients [67] . in norway, cohort isolation was utilized in trondheim during the first national outbreak of mrsa (1970) (1971) (1972) (1973) (1974) . a total of 144 patients were infected (including 15 personnel), and 48 died (31.6%) directly or indirectly from the hospital epidemic [68] . the control of the outbreak came first when infected patients and personnel were cohort isolated in a separate isolation ward [68] . barrier care of one infectious patient among other patients without infections on the same bedroom in dormitory is not recommended. a good standard of patient isolates depends on good daily cleaning and a thorough disinfection by termination of the isolation. long-time surviving bacteria, viruses and fungi should not be a risk for the next patient to be isolated, which may happen [69, 70] . mrsa, multiresistant acinetobacter baumannii and other mdros are contaminating bedding, bed and environment for months, if not removed [71] . disinfection of rooms and isolates are often not well enough done [64] . the terminal disinfection, removal and disinfection of all equipment in the room and disinfecting of all surfaces are important. disinfectants (liquid) that are effective against all microbial agents are chloramine 5%, household bleach 10% or peracetic acid for 1 h. treatment with hot water (>85 °c) for 10 min is a very effective disinfection of equipment and textiles. different types of gas or steam disinfection (hydrogen-peroxide dry gas, chlorine gas, formaldehyde gas) may be effective for equipment and room disinfection [72] [73] [74] [75] . note that there is yet no gas treatment that is documented to kill mycobacteria [76] . the isolates are often used incorrectly. in the same ward and at the same time, patients without infection are placed on the isolates while infectious patients are not isolated. although defined isolate treatment is determined and ppe used, it turns out that the patient is walking freely around in the ward, not really isolated. knowledge and practices increases understanding of disease control measures. some patients that are both prone to infection and have serious infections, like cystic fibrosis patients, should be treated in isolates [77] . depression, anxiety and anger get less visits and information by health professionals; more often fall injuries or other injuries, inferior care, etc. are claimed to be side effects of isolation [41, 43] . however, patients previously depressed and anxious at admission suffer no more under an isolation stay [39] . there is not shown differences between isolated and non-isolated patient, according to the patient's view of treatment, on the contrary [42] . but the patient satisfaction is the highest among those who are well informed [38] . most studies show that contact time with the medical doctor is approximately equal for isolated and non-isolated patients and approximately equally frequent [40, 43] . although mrsa or vre infected patients, in one study, had more frequent falls and pressure ulcers, this was not reduced by removing the contact isolation, so that it was likely other factors which could contribute to such complications [78] . outbreaks are very costly and take up significant resources and healthcare for other patients [79] . a patient infected with mrsa was at ullevål university hospital, norway, estimated to generate an additional charge of 526,000 nkr (ca. $75,100), including extended length, spread to a hospital employee, screening tests and antibacterial treatment in 1999 [36] . in 2002, in connection with a mrsa outbreak in neonatal ward at ullevål, the calculated additional costs were 375,000 nkr (ca. $53,500) per mrsa-positive patient, including isolation expenditure, disinfection and cleaning, screening and extra days in the hospital [24] . active screening and isolation of patients with mrsa are significantly and markedly cheaper for the healthcare than uncontrolled transmission which can result in serious infection and death [80] . anchoring isolation procedures in laws is essential to the implementation of isolation. norway has several laws, regulations and guidance to control infections in healthcare facilities [2] [3] [4] [5] [6] [7] . international studies have laid the basis for the norwegian isolation measures and techniques [9-14, 17-20, 27] . a number of studies regarding the design and function of isolation units exist [25, 26, [63] [64] [65] [66] . simply to place patients in single rooms provides noticeable improvement in the spread of infection [81] . the implementation of good isolation measures and compliance is often weakened by confusion, lack of written procedures, lack of training, competence and understaffing. a number of other factors also influence the attitude of healthcare professionals with regard to follow the hospital's infection control procedures, not least professional disagreement about the procedures. • in emergency wards in the united states, only 45% of the wards used contact protection (gloves and gown) upon receipt of patients with diarrhoea or faecal incontinence, 84% for suspected cd, 49% for purulent skin infections, 79% for suspected mrsa and 63% by multiresistant gram-negative rods [21] . • it may be a big difference between what staff and others say they do and what happens in reality [82] . among 470 persons who entered isolates, the compliance was higher by strict isolation infections (65%) than with other infectious conditions [83] . visitors followed the measures better than personnel (88% versus 41%) [83] . the compliance with isolation procedures increased significantly when more time was spent with the patient, being a visitor, and when the patient was on a strict isolation [83] . the study pointed out the danger of increased demands for doctors and nurses due to savings can result in adverse effects such as reduced compliance with key routines as isolation [83] . morgan et al. observed 7750 visits of a healthcare professional to isolated and non-isolated patients. patients on contact isolation had 36% fewer visits and 18% less contact, and there were fewer visitors than non-isolated patients [84] . however, the staff implemented significantly more frequent handwashing after visit in isolates, 63%, than by visits with non-isolated patients, 47% [84] . • during the mrsa outbreak with ca 150 patients in trondheim, norway, 1970-1974, kvittingen et al. described problems that may occur during epidemic and endemic conditions as "doctors more or less wholehearted support and scepticism to the measures made by the hygiene committee, discussions concerning the virulence and resistance development, what to do when a local outbreak paralyzes the department completely, information problems, lack of qualified personnel, etc." [68, 85] . they stressed that "larger hospitals should dispose well-planned isolation opportunities, both for patients who are infectious spreaders and perhaps equally important, to protect patients with reduced infection defence," and that "personnel in management positions (should) provide effective and loyal support for infection control personnel" [68] . • this was a very important observation done for more than 40 years ago. it is still causing major problems during outbreaks and endemic situations when there may be a choice between shortcut thoughts about "economy" and infection control. still, impression and experience is that staff working directly with the patients have great respect for contact and airborne routines, at least in norway. isolation regimes and interpretation of these are an almost perpetual discussion which changes with endemic and epidemic conditions, economy and guesswork. although isolation of patients with infectious diseases has been used for hundreds of years, there is little evidence in terms of endemic conditions [19] [20] [21] [22] . the norwegian isolation regimes are based mostly on guidelines from cdc [9] [10] [11] [12] [17] [18] [19] [20] [21] . in addition, an increasing number of guidelines are focusing on special isolation factors [19, 64-66, 69-73, 86 ]. cdc's isolation guideline is graded from high evidence, highly recommended as the ia, to no recommendation and unresolved questions or professional disagreement [19] . category ic is included in the legislation for patients and caregivers [19] . classification into categories is essential for the most important infection control measures in hospitals. "these recommendations are designed to prevent transmission of infectious agents among patients and healthcare personnel in all settings where healthcare is delivered. as in other cdc/hicpac guidelines, each recommendation is categorized on the basis of existing scientific data, theoretical rationale, applicability and, when possible, economic impact" [19] . the cdc recommendations are changed over time [9] [10] [11] [12] [17] [18] [19] . cdc/hicpac system recommendations in categories in correspondence with investigations, theory and economic possibility; quoted [19] . cdc's isolation guideline refers to the administrative responsibility around isolation treatment [19] . the hospital's management is responsible for ensuring the implementation of good practical measures and to be the driving force in the hospital's infection control preparedness. this should particularly be done with regard to isolate patients, an extra burden, and also for the economy. enough isolate capacity is to be provided in relation to citizens, patient categories, type of hospital activity and endemic/epidemic conditions. the isolates must be of good quality and design, and it should be enough airborne infection isolation units [19] . some of the cdc-recommended administrative responsibilities are quoted here: [19] citation: "healthcare organization administrators should ensure the implementation of recommendations in this section: transmission-based precautions), establish processes to monitor adherence to those performance measures and provide feedback to staff members. category ib" [19] . citation ended. a good and active hospital management is of major importance to stop the outbreak [23, 24, 37, 49, 50, 68, 87, 88] . ransjø et al. attached the importance to the activity of the hospital management during outbreaks of multidrug-resistant microbes in sweden [87] . in norway, an outbreak of vre by a dialysis department spring 2011, at ullevål university hospital, was expected from the department's critical building condition [23] . after great effort from the staff, the outbreak stopped within 14 days [23] . a structural rehabilitation started then, with expansion of bed numbers, assisted by the hospital management. one year later, all patients were vre negative [23] . a number of outbreaks at ullevål's premature intensive care unit during years have been linked to chronic understaffing, overcrowding, part-time work, lack of infection control practices and isolates, lack of information and a lack of management responsibilities [24, 37] . the responsibility of the hospital management to prevent and combat outbreaks of infection should be clearly stated in the hospital's infection control program. cdc defines contact isolation, using gown and gloves when in contact with patients infected with resistant bacteria like mrsa and other mdros (multidrug-resistant organisms), and single rooms are recommended [19] . direct contact contamination occurs when microbes are transmitted from an infected person to another without the "intermediate stage" in the environment. microbes transferred to the hands can further spread to the own mouth, nose, hair, ears, clothing, etc. [89] healthcare personnel is likely to be a "source, vector or a victim" of infections like mrsa [90, 91] . patients bring their own microbes into the hospital, with often large contact before a known infection [92] [93] [94] [95] [96] . therefore, rapid tests are recommended where possible to detect pathogens [80, 97] . indirect contact contamination happens when the microbes are transferred to an object in the environment and from there transmitted to other objects or people. infectious patients themselves will have the agent on the body and clothes and transmit it to the environment. the undetected microbes can spread quietly and calmly in many directions via equipment, rooms, surfaces and missing/incorrect procedures [98] . this is often a large and persistent environmental problem and a threat to other patients [23, 24, 37, 49, 50, 57, [68] [69] [70] [71] [72] 87] . the most typical spread occurs when people with contaminated hands are depositing microbes on equipment, machinery, knobs, door handles, uniforms, other textiles, furniture, toys, etc. [89] [90] [91] fellow-patients on the same room are highly susceptible to infection [16, 20, 23, 24, 37, 57, [96] [97] [98] [99] . infection can be spread further via equipment (bp apparatus, stethoscope, medicine tray, blood sugar test equipment, journal, etc.) and the personnel's uniform. it is often brought to the ward office, to service rooms and to other personnel or patients. a long "domino" chain of infections may follow infectious agents not stopped by simple hygienic measures and isolation [98] . contact infection may also be an airborne infection from patients coughing and sneezing by certain aerosol procedures, when cleaning the surfaces, by bedding and by strong air currents (e.g. by rapidly opening and closing doors) which dislodges dust from the lamps, shelves, etc. [44, 46, 47, 52, [100] [101] [102] therefore, a daily good housekeeping and good routines for final disinfection in isolates is very important to reduce the burden and risk of pathogenic microbes [22, 74] . contact infection prevention (cp)-recommended by cdc [19] . some citations selected from siegel et al. 2007 ; cdc guideline for isolation, where recommendations are in categories where ia is strongest recommendation. selected citations: [19] " 1. in acute care hospitals, place patients who require cp in a single-patient room when available. category ib. 2. when single-patient rooms are in short supply, apply the following principles for making decisions on patient placement: ----place together in the same room (cohort) patients who are infected or colonized with the same pathogen--. category ib. in close proximity to the patient (e.g., medical equipment, bed rails). don gloves upon entry into the room or cubicle. category ib. 4. wear a gown whenever anticipating that clothing will have direct contact with the patient or potentially contaminated environmental surfaces or equipment in close proximity to the patient. don gown upon entry into the room or cubicle. remove gown and observe hand hygiene before leaving the patient-care environment. category ib. 5. patient transport. in acute care hospitals and long-term care and other residential settings, limit transport and movement of patients outside of the room to medically-necessary purposes. category ii. 6. when transport or movement in any healthcare setting is necessary, ensure that infected or colonized areas of the patient's body are contained and covered. category ii. 7. remove and dispose of contaminated ppe and perform hand hygiene prior to transporting patients on cp. category ii. 8. don clean ppe to handle the patient at the transport destination. category ii. precautions. category ib/ic. 10. in acute care hospitals and long-term care and other residential settings, use disposable noncritical patient-care equipment (e.g., blood pressure cuffs) or implement patient-dedicated use of such equipment. if common use of equipment for multiple patients is unavoidable, clean and disinfect such equipment before use on another patient. category ib [19] ." selected citation ended. spread of pathogenic infectious agents through the air and droplets requires a defined negative pressure ventilation isolate and a system which reduces airborne infection in the patient's room. it is best done by air replacement 6-12 times an hour [19, 61] . the cause of airborne, pathogenic microbes may include: and the urinary tract. 6. by rapid removal of the bandage from pus and discharging wounds. 7. by raise up of infected dust, skin cells and other contaminants by activities that create air currents, often in poorly cleaned rooms [44] . 8. re-aerosolized pathogens from used textiles and equipment in the patient room or where these contaminated items are brought, for instance, to the ward's disinfection room or to the laundry. by massive contamination and poor routines for washing equipment and hospital textiles, this may cause a special problem of re-aerosols [105] . 9. construction activity of all types without measures against airborne infection is influencing the airborne spread of bacteria, virus and fungi. this must be taken into account when rebuilding and repairing buildings and ventilation ducts etc. [22, 105] . if ignored, it may cause large problems like the outbreak of bacillus cereus among 171 patients, most with sepsis, during 6 months of a large construction project next to the hospital [105] . air samples showed growth of large quantities of bacillus inside the hospital, the units and also isolates, and hospital textiles were heavily contaminated [105] . on a daily basis, the human inhales 10,000 particles or more via airways; many of these are bacteria, viruses and fungi but mostly come up again without causing disease. a solid documentation shows that airborne infection may occur at a variety of infectious diseases [12, 22, 31, 47, 52, 54, 58, 59, 85, [100] [101] [102] [103] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [119] . rooms with mrsa-infected cases may have larger numbers of mrsa bacteria in air samples than in samples from surfaces [113, 114] . mrsa may even be spread to nearby located rooms, to patients not infected [113, 114] . the few existing units for airborne infection isolation mean that most patients, except for pulmonary tuberculosis, most often are admitted to contact isolation or single-patient rooms, with the use of ppe corresponding to airborne infection: gloves, infection-protecting gown, cap and surgical mask and sometimes room-bound shoes [22] . airborne precautions (ap)-recommended by cdc [19] . an aiir directly to the outside, the air may be returned to the air-handling system or adjacent spaces if all air is directed through hepa filters. (c) whenever an aiir is in use for a patient on airborne precautions, monitor air pressure daily with visual indicators (e.g., smoke tubes, flutter strips), regardless of the presence of differential pressure sensing devices (e.g., manometers). (d) keep the aiir door closed when not required for entry and exit. 2. when an aiir is not available, transfer the patient to a facility that has an available aiir. category ii. 3. in the event of an outbreak or exposure involving large numbers of patients who require airborne precautions: (a) consult infection control professionals before patient placement to determine the safety of alternative room that do not meet engineering requirements for an aiir. (b) place together (cohort) patients who are presumed to have the same infection(based on clinical presentation and diagnosis when known) in areas of the facility that are away from other patients, especially patients who are at increased risk for infection (e.g., immune-compromised patients). (c) use temporary portable solutions (e.g., exhaust fan) to create a negative pressure environment in the converted area of the facility. discharge air directly to the outside, away from people and air intakes, or direct all the air through hepa filters before it is introduced to other air spaces. category ii. 4. place the patient in an aiir as soon as possible. if an aiir is not available, place a surgical mask on the patient and place him/her in an examination room. once the patient leaves, the room should remain vacant for the appropriate time, generally 1 h, to allow for a full exchange of air. category ib/ic. instruct patients with a known or suspected airborne infection to wear a surgical mask and observe respiratory hygiene/cough etiquette. once in an aiir, the mask may be removed; the mask should remain on if the patient is not in an aiir. category ib/ic. 6. restrict susceptible healthcare personnel from entering the rooms of patients known or suspected to have measles (rubeola), varicella (chickenpox), disseminated zoster, or smallpox if other immune healthcare personnel are available. category ib. 7. wear a fit-tested niosh-approved n95 or higher level respirator for respiratory protection when entering the room or home of a patient when the following diseases are suspected or confirmed: (a) infectious pulmonary or laryngeal tuberculosis or when infectious tuberculosis skin lesions are present and procedures that would aerosolize viable organisms (e.g., irrigation, incision and drainage, whirlpool treatments) are performed. category ib. (b) smallpox (vaccinated and unvaccinated) ---category ii. (c) no recommendation is made regarding the type of personal protective equipment (i.e., surgical mask or respiratory protection with a n95 or higher respirator) to be worn by susceptible healthcare personnel who must have contact with patients with known or suspected measles, chickenpox or disseminated herpes zoster. unresolved issue. 8. in acute care hospitals and long-term care and other residential settings, limit transport and movement of patients outside of the room to medically-necessary purposes. category ii. 9. if transport or movement outside an aiir is necessary, instruct patients to wear a surgical mask, if possible, and observe respiratory hygiene/cough etiquette. category ii. 10. for patients with skin lesions associated with varicella or smallpox or draining skin lesions caused by m. tuberculosis, cover the affected areas to prevent aerosolization or contact with the infectious agent in skin lesions. category ib. 11. healthcare personnel transporting patients who are on airborne precautions do not need to wear a mask or respirator during transport if the patient is wearing a mask and infectious skin lesions are covered. category ii. 12. exposure management immunize or provide the appropriate immune globulin to susceptible persons as soon as possible following unprotected contact (i.e., exposed) to a patient with measles, varicella or smallpox: category ia. (a) administer measles vaccine to exposed susceptible persons within 72 h after the exposure or administer immune globulin within 6 days of the exposure event for high-risk persons in whom vaccine is contraindicated. (b) administer varicella vaccine to exposed susceptible persons within 120 h after the exposure or administer varicella immune globulin (vzig or alternative product), when available, within 96 h for high-risk persons in whom vaccine is contraindicated (e.g., immune compromised patients, pregnant women, newborns whose mother's varicella onset was <5 days before or within 48 h after delivery. (c) administer smallpox vaccine to exposed susceptible persons within 4 days after exposure." citation ended. "--the environmental recommendations in these guidelines may be applied to patients with other infections that require airborne precautions". selected citation ended. particularly dangerous or unknown infectious agents with high mortality need special treatment in high-risk isolates with separately, controlled ventilation and disinfection of air extraction/waste/water, etc. or in buildings separated from other patients and without common ventilation [22, [30] [31] [32] [33] [34] . during the ebola epidemic from march 2014, health professionals and patients were not protected well enough since the who recommended procedures for close contact/droplet infection [120] . the who excluded risk of airborne transmission of ebola, even though it was documented to be transferred via air to primates [120, 121] . the uncertain infection control procedures of who were upgraded from september to october 2014 by the who and cdc, from contact/droplet infection 1 metre from the patient to strict isolation, but still without measures against airborne infection [121, 122] . the principle of "prevention is better than cure" was not in use. the same happened in connection with sars in 2003 where who was recommending a low infection control level, contact/droplets 1 metre from the patient, and had to raise the disease control level up to strict isolation ca. 2 months later on [33] . high-risk infections are recorded almost daily around the world, like the pneumonic plague in madagascar, outbreaks (partly nosocomial) of multidrug-resistant tuberculosis in all parts of the world and nosocomial outbreaks of crimean-congo haemorrhagic fever in germany after import of ill us soldier from afghanistan [123] [124] [125] . nearly each day, there are reported new cases of avian influenza, mers, various haemorrhagic viruses or other dangerous microbes [1, [31] [32] [33] [34] . since 1945, there has been no need for strict isolation in norway, with the exception of the imported healthcare professionals with ebola in autumn 2014. risk of infection may occur when patients with greatly reduced infection defence are coming into contact with infected equipment, textiles or other patients, staff or visitors who have infection or are carriers of possible pathogenic microbes [19, 22, 126] . the following conditions are particularly vulnerable to contamination, agranulocytosis, neutropenia; <0.5 in granulocytes or rapidly decreasing number < 2, severe burns, patients with severely weakened immune system due to the treatment with immunosuppressive, chemotherapy or radiation (transplants, cancer or leukaemia). this heterogeneous group is growing due to a more advanced treatment and more cancer and transplant cases. they should be protected against infection from "outside" by treatment in protective isolation in a clean room with anteroom or sluice and with hepa-filtered air in a positive pressure room [19, 22, [126] [127] [128] [129] [130] [131] . cdc recommends that by allogeneic haematopoietic stem cell transplantation (hsct), the patients should be treated in protective isolation, especially to protect against fungal infections such as aspergillus; category ib. [19] "as defined by the american institute of architecture, air quality for hsct patients is improved through a combination of environmental controls that include [1] hepa filtration of incoming air, [2] directed room air flow, [3] positive room air pressure relative to the corridor, [4] well-sealed rooms (including sealed walls, floors, ceilings, windows, electrical outlets) to prevent flow of air from the outside, [5] ventilation to provide ≥12 air changes per hour, [6] strategies to minimize dust (e.g., scrub-able surfaces rather than upholstery and carpet, and routinely cleaning crevices and sprinkler heads), and [7] prohibiting dried and fresh flowers and potted plants in the rooms of hsct patients. the latter is based on molecular typing studies that have found indistinguishable strains of aspergillus terreus in patients with haematologic malignancies and in potted plants in the vicinity of the patients" [19] . • in protective isolate should intake air be hepa filtered which removes 99.97% of particles larger than or equal 0.3 μm in diameter; category ib. [19] • the room should be at positive pressure relative to the corridor with a pressure difference of 12.5 pascal or more; category ib. [19] • the pressure should be monitored visually every day; category ia [19] . • the room must be sealed so that no outside air to seep in; category ib. [19] • there should be a minimum of 12 air changes per hour; category ib. [19] • use smooth surfaces, avoiding textile furniture and the like, and perform good cleaning; category ii [19] . • do not use carpeting in corridors or rooms in the area; category ib. [19] • the patient should be at least possible out of the room, the necessary examinations, etc., to be implemented in the shortest possible time; category ib. [19, 22, 126, 130, 131] • if necessary, use respiratory protection if out of isolation; category ii [19, 130] . • staff are using adequate infection control equipment if the patient has infection; category ia/ib. [19] • patients with respiratory infection-at the same time they need protective isolation-should be transferred to a defined airborne isolate. if hepa filters are lacking on the air intake to the airborne infection isolation units, a portable system of the hepa filter may be used in the room to remove fungal spores and bacteria; category ii [19] . avoid transfers outside the isolate [130, 131] . a retrospective, case-control study of immunocompromised patients in switzerland showed that when invasive fungal infection occurred, the mortality was over 20% [131] . an outbreak of fungal infection among 29 patients on a haematological department showed that the more often 21.11 protective isolation the patient was transferred to other departments/was outside the isolate, the more frequently he was infected with invasive fungi [131] . with more than five patient transfers outside protective isolates, the risk of fungal infection was sixfold higher than if staying in protective isolation. if the patient was transferred during neutropenia, the risk increased by nearly sevenfold [131] . building activities. fungal spores are always released during construction activity and may become airborne also in risk areas where severely immunocompromised patients are located [105, 126, 130] . infectious agents may be spread via air, textiles and equipment with deposition of fungal spores and other microbes during the construction period. fungal spores follow air currents far away [105] . construction activity must therefore be coordinated with appropriate departments and wards so that the patient area is shielded. air from construction places should not leak into patient areas and be taken out directly from the building in such a way that it does not re-enter through the systems for intake of air. leakage of water in buildings. fungal growth in patient room walls is due to neglected maintenance of buildings for many years and usually comes from water leakage from the old pipes, roof leaks or poorly maintained bathrooms. patients should not stay in rooms with suspected fungal growth and decay! control of fungi should be implemented in protective isolates and storages for such isolates once a year or more frequently when suspected water leaks and rot. the requirement is 0 fungal spores per 1000 litres of air using the sas-slit sampler. patients must be protected against infections during care and treatment [19, 22, [132] [133] [134] . cleaning and disinfection of rooms, surfaces and equipment between infection susceptible patients must be extra careful since many-at the same time-have serious infections, including resistant bacteria. despite a good cleaning, abundant amounts of microbes may be detected in the environment and the equipment after cleaning [135, 136] . healthcare professionals who treat infectious patients may be highly exposed to infection, although they really should be protected [3, 6-8, 19, 22, 137, 138] . at the beginning of the sars epidemic in 2003, 90% of sars patients were the hospital personnel who had taken care of the first sars patients [33] . during the ebola outbreak in 2014, healthcare workers were again severely attacked by the disease and death in the first phase, until a more protective personal protective equipment system was in place [34, [120] [121] [122] . there is still a great variety in international guidelines in terms of personal protection equipment (ppe) by ebola virus for healthcare workers [121] . the who, united kingdom and cdc defined ebola as non-airborne infection in 2014 and still does the same in 2017 [121] . none of these recommended respiratory protection for suspected ebola infection in august-september 2014, at least half a year after the start of the epidemic. only the united kingdom recommended respirator mask for confirmed ebola. there was no covering of the head or neck, not either during aerosol-generating procedures! in october 2014, the cdc upgraded the ppe measures to strict isolation measures for proven ebola infection, while the earlier ppe measures were recommended for suspected ebola infection (table 21 .1) [120] [121] [122] . during the pandemic flu in 2009, norwegian health authorities did not allow the staff at hospitals to use respirator masks (p3 masks) when taking care of the flu patients; they should use surgical masks [138] . exceptions were for aerosol-generating procedures. the same health authorities went public and said that surgical masks did not have any protective effect! [138] recurrent problems are that the health authorities are not able to see the importance in protecting health professionals who are-and will always be-the frontline workers, even at the most serious infectious diseases [137] . governments that are not willing to take care of their healthcare employees create unnecessary fear and reluctance to make a good contribution during outbreaks. if the disease is not as dangerous as sars and ebola, a highly contagious pandemic influenza or a large norovirus outbreak could paralyse the healthcare system when a high number of the staff get sick at the same time. by preventing disease transmission to health professionals, the hospital can be operatively enough to handle properly other serious diseases such as traffic accidents, myocardial infarction, stroke, etc., even at largescale epidemics. in spite of problems concerning isolation and prevention of spread of emerging and new serious infections, new guidelines are now appearing, which works with reduction of infection control in severely affected countries with endemic infections [139, 140] . [19] handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control no. 55 of protection against infectious diseases regulations in infection control in health facilities -hospital infections, established by the health and social affairs action plan for infection control in norwegian hospitals. health directorate's guidance series 2-92. directorate of health use of isolation to prevent spread of infection in hospitals. health directorate's guidance series: directorate of health regulations for protection against exposure to biological agents (bacteria, viruses, fungi, etc.) in the workplace. directorate of labour inspection on the protection of workers from the risks related to exposure to biological agents at work cdc draft guideline for isolation precautions in hospital guideline for isolation precautions in hospitals cdc draft guideline for preventing the transmission of mycobacterium tuberculosis in health care facilities the use of adult isolation facilities in a uk infectious disease unit an audit of the use of isolation facilities in a uk national health service trust risk factors for the isolation of multi-drug-resistant acinetobacter baumannii and pseudomonas aeruginosa: a systematic review of the literature closure of medical departments during nosocomial outbreak: data from systematic analysis of the literature management of multidrug-resistant organisms in healthcare settings prevention of transmission of multidrug resistant organisms guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings. cdc strategies to prevent methicillin-resistant staphylococcus aureus transmission and infection in acute care hospitals: 2014 update variability of contact precaution policies in us emergency departments in: handbook of hygiene and infection control in hospitals. oslo: ullevål university hospital a predicted outbreak in an overcrowded, administratively neglected and run-down haemodialysis unit as an offer of "new public management" in norwegian hospitals spread of methicillin-resistant staphylococcus aureus in a neonatal intensive unit associated with understaffing, overcrowding and mixing of patients the design of isolation rooms hospital and community acquired infection and the built environment-design and testing of infection control rooms evaluation of the contribution of isolation precautions in prevention and control of multi-resistant bacteria in a teaching hospital a three-year survey of nosocomial and community-acquired infections, antibiotic treatment and re-hospitalization in a norwegian health region hospital-acquired infections before and after healthcare reorganization in a tertiary university hospital in norway isolation of dangerous infections. in: handbook of hygiene and infection control in hospitals. oslo: ullevål university hospital in: handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control in: handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control. fagbokforlaget hospital infections at ullevål hospital. occurrence and additional expenses a norwegian nosocomial outbreak of methicillin-resistant staphylococcus aureus resistant to fusidic acid and susceptible two other anti-staphylococcal agents a multidrug resistant, methicillin-susceptible strain of staphylococcus aureus from a neonatal intensive care unit in oslo psychological effects of source isolation nursing (2) patient satisfactory depression, anxiety, and moods of hospitalized patients under contact precaution care of children isolated for infection control: a prospective observational cohort study adverse outcomes associated with contactless precautions: a review of the literature contact isolation for infection control in hospitalized patients: is patients satisfaction affected? adverse effects of isolation in hospitalized patients: a systematic review door-opening motion can potentially lead to a transient breakdown in negative-pressure isolation conditions: the importance of vorticity and buoyancy airflows lessons from the history of quarantine textbook of hygiene. oslo: fabritius & sønners forlag violent expiratory events: on coughing and sneezing usage of antimicrobial agents and occurrence of antimicrobial resistance in norway nosocomial infection with resistant enterobacter cloacae an outbreak of multidrug-resistant pseudomonas aeruginosa associated with increased risk of patient death in an intensive care unit food contamination is a neglected problem the potential for airborne dispersal of clostridium difficile from symptomatic patients burden of clostridium difficile infection in the united states outbreaks of acute gastroenteritis in and outside the ullevål hospital in norovirus gastro-enteritis at ullevål university cross-transmission of nosocomial pathogens in an adult intensive care unit: incidence and risk factors virus associated with skin rash and children's diseases. in: handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control bordetella pertussis -whooping cough healthcare professional arguments and sick people die isolation rooms for highly infectious diseases: an inventory of capabilities in european countries patient care in a biological safety level 4 (bsl-4) environment evidence-based design of health care facilities: opportunities for research and practice in infection prevention the role of facility design in preventing the transmission of healthcare-associated infections: background and conceptual framework the role of the hospital environment in preventing healthcare-associated infections caused by pathogens transmitted through the air the role of the hospital environment in the prevention of healthcare-associated infections by contact transmission the impact of a single ward for cohorting patients with infection due two multidrug-resistant organisms hospital endemic with methicillin-resistant staphylococci significances of fomites in the spread of respiratory and enteric viral disease predictors of hospital surface contamination with extended-spectrum beta-lactamase-producing escherichia coli and klebsiella pneumoniae: patient and organisms factors comparing the transmission potential of methicillin-resistant staphylococcus aureus and multidrug-resistant acinetobacter baumannii among inpatients overusing target environmental monitoring role of hospital surfaces in the transmission of emerging healthcare-associated pathogens: norovirus, clostridium difficile, and acinetobacter species mopping up hospital infection decontamination of rooms, medical equipment and ambulances, overusing a dry mist of hydrogen peroxide disinfectant cleaning and decontamination of reusable medical equipment, including the use of hydrogen peroxide dry-mist gas decontamination failure of dry mist of hydrogen peroxide 5% to kill mycobacterium tuberculosis isolation measures for prevention of infection with respiratory pathogens in cystitis fibrosis: a systematic review impact of contact precautions on falls, pressure ulcers and transmission of mrsa and vre in hospitalized patients economic consequences of hospital infections in a 1000 bed university hospital in norway rapid mrsa test in exposed persons: costs and savings in hospitals infection acquisition following intensive care unit room privatization compliance with routine use of gowns by healthcare workers (hcw) and non-hcw visitors on entry into the rooms of patients under contact precautions epidemiology of isolation precautions the effect of contact precautions on healthcare worker activity in acute care hospitals oslo: gyldendal norsk forlag guideline for hand washing in healthcare settings hospital outbreak control requires joint efforts from hospital management, microbiology and infection control changing epidemiology of methicillin-resistant staphylococcus aureus in the veterans affairs healthcare system comparative surface -to-hand and fingertip-to-mouth transfer efficiency of gram-positive bacteria, gram-negative bacteria, and phage evidence from a uk teaching hospital that mrsa is primarily transmitted by the hands of healthcare workers health-care workers: source, vector or victim of mrsa? patient empowerment and hand washing a randomized trial of soap and water hand wash versus alcohol hand rub for removal of clostridium difficile spores from hands of patients could hospital patients' hands constitute a missing link? patient-centred hand washing: the next step in infection prevention systematic patients' hand disinfection: impact on methicillin-resistant staphylococcus aureus infection rates in a community hospital occurrence of skin and environmental contamination with methicillin-resistant staphylococcus aureus before results of polymerase chain reaction at hospital admission become available what may be lurking in the hospital undergrowth? inapparent cross-transmission of extended-spectrum beta-lactamase-producing klebsiella pneumoniae the exposure to hospital roommates as a risk factor for healthcare-associated infection evaluation of bedmaking-related airborne and surface methicillin-resistant staphylococcus aureus contamination effect of floor cleaning bacteria and organic materials in patient floor cleaning: effect on bacteria and organic materials in hospital rooms air quality and microbiological contamination in operating theaters with and without laminar air flow occlusive scrub suits in operating theaters during cataract surgery hot and steamy: outbreak of bacillus cereus in singapore associated with construction work and laundry practices the airborne transmission of infection in hospital buildings: fact or fiction? role of ventilation in airborne transmission of infectious agents in the built environment-a multi-disiplinary systematic review virus diffusion in isolation rooms an outbreak of influenza abroad a commercial airliner review of aerosol transmission of influenza a virus detection of bordetella pertussis and respiratory syncytial virus in air samples from hospital rooms airborne dispersal as a novel transmission route of coagulase-negative staphylococci, interaction between coagulase-negative staphylococci and rhinovirus infection aerial dispersal of methicillin-resistant staphylococcus aureus in hospital rooms by infected and colonised patients air and surface contamination patterns of methicillinresistant staphylococcus aureus on eight acute hospital wards air contamination around patients colonized with multidrug-resistant organisms protecting staff against airborne viral particles: in vivo efficiency of laser masks association of private isolation rooms with ventilator-associated acinetobacter baumannii pneumonia in surgical intensive-care unit droplet fate in indoor environments, or can we prevent the spread of infection environmental survey to assess viral contamination of air and surfaces in hospital settings international infection control guidelines may not protect against ebola. hospital health care guidelines does not protect against ebola. bit's 5th annual wold congress of microbes -2015 (wcm 2015) pneumonic plague outbreak multi drug-resistant tuberculosis outbreak in gaming centres health care response to cchf in us soldier and nosocomial transmission to health care providers incidence of, and risk factors for, nosocomial infections among haematopoietic stem cell transplantation recipients, with impact on procedure-related mortality ventilation systems for hospital rooms devoted two immune compromised and/or infectious patients invasive aspergillosis in severely neutropenic patients over 18 years: impact of intranasal amphotericin b and hepa filtration hospital infection control in haematopoietic stem cell transplant recipients masking of neutropenic patients transport from hospital rooms is associated with a decrease in nosocomial aspergillosis during construction in-hospital transfer is a risk factor for invasive filamentous fungal infection among hospitalized patients with haematological malignancies: a matched case-control study rikshospitalet: daily work in protective isolation rikshospitalet: for you to be a stem cell transplant. guideline for adult patients and their families protective isolation. in: handbook of hygiene and infection control for hospitals. oslo: ullevål university hospital risk factors for acquiring vancomycin-resistant enterococcus and methicillin-resistant staphylococcus aureus on a burn surgery step-down unit residual viral and bacterial contamination of surfaces after cleaning and disinfection protecting the frontline: designing an infection prevention platform for preventing emerging respiratory viral illness in healthcare personnel in: handbook of hygiene and infection control in hospitals. part 1 microbiology and infection control reconsidering contact precautions for endemic methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococcus duration of contact precautions for acute-care settings comparing the recommended ppe equipment by ebola infection in guidelines from the who, united kingdom and cdc in 2014 [120] [121] [122] who key: cord-019490-m1cuuehi authors: nan title: abstracts cont. date: 2015-12-28 journal: clin microbiol infect doi: 10.1111/j.1469-0691.2005.clm_1134_02.x sha: doc_id: 19490 cord_uid: m1cuuehi nan addition of a biocatalytic oxygen-reducing agent may be required in the absence of fresh media (<12 hours old) when testing tigecycline using broth microdilution t. stevens, b. johnson, s. bouchillon, j. johnson, d. hoban, m. dowzicky, p. bradford (schaumburg, pearl river, usa) objectives: tigecycline (tgc), a new glycylcycline antimicrobial in development has demonstrated excellent in vitro activity against gram-positive and -negative pathogens. recent reports suggest that tgc mic values, against some organisms, may be elevated if broth used in microdilution panels is (>12 hours old (aged). the nccls has tentatively recommended that testing of tgc be performed in broth that is (<12 hours old (fresh). this study looks at the difference between panels using fresh broth, aged broth and broth with a biocatalytic oxygen-reducing agent (bora) added to compensate for any potential broth differences. materials and methods: testing was performed on approximately 120 organisms including: e. coli; k. pneumoniae; m. catarrhalis; s. epidermidis; and s. pneumoniae. the bora used in this study is oxyrase ò for broth at 2% concentration. tig microdilution panels evaluated in this study include: panels and aged broth without oxyrase (aged); panels and aged broth with oxyrase (ao); panels and fresh broth without oxyrase (fresh); and panels and fresh broth with oxyrase (fo). panels and aged broth were prepared by microscan. fresh broth was prepared internally. each organism was tested on all four panel types. quality controls were performed using nccls approved atcc strains. results: combined test results showed an mic correlation (with in 1 log2 dilution) as follows: 97.5% between fresh/ao; 85.6% between fresh/aged; 94.3% between fo/ao; and 87.9% between fo/aged. quality controls ranges for fo, fresh and ao were all in compliance, but aged panels were out of range 29.3% of the time. conclusion: the addition of a bora to aged broth produced results equivalent to fresh broth without a bora. objectives: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many grampositive pathogens including methicillin-resistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of 9 comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillin-tazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from multinational evaluation centres in the test program. methods: a total of 1245 clinical isolates were identified to the species level at each of 15 sites in 8 countries and confirmed by the central laboratory. isolates were collected between january 2004 and november 2004. mics were determined by each participating laboratory using supplied broth microdilution panels from dade microscan. all testing was performed according to nccls guidelines and manufacturer's instructions. results: the mics of tigecycline ranged from 0.06 to 1 mcg/ml for all isolates of s. aureus. tigecycline's mic50/mic90 of 0.12/ 0.25 mcg/ml, respectively, against mssa was similar to imipenem and minocycline and 4/8 fold lower than the remaining comparative agents. tigecycline's mic50/mic90 of 0.25/0.5 mcg/ml, respectively, against mrsa was 8/4 fold lower than vancomycin, 2/4 fold lower than minocycline and 4/8 fold lower than linezolid. all isolates of s. aureus were inhibited by tigecycline at an mic of 1 mcg/ml regardless of methicillin phenotype. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. background: rapid increasing resistance in nosocomial pathogens has always been a challenge for clinicians and hospital infection control. tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of enterobacteriaceae (mainly e. coli, klebsiella spp., enterobacter spp., and serratia spp.) collected from hospitals in north america, europe and asia. methods: a total of 3204 clinical isolates of enterobacteriaceae were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity was equivalent to imipenem presenting a mic50/mic90 of 0.25/1 mcg/ml against all strains of enterobacteriaceae. in comparison to other antimicrobials tested, the mic90 of 1 mcg/ml for tigecycline was also the lowest being 8 fold lower than commonly prescribed broad spectrum antimicrobials such as ceftriaxone, levofloxacin, and minocycline and 16 fold lower than ceftazidime and piperacillin/tazobactam. the frequency of esbl production among k. pneumoniae and e. coli was found to be 10.3% and 2.2%, respectively. tigecycline inhibited >98% of all e. coli and k. pneumoniae esbl producers at an mic of 2 mcg/ml. approximately 20% of enterobacter spp. and serratia spp. presented resistance to third generation cephalosporins (ceftazidime and ceftriaxone) suggestive of ampc-type resistance. tigecycline also inhibited a majority of these isolates with an mic90 of 2 mcg/ml. conclusion: tigecyclines in vitro activity was comparable to the activity of a broad spectrum antimicrobial, carbapenem (imipenem) , and greater than other commonly prescribed broad spectrum agents tested in this study. the presented data suggest that tigecycline may be an effective therapeutic option against both susceptible strains of enterobacteriaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have a potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram-positive, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against isolates of enterobacteriaceae collected from hospitals across the usa. methods: a total of 2446 clinical isolates, collected in 2004, were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: the efficacy of all broad spectrum antimicrobial agents still remain highly active against enterobacteriaceae in the united states. the susceptibility rates for amikacin, cefepime, ceftazidime, ceftriaxone, imipenem, levofloxacin, minocycline and piperacillin/tazabactam are 99.3%, 97.5%, 89%, 92.2%, 98.8%, 85.5%, 85.7% and 91.8%, respectively. tigecycline's activity was similar to imipenem presenting a mic50/mic90 of 0.25/1 mcg/ ml against all strains of enterobacteriaceae. the frequency of esbl production among k. pneumoniae and e. coli was found to be 8.5% and 2.2%, respectively. tigecycline successfully inhibited >98% of all e. coli and k. pneumoniae esbl producers at a mic of 2 mcg/ml. it was also noticed unusual resistance to imipenem in 29 of these isolates. while still under more detailed analysis, preliminary data have shown that tigecycline presented a mic50/ mic90 of 1/2 mcg/ml against these multi-resistant isolates. conclusion: most of broad spectrum antimicrobial agents still remain active against enterobacteriaceae from the us. tigecycline's activity was comparable to the activities of broad spectrum antimicrobials and with greater activity against most esbl and ampc producing isolates. tigecycline also showed in vitro activity against isolates that were intermediate or resistant to imipenem, which in many instances is considered as a last therapeutic option. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible strains of enterobacterieaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered gram-positive and gram-negative species, including anaerobic pathogens responsible for community and hospital infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/ tazobactam against gram negative rods in addition to linezolid, penicillin and vancomycin for the gram positive species. isolates were collected from hospitals in the united states throughout 2004. methods: a total of 4100 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with a mic equal or lesser than 2 mcg/ml. tigecycline also showed in vitro activity with a mic90 2 mcg/ml against 29 imipenem resistant enterobacteriaceae strains. although similar to other classes of broad spectrum antimicrobial agents against non-fermenters, tigecycline was especially active against acinetobacter spp. with the lowest mic90 of 2 mcg/ml. tigecycline inhibited s. aureus with mic90 of 0.25 mcg/ml for both mssa and mrsa isolates. against enterococci, tigecycline's mic90 was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable or greater than most commonly prescribed broad spectrum antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible common gram-positive and gram-negative pathogens, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline, and piperacillin/tazobactam against gram negative rods in addition to linezolid, penicillin, and vancomycin for the gram positive species. isolates were collected from hospitals located in germany, italy, spain, and united kingdom throughout 2004. methods: a total of 1064 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with mics equal or lesser than 2 mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against glucose non-fermenters, tigecycline was especially active against acinetobacter spp. presenting the lowest mic90 of 1 mcg/ml. tigecycline inhibited s. aureus with a mic90 of 0.25 mcg/ml regardless of sensitivity or resistance to methicillin. the same results were noticed against enterococci where tigecycline's mic90 of 0.25 mcg/ml was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered spe-cies responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/ tazobactam against gram negative rods in addition to linezolid, penicillin and vancomycin for the gram positive species. isolates were collected from hospitals located in asia throughout 2004. methods: a total of 424 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity was similar to imipenem against enterobacteriaceae with mic50/mic90 of 0.25/1 mcg/ml. resistance to third generation cephalosporin was found in 63.2% of e. coli and 77.8% of k. pneumoniae consistent with esbl phenotype. tigecycline inhibited esbl and ampc producers with mics equal or lesser than 1 mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against glucose non-fermenters, tigecycline was especially active against acinetobacter spp. presenting the lowest mic90 of 1 mcg/ml. tigecycline successfully inhibited s. aureus with mic90 of 0.25 mcg/ml regardless of sensitivity or resistance to methicillin. same phenomenon was noticed against enterococci where tigecycline's mic90 of 0.12 mcg/ml was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae as well as gram-positive, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against isolates of enterobacteriaceae collected from hospitals in germany, italy, spain and united kingdom. methods: a total of 627 clinical isolates, collected in 2004, were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory abstracts using supplied broth microdilution panels and interpreted according to nccls guidelines. results: it was observed that the in vitro activity of all broad spectrum antimicrobial agents still remains highly active against enterobacteriaceae in europe. the susceptibility rates for amikacin, cefepime, ceftazidime, ceftriaxone, imipenem, levofloxacin, minocycline,andpiperacillin/tazobactamare99.8%,94.6%,84.8%, 87.6%, 100%, 86.1%, 84.5%, and 92.2%, respectively. tigecycline's activity was similar to the most effective antimicrobial agent, imipenem (100% susceptibility) presenting a mic50/mic90 of 0.25/1 mcg/ml against all strains of enterobacteriaceae. the frequency of esbl production among k. pneumoniae and e. coli was found to be 21.7% and 2.4%, respectively. tigecycline inhibited 100% of all esbl producing e. coli at a mic of 0.5 mcg/ml and at a mic of 4 mcg/ml for esbl producing k. pneumoniae. approximately 30% of enterobacter spp. and 6% of serratia marcescens presented resistance to third generation cephalosporins (ceftazidime and ceftriaxone) suggestive of ampc-type resistance. conclusion: most of the broad spectrum antimicrobial agents still remain active against european representatives of enterobacteriaceae. tigecycline's in vitro activity was comparable to the activities of all broad spectrum antimicrobials with greater activity against esbl and ampc producing isolates with a mic 90 of 2 mcg/ml. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both susceptible strains of enterobacterieaceae and multi-drug resistant strains regardless of degree or type of resistance. background: tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline, and piperacillin/tazobactam against gram negative rods in addition to linezolid, penicillin, and vancomycin for the gram positive species. isolates were collected from hospitals in north america, europe and asia throughout 2004. methods: a total of 6383 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: as shown in the table below, tigecycline's activity was similar to imipenem against enterobacteriaceae. it inhibited multi-resistant esbl and ampc producers with a mic equal or lesser than 2 mcg/ml. although similar to other classes of broad spectrum antimicrobial agents against non-fermenters, tigecycline was especially active against acinetobacter spp. demonstrating the lowest mic90 of 2 mcg/ml. tigecycline successfully inhibited s. aureus with mic90 of 0.25 mcg/ml regardless of sensitivity or resistance to methicillin. the same results were noticed against enterococci. tigecycline's mic90 was consistent regardless of vancomycin susceptibility. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against both aerobic gram-positive and aerobic gram-negative bacteria, including multi-drug resistant strains regardless of degree or type of resistance. background: resistance to glycopeptides in enterococci was first recognized in the late 1980s, and since then has been a major challenge to clinicians and infection control. tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to vancomycin, linezolid, ampicillin, imipenem, ceftriaxone, levofloxacin, minocycline, penicillin and piperacillin/tazobactam against members of enterococcus spp. collected from hospitals in north america, europe and asia. methods: a total of 685 clinical isolates of enterococcus spp. were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using supplied broth microdilution panels and interpreted according to nccls guidelines. results: of 437 e. faecalis evaluated, resistance to vancomycin in 15 (3.4%) isolates was observed. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic50/mic90 (0.06/0.12 mcg/ml) among all antimicrobial agents evaluated. among 248 e. faecium, 50 (20.2%) were resistant to vancomycin, of which three isolates were also resistant to linezolid. tigecycline also presented the lowest mic50/mic90 of 0.03/0.06 mcg/ml. five isolates of vancomycin susceptible e. faecalis and one vancomycin susceptible e. faecium were nonsusceptible to linezolid. no abnormal resistance phenotype was observed in other enterococcus species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multidrug resistant strains regardless of degree or type of resistance. tigecycline evaluation surveillance trial (test) -global in vitro antibacterial activity against selected species of glucose non-fermenting organisms objective: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many gram-positive pathogens including methicillinresistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of 9 comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillintazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from 23 us centres in the test program. methods: a total of 794 clinical isolates were identified to the species level at each of participating sites and confirmed by the central laboratory. isolates were collected throughout 2004. mics were determined by each participating laboratory using broth microdilution panels from dade microscan. all testing was performed and interpreted according to nccls guidelines and manufacturer's instructions. results: among the 794 isolates, 389 (49.0%) were found to be resistant to methicillin (mrsa). besides the cross resistance of mrsa isolates to imipenem, ceftriaxone, penicillin, ampicillin, and piperacillin/tazobactam, it was observed a high rate of nonsusceptibility to levofloxacin (74.9%). no resistance was observed against vancomycin and linezolid. the mics of tigecycline ranged from 0.03 to 1 mcg/ml for all isolates of s. aureus, and tigecycline presented the lowest mic50/mic90 of 0.12/0.25 mcg/ml against mrsa isolates, being several folds lower than all the comparator agents. the mssa isolates showed the expected profile of high resistance to ampicillin and penicillin. the only unusual pattern was the 20.4% nonsusceptibility to levofloxacin. tigecycline's mic50/mic90 of 0.12/0.12 was also the lowest among all mssa isolates. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. tigecycline evaluation surveillance trial (test) -united states in vitro antibacterial activity against selected species of enterococcus spp. results: of 276 e. faecalis evaluated, it was observed resistance to vancomycin in 8 (4.5%) isolates. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic50/mic90 (0.06/0.12 mcg/ml) among all antimicrobial agents evaluated. as a typical profile of e. faecalis, fluoroquinolone (levofloxacin) and tetracycline (minocycline) had limited activities against this species. among 144 e. faecium, 35 (24.3%) were resistant to vancomycin, of which three isolates were also resistant to linezolid. tigecycline also presented the lowest mic50/mic90 of 0.06/0.12 mcg/ml. five isolates of vancomycin susceptible e. faecalis and one vancomycin susceptible e. faecium were non-susceptible to linezolid. no abnormal resistance phenotype was observed in other enterococcus species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multi-drug resistant strains regardless of degree or type of resistance. background: glucose non-fermenting gram negative rods are known to be highly resistant in hospital settings and have always been a challenge for clinicians and hospital infection control. the degree or type of resistance may be due to several sophisticated mechanisms such as production of broad spectrum beta-lactamases, efflux pumps and altered membrane permeability, inactivating most classes of antimicrobials that are available for treatment (cephalosporins, carbapenems, aminoglycosides, fluoruquinolones). tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae and selected species of non-fermenters, as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of acinetobacter spp. and pseudomonas aeruginosa collected from hospitals in the united states. methods: a total of 1687 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity against p. aeruginosa showed a mic90 of (16 mcg/ml. towards a. baumannii (n = 849), which cephalosporins were ineffective, tigecycline showed the lowest mic50/mic90 of 0.5/2 mcg/ml outperforming amikacin mic50/mic90 4/32, imipenem mic50/mic90 0.5/16 and minocycline mic50/mic90 1/8. similar findings were found in other species of acinetobacter genus. conclusion: the presented data suggest that tigecycline may be an effective and reliable therapeutic option against strains of acinetobacter spp., including multi-drug resistant strains regardless of degree or type of resistance. objective: despite the introduction of new antimicrobials to treat resistant gram-positive bacteria, staphylococcus aureus continues to be a therapeutic challenge for the clinician. glycylcyclines are showing the promise of significant activity against many gram-positive pathogens including methicillinresistant s. aureus. tigecycline, the first glycylcycline to enter clinical trials, has shown excellent activity against staphylococcus spp. this study was initiated to evaluate the in vitro activity of tigecycline as compared with those of 9 comparator agents (ampicillin, amoxicillin-clavulanic acid, imipenem, ceftriaxone, levofloxacin, minocycline, vancomycin, linezolid, piperacillintazobactam) against s. aureus including methicillin-resistant staphylococcus aureus (mrsa) and methicillin-sensitive staphylococcus aureus (mssa) from 5 european centres in the test program. methods: a total of 216 clinical isolates were identified to the species level at each of participating sites and confirmed by the central laboratory. isolates were collected throughout 2004. mics were determined by each participating laboratory using broth microdilution panels from dade microscan. all testing was performed and interpreted according to nccls guidelines and manufacturer's instructions. results: among the 216 isolates, 76 (35.2%) were found to be resistant to methicillin (mrsa). besides the cross resistance of mrsa isolates to imipenem, ceftriaxone, penicillin, ampicillin, and piperacillin/tazobactam, it was observed that all of mrsa isolates were also non-susceptible to levofloxacin. no resistance was observed against vancomycin and linezolid. the mics of tigecycline ranged from 0.06 to 0.5 mcg/ml for all isolates of s. aureus, and tigecycline presented the lowest mic50/mic90 of 0.25/0.25 mcg/ml against mrsa isolates, being several folds lower than all the comparator agents. the mssa isolates showed the expected profile of high resistance to ampicillin and penicillin. opposite to mrsa isolates, mssa presented very little resistance to levofloxacin (2.5%). tigecycline's mic50/mic90 of 0.12/0.25 was also the lowest among all mssa isolates. conclusion: the in vitro activity of tigecycline was comparable in all s. aureus tested regardless of methicillin phenotype. tigecycline activity against mrsa was more potent than all antimicrobial agents tested in this study including imipenem, minocycline, linezolid, and vancomycin. tigecycline evaluation surveillance trial (test) -european in vitro antibacterial activity against selected species of enterococcus spp. been shown to have potent expanded broad spectrum activity against most commonly encountered species responsible for community and hospital acquired infections. the test program determined the in vitro activity of tigecycline compared to vancomycin, linezolid, ampicillin, imipenem, ceftriaxone, levofloxacin, minocycline, penicillin and piperacillin/tazobactam against members of enterococcus spp. collected from hospitals in germany, italy, spain and united kingdom. methods: a total of 146 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: of 82 e. faecalis evaluated, resistance to vancomycin in 3 (3.6%) isolates was observed. these isolates were all susceptible to linezolid and tigecycline. tigecycline presented the lowest mic50/mic90 (0.12/0.25 mcg/ml) among all antimicrobial agents evaluated. among 40 e. faecium, 1 (5.0%) was resistant to vancomycin. tigecycline also presented the lowest mic50/ mic90 of 0.03/0.06 mcg/ml. no abnormal resistance phenotype was observed in other enterococci species tested. conclusion: tigecycline's in vitro activity was comparable to or greater than most commonly prescribed antimicrobials. the presented data suggest that tigecycline may be an effective and reliable therapeutic option against enterococcus spp. including multi-drug resistant strains regardless of degree or type of resistance. tigecycline evaluation surveillance trial (test) -european in vitro antibacterial activity against selected species of glucose non-fermenting organisms background: glucose non-fermenting gram negative rods are known to be highly resistant in hospital settings and have always been a challenge for clinicians and hospital infection control. the degree or type of resistance may be due to several sophisticated mechanisms such as production of broad spectrum beta-lactamases, efflux pumps and altered membrane permeability, inactivating most classes of antimicrobials that are available for treatment (cephalosporins, carbapenems, aminoglycosides, fluoruquinolones). tigecycline, a member of a new class of antimicrobials (glycylcyclines), has been shown to have potent expanded broad spectrum activity against most species of enterobacteriaceae and selected species of non-fermenters, as well as gram positives, atypicals and anaerobes. the test program determined the in vitro activity of tigecycline compared to amikacin, ampicillin, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline and piperacillin/tazobactam against members of acinetobacter spp. and pseudomonas aeruginosa collected from hospitals in germany, italy, spain, and united kingdom. methods: a total of 826 clinical isolates were identified to the species level at each participating site and confirmed by the central laboratory. isolates were collected throughout 2004. minimum inhibitory concentration (mics) were determined by the local laboratory using broth microdilution panels and interpreted according to nccls guidelines. results: tigecycline's activity against p. aeruginosa showed a mic90 of (16 mcg/ml. the cephalosporins were ineffective towards a. baumannii (n = 552). tigecycline showed the lowest mics against a. baumannii mic50/mic90 of 0.25/1 mcg/ml, outperforming amikacin mic50/mic90 4/>64, imipenem mic50/mic90 0.5/16 and minocycline mic50/mic90 1/8. similar findings were found in other species of acinetobacter genus. conclusion: the presented data suggest that tigecycline may be an effective and reliable therapeutic option against strains of acinetobacter spp., including multi-drug resistant strains regardless of degree or type of resistance. p818 antimicrobial activity of tigecycline tested against bacterial pathogens from intensive care units h. sader, t. fritsche, r. jones (north liberty, usa) objectives: to evaluate the antimicrobial activity of tigecycline (tig) and selected antimicrobials against bacterial pathogens isolated from patients hospitalized in intensive care units (icus) worldwide. methods: a total of 7129 were consecutively collected in >70 medical centres located in north america (3164), south america (1465), europe (2428) and the asia-australia region (72). the isolates were collected from (no. of isolates/%): bloodstream (5349/75%), respiratory tract (746/10%), skin/soft tissue (323/ 5%), and urinary tract (182/3%) infections in the 2000-2004 period, and susceptibility tested by nccls broth microdilution methods. results: the antimicrobial activity of tig and the frequency of occurrence of bacterial pathogens are summarized in the table: all gram-positive pathogens (4817) were inhibited at <1 mg/l of tig. resistance (r) to oxacillin was detected in 43% of sa and 84% of cons, and r to vancomycin was detected in 19% of enterococci. tig was very active against enterobacteriaceae (ent; 1468) with a mic90 <1 mg/l, except for serratia spp. 10% of e. coli and 30% of klebsiella spp. showed an esbl phenotype while 28% of enterobacter spp. were r to ceftazidime. 14% of ent showed r to ciprofloxacin. tig and trimethoprim/sulfamethoxazole were the most active compounds against s. maltophilia (mic90, 2 and 1 mg/l respectively). tig was also highly active against asp (mic90, 1 mg/l), but psa showed decreased s to tig (mic90, 16 mg/l). non-s to imipenem (mic, >8 mg/l) was observed in 16% of asp and 31% of psa isolates. conclusions: isolates from icu patients showed high rates of antimicrobial r. the most alarming problems detected were vancomycin r among enterococci, esbl mediated beta-lactam r and fluoroquinolone r among ent, and carbapenem r among psa and asp. tig exhibited potent in vitro activity against the vast majority of clinically important pathogenic bacteria (except psa) isolated from icu patients and may represent an excellent option for the treatment of infections in this clinical environment. potency and spectrum of tigecycline tested against an international collection (1999) (2000) (2001) (2002) (2003) of bacterial pathogens producing skin and soft tissue infections t. fritsche, h. sader, m. stilwell, r. jones (north liberty, usa) objective: tigecycline (tig) is the sentinel representative of the glycylcycline class to be developed as a parenteral agent targeting bacterial pathogens responsible for pneumonia, intraabdominal sepsis and ssti. the aim of this study was to evaluate the activity and potency of tig when tested against a large collection of bacterial pathogens causing ssti. methods: consecutive, non-duplicate bacterial isolates (3,421 strains) were collected from 1999 to 2003 from patients with documented community-acquired or nosocomial ssti in >70 medical centres participating in the tig surveillance program in north america (38.6%), europe (54.0%), latin america (3.7%) and the asia-pacific region (3.7%). all isolates were tested using nccls broth microdilution methods against tig and representative comparator agents used for empiric and directed therapy of ssti. results: ssti pathogen rank order (top ten), potency and cumulative inhibition rates for tig are in the table: all sa, streptococci, enterococci, cons, ksp and ec were inhibited by £2 mg/l of tig, along with 98% of asp and 96% of ent. the broad-spectrum of activity exhibited by tig included tetracycline resistant subsets as well as mrsa, vre, and esbl-producing strains. only pm and psa isolates were less susceptible (mic90 values at 8 and 16 mg/l, respectively). conclusions: among the top ten-ranked pathogens producing ssti, 94% of isolates were inhibited by £2 mg/l of tig and 98% were inhibited by £4 mg/l (the current nccls breakpoint for tetracyclines). tig may represent a welcome choice among newer parenteral agents for the common gram-positive and negative pathogens producing serious ssti given in vitro testing results, thus warranting continued investigation for this indication. objective: to assess the activity of tigecycline (formerly gar936), a novel glycylcycline, against recent bloodstream infection (bsi) pathogen isolates from six continents. frequency of clinical occurrence of these pathogens was determined and their antibiograms assessed using nccls reference broth microdilution methods. methods: a total of 22,950 strains were tested by the m7-a6 (2003) method with interpretations from m100-s14 (2004) . a tigecycline susceptible (s) breakpoint was defined as £2 mg/l for comparison purposes only, although £4 mg/l has been used for tetracyclines. the rank order of pathogens was: s. aureus (sa; 34.2%), coagulase-negative staphylococci (cons; 14.1%), e. coli (ec; 13.2%), enterococci (ent; 12.7%), klebsiella spp. (ksp; 5.3%), p. aeruginosa (psa; 4.0%), enterobacter spp. (ebs; 2.9), beta-haemolyticstreptococci (bst; 2.8%); s. pneumoniae (spn; 2.4%), and viridans group streptococci (vgs; 1.6%). more than 20 comparison agents were tested including tetracycline (tc) and ciprofloxacin (cip) . results: bsi pathogens (gram-positive and enterobacteriaceae) tested against tigecycline are shown in the table. tigecycline was consistently active against tc-resistant (r) strains (89-100% s versus 45-90%). mic50:% s results for other bsi species were: p. mirabilis (4 mg/l:10), acinetobacter spp. (0.5 mg/l:77), serratia spp. (1 mg/l:81), s. maltophilia (1 mg/l:77) and indole-positive proteae (1 mg/l:54). tigecycline exhibited a broader spectrum of activity against bsi isolates when compared to cip, tc, older aminoglycosides and imipenem. tigecycline was not active against psa (mic50, 8 mg/l; 4.0% of bsi isolates). conclusions: tigecycline exhibited a wide spectrum of antimicrobial potency versus bsi isolates collected worldwide. serious infections in nosocomial environments should benefit from tigecycline among the investigational phase 3 agents focused on r strains. evaluation of the in vitro activity of tigecycline against gram-negative anaerobic bacteria objectives: the evaluation of the in vitro activity of tigecycline in comparison to tetracycline, penicillin, piperacillin ( tazobactam, cefoxitin, clindamycin, metronidazole and imipenem against recently isolated gram-negative anaerobic bacteria. materials and methods: a total of 185 gram-negative anaerobic clinical strains (116 bacteroides fragilis group, 20 other bacteroides spp. non-fragilis, 34 prevotella spp. and 15 miscellaneous) isolated during the period 11/2002-11/2004 were tested using the e-test and the agar dilution methods on brucella agar plates supplemented with 5% horse blood, vitamin k1 and hemin. incubation in a chellab anaerobic chamber was performed for 48 hours. interpretation of the results was according to nccls guidelines. for quality control the strains b. fragilis atcc25285 and b. thetaiotaomicron atcc29741 were used. results: overall mic90 to tigecycline, tetracycline, penicillin, piperacillin + tazobactam, cefoxitin, clindamycin, metronidazole and imipenem were 8, 128, 256, 2, 64, 256, 1 and 0.5 mg/l, respectively, whereas mic50 were 0.5, 16, 32, 0.125, 8, 1, 0.5 and 0. 064 mg/l, respectively. bacteroides fragilis group mic90 were 8, 256, 256, 4, 128, 256 , 1 and 0.5 mg/l, respectively, whereas mic50 were 0.5, 16, 64, 0.25, 16, 1, 0.5 and 0. 064 mg/l, respectively. at the tigecycline mic90 concentration of 8 mg/ l the compound inhibited a higher percentage of isolates than clindamycin, cefoxitin or tetracycline (which inhibited 76, 56 and 45% of the isolates, respectively). only one strain was resistant to imipenem (mic > 32 mg/l), having a tigecycline mic of 8 mg/l. in addition, six of the eight strains with a metronidazole mic > 16 mg/l had a tigecycline mic < 1 mg/l. conclusions: in general, tigecycline showed better in vitro activity than clindamycin, cefoxitin, tetracycline or penicillin against gram-negative anaerobic bacteria and somewhat lower clinical microbiology and infection, volume 11, supplement 2, 2005 activity than imipenem, metronidazole or piperacillin + tazobactam. nevertheless, among most metronidazole and imipenem resistant isolates tigecycline displayed good activity. the results of this in-vitro evaluation show that tigecycline should be considered as a possible alternative for the treatment of mixed aerobicanaerobic infections. objectives: to ascertain the variation between mics of tigecycline determined in broth and agar, and by nccls and bsac methods. to assess the effect of adding tigecycline to the medium one day before use and of using media that had been aged for one week before addition of tigecycline and inoculation. method: mics for 96 non-fastidious bacteria and 20 streptococci were determined in mueller-hinton broth (mhb), mueller-hinton agar (mha), iso-sensitest broth (isb) or iso-sensitest agar (isa). fresh antibiotic stock solutions (tigecycline, tetracycline and minocycline) were incorporated into bulk media by rolling, to minimise aeration. preparation conditions otherwise were as fol-lows: (a) freshly-prepared media, antibiotic added upon cooling, then inoculated; (b) freshly-prepared media, antibiotic added upon cooling thenstored for 1 day before inoculationand (c) media stored for 7 days before addition of the antibiotic and inoculation. results: mics in fresh mhb (the nccls reference method) were taken as a standard. for all the tetracyclines, these mics were 0-2 doubling dilutions higher than on mha or isa (bsac reference method), or in isb. media with tetracyclines added a day before use gave raised mics, though rarely by more than one dilution. tigecycline mics were increased in 7-day-old mhb or isb. this effect was greatest (2-3 dilutions) for the most susceptible strains (mic £ 0.5 mg/l) and was absent for the resistant organisms (mic ‡ 8 mg/l); it did not occur in agar. minocycline mics were not raised in aged media, and tetracycline mics were only marginally affected. the addition of blood to the mhb largely abrogated the effect on tigecycline mic, as did boiling the broth prior to adding the antibiotic. conclusion: the raised mics of tigecycline in aged broth probably reflect inactivation by dissolved oxygen. this accords with lack of any mic increase in newly boiled (i.e. degassed) mhb or on aged agar (which is boiled to melt before use). the effect of blood may be to add to the reducing capacity, protecting the tigecycline. at a practical level, broth mic methods for tigecycline (e.g. the nccls reference method) require, freshly prepared or steamed medium; this is not a concern if agar dilution methods are used, such as that of the bsac. sexually transmitted diseases p823 prevalence of chlamydia trachomatis and neisseria gonorrhoeae in native and immigrant population (ambits study) in barcelona (spain) testing of all danish gc strains isolated in 2002-2004 (n = 953) . clinical and epidemiological data were obtained when possible. results: the 25 pip-negative gc isolates were all designated as serovar ib-4 and similar mic values were identified in the antibiograms of these isolates fingerprints were identified using spei and three using bglii among the 25 ib-4 isolates. four distinguishable pfge . however, all these isolates differed by less than six bands in both fingerprints. the phylogenetic tree analysis of the porb1b sequences, suggested that these minor differences represented the ongoing evolution of the same strain. during the period jan. 2002 to nov. 2004 the proportions of pip-neg gc per year in denmark were 15/332, 47/261 and 27/360, respectively, and the pip-neg gc infected mostly men (only 5 women). the antibiograms of all danish isolates 2002-2004 indicate a spread of at least one pip-neg gc strain. the results of the present study indicate a circulation of at least one n. gonorrhoeae prolyliminopeptidasenegative strain in the danish homosexual community. an increased awareness for pip-negative n. gonorrhoeae, which create large problems in diagnosis using e.g. api nh, rapid nh, gonocheck ii or neisseria pet, is essential. a new recombinant antigen haemagglutination test for the serological diagnosis of syphilis n. chepurchenko, t. ulanova, v. puzyrev, l. loginova, a. burkov, a. obriadina (nizhniy novgorod, rus) introduction: passive treponema pallidum hemagglutination (ha) -the routine treponemal test has equal sensitivity with the fluorescent treponemal antibody absorption (fta-abs) test and with the enzyme immunoassay (eia) in later stages of syphilis, but some studies have indicated that it is less sensitive in the primary stage of the disease. the use of recombinant t. pallidum antigens instead of a poorly defined mixture of antigens from wild-type t. pallidum has the potential for improving the sensitivity of hemagglutination test. objectives: the purpose of this study was to evaluate the diagnostic relevance of 4 recombinant proteins that efficiently model the antigenic epitope(s) of the treponema pallidum proteins. methods: four full-length recombinant proteins (tmpa, 47, 17, 15 kda) were expressed in e. coli and then used individually to develop the hemagglutination test (ht) for the detection of anti-treponema pallidum (anti-tp) activity in serum specimens. serum samples (n = 267) from patients with clinically proven syphilis in various stages of the disease (primary (n = 36), secondary (n = 70), latent (n = 161)) and from normal blood donors (n = 505) were tested. 24 samples from patients with primary syphilis, 21 samples from patients with secondary and 17 samples from patients with latent stage were tested initially with commercially available hemagglutination test based on mixture of treponema pallidum antigens (http). all specimens were additionally tested for specific antibodies by commercially available enzyme immunoassay (eia). results: the sensitivity of the ht for the detection of anti-tp activity in human serum specimens varied from 38.9% to 97.2% with primary syphilis sera, from 87.1% to 100% with secondary, and from 96.9% to 98.1% with latent stage sera for each protein. the tmpa was found as the most immunoreactive when serum samples from patients with untreated syphilis were tested. the sensitivity of tmpa ht was identical to those of the http in cases of untreated secondary and latent syphilis and significantly higher with specimens from primary stage of disease. the overall specificity and sensitivity of the tmpa ht were comparable to those of eia 99% and 99.6%; 98% and 99.6% respectively. the results of this study indicate that the tmpa ht may be a reasonable alternative to the routine hemagglutination test and the elisa as a confirmatory test for syphilis. use of treponema + vdrl virablot test as a useful ôall in oneõ confirmatory assay for treponema pallidum antibodies a. kostoula, g. vrioni, c. bobogianni, c. stergiopoulou, e. papantoniou, s. levidiotou (ioannina, gr) objectives: as detection of treponema pallidum subsp. pallidum by dark-field microscopy or direct immunofluorescence is possible only in the early stages of the disease, serology has become the method of choice for syphilis diagnosis. except for conventional treponemal tests, the t. pallidum western immunoblot assay has also been used as an alternative confirmatory assay for t. pallidum antibodies. the aim of the present study was the use of a multiparameter immunoassay, which simultaneously detects treponemal specific and lipoid antibodies, for the confirmation of syphilis antibodies. methods: a total of 86 serum specimens were tested including, 56 reactive sera from patients with documented treponemal infection; a specificity panel of 10 sera from normal subjects, 10 sera with rapid plasma reagin (rpr) reactivity (fta-abs and tpha negative), 5 leptospira igm serology-positive sera and 5 borrelia burgdorferi igg or igm serology-positive sera. all serum samples were tested with conventional syphilis tests according to the manufacturersõ instructions: rpr, venereal disease research laboratory (vdrl), t. pallidum haemagglutination (tpha) at a serum dilution of 1:80, fluorescent t. pallidum absorption (fta-abs) and igm capture enzyme immunoassay (eia). subsequently, the results were compared to those of the treponema + vdrl virablot igg, igm test (viramed, labor -diagnostika), a immunoassay for the qualitative detection of specific igg or igm antibodies to t. pallidum using the specific treponemal proteins p47, p44.5, p17 and p15, and the quantitative determination of vdrl reaction on one nitrocellulose strip. results: treponema + vdrl virablot igg and igm tests confirmed the results for 56 serum specimens positive for t. pallidum antibodies: reactive vdrl band and at least two clear bands from p47, p44.5, p17 or p15 for igg treponemal antibodies or at least one clear band p47, p17 or p15 for igm treponemal antibodies. the 10 rpr reactive sera were vdrl virablot reactive, but treponema virablot specific antibodies negative. none of the remaining sera reacted in tests with the treponema + vdrl virablot assay. conclusions: the treponema + vdrl virablot assay combining the necessary serologic markers in one test strip (i.e. specific and non-specific treponemal antigens) is a useful confirmatory test for syphilis because it increases the reliability of syphilis diagnosis with respect to current conventional techniques. the great imitator: syphilis with predominant acute severe hepatic involvement the ground of a significant increase of serum transaminiases (up to 2200 and 900 u/l of gpt, respectively), frank jaundice, elevated bilirubin (up to 11.7 and 9.6 mg/dl, respectively), and evident rise of serum alkaline phosphatase and gamma-gt. clinical history did not show risk factors for viral hepatitis, and all laboratory examinations for major and minor hepatotropic viruses proved negative, as well as autoimmmune, dysmetabolic, or drug-induced liver damage. repeated ultrasonography detected increased liver and spleen dimensions, in absence of other abnormalities. in the second p, histopathologic study performed after liver biopsy, showed an aspecific cholestatic hepatopathy, a moderate granulocyte and lympho-mononocyte intralobular infiltrate, appreciable necrotic foci, and a mild portal fibrosis (knodell score 5). only syphilis serology, performed despite the absence of significant history or clinical clues, allowed to recognize an igm positive serology associated with tpha titres ranging from 1:1250 and 1:680, respectively. specific chemotherapy (i.v. penicillin g at 24 mu daily for 7 days, followed by 12 mu/day in the second p),led to a complete resolution of hepatic involvement, associated with an improvement of serology after 1 month. discussion: while in the majority of episodes of syphilis liver involvement remains missed or subclinical, an early diagnosis of syphilis with predominant hepatic disease remains an unresolved problem of differential diagnosis, due to the rare occurrence of isolated organ involvement compared with typical syphilis signs and symptoms, and the aspecific clinical, histopathological, and imaging picture (jozsa l,acta hepatogastroenterol 1977; 24:344-7; young mf, j clin gastroenterol 1992; 15:174-6) . during the recent recrudescence of syphilis in italy, a diagnostic suspicion should be maintained by clinicians who face an apparent acute icteric hepatitis, and syphilis serology should be included among initial laboratory workout. the great imitator: syphilis with predominant meningoencephalitic features r. manfredi, s. sabbatani, d. pocaterra, f. chiodo (bologna, i) introduction: a significant recrudescence of syphilis was observed in recent years, not contained by prophylactic measures against hiv and other stds. meningeal and meningoencephalitic involvement may occur also in early stages of syphilis, thus posing problems of differential diagnosis. case reports: a young woman and an hiv-infected male developed a syphilis with predominant meningoencephalitis expression. in the first p, based on an isolated positive borrelia burgdorferi serology (later deemed as a cross-reaction), early ceftriaxone was started, due to a suspected neurological lyme disease. also after the diagnosis of neurosyphilis (on the ground of positive csf serology), antimicrobial therapy was left unchanged for 3 weeks at our day-hospital facilities, until complete clinical-microbiological cure. a second, hiv-infected p was hospitalized owing to mild fever lasting 2 weeks, associated with cephalalgia and anxiety.the favorable virological-immunological status (cd4 + count 439 cells/ll and undetectable hiv viraemia, under an effective haart regimen), did not exclude all searchs for possible hiv-related disorders.although serum examination detected a potential latent syphilis (isolated positive serum treponema pallidum igg, tpha 1:640,igg-positive rpr, and mute history for syphilis), only csf examination disclosed an increased cell content (50/ll), altered brain-blood barrier indexes with increased intrathecal ig synthesis, and frank vdrl,tpha (1:1520), and borderline t. pallidum igm serology.penicillin g at 24 mu/day for 14 days led to a slow resolution of neurological signs and symptoms, and a tendency to improvement of specific csf-serum syphilis serology during subsequent controls. discussion: focusing on differential diagnosis, a luetic etiology should not be underestimated, when facing young p suffering from a meningoencephalitis of unclear origin.our cases were characterized by a young age (34 and 44 years, respectively), when compared with usual mean age of tertiary neurosyphilis. in absence of suggestive history and other syphilis signs, the diagnosis was achieved only after the retrieval of elevated syphilis serology positivity on both csf and serum, together with some clinical signs, such as seizures, altered mentation, cognitive abnormalities, and anisochoria in the first p, and persisting headache and anxiety in the second p. our experience with ceftriaxone (started in the first p when neuroborreliosis was suspected), was favorable like that with high-dose i.v. penicillin g. comparison of the genomes of pathogenic treponemes, treponema pallidum ssp. pallidum, ssp. pertenue and treponema paraluiscuniculi objectives: the cervical human papillomavirus (hpv) infection is common among young sexually active women. because the cytology tests have some limitations, including up to 30% false-negative results, molecular techniques are increasingly used for identification of hpv-induced cell changes in the cervix. the polymerase chain reaction (pcr) for hpv diagnostics was successfully introduced recently in bulgaria. however limited data on specific prevalence and determinants of subclinical hpv infections are available. in this study we designed a multiplex primers pcr system for simultaneous identification of the most common low-and high-risk hpvs in women with cytologically normal cervical smears. materials & methods: dna from 102 cervical cell samples from women aged between 18 and 40 years with normal pap smear tests was extracted by standard procedure and studied for hpv presence. six type-specific primer sets for e6 hpv gene were used in a single-tube reaction for detection and identification of . respective controls and statistic analysis were included. results: the overall hpv dna prevalence was 23.5% (24/102). ten of the hpv positive women (41.6%) were infected with high-risk (hpv-16 or hpv-18) virus types; eight (33.3%) with low-risk types (hpv-6 or hpv-11), and six (25%) were infected with both high-and low-risk types (3 hpv-6/-16; 2 hpv-6/-18 or 1 hpv-6/-33). hpv prevalence among women younger than 23 years was 58.3% (14/24). for low-risk types the peak prevalence was observed in women between 35-40 years. besides age, there was a positive association between the detection rates of hpv infection and number of sexual partners and oral contraceptive use. conclusion: the used technique is simple, economic, rapid and reliable for screening studies of hpv infections. the prevalence of hpv in cytologically normal bulgarian women is similar to the reported in other countries. our findings suggest that molecular techniques for hpv detection might be useful as an adjunct to pap smear screening. were examined. mycoplasmas were determined by use of a commercially available system (liofilchem s.r.l, italy) and c. trachomatis antigen by dif (cellabs pty ltd, australia). results: of 1284 specimens, 1121 were positive. the isolated species in each age group are summarized in table 1 . in 818 cases of lower genital tract (lgt) infection, only one species was isolated. in all three age groups, mycoplasmas were the most commonly identified. in particular, the species of the isolated pathogens were: (1) group a (n = 43): mycoplasmas 52%, streptococcus spp 24%, candida spp 8%, c. trachomatis 8% and anaerobes 8%, (2) in 39 women of this group a mixed infection with three species was found. from 818 women with positive cultures, 562 (68.70%) were symptomatic, while in 256 asymptomatic women (31.20%), sexually transmitted pathogens were identified. noticeably 40.58% and 37.5% of the infected women with candida spp and c. trachomatis respectively, presented no symptoms. conclusions: (1) the rate of mixed lgt infections was 27.02% (2) asymptomatic infections were 31.20% (3) all teenagers in group a were symptomatic. ureaplasma mba size variants in woman with spontaneous preterm delivery j. spergser, a. witt, l. petricevic, p. husslein, a.m. hirschl, r. rosengarten (vienna, a) objectives: despite colonization of the lower urogenital tract, ureaplasmas reach the upper genital tract only in a subpopulation of women, and only in some of these individuals symptoms like preterm delivery ensue. while some studies indicated that certain serovars are more frequently implicated with particular diseases, others have suggested that size variability of the multiple banded antigen (mba) may contribute to ureaplasmal pathogenesis. to address these hypotheses, vaginal and placental samples from women with spontaneous preterm delivery were tested for ureaplasma species, serovars and mba size variants. methods: placenta/amnion specimens and corresponding vaginal samples from women that delivered via caesarean section less than 34 weeks of pregnancy were collected. samples were examined for ureaplasma species and serovars by cultivation and pcr assays. to determine mba size variants, pcr with primers amplifying the non-repetitive and the c-terminal repeat region of the mba gene were performed. results: out of 100 cases examined so far, 32 placenta/amnion specimens and 40 corresponding vaginal samples were positive for ureaplasmas by cultivation and pcr. while ureaplasma positive samples from the lower genital tract were carrying u. parvum (100%) and u. urealyticum (50%), colonization of the placenta/amnion was restricted to u. parvum which was only recovered from women with preterm labor or preterm prom. in contrast, ureaplasmas were not detected in samples from women with hellp syndrome, iugr and preeclampsia. as opposed to some previous studies, particular serovars were not implicated in adverse pregnancy outcome. however, significant differences between number and lengths of mba pcr products among placental and vaginal ureaplasma isolates were obvious. ureaplasma isolates from placenta/amnion membranes gave shorter pcr products than did corresponding vaginal isolates and while a majority of vaginal ureaplasma isolates were composed of multiple size variants, placental isolates were predominantly restricted to a singular mba size variant. conclusion: only u. parvum was found to be frequently present in the placenta/amnion of women with preterm delivery and certain serovars were not more frequently implicated with particular diseases. in addition, mba size variation represents an additional level of phenotypic and genetic variability beyond the serovar category which may provide an important contribution to ureaplasmal pathogenesis. use of kanamicin in the topical therapy of aerobic vaginitis g. tempera, g. bonfiglio, e. cammarata, s. corsello, a. cianci (catania, paternò, i) objectives: demonstrate the efficacy of the topical therapy with kanamicin in a new pathology called aerobic vaginitis (av). methods: eighty-one patients with clinical diagnosis of av in accordance to donders's criteria, have been included in the study. the presence at least of four of the following symptoms were taken as diagnosis of av: objective abnormal yellow vaginal discharge, foul smell (but negative to koh test), elevated vaginal ph (>5), abundant vaginal leukocytes and lactobacillary grade iia, iib and iii in accordance to donder's classification. other characteristics such as vaginal dyspareunia, vulva-vaginal itching, cervical erosion as well as the isolament of vaginal microorganisms were also documented. the patients were randomised treated, 45 with kanamycin (100 mg vaginal ovules per 6 days, consecutively) whereas, 36 with meclocycline (35 mg vaginal ovules per 6 days, consecutively). the patients were visited before starting the study, 1-2 days and 30 days after the end of the study. results: at the first follow-up the patients showed different level of symptoms reduction. particularly, reduction of presence of leukocytes, burning of vaginal mucosa and itching were statistically significant in the group treated with kanamycin with respect to the group treated with meclocycline. moreover, there was also a reduction of isolament of enterobacteriaeae (97%) in the group treated with kanamycin versus 76% treated with meclocycline. at the second follow-up, vaginal homeostasis (normalisation of ph and presence of lactobacilli) was demonstrated only in kanamycin treated group. conclusion: our data suggested that the topic use of kanamycin could be considered a specific antibiotic for the therapy of this new pathology. objectives: establishing the identity of lactobacillus species colonizing the vagina of women is of importance, because clinical studies have demonstrated an association between the presence of h 2 o 2 -producing strains of lactobacillus and a decreased prevalence of bacterial vaginosis (bv). recently, culture based studies using molecular identification methods showed that l. crispatus and l. jensenii are the most common species of vaginal lactobacilli and that colonization by these species was positively associated with a lower frequency of bv. here were report that l. crispatus can be distinguished from other lactobacilli using gram staining of vaginal smears. methods: several approaches were used to characterize 515 vaginal microflora samples obtained from 197 pregnant women at three time points in pregnancy: 1/gram stained smears from vaginal swabs, scored according to modified ison and hay criteria; 2/identification of cultured isolates obtained after anaerobic culture, identified using tdna pcr; and 3/species specific pcr for atopobium vaginae and gardnerella vaginalis. grade i specimens, representing a normal microflora, were further characterized as grade ia when only l. crispatus cell types were present, grade ib when other lactobacillus cell types were present, grade iab when both l. crispatus and other lactobacilli were present and grade ic when either gram-positive rods, small and short, or irregularly shaped gram-positive rods, with clubbing, curved edges and irregular staining (ôdiphteroid cell typesõ) were seen. results: out of the 515 samples, 86.4% showed a normal vaginal microflora (grade i), 8.9% were grade ii, 3.7% grade iii and 1.0% grade iv. based on the presence of different lactobacillus species, grade i specimens were further characterized as grade ia (36.4%), grade ib (40.9%), grade iab (13.5%) and grade ic (8.5%). this classification was supported by the finding that out of respectively 86.0% and 76.6% of grade ia and iab specimens l. crispatus was cultured while this species was present in only 13.1% and 2.6% of respectively ib and ic specimens. in addition, 55.1% of grade ic specimens contained bifidobacterium spp. conclusion: further refinement of gram stain based grading of vaginal smears is possible by distinguishing additional classes of normal microflora. these categories of gram scores may facilitate and improve future studies regarding the interpretation of clinical data and therapeutic outcome. objectives: the importance of the isolation of gardnerella vaginalis in bacterial vaginosis is well known. the involvement in male urogenital infection is uncertain, probably due to low rate of isolation for inadequated specimens process. the objective of this study is to evaluate the possible pathogenic rol of g. vaginalis in male urogenital pathology. semen is a recommended specimen in diagnosing of prostatitis. at the same time it also reflects normal genital tract microflora. although its composition may be affected by the sexual activity, there are no data regarding the changes in genital tract microflora during the sexual debut. objective: to identify possible associations between sexual experience and the reproductive tract inflammatory parameters and microflora in healthy young men. methods: a total of 72 healthy men aged 17-22 years were included and divided into 3 groups based on their sexual experience (virgins n = 13, monogamous n = 11 and polygamous n = 48). a semen sample of each subject was assessed for basic semen parameters (semen volume, sperm concentration, sperm motility), white blood cell (wbc) counts and quantitative cultures for aerobic, microaerobic and anaerobic bacteria, and possible correlations between the type of sexual experience, wbc counts and seminal microflora were calculated. results: basic semen parameters were similar in all groups. 5.5% of men had high (>1 m/ml) and 19.4% of men had medium (0.2-1 m/ml) semen wbc counts. there were no correlations between the type of sexual experience and semen wbc counts. in comparison to sexually active men in virgin subjects the total microbial counts in semen (4.4 vs 5.0 log cfu p < 0.05) and the number of different species (3 vs 5, p ¼ 0.05) were lower, however, no differences between mono-and polygamous men were found. there was a positive correlation between the total microbial concentrations and number of different species (r = 0.54; p < 0.0001). staphylococci, corynebacterium seminale, other coryneforms, gamma-and alpha-haemolytic streptococci and peptostreptococci were the most prevalent organisms. no significant differences in microflora were observed between the groups yet the gram-negative anaerobic rods were more often seen in sexually experienced men than in virgins. the subjects with high wbc counts (>1 m/ml) tended to have higher number of different microorganisms than the rest of the men (median 7 vs 4, p = 0.05). the sexual debut is associated with the enrichment of seminal microflora but not with the influx of wbc or changes in basic seminal parameters. strong positive correlation can be observed between the total microbial concentration and the number of different species in semen. sexually transmitted infections among registered female sex workers in athens under investigation were 122 sexually active non-pregnant women aged from 18 to 40 years with diagnosed cervicitis by cytological examination and presence of muco-purulent discharge. fifty five women were included in studied group -with confirmed c. trachomatis cervicitis and 67 women -in control group (with non-chlamydial/non-gonococcal cervicitis). bv was diagnosed using amsel and nugent (0-3 negative; 4-6 intermediate; 7-10 positive) criteria. by using amsel criteria bv was suggested in 11% and 6% correspondingly among women in studied and control group. by using nugent criteria bv was suggested correspondingly in 5.5% and 3%. these differences were not statistically significant and can be explained by the presence of muco-purulent discharge among studied women. objectives: helicobacter pylori is recognized as an important human pathogen but the differentiation of the species by classical phenotypic methods is really difficult. therefore the objective of this study was the development of an identification method for helicobacter species based on pcr-restriction fragment length polymorphism (pcr-rflp) analysis of the 16s and 23s rrna genes. objectives: since the first identification of helicobacter pylori by warren and marshal in 1983, members of the genus helicobacter increased to more than 26 species, which have been detected in human and animals. the construction of species specific pcr assays is difficult due to the close relatedness between different helicobacter species. the pcr-dgge technique was developed for the identification of helicobacter colonization. the aim of this study was to investigate the effect of multiple regions of the 16 rrna gene on the diagnostic efficiency pcr-dgge. methods: dna was extracted from 40 helicobacter strains, which represent 20 helicobacter species. amplification of 1.2 kb of helicobacter 16s rdna, which contains v3 and v6-7 regions, was done using previously published helicobacter genus specific primers c97 and c05. the pcr product can be used as a template for two helicobacter genus pcr assays, the v6-7 regions 16s rdna was amplified using the primers 1fc254 and 2rc686, the v-3 region was amplified using the primers bsf917 and bsr1114 published at (http://rrna.uia.ac.be/ primers). dgge analysis of the v3 and v6-7 regions was performed on 9% polyacrylamide gels containing urea and formamide gradient from 20-40% or 15-30%, respectively. electrophoresis was performed in a dcode electrophoresis unit (biorad) at constant voltage (200 v) at 60°c for 4 hours. results: dgge analysis of two regions of helicobacter 16srrna gene showed mobility patterns that allowed discrimination of most helicobacter species except those which are closely related such as h. pullorum-h. pametensis, h. ganmani-h. rodentium and h. bizozeroni-h. felis-h. salmonis. conclusions: helicobacter species are widely distributed in the gastrointestinal tract of mammals, birds and other animals. the pcr-dgge technique has proven to be an easy, inexpensive and efficient tool for the identification of helicobacter species and for the detection of colonisation by more than one helicobacter species, without the need for species-specific pcr assays. in addition, the diagnostic efficiency of this technique was increased by analysis of multiple regions of the 16s rrna gene. helicobacter spp. in chronic pancreatitis, pancreatic adenoma and pancreas cancer objectives: helicobacter spp. efficiently colonise various hostile habitats such as the stomach, colon and biliary tract of animals and man. with the exception of h. pylori, many helicobacter spp. are difficult to culture. nucleic acid based detection has thus become a method of choice for analysis of helicobacter spp. in chronic inflammatory gastrointestinal (gi) tract diseases and cancers. pancreatic exocrine cancer, with few established risk factors and very poor prognosis, is a common cause of cancer death. previously, serological studies found an association between h. pylori and pancreas cancer (pc). methods: pancreatic tumour specimens of patients with pc (n = 40), tissues of chronic pancreatitis (n = 6), pancreatic adenoma (n = 8), and benign pancreatic tissue of patients with cancers of the choledochus, colon and duodenum (n = 6), were analysed by semi-nested helicobacter-specific 16s rdna pcr. stomach (n = 23) and gallbladder (n = 12) specimens of the pc-patients were also analysed. were characterized by dna-sequence analysis. results: the helicobacter spp. pcr was positive in 19 of 40 (47.5%) pancreas cancer patients, and in the pancreas in 4 of 6 (66.7%) patients with chronic pancreatitis. adenoma and pancreas tissues of other gi-tract cancers, as well as gallbladder specimens, were all negative. four of 23 (17.4%) stomach samples of the pc-patients were helicobacter positive with sequences homologues to h. bilis by blastn analysis. dna-sequencing of 13 pcr-products amplified in pcs were closely related to h. pylori (n = 9), h. flexispira sp. (n = 3) and h. cinaedi (n = 1). two pancreatitis pcr-products matched h. pylori. conclusions: helicobacter spp. were common in pc compared with benign pancreatic tissue. helicobacter pylori infection and low igg immunoglobulin s. kokkinou, k. pavlou, a. varouta, j. villiotou, k. papaefstathiou, a. moutsopoulou, e. papafrangas (halandri athens, athens, gr) it is established that helicobacter pylori (hp) may cause haematological disturbances such as iron malabsorption and idiopathic thrombocytopenic purpura (itp). the aim of this study is to show that hp is involved in human disease in a more complicated way, producing an abnormal immunologic profile. patients and methods: this work reports the presence of a significant low value of igg immunoglobulin in 52 pts suffering from unidentified iron deficiency (id)or iron deficiency anemia (ida coexisting with megaloblastic anemia(ma), in 12 leukopenic pts and in 3 more pts having thrombocytosis, itp and polycythemia respectively. the ratio male/female was 25/42, and their age ranged from14-80 years. in the blood examination there was a10-fold titre of hp-igg type antibody in all, while in 23 there was also a high titre of hp-iga type using elisa technique. the histopathological examination of an antral biopsy showed diffuse corpus gastritis, atrophic gastritis and achlorydria secondary to hp infection that take place in gastric area. results: 52 pts had unidentified id or ida coexisted with megaloblastic anemia. serum iron, transferrin saturation, serum ferritin and b12levels were found to be significantly low. all of them had also hp infection. their anemia existed for more than 10 years, was unresponsive to treatment and recurrent in nature. twelve pts with undefined eukopenia (l:3.3 · 10 9 /l) lasting for at least 8 years, had also an hp infection. all of our pts had a significantly low value of igg immunoglobulin. mean value ranged from 551 to 868 mg/dl(normal value:850-1517 mg/dl). they subjected to eradication therapy. all but one responded well. conclusions: (1) hp infection was found to coincide with id, ida, ma and leukopenia. (2) the normalization of haematological parameters after eradication started 6 months later. (3) the essential mechanism between the hp infection and low igg is unclear and its value remains low and after the eradication therapy. (4) studies with larger samples should be done for a better evaluation of the hp infection and it's involvement in human disease. (5) hp infection acts in a catabolic way for the humans. it must be considered to search for it in haematological conditions of undefined origin. helicobacter pylori from the dyspeptic patients in thailand: diagnosis, antimicrobial susceptibility and correlation to clinical outcomes c. chomvarin, p. kulsuntiwong, p. mairiang, c. kulabkhow (khon kaen, th) objectives: to evaluate methods for routine clinical diagnostic use of helicobacter pylori infection of gastric biopsies, to study the correlation of h. pylori infection to the clinical outcomes and to study antimicrobial susceptibility by disk agar diffusion in thailand. methods: gastric biopsies, obtained from 210 patients underwent at the endoscopy unit of srinagarind hospital, faculty of medicine, khon kaen university, were diagnosed by culture, rapid urease test (rut, pronto dry) and histological examination. a true positive criteria was indicated when culture or both rut and histology tests were positive. one hundred and fifteen isolates of h. pylori isolates were tested for six antimicrobial agents by disk agar diffusion method. results: the h. pylori infection rate was 44.3% (93/210). the sensitivity vs. specificity of the culture, rut and histology were 88.2% and 100%, 95.7% and 98.3%, and 96.8% and 59.8% respectively. the prevalence of h. pylori in gastritis (gt), duodenal ulcer (du) and gastric ulcer (gu); and gastric cancer (gca) patients were 41.2%, 57.9% and 70.6%, respectively. the chi-squared test showed that gca patients were significantly more often infected with h. pylori than gt patients. the resistance of 115 h. pylori isolates to single antimicrobial agent as metronidazole (mtz), clarithromycin (clr),ciprofloxacin (cip), amoxycillin (aml), and tetracycline (te) were detected in 21.7, 0, 5.2, 1.7 and 0 percent, respectively. the combination resistance to mtz & clr, mtz & cip, mtz & aml, mtz & te; and clr & cip were detected in 1.7, 1.7, 0.8, 1.7 and 0.8 percent, respectively. the 2.5 per cent of h. pylori isolates were resistance to three or more antimicrobial agents. conclusion: rut method was highly sensitive and specific and appropriate for routine laboratory use. the correlation of the gca patients to h. pylori infection was significant difference compared to gt patients. the high resistance rate to metronidazole indicated that the effective for eradication of h. pylori by metronidazole should be considered in the clinical management of h. pylori infection. use of an indirect immunofluorescence test for the detection of caga in h. pylori c. scherer (essen, d) objectives: the cytotoxin associated gene (caga) of h. pylori is a frequently discussed virulence factor which codes for a 128-kda cytotoxin associated antigen (caga). the caga-gene is usually detected by pcr methods. publications about phenotypic methods for the detection of caga are rare. aim of our investigation was to compare a slightly modified, recently published indirect immunofluorescence test (iift) for the detection of caga with commonly used pcr assays. methods: h. pylori atcc 43504 (caga+/caga+) was used as positive control strain, atcc 51932 (caga+/caga+) as negative control strain. nineteen clinical isolates of h. pylori were examined for their caga-and caga-status. caga was detected by iift using monoclonal anti-caga-antibody from mice, which were visualised for immunofluorescence microscopy with fitc-conjugated anti-mouse-antibody from rabbits. dna was extracted by a standard phenol-chlorophorm procedure after proteinase k treatment of the cells from a 4-day old culture. 7 primer pairs and their published protocols were used: f1/b1 and d008/r008 as the most described primer pairs and further five primer pairs. all tests were performed in duplicate. results: 4 from 19 (22%) strains were caga positive in the iift, and also caga positive in each pcr. 13 from 19 (68%) strains were negative in the iift. 10 from these 13 were caga-negative in each pcr and 3 showed varying results in the pcr-assays. 2 from 19 ( 10%) strains could not be interpreted by iift, both showed varying results in the pcr-assays. the iift might be a simple to use tool in the determination of the caga-status, since it showed no false positive result in neither tested pcr. the differing results in iift and pcr suggest that genotypic positive strains may not always express caga in vitro and maybe also not in vivo. therefore the caga-iift may be an interesting parameter for the determination of strain virulence in addition to pcr-assays. evaluation of elisa serology for the primary diagnosis of helicobacter pylori infection in turkish dyspeptic patients: a prospective study from a developing country b. kocazeybek, y. erzin, s. altun, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: hp is recognized as an important human pathogen by virtue of it's association with peptic ulcer disease, gastric cancer and gastric lymphoma and the high prevalence of infection worldwide. both invasive and noninvasive tests are available to diagnose hp infection but there is still no single gold standard. the aim of the present study was to evaluate the diagnostic accuracy of a commercially available anti-hp igg elisa kit for the primary diagnosis of hp infection. materials and methods: a total of 185 patients who were referred to our endoscopy unit were included. according to our gold standard, a patient was classified as being hp(+) if the culture and/or both histology, rapid urease test were positive and as hp()) only if all of these tests remained negative. standard methods were used to calculate sensitivity, specificity, predictive values of positive and negative results and 95% confidence intervals of these values. results: after excluding patients with discordant results131 of 152 patients (86%) were hp (+). the sensitivity, specificity and diagnostic accuracy of the igg-elisa were 97%, 67%, 93% respectively. conclusion: due to it's lack for specificity, we conclude that quantitative anti-hp elisa test may not be a suitable alternative for primary diagnosis of hp in our country, where the prevalence of infection is still very high. comparison of two different stool antigen tests for the primary diagnosis of helicobacter pylori infection in turkish dyspeptic patients b. kocazeybek, y. erzin, s. altun, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: to assess the reliability of two different enzyme immunoassays in detecting the helicobacter pylori(hp) status in stool specimens of turkish dyspeptic patients. materials and methods: 151 patients [74 with non-ulcer dyspepsia(nud), 64 with duodenal ulcer(du),13 with gastric cancer]who were admitted to the endoscopy unit of istanbul university, cerrahpasa medical faculty for upper gastrointestinal endoscopy due to dyspepsia were enrolled in the study. hp infection was confirmed in all patients by histology, rapid urease test(rut) and culture. a patient was classified as being hp-positive if the culture alone or both histology, rut were positive in the absence of a positive culture and as negative only if all of these tests remained negative. stool samples of patients were obtained in order to assess the reliability of a monoclonal (femtolab h. pylori, connex, martinsried, germany) and a polyclonal (premier platinum hpsa, meridian diagnostics inc., cincinnati, usa) stool antigen test and to compare their diagnostic accuracies. the v 2 test was used for statistical comparison of the values. results: using a cutoff 0.19 for femtolab h. pylori and 0.16 for premier platinum hpsa the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy of the former and latter tests were 93%, 90%, 98%, 68%, 93% and 84 %, 67%, 94%, 40%, 81% respectively. the sensitivity, specificity, npv and diagnostic accuracy of the former test were significantly superior to the latter one's (v 2 = 3.98; p < 0.05 for sensitivity and v 2 = 15.67; p = 0.000 for specificity, v 2 = 15.78; p = 0.000 for npv and v 2 = 6.37; p = 0.012 for diagnostic accuracy). the bacterial load did not affect the sensitivity of both tests. conclusions: the monoclonal femtolab h. pylori, using a cutoff 0.19 is a very sensitive, specific and easy to perform diagnostic tool for the primary diagnosis of hp infection in turkish, dyspeptic patients. evaluation of two different enzyme immunoassays for detection of helicobacter pylori in stool specimens of turkish dyspeptic patients after eradication therapy b. kocazeybek, y. erzin, s. altun, s. saribas, a. dobrucali, m. aslan, s. erdamar, a. dirican (istanbul, tr) aim: to assess the reliability of two different enzyme immunoassays(eias) in detecting the hp status in stool specimens of turkish, dyspeptic patients in the post-treatment period. materials and methods: 48 patients with non-ulcer dyspepsia(nud) who were positive for hp underwent a one week regimen of triple therapy. stool samples of patients were obtained 2-6 weeks and 13c-urea breath test(ubt) was performed 6 weeks after eradication therapy in order to assess the reliability of a monoclonal (femtolab h. pylori, connex, martinsried, germany) and a polyclonal (premier platinum hpsa, meridian diagnostics inc., cincinnati, usa) stool antigen test and to compare their diagnostic accuracies. the v 2 and fisher's exact tests were used for statistical comparison of the values. results: using a cutoff 0.19 for femtolab h. pylori and 0.16 for premier platinum hpsa the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy of the former and latter tests were 93%, 67%, 56%, 96%, 75% versus 87%, 33%, 37%, 85%, 50% 2 weeks and 93%, 88%, 78%, 97%, 90% versus 67%, 70%, 50%, 82%, 69% 6 weeks after completion of eradication therapy, respectively. both at the 2nd and 6th weeks in the post-treatment period the diagnostic accuracy of femtolab h. pylori was significantly superior to premier platinum's (75% versus 50%, v 2 = 6.4 ;p = 0.011, and 90% versus 69%, v 2 = 6.316; p = 0.012 respectively). conclusions: the monoclonal femtolab h. pylori, using a cutoff 0.19 is sensitive and specific enough to monitor hp infection in turkish, dyspeptic patients 6 weeks after completion of eradication therapy. carriers develop serious gastroduodenal diseases. so, it is important to define whether strains with specific genotype are associated with the clinical outcome. the aim of this study was to determine the distribution of the caga, cage and vaca subtypes of hp in patients with various gastroduodenal diseases in turkey, and to explore the association between genotype and the clinical outcome of infection. methods: 93 strains of hp were isolated from cultures and pathological archives of 30 patients with non-ulcer dyspepsia (nud), 30 with duodenal ulcer(du) and 33 with gastric carcinoma. the caga, cage and vacas1a/s1b, m1/m2 genotypes were determined by polymerase chain reaction methodology. results: there were no statistically significant difference among three different clinical outcomes according to the positive rates of vaca s1b, s2 and vaca m1/m2. but the positive rates of vaca s1a, caga and cage were 66.7% (20/30), 50% (15/30) and 26.7% (8/30) in nud; 93.3% (28/30), 83.3% (25/30) and 66.7% (20/30) in du; 87.9% (29/33), 84.8% (28/33) and 81.8% (27/33) in gastric cancer group, respectively, showing statistically significant difference (p = 0.015, p = 0.002, p = 0.001, respectively). the prevalence of vacas2 and m2 in nud patients was higher than that in duodenal ulcer group, but this difference was not statistically significant. conclusions: helicobacter pylori caga, cage and vacas1a genotypes have a significant relation with duodenal ulcer and gastric carcinoma in turkish population. there were no statistically significant association of the vaca s1b, s2 and vaca m1/m2 strains among disease groups. clinical relevance of icea1, icea2 and baba2 genotypes of helicobacter pylori in turkish patients with non-ulcer dyspepsia, duodenal ulcer and gastric cancer b. kocazeybek, y. erzin, v. koksal, s. erdamar, s. altun, a. dobrucali, a. oner (istanbul, tr) objectives: the clinical relevance of the helicobacter pylori(hp) adherence factor, baba gene, and the other virulence determinant, icea gene, has not yet been determined in turkish clinical isolates to date. therefore, the aim of this study was to evaluate the prevalence of icea1, icea2 and baba2 hp genotypes in turkish patients with non-ulcer dyspepsia(nud), duodenal ulcer(du) and gastric cancer. methods: 30 strains were isolated from patients with nud, 30 were isolated from individuals with du and 33 were isolated from patients with gastric cancer. bacterial dna was extracted from cultures and hp positive paraffin-embedded biopsy samples and genotyping of icea and baba2 was carried out utilizing polymerase chain reaction molecular technique by using specific primers. results: the icea1, icea2 and baba2 genotypes were detected in 75.3% (70/93), 24.7% (23/93), 53.8% (50/93) respectively, of the hp strains studied. the prevalence of icea1 in gastric cancer patients was higher than that in nud group, 84.8% (28/33) and 60% (18/30), respectively (p = 0.045). the prevalence of icea2 in nud group was higher than that gastric cancer patients, 40% (12/30) and 15.2% (5/33), respectively (p = 0.045). the positive rates of baba2 were 23.3% (7/30) in nud; 46.7% (14/30) in du and 87.9% (29/33) in gastric cancer group, showing statistically significant difference (p = 0.0001). conclusions: baba2 genotype distribution was evaluated in different gastric pathologies in turkish population and was a good marker for the presence of du and gastric adenocarcinoma. thus, our current results confirm previous experimental studies indicating a central role of hp's adhesin and lewis antigens in the pathogenesis of ulcer disease and adenocarcinoma. influences of interleukin 1b and interleukin 1rn polymorphisms on the development of non-ulcer dyspepsia, duodenal ulcer and gastric cancer in turkish population b. kocazeybek, y. erzin, v. koksal, s. erdamar, s. altun, a. dobrucali (istanbul, tr) objectives: il-1b and il-1rn gene polymorphisms (which encode interleukin 1b and interleukin-1 receptor antagonist, respectively) have been associated with development of gastric atrophy and with increased risk of gastric carcinoma. the aim of this study was to determine the influences of host il-1b -31tc, il-1b -511ct and il-1b rn gene polymorphisms on the development of various gastroduodenal pathologies in 93 helicobacter pylori (hp) positive turkish patients [(30 with nonulcer dyspepsia (nud), 30 with duodenal ulcer (du) and 33 with gastric cancer (gc)]. methods: genomic dna was extracted from hp positive paraffin embedded gastric biopsy samples of 30 nud, 30 du and 33 gc patients. il-1b and il-1b rn gene polymorphisms were analysed by polymerase chain reaction-restriction fragment length polymorphism. these polymorphic sites include promoter regions of il-1b at positions-511 (c-t transition) and -31 (t-c transition), and il-1 rn variable tandem repeats (intron2 vntr). results: there were no statistically significant difference among three clinical outcomes in the frequencies of il-1b -511ct + tt and il-1b -31 tc + cc genotypes (as called proinflamatory genotypes) (p = 0.161 and p = 0.077, respectively). the prevalence of the proinflamatory il-1b rn 1/2 + 2/ 2 alleles in gc group was higher than that in nud and du groups. the frequencies of il-1b rn 1/2 + 2/2 alleles were 20.0% (6/30) in nud; 31.0% (9/30) in du; and 57.6% (19/33) in gc group, showing statistically significant differences (p = 0.006). conclusions: there were no statistically significant difference among three clinical outcomes in the frequencies of il-1b -511ct + tt and il-1b -31 tc + cc genotypes, whereas proinflamatory il-1b rn *2 allele (1/2 + 2/2) was associated with gastric cancer in turkey. rapid detection of clarithromycin-resistant helicobacter pylori in patients with dyspeptic by fluorescent in situ hybridisation (fish), in comparison with e-test m. moosavian, s. tajbakhsh, a. samarbafzadeh (ahwaz, ir) objectives: isolation of h. pylori from more than 90% of the patients with peptic ulcer have demonstrated a strong relationship between these bacteria and created ulcers or dyspeptic. recovery of the patients will be accelerated, if clarithromycin be added to therapeutic protocol. the objectives of this study are: 1-rapid detection of the susceptible or resistant strains of helicobacter pylori to clarithromycin, in patients with dyspeptic by fish technique. 2-comparison of the results of fish & epsilometer test ( e-test) techniques. methods: frozen -sections of gastric biopsies of 50 patients with dyspeptic were hybridized in situ by 5 fluorescent oligonucleotide probes (fish). the prepared slides were examined under a fluorescent microscope, after staining with dapi. also, susceptibility and resistance of isolated strains of h. pylori to clarithromycin were determined by e-test. both results of e-test and fish techniques were compared in final. results: 25 out of 50 examined gastric biopsy samples were positive for h. pylori by fish. out of these 25 h. pylori strains, 17 strains (68%) were susceptible, 6 strains (24%) were resistant and 2 strains (8%) were mixture of susceptible and resistance strains to clarithromycin. this study showed no significant different between fish and e-test results, in view of number in susceptible or resistance strains. conclusions: since the patient gastric biopsies are not routinely cultured in the most of clinical laboratories, also, for isolation of h. pylori, it is needed to enriched and selective media and the other way, clarithromycin is an expensive drug, so, it looks like that fish technique is a suitable method for replacing of the culture, antibiotic sensitivity test and or e-test method. diagnosis of atrophic gastritis from a blood sample p. douramani, s. mavrea, a. arkas, a. adamopoulos, e. anastasakou (athens, paros, gr) introduction: the majority of gastritis is related to infectious agents, where h. pylori is the most important and common. h. pylori gastritis could proceed, over time, to atrophic form of gastritis a serious disease that increases the risk of peptic ulcers and gastric cancer. a new test panel was developed for nonendoscopic diagnosis of atrophic antrum and corpus gastritis from a blood sample. aim: to investigate whether atrophic gastritis can be diagnosed and typed non-endoscopically if the serum levels of pepsinogen i (s-pgi) and gastrin-17 are assayed in connection with h. pylori testing. materials and methods: the study population consists of 50 selected dyspeptic outpatients with (cases) or without (controls) advanced (moderate or severe atrophic gastritis) who underwent a diagnostic gastroscopy for dyspeptic symptoms. of the 50 selected patients,28 (cases) had an advanced atrophic gastritis or a resected stomach. of these 28 cases, 4 had an advanced antrum-limited atrophic gastritis, 6 had resected antrum,15 had a corpus limited advanced atrophic gastritis. two patients had an advanced atrophic gastritis in both the antrum and corpus (multofocal atrophic gastritis) and the whole stomach was removed in one patient. the controls comprise 22 patients of whom 10 had a non atrophic h. pylori gastritis; the antrum and corpus were normal and healthy in 12 patients. the sample for ôpostprandialõ gastrin -17 (s-g-17 prand) was taken 20 min after a protein drink. the s-g-17,s-pgi and igg class antibodies to h. pylori were determined using elisa methods. results: a low s-pgi (<25 mg/l) was found in 15 of 18 patients (83%) with and in 2 of 32 patients (6%) without corpus atrophy. a low s-g-17 prand (<5 pmol/l) was found in all 4 patients with h. pylori associated antral-atrophy and in 5 of 7 patients (71%) with resected antrum but in one of 10 patients (10%) with h. pylori-related non-atrophic gastritis. the mean values of both s-g-17 prand and s-pgi decreased with increasing grade of antral and corpus atrophy respectively. among all patients with atrophic gastritis,24 of 28 patients (86%) had a low s-pgi and/or a low s-g-17 prand with positive igg h. pylori antibodies. such low values were found only in one of the 22 (4.6%) control patients. conclusions: low serum levels of g-17 and s-pgi are diagnostic markers of atrophic antral and corpus gastritis, respectively. a low s-g-17prand is a sigh of the multifocal or antrum-limited atrophic gastritis in patients infected with h. pylori. helicobacter pylori in gastric mucosa: an ultrastructural study e. ozbek, a. ozbek, h. dursun, o. vuraler (erzurum, tr) objectives: helicobacter pylori are extraordinary among bacteria in their ability to colonize the human gastric mucosa, an inhospitable acidic environment, and to stay on there for long periods as decades in despite of host immune and inflammatory responses. in this study, we aimed to research where h. pylori are found within the stomach by transmission electron microscopy (tem). methods: gastroscopic biopsy samples from patients suspected with gastritis or peptic ulcer were processed for both of pcr and tem. the presence of h. pylori in biopsy samples was investigated with pcr method by using specific 23 s ribosomal rna primers. the samples for electron microscopic examination were fixed in 3% glutaraldehyde, postfixed in 1% osmic acid, dehydrated in acetone, and embedded in araldite. plastic blocks of positive samples for the presence of h. pylori confirmed by pcr were cut with a nova lkb bromma ultratome. thin sections were stained with uranyl acetate and lead citrate, and examined by using a jeol 100 sx transmission electron microscope. results: we observed that cross-and longitudinal-sectioned h. pylori within the mucus layer overlying gastric epithelium, in the intercellular spaces between gastric epithelial cells (figure 1), and between two microvilli protruding from the apical surface of an epithelial cell into the gastric lumen. apart from the extracellular h. pylori, we found also intravacuolar h. pylori within the epithelial cells of the gastric mucosa. we observed h. pylori engulfed by pseudopod-like structures at the apical part of the epithelial cells. there were also late endosomal vacuoles containing h. pylori within the deeper cytoplasmic parts of the cell. conclusion: although h. pylori is considered generally a noninvasive pathogen, our electron microscopic observations prove that h. pylori is able to invade gastric epithelial cells, and to enter large cytoplasmic vacuoles. that h. pylori is able to be found intracellularly might explain why the eradication of h. pylori infection is difficult with the antibiotics such as gentamicin that cannot easily cross the eukaryotic cell membrane. abbreviations for figure 1: h, helicobacter pylori; n, nucleus of a gastric epithelial cell; e, cross-sectioned epithelial cells; nm, nuclear membrane; is, intercellular space. bar = 0.5 micrometre helicobacter pylori and association with histological findings in endoscopic biopsies in western greece c. petropoulos, e. jelastopulu, m. kardara, s. spiropoulos (erymanthia, patras, gr) objectives: there is evidence that helicobacter pylori (h. pylori) infection is strongly associated with gastric and duodenal lesions in general population. in this study we reviewed gastroscopic and histological records of patients who underwent gastroscopy for multiple reasons in a tertiary hospital in order to evaluate the frequency of h. pylori colonization in biopsy specimens and to examine the association between h. pylori infection and histological findings. methods: the medical records of 251 patients who presented to the general hospital ôagios andreasõ in patras, western greece, during the period 2002-03 for upper gastrointestinal complaints and other specified symptoms were reviewed for the presence of h. pylori. all patients have been submitted to endoscopy with biopsy mainly of the gastric mucosa. the statistical analysis was performed using the spss. results: gastroscopies and biopsies were undertaken in 251 patients (149 men, 102 women; mean age 58.5, range 16-95 years). in 140 (59.1%) of the patients dyspepsia was the main indication for gastroscopy, followed by 44 (18.6%) patients with anaemia, 12 (5.1%) patients with black faeces and 11 (4.6%) patients with reflux. usual macroscopic findings were oedema (70%), hyperaemia (50%), erythema (22.3%), ulcer (16.8%) and erosions (7.5%). the histological examination revealed chronic gastritis in 209 (83.3%) subjects (in 39% of mild form, in 50% of moderate and in 11% of severe form), atrophic chronic gastritis in 8 (3.1%) and adenocarcinoma in 10 (3.9%) patients. the overall colonization of h. pylori was detected in 80 (34.2%) subjects. in case of h. pylori positive patients, chronic gastritis was found in 15.4% of mild form, in 61.5% of moderate and in 23.1% of severe form, whereas in absence of h. pylori infection the histological type of chronic gastritis was 52.5%, 43.2% and 4.3% respectively. in case of chronic gastritis of severe form h. pylori colonization was found in 75% (p < 0.001), whereas regarding the mild form h. pylori presence was observed only in 14% (p < 0.001). conclusions: the study reveals that h. pylori infection is strongly associated with the severe form of chronic gastritis. thus, for the optimal management and treatment of the patients with gastritis a biopsy of the mucosa appears to be necessary. objective: the ultimate aim of the study is to explore potential natural products as alternative for treatment of infections from staphylococcus aureus including methicillin resistant staphylococcus aureus (mrsa). the specific objective of the paper is to assay for molecular changes in the genes of mrsa isolates upon inhibition by natural products. five genes of mrsa isolates study include meca, meci and mecri adab and sav1017 and the non-mrsa isolates were adab and sav1017 genes. the adab gene encodes for methyltransferase activity for dna repair mechanism while the sav1017 is the cell wall protein gene. methods: mrsa and non-mrsa isolates were obtained from patients receiving treatment in malaysian hospitals. these isolates were subjected to growth in methanol extract of marine seaweed. inhibition assay of extract on isolates including several genera of the gram-negative bacteria, were determined by the disc diffusion and minimum inhibitory (mic) methods. treated and untreated isolates inhibited by extract were subjected to pcr amplification, followed by commercial sequencing, rt-pcr assay of the mrna followed by sequencing of the cdna and blastn analysis with the genbank sequences. results: the inhibition assay of methanol extract showed activity only in mrsa and non-mrsa isolates but not in gram negative isolates. the nucleotide sequences changes were seen in four genes of treated mrsa isolates which were the meca, mecri, meci and the adab. the nucleotide changes in non-mrsa and mrsa were only seen in the adab gene but not the sav1017. the blastn analysis showed variation in nucleotide changes in all genes involved in mrsa phenomenon. conclusions: these preliminary results utilizing genomics for study on nucleotide sequences of pathogen can aim at utilizing the biomolecules from natural products to target the affected nucleotides so as to inhibit growth of the organism. the research activity has the potential of speeding up drug discovery programme and the nucleotide changes in several genes treated with the extract indicates the potential target sites of the extracts. the inhibitory effect of extract on the nucleotide of some genes remained unchanged in the pcr and rt_pcr assay, indicating selective effect of extracts on the genes and the extracts has the potential to be applied as antibacterial agents. implication of the findings is directed towards discovery of antibacterial drug target sites, which warrants further study. phenotypic and genotypic characteristics of staphylococcus aureus strains defective in species-specific proteins a. luczak, j. krzyszton-russjan, k. nowak, w. hryniewicz (warsaw, pl) objectives: the objective of this study was to characterise phenotypically clumping factor and/or coagulase defective s. aureus. methods: the study was performed on 170 s. aureus isolates identified between 1996 and 2004 as clumping factor (cf) or free coagulase (fc) defective by clinical microbiology laboratories and submitted to national institute of public health in warsaw for verification. reidentification included detection of clumping factor, coagulase, thermonuclease, and ability to ferment mannitol. antimicrobial susceptibility testing was performed by diskdiffusion method according to the nccls. glycopeptides susceptibility was determined by screening method, mic evaluation and population analysis. pcr reactions were performed to confirm the presence of species specific genes, as cfa, cfb, coa, nuc, spa. to obtain isolates clonality, multiple-locus variable-number tandem repeat analysis (mlva) was applied. results: based on the phenotypic reidentification methods, 114 (67%) isolates were phenotypically defective in respect to clumping-factor or coagulase production. all but one isolate exhibited the presence of the genes encoding cf or fc. seventyfive (67%) isolates were resistant to methicilin (mrsa) and multiresistant. twenty-six (22.8%) isolates showed reduced glycopeptides susceptibility in pap. all hvisa and hgisa isolates were mrsa and all were defective mainly in clumpingfactor production. thirty-one different mlva groups were distinguished. the a and l groups were the most common and variable. the a type was the most prevalent in hvisa/hgisa isolates and found in 18 centres. conclusion: the clonal spread of s. aureus defective in species specific proteins was revealed. the correlation between decreased susceptibility to glycopeptides and defective species specific proteins was observed. detection of panton-valentine leucocidin and other staphylococcal toxins using oligonucleotide arrays s. monecke, r. ehricht (dresden, jena, d) objectives: the recent outbreaks of community acquired mrsa (cmrsa) harbouring panton-valentine leucocidin (pvl) caused serious concern worldwide. in order to screen clinical isolates, we designed and tested an oligonucleotide array which can detect both components of this virulence factor, six staphylococcal superantigens and the two exfoliative toxins as well as several resistance genes and species-specific controls. methods: twenty-two clinical isolates (two suspected cmrsa and twenty randomly selected) from swabs obtained from a dermatological clinic in saxony/germany were cultured. dna was isolated and subjected to a linear multiplex amplification incorporating biotin-labeled dutp into the amplicon. hybridization of the amplicons to the array was detected using an enzymatic precipitation reaction. results: in two cases of chronical furunculosis, pvl-positive cmrsa were identified. one case was a soldier returning from a peace mission to kosovo where he apparently contracted the disease, the other one was his spouse. in three other cases of furunculosis/abscesses, pvl-positive but methicillin-susceptible s. aureus were found. staphylococcal enterotoxins were found in four, toxic shock syndrome toxin in three isolates, and exfoliative toxin a in one isolate. in two cases, multiple toxins were found (pvl + tst + entc and entb + entk + entq + eta). conclusions: while epidemic cmrsa strains often can presumably be identified due to their resistance profile (methicillin, tetracycline, fusidic acid), there is no easy method to detect pvlpositive, methicillin-susceptible s. aureus. therefore, strains carrying pvl are more common than previously suspected. the high prevalence of this virulence factor as well as of other toxins found in the present study warrants further investigations. characterisation of methicillin-resistant staphylococcus aureus harbouring the panton-valentine leucocidine a. anders, m. kaase, s. friedrich, s.g. gatermann (bochum, d) objectives: until now methicillin-resistant staphylococcus aureus (mrsa) have been known only as a major problem in hospitals. lately, a new kind of mrsa has been described. this mrsa is responsible for community-acquired infections (c-mrsa) and harbours the determinant for the panton-valentine leucocidine (pvl).as there are descriptions of c-mrsa possessing the pvl throughout the world with different mlst types we wanted to characterize eleven c-mrsa from our area possessing the gene for panton-valentine leucocidine. methods: the isolates were found from 1999 until the summer of 2004, the gene encoding pvl was verified by pcr.we determined the pfge pattern, the mlst type, the sccmec type and the agr allele type of the eleven isolates. results: so far all isolates belong to the st 80 which has been also described elsewhere in germany and in france but differs from the types found in the us (1 and 8) or in australia (30 and 298). they possess a sccmec element of type ivb and the agr3 allele type. all isolates were resistant to oxacillin, which was determined by meca pcr, and all were resistant to fusidic acid. only two were additionally resistant to erythromycin. no resistance could be found to fluoroquinolones.the average age of patients was 47 years and they all had severe communityacquired abscesses of the skin or soft tissue that caused hospital admission. conclusion: mrsa cannot be longer considered as affecting only hospitalized and elderly patients but we should also be aware of them in community-acquired infections. accessory gene regulator (agr), and its locus of s. aureus is a quorum-sensing gene cluster of five genes (hld, agrb, agrd, agrc, and agra) that upregulates production of secreted virulence factors, including the alpha-, beta-, delta-hemolysins, and downregulates production of cell-associated virulence factors. s. aureus strains can be divided into 4 major agr groups (agr i-iv) on the basis of a polymorphism in agrd and agrc. the purpose of this study is to know the proportion of agr i, ii, and iii polymorphisms and to compare clinical characteristics between group ii and non-group ii polymorphism of methicillin-resistant staphylococcus aureus (mrsa) strains in a tertiary care teaching hospital in korea. methods: isolates were identified as s. aureus by conventional methods. susceptibility to methicillin was determined by the nccls guideline. agr locus was identified using restriction fragment length polymorphisms (rflps) analysis of agr bdc amplicons with drai digestion. the factors assessed included the patients' demographics, comorbidities (diabetes mellitus, congestive heart failure, peripheral vascular disease, dialysisdependent renal failure, cirrhosis, malignancy, and alcoholism), infection site (central catheter-related bacteraemia, bacteraemia of unknown origin, device, endocarditis, intraabdominal, respiratory, skin, urine, and ear), receipt of mechanical ventilation and operation, the presence of nosocomial infection and treatment failure, creatinine level, and mortality. results: a total of 18 strains from 18 mrsa-infected patients (7 males and 11 females) were evaluated. their mean age was 50.5 ± 24.3 years old. there were 4 (22.2%), 12 (66.7%), and 2 (11.1%) strains in agr group i, ii, and iii polymorphism, respectively. only nosocomial infections were statistically significant clinical parameter according to univariate analysis (p = 0.009) and multivariate analysis (or, 22.0 (1.540 ) 314.292); p, 0.023). conclusion: agr group ii was most prevalent in this study and nosocomial infections were correlated with group ii polymorphism. this result suggests virulence and nosocomial spread of mrsa strains are originated from group ii polymorphism. a recruit of more strains will increase and clarify the clinical difference between agr groups. distribution of toxic shock syndrome toxin-1, exfoliative toxins and enterotoxins seg and sei among methicillin-resistant staphylococcus aureus clonal types v. chini, g. dimitracopoulos, i. spiliopoulou (patras, gr) objectives: staphylococcus aureus produces a variety of exotoxins including the toxic shock syndrome toxin-1 (tsst-1), the exfoliative toxins eta and etb and the enterotoxins seg and sei. the seg and sei genes (seg and sei) coexist in the same operon. the aim of this study was to investigate the presence of genes encoding the tsst-1 (tst), eta (eta), etb (etb), seg (seg) and sei (sei) among methicillin-resistant s. aureus (mrsa) clinical isolates, in relation to their clonal types. methods: the mic of oxacillin was determined by the agar dilution method in mueller-hinton agar supplemented with 2% nacl according to the guidelines of nccls. pbp2a production was investigated by a latex agglutination test (biomerieux) in all s. aureus isolates with mic >0.5 mg/l. pcr amplification was performed for the detection of meca gene to the aforementioned isolates, from different patients, during 2001-2003, resulting to 160 mrsa. the presence of tst, eta, etb, seg and sei genes was also investigated by pcr. clonal types were determined by pfge of chromosomal dna smai digests. results: among the 160 mrsa, 35 (22%) carried both seg and sei genes, 24 (15%) isolates carried the tst, 2 (1%) the eta, whereas no strains exhibited the etb gene. precisely, during 2001 the tst gene coexisted in 12 out of 13 seg/sei-positive strains. eleven strains belonged to pfge type a, whereas the other two to type b, mainly associated with wound infections. eight mrsa in 2002 belonging to pfge type a, carried tst, seg and sei. two more strains carrying seg/sei, belonged to pfge type c. two out of 3 seg/sei-positive additional mrsa which were characterized as a new clone f, carried also eta. in 2003, four mrsa carrying the tst, seg and sei belonged to clone a, whereas 5 more carrying the seg/sei were characterized as pfge type c. most isolates were associated with wound infections and abscesses. conclusions: during the study period (2001) (2002) (2003) the incidence of the tst gene carriage among mrsa was reduced from 26% to 6%, while the seg/sei genes were almost constantly present. the distribution of these genes was mainly associated with strains of clonal type a, exhibiting a drift towards clone type c predominant during the last two years. detection of decreased susceptibility to glycopeptides in staphylococcus aureus using tablet (disc) prediffusion s.v. nielsen, j.b. casals (taastrup, dk) objectives: the aim of the study was to develop a screening and confirmatory method to detect staphylococcus aureus with decreased susceptibility to glycopeptides (hvisa/visa and gisa). methods: the glycopeptide resistance of 20 staphylococcus aureus strains from our strain collection including well-known resistant strains (atcc 700788, atcc 700789, atcc 700698, atcc 700699) was examined. we compared: (i) zone sizes of vancomycin 5 lg and teicoplanin 30 lg neo-sensitabs (rosco diagnostica) when prediffused for 2 h (with disc) + 18 h (without disc) on mueller-hinton and bhi, both media supplemented with 5% horse blood, (ii) agar dilution mics (iii) zone sizes around cefoxitin 60 lg neo-sensitabs. the inoculum used was mcfarland 0.5 and the plates were incubated for 24 h at 35°c. results: regression lines were prepared and there was a good correlation between mics for vancomycin and teicoplanin and the corresponding zone sizes. all hvisa/visa/gisa strains tested showed zones less than 15 mm with cefoxitin 60 lg and zones £ 21 mm (teicoplanin) and/or £ 23 mm (vancomycin) on mh blood agar, corresponding to mics > 2 lg/ml for both antimicrobials. on bhi agar the zones were £ 17 mm (teicoplanin) and/or £ 21 mm (vancomycin) for the hvisa/visa/gisa strains corresponding to mics > 4 lg/ml for both antimicrobials. conclusion: zones obtained by agar diffusion methods using prediffusion with vancomycin and teicoplanin neo-sensitabs correlated with mics and predicted hvisa/visa/gisa strains. for screening high resistance to cefoxitin (zones <15 mm) may be used to select strains for further testing in a daily routine laboratory. the system should be evaluated further in the routine diagnostic laboratory. high rate of vancomycin intermediate staphylococcus aureus among methicillinresistant isolates in taiwan from 637 patients in the tri-service general hospital in taiwan were screened for vancomycin resistance and isolates with reduced susceptibility to vancomycin were further studied for their characteristics. methods: brain heart infusion agar plates containing vancomycin (5 lg/ml) (bhia-va5) were using for vancomycin resistance screening. minimum inhibitory concentration (mic) of vancomycin was determined on both bhia and the standard mueller-hinton agar (mha) on screened-positive isolates. for strains with mics equaled to 8 lg/ml, characteristics including population analysis, susceptibility to triton x-100 induced autolysis, van gene and pulsed field gel electrophoresis (pfge) typing were further performed as previously described. background: mrsa isolates with decreased susceptibility to glycopeptides (gisa) have been reported worldwide since 1997. most strains were isolated from catheters and other foreign bodies in patients treated with glycopeptides. we aimed to determine the prevalence of gisa isolates in a belgian hospital where mrsa has been endemic for many years. methods: all mrsa isolates collected between 09/98 and 06/ 04 were screened for reduced susceptibility to glycopeptides on mueller-hinton agar and 5 mg/l teicoplanin (mh-teico) as recommended by the casfm. the macromethod etest (bhi agar, 2 mcfarland (mf) inoculum, 48 h incubation) was performed as secondary screening for all isolates which grew 4 colonies on mh-teico. gisa/h-gisa phenotypes were confirmed by e-test mic determination on mh agar with 0.5 mf and 24 h incubation as well as by vancomycin (v) and teicoplanin (t) population analysis profiles (pap). all confirmed gisa/h-gisa isolates identified were compared by pfge to other strains identified in belgium in order to delineate their clonality. results: among 520 mrsa strains (from 468 patients), 14 isolates showed growth on mh-teico and 8 of these (from 7 patients) yielded a positive macromethod e-test (mic 8 lg/ml for v and t). seven were confirmed by pap as h-gisa and one as a gisa (t mic: 32 lg/ml; v mic: 8 lg/ml). origins of the isolates were: lower respiratory tract (4), urine (2) and wound (2). all h-gisa strains had a similar antibiotic resistance phenotype by disk diffusion (gentamicin, rifampin, erythromycin, clindamycin, ofloxacin) . by molecular analysis, the h-gisa/ gisa isolates clustered in two different pfge groups a (n = 6) and g (n = 1). the a pfge group was previously described in h-gisa isolates in belgium. in this study, it was subdivided in 2 types, both including 3 strains. one pfge type resulted in a small outbreak in 3 patients during a stay in icu. all 5 other isolates were sporadic with no apparent geographic or temporal relationship between cases. conclusion: gisa/h-gisa were found in only 7 out of 468 (1.5%) mrsa-positive patients. none of them presented with severe infection nor had any previous known exposure to glycopeptides. in view of the low prevalence of gisa/h-gisa in our hospital, screening should not be performed systematically but rather on an individual basis taking into account clinical data and risk factors. whenever the occurrence of gisa is considered, teico-mh agar appears as a suitable first screening approach. a case-control study to evaluate the economic outcome of early po linezolid in patients with methicillin-resistant staphylococcus aureus infections oral therapy with linezolid (lzd) has the potential to decrease hospital costs and shorten length of stay of patients with grampositive infections who would otherwise continue to receive iv therapy. objective: to evaluate the impact of early po conversion to lzd in patients receiving traditional iv therapy for methicillinresistant s. aureus (mrsa) infections. methods: patients with documented mrsa infection between 2001 and 2004 were evaluated in a matched case-control study. cases included patients who were converted to po lzd from iv table 1 includes comparisons of cost, duration of therapy and los. predictably, patients receiving lzd in the hospital had higher drug costs; however, a decreased los contributed to a $7000 lower cost of hospitalization. lzd or iv vancomycin (vanc) , and controls were patients who received only iv vanc. patients were paired in a 1:2 ratio of cases to controls and matched based on infection type, age and comorbidities. demographics, antimicrobial agents, concomitant infections, and clinical status were assessed daily from the onset of infection through the end of treatment or hospitalization. antibiotic cost, length of stay (los), and clinical success rates were compared between the groups. results: 78 patients were assessed. demographic characteristics were similar between groups. average age (mean ± sd) was 46 ± 14; apache ii (median, range) was 6 , charlson comorbidity score 2 [0] [1] [2] [3] [4] [5] [6] [7] [8] , and 60% were male. 55% of patients were treated for skin/soft tissue infection, 19% pneumonia, 13% bone/joint infections, 11% bacteraemia, and 2% other infections. clinical and microbiologic outcomes were similar between groups, with 100% clinical success at the end of therapy. objectives: to compare the efficacy and safety of linezolid (lzd) with those of vancomycin (van) for the treatment of infections caused by methicillin-resistant staphylococcus aureus (mrsa) in japan. methods: this was a randomized, open-label study conducted in japan. patients with nosocomial pneumonia, complicated skin and soft tissue infections, or sepsis syndrome, caused by mrsa, were randomized (2:1 lzd: van) to receive lzd, 600 mg q12 h, or van, 1 g q12 h. outcomes were evaluated at end of therapy (eot) and at a follow-up (fu) evaluation 7-14 days after completion of therapy. results: 151 patients received study drug (100 lzd, 51 van); the treatment groups were similar with regards to demographics and risk factors. at eot, clinical success rates in the mrsa-microbiologically evaluable population were 62.9% and 50.0% for the lzd and van groups, respectively; microbiologic eradication rates were 79.0% and 30.0% in the two groups, respectively (p < 0.0001). at fu, the clinical success rates were 36.7% for both groups, and the microbiologic eradication rates were 46.8%, and 36.7%, respectively. clinical success rates in both groups reflect an automatic outcome of failure for patients receiving prohibited antimicrobials prior to fu. reversible anemia (13%) and thrombocyotopenia (19%) were reported as adverse events more frequently in lzd patients. analysis of laboratory data showed that platelet counts decreased more frequently in lzd patients with recovery by fu. the mean platelet count in lzd patients with an adverse event of thrombocytopenia was 101,000/mm 3 . significantly low platelet counts (<50,000/mm 3 ) were more frequently observed in van patients (6 % vs 3%). no difference was observed in mean changes in hemoglobin levels between the treatment groups. conclusions: lzd is as effective as van for the treatment of mrsa infections in seriously ill patients, and may be more effective in achieving microbiologic eradication. although hematologic adverse events were reported more frequently in lzd-treated patients, analysis of laboratory data showed only a mild reversible trend toward lower platelet counts. cost-effectiveness of linezolid versus vancomycin in complicated skin and soft-tissue infection due to suspected methicillin-resistant staphylococcus aureus infection in germany -12, 2003; san diego, calif) . the objective of the present analysis was to evaluate the cost-effectiveness of linezolid compared with vancomycin in the treatment of cssti due to suspected mrsa infection from the german perspective. methods: a decision-analytic model was developed to examine the costs and outcomes of using linezolid versus vancomycin in hospitalized patients with cssti in germany. an expert panel of german physicians experienced in treating cssti provided resource-utilization data through structured interviews. costs from published sources (rote liste, dkg-nt, ebm) were applied to clinical and laboratory tests, adverse events, isolation procedures, intravenous (iv) or oral (linezolid only) drug treatment, hospitalization by ward type (medical, intensive care), and outpatient physician consultation. if appropriate, patients could be discharged from hospital and treated in an ambulant setting. the model assumed 50% of suspected mrsa patients had proven mrsa. outcomes included total costs per patient, cost per death avoided, cost per life-year gained, and cost per cure. results: an additional 4.4% of patients treated with linezolid (98.4%) versus vancomycin (94.1%) were cured. average total cost per episode was 9352 versus 9474 for linezolid-versus vancomycin-treated patients, giving a 121 saving per episode. the model was sensitive to the los in hospital, days in isolation, duration of oral versus iv treatment, percentage of mrsa patients, and price of linezolid. conclusion: in this decision analytic model, more patients with cssti due to suspected mrsa achieved clinical cure in the linezolid group compared with the vancomycin group. linezolid was cost saving versus vancomycin for the treatment of cssti. linezolid resulted in a shorter treatment duration and shorter los that offset the higher acquisition cost of linezolid versus vancomycin in cssti in germany. clinical microbiology and infection, volume 11, supplement 2, 2005 training, scientific output in infectious disease p874 a bibliometric analysis of worldwide trends in research productivity in microbiology p. vergidis, a. karavasiou, k. paraschakis, i. bliziotis, p. papastamataki, m. falagas (athens, gr) background: microbiology contributes significantly to the understanding and control of infectious diseases and has always been a field of extensive research. however, the literature lacks studies estimating the quantity and quality of worldwide research production. we evaluated the contribution of different world regions in research production in the field of microbiology. methods: using the medline database we retrieved articles from 64 journals included in the ômicrobiologyõ category of the ôjournal citation reportsõ database of the institute for scientific information for the period 1995-2002. the world was divided into 9 regions based on geographic, economic and scientific criteria. using an elaborate retrieval system we obtained data on published articles from different world regions. in our evaluation we introduced an estimate of both quantity and quality of research produced from each world region per year using: (1) the total number of publications, (2) the mean impact factor of publications, and (3) the product of the above two parameters. results: data on the country of origin of the research was available for 76,118 out of 77,080 retrieved articles (98.8%). western europe exceeds all other world regions in research production for the period studied, with usa ranking second (table). the difference in production between these two regions increased gradually from 1995 to 2002. however, the mean impact factor for articles published in microbiology journals was highest for the usa (3.34), while it was 2.79 for western europe and 2.31 for the rest of the world (7 regions combined). conclusions: usa and western europe make up a striking 78% of the world's research production in terms of both quantity and quality. all world regions have increased their research production during the period studied. the highest increase was achieved by asia (excluding japan), latin america, and eastern europe. from conference abstract to full paper: differences between data presented in conferences and journals e. rosmarakis, p. vergidis, a. kapaskelis, k. paraschakis, m. falagas (athens, gr) background: on some occasions, we noted differences between data presented in conference abstracts and subsequent published papers. we studied the frequency and the type of these differences in the fields of infectious diseases and microbiology. methods: we reviewed all abstracts from the first session of 7 out of 15 major research categories presented in the 1999 and 2000 icaac. for each selected pair of icaac abstract and related full paper published in journals indexed by index medicus, two independent investigators performed a comparison of all data in the abstract and the corresponding information in the published paper. using cox logistic regression models we analysed variables for association with differences of data. results: from 190 abstracts that were reviewed, 68 (35.8%) were subsequently published as papers by march 2004. from them, 52 referred to the same study period and population for both the abstract and the paper. differences between data presented in conference abstracts and published papers were found in 28 out of 51 pairs which were analysed further (54.9%, 95% c.i.: 41.2%-68.6%). the identified differences were related to the numbers and/or rates of the studied patients (11/28), numbers or rates of isolates (9/28), mics values or ki values (5/ 28), other chemical properties of antibiotics (2/28), odds ratio (1/28), and duration of observation (1/28). the differences were substantial in several pairs. time to publication of full paper was found to be independently associated with presence of differences (p = 0.029, or = 2.043 per year), while the research category, type of presentation (oral or poster), number of publications of the presenting and last author, impact factor of the journal, and country of origin were not. conclusions: while there are several explanations for the noted differences between data presented in conference abstracts and full papers, it is likely that the research community may improve the accuracy of presentation of data. worldwide trends in quantity and quality of published articles in the field of infectious diseases i. bliziotis, k. paraschakis, p. vergidis, a. karavasiou, a. kapaskelis, m. falagas (athens, gr) background: trying to confront with the widespread burden of infectious diseases, the society worldwide invests considerably on research. however, the literature lacks studies estimating the quantity and quality of worldwide research production. we evaluated the contribution of different world regions in research production in infectious diseases. divided into 9 regions based on geographic, economic and scientific criteria. using an elaborate retrieval system we obtained data on published articles from different world regions. in our evaluation we introduced an estimate of both quantity and quality of research produced from each world region per year using: (1) the total number of publications, (2) the mean impact factor of publications, and (3) the product of the above two parameters. results: data on the country of origin of the research was available for 45,232 out of 45,922 retrieved articles (98.5 %). usa and western europe are by far the most productive regions concerning publications of research articles, as displayed in the table. however, the rate of increase in the production of articles was higher in the developing world regions during the study period. the mean impact factor is highest for articles originating in the usa (3.42), while it was 2.82 for western europe and 2.73 for the rest of the world (7 regions combined). conclusions: usa and western europe make up a striking 80% of the world's research production in infectious diseases in terms of both quantity and quality. however, all world regions present a steady increase in the production of infectious disease articles with the developing countries displaying the highest rate of increase. . financial support was obtained from a pharmaceutical company. however, the sponsor had no role in organizing the scientific content of the programme. results: 146 physicians including id specialists were selected from several university hospitals or tertiary care government hospitals where running clinical trials has been a common practice. pre-training queries revealed that among the trainee population, 30.5% never obtained an informed consent from a patient, 47% did not randomize any patients for trials, 44% did not run any phase i-iv trials, 55% did not use placebo for trial purposes and 49% did not report any serious adverse events to local and or international authorities. a 24-question pre-and post-course tests about basic principles of gcp and local and eu regulations for running clinical trials were applied to determine and compare the knowledge of the trainees on these subjects prior and after the course. the percentage of correct answers ranged 35-91% before, and 51-98% after the course was completed (p < 0.01). conclusions: training in gcp and other regulations about running clinical trials may increase the awareness and knowledge of physicians. these educational activities will also help to raise the quality of clinical trials. learning appropriate use of antibiotics (pk/pd and guidelines): a cd-rom course for healthcare professionals and students e. ampe, y. glupczynski, p.m. tulkens, f. van bambeke (brussels, mont-godinne, b) objectives: in a context of growing resistance and limited supply of new molecules, a rational use of antibiotics should be a high priority. our objective is to train healthcare professionals and students in pk/pd and in a correct implementation of guidelines, since this could help to improve antibiotic use in both short and mid-terms. methods: we developed a pk/pd -guidelines course on cd-rom, targeted to both physicians and pharmacists but also usable by students. the course was prepared by a team of 2 pharmacists, 1 clinical microbiologist, and 1 pharmacologist. sources of information were (i) textbooks, review papers and primary papers by internationally recognized experts (ii) materials presented at training workshops of the international society for antiinfective pharmacology (isap; www.isap.org) during the last 3 years, (iii) national and, if not available, international guidelines for the management of respiratory tract or urinary tract infections. results: the course is organized as a series of power point presentations covering in a progressive fashion the followings topics: (1) bases in microbiology (in vitro properties of antibiotics); (2) pharmacokinetics (definition of the main parameters); (3) pharmacodynamics, with (a) the concepts, (b) the methods and pertinent models, and (c) the data, including the parameters to take into account to optimize the dosage of the main antibiotic classes; (4) resistance, including (a) the main mechanisms and (b) the use of pharmacodynamics to avoid the selection of resistance; (5) the appropriate use (including appropriate dosages) of antibiotics in (a) respiratory tract and (b) urinary tract infections. conclusions: this course promotes continuous education in the pharmacology and pharmacotherapy of antibiotics, in a format easily usable for courses and seminars to both students and professionals. background: although macrolides have no intrinsic antipseudomonal activity, these drugs appear effective in controlling infection by p. aeruginosa through mechanisms such as disruption of quorum sensing or anti-inflammatory effects. objectives: the aim of our prospective study was to evaluate the efficacy of macrolides in patients with bronchiectasis infected and colonised by p. aeruginosa. methods: the study included hospitalised patients with ct evidence of bronchiectasis and presenting an acute exacerbation by p. aeruginosa, isolated in sputum. microbiological findings, clinical data and treatment recommendations were recorded at admission and at the end of therapy. patients completed daily diary cards for symptoms and pef values. patients were followed for 1 year. results: twenty-two patients (14 men, mean age 57.3 ± 14.5) with bronchiectasis and p. aeruginosa infection were included. during hospital stay, patients were treated with at least two susceptible antipseudomonal drugs, according to the antibiogram (usually beta-lactam plus aminoglycoside) for a period of 16.9 ± 1.6 days. an oral macrolide (azithromycin 250 mg x 3 days/week in 10 patients, clarithromycin 500 mg daily in 12 patients) was further administrated for 4.9 ± 1.9 months. at the end of long-term therapy, 11 (50%) patients showed no evidence of p. aeruginosa in sputum, 8 (36.3%) patients still presented p. aeruginosa in sputum and 3 (13.6%) discontinued the treatment after less that 1 month because of adverse events. all patients had a significant reduction of sputum volume (83.3 ml/day before therapy vs. 22.5 ml/day at the end of therapy, p = 0.03) and of the mean number of exacerbations during follow-up (2.4 in the previous year vs. 1.2 in the follow-up year, p = 0.01). an increase of the mean pef value was also noted, although statistically not significant (373.2 ± 43.5 l/min before therapy vs. 489.2 ± 23.5 l/ min at the end of therapy, p = 0.3) conclusion: these observational findings suggest that macrolides may have a role in modulation of p. aeruginosa colonisation in patients with bronchiectasis. objectives: there is little information on cap requiring hospitalization in young adults (£ 40 years). we analysed clinical features, causative organisms, and outcomes of cap in this patient population. methods: prospective observational study of non-immunocompromised adult patients with cap (1995 cap ( -2003 . patients with hiv infection, transplantation, and neutropenia were not included. for the purposes of the study, patients were divided into 2 age-groups, £ 40 years and > 40 years. results: we documented 1732 consecutive patients with cap; 150 patients (8.7%) were £ 40 years and 1582 were > 40 years. mean patient ages were 30.3 years in the former group and 69.6 years in the latter group. main reasons for hospitalization in young adults were: hypoxia (po2 < 60) 59 patients, port high severity risk class (iv-v) 13 patients, empyema 9 patients, and/ or shock 7 patients. young patients were more frequently current smokers (63% vs 22%, p < 0.001). comorbid conditions were more common in older patients (20% vs 66%, p < 0.001), mainly diabetes mellitus, copd, and chronic heart disease. multilobar pneumonia was more frequent in young patients (56% vs 32%, p < 0.001). the most common causative organisms were streptococcus pneumoniae (19% vs 24%, ns) and legionella pneumophila (9% vs 7%, ns). atypical agents were more frequent in young adults (11% vs 4%, p = 0.001), while haemophilus influenzae (2% vs 7%, p = 0.026) and gram-negative bacilli (0% vs 1.4%, ns) were rarely identified in this group. the frequency of bacteraemia was similar in both groups (8% vs 12%, ns). icu admission was more frequent in young adults (13% vs 7%, p = 0.006), but no differences were found regarding the need of mechanical ventilation (5% vs 4%). median los was shorter in young patients (7 vs 9 days, p = 0.002). five young patients died; shock (2 patients) and multiorganic failure (3). early mortality (< 48 hours) was similar in both groups (1.4% vs 2.8%). overall mortality (< 30 days) was higher in older patients (3.4% vs 8.7%, p = 0.026). conclusions: cap requiring hospitalization in young adults, mainly smokers, is not uncommon, being respiratory failure the most frequent reason for admission. s. pneumoniae and atypical agents are the most frequent causative organisms in this group. although overall mortality is relatively low, a number of young patients require icu admission and have a complicated course. corticosteroids in patients with severe community-acquired pneumonia: impact on mortality c. garcia vidal, e. calbo, c. ferrer, v. pascual, s. quintana, j. garau (terrassa, e) background: mortality in patients with severe cap has been traditionally related to microorganism virulence and to host characteristics. recently, there has been an increasing interest host-pathogen interaction, and inadequate immunologic response has been shown to be associated with a higher mortality. immune modulation concomitant to antibiotic therapy has been postulated to improve outcome. the aim of our study was to determine the risk factors for increased mortality in patients with severe cap and to evaluate the impact of administration of corticosteroids in outcome. methods: we reviewed the charts of all hospitalized cap patients in our centre between october 2001 and december 2003. severe cap defined by pneumonia severity index (psi) categories 4 and 5 were included. data on demographics, comorbidity measured by charlson score, bacterial aetiology, the presence of immunosuppression, copd, icu admission, antibiotic therapy, use of corticosteroids and mortality were recorded. results: of the 375 severe cap patients included, 258 and 117,were categories 4 and 5, respectively. 256 patients(69%) were treated with standard antimicrobial therapy and116 (31%) received also corticosteroids. mean age (78 vs 77), charlson score (2.5 vs. 2.6), neoplasm (22% vs. 21%), hiv (0.7% vs. 0.8%), chronic liver disease (9% vs.7%), diabetes mellitus ( 19% vs.22%), icu admission (4% vs. 4%), monotherapy (63% vs. 66%), and aetiology (s. pneumoniae 34% vs. 36%; l. pneumophila 3% vs.2%; others 8% vs.11%; unknown 54% vs.51%) were similar. patients receiving corticotherapy had been exposed to another immunosuppressive treatment more frequently (6% vs. 12%; p = 0.04), copd was commoner ( 20% vs. 66%; p < 0.001) and mortality was higher (5% vs. 11%; p = 0.03) than in the group not treated with corticosteroids. in multivariate analysis, severity of disease (or 3.1; ic95% 1.3-7.1; p = 0.07) and the use of corticosteroids (or 3.3; ic 95%, 1.3-8.0;p = 0.009) were found to be related to mortality. conclusions: in our experience corticosteroid treatment in patients with severe cap appears to be associated with higher mortality. use of levofloxacin in community-acquired pneumonia j. olalla, i. escot, j.j. garcía-alegría (marbella, e) introduction and objectives: community acquired pneumonia (cap) is a prevalent and important disease, with an important consume of resources at hospitals. our aim is to study the profile of income patients with cap diagnosis, the year of introduction of levofloxacin in our centre and compare the length of stay in order to different antibiotics treatments, adjusting the results by fine's scale. methods: in our hospital (a second level hospital in marbella, spain) all the diagnosis at discharge are codified and included in the hospital database, so, all the diagnosis of cap between september 2001 and march 2002 were collected, and clinical reports were examined. demographic data were registered, so were all the parameters affecting fine classification, antibiotic treatment, intensive care unit (icu) incomes, deaths and length of stay. results: 129 cases of cap were found (86 males, 43 females), with a mean age of 67 years (ys)-ci 95%: 64-70), no difference between gender. 4 patients (pts) were living in a residence. 11 pts (8.5%) had a previous diagnosis of heart failure -hf-(mean of age 78 vs 66, p < 0.005) and other 11 pts had cerebrovascular disease -cvd-(mean of age 80.7 vs 66, p < 0.01), 12 pts had chronic hepatopathy, 15 -11.5%-any form of cancer, 23-17.8%-pts chronic renal failure (mean of age 74 vs 66, p = 0.049), 41 pts (32%) chronic obstructive pulmonary disease (copd). 3 pts were admitted at icu and 3 deaths were registered. blood cultures were collected in 30% of cases, culture of sputum in 21%, legionella antigen in urine in 13.2%. in according with fine's classification the patients were stratified in group i (10.9%), ii (16.3%), iii (22.5%), iv (27.9%) and v (22.5%). in 40% cases corresponding to fine i and ii levofloxacin was used alone, vs 19% in fine iii, iv and v (p=0.021). when length of stay was analysed in patients fine i and ii, the use of levofloxacin was associated with a non significant reduction (mean + se: 5.86 + 2.7 days vs 7.67 + 4.9 days), in people on groups iii, iv and v use of levofloxacin do not showed any reduction of length of stay (6.67 + 5.06 days in people using levofloxacin vs 6.11 + 4.5 days). no readmissions in relationship with recurrent cap was registered. conclusions: in our experience, use of levofloxacin is associated with less severe cap, in which a non significant reduction in length of stay is observed. telithromycin versus other first-line single-agent antibiotics in the treatment of communityacquired pneumonia: a randomised superiority trial y. mouton, v. thamlikitkul, r.b. nieman, c. janus (tourcoing, f; bangkok, th; bridgewater, usa; paris, f) objectives: macrolides and beta-lactams are commonly recommended for the treatment of outpatients with communityacquired pneumonia (cap). the aim of this study was to demonstrate the superiority of the ketolide telithromycin (tel) to other first-line, oral, single-agent antibiotics (comparators, comp) in achieving clinical cure in outpatients with mild to moderate cap in geographical areas of high pneumococcal resistance (erythromycin resistance rate ‡ 30%). methods: patients with clinical and radiological evidence of cap were randomised centrally to receive either tel 800 mg once daily for 7-10 days or comp (chosen by the investigator in accordance with local treatment guidelines/practices). efficacy was assessed post-therapy (days 17-21). clinical outcomes in this open-label trial were validated by three independent experts who were fully blinded to treatment assigned. results: of the 505 patients enrolled, 482 were included in the modified intent to treat (mitt) and 438 in the per protocol (pp) efficacy analyses. in the comp group, 39% of patients had received macrolides, 44% beta-lactams and 17% fluoroquinolones. resistance to penicillin and erythromycin among streptococcus pneumoniae isolates was 28% and 31%, respectively. clinical cure rates in the tel group were significantly higher than those in the comp group (as shown in the following table). tel and comp were similarly well tolerated. conclusions: in this outpatient cap study, post-therapy clinical cure rates achieved with tel were shown to be statistically superior to those achieved with other usual care first-line antibiotics -in both the mitt and pp populations, and according to evaluations by both the investigators and blinded experts. changing epidemiology, clinical features, and outcome of acute community-acquired pneumonia among adults in india background: acute cap is the third leading cause of mortality in india. two prospective studies from our centre identified common causes of cap in india to be mycoplasma pneumoniae [mp] and legionella pneumophila [lp] by serology in 11% each, and spn in 10% by culture of respiratory secretions/blood/ conclusion: although spn is the most common isolate, the rising numbers of gram negative organisms (38%) and atypical pathogens associated with increasing mortality stress the need for review of initial antibiotic choice for adults with higher port classes. in view of enduring susceptibility of spn to pcn and minimization of resistance with narrow spectrum activity, this should be the drug of choice for spn in india. fqr that has a wide coverage against most respiratory pathogens including atypicals, can be considered as an initial choice in both hospitalized and ambulatory setting. continued surveillance of respiratory pathogens is needed. fluoroquinolones in the treatment of community and hospital-acquired legionella pneumonia objective: community-acquired pneumonia (cap) is a major cause of morbidity and mortality worldwide. inability or failure to comply with standard antibiotic regimens, which may last up to 10 days, may result in patients receiving suboptimal antibiotic treatment. treatment with a single-dose antibiotic regimen maximizes compliance with prescribed therapy. a novel microsphere formulation of azithromycin makes it possible to administer more drug in a single dose while maintaining tolerability. the objective of this study was to test the hypothesis that a single 2.0 g oral dose of azithromycin microspheres was as effective as a 7-day regimen of oral levofloxacin 500 mg/day when used to treat adult patients with mild to moderate cap (fine classes i, ii, iii objectives: many guidelines have been issued for the treatment of cap. we wanted to assess how frequently the national guidelines for treatment of cap were followed in 10 spanish acute care hospitals in patients admitted with cap. methods: retrospective review of the charts of patients admitted to the hospital with the diagnosis of cap over a 1-year period (1nov 01 to 31oct 01) in 10 geographically scattered hospitals in spain. data were available from 3233 patients. results: amoxicillin/clavulanate was the most common antibiotic choice (79.5% was given as monotherapy and 20.4% in association with clarithromycin). levofloxacin (93.1% given only as monotherapy) follows closely. they were followed by third generation cephalosporins (ceftriaxone and cefotaxime) but up to 60% of them were given in association with clarithromycin. clarithromycin was used mainly as a complement to both amoxicillin/clavulanate and cephalosporins since only 8.6% of its 25.8% was given as monotherapy. other antibiotics were very uncommonly prescribed (cefepime 1.9%, and ciprofloxacin 1.4%). conclusions: 1) monotherapy with levofloxacin, followed by amoxicillin/clavulanic acid were the most common first line antibiotic choices in these 10 spanish hospitals. however, amoxicillin/clav alone or in combination with clarithromycin was the most common antibiotic prescribed. 2) monotherapy with clarithromycin was relatively uncommon (2.2%). 3) these choices reflect quite well the recommendations of the different national guidelines issued in our country in an environment of high prevalence of resistance to penicillin and to macrolides in s. pneumoniae in spain and the concomitant coverage of legionella and other atypical pathogens. the microbiology of abecb -potential predictive value of clinical criteria j. kahn (raritan, usa) objectives: to explore the relationship between specific pulmonary disease severity criteria and the microbiology of acute bacterial exacerbations of chronic bronchitis (abecb). methods: this was a double-blind, randomized clinical trial evaluating levofloxacin 750 mg qd for 3-5 days in abecb. all potential subjects had to meet the ats definition of chronic bronchitis but only those patients with anthonisen type 1 or 2 exacerbations (n = 763) were enrolled. stratification by disease severity was determined using parameters suggested by grossman: fev1 % of predicted value, defined co-morbidities, and number of exacerbations during the previous 12 months. because different spectra of etiologic organisms were expected on the basis of the stratification, different comparator agents were used for uncomplicated patients (azithromycin for five days) and those considered complicated (amoxicillin/clavulanate for ten days). results: initial microbiology from all intent-to-treat patients revealed notable differences between the two strata. among isolates from uncomplicated cases, 55% were the ôtraditionalõ triad of s. pneumoniae, h. influenzae, and m. catarrhalis. inclusion of h. parainfluenzae raises this figure to 79%. in the complicated arm the figures were 48% and 68%, respectively. gram-negative bacilli, primarily enterobacteriaceae spp. and several pseudomonads, represented 15% of the uncomplicated and 23% of the complicated isolates. microbiological eradication rates and the percentage of organism persisting after therapy from the uncomplicated stratum were compatible with the unexpectedly large number of macrolideresistant organisms isolated. in the complicated arm, both levofloxacin and amoxicillin/clavulanate had lower, but comparable, eradication and persistence rates. clinical efficacy in microbiologcally-evaluable patients was consistent with these figures. conclusion: while there was clearly overlap among the flora isolated from the two strata defined by application of the grossman criteria, there was separation of the populations. this predictive approach seems to be of value in identifying the optimal antimicrobial regimen, especially for uncomplicated exacerbations. further work to define and validate predictive clinical parameters may help optimize the choice and duration of antimicrobial agents for abecb. objectives: nosocomial pneumonia is the second most common nosocomial infection and, along with primary bacteraemia, the leading cause of death from infection acquired in the hospital. despite the frequency of ventilator-associated pneumonia (vap) and the threat it poses to patient survival, consensus on an appropriate antibiotic treatment of vap has yet to be established. the uncertainty is compounded by a lack of well-controlled comparisons of specific treatment regimens and their impact on relevant outcomes, such as morbidity, mortality, and cost. the comparable analysis of clinical and microbiological efficacy of cefepime and a combination ceftazidime and amikacin in the treatment of vap in severe trauma patients was the aim of present study. methods: this prospective, randomized study was approved by the institutional review board for human research. thirty adult patients who admitted in icu of an 1850-bed emergency municipal hospital with severe trauma complicated with vap were included. the cpis was used for diagnostics of vap. a combination of ceftazidime (2 g tid, iv) and amikacin (1 g once daily, iv) was used as initial empiric therapy of vap in 17 patients and monotherapy with cefepime (2 g bid, iv) -in 13 patients. these regimes of empiric therapy have been chosen according our local microbiological monitoring and p. aeruginosa was the prevalent pathogen of vap. the cost-effectiveness analysis was performed to compare both costs and outcomes of competing regimes. results: there was no difference between groups in patientsõ mean age, average time of mechanical ventilation before onset of vap and cpis. mean apache ii score was 23.1 in the group of combination therapy and 24.0 in the group of monotherapy. the positive clinical outcome was registered in 92.3% patients on cefepime and 88.2% patients on ceftazidime plus amikacin. the rate of eradication was 53.8 and 58.8% respectively. the duration of effective treatment with cefepime was from 7 to 12 days (mean 8.5 days), with combination therapy -from 7 to 18 days (mean 9.1 days). the mean cost of vap treatment with cefepime was 452.8 euro in comparison with 585.4 euro when the empiric therapy was initiated with ceftazidime and amikacin. conclusions: cefepime in a monotherapy regimen has the same clinical and bacteriological efficacy in comparison with a combination of ceftazidime and amikacin in vap patients with severe trauma but the use of cefepime results in lower costs. influence of antibacterial protocol in multiple trauma patients on incidence and mortality of vap d. protsenko, a. yaroshetskiy, s. yakovlev, b. gelfand (moscow, rus) objectives: the aim of present study was the evaluation of antibacterial protocol in multiple trauma patients on incidence and mortality of vap. methods: a comparable analysis of incidence, attributive mortality (chi-square test) and pathogens of vap in multiple trauma patients was performed during two periods: 2001 (before introduction of protocol) and 2003 (after introduction of protocol) years. the developed protocol included: 1. abandoning of antibiotic prophylaxis of vap; 2. intrusion criteria of early diagnostics of vap; 3. exclusion of 1st, 2nd and 3rd generation cephalosporines , aminoglycosides and fluoroquinolones as empiric therapy of vap; 4. cefepime (apache ii <20) or carbapenems (apache ii >20) were used as initial empiric therapy of vap; 5. efficacy of antibiotic treatment was evaluated within 48 hours; 6. carbapenems and/or vancomycin was added if initial therapy was inefficient. in the case of suspected diagnosis of vap microbiological analysis of bronco-alveolar lavage fluid (bal) was performed. a widespread use of broad spectrum antibiotics shifted the structure of nosocomial pathogens. we observed decrease of mrsa rate and significant increase (more than 10 times) in rate of klebsiella pneumoniae (from 0.6 to 18.1%, most of strains were resistant to 3rd generation cephalosporines) and in rate of acinetobacter baumanii (from 1.2 to 12.3%, most of strains were resistant to ceftazidime). conclusions: introduction of strict antibacterial protocol in patients with multiple trauma and vap results in significant decrease of attributive mortality without any change in incidence of vap. antimicrobial prophylaxis in cardiac surgery and postoperative pneumonia rate objective: the purpose of this study was to assess the postoperative pneumonia rate according to antimicrobial prophylaxis regimens in patients after cardiac surgery with cardiopulmonary bypass. patients and methods: cardiac surgery patients were included (n = 86) in the retrospective study. we observed 44 patients with postoperative pneumonia (pneumonia group) and 42 patients without infectious complications in postoperative period. the groups were compared by age, sex, volume of transaction, severity of disease. in a studied period is from 2001 to 2003 two main regimens of antimicrobial prophylaxis were used: ceftriaxone 1-2 g iv once before operation or cefuroxime 1.5 g iv before operation and additional dosage 0.75 mg in 2-3 hours after surgery beginning; then prophylaxis were conducted after operation during 48-72 hours. data were compared by fisher exact; we determined about significantly differences between groups when p < 0.05. results: preoperative prophylaxis by cefrtiaxone was administered more often in pneumonia group than in patients without infection complications (56.3% vs. 33.3%, ns). ceftriaxone was used significantly more often in comparison to cefuroxime in subgroup of patients with cardiopulmonary bypass less than 180 min who developed postoperative pneumonia (63.2% vs 30%, p < 0.05). during cardiopulmonary bypass longer than 180 min we did not reveal difference between antimicrobial prophylaxis regimens. conclusion: cefuroxime is more preferable than ceftriaxone for antimicrobial prophylaxis of postoperative pneumonia in patients after cardiac surgery with cardiopulmonary bypass less than 180 min duration. closed versus open tracheal suction system to prevent ventilator-associated pneumonia objective: the aim of this study was to analyze the incidence of ventilator-associated pneumonia (vap) using a closed tracheal suction system ( objectives: dalbavancin is a novel, second generation lipoglycopeptide that has been shown to have clinical efficacy as a once-weekly treatment for complicated skin and skin structure infections (csssi). the dosage used in clinical studies is 1000 mg day 1/500 mg day 8. in vitro studies have determined dalbavancin mic90s of less than or equal to 0.06 mg/l for target bacteria, principally staphylococci and streptococci, including isolates resistant to other antibiotics. the plasma protein binding of dalbavancin is 93%. as skin infections occur in extravascular space, antibiotic concentrations are assessed in blister fluid as a measure of skin penetration and to obtain pharmacokinetic observations that may correlate with efficacy. methods: a phase i, open label, single-dose study was conducted in 9 healthy subjects. subjects were administered a 1000 mg iv dose of dalbavancin. blisters were induced by applying 0.25% cantharidin ointment to the skin. blister fluid was collected prior to dose, and at 12 hours, day 3, day 5 and day 7. blood samples were collected throughout the study. blister fluid and plasma were assayed for dalbavancin using a validated lc-ms/ms method and pharmacokinetic parameters were determined. area under the concentration-time curve (auc) was determined for blister fluid (aucbf) and plasma (aucp) through the first week. the degree of penetration into skin was determined by aucbf/aucp (%). results: dalbavancin was well tolerated with no serious adverse events; most adverse events were mild and self-limited. maximum observed dalbavancin blister fluid concentrations were achieved by the first collection (12 hours) and concentrations were maintained above 30.3 (±4.4) mg/l through day 7. the mean (sd) aucbf and aucp were 6438 ± 1238 and 10806 ± 1926 mg h/l, respectively. skin penetration was approximately 60%. dalbavancin blister fluid concentrations were maintained well above mics throughout the treatment interval, even when considering the possible effects of protein binding. conclusions: blister fluid concentrations are maintained well above mics of csssi target organisms for a week following a single dose of dalbavancin. these data support a once-weekly regimen and are consistent with the efficacy observed in clinical studies. the safety and pharmacokinetics of dalbavancin in subjects with renal impairment or end-stage renal disease j.a. dowell, e. seltzer, d. krause, t. henkel (king of prussia, usa) objectives: dalbavancin is a novel, second generation lipoglycopeptide antibiotic in late stage clinical development for complicated skin and skin structure infections (csssi). the weekly dosage used in clinical studies is 1000 mg day 1/500 mg day 8. since dalbavancin will likely be used in patients with various degrees of renal impairment, it is important to determine the safety and pharmacokinetics in this population, as well as to evaluate if a dosage adjustment is necessary. methods: single intravenous doses of 1000 mg dalbavancin were examined in subjects with mild (clcr of 50-79 ml/min) and moderate (clcr of 30-49 ml/min) renal impairment. doses of 500 mg dalbavancin were studied in subjects with endstage renal disease (esrd; dialysis-dependent). single doses of 500 mg and 1000 mg dalbavancin were studied in subjects with severe renal impairment (clcr <30 ml/min). subjects with normal renal function were studied and used as controls. pharmacokinetic data were analysed using non-compartmental methods; parameters included maximum concentration (cmax) and area under the plasma concentration time curve (auc). results: a total of 43 subjects received dalbavancin and were included in the pharmacokinetic analysis (6 mild, 6 moderate, 10 severe, 6 esrd, and 15 normal). dalbavancin was well tolerated in each of the renal impairment groups. the majority of adverse events were mild or moderate in severity and unrelated to study drug. there were no related serious adverse events. dalbavancin pharmacokinetics were similar between subjects with mild and moderate renal impairment and subjects with normal renal function. concentrations in subjects with esrd were similar to subjects with normal renal function, indicating compensation in renal insufficiency due to regular dialysis (3 times/week). subjects with severe renal impairment had increased concentrations and exposure. concentrations were increased by no more than 40% through the first week post dose, but differences continued to increase through the rest of the profile. auc was increased almost 2-fold. background: tlv, a novel lipoglycopeptide antibiotic with multiple mechanisms of action, exerts rapid and concentration-dependent bactericidal activity against clinically important gram-positive bacteria, including methicillin-resistant s. aureus. the kidney is the primary route of elimination of tlv in man. phase 3 studies are ongoing in skin and skin structure infections and hospital acquired pneumonia. the lines represents the model-based predictedprobability of the first occurrence of naused. the boxes represent the 25th and 75th percentiles of auc for each dose group. the wiskers extend to the minimum and maximum values. objective: to compare the single dose pk of tlv in subjects maintained on hemodialysis with an aged and sex-matched group of healthy subjects without renal dysfunction. methods: six hemodialysis subjects (5 males, 1 female) received a single 7.5-mg/kg dose of tlv intravenously over 1 hour approximately 2-4 hours prior to a 4-hour hemodialysis session. six subjects (5 males, 1 female) with normal renal function (mean creatinine clearance 94 ml/min) also received the same dose of tlv over 1 hour. blood and dialysate samples were obtained at specified intervals post dose and assayed for tlv with a validated lc/ms/ms assay. pk parameters were determined using non-compartmental methods. results: average values for plasma pk parameters for tlv in both groups of subjects are shown below.the average (range) clearance of tlv via dialysis was 4.6 ml/min (4. 0-5.8 ) and the per cent of dose removed by dialysis was 5.9% (3.0-7.6). subjects tolerated tlv well. conclusions: dose reductions of tlv of 50% are recommended in patients maintained on hemodialysis. supplementation of a tlv dose post dialysis is not needed. pharmacokinetics and tissue penetration of telavancin in healthy subjects background: tlv, a novel lipoglycopeptide antibiotic with multiple mechanisms of action, exerts rapid and concentration-dependent bactericidal activity against clinically important gram-positive bacteria, including methicillin-resistant s. aureus. the mic90 for s. aureus, including mrsa and gisa, from pooled studies is 0.5 lg/ml. phase 3 studies are ongoing in skin and skin structure infections and hospital acquired pneumonia. objectives: to determine the steady-state pk profile of tlv in plasma and skin blister fluid in healthy subjects. methods: 8 subjects (7 males, 1 female) received three daily doses of tlv (7.5 mg/kg) intravenously over 1 hour. cantharidin ointment (0.25%) was applied to the forearms to produce 7 blisters/subject beginning 12-14 hours prior to the third dose of tlv. blood and blister fluid samples were obtained at specified intervals on days 3 and 4 and assayed for tlv with a validated lc/ms/ms assay. pk parameters in both matrices were determined using non-compartmental methods. the average values for pk parameters for tlv in plasma and in blister fluid are shown below. results: after single intravenous infusions of 750 mg equivalent (eq) ceftobiprole for 30 minutes or 500 mg eq ceftobiprole for 60 minutes, mean cmax-values at infusion endpoint are 57.9 lg/ ml and 34.2 lg/ml, respectively. the corresponding mean auc0-inf-values are 154 lg h/ml and 116 lg h/ml. intersubject variability of the parameters auc and cmax of ceftobiprole is below 20%. mean systemic clearance is 4.9 and 4.5 l/h and mean steady-state volume of distribution is 13.7 and 11.0 l for the 750 mg and 500 mg dosing regimen, respectively. after infusion of 500 mg for 60 minutes or 750 mg for 30 minutes, the ôtime above micõ (target mic of 4 lg/ml for methicillin resistant staphylococcus aureus) is 6 and 7 hours for the total plasma concentrations and 3.5 and 5.2 hours for the corrected unbound plasma concentrations, respectively. with twice-daily administration of 500 mg for 60 minutes or 750 mg for 30 minutes, the proportion of the dosing interval above the mic of 4 lg/ml, would be 75% and 87% for the total plasma concentrations and 55% and 65% for the unbound plasma concentrations, respectively. conclusions: ceftobiprole infusions of 500 mg eq for 60 minutes or 750 mg eq for 30 minutes, results in similar time above target concentration of 4 lg/ml well above the minimum efficacy requirement 25-30% required for mrsa. methods: the study was a randomized, three-way crossover study with 30 hours wash-out period in 8 healthy volunteers. each subject received imipenem in three regimens : (i) 0.5 h infusion of 0.5 g every 6 h for 3 doses; (ii) 2 h infusion of 0.5 g every 6 h for 3 doses; (iii) 2 h infusion of 1 g every 6 h for 3 doses. conclusion: the 2 h infusion of 0.5 or 1 g of imipenem both give greater values for t > mic than a 0.5 h infusion and that a 2 h infusion may be a useful mode of administration in tropical countries where drug instability may prevent the use of continuous infusion. pharmacodynamic characterisation of lmb415 against haemophilus influenzae in an in vitro hollow-fibre system a. meagher, p. smith, a. forrest, a. odundele, p. ambrose (buffalo, albany, usa) objectives: lbm415 is a peptide deformylase inhibitor with in vitro activity against those pathogens commonly associated with community-acquired respiratory tract infections. the purpose of these studies was to determine which pharmacokineticpharmacodynamic (pk-pd) measure is most strongly associated with drug response and to examine the relationship between drug exposure and response for lmb415 against haemophilus influenzae (hi). methods: two wild-type hi strains (mic 2 and 8 mg/l) were studied. the hollow-fiber system (hfs) was inoculated with approximately 10e7-10e8 cfu/ml in log-phase growth. simulating human pharmacokinetics (t ½ = 2 hours), bacteria were exposed to escalating free drug lmb415 exposures (auc ranging from 0 to 200 mg · h/l) using a dose fractionation study design and delivering drug q12 hours, q24 hours, and by continuous infusion (ci). serial samples were collected to determine bacterial counts (cfu/ml) and drug concentrations. drug effect was quantified as the log 10 ratio (lr) of the 24 hour area under the bacterial growth/kill curves for drug and growth control (lr = log 10 aucdrug/aucgrowth control). results: overall, the greatest activity (at 6xmic) was seen with the ci regimen (ci > q12 >> q24). due to the short half-life, dosing q24 hours yielded no net kill compared to baseline. neither per cent time above mic, auc:mic ratio, nor peak:mic ratio could co-model the q12 and q24 hour regimens with ci results. separating these into 2 datasets (q12/24 vs ci), only the auc:mic ratio could adequately describe the ci results (emax lr of (2 at an auc:mic ratio ‡50). the q12/24 dataset was reasonably fit by the auc:mic ratio and per cent time above mic (emax lr of (2 to (3 at an auc:mic ratio ‡120 and per cent time above mic of ‡50-60%, respectively). the peak:mic ratio was not informative. conclusion: for intermittent dosing regimens, per cent time above mic and the auc:mic ratio adequately described drug response. percent time above mic and the auc:mic ratio associated with maximal decline were ‡50-60% and ‡120, respectively. ci regimens could not be co-modeled with intermittent regimens, suggesting that neither per cent time above mic nor the auc:mic ratio completely described drug effect. drug effect continued to increase beyond 100% time above mic. q12 hour dosing had more effect than q24 hour dosing, and would probably be an effective lbm415 regimen in humans for hi. pharmacodynamics of antimicrobials for the empiric treatment of nosocomial pneumonia: a report from the optama program objectives: appropriate empiric antibiotic therapy is vital for maximizing patient outcomes in the treatment of nosocomial pneumonia and is dependent on the drug exposure achieved in a patient and the causative pathogen's mic. the purpose of this study was to compare the probability of achieving bactericidal exposures for commonly used intravenous antibiotics against the bacteria most commonly implicated in nosocomial pneumonia. was high, target attainments for fep, caz, and tzp decreased, but carbapenem target attainments remained the same regardless of pathogen prevalence. conclusions: target attainments were greatest for ipm and mem, followed by higher doses of fep, caz and tzp. because these antibiotic regimens provide optimal bactericidal exposure, they would be most suitable for the empiric treatment of nosocomial pneumonia along with an anti-mrsa antibiotic until pathogen identification and susceptibility results are available. minimal inhibitory concentration effect (sme) of 6 antibacterial agents against two strains of b. anthracis. methods: the pa-sub mic and sme were determined by exposing the bacteria to antibiotic concentrations 10 times the mic for 2 h at 37°c. following repeated washings and centrifugations to remove the antibiotic, cultures were divided into four tubes. to three tubes, the tested antibiotic was added to make a final concentration of 0.5 mic, 0.25 mic and 0.125 mic to the fourth tube no antibiotic was added. optical density was determined before exposure, immediately after washing and then hourly up to 9 h. for the determination of sme, the same procedure was performed except that the organisms were not pre-exposed to any antibiotic. the ods were converted to cfus by using a standard curve. the pa-sub mic was defined as pa-sub mic = tpa ) c, where tpa is the time for cultures previously exposed to an antibiotic and then reexposed to different sub-mics to increase 1 log 10 above the counts determined immediately after washing and c is the corresponding time for the antibiotic unexposed control. the sme was defined as sme = ts ) c, where ts is the time for the cultures exposed only to sub-mics to increase 1 log 10 above the counts determined immediately after washing and c is the corresponding time for the unexposed control. methods: serum and csf cefepime pk data was obtained from 7 hospitalized patients with pneumonia and external ventricular drains. the concentration-time profiles in serum and csf were modeled using a three-compartment model with zero-order infusion and first order elimination and transfer. the apparent volume of the central compartment (vc), apparent volume in the csf (vcsf), intercompartmental transfer rate constants (k12, k21, k13, and k31) and plasma clearance (cl) were identified in a population pk analysis (npag) [table 1]. for cefepime 2 g iv q8 h (0.5 h infusion), a monte carlo simulation of 10,000 subjects (adapt ii) was performed to estimate the probability of attaining the targets of free cefepime serum concentration (assumed 20% protein binding) and total cefepime csf concentration 40-100% t > mic for mics 0.25-8 mg/l (table 2) . csf/serum median penetration ratio was calculated. post map-bayesian observed-predicted regression and r 2 for serum and csf were as follows: (serum) observed = 0.984 · predicted + 2.570; r 2 = 0.944. (csf) observed = 0.785 · predicted + 0.868; r 2 = 0.821. the penetration of cefepime as measured by median auccsf/auc serum was 7.8% (25th-75th percentiles 2.9-21.4%). results of serum and csf target attainment analysis for cefepime 2 g iv q8h conclusion: in the setting of non-inflamed meninges, cefepime 2 g iv q8 h does not provide adequate t > mic in the csf for >80% of patients for mics ‡0.5 mg/l. the influence of inflammation on the calculated csf target attainment rates is unknown. the definitive pharmacodynamic target in the csf has not been elucidated and further research is needed. application of microbiological and capillary electrophoresis methods to phenoxymethylpenicillin dissolution assay w. grzybowska, g. pajchel, m. zapasnik, s. tyski (warsaw, pl) objectives: drug absorption from a solid dosage form after oral administration depend on the release of the active substance from the medicinal product. in some cases of drug analysis, especially when the results of tests are out of specification, it is necessary to use another assay, simultaneously with the reference method. in case of phenoxymethylpenicillin tablets, the reference method for dissolution assay is spectrophotometric method. the aim of the study was to apply parallel microbiological and capillary electrophoresis methods and compare the results with those obtained using uv assay. methods: two preparations (tablets), containing 1 00 000 iu/mg of penicillin v: taropen and ospen were examined. the dissolution tests were performed at temperature 37°c ± 0.5°c, in phosphate buffer ph 6.8 (900 ml for ospen and 1000 ml for taropen) using baskets, rotation speed 100 rpm; samples were taken only at: 30 minute or at 10, 20 and 30 minutes in case of profile of dissolution analysis. the amount of penicillin v dissolved was assayed spectrophotometrically at 268 nm. hanson research dissolution system and ce apparatus: quanta 4000 of (waters) were used. pharmacopoeal, microbiological agar diffusion method and staphylococcus aureus atcc 6538 p strain, were applied. results: the dissolution of penicillin v from taropen preparation after 30 minutes was 100%, independently on method applied. in case of ospen tablets these values were different, depending on the analytical assay. the statistical fisher-snedecor test for comparison of dissolution data obtained using three methods was performed. the fisher fcalc. value was lower than theoretical only in taropen case. the dissolution profiles of two examined preparations were also compared and statistically evaluated according to the fda method. the results of these calculations showed that dissolution profiles of phenoxymethylpenicillin from taropen and ospen tablets differ significantly. conclusion: performed analysis proved that capillary electrophoresis and microbiological methods can be used alternatively but only for determination of taropen dissolution. penetration of penicillin g in combination with sulbactam into nasal tissues u. frank, s. wenzler-rö ttele, w. maier, f. knapp, r. trittler, h. egle, k. kuemmerer (freiburg, d) objective: the penetration of antimicrobial agents into target tissues is essential for treatment at the site of infection. we investigated the distribution of penicillin g in combination with sulbactam in human nasal tissues. methods: to determine the pharmacokinetics of penicillin g/ sulbactam, informed written consent was obtained from 22 patients aged between 19 and 73 years scheduled for endonasal operations such as septoplasty and conchotomy. after infusion of 5 m iu of penicillin g and 1 g of sulbactam, the concentrations of these agents were determined in serum and various nasal tissues such as septal mucosa, septal cartilage and bone. serum and nasal tissue concentrations were determined by liquid chromatography-mass spectrometry (lc-ms). results: up to 1, 2, 3, 4 and 5 hours after infusion, the mean serum concentrations of penicillin g were 114.9 lg/ml (sd ± 24.2), 54.0 lg/ml (sd ± 29.8), 33.6 (sd ± 26.9), 12.7 lg/ml (sd ± 9.6), 17.6 lg/ml (sd ± 8.2) and 2.8 lg/ml, while the mean serum concentrations were 17.6 lg/ml (sd ± 8.2) 24.3 lg/ml (sd ± 15.2), 9.3 lg/ml (sd ± 3.5), 3.5 lg/ml (sd ± 0.6), and 1.9 lg/ml (sd ± 0.4). mean penicillin g and sulbactam concentrations in septal mucosa decreased from 141.7 and 128.3 lg/g (1st hour) to 3.0 and 2.7 lg/g (5th hour), respectively. in septal cartilage, the highest tissue concentrations were observed 2 hours after infusion, with mean penicillin g and sulbactam concentrations of 59.4 and 80.1 lg/g, respectively. the mean penicillin g and sulbactam concentrations in bone decreased from 31.2 and 14.8 lg/g (1st hour) to 1.4 and 1.8 lg/g (5th hour), respectively. the regression analysis of the data showed that 3 h after administration of the drug combination, the levels still exceeded the 4-fold mic90 of methicillin-susceptible staphylococcus aureus, streptococcus pyogenes, moraxella catarrhalis and haemophilus influenzae. conclusions: our data support the use of the drug combination in perioperative prophylaxis and the treatment of ent (nasal and paranasal) infections due to common bacterial pathogens. mic vs. kill-curve based pharmacokinetic/ pharmacodynamic modelling of activities of cefpodoxime and cefixime h. derendorf, p. liu, k. rand, b. obermann (gainesville, usa; munich, d) objectives: pharmacokinetic (pk)/pharmacodynamic (pd) modeling of antibiotics usually consists of the comparison between plasma pk and the mic, such as the t > mic, cmax/ mic, and auc/mic. these indices are limited due to the innate inaccuracy of the mic and the fact that it does not reflect the in vivo scenario where concentrations are not static but fluctuate between doses. an alternative is to use time-kill curves that follow bacterial killing and growth as a function of time and concentration. this study provides a systematic comparison of mic and kill-curve approaches to show the potential of both methods for antibiotic evaluation. in an example, we developed a mathematical pharmacokinetic/pharmacodynamic (pk/pd) cefepime population on pk mean (sd) parameter estimates obtained by npag analysis model to integrate the in vitro antimicrobial activity with the pk profile of two oral cephalosporines at the tissue site. methods: kill curves could be described with different combinations of maximum kill rate (kmax), drug concentration at half-maximum effect (ec50) and bacterial growth rate (k0) that resulted in the same mic in each scenario. in the experimental part, bacterial time-kill curves of cefpodoxime and cefixime against four bacterial strains were compared in in vitro kinetic models in which previously measured human pharmacokinetic profiles of unbound antibiotic were integrated. results: different combinations of kmax, ec50 and k0 can yield the same mic and consequently the same mic-based index. however, depending on the combination of pd parameters, kill curves may predict quite opposite outcomes in both scenarios. ec50 values of cefpodoxime and cefixime were consistent with their respective mic values. both antibiotics had similar high potency against h. influenzae (ec50: 0.04 mg/l) and m. catarrhalis (ec50: 0.12 mg/l), while the potency of cefpodoxime against s. pneumoniae strains was about 10-fold higher than that of cefixime (ec50s/ sensitive: 0.02 vs 0.27 mg/l; ec50s/intermediate: 0.09 vs 0.69 mg/ l). simulations showed that cefpodoxime will have higher bacteriological success against s. pneumoniae than cefixime. conclusions: simple comparison of exposure and mic may not be sufficient to evaluate anti-infective efficacy. kill curves provide a more detailed approach in predicting antimicrobial effects. the developed emax model effectively described the pharmacodynamics of cefpodoxime and cefixime. cefpodoxime (200 mg bid) has higher tissue penetration and antimicrobial efficacy than cefixime (400 mg qd) against s. pneumoniae. bacteriologic efficacy of intravenous piperacillin/ tazobactam and ampicillin/sulbactam for infected diabetic foot ulcers objectives: to test the efficacy of single-agent empiric treatment, either intravenously administered piperacillin/tazobactam (tzp) (4 g/0.5 g q8 h) or ampicillin/sulbactam (sam) (2 g/1 g q6 h), of moderate to severe infected foot ulcers in patients with diabetes. in this open-label trial, 314 adults were randomized to receive tzp or sam for up to 21 days. patients with polymicrobial infections involving methicillin-resistant staphylococcus aureus also received vancomycin 1 g q12 h. samples for bacteriologic evaluation were taken at baseline from infected ulcers and blood to document causative pathogen(s) and test for antimicrobial susceptibility; samples were taken as clinically indicated at the end-oftreatment and test-of-cure visits (14-21 days post-treatment). to minimize risk of contamination, samples were to be obtained by aspirate, curettage, or biopsy rather than by swabbing. results: a total of 123 of the 314 patients in the study were bacteriologically evaluable. the most common causative pathogens in monomicrobial infections were s. aureus, streptococcus agalactiae, enterococcus faecalis, and pseudomonas aeruginosa. about 30% of patients in each treatment arm had polymicrobic infections. the combination of s. aureus plus s. agalactiae was most common. the median duration of treatment was 8 days for both groups. the bypathogen bacteriological success rates were similar in both treatment groups for s. aureus, s. agalactiae, and e. faecalis. tzp had an eradication rate of 85.7% for p. aeruginosa; sam is not active against p. aeruginosa, and patients with p. aeruginosa in the sam group (n = 5) were discontinued from the study . conclusions: previous studies have shown that tzp at a dose of 3 g/0.375 g q6 h was effective for treatment of diabetic foot infections. our study confirmed that less frequent administra-tion of tzp at 4 g/0.5 g q8 h was sufficient to attain bacteriologic success rates of 76.9% for s. aureus, 83.3% for s. agalactiae, 71.4% for e. faecalis, and 85.7% for p. aeruginosa in the bacteriologically evaluable population. this study differed from previous studies of infected diabetic foot ulcers, which found gram-negative enterics to be more common than p. aeruginosa as causative pathogens. however, it is consistent with data showing that p. aeruginosa has recently become the gramnegative pathogen most frequently isolated from soft tissue infections in europe, north american, and latin america. penetration of beta-lactamase inhibitors tazobactam and sulbactam in severe acute pancreatitis objectives: despite of high standard intensive care and surgical management, acute necrotising pancreatitis is still related with an extremely high mortality rate. this is determined by local infectious complications, especially in necrotising areas. limited penetration of antimicrobial drugs in these areas is considered to be a major cause for failure of therapy of severe infections. combinations of beta-lactamase inhibitors (bli) and beta-lactam antibiotics like broad-spectrum penicillines (bsp) have antibacterial activity against most of the common pathogens in severe necrotising pancreatitis. co-administration leads to an increase of antibacterial activity due to an inhibition of beta-lactamases compared to those of beta-lactam antibiotics alone. some bsp has been shown to penetrate rapidly and efficiently into pancreatic tissue. the penetration of bli into inflamed pancreatic tissue has not been investigated yet. methods: addressing the penetration capability of bli, a clinical trial was designed to investigate the penetration of tazobactam (taz, n = 8) and sulbactam (sul, n = 5) in patients with severe necrotising pancreatitis undergoing pancreas surgery. samples were taken from blood, necrotic areas of pancreatic tissue (pn), peripancreatic fatty tissue (pft) and bursa secretion (bs) following intravenous administration of 0.5 g taz or 1.0 g sul. concentrations of bli were determined by hplc/ uv. the aimed concentration for full enzymatic effect of bli should be 4 lg/mg (taz) and 8 lg/mg (sul), respectively. results: mean plasma concentrations at 1.0 h after application were 20.6 ± 9.58 lg/ml (taz) and 48.6 ± 18.8 lg/ml (sul background: uti is one of the most common infectious conditions treated in general practice and represents a major part of abstracts antibiotic use in the community. it is of major importance to know, how we treat these infections correctly, i.e. maximum efficacy with the least amount of drug in order to reduce the risk of development of resistance. materials and methods: uti was induced in anesthetized female nmri mice via intraurethral inoculation of 10-8 cfu of e. coli. one day later, treatment was started with 12-24 hour dosing schedules with 12-16 different doses of each drug securing a wide variation of time > mic and auc/mic. 24 h after the last dose mice were sacrificed and urine collected, and the bladder and both kidneys removed. bladder and kidneys were homogenized before cfu determination. the drugs used were the cephalosporins, cefuroxime (cef, mic = 0 3 mg/l) and ceftriaxone (cro, mic = 0.094 mg/l), and the penicillin, mecillinam (mic = 0.1 mg/l). the pk of the drugs in serum was determined in similar mice as well. the two cephalosporins were studied together (cef has a t ½ of 14 min and cro a t ½ of 56 min, respectively, in mice). pd parameters were calculated from serum total antibiotic concentrations, since protein-binding is a minor issue in the urine, and the relation to cfú s estimated by the hill-equation. results: all drugs reduced cfú s in urine and kidney tissue by 3-4 logs, but little effect was found in the bladder tissue. for the two cephalosporins combined time > mic in % of dosing interval best described the correlation with cfú s in urine (r 2 = 0.69, p < 0.05) and in kidney tissue (r 2 = 0.89, p < 0.05), respectively, while no such correlation was found in the bladder tissue, neither did auc/mic reveal any correlation with cfú s in urine or any organs. the same was found for mecillinam, i.e. no correlation for auc/mic vs. cfú s in urine or organs, while time > mic % significantly (p < 0.05) correlated with cfú s in urine (r 2 = 0.48), and kidney tissue (r 2 = 0.82), respectively. the time > mic% for maximum efficacy was around 30-40% for all three antibiotics. conclusion: the optimal pkpd parameter for efficacy of betalactam antibiotics in uti is the time > mic. maximum effect is seen for time > mic for 30-40% of the dosing interval, when serum total drug concentration is used as surrogate parameter. in humans this would call for tid dosing of mecillinam and cefuroxime, and od dosing for ceftriaxone. comparative performance of different methods to simulate drug exposure variability in a population v.h. tam, g.l. rosner (houston, usa) objectives: stochastic pharmacokinetic (pk) forecasting such as monte carlo simulations (mcs) are increasingly being used to predict the pk variability of antimicrobials in a population, based on data from relatively few subjects. however, various mcs approaches may significantly differ in the accuracy and precision of the predictions. we compared the performance of 4 different mcs approaches using a dataset with known parameter values and dispersion. methods: concentration-time profiles for 10 subjects after an intravenous bolus of 1000 mg were randomly generated using elimination rate constant (k) 0.1 ± 0.025 h)1(mean ± sd) and volume of distribution (v) 20 ± 5 l. normal distribution of parameter values and no correlation between k and v were assumed. system noise was incorporated as a linear function of drug concentration. using these concentration-time profiles, the best-fit parameter estimates were determined by the standard two-stage method. four methods were subsequently used to simulate the auc0-inf of the population by mcs, using the central tendency and dispersion of the following in the subject sample: (1) k and v; (2) clearance and v; (3) dose/clearance; (4) auc0-inf. in each scenario, 10,000 subject simulations were performed with adapt ii, using normally distributed input parameter(s) with means and variances set to the fitted values. results: reasonably good parameter estimates were obtained (k = 0.101 ± 0.034 h)1; v = 18.0 ± 7.2 l). compared to true auc0-inf of the population (581.1 ± 314.9 mg h/l), the simulated auc0-inf by various methods were: (1) 911.1 ± 2416, (2) 1348 ± 23510, (3) 1348 ± 23510, and (4) 713.7 ± 430.1 mg h/l, respectively. conclusions: our results suggest that various mcs approaches may predict pk variability in a population differently. the most realistic approach appeared to be based on the variability of auc0-inf in the subject sample. our observations are consistent with statistical principles concerning estimation. this method did not amplify variability of the model parameters and was the least likely to be associated with model misspecification. in objectives: fluoroquinolone treatment of humans and animals can rapidly select for organisms with increased resistance to these antibiotics. animal models are key to investigating in vivo antibiotic concentrations that prevent selection of resistance (e.g. the mutant prevention concentration (mpc) concept), but use of such models requires validated procedures for extraction and analysis of fluoroquinolones from relevant tissues. here, we report the validation of a method to quantify the concentration of enrofloxacin, and its metabolite ciprofloxacin, extracted from chicken liver, caecal contents and serum following treatment with baytril tm (enrofloxacin). methods: the extraction procedure described by wiuff et al. 2002 was validated for fluoroquinolone analysis, in pig tissues, by fortification of target matrices with enrofloxacin and ciprofloxacin. norfloxacin was added as an internal standard to all samples. samples were homogenised and extracted with acetonitrile (6 ml), centrifuged and the supernatants retained for analysis. the extracts were diluted with distilled water and analysed by hplc equipped with fluorescence detection. the analytes were chromatographed on a c18 reverse phase column and eluted with an isocratic potassium phosphate buffer/ acetonitrile system. results: liver samples were fortified with 0.1, 1.0 and 5.0 lg/g enrofloxacin and provided recoveries of 114.5, 78.6 and 78.2% respectively. quantification of ciprofloxacin at the 0.1 lg/g level was not possible due to interference from sample co-extractives, but the recoveries at 1.0 and 5.0lg/g were 73.7 and 91.1%. for caecal content samples, enrofloxacin recoveries were 70.6 and 69.1% at 1.0 and 5.0lg/g respectively, and ciprofloxacin 65.4 and 63.3%. the recovery from serum was much higher, samples fortified at 0.1, 1.0 and 5.0lg/ml, enrofloxacin and ciprofloxacin yielded recoveries of 94.7, 80.9 and 86.1%, and 91.1, 85.6 and 87.9% respectively. selected samples were also analysed by zonal microbial growth inhibition bioassay and hplc equipped with ms/ms detection; the data were broadly similar between all methods. conclusion: a valid method for the analysis of fluoroquinolones in chicken liver, caecal content and serum has been established, and will be applied to in vivo mpc studies. where sample matrices interfere (e.g. liver, caeca), bioassay or lc-ms/ ms must be used to provide valid results. the ecological effects of norfloxacin and pivmecillinam (piv) on the periurethral and vaginal flora in women with recurrent urinary tract infection objectives: to compare the ecological effects on the periurethral and vaginal microflora and time to normalisation of orally administered norfloxacin (nflx) and pivmecillinam (piv) in women with recurrent lower uti. methods: women with recurrent lower uti participated in a randomized, single blind parallel multi-centre study. twentyfive women, aged 18-55 years, with a positive nitrite test and symptoms (urgency, frequency, dysuria and/or suprapubic pain) of lower uti were included. only patients with an uti caused by e. coli or klebsiella spp were evaluated. key exclusion criteria were; menopause; pregnancy or breast feeding, known hypersensitivity to study drugs, antibiotics within the preceding month, impaired liver or kidney function, known or clinically suspected pyelonephritis, complicated uti and/or gastrointestinal infection. study drugs: seven days treatment of either nflx 400 mg bid or piv 400 mg tid. samples from midstream urine, periurethra and vagina were obtained before start of treatment day 1 and at two follow-ups day 12-4 and 28-35. informed consent was obtained. the ethical review committee and medical product agency approved the study protocol. results: nineteen patients (11 nflx and 8 piv) fulfilled the inclusion and exclusion criteria. no differences of patient characteristics were seen between the two groups. at the initial visit, more nflx patients were colonized with aerobic bacteria, although no differences were seen for anaerobic bacteria. the e. coli strains were suppressed markedly by nflx and piv in both locations. s. epidermidis increased more markedly following piv treatment compared to nflx in the periurethral location. in the piv group e. faecalis was less frequent initially, increased at visit 2, and returned to pre-treatment numbers at visit 3. for the nflx group, more patients were colonized initially with e. faecalis, with a decrease at visit 3. lactobacilli decreased slightly in the periurethra in the nflx group, whilst no changes were seen for piv. bacteroides spp. decreased more markedly for nflx. restoration of the pre-treatment colonization levels occurred gradually, except for e. coli being markedly suppressed by nflx throughout the study period. the bacterio-logical outcome of the urinary tract infection both at the shortterm and long-term follow up was successful. conclusions: limited ecological effects on the microflora were seen following treatment with nflx and piv, a benefit for antibiotics used for treatment of patients with uti. antibiotics influence release of il-6 and il-8 by kb cells and gingival fibroblasts s. eick, m. lausse, s. schwarz, p. wachter, w. pfister, e. straube (jena, d) objectives: in general, antibiotics are used in defense against pathogenic bacteria. nevertheless, side effects such as immunomodulatory activity should be considered. the purpose of this study was to determine the effect of antibiotics normally used in periodontal treatment on the release of il-6 and il-8 by kb cells and gingival fibroblasts. methods: kb cells and primary gingival fibroblasts were infected with actinobacillus actinomycetemcomitans y4 (a.a.) and porphyromonas gingivalis atcc 33277 (p.g.). the antibiotics doxycycline, tetracycline, minocycline, metronidazole, and moxifloxacin were added in concentrations ranging from 0.25 mic to 100 lg/ml. after 1, 6 and 18 h supernatants were obtained and the levels of il-6 and il-8 were measured by elisa technique. additionally, after 18 h the number of surviving bacteria was enumerated. results: minocycline and moxifloxacin in concentrations predicted in serum as well as 100 lg/ml were effective in killing planctonic a.a. and p.g. as well as adherent and intracellular bacteria. low levels of both interleukines released from kb cells and fibroblasts infected by p.g. were found only after addition of tetracycline up to 10 lg/ml. metronidazole, moxifloxacin and tetracycline in subinhibitory concentrations enhanced the release of il-6 from non-infected and a.a.-infected fibroblasts (up to 3200 pg/ml) after 18 h, contradictory 100 lg/ml tetracycline and minocyline reduced the release of il-6. il-6 in the supernatants of kb-cells was detectable only after addition of 100 lg/ml moxifloxacin. each 100 lg/ml of tetracycline and minocycline suppressed totally the release of il-6 and il-8 from non infected and infected fibroblasts, but not from kb cells. metronidazole and moxifloxaxin in high concentrations promoted the release of il-8. conclusions: antibiotics influence the release of il-6 and il-8. besides the good antibacterial effect especially minocycline might suppress inflammation. nevertheless, the killing of bacteria as well as a possible inhibition of virulence factors (bacterial proteases of p.g. by tetracycline) might influence the enhanced or reduced release of these cytokines. exceedingly infrequent infectious complications during orthotopic liver transplantation: tubercular peritonitis introduction: tuberculosis (t) is a very infrequent complication in patients (p) who undergo solid organ transplantation: in liver transplant recipients a 0.9-2.3% rate has been retrieved by a literature update. in these p t occurs within 12 months, and less than 10% of cases are observed after a repeated transplantation. pulmonary localization predominates (50% of p), followed by disseminated t (30%), while extrapulmonary t is an exceedingly rare event, reported only nu anectdotal p descriptions. pathogenetically, a t peritonitis follows disseminated infection or a primary t enteric localization, and is borne by a 80% mortality rate. case report: a 57-year-old female p with a mute t personal and familial history and normal chest x-ray, underwent liver transplantation 9 years ago, because of a hcv-related decompensated cirrhosias. four years later a novel graft was needed, after the development of end-stage hepatic insufficiency due to massive hcv re-infection. one year later (while of cyclosporin treatment), abdominal pain and ascites formation prompted admission, and m. tuberculosis sensible to all tested drugs was repeatedly isolated from an abdominal fluid with an elevated lymphocyte-monocyte count. associated ethambutol, isoniazid, streptomycin and levofloxacin (this one replaced by cycloserine after 2 months), led to a complete clinical and bacteriological cure already achieved after 3 months, although a 3-drug anti-t treatment was continued for 1 year. conclusionas: in our rare case report, the selected anti-t chemotherapy showed a successful efficacy and tolerability profile, with contained untoward events despite a very critical clinical context, due to the severity of t complication, and the broad spectrum of problems related to graft and all associated issues. in order to obtain an early diagnosis, an elevated clinical suspicion should be maintained for this rare complication too, by recommending microscopy and culture search of mycobacteria on ascitic fluid. methods: from january 2000 through december 2001, a prospective cohort study was conducted to determine the rate of bacterial nosocomial infection in renal transplant recipients. the patients were divided in two groups according to the origin of the allograft: cadaver or living donors. enterobacter cloacae strains, the most prevalent multidrug-resistant bacterial organisms isolated from uti were determined using genomic analysis with pulsed-field gel electrophoresis (pfge). results: one hundred sixty-three patients who received renal transplants were reviewed during the hospitalization. one hundred and ten (67.5%) renal transplanted were from cadaver and 53 (32.5%) from living donor. the median of length of stay in hospital was 12 days (range, 9-75 days) from transplant of living donor and 26 days (range, 2-28 days) from transplant of cadaver (p < 0.0001). twenty-one (39.6%) living donors recipients and 68 (61.8%) cadaver donors recipients had bacterial infection episodes (p = 0.019). post-transplant nosocomial infections diagnosed during the hospitalization were uti (44.8%), ssi (11%), pneumonia (6.1%), bloodstream catheter-related infection (4.2%) and others (1.8%). risk factors for uti in the multivariate analysis included cadaver donor recipient; substitution of the initial immunosuppressive regimen; days of urinary bladder catheterization and length of stay in hospital before the infection. substitution of initial protocol of the post-transplant immunosuppressive regimen and the surgeon was also associated with ssi. six different e. cloacae multidrug-resistant to antibiotics dna profiles were detected in uti of our recipients and hospital dissemination was documented. conclusions: uti was the single most important type of hospital infection in renal transplant recipient and a significant difference in the incidence of uti was found comparing living donor and cadaver donor recipient. the high incidence of uti in the early period post-transplant suggested that the operative manipulation of the urinary tract may be an important causative factor for the development of uti. usefulness of cmv antigenaemia assay after liver transplantation recurrence rates were higher (p < 0.001) in symptomatic infections. underlying liver diseases of the recipients, infectious complications other than cmv, rejection rates, and survival rates were not different between symptomatic and asymptomatic cmv infections (p > 0.05).conclusion: about an half of the recipients experienced cmv reactivation, mostly within 180 days post-transplantation. thirty percents of reactivation were symptomatic. peak value and duration of cmv antigenaemia were significantly higher in symptomatic infections than those in asymptomatic infections. toxoplasma gondii infection in bone marrow transplant recipients: the polymerase chain reaction-blood sample combination in diagnosis and early detection a. sensini, r. castronari, c. ferranti, t. aloisi, f. aversa, f. bistoni (perugia, i) objectives: toxoplasmosis is a rare and frequently fatal complication in bone marrow transplant recipients. it presents with local brain lesions or disseminated infection. usually, the diagnosis is based on histologic confirmation and observation of typical lesions on radiologic imaging, which resolve after appropriate therapy. the aim of this study was to stress the pivotal role of the polymerase chain reaction (pcr) in diagnosis and early detection of toxoplasma infection. clinical microbiology and infection, volume 11, supplement 2, 2005 methods: from january 1999 to july 2004 two hundred and thirteen patients underwent allogeneic peripheral blood stem cell transplantation (pbsct). in the presence of symptoms suggestive of cerebral, pulmonary and disseminated toxoplasma infection, 992 biological samples (blood, serum, csf, pleural fluid, bal) were collected and examined by nested pcr. results: fourteen cases of toxoplasmosis were identified, having an incidence of 6.6%. ten patients manifested cerebral toxoplasmosis, two had pulmonary infection and two disseminated. the overall mortality in this group, not necessarily due to toxoplasmosis, was 57% (8/14). the pre-transplant recipient serostatus was known in 9 patients: 5 were seropositive, but it is to be noted that 4 were seronegative, whereas 5 were unknown. mri study was performed in 12 patients. typical lesions were observed in 7 patients (sensitivity: 58%). the pulmonary toxoplasmosis occurred very early (day 9 and 10) and cerebral toxoplasmosis from day 29 to day 240 (mean 80.4) after pbsct. one case of disseminated infection had cns localization (day 21) and then pulmonary (day 42), whereas the second case had first pulmonary involvement (day 8) immediately followed by cns. blood samples demonstrated higher sensitivity (10 pcrpositive/12) than csf (6 pcr-positive/11) in identification of the etiologic agent. all patients with toxoplasmosis were treated with pyrimethamine and sulfadiazine. the study confirms the pivotal role of pcr in early diagnosis of toxoplasmosis after pbsct. in our experience, a positive pcr signal in blood was an early sign of toxoplasma infection, therefore blood appears to be a suitable and reliable biological sample for diagnosis. moreover, the samples were pcr-negative after the start of therapy and therefore pcr can be useful to monitor the effect of treatment. finally, our study indicates that toxoplasmosis is a potential pathology even in pre-transplant seronegative subjects, suggesting primary infection. cryptosporidium associated cholangitis in a livertransplant patient c. denkinger, p. harigopal, p. ruiz, a. tzakis, l. dowdy (wü rzburg, d; miami, usa) cryptosporidium parvum is a parasite of the group coccidia. c. parvum causes self-limiting diarrhoeal illness in immunocompetent hosts and severe long standing diarrhoea in immunocompromised patients. sclerosing cholangitis (sc) caused by c. parvum is frequently found in hiv-infected patients. in the transplant recipients only two case reports exist describing sc due to c. parvum; one in an adult renal transplant patient and the others in three children who underwent liver transplantation. we report herein the first case of c. parvum associated cholangitis in an adult liver transplant patient. this 52-year-old male patient underwent a liver transplantation three years prior to presentation. his initial transplant failed due to acute rejection requiring retransplant within one year. the second transplant was complicated by sepsis, tacrolimus induced hemolytic uremic syndrome, leaving the patient hemodialysis dependent. in 2004, he presented with pruritus, hypotension, severe dehydration and a three-week history of severe diarrhoea. endoscopic biopsy revealed numerous organisms in the stomach and changes in the colon consistent with cryptosporidiosis. stool cultures were negative. in addition, gamma gluytamyl transferase and alkaline phosphatase were elevated while bilirubin and ast were normal. the patient was diagnosed with colitis due to cryptosporidium and treated with azithromycin. immunosuppression with tacrolimus was continued. the patient continued to have diarrhoea, low-grade fevers and fatigue. two weeks later a cholangiogram and liver biopsy were performed. the liver biopsy revealed c. parvum lining the ductal epithelium. sc was diagnosed. the cholangiogram showed no sclerosing changes in the biliary tree, suggesting an early phase of disease. paromomycin was added to the regimen. the patient improved, and was discharged home. we believe this to be the first case of sc associated with c. parvum in an adult liver transplant recipient. objective: human herpesvirus 6 (hhv-6) infection usually occurs during the first 2 years of life, and its prevalence is higher than 95% in adult population. negative serostatus for hhv-6 in solid organ transplant recipients is a risk factor for fungal infection, but the incidence and severity of hhv-6 infections in seronegative patients has not been evaluated. our objective is evaluate prospectively seronegative hhv-6 solid organ transplant patients for hhv-6 infection by means of quantitative pcr for hhv-6. methods: from february to august 2004, we prospectly evaluated all solid organ transplant patients. patients must have a minimum follow-up of 3 months. patients underwent basal determination of igg for hhv-6. seronegative patients were included for follow-up. blood samples were obtained at 7, 14, 21, 28, 45, 60, 75, and 90 days post-transplant. for viral dna analysis, following dna amplification, it was determined by hibridation in microplaque (affigene). results: in the study period, 112 patients were evaluated (58 kidney, 35 liver, 11 heart, 4 kidney and pancreas, liver and kidney 2, pancreas 1, heart and kidney 1). nine patients were seronegative (7 kidney, 1 liver, and 1 heart). we analysed 67 plasma samples. all patients but one had a negative pcr for hhv-6. the patient with primary infection had a maximum of 78,002 hhv-6 dna copies/ml. clinically, the patient suffered from slight abdominal pain and discrete cholestasis in blood analyses; no fever or serious complications were detected. he did not receive pre-emptive treatment with ganciclovir. no fungal infections were detected. conclusion: primary infection by hhv-6 in seronegative solid organ transplant patients is not frequent, and in 1 patient it appeared with slight symptoms. however, a larger study with more seronegative patients is needed. development and validation of a new real-time pcr assay for hhv-6 detection and differentiation of hhv-6a and hhv-6b d. becker, s. ziegelmaier, s. wach, t. laue, t. grewing (hamburg, d) objectives: human herpesvirus 6 (hhv-6) has been identified as the etiologic agent of roseola infantum in infants. in adults hhv-6 causes complications in immunocompromised patients such as aids patients and transplant recipients. two virus variants can be distinguished: hhv-6a and hhv-6b. several studies have shown variant specific clinical outcome. the objective of this work was the development of a reliable, sensitive and specific hhv-6 detection assay, based on real-time pcr technology, which enables the discrimination between hhv-6a and b. this assay ought to run with the same temperature profile as the realart lc pcr assays for hsv1/2, ebv, cmv and vzv (artus gmbh). methods: by sequence alignments, a region of the hhv-6 genome was defined, which allows the specific amplification by real-time pcr, and discrimination between hhv-6a and b by melting curve analysis. assay specificity has been tested by analysing hhv-6 a/b reference material (abi) and cell culture supernatant from clinical isolates. cross reactivity was excluded by testing dna from related viruses and bacteria. analytical sensitivity was determined by probit analysis. a heterologous pcr system was incorporated, which serves as an internal control. a set of quantitation standards was established, for exact quantitation of the viral load. results: with the realart hhv-6 a/b lc pcr assay artus gmbh developed a ready-to-use assay for the detection and distinct differentiation of hhv-6a and b. quantitation of the viral load within a broad linear range is possible for both variants. simultaneous amplification and parallel detection of internal control and specific sample in one reaction tube excludes false negative results. the realart hhv-6 a/b lc pcr assay has the same temperature profile as the realart lc pcr assays for hsv1/2, ebv, cmv and vzv. 0 conclusion: the realart hhv-6 a/b lc pcr assay allows sensitive and specific detection, as well as subtyping of hhv-6. this leads not only to a reliable hhv-6 diagnosis, but also to a better understanding of the role of hhv-6 variants in pathogenesis. due to the same temperature profile, the realart hhv-6 a/b pcr assay completes the realart lc pcr assays for the detection of different herpes viruses. now the parallel detection of hsv1/2, ebv, cmv, vzv, and hhv-6 a/b is possible in a single real-time pcr run. thus, clinicians receive a reliable diagnostic tool for rapid detection of herpes viruses, and especially in transplant recipients. quantitative cmv pcr in allogenic stem-cell transplant patients l. cardeñ oso, c. blazquez, r. rodriguez, j. diaz-regañ on, e. lomas, p. sanchez, r. cámara (madrid, e) objective: to assess the clinical value of a commercial quantitative plasma cmv-pcr assay (cobas amplicor cmv monitor test, roche molecular system) in allogeneic sct patients by comparing the results obtained with the pcr with those obtained with antigenaemia. to evaluate the impact of an automatised dna extraction method and a lower cut-off on pcr sensibility. methods: all patients were monitored until day 100 post-sct with weekly antigenaemia and pcr. a total of 562 blood samples from 30 patients (23 mieloablative and 7 non-mieloablative; 25 hla identical siblings and 5 from unrelated donors) were tested prospectively. twenty-seven cmv seropositive patients (or negative with a seropositive donor) received high dose of acyclovir as prophylaxis. pcr was considered positive when more or equal 600 dna copies/ml were detected. antigenemia was considered positive when one or more positive cells were detected (in 2x105 pmns). positive samples by antigenaemia and/or pcr (n = 54 samples) were tested retrospectively with an automatised extraction method (magnapure). results: all cmv seronegative patients with a seronegative donor (n = 3) were antigenaemia and pcr negative. seropositive patients (n = 27): 14 had a positive antigenaemia and/or pcr, with a total of 23 cmv infection episodes. none developed cmv disease. pcr detected 10 out of 23 cmv episodes. overall there were 44 positive antigenemias (belonging to 13 patients): 23 were pcr+, 20 pcr negative and 1 pcr inhibited. only 1 patient had a positive pcr with a negative antigenaemia. automatised magnapure extraction increased the sensibility of the pcr. with this method pcr detected 19 out of 23 episodes in contrast with only 10/23 with the manual extraction. for samples with less than 600 dna copies/ml a manual calculation of the number of copies was retrospectively done (using a new cut-off of 50 copies/ml). with this lower cut-off, pcr detected 20 out of 23 cmv episodes. none of the negative patients by antigenaemia and/ or pcr gave a positive result with the automatised extraction method or with the lowered cut-off pcr. conclusion: quantitative cmv pcr assay showed a lower sensibility for the detection of cmv infection in allogeneic sct compared with antigenaemia. two technical modifications increased the sensibility without a decrease in the specificity: automatised magnapure extraction, and lowering the cutoff. trichosporon beigelii as a life-threatening pathogen in bone marrow transplantation recipients: the first case report from iran patients undergoing bone marrow transplantation (bmt) are profoundly immunosuppressed as a result of their intensive chemotherapy and are at high risk for opportunistic fungal infections mainly caused by candida spp and aspergillus spp. trichosporon beigelii (t. beigelii) has emerged as a life-threatening opportunistic pathogen in immunocompromised hosts. response to antifungal agents is poor, mortality is high and immunological recovery is the most important factor for a favorable outcome in patients with trichosporonosis. we present the first case of t. beigelii infection in patients undergoing allogeneic bmt in iran. a 12-year-old female presented with aplastic anemia, cough and fever. she had received cytotoxic drug therapy, broad spectrum antibiotics and was neutropenic. t. beigelii was repeatedly demonstrated by appropriate morphological and physiological characteristics in her sputum, nose and mouth ulcers by direct microscopy and culture, and also isolated and histopathologically recognized in post-mortem from lung and liver. trichosporonosis is likely to be recognized more frequently than apparent from the available reports. objectives: trichinellosis is a well-known problem in greenland. in west greenland a number of large outbreaks took place during the 1940's and 1950's, but since then only sporadic cases have been registered. it is unknown whether the decrease in trichinellosis cases reflects the general transition in greenland towards a more western lifestyle with less consumption of meat from wildlife, or whether it reflects insufficient case registration. the objectives of the present study were to determine the prevalence of human trichinellosis from the 1980õies to present times in nuuk, the capital of greenland, and in ammassalik district on the east coast of greenland, where more traditional food is still eaten, and to evaluate if changes in lifestyle had an effect on prevalence. methods: blood samples from 86 persons collected in 1981 and 533 persons collected in 1998 in nuuk and ammassalik district were tested for trichinella-specific igg antibodies using elisa and western blot analyses. background information was obtained from questionnaires from the 1998 study. results: in 1981 the trichinellosis prevalence was 8.7% in nuuk and 23.8% in ammassalik district, while the prevalence in 1998 was 5.2% in nuuk and 19.8% in ammassalik district. persons living in the settlements of ammassalik district had higher trichinella-specific antibody prevalence than persons living in the town of tasiilaq. the following risk factors were significant in a univariate analysis: ethnicity, age, place of living, dietary habits, intake of seal meat, and hunting. a multivariate model was constructed consisting of the variables: ethnicity, age, place of living, diet group, intake of seal meat, and hunting. the variables were removed stepwise in the following order: seal meat (p = 0.73), hunting (p = 0.36), age (p = 0.3), and ethnicity (p = 0.16). the final multivariate model consisted of diet group and place of living, both being significant. thus, living on a greenlandic diet and living in the settlements in ammassalik district implied higher relative risk of being trichinella seropositive than living on a diet dominated by imported food items and living in nuuk. conclusion: as lifestyle in the settlements is more traditional compared both with tasiilaq and nuuk, these results indicate that the decline in human trichinellosis cases in the 19th century most likely reflects the transition from traditional greenlandic lifestyle to more western dietary habits. study on prevalence of toxocara cati in vagrant cats of sari township, iran, 2004 m sharif, p. ziapoor, h. ziaee, s. kholami (sari, ir) objectives: toxocara cati is one of the important and prevalent parasites in cats and common between human and animals. ingestion of larvae of this parasite and lack of it's maturation in human body , the infected persons develop visceral larvae migrans which is known as toxocariasis. considering the significance of transmission of this parasite in human and due to the increase in vagrant cats in sari township, this study was performed in order to determine the severity and prevalence of toxocara cati in these cats in sari township. methods: this cross sectional descriptive study was done on 100 vagrant cats (28 males and 72 females) during april to october 2004. sampling was done randomly at different regions by net. the specification of vagant cats were recorded after hunting and were deeply anaesthetized by chloroform to annihilate them. the intestinal content were examined for the presence of parasites and counting of their number was done finally after fixation and staining by recommended kits of reference book for genus and species identification. results: in all, 42 (42 %) cats were infected with toxocara cati, of which 8(19%)were males and 34 (81%)were females. range of infection was 1-32 and mean severity of infection was 3.14 for each cat. the rate of infection at the west region was more than the other sites, and it was more in the females than males and also it was more in immature than the mature cats. conclusion: considering the merely high prevalence of this zoonotic parasite and it's hygienic significance in causing toxocariasis in human particularly in children, who are in more contact with soil and also lack of control on vagrant cats population, which are the potential risk factors, it is recommended to control personal and dietary hygiene and avoid storing the garbages in unprotected wire boxes on the streets in order to prevent the gathering of vagrant street cats. imaging in 100 patients of pulmonary hydatid disease; review of unusual imaging appearances s. zahirifard, m. bakhshayeshkaram, m. tahbaz, k. kaynama (tehran, ir) objective: to evaluate the chest radiography and ct scan characteristics of pulmonary hydatid disease. material and methods: 100 patients with surgically proven pulmonary hydatid cysts were enrolled for study. we reviewed clinical findings and imaging of patients. the radiological features (localization, diameter, architecture, density and other radiological signs and appearances) were determined. results: on cxr 124 cysts were determined. on 82 available ct scans a total of 112 cysts were detected. no discrete cyst was detected on 11 cxr. on 82 available ct, a total of 112 cysts were detected and in 5 ct scans no cyst was detected. 57 cysts were ruptured and 25 patients with ruptured cysts had hemoptysis. single cysts were in 63 patients while multiple cysts were in 37. median ct density was 24hu. respectively, it was seen on ct and cxr waterlily sign in 18 and 22 patients, air-fluid level in 12 and 17 patients and cresent sign on 11 and 5 of patients. inverse cresent sign and calcification were not observed on cxr s but each one was recorded on 4 ct scans. on ct scan 90% of cysts were smooth and 89% were uniloculated. 19% of cysts were infected. conclusion: ct scan should be done to elucidate cystic nature of the lung masses and for accurate localization in the preoperative period. in endemic regions like iran, atypical imaging presentation of complicated pulmonary hydatid disease such as solid masses should be considered in differential diagnosis of pulmonary lesions (abstract truncated at 250 words) the seroprevalence of coxiellosis in farmers and cattle in north-eastern turkey s. seyitoglu, z. ozkurt, u. dinler, b. okumus (erzurum, tr) objectives: coxiellosis is a worldwide zoonosis caused by coxiella burnetii. the aim of this study was to determine the abstracts prevalence of coxiellosis in cattle and farmers in north eastern turkey. methods: a total of 230 cattle and 92 human sera were collected in 2003, and tested for antibodies against c.burnetii by commercial elisa test. results: the antibodies of c. burnetii were detected in 22 (9.56%) cattle and 18 (19.5%) healthy farmers. seropositivity was found in 12 of 53 (22.6%) cattle with abortion history, and 10 of 177 (5.6%) cattle without abortion history (p < 0.05). there was correlation between animals and human seroprevalance in same district. thirteen of 18 farmers who were antibody positive had seropositive animals, and there were seropositive animals in the villages of remaining 5 cases. it was observed that seroprevalance of coxiellosis was higher in north districts (in animals 15.4%, in humans 32.4%), have drier and warmer climate, than in other districts (in animals 6.5%, in humans 12.1%) both for humans and animals (p < 0.05). conclusions: the study show that coxiellosis was an important health problem both in humans and in animals in our region. an unusual case of gramineae in sublingual salivary gland l. doganci, m. calgurel, y. gungen, g. kiran (ankara, tr) foreign bodies of organic nature and parasitic elements are very rare in oral cavity and in salivary glands in humans because of their structural and functional features. here we report a very interesting case, with sublingual salivary gland involvement of gramineae (brachiaria spp.) which was thought to be a parasitic life cycle in the gland. case: 40 y/o female teacher with a recent history of travel to mountains where she drunk and had an exposure to unsafe spring water. she had a strong stinging pain right after she drunk the water in her mouth, radiating to her neck. she developed fever, pain, lymph node swelling, difficulty of eating, weight lost and remarkable eosinophilia. she also noted a self moving small tail-like object on the orifice of the sublingual salivary gland. removal of the object revealed an interesting unusual living object. then, definitive diagnosis is gramineae (brachiaria spp) after parasitological and pathological consultation. comment: to our best knowledge, this unusual case is the first presented patient related to travel in our region. differential diagnosis has several entities including squid sperm bag sting, marine animal larvae and nematod and trematod life stages. evaluation of echinococcus granulosus prevalence using elisa and abdominal ultrasonography in a group of students staying in a state hostel in turkey objective: cystic echinococcosis, caused by larval form of echinococcus granulosus is one of the most important and widely distributed parasitic zoonotic diseases in the world. echinococcosis is a major problem in all regions of turkey but particularly prevalent in rural areas, where domestic livestock raising is common. the reported epidemiologic findings are usually based on retrospective evaluation pf surgical findings or hospital charts. well planned epidemiologic studies are limited. in this study our aim was to analyse the seroprevalance of cystic echinococosis and prevalance of lesions (with abdominal ultrasonography) in a group of young adult university students who were staying in a state hostel in bornova-izmir-turkey. method: the study group consisted 750 students (360 woman, 390 men, aged 20.92 ± 1.82, min 17, max 29). informed written consent was received from each student and they were requested to fill a questionnaire form. blood sampling was performed by iv puncture and sera were obtained after centrifugation. anti echinococcus granulosus antibodies were detected by using elisa tecnique. all participants were gven an appointment for abdominal ultrasonography. results: of 750 students 99 (13.2%) were seropositive for anti echinococcus granulosus ig g (table 1). a total of 250 students (49 seropositive-201 seronegative) were performed abdominal ultrasonography (3.5 mhz transducer, sonoline, elegra-siemens). two of 250 students (1 in liver, 1 in kidney both seropositive) had cystic lesions and were referred to surgery. well designed epidemiologic studies about cystic echinococcosis are lacking in turkey. a previous study showed seropositivity rate of 13.7% for echinococcus granulosus. our findings suggest that cystic echinococcosis is prevalent in turkey and epidemiologic studies combining elisa and abdominal ultrasonograpy are warranted. objective: anthrax is an epidemic zoonosis in eastern part of turkey. this study was aimed to investigate the epizootiology and epidemiology of anthrax in this region. methods: animal and human anthrax cases during 1992-2004 period were included in this study. data were obtained formal records of health directorate, institute of veterinary control and investigation. the diagnosis of anthrax had based on clinical findings and/or microbiological procedures, including gram strains and culture of materials obtained from lesions. results: in a 13-years period, 464 animal cases and 503 human cases with anthrax were detected. of 464 animal cases, 20 (4.3%) were sheep, others (95.7%) were cattle. most cases occurred between july and september in both animals and humans. anthrax was seen most frequently in erzurum, which is the important centre for animal commencement. when the first 6 years period (1992) (1993) (1994) (1995) (1996) (1997) was compared with last 7 years period (1998) (1999) (2000) (2001) (2002) (2003) (2004) ) the number of animal cases were 161 vs. 230 and human cases were 234 vs. 269. an analysis of the yearly case distribution shows that the incidence of anthrax in this region had not been changed. conclusion: anthrax is an important health problem both animals and humans in this region. the preventive measures should be taken for decreasing the incidence of anthrax. objectives: anthrax is endemic in kazakhstan and occurs among humans at an incidence rate of 0.1 to 1 per 100,000 population per year. during the late 1990s, several epidemics occurred associated with home slaughter of infected animals, and a general increase in incidence was seen among animals and humans. historically, kazakhstan accounted for 15% of the anthrax cases reported from the soviet union; and the plains of central asia may have served as the historical source of b. anthracis for the rest of the world. in kazakhstan, the geographic distribution of anthrax among animals and humans is focal and generally associated with areas considered to be ôhigh-riskõ. the determinants of this focal distribution are poorly understood. in addition, strains of b. anthracis isolated from outbreaks within these foci have not been compared on a molecular or pathogenic basis. methods: work has been initiated to study the geographic distribution and potential environmental preferences of b. anthracis using geographic information system (gis) technologies. results: the gis was used to map a set of b. anthracis strains from several outbreaks between 1997 and 2003 to evaluate geographic patterns. the gis is also being used to derive a historical database of spatially explicit human and livestock outbreaks from 1948 to 2003 for spatial analyses on the nationwide distribution of b. anthracis. this database contains information on the location of the anthrax incident, the year and month of the occurrence, whether the incident involved livestock or human, the outcome of the disease, and biological information about the strains. conclusions: although in a preliminary stage of application, the arcgis software can be used to map loci of anthrax occurrence. in the future, these maps will be used to identify factors that influence the occurrence and spread of the pathogen and to prevent infection of livestock or humans. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency (dtra) under the project «kb0-1950-al-03» and administered by u.s. civilian research and development foundation (crdf). objectives: the anthrax situation in the country was unfavorable from the 1950s through the 1970s. until the 1990s, anthrax in kazakhstan was an occupational disease in animal husbandry. after 1991, the practice of raising livestock for private use increased. this report shows that, at the same time, the incidence of anthrax in persons involved in this practice increased significantly. methods: in order to characterize the epidemic process, we used methods of descriptive epidemiology, using epidemiological variables (who became ill, where and when, trends of disease by time, sources, transmission factors of b. anthracis, and the age, gender, and occupation of persons who become ill with anthrax). results: anthrax occurs in rural areas among private owners of livestock and their families and persons hired to assist with the slaughter. in the last decade, 89.4% of cases of anthrax illness in humans were caused by the slaughter of domestic livestock. of these cases, 62.2% were unemployed rural residents, 2.3% were shepherds working for cooperatives, 11.7% were blue-collar workers, 5.4% were white-collar workers, and 18.1% were students. a single source, path and factors of transmission of b. anthracis are typical of this type of the disease. in recent years, the majority of cases involve infection of several persons during the slaughter of a single diseased animal. the source of infection is cattle in 48.2% of cases, sheep and goats in 15.7% of cases, horses in 31.2% of cases, and soil in 4.7% of cases. the factors of transmission of b. anthracis to humans are contact with the meat of livestock during slaughter and butchering in 91.9% of cases, vector-borne transmission in 2.7% of cases, and soil in 5.4% of cases. a large number of human cases occur in july through september. conclusion: in kazakhstan today, the prevalent sub-type of anthrax disease in humans involves non-professional activity at home. this requires changes in the tactics of preventing the disease. the research described in this abstract was objectives: severe acute respiratory syndrome (sars) is an emerging infection caused by a novel coronavirus known as sars-cov. during the early phases of the disease, the presence of the replicative intermediates of sars-cov has been shown in pbmc from patients. no information is available on the ability of sars-cov to stimulate ifn induction, although ifn-gamma may be activated in sars patients. we investigate the capability of sars-cov to give rise to a productive infection in normal pbmc, in parallel with the ability to activate ifn-alpha andgamma gene expression. methods: normal pbmc were infected with a moi 0.1. virus progeny formation was measured at subsequent time points, by back-titration of cell lysates on vero cells. cell mortality and apoptosis were detected by trypan blue and propidium iodide staining. in order to detect virus-specific plus-and minus-rna strand, total rna extracted from sars-cov-infected pbmc was retrotranscribed in the presence of the sense and antisense primers, respectively, targeting the replicase gene. measurement of sars-cov genomic rna was performed by quantitative real time rt-pcr. ifn induction was performed by exposing pbmc to sars-cov at different moi, ranging from 0.1 to 10. induction of ifn-alpha and -gamma was analysed by measuring their mrna levels by limiting dilution rt-pcr. sensitivity of sars-cov replication to exogenous ifn-alpha and -gamma was tested in vero cells pre-exposed to the individual cytokines of to a combination of both. results: in unstimulated pbmc, no infectious viral progeny formation was detected up to day 13 post-infection. nevertheless, minus-strand and genomic sars-cov rna peaked at 24 hours post-infection. dose dependent induction of both ifnalpha and ifn-gamma mrna was observed, that was most evident at moi 10. a combination of these two cytokines was shown to strongly inhibit sars-cov replication in vero cells. our results show that sars-cov is able to infect normal pbmc. however, this appears to be only a transient phenomenon, followed by a progressive disappearance of both virus-specific rna strands, with no infectious virus progeny production. moreover, sars-cov appears to have the intrinsic ability to activate dose-dependently both ifn-alpha andgamma gene expression in pbmc cultures. our results can be pathogenically relevant to the inflammatory events occurring in the diseased tissues that can be mediated by the endogenous activation of inf system. objectives: trichoderma species are potential candidates for biocontrol of plant pathogenic fungi and cellulase producers of biotechnological importance. on the other hand, they are emerging as opportunistic pathogens of immunocompromised and dialized patients. this study was designed to examine the ability of 10 clinical trichoderma isolates to produce extracellular proteases and to screen them for toxicity to mammalian cells. methods: supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. trypsin-and chymotrypsin-like activities were separated by sephadex g-100 column chromatography and their ph-dependence was studied. sperm toxicity bioassays were performed by exposing boar sperm suspension to trichoderma-methanol extracts, the motility of spermatozoa was estimated by phasecontrast microscopy. mitochondrial membrane damage in spermatozoa was studied by epiflurescence microscopy using the jc-1/propidium iodide staining. results: the production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities was common among the examined strains. separation of trypsin-and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. relatively high activity levels were detected between ph 5 and 9 suggesting that the different isoenzymes have different ph characteristics. more than 90% of the spermatozoa were motile in a non-exposed control sample and in a sample exposed to 5 microliter of methanol. more than 90% of the spermatozoa were immotile in samples exposed to t. longibrachiatum uamh 9515, atcc 201044, cm382 and t. harzianum cbs 102174 in a concentration of 1.25 mg biomass ml-1, indicating that these strains produce heat-stable substances toxic to sperm cells. these four strains were found to inhibit the motility of boar spermatozoa even at a low concentration (50% reduction of motility in the case of 5 microgramm ml)1 of extended boar sperm) and to induce dissipation of mitochondrial membrane potential. other studied strains had no toxic effect. conclusions: production of extracellular proteolytic enzymes and toxic metabolites are among the potential virulence factors of trichoderma strains as emerging fungal pathogens. this work was supported by grants f037663 of the hungarian scientific research fund and 53305 of the academy of finland. occurrence of scabies in patients and in the staff of healthcare facilities objectives: scabies is an itching dermatosis which continues to be a frequent health problem. long-term follow-up of the epidemiological situation confirms a thirty-year cycle for which there is no satisfactory explanation. methods: standard methodology of diagnostics is based on a) subjective sensations in the patient b) objective dermatological finding c) positive epidemiological history d) laboratory demonstrations of causative agent e) remission of symptoms upon specific therapy. cases of scabies diagnosed and reported by dermatologists nationwide are included by the public health service in the central system epidat (niph, prague). results: the last two documented epidemic waves of scabies affecting the czech republic peaked in 1970 and in 1993. specific morbidity is the highest among persons 15-24 years of age (it closely connected with sexual activity-the source of infestation in 60% of adults was a sexual partner and that is confirmed by an almost identical trend in reported scabies morbidity and fresh cases of syphilis in 1965-1999 years), but the morbidity trend has been falling sharply after the year 1993. however, morbidity in persons over 65 years of age has been gradually rising over recent years. group epidemics (86) have been repeatedly reported in such facilities as departments of gerio-psychiatry, institutions for long-term chronic patients, old folks homes, social care institutes and charity facilities. that occurs in bed-ridden subjects hospitalized on a long-term basis, the mentally retarded and the superannuated, in whom the scabies is of a nosocomial character. the affected staff of healthcare institution is very often the source of further transmission to others, namely to immobile patients, as well as to especially vulnerable individuals under higher risk of infection. analysis of the reported cases revealed a 13% share of healthcare workers in this infection. under greater risk are senior nurses (62%), junior nurses (21%), auxiliary paramedical staff (13%)and the last of all physicians (4%). scabies as an occupational disease occurs almost exclusively in the healthcare sector. conclusions: scabies poses on increased risk for the younger population (active scabies) and the elderly population (passive scabies). it poses a great occupational risk in the paramedical healthcare staff carrying out nursing or managing services. it is necessary to consistently observe preventive and repressive epidemiological measures in the population. fire ants, the new public health problem in iran k. akbarzadeh, m. nateghpour, s. tirgari (tehran, ir) ants are probably the most successful of all insects. they are present almost in all countries and all places. a few of them can bite, sting and squirt formic acid. formica rufibarbis and a few the others are secondary hosts of dicrocoelium dentriticum in iran. but the newest public health problem depending on ants in the country is biting and stinging of fire ant pachycondyla sennaarensis (formicidae; hymenoptera) in south and southeastern iran. ecology, biology and morphology of p. sennaarensis were considered in iranshahr county (southeast of iran) where has been much infected because of the ant. according to the survey over 92% of questioned individuals had bitten at least once with the ant. usually the effects of the stings are mild but this ant is capable of multiple stinging and this can induce annoyance people especially in children. objective: tick play as important role in diseases transmission to human being. different tick-borne diseasea including borreliasis, cchf have been reported from iran. method: an attempt was made to determine the fauna and geographical distribution of soft and hard ticks in this area from october 2002 until april 2004. about 10% of the villages were selected randomly and tick collection was carried out using standard method. ticks were collected from indoor resting and hiding places in 70 villages as well as inside stables, rodent borrows, on sheep, lamps, goat and cattel by monthly in each season. result: 3382 ticks were collected and identified using national systematic key. the copmosition and frequencies of them were as follows: ornithodoros lahorensis (11%), argas persicus (23.12%), rhipicephalus bursa (4.3%), rhipicephalus sanguines (1.53%), haemaphysialis concinna (9.6%), haemaphysialis sulcata (26.8%), haemaphysialis punctata (1.54%), hyaloma schulzei (8.16%), hyaloma marginatum (7.21%), hyaloma dentriticum (3.08%), hyaloma asiaticum(1.53%). conclusion: distribution and ferquency of tlck was differ in various season and location. evaluation of the use of saliva to soothe bloodsucking arthropod bites (dedicoat m et al, 2004 , j infect dis 190:1068 . the conditions for hhv-8 transmission are met when i) a child's skin responds to the bite of a blood-sucking arthropods, and ii) an hhv-8-seropositive mother (or caregiver) attempting to relieve the child's itching and to reduce scratching applies her infective saliva to the bite's site. the use of saliva is a traditional behavioural practice in sub-saharan africa, for healing with herbal leaves crushed and mixed with saliva and for medical practices and in the premastication of food (wojcicki jm, 2003 , br j cancer 89:2016 . the human behaviour associated with the transfer of saliva from parents to infants and the use of herbal leaves treated with saliva in relation to arthropod bite could be a risk factor for hhv-8 infection and perhaps other infections too (such as the hepatitis b virus). methods: to evaluate to what extent saliva is used to heal insect bites on children's and adolescentsõ skin, we draw up a questionnaire directed at students of elementary and intermediate school. two groups were tested, one from italy (a school near rome, latium, and schools in veneto) and one from a sub-saharan african country known for high transmission levels (uganda, between kampala and entebbe by lake victoria). results: the frequencies of the use of saliva were not significantly different in latium (12.5%) and in veneto (14.6%) so that an average frequency of 13.5% (96 / 712) has been compared with the frequency of 74.8% (119 / 159) from uganda (p << .0001). the frequencies in the two groups are related also to a different cultural and economic development and improvement in hygiene. conclusion: we suggest that blood-sucking promoter arthropods can play a role in the spread of hhv-8 infection particularly in africa. therefore, a behavioural change limiting the use of saliva on the skin and to prepare herbal leaves, could be an attempt to control the spread of hhv-8 infection. aim of study -to find out in csf some baseline markers of inflammation and redox status for the estimation of development tendences and clinical course of pathological process in tbe. patients and methods: 60 patients with meningeal and meningoencephalitic form of tbe treated at the infectology center of latvia were included in this study. diagnosis of tbe was confirmed by elisa, enzygnost, anti-tbe igm in blood and/or csf. traditional clinical criteria were used for the characterization of tbe severity degree. some nontraditional lab methods were used: c-reactive protein (crp) (immunoturbidimetrically) and reduced glutathione (gsh) concentration (colorimetrically) detection in csf. results: concentration of crp in csf was significantly higher in tbe patients in acute phase (5.55 ± 1.0 mg/l; n = 34) than in reconvalescence (2.41 ± 0.5 mg/l; n = 26), p = 99%. crp and gsh in about 50% of tbe cases in csf was under detectable values. the second part of tbe patients demonstrated tendency of the increasing of gsh during acute stage of disease (1.64 ± 0.20 mg%; n = 32) if compared with reconvalescence (1.41 ± 0.20 mg%; n = 24), p = 61%. no statistically significant differences of crp and gsh concentrations in csf were found in cases of moderate meningitis form of tbe if compared to severe meningoencephalitic form of tbe. conclusion: 1) analysis of data obtained from patients with tbe gives evidence of substantial crp and gsh changes in csf in dependence on disease stage, but not on its severity degree. 2) these objective biochemical data from csf investigation give proof that there are no separately moderate and severe tbe cases in clinic: they have to be interpreted always as equally severe. 3) the origin of crp and gsh in csf is unknown (production in cns and/or from blood due to blood-brain barrier permeability increasing?). background: cholera is a severe disease caused by vibrio cholerae. the natural reservoir of this aquatic pathogenic bacterium is referred to as the ôaquatic environmentõ. our group showed that egg masses of the non-biting midge chironomidae spp. (diptera) harbour v. cholerae and provide nutrient source for their multiplication, thereby offering a new natural reservoir for the cholera bacterium. objective: the presence of viable v. cholerae on flying adult chironomids will suggest that v. cholerae can be carried through the air to other water bodies by the adult flying insects and hence a new route of infestation of water sources by the bacteria is suggested. methods: in field studies chironomid egg masses and adults were simultaneously collected from the same environment. in addition fresh, drinking quality water was left exposed or netted in the vicinity of adult chironomid hatching foci. the exposed water was monitored for chironomid egg masses and v. cholerae presence. in the laboratory those experiments were repeated with v. cholerae o9 tagged with green fluorescent protein. results: to date, more then 30 v. cholerae serogroups have been isolated from chironomids egg masses and adults. in the field experiments, a clear-cut connection between netting and v. cholerae absence was noted implying that a relationship exist, between entrance of large invertebrate to water bodies and presence of v. cholerae in that water. in the laboratory experiments, the gfp tagged bacteria were found on the outer surface of the adult chironomid, in locations prone to microbial attachment. conclusions: the evidence shown here suggests that aerial transport of v. cholerae by chironomids flying adults is feasible and does lead to v. cholerae contamination of unprotected water bodies. objectives: cholera is a severe and potentially life-threatening diarrheal disease caused by certain spices of the gram-negative bacterium vibrio cholerae. many millions of cases of cholera occur annually, in epidemic and pandemic forms due to v. cholerae o1 and o139, and also sporadically due to non-o1 and non-o139 v. cholerae strains. in this context, the survival of v. cholerae in nature is of interest from an epidemiological perspective. although the natural reservoirs for survival and multiplication of v. cholerae are far from compeletly disclosed, our previous study has shown that free-living amoebae can be reservoirs for the seventh-pandemic v. cholerae o1 el tor-inaba strain n16961, which can survive and grow intracellularly in acanthamoeba castellanii.the aim of this study was to examine the ability of different strains of v. cholerae o1 el tor to grow and survive in acanthamoeba castellanii, and to examine whether intra-amoebic survival of bacteria alter their pattern of resistance and sensitivity to different antibiotics. methods: v. cholerae o1 el tor strains were co-cultured with a. castellanii for more than two weeks. the interaction between these microorganisms was followed by viable counts of alone-and co-cultivated microorganisms. intra-amoebic growth and localization of each bacterial strain were estimated by gentamicin assay, viable count, microscopy, and pcr to detect cholera toxin gene and amoebic 18s rna gene disclosing symbiont-host association. results: the results show that examined v. cholerae o1 el tor strains multiplied and survived inside trophozoites and cysts of a. castellanii. the bacterial internalization was in cytoplasmic compartment of the amoebae cells. the relation between these microorganisms in co-cultures could be classified as symbiosis, since presence of the amoebae enhanced growth of bacterial strains, and presence of the bacteria did not affect amoebic growth. the intra-amoebic survival of bacteria did not alter their pattern of resistance and sensitivity to different antibiotics such as ampicillin, gentamicin, and tetracycline. conclusions: this study shows a facultative intracellular behaviour of examined v. cholerae o1 el tor strains, which is in contrast to the general held view, which considers the bacterium to be extracellular microorganisms. the clinical importance of free-living amoebae is their possible role as ôtrojan horseõ. the genetic variation of vp7 and vp4 antigenic proteins was studied in 38 g4 strains recovered in palermo from 1985 to 2003. vp7 sequence analysis showed that 14 strains, recovered during 1999-2003, though cross-reacting with g4-st3:1 mab, belonged in fact to genotype g9 and were 99% identical to g9 strains recovered in palermo from 1999. sequence comparison of 23 g4 strains grouped them into three sublineages (ia to ic) containing respectively viral strains collected in 1990-1994, 1995 and 1999-2003 . amino acid sequences were well conserved within each sublineage and a peculiar single amino acid insertion was observed in the variable region 4 of all sublineage ic strains. both g4 and g9 italian strains exhibited p[8] genotype and their vp4-encoding sequences clustered in previously defined lineages following a temporal distribution: strains collected in 1989-1993 fell within lineage p[8]-1, while those recovered in 1995-2003 clustered in lineage p[8]-3. the variety of their deduced amino acid sequences enabled us to describe, respectively, two (1a and 1b) and six (3a-3f) different patterns of substitutions with regard to lineage p[8]-1 and p[8]-3 reference strains. comparison of vp7 and vp4 gene sequences of italian and european g4 strains indicates that different g4p[8] populations have been circulating in europe over the years and that the latest italian strains are more closely related to recent isolates from other countries than to the reference vaccinal st3 strain. these results will help increase knowledge about g4 rotavirus epidemiology and evolution, and might be useful for future rotavirus vaccine formulation. detection of rotavirus, adenoviruses and astrovirus in gastroenteritis cases among young children, first experience with adenovirus identification by pcr and microplate hybridisation assay , adeno (all human serotypes) and astroviruses antigens (seven human serotypes) was performed by enzyme immunoassay method (dako ideia rotavirus, adenovirus and astrovirus tests, respectively). all specimens positive for adenoviruses were confirmed and identified by a pcr-microplate hybridization assay (pcr adenovirus consensus, argene, biosoft), which used adenovirus genus-specific primers and one generic and six species-specific probes defined in the va rna gene. according to our results: rotavirus was detected in 63 cases (9.2%), adenovirus in 15 cases (2.2%) and astrovirus in 15 cases (2.2%). pcr adenovirus consensus assay identified the adenovirus species from all the 15 cases: adenovirus species f (enteric adenovirus serotype 40 / 41) in 11 cases (1.6%), adenovirus species c in 2 cases (0.3%) and adenovirus species a in 2 cases (0.3%). rotavirus gastroenteritis presented a seasonal distribution in spring, while adenovirus and astrovirus gastroenteritis presented no seasonality. conclusions: rotavirus gastroenteritis was significantly more common than adenovirus and astrovirus infections in young children in our territory. pcr adenovirus consensus technique was a useful method for the identification of adenovirus in stool specimens. eventhought enteric adenoviruses 40 / 41 (species f) caused the majority of acute gastroenteritis due to adenovirus, non-enteric serotypes (species a and c) were occasionally involved in the aetiology of acute diarrhoea. the study continues with the examination of great number of specimens and patients for the investigation of more useful and specific results. immigrant inpatients from 1999 until today: 'pressure' exerted over infectious disease wards r. manfredi (bologna, i) objective: foreign individuals officially living in bologna and province are 39186 with predominant origin from northern africa (31.5%), eastern europe (22%), and far east (12%). the temporal trend of admissions of patients coming from outside of the european union was examined according to a broad series of variables. methods: through the electronic databases of our hospital located in a metropolitan area of northern italy, informations related to all foreign patients (p) hospitalized or followed by day-hospital centres have been collected from january 1999 up to march 31, 2004. after recording all discharge diagnosisrelated group (drg) codes we focused attention on those regarding infectious diseases. results: overall discharges were 339051, with 6670 regarding foreign p:a prevalence of the female sex (64.4%) was noticed. in 49.6% of foreign p,the mean age ranged from 20 to 35 years. the rate of foreigners compared with overall admitted p varied from 0.3 of year 1999 to a. maximum of 0.47% of year 2002. a major involvement was borne by obstetrics-gynecology (39.3%), internal medicine (13.1%), specialistic surgery (7.3%), specialistic medicine and pediatrics (7.3% each), and emergency medicine (7.1%). when considering infectious disease-related drgs, tuberculosis (142 cases), hiv-aids (25), chronic viral hepatitis and related complications (22), septicemia, central nervous system infection, and acute viral hepatitis (12 episodes each), were the most frequent discharge codes followed by other infectious disease drgs (72 cases). the duration of admission of foreign p was very short (1-3 days) in 42.1% of cases,while it reached 4-7 days in 22.6% of p,and 8-14 days in 11.2% of cases, while the mean hospitalization time at infectious diseases ward was 8.6 ± 3.2 days (p < .002). even 549 hospitalizations ended with voluntary discharge which occurred during the first day of admission in 61.8% of p. discussion: our pilot study demostrates that the need for health care expressed by foreign p is increasingly frequent and covers a very broad spectrum of disorders with infectious diseases-associated illnesses gaining a increasing role through time. a significant problem is represented by the tendency to very short duration of hospitalization for foreign p, often ending with voluntary discharge while admissions carried out at our specialized infectious disease ward are significantly more prolonged, although they are expected to give an increased benefit in terms of effective management. change of the characteristics of natural focuses of plague and microbiological properties of plague microbes as a result of consequences of the natural calamity on aral sea enzootics of plague as that of any other transmissible feral herd [feral nidal] infection are determined by the interaction of a disease's causative agent and its warm-blooded carriers under specific ecological environment. introduction and objectives: change of the characteristics of natural focuses of plague and microbiological properties of plague microbes as a result of consequences of the natural calamity on aral sea. methods: conventional microbiological research methods were used, when studying microbiological properties of 116 strains of plague microbe from kyzylkum natural focuses of plague isolated from rodents, ticks and fleas. results: strains from the kyzylkum natural focuses of plague have all determinants of virulence, show characteristics that allow attributing them to the continental variety, i.e. glycerin digestion, do not ferment rhamnose and decompose arabinose; they have nutrient need that is typical for the strains from the central asian desert focus. there are differences in the need for amino acid leucine, whereas strains from ustyurt and gissar focuses are dependant on it. out of 116 strains from the kyzylkum natural focus only 8 turned out to be dependant on luecine. there were identified stains from the kyzylkum natural focus that had essentially different properties as compared to the majority of isolated cultures, in particular, those can not synthesize fraction 1 of the plague agent; have growth factor of different nature, i.e. lower dependence on phenylalanine. plague bacteria strains circulating in rodent populations in the kyzylkum natural focus are characterized by low virulence in white mice and guinea-pig, although they have all the determinants of virulence. when studying resistance to antibiotics, there were identified strains non-susceptible to streptomycin (5%). conclusions: thus, studying the properties of the causative agent of plague is of great importance for solving problems of natural focuses and understanding patterns of fluctuations in the epizootic situation in a certain territory. this understanding is an integral part of determining the epidemic potential of natural focuses, and is used in organizing epidemiological surveillance of the infection. objectives: toll-like receptors (tlr) play a key role in the recognition of products from virtually all classes of pathogenic organisms. amyloid peptides also can stimulate the innate immune system. for this reason, we hypothetized that bacterial compounds and amyloid peptides may jointly stimulate the innate immune system. methods: the interaction of endogenous and exogenous stimulators of innate immunity was examined in primary cultures of mouse microglial cells after application of defined toll-like receptor (tlr) agonists [lipopolysaccharide (lps) (tlr4), the synthetic lipopeptide tripalmytoyl-cysteinyl-seryl-(lysyl)3-lysine (pam3cys) (tlr2) and single-stranded unmethylated cytosineguanosine (cpg) oligodesoxynucleotide (tlr9)] alone and in combination with amyloid beta peptide (abeta) 1-40. supernatants from stimulated glial cultures and unstimulated controls were analysed for nitric oxide (no), tumor necrosis factor-alpha (tnf-alpha) and interleukin-10 (il-10) production. results: co-administration of abeta1-40 with lps or pam3cys led to an additive release of no and tnf-alpha. in contrast, coapplication of abeta1-40 with cpg led to a substantial decrease of no and tnf-alpha release compared to stimulation with cpg alone. cpg was the only compound investigated, which induced the release of the anti-inflammatory cytokine il-10. conclusion: the additive effect of lps and pam3cys and abeta may be one reason for the clinical deterioration frequently observed in patients with alzheimer's disease during infections. the substantial decrease of no and tnf-alpha release after cpg and abeta application compared to stimulation with cpg alone suggests that not all microbial products enhance the stimulatory effect of abeta on innate immunity. the cause for the divergent behaviour after activation of tlr9 versus stimulation of tlr2 and tlr4 probably lies in differences of the signaling cascade. hla-b27 is a marker for susceptibility and severity of reactive arthritis after salmonella, shigella, and yersinia infections but not after campylobacter and e. coli enteritis p. schiellerup, h. locht, k.a. krogfelt (copenhagen, dk) objectives: reactive arthritis (rea) is a well-known complication after infections with salmonella, yersinia, shigella and campylobacter. the presence of tissue-antigen hla-b27 is associated with rea, and hla-b27 may be a marker of a prolonged disease course. we designed a case-case comparison study of rea after gastrointestinal infections caused by salmonella, yersinia, campylobacter, shigella and e coli, to evaluate the impact of hla-b27 on attack rate and severity of rea between these 5 bacteria. methods: between january 2001 and november 2002 consecutive patients were included from the danish registry of gastrointestinal infections, when culture positive for salmonella, yersinia, campylobacter, shigella, or e coli. all patients were addressed by a questionnaire. in this study rea includes reactive arthritis and arthralgia. debut of pain in a previously healthy joint was registered and patients were asked to mark swollen or tender joints on a drawing. a vas-scale was used for assessment of pain caused by rea. all patients were encouraged to donate blood samples for serology and hla-b27 typing (pcr). results: a total of 2106 patients returned the questionnaire (response rate 67%). only subjects with mono-infections were included. the male/female ratio was 40/60. blood samples were obtained from 1558 (74%): campylobacter (n = 693), salmonella (n = 503), e. coli (n = 213), shigella (n = 74) and yersinia (n = 75). the overall prevalence of hla-b27 was 9%, corresponding to the rate within the danish population. odds ratios for contracting rea when the individual was hla-b27 positive were; campylobacter 1.70 (ci: 0.89; 3.43), salmonella 3.4 (ci: 1.83; 6.37), e. coli 1.1 (ci: 0.23; 5.04), shigella 33.5 (ci: 2.48; 452.78) and yersinia 5.1 (ci: 1.03; 25.68). thus, hla-b27 was significantly more frequent among rea-patients with salmonella, yersinia, and shigella compared to campylobacter and e coli. the vasscore for rea was significantly higher in the groups with yersinia and salmonella. there was a significant positive correlation between vas-score and the fractions of patients that were hla-b27 + . conclusions: this large comparative study shows that hla-b27 has a significant impact on the susceptibility to contract rea after salmonella, shigella, and yersinia, but not after campylobacter or e. coli. furthermore the severity of rea as measured by vas differed among bacterial species and was strongly correlated to the presence of the hla-b27 antigen. objectives: the normal gut microbiota triggers experimental colitis and large intestinal manifestations of human inflammatory bowel diseases (ibd). however, the availability of animal models for the study of ileitis is limited. thus, only little is known hitherto in small intestinal ibd. recently, parasiteinduced ileitis in the mouse, mimicking characteristics of ileitis in human ibd, was proposed as a model for crohn's disease (cd). oral infection of susceptible c57bl / 6 mice with toxoplasma gondii induces a severe th1-type immunopathology, which is restricted to the terminal ileum. in order to unravel the mechanisms underlying ileitis, we used this model to monitor microflora changes during ileal inflammation and determined contributions of the gut microbiota to ileal disease. methods: c57bl / 6 mice were infected perorally with 100 cysts of t. gondii (me49 strain). ciprofloxacin, metronidazole, ciprofloxacin plus metronidazole (each 50 mg / kg body weight / d, p.o.), piperacillin plus tazobactam (200 mg/kg body weight/d, i.p.) were applied from day 0 until day 8 after t. gondii-infection. the ileal bacterial flora was analysed on day 8 p.i. by microbiological and molecular methods (16s rdna-targeted denaturing gel electrophoresis (dgge), sequencing of clone libraries). results: microbiological and molecular approaches revealed that ileal inflammation leads to an increased total bacterial load and to drastic changes in the flora composition. in the acute stage of disease, the grampositive cocci and rods -predominant in the ilea of healthy mice -are displaced by gramnegatives, identified as escherichia coli and bacteroides sp., respectively. antibacterial treatment (ciprofloxacin, metronidazole, a combination of both, or piperacillin plus tazobactam) ameliorated disease symptoms and reduced ileal inflammation. this was also seen in spf-mice colonized by only one grampositive bacterial species. conclusions: the fact that gramnegative bacteria accumulate during acute ileitis and contribute profoundly to intestinal inflammation is well in line with similar observations in experimental colitis and in human ibd. this provides evidence that gut flora modulation is a valuable therapeutical strategy for ibd treatment. finally, the contribution of gut microbiota to ileal disease supports the use of the parasite-induced small intestinal inflammation as a model for ibd research. objectives: pathophysiologic mechanisms that contribute to brain injury in neuroinflammatory disorders include breakdown of the blood-brain-barrier, extravasation of leukocytes, cerebral hypoperfusion and vasculitis. matrix-metalloproteinases (mmps) including the collagenases mmp-2 and -9 are crucially involved in all these steps. in this study we aimed to assess the extent of in-vivo collagenolytic activity in cerebrospinal fluid (csf) by determination of the amino acid hydroxyproline, a major and exclusive degradation product of collagen. methods: paired serum and csf samples from patients with bacterial meningitis (n = 11), aseptic meningitis/encephalitis (n = 17), multiple sclerosis (n = 13), and normal csf (n = 12) were assessed. degraded collagen was hydrolysed to dissolve its major component hydroxyproline, which subsequently was determined spectrophotometrically. csf levels of mmp-2 and -9 were studied by gel zymography. in a rat model of pneumococcal meningitis localization of collagenolytic activity was performed by in-situ zymography with intramolecularly quenched gelatin. results: hydroxyproline in csf from patients with bacterial meningitis was significantly increased compared to all studied groups (p < 0.001) while serum hydroxyproline did not differ significantly between the groups. the amount of hydroxyproline in csf correlated significantly with the amount of mmp-9 (r = 0.8; p < 0.001). in the rat model in-situ zymography localized gelatinolytic activity to the subarachnoidal and ventricular space inflammation and in association to cortical lesions. the study documents a significant increase of the collagen degradation product hydroxyproline in csf of patients with bacterial meningitis. the close correlation of hydroxyproline and mmp-9 in the csf validates the assessment of hydroxyproline as an index for csf collagenolytic activity in neuroinflammatory diseases and supports a role for collagenases in the pathogenesis of bacterial meningitis. introduction: toxic shock syndrome (tss), is an acute, noncontagious systemic illness. it is caused by the toxin producing strains of s. aureus and the b-haemolytic streptococci and can occur in any non-immune person exposed to a tss toxin. tss is commonly associated with menstruation and tampon use, however can also be related to skin or soft tissue infections, particularly post surgical, skeletal infections or respiratory tract infection. tss is often non-immunising and recurrent menstrualassociated tss is well-described. literature suggests that tss is extremely rare, but diagnostic difficulties can lead to misdiagnosis and tss can be fatal if left undiagnosed. we report a series of three cases of tss, presenting within a short period of time. case reports: case 1. 17 year old female, presented with sudden onset collapse, diarrhoea, vomiting and abdominal pains. she gave no history of menstruation and an initial diagnosis of severe gastroenteritis was made. she failed to respond to conservative management and required itu support. she was discharged with no firm diagnosis and re-presented one month later with similar symptoms, when a diagnosis of staphylococcal tss was confirmed. case 2. 15 year old female, presented during menses with sudden onset rash, rigors, severe diarhhoea, vomiting and abdominal pains. she was diagnosed with staphylococcal tss on admission. case 3. 28 year old female. presented with sudden onset severe diarrhoea, vomiting, pyrexia and rash. she gave no history of menstruation. she responded poorly to treatment, required itu support, high dose steroids and was eventually diagnosed with streptococcal tss. discussion: diagnostic criteria for tss include high fever, hypotension, erythematous rash and a complicated multisystem disfunction. patients often require aggressive management. the public health laboratory service reports an average of 18 cases of diagnosed tss in the uk per year. however, because of the uncommon and difficult nature of the diagnosis, many cases are misdiagnosed and therefore go unreported. it is essential to maintain a high level of suspicion for patients who are epidemiologically at high risk, but importantly, also the less ill patient with suggestive symptoms who fails to meet all diagnostic criteria but are in an at-risk group. high morbidity and mortality has been reported for undiagnosed and untreated cases. objectives: staphylococcus aureus colonization and infection of burn wounds increases morbidity and delays wound healing of patients suffering from burn wounds. a vulnerable moment in the route of transmission of s. aureus is the exposed burn wound during dressing changes. the aim of this study was to evaluate the effect of dressing changes under laminar flow conditions on burn wound patients with regard to s. aureus colonization. methods: from the first of february 2003 to the first of june 2003, 25 patients were included in this study at our 10-bed burn centre. in 12 of these patients the dressings were changed under laminar flow conditions. all patients were frequently screened for s. aureus in nose, throat and burn wounds. all s. aureus isolates were genotyped with pulsed field gel electrophoresis. results: at admission 17 / 25 (68%) patients had no s. aureus colonization in their nose, throat or burn wounds. dressing changes under laminar flow conditions were carried out on 8 / 17 patients of this group. however, 6 / 8 (75%) patients acquired burn wound colonization with s. aureus during their stay at the centre. six out of 9 patients (67%) whose dressing changes were not carried out under laminar flow conditions acquired burn wound colonization with s. aureus. a total of 12 patients acquired burn wound colonization; these patients also acquired carriage of s. aureus in nose or throat. the same type of s. aureus was carried in the nose or throat as well as the burn wounds in 7 (58%) of these patients. the results of this study suggest firstly that dressing changes under laminar flow conditions does not prevent colonization of burn wounds with s. aureus, and secondly, that the exogenous route plays an important role in the transmission dynamics of this pathogen in burn wound centres. future research should be focused on this mode of transmission of s. aureus in this special circumstance. mupirocin prophylaxis to prevent staphylococcus aureus wound colonisation in patients admitted to a burn centre objectives: staphylococcus aureus (s. aureus) colonisation of burn wounds increases morbidity and delays wound healing. many burn wound colonisations with s. aureus are endogenous in nature. the aim of this study was to evaluate the effect of eradication of nasal s. aureus with mupirocin in patients with regard to s. aureus colonisation of their burn wounds. methods: from september 2000 to march 2001, 42 patients admitted to our 10-bed burn centre were screened for s. aureus in nose and burn wounds. isolates were genotyped with pfge. all patients received nasal mupirocin at the time of admission to the burn centre. fifty-five patients from the same unit who were followed from september 1997 till may 1998 and had not received mupirocin prophylaxis served as a historical control group. results: at admission 29 / 42 (69%) patients in the mupirocin trial had no s. aureus colonisation in their burn wounds. of this group 25 patients were non-carriers and 4 patients were nasal carriers of s. aureus. seven (28%) non-carriers and 2 (50%) nasal carriers acquired burn wound colonisation with s. aureus during their stay at the centre. of the historical control group, 39 (71%) patients had no s. aureus colonisation of their burn wounds at the time of admission. of this group 31 patients were noncarriers and 8 patients were nasal carriers; 18 (58%) non-carriers and 7(88%) nasal carriers acquired s. aureus wound colonisation during their stay. the overall probability of wound colonisation in the patients treated with mupirocin was significantly lower than in the historical non-treated group (p = 0.01, chi-square test with yates correction). conclusion: the results of this study suggest that application of nasal mupirocin to all patients upon admission to a burn centre may reduce but not eliminate the risk of subsequent s. aureus colonisation of burn wounds. other measures including improved infection control practices and eradication of exogenous s. aureus reservoirs, including s. aureus carriage among healthcare workers, may be necessary to further reduce the incidence of s. aureus burn wound colonisation. controlling an outbreak of staphylococcus aureus on a surgical ward results: the characterisation of the s. aureus in february showed a cluster of identical strains in three patients and hcw a. the strains of two patients were similar to the strain of hcw b. three patients had a unique strain. in the surveillance period a total of eighteen isolates in sixteen patients were found. in the surveillance period the epidemic-strains were found in other patients but only in patients who have been on the ward during the epidemic period. two patients had an identical new isolate. that indicates that the exchange between patients of s. aureus may occur without notion. another remarkable observation was that some of the patients lost their original strain and acquired a new isolate of s. aureus during hospitalisation. conclusions: a small outbreak of s. aureus could be traced to 2 hcw who proved to be carriers. relieving them from patient contact stopped the outbreak. molecular surveillance is an appropriate way to evaluate the success of measurements taken and to underscore the importance of hygienic measures. risk factors for s. aureus carriage in health care workers a. widmer, m. lang, r. frei (basel, ch) introduction: s. aureus (mssa) carriage is common among health care workers (hcws)and might be associated with type of care provided and other potential risk factors. transmission of mssa has been observed by ''cloud adults''. objectives: to determine risk factors for mssa carriage in hcws, and the percentage of methicillin-resistant s. aureus (mrsa) in a tertiary care centre. methods: retrospective review of all data of staff health department and the microbiology laboratory. all hcws who were working in a high mrsa prevalence country were routinely screened at the time they were hired by the institution. in addition, data were generated from mrsa screenings of hcws if there was evidence for nosocomial transmission of mrsa in patients. mrsa was defined as s. aureus being oxacillin-resistant and expressing the pbp2' and/or being positive for the meca gene by pcr. data of hcws were collected by chart review in a case-report form that included demographic data, work place, skin diseases and other variables. data were compared by univariate and multiple logistic regression analyses. results: a total of 3474 swabs were taken from 1217 hcws between jan 1997 and dec.2001. data were available from 1182 hcws:50% originated from switzerland, 27% from germany, 4% from france, and 19% from other countries. the overall prevalence of mssa and mrsa was 53% and 1%, respectively. more than one sample was available from 332 individuals: of the repeat samples, 38% were negative, 32% were negative, but had one single positive culture, and 30% were consistently positive. sex, profession, work place, number of years at the institution was not associated with s. aureus carriage (p > 0.2). the only significant risk factor for s. aureus carriage was young age, defined <35years (p = 0.02). the prevalence of mssa increased from 51% (1997) to 63% (2001) . eight hcws were carriers of mrsa: they were young and half of the them reported skin diseases. conclusion: s. aureus carriage among hcws is common: only young age remained a risk factor even after adjusting for other variables. mrsa was rare among hcws. prevalence of nasal carriage of methicillinsusceptible and methicillin-resistant staphylococcus aureus among hospital personnel and outpatients st. fokas, sp. fokas, c. tsamolia, m. skoutari (eghio, gr) objectives: to assess the nasal colonization of staphylococcus aureus among hospital staff in the absence of an epidemic and to compare it with the nasal carrier status among outpatients, focusing on the mrsa rate. methods: the study included 150 hospital personnel and 150 adults admitted to outpatient clinics of our hospital. given that a history of hospitalization is the most important facilitative factor for colonization of staphylococcus aureus we exclude individuals with a history of hospitalization in the preceding year of our study. the specimens were obtained with swabs from the anterior nares-ecological niches of s. aureus-and cultured according nccls guidelines. id 32 staph strips were used for identification and confirmed with slidex staph plus kit (both by biomerieux). mrsa strains detection was achieved with slidex mrsa detection kit of the same company. antibiotic susceptibility of mrsa strains was determined with atb staph method (version 2000) according to the recommendations of the producer (biomerieux). results: among hospital staff the carriage rates of mrsa and methicillin susceptible s. aureus were 2.7% and 20% respectively. none of the outpatients was found to be a nasal carrier of mrsa. in addition mssa carriage in this group was 11%. the prevalence of mrsa nasal carriage rate among personnel working in the different medical wards was also examined. the difference in the rates among personnel working in the surgical ward, operating theaters and the rest of the staff was statistically significant (p < 0.001). all mrsa strains were susceptible to oral antibiotics. conclusions: although the prevalence of mrsa is low among hospital personnel, education is needed as they are one of most important reservoirs for nosocomial mrsa infections. the low incidence of mrsa infections around the time of the study suggests that with usual infection control practices mrsa spread was avoided. all mrsa carriers were treated with intranasal muripocin for eliminating the nasal carrier status. continuous monitoring of mrsa carriage is needed to avoid nosocomial spread. aids patients are a special population related with high risk of infection due to staphylococcus aureus. previous colonization is a recognized predisposing factor for development of infection due to this organism. however, nasal carriage of s. aureus in hivpatients is still not completely characterized. objectives: to describe the epidemiological features of community-acquired s. aureus nasal colonization in out care hivpatients based on the molecular genotypes. methods: hiv-outpatients without previous hospitalization within the last two years were screened for s. aureus nasal colonization. three samples from each patient were collected, and the variables associated with colonization were assessed. nasal carriage was classified as: absent, transient colonization or persistent colonization. the persistent colonization was assessed according to the molecular profiles performed by pulsed-field gel electrophoresis, in: simple (same profile), multiple (different profiles) or combined (two with the same e and one with different profiles). results: 111 patients were enrolled in the study. seventy patients (63%) had at least one culture positive for s. aureus and 99 patients concluded three samples collection. only one isolate was a community acquired mrsa. the rates of colonization was higher when two or more samples were taken, ranging from 45.5% in the first sample up to 65.6% when 3 samples were collected. hiv-patients with aids were more likely to be colonized than non-aids patients (p=0.02). among the patients with s. aureus nasal carriage, 39% were transient and 61% were persistent carriers, whose genomic subtype was: 61.5% simple, 20.5% combined and 18% multiple persistent colonization. previous use of antibiotics was associated with absence of s. aureus colonization (p < 0.05). conclusions: hiv out care patients had high rates of oxacillinsusceptible s. aureus nasal colonization. regardless, the recent literature, only one isolate was community-acquired mrsa. the most common characteristic of colonization is simple persistent colonization showing the same genomic profile. objectives: s. aureus bacteraemia (sab) continues to be a major problem related to both community-acquired and nosocomial infection. we undertook an evaluation of a large cohort of patients with sab to assess features of the infection and predictors of mortality. patients and methods: 238 cases of sab admitted to our hospital (2000-3) were prospectively studied. all patients had at least one positive blood culture for s. aureus and clinical symptoms of bacteraemia. sab was considered to be nosocomial if the first positive blood culture specimen was obtained > 72 h after admission and there was no clinical evidence of infection on admission. microbiological studies were carried under standard protocols. epidemiological and clinical characteristics, antimicrobial treatment and outcome (relapse or mortality) were analysed. results: 434 cases of sab were identified but only 238 were prospectively included in the study protocol. incidence was the highest in year 2001 (4.21 per 1000). out of the 238 cases, 167 (70%) were male with a mean age of 51 year (sd 24.89 have not yet implemented similar guidelines. this study evaluated the detection and reporting of mlsb results in staphylococcus using the bd phoenix tm automated microbiology system and bdxpert system with nccls, sfm or din as interpretative standards. methods: comments regarding mlsb resistance as listed in the nccls m100-s14 were converted into expert rules. special bdxpert rules were also constructed for the detection and reporting of mlsb resistance when applying sfm or din guidelines. a total of 177 strains of staphylococcus (146 s. aureus, 12 s. epidermidis, and 19 coagulase-negative staphylococci) were tested in phoenix panels containing erythromycin (e) and clindamycin (cc). nccls, sfm or din breakpoints were used to interpret the phoenix mic results. the double disk diffusion d-test (e and cc) was used as the reference method for the determination of mlsb resistance phenotypes. results: the phoenix e and cc mic values were interpreted based on the standard selected. the bdxpert rules were executed and applicable expert messages were displayed. the phoenix correctly detected 38 out of 43 constitutive mlsb (cmlsb) phenotypes as compared to the d-test results. the remaining 5 cmlsb strains were interpreted by the bdxpert as potential inducible mlsb (imlsb)/efflux phenotypes. a total of 69 imlsb and 22 efflux phenotype isolates were all reported by the bdxpert as imlsb/efflux phenotype and the users were alerted to perform d-test before reporting the cc results. the cc interpretation was suppressed in these isolates. the nccls, sfm, or din showed identical detection and interpretation of mlsb resistant phenotypes by the phoenix and bdxpert systems. conclusions: the phoenix and bdxpert systems can assist laboratories in rapid detection and accurate interpretation of mlsb results for staphylococcus. special messages can be used to communicate timely and accurate information to clinicians for proper therapy of staphylococcal infections with mlsb resistant phenotypes. in vitro activity of new compounds tested against multi-drug resistant s. aureus isolated in latin american medical centres background: oxacillin-resistant s. aureus (orsa) isolated in lamcs shows high rates of co-resistance (r) with most antimicrobial classes used in the clinical setting. we evaluated the mdr patterns of orsa strains collected in lamc by the sentry antimicrobial surveillance program in 2003. we also evaluated the susceptibilities (s) of new, investigational compounds against these important endemic pathogens. methods: among 1.437 s. aureus collected, 507 (35.3%) were r to oxacillin. these strains were isolated from 10 lamcs (5 countries) and tested against > 30 antimicrobials by nccls broth microdilution methods. orsa strains were grouped by the mdr patterns for the 8 primary drugs (47). conclusions: all newer compounds (lzd, q/d, dap and dal), tei and van were very active against endemic and epidemic orsa in lamcs. these s. aureus having mdr patterns (ave. r to 6 agents) will require greater use of newer compounds particularly in brazil. objectives: coagulase negative staphylococci (cons) represent a part of physiological microflora. however, in recent years, they have also emerged as nosocomial pathogens of infections associated with indwelling medical devices (e. g. pleural drains), mainly in immuno-compromised patients. the aim of this study was to determine dynamics of nasopharyngeal colonization by cons in patients with resectable lung cancer during short-term hospitalization, especially those receiving routinely preoperative antimicrobial prophylaxis. methods: in present study cons species were selected from patients divided into two groups: (i) patients who were undergoing pulmonary resection and receiving preoperative prophylaxis (piperacillin, cefuroxime alone or in combination with amikacin) -ôprophylaxisõ group and (ii) control group. throat and nasal specimens were taken from each patient twice, in four-days interval. all isolates were identified by the biochemical microtests api staph (biomerieux) and tested for drugs susceptibility according to nccls reccomendations. results: 187 strains of cons strains were selected from clinical specimens: 137 strains from ôprophylaxisõ group and 50 strains from control group. the most often isolated species was staphylococcus epidermidis with the predominated api numerical profile 6606113. a detailed analyses of the api numerical profiles and resistance to antimicrobial agents indicated that during four-days interval of hospitalization, changes in phenotypes among cons colonizing nasopharynx were observed in both groups of patients. the changes in drug resistance patterns were found more often in ôprophylaxisõ group compared to those in control group -48 / 54 (88.9%) and 11 / 22 (50%), respectively. this difference was statistically significant (p = 0.0007 -chi2 test with yates correction). conclusion: our data suggest that preoperative antimicrobial prophylaxis routinely used in patients with resectable lung cancer may be regarded as an important factor predisposing to changes in drug resistance patterns among cons isolates colonizing nasopharynx. the susceptibility of coagulase-negative staphylococci in a teaching hospital compared with a secondary care hospital results: in total, six different species were identified in 71 isolates, 72% of which were staphylococcus epidermidis. in the lumc the species diversity was greater than in the mca, with five isolates of staphylococcus haemolyticus found in the first. no difference between the two hospitals was found in activity of the antibiotics tested, except for li. the mic90s were as follows: v 2 mg / l, t 4 mg / l, c 16 mg / l, m 2 mg / l, l 8 mg / l. of li the mic90 was 2 mg / l in the lumc and 1 mg / l in the mca. according to dutch guidelines, in total 7 / 71 isolates were found resistant to t and 21 intermediate resistant. all isolates were susceptible for v. conclusion: the first choice glycopeptide for serious cons infections, t or v, has no influence on the susceptibility of cons for these antibiotics. according to dutch guidelines, the activity of t, c, and l against cons is relatively low, whereas the activity of m and le is in the susceptible range. objective: staphylococcus epidermidis is one of the bacterial species mainly implicated in foreign body associated infections. we have characterized several s.epidermidis clinical isolates for their ability to form biofilms and their resistance to antibiotics. methods: 76 s.epidermidis strains isolated from implantable medical devices have been collected from hospitals of central italy. susceptibility to penicillin, methicillin, erythromycin and tetracycline has been determined in vitro by e-test according to nccls guidelines. the specific antibiotic resistance determinants have been checked by pcr (blaz, meca, erma, ermb, ermc, msra, tetk and tetm). the ability to form biofilms has been determined: (i) by pcr, detecting genes specific for attachment and biofilm development (icaadbc operon, aap, and atle); (ii) by congo red agar (cra) plate test to assay the production of polisaccaridic intercellular adhesin (pia); (iii) by crystal violet (cv) stain to determine the biofilm biomass development on polystyrene microtiter plates; (iv) and by cslm microscopy observations to investigate biofilm structure. results: 94% of the strains under study was resistant to penicillin, 87% to methicillin, 72% to erythromycin and 25% to tetracycline. on the side of biofilm-specific genes detection, 66% of strains was positive to ica operon genes, 82% possessed atle gene, and 42% aap determinant. in 89% of the population, the cra test confirmed the correlation between the presence of ica genes and slime expression. the cv assay classified the quasitotality of our strains (97%) as biofilm producers on plastic surface. in addition, the distribution of optical density values (od540) obtained after cv stain, showed a significant statistical difference in biofilm biomass development between the ica-adbc-positive strains and the icaadbc-negative ones. finally, a correlation, although not always present, has been observed between ability of the strains to develop in a high-structureted biofilm and specific biofilm-formation determinants. conclusions: analysing the origin of colonization or infection in 6 patients with cons isolated from the operative site, the bacteria could only be traced in one patient who developed an infection. transmission most probably occurred by direct contact. the low level of bacterial contamination in the uca together with the lack of correlation of genotypes between isolates found in the uca and in the wounds suggest that airborne transmission does not play a major role in the development of swi. more than 90% of staphylococci found in air samples were not traceable to any investigated sources. most probably, they originated from non swabbed parts of the hcw's bodies. since 50% of the traceable isolates from the uca originated from non scrubbed team members, protection of the operative site by lfv appears to be suboptimal. clonality of endemic methicillin-resistant staphylococcus epidermidis strains isolated in a neonatal intensive care unit n. ben saida, a. ferjani, n. sfar, j. boukadida (sousse, tn) objectives: methicillin-resistant staphylococcus epidermis (mrse) is one of the important pathogen responsible for nosocomial infections particularly in newborns. the aim of our study was to establish if these infections are caused by endemic clones or by incidentally occurring bacterial strains of this ubiquitous species and to evaluate the performance of pcr iner-is256 as a novel method for typing mrse strains using pulsed-field gel electrophoresis (pfge) as the reference method. methods: twenty strains of mrse (meca+) has been collected during the period of survey (december 2003 to september 2004) from different pathological products of newborns hospitalized in a neonatal intensive care unit of a public maternity hospital in sousse, tunisia. identification of strains was done by conventional procedures and the api staph (api-biomerieux. sa). susceptibility testing to antibiotics was determined according to ca-sfm recommendations. all strains have been characterized by genotypage in pulsed-field gel electrophoresis (pfge) variety chef after smai macrorestriction. genomic profiles have been interpreted according to criteria of tenover (j. clin. microbiol 1995; 33:2223 -2239 . strains were also characterized by electrophoretic profiles obtained by pcr-based analysis of inter-is256 spacer polymorphisms. results: these mrse represent 41.6% of the total strain of s.epidermidis collected from the neonatology ward during the period of survey. majority of these strains come from blood sample (75%), other strains from vascular catheter (5%), pus (10%), sound (10%). 17 antibiotypes and 6 genotypic profiles according tenover criteria were individualized: type a (with 5 subtypes), type b, type c (with 1 subtypes), type d (with 1 subtypes), type e (with 2 subtypes)and type f. a total of four pcr patterns were obtained based on the interpretation criteria that pcr patterns exhibiting more than one band difference were considered to represent distinct types, type 1, type2, type3, and type4. conclusion: mrse appear to be endemic and multiclonal with a dominant clone in the neonatal intensive care unit. our study demonstrates that a significant proportion of mrse infections may be attributable to transmission among newborns and that certain strain can become endemic over long periods in this setting. clonality of staphylococcus haemolyticus strains isolated in a neonatal intensive care unit n. ben saida, a. ferjani, j. boukadida (sousse, tn) objective: methicilllin-resistant staphylococcus haemolyticus (mrsh) was increasingly important nosocomial pathogens particularly in critically ill neonates. the aim of our study was to establish if these infections are caused by endemic clone or by incidentally occurring bacterial strains of this species. methods: thirteen strains of mrsh according ca-sfm recommendations and meca+ (murakami k. j.clin.microbiol 1991 , 29:2240 has been collected during the period of survey (april 2004 to october 2004). eleven strains come from different pathological products of newborns hospitalized in a neonatal intensive care unit of a public maternity hospital. tow strains come from paediatric and internal medicine wards were used as control to ensure that endemic strains can be differentiated from outbreak strains. all strains were identified with the api-staph method (api biomerieux). strains have been characterized by genotypage in pulsed-field gel electrophoresis (pfge variety chef) after smai macrorestriction. genomic profiles have been interpreted according to tenover criteria (j. clin.microbiol.1995 ; 33 :2223 -2239 . results: s. haemolyticus was quantitatively the second staphylococci (23.6 %) after s. epidermidis isolated in neonatology ward. mrsh represent 85. 7 % of the total strains of s. haemolyticus collected from the neonatology ward during the period of study. the majority of the strains come from blood sample 7 (63.63%), 4 (36.36%) from pus and 1 (9%) from csf. eight resistant antibiotyps profiles and 3 genotypic pattern according tenover criteria were individualized: type a, type b (with 3 subtypes), and type c. the strains coming from paediatric ward present pattern type b3, but strains coming from internal medicine ward present distinct pattern. conclusion: mrsh is a major bacterium of nosocomial infection in neonatal intensive care unit. this bacterium is responsible as shown in our study, of endemic multiclonal nosocomial infections with a predominant clone. results: s. haemolyticus was isolated from 21% of all positive blood cultures and represented the 37.9% of the coagulase negative staphylococci. twenty-nine cases of shb occurred during the study period (5.6 episodes per 1000 patients-days). all cases were hospital-acquired and occurred after a mean hospitalisation of 24.5 days. the mean age was 68.4 years (range 20-90) and 16 patients (55.2%) were males. twenty-eight patients (96.5%) had a central venous catheter (cvc). all patients received previously antimicrobial therapy. thirteen patients (44.8%) had polymicrobial bacteraemia. in 18 cases (62.1%) the source of bacteraemia was the cvc. all isolates were resistant to oxacillin and gentamicin, 94.3% and 34.3% were resistant to ciprofloxacin and teicoplanin, respectively; all strains were susceptible to vancomycin. sixteen patients (55.2%) had sepsis, 3 (10.3%) severe sepsis, and 7 (24.7%), all polymicrobial, septic shock.; in 3 patients (10.3%) shb had no clinical significance. twenty patients died (69%) and deaths were directly related to shb in 1 patient (3.4%), and partially related in 2 (6.8%). nine patients (31%) survived.the patient who died for shb, developed a persistent cvc-related bacteraemia after a 6-week course therapy for streptococcal endocarditis. s. haemolyticus become resistant to teicoplanin during a teicoplanin-based therapy and the patient died 47 days after the onset of bacteraemia. the 2 patients, in which death was partially related to shb, had a polimicrobial bacteraemia due to c. tropicalis and enterococcus spp, respectively, and died after 22 and 6 days respectively, for septic shock, during adequate therapy. among the 17 patients in which death was not related to shb, 15 (88.2%) received adequate therapy that cleared the blood cultures, 2 patients improved after the removal of cvc without antimicrobial therapy. conclusions: s. haemolyticus is an emerging pathogen, responsible of sepsis, frequently cvc-related; most of strains are resistant to many antistaphylococcal agents currently used as empiric therapy, thus constituting a clinical concern. nevertheless, this pathogen shows a low virulence and is associated with a low related mortality, probably representing a marker of severe underlying diseases. staphylococci from the skin of patients: changes associated with treatment by isotretinoin a. thomas, r. williams, n. hepburn, t. watts, r. dixon (lincoln, uk) objectives: isotretinoin, a retinoid has been used to treat patients with moderate to severe acne despite the adverse effects of mucosal surface drying and the contraindication of use during pregnancy. in clinical practice, patients are frequently prescribed systemic isotretinoin because they have not responded to previous treatment with oral or topical antibiotics. broad-spectrum antibacterial therapy has nevertheless promoted changes in the diversity and antibiotic resistance status of the patientsõ skin microbiota and we are studying the effect of isotretinoin on these changes. methods: the present study investigated the recovery and analysis of skin organisms from 53 patients (mean age 23 y range 15-37y) before, during and after treatment with a 16-week course of isotretinoin (1 mg / kg body weight). the number of aerobic bacterial isolates (presumptive staphylococci) recovered from baird-parker agar from each of three specific sites per patient were compared with age-matched controls of healthy volunteers. results: both patients and controls had generally similar numbers of organisms on the nares, cheek and toe webs before treatment, whereas all patients showed a significant reduction in staphylococci recovered from one site during and after 16 weeks of isotretinoin treatment. the majority of the patients had a greater reduction (1-2 log) for the cheek site than either nares or toe webs both of which showed minimal reduction. conclusions: preliminary results from this study show a pronounced reduction in the number of presumptive staphylococci recovered from the treated group immediately following isotretinoin compared to untreated controls. since isotretinoin has little known antibacterial activity against the staphylococci the observed reductions would suggest that the effect is mediated through changes in the skin nutritional micro-environment. objectives: emergence of mutational resistance to linezolid (lin) in staphylococcus aureus has been associated with loss of erythromycin resistance methylase (erm) genes, both in vivo and in vitro. we tested the general validity of this claim, and examined whether the emergence of lin resistance is delayed in the presence of ery. methods: six lin-susceptible s. aureus strains were used: 2 genetically-related, ery-resistant (ermc) and susceptible pairs of emrsa-15, also the isogenic pair rn4220 and its hypermutable rn422muts mutant, which harbours ermb. in 4 replicated experiments, ery-susceptible and ery-resistant isolates were grown on agar containing increasing concentrations of lin. in addition, ery-resistant isolates were grown with 100 mg / l ery and increasing concentrations of lin. the number of days for isolates to develop the ability to grow in the presence of 6 mg / l lin was recorded. the absence of erm genes was checked by pcr; mics of lin and ery were determined by agar dilution, and pfge profiles of mutants were verified. results: thirty-three mutants were obtained from the 4 experiments; 24 from the 4 clinical emrsa-15 isolates, 3 from rn4220 and 6 from rn422muts. lin-resistance emerged more slowly in ery-resistant clinical isolates grown with lin and ery than with the same isolates grown with lin alone: mean 20 days (sd 26.8) to emerge on 6 mg / l lin versus 30 days (sd 18.4) in the presence of lin and ery. growing the hypermutable strain, rn422muts, in the presence of lin and ery did not delay the emergence of lin-resistance (11 days with sd 8.7, in either case). in the absence of ery selection, loss of erm with the emergence of lin-resistance was only noted for one emrsa-15 isolate in 1 of 4 experiments. all isolates grown in the presence of ery retained erm determinants. ery 100 mg / l did not supress growth of ery-resistant strains and linezolid was stable within the ambit of the long-selection experiments. conclusions: growing the clinical isolates in the presence of ery and lin slowed the emergence of lin-resistance. this merits further study with lower, clinically-relevant concentrations of ery. in vitro selection of linezolid resistance in hypermutable and wild-type staphylococcus aureus objectives: resistance to linezolid (lin) is rare, but can arise via mutations in the 23s rrna genes. we hypothesised (a) that resistance would emerge preferentially in staphylococci with a hypermutable phenotype engineered by mutations in the muts gene, and (b) that hypermutability might be co-selected with linezolid resistance. muts is part of the dna mismatch repair (mmr) system, which identifies and corrects genome alterations post-replication, thereby influencing the net mutation rate. method: linezolid-resistant (linr) mutants of staphylococcus aureus rn4220, its hypermutable muts deletion mutant rn4220muts, harbouring an erythromycin resistance marker gene (ermb), and a genetically-related pair of clinical isolates were selected by repeated passage with increasing concentrations of lin. mutant parentage was confirmed by pfge and susceptibility was tested by agar dilution. an amplified 694-bp fragment of 23s rrna genes was studied by sequencing and by rflp analysis. frequencies at which linr mutants yielded variants resistant to fusidic acid and rifampin were calculated in triplicate experiments. results: seventeen linr mutants (mics 8-64 lg/ml) were raised. ten mutants had a g2447t mutation in their 23s rrna genes; 4, all from rn4220muts, had g2576t, typically seen in linr gram-positive cocci from the clinic; 1 had t2504c; 2 had no identifiable mutations. curiously 5 / 7 rn4220muts mutants lost ermb during the course of the experiment, reverting to macrolide susceptible. linr mutants had mutation frequencies for rifampicin and fusidic acid resistance comparable with those of their parents (10-6 to 10-9), implying no co-selection of stable hypermutability. conclusion: more linr mutants were obtained from the hypermutable (rn4220muts) strain than from the wild type strain, and g2576t mutants were only obtained from the hypermutable organism. these data suggest hypermutability facilitates the emergence of linr, nevertheless, linr mutants derived from wild-type parents did not have elevated mutation frequencies. objectives: the mechanism of resistance in glycopeptide intermediate staphylococcus aureus (gisa) and hgisa isolates is unknown. however the inactivation of certain genes has been shown to confer an increase in glycopeptide resistance. two genes, tcaa and mprf, when inactivated in gisa strains cause a reduction in glycopeptide mic. overexpression of tcaa causes an increase in teicoplanin susceptibility and so both genes may play a role in the development of glycopeptide resistance. this study aims to investigate the expression levels of tcaa and mprf in a set of gisa, hgisa and glycopeptide-susceptible s. aureus (gssa). methods: rna was extracted from the following: 1 set of clinically related (cr) gssa and hgisa (lla and lle), 2 sets of cr hgisa and gisa (pc1 and pc3, lim1 and lim3) harvested during exponential phase growth (ex) and during exponential phase growth in the presence of sub-mic levels of vancomycin (exv). rna was extracted from a further hgisa, mu3 and gisa, mu50 as well as 4 clinical gssa strains. rt-pcr was performed with customised primers and the products visualised by electrophoresis. band intensity was taken as indicative of mrna quantity, and hence expression level, at time of cell harvest. results: the expression of tcaa is similar in all strains tested. each strain within the related strain sets all show similar band intensities, despite differing glycopeptide resistance phenotype. both mu3 and mu50 also exhibited similar expression levels to those found for all other gisa, hgisa and gssa strains. levels of expression were not affected by the presence of sub-mic levels of vancomycin in any strain tested. sequence analysis of tcaa in these strains showed no mutations present as previously thought. expression levels of mprf were found to be strain specific. no difference in gene expression was found in the cr related strain sets. conclusions: no reduction in expression levels of tcaa and mprf were found to correlate with clinical strains exhibiting higher glycopeptide resistance. thus it it uncertain what role tcaa ot mprf play with mediating glycopeptide resistance in staphylococcus aureus. objectives: gisa and hgisa strains all have a characteristic thickened cell wall with reportedly fewer cross-links. fewer peptidoglycan cross-links create increased d-ala-d-ala pentapeptide termini and hence more vancomycin targets. pbp4 has been shown to have both transpeptidase and d,d-carboxypeptidase activity, cleaving terminal d-alanine residues from un-cross-linked muropeptides. reduced levels of pbp4 have been reported in laboratory-generated gisa mutants and a few clinical gisa strains. this study aims to investigate the expression levels of pbp4 in a set of gisa, hgisa and glycopeptide-susceptible s. aureus (gssa). methods: rna was extracted from the following: 1 set of clinically related (cr) vssa and hgisa (lla and lle), 2 sets of cr hgisa and gisa (pc1 and pc3, lim1 and lim3) harvested during exponential phase growth (ex), after overnight growth (on) and during exponential phase growth in the presence of sub-mic levels of vancomycin (exv). rna was extracted from a further 8 clinically unrelated (cu) gisa (including mu50), 8 cu hgisa (including mu3) and 8 gssa (ex). rt-pcr was performed with customised primers and the products visualised by electrophoresis. band intensity was taken as indicative of mrna quantity at time of harvest and hence gene expression. results: the expression of pbp4 varies according to strain. the band intensities of the related strain set lla (gssa) and lle (hgisa) reduce slightly with increasing intermediate vancomycin resistance. however in two other cr hgisa and gisa (pc1/ pc3, lim1/lim3) strain sets no variation in expression was observed. both mu3 and mu50 exhibited lower pbp4 expression than any other strain, with the presence of sub-mic levels of vancomycin reducing expression further. pbp4 expression was found to be reduced in 3 other gisa and 1 other hgisa, which showed lower band intensities than gssa. conclusions: the reduced levels of pbp4 suspected to be associated with reduced susceptibility to vancomycin in s. aureus is not exclusive. the expression of pbp4 is reduced in mu50 and 3 clinical gisa, as reported previously. however 5 other gisa strains exhibit pbp4 expression levels similar to gssa isolates. therefore pbp4 expression levels appear to be strain specific and a lowered expression level is not an essential requirement in the development of the gisa phenotype. objectives: the prolonged use of vancomycin is known to be the most important factor in inducing vancomycin resistance to s. aureus. methicillin-resistant s. aureus (mrsa) colonizing in respiratory tracts might be chronically exposed to subtherapeutic concentrations of vancomycin because the level of vancomycin within pulmonary lining fluids was much lower than the concentration of serum. despite the continuous use of vancomycin mrsas were still found to be presented in respiratory specimens of some patients. methods: all mrsas which had been continuously isolated in respiratory specimens during vancomycin therapy were collected from jul 2003-mar 2004 at seoul paik hospital. mrsas were screened on brain heart infusion plates containing vancomycin 4 lg / ml (bhi-4) and 6 lg / ml (bhi-6) for vancomycin resistance. minimal inhibitory concentrations (mics) were determined by e-tests, agar dilution methods, and broth dilution methods. the patterns of pulsed field gel electrophoresis were analysed. results: nine patients and 27 isolates were assessed. five patients had pneumonia and four patients had other mrsa infections. twelve mrsas were isolated from tracheal aspirates and 15 from expectorated sputum specimens. the duration of vancomycin use ranged from 7-22 days. five isolates and one isolate were cultured on bhi-4 and bhi-6, respectively; the vancomycin mics of these isolates revealed 3 lg / ml by e-tests. the mics from agar dilution methods and broth dilution methods showed from 0.5-2 lg / ml. conclusion: vancomycin resistance during the treatment of patients did not develop. objectives: fusidic acid resistance (fusr) in staphylococcus aureus usually results from acquisition of the fusb resistance determinant, or from mutations in the fusa gene encoding the drug target (elongation factor g). following a recent report of fusr in s. intermedius, and the detection in our own studies of fusr s. lugdunensis (unpublished), we have examined whether resistance in these strains results from fusa / fusb-type mechanisms. methods: standard methodology was employed for strain identification, susceptibility testing, southern hybridization (sh), pcr amplification and dna sequencing. primers for pcr amplification of the previously-unsequenced s. intermedius fusa gene were based on portions of the flanking genes (rpsg, tufa) that are conserved between b. subtilis and s. aureus. results: three fusr s. lugdunensis (fus mic = 4 ug / ml) and two fusr s. intermedius (fus mic = 2 ug / ml) were probed for the presence of fusb by sh alongside their wild-type, fuss counterparts (fus mics of 0.064 and 0.125 ug / ml, respectively). the fusr s. lugdunensis all carried fusb, but this gene was not detected in s. intermedius, or in the fuss strains. furthermore, fusb appeared to be chromosomally-located, since no hybridization was observed with purified plasmid dna. pcr analysis mapped fusb to a chromosomal region upstream of the groel gene, the same locus at which this gene is carried in the epidemic fusr european s. aureus clone. to examine whether mutations in fusa are responsible for fusr in s. intermedius, pcr amplification and sequencing of fusa from the resistant and sensitive strains was performed. relative to fuss s. intermedius nctc 11048, the resistant strains carried six coding polymorphisms in fusa, although none of these correspond to reported fusr mutations in the fusa gene of s. aureus. conclusions: fus resistance in recent strains of s. lugdunensis results from carriage of the fusb determinant on the chromosome, probably acquired horizontally from fusr s. aureus strains. the fusb gene was not detected in the s. intermedius strains, although multiple fusa polymorphisms were identi-fied which may be responsible for the observed fusr phenotype. objectives: methicillin-resistant s. aureus (mrsa) and methicillin-resistant coagulase-negative staphylococci (mrcns) are the most important pathogens that cause nosocomial infections worldwide. one of the few antibiotics which is still effective against mrsa is mupirocin. but mupirocin-resistant s. aureus has been increased since first reported in 1987. and more than 5-10% of s. aureus isolated in tertiary hospitals in korea was reported to be high level mupirocin resistant. in this study, we investigated the restriction fragment length polymorphism (rflp) type for mupa gene loci of plasmid dna and sequenced mupr plasmid containing mupa gene of high level mupirocin resistant (muh) staphylococci from tertiary hospitals. methods: susceptibility to antimicrobial agents including mupirocin was tested by the disk diffusion and mic of mupirocin was determined by agar dilution method. the plasmid-encoded mupa gene was detected by pcr. mupa polymorphisms of plasmid dna digested by hind iii, ecor i, and cla i restriction enzymes was investigated with southern hybridization using mupa probe. s. aureus isolate showing the most prevalent rflp type was conjugated by filter mating method and selected the transconjugant showing only mupirocin resistant phenotype. the mupr plasmid of selected transconjugant was purified and analysed the plasmid dna sequence. results: 9 muh-staphylococci were isolated among the 680 staphylococci from tertiary hospital in korea. muh-mrsa (6 isolates), s. haemolyticus (1 isolates), s. hominis (1 isolates) and s. epidermidis (1 isolates) were isolated. 1.65kb mupa pcr product was detected in plasmid of muh isolate. all isolates contained more than two plasmid molecules that were repeatedly purified with different efficiences. three different polymorphs of the mupa loci were investigated. the most prevalent mupa polymorph was 4.2kb ecori-8kb hindiii-2.1kb clai hybridizing fragment. the mupirocin resistant plasmid dna was about 52kb and is257-mupa-is257 sequence was located. conclusion: the high-level mupirocin resistant staphylococci had the multiple plasmids of various size and the diverse rflp type suggested the variation of mupa gene loci. the mupr plasmid contained transferable element such as is257 with mupa gene. genetic basis of macrolide, lincosamide, streptogramin resistance in coagulase-negative staphylococci s. gatermann, t. koschinski, s. friedrich (bochum, d) objective: in s. aureus erythromycin resistance is almost exclusively caused by erma or ermc genes whereas export of macrolide antibiotics by msra or inactivation of lincosamides by lina is rare. resistance may be inducibly (clindamycin appears susceptible) or constitutively (clindamycin appears resistant) expressed. during therapy of inducibly resistant strains with clindamycin mutations may occur that render them objectives: lysostaphin is an antibacterial enzyme which is specifically capable of cleaving the cross-linking pentaglycine bridges in the cell walls of staphylococci. s. aureus cell walls contain high proportions of pentaglycine, making lysostaphin a highly effective agent against both actively growing and quiescent bacteria. relationship between lysostaphin susceptibility, vancomycin susceptibility and cell wall thickness of passage-selected vancomycin -resistant staphylococcus aureus isolates (vrsa) and their parent strains were studied in order to investigate characterization of isolates with decreased susceptibility to vancomycin. methods: the susceptibility of lysostaphin for six vrsa and their parent strains were determined using the spectrophotometric and the macrodilution methods. spectrophotometric determination of turbidity was measured as a decrease in a620 during incubation of the isolates samples at 37°c. mics were determined by a broth macrodilution method in cationadjusted mueller-hinton broth according to standards of the national committee for clinical laboratory standards. cell wall measurement were determined using transmission electron microscopy. results: determination of lysostaphin activity revealed that there are significant decreases in per cent reduction in turbidity (activity of lysostaphin) of the vrsa. vrsa shown to be more resistant to lysostaphin than parent strains. the mics of lysostaphin for the vrsa strains also increase. there were either a 2 or 4 fold increases in the mic to lysostaphin. electron microscopy of the vrsa showed enhanced cell wall thickness, uneven surfaces and irregular shape in contrast of the thin and regular cell wall morphology of the parent strains. those strains acquiring the highest thickness in cell wall demonstrating the highest resistant to lysostaphin. conclusion: the mechanism of lysostaphin resistance and its relationship to vancomycin resistance remains unclear. it is possible that the increase in cell wall thickness we observed prevented lysostaphin access to all pentaglycine targets or increased the number of cross-linkers requiring cleavage before the strain could lyse. typing and epidemiology of mrsa methods: creation and distribution of a consensus document detailing the needed harmonization of sequencing technology in diagnostic microbiology will be followed by a certification program for sequence based typing, using methicillin-resistant s. aureus (mrsa) as a prototype organism. as typing method spatyping will be used. five strains, 5 dnas, and 5 forward and reverse chromatogram files of well characterized mrsa strains will be distributed to all participating laboratories. an annual proficiency testing for sequence based typing of mrsa is planned. staphylococcus aureus is an important human pathogen related to its ability to develop resistance to many antimicrobial agents. methicillin resistance of s. aureus (mrsa) has become a major public health problem especially in nosocomial infectious. more over community acquired mrsa is a growing concern, the rate of mrsa vary importantly within countries, the highest rates were reported especially in southern european countries. a multicentric study was done to establish antimicrobial resistance of s. aureus isolated from clinical specimens of hospitalized patients over five years period (1999) (2000) (2001) (2002) (2003) . s. aureus was identified by conventional methods and antibiotic susceptibility testing was performed by disk diffusion method according to nccls standards. a total of 5186 strains of s. aureus were collected, they were recovered mainly from pus (60%), blood cultures (15%), respiratory samples (8%) and urines (7%). 34% of the strains were from in medicine, 18% from surgery, 16% from orthopaedics, 14% from paediatrics and 13% from intensive care units. the rates of resistance to different antibiotics was 15% to oxacilline,9% to gentamicin, 23% to amikacin,8% to ofloxacin, 23% to erythromycin and 8% to clindamycin. all isolates were susceptible to vancomycin. according to this data the rate of mrsa remained relatively low (< 20%) with no significant change during this time period, however, mrsa presented high rates of associated resistance to gentamicin (48%), to amikacin, (91%) to erythromycin (38%), to clindamycin (20%), to ofloxacin (40%) and to trimethoprim-sulfamethoxazole (33%).mrsa were not of great concern in our country and meticillin remains an effective antibiotic for treatment of s. aureus infections. characterisation of gentamicin-susceptible strains of methicillin-resistant staphylococcus aureus in two spanish hospitals c. potel, m. alvarez, c. lopez-melendez, i. otero (vigo, e) objectives: characterize gentamicin-susceptible methicillinresistant staphylococcus aureus(gs-mrsa)by two molecular typing methods. find out whether some gs-mrsa clones are related to gr-mrsa. describe the spread of these clones. methods: one hundred and twelve samr were isolated in two hospitals between 1997-2001. the epidemiological relatedness was studied by repetitive element sequence-based pcr and coa gene restriction fragment length polymorphism. the genes ant(4) and aac(6)-aph(2õ), that codify two aminoglycoside modifying enzymes, were detected by pcr.we analysed the gentamicin consumption and its relation with the emergence of gs-mrsa. results: thirty nine strains were gs-mrsa. the 80% of these gs-mrsa belonged to two epidemic clones, the clone a was tobramycin sensitive and the clone b was tobramycin resistant. the clone a was only isolated at hospital-i and was replaced by an epidemic gr-samr clone (clone c). the clone b was only isolated at hospital-ii and displaced the clone c. objectives: the long-term care facilities (ltcfs) patients are those with serious underlying disease, poor functional status, wounds such as pressure sores, invasive devices of urinary catheters. there is increasing concern about the emergence of resistance to antimicrobial agents including methicillin resistant s. aureus in ltcfs and mupirocin has been used in ltcfs to prevent the occurrence and spread of mrsa. for inducible mls-resistance. nitrocefin discs were used for beta-laktamase detection after induction with cefoxitin and/or oxacillin. the oxacillin (4 mg / l) agar method was used for mrsa screening. mrsa was confirmed by meca and nuc pcr and further analysed by smai-pulsed-field gel electrophoresis (pfge). results: a total of 102 strains (78%) were beta-lactamase positive. other resistance prevalence rates were: tc (29%), em (19%), cm (19%), ci (11%), gm (11%), ts (0%) and fu (0%). thirteen strains (10%) were mrsa-screen positive, both with the oxacillin-agar test and the cefoxitin disc, and confirmed mrsa by meca and nuc pcr. all mrsa strains were isolated from hospitalized patients and expressed multiple resistances. pfgeanalysis revealed that the mrsa-strains belonged three different pfge-types. type 1 consists of 8 strains from the department of general surgery and icu. the type 2 group consisted of 3 strains from general surgery department. finally, one strain from a patient at the department of pulmonary medicine belonged to the type 3 group. we were not able to type one strain. mlsttyping of mrsa-strains will be performed. results: a total of 793 infants were included in this study. mrsa colonization was detected in 331 infants (42%) during nicu stay and 89% were detected by the first 2 samplings. naris (71%) and umbilicus (60%) were the 2 most common sites of colonization. clinical mrsa isolates were identified from 92 infants (12%). among the 92 infants, mrsa colonization, either preceding or later acquiring, was noted in 84 infants (91%). 121 clinical isolates from 64 infants with 84 episodes of possible mrsa infection were available for genotyping analysis. prior colonization was detected in 68 episodes (81%), among which both the colonized and clinical isolates were indistinguishable in 63 episodes (93%), highly related in 2 episodes and distinct in 3 episodes. among the 16 episodes without prior colonization, colonization was acquired following infection in 9 episodes (53%), among which indistinguishable or highly related colonized strain was acquired in 6 episodes. conclusion: surveillance of mrsa strains as a basis for active antibiotic policy has become of increasing concern to both health care providers in hospitals and community general practitioners. there is a need for better awareness of mrsa infections among both health care professionals and the public. the incidence of mrsa infections in last years in the czech republic shows a slightly increasing trend. very interesting is local distribution of mrsa strains. the development of the incidence of mrsa infections in their spread will be the subject of further study. comparison of phenotypic typing methods and pfge of methicillin resistant s. aureus isolated from two university hospitals in thailand c. chomvarin, k. chaicumpar, s. srigulbutr (khon kaen, th) objectives: comparison of phenotypic typing methods and genotypic typing method to differentiate mrsa isolates obtained from the two university hospital in thailand. methods: seventy-four mrsa isolates were randomly collected from the two university hospital (central and northeastern) in thailand. they were confirmed with the presence of meca gene. antibiograms, phage typing and enterotoxin productions were used for the phenotypic typing analysis. pulsed-field gel electrophoresis (pfge) with smai digestion of chromosomal dna was used for the genotypic typing analysis. results: we found 19 distinct profiles by phenotypic typing methods and 18 pfge types designed as 5 major types (a to e) and 13 subtypes. the most frequently types and their related subtypes found in both hospital, were a and c with represented 54.1% and 27%, respectively. all isolates resistant to penicillin, cephalosporin, erythromycin, gentamicin, kanamycin, tetracycline and oxacillin; and variably resistant to cotrimoxazole, lincomycin, chloramphenicol, ciprofloxacin. all isolates were susceptible to vancomycin and fosfomycin. ten (13.5%) mrsa isolates produced enterotoxin a. forty-five (60.8%) isolates were nontypable by phage typing method. no significant correlation between the phenotypic typing methods and the genotypic typing method were found. conclusions: the phenotypic typing methods were not significantly correlated with the genotypic typing (pfge). our results demonstrated that pfge types a and c were the common endemic mrsa clone of both hospitals in thailand. objective: to investigate the molecular characteristics of methicillin-resistant staphylococcus aureus (mrsa) strains isolated from neonates transferred from primary care obstetric clinics. methods: twelve mrsa strains were isolated from 11 neonates in 2004. ten mrsa strains were also isolated from nurses and facilities in corresponding primary care obstetric clinics. molecular features of mrsa strains were analysed by using multilocus sequence typing (mlst, arcc-aroe-glpf-gmk-ptatpi-yqil), spa typing, and sccmec typing. presence of panton-valentine leukocidin (pvl) gene was investigated by pcr method. results: all 22 mrsa strains analysed in this study contained sccmec type iva. of six isolates from neonates of clinic a, five showed st1 (1-1-1-1-1-1-1) and resistance to only gentamicin and tetracycline. the remaining one showed novel st (25-1-1-1-1-1-1), shared with one isolate from nurse of clinic a. four mrsa strains from neonates of clinic b showed st1 (1-1-1-1-1-1-1) and resistance to erythromycin and gentamicin, which is the same characteristics with three isolates from nurses and environment of clinic b. the other two mrsa strains from neonates of clinic b showed st493 (62-1-1-1-1-1-1), which is shared by six strains from nurses and environment of clinic b. spa type of these 22 mrsa strains was identical as ujebkbp and all strains were negative for pvl gene. objectives: over the last years there has been a dramatic increase in the prevalence of methicillin-and multiresistant staphylococcus aureus (mrsa) leading to a major health problem, especially in the nosocomial setting. to support infection control measures typing of mrsa is essential. the present ôgold standardõ for strain typing is pulsed field gel electrophoresis (pfge), but several sequence based methods including spa typing have recently been introduced. therefore this study was initiated to compare typing results obtained from pfge with those from spa typing. methods: 90 well characterized s. aureus strains of different evolutionary origin including methicillin sensitive and resistant isolates were included. strains were selected from a variety of clonal groups prevalent in germany and middle europe during the last ten years. the collection comprised hospital derived strains as well as isolates from the community, including the recently emerging community acquired mrsa. all isolates were subjected to pfge with subsequent cluster analysis; additionally the polymorphic x-region of the protein a gene was sequenced and a spa type was assigned using the ridom staphtype software. the newly developed algorithm burp (based upon repeat patterns) was applied to the spa sequences to cluster the resulting spa types into different groups. clustering results were compared to those obtained from pfge and mlst. results: overall the results obtained from the different typing approaches were in agreement. in some cases where pfge results were inconsistent despite uniform epidemiological background spa typing was able to group the corresponding strains together. only two strongly related epidemic clones could not be separated by spa typing. conclusion: spa typing in combination with burp analysis is an easy, rapid and reliable method for epidemiological analyses in s. aureus. it is superior to pfge in cases where pfge might be ôoverdiscriminatoryõ. in rare cases a second discriminatory locus (for example a single mlst locus) might be helpful for an ultimate classification. the antibiotic susceptibility of methicillinresistant staphylococcus aureus methicillin-resistant staphylococci (mrsa) are often multidrug resistant and represent a major problem for the antimicrobial therapy. glicopeptides are the golden standard for these infections but resistance and toxicity concerns limit their usage. mrsa antibiotic resistance may be divided into two distinctive profiles: multidrug resistance (probably nosocomial infections) and variable resistance, usually to 1 or 2 non-betalactamic antibiotics (often correlated with community-related mrsa infections results: in total, 4.5 % (98) of s. pn was resistant to erythromycin and 2.9% (8) of invasive and 4.6% (10) of non-invasive s. pyo were resistant to erythromycin. none of the isolates were resistant to clindamycin. among the ery-res s. pn, the most frequent gene was mef(a) 83.7%, while erm(b) was found in 13.3%. in two isolates no resistance genes were identified. 81 out of 82 mef(a) carrying isolates had serotype 14.erm(a) and mef(a) was present in 62.5% and 37.5% of the invasive ery-res. s. pyo isolates. none of the isolates were positive for erm(b). in non-invasive ery-res. s. pyo isolates 40%, 10%, 50% were pcrpositive for erm(a), erm(b) and mef(a) respectively. conclusion: in contrast to s. pyo, ery-res. s. pn is dominated by one serotype, carrying mef(a). the overall resistance profile of s. pn and s. pyo is still favourable, but the resistance to macrolides is of growing concern in denmark. resistance in streptococcus pneumoniae: auc/ mic breakpoints differ between gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin k. laplante, m. rybak, b. tsuji, g. kaatz (detroit, usa) objective: the potential for resistance development in streptococcus pneumoniae (sp) secondary to varying exposure to gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin was examined at high inoculum (108.5-9 log 10 cfu/ml) over 96 hours in an in vitro pharmacodynamic model. results: the molecular analysis of one hundred and forty serotype 11a dspn isolates revealed six clonal types. however, the vast majority (95%) belonged to a single clone. the prevalence of this dominant clone during the study ranged from 6.3% to 13.9% and it was disseminated in all day-care centres. conclusions: serotype 11a is not a capsular type with an important representation in drug-resistant collections which are the most commonly studied worldwide. however, it seems to be common among dspn strains. this serotype 11a collection showed little genetic variability. in fact, one genotype was found to be dominant and disseminated successfully in all day-care centres studied. these findings suggest that this serotype 11a clone is frequent in colonization and thus its monitoring is of importance particularly after the introduction of the sevenvalent pneumococcal conjugate vaccine. diseases. an abrupt antimicrobial resistance increase and clonal spread of resistant pneumococcal strains has resulted in serious therapeutic problems in recent years. the aim of this study was to analyze resistance patterns and genetic diversity of s. pneumoniae resistant strains isolated in our region during three years (2001) (2002) (2003) . methods: using e-test method and the nccls criteria for benzylpenicillin (p), erythromycin (e), clindamycin (l), tetracycline (t), cotrimoxazole (s), ceftriaxone (c), chloramphenicol (h), vankomycin (w), imipenem (i), fifty nine resistant or intermediate to at least one drug strains were obtained. strains were submitted to molecular characteristic by pfge with smai restriction enzyme and computer analysis using molecular analyst software application. for each strain resistance pattern and pfge profile was determined. results: resistance to 8 out of 9 determined antibiotics (except vancomycin) was described. strains showed 22 different resistance patterns and tsh (13%strains), psi (11.8%) and elts (10.1%) were the most often. resistance degree reached 7 drugs (5% strains) and 69% strains were mdr. we have found 26 pfge profiles: 15 of them (u 1-15) were unique single isolates, 11 clusters (a-k) were represented by 2-10 strains, which were more than 78% of similar. the most numerous clusters (a-k) consists of strains that were isolated over three years of study and showed the same or very similar resistant patterns: a-tsh (8 of 10 strains), th (2); b-psi (7 of 8), s (1); c-pelshi (3 of 6), ptshi (2), ptschi (1); d-elts (3 of 4), els (1); e-sh (2 of 4), ts (1), t (1). all strains of tsh and psi resistance patterns were found in one cluster a(a1-a9) or d (d1-d6) respectively but strains with elts and pelshi patterns belonged to different (2-3) clusters and unique pfge profiles. another clusters (f-k) were represented by two strains with different resistant patterns each. conclusions: population of s. pneumoniae resistant strains in our region presents high genetic diversity and numerous different resistance patterns. the majority of strains with the same resistance pattern showed different pfge profiles but there were observed some strains of the same resistance pattern, which belonged to only one cluster and were isolated over three years of study. ) hlpr strains showed a profile identical or related to two epidemic clones: spain23f-1 and spain9v-3. the remaining hlpr pneumococci belonged to unique or rare clones. llpr isolates were characterised by more variable backgrounds in terms of pfge patterns and serotypes than hlpr strains. in fact, 27.7% showed an identical or related profile (arbitrarily called d, serotype 15a) previously not described in italy and 10 (15.4%) exhibited the pfge patterns typical or related to the spain23f-1 and spain9v-3 clones. the remaining 37 llpr isolates (56.9%) belonged to 22 different profiles and to 11 distinct serotypes. in this study, the 23f ôitalian cloneõ circulating in 1993-1996 was not found. conclusions: the recent increase of total penicillin-resistance in italy can be ascribed to the emergence of a new clone and to the diffusion of two well known international clones, whose ability to spread is higher than that of the autochthonous italian clone described in 1996. changing antibiotic prescribing habits, including the recent strict limitation to the consumption of parenteral drugs, may also explain the variations described. results: during the study period, 143 strains of s. pneumoniae were isolated: 6 (4.2 %) in csf and 137 (95.8%) in blood. total incidence was 6 cases/100,000 inhabitants/year, with maximum incidence in winter and spring. the largest number of cases, 76 (54 %), were in the over 65s. in children, 11 (7.7 %) were detected, all in the under 5s. the total number of strains whose sensitivity to penicillin fell was 43 (30 %), of which 20 (46.5 %) had an mic 2 mg/ml. 46.5% of the penicillin-resistant strains were also resistant to erythromycin. 16 (11%)/ 143 had reduced sensitivity to cefotaxime, and most of these (63%) had intermediate sensitivity. resistance to erythromycin was detected in 34 (24%). during the study period, resistance to antibiotics decreased gradually in such a way that the resistance percentages each year were: penicillin 41%, 30%, 27%, 23%; cefotaxime 28%, 14%, 4%, 4%; erythromycin 34%, 19%, 29%, 8%. no differences were observed in the resistance percentage in the different age groups. conclusions: the prevalence of csf and blood strains of s. pneumoniae with reduced sensitivity to penicillin in this study was 30%. the treatment of choice in s. pneumoniae invasive disease was third-generation cephalosporins, due to their low level of resistance. resistance to the main antimicrobials from this type of strain has fallen. during the study period, resistance to penicillin fell by 43.9 %, 58.5 % for cefotaxime 63.4 % for erythromycin. the fall in resistance among invasive s. pneumoniae could be due to vaccination in children and the elderly from 2002 onwards, since the vaccination includes the most common and resistant serotypes. the low number of isolates in children could be due to the low number of samples processed. change in distribution of macrolide-resistant streptococcus pneumoniae genotypes over 4 years -data from protekt 1999-2003 objectives: this analysis of data from 39 countries over the first 4 years (1999) (2000) (2001) (2002) (2003) of the protekt global surveillance study was undertaken to investigate longitudinal and regional trends in the distribution of macrolide resistance mechanisms among streptococcus pneumoniae, and the susceptibility of these isolates to antibacterials, including the ketolide telithromycin. objectives: the 7-valent formulation of the pneumococcal conjugate vaccine (pcv7) was introduced in the usa in february 2000. there is a general recommendation for its use in all children <2 years of age, and the vaccine is licensed for children up to 5 years of age. in europe, the vaccine is licensed for children up to 5 years of age, but most countries do not have a general recommendation for use. protekt -a global, longitudinal study of the antimicrobial susceptibility of bacterial respiratory tract pathogens -has now completed its fourth year. a comparison was made between serotype distribution and pcv7 coverage between y1 and y4. methods: analysis was performed only on streptococcus pneumoniae isolates obtained from paediatric patients at the 31 centres common to both years (y1, n = 553; y4, n = 682). serotyping was performed by neufeld's quellung reaction using statens serum institute antisera (ssi, denmark). results: pcv7 coverage decreased from 67.7% and 62.5% in y1 to 51.0% and 41.0% in y4 in the usa and latin america, respectively (chi-squared; p = <0.001 and 0.007, respectively). serotypes that showed the greatest increase (y1, y4) in the usa were 19a (7.0%, 22.2%), 6a (1.8%, 9.9%), 3 (1.8%, 6.2%), 15 (1.8%, 6.2%), and 11 (0%, 6.2%). in brazil, a similar pattern was seen although there was no change in the proportion of isolates with the 19a serotype. in contrast, no change in serotype distribution was observed in canada, western europe, and the far east, where the pcv7 vaccine is not routinely used for paediatric vaccination. conclusions: results from this analysis of protekt data indicate that the proportion of s. pneumoniae isolates from paediatric patients covered by the pcv7 vaccine has decreased significantly over 4 years in regions where the vaccine is routinely used. other serotypes, such as 19a, which is known to be highly resistant to antimicrobials and is not covered by pcv7, have increased in these regions. this study demonstrates the importance of serotyping antimicrobial surveillance study isolates in order to monitor such changes and any potential future implications for therapy and vaccine formulations. background: sequential alterations in pbp sequences constitute the classical mechanism to acquire penicillin resistance in s. pneumoniae, that is reflected in changes in the cell wall peptidoglycan structure. murm gene controls the addition of the first amino acid of the dipeptide bridge of the pneumococcal muropeptide. mutations in this gene are apparently required for high penicillin and cefotaxime resistance. the aim of this study was to compare the murm gene sequence within the earlier and the latest highly penicillin resistant pneumococcal populations recovered in spain. material and methods: a total of 56 s. pneumoniae isolates (10 of them from 1987-88 and 46 from 2002-04) with resistance to penicillin (mic 2-8 lg/ml) were included. susceptibility to different b-lactams, macrolides, tetracycline, levofloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole (sxt) were determined by the agar dilution method. all isolates were grouped according to serotype and pulsotype (pfge-smai pattern) and the murm gene was sequenced. results: a high percentage of resistance (intermediate plus resistant) was observed for cefotaxime (100%) and sxt (96.6%). all isolates remained susceptible to telithromycin but not to levofloxacin (93% of susceptibility). all isolates recovered during 1987-88 were genetically unrelated and mainly belonged to serotypes 23f (40%) and 6b (40%). in contrast, the most frequent serotypes during 2002-04 were 14 (30%), 9v (26%), and 6b (17%). in this period, a predominant spain9v-3clone was detected in 10 out of 14 isolates of serotype 14, and in 11 out of 12 isolates of serotype 9v (capsular switch). all 8 isolates of serotype 6b belonged to a single clone (spain6b-2). the analysis of murm sequences of isolates from both periods revealed the existence of different alleles. homologous sequences to the murma (identical to that described for r6 penicillin susceptible strain), were replaced in part by murmb5, and murmb6 (different variants of these alleles were detected in our collection). conclusion: early high penicillin resistant s. pneumoniae isolates in spain were genetically unrelated and corresponding to 23f and 6b serotypes. on the contrary, recent isolates showed dissemination of selected clones, particularly spain9v-3. many s. pneumoniae isolates with high-level resistance to penicillin retain (or reacquired) the murm gene of susceptible strains, but different allelic variants was also detected. objectives: to determine among 712 s. pneumoniae clinical isolates recovered from different spanish hospitals during three different periods (1999-2000, 2001-2002 and 2002-2003) the prevalence of erythromycin resistance phenotypes and resistance genes associated to this resistance. methods: susceptibility testing was performed by the standard microdilution technique (nccls). a pcr assay was carried out to identified erythromycin resistance genes (erm or mef) and a multiplex-pcr was designed to distinguish between mef(a) and mef(e) genes within mef positive isolates. clonality was studied using the sma-i-pfge technique. during the studied period ranged from 48.3% (first studied period) to 45.2% (third studied period) for penicillin, 39% (first studied period) to 32.9% (third studied period) for erythromycin, and 32% (first studied period) to 30.3% (third studied period) for clindamycin. levofloxacin resistance was only found in 2.4% of isolates and were recovered during the third studied period. m phenotype was observed in 3.2% of all tested isolates. considering non-susceptible erythromycin isolates, ermb and mef genes was found in 87.1% and 9.5% of isolates, respectively, as a sole erythromycin resistance gene. the concomitant presence of both determinants was found in 3.3% of isolates. interestingly, among the isolates with mef pcr positive result, only one (3.2%) carried the mef(a) gene whereas the mef(e) gene was detected in the others (96.8%). an increase in mef prevalence was observed between the first and the second studied periods (2% to 5.8%) and a slightly decrease (4.9%) in the third period. pfge analysis revealed a polyclonal structure of mef positive isolates. conclusions: prevalence of mef positive isolates among erythromycin-resistant strains remains low in our country (4.3%) being the mef(e) gene the most prevalent mef determinant among these isolates (96.8%). results: among young children, single ery resistance was most prominent in all countries, (from 6% in sweden to 38% in belgium), except for spain where the proportion of dual resistance was highest (33%). for the other countries dual resistance among young children ranged from 0% in denmark to 13% in belgium. except for ireland (12%) and spain (21%), single pen resistance remained below 5%. conclusion: between countries, large differences in the patterns of s. pneumoniae resistance were found among young children. ery resistance was most common among young children, which may indicate greater use of macrolides in this age group. in order to assess the effectiveness of interventions like vaccination, resistance and serotype data should be monitored carefully. evolution in the pattern of sensitivity to penicillin and cefotaxime of streptococcus pneumoniae background: the increase in resistance to penicillin, and more recently to cefotaxime, means that it is necessary to study the prevalence of resistance to these antibiotics, giving them huge clinical importance. this study compares the sensitivity of s. pneumoniae to penicillin and cefotaxime during 1991,1995,2000,2001,2002,2003 and 2004. methods: a total of 892 strains of s.pneumoniae from significant respiratory tract isolates were studied. these corresponded to the years 1991 (130) conclusions: in 2004, (1) 14.9 % of the isolates were erythromycin-resistant and clindamycin-and miocamycin susceptible (m phenotype), a smaller percentage than in previous studies; (2) there was a significant increase in 2004 of isolates with the mlsb constitutive phenotype; (3) there was a high prevalence of resistance to telithromycin (88.6 %) in the 35 strains with mlsb constitutive phenotype. patterns of macrolide resistance determinants among s. pyogenes and s. pneumoniae isolates in saudi arabia objective: to characterize the macrolide sensitivity of recent isolates of s. pyogenes and s. pneumoniae collected from different hospitals around saudi arabia and to investigate the resistance determinants carried by macrolide-resistant isolates. methods: susceptibility testing was performed using standard nccls methodology on 335 s. pyogenes and 350 s. pneumoniae isolates. macrolide resistance mechanism phenotypes were identified using double disk diffusion. results: all s. pyogenes were penicillin sensitive, while 6.4% were macrolide resistant, the main mechanism of which was of m-phenotype (96%). approximately 51% of s. pneumoniae was penicillin non-susceptible. macrolide resistance in s. pneumoniae accounted for 18.8%, the majority of which was m phenotype (92%). low-level resistance mediated by mef-bearing strains predominated. conclusions: efforts should focus not only on antibiotic resistance surveillance and the development of guidelines but also on appropriate use of antibiotics. strategies have been proposed which include restricting access, compliance promotion and reduction in our prescriptions and inappropriate use of antibiotics. newer macrolides, including azithromycin, are still considered drugs of choice for empirical treatment of respiratory infection in such circumstances. objectives: mlsb phenotype erythromycin resistance (eryr) is often associated with tetracycline resistance (tetr) and in streptococci is mediated by the methylase genes erm(b) or erm(tr). in streptococcus pneumoniae, erm(b) is carried in transposons such as tn1545 which also carry the tetr gene tet(m). m phenotype eryr is associated with the efflux genes mef(a) or mef(e). these genes are carried in genetic elements that do not commonly include tetr genes, but the mef(e) element in s. pneumoniae can be inserted in a transposon that carries tet(m). our objective was to investigate tetr in eryr beta-haemolytic streptococci of groups a, b, c and g. methods: 129 eryr beta-haemolytic streptococci of groups a (26), b (42), c (9) and g (52) were collected over two years. tetr was determined by disc diffusion and mics by e-test. pcr was performed for tet(m), tet(o) and tet(l); eryr genes had been characterised previously. results: the prevalence of tetr amongst eryr mlsb isolates was high in groups a (73%, 11 of 15), b (92%, 34 of 37) and c (100%, 2 of 2) but lower in group g (38%, 18 of 47). the range of mics in tetr isolates was 4 ‡ 256 (mg/l). the tet(m) gene was responsible for most tetr in groups a (100%, 11 of 11), b (85%, 29 of 34), c (100%, 2 of 2) and g (56%, 10 of 18); tet(l) and tet(m) were both found in the same isolates in groups b (2) and g (1). at least 80% of erm(b) isolates and 57% of erm(tr) isolates of groups a, b and c carried tet(m). in contrast, only 21% of erm(tr) isolates in group g carried tet(m). amongst m phenotype isolates, the prevalence of tetr was high in groups b (100%, 5 of 5) and g (80%, 4 of 5) and lower in group c (14%, 1 of 7). tetr was not detected in m phenotypes of group a. in m phenotypes, tet(m) was responsible for 100% tetr in group b (5 of 5) and 25% in group g (1 of 4); 1 isolate of group c carried tet(o). tet(m) and tet(o) were found in mef(e) isolates but not in mef(a) isolates. there were tetr isolates in groups b (5) and g (11) in which no tetr gene was identified. conclusions: 1) the prevalence of tetr in eryr beta-haemolytic streptococci was high in mlsb isolates of groups a, b and c and in m isolates of groups b and g. 2) tet(m) was the predominant tetr gene. 3) tet(m) was strongly associated with erm(b) and to a lesser extent with erm(tr). 4) tet(m) was found in association with mef(e) but not mef(a). results: reduced susceptibility (mic >1 mg/l) was detected in 44 (2.7%) s. pyogenes isolates. a total of 32 (73%) s. pyogenes isolates demonstrated a mlsb phenotype resistance. the dominating gene was erm(a) (n = 27), encoding resistance in 27 strains, whereas erm(b) was detected in only five of the strains isolates. while one of the cmlsb strains carried both erm(a) and the mef(a) gene, another cmlsb strain was reproducibly negative in erm and mef pcr analyses, and will be further investigated. the remaining 11 (25 %) isolates all carried the mef(a) gene and showed a consistent with their m phenotype resistance. thirty (68%) strains were resistant to tetracycline and carried the erm-positive strains (n = 30) genes, while all the mtype strains were susceptible to tetracycline. typing analyses revealed t-type 4 and st39 dominated in nine 9 out of 10 with mtype phenotype, whereas t-type 3 was associated with st46 in 8 of 9 erm(a)-positive and tetracycline resistant strains. results from emm typing supported these clonal observations. conclusions: (i) the prevalence of macrolide resistance among s. pyogenes in norway is low, and the (ii) mlsb resistance, erm(a) or erm(b) was the most prevalent resistance type and co-resistance to tetracycline was frequently observed. rates of resistance to macrolides vary from region to region and these rates are important in defining the role of macrolides in empiric therapy. macrolide and lincosamide resistance are mediated by 3 distinct mechanisms, (1) mefa encodes resistance to erythromycin but not clindamycin, (2) ermb encodes resistance to erythromycin and clindamycin, (3) erma encodes resistance to erythromycin and inducible resistance to clindamycin. methods: consecutive pharyngeal isolates of streptococcus pyogenes isolates were collected between january 2003 and april 2004. isolates were confirmed as s. pyogenes by lancefield grouping using the pastorex strep rapid agglutination kit. erythromycin susceptibility testing was performed by nccls disc diffusion. erythromycin resistant isolates were tested for susceptibility to erythromycin and azithromycin by etest. the mechanisms of resistance was assessed using the erythromycin/ clindamycin disc approximation test and by pcr, with primers specific for the erma, ermb and mefa genes. positive controls were kindly provided by ralf r. reinert. results: four (4) of 360 consecutive isolates (1%) were resistant to erythromycin. erythromycin resistance was confirmed by etest and these isolates were also resistant to azithromycin. all 4 erythromycin-resistant study isolates and 3 of 4 previously collected erythromycin-resistant isolates were confirmed as erma positive by pcr and exhibited inducible resistance by disc approximation. one previously collected isolate was susceptible to clindamycin and was mefa positive by pcr. conclusion: these data show that macrolide resistance in s. pyogenes is very uncommon in this area and is encoded primarily by erma. objective: aim of this study was to evaluate antimicrobial prophylaxis (ap) practice and its cost in intensive care unit of heart surgery depertment in our hospital. method: study was performed prospectively between january-december 2002. ap lasting >1 day was considered inappropriate, unless the patient was in high risk group (heart transplantation or repeated by-pass). in addition ap with two beta lactam agents; beta lactam+aminoglycoside (only beta lactam+gentamicin was allowed); 3rd generation cephalosporins; quinolones or glycopeptides were considered inappropriate. in high risk patients, ap lasting up to 3 days and ap with glycopeptides were considered appropriate. cost of inappropriate ap was calculated by using august-2004 prices in euro. cost of inappropriate ap in patients who received cefazolin or beta lactam/beta lactamase inhibitors (ampicillin/sulbactam or amoxycillin/clavulanate) was calculated by substraction of cost of 1 day lasting ap from total ap cost. in case of inappropriate glycopeptide or quinolone or aminoglycoside usage, one day lasting cefazolin cost was substracted from total ap cost. in high risk patients cost of inappropriate glycopeptide usage was calculated by substracting 3 days lasting ap cost from total cost. infections were diagnosed according to the criteria of center for diseases control and prevention (cdc). data were analysed with chi square and student's t-tests. objectives: the aim of the present study was to investigate the frequency and the type of wound infections, as well as the bacteria involved, after posterior spinal fusion in children, especially with spinal deformities, over a 6-year period (1999) (2000) (2001) (2002) (2003) (2004) . materials and methods: a total of 133 spinal fusions were performed on 110 children, with or without instrumentation, because of spinal scoliosis (120), spondylolisthesis (4), fractures (4), and for other reasons (5 objectives: therapy of chronic wounds with lucilia sericata presents a promising alternative to the classical approach with antibiotics. larvae are placed in wounds free-roaming or in biobags. the therapy often stimulates the process of wound healing. this effect is based on different mechanisms. maggotsõ saliva contains collagenases, trypsin and chymotrypsin-like enzymes which dilute necrotic tissue. furthermore, an antibacterial effect has been described. in our study, we focused on the question whether this effect could be explained by the ingestion of bacteria by larvae. methods: free roaming maggots were placed on agar plates with escherichia coli. these bacteria had been transformed with green fluorescent protein (gfp)-containing plasmids. gfp-free escherichia coli xl1 blue served as a control. two days old maggots were put on agar plates in groups of ten at room temperature. every thirty seconds, a maggot was displaced from the agar plate and washed with sterile saline (0.9%). dead larvae which could not have taken up bacteria served as a control. maggots were tested for flourescence under a fluorescence microscope [zeiss axioskop; hpo50/ac, axiocam mrmzeiss]. larvae were examined by an independent observer as well. results: maggots that had been placed on gfp-labelled escherichia coli showed an intensive green fluorescence, especially at the back of the head and in the intestines. the controls did not show any fluorescence. after three to four minutes gfp-labelled escherichia coli could be detected inside the maggots' body. at this early stage of digestion gfp-fluorescence could mainly be examined in the area of the head. this fluorescence results of the ingestion of gfp-labelled escherichia coli by larvae. we have proven that the uptake of bacteria represents a further mechanism of antibacterial activity of larvae. not only diluting necrotic tissue, but also minimizing the rate of infections, maggot therapy which is also well-known as biosurgery could be a beneficial alternative to the use of antibiotics. as the mechanical uptake is a non-specific process, there might not be a risk of resistance. antibiotic resistance: nosocomial pathogens p1055 antimicrobial resistance patterns of bacterial pathogens from blood culture of cancer patients in a single cancer institution m. faiz, h. aziz, t. bashir, s. asghar (lahore, pak) objective: the widespread emergence of resistance to antimicrobial agents among bacterial pathogens is well known and has an impact on our ability to treat patients effectively. blood stream infections (bacteraemia) among cancer patients that develop during the course of disease are potentially life threatening because of suppression in their immune systems. the changing spectrum in the incidence and epidemiology of microbial pathogens has resulted in an increase in resistance to many antibiotic compounds emphasizing the need to monitor the prevalence of resistance in these strains. methods: susceptibility and resistance pattern of clinically significant bacterial isolates from positive blood cultures collected during 2000-2004 was studied. the isolated strains were tested against a wide range of antibiotics belonging to cephalosporins, aminoglycosides, carbapenems and quinolones derivative groups by disc diffusion method and the results were interpreted according to british standard for antimicrobial chemotherapy. results: a total of 250 bacterial pathogens were isolated with 60% gram positive and 40% gram negative bacteria. the dominating pathogens were staphylococcus aureus, streptococci, pseudomonas , enterobacter and klebsiella. among gram negative strains, highest level of resistance (98%) to third generation cephalosporins was observed followed by carbapenems and penicillin (79%, 80%) respectively. similarly, high resistance to aminoglycosides were found (69% to ampicillin, 50% to tobramycin and 62% resistant to quinolone derivates group of antibiotics. however only 29% were resistant to ciprofloxacin. in gram positive bacteria, high resistance to ciprofloxacin (40%) was observed as compared to gram negative bacteria. a still higher resistance level (100%) was observed for aminoglycosides and third generation cephalosporins (97%) respectively. conclusion: the spectrum of isolates among our patients were shifting towards gram positive bacteria with high resistance to different groups of antimicrobial agents limiting few choices for alternative therapies for infection control. antimicrobial resistance continues to increase and ongoing surveillance of microbial pathogens is essential. this study also warrants the need of infection control measures, rational antibiotic policies and rapid laboratory detection of resistance to prevent the spread of resistance among these strains. (9), and epidemiological swabs from throat and rectum (21). we found two susceptibility groups: group 1 (7 patients) resistant to amox/ clavul.acid, pip/tazobactam, aztreonam, cefuroxime, and group 2 (11 patients) susceptible for these antibiotics. rapd profiles corresponded with the susceptibility testing results. drug resistance profile in group 1 could be attributed to overproduction of chromosomal encoded k1 beta-lactamase. group 1 was from ru and group 2 from nicu, pu and su. analysis of sociodemographic variables and potential risk factors showed differences between analysed groups in mean birth weight, length of hospital stay, antimicrobial treatment and invasive therapeutic procedures usage. the outbreak analysis revealed concurrent isolation of two k. oxytoca clones, normal and k1 overproducer. rapd results were supported antibiotyping results. results: a total of 3077 isolates was analysed. 51.4% of these isolates were non-susceptible to at least one agent, the highest proportion is due to mono-drug resistance to piperacillin (39.6%). 4.8% of the strains were classified as multi-resistant, the most frequent patterns were pip/caz/ctx/gen (2.1%) and pip/caz/ctx/gen/cip (1.7%). two strains were resistant to all six antibiotics. significant differences in multi-drug resistance rates were associated with ward type with highest rates for icu-patients (8.8%). furthermore, multi-resistance rates varied significantly between the centres involved with a range from 0.9% to 7.2%. the centres differed not only in respect to the multi-drug resistance rates, but also in respect to the dominating phenotype. conclusions: the relevance of multi-resistance in k. pneumoniae as a major clinical problem is proved by an overall rate of almost five per cent for german university hospitals and an even higher proportion for icu-patients. among the agents tested piperacillin plays an eminent role in regard to its mono-resistant rate as well as a component of the most frequent resistance patterns. resistance to the combination of pip with tazobactam however is rare. the development of antimicrobial resistance in german hospitals objectives: quinolones (q) are among the most frequently used agents for treating ltcf-acquired infections. consequently, bacteria exhibit increasing resistance to q. a multicentre survey was conducted in order to determine (i) the prevalence and risk factors for colonization with qrgn in greek ltcf (ii) the corresponding antimicrobial resistant rates (á r). methods: a total of 28 ltcf were randomly selected from the public sanitation list of attica province. urine, nasopharyngeal and wound samples were collected from 668 elderly residents. we chose randomly 35% of the existing population from each ltcf (minimum sum 25 residents, age above 65 years). gram negative bacteria were identified by api strips and underwent antimicrobial disk susceptibility testing, following the nccls guidelines. data were also collected on resident factors and institutional variables. univariate and multivariate analyses were performed. odds ratios (or) and p values were calculated. results: 412 gram negative (gn) strains were isolated from 1448 samples (prevalence rate 28.5%). the majority of them were recovered from urine samples (86.8%). 53% of the residents had been taking systemic antibiotics during the preceding month. q were the leading class (38%). á r of gn to ciprofloxacin (cip) were estimated (table) . multi-drug qrgn accounted for 11.5% of the isolated strains. the most prevalent phenotype was resistance to ampicilline, cip and trimethoprim-sulfamethoxazole. in the multivariate analysis, only prior exposure to antimicrobial agents (p < 0.001; or = 2), specifically to q (p < 0.001; or = 14), and the presence of a urinary catheter (p < 0.001; or = 2) were significantly associated with colonization with qrgn. objectives: colonization and resistance dynamics of gramnegative bacteria in the intestinal and oropharyngeal microflora of patients admitted to intensive care units (icu) and general wards (gw) were investigated during and after hospitalization. methods: specimens were obtained on admission, once weekly during hospitalization, at discharge from the icu, at discharge from the hospital and one and three months after discharge from the hospital. five colonies per specimen were selected for identification and susceptibility testing by the vitek 2 system. results: a total of 3316 specimens from 411 patients were collected. in both patient populations, the gram-negative colonization rates in oropharyngeal specimens increased during hospitalization and did not decrease in the three months after discharge. in rectal specimens, colonization rates decreased during hospitalization and increased after discharge. there was a change in species distribution among the dominant microflora during hospitalization. klebsiella spp., enterobacter spp., serratia marcescens and pseudomonas aeruginosa were more often isolated, whereas the frequency of e. coli declined. the percentage of icu patients colonized with ampicillin and/or cephalothin resistant faecal e. coli was significantly increased at discharge from the hospital and did not change in the three months after discharge. emergence of multi-drug resistance was observed in many gram-negative species during stay on the icu. resistance frequencies in e. coli significantly increased with length of stay on the icu. for the gw population, no significant changes in resistance frequencies were found during hospitalization. conclusion: from a population perspective, the risk of dissemination of resistant gram-negative bacteria into the community through hospitalized patients appears to be low in gw patients, but is noticeably higher among icu patients. association of antibiotic resistance phenotypes and the presence of class i integrons in a sample of bacterial isolates from u.s. hospitals objective: we assessed the association between class 1 integrons and patterns of resistance to aminoglycosides, beta-lactams, quinolones, and chloramphenicol in escherichia coli and klebsiella species isolates from u.s. hospitals participating in project icare. methods: a convenience sample of 320 e. coli and klebsiella species isolates was collected between october 2002 and december 2003 from 19 u.s. hospitals as part of phase iv of project icare and tested for susceptibility to a variety of antimicrobial agents. these organisms were submitted in response to a request for organisms resistant to 3rd generation cephalosporins (ceftazidime, ceftriaxone, cefotaxime) and/or fluoroquinolones. the isolates were screened for the presence of class i integrons using sets of primers specific for 5¢ and 3¢ conserved segment of the integrase gene. we obtained measures of association between the presence and absence of integrons and resistance phenotypes. results: of the 320 e. coli and klebsiella species isolates screened, 181 (57%) contained a class i integron. a significant association was seen between the presence of the class i integron and resistance to amikacin, gentamycin, tobramycin, ampillicin, chloramphenicol, aztreonam, ceftazidime, cefotaxime, and cefpodoxime. resistance to ciprofloxacin and cefepime was not significantly associated with the presence of an integron. the presence of class i integrons in 57% of our convenience sample of e. coli and klebsiella species isolates, along with the differential association of those integrons with resistance to some drugs, but not others, suggests that integrons may play an important role in determining how the epidemiology of bacterial resistance results from antimicrobial agent selective pressure. objectives: the main purpose of this study was to investigate the mechanisms of resistance to fluoroquinolones in two isogenic citrobacter freundii clinical isolates, which led us to characterize the acra and acrb genes of this microorganism. methods: two c. freundii strains (1.44 and 1.38) sequentially isolated from the same patient were characterized. the relationship was determined by rep-pcr and pfge. the susceptibility to ciprofloxacin and chloramphenicol was determined by e-test. mutations in the quinolone resistance determining region of the gyra and parc genes, the outer membrane protein profile and the accumulation of ciprofloxacin was also investigated. the expression of genes in both strains was analysed using dna microarrays for e. coli and the expression of the acra and acrb genes was verified by rt-pcr using the gapa gene as the control. the acra gene was cloned and dna sequenced. results: both c. freundii belong to the same clone by both rep-pcr and pfge. the mic of ciprofloxacin was of 8 (strain 1.44) and 32 mg/l (strain 1.38). the mic of chloramphenicol was of 16 and 96 mg/l, respectively. the two strains showed the same substitutions in the gyra and parc (thr-83-ile and asp-87-tyr in gyra and ser-83-ile in parc). no major differences were found between the outer membrane protein profiles. however, differences were observed in the amount of ciprofloxacin accumulated, with strain 1.38 showing less accumulation. eleven genes were overexpressed in strain 1.38 compared to strain 1.44. among these genes the acra was overexpressed. this result was further corroborated by rt-pcr. the nucleotide similarity between the partially sequenced acra (1027 bp) and acrb (420 bp) genes of c. freundii and e. coli was of 81.5% and 86%, respectively. conclusion: the acra and acrb genes of c. freundii are similar to those described in e. coli and in collaboration with mutations in the gyra and parc genes, their overexpression may play an important role in modulating the final mic of fluoroquinolones. high prevalence of aac (3) objectives: multidrug resistance of gram negative bacilli (gnb) has become a major public health problem in tunisia. surveillance of both antimicrobial resistance and antimicrobial consumption has now been recognised as essential for planning future strategies to control resistance. the aim of our study is to examine for the whole hospital and wards with risks the correlation between resistance of gnb and the consumption of third generation cephalosporins (3rd gc) and imipenem for the period from january 2003 to september 2004. methods: a surveillance of antibiotic resistance and antibiotic consumption was established respectively by the laboratory and the department of pharmacy. data on antimicrobial resistance and antibiotic consumption were collected quarterly and antibiotic consumption was calculated in defined daily doses (ddd) using abc calc. results were expressed per 1000 bed-days (bd). statistical analysis was done using spss software for the calculation of the spearman rank correlation (rs) with an alpha risk of 5%. results: for the whole hospital, resistance to 3rd gc ranged from 0.9 to 1.7 gnb/1000 bd. the highest rates were observed in intensive care unit (icu: 4.7-12.7 gnb/1000 bd) and the lowest rates in orthopaedic ward (0-2 gnb/1000 bd all mrsa isolates were resistant to multiple classes of antimicrobials whereas mssa were sensitive (p < 0.001). all esbl+ bacteria were resistant to multiple classes but sensitive to meropenem, colistin, amikacin (82%), cotrimoxazole (50%) and chloramphenicol (50%). resistance in non-esbl+ bacteria was detected to ampicillin (73%), cefalexin (32%), 2nd/3rd generation cephalosporin (0%), chloramphenicol (32%), cotrimoxazole (11%), ciprofloxacin (5%), trimethoprim (21%), gentamicin (5%), netilmicin (53%) and tobramycin (11%). potentially community acquired isolates showed lower rates of resistance (0% to trimethoprim, ciprofloxacin, gentamicin, amikacin, tobramycin, 2nd/3rd generation cephalosporin).duration of hospital stay was not a risk for acquiring either esbl+ bacteria or mrsa whereas having a surgery was associated. exposure to quinolone was a risk factor for mrsa but not esbl+ bacteria. aminoglycoside and cephalosporin were risks for esbl+ bacteria. acquiring both mrsa and esbl+ enterobacteriacae together did correlated with the duration of hospital stay in addition to exposure to aminoglycoside and cephalosporin. conclusion: majority of patients remained free from resistant bacteria implying that cross-transmission alone was not sufficient in absence of risk factors, particularly exposure to antimicrobials. good hand-hygiene and prudent use of antimicrobials are realistic options for resource poor countries to reduce the burden of resistant bacteria. there is a unique opportunity to sequentially introduce specific infection control measure and evaluate its effectiveness. antimicrobial resistance in an intensive care unit before adopting an antibiotic rotation programme for empiric therapy of ventilator-associated pneumonia results: among gram-negative aerobic bacilli, we found p. aeruginosa strains to be meropenem and piperacillin/tazobactam-resistant in 22% and 14.6% of cases, respectively; p. aeruginosa, e. coli and k. pneumoniae were resistant to ceftazidime in 29.3%, 6.25%, and 0% of cases, respectively, and resistant to ciprofloxacin in 54.9%, 12.5%, and 5.6% of cases, respectively. neither e. coli nor k. pneumoniae were resistant to either meropenem or piperacillin/tazobactam. among gram-positive aerobic cocci, oxacillin-resistant s. aureus and cns were 55.8% and 82.7%, respectively; s. aureus isolates were very susceptible to cotrimoxazole (resistance 7.65%) and rifampin (11%), whereas resistance exceeded 40% for clindamycin, gentamicin, and norfloxacin. ampicillin-and penicillin-resistant e. faecalis were 20.8% and 33.3%, respectively. neither s. aureus nor e. faecalis were resistant to vancomycin, whereas one cns strain was resistant to it (1.3%) and 6 out of 76 to teicoplanin (7.9%). conclusion: in our icu, where a careful policy of antibiotic use with no predetermined restrictions has been applied for years, ar among both gram-negative and gram-positive microorganisms is generally lower than in the icus of italy and other mediterranean countries. we expect that the institution of an arp for empiric therapy of vap can further minimize ar in our icu. background: although newer antibiotics have been introduced to the market during the last years, they have not solved the problems arising in the management of infections due to multiresistant gram-negative bacteria. colistin, an antibiotic almost forgotten for decades has proved itself helpful, when used parenterally in patients where a lot of the classic and newer antibiotics fail. methods: we report our experience with the management of the case of a young patient who, after head trauma, had five episodes of meningitis due to multidrug-resistant gram-negative microorganisms. results: a 28-year-old patient was admitted in our hospital because of coma. three months prior to his admission, he sustained acute brain injury due to a fall and was hospitalized elsewhere for this entire period. a computerized tomography scan of his brain at his admission in the first hospital, showed multiple cerebral contusions, and an intracerebral hematoma causing deviation of the midline. he underwent a decompressive craniotomy with removal of the hematoma. during his long-standing hospitalization, he developed 5 episodes of meningitis, i.e. on day 65, 96, 108, 147, and 165 after head trauma. the pathogens isolated from each episode of meningitis were multiresistant strains of pseudomonas aeruginosa (1st and 2nd episodes), acinetobacter baumannii (3rd episode), enterobacter cloacae (4th episode), and acinetobacter baumannii (5th episode). the two episodes of acinetobacter baumannii meningitis were managed only when colistin and amikacin were given by the intraventricular in addition to the intravenous route for long period of time (six weeks for the last episode) and in high doses (40,000 iu of colistin and 10 mg of amikacin results: eight patients received aerosolized colistin. all were admitted to the icu with a mean acute physiological and chronic health evaluation ii (apache ii) score on the day of icu admission and on the 1st day of aerosolized colistin administration of 14.6 and 17.1, respectively. six of the 8 patients had ventilator-associated pneumonia. the responsible pathogens were acinetobacter baumannii (7/8 cases) and pseudomonas aeruginosa (1/8 cases) strains. half of the isolated pathogens were sensitive only to colistin. daily dosage of aerosolized colistin ranged from 1.5 to 6 million iu (divided into 3 or 4 doses) and the mean duration of administration was 10.5 days. seven out of 8 patients received concomitant intravenous treatment with colistin or other antimicrobial agents. clinical response of pneumonia was observed in 7 out of 8 patients [4 cured, 3 improved (they were transferred to another facility)]. one patient deteriorated and died due to septic shock and multiple organ failure. aerosolized colistin was well tolerated by all patients; no bronchoconstriction or chest tightness was reported. conclusions: aerosolized colistin may be a beneficial adjunctive treatment in the management of nosocomial pneumonia (vap or not) due to multidrug-resistant gram-negative bacteria. to date, susceptibility rates to imipenem and colistin of nosocomial strains are reported as high as 88% and >98%, respectively. in particular, resistance to colistin has been rarely described and the mechanisms of this resistance are poorly understood. we report a case of colistin resistant a. baumannii infection in a patient without previous administration of colistin. case report -methods: a colistin resistant (mic >2 mcg/ml) a. baumannii was isolated from 3 sets of blood cultures in a patient admitted in a burn unit without previous colistin exposure. the patient was treated with ampicillin/sulbactam and colistin, despite the documented resistance to all antibiotics. mdr a. baumannii was repeatedly isolated from different sites and the patient died after 61 days of hospitalization. strain identification and susceptibility tests were performed using phoenix automated microbiology system ò . resistance to ampicillin, ampicillin/sulbactam, ceftazidime, imipenem, meropenem, cotrimoxazole, amikacin, ciprofloxacin and colistin was confirmed by disk diffusion test and broth microdilution, in accordance with the nccls guidelines. the susceptibility results were confirmed by a reference laboratory. conclusion: colistin is one of the few effective drug available against mdr acinetobacter infections and acquired resistance is exceptional. our report points out that colistin resistant a. baumannii may be found in hospitalized patients without previous exposure to colistin. nosocomial infections by colistinresistant a. baumannii could represent a new therapeutic challenge. programmes of active surveillance cultures and contact precautions should be implemented to control the spread of this mdr microorganism. intrathecal colistin treatment of central nervous system infections due to multi-resistant acinetobacter baumannii c. suárez fernández, p. fernández-viladrich, f. tubau, l. corral , a. mateu, j. ariza (barcelona, e) objectives: the optimal therapy of cns infections due to multi-resistant acinetobacter baumannii (mrab) is not established. in 1999 we published the first two cases of ventriculitis due to mrab treated with intrathecal (it) colistin. one patient received 10 mg colistin base/12 hours after delayed csf sterilization was documented with initial dosage of 5 mg/12 hours. here we describe our extended experience with this kind of treatment . methods: we retrospectively reviewed all documented cns infections due to mrab diagnosed in our terciary 1000-bed hospital from 1999 to 2004. mrab was defined as those strains with only susceptibility to colistin (mic < 4 mg/l). cns infections were defined as those cases with suggestive clinical features plus ventricular or lumbar csf with both characteristic cytochemical alterations and positive culture. results: there were 7 cases with documented nosocomial cns infection due to mrab. six of them were acquired after neurosurgical procedure through surgical wound infection or local catheter infection. one case was caused by hematogenous spread after urological manipulation. intrathecal therapy was administered through a ventricular catheter in five cases, lumbar catheter in one case and both ventricular and lumbar catheter in one case. the usual doses administered was colistin base 10 mg (equivalent to colistimethate sodium 24 mg) every 12 hours with csf continuous drainage being interrupted for about 3 hours. length of treatment were 8-21 days with any apparent adverse effect. six of these patients received simultaneous intravenous colistin therapy. culture sterilization was documented in all patients. two patients died (one from unclear cause after initial favorable evolution; one after receiving only two doses of it colistin). the rest of them cured with sequela attributable to their previous neurological diseases. conclusions: in our experience, combined treatment with both intrathecal and intravenous colimicin seems safe and effective. when administered by local route to patients with continuous csf drainage we suggest a dosage of colistin base of 10 mg every 12 hours with temporary interruptions of the drainage. bacillus cereus -the forgotten pathogen. an unusual infection in an orthopaedic patient s. thomas, a. pillai, j. arora, a.c. ogden (dumfries, uk) background: infection with bacillus cereus has been well documented in the literature for over a century. infection is generally associated with the gastrointestinal effects of food poisoning and linked to the consumption of infected rice dishes. however, the bacillus genus is extremely heterogenic. it can occupy a wide variety of ecological niches and bacillus spores are found ubiquitously in the environment. even so, bacillus cereus isolated from the hospital and clinic setting in any material other than vomitus or faeces is commonly dismissed since it is a known contaminant of blood cultures and most bacillus bactereamias are transient and usually insignificant. case report: we report a case of bacillus cereus wound infection in a previously healthy 31-year-old male admitted to the orthopaedic ward with a comminuted fracture of the right distal tibia. he developed compartment syndrome one day later and was taken to theatre for fasciotomy. within 48 hours, he developed chest complications and was admitted to the high dependency unit. after three days he was taken back to theatre for wound inspection and change of dressing. seven days later external fixation was applied. subsequently, the wound site became red, inflamed and tender and was associated with an acute rise in inflammatory markers. microbiological reports showed bacillus cereus growth from the suture sites, sensitive to ciprofloxacin. no source was identified. discussion: this report merits concern, because it highlights the risk of wound cross infection with this unlikely pathogenic species. bacillus cereus has been known to be a contaminant of dressings, intravenous catheters, theatre scrub suits and linens pointing to an array of possible infective sources within the hospital. it emphasizes the need for theatre and hospital sterility and stresses the importance of vigilance against this infrequent cause of a potentially serious non-gastrointestinal bacillus infection. this is of particular importance because alcoholbased cleaning solutions are known to be ineffective against bacillus spores and infections dismissed as contaminants may lead to rapid clinical deterioration. antibacterial effect of phenytoin in wound healing objectives: phenytoin is an antiseizure medication, has since been reported to promote wound healing when applied as a topical agent. this effect is due to rapid infiltration of fibroblasts, collagen deposition, new vessel formation as well as antibacterial activity. this study was undertaken to evaluate it's effect on chronic skin ulcers with different causes and compare it with normal saline. methods: fifty inpatients with chronic skin ulcers were included in this case-control study: diabetic foot (20%), fracture and surgery wounds (40%), decubitus ulcers resulting from warrelated (40%). the patients were matched for age, sex, severity and size of wounds and randomly assigned to two group: 25 phenytoin treated (pht) and 25 control (ctl). each group included 15 men and 10 women, age range was between 20 and 60. surface areas of the ulcers was 12-200 cm 2 . the ulcers were debraded of necrotic tissue (if required). cultures of ulcers were taken at the beginning of treatment and on day 7, 14. in pht, thin dusting of phenytoin powder and dry gauze dressing were applied daily after washing with normal saline. ctl group received only daily washing with normal saline and plain dressing. results: bacteriologic culture in both group confirmed some strains (staphylococcus aureus, kelebsiella, proteus and psudomonas). at the beginning of treatment, there was 17 culture positive in pht. 13 case became negative by day 7 and 4 case became negative by day 14. in ctl, we had 15 culture positive that they didn't become negative by day 7 and 14 (seventy-six per cent of pht had negative cultures by day 7 compared to 0% of ctl). the mean time for appearance of granulation tissue was 13.48 days in pht compared with ctl that was 37.5 (p = 0.006). the average time to complete healing in pht was 24.76 days compared to 43.36 in the ctl (p = 0.0001). in pht only 13 patients required to analgesics (mean 1/day) compared with ctl that all patients required to analgesics about 3-4/day (p = 0.0001). age, sex and kind of wounds didn't effect the healing time in both group. conclusions: this study with statistical analysis demonstrated good improvement and efficacy of phenytoin in treatment of chronic ulcers compared with normal saline. 1. prompt pain relief, 2. reduction in ulcer size, 3. changing bacterial culture to negative, 4. more rapid granulation and healing time, characterized the pht group. we recommend wider use of this safe, inexpensive, readily available and easy to use agent because of it's positive effect on wound healing especially in our country that a lot of patients suffer from decubitus ulcers resulting from war-related wounds and limited access to more expensive wound care therapies. recurrent osteomyelitis secondary to different bacteria i. uckay, m. assal, l. legout, p. rohner, r. stern, d. lew, p. hoffmeyer, l. bernard on behalf of the osteomyelitis group study objective: recurrence of osteomyelitis due to the same bacteria from a prior site of infection without evidence of trauma or bacteraemia is an entity well-known in the literature and clinical practice. the objective of this study is to try and determine if all these reactivations are actually due to the same bacteria, as has been assumed in previous case reports. case reports: we report three cases of osteomyelitis recurring in the same bone in otherwise healthy patients. in all three patients, there was no history of illness or trauma to assume another origin. surprisingly, the strains were different from the two infectious episodes. conclusion: reactivation of osteomyelitis can be caused by different strains of bacteria, many years after the initial episode without trauma or evident bacteraemia. former infected and therefore altered bone surface might be a region of diminished resistance for a new infection during silent transient bacteraemia, reminiscent of the clinical entity of (recurrent) infectious endocarditis on altered valve surfaces. objectives: vascular graft infection proved to be the most dangerous complication in vascular surgery patients. the aim of our study was the identification of microorganisms causing vascular graft infections and the evaluation of their antimicrobial susceptibility. 25 patients with infected vascular grafts, treated in vascular surgery department, took part in our research. in 76% of patients, the late type of infection was recognized, in 24% of patients the infection was qualified as early. methods: purulent discharge obtained from the fistula was inoculated on the bacteriological media. antimicrobial susceptibility was assessed by disc-diffusion method. results: staphylococcus aureus and pseudomonas aeruginosa, present in 16,7% of patients, proved to be the most frequently isolated microorganisms. staphylococcus epidermidis and e. coli were isolated in 13.3% and 10% of patients, respectively. mixed infection, caused by two distinct bacteria, occurred in 20% of patients; in all cases one species belonged to gram-positive, and the second one to gram-negative bacteria. in 50% of patients with early type infection different species of gram-negative rods were present, in 37.5% of patients, staph. aureus and staph. epidermidis were isolated, enterococcus faecalis occurred in 12.5% of patients. in late type infection gram-negative rods were isolated from 54.5% of patients and gram-positive bacteria from 31.5% of patients. the most frequently isolated species appeared to be pseudomonas aeruginosa. in one patient candida crusei was isolated. the isolated species of bacteria varied depending on the degree of infection (according to shilagy and samson). in iiia degree of infection staph. epidermidis and e. coli were present in 67.7% and 33.0% of patients, respectively. pseudomonas aeruginosa and staph. aureus were isolated in 21,1% of patients with iiib degree of infection. various species of gramnegative rods were isolated from 80% of patients with iiic degree of graft infection. conclusions: most isolated bacteria appeared to possess the resistance patterns typical for hospital flora; among them methicillin-resistant staph. aureus (mrsa) and methicillin-resistant coagulase-negative saphylococci (mrcns) strains, gramnegative rods producing extended spectrum of beta-lactamases (esbl) or ampc beta-lactamases, and enterococcus faecalis with high level of aminoglicosides resistance (hlar). treatment failure due to emergence of resistance to imipenem during therapy for shewanella algae bacteraemia objective: we describe here a case of bacteraemia caused by s. algae, which was initially susceptible to imipenem, but the bacteria later became resistant during the treatment. in addition, we investigated the propensity of s. algae to develop resistance to imipenem by using a serial passage technique. method: in vitro test for resistance induction 1. bacterial strains resistant variants selection (a) single step resistant variants were obtained from clinical isolate on mueller-hinton agar containing increasing amounts of imipenem (1xmic, 2xmic, 4xmic, 8xmic), (b) serial passage experiment s. algae strain and p. aeruginosa atcc 27853 were grown overnight in mueller-hinton agar and then grown overnight in mueller-hinton agar and then swabbed onto mueller-hinton agar plates containing one-half the mic of imipenem. the surface growth at 24 h was swabbed to mueller-hinton agar containing twice the prior concentration of imipenem. results: single step resistant variants were selected from isolate 1 at up to 4 times the mic, whereas the resistant variant from p. aeruginosa atcc 27853 could be selected at up to 2 times the mic. all the resistant variants of s. algae selected either by a single step or by a sequential stepwise passage exhibited at up to 8-16 lg/ml of mic, whereas those of p. aeruginosa atcc 27853 showed up to 16 lg/ml of mic conclusion: we documented that s. algae, which was initially susceptible to imipenem, subsequently became resistant to imipenem during the treatment. we also demonstrated in vitro that the initial isolate of s. algae could easily develop resistance to imipenem when the organism was exposed to imipenem, suggesting that s. algae organisms have a propensity toward resistance to imipenem. objective: peritonitis remains a common and serious complication of peritoneal dialysis. in this study, we evaluated the frequency of microorganisms isolated from peritoneal fluids of patients on continuous ambulatory peritoneal dialysis (capd). methods: during a 2.5 year period, 321 peritoneal fluid samples were collected from 107 capd patients with peritonitis symptoms. the specimens were inoculated on appropriate media after 10-min centrifugation. gram stained smears and aerobic, anaerobic and broth cultures were performed. the isolates were identified by commercial id panels. mics were determined with a microdilution method according to nccls guidelines. results: fifty two out of 321 samples were positive (16.2%), six of the positives were polymicrobial. the following microorganisms were obtained: pseudomonas aeruginosa (8), staphylococcus epidermidis (7), escherichia coli (7), staphylococcus aureus (6), streptococcus viridans (6), klebsiella pneumoniae (4), enterococcus faecalis (4), stenotrophomonas maltophilia (4), enterobacter aerogenes (2), acinetobacter baumannii (1), enterococcus faecium (1), proteus mirabilis (1), pseudomonas fluorescens/putida (1), acinetobacter lwoffii (1), salmonella group d (1), bacteroides spp. (2). fungi were isolated in three patients: candida tropicalis (1), candida krusei (1) and candida parapsilosis (1). three out of 6 of s. aureus strains and three out of 7 of s. epidermidis isolates were found resistant to methicillin. all saphylococci and enterococci were susceptible to vancomycin and linezolid. gram-negative pathogens were sensitive to used antibiotics. conclusion: thirty five patients (32.7%) developed peritonitis the most prevalent etiologic agents of peritonitis on capd patients were pseudomonas aeruginosa, staphylococcus epidermidis, escherichia coli, staphylococcus aureus and streptococcus viridans. since capd patients are commonly outpatients, the antimicrobial resistance in the gram-negative strains is low compared to the nosocomial isolates. appropriate antibiotic therapy based on microbiologic results needs for the management of peritonitis on capd patients. objectives: cefepime is a fourth generation cephalosporin with a broader spectrum of activity against gram-negative and gram-positive pathogens in comparison with all other cephalosporins. due to the excellent antibacterial activity including pseudomonas aeruginosa and enterobacter spp. together with low rates of resistance and favourable pharmacokinetic and clinical properties, cefepime is a drug of choice for initial empiric treatment of severe nosocomial infections. nevertheless, in addition to recommendations in guidelines, the surveillance of local resistance data is important for empiric treatment decisions in hospitals. methods: in this study, the in vitro activity of cefepime (cep), imipenem (imi), piperacillin tazobactam (p/t), ceftazidime (caz) and cefotaxime (cft) has been investigated against 1130 bacterial strains, isolated in the year 2004. mics have been determined by microdilution method according to din 58940. reference test strains were e.coli atcc 25922, s. aureus atcc 29213, p.aeruginosa atcc 27853, and s.pneumoniae atcc 49619. results: highest in vitro activity against gram negative enterobacteriaceae were determined for cep and imi with susceptibility rates for enterobacter spp. 98%,98%, m.morganii 96%,92%, proteus spp. 98%,96% and citrobacter spp. 96%,100% whereas the susceptibility rates for p/t, caz and cft were much lower (e.spp. 68%,72%,68%; m.m. 88%,72%,66%; p.spp. 96%,96%,66%; c.spp. 68%,76%,72%). for pseudomonas aeruginosa (n = 100), susceptibility rates were 76% (cep), 73% (caz), 72% (imi) and 55% (p/t) with lowest resistance rates for cep (6%) followed by caz (11%), p/t (14%), imi (23%). the study confirmed excellent cefepime activity against gram positive isolates (pneumococci, streptococcus viridans and b-hemolytic streptococci) and high cefepime susceptibility rates for staphylococci (mssa 99% and msse 98%) on contrary to ceftazidime (mssa 44% and msse 42%). conclusion: regarding our in vitro data, cep is a reliable treatment option for empiric therapy in patients with severe nosocomial infections, demonstrating high activity against gram positive isolates, comparable activity in gram negative enterobacteriaceae to carbapenems (imi) and lowest resistance rates in pseudomonas aeruginosa. antimicrobial activity and spectrum of cefepime tested against 65,746 clinical strains from north american medical centres: report from the sentry antimicrobial surveillance program, 1998 program, -2004 h. sader, d. biedenbach, t. fritsche, r. jones (north liberty, usa) objectives: to evaluate antimicrobial spectrum and potency of cefepime (cpm) and selected comparators against clinical bacterial strains collected in north america (na) over a 7-year period (1998) (1999) (2000) (2001) (2002) (2003) (2004) . methods: isolates were consecutively collected from bloodstream (44%), respiratory tract (41%), urinary tract (6%) and skin/soft tissue (5%) infections in 48 medical centres. 75% of isolates were from hospitalized patients. isolates were susceptibility (s) tested by reference nccls broth microdilution methods in a central laboratory. oxacillin-resistant (r) staphylococci (ors) and enterococci were excluded. results: the activity of cpm against the key organisms tested is summarized in the table. overall, 99.8% of gram-positive cocci (gp) tested were s to cpm. imipenem (imp; mic90, 1 mg/l; 99.9% s) was the most active compound tested against ent, followed by cpm (mic90, 0.25 mg/l; 99.5% s) > amikacin (amk; 99.4% s) > ceftriaxone (95.6% s) > aztreonam (95.1% s). the lowest s rate for ent was observed with ciprofloxacin (cipro; 92.8%). imp was also the most active compound against esbl-producing ksp and e. coli (99.3 and 100% s, respectively), followed by amk (81.4 and 97.2% s) and cpm (92.5 and 93.8% s). cpm activity against psa (85.2% s) was similar to that of imp (86.9% s). against ossa, cpm was 4-fold more potent than ceftazidime (caz; mic90, 16 mg/l, 86.4% s) and showed higher activity than cipro (93.2% s). cpm was the most active compound against spn after gatifloxacin and levofloxacin (99.2% s). against vgs, cpm was 8-fold more potent than caz and 4-fold more potent than piperacillin/tazobactam. regionally, mdr ranged from 9.1% in los angeles to 32.2% in south florida. r to pen, ery, and sxt was the most common mdr phenotype in all regions except baltimore/dc (r to ery and sxt was most common). by specimen source, 15.5% of blood, 21.0% of lr, and 29.0% of ur isolates were mdr with r to pen, ery, and sxt being the most common phenotype regardless of specimen source. for the 30 strains tested, the fluoroquinolone mic ranges were: 0.008-0.03 mg/l (gem), 0.12-0.25 mg/l (gat), 0.06-0.25 mg/l (mxf), 0.5-1 mg/l (lfx). conclusions: mdr among sp is a phenotype that is widely dispersed geographically and is likely to be encountered regardless of the site of infection. fluoroquinolones show activity against most mdr isolates. as the use of fluoroquinolone compounds increases, surveillance to monitor the prevalence of mdr and track the in vitro activity of agents such as gem used for these resistant strains must be continued. background: multi-drug resistant (mdr) s. pneumoniae strains are increasing at an alarming rate worldwide. the therapy of respiratory tract infections due to these strains is challenging with an urgent need for antimicrobials with reliable activity against the mdr strains. comparative activity of oral cephalosporins and parenteral cephalosporins against various pneumococcal mdr phenotypes was analysed in a large, multi-year international collection of clinical strains of s. pneumoniae. methods: s. pneumoniae strains (21,605) collected during 1997-2003 from worldwide participants of the sentry program were tested and interpreted using nccls (m7-a6, m100-s14) guidelines. the antimicrobial agents analysed included penicillin (pen), erythromycin (er), clindamycin (cm), tetracycline (tet) and trimethoprim/sulfamethoxazole (ts); cephalosporins monitored included oral (cefpodoxime, cefuroxime) and parenteral (ceftriaxone, cefepime) agents. results: the rank order of occurrence rates of the various resistance phenotypes were: pen only (32.0%) > pen and er (17.6%) > pen, er and cm (8.6%) > pen, er, cm and tet (7.6%) > pen, er, cm and ts (6.5%) > all five drugs (5.7%). the susceptibility rate of all strains to the orally administered cephalosporins, cefpodoxime (77.6%) and cefuroxime (77.3%), dropped to only 16.1% and 14.5% respectively, for the five-drug mdr phenotype. the parenteral cephalosporins retained excellent activity for all mdr phenotypes, with resistance rates being lower for cefepime than ceftriaxone (cefepime, 1.3-1.9%; ceftriaxone, 3.0-4.4%) or the oral cephalosporins (cefpodoxime, 64.4-74.1%; cefuroxime, 69.3-79.1%) using respiratory infection breakpoints (nccls). conclusions: our in vitro findings confirm that the parenteral cephalosporins, cefepime and ceftriaxone, retain excellent activity against the mdr phenotypes analysed, and remain useful drugs in the armamentarium to treat mdr pneumococcal respiratory tract infections. antibiotic resistance surveillance over a 5-year period in spain: results of the mystic programme results: three icus, two neutropenia units and two general wards provided a total of 4.022 gram-negative and grampositive isolates during the period 1999-2003. the most common species tested were escherichia coli (14.3%), methicillin-susceptible staphylococcus aureus (13.8%) and coagulase-negative staphylococci (13.5%), pseudomonas aeruginosa (12.2%), enterobacter cloacae (7.2%), klebsiella pneumoniae (5.7%) and a.baumannii (5.6%). in general, the carbapenems (mem and ipm) were the most active antimicrobial agents tested against the common organisms (range of %s:100% to 71% and 100% to 67%,respectively). among enterobacteriaceae, 100% of enterobacter spp, citrobacter spp and serratia spp were susceptible to carbapenems. e. coli and k. pneumoniae susceptibility to carbapenems were 100% and >98% respectively. the %s of mem was the same as or higher than ipm against every organism tested. cip resistance in e. coli was around 20%. caz and taz were the least active antimicrobial agents against enterobacter sp (74% and 77% s, respectively) and citrobacter spp (67% and 68% s, respectively). mem and taz were the most active agents against p. aeruginosa (89% and 88%s, respectively). a. baumannii were 61-90% susceptible to carbapenems, but only 6-1% susceptible to cip. in this period, around 100% of methicillinsusceptible staphylococci were susceptible to mem. conclusion: there was no significant decrease in susceptibility to the carbapenems throughout the five-year period. mem and imp appear to remain reliable options for the treatment of serious nosocomial infections. the clinical use of mem did not increase bacterial resistance to this agent in the spanish centres evaluated. surveillance of antimicrobial susceptibility of anaerobes in a belgian university hospital y. glupczynski, h. nizet, c. berhin (yvoir, b) background: antimicrobial resistance is becoming a growing concern among anaerobes involved in human infections. since there are only scarce data on the in vitro susceptibility of anaerobes in our country, we aimed to determine the susceptibility and resistance profiles of anaerobic bacteria isolated in our hospital. table. conclusion: b-lactams-b-lactamases inhibitors, carbapenems, and met remain highly active against anaerobes including the more resistant bfg of organism. resistance in cli actually almost reaches 30% and is observed among almost all different species of anaerobes. mox, as a representative of the newer fluoroquinolones exhibits a broad spectrum and potent activity in vitro against all anaerobes tested and looks promising for the treatment of mixed infections involving anaerobes. pharmacokinetics/pharmacodynamics of quinolones objective: garenoxacin (grn) is a novel des-f(6)-quinolone effective against a broad spectrum of pathogens, including anaerobes. some of the fluoroquinolones have the potential to prolong qtc. to determine the effect of grn on qtc, a retrospective analysis was performed on manually read ecgs from five phase i studies. methods: serial ecgs were collected in 5 randomized, doubleblind, placebo-controlled studies in healthy adult subjects administered oral (po) or intravenous (iv) grn 50-to 1200mg doses (therapeutic dose £600 mg) with dosing duration 1 to 28 days (therapeutic duration £14 days). the qt interval was corrected for heart rate using bazett's (qtcb) and fridericia's (qtcf) formulas, and the effect of grn was assessed by counts of outliers and linear regressions. single-and multiple-dose pharmacokinetics of po and iv grn were derived from plasma concentration versus time data. results: a total of 149 subjects received grn and 55 received placebo. grn plasma exposure (auc and cmax) increased with increasing dose. no subjects experienced a prolonged qtcb interval (>450 msec for males, >470 msec for females). only one subject experienced a prolonged (>60 msec) qtcb change from baseline which occurred on day 7, but not on subsequent days despite continued dosing. the incidence of borderline qtcb (change between 30-60 msec) was comparable between placebo and grn. means for qtcb max, qtcb avg and qtcb at tmax and their changes from baseline for grn were similar to those for placebo, with the exception of qtcb max, qtcb at tmax, and their changes for 600 mg iv grn on day 14. although 1200 mg po and 800 mg iv grn produced higher plasma exposures than 600 mg iv on day 14, the means for the derived qtcb values were comparable to placebo. all 95% confidence intervals for the linear regression slopes of derived (delta)qtcb on cavg(0-12 h) were equal to or less than 0, except for (delta)qtcb at tmax on day 7, indicating that grn had no effect on qtcb. results obtained for qtcf were similar to those for qtcb. conclusions: grn does not appear to have dose-, route of administration-or concentration-dependent effects on qtc interval in healthy subjects. objective: garenoxacin (grn) is a novel des-f(6)-quinolone being developed for a variety of indications because of its efficacy against a broad spectrum of pathogens, including anaerobes. the objective of this study was to assess the tissue or fluid to plasma ratios of grn following a single oral dose. method: an open-label study was conducted in subjects ‡18 years of age with a body mass index £33 kg/m2 undergoing abdominal surgical procedures that would permit the removal of tissue or fluid without increased risk to the subject. a single 600 mg oral dose of grn was administered based on the scheduled operative time. the tissue or fluid (with corresponding plasma sample) was collected 3 to 5 hours post-dose. concentrations of grn in biological fluids and tissues were determined using validated lc/ms/ms assays. safety was assessed by measurement of vital signs, physical examination, and electrocardiographic and clinical laboratory evaluations. adverse event (ae) monitoring was performed from time of consent until discharge from the study. results: thirty-one subjects were enrolled in and completed the study. mean fluid or tissue grn concentrations greater than that found in plasma (mean fluid or tissue to plasma ratio >1) occurred in large and small bowel tissue, gallbladder, and liver. mean fluid or tissue grn concentrations less than that found in plasma occurred in adipose tissue, bone, sinus mucosa, striated muscle, incisional skin, and subcutaneous tissue. mean grn concentrations in bile and lymph node tissue were roughly similar to that found in plasma. tissue and fluid concentrations of grn exceeded the mic90 of most target organisms involved in skin and soft tissue, bone, and intra-abdominal infections and sinusitis ‡2-fold. no treatment-emergent aes or serious aes were considered to be related to grn. conclusion: a 600 mg oral dose of grn penetrates well into most of the tissues and fluids studied, with concentrations exceeding the mic90 of most pathogens causing sinusitis, skin and soft tissue infections, bone infections, and intra-abdominal infections. these results suggest that adequate concentrations of grn can be achieved to treat infections at these sites. penetration of garenoxacin into lung tissues in patients undergoing lung biopsy or resection objective: garenoxacin (grn) is a novel des-f(6)-quinolone effective against a broad spectrum of pathogens, including those commonly found in the respiratory tract (rt). this study was conducted to determine the penetration of grn into lung parenchyma (lp) and bronchial mucosa (bm) following a single 600 mg oral dose. penetration of grn into bone was also assessed. this was an open-label, nonrandomized study in subjects undergoing an invasive procedure to the lung (other than percutaneous lung biopsy) which facilitated the removal of macroscopically normal healthy lung tissue without exposing the subject to undue risk. penetrance of grn into bone was determined when removal of rib bone was part of the normal surgical procedure. a single 600 mg oral grn dose was administered based on the scheduled operative time. lp and, if possible, bm and/or bone samples and corresponding plasma samples were removed at 2 to 4, 4 to 6, 10 to 12, or 20 to 24 h post-dose. concentrations of grn in these samples were determined using validated lc/ms/ms assays. safety was assessed based on the occurrence of adverse events (aes) and changes in physical examinations, vital signs, clinical laboratory results, and electrocardiographic results. results: twenty-seven subjects were enrolled; samples were taken from a minimum of 6 patients at each time interval. mean grn concentration in lp increased between the 2-4 and 4-6 h post-dose, suggesting rapid penetration into lp, then declined similar to the decrease seen in plasma concentration. concentrations of grn in lp exceeded the mic90 of organisms associated with rt infections by 62-to 466-fold over a 24 h period. grn concentrations in bm over a 24 h period exceeded the mic90 of respiratory pathogens 51-to 380-fold. concentrations of grn in bone exceeded the mic90 of the organisms associated with bone infections (except pseudomonas aeruginosa, mrsa, fusobacterium species, and enterobacter species) by 6-to 101-fold over a 12 h period. across the 4 time intervals, mean ratios of tissue to plasma grn concentration in lp, bm, and bone reached 2.80, 0.99, and 0.56, respectively, suggesting adequate penetration. no grn-related aes were reported, indicating that a single 600 mg oral grn dose was well tolerated in subjects undergoing invasive procedures. conclusions: grn penetrates rapidly into lp, bm, and bone tissue, producing sustained concentrations that predict adequate coverage of grn-susceptible pathogens at these sites. bioavailability and safety in healthy volunteers unaltered when crushed garenoxacin tablets are administered via a nasogastric tube with or without enteral feeding r. noveck, r. vargas, g. krishna, d. grasela (new orleans, kenilworth, princeton, usa) objective: the purpose of this trial was to assess, in healthy volunteers, the bioavailability and safety of single doses of the novel des-f(6)-quinolone garenoxacin (grn) administered as crushed tablets with and without concomitant enteral feeding compared with intact tablets. methods: in this randomized, crossover, single-dose study, healthy adult volunteers (aged 18 to 45 y) received grn 600 mg (one 200 mg and one 400 mg tablet) orally in 3 regimens: a) intact tablets; b) crushed tablets suspended in water and delivered via nasogastric (ng) tube; c) regimen b plus concomitant enteral feeding (osmolite, 600 ml at 100 ml/hr). subjects were randomized to receive all 3 regimens in 1 of 6 crossover sequences (abc, acb, bac, bca, cab, cba) separated by 7-day washout intervals. for each treatment, subjects entered the facility 1 day before and were confined for 72 hours after drug administration. subjects were discharged from the study after final assessments on day 4 of the third treatment. post-screening assessments included vital signs; plasma drug levels for pk analysis; and physical, laboratory, and ecg examinations for drug safety. results: 18 male subjects were enrolled (4 white, 14 black; mean age, 30 y). pk analysis for grn administered as crushed tablets with and without concomitant enteral feeding vs intact tablets showed no differences in the adjusted geometric means for cmax (8.5 and 8.3 lg/ml vs 8.3 lg/ml) or auc(inf) (97.2 and 93.4 lg*hr/ml vs 103.3 lg*hr/ml). the 90% ci for logtransformed cmax and auc(inf) for b and c compared to a were contained within the protocol-specified ôno effectsõ limit of 70% to 143%. mean grn half-life was similar among the 3 groups (mean range of 12-13 h). tmax was 1 hour following administration of intact tablets compared with 0.5 hour in the other 2 groups. a single oral 600 mg dose of grn was well tolerated whether administered as crushed tablets suspended in water and delivered via ng tube with or without enteral feeding, or as intact tablets. three adverse events (aes) were reported; 1 (nausea) was deemed probably related to study drug. there were no serious aes, meaningful changes on safety examinations, or discontinuations due to aes. conclusions: the bioavailability of grn was similar for crushed versus intact tablets, regardless of whether an enteral feeding was given with crushed tablets. these results show that grn may be administered as crushed tablets with or without concomitant feeding. garenoxacin pharmacokinetics in subjects with severe renal impairment objective: garenoxacin (grn) is a novel des-f(6)-quinolone effective against a broad spectrum of pathogens. the absolute bioavailability of grn after oral intake is 92% and approximately 40% of grn is excreted unchanged in the urine. the objective of this study was to evaluate the pharmacokinetics of grn in subjects with severe renal impairment. methods: this non-randomized, open-label study enrolled patients into 1 of 4 groups: healthy control subjects (hc) with normal renal function (clcr > 80 ml/min); subjects with severe renal impairment (sri) (clcr < 30 ml/min) not requiring dialysis; subjects with sri receiving hemodialysis (hd); and subjects with sri receiving continuous ambulatory peritoneal dialysis (capd). a single 600 mg oral dose of grn was administered on day 1 for all groups except hd patients. hd patients received a single 600 mg oral grn dose both before (hd1) and after (hd2) hd, with dosings separated by a 14-day washout. blood, urine, and dialysate samples were collected up to 144 h post-dose and concentrations of grn were determined using validated lc/ms/ms assays. safety was assessed by physical examination, vital signs, and electrocardiographic and laboratory evaluations. results: twenty-five subjects received grn (hc, n = 6; sri, n = 6; hd1, n = 7; capd, n = 6). six subjects in hd2 received grn. mean grn exposure [auc(i)] was similar in hc and hd1 but was 51%, 15%, and 21% higher in sri, hd2 and capd, respectively, than in hc. however, many of the individual values were within the range observed for hc, and these average increases in auc(i) did not exceed values previously shown to be well tolerated. decreases in cmax were observed in sri, hd1, capd, and hd2, but were not considered clinically relevant. approximately 11%, 1.5%, and 3% of grn dose was removed in the dialysate for hd1, hd2, and capd, respectively. a total of 34 treatment-emergent adverse events (aes) were reported, all mild or moderate in severity; 5 were considered to be probably or possibly related to grn. one treatment-emergent but not treatment-related serious ae was reported. conclusions: a single dose of 600 mg grn was well tolerated in patients with sri, including those requiring hd and capd. there was no clinically significant increase of grn exposure in patients with sri based on the broad therapeutic index of grn. therefore, no grn dosage adjustment is recommended in subjects with sri. the effect of omeprazole on the bioavailability and safety of garenoxacin in healthy volunteers j. kisicki, g. krishna, s. olsen, d. grasela (lincoln, kenilworth, princeton, usa) objective: the novel des-f(6)-quinolone garenoxacin (grn) is more soluble in acidic conditions than at neutral ph. therefore, this study was designed to determine if omeprazole affects the bioavailability of grn. methods: this non-randomized, open-label pharmacokinetic study was conducted in healthy adult subjects. on day 1, the single-dose pharmacokinetics of 600 mg of oral grn (one 200 and one 400 mg tablet) were determined in fasting subjects, with serial blood samples obtained through 72 hours post-dose. omeprazole 40 mg was administered once daily on days 4 to 7 to achieve steady-state inhibition of gastric acid secretion. on day 8, single doses of grn and omeprazole were administered concomitantly. omeprazole treatment was continued on days 9 and 10 throughout the period of pharmacokinetic blood sampling. study assessments included vital signs and physical, laboratory, and electrocardiographic examinations for safety. the gm cmax value for grn and grn/omeprazole co-administration was 9.6 and 9.3 mcg/ml, respectively, with 90% ci of 90% to 104%. half-life (mean range 12.9 to 14 h) and tmax (median range 1.5 to 1.8 h) were similar after administration of grn either alone or concomitantly with omeprazole. concomitant administration of garenoxacin and omeprazole was well tolerated. nine of 14 subjects (64%) experienced a total of 33 adverse events (aes). the majority of aes were mild, and only 12 were deemed possibly or probably related to the study drug. the most frequently cited aes, headache, nausea and abdominal pain, were mild to moderate in severity. two subjects withdrew from the study; neither discontinuation was due to aes. conclusions: the concomitant administration of omeprazole had no effect on garenoxacin bioavailability. these findings indicate that garenoxacin can be administered with omeprazole or other agents that affect gastric ph to a similar or lesser extent. the effect of maalox on bioavailability and safety of garenoxacin in healthy volunteers j. kisicki, g. krishna, s. olsen, d. grasela (lincoln, kenilworth, princeton, usa) objective: garenoxacin (grn) is a novel broad-spectrum des-f(6)-quinolone antibiotic. quinolones are known to chelate with cations such as aluminum, impairing antibiotic absorption. this study was therefore designed to assess the effect of a 20-ml dose of maaloxò (containing 900 mg aluminum hydroxide and 800 mg magnesium hydroxide) on the pharmacokinetics of grn when administered concomitantly with, 2 and 4 hours before, and 2 and 4 hours after, maalox. methods: this was a randomized, open-label, 6-treatment, control-balanced, residual effects design pharmacokinetic study. the 6 oral treatments consisted of 600 mg grn (one 200 and one 400 mg grn tablet) given alone, with concomitant maalox, 2 h before maalox, 4 h before maalox, 2 h after maalox, and 4 h after maalox. each healthy adult subject received 3 of the above treatments, with a 7-day washout period between treatments. in each treatment, serial blood samples for pharmacokinetic analysis were collected before and up to 72 h after grn dosing. study assessments included vital signs and physical, laboratory, and electrocardiographic examinations for drug safety. results: twenty subjects (12 male, 8 females; mean age, 27 years) were enrolled. exposure to grn [auc(inf)] was reduced by 58% when coadministered with maalox. exposure to grn was also reduced when administered 2 or 4 hours after maalox, by 22% and 16%, respectively. in contrast, when administered 4 hours before maalox, grn exposure was not affected. when grn was administered 2 hours before maalox, a small reduction (12%) in grn exposure was observed that is unlikely to be clinically relevant. half-life (mean range 11.5 to 14.2 hours) and tmax (median range 1.5 to 2 hours) were similar across treatment groups. a single oral 600 mg dose of grn was well tolerated. mild adverse events were reported by 2 subjects; 1 subject discontinued due to mild abdominal pain and blood in the stool. conclusions: maalox does not affect grn bioavailability when grn is administered at least 2 h prior to maalox. however, a reduction in grn exposure is observed when grn is administered concomitantly or up to 4 h after maalox. therefore, grn can be administered 2 h before or 4 h after administration of maalox or other products containing a high content of cations, particularly aluminum. objectives: it has been demonstrated that fluoroquinolone treatment of humans and animals can rapidly select for bacteria with reduced susceptibility to fluoroquinolone antibiotics. in recent years, there has been considerably interest in the concept of the mutant prevention concentration (mpc). the mpc has been defined as that concentration of antibiotic at which no mutants are selected from 10,000,000,000 organisms. in previous studies mpcs of ciprofloxacin and enrofloxacin were determined in vitro against salmonella enterica serovar typhimurium dt 104. here the use of a chick model to investigate the mpc of enrofloxacin in vivo is reported. methods: chicks were experimentally infected with s. typhimurium and treated with baytril (enrofloxacin) at 50 ppm/10 mg/kg for 5 days (recommended therapeutic dose) or 2 days or 125 ppm/25 mg/kg for 2 days or 250 ppm/50 mg/kg for 1 day. chicks received the dose in the drinking water (ppm) or by oral gavage (mg/kg). during and 24 hours after dosing, birds were killed and tissue samples (caecal contents, liver and sera) were taken and the levels of enrofloxacin and ciprofloxacin were determined in these tissues by hplc or bio-assay. caecal contents were also monitored for the presence of salmonella during dosing and for up to 4 weeks after dosing. selection of resistance was monitored by replica plating of colonies to media clinical microbiology and infection, volume 11, supplement 2, 2005 containing 4x the ciprofloxacin and nalidixic acid mic of the strain before treatment.results: the only conditions where the antibiotic treatments did not select for reduced susceptibility (e.g. 4x mic of parent strains) were in birds that received the antibiotic at 2.5x the dose for 2 days by oral gavage or 4x the dose for 1 day in the water. this oral gavage treatment also significantly reduced the counts of salmonella compared to all other oral gavage treatments. for the oral gavage study, concentrations of fluoroquinolones in caecal contents were above the mpc for at least 6 hours after dosing for all treatment regimes. conclusion: it was of interest to note that whilst the fluoroquinlone antibiotics are regarded as concentration rather time dependent antibiotics, the 2 day at 2.5x the dose was more effective at reducing the counts of salmonella in both experiments than the 1 day at 5x the dose. these results would suggest that length of treatment as well as dose and route of administration, is critical in minimising the selection of fluoroquinolone resistance in vivo. effect of levofloxacin coadministration on the international normalised ratios during acenocumarol therapy j. de otero, m. payeras, c. carratala, a. artigues, m. sanz, p. nadal (manacor, e) levofloxacin (l) showed no effect on warfarin pharmacokinetics in healthy volunteers but several recent descriptions reveal elevated international normalised ratios (inr) in patients receiving concurrent l and warfarin. to our knowledge no cases of l-acenocumarol (a) interaction have been reported. objectives: to report 6 cases of hypoprothrombotic response resulting from addition of l therapy to chronic a therapy. methods: a 9-month retrospective analysis of adult patients receiving a who were admitted to our ward under l prescription on the basis of clinical diagnosis and judgement. descriptive statistical measurements were performed. results: six patients, 74-89 years old (median 81.5), were included. four were men (66.6%). they suffered from a median of 4 (range 2-6) concurrent chronic medical problems (including coronary heart disease, atrial fibrillation, hypertension, obstructive pulmonary disease, renal insufficiency, cardiomyopathy and heart failure) and took a median of 7 (range 4-10) different kind of drugs (including digoxin, furosemide, antihypertensives and inhaled bronchodilators). types of infection for which the patients were treated with l included acute bronchitis (4 cases) and pneumonia (2 cases objectives: cip is substrate for an mrp efflux pump in j774 mouse macrophages (michot et al, aac 48:2673-82), which reduces its cellular accumulation. we have recently shown that cip-ôresistantõ macrophages can be selected upon chronical exposure of these cells to cip, in which cip accumulation becomes negligible because of an increased activity and/or expression of the efflux transporter (icaac 2004 (icaac , abstract a-1304 . we have now examined whether this reduced accumulation affects cip intracellular activity using a model of intracellular infection by l. monocytogenes (l.m.) and comparing resistant cells to the wild type parent cell line. methods: infection was carried out using a bacteria: macrophage ratio of 7:1, according to the procedure described in seral et al (jac 51:1167-73) . cfu/mg cell protein was determined after 5 h exposure to increasing concentrations of cip ± 10 mm probenecid (inhibitor of mrp transporters). cip cellular concentration was measured by fluorimetry (aac 48:2673-82) . results: in wild cells, an extracellular concentration corresponding to 3 x the mic was sufficient to obtain a static effect, and this value rose to 30 x in cip-resistant cells. this value was decreased to 1 x and 1.5 x the mic, for wild and cip-resistant cells respectively, in the presence of probenecid. in cells incubated with 20 mg/l cip (10 x mic), cip accumulation was 4.8 ± 0.3 and 0.3 ± 0.1 in wild and cip resistant cells, but increased to 21.3 ± 0.8 and 10.6 ± 0.5 in the presence of probenecid. conclusions: increase in expression and/or activity of the cip efflux transporter causes a reduction of the antibiotic efficacy against intracellular infection, which can be reversed upon inhibition of the efflux transporter. objectives: quinolones can cause tendinitis and tendon ruptures. retrospective studies and case reports reveal that an additional therapy with glucocorticoids increases the risk of quinolone-induced tendon disorders. methods: we investigated possible combination effects of quinolones and glucocorticoids in an in vitro model with human tendon cells. tenocytes were cultured for 1, 2, 3 and 4 days as monolayers and incubated with (a) ciprofloxacin or levofloxacin (3 mg/l), (b) 0.1 nm dexamethasone, and (c) ciprofloxacin or levofloxacin (3 mg/l) plus 0.1 nm dexamethasone. immunoblotting was used to quantify changes in the amount of beta1-integrin, activated src homology collagen (shc) and of activated caspase-3 (casp-3). furthermore, ultrastructural alterations were analysed by electron microscopy. results: at 3 mg/l, ciprofloxacin caused a significant decrease of the amount of beta1-integrin from day 3 onwards, while no effect was seen with 3 mg levofloxacin/l medium or 0.1 nm dexamethasone compared to the untreated control. the combination of both quinolones (3 mg/l) with 0.1 nm dexamethasone resulted in an earlier onset of the beta1-integrin reduction: for ciprofloxacin + dexamethasone from the first day of incubation, for levofloxacin + dexamethasone from day 3 onwards. quinolone-induced changes in signalling proteins, such as activated shc, are not fortified by dexamethasone at the concentration tested. interestingly, the time and concentration dependent increase of the apoptosis marker activated casp-3 was intensified with dexamethasone. the results of immunoblotting with respect to the induction of apoptosis were confirmed by electron microscopy. conclusions: our results show that quinolone-induced changes in the amount of the beta1-integrin as well as of the apoptosis marker activated casp-3 are enhanced by dexamethasone in vitro. the addition of the glucocorticoid caused an earlier onset of the quinolone-induced changes. our results support the clinical observations that glucocorticoids are an additional risk factor for quinolone-induced tendopathies. pharmacokinetics and pharmacodynamics of a novel extended release formulation of ciprofloxacin as compared to levofloxacin against extended spectrum beta-lactamase producing enterobacteriaceae s. schubert, h. stass, u. ullmann, a. dalhoff (kiel, wuppertal, d) background: an extended release formulation of ciprofloxacin (cip xr, 1000 mg qd, p.o.) has been developed, delivering peak concentrations which are 40% to 50% higher than following the administration of the conventional formulation (cip ir, 500 mg bid, p.o.) while the areas under the curve (auc) were comparable. objectives: first, cip is used for the treatment of infections due to enterobacteriaceae, frequently being esbl producers. thus, the pk/pd characteristics of cip xr vs. ir against esbl strains was examined. second, cip is used in urinary tract infections (utis). thus, the pk/pd profiles of cip xr vs. ir in serum and urine were studied. third, the pk/pd characteristics in either bhi or artificial urine were compared. methods: genotypically characterised esbl producing e. coli (ec) and k. pneumoniae (kpn) and their wild type counterparts were exposed to the geometric mean plasma and urine concentration profiles following cip 1000 mg xr qd, 500 mg ir bid and lev (500 mg qd). the cip and lev urine concentrations were fitted from phase i study data by compartmental modeling using topfit 2.0. bacteria were cultivated in a modified grasso model at 37°c in brain-heart infusion broth as well as artificial urine acc. to griffith et al.; viable counts were quantitated at 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 24 h. the areas under the bacterial kill curves normalised to the inoculum were calculated. results: first, irrespective of the pk profiles simulated, the wt were eliminated rapidly. the esbl producing ec and kpn were moderately to negligibly affected upon simulation of serum pk profiles of both cip and lev. second, simulation of urine concentrations by using artificial urine ir and xr resulted in an elimination of the esbl producers from the test system. lev eliminated the esbl producing k. pneumoniae but not the e.coli strain, which regrew. cip xr killed the esbl kpn strain more rapidly than cip ir or lev. third, pk/pd surrogates derived from studies in conventional media or artificial urine are significantly different. conclusions: this model and the use of artificial urine predicts a more rapid and pronounced effect of cip xr as compared to cip ir or lev. objectives: the antibacterial spectrum of moxifloxacin includes all the major respiratory pathogens and its pharmacokinetics demonstrates high peak concentrations in plasma as well as at respiratory sites. nevertheless, the tonsillar tissue concentrations have never been investigated. in the present study we determined the moxifloxacin concentrations in plasma and tonsils after the administration of three doses of 400 mg to adult patients with chronic or recurrent tonsillitis undergoing tonsillectomy. methods: this was an uncontrolled, open-label, randomised, parallel group study including 35 patients randomly assigned to 5 groups of 7 patients each, depending on the time between the last dose of moxifloxacin and that of plasma and tissue sampling (2, 3, 6, 12 and 24 h). moxifloxacin was given orally od for 3 days. moxifloxacin concentrations were measured by a validated hplc assay and fluorescence detection. each sample was analysed twice and the mean value obtained used for statistical analysis. pharmacokinetic data were analysed by presenting descriptive statistics of moxifloxacin levels in plasma and tonsillar tissue. results: cmax occurred at 3h in plasma (mean value 3.20 mg/ l) and in tonsillar tissue (mean 8.96 mg/l). tonsillar tissue/ plasma ratios (mean values) were constantly >2 mg/l at any collecting time, ranging between 2.37 mg/l (after 2 h) and 2.93 mg/l (after 24 h), which indicates a prolonged maintenance of moxifloxacin level in tonsillar tissue compared to plasma. variability among pts was present at 6 h, the tonsillar tissue / plasma ratio ranging between 0.8 and 3.4 mg/l. conclusions: moxifloxacin achieves a good penetration in tonsillar tissue, which favourably compares with that reported for other fluoroquinolones. the moxifloxacin concentrations that we observed exceed the mics of the usual respiratory tract pathogens. objectives: acute necrotizing pancreatitis is still related to a high mortality rate, based on local infectious complications, particularly due to bacterial infections of the necrotic pancreatic and parapancreatic areas. limited penetration of antimicrobial drugs in these areas/compartments is considered to be a major cause of failure of therapy of severe local pancreatic infections. however, fluoroquinolones (e.g. ciprofloxacin, levofloxacin) have been shown to penetrate sufficiently into pancreatic tissue. on that score, the value of new quinolones such as moxifloxacin (mxf) has not been investigated yet. using a rat model of acute necrotizing pancreatitis mxf has been demonstrated to penetrate rapidly and efficiently into pancreatic tissue, also into the inflamed and necrotic pancreas. methods: addressing the penetration capability of mxf following intravenous (iv) or oral (po) administration with respect to the human pancreas, a prospective clinical trial was designed using a single iv (20 patients) or po dose (40 patients) of 400 mg mxf for antimicrobial prophylaxis in patients undergoing pancreas resection. samples were taken from blood and from resection area of pancreatic tissue at two time points after application (1st sample at the beginning, 2nd sample at the end of resection). concentrations of mxf were determined by hplc/uv using ofloxacin as internal standard. results: mean plasma concentrations of mxf iv and po at first sampling time (3 -3.7 h) were 1.8 ± 0.5 and 1.2 ± 0.6 lg/ml, respectively. at the end of resection (4.3-5.3 h) 1.5 ± 0.4 and 1.0 ± 0.5 lg/ml were measured. corresponding mean concentrations of mxf in pancreatic tissue were 2 to 3times higher (3.1 ± 0.9 and 2.7 ± 1.4 lg/g 1st sample; 3.6 ± 1.5 and 3.1 ± 1.8 lg/g 2nd sample). the mxf concentrations in pancreatic tissue exceeded the mic90 s of the relevant pathogens encompassed by mxf (e.g. e. coli 0.008-0.06 lg/ml, klebsiella spp. 0.13 lg/ml, and s. aureus 0.06-0.12 lg/ml) for at least five hours at the dosing interval. conclusion: mxf has been demonstrated to penetrate efficiently into human pancreatic tissue following iv as well as po administration. in this study mxf concentrations after po administration were found to be slightly lower than those observed in healthy subjects probably due to diminished or delayed intestinal absorption prior and during surgery. objective: in clinical practice on icu wards moxifloxacin (mfx) may be occasionally administered through a nasogastric (ng) feeding tube. in absence of an oral liquid formulation and since the divalent cations contained in enteral feeds may potentially impair absorption of mfx given via this way, we wanted to study the effect of concomitant enteral feeding on the pharmacokinetics and tolerability of mfx passed as a crushed tablet through a ng tube, firstly in healthy volunteers. methods: in a randomized 3-way crossover study the oral bioavailability of mfx was investigated in 12 healthy volunteers (9 f, 3 m). each subject received separately an intact 400 mg mfx tablet (a), a crushed tablet as a suspension through an ng tube with water (b), and while receiving enteral feeding (c). the concentrations of mfx in serum were measured by a validated hplc. auc and cmax were analysed statistically assuming log normally distributed data using anova. results: all mfx treatments were well tolerated. the auc was slightly, but not significantly decreased in treatments b and c [geo means ( . thus, rate of absorption was not affected by ng tube administration. this route of ingestion seems to be associated only with a slight loss of bioavailabilityindependent of the carrier medium used (water vs. enteral feed). no clinically relevant interaction with the multivalent cations contained in the enteral feed was observed indicating that mfx and enteral nutrition can be administered concomitantly. conclusion: there was no clinically relevant effect of enteral feeding on the pharmacokinetics of oral mfx in healthy volunteers. this results has to be evaluated in patients, especially those from icu, who are characterised by severe infectious and/or concomitant diseases, which might influence absorption of mfx. in the healthy volunteer studies, compliance with dosing regimens was strictly enforced and participants had no significant underlying diseases or concomitant medications that could confound interpretation of safety data. therefore, for the purpose of this analysis, the healthy volunteers served as a control group for the patient population. in both groups, most subjects received pos 800 mg/d in divided doses. results: treatment-related aes (traes) occurred in 44% (196/ 449) of healthy volunteers; the most common were headache (17%), dry mouth (9%), and dizziness (6%). in patients, many aes were consistent with underlying diseases. traes occurred in 38% of patients (164/428); the most common were nausea (8%), vomiting (6%), headache (5%), abdominal pain (4%), and diarrhoea (4%). notably, gastrointestinal (gi) traes occurred in 18% of healthy volunteers and 19% of patients. treatmentrelated abnormal liver function test results were observed in 2% of healthy volunteers and up to 3% of patients. there were no clinically significant differences in mean qtc interval change from baseline in either population. serious aes (sae) considered possibly or probably related to pos occurred in 35 (8%) patients and 1 healthy volunteer. the most common saes in patients were altered drug level, increased hepatic enzymes, nausea, rash, and vomiting (1% each). no significant trends related to age, sex, or race were observed in either group. additionally, no unique traes were identified in patients during long-term exposure (>6 months) compared with those identified during shorter-duration therapy. conclusion: the safety profile of pos in patients was similar to that observed in a controlled population of healthy volunteers and is likely indicative of what will be observed in the clinical setting. headache and gi events (nausea, vomiting, abdominal pain, diarrhoea) were the most common traes observed in patients. given its favorable safety profile, pos should provide an additional treatment option for severely ill patients with ifis. objective: we evaluated the drug interaction (di) potential of posaconazole (pos), an extended-spectrum triazole antifungal agent, in 7 phase 1 studies. pos is an inhibitor of cytochrome p450 (cyp) 3a4 activity, but it does not affect the activity of other cyp enzymes. these studies were conducted to evaluate potential changes in the pharmacokinetics of pos and the coadministered medication, when given in combination. methods: seven open-label di studies were conducted in which subjects received pos (200-800 mg) for 8 to 14 consecutive days, alone or in combination with single or multiple doses of cimetidine, cyclosporine, glipizide, phenytoin, rifabutin, tacrolimus, or antiretroviral medications (zidovudine, lamivudine, ritonavir, and indinavir). in the cyclosporine and antiretroviral studies, patients received established doses (ed) of these drugs, and plasma concentrations were presumed to be at steady state (ss) before pos administration. bioavailability, based on logtransformed auc values, was expressed as the ratio of the combination treatment to pos or concomitant drug given alone. the effects of coadministration on the auc values of pos and each concomitant medication are summarized in the table. conclusion: no dose adjustments are required for glipizide, zidovudine, lamivudine, indinavir, or ritonavir when coadministered with pos, as the small differences in exposure are not considered clinically significant. however, when pos was given with glipizide, glucose concentrations decreased in some healthy volunteers more so than after glipizide administered alone. monitoring of cyclosporine and tacrolimus blood levels is warranted with pos coadministration, and dose adjustments of cyclosporine and tacrolimus should be made accordingly. concomitant use of pos with rifabutin, phenytoin, or cimetidine should be avoided unless the benefits outweigh the risks, due to the decrease in pos concentrations. objectives: the pharmacokinetics (pk) of many medications may be altered in the elderly population. because alterations in pk can potentially affect clinical outcomes, the pk parameters of posaconazole (pos), an extended-spectrum investigational triazole antifungal agent, were examined in healthy elderly persons ( ‡65 years), and the clinical implications of age-related pk differences were evaluated. methods: we conducted a meta-analysis of pos steady-state pk data from 5 clinical studies in healthy volunteers. pk data from elderly healthy volunteers were compared with those of elderly patients with invasive fungal infections ( objective: to develop a bioassay for measurement of csp blood levels using the hypersusceptible c. albicans cell wall sensor mutant delta-mid2. methods: the c. albicans mutant delta-mid2 (mic for csp: 0.008 mg/l) was constructed by targeted deletion of the gene encoding for a cell wall sensor putatively involved in the maintenance of cell wall integrity. the bioassay was validated according to international guidelines (shah et al, 2000; fda, 2001) . standard curve included 8 csp concentrations over the range 0.4 to 25 mg/l. four quality controls were used (0.5, 2, 8, 16 mg/l) to study: i) stability of csp concentrations over time at different pre-analytical storage conditions, ii) accuracy (measured/nominal value x 100, validation range 85-115%), iii) precision (coefficient of variation: sd/mean of measured values x 100, validation range 15%). the validation procedure included 6 intra-run and 6 inter-run measurements. results: the limit of detection and quantification was 0.2 and 0.4 mg/l (corresponding to 5 and 10 ng of csp in a sample volume of 25 ml), respectively. reproducible standard curves were obtained over the clinically relevant concentration range (0.4 to 25 mg/l) (r ‡ 0.99). analytical time was 16 h. csp concentrations with a deviation from nominal values < 15% were measured: i) over 4 days in plasma, and 3 days in whole blood, at 4 c, ii) over 3 and 2 days, respectively, at 21 c, iii) over 6 months in plasma at -80 c, iv) after 5 freeze/thaw cycles. intra-and inter-run validation with the four quality controls (mean % value, ± sd) are shown in objective: to identify the pharmacokinetic and pharmacodynamic (pk-pd) parameters of antifungal therapy predictive for intra-and inter-run bioassay validation i intra (n = 6) inter (n = 6) accuracy 93.1 ± 1.9 96.0 ± 2.5 precision 1.3 ± 1.7 5.8 ± 3.0 invasive fungal infections in patients admitted to an intensive care unit (icu). the medical records of all ptt admitted to a tertiary hospital icu in a 6 month period in 2002 were retrospectively reviewed, and the antifungal therapies were recorded and correlated to the findings of fungi from the ptt. the pk-pdparameters for each pt was estimated from the treatment given and the mic of the isolated candida, i.e. auc/mic-ratio (in h) for the azoles, and peak/mic ratio for the free fraction of amphotericinb. candida isolated from various infectious foci, or from the surveillance samples taken routinely three times a week, were susceptibility tested using e-tests for fluconazole, itraconazole and amphotericinb. results: in total 120 ptt were included.among the 14 ptt with invasive fungal infections, 3 ptt had bloodstream infections at admission. one of the remaining 11 (9%) received systemic prophylactic therapy before getting colonized, and 3 ptt (27%) were prophylactic treated when colonized, 3 ptt with fluconazole (auc/mic: 66-1600), and one pt with liposomal ampho-tericinb (peak/mic = 3 objective: to predict the pharmacokinetic properties of bal4815 a new azole antifungal, in humans.methods: bal8557 (the water-soluble pro-drug of bal4815) was incubated at 10 lg/ml in pooled heparinized rat, cynomolgus monkey and human plasma for 5 minutes at 37°c. bal4815 was incubated at 1,10 and 100 lg/ml equivalent bal4815 with rat, cynomolgus monkey and human liver microsomes for 120 min at 37°c in the presence of 1 mg/ml proteins. the extent of plasma protein binding of bal4815 in rat, cynomolgus and human plasma was measured using the red blood cell partitioning method and 14c-bal4815. rats received a single oral dose of 10 mg/kg equivalent bal4815 and a single intravenous dose of 5 mg/kg equivalent bal4815 as bal8557. cynomolgus monkeys received single oral and intravenous doses of 3 mg/kg as bal8557. serial blood samples were obtained. plasma concentrations of bal4815 and bal8557 were quantified using an hplc/fluorescence method or a lc-ms/ms assay. results: in plasma, bal8557 is converted within minutes to bal4815 in all species investigated. in the presence of rat, cynomolgus monkey and human liver microsomes, less than 10% of bal4815 is metabolized during a 120 min period. bal4815 is bound to plasma proteins with a free fraction of 3.5%, 3.6% and 2.2% in rat, cynomolgus monkey and human, respectively. after intravenous administration, the pro-drug bal8557 is converted to bal4815 within minutes. bal4815 has a large volume of distribution with values greater than the total body water: 15 l/kg and 5 l/kg in rat and cynomolgus monkey, respectively. bal4815 is slowly eliminated with halflives of 5 h in rat and 10 h in cynomolgus monkey. after oral administration of the pro-drug, bal4815 reaches cmax-values between 2.0 to 3.5 hours. we observed high bioavailability of bal4815: 62% in rat and 87% in cynomolgus monkey. conclusions: based on these in vitro results and animal data, we predict that the pro-drug bal4815 is very rapidly converted to bal4815 in man. due to the protein binding and in vitro metabolic stability, bal4815 is expected to behave as a low intrinsic clearance drug in human (clearance < 10 % of liver blood flow). this combined with a large volume of distribution explains the long half-life > 30 hours observed in man. animal data suggest a good oral bioavailability as is confirmed in humans. pharmacokinetics and fungicidal activity of aminocandin (hmr3270), a novel echinocandin in healthy volunteers b. sandage, g. cooper, n. najarian, j. lowther (lexington, usa; romainville, f) objective: aminocandin (ac), an echinocandin, is being developed for treatment of systemic fungal infections. a study was carried out to determine the pharmacokinetic (pk) and fungicidal activity of ac in plasma samples against 2 strains of c. albicans and 1 strain of a. fumigatus following single iv doses. methods: single iv doses of 75-300 mg of ac were administered to 12 healthy, male volunteers in a single-blind, randomized, placebo-controlled trial. individual plasma samples were collected from active and placebo (pbo) treated volunteers, serially diluted in 25% control plasma/75% growth medium, and the fungicidal titre determined on 2 c. albicans strains and inhibitory titre against 1 a. fumigatus strain. titres and pk results are the mean values (n = 3). due to the dilution factor in test medium, the limit of detection was a titre of 4 the germicidal effect of ceiling-and wall mounted ultraviolet c (uvc) light at 254 nm in isolation units with negative pressure ()45 pascal) was examined and compared with disinfection with chloramine during end-disinfection after patient stay. microbial samples were taken from surfaces before and after disinfection with uvc (33-47 min) and chloramine (5%, 1 h exposure) using standard contact plates (20cm 2 ). the uvc-distribution in the isolation units was monitored at 165 positions. the output on the floor varied between 0.08 and 3.2 w/m 2 , with an average ( ± sd) of 2.2 ± 0.5 w/m 2 in the patient room, 2.0 ± 0.7 w/m 2 in the sluice and 1.4 ± 0.5 w/m 2 in the bath/ decontamination room. on other places, the values varied from 0.08 to 6.82 w/ m 2 . the units were uvc-disinfected for 33-47 min, corresponding to doses ranging from 160 j/m 2 in shadowed area to 19 230 j/m 2 at the highest exposed site. according to published uvc-dosimetry, the survival of bacteria and bacterial spores are reduced by 90% with doses ranging from 4-120 j/m 2 and 100-365 j/m 2 , respectively. thus, uvc doses used in this study should be high enough to inactivate most bacterial organisms, including spores, even on surfaces not directly exposed to uvc. uvc-disinfection significantly reduced the bacteria on surfaces directly or indirectly exposed to uvc to a very low number (from ca 30 to 1-2 cfu/plate), as did 5% chloramine disinfection (fom ca 30 to 1-2 cfu per plate) alone; p < 0.001,and p < 0.001, respectively. since cleaning before disinfection may be a risk for the staff in isolation units, disinfection with uvc-or chemicals should always be performed first. the presence of completely shadowed areas in the isolation unit (the bed rail, lockers, matresses etc.) still needs disinfection by chemicals before cleaning. therefore, uvc may not be used alone, but is a good additive to chemical disinfection, to lower the biological burden of infectious agents in isolation units for high risk infectious patients. disinfection of medical equipment, using dry mist of hydrogen peroxide objective: to compare the antimicrobial efficacy before and after disinfection of pine core wood surfaces with surfaces made of polyethylene and synthetic laminate against organisms typically causing hospital-acquired infections. methods: s. aureus (mrsa), e. faecium, e. coli, p. aeruginosa. c.albicans, m. terrae and p camembertii were uses as test organisms. after inoculation of the test organisms on surfaces made of pine core wood, polypropylene and synthetic laminate, samples were collected on rodac plates before and after disinfection with commonly used hospital germicides (alcohol, aldehyde, glucoprotamine and a quaternary ammonium compound). bacterial colonies were counted after 0 min, 30min, 1 h, results: substantially lower colony counts were measured on pine core wood surfaces before application of the disinfectant compared with counts on polypropylene and synthetic laminate surfaces. after bacterial contamination of the surfaces, significant colony counts were not observed on the surfaces after disinfection with alcohol, aldehyde and glucoprotamine. however, after disinfection with a quaternary ammonium compound, significant elevations in colony counts were found on the pine core wood surfaces in comparison to the polypropylene and synthetic laminate surfaces. discussion: the poor efficacy of quaternary ammonium compound on wooden material might be explained by the interaction between wood and the anionic polyphenols and cationic surfactants contained in the disinfectant. the study findings corroborate the antimicrobial effect of pine core wood against organisms typically causing nosocomial infections. with the exception of a quaternary compound, all the germicides displayed good disinfecting properties. from a hygienic point of view, pine core wood is suitable for use in hospitals. survey of the microbiological quality of drinking water supply in hospitals. drinking water cooler vs tap water fed dispenser vs bottled water objectives: hygienic, economic and ecologic comparison of drinking water coolers versus tap (mains) water-fed dispensers versus bottled drinking water. methods: samples were taken from each system without preflow or cleaning of the nozzle. these comprised 2 samples from 20 drinking water coolers each over 2 months, 1 sample per week from the tap water dispenser over 6 months, and samples from 6 different bottles of drinking water. total colony counts were analysed at 22°c and 36°c, respectively. total p. aeruginosa, e. coli, and coliforms, as well as sulphite-reducing clostridia were examined according to european norms (en iso) for compliance with the german drinking water ordinance 2001 (limit value for total colony counts at 22 ± 2°c: 100 cfu/ml, at 36 ± 1°c: 100 cfu/ml and 20 cfu/ml for bottled water). costs per litre were calculated and compared based on the economic data supplied by the manufacturers. the ecological impact of the different systems was evaluated based on expert opinion. results: all the samples from the water cooler system exceeded german drinking water ordinance threshold values. all the samples taken from the tap water dispenser, and, with one exception, all the samples from the bottled water complied with the german drinking water ordinance. if more than 400 l of water are consumed per month, the tap water dispensing system and bottled water are more economical than the water cooler system ( 0.57 per l for water cooler, 0.45 for month tap water dispenser, 0.49 per l for bottled water). in economic terms, the water cooler system can only be recommended for consumption levels below 400 l per month. from an ecological point of view, the tap water dispenser is most favourable (expressed in co2emissions per year for 403.720 l at freiburg university hospital), followed by the water cooler system and finally the bottled water. conclusion: for reasons of hygiene, the use of water cooler systems cannot be recommended in hospitals. furthermore, drinking water cooling systems are only economic for low consumption rates. tap (mains) water-fed dispensers feature the best hygienic, economic and ecologic properties. objectives: the application of antimicrobial finishes to textiles can prevent bacterial growth and might reduce the risk of infection resulting from fabrics that are contaminated with pathogenic microorganisms in hospitals. the main aim of this study was the determination of the antibacterial activities of chemical treatments applied to textiles. comparison of testing methods assessing antibacterial efficiency was conducted. these studies were performed in order to select the right methods of evaluating the antibacterial and bacteriostatic activity of finishes. methods: fibers and fabrics made from cotton (100%) applied with quaternary ammonium salts were tested. finishes treated with bactericidal agent were compared with samples (not treated with the disinfectant). the european standards iso / dis 20645/2002 and aatcc 147/1998 suitable for assessment of antibacterial activity were applied. the bacterial strains recommended by the above standards such as: klebsiella pneumoniae (atcc 4352), staphylococcus aureus (atcc 6538), escherichia coli (atcc 11229) were tested. due to its high resistance to quaternary ammonium salts, additionally p. aeruginosa (atcc 9027) was examined. bacterial suspension 1 -2,9 x 10 8 cfu/ml was prepared according to the standardsõ requirement. the samples impregnated with biocidal products were examined with agar diffusion plate test. the specimens were located transversely across the bacterial inoculum on the nutrient agar. the level of antibacterial activity was assessed by examining the extent of bacterial growth in the contact zone between agar and the specimens. additionally, the extent zone of inhibition (minimal 1 mm) around the specimens was considerate. results: tested fibers did not show antibacterial activity. the fabrics showed antibacterial activity against k. pneumoniae and s. aureus (inhibition zone 4 mm). the examined specimens showed no bacteriocidal activity against escherichia coli and pseudomonas aeruginosa. the results obtained in two applied methods were comparable in assessing antibacterial activity of finishes. the antymicrobial finishes did not fulfill requirements of the standards. conclusion: based on the obtained result the tested antimicrobial finishes may not be considered effective in preventing bacterial transfer at contaminated areas in hospitals. a new interactive approch to improve hand hygiene compliance j. holt, e. tvenstrup jensen, d. buhl (copenhagen, dk) introduction: the compliance of guidelines for hand hygiene is well below 50 % despite several at-tempts to improve. when planning an educational outline to promote a behavioural change in the hand hygiene practice in the aim of lowering the incidence of nosoco-mial infections the mapping of critical factors and implications that affect this practice is of utmost importance. objectives: this study was an empirical investigation of the aspects of the hand hygienic practice as it takes place in the danish health care system today. the study attempted to answer the question:''how can the health staff's lack of compliance with hand hygiene be explained and understood as basis for planning an educational material to support a behavioural change.'' methods: literature studies of hand hygiene, a questionnaire based done survey (1700 per-sons/294 respondents) and qualitative interviews (n = 15) was performed in order to uncover the explanations for this low rate of compliance.the data was analysed and discussed on the basis of theories on action, experience, reflective thinking, control and rituals in order to aim for a broader view and under-standing of this field. results: both the questionnaire and interviews showed that hygiene still is a field that has great implications on the interactions between individuals. furthermore it seemed difficult for the staff to draw a professional and not a private line between ''the clean and the unclean'' and thereby perform hand hygiene without compromising each other. further the interviews showed that it is difficult for the staff to react on something they cannot see and that not gives an immediate result when staff does not act as pre-scribed. introduction: leishmaniasis has increased in importance in recent years as hiv-infected patients have emerged as a risk group for the disease. however, real prevalence in the general population is unknown. objetive: the objective is to know the antibody seroprevalence for infection by leishmania infantum in the general population of castilla-leon (spain). methods: a random sample from the general population (4825 sera) and from hiv-infected patients in the autonomous community of castilla-leon was collected in 1996. seroprevalence of antibodies against l. infantum was determined by an indirect enzymoimmunoanalysis (eia) test designed in the laboratory. results: anti-leishmania infantum antibody seroprevalence in the general population was 4.9%. there is a significant increase in seroprevalence with age (p = 0.001), from 3.96% in the 14-20 years group up to 7.2% for those over 70 years old. there were no significant differences between women and men (5.0% versus 4.9%; p = 0.9534). seroprevalence was significantly higher in people from rural areas than in those from cities (6.0% versus 3.4%; p = 0.001). hiv-infected patients had a seroprevalence against l. infantum of 64.0%. no differences were observed between women and men, nor was there a prevalence increase with age. efficacy of intralesional pentavalent antimony in combination with oral azole for treatment of oldworld cutaneous leishmaniasis results: a 30-year-old man presented with a 6-month history of slowly growing round lesions on his right arm and leg. he had a history of travel in the mediterranean area (including north african and middle east countries) 6 months prior to presentation. examination showed one nodular lesion (1 · 1 cm) with margin induration and depressed central ulceration on the right arm, a second nodular lesion (0.5 · 0.5 cm) on the right leg, and a red papule (0.5 · 0.5 cm) on the left arm. a 52-year-old man with an history of a 4-month stay in iraq until 1 months prior to presentation, showed 6 lesions, which had grown during the last three months: two nodular lesions (3.5 · 1 cm, 4.5 · 2 cm) with erythematous margins and a thick crust on the left arm, 4 round lesion (2.5 · 2.5 cm) with depressed central ulceration, at the left arm, right arm, right leg and second finger of the right hand. a 49-year-old man with a 5-month stay in iraq until 5 months prior to presentation, showed two painless confluent nodular lesions (5.5 · 3.5 cm) with depressed central ulceration at the right elbow, which had grown over 4 months, two round lesions (1 · 0.5 cm and 0.8 · 0.7cm) with a thick crust on the left poplitea fossa and one nodular lesion (1.5 · 1 cm) at right leg. all patients received 12 alternate-days intralesional injections of meglumine antimoniate and 6 weeks of oral fluconazole. all lesions healed completely at the end of therapy and at the 3-month follow-up. none of the patients complained of any adverse events during treatment or follow up. conclusion: treatment with intralesional pentavalent antimony in combination with azole is effective and absolutely safe, in that it reduces the total amount of antimonial exposure. entomological study of sandflies (dipthera: psychodidea) in three foci of endemic cutaneous leishmaniasis in iran human leishmaniasis is a globally widespread parasitic disease caused by members of the genus leishmania. currently, leishmaniasis is considered to be endemic in 82 countries including iran. phlebotomine sandflies the vectors of leishmaniasis have received considerable attention in recent years due to the resurgence of leishmaniasis in some endemic areas of iran ,so extensive studies have been conducted on the ecology of sandflies in different parts of the country in recent years. a total 8026 sandflies were collected by using funnel traps of rodent burrows in rural district of the 3 province of iran (shiraz, yazd, khozestan). these sandflies identified in 3 maine croups: phlebotomus. papatasi(61%) , p. sergenti (10%) , p. sergentomya (29%). these findings indicate that p. papatasi could be a vector of humans and the gerbils (merion libycus). the close contact between vectors and resevoirs have created a very efficient cycle for the transmission of the disease in these areas and the villages around these 3 provinces. erysipelas like cutaneous leishmaniasis: a case report a. erdogan, d. balaban, e. dervis, g. sengoz, g. barut, a. karaoglu (istanbul, tr) cutaneous leishmaniasis is an infectious disorder of the skin caused by leishmania major, tropica, aethiopica and infantum which are transmitted by phlebotomine sandflies. we present here a patient who demonstrated a morphologically rare form of erysipelas like cutaneous leishmaniasis. a 74-year-old woman with a four-month history of erythema and edema on her nasal and left malar area was referred to our clinic for further evaluation. her dermatological examination revealed an erythematous, edematous and fine desquamative plaque on her nose and left malar region resembling erysipelas. other physical and laboratory findings were normal. punch biopsy taken from the lesion and dyed with h.e revealed dense lymphocytic infiltration between the layers of just beneath the epidermis and deep dermis. in addition, a huge number of amastigots were found in macrophages and histiocytes that formed granulomas. in light of these findings the patient was diagnosed with cutaneous leishmaniasis and commenced a regimen of meglumin antimonat 425 mg b.i.d. after two weeks of therapy the lesion was gradually disappeared without any scar formation. in conclusion, cases with cutaneous leishmaniasis are still observed in our country not always with usual clinical appearance, but also like the present case, it may clinically resemble erysipelas and provides difficulties in the differential diagnosis. study of human infection of cutaneous leishmaniasis in a focus of the disease, southern iran ) with scar and 14 cases (1%) with ulcers. the mean of acute lesions and scars per infected cases was 1.64 and 1.87, respectively. totally 129 cases were observed with sores and scars, and 122 out of them were infected in the area. the highest rate of the acute lesion was observed in 0-4 years old age group (4.5%); meanwhile the highest rate of scars was in 10-14 years old age group (12.9%). sores were located on hands (8.7%), foot (65.2%) face (4.4%) and other parts of body (21.7%). in the study of schools, 1201 students (53.5% boys, 46.5% girls) were visited. the infection rate to acute lesion was 0.6% and 22.6% of students had scar. there is no significant difference between males and females based on the acute infection (p > 0.05), but this difference was significant based on scar (x 2 = 9.84, p < 0.05). the highest infection rate was observed in tashkooieh village (2.7%). two injected mice were developed the acute lesion and the agent of the disease was identified as leishmania major by pcr test. conclusion: the infection rate of the sores and scars shows that the disease is located in the studied area in recent years. the disease had one peak during 1995-2000 and has been increased from 2002 up to now. this is the first time that the parasite isolated from human in the area. therefore, this focus must be added to the zoonotic cutaneous leishmaniasis foci of iran. the disease is endemic in the area because more than 95% of cases were infected locally. giardia and cryptosporidium in the netherlands objectives: • the studies were designed to get an insight into the incidence of protozoal-, bacterial-, and viral infections in patients with diarrheal complaints in different groups: patients consulting their general practioner and the dutch population. here we focus on giardia and cryptosporidium • to decrease diagnostic deficit • study the risk factors methods: three studies were designed and conducted : • ôhaarlemõstudy: 1994-1996, general practitioners, haarlem region • nivel: case-control study in sentinel general practitioners practices (1996 general practitioners practices ( -1999 • sensor: prospective population based cohort study with a nested case-control study in the dutch population. (1999)the studies differ in inclusion criteria and the diagnostic laboratory techniques used, esp. virological stool examinations results: incidence of gastroenteritis in the nivel (gp) study (after correction) was 79.5 per 10,000 personyears. this means that 80.000-130.000 persons will consult a gp annually for gastroenteritis. incidence of gastroenteritis in the populationbased study was 283 per 1000 personyears. giardia was detected in 14.8% of the cases in haarlem, in 5.4% of the cases in the nivel study and 3.3% of the controls. for cryptosporidium this was resp 3.3%, 2.1% and 0.2%. the diagnostic deficit decrease substantialy by testing for viral pathogens like nlv. detection of pathogens was influenced by age, season and duration of symptoms. we were able to construct an algoritm for diagnostic workup in gi patients. statistical surveys have been made for the effect of the climate on the epidemic diseases in tropical area, but not so much has been clarified on the relation between the variations of meteorological elements and of the number of patients. in this paper, we apply eof analysis method to time series of diarrhoea patients and meteorological elements in bangladesh, to understand effects of the meteorological variation to the prevalence of the diarrhoea disease. the eof analysis of the time series of patients and meteorological elements averaged every two weeks for 21 years from 1981 to 2001 shows that in the dominating component, the anomaly of the number of the diarrhoea patients has different signs for the periods before and after june, corresponding to the two seasonal peaks of the number of the patients. higher maximum temperature and more humidity in the pre-monsoon period are found to have a tendency to enhance the first peak of the diarrheal occurrence. we will also report the result for the different types of diarrhoea as v. cholera, rota and etec. serologic evidence for babesiosis in the northern and eastern tyrol (austria) and the southern tyrol (italy) methods: leptospirosis was diagnosed by leptospiral igm enzyme linked immunosorbent assay (elisa) and the serovar was confirmed by the microscopic agglutination test (mat) using a battery of 12 live leptospiral strains as antigens. results: most of them were outdoors manual workers (52%), housewives (29%), indoors non-manual workers (16%) and unknown (3%). mean duration of symptoms was 12.4±21.2 days. majority of the patients presented with fever (89%), jaundice (55%), chills and rigors, vomiting (47% each), cough (28%), abdominal pain (25%), diarrhoea, and altered sensorium (19% each), purpura/bleeding (11%). salient laboratory abnormalities included anaemia (50%), leucocytosis (59%), thrombocytopaenia (64%), elevated erythrocyte sedimentation rate (esr) (55%), hyponatremia (50%), hypokalemia (23%). acute renal failure occurred in 47%. hepatic function derangement occurred in 64%. three patients had pulmonary infiltrates and sputum revealed haemosiderin laden macropahges in them. two others manifested exudative pleural effusion which was bilateral and sequential in one of them. the common serovars encountered included l. autumnalis (30%), l. hebdomadis (22%), l. grippotyphosa (18%), l. icterohaemorrhagiae and l. javanica (11% each), l. australis (8%). all patients were treated with parenteral crystalline penicillin, oral doxycycline and were managed conservatively. haemodialysis was required in four patients. two patients died, both of whom developed multiorgan system failure. conclusions: leptospirosis is an important cause of acute febrile illness with renal, hepatic dysfunction and bleeding abnormalities. a high index of clinical suspicion is required confirm the diagnosis early as the condition responds well to conservative management. determination of fr900098, a promising antimalarial agent, in human serum by capillary electrophoresis for pharmacokinetic studies the acetyl derivative of fosmidomycin, fr900098, was demonstrated to be twice as active against plasmodium falciparum in vitro and in the plasmodium vinckei mouse model. fr900098, as fosmidomycin, is an inhibitor of 1-deoxy-d-xylulose-5-phosphate (doxp) reductoisomerase, an essential enzyme of the non-mevalonate pathway of isoprenoids biosynthesis. the biosynthesis of isoprenoids in plasmodium is depending on this doxp pathway, as found in eubacteria, algae and plants, but not in human. in plasmodium, the doxp pathway is localised inside a plastid like organelle, the so-called apicoplast. here, we report on the development of a high-performance capillary electrophoresis (hpce) technique for the determination of fr900098 in serum. various instrumental setting for migration (voltage, capillary temperature) were tested and the buffer properties (ph values, component molarities) were optimized. the assay was submitted to standard quality control procedures: within-, between-days reproducibility, accuracy, limits of detection (lod) and quantification (loq), linearity, short and long term stability. finally, the working buffer used was 14 mm kh2po4 / 56 mm k2hpo4, 15% methanol, and 0.2 mm hexadecyltrimethylammonium bromide (htab). the ph was adjusted to 10.9. the electroosmotic flow was modified by the cationic ion pairing reagent, htab. the assays were linear over the large concentration range tested, from 0.25 to 100 lg/ml. the recovery of the sample pre-treatments was higher than 100%. good precision was obtained, with betweendays reproducibilities resulting in coefficients of variation (cv%) of 2.1, 2.0 and 3.2%, and within-days reproducibilities of 0.6, 1.8, and 4.9% (cv%), for 100, 10, and 1 lg/ml, respectively. the lod was 0.25 lg/ml, and the loq was 0.5 lg/ml. the studies of short and long-term stability of fr900098 in serum showed a good stability of the molecule, at room temperature, +4°c or )80°c. moreover, fr900098 was resistant to numerous cycles of freezing and thawing. indeed, after four freeze-thaw cycles (4 days), 94.9%, 101.0%, and 97.5% of fr900098 were recovered from the 100, 10, and 1 lg/ml samples, respectively. in conclusion, we have developed a convenient ce technique for the determination of fr900098 in serum which offers advantages of speed, sensitivity, and accuracy. at present, the procedure is applied for pharmacokinetic studies in an animal model of gö ttingen mini-pig. present and future of malaria in kahnouj endemic area, southern iran objective: kahnouj district is associated with one of the malaria regions in southeast of iran. the anopheleine fauna does not appear to have changed much over several decades. methods: entomological studies and mosquito collection were performed every 15 days from indoor and out door shelters. malaria surveillance was carried out by health centers, ministry of health. microscopy was performed on out patients with fever or suspected malaria. malaria cases detection were mostly performed passive and rarely actively. the positive cases were treated with chorolquien/primaquen. results: in the present study five vectors species of malaria were found in this study which had been previously recorded 3 decades ago. this district like other malaria endemic areas in iran has been under pressure of anti malaria programmes including case finding and residual spraying insecticide as well as larviciding against the vectors since 1958. annual incidence of malaria declined from 10.27 to 3.86 during ten years. the most transmission occurred in october to december when the temperature was suitable for vector activity in kahnouj area. electricity was recently supplied into the rural area where most malaria cases were found. therefore, most houses equipped with air conditioner and the resident keep windows close, so they are secured from mosquitoes bites during hot season but not in beginning of spring and autumn when temperature is mild enough, in order to save the electricity cost, the residents do not use air conditioners whereas they leave windows open and no other protection against mosquito bites is provided. residual spraying insecticide of indoor shelters have been stopped for five years and the most activity of antimalaria programme set up base on case finding. conclusion; in order to control malaria, indoor residual spraying of insecticide would not assist effectively, so given knowledge to people to use bed net particularly in season people are more expose to mosquitoes bite may be considered as an effective measure in controlling malaria in khanouj district. also development in this area is effectively pushing back malaria in near future. entomological studies and mosquitoes collection were performed every 15 days from indoor and outdoor shelters as well as breeding places with the aid of suction tube and dipper. results: entomological researches were found that five vectors species of malaria in this study had been previously recorded 2 decades ago. anopheles stephensi was recognized as the main vector of malaria in this area with two peaks, one in may and the other in december. the most malaria transmission occurred in june and december. the larval habitats includes draying river bed with pools, rocky river pools, stagnant streams, slow foothill streams, temporary pools, slow moving water with or without vegetation. conclusion: operational of insecticides for adult and larval control, as well as surveillance of malaria cases, would not assist effectively to control of malaria, so given another malaria control methods as impregnation of bed nets as well as repellent particularly in seasons that people are more expose to mosquitoes bite, may be considered as an effective measure in controlling malaria in this area . case report: a 16 year old boy presented with fever and a generalized exanthema at the local dermatology clinic. the mixed appearance of pustules and umbilicated non purulent vesicles led to the suspicion of an orthopoxvirus infection. the patient reported that little red dots had occurred 2 weeks earlier at the extremities after contact with a sick pet rat. 1-2 weeks later a generalized pustular exanthema appeared, which was associated with high fever (>39°c) and a pronounced feeling of sickness. histologically no viral inclusion bodies were found in pustule material but poxvirus particles were detected by elmi in swabs from pustule ground. following the anamnestic suspicion of transmission by an infected rat, which unfortunately had perished soon afterwards, and the experience made at the recent monkeypox outbreak in the usa initiated by imported rats, one of the major aims was the exclusion of monkeypoxvirus. this was accomplished by pcr typing, which like elmi had been established on occasion of the implementation of the austrian plan for poxvirus preparedness (ôpocken-alarmplanõ) and despite the diagnostic means for detection of variola vera also included the ability to discriminate between animal orthopoxviruses. additionally serological diagnosis was performed and presumably due to the protracted course of infection cowpox specific igg antibodies were already present at admission with a titre of 1:800. interestingly the humoral immune response initially seemed to be rather cowpox specific as there were no antibodies crossreacting with vaccinia virus detectable in indirect immunofluorescence. as an exclusive immune reaction with one species alone would be a very rare situation observed with orthopoxviruses we did more extensive testing by western blot analysis. although not exclusively directed against cowpoxvirus antigens a very restricted reactivity of the patient serum was observed in the early course of infection using different recombinant and virus-derived orthopoxvirus antigens. this would suggest that also serological methods might be able to give a hint towards rapid determination of orthopoxvirus species, which certainly is also the case with immunological antigen detection methods. crimean-congo haemorrhagic fever in results: forty veterinerians from endemic region (tokat), and 43 from non-endemic region (aydin) were included. demographic characteristics in two groups were similar, whereas professional activities of veterinarians in non-endemic region were more intense (p = 0.001). the cchf igg positivity (2.5% vs 0%), brucella agglutination titre of >1/160 (27.5 vs 4.6%) were more common in endemic region than non-endemic region. three veterinarians in tokat had malaise, myalgia, and fever. in multivariate analysis to detect the risk factors for serum tube agglutination of > 160, the veterinarians living in endemic area were found to have 7.8 times higher risk of brucella infection than the ones living in non-endemic area (or; 7.8, confidence interval; 1.4-40.9, p = 0.015). the prevalence of coxiella burnetii serology was equal in both regions as 7%, and none of the seropositives had complaints. the history of tick bite was significantly more common in the endemic region than the nonendemic region (35% versus 12%, p = 0.011). conclusions: cchf and brucellosis compose infection risks for veterinarians in endemic region, despite the veterinarians in endemic region perform less riskfull professional activities. veterinarians in cchf endemic regions should be warned to protect themselves against tick bites according to universal precautions. the use of masks should be employed to prevent inhalational transmission of brucellosis in endemic regions. changes in temperature and the crimean congo haemorrhagic fever outbreak in turkey o. ergonul, s. akgunduz, i. kocaman, z. vatansever, v. korten (ankara, istanbul, tr) objective: to investigate the role of the climatic factors that could effect the activation of hyalomma marginatum marginatum population, and consequently the emergence of crimean-congo hemorrhagic fever (cchf) epidemic. methods: the meteorological data were obtained from three meteorology stations (tokat, sivas, and yozgat), where the majority of the cchf cases were reported in the last 2 years. these 3 provinces are located at the northern parts of eastern anatolia and southern parts of black sea region. temperature data have been observed and recorded by the turkish state meteorological service (tsms), and were available for 70 years in sivas, for 40 years in tokat, and for 60 years in yozgat. meteorology stations are located at the city centres, not at the airports. temperature variations and trends for turkey were analysed by using a data set including monthly averages of daily mean, and minimum temperatures. annual rainfall, and the number of days in april with the temperature of > 5°c were also included in the analysis. in order to detect homogeneity in mean annual series, first a homogeneity analysis was performed by using the non-parametric kruskal-wallis (k-w) test. the non-parametric mann-kendall (m-k) rank correlation test (13) the interest in the coordination properties of acrylate acid and its homologues was generated by the facile synthesis of the ômetal-containing monomersõ (mcm) materials. these compounds can be polymerised with a lot of organic monomers leading to various metal-containing polymers. polymeric transformations of mcm led to a new research field of current interest due to the practical importance of the obtained products which exhibit a number of unique features: high catalytic activity, unusual magnetic, electro-physical and biological properties.these polymers are especially appropriate for biological applications (tissue engineering, implantation of medical devices, dentistry, bone repair etc.) because of their molecular weight, compositions and architectures which can be regulated through controlled reactions. we report here the antibacterial and antifungal activity of new complexes of type m(phen)(c3h3o2)2(h2o)y ((1) m: mn, y = 0; (2) m: ni, y = 2;(3) m: cu, y = 1;(4) m: zn, y = 2; phen = phenantroline and c3h3o2 is acrylate anion), representing the first step products in the synthesis of polymeric materials. the in vitro antimicrobial testings were performed by broth microdilution method, in order to establish the minimal inhibitory concentration (mic), against gram-positive (bacillus subtilis, listeria monocytogenes, staphylococcus aureus), gram-negative (psedomonas aeruginosa, escherichia coli, klebsiella pneumoniae, salmonella enteritidis), as well as candida sp., using both reference and clinical, multidrug resistant strains. our results showed that the tested compounds exhibited a specific antimicrobial activity, both concerning the microbial spectrum and the mic value. the mics values widely ranged between 4 mg/ml and 16 mcg/ml. all the tested compunds were highly active against salmonella and listeria (mic = 16 mcg/ml objectives: c. glabrata is innately less susceptible to azole than most other species of candida and it acquires azole resistance after short-term exposure to fluconazole, as recently noted in oropharyngeal isolates. since c. glabrata has emerged as a significant cause of candidemia, we examined the change in azole mic and karyotype in sequential isolates of c. glabrata during the course of fungaemia, and its relationship to antifungal therapy. methods: serial bloodstream isolates of c. glabrata were obtained from 15 patients with fungaemia over periods of up to 38 days. forty-seven c. glabrata isolates from 15 patients (6 patients who received antifungal therapy and 9 patients who did not receive antifungal therapy) were analysed using electrophoretic karyotyping (ek) and tested for antifungal susceptibility to fluconazole, voriconazole, and itraconazole. results: the overall rates of resistance to fluconazole (mic ‡ 64 lg/ml) and itraconazole (mic ‡ 1 lg/ml) for all 47 isolates were 8 and 50%, respectively. for most patients, the sequential strains from each patient exhibited the same or similar azole susceptibilities. however, sequential isolates from two patients showed three-or four-fold increases in the mics of all three azole antifungals, while they retained the same karyotypes. azole-resistant strains were isolated from both patients after fluconazole therapy was discontinued and the intervals after the first blood isolation were 24 and 38 days, respectively. the isolates from one of these patients exhibited increased expression of the cgcdr1 efflux pump. the sequential strains from each patient had identical karyotypes in 10 (67%) patients, but two or four different karyotypes in 5 (33%) patients. the sequential isolates from these five patients exhibited the same or similar antifungal susceptibilities, showed only one or two chromosome band differences, and had no association with previous antifungal therapy. conclusion: this study showed that sequential bloodstream isolates of c. glabrata were able to acquire azole resistance in association with fluconazole therapy, and that they developed two or four different karyotypes in some patients, during the course of fungaemia. results: the next table shows the mics obtained for the tested drugs. from the three isolates with voriconazole mic > 1 mg/l, two of them had a fluconazole mic ‡ 16 mg/l. six isolates with voriconazole mic = 1 mg/l were found: the fluconazole mic for one of them was 4 mg/l, for another four was 8 mg/l and for the last one was 32 mg/l. conclusions: a. there wasn't found any fluconazole or voriconazole ôresistantõ isolate in the haart era. b. the percentage (11.84%) of isolates with voriconazole mic ‡ 1 mg/l is higher than it has been described in other works. c. the 75% of the voriconazole ôresistantõ isolates were fluconazole ôresistantõ too, so a cross resistance mechanism could be implicated. in vitro susceptibiility testing of micafungina (fk-463) and anidulafungin (ly303366) against candida spp. objectives: despite antifungal therapy, mortality in disseminated zygomycosis is still too high. we analysed the in vitro activity of posaconazole (pos), voriconazole (vrc) and caspofungin (cas) against 59 strains of the genera rhizopus, mucor, absidia, and cunninghamella in different media compositions. methods: the following five media compositions were compared: rpmi 1640 ± 2% glucose, am3 ± 2 % glucose and hr-medium. mics were determined by microdilution method following nccls guidelines with minor modifications. each well was read visually, the growth in each well was compared with the growth control. two endpoints were evaluated for each drug: an inhibition of growth >90% war recorded as mic1 and an inhibition of growth >50% was recorded as mic 2. the final concentrations of the antifungal agents were 0.015-8 mg/l for pos and vrc, and 0.03-16 mg/l for cas. results: pos was significantly more active than cas and vor, both in r + g and in hr media (p < 0.05 when comparing the mic1 of pos in hr medium to that of vrc; p < 0.001 for all other comparisons). growth in rpmi and am3 media supplemented with glucose was more robust than in the corresponding media lacking glucose. glucose had little influence on mic values. the agreement when comparing the mic1 evaluated in am3 ± glucose was > 85%, while the use of mic2 endpoint yielded 100% agreement for all genera. when comparing the data obtained in rpmi ± glucose to that in hr, the agreement was good. the percentage agreements using the mic2 and mic1 endpoints were 100% and > 70%, in contrast, the agreement between am3 ± glucose and the other media was generally poor. moreover, the average mics obtained in am3 medium were lower than those obtained in either hr or rpmi which was due to a difference among genera. conclusions: our results suggest the following: a) pos is active in vitro against zygomycetes at clinically relevant concentrations. b) within zygomycetes, there are differences between genera in terms of their antifungal susceptibilies. c) growth medium is an important variable for mic determination in zygomycetes, and the more relevant medium appears to be rpmi supplemented with 2% glucose. objectives: here we describe first clinical case of a caspofungin resistant c. glabrata infection. the patient was a 43 year old male with aml. he received a matched unrelated hsct, 9 months prior to his death. he had a complicated hospital course which included c. glabrata sepsis. c. glabrata was cultured from his stool, from the time of transplant to death. initial blood culture isolates were azole resistant and amphotericin b was started. this was stopped due to renal insufficiency and caspofungin was started (mic 0.12 lg/ml). he had a prolonged duration of therapy which comprised of alternating courses of iv voriconazole and caspofungin. four months after initial fungaemia c. glabrata was cultured which was both caspofungin resistant (mic > 8lg/ml) and azole resistant. despite the addition of amphotericin b the patient died 8 weeks later. c. glabrata was isolated from the bone marrow at autopsy. methods: the series of patient isolates, from time of transplant to death, had susceptibilities performed as per nccls document m27-a. the isolates were typed using cg6 probe and mlst. results: he susceptibilities demonstrated caspofungin resistance in the blood isolate ( > 8 lg/ml) and azole resistance in all but a few of the initial stool isolates. all the isolates were shown to be identical by cg6 and were determined to be mlst group 1, which has previously been shown to be the most prevalent clade of c. glabrata worldwide. discussion: this describes the first caspofungin resistant clinical isolate of c. glabrata. it has been recently reported in c. albicans. this data details an isolate that has developed resistance in the presence of therapy. resistance to caspofungin is not common and is thought to be due to mutations in the beta-1-3-glucan synthase (fks) gene. there are 3 fks genes in c. glabrata. preliminary sequencing data of one of these genes has so far revealed no non-conservative mutations. the 2 remaining genes are yet to be sequenced and the presence of other mechanisms cannot be ruled out. objectives: the aim of this study was to investigate the production of slime factor among candida species which were isolated from hospitalized patients. another aim of this study was to see in vitro activities of antifungal agents and to compare these results with slime production. methods: total 116 candida spp (79 c. albicans and 37 nonalbicans candida spp) isolated from various specimens were included to the study. fluconazole, itraconazole, amphotericin b and caspofungin susceptibilities of these strains were determined by broth microdilution method according to nccls m27-a2 standards. biofilm production of candida spp. was determined by microplate method on polystyrene microtiter plates using brain heart infusion broth supplemented with 0.25% glucose as a growth medium. results: caspofungin and amphotericin b was the most active agents which mic90 values 1mg/ml and 0.5 mg/ml respectively. fluconazole resistance (mics > 64) was obtained from 24 of the isolates (21%). biofilm formation was detected in 33 of the total strains (28%). statistically important difference (p < 0.05) was determined between the biofilm production of c. albicans (21.5%) and nonalbicans species (%48.4). significant correlation between biofilm production and amphotericin b mic values was established (p < 0.05) conclusion: candida species are one of the most important etiologic agents of catheter related infections especially biolfilm producing strains. this study showed us biofilm production rates are too high particularly in non-albicans strains. this will explain the rising incidences of non albicans strains especially c. parapsilosis in serious infections. our study also implicated that the mic values amphotericin b which was one of the most active agent against candida infections had a significant correlation with slime production. this will be a problem in treatment of slime producing candida infections with this drug. conclusions: (i) scedosporium spp. carry on with high mics of antifungal agents, even for new antifungal agent as pos. (ii) s. prolificans is a multi-resistant organism. (iii) s. apiospermum is resistant to itc and tbf, but 15% of strains are susceptible in vitro to amb (mic < 2 mg/l), 75% to vrc (mic < 2 mg/l), and 60% to pos (mic < 2 mg/l). (iv) these findings reinforced the need of continued surveillance programmes that analyze antifungal susceptibility profiles of medically important fungal isolates. assessing susceptibility of fungi isolated from hospital environment to disinfectants objectives: the past decade has witnessed a worldwide increase in serve invasive gas infections. these rapidly progressive infections are associated with high mortality rates despite prompt antimicrobial therapy. the aim of this study was to characterize gas isolates causing severe invasive disease in different regions of poland by emm-typing, multilocus sequence typing (mlst), pfge, virulence genes distribution and their susceptibility to antimicrobial agents. methods: a total of 42 gas isolates from blood (54.7%), pus (33.3%), sputum (7.1%), peritoneum fluid (4.7%) and other sources were examined. susceptibility to penicillin, erythromycin, clindamycin, telithromycin, tetracycline, levofloxacin, chloramphenicol, quinupristin/dalfopristin and linezolid was determined by the microdilution method according to the nccls guidelines. clonality of all isolates was studied by emm typing, pfge of sma i-restricted bacterial dna as well as mlst. the strains were also tested for the presence of spea, speb, spec, spef and ssa genes by pcr. results: resistance to erythromycin was found in four isolates (9.5%), two of them exhibited the imlsb and two the cmlsb phenotype. twenty-one (50%) and five (11.9%) were resistant to tetracycline and chloramphenicol, respectively. all tested isolates were fully susceptible to penicillin, levofloxacin, quinupristin/ dalfopristin and linezolid. twenty different emm types were detected, of which emm1 (23.8%) and emm12 (21.4%) were most common, followed by emm85, emm60 and emm81. all emm1 types and emm12 types corresponded to the st28 and st36, respectively. altogether, 24 different pfge patterns, designated a-y were discerned among the isolates, with two predominant profiles a (n = 10) and b (n = 9). our study showed that all isolates possessed speb and spef genes, while the frequencies of spec and spea were 59.5% and 26%, respectively. conclusion: two clones predominated among gas strains causing severe invasive disease in poland; these clones were of emm type 1 and 12. we present 45 actinomyces spp isolations, their identification to a species level and their clinical sources. in addition, we perform susceptibility testing of 23 of those strains to 13 drugs. the identification of actinomyces spp was done taking into account their cultural features, growthõs atmosphere and biochemical and enzimatical tests, according to schemes proposed by sarkonen n., funke g., moncla b.,hillier s., and bernard k. we studied too, the clinical sources of actinomyces spp, if they were isolated lonely or in association with mucosa's normal flora. the susceptibility testing was performed by the agar dilution method, with mueller hinton agar supplemented with 5% sheep blood. the reading of minimum inhibitory concentration (mics) were done after 48 hours incubation at 37°c in at atmosphere enriched with co 2 . all strains were grown at 37°c on sheep blood agar plates with co 2 added. a. radingae, a. europaeus, and a. odontolyticus were the most frequent isolated species ( 7 each one) followed by a. israelii, a. graevenitzii, a. turicensis and a. viscosus. in 27 isolations, actinomyces spp were recovered as sole microorganism, and in the 18 remainder, in association with mucosa's normal flora. there were not relations between actinomyces species and the clinical sources of the samples. mics for penicilin, ampicilin and cefotaxime were from <0.016 to 0.25 lg/ml. there was a bimodal behaviour with macrolides : erythromycin, azithromycin and clarithromycin (mics from 0.032 to 128 lg/ml) and the same was observed with quinolones: levofloxacin, ciprofloxacin and moxifloxacin (mics from 0.032 to 8 lg/ml). all isolations presented mics for vancomycin <0.125 lg/ml. the identification of the actinomyces genus presents diagnostic difficulties due to its growth requirements. some species are not so infrequent, and the sources from which they are recovered suggest that they may be of clinical relevance, for they are often isolated as sole pathogen. isotretinoin versus tetracycline: a comparative study with regard to efficiency in the treatment of acne vulgaris c. oprica, l. emtestam, c.e. nord (stockholm, s) tetracyclines are most commonly used for treatment of moderate and severe inflammatory acne, and systemically administered isotretinoin has proved to be the most efficient treatment, used in patients with moderate or severe acne that fails to respond to other therapies. objectives: a randomized clinical trial was conducted to compare the clinical efficacy and the antimicrobial susceptibility of propionibacterium acnes strains isolated before, during and after treatment with either tetracycline or isotretinoin in patients with acne vulgaris. methods: 52 male and female patients, 15-35 years of age, with moderate or severe acne, were randomized into two groups of 26 patients each. they received oral tetracycline hydrochloride 1 gram/day together with topical retinoid (differine gel 0.1%) or isotretinoin (roaccutane) 1 mg/kg/day. the therapy was given for a 6-month period. clinical evaluation (leeds acne grading system and lesions counting) and bacterial samples were taken before the treatment started, during the treatment and 2 months after the treatment had stopped. dermatology life quality index questionnaire was completed by patients before and after treatment, in order to determine impairment of life quality. results: acne severity was significantly reduced by both regimens during therapy, and patients in the isotretinoin group continuously improved the acne scores after the treatment had stopped. after 6 months of treatment, isotretinoin produced greater lesion reductions than tetracycline. the mean per cent reduction in the different lesion counts was as follows: 83% versus 45% for non-inflammatory lesions (p < 0.01) and 80% versus 56% for inflammatory lesions (p < 0.01) in isotretinoin or tetracycline group, respectively. in the drug-free period, the group of patients treated with isotretinoin presented significantly less inflammatory lesions compared to the group treated with tetracycline (p < 0.001). both treatments had improved the life quality (p < 0.01), independent of acne severity. resistant p. acnes strains were isolated after treatment in both the tetracycline group (53%) and isotretinoin group (25%). conclusions: both treatments were effective during treatment period. more resistant strains were recovered from the tetracycline group. psoas abscesses. differences between pyogenic and tuberculous abscess objectives: to compare the demographic characteristics, clinical features, laboratory, microbiologic and imaging data, therapeutic options and outcome of pyogenic and tuberculous psoas abscesses. patients and methods: retrospective descriptive study of the medical records of the patients diagnosed of psoas abscess in our institution, in the period between january/1994 and october/2004. results: in this period 14 patients were diagnosed of psoas abscess, all of them secondary to a adjacent source of infection. nine patients had pyogenic abscess, and m. tuberculosis was the causal microorganism in 5 patients. in 3 patients there were bilateral involvement, and all three were tuberculous abscess. an underlying disease was present in 67% patients of pyogenic abscess, and only in 20% of tuberculous abscess. these one were malignancy, intravenous drug use, cirrhosis, use of steroids, and 3 with inflammatory bowel disease. patients with tuberculous abscess were younger (37 vs 49 years), and they presented with a longer duration of symptoms from presentation to diagnosis (145 vs 15 days) than patients with pyogenic abscess. abdominal pain was the most frequent symptom at diagnosis in pyogenic abscess (78%), whereas lumbar pain (80%) was in tuberculous abscess. other clinical manifestations were similar in both groups. the source of infection in pyogenic abscess was gastrointestinal in 7 patients (4 polymicrobial), and in 1 patient infection of an aortobifemoral bypass and sacroilitis (both caused by s. aureus). all tuberculous abscess were secondary to spondilitis. there were no differences between both groups in analysis or imaging alterations. culture of the abscess was positive in all cases practised. drainage was performed in 12 patients (7 percutaneous), without differences in both groups. clinical improvement was more delayed in patients with tuberculous abscess than in patients with pyogenic abscess (18 vs 10 days). there were no deaths in any group. conclusions: tuberculous psoas abscesses presents in younger patients with lesser underlying diseases and a more prolonged clinical presentation than pyogenic abscesses. these tuberculous abscesses are secondary to spondilitis, usually with bilateral involvement. prevalence of micro-organisms isolated from wounds of inpatients versus outpatients in a spanish teaching hospital objectives: hidradenitis suppurativa (hd) is a chronic disorder characterized from dilatation of sweat glands and recurrent bacterial infections. vitamin e was administered in several patients as an antioxidant in an attempt to relieve tissue function probably altered by the locally increased oxidant status. methods: twenty nine patients with hd, 15 male and 19 female, were enrolled over a period of twelve months. all have presented with more than three episodes of bacterial exacerbations for at least two years. a detailed medical history was taken upon first evaluation and patients were examined for areas affected by the disease. they were asked to self-evaluate the severity of their condition on a scale of 1 to 10 (1 representing intact skin and 10 maximum severity). patients were divided into three groups of treatment: a (n = 6), controls; b (n = 8), vitamin e orally 200 mg bid; and c (n = 15), vitamin e 200 mg tid orally. patients were withhold from any antimicrobial regime. patients were followed-up at three-month intervals; they were asked to re-evaluate their condition, and to provide details regarding frequency of relapses before and after the initiation of treatment. results: mean ± se duration of the disease was 11.7 ± 1.9) years and of involved areas 3.84 ± 035, with axillas and groin being involved in the majority of cases. mean interval between exacerbations before initiation of therapy with vitamin e was 33.98 ± 9.43 days and after initiation of therapy 91.35 ± 21.64 days (p: 0.042). for group b, mean ± se time interval between exacerbations before initiation of treatment was 45.00 ± 27.39 days and after initiation of treatment 69.00 ± 38.42 days. for group c, respective values were 36.30 ± 9.42 days and 132 ± 12 days. mean ± se of self-evaluation scores before therapy with vitamin e was 9.00 ± 0.71 and 4.33 ± 1.76 for patients of groups b and c respectively. they were 4.75 ± 1.31 and 2.75 ± 1.55 respectively after twelve months of follow-up. the latter changes constituted a significant improvement (p: 0.027). conclusion: vitamin e seems to improve the overall clinical condition of patients with hd substantially. further placebocontrolled studies are necessary to confirm these results. clonal diversity and toxin genes of staphylococcus aureus causing infections in a clinical ward over a 12-year period (1992-2003) s. pollini, g. zanelli, a. sansoni, s. cresti, c. cellesi, g.m. rossolini (siena, i) objectives: s. aureus remains one of the leading bacterial pathogens worldwide, representing a major challenge for antimicrobial chemotherapy. the aim of this study was to evaluate the molecular epidemiology of s. aureus infections observed in an infectious disease ward during the past 12 years. clinical microbiology and infection, volume 11, supplement 2, 2005 methods: 38 nonreplicate s. aureus isolates were collected from patients with staphylococcal infections (including food-borne infections, osteomyelitis, skin and soft tissue infections [ssti], pneumonia, meningitis and bacteraemia/endocarditis; mostly community-acquired) at the infectious diseases clinic, university of siena, during the period 1st feb 1992-31st march 2003. all isolates were analysed for antimicrobial resistance, clonal diversity and toxin genes (sea-e, eta-d, tst, luks-pv and lukf-pv). susceptibility testing was performed according to the nccls guidelines. clonal diversity was determined by pfge and by analysis of the coagulase (coa) and protein a (spa) genes polymorphism. toxin genes were analysed by pcr and restriction mapping. results: most isolates (35, 92%) were methicillin-susceptible (mssa), while 2 were methicillin-resistant (mrsa, mecapositive) and 1 borderline. genotyping revealed a single mrsa clone and remarkable clonal diversity among the mssa isolates (at least 20 distinct clonal lineages). several clones (including 6 mssa and the mrsa clone) were detected over prolonged times (2-8 years). the most prevalent clone was detected over a 8-year period and was exclusively associated with ssti (mostly bullous impetigo). toxin genes were overall detected in 87% of isolates, the most frequent being sea (45%), tst (32%) and eta and/or etb genes (29%). six isolates (all mssa) harboured the luks-pv and lukf-pv genes. conclusions: mrsa were rare and appeared only since 1997. significant clonal diversity was observed within mssa. a significant relationship was observed between toxin production and some infections (e.g. ssti). severe group a streptococcal infections in romania. a surveillance within the strep-euro project b. luca, m. straut, v. ungureanu, c. schalen, a. jasir on behalf of the strep-euro study group objectives: in the last 20 years, severe invasive streptococcal infections, often afflicting otherwise healthy subjects and yielding high mortality rates, have been increasingly recorded in europe and other areas. our main objective was to investigate the situation in romania lacking earlier data in the field. methods: the strains used in this study were clinical isolates from nine regions in romania. the strains were mainly blood isolates, but some were from other sterile sites. the in vitro susceptibility to antibiotics was tested by disk diffusion on pdm agar following the standard instruction. t-typing was performed by slide agglutination using sera from sevapharma, prague. spe gene detection and emm sequence typing was performed according to provious publications. results: during eighteen months 33 cases were reported. most prevalent t-type was 3,13 followed by types 12 and 1. more than fifteen different emm types were recognised, most of them unusual types; only four were type 1. half of the reported cases were from children below 12 years. out of all strains, 50% harboured spec gene, and 25 harboured spea. erythromycin resistance was uncommon (one isolate), whereas an overall high rate of tetracycline resistance was found (54%). conclusion: this is the first report on severe invasive streptococcal infections since no surveillance has been previously done in romania. the population covered was from eight out of 42 provinces. the emm type distribution might be of concern since many unusual types, not previously associated with severe disease, were found. this may be partly related to the dissemination of new types in populations immune to classical types. since tetracycline is not used in the treatment of streptococcal infections the level of tetracycline resistance among clinical isolates, appeared comparatively high. in contrast to several european countries, macrolide resistance seems not to be common among invasive group a streptococci in romania. acknowledgment: the strep-euro project is funded by the european commission. severe soft tissue infection and secondary bacteraemia due to community-acquired mrsa in a traveller returning from the congo r. padmanabhan, r. shrestha, g. hall, s. gordon, l. saravolatz (cleveland, detroit, usa) objectives: community acquired mrsa is increasingly becoming a global problem. these isolates possess the staphylococcal cassette chromosome (scc) mec type iv in their genome and a different pattern of antibiotic susceptibilities than hospital acquired strains. they are frequently virulent and cause predominantly skin and soft tissue infections by virtue of a panton valentine leukocidin (pvl) toxin gene. a returning traveler, who had spent the preceding four weeks in congo, presented with a staphylococcal bacteraemia and a large cutaneous ulcer of the right lower extremity that progressed to extensive soft tissue necrosis. he first experienced symptoms in kinshasa international airport, while awaiting his return flight to the united states, approximately 48 hours prior to presentation at our facility. methods: mrsa that was isolated from blood was susceptible to vancomycin, clindamycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole and gentamicin. pulse field gel electrophoresis (pfge) analysis was performed using sma1 on the patient's isolate along with another strain from our hospital and compared to other strains isolated from the midwestern united states. pcr amplification of the genes encoding panton-valentin leukocidin (pvl) was performed using primers described elsewhere. results: pfge demonstrated that the congo strain was different from any of the other midwestern united states strains. scc mec typing of this isolate revealed that this strain was type iv. pcr analysis did not detect sequences specific for the pvl gene. conclusions: the differential diagnosis of an ulcer in a returning traveler is large and includes bacterial, fungal, helminthic, protozoal, viral and arthropod borne causes. mrsa should be added to this list. the widespread occurrence of ca-mrsa in many parts of the world would support the finding of this organism on the african continent. to our knowledge, this represents the first ca-mrsa with the scc type iv mec gene reported from congo. ten-year of community-acquired methicillinresistant staphylococcus aureus st80-iv clone in denmark objectives: to determine the epidemiology in denmark of the pandemic european ca-mrsa clone st80-iv with respect to subtypes, dissemination, acquisition and types of infection. methods: all methicillin resistant staphylococcus aureus (mrsa) isolated in denmark between 1999-2003 were referred to and stored at the staphylococcus laboratory, statens serum institut (n = 617). the isolates were characterized by macro-restriction (smai) analysis using pfge, sccmec typing, dru sequence typing, antibiotic susceptibility testing and pcr amplification of the panton valentine leukocidin gene (pvl). clinical and epidemiological information from all patients were obtained from discharge summaries and registered. results: between 1999 and 2003, the mrsa st80-iv clone accounted for 28% (172/617) of the total number of mrsa in denmark and 30% (146/490) of infections due to st80-iv had primarily community onset (122/146) and thereby st80-iv accounted for 67% of all community onset mrsa in the period. more than 80% of the st80-iv infections were skin and soft tissue infections. by comparing the antibiogram of st80-iv (streptomycin-, kanamycin-, tetra-cycline-and fusidic acidresistant and gentamicin sensitive) with the isolates stored in our s. aureus bacteraemia collection, we traced the st80-iv clone back to 1993. before 1999 one to five st80-iv isolates were encountered pr. year, which increased to 25, 26, 50, 26 and 45 isolates in the years after. by pfge, the st80-iv isolates exhibited nine different patterns (a1-9), of which 60% were type a1. nine type a1 isolates, isolated 1993-2003 were subjected to dru typing, which has earlier enabled separation of pfge clones into subtypes. this was however not possible for the st80-iv isolates. conclusions: st80-iv isolates cause a large proportion of the mrsa infections in denmark and in particular among the infections with a community onset. the success of st80-iv as an infective clone outside the hospital environment may in part be due to its small sccmec type iv and its ability to cause skin and soft tissue infections probably associated with the expression of pvl. the dru sequence typing could not subdivide the major type of st80-iv found in denmark pointing out the strict clonality of this clone, which may indicate that it is a very well adapted pathogen. epidemiology and management of pressure ulcers in an acute care hospital objectives: the aims of the study were to determine pressure ulcer prevalence in an acute care hospital, to identify risk factors, to evaluate the medical and non medical management of pressure ulcers and to study the microbiological contamination of pressure ulcers. methods: for each patient, a standardized questionnaire was completed. the questionnaire included demographic data (age, sex, previous hospitalizations…) and the risk factors of the braden scale. detection of pressure ulcer was performed by skin examination of patients by two experts in skin care. management of ulcer pressure was evaluated by reviewing the clinical charts of each patient with pressure ulcer. each pressure ulcer was swabbed and inoculated on selective media. all the data were entered and analysed with epiinfo 6.04d (cdc, atlanta, ga). results: a total of 549 patients (59 ± 19 years old) were included : 75 ulcer pressures were observed in 37 patients (prevalence = 6.9%). heel anckle was the most frequent localization (40%), followed by sacrum (20%), elbow (11%), spinous processes (7%) and ischial tuberosities (7%). pressure ulcers were stage i (24%), stage ii (29%), stage iii (31%) and stage iv (16%). eighty percents of pressure ulcers were acquired within the hospital. using univariate analysis, risk factors significantly associated with pressure ulcer were : braden cutoff < 15 (or = 6.56, p < 0.0001), neurological disorders (or = 2.30, p = 0.009), previous ulcer pressures (or = 6.36, p < 0.0001), and hospitalization in an intensive care unit (p = 0.0003). among the criteria used for braden scale, humidity, activity, motility, nutrition friction and shear were significantly associated with ulcer pressures (p < 0.05). among the 75 pressure ulcers, 9.3% were diagnosed only by the experts in skin care and 83.8% were treated. treatment was considered inappropriate in 31.5% (mostly in stage i and iii) according to the french guidelines. microbiological results showed that 24.5 % of ulcer pressures, mostly from stage iii and iv were colonized with multiple resistant bacteria (i.e. methicillin resistant staphylococcus aureus, extended spectrum beta-lactamase enterobacteriaceae). conclusion: this prospective prevalence study led to a better awareness of patients at risk for pressure ulcer. this surveillance also contributed to a better knowledge of the mobile unit of geriatrics recently created within the hospital. background: following surgery for a musculoskeletal infection (mi), a positive suction drainage culture (sdc) is consistent with persistent sepsis and an unfavorable evolution in the majority of cases. the objective of this study was to determine the effect of a negative sdc obtained in a subsequent operation or repeated operations on the outcome of mi. methods: one hundred patients treated surgically for mi utilizing suction drainage for 24-48 hours postoperative and appropriate antimicrobial therapy were enrolled in this prospective study. surgical treatment consisted of the routine practice developed for the treatment of mi, consisting of drainage of purulent material, débridement, and prosthetic exchange or implant removal. the accumulated drainage fluid in the reservoir was cultured. sdc was considered negative if all bottles resulted in negative cultures. patients were placed into one of three possible mi treatment groups according to the sdc results identified after the first surgical procedure, as follows: (i) patients with a negative sdc and no new operation was performed; (ii) patients with a positive sdc and a new operation(s) was performed until the sdc was negative; and (iii) patients with a positive sdc and there was no new operation. the duration of antibiotic therapy for those with osteomyelitis ranged from 6 to 12 weeks, while others with mi (soft tissue infection) were treated for 10-21 days. results: the 3 groups were similar with regards to gender, age, underlying conditions, mi, bacterial organism, surgery, and antibiotic therapy. the majority of patients (84%) had osteomyelitis in the presence of an implant. at final review, a cure was obtained in 90% of patients (45 of 50) in group i, 87% of patients (20 of 23) in group 2, and 44% of patients (4 of 9) in group 3. conclusion: a negative sdc following mi surgery is a strong indication as to the eventual outcome of the infectious process. microbial aetiology of osteomyelitis of the foot in diabetic patients related hospitalization and lower-extremity amputation.acute infections in pts who have not recently received antibiotic therapy are predominantly caused by aerobic gram-positive cocci, often as a monomicrobial infection. although chronic wounds tend to develop polymicrobial flora. objective: to know the microbial etiology of osteomyelitis of the foot in diabetic pts, to determine the best choice empirical antibiotics combination. methods: diabetic pts with osteomyelitis of the foot admitted in our unit between 2002 and 2004 were included. the infection was documented with intraoperative samples and osteomyelitis was histolopathologically prouved. clinical and biological data were collected. results: 87 diabetics pts (64 males, 23 females) with a median age 65 years(23-94) were included.the co-morbidities were chronic renal failure (n = 35), arteriopathy (n = 34), obesity (n = 13), alcoholism (n = 10), immunodepression (n = 10). the clinical data were fever (40%), shock (2%), local signs as erythema (90%), fistula (86%), necrosis (43%) and foul odor (54%). the median rates of neutrophils polynuclear, c-reactive protein and erythrocyte sedimentation were respectively 9.7 g/l, 131 mg/l, 82 mm. 59 pts (68%) had a polymicrobial infection.the intra-operative samples (n = 87) yieled a majority of gram positive cocci [mssa:n = 30;mrsa:n = 15; cns:n = 15; streptococcus sp:n = 34; enterococcus sp: n = 15], following with gram negative bacteria [ pseudomonas sp: n = 12; proteus sp: n = 11; morganella sp: n = 11; e. coli: n = 8; klebsiella sp: n = 6; e. cloacae: n = 6; citrobacter sp: n = 4; serratia sp: n = 3, others: n = 3] and anaerobes (n = 17). 10 pts had positive blood samples. s. aureus is the most frequent pathogen isolated especially in monomicrobial infections and infections with positive blood samples. pseudomonas spp. seems to be correlated with chronic renal failure and immunodepression. no clinical data was significantly associated with a special bacteria , except necrosis and foul odor with gram negative bacteria or anaerobes. conclusion: our results are according with clinical studies in the literature. the major difficulty to treat osteomyelitis of diabetics foot is to well documented the infection. selecting an appropriate antibiotic to treat is particularly important because of the prolonged duration of therapy required and potential resistance of pathogens. the duration of hospitalisation (length of stay) in patients hospitalised with complicated skin and skin structure infections: identifying clinical and microbiologic risk factors in a comparison of tigecycline with vancomycin/aztreonam 42% of patients had polymicrobial infections. about 18% had a gram-negative causative pathogen, mostly escherichia coli. gram-negative causative pathogen prevalence was greatest in asia (39%). conclusions: this analysis showed significant global variations in csssi subdiagnoses, etiologies, comorbidities and causative pathogens. consistent with findings of the sentry antimicrobial surveillance program, s. aureus was identified as the predominant pathogen in csssi, especially in the us. gramnegative pathogens were also identified as causative in a substantial proportion of complicated skin infections. these findings may offer guidance in selecting effective empiric therapy, especially regarding newer agents with expanded broad-spectrum activity. a novel approach for evaluating the microbiological efficacy of tigecycline in patients with complicated skin and skin-structure infections a. meagher, p. ambrose, j. passarell, b. cirincione, t. babinchak, e.j. ellis-grosse (buffalo, albany, collegeville, usa) objectives: tigecycline (t) is a glycylcycline in development for the treatment of patients (pts) with serious infections, including complicated skin and skin-structure infections (csssi). while csssi can be caused by a mixture of grampositive and -negative bacteria, staphylococcus aureus and streptococci are the predominant pathogens. previous analyses combining all pathogens have failed to identify an exposureresponse (er) relationship. a method was developed to create more homogenous pt populations for the microbiological (m) er analysis of t in the treatment of csssi. methods: pts from 3 csssi clinical trials (one phase 2 & two phase 3) with t pharmacokinetic data and classified as both clinically and m evaluable, were pooled for analysis. pts received 100 mg loading dose (ld)/50 mg q12h (100/50) or 50 mg ld/25 mg q12h (50/25). at the test of cure visit, m (eradication or persistence) response was evaluated. indeterminate responses were excluded. non pathogenic baseline isolates were excluded. five homogeneous pt cohorts (c) were created based on baseline pathogens: s. aureus only (c1); s. aureus or streptococci (c2); 2 gram-positive pathogens (c3); polymicrobial (c4); other monomicrobial infections (c5). prospective step-wise procedures for combining c to increase sample size were used. logistic regression was used to evaluate steady-state 24 hr area under the concentration-time curve (auc) to mic ratio (auc/mic) to predict response. results: the dataset included 58 pts with 88 observations. c1 (n = 20) and c2 (n = 9) could not be evaluated due to small sample size. analysis began with pooled c2 + c3. continuous auc/mic ratio was marginally significant (p = 0.1130); a pt was 5.1% more likely to have successful response for every oneunit increase in auc/mic. adding c4, including pathogens with mic values up to 16 mcg/ml, decreased auc/mic, added cures to the lower end of the distribution, and added significant noise to the analysis. adding c5 increased sample size and further decreased the ability to detect a relationship. conclusion: analysis of all pathogens combined could not identify an er relationship. polymicrobial infections with gramnegative and anaerobic pathogens, associated with high mic values, added noise to the analysis and decreased the predictive capability of the model. the approach of creating homogenous populations based on two key pathogens in csssi, s. aureus and streptococci, was critical for identifying significant er relationships. exposure-response analysis of the efficacy of tigecycline in patients with complicated skin and skin-structure infections a. meagher, j. passarell, b. cirincione, s. van wart, k. liolios, t. babinchak, e.j. ellis-grosse, p. ambrose (buffalo, collegeville, albany, usa) objectives: tigecycline (t), the first glycylcycline to reach clinical trials, is in development for the treatment of patients (pts) with serious infections, including complicated skin and skin-structure infections (csssi). pharmacokinetic-pharmacodynamic (pk-pd) relationships, including pt covariates, for microbiological (m) & clinical (c) efficacy of t were evaluated in pts with csssi. methods: pts from 3 csssi clinical trials (one phase 2 & two phase 3), with pk data and classified as both c & m evaluable, were pooled for analysis. only those pts with infections due to staphylococcus aureus and/or streptococci, the predominant pathogens in csssi, were prospectively evaluated. pts received 100 mg loading dose (ld)/50 mg q12h (100/50) or 50 mg ld/ 25 mg q12h (50/25). at the test of cure visit, m (eradication or persistence) & c (cure or failure) outcomes were assessed. indeterminate responses were excluded. steady-state 24 hr area under the concentration-time curve (auc) and auc/mic ratio were evaluated as predictors of response. pt covariates included: age, weight, country, baseline pseudomonas aeruginosa or anaerobes, & comorbidities (diabetes, peripheral vascular disease). classification and regression tree (cart) analyses determined auc/mic breakpoints (bp). logistic regression (one observation/pt) was performed to determine predictors of efficacy. results: the dataset included 35 pts with 40 s. aureus and/or streptococcal baseline pathogens. mic values ranged from 0.06 to 0.5 mcg/ml. clinical cure was achieved in 30 (85.7%) pts and 35 (87.5%) pathogens were successfully eradicated. the median auc/mic ratio was 13.5 and 29 for the 25 and 50 mg dose groups, respectively. covariates were not significant predictors of efficacy. cart identified a significant auc/mic bp of 12.5 (p = 0.0177 for m and 0.0341 for c). the continuous auc/mic ratio was marginally significant based on sample size (p = 0.0563 for m & 0.1960 for c) and was deemed the most informative model. for each unit increase in auc/mic, within the observed range, pts were 3.7% more likely to have a successful c response and 17.1% more likely to have a successful m response. conclusion: pts with auc/mic ratios ‡12.5 were 13.5 times more likely to have successful m response. at the median auc/ mic ratio of 13.5 & 29 for the 50/25 & 100/50 dose groups, the model-predicted probability of c success was 0.66 & 0.96, respectively. t is likely to be an important treatment option for csssi. introduction: vertebral osteomyelitis and/or sacroiliitis due to streptococcus pneumoniae are extremely rare. although it is one of the most common pathogen isolated in blood cultures, there are only about 30 cases reported in the literature. in the main series of vertebral osteomyelitis (vo) spsi represents less than 1% of cases (1/587). material and methods: we report 6 cases of spinal infection due to s. pneumoniae (5 vo and 1 si with psoas abscess) seen between 1999 and 2004 at 4 different acute care hospitals. results: the 6 cases reported were part of a total of 1227 episodes of pneumococcal bacteraemia (0.48%).mean age of the patients was 57.1 years (range 37-82); there were 4 men. alcohol abuse 2/6, immunosupression 2/6 and smoking 4/6 were the most common comorbilities. blood cultures were positive in all cases, epidural pus and vertebral biopsy were also positive in 3/ 6. all strains were susceptible to penicillin. diagnoses were made by mri in 4 and ct scanning 2. all patients recived betalactam agents during a mean of seven weeks (3.8-10.7) in monotherapy (cefotaxime 4/6, ampicillin 1/6) or in combination (with vancomicin+gentamicin in 1 case). one case needed surgery, because of progression of a psoas abscess. other foci were found in three cases (psoas and paravertebral abscesses and acromio-clavicular arthritis). conclusions: spsi is a rare presentation of invasive pneumococcal disease. underlying disease is comnmon and other territories are frequently involved. infective endocarditis must be ruled out. its course appears to be benign. beta-lactam antibiotics are a good option, and surgery is occasionally indicated for progressive disease despite appropriate antimicrobial therapy. lumbar spine spondylitis due to staphylococcus schleiferi following coronary angioplasty: isolation of the pathogen in peripheral blood cultures a. karakolios, c. karagiannidou, t. kallinikidis, f. markou, v. kontos, g. tsinopoulos (serres, gr) a 70-year old man presented with acute, severe lower back pain 3 weeks after having been subjected to coronary angioplasty for ischaemic heart disease. he was treated with nsaids but not antibiotics without any improvement. ct scan of the lumbar spine showed changes in l5-s1 vertebrae that were consistent with spondylitis. mri imaging, as well as confirming spondylitis at l5-s1, it also revealed the presence of a left paravertebral abscess. on admission to hospital, low grade fever was documented. clinical examination of the lower limbs was normal. staphylococcus schleiferi, sensitive to several antibiotics, was isolated from 3 sets of blood cultures taken from both arms. initially, ciprofloxacin 200 mg bd iv was administered for 4 weeks. despite continuous clinical improvement, mri imaging 8 weeks after the commencement of antibiotic treatment showed no improvement of the radiological findings. thus, levofloxacin 500 mg bd p.o. rifampicin 300 mg bd p.o. were given for further 16 weeks. at the end of the treatment, the patient was symptom-free, resumed full activity and mri imaging showed considerable improvement. multiple blood cultures taken from both arms at different time points can help isolate pathogens causing spondylitis averting thus the need for difficult, interventional sampling of the focus of infection. diabetes and use of topical ocular antibiotics: a population-based case-control study objectives: we conducted this population-based case-control study to examine whether patients with diabetes mellitus have an increased relative risk of being treated with topical ocular antibiotics, as compared with population controls. methods: incident cases were defined as 24,559 individuals who redeemed a prescription for a topical ocular antibiotic in 1999 in north jutland county, denmark. ten gender-and agematched population controls per case were selected, using a unique personal identifier. diabetes prior to the topical ocular antibiotic prescription was determined by record-linkage with the county prescription database and hospital discharge registry. we did conditional logistic regression to estimate odds ratios (ors) for ocular antibiotic use among diabetic individuals and population controls, with adjustment for a range of comorbid diseases. results: among individuals treated with topical ocular antibiotics, 2.5% had diabetes as compared with 2.0% among control subjects. the overall adjusted or for use of ocular antibiotics in diabetic individuals was 1.24 (95% confidence interval [ci]: 1.13-1.35). stratified analyses showed that children between 3 and 15 years with diabetes had a 60% increased relative risk for ocular antibiotic use, whereas the effect of diabetes as a risk factor was low in individuals over 65 years and in those with presence of other diseases. conclusions: these results suggest that diabetes is a risk factor for the use of topical ocular antibiotics, especially in younger individuals. objectives: determination of bacterial species and their susceptibility to antibiotics in infectious conjunctivitis in general practice. methods: 179 patients suspected of having infectious conjunctivitis were included from 25 general care centres in the netherlands. from both eyes a swab was taken and pathogens found in the bacterial cultures were tested for their susceptibility using the etest for chloramphenicol, fusidic acid, trimethoprim, ciprofloxacin, and gentamicin (mics according to dutch national criteria). results: 80/220 affected eyes were culture positive. streptococcus pneumoniae (n = 55) was the most predominant pathogen, followed by haemophilus influenzae (n = 19), and staphylococcus aureus (n = 18). only for chloramphenicol and ciprofloxacin the mic90s were in the susceptible or intermediate susceptible range for all three pathogens, whereas this was not the case for the other antibiotics tested, of which at least one mic90 was in the resistant range. conclusion: in only one thirdth of the patients suspected of infectious conjunctivis in general practice a bacterial pathogen was found. although the prescription of antibiotics can be debated on the basis of our results, chloramphenicol and ciprofloxacin seems to be superior above fusidic acid, trimethoprim, and gentamicin. investigation of the aetiology of a trachoma-like condition in a rural population in guangxi province, prc p. cho, m.v. boost, w. ng (hong kong, hk) objectives: to determine the presence of trachoma in primary school children of the zhuang and yiao ethnic minority groups in du¡an county in guangxi, china. method: primary school children were examined using a slit lamp and several were noted to display follicular/papillary conjunctivitis suggestive of trachoma. to confirm the etiology of the condition, children were examined on a follow-up visit. for each subject, after examination with the slit lamp, the upper lid was everted after the application of a topical anesthetic (novesin), and a dacron swab was passed along the upper tarsal lengthwise four times. strict aseptic precautions were employed to prevent cross-contamination during sample collection. swabs were placed in dna-free tubes, transported to hong kong, and tested using the roche amplicor kits for detection of chlamydia trachomatis following the manufacturer¡s instructions. twenty children displayed significant conjunctival signs and photographs of the everted upper lids were taken to determine if these visual signs correlated with presence of trachoma as confirmed by pcr. results: sixty-five of 120 primary school students were willing to be tested, and 29% of these were positive for trachoma by pcr. comparison of photographs and pcr result indicated that not all children who showed signs of conjunctivitis were positive for trachoma. in addition, some children whose eyelids displayed milder form for conjunctivitis were positive by pcr. no obvious corneal involvement was observed for these children. conclusions: it was confirmed that hyper-epidemic levels of trachoma can be found among ethnic minority children in du¡an county. this may be related to the poor hygiene standards and the scarcity of water in this limestone region. pcr was not positive for all children showing signs of follicular/papillary conjunctivitis, which may indicate that some cases were in a dormant stage. it is also possible that failure to detect the organism was due to sampling error or delay in pcr analysis. nevertheless, the high level of infection is a cause for concern as trachoma remains one of the leading causes of blindness, and further investigation and treatment are warranted. clinical and epidemiological considerations about anthrax in constanta county, romania s. rugina, i.m. dumitru, c.n. rugina, e. dumea, e. basca (constanta, ro) introduction: anthrax is by primarily a disease of herbivores but humans can become infected as they come into contact with infected animals or their products. objective: to study the cases of human anthrax diagnosed in the last twelve years and their characteristics: epidemiology, clinical forms and evolution. material and method: it is a retrospective study on a period of twelve years performed in infectious diseases clinical hospital. the diagnosis was based by smear, and culture of vesicular fluid or csf. results: during this period fifteen cases of anthrax in humans were identified. the repartition of cases according to sex groups revealed a high predominance of male cases (13 cases). the disease was most prevalent in 35-44 age group and after 55years old (6 cases), although anthrax can be observed between 15 years and 65 years. in all cases anthrax was occupational disease and the provenience of patients was rural area. the annual incidence of anthrax was characterized by a low value and irregular frequency of the disease. the most cases were in 1994 (6 cases) and 2004 (5 cases). according to the seasonal repartition, anthrax was most prevalent in the summer months (11 cases). the most frequent clinical form of anthrax was the cutaneous anthrax (14 cases), and only one patient achieved anthrax meningitis. under specific treatment with penicillin g, all the patients with cutaneous anthrax recovery complete, only the patient with anthrax meningitis died. conclusions: in constantza county, anthrax remained sporadic in the last years. after a free period (2001) (2002) (2003) 5 cases of coutaneous anthrax were registered in 2004. a good cooperation between veterinary and physicians and individual awareness of the materials is the key to prevention of anthrax. hemolytic activity. all of the 30 strains tested have hemopeptic activity, which is indirect evidence of their virulence. the strains have no phosphatase enzymes or lecithinase. the strains we studied ferment glucose, sucrose, glycerin, and rhamnose, and they form acid without gas. the strains all grew in synthetic "a" medium, although growth was weak. when l-tyrosine, ltryptophan, l-threonine, and l-methionine were added to the synthetic "a" medium, the pathogen grows in the typical manner. two anthrax strains are moderately resistant to rifampicin (mic 1.5-16), and three are not sensitive. the anthrax strains are highly sensitive to tetracycline, benzylpenicillin, gentamycin, and oxacillin. conclusions: regardless of their source of isolation and time of storage, the anthrax strains isolated during outbreaks are virulent, spore-forming, and capsule-forming cultures. this study confirms that virulent forms of b. anthracis are still circulating in kazakhstan. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency under the project «kb0-1950-al-03» and administered by u.s. crdf. the importance of laboratory methods in diagnosing anthrax in humans l. lukhnova, a. aikimbayev, t. meka-mechenko, g. temiraliyeva, s. zakaryan, m. sagatova (almaty, kaz) objectives: isolation of anthrax bacillus using bacteriological methods is slow and frequently misleading. the literature suggests anthrax bacillus is isolated in 13% to 48% of patients infected with bacillus anthracis. the pathogen quickly perishes or transforms into atypical forms. the number of bacilli in a clinical specimen is often low or below the sensitivity of culture. this suggests other methods would be useful in establishing the diagnosis of anthrax, specifically; serological methods would be beneficial in diagnosing infection with bacillus anthracis. methods: we conducted studies to assess various methods for diagnosis of bacillus anthracis infection in patients. we reviewed archival data and our own results on pathological material from patients infected with bacillus anthracis in the southern and eastern kazakhstan oblasts. results: the most informative test for detection of anthrax in patients was the delayed type hypersensitivity (dth) or dermatoallergic test with anthraxin. eighty one per cent of patients with signs and symptoms of anthrax gave a positive reaction with anthraxin. the ascoli reaction complete with scab was positive in 72.1% of patients, and the indirect hemeagglutination (iha) test was positive in 65.6% of patients. the clinicoepidemiological diagnosis of anthrax was confirmed with culture in 44% of cases. serum samples from patients diagnosed with anthrax in the eastern kazakhstan oblast were evaluated using iha in 2001-2004. high titres of specific antibodies (1:640-1:5120) were detected in patients with dermatologic anthrax lesions from which anthrax was isolated. conclusion: our data suggest the anthraxin dth test and iha are good methods for establishing a diagnosis of anthrax. cultures for anthrax bacillus were the least efficient method for establishing the diagnosis. when results of laboratory tests are negative or inconclusive, one still has to rely on the clinicoepidemiological diagnosis. the research described in this abstract was made possible in part by support provided by the u.s. defense threat reduction agency (dtra) under the project «kb0-1950-al-03» and administered by u.s. civilian research and development foundation (crdf). objectives: the fluoroquinolones are bactericidal agents frequently used in the treatment of respiratory tract infections. the viridans streptococci (vgs), although associated with disease, generally exist as part of the normal human oral flora. as such, they may exposed to antibiotics with greater frequency than oppurtunistic pathogens. preliminary data suggest that the vgs may act as a reservoir for fluoroquinolone resistance in s.pneumoniae through horizontal transfer. here, we compared the killing kinetics of moxifloxacin (mxf) and levofloxacin (lev), against vgs at simulated saliva concentrations. methods: eight strains of vgs, 4 s. mitis (2 with parc mutation) and 4 s.oralis, (2 with parc mutation) were challenged with mxf and lev in a pharmacodynamic model utilizing mueller-hinton broth supplemented with 5% lysed horse blood. cultures were inoculated at a density of 1 · 108 cfu/ml, incubated at 35ordm;c, and examined for viable growth at 0, 1, 2, 4, 6, 12, and 24 hrs after exposure to the antibiotics at concentrations simulating salivary levels. salivary concentrations of mxf (400 mg), and lev (500 mg) were 3.19 and 3.71 lg/ml respectively. protein binding of mfx and lev were taken to be 50% and 30%. adjusted salivary levels were 1.6 (mxf) and 2.6 (lev) ug/ml. pcr was used to amplify parc and gyra, and mutations detected by dna sequencing. results: both mxf and lev were highly bactericidal against susceptible strains of vgs at salivary concentrations. mfx typically eradicated the vgs by 6 hours and lev by 12-24 hours. for parc mutants, both mxf and lev were consistently bactericidal resulting in eradication after12 hours for mxf and 24 hours for lev. regrowth was observed in both strains of s.mitis (parc) exposed to lev (2 of 10 experiments). the mic of these strains to lev increased 4 fold and dna sequencing revealed the selection of a gyra mutation in each strain. no regrowth was detected in the s. oralis strains. conclusions: both mxf and lev are extremely active against susceptible vgs at concentrations of drug found in saliva, however, superior killing kinetics were consistently observed with mxf. mxf consistently eradicated strains with a parc mutation, however, regrowth was observed on 2 occasions with lev suggesting that mxf may be less likely to drive resistance among vgs. again, this may have implications due to the ability of vgs to exchange its genetic determinants with s. pneumoniae. moxifloxacin and ciprofloxacin uptake by human neutrophils and their influence on viability, phagocytosis, oxidative burst, and chemotaxis a.m. berg, j. altrichter, b. drewelow, r.g. mundkowski (rostock, d) objectives: the influence of antibiotics on the immune defence is of increasing interest. the purpose of this study was to set up a cellular model for correlating the intracellular concentrations of the fluorochinolones moxifloxacin and ciprofloxacin with selected cell functions of early immune response. a human promyelotic cell line which is used for sepsis treatment by the extracorporeal immune support system (eissò, teraklin gmbh) was chosen to establish the model. the second stage involved native human polymorphonuclear neutrophiles (pmns) as target cells. methods: the promyelotic model cell line was differentiated towards neutrophils with all-trans retinoic acid, human pmns were isolated using dextran sulfate sedimentation and density gradient centrifugation. the cells were incubated with antibiotic concentrations within the therapeutic range and above (0.5-2 cmax) for time periods up to 8 hours. the extra-and intracellular concentrations were determined by hplc-uv. the viability of the cells was controlled using the trypanblue-exclusion assay. phagocytosis and oxidative burst were assessed by a combined flow cytometric assay after stimulation of the cells with serum-opsonised fluorescein-isothiocyanate-conjugated e. coli in the presence of dihydroethidium as an indicator of superoxid production. a two-compartement chamber system was used to study the influence of either fluorochinolone on the chemotaxis. results: with either cell type, moxifloxacin accumulated rapidly with peak concentrations within 15 min whereas intracellular maximum concentrations of ciprofloxacin were achieved after 2 h. the accumulation rate of both antibiotics inside the native pmns was more than twice as high as in differentiated eiss ò cells. neither moxifloxacin nor ciprofloxacin showed a significant influence on viability and the immune response parameters phagocytosis and oxidative burst of native pmns; chemotaxis ability was reduced to a minor degree.viability and phagocytosis of the diffentiated eiss ò -cells remain unaffected whereas the oxidative burst appeared decreased. chemotactic acticity of eiss ò -cells could not be stimulated. conclusions: a significant accumulation of moxifloxacin and ciprofloxacin was determined in both cell types. however, no relevant influence on viability, phagocytosis, oxidative burst, and chemotaxis was observed. hence no hints were found suggesting a restricted use of moxifloxacin and ciprofloxacin for immunocompromised patients. prospective, multicentre in vitro study to determine resistance rates and comparative activity of moxifloxacin vs. clinical bacterial isolates from patients with respiratory tract infections (moxiaktiv study) a. dalhoff, g. korfmann, e. jacobs (kiel, leverkusen, dresden, d) objective: to determine the prevalence of resistance in current clinical isolates of s. pneumoniae, h. influenzae, m. catarrhalis, s. aureus and k. pneumoniae as well as the in vitro susceptibility to moxifloxacin and other iv antibiotics in germany. methods: up to 20 isolates of each of the above-mentioned species were cultured on standard media and tested for in vitro susceptibility using etestò in each of the 29 study centres in germany. for validation, an atcc control strain was included for each species. drugs tested were moxifloxacin, levofloxacin, amoxicillin/clavulanate, cefuroxime, clarithromycin and penicillin g. the latter two were not tested for klebsiellae. s. aureus was additionally tested for oxacillin resistance and k. pneumoniae was tested for production of extended spectrum beta-lactamases (esbl). din breakpoints were used where applicable. results: overall, 1859 pathogens of 1,811 hospitalised patients with respiratory tract infections were analysed. results for moxifloxacin are summarized in table 1. of s. pneumoniae isolates 93.3% were susceptible to penicillin g and 85.7% to clarithromycin. with an mic90 of 1 mg/l, the in vitro activity of levofloxacin was markedly lower than that of moxifloxacin. h. influenzae showed almost 100% susceptibility to fluoroquinolones, but only 4.5% to clarithromycin (mic90 32 mg/l). as beta-lactamase production is common among h. influenzae isolates in germany, amoxicillin/clavulanate showed far higher susceptibility rates than penicillin g (87.9% vs. 1.0%). due to a high rate of beta-lactamase production only 9.8% of moraxella isolates were susceptible to penicillin g, compared with 99.1% to amoxicillin/clavulanate. moraxella isolates were fully susceptible to the fluoroquinolones. overall, 13.0% of klebsiella isolates produced an esbl. interestingly, esbl prevalence was 38.8% in eastern germany, but below 10% in western germany. esbl producers of klebsiella were less susceptible to antibiotics other than cefotaxime or ceftazidime than non-esbl producers. conclusion: for all species tested, the fluoroquinolones achieved the highest overall susceptibility rate (92.8%) compared to the other antibiotics (penicillin g: 36.9%, clarithromycin: 60.5%, ampicillin/clavulanate: 85.7%, cefuroxime: 89.6%). moxifloxacin showed a high activity against current respiratory pathogens in germany and was the most active fluoroquinolone, in particular against gram-positive pathogens. objectives: drug combinations have become the best choice in treatment of serious infections with resistant microorganisms including p. aeruginosa (pa). we tested ciprofloxacin (cip) in double and triple combinations with ceftazidime (caz), imipenem (imp), piperacillin (pip), and amikacin (amk) against 20 mdr clinical isolates of pa. methods: the mic for each drug alone was determined by broth microdilution technique described by nccls. double and triple combinations of cip with other drugs were tested by using 96-well plates and by time-kill assay. rows a to g were activity of moxifloxacin on s. pneumoniae (sp), h. influenzae (hi) and m. catarrhalis (mc) clinical isolates collected from the respiratory tract by determination of the mic's using the etest and to compare these results against 9 other antimicrobial agents.these results were also compared for sp versus those of the previous study (winter periods 2000-2002) for interpretive criteria s -i -r (according the nccls -breakpoints). methods: 10 belgian university and non-university medical microbiology laboratories participated during the past winter period in this multicentre in-vitro evaluation of moxifloxacin. each laboratory included 40 strains of sp and 20 strains of hi and mc. the total number of evaluable strains (with the exclusion of duplicate isolates) was 758. testing conditions: method and antibiotics: susceptibility testing was performed in the participating centres by using the e-test. the antibiotics tested were penicillin, ampicillin, amoxi/clav., doxycycline, clarithromycin, cefuroxime, ceftriaxone, ciprofloxacin, levofloxacin and moxifloxacin.medium : sp and mc : mueller-hinton agar with 5% blood; hi : htm agar. inoculum : direct suspension and 0.5 mc farland standard.incubation : 35°c for 24 hours in 5% co 2 . quality control was performed using atcc reference strains (s. pneumoniae atcc 49619 ; h. influenzae atcc 49766). results: see graphs 1 and 2. conclusions: this follow-up belgian multicentre study confirms the excellent in-vitro potency of moxifloxacin against clinical isolates of sp, hi and mc and it demonstrates that it is the most potent fluoroquinolone available in belgium for the treatment of lower respiratory tract infections. according the nccls -breakpoints, there are no major s -i -r differences for sp between these results (2003) (2004) and the results of the previous study (2000) (2001) (2002) . this means that the use of new fluoroquinolones like moxifloxacin and levofloxacin has not led, to date, to an increase of resistance in sp. however, repeated follow-up surveys will continue to be needed in order to assess the activity of fluoroquinolones and to monitor the ecological impact of the recently increased fluoroquinolone usage for respiratory tract infections in belgium in the forthcoming years. objectives: the objective of several experiments was to evaluate the in vitro activity of moxifloxacin against porphyromonas gingivalis (p.g.). methods: mics of moxifloxacin against 16 strains of p.g. were determined by e-test (ab biodisk, solna, sweden). furthermore the spontaneous mutation rate and the induction of resistant strains by the 0.25-fold mic of the antibiotic were determined. to find the target of resistance fragments of gyra and gyrb were sequenced. finally the efficacy of moxifloxacin against p. gingivalis atcc 33277 considering special conditions such as effects on the strain in a biofilm or within epithelial cells (kb cells) was evaluated. results: moxifloxacin had very low mic values (ranging from 0.006-0.032 lg/ml), but subinhibitory concentrations of the fluoroquinolone induced very fast mutations. the spontaneous mutation rate was up to 5 · 10 )8 after the two-fold mic and 1.2 · 10 )8 after the eight-fold mic. often the mutants exhibited a high resistance mics >32 mg/l. all these mutants bore ser-83->phe substitution in gyra. the 5-fold mic eliminated p.g. atcc 33277 within biofilms after 24 h, and the 100-fold mic was able to kill all intracellular p. gingivalis within kb cells. conclusions: moxifloxacin showed a very good activity against planctonic p. gingivalis and p.g. within a biofilm. this antibiotic in a higher concentration was also efficient to intracellular bacteria. but a rapid development of resistance was observed in laboratory. moxifloxacin might be an alternative in the antibiotic treatment of p. gingivalis associated periodontitis, nevertheless clinical studies should focus not only on improvement of clinical parameters but also on occurrence of resistant strains. levofloxacin activity on respiratory isolates of streptococcus pneumoniae with decreased susceptibility to ciprofloxacin in spain j. garcía-de-lomas, j.l. juan-bañ ó n, c. garcía-rey, r. dal-ré on behalf of the spanish surveillance group for respiratory pathogens objectives: to test the activity of levofloxacin against recent respiratory isolates of s. pneumoniae with reduced susceptibility to ciprofloxacin (mic ‡ 2 mg/l; n = 607) belonging to the national surveillance sauce-3 (n = 2721; nov01-oct02) and to identify chromosomal mutations in isolates with a levofloxacin mic ‡ 4 mg/l. methods: the activity of levofloxacin was tested by broth microdilution following nccls recommendations. genotypic sequenciation of the qrdr (gyra, gyrb, parc and pare) was done on all the strains with a levofloxacin mic ‡ 4 mg/l. results: levofloxacin mic distribution, mic50 and mic90 (in bold) are given in the table for the whole sample and broken into ciprofloxacin mic categories. mutations in gyra (e85k) were found in 8 (18.6%) strains. no gyrb mutations were found. mutation in parc occurred in 100% these strains (26 s79f; 12 s79y; 2 s79i; 1 s79r; 1 d831; 1 d83n). finally, mutation of pare was found in 17 (39.5%) strains and all of them had the change i460v. one single parc mutation occurred in 24 (55.8%) strains, (1999) (2000) (2001) (2002) ; >99.9% were at £0.12 mg/l conclusions: gemi continues to be a highly potent fq when tested against the two most commonly isolated gram-negative carti pathogens; h. influenzae and m. catarrhalis. this potency and spectrum was consistent across six years and three continents. norfloxacin as a marker of decreased susceptibility to fluoroquinolones in enterobacteria using the vitek 2 system l. ló pez, j. alcala, f. torres, e.j. perea, a. pascual (seville, e) objective: the detection of nalidixic acid resistance acquires importance in enterobacteria since already resistant strains develop more frequently resistance to fluorquinolones than susceptible strains. many clinical laboratories have incorporated the vitek 2 system because their benefit in routine, but nalidixic testing is not included for enterobacteria cards. the aim of this study was the use of norfloxacin vitek mics value as alternative marker to detect nalidixic acid resistant enterobacteria strains. material and methods: a total of 1013 isolates of enterobacteriaceae were analyzed and routinely inoculated into the vitek 2 id-gnb and ast-n020 cards (biomérieux) and then loaded into the vitek 2 system, as recommended by the manufacturer. ninety-six strains with a higher norfloxacin vitek mic value (1-4 mg/l), but still within the susceptibility range established by the nccls breakpoints, and 100 strains with a norfloxacin vitek mic £0.5 mg/l were selected. they were further tested with a 30 microg-nalidixic acid-disk (nal) (oxoid) according to nccls reference. results: the norfloxacin 1-4 mg/l and susceptible to ciprofloxacin phenotype represented the 9% of total isolates loaded in the vitek system during the study, similar to the prevalence observed in previous analysis in spain. both selected groups, one with the highest and the other with the lowest norfloxacin vitek mic value, included 60% and 58% e. coli isolates, 8% and 20% s. enterica isolates, respectively. among the norfloxacin vitek mic 1-4 mg/l strains 93 isolates (98%) were nal resistant, and 76 (79%) were ciprofloxacin susceptible. the agreement between norfloxacin vitek mic £0.5 mg/l and nal susceptibility was foun in 97 (97%) strains. two isolates (one e. cloacae and one k. pneumoniae) with norfloxacin vitek mic 1-4 mg/l were nal-s and 3 isolates (two e. coli and one p. mirabilis) with norfloxacin vitek mic £0.5 mg/l were nal-r. conclusion: when using vitek 2 system for enterobacteria, the consideration of mic of norfloxacin >0.5 mg/l could be an alternative marker of nalidixic acid resistance (and decreased susceptibility to fluoroquinolones). antibacterial susceptibility studies -i objectives: p. aeruginosa is an important nosocomial pathogen, which causes serious and often lethal infections in immunocompromised hosts. this pathogen is intrinsically resistant to many antibiotics and can easily develop resistance towards many currently available agents. natural resistance can be attributed to the low permeability of the p.aeruginosa outer membrane to a variety of antibiotics including glycopeptides (glys). since glys are powerful antibiotics against grampositive bacteria and resistance is very rarely develop, it seemed interesting to evaluate the effect of combining these antimicrobial agents with antibiotics that might disorganize the structure of the outer membrane allowing the entrance of glicopeptides into the gram-negative cells. in order to verify this hypothesis, ceftazidime (caz) has been tested in association with vancomycin (van) or teicoplanin (tei). the same experiments have been carried out also in the presence of azithromycin (azi), which is normally a non anti-pseudomonal agents but has been shown to interfere with some virulence factors. methods: a bacterial suspension of about 10 9 cfu/ml was seeded on plates containing a fixed concentration of glys (500 mg/l) and increasing doses (2x,4x,8x,16x) of caz. survivors were counted after 48 hr at 37°c. results were interpreted as synergism (99%), additivity (90%), and indifference (10%) of the cfu/ml reduction found in the drugs combination in comparison to the drug alone.the same experiments have been repeated adding azi (16 mg/l) and using glys at concentrations ranging from 500 to 300 mg/l. results: caz in combination with glys reacted synergically in 20 out of 59 cases, additivity was found in 31/59 interactions and indifference was noted in 8/59 tests. preliminary results (12 tests performed) indicated that the addition of azi increased the incidence of synergisms and additivities even when using glys concentration of 300 mg/l. conclusions: caz combined with glys gave additive or synergistic results in the great majority of experiments, while the simultaneous combination of azi, caz and a gly always produce an additive or synergistic effect against p. aeruginosa. these data, given the high concentration of glys employed, could be of particular interest in clinical situations where the drugs could be topically administered. a multicentre correlation study of vitek 2 with reference methods for telithromycin, mupirocin, and daptomycin against staphylococcus spp. k.e. aldridge, a. dizney, k. thomson, g. procop (new orleans, omaha, cleveland, usa) objective: clinical studies have shown that appropriate antimicrobial therapy based on in vitro susceptibility data correlates with better patient outcome. vitek 2 is a fully automated susceptibility testing methodology providing rapid(6-8 h) results for a variety of aerobic and facultatively anaerobic gram-positive and -negative pathogens which have been shown to correlate to established reference methods. the objective of this study was to correlate the susceptibility results of vitek 2 testing of telithromycin, mupirocin, and daptomycin against staphylococcus spp. with those from reference methodology. methods: three geographically distinct clinical microbiology laboratories participated in the testing with each site testing 100 consecutive clinical islates and 84 blinded challenge isolates of staphylococci sent to each site.vitek 2 testing was performed according to the manufacturer's recommendations. the challenge isolates were also tested using a manual preparation of the inoculum. nccls recommended reference agar and broth microdilution methods were used to test telithromycin, mupirocin, and daptomycin. mic results were collated to determine the per cent (%) of isolates susceptible ( in vitro efficacy of tigecycline tested against community-acquired respiratory tract infection and nosocomial pneumonia pathogens conclusions: among r subsets of commonly occurring pathogens, 97% were inhibited by £2 mg/l of tig and >99% were inhibited by £4 mg/l (the current nccls breakpoint for tet). tig is highly stable to most tet-r determinants, including protected ribosomes and efflux mechanisms, and may represent a superior choice among parenteral agents for broad-spectrum coverage, including the most commonly occurring-and problematic-r phenotypes. determination of the minimum inhibitory and mutant prevention concentration of telithromycin against clinical isolates of streptococcus pneumoniae s. borsos, c. hesje, l. blondeau, j. blondeau saskatoon, can objective: telithromycin (tl) is a novel ketolide antimicrobial agent that has recently been approved for use in north america for respiratory tract infections of which sp is a principal pathogen. the global escalation of penicillin (p) and macrolide (m) resistant sp has compromised the use of beta-lactam and macrolide compounds. tl was reported to be active against p and m resistant sp. objectives: garenoxacin (grn) is an investigational des-f(6)quinolone with enhanced in vitro activity against gram-positive cocci. the global escalation in antimicrobial resistance amongst respiratory tract pathogens -particularly sp-has highlighted the importance of quinolone compounds for treating sp infections. the mutant prevention concentration (mpc) is a novel in vitro measurement that defines the drug concentration threshold that would require an organism to simultaneously acquire two or more resistance mutations for growth in the presence of the drug. we measured the mics and mpcs for grn against clinical isolates of sp, including multidrug resistant strains and those with elevated mpcs to levofloxacingrn. methods: microbroth dilution in accordance with nccls guidelines was followed for mic testing using todd-hewitt broth and two-fold concentration drug increments. for mpc testing, ‡1 billion organisms were applied to agar plates containing grn, incubated at 35-37°c and 5% co 2 and screened for colony growth at 24 and 48 hours. the mpc was the lowest drug concentration that completely prevented growth. select organisms with grn mpc values >2 lg/ml were screened for amino acid substitution in the quinolone resistance determining region. great concern nowadays because the therapeutical alternatives are limited. fusidic acid inhibits protein synthesis. it has only a few side effects and is usually well-tolerated by patients as an oral drug. fusidic acid can be considered as an alternative drug for the treatment of infections due to both methicillin susceptible and resistant s. aureus strains. evaluation of synergy between glycopeptides, levofloxacin and beta-lactams against methicillinresistant staphylococcus aureus (imi) + va/tp, lvx + piperacillin/tazobactam (ptz) + va/tp] were considered. synergy was evaluated by means of checkerboard assay against 50 (double combinations) and 25 (triple combination) mrsa strains isolated from respiratory tract infections and by means of time kill curves (5 strains). in checkerboard assay synergy was defined as fractional inhibitory concentration index (fici) of <0.5, additivity as fici >0.5 and >1, indifference as fici >1 and antagonism as fici >2, while in time kill studies synergy was defined as a >2 log decrease in bacterial count of combinations in respect to the most active single drug. moreover, mutational rates of single and combined drugs at antimicrobial concentrations equal to the resistance breakpoints were calculated in 10 strains results: synergy and additivity were the prevalent effects, while no antagonism was observed in checkerboard assay. in particular, lvx + va/tp and ctx + tp/va gave synergy in 16/50, 9/50, 43/50 and 23/50 strains, respectively. combinations of lvx + va + caz/cpm/ptz and of lvx + tp + caz/cpm/ptz yielded synergy or additivity in all the tested strains, with combinations of lvx + tp + caz / ptz giving synergy in 20/25 strains. time kill curves evidenced synergy for lvx/ctx + va/tp after 12 and 24 h of incubation. synergistic triple combinations occurred more frequently with lvx + tp than with lvx + va after 12 and 24 h. for single antibiotics, mutational frequencies ranged between 10-5 and <10-9 for lvx, ctx, amk and imi, and <10-9 for va and tp. when tested in double and triple combinations, mutational frequencies fell below 10-9 for all the combinations. conclusion: the study provide in vitro evidence of synergy between glycopeptides with fluoroquinolones, cephalosporins and beta-lactams and of limitations of occurrence of mutations in mrsa strains responsible of respiratory infections, thus underlining a potential role in therapy of such infections. comparative activity (mic and mbc) of daptomycin, quinupristin/dalfopristin, linezolid, vancomycin and teicoplanin against a large worldwide collection of visa/hvisa a. bolmströ m, a. engelhardt, å . karlsson, e. edling, p. ho (solna, s) objective: the worldwide emergence and possible dissemination of vancomycin (va) resistance in staphylococci involving vana (vrs), visa and hetero-visa phenotypes comprise formidable clinical threats. comparisons of the in vitro activity of newer gram positive agents using larger worldwide collections of visa strains become increasingly important both for individual therapy guidance and for epidemiologic purposes. method: a 10 country collection of 243 staphylococci (217 mrsa, 26 mssa), of which 150 were visa (e-test macromethod and pap-auc, walsh et al, jcm 2001) was assessed. mic of dp, qd, lz, va and tp were determined using the nccls broth microdilution (bmd) and e-test. mbc for all agents for a subset of 46 visa and non-visa strains were determined using an etest mbc procedure and bmd. results: inter-method agreement (n = 2430) for all antibiotics were ±95% ±1 dilution for mic and ±80% for mbc results. the continuous gradient format in e-test allowed detection of subtle upward shifts in mic and mbc distributions of visa, as well as the detection of persister colonies in mbc assays seen with the less bactericidal compounds. conclusion: mic distributions showed that daptomycin and linezolid maintained potency against the large collection of visa while quinupristin/dalfopristin and glycopeptides showed a shift towards reduced activity. mbc results showed daptomycin to be superior to linezolid (bacteriostatic) and the other agents. the potential usefulness of newer agents for visa should be continually assessed with respect to both their static and cidal activities in order to detect subtle shifts in susceptibility as early as possible. performance of a unique e-test daptomycin gradient + calcium gradient on isosensitest compared to nccls broth microdilution in mueller hinton objective: daptomycin (dp) requires physiologic levels of free calcium ions (ca 2+ ) for expression of adequate activity. susceptibility testing methods can be significantly influenced by ca 2+ variations in agar and broth. nccls broth microdilution (bmd) uses a final physiologic level of 50 lg/ml calcium for dp testing. e-test dp is a unique gradient which incorporates a constant level of calcium. e-test dp has been shown to be equivalent to bmd when tested with commercially available mueller hinton (mh) agar. since isosensitest (iso) is used in some parts of europe, and has an inherently low level of calcium (approximately 5-10 lg/ml), it is important to evaluate the performance of e-test dp on iso in comparison to bmd. method: a 3 lab study compared e-test dp on iso to nccls bmd. a total of 760 clinical and stock strains comprising clindamycin (cc), ciprofloxacin (c) and tetracycline (t) and also to determine whether there are any differences in the antimicrobial susceptibility patters among the difference species of the vgs and s. bovis. methods: the 145 vgs and 10 s. bovis were isolated from blood and identified at species level by id 32-strep system (68 s. mitis, 44 s. anginosus, 25 s. sanguis, 7 s. salivarius and 1 s. mutans). mics were determined by the agar dilution method and the percentages of resistance determined according to nccls criteria for streptococci other than s. pneumoniae. results: overall, 64.5% of the streptococci were resistant at least to one of the antibiotic tested and of these, 32% was resistant to 3 or more. the most susceptible species were s. anginosus and s. salivarius (57% susceptibility to all antibiotics tested). the only s. mutans strain was susceptible to all the antibiotics tested. the resistance phenotypes for the different species are shown in the table. conclusions: as expected cc resistance was always associated with e resistance. overall, the phenotype most frequently founded was ecct in 24 strains (15.5%) and it was present in the 70% of s. bovis and 27% of s. anginosus. the pattern eccpt was the second more common phenotype (12.3%) and the most frequent in s. sanguis and s. mitis with 20% and 17.6% respectively. s. mitis was the specie that showed more number of phenotypes (17). heterogeneity in the susceptibility patterns in the species of vgs indicates the need for accurate identification. in vitro antibacterial activity of temocillin against extended spectrum beta-lactamases enterobacteriaceae producing clinical isolates objectives: temocillin is a methoxy-derivative of ticarcillin, active on enterobacteriaceae and stable against b-lactamases, including ampc and extended spectrum b-lactamases (esbl). however data concerning its activity on esbl producing strains are limited. the aim of this study was describe the range of minimal inhibitory concentration (mic) of temocillin in different species of esbl producing enterobacteriaceae from a single tertiary care centre in belgium . methods: esbl producing enterobacteriaceae (172 e. aerogenes, 164 e. coli, 104 e. cloacae, 58 k. pneumoniae and 35 other ) strains from patients hospitalised in our institution during the period of 1 january 2000 to 31 december 2003. esbl production was screened by double-disk test and confirmed by oxoid combined disk method. characterisation of enzymes was performed by multiplex pcr for bla tem, bla shv and bla ctx-m gene families detection and by dna sequencing and/or iso-electric focusing. mics (mg/l) were determined by agar dilution method according to nccls guidelines. susceptibility was determined according to breakpoints provided by fuchs et al: susceptible mic £ 16 mg/l. (eur j clin microbiol 1985) results: isolates originated from screening rectal swabs (28%), urinary tract (25%), respiratory tract (19%), wound (10%), blood (6%), gastrointestinal tract (4%), or other sites (8%) from 135 male and 233 female patients with a mean age of 61 (range, 0-94) years. the antimicrobial activity of temocillin against esbl producing enterobacteriaceae is summarised in the table. conclusions: this study confirmed the good in vitro activity of temocillin against multiresistant esbl-producing clinical isolates of enterobacteriaceae. its activity included enterobacter ampc depressed mutant strains that co-produced multiple esbl. these data suggest that temocillin could be a valuable drug to be considered in the treatment of infections by some esbl-producing enterobacteriaceae. in vitro activity of ertapenem against cefpodoxime-resistant gram-negative bacilli from urine introduction: resistance to commonly used antibiotics for urinary tract infection (uti) is of growing concern. this is due to the emergence of extended-spectrum beta-lactamase (esbl) producing bacteria in the community with reports from different parts of the world. the appearance and dissemination of these resistant bacteria cause serious concerns for the treatment of urinary tract infections (utis). objectives: ertapenem is the latest member of the carbapenem class of antimicrobials. the aim of this study was to assess the activity of ertapenem against a collection of cefpodoximeresistant gram-negative bacilli isolated from urine samples of community and hospital-based patients. materials and methods: between june and october 2004, our laboratory received 10,234 urine samples. of these 2246 gave a positive culture, of which 446 from hospitalised patients and 1800 from the community. of these 258 were gram negative bacilli resistant to cefpodoxime 1 mg/l. the identity of the isolates was confirmed using api 20e or api 20ne (biomerieux, france). esbl production was sought using the mastdd and the identity of the resistant determinant(s) was carried out using a combination of pcr and nucleotide sequence analysis techdifferent antibiotics in order to evaluate alternatives for intrapartum chemoprophylaxis to women allergic to penicillin and colonized by a gbs strain resistant to macrolides and/or lincosamides; (2) characterize the mechanisms of resistance to macrolides and lincosamide; (3) evaluate the clonal relationship between these strains. methods: a total of 610 strains collected in a multicentre study: 131 isolated between 1997 and 2002 from newborns diagnosed of early-onset gbs disease and 479 strains collected in 2002 from vagina or rectum of pregnant women. microdilution method was used to study the antimicrobial susceptibility and disk diffusion to define the macrolide-lincosamide resistance phenotype. pcr was performed to determine the presence of ermb, ermtr and mefa resistance genes. to evaluate the clonal relationship, pfge of total dna was done using smai. results: all the strains were susceptible to penicillin, ampicillin, vancomycin, quinupristin/dalfopristin, levofloxacin and teicoplanin. the 12.45% were resistant to erythromycin and azithromycin, 11.80% to josamycin, 11.31% to clindamycin, 1.80% to telithromycin and 0.32% to fosfomycin. among the macrolide resistant strains, the 90.8% presented a constitutive mlsb phenotype (cmlsb), 5.26% an inducible mlsb phenotype (imlsb) and the remaining 3.94% a m phenotype. three strains were susceptible to macrolides and resistant to clindamycin. the ermb, ermtr and mefa genes were presented in 63.2%, 30.3% and 3.9% of the macrolide resistant strains. two strains did not amplified none of the studied genes. the 100% of the strains harbouring the ermb gene showed a constitutive phenotype. all the strains showing an inducible phenotype harboured the ermtr gene. the 11 telithromycin resistant strains presented a cmlsb phenotype; of them 10 posses the ermb gene and one the ermtr. conclusion: in spain, there is a high rate of resistance to macrolides and lincosamides that makes mandatory to perform susceptibility testing to gbs strains isolated from pregnant women allergic to penicillin. in our region ermb methilase is the main cause of macrolide resistance, followed by the ermtr and the macrolide eflux pump in a low proportion. there is a wide clonal diversity among resistant strains to macrolides, lincosamides and telithromycin. detection of the ermx determinant of macrolide resistance in clinical strains of corynebacterium urealyticum a. ortiz, j.i. garcía-cía, r. fernández-roblas, j. esteban (madrid, e) objectives: to detect the presence of the ermx macrolide resistance gene in clinical isolates of c. urealyticum. methods: 57 c. urealyticum strains were isolated from clinical samples in the fundació n jiménez díaz hospital. the strains were maintained frozen until the studies to evaluate macrolide resistance were performed. antibiotic susceptibility testing was performed by agar dilution assay in 14 cases and by disc diffusion assay in 43 cases. detection of ermx gene was performed by using primers cerm-1 and cerm-2 according to the protocol described by rosato et al. dna was extracted by boiling a suspension of bacteria in distilled water. amplification products were analysed by agarose gel electrophoresis and molecular weight of the amplicons were calculated by using the photocapt software (biogene, usa). results: 46 strains were resistant to erythromycin and clindamycin, and 11 were susceptible to both antimicrobials. we detected the gen ermx in 34 cases (all of them resistant). 12 macrolide-resistant strains gave negative results for ermx detection. the susceptible strains did not show any amplification product. the ermx gene is detected in 73.9 % of macrolideresistant corynebacterium urealyticum strains. however, there were 26.1 % macrolide-resistant strains in which ermx gene was not detected. other resistance determinants must be involved in macrolide resistance among corynebacterium urealyticum. characterisation of phenotype and genotype of macrolide-resistant streptococcus pyogenes objectives: the resistance of streptococcus pyogenes to macrolide is a world wide problem. the aim of present study was to determine the prevalence of antibiotic resistance rate of erythromycin (em) resistant isolates from korea, and evaluate their genetic mechanism, phenotype distribution and clonal relationship. methods: total 51 em resistant s. pyogenes from clinical specimens from 1996 to 2003 were studied; agar dilution method to determine minimal inhibitory concentrations (mics) to 9 antimicrobial agents was performed. resistance phenotypes were determined by triple-disc test and resistance induction experiment with sub-inhibitory concentration of erythromycin (0.05 lg/ml). their genetic mechanisms of resistance were determined by pcr. the genetic relatedness of them was also investigated by means of emm genotyping and random amplified polymorphic dna (rapd) analysis. results: an average em resistance rate was 23% and their cross-resistance rate to clarithromycin, azithromycin, and spiramycin were 100%, 98%, and 67%, respectively. the most common resistance phenotype was inducible mls (imls; 51%), followed by constitutive mls (cmls;31%), and m type (18%). 18 of 26 imls isolates (69% ) showed novel imlsd phenotype, which had small (9-12 mm) inhibition zones around all three discs on triple-disc test and high level resistance to clinamycin and spiramycin after erythromycin induction. all isolates of the cmlsa and imlsd harboured ermb gene, while imlsc and m isolates harboured erma and mefa gene, respectively. all imlsd isolates were emm12 except one while all multidrug resistance cmlsa isolates were emm28 genotype. imlsd isolates made a tight cluster on phylogenetic tree and 78% of them showed a common pattern by rapd analysis. conclusion: imlsd was most common macrolide resistance phenotype in korea and emm gene and rapd analysis was suggestive of its clonal relationship. detection of inducible clindamycin resistance in cutaneous staphylococci clinical isolates by phenotypic and genotypic method , and 17 (14.5%) isolates with constitutive clindamycin resistance (7 s. aureus and 10 coagulase-negative staphylococci). the mrsa gene was present in all the clindamycin susceptible isolates except for 1 cns. among the 40 isolates with clindamycin inducible resistance, 37 isolates possess either erma or ermc while 1 cns isolate possess both ermc and mrsa. one cns and 1 s. aureus isolates were negative for all the genes tested. all 17 isolates with constitutive clindamycin resistance amplified to any of the genes tested. among the s. aureus isolates, 2 have the erma gene, 4 the ermc and 1 both ermc and mrsa genes. all the cns isolates possess the ermc but 3 of them possess also the mrsa gene (table 1) . the disk-diffusion test detected all the clindamycin resistant strains, and none of the clindamycin inducible resistant strains was detected by the microscan. the ermc gen was the most prevalent among the clindamycin resistant, in both cns and s. aureus isolates. ribosomal protein l22 mutations in bacillus anthracis associated with cross-resistance between macrolide, lincosamide, streptogramin and ketolide antibiotics rob chemother 2004; 54: 424) . the aim of this study was to determine ribosomal mutations associated with this resistance. methods: primers were designed and used to amplify and sequence the l4, l22 riboprotein genes and the 11 copies of the 23s rrna gene using b. anthracis str. ames complete genome (genbank accession no. nc_003997). results: (mics in mg/l) two significant mutations associated with resistance were found. a 4 amino acid (aa) insertion (a tandem duplication of 90mgra93) into the l22 gene at position 94 was found in the st-1 strain with a quinupristin/dalfopristin (q/d) mic of 128 selected after 18 passages in q/d. this strain was cross-resistant to telithromycin, erythromycin, clarithromycin and clindamycin with mics of 16, 64, 16 and 8 respectively. an 8 amino acid insertion (mgramgra) into the l22 gene (also at position 94) was found in the sterne strain with a clarithromycin mic of 64 after 18 passages in clarithromycin. this strain was cross-resistant to telithromycin, q/d, erythromycin and clindamycin with mics of 16, 8, 32 and 1 respectively. conclusions: tandem duplications of l22 90mgra93 were found to be important in mlsk resistance in 2 separate strains of b. anthracis demonstrating that this locus is important for resistance development under selective pressure of q/d and clarithromycin. importantly, these mutations were also associated with cross-resistance to other mlsk antibiotics. horizontal transfer of the mlsb resistance gene erm(b) between the human pathogen clostridium difficile and the ruminal anaerobe butyrivibrio fibrisolvens p. mastrantonio, f. barbanti, p. spigaglia (rome, i) objectives: the aim of this study was to investigate the possibility of in vitro transfer of the mlsb resistance gene, erm(b) between the human pathogen clostridium difficile and the ruminal commensal butyrivibrio fibrisolvens. methods: the erm(b)-positive c. difficile strain cd51 was used in filter matings as donor, whereasthe b. fibrisolvens strain 1.230, tetracycline resistant, and the b. fibrisolvens strain 2221r, rifampicin resistant, were used as recipients. the strains were cultured in m2gsc broth, with 30% of sterile rumen, in anaerobic conditions and the filter matings were performed on sheep blood agar plates, supplemented with hemin (0.1%) and vitamin k (0.1%). the same medium, supplemented with erythromycin (20 mcg/ml) and tetracycline (10 mcg/ml), were used to select the transconjugants. mic values were assessed by e-tests. the erm(b) transfer was confirmed by pcr and by hybridisation assays, using an erm(b) probe on transconjugant genomes digested with pvuii and with smai (pfge). results: the transfer average frequency was 5 · 10 )7 . mic values for erythromycin were >256 mg/l both in donor and transconjugants. an internal fragment of the erm(b) gene (730 bp) was amplified in all the transconjugants and a specific band was visualised in hybridisation assays on transconjugant genomes digested with both pvuii and smai. the re-transfer of the erm(b) gene from b. fibrisolvens transconjugants to c. difficile 1977, an erythromycin susceptible strain, was also obtained. conclusion: the in vitro transfer of the mlsb resistance gene erm(b) from c. difficile to b. fibrisolvens and vice-versa, is an important result supporting the possibility that horizontal transfer of resistant genes between human pathogenic bacteria and animal commensal microorganisms could occur, under natural conditions, more easily than expected. candida spp. attach on polymers, create a biofilm protecting the yeast and pose a reservoir for entering the bloodstream. candida spp. in biofilm are less susceptible to the antifungal drugs currently in use. we investigated the in vitro antifungal activity of micafungin (mcf) against biofilms of c. albicans, c. parapsilosis, c. dubliniensis, c. tropicalis, c. glabrata and c. kefyr. methods: biofilm formation was performed in vitro on polystyrene 96-well plates and on hickman catheter discs with minor modifications to kuhn (1). mcf was tested at six concentrations (0.25-32 lg/ml). measurements were done by xtt reduction assay at 490nm, % inhibition was calculated in comparison to the growth control. the biphasic structure of biofilms were imaged by reverse light-through microscopy. results: on polystyrene plates and on hickman catheters all candida spp. produced intermediate biofilm after 24-48h incubation. at all concentrations on polystyrene plates micafungin reached at maximum 50% inhibition in biofilm against all candida spp.: c. albicans (47% at 16 lg/ml), c. parapsilosis (49% at 16 lg/ml), c. dubliniensis (42% at 16 lg/ml), c. tropicalis (28% at 16 lg/ml), except for c. glabrata (73% at 8 lg/ml) and c. kefyr (53% at 1 lg/ml) methods: this multicentre, randomized, double-blind study compared mem with ipm (both 500 mg iv every 8 hours) in patients hospitalized with csssi. the primary efficacy endpoints were clinical and bacteriological responses at follow-up in the fully evaluable (fe) patient population (all patients meeting eligibility criteria and who had an identified pathogen prior to treatment) polymicrobial infections were seen in 38% of all documented infection s. agalactiae (n = 59; 100% s to mem and ipm), and e. faecalis (n = 52; 82% s to mem and 100% s to ipm) pseudomonas aeruginosa (n = 53; 94% s to mem and 92% s to ipm), bacteroides spp. (n = 74; 97% s to mem and 100% s to ipm), and peptostreptococcus spp 3% (mem) and 81.2% (ipm) in patients with polymicrobial infections; bacteriological (documented or presumed eradication, colonization) response rates were 79 we used a cox proportional hazard (cph) model adjusting for potential clinical and microbiologic risk factors, to evaluate los. results: among the 1116 modified intent-to-treat patients, the most common infection diagnoses at baseline were cellulitis (58.9%) and major abscess (27.9%). the most common underlying etiologies were spontaneous infection (52.3%) and trauma (27.6%). about 20.2% of patients had diabetes; 6.9% had peripheral vascular disease. staphylococcus aureus was the most common causative pathogen (about 50% of patients) among 540 patients with confirmed microbiology and complete hospitalization data. however, about 18% of patients had a gramnegative causative pathogen among 540 patients with confirmed microbiology, staphylococcus aureus was the most common causative pathogen but varied significantly in prevalence across regions (about 50% of patients overall, 82% in the us, 67 to 68% in europe and asia, and 39% in south africa). about assigned for the drugs as follows: row a for cip alone, b for second drug, c for third drug, d for cip + second drug, e for cip + third drug, f for second drug + third drug and g for the three drugs together. row h was left for drug-free control. briefly, all wells were filled with 50 ll of cation adjusted muller hinton broth. the drugs at 2x of the highest tested concentrations in 50 ll of the medium (alone, in double or in triple combinations) were delivered to wells a1-g1. two-fold serial drugs. results: the best synergistic effects were observed in combination of amk with caz (80%) or pip (80%) and in combination of caz with pip (60%) or cip (60%) as quinolones are widely used for treating sp infections and quinolone resistance is a concern, we tested gm-a newly approved compound-by mic and mpc against ps and prsp. methods: mic testing was by microbroth dilution in accordance with nccls guidelines. for mpc testing, 10 billion organisms were applied to agar plates containing drug and read at 24 and 48 hours following incubation. the lowest concentration preventing any growth was the mpc. organisms with high mpc values ( ‡2 lg/ml) were screened for amino acid substitution in the quinolone resistance determining region. results: a total of 402 clinical sp isolates were tested: 320 ps, 69 penicillin intermediate (pi) and 13 pr. the following represents the mic50/90 (lg/ml) and range (lg/ml) respectively against pr tr) objective: to determine the in-vitro activities of moxifloxacin and ciprofloxacin against methicillin-susceptible s. aureus (mssa) and methicillin-resistant s. aureus (mrsa) strains. methods: the study was conducted in a university hospital in turkey. a total of 440 non-repeat isolates (196 mssa and 244 mrsa) from various clinical specimens were included in the study. all isolates were identified as s. aureus by phenotypic characteristics, gram staining, catalase and tube coagulase tests. mics of the drugs were determined by an agar dilution method, according to the nccls criteria. the drugs were obtained from their manufacturer (bayer inc). s. aureus atcc 29213 was used as quality control strain. results: twenty-seven (14%) of 196 mssa strains and 239 (98%) of 244 mrsa strains were resistant to ciprofloxacin. mic50 and mic90 values of 27 ciprofloxacin-resistant mssa strains for moxifloxacin were 0.25 mg/l and 4 mg/l, respectively. mic50 and mic90 values of ciprofloxacin-susceptible mssa strains for moxifloxacin were £0.125 mg/l and 0.250 mg/l, respectively. moxifloxacin was 4-fold more active than ciprofloxacin against mssa strains. there was not any difference between these two quinolones against mrsa strains (mic90s: ‡16 mg/l). conclusion: mrsa strains are major causes of nosocomial infections. despite advances in antibacterial therapy, treatment of infections caused by mrsa is still troublesome. new quinolone-derived compounds such as moxifloxacin are reported to have improved activities against s. aureus including mrsa. in this first study on in-vitro activity of moxifloxacin against s. aureus from turkey, the moxifloxacin mics were higher than those reported from different countries objectives: a multicentric study during the winter period of 2003-2004 was performed in belgium to assess the in-vitro two mutations in 15 (34.8%) (3 parc+gyra and 13 parc+pare), and 3 mutations in parc+pare+gyra in 5 (11.6%) strains. conclusions: 1) levofloxacin mic50 and mic90 for pneumococcal strains with decreased susceptibility to ciprofloxacin were 0.5 and 1 mg/l, respectively. resistance to levofloxacin (mic ‡ 8) occurred in 32 isolates (1.2% of the sauce-3 collection). 2) activity of levofloxacin was negatively affected only among pneumococcal isolates with a very high mic of ciprofloxacin ( ‡8 mg/l). in this case, levofloxacin mic50 and mic90 were 4 and 8, respectively, and 44.4% of these strains were resistant to levofloxacin. 3) all sequenced strains with a levofloxacin mic ‡ 4 mg/l had a mutation in the parc gene (single in 56% strains), but none of them in the gyrb. the most common mutations identified were parc/s79f, pare/i460v, parc/s79y and gyra/e85k. 4) one single parc mutation was organism/year (no. tested) gemi cipro levo gati moxi 008/2 (na) methods: the organisms numbered 9,347, a total of 2,004 from np and 7,343 from carti. the most frequent carti pathogens were: s. pneumoniae (spn; 2,865), h. influenzae (hi; 4,011) and m. catarrhalis (mcat; 607) usa) objective: to evaluate the activity and potency of tigecycline (tig) when tested against a large international collection of common bacterial pathogens displaying increasing and worrisome resistance (r) profiles. tig is a novel glycylcycline derivative of minocycline that has demonstrated activity against a variety of gram-positive and -negative pathogens mic 50/90 (mg/l) 275 strains) were collected from 2000 to 2004 in >70 medical centres participating in the global tig surveillance program results: tig results for the r organism subsets are in the table: tig was highly active (mic50 and 90, £0.25 and £0.5 mg/l, respectively) against all resistant sa, cons, esp, spn and vgs with >99% of strains inhibited by £2 mg/l. while potency of tig against r subsets of ent was less (mic50 and 90, £0.5 and £4 mg/l, respectively), the vast majority of tet-r isolates remained s to tig (>98% of isolates were inhibited by £4 mg/l). no geographic differences in tig potency among esbl-producing, cip-r or tet-r ent strains were noted. grn drug concentrations in serum would be expected to remain above the mpc90 value for >24 hours. conclusion: grn exhibits potent activity against sp, as measured by both mic and mpc, and is active against strains with high mic/mpc values for other quinolones. grn is less likely to select for quinolone resistant sp isolates s. agalactiae (119) and s. dysagalactiae (30) were used. interlaboratory reproducibility was evaluated using 25 bias/precision strains tested at all sites and 3 nccls qc strains were used to compare e-test dp on iso values to nccls mh values. the various methods were used according to standard recommendations. nccls susceptible breakpoints ‡1 lg/ml for staphylococci and streptococci and ‡4 lg/ml for enterococci were used as is and as adjusted upwards by 1 dilution. results: percentage essential agreement (ea), category agreement methods: e test strips were used to establish the fici of the antibiotic combination pairs. combination testing was carried out on 125 strains of pseudomonas aeruginosa (pa), 69 strains of burkholderia cepacia (bc) and 29 strains of stenotrophomonas maltophilia (sm) from 96 cf patients. 19, 13 and 6 combination pairs were tested between 10 and 69 times against pa, bc and sm respectively. the fici results were categorised as synergistic (fici £ 0.5), additive (fici = 0.5 to £1.0), indifferent (fici = 1.0 to £2.0) and antagonistic (fici > 2.0). results: 1107 combinations were tested. synergy was found in 3.1% of pa, 9.7% of bc and 8.6% of sm. 31.6%, 32.6% and 25.8% of pa, bc and sm respectively demonstrated an additive effect. 64.2% of pa, 54.5% of bc and 62.4% of sm demonstrated an indifferent effect. antagonism was found in 1.1%, 3.2% and 3.2% of pa, bc and sm respectively results: of the 258 isolates resistant to cefpodoxime, 83 were positive for esbl production and the enzymes produced were: tem-1, shv-5, shv-12, ctx-m-15, ctx-m3 and ctx-m9 singly or in combination. ertapenem proved active against all enterobacteriaceae regardless of production of esbls (mic range 0.003-0.125 mg/l). for acinetobacter sp giamarellou on behalf of the hellenic study group for the susceptibility of streptococcus pneumoniae results: of the 275 strains studied, 76 were ery s and 159 were ery r. all strains but two (mic 4 and 8 mg/l) were susceptible to tel. in particular, results for telithromycin for the ery s strains, showed an mic range for tel between 0.008-0.5, with mic50 of 0.015 and mic90 of 0.06. for ery r in total, mic50 and mic90 were 0.125 and 0.5 (range 0.008-8). with respect to macrolide resistance phenotype, the distribution per type was: m 54.5% (ery mic range 1-32), and cmlsb and imlsb (ery mic range 4 to (256) 39% and 6.5% respectively. the two strains resistant to tel displayed the cmlsb phenotype (ery mic ( 256). for each different macrolide resistance phenotype no isolates demonstrated an inducible mlsb phenotype by this method. forty-nine of these carried the ermam gene (34.3%) and three carried both ermam and mefe genes (2.18%). eighty-seven isolates (63.5%) had the m phenotype (macrolide resistance) and all of these contained the mefe gene. conclusions: our findings are in contrast to european studies and to previous greek studies, where the erm gene prevailed or at least equaled the prevalence of mef gene. this is the first report of isolates carrying both ermam and mef genes in greece. knowledge of the resistant mechanisms to macrolides is crucial for the empiric antibiotic treatment of community acquired infections. p1225 antibiotic susceptibility, mechanisms of macrolide resistance and clonal relationship in group b streptococci microbiological characteristics of anthrax strains l. lukhnova, g. temiraliyeva, t. meka-mechenko, y. pazylov, s. zakaryan, j. denissov (almaty, kaz)objectives: the kazakh science center for quarantine and zoonotic diseases (kscqzd) has a collection of pathogens of especially dangerous infections that have been isolated in kazakhstan. by kazakh government resolution, the kscqzd's collection is the official collection of pathogenic bacteria for kazakhstan. kscqzd has 71 strains of b. anthracis that were isolated in various years . methods: we studied the biological properties of 30 anthrax strains from our collection: 16 from humans, 4 from soil, 5 from sheepskins, 2 from sheep organs, and 3 from the external environment. we tested the anthrax strains using standard identification tests. results: the growth of the bacillus anthracis strains on the hottinger's agar was typical (r -form), and no distinctions based on the origin of the strains were noted. all of the strains form spores and are immobile. when stained with specific antibody directed toward anthrax antigens, all strains stained positive. three of the b. anthracis strains did not have capsules when originally studied, but after 7 cultures on the hottinger's agar, the strains' ability to form capsules in a co 2 atmosphere was restored. the anthrax strains are sensitive to anthrax bacteriophage and penicillin. the strains have lipolytic and background: we studied the application of pk/pd modeling techniques as an alternative method to define pharmacodynamic breakpoints complementary to conventional microbiological breakpoints for fluoroquinolones (fqs). methods: 8 staphylococcus aureus strains with mics ranging from 0.03-8 mg/l were exposed to fluctuating concentrations of levo-(lfx) gati-(gfx) and moxifloxacin (mfx) mimicking their total serum concentration profiles following oral doses of 400 mg mfx, 400 mg gfx, 500 and 750 mg lfx over 24 h using the sedimentation model. strains were cultivated in brain heart infusion broth at 37°c. viable counts were quantitated at 14 sampling points. as pd-measure the aubkcnorm (i.e. area under the bacterial kill curve normalized to the initial inoculum [h] ; aubkcnorm <24 indicates a bactericidal activity, >24 bacterial growth) was determined for the time kill experiment and plotted as a function of mic for each treatment. results: the aubkcnorm vs mic function was similar for all treatments following a bimodal profile. while the slope of all fqs studied was shallow for mics below 1 mg/l with au-bkcnorm <5 h, aubkcnorm started increasing steeply beyond this point. it intercepted the 24 cut off line at concentrations of 1.5 mg/l for lfx 500 mg, 2 mg/l for gfx, 2.4 mg/l for mfx and 3.1 mg/l for lfx 750 mg. this indicates that s. aureus with susceptibilites beyond these mics would no longer be eradicated by the corresponding treatments. thus, according to our investigations a breakpoint for fqs integrating both microbiological and clinical pharmacokinetic relation-ships results in a value of 1 mg/l for lfx 500 mg, and 2 mg/l for mfx, gfx, and lfx 750 mg. conclusion: pharmacodynamic breakpoints determined by pk/pd m+s which are taking into respect the fluctuating concentration time profiles encountered in patients are suitable surrogates complementing the data obtained in microbiological experiments. a multicentre agar and broth dilution susceptibility testing study of fluoroquinolones against the bacteroides fragilis group k.e. aldridge, d. snydman, d. hecht, c. edlund, j. herrington (new orleans, boston, chicago, usa; stockholm, s; west haven, usa)objectives: the development of fluoroquinolones (fq) antimicrobials has resulted in effective therapy for a variety of infections due to aerobic and facultatively anaerobic grampositive and -negative pathogens. fq activity against anaerobes has not been as promising with only trovafloxacin receiving fda approval for anaerobic infections. moreover, more recent reports of increased resistance to fq among the b. fragilis group has prompted interested to determine if these increases are due to susceptibility testing methodology problems. this study was designed to compare the susceptibility results from five laboratories that tested the same 70 isolates against fq using agar dilution(ad) and broth microdilution (bmd) testing. methods: seventy clinical isolates of the b. fragilis group identified by only a sequential number were distributed to each of the five participating laboratories. four of the laboratories performed ad and one laboratory performed bmd susceptibility testing using nccls recommendations including breakpoints where appropriate. serial two-fold dilutions of moxifloxacin (mox), gatifloxacin (gate), trovafloxacin (trov), and metronidzaole (metro) were prepared in the test medium within a range of 0.03 to 64 mg/l. plates were inoculated with 1 · 10 5 cfu per spot or well, incubated anaerobically for 48 hours and the mic read. qc was performed using nccls-recommended atcc strains. results: mic values for each antimicrobial were collated as mic90 and per cent (%) of isolates inhibited at £2 and £4 mg/l: these data indicate that inter-laboratory testing of the same test isolates gave very similar results for each antimicrobial. bmd gave slightly lower mic results than ad but was not significantly different. we conclude that susceptibility methodology is not responsible for unusually high resistance rates. global evaluation of gemifloxacin activity tested against community-acquired respiratory tract pathogens, haemophilus influenzae and moraxella catarrhalis (1999) (2000) (2001) (2002) (2003) (2004) fluoroquinolones are a therapeutic option in treatment of infections caused by non-fermentative gram-negative bacteria.however, resistance to these antibiotics rapidly increase in clinical isolates. the efflux mechanism is the most common cause of multidrug resistance of strains p. aeruginosa, a. baumanii and s. maltophilia. these mdr pumps belonging to the rnd family are inhibited by phe-arg-beta-naphthylamide [mc207, 110] . however, different results of reserpine effect on the activity of rnd pumps was established. in our study, we searched for effect of the efflux pump inhibitors (mc207,110, reserpine and rescinnamine) on the mic of quinolones for mdr clinical isolates of p. aeruginosa, a. baumanii and s. maltophilia. we looked for a interdependence between structure of quinolones and ability of tested inhibitors to inhibit efflux of these antibiotics. results: the mdr strains were resistant to all tested quinolones (nalidixic acid-nal, pipemidic acid-kp, norfloxacin-nor, ciprofloxacin-cip and ofloxacin-of). among studied inhibitors only mc207,110 affected the susceptibility of all tested strains to quinolones, first of all to nal. effect of mc207,110 on the mic depended on its concentration, but did not depend on the temperature. for majority of p. aeruginosa and a. baumanii strains the mic of all quinolones decreased in the presence of mc207,110. the highest decrease of the mic of quinolones was obtained for p. aeruginosa (4-512-fold). for a. baumanii strains the mic of kp, nor, cip and of decreased only from 2-to 4-fold but the mic of nal decreased to 16-fold. moreover, the presence of this inhibitor increased the sensitivity of 80% of s. maltophilia only to nal (4-to 16-fold). reserpine and rescinnamine did not effect on the mic of quinolones for all studied mdr strains. however, reserpine alkaloides showed inhibitors activity against control strains of s. aureus (decrease mic of nor). conclusions: our data confirm that mc207,110 inhibited efflux pumps not only in p. aeruginosa but also in a. baumanii and s. maltophilia strains. additionally, obtained results suggested possibility that for quinolones, depending on their structure, are different binding sites in efflux pumps. furthermore, probably also mc207,110 binds to specific site of pumps depending on the efflux systems. so, the effect of this inhibitor on mic of quinolones depends on genus of bacteria, kind of pumps and structure of agents. in vitro activity of fucidic acid against staphylococcus aureus strains objectives: viridans group streptococci (vgs) resistant to penicillin, macrolides and other antimicrobial are increasingly found. linezolid and quinupristin-dalfopristin exert their mechanism of action by protein synthesis inhibition and are agents with activity against multidrug resistant gram-positive cocci. this study was aimed to determine, by time-kill curves, the activity of linezolid and quinupristin-dalfopristin against five vgs expressing various degrees of resistance to erythromycin and harbouring different erythromycin resistance determinants methods: the species, erythromycin mics (mg/l) and the erythromycin resistance genes present for each of the five isolates tested were: s. anginosus/0.06/none, s. sanguis/4/mef(a), s. sanguis/64/erm(b), s. mitis/256/erm(b) +mef(a), s. anginosus/256/erm(b) +mef(a). quinupristin-dalfopristin and linezolid mics for all strains were 1 and 0.5 mg/l respectively. strains were incubated in cation-adjusted mueller-hinton broth supplemented with 5% lysed horse blood. each of the five isolates were exposed to linezolid and quinupristin-dalfopristin for 24 hours in a shacking water bath in presence of 2 · mic. colony counts were performed at 0, 4, 8 and 24 hours. results: time-kill studies showed that for linezolid, the antibiotic concentration of 2 · mic was bacteriostatic for each strain irrespectively of the resistance to erythromycin and the genes harboured, showing a 90% of killing at 24 h. the in vitro activity of quinupristin-dalfopristin was predominantly bactericidal with a 99.9% of killing at 3 h. the exception were the 2 strains with erm(b) + mef(a) determinants, in one of them, a s. milleri, the bactericidal activity was delayed to 24 h, and in the other strain, a s. mitis, the activity was bactericidal at 3 h but with regrowth at 24 h. conclusions: when vgs are implicated in infections in neutropenic patients and in cases of endocarditis where bactericidal activity is usually considered mandatory, we have to considered that: linezolid was bacteriostatic, at the concentration tested, against all strains and the rapid killing of quinupristin-dalfopristin against vgs was lost when the strain presented the erm(b) and mef(a) genes together. comparative antimicrobial susceptibility patterns in different groups of species of viridans group streptococci and streptococcus bovis isolated fom blood cultures i. rodriguez-avial, c. rodriguez-avial, j.j picazo (madrid, e)objectives: viridans group streptococci (vgs) and s. bovis are part of the normal human microbiota and are implicated as a cause of endocarditis and sepsis in neutropenic patients. because of their resident nature are frequently exposed to the antimicrobial agents. this study was performed to study the resistance phenotypes to penicillin (p), erythromycin (e), key: cord-017831-anadq4j9 authors: lai, yi-horng title: network analysis of comorbidities: case study of hiv/aids in taiwan date: 2015-07-30 journal: multidisciplinary social networks research doi: 10.1007/978-3-662-48319-0_14 sha: doc_id: 17831 cord_uid: anadq4j9 comorbidities are the presence of one or more additional disorders or diseases co-occurring with a primary disease or disorder. the purpose of this study is to identify diseases that co-occur with hiv/aids and analyze the gender differences. data was collected from 536 hiv/aids admission medical records out of 1,377,469 admission medical records from 1997 to 2010 in taiwan. in this study, the comorbidity relationships are presented in the phenotypic disease network (pdn), and φ-correlation is used to measure the distance between two diseases on the network. the results show that there is a high correlation in the following pairs/triad of diseases: human immunodeficiency virus infection with specified conditions (042) and pneumocystosis pneumonia (1363), human immunodeficiency virus infection with specified malignant neoplasms (0422) and kaposi’s sarcoma of other specified sites (1768), human immunodeficiency virus acquired immunodeficiency syndrome, and unspecified (0429) and progressive multifocal leukoencephalopathy (0463), and lastly, human immunodeficiency virus infection with specified infections (0420), meningoencephalitis due to toxoplasmosis (1300), and human immunodeficiency virus infection specified infections causing other specified infections (0421). the human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids) epidemic is one of the most important and crucial public health risks facing governments and civil societies in the world. adolescents were at the center of the pandemic in terms of transmission, impact, and potential for changing the attitudes and behaviors that underlie this disease. therefore, hiv/aids prevention has become a priority all over the world. the first hiv/aids case in taiwan was reported in 1984. as of the end of 2013, the total number of hiv/aids cases had been accumulated to 26,475. faced with this serious situation, taiwan's centers for disease control worked with other departments and dedicated a tremendous amount of effort and resources to introduce harm reduction programs. total reported cases dropped in 2006, which was the first trend reversal since 1984. in 2008 and thereafter, the epidemic took a turn; infections mainly occurred through sexual encounter [1] . there are no clear boundaries between many diseases, as diseases can have multiple causes and can be related in several dimensions. from a genetic perspective, a pair of diseases can be related because they have both been associated with the same gene, whereas from a proteomic perspective, diseases can be related because diseaseassociated proteins act on the same pathway [2] . during the past half-decade, several resources have been constructed to help understand the entangled origins of many diseases. many of these resources have been presented as networks in which interactions between disease-associated genes, proteins, and expression patterns have been summarized. goh, cusick, valle, barton, vidal, and barabási created a network of mendelian gene-disease associations by connecting diseases that have been associated with the same genes [3] . besides, more and more studies have applied the network approach in diseases, such as neurodegenerative diseases [4] , infertility etiologies [5] , sars, and hiv/aids [6] . a comorbidity relationship exists between two diseases whenever they affect the same individual substantially more than chance alone. in the past, comorbidities have been used extensively to construct synthetic scales for mortality prediction [7, 8] , yet their utility exceed their current use. studying the structure defined by entire sets of comorbidities might help the understanding of many biological and medical questions from a perspective that is complementary to other approaches. for example, a recent study built a comorbidity network in an attempt to elucidate neurological diseases' common genetic origins [9] . heretofore, however, neither this data nor the data necessary to explore relationships between all diseases is currently available to the research community. this present study decided to provide this data in the form of a phenotypic disease network (pdn) that includes all diseases recorded in the medical claims. additionally, this study illustrates how a pdn can be used to study disease progression from a network perspective by interpreting the pdn as the landscape where disease progression occurs and shows how the network can be used to study phenotypic differences between patients of different demographic backgrounds. this study indicates the directionality of disease progression, as observed in our dataset, and finds out that more central disease in the pdn are more likely to occur after other diseases and that more peripheral diseases tend to precede other illnesses. in order to guide hiv/aids-related diseases prevention program, this study conducted the pdn of hiv/aids to explore the relationship between hiv/aids and other diseases. the objective of this study is to identify diseases that are highly correlated with hiv/aids, and discuss gender differences. the national health insurance (nhi) program was initiated in taiwan in 1995 and covers nearly all residents. in 1999, the bureau of nhi began to release all claims data in electronic form to the public under the national health insurance research database (nhird) project. the structure of the claim files is described in detail on the nhird website and in other publications [10]. nhird offers reliable, systematic, and complete data for disease detection. the datasets contained only the visit files, including dates, medical care facilities and specialties, patients' genders, dates of birth, and the four major diagnoses coded in the international classification of disease, 9th revision, clinical modification (icd-9-cm) format [10, 11] . in total, the icd-9-cm classification consists of 657 different categories at the 3 digit level and 16,459 categories at 5 digits. human immunodeficiency virus infection and acquired immune deficiency syndrome (hiv/aids) is coded 042 as human immunodeficiency virus (hiv) infection disease, 0420 as human immunodeficiency virus infection with specified infections, 0421 as specified infections causing other specified infections, 0422 as human immunodeficiency virus infection with specified malignant neoplasms, and 0429 as acquired immunodeficiency syndrome, and unspecified. to protect privacy, the data on patient identities and institutions had been scrambled cryptographically. the visit files in this study represented 1,377,469 admission activities within the nhi from 1997 to 2010. demographically, the data set consists of 1,377,469 admission medical records from 1,000,000 patients. of all these patients, 50.93% were females, 48.93 were males, 20.67% were over 71 years of age, and 536 persons were diagnosed with hiv/aids (table 1) . these hiv/aids admission medical records included 45 females (8.40%) and 491 males (91.60%). of all the 536 hiv/aids records, 461 (86.01%) had major diagnoses coded 042, 21 (3.92%) had major diagnoses coded 0420, 3 (.56%) had primary diagnoses coded 0421, and 7 (8.77%) had major diagnoses coded 0429 (table 2 ). there are several limitations to the current study. first, although data gathered from nhird is comprehensive and reliable, there are still some mistakes that the system couldn't find, such as code entry errors. these errors may be carried into data y.-h. lai pre-processing, and it is beyond the control of this study. second, the data was not upto-date. although future researchers are still recommended to apply the method of this study to analyze the characteristics of patients for the purpose of disease prevention, changes of medical treatments and other factors should be considered. third, this study does not have a global sample, so there might be limits to replicate the findings of this study in all the other countries. it might, however, be generalized to other ethnic chinese population due to the similarity in genes and physiology. to measure the comorbidity relationships, it is necessary to quantify the strength of comorbidities by introducing a notion of distance between two diseases. a difficulty of this approach is that different statistical distance measures have biases that over-or under-estimate the relationships between rare or prevalent diseases. these biases are important given that the number of times a particular disease is diagnosed, such as its prevalence, follows a heavy tailed distribution [2] , meaning that while most diseases are rarely diagnosed, a few diseases have been diagnosed in a large fraction of the population. in this study, the φ-correlation is used to quantify the distance between two diseases. the φ-correlation, which is pearson's correlation for binary variables, can be expressed mathematically as [2, 12] : where c ij is the number of patients affected by both diseases, n is the total number of patients in the population and p i and p j are the prevalence of diseases i and j. the distribution of φ values representing all disease pairs where c ij >0 is presented in fig 1, 3 and 5. this research utilizes data from nhird to obtain the four major diagnoses codes of all patients. this study calculates φ-correlation with equation 1. pajek 4.03 program was used to compute the compute the degree of centrality and betweenness of each node and the path value (φ-correlation). this study is focused on the path between each disease as network and the correlation as the value of line (path weight), and they could be affected by different populations, which indicates differences in gender for each population. it can be summarized the set of all comorbidity associations between all diseases expressed in the study population by constructing a phenotypic disease network (pdn). in the pdn, nodes are disease phenotypes identified by unique icd9 codes, and links phenotypes that show significant comorbidity according to the measures introduced above. in principle, the number of disease-disease associations in the pdn is proportional to the square of the number of phenotypes, yet many of these associations are either not strong or are not statistically significant [2] . the structure of the pdn can be explored by focusing on the strongest and the most significant of these associations. the pdn can be seen as a network of the phenotypic space. this network allows people to understand the relationship between illnesses. the distribution of φ-values representing all disease pairs is presented in figure 1 . most of them are between .000 and .005. a discussion on the confidence interval and statistical significance of these measures can be found in hidalgo, blumm, barabási, and christakis's study, and φ-correlation > .06 is statistically significant [2] . in figure 2 , nodes are diseases; links are correlations. node color identifies the icd9 category; node size is proportional to disease prevalence. link color indicates correlation strength. the pdn built using φ-correlation. all statistically significant links where φ-correlation>.06 are shown here. human immunodeficiency virus infection with specified conditions (042) and pneumocystosis pneumonia (1363), cryptococcal meningitis (3210), candidiasis of mouth (1120), unspecified secondary syphilis (0919), kaposi's sarcoma of unspecified (1769), cryptococcosis (1175), kaposi's sarcoma of palate (1762), neurosyphilis, unspecified (0949), kaposi's sarcoma of lung (1764), kaposi's sarcoma of lymph nodes (1765), and late syphilis, latent (096) are highly correlated. those φ-correlations are all above .06. the relationship between human immunodeficiency virus infection with specified conditions (042) and pneumocystosis pneumonia (1363) is the strongest. pneumocystis pneumonia is a form of pneumonia, caused by the yeast-like fungus pneumocystis jirovecii. pneumocystis pneumonia is not commonly found in the lungs of healthy people, but, being a source of opportunistic infection, it can cause a lung infection in people with a weak immune system [13] . human immunodeficiency virus infection with specified infections (0420) and human immunodeficiency virus infection specified infections causing other specified infections (0421), kaschin-beck disease (7160), pneumocystosis (1363), meningoencephalitis due to toxoplasmosis (1300), falciparum malaria (0840), kaposi's sarcoma of other specified sites (1768), with specified malignant neoplasms (0422) are highly correlated. those φ-correlations are all above .06. y.-h. lai the φ-correlation of human immunodeficiency virus infection with specified infections (0420) and meningoencephalitis due to toxoplasmosis (1300) is highest. toxoplasmosis is a parasitic disease caused by the protozoan toxoplasma gondii. the parasite infects most genera of warm-blooded animals, including humans, but the primary host is the felid family. infection occurs by eating infected meat, particularly swine products. by ingesting water, soil, or food that has come into contact with infected animals' fecal matter [14] . besides, human immunodeficiency virus infection with specified infections (0420) and specified infections causing other specified infections (0421) is highly correlated, and the φ-correlation is .11. human immunodeficiency and kaposi's sarcoma of sk specified infections (0420) highly correlated. those φthe φ-correlation of hu lignant neoplasms (0422) a highest. human immunodeficien specified (0429) and prog correlated. those φ-correlat the distribution of w va most of them are between . in pdns for females (figure 4 ), human immunodeficiency virus infection with specified conditions (042) and cryptococcal meningitis (3210), kaposi's sarcoma of unspecified (1769), and pneumocystosis (1363) are highly correlated. human immunodeficiency virus acquired immunodeficiency syndrome, unspecified (0429) and other cerebellar ataxia (3343), and progressive multifocal leukoencephalopathy (0463) are highly correlated. those φ-correlations are all above .06. the distribution of w values representing all disease pairs is presented as fig. 5 . most of them are between .000 and .005. in pdns for males ( figure 6 ), human immunodeficiency virus infection with specified conditions (042) and unspecified secondary syphilis (0919), cytomegaloviral disease (0785), kaposi's sarcoma of palate (1762), amebic liver abscess (0063), kaposi's sarcoma of lung (1764), kaposi's sarcoma of lymph nodes (1765), cryptococcosis (1175), late syphilis, latent (096), candidiasis of mouth (1120), cryptococcal meningitis (3210), neurosyphilis, unspecified (0949), kaposi's sarcoma of unspecified (1769), and pneumocystosis (1363) are highly correlated. those φ-correlations are all above .06. the φ-correlation of human immunodeficiency virus infection with specified conditions (042) with pneumocystosis (1363) is highest. human immunodeficiency virus infection with specified infections (0420) and specified infections causing other specified infections (0421), meningoencephalitis due to toxoplasmosis (1300), pneumocystosis (1363), kaschin-beck disease (7160), kaposi's sarcoma of other specified sites (1768), with specified malignant neoplasms (0422), and falciparum malaria (0840) are highly correlation. those φ-correlations are all above .06. the φ-correlation of specified infections causing other specified infections (0421) with human immunodeficiency virus infection with specified infections (0420) is highest. human immunodeficiency virus acquired immunodeficiency syndrome, unspecified (0429) and hiv infection, unspecified (0449) is highly correlation, and the φcorrelation is .09. through the pdn, this paper has identified the diseases that are associated with hiv/aids. it could be showed that the pdn has a complex structure where some diseases are highly connected while others are barely connected at all. while not conclusive, these observations can explain the observation that more connected diseases are seen to be more lethal, as patients developing highly connected diseases are more likely those at an advanced stage of disease, which can be reached through multiple paths in the pdn. the findings suggest that human immunodeficiency virus infection with specified conditions (042) and pneumocystosis pneumonia is highly correlated (1363). this result is consistent with aliouat-denis, chabé, demanche, aliouat el, viscogliosi, guillot, delhaes, and dei-cas's study [13] . pneumocystis pneumonia is especially seen in people with cancer undergoing chemotherapy, hiv/aids, and the use of medications that suppress the immune system. human immunodeficiency virus infection with specified infections (0420) and meningoencephalitis due to toxoplasmosis (1300) is highly correlation. besides, it is also highly correlation with human immunodeficiency virus infection specified infections causing other specified infections (0421). the result is the same as dubey, hill, jones, hightower, kirkland, roberts, marcet, lehmann, vianna, miska, sreekumar, kwok, shen, and gamble''s study [14] . human immunodeficiency virus infection with specified malignant neoplasms (0422) and kaposi's sarcoma of other specified sites (1768) is highly correlation. the result is the same as holmes, hawson, liu, friedman, khiabanian, and rabadan's study [15] . since patients infected with hiv/aids have a high risk of developing kaposi sarcoma, the prevention of this malignant disease should be prioritized. human immunodeficiency virus acquired immunodeficiency syndrome, and unspecified (0429) and progressive multifocal leukoencephalopathy (0463) is highly correlation. the result is the same as casado, corral, garcía, martinez-san millán, navas, moreno, and moreno's study [16] and sano, nakano, omoto, takao, ikeda, oga, nakamichi, saijo, maoka, sano, kawai, kanda [17] . progressive multifocal leukoencephalopathy is a usually fatal viral disease characterized by progressive damage or inflammation of the white matter of the brain at multiple locations. it is caused by the jc virus, which is normally present and kept under control by the immune system. jc virus is harmless except in cases of weakened immune systems. progressive multifocal leukoencephalopathy occurs almost exclusively in patients with severe immune deficiency, most commonly among patients with acquired immune deficiency syndrome, but people on chronic immunosuppressive medications including chemotherapy are also at increased risk of progressive multifocal leukoencephalopathy [16, 17] . for females, human immunodeficiency virus infection with specified conditions (042) and cryptococcal meningitis (3210), kaposi's sarcoma of unspecified (1769), and pneumocystosis (1363) are highly correlation. human immunodeficiency virus acquired immunodeficiency syndrome, unspecified (0429) and other cerebellar ataxia (3343), and progressive multifocal leukoencephalopathy (0463) are highly correlation. for males, human immunodeficiency virus infection with specified conditions (042) and pneumocystosis (1363) is highly correlation. human immunodeficiency virus infection with specified infections (0420) and meningoencephalitis due to toxoplasmosis (1300), and falciparum malaria (0840) are highly correlation. human immunodeficiency virus infection with specified malignant neoplasms (0422) and kaposi's sarcoma of other specified sites (1768) is highly correlation. human immunodeficiency virus acquired immunodeficiency syndrome, unspecified (0429) and hiv infection, unspecified (0449) is highly correlation. exploring comorbidities from a network perspective could help determine whether differences in the comorbidity patterns expressed in different populations indicate differences in races, country, or socioeconomic status for each population. here this study show as an initially stage that there are differences in the strength of comorbidities measured for patients of different gender. the pdn could be the starting point of studies exploring these and related questions. communicable diseases & prevention-hiv/aids, health topics a dynamic network approach for the study of human phenotypes the human disease network a network approach to clinical intervention in neurodegenerative diseases infertility etiologies are genetically and clinically linked with other diseases in single meta-diseases network-based analysis of comorbidities risk during an infection: sars and hiv case studies chronic conditions and risk of in-hospital death improved comorbidity adjustment for predicting mortality in medicare populations probing genetic overlap among complex human phenotypes international classification of diseases, ninth revision the phi-coefficient, the tetrachoric correlation coefficient, and the pearson-yule debate pneumocystis species, co-evolution and pathogenic power. infection prevalence of viable toxoplasma gondii in beef, chicken, and pork from retail meat stores in the united states: risk assessment to consumers discovering disease associations by integrating electronic clinical data and medical literature continued declining incidence and improved survival of progressive multifocal leukoencephalopathy in hiv/aids patients in the current era rituximab-associated progressive multifocal leukoencephalopathy derived from non-hodgkin lymphoma: neuropathological findings and results of mefloquine treatment acknowledgements. this study is based in part on data from the national health insurance research database provided by the bureau of national health insurance, department of health and managed by national health research institutes (nhri). the interpretation and conclusions contained herein do not represent those of bureau of national health insurance, department of health or national health research institutes. key: cord-009664-kb9fnbgy authors: nan title: oral presentations date: 2014-12-24 journal: clin microbiol infect doi: 10.1111/j.1469-0691.2009.02857.x sha: doc_id: 9664 cord_uid: kb9fnbgy nan [ primary immunodeficiency diseases are a heterogeneous group of disorders, caused by inherited defects in the immune system, and characterised by wide spectrum of clinical manifestations, particularly an increased susceptibility to infections and a predisposition to autoimmune diseases and malignancies. recurrent infections or infection with unusual organisms are the most commonly presentation of primary immunodeficiency diseases. although recurrent respiratory tract infections and gastrointestinal manifestations are the most common features of these diseases, especially in predominantly antibody deficiencies and combined immunodeficiencies, other organs can be involved as well. recurrent cutaneous abscesses with unusual organisms or deep abscesses may represent infections with an association with immunodeficiencies, particularly in phagocytes defects. meningococcal infections could have an association with complement deficiencies. meanwhile other bacterial infections, mainly streptococcus pneumoniae and staphylococcus aureus, as well as infections with viruses, fungi and parasites are also common in several primary immunodeficiency diseases. autoimmune diseases such as idiopathic thrombocytopenic purpura, autoimmune haemolytic anaemia, systemic lupus erythematosus, juvenile arthritis, sclerosing cholangitis, and vasculitis are common in primary immunodeficiency diseases. whilst some syndromic immunodeficiencies (e.g., wiskott aldrich syndrome, di george syndrome) have a strong association with autoimmunity, there are a group of disorders (e.g., alps, apeced, ipex) that the autoimmune manifestations are typically the first and most significant findings. malignancies are also common in some primary immunodeficiency diseases (e.g., cvid, alps, xlp, and dna repair defects). other manifestations such as dysmorphic features, associated anomalies, skeletal dysplasia, and oculocutaneous hypopigmentation can be unique characteristics of some cases with primary immunodeficiency diseases. the clinical manifestations of these diseases are often helpful in guiding the appropriate evaluation of the patients. prompt and precise diagnostic laboratory evaluation should be performed in the patients with such features, whereas early diagnosis and successful management of these patients prevent irreparable organ system damage and improve the prognosis. immunodeficiency specialists from all over europe have composed a multistage diagnostic protocol that is based on their expert opinion, in order to increase the awareness of pid among doctors working in different fields. the protocol starts from the clinical presentation of the patient; immunological skills are not needed for its use. a list of relevant symptoms and signs from the history and physical examination that should alert any physician to potential pid is given. these are grouped together to form eight typical clinical presentations of pid: recurrent ent and airway infections; failure to thrive from early infancy; recurrent pyogenic infections; unusual infections or unusually severe course of infections; recurrent infections with the same type of pathogen; autoimmune or chronic inflammatory disease, or lymphoproliferation; characteristic combinations of clinical features in eponymous syndromes; and angioneurotic edema. these presentations lead the user towards different algorithms, which in fact represent the traditional division into antibody, complement, lymphocyte, and phagocyte deficiencies, respectively. the algorithms each are comprised of several steps. this multistage design allows cost-effective screening for pid within the large pool of potential cases in all hospitals in the early phases, while more expensive tests are reserved for definitive classification in collaboration with an immunologist at a later stage. g. schmid°(geneva, ch) in 1986, articles suggesting that male circumcision (mc) decreased the risk of hiv infection appeared. over the next 15 years, studies of two epidemiologic types − ecologic and observational − increasingly supported this contention. ecologic studies showed strong correlations between prevalences of mc and hiv, e.g., tribes with low prevalences of mc had high prevalences of hiv infection. observational cross-sectional studies showed that uncircumcised men had higher rates of hiv than circumcised men. observational cohort studies confirmed these weaker study design findings. a systematic review of observational studies in 2000 found a relative risk (rr) of 0.42 (95% ci, 0.34−0.54), a 58% protective effect. in 2005 and 2007, results from three randomised controlled trials, all from sub-saharan africa, were reported. results were consistent, and the pooled rr of 0.42 (95% ci, 0.31−0.57) was identical to that of the observational studies. the protective effect in the three trials, found at about 21−24 months' follow-up, has been extended in one trial to a protective effect of 64% at 42 months of follow-up. who and unaids have strongly endorsed mc as an effective hiv prevention strategy in generalised hiv epidemics where mc is uncommon. what about europe? mc is uncommon with an adult male prevalence of <20%. hiv incidence is low enough that mc for hiv prevention purposes is unlikely to have much impact. no public health authority recommends routine neonatal circumcision. increasingly, however, data are showing benefits of mc in addition to hiv prevention. lessened risk of urinary tract infection in infants (rr 0.13, 95% ci 0.08−0.20) and lifetime avoidance of phimosis and associated conditions occur when mc is performed neonatally. other benefits occur in males circumcised at any age. mc protects against acquiring sexually transmitted infections characterised by genital ulcers-syphilis, chancroid and herpes-and possibly trichomoniasis. circumcised men may be less likely to acquire hpv and are more likely to clear the infection. through the protective effect against hpv, mc halves risk of penile cancer (rr 0.52, 95% ci 0.33−0.82) and partners of circumcised men are at lessened risk of cervical cancer. other issues must be considered in making public health decisions about mc. cultural objections may occur, but mc in the developing world is readily accepted in non-circumcising societies. studies of sexual pleasure and function have found no relationship to circumcision status. mc may be advised for subgroups, even if not for the entire population. and, surgical risk and cost must be considered. while many sub-saharan african countries are scaling up mc services to prevent hiv infection, public health agencies in many industrialised countries are reconsidering mc policies-the outcomes of both efforts are being followed with interest. acute otitis media (aom) is generally considered a bacterial infection that is treated with antibiotics. however, despite extensive use of broadspectrum antibiotics for this condition, the clinical response to the treatment is often poor. this fact, together with vast clinical experience connecting aom with viral respiratory infections, has prompted research into the role of viruses in aom. to date, ample evidence from studies ranging from animal experiments to large clinical trials supports a crucial role for respiratory viruses in the aetiology and pathogenesis of aom. in most cases, viral infection of the upper respiratory mucosa initiates the whole cascade of events that finally leads to the development of aom as a complication. the pathogenesis of aom involves a complex interplay between viruses, bacteria, and the host's inflammatory response. recent studies indicate that with sensitive techniques viruses can be found in the middle-ear fluid in most children with aom, either alone or together with bacteria. viruses appear to enhance the inflammatory process in the middle ear, and they may profoundly impair the resolution of otitis media. it is important to understand, however, that our increasing knowledge of the importance of viruses in the etiopathogenesis of aom does not diminish the central role of bacteria in aom. therefore, while viruses may explain many of the problems encountered in treating aom, the ultimate decision on whether or not to treat aom with antibiotics cannot be based solely on the degree of viral involvement in aom. the non-judicious use of antibiotics has lead to an epidemic in antimicrobial resistance. acute otitis media (aom) is the most common indication for use of antibiotics in children in the united states (us). despite available evidence that supports a wait and see approach, most us physicians immediately prescribe antibiotics for the treatment of aom. the american academy of pediatrics published a guideline in 2004 that addressed the diagnosis and treatment of aom. this guideline recommends the use of observation as a potential strategy for the treatment of aom. the key components of this published guideline will be discussed, as well as the evidence and rationale that supports the use of observation as an initial strategy to treat aom. otitis media (om) is the most common bacterial infection in children aged <5 years for which antibiotic treatment is prescribed worldwide. although most of the time this entity resolves spontaneously it is associated with morbidity, family dysfunction, antibiotic use and burden on the medical system. efforts to reduce the burden of om by vaccination have not been extremely rewarding, but some progress has been made. the first obvious step would be to reduce viral infections leading secondarily to om. in the modern era, the only viral vaccine with proven effect on aom is the influenza virus vaccine. both the inactivated and the live virus showed some effect, but since influenza virus has only a limited season yearly the effect on the overall om rate is far from being remarkable. haemophilus influenzae (hi) b vaccine did not reduce om since most hi causing om are nontypable (nthi) and not hib. the newly developed pneumococcal conjugate vaccines (pcvs) have all been shown to reduce >50% of the om caused by the serotypes included in the vaccines, but some replacement with serotypes not included in the vaccines and non pneumococcal organisms was demonstrated to reduce the overall effect of pneumococcal vaccines. the effect of pcv on the reduction of recurrent om, om with effusion, the need for ventilation tubes and frequent visits for aom has been suggested, and the real impact is still being studied. aiming with pcv at those with established recurrent om has proved disappointing. pcvs can reduce om caused by antibiotic-resistant s. pneumoniae but the continued overuse of antibiotics is responsible for the increase in antibiotic resistance in non-vaccine serotypes. a newly developed pcv with an outer membrane protein for hi (pnpd) is suggested to reduce also om caused by hi, but confirmation studies are needed. the expansion of the 7 serotypes included in the current licensed pcv to 10 or more serotypes may add to the prevention of om in the near future. in the next decade, om will continue to be an important disease in children. however, we can expect it to be modified in terms of bacteriologic aetiologies, antibiotic resistance and hopefully short and long term consequences. v. korten°(istanbul, tr) infectious consequences of an earthquake mainly involve several types of communicable diseases and crush related infections. water-borne and food-borne illnesses often result from the disruption of the public water and sewage systems and contamination of water supply. overcrowding, poor hygiene and sanitation in temporary shelters also may be factors. the type of infectious diseases are associated with the epidemiology of communicable diseases in the area where the earthquake occurred. the most common outbreaks associated with earthquakes are gastroenteritis, infectious hepatitis and pulmonary infections. in unvaccinated populations, there are reports of increased measles. tetanus can be seen in populations where vaccination coverage levels are low. the risk for diarrhoeal disease outbreaks following earthquakes is higher in developing countries than in industrialised countries. an outbreak of acute watery diarrhoea involved >750 cases occurred in a camp after the 2005 earthquake in pakistan. acute respiratory infections, hepatitis e clusters and measles (>400 clinical cases in the 6 months) also occurred among the displaced victims after the same earthquake. contamination of drinking water led to an outbreak of rotavirus after the 2005 earthquake in kashmir, india. an unusual outbreak of coccidiomycosis associated with exposure to increased levels of airborne dust occurred after the 1994 southern california earthquake. persons who have been trapped by rubble for several hours or days may develop compartment syndromes requiring fasciotomy or amputation. infectious complications were common in renal victims of the1999 marmara earthquake in turkey and were associated with increased mortality when complicated by sepsis. of 639 renal victims, 223 (34.9%) had infectious complications, mainly sepsis and wound infections. most of the infections were nosocomial in origin and caused by gram-negative aerobic bacteria and staphylococcus spp. multivariate analysis of the risk-factors for nosocomial infections revealed a significant association with fasciotomy and length of hospital stay in a back up university hospital. the most frequent pathogens isolated from pus and/or wounds culture in 2008 wenchuan earthquake survivors were s. aureus, e. coli, a. baumannii, e. cloacae, and p. aeruginosa. disaster-preparedness plans, focused on trauma and mass casualty management and also on health needs of the surviving affected populations may decrease the health impact of earthquakes. s16 infections in the disaster setting: famine. experience from darfour, sudan clinic malnutrition is a known risk factor for id worldwide. subsaharan africa and india is at higher risk due to vegetarian habits on absolute absence of animal meat proteins, resulting to depletion of micronutritients (zinc, iron, selenium), responsible for recovery of postmalarial anaemia. in addition, depletion of proteins results to immunoglobulinaemia and to delayed response to many bacterial pathogens causing id in topics (pneumococci, salmonella, etc.) . third problem is absence of vitamins dissolved in oil and fat, resulting to delayed phagocytic activity. therefore proteinocaloric malnutrition results to significant adverse outcome in hiv, tb (diarrhoea, pneumonia), the major killers of children under five. st. elizabeth university tropical programme runs 4 antimalnutrition centres: 1 in sudan, darfour and 2 in kenya amaong upcountry refugees from major conflict areas (sudan − turrana border) and 1 in uganda trying to rehabilitate malnourished children under 5 and helping them to combat disease, responsible for 12.5 million deaths in children mean 5 a year − malaria (1.2 mil), tb (1.1 mil), hiv (2.0 mil), pneumonia (7.5 mil) and diarrhoea (0.5 mil. children deaths approximately a year). h. giamarellou°(athens, gr) for the last six years greece has faced a large number of infections, mainly in the intensive care units (icu), due to carbapenemsresistant klebsiella pneumoniae. the proportion of imipenem-resistant k. pneumoniae has increased from less than 1% in 2001, to 23% in isolates from hospital wards and to 53% in isolates from icus in 2008. likewise, in 2002, these strains were identified in only three hospitals, whereas now they are isolated in at least 32 of the 40 hospitals participating in the greek surveillance system. until 2007 this situation was due to the spread of the blavim-1 cassette among the rapidly evolving multiresistant plasmids and multiresistant or even panresistant strains of mainly k. pneumoniae and also other enterobacterial species. however, the fact that most strains display mic values below or near the clsi resistance breakpoint create diagnostic and therapeutic problems, and possibly obstruct the assessment of the real incidence of these strains. as of 2007, the emergence of kpc-producing k. pneumoniae has been noted in icus of some greek hospitals and has now spread to most hospitals throughout the country creating a countywide outbreak in 2008. in attikon university hospital we recently described the icu outbreak of kpc-producing k. pneumoniae. twenty-nine patients (admitted from february to december 2008) were colonised mainly in gi tract. fifteen patients were male (52%) and the median apache ii was 19. patients had already long hospital stays preceding icu admission with a median of 25 (17−40) days. in twenty-two of these patients (76%) kpc-producing k. pneumoniae colonisation was definitely icuacquired while in 7 (24%) acquisition in other wards or other hospitals was hypothesized. five of these patients are still hospitalised in the icu and, of the remaining 24, 11 died (icu mortality 46%). ten of the 29 colonised patients were clinically infected. fifteen infections were documented, mostly bsi (11/15), followed by vap (2/15) and ssi (2/15). only 1 patient died from this infection (1/15, 6.7%). an evidence-based consensus on the therapeutic strategy for these infections has been reached by keelpno and the greek ministry of health which proposed the use of high dose meropenem (6−8 g/day) combined with an active aminoglycoside or colistin for strains with an mic 4 mg/ml whereas for strains with a higher mic the use of carbapenems is contraindicated and active alternatives (monotherapy with tigecycline, colistin, or an aminoglycoside or aztreonam-based combinations) could be used. antibiotic stewardship is of great importance in such a dismal situation but stringent adherence to infection control measures is probably of even greater importance for the effective containment of these pandrugresistant strains. the presentation of clostridium difficile infection (cdi) varies from mild diarrhoea to a potentially fatal pseudomembranous colitis. the recent emergence of types 027 and 078 of c. difficile has been associated with increased virulence. c. difficile takes advantage of disruption of the normal intestinal flora as caused by antibiotic therapy. the antibiotical class and the antimicrobial resistance pattern of c. difficile influence the development of disease. in the netherlands, significantly more patients with cdi due to type 027 used fluoroquinolones (or, 2.88; 95% ci, 1.01−8.20) compared with those who were infected with other pcr ribotypes. similar as type 027 cdi, patients infected with type 078 also more frequently received fluoroquinolones therapy (or, 2.17; . the risk to develop cdi due to type 027 was particularly high in persons receiving a combination of cephalosporin and fluoroquinolone (or 57.5, ). this association was also strongly dependent on the duration of therapy. the use of clindamycin was found as a protective factor. however, the recent detection of clindamycin-resistant c. difficile type 027 strains in other european countries is an important and worrying development. since the association of cdi with fluoroquinolones has only been investigated at patient level, a study was performed to investigate the relationship between cdi incidence and the preceding use of different antibiotic classes at hospital level in the netherlands. comparisons were made between hospitals where type 027 caused an epidemic, hospitals where only isolated cases of type 027 were observed and hospitals where no outbreak of cdi or type 027 were encountered. in the pre-epidemic period, the total use antibiotics was comparable between affected and unaffected hospitals. higher use of secondgeneration cephalosporins, macrolides and all other studied antibiotics were independently associated with a small increase in cdi incidence, but the effect was too small to predict which hospitals might be more prone to 027-associated outbreaks. despite the fact that the netherlands is known by its restrictive and conservative use of antibiotics, outbreaks of cdi due to new emerging types have been recognized. this is probably associated with the use of antibiotics at patient level and hospital department level rather than the use of antibiotics at the level of the healthcare institute. m. peiffer, j. bulitta, h.a. haeberle, m. kinzig-schippers, m. rodamer, v. jakob, b. nohé, f. sörgel, w.a. krueger°(trier, de; albany, us; tubingen, nuremberg, constance, de) piperacillin-tazobactam (pip-tazo) is a broad spectrum antibiotic, used for treatment of severe infections such as ventilator-associated pneumonia (vap). the effectiveness of betalactams is best predicted by the duration of free drug concentrations above the minimal inhibitory concentration (t > mic) of infecting pathogens [1] . animal experiments suggest that more than 50% of t > mic should be reached. continuous infusion (ci) of pip-tazo may enhance the therapeutic performance, but there is little data on pharmacokinetic/-dynamic (pk/pd) parameters, when ci is used in critically ill patients. objectives: the aim of our study was to determine concentrations of pip-tazo in plasma and broncho-alveolar epithelial lining fluid (elf) at steady state during ci. based on these results, the penetration ratio (plasma/elf) and pk/pd parameters for pip-tazo are derived. methods: after approval by the ethics committee, 16 mechanically ventilated critically ill patients were enrolled during treatment in 3 intensive care units. each patient received a loading dose of 4 g/0.5 g of pip-tazo, followed by ci of 12 g/1.5 g over 24 h. at steady state (67.8 + 39.5 h after loading dose), a total of 30 blood samples were drawn and bronchoalveolar lavage (bal) was simultaneously performed in 8 cases (1 sample discarded for technical reasons). samples were stored at −80ºc until analysis by liquid chromatography coupled with mass-spectrometry (lc-ms). elf-concentrations were calculated from bal-samples using the relation of ureaplasma:ureabal as dilution factor. results: plasma concentrations of pip and tazo (n = 30 in 16 pts.) amounted to 15.38+8.89 mg/ml, and 1.31+0.95 mg/ml, respectively. elflevels (n = 7) were 56.63+27.24 mg/ml, and 5.95+3.74 mg/ml. elf-levels were 368+236%, and 587+584% of corresponding plasma levels (n = 7) for pip and tazo, respectively. the ratio pip:tazo was 11.74:1 in plasma, and 9.52:1 in elf. conclusions: using advanced analytical techniques, elf concentrations were higher compared to traditional bolus administration [2] . ci yielded steady state plasma concentrations in excess of mics of susceptible bacteria (<8 mg/ml, according to eucast) in 76.6% of measurements, respectively, but elf levels exceeded 8 mg/ml in all cases. taken together, our data provide further arguments for ci being the preferred mode of administration for pip-tazo in critically ill patients with suspected vap. [ objectives: staphylococcus aureus is a potential pathogenic microorganism and a causative agent of~25% of infections in intensive care patients. an optimal empiric choice for the treatment of these infections will result in a reduction in morbidity and mortality. therefore, it is essential to provide the clinician with resistance data of the bacterial population to be treated. to optimise the empiric choice and to monitor the emergence of microbial resistance, a national surveillance program of the swab was started in the netherlands in 1996.this study describes the results of the resistance development of s. aureus from icu's of 14 hospitals all over the netherlands over a ten year period. methods: in the first 6 months of each year, the participating hospitals collected clinical isolates from among others blood and respiratory samples. in total 943 isolates were collected: 250 from 3 hospitals in the north, 187 from 2 in the east, 229 from five in the west and 280 from four in the south. the antimicrobial susceptibility was determined as a micro broth dilution method according to the clsi guidelines. results: an increase in resistance to ciprofloxacin was observed from 4% until 2002 to 14% from in 2005, which dropped again to 7% in 2006. the resistance to moxifloxacin was rather constant over time, i.e. 2%, only in 2003 8% resistance was found. resistance to clarithromycin increased to 10% in 2003, but decreased in 2006 to 6% the level before 2003. resistance to penicillin, clindamycin and tetracycline fluctuated over time at~75%, 4−8% and 2−10% respectively. during the study period seven methicillin resistant s. aureus were isolated, no resistance to vancomycin, teicoplanin and linezolid was observed. resistance to gentamicin and rifampicin was sporadicly found. regional differences were observed for ciprofloxacin, being the highest in the western and southern part and tetracycline being the lowest in the northern part. conclusion: during the 10 year study period only an increase in resistance to ciprofloxacin was observed. the data presented justify the empiric choice of flucloxacillin, (with rifampicin or gentamicin depending on the indication) in case of an infection in icu patients probably caused by s. aureus. j.j. lu°, p.r. hsueh, s.y. lee (taichung, taipei, tw) objectives: to investigate the prevalence of visa in hospitalised patients with mrsa infections or colonisations at a teaching hospital in taiwan and to evaluate the possible clonal spread of visa in the hospital. methods: from september 2001 to august 2002, 1500 consecutive mrsa isolates were collected from various clinical specimens of 637 patients hospitalised at a teaching hospital in taiwan. minimum inhibitory concentrations (mics) of vancomycin for all mrsa isolates were determined by the broth microdilution method in accordance with clsi guidelines. molecular characteristics and antimicrobial susceptibilities of visa isolates were investigated and pulsed-field gel electrophoresis was used to evaluate the clonality of the isolates. results: among the 1500 mrsa isolates, 43 (2.9%) were visa. of the 43 visa isolates, 35 had vancomycin mics of 4 microgram/ml and 8 had vancomycin mics of 8 microgram/ml. all isolates were inhibited by tigecycline at 0.5 microgram/ml, linezolid at 1 microgram/ml, and ceftobiprole at 2 microgram/ml. five (11.6%) isolates had reduced susceptibility to daptomycin (mics of 1−2 microgram/ml). six of the 43 visa isolates had decreased susceptibility to autolysis in 0.05% triton x-100. the 43 visa isolates were recovered from 21 patients; 13 of these patients had received glycopeptide treatment prior to the isolation of visa. five (23.8%) patients died despite vancomycin therapy. all 43 visa isolates carried sccmec type iii and agr group i but were negative for pvl gene (luks-lukf). none of the enterococcal van genes were detected in the 43 visa isolates. results of pfge analysis revealed that one major clone of visa isolates (90.5%, clone a exhibiting sccmec type iii, agr group i, and absence of pvl gene) had disseminated in the hospital. conclusion: this retrospective study demonstrated that clonal dissemination of visa had occurred in the hospital. rapid and correct detection of visa and proper use of antibiotics are the most effective approaches for preventing its emergence and spread. x. zheng°, c. qi, a. o'leary, m. arrieta, s. shulman (chicago, us) objectives: vancomycin remains one of the major options for treating methicillin-resistant s. aureus (mrsa) related infections. some but not all studies have shown an increase in prevalence of mrsa isolates with elevated vancomycin mic values among recent clinical isolates, so called "mic creep". although still within the susceptible range, higher mics may be associated with increased chance of treatment failure. because of the conflicting reports and lack of published data from paediatric patients, we sought to assess possible mic change over time and to compare results generated by using different methodologies including etest, agar dilution, and broth microdilution (microscan) methods. methods: we studied 318 mrsa isolates predominantly community acquired including all blood and normally sterile site isolates collected in our large children's hospital in 2000/2001, 2003, 2005, and 2007 molecular bacteriology o41 genome sequence of a virulent, methicillin-sensitive staphylococcus aureus clinical isolate that encodes the panton-valentine leukocidin toxin l. faraj, l.a.s. snyder, n.j. loman, d.p. turner, m.j. pallen, d. ala'aldeen, r. james°(nottingham, birmingham, uk) objective: to determine the genome sequence of a virulent meticillinsensitive staphylococcus aureus (mssa) clinical isolate sanot01. methods: roche 454 sequencing determined the genome sequence of the clinical isolate at 12 times coverage. newbler sequence assembly (roche) generated 10 scaffolds that were annotated using gendb and compared with other s. aureus genome sequences. results: an 11-year-old asian girl presented with fever and a 1-week history of knee pain following a trivial fall. an mr scan revealed a large subperiosteal abscess around the upper tibia secondary to metaphyseal osteomyelitis. a pvl-positive, mssa was isolated from blood cultures and pus. the child deteriorated, required repeated debridement and developed septic shock. further investigation revealed aortic valve endocarditis with an aortic root abscess. whole genome sequencing revealed that sanot01 is the first sequence of an st30 s. aureus isolate to be determined. sanot01 is agr type iii and carries three coding regions that are not found in any other s. aureus genome sequences. amongst the unique genes present in these regions is a dihydrofolate reductase gene (dfrg) which is present in addition to the usual dfrb gene. downstream of the orfx gene, a 6.5 kb remnant of sccmec type ivc was found. this sequence has only previously been found in the mrsa252 genome sequence where it is located between the orfx and sccmec type ii sequences. mrsa252 is unique in sharing 14 genome regions with s. aureus strain rf122, a causative agent of contagious bovine mastitis. all but one of these 14 genome regions are also present in sanot01. conclusions: comparison of the genome sequence of sanot01 and the closely related mrsa252 ha-mrsa (emrsa-16) isolate reveals new insights in the evolution of both ca-mrsa and ha-mrsa isolates and the link to s. aureus rf122. pvl-encoding mssa strains can be significant pathogens but are not currently under mandatory surveillance in uk. as the cost of whole genome sequencing falls further it will become feasible to use this technology to monitor the evolution of both mssa and mrsa in healthcare settings and reveal clinically relevant information that will help to improve patient outcomes. objectives: ca-mrsa often produce panton-valentine leukocidin (pvl), a leukocidin encoded by two co-transcribed genes located on lysogenised phages. five pvl-encoding phages have been described in s. aureus: phipvl, phi108pvl, phislt, phisa2mw and phisa2958. single nucleotide polymorphisms (snps) in the pvl genes tend to vary with lineage and may have structural and functional implications. we examined a selection of pvl-positive ca-mrsa reported in our hospital to determine whether sequence variation and the pvl-encoding phage vary with lineage. methods: twenty-two pvl-positive isolates were chosen to reflect mlst clonal complexes identified in our hospital: cc1, 5, 8, 59, 80, 88 and 154 . isolates were characterised by antimicrobial resistance profile, sccmec and spa type, pulsed-field gel electrophoresis (pfge) profile and multilocus sequence typing (mlst); an oligonuleotide array (clondiag arraytube) was used to detect a range of toxin and antimicrobial resistance genes. primers were designed to amplify and sequence the luksf-pv genes. the pvl-encoding phage was characterised using a recently described pcr-based assay (ma et al. j clin microbiol 2008; 40:3246−58) . results: snps were identified at seven positions in the luksf-pv genes and the snp profile varied with lineage. three of the snps were coding mutations, which may have structural and functional implications. cc1 and cc80 isolates were both found to carry phisa2mw. the pvlencoding phage was not definitively identified in the other lineages, although the cc59 isolates carried a phisa2958-like phage and the cc8, cc80 and cc154 isolates carried elongated head-type phages. one of the cc1 isolates had an unexpected snp pattern compared with other cc1 isolates; this isolate also carried a novel or variant phage. conclusion: pvl gene sequence and the pvl-encoding phage vary with lineage in pvl-positive ca-mrsa isolates. this suggests that certain lineages are susceptible to infection or lysogeny with certain phage types. although ca-mrsa commonly carry pvl genes, some strains do not; it is possible that some pvl-negative types are resistant to infection with pvl-encoding phage, perhaps via restriction modification systems. crucially, our findings suggest the pvl genes have co-evolved with their phage and are not freely transmitted between different phages. further work is required to characterise the pvl-encoding phage in other isolates and to investigate whether the pvl sequence variants result in biological differences. objectives: community-associated mrsa (ca-mrsa) of many different mlst clonal complexes (ccs) can harbour lysogenised bacteriophage dna (prophage) encoding panton-valentine leukocidin (pvl). five pvl phages (phipvl, phislt, phisa2mw, phi108pvl, and phisa2958) have been reported to date. we sought to determine the distribution of chromosomally integrated copies of these lysogenised pvl-phages amongst dominant clones of pvl mrsa in england and wales. methods: seventy isolates of previously characterised pvl-mrsa were analysed by pcrs developed by ma et. al, (jcm, 2008) , to identify and discriminate between the five known pvl phages. to maximise any underlying diversity, representatives of each cc were selected based upon their spa, staphylococcal cassette chromosome mec (sccmec), toxin gene and pulsed-field gel electrophoresis (pfge) profiles. these included isolates of internationally disseminated pvl-mrsa lineages ccs 8, 30 and 80 which resemble the usa300, south west pacific (swp) and european clones, respectively. in addition we analysed pvl-mrsa from ccs 1, 5, 22, 59, 88 and st93. results: all seven cc80 isolates, which included representatives of the european clone, possessed an elongated-head-type phage and were positive by the pcr specific for the phisa2mw phage. one of the cc30 isolates possessed a phi108pvl phage, four swp representatives had elongated head type phages, whilst the remaining four cc30 isolates harboured an icosahedral-head-type phage. one cc30 was positive for both head shapes. the 12 cc8 (including representatives of usa300), eight cc1, six cc88 isolates and the st93 isolate were all positive for elongated-head-type phage. nine cc5 isolates were non-typeable for phage head shape and specific phage pcrs. three of four cc59 isolates, harboured a phisa2958-like phage of an unknown head type and the other cc59 isolate was non-typeable. all 14 cc22 isolates possessed an icosahedral-head-type phage, 13 were positive for the phipvl phage type and one possessed phi108pvl type. we have determined the pvl phages present in a diverse panel of distinct pvl-mrsa clones and found considerable inter-lineage variation in the pvl prophage present. there was also evidence of intra lineage variation in some major ccs such as ccs 22, 30 and 59. together with variation in mlst cc and sccmec, these data suggest pvl-mrsa have evolved on multiple occasions, sometimes within the same lineage. o44 transcriptional profiling of klebsiella pneumoniae genes controlled by the transcription factor, rama objectives: rama is an arac/xyls family transcriptional activator where over expression is associated with a multidrug resistance phenotype. in both multidrug resistant klebsiella and salmonella isolates, the rama gene has been associated with increase in expression of the acrab efflux pump. in salmonella it has been shown that a deletion of the rama locus prevents the emergence of multidrug resistant mutants. therefore in order to understand the role of this key regulator in the emergence and development of antibiotic resistance, transcriptomic analyses of its regulon were undertaken in k. pneumoniae. methods: rna was extracted from a combination of isogenic mutants and clinical isolates using the qiagen or ribopure kits. rna integrity was assessed using nanodrop and agilent nanochip systems. the rna was transcribed into double stranded cdna prior to labelling with cy3. the cdna was hybridised to the nimblegen expression array platform designed from the k. pneumoniae mgh 78578 genome. results: approximately 50 genes were found to be affected by rama expression, of which twenty (involved in metabolism, physiology, transcription, drug efflux, protection responses and the cell envelope) were confirmed by rt-pcr. the rama protein appears to affect drug efflux operons not previously shown to be associated with multidrug resistance and or affected by similar proteins such as mara. comparative transcriptome analyses of different k. pneumoniae clinical isolates overexpressing rama showed that variations exist in the levels of expression of the drug efflux genes. of note genes shown to be directly regulated by rama have a marbox-like sequence within the promoter sequences. conclusion: in this study, the transcriptome of the regulatory protein, rama, was determined in the pathogen k. pneumoniae. drug efflux proteins not previously associated with rama overexpression were found to be directly affected. the rama regulon overlaps with the mara and soxs regulons in e. coli and salmonella but is directly associated with regulating the expression of a subset of genes via a marbox sequence. interestingly, variations in the levels of the expression of the regulon genes were found in the different rama overexpressing strains. m. eshoo°, c. crowder, h. li, h. matthews, s. meng, s. sefers, r. sampath, c. stratton, d. ecker, y.w. tang (carlsbad, nashville, us) objectives: the potential for fatal outcome from tick-borne human infections such as ehrlichiosis emphasizes the need for rapid diagnosis. we developed and validated an ibis t5000 assay (ibis biosciences, inc., carlsbad, ca) that can detect and identify a wide range of tick-borne pathogens from clinical samples. methods: a multi-locus assay was used that employs 16 broadrange pcr primer pairs targeting all known bacterial tick-borne pathogen families. electrospray ionisation mass spectrometry of the pcr amplicons was used to determine their base composition. these base composition signatures were subsequently used to identify the organisms found in the samples. the assay was developed using field collected ticks and a wide range of clinical sample types and has been shown to be sensitive to the stochastic limits of pcr. results: whole blood (198) , cerebrospinal fluid (20) and plasma (1) samples, which were originally submitted for ehrlichia species detection by a colorimetric microtiter plate pcr (pcr-eia), were collected consecutively from january 5 to august 1, 2008 at vanderbilt university hospital. among the total 219 specimens, pcr-eia detected 40 ehrlichia species with a positive rate of 18.3%. the ibis system detected ehrlichia in 38 of the 40 pcr-eia-positive samples and 1 in 179 of the pcr-eia-negative specimens, giving sensitivity and specificity of 95.0% and 99.4%, respectively. the ibis system further characterised the 38 ehrlichia-dual positive specimens to the species level (e. cheffeensis, 35; e. ewingii, 3) with a 100% agreement to that identified by pcr-eia using additional species-specific probes. in addition we demonstrated the detection of borrelia burgdorferi from the blood and skin of a patient with lyme disease. conclusions: we demonstrate broad-range detection of tick-borne pathogens in a single assay using skin, whole blood, plasma, skin and csf. in addition to ehrlichia, the ibis system detected 4 rickettsia rickettsii positive specimens, which were confirmed by serology and clinical findings. the ibis t5000 system, which can be completed within five hours from specimen processing to result reporting, provides rapid and accurate detection and identification of a broad range of pathogens causing tick-borne human infections. r. sampath°, l. blyn, r. ranken, c. massire, t. hall, m. eshoo, r. lovari, h. matthews, d. toleno, r. housley, s. hofstadler, d. ecker (carlsbad, us) objective: to investigate the use of a novel platform-based approach for rapid characterisation of hai organisms. pathogens that cause healthcare-associated infections (hais) pose an ongoing and increasing challenge to hospitals, both in the clinical treatment and in the prevention of the cross-transmission of these problematic pathogens. here we describe the utility of a pcr electrospray ionization mass spectrometry (pcr/esi-ms) detection platform as an innovative, rapid approach for detection and complete characterisation of important hai pathogens. methods: we have developed pcr/esi-ms based methods to rapidly identify and characterise mrsa, vre, c. difficile (nap-1 strain), p. aeruginosa and a. baumannii. each target organism can be analyzed using an independent 8-well assay that can be run on the same platform and can provide species and strain id, virulence factors, antibiotic resistance and genotyping as appropriate. validation studies were performed using 100-300 retrospective, well-characterised clinical isolates for each organism. this was followed by a prospective study for one of the 5 organisms, mrsa, that included screening of 557 clinical specimens (nares swab) from patients who were admitted to a medical unit with a high prevalence of mrsa clinical infections. results: for each of the five hai organisms, pcr/esi-ms species identifications were compared to gold standard testing results from the clinical microbiology laboratory and showed 100% concordance. for s. aureus, p. aeruginosa and a. baumannii, molecular genotyping by pcr/esi-ms was compared to pulse field gel electrophoresis (pfge) clusters and showed >95% concordance. characterisation of virulence and/or drug resistance was performed for mrsa, vre and c. difficile and showed 90−95% correct detection compared to existing testing methods. analysis of clinical specimens for mrsa showed that of the 557 swabs, 95 (15%) contained mrsa, either singly or as a dual infection with cons, 33 (5%) were mssa and 358 (58%) contained meca+ coagulase negative staphylococcus (mr-cons). comparison to gold standard analysis showed 100% sensitivity for mrsa detection with 96.8% specificity, 84% ppv and 100%npv. the pcr/esi-ms technology is a high throughput assay system useful for infection control and for epidemiological studies. it is capable of simultaneous identification of hai organisms while detecting presence of key phenotypic markers and genotypic strain characterisation. m. reijans°, j. ossel, j. keijdener, g. simons (maastricht, nl) objective: molecular diagnostics play an increasingly important role in the detection of infectious agents in cerebrospinal fluids. however, the growing list of targets and the relatively small sample volumes are challenges that demand an improved molecular diagnostic approach. the meningofinder is a multifinder assay allowing the simultaneous detection of 7 viruses and 1 internal control in 1 reaction. until now, the analysis of multifinder assays was based on size-fractionation, identifying each multifinder probe due to its specific length. here we present an alternative approach allowing realtime detection of eight meningofinder probes in a single tube. the realtime detection enables a faster analysis, less handling and lowers the risk of contamination. method: the meningofinder assay is a multifinder assay which detects herpes simplex virus 1 and 2 (hsv1−2), human parechovirus (hpev), cytomegalovirus (cmv), epstein-barr virus (ebv), enterovirus (ev) and varicella-zoster virus (vzv) plus an internal control in a single reaction. each meningofinder probe can be distinguished based upon the specific length of each probe by size-fractionation using gel or capillary electrophoresis. we developed an alternative detection method using fluorescently labelled probes which allow specific identification of 8 multifinder probes in a realtime pcr machine. results: a large number of qcmd samples (n = 44), several enterovirus types (n = 27) and characterised clinical samples (n = 66) were analyzed using the meningofinder. all meningofinder reactions were analyzed by capillary electrophoresis and by fluorescently labelled probes in a realtime pcr machine. the results of the meningofinder showed a very good correlation with the expected results (>95%). furthermore, the results of both meningofinder analyses showed a high degree of correlation. the realtime detection of the meningofinder probes decreases the analysis time and post pcr handling dramatically. we developed a new assay for the realtime detection of 8 meningofinder probes. the realtime analysis showed a very good correlation with the conventional capillary electrophoresis analysis. in addition, the realtime detection reduced contamination risk and patient results became available more quickly. the combination of multifinder technology combined with realtime detection shows great potential in fast and easy multiparameter screening of clinical samples for infectious pathogens. in-house naats were applied to nucleic acid extracts obtained by own in-house methodology in each centre. results: sensitivities for the detection of the respiratory viruses were 40% for commercial mx naat, 86% for in-house mw naat, and 90% for mono in-house naat. the viral load was low each time false-negative results were obtained. false positive results were obtained by all methods used, resulting in specificities ranging from 88%-97%. for the atypical bacteria, the 2 multiplex naats failed to detect low l. pneumophila positive samples and low m. pneumoniae positive sample resulting in sensitivities of 25% and 75% compared to 100% in the inhouse mono naats. the commercial mx naat also failed to detect strong positive samples. no false positive results were obtained for the atypical bacteria. revisiting phage therapy against problematic pathogens s61 how the past feeds the future: from d'herelle to modern phagotherapy the increasing antibiotic resistance problem boosts the interest in alternative treatments for infections. a prominent example for this is the so-called phagotherapy. it makes use of bacterial viruses − bacteriophages − as drugs against bacterial agents. these bacteriophages are isolated from nature, characterised and then tested against the bacterial strains that are targeted. in theory, this approach has several advantages. for instance, bacteriophages infect, as a rule, their bacterial prey very specifically. therefore, they do not harm the commensal bacteria of the patient. additionally, if a bacterial strain becomes resistant against a certain bacteriophage strain, evolution will provide for new and active bacteriophage strains. in practice, phagotherapy has been used for a long time. already one of the two discoverers of bacteriophages, félix d'herelle, was an ardent advocate of this method. in fact, he was the first to use bacteriophages against infections − 1919 against bacterial diarrhoea (shigella spp.). after that, phagotherapy has been used to quite some extent in europe, the us and other parts of the world until penicillin entered the market in the 1940 s. in some parts of the former soviet union and the eastern bloc, the method has been utilised until today. now, several companies and university researchers are developing bacteriophages for therapeutical purposes again. historical documents related to phagotherapy and oral history reveal a fascinating past. bacteriophages have been employed against a wide variety of bacterial diseases in a time in which there were virtually no other anti-infectives. for example, in india, millions of cholera patients were treated with bacteriophages in the 1930 s. anti-cholera phages were also poured into drinking wells as prophylactics. bacterial viruses have also been utilised by the german and soviet armies in the second world war. the history of phagotherapy makes for more than an exciting story, however. analysis of the old literature helps identify important factors for success and failure. this is especially relevant for a field which holds promise but which has had limited funds at its disposal in the past few years − and which, therefore, has been making rather slow progress. additionally, examination of the strategy used for phagotherapy in the soviet union and poland also contributes to a better application of this method today. the discovery of bacteriophages, particularly their ability to replicate and lyse pathogenic bacteria may have been among the most important milestones in the history of biomedical sciences. in the pre-antibiotic era of the early 20th century, phage therapy was becoming a powerful weapon against infectious diseases of bacterial aetiology. unfortunately, phage treatment and research was largely forgotten in the western world as antibiotics became widely available. nowadays, the rapid propagation of multi-drug resistant bacterial strains is leading to renewed interest in phage therapy. in contrast to its decline in the west, phage therapy remained a standard part of the healthcare systems in eastern europe and the ussr during the second half of the 20th century. phage preparations were used for diagnostic, therapeutic and prophylactic purposes to combat various bacterial infections. the eliava institute of bacteriophages, microbiology and virology (tbilisi, georgia) is perhaps the most famous institution in the world focused on the study of bacteriophages, particularly the isolation and selection of phages active against various bacterial pathogens. phages have been isolated against bacterial strains received from all over the former ussr and socialist east european countries; consequently, a huge collection of phages and pathogenic bacterial strains has been constructed at the institute. thousands of people were treated with individual phages and phage mixtures during the soviet era. the preparations developed in tbilisi have been studied through extensive preclinical and clinical trials. however, little of this information has ever been published and even when details are available, the trial reports do not meet internationally approved regulations and standards. bacteriophages have a number of advantages in comparison to antibiotics. phage therapy as an alternative approach for treatment of infections has become an evident and promising remedy. today, many people from various parts of the world express their willingness to take phage treatment against different infections, including those that are caused by antibiotic-resistant bacterial pathogens. the eliava institute has elaborated new, phage-based products and technological schemes for their production. strong collaboration with the medical community in the design of clinical trials according to international standards is absolutely critical to supporting the broader implementation of phage therapy. an australian male aged 57 years died from an intracerebral haemorrhage ten days after he returned from a trip to rural yugoslavia. his kidneys and liver were donated to three female recipients aged 44 years (kidney), 63 years (kidney), and 64 years (liver). four to five weeks after the organ donation, all three recipients died. all had febrile illnesses with altered mental status. subsequent testing of post-mortem tissues from the recipients identified a novel arenavirus, which was related to lymphocyctic choriomeningitis virus (lcmv). this viral detection process involved the use of high-throughput sequencing techniques to identify novel microbial rna sequences. confirmatory testing was performed using the techniques of reverse transcriptasepolymerase chain reaction, immunohistochemical analysis for arenavirus antigens, and immunofluorescent testing for igg and igm antibodies. the clinical features in these four patients as well as other similar problems with transplant-related illness from classic lcmv will be discussed, as well as details of the laboratory identification of this new virus, and implications for organ transplantation protocols in future. successful management of invasive fungal infections depends on timely and correct treatment. over the last decades a number of new tests have become available which have improved the diagnostic options. in contrast to the scenario for bacterial infections, acquired resistance in fungi is rare and thus species identification is a valuable tool guiding choice of treatment. therefore, microscopy & culture is still a corner stone in diagnosis, but culture and identification are time consuming (app. 1−5 and 1−3 days, respectively). the sensitivity and speed of microscopy have been improved by the use of fluorescent brighteners such as calcofluor white or blankophor. but only with the recent development of pna probes specific for a number of the candida spp. has species identification become possible directly from a positive blood culture before subculture on agar media. chromogenic agars allow a presumptive identification of several candida spp. and facilitate the recognition of yeast isolates in samples containing several yeasts or yeast and bacteria in combination. the use of such plates has been shown to lead to a better identification of mixed cultures in a recent nordic eqa scheme including more than 50 laboratories. rapid species identification of the most important candida spp. is possible in the routine laboratory using easy commercially available kits. thus, a species identification of c. albicans, c. dubliniensis and c. krusei can be obtained within minutes using latex agglutination kits (bichro-dubli, krusei-color; fumouze diagnostics) and c. glabrata can be rapidly identified due to its high amounts of preformed intracellular trehalase enzyme (glabrata rtt; fumouze diagnostics). finally, pna probes and fluorescence microscopy can also be used for a same day identification of a range of the clinically relevant candida spp. (advandx). susceptibility testing is possible using etest and the results are comparable with those obtained by reference methodologies in head to head comparisons. however, recent data from eqa distributions suggest that detection of isolates with acquired resistance causes many laboratories difficulties. this illustrates that a critical number of isolates should be tested per technician per week and quality control strains should be included on a regular basis. in conclusion, a number of new diagnostic tests have become available over the last decade and the diagnostic laboratories are encouraged to take advantage of these new options. 19th eccmid, oral presentations since the introduction of newer antifungals with different in vitro spectra, the aetiology of invasive fungal infections (ifi) has become a major diagnostic issue as a prerequisite for a guided antifungal therapy. while molecular methods, such as pcr and sequencing for the diagnosis of ifi have been evaluated from specimens such as blood and bronchoalveolar lavage fluid for some years, they have been less studied for biopsies. characteristics inherent to these molecular methods, e.g. sensitivity, specificity and short turnaround time makes them promising as adjuncts to conventional diagnostic tests, e.g. culture and histopathology from organ biopsies. studies using tissue from animal models of mould infections suggest that pcr might be more sensitive than culture and allows for a better species identification than histopathology. however, most of these studies used assays detecting only a small range of agents or even single organisms. while this may increase the sensitivity of the assays and reduces the likelihood of contaminations it limits the usefulness in the clinical setting, given the broad range of potential fungal pathogens. studies using fresh clinical samples suggest that the detection and identification of a wide range of fungi is possible using broad range assays in combination with sequencing or by combining more specific pcr assays. further studies are needed to optimise dna extraction, define the best molecular targets and the best method for amplicon detection. the prevention of contaminations due to ubiquitous fungi and unspecific amplifications are a major problem, especially when using broad range assays. in contrast, fish probes may potentially be more specific than pcr due to the visualisation of fungal elements in tissue. in contrast to pcr, they appear to work well with formalin fixed specimens. species identification might be more challenging than by pcr and sequencing. direct comparisons between fish and pcr are needed to characterise the pros and cons of each method in determining the aetiology of ifi. molecular tissue diagnosis has the potential to evolve into a useful method to describe the aetiology of ifi even in culture negative samples. results might be obtained fast enough to guide the antifungal therapy in patients with ifi progressive to empiric antifungal therapy. in these patients, the risk associated with invasive tissue sampling might be outweighed by potential benefits of a guided antifungal therapy. the two groups of carbapenemases (serine carbapenemases and metallobeta-lactamases (mbls)) can be encoded by genes that can be carried on plasmids. the serine carbapenemases are distinctly either class a or oxa (class d); the latter being mainly associated with acinetobacter spp. the dominant mbl subgroups, vim and imp have genes that are reportedly carried on plasmids and chromosomes. recent evidence has shown that the majority of blavim-2, even those initially reported, are indeed plasmid mediated and probably accounts for their rapid dissemination. blavim-1 genes have been recently shown to be carried on incn and incw plasmids. the "brazilian" mbl gene, blaspm-1, is exclusively chromosomally encoded. the mbls sim-1 and aim-1 are both chromosomally encoded whereas gim-1 is encoded from a plasmid of approx. 48 kb. the recently described blakmh-1 gene is also carried on a plasmid (200 kb). hitherto, only two mbl-positive plasmid sequences are available thus far -those carrying blaimp-8 and blavim-7. the former carries other resistance genes and are approx. 302 kb (inchi2), whereas the latter is a small plasmid (24 kb) and shows similarities with incp plasmids. oxa carbapenemase genes have been shown to be both plasmid and chromosomally mediated. thus far, the blaoxa-23 and blaoxa-24/40 clusters can be both plasmid and chromosomal and have mainly been found in acinetobacter spp. the blaoxa-48 and blaoxa-58 clusters have been found in k. pneumoniae and acinetobacter spp., respectively, and both are plasmid mediated. blaoxa-48 and blaoxa-58 have been shown to be carried on 70 kb and 28-100 kb plasmids, respectively. a blaoxa-58 plasmid has been recently sequenced and shown to carry two different replicases. the class a carbapenemase genes, blakpc, blaimi-2 and blages are all carried on plasmids. blakpc is found mainly in k. pneumoniae and carried on plasmids that vary in size 12−95 kb and mostly possessing the origin of replication incn. however, kpc-2 has recently described in a pseudomonas as being chromosomally mediated. blaimi-2 is exclusive to the usa and carried on a 66 kb plasmid although blaimi-1 is chromosomal. the blages genes have been found in p. aeruginosa and enterobacteriaceae of which ges-2, 4, 5 and 6 have been shown to be plasmid mediated although little else in known. this lecture will provide a synopsis, discuss the evolution of resistance due to plasmids and briefly predict what we may face in the 21c with respect to carbapenemase resistance. nosocomial infections caused by multidrug-resistant pathogens, especially gram-negative bacilli, have become a serious clinical concern in every healthcare setting worldwide. as well as carpapenemhydrolysing metallo-b-lactamases, ctx-m-type b-lactamases, and qunolone-resistance genetic determinants such as qnr, aac(6 )-ib-cr, and qepa, plasmid-mediated novel molecular mechanisms such as rmta, rmtb, rmtc, rmtd, arma, and npma responsible for pan-resistance to aminoglycosides have recently been identified in pseudomonas aeruginosa, acinetobacter spp., serratia marcescens, esherichia coli, klebsiella pneumoniae, proteus mirabilis etc. since 2003, and these enzymes have indeed methylation activity of 1405g or 1408a at the a-site of the bacterial 16s rrna as found in aminoglycoside-producing actinomycetes. these plasmid-mediated 16s rrna methylases are speculated to be originated from some nonpathogenic environmental microbes that produce aminoglycosides or some similar compounds, so it is quite natural that several new enzymes would be further identified hereafter in both clinical and livestock farming environments. rmtb and arma have widely spread in asia, europe, america and australia via various pathogenic gram-negative bacilli, we should pay special attention to the further spread of such hazardous microbes. in my talk, i would like to give an outline of newly identified molecular mechanisms that confer pan-resistance to aminoglycosides in pathogenic microbes isolated from both human and veterinary environments. [ acquired resistance to quinolones mainly results from chromosomal mutations responsible for modification(s) of dna gyrase and topoisomerase iv, and for a decrease of drug accumulation into bacteria due to decreased permeability and/or overexpression of efflux systems. plasmid-mediated quinolone resistance (pmqr) was first reported in 1998 from the usa, and two other mechanisms have been identified to date. the first pmqr determinants, qnr proteins, belong to the family of pentapeptide repeat proteins. five determinants have been identified: qnra, qnrb, qnrc, qnrd, and qnrs with 6, 20, 1, 1, and 3 different variants, respectively. they may act by binding directly to both dna gyrase and topoisomerase iv leading to protect them from quinolone inhibition. they confer resistance to nalidixic acid and reduced susceptibility to fluoroquinolones (fqs), but may facilitate recovery of mutants with higher level of resistance. the overall prevalence of qnra, qnrb, and qnrs determinants generally ranges from 1 to 5%, and they have been identified worldwide mostly in esbl-producing enterobacterial isolates. the origin of the qnra and qnrs genes were identified as shewanella algae and vibrio splendidus, respectively. the second type of pmqr determinant, aac(6 )-ib-cr, is a variant of the aminoglycoside acetyltransferase aac(6 )-ib which confers resistance to kanamycin, tobramycin and amikacin. this variant possesses two substitutions (trp102arg and asp179tyr) that are sufficient to acetylation of ciprofloxacin and norfloxacin with a 2-to-4-fold mic increase. the overall prevalence of aac(6 )-ib-cr may range from 0.4 to up to 34%, and it has been reported mainly in escherichia coli and klebsiella pneumoniae. the third type of pmqr determinant, qepa, has been identified in two e. coli clinical isolates from japan and belgium. the qepa gene encodes a 14-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily. this protein confers decreased susceptibility to hydrophilic fqs (e.g. norfloxacin, ciprofloxacin and enrofloxacin) with an 8-to-32-fold mic increase. the two epidemiological surveys for qepa may indicate its low prevalence (<1%). the natural reservoir of qepa remains unknown but might be an actinomycetal species. discovering of three main mechanisms of pmqr within the last ten years is peculiar. it may reflect the emergence of novel mechanisms of resistance but also a deeper investigation of resistance mechanisms in clinical isolates. emerging infections: can we cope with them? a. kühn°, c. schulze, h. ranisch, p. kutzer, h. nattermann, r. grunow (berlin, frankfurt-oder, de) objective: little is known about the prevalence of francisella tularensis in humans and animals in germany. interestingly, the pathogen emerged recently when several marmosets (callithrix jacchus) died from tularaemia and a group of hunters became infected in the areas of western germany. to find out more about the distribution of the pathogen also in eastern germany we investigated the seroprevalence of tularaemia under foxes (vulpes vulpes) and raccoon dogs (nyctereutes procyonoides) in the area of brandenburg (around berlin). methods: sera of animals (n = 351 and n = 32, respectively) from the years 2007 and 2008 were tested for f. tularensis − lps antibodies in an indirect elisa and suspicious samples were confirmed by western blot for lps ladder recognition using protein g − pod conjugate. furthermore we investigated the serum samples by a competitive elisa using a peroxidase-conjugated anti − lps monoclonal antibody. results: from the serum collection, we tested 31 (8.8%) foxes and 3 raccoon dogs (9.4%) positive for specific f. tularensis antibodies. the geographical distribution showed hot spots in the area of the investigated region. our results indicate for a higher seroprevalence in wildlife for tularaemia in eastern regions of germany than assumed. since the reported human cases for the last decade seem to be underestimated, the real prevalence of the pathogen is unknown. the high number of tularaemia antibody positive foxes and raccoon dogs indicates that this zoonose is present in wildlife in eastern germany. however, the impact of transmission of zoonotic pathogens from wildlife to domestic animals and humans is not yet well studied. in conclusion, the obtained data will contribute for creating of up-to-date strategy for more efficient control of the two rickettsial zoonoses. objective: helicobacter pylori is established as the primary cause of gastritis and peptic ulceration in humans. in a minority of patients with upper gastrointestinal symptoms long tightly coiled spiral bacteria, clearly distinct from h. pylori, and provisionally named as "h. heilmannii", can be observed in gastric biopsies. our objective was to isolate and identify the spiral organism, resembling "h. heilmannii" from the gastric mucosa of a finnish patient presenting with severe dyspeptic symptoms. methods: we used two different selective media for the isolation of the bacteria from gastric biopsy samples before and after treatment of the patient with a 7-day course with lansoprazole, tetracycline and metronidazole. the isolates were characterised by testing for urease and catalase activity, light and electron microscopy, and sequencing the partial 16s rrna and ureab genes. single enzyme aflp was used to analyse the genetic diversity among the isolates. results: growth of long spiral organisms was obtained from 7 out of 8 antrum and all 8 corpus biopsies before and all three antrum biopsies after treatment of the patient. the partial 16s rrna gene sequence showed high sequence similarities with other gastric helicobacter species. the partial ureab gene showed high sequence similarity with h. bizzozeronii and was clearly distinct from other gastric helicobacter species. aflp indicated that the isolates belonged to the same clone however some minor genetic diversity was observed among the isolates. results: b. pseudomallei was primarily found in close proximity to streams and in grass-rich areas but was also correlated with environmentally disturbed soil such as caused by the presence of animals, farming or irrigation. prediction maps are currently being verified by sampling predicted b. pseudomallei "hot-" and "cold-spots". see in figure a prediction map for rural darwin with red areas indicating high probability for presence of b. pseudomallei. this study contributes to the elucidation of the environmental distribution of b. pseudomallei in endemic tropical australia and to the clarification of environmental factors influencing its occurrence. it also raises concerns that b. pseudomallei are spreading due to changes in land management. o82 concurrent multi-serotypic dengue infections in various body fluids w. kulwichit°, s. krajiw, d. chansinghakul, g. suwanpimolkul, o. prommalikit, p. suandork, j. pupaibool, k. arunyingmongkol, c. pancharoen, u. thisyakorn (bangkok, th) objectives: dengue virus infection is one of the rapidly-spreading emerging diseases worldwide. the virus is divided into 4 distinct serotypes with limited cross-protective immunity; therefore, one can be reinfected with different serotypes. while each episode is usually caused by a single serotype, an individual can occasionally be infected by concurrent multiple ones. our group has previously detected dengue virus from urine and oral specimens of some patients. in this study, we sought to determine the characteristics of multi-serotype infections when analysing beyond the patients' blood compartments. methods: during 2003 during -2007 and adult patients suspected of dengue infections were enrolled. plasma, peripheral blood mononuclear cells (pbmc), urine pellets, buccal brushes, and saliva were collected during and after the febrile episode. only specimens from patients with both positive dengue serology and pan-dengue-specific rt-pcr were included. serotype-specific rt-pcr was then performed on the aforementioned various specimens of each patient. results: 95 patients met the above criteria. serotyping was successful in 85 patients. den-4 was the most common serotype, accounting for half of the cases. 20 of these 85 (23.5%) demonstrated multiserotypic infections when combining data from all specimen types in each individual. serotyping using single, conventional serum/plasma specimens, however, would detect only half of the cases. the phenomenon of concurrent multi-serotypic infections was present in all examined specimen types, including urine pellets, buccal brushes, and saliva. the most frequent combinations were den-1 + den-4 and den-2 + den-4 (5 cases each). two patients were simultaneously infected by serotypes 1, 2, and 4 and one by serotypes 1, 3, and 4. there was no demonstrable significant difference in clinical severity between single-and multi-serotypic infections. conclusion: in a dengue-hyperendemic country with simultaneous circulation of all four serotypes, the phenomenon of concurrent multiserotypic infections are more common than previously demonstrated by traditional serotyping on single serum/plasma specimens. this may be explained by the sensitivity limitation of the detection method or by biological behaviour of the virus. our findings have an implication for potentially more accurate epidemiologic studies in the future, and for further exploratory investigations regarding dengue virus in various secretions and excretions. o83 emerging concepts about the evolutionary history of hantaviruses h.j. kang, s.n. bennett, l. sumibcay, s. arai, a.g. hope, j.a. cook, j.w. song, r. yanagihara°(honolulu, albuquerque, us; tokyo, jp; seoul, kr) objective: recent discovery of genetically distinct hantaviruses in shrews (family soricidae), captured in widely separated geographic regions, challenges the conventional view that rodents are the principal and progenitor reservoir hosts of hantaviruses, and raises the possibility that other soricomorphs, notably moles (family talpidae), harbour hantaviruses. methods: using oligonucleotide primers based on conserved genomic regions of rodent-and soricid-borne hantaviruses, rna extracts from tissues of the japanese shrew mole (urotrichus talpoides), american shrew mole (neurotrichus gibbsii) and european common mole (talpa europaea) were analyzed for hantavirus sequences by rt-pcr. newfound s-, m-and l-segment sequences were aligned using clustal w and were analyzed phylogenetically by the maximum-likelihood and markov chain monte carlo tree-sampling methods, with the gtr+i+g model of evolution. results: novel hantavirus genomes, designated asama virus (asav), oxbow virus (oxbv) and nova virus (nvav), were detected in tissues of urotrichus talpoides, neurotrichus gibbsii and talpa europaea, respectively. sequence and phylogenetic analyses indicated that asav and oxbv were related to hantaviruses harboured by soricine shrews in eurasia and north america, respectively. by contrast, phylogenetic analyses of full-length s-and l-segment sequences showed that nvav formed a unique clade, clearly distinct and evolutionarily distant from all other hantaviruses. despite the high degree of sequence divergence at the nucleotide and amino acid levels, the secondary structures of the nucleocapsid proteins, as well as the l-segment motifs, of the moleassociated hantaviruses were well conserved. conclusions: while cross-species transmission has influenced the course of hantavirus evolution, such host-switching events alone do not satisfactorily explain the co-existence and distribution of genetically distinct hantaviruses among species in two taxonomic orders of small mammals spanning four continents. when viewed within the context of molecular phylogeny and zoogeography, the close association between distinct hantavirus clades and specific subfamilies of rodents, shrews and moles is likely the result of alternating and variable periodic codivergence at certain taxonomic levels through evolutionary time. thus, the primeval hantavirus might have arisen from an insect-borne virus, with ancestral soricomorphs, rather than rodents, serving as the original mammalian hosts. from south-eastern france m. kaba, b. davoust, j.l. marié, m. barthet, m. henry, c. tamalet, j.m. rolain, d. raoult, p. colson°(marseille, toulon, fr) objectives: autochthonous hepatitis e is currently considered as an emerging disease in industrialised countries and several studies suggest that hepatitis e is a zoonosis, especially in pigs, boars and deer. we aimed to study whether hepatitis e virus (hev) is commonly present in domestic pigs in southern france, and to determine the relationship between hev sequences detected from pigs and those described in human hepatitis e cases. methods: serum and stools samples were collected from 207 three or six-month-old pigs from different regions of southern france. 107 sixmonth-old pigs were from a slaughterhouse, and 100 three-month-old pigs were from a pig farm. swine igg anti-hev antibodies testing was performed using a commercial elisa kit for clinical diagnosis with minor modifications. swine hev rna detection was conducted by realtime pcr and amplification/sequencing assays using in house protocols targeting the 5 orf2 region of the hev genome. results: 40% of pigs were seropositive, and 65% of three-monthold pigs were hev rna-positive, whereas none of the six-monthold pigs were hev rna-positive. hev rna was significantly more frequently detected from stools than from serum (65% versus 22%; p < 0.001). phylogenetic analysis showed that swine hev sequences belong to genotype 3f or 3e and formed two clusters within which sequences showed high nucleotide homology (>97%). these clusters were correlated with the geographical origin of pigs as well as with their repartition into pens and buildings in the pig farm where samples were collected. swine hev sequences from the present study were genetically close to hev sequences found from humans or swine in europe, although no strong phylogenetic link could be observed neither with these latter sequences nor with those from human hepatitis e cases diagnosed in the laboratory. conclusion: our data indicate that three-month-old farm pigs from southern france might represent a potential source of contamination to humans, and they underscore the great potential of hev to cause epizootic infections in populations of farm pigs. o85 clostridium difficile: changing epidemiology trends, 2000 -2007 objectives: clostridium difficile infection (cdi) has become a growing concern world-wide with an increased reported incidence and an increase in the associated financial burden. our aim therefore was to review trends in cdi occurring from 2000-2007 inclusive. methods: all patients admitted to lothian university hospitals division (luhd) tested for c. difficile toxins a+b by eia were included. retrospective analysis of prospectively collected data was performed. the number of occupied bed days was provided by nhs-lothian statistics department. the most recent published costs associated with cdi were used to estimate potential costs to lothian nhs trust. results: 50,590 faecal samples were tested for c. difficile toxins from 2000-2007 inclusive; of these 7301 samples were positive. overall cdi was identified in 15.2 cases/10000 patient days and 5.8 cases/1000 inpatient hospital admissions. the incidence of identified cdi rose from 3.6cases/10000 patient days in 2000 to 14.8cases/10000 patient days in 2007. incidence also increased with age from 3.3cases/10000 patient days in the 0−20 years age group to 18.1cases/10000 patient days in the 61−80 years age group. renal medicine and intensive care had the highest incidences of identified cdi with greater than 57cases/10000 patient days each followed by infectious diseases and gastrointestinal medicine whose rates were 47.5 and 42.6 cases/10000 patient days respectively. medicine of the elderly in comparison had an incidence of 19.5cases/10000 patient days. of note 10% of all patients were transferred through a minimum of two specialties during the period in which they remained positive for c. difficile toxins. estimated costs over the study period for toxin testing alone were in the region of £126,500 and the minimal potential hospitalisation costs of patients with cdi was in the region of £20,000,000. conclusion: the incidence of patients identified with cdi has risen markedly and not surprisingly the incidence has also been noted to increase with age. medicine of the elderly however had a much lower incidence than several other specialties and therefore risk assessment of cdi development and containment should now also be targeted within other specialties. with 10% of identified cdi patients transferred through different specialties and the significant financial burden cdi imposes on healthcare institutions judicious application of infection control measures remains an important factor to prevent cdi spread. isolates of this strain were pvl negative, but positive for enterotoxin a (sea) and, in most cases, also for seb, sek and seq. a fifth strain was the "taiwan clone", st59/952-mrsa-v (wa mrsa-9 and -52) which also comprised two closely related sequence types. this strain carried a sccmec element of type v(t) or vii as well as pvl and, usually, seb, sek and seq. it was the most common cc59 strain in wa. the sixth strain differed from the "taiwan clone" in the presence of a sccmec type v element and in the absence of pvl. the differentiation of this clonal complex into various different strains indicates a rapid evolution and spread of sccmec elements, and the diagnostic microarray technology allows one to distinguish beyond mlst level and hence to accurately trace outbreaks and spread of these strains. a sample taker 12 has daily contact with poultry and is excluded from analysis. b sample taker 5 reported no contact with livestock elsewhere than in this study at that moment (spa-types of sample taker 5 and farm are not corresponding). c sample taker 6 tested mrsa-negative in following tests. d sample taker 9 was not tested again. complete data sets (samples taken before, directly after and 24 hours after a visit) were collected on 141 visits by 29 sample takers visiting 50 farms. on 28 farms mrsa was collected from pigs or stabledust (56%). these farms were visited 78 times by 23 different sample takers. one sample taker (#12) was positive for mrsa before visiting a farm, he was removed from the following analysis. fifteen of the 78 (19%) visits to mrsa-positive farms resulted in acquisition of mrsa and 11/23 (48%) sample takers acquired mrsa at least once after visiting a positive farm. of these 11 positive sample takers 2 acquired mrsa twice and 1 sample taker acquired mrsa three times after separate visits. of the 15 acquisitions of mrsa, 13 were negative after 24 hours. the spa-types of mrsa isolates found on the farms and sample takers were grossly comparable. on the 32 negative farms, none of the 60 visits resulted in mrsa acquisition. for further information see the table. discussion: mrsa-cc398 was acquired by 48% of the sample takers after occupational exposure in this study. however, in 11 of the 13 cases the strain was not recovered the next day, therefore acquisition was of short duration, posing a limited treat to human health. some persons seemed to be more vulnerable to acquire mrsa during their work. the sample size of this study was too small to draw final conclusions concerning this inter-personal variation. this requires a more extensive study. [ objectives: community-associated mrsa is an increasing problem and an association with food animal contact has been made in some regions. this has led to concerns about the potential role of food in mrsa transmission. the objective of this study was to evaluate the prevalence of mrsa colonisation of retail pork in canada. methods: pork chops, ground pork and pork shoulders were purchased at retail outlets in four canadian provinces in conjunction with the canadian integrated program for antimicrobial resistance surveillance. both direct inoculation of meat into enrichment broth and rinsing of meat in broth were performed for pork chops and shoulders, followed by inoculation onto chromogenic agar. ground pork was tested only using the direct method. mrsa isolates were typed by pfge and spa typing. real time pcr was used to detect panton-valentine leukocidin genes. results: mrsa was isolated from 31/402 (7.7%, 95% ci 5.5−10.7%) of samples. there was a significant difference between provinces (p < 0.001) but no difference between different products, with mrsa isolated from 23/296 (7.7%) pork chops, 7/94 (7.4%) ground pork and 1/12 (8.3%) pork shoulders (p = 0.99). 21/403 (5.2%) samples were positive using direct culture while mrsa was isolated from 15/355 (4.2%) of samples testing using the rinse method. nine samples were positive on direct culture but negative using the rinse method, while 10 others were positive only with the rinse method and only 5 were positive with both methods. seven samples (ground pork) that were positive on direct culture were not tested using the rinse method. 3 main clones were present. the most common (40% of isolates) was a group of 3 related spa types (t064, t008 and new related type) were classified as canadian epidemic mrsa-5 by pfge, an st8 human epidemic clone that has been associated with horses. pfge-non-typable spa t034 were not surprisingly common, accounting for 30% of isolates. the 3rd main group was 3 related spa types (t002, t045 and new type) that were cmrsa-2 (usa100), an st5 clone that is common in humans in canada, that also accounted for 30% of isolates. the clinical relevance of mrsa contamination of pork is currently unclear. it is possible that contact with contaminated food could be a mode of mrsa transmission in the community, although further study of the prevalence of contamination, amount of mrsa in contaminated samples, sources of contamination and implications on human health are required. o95 prevalence of the novel trimethoprim resistance gene dfrk among german staphylococcal isolates of the bft-germvet monitoring study k. kadlec°, s. schwarz (neustadt-mariensee, de) objectives: very recently a novel trimethoprim resistance gene, dfrk, was identified on a tet(l)-harbouring plasmid in a porcine mrsa isolate from the bft-germvet monitoring study. this study included in total 248 independent coagulase-positive and coagulase-variable staphylococci collected between 2004 and 2006 all over germany: 46 isolates from infections of the urinary-genital tract of pigs, 44 isolates from skin infections of pigs, 57 isolates from respiratory tract infections of dogs/cats, and 101 isolates from infections of skin/ear/mouth of dogs/cats. in this study, we investigated the prevalence and the plasmid location of the dfrk gene among these isolates. methods: pcr primers were designed and a pcr with subsequent restriction analysis of the pcr product was established to detect dfrk. isolates with positive results were tested for a plasmid location of dfrk by transfer experiments and dfrk-carrying plasmids were further analysed. the trimethoprim resistance gene dfrk was detected in another 10 isolates. all isolates were from pigs: 9 from skin infections and the remaining 1 from a urinary-genital tract infection. six staphylococcus hyicus subsp. hyicus isolates, 3 s. aureus isolates (2 mrsa and 1 mssa) and 1 s. pseudintermedius. all these isolates harboured plasmids. in 7 isolates (4 s. hyicus, 2 mrsa and the single s. pseudintermedius), the plasmid location of dfrk was confirmed by protoplast transformation with subsequent susceptibility testing and pcr analysis of the transformants. in all 7 cases, the plasmids harbouring dfrk also carried a tet(l) tetracycline resistance gene. the results of a combined pcr assay with primers from tet(l) and dfrk confirmed that the dfrk gene was always located immediately downstream of the tet(l) gene. further analysis of these dfrk-and tet(l)-harbouring plasmids showed that they varied in size between 6 and 40 kb and that similar sized plasmids differed in their ecorv and hindiii restriction patterns. the novel trimethoprim resistance gene dfrk occurred in 11 (12.2%) of the 90 porcine staphylococcal isolates from the bft-germvet study. in 8 (72.7%) of the 11 isolates, it was located on structurally diverse plasmids, however, always in close proximity to a tet(l) gene. the linkage of the dfrk and tet(l) genes allows the maintenance and coselection of such plasmids under selective pressure by either tetracyclines or trimethoprim, both of which are widely used in veterinary medicine. (table) . the isolates were resistant to ciprofloxacin, clindamycin, erythromycin, gentamicin but susceptible to vancomycin. only one se was methicillin-susceptible and two isolates were quinupristin/dalfopristin non-susceptible. all strains were clonally related and clustered into three subtypes (a, a1 and a2). cfr gene was detected in a linezolid non-susceptible strain (mic, 64 mg/l), which was recovered from a 57 y/o male who underwent liver transplantation. plasmid analysis identified six plasmid bands ranging from c.a. 1.5-to 154-kb in the cfrcarrying strain. hybridisation signals were observed from the 154-kb plasmid band as well as from a chromosomal band after i-ceui digestion. mutations at the 23s rrna, l4 or l22 were not detected. the cfr increased the linezolid mic value between 8-and 16-fold. this report highlights the ability of se to acquire linezolid resistances. the potential mobility of cfr combined with the clonal tendency for dissemination among staphylococcus spp., represent a serious threat to several potent gram-positive-active agents, including oxazolidinones. active surveillance combined with effective infection control and molecular studies seem prudent to minimise the spread of these resistance mechanisms. the objective is to get a glimpse of the potential impact of infectious diseases on music, as regards to the composer's or performing musician's own disease, living conditions or other relevant elements which might have affected the end result, the music we enjoy today. as music is an art of senses, full of drama, despair, realities of life − or just the opposite, blissful ignorance of those realities, full of romance, beauty, and delicacy − various forms of music was researched paying special attention to infections which potentially have played a significant role in the birth of that particular piece or performance. the entire research process was subjective, biased, and emotional, but done wholeheartedly. it aimed at to taking into account, not only the personal life of a composer or performing musician, but also the historical context in which the music was born. musical examples, served to the audience along with the essential background data, will show the extent to which infections have impacted music. regarding the aetiology of those infections, bacterial, viral and parasitic agents are well represented. in addition, many epochs in history have played their role. sometimes, the connections are surprising, even dramatic. if listened to with a tender ear, music quite often turns out to be affected also by infectious diseases. as physicians we should realise the strength with which some people are driven by this demonic, divine − but altogether beautiful force: music. the prevalence of antibiotic resistance has been increasing in asian countries in recent years. this problem has most likely arisen due to a combination of inadequate infection control practices particularly in hospital settings and the widespread misuse of antibiotics in hospital and community settings. factors that lead to antibiotic misuse include inappropriate antibiotic prescription due to a lack of clinical, microbiological and/or imaging data in many clinical settings in the asian region. a lack of separation of prescribing and dispensing by medical practitioners as practised in many countries in asia as well as the easy availability of over the counter medications also contribute to antibiotic misuse. optimal control of antibiotic use can only be achieved through a multipronged approach that includes better education of the public and medical practitioners on rational use of antibiotic, a review of the health system structure, as well as better control of over the counter sales of antibiotics. upgrading of microbiology and other laboratories and radiological facilities that will enhance the accuracy of clinical diagnosis is also urgently needed in most developing countries to keep pace with the complexities of managing patients in this new era to minimise the widespread practise of inappropriate antibiotic use. examination of the csf for microorganisms, wbc and differential counts, and concentrations of glucose and protein is the primary investigation to diagnose meningitis. however, this csf examination may not always be conclusive, and it can be difficult to distinguish bacterial from viral meningitis. therefore, improvement in diagnostic sensitivity and specificity of bacterial meningitis and development of rapid test for a bacterial aetiology are still needed. this presentation gives a review of the strength and weakness of several analyses and methods to reveal the microbiological agent (i.e. csf microscopy and culture, antigen or antibody detection, molecular methods to detect dna or rna) and the use of several mediators of the host immune response for diagnostic and prognostic purposes. bacterial meningitis is a medical emergency that requires a multidisciplinary approach. a diagnosis of bacterial meningitis is often considered, but the disease can be difficult to recognize. recommendations for antimicrobial therapy are changing as a result of the emergence of antimicrobial resistance. in this lecture, current concepts of the initial approach to the treatment of adults with bacterial meningitis will be summarised. the management of the critically ill patient with bacterial meningitis poses important dilemmas. controversial areas (i.e., prehospital admission antibiotics) will be reviewed and relevant literature will be discussed in the framework of current treatment guidelines, highlighting new developments in adjunctive dexamethasone therapy. acute bacterial meningitis (abm), especifically when caused by infection with streptococcus pneumoniae, still has an unacceptably poor prognosis with a mortality of 10−30%. bacterial infection of the meninges causes one of the most powerful inflammatory reactions known in medicine. yet 50 years ago, this inflammatory reaction was suggested to contribute substantially to brain damage. this concept underlies the use of anti-inflammatory agents as adjunctive therapy in abm. of all adjunctive treatments in abm, only corticosteroids have been properly evaluated in clinical trials. these trials recommend corticosteroids in patients with haemophilus influenzae type b and pneumococcal meningitis (pm). however, adjunctive corticosteroid therapy has several weaknesses such as a narrow treatment window and borderline effects on neurologic sequelae. thus, there is still the need for additional or alternate adjuvants in the therapy of abm. experimental studies using animal models (predominantly of pm) have provided insight into the pathogenic mechanisms underlying brain injury in abm. it is now clear that the autodestructive inflammatory reaction is initiated by the interaction of bacterial components with host pattern recognition receptors (prr) like toll-like receptors (tlr). prr signaling results in the activation of transcription factors like nf-kb which up-regulate the production of proinflammatory cytokines. cytokines like il-1b are also potent triggers of nf-kb activation and therefore can exaggerate the inflammatory reaction (via positive feedback loops). as a consequence, great numbers of neutrophils are recruited to the meninges. activated neutrophils release many potentially cytotoxic agents including oxidants and matrix metalloproteinases that can cause collateral damage to brain tissue. additionally to the inflammatory response, direct bacterial cytotoxicty has been identified as a contributor to tissue damage in abm. thus, experimental studies point at four different targets of adjunctive therapy, namely interference with (i) the induction of inflammation (e.g., tlr blockade), (ii) the exaggeration of inflammation (e.g., il-1 antagonism), and (iii+iv) the generation of cytotoxic factors (either of host or bacterial origin, e.g., scavenging of oxidants). this presentation will give an overview of the pathophysiology of abm (with special emphasis on pm) and highlight promising targets for adjunctive therapy in abm, as deduced from experimental studies. a clinician's approach to managing difficult infections s120 acute post-surgical prosthetic joint infection optimal management of prosthetic joint infections (pji) remains undefined. important issues such us when the implant can be retained (conservative strategy), optimal duration of antimicrobial therapy (at) or the role of rifampin are yet matter of controversy. in spite of a number of reports, literature appears confusing. among the limitations of the literature we must emphasize: 1) different criteria to classify pji; 2) different criteria to select for conservative strategy (cs); 3) no description of the initial population from which patients were selected for cs; 4) very different at (from 4 weeks to chronic suppressive therapy); 5) low numbers of patients or short follow-up; 6) absence of clinical trials. it is not so surprising that the rates of cs success have varied from 0 to almost 100%. the most useful classification to approach pji was proposed by tsukayama (1996) . in his series 25 out of 35 patients with early pji managed by a cs (debridement, exchange of polyethylen and implant retention) were cured after 4 weeks of at. the spanish group for the study of pji was constituted in 2003 within the spanish network for the study of infectious pathology (reipi), a public funded initiative. data from 139 consecutive cases of early pji attended in 10 hospitals were recorded in an online database. 117 cases managed with cs could be analysed (mean followup of 2 years). sixty-seven patients (57.3%) were cured after a mean of 81 days of at. in 35 (29.9%) the infection was not controlled (or relapsed) after a mean of 84 days of at, and the implant had to be removed. in other 15 patients (12.8%) the implant was not removed, but suppressive at was given because of suspected ongoing infection. results were significantly worse in one hospital. no other factors resulted statistically significant, but there was a trend of worse results for mrsa produced infections (p = 0.06). time from the symptoms appearance to debridement was shorter in successfully treated cases (median, 7 days) than in failures (median, 10 days); p = 0.08. good functional results were obtained in patients with successfully cs. in summary, a substantial proportion of early pji can be managed with cs strategy and a definite (non suppressive) at. it is difficult to identify patients at higher risk for failure, although mrsa aetiology and longer time until debridement seem to predict failures. different outcomes in some centres suggest that surgical technique could be an important factor for failure. more than 3 million cardiac pacing systems are implanted worldwide and the estimated rate of infections after implantation of permanent endocardial leads is 1% to 2%, but varies between 0.1 to 20%. pacemaker infections correspond to different clinical situations including localised infection in the device pocket, pacemaker leads to systemic infection associated with bacteraemia and lead-associated endocarditis. this latter represents 10 to 25% of all cases of pacemaker infections. the severity of pacemaker related infective endocarditis is sustained by a mortality range between 10 to 20%. risk factors related to infections of implanted pacemakers are correlated with fever before 24 h before implantation, temporary pacing before implantation and early re-interventions (haematoma, lead dislodgment). in contrast, an inverse correlation is observed between development of infection and antibiotic prophylaxis and implantation of a new system. data to guide therapy in patients with pacemaker infection are limited and the most appropriate management remains to be determined. according to different series, staphylococci accounted for 60 to >90% of the responsible organisms. coagulase-negative staphylococci (cns) are reported as predominant pathogens following by staphylocococcus aureus. the biofilm production, responsible for bacterial survival, and the emergence of methicillin-resistant in s. aureus and cns have complicated the management of pacemaker infections. this implies that empiric treatment of suspected pacemaker infection should coverage for staphylococci including methicillin-resistant strains. streptococci, corynebacterium spp, propionibacterium acnes, gram-negative bacilli and candida spp can cause occasional infections. the optimal therapy combines complete device extraction (percutaneous ablation or surgical removal during extracorporeal circulation) and prolonged course of antibiotics, in particular in case of multiresistant bacteria. leaving the device intact is associated with increased mortality and risk of relapsing or persistent infections. in absence of prospective studies, the duration of antibiotic treatment remains to be determined but 1 month has been shown not to be associated with an increased incidence of relapse. shortest course of treatment (2 weeks) has been proposed in case of vegetations strictly localised to leads without affecting cardiac valves. antibiotic therapy working alone should be reserved for highly selected patients. infection remains the most critical complication of ventriculoperitoneal shunt placement with an incidence of 2.2−39%. factors as the age of patient, aetiology of hydrocephalus, the type of shunt implanted, and the surgeon's experience are determined to be associated with increased risk of infection. children are more likely than adults to acquire shunt infection. the possible reasons are longer hospital stay, higher skin bacterial concentrations, immature immune systems, or more adherent strains of bacteria. staphylococci, as skin commensals, are the main causative organisms. nevertheless, in recent years a change in the epidemiology of microorganisms was observed with an increase of gram-negative bacteria. appropriate systemic antibiotics according to the antimicrobial susceptibility testing and surgical removal of the shunt with temporary external cerebrospinal fluid drainage and shunt replacement following the eradication of the infection are the cornerstone of the treatment of cerebrospinal fluid shunt infections. good compliance with infection control practices, inserion of the catheter under aseptic techniques and short-term perioperative antimicrobial prophylaxis in order to prevent the emergence of drug-resistant subpopulations are important steps in the prevention of shunt infections. o125 influenza in adults admitted to canadian hospitals: data from two seasons a. mcgeer, d. gravel, g. taylor°, c. weir, c. frenette, j. vayalumkal, a. wong, d. moore, s. michaud, b. amihod (toronto, ottawa, edmonton, montreal, saskatoon, sherbrooke, ca) objective: seasonal influenza (flu) remains a cause of substantial morbidity and mortality. antiviral treatment should be considered for all hospitalised patients with influenza. to better understand the epidemiology and burden of illness within the hospital sector in canada and the current use of antiviral therapy, we carried out a multihospital survey of virologically confirmed flu in hospitalised adults. methods: cnisp is a network of largely teaching hospitals across canada that collaborates to collect data on infections in hospitalised patients. during two consecutive years (2006/2007 and 2007/2008) hospitals within cnisp identified inpatients >16 years who had virologically confirmed flu. case patient charts were reviewed to capture demographic and clinical data and to determine whether flu was community (ca) or hospital acquired (ha). cases were reviewed at 30 days to determine outcomes. deaths at 30 days were reviewed to determine whether flu was a main or contributing cause. results: fifteen (06/07) and 11 (07/08) hospitals were recruited from the cnisp network. 532 virologically confirmed cases of flu were found, 182 in 06/07 (95% flu a) and 358 in 07/08 (56% flu a). mean patient age was 67 years, 52% were male. there was documentation of patient vaccination that season in 29%. incidence of ca flu was 11/10,000 admissions in 06/07 (range by hospital 2 − 23) and 27 in 07/08 (1 − 47). admitting diagnoses in ca cases were: pneumonia or influenza 48%, exacerbation of copd 20%, sepsis or fever not otherwise specified 9%, cardiac diagnoses 7%, other diagnoses 16%. 24% of cases were ha, range by hospital 3.9 − 5.4/100,000 patient days. 68% of patients were managed with droplet and contact isolation practices, an n-95 mask was used in 19%. 29% of ca cases but 75% of ha cases received antiviral therapy p < 0.01, almost entirely oseltamivir. 9% of cases were admitted to an icu; 30-day mortality was 8% with 2.6% attributed to influenza. conclusion: there is considerable season-season and hospital-hospital variation in flu in patients in canadian hospitals. hospitalised patients ca flu present with a wide spectrum of clinical diagnoses; nearly a quarter of all cases were ha. few ca cases but most ha cases were treated with antiviral drugs. attributable 30 day mortality was 2.6%. v. papastamopoulos, e. kakalou°, t. panagiotopoulos, j. baraboutis, m. samarkos, a. skoutelis (athens, gr) objectives: our study sought to describe influenza vaccination coverage among adults in greece for the season 2007/08. methods: we conducted a random-sampling, telephone based household survey among adult individuals in greece. for this purpose a sample of 1104 adults representative of the basic demographic, social and geographical characteristics of the overall greek population according to the latest national survey, was used. two target groups were determined for analysis: persons >65 years of age and persons with chronic conditions such as respiratory and heart conditions (other than hypertension), diabetes mellitus and other conditions. results: the influenza vaccination rate for the season 2007/08 among the adult population in greece was: 16% for the overall adult population (19.5% for men, 12.7% for women), 48.1% for people >65 years of age, 31% for persons with chronic illness (32.5% for persons with respiratory illness, 50.2 for persons with heart conditions, 35% for persons with diabetes mellitus). a high rate of 81% of the overall population reaching 88% among persons with chronic conditions report having had any type of contact with the national health system or a private physician within the last three years. among them only 20.1% had been recommended to get vaccinated. among the ones recommended any vaccination, 80.5% of persons with respiratory illness, 100% of persons with diabetes mellitus and 89.1% of persons with heart conditions had been recommended to get the influenza vaccine. conclusions: available data show unacceptably low levels of influenza vaccination coverage among vulnerable groups such as the population over 65 years of age and people living with chronic illness. influenza vaccination is the only preventive measure reducing influenza morbidity and mortality and its use has proven cost-effective among high risk groups. it is also the main vaccine recommended by physicians. however the overall rate of physicians recommendation of vaccination is very low. dynamic efforts are thus needed to design and implement strategies and policies that have demonstrated their rigorous effectiveness in enhancing influenza vaccination coverage rates. conclusions: nasopharyngeal sampling with flocked swabs is well tolerated and suitable to be used in an outpatient setting. implementation of real-time mono and multiplex naats results in a significant improvement of the rate in diagnosing lrti. hrv account for the majority of viral lrti in primary care followed by influenza and coronaviruses but also rsv and hmpv are prevalent in an adult population. in this study, 19 polyomaviruses were detected of which 10 were involved in a double infection. methods: observational analysis of a prospective cohort of 1041 nonseverely immunosuppressed adults with pp requiring hospitalisation (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) . of them, 556 were diagnosed by urinary antigen and/or 650 were diagnosed by culture. overall, 86% of pneumococcal strains were available for serotyping (quellung) and 58% for pfge (smal) and or mlst. the diagnosis of septic shock was based on a systolic blood pressure <90 mmhg and peripheral hypoperfusion with clinical or bacteriologic evidence of uncontrolled infection. results: a total of 114 (11%) patients with pp had septic shock at presentation. patients with shock were younger (61 vs 66 yrs; p = 0.003), were more frequently current smokers (45% vs 28%; p = 0.002), had received more commonly corticosteroid therapy (13% vs 6%; p = 0.015), and were more frequently classified into high-risk psi classes (81% vs 60%; p < 0.001) than those who did not have this complication. they were also less likely to have received prior influenza vaccine (31% vs 48%; p = 0.007) and had more frequently bacteraemia (41% vs 30%; p = 0.014). no significant differences were found in rates of penicillin-(2% vs 2%) and erythromycin-resistance (16% vs 12%). serotype 3 was more commonly associated with shock (40% vs 24%; p = 0.007), whereas serotype 1 was rarely associated with this complication (2% vs 9%; p = 0.041). no significant differences were found regarding genotypes: st2603 (26% vs 16%), netherlands-ser8-st53 (10% vs 3%), netherlands-ser3-st180 (10% vs 8%), spain-ser9v-st156 (10% vs 12%). patients with shock required more frequently mechanical ventilation (38% vs 4%; p < 0.001), and had longer los (19 vs 10 days; p < 0.001). early (10% vs 1%; p < 0.001) and overall case-fatality rates (25% vs 5%; p < 0.001) were higher in patients with shock. conclusions: pp presenting with septic shock is still associated with a poor outcome. it occurs mainly in current smokers, patients receiving corticosteroids, and in those infections caused by serotype 3. prior influenza vaccination and pp caused by serotype 1 are associated with a lower risk of shock. o131 high long-term mortality rate after initial recovery from severe community-acquired pneumonia background: despite the presence of antibiotics and vaccination strategies against pneumocci, community-acquired pneumonia (cap) is still a major cause for mortality in developed countries. however, it is unclear how an episode of cap influences long-term survival after initial recovery. therefore, we determined mortality up to 5 years after discharge in patients hospitalised because of an episode of severe cap in a non-intensive care setting. methods: in 5 hospitals in the netherlands, patients (pts) with severe cap (psi class iv and v without need for treatment in icu) were prospectively followed for 28 days and mortality up to 5 years after discharge was determined using the dutch municipal public records database. we used cox regression analysis to examine predictors for mortality. results: compared to strategy 2, strategy 1 resulted in slightly higher costs (chf 8,748 vs. 8,981) but fewer infections (.008 vs. 0.006) during patients' mean length-of-stay, producing an incremental costeffectiveness ratio (icer) of chf 83,303 per mrsa infection avoided. strategy 3 was dominated by strategies 1 and 2 (both more costly and less effective). sensitivity analyses suggest that prevalence of colonisation on admission is a stronger predictor of cost-effectiveness than the costs of infection or rapid screening, the probability of cross-transmission, or the incremental costs of isolation and contact precautions. increasing the relatively low on-admission prevalence at our centre by 20% lowers the icer to chf 60,973 per infection avoided. in contrast, increasing the cost of each infection, the cost of rapid screening, or the risk of cross-transmission by 20% only marginally affects the icer. conclusion: this analysis suggests that compared to risk factor identification and pre-emptive isolation, universal rapid screening upon surgical admission is not strongly cost-effective at our centre. however, local epidemiology plays an important role. in particular, settings with higher prevalence of colonisation on admission may find universal rapid screening more cost-effective. of note, no screening is undesirable, as costs and infections would be higher. results: admission and weekly screening coupled with patient isolation was found to dramatically reduce the number of mrsa acquisitions. the largest reductions were obtained with pcr technology, followed by chromogenic agar. the differences, however, were surprisingly small, and all screening technologies achieved reductions in mrsa acquisition of close to 80% compared with the no-intervention scenario. nonetheless, chromogenic and pcr-based systems were able to decrease the number of unisolated mrsa-bed-days by approximately 15 and 35% respectively. conclusions: the small differences in the ability of the screening technologies to reduce mrsa acquisition reflect both a relatively low estimated isolation efficacy and the observed highly skewed distribution of icu-stays, and may provide some important insights into the reasons for recent disappointing trial results. in particular, the skewed length of stay distribution means that most mrsa-bed days are accounted for by relatively long-stay patients for whom rapid detection will make the least difference. key sources of uncertainty were found to be isolation effectiveness and attributable mortality due to mrsa infections, both of which are difficult to accurately estimate with currently available data. the model results allow us to quantify the expected value of reducing these key uncertainties, and help to provide a rational basis for setting future research priorities. objectives: we have shown that there is substantial colonisation of mrsa among nursing home residents and staff with our recently conducted point prevalence study in 45 nursing homes which revealed an overall prevalence rate of 24% in residents and 7.6% in staff.1 the aim of this study was, therefore, to test the effectiveness of an intervention in nursing homes which sought to improve standards of infection control as a means of reducing mrsa prevalence. methods: a cluster randomised controlled trial (crct) involving 32 nursing homes, with each home representing the unit of analysis, was performed. the study ran for 12 months with data collected at baseline, 3, 6 and 12 months. nasal swabs were taken at baseline from consenting residents and staff in all homes prior to randomisation with an audit of infection control procedures also undertaken. following collection of these baseline data, nursing homes were allocated to the intervention or control arm (1:1). intervention home staff were trained in infection control, specifically hand hygiene, catheter care, barrier approaches such as use of gloves, aprons and masks, and decontamination of equipment and the environment with usual practice continuing in control homes. after each data collection timepoint, feedback was given to the intervention homes in terms of their performance and further education and training provided as required. the primary outcome was the prevalence of mrsa in intervention homes compared to control sites. results: preliminary analysis of the data has revealed no significant change in the prevalence of mrsa in the intervention and control homes, taking account of the clustering, over the one-year intervention period [risk ratio 0.83; 95% confidence intervals (ci) 0.53−1.29]. however, there was an improvement in infection control audit scores in the intervention homes, with a mean score in control homes at 12 months of 64.4% compared with 81.7% in the intervention sites; these scores were significantly different (paired t-test, p < 0.0001). the results suggest that infection control education and training as implemented in this study was not sufficient to affect mrsa prevalence. therefore, a more detailed education and training package either alone or in combination with mrsa decolonisation of staff and residents, may be required to reduce mrsa prevalence within this unique environment. [ objectives: in a response to the rapid global increase in the nosocomial prevalence of multi-resistant micro-organisms, infection control measures, such as patient isolation, are increasingly used. it is unknown how these measures influence the quality of life (qol) of patients during short-term isolation, and this was determined in a prospective matched cohort study. methods: all adult patients needing isolation in a single-patient room between 11/06 and 03/07 in the umc utrecht were eligible and included 24−48 hours after start of isolation (after giving informed consent and being able to fulfil study requirements). for each index patient we identified two control patients, admitted to the same wards at the same time, yet not subjected to any isolation measure. anxiety and depression and qol were assessed using the hospital anxiety and depression scale (hads) and visual analogue scale (eq-5d-vas) in all patients. opinions on and experiences with isolation were measured in isolated patients by means of a self-developed 'isolation evaluation questionnaire'. results: 42 isolated patients and 84 controls were included, with comparable baseline characteristics (age, sex, nationality, level of education, length of hospital stay and severity of underlying disease and co-morbidity (using the cumulative illness rating scale)). reasons for isolation were clostridium difficile-associated disease (n = 17, 40%), high risk for mrsa carriage (n = 12, 29%), or resistant gram-negative bacteria (n = 7, 17%). mean scores of questionnaires are presented in table 1. isin univariate analysis only duration of isolation of 48 hours (compared to 24 hours) was associated with a reduced quality of life (vas 57.7 compared to 68.7, p 0.02). on a visual analogue score of opposite terms isolation measures were rated with means of 87.5, 83.3 and 70.8 for safety, usefulness and quietness, respectively. conclusion: short-term isolation (up to 48 hours) is not associated with anxiousness or depression, but with positive feelings about safety, usefulness and quietness. index patients (n = 42), mean (sd) 4.7 (3.5) 5.3 (3.5) 9.9 (6.0) 62.3 (15.5) control patients (n = 84), mean (sd) 5.4 (3.7) 5.2 (3.6) 10.6 (6. objectives: there is a lack of data about the impact of healthcare associated infection (hai) on the experience of individual patients. this information is essential to empower health organisations to understand, prioritise, develop and implement solutions that will minimise risks to patients. this study explored comparable narratives from patients who had experienced a staphylococcus aureus blood stream infection with patients who had not. we conducted qualitative semi-structured interviews with eighteen adults who had previously been an in-patient in an acute teaching hospital in scotland. nine patients had had a laboratory diagnosed staphylococcus aureus blood stream infection and nine had no blood stream infection. all patients were interviewed for 20−40 minutes. the interviewer asked patients about their thoughts around hai, what concerns they had or still do, what measures they took to safeguard themselves from hai and how their experience impacted on their confidence of the nhs. probing questions were then asked depending on the responses given to the initial questions. all interviews were recorded, transcribed and analysed thematically. results: analysis of transcribed interviews is ongoing. preliminary analysis showed that all patients had positive and negative comments about infection prevention and control practice in the hospital. specific concerns included poor communication, poor cleanliness, awareness of patient boarding, lack of facilities, staff shortages and multi-tasking. some patients who had experienced bacteraemia said they had not been informed about the infection. those who had been informed were not given clear information about treatment or subsequent results. most patients were not specifically told what they or their family should do to safeguard them from infection and little or no written information about hai was provided. most patients are worried about hai on future admissions. the concerns of patients were not fundamentally different if they did or did not experience blood stream infection. the patient's reported experiences show that they have a broad awareness of systems issues that may increase risk of infection. consequently we need to involve patients in the design and evaluation of systems change and information that will improve patient experience. improving the safety and reliability of the system will have direct benefits for all patients in the hospital, not just the ones at risk of hai. analysis of surgical specialties separately revealed a significant reduction of mortality in cardiothoracic surgery who had been treated with mup-chx (2.3% (5/218) vs. 6.5% (11/170), p = 0.040, figure) . in other surgical specialties no significant difference was found. conclusion: peri-operative application of mup-chx in nasal carriers of s. aureus undergoing cardiothoracic surgery results in a threefold reduction of mortality after one year. o142 a lot done, more to do − a survey of teaching about healthcare-associated infections in uk and irish medical schools h. humphreys°, d. o'brien, j. richards, k. walton, g. phillips (dublin, ie; norwich, newcastle-upon-tyne, dundee, uk) objectives: patient safety and the prevention of healthcare-associated infections (hcai) are increasingly important health issues. medical doctors have traditionally been poor in complying with preventative measures to minimise hcai such as hand hygiene compliance. we surveyed medical schools in the uk and ireland to assess what is being taught and assessed in this area. methods: a questionnaire was drafted, piloted and then subsequently forwarded to the heads of medical schools as well as to known contact professionals with an interest in hcai in 38 medical schools. the questionnaire surveyed topics covered in the curricula, the modalities used to assess knowledge and practice, the usefulness of various teaching methods and materials, e.g. lectures, and what education resources were available. results: replies were received from 31 (82%) medical schools; two supplied data on their undergraduate and postgraduate courses. only 18 (60%) covered hcai as a quality and safety issue but over 90% covered prevalence, recognised risk factors, transmission, and preventative measures. 24 (80%) medical schools assessed competence in undertaking aseptic techniques and the disposal of sharps and mcqs were the most common (87%) means of assessment. case scenarios, resource materials and clinical skills stations were used in educating students in 26 (87%), 22 (73%) and 22 (73%) medicals schools respectively. 25 (83%) medical schools would be willing to share educational resources on hcai with other medical schools. conclusions: medical schools in the uk and ireland include hcai in their curricula but its importance as a safety and quality issue needs to be further emphasized. there is potential for agreeing a core curriculum on hcai and for sharing teaching resources such as videos and e-learning material. objectives: noroviruses are most common cause of outbreaks of gastroenteritis in uk national health service hospitals, leading to ward closure costing as much as £115 million per annum. using a detailed data set on norovirus outbreaks from three hospital systems in the south west of england, we estimated (1) the relative importance of introduction of norovirus from the community and within the hospital and (2) the cost effectiveness of ward closure at different time points during an outbreak. methods: using regression models we examined the association between number of new outbreaks in a hospital and community levels of activity and number of outbreaks currently occurring in other wards within the hospital. we examined the effect of different ward types (admission, general and long stay units) and whether the ward was open or closed to new admissions on a given day. we then undertook as analysis of cost (-effectiveness) of unit closure by developing a dynamic transmission model taking into account that ward closure may reduce norovirus transmission within and between wards. the stochastic simulation model was based on the actual characteristics of an acute hospital and the norovirus transmission parameters quantified in the statistical analysis. we measured the costs and benefits of closing affected wards at 1, 3 and 5 days after the onset of symptoms in the first case. results: community level of norovirus infection had a significant effect on the occurrence of new outbreaks as did outbreaks in admission and general medical units. the cost of closing wards to new admissions varied between £0.5 million to £0.9 million depending on the assumed effectiveness of closure in curtailing transmission. cost of bed day loss − compared with staff illness -accounted for around 90% of the total cost of closure. although the total number of cases tends to fall with rapid ward closure (by around 50% compared with no closure), the actual cost of control is similar regardless of when the closure is performed. we have developed a modelling framework to assess the effectiveness and cost-effectiveness of strategies to control norovirus outbreaks in hospital settings. ward closure is effective at preventing cases but since closure itself is an expensive intervention, it may not always be cost-effective. . other prevalent ribotypes were 001 (25%) and 106 (36%). 76% of the 027 isolates originated from 5 hospitals located in 2 healthboard areas. the remaining 18 isolates of ribotype 027 originated from 11 hospitals across scotland. in vitro 96% of 027 isolates were resistant to clindamycin with a mic range of 8−24 mg/l, mic50 of 12 mg/l and mic90 of 16 mg/l. furthermore 100% of the 027isolates were highly resistant to erythromycin (mic50 256 mg/l, mic90 256 mg/l), and to levofloxacin and moxifloxacin (mic50 32 mg/l, mic90 32 mg/l for both), while 65% of these isolates were resistant to cefotaxime (mic50=64 mg/l, mic90=96 mg/l). all 027isolates were susceptible to metronidazole, vancomycin, meropenem and piperacillin-tazobactam. high frequencies of clindamycin, erythromycin, levofloxacin, moxifloxacin and cefotaxime resistance were also found among isolates of ribotype 001 (90−99%) and 106 (94-100%). conclusion: until 2008 c. difficile ribotype 027 was only reported infrequently in scotland. in 2008, reports of ribotype 027 became more frequent and clusters were detected in 5 hospitals. the majority (96%) of ribotype 027 isolates were resistant to clindamycin. three other european countries have previously reported clindamycin resistance in pcr ribotype 027, albeit with a higher mic90 of >256 mg/l. objectives: to analyze trends in mortality due to clostridium difficile enterocolitis and to describe the most affected groups in order to better understand current clostridium difficile changing epidemiology. methods: we reviewed mortality data from the flanders and brussels regions in belgium (about 7 million inhabitants). we selected those records in which icd-10 code a04.7 (enterocolitis due to clostridium difficile) appeared as underlying cause of death within the death certificate. age-and sex-specific mortality rates were calculated for the period 1998-2006. direct standardisation was performed using the european standard population and 95% confidence intervals were calculated. stata 10 ® and excel ® were used as statistical software. objectives: toxigenic clostridium difficile is an enteric pathogen typical in the hospital environment but also community-acquired cases have been reported. however, relatively few attempts have been made to clarify the role of soil or water as a source of c. difficile infection. in november-december 2007, the drinking water distribution system in the town of nokia, finland was massively contaminated with treated sewage effluent resulting in a large gastroenteritis outbreak. the aim of the present study was to evaluate if contaminated water in this outbreak was also a potential source of c. difficile infection. a sample from the contaminated tap water and a treated sewage effluent sample were collected as soon as possible after the massive faecal contamination of the drinking water distribution system had occurred. c. difficile was isolated from heat-treated water samples by filtrating of 100 ml, 10 ml and 1 ml volumes of water and placing the membranes on selective ccey agar plates, which were anaerobically incubated for 3 d. stool samples from the patients fallen ill during the epidemic were examined for enteric pathogens, including c. difficile. all potential c. difficile colonies were subcultured on ccfa agar plates and toxin-positive isolates were identified by pcr. pcr ribotyping was performed according to the protocol of the anaerobe reference unit in cardiff, uk, using the cardiff-ecdc culture collection as a set of reference strains. after gel electrophoresis, the band patterns were analyzed using the bionumerics software. results: altogether 22 c. difficile isolates were found in water samples. twelve isolates were toxin-positive; 5 isolates were from contaminated tap water and 7 isolates from treated sewage effluent, the latter being the contamination source. among the tap water and sewage effluent isolates, 4 and 5 distinct pcr ribotype profiles were identified, respectively. the 9 human faecal c. difficile isolates detected were divided into 4 distinct pcr ribotype profiles. none of the profiles were identical with that of the hypervirulent pcr ribotype 027. two isolates, one from tap water and another from a patient, had an indistinguishable pcr ribotype profile. conclusion: our observation implies that c. difficile contamination of a tap water distribution system had occurred. waterborne transmission of toxigenic c. difficile and subsequent c. difficile infection seems possible. objectives: an accurate and rapid method is needed for typing of toxigenic clostridium difficile. a commercial automated repetitive pcr system (rep-pcr; diversilab ® , biomérieux inc., st louis, usa) utilises amplification and subsequent automated electrophoretic separation of the repetitive extragenic palindromic sequences of c. difficile. our aim was to evaluate the performance of this rep-pcr method for genotyping of c. difficile isolates and to compare it to pcr ribotyping. in addition, the correlation between the rep-pcr and the virulence gene profiles of c. difficile strains was studied. methods: a total of 195 toxin-positive c. difficile isolates were studied. we included consecutive isolates from two laboratories in finland, containing also strains of the hypervirulent c. difficile ribotype 027. in addition, selected c. difficile strains with >18 bp deletions in their tcdc genes were analyzed. the dna was extracted and the rep-pcr performed according to the manufacturer's instructions. the amplification products of rep-pcr were detected and analyzed using the diversilab system. further analysis was performed with the web-based software accompanying the system. the usefulness of the library construction option of the diverslab system for isolate comparison was tested. the virulence genes (tcda, tcdb, cdta, cdtb and tcdc) were analyzed by conventional pcr and the whole gene sequencing of tcdc was performed from isolates with deletions >18 bp. pcr ribotyping was performed using the protocol of the anaerobe reference unit in cardiff, uk. the correlation between the rep-pcr profile and the ribotype was excellent. all major ribotype groups were clustered in their own rep-pcr groups. interestingly, subgroups could be found with rep-pcr within two most prevalent ribotypes 001 and 027. the automated rep-pcr proved to be reproducible; the results from separate dna isolations and pcr-runs/microfluid electrophoresis as well as the results performed by different individuals of laboratory personnel were comparable. the rep-pcr profiles and pcr ribotypes correlated also with the virulence gene profiles. conclusion: this automated rep-pcr represents an effective and reproducible method for the genetic characterisation of c. difficile strains in clinical laboratories with molecular biology facilities. the constructed c. difficile library allows comparing the relatedness of c. difficile strains and their fingerprints over time. objectives: clostridium difficile infection (cdi) is a serious diarrhoeal illness associated with high morbidity and mortality. currently available treatments (oral vancomycin or metronidazole) usually produce good resolution of diarrhoea but are associated with a 20% to 30% incidence of recurrence. opt-80, the first in a new class of macrocyclic antibiotics, is bactericidal via unique inhibition of rna polymerase. this phase 3, non-inferiority clinical trial was conducted in more than 100 sites in north america and compared the efficacy and safety of opt-80 and vancomycin in treating cdi. methods: eligible patients were adults with acute cdi symptoms and a positive stool toxin test. patients received oral opt-80 (200 mg twice daily) or oral vancomycin (125 mg 4 times daily) for 10 days. primary end point was clinical cure (resolution of symptoms and no further need for cdi therapy 2 days after stopping study drug). secondary end point was cdi recurrence (diarrhoea and positive stool toxin test within 4 weeks after treatment). global cure was defined as a clinical cure with no recurrence. results: 629 patients were enrolled and 87% were evaluable. in the per protocol (pp) population (n = 548), mean age was 61.3±17.1 years and 44.0% of patients were male. equivalent rates of clinical cure were observed with opt-80 (92%) and vancomycin (90%) in the pp analysis; similar outcomes were observed in a modified intent-to-treat (mitt) analysis. significantly fewer patients treated with opt-80 (13%) than vancomycin (24%) experienced recurrence in the pp analysis (p = 0.004) and in the mitt analysis (15% vs 25%; p = 0.005). significantly more opt-80-treated patients achieved global cure (78%) than vancomycintreated patients in the pp analysis (67%; p = 0.006) and in the mitt analysis (75% vs 64%; p = 0.006). opt-80 was well tolerated with an adverse event profile similar to that of vancomycin. in this study -the largest comparative trial of a new antimicrobial agent versus vancomycin for the treatment of cdi -clinical cure rates after treatment with opt-80 or vancomycin were equivalent. however, opt-80 was associated with a significantly lower recurrence rate and a higher global cure rate than vancomycin. opt-80 is an oral, non-absorbed agent that has a convenient (twice daily) dosing schedule and low risk of adverse events. opt-80 represents a potential new treatment option for cdi that is associated with a lower recurrence rate than currently available treatments. results: sequence analysis (sa) revealed that locus a is absent in type 078 and that some mismatches are present in the primer annealing sites for loci b, c and g. lowering the annealing temperature and increasing the magnesium chloride concentration for loci b, c and g resolved the low yield of pcr products. applying the mlva on 54 type 078 strains revealed that 42 (80%) strains, encompassing isolates from human (n = 42) and porcine (n = 11) origin, are genetically related with a summed tandem repeat differences (strd) 10). three clonal complexes (cc, defined by strd 2) were recognized; one cc contained both human (n = 4) and porcine (n = 3) strains. the optimised mlva identified 3 genetically related clusters and 6 cc among the 67 isolates from e and ni. ccs contain isolates from more than one hospital and indeed for several clusters isolates from both e and ni. 2 isolates obtained from ni 8 years earlier were part of one large cc. the optimised mlva can distinguish and/or group type 078 strains from distinct settings. type 078 strains from human and animal origin are genetically related. the clustering of some isolates from distinct settings is consistent with community sources for type 078. the last 2 observations suggest zoonotic transmission. objectives: this paper updates our assessment of the contribution that community-associated clostridium difficile infection (cdi), as reported to the english mandatory surveillance scheme since 2007, makes to both the acute and community sectors of the national health service (nhs) in england. methods: nhs acute trusts (hospital groups) in england are required to report all c. difficile toxin positive diarrhoeal specimens processed by their laboratories whether the patients were in hospital or the community at the time of onset of the illness or when the specimen was taken via a web enabled reporting system. positive specimens from the same patient within 28 days are not reported. reported cases in patients under 2 years of age were omitted from this analysis. enhanced surveillance data (including information on date of admission, patient location prior to testing, sex, age and patient category) on cdi have been collected through a web-enabled reporting system since april 2007. risk factor information is completed on a voluntary basis. results: more than 75,000 cases of cdi in patients aged >2 years were reported, 23% of these cases were taken in non-acute settings of which 74% were taken by a general practitioner. a further 17% of specimens were taken on presentation or <2 days of admission into an acute trust. approximately 32% of all cases had at least one risk factor field completed, >19,000 cases reported risk factor information on episode category; 23% of these cases were community associated and 77% were hospital acquired. the information reported suggests that only 3% of the community associated cases were from patients with continued infection or relapsed episodes of cdi, this is compared to 8% of the hospital acquired cases who had continued infection or relapsed episodes of cdi. conclusions: 23% of the c. difficile specimens reported by acute trusts were diagnosed in a community setting. published studies suggest that 12−15% of these might be expected to have been acquired during a hospital stay within the previous month (i.e. were community onset hospital acquired cases). future work is required to investigate whether there are differences in the epidemiology, risk factors e.g. antibiotic exposure and outcome of patients with community onset disease. o152 clostridium difficile-associated disease: a newly notifiable disease in ireland m. skally, f. roche, d. o'flanagan, p. mckeown, f. fitzpatrick°( dublin, ie) new cases of clostridium difficile-associated disease (cdad) became notifiable in ireland on 4th may 2008. the main objective of this new notification process was to provide a national overview of the epidemiology and burden of cdad. this paper review the first six months of preliminary data notified. methods: the interim case definitions for new and recurrent cdad cases proposed by the european society for clinical microbiology and infectious diseases (escmid) study group for c. difficile were employed. this report reviews the weekly events of cdad extracted from the computerised infectious disease reporting (cidr) system in january 2009. census of population 2006 figures were used as denominator data in the calculation of incidence rates. results presented represent 34 weeks of data submitted. results: there were 1581 new cdad cases notified on cidr between the 4th may 2008 and 27th december 2008, representing a crude incidence rate (cir) of 37.3 cases/100000 population (estimated annual cir is 57.0 cases/100,000). all cases were laboratory confirmed. there was a higher occurrence of cases in females. the male:female ratio for the period was 1:1.6. in 0.4% of cases the sex was unknown. 71.4% of cases were in the greater than 65 years age category. the preliminary data submitted on cidr indicate that 63.0% of cases were hospital inpatients and 8.9% of cases were either gp patients or outpatients. the origin of 28.1% of samples is unknown. there was large variation between the 8 public health regions (table 1) . the incidence of cdad in ireland is prominent in older age groups and in healthcare settings. what is more remarkable is the regional variation of cases reported. this varies from 9.1 per 100,000 in the north east to 52.4 per 100,000 in the west. the seasonal trend is indistinguishable at present due to late and batch notifications from institutions. o153 clostridium difficile-associated diarrhoea in immunosuppressed patients with cancer objective: to assess the epidemiology, clinical features and outcome of clostridium difficile (cd) associated diarrhoea in immunosuppressed patients with cancer. methods: review of all episodes of cd associated diarrhoea documented in adults with cancer and haematopoietic stem cell recipients (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) . microbiologic diagnosis included cd isolation from stool samples, direct detection of cd toxin, and testing for cytotoxin production by the isolated strain. we documented a significant increase of cd associated diarrhoea, from 0.34/1000 admissions in 2000 to 4.05/1000 admissions in 2008 (p < 0.01). there were 56 episodes in 54 patients. thirty-one patients were male (55%) with a mean age of 52 years (± 16). forty three (77%) patients had an haematological underlying disease and 13 had solid tumour; 41 (73%) had received previous chemotherapy, 14 (25%) were stem cell transplant recipients (3 presenting with gvhd) and 17 (30%) were neutropenic (<500). in the previous month 52 patients (93%) had received one or more antibiotics (cephalosporins 63.5%, glycopeptides 40%, carbapenems 38.5%, betalactam + betalactam inhibitors 29%, quinolones 19%). fever >38ºc (71%) and abdominal pain (44%) were the most frequent manifestations, and the diarrhoea was hemorrhagic in 8% of the cases. most patients (77%) were treated with metronidazole (median 11 days), and the antibiotic therapy was discontinued in 56%. in 5 patients who had recovered from neutropenia, the diarrhoea resolved just by discontinuing the antibiotic therapy. no patient developed toxic megacolon or needed surgery. three patients (5.5%) had relapses. overall mortality (<30 days) was 22% (12 patients). the incidence of cd associated diarrhoea in cancer patients has increased significantly in recent years. it is related with important morbidity and mortality. better strategies to improve its prevention and treatment are needed. s154 linking research to the clinic: how laboratory findings relate to management of invasive candida infections the role of the research laboratory in the management of invasive candida infections goes beyond routinely available tests for identification of candida species and susceptibility testing of antifungal agents. cutting-edge molecular epidemiology technologies have been used to type isolates of candida species based on their dna sequences. multilocus sequence typing schemes have been designed for c. albicans, c. dubliniensis, c. glabrata, c. krusei and c. tropicalis. multi-locus sequence typing can be used to investigate possible hospital outbreaks of infection (finding widely different strain types within a unit indicates no outbreak, although the converse is not true). for c. albicans, typing multiple isolates from the same patient has shown that people tend to harbour as commensals a mixture of closely related but different strain types, which may provide for selection of the most appropriate type for invasion of a particular tissue or in response to antifungal treatment. strains in c. albicans clade 1, the largest group of related strain types, have a higher proportion of isolates resistant to flucytosine than other clades, and they all share a common resistance mechanism. research on mechanisms of resistance of candida species to many types of antifungal has progressed to the point that some investigators are looking to design dna chips that could be used both for identification and for susceptibility testing of a candida isolate. much research effort goes into detailed study of host-fungus crosstalk in experimental candida infections. animal models of infection have been greatly refined and the latest research shows how early release of chemokines that attract neutrophils into infected tissues contributes to the immunopathology of candida infection. this rapid, innate immune response also emphasizes the need for antifungal intervention at the earliest possible stage to provide the best chance for successful treatment of a disseminated candida infection − a finding now supported by clinical data as well as experimental models. translation of the latest research advances into practical diagnostic tests and new therapeutic approaches for candida infections always takes a long time − typically years − and not all research results find clinical applications. however, the level of effort invested in basic candida research ensures support for steady progress in diagnosis and management. the echinocandins are semi-synthetic lipopeptides that are increasingly used for the prevention and treatment of invasive fungal infections. understanding the pharmacokinetic and pharmacodynamic (pk/pd) characteristics of these compounds is critical for their optimal clinical use. the echinocandins have potent in vitro activity against candida spp., although c. parapsilosis is less susceptible than other candida species. the molecular mechanisms of resistance in candida species, which relate to amino acid substitutions in 'hot spots' within the fks1 gene, are becoming well characterised. susceptibility breakpoints for all three clinically available compounds have been determined recently by the clinical laboratory standards institute, with a 'susceptible-only' breakpoint of >2 mg/l suggested. the pk/pd of the echinocandins have been determined in experimental models of disseminated candidiasis, and of both disseminated and pulmonary invasive aspergillosis. these studies suggest that the echinocandins: (1) display concentration-dependent antifungal killing (or effect); (2) are extensively distributed into peripheral tissues, where they exhibit prolonged mean residence times at the site of infection; (3) are fungicidal against candida spp. and induce dose-dependent morphological changes in aspergillus spp.; and (4) result in a diminished propensity for angioinvasion by aspergillus spp. recent evidence also suggests that the echinocandins have important immunomodulatory properties, which may contribute significantly to their observed antifungal effect. pk/pd modelling and laboratory animal-to-human bridging techniques have been used to identify safe and effective dosages for the echinocandins for relatively uncommon clinical syndromes such as neonatal haematogenous candida meningoencephalitis. these techniques are an efficient method of identifying effective regimens for humans that can be expedited for study in clinical trials. pk/pd modelling techniques can and should be used to address outstanding clinical queries in relation to these compounds, including optimal dosages, decision-support analysis for the setting of in vitro antifungal susceptibility breakpoints and the clinical relevance of inherent or acquired reduced antifungal susceptibility. s156 invasive candidiasis: which antifungal treatment for which patient? management of patients with invasive candidiasis represents a complex issue owing to the heterogeneity of patients in whom these infections occur. established risk factors for invasive candidiasis, which include total parenteral nutrition, multiple organ failure and candida colonisation, are common to many types of patients that are treated within the critical care setting. furthermore, the severity of the underlying condition in these patients necessitates swift antifungal treatment to ensure optimal outcomes. an additional factor for consideration when treating candida infections is the changing epidemiology of candida species; potentially fluconazole-resistant species such as c. glabrata and c. krusei are becoming more common, particularly in patients with prior fluconazole exposure. a range of antifungal agents is available with in vitro activity against candida species. however, not all of these agents are suitable options for the clinical management of invasive candidiasis because of the overall complexity of both infection and underlying condition. for example, the position of the polyenes, particularly amphotericin b deoxycholate, is becoming less tenable as the risk of renal complications is increasingly regarded as unacceptable in patients that are likely to have or be at risk of multiple organ failure. furthermore, because of the increasing prevalence of fluconazole-resistant species, recent guidelines no longer recommend the use of azoles as first-line treatment for invasive candidiasis except in special cases, focusing instead on the echinocandin agents. there is now a wealth of clinical data available for the echinocandins. micafungin, for example, has been assessed in invasive candidiasis in clinical trials that included a wide variety of underlying conditions and patterns of infection, including neutropenic patients and those with deep infections such as peritonitis. furthermore, micafungin is the most extensively evaluated of the echinocandins in paediatric patients, having been tested both in children up to the age of 16 years and in premature infants and neonates. optimal management of patients with invasive candidiasis depends on a strategy that takes into account the complex nature of the disease. judicious selection of antifungal treatment should be accompanied by consideration of non-drug-related factors that improve survival, such as careful assessment of intravenous catheters and their potential involvement in candida infections. patients with invasive candidiasis often have underlying conditions that are severe illnesses in themselves. these range from neutropenia during cancer chemotherapy to the multi-organ failure of intensive care unit patients. against this background of severe underlying illness, it can be difficult to appreciate the success or otherwise of treatment strategies for candida infections. in the last decade, major advances have been made in antifungal therapy with the introduction of 1. echinocandins; 2. extended-spectrum azoles; and 3. lipid formulations of amphotericin b. robust clinical studies for their successful use in candidaemia have been published. however, it is important to translate these studies into practical strategies for the care of individual patients. in this presentation, individual cases will be used to provide insights into the successes and failures of these antifungal classes for the management of invasive candidiasis. specific interest will be focused on the use of fluconazole versus the echinocandins. these micafungin-based cases will be supported by insights from the evidence-based literature combined with practical experiences at the bedside. the factors to be considered are: 1. spectrum of activity; 2. drug toxicity; 3. drug interactions; 4. drug resistance; 5. pharmacology; 6. diagnosis; 7. site of infection; 8. use of biomarkers/cultures in treatment strategies; and 9. costs. it is important to realise that large clinical trials exclude many patients with invasive candidiasis. therefore, with the use of individual cases, it is possible to provide further insights into the clinical use of these outstanding antifungal agents. patient management: the era of rapid diagnostic results (symposium organised by cepheid) s161 will community mrsa and clostridium difficile change infection control in hospitals? infections caused by methicillin-resistant staphylococcus aureus (mrsa), vancomycin-resistant enterococci, and clostridium difficile are inter-related in healthcare institutions. the emergence of epidemic mrsa and c. difficile strains has placed a greater burden on infection control systems in healthcare facilities, which often must increase surveillance and change disinfection strategies to halt the transmission of these pathogens in hospitals. ironically, the usa300 mrsa strain arose in the community but now is being transmitted frequently in healthcare settings, while the epidemic nap1/bi/027 c. difficile strain was originally a healthcare-associated pathogen, which now is causing considerable morbidity in community settings. to successfully slow the spread of these pathogens, infection control must work closely with both the laboratory and pharmacy services to ensure that these organisms are detected rapidly and that the selective pressure to maintain the organisms in the institution are reduced. clearly, bundles of interventions, rather than single approaches, are necessary to contain the spread of these organisms in hospitals. the continued influx of patients with communityacquired mrsa and c. difficile infections into healthcare institutions is a challenge for infection control practitioners that will clearly increase in the future. the food borne pathogen l. monocytogenes discovered by murray in 1926 is responsible for a severe infection with various clinical features (gastroenteritis, meningitis, meningoencephalitis and materno foetal infections) and a high mortality rate (30%). the disease is due to the ability of listeria to cross three host barriers during infection: the intestinal barrier, the placental barrier and the blood brain barrier. it is also due to listeria capacity to survive in macrophages and to enter into non phagocytic cells, such epithelial cells. recovery from infection and protection against reinfection are due to a t-cell response, explaining why listeria has since many years has become a model in immunology. nearly three decades of molecular biology and cell biology approaches coupled to genetic and post-genomic studies have promoted listeria among the best models in infection biology. in depth studies of the mechanism of entry into cells has help unraveling how listeria crosses the intestinal and placental barrier. unsuspected concepts in cell biology were discovered. post-genomic studies have recently allowed to unveil the listeria transcriptional landscape during switch from saprophytism to virulence. the talk will give an overview highlighting recent results in the frame work of well established data. the last several decades of research in medical mycology have offered great insights into fungal cell biology, epidemiology, phylogenetics and the cells and molecules involved in the pathogenesis of fungal disease. a legitimate question is to ask to what extent our extensive advances in comprehension of the biology of fungal pathogens have contributed to improvements in diagnosis and treatment. to what extent do patients benefit from translation of basic research into tools for clinical management? and the equally valid question: to what extent does biological science benefit from study of fungi that are opportunistic pathogens? the speaker will examine some of these questions from the perspective of long experience in the field and the curmudgeonly attitude that develops with age. objectives: the incidence of invasive meningococcal disease (imd) has been reported in the czech republic since 1943. in response to the emergence of a new hypervirulent clonal complex, cc11, nationwide enhanced surveillance of invasive meningococcal disease was implemented by the national reference laboratory for meningococcal infections (nrl) in 1993. the case definition is consistent with the ecdc guidelines. culture and pcr are used for confirmation of cases. notification is compulsory and is performed by local epidemiologists. strains of neisseria meningitidis isolated from imd cases are referred by the field laboratories to the nrl to be characterised by serogrouping, pora and feta sequencing (http://neisseria.org/nm/typing/) and multilocus sequence typing (mlst) (http://pubmlst.org/neisseria/). in the nrl, the epidemiological database is matched against that of strains to avoid duplicate reporting in the final enhanced surveillance database. results: despite the stable trend in imd incidence (0.8/100 000) since 2005, the case fatality rate was high (11.8%) in 2007. the disease was caused mainly by serogroup b meningococci (67.4%) in 2007, followed by serogroups c (20.9%) and y (9.3%). the most frequent clonal complexes were cc18, cc41/44 and cc32 (typical for serogroup b) and cc11 (typical for serogroup c). the highest age-specific morbidity rates were observed in the lowest age groups, i.e. 0−11 months and 1−4 years (11.4/100 000 and 4.5/100 000, respectively), and were associated with high prevalence of serogroup b. the case fatality rate was the highest in infants under 1 year of age (38.5%). the incidence of imd caused by serogroup c is currently low and there is no indication for mass vaccination with menc conjugate vaccine. menb vaccine is needed for infants, but the sero/subtype coverage by the currently developed porin-based vaccines is low for czech meningococcal isolates (maximum 56.8% for nine-valent meningococcal pora vaccine). methods:the vaccination programme incorporates dedicated vaccine clinic with a multi-disciplinary team including a nurse, data manager, a pharmacist specifically appointed to the unit. additional interventions to improve vaccine uptake and outcome have included use of sms texting to announce availability of influenza annually and improve adherence to completion of hepatitis b vaccination, educational programmes changes in guidelines e.g. varicella vaccination and creation of a vaccine passport. we reviewed vaccination clinic activity in the cohort of 1,700 hiv positive patients since introduction of a dedicated vaccine service. results:there has been a large increase in the uptake of vaccinations since introduction of this service. the varicella vaccination uptake increased from 8 (2007) to 43 (2008) due to targeted vaccine programme.(see graphic, legend reads left to right) conclusion: strategies implemented increased the uptake of recommended vaccinations in our hiv population. these included appointment of a dedicated health professional team, use of it supports, education of staff and patients and development of a vaccine passport. we developed the vaccine passport to help with patient education and awareness and it will serve as a record of vaccine administration for physicians off site. in the latter year, post guideline change, we have targeted our varicella non immune population. the next intervention planned is to assess all late entrants to our healthcare system to determine need for catch up vaccines, including mmr. results: column purified recombinant protein sspb1 was found to be a good antigen for both groups of animals used for immunisation. antibodies against the recombinant sspb1 tested by opsonophagocytosis were found to enhance phagocytosis of 4 gbs strains belonging to different serotypes at the average 5.5 times relatively to control. affect against gas strains was less pronounced (2.5 times) but still statistically significant. antibodies were also capable to interfere with adherence of gbs strains carrying sspb1 relatively to the strain without the protein. adherence of the strain with sspb1 towards different cell lines was dramatically higher which proves the function of the protein as adhesin. in passive protection test carried out with mice challenged with virulent gbs or gas strains introduced intranasaly were eliminated from the lungs of the animals 20 times faster in case of the usage of anti sspb1 serum relatively the control. in the experiments with active protection sspb1 immunised animals were found be significantly better protected against gbs and gas infection. (table 1) . similar results were obtained in the analysis of factors associated with 90-day mortality. conclusion: these data suggest that outcomes of both community-onset and nosocomial bloodstream infections due to s. aureus may be improved by an expert consultation service. the factors most critical for better outcomes and modifiable in time by id specialist consultation remain to be determined and may be explored as process of care quality indicators. objective: worldwide, the present tuberculosis epidemic is characterised by an alarming emergence in drug resistance. given the limited therapeutic options in mdr (and especially xdr) tuberculosis, there is a need to define the resistance levels and mechanisms present in clinical isolates categorised as drug resistant on the basis of critical concentration testing, so as to facilitate rapid therapeutic decisions. methods: we determined quantitative resistance levels of drug resistant isolates of mycobacterium tuberculosis sampled in switzerland over the past 3 years. resistance-conferring genetic alterations were identified by probe assays and pcr-mediated gene sequencing. results: rifampicin resistant isolates unanimously showed a high-level resistant phenotype (>50 mg/l) associated with mutations in rpob. in contrast, a significant fraction of clinical tb isolates categorised as isoniazid resistant on the basis of critical concentration testing showed a low-level resistant phenotype (mostly mutations in inha); heterogeneous phenotypic resistance levels were associated with mutations in katg. one third of streptomycin resistant clinical isolates had a low-level resistance phenotype (<10 mg/l). ethambutol resistance occurred mostly in mdr strains and was linked to alterations in embb, but resistance never exceeded 25 mg/l. our data indicate that some first line agents may be considered as therapeutic treatment option despite in vitro resistance at the critical concentration. diagnostic mycobacteriology would benefit from standardised measures of quantitative drug susceptibility testing in particular for those drugs were significant variations in phenotypic resistance levels are found in clinical isolates, e.g. isoniazid, ethambutol and streptomycin. introduction recent advances in the diagnostics of varicella zoster virus (vzv) infections have changed the perception of this virus as a cns pathogen. a real-time pcr method amplifying a 70 nt segment of the vzv gb region gave 0.5 log improved sensitivity over conventional pcr and was employed for routine diagnosis of vzv dna in samples of cerebrospinal fluid (csf). in addition, a new elisa method for detection of antibodies in the csf to glycoprotein e was developed, using a mammalian cell expression system for optimal glycosylation of the antigen. these methods were utilised for studies of vzv-induced cns infections. in a retrospective study, almost all patients had a reactivated vzv infection, but only 60% showed skin lesions. the following diagnoses were made: acute aseptic meningitis (aam), n = 34; encephalitis, n= 22; meningoencephalitis, n = 6; cranial nerve affections, n = 20; encephalopathy, n = 5; and cerebrovascular disease, n = 6. in 66 patients in whom vzv dna levels were determined, significantly higher viral loads were found in those with aam and encephalitis compared to patients with cranial nerve affection (including ramsay hunt syndrome). of the 50% (n = 50) who had a follow-up, 50% (n = 25) had neurological complications after 3 months. sixty-two percent had a ct/mri scan of the brain performed and 46% of these had pathological findings. vzv encephalitis showed a more broad disease spectrum as compared with herpes simplex encephalitis (hse), as will be presented. detection of intrathecal synthesis of vzv ge antibodies was positive in the vzv encephalitis patients, as well as in some of the hse patients, arguing for a previous suggested role for vzv as a co-pathogen at least in some cases of the latter disease. vzv vasculitis was a more common finding (6% of all cases) than expected from the literature of case reports. mr findings showed that middle and posterior cerebral arteries were targeted. surprisingly, despite substantial vzv dna loads in the csf of these patients, investigated serum samples were pcr negative. thus, vzv might be suggested to be neuronally transported to the arterial walls rather than haematogenously spread. conclusions: vzv is a serious and underestimated cause of cns infection. a substantial number of the patients presented with serious neurological symptoms and sequela, and pathological findings on ct/mri of the brain were abundant, especially in patients with encephalitis and vasculitis. pk/pd controversies for the clinician s190 pk/pd and azoles the triazoles have revolutionised the treatment of invasive and allergic fungal diseases. fluconazole, itraconazole, voriconazole and posaconazole are available for clinical use. isavuconazole and ravuconazole are in development. the triazoles have broad spectrum antifungal activity. the pharmacokinetics and pharmacodynamics (pk-pd) of the triazoles have been extensively investigated in murine models of disseminated candidiasis. the pd parameter that optimally links drug exposure with the observed antifungal effect is the ratio of the area under the concentration-time curve (auc) to mic (auc:mic). there is increasing information on the magnitude of the auc:mic that is required for optimal antifungal effect. pk-pd principles have been used to define in vitro susceptibility breakpoints. the triazoles are fungistatic against candida spp. their mode of action against aspergillus spp. is less well defined, although they clearly exhibit dose-dependant decrement in fungal burden in laboratory animal models of invasive pulmonary aspergillosis. the triazoles accumulate in tissues and this is important for an understanding of their antifungal effect. in humans, the triazoles are characterised by complicated pharmacokinetic properties. both itraconazole and voriconazole exhibit nonlinear pharmacokinetics. the triazoles all exhibit clinically relevant exposureresponse relationships. recent work from our laboratory suggests that itraconazole exhibits clinically relevant concentration-toxicity relationships. higher concentrations of voriconazole are associated with a progressively higher probability of hepatotoxicity, photopsia and central nervous system toxicity. because of the significant pharmacokinetic variability and clinically relevant drug exposure-response relationships, therapeutic drug monitoring (tdm) is frequently used. a strong argument can be made for the routine monitoring of itraconazole and voriconazole. there may also be grounds to consider monitoring posaconazole levels. tdm should be considered for all patients receiving triazoles who have refractory disease. furthermore, tdm should be considered when compliance, drug interactions and variable pharmacokinetics result in uncertainty about resultant drug exposures. an understanding of the pk-pd relationships of the triazoles has been instrumental in optimising their clinical efficacy. innate immunity s192 the inflammasomes: danger sensing complexes triggering innate immunity the nod-like receptors (nlr) are a family of intracellular sensors of microbial motifs and 'danger signals' that have emerged as being crucial components of the innate immune responses and inflammation. several nlrs (nalps and ipaf) form a caspase-1-activating multiprotein complex, termed inflammasome, that processes proinflammatory cytokines including il-1beta. amongst the various inflammasomes, the nalp3 inflammasome is particularly qualified to sense a plethora of diverse molecules, ranging from bacterial muramyldipeptide to monosodium urate crystals. the important role of the nalp3 inflammasome is emphasized by the identification of mutations in the nalp3 gene that are associated with a susceptibility to inflammatory disorders. these and other issues related to the inflammasome will be presented. it is now 20 years since charles janeway hypothesized the existence of clonally derived pattern recognition receptors and pointed to the importance of these in initial responses to bacterial and viral infections. janeway's hypothesis has been validated by the discovery of three groups of prrs. first, are the toll-like receptors which detect microbial lipids and non-self nucleic acids at the cell surface an in intracellular compartments. in addition cytoplasmic sensors of bacteria (nods) and of viral nucleic acids (rigs) have also been characterised. as well as being critical for responses to infections, these prrs also underlie a large burden of autoimmune and inflammatory disease in the human population and are thus important targets for therapy. in my talk i will describe the molecular mechanisms by which these conserved pathogen associated moecules are recognized by the tlrs with particular reference to lipo polysaccharide and single stranded viral rnas. i will also present new results which show how receptor activation is coupled to downstream signal transduction and in particular the role played by oligomeric signaling platforms assembled form adaptors and other signaling molecules involved in the pathway. i will discuss the potential for structural analysis to be used in the rational design of new drugs. this session proposes a critical review of the most salient recently published papers in the field with a special focus on control of multi drug-resistant organisms, prevention of infections in the intensive care unit, surgery etc. and highlights the need for validity/scope assessment. it emphasizes also the importance to prioritise information published in the abundant literature available so as to be able to summarise and understand the potential changes in clinical practice, and identify unresolved issues and areas of possible future clinical research. tourism is europe's face to the world. it is also a major source of revenue, employment and productivity. each year over 450 million arrivals are recorded into the continent, and of those, approximately 4 million are from latin america. returning travelers are even more numerous and more often associated with disease transmission into europe. within countries of the european continent, imported cases of environmental and zoonotic illnesses such as cholera, dengue, malaria, viral haemorrhagic fevers and west nile virus infections are a rare but established fact. diseases imported from latin america with the potential for autochthonous transmission (chikungunya, malaria, yellow fever) and or high infectivity (viral haemorrhagic fevers) will be described in detail and the possibility of european outbreaks from latin american countries will be discussed. cutaneous leishmaniasis (cl) is a worldwide disease, endemic in 88 countries, that has shown an increasing incidence over the last two decades. so far, pentavalent antimony compounds have been considered the treatment of choice, with rates of curing close to 85%. however, the high efficacy of these drugs is counteracted by their adverse events. recently, in vitro and in vivo studies have shown that no plays a key role in the eradication of the leishmania parasite objective: to determine whether a no donor patch (developed by electrospinning technique) is as effective as meglumine antimoniate in the treatment of cl while causing less adverse events methods: a double-blind, randomised, placebo-controlled clinical trial was conducted with 178 patients diagnosed with cl in santander, colombia, south-america. the patients were randomly assigned to two groups. during 20 days group 1 received simultaneously meglumine antimoniate and placebo of nitric oxide patches while group 2 received active nitric oxide patches and placebo of meglumine antimoniate. biochemical determinations (aspartate aminotransferase, alanine aminotransferase, creatinine and pancreatic amilase) were measured at he beginning and at the end of the treatment. a follow up was realised 21, 45 and 90 days after the beginning of the treatment results: the study included 69 (38.77%) women and 109 (61.23%) men. the average age in group 1 was 30.80±14.23 years; while in group 2 it was 27.88±13.79 years. clinical and demographic data were similar in the two groups. after the follow up period, the complete clinical healing of group 1 was 94.81% versus 37.14% for group 2 (p= 0.0001). treatment with no patches generated both, a lower frequency of non-serious adverse events (fever, anorexia, myalgia, arthralgia, headache), and a reduced variation in biochemistry determinations (asat 26 the treatment with no patches resulted in a lower percentage of complete clinical response compared with meglumine antimoniate. despite its inferior effectiveness, the safety, the lower frequency of adverse events, the facility of administration (topical) and the low cost of the patches justifies its evaluation in further poblational studies, especially in populations as the colombian ones, where the serious adverse events due to glucantime have increased dramatically. objectives: trichinellosis is a zoonotic disease which has never been reported in taiwan and is rarely linked to consumption of reptiles. we investigated the first documented outbreak of trichinellosis in taiwan consisting of 8 patients who became acutely ill after eating at the same restaurant in may 2008. we conducted a retrospective cohort study by interviewing the patients and persons who ate together with them. a case was defined as illness in an attendee who had fever (>38.0ºc) or myalgia 4 weeks after the festivals and was seropositive to trichinella antigen using an enzymelinked immunoassay and immunohistochemical staining. environmental study of the soft-shelled turtle farm was performed. results: of the 23 attendees, 8 persons met the case definition (attack rate = 35%). the most common presenting symptoms were myalgia (88%), fever (88%), and periorbital swelling (38%). all 8 patients sought medical care; five were hospitalised. of the 7 patients who underwent blood test, all had moderate eosinophilia. all 8 patients' serum samples were strongly reactive to trichinella excretory-secretory antigen. the only food item significantly associated with illness was the raw softshelled turtle meat (relative risk undefined; p = 0.005). traced back to the farm, histological examination of soft-shelled turtles was negative for trichinella species. the most likely cause of this outbreak was consumption of raw soft-shelled turtle served in the festivals. this investigation indicates taiwan is not free of trichinellosis. prevention and control programs of trichinellosis should be established. the public should be aware of the risk of acquiring trichinellosis from consumption of raw soft-shelled turtle. objective: to develop and evaluate a modified, rapid giemsa staining procedure for detection of malaria parasites in blood smears. disadvantage of the rapid commercially available staining methods is that they require highly experienced technicians for interpretation of results because the interpretation can be difficult. for this reason, many laboratories use the giemsa stain. shorter giemsa staining times have been reported previously, however, to our knowledge, the effect of 5 and 10 minute staining in different giemsa dilutions have not been evaluated. the stock solution of giemsa stain (merck, darmstadt, germany) was used in different dilutions (1:10 and 1:5) and incubated for different lengths of time (10 min and 5 min). the staining effect was compared to our standard giemsa stain (1:40, 45 min). sensitivity was determined by examining smears of p. falciparum from fresh and edta blood. the level of parasitaemia was followed in two patients admitted to our hospital with p. falciparum parasitaemia's of 21.5% and 28.8% (see table; patient a and b) by examination of blood smears taken at different time points after initiation of therapy. these samples were used to evaluate the different giemsa dilutions and staining times. smears were read by three independent observers (a clinical microbiologist, a laboratory technician specialised in parasitology, and a resident in clinical microbiology). in the table results of the three staining methods on blood from two patients from ghana with high parasitaemia's on admission and during follow-up are shown. all smears were equally easy to read and yielded parasite counts within internationally accepted ranges of variation (see united kingdom national external quality assessment service). conclusion: staining blood smears for detection of plasmodium falciparum parasites with a 1:5 dilution of giemsa stain for five minutes provides easy to read slides and results comparable to those obtained with the standard giemsa staining. advantage of the rapid method is the shorter turnaround time, disadvantage is the larger amount of stain used. objectives: diarrhoeal diseases are common in developed and developing countries and are major causes of morbidity and mortality worldwide. the need to differentially diagnose protozoan parasites versus other gastrointestinal (gi) aetiologies is well recognized. the most common gi protozoan parasites infecting humans worldwide are considered to be entamoeba histolytica, giardia lamblia, blastocystis hominis, dientamoeba fragilis and cryptosporidium spp. laboratory detection of these parasites is relying on microscopic analysis of stool samples and water concentrates, as well as enzyme immunoassay (eia) tests. utilising the microscopic examination usually results in underdetection of gi parasites, while usage of eia is often not cost-effective. methods: savyon diagnostics is currently engaged with developing an approach aiming to address the unmet needs and the current limitations in this field. this approach includes 3 major aspects: (1) the ability to detect a panel of all the above 5 organisms in one test kit, (2) the possibility to perform the diagnosis in two steps − first, simultaneous detection of these organisms without distinguishing between the different species for screening of large number of specimens, and second, distinctive detection of the specific aetiology in the positively-found specimens, and (3) the ability to apply eia diagnosis in formalin-preserved specimens for all the mentioned parasites. results: polyclonal antibodies were produced in-house based on native antigen extracts, recombinant antigens and synthetic peptides. the resulted inventory of antibodies enabled finding the optimal combination that provided the desired performance parameters for separate detection of each of the parasites in fresh, frozen or formalin preserved faeces specimens. the analytical limit of detection and the performance in characterised clinical specimens were comparable to microscopy or to reference eia, when available. the results show unique detection of e. histolytica in formalin-preserved specimens, which is comparable to detection in fresh specimens. furthermore, we demonstrate simultaneous detection of the parasites without compromising performance characteristics in fresh or preserved specimens. the presented work is a paradigm of an innovative approach, expected to advance the diagnosis of protozoan parasites in gi patients, thus, enabling appropriate and cost-effective diagnosis and treatment. objectives: systemic administration of certain facultative anaerob bacteria to mice bearing solid tumours leads to accumulation in tumours compared to normal target organs, like spleen and liver, and to retardation of tumour growth. salmonella enterica serovar typhimurium (s. typhimurium) as well as escherichia coli 1917 nissle (ecn) are such bacteria. preliminary experiments showed that such bacteria that exhibit the ability to form biofilms in vitro might also do so in tumours. in the present study this was systematically investigated. methods: biofilm formation of bacteria were detected on low-salt biofilm plates. additionally, salmonella-or e. coli-infected ct26tumours of balb/c mice that were left untreated or were treated with anti-gr1 to deplete neutrophilic granulocytes were removed two days post infection, fixed and prepared for electron microscope analysis. the expression of different genes which are probably involved in the biofilm formation were tested via real-time pcr. results: when examined after colonising tumours s. typhimurium sl7207 and sl1344 as well as ecn are almost exclusively found extracellular although they are able to invade the ct26 cells in vitro. interestingly, like in vitro all three bacteria form biofilms to various extend when residing in the tumours. this was followed in more detail for s. typhimurium sl7207. biofilms were not formed by sl7207 when neutrophils had been removed by antibodies. in addition, when arda a central switch for biofilm formation in the salmonellla had been deleted no biofilms could be found. importantly, now bacteria could be found intracellularly most likely in neutrophilic granulocytes. conclusion: the formation of biofilms by facultative anaerobic bacteria when residing in solid tumours is a novel and surprising finding. when neutrophils were removed, no biofilms are formed, while uptake into neutrophils is allowed when the ability of the bacteria to form biofilms was blocked. hence, it appears that the bacteria use biofilm formation as a defence system against the immune system of the host. objectives: rama is an arac/xyls family transcriptional activator found in klebsiella pneumoniae, salmonella spp. and enterobacter spp., the overexpression of which is associated with an mdr phenotype. recently a tetr-like gene that lies upstream of rama, known as ramr, has been identified as a repressor of rama. k. pneumoniae kp342 is a diazotrophic endophyte strain which has been reported to exhibit notable resistance to antibiotics. despite its mdr phenotype kp342 has been shown to exhibit attenuated pathogenicity in mouse models in comparison to clinical k. pneumoniae strains. the aims of this study were to: determine the levels of rama expression and establish its role in kp342's mdr phenotype; determine the effect of ramr complementation on rama expression and antibiotic susceptibility. methods: genome and sequence analysis performed in k. pneumoniae strain kp342 demonstrated a 96 bp deletion within the ramr gene. cloning and complementation with full size wild type ramr was performed in kp342 (hereby known as kp342/ramr). rt-pcr was used to assess levels of gene expression which were subsequently quantified using bio-rad quantity one software. mic testing was performed against chloramphenicol (cm), norfloxacin (nor) and tetracycline (tet) according to bsac guidelines. biofilm formation was measured using a modified protocol of o'toole and kolter. results: kp342 containing the mutated ramr gene (96 bp deletion) was shown to overexpress rama and the putative outer membrane protein roma. complementation of the ramr gene resulted in the repression of both rama and roma transcription by 3−4 fold. interestingly, the ramr complemented strain demonstrated increased biofilm formation (up to 9-fold increase) over a 72 hour period in both lb and m9 medium after static growth at 37ºc. mics of the tested antibiotics were reduced up to 16-fold in kp342/ramr compared to the ramr mutated kp342. conclusions: this result demonstrates that ramr acts as a repressor of both rama and putative outer membrane protein roma thereby increasing its susceptibility to antibiotics. however the restoration of a functional ramr in kp342 also increases biofilm formation significantly, suggesting that ramr plays a role in the regulation of biofilm formation genes and possibly bacterial virulence. rifampicin showed the highest activity on biofilm matrix and bacteria in sa and pa biofilms. results also indicated that biofilm viable mass was more susceptible to treatment than the biofilm matrix, which is mainly responsible for biofilm persistence. further research should specifically focus on compounds destroying matrix and which can be used as an adjunct to antibiotic therapy. [ objectives: staphylococcus epidermidis is a common cause of foreignbody infections (fbi) because of its ability to form biofilms. biofilms are very resistant to antibiotics. active and passive immunisation against biofilm-associated bacterial antigens may be an alternative. we studied the effect of immunisation against the lpxtg protein sesc in s. epidermidis biofilms in vitro and in vivo. we previously reported that sesc is present in all s. epidermidis strains tested. sesc is mainly expressed during the early and late fbi and at a higher level in sessile cells than in planktonic cells. methods: we used rabbit polyclonal anti-sesc-iggs (4 mg/ml) to study biofilm inhibition in vitro and in vivo in our rat model (50 mg igg per rat) on 1-day old biofilms. we also vaccinated rats twice with sesc according to standard protocols. serum samples taken at day 0 and 2 weeks after the 1st and 2nd immunisation were tested by elisa and showed an increase in anti-sesc antibody levels. s. epidermidis strains 10b and 1457 are biofilm forming strains and have been described before. for in vitro experiments, s. epidermidis 10b or 1457 were mixed with anti-sesc-iggs and incubated for 2 hours at 4ºc. subsequently 10 6 cells were added to each well. after 24 h at 37ºc biofilms were washed and stained with crystal violet and od595 was measured. for in vivo experiments, catheter fragments were pre-incubated with s. epidermidis 10b and implanted subcutaneously in each rat. after explantation, the average number of cfu was determined after 24 hrs. results: our data show that rabbit anti-sesc-iggs inhibit in vitro biofilm formation by s. epidermidis strains 10b and 1457 by 74% and 65%, respectively (n = 9). in the in vivo rat model, rabbit anti-sesc-iggs reduced the bacteria in a 1-day old biofilm 60-fold (n = 18). active immunisation with recombinant sesc led to a 10-fold reduction of cfu compared to control rats in 1 day-old biofilms (n = 10). after 3 days, the reduction in biofilm-associated bacteria in the immunised rats was 15-fold (n = 10) (fig 1.) . conclusion: sesc represents a promising target for prevention of s. epidermidis biofilm formation. the higher effect of passive immunisation compared with active immunisation is probably due to the subcutaneous injection of anti-sesc-iggs at the place of catheter insertion. objectives: staphylococcus epidermidis has emerged as a pathogen associated with infections of implanted medical devices impeding their long-term use. characteristics of s. epidermidis that allow persistence of infection are the ability of bacteria to adhere to surfaces in multilayered cell clusters, followed by the production of a mucoid substance more commonly known as slime, encoded by the ica operon. the adherent bacteria and slime are collectively known as biofilm. the coupled effects of specific chemical terminal surface groups and flow conditions on slime production and biofilm formation by s. epidermidis were investigated in correlation to the expression of two genes of the ica operon. methods: reference control strains (atcc35984, slime-positive and atcc12228, slime-negative), and two clinical strains isolated from different hospitalised patients, (one ica-positive/slime-positive and one ica-positive/slime-negative) were tested. bacteria grown in bhi medium were suspended in physiological saline at a concentration of~3×10 9 cells/ml. hydroxyl (oh)-terminated (hydrophilic) and methyl (ch3)terminated (hydrophobic) glass surfaces were used as substrates in a parallel plate flow chamber. bacterial adhesion was examined under two flow rates: 2 ml/min and 20 ml/min for two and four hours. total rna from both planktonic (p) and adherent (a) bacteria, after detachment with trypsin, was isolated by the trizol method. reverse transcription followed by relative real-time pcr (rrt-pcr) towards a 207 bp part of 23s rrna gene, allowed the detection of expression levels of icaa and icad. adherent bacteria were investigated with scanning electron and confocal laser microscopes. results: higher expression levels of both icaa and icad genes onto glass and especially methyl-terminated glass surfaces were calculated by rrt-pcr, under higher flow rate in two hours by the reference and the clinical slime-positive strains. these results correlate well with adherent bacterial cell counts and images taken by both microscopes. the icapositive slime-negative clinical strain showed lower expression levels of ica genes, less adherent ability and pia production on glass surfaces, as observed by microscopes. higher flow rate enhances the expression level of both ica genes, with a peak in two hours. hydrophobic biomaterial surfaces seem to play a crucial role to initial adherence, increasing ica gene expression and pia synthesis. consenting men and women with dfi (predefined by clinical signs and symptoms) caused by mrsa were potentially eligible including those associated with bacteraemia. patients with initial osteomyelitis were excluded. patients could receive l 600 mg bid either iv or po. primary end point were cure or improvement rates (c+i) and microbiologic eradication (me) at 60 days after the beginning of l. secondary end points were c+i on days 5 and 30 after the beginning of treatment and hospital discharge day, need of amputation, duration of therapy and mortality rates. all the adverse events were collected. results: 70 patients were enrolled. relation men:women was 2.1.the age of patients was 63.2±13 years and the average period from the diagnosis of diabetes was 16.5±9.7 years. associated bacteraemia was present in 27.1% of patients included. primary end points: c+i 60 days after the beginning of l was achieved in 91.4% of patients and me was obtained in 84.3% of patients. secondary end points: c+i on day 5, hospital discharge day and day 30 after the beginning of treatment and were; 70%; 84.3% and 88.6% respectively. only 8 patients needed a minor amputation. the primary and secondary end points in the subgroup of bacteraemic episodes were not statistically different of those previously described. the mean duration of therapy was 29.5±18.4 days. global mortality was 4.3%. only one episode of polineuropathy was reported. neither thrombocytopenia nor lactic acidosis was found. conclusions: l achieved excellent c+i even at first evaluation visit in documented dfi caused by mrsa. l also showed high me rates. although patients received prolonged periods of treatment, l was a safe drug. objectives: azithromycin microspheres formulation (azm) was developed to enable a higher dosage of 2 g to be administered as a single oral dose without decreasing the safety profile. this study compared azm with moxifloxacin (mox) aimed at confirming the efficacy and safety of azm in acute exacerbations of chronic bronchitis (aecb). methods: this prospective, multicentre, randomised, double-blind, double dummy study compared azm 2 g single dose with mox 400 mg once daily for 5 days, enrolled aecb patients 50 years old and above, with anthonisen type 1 exacerbations, and with at least 2 exacerbations of aecb in the past 12 months. subjects were to have a history of smoking of at least 20 pack-years and documented forced expiratory volume in 1 second (fev1) less than 80% of predicted. they were followed up for up to 9 months. results: a total of 396 patients were treated (198 in each of the treatment groups) the distribution of the age, and mean fev1 were similar for the 2 treatment groups. pathogens were isolated from 62.9% of the patients (61.1% of patients on azm and 62.9% of patients on mox). the clinical success (signs and symptoms related to the acute infection had returned to the subject's normal baseline level, or clinical improvement was such that no additional antibiotics were deemed necessary) rate for the per protocol population at test of cure (toc) at day 12−19 was 93.0% for azm and 94.2% for mox group (95% ci −5.8, 3.9). bacterial eradication rate (bacteriologic pre protocol population) at toc was 96.0% for azm group and 96.7% for mox group (95% ci −4.5, 3.3). although the study population had history of at least 2 exacerbation in the past 12 months, less than half of the subjects experienced a recurrence during the follow-up, and there was no statistically significant treatment difference in time taken to first occurrence of aecb. both treatments were well tolerated. the incidence of treatment related adverse events was low, being reported by 17% of subjects receiving azm and 12% of subjects receiving mox. most aes were mild or moderate in severity. the most common aes were gastrointestinal disorders, being reported by 14% of subjects receiving azm and 8% of subjects receiving mox. conclusions: a single oral dose of azm was as effective as a 5-day course of mox in the treatment of aecb and was well tolerated. objectives: optimal duration of gentamicin containing regimen for therapy of human brucellosis is not clearly determined. methods: this randomised clinical study was conducted to compare the efficacy of gentamicin 5 mg/day for 5 days plus doxycycline 100 mg twice daily for eight weeks (gd group) versus streptomycin 1gr im for 2 weeks plus the same dose of doxycycline for 45 days (sd group). all cases were followed for one year after cessation of therapy. efficacy of both regimens (failure of therapy or relapse) were compared. results: seventy-nine patients with the mean age of 35±14.5 years and 75 cases with the mean age of 36.7±13.9 years were treated with regimen of gd or sd, respectively. the clinical manifestations in these two treated groups were similar. failure of therapy was seen in one patient in gd group and in 2 cases in sd group ( objectives: to study the efficacy of telavancin (tlv), an investigational bactericidal lipoglycopeptide, for the treatment of complicated skin and soft tissue infections (cssti) caused by presumed or confirmed grampositive organisms. methods: atlas 1 and atlas 2 were methodologically identical, double-blind, randomised, multinational, phase 3 studies. adult men and women presenting with cssti including major abscess were randomised 1:1 to tlv 10 mg/kg intravenous (iv) q24 h or vancomycin (van) 1 g iv q12 h for 7 to 14 days. test-of-cure (toc) visit was conducted 7 to 14 days after end of study treatment. the all-treated population (at) included patients with confirmed diagnosis of cssti who received 1 dose of study medication. this analysis examined the baseline characteristics and cure rates at toc for patients with major abscess in the combined atlas at population. results: in the pooled at population of atlas, 772 patients presented with major abscess. more than 60% of these patients required hospitalisation. the baseline lesion surface area exceeded 5 cm 2 in 98% of the cases, while 65% of the patients presented with lesions exceeding 50 cm 2 (table 1) . elevated white blood cell counts were found in more than 40% of the cases (table 1) . nearly all patients required surgical drainage, with approximately 2/3 performed prior to the first dose of study medication. very few patients required a surgical procedure more than 4 days after the start of study medication. clinical cure rates at toc are presented in table 1 . overall, adverse events in the at population were similar between the treatment groups with regard to type and severity. conclusion: telavancin administered once daily was non-inferior to vancomycin for the treatment of major abscess. objectives: b. fragilis and related species, members of the normal bowel flora, are the most widely isolated anaerobic bacteria from different infections. to follow the development and spread of the resistance among these strains is difficult, as antibiotic susceptibility testing of clinically relevant anaerobes in different routine laboratories in europe is less and less frequently carried out due to the fact, that clinicians treat many presumed anaerobic infections empirically. to follow the changes in the antibiotic resistance of bacteroides strains three europe-wide studies were organised during the past twenty years. the evaluation of the results of these studies may show changes in the resistance to different antianaerobic drugs. only clinical isolates and no normal flora members of bacteroides strains belonging to different species were collected from different countries throughout europe during these studies. agar dilution method was used for the antibiotic susceptibility determination. actual breakpoints accepted by nccls (clsi) and eucast were used. molecular genetic investigations were carried out to detect resistance mechanisms. since the first study the chromosomally mediated beta-lactamase production and tetracyclin resistance is the most prevalent among bacteroides strains in europe. clindamycin resistance in bacteroides is mediated by a macrolide-lincomycin-streptogramin (mls) mechanism and its frequency differs in different countries in europe. resistance to beta-lactam-beta-lactamase inhibitor combinations was studied using amoxicillin-clavulanic acid and/or piperacillintazobactam. increase in resistance was observed to both combinations throughout the years. the same is true for cefoxitine and in the third study several hetero-resistant isolates were found. the occurrence and spread of resistance to imipenem and metronidazole among bacteroides strains merit special clinical importance. the presence of the cfia gene is much more prevalent than the expression of the imipenem resistance; however the spread of the cfia gene among species other than b. fragilis is still very rare. the molecular genetic methods looking for the resistance genes among strains with elevated mics against these antibiotics prove that resistance breakpoints should be reconsidered. the resistance to moxifloxacin shows great differences in different countries. the lowest resistance rate was observed in the case of tigecyclin. many factors may affect the response to treatment such as site of infection, surgical procedures, severity of the illness, patient status, presence of other pathogens (mixed infection), pk/pd parameters of the antibacterial drugs. thus, correlation between treatment failure and antibiotic resistance among anaerobes remains difficult to assess. the main discrepancies came from intra-abdominal infections and a worrisome disjunction between surgeon and microbiologist opinions emerged in the 1990's. but, patients in whom primary therapy failed had more resistant strains compared with patients in whom therapy succeeded. in contrast many failures may be due to the lack of isolation of anaerobes from clinical samples! during anaerobic bacteraemia, salonen et al. demonstrated that mortality increased dramatically from 17% for initially effective treatment to 55% when an ineffective treatment was started. facing new mechanisms of resistance and global increase resistance to many antibiotics among anaerobes may lead nowadays to a different answer. clindamycin vs. penicillin studies for the treatment of lung infections pointed out the failure due to b-lactamase production among gram-negative anaerobes. we found many reports of failure after clindamycin treatment in osteomyelitis, septic arthritis, brain abscess in presence of clindamycin-resistant anaerobes (bacteroides fragilis group and prevotella), probably because when resistance occurs, clindamycin mic's are high. similarly, the lack of coverage of an undetected resistant anaerobe allows the selection of an anaerobic strain resistant to the treatment chosen against the associated aerobes such as imipenemresistant eghertella lenta or metronidazole-resistant strains of prevotella or bacteroides fragilis. the later failures may give opportunity to set up a new metronidazole breakpoint for resistance (mic > 4 mg/l). the main problem is related to the difficulty to detect some heterogeneous resistant strains, that needs prolonged incubation period on agar medium. this kind of situation is probably the most suitable to correlate the bacterial antibiotic resistance with the failure of the antibiotic treatment. methicillin-resistant s. aureus isolates causing community-acquired infections (ca-mrsa) in children is a major problem in several areas around the world. ca-mrsa are associated with both skin and soft tissue infections and invasive infections. recurrent soft tissue infections and infections within the family caused by ca-mrsa isolates are common. ca-mrsa s. aureus isolates containing gene coding for pvl have been associated with serious staphylococcal pneumonia as well as osteomyelitis complicated by subperiosteal abscesses or venous thromboses. in addition to vancomycin, ca-mrsa generally are susceptible to clindamycin and trimethoprimsulfamethoxazole. treatment of superficial skin and soft tissue infections involves surgical drainage of abscesses followed by an oral agent such as tmp-smx or clindamycin. minocycline or doxycycline is a consideration for children >8 years old. empiric vancomycin is typically administered for more serious and invasive infections such as osteomyelitis, septic arthritis, serious head and neck infections or suspected staphylococcal pneumonia. clindamycin is efficacious in treating invasive ca-mrsa infections caused by susceptible organisms. linezolid or daptomycin is another option in selected circumstances. mri is the optimal imaging modality for assessing children with ca-mrsa osteomyelitis. aggressive surgical drainage of subperiosteal abscesses or sites of pyomyositis is recommended. venous thombosis is increasingly recognized as a complication of ca-mrsa osteomyelitis. anti-coagulation until the thrombus has resolved is recommended. the optimal approach to prevention of recurrent ca-mrsa infections is unclear but a strategy that includes emphasizing personal hygiene, plus/minus antimicrobial soaps, mupirocin to the nose or "bleach baths" is frequently suggested. s226 understanding the pathogenesis of group a streptococcal disease: the bedside-to-bench approach invasive group a streptococcal (gas) infection presents itself in a range of guises, most notoriously necrotising fasciitis and the streptococcal toxic shock syndrome. as a human pathogen, gas pathogenesis research should ideally be shaped by clinical questions arising from either epidemiological or case-based investigation of human disease. in the mid 1990 s, large epidemiological studies pointed to a central role for specific t cell-stimulating superantigens in the aetiology of streptococcal toxic shock. this sparked a series of clinical and laboratory investigations that demonstrated production of superantigens during infection which were indeed capable of triggering massive t cell activation in patients but were unlikely, alone, to account for all the features observed in toxic shock. genomic, clinical and laboratory-based investigations have identified novel and highly potent superantigens that appear to directly contribute to sepsis pathogenesis and, together, may constitute targets for adjunctive treatments in invasive disease. epidemiological, clinical, and laboratory studies have highlighted a role for blunt trauma in the aetiology of at least a quarter of cases of gas necrotising fasciitis. one of the most striking findings on examination of tissues from patients suffering with necrotising fasciitis is the failure of neutrophils to migrate to the focus of infection. investigation of patients with invasive gas infection led to the discovery that gas produces an enzyme that can cleave and inactivate human chemokines and study of patients with bacteraemia has highlighted a likely role for the causal enzyme spycep in disease pathogenesis; this bacterial surface enzyme has also shown promise as a potential vaccine antigen. notwithstanding a potential role for individual virulence factors in disease causation, clinical studies have demonstrated that gas bacteria may persist at the site of infection despite high concentrations of bactericidal antibiotics, and this has been borne out by experimental studies; the reasons behind such persistence are unclear but may include internalisation of gas by immune cells, formation of biofilm, and antibiotic penetration of necrotic tissues. the persistence of viable bacteria in such cases is not widely recognized and deserves focused consideration in the research laboratory. genome-wide analysis of microbial pathogens and molecular pathogenesis processes has become an area of considerable activity in the last 10 years. these studies have been made possible by several advances, including completion of the human genome sequence, publication of genome sequences for many human pathogens, development of microarray technology and high-throughput proteomics, and maturation of bioinformatics. despite these advances, relatively little effort has been expended in the bacterial pathogenesis arena to develop and use integrated research platforms in a systems biology approach to enhance our understanding of disease processes. we have exploited an integrated genome-wide research platform to gain new knowledge about how the human bacterial pathogen group a streptococcus causes disease. results of these studies have provided many new avenues for basic pathogenesis research and translational research focused on development of an efficacious human vaccine and novel therapeutics. new data stemming from use of a systems biology approach to provide new data about group a streptococcus pathogenesis will be presented. streptococcal toxic shock syndrome and necrotising fasciitis caused by group a streptococcus are rapidly progressive invasive diseases that are associated with significant morbidity and mortality, ranging from 30−80% despite prompt antibiotic therapy and surgical debridement. s. pyogenes is known to primarily cause disease by activating and modulating host immune responses. the exotoxins with superantigenic activities have been demonstrated to be crucial triggers of excessive inflammatory responses and consequently systemic toxicity, organ dysfunction, tissue necrosis and shock. another important virulence determinant is the m-protein, which is classically known for its antiphagocytic properties, and lately, was shown to trigger pro-inflammatory responses as well as induction of vascular leakage and shock. this likely represents important mechanisms contributing to the rapid development of shock and systemic toxicity in patients with severe invasive group a streptococcal infections. the understanding of these infections as hyperinflammatory diseases highlighted the potential of immunotherapy to improve outcome. one such strategy includes the administration of intravenous polyspecific immunoglobulin (ivig) as adjunctive therapy. the mechanistic actions of ivig in this setting are believed to include opsonisation of the bacteria, neutralisation of the superantigens and suppression of the pro-inflammatory responses. there is growing evidence to support the use of ivig in patients with streptococcal toxic shock syndrome. these studies include one observational cohort study based on canadian patients identified through active surveillance of invasive group a streptococcal infections, and one european multicentre placebo-controlled trial. however, the question remains whether ivig is efficacious also for the severe streptococcal deep tissue infections. an observational study of seven patients with severe streptococcal deep tissue infections suggested that the use of high-dose ivig in patients with severe gas soft tissue infections may allow an initial non-operative or minimally invasive approach, which can limit the need to perform immediate wide debridements and amputations in unstable patients. the fact that seven patients with severe group a streptococcal infections survived with this approach definitely warrants further studies to be conducted on the use of ivig in these severe infections. hepatitis o229 prevalence and outcome of pregnancy in chronic hepatitis c virus infection i. julkunen°, a. sariola, m. sillanpää, k. melen, p. koskela, p. finne, a.l. järvenpää, s. riikonen, h.m. surcel (helsinki, oulu, fi) objectives: in the western countries the incidence of hepatitis c virus (hcv) infection has steadily been increasing especially among young adults. it is thus likely that an increasing prevalence of hcv infection is also found in pregnant women. methods: to assess the frequency of hcv infection in the metropolitan area of helsinki selected anti-hcv antibody testing was carried out for pregnant women during the years 1991-1999. in addition, hcv prevalence was analysed in serum specimens collected from pregnant maters during the years of 1985-2005. results: altogether 145 mothers were identified among 44680 mothers. the frequency of anti-hcv positivity rose from 0.13% in 1991 to 0.43−0.53 in 1997-1999. in early 90's only 20% of mothers knew about their seropositivity, whereas by the end of the follow-up period almost 70% of mothers knew about their hcv infection already before the pregnancy. intravenous drug abuse was the major risk factor (71% of cases) for contracting the disease. in 90% of the mothers chronic hcv infection was well under control and in this population the mean serum alanine aminotransferase (alt) values decreased towards the end of the pregnancy. however, 10% of anti-hcv ab positive mothers developed intrahepatic cholestasis (odds ratio 16.4) as characterised by itching and elevated serum bile acid levels. the correspondig value in the control pregnancies was only 0.7%. anti-hcv ab positive mothers were younger, delivered earlier and gave birth to babies with smaller birth weight as compared to control deliveries. to have a more comprehensive view of the problem of hcv infection during pregnancy randomly selected serum specimens from the finnish maternity cohort were tested. 2000-5000 serum specimens were tested in selected cohorts (1985, 1990, 1995, 2000 and 2005) . in 1985 the nationwide prevalence was 0.19% and it steadily role to 0.50% in 2005. in the metropolitan area of helsinki the prevalence was higher being 0.68% and 0.70 in 1997 and 2002, respectively. conclusion: our study indicates that there is an increasing problem of hcv infection in pregnant women in finland. although most women cope well with their disease during pregnancy there is a subpopulation of mothers who develop cholestasis and their liver status should thus be followed-up carefully. testing of all mothers for serum anti-hcv antibodies is recommended. objectives: the viral genome of hepatitis c virus constitutes a 9.6kb single-stranded positive-sense rna which encodes altogether 11 viral proteins. in order to study the humoral immune responses against different hcv proteins in patients suffering from chronic hcv infection, we produced three structural (c, e1 and e2) and six nonstructural proteins (ns2, ns3, ns4a, ns4b, ns5a and ns5b) in sf9 insect cells by using the baculovirus expression system. the recombinant hcv proteins were purified and used in western blot analysis to determine antibody responses against individual hcv protein in 68 hcv rna and antibody positive human sera that were obtained from patients suffering from genotype 1, 2, 3 or 4 infection. results: these sera were also analysed with inno-lia score test for hcv antibodies against core, ns3, ns4ab and ns5a, and the results were similar to our western blot method. based on our western blot analyses we found that the major viral antigens were the core, ns4b, ns3 and ns5a proteins and they were recognized in 97%, 86%, 68% and 53% of patient sera, respectively. there were no major genotype specific differences in antibody responses to individual hcv proteins. a common feature within the studied sera was that all except two sera recognized the core protein in high titers, whereas none of the sera recognized ns2 protein and only three sera (from genotype 3) recognised ns5b. the data shows significant variation in the specificity in humoral immunity in chronic hcv patients. anti-hcv antibody pattern also remains very stable within one individual. alt and ast levels were tested in all subjects. the presence of hbv-dna was determined quantitatively in plasma samples of hd patients with anti-hbc alone (hbsag negative, anti-hbs negative and anti-hbc positive) by real-time pcr using the artus hbv rg pcr kit on the rotor-gene 3000 real-time thermal cycler. results: of 289 patients enrolled in this study, 18 subjects (6.2%, 95% ci, 3.5%-8.9%) had anti-hbc alone. hbv-dna was detectable in 9 of 18 hd patients (50%, 95% ci, 27%-73%) with anti-hbc alone. plasma hbv-dna load was less than 50 iu/ml in all of these patients. our study showed that detection of anti-hbc alone could reflect unrecognized occult hbv infection in hd patients. the majority of these infections are associated with low viral loads. were included in the study. all the subjects had never been exposed to antiretroviral therapy. genotypic resistance testing was performed at the time of diagnosis with a sequence-based assay (trugene hiv-1 genotyping test) targeted at the protease region (codons 1 to 99) and rt region (codon 40 to 247) of the hiv-l genome. results: 21 of 218 patients (9.63%) harboured a virus with at least one mutation associated with phenotypic resistance; 1/218 with mutations associated with resistance to nucleoside reverse-transcriptase inhibitors (nrtis), 17/218 to non-nucleoside reverse-transcriptase inhibitors (nnrtis) and 3/218 to protease inhibitors (pi). resistance to nrtis was associated with the key mutation m184v, while resistance to nnrtis was associated with y181c and k103n mutations. among mutations to pi, major resistance mutations l90m and d30n were found in three patients, whereas there was a high prevalence of accessory pi resistance mutations at positions 10, 20, 36 and 63. conclusion: our data estimate the prevalence of primary resistance and mutations patterns among naive hiv patients, underlining the importance of genotypic resistance testing in hiv patients before starting treatment, especially when nnrtis would be included in the initial antiretroviral therapy. objectives: few data are available on the genetic mechanisms of protease inhibitor (pi) resistance in non-b hiv-1, and pi resistanceassociated mutations (rams) are commonly observed in pi-naive patients with subtype a/e infection. this study aimed to compare pi-rams between pi-naive and -experienced patients. methods: genotypic resistance testing was conducted among a cohort of hiv-1 infected patients who had virologic failure. patients were categorised into 2 groups: pi-naive and pi-experienced. we focused on pi-rams previously described by ias-usa 2008. results: we studied 137 patients (mean age, 41.8 years; 64% male). median cd4 cell count and hiv-1 rna at virologic failure were 169 cells/cu.mm. and 14100 copies/ml, respectively. 85% of patients were infected with subtype a/e; the others had subtype b (12%), ab (2%), and c (1%). there were 75 patients in pi-naive group and 62 patients in pi-experienced group. the clinical characteristics between 2 groups were similar (p > 0.05) except for the duration of antiretroviral therapy which was shorter in pi-naive group (31.5 vs. 46.8 months, p = 0.028). percentage of patients who had primary pi-rams was 1% in pinaive and 19% in pi-experienced groups (p = 0.001). the most common primary pi-rams in the latter group were v82a (10%) and i54v (7%). percentage of patients with secondary pi-rams in the corresponding groups was 99% and 98%, respectively (p = 1.000). median number of secondary pi-rams was also similar between 2 groups (p = 0.244). the most common secondary pi-rams in both groups were m36i (91%), h69k (34%), l89m (30%), i13v (26%), l63p (25%), l10i we also defined a "silent score" (ss) and a "resistance score" (rs) as the number of synonymous mutations and of resistance mutations (in the second sequence in comparison with the first one) divided by number of days between the two tests, respectively. (12); pts with drms in non-b-st (%) were 7 (23.3), 6 (14), 5 (7) and 3 (4.2). a significant increase of non-b-st (p = 0.021) and a significant decrease in drms (p < 0.001) were observed. crf02_ag was the prevalent non-b st (44%). 35.3% of non-b st pts were italians. among b-st, drms predicted a reduced susceptibility to one drug class in 23, 17, 15 and 14 cases in the different periods; to two drug classes in 4, 6, 5 and 8; to three classes in 3, 2, 0 and 0. in non-b-st, a reduced susceptibility to one drug class was found in 6, 6, 4 and 0 cases; to two drug classes in 1, 0, 0 and 2; to three drug classes in 0, 0, 1 and 1, respectively. among pts with one or two classes of resistance, a decrease of percentage of protease inhibitors related drms, and a persistence of non nucleoside rt inhibitors involving drms, mainly 103n and 190a, were observed. methods: from hiv+ persons with a history of, or an acute episode of opc, oral fungal burden was evaluated bi-weekly and buccal mucosa tissue was collected bimonthly for a period of one year. tissue was evaluated for the presence of cd8+ t cells and e-cadherin by immunohistochemistry or flow cytometry. objectives: to define the secular trends in the epidemiology of candidaemia in queensland, australia (population, 4.1 million) over a 10-year period. methods: all episodes of candidaemia within queensland public hospitals from 1999-2008 were identified from laboratory information systems. data on species identification, antifungal susceptibility, demographics, and hospital ward of diagnosis, and denominator data (hospital admissions, accrued patient-days (pt-days) and fluconazole usage) were collected. results: over the 10-year period, 1137 unique episodes (100% case ascertainment) were identified from 42 healthcare facilities (8 tertiary, 2 paediatric, 11 secondary and 21 smaller hospitals). the median patient age was 56.4 years. the overall incidence-density was 0.45/10000 ptdays, highest in paediatric (1.28/10000 pt-days) and tertiary hospitals (0.62/10000 pt-days). over the 10 years, the incidence-density increased 3.2-fold in tertiary hospitals and 6.6-fold in secondary hospitals (both p < 0.0001 for trend), but not in paediatric or smaller hospitals. the incidence-density in icus (5.2/10000 pt-days) was 10-fold higher than in non-icu wards, but did not significantly increase over the study period. the relative proportion of episodes occurring in adult general medical/surgical (ie non-oncology/non-icu) wards significantly increased (p < 0.001), accounting for 62% of episodes at the end of the 10-year period, whereas that occurring in paediatric and adult oncology wards decreased (p < 0.001 and p = 0.07 respectively). overall, c. albicans accounted for 44%, c. parapsilosis 27% and c. glabrata 13%. although the incidence-density of all species increased over the study period, the relative proportion caused by c. albicans decreased (p = 0.007) and c. parapsilosis increased (p = 0.01). despite significantly increased fluconazole usage (from 19.7 to 30.6 ddd/1000 pt-days, p < 0.0001), the relative proportion caused by c. glabrata/c. krusei did not change (p = 0.5). the overall incidence of candidaemia has increased almost 400% in queensland public hospitals over the last 10 years. the relative proportion of episodes occurring among general medical/surgical patients and caused by c. parapsilosis has increased. candidaemia is an increasing problem the epidemiology of which continues to evolve. it is increasingly affecting patients outside traditional risk groups. conclusions: this surveillance study and pharmaco-economic modelling has proved immensely beneficial in setting up inhouse processing, improved tat, reduced costs of outsourcing and subsequent use of expensive antifungals. reduction in mortality has been noted but is not statistically significant. c. albicans was the commonest isolate; fluconazole resistance is minimal and associated mortality is lower than reported from europe. many pts received systemic prophylaxis (72%); itraconazole and fluconazole were used in 68 and 33 pts respectively. no differences emerged between empirical vs pre-emptive therapy and none of the drugs resulted to significantly influence outcome. in 66% of pts initial empirical/pre-emptive drug remained unchanged after ia diagnosis, while in 16% clinicians shifted to a combined treatment. conclusion: this study allows as to analyzed multiple factors as potentially influencing outcome. we confirmed that aml phase and neutropenia influence ia outcome. present data confirm the perception that during last years the application of a correct and timely diagnostic work-up and the availability of more efficacious and less toxic drugs (i.e. voriconazole, liposomal amphotericin b, caspofungin) have modified the course of ia. however none of the new drugs emerged as the most efficacious in our series. even combined treatment did not confer any advantage in survival analysis. (<3% each). the first line therapy was monotherapy with voriconazole (49%), caspofungin (14%), lipid formulations of amb (9%) or used antifungal drugs combination (20%). the mortality rate at day 90 was 41% when first line therapy included voriconazole compared to 60% when it did not (p < 0.001). conclusion: comprehensive collections of cases based on systematic reporting and description of cases using a dedicated network of hospitals in selected regions and stringent definition criteria applied by trained clinicians and microbiologists are useful to describe ia, to assess its burden and secular trends, and to identify potential changes in diagnostic and therapeutic procedures. this network will expand to other regions in the near future, and data will help assessing the impact of new management strategies such as prophylaxis with posaconazole, the impact of modification of new diagnostic criteria as recently proposed (clin infect dis, 2008), and identifying new populations at risk for ia. nosocomial aspergillosis represents a serious threat for severely immunocompromised patients and outbreaks have been attributed to airborne sources. the role of hospital-independent fungal spread sources e.g. the private homes or business suites are not known. we investigated the relationship between fungal exposure prior hospitalisation and the ensuing onset of invasive mould infections (imi) in patients at risk. patients admitted to the department of haematology and oncology or to the department of transplant surgery of the innsbruck medical university received a structured questionnaire regarding their fungal exposure prior hospitalisation. questions inquired heavy fungal exposures up to five days prior hospitalisation. 234 patients were enrolled in this study and 19% were smokers, 22% suffered from an airborne allergy, 62% lived in old buildings, 73% were ruralists, 82% and 92% were exposed to any outdoor or indoor fungus sources. poor housing conditions and other fungus exposures were associated with the onset of community-acquired imi only in patients with acute myelogenous leukaemia (p < 0.01). aml patients being more at risk for imi when smoking cigarettes (p < 0.05), living on the country site (p < 0.05), having two or more fungus exposures (p < 0.05) and suffering from allergy to dust, pollen and/or moulds (p < 0.05). a similar trend was for lung transplant recipients receiving extensive immunosuppressive agents to treat allograft rejection. overall, 88% of imi were community-acquired cases. hospital-independent fungal sources highlight risk-factors for imi in severe immunocompromised patients and the rate of communityacquired imi does increase. an analysis of an individual patient's risk factors for fungal infection and the type of fungus to which they are most susceptible, indicates the preventative strategies that are likely to be successful. to the icu-mhs with aspergillus spp detected in significant amounts in clinical samples. the underlying conditions of the patients were heart transplantation (n = 5), major heart surgery (n = 4), and other (n = 2). eight (72.7%) patients developed proven/probable ia (4 with lung infection, 2 with mediastinitis, 1 with disseminated ia, and 1 with prostate involvement). the mortality of patients with ia was 87.5%. the icu-mhs is divided into 3 areas, one of which is equipped with hepa filters. only 1 case of ia occurred in the protected area. we measured the fungal conidia levels in the air of each of the 3 areas (508 samples analyzed) monthly. a total of 172 strains of a. fumigatus (110 clinical strains from 10 patients and 62 environmental strains) were genotyped using microsatellites (de valk et al, jcm 2005) . the mean airborne conidia levels (6 months) before and after the outbreak were, respectively, 5.6 (0−15) cfu/m3 and 1.8 (0−10) cfu/m3. no cases of ia occurred during these periods. however, all cases of ia were linked to 4 peaks of abnormally high airborne conidia levels (65, 70, 200 and 500 cfu/m3). a. fumigatus was involved in 7 cases of ia; 1 patient was infected by non-fumigatus aspergillus (not further genotyped). in 4 patients (1 mediastinitis, 2 pulmonary ia and 1 colonisation), we demonstrated similar genotypes in the air and in clinical samples. patient 1 was located in the protected area and had a unique genotype. patient 2 had two different clusters of genotypes: one cluster was similar to that of patient 3 and the other was also found in patient 4 and in the air. the genotype present in patients 2 and 4 was also detected in the air during a 6-month period. conclusions: epidemiologic and molecular typing suggests that there is a causal relationship between aspergillus causing ia and those present in the air. our finding also supports the need for hepa filtration in icu-mhs. j. guinea is contracted by fis (cm05/00171). sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) were calculated in reference to proven and probable cases of ia. reasons for performing bronchoscopy on patients were also recorded. the protocol received approval by the local ethic committee. results: from the 117 samples studied, 5 (4.3%) were classified as proven, 6 (5.1%) as probable, and 35 (29.9%) as possible cases of aspergillosis. twelve samples (10.3%) represented colonisation, and 59 bal samples were obtained during routine surveillance. pulmonary aspergillosis was the main clinical presentation of ia (63.6%). using roc analysis, the best cut-off for galactomannan testing in bal was defined as 1.5 (sensitivity 90.9%, specificity 90.6%, ppv 48% and npv 99.1%). median bal gm index for the group of patients with proven/probable aspergillosis and for 'negative cases' were 3.3 and 0.5, respectively (p < 0.001). overall mortality was 20% (n = 12). the odds for death for patients diagnosed with ia were 11.8, in comparison to patients who did not have this infection (95% ci 2.9−48.4). conclusion: gm testing in the bal added to the diagnosis of ia in lung transplant recipients. in order to avoid false-positive results, a higher test cut-off should be applied to bal samples, in comparison to sera. increasing the cut-off to 1.5 resulted in a very high npv, with an associated sensitivity of >90%. objectives: 1) determine the performance characteristics of the galactomannan (gm) assay in broncho-alveolar lavage (bal) in haematology-oncology patients; 2) evaluate the prognostic value of the gm assay in this particular population. methods: the platelia gm eia assay (bio-rad) was performed on all bal specimens obtained from haematology-oncology patients at our institution between march 2005 and april 2008, in addition to routine laboratory stains and cultures. all results were reported to physicians. we conducted chart reviews to classify cases as proven, probable, possible or without invasive pulmonary aspergillosis (ipa) according to the revised definitions of invasive fungal disease from the eortc/msg consensus group. for performance characteristics, proven and probable cases were considered as ipa; possible cases were considered as without ipa. the result of bal gm was not considered as a criterium to classify cases in order to avoid incorporation bias. in patients with >1 positive (gm index >0.5) specimen, only the first one was considered for the analysis. mortality was calculated at 60 days following the first bal procurement. data were analyzed with stata 8.0. results: there were 173 bal samples from 145 patients, including 101 haematopoietic stem cell transplant (hsct) recipients. we found 5 proven, 7 probable and 35 possible cases of ipa (total of 12 ipa cases; 6.9%). gm on bal was positive in 47 (27.2%) specimens. the sensitivity and specificity of the gm assay in bal were 100% and 78.3% respectively. positive predictive and negative predictive values were 25.5% and 100%, respectively. false-positive results were found in 21 patients without ipa and in 14 with possible ipa. an index value 0.5 was significantly associated with a 60-day mortality risk (12/39 patients with a positive gm died within 60 days after bal compared to 13/106 with a negative gm (or = 3.2, 95%ci 1.3−7.8; p = 0.01). this association was even stronger when restricted to hsc recipients (or =4.6, 95%ci 1.5−13.6; p = 0.006). the clinical utility of gm assay in bal mainly lies in its negative predictive value, identifying patients at low risk of ipa. this test also carries a prognostic value in predicting patients at higher risk of mortality. (see table below) . not significant differences have been found among pneumocystis colonisation and copd status evaluated by fev-1%. as well as no significant differences respect to age, sex or lymphocytes and leucocytes blood count were found. background: infliximab, a monoclonal antibody targeting tumour necrosis factor alpha (tnf-a), is indicated for the treatment of rheumatoid arthritis (ra) and other autoimmune diseases. however, its use has been associated with opportunistic infections, including pneumocystis jirovecii pneumonia (pcp). moreover, p. jirovecii has been observed colonising to humans with several disorders. objectives: to obtain information about p. jirovecii colonisation among patients with rheumatologic disease treated with infliximab. this information could be useful for assessing new strategies in the prevention of pcp in patients at risk. methods: 62 consecutive patients treated with infliximab for rheumatic disorders were included in the study. oropharyngeal washes (ow) samples were collected for p. jirovecii detection. clinical and demographic data were collected (sex, age, rheumatologic diagnosis, duration of infliximab use, concomitant use of other drugs for rheumatologic treatment, use of any other anti-tnf-a agent, use of anti-pc drugs in the last six months, smoking, and diagnosis of chronic pulmonary respiratory disease). p. jirovecii colonisation was identify in ow samples by pcr at mtlsu-rrna gene, with primers paz102-x and paz102-y. we adapted a method previously described to a real-time pcr setting, using a lightcycler 1.5 (roche, germany). individuals in whom the presence of p. jirovecii was detected at two independent assay in the absence of respiratory symptoms or radiological findings suggestive of pcp were considered to be colonised. results: clinical and demographic data for 62 patients treated with infliximab are presented in table 1 objectives: most research with human bocavirus, a recently found respiratory pathogen, has been done by molecular biology (polymerase chain reaction, pcr). the results have been ambiguous because the virus has often been found in co-infection with other viruses, and also in clinically healthy subjects. it has been proposed that, for bocavirus, antigen detection could better indicate the aetiology than qualitative nucleic acid detection. we have developed a rapid antigen detection test for the virus. the one-step test for bocavirus vp2 antigen is based on a separation-free two-photon excitation fluorometry (arcdia tpx assay technique). the assay protocol is simple; the swab sample is dissolved in sample buffer, and the solution is dispensed (20 ml) onto a 384-well microtitre plate (containing the reagents in dry form) for incubation and automated quantitative measurement. the immunoassay applies microspheres as solid-phase carriers of purified bocavirus-specific polyclonal antibodies. the virus antigens concentrate onto the solid-phase which is probed in real-time with fluorescently labelled antibody reagents. strong positive samples are reportable in 15 minutes, while low positive and negative samples are reported in 2 hours. the performance of the method was studied with recombinant human bocavirus-like particles (vp2), and purified respiratory pathogens (group a streptococci, streptococcus pneumoniae, and influenza a and b, respiratory syncytial, metapneumo, adeno, and parainfluenza 1−3 viruses). results: analytical detection sensitivity of the method (lowest limit of detection, 0-control + 3sds) was 3 ng/ml, dynamic concentration range was three orders of magnitude, and intra-assay imprecision was 5−10%. cross-reactions with the other respiratory pathogens were not found. the new method enables rapid detection of bocavirus antigens. the new test is very easy to perform in comparison to standard elisas. the analytical sensitivity of the method is expected to allow analysis of clinical samples. the sensitivity of the antigen detection test could be significantly increased by the use of monoclonal antibodies (10-100 fold). our future objectives include increasing the detection sensitivity, and analysis of clinical samples in order to study the correlation of antigen detection and the clinical aetiology. life-year for patients who survived. all analyses were performed using treeage software (2008). results: the overall mortality rates for empiric vancomycin (v) and semi-synthetic-penicillin (ssp) was 30% and 35%, respectively, as apposed to 24% for those receiving the rapid mrsa pcr testing. these mortality rates were similar in both the eu and us subsets. furthermore, the number needed to test in order to save one life was 20 and 11 for empiric v and ssp, respectively. using sensitivity analysis the prevalence of mrsa was varied from 5% to 80% and yielded an absolute mortality difference favouring the pcr testing group of 10% and 2%, respectively as compared to empiric v and 1% and 18% compared to empiric ssp. in eu the c/e for empiric v and ssp treated patients was €873 and €949, respectively as compared to €807 for rapid pcr testing. in the us the c/e for empiric v was $1,049 as compared to $971 for rapid pcr testing. using sensitivity analysis the prevalence of mrsa was varied from 5% to 80% and yielded favourable c/e in both the eu and us for rapid pcr testing regardless of the empiric treatment regimen. conclusion: rapid mrsa pcr testing using the xpert mrsa/sa blood culture pcr assay appears to improve mortality rates and is cost effective in the eu and us across a wide range of mrsa prevalence rates. background: rapid detection of gastro-intestinal carriage of glycopeptide-resistant enterococci (gre) from screening cultures is crucial for an efficient control of their spread. we assessed 4 media − 2 chromogenic, chromid, (biomérieux), and chromagar (chromagar microbiology), and 2 selective, vre selective (oxoid) and eccv (bd) − for their ability to detect gre using well-characterised isolates and stool samples from hospitalised patients at high risk of gre colonisation. methods: twenty-five isolates consisting of 13 gre. faecalis/faecium carrying various van genes and 12 non-vre at concentrations of 10 6 -10 1 cfu/ml and 10 6 cfu/ml, respectively, and 37 stool samples were randomised and spiral plated on all media and scored by 5 blinded investigators for characteristic colonies after 24 hrs incubation. standard confirmatory tests were done on 1 putative gre colony or on 1 characteristically coloured colony each for e. faecalis/faecium from the selective and chromogenic media, respectively. detection of van genes, and ddl or soda based speciation was done on pcr-sequencing. mean sensitivity (sen) and specificity (spec), and confidence intervals (cis) were estimated for each medium by a logistic regression model using a penalised likelihood approach based on the reader response for the stool samples and isolates, and additionally on confirmation test results for the stool samples, both at the aggregated (gre detected) and penalised level (correct species-colony colour correlation). results: chromagar showed the highest sen based on reader response at the aggregated and penalised level for both stool samples and isolates (table) . using confirmation test results at the aggregated level, sen for eccv was highest while the two chromogenic media showed a decrease in sen by at least 11% in comparison to the values obtained based on reader response. sens for the 2 chromogenic media were even lower (<70%) based on confirmation test results at the penalised level. eccv and chromid showed the highest specs with both reader response (stool samples) and confirmation test results at the aggregated level, and chromid also at the penalised level, with narrow cis indicating a high precision of this parameter estimate. for isolates, specs were highest for chromagar at both levels. conclusions: chromagar showed the best overall performance considering both sen and spec estimates. eccv performed well as a selective medium for gre detection from stool samples. objectives: metallo-beta-lactamases (mbls) expressed from pseudomonas are able to confer resistance to all beta-lactams with the exception of aztreonam. however, enterobacteriaceae possessing mbls exhibit moderate cephalosporin and low carbapenem mics and thus are often underestimated. herein, we describe data from new etest prototypes specifically designed to detect this problematic resistance mechanism. methods: 82 mbl-positive (vim or imp derivatives) enterobacteriaceae clinical isolates from 8 countries and 27 randomly selected enterobacteriaceae negative controls (including the atcc type strains) were tested against the 4 different etest mbl prototypes. beta-lactam substrates used were imipenem (ip), meropenem (mp), ceftazidime (tz) and cefotaxime (ct) with or without the inhibitors dipicolinic acid (dpa) and edta. the etest standard procedure for gram negative aerobes was used and a reduction of beta-lactam mic by equal to or greater than 3 dilutions by edta or dpa was interpreted as positive for mbl. presence of esbls was tested using the etest ct/ctl, tz/tzl and cefepime (pm)/pml strips. ampc production was detected using the etest cefoxitin (fx)/fxi and cefotetan (cn)/cni strips. of the 784 select specimens that were negative for gbs, 345 grew turquoise-blue colonies, but the majority that required further work to rule out gbs grew after 48 hours. two strains of gbs that were missed grew as white colonies on select, and even at 48 h, did not exhibit the characteristic turquoise-blue colour. conclusion: ssb enrichment followed by select subculture was extremely sensitive (99.2%) and superior to cna/ssb for detection of gbs from genital specimens. however, non-gbs organisms can produce turquoise-blue colonies on select and further work must be performed to rule out the presence of gbs. objectives: screening for chlamydia trachomatis (ct) specific antibodies is valuable in investigating recurrent cause of miscarriage, pelvic inflammatory disease and tubal damage following repeated episodes of pelvic inflammatory disease. immunofluorescence (if) is considered the gold standard for detection of ct antibodies. the present study aims to compare the performance of 4 other commercial tests for the detection of serum igg antibodies specific for ct: two ct igg pelisa both using major outer membrane protein (momp; ["momp-medac", ct-igg-pelisa; medac, wedel, germany and "momp-ruwag", ct pelisa; ruwag, bettlach, switzerland), one ct hsp-60 igg pelisa ("hsp60-medac", chsp60-igg-pelisa; medac, wedel, germany), and a new automated epifluorescence immunoassay ("inodiag", "must chlamydiae; inodiag, signes, france). methods: a total of 405 patients with (n = 251) and without (n = 154) miscarriages were tested by all 5 serological tests described above. sensitivity and specificity were calculated using if as gold standard. a second standard, defining true positive or negative samples as sera respectively positive and negative in all 4 others tests, was also used (see table) . objectives: participation in diagnostic microbiology internal and external quality control (qc) processes is good laboratory practice, an essential component of a quality management system and compulsory in some european countries. currently, there is no qc scheme for diagnostic oral microbiology. the aim of this study was to collate information on current qc needs, and processes undertaken in diagnostic oral microbiology laboratories. method: an on-line questionnaire was devised to ascertain interest in participating in an oral microbiology qc scheme and sent to oral microbiology diagnostic laboratories. the laboratories were identified from participants attending the european oral microbiology workshop in helsinki, 2008. following this, a pilot round of qc samples was distributed to all interested laboratories. results: we identified 12 individuals that worked in diagnostic oral microbiology laboratories and received 7 (58%) positive responses. of these 7 laboratories (representing 6 european countries) 71% did not participate in either internal or external qc. each laboratory processed on average a total of 4135 samples annually. 86% of participants were in favour of a european-wide oral microbiology qc scheme. the preferred frequency for receiving external qc specimen was once in 3−4 months. the most preferred specimen types were periodontal pocket and oral pus specimens (both 29%), followed by oral mucosal swabs and caries activity tests. all participating laboratories were willing to share and harmonise their specimen processing and interpretation standard operating procedures. the pilot round specimen was a periodontal pocket sample. six laboratories reported their findings in the specified time. the predominant pathogens (aggregatibacter actinomycetemcomitans, porphyromonas gingivalis) were identified by 5 of 6 laboratories. in addition to conventional culture, one laboratory used pcr. 5 laboratories performed antibacterial sensitivity testing primarily by disc diffusion. conclusions: this is the first attempt to a standardised europeanwide approach to diagnostic oral microbiology. the findings from this feasibility study have indicated that a qc scheme for oral microbiology is of interest and have raised a number a pointers for subsequent rounds of specimens. further work to improve the quality, to standardise the methodology and the interpretation of diagnostic oral microbiology at the european level is on-going. objectives: since severe sepsis with acute organ dysfunction can be fatal within hours, it is customary to start empirical broad-spectrum antimicrobial therapy in all patients hospitalised for a suspicion of systemic inflammatory response syndrome. however, increased use of broad-spectrum antimicrobials over the years has contributed to the emergence of drug resistant strains of bacteria. especially, drug resistance among gram-positive bacteria, the leading cause of sepsis, is now a serious problem. the objective of this preliminary study was to develop a method for distinguishing between gram− and gram+ bacterial infection. methods: in this prospective study, leukocyte and neutrophil counts, crp, esr, and quantitative flow cytometric analysis of neutrophil complement receptors 1 (cr1/cd35) and 3 (cr3/cd11b), were obtained from 289 hospitalised febrile patients, of which 89 had bacterial and 38 viral infection. the patient data were compared to 60 healthy controls. results: it was noticed that in gram− infection (n = 21) the average amount of cd11b on neutrophils was significantly higher than in gram+ infection (n = 22). on the contrary, serum crp level was significantly higher in gram+ than in gram− infection. other measured parameters did not differ significantly between gram+ and gram− infections. we derived a crp/cd11b ratio dividing the serum crp value by amount of cd11b on neutrophils. in thirteen (76%) out of 17 patients with gram+ sepsis had crp/cd11b ratio cutoff value of 3.1 (figure 1 ). of these 13 patients, 9 (70%) were diagnosed with streptococcus pneumoniae, 2 with staphylococcus aureus, 1 with enterococcus faecalis, and 1 with both streptococcus intermedius and streptococcus oralis. corresponding percentages in patients with local gram+ infection, gram− infection, clinical pneumonia, other clinical infection, and viral infection were 20%, 14%, 30%, 15%, and 0%, respectively. conclusion: the detection of gram+ sepsis is possible after combination of neutrophil cd11b data and serum crp level. crp/cd11b ratio viral infections of the central nervous system s61 displayed 76% sensitivity and 80% specificity for detection of gram+ sepsis. the proposed crp/cd11b ratio test could, for its part, assist physicians to decide appropriate antibiotic treatment in patients with severe bacterial infection. a bacterial biofilm is a structured consortium of bacteria cells surrounded by a self-produced polymer matrix. biofilms may be monospecies or polyspecies biofilms. biofilm growing bacteria give rise to chronic infections, which persist in spite of therapy and in spite of the host's immune-and inflammatory responses. biofilm infections are characterised by persisting pathology and immune response (in contrast to colonisation). bacterial biofilms use both biofilm specific (b) and conventional (planktonic) resistance mechanisms (p) when they are exposed to antibiotics. the following resistance mechanisms have been described in bacterial biofilms: 1. stationary phase physiology (b), low oxygen tension (b) and slow growth (b) especially inside biofilms whereas the surface of biofilms is more similar to planktonic growth. 2. penetration barriers (b), binding to the polymer matrix (b). 3. mutations, hypermutators (b, p). 4. chromosomal betalactamase is upregulated (b, p). 5. antibiotic tolerance/adaptive resistance (b). 6. efflux pumps (b, p). 7. alginate production (b). 8. high cell density and quorum sensing (b, p). 9. pbp 3 − sos response ? (b). the knowledge of these resistance mechanisms can, however, be used to design new therapeutica approaches especially as regards quorum sensing inhibitors. we consider two factors that contribute to treatment failure in the absence of inherited resistance, the density of the population being treated and the physiological state of the bacteria. we also explore how these factors might contribute to the evolution of inherited resistance during the course of treatment. we conclude with a computer-and chemostat-assisted consideration of the potential clinical implication of these density and physiology effects and make suggestions for treatment protocols to deal with them. using in vitro cultures of staphylococcus aureus atcc25923 or the clinical isolate ps80 and antibiotics of six different classes we determined the functional relationship between the inoculum density and the efficacy of the antibiotics. as measured by the rates and extent of kill and/or the minimum inhibitory concentration (mic), the efficacy of all of these antibiotics declined with increases in the density of bacteria, albeit to different extents. for daptomycin and vancomycin, much of this density effect can be attributed to bacteria-associated declines in the effective concentration of the antibiotic in the medium. for gentamicin, vancomycin, ciprofloxacin and oxacillin, our bioassays failed to reveal significant reductions in their effective concentration in the medium. the effects of the physiological state of s. aureus on the efficacy of these antibiotics were examined for bacteria from cultures in "stationary phase" for different times and from chemostats run at different generation times. these experiments are currently under way but by the time of the symposium we will have the full (and true) story. it is, however, clear that the efficacy of all of these antibiotics declines with the time in stationary phase (its "age"). and, even slowly dividing cultures from chemostats are more susceptible to antibiotic-mediated killing that early stationary phase batch cultures. the efficacy in killing non-growing bacteria varies among the bactericidal antibiotics examined. to ascertain the potential clinical implications of these density and physiological effects, we use both computer and in vitro simulations of antibiotic treatment. the results of these simulations provide compelling support for the proposition that antibiotic treatment regimes, including those designed to prevent the ascent of resistance, should take into account the anticipated density and physiological state of the target population of susceptible bacteria. there have been an increasing number of neurotrophic viral infections playing an important role in the world over the last decade. the list includes west nile virus, nipah and hendra virus (both paramyxoviruses), as well as chikungunya virus which suddenly emerged. furthermore, the relation between jc virus in progressive multifocal leukoencephalopathy (pml) in patients with multiple sclerosis treated with a new immunosuppressive drug, has triggered our attention. the development and implementation of molecular based amplification method has assisted us to detect these viruses more efficiently. these technologies have been used now routinely in a large number of laboratories to enable the detection of more commonly known neurotrophic viruses, like hsv, vzv and the neurotrophic picornaviruses like enterovirus and parechovirus. the pitfalls of these molecular methods have been generally solved by implementing regular quality control testing schemes, like organised by qcmd (quality control of molecular diagnostics) and the introduction of internal controls during the whole diagnostic process. finally, with the ability to quantify the amount of nucleic acid present in csf, more information on the pathogenesis of these viral infections, as well as significant tool to monitor the antiviral effect of treatment options for these viruses, has become available. to as a rare disease in europe restricted to some endemic foci. however, current data suggest that the incidence of ae has significantly increased, and the disease is spreading to the north, west, and east. ae has become an emerging disease in the baltic countries. thus, human infections with e. multilocularis have arrived in the "centre" of europe. ae is a lifethreatening disease, and is characterised by a tumour-like lesion in the liver. the larva can infiltrate the surrounding tissues and metastasize to distant organs. in an attempt to classify the large variety of anatomical findings in ae, the pnm-classification system was developed and serves as a benchmark for standardised evaluation of diagnostic and therapeutic measures. modern imaging techniques, such as ultrasound, ct or mri and pet/ct contributed not only to a much better description of the lesions, but also to a judgment upon the activity of the metacestode. the differential diagnosis of ae varies from haemangioma-like lesion of the liver or cancer. the diagnostic skills are limited, and are the reason for frequent misdiagnosis in geographic areas where ae is rather unknown. continuous treatment with benzimidazoles is the backbone of a lifelong management of ae. however, radical resection is the procedure of choice and should always be strived for. ae is still a rare disease in europe, but where it occurs, it is often diagnosed too late. patients are misdiagnosed for months and years, before receiving the correct treatment. at that late stage the disease has progressed, and radical cure of the liver lesion(s) is not anymore possible. recent reports provided hints for an accelerated larval growth of echinococcus spp. in the immunodeficient host. a careful monitoring of patients receiving immune-modifying drugs is warranted. the modern clinical management and long-term parasitostatic treatment with benzimidazoles are highly effective. thus, a higher alertness for the "tumours from the centre" would increase the prognosis of this hepatic disease resembling liver cancer. the percutaneous treatment of liver hydatid cysts were considered to be contraindicated due to two main potential risks: anaphylactic shock and abdominal dissemination of the disease. since the first case percutaneously treated was published, several series of successful percutaneous treatment of the liver and the other abdominal organs, peritoneum, thorax, soft tissue and orbital cavity hydatid cysts have appeared in the literature. percutaneous treatment of hydatid liver disease is an effective and safe procedure with its unique advantages (e.g., shorter hospital stay, low complication rate). today, the percutaneous approach has an important role in treatment of hydatid cysts not only in the liver but also in the other organs and tissue. therefore it must be first treatment option whenever it is indicated. in europe, dirofilaria immitis and dirofilaria repens are responsible of autochthonous filariases in dogs. adults of d. immitis kills the dogs with an heart location and d. repens is often found in subcutaneous nodules in dogs and cats. the microfilariae are present in the blood of these animals. dirofilariasis is due to the transmission of microfilariae by some mosquito bites (aedes, culex, anopheles, mansonia, psorophora and taeniorhynchus). usually non pathogenic to humans, these parasites are particularly present around the mediterranean basin. d. immitis is very rare in humans in europe, sometimes found in a pulmonary nodule and the heart location is not described. d. repens is more frequent and emerging in humans. usually, only one larva develops, producing an immature adult worm inside a subcutaneous nodule. ultrasound examination may suggest the parasitic origin of the lesion before an extraction and a parasitological diagnosis of the worm. more often, a fortuitous diagnosis is made on histological examination. very rarely, an adult worm may mature and produce systemic diffusion of microfilariae. dirofilariasis due to d. repens can present problems in diagnosis and treatment. an ocular and subconjunctival location of the worm and a subcutaneous nodule enclosing an immature adult are the commonest clinical forms. exceptional pulmonary locations are described. the subcutaneous locations described are: skull, cheek, breast, inguinal area, buttocks, arms and legs. cases of testicular location with painful symptoms have been observed. blood hypereosinophilia was exceptionally observed in human. it is treated surgically, by excision, without chemotherapy. while the majority of esbls, isolated in clinically-relevant gram negative bacteria (gnb) (mostly enterobacteriaceae, p. aeruginosa, a. baumannii) are tem-, shv-or ctx-m-types, a few others have been reported (sfo, bes, bel, tla, ges, bel, per, veb-types, and some oxa-esbls). laboratory detection of esbl-producers is important to avoid clinical failure due to inappropriate antimicrobial therapy and to prevent nosocomial outbreaks. selective culture media (macconkey and drigalski agar supplemented with cefotaxime and/or ceftazidime) have been proposed for detection of gnb resistant to expanded-spectrum cephalosporins (esc). media using chromogenic based substrates and selective antibiotics have been developed recently for the detection and presumptive identification of esbl-producing enterobacteriaceae directly from clinical specimens. detection of esbls based only on susceptibility testing is not easy due to the variety of b-lactamases and their variable expression of blactam resistance. commercially available esbl detection methods yield at most 90% accurate esbl identification, since some esbl-producers may appear susceptible to some escs. therefore, any organism showing reduced susceptibility to esc should be investigated using esbl confirmatory tests. these tests should be able to discriminate between esbl-producers and those with other mechanisms conferring esc resistance. these phenotypic tests (double-disk synergy test, esbl etest, and the combination disk method) are based on clavulanate inhibition and esc susceptibility testing. they often need slight changes by either reducing the distance between the disks of esc and clavulanate, the use of cefepime (not hydrolysed by ampcs), the use of cloxacillincontaining plates (that inhibits ampc), or by double inhibition by edta and clavulanate (masking metallo-enzymes). enzymatic tests have also been proposed for identification of esbl-producers. several pcr-based techniques (end-point or real time) have been developed on clinical samples or on colonies. several esbl genes have been detected using pcr coupled to either pyrosequencing, inverse hybridisation, to dhplc, or to fluorescent probes. these techniques even though more specific require technical knowledge, special equipment, are costly and detect only known genes, regardless of their expression. detection of esbl-producer remains a challenge for the microbiology laboratory and one shall be aware that esbl screening media are now available. resistance to antimicrobial agents has become common in many bacterial species, particularly those that cause human infections. the rapid detection of resistant organisms directly in clinical samples by real-time pcr coupled with molecular beacons, or of potentially resistant bacteria and yeast in blood culture bottles by peptide nucleic acid-fluorescence in situ hybridisation (pna-fish) is already having a positive impact on antimicrobial therapy. the direct detection of mycobacterium tuberculosis in sputum in approximately 2 hours with concomitant detection of mutations in rpob indicating rifampin resistance (as a surrogate for multidrug resistance) in the near future will likely improve the outcomes for tuberculosis patients in many developing and developed countries. several molecular technologies, including microarrays, bacterial tag encoded flx amplicon pyrosequencing (btefap), and ultra deep sequencing, have not yet transitioned to clinical laboratories but will likely provide even greater information about antimicrobial resistance not in just a single species, but in a whole community of microorganisms. complex wounds, like diabetic foot ulcers, containing multiple resistance genotypes are amenable to analysis by btefap. the implementation of these technologies in the clinical laboratory will be expensive but the potential to dramatically improve therapeutic outcomes especially for life-threatening diseases is unprecedented. objective: to determine the appropriateness of antimicrobial therapy (amt) in 11 dutch hospitals. method: data were obtained from a prevalence survey performed within the dutch surveillance network for nosocomial infections (prezies). amt administrated on the day of the survey was registered. antiviral and antifungal drugs, tuberculostatics, cements containing amt and prophylaxis administrated in the operation-theatre were excluded. the appropriateness of amt was assessed according to a standardised algorithm based on the local antimicrobial prescription guidelines. per patient a classification in appropriate use, inappropriate use and insufficient information was made. figure: relative risk of ia use of amt against largest hospital (hospital c). results: a total of 3,546 patients were included of which 1,075 (30%, range per centre (rpc): 23−37%) received amt. in the latter group, amt was considered appropriate in 70% (rpc: 57−84%), inappropriate in 17% (rpc: 3−32%) and was not judged because of insufficient information in 13% (rpc: 1−30%). there was considerable variation in inappropriate use among the participating centres (figure). in univariate analysis older age, the use of quinolones, being on the urology ward and presence of a suprapubical catheter were associated significantly with inappropriate use. admission on the icu and presence of an intravascular catheter were associated significantly with appropriate use. in a multivariate analyses the presence of suprapubical catheter, being on the urology ward and the use of quinolones were determinants for inappropriate use. this study showed large differences in overall use and appropriateness of use of amt between hospitals. based on these results it is possible to define targets for intervention to improve the prudent use of amt. the high fraction of patients with insufficient information in several centres may have influenced the analyses and should be addressed in future studies. m. struelens°, s. metz-gercek, r. mechtler, f. buyle, a. lechner, h. mittermayer, f. allerberger, w. kern objectives: the eu-project antibiotic strategy international (abs) qi team developed process qis for auditing the performance of key treatment and prophylactic practices. an international network of pilot hospitals tested these tools for feasibility, reliability and sensitivity to improvement. methods: qis included: 1. surgical prophylaxis (indication, drug choice, timing and duration of administration); 2. management of community-acquired pneumonia (cap) (blood culture and legionella antigen tests and drug choice for empirical treatment); 3. management of s. aureus bacteraemia (echocardiography, iv catheter removal and duration of therapy); and 4. iv-po switch for bio-available antibiotics. a minimum of 40 consecutive cases per centre and qi were retrospectively reviewed from clinical, laboratory and administrative records and assessed for data availability, inter-observer reliability, data collection workload and performance score. results: a total of 1240 patients were evaluated in 11 acute care hospitals from 5 countries, with a range of 80 to 500 cases and 2 to 9 centres per indicator. seven centres had already implemented antibiotic quality improvement and audit programmes. availability of data was >85% of cases and ranged between 87% (catheter removal in s. aureus bacteraemia) and 100% (diagnostic tests for cap). 13/14 indicators were found to be reliable with kappa 0.60 (good to excellent agreement). the workload per case ranged from a median time of 16 (cap) to 35 min (iv-po switch). the intention to treat qi scores showed high levels of adherence to the surgical prophylaxis qi bundle, with median values of 81 to 97% for hip prosthesis and 65 to 92% for colo-rectal surgery. for cap management, diagnostic testing appeared sub-optimal (<56% compliance with idsa guidelines). for s. aureus bacteraemia management, indicator results ranged from 60 to 65%. for use of bio available antibiotics, a median of 45% iv administrations were avoidable. there were marked differences of scores between centres for all qis. conclusions: the abs qis are reliable and broadly applicable tools for auditing antibiotic treatment and prophylactic practices. inter-hospital variation in adherence to recommended practice indicates substantial potential for improvement with different local priorities. these qis can be recommended for assessing the effect of quality of care interventions at either local or multi-centre level. d.j. noimark°, e. charani, s. smith, b. cooper, i. balakrishnan, s.p. stone (london, uk) introduction: reduction of clostridium difficile infection (cdi), which often follows use of third generation cephalosporins, is a national priority. over a three year period, antibiotic policies were reviewed and changed in an elderly medicine department according to local sensitivities of common pathogens and levels of cdi. a laminated pocket-sized card describing antibiotic policies was given to all doctors in the department on induction with instructions not to depart from these without microbiologists' approval. this prospective controlled interrupted time series examines whether this intervention increased compliance with antibiotic policy and decreased cdi incidence. methods: the department's "narrow-spectrum, no cephalosporin" antibiotic policy was changed on 1st august 2006 to replace trimethoprim with cephradine (1st generation cephalopsporin) as empiric treatment for urinary tract infection, reflecting local escheriscia coli sensitivities. in october 2007, all cephalosporins and quinolones were removed from the policy as cdi levels had increased. notional 7 day antibiotic usage was calculated from prospective pharmacy generated data with aspirin, calcium, bisphosphonate & laxative prescription use as a non-antibiotic control, and analysed by segmented regression with a robust variance estimator. cdi rates were prospectively collected separately & analysed by a poisson regression model. results: an immediate response to change in antibiotic guidelines was observed (figure) . from august 06-sep 07 there was a highly significant increase in cephalosporins (85-100% of which was cephradine alone) (p < 0.001), a significant fall in trimethoprim (p < 0.004) and a significant increasing trend in cdi ( no tools existed to assess the readiness of public hospitals to receive this technology, and therefore guide resource allocation to facilitate implementation. aim: to assess the readiness of victorian public hospitals to introduce electronic antimicrobial stewardship. method: literature on readiness for change, organisational culture and information technology acceptance were reviewed. group interviews with project teams at site initiation meetings, one on one interviews with project officers at subsequent meetings, and observation where appropriate were all used to determine potential barriers and enablers. this information was recorded using a 'readiness assessment tool' and analysed to identify a number of key domains. to triangulate the data, questionnaires were distributed to project officers asking them to assess their sites' readiness to implement the system. results: a novel 'readiness assessment tool' was developed. it covered the domains of technical readiness, skills readiness, process readiness, administrative support readiness, resource readiness and hospital organisational characteristics. assessments at several hospitals highlighted a variety of issues at different sites and allowed early efforts to address these. a formative readiness assessment can be used to identify systematic problems that might facilitate or hinder uptake of electronic antimicrobial stewardship and to inform the adopters of potential resources required. [1] buising, k, thursky, k, robertson, m, black, j, street, a, richards, m & brown, g (2008) . electronic antibiotic stewardship-reduced consumption of broad-spectrum antibiotics using a computerised antimicrobial approval system in a hospital setting. j antimicrob chemother. w.v. kern°, m. steib-bauert, a. pritzkow, g. peyerl-hoffmann, h. von baum, u. frank, m. dettenkofer, c. schneider, k. de with, h. bertz (freiburg, ulm, de) objectives: fluoroquinolone prophylaxis (fqpx) may reduce morbidity and mortality in cancer patients (pts) with neutropenia, but the development of fluoroquinolone resistance (fqr) in escherichia coli and other target organisms limits its usefulness. we evaluated changes in the incidence density of gram-negative bloodstream infection (gnb) and in the in vitro fqr rates after the introduction of fqpx (with levofloxacin) as a standard of care for pts with high risk neutropenia in a university hospital. methods: we collected individual data for 357 pts admitted during baseline and during the first months following the intervention to assess clinical outcomes. individual pt data were compared with aggregate data (3-month periods). aggregate data analysis (unit-wide antibiotic consumption, gnb and numbers of in vitro fqr bloodstream isolates) was continued for a total of eight 3-month periods for both the haematology-oncology service and for general internal medicine. the new policy was introduced in the second half of the year 2005 when unit-wide baseline fqr of e. coli and of coagulase-negative staphylococcal (cons) bloodstream isolates had been 15% and 80% in the haematology-oncology unit, and 8% and 60% in general internal medicine, respectively. the individual pt data analysis revealed that pts not given fqpx had a much higher incidence of gnb than those given fqpx ( -2007) . the monthly use of iv and oral quin was calculated based on data from the pharmacy department. statistical analyses were performed using segmented linear regression analysis. bayesian model averaging was used to account for model uncertainty. results: before the interventions the use of quin (both iv and total) was stable. the best fitting models indicated that the first intervention was associated with a stepwise reduction in iv use of 71 prescribed daily doses (pdd) (95% ci: 47, 95 (p < 0.001)). there was also an indication of smaller reduction in iv use associated with intervention 4, but only the intervention 1 effect was robust to model uncertainty. the overall use of quin was also significantly reduced (figure) with a large stepwise reduction of 107 pdd (95% ci: 58, 156) associated with intervention 2. this study showed that the hospital-wide use of quin can be significantly improved (and decreased) by an active policy consisting of multiple interventions. marwick°, j. broomhall, c. mccowan, s. gonzalez-mcquire, k. akhras, s. merchant, p. davey (dundee, high wycombe, uk; raritan, us) aim and objectives: to describe the antibiotic treatment and outcomes stratified by severity in a representative sample of adult patients aged 18 or older who were treated in hospital for skin and soft tissue infections. inadequate. we also judged that 43% of patients received unnecessarily broad spectrum therapy. conclusions: ssti is common and is associated with significant mortality. however, choice of empirical therapy is not evidence based, with significant under treatment of high risk patients. ab were mostly (16/17) prescribed by gps and delivered by public (n = 14) or hospital pharmacies (n = 3). surveillance of ab use in nhs was organised in only 4 ms. in 3 countries a nh specific pharmaceutical formulary was available. prescription profiles by prescriber were available in 5 countries. other quality improvement initiatives in nhs such as regular training of prescribers, promoting microbiological sampling, collection of antimicrobial resistance profiles or pharmacist advice on ab prescription were scarce. guidelines for ab treatment of most frequent infections were available in many countries but were focussing on ambulatory care and did not consider the specific nh situation. only in 1 country the presence of an infection control practitioner was compulsory and partnership with hospital infection control teams was legally imposed in 3 ms. conclusion: important structural, functional and regulatory nh differences exist between eu countries. specific tools to improve infection prevention and ab therapy in nhs should take into account these differences. a european nh network was created in the framework of the esac nh subproject, which will organise point prevalence surveys on ab use in 2009. c. escherichia coli in south-western finland j. jalava°, o. meurman, h. marttila, a. hakanen, m. lindgren, k. rantakokko-jalava (turku, fi) objectives: extended-spectrum betalactamases (esbls), especially enzymes of the ctx-m group, are spreading rapidly in europe. enterobacteriaceae with reduced susceptibility to third generation cephalosporins and a positive esbl confirmatory test are also increasing in southwest finland. the purpose of this work was to study the resistance genetics of these esbl-positive enterobacteriaceae. methods: the study comprises a total of 271 clinical enterobacteriaceae strains isolated from both inpatient and outpatient specimens. all enterobacteriaceae strains that were esbl confirmatory test positive between january 2004 and december 2008 were included in this study (263 escherichia coli, 8 klebsiella pneumoniae, one isolate per patient). of these strains, 225 (83%) were urine isolates. resistance determinations were done using disk diffusion method (clsi) or vitek 2 and esbl confirmations by the double disk method using cefotaxime and ceftatzidime with and without clavulanate. thus far, 219 strains (those collected by end of june 2008) have been analysed for the presence of the most important esbl genes (tem, shv and ctx-m) using pcr and pyrosequencing as described before (haanpera et al. aac, 52:2632; 2008) . results: in 2004 only 10 esbl-positive strains were found. all of them harboured a ctx-m type esbl gene. since then, the number esblproducing enterobacteriaceae strains has increased significantly being tenfold in 2008 compared to year 2004 (figure) . a high majority, 197 (90%) of the 219 strains analysed thus far had a ctx-m-type esbl gene. most of those (79%) belonged to the ctx-m-1 group according to the pyrosequencing results. ctx-m-9 group was the next common, with 20% of the ctx-m genes belonging to this group. only two strains with ctx-m group 2 enzyme were found. conclusions: enterobacteriaceae strains which produce esbl are increasing rapidly in southwest finland. this is especially true with e. coli strains isolated from urine. towards the end of the study period, the esbl enzymes were almost exclusively ctx-m, ctx-m-1 group being the most common. further research is needed to characterise genetic elements that carry these esbl genes. esbl strains and the proportion of ctx-m genes in 2004-2008. (2000) (2001) (2002) (2003) (2004) (2005) (2006) in france (n = 6), spain (n = 4), portugal (n = 6), uk (n = 11), kuwait (n = 2), canada (n = 13) and china (n = 10), including hong kong (n = 3) were studied. clonality was established by pfge and phylogenetic groups of ec and kp were determined as reported. susceptibility testing (clsi), blactx-m-14 transferability and location (i-ceu-i/s1 nuclease) were investigated. plasmid analysis included determination of inc group (pcr-replicon typing, hybridisation, sequencing) and comparison of rflp patterns. association of blactx-m-14 with isecp1, isecp1-is10 or iscr1 was established by pcr and sequencing. we identified 42 pfge types among 52 isolates: 38/47 ec, 3/4 kp and 1/1 cf. distribution among phylogroups were as follows: i) ec: a (n = 7), b1 (n = 3), b2 (n = 5) and d (n = 23), and ii) kp: kpi (n = 2) and kpii (n = 1). resistance to tetracycline (76%), nalidixic (74%), streptomycin (67%), sulfonamides (67%), ciprofloxacin (60%) and trimetroprim (43%) was common. were spreading horizontally in our hospitals and, here, we characterised the plasmids responsible in the major k. pneumoniae strains identified during the survey. methods: plasmids from representative k. pneumoniae strains with ctx-m-15 enzyme were extracted by alkaline lysis and compared by apai, psti and ecori restriction analysis. they were transferred into e. coli dh5a by electroporation. transformants were selected on cefotaxime-containing agar and were screened by pcr for beta-lactamase genes, the aminoglycoside resistance genes aac(6 )-ib and aac3-iib, and the plasmid-mediated quinolone resistance genes qnra/b/s. results: twelve isolates were characterised, representing 5 major strains (a-d, and f) found in the most-affected hospitals. restriction analysis divided their plasmids into several groups. representatives of strain a (n = 4) had essentially the same plasmid (group 1), as did the two representatives of strain d (group 2a). one strain f isolate had a plasmid (group 2b) very similar to plasmid 2a from strain d, indicating possible horizontal transfer. plasmids of group 3 were retrieved from representatives of strains b and c, again indicating probable transfer. plasmids from three other strains differed substantially from each other and from plasmids 1, 2a, 2b and 3. nevertheless, on all plasmids, blactx-m genes were linked to an upstream isecp1 element, known to be involved in their mobilisation. all encoded multi-resistance: all but one group 1 and one ungrouped plasmid carried aac(6 )-ib; blaoxa-1 and aac(3)-iia were detected on all except group 1 plasmids; blatem was found on group 1, 2b, one group 3 and two ungrouped plasmids. blashv and qnra/b/s genes were not detected. the considerable diversity of plasmids encoding ctx-m-15 enzyme in major slovenian k. pneumoniae strains suggested only limited transfer, even when multiple strains were present in the same hospital. evidence of plasmid transfer was between strains b and c, and possibly between strains d and f, although these plasmids were not strictly identical. analysis of resistance genes encoded by the plasmids revealed diversity, with groupings coinciding largely with those based on restriction profiles. a. ingold, g. borthagaray, a.k. merkier, d. centrón, h. bello, c.m. márquez°(montevideo, uy; buenos aires, ar; concepción, cl) objectives: to examine the genetic context of class 1 integron harbouring blactx-m-2 in fifteen nosocomial k. pneumoniae isolates from south america in order to enhance the understanding of the antibiotic resistance spread among the region. methods: dna was extracted with the use of axypreptm bacterial genomic dna miniprep kit. the analysis of the cassette array was carried out with the use of primers hs458/hs459 targeting adjacent conserved regions. the examination of the surroundings were performed using two pcr primer pairs, hs817/hs818 and hs825/hs826, to amplify the initial(iri) and the terminal(irt), inverted repeat boundary, respectively. the primer pair hs825/hs911 was used whenever a negative result was obtained with hs825/hs826. all pcr products were purified and sequenced and the data was analyzed with ncbi blast tool. the sequence obtained with primers hs817/hs818 revealed the presence of three different transposons backbones at the iri end. the tn5036-like module and the tn21-like module were present in 4 isolates, the tn1696-like module was present in 7 isolates. no amplicons were obtained with the use of primers hs825/hs826 that amplify a tn21-like insertion. two uruguayan isolates with a tn5036 boundary at the iri end were tested with hs825/hs911 that target a tn5036-like backbone and one generated a product consistent with a tn5036-like mer region. uruguayan isolates carried a single aada1 cassette (4/5) and the other one contained a dfra17-aada5 array, while the four argentinian isolates carried the combination aaca4-aada1-orfd. chilean isolates arrays are in process. conclusions: among the extended-spectrum beta-lactamases, the cefotaximases constitute a rapidly growing cluster of enzymes that have disseminated geographically. there is a high frequency of isolation of ctx-m-2 producing k. pneumoniae associated with a class 1 integron in the region. despite being common the presence of iscr1 linked to blactx-m-2 in k. pneumoniae isolates, this study provides new and relevant information in the sequence context at the iri. here we report about the cassette array diversity and the diversity of elements in which the class 1 integron are embedded. different integron/transposons carrying the blactx-m-2 gene seem to be circulating and different regional patterns could be emerging, this study highlights the ability of different genetic elements to act cooperatively to spread and rearrange antibiotic resistance. l. vinué, a. garcía-fernández, d. fortini, p. poeta, m.a. moreno, c. torres, a. carattoli°(logroño, es; rome, it; vila real, pt; madrid, es) objectives: ctx-m enzymes are frequently detected in europe. in particular, ctx-m-1 and ctx-m-32-producing strains have been recovered from both humans and farm animals in spain, italy, greece, and portugal, suggesting the existence of community reservoirs for these enzymes. the aim of this study was to compare escherichia coli strains and plasmids harbouring blactx-m-1 and blactx-m-32 genes isolated from human and animals. methods: four e. coli ctx-m-1 and eight ctx-m-32 epidemiologically unrelated producers from sick or healthy animals (pig, dog, cow and chickens) and from humans (urine, blood and faecal samples) were analysed by xbai-pfge, plasmid transferability, pcr-based replicon typing, plasmid restriction analysis and southern blot hybridisation. all isolates were from spain but the dog isolate was from portugal. the genetic context of the blactx-m genes was previously investigated for all the strains. results: three ctx-m-32 strains (one from healthy chicken and two from hospitalised patients) showed the same pfge pattern. a chromosomal localisation of the blactx-m-32 gene was suspected in these strains. the five remaining ctx-m-32 producers showed the blactx-m-32 gene on plasmids belonging to the incn (4 strains) or untypable groups (1 strain). two incn plasmids showed identical pvuiirestriction patterns: one was identified in a strain from a healthy chicken and one was from a hospitalised human patient; these two strains were isolated in 2002 and 2004, respectively and showed different pfge patterns. ctx-m-1 producers (three from animal strains and one a healthy human) did not show clonality by pfge and the blactx-m-1 gene was always located on plasmids, three belonging to the incn and one to the inci1 groups. two of the incn plasmids carrying the blactx-m-1 gene showed highly related restriction patterns: one was from a healthy dog and one from a healthy human. conclusion: this study demonstrated the presence of clonal e. coli ctx-m-32 producers in animal and human sources and also detected epidemic incn plasmids disseminating among unrelated isolates from humans and animals, clearly suggesting a potential animal reservoir for the blactx-m-1/32 genes. o309 characterisation of bladim-1, a novel integron-located metallo-beta-lactamase gene from a pseudomonas stutzeri clinical isolate in the netherlands l. poirel°, j. rodriguez-martinez, n. al naiemi, y. debets-ossenkopp, p. nordmann (k.-bicetre, fr; amsterdam, nl) objectives: characterisation of the mechanism involved in the uncommon resistance to carbapenems observed from a pseudomonas stutzeri isolate recovered from a patient hospitalised in the netherlands with a chronic tibia osteomyelitis. that strain was resistant to ticarcillin, piperacillin-tazobactam, imipenem and meropenem, of intermediate susceptibility to ceftazidime and cefepime, and susceptible to aztreonam. methods: screening for metallo-beta-lactamase (mbl) production was performed using the e-test method with a strip combining imipenem and edta. shotgun cloning was performed with xbai-digested dna of p. stutzeri and pbk-cmv cloning vector. selection was performed on amoxicillin and kanamycin-containing plates. results: e. coli top10 (pdim-1) recombinant strains were obtained, displaying resistance to penicillins and ceftazidime, reduced susceptibility to cefepime, imipenem and meropenem, and full susceptibility to aztreonam. sequence analysis identified a novel ambler class b betalactamase dim-1 for "dutch imipenemase" (pi 6.1) weakly related to all other mbls. dim-1 shared 52% amino acid identity with the most closely related mbl gim-1, and 45 and 30% identity with the imp and vim subgroups, respectively. dim-1 hydrolyzes very efficiently imipenem and meropenem, expanded-spectrum cephalosporins, but spares aztreonam. the bladim-1 gene was as a form of a gene cassette located at the first position in a class 1 integron, but the 59be of that gene cassette was truncated giving rise to a fusion with an aadb gene cassette encoding an aminoglycoside adenylyltransferase. the third and last gene cassette corresponded to the qach cassette encoding resistance to disinfectants. conclusion: a novel mbl gene was identified in p. stutzeri further underlining (i) the diversity of acquired mbl genes, especially among non-fermenters, (ii) that pseudomonas sp. may be a reservoir of these genes and (iii) the possibility of spread of important resistance determinants in northern part of europe. isolates in greece p. giakkoupi, o. pappa, m. polemis, a. bakosi, a. vatopoulos°( athens, gr) objectives: metallo-beta-lactamases of the vim family are the main mechanism of carbapenem resistance in p. aeruginosa in greece. in this preliminary report we attempted to survey the subtypes of vim betalactamase currently prevailing in p. aeruginosa clinical isolates in greek hospitals, the genetic relatedness of the respective isolates, as well as the genetic environment of the blavim gene. methods: fifteen mbl producing and epidemiologically unrelated p. aeruginosa clinical isolates were collected in september 2006 from fifteen different hospitals around greece. mbl production was initially identified by an edta synergy test. identification of blavim gene, as well as mapping of the blavim cassette carrying integrons were performed by pcr and sequencing of the products. the o serotypes of the isolates were determined by a slide agglutination test using p. aeruginosa antisera (biorad). molecular typing was performed by pulse-field gel electrophoresis of spei-restricted genomic dna. results: blavim-2 gene was detected in nine isolates, blavim-4 in five and blavim-1 in only one isolate. the blavim-2 cassette of all nine isolates was located on the 1600 bp variable region of a class i integron, preceded by aaca29 gene cassette. blavim-4 cassette of all five isolates was the first cassette of the 3200 bp variable region of a class i integron, followed by the aaca4 and blapse-1 gene cassettes. blavim-1 was the unique cassette of a class i integron. vim-2 producers belonged to o8, o11 and o12 serotypes, whereas four isolates were non-typeable. vim-4 producers belonged to the same three serotypes, whereas only one was non-typeable. the vim-1 producer belonged to o12 serotype. the nine vim-2 producing p. aeruginosa isolates revealed a great degree of variability in pfge molecular typing, belonging to seven types. contrary, the five vim-4 producing p. aeruginosa isolates displayed higher genetic similarity and fell into one major type with 85% homology, which also included the vim-1 producing isolate. there was no correlation between the results of serotyping and molecular typing. conclusions: mbl production in p. aeruginosa in greece seems to be mainly due to specific class i integrons harbouring either blavim-2 or blavim-4 genes. genetic variability was higher among bacteria carrying vim-2 beta-lactamase, a fact indicating wider intraclonar spread of the respective integron. j.m. rodriguez-martinez, l. poirel°, p. nordmann (k.-bicetre, fr) objectives: extended-spectrum beta-lactamases of ampc-type (esacs) contributing to reduced susceptibility to imipenem have been recently reported from enterobacteriaceae. the aim of the study was to evaluate the putative role of natural ampc-type beta-lactamases of p. aeruginosa in a similar resistance profile. methods: thirty-two non-repetitive p. aeruginosa clinical isolates recovered in our hospital in 2007 were included. they were selected on the basis of criteria of intermediate susceptibility or resistance to ceftazidime and intermediate susceptibility or resistance to imipenem. mics were determined by agar dilution and e-test techniques. the level of expression of the ampc beta-lactamases was evaluated by measuring specific activities. pcr, sequencing, and cloning allowed to characterise the different bla(ampc) genes. identified esacs were purified and their km and kcat values for beta-lactams determined by spectrophotometry. results: using cloxacillin-containing (an ampc beta-lactamase inhibitor) plates, the susceptibility to ceftazidime was restored for 25 out of 32 isolates, suggesting overproduction of the ampc. in addition, in presence of cloxacillin, reduced mic values were also observed with ceftazidime, cefepime and imipenem for 21 out of those 25 isolates. cloning and sequencing identified 10 distinct ampc b-lactamase variants among the 32 isolates. recombinant plasmids expressing the ampcs were transformed into reference p. aeruginosa strain and reduced susceptibility to cefepime and imipenem was observed only with recombinant p. aeruginosa strains expressing ampc beta-lactamases that had an arginine residue at position 105. the catalytic efficiencies (kcat/km) of the ampc variants possessing this arginine residue were increased against oxyiminocephalosporins and imipenem. in addition, in-vitro assays demonstrated that those ampc variants constituted a favourable background for selection of additional degree of carbapenem resistance. conclusions: some ampcs of p. aeruginosa possessing extended activity torward carbapenems may contribute to carbapenem resistance. background: most oxa-type esbls are oxa-10, oxa-2 or oxa-1 derivatives. they display a very low homology, the percentage of which is between 20% and 30%. oxa-type esbls are divided into five groups according to the different homology by frederic bert, etc. group 1 includes oxa-5, oxa-7, oxa-10 and its derivants;group 2 includes oxa-2, oxa-3, oxa-15 and oxa-20;group 3 includes oxa-1, oxa-4, oxa-30 and oxa-31; group 4 is named after oxa-9; group 5 only includes a single enzyme called lcr-1. oxa-type esbls has been reported widespread in the world since the first report in 1987, such as turkey, france, england and so on. but there is few report about it in china. objective: to investigate the prevalence and genotype distribution of oxa-type extended-spectrum beta-lactamases (esbls) in clinical pseudomonas aeruginosa strains isolated from xiangya hospital of central south university in changsha city, hunan province, china. methods: ninety-seven non-repetitive clinical isolates of p. aeruginosa were collected between october 2006 and january 2007 from the hospital. they were screened for oxa-type esbls production by polymerase chain reaction pcr with five pairs of primes specific for blaoxa genes, respectively. then amplification of oxa-type esbls production was performed by pcr with specific primers. the purified and amplified products were sequenced to confirm the genotype of the oxa-type esbls. results: the sequences of the three oxa-type esbls pcr products were then compared in genbank database and there were no the completely same ribonucleotide and amino acid sequence with them. they were two novel oxa-type esbls, named as blaoxa-128 and blaoxa-129, which have been registered in genbank database under accession numbers eu573214 and eu573215, respectively. conclusions: there have occurred infections caused by p. aeruginosa producing oxa-type esbls in xiangya hospital of central south university. two novel oxa-type esbls in p. aeruginosa strains have been discovered in our study, which are named blaoxa-128 and blaoxa-129, respectively. pneumonia is one of the most common nosocomial infections and is associated with high mortality. in the last 15 years, gram-positive bacterial pathogens have risen in prevalence as a cause of hospitalacquired pneumonia (hap), including that occurring during mechanical ventilation (ventilator-associated pneumonia; vap). in particular, staphylococcus aureus is a major cause of hap, including vap. the rise of multidrug-resistant infections is a source of concern, with methicillinresistant s. aureus (mrsa) accounting for >40% of s. aureus isolates in some european hospitals. this symposium will take the format of a question-and-answer roundtable session in which experts will answer questions and initiate discussion surrounding emerging concerns and appropriate therapeutic strategies in nosocomial pneumonia, including that caused by multidrug-resistant gram-positive pathogens. recently, shifts in the susceptibility of s. aureus to established therapeutic agents for nosocomial pneumonia have added to the challenge of selecting appropriate empiric therapy. in patients with suspected multidrug-resistant infections or those who are mechanically ventilated, prompt initiation of therapy, often before the pathogen has been confirmed, is critical. vancomycin is the gold-standard treatment for multidrug-resistant infections and resistance has been remarkably slow to emerge. however, clinical reports in europe of 'mic creep' and the emergence of vancomycin-intermediate s. aureus (visa), hvisa and linezolid-resistant mrsa have presented new clinical dilemmas. elevated vancomycin mics are linked to treatment failure and increased mortality. hence, while vancomycin remains a useful therapeutic tool, treatment decisions present an increasing challenge, especially in groups of patients in whom rapid eradication of infection with appropriate agents is critical. telavancin is a novel lipoglycopeptide under investigation for treatment of nosocomial pneumonia. a number of key features suggest telavancin as a potentially attractive option for nosocomial pneumonia. telavancin has a unique dual mechanism of action that disrupts both bacterial cell wall biosynthesis and cell membrane integrity. the agent is rapidly bactericidal against a broad range of clinically relevant grampositive bacteria, including mrsa. two pivotal phase iii studies have demonstrated telavancin efficacy equivalent to vancomycin in hap, including vap, including in seriously ill patient subgroups and in that caused by mrsa. hantaviruses are enveloped rna viruses, each carried primarily by rodents or insectivores of specific host species. they have coevolved with the hosts in which they cause almost asymptomatic and persistent infections. in humans some hantaviruses cause disease: haemorrhagic fever with renal syndrome (hfrs) in eurasia. in europe puumala (puuv) from bank voles and saaremaa (saav) from field mice cause mild hfrs and dobrava (dobv) from yellow-necked mice severe hfrs. in asia hfrs is caused mainly by hantaan and seoul viruses. in americas some viruses cause hantavirus cardiopulmonary syndrome: sin nombre, andes and other viruses carried by sigmodontine rodents, not found in eurasia. in addition, in europe the common vole carries tula and rats seoul virus. however, they have not been definitely associated with disease in europe, although both can infect humans. we discuss the epidemiology, molecular genetics, detection of infection in carrier hosts and humans (including rt-pcr and 5-min serological tests), functions of hantaviral proteins, risk factors for humans to catch hantavirus infection (including smoking) and disease (including risk and protective hla haplotypes), role and mapping of epitopes of cytotoxic t-cells, mechanisms of hantavirus-induced apoptosis, newly discovered clinical features (including hypophyseal haemorrhages in puuv infection), and long-term consequences and pathogenesis of hfrs (endothelial permeability, thrombocytopenia, tnf-alpha and il-6). puuv occurs widely in europe except in the far north and mediterranean regions, saav in northern, eastern and central europe and dobv mainly in the balkans. the epidemiological patterns differ: in western and central europe hfrs epidemics follow mast years with increased oak and beech seed production promoting rodent breeding. in the north, hantavirus infections and hfrs epidemics occur in 3−4 year cycles, driven by prey-predator interactions. the infections and hfrs are on the increase in europe, partly because of better diagnostics and partly perhaps due to environmental changes. in several european countries hantavirus infections are notifiable and in some countries (e.g. belgium, finland, france, germany, scandinavian countries, slovenia) their epidemiology is relatively well studied. in large areas of europe, however, hantavirus infections and hfrs have not been studied systematically and they are still heavily under-diagnosed. mrsa screening − will we ever agree? s330 mrsa: universal screening! the successful control of any outbreak or epidemic relies on detection of those harbouring the pathogen (infected and colonised persons) combined with eliminating spread to new individuals. the approach to containment and reduction of the global mrsa pandemic is now being discussed. a challenge for this infection is that most persons harbouring mrsa do not exhibit signs of disease and thus in order to detect all potential spreaders of this organism some surveillance must be done. the required level of detection (surveillance through screening) is not known and likely varies with the prevalence of colonisation and disease. for a given mrsa prevalence, the factor that seems most crucial in reducing spread is the percentage of potential isolation days captured. the operational processes that highly influence this are 1) the sensitivity of screening detection (including sites tested and laboratory methods used), 2) the speed at which results of newly detected positive patients are reported from the laboratory (assuming pre-emptive isolation is not employed), and 3) the selection of patient populations who are to undergo screening. laboratory testing has a major impact on detecting mrsa colonised patients with real-time pcr having a sensitivity of 98% and a possible 2 hour reporting time compared to direct chromogenic agar cultures with a sensitivity of 80% and >24 hour reporting and enriched chromogenic agar testing with a sensitivity of 90% and >48 hour reporting (am j clin pathol, 2009); both reduced sensitivity and prolonged reporting time negatively impacting the success of mrsa timely isolation. we have shown that capturing 33% of mrsa isolation days in a modest mrsa prevalence setting (9 infections/10,000 patient days) with a high sensitivity test having a >24 hour result reporting time did not reduce hospital-wide mrsa disease (ann int med 148:209, 2008) . others have demonstrated that surveillance in an icu with similar mrsa prevalence, again with a high sensitivity test having 1 day result reporting, did not reduce icu disease until preemptive isolation was initiated (crit care 10: r25, 2006) . finally, we demonstrated that universal admission surveillance and decolonisation capturing 85% of possible mrsa isolation days had a dramatic impact by reducing 70% of all in-hospital infections from mrsa. future research in this area should focus on better defining those patients that benefit from mrsa screening and the role of decolonisation in these programs. clostridium difficile infection (cdi) is a toxin-mediated intestinal disease and extraintestinal manifestations are exceptional. clinical outcomes can range from asymptomatic colonisation to mild diarrhoea and more severe disease characterised by inflammatory lesions and pseudomembranes in the colon, toxic megacolon or bowel perforation, sepsis, shock, and death. the main clinical symptoms, secretory diarrhoea and inflammation of colonic mucosa, can be in great part explained by the actions of two large protein toxins, toxin a (tcda) and toxin b (tcdb). both toxins are cytotoxic, destroy the intestinal epithelium and decrease colonic barrier function by disruption of the actin cytoskeleton and tight junctions resulting in a decreased transepithelial resistance allowing fluid accumulation. in addition, c. difficile toxins also cause release of various inflammatory mediators which affect enteric nerves, sensory neurons and promote inflammatory cells, adding to the fluid secretion, inflammation and transmigration of neutrophils. some experimental evidence points also to possible extraintestinal action of c. difficile toxin b. in zebrafish embryos tcdb caused damage and edema in cardiac tissue and in hamsters the same toxin caused lung damage. only recently efficient systems have been developed to genetically manipulate c. difficile. comparison of knock-out mutants producing only one of both toxins have shown that tcdb-positive-only mutants retain the ability to kill hamsters, whereas tcda-positive-only mutants were not virulent for hamsters. these results are in concordance with epidemiological findings that naturally occurring a-b+ strains still cause the entire spectrum of cdi, but are not in concordance with effects observed after intragastric challenge of hamsters with purified toxins tcda and tcdb. the role of the third toxin produced by c. difficile, binary toxin cdt in the development of human disease is not well understood. cdt was shown to have enterotoxic effect in rabbit ileal loop assay, but natural strains producing cdt but neither tcda nor tcdb colonised animals but were not lethal in hamsters. comparative genomic analysis will most likely reveal additional factors involved in pathogenesis and in increased virulence (including cell surface layer proteins, sporulation characteristics and antibiotic resistance). additionally, the role of the host immune response in cdi has just started to be better understood. since 2002, there has been an escalation in rates of clostridium difficile infection (cdi) with epidemic c. difficile (pcr ribotype 027/north american pulsed-field type 1 [nap1]) responsible for outbreaks of severe infection in north america and europe. while fluoroqinolone resistance and over-use are thought to be driving the epidemic, the ageing population and improved case ascertainment are contributing to the dramatic increase in cases. other factors may also be important, such as the increase in prescription of proton pump inhibitors. in the netherlands, since 2005, there has been an increase in prevalence of human cdi with ribotype 078 strains usually found in animals. these infections were in a younger population and more frequently community acquired. there was alarm when it was reported that 20% of retail beef samples in canada contained c. difficile. the figure is higher in the usa where more than 40% of packaged meats (beef, pork and turkey) from 3 arizona stores contained c. difficile. most animal isolates of c. difficile produce binary toxin, and both pigs and cattle harbour pcr ribotype 078 a strain that, like ribotype 027, also produces more toxins a and b, and binary toxin. in the eastern part of the netherlands where >90% of pig farms are located, >20% of human isolates are now ribotype 078, and human and pig strains of c. difficile are highly genetically related. it has been suggested that the overlap between the location of pig farms in the netherlands and the occurrence of human ribotype 078 infections involves a common source. that source is likely to be the environment. the upsurge in cdi has prompted diagnostic companies to try to either improve current tests or develop new ones. laboratory diagnostic methods can be divided into 3 groups; traditional faecal cytotoxin detection (with or without culture), enzyme immunoassays (eias) and molecular methods. faecal cytotoxin detection is specific but lacks sensitivity, culture is sensitive but lacks specificity. new eias should find a niche in medium sized laboratories. current in-house pcr methods have the potential for great sensitivity and specificity but have been available only in larger laboratories. new commerciallyavailable platforms will make this methodology more accessible to smaller laboratories. whatever method is chosen, it is necessary for the laboratory to have as fast a turn-around-time as possible, particularly in an outbreak situation. d. lévy-bruhl°(saint-maurice, fr) in 2005, the advisory board on immunisation (abi) has been asked to make recommendations to the ministry of health regarding the inclusion or not in the french immunisation schedule of the soon to be licensed first hpv vaccine. the main elements considered in the establishment of the benefit-risk balance of routine hpv vaccination were: on the benefit side: -the very significant potentially preventable burden of diseases; -the very high efficacy of the vaccine against persistent hpv 16/18 infections in naive subjects; -the expected additional impact on other hpv16/18 related lesions and cancers; -the fact that vaccination, by preventing the pre-cancerous lesions, has the advantage over screening to reduce the cost and anxiety related to their detection and management; -the available data in favour of a satisfactory safety profile; -the benefit of vaccination for the women not covered by the opportunistic screening program. on the "risk" side: -the high cost of vaccination; -the unknown duration of protection; -the need for continuation of screening, even for vaccinated women; -the fact that the majority of residual cervical cancers could be prevented by the organisation of the screening program; -the risk of a decrease in compliance to screening for vaccinated women; -the low benefit if vaccinated and screened women were the same. a cost effectiveness analysis, carried out on a multi cohort markov model, showed that, over a 70 years period, the impact of vaccinating 80% of 14 years old girls or of organising the screening were comparable (reduction of cancer deaths close to 20%). however, the cost-effectiveness ratio of the vaccination was higher than that of the screening organisation, resp. 45,200 and 22,700 € per life year saved (at a 3% discount rate). on the basis of the economical analysis, the screening organisation was therefore the first priority. however, if both interventions were implemented, the overall reduction in cervical cancer deaths was estimated at 32%. the cost-effectiveness of the addition of vaccination on the top of the organisation of the screening appeared acceptable (55,000 € per life year saved). based on those results, the abi issued in march 2007 a recommendation to include the hpv vaccination in the immunisation schedule for 14 years old girls, together with a catch up for 15 to 23 years old women not having started their sexual life more than one year ago. the vaccine cost has been reimbursed since july 2007. clinical microbiology − is outsourcing the way to go? s338 the (r)evolution of clinical microbiology in europe − is it good or bad? laboratory medicine in general and clinical microbiology in particular is presently subject to rapid (r)evolution. are we aware? are we in command? do we know where we are going? should we oppose or cooperate? do we have a choice? do we recognise a driving force other than money? is it good, bad or just plain necessary? and are we gaining or losing? it is not one evolutionary process -it is several parallel processes with varying emphasis in different areas. there are at least four distinctive major trends over the last 15 years; the gradual formation of bigger and bigger units (concentration), the amalgamation of many different laboratory services into one (laboratory medicine), accreditation and an explosion of professional proficiencies and backgrounds of staff in microbiological laboratories. personally i have withstood the first two, with pleasure succumbed to the latter. a recent 5th trend, outsourcing microbiology services to large private consortiums, is splitting clinical microbiology into a purely analytical high-throughput money-saving activity, often leaving the consultative, clinical part of microbiology and health care infection control adrift. what is driving the evolution? not only cost-saving but also our inability to recruit medically trained microbiologists, the need to broaden the knowledge base of microbiology laboratories, automation, the development of new techniques and apparatus common to many laboratory disciplines, computerised medicine, political trendiness, power struggles, and much more. there is much to be gained by both concentration and amalgamation but much to be lost as well and many consider the heart and soul of clinical microbiology at risk. over a period of years, rational high-throughput production has won over consultation and personalised microbiology. that may be fine for the production of negative hiv-antibody/antigen analysis as for the screening of blood-donors but certainly not for the bacteriological cultures taken in conjunction with a hip replacement. or when it comes to understand and advise on the intricacies of antimicrobial resistance development. in other cases "outsourcing" and/or "amalgamation" mean that blood cultures are sent to x-town, cmv-antibodies to y-town and everything else to z-ville. when that happens clinical microbiology is lost. there are several instances where concentration, amalgamation and/or outsourcing of clinical microbiological services, alone or with other services, have meant that the tie between clinical microbiology and infection control has been severed and that many, both small and large hospitals have lost the personalised service so necessary to control outbreaks of multi-resistant bacteria and other health care related infections. a good service requires a strong knowledgeable and enthusiastic champion. a service which encompasses too many branches of laboratory medicine cannot be expected to champion each and every one with equal strength and fervour. and when outsourced to "big companies", there is no "clinical", only "microbiology". in 2008 "medical microbiology" broke out from "laboratory medicine" in uems. we are now striving towards a strong "medical microbiology" service in europe. it will have many facets, much strength, some weakness, great opportunities, but many threats. escmid certainly intends to help shape microbiology in europe. the optimal organisation of microbiology laboratories in european metropolis is an evolutionary task, driven by the evolution in laboratory tasks, laboratory technologies, communication technologies, regulations and financial issues. in the past five-ten years, medical and societal query for a more rapid and refined detection and identification of pathogens and antimicrobial resistance determinants coincided with the expansion of internet-based and remote tools for communication, an unprecedented revolution in laboratory technologies and new financial constraints. the concentration of laboratory workforces into one unique laboratory is one way to address these apparently contradictory issues. the tertiary medical school hospital system in marseille, a 2-million metropolitan area in france, comprises four hospitals for a total of 3,500 beds. the system had once four microbiology laboratories which have been progressively embedded into a unique, 600,000 acts per year, laboratory which deals with bacteriology, virology and environmental microbiology and hygiene. the medical staff comprises of 17, the ingenior staff of 11, technical staff of 88 and support staff of 13 persons for a total of 129 persons. this organisation allowed reducing labour time for routine microbiology, to develop prospective and sophisticated time-consuming diagnostic methods and to develop advanced diagnostic methods such as molecular methods (real-time pcr-based tests, sequencing, and mass spectrometry identification) and new generation serology. new, sophisticated technologies such as automated serology and mass spectrometry were corner-stones on which to base the constant diminution of routine labour time and the development of time-consuming tasks such as fastidious organisms' isolation. these evolutions paralleled the exponential increase in the ratio of ingeniors in the laboratory. this paradigm allowed for the constitution of large collections of biological specimens for retrospective analyses, the specialisation of every medical senior in one particular field of internationally recognized expertise and the increase in knowledge output in terms of peer-reviewed papers, patents and grants. implantation of point-of-care in the emergency department, in permanent internetbased connection with the central laboratory, was the last, but not least, evolution of this system. when tuberculosis epidemiology is seen in a global perspective, and the millennium development goals are considered, it is clear that two regions of the world, africa and europe, are severely behind in the control of the disease. in africa, especially sub-saharan africa, the tb problem is closely related to the endemic hiv/aids situation. in europe, especially the eastern part and in parts of the former soviet union, the main obstacle to an effective tb control is related to drug resistant forms of m. tuberculosis. the prevalence of the most severe forms of resistance, mdr-and xdr-tb, is so high that it makes control efforts both extremely complicated and very expensive. unfortunately, increasing levels of drug resistant tb are today also seen in many african countries, and hiv infection is spreading in eastern europe. during the last ten-year period new tools, based on molecular fingerprinting of m. tuberculosis strains, have been increasingly adapted to study tb transmission. with such molecular methods to characterise clinical isolates of m. tuberculosis it is now possible to study the spread of individual strains of the bacteria in detail. the laboratory tools used, rflp, miru/vntr, spoligotyping and others, will be presented and their use exemplified. how molecular epidemiology contributed to the detection and characterisation of a major outbreak of drug resistant tb in the stockholm area will be discussed. molecular characterisation of clinical isolates from different parts of the world has led to an increased recognition of the differences between different families of m. tuberculosis strains. to further describe and understand the role of these differences in the clinical field as well as for tb epidemiology is an ongoing and interesting field of research. an increased understanding of how tb is transmitted will hopefully help in the efforts to control this global health threat both on the local level and in a global perspective. living in the era of increasing tuberculosis drug resistance, the importance of making an early and accurate diagnosis with drug sensitivities has never been greater. the epidemiology of tuberculosis defines the extent of latent disease and the proportion which becomes active. accurate diagnosis is vital if patients are to be treated in a timely manner and to reduce the amount of time infectious individuals go untreated in the community disseminating disease. in many areas of the world, dots programmes are at the forefront of tuberculosis control. however, as a diagnostic this currently relies on sputum smear microscopy which is known to miss 50% of cases of tuberculosis and provides no data on drug sensitivity. the second major issue around tb is the lack of worldwide diagnostic facilities. there is a need for a simple, low cost, easily implemented diagnostic test. this talk will briefly consider the issues around the diagnosis of latent and active disease which are quite distinct. the focus will be on the diagnosis of active infection. in particular, the use of mods (microscopic observation drug-susceptibility) assay in diagnosis of tuberculosis will be discussed. the potential for using this in resource poor countries will be reviewed as well as the way sophisticated technology maybe harnessed to improve reporting and allow translation to all parts of the world. the important issue of how to distinguish patients with latent and active disease will also be considered. key issues and principles in diagnosis both now and in the future will be reviewed. in terms of treatment, there are 2 main issues. the first is that even short-course therapy is prolonged being a minimum of 6 months leading to issue of compliance. this may result in drug resistance. the massive rise of multi-drug resistant tuberculosis to approximately 500,000 cases world-wide with around 50 countries reporting extensively drug-resistant disease means that the need for new approaches to therapy are urgent. the second part of this talk will review different approaches to using current anti-mycobacterial drugs, the emergence of a small number of new drugs such as the diarylquinolones and entirely novel approaches to control and treat tuberculosis. there has been great success and also many threats in the field of infectious diseases during the previous year. the antimicrobial resistance, especially increasing carbapenem resistance among aerobic gram-negative rods and xdr mycobacterial tuberculosis strains are already big threats in some countries and they will probably spread to many other areas all over the world in the future and we will need new drugs for these indications but unfortunately very few new promising drugs seem to be in the pipeline at the moment for these purposes. the virulent clostridium difficile 027 strain spreads rapidly to many new countries and e.g. in finland it killed many times more people compared with mrsa and esbl strains in 2008. however, it is possible to stop its spreading but it needs new thinking in antibiotic use policy and infection control policy in hospitals. clostridium difficile 027 infection has a high relapse rate after metronidazole or vancomycin therapy, but an experimental "stool exchange treatment" is a promising therapy although controlled studies are needed to prove this assumption. an interesting research area during the last years has been the role of infections in the etiopathogenesis of chronic diseases like cancer, atherosclerosis, cardiovascular diseases and many autoimmune diseases. we can fight against many cancers like liver cancer and cervix cancer with virus vaccines and gastric cancer with antimicrobial drugs. also the high incidence of malignant tumours seems to decrease during haart treatment in hiv patients. the role of infections in the etiopathogenesis of cardiovascular diseases and atherosclerosis is complex. it is obvious that infections play a role in the etiopathogenesis of atherosclerosis, stroke and myocardial infarction but the undirected routine antimicrobial treatment is not recommended for these patients but there seems to be subgroups in patients with various cardiovascular diseases which may benefit from antimicrobial treatment. recent studies seem to suggest that there are hla types which protect or make people susceptible for coronary heart disease. the hla type hla-b*35 seems to be a risk factor for coronary heart disease but it is also a risk factor for chronic chlamydia pneumoniae infection. the feared pandemia due to h5n1 influenza a did not appear during the recent year and the world is now much more prepared to meet the next pandemia which, however, hopefully does not come during the next year. ø. samuelsen°, c. giske, u. naseer, s. tofteland, d.h. skutlaberg, a. onken, r. hjetland, a. sundsfjord (tromsø, no; stockholm, se; kristiansand, bergen, oslo, førde, no) objectives: the worldwide dissemination of kpc-producing multidrugresistant enterobacteriaceae is worrisome. the first kpc-producing klebsiella pneumoniae in norway was isolated late 2007 from a patient after hospitalisation in greece. throughout the following year seven additional kpc-producing k. pneumoniae isolates have been detected in clinical samples from six new patients. the aim of this study was to perform molecular characterisation of the strains and examine their epidemiological relatedness. materials and methods: antimicrobial susceptibility was examined by etest. molecular characterisation was performed by mlst, pfge and sequencing of the blakpc genetic structure. plasmid analysis was carried out by pfge of s1 nuclease-digested total dna and southern blot hybridisation using a blakpc probe. relevant epidemiological data were collected retrospectively. results: eight kpc-producing clinical isolates of k. pneumoniae have been identified from seven patients in two different regions of norway from the following specimens: blood culture (n = 3), urine (n = 2), expectorate (n = 1), perineal swab (n = 1) and wound secretion (n = 1). two blood culture isolates with clonally related but different pfgeprofiles were observed in one patient. the detection of kpc-producing k. pneumoniae isolates in norwegian patients was associated with import in four cases after hospitalisation in greece. two patients had been hospitalised at the same hospital in greece. isolation of a kpc-producing isolate in a fifth patient was epidemiologically linked to one of these imported cases and was a case of nosocomial transmission in norway. for the latter two cases no risk factors were identified with respect to recent hospitalisation or travel abroad. molecular analysis of six isolates has shown genetically related pfge-patterns and a common sequence type (st258). st258 has been associated with dissemination of ctx-m-15 in hungary. the blakpc gene was localised in tn4401 on a~97 kb plasmid. the two most recent isolates are currently undergoing similar analysis. conclusion: the first seven cases of kpc-producing k. pneumoniae in norway are associated with hospitalisation abroad, nosocomial transmission in norway, or urinary tract infections in outpatients without obvious risk factors. the clonal relationship between isolates underlines the existence a biological fit genetic lineage of kpcproducing k. pneumoniae with an epidemic potential. objectives: two recent publications have reported the isolation of kpc producing k. pneumoniae from infections in two patients, one in france and one in sweden, who originally had been hospitalised in greece. since this resistant mechanism had not been identified before in this country, the purpose of this report was to confirm the presence of blakpc producing k. pneumoniae in greece, to assess the extent of its spread and to study the genetic relatedness of the respective bacterial strains and the transferability of the blakpc harbouring plasmids. methods: for a three month period (february to april 2008) 40 hospitals participating in the greek system for surveillance of antibiotic resistance (www.mednet.gr/whonet) were asked to seek for possible kpc producers among k pneumoniae isolates displaying reduced susceptibility to imipenem (equal or higher than 1 mg/l), a positive hodge test for the presence of carbapenemase and a negative edta synergy test for the presence of metalloenzymes. the presence of blakpc gene in these strains was confirmed by pcr and sequencing. mics to carbapenems were determined by etest. conjugation experiments were carried out both in broth and on agar. the possible absence of ompk36 porin was detected by pcr. molecular typing was performed by pulse-field gel electrophoresis of xbai-restricted genomic dna. results: ninety two k. pneumoniae clinical isolates (one per patient) from 13 hospitals all over greece were found to harbour blakpc-2 gene. although colonies present in the inhibition zone made the exact determination of imipenem mic difficult, the absence of ompk36 porin was always associated with mic of imipenem higher than 32 mg/l. all isolates exhibited resistance to all other drug classes except colistin, tetracycline and tigecycline. pfge analysis revealed that 85 isolates from 12 hospitals displayed more than 95% similarity and were classified into one pulsotype, whereas the remaining seven isolates belonged into four different pulsotypes. blakpc-2 gene could not be transferred by conjugation from strains belonging to the main pulsotype. however, it was transferred from strains belonging to three out of the four remaining pulsotypes. conclusion: production of kpc-2 betalactamase seems to be a new emerging resistance mechanism in klebsiella pneumoniae in greece. blakpc-2 gene's possible clonal spread imposes the urgent need of implication of infection control practices in the affected hospitals. i. galani, m. souli, e. papadomichelakis, f. panayea, n. mitchell, a. antoniadou, g. poulakou, f. kontopidou, h. giamarellou°(athens, gr) background: until now, carbapenem resistance among klebsiella pneumoniae (kp) clinical isolates in greek hospitals has been attributed to the dissemination of vim-1 metallo-beta-lactamase. we describe the first outbreak of kpc-producing kp in greece; the first to occur outside the usa or israel. setting: 21-bed icu of attikon university hospital, athens. methods: kp isolates with an imipenem mic > 1 mg/l and a negative edta-imipenem disk synergy test were submitted to boronic acid disk test, to pcr for a kpc gene with specific primers and sequencing. records from patients colonised or infected with a kpc-producing kp were retrospectively reviewed for clinical and epidemiological data. environmental cultures for kpc-producers were performed. clinical isolates were submitted to molecular typing using pfge. results: from february to november 2008, 552 kp were isolated from 95 patients, 132 (23.9%) of which were boronic acid positive and produced kpc-2. most of them (126/132, 95.5%) were isolated since august. a total of 24 patients were identified as colonised or infected by a kpc producer which in 22 of them belonged to the same genetic clone. the source was faeces (73), bronchial secretions (26), blood (7), cvc tip (5), urine (15), pus (4) and throat (2). among patients whose medical records were available, median age was 74, apache ii score; 21, length of preceding hospital stay; 28 days, total length of stay; 50 days, immunosuppresion was identified in one and crude mortality was 71%. the kpc-producing kp was more frequently icu acquired whereas in a minority of patients it was already present on icu admission. seventy percent of the patients had previously received a carbapenem for a median of 15 days. environmental colonisation was not identified. ten (7.6%) of the kpcproducers from 8 (33.3%) patients were identified as the cause of an infection: bacteraemia (7), ventilator-associated pneumonia (2) and surgical site infection (1) and exhibited mic90 (mg/l) for imipenem, >8; meropenem, >8; gentamicin, 4; ciprofloxacin, >2; fosfomycin, >128; colistin, 0.5 and tigecycline, 4. most patients were successfully treated with a colistin-containing combination mostly with a beta-lactam. there was no attributed mortality. isolates from the same bacterial species were typed by pfge or automated ribotyping. kpc-encoding gene was fully sequenced. plasmid preparations and i-ceu digestion of total dna were resolved in agarose gels, blotted and hybridised with a blakpc probe. the blakpc-carrying element (tn4401) was amplified with various primer pairs, digested with eag i and sequenced. results: 30 strains each carried kpc-2 and kpc-3. one e. cloacae carried kpc-4. 13 k. oxytoca were kpc-2-producers and 2 s. marcescens harboured blakpc-3, all from usa. great genetic diversity was observed among the isolates (41 different types). one clone of 10 e. cloacae was detected in new york state (2006) (2007) . small clusters of 2 and 3 strains were detected among e. coli, e. cloacae, k. oxytoca. plasmids were present in all but 3 isolates. persistence of clones throughout the years was not observed. in 35 isolates the kpc-encoding gene was located in high molecular weight plasmids (>54 kb). blakpc was located in the chromosome of 11 strains (e. cloacae, e. coli and k. oxytoca) and the location of this gene could not be determined in 15 strains. small plasmids were present in several strains, but did not harbour blakpc. tn4401 carried blakpc in 46 isolates, and the transposon element was conserved. this structure was not detected in 12 strains. conclusions: kpc-encoding genes were most often located in tn4401 among several enterobacteriaceae species collected in usa and israel. this blakpc-carrying element was located in plasmids and on the chromosome. this study highlights the importance of tn4401 in the dissemination of blakpc genes in several genetically diverse bacterial species. blakpc was not associated with tn4401 in only 12 of 61 strains. these strains are under further investigation. objective: to evaluate the carbapenem resistance mechanism in a raoultella planticola bacteraemia isolate recovered from a patient hospitalised in ohio, usa. methods: species identification was performed by vitek 2 and confirmed by 16s rrna sequencing. susceptibility testing used clsi broth microdilution method. blakpc was amplified and sequenced. the blakpc genetic element (tn4401) was amplified and sequenced. plasmid extractions and conjugation experiments were carried out and the isolate was screened for esbl-encoding genes, qnr and qepa. a 83 year old female patient was admitted to a hospital located in akron with a diagnosis of cap in may/2008. sputum, paracentesis and blood cultures were negative. urine culture grew e. coli and patient received courses of moxifloxacin, ceftriaxone, azithromycin and meropenem. the patient was discharged and returned after three weeks with respiratory problems. tracheal aspirate grew a multidrug resistant a. baumannii and the blood culture grew the enteric-like gramnegative bacillus. the isolate was identified as r. planticola by the vitek 2, which was confirmed by 16s sequencing. r. planticola strain demonstrated resistance against most b-lactams, including carbapenems. screening for kpc-encoding genes was positive and this strain carried blakpc-2. fluoroquinolone and aminoglycoside mic values were elevated. kpc-2-encoding gene was located in tn4401, but conjugation experiments failed. esbl and qnr/qepa genes were not detected. conclusions: kpc serine-carbapenemases have been detected in several gram-negative species commonly isolated from clinical specimens. kpc genes are embedded in transposon-like structure usually harboured in conjugative plasmids carrying multiple antimicrobial resistance mechanisms. this is the first report of kpc-producing r. planticola that is an environmental organism related to klebsiella spp. the similarity between these organisms could facilitate the transfer of genetic material. kpc-producing isolates appear to be prevalent among different enterobacteriaceae species in usa hospitals and was detected in an isolates of apparent environmental origin. objectives: it is long known that not all individuals with a specific disease present with the same clinical manifestations, nor do they have identical prognoses or responses to treatments. it has become clear that variations in the human genome are likely to have an impact on these aspects. tank-binding kinase 1 (tbk1) is a central molecule in the induction of a.o. the type i interferon response to pathogens. our goals for this study were 1) to investigate the frequency of single nucleotide polymorphisms (snps) in the promoter and coding region of tbk1 in a dutch caucasian population and 2) to search for potential associations between these snps and bloodstream infections. methods: whole blood samples or samples of positive blood cultures were collected and after genomic dna was isolated, pcr and sequencing were performed for snp identification. functional studies included promoter activity measurements using a luciferase assay as well as electrophoretic mobility shift assays (emsa) to study binding of the transcription factor usf1 to the wt and mutant promoter. snp incidences were studied in a case control study. results: in samples from dutch caucasian healthy volunteers, 4 snps were found with allele frequencies higher than 5% whereas 6 other known snps had frequencies lower than 5% in our cohort. two snps (rs89208169 and rs89208163) located in the promoter region were studied in a larger cohort of 350 anonymised patients from the maastricht university medical center with either gram-positive or gram-negative blood cultures. we found that the prevalence of rs89208169 was significantly increased in patients with positive blood cultures in comparison with those with negative blood cultures or healthy volunteers. further investigation of this snp showed that it is located just outside a usf1-binding site. measuring the promoter activity using luciferase assays, the mutant promoter exhibited a decreased activity of <35%. this observation was confirmed by emsa which showed that recombinant usf1 protein had a reduced binding affinity to the mutant promoter. conclusions: snp rs89208169 in the promoter region of tbk1 has a significant association with gram-positive infections. our results demonstrate that this is likely due to a decreased expression of tbk1 due to reduced binding of the transcription factor usf1 to the mutant promoter. our results support recent findings that tbk1 plays also an important role in the host response to gram-positive infections. objective: lymphocyte apoptosis has been recognized as an important factor contributing to both the onset of sepsis post infection and to the progression into septic shock. animal data suggest that prevention of lymphocyte apoptosis by caspase inhibition stabilises the immune system, improves bacterial clearance and decreases mortality in experimental sepsis. the present study evaluated the potential of vx-166, a novel broad caspase inhibitor, as a therapy for sepsis. methods and results: initial characterisation of vx-166 in a number of enzymatic and cellular assays clearly demonstrated that the compound is a broad caspase inhibitor with potent anti-apoptotic activity in vitro. in vivo, vx-166 was tested in a murine model of endotoxic shock and a clinically relevant model of peritonitis. in the endotoxic shock model, male cd-1 mice (n = 28 per group) were administered lps (20 mg/kg iv) and survival was monitored for 96 h. vx-166 administered by repeat iv bolus (0, 4, 8 and 12 h post-lps) significantly improved survival in a dose-dependent fashion (p < 0.0001). in the rat peritonitis model, adult male sprague-dawley rats (n = 12 per group) underwent caecal ligation and puncture (clp) and survival was monitored over 10d. continuous administration of vx-166 by mini-osmotic pump (0.9 mg/kg/h) immediately following surgery significantly improved survival (p < 0.01) from 38% in the control group to 88% in the compound-treated group. mode of action studies in the rat clp model confirmed that vx-166 reduced thymic atrophy and lymphocyte apoptosis (p < 0.01), supporting the anti-apoptotic activity of the compound in vivo. in addition, vx-166 reduced plasma endotoxin levels (p < 0.05), strongly suggesting an improved clearance of bacteria from the bloodstream. most importantly, we demonstrated that vx-166 fully retained its efficacy when dosed 3 hours after insult (p < 0.01) by improving survival to 92% versus 42% in control animals, further highlighting the potential of anti-apoptotic therapy in sepsis. overall these data demonstrate that vx-166 inhibits lymphocyte apoptosis, improves the clearance of bacterial endotoxin and improves survival in experimental sepsis. importantly vx-166 improves survival in the clp model when dosed post insult, and therefore represents significant progress in the development of therapeutically viable broad caspase inhibitors for the treatment of this disease. v. vankerckhoven°, s. van voorden, n. hens, h. goossens, g. molenberghs, e. wiertz (wilrijk, be; leiden, nl; hasselt, be) objectives: toll-like receptors function as key regulators of both innate and adaptive immunity. lactobacilli modulate the immune system in different ways. the aim of this study was to examine toll-like receptor (tlr2, tlr2/6 and tlr4) signalling induced by clinical and probiotic lactobacillus strains. methods: a total of 45 lactobacillus strains (19 l. paracasei and 26 l. rhamnosus) of different origin (22 probiotic, 2 faecal, and 21 clinical) were tested for tlr2, tlr2 in combination with tlr6, and tlr4. tlr signalling was measured as relative il-8 promotor activation in transfected human embryonic kidney (hek) 293 cells. il-8 concentrations were measured using an enzyme-linked immunosorbent assay. heat-killed listeria monocytogenes (hklm) was used as positive control in all assays, whereas pam3, pam2, and lps were used as positive controls for, respectively, tlr2, tlr2/6, and tlr4. all assays were performed at least in duplicate. linear mixed model analyses and stepwise model selection were used to identify the statistically significant effects. random effects were used to account for heterogeneity across and homogeneity within isolates. p < 0.05 was considered statistically significant. results: hek-tlr2 and hek-tlr2/6, but not hek-tlr4, cells released il-8 upon stimulation with uv-inactivated lactobacilli, which was enhanced by co-transfection with cd-14. interestingly, the production of il-8 was shown to be variable for the different lactobacillus isolates. although similar results were seen for all isolates for tlr2 and tlr2/6, il-8 production was significantly higher for tlr2 (8.4 log pg/ml) compared to tlr2/6 (6.05 log pg/ml) (p < 0.0001). no significant differences in il-8 production were seen between clinical and probiotic isolates. however, l. rhamnosus isolates induced a significantly higher il-8 production compared to l. paracasei isolates in both cell lines, 7.88 and 6.84 log pg/ml, respectively (p = 0.0004). intra-isolate correlation was found significant (p < 0.0022). conclusions: our study shows that lactobacilli activate both tlr2 and tlr2 in combination with tlr6. our results also indicate that heterodimerisation of tlr2 with tlr6 does not lead to an improved recognition of lactobacilli. furthermore, taking intra-isolate correlation into consideration proved to be important. finally, our results suggest that differences in immunomodulation by lactobacilli may be related to differential signalling through tlrs, including tlr2 and tlr2/6. m.c. gagliardi, v. sargentini, r. teloni, m.e. remoli, g. federico, m. videtta, g. de libero, e. coccia, r. nisini°(rome, it; basel, ch) objective: to gain insights into the mechanisms used by mycobacterium tuberculosis and bacillus calmette guérin to cause human monocytes differentiation into cd1 negative dendritic cells (my-modc), unable to present lipid antigens to specific t cells. methods: human monocytes infected or not with mycobacteria were induced to differentiate into dc with gm-csf and il-4 in the presence or absence of p38 or erk specific inhibitors. kinases activation was detected by western blot using antibodies specific for phosphorylated and non phosphorylated isoforms. differentiation of monocytes into dc and the cd1a, cd1b and cd1c expression was evaluated by flow cytometry and by real time pcr at different time points from infection. functional expression of cd1 molecules was assessed by recognition of lipid antigens by cd1 restricted t cell clones. results: we show that mycobacteria trigger phosphorylation of erk and p38 mitogen-activated protein kinase in human monocytes as well as of activating transcription factor (atf)-2. mycobacteria-infected monocytes treated with a specific p38 inhibitor, but not with a specific erk inhibitor become insensitive to mycobacterial subversion and differentiate into cd1 positive my-modc, which are fully capable of presenting lipid antigens. data indicate that phosphorylation of p38 is directly involved in cd1 inhibition. conclusions: we propose p38 signaling as a pathway exploited by mycobacteria to affect cd1 expression, thus representing a novel target of possible pharmacological intervention in the treatment of mycobacterial infections. s. ebert°, s. ribes, r. nau, u. michel (gottingen, de) objective: activin a (act a) is a multifunctional cytokine with roles in the immune system and the inflammatory response. act a levels are elevated in the cerebrospinal fluid of patients with meningitis. microglial cells, the major constituents of innate immunity within the brain, express toll-like receptors (tlrs) recognising exogenous and endogenous ligands. upon stimulation with tlr agonists, primary mouse microglial cells become activated and release nitric oxide (no), cytokines, and also act a, suggesting that they are a source of elevated conclusions: pre-treatment with act a enhances no release from microglial cells activated by agonists of the principal tlrs involved in the recognition of bacteria. these findings provide further evidence for a role of act a in the innate immune response and suggest that act a acts as an pro-inflammatory modulator during infection and inflammatory processes in the cns. insertion sequences (is) are genetic tools that can mediate expression of previously silent genes or be responsible for the overexpression of certain genes (in each case by providing promoter sequences). in addition to be involved in gene transcription levels, is elements also play a very important role for gene acquisition/mobilisation. an is is usually made of of two inverted-repeat sequences (irs) bracketing a gene encoding the transposase which activity enables this entity to replicate and target another sequence. the is-related mechanisms at the origin of antibiotic resistance gene acquisition are diverse, including composite-transposition, rolling-circle transposition, one-ended transposition. is elements may be also involved in gene acquisition by mediating co-integration processes, or recombination events as hypothetized for is26 in relation with blashv extendedspectrum b-lactamase (esbl) genes originating from the chromosome of klebsiella pneumoniae. the blactx-m esbl genes known to be extremely widespread worldwide are encoded on plasmids, and have been found in association with isecp1 (acting by one-ended transposition) or iscr1 (acting by rolling-circle transposition). in that case, iss have played a role in the mobilisation from the chromosome of kluyvera spp. being the blactx-m progenitors and then in their expression. also, genes encoding acquired ampc b-lactamases, being of the blaacc, bladha, and blacmy-types, are mostly found in association with iscr1 or isecp1. sometimes antibiotic resistance genes are mobilised by composite transposons which are made of two copies of a given is bracketing the mobilised fragment. in acinetobacter baumannii, the worldwide disseminated blaoxa-23 carbapenemase gene is part of a composite transposon structure made of two copies of isaba1, forming transposon tn2006 which had mobilised a chromosomal fragment from acinetobacter radioresistens that actually corresponds to the progenitor of blaoxa-23. another possibility can be the forming of composite transposon structure bracketed by two different is (sharing similar irs) as observed with the blaper-1 esbl gene in pseudomonas aeruginosa. this diversity of iss elements at the origin of mobilisation/acquisition of antibiotic resistance genes is therefore responsible for the very efficient dissemination of many of them. s362 resistance islands − their role in the accumulation and spread of antimicrobial resistance genes historically, multi-antibiotic resistance in many bacterial species was largely attributed to the acquisition of resistance (r)-plasmids encoding one or more resistance determinants. however, over the last decade the r-plasmid paradigm has begun to be challenged. 'resistance islands' comprising large, chromosomally-integrated spans of alien dna harbouring multiple antibiotic resistance genes have been identified in the major hospital pathogens methicillin-resistant staphylococcus aureus (mrsa) and multi-resistant acinetobacter baumannii, and the foodand water-borne diarrhoeal pathogens shigella, salmonella and vibrio cholerae. in addition, comparative genomics analysis of the archetypal haemophilus influenzae conjugative resistance element that had spread worldwide revealed that it belonged to a large syntenic family of integrative islands, members of which could be found in at least 15 other b-and g-proteobacteria. with the exception of the a. baumannii island, these elements can be described as classic self-excising, -circularising and -integrative elements. all three functions are mediated by short island-flanking direct repeats and cognate integrase proteins encoded by the islands. in 2006 fournier et al. described an 86 kb a. baumannii island (abar1) which harboured 45 resistance genes packaged within a highly mosaic, integron-rich element that had almost certainly evolved via recombination, transposition and integron-mediated cassette capture from an 'empty' ancestral prototype. abar1 probably represents a new class of resistance island as it exhibits several features reminiscent of complex nested transposons, suggesting a distinct functional natute. however, despite the widespread distribution of resistance and genomic islands only a minority are known to code for part or all of the conjugative machinery necessary for their dissemination; others have been mobilised by helper plasmids or bacteriophages. regardless, data on the mechanisms of mobilisation of the vast majority of similar nonresistance islands remain sparse. importantly, resistance islands may not consists merely of packages of resistance genes. on the contrary, these diverse and frequently hybrid entities could potentially confer upon their hosts other advantageous traits relating to host-pathogen interaction, virulence, survival in the environment and/or transmissibility, truly justifying the label 'selfish islands' and further explaining their evolutionary success. due to the availability of new techniques, genome sequencing of bacteria has become fast and inexpensive. furthermore, recent methods using paired-end reads located several kb apart in the genome eases the assembling process, even though no reference sequence is available. in a reasonably close future, it should be possible to obtain the fully assembled sequence of a bacterial isolate overnight. the new sequencing techniques generate enormous amounts of genomic data and, thereby, a need for new tools. these should able to quickly analyze genomes and point to zones of interest, prompting further analysis on a reduced number of regions or genes, such as genomic islands. pathogenicity islands, a subset of genomic islands, carry genes such as toxins or resistance genes and have the particularity to be mobile, i.e. they may transfer to other species or strains. thereby, they confer their new hosts a more resistant or infectious phenotype, making this phenomenon particularly important to study. nucleotide composition of genomes is fairly homogeneous inside bacterial genomes. in general, horizontally transferred regions can be spotted due to their particular nucleotide content, because they tend to retain the composition of their original host and don't share the one of their new hosts. to do an analogy with languages, genomes speak dialects, and as one would easily spot a paragraph in finnish in an english text while not knowing finnish, one can spot genomic and pathogenicity islands transfers in a given genome. several techniques relying on various compositional aspects and on different algorithmic methods have been recently developed to detect pathogenicity islands in bacterial genomes. even very simple measures of the genome composition, such as the variation in t vs. a bias (ta skew) can lead to the identification of all known prophages in streptococcus pyogenes. it can even trigger the discovery of a putative ancient genomic island carrying a large number of genes related to pathogenicity in all strains of that species. in conclusion, with the rise of fast and inexpensive genome sequencing, new quick and simple methods are being developed. they take the advantage of the homogeneous nucleotide composition of bacterial genomes to uncover mobile genetic elements carrying genes involved in pathogenicity. in the past 10 years, significant progress has been achieved in the management of chronic hepatitis b with the successive development of six potent antiviral medications (lamivudine, adefovir dipivoxil, pegylated interferon alpha, entecavir, telbivudine and tenofovir). however, the clinical results of antiviral therapy have been limited by the emergence of antiviral drug resistance especially with the first generation of nucleoside analogs (lamivudine, adefovir and telbivudine). furthermore, the unique mechanism of viral genome replication and persistence within infected cells is responsible for viral persistence even after prolonged therapy with the newer antivirals (entecavir and tenofovir). this is the major reason why life-long treatment is envisaged in the majority of patients, which may expose them to long-term risk of developing resistance. the use of in vitro phenotypic assays has been crucial for the characterisation of newly identified resistant mutants and determine their cross-resistance profile. results allowed to understand the different mechanism of viral resistance to lamivudine and adefovir, the mechanism of primary failure to adefovir therapy, the unique mechanism of entecavir resistance, and to characterise the emergence of multi-drug-resistant strains in patients receiving sequential antiviral therapy. the crossresistance profile for the main resistant mutants was determined which allowed to provide recommendation to clinicians for treatment adaptation based on molecular virology data. the understanding of the development of hbv drug resistance has allowed to significantly improve the management of antiviral resistance and to design better treatment strategies to prevent resistance. the current standard of care relies on treatment initiation with antivirals combining a strong antiviral potency and a high barrier to resistance. a precise virologic monitoring is required to measure antiviral efficacy, and to diagnose partial response or viral breathrough at an early stage. this allows to adapt antiviral treatment preferrably using an add-on strategy with a drug having a complementary cross-resistance profile. this strategy has been shown to be efficient in controling viral replication and preventing liver disease progression in the majority of patients. treatment of chronic hepatitis b virus (hbv) infection is aimed at suppressing viral replication to the lowest possible level. in many prospective clinical trials it has been shown that a sustained hbv dna response was correlated with serologic, histologic, or biochemical responses. despite the recent progress in hepatitis b antiviral treatment, it is shown that antiviral drug resistance is inevitable against many of the nucleoside analogs. the emergence of antiviral-resistant strains of hbv leads to viral and subsequently biochemical breakthrough and may lead to disease progression and increased death. most of the data on the clinical impact of antiviral resistant hbv came from the data derived from studies of lamivudine therapy. there is limited data on other hbv antiviral drugs like adefovir. it is shown in several studies that treatment of hbeag-negative chronic hepatitis b with lamivudine effectively suppresses hbv replication and results in biochemical remission and histologic improvement in more than two thirds of patients. however, relapse has occurred in the majority of hbeag-negative patients after the cessation of therapy. there are several studies to support the occurrence of severe hepatic flares, and liver failure after the emergence of lamivudine resistance. several studies, where liver biopsies were taken, demonstrated that histological improvement was reduced in those patients experiencing lamivudine resistance. the clinical outcome for patients with antiviral resistance is related to their age, the severity of the underlying liver disease and the severity of the hepatic flares. on the other hand in a different study it was found that long-term lamuvidine treatment was associated with a reduced chance of developing cirrhosis and hcc in patients without advanced disease but, although resistant mutants reduced the benefits from lamivudine therapy, the outcome of these patients was still better than untreated patients. results of several clinical trials have shown that the addition or substitution of newer antiviral agents can restore suppression of viral replication, normalisation of liver function and reverse histological progression in patients with antiviral resistance. consequently, well-tolerated, potent therapies that offer a strong genetic barrier against the development of resistance are desirable, since antiviral resistance and poor adherence are key risk factors for treatment failure and subsequent reversal of clinical improvement. resistance of enteric fever-causing and non-typhoid salmonella serovars to agents traditionally used to treat these infections in the past shows extensive geographical variation. decreased susceptibility to ciprofloxacin is rapidly increasing all over the world with target alteration and increased efflux being the most important mechanisms behind. infections with such strains often result in extended hospitalisation or even in therapeutic failures. furthermore, it is likely that moderately increased mic values facilitate the development of strains with higher level of resistance, i.e. a pattern described at various locations. screening methods based on quinolone sensitivity testing may fail to indentify decreased fluoroquinolone susceptibility both in typhoid, as well as in non-typhoid salmonella. plasmid mediated quinolone resistance genes are detected increasingly all around the world although neither the frequency nor the variety of genes identified has approached that seen in some other members of enterobacteriaceae. treatment with gatifloxacin or azithromycin are alternative options for invasive and systemic infections caused by strains with decreased susceptibility to ciprofloxacin. at some parts of the world resistance to extended spectrum cephalosporins reached such incidence that may have therapeutic implications particularly when initial, empiric treatment of invasive infections is concerned. resistance is due to plasmid coded ampc type beta lactamases (particularly to cmy-2), and most often to esbls of which usually some of ctx-m types are the frequently encountered ones. carbapenem resistance is still rare, albeit does occur, among salmonella isolates. the recent description of a non-typhoid salmonella strain with the blaimp-4 gene co-located on a class-1 integron with several other resistance determinants on a conjugative plasmid is of particular concern. campylobacters exhibit natural resistance to a variety of antimicrobials. the drugs of choice used to be fluoroqunolones or macrolides. however, the current incidence of ciprofloxacin resistance made the former drugs already obsolete or seriously limited their use at several parts of the world. with the exception of few locations the incidence of macrolide resistance is still relatively low and is seen more frequently in c. coli than in c. jejuni. however, strains exhibiting resistance against both groups of drugs have been emerging, particularly in south-east asia. neisseria meningitidis, the meningococcus, is a major cause of meningitis and septicaemia worldwide while neisseria gonorrhoeae, the gonococcus, is responsible for one of the most widespread sexually trasmitted disease. the behaviour of these two species towards antibiotics is very different: resistance in n. gonorrhoeae is now widespread occuring as both chromosomally and plasmid mediated to a variety of drugs, whereas, besides resistance to sulphonamides, n. meningitidis remains largely susceptible to antibiotics used both for therapy and prophylaxis. however, as in the gonococcus, the resistance to antibiotics of n. meningitidis is also evolving, as documented by the ever higher frequency of strains with intermediate resistance to penicillin in many countries. transformation has apparently provided both species with a mechanism by which they can increase resistance to penicillin by replacing part of their pena gene, which encodes pbp2, with part of the pena gene of related species that fortuitously produces forms of pbp2 less susceptible to the antibiotic. n. meningitidis is still at this step, whereas n. gonorrhoeae has acquired also mutation in the pona gene that encodes pbp1, mutation in porin ib, increased expression of efflux pump and the tem-1 b-lactamase plasmid. the emergence and the spread of gonococci fully resistant to penicillin since the second half of the 1980 s years led to the recommended use of fluoroquinolones as primary therapy. however, this class of antibiotics became rapidly unefficacious, mainly in asia, due to the emergence of mutations in gyra and parc which are able to block the activity of the quinolones on gyrase and topoisomerase iv. since 2006, cdc no longer recommends their use for treatment of gonococcal diseases. fortunately, the occurrence of quinolone resistant meningococci, due to mutations in gyra, is still rare but even if cases are still few they are of great concern for the epidemic potential of this pathogen and the required prophylaxis of contacts. also for the other antibiotic, frequently used to this aim, rifampicin, some meningococci have showed to be resistant, again for the presence of mutations, in this case in the rpob gene coding for the b-subunit of the meningococcal rna polymerase. the molecular epidemiological identification of clonal clusters for both neisseria species with distinct resistance profiles is required to monitor ongoing trends that may pose problems both in therapy and prophylaxis. l. brookes-howell°, c. butler, k. hood, l. cooper, h. goossens (cardiff, uk; antwerp, be) introduction: grace is a european network of excellence established to focus on antibiotic use for community-acquired lower respiratory tract infection (lrti) and antimicrobial resistance across europe. grace-02, the second study to begin within grace, is a large qualitative study that explores the attitudes of clinicians and patients to antibiotic use for lrti and antibiotic resistance. aims: this presentation will focus on clinicians' accounts of the factors that contribute to variation in management of lrti and patient views on when antibiotics are necessary. methods: semi-structured interviews with 81 clinicians and 121 patients were conducted in primary care networks in nine european countries. interviews were audio-recorded, transcribed and, where necessary, translated into english for analysis. themes were identified, organised and compared using a framework approach. results: analysis of clinician interviews shows that, beside clinical findings, factors which influence the management decision for patients can be divided into two main areas. firstly, within each european network there is a group of country specific factors imposed by the system in which consultations take place. these factors include: near patient test usage, self-medication, patients' finances and lack of consistent, local prescribing guidelines. secondly, there is a group of factors, similar across all networks, that relate to personal characteristics of certain groups of clinicians. these include clinicians' professional ethos, self-belief in decision making and attitude towards the doctorpatient relationship. analysis of patient interviews shows that beliefs about antibiotic use tend to draw on clinical factors, namely the severity of specific symptoms (fever and/or coughing). many patients also implied a period of waiting or alternative action required before antibiotics are used − to identify whether the immune system would fight the infection or whether nonantibiotic management was effective before turning to antibiotics. discussion and conclusion: with a greater understanding of the factors that contribute to the decision to prescribe, we discuss ideas to enhance appropriate prescribing. this analysis highlights the need for interventions to be sensitive to factors relating to the systems in which different european networks operate, to target the individual characteristics of specific groups of clinicians and to build on the clinical beliefs already held by patients. o377 pre-treatment with low-dose endotoxin prolongs survival from experimental lethal endotoxic shock k. kopanakis, i. tzepi, e.j. giamarellos-bourboulis°, a. macheras (athens, gr) objective: clinical trials of immunointervention with anti-endotoxin antibodies in patients with severe sepsis have failed to disclose survival benefit. these failures led us to the assumption that the opposite approach with a low endotoxin stimulus may result to low level immunoaralysis and subsequent survival benefit. this approach was tested in an experimental setting. methods: a total of 36 male c57b6 mice were studied divided into two groups: group a stimulated with the ip injection of sodium saline followed after one day by the ip injection of 30 mg/kg of lipopolysaccharide (lps) of escherichia coli o155:h5; and group b stimulated with the ip injection of 3 mg/kg of lps of the same isolate followed after one day by the ip injection of 30 mg/kg lps. lps was diluted in sodium saline and the volume of each injection was 0.2 ml. survival was recorded at six hour time intervals. results: survival of group b was considerable prolonged compared with group a (log-rank: 5.435, p: 0.020) as shown in figure 1 . thirteen mice of group a died (72.2%) compared with seven mice of group b (38.9%, p: 0.044 between group). conclusions: administration of low doses of lps prolongs survival after lethal endotoxic shock. this approach opens a promising novel pathway for immunointervention in sepsis. fragilis isolates with an mxf mic of 2 mg/ml (n = 5), 4 mg/ml (n = 20) and 8 mg/ml (n = 8), which were virulent in the mgp model, were used to determine the efficacy of mxf. for the mgp model, pouches were created by injecting 5 ml of air and 0.5 ml of 0.1% croton oil in olive oil under the skin of the back. on day 3, the air was withdrawn and replaced by 1 ml soft agar. on day 5, a bacterial suspension was injected into the pouch. infected mice (n = 6 mice/group) were treated with mxf 100 mg/kg iv, b.i.d. for 2 days. this dose simulates the auc of the human 400 mg once-daily mxf iv dosage. efficacy was assessed by the reduction in colony forming units (cfus) in pouch exudates 48 hours post-infection compared with the untreated infection control. results: in the mgp model, mxf, 100 mg/kg b.i.d., displayed good efficacy in term of cfu reduction against all used strains in this study. there were no non-responders in terms of cfu reductions. conclusion: the loss of atle had no impact on the mics of cloxacillin and vancomycin. conversely, the mutant atle(−) strain was less susceptible to bactericidal activity of both antibiotics, supporting the implication of atle in the tolerance of s. epidermidis to cell wall active antibiotics. the loss of atle did not alter the virulence of s. epidermidis in the mouse peritonitis model, whereas it decreased virulence in previously published experiments using an intravenous catheter infection model. therefore, the mouse peritonitis model was suited to compare antibiotics efficacy against atle(+) and atle(−) strains. our results showed that the loss of atle did not alter significantly the activity of cloxacillin and vancomycin in the mouse peritonitis model. this study shows that the loss of atle results in decreased susceptibility to bactericidal activity of cell wall active antibiotics, with no apparent impact on the activity of these antibiotics in the mouse peritonitis model. in infant rat pneumococcal meningitis, ceftriaxone plus daptomycin versus ceftriaxone attenuates brain damage and hearing loss while ceftriaxone plus rifampicin versus ceftriaxone does not d. grandgirard, m. burri, k. oberson, a. bühlmann, f. simon, s.l. leib°(berne, ch) objectives: lytic antibiotics for therapy of bacterial meningitis (bm) increase the release of pro-inflammatory bacterial compounds which, in turns, induce inflammation. exacerbation of the inflammatory response in cerebrospinal fluid (csf) contributes to the development of neurological sequelae in survivors of bm. daptomycin, a nonlytic antibiotic acting on gram-positive bacteria has been shown to decrease inflammation and brain injury vs. ceftriaxone in experimental pneumococcal meningitis. with a view on the clinical application for empiric therapy of paediatric bacterial meningitis we investigated, whether therapies combining daptomycin or rifampicin with ceftriaxone are beneficial when compared to ceftriaxone monotherapy in infant rat pneumococcal meningitis. methods: eleven day old wistar rats were infected by intracisternal injection of s. pneumoniae and animals were treated with daptomycin (10 mg/kg, s.c., daily) plus ceftriaxone (100 mg/kg, s.c., bid), rifampicin (20 mg/kg, i.p., bid) plus ceftriaxone or ceftriaxone alone. csf was sampled at 6 h and 22 h after the initiation of therapy and assessed for concentrations of chemo-and cytokines (mcp-1, mip-1a, il-1b, il-6, il-10; il-18 and tnf-a). a subset of animals was sacrificed 40 h post infection (h pi) and brain damage quantified by histomorphometry. the remaining animals were treated for 3 d and were tested for hearing loss, by assessing the auditory brainstem response (abr) at 3 weeks after infection. results: compared to ceftriaxone alone, daptomycin plus ceftriaxone significantly (p < 0.04) lowered csf concentrations of mcp-1, mip-1alpha and il-6 at 6 h and mip-1a and il-1b at 22 h after initiation of therapy, led to significantly (p < 0.01) less apoptosis assessed at 40 h pi, and significantly (p < 0.01) improved hearing capacity. while rifampicin plus ceftriaxone also led to lower csf inflammation (p < 0.02 for il-6 at 6 h), apoptosis and hearing loss were not significantly different from the ceftriaxone group. conclusion: compared to ceftriaxone monotherapy, daptomycin plus ceftriaxone lowers the level of pro-inflammatory mediators in the csf and reduces hippocampal apoptosis and hearing loss in infant rat pneumococcal meningitis. d. croisier-bertin°, l. piroth, p.e. charles, d. biek, y. ge, p. chavanet (dijon, fr; alameda, us) objectives: ceftaroline (cpt) is a novel, parenteral, broad-spectrum cephalosporin exhibiting bactericidal activity against gram-positive organisms, including methicillin-resistant s. aureus (mrsa) and multidrug-resistant s. pneumoniae, as well as common gram-negative pathogens. the efficacy of simulated human dosing with cpt or ceftriaxone (cro) was evaluated in a rabbit model of penicillin-resistant pneumococcal pneumonia. methods: 3 s. pneumoniae strains were used to induce pneumonia in rabbits: pssp, pisp, and prsp. mics (mg/l) were 0.06/0.015, 1/0.125, and 4/0.25 for cro and cpt, respectively. the animals were randomised to no treatment (controls), intravenous (iv) cpt human equivalent (he) dosage (600 mg/12 h), iv cro he dosage (1 g/24 h), or intramuscular (im) cpt (5 or 20 mg/kg) for prsp-infected rabbits. serum levels were measured by microbiological assay and pk data were obtained. evaluation of efficacy was based on bacterial counts in lungs and spleen (per gram tissue). results: 5−7 animals/group were tested. for iv cpt/iv cro, mean auc0−24 was 155/938 mg.h/l, cmax was 20/158 mg/l and cmin was 1.3/6 mg/l, respectively. bacterial counts in target tissues are listed in the iv cpt and iv cro were highly efficacious against pssp and pisp. iv and im cpt were superior to iv cro against prsp with a quasi sterilisation of lungs and spleen. combined results from the iv and im studies indicated that %t > mic for cpt of 30% and 45% were associated with 50% and 100% bacterial count reductions, respectively. in this rabbit model of penicillin-resistant pneumococcal pneumonia, cpt administered iv (with he dosing) or by im administration was more effective against prsp than iv cro. r. endermann°, d. hoepker, k. merfort, m. glenschek-sieberth (wuppertal, de) objective: moxifloxacin (mxf) is approved in the usa and other countries for the treatment of complicated intra-abdominal infections (ciais). we compared the efficacy of mxf with piperacillin/tazobactam (pip/taz), a commonly used treatment for ciais, in three different models: ( . c. clp model: survival over 10 days was significantly higher in the mxf group than in the pip/taz group (p < 0.0001). conclusions: using humanised dosages, mxf had greater antimicrobial activity and provided higher survival rates that pip/taz in three different models for ciai. m. nairz, i. theurl, a. schroll, m. theurl, s. mair, t. sonnweber, g. fritsche, r. bellmann-weiler, g. weiss°(innsbruck, at) mutations in hfe predispose to hereditary haemochromatosis type i, a frequent genetic disorder characterised by progressive parenchymal iron deposition and eventual organ failure. since hfe mutations are associated with reduced iron levels within mononuclear phagocytes, we hypothesized that hfe deficiency may be beneficial in infections with intramacrophage pathogens. using hfe+/+, hfe+/− and hfe−/− mice in a model of typhoid fever, we found that animals lacking one or both hfe alleles are protected from systemic infection with salmonella typhimurium, displaying prolonged survival and improved bacterial control. this increased resistance can be referred to an enhanced production of the siderophore-binding peptide lipocalin 2 and the reduced availability of iron for salmonella engulfed by hfe deficient macrophages. this effect is mediated via stimulation of lipocalin 2-dependent iron export from infected cells since hfe−/− macrophages concurrently knocked out for lipocalin 2 are unable to efficiently control the infection or to withhold iron from intracellular salmonella. correspondingly, infection of hfe+/+ and hfe−/− mice with siderophore deficient salmonella abolishes the protection conferred by the hfe defect. thus, by inducing the formation of the iron-capturing peptide lipocalin 2, the hfe mutation harbours a genetically determined immunological advantage towards infections with intracellular pathogens such as salmonella. i. koutelidakis, a. kotsaki, p.d. carrer, k. louis, a. savva, e.j. giamarellos-bourboulis°(thessaloniki, athens, gr) objective: the majority of clinical trials of immunointervention in severe sepsis have failed to disclose survival benefit. a likely explanation may be administration of therapy when immunoparalysis of the septic host supervenes. in an attempt to reverse immunoparalysis, injection of mononuclear cells was attempted in experimental sepsis by multidrugresistant pseudomonas aeruginosa (mdrpa). methods: peripheral blood mononuclear cells (pbmcs) diluted in rpmi were isolated from five healthy human volunteers after gradient centrifugation over ficoll. 1×10 7 /kg of one mdrpa live or heat-killed isolate from one patient with severe sepsis was injected intraperitoneally for bacterial challenge. a total of 72 male c57b6 mice were studied divided into four groups: group a (n = 26) pre-treated with rpmi and challenged after one hour with live isolate; group b (n = 26) pretreated with 5×10 7 pbmcs/kg and challenged after one hour with live isolate; group c (n = 10) pre-treated with rpmi and challenged after one hour with heat-killed isolate; group d (n = 10) pre-treated with 5×10 7 pbmcs/kg and challenged after one hour with heat-killed isolate. survival was recorded for 20 mice of groups a and b and for all mice of groups c and d. six mice of groups a and b were sacrificed six hours after challenge. blood was collected from the lower vena cava and tnfalpha and il-6 were estimated in serum by an enzyme immunoassay. bacterial growth of liver and lung at the same time was assessed. results: median survival of group a was 24 hours and of group b 88 hours (log-rank: 4.524, p: 0.033). nineteen animals of group a died (95%) compared with eight animals of group b (40%, p: 0.038). four animals of group c died (40%) compared with nil animals of group d (0%, log-rank: 4.274, p: 0.03). median serum tnf-a of groups a and b at sacrifice was 31 and 184 pg/ml respectively (p: 0.048). respective values for il-6 were 2084 and 2231 pg/ml (pns); for liver bacterial cells 3.63 and 4.99 log10 cfu/g (pns); and for lung bacterial cells 2.56 and 4.22 log10 cfu/g (pns). conclusions: allogeneic transplantation with pbmcs prolonged survival in experimental sepsis by mdrpa. its mechanism of action was related with a) blockade of cell wall structures as shown by survival experiments with heat killed isolate; and b) reversal of immunoparalysis as evidenced by increase of serum tnf-a. this approach creates a promising novel perspective for immunointervention in sepsis. a. marangoni°, c. nanni, m. donati, r. aldini, d. di pierro, s. trespidi, s. accardo, s. fanti, r. cevenini (bologna, it) objectives: chlamydia trachomatis is one of the world's major causes of sexually transmitted diseases of the cervix and urethra and it is a major agent of pelvic inflammatory disease. genital tract infection of female mice with chlamydia muridarum closely mimics acute genital tract infection in women. aim of this study was to assess the predictivity of 68ga-chloride small animal positron emission tomography ( o387 inadequate statistical power of published comparative cohort studies on ventilator-associated pneumonia to detect mortality differences between the compared groups m. falagas°, v. kouranos, a. michalopoulos, s. rodopoulou, a. athanasoulia, d. karageorgopoulos (athens, gr) objective: comparative cohort studies are often conducted to identify novel therapeutic strategies or prognostic factors for ventilator-associated pneumonia (vap). we aimed to evaluate the statistical power of such studies to provide statistically and clinically significant conclusions. methods: we searched in pubmed and scopus for comparative cohort studies evaluating the mortality of patients with vap. we calculated for each of the included studies the statistical power to detect the observed difference in mortality between the compared groups (observed power), as well as 3 expected, clinically relevant, effect sizes (expected power). we identified 39 (20 prospective) comparative cohort studies on vap as eligible for inclusion in this analysis. the median observed power of these studies was 17.9% [interquartile range (iqr), 9.8−52.4%]. the median expected power was 10.0% (iqr, 7.2−13.6%) for a risk ratio for mortality of 0.85 between the compared groups; 14.7% (iqr, 10.6−21.8%) for a risk ratio of 0.80; and 7.9% (iqr, 6.3−10.2%) for a reduction in mortality from 30% to 25%. all expected power measures were significantly lower than the observed power. the statistical power of most cohort studies to detect the observed difference in mortality between compared groups of patients with vap is low. the power is even lower when expected, clinically relevant, differences in mortality are considered. for a wiser utilisation of resources allocated to research, we favour the conduction of cohort studies with larger sample size so that potential differences between the compared groups are more likely to be shown. objective: to clarify issues regarding the frequency, prevention, outcome, and treatment of patients with ventilator-associated tracheobronchitis (vat), which is a lower respiratory tract infection involving the tracheobronchial tree, while sparing the lung parenchyma. methods: we performed a systematic review and meta-analysis of relevant available data, gathered though searches of pubmed, scopus, and reference lists, without time restrictions. a conservative random effects model was used to calculate pooled odds ratios (or) and 95% confidence intervals (ci). results: out of the 564 initially retrieved articles, 30 papers were included. frequency of vat was 10.2%. selective digestive decontamination was proved an effective preventive strategy against vat. presence, as opposed to the absence, of vat was not associated with higher mortality (or: 1.18, 95% ci 0.90−1.53). administration of systemic antimicrobials (with or without inhaled ones), as opposed to placebo or no treatment, in patients with vat was not associated with lower mortality (or: 0.56, 95% ci 0.27−1.14). most of the studies providing relevant data noted that administration of antimicrobial agents, as opposed to placebo or no treatment, in patients with vat was associated with more ventilator-free days and lower frequency of subsequent pneumonia, but without shorter length of intensive care unit stay or shorter duration of mechanical ventilation. conclusions: approximately one tenth of mechanically ventilated patients suffer from vat; an infection potentially prevented by the implementation of selective digestive decontamination. antimicrobial treatment of patients with vat may protect against the development of subsequent ventilator-associated pneumonia. degranulation. subsequently, allergen specific ige to chlorhexidine was demonstrated and skin prick/intradermal testing was positive to chlorhexidine, confirming the diagnosis of chlorhexidine-precipitated anaphylaxis in each patient. a detailed review of the case-notes revealed that each patient had manifest evidence of minor cutaneous reactions to pre-operative chlorhexidine use that had not been ascribed to chlorhexidine at the time. discussion: fda issued a public health notice [1998] following 1st description of anaphylaxis to chlorhexidine coated central venous catheter. a recent case cluster has also been reported from another cardiac centre in the uk [3-cases over a 9-month period]. references to be presented. it is interesting that these reports of chlorhexidine anaphylaxis have all occurred in patients undergoing cardiac surgery. these patients receive multiple exposures to chlorhexidine during their pre-operative investigations and preparation. this has increased recently as a result of the drive to reduce the incidence of hospital-acquired infections. we wish to postulate that these patients have been sensitised by repeated topical exposure to chlorhexidine and have exhibited anaphylaxis when this allergen was presented to the patient in the form of the chlorhexidine coated central venous catheter. type i strains of helicobacter pylori possess the cag pathogenicity island to deliver virulence factors. cag is a specialised type iv secretion machinery that is activated during infection and comprises 31 genes originated from a distant event of horizontal transfer. after translocation the effector protein caga is phosphorylated on tyrosine residues restricted to a previously identified repeated sequence called d1. this sequence is located in the c-terminal half of the protein and contains the five amino acid motif epiya, which is amplified by duplications in a large fraction of clinical isolates. tyrosine-phosphorylation of caga is essential for the activation process that leads to dramatic changes in the morphology of cells growing in culture. in addition, we observed that two members of the src kinases family, c-src and lyn, account for most of the caga-specific kinase activity in ags cell lysates. translocated caga interacts with the zo-1 and jam host-cell proteins causing disruption of the apical junctional complex. transfection of the caga gene into polarised epithelial cells induces disruption of cell-to-cell contacts and altered morphology. strikingly caga-expressing cells become migratory and invasive penetrating into collagen gel. the study of different portions of the molecule revealed the presence of two distinct functional domains and both are necessary to induce abnormal cell differentiation through interactions with host cell morphogens. cell polarity and invasion have been suggested to contribute to both early and late stages of cancer formation. these results suggest a mechanism by which caga may acts at the early stage of tumorigenic progression causing loss of cell polarity, increased cell motility and invasiveness of epithelial cells. after a period of 50 years of silence, a disease with an unpronouncable name, "chikungunya" (chik), has recently become a medical reality and reached the public throughout the world. conclusion: low mhla-dr expression after septic shock independently predicts ni. this promising biomarker may be of major interest in identifying patients at increased ni risk who could benefit from targeted and tailored therapy aimed at restoring immune functions. pneumonia, the leading infectious cause of death in the us, kills more people annually than aids, tuberculosis, meningitis and endocarditis combined. from a wide range of observational studies of communityacquired pneumonia (cap), only half of the cases had an aetiologic agent identified. streptococcus pneumoniae was consistently the predominant bacterial aetiology. this lecture will primarily focus on the innate immune response to pneumococcal pneumonia. toll-like receptors (tlrs) are key molecules that recognize pathogen associated molecular patterns (pamps) and induce an inflammatory response. pneumolysin, an intracellular toxin found in all s. pneumoniae clinical isolates, is an important virulence factor of the pneumococcus that is recognized by tlr4. although tlr2 is considered the most important receptor for gram-positive bacteria, tlr2 does not play a decisive role in host defence against s. pneumoniae pneumonia; likely, pneumolysin-induced tlr4 signalling can compensate for tlr2 deficiency during respiratory tract infection with s. pneumoniae. besides tlr2 and tlr4, tlr9 contributes to an effective host defence against s. pneumoniae in the airways. the importance of tlr signaling for host defence against pneumococcal pneumonia is illustrated by the fact that mice lacking the common tlr adaptor protein myd88 are highly susceptible to this infection. activation of tlrs results in the production of proinflammatory cytokines. there is ample evidence that underlines the importance of tumour necrosis factor (tnf) and interleukin (il)-1 in host defence in bacterial pneumonia: in a murine s. pneumoniae pneumonia model, treatment with a neutralising anti-tnf mab strongly impaired antibacterial defence. in addition, il-1a receptor type i deficient mice infected with s. pneumoniae displayed an increased bacterial outgrowth. of considerable interest, treating il-1 receptor deficient mice with a neutralising anti-tnf antibody made them extremely susceptible to pneumococcal pneumonia. infection of the lower airways by s. pneumoniae is associated with complex interaction between the pathogen (e.g. cell wall components, pneumolysin) and the host (e.g. tlrs, cytokines). these interactions play a crucial role in the outcome of this clinically important infection. severe bacterial pneumonia remains uncommon unless specific conditions exist that tip the balance between the host and pathogen in favour of the microorganism. such conditions include: persons at the extremes of age; exposure to especially virulent organisms; patients with concomitant illness impairing pulmonary clearance mechanisms; and immunocompromised hosts. pathogens overcome an array of innate and acquired host defences to successfully invade the host. the known virulence traits of three common respiratory pathogens (streptococcus pneumoniae, staphylococcus aureus, and pseudomonas aeruginosa) will be briefly reviewed. the capsular polysaccharide of pneumococci is the major anti-phagocytic virulence trait but many other factors contribute to disease pathogenesis including the critically important exotoxin known as pneumolysin, bacteriocins, adherence factors, choline binding proteins, lipoteichoic acid, iron, manganese and magnesium transporters, pili, competence and biofilm capacity, and virulence genes that promote invasion and impair clearance once the organism has entered the blood stream. s. aureus is notorious for the numerous a/b type toxins, cytotoxins, and superantigens it generates during the course of invasion. staphylococci deploy a complex series of quorum sensing signals that coordinate adhesin and invasion genes within biofilms or between planktonic organisms and likely contribute to the success of this pathogen. p. aeruginosa produces an array of extracellular exotoxins and cytotoxins delivered by type iii secretion systems. these include elastase, phospholipases c, a series of apoptotic and anti-phagocytic exotoxins, along with an alginate capsule and an unusual and variable lps structure that participate in microbial invasion. the pathogen expresses at least three interacting, quorum sensing systems to coordinate virulence and biofilm formation. a detailed understanding of these virulence factors is now providing therapeutic options to control these respiratory pathogens. surface expressed and extracellular toxins of pneumococci have been selected as new vaccine targets. inhibitory peptides and small molecule inhibitors of quorum sensing and biofilm formation are under investigation for staphylococcal and p. aeruginosa infections. these innovative and non-antibiotic treatment strategies are gaining greater importance as progressive antibiotic resistance threatens the management of these severe bacterial infections in the future. brucellosis, possibly the commonest zoonotic infection worldwide, has troubled humans since antiquity. recent years have seen the expansion of the animal reservoir of the disease to a wide spectrum of wildlife species, extending to marine mammals, and the recognition of novel brucella species. furthermore, animal and human disease has re-emerged in numerous countries which were brucellosis-free, and currently the most important endemic foci include near east and central asia. complex socioeconomic and political factors may be incriminated for these alterations in endemicity. the complex mechanisms by which brucella evades immune response and survives intracellularly are progressively clarified. novel diagnostic techniques as real time pcr may shed light in the life cycle of brucella inside the human host; preliminary studies have indicated that the pathogen may persist in latent form for years after apparent clinical cure, in asymptomatic individuals. treatment principles have not evolved significantly. the expert guidelines issued recently under the name of "ioannina recommendations" support the need for a six-week combined treatment that includes traditional antibacterials and is modified accordingly in serious complications as spondylitis and central nervous system involvement. the road to the development of a vaccine for humans seems long though. anthrax is ancient diseases and relatively a forgotten disease in western world until 2001 when spores were mailed in usa causing five deaths. currently, human anthrax is seen most commonly in agricultural regions of the world where anthrax in animals is prevalent, in which countries of middle east, in africa, central asia and south america. it is also an endemic disease in turkey. human cases may occur in an agricultural or an industrial environment. the infection is an occupational hazard of workers who process hides, hair, bone and bone products, and wool and of veterinarians and agricultural workers who handle infected animals. the main route of transmission is contact with or ingestion of contaminated meal with or inhalation of bacillus anthracis spores. leptospirosis is a very old disease that has been known for more than a hundred years and possibly even longer since the time of hippocrates. it remains a major cause of illness in many tropical and subtropical countries and thus in travellers. it has also been identified as a zoonosis in europe and north america. it is a disease that can surprise us because the clinical presentations are not always typical. in recent years, pulmonary and other atypical presentations have been more widely recognised. there is no effective vaccine but chemoprophylaxis is effective in selected populations. prompt recognition and early institution of appropriate treatment as with most other infectious diseases appear to be critical in ensuring a good outcome for our patients. there are interesting new developments in diagnostics and molecular epidemiology but clearly there are many challenges remaining in this field. objectives: the spread of carbapenemase genes within gram negative bacteria is of great cause for concern. in 2008, the first report of a blaoxa-58 gene outwith acinetobacter baumannii was reported in acinetobacter genospecies 3. we had also identified a genospecies 3 isolate encoding a blaoxa-58-like gene, and the aim of this study was to examine the genetic environment of the gene to investigate the mobilisation between species. methods: restriction analysis of rrna was used to confirm identity to the species level. susceptibility to imipenem and meropenem was determined through the plate doubling dilution method. screening by pcr for blaoxa-51-like, blaoxa-23-like, blaoxa-40-like and blaoxa-58-like genes was carried out. analysis of the genetic environment surrounding the blaoxa-58-like gene was conducted by sequencing inverse pcr products and gene-walking fragments. the structure of the surrounding sequence was confirmed using internal primers, which were also used to screen other blaoxa-58-like positive isolates in our collection. results: restriction analysis confirmed the isolate belonged to acinetobacter genospecies 3. the isolate showed reduced susceptibility to imipenem and meropenem with mics of 2 mg/l for both antibiotics. the isolate was negative for a blaoxa-51-like, blaoxa-23-like or blaoxa-40-like gene, but positive for a blaoxa-58-like gene. analysis of the genetic environment of the blaoxa-58-like gene revealed the gene was within a novel genetic structure. upstream of the blaoxa-58-like gene was the left-hand end of an isaba3 element, interrupted by an isaba125 element. the elements contained putative promoter sequences. downstream was an arac1 and a lyse gene, followed by a sequence similar to the re27 element described previously. following this was a complex region containing the right-hand end of an isaba3 tnpa gene, interrupted by an incomplete tnpa gene with 99% similarity to isaba3, itself interrupted by an isaba125 sequence. this region was followed by a second blaoxa-58-like gene. all other blaoxa-58-like positive isolates in our collection were negative for isaba125 upstream of blaoxa-58. this study is the first to report multiple copies of a blaoxa-58-like gene in an acinetobacter genospecies 3 isolate, and has identified a novel structure containing two blaoxa-58-like genes and two isaba125 sequences. the isaba125 elements may be responsible for the duplication of the blaoxa-58-like gene. objective: acinetobacter baumannii is an important nosocomial pathogen with wide intrinsic resistance. however, due to the dissemination of the acquired resistance mechanisms; such as extended-spectrum beta-lactamase (esbl) and metallo betalactamase (mbl) production, multidrug resistant strains have been isolated more often. per-1 was first detected in turkey and was found to be widespread among acinetobacter spp. and p. aeruginosa. since then, per-1 has been discovered in other countries, and most recently found in northern italy and in korea. in this study, the presence of per-1 type esbl was investigated in caftazidime resistant a. baumannii strains isolated from bloodstream infections by pcr and also the clonal relatedness of the isolates were investigated by random amplified polymorphic dna (rapd) and pulsed field gel electrophoresis (pfge) in all per-1 producing a. baumannii strains. methods: a. baumannii strains isolated from bloodstream infections was included in this study. the isolates were identified as a. baumannii by conventional methods and phoenix 100 bd automated system system (becton dickinson diagnostic systems, sparks). ceftazidime resistance was determined by e-test. per-1 genes were screened by these clusters encode: (i) resistance genes and transporters plausibly involved in drug efflux (30 transporters of the mfs, dmt, abc, rnd, mop and acr3 families were unique of drug resistant strains and absent in the susceptible sdf strain); (ii) pili and fimbriae systems related to biofilm formation and motility; (iii) haemolysin-and haemagglutininrelated proteins differently distributed among the four genomes, (iv) iron uptake and other metabolic genes. conclusion: genome comparison identified unique features of a. baumannii epidemic clones and provided novel insights into the genetic basis of multidrug resistance and pathogenesis in this species. this study may contribute to understand the concept of infection, invasiveness and colonisation in the emergent pathogen a. baumannii. hard to swallow − emerging and re-emerging issues in food-borne infection (symposium arranged with efwisg) s460 mrsa in food products: cause for concern or case for complacency? in 2003 first, a switch from intravenous-to oral medication (01-2006); second, education programs for interns/residents and physicians and the release of a new antimicrobial formulary (05-2006); third, a restriction note was printed on all laboratory rapports (10-2006) and fourth, active monitoring and giving feedback on prescriptions (01-2007). susceptibility patterns for e. coli including ciprofloxacin, cefuroxim, ceftazidim, co-trimoxazole and tobramycin from hospitalised patients were analyzed starting in 2004. statistical analyses were performed using segmented poisson regression models to look at effect of interventions on resistance (both sudden stepwise changes and changes in trends). bayesian model averaging was used to account for model uncertainty. results: before the start of the interventions the resistance rate was increasing by an average of 2.6% per year. the interventions resulted in a significant reduction of quin use from on average 550 prescribed daily doses to 350 pdd per month. in the best fitting poisson model for the resistance data, a significant stepwise decrease was found to be associated with interventions 2 and 4. however, there was substantial uncertainty in the model choice, and after accounting for this there was no conclusive evidence in support of any particular intervention, although there was evidence that at least one of the interventions was associated with the observed reduction in resistance. there were no stepwise decreases or decreasing trends in resistance rates to other antimicrobials during the study period. conclusion: many mds prescribe antibiotics often and believe their practice may have an effect on antibiotic resistance. results indicate that mds value information, interventions and surveillance in order to support responsible use of antibiotics. there is an ongoing effort in germany to address these findings at the national level e.g. by establishing a surveillance system for antibiotic resistance and antibiotic usage. table) . . ir for pn, er and tt were always higher in children (ch) than in adults (ad). significant differences were found for pn (1995), er (1997 er ( , 2004 er ( , 2006 er ( , 2007 er ( , 2008 , tt (2004 tt ( , 2006 . generally, cp-ir was higher in ad than in ch. ir was lower in the north (n) than in the south (s). significant differences: pn (2005 pn ( , 2006 , er (2003 er ( , 2004 er ( , 2005 , tt (2005) . both n and s knew a deceasing ir tendency: pn= n (12.1−8.1), s (18.8−13.2); cp= n (11.6−5.9), s (18.9−5.9); tt= n (27.4−21.5), s (35.9−23.5). er increased in the n (20.9−29.7). total outpatient antibiotic use (did) decreased from 26.2 (1999) to 22.7 (2004) and increased to 24.2 (2006) . did for pn and fq increased, mls stabilised and tt decreased. conclusions: since 2001-2003 an ir decrease was noted for pn, cp and tt. er-ir increased further over the years. the decrease paralleled the start of public campaigns on antibiotic use. ir rates remain higher in ch than in ad. the n/s difference became less marked. objectives: parachlamydia acanthamoebae is a new recognized member of the order chlamydiales. growing evidences suggest that this bacteria may have a pathogenic role in humans causing respiratory diseases. it has also been recently identified as an agent of bovine abortion and may be a cause of miscarriage in women. in contrast, little is known about the pathogenic role of rhabdochlamydia crassificans, another related chlamydiales. molecular diagnostic tools are useful to detect these obligate intracellular bacteria because of their inability to grow on conventional culture media. the aim of this work was (i) to develop a real-time pcr for the diagnosis of rhabdochlamydia infection and (ii) to study respiratory secretions of newborns for the presence of parachlamydia and rhabdochlamydia dna. methods: a new quantitative real-time taqman pcr (q-pcr) to be used on abi prism 7900 was developed. the q-pcr was then blindly applied to 41 consecutive respiratory samples (endotracheal or nasopharyngeal secretions) taken from 29 critically-ill newborns admitted in the neonatology ward of our university hospital. these samples were also tested using a previously developed parachlamydiaspecific pcr. results: most newborns (28/29) were premature (median gestational age: 28.6 weeks; range: 24.6−41.2). initial respiratory distress syndrome was present in 86% of them. positive pcr results were obtained in 12/29 (41%) patients (8 parachlamydia, 3 rhabdochlamydia, 1 both species) at a median of 17.5 days (range: 2-230) after birth. when compared to the control group (17 patients with negative pcr), these 12 newborns had a significantly worse primary adaptation and a higher incidence of resuscitation maneuvers at birth (table) . duration of noninvasive mechanical ventilation and stay in neonatology ward were also significantly longer. a fatal issue was observed in 3 infected cases, as compared to no death in controls (p = 0.06). gestational age at birth as well as the incidence of pulmonary or systemic infections did not differ between cases and controls. conclusion: a high prevalence of parachlamydia and rhabdochlamydia dna was observed in respiratory secretions of premature critically-ill newborns. the presence of dna of these microorganisms was associated with a worse primary adaptation, a more severe respiratory distress syndrome and a trend towards a higher mortality. their pathogenic role should be further investigated. the genus kingella consists of 3 species, k. kingae, k. oralis and k. denitrificans. all are gram negative, sometimes difficult to stain, rod shaped bacteria that are normal respiratory and genitourinary flora. they are slow-growing and fastidious. although improved recovery was shown when using fan or peds-f blood culture bottles, the majority of these infections remain undetected, especially in pre-treated patients. we report the use of real time polymerase chain reaction (rt-pcr) assays for detection of k. kingae and s. aureus in paediatric osteoarticular infections. methods: 116 synovial fluid samples from 97 patients, 1 month and 17 years of age, were collected over 19 months (03/2006 to 10/2007). the samples were from 54 knees, 39 hips, 9 ankles, 6 elbows, 4 shoulders, 2 wrists and 2 femur abscesses. after automated dna/rna extraction, specimens were subjected to 4 hour pathogen-specific rt-pcr. samples were inoculated onto sheep blood and chocolate agar as well as a peds-f bottle. final species identification and antimicrobial susceptibilities were determined by phoenix (tm). results: 45 patients (56 specimens) had positive culture and/or rt-pcr, resulting in an overall positivity rate of 46%. s. aureus was the predominant pathogen accounting for 31 specimens of 23 patients (12 mrsa, 11 mssa) and. 37% of positive specimens (18 patients) were due to k. kingae (n = 21). among children 0−2 years (n = 35), k. kingae was the predominant pathogen accounting for 16 positive patients (46%), followed by mssa in 4 patients (11%). the positivity rate for this age group was 57%. only 2 children >2 years (5 and 9 years) were positive for k. kingae. mrsa was the predominant pathogen in 6−12 year olds, and mssa was evenly distributed among children 3−17 years old. culture detected only 5 of 21 specimens positive for k. kingae and 25 of 31 s. aureus. 4 other pathogens were detected by culture only. the use of these molecular assays enhances detection of organisms, especially for k. kingae (19% vs. 5% for culture). additionally, faster identification (tat 4 hrs) allows for rapid targeted therapy. this improvement in tat could lead to shorter hospital stays in about 54% of cases. results: genotyping revealed a high degree of diversity, indicative of a panmictic bacterial population. further, there was no association between genotype and colonisation frequency, or year of isolation. pcr screening for virulence genes revealed an incidence of 98% for uspa1, 81% for hag, 82% for uspa2 and 18% for uspa2h. no significant difference was observed in the prevalence of virulence-associated genes between isolates originating from children who were colonised only once or children colonised on all 3 occasions (p = 1). pcr-rflp analysis of uspa1, hag and uspa2 showed many gene variants, with no association between pcr-rflp patterns and colonisation frequency, or year of isolation. conclusion: even in relatively localised geographical settings, the genotypic diversity of m. catarrhalis isolates colonising children is large, with no yearly pattern of genotype predominance. children serially colonised with m. catarrhalis isolates appear to clear a particular genotype only to become subsequently colonised with a different genotype. the incidence of virulence genes in this relatively localised study group is remarkably similar to that reported in global m. catarrhalis isolates, possibly indicating that similar selection pressure exists for m. catarrhalis at both the local and global level. virulence gene variation appears to be high, even in this relatively restricted geographical group. these results could have consequences for vaccines designed against virulence genes. a. naessens°, i. foulon, a. casteels, w. foulon (brussels, be) objectives: to evaluate the epidemiology of cytomegalovirus in pregnancy and to evaluate the risk for delivering a child with congenital cmv (ccmv). methods: between 1996-2006, 11825 unselected mother-infant pairs were included. in the mother a serological screening was performed consisting in the detection of cmv igg and igm antibodies at the first prenatal visit and at birth. in the neonate cmv urine culture was performed to diagnose congenital infection. when a pregnant woman was found to have a second trimester spontaneous abortion or a death in utero, an investigation for possible congenital cmv infection was carried out. results: serological screening at the first prenatal visit showed no immunity in 4701 women, evidence of past infection (igg positive igm negative) in 6877 women (58.2%) and in 250 women (2.0%) both igg and igm antibodies were detected. after investigation of stored and follow up samples from these 250 patients, 14 could be classified as having a primary cmv infection during pregnancy, 99 patients had previous immunity before the current pregnancy and from 137 patients the type of the maternal cmv infection could not be determined. follow-up serology of the 4701 women without immunity revealed a seroconversion in 58 of them (1.2%). a total of 61 (0.52%) congenital infections (ccmv) were diagnosed. the incidence of the ccmv among the different groups of women are summarised in the table. conclusion: ccmv infection occurs in 0.52% of our population of pregnant women. ccmv was considered to be due to a primary maternal cmv infection in 54% of the infants; 33% due to a recurrent maternal cmv infection and in 13% the type of maternal infection could not be determined. the risk for a seronegative pregnant woman of acquiring cmv during pregnancy is 1.2%. the transmission risk after a maternal primary infection is 45%. women with prior immunity have a very low risk (0.20%) for ccmv, this risk increases to 3% when igm are find in women with know prior immunity. the risk for women with undetermined infectious status in early pregnancy to give birth to a congenitally infected neonate is 5.8%. this report provides the first data on rotavirus epidemiology and disease burden in norway. further studies are needed to assess the economic impact of rotavirus disease and the cost-effectiveness of vaccination to inform decisions on introduction of rotavirus vaccines into the national program of childhood immunisation. pseudomonas aeruginosa may colonise the lungs of cystic fibrosis patients over years but may also cause acute infections in mechanically ventilated patients and immuno-compromised hosts within a matter of days. despite aggressive antibiotic treatments the organism is rarely eradicated. instead p. aeruginosa adapts to its host environment by developing resistance mechanisms and changing its lifestyle and virulence properties. focusing on mechanically ventilated patients, we will detail the dynamics of resistance emergence and persistence of p. aeruginosa lung populations during antibiotic therapy. we further discuss how p. aeruginosa populations evolve naturally in the absence of any antimicrobial treatment within the lungs of intubated patients by changing their virulence properties. the relevance of these findings both with respect to concepts of social evolution and the development of novel anti-infective strategies will be highlighted. the genome of p. aeruginosa encodes many potential efflux systems. however, only a few of them appear to play a significant role in antibiotic resistance. in this respect, the mex (for multiple efflux) systems are of particular interest because of their ability to extrude a wide range of antimicrobials. these polyspecific machineries result from the assembly of (i) a drug/proton antiporter, (ii) a periplasmic adaptor protein, and (iii) an outer membrane gated channel. it is now well established that the constitutive expression of the tripartite pump mexab-oprm provides p. aeruginosa with a relatively high intrinsic resistance to quinolones, blactams (except imipenem), tetracyclines, macrolides, chloramphenicol, trimethoprim, and novobiocin. this protective mechanism is potentiated by the poor permeability of the outer membrane and activity of another pump, mexxy/oprm, whose expression is induced by substrates targeting the ribosome (e.g., tetracyclines, macrolides, aminoglycosides). accumulating reports indicate that multidrug resistant mutants upregulating one or both of these systems are quite common in the clinical setting. such mutants, which are readily selected by sub-optimal treatments with fluoroquinolones, b-lactams or aminoglycosides, tend to accumulate various resistance mechanisms without loosing the wildtype pathogenicity of p. aeruginosa. whether the low resistance levels (mic x 2-to 8-fold) conferred by efflux may promote second-step mutants with altered drug targets (gyra, gyrb, parc) or derepressed ampc b-lactamase has not been confirmed in vitro. in the specific context of cystic fibrosis (cf), a recent study from our laboratory showed that the mexxy/oprm pump can be responsible for much higher resistance levels to aminoglycosides (64-to 128-fold). this increased efficacy of the system partially results from adaptive mutations in the mexy gene. in contrast, subpopulations deficient in mexab-oprm tend to emerge during long-term colonisation of cf airways. while easily selected in vitro on selective media, mutants overexpressing other mex systems (mexcd-oprj, mexef-oprn, mexghi-opmd, mexjk/oprm, mexvw/oprm) have been rarely described in cf and non-cf patients. some data support the notion that up-regulation of mexcd-oprj or mexef-oprn might be detrimental to the virulence of p. aeruginosa. in conclusion, therapeutic strategies based on efflux inhibitors should target the mexab-oprm and the mexxy/oprm systems in priority. european aspects of malaria s478 rapid diagnostic tests for malaria: twenty years to convince . . . prompt diagnosis and treatment of malaria are critical factors in reducing morbidity and mortality. microscopy has long been the gold standard for malaria diagnosis, but the newer rapid diagnostic tests (rdts) now offer considerable advantages, especially so in endemic countries. after close to twenty years of development and operational research, the diagnostic performance of rdts is now established in all settings. meta-analyses have clearly demonstrated equivalence of rdts over expert microscopy to detect parasites, and clear superiority over routine microscopy. actually, one of the major reasons that have delayed successful implementation of rdt in endemic areas was the use of poor quality microscopy that has impeded reliable measurement of sensitivity and specificity and undermined confidence of health workers in rdts. other factors were poor product performance, inadequate methods to determine the quality of products and a lack of emphasis and capacity to deal with these issues. for the potential of rdts to be realised, it is crucial that high-quality products that perform reliably and accurately under field conditions are made available and that quality insurance is performed on all steps of the procedure. in achieving this goal, the shift from symptom-based diagnosis to parasite-based management of malaria can bring significant improvement for the management of fever in endemic areas. for travelers returning in temperate climates with fever, rdts have also the potential to improve diagnostic procedures, especially so in hospitals where reliable microscopy is not available out of hours. in patients with no danger sign or significant thrombopenia, a negative rdt is sufficient to exclude malaria and allows waiting 12−24 hours for performing or reading the microscopy slide. rdts should be repeated every 12−24 hours for three consecutive days if fever persists and in the absence of alternative diagnosis. rdts represent a revolution in the fight against malaria and will tremendously help to manage appropriately patients with fever, especially so when malaria is declining and hence other causes of fever increasing. the ambitious deployment that is foreseen in the coming years in africa through large grants from the global fund should contribute to achieving the millennium goals. fever is the key symptom of malaria among returning travellers (97%). headache, chills, myalgia, sweating and lack of a focus are frequently recorded, but non-specific. nausea and vomiting are often seen in children. the differential diagnosis of other infections, mainly of viral origin, is further difficult because (dry) cough and (mild) diarrhoea are often present. laboratory findings (thrombocytopenia, low or normal leucocyte count) can be helpful in the assessment of mild to moderate malaria. clinical signs and symptoms, e.g. fever, may be mitigated in semiimmune patients (visiting friends and relatives, foreign visitors) seen in non-endemic countries who represent the majority of cases diagnosed in industrialised countries. caution is warranted in assessing such patients as many of them may no longer be exposed to malaria in their countries of origin, thus no longer partially protected and also at risk of suffering from severe complications. up to 10% of all imported malaria cases may be severe, presenting with jaundice, impaired consciousness to coma, acute renal failure, and, in the course of events, acute respiratory failure. delay in diagnosis and start of treatment is partly responsible for fatality rates of 1% and more in some countries. if you don't look for them, you won't find them: anaerobes revisited s481 anaerobic microbiota of the mouth − friend or foe? anaerobes form a major part of the commensal microbiota in the digestive tract where they constitute an integral component of the function on mucosal surfaces. in the mouth, teeth create a unique, non-shedding environment for bacteria to attach and to form biofilms. there is an age-related succession order of species in bacterial colonisation of the mouth, and once established, individual anaerobic species tend to remain as members of the oral microbiota. the agerelated pattern of the colonisation of anaerobic bacteria is partly connected with the development (or loss) of the dentition. interactions between different bacteria residing in the same microenvironment influence the composition of the microbiota − or the development of pathologic conditions. although commensal bacteria are regarded beneficial to the host, some anaerobic members of the oral microbiota contain characteristics potentially detrimental for the health status of an individual. molecular means of characterisation have resulted in increased knowledge about the "normal" microbiota of the mouth and in detection of new species and genera as well as phylotypes, which can be associated with infectious situations in the mouth. oral infections are multifactorial and polymicrobial in nature, and their aetiologic organisms originate mainly from the oral resident microbiota. the involvement of anaerobes is most obvious in infections of root canals, periodontal tissues, and tissues surrounding erupting wisdom teeth where typical anaerobic findings are gram-negative rods. in addition, gram-positive anaerobic cocci and non-spore-forming gram-positive anaerobic rods are common in odontogenic infections. on some occasions, anaerobes of localised dentoalveolar infections can spread to adjacent tissues and even to the bloodstream, resulting in severe complications in extraoral sites. interestingly, a relatively limited number of anaerobic species are involved in clinically severe infections, however, microbial findings seem to vary depending on geography. concomitant with the increase in the number of immunosuppressed patients, the number of opportunistic infections caused by commensal anaerobes may increase. identification to the species level will help to establish associations between individual anaerobic species and specific disease states. studies on the bacteriology of diabetic foot infections (dfi) have yielded varied and often contradictory results. the role of anaerobes is particularly unclear, often because the type and severity of the infection is poorly defined, recent antibiotic therapy is unknown, and specimen collection and culture techniques are inadequate. when optimal collection, transport, and culture techniques are used, multiple organisms including aerobes and anaerobes are usually recovered from severe dfi. interactions within these polymicrobial soups lead to production of virulence factors, such as haemolysins, proteases, collagenases, and short chain fatty acids, which promote inflammation, impede healing and contribute to the chronicity of the infection. to better define the bacteriology of diabetic foot infections, we analyzed our data from a large prospective u.s. multicentre trial of patients with moderate to severe infection that required initial parenteral antibiotic therapy and used optimal post-debridement sample collection, transport and culture procedures. of the 427 culture-positive specimens (of 454 total), only 16.2% were pure cultures while 30.4% yielded 5 or more organisms. a total of 462 anaerobes (range 0−9, average 2.3, per specimen) were recovered from 49% of patients, with gram-positive cocci (gpc) accounting for 45.5% of all anaerobic strains. s485 is culture still the gold standard, really? tremendous technological advances are made in culture-independent methods of detection and identification of human bacterial pathogens, such as pcr or hybridisation of their genomic dna. yet, time honoured pastorian bacterial culture in liquid and solid nutritive media still remains the gold standard for the laboratory diagnosis of a majority of bacterial infections. this unusual robustness of a 19th century technology stems from its unmatched operational characteristics: 1. broad range of detected agents, depending on adequate combination of media/incubation conditions; 2. unlimited source of clonal population for individual isolate, allowing versatile characterisation of antibiotic susceptibility and/or pathogenic factor production and/or epidemiological subtyping; 3. possibility of storage/bio-banking of cells for complementary clinical testing, research and diseases surveillance collections; 4. proof of pathogenic role of agent at the time of viable cell isolation from the site of infection, in contrast to false-positive results with molecular tests (tissue translocation or persistence of bacterial dna, soluble antigen,. . . ). major drawbacks of bacteriological culture include long turn-around time, cost and labour/skill intensity. these are partly alleviated by new technologies, including automated processing, physical/chemical growth detection and rapid molecular fingerprinting (maldi-tof, raman spectrometry, 16s rdna snp detection). it is likely that the next decade will see a complete redefinition of the place of direct detection methods and culture-based confirmation methods in clinical bacteriology, enabling a rejuvenation rather than elimination of culture as a daily diagnostic tool. the advent of real-time pcr revealed instrumental to the successful implementation of molecular methods in routine clinical microbiology laboratories. automated nucleic extraction platforms can now be coupled to robotic handling for large-scale detection and quantification purposes, mostly in virology. i will review here the attempts of implementing home-brew and commercial nucleic-acid based detection methods directly from blood samples and highlight hopes and pitfalls. i will then expand on two promising nucleic acid amplification methods: lamp (loop mediated isothermal amplification) and a protein-free method called dnazyme. these isothermal amplification methods share several strengths: robustness across highly diversified physico-chemical conditions, versatility in assay development and minimal requirements (if any) for sample preparation. they will definitely compete against current real-time pcr assays and might become a novel standard, due to lower costs and improved performances. the ribosomal rna (rrna) approach to microbial evolution and ecology has become an integral part of microbiology. rapidly growing databases exist that encompass besides the 16s rrna sequences of almost all validly described bacteria and archaea also numerous 16s rrna sequences of so far uncultivated microbes, directly retrieved from the environment by pcr or metagenomics. based on the patchy evolutionary conservation of rrna genes oligonucleotide probes can be designed in a directed way with specificities ranging from species up to large evolutionary entities like phyla or even domains. when such probes are labeled with fluorescent dyes or the enzyme horseradish peroxidase they can be used to identify single microbial cells by fluorescence in situ hybridisation (fish) directly in complex environmental samples. an update on recent applications and methodological improvements will be given which includes the identification of small bacterial cells by catalyzed reporter deposition (card)-fish. with optimised methods and proper controls fish yields exact cell numbers and spatial distributions for defined bacterial populations also in highly complex mixed microbial communities. r. amann & b.m. fuchs (2008) nature reviews microbiology 6:339-348. quick and reliable species identification of microorganisms is of great importance in medical microbiology. several bacterial and fungal species can be identified only using laborious and time-consuming methods. furthermore, in many cases misidentification occurs due to e.g. limited biochemical reactivity, different morphotypes or limited information in reference panels. in this talk, matrix-assisted laser desorption/ionisation time-of-flight (maldi-tof) mass spectrometry will be presented as a method for species identification. this technology applies protein pattern matching based on mass spectrometry. during the identification process, a mass pattern is generated for each organism. the subsequent comparison of this pattern with a database comprising reference patterns derived from well-characterised reference strains leads to species identification. as examples, the identification of various nonfermenting bacterial strains isolated from clinical specimens in comparison to partial 16s rdna sequencing will be shown. moreover, speed, accuracy in comparison to other methods, and inter-and intra-laboratory reproducibility of maldi-tof ms-based species identification will be discussed. o489 trends in invasive streptococcus pneumoniae serogroup 1 sequence types in belgium t. goegebuer, k. van pelt, j. verhaegen, j. van eldere°(leuven, be) objectives: s. pneumoniae serogroup 1 (sg1) isolates frequently cause invasive pneumococcal disease, particularly in children. from 2003 onwards a marked increase in sg1 isolates was observed; overall prevalence increased from 8. 2% (1998-2002) to 13.6% (2003) (2004) (2005) (2006) . we determined the sequence types (st) in sg1 isolates in order to better understand trends in sg1 resistance and spread. methods: as national reference centre, we receive all invasive isolates from more than 100 of 182 laboratories in belgium. 124 randomly chosen sg1 isolates from all ages from 1998 to 2006 were analysed via multi-locus sequence typing (mlst) as described by enright & spratt (microbiol. 1998; 144: 3049−60) . we also included data on strain characteristics and patient characteristics. results: 10 different sequence types (st) were identified: st350 (n = 66), st306 (n = 24), st304 (n = 13), st227 (n = 10), st228 (n = 5), st2915 (n = 2), st305 (n = 2), st612 (n = 1), and st217 (n = 1 mutations usually increase the mic slightly, but enhance the probability of further mutations. efflux pumps like pmra reduce antibiotic concentrations in the bacterial cell, enabling longer survival. we hypothesised that efflux positive bacteria are more likely to develop resistance than efflux negative bacteria. the following questions were addressed: 1. do the efflux pump inhibitors reserpine and verapamil reduce the mutation frequency? 2. do fluoroquinolone-susceptible efflux positive pneumococci exhibit higher parc or gyra qrdr mutation frequencies than efflux negative isolates? 3. does efflux phenotype impose a fitness cost? methods: matched efflux positive and negative pneumococcal isolates with identical or similar genotype according to multi-locus sequence typing collected by the german community acquired pneumonia network capnetz were analysed (n = 17). strains tigr4 and r6 were included as efflux negative controls. ciprofloxacin (cip) mics and efflux phenotype were measured by agar dilution method, for efflux detection reserpine (10 mg/l) was added and a fourfold decrease in mic was considered as efflux positive. mutation frequencies were determined by plating bacterial suspensions onto agar with and without cip. after incubation colonies were counted and the ratio of cfu/ml yielded the mutation frequency. equally, the mutation frequency was determined adding different concentrations of verapamil (10, 25, 50, 100, 500 mg/l) or reserpine (0.01, 0.1, 1, 5, 10 mg/l). biological fitness was calculated as the maximum slope of growth curves recorded in a microtitre plate reader. results: 1) even at low concentrations, reserpine clearly reduced the mutation frequency of efflux positive and, to a lesser extent, efflux negative pneumococci when exposed to cip (figure 1); verapamil exhibited this effect merely at high concentrations. 2) efflux positive isolates produced more frequently mutants (8/9) than efflux negative isolates (2/10) (p = 0.005, fisher's exact test). 3) efflux phenotype had no measurable impact on the biological fitness. conclusion: a positive efflux phenotype increases the qrdr mutation frequencies in the presence of fluoroquinolones and this effect can be inhibited by very low concentrations of reserpine. as a matter of concern, efflux is not associated with decreased biological fitness. background: use of fluoroquinolone (fq) has been associated with increasing fq resistance in s. pneumoniae. because respiratory fqs (levofloxacin (levo) and moxifloxacin (moxi)) are first line therapy for serious respiratory infections, increasing fq resistance (fqr) in sp is a concern. levo targets parc, and moxi targets gyra, which may permit differentiation of degree of selective pressure. we examined fq use, and changes in the prevalence of fqr and qrdr mutations in canadian isolates of sp. methods: cbsn is a canadian collaborative network of microbiology laboratories that has performed surveillance for antibiotic resistance in sp since 1988. antimicrobial resistance is performed in a central lab to clsi standards. we sequenced qrdr regions of all fqr isolates and a stratified sample of fq susceptible isolates. population fq use was obtained from ims canada. results: from 1995 to 2007, fq use increased from 53 to 97 rx/ 1000pop/yr; levo use from 0 to 10 rx/1000pop/yr, and moxi use from 0 to 17 rx/1000pop/yr. 31081 isolates were available for testing. levo r rates increased from 0 in 1993 to 1.8% in 2002 then remained stable until 2008 (1.6% in 2008). moxi r rates increased to 0.6% in 2004, then stabilised (0.7% in 2008). the prevalence of parc only mutations has not increased significantly in the last decade (see table) . the prevalence of isolates with both parc and gyra mutations increased until 2002, but has decreased in 2008. the first gyra only mutant was detected in 2000; the prevalence of gyra only mutants since then has increased, but remains very low (7/2044, 0.34% in 2007) . conclusion: despite increasing use of respiratory fqs, fqr in pneumococci is very low and not increasing in canada. the prevalence of isolates with parc mutations is decreasing. isolates with mutations in gyra alone remain extremely rare, suggesting that moxi exerts minimal selective pressure for resistance. in streptococci, two well characterised macrolide resistance have been described: target modification and active drug efflux. target site modification is mediated by the erm genes -erm(b), erm(a), erm(c)which confers the mlsb phenotype. target modification by mutations in 23s rrna as well as mutation in l4 and l22 ribosomal proteins have also been reported. expression of mef(a) genes activate an efflux mechanism responsible for m-type resistance we characterised a clinical isolate of s. agalactiae mb56gbs022 exhibiting the mlsb phenotype and tetracycline resistance. in this study, we determined the resistance genes, their association, and their localisation and mobility by conjugation. methods: the macrolide and tetracycline resistance genes were confirmed by pcr. the association between macrolide and tetracycline genes was investigated by long-pcr and sequencing. conjugation experiments were performed by filter matings. the genetic localisation of resistance genes was determined by endonuclease i ceui -followed by pfge and southern blot. the hybridisation study was performed using three specific probes for the 16s and 23s rrna genes, for erm(b) and tet(o) genes. results: s. agalactiae mb56gbs022 carried erm(b) and tet(o) genes on the same amplicon of 7 kb in size. the nucleotide sequence analysis of the entire product was identical to the peoc01 of 11 kb from pediococcus acidilactici that contains four orfs, of which orf2 and orf3 encode a putative resolvase and topoisomerase type i, respectively. the endonuclease i ceui method, that easily distinguishes between plasmid and chromosomal localisations as i-ceui only cuts chromosomal dna, revealed the localisation of resistance genes on the plasmid. all attempts to transfer erm(b)-tet(o) structure by conjugation from s. agalactiae mb56gbs022 to og1ss e. faecalis as recipient failed. conclusion: our results show the first case of the association between erm(b) and tet(o) genes on the unique mosaic structure in s. agalactiae, probably on the plasmid, as demonstrated by the i ceui-assay. further studies are on going to characterise the entire genetic element carrying resistance genes. o499 improving influenza pre-analytic collection systems: alternative collection systems to inactivate, preserve, or extract influenza for rapid testing s. castriciano°, k. luinstra, m. ackerman, a. petrich, m. smieja (brescia, it; hamilton, ca) objectives: in this study, 3 alternative influenza sample collection systems were evaluated for potential use in a pandemic situation. the objectives were to develop: 1) a non-temperature dependent swab collection and transport system, that inactivates influenza virus infectivity but preserves cell morphology and nucleic acid (na) for the detection of suspected influenza infections and/or 2) a system compatible with direct na testing without the need for purification prior to detection by a rapid real-time rt-pcr. methods: flocked nasopharyngeal swabs (nps) collected in utm (u) were compared to nps collected in a cymol (c), m-swab (m) or dry (d) flocked swab collection system (copan, italia). cymol is an alcoholbased medium that preserves cells for dfa testing. the m-swab contains 600 ul of medium and 150 ul of glass beads, and requires no na purification step. shell vial culture was used to assess influenza virus inactivation after 30 minutes exposure to the collection media. a mockinfected influenza a virus sample was absorbed to duplicate swabs then placed into the 4 collection systems. the infected collection media were held at rt for 30 minutes and then inoculated in duplicate into shell vial culture and stained after 48 hours. influenza a stability and na recovery after mock infection of each collection system was assessed after 1, 7, 14 and 21 days (d) at 4ºc, −20ºc, room temperature (rt) and 37ºc. aliquots of infected collection media were extracted by easymag and 5 ul of purified na tested by a quantitative influenza a rt-pcr on the roche lightcycler. m-swab collected samples were also tested directly or after boiling, without na purification. results: shell vial culture found that influenza a virus was inactivated after 30 minutes exposure to the c medium but not when exposed to the u and m media. influenza a was detected by dfa from the u and c cell smears. quantitation of influenza a rna was constant after 1, 7 and 14 d in u, c, m and d collection systems at −20, 4ºc and rt. the quantity of rna recovered declined significantly after 14 d at 37ºc in all 4 collection systems. m with boiling yielded data comparable to the easymag extraction. the copan cymol medium inactivates influenza infectivity, preserves cells and stabilises rna up to 14 days at −20, 4ºc and rt. cymol medium is a potential alternative for safe sample collection during a pandemic influenza situation. the m-swab presents a rapid testing alternative. luminex respiratory viral panel in respiratory specimens from children r. selvarangan°, s. selvaraju, d. baker, k. estes, l. hays, d. abel, s. hiraki (kansas city, us) objective: luminex respiratory viral panel (rvp) is a multiplex pcr capable of detecting and differentiating twelve different respiratory viruses and their subtypes; influenza a (flu a) (subtypes h1 and h3), influenza b (flu b), respiratory syncytial virus (rsv) (subtypes a and b), adenovirus (adv), parainfluenza 1 (piv 1), parainfluenza 2 (piv 2), parainfluenza 3 (piv 3), human metapneumovirus (hmpv) and rhinovirus (rhv). the aim of this study was to evaluate the analytical performance characteristics of rvp assay and to evaluate its ability to detect respiratory viruses from nasopharyngeal aspirates obtained from children. method: analytical sensitivity, specificity, accuracy and precision of the luminex rvp assay were determined using control viral stocks and respiratory specimens previously tested by rmix shell vial culture. result: luminex rvp assay reliably detected atcc viral stocks of flu a, flu b, rsvb, rhv and piv3 in the range of 10e-2 to 10e-4 tcid50/ml. no cross reactivity was noted with cmv, hsv, hhv6, ebv, vzv, piv4, cornoavirus 229e and oc43. among 146 respiratory specimens previously characterised by culture 138 specimens were accurately detected with overall accuracy of 95%. the median coefficient of variation in mean fluorescent index values of signals from replicate analyses of influenza a, b and rsv was 9% (7% to 25%). the 146 clinical specimens tested by rvp assay included 109 culture positive and 37 culture negative specimens. respiratory viruses isolated from the culture positive specimens include the following; 19 adv, 11 flu a, 10 flu b, 19 rsv, 9 piv1, 6 piv2, 5 piv3, 19 hmpv and 14 rhv. rvp assay detected all of the respiratory viruses except one each of rsv, piv1 and piv2 virus with overall sensitivity ranging from 88% to 100% for the different respiratory viral groups. among the 37 culture negative specimens 20 respiratory viruses were detected by rvp of which 15 were subsequently confirmed by repeat analyses. conclusion: luminex rvp assay is a highly sensitive and specific test useful in the detection of commonly encountered respiratory viruses in respiratory specimens. the addition of rvp assay to the viral testing algorithm of respiratory infections in children provides rapid results, improves diagnostic yield and may result in decreased antibiotic usage, reduced diagnostic testing and reduced hospital stay. m. savvala, i. daniil, i. berberidou, a. koutsibiri, a. stambolidi, m. papachristodoulou, n. spanakis, d. petropoulou°, a. tsakris (athens, gr) objective: in developed countries, viruses, particularly noroviruses, are recognized as the leading cause of acute gastroenteritis. we determined the aetiology, prevalence and seasonal distribution of viral gastrointestinal infections in hospitalised patients with acute diarrhoea. methods: during one-year period (november 2007-november 2008), a total of 201 faecal specimens were collected from 165 children, 21 premature neonates and 15 adults who were hospitalised with symptoms and signs of acute gastroenteritis. stool samples were tested for the presence of rotavirus, adenovirus, astrovirus and norovirus. rotavirus, adenovirus and astrovirus antigen detection was performed by chromatographic immunoassays (rotavirus and adenovirus, vikia ® -biomerieux, france; h&r astrovirus-vegal farmaceutica, spain). noroviruses were detected by an enzyme immunoassay (ridascreen ® rbiopharm, germany) and confirmed by reverse transcription-pcr. data were analyzed for seasonality of infection and possible transmission mode. the overall incidence of viral identification in acute diarrhoeal stool was 24% (48 of 201 patients). fifty one viral antigens were detected one patient with positive antigen detection is suffering from a disease of unclear aetiology. so, an association of replication of cihhv-6 with the disease might be considered. in contrast, the other patient did not show any symptoms at the time of antigen detection. this patient shows a special mode of acquisition of cihhv-6 (by bmt) possibly resulting in differences in the immunological priming and response. in addition, in the latter patient cihhv-6 is restricted to blood cells. two other patients did not show antigen expression. so, it is unclear how the transcription and translation of viral genes is influenced? furthermore, is there a pathophysiological impact of viral replication in individuals with cihhv-6? objectives: several case studies have reported on meningo-encephalitis caused by a primary epstein-barr virus (ebv) infection. we aimed to investigate the viral loads, and the inflammatory characteristics of this thus far poorly defined disease entity. we evaluated all cases from 2003-2008, in which an ebv polymerase chain reaction test (pcr) was requested on a cerebro spinal fluid (csf) sample. primary infection was defined as a clinical presentation with sore throat/pharyngitis/malaise in combination with lymphocytosis, and detectable heterophile antibodies or positive ebv igm antibodies. patients with proven neuroborreliosis served as control group. leukocyte response and ebv viral loads in csf, and serum were compared between primary ebv and neuroborreliosis cases. results: we identified six cases with a primary ebv infection (median age: 22, male: 4) with neurological symptoms ranging from meningeal signs to encephalitis. these were compared to 14 patients with neuroborreliosis (median age: 27, male: 6). in four out of six patients with a primary ebv infection with neurological symptoms ebv dna was detected in csf and in serum, whereas all neuroborreliosis cases were ebv pcr negative in both compartments. viral loads were lower in csf as compared to serum. in blood, leukocytes, lymphocyte, and monocyte counts were significantly increased as compared to the neuroborreliosis cases (see table 1 ). specific for vp7 and vp4 genes, using pools of g and p type specific primers. all strains (niv/brv/68, niv/brv/79, and niv/brv/86) were not typeable for the vp4 and vp7 genes. after purification by "qiaquick gel extraction kit" (qiagen, germany), the vp4, vp6, vp7, and nsp4 first amplicons of the borv-a strains were subjected to sequence analysis with automated sequencer abi 3130 xl dna analyzer (applied biosystems, usa). phylogenetic analysis was performed using mega version 4.0. objectives: dengue is a flavivirus and is among the most widely-spread viral diseases. our previous report demonstrates existence of live dengue virus in blood and urine even in the convalescent postfebrile period. in some cases, excretion in the patient's urine can be detected as late as 28 days after the onset of illness. this goes along with the model of west nile virus, another type of flavivirus, which can be excreted in the urine for months after acute infection in both animal studies and human case report. here we report a pilot study to address a magnitude of such findings. methods: between april 2007 and october 2008, paediatric and adult febrile patients suspected of dengue infection were enrolled. diagnosis of dengue was based on standard specific serology on paired sera. patients with negative serology served as controls. blood and urine specimens were collected at several time points. whole blood was separated into plasma and peripheral blood mononuclear cells (pbmc). these have been aliquoted and used for earlier studies and some stored in freezers. available plasma, pbmc, and urine were processed and inoculated into aedes aegypti. surviving mosquitoes at 14 days after inoculation were employed for viral detection by dengue-specific rt-pcr. indirect fluorescence antibody (ifa) staining of mosquito heads was performed on all positive rt-pcr specimens, except for the one from pbmc (awaiting ifa result). results: 5 and 45 cases of primary and secondary infections, respectively, and 4 negative controls were included. these translated into 55 and 59 early and late dengue specimens, and 6 and 4 early and late negative-control counterparts, respectively. dengue virus were isolated in some blood and urine specimens as late as 46 days after the onset of illness. no virus was isolated from control specimens. all but 5 positive rt-pcr specimens also demonstrated positive ifa. 4 out of 5 negatives were from early-phase specimens. conclusion: our study demonstrates prolonged survival of dengue virus after clinical recovery. this finding has pathologic and epidemiologic significance, adding a potential role of urine in the transmission of the disease. spread of the virus to humans might occur through infectious urine with help from arthropod vectors. this research could provide new insights into our understanding of the pathogenesis of denv infection. isolation of dengue virus from blood and urine specimens during early (days 1−7 after onset of illness) and late (days 8−46) phases of infection (specimens with dengue isolated/total specimens for mosquito inoculation) early phase late phase plasma 16/25 (64%) 0/13 (0%) pbmc not performed 1/2 (50%) urine 8/29 (28%) 12/44 (27%) all specimens 24/54 (44%) 13/59 (22%) dna copies) 226 (<50-1461) † ; n = 2 <dl all <dl ebv pcr in serum (dna copies) 1507 (<50-14127) † ; n = 2 <dl all <dl data presented as median (interquartile range). *p < 0.05; † p < 0.01; comparison by mann-whitney u-test. csf: cerebrospinal fluid. ebv: epstein-barr virus y. achermann°, c. spormann, c. kolling, c. remschmidt, j. wüst, b. simmen, m. vogt (baar, zurich, ch) objectives: elbow arthroplasty is increasingly used for treatment of rheumatic or posttraumatic arthritis. data on epidemiology, characteristics and treatment outcome of elbow prosthetic joint infection (pji) is limited. furthermore, no validated therapeutic algorithm exist as for hip or knee pji. we analyzed all pji, which occurred in a cohort of implanted elbow prostheses during a 13-year-period. methods: between 01/1994 and 12/2007, all cases with implanted elbow prosthesis at our institution were retrospectively included. elbow pji was defined as visible purulence, acute inflammation on histopathology, sinus tract or microbial growth in periprosthetic tissue. patients were regularly follow-up at outpatient orthopedic visits and were in addition recently contacted by phone. a kaplan-meier survival analysis was performed. results: during the study period, 358 elbow prostheses were implanted. overall, 25 of 358 cases (7%) developed elbow pji (median age 61 y, range 41−82 y, 40% males). among them, 14 (56%) had a rheumatic disorder and 11 (44%) a posttraumatic arthritis. 12 infections were early ( 3 months), 3 were delayed (3−24 months) and 10 were late ( 24 months). more infections were acquired intraoperatively (n = 15, 60%) than haematogenously (n = 10, 40%). the following pathogens were cultured: staphylococcus aureus (n = 11), coagulase-negative staphylococci (n = 7), streptococcus agalactiae (n = 2), corynebacterium sp. (n = 1), enterococcus sp. (n = 1), enterobacter cloacae (n = 1), mixed infections (n = 1) and culture-negative (n = 1). treatment approaches included débridement with implant retention (n = 19), one-stage exchange (n = 2), two-stage exchange (n = 1), resection arthroplasty (1) and antibiotics only (n = 2). at follow-up, 16 (64%) patients were free of infection (median follow-up time 2.6 y, range 0.7−11.3 y) and 9 (36%) had a relapse (median time to infection 0.4 y, range 0.1−4.0 y). one patient died due to infectious endocarditis with secondary haematogenous elbow pji. the relapse-free survival (95% confidence interval) was 75% (58%-93%) after 1 year, 70% (52%-89%) after 2 years, 62% (39%-85%) after 3 years and 52% (27%-78%) after 4 years. the infection incidence after elbow arthroplasty was higher (7%) than reported after hip (<1%) or knee arthroplasty (<2%). most infections (48%) manifested early ( 3 months). underlying rheumatic disorder were common in elbow pji (58%). the relapse-free survival after elbow pji was 75%, 70%, 62% and 52% after 1, 2, 3 and 4 years. e.a.n. oostdijk°, a.m.g.a. de smet, h.e.m. blok, e.s. thieme groen, j.a.j.w. kluytmans, m.j.m. bonten (utrecht, breda, nl) introduction: selective digestive tract decontamination (sdd) and selective oropharyngeal decontamination (sod) aim to eradicate gram-negative bacteria (gnb) from the intestinal en respiratory tract in intensive-care-unit (icu) patients. we aimed to quantify effects of sdd and sod on bacterial ecology in 13 icus that participated in a clustered group-randomised cross over study in the netherlands, in which sdd, sod or standard care (sc) was used during consecutive periods of 6 months (nejm 2009; 360:20) . the order of periods was randomised per icu. methods: point prevalence surveys of rectal and respiratory samples were performed once monthly in all patients in icu (receiving or not receiving sod/sdd). selective media were used to detect gnb and mic testing was done for ceftazidime (cft), ciprofloxacin (cip) and tobramycin (tob). effects of sdd on rectal carriage were determined by comparing data from 6 sdd months in each icu to data from all months preceding and following (in which sod or sc was given). combined effects of sdd/sod on respiratory tract carriage were determined by comparing data from 12 months sdd/sod (in any order) to data from the 6 sc months before and afterwards. results: 2583 rectal and 2023 respiratory tract samples were analysed. during sdd average proportions of patients colonised with gnb resistant to either cft, tob, cip were 5%, 7% and 7% and these proportions were 15%, 13% and 13% during the post-intervention period (p < 0.05 in each case). this effect is most explicit for cft with resistance levels increasing from 2.6% in the last month of sdd to 11.4% in the 1st month after sdd (p < 0.05). during sdd and sod resistance levels in respiratory tract samples were <6% for all three antibiotics, but prevalence increased gradually (for cft and tob resistance; p < 0.05 for trend). after discontinuation of sdd and sod average proportions increased to levels >10% for all three antibiotics (p < 0.05 in each case). cft resistant isolates in rectal samples mainly included enterobacteriaceae not being escherichia coli and klebsiella spp, whereas tob and cip resistant isolates mainly included e. coli. conclusion: sod and sdd have marked effects on the bacterial ecology in an icu with a rapid and persistent increase in resistance after intervention. antibiotic resistance remains a major concern associated with these infection control measures. o391 throwing caution to the winds? three cases of anaphylaxis to chlorhexidine coated central venous catheters from a regional cardiac centre in northwestern england this blaoxa-58-producing clone showed resistance to several b-lactams (including imipemem), susceptibility to ceftazidime, netilmicin and minocycline, and variable susceptibility to meropenem, cefepime, and aztreonam. mics for colistin and tigecycline ranged from >16 mg/l and from 0.25−4 mg/l, respectively. all oxa-58-producing isolates presented the isaba3 downstream of the blaoxa-58 gene. hybridisation assays revealed a plasmidic location for the blaoxa-58 gene with ca 90kb. plasmid sequencing showed an isaba3-like truncated at the 3 end upstream of the blaoxa-58 gene, a fact that may explain the observed negative carbapenemase-production bioassay. conclusion: blaoxa-58-carrying a. baumannii is, apparently, more ancient than initially imagined. although undetected from 2004 onwards, the fact that it possessed a non-expressible gene, due to alterations in the promoter region, suggests that this information might have been incorporated from a still unidentified source. twenty-seven (45%) were male. isolates were recovered from respiratory secretions (33 isolates, 55.0%), blood (11, 18.3%), urine (7, 11.7%), catheter (5, 8.3%) and other secretions (4, 6.7%). only 24 (40.0%) of 60 patients received appropriate antimicrobial therapy either with polymyxin b (79.2%), ampicillin-sulbactam (12.5%) or tigecycline (8.3%). overall 30-day mortality of patients with crab was 50%. mortality rates were 3.2 per 1000-patient/day. these rates were significantly higher among patients who have not received appropriate therapy (1.2 per 1000-patient/day) compared with those who have received it (0.3 per 1000-patient/day; p = 0.001; figure 1 ). in the cox regression model only receiving appropriate treatment (hazard ratio [hr] 3.29; 95% confidence interval [ci] 1.35−8.02); p = 0.009) was independently associated with 30-mortality. positive blood culture for crab remained in the final model (hr 1.85; 95% ci 0.86−4.00; p = 0.12). all 25 isolates submitted to pcr were positive for blaoxa-23. all these isolates were susceptible to polymyxin b and tigecycline. conclusion: high 30-day mortality occurred in this icu outbreak. many patients did not receive appropriate therapy, which significantly increased mortality. other clinical risk factors for mortality in this outbreak are currently under investigation. acinetobacter baumannii in norwegian strain collections reveal major discrepancies to phenotypic identification and the presence of carbapenemase-producing clonal lineages baumannii isolates the per-1 gene was identified in 18 (23%). the similarity of the bands were calculated according to "dice smilarity coefficients" and all per-1 positive isolates were found as clonally related. conclusion: in our study the prevalence of per-1 was lower than the previous studies. but the presence of high ceftazidime resistance rates among these isolates may indicate the presence of other beta-lactamases. dna analysis by pfge and rapd revealed an outbreak caused by a unique clone. detection of clonal related isolates among different services may be because of the treatment of these patients at the same services before and this may explain the spread of per-1 positive strains.o442 resistance genomic islands related to abar1 are common in acinetobacter baumannii strains belonging to european clone i l. krizova°, m. maixnerova, l. dijkshoorn, a. nemec (prague, cz; leiden, nl) objective: acinetobacter baumannii strains belonging to european (eu) clone i are commonly resistant to multiple antimicrobial agents. a number of resistance genes were recently detected on an 86-kb genomic resistance island (abar1) inserted in the atpase gene of eu clone i strain aye. the aim of this study was to assess the presence of abar1related structures in epidemiologically unrelated strains of eu clone i. methods: the study set included 25 multi-drug resistant (mdr) strains of eu clone i collected in 19 european countries in 1978-2004 and 10 genotypically unique, fully susceptible strains. using pcr, all strains were investigated for the presence of the atpase gene and for nine genes found to be associated with abar1. furthermore, the strains were tested for the disruption of the atpase gene using pcr primers directed against the 3 and 5 ends of this gene. strains with the disrupted gene were investigated for the presence and structure of the atpase gene-abar1 connecting regions using pcr mapping and rflp. pcr primers were derived from the known sequence of strain aye. results: all strains were positive for the atpase gene. the 10 susceptible strains had an intact atpase gene whereas all mdr strains failed to produce the expected amplicon in the atpase disruption test. all eu clone i strains yielded positive results for the atpase gene-abar1 connecting regions, the structure of which corresponded to those of aye. these findings suggest the presence of atpase integrated elements in clone i strains, the integration of which had invariably taken place at the same locus site. none of the abar1-associated resistance genes were found in any of the susceptible strains. in contrast, the mdr strains harboured the following abar1-associated genes (% positive strains): aacc1 (21), aada1 (21), aadb (4), apha1 (21) stra (3), mera (20), teta (18), cat (23), the gene encoding heavy metal detoxification protein (25). individual mdr strains carried from one to nine abar1-associated genes in 11 different combinations. there was a good correlation between the content of resistance genes and resistance phenotypes. conclusion: genetic structures related to abar1 are common in strains belonging to eu clone i. the heterogeneity of resistance patterns in this clone is likely to result from the variations in the content of abar1related structures. supported by grant 310/08/1747 of the grant agency of the czech republic. objectives: to study the differences in mutation frequency and evaluate the possible correlations between drug resistance development and mutation rate in acinetobacter baumannii (ab). the mutation frequency (mf) of rifampicin (rif) resistance was used as a surrogate measure of differences in mutation rate and for detection of the presence of mutator phenotype. 10-and 100-fold higher when larvae were infected with atcc17978 and sdf, respectively. thus, the sdf genome was used as reference genome to identify functions acquired by pathogenic strains with a possible role in antibiotic resistance and pathogenicity. sixty-two clusters, corresponding to almost 870 cdss, were identified in the acicu and aye genomes (and partially in atcc17978) that were absent in sdf. this study found that targeted interventions that reduce the use of quin were associated with a decrease of the quin resistance rate in e. coli. e. velasco°, w. espelage, i. noll, a. barger, t. eckmanns (berlin, de) objectives: growing populations of older and immunocompromised patients, changes in epidemiology and unchecked use of antibiotics can led to a rise in consumption as well as resistance to certain treatments. medical doctors (mds) often have an important role alongside contributing factors. we conducted a national survey of mds in germany on their behaviours and expectations for intervention. we aimed to assess md behaviours with and influences on antibiotic prescribing and the potential for related interventions that address antibiotic resistance.methods: a representative sample comprised 10,610 mds with differing practice specialties, from both stationary and ambulatory settings (respectively: 36% and 0% internists, 0% and 54% general practitioners, 32% and 11% surgery, 3% and 4% ear/nose/throat, 10% and 10% paediatrics, 5% and 3% urology, 9% and 13% gynaecology, 2% and 4% dermatology, 3% and <1% other) in 15 federal states. we developed study questions to capture baseline information on mds and their practice with antibiotics. questions also focused on selected influences that may affect behaviour in practice. other questions solicited opinions about interventions that may improve practice. mailed questionnaires were distributed to participants via state medical associations. results: among survey respondents (n = 3,613; response rate = 34%), 66% reported that they prescribe antibiotics daily, and 90% indicated they do so at least weekly. of all surveyed mds, 60% reported that they think their own prescribing practice has an influence on antibiotic resistance in their region. of all mds, 83% found it "important" to continually improve use of antibiotics through industry independent experts providing consultation, audits and feedback. of all mds, 96% found it "important" to have provision of regional coverage of antibiotic resistance with appropriate feedback for practicing mds, and 82% found it "important" to have provision of antibiotic regulations of prescriptions with appropriate feedback for practicing mds. (results in table 1 .) -a not all results shown and remaining percentages are as follows: a closed three category scale was used for options "yes", "no", "do not know". a closed four category scale was used with options "very important", "important", "less important" and "not important". a closed five category scale was used for options "daily", "weekly", "monthly", "seldom" and "never". a closed five category scale was used for options "strongly agree", "agree", "neutral", "disagree" and "strongly disagree". objectives: to investigate the mlsb and tetracycline resistance and the emm gene distribution among the invasive streptococcus pyogenes (gas) strains. methods: between january 2006 and december 2008, a total of 991 strains responsible for invasive infections for adult patients were sent to the french national reference center for streptococci to be studied. antibiotic susceptibility testing was done by disk diffusion method according to the ca-sfm guidelines. mics were determined by e-test method. streptococcal emm sequence was done according to the cdc protocol. detection of macrolide and tetracycline resistance genes: erm(b), erm(tr), mef(a), tet(m), tet(o), tet(k), and tet(l) was performed by pcr. results: among the 991 streptococcus pyogenes invasive strains; more than ten different emm-types were identified. the most frequent emm sequence types were emm1, emm28 and emm89. a total of 80 strains (8%) were resistant to erythromycin. erythromycin resistance prevalence had decreased during the three years period (12. 2%-2006, 7.6%-2007, 5.5%-2008) . 69 had an mlsb constitutive (65 strains) or inducible (4 strains) phenotype due to erm(b) or erm(tr) resistance gene. 11 with the m phenotype and mef(a) gene were susceptible to clindamycin. among the 132 (13.3%) tetracycline resistant isolates tet(m), tet(o) and tet(l) genes were detected in 86, 27 and 19 strains, respectively. tetracycline resistance prevalence had also decreased during the three years period (16. 8%-2006, 14%-2007, 8.7%-2008) . conclusion: most of the invasive french gas isolates remained erythromycin and tetracycline susceptiple during three years. nontheless, the resistance rates have had the tendency to decrease slightly. taking into account the resistance trends helps to guide the therapy for penicillin-allergic patients. objectives: during a survey on antimicrobial susceptibility in betahaemolytic group c and g streptococci (gcgs) from portugal, a macrolide resistance rate higher than previously reported in other european countries was found (22%) among s. dysgalactiae subsp. equisimilis isolates. to gain further insights into the resistance mechanisms involved and the clonal structure of the resistant population, we undertook the phenotypic and molecular characterisation of macrolide resistant s. dysgalactiae subsp. equisimilis isolates and compared it with the susceptible population. methods: antimicrobial susceptibility testing and macrolide resistance phenotype were determined by disk diffusion. all the macrolideresistant isolates were further characterised by mic testing and genotype determination by pcr. a combination of emm typing and pulsed-field gel electrophoresis (pfge) was used to type the population and the simpson's index of diversity (sid) with 95% confidence intervals was calculated as previously described.results: a total of 69 isolates were resistant to erythromycin (mic range, 4 to >256 ug/ml). the vast majority of isolates presented a mlsb phenotype (n = 64) and carried the erm(a) gene (n = 55), while the mefencoded m-phenotype was expressed by only 5 isolates. among resistant isolates, 13 distinct emm types were found distributed by 10 pfge clusters that overlapped with the main clusters detected in the susceptible population. the emm types stg480, stg6, stg485 and stg2078 accounted for approximately two thirds of the resistant isolates. pfge did not always separate neither macrolide-resistant from susceptible isolates nor erm(b) and mef(a) from the prevailing erm(a) isolates. the sids of emm and pfge calculated for resistant isolates were not statistical different from the overall population. the two most prominent mls resistant lineages were one with stg480/erm(a) isolates (n = 8) and stg485/mef(a) (n = 3), and another including stg2078/erm(a) (n = 8).conclusion: although most of the resistant isolates presented a mlsb phenotype and carried an erm(a) gene, molecular typing revealed extensive diversity in both emm types and pfge clones. macrolide resistance had a polyclonal origin, with resistance emerging among most susceptible clones. monitoring of macrolide resistance patterns in s. dysgalactiae subsp. equisimilis is essential as this pathogen is increasingly recognised as an important human pathogen. a.s. simões°, r. sá-leão, s. nunes, n. frazão, a. tavares, h. de lencastre (oeiras, pt) while performing pneumococcal nasopharyngeal colonisation surveillance studies among children attending day care centres (dcc) in portugal, we observed that the rate of strains with penicillin mic 2 ug/ml more than tripled from 1.8% in 2006 to 6% in 2007 (p = 0.002). the aim of this study was to characterise the 20 isolates recovered in 2007 which had a mic to penicillin 2 ug/ml. methods: pneumococci were isolated and identified on the basis of selective growth on gentamycin blood agar plates, optochin susceptibility, colony morphology, and alfa-haemolysis. susceptibility to antimicrobials agents was performed according to the clsi recommendations and definitions. strains were serotyped by the quellung reaction and/or multiplex pcr using specific primers for each serotype. pulsed-field gel electrophoresis (pfge), after restriction of the total dna with smai, was performed to compare genetic backgrounds. results: sixteen of the 20 isolates belonged to serotype 14, three were serotype 19a and one was of serotype 15a. strains of serotype 14 were also resistant to sulfamethoxazole-trimethoprim and belonged to a single pfge cluster identified as clone spain 9v st156. the penicillin resistant serotype 14 strains were isolated in two dcc, from nine children vaccinated with the 7-valent pneumococcal conjugate vaccine (pcv7), four non-vaccinated children and three children with unknown vaccination status. five of these carriers had received antibiotics recently. in these two dcc the overall proportion of children vaccinated with pcv7 was 64%; 27% of the children had received antibiotics within the previous month and 16% had received three or more courses of antibiotics in the last six months. since the introduction of the pcv7 in portugal, in june 2001, the proportion of penicillin resistant pneumococci recovered from colonisation has been stable (c.a. 2%). the sudden increase in the levels of penicillin resistance observed in the 2007 surveillance study was found to be largely due to the dissemination of clone spain 9v st156 serotype 14 variant in two dcc with high consumption of antibiotics. the observations suggest a combination of high antibiotic selective pressure and transmission rates resulting in an outbreak-like situation with a penicillin resistant vaccine type clone being disseminated among children in day care despite use of pcv7. background: beside target mutation, active efflux is another common resistance mechanism to fluoroquinolones (fq) in s. pneumoniae. two main efflux systems have been described so far, namely pmra (member of the mfs superfamily) and the two abc transporters pata/patb. we have studied the inducibility of pmra, pata and patb genes expression when bacteria are exposed to subinhibitory concentrations of fq. we used a wild-type sensitive strain (atcc49619), two clinical strains resistant to fq (sp13 and sp295), and two efflux mutants (sp334 and sp335; selected in vitro after exposure to ciprofloxacin [jac 2007, 60:965-972] ). mic were determined according to clsi. induction was obtained by growing bacteria in todd-hewitt broth added by half the mic of each fq (cip, nor, lvx, mxf, gmf) for 4 h at 37ºc in 5% co2 atmosphere. expression levels of pmra, pata and patb genes were determined by real-time pcr. reversibility of induction was tested by re-cultivating bacteria for 4 h in drug-free medium. results: antimicrobial susceptibilities for cip and mxf and gene expression at basal level and after exposure to these fq are shown in the in women with single infection, the most common hpv types were hpv-6 and hpv-16, followed by hpv-51, hpv-31, hpv-53 and hpv-66, whereas in women with multiple infections hpv-6 was the most commonly detected type, followed by hpv-66, hpv-31, hpv-16 and hpv-51. a different distribution of hpv types and a higher rate of multiple infections were observed in young vs. older women, suggesting the existence of a natural selection of hpvs which preserve a better fitness. high-risk hpvs were detected in all high-grade cervical intraepithelial lesions, with hpv-16, hpv-18, hpv-31, and hpv-51 as the most frequent types. however, hr-hpv types were detected also in a high rate of women with a negative pap test as well as in women with a negative cervical biopsy, suggesting the need to improve the accuracy of available cervical cancer screening tests. the results of this study, which provide information on the epidemiology of hpv infection and type distribution in women from south italy, should be taken into consideration in the implementation of local vaccination programs. objectives: chromosomal integration of the hhv-6 genome (cihhv-6) into the human genome occurs in 1−2% of healthy individuals and leads to persistently high levels of hhv-6 pcr copy numbers in blood and tissue. consequently, this may be interpreted as persistent active hhv-6 infection. although hhv-6 mrna has been detected in a few individuals with cihhv-6, there is no evidence of replication of viral particles up to now. viral cultures have shown negative results. so, cihhv-6 is thought not to be linked to any disease. methods: we performed hhv-6 antigen detection in pbmcs of 4 individuals with fish proven cihhv-6 by means of antibodies directed against hhv-6 variant a and b (indirect immunoperoxidase staining). results: in 2 unrelated female adolescents (both with cihhv-6 variant a) we detected hhv-6 antigen. one patient is suffering from recurrent parotitis since 5 years and from hypoimmunoglobulinaemia. the other patient (15a) was treated with allogeneic bone marrow transplantation (bmt) for acute myeloid leukaemia (aml) and acquired cihhv-6 from the healthy donor. so, cihhv-6 is only found in blood cells. in the latter patient only symptoms attributable to the post bmt course have been observed (prolonged mixed haematological chimerism, protracted mucositis, transient hypertension and transient neuropathy). at the time of antigen detection 5 years after bmt the patient was clinically well. in 2 individuals (a girl after fatal myocarditis and her healthy father − both with variant b) no hhv-6 antigen has been detected. discussion: up to now cihhv-6 is considered not to cause any disease. for the first time we show the expression of hhv-6 antigen, which indicates the replication of viral particles. this might have a pathophysiological impact. sixty-seven % of cases with ebv meningo-encephalitis have detectable viral dna amounts in csf and serum, whereas neuroborreliosis patients do not. cases with primary ebv meningoencephalitis have increased systemic leukocytosis, with higher lymphocyte, and monocyte levels compared to neuroborreliosis patients.o506 incidence of post-herpetic neuralgia in treated and untreated patients with herpes zoster followed for 1 year in an italian prospective cohort: preliminary results g. parruti°, f. sozio, c. rebuzzi, m. tontodonati, e. polilli, a. agostinone, a. manna, f. di masi, a. consorte, g. congedo, l. cosentino, d. d'antonio, l. pippa, l. manzoli, c. granchelli (pescara, chieti, it) objectives: a large prospective cohort of patients with herpes zoster (hz) was enrolled between may 2006 and june 2008 in pescara, italy, with a planned 1-year follow-up after clinically and/or molecularly assessed diagnosis. aim of the study was to evaluate predictors of prolonged acute course and/or incidence of post-herpetic nevralgia (phn). methods: data from all enrolled patients were collected by a network of 51 general practitioners. suspected cases and patients with intense acute pain were referred to our institution for immediate evaluation. clinical and demographic information was mandatory at baseline, as photographs of enrolled patients. for uncertain cases, varicella-zoster virus (vzv) antibodies and vzv dna pcr on plasma and/or vesicular eluates (whenever available) were performed. follow-up data were collected at outpatient control visits or by phone calls at 1, 3, 6 and 12 months after onset of hz. phn was diagnosed when pain persisted or relapsed at least one month after complete clearing of dermatomeric lesions. adverse events other than pain were classified according to who grading scale and reported if 2. all statistical calculations were performed by stata 8.0 software package. results: 523 patients were enrolled, 306 (58.5%) females, with a mean age of 57.7 years, 1-year follow-up data being now available for 489. hz was localised at thorax in 45.4% and head in 20.7%; pain in the acute phase was reported as intense or very intense by 127 (25.97%) patients; 54 (11.04%) patients were referred for molecular diagnosis as clinically uncertain, 37 (68.5%) being confirmed as vzv-related cases. forty eight (9.82%) patients were not prescribed any antiviral drug at diagnosis by referring physicians, in spite of extensive support in the study plan. during follow-up, 163 (33.3%) patients reported any type of adverse event (at a mean of 91.2±74.8 days), including 93 (19.02%) patients reporting phn. phn was significantly more frequent in untreated vs treated patients (37.5% vs 17.0%, p = 0.001), as were total adverse events (54.2% vs 31.1%, p = 0.001). untreated patients did not significantly differ from those treated by age (56.3% vs 57.7%, p = 0.66) and sex (females vs males 11.9% vs 6.9%, p = 0.056), whereas they complained for more intense pain (15.0% vs 8.0%, p = 0.024) at presentation. conclusion: our study confirms the importance of early diagnosis and prompt antiviral treatment at the onset of hz in order to minimise the risk of phn. methods: faecal specimens (n = 78) from apparently healthy and diarrheic calves (aged <1 year) were collected per-rectally and investigated for detection of group a rotavirus by antigen capture elisa (generic assay, germany). elisa positive specimens (n = 3) were investigated further for molecular characterisation. genotyping of borv-a strains was carried out on dsrna extracted from 10% pbs faecal suspensions by a nested and/or heminested rt-pcr key: cord-004675-n8mlxe7p authors: nan title: 2019 cis annual meeting: immune deficiency & dysregulation north american conference date: 2019-02-26 journal: j clin immunol doi: 10.1007/s10875-019-00597-5 sha: doc_id: 4675 cord_uid: n8mlxe7p nan a 34 y.o. female was referred to our clinic with a history of multilineage cytopenias/evans syndrome, a history of idiopathic thrombocytopenic purpura, hemolytic anemia, chronic neutropenia, lymphopenia, and hypogammaglobulinemia treated with ivig. our patient was healthy until she was 8 years old; at that time, she developed joint pain, rash, and bruising. she was found to have evans syndrome with idiopathic thrombocytopenic purpura (itp), neutropenia, and lymphopenia. she was initially diagnosed with lupus and was given steroids. her bone marrow biopsy did not conclude myelokathesis. when she was 15 years old, she remained thrombopenic and was started on high dose of immunoglobulin replacement therapy. in 2012 (29 years old), she developed polyarthritis in her upper and lower extremities. in 2013 (30 years old), she had a severe nosebleed, for which she was admitted and treated with amicar twice; her platelets were found to be 2,000 k/ul. she received rituximab weekly for 4 weeks resulting in an increase of platelet count to 90-100k/ul. she recently (march 2017) had a splenectomy to remove her large spleen, and since then, her platelets have rebounded to 400-500k/ul. in 2015, she was placed on long-term immunoglobulin replacement therapy after being hospitalized for bilateral pneumonia for 5 nights requiring iv antibiotics for treatment. in 2017, she developed and was treated for another pneumonia. her family history is characterized by multiple members with autoimmune multilineage cytopenia as well as autoimmune diseases such as multiple sclerosis (mother), thyroiditis and enteropathy. on physical examination, she did not present with any warts and the remainder of her physical examination being unremarkable, except for her scar from her splenectomy and a cervical lymphadenopathy. immunologic evaluations showed igg 601 mg/dl, iga <5 mg/dl, and igm 208 mg/dl. cbc with differential and lymphocyte screen were as follows (cell/mm3): wbc 12.3 x103, hemoglobin 10.2 g/dl, platelets 503 x 103; 3 % neutrophils (anc: 300), 82% lymphocytes, 10% monocytes, 0% eosinophils; absolute total t-cell number was 8884 (750-2500 cells/mcl), cd4+ t-cells 6554 (480-1700cells/mcl), cd8+ t-cells 2185 (180-1000cells/mcl), natural killer cells 206 (135-525 cells/ mcl), and absolute number of b cells was 996 (75-375 cells/ mcl). she came to our clinic with her sister, who also had multilineage cytopenia and hypogammaglobulinemia, treated with monthly ivig; and her nephew whom had neutropenia. based on this family presentation all three underwent whole exome sequencing (wes). the patient, the patients sister and the patients nephew were all found to have a variant on cxcr4 (frameshift mutation on chromosome 2, p.val324fs; refnt: tca; altnt: t). as an important note, the patient had a bone marrow biopsy, which did not conclude myelokathesis. in summary, our patient with trilineage cytopenia and hypogammaglobulinemia, without any warts or myelokathexis, had whim syndrome (warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis), which was discovered by studying her wes. with the identification of her specific diagnosis, this allowed us to discuss the potential future indication of plerifaxor (antagonist of the alpha chemokine receptor cxcr4). and equally important, we discussed family planning and future pregnancies given that the mutation is autosomal-dominant. (4) submission id#555017 taha al-shaikhly, mbchb 1 , kathleen mohan, arnp 2 , matthew basiaga, do, msce 3 introduction: complement component-3 (c3) is shared by the classical, lectin and alternative complement activation pathways. c3, a major opsonin, facilitates phagocytosis of encapsulated microorganisms. inherited c3 deficiency is rare and is associated with increased risk of bacterial infections. subjects with connective tissue diseases (ctd) and c3 nephritic factors can have low and occasionally undetectable c3 levels, yet they are at an underappreciated infectious risk. we hypothesize that excessive c3 consumption in secondary complement deficiency disorders (scd) is associated with higher risk of bacterial infections similar to primary complement deficiency disorders (pcd). objectives: to compare the rate of bacterial infections between pcd and scd patients and evaluate the association between c3 level and bacterial infection risk. methods: we performed a retrospective cohort study. subjects with an undetectable complement activity (ch50) or any of the complement components measured at seattle childrens hospital from 2002-2018 were included in our study. we recorded the number of infections, observation periods, diagnosis (pcd, scd and its underlying etiology), lowest complement component levels, and the immunosuppressive agents used. the date of birth, and date of lowest c3 level were considered as start points to calculate the observation periods for pcd and scd subjects respectively. infections requiring hospitalization or parenteral antibiotics were categorized as serious bacterial infections (sbis). descriptive analyses were performed to determine medians and ranges for continuous variables. differences in rates of bacterial infection were assessed using the chi-square and kruskal-wallis tests when appropriate. among subjects with ctds, we treated every c3 measurement as a single observation (n=1,197) and studied the association between c3 concentration and the 30-day odds of having a sbi. multivariable logistic regression was performed to determine infection risk based on c3 level while controlling for contributing factors. results: we identified 14 subjects with pcd, and 52 subjects with scd. scd consisted of three subgroups (ctd-related (n=44), nephritic factor-related (n=2), and infection-related (n=6)). collectively, ctd subjects had a lower median rate of sbi compared to pcd subjects (p = 0.004). subjects with ctd and c3 level <40 have higher rate of bacterial infection (of any severity) (p = 0.002) and of sbi (p = 0.004) when compared to ctd subjects with c3 >=40 at the beginning of observation period ( figure 1 ). while controlling for immunosuppression level 1 pediatric resident, baystate medical center 2 faculty advisor, baystate medical center introduction: zap70 codes for a 619-amino acid enzyme, zap70, a member of the syk-protein tyrosine kinase family that plays an important role in t cell development and activation. zap70 is phosphorylated at tyrosine kinase residues upon t cell receptor (tcr) stimulation resulting in tcr-mediated signal transduction with src family kinases. zap70 deficiency results in a rare t+b+ nk+ severe combined immunodeficiency (scid). we report a novel compound heterozygous mutation in zap70 leading to presumed absent zap70 function in an infant with a normal trec newborn screen and scid. case description: the patient is a term, fully immunized female, born to non-consanguineous parents who was hospitalized for rsv bronchiolitis at 2 mo. at 4 mo she developed an erythematous, papular rash on her face and extremities, nonresponsive to topical antifungal therapy. at 6 mo she was re-hospitalized with rsv bronchiolitis and subsequently treated with multiple courses of antibiotics for presumed bacterial pneumonia followed by albuterol and oral steroids for possible reactive airways disease. during this course of treatment, her rash resolved. at 8 mo she presented with failure to thrive (wt <0.1% for age), multifocal pneumonia and respiratory failure requiring intubation. bronchial alveolar lavage confirmed pneumocystis jiroveci pneumonia prompting an immune evaluation. total immunoglobulins were normal for age, however antibody titers to tetanus, diphtheria and streptococcus pneumoniae were absent. lymphocyte enumeration revealed elevated cd4 t cells and markedly diminished cd8 t cells, normal b and nk cells. t cell proliferation to mitogens (pha, pwm) and antigens (candida, tetanus) was absent, however t cells proliferated normally to stimulation with pma and ionomycin. trec number was normal by newborn screening, but was 2 std deviations below the mean and would have resulted in a positive screen upon repeat. invitae 18 gene scid panel revealed two variants of unknown significance, c.109c>g (p.arg37gly) leading to substitution of arg with gly and c.1529_1532dupgcat (p.ile511metfs*65) resulting in a premature translational stop signal expected to disrupt the last 109 amino acids of zap70 protein. parental sequencing revealed these variants to be on opposite chromosomes. the patient was successfully treated for pjp pneumonia and has since successfully engrafted a 9/10 matched unrelated donor stem cell transplant. discussion: we report a novel compound heterozygous mutation in zap70 which we presume led to t+ b+ nk+ scid. our patients clinical presentation of failure to thrive, recurrent lower respiratory tract infections, dermatologic findings and pjp pneumonia are consistent with previously reported cases of zap70 scid. her paucity of cd8 t cells, abundance of cd4 t cells and absent proliferation to mitogens are also consistent with previously described cases of zap70. normal proliferation of t cells when bypassing the tcr by stimulating cells with ionomycin and pma confirms a defect in the tcr. we believe this is the second documented case of missed scid by newborn screen in ma since the implementation of trec screening in 2008. pediatric resident (pgy iii), goryeb children's hospital 2 attending physician, pediatric and adult asthma, allergy and immunology, llc introduction: acute otitis media (aom) is one of the most common reasons for antibiotic use in early childhood. we explored the challenges when aom fails traditional therapies and immunologic evaluation does not identify a commonly described immunodeficiency. case description: an eighteen-month-old male presented with 12 episodes of aom and recurrent purulent otorrhea requiring intravenous antibiotics. laboratory evaluation revealed a normal cbc, normal immunoglobulins (igg 588, iga 76, igm 63, ige 12) and igg subclasses. lymphocyte subset panel was normal. initial responses to dtap and prevnar boosters were normal, however, there was rapid decline to tetanus and pneumococcal antibody titers. a sub optimal response to haemophilus influenza type b vaccine was noted. although vaccinated twice for mmr, he never mounted mumps specific igg. mitogen response to pha was normal with decreased responses to cona and pokeweed and no detectable tetanus nor candida responses. further investigation revealed decreased non-class and class switched memory b-cells. the patient was recently vaccinated to pcv23 and at the present time has protective titers. discussion: it has been previously suggested that decreased memory b cells may contribute to decreased antibody responses to select vaccine antigens resulting in recurrent aom in children. our case supports the need to investigate beyond typical immunologic screening for immunodeficiencies. introduction: dna mismatch repair (mmr) system corrects replication errors in newly synthesized dna, and prevent recombination between dna sequences when they were not identical (1) . msh6 is a part of mmr genes, (2) (3) (4) . case: a ten-year-old girl presented with fever, brown spots on her skin, hair loss, recurrent pulmonary infections, arthritis on the left hand and right ankle. she has also been followed up with nf ( figure 1 ). there was a first-degree cousin marriage between her parents. physical examination revealed findings of pneumonia and nf. anti-nuclear antibody, anti-ndna, anti-dsdna, anti-histone, anti ro52 and anti-nucleosome antibodies were positive. in her immunologic assessment showed low igg and iga levels associated with high igm level ( table 1 ). the coexistence of nf, hyper igm syndrome, sle, were considered in the patient. intravenous ig (400 mg/kg, every 3 weeks) treatment was started due to hypogammaglobinemia. the frame shift mutation in exon 2 of the msh6 gene was detected in the boztug's laboratory. in the follow up period, she admitted at 11 years old with back pain. a mass in the left paravertebral area, related to the spinal canal and neural foramina, was detected at the l4-l5 levels in spinal mri. the lymphadenopathy around the liver and hilum and the left parietal bone lesions were developed within two months despite surgical excision of primary mass ( figure 2 ). as a result of pet examination; suvmax was found to be around 6.5 in the mass lesion in the paravertebral region and suvmax values did not exceed 2.5 in other lymphadenopathy and masses. atypical cellular infiltration suggesting neoplastic events, which were including small-medium size atypical pleomorphic mononuclear cells and t cells. since all these formations did not indicate definite cancer, chemotherapy was not started. interestingly, although chemotherapy was not given, progression stopped, and partial spontaneous regression was observed. discussion: the effect of msh6 mutations on patients may significantly vary with the inheritance pattern (2) . leukemias or lymphomas are not common in heterozygote mmr gene defects (5, 6) . however, homozygote mutations in mmr genes show a different pattern. wimmer and etzler proposed the new term constitutional mismatch repair-deficiency syndrome (cmmr-d) for patients who have a homozygous mutation in mmr (3) . cmmr-d characterized by development of childhood cancers, mainly hematological malignancies and/or brain tumors, as well as early-onset colorectal cancers, and neurofibromatosis type 1 (3) . bi-allelic germline mutations in any of the mmr genes in which msh6 is involved increases hematological malignancies by 15% (7, 8) . msh6 mutation has been associated with many cancers since its identification. leukemia, lymphoma, colorectal cancer, endometrial cancer, brain tumors are some of these cancer types (2) (3) (4) 9) . msh6 deficiency is an important disease that can affect different systems at the same time. there is a high risk of malignancy in the cases and therefore they must be closely monitored. this case has also shown that atypical lymphoproliferation may occur in msh6 homozygous mutant cases. (normal rage: 842-1943) background: advances in inborn errors of human immunity have supported the discovery of new syndromes that are marked by striking features of autoimmunity and immune dysregulation often associated with cytopenias, lymphoproliferation, and a predisposition to reticuloendothelial malignancies leading to evaluation with hematologists/oncologists. moreover, hematologists/oncologists have also seen an increasing use of effector cell-based therapies, checkpoint inhibitors, immunomodulatory and targeted therapies resulting in autoimmunity and hyperinflammatory complications. a working knowledge of clinical immunology could help practicing hematologists/oncologists in the identification and management of these conditions. objectives: to support the advancement of aspho members and the field by facilitating education regarding the best practices in diagnosis and management of immunological disorders. to create a platform for the development of collaborative clinical research in patients with hematological/oncological manifestations of immunological disorders or those requiring hematopoietic stem cell transplantation for a underlying immunological disorder. design/methods the aspho clinical immunology sig was initiated based on collaboration with the clinical immunology society (cis). aspho members who are pediatric hematology/ oncology clinicians, clinical researchers, and trainees are eligible to participate. we have established a steering committee with representatives from across the united states and canada with diverse clinical and research expertise. through regular teleconferences and annual in-person meetings, we have developed a platform to provide our members with a network of immunology resources to ensure a strong foundation of knowledge and tools to conduct clinical care and research pertaining to the diagnosis, evaluation, and treatment of patients with immunological disorders. results we currently support over 50 members within our online community. several educational initiatives have been successfully launched. we have submitted an invited review to pediatric blood and cancer which provides a case-based review of primary immune regulatory disorders. we hosted the first immunology for hematology oncology practice (i-hop) cased-based webinar series. this series features case-based discussions of patients with primary immunodeficiency disorders presented by fellow trainees and mentored by senior clinicians. we will also be hosting an aspho webinar focusing on the laboratory evaluation of primary immunodeficiencies and immune dysregulation syndromes. we have also begun the process of laying the groundwork for clinical research initiatives. conclusion: the aspho clinical immunology sig seeks to serve as a collaborative resource for pediatric hematology/oncology clinicians and researchers. through the development of educational and research initiatives, we envision improving the care of patients with immunological disorders that are often managed by pediatric hematologists/oncologists. moreover, we hope to broaden our understanding and application of clinical immunology within pediatric hematology/oncology. we hope that this successful initiative will serve as a blueprint for the development of future collaborations with other specialty societies and patient groups. autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced) is a rare autosomal recessive disease caused by aire gene mutations. clinical diagnosis is established by the presence of at least two components of the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and addisons disease. in europe, the classic presentation is widely recognized and nonendocrine autoimmune manifestations are rarely reported. a recent study of 35 american apeced patients demonstrated a more heterologous presentation, with many nonendocrine manifestations including urticarial eruption, hepatitis, gastritis, intestinal dysfunction, pneumonitis and sjogrens-like syndrome, all uncommon in european reports. within the american cohort, 80% of patients developed a mean of three non-triad manifestations before reaching the classic triad. finding of aire mutations and high-titer antiifn-autoantibodies is seen in both european and american cohorts. we present the case of two siblings, who demonstrate an apeced-like phenotype with both classical and atypical features. they share the same heterozygous c132+1_132+3delinsct aire mutation. the older, an eight-year-old boy, with history of prematurity, bronchopulmonary dysplasia and onychomadesis in infancy, came to medical attention at 16 months of age due to failure to thrive (ftt), in addition to fevers and urticarial rash lasting months after his mmr vaccine. the fevers resolved with anakinra, which was discontinued two years later due to pneumonia. from age 2-4 he developed an alps negative lymphadenopathy which self-resolved. lung issues include chronic cough, initially treated as asthma but with poor bronchodilator response, and frequent lung infections, including 1-2 pneumonias per year. at age five evaluation for ftt revealed growth hormone deficiency. two years later he was diagnosed with primary addisons disease. chronic abdominal discomfort, bloating, cyclical constipation/diarrhea, recurrent rashes, dystrophic nails, and sicca symptoms are also present. his sister, age five, shows ftt, but no growth hormone deficiency. at age one, she too developed a fever and rash syndrome lasting 3 months. severe gerd and constipation started in infancy and are ongoing. at age three she developed a transaminitis, initially diagnosed as ebv, but later thought to be autoimmune hepatitis. she has frequent viral respiratory infections, and pneumonia at age two. she has had a chronic cough, with poor bronchodilator response, for most of her life. evaluation of seizure at age three showed normal brain activity. brain mri revealed partial agenesis of the corpus callosum and microgyria. her brother has similar mri findings. both children have had developmental motor delay and poor tone. brain dysgenesis and neurodevelopmental delay has not previously been described in apeced. although there were both typical and atypical symptoms, the history in combination with genetic findings led to further investigation of an apeced-like syndrome. autoantibody testing confirmed high-titer antiifn-autoantibody typical of apeced in both children and hightiter bpifb1 autoantibodies found almost exclusively in apeced pneumonitis in the brother. whole exome sequencing and copy number variation analyses are underway to further evaluate the patients condition. this case demonstrates the importance of clinical presentation in the evaluation of genetic results and in the guidance of therapeutic management. (12) submission id#570047 rationale: infants with low t cell receptor excision circles (trec) born in queens, nassau, and suffolk counties are referred to our center for further evaluation. this study elucidates the demographic and laboratory characteristics of referred infants with transient or persistent idiopathic t cell lymphopenia (tcl) without clearly identified genetic or acquired etiology. methods: a retrospective analysis was performed from september 2010 (when trec screening started) through the end of december 2017. descriptive statistics were calculated for demographic and laboratory characteristics. t-test or mann-whitney tests were used to compare laboratory variables. pearson or spearman tests were used to determine correlation between initial trec levels and t cell counts. by definition, the cd3+, cd4+, and cd8+ populations of transient tcl patients normalize by age 1 year. results: eighteen infants with transient and 17 with persistent tcl were identified. males comprised 61.1% of the transient and 47.1% of the persistent tcl cohorts. whites comprised 11.1% of the transient and 35.3% of the persistent tcl cohorts. the mean initial trec levels did not differ between the transient and persistent cohorts (67.7 vs. 78.5 trecs/l of blood, p = 0.56). mean initial absolute counts of cd3+ (2149 vs. 1300 cells/l, p <0.0001), cd4+ (1462 vs. 922 cells/l, p <0.0001), and median initial absolute counts of cd8+ (524 vs. 309 cells/l, p = 0.0075), were higher for transient vs persistent cohorts. initial trec level did not correlate with initial cd3+, cd4+, or cd8+ absolute counts. the median age of resolution for the transient cohort was 121.5 days (range 23-244). the absolute cd3+, cd4+, or cd8+ counts rarely exceeded the reported median values for age, and remained closer or below the 5th percentile for age up to 1000 days of life. the majority of both transient and persistent tcl patients demonstrated unremarkable lymphocyte proliferation to mitogens. conclusion: our centers transient tcl cohort appears to be predominantly male and non-white, whereas the persistent tcl cohort is more evenly distributed by sex but still predominantly non-white. the transient cohort had lower initial trec levels, but higher initial t cell counts. both cohorts appear to have relatively intact in vitro function. introduction: primary immune deficiency disease (pidd) is typically considered a pediatric illness, although advances in treatment and diagnosis are changing this paradigm. currently, data on pidd in older patients are very limited. objectives: to characterize the prevalence of pidd among older individuals using a patient database maintained by the consortium of independent immunology clinics (ciic), comprised of 17 specialty immunology outpatient practices in the us. methods: patients with pidd were identified in the ciic database using icd-10 codes d80, d.80. 3, d80.4, d80.5, d80.6, d81.1, d81.2, d82.0, d82.3, and d83 .0. a total of 235 records from 11 geographically-diverse clinics were identified and characterized by age, gender, and pidd diagnosis. results: of the 235 pidd patients in the ciic registry, 73 (31%) were between 60-87 years of age (see figure) . within this age group, most patients were female (n=56, 77%). the most common diagnoses among patients >60 years of age included common variable immunodeficiency with predominant abnormalities of b-cell numbers and function (d83.0; n=41, 56%) and antibody deficiency with near normal immunoglobulins (d80. 6; n=14, 19%) . in comparison, the registry included 36 (15%) patients aged 0-19 years; this age group was predominantly male (n=23; 64%). the most common icd-10 codes within the younger cohort were relatively evenly distributed between hereditary hypogammaglobulinemia (d80.0), antibody deficiency with near normal immunoglobulins (d80. 6) , and common variable immunodeficiency with predominant abnormalities of b-cell numbers and function (d83.0). conclusions: our data suggest that pidd in patients over age 60 may be more prevalent than previously reported. additional research is needed to corroborate these findings, further characterize the nature of pidd in this population, and determine whether there are unique diagnostic and treatment considerations within this demographic. introduction/background: increased susceptibility to invasive infections with neisseria has been well documented in patients with deficiency of terminal complement proteins. the molecular attack complex is constructed with complement components c5 to c9. a deficiency in complement c6 has been described previously in both african american and south african populations. complement c6 deficiency is inherited in a co-dominant pattern, with multiple known mutations. we present a case of a 19-year-old, previously healthy male, who presented with invasive n. meningitides infection. he was found to have a novel mutation noted on genetic sequencing of the complement c6 gene. objective: we present the case of a 19-year-old, previously healthy male, who presented with invasive n. meningitides infection. on genetic sequencing, he was found to have three mutations of the complement c6 gene. two of which have been described previously, and a third novel mutation. methods: a 19-year-old male with no known history presented to us with a 3-hour history of emesis. he was found to be febrile, and quickly decompensated, developing septic shock. blood cultures were drawn, and within 12 hours grew n. meningitides. he was treated with broad spectrum antibiotics upon arrival, and subsequently narrowed to ceftriaxone. his hospital course was complicated by disseminated intravascular coagulation, as well as acute tubular necrosis, leading to endstage renal disease for which he is listed for kidney transplant. results: on immunodeficiency evaluation, he was noted to have an undetectable ch50 (<13, reference range 31-60). complement levels returned with c6 of 10.8 (reference range 28-69) and c1r of 41.5% (reference range 61-102%). complement c6 function screen returned at 0% (reference range 40.7-169%). all other complement levels were within normal limits. genetic sequencing showed the patient to be compound heterozygous for two of known four variants which have been reported to recur in african patients with complement c6 deficiency. this included c.821del and c.1879del, which are predicted to result in frameshift and premature protein termination. he was also found to be heterozygous for sequence c1202g>a, which results in amino acid substitution p.arg401lys. this variant is rare, with one large database reporting it in 6 of 276000 alleles, and not in a homozygous state. it has not been reported in a case of c6 complement deficiency previously. conclusions: we present the case of a previously healthy 19-year-old male with invasive meningococcal disease. he is compound heterozygous for two mutations that have been associated with total complement c6 deficiency; however, he was found to have subtotal c6 deficiency. furthermore, he has a third novel mutation of the complement c6 gene. further investigation is warranted on the significance of this finding and impact on relevance to possible kidney transplant. background: measuring the function of the classical pathway of complement activation is useful in several disease states, including complement deficiency, autoimmune conditions such as systemic lupus erythematosus and certain forms of nephritis. the original method for assessing classical pathway activity was the haemolytic ch50 method, but this assay can be time consuming and has reagent stability issues due to the use of sheep red blood cells. there can also be high lab-to-lab variability due to differences in the protocols used. here we report the assay characteristics of an automated, commercial, liposome-based assay to measure ch50 activity. we also compare the results obtained using the traditional haemolytic method with the automated, liposome-based method used on the spaplus turbidimetric analyser. methods: a linearity study was performed based on clsi guideline ep06-a. the linear range of the spaplus ch50 liposome assay was established by analysis of a series of sample dilutions and evaluation of results against pre-defined goals for recovery and %cv. precision was assessed based on clsi guideline ep05-a2 over 21 days. 4 samples with different ch50 activities (23.7-65.1 u/ml) were run in duplicate, with two runs per day using 3 reagent lots and 3 different analysers. interference analysis was performed by spiking haemoglobin, bilirubin, chyle, ascorbic acid or saline (as a control) into samples before measuring the ch50 activity. for the assay comparison study, sera from 125 routine patient samples were used. samples were collected from chulalongkorn hospital, faculty of medicine, chulalongkorn university, thailand. ch50 classical pathway activity was assessed using a haemolytic method and also using the liposome based ch50 assay for use on the spaplus turbidimetric analyser (the binding site ltd., birmingham, uk). c3 protein concentrations were also available for 116 of these samples. results: the liposome ch50 assay gives a linear response over the range 11.8-95.5 u/ml, covering the measuring range of the assay (12.0-95.0 u/ml) at the standard analyser dilution (neat). the within run, between run and between day %cvs were all 5.4%. the total %cv was 6.8% in all 4 samples. minimal interference was observed with the four common interferents tested. a significant correlation was observed between the two ch50 methods (p<0.0001, r=0.66, y=1.1xâ±0.1), with 90.4% agreement between the methods in determining whether patients were above or below the lower limit of the assay normal range. the 12 individuals in disagreement had normal ch50 results using the haemolytic method, and low ch50 values in the liposome assay. of these, c3 values were available for 10/12, and 5 had c3 concentrations below the lower limit of the assay normal range. conclusion: the liposome ch50 assay for use on the spaplus analyser has passed assay development guidelines based on those set out by the clsi for linearity, precision and interference, and there is a strong correlation between this automated assay and the haemolytic ch50 method used here. five additional patients with low c3 concentrations were defined as having a low ch50 using the spaplus liposome method compared to the haemolytic method. (18) submission id#580179 background/aims: rotavirus vaccine is a live viral vaccine that is part of the routine u.s. childhood immunization schedule. live viral vaccines administered to infants of mothers who received biologic medications during pregnancy can potentially cause vaccine-associated disease. infant death from disseminated mycobacterial infection after vaccination with bacille calmette-guerin (bcg) in infants whose mothers received infliximab during pregnancy has been reported. it is currently recommended that infants born to women who received biologic therapy during pregnancy not receive live viral vaccines, however there is a paucity of information regarding adverse events from live viral vaccines. we report two infants, born to mothers receiving infliximab during pregnancy, who tolerated the complete series of rotavirus vaccine. methods: two infants who received rotavirus vaccine and whose mothers received infliximab (monoclonal antibody against tumor necrosis factor alpha which blocks the inflammatory response) during pregnancy were identified and their charts were reviewed. each mothers chart was assessed for timing of the biologic doses during pregnancy and concurrent immunosuppressant therapy. results: the mother of the first infant had crohn's disease and received infliximab every 6 weeks throughout her pregnancy (final infusion at approximately 35 weeks estimated gestational age [ega] ). she did not take additional immunosuppressive drugs throughout her pregnancy. the infant was born at 39 weeks ega. the infant received rotavirus vaccine at 2, 4, and 6 months of age. the infant did not have coexisting medical conditions or recorded hospitalizations during the first year of life. there were no side effects from rotavirus vaccine documented during well child examinations. the childs growth was normal during the first year of life. the mother of the second infant also had crohn's disease and received infliximab infusions every six weeks during pregnancy until 27 weeks ega. additionally, she took mesalamine (anti-inflammatory) daily. the infant was born at 33 weeks ega. the baby had a brief and uncomplicated neonatal intensive care unit stay. she did not have medical conditions diagnosed at the time of birth, or in the first year of life. the child received rotavirus vaccination at 2, 4, and 6 months of chronological age, and the infant did not experience documented adverse reactions. the child presented to the emergency department twice in the first year of life: once for thrush at 10 months of age and once for viral gastroenteritis at 11 months of age. the childs growth curve was unremarkable. conclusions: we report two infants, whose mothers received infliximab during pregnancy, who safely tolerated the 3-dose series of rotavirus vaccination. neither infant in this case series suffered from minor or severe adverse events as a direct consequence of receiving rotavirus vaccine. this suggests that administration of rotavirus vaccine may be safe in infants whose mothers received biologic therapy. introduction: combined immunodeficiencies (cids) can arise from partial loss of function variants in recognized scid genes, which can lead to relative lymphopenia with poorly functioning and oligoclonal t cells. cids have been most commonly associated with variants of the rag genes, but other genes are also implicated. clinical symptoms may be less severe, and the onset generally is delayed, compared to typical scid presentations. case report: a 28-year-old female presented with a history of recurrent and progressively worsening infections involving multiple microorganisms and organs, starting in infancy and requiring frequent hospitalizations. bacterial or viral infections included rhinosinusitis, otitis media, herpetic stomatitis, dental abscesses, pneumonias, pulmonary mycobacterial abscesses, cmv hepatitis, urinary tract infections, dermal abscesses, and groin hidradenitis. fungal and yeast infections included cryptococcal meningitis, oral thrush, dermatophytosis of the face, osteomyelitis of a finger, and onychomycosis. laboratory tests in 2018 showed: mildly low t cell counts (791/ul) with a reversed ratio of cd4/cd8 t cells (0.22); almost absent b cells (2/ul) ; and low nk cell counts (19/ul). cd4+ t cells were mostly of the memory phenotype (87%). t cell development showed low counts of th17 cells. t-cell stimulation tests demonstrated poor proliferation responses (<30%) to concanavalin a, tetanus toxoid, and candida albicans, with near-normal responses to pokeweed (>13%) and pha (>84%). she had low ig levels (iga 72, igm 23, ige <2), except for igg (872mg /ml; due to replacement since early childhood). limited genetic evaluation at age 9 showed a heterozygous variant in the rag1 gene (g.36595918t>c, c.1064t>c, p.met355thr; nm_000448.2). discussion: loss of function variants in rag1 or rag2 genes are known to cause a t-b-nk+ type scid. more than 100 missense variants have been reported for rag1, with disease-associated variants predominantly in zinc binding regions. the rag1 missense variant in our patient also lies within the zinc binding region (amino acids 354-383). the variant is rare (mean allele frequency 0.0001521 in gnomad) and has been identified in at least one other individual with scid (t-, b cell-, nk+). although classified as a variant of unknown significance, occurrence in at least two individuals with deficiencies of t and b cells-within a functionally important rag1 domainsupports an interpretation that the variant may be pathogenic. most patients with cid with rag variants are either homozygous for a poorly functional allele or have one nonunfucitonal and a second, poorly functional allele. we detected only a single potentially pathogenic allele. our patient has decreased nk cells in addition to t and b cell defects. further genetic studies including whole exome sequencing, are planned to identify further variants in rag1 or other relevant genes. rationale: infants with low t cell receptor excision circles (trec) born in queens, nassau, and suffolk counties in new york were referred to northwell health for further evaluation after abnormal newborn screens. the demographic and immune parameters of infants with transient t cell lymphopenia (ttcl) without clearly identified genetic or acquired etiology are described. tcl is considered transient if the lymphopenia resolves by 12 months of age. similar data from the following infants with low lymphocytes (fill) program of the united states immunodeficiency network (usidnet) are presented. methods: a retrospective analysis of two separate patient cohorts with ttcl are described. cohorts include patients referred to a single center, northwell health, in ny from september 2010 to december 2017 and at usidnet using data tracked by fill from june 2011 to july 2018. results: out of 1,234 referrals at northwell, 18 infants with ttcl were identified. infants were predominantly male (61.1%) and non-caucasian (89.9%). out of 71 fill participants, 9 infants with ttcl were identified. infants were predominantly male (55.6%) and non-caucasian (55.6%). initial laboratory parameters for the northwell versus fill cohorts are summarized: a) median trec levels: 54.5 vs. 47.0 trec/l of blood; b) median absolute cd3+ count: 2135 vs. 1166 cells/l; c) median cd4+ count: 1460 vs. 777.0 cells/l; d) median absolute cd8+ count: 524.5 vs. 440.0 cells/l. initial naã¯ve cd4+ t cell information was available for 0 northwell and 5 fill infants (median 52%). mitogen proliferation studies were performed in 10 (55.6%) northwell and 6 (66.7%) fill infants with 90% of these northwell and 50% of these fill infants demonstrating normal proliferation. genetic testing, such as targeted genetic panels or chromosomal microarrays (cma), was performed in 5 northwell and 0 fill infants. no genetic or chromosomal aberrations were identified. whole exome sequencing (wes) was not performed in either cohort. 11 of 18 (61.1%) northwell and 7 of 9 (77.8%) fill infants did not receive the initial rotavirus vaccine. no fill infants were vaccinated but no adverse effects were reported in 5 of 18 (27.8%) northwell infants who received the first rotavirus dose. of these, 3 of 5 (60.0%) had normal mitogen proliferation while 1 (20.0%) had decreased proliferation to phytohemagglutinin. conclusions: identifying biomarkers for ttcl and developing evidencebased guidelines for the diagnosis and management of ttcl are important knowledge gaps. this descriptive study is limited by small sample size and the constraints of registry-based research. although there appear to be differences between these cohorts, our findings suggest that ttcl may disproportionately affect different segments of the population. ttcl infants with normal mitogen proliferation may be able to tolerate rotavirus vaccination. thus, routinely checking proliferation studies in all ttcl infants may help risk stratify these patients and minimize vaccinerelated adverse events. currently, there is insufficient evidence to recommend more extensive genetic testing such as genetic panels, cma, or wes. systematically collecting information about patient characteristics and outcomes, as well as encouraging increased participation in registries such as fill, may help address these shortcomings. background: systemic lupus erythematosus (sle) is a chronic, inflammatory disease that affects multiple organs. the measurement of anti-dsdna antibodies (abs) is a gold standard serological test used in the diagnosis and monitoring of sle, with higher serum levels associated with worse prognosis. however, not all anti-dsdna abs are pathogenic, and some patients have consistently high levels with low disease activity. one mechanism suggested for the pathogenicity of these antibodies is complement activation. here we describe an assay to measure the c1q binding activities of anti-dsdna abs in sle patients. materials & methods: the concentration of anti-dsdna abs was determined using the quantalite dsdna elisa kit (inova) as per the manufacturers instructions. in order to determine the c1q binding capacity of bound abs, samples were added to the pre-coated plate and incubated. bound anti-dsdna ab/c1q complexes were then detected using a biotinylated anti-c1q antibody (570 ng/ml) and streptavidin peroxidase (1 mg/ml). normal reference ranges were developed in serum samples from healthy controls, and upper limits of these normal ranges were used as cut-offs. the dsdna abs and c1q binding capacity of bound abs was then assessed in 49 sle patients, and compared to other markers and the sle disease activity index (sledai) score. results are displayed as absorbance at 450nm (au). results and conclusions: the 95th percentile ranges for anti-dsdna abs (0.068-0.137 au) and c1q binding activities (0.207-0.313 au) were developed from the measurements generated in 17 healthy serum samples. sle patients with an increased anti dsdna ab concentration (>0.137 au) were then separated into those with low (<0.313 au) and high (>0.313 au) c1q binding activities. patients whose dsdna abs had high c1q binding activity were found to have significantly higher sledai scores (mean 6.70 vs 3.19) . serum c1q concentration, serum dsdna abs (measured by another method) and serum c3 and c4 concentrations were not significantly different between the two groups. this assay suggests that dsdna abs from sle patients differ in their ability to bind complement, and that high complement binding activity of these antibodies may be linked to a more active form of disease. x-linked lymphoproliferative (xlp) is a primary immunodeficiency, caused by signaling lymphocyte activation molecule (slam)-associated protein (sap) deficiency. patients with xlp have severe immune dysregulation, usually triggered by ebv infection, leading to fulminant infectious mononucleosis, dysgammaglobulinemia and lymphoproliferation. without hematopoietic stem cell transplant (hsct) fatality is reportedly 100% by age 40. we report the natural history of xlp in a patient, and describe the lessons learned. our patient was healthy and developed normally until 6-years of age, when he developed progressive respiratory symptoms. lung biopsy revealed mature lymphoplasmacytic infiltrate in the alveolar septa, consistent with lymphoid interstitial pneumonia (lip). he received corticosteroids and cyclophosphamide with significant improvement. at age 12, he developed severe infectious mononucleosis (fever, hepatosplenomegaly, lymphadenopathy, lymphocytosis). he had a protracted clinical course, but eventually recovered and seroconverted to a typical convalescent pattern. he subsequently developed hypogammaglobulinemia, and was started on intravenous immunoglobulin (ivig). during the same year, his 10-year-old brother developed lip, and subsequently hemophagocytic lymphohistocytosis (hlh) and died within 4 months from overwhelming candidiasis. unfortunately, his youngest brother (age 7) then developed lip and died 2 months later from a massive gastrointestinal bleed. both siblings were treated with corticosteroids and cyclophosphamide; they did not have detectable ebv infection. at age 13 years, our patient experienced recurrent strokes and was found to have biopsy-proven cns vasculitis. he was treated with interferon-and recovered with residual left sided weakness, but was lost to follow-up. he continued on ivig, with no other immunomodulatory agents for several decades. he had progressive lung disease and recurrent seizures controlled with anti-epileptics. at age 43, he developed sudden vision change, headache and right-sided weakness, followed by a seizure. mri of the brain revealed small bilateral areas of acute infarction suggestive of a central embolic event, however, no primary thrombus was identified. he did not receive any immunosuppression but was anti-coagulated. eventually he was discharged home with resolution of weakness to his baseline. the patient was referred to our clinic after discharge and we re-evaluated him after 31 years. immune profiles at the time showed therapeutic igg troughs, low/undetectable igm/a/e, normal t/b/nk-cell counts, normal spontaneous, but decreased antibody-dependent nk cytotoxicity, 0% sap protein expression (on cd3+cd8+, cd3-cd56+ and cd3+ cd56+ cells), and deletion on the x chromosome encompassing the sh2d1a gene which encodes sap. his mother was a carrier of the same deletion. his functional status excluded the option of hsct. a year later, he had rapid deterioration with recurrent lung infections, liver failure, and thrombocytopenia. bone marrow biopsy revealed hodgkins lymphoma. he declined chemotherapy and died few days after diagnosis. our case represents a rare patient with xlp surviving to the fifth decade without hsct, particularly having experienced mononucleosis and non-ebv related cns vasculitis. our patient survived decades longer than his brothers (who most likely shared the same genetic defect) without evidence of somatic reversion (0% sap expression in cd3+cd8+) to explain his milder clinical phenotype. this case may help in understanding the natural history of xlp, and confirms that prognosis remains poor without hsct. haematology and oncology, chu de quã©bec ctla-4 is a major negative regulator of immune responses, and ctla-4 haploinsufficiency has been identified as a monogenic cause of primary immunodeficiency in patients presenting with a common variable immunodeficiency (cvid) phenotype with autoimmunity. here we present the case of pb, a 40-year-old man who had been followed by the immunology service of our center for 17 years. a diagnosis of cvid had first been made when the patient presented with atypical transverse myelitis, low immunoglobulin levels, and lymphopenia. over the years, his clinical picture was dominated by various forms of autoimmunity, namely inflammatory demyelinating disorder of the central nervous system, autoimmune haemolytic anemia, immune thrombocytopenia, cryptogenic organizing pneumonia, rheumatoidlike polyarthritis, chronic liver transaminitis with biopsy-proven moderate fibrosis, and lymphocytic colitis with malabsorption. immunoglobulin replacement therapy was started at diagnosis, and autoimmunity was sequentially treated with methotrexate, interferon beta 1-a, cyclophosphamide, mycophenolate mofetil, rituximab, and finally a combination of low-dose prednisone and sirolimus, with stabilization of his neurological condition, the most debilitating complication of his immune dysregulation syndrome. bone marrow transplant had been offered, but declined by the patient due to perceived good quality of life compared to transplant-associated risks. the patient was later referred to our hematology ward in july of 2018 for septic shock complicating febrile neutropenia, which was part of a twomonth, gradual-onset pancytopenia. the diagnosis of immune-mediated aplastic anemia soon became apparent, as demonstrated by a bone marrow biopsy performed in a peripheral center two days prior to admission. the underlying pneumonia and thereafter biopsy-induced staphylococcus aureus iliac osteomyelitis and soft-tissue abscess were treated with broad-spectrum antibiotics as well as multiple surgical interventions. the patient was started on eltrombopag, high-dose corticosteroids and cyclosporin a, the latter promptly switched to tacrolimus due to liver enzymes disturbances, all of which resulted in no significant hematologic response despite over seven weeks of treatment (with concurrent treatment of complicating infection, upper gastrointestinal bleeding, and intensive-care-unite myopathy). during that time, genetic confirmation of ctla-4 haploinsufficiency was received, and the patient was thereafter started on abatacept on day 48 of current hospitalization. administration of equine anti-thymocyte was initially foregone because of perceived infectious risk in the setting of poor iliac wound healing and superimposed adenovirus viremia; however, given the lack of response, it was given on days 52 through 54 of hospitalization. haematologic response began on day 67 of hospitalization with a steady rise in alllineage myelopoiesis up to a complete neutrophil response, platelet near-complete response as well as resolution of transfusion needs by day 101. while waiting for a well-matched bone marrow donor, isolated platelet decrease was observed and attributed to multiple factors, including low-grade thrombotic microangiopathy, inflammatory consumption and drug-related thrombocytopenia, but the patient remained well. to our knowledge, our patients presentation is one of the most severe manifestation of ctla-4 haploinsufficiency to have responded to targeted therapy with abatacept, as a bridge to hematopoietic stem cell transplantation, with resolution of both immune and infectious complications, showing that genetic diagnosis is helpful in optimizing the management of presumed cvid patients. hospital 12 de octubre health research institute (i+12), madrid, spain, dept. of immunology, university hospital 12 octubre. madrid. spain background: xlf/cernnunos deficiency is a rare primary immunodeficiency classified within the dna repair defects. these patients present severe growth retardation, microcephaly, lymphopenia and increased cellular sensitivity to ionizing radiation. here, we describe two unrelated cases with the same nonsense mutation in the nhej1 gene showing significant differences in clinical presentation and immunological profile but a similar dna repair defect. methods: missense nhej1 mutation was identified by targeted next-generation sequencing with an in-house designed panel of 192 genes. for foci experiments, primary skin fibroblasts were irradiated with ionizing irradiation (137cs) or treated with 20mm etoposide for 1 hour. after irradiation, the cells were seeded at a density of 1x104 cells/ml in t75 flasks in triplicate. to evaluate cell sensitivity to gamma-ir (1 and 3 gy),adherent cells were trypsinized and counted 11 days later. pbmcs from patient and healthy controls were irradiated with 10gy, fixed and stained for cd3, cd19 and phospho-histone h2ax. mean fluorescence intensities (mfi) of gamma-h2ax were evaluated on gated cd3+ lymphocytes. results:we report two patients harboring the same homozygous mutation in cernunnos/xlf/nhej1 gene. strikingly, their clinical phenotype ranges from severe combined immunodeficiency to isolated thrombocytopenia followed until escolar age (table 1) . they harbour the same c.169c>t mutation in nhej1 gene but different immunologic features (table 2) . p2 presented with mild t lymphopenia, hypersensitivity and nhej repair defect, typical for patients with xlf/nhej1 defects. on the other hand, p1 presented a more severe phenotype (t-b-) , however hypersensitivity and nhej repair defect was similar to p2.of note, p2 has survived into the first decade of live. both patients are alive and well after hsct. discussion: usually the repair defect in these disorders is assessed by immunofluorescence assays of irradiation-induced gamma-h2ax foci using skin fibroblasts. a high throughput, sensitive and reliable assay to quantify gamma-h2ax foci in pbmcs isolated from blood samples would be a valuable tool to diagnose these patients and perform hsct early. flow cytometry (fc) can be applied as a rapid diagnostic tool for dna repair disorders. patients with the same homozygous mutation (p.r178x) in nhej1 gene have been previously reported. two patients died at 1.5 and 4 years while another of the patients is already 8 years old and is alive (without hsct). however,none of these patients presented severe t lymphopenia as it has been observed in our first patient. conclusions: the assignment of a timely and accurate diagnosis is of paramount importance in the management of patients with defects in dna repair. in the era of nbs an abnormal trec assay should be followed by ngs approach as cernunnos deficiency may present early in life as scid,as other rs-scid defects. since genetic diagnosis takes time,functional radiosensitivity assays in peripheral blood may lead to the correct diagnosis and avoid exposure to alkylating agents during the conditioning regimen prior to genetic diagnosis. it would also be helpful in cancer patients to individualize and to guide the dosing of ionizing radiation (ir) and/or genotoxic agents to avoid accumulation of cells with genomic instability that could accelerate cancer development. figure 1 ). her skin lesions also significantly improved after starting the medication ( figure 2 ). her hospitalizations were complicated by fluid overload and hypertension. both fluid overload and hypertension resolved prior to discharge. she remains on 2mg prednisone daily, cetirizine, ranitidine, cromolyn and benadryl and hydroxyzine prn. to our knowledge, this is the youngest patient successfully treated with midostaurin and she is doing very well on therapy with no apparent side effects. she has had resolution of many of her systemic mastocytosis symptoms including skin lesions, axillary mass and improvement in her diarrhea and growth as well as objective improvements in her tryptase levels. case report: a two-year-old male presented to the hospital with a painful, non-pruritic facial and groin rash. the rash started one week prior to presentation. he had no associated fevers. his history was remarkable for failure to thrive (ftt) and chronic bilateral leg pain with antalgic gait. over the preceding months, he had been diagnosed with hand-foot-mouth disease and varicella. he had also had recurrent cervical lymphadenopathy (lad) for greater than one year requiring incision and drainage. gram stain and gomori methenamine-silver nitrate stain (gms) were negative and pathology showed only acute and chronic inflammation with areas of necrosis. his family history was negative for autoimmune disease or immunodeficiency. infectious exposure history was significant for an incarcerated father with unknown tuberculosis status and history of living in a shelter. on physical examination, the patient was well appearing with multiple erythematous papules, with superficial erosions and scabbing on the face (figure 1 ), lower abdomen, genital area, buttocks and proximal lower extremities. he had large, firm, non-tender submandibular lymph nodes. he also had small palpable axillary and inguinal lymph nodes bilaterally. his laboratory workup revealed normal white blood cell and platelet counts, but microcytic anemia, an erythrocyte sedimentation rate of 140 mm/hr, and c-reactive protein of 7.5 mg/dl. full body magnetic resonance imaging (mri) revealed bilateral cervical, supraclavicular, right hilar and inguinal lymphadenopathy and a patchy right upper lobe consolidation with at least one small area of cavitation ( figure 2 ) and an adjacent smaller area of ring enhancement. it also revealed three small nonspecific hypodense foci within the right lobe of the liver and borderline splenomegaly. given these findings, there was concern for granulomatous diseases. the patient underwent a liver biopsy ( figure 3 ) which showed non-specific evidence of necrotizing granulomatous disease. microbiological cultures and stains for bacteria, acid-fast bacilli and fungi were negative. his infectious work-up was negative for hsv, tuberculosis, hiv, syphilis, histoplasmosis, and toxoplasmosis. superficial bacterial cultures from the face and groin grew mixed gram positive and negative organisms, including methicillin-susceptible staphylococcus aureus (mssa). his immunologic workup revealed borderline elevated iga and igg with normal igm, normal t,b, nk-cell counts and pneumococcal and tetanus titers. a dihydrorhodamine (dhr) flow cytometric test was positive, consistent with a diagnosis of chronic granulomatous disease (cgd). genetic testing confirmed x-linked disease. he was treated with acyclovir and ceftriaxone with resolution of his rash. conclusion: we present a case of a two-year-old male with newly diagnosed x-linked cgd. though he had been seen by multiple healthcare providers for recurrent lymphadenopathy over the preceding year, he had no other history of recurrent viral or bacterial infections or significant family history that might implicate a primary immunodeficiency. at time of presentation, he had diffuse rash which could have caused his palpable lymphadenopathy on exam. a high index of suspicion for cgd in the setting of recurrent lad and ftt prompted sending the dhr, which led to the diagnosis. chronic granulomatous disease (cgd) is an inherited primary immunodeficiency (pid) which results in both inflammatory response dysregulation and an increase in susceptibility to certain bacterial and fungal infections. without curative treatment such as a bone marrow transplant, it remains a chronic disease with daily medication management, intermittent treatment and life-long surveillance. in general, chronic disease involves physical, psychological and social effects which can affect the patients quality of life. although some research has been done on how pid affects quality of life, there is little research in the united states about how cgd affects patients quality of life. to examine the effect of cgd on patients quality of life, as a part of a voluntary research protocol examining the natural history of immune deficiencies, we administered the who qol-bref instrument to adult cgd patients enrolled on a nih irb approved protocol and seen in the infectious disease clinic at the national institutes of health (nih) over a five-month period. the who qol-bref is comprised of 26 items, which measure the following broad domains: physical health, psychological health, social relationships and environment. each item is rated on 5point likert scale. it has been validated cross culturally and has been widely field tested. the survey was interview administered to 35 patients (23 males, 12 females) with genetically confirmed cgd. the age range was 18 -60 years old (mean age 37.6 years) with a distribution of 57 % x-linked cgd and 43% autosomal recessive cgd. results have been obtained and will be presented. rationale: common variable immunodeficiency (cvid) is the most common primary immunodeficiency with an estimated prevalence of 1:25,000. we aimed to analyze the clinical presentations and their associated comorbidities amongst cvid patients in usa. methods: data on 1,546 cvid patients reported in the united states immunodeficiency network (usidnet) from 1992 to 2018 were analyzed based on clinical, immunological and genetic factors. univariate analysis with spearman rank coefficients was done to analyze correlations between disease outcomes. observed survival was estimated using the kaplan-meier method. results: among the 1546 patients, 908 (58.7%) were female and 638 (41.3%) were male. median age at diagnosis was 29 years [mean (sd), 30.1 (20.2); range, 0-82; iqr, 12-47] with median age of onset of 14 years (mean (sd), 20. 3 (19.2) ; range, 0-81; iqr, . females showed a longer delay in diagnosis (9.5 vs. 6.6 years, p=0.006). higher body mass index (bmi) linearly correlated with the age of diagnosis (r= 0.46). in survival analysis, a 5-year delay in age at diagnosis increased the risk of death by 7.4% (hr: 1.07, 95% ci: 0.98-1.18, p=0.14). conclusions: our study suggests a longer delay in diagnosis in female subjects and a strong association with diagnosis of cvid in patients with higher bmi. females may have a longer period without symptoms leading to a diagnostic delay. gender-based and disparities-based inquiry into these trends may need additional study. the physical well-being of those with primary immunodeficiency (pi) and the physical maladies of those with pi are well-documented. since the 1950s, advances in identification and treatment of pi has for many led to lives where the physical infections of these groups of diseases are manageable. however, not as well understood are the emotional and mental health aspects of living with pi. as part of a larger survey project the idf 2017 national patient survey, this study aims to quantify any potential mental health issues or challenges faced by adults with pi. our hypothesis-those with pi, suffer from statistically higher rates of depression when compared to the u.s. general population. the 2017 idf national patient survey was a nationally distributed, unincentivized, mail-based survey of 4,500 persons in the idf patient database identified as being either adults with pi or the parent/caretaker of a child with pi. the questionnaire comprised approximately 44 main questions about pi as well as the validated sf-12v2, brief fatigue inventory and the patient health questionnaire-2 (phq-2) instruments. additional questions asked about current use of prescription medications for anxiety, depression, stress and pain. for the purpose of this study, only adult respondents with pi are included as the basis for analysis. the two-item patient health questionnaire (phq-2) meets the criteria for general screening of depression suggested by the u.s. preventive services task force. scored on a scale of 0-6, a score of three or higher is suggested as the cut-point for depressive screening. according to a 2014 ahrq study that utilized meps data, 2,139 of the 23,770 (9%) respondents scored three or greater. in our survey 211 of the 925 (23%) adults scored three or greater (2 <.05.) overall, those in our survey scored lower on the sf-12v2 mcs scale when compared to the u.s. population (44.3 v.50.0, p<.05) . further, adults with pi who scored three or higher on the phq-2 had an average mcs of 31.8. those who met the phq threshold in our survey were also more likely to report moderate to severe limitations in normal activities as a result of emotional problems than those that fell below the threshold (74% versus 13%, p <.05). not surprisingly, those that met the phq threshold reported much higher use of prescription medications for anxiety, depression, stress (69% versus 33% below threshold, p <.05) as well as a higher reported use of prescription pain medications (33% versus 17% below threshold, p <.05). though moderate to severe fatigue was reported by 68% of those below threshold, 99% of those with phq scores at threshold reported experiencing moderate to severe fatigue (p <.05). health care providers should consider including the phq-2 in the overall health assessments of their patients with pi. those scoring three or higher should be referred to the appropriate professional for further evaluation. (lek et al., 2016) . the w623l is a semi-conservative amino acid substitution, which may impact secondary protein structure. in-silico analyses supported a deleterious effect, located within the sh2 domain, which is a critical functional domain (chandesris et al., 2012; koskela et al., 2012) . it was thus determined that this variant is likely pathogenic. the patients prophylactic treatment was optimized with tmp-smx (800mg-160mg) twice daily for prevention of infections. she was also started on hibiclens (chlorhexidine) baths once per week. she was referred to pulmonology for optimization of pulmonary health in the setting of bronchiectasis and mild decline in dlco. she was advised to followup on a yearly basis to the primary immunodeficiency clinic to assess for recurrent infections and for changes in pulmonary health. finally, targeted testing and clinical evaluation of both of the patients parents was recommended to determine if w623l was inherited or arose de novo. the pathogenic role of the w623l missense change would be further supported if it had occurred de novo or if it segregates with the disease in the family. uploaded file(s) uploads pulmonary function testing results.pdf j clin immunol (2019) 39 (suppl 1):s1-s151 s20 introduction: lipopolysaccharide-responsive and beige-like anchor protein (lrba) deficiency is a rare autosomal recessive disease of the immune systems characterized by hypogammaglobulinemia and decreased ctla4 expression on t regulatory cell (t regs) due to defective intracellular trafficking of ctla4. previous in vitro study has shown a significant increase of ctla4 expression on lrba deficient t cells after overnight culture with chloroquine, an older anti-malarial agent. this effect is likely due to increasing lysosomal ph. however, there is no evidence of such effect in human subjects after administration of weight appropriate doses anti-malarial agents. we are presenting a set of siblings with lrba deficiency who had ctla4 expression measured before and four weeks after starting hydroxychloroquine. case reports: case 1 is a 14-year-old east-indian boy with autoimmune thyroiditis, type 1 diabetes mellitus (dm), short stature, autoimmune cytopenias, and lymphadenopathy. he was referred to immunology clinic at 9 years of age for suspicion of autoimmune lymphoproliferative disorder. primary immunodeficiency genetic panel was sent which revealed a homozygous mutation in lrba gene (c.6480_6481del). this novel variant resulted in a frameshift and created a premature stop codon 18 amino acids downstream from this location which may lead to absent or abnormal protein. lung ct scan showed interstitial lung disease. lung biopsy showed interstitial nodular and diffuse lymphoid proliferation. this diagnosis led to the testing of his sister (case 2) given her history of autoimmune illnesses and the family history of consanguinity. case 2 is a now 13-year-old girl with type 1 dm, autoimmune thyroiditis, lymphadenopathy, psoriatic arthritis, and seizures. her lung imaging showed pulmonary nodules without interstitial lung disease. both cases received hydroxychloroquine while waiting for insurance approval of abatacept. ctla4 expression on tregs was measured prior to and four weeks after starting hydroxychloroquine treatment. at baseline, 8.6% of case 1s cd4 cells were treg (foxp3+ve, cd25hi) and 51.4% of them expressed ctla-4 (in contrast to 94.1% tregs in the healthy control) with mean fluorescence intensity (mfi) of 335. this ratio and mfi did not change after 4 weeks of hydroxychloroquine treatment (6 mg/kg/day). soluble interleukin-2 receptor levels were measured: case 1 had a baseline level of 8510 pg/ml, which decreased to 2228 pg/ml after 4 weeks of hydroxychloroquine treatment. for case 2: 8.4% of her cd4+ t cells were found to be foxp3+cd25hi and 36.1% of these tregs expressed ctla-4. this ratio increased by 7% after one month of hydroxychloroquine. increase in mfi was also noted from 298 to 386. case 2 had a drop in soluble interleukin-2 receptor level from 1265 pg/ml to 950pg/ml after treatment. conclusion: in contrast to the previous in vitro assays, we did not find a significant increase in ctla4 expression on t regulatory cells in vivo after 4 weeks of 6mg/kg/day hydroxychloroquine. interestingly, soluble il-2 receptor levels improved dramatically with hydroxychloroquine. (36) submission id#592574 human nf-kappab2 defect results in defective intrinsic b-cell differentiation, function and class switching introduction/background: autosomal dominant heterozygous mutations in nfkb2 (encoding for the protein nf-kb2) have been identified in the etiology of a form of primary immunodeficiency disorder that presents with hypogammaglobulinemia, defects in b-cell maturation, endocrinopathy, and autoimmune manifestations. in humans, the effects of altered nf-kb2 and mechanisms of immune system impairment have not been fully delineated. objectives: to understand the mechanism of the antibody deficiency in patients with hypomorphic mutations in nfkb2 (c.2564dela; p.lys855serfs*7) by evaluating b-lymphocyte proliferation, differentiation, function, and gene expression. methods: immunophenotyping of primary b-cells from subjects with mutant nfkb2 was completed by flow cytometry. proliferation of b-cells was assessed by cfse stimulation of primary cd19+ b-cells from healthy and nfkb2 mutant subjects. differentiation of healthy and affected naã¯ve b-cells (cd27-cd38-) into plasmablasts (cd27+cd38+) following stimulation was assessed by flow cytometry. the supernatant from these cells were assayed for iga, igg and igm production by elisa. to study the defect in class-switch recombination, naã¯ve b-cells and ebvtransformed b-cells from affected and healthy individuals were stimulated and expression of the aicda gene was quantified by qpcr. in parallel experiments, ebv b-cells from wildtype and nfnb2 mutant individuals were stimulated and aid (activationinduced cytidine deaminase) protein levels were determined by western blot. results: patients with hypomorphic mutations in nfkb2 (c.2564dela) had low memory b-cell (cd19+ cd27+ igd-igm+) and class-switched memory b-cell (cd19+ cd27+ igd-igm-) numbers. in vitro, primary bcells from these patients demonstrated a 50% reduction in proliferation and cell division in response to cd40l and il-10 (p =0.01). compared to healthy naã¯ve b-cells, mutant naã¯ve b-cells had a significant reduction in plasmablast differentiation (p = 0.002) and secreted significantly lower levels of immunoglobulins in response to cd40l and il-21 stimulation. mutant naã¯ve b-cells and mutant ebv b-cells failed to increase aicda expression and aid protein levels in response to cd40l and il-21 stimulation. conclusions: our studies demonstrate that a hypomorphic nfkb2 mutation in humans affects intrinsic b-cell proliferation and differentiation. the mutation impairs transcription of the aicda gene that encodes aid, a key protein involved in b-cell class-switch recombination. the nfkb2 gene defect also impairs immunoglobulin production, as seen in common variable immunodeficiency-like cases. these studies provide unique translational insights into physiological activities of nf-kb2 in downstream immunologic outputs in humans, expanding those suggested by experimental observations in mice. background: few studies have evaluated the quality of life (qol) and patient reported outcomes of primary immunodeficiency disease (pidd) patients, and no studies have assessed medical provider perceptions of their pidd patients qol, neurocognition, physical well-being and psychosocial health. understanding provider beliefs regarding patient reported outcomes is essential to improving clinical management of pidds. here we report our pidd medical provider survey results. methods: providers were contacted via email with the assistance of the clinical immunology society. participants completed adult and/or pediatric-based likert scale survey questions via a secure online survey service. in addition to demographic information, survey questions assessed provider perceptions of patients overall qol and their impression of the impact of disease or its associated treatment on mental health, physical well-being, neurocognition, social relationships and school/work performance. clinicians were expected to make their assessments based on their pidd patient cohort as a whole rather than on specific diagnoses or patients. given the small sample size, a p-value < 0.1 was considered statistically significant; repeated measures anova and paired t-test analyses were used. results: study participants (n=58) were primarily from the united states (64%), born between 1965-1979 (44%) , and trained in allergy/ immunology (77%). 85% of survey takers practiced within an academic center, 52% were female and 95% cared for children with 42% of providers concurrently caring for adults. there was a statistically significant difference (p=0.07) in the perceived overall qol of pediatric versus adult pidd patients with 41% of providers feeling as though their pediatric patients had a good qol while only 25% believed their adult patients had a good qol. clinicians believed adult pidd individuals had more difficulties related to associated co-morbidities rather than their actual pidd compared to pediatric pidd patients (p=0.046). providers felt that the neurocognition and school performance of children were more often negatively affected by a pidd than the neurocognition and work performance of immunodeficient adults (p=0.1). clinicians believe children with pidd more frequently had difficulties related to their concentration than memory (p<0.01). 96% of those who care for pidd adults believe their patients work performance or daily mental functioning is at times negatively impacted. anxiety symptoms and social relationships were viewed as being more negatively impacted by a pidd diagnosis or treatment than anger or depressive symptoms in both children and adults (p<0.01). 38% of pediatric clinicians feel their pidd patients experience anxiety symptoms often or almost always. of physical health parameters, energy, rather than mobility or pain, was deemed to be more deleteriously influenced by an immunodeficiency in adult and pediatric patients (p<0.01). conclusions: our results show that medical providers perceive the overall qol of pediatric pidd patients to be superior to that of adults with pidd, but most clinicians feel a diagnosis or associated treatment regimen for pidd can negatively impact the physical well-being, psychosocial health, school/work performance and neurocognition of both children and adults. [cbm] complex is a critical signalling adaptor that regulates lymphocyte activation, proliferation, survival, and metabolism. primary immunodeficiencies affecting each component (termed cbmopathies) result in broad clinical manifestations ranging from severe combined immunodeficiency (scid) to lymphoproliferation. we present the laboratory and clinical findings of two canadian first nations patients found to be homozygous for the same novel card11 mutation (c.2509c>t; p.r837*). results: we have identified an 8-month-old boy who presented with a severe case of entero/rhinovirus bronchiolitis with interstitial lung disease and a 17-year-old boy with a history of severe pulmonary infections (including pjp), chronic sinusitis, candidiasis, invasive bacteremia, and severe ileo-colitis and oral ulceration requiring total colectomy. both patients possessed absent tregs, absent memory b cells, and hypogammaglobulinemia. however, only the 8-month-old had poor t cell proliferation to pha, cona, and cd3. both patients were found to be homozygous for the same novel variant of card11 (c.2509c>t; p.r837*). the mutation rendered card11 protein expression unstable and it was undetectable by immunoblot. to confirm card11 deficiency, we stimulated patient b cells with phorbol 12-myristate 13 acetate (pma) and ionomycin across a time-course and immunoblotted for various signalling proteins in both the nf-b (ikk/, ib, p65) and mapk (mek1/2, mkk4, jnk1/2, erk1/2) pathways as well as various cleavage substrates of the malt1 paracaspase (relb, cyld, bcl10, hoil1). nf-b and jnk activation were completely absent and malt paracapase activity was lost, but surprisingly, mkk4 (which acts upstream of jnk) was intact. furthermore, co-immunoprecipitation experiments revealed that card11 was required for optimal malt1 association with bcl10 in response to stimulation. conclusions: these two cases highlight the crucial role of card11 in regulating lymphocyte development, function, and humoral responses. in addition, we have identified the oldest known living individual with card11 deficiency and he presented uniquely with inflammatory gastrointestinal disease in addition to scid, further adding to the spectrum of phenotypes associated with card11-related primary immunodeficiencies. abstract: the usidnet registry began in 1992 with an niaid contract with the immune deficiency foundation, which continues today. it aims to provide a resource for clinical and lab research through enrollment of known immunodeficiency patients into a national registry, the usidnet. nih is a major national and international referral center for clinical trials on inborn errors of immunity, or primary immunodeficiency diseases. it is a mechanism for depositing nih data into usidnet. a registry of patient information may help us understand how many people have each disease. the information may improve how we diagnose and treat these conditions. the patient registry is designed to obtain longitudinal data on a large number of patients with primary immunodeficiency diseases who come to nih to participate in research. the data is collected from the nih electronic medical record system, cris and is deposited into a secure registry with restricted and monitored access. all medical information is anonymized for patient privacy. department of biochemistry, emory university, atlanta, ga oas1 is an intracellular sensor for dsrna that generates the second messenger 2'-5'-oligoadenylate to activate rnase-l as a means of antiviral defense. we describe four patients with a complex early-onset autoinflammatory and immunodeficiency disease caused by heterozygous de novo oas1 mutations. patients presented early in life with lung inflammation including pulmonary alveolar proteinosis and interstitial lung disease. they had febrile flares with dermatitis specifically with macular, pustule and bullous features often progressing to ulceration. infants had episodes of bloody diarrhea in 3 patients (assoc. with villous blunting and cryptitis in two patients and oesophagitis in one patient). immunoglobulin igm, igg, and iga levels were low while t cell, b cell, and nk cell numbers were generally in the normal range. exome sequencing identified de novo heterozygous oas1 missense mutations in all patients. one patient had a heterozygous de novo oas1 mutation p.ala76val, with mutant oas1 protein being expressed in ex vivo generated t cell blasts. in sorted primary patient monocytes and b cells, oas1 p.ala76val was associated with spontaneous rna degradation and apoptosis as determined by rna chip technology and flow cytometry, respectively, while t cells were not affected. monocytes displayed disturbed terminal differentiation and functioning as indicated by reduced gm-csf-r expression and signaling. b-cells display reduced class-switch-recombination. proliferation of allogeneic t-cells was reduced in response to sorted oas1 mutated monocytes and b-cells. activation of interferon response genes in pbmcs was detected. two further unrelated patients had a heterozygous de novo oas1 mutation p.cys109tyr, which appeared to compromise protein stability in transformed patient fibroblasts and when transfected. cells transfected with this mutant protein had reduced 2-5 oligoadenylate synthesis compared to wild type transfected cells. immortalized fibroblast lines demonstrated higher levels of inflammatory cytokines and spontaneous cleavage of rnas. a 4th patient with the clinical phenotype had a heterozygous de novo oas1 variant p.val121gly, but has yet to have formal validation of the variant. three patients underwent hematopoietic stem cell transplants in an effort to control their diarrhea and skin inflammation. one patient died with ongoing chronic graft versus host disease, while the two others (p.ala76val, cys109tyr) are alive and reasonably well with a followup of 0.5-7 years. the untransplanted patient died as a result of respiratory failure. in summary, patients with de novo heterozygous oas1 mutations have chronic ongoing inflammation of multiple organs. this is at least in part due to spontaneous rna cleavage, apoptosis and production of inflammatory cytokines and type i interferons. this defines a new category of autoinflammatory disorder. introduction: increased susceptibility to infections is the most common complication of chronic granulomatous disease (cgd). hemophagocytic lymphohistiocytosis (hlh) is a severe disorder resulting from hyperinflammation and hypercytokinemia that can lead to multi-organ system dysfunction (1) characterized by certain criteria: fever, splenomegaly, cytopenias, hypofibrinogenemia or hypertriglyceridemia, hyperferritinemia, increased soluble cd25/il-2ra, evidence of hemophagocytosis, or decreased/absent nk cell cytotoxicity (2) . secondary hlh occurs infrequently but often is preceded by smoldering infection in cgd (3, 4, 5) . we present a case of hlh in a 38-day old male, the youngest reported case with cgd. case: a 38-day old male with previously diagnosed x-linked cgd, due to known family history, presented with fevers. initial evaluation was unrevealing including chest x-ray, urinalysis, and blood and csf cultures. he was admitted and treated empirically with cefepime. ct demonstrated multiple multifocal nodules of the lungs and spleen. after lung nodule biopsy was performed, antimicrobial therapy was broadened to iv meropenem, voriconazole, and micafungin. despite this, he continued to have fever and developed new onset tachycardia, respiratory distress, and lactic acidosis. further decompensation with vasoactive refractory shock was treated with vasopressors and stress dose hydrocortisone. additional laboratory evaluation revealed rising liver enzymes (ast 1670u/l, alt 307u/l), cytopenias (hemoglobin 7 g/dl, anc 90/ul, platelets 96,000/ul), and coagulopathy (fibrinogen 93-135mg/dl). splenomegaly was present on abdominal ultrasound. a diagnosis of evolving hlh was considered and dexamethasone was administered. within 24 hours of clinical decompensation, the patient died of multiorgan failure. subsequent blood cultures returned with gram-negative rods (and ultimately burkholderia cepacia). autopsy confirmed hemophagocytosis within the bone marrow. no mutations were found in genes associated with primary hlh. discussion: patients with cgd are susceptible to infectious complications and auto-inflammation most commonly involving the lungs, gi, and gu systems (6, 7) . patients with cgd can be at increased risk of hyperinflammatory syndromes secondary to infections and chronic inflammation. as shown in the included case, hlh can present in infancy and can be deadly. early consideration and directed treatment of hlh is imperative, even in the setting of sepsis malignant proliferation of gamma-delta t cells include hepatosplenic t-cell lymphoma (hstl), primary cutaneous t-cell lymphoma and t-cell large granular lymphocytic leukemia (t-lgl). the former two have often been associated with splenomegaly and cytopenias. however, reactive proliferation of gamma-delta t cells in spleen mimicking malignancy has only been reported once and has a significant risk of misdiagnosis. a 30-year-old female presented with two years of unintentional weight loss, persistent leukopenia and thrombocytopenia, with leucocytes around 1-2 x 10^9/l and platelets around 100 x 10^9/ l. she also had associated macrocytic anemia (hemoglobin=10-11g/dl) with laboratory evidence of dat (direct anti-globin test) negative hemolysis. physical examination and computed tomography (ct) imaging showed splenomegaly. there was no hepatomegaly or lymphadenopathy. serum liver function test, auto-immune studies, hemolysis and hereditary diseases workup, viral and bacterial serologies were all normal or negative, except for mild hyperbilirubinemia and ldh elevation. bone marrow examination performed four months prior to the splenectomy revealed mildly hypocellular marrow (50%) with trilineage hematopoiesis. flow cytometric analysis and cytogenetics of the bone marrow aspirate and peripheral blood were normal except for small population of large granular lymphocyte and mild low absolute b cell counts in peripheral blood. a laparoscopic splenectomy was performed for diagnostic and therapeutic purposes due to patients worsening luq pain. there was no other treatment given prior to surgery. 24 hours postsplenectomy her leucocytes increased to 13.1 and platelets to 247. her three-month post-splenectomy wbc count and platelet count was 8.9 and 391, respectively. hemoglobin also improved to 14.9. pathology showed red pulp expansion by small lymphocytes (fig. 1 ) and subsequent ihc (immunohistochemistry) was positive for cd3 ( fig. 2) , cd2, cd7, tia-1 and negative for cd8, cd5 and cd56. cd4 was difficult to interpret. eber was negative. flow cytometry ( fig. 3) showed increased gamma-delta t-cell population (20%) with positive cd3, cd2 and cd 7 and negative cd 5, cd4 and cd 8. molecular studies by pcr didnt reveal any t-cell receptor gamma or beta gene rearrangement. cytogenetics was negative for isochromosome 7q or any other abnormalities. she was symptom free at 6 months from her splenectomy. the morphology and immuno-phenotype of these gamma-delta t cells show significant overlap with the malignant cells seen in hstl and t-lgl, such as loss or downregulation of cd5, cd4 and cd8. awareness of this reactive condition is necessary to prevent making a wrong diagnosis of a malignant disease with a potentially benign, spontaneously resolving disease. additional studies of similar cases is needed in order to establish more definitive criterion to separate benign from malignant processes and delineate the role of gamma-delta t cells. uploaded file(s) uploads fig 3. flow cytomtery.pptx background: sex steroids in the human thymic environment influence aire expression as well as interactions with its partners, i.e. genes coding for aire interactors. here we investigated the effects of sex steroids on these interactions during minipuberty the surge of sex hormones that occur along the first six months of life -and up to 18 months of life. we employed a network-based approach for investigating aire-interactors gene-gene relationships and how abundantly co-expressed thymic mirnas covariate with those genes. aire-interactors networks allowed the measuring of gender-related differences in gene-gene expression correlation disclosing relevant differences between minipuberty groups. methods: total rna was extracted from thymic surgical explants obtained from male (m) and female (f) infants -aged 0-6 months (groups mm and mf, for minipuberty) and 7-18 months (group nm and nf, for nonpuberty) and used in dna microarray assays. gene coexpression network (gcn) analyses were performed for aire and its interactors and for mirna-gene coexpression analysis. the set of genes coding for the aire-targeted proteins was previously identified in tecs by abramson et al. (cell 140:123-35, 2010) . aire-interactors networks were obtained for all groups (link strength cut-off for gene-gene > |0.80| and for mirnagene < -0.80). aire expression in mtecs was quantified by immunohistochemistry. these methodologies are described in moreira-filho et al. (sci rep 8:13169, 2018) . results: the mm x mf networks comparison showed that 16 abundantly expressed mirnas are interacting with the different aire interactor genes in both networks. it is interesting to note that network topology were more similar between nm and nf groups, although aire interacts with only one distinct mirna in each network (mir-150-5p in the nm group or mir-7977 in the nf group). conversely, in the non-puberty networks the sets of mirnas and their interacting genes are distinct for each network. immunohistochemistry analysis revealed a higher percentage of mtec aire positive cells in the minipuberty groups: i.e. there is a significant difference between mm x nm (p = 0.0006) and between mf x nf (p = 0.0060). conclusions minipuberty and genomic mechanisms shape thymic sexual dimorphism along the first 6 months of life. this process does not involve changes in aire expression between genders, but differences in the interactions of aire with its partners that persist throughout the non-puberty period, probably regulated by mirnas and also by genetic and epigenetic factors. introduction: neutrophils are presumed to defend against aspergillus species by releasing reactive oxygen species (ros) and neutrophil extracellular traps (nets) to degrade fungal hyphae. triazole antifungals synergistically enhance neutrophil mediated hyphal degradation. patients with cgd are particularly susceptible to aspergillus species likely due to their inability to create ros and nets, and in severe cases may not be amenable to antifungal therapy alone. objective: we present a case of severe disseminated aspergillosis in a patient with cgd in whom gt served as an important adjunct to antifungal therapy and bridge to transplant. results: a 6-year-old boy with known cgd, lost to follow up and nonadherent to prophylaxis, presented acutely with right-sided hemiparesis. neuroimaging revealed an embolic left middle cerebral artery infarction and cardiac magnetic resonance imaging showed extensive vegetations involving both right and left ventricles and atria, with an ejection fraction of 28%. the patient was admitted to intensive care, started on liposomal amphotericin b, meropenem and vancomycin, and underwent debulking of the intracardiac masses on post admission day (pad) 1. operative findings showed severe constrictive pericarditis with multiple abscesses and intracardiac vegetations. thorough debridement of the vegetations was undertaken, however some deep seated abscesses in the myocardium were not amenable. operative cultures were positive for aspergillus fumigatus. clinical status remained precarious, with ongoing requirement for inotropic and ventilator support. antimicrobial therapy was refined to voriconazole, with amphotericin b remaining on board until therapeutic levels of voriconazole were achieved. as effective neutrophils are integral in the immune response against aspergillus, the decision was made to start granulocyte transfusions to aid in clinical stabilization prior to hsct. interferon gamma infusions were not administered because of the risks of adverse effects and potentially increasing transplant rejection. gts were started on pad 6, at a dose of approximately 1x10^10 granulocytes, three times a week. the patient tolerated the infusions well, with no allergic or inflammatory response. neutrophil oxidative burst measured one hour post infusion showed 23.9% mean fluorescent intensity, compared to a baseline of 0% ( figure 1 ). clinical improvement was seen, with inotrope cessation on pad 12 and extubation to bipap on pad 41. human leukocyte antigen (hla) allosensitizaton was tested on pad 12, 6 days after the first gt, with no evidence of hla antibodies. a total of 28 gts were given over 3 months, prior to proceeding to a 10/10 hla matched related donor transplant (pad 69), with two transfusions given before neutrophil engraftment (anc 500) on day +14. the patient is now stable 13 months post transplant, with no evidence of graft rejection. he remains on chronic suppressive antifungal therapy, to continue until full lymphoid reconstitution. conclusion: gt may be a useful adjunct to antifungal therapy in patients with impaired neutrophil function with severe invasive aspergillosis, and potentially provide a life sustaining bridge to hsct. methods: subjects were enrolled in irb protocol 00051692 for rvt-802. rvt-802 was implanted into the quadriceps with immunosuppression. results: subject 1 was normal at 22q11.2 but had hypocalcemia, an asd, pda, and abnormal ears. the subject received a cord blood transplant mismatched at hla-b and hla-c alleles at age 3 months. subsequently mild graft-versus-host disease (gvhd) developed and was treated with antithymocyte globulin, steroids and cyclosporine. donor t cells developed in low numbers. twelve years later, the subject developed epstein barr virus lymphoma and suffered two relapses. while in remission, subject 1 received unmatched rvt-802. two weeks after rvt-802 implantation, the subject developed an adenovirus infection resulting in skin and gut gvhd, presumably from activation of the cord blood t cells. subject 1 was treated with corticosteroids, cyclosporine, cidofovir and infliximab. four years post rvt-802, subject 1 is healthy with 609 genetically recipient t cells/mm3 and 40% naã¯ve cd4 t cells. subject 2 was normal at 22q11.2 but had an asd, pda, hypoparathyroidism, and no t cells at birth. his genetic defect is unknown. subject 2 was treated with a ric myeloablative, allogenic, unrelated, 10/10 cord blood transplant, and a subsequent myeloablative, unrelated 9/10 cord blood transplant. hematopoietic chimerism was established without t cell development. rvt-802 expressed the one allele in the recipient that was not expressed by the second cord donor. the post-thymic transplant course included immune thrombocytopenia requiring rituximab and splenectomy and generalized adenopathy for 3 years but no gvhd. he failed weaning of immunoglobulin replacement. three years post rvt-802, he has 930 cd3, 750 cd4, and 105 cd8 t cells/mm3. he is active in school. subject 3 had absent trecs on newborn screening with 7 cd3+ t cells/ mm.3 a single mutation in foxn1 was identified; she has sparse scalp hair. subject 3 received a 9/10 matched unrelated umbilical cord transplant. the post-transplant course was complicated by significant morbidity, and no naã¯ve t cell development. rvt-802 expressed the one allele in the recipient that was not in the cord blood donor. the subject did not develop gvhd, is healthy and at 9 months has 98 naã¯ve cd4+ t cells. she had resolution of longstanding norovirus and sapovirus gastroenteritis. conclusion: rvt-802 can improve t cell immunity after poor or failed correction with allogeneic hematopoietic transplants. in subject 1, gvhd post rvt-802 was related to an acute viral infection; cord t cells attacked hla mismatches in the recipient. subjects 2 and 3 were given rvt-802 matched to recipient alleles that were not expressed in the hematopoietic donor. we hypothesize that thymocytes developing in rvt-802, if strongly reactive to the recipient-mismatched allele, are deleted by the bonemarrow-donor dendritic cells (that acquire recipient mhc from the recipient-allele-matched thymic epithelial cells) thereby preventing gvhd. rationale: ctla4 haploinsufficiency is an autosomal dominant immune dysregulation syndrome characterized by variable phenotypes. here we present a young woman diagnosed with evans syndrome and lymphoproliferation as a child, found to have a novel ctla4 variant as a young adult, and who developed hypogammaglobulinemia and a bacterial endocarditis while stabilized on ctla-4 replacement therapy. methods: sequencing of 207 genes, including ctla4, in primary immunodeficiency panel. results: our patient was diagnosed with evans syndrome at age 2 with manifestations of anemia and thrombocytopenia recalcitrant to treatment over many years with steroids, cyclosporine, and vincristine. bone marrow biopsy reportedly showed normal trilineage maturation and her symptoms responded for a short time to splenectomy at age 14. symptoms recurred at age 16 when she was also found to have pulmonary reticular opacities, prominent lymph nodes, and elevated b cells. repeat bone marrow and lymph node biopsies at that time were unrevealing. minor responses to treatment with ivig, rituximab, mycophenolate mofetil and gcsf were noted. at age 17, she developed varicella-related encephalitis shortly after vaccination. with a strong suspicion of an immune dysregulation syndrome, immune evaluation revealed normal immunoglobulins with good vaccine responses, elevated b cell numbers, normal t cell numbers, and normal mitogen proliferation. ctla4 sequencing revealed a mutation in exon 2 [c.420c>a, p.tyr140*] causing a premature translational stop signal, which was consistent with previously reported cases of ctla4 haploinsufficiency. she was started on rapamycin initially for her cytopenias but was then transitioned successfully to abatacept with almost complete resolution of her anemia, neutropenia, and pulmonary opacities. after 6 months of stable control, she developed a precipitous drop in her platelets and was eventually diagnosed with streptococcus viridans endocarditis of her native mitral valve. this responded to antimicrobial therapy, but eventually needed surgical intervention due to ongoing insufficiency. around this time, she was also found to be newly hypogammaglobulinemic, necessitating ongoing igg supplementation therapy. during successful replacement of her mitral valve with a biosynthetic prosthesis, it was noted that her aortic valve also had evidence of previous disease, implicating a prior endocarditis as part of her clinical syndrome as well. conclusions: in this patient, the presentation of recalcitrant cytopenias, lymphadenopathy, elevated b cells, vaccine-induced viral infections and lung findings precipitated concern for immune dysregulation syndromes and allowed for identification of a novel deleterious ctla4 mutation. in addition to previously reported clinical findings, our patient presents with the first reported case of repeated endocarditis in the setting of ctla4 insufficiency disease. given the finding in this patient of prior (unrecognized) disease, regularly screening patients with ctla4 insufficiency for evidence of cardiac affectation may be prudent. clinical research nurse, johns hopkins university background: the relationship between elevated serum alpha fetoprotein (afp) concentration and age, mortality, genotype and neurologic outcome in ataxia telangiectasia (a-t) patients has remained inconclusive over the past decades, leaving afp as a useful marker for disease diagnosis without further clinical significance. objective: to examine the relationship between afp levels and age, mortality, genotype and neurologic outcome using a data set larger than any prior study. methods: we retrospectively collected data on 280 a-t patients at johns hopkins medical center (0-34 years of age) with both classical (predicted protein null) and variant a-t. this included 459 serum afp measurements (179 serial levels in 50 a-t patients, max observations 9 per patient). mixed model compound symmetry covariance was used for statistical analysis to examine the effect of age at visit on afp levels. subgroup analysis by mutation type, mortality, feeding/swallowing scores as a surrogate for neurologic function, x-ray induced in vitro chromosomal breakage and serum transaminase levels were similarly analyzed. results: significant association between age and afp level was found such that for every 1 year increase in age, afp level increases 20 ng/ml (p<0.0001). subgroup analysis by mutation type found that the 12 patients with missense mutations showed a negative linear relationship be-tween log afp levels and age (r= -0.10, p=0.03). we found greater afp levels in patients who subsequently died, after controlling for age (least square mean afp level in log scale 0.67 greater in deceased patients versus living patients, p=0.002). we found a significant decline in feeding score by 0.18 units (score range 0-5) per 100 ng/ml afp increase (p=0.05) after adjusting for age. there was no significant relationship between afp levels and serum transaminase levels. conclusion: afp increases with age in a-t patients, though this may not apply to patients with missense mutations. there is a statistically significant increase in mortality and worsened swallowing scores with increasing afp levels, but this remains to be proven clinically significant. here we present a pediatric hae patient who had recurrent abdominal attacks in which constipation, secondary to the adhd medication dexmethylphenidate (focalin), appears to be a trigger. of importance, this is the first pediatric patient with hae to be described as having safely undergone a capsule endoscopy for direct visualization of the gastrointestinal tract. this was done to decrease the risks associated with the more invasive procedure of traditional endoscopy and colonoscopy. case presentation: the patient was an 8-year-old male with hereditary angioedema who presented with 1 day history of diffuse abdominal pain and nausea. in the ed, patient was in no acute distress. abdominal ultrasound showed severe circumferential thickening of the wall of multiple bowel loops and a large amount of simple ascites. x-ray revealed stool in the colon. he was admitted for pain control and hydration. in the next year, he visited the ed five more times for exacerbations of angioedema of his hand, penis, and bowel. each time, he presented he had underlying abdominal pain and constipation. he was seen by gastroenterology and had a workup that was negative for helicobacter pylori, parasites, and other gastrointestinal infections. to further evaluate his abdominal pain, capsule endoscopy was performed and well tolerated. during an admission in january 2016 he received a full inpatient bowel cleanout, after which, his angioedema finally improved. of note, he was diagnosed with adhd and started on dexmethylphenidate (focalin) just prior to this period of recurrent angioedema attacks, and he did not have attacks during the summer months when he was off the medication. discussion: abdominal pain is a common complaint in pediatric hospitals, and further workup consists of endoscopy and colonoscopy. this may be easily accomplished in the general population, however, in patients with hae, these procedures carry greater risk and may be avoided, leading to delayed diagnosis and treatment (2, 4) . a newer and less commonly used alternative for direct visualization of the gastrointestinal tract is capsule endoscopy. some benefits are that it does not require sedation, is less invasive, and is less likely to be irritating to the mucosa (3). additionally, since psychological stress may be a trigger for angioedema attacks, the decreased stress associated with a noninvasive procedure such as capsule endoscopy, makes it safer to use (1) . limitations of capsule endoscopy include dependence on battery life and its inability to biopsy or administer therapy if needed (3) . hereditary angioedema treatment consists primarily of avoiding triggers and managing acute episodes. in this first case of hae in a pediatric patient where capsule endoscopy was used, the procedure was well tolerated without any complications. recognizing constipation as a trigger and capsule endoscopy as a safe method of direct visualization of the gastrointestinal tract will help others to control and decrease the severity of their hae attacks as well. a 45 year old male with past medical history of common variable immune deficiency (cvid) and related autoimmune complications, including granulomatous-lymphocytic interstitial lung disease (glild), hepatosplenomegaly, leukopenia, and thrombocytopenia tolerated monthly subcutaneous immunoglobulin replacement as outpatient for several years with infrequent infectious complications. four months ago, he was found to have elevated liver enzymes on routine chemistry. a liver biopsy two months later showed pathology consistent with nodular regenerative hyperplasia (nrh) without overt cirrhosis. a hepatic venous pressure gradient (hvpg) of 21 mmhg was found, consistent with portal hypertension. his hepatitis viral markers were negative, he did not drink, and portal venogram was negative for thrombosis. in early october, the patient was admitted to the hospital with anasarca and tense ascites. he underwent a diagnostic and therapeutic large volume paracentesis and was also found to have spontaneous bacterial peritonitis (sbp) and bacteremia with group b streptococcus. the patients course was complicated by polymicrobial peritonitis, vre bacteremia, fungemia, variceal hemorrhage, hepatic encephalopathy, and hepatorenal syndrome. his hepatic complications from portal hypertension were out of proportion to his liver parenchymal disease. transjugular intrahepatic portosystemic shunt (tips) was considered to alleviate portal hypertension but was not feasible due to his degree of encephalopathy. immunosuppressants such as high dose steroids were given while in the hospital with plans to start rituximab to treat patients glild after he had recovered from the acute infections. unfortunately, after two months in the hospital, the patient succumbed to sepsis and progressive liver failure. this case emphasizes the importance of systematic screening and continued vigilance for hepatic complications in patients of cvid as studies have shown that nrh of the liver is present in more than 80% of cvid patients who undergo a liver biopsy (pmid: 23219764). a cross-sectional study of patients with primary hypogammaglobulinemia and hepatic dysfunction found that histological findings of nrh were present in 84% of cvid patients and was associated with portal hypertension in 75% of cases (pmid: 17998147). another study estimated the minimal prevalence of nrh in cvid patients as 12% (pmid: 18647320), stating that this was likely a gross underestimate as nrh may also be present in patients with normal liver function tests that are not routinely biopsied. therefore, liver enzyme levels may not anticipate the severity of liver involvement. there is currently no treatment for cvid-related liver disease. other causes of non-cirrhotic portal hypertension, including hepatic veno-occlusive disease and budd-chiari syndrome should be ruled out or treated in cvid patients presenting with hepatic disease. in the case of hepatic nrh in cvid patients, early detection could lead to earlier interventions (such as tips prior to hepatic encephalopathy), to mitigate complications. we describe the application of epigenetic quantification of t regulatory (treg) cells in addition to cd3+, cd4+, cd8+ t cells, b cells, nk cells, monocytes and neutrophils from as little as 50 î¼l of fresh, frozen or dried blood. the method yields identical results to flow cytometry from fresh blood samples of a healthy donor cohort, with the advantage of being more sensitive and precise with limited amount of blood and minimal sample preparation (sci transl med 2018). we have used this method 1) to immunophenotype patients with early onset immune regulatory disorders (pird) and primary immune deficiency (pid), and 2) to evaluate cell subsets reconstitution early after hematopoietic stem cell transplantation (hsct). patients with immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) and ipex-like pird were evaluated by analyzing the treg-specific demethylated region (tsdr) of the foxp3 locus in the total of cd3+ t-cells. despite the dysfunctional foxp3 mutated protein, ipex patients exhibited elevated treg/cd3+ cell ratios which seemed to correlate with disease severity. in contrast, most of the patients with ipex-like symptoms without foxp3 mutations exhibited decreased treg/cd3+ cell ratios -in line with the possible central pathogenic role of treg function and number in pird. using epigenetic quantification of cd3+/b-and nk cells, 23 out of 24 confirmed scid and xla cases were correctly identified within a cohort of 250 newborn dried blood spot (dbs) samples (96% sensitivity, 100% specificity). the method identified one delayed onset scid as well as a xla case that were missed by combined trec/krec testing. epigenetic immune cell quantification missed one scid case with maternal engraftment that was identified by combined trec/krec testing. abnormally elevated treg/cd3+ ratio was also detected in a dbs from a newborn who was subsequently confirmed to be affected with ipex syndrome. when applied to serial blood samples during engraftment and reconstitution post-hsct, the epigenetic method allowed identification of the different blood cell subsets, including treg cells, at earlier time points than flow cytometry according to current clinical practice. this opens the way to a better understanding of the correlation between early immune reconstitution events and graft vs. host disease or viral reactivation, earlier than with the current methods, in different types of hsct. these studies underscore the suitability of epigenetic immune cell quantification for accurately measuring multiple immune cell types from limited blood sample sources. we propose this method as uniquely suitable for novel molecular diagnostic applications in settings with limited fresh blood sample or limited cell number, at the point of care as well as for newborn screening. we evaluated a 5-year-old male with hyperpyrexia, hypertrichosis, conical hypodontia, and a history of illnesses concerning for nemodeficiency syndrome. starting at six months of age, he suffered recurrent episodes of acute otitis media (non-typeable hib and actinobacter iwolffli), pneumonia, and rsv bronchiolitis. whole exome sequencing demonstrated a de novo heterozygous c.1259g>a (p.r420q) mutation in the eda-receptor (edar) gene not present in the parental dna. his physical exam findings and mutation were consistent with hypohidrotic ectodermal dysplasia (hed), a rare genetic condition characterized by abnormal development of skin, teeth, hair, and sweat glands. hed is caused by defects in the ectodysplasin-a (eda)-nfkb signaling pathway but is not typically associated with immune deficiency. consistent with this, immunophenotyping showed normal sub-populations of t-, b-, and nk-cells. immunoglobulin and complement levels were quantitatively appropriate. he had normal mitogen-induced lymphocyte proliferation and normal antibody response to pneumococcal vaccination. nk-cell studies demonstrated robust cytotoxicity. however, nasal mucosa biopsy showed diffuse squamous metaplasia and the absence of ciliated epithelial cells. we hypothesize that recurrent infections in our patient arose from impaired mucociliary clearance due to a ciliary defect. this case raises the possible association between edar variants and ciliary dysfunction. it also underscores the importance of evaluating the immune status of hed patients with recurrent infections which could mimic nemo-deficiency and have broad implications about clinical management. the rapid pace of new gene discovery and phenotype expansion for primary immunodeficiency diseases (pidds) creates challenges for genetic testing and variant interpretation. whereas well-described clinical case reports in published literature have traditionally served as the source of phenotypic data used for variant interpretation, for pidds the causal variants are often private to the patients family and thus the sole source of phenotypic information for a novel genetic variant is frequently the history provided by the clinician on the test requisition form. taking into account such heterogeneous information during variant interpretation requires establishing objective criteria for its inclusion as part of the variant interpretation process. to this end, we adapted our laboratorys preexisting, evidence-based variant classification framework, called sherloc, by developing point-based criteria for the inclusion of clinical information such as a patients phenotype, familial segregation patterns, and whether the variant is inherited or de novo in the patient. as part of this process, we defined clinical criteria for 154 pidd genes. here, we illustrate the application of this method and the importance of integrating clinical information into variant interpretation. between april 2017 and october 2018, our commercial diagnostic laboratory performed 4057 immunological genetic tests, and information about the patients clinical history was provided in 2849 (70%) of these orders. restricting our analysis to just the 154 genes for which case report information is currently used in variant interpretation, these tests revealed 3868 variants, 370 (10%) of which were classified as pathogenic or likely pathogenic (p/lp). information from case report descriptions, segregation patterns, and de novo status were applied for 32%,15% and 4% of p/lp variants, respectively. in 37 (10%) cases, the clinical information provided by the clinician on the test requisition form was used as evidence in the classification of the patients variant as p/lp. ten variants were initially classified as being of uncertain significance and reclassified following receipt of further clinical information or testing of additional relatives. in addition, 35 suspicious variants of uncertain significance were identified in which one or two additional patient case reports would allow for reclassification from uncertain significance to p/lp. these data illustrate the importance of providing good quality clinical information to the genetic testing laboratory both at the time of sample submission and following the receipt of genetic test results. background: cartilage-hair hypoplasia (chh) is a skeletal dysplasia with combined immunodeficiency, variable clinical course and increased risk of malignancy, mostly non-hodgkin lymphoma and basal cell carcinoma. there is a paucity of long-term follow-up data, as well as knowledge on prognostic factors in chh. objective: we conducted a prospective cohort study in finnish patients with chh to describe clinical course and analyze risk factors for adverse outcomes. methods: we recruited 80 finnish patients with chh in 1985-1991 and performed clinical follow-up in 2011-2015. we obtained health information from finnish national medical databases (covering time period of 1969-2016), the finnish cancer registry and the cause-of-death registry of the statistics finland and analyzed all patients' health records. standardized mortality ratios (smrs) were calculated based on the population data. primary outcomes included immunodeficiencyrelated death (from infections, respiratory diseases or malignancies), the development of lymphoma and the development of skin cancer. results: the study cohort included 35 males and 45 females. median age at recruitment was 14.6 yrs (range 2 weeks -49.6 yrs) and median duration of follow-up for the surviving patients was 29.2 yrs (range 25.6 -31.0 yrs). half of the patients (46/80, 57%) had no symptoms of immunodeficiency, while 15 (19%) and 19 (24%) patients manifested symptoms of humoral or combined immunodeficiency respectively, including six cases of late-onset immunodeficiency. in a significant proportion of patients (17/79, 22%), clinical features of immunodeficiency progressed over time. of the 15 patients with non-skin cancer, eight had no preceding symptoms of immunodeficiency. altogether 20 patients had deceased (smr=7.0, 95% confidence interval (ci)=4. [3] [4] [5] [6] [7] [8] [9] [10] [11] including deaths due to pneumonia (n=4), malignancy (n=7, smr=10, 95%ci=4.1-21) and lung disease (n=4, smr=46, 95%ci=9. . malignancy was diagnosed in 21/80 (31%) patients, mostly lymphoma (n=9) and skin cancer (n=15). severe short stature at birth (compared to normal, smr/smr ratio=5. 4, , symptoms of combined immunodeficiency (compared to asymptomatic, smr=19 (95%ci=8.0-36) vs smr=4.8 (95%ci=2. 3-8.9 ), hirschsprung disease (odds ratio (or) 7.2, 95%ci=1.04-55), pneumonia in the first year of life or recurrently in adulthood (or=7. 6/19, , and autoimmunity (or=39, 95%ci=3.5-430) in adulthood associated with early mortality. in addition, recurrent pneumonia in childhood was associated with the development of lymphoma, while warts and actinic keratosis were associated with the development of skin cancer. birth length standard deviation score correlated significantly with the age at the diagnosis of first malignancy (p=0.0029), lymphoma (p=0.011) and skin cancer (p=0.014), demonstrating that patients with shorter birth length developed malignancies at an earlier age. conclusions: patients with chh have high mortality due to infections and malignancies, but also from lung disease. some subjects present with late-onset immunodeficiency or malignancy without preceding symptoms of immune defect, warranting careful follow-up and screening for cancer even in asymptomatic patients. we provide clinicians with the risk factors for adverse outcomes to assist in management decisions. autoimmune lymphoproliferative syndromes (alps and related disorders) are characterized by insufficient apoptosis due to defects in the fas apoptosis pathway. fadd deficiency (omim 602457) is an autosomal recessive disorder resulting from a mutation in fas-associated protein with death domain (fadd), the adaptor protein involved in fas signaling to caspases 8 and 10. we present a case of fadd deficiency identified by whole exome sequencing with a novel genetic mutation we describe two brothers with recurrent febrile episodes accompanied by seizures and respiratory compromise. the older sibling initially presented with status epilepticus following the measles mumps rubella vaccination later experiencing similar episodes until his demise at 18 months of age. the younger sibling, who is unvaccinated, presented at 14 months with fever, rash, vomiting, and diarrhea. he developed status epilepticus with respiratory depression that required intubation. he also had enlarged cervical lymph nodes that regressed with antibiotics and steroids. he recovered from that episode but subsequently had a series of similar illnesses with fevers, altered mental status and seizures. with the exception of elevated hhv6 igg, extensive infectious workup up in all instances was negative. previously described fadd deficiency patients demonstrate an alps like phenotype with increased circulating double negative t cells, lymphocyte apoptosis defects, elevated fas ligand and il10, encephalopathy, functional asplenism but no splenomegaly or lymphadenopathy. our patients clinical and laboratory findings were similar. he had normal igg and iga, decreased igm, and lack of isohemagglutinins. absolute cd3+ count is elevated, with elevated percent of cd3+ tcr+ cd4-cd8-. normal mitogen and antigen t lymphocyte stimulation, but with defect in pokeweed induced b cell proliferation. fas ligand and il10 level are increased (see table 1 ). no hepatosplenomegaly, but howell jolly bodies were detected in peripheral blood indicating functional hyposplenism. whole-exome sequencing revealed two different genetic alterations in the fadd gene: a maternally inherited nonsense mutation predicted to severely truncate the protein and a paternally inherited missense mutation in codon 105. although this paternal mutation has not been described as pathogenic, a different variant in same nucleotide of fadd has been associated with fadd deficiency (reference1). there are very few cases in the literature of fadd deficiency patients and the overall prognosis is poor compared to classical alps patients, as these patients are at significant risk of deadly sepsis from encapsulated organisms or death from neurologic complications. of the fadd deficiency patients described in the literature, several died prior to 5 years old. while pneumococcal prophylaxis may reduce the risk of sepsis, hematopoietic stem cell transplant has been reported for patients with fadd deficiency (reference2), and is being considered for our patient. rationale: hcuvp is a patient product-introduction program that provides cuvitruâ® (immune globulin subcutaneous [human], 20% solution [ig20gly]) free of charge for the first 4 infusions to eligible patients with primary immunodeficiency disease (pid). using patient data from this ongoing program, our analysis described the clinical characteristics and infusion parameters of pediatric and adolescent patients who were initiated on ig20gly through hcuvp. methods: hcuvp eligibility criteria were: patients aged 2 years old, with a primary icd-10-cm code verifying diagnosis of pid, and no current or prior use of ig20gly at program initiation. data from patients who received the first ig20gly infusion between january 1, 2017, and september 1, 2017 were included. data from patients receiving infusions after october 31, 2017 were censored. descriptive statistics were calculated for patients demographic and clinical characteristics and prescribed and actual infusion characteristics by age group (<18 years and 18 years). results: in total, 817 patients who completed all 4 infusions were included in the analysis, of whom 97 were aged <18 years. among those who previously received immunoglobulin (ig) therapy, a greater percentage of patients aged <18 years were treated with intravenous ig therapy (n=46; 73%) compared with adult patients (n=222; 62%) before initiating ig20gly. nine patients aged <18 years were treatment naã¯ve. the mean infusion volume per site was lower among patients aged <18 years (25 years: 17.9 ml; 611 years: 26.4 ml; and 1217 years: 34.6 ml) than among patients aged 18 years (1864 years: 38.5 ml and 65 years: 38.9 ml). however, the mean infusion rate per site was similar between patients aged <18 years ( xmen disease (x-linked immunodeficency with magnesium defect, epstein-barr virus infection and neoplasia) is a primary immune deficiency caused by mutations in magt1 and characterized by chronic infection with epstein-barr virus (ebv), ebv-driven lymphoma, cd4 t-cell lymphopenia, and dysgammaglobulinemia. magt1 gene codifies to magt1 protein, a mg2+-selective transporter, expressed in the human immune system, specifically in the spleen and the thymus. functional studies have established the key role of magt1 in t cells and natural killer (nk) cell activation. upon cd4+ t-cell receptor stimulation, magt1 mediates a transient mg2+ influx that is necessary for phospholipase c gamma 1 (plcy1) activation, which drives ca2+ rise and downstream signaling. this mg2+ influx also regulates cytotoxic functions of nk and cd8 t cells through nkgd2, reason why these patients have impaired cytolytic responses against ebv. eleven male xmen patients have been described. we present the case of a 1-year old hispanic infant with a pathogenic variant in magt1 gene that clinically manifested with early pneumocystis jirovecii and cytomegalovirus (cmv) interstitial pneumonia, and ebv chronic infection with good response to intravenous immunoglobulins supplementation without hematopoietic stem cell transplantation or gene therapy. laboratory study highlights low levels of nkg2d ligands. the objective of this case report is to broaden the spectrum of clinical presentation of xmen disease, that manifests initially as a combined immune deficiency (cid) and evolved with a favorable course of the disease with intravenous immunoglobulins supplementation therapy and chemoprophylaxis with trimethoprim-sulfamethoxazole. introduction: lysinuric protein intolerance (lpi) is a recessively inherited disorder of the cationic amino acids transporter subunit y+lat1 caused by variants in the slc7a7 gene. the disease is characterized by protein-rich food intolerance has a heterogeneous presentation. the clinical findings are a result of depletion of lysine, ornithine, and arginine. symptoms can include hyperammonemia, failure to thrive, protein aversion, neurologic disease, and lung disease. there is also evidence that inflammatory manifestations are mediated through upregulation of nfb, il1, and tnf that occur independent of intracellular arginine levels and can lead to lifethreatening episodes of hemophagocytic lymphohistiocytosis (hlh). case presentation: a 17-year-old male presented with history of anxiety, depression, eating disorder, delayed puberty and complex partial seizures. due to poor nutrition and failure to thrive, a gastrostomy tube was placed. following commencement of enteral feeds, he presented with altered mental status, bilateral mydriasis, hyperreflexia, and agitation which lead to a picu admission. ammonia peaked as high as 181 î¼mol/l and episodes ceased with cessation of enteral feedings. prior to enteral feeds, he had been self-restricting protein in his diet. biochemical testing was consistent with lpi and illumina next-generation sequencing revealed compound heterozygous variants in slc7a7 (p.s396lfs*122 and p.e465dfs*54). hyperammonemia resolved quickly with cessation of protein intake and high rate dextrose infusion without the need for ammonia scavenging agents. he was subsequently started on proteinrestricted enteral feeds. at diagnosis he did not have any respiratory symptoms, ct scan of chest showed patchy areas of groundglass opacification that was suggestive of early pulmonary alveolar proteinosis (pap). bronchoalveolar lavage demonstrated foamy, cloudy pink fluid and elevated bronchioalveolar macrophages on cell differential. his clinical course and slc7a7 genotype led to suspicion for smoldering hlh. the findings of elevated ferritin, hypertriglyceridemia, decreased fibrinogen, splenomegaly, elevated il-2 receptor, decreased nk cell function, along with hemophagocytosis on bone marrow biopsy confirmed the diagnosis. because of his pap and hlh, in addition to dietary modifications, a trial of il-1 beta inhibition (anakinra) at 3 mg/kg/day was initiated. follow up ct scan of chest 2 months after initiation of anakinra showed complete resolution of pulmonary groundglass opacifications and pap. bone marrow evaluation showed continued hemophagocytosis in spite of the normalization in ferritin, soluble il-2 receptor, nk function, and triglycerides levels. overall, he is significantly improved on daily anakinra and no longer meets criteria for hlh or pap. discussion: recent data has shown in y+lat1 models that thp-1 macrophages and a549 airway epithelial cells upregulate il1 and tnf regardless of intracellular arginine content. this suggests that inflammatory manifestations may continue independent of dietary modifications. we present a 17 year old patient with newly diagnosed lpi who was treated dietary modification and anti-il1 therapy resulting in resolution of hlh and pap. more research is needed to see if long-term il1 blockade that can consistently control both the immunologic and pulmonary manifestations of lpi and positively impact morbidity and mortality. learning objective: recognize that symptoms of bartonella endocarditis and associated complications can share features of certain immunocompromising conditions. case description: an 8-year-old caucasian boy with history of repaired pulmonary atresia and aortic root dilation was diagnosed with pancytopenia and splenomegaly during a brief hospitalization for atypical pneumonia. pancytopenia persisted, splenomegaly worsened, and five months after presentation, he developed hypertension and renal insufficiency. he was diagnosed with hypocomplementemic, diffuse sclerosing and crescentic glomerulonephritis and was started on mycophenolate mofetil with improvement in kidney function and stabilization of cytopenias. as part of a comprehensive immune work-up, alps (autoimmune lymphoproliferative syndrome) panel was sent and demonstrated elevated double-negative t (dnt) cells with 3 out of 4 positive immunologic criteria for alps. neither targeted sequencing for alps and alpslike disorders nor whole exome sequencing revealed pathogenic mutations. by age 10, the patient remained on mycophenolate, but developed failure to thrive, with weight dropping from 37th percentile to less than 3rd percentile. he was hospitalized again for low-grade fever, increased work of breathing, left shoulder pain and fatigue and was found to have right lower lobe pneumonia. pancytopenia worsened, and he was started on cefepime and azithromycin without improvement in symptoms. echocardiogram revealed vegetations in his pulmonary conduit and bilateral branch pulmonary arteries, but multiple blood cultures were negative. upon further history, the patient reported contact with kittens. bartonella henselae titers and polymerase chain reaction (pcr) from blood were sent and were both positive. he completed a 2-week course of gentamicin, 1-month course of ceftriaxone, and was transitioned to doxycycline and rifabutin. after initiating antimicrobial therapy, his weight and energy significantly improved, his blood bartonella pcr became negative, and his splenomegaly resolved. approximately one year later, the patient underwent pulmonary artery conduit replacement and bartonella pcr testing of the tissue specimen was positive. he has had sustained weight increase, resolution of hypocomplementemia and splenomegaly, decrease in dnt cell frequency from >2% to 0.9%, and improvement though not resolution of cytopenias. he currently remains on doxycycline and rifabutin and continues treatment with mycophenolate. discussion: alps is characterized by defective lymphocyte apoptosis and clinical features such as lymphadenopathy, splenomegaly, hepatomegaly, cytopenias, and glomerulonephritis. the hallmark laboratory finding is expansion of dnts. our patient met criteria for a probable alps diagnosis based on the presence of both required criteria (chronic splenomegaly and elevated dnt cells) and secondary additional criteria (typical immunologic findings noted on alps panel). pediatric cases of bartonella henselae endocarditis have been associated with splenomegaly, cytopenias, and glomerulonephritis which mimic many features of monogenic immune dysregulatory disorders. the diagnosis of bartonella endocarditis in our patient therefore raises the question of whether his immunosuppression predisposed him to infection or if his entire clinical presentation can be explained by bartonella endocarditis. physicians taking care of patients with immune dysregulatory disorders should consider bartonella endocarditis in the differential diagnosis of onset or exacerbations of immune dysregulation. rationale: while fever is considered a sign of infection, many individuals with primary immunodeficiency (pi) anecdotally report a lower than normal average body temperature. on immune deficiency foundation (idf) friends and idf pi connect research forum online, pi patients report a diminished fever response even when other signs of infection are present. there is limited knowledge about the average body temperature in persons with pi. however, the implications of missing an infection in those with pi is well established. methods: study investigators partnered with patient investigators to design a prospective cohort study to determine whether body temperature differed between persons living with and without pi. three hundred fifty adults with pi were recruited from idf and one adult household member without pi was also recruited. mckesson digital oral thermometers (model 01-413bgm) were provided and used to record temperatures in all participants three times a day for five consecutive days. descriptive statistics were calculated. median body temperatures were compared between the two cohorts at each time point using mann-whitney test. results: data from 254 households were used for analysis (72.6% participation rate). the pi population was largely female (85.8%) with a median age of 49 years and largely caucasian population (97.6%). the non-pi population was largely male (66.9%) with a median age of 53 years and largely caucasian population (92.9%). pi diagnoses included cvid (74.8%), hypogammaglobulinemia (12.6%), igg subclass deficiency (4.7%), selective iga deficiency (3.1%), specific antibody deficiency (3.1%), agammaglobulinemia (0.4%), chronic granulomatous disease (0.4%), combined immunodeficiency (0.4%), and complement deficiency (0.4%). a total of 123 individuals with pi (48.4%) reported a lower than normal non-sick body temperature, while 108 individuals with pi (42.5%) reported a normal (between 97â°f -99â°f) non-sick body temperature. a total of 172 individuals with pi (67.7%) reported absence of fever with infection, while 50 individuals (19.7%) reported a normal fever response with infection. the median body temperature was significantly higher for the pi patients in the morning, but not evening or bedtime, reading in 4 of the 5 days (monday: pi = 97.5â°f vs. non-pi = 97.2â°f, p = 0.0291; tuesday: pi = 97.4â°f vs. non-pi = 97.2â°f, p = 0.0020; wednesday: pi = 97.5â°f vs. non-pi = 97.2â°f, p = 0.0009; thursday: pi = 97.4â°f vs. non-pi = 97.2â°f, p = 0.0575; friday: pi = 97.4â°f vs. non-pi = 97.2â°f, p= 0.0008). conclusions: despite the limitations of this non-clinical study, individuals with pi are knowledgeable about their conditions and can offer unique insights and direction to researchers. this study demonstrates that collaboration with patient advocacy groups may facilitate patient-centered and patient-driven research with high participation among the target population. introduction: familial mediterranean fever (fmf) is a hereditary condition characterized by recurrent episodes of painful inflammation caused by mutations in the pyrin (mefv) gene. alterations in the mefv gene affect pyrin production leading to recurrent fevers and painful inflammation in the peritoneum, synovium, and pleura. amyloidosis may also develop as a complication. arabic, turkish, armenian, and sephardic jewish populations are most commonly affected. homozygosity for mefv mutations are associated with a more severe course. there is a paucity of information regarding pediatric fmf in the literature. case: we present a case of a 2-year-old male with minor speech delay diagnosed with compound heterozygous fmf. patient was initially referred due to recurrent fevers and infections. at 4 months of age, he was hospitalized with septic shock requiring intubation secondary to adenovirus. at 5 months of age, the patient began to have recurrent fevers every 3 to 4 weeks, leading to multiple blood draws and courses of antibiotics prior to referral. at 11, 12, and 22 months of age, he developed three separate episodes of febrile seizures. a total of 10-15 lifetime episodes of acute otitis media occurred prior to bilateral myringotomy tube placement. four episodes of streptococcus pyogenes pharyngitis confirmed by throat culture preceded tonsillectomy. no oral ulcers, joint pain, or abdominal pain were reported. no other infections such as pneumonia, sinusitis, uti, non-viral gastroenteritis, fungal infections, or skin infections were reported. both parents are ashkenazi jewish and a maternal history of early miscarriage was noted. family history was negative for immunodeficiency, malignancy, and autoimmunity. the patients vital signs and physical exam were unremarkable. serology indicated leukocytosis of 18.53 k/l with elevated monocytes of 1390 cells/l, elevated eosinophils of 1200 cells/l, and slightly elevated cd8 t cell count of 2653 cells/l. neutrophil, cd4 t cell, b cell, nk cell enumeration, and immunoglobulin panel were normal for age. tetanus, diphtheria, rubella, streptococcus pneumoniae, and haemophilus influenzae b titers were protective. genetic analysis identified that the patient was compound heterozygous for the e148q and v726 mutations in the mefv gene. family was instructed to keep a fever diary. colchicine 0.6mg once a day was given initially, then increased to 1.2mg once a day for inadequate response. loose stools were observed while patient was maintaining a lactose free diet so he was switched to colchicine 0.6mg bid with resolution of loose stools. apart from two occasions when his colchicine dose was missed, the patient remained afebrile at his follow up visits. conclusion: we present a pediatric case of compound heterozygous fmf (e148q and v726 mefv mutations) in an otherwise healthy 2-year-old male of ashkenazi jewish background, initially symptomatic at 5 months of age. individuals who are compound heterozygous for the e148q and a second mevf mutation are generally symptomatic, although severity cannot be predicted. additional pediatric research on symptomatic heterozygous and compound heterozygous fmf is recommended. natural killer (nk) cells are innate lymphocytes that play a key role in defense against virally-infected cells and in tumor surveillance. nk cells can be divided in two subsets. the majority of nk cells in peripheral blood expressed intermediate levels of cd56 and are referred to as cd56(dim). these nk cells are responsible for nk cell cytotoxicity. a minor population of nk cells express very high expression of cd56 and are referred to as cd56(bright). these nk cells are responsible for cytokine production and are precursors to cd56(dim) nk cells. a few immunodeficiencies have been described in which there are abnormal nk cell subsets, such as autosomal dominant gata2 deficiency where cd56(bright) nk cells are absent and irf8 where there is a paucity of cd56(dim) nk cells and relative expansion of cd56(bright) nk cells. here we present a patient with an absence in cd56(bright) nk cells secondary to cd27 deficiency. our patient is a 6-year-old african american female born to non-consanguineous parents. the patients past medical history is significant for chronic lung disease secondary to prematurity, recurrent acute otitis media, failure to thrive and congenital hypothyroidism. family history is significant for an older sister that presented at age 3 with ebv-associated hodgkin lymphoma whose treatment was complicated by chronic activated ebv infection and who ultimately underwent hematopoietic stem cell transplantation (hsct). our patient presented with pancytopenia, fever, lymphadenopathy and splenomegaly. she was found to have ebv viremia with greater than 550,000 copies in whole blood by pcr. she was treated with two doses of rituximab followed by etoposide and dexamethasone as a bridge to hsct. whole exome sequencing demonstrated a homozygous mutation in cd27. cd27 is a member of the tumor necrosis factor receptor family and influences the function of t cells, b cells and nk cells. in nk cells, cd27 is primarily expressed in cd56(bright) nk cells. cd27 deficiency is an autosomal recessive disorder associated with persistent symptomatic ebv viremia, including ebv-driven hemophagocytosis and lymphoma, hypogammaglobulonemia and specific antibody deficiency. our patients immune evaluation prior to initiation of chemotherapy and immunosuppression was notable for very elevated igg, iga and igm. despite hypergammaglobulonemia patient had only 3 out of 11 protective titers against streptococcus pneumoniae. the patient had pan-lymphopenia with appropriate percentages of lymphocyte subsets. assessment of her b cell subsets showed a slight increase in the percentage of transitional b cells/plasmablast and a nearly complete absence of cd27-expressing b cells. her nk cell phenotyping demonstrated a complete loss of cd56(bright) nk cells with reduced nk cell cytotoxicity, comparable to what has been previously reported in patients with gata2 deficiency. previous reports of patients with cd27 deficiency denote normal nk cell numbers with normal to moderately reduced nk cell cytotoxicity, however, cd27 deficiency causing a specific loss of the cd56(bright) nk cell subset has not been previously reported. cd27 deficiency should be consider in patients with ebv driven disease and abnormal nk cell studies. introduction/background: the transcription factor ikaros is encoded by the ikzf1 gene and plays a crucial role in lymphopoiesis. somatic, and more recently also germline mutations of ikzf1 are associated with a hematologic malignancies, most notably b-cell precursor acute lymphoblastic leukemia. germline mutation in ikzf1 was first reported as a monogenic cause of human disease characterized by marrow failure and immune deficiency in a single neonate in 2012. subsequently, mutations leading to haploinsufficiency were discovered to underlie a proportion of patients with cvid and low b cell numbers, and dominant-negative mutations have been observed to cause more severe combined immune deficiency phenotypes. at this time, there is very little known regarding allogeneic hematopoietic cell transplantation (hct) outcomes for patients with severe dominant-negative ikzf1 mutations. concerningly, ikaros deficiency has been observed to have a negative impact on graft versus host disease in mouse models. objective: to describe allogeneic stem cell transplant outcomes in patients with the dominant-negative ikaros mutation. methods: we collected transplant data from 4 patients who underwent allogeneic hct at transplant centers around the world. results: patients underwent allogeneic hct using a variety of conditioning regimens. patients received bone marrow (n=3) or cord blood (n=1) grafts from an hla-matched sibling donor (n=1) or single allele hlamismatched unrelated donor (n=3). neutrophil engraftment occurred between day +12 and +51 post-transplant. platelet engraftment occurred between day +8 and +167 except in one patient who did not have return of normal platelet counts due to underlying liver dysfunction. all patients were documented to have greater than 99% whole blood donor chimerism at a median of 28 days (range 12-51 days) following transplant and maintained >95% donor chimerism until last follow-up. only one patient developed grade ii acute gvhd. no patients developed chronic gvhd. one patient died approximately 1 year post transplant related to cryptosporidium cholangitis which existed prior to hct. at the most recent follow up of the 3 surviving patients (range: 0.99-7.2y), ivig had been discontinued, antimicrobial prophylaxis had been stopped, and patients had received routine vaccinations. they all had excellent performance status. conclusions: allogeneic hct may be a safe option to consider for patients with dominant-negative ikaros mutation as there does not appear to be an increased risk of death or gvhd. moreover, 3-out-of-4 of the transplanted patients are alive and well and show no features of the disease. however, because of the limited number of patients evaluated and the retrospective nature of this analysis, our data do not allow firm conclusions to be made, and further studies will be needed to evaluate outcomes in larger cohorts. introduction: when evaluating patients with t-cell lymphopenia, we often are concerned about defects in lymphocyte production and function, especially in the setting of frequent infections. here we outline a case demonstrating t-cell lymphopenia due to increased loss, which should be considered in the differential diagnosis. case report: we report a 13-year-old male who initially presented with recurrent, right-sided pneumonias requiring frequent hospital admissions including severe episodes necessitating intensive care unit admission. his work up for the pneumonias included a bronchoscopy revealing normal anatomy with minimal inflammation, and a chest ct with mild peribronchial wall thickening. as his pulmonary disease progressed, he developed a persistent, productive cough with expectorated mucous plugs that were plastic-like in appearance. while his pulmonary symptoms responded to steroids, his mucous plug production persisted. sputum cultures were intermittently positive, isolating cryptococcus neoformans and aspergillus niger. he underwent vats and wedge biopsy, concerning for recurrent aspiration. an immunologic evaluation initially demonstrated normal t-and b-cell counts, but serial evaluation of his lymphocyte population demonstrated low cd4+ cells (ranging 151-367 cells/cumm), and low normal cd8 cells (ranging 101-177 cells/cumm) with normal b-and nk-cell numbers. further t-cell evaluation revealed normal ratios of naive and memory p o p u l a t i o n s ( c d 4 c d 4 5 r a + 6 1 % , c d 4 c d 4 5 r o + 3 9 % , cd8cd45ra+ 74%, cd8cd45ro 33%), normal trec (7768 copies per 10^6 cd3 cells) and normal thymic emigrants (cd4cd31cd45ra+ : 158, normal 150-1500), indicative of sufficient thymopoiesis. mitogen and antigen stimulation assays demonstrated normal responses to phytohemagglutin, concanavalin a, and pokeweed mitogen, with a low lymphocyte response to candida. he had normal quantitative immunologlobulins, normal diphtheria, tetanus and streptococcus pneumonia titers. his dihydrorhodamine flow cytometry and fish for chromosome 21q11.2 deletion were negative. given normal function and thymic output, his immunologic profile was concerning for t-cell loss. our patient was registered with the undiagnosed disease network, and had a second review of his lung biopsy, concerning for plastic bronchitis. subsequent lymphatic imaging demonstrated abnormal lymphatics within the bilateral clavicular space, right greater than left, with questionable partial thoracic duct, explaining his unilateral symptoms. he was diagnosed with plastic bronchitis secondary to abnormal lymphatic drainage, with lymphatic fluid filling his airways and secondary t-cell loss. discussion: plastic bronchitis is a rare and potentially fatal disorder, seen commonly after the fontan procedure for congenital heart disease. this process has resulted in t-cell loss into the airway and subsequent t-cell lymphopenia. in patients with fontan-related protein losing enteropathy, multiple immune abnormalities have been described including reduced immunoglobulins, lymphopenia, and selective cd4 lymphocyte deficiency. similar findings have been reported in patients with lymphatic malformations. although the impact of t-cell loss on adaptive immunity is not entirely known, there is no indication of increased risk for atypical infections. given his normal mitogen assay, our patient did not start prophylactic antibiotics. he continues to have symptomatic episodes with lymphopenia, but has had no opportunistic infections, and remains stable with an aggressive pulmonary regimen. we conclude by reiterating the importance of considering t-cell loss in patients presenting with lymphopenia, particularly with evidence of normal thymopoeisis and t-cell function. introduction: granulomatous disease (gd) has been described with a variable incidence (8.0-22.0%) in patients with common variable immunodeficiency (cvid). an increase in malignancies has been reported in cvid patient cohorts, particularly for lymphoma, reported in 1.6-8.2% of the cvid patients depending on the cohorts. prior analysis of a cohort of 436 cvid patients included 59 patients with gd (gd+). in these, there was a suggestion of more cases of lymphoma (12.5%) when compared to cases without (gd-) (5.0%) although the difference was not statistically significant (p=.07). objectives: compare the frequency of lymphoma in gd+ and gd-patients in the cvid patient cohort from the usidnet registry. methods: we submitted a query to the usidnet registry requesting deidentified data for patients with the diagnosis of cvid, through august 2018. statistical analysis was performed on spss, with comparisons done with pearson chi-square or fisher's exact test, depending on the sample sizes, using an alpha level of .05. results: a cohort of 1395 cvid patients from the usidnet registry was analyzed. ninety-one patients (6.5%) were gd+. overall, 152 patients (10.9%) had a malignancy diagnosis, 47 of these (3.4%) with lymphoma. lymphoma was present in 6/91 gd+ patients (6.6%) versus 41/1304 gdpatients (3.1%) (p=.12). overall malignancy was present in 15/91 gd+ (16.5%) versus 137/1304 (10.5%) (p=.08). discussion: in the cohort of 1395 cvid patients from the usidnet registry, we found a frequency of lymphoma of 3.4%, which is in the range of previously described cohorts. the frequency of lymphoma was 6.6% in patients with gd, higher than the 3.1% frequency for gd-patients, but these differences were not statistically significant. our identified frequency of lymphoma in gd+ patients was lower than the one previously identified in the 436 cvid patient cohort, but with similar proportional differences between gd+ and gd-patients. despite no statistical significance, the frequency of lymphoma, as shown here and elsewhere, was higher in cvid patients gd+ than gd-in both studies, with no full understanding of this increased risk of lymphoma. expanding this analysis to larger groups of cvid patients may help to confirm, or deny a more robust association, which may have a meaningful impact in the outcomes of this particular population. introduction: patients with refractory pericarditis have been treated with intravenous immunoglobulin (ivig) or interleukin 1 receptor antagonist (anakinra) with limited and transient benefit. separate or combined therapy with subcutaneous immunoglobulin (scig) and interleukin (il) 1 inhibitor (rilonacept) for refractory pericarditis in a cohort of patients has not been previously described. case descriptions: 4 patients were referred for recurrent pericarditis refractory to traditional therapies at ages ranging from 16 to 54 years. they all had multiple serious sequelae of their pericarditis and abnormal immune parameters including hypogammaglobulinemia, poor responses to vaccines, poor mitogen induced lymphocyte proliferation, and/or b cell lymphopenia. the patients had varied past medical histories and associated conditions. patients were started on ig, with some initiated on ivig, though all were transitioned to hyaluronidase-facilitated scig (hyqvia). patients were then started on either anakinra or rilonacept with 3 patients continuing on rilonacept and 1 remaining on anakinra. all patients had complete or near complete resolution of their pericarditis on dual therapy for greater than 1 year. the markedly elevated il1 prior to therapy seen in all of the patients normalized post-therapy. some patients had elevated il6 prior to therapy that also improved post-therapy. 1 patient who has also been diagnosed with familial mediterranean fever (fmf) has stopped both therapies for greater than 1 year with no further episodes of her pericarditis. discussion: 4 patients with recurrent refractory pericarditis and signs of immunodeficiency and autoinflammatory disease on laboratory testing responded to dual therapy with hyqvia and rilonacept or anakinra resulting in resolution of pericarditis. inflammasome and immune abnormalities may be implicated or associated with recurrent pericarditis and may respond to targeted therapies. chief, laboratory of clinical immunology and microbiology, idgs, dir, niaid, nih, bethesda, md, usa hypomorphic recombination activating gene 1 (rag1) mutations result in residual t-and b-cell development in both humans and mice and have been found in patients presenting with delayed-onset combined immune deficiency with granulomas and/or autoimmunity (cid-g/ai). recent studies have shed light on how hypomorphic rag1 mutations alter the primary repertoire of t and b cells, but less is known about their effect on immune dysregulation in targeted organs. in order to investigate the role of these mutations in determining intestinal disease, we set out to evaluate gut immunity and microbiota interplay in rag1 mutant hypomorphic mice. we evaluated two mouse models carrying homozygous rag1 mutations (r972q and r972w), corresponding to human mutations (r975q and r975w, respectively) described in patients with cid-g/ai. both mutations fall in the coding flanksensitive region of the rag1 c-terminal domain. on the basis of aminoacid properties and in vitro studies, the r972q mutation has demonstrated a moderate effect on rag1 protein stability while the r972w mutation resulted highly disruptive. analysis of intestinal pathology in rag1 mutant mice (niaid animal protocol lcim 6e) revealed different degrees of spontaneous colitis, with the most severe inflammatory infiltrate observed in mice carrying the most disruptive mutation, r972w. colonic inflammation was characterized by crypt elongation, epithelial hyperplasia, and an abundant inflammatory infiltrate extending to the colonic lamina propria, with occasional crypt abscesses. a significant increase in activated cd44hicd62lcd4+ t cells expressing the gut homing receptor 47 was observed in mesenteric lymph nodes (mlns) of both mutant strains, and was especially prominent in r972w mutant mice. additionally, the proportion of mln cd4+ t regulatory (treg) cells was increased in both mouse models. finally, mln of mutant mice contained a high number of myeloid cells (cd11b+ ) along with a decreased number of b220+ b cells, and these abnormalities were also more prominent in r972w than in r972q mice. in summary, we have shown that rag1 mutant hypomorphic mice present with different degrees of inflammatory bowel disease, with the mouse model carrying the most disruptive mutation presenting with the most severe phenotype. we are currently performing studies to evaluate the impact of rag1 mutations on microbiome composition and diversity in these mouse models of cid-g/ai. background: hypogammaglobulinemia or low serum immunoglobulin g (igg) levels either inherited (primary) or acquired (secondary) is associa t e d w i t h i n c r e a s e d i n f e c t i o n r a t e s . p r i m a r y ( 1â°) hypogammaglobluinemia can be caused by many primary immune deficiencies (pid) including combined variable immune deficiency (cvid), while secondary (2â°) hypogammaglobluinemia can be caused by many acquired conditions such as lymphomas, leukemias, or chemotherapies and other immunosuppressive drugs. immunoglobulin replacement therapy (irt) has been the mainstay of treatment in patients with hypogammaglobulinemia by reducing infection through replenishing the quantitative igg. there are other applications of ig therapy such as in autoimmune diseases, where the mechanism of action is thought to be ig mediated immunomodulation. innate immune cells have shown to be involved in such mechanism, but whether irt modulates adaptive immune cells in patients with hypogammaglobulinemia is not well known. hypothesis: irt has an immunomodulatory effect on t-cell function and proliferation in patients with hypogammaglobulinemia. methods: blood from thirty patients with 1â°(n=12) or 2â°(n=18) hypogammaglobulinemia recruited from the immunodeficiency clinic at the ottawa hospital was drawn for peripheral blood mononuclear cell (pbmc) isolation, before starting irt and minimum 8 weeks after starting irt. data regarding igg level, number and type of infections after receiving irt was collected. pbmcs were analyzed using flow cytometry for quantitation of t-cell subset. cultured and anti-cd3/cd28 stimulated pbmc were also analyzed for extracellular and intracellular cytokine production, measured by e l i s a a n d f l o w c y t o m e t r y, r e s p e c t i v e l y. c o m b i n e d cytomegalovirus, epstein-barr virus and influenza virus (cef) peptides were used to study specific t-cell responses. anti-cd3/ cd28 stimulated pbmc were used for celltrace t-cell proliferation a s s a y s . d a t a w a s g r o u p e d b a s e d o n n a t u r e o f hypogammaglobulinemia i.e. 1â°or 2â°. results were compared between before and after irt using wilcoxon matched-pairs signed rank test. results: irt was not found to significantly alter proportion of treg, cd4+, or cd8+ t-cell populations or activation state as measured by cd45ra/r0 expression. however, irt was found to significantly increase expression of intracellular ifn-y in cd4+ and cd8+ t-cells post-cd3/cd28 stimulation in 2â°(p = 0.007), but not in 1â°h ypogammaglobulinemia patients. there was no change in extracellular il-10 and il-17 cytokine production in both groups. in contrast, cd8+ tcells in 1â°hypogammaglobulinemia patients showed significantly higher expression of intracellular ifn-y and tnf-a post-cef viral peptide stimulation (p = 0.027). cd3+ and cd8+ t-cell proliferation after cd3/cd28 stimulation was found to be decreased after irt for both groups (p = 0.025 & p = 0.049). conclusions: our results suggest that irt can alter cd4+ and cd8+ t-cell function with differential effect in patients with 1â°o r 2â°hypogammaglobulinemia in addition to replenishing serum igg level. more experiments assessing cytotoxicity of t-cells will be conducted to further study t-cell subset function as well as bcell function. these laboratory results will be analyzed for association with clinical outcomes. uploaded file(s) uploads background: severe congenital neutropenia (scn) is a rare immunodeficiency disorder characterised by the extremely low absolute neutrophils count (anc) less than 0.5x109/l. the clinical feature of scn is recurrent bacterial infections and the patients the risk of leukemia development. the incidence of scn is estimated to be 1 in 200 000 individuals. mutations in more than 20 genes have been described causing scn and it is either recessive, dominant or x-linked inheritance. case presentation: we described an 11 years old malaysian girl who presented with recurrent abscesses over the whole part of the body, recurrent oral candidiasis, growth failure and recurrent pneumonias since 4 months old. she also had history of a few episodes of acute tonsillitis, chronic suppurative otitis media and herpes zoster infections. throughout her age, she had persistent neutropenia less than 0.5x109/l but in few occasions, her anc elevated up to more than 1.0x109/l . she was treated as autoimmune neutropenia, respectively due to few positive results of autoimmunity workout such as antinuclear antibodies (ana) and double stranded dna (dsdna) but eventually later to be negative. later at the age 9 years old, whole exome sequencing was performed and confirmed by sanger s e q u e n c i n g , f o u n d a h e t e r o z y g o u s v a r i a n t i n e l a n e gene(c.640g>t; p.gly214ter), an autosomal dominant which was described to cause scn. both parents do not carry this mutation, hence, it is a de novo mutation. currently, she had few on and off recurrent infections. despite that, she is relatively well and on prophylaxis antibiotic. conclusion: to our knowledge, we report for the first time a malaysian girl with scn, with confirmed mutational analysis of the elane gene. the delayed diagnosis might be due to the insufficient awareness of the phenotypic presentation of this rare disease. moreover, the genetic analysis is not available in malaysia and need to be done outside of the country. this case demonstrates the importance of the genetic analysis which may help in improving the diagnosis and management of the patient. (69) submission id#600360 professor of paediatrics and immunology, university college london; great ormond street hospital nhs trust; orchard therapeutics, london, uk background: ada-scid is a rare genetic disorder which causes severe combined immunodeficiency. historically, ada-scid has been treated using enzyme replacement therapy (ert) followed by allogeneic hematopoietic stem cell (hsc) transplant (hsct) from a matched related donor (mrd) or, if none is identified, a non-mrd (matched/mismatched unrelated or mismatched related donor). we developed a self-inactivating lentiviral vector (lv), in which a codon optimized human ada cdna is driven by the short form of the elongation factor-1alpha (efs) promoter (efs-ada lv). the drug product (otl-101), composed of autologous hscs transduced ex vivo with the efs-ada lv, was evaluated in a prospective, historically-controlled phase i/ii clinical trial in ada-scid pediatric subjects. we report safety and efficacy at 24 months in 20 ada-scid subjects treated with lentiviral gene therapy (gt) compared to a historical cohort of 26 ada-scid patients treated with hsct. methods: twenty subjects (9 male, 11 female; 4 mo 4.3 yrs) were treated with gt. autologous cd34+ hscs were isolated from bone marrow and pre-stimulated with cytokines before transduction with efs-ada lv. busulfan was administered at a single dose (4 mg/kg) prior to infusion of otl-101. the control group included 26 patients (0.2 mo 9.8 yrs) treated with allogeneic hsct (mrds n=12, non-mrds n=14) at great ormond street hospital, uk (n=16) or duke university childrens hospital, usa (n=10) between 20002016. results: at 24 months, overall survival (os) and event-free survival (evfs), defined as survival in the absence of ert reinstitution or rescue allogeneic hsct) were statistically significantly higher in the gt group compared with the hsct group (table) . successful engraftment of genetically modified hsc was observed in all gt subjects at 6 months, which persisted over 24 months, based on vector gene marking in granulocytes (median 0.085 copies/cell [range 0.04-2.50] at 24 months) and peripheral blood mononuclear cells (median 0.843 copies/cell [range 0.13-1.86] at 24 months), and was associated with increased red blood cell ada enzyme activity and metabolic detoxification from deoxyadenosine nucleotides. over 24 months, none of the gt subjects required peg-ada ert reinstitution and 90% were able to stop receiving immunoglobin replacement therapy (igrt), whereas 38% hsct patients required rescue hsct or reinstitution of peg-ada ert, and 52% were able to stop receiving igrt (table) . nine subjects in the gt group experienced a serious adverse event (sae), most frequently infections and gastrointestinal events; only one was considered treatment-related. in the gt group, there were no events of autoimmunity during the study. due to the autologous nature of the product, there was no incidence of graft vs host disease (gvhd) in the gt group; whereas 5 patients in the hsct group experienced acute gvhd and 3 experienced chronic gvhd events, one of whom died. conclusions: treatment with lentiviral gt for ada-scid is well tolerated and has a favorable benefit-risk profile at 24 months based on sustained gene correction and restoration of immune function, as well as improved os and evfs compared with hsct (mrd or non-mrd) at 24 months. background: ada-scid is a rare genetic disorder that causes severe combined immunodeficiency, with minimal or absent b cell function. prior to, and often after, treatment with allogeneic hematopoietic stem cell (hsc) transplant (hsct) or autologous ex vivo hsc gene therapy (gt), patients are managed with enzyme replacement therapy (ert) and immunoglobulin (ig) replacement therapy (igrt). we evaluated a gt treatment with autologous hscs transduced ex vivo with a self-inactivating lentiviral vector (lv), in which a codon optimized human ada cdna is driven by an internal short form of the elongation factor-1alpha (efs) promoter ("efs-ada lv"). at 24 months follow-up, 20 pediatric ada-scid subjects treated with gt were compared to a historical cohort of 26 ada-scid patients treated with hsct. here, we report on b cell reconstitution in these cohorts. methods: twenty subjects (9 male, 11 female) aged 4 mo -4.3 yrs received gt. autologous cd34+ hscs were isolated from bone marrow and pre-stimulated with cytokines before transduction with efs-ada lv. genetically modified cells were administered after conditioning with single dose busulfan (4 mg/ kg). the control group included 26 patients aged 0.2 mo to 9.8 yrs treated with hsct at great ormond street hospital (uk) (n=16) or duke university children's hospital (us) (n=10) between 2000 -2016. the hsct patients received an allogeneic transplant from matched related donors (mrds) (n=12) or non-mrds (n=14). subjects continued to receive igrt post-gt until a clinical decision was made to stop, factoring in b cell reconstitution, general medical condition and seasonal infections. results: by month 12, in the gt group, 45% had stopped treatment with igrt compared to 38% in the hsct group overall. by months 18 and 24, higher proportions of gt-treated subjects had stopped igrt (70% and 90%, respectively) compared with mrd hsct patients (55% and 70%, respectively) and non-mrd hsct patients (42% at both timepoints) (table) . in the gt group, vector gene marking was detectable in peripheral blood mononuclear cells within 3 months and persisted at 24 months post-infusion (median 0.843 copies/cell [range 0.13-1.86]), suggesting successful gene modification. as evidence of b cell reconstitution, iga and igm levels in peripheral blood sera more than doubled by 18 months, from 18.5 mg/dl (range 8 to 95) to 48.0 mg/dl (range 20 to 110) and 32.5 mg/dl (range 16 to 107) to 69.0 mg/dl (range 20 to 180), respectively. additionally, antibody response following tetanus vaccination, was evaluated in 3 subjects. all 3 subjects mounted a protective response to the vaccine (median antibody response 3.2 iu/ml [range 0.1 to 3.5]), based on a normal threshold of 0.01 iu/ml (hammarlund clin infect dis 2016) and a laboratory reference range (0.10 to 2.9 iu/ml). conclusions: gt with autologous hscs transduced ex vivo with efs-ada lv resulted in b cell reconstitution, as evidenced by doubled iga and igm production at 18 months, cessation of igrt in 90% of patients by 24 months, and protective specific antibody responses to tetanus vaccine in patients that were evaluated. background: x-linked chronic granulomatous disease (xcgd) results from mutations in cybb encoding the gp91phox subunit of phagocyte nadph-oxidase. attempts to treat xcgd with gene therapy (gt) using transduced autologous hematopoietic stem cells (hsc) transduced ex vivo with a gammaretroviral vector have met with limited efficacy due to transient engraftment of gene corrected hscs, gene silencing, and vector insertion-mediated activation of oncogenes leading to myelodysplasia. we developed a novel self-inactivating (sin) lentiviral vector (g1xcgd lv) with a chimeric cathepsin g/cfes myeloid-specific promoter driving gp91phox expression from a codon optimized cdna. following transplant of g1xcgd lv ex vivo transduced autologous hscs into busulfanconditioned xcgd patients, there was long-term restoration of oxidase activity in peripheral blood polymorphonuclear neutrophils (pmn) at 12 months in 6 of 9 severely affected xcgd patients without evidence of genotoxicity. here we present data about the multiple assays used to assess quality and quantity of restoration of pmn oxidase activity. methods: similar trials of gt with g1xcgd lv were initiated in the uk (n=3, plus 1 compassionate use patient) and usa (n=5). all patients had histories of inflammatory disease and severe, persistent infections (some non-responsive to conventional therapy at time of gt). g-csf plus plerixafor-mobilized cd34+ hscs were transduced with ex vivo g1xcgdlv. subjects received myeloablative conditioning with singleagent busulfan, targeted to net area-under-the-curve of 70,000 ng/ml*hr. freshly prepared or cryopreserved quality-tested genetically-modified hsc, manufactured on-site, were administered intravenously. pmn oxidase activity post-gt was assessed by p-nitroblue tetrazolium (nbt) reduction, dihydrorhodamine (dhr) flow cytometry assay, and quantitative ferricytochrome c assay (ferric) measurement of superoxide generation. results: we report results for 7 patients (aged 2-27 years) with 1-2.5 years of follow-up; two additional patients were treated but died within three months of gt from complications deemed related to pre-existing diseaserelated co-morbidities (severe pulmonary disease and anti-platelet antibodies). within 1 month post-gt, oxidase (+) pmn were present in peripheral blood based on nbt testing and dhr flow cytometry. expression of the corrective transgene was confirmed by flow cytometry using antibody detection of gp91phox. quantitative biochemical measurements of oxidase activity were also confirmed in some samples using the ferric assay, demonstrating quantitative levels of superoxide production per corrected cell that were within the normal range. functional testing of oxidase burst activity using dhr fluorescent assays was applied serially to follow levels of corrected pmn where oxidase activity per corrected cell also were in the normal range. all patients had >15% pmn dhr+ within one month, which remained stable for most patients over the follow-up period ( figure) . follow-up demonstrated sustained stable persistence of 12-46% oxidase burst positive neutrophils in 6 of 7 surviving subjects at 12 months, with restoration to clinically beneficial levels (defined as 10% of pmn being dhr+) in these patients as of december 2018. conclusion: these results demonstrate corrected pmn function within 1 month in x-cgd patients treated with autologous gt. pmn oxidase activity was sustained at levels which restore biochemical function and provide clinically beneficial levels of immunity for 12 months in 6/7 patients. the formulation for igsc 20% was developed based on the knowledge acquired from the formulation of grifols currently licensed 10% immune globulin (human), gamunexâ®-c; however, the protein concentration was increased from 10% to 20% to facilitate efficient subcutaneous administration. gamunex-c has an extensive record of safety and tolerability when administered intravenously and subcutaneously for greater than 15 years in diverse patient populations. the igsc 20% manufacturing process employs the same purification steps as gamunex-c and was demonstrated to be robust and to provide an igg product with the required potency, purity, and quality. the formulation excipient characteristics and compatibility with the drug product have been well established. glycine has been an excipient of intramuscular immune globulin (human) for fifty years and intravenous immune globulin (igiv) for over twenty years. the igsc 20% formulation has low buffering capacity, and a low ph was selected to achieve a product with low aggregates, low fragments and viscosity suitable for subcutaneous administration. to improve visual clarity, the igsc 20% formulation contains a small amount of polysorbate 80 (ps80), which is widely used in biopharmaceutical products. subcutaneous administration of the igsc 20% formulation has been well tolerated in clinical studies. objectives: the goal was to provide the pid population with a new 20% immunoglobulin liquid product for subcutaneous administration (igsc 20%). methods: igsc 20% is manufactured using the current manufacturing process for gamunex-c, followed by an additional concentration step so that the product can be formulated at a higher protein concentration. igsc 20% and gamunex-c batches were produced at full industrial scale and then subjected to a series of analytical testing including assessment of purity, composition and neutralizing activity. results: the igsc 20% and gamunex-c manufacturing processes and formulations have preserved the igg integrity, molecular characteristics and potency. the manufacturing processes have eliminated lipids, alcohols, and acetate and coagulation factor impurities, including fxia, which were undetectable by either specific or global methods. the igsc 20% and gamunex-c batches were 100% gamma globulin by agarose membrane electrophoresis, and have a subclass distribution similar to normal plasma and acceptable specific antibody content. igsc 20% was shown to be primarily monomer plus dimer igg (99â±1%) with minimal aggregate or fragment, which confirms that appropriately gentle processing conditions were used during the concentration of 10% igg solutions to 20% igg. conclusions: igsc 20% is a highly concentrated igg solution with characteristics comparable to gamunex-c, but with twice the igg concentration in order to facilitate subcutaneous administration with reduced volumes and shorter infusion times. analytical testing demonstrates suitable potency, purity, and neutralizing activity for a number of specific antigens. funding: this study was funded and conducted by grifols, a manufacturer of 20% immunoglobulin for subcutaneous administration. disclosure: all authors are employees of grifols. frequent respiratory tract infections and seizures cause recurrent hospitalizations in these children and are typically considered a result of neurological impairment and poor airway clearance. evaluation of these patients for immunodeficiency is not a common clinical practice. here we report combined immune deficiency in 2 patients with mds and recurrent respiratory tract infections. case presentation case 1: a boy with mds was initially referred at age 2 months for an abnormal newborn screen with low t cell receptor excision circles (trec) for severe combined immunodeficiency (scid). initial evaluation revealed moderate cd3+ and cd4+ t cell lymphopenia (figure 1). initial immunoglobulins levels were normal. he was placed on antiseizure medications. he later developed recurrent and severe respiratory tract infections starting in infancy. at 12 months of age, he developed hypogammaglobulinemia ( figure 2 ). in addition, t cell counts progressively decreased and stayed around 600 cells/ul. immunoglobulin replacement therapy started at 18 months of age. hospitalizations due to respiratory tract infections significantly decreased. case 2: a 3-year-old boy with mds had recurrent bacterial and viral respiratory infections which required numerous hospitalizations including intensive care unit stays. newborn screening for scid was negative. he had been on anti-seizure medications. immunologic evaluation at 3 years of age revealed low total cd3+ cells and cd8+ t cells (cd3+: 1284cells/ul[normal range 1400-3700cells/ul], cd8+:278cells/ ul[normal range 490-1300cells/ul]), hypogammaglobinemia (igg: 252mg/dl[normal range 453-916mg/dl]), and non-protective igg levels to tetanus, varicella and pneumococcus serotypes. immunoglobulin replacement therapy started at 3 years of age which resulted in reduced frequency and severity of respiratory infections, and improved quality of life. discussions: t cell lymphopenia and hypogammaglobulinemia were seen in both our cases of miller-dieker syndrome. to our knowledge, immune deficiency has never been reported in mds. one of our cases suggests that low t cell counts may start as early as at birth and may be detected by newborn screening. hypogammaglobulinemia may be primary or secondary due to antiepileptics. both children had reduced frequency and severity of respiratory infections and improved quality of life after immunoglobulin replacement highlighting the importance of screening and early management of immunodeficiency. conclusion: miller-dieker syndrome is likely another syndromic primary immune deficiency disorder. a high index of suspicion with early screening and management of immunodeficiency may be beneficial for children with miller-dieker syndrome. uploaded file(s) uploads this prospective, multi-center, open-label study assessed the pharmacokinetic (pk), safety, and tolerability of immune globulin subcutaneous (human), 20% caprylate/chromatography purified (igsc 20%) in subjects with primary immunodeficiency (pi). the objectives were to determine a weekly subcutaneous (sc) dose of igsc 20% that is noninferior to the intravenous (iv) dose of immune globulin injection (human), 10% caprylate/chromatography purified (igiv-c 10%) and to determine the steady state trough igg levels after igsc 20% and igiv-c 10% infusions. there were 3 possible phases. if not on a qualifying igg regimen at enrollment, subjects (n=44) were required to enter the run-in phase, receiving igiv-c 10% to achieve steady-state before entering the iv phase to determine steady-state area-under-the-curve (auc) of iv infusions. subjects with a qualifying igiv-c 10% regimen (300-800 mg/kg) (n=9) directly entered the iv phase for steady-state iv pk assessments. upon completion of the iv pk assessments subjects entered the sc phase, receiving weekly doses of igsc 20% for up to24 weeks, with steady-state auc determined at the 13th dose. igsc 20% was not associated with any reports of serious local infusion site reactions (isrs). the majority of local isrs were mild-to-moderate. igsc 20% (at a dose conversion factor of 1.37) provided equivalent exposure to igiv-c 10% as assessed by steady-state auc0-7 days, with 33% higher mean igg trough values, lower fluctuations in igg concentrations and the flexibility of at home administration. igsc 20% was well tolerated with a safety profile comparable to igiv-c 10%. clinicaltrials.gov identifier: nct02604810 disclosure: kecia courtney, elsa mondou, and jiang lin are employees of grifols, a manufacturer of igsc 20%. grifols is the sponsor of this study. background: in 2014 two reports described the deficiency of adenosine deaminase 2 (dada2) as early-onset lacunar strokes, intermittent fevers, livedoid rash, and early onset polyarteritis nodosa (pan). since these first reports, the clinical spectrum has dramatically expanded to include antibody deficiency, liver disease, vasculopathy, pure red cell aplasia, cytopenias, and lymphoproliferative disease. methods: forty-two patients were enrolled in an irb approved study at the nih. sequencing of ada2, the gene encoding adenosine deaminase 2 (ada2), was performed in all patients. information was obtained by chart review of all clinical, serologic, and radiographic testing. results: all 42 patients had germline biallelic loss of function mutations in ada2, leading to absent or significantly decreased protein expression and function of ada2. the cohort comprises 20 females (48%) and 22 males (52%). there were 6 sibling pairs and 2 families with 3 affected individuals. twenty-seven patients had a history of at least one ischemic stroke and 6 experienced a hemorrhagic stroke. the average age at the time of first stroke is 5.6 years (range 4 months -24 years), and the average number of strokes is 3 (range 1-11). no new strokes have occurred in patients on anti-tnf therapy. skin manifestations occurred in 86% of patients and include livedo (74%), cutaneous vasculitis resembling pan (64%), and raynauds (19%). hepatomegaly (43%) and splenomegaly (55%) were also notable. portal hypertension was observed in 6 (14%) patients, with 1 patient requiring a spleno-renal shunt for a massive variceal bleed. abdominal mra revealed arteritis and aneurysm in 7/13 patients evaluated; 3 patients developed bowel necrosis. peripheral vasculopathy was seen in 3 patients, with one requiring amputation of gangrenous digits. the most common immune abnormality seen in this cohort is hypogammaglobulinemia (62%); 20 patients have low igg, 20 patients have low igm, and 14 patients have low iga. ten of these patients are on immunoglobulin replacement. specific antibody responses to vaccines were inadequate in 5/16 patients challenged. lymphocyte phenotyping revealed decreased class-switched memory b cells in 23/32 patients (72%) tested. however, there was no relationship between absolute number of class switched memory b cells and hypogammaglobulinemia or infection frequency. hematologic abnormalities include transfusion depended anemia (7%), neutropenia (7%), lymphopenia (5%), and thrombocytopenia (2%). seven patients developed pancytopenia, 1 presented with pure red cell aplasia, and 1 developed aplastic anemia. three patients have undergone bone marrow transplant, with two of those patients requiring a second transplant for graft failure. conclusions: the spectrum of dada2 has expanded from strokes, intermittent fever, and cutaneous manifestations to include portal and systemic hypertension, immune deficiency, cytopenias, vascular abnormalities, and bone marrow failure. while initiation of anti-tnf therapy improves inflammatory markers, and no new strokes have occurred while on therapy, cytopenias do not seem to improve. bone marrow transplantation should be considered in patients with findings of bone marrow failure, although transplant of our patients has been complicated by immune mediated neutropenia. disease manifestations are heterogenous, making a comprehensive evaluation critical to our understanding of this disease. given the increase in neonatal diagnosis of athymia, clinical care is provided by the referring medical centers prior to rvt-802 implantation and patients return to the referring centers earlier after rvt-802. this creates the need for clear, concise guidelines for the care of these patients. primary goals of pre-transplantation clinical care are (1) management of pre-existing medical needs such as feeding difficulties, airway obstruction, congenital cardiac defects and developmental disabilities; (2) management of symptoms related to oligoclonal recipient t cell expansion (autologous gvhd/atypical complete digeorge anomaly) and (3) prevention of infections. most deaths in the pre and early post-transplantation period are secondary to pre-existing infections. necessary surgical and medical procedures (ie cardiac surgery, hearing aids) should not be delayed. for the first 6 to 9 months after rvt802, patients have profoundly low naã¯ve t cell numbers and may require immunosuppression to prevent rejection of rvt-802 by oligoclonal recipient t cells. immunosuppression needs to be closely monitored and titrated for desired effect while minimizing side effects such as renal toxicity, electrolyte abnormalities and hypertension. t cell counts should be performed every 3 months and are used to guide weaning of immunosuppression. most patients with successful transplants develop greater than 100/mm3 naã¯ve t cells by 12 months post rvt-802. infection prevention, clinical stability and optimal nutrition are critical for lasting engraftment. clinical guidelines have been developed to address immunosuppression, management of autologous gvhd symptoms (gut, skin and liver), preservation of renal function, and developmental considerations. after the development of naã¯ve t cells, patients should continue to be monitored regularly by an immunologist. patients may develop autoimmune complications such as thyroid disease and transient cytopenias. while risk of complications related to viral infections is greatly decreased after development of naã¯ve t cells, patients with comorbidities (central venous access device dependence, tracheostomy, chronic lung disease) continue to require complex care from multidisciplinary teams. medical conditions associated with athymia but not alleviated by thymus transplantation, such as hypoparathyroidism or cardiac defects, may require lifelong medical care. lastly, patients must be evaluated for readiness for killed and live vaccines. transplant outcomes are influenced by the clinical condition at the time of rvt-802 implantation and optimization of immunosuppression, nutrition and clinical stability in the first 9 months following rvt-802. clinical care that maintains a well-nourished, clinically stable, infection free patient yields the best chance for successful t cell development. guidance documents supporting these goals ensure patients are best prepared to receive rvt-802 and develop long lasting thymic function. hemophagocytic lymphohistiocytosis (hlh) is a life-threatening disease of immune dysregulation characterized by unchecked inflammatory responses leading to end-organ dysfunction. primary hlh results from inherited mutations that impair capacity for immune regulation whereas secondary hlh arises from inappropriate response to an immune stimulus such as infection, malignancy or autoimmunity. we report a 9-monthold male who presented with symptoms of hlh as an initial manifestation of congenital disorder of glycosylation (cdg) due to mutations in the gene component of oligomeric golgi complex 4 (cog4) resulting in cog4-cdg (cdg-iij). a 9-month-old male with history of mild motor delay presented with 3 days of fever, vomiting, and diarrhea. initial evaluation identified highly elevated ferritin and triglycerides, transaminitis, coagulopathy, and hyperammonemia. he subsequently developed generalized seizures. liver and bone marrow biopsies demonstrated erythrophagocytosis consistent with hlh. immunologic evaluation was notable for mild hypogammaglobulinemia, neutropenia, thrombocytopenia, and anemia. serum cd25 levels and nk functional studies were later found to be normal. the patient was initially treated with ammonia-scavenger therapy and fresh frozen plasma (ffp) for coagulopathy with subsequent intravenous immunoglobulin and dexamethasone several days later. within 24 hours after starting ffp, the patients ferritin level declined sharply. hyperammonemia and transaminitis also resolved, and his fever curve improved. additional immunosuppression was considered, but not initiated due to the patients ongoing clinical improvement. over the next 3 months, the patient experienced two further acute episodes of fever, liver dysfunction, coagulopathy, and sepsis physiology. the second episode was successfully treated with ffp, though no clear infectious trigger was identified. the third episode occurred 4 days after routine vaccinations. the patient had prolonged hypotension requiring ionotropic support that resolved after receiving daily ffp, and hypoxia with pleural effusions that resolved after a single treatment with protein c concentrate. as the patient had met 5/8 clinical diagnostic criteria for hlh, but also had a history of hyperammonemia, he underwent concurrent biochemical and genetic evaluation for both primary hlh and inborn errors of metabolism. whole exome sequencing identified compound heterozygous mutations in cog4, part of an oligomeric protein complex involved in golgi apparatus structure and function. cog4 mutations have previously been reported in two patients with autosomal recessive cog4-cdg (cdg-iij), who were described to have similar clinical symptoms of hypotonia, seizures, coagulopathy, and liver dysfunction, as well as recurrent infections. subsequent immune phenotyping while the patient was healthy was notable for slightly low numbers of nk cells, but normal cd107a mobilization and perforin/granzyme b expression in vitro. our patient represents a novel presentation of cdg due to cog4 defect with associated immune dysfunction manifesting as recurrent episodes of inflammatory crisis with features of hlh. cdg and inborn errors of metabolism should be considered during diagnostic evaluation for patients with hlh symptoms, as cdg patients may develop acute episodes of severe inflammation, in the absence of cellular regulatory defects, for which ffp and protein c concentrate may have therapeutic value. of the 14 deaths with identifiable causes, 10 (71%) were related to infections. the rate of death per person-year was 0.044. the most common autoimmunity-related complication was sweets syndrome, seen in 29 patients (39%) with anti-ifn-g autoantibodies. sixteen of those patients (55%) had recurring sweets syndrome. additionally, 14 patients (19%) developed lymphatic obstruction, which continued to recur in 12 patients (86%). seven patients (9%) in this study did not have anti-ifn-g autoantibodies. the median [iqr] age of autoantibody-negative patients was 38 [27, 54] years and 3 patients (43%) were female. none of the autoantibody-negative patients developed new infections during follow-up. at the end of the follow-up period, none of the patients had active/progressive disease and 2 patients (29%) had died. conclusions: ninety-one percent of hiv uninfected thai patients with disseminated ntm infection with or without other opportunistic infections had detectable anti-ifn-g autoantibodies. about one third of patients with autoantibodies to ifn-g had recurrent infections during follow-up. after approximately 7 years of follow-up, 55% of patients with anti-ifn-g autoantibodies had inactive disease following multi-drug antibiotic therapy while 8% had active/progressive disease and 24% had died. patients with anti-ifn-g autoantibodies are at risk for recurrent infections and autoimmunity-related complications. therefore, longterm follow-up is recommended. life-long secondary antibiotic prophylaxis may be required to prevent recurrence of infection in the setting of persistent anti-ifn-g autoantibodies. the study of early t cell development in patients with severe t cell immunodeficiencies is challenging because of the rarity of these diseases, the difficulty to obtain hematopoietic stem cells (hscs), and limitations in the assays to assess in vitro differentiation of hscs to mature t cells. we recently developed a serum-free system that allows faithful analysis of sequential steps of t cell differentiation. in this system, artificial thymic organoids (atos) are generated, based on the 3d aggregation and culture of a delta-like canonical notch ligand 4 (dll4)-expressing stromal cell line (ms5-dll4) with cd34+ cells isolated from bone marrow (bm) samples of normal donors (nd). in this project, we set out to evaluate the possibility of using the ato system to study t cell differentiation in patients carrying t cell defects, in order to define the exact steps of t cell development affected by different genetic defects. using the ato system, we studied in vitro t cell differentiation from cd34+ cells obtained from patients carrying defects that are intrinsic to hematopoietic cells (rag1, rag2, ak2, il2rg) or that affect thymus development (digeorge syndrome, dgs). the ak2-deficient patient showed a markedly decreased viability in cd34+ cells and a very early defect in t cell development, already at the pro-t cell stage. this defect was very similar to that observed in a patient carrying a null il2rg mutation who was reported to show autologous reconstitution after unconditioned haploidentical hsc transplantation. in contrast, cd34+ cells from a patient carrying a missense il2rg mutation and with a leaky scid phenotype were capable of differentiating into mature t cells in vitro, although with 100-fold decreased efficiency as compared to normal donors (nd). interestingly, in the patient carrying the null il2rg mutation, we noticed very few cells that could reach full maturation, with an absolute number of cd3+ tcrab+ cells around 1000-times less than in nd. at variance with pro-t cells (that failed to express the gc protein), these mature t cells did express normal levels of gc, suggesting that they may have derived from residual cd34+ cells from the bm donor. in addition, cd34+ cells from the patients carrying rag1 and rag2 hypomorphic mutations were able to differentiate to cd4+cd8+ double positive cells, but not to cd3+tcrab+ cells. finally, the dgs patient showed a completely normal in vitro t cell differentiation, confirming that t cell deficiency reflected thymic abnormalities. in summary, our data show that the ato system could be extremely useful in determining whether the lack of t cells in patients with unknown gene defects reflect hematopoietic or thymic intrinsic problems, and may therefore provide critical evidence in deciding whether hsc or thymus transplantation is warranted, even without knowing the actual gene defect. introduction: ataxia-telangiectasia (at) is an autosomal recessive disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene, which aids in detection and repair of dna damage. at is characterized by progressive cerebellar ataxia, oculomotor apraxia, choreoathetosis, conjunctival telangiectasias, variable degrees of t-cell lymphopenia (tcl) and immune compromise. patients are at an increased risk for malignancy, particularly leukemia and lymphoma, and are unusually sensitive to ionizing radiation. with the advent of trecbased newborn screening (nbs) for scid, at patients are being recognized with asymptomatic tcl in early infancy. objectives: we present an older child with at and chronic granulomatous lesions and discuss how this may be avoided in individuals with at diagnosed following abnormal nbs. case report: a 12 y/o male was born at term following an uncomplicated twin pregnancy and delivery, prior to institution of scid nbs. he demonstrated mild gross motor and speech delay as an infant and was diagnosed with at at age 3. he had received all routine immunizations, including live vaccinations. he developed granulomatous skin lesions at age 1, initially small papules on his cheeks and ears, which subsequently formed large disfiguring plaques on sun-exposed areascheeks, arms and hands (fig 1) . following an extensive workup, his lesions were found to be secondary to a mutated vaccine-strain rubella (ra27/3) based on 739bp genotyping, previously described in other immunocompromised individuals [perelygina/sullivan et al. jaci 2016] . his lesions have been refractory to multiple treatments including nitazoxanide. he is currently on daily oral and topical steroids, tmp/smx and ivig. retrieval of his nbs for trec determination revealed that he would have screened positive [mallot/puck et al. j clin immunol 2013] . when first measured at age 3, cd3 t-cells were low, 443/ul, with cd4 227/ul and cd8 140/ul. b and nk cell numbers were normal. since april 2017, 4 cases of at were seen at ucsf in infants with non-scid tcl on nbs. these 3 males and 1 female were all born at term and discharged from well-infant nurseries. at was diagnosed at 2-7 months of age. their initial trecs ranged from 5-12/ul (normal with perkinelmer enlite kit >18), and all had low t-cells on initial flow cytometry (242-1612 cd3/ul, ref range>2500) with decreased cd4 (146-1178/ul) and cd8 (87-403/ul) t-cells; however naã¯ve t-cells were present, ruling out typical scid and raising concern for non-scid tcl. three infants also demonstrated low b-cells (<20-77/ul), while nk cells were normal in all. two are currently receiving ivig, one of whom is also on tmp/smx. all have avoided not only rotavirus but also mmr and varicella live vaccinations. conclusions: at is now often diagnosed in infants with low trecs on scid nbs, prior to neurologic manifestations. benefits of early diagnosis include avoidance of live vaccines, including mmr, which led to the debilitating granulomas in our older patient. additionally, patients receive prompt immunologic monitoring and treatment, avoidance of unnecessary radiation, specialty referrals and family genetic counseling. while there is no cure for at, ongoing research may bring neuroprotective treatments in the future. introduction: subcutaneous immune globulin 20%, ig20gly, was well tolerated in the phase 2/3 north american study in patients with primary immunodeficiency diseases (pidd). here we assess comorbidities, use of concomitant medications, infusion parameters, and tolerability in advanced age patients (60 y) treated with ig20gly in the north american study. methods: patients aged 2 years with pidd received weekly ig20gly infusions at volumes 60 ml/site and rates 60 ml/h/site for~1.3 years in the north american study (nct01218438). the medical history at baseline, medical conditions that were ongoing (defined as comorbid events), use of concomitant medications, adverse events (aes), tolerability, and infusion parameters were assessed by age: in advanced age patients (60 y; n=14), adult (16<60 y; n=39), and pediatric/adolescent patients (<16 y; n=21). results: the mean number of medical history events at baseline was higher in advanced age patients (28.7 events/patient; 402 events in 14 patients) versus adult (16.8 events/patient; 657 events in 39 patients), and pediatric/adolescent patients (6.5 events/patient; 137 events in 21 patients). of these, the medical conditions that were ongoing at baseline (comorbid events) were also higher in the advanced age patients (20.9 events/patient; 292 events in 14 patients) versus adult (12.4 events/ patient; 482 events in 39 patients), and pediatric/adolescent patients (3.4 events/patient; 71 events in 21 patients). in the advanced age patients, neurological comorbidities (51 events) were the most common, followed by those related to eyes, ears, nose, and throat (49 events), gastrointestinal (43 events), and musculoskeletal comorbidities (43 events). concomitant medications were given to treat a preexisting condition in all patients in the advanced age group (225 medications in 14 patients). despite the higher mean number of comorbid conditions, infusion parameters in the advanced age patients were comparable to those in the adult age group. median maximum infusion rates and infusion volumes/site were comparable in the advanced age patients (60 ml/h/site; 47.5 ml/site) and adults (60 ml/h/site; 44 ml/site); lower infusion rates and volumes/site were reported in the pediatric/adolescent patients ( . larger infusion volumes and faster infusion rates were not associated with increases in causally related local aes in the advanced age group, consistent with the trends seen in the pediatric/ adolescent and adult patients. conclusions: despite the higher mean number of comorbidities in advanced age patients with pidd, ig20gly was infused at relatively high rates and volumes and was well tolerated. introduction: hyqvia (ighy; immunoglobulin infusion 10% with recombinant human hyaluronidase [rhuph20]) is an immunoglobulin (ig) replacement therapy approved for patients with primary immunodeficiency diseases (pidd) that allows larger infusion volumes, up to 600 ml/site, and has improved ig bioavailability compared with conventional subcutaneous ig products. a post-authorization safety study is being conducted in the united states to acquire long-term safety data on ighy and to assess prescribed administration regimens in routine clinical practice. infusion characteristics and treatment-related adverse events from an interim analysis are reported here. methods: patients aged 16 years with pidd receiving ighy were included in this ongoing, prospective, non-interventional, open-label, uncontrolled, multicenter study. as a part of routine clinical practice, patients are treated with ighy according to standard medical care and their treatment regimen is at the discretion of the treating physician. adverse events (aes) are collected from enrollment to study completion/discontinuation using a subject diary and assessed at every study visit (every 3 months or standard practice). aes are assessed based on seriousness, severity, and causal relatedness to ighy. the presence of anti-rhuph20 antibody is evaluated on a voluntary basis. treatment preferences for various attributes of ig therapy were assessed annually using a treatment preference questionnaire. results: a total of 175 patients were enrolled at 26 us study sites (data cutoff date: august 21, 2017). infusions were self-administered at home (56%) or at the clinical site (44%) most commonly using 4-week infusion intervals (56.6%). the mean maximum ig infusion rate was 302.8 ml/h and the mean ig dose was 418 mg/kg bodyweight/4weeks. the mean number of infusion sites used for administration was 1.9 and mean infusion duration was 2.8 hours. most infusions (97.3%) were administered without a rate reduction, interruption, or discontinuation due to aes. there were no serious aes (saes) related to ighy. sixteen patients experienced a causally related non-serious local ae (9.1%; 0.43 events/patient-year, 0.07 events per infusion) and 25 patients experienced a causally related non-serious systemic ae (14.3%, 0.88 events/patient year, 0.14 events per infusion). seven of 113 patients who were tested for anti-rhuph20 antibody had 1 positive binding antibody test to rhuph20 (titer 1:160; maximum titer 1:10240 at enrollment, 1:5120 during the study); no neutralizing rhuph20 antibodies were detected. of the patients who responded to the treatment preference questionnaire at the end of year 1, the majority (38/52 [73.1%]) preferred to receive their ig therapy at home; 21.2% (11/52) preferred the doctors office; 3 patients preferred treatment at the hospital, had no preference, or indicated other. almost all patients (51/52 [98.1%]) indicated a preference to continue treatment with ighy. conclusion: this interim analysis of 175 patients with pidd treated with ighy in routine clinical practice supports previous observations that ighy is a well-tolerated and preferred therapy with no reports of treatment-related saes or neutralizing anti-rhuph20 antibodies. background: cartilage hair hypoplasia (chh) is an autosomal recessive chondrodysplasia associated with variable immunodeficiency. pathogenic defects in rmrp, encoding the untranslated rna subunit of ribonucleoprotein endoribonuclease complex (rmrp), result in reduced mrna and rrna cleavage. rmrp c.70a>g is the most common variant, increased in finnish and amish populations. while cellular immunodeficiency is associated with increased morbidity and mortality, there is no established correlation between clinical and immunological phenotype. lymphocyte radiosensitivity has not been described. case: a full-term amish female infant had low trec copies on newborn scid screen. flow cytometry at 3 months-old demonstrated severe t and b cell lymphopenia (cd3+t-cells 413 cells/mcl, range: 2,300-6,500 cells/mcl; cd19+b-cells 214 cells/mcl, range: 600-3,000 cells/mcl) with normal nk quantitation (cd16/56+ 340 cells/mcl, range: 100-1,300 cells/mcl) and cd4+ memory t-cell expansion (33.2%) relative to the naã¯ve subset (67.0%). t-cell functional mitogen responses were normal. she was diagnosed with chh with homozygous rmrp c.70a>g mutation. lymphocyte subset (t, b and nk cells) radiosensitivity was evaluated by flow cytometric analysis of phosphorylated (p) atm, smc1 and gamma-h2ax after low-dose (2gy) irradiation. an increase in gamma-h2ax level was observed in a subset of non-irradiated t cells (17.66% v. 1.36% gamma-h2ax+) and nk cells (23.07% v. 1.04% gamma-h2ax+) in the patient, suggestive of a constitutive defect in dna repair. the relative distribution of t, b and nk cells expressing patm, psmc1 and gamma-h2ax at 1 hour postirradiation (ir) was not significantly different from the experimental healthy control (ehc) or pediatric reference range (prr). however, the kinetics of dephosphorylation at 24 hours post-ir was altered with residual gamma-h2ax expression in a subset of the patients t cells (delta 3.84%, mode ratio mean fluorescence intensity (mfi)=2.58; ehc: delta 0.10%, mode ratio mfi=1.39; prr: delta 2.16%, mode ratio mfi=2.42). a similar finding was observed in a subset of patient b-cells for gamma-h2ax (delta 11.35%, mode ratio mfi=1.48; ehc: delta 0.82%, mode ratio mfi=0.86; prr: delta 1.95%, mode ratio mfi=1.19). the frequency of the patient's lymphocytes with residual gamma-h2ax persistence at 24h post-ir was prominent, with 8.29% t-cells demonstrating persistence of gamma-h2ax (compared to 0.82% in the ehc, and 2.60% in the prr), and 18.02% b-cells gamma-h2ax+ (compared to 1.80% in the ehc, and 2.96% in the prr). there has been lack of follow-up, but verbal report suggests no significant immunological or infectious concerns at 1 year of age. discussion: lymphocyte radiosensitivity is a novel finding in chh with t and b cell lymphopenia. the ability of rmrp to associate with telomerase reverse transcriptase (tert) and function as an rna-dependent rna polymerase, yielding distinct silencing rna sequences, may underlie radiosensitivity in rmrp mutants. systematic characterization of lymphocyte radiosensitivity and immunological phenotype could provide useful information on whether this could serve as a biomarker for the magnitude or complexity of immunodeficiency. assessment of radiosensitivity has implications in conditioning regimen selection for patients requiring allogeneic hematopoietic cell transplantation. we recommend lymphocyte radiosensitivity assessment in chh infants identified by nbs scid and chh patients with significant immunodeficiency and/or malignancy. novel primary immunodeficiency with lymphoproliferative disease due to biallelic defects in nckap1l background: three children from 2 non-consanguineous families and different ethnic backgrounds developed lymphoproliferative disease by 2 years of age. they also had recurrent infections, including pneumonia and bronchiectasis, otitis media, and skin pustules. immune phenotyping revealed low cd4+ t cell percentages, an accumulation of memory-like cd8+ t cells, impaired t cell proliferation, and low total nk cell numbers. methods: the affected individuals, unaffected parents, and other unaffected family members underwent exome sequencing. results: all 3 affected cases had rare and bioinformatically damaging biallelic variants, with appropriate familial segregation, in nckap1l, which encodes hem1. hem1 is an essential component of the wave2 regulatory complex (wrc). immunoblotting confirmed destabilization of the wrc in all patients. immunofluorescence microscopy demonstrated defective f-actin and wave2 localization to immune synapses in nk cells. significant abnormalities were identified in patient lymphocyte and neutrophil migration and morphology, consistent with altered wrc-mediated cytoskeletal dynamics. all patients exhibited impaired inside-out integrin activation. knockdown of hem1 produced deficient proliferative responses and mtorc2-mediated akt activation in control t cells. conclusions: the immunologic and clinical phenotype in the affected individuals recapitulates the phenotype observed in hem1-deficient mice. biallelic defects in nckap1l therefore result in a novel human primary immunodeficiency disease characterized by lymphoproliferation and susceptibility to infections. background: concurrent existence/significance of immunodeficiency with new onset lymphoproliferative disease remains understudied. just two studies to date have evaluated the prevalence of hypogammaglobulinemia in chronic lymphocytic leukemia (cll) and neither studied prevalence and impact of ige deficiency on outcomes in cll [1, 2] . therefore, the objective of this study was to examine the prevalence of hypogammaglobulinemia, examining all isotypes, in newly diagnosed cll patients and to test the hypothesis that patients with hypogammaglobulinemia have a distinct clinical profile and outcome. methods: using the banked sera of 150 newly diagnosed, treatmentnaã¯ve, cll adult patients from the lymphoma molecular epidemiology resource (l-mer), ig (igg, iga, igm and ige) levels were measured. the l-mer was initiated as an observational cohort study of prospectively enrolled newly diagnosed lymphoma patients evaluated at the mayo clinic (rochester, mn) and the university of iowa (iowa city, ia) [3] . igg/a/m levels were measured using immunoturbidimetric assay whereas the ige level was determined using electrochemiluminescence immunoassay. the associations between ig deficiencies and clinical factors were evaluated with wilcoxon rank sum and chi-squared (fishers exact, where appropriate) tests. cox regression models were used to assess the effects of clinical variables on overall survival (os). time was calculated from biopsy to death due to any cause; patients still alive were censored at last contact. all tests were two-sided and assessed for significance at the 5% level using sas v9.4 (sas institute, cary, nc). results: the mean age (sd) of the selected cll cohort was 63.8 (11.0) years with a male predominance (69.3%). 96.2% of the patients were white. with a median follow-up of five years, there were 50 deaths. hypogammaglobulinemia in newly diagnosed, treatmentnaã¯ve cll was common in our cohort with 88 (58.7%) patients having a measurable isotype deficiency. the most common ig deficiency was igm (44.0%, 95% ci 35.9-52.3%), followed by igg (34.7%, 95% ci 27.1-42.9%), ige (16.7%, 95% ci 11.1-23.6%) and iga (12.0%, 95% ci 7.3-18.3%). multiple deficiencies in the same patient were common ( figure 1 ). iga and ige deficiency were associated with higher rai stages (grading system for cll) at presentation (p<0.01 and 0.04 respectively) as well as with higher white blood cell counts at presentation (p=0.02 and 0.01 respectively). a higher proportion of iga deficient patients needed second treatment during follow-up (61% compared to 36%, p=0.04). when comparing predictors of overall survival, higher rai stage [3-4 vs 0, hazard ratio (hr) 2.43, 95% ci 1.08-5.46, p=0.03] and age (hr 1.08, 95% ci 1.05-1.12, p<0.01) correlated with worse overall survival. individual immunoglobulin deficiencies did not correlate with overall survival. conclusions: a significant proportion of treatment-naã¯ve patients with cll have underlying ig deficiencies-both in isolation and a combination of different isotypes. a deficiency of iga or ige was associated with severe disease at presentation. the underlying relationship between these two immunologic disorders deserves further study. background: patients with primary immunodeficiency (pid) have an increased risk of developing autoimmune diseases, including rheumatoid arthritis (ra). management of these patients is challenging as immunomodulators can further increase their risk for infections. additionally, patients with ra that undergo therapy with drug modifying antirheumatic drugs (dmards) may develop a secondary immunodeficiency. there are few studies reviewing the characteristics of patients with a pid who later develop ra, and no studies have been reported comparing these patients to those who develop an immunodeficiency after starting dmard therapy for ra. methods: 65 patients were identified as having inflammatory arthritis and a concomitant immunodeficiency (id) at our institution between 1/1/2000-10/03/2017 using icd-9 and 10 codes. manual chart review was performed to confirm and identify the timing of diagnosis of these disorders. patients were excluded if either there was no definitive diagnosis of id or ra (clinically diagnosed by a practicing allergist/immunologist and meeting acr 2010 criteria for ra with a score of 6 or higher, respectively), or rituximab was administered prior to diagnosis of id . clinical symptoms, treatment, and laboratory data were extracted. fishers exact test was used to compare the categorical variables between the groups; ttest was used to compare the continuous variables. results: 10 patients met the inclusion criteria. 5 patients were diagnosed with an id and developed ra later in life (group 1), and 5 patients were diagnosed with ra and subsequently developed a clinically significant id (group 2). the mean ages of diagnosis of id and ra in group 1 patients were 32.0 years (sd â± 26.9) and 42.6 years (sd â± 19.0), respectively. in group 2, the mean age of diagnosis of ra was 37.8 (sd â± 14.2), compared to 54.8 years (sd â± 12.7) for the diagnosis of id. most patients in both groups were female (60% in group 1 and 80% in group 2). all patients in both groups had a humoral id, including common variable immunodeficiency (cvid) (40% of group 1 patients), specific antibody deficiency (sad) (20% of group 1 and 60% of group 2 patients), and hypogammaglobulinemia (20% of group 1 and 40% of group 2 patients). all patients in group 2 were seropositive for rheumatoid factor (rf) or anti-cyclic citrullinated peptide (anti-ccp), whereas only 20% of patients in group 1 were positive for rf or anti-ccp (table 1 ). most patients in both groups were treated with immunoglobulin replacement therapy. treatment of ra in both groups was similar, but combination dmard therapy was not used in group 1 patients in contrast to group 2 patients. conclusions: our study indicates that even though clinical characteristics and management are similar in patients with coexisting id and ra, rf and anti-ccp are usually negative in patients who develop ra after id, possibly due to impaired antibody production in immunodeficient patients. assistant professor of allergy and immunology, arkansas children's hospital, university of arkansas medical sciences introduction/background: complement deficiencies are relatively rare, comprising less than 1% of primary immunodeficiencies. they are associated with increased risk for infections with encapsulated organisms and autoimmunity. of all complement deficiencies, the rarest are defects in the alternative complement pathway. properdin deficiency is the most commonly described alternative pathway deficiency, with factor b and factor d deficiency more rarely described. fewer than 5 patients with factor d deficiency have been reported with all reported cases being children of consanguineous parents who succumbed to meningococcal sepsis. objectives: to describe a case of factor d deficiency associated with recurrent respiratory infections with streptococcus pneumoniae pneumonia with associated lung abscess and empyema. methods: retrospective chart review was conducted. laboratory investigations included lymphocyte immunophenotyping by flow cytometry, lymphocyte proliferation to mitogen, quantitative serum immunoglobulins, vaccine titers, complement assays and functional evaluation, and genetic evaluation by next generation sequencing. results: a 2 year old marshallese male was transferred from an outside hospital to our facility for further evaluation of worsening pneumonia and was found to have right-sided pleural effusion and pulmonary abscess in the right lower lobe. the abscess was drained and was found to be positive for streptococcus pneumoniae via polymerase chain reaction. he improved after chest tube placement and treatment with intravenous antibiotics. his medical history was significant for recurrent acute otitis media and prior hospitalization out-of-state for pneumonia with empyema secondary to streptococcus pneumoniae, which required chest tube placement and admission to the pediatric intensive care unit at 18 months of age. immunologic work up revealed age-appropriate lymphocyte subpopulations, lymphocyte proliferative responses to mitogens, quantitative immunoglobulin levels, pneumococcal/tetanus/diphtheria titers, and ch50 complement assay. ah50 complement assay was decreased to 44 units/ml. complement testing was repeated -with normal ch50 and ah50 of 0 units/ml. further evaluation revealed normal levels of factors b, h, i and properdin. factor d level was 0.12 mcg/ml, and factor d function was decreased to 2 units/ml, indicating a diagnosis of factor d deficiency. sequencing of the cfd gene revealed a previously undescribed homozygous deletion (c.721_723del and p.lys241del). the parents were not agreeable to personally undergoing genetic evaluation to determine if this was a de novo mutation. the patient was managed with pneumococcal and meningococcal immunizations, prophylactic amoxicillin and intravenous gamma globulin (ivig) without any further infections. unfortunately, after two ivig infusions, he was lost to follow up. conclusion: factor d deficiency is an extremely rare alternative complement pathway deficiency, described in less than 5 patients. all infections described thus far have been secondary to neisseria meningitidis. this case represents not only a novel mutation in the cfd gene leading to factor d deficiency, but also the first description of a patient with factor d deficiency developing invasive infection secondary to streptococcus pneumoniae. background: viral infections are a significant cause of morbidity and mortality in patients with primary immunodeficiency disorders and following hematopoietic stem cell transplantation. adoptive immunotherapy using virus specific t-cells (vsts) has been shown to prevent and treat viral infections in immunocompromised hosts. human parainfluenza virus-3 (hpiv3) is a common cause of severe respiratory illness in immunocompromised patients and has no approved antiviral therapies and has not previously been used as a target for t cell therapeutics. introduction: we previously reported that fatigue is increased in common variable immunodeficiency (cvid). however, in previous studies, fatigue was not defined using validated tools. our aim from this study is to identify the prevalence of patient-reported fatigue, using validated questionnaires, and determine the factors predisposing to fatigue in cvid methods: data from cvid who responded to the idf 2017 patient national survey a were analyzed. fatigue was measured using the brief fatigue inventory (bfi) questionnaire, which includes seven items to identify fatigue, and measure fatigue severity. a total of 555 patients with cvid and responses to bfi were enrolled. demographics, co-morbidities, immunoglobulin replacement therapy (iggrt) route and dose, co-morbidities, infections, depression, quality of life (qol) (using the sf-12v2) and disability were compared between fatigued and non-fatigued. logistic regression was used to identify the significant variables. ebv reactivation without ptld, treated with rituximab. alive and well. j clin immunol (2019) 39 (suppl 1):s1-s151 s58 granulomas are the most significant day-to-day problem for cvid patient management. currently, there are limited options for their treatment and the optimal therapy is unknown. in case reports and small series, infliximab has been reported effective while others found it useless. we here describe a 26yo white male referred for monthly ivig in august 2016. at age 1, he developed large areas of erythematous polymorphic plaques in his cheeks, arms and legs. a skin biopsy showed tuberculoid granulomas negative for bacteria, baar and fungi, with infiltrating cd4+ lymphocytes. a prolonged course of steroids did not improve his skin. he also had multiple pneumonias and bronchiectasis, and oral candidiasis. he received all vaccines, including bcg with no complications. with low immunoglobulins and a poor response to pneumococcal polysaccharides and tetanus toxoid he was diagnosed as cvid and placed on ivig at 7yo with excellent infectious control since then. at age 8, his skin lesions persisted and deepened to the bone on his left leg. broad spectrum antibiotics for 3 months were unsuccessful. at 16yo to 18yo, skin grafts were performed on his arms, legs and both cheeks. two ulcers persisted on his left leg until august 2018 that increased in size, deepened and became erythematous and extremely painful (fig. 1) . in september, two new ulcers appeared on his right cheek and right gluteus, respectively. one week later a third ulcer was found on his left calf. on september 28th, infliximab 5mg/kg (300mg) was administered. on the second infliximab dose, october 12th, the pain was completely gone and all ulcers were shrinking, and those ones in the cheek, gluteus and calf almost completely resolved. by the third dose, on november 23rd the ulcers in his right leg were almost closed (fig. 2) . infliximab 300mg treatment continues every 8 weeks. lab test remained unchanged from 2016 till 2018, when his wounds got worsened. (table 1 ) granulomatous disease in cvid is a challenge. both b and t cell directed therapies are encouraged. we add a new case of an infliximab responsive patient to others already reported. over 20 genes have been reported to cause monogenic cvid. a 4 year old girl presented with recurrent pneumonias and a diagnosis of cvid. the parents sought a second opinion. born at 33 weeks gestational age, she was "always smaller and sicker than her friends," and in the prior 8 months she had 3 episodes of pneumonia with fever to 104f requiring emergency department treatment. two of these were associated with rsv and metapneumovirus, respectively. laboratory evaluation confirmed low levels of igg (326 mg/dl) iga (7) and igm (6) congenital tuberculosis (ctb) is a rare disease most often associated with maternal genitourinary (gu) tuberculosis (tb) or disseminated tb. due to infertility caused by gu tb, ctb is rarely reported even in endemic countries. infants can acquire tb hematogenously via the placenta or umbilical vein or by fetal aspiration of infected amniotic fluid. presenting symptoms include respiratory distress, fever, hepatosplenomegaly, poor feeding, lethargy, and low birth weight. we report a premature female infant conceived via in vitro fertilization (ivf), who was born to indian immigrant parents at 29 weeks of gestation due to preterm premature rupture of membranes. maternal history was significant for pulmonary tb at 9 years of age. she denied abdominal or gu symptoms. infants nicu course was complicated by opacifications in the right lung and leukocytosis with neutrophil predominance, identified during evaluation of frequent apnea and bradycardia episodes at 1 month of age. clinical improvement was noted after treatment with vancomycin, amikacin and piperacillin-tazobactam; however, leukocytosis of unknown etiology persisted. at 1.5 months of age she was discharged to inpatient rehabilitation. at 3 months of age, she was readmitted for fever and respiratory distress. during this admission, an immune evaluation was undertaken due to persistence of symptoms along with unresolved leukocytosis with a peak of 58,000 cells/l with neutrophilia to 42,850 cells/l, and chest ct evidence of progressive multifocal lung disease worse in the right upper lobe despite empiric treatment with broadspectrum antibiotics. infectious work-up was negative, including acid-fast bacilli testing from bronchoalveolar lavage. due to the pronounced and persistent leukocytosis and neutrophilia, a primary immune defect was suspected. immune evaluation included: normal immunoglobulins (ig) g, a, and e, elevated igm, vaccine-specific antibody titers protective to diphtheria and 9 of 13 streptococcus pneumonia strains, mildly elevated t and b cells, a normal flow cytometry for dihydrorhodamine, myeloperoxidase stain and glucose-6-phosphate dehydrogenase level, as well as a peripheral smear with no giant azurophilic granules. her primary immunodeficiency genetic panel was unrevealing. she underwent lung biopsy via video-assisted thoracoscopic surgery, which showed noncaseating granulomas and eventual growth of multi-drug-resistant mycobacterium tuberculosis (mtb). upon treatment with an appropriately adjusted anti-tuberculosis regimen, she showed rapid clinical and laboratory improvement. endometrial samples obtained from mother showed gu tb, confirming the diagnosis of ctb. the slow-growing nature of mtb that resulted in delayed diagnosis, along with the presence of non-caseating granulomas and persistent neutrophilia, prompted an immune work up that was completely normal. this case demonstrates the importance of considering ctb in the differential diagnosis of an infant presenting with severe lung infection, persistent neutrophilia, suboptimal response to broad-spectrum antibiotics and relevant epidemiologic risk factors. furthermore, in the setting of appropriate parental exposures and infertility prompting the use of ivf, maintaining a high level of suspicion of ctb can aid in earlier diagnosis of affected neonates. 15-year-old caucasian male who initially presented with recurrent otitis media, persistent hsm, lad, and hypogammaglobinemia (igg <170 mg/dl) at 2 years of age. he was diagnosed with common variable immunodeficiency (cvid) and chronic arthritis when he was 6 and 9 years of age, respectively. subsequently, he developed hepatitis and recurrent pneumonia with mycobacterium avium complex (mac). his arthritis partially responded to anti-tumor necrosis factor (tnf) agents and tofacitinib, but did not respond to anti-interleukin-6 treatment. a combination of anti-tnf inhibitor, tofacitinib, and low dose prednisone was required to control his arthritis. hypogammaglobulinemia (igg <110 mg/dl), recurrent otitis media, pneumonia, crohn's disease, celiac disease, lad and failure to thrive at 2 years of age with more recent development of hsm. he required only immunoglobulin replacement therapy. case#3 is a 9-year-old caucasian male, the half-brother of case#2, who initially presented with recurrent pleural effusion and bilateral pulmonary infiltrates, hsm, lad, abdominal distension and ascites at 7 years of age. a transbronchial lung biopsy revealed chronic eosinophilic pneumonitis. liver biopsy showed increased eosinophils in the sinusoids with diffuse enlargement of hepatocytes, but without hepatitis. colon biopsy revealed minimal colonic eo-sinophilia. his pulmonary infiltrates and pleural effusion responded to prednisone, and he has not required additional treatment for past 1.5 years. conclusions: the clinical manifestations of the same genetic variant may be variable and unpredictable even in the same family. stat3 gof syndrome should be considered in children with multisystem autoimmune diseases, lad, hsm and low switched memory b cells regardless of presence of hypogammaglobulinemia or history of recurrent infections. background: patients with primary immune deficiencies characterized by severe t lymphopenia and/or poor t cell function and patients posthematopoietic cell transplantation are at high risk of severe viral infections. antiviral medications are expensive, not always effective and associated with significant toxicity and/or long-term side effects. as such, there has been increasing interest in the use of donor-derived or thirdparty virus-specific t cells (vsts), and several studies have demonstrated efficacy of vsts generated using various manufacture strategies. however, in depth immunologic and metabolic characterization of vsts has not been reported, limiting correlative investigations into efficacy. methods: ebv-vsts were generated from apheresis t cells collected from healthy donors using three methods: (1) stimulation and expansion with hla-matched ebv-lymphoblastoid cell lines (lcls) purchased from astarte biologics or sigma-aldrich over a period of 4 weeks, (2) stimulation with ebv peptivator from miltenyi followed by expansion over 9-12 days with different cytokines, and (3) stimulation with ebv peptivator followed by isolation of activated cells using the ifn-gamma capture system from miltenyi. immunophenotyping by flow cytometry was performed using the miltneyi macsquant analyzer. the nanostring ncounter system was used to measure gene expression for metabolic pathway analysis, and the agilent seahorse xf cell mito stress test system was used to measure mitochondrial respiration. results: ebv-vsts generated using lcls or peptivator plus il-15 both resulted in a high percentage of cd8 t cells skewed to the effector memory and terminal effector memory phenotype with high expression of the exhaustion markers pd-1, tim-3, and lag-3. conversely, ebv-vsts generated using peptivator plus il-4 and il-7 and the ifn-gamma capture system resulted in a mixed cd4 and cd8 t cell population with a high number of central memory t cells and lower percentage of cells positive for pd-1, tim-3, and lag-3. stimulation with peptivator followed by expansion with il-2 resulted in an intermediate immunophenotype. nanostring results demonstrated upregulation of the glycolytic pathway in ebv-vsts stimulated with peptivator followed by expansion with il-2 or il-15 compared to ebv-vsts generated using the other manufacture approaches. the seahorse mito stress test demonstrated that the peptivator plus il-2 ebv-vsts had a significantly lower spare respiratory capacity than other ebv-vsts and a low extracellular acidification rate despite upregulation of the glycolytic pathway. the peptivator plus il-4 and il-7 ebv-vsts had the highest basal oxygen consumption rate, atp-linked respiration, and extracellular acidification rate. conclusions: manufacture of ebv-vsts using the various approaches currently employed clinically results in t cell pools with different immunophenotypes and different metabolic profiles. ebv-vsts stimulated with peptivator followed by expansion in il-4 and il-7 and ebv-vsts isolated using the ifn-gamma capture system have immunophenotypes and metabolic phenotypes suggestive of potential greater in vivo persistence, whereas ebv-vsts expanded in il-2 and il-15 have characteristics correlated with increased effector function. however, these vsts are more likely to be short-lived and to have impaired metabolic fitness. these phenotypes will enable better correlation with clinical results and suggest combinatorial approaches depending on clinical indication. introduction: majority of patients with primary immunodeficiencies (pid) require life-long replacement therapy with immunoglobulins (ig) to prevent severe infections and irreversible complications. in addition to safety and efficacy, tolerability and convenience of administration of ig products are essential factors for patients. a new 16.5% ig preparation octanorm (octapharma, lachen; tradename cutaquigâ® in north america) has been developed for subcutaneous administration (scig) derived from the established manufacturing process of octapharmas intravenous ig (ivig) brand octagamâ®. objectives: primary outcome was assessment of the efficacy of octanorm in preventing serious bacterial infections. main secondary endpoints included (among others) evaluation of tolerability and safety of octanorm, the number and rate of other infections, number of days missed at work, and use of antibiotics. methods: a prospective, open-label, non-controlled, single-arm phase 3 study involving 25 adult patients with pid was conducted at 5 russian centers. patients treated with at least 4 infusions of ivig prior to enrollment and with igg trough levels 5.0 g/l underwent an 8-week wash-in/wash-out period followed by a 24week efficacy period. during the study, patients received weekly administrations of octanorm at the same monthly dose as during previous ivig treatment (monthly ivig dose divided by 4 for weekly dose). in total, each patient received 32 scig infusions. results: twenty-four patients completed the study. one patient terminated early (after infusion 7, during wash-in/wash-out phase; personal reasons). mean age was 35.24 years (range 18-64 years). fifteen patients (60%) were female and 10 patients (40%) male. no serious bacterial infections were recorded. during the efficacy period a total of 26 non-serious infections was observed in 14 patients. seventeen infections in 11 patients were of mild and 9 infections in 5 patients of moderate intensity. the infection rate per person-year was 2.37. in total 25 patients received 775 infusions of study drug. the average dose of cutaquigâ® was 0.11 g/kg/week. during the entire study, 59 systemic adverse events were reported (including 34 infections). three of these systemic adverse events were rated as related to study drug, all were non-serious. there was no serious or significant adverse event nor was there an adverse event leading to withdrawal. infusion site reactions were reported for 15% of infusions. serum igg trough levels were nearly constant during the efficacy period. median igg trough levels were 8.15 g/l at screening, 9.52 g/l at the end of wash-in/wash-out period and 10.71 g/l at the termination visit. one patient had a trough level 5g/l at 2 visits during the efficacy period and the dosing was subsequently adjusted for this patient. during the primary treatment period 10 patients (41.7%) used antibiotics in 19 treatment episodes (total of 229 treatment days; range 5-76 days) and 3 patients had 4 absences from work or school due to infections (total of 14 days of absence). conclusion: this study demonstrated that the new subcutaneous human normal immunoglobulin 16.5% is well tolerated, safe and effective in adult patients with pid. background: children with chronic granulomatous disease (cgd) are at high risk for fungal infections (especially with aspergillus species) and these infections usually have contiguous site involvement. most patients have pulmonary presentation. infective endocarditis and fungal osteomyelitis of skull are distinctly unusual. we report one such case. case: a 6-year-old boy, born out of a non-consanguineous marriage, presented with soft tissue swellings of skull for 2 months. his past history was significant with an episode of pneumonia at 1 year and recurrent soft tissue swellings all over the body since 1â½ years of age. on examination he was wasted, had signs of micronutrient deficiency, rickets, pallor, cervical lymphadenopathy and two abscesses, 12x4 cm on right temporo-parietal region and 4x3 cm over left frontal region. he was also found to have hyperdynamic precordium with an ejection systolic murmur. investigations revealed hemoglobin 85g/l; platelet count 7.34x109/l; total leukocyte count 13x109/l(n60/l23/m13/e1); elevated c-reactive protein( 244 mg/l) and a raised erythrocyte sedimentation rate(104 mm 1sthr). chest x ray revealed cardiomegaly (cardiothoracic ratio 67%) and 2d echocardiography showed vegetation of 6x3 mm over the anterior mitral leaflet suggestive of infective endocarditis. blood and urine cultures were sterile. culture from pus over the temporo-parietal abscess showed growth of aspergillus fumigatus. human immunodeficiency virus serology was non-reactive. immunoglobulin profile revealed elevated igg 21.20g/l (5.40-16.10g/l) and iga 5.66 g/l(0.5-2.4g/l); igm was 1.63 g/l(0.50-1.8g/l). in view of strong suspicion of cgd, nitroblue tetrazolium dye reduction test (nbt) was carried out-it revealed no reduction and dihydrorhodamine (dhr) assay showed a low stimulation index (4.34). flow cytometry for gp 47 phox and gp 67 phox was normal and dhr of mother did not reveal x linked carrier state. contrast enhanced computerized tomography (cect) of head showed osteomyelitis of the calvarial bones. contrast enhanced magnetic resonance imaging (cemri) brain showed heterogeneously enchancing soft tissue lesion in the scalp at right fronto-parietal region and left frontal region with underlying bony destruction suggestive of osteomyelitis. he was given intravenous antimicrobials (ceftriaxone, gentamycin, cloxacillin, voriconazole). after 6 weeks of therapy, he showed resolution of findings on mri brain and a repeat 2d echocardiography showed significant decrease in size of mitral leaflet vegetation. conclusion: this case highlights a rare presentation of cgd with infective endocarditis and skull osteomyelitis due to aspergillus fumigatus. to the best of our knowledge, this has not been reported previously. background: genetic defect in il12r1 affect cellular immunity, underlie mendelian susceptibility to mycobacterial disease (msmd) and inflammatory bowel disease (ibd) through different pathways. we present for the first time a patient with il-12r1 deficiency from a consanguine family with two different phenotypes. initially diagnosed as crohn's disease prior to the msmd diagnosis. method and material:patient was referred to the clinical immunology and allergy clinic at the at alzahra university hospital for immunological and genetic evaluation . blood samples from patient, his family and healthy donor controls were collected upon informed consent. in this study, we investigated effect of il12r1 mutation in il-12/ifnaxis by evaluation of patients whole blood cell response to il-12 and ifn-, il-12r1 expression in pbmcs and t cell blasts. also wholeexome sequencing has been performed. result and discussion: a 26 years old male from consanguine family , with history of right sub-axillary bcg lymphadenitis, recurrent mouth ulcers , chronic diarrhea in childhood and appendectomy at age of 5 was investigated. based on his clinical presentation abdominal pain, significant weight loss, chronic and bloody diarrhea , endoscopic and pathological findings treatment for crohn's disease (cd) was started at the age of seven . unfortunately, protracted patient's symptoms ends up to resection of his colon and colostomy two years later. he was presented with multi focal osteomyelitis at the age of 13 . although no bacteria was detected in pcr and tissue culture of the bone biopsy and the patient was not responded to antibacterials , he had a dramatic response to empirical anti mycobacterial treatment and his severe bone pain and lesions were healed. even though the bone manifestations were completely controlled, he continuously was under treatment for his gastrointestinal symptoms. genetic analysis was confirmed segregation of homozygous mutation in 3splice site of exon 15 in il-12r1. expression of gene was completely abolished in pbmcs of patient and the surface expression of il12rb1 was not detectable in t cell derived pbmcs of the patient compared to healthy control. furthermore, did not response to il12 stimulation since we could not detect increase of inf-after stimulation with il12 and bcg. our patient received bcg vaccination at birth and had bcg lymphadenitis as an infant, cd and mycobacterial multifocal osteomyelitis as a child. furthermore there are some evidences which indicate the role of atypical mycobacterial infections as a trigger for cd. conclusion: we reported for the first time contemporary msmd and ibd in 26 years old patient, who had impaired il-12 signaling and abolished il12 r1 expression in pbmcs and t cell blast. however, mycobacterial osteomyelitis is a typical phenotype of msmd patients with deficiency in ifn-r1 or stat, there were no mycobacterial osteomyelitis reported in il-12r1 deficient patients. background: advanced genetic studies help explain the occurrence of many undiagnosed, rare conditions. recently, nbas variants were identified as a causative basis of recurrent liver failure in infants (infantile liver failure syndrome 2, ilfs2). the nbas (neuroblastoma amplified sequence) gene encodes a protein involved in golgi to er retrograde transport. nbas functions seem to be broad and loss of function variants in nbas have been associated with multisystem manifestations. case report: a 5y 9m old chilean male presented to the er with a three day history of vomiting, diarrhea and one day of fever (38.3â°f). on examination he was pale, lethargic, and tachycardic. a chemistry profile revealed markedly elevated liver enzymes, increased bilirubin, and coagulopathy, consistent with the acute hepatic failure (alt 6291, ast >4000, total bilirubin 3.49 (2.82 db), ggt 52, and inr of 2.1). he was hospitalized, given vitamin k, and kept on intravenous fluids, ursodiol, and antipyretics. his liver function improved significantly within 5 days of admission (alt was down to 980, ast 45, total bilirubin 1.62). work-up of possible etiologies including autoimmunity and infectious hepatitis was negative. liver sonogram was normal, but liver biopsy was consistent with acute hepatitis with some necrosis. urine organic acid and plasma amino acid screens were not consistent with any inherited metabolic disorders. his parents recalled two previous episodes of liver failure at ages 3 and 4 years. both were preceded with a mild febrile illness and non-specific symptoms including fever, coughing, vomiting, diarrhea, lethargy, and decreased po intake. these subsequently were followed by jaundice and marked elevation of liver enzymes. flu a and adenovirus were identified as causes of febrile illnesses of the two previous episodes. for this admission, adenovirus was found in the respiratory secretions and a mild ebv viremia was also detected. genetic evaluation in chile was reportedly normal. after a literature review we obtained sequencing of nbas which revealed two variants: c.2827g>t,p.glu943* and nbas c.2951t>g, p.iie984ser. both variants have been previously reported in patients with an infantile onset, recurrent liver failure syndrome. his other clinical features include developmental and speech delays, failure to thrive, and facial dysmorphism. he also has a history of recurrent ear infections and has had 3 sets of tympanostomy tubes. further testing was limited due to the lack of insurance coverage. conclusion: nbas deficiency is a newly described syndrome of recurrent acute liver failure that occurs early in life. once individuals have survived to adulthood they do not seem to develop liver failure with illness. typically, liver crisis is triggered by a common childhood febrile illness. the mechanism of disease is thought to be thermal instability of hepatocytes which improves over time in most cases. however, although spontaneous recovery can occur following the crises, each episode can be fatal or result in permanent liver damage required liver transplantation. increased awareness of this disease will lead to the early establishment of the diagnosis. appropriate and timely management of fever at the onset of illness can significantly improve outcome in this potentially fatal disease. associate prof., infectious diseases and tropical medicine research center, isfahan university of medical sciences, isfahan, iran background: pre-eclampsia, a pregnancy-specific complication, has been shown to be associated with cytomegalovirus (cmv) infection. cmv specific t-cell response plays the major role in cmv infection or disease .we explored whether a change in cmv-specific cell-mediated immunity (cmi) is related to the development of preeclampsia. method: cmv-specific cmi was assessed using cmv-quantiferon (qf-cmv) assay in serum from 35 women with pre-eclampsia as well as 35 normal pregnancy controls retrospectively. participants were matched for gestational age individually. proportion of reactive results, mean value of interferon-level produced in mitogen and antigen tubes were compared between the cases and controls via chi-square, wilcoxon rank-sum tests, respectively. odds ratio (or) and confidence interval (ci) were calculated as well. result: no significant differences observed between demographic characteristics of the case and control groups. the qf-cmv assay turned reactive (qf-cmv [+]) in 22 of 35 of patients (63%) vs. 32 of 35 controls (91.4%) (p = 0.004). women with pre-eclampsia had lower mean ifn-levels in antigen tube (1.57 â± 1.79) compared with normal pregnancy controls (2.40 â± 2.21) (p = 0.028). there was no statistically significant differences in this value of mitogen tube between cases (3.53 â± 1.67) and controls (3.53 â± 1.67) (p = 0.209). women with suppressed cmv-cmi were 6.3 times more likely to manifest pre-eclampsia (or= 6.30, 95% ci: 1.60-24.7). this result even strengthened after adjustment for age, gestational age and gravidity (or = 12.86, 95% ci: 2.68-61.6). conclusion: our finding support an association between suppressed cmv specific cmi and pre-eclampsia. introduction: the triad of susceptibility to infections, auto-inflammation, and cancer in a patients personal and family history are always suggestive of an underlying primary immunodeficiency; however, in some cases the diagnosis might be delayed for years. furthermore, the results of immunological and inflammatory evaluation can also be affected by ongoing immunomodulatory therapy initiated by different specialists upon clinical diagnosis. objective: to describe a unique presentation of auto-inflammatory disease with combined immunodeficiency in an adult patient. case presentation: we report here the case of a 64 year old male, who had a long history of infections including recurrent sino-pulmonary bacterial infections starting during childhood, osteomyelitis at 7 years of age, recurrent tonsillitis requiring tonsillectomy at 21 years of age, recurrent cellulitis, an episode of prostatitis with septicaemia, as well as recurrent varicella zoster and warts. the patient was also diagnosed with sclerosing mesentheritis, and reynauds phenomenon, recurrent oral ulcers, arthritis, uveitis, autoimmune thyroiditis, lung fibrosis and suffered repeated episodes of abdominal pain. furthermore, there is a family history of early childhood death, multiple soft tissue cancers, crohns disease, and autoimmune thyroiditis. upon physical examination, the patient had multiple telangiectasia, baseline erythroderma, and flushing. immunological evaluation showed lymphopenia with significant reduction in both circulating b and t cells, however, assessment of humoral immunity revealed low igg and decreased igm with normal iga levels. at the time of the evaluation he had been on low dose daily prednisone (7.5mg), colchicine, and methotrexate as immuno-modifying therapy. genetic evaluation revealed a heterozygous mutation in nod2 as well as compound heterozygous mutations in the mefv gene. discussion: mutations in nod2 have been described in association with blau syndrome a multisystem auto-inflammatory syndrome which may explain many of the features experienced by our patient. to our surprise next generation sequencing revealed a second aberration in the mefv gene which causes familiar mediterranean fever, another multisystem auto-inflammatory disease, which might lead to the phenotype observed in the patient. conclusion: this is the first report of genetic lesions in two different genes leading to a severe course of auto inflammation. monogenic autoinflammatory syndromes (mais) are a diverse group of disorders characterized by primary over-activation of the innate immune system. induction of the inflammasome complex by innate immune sensors and increased production of il-1b are implicated in the pathogenesis of mais. macrophage activation syndrome (mas) is a life-threatening illness defined by acute hyper-inflammation and unopposed cytokine release. it is considered an acquired condition secondary to infection, rheumatoid disease or malignancy. the early therapeutic use of il-1b inhibition has profoundly improved the prognosis mas. it has recently been shown that increased free il-18 levels in the blood are causatively linked to the development of mas. significant overlap in clinical presentation and laboratory markers between patients with mais and mas led us to explore the role of free il-18 and therapeutic use of il-1b inhibition in a patient with cdc42 mutation. here, we report the case of an 18 months-old female who presented with hydrops fetalis in utero, and later developed failure-to-thrive, splenomegaly, anemia, thrombocytopenia, arthralgias, rashes, frequent febrile episodes and mild facial dysmorphism along with massive increase in crp, esr and ferritin. whole exome sequencing (wes) identified a heterogenous likely pathogenic de novo variant in cell division control protein 42 homolog (cdc42) c.563g>a (p.c188y). cdc42 encodes a small rho family gtpase that regulates multiple signaling pathways controlling cell polarity, migration, endocytosis and cell cycle progression. single allele mutations in the cdc42 gene were recently reported to cause takenouchi-kosaki syndrome manifesting with growth retardation, developmental delay, facial dysmorphism, and thrombocytopenia however systemic autoinflammation has not been described. cdc42 closely interacts with the wiskott-aldrich syndrome protein but little is known about the mechanism underlying immune abnormalities associated with cdc42 mutations. our patient had an inflammamosopathy-like syndrome. because of significant clinical overlap to mas, we measured il-6, il-18, free il-18 and il-18 binding protein, all of which were significantly increased. this increase in free il-18 heightened her risk of developing mas. her il1b level was normal, but an increase in il-1b is hardly ever detectable in the serum despite playing a critical role in this type of inflammation. indeed, chronic il-1b excess in the tissues promotes systemic inflammation and is associated with chronically elevated crp and esr. with this rationale we started the il-1 receptor antagonist anakinra. within 48 hours from starting anakinra, the parents observed an increase in appetite, resolution of arthralgias and improved mobility. over the course of the following weeks, fever, anemia, thrombocytopenia and rash disappeared, the spleen massively decreased in size and the patient started to meet developmental milestones. crp, esr eventually normalized while ferritin and free il-18 are still trending down. conclusions: significant increase in free il-18 and extremely encouraging clinical response to therapy with anakinra in a patient with novel cdc42 mutation suggests a link between mas and defects in cdc42. elucidating the mechanism of inflammasome activation and the drivers of il-18 increase in mas and mais more broadly may shed light on novel therapeutic targets like the use of human recombinant il-18 binding protein. j clin immunol (2019) 39 (suppl 1):s1-s151 s69 maintenance; smarcal1 is enriched in cells that maintain telomeres via the alternative lengthening of telomeres pathway and smarcal1decifient cells demonstrate telomere instability with replication fork collapse and increased telomere-associated dna damage. [1, 2] telomere analysis of 4 siod patients, including one patient who received a hematopoietic stem cell transplant (hsct) 20 years prior, as well as 5 heterozygous family members revealed significantly shorter telomeres in siod patients compared to heterozygous family members and compared to agematched, healthy controls. methods: peripheral blood mononuclear cells were isolated using a ficoll-hypaque density gradient, cryopreserved, then sent to repeat diagnostics in north vancouver, bc. telomere length measurements were performed at a single-cell level using flow-fluorescence in situ hybridization as previously described. [3] telomere length was measured in total lymphocytes, naive and memory enriched t cells, b cells, and nk cells and compared to reference samples from age-matched, healthy individuals. results: compared to age-matched healthy controls, three siod individuals had mean telomere lengths (mtls) less than the 1st percentile for age across all lymphocyte subsets (total lymphocytes, b cells, nk cells, naã¯ve and memory t cells). in comparison, three unaffected family members had normal mtls (10th percentile< x <90th percentile) across all subsets, and two unaffected family members had low mtls (1st< x <10th percentile) in some subsets. the siod individual who received a matched-sibling hsct 20 years prior, had normal mtl in nk cells (10th < x <90th percentile) but low mtls (1st< x <10th percentile) for all other subsets. conclusions: these data show that siod patients have significantly impaired telomere lengths across multiple lymphocyte lineages and support a limiting role for smarcal1 deficiency in telomere maintenance. in comparison, unaffected family members, heterozygous for smarcal1 mutations, have mean telomere lengths that are normal or slightly low for age. this suggests that abnormally short telomeres are seen in individuals with homozygous but not heterozygous smarcal1 mutations. for the individual who received a hsct, we do not have pre and post-hsct telomere data, but these results support obtaining pre and post-hsct telomere length analysis in future cases. abnormally short telomeres have been linked to widespread perturbation of gene expression. [4] we hypothesize that smarcal1 deficiency, by the effect of stalled forks and shortened telomeres, leads to perturbation in the transcriptome of affected tissues. shortened telomeres may explain the reduced hematopoietic bone marrow production in siod, as bone marrow failure is a cardinal feature of dyskeratosis congenita, a disorder of impaired telomere maintenance. future studies to investigate the role of telomere maintenance in siod include measurement of telomerase activity in polyclonally activated t cells and transcriptome analysis using rna-seq background: yellow fever is a potentially fatal disease for which only supportive treatment is available. vaccination is the primary strategy for prevention of this disease and the vaccine is extremely effective, but there are a few specific populations where it is contraindicated. regarding iga deficiency (the most frequent primary immunodeficiency), current recommendations in the literature are controversial. there are no specific studies in this disease, so case series addressing the safety or possible adverse events after vaccination are essential for decisionmaking during epidemic scenarios, as experienced in brazil in the last years. in this context, this study aimed to describe adverse events after the use of the yellow fever vaccine in iga deficient patients. method: a retrospective cross-sectional study was conducted including iga deficient patients followed at a specialized pediatric outpatient clinic between 2017 and 2018. all patients had at least one year of follow-up. immunoglobulin levels, antibody response to vaccines and lymphocyte subset count were evaluated to exclude other immunodeficiencies or the presence of abnormalities that could contraindicate vaccination. demographic data, the presence of infections and comorbidities, use of immunosuppressive medication and adverse events after vaccine administration of the vaccine were described. results: thirty-eight patients with iga deficiency were included in the study and 18 received the vaccine. vaccinated patients had a mean age at the time of the study of 13.7 years (sd â± 3.5y). six out of the 18 presented comorbidities: thyroiditis (n=3), type 1 diabetes mellitus (n=1), celiac disease (n=1) and juvenile rheumatoid arthritis (n=1). all patients were atopic and only one had recurrent infections in the last year despite the use of antibiotic prophylaxis. all 18 patients had normal igg and igm levels for their age, positive vaccine responses for measles, rubella and mumps, and age-appropriate lymphocyte subset count. after 6 months of observation, no immediate or late adverse events were reported. among the 20 non-vaccinated patients, only one had a formal contraindication (systemic erythematosus lupus using immunosuppressive therapy). five out of the 20 non-vaccinated patients reported being afraid of receiving the vaccine, 7 still intended to receive it and for other 7 patients data regarding vaccination was unavailable. conclusion: despite the small number of patients, the absence of adverse events in this case series suggests that immunization with yellow fever vaccine may be safe in iga deficient patients, excluded other contraindications. more studies are essential to confirm the safety and help the decision-making process regarding the vaccine administration for iga deficient patients, especially in this yellow fever outbreak scenario. introduction/backround: immunodeficiency, centromeric instability, and facial anomalies syndrome (icf) is a rare group of autosomal recessive disorders involving the triad of hypogammaglobulinemia, centromeric instability, and facial anomalies. the majority of patients have hypo-or agammaglobulinemia, but t cell defects have also been reported. we present the case of a child with icf-2 who presented with nk deficiency and ultimately developed an ebv-driven malignancy and was successfully treated with bone marrow transplant. methods: whole exome sequencing and nk cell function via 51-cr cytotoxicity assay and phenotyping via flow cytometry were performed at baylor college of medicine and texas childrens hospital. centromeric banding studies were performed at university of pittsburgh medical center. results: the female patient presented at 3 months of age with cmv pneumonitis and persistent cmv viremia requiring treatment followed by prophylaxis with valgancyclovir. she initially had hypogammaglobulinemia and low t, b, and nk cells; she had normal trecs, lymphocyte mitogen proliferation responses and zap 70, mhci and mhcii expression. the hypogammaglobulinemia and t-and b-cell lymphopenia resolved within 9 months after initial presentation as she clinically improved from her cmv infection. she was found to have nk cell deficiency on three separate commercially tested samples. whole exome sequencing revealed a homozygous variant in zbtb24 indicative of icf-2 syndrome that was confirmed with sanger sequencing (c.1492_1493del, p.q498vfs). repeat nk cell studies confirmed impaired function, and phenotyping showed an increase in cd56-bright and a decrease in cd16-positive cells, suggesting either impaired transition from immature to mature nk cells or impaired survival of mature cells. her karyotype and centromeric banding studies were normal, as were centromeric instability studies. she later developed a memory b-cell defect and presented at 34 months of age with persistent fever, respiratory distress, loss of vaccine titers, hypogammaglobulinemia and low b and t cells. she was found to have ebv viremia and an eber-positive diffuse large b-cell lymphoma in her right lung. due to tenuous clinical status, she received rituximab for treatment of ebv prior to definitive lymphoma diagnosis. she was treated with chemotherapy per protocol anhl1131, group b (pre-phase with cop, courses 1 and 2 with copadm, and courses 3 and 4 with cym) and her course was complicated by seizures attributed to methotrexate toxicity. she ultimately underwent reduced intensity conditioning with hydroxyurea, alemtuzumab, fludarabine, mephalan, and thiotepa followed by a cd-34 selected, hla-matched, unrelated donor peripheral blood stem cell transplant. her early post-transplant course was complicated by adeno-, ebv, and cmv viremia, all successfully treated with antivirals and a donor lymphocyte infusion. she is now greater than 8 months posttransplant, off immunosuppression with 100% donor engraftment, no evidence of organ toxicity or gvhd, and with excellent immune reconstitution. conclusions: this is the first reported case of impaired nk cell function and phenotype and ebv-driven malignancy in a patient with icf-2. this case expands the phenotype of icf-2 and suggests that early bone marrow transplant should be considered in these children. it also demonstrates a novel requirement for zbtb24 in human nk cell maturation and function. rationale: common variable immunodeficiency (cvid) is a disorder that affects the production of immunoglobulins and is associated with development of autoimmunity. multiple mutations have been described that are associated with cvid, but plcg2 mutations have only been described in patients with phospholipase c gamma 2 (plc2) associated antibody deficiency and immune dysregulation (plaid) and autoinflammatory plc2 associated antibody deficiency and immune dysregulation (aplaid). we present a case of a 44 y/o male cvid patient with recurrent upper respiratory tract infections, steroid-dependent autoimmune thrombocytopenia, low b cell count, hepatosplenomegaly, and restrictive lung disease. he was found with a variant of unknown significance at the plcg2 gene. in contrast to plaid our patient does not exhibit cold urticaria. method: case presentation of a cvid patient followed in our clinics. patients chart and previous laboratories were reviewed. sequence analysis and deletion/duplication cvid panel testing was performed using invitaeâ© discussion: genetic testing has revolutionized the diagnosis of immune deficiencies, but variants of unknown significance are being increasingly reported. in this case, a variant of uncertain significance was identified which replaces threonine for alanine at codon 829 of the plcg2 protein. this codon is located at the sh3 domain, which is part of a region that provides auto-inhibitory enzymatic functions. plaid mutations have been identified in sh2 domain, but it has been known that both sh2 and sh3 domains facilitate plcg association with other proteins. studies with deletion of plcg2 gene have shown functional abnormalities in b cells, natural killer cells and mast cells. to our knowledge, there has not been any previous report of a cvid patient with a variant mutation at the sh3 domain of the plcg2 gene without being diagnosed as plaid or aplaid. our patient has immunodeficiency, recurrent upper respiratory tract infections, steroid-dependent recurrent autoimmune thrombocytopenia, rheumatoid arthritis, hepatosplenomegaly, early-osteoporosis and restrictive lung disease. he does not have cold urticaria as seen in plaid, but exhibits autoimmunity not observed in aplaid. conclusion: conclusion: plcg2 is an important protein in the pathway of b cell development. a novel mutation in the sh3 domain of the plcg2 gene may be associated with the cvid phenotype of low b cells and autoimmunity. this could lead to a gain-of-function mutation as seen in plaid but without early-onset cold urticaria. functional studies are required to confirm the significance of this mutation. primary (or familial) hemophagocytic lymphohistiocytosis (hlh) is a rare, life-threatening hyper-inflammatory disease affecting mainly young children. it is caused by mutations in genes involved in the granule-dependent cytotoxic pathway, and is characterized by extreme inflammation and massive tissue infiltration by activated t cells and macrophages. to this day, hematopoietic stem cell transplantation is the only available curative treatment with a transplantrelated mortality of 30%. thus, the development of new, more efficient anti-inflammatory treatments would be a significant advancement in the treatment of hlh. here, we hypothesize that combination therapies targeting both jak-dependent and independent cytokines will be more effective than either one alone to reduce the lifethreatening symptoms induced by this pathology. using a perforin-deficient mouse model of hlh, we first compared the effect of targeting individual cytokines with blocking antibodies on the progression of the disease. we show that blocking ifng and il-18, but not il-6, significantly reduces the severity of hlh. targeting the jak-stat signalling pathway with ruxolitinib, a specific inhibitor of jak1 and jak2, downstream of ifng and il-6, but not il-18, is similarly beneficial. more importantly, combination therapies using ruxolitinib and blocking antibodies to either ifng or il-18 show synergistic effects, further mitigating the progression of the disease. these results suggest that jak-dependent and independent cytokines drive the pathogenicity of hlh in perforin-deficient mice. it further supports that ruxolitinib, although effective in reducing the symptoms of hlh, should be used in combination with anti-ifng and/or anti-il-18 antibodies to prevent hlh progression. this is particular relevant since the former were recently approved for the treatment of hlh while the latter (il-18 binding proteins) are in clinical trials for il-18-dependent macrophage activation syndromes. despite the increased risk of opportunistic lung infection in patients with severe t cell dysfunction (e.g. cd40l deficiency) and/or severe cd4 t cell lymphopenia, we are not aware of any reports of disseminated pneumocystis jiroveci infection in non-human immunodeficiency virus (hiv) patients with primary immunodeficiency (pid). we report the first case, to our knowledge, of disseminated pjp in a patient with cvid like/ctla4 haploinsufficiency. he had been diagnosed with common variable immunodeficiency (cvid) in 2009, approximately eight years prior to being referred to us, and was on intravenous immunoglobulin (ivig). he was also diagnosed with multilineage evans syndrome in 2015. his medical history was also significant for potential granulomatous lymphocytic interstitial lung disease (glild) (lung biopsy in the remote past with interstitial disease), significant splenomegaly (29.4 cm), severe portal hypertension, nodular liver disease (likely nodular regenerative hyperplasia) complicated by anasarca, history of chronic diarrhea (potential enteropathy), lymphadenopathy s/p biopsy with nodular lymphoid hyperplasia, and a history of multiple pneumonias. in 2017, he had developed disseminated pjp with lung, liver, and bone involvement. the t2 vertebra pjp invasion was confirmed with a bone biopsy; gomori methenamine silver staining and pcr were performed and concluded pjp. he was treated with trimethoprim sulfamethoxazole (tmp-smx) and steroids, then was continued on tmp-smx prophylaxis. due to his liver damage and his chronic neutropenia, tmp-smx was replaced by atovaquone as a secondary prophylaxis for pjp. his laboratory studies were significant for an absolute neutrophil count of 1.54 k/ul, absolute lymphocyte count of 0.61 k/ul, hemoglobin of 12.7 g/dl, platelets of 78 k/ul, total bilirubin of 2 . t-cell receptor beta chain repertoire analysis showed an oligoclonal distribution. severe combined immunodeficiency panel through ambry genetic testing was negative as was genetic testing for cd40l deficiency. given his complex clinical history, whole exome sequencing was obtained and detected an autosomal dominant heterozygous missense mutation (c.436g>a) implicated in ctla-4 haploinsufficiency and previously reported by schwab et al. our patient is currently undergoing therapy with abatacept (ctla-4 fusion protein), which has been reported to improve glild, splenomegaly and enteropathy in patients with ctla-4 haploinsufficiency. he is improving on this regimen. he has met with the stem cell transplant team, but at this point of time, due to his abnormal lung function, his liver damage and his significant splenomegaly, he is not a good candidate. defects in the nf-b signaling pathway are implicated in the pathogenesis of several primary immune deficiencies in humans. the clinical features of these conditions vary significantly, reflecting the complexity of the pathway, and its broad role in innate and adaptive immune responses, and the development and differentiation of lymphoid organs. here we report the first case of a human pid caused by a homozygous mutation in nfkbid in a 30 year-old male. he was the second child of consanguineous parents, and was diagnosed with possible cvid at the age of 16, after recurrent episodes of pneumococcal pneumonia. however the clinical features have evolved over time; he developed severe ebv infection at age 18, causing hepatitis and pancreatitis. at the age of 20, he presented with an anca-negative systemic vasculitis, manifesting as pulmonary haemorrhage, and acute necrotizing pauci-immune glomerulonephritis. pulsed methylprednisolone and cyclophosphamide induced an initial remission, however, relapse a year later led to end-stage renal failure. he is now dialysis-dependent, and due to the underlying pid, and chronic cmv viraemia, is not a candidate for renal transplantation. genomic dna was subjected to whole-exome sequencing. variants were filtered using a model of autosomal-recessive inheritance and functional analysis of primary cells was performed. we identified a novel, homozygous, single-base deletion resulting in a frame-shift, and premature stop in nfkbid. nfkbid encodes ibns, a non-classical inhibitor of nf-b signaling. at diagnosis the patient had reduced levels of igg2, iga and igm, elevated ige, with absent humoral immune responses to pneumococcal polysaccharide vaccine, and an intact response to tetanus. lymphocyte numbers were initially within normal reference ranges, albeit with an increased proportion of cd4+:cd8+ t cells. however, over time there has been a significant reduction in b cells and cd8+ t cells. cd4+ t cells demonstrated a skewing towards a central memory phenotype (cd45ro+/ccr7+), and cd4 t cell proliferative responses to pha were comparable to a healthy control. functional analysis of primary cells from the proband revealed a complete absence of bns protein expression, dysregulated nf-b signaling, and elevated pro-inflammatory cytokine production. the patient is currently receiving a trial of targeted therapy to modulate the aberrant immune responses. this novel pid highlights the importance of regulation of nf-b signalling, in orchestrating an appropriate immune response, maintenance of self-tolerance, and protection against viral pathogens. primary immunodeficiency diseases (pid) are a heterogeneous group of conditions with variable clinical features that are frequently associated with significant diagnostic delay. accurate diagnosis has significant therapeutic benefit and may lead to personalized therapies. we established the immunology flagship of melbourne genomics health alliance in australia to determine the clinical utility of genomic sequencing for diagnosis and management of individuals with suspected and confirmed cases of pid. 198 adults and children with suspected or confirmed pid (n=153), autoinflammatory disease (n=33) and hereditary angioedema (hae, n=11) were recruited to the melbourne genomics immunology flagship. whole-exome sequencing (wes) was performed, with targeted gene analysis. variant curation and reporting was performed according to the american council of medical genetics guidelines. overall, wes was diagnostic in 15% (30/198), confirming a preexisting diagnosis in 7% (14/198), and offering a new or more specific diagnosis in 8% (16/198). variants of uncertain significance were identified in a further 28 patients (14%) in genes known to be associated with their clinical diagnosis, that warrant further functional validation. in the hae group, diagnosis was confirmed in only 5 patients (45%), suggesting that wes may not be the appropriate technique for genetic diagnosis in this condition. a higher diagnostic rate was observed for autoinflammatory disorders (20%; 8/40) compared to pid (12%; 18/146). of those who received a diagnosis, immediate changes to patient management and treatment occurred for 17/29 patients (59%), including hsct for 3 and specific targeted therapy for 11 (38%) individuals. we have demonstrated the utility of wes for accurate diagnosis of complex immune diseases, with the potential to change diagnoses, guide therapeutic intervention and provide opportunities for genetic counseling. further longitudinal analysis will determine clinical outcomes and health economic implications of genomic sequencing for diagnosis and management of immunological conditions in australia. at birth he had neonatal asphyxia and cerebral palsy. at 4 years old he had presented involuntary movements, left paresis, bilateral horizontal nystagmus. at 8 years of age, he had a right nasal obstruction. it was resected by otorhinolist and informed by biopsy: inflammatory polyp and chronic sinusitis. he has had 3 pneumonias, sinusitis and diarrhea. at the age of 13 years, the ataxia telangiectasia was confirmed by sequencing with pcr (62 exons, 91711 bp) of the atm gene: transition g> a, nucleotide position 2250, codon 750, affecting splicing. alpha fetoprotein 572-606.90 u/ml. brain mri, say cerebellar atrophy. he had igg 685 mg / dl -734 mg / dl, iga 0.00 mg / dl, <1 mg / dl, igm 268 mg / dl -315 mg / dl, ige 0.10 -<1 iu / ml. subclasses of igg: igg3: 0.05 g / dl, igg4: 0.04 gr/dl, low. igg anti hepatitis b 6,22. no seroconversion. hiv negative tcd3 + lymphocytes: 32,40%, = 553 cells / mm3, ltcd4 +: 23,78% = 413,21 cel / mm3, ltcd8 +: 7,69% = 133,5 cells / mm3, cd4 / cd8: 3.09. for all of the above, common variable immunodeficiency was diagnosed. he receives human immunoglobulin. at 16, i arrived at this hospital due to fever, respiratory distress and lymphadenopathy in the neck. ct showed ganglionic conglomerate on right side neck. lymph node biopsy: strong tumors with cd20 and bcl2, weak and moderate diffuse pax-5; negativity with cd68, cd3 and cd10, and a cell proliferation index with ki67 of 50%, diagnosis: diffuse large b cell lymphoma. treated with rituximab and chemotherapy. lymphoma completely remitted. conclusion: the association ataxia telangiectasia and lymphoma is frequent. by contrast, cvid and ataxia telangiectasia are extraordinarily rare. introduction: chronic granulomatous disease (cgd) is a primary immunodeficiency wherein affected patients are susceptible recurrent infections caused by specific bacteria and fungi as a result of defective nadph activity. additionally, inflammatory complications involving the bowel and lungs can cause significant morbidity. currently the only proven permanent cure to cgd remains hematopoietic stem cell transplant. case: a 25-year-old patient was diagnosed in infancy with x-linked cgd. at age 5yrs he received a nonmyeloablative peripheral blood stem cell transplant from his 10/10 non-carrier sister as previously reported (nejm 344:881, 2001) . conditioning was cyclophosphamide (60 mg/kg) on d-6 and d-7; daily fludarabine (25mg/m2) on d-5 through d-1; antithymocyte globulin at 40mg/kg on d-5 through d-2. posttransplant immunosuppression consisted of cyclosporine on d-4 through d+100. he received 7.8x106 cd34+ peripheral blood stem cells which were t-cell depleted with 1x105 add back of cd3+ cells on day 0. after 10 days of neutropenia (anc <500) there were signs of engraftment. per protocol, he received donor peripheral-blood lymphocytes containing 2.0x106 cd3+ cells/kg on d+ 30 after transplantation. since donor t cells constituted less than 60 percent of his circulating cd3+ t cells and he had no graft versus-host disease, he received 1.0â¬107 cd3+ cells/kg on d+60. after the discontinuation of cyclosporine, he received a total of three donor-lymphocyte infusions (1.0â¬107 cd3+ cells/kg) at 90-day intervals achieving 100% t cell and myeloid engraftment at 26 months post-transplant with no acute nor chronic gvhd. at last follow-up 6 years post-transplant (2004) he had 100% and 98% lymphoid and myeloid peripheral chimerisms, respectively. the patient and family declined further periodic followup. then, in october 2018 he presented with malaise, cough and fevers. he eventually was found to have a large consolidation and a bal grew burkholderia cepacia. his dhr showed 12% activity and peripheral blood myeloid and lymphoid chimerisms were 12% and 60%, respectively. discussion: this late graft failure following peripheral blood transplant occurred following a conditioning regimen which is not the current standard for transplant in cgd. in the case series in which this patients transplant is reported (nejm 2001), another patients myeloid chimerism fell to 15% by 3 years post-transplant, remaining stable at that level of chimerism without any serious infections over regular periodic follow up to the present time. current regimens typically include busulfan to enhance engraftment and prevent graft failure. this case reinforces the need for prolonged monitoring of primary immune deficiency patients after transplantation. introduction: with the introduction of severe combined immunodeficiency (scid) newborn screen (nbs) in the state of kansas in 2017, a case of complete digeorge syndrome (dgs) was discovered in an infant born to a diabetic mother with atypical features. this is the first dgs case diagnosed after adding the scid nbs, which emphasizes the need to establish scid nbs in all 50 states. case presentation: the female infant was born via spontaneous vaginal delivery at 39 1/7 weeks to a 31 year old g1 now p1 mother. maternal history was significant for chronic hypertension, obesity, insulin dependent type 2 diabetes, anxiety, depression, and scoliosis. the infant was noted to have a left sided abdominal wall defect and hernia, imaging identifying left renal agenesis, and was initially suspicious for vater syndrome. fortunately, the infant's scid nbs revealed low t cell receptor excision circles (trecs). her initial white blood cell count was 14.1 with an absolute lymphocyte count of 2.679 k/ul. ebv pcr, cmv pcr, and hiv studies were negative. chest imaging discovered absent thymus, abnormal vertebrae with only 10 ribs on the right and 12 ribs on the left, and abnormally formed thoracic vertebrae (t7). echocardiogram detected an atrial septal defect measuring 0.32 cm, possible pfo versus secundum asd. endocrinology was consulted for management of labile calcium and phosphorus levels. fish was negative for 22q11.2 deletion. microarray r evealed a variant of unknown signif icance arr[grch37]2p11.2(86285942_86506132)x3. sequence analysis of combined and severe immune deficiency genes showed a variant of uncertain significance c.544c>a (p.leu182met). management and outcome: additional evaluation included: cd3 67ul (1700-3600ul), cd4 51ul (1700-2800ul), cd8 19ul (800-1200ul), cd45ra 14 cells/ul (1100-5200cells/ul), normal cd 19, and cd 16/ 56, normal immunoglobulin g level, and normal dihydrorhodamine assay. skeletal survey, ct abdomen and chest, and hla typing were performed in preparation for thymic transplant. discussion: patients with complete dgs, a form of scid found in less than 1 percent of patients with 22qds, have absent thymus and a t cell count <3 standard deviations below normal for age (typically <50 naã¯ve cd3+ t cells/mm3). in a large series of patients with complete dgs, only 52 percent had an identifiable 22q11.2 deletion [1] . infants of a diabetic mother have various genetic and syndromic associations including diabetic embryopathy. [2] despite the importance of immunological aspects in pregnancy, few studies have reported on the cellular immune modifications of diabetic embryopathy. diabetes during pregnancy may affect the development of the thymus and thus maturation of the immune system in the offspring. [3] the recent addition of a trec assay to newborn screening can identify such a subset of infants with atypical presentations. scid nbs uses an assay for trecs, a biomarker of t cell development. [4] [5] [6] this initial presentation now places the immunologist in the role of "first responder" with regard to diagnosis and management of these patients, who may present with atypical features. newer genetic and molecular techniques now allow for earlier identification of immune defects in such disorders with life-long clinical concerns. [7] references: introduction/background: goods syndrome is a rare cause of combined b-and t-cell immunodeficiency occurring in association with a thymoma. affected patents are susceptible to bacterial, fungal, viral, and opportunistic infections. an association with autoimmunity has also been reported. current knowledge of goods syndrome is primarily limited to case reports and small series. objectives: to examine the spectrum of clinical and laboratory features of a major cohort of goods syndrome patients in the us. methods: we conducted a retrospective analysis of patients with goods syndrome in the usidnet registry and the mount sinai hospital (msh) cohort. r e s u l t s : we i d e n t i f i e d 2 0 p a t i e n t s w i t h t h y m o m a a n d hypogammaglobulinemia (usidnet, n=11; msh, n=9; median age: 60 years; female: 45%), representing data from 151 patient years. the median age at diagnosis of thymoma and hypogammaglobulinemia were 52 years (range 31-85), and 50.5 years (range 28-86), respectively. two patients were deceased (at age 65 and 70 years, cause unspecified). all patients had low igg (median 313mg/dl, range 47-699). iga and igm were reduced in 90% and 45% of patients, respectively. low cd19+ b cells (median 0.5/mm^3, range 0-28) were reported in all available records. the absence of cd19+ b cells was observed up to 21 years postthymectomy. a wide range of additional laboratory abnormalities were identified: low cd4+ t cells (n=5), low cd8+ t cells (n=2), low cd4/ cd8 ratio (n=6), low nk cells (n=6), and absent peripheral eosinophils (n=8). the most common sites of infections were lower respiratory (70%), upper respiratory (55%), and gastrointestinal (35%). in addition, sepsis (15%), meningoencephalitis (5%), osteomyelitis (5%), and urinary tract infection (5%) were also observed. identifiable infectious agents included: bacteria (35%), virus (35%), fungus (25%), parasites (10%), and protozoa (5%), with opportunistic infections recorded in 25% of patients. opportunistic infections were significantly associated with absolute cd4 lymphopenia (p=0.048, fishers exact test). enterovirus was identified as a previously unreported cause of meningoencephalitis in this population. autoimmune manifestations were reported in 45% of patients, with a higher prevalence of inflammatory colitis (20%) than previously reported. hashimoto thyroiditis, fibromyositis, and bronchiolitis obliterans organizing pneumonia (n=1 each) were identified as previously unreported autoimmune/inflammatory conditions in this population. a case of alopecia areata was also observed. additionally, bronchiectasis was recorded in 20% of patients. all patients were initiated on immunoglobulin replacement, with antibiotics prophylaxis in 20%, and immunosuppressive medications employed in 10% of patients post diagnosis of immunodeficiency. conclusion: goods syndrome is a combined immunodeficiency, with a wide range of autoimmunity in a subset of patients. we expanded upon the spectrum of associated infectious and inflammatory complications through a major us cohort. persistent immune dysregulation was observed up to 2 decades post-thymectomy. introduction: primary immunodeficiencies (pids) constitute a large group of rare disorders that affect the immune systems function. some pid patients develop autoimmunity in addition to having increased susceptibility to infections due to their impaired immunity [917] . (1) case presentation/ management: a 43 year old caucasian female with history of bipolar disorder, factor v leiden deficiency, anti thrombin 3 deficiency, pulmonary embolism, endometriosis, and seasonal allergies was evaluated for chronic granulomatous disease (cgd) in 2007. the main symptoms were inflammatory breast lesions necessitating 4 surgeries on the right breast, and back, facial, genital, ocular, mouth, and scalp sores. biopsy with cultures of the wounds was positive for corynebacterium, coagulase-negative staphylococcus, enterococcus, bacteroides species, and provatella. neutrophil oxidative burst was ordered by the infectious disease specialist and showed normal and abnormal neutrophil populations, a finding consistent with cgd carrier. patient was started on interferon gamma-1b after failing multiple courses of antibiotics. her symptoms were well controlled on interferon gamma-1b 100mcg/0.5ml sq every other day, trimethoprim 100mg tab (2tabs in am and 1 tab in pm), cefixime 400mg once daily, and topical mupirocin as needed except for her recurrent genital ulcers. cgd can be rarely associated with oral ulcers however there is a limited literature describing associated genital ulcers. according to the international study group diagnostic criteria published in 1990 (2), the patient was diagnosed by a rheumatologist as having behcets disease (bd). there are no pathognomonic laboratory tests in bd; as a result, the diagnosis is made clinically. patient failed a trial of colchicine and was later started on cyclosporine, which resulted in decrease of her mouth and genital ulcers. discussion: bd is a rare disease mostly seen along the silk road. the prevalence has been reported as 0.12 (usa) to 370 (in a single village, northern turkey) for 100 000 inhabitants. (3) cgd is a primary immunodeficiency caused by defects in any of the five subunits of the nadph oxidase complex responsible for the respiratory burst in phagocytic leukocytes. patients with cgd are at increased risk of life-threatening infections with catalase-positive bacteria and fungi, and inflammatory complications such as cgd colitis. (4) reports of cgd female carriers with discoid lupus erythematosus, photosensitivity rashes, and other autoimmune phenomena have been published [48, 49] (4) . to the best of our knowledge, this is the first case to report bd in an affected cgd carrier. the treatment of inflammatory disease in patients with cgd poses a difficult balance between therapeutic immunosuppression and the increased risk of severe infection. (5) . high dose intravenous immunoglobulin, and targeted therapies such as ctla4-ig for t cell mediated pathologies, rituximab for b-cell mediated pathologies, and anti-tnf for ibd, may be preferable over the broad immunosuppressive activity of glucocorticoids. in addition, emerging evidence suggests that hematopoietic stem cell transplantation has indication for cases that have been difficult to control using immunosuppression. (1) given all that, our case emphasizes the need to maintain suspicion for autoimmune disorders / immune dysregulation in patients with pid. introduction: cd40-ligand deficiency is an x-linked combined immunodeficiency, characterized by susceptibility to infection, often with associated neutropenia, malignancy, and autoimmunity. central nervous system (cns) manifestations are less commonly reported than respiratory or gastrointestinal complications, but are most often attributed to infection. herein we describe a challenging case of gradual onset episodic memory loss, confusion, and unilateral hemiplegia in a young male with cd40ligand deficiency. case presentation: the patient is a 13-year-old male with cd40-ligand deficiency on immunoglobulin replacement therapy presenting with recurrent, episodic altered mental status (ams) and gradual neurocognitive decline. initial neurologic symptoms began at age 11 years, and included fever, nausea, and eyelid fluttering. initial comprehensive infectious workup at this time, including blood and urine cultures, lyme antibody, serum pcr for hsv, cmv, ebv, respiratory viral pcr including atypical viruses, csf studies including culture, lyme eia, pcrs for enterov i r u s , v z v, e b v, c m v, h s v 1 / 2 w e r e u n r e v e a l i n g . electroencephalogram (eeg) and mri displayed generalized slowing and global atrophy, respectively. definitive diagnosis was not made. the patient continued to decline with worsening developmental delay and memory loss. one year later, at age 12 years, he had a recurrent episode of ams with repeat negative infectious workup including blood and urine cultures, respiratory virus pcr including atypical viruses, csf culture including acid fast bacillus and fungi, cryptococcal antigen, viral encephalitis panel by pcr, and serum pcr for ebv and hhv-6. eeg at this time showed left hemispheric epileptogenic potential, consistent with seizure activity. his presentation, at age 13 years, was notable for right-sided hemiplegia with facial numbness, dysarthria, nausea, and fever. he was found to have anello virus on pcr of csf, abnormal left temporal region on eeg, and global atrophy with stable, diffuse generalized volume loss on mri. he was diagnosed with occult anello virus-induced encephalitis with hemiplegic migraine and discharged on valproate. discussion: here we present the first reported case of anello virus detected by pcr in a cd40-ligand deficient male with neurocognitive manifestations, attributed primarily to hemiplegic migraine. given the anello virus prevalence and relatively avirulent character, it is presumed to be unlikely culprit for encephalitis; however, the significance of this finding is as yet unknown. this case highlights diagnostic challenges in immunodeficiency: infection may go undetected by standard diagnostic techniques; however, the significance of infections identified with advanced techniques may not yet be understood. background: henoch-shã¶nlein purpura (hsp) is an iga-mediated small vessel vasculitis that presents with a tetrad of abdominal pain, arthritis, glomerulonephritis, and purpura. hsp is typically a selflimiting disease of childhood following a viral illness. there is no universal treatment for patients with chronic or recurrent hsp. we report a chronic refractory case of hsp that was successfully treated with a tumor necrosis factor inhibitor (tnfi), etanercept. etanercept functions as recombinant protein that consists of a tnf-alpha receptor ligand-binding region that links to the fc portion of human igg. it is currently approved for use in 5 diseases: juvenile rheumatoid arthritis, rheumatoid arthritis, ankylosing spondylitis, plaque psoriasis, psoriatic arthritis. tnfi are categorized into two broad categories, recombinant receptors (etanercept) and neutralizing antibodies (ex. infliximab and adalimumab). there have been prior case reports of hsp associated with tnfi agents during the treatment of other autoimmune conditions in the adult population. to our knowledge, there have been 3 prior etanercept related hsp reports, one report associated with adalimumab, and one with infliximab. however, there has been no prior report of etanercept use successfully treating chronic refractory hsp. case presentation: a 16-year-old native american male with 3 year history of chronic hsp, hla-b27 positive, and enthesitis related arthritis who was initially treated with steroids, sulfasalazine and methotrexate for symptoms of joint pain and purpura. his iga level was 545 mg/dl prior to therapy. despite treatment for one month of steroids, eight months of sulfasalazine exclusively and eight months of methotrexate and sulfasalazine, he continued to have persistent purpura on bilateral extremities without improvement. he was subsequently initiated on etanercept 50mg weekly and methotrexate was discontinued. approximately one month later, his rash significantly improved. his rash and joint pain recurs when he misses a dose of etanercept. punch biopsies were taken 3 months after initiation of etanercept. the biopsies of a lesion from his left arm showed early leukocytoclastic vasculitis and from his left leg showed weak granular deposition of iga, igm and c3 within vessel walls. there is controversy whether this is a true iga vasculitis. however, we believe that his clinical presentation and the deposition of iga and c3 within blood vessel walls seen on biopsy correlates with chronic henoch-shã¶nlein purpura. conclusion: there is no standard treatment of chronic hsp, but there are reports of benefit with nsaid and corticosteroids. per our literature review, there are no prior reports of etanercept use in the treatment of chronic hsp. tnf inhibitor, etanercept should be considered as a treatment for chronic refractory hsp in the pediatric population as it has showed rapid resolution of purpura in this case report. further studies of etanercept in the treatment of chronic hsp should be conducted given the controversial literature of anti-tnf ab induced hsp during the treatment of other autoimmune diseases. although clinical manifestations of iron overload appear to be quite uncommon in patients who are heterozygous carriers of hfa mutation, we present cases that appear to suggest an increased risk non allergic rhino-sinusitis. case report: we present a 66 year old gentleman with perennial colored rhinorrhea, with facial pressure and tenderness, constant post nasal drip, dry cough and bilateral congestion that had been going on for the past several years. he also had a frequent urge to clear his throat and had frequent episodes of sore throat despite having no history of gerd or lpr. he reported to have multiple sinus infections every year that would progress to pneumonia and eventually require long courses of oral antibiotics. all started in his 40s intensified in the recent past. he had 3 other siblings; one died in his 40s due to liver complications of hh and had a carrier sister and brother with a hx of sino nasal problems exactly similar to the patients. his exam was remarkable for bilateral narrowed nasal passages and moderate edema of the mucosa. his rhinolaryngoscopy showed significant edema and purulent drainage, most notably from bilateral middle meati. his skin test was negative. his cbc showed a wbc count of 6.7/ml with 2% eosinophils and his immunoglobulin panel showed an iga of 236 mg/dl, igg of 1190 mg/dl and ige of 31 mg/dl. patient was placed on alkalol sinus rinses and azelastine nasal spray, which he reported to work pretty well. he left for costa rica and is expected to return back with his siblings to a&i clinic in the coming months. discussion: hh is one of the most common inherited disorders in people of northern european descent with an incidence of 1:200 and carrier rate of 1:10.. most affected hh patients are homozygous for the mutation designated c282y at the hfe gene located at the 6th chromosome. unlike hereditary hemochromatosis, clinical manifestations of iron overload appear to be quite uncommon in patients who are heterozygous carriers. hh patients are at risk for a number of infections with bacteria whose virulence is increased in the presence of excess tissue iron. hh is also a risk factor for acute fulminant frs . here the mechanism is postulated to be due to quantitative or qualitative neutrophil defects as this condition is mostly seen in patients with dm, aplastic anemia, and can happen in patients undergoing antineoplastic chemotherapy. no known increased susceptibility for infections through either mechanism is postulated for patients with the heterozygous carrier state. here we present 3 hh carrier patients who present with recurrent rhinosinusitis with no allergen sensitizations and normal ige levels. since most fungal immunity is at the tissue level and is cytokine driven, it can be speculated that increased tissue levels of iron might interfere with mechanisms of innate immunity. chief, human immunological diseases section, laboratory of clinical immunology and microbiology, niaid, nih, bethesda, md background: dedicator of cytokinesis 8 (dock8) mutations are associated with a combined immunodeficiency disorder marked by atopic features, infectious susceptibility with a striking preponderance of cutaneous viral disease, and a risk for the development of malignancy including lymphoma. almost all cases can be diagnosed by documentation of the loss of dock8 protein expression. methods: we describe a 22-year-old male with a diagnosis of pre-b cell acute lymphoblastic leukemia (all) followed by epstein-barr virus (ebv) associated diffuse large b cell lymphoma (dlbcl). compound heterozygous mutations in dock8 were documented following the completion of whole exome sequencing (wes). the pathogenicity of the variants was assessed. flow cytometric quantification of intracellular dock8 protein was completed. dock8 protein function was assessed by evaluating the morphology of patient lymphocytes when migrating in a 3d collagen matrix. results: a concern for a primary immunodeficiency was raised due to a history of recurrent otitis media which began at 12 months of age. by 4 years of age, numerous warts were noted on his fingers; however, they were transient for a duration of only 2 years. no atopic features were appreciated. at 15 years of age, a diagnosis of pre-b cell all was made. during all therapy, infectious complications were severe including an intestinal perforation, osteomyelitis, and sepsis. at 22 years of age, still in an ongoing remission from his all, an incidental finding of a lung nodule led to a diagnosis of ebv-associated dlbcl. during therapy, however, infectious complications were again severe including a soft tissue infection and sepsis. wes was performed and compound heterozygous mutations in dock8 (c.1128_1132del and c.4474-1g>c) were documented. flow cytometric quantification of intracellular dock8 protein was normal when compared to a normal control. nevertheless, additional functional assessment of dock8 protein was completed. when migrating through a 3d collagen matrix, 45% of the patient lymphocytes studied demonstrated abnormal elongation (stretch ratio > 8 defined by length/width) compared with 10% of lymphocytes from a normal control. he is being evaluated for hematopoietic stem cell transplant. conclusion: autosomal recessive mutations in dock8 are a rare cause of a combined immunodeficiency marked by atopic features, infectious susceptibility with a striking preponderance of cutaneous viral disease, and a risk for the development of malignancy including lymphoma. here, pre-b cell all followed by the development of a subsequent malignant neoplasm (ebv-associated dlbcl) led to the discovery of dock8 deficiency. hence, as our case underscores, for rare instances of high clinical suspicion despite normal dock8 protein expression, additional functional testing is crucial to make a definitive diagnosis and plan treatment. understanding the spectrum of dock8 mutants and their phenotypes will improve our understanding of dock8 deficiency. background: autosomal dominant hyperimmunoglobulin e syndrome (ad-hies) is a rare primary immunodeficiency caused by heterozygous loss-of-function mutations in the signal transducer and activator of transcription 3 (stat3) gene. ad-hies classically characterized by recurrent cold staphylococcal abscesses, pneumonia, eczema, and an elevation of ige level. other additional clinical manifestations of hies have been recognized including skeletal dysplasia (scoliosis, pathologic fractures, delayed dental deciduation), pneumatoceles, coronary-artery aneurysms, brain lesions, and chiari malformations. objective: to describe a unique case of abdominal abscesses in a patient with ad-hies. method: a 22-year-old female with known ad-hies (c.1144 c>t (p.arg382trp)) and a complicated history of early pneumococcal pneumonia and meningococcemia resulting in bilateral amputation below the knees along with loss of several digits, presented for evaluation of skin infection. she had a history of recurrent staphylococcal skin abscesses and presented with inability to use her prostheses due to pain from inflammation around her amputation sites. she underwent imaging and was found to have bilateral extremity abscesses with an associated osteomyelitis of her l tibia (which was found to be mrsa after incision and drainage). while receiving intravenous antibiotics for her osteomyelitis, she developed intractable abdominal pain. imaging showed a thick-walled, multi-septated, paranephric abscess as well as several smaller abscesses scattered throughout her abdomen. she underwent multiple drain placements and drainage of retroperitoneal fluid collections via interventional radiology (ir). purulent fluid from the abdominal abscess drainage grew mrsa. the patient continued to have re-accumulation of abscesses despite multiple drainages. repeat imaging noted increased paranephric abscesses which were not communicating with drains. given lack of response to several ir-placed abdominal drains and to 6 weeks of intravenous antibiotics, she had an open surgical washout with minimal improvement. hospital course was further complicated by development of a left lower lung lobe consolidation and sub-segmental pulmonary embolism necessitating treatment with heparin. finally, after several weeks of escalating antimicrobial therapy and with additional drain placements, the retroperitoneal abscesses started to recede. repeat abdominal imaging several months later while asymptomatic revealed slow but continuing resolution of the abscesses. conclusion: the present case raises awareness of an unusual location for infection in a patient with ad-hies. although the majority of complications of ad-hies are sinopulmonary and skin infections, recalcitrant intra-abdominal abscesses should be considered in the differential of infections in hies. introduction/background: the recent epidemiologic studies have revealed that primary immunodeficiencies (pids) are more common than previously thought. however, there are very few data on epidemiology of pids in korea. objectives: we attempted to estimate the pid epidemiology and disease burden in korea and provide the background information for pid registry for future. methods: to review the previously reported scientific studies, pubmed, koreanmed, google scholar were searched. any studies on pids reported in scientific journal (korean or international) from january 2001 to november 2018 were searched. both korean and english reports were searched. diagnosis for pid was categorized from group i to group xi according to 2017 iuis phenotypic classification. study period was divided into two periods: period 1 from 2001 to 2005 and period 2 from 2006 to 2018, because there was a multicenter study to estimate pid epidemiology from 2001 to 2005. in addition, the number of pid patients and the cost for care were estimated among patients who requested reimbursement to health insurance review and assessment service (hira) korea for one year in 2017. results: a total of 334 pid patients were identified in 75 reports. one hundred and ninety-nine patients (20 reports) and 135 patients (55 reports) were found in period 1 and period 2, respectively. the pids were reported in 11 patients for immunodeficiencies affecting cellular and humoral immunity, 23 patients for combined immunodeficiency with associated or syndromic features, 143 patients for predominantly antibody deficiencies, 33 patients for diseases of immune dysregulation, 113 patients for congenital defects of phagocyte, 1 patient for defects in intrinsic and innate immunity, 4 patients for auto-inflammatory disorders, 6 patients for complement deficiencies, and none for phenocopies of pid. from hira reimbursement data, the number of pid patients were 42 for combined immunodeficiency, 486 for predominantly antibody deficiency, 47 for common variable immunodeficiency, 135 for functional defect of neutrophils, 238 for immunodeficiency associated with other major defects, 272 for other immunodeficiencies. a total of 1,220 pid patients were treated for 14,316 days and $3,351,678 was reimbursed in 2017. conclusions: we performed a systematic review on published studies for pid in medical journals and national open data system of hira to estimate the pid disease burden for the first time in korea. to obtain more information on true pid epidemiology and disease burden in korea, a national multicenter study for pid registry is required in the future. micro-thrombocytopenia is one of the most serious challenges for wiskott-aldrich syndrome (was) and x-linked thrombocytopenia (xlt) patients. thrombocytopenia leads to severe, potentially life-threatening, bleeding episodes, which require frequent transfusions and account for 23% of deaths in patients experiencing was mutations. the gold standard treatment for was patients is hematopoietic stem cell transplantation (hsct) from an hla-identical donor but more recently a number of gene therapy (gt) trials in europe and usa showed promising results. in particular, it has been shown that was patients receiving lentiviral mediated gt, consisting of autologous cd34+ cells transduced with lentiviral vector encoding the human was gene under the control of the endogenous promoter, in combination with a reduced intensity conditioning regimen, have a significant increase in platelet (plt) counts. even though plt counts do not reach normal levels, treated patients decreased the severity and frequency of bleedings. here, in a cohort of 4 xlt and 16 was patients, fifteen treated with gt, the plt phenotype and function were analyzed by electron microscopy, flow cytometry and proteomic profile. the aim of the project is to assess the presence of plt defects in was untreated patients and the impact of gt treatment on the correction of plt behavior. we demonstrate that plts of untreated was patients have reduced size and abnormal ultrastructure along with hyperactivated phenotype at steady state, showing increased expression of cd62p, activated iib3 integrin and cd40l; conversely, activation response to agonist and aggregation capacity are both decreased. analyzing plt samples isolated from treated patients, we found that gt restores plt size and ultrastructure very early after treatment and reduces the hyperactivated phenotype proportionally to was protein (wasp) expression and follow-up length. plts isolated from gt treated patients showed a normal activation response to agonists and restored aggregation capacity in 5 out of 7 analysed patients. by proteomics, various protein pathways were found downregulated in untreated plt samples, mainly involving cytoskeletal-rearrangement proteins, integrins, signal transduction molecules, vesicles-transport proteins; additionally, decreased metabolic capacity were observed. these results are in line with the functional defects observed in plts in terms of activation and aggregation. conversely, the expression of protein-pathways found downregulated in untreated patients is comparable to healthy controls in gt-treated plt samples, reflecting the amelioration of plt phenotype and function. overall, our study highlights the coexistence of multiple defects in the activation and aggregation responses occurring in was patient plts in absence of wasp. gt was able to normalize the plt proteomic profile followed by consequent restoration of plt ultrastructure and phenotype, suggesting gt is responsible for the observed reduction of bleeding episodes in treated patients. introduction: pik3cd is an autosomal dominant genetic disorder of the immune system that results in persistent activation of pi3k. signaling through pi3k is essential for immune cell regulation of metabolism, migration, proliferation and differentiation, leading patient to present with lymphadenopathy, immunodeficiency and senescent t cells. the mutated protein causes t cells to over activate and mature too quickly leading to their death, this over activation also blocks the maturation of b cells. case presentation: a 51-year-old female with a childhood history of failure to thrive, asthma, chronic rhinitis and common variable immunodeficiency on intravenous immunoglobulin replacement, was seen in immunology clinic to establish care. she reported frequent episodes of pneumonia and bronchitis in her childhood. her family history was significant for family members with leukopenia, but no diagnosed immunodeficiency. patient had 1 son who did not report symptoms concerning for immunodeficiency. physical exam was within normal limits with no lymphadenopathy. laboratory examinations exhibited normal iga (185 mg/dl), igg (800 mg/dl), and igm (100 mg/dl). while flow cytometry showed normal absolute cd3 687 (570-2400 cells/ul), cd4 (540 cells/ul), nk cells (151 cells/ul), cd19 (179 cells/ul), cd45ra (160 cells/ul), cd45ro (311 cells/ul), cd2 (757 cells/ul), and hla-dr (173 cells/ul), nonswitched memory cells (9 cell/ul) and class-switched memory cells: (15 cells/ul). (4-62 cells/ul). vaccine response was not pursued as patient had been on ivig. genetic testing was pursued, and revealed a mutation in pik3cd gene, specifically a mutation in the c.2320g>a; p.val774met variant (rs370932461). this mutation though seen in databases, is not currently reported in medical literature as associated with this condition. based on these, ct chest was ordered to screen for bronchiectasis, adenopathy and lymphoma. ct showed no cardiopulmonary disease or adenopathy, but did show an incidental adrenal mass which is now being worked up. while the pattern of inheritance of this mutation is autosomal dominant, her son is asymptomatic and testing of her son has not been pursued, though it was advised for her cousins given history of leukopenia. patient has continued on igg replacement therapy. conclusion: recent publication by the clinical immunology society suggests consideration for next generation sequencing when it can affect future family planning or it has treatment and prognostic implications. this case highlights all aspects of the importance of genetic testing as part of the diagnosis of cvid, since it can affect progeny, it offers the possibility of treatment with immune modulating agents and has implications on screening, since patients are at increased risk for malignancies. background: abnormal v(d) j recombination activity in patients with mutations in the recombination-activating genes 1 and 2 (rag1/2) results in markedly reduced usage of distal vand j genes at the t cell receptor alpha (tra) locus. mucosa-associated invariant t (mait) cells express a semi-invariant t cell receptor containing the distal trav1-2 gene. mait cells can be identified by flow cytometry using a mab directed against valpha 7.2, which recognizes the product of the trav1-2 gene. by performing high throughput sequencing (hts) of tra rearrangements and flow cytometry, we have confirmed lack of t cells using distal valpha genes in patients with known rag mutations. we now report that flow cytometry with mab against valpha 7.2 successfully identified rag deficiency in two patients with an atypical presentation. methods: tra rearrangements were analyzed by hts using gdna from sorted t cell subsets from rag-mutated patients and healthy donors. distal valpha usage was measured in whole blood by flow cytometric analysis with an anti-valpha 7.2 antibody. rag mutations were detected by sanger sequencing. patients were enrolled in niaid protocol 18-i-0041. results: hts of tra rearrangements revealed lack of distal trav and traj gene usage in patients with rag1/2 mutations. the presence of circulating mait cells in controls and patients with known rag1/2 mutations and various clinical phenotypes was analyzed by flow cytometry using mab against valpha 7.2. we found a virtual lack of valpha 7.2 expression in rag mutated patients (<0.5%) compared to controls (2-8%) . we used the valpha 7.2 assay to test two patients with unknown immunodeficiency manifesting as skin granulomas and autoimmune cytopenia, and found nearly absent expression (0.14% and 0.08%). targeted sequencing of rag1/2 revealed that both patients were compound heterozygous for rag1 mutations: p.r112h/p.c328y and p.r410w/p.r507q, respectively. conclusions: patients with mutations in rag1/2 demonstrate a skewing of their tcralpha repertoire. the reduction in recombinase activity in these patients does not allow for rearrangements of the most distal valpha segments. rapid identification of patients lacking valpha 7.2+ t cells by flow cytometry may prompt sanger sequencing and identification of rag1/2 mutations in a matter of days. this assay represents a simple but powerful tool to reduce the cost and time associated with other analysis methods. acknowledgements: supported by dir/niaid/nih. director, centro de inmunologã­a clã­nica dra.bezrodnik y equipo introduction: the fate of effector t cells is strongly dependent on the expression of bcl-6 or blimp-1, which are inhibited reciprocally through a complex signaling pathway. several studies have shown that bcl-6 is a key transcription factor for differentiation towards the follicular helper t cells (tfh) lineage able to collaborate with b lymphocytes (bl). on the contrary, the transcription factor blimp-1 is highly expressed in t lymphocytes th1, th2 and treg, thus regulating the differentiation towards tfh. materials and methods: whole fresh blood and peripheral mononuclear cells from a patient with homozygous mutation in stat5b were analysed by flow cytometry. analysis of ctfh (cd4+cd45ra-cxcr5+), ctfh1 (cxcr3+), ctfh17 (ccr6+), ctfh2 (cxcr3-ccr6-), naã¯ve bl (lb igm+igd+cd27-), memory (mbl) (lb igm+ igd-cd27+), switched (mbl-sw) (igd-igm-) and plasmablast (pbc) (cd27+cd38++) cells was performed. immunoglobulins were measured in serum. results: the patient with stat5b deficiency showed increased values of ctfh (38%) (healthy donors p10-p90: 7,9-17,8 %) that presented an activated phenotype (icos+ and pd-1+) with a skewed to a th17 profile (ccr6+), consistent with her hipergammaglobulinemia and the marked and sustained increase in the switched mbl and pbc subpopulations in peripheral blood over the years. discusion: this immunological phenotype described in the patient with stat5b deficiency could explain in part the pathophysiology of the autoimmune disorders. this patient (as well as the other two patients with mutations in stat5b previously described by our group), have had chronic hypergammaglobulinemia, autoantibodies and consequently autoimmune processes (psoriasis, hypothyroidism, eczema, alopecia and celiac disease, among others). we believe that the link between this clinical symptomatology and the molecular defect relies in the fact that the absence of stat5b promotes a greater expression of bcl-6, which generates a bias towards the production of ctfh cells, that give rise to a greater activation of lb, generation of lbm and plasma cells (dysregulation in the cg), events that manifest as hypergammaglobulinemia and autoimmunity. in summary, we provide promising evidence of the mechanisms that lead to autoimmunity in this type of patients that could also be a consequence of the defect in the regulation of gc, highlighting the crucial role of stat5b in the humoral immune response and maintenance of the tolerance of the immune system. background/introduction: the term primary immunodeficiencies (pid) encompasses a phenotypically and genetically diverse group of conditions. genetic testing for these conditions can guide treatment, reduce morbidity and mortality, allow for genetic counseling, and identification of additional at-risk family members. however, this testing can be complicated by a number of factors, including pseudogenes, high homology, methodology limitations, and the heterogeneous nature of pids. methods: mayo clinic laboratories launched their first set of nine pid next generation sequencing (ngs) tests approximately one year ago. these tests include one single gene assay for gata2 deficiency and eight targeted next generation sequencing panels for: atypical hemolytic uremic syndrome (ahus), autoinflammatory disorders, b-cell disorders, monogenic irritable bowel disease (ibd), phagocytic defects, severe combined immunodeficiencies (scid), and severe or cyclic neutropenia. herein we summarize our first year of experience with these ngs tests, with a focus on the eight targeted panel tests. results: from march 2018 through november 2018 we performed testing for 341 cases. our highest volume of tests was for the ahus panel (127/341 cases, 41%). a variant was reported in 76/341 cases (22.29%). these variants included variants of uncertain significance, likely pathogenic variants and pathogenic variants. the indication with the highest percentage of cases where a variant was reported was scid (9/13 cases, 69.23%). the number of cases that were considered solved, where the genotype likely explains the patients phenotype, varied widely by indication. twenty cases were found to have a pathogenic or likely pathogenic variant or variants; however 2/20 cases were heterozygotes for an autosomal recessive condition and were not considered solved cases. the panel with the highest percentage of solved cases is our scid panel (4/13 cases, 30.77%). conversely, we have yet to solve an autoinflammatory, irritable bowel disease, or telomere defects case; however 20% of cases in each of those three panels have had a variant of uncertain significance reported. we hypothesize that one of the reasons for the low detection rate for these three panels is inappropriate test orders. we are also actively looking for ways to update all 8 panels to increase detection rates and clinical utility, for example expanding the gene list of our ibd panel, including large deletion/duplication detection, and including ncf1, a difficult gene to capture by ngs, on the phagocytic panel. finally, we present the molecular findings from a number of interesting cases that were solved using our targeted ngs panels. conclusions: the launch of our pid ngs tests in march of 2018 has allowed us to aid patients by confirming diagnoses and providing molecular diagnoses that will enable more accurate genetic counseling and risk assessment. we have also uncovered areas for improvement, both on the clinical side: provider education is important to enable better identification of patients who can benefit from molecular genetic testing for pids, and on the laboratory side: introduction of more expanded panels and additional methodologies. the progressive decrease of red blood cells, platelets or neutrophils via a self-directed immune process is jointly termed as autoimmune cytopenias. while autoimmune cytopenias, including autoimmune hemolytic anemia (aiha), immune thrombocytopenic purpura (itp), and autoimmune neutropenia (an), are a common presentation of autoimmunity in the general population, they are particularly frequent and can appear as the first sign in patients with primary immunodeficiencies (pids). possible causes of cytopenia in pids comprise mainly immune dysregulation, bone marrow failure (bmf) and myelodysplasia. our goal is to investigate possible immune mediated mechanisms underlying chronic cytopenia in children in order to achieve an early diagnosis and consequently offer timely and appropriate therapy. we selected 24 patients affected by chronic cytopenia, evaluated with immunophenotyping by flow-cytometry; data were subjected to multivariate analysis by principal component analysis (pca). next generation sequencing (ngs) analysis of genes frequently implicated in pids was performed. among the patients, 5 were affected by bone marrow failure, of which 2 were diagnosed with fanconi anemia and severe congenital neutropenia; 12 were affected by immune-mediated cytopenia and 7 by idiopathic cytopenia. the immunephenotyping showed a typical pattern of cd8 t cell subpopulations expression in patients compared with healthy donors with an increase of naã¯ve t cells and a reduction of central memory (cm) and effector memory (em) t cells levels. we observed a decrease in total b cells, b switched and b memory cells and an increase in cd21low cells. pca showed an overlap between groups, however it revealed a peculiar trend of some single patient, suggesting the pathway involved in immune defect. preliminary results from ngs studies revealed genetic variations in genes previously associated with pids in 10 out of 11 patients investigated. in particular we identify one patient with a mutation in fas, one with a mutation in aire and one with a mutation in ikaros. concerning the remaining patients further studies are ongoing to validate the pathogenicity of the genetic variations. pca is a very effective tool to analyze several parameters at the same time, highlighting patients whose phenotype shows the main peculiarities. the presence of specific lymphocyte subpopulation patterns can be important indicators of immune-mediated cytopenias and helpful signs of specific pids that should promptly be investigated with genetic analysis. the rapid of discovery of novel, monogenic primary immunodeficiencies has been made possible by the broad availability of clinical whole exome sequencing (wes). however, clinical wes has major shortcomings that should be understood by practicing immunologists. focusing on the 2017 iuis list of~330 monogenic primary immunodeficiency genes, we show here limitations in coverage that could significantly impact clinical interpretation. on the agilent whole exome capture kit, the most common wes platform, there are a number of genes with exons that are poorly covered. specifically, there are at least 94 genes with less than 100% exonic coverage, 26 with less than 99% coverage and 5 with less than 90% coverage (e.g. ikbkb, ncf1, taci, unc93b1 and tbx1). beyond this challenging technical issue, there are more subtle issues as well. these include the presence of pseudogenes in at least 17 of our genes (e.g. ak2, c1qbp, cd46, cftr, cr2, msn, ncf1, ncstn, ikbkg, nhp2, pms2, pten, rnaseh2c, rps, sbds and was), which can make accurate sequencing very challenging. finally, there are many known causative intronic (e.g. btk, ctla-4, wasp) and copy number variant mutations (e.g. rag1 and xiap) as well as large deletions (e.g. dock8) that we cannot expect to be optimally covered using wes. this list of genes requires consideration even with a negative exome and may require additional approaches including whole genome sequencing, sanger sequencing, cnv arrays and/or long-read ngs sequencing. wes is a powerful genomic diagnostic tool, but to avoid missing key diagnostic insights using these alternative approaches may be critical when certain genes are in the differential diagnosis. going forward, as pid phenotypes continue to broaden, these issues remain fundamentally important even if these genes are not obviously implicated in a given clinical phenotype. more physicians are utilizing targeted genetic panels to reach a definitive diagnosis for their patients with immunodeficiency. however, this increase in testing also has led to the discovery of many more variants of uncertain significance (vus) in the genes tested. these findings can often leave the patient and the physician with more questions than answers. we present a patient with recurrent infections found to have multiple variants of uncertain significance in several genes associated with primary immunodeficiency. a 13-year-old female who was diagnosed with crohns disease at age 9 after intestinal perforation and jejunal resection experienced two discrete episodes of epstein barr virus (ebv) meningoencephalitis and septic shock. the first episode was diagnosed when patient had fever and altered mental status and occurred prior to her crohns disease diagnosis and the second episode was complicated with altered mental status, disseminated intravascular coagulation (dic) and hypotension requiring picu admission. aside from these two major infections, the family denied any other infections requiring antibiotics in the last 5 years and reported a remote history of repeated streptococcal pharyngitis that have not recurred. immunology was consulted at the time of the second episode of meningoencephalitis and work up was mainly unremarkable with normal immunoglobulins, adequate vaccine response to hib, tetanus, diphtheria, rubella, measles and pneumococcus (18 out of 22 protective titers). she had normal t cell numbers with slightly decreased natural killer numbers for age. neutrophil studies showed normal dihydrorhodamine (dhr) analysis, glucose-6-phosphate dehydrogenase levels and myeloperoxidase (mpo) stain. commercial testing of her toll like receptors (1) (2) (3) (4) (5) (6) (7) (8) showed normal function. invitae primary immunodeficiency panel demonstrated a heterozygous variant in nod2 (c2.104c>t; p.arg702trp) as well as heterozygous variants of uncertain significance in il7r (c.662g>t; p.ser221ile) and tlr3 (c.889c>g; p.leu297val). the patients nod2 variant is known to be associated with an increased risk for crohns disease. even with our patients presentation with recurrent severe viral infections and ibd, it is not immediately clear how these genetic results explain the pathology. innate immune defects probably contribute to her presentation and it is currently unclear if and how the combination of multiple genetic variants has left her immunologically vulnerable. we use this case to demonstrate that even when genetic testing does not elucidate a clearcut diagnosis of primary immunodeficiency, it can still provide helpful insight into a patients underlying immune phenotype. introduction: xiap deficiency is a rare primary immune deficiency characterized by hemophagocytic lymphohistiocytosis, recurrent fever and inflammatory syndromes, inflammatory bowel disease, hypogammaglobulinemia, recurrent infections, and other manifestations. loss of xiap results in abnormal tnf receptor signaling and nlrp3 inflammasome actvity which leads to dysregulated production of il-1beta and il-18. we hypothesized that suppressing the nlrp3 inflammasome with either targeted deletion or pharmacologic inhibition would suppress abnormal production and secretion of inflammatory il-1beta and il-18. methods: bone marrow derived macrophages (bmdms) from control, xiap-deficient, and xiap and nlrp3 double knock-out mice were derived with 1 week of culture in l929-cell conditioned media. bmdms were stimulated with a variety of tlr agonists or tnf-alpha, with or without a variety of inhibitors including the nlrp3 inhibitor mcc950, the cathepsin b inhibitor ca-074, and quercetin, which is a natural flavonoid (antioxidant) found in many fruits and vegetables, and available as a nutritional supplement. il-1beta, il-18, and tnf-alpha were measured in supernatants by elisa, and cell death was evaluated by flow cytometry using pi exclusion. results: as expected, bmdms from xiap deficient mice had markedly increased tlr-agonist-or tnf-alpha-induced il-1beta production compared to normal bmdms. genetic deletion of nlrp3 and the pretreatment of cells with the nlrp3 inhibitor mcc950 greatly reduced abnormal il-1beta production; residual production of il-1beta could be inhibited by caspase-8 inhibition. pre-treatment of cells with the cathepsin b inhibitor ca-074 also decreased cytokine production but was toxic at higher concentrations. quercetin reliably abrogated il-1beta, and also il-18. quercetin was found to inhibit priming of the nlrp3 inflammasome (decreased upregulation of pro-il1beta and nlrp3) and also decreased tnf-alpha secretion following tlr agonist stimulation. conclusion: quercetin suppresses the nlrp3 inflammasome and may be a promising therapeutic option for patients with xiap deficiency. it prevents il-1beta and il-18 secretion. it is a particularly appealing option given that it is a naturally occurring antioxidant, has a great safety profile, and is readily available as a nutritional supplement. human studies are needed. recently, single cell rna sequencing (scrnaseq) analysis in mice has disclosed an unexpected complexity of thymic stromal cells, and medullary thymic epithelial cells (mtecs) in particular. however, the developmental origin, hierarchy, and function of these subpopulations remain ill-defined. moreover, although cortical tecs (ctecs) are thought to represent a more homogeneous population, their characterization has been largely restricted to the adult thymus. we have previously shown that impaired lymphostromal cross-talk in the thymus of patients with combined immunodeficiency (and of corresponding mouse models) is associated with abnormalities of thymic architecture and tec maturation. here, we sought to compare tec distribution and gene expression in wild-type (wt) and in mice carrying rag1 hypomorphic mutations observed in patients with combined immune deficiency and immune dysregulation. methods: multi-color flow cytometry and scrnaseq were used to analyze composition and distribution of ctec and mtec subpopulations in wt and rag1 mutant mice at various weeks of age (niaid animal protocol: lcim-6e). results: we observed that rag1 mutant mice have an excess of ctecs, and that their mtec compartment is predominantly represented by cells with high levels of mhc class ii (mhc-ii) expression, recapitulating the phenotype of neonatal wt thymi. while mhc-iihi mtecs are thought to represent a minor fraction of mtecs in adult wt mice and include mature aire+ cells, a relative abundance of mhc-iihi mtecs is observed also at neonatal age, where they are thought to represent immature mtecs. to define more precisely tec maturation, we performed scrnaseq on sorted cd45-epcam+ cells, and identified 8 and 10 distinct clusters of tecs in wt and rag1 mutant mice, respectively. a large proportion of cells in rag1 mutant mice could be ascribed to the ctec compartment, confirming our previous flow cytometry and histopathology results. furthermore, scrnaseq analysis also disclosed a different distribution of mtec subsets in wt and rag1 mutant mice. to address the hypothesis that this difference in ctec and mtec abundance and subset distribution may reflect different maturation stages in tec development in wt and rag1 mutant mice, we will perform lineage tracing and transplantation experiments, and we will also extend tec scrnaseq analysis to wt and mutant mice of embryonic and neonatal age. in parallel, to evaluate the contribution of thymocyte maturation in shaping the stromal populations, scrnaseq will be performed on thymocytes. conclusions: we have further refined the complexity of tecs, and shown that impaired development of t cells in combined immune deficiency (as exemplified by rag1 mutant mice) has profound effects on the composition and maturation of tecs and may thus contribute to abnormalities of immune tolerance that are often associated with these conditions. the advent of next-generation sequencing (ngs), with the development of whole-exome sequencing (wes) in particular, has allowed the identification of unknown genetic lesions for many diseases and the implementation of specific therapeutic strategies. primary immunodeficiencies (pids) are a group of rare diseases which have benefited from ngs, with the discovery and molecular characterization of previously genetically undefined diseases and the identification of novel molecules involved in the regulation of the immune system. pids are often associated with autoimmune disease due to the dysregulation of the immune system as a whole. the clinical phenotypes are heterogeneous and often overlapping. while a monogenic cause of disease has been identified in a most subsets of patients, the recent application of whole-genome sequencing has found that a polygenic cause is likely. our aim is to investigate the genetic background of patients with immunedysregulations and autoimmunity and to evaluate the possible pathogenicity of the identified gene variants through extensive functional studies. we select 19 patients with sign of immunedysregulation and autoimmunity, extended immunophenotyping and next-generation sequencing (ngs) analysis of 50 genes frequently implicated in pids was performed. in six of them we identify a single gene as responsible of the clinical feature. in particular, we identify two patients with gain of function mutation in stat3, one patient with a mutation in ctla4, one patient with an activating pik3cd mutation, one with a rag1 mutation and one with a fas mutation. in most of them variants in multiple genes have been detected. interestingly, we find that some genes are recurrently mutated in more then one patient such as was, dock8, casp10, casp8, nfatc2 and fcgr3a. further studies are ongoing to validate the effect of the variations identified. our results strongly suggest that the old hypothesis, based on a single gene mutation as a cause of illness, should be revised in favor of the concept that "is the sum that causes the effect" and that a different point of view on pids now seems inevitable. physician, omni allergy, immunology, and asthma introduction/background: immunoglobulin replacement therapy (igrt) may be optimized to reduce the severity and incidence of infections and potentially delay or abrogate the development of pulmonary complications of primary immune deficiencies. pulmonary complications including bronchiectasis are common in common variable immune deficiency (cvid) and contribute significantly to morbidity and mortality in these patients. it remains unclear whether continued obstructive bronchial changes are a result of repeated respiratory infections, associated inflammation and immune dysregulation, or simply lung-damage that is irreversible by the time therapy is initiated. it has also been suggested that under-treatment in addition to the diagnostic delay may contribute to the development of bronchiectasis in patients with pid. lower serum igg levels with any given dose of immunoglobulin replacement therapy have been demonstrated in patients with bronchiectasis compared to those pid patients without this complication. in addition, earlier studies have shown that greater doses of ig (600 mg/kg/ month) may reduce the frequency and duration of infections and help prevent or slow progression of chronic lung disease. objective: to evaluate the prevalence of bronchiectasis in a cohort of patients with a diagnosis of cvid and identify associated ig dosing patterns and clinical outcomes. methods: data were analyzed from the ideal (immunoglobulin, diagnosis, evaluation, and key learnings) patient registry. this is a prospective, longitudinal registry study of patients receiving ig replacement therapy in the home or ambulatory infusion suite with one national home infusion provider. nursing and pharmacy standard of care forms were collected, and dose, infection rate, and prevalence of bronchiectasis were evaluated in patients with a diagnosis of cvid (icd-10 codes: d83.9, d83.1) results: there were 310 patients in the registry with cvid, 14 (4.5%) of which bronchiectasis was also observed. seventy-nine percent (n=246) of the study population was female, and 50% (n=7) of the cases of bronchiectasis were observed in females. the mean age of the patients with concurrent bronchiectasis was 65â±15.8 at start of care compared to 57â±15.8 in those without this observed bronchial obstruction. most bronchiectasis patients (n=11) received igrt subcutaneously every week with a mean dose of 123.8â±22.8 mg/kg/wk. the mean dose of ig in the 3 remaining patients receiving ig intravenously was 506.8â±82.0 mg/kg/month. the average annual rate of infection in ivig and scig patients with bronchiectasis was 1.6â±1.0 and 2.2â±1.3, respectively, however many were serious bacterial infections. at time of analysis, 7 of the bronchiectasis patients remained active in the registry and 7 had withdrawn. reasons for withdrawal included stopping igrt due to the following: patient decision (n=3), physician decision (n=1) insurance change (n=1), and patient expired (n=2). conclusions: there were 14 documented cases of bronchiectasis in our cohort of cvid registry patients, and dosing patterns aligned with standard doses despite the presence of bronchial obstruction. further studies are necessary to assess evolution of lung damage with respect to ig dosing in patients with cvid. background: activated phosphoinositide 3-kinase syndrome type 1 (apds1) is a combined immunodeficiency resulting from gain-offunction (gof) mutations in pik3cd, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (pi3k). this form of pid is characterized by recurrent respiratory tract infections, susceptibility to herpes virus infections, impaired antibody responses, lymphoproliferation and autoimmunity. previous studies showed that patients with apds1 have b cell defects that contribute to the clinical phenotype. furthermore, these patients display t cell abnormalities, including increased numbers of memory t cells and t follicular helper cells (tfh), reduction of naã¯ve t cells and impaired t regulatory cell (treg) function. whether these t cell abnormalities are also associated with perturbations of t cell repertoire in unknown. objective: we aimed to investigate the effects of increased pi3k signaling on the t-cell repertoire of patients with apds. methods: high throughput sequencing was used to study composition and diversity of t-cell receptor (tra) and t-cell receptor (trb) repertoire in sorted treg, tfh, conventional cd4+ (tconv), and cd8+ t cells from 4 patients with pik3cd gof mutations and healthy controls. results: treg cells of patients with apds1 show restriction of tra and trb repertoire diversity, and increased clonality. no repertoire restriction was detected in tfh, tconv, and cd8+ t cells from the same patients. however, the trb repertoire of treg and cd8+ cells was enriched for the presence of hydrophobic amino acids in position 6 and 7 of the cdr3, a biomarker of self-reactivity. conclusion: these data demonstrate that the t-cell repertoire of patients with apds1 is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition. furthermore, our result support the notion that the pi3k pathway is a key regulator of treg cell development and homeostasis in humans. j clin immunol (2019) 39 (suppl 1):s1-s151 s87 (4), iii. predominantly antibody deficiencies (2), i. immunodeficiencies affecting cellular and humoral immunity (1), vii. auto-inflammatory disorders (2), ix. phenocopies of pid (1) . two non related cases of ataxia-telangiectasia and one case of schimke syndrome (smarcal1 compound heterozygous mutation) were diagnosed in the last year. we observed a wide range of age (we evaluate adult and pediatric population) with a male:female ratio close to 1: immunodeficiency, immune dysregulation, and systemic autoimmunity. clinical diagnosis of these disorders is complicated by overlapping phenotypes. in april 2017, a 207-gene next generation sequencing (ngs) panel inclusive of copy number variation analysis was launched by a commercial laboratory to facilitate clinical diagnosis of primary immunodeficiency (pid), monogenic autoimmunity and autoinflammatory disorders. we assessed the outcomes of genetic testing utilizing this panel on a cohort of pediatric patients with immunohematologic phenotypes evaluated at our tertiary care center during an 18-month period (5/1/17-10/31/18). eligible subjects were evaluated by at least two of three providers from a multidisciplinary pediatric hematology-immunology team, including a hematology physician, immunology physician and a geneticist or genetic counselor. twenty-three patients met inclusion criteria; 20 (87%) were caucasian, 12 (52%) were male with an average age of 11.7 years. the two most common phenotypic diagnoses included cytopenias, single-or multilineage (leukopenia, neutropenia, anemia, thrombocytopenia) primarily attributed to autoimmune causes or hypogammaglobulinemia. five (22%) were given a definitive genetic diagnosis as a result of panel testing, though in two of these cases, the causative mutations were listed as variants of uncertain significance (vus). diagnoses included common variable immunodeficiency due to a pathogenic variant in nfkb2, stat3 multiorgan autoimmunity due to gain-of-function mutation, and familial cold autoinflammatory syndrome due to a pathogenic mutation in nlrp12. biallelic dnmt3b vus were found in a patient whose phenotype and further laboratory studies (including karyotype) were consistent with immunodeficiency-centromeric instability, facial anomalies syndrome. further, a stat3 vus was identified in a patient with multiorgan autoimmunity and his father with hypothyroidism; studies from an outside research laboratory were consistent with gain-of-function with this variant (private communication). an additional three patients had vus identified that were suspected to be related to their phenotype, prompting eligibility for research studies. four (17%) patients had increased risk alleles in nod2, conferring an increased risk of crohns disease. three (13%) patients had pathogenic or likely pathogenic carrier findings warranting genetic counseling. in addition, 47 vus (an average of 2 per patient) thought to be unrelated to phenotype were identified, necessitating further investigation and counseling. the use of an ngs panel in a cohort of pediatric patients with immunohematologic disorders led to a definitive diagnosis in 22% of previously undiagnosed patients and prompted further research investigation in several more. genetic testing also led to the identification of clinically significant carrier findings, risk alleles and 47 vus unrelated to phenotype, necessitating genetic counseling. our experience illustrates the value of genetic testing for diagnosis of immunohematologic disorders, and the importance of multidisciplinary care, including genetic counseling, for the proper evaluation and management of these patients. background: allogeneic hematopoietic cell transplantation (allohct) is curative for primary immune deficiencies (pid). however, many patients lack a fully-matched unaffected sibling, or may have an unknown underlying genetic defect, rendering it undesirable to use related donors. many pid patients have significant comorbidities at the time they are referred to allohct, precluding the use of myeloablative conditioning. the use of alternative donors with reduced-intensity conditioning (ric) has historically led to increased rates of graft failure, graft-versus-host disease (gvhd), and transplant-related mortality (trm). posttransplantation cyclophosphamide (ptcy) as gvhd prophylaxis immunomodulates the graft through the preferential sparing of regulatory t cells and hematopoietic stem cells from its cytotoxic effects, thus allowing for robust donor engraftment that overcomes the hla barrier while effectively preventing severe acute and chronic gvhd. we report the outcomes of two institutions using a ric allohct regimen with alternative donors and ptcy in patients with pid. design: we transplanted 35 pid patients (table 1) using alternative donors and ric, either serotherapy-free (n=21) or alemtuzumab-based (n=14). all patients received ptcy for gvhd prophylaxis on days +3 and +4, either alone (n=3), or combined with mycophenolate mofetil and either sirolimus (n=21) or tacrolimus (n=11). donors included haploidentical family members (n=16), matched unrelated (n=15), and mismatched unrelated (n=4). stem cell source was t cell-replete bone marrow (n=33) or peripheral blood stem cells (n=2). results: the median follow-up is 17 months (range 0.5-8 years). at 17 months, overall survival is 91%, and event-free survival (defined as alive without graft failure) is 83%. the median days of neutrophil and platelet engraftment are 17 (range 14-42) and 28 (range 15-110), respectively. there were 10 patients who developed acute gvhd, grade 1 (n=5) or grade 2 (n=5), and there were no cases of grade 3 or 4 agvhd. seven of eight patients treated with systemic corticosteroids responded, and one was corticosteroid-dependent, then responded to second-line therapy. one patient developed skin-only chronic gvhd, which responded to corticosteroids and puva light therapy. five patients developed graft failure, either primary (n=1) or secondary (n=4), and four were successfully re-transplanted and remain engrafted. one patient with secondary graft failure had autologous recovery and has not required a second allohct given some durable infection control gained during initial engraftment. there were three deaths prior to day 180 due to infection, and one death at 1.5 years secondary to presumed overdose. in ongoing follow-up of engrafted survivors (n=30), evidence of phenotype reversal has been demonstrated in all patients, with complete or ongoing resolution of some or all of their underlying disease manifestations, including infection, transfusion-dependence, autoimmunity, malignancy, and/or immune dysregulation. discussion: we have observed high rates of engraftment, low rates and severity of acute and chronic gvhd, and low trm in 35 patients with pid transplanted using alternative donors, ric, and ptcy-based gvhd prophylaxis. ric allohct with ptcy shows promise for curing pid, and its use minimizes toxicity and widely expands the donor pool, thus allowing us to offer this curative therapy to many more patients with pid. chronic granulomatous disease (cgd) is a primary immune disorder that involves mutations in the nicotinamide adenine dinucleotides (nadph) oxidase complex (deffert, cachat, & krause, 2014) . two-third of cgd cases are caused by loss-of-function mutations in the cybb gene that encodes the gp91pox subunit of the nadph. the increased in patients' life expectancy thanks to progress in diagnosis and management has underlined the burden of inflammatory manifestations occurring independently of infectious agents (dunogue et al., 2017; marciano et al., 2018) . cgd patients develop inflammatory granulomatous disorders, notably colitis, as a consequence of a dysregulated inflammasome activation. the treatment of inflammatory manifestations remains challenging, as it can be associated with an increased risk of infections. thus, understanding the pathophysiological mechanism of auto-inflammation in cgd could help improve the therapeutic arsenal for the management of these manifestations. to reveal the precise pathophysiological mechanism of auto-inflammation in cgd, we have developed a cellular model that reproduces the cgd phenotype in phagocytic cell. through crispr-cas9 gene-editing we generated a thp-1 c e l l l i n e h a r b o r i n g t h e p r e v i o u s l y d e s c r i b e d mu t a t i o n c.90_92delccginsggt (p.tyr30ter) in the cybb gene responsible for gp91phox knock-out by early termination of translation. this cell line recapitulates the phenotype of cgd phagocytes: (i) decreased h2o2 production (ii) and enhanced inflammatory responses after pma stimulation as evidenced by increased il-1, il-6 and tnfa secretion levels (kuijpers & lutter, 2012) . these features were rescued by complementation through lentiviral transduction of a wild type cybb gene. this new model will help us to investigate the auto-inflammation reported in cgd patients and also to propose new therapeutic targets of inflammatory manifestations in this disorder. interleukin-1 (il-1) driven responses. children with irak-4 deficiency are predisposed to recurrent and invasive infections secondary to streptococcus pneumoniae, staphylococcus aureus and other pyogenic bacteria with high mortality rates in early childhood. the frequency and severity of infections is thought to decrease with age due to the acquisition of humoral immunity and immunologic memory, however due to the rarity of the disease, the natural history of this condition beyond early childhood is not well described. objectives: we present three unrelated irak-4 deficient patients with persistent chronic rhinosinusitis with nasal polyposis that developed in childhood. cases: patient 1 is a 15 y/o male with compound heterozygous mutations in irak4 (p.g75afs*14/c.717-1g>t) with a history of recurrent s. pneumoniae osteomyelitis (left hip at age 9 and left knee at age 10) and c. septicum sepsis at age 9 following acute bowel perforation. additionally, he experienced recurrent aom during infancy and recurrent uti since age 9. despite prophylactic antibiotics and ivig, he has had recurrent polymicrobial (mrsa, s. pneumoniae, h. influenzae, p. aeruginosa, a. fumigatus) rhinosinusitis with nasal polyposis since age 4 refractory to medical management requiring surgical intervention and prolonged courses of iv antibiotics. patient 2 is an 11 y/o female with homozygous deletions (exons 10-12) in irak4 with a history of ruptured appendicitis complicated by pseudomonas abscess and bacteremia at age 2, culturenegative sepsis with septic arthritis and osteomyelitis of the right leg at age 3, and septic shock secondary to mssa bacteremia complicated by rhabdomyolysis and dic at age 5. she has a history of chronic rhinosinusitis, and despite ivig and prophylactic antibiotics, she developed polymicrobial (h. influenzae, b. fragilis) rhinosinusitis with associated nasal polyposis pending surgical management. patient 3 is a 10 y/o female with homozygous mutations in irak4 (q293x/q293x on exon 8) with a history of s. pneumoniae meningitis at 3 months, m. catarrhalis epiglottitis and neck cellulitis at 4 months, rsv bronchiolitis at 6 months, enterococcus bacteremia at 8 months, s. pneumoniae sepsis at age 2 and streptococcus lymphadenitis at age 9. despite ivig and prophylactic antibiotics, she developed recurrent polymicrobial (h. influenzae, b. fragilis, mssa, v. cholera, p. aeruginosa, a. fumigatus) rhinosinusitis refractory to medical management requiring surgical intervention and iv antibiotics. conclusions: in our centers experience, irak-4 deficient patients continue to suffer from infectious complications, most prominently recurrent polymicrobial sinus infections beyond early childhood. the consistent presence of sinonasal polyps in these children is unusual, as it is not typically found in uncomplicated pediatric chronic rhinosinusitis. these infections have occurred despite antimicrobial prophylaxis and ivig, highlighting the role of irak-4 in sinopulmonary epithelium. additionally, the infectious organisms identified in our patient cohort are not commonly associated with irak-4 deficiency. further study of chronic rhinosinusitis and nasal polyposis in a larger cohort of irak-4 deficient patients and other innate immunodeficiencies may help identify pathways for targeted treatment of these patients. introduction: chronic granulomatous disease (cgd) is an inherited phagocytic defect associated with inability to clear catalase positive organisms. infections in patients with cgd are severe and recalcitrant. commonest infections are pulmonary followed by soft tissue infections and suppurative lymphadenitis. osteomyelitis is an uncommon infection in patients with cgd. it poses several diagnostic and therapeutic challenge. we herein report our experience of osteomyelitis in cgd over the last 10 years. material and methods: review of records was carried out to describe the profile of osteomyelitis in cohort of patients with cgd at pediatric immunodeficiency clinic, advanced pediatrics centre, postgraduate institute of medical education and research, chandigarh, india. the diagnosis of cgd was based on nitroblue tetrazolium dye reduction test (nbt) and dihydrorhodamine reduction (dhr) assay. results: of the 63 patients with cgd, 8 (12.7%) had osteomyelitis (6 males and 2 females; age range 1-10 years). most patients had their first episode of serious infection in early childhood (mean age: 1.5 years). stimulation index (si) of dhr assay ranged from 1 to 4.58. mutational analysis was done in 5/8 patients (3 x-linked; 2 autosomal recessive). site of involvement was variable ribs-4; vertebrae-2; radius-1; skull-2; tibia-1. aspergillus fumigatus was the most common isolate (62%; 5/8); others had aspergillus flavus, aspergillus terreus and serratia marcescens each. all 4 patients with rib osteomyelitis had concurrent pneumonia, and fungus was isolated in all of them (aspergillus fumigatus-2, aspergillus flavus-1, zygomyces spp.-1). antifungals (intravenous amphotericin b) were given for a duration of 4-6 weeks and were followed by oral voriconazole in therapeutic doses for 3 to 6 months in majority of them. debridement and resection of ribs was required in one patient, while other patients were managed conservatively. out of 8 patients, 2 (25%) succumbed to pneumonia and respiratory failure. conclusion: osteomyelitis in the context of cgd is usually caused by aspergillus spp. involvement of ribs and vertebra usually occurs with the contiguous spread of infection from the lungs. therapy often requires prolonged duration of anti-microbials, and may require surgical debridement in addition to it. a 29-year-old woman with history of hypogammaglobulinemia and acute liver failure a 29-year-old woman with a 7-month history of nausea, vomiting, and abdominal pain was admitted to an outside hospital with new onset of jaundice and anasarca. liver biopsy was thought most consistent with alcoholic steatohepatitis, and she was discharged with counseling on alcohol cessation and medical management of liver disease. she presented to our facility for a second opinion. over the following days, she developed further rise in direct hyperbilirubinemia up to 19.2 mg/dl, new coagulopathy with an inr 2.06 and hypoalbuminemia to 1.7 mg/ dl in the absence of ongoing alcohol consumption. liver sonography revealed course echotexture and patent vessels. pcrs directed against multiple hepatotropic viruses were negative and copper studies were normal. due to a history of moderate alcohol consumption, she was started on high-dose corticosteroids due to a presumptive diagnosis of alcoholic hepatitis. additional history raised concern for a possible primary immunodeficiency, including idiopathic thrombocytopenic purpura at 11 years of age, multiple episodes of sinusitis treated with antibiotics and sinus surgery, one episode of suspected bacterial pneumonia, and one hospitalization for influenza a during which she developed neutropenia. in her 20s, she developed refractory genital warts, prompting infectious diseases evaluation. initial immune evaluation had revealed low immunoglobulins (iga <7 mg/dl, igg 198 mg/dl, igm 13 mg/dl) with very low responses to tetanus and diphtheria, despite a recent booster dose, and b and t cell lymphopenia (cd19+ 89 cells/î¼l, cd3+ 567 cells/î¼l, cd4+ 345v, cd 8+ 244 cells/î¼l, cd16/56+ 236 cells/î¼l); antigen and mitogen proliferation were not assessed. intravenous immunoglobulin replacement was initiated but discontinued by the patient due to infusion-related adverse effects, and she was lost to follow up until she presented with liver failure. both parents were deceased from cardiovascular disease in their 40s and she had no siblings. she had limited knowledge of family history but no known immune diseases. due to suspicion for genetic etiology of immune disorder and liver disease, we performed next-generation sequencing of a panel of over 200 genes implicated in primary immune deficiencies. patient was heterozygous for a nucleotide substation (c.1752+1g>a) within a splice site at the exon 16/intron 16 boundary of the nfkb1 gene. during the hospitalization, immunoglobulin replacement and trimethoprim-sulfamethoxazole prophylaxis were initiated. an attempt was made to refer the patient for additional immunological evaluation and transplantation evaluation but unfortunately, she developed worsening liver failure and multiple complications, including extended-spectrum beta-lactamase (esbl)-producing e. coli bacteremia, hypotension requiring vasopressors and extensive bowel ischemia, and died in the hospital. in summary, this case highlights both the risk of diagnostic delay in adult patients presenting with a primary immune deficiency and potential for genetic testing to clarify the diagnosis. while the particular genetic change has not been described, other splice site and predicted loss-offunction mutations have been reported as pathogenic in this gene, which have been implicated in autosomal dominant common variable immunodeficiency. this case further expands on the genetic causes and spectrum of disease associated with changes in the nfkb1 gene. introduction: malnutrition and micronutrient deficiency are underrecognized causes of acquired immunodeficiency in adults, and may occur even in patients with high body mass index (bmi). methods: a 46-year-old woman with a medical history significant for one remote urinary tract infection presented to the emergency department after sudden onset of severe right flank pain. the pain was accompanied by urinary frequency and not relieved by ibuprofen; she denied fevers or chills. she was diagnosed with pyelonephritis and discharged on ciprofloxacin, which was later changed to trimethoprim-sulfamethoxazole after her culture grew resistant e. coli. her pain continued despite treatment, prompting her to return to the hospital three days later. upon presentation, she was afebrile with blood pressure of 128/88 mmhg and heart rate of 86 bpm. her body mass index was 32.4 kg/m^2. her physical exam was otherwise notable for right costovertebral angle tenderness. laboratory studies revealed a leukocyte count of 14,300/ul with 83% neutrophils; alkaline phosphatase of 146 units/l and albumin of 2.7 g/dl, but otherwise normal liver function tests; normal lactic acid; and urinalysis with 3,000 wbc/hpf, 40 rbc/hpf, moderate bacteria, and the presence of wbc clumps. ct scan of the abdomen and pelvis demonstrated an obstructing 13 mm right renal stone with hydronephrosis and a right renal abscess contiguous with a right-sided hepatic abscess measuring 7.8 x 6.0 x 7.5 cm. she was treated with ceftriaxone and metronidazole, and underwent imaging-guided drainage of the abscesses. abscess cultures again grew resistant e. coli. she was discharged from the hospital with drains in place and a plan to continue trimethoprim-sulfamethoxazole until definitive management of her nephrolithiasis with ureteroscopy and lithotripsy. discussion: there remained the question of how an ostensibly immunocompetent patient had developed such severe intraabdominal infection with little systemic inflammatory response (e.g. no fever and only mild leukocytosis). a hiv antibody screen was negative. on further interview, she described a 200lb intentional weight loss over the preceding 2 years, accomplished by dietary restriction to less than 600 calories per day. nutritional assays revealed prealbumin, vitamin c, and vitamin b6 levels below the threshold of detection. she had low-normal b12 and b1. out of concern for an acquired immunodeficiency resulting from malnutrition with micronutrient deficiency, balanced nutrition was discussed with the patient who agreed to liberalize her diet. background: the past decade has brought dozens of new mendelian disorders of immunity. yet, the genetic contribution(s) to diverse disorders of the immune system remain largely unelucidated. the majority of research participants referred to the national institute of allergy and infectious diseases (niaid) for what may be a mendelian disorder evade molecular diagnosis. making progress in this area requires a coordinated, systematic, and transparent approach to clinical genomics research which leverages the unique environment at the national institutes of health clinical center (nih cc). methods/design: this study is designed to systematically apply exome sequencing and related technologies with clinical grade interpretation and reporting to niaid research participants at the nih cc under a single protocol in order to facilitate research and clinical genetics care across niaid. we are recruiting approximately 1000 participants per year from approximately 35 intramural clinical investigators. we generate genomic data, collect standardized phenotyping and report clinical interpretation in the medical record, all while providing linked genetic counseling. results: to date, we consented 1287 participants, we sent out 1058 samples for exome sequencing and 183 samples underwent copy number variant analysis. we have completed analysis for 359 families (502 individuals) and finalized and resulted 177 cases. here we present a case series illustrating some of our findings. case 1: a 10year-old female was referred to niaid for neonatal onset multisystem inflammatory disease (nomid). developmental delay and mild intellectual disability were appreciated on clinical evaluation. exome sequencing detected a mosaic novel likely pathogenic variant in nlrp3. chromosomal microarray analysis (cma) showed 㣠5 mb interstitial deletion of chromosome 12 previously associated with developmental delay and intellectual disability. case 2: a 10year-old ukrainian male was referred to niaid for the clinical diagnosis of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced). exome sequencing and cma did not detect pathogenic variants in aire, but did find a de novo variant in fam111b. defects in fam111b are associated with poikiloderma with tendon contractures, myopathy, and pulmonary fibrosis (poiktmp). the clinical features of the patient were consistent with poikmp. case 3: a 63-year-old man had a history of brain, liver and kidney nocardiosis, disseminated mac infection, prostate cancer and lymphoma. family history was significant for prostate cancer. exome sequencing showed a heterozygous pathogenic variant in brca2, associated with susceptibility to breast-ovarian, male breast, pancreatic and prostate cancer. conclusion: this case series illustrates that multiple diagnoses, unexpected diagnoses, secondary genomic findings, and data sharing helped identify variants in candidate genes. process standardization supports data integrity and efficiency while accommodating the need for investigator flexibility and providing tailored patient care. rationale: activated pi3 kinase delta syndrome (apds) is a primary immunodeficiency caused by dominant mutations that increase activity of phosphoinositide-3-kinase (pi3k). the catalytic subunit p110 is mainly expressed in cells of the hematopoietic system, primarily lymphocytes and myeloid cells, and mutations affect both b-and t-cells. we sought to further evaluate the role of the t-cell receptor (tcr) repertoire in immune dysregulation and the pathogenesis of autoimmunity and lymphoproliferation in patients with apds. methods: we evaluated the tcr repertoire in the peripheral blood in 3 patients with pik3cd mutations and compared these to the peripheral tcr repertoire in 26 patients with common variable immunodeficiency (cvid) and 50 healthy controls to investigate the role of the tcr in disease. the tcr repertoire in affected tissue of 2 patients with pik3cd mutations was also evaluated (tissue included lymph nodes for both patients, in addition to gastrointestinal tract and lung tissue in one patient). a fixed number of tcrs were subsampled (35,000 for blood and 5,000 for tissue) and diversity was calculated using the gini and shannon indexes. results: using the shannon and gini diversity indexes, the tcr repertoire in patients with pik3cd mutations had less diversity/ increased clonality as compared to healthy controls and those with cvid ( figure 1 ). for the two apds patients with biopsy tissue available for analysis, the diversity of the tcrs in tissue was increased as compared to the peripheral blood tcr repertoire ( figure 2 ). conclusions: pi3k plays an important role in the development and function of both b-and t-cells. patients with apds were found to have decreased tcr repertoire diversity in the circulating t-cell compartment compared to healthy controls and other cvid patients. the increased tcr diversity in the affected tissues compared to peripheral blood implicates the pi3k/akt signaling pathway with t-cell trafficking and tissue immune homeostasis, and suggests this pathway may play a role in the development of inflammatory and lymphoproliferative complications in these patients. gain-of-function mutations in pi3kd result in a human primary immunodeficiency, named apds (activated pi3k-delta syndrome), characterized by lymphopenia, lymphoproliferation, respiratory infections and inefficient responses to vaccination. however, what promotes these immune disturbances at the cellular and molecular level remains unknown. we have recently published a mouse model that recapitulates major features of this disease and used this model and patient samples to probe how hyperactive pi3kd fosters aberrant humoral immunity. we found that mutant pi3kd alters the intrinsic function of t and b cells, leading to icos-independent increases in t follicular helper (tfh) and germinal center (gc) b cells, disorganized gcs, and poor class-switched antigen-specific responses to immunization. these phenotypes were associated with increased phosphorylation of akt and s6 in t and b cells, and lower threshold of activation, with altered regulation of foxo1 and bcl2 family members. moreover, b cells showed enhanced responsiveness and proliferation to both antigens and innate stimuli, accompanied by reduced cell death. strikingly, aberrant responses were accompanied by increased reactivity to gut bacteria, and a broad increase in autoantibodies that were dependent on commensal microbial stimulation, as demonstrated by striking reduction of self-reactivity upon antibiotic treatment in mutant mice. we now have further examined b cell function in these mice and demonstrate that altered foxo1 plays a major role in disruption of both b and t cell function. we further provide evidence for altered activation of metabolic pathways in b cells, compared to wt cells, that may contribute to the dysregulated b cell reactivity. our findings suggest that proper pi3kd regulation is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome. this research was supported in part by the intramural research program of the nih, nhgri and niaid. autoimmune cytopenias are seen in a significant proportion of patients with immunodeficiencies affecting antibody production. previous b-cell maturation studies using fluorescence-activated cell sorting (facs) have associated various phenotypes of primary immunodeficiency diseases affecting antibody production with differing levels of b-cell differentiation. in this study we analyzed the peripheral b-cell compartment of 84 patients with a hypogammaglobulinemia and >1% b-cells with and without a history of autoimmune cytopenias. b-cells were isolated from peripheral blood using monoclonal anti-cd19 and these cells were gated to identify the proportion of memory b cell (cd19+cd27+ ), igm+ memory b (cd27+igm+), marginal zone b-cells (igm+ igd+), isotype-switched memory b-cells (cd27+igm-igd-) and transitional cells (igmhicd38hi). pid patients with a history of aic had decreased proportions of total cd27+ b-cell (11.6% vs 25.6%; p=0.0003) and igm memory b cells (8.3% vs 18.4%; p = 0.0018). conversely, the proportion of marginal zone b-cells was increased in this group (82.0% vs 66.5%; p = 0.0043). consistent with previous reporting, the proportion of isotype-switched memory b-cells was significantly lower in the aic group (0.75% vs 2.8%; p = 0.0003). statistically significant inter-group difference was not seen within the transitional b-cell subset. our data suggest that maturation arrest of marginal zone (cd27+igm+ igd+) b-cells may be implicated in the development of autoimmune cytopenias in humoral immunodeficiency. (159) submission id#601984 taissa de matos. kasahara 1 , sudhir gupta, md 2 1 phd student, state university of rio de janeiro and university of californis irvine 2 professor, university of california at irvine, irvine, ca, usa introduction/background: common variable immunodeficiency (cvid) is the most frequent form of primary hypogammaglobulinemia with decreased serum igg and iga levels and variable levels of igm in adults. in addition to decreased serum immunoglobulins, 25-30% of cvid patients present autoimmune manifestations. the mechanisms that lead to a breakdown of selftolerance in cvid are not completely understood. however some differences in b and t cells subsets and autoreactive b and t cells can be detected. elevated expression of surface igd and downregulation of igm receptor are hallmarks of anergic naã¯ve b cells that contain autoreactive receptors in human peripheral blood. moreover, memory b cells that have class switched to igd and present an igd+igm-phenotype are also highly reactive to self-antigens in healthy individuals. the role of these autoreactive naã¯ve and memory b cells in the immunopathogenesis of cvid has not been evaluated. here we investigated the frequency of cd27-and cd27+ b cells expressing igd and igm in peripheral blood of cvid patients. methods: peripheral blood mononuclear cells (pbmc) from cvid patients (n=29) and health subjects (n=32) were separated by ficollhypaque and incubated with anti-human cd19-percp, cd27-fitc, igd-bv510 and igm-apc to identify different subsets of b cells by flow cytometry. cd19+cd27-igd+igm-and cd19+cd27-igd+ igm+ b cells were sorted, loaded with cfse and cultured with cpg and ant-cd40 for 5 days to evaluate the proliferation. results: among the compartment of cd27-b cells, cvid patients showed an increased frequency of igd+igm+ cells and a lower frequency of igd-igm-cells as compared to control group. no differences were observed in the frequency of igd+igm-cells in cd27-b cells between cvid patients and controls. in contrast, in the compartment of cd27+ b cells, cvid patients showed an increased frequency of igd+igm-, igd+ igm+ and igd-igm+ cells and a lower frequency of igd-igm-cells when compared to health subjects. when the patients were divided in two groups based on autoimmune manifestations, the group with autoimmune disease showed an increased frequency of igd+igm+ and igd-igm+ cells in cd27-b cells when compared to the control groups. both patient groups showed an increased frequency of igd+igm-, igd+igm+ and igd-igm+ cells and a lower frequency of igd-igm-cells when compared to health subjects. regarding the proliferation, naã¯ve b cells from cvid patients showed a reduced proliferative capacity in response to in vitro stimulation as compared with naã¯ve b cells from health subjects. conclusion: our results suggest that the increase of cd27+igd+igm-b cells can be related to the susceptibility of autoimmunity in cvid patients. introduction: immunoglobulin g4-related disease (igg4-rd) is a group of immune-mediated conditions where tissues are affected with dense lymphoplasmacytic infiltrations with a predominance of igg4-positive plasma cells and storiform fibrosis, usually in the setting of elevated serum concentrations of igg4. common presentations include autoimmune pancreatitis, sclerosing cholangitis, retroperitoneal fibrosis, salivary gland disease, and orbital disease, among others. symptoms of asthma or allergy are present in approximately 40 percent of patients and they typically exhibit a good initial therapeutic response to glucocorticoids. case presentation: a 61-year-old female with a history of gastroparesis, cutaneous lupus erythematosus and suspected autoimmune pancreatitis was referred to allergy/immunology clinic for evaluation of elevated igg4. she reported a 15-year history of recurrent abdominal pain attributed to recurrent pancreatitis based on previous mild lipase elevations. prior endoscopic ultrasound (eus) of the pancreas revealed edema. there was concern for gallstone pancreatitis but ercp followed by cholecystectomy, biliary and pancreatic sphincterotomy had no change in her symptoms. in 2016, she was noted to have a positive ana and high serum igg4, per patient (values from osh records could not be obtained). symptoms improved with a course of steroids, hence suspicion for autoimmune pancreatitis. in 2018 she developed a rash on her arms and face. biopsies of the affected areas revealed cutaneous lupus erythematosus on the arms and a basal cell carcinoma on the face, which was excised. ana was only 1:80 at that time. at the visit, she complained of severe allergic rhinitis, joint pains, as well as a malar rash, which responded to intermittent courses of prednisone by prior providers. laboratories obtained at initial visit were significant for thrombocytopenia (135 thou/cu mm), positive lupus anticoagulant (56 sec) and elevated igg4 (95 mg/dl; normal range 4-86 mg/dl). c3, c4, c1q, ana, anti-double stranded dna, anti-smith antibodies, antiphospholipid panel, upep and spep were all unremarkable. ct chest and abdomen were also normal. given the patient's history of cutaneous lupus erythematosus, plaquenil was started as a steroid sparing agent. eus of the pancreas with possible biopsy was ordered in an attempt to obtain a histopathologic diagnosis of igg4-rd. conclusion: this case exhibits the association between elevated igg4, pancreatitis of unknown origin, allergic rhinitis, and cutaneous lupus erythematosus, highlighting the value of identifying a pathologic connection between seemingly unrelated disorders in patients with elevated igg4, as they may be manifestations of igg4-rd. in order to make the diagnosis, histopathologic findings showcasing lymphoplasmacytic tissue infiltration consisting mainly of igg4-positive plasma cells and small lymphocytes is essential. the majority of patients respond to glucocorticoids, and while the duration of response is variable, most patients flare during or after glucocorticoids are tapered, as noted in this patient. rituximab has been shown to be effective in some patients and will be considered in this patient if symptoms persist. (161) submission id#602042 rationale: pnp deficiency is an autosomal recessive disorder due to defective purine metabolism leading to severe combined immunodeficiency (scid) and neurological deterioration. newborn screening utilizing t-cell receptor excision circle (trec) assay can detect affected patients before complications arise. herein, we describe an infant initially identified by newborn screening with pnp deficiency and congenital cmv, a previously unreported presentation. methods: cmv quantitative pcr (qpcr) was performed by nebraska medicine, pnp enzyme activity by duke and genetic sequencing by invitae. results: a small for gestational age (sga) male infant was reported to have an abnormal trec assay on day of life (dol) 7. he was hospitalized for further evaluation. initial studies revealed profound lymphopenia, normal lymphocyte proliferation to mitogens and no evidence of maternal engraftment. additionally on dol 10, he had cmv viremia and viruria; thus with sga, failed unilateral hearing screen and head ultrasound with bilateral parenchymal calcifications, congenital cmv was suspected. pnp enzyme activity was abnormal. cmv treatment was initiated with ganciclovir on dol 10. foscarnet was added on dol 13. cmv qpcr levels decreased below the limit of detection by dol 30. genetic testing found a pathogenic homozygous mutation in pnp (c.286-18g>a). the infant has a 10/10 hla-matched, unaffected, cmv positive sibling and will proceed to hematopoietic stem cell transplantation. conclusions: to our knowledge, this is the first reported case of pnp deficiency identified through newborn screening. this novel case of congenital cmv and pnp deficiency highlights the importance of cmv screening and need for treatment strategies for congenital cmv in scid. despite a dramatic increase in the use of next generation sequencing over the last decade, the majority of the more than 50 million identified human genomic variants do not have well-established clinical implications. progress is being made on this complex challenge through multiple approaches, including data sharing. to maximize our understanding of genomic data, platforms that enable effective and responsible data-sharing are essential. this means that genotypic and phenotypic data must be findable, accessible, interoperable, and reusable under conditions that are ethical and transparent. to highlight innovations in data-sharing and their potential to advance discovery, we present three data-sharing mechanisms. for each platform, we will present a case highlighting its key functionality and discuss opportunities and challenges that may arise as each platform is scaled up. (1.) genomic research integration system (gris) is a collaborationengendering web application that facilitates the identification of genetic variants associated with rare immunological disorders. users can access integrated and standardized phenotypic and genomic data that is analyzable within the platform. gris enables systematic and automated capturing, and links patient data from disconnected systems and paperbased records. standardized annotations allow for the comparison of data from different clinical studies. the main goal of this tool is discoverability of other affected individuals enrolled in separate protocols within the niaid intramural research program. this internal database was used to find a second family with a rare variant in a candidate gene. (2.) the genomic ascertainment cohort (tgac) is a resource that aims to improve our understanding of the phenotypic consequence of genetic variation by providing access to aggregate, de-identified genomic data from large nih intramural and related cohorts. participants have provided informed consent to be re-contacted for additional phenotyping in the future. the main goal of this tool is to enable further study of the clinical consequence of variants in a large, unbiased cohort of patients ascertained for many indications. this database was used to investigate findings in participants with previously published pathogenic variants in genes associated with primary immune deficiency based on medical record review. (3.) clingen is dedicated to building an authoritative central resource that defines the clinical relevance of genes and variants for precision medicine and research. through the sharing of genetic and health data, clingen seeks to answer whether a given gene is associated with a disease (clinical validity)?; whether a given variant is causative (pathogenicity)?; and whether the information is actionable (clinical utility)? this resource is meant to convene disease-and gene-specific expert groups to curate the medical literature on mendelian disease to better define gene-disease and variant-disease relationships using many lines of evidence. this resource was used to clarify clinical validity of disease-gene assertions. together these efforts help create a clinical research ecosystem that maximizes the value of clinical research data and ultimately improves patient care. this research was supported by the intramural research program of the nih, niaid. introduction: according to the population reference bureau, the number of elderly americans, defined as age 65 and older, is projected to more than double from 46 million to 98 million by 2060, rising from 15% to 24% of the total population. the impact of immunodeficiency in this important segment of the population remains understudied. methods: the usidnet registry was queried to obtain demographic, clinical data of elderly patients defined as age 65 and older. descriptive analyses were performed on the data. results: 373 participants (7.2%) were eligible out of 5176 total registry participants. the median age of the cohort was 70 years and predominantly female (74.7%) and white (78.0%) with a median bmi of 26.6 â± 6.6.the majority (81.8%) of subjects were living. humoral deficiencies comprised the majority of diagnoses (94.6%), with common variable immune deficiency being the most frequent (76.9%). of the remaining non-humoral diagnoses, immune dysregulation (1.3%) and immunodeficiency with myelodysplasia (1.1%) were the most frequent. the majority (79.1%) of subjects reported having received immunoglobulin replacement therapy (igrt) at some point, with 51.7% reporting via iv route. of the 1275 infections that occurred in this cohort, sinopulmonary infections were the most commonly reported, specifically sinusitis (18.5%), pneumonia (13.8%), upper respiratory infection (6.7%), and otitis media (5.5%). in this cohort, 107 autoimmune, 49 cardiovascular, and 11 granulomatous complications were reported . the number of patients with malignancy was 89, with some patients diagnosed with multiple malignant disorders. of the reported malignancies, the majority (69.9%) were solid tumors. conclusions: compared to the age-matched non-immunodeficiency united states population, this cohort had more females 74.7% (usidnet) versus 56.0% (us population) and fewer whites 78.0% (usidnet) vs 86.0% (us population. humoral immunodeficiencies, specifically cvid, were most common diagnoses, similar to other age groups of immunodeficiency patients. majority of these patients have received igrt, with approximately half via iv route. this cohort reported living with a variety of non-infectious complications, including autoimmunity and malignancies. more research which specifically focuses on elderly patients with immunodeficiency is needed. clinical microbiologist and infectious disease physician, university of calgary x-linked agammaglobulinemia (xla) is a primary immunodeficiency caused by mutations in the bruton tyrosine kinase gene which leads to b cell maturation failure and defective antibody production. this puts patients at risk of recurrent sinopulmonary infections, gastrointestinal infections, and recurrent skin infections including infections caused by helicobacter sp. helicobacter sp are gram negative bacilli commonly found in the gastrointestinal tract of various animals. helicobacter sp. have been linked with gastritis most notably helicobacter pylori causing gastric ulcers in humans. helicobacter sp. has been found in rare cases to cause disseminated infections including pyodermic gangrenosum and cellulitis notably in patients with agammaglobulinemia. infections caused by helicobacter bilis are challenging to diagnosis due to difficulties with culturing the pathogen as well as poor guidelines for antimicrobial management. case report: the patient was diagnosed with x-linked agammaglobulinemia at the age of 16 months with a history of recurrent sinusitis and was started on ivig q3weeks. despite regular ivig, he developed bronchiectasis. at 11 years of age in 2013, he developed a chronic rash around his left knee resembling erythema nodosum. by 2014, he had developed a left knee effusion associated with left sided calf pain. his knee pain was found to improve during courses of ciprofloxacin to treat recurrent lung infections. given case report data of h. pylori causing erythema nodosum in patients with agammaglobulinemia, he was treated empirically for an h. pylori infections with no improvement. in 2015 he was found to have progressive cellulitis with pyomyositis of the left leg. a skin biopsy of a calf nodule was found to be culture negative but 16s pcr was positive for h. bilis. he was started on treatment with ertapenem and levofloxacin with subsequent resolution of his rash. his left ankle pain progressed and by late 2015 and was found to have possible osteomyelitis of the left ankle on mri. in 2016 he was found to be bacteremic with h bilis. due to progressive symptoms with significant impact on function and rising inflammatory markers despite 12 months of antimicrobial treatment, doxycycline and flagyl were added leading to clinical improvement and normalization of his inflammatory markers. he was continued on oral doxycycline and flagyl for 12 months for a chronic osteomyelitis. discussion: h. bilis is a slow growing pathogen which is challenging to culture in the laboratory often requiring special agar plates and prolonged incubation. in patients with agammaglobulinemia and associated chronic skin infections or erythema nodosuma, h bilis should be suspected as a possible pathogen. due to challenges with culturing, 16s pcr or amplification of the 16s ribosomal subunit should be considered to try to identify the pathogen. there are poorly delineated clinical antimicrobial breakpoints to help guide therapy with minimal evidence. case reports suggest prolonged therapy with aminoglycosides and penicillin. other studies have successfully treated patients with a carbapenem, azithromycin and levofloxacin. in the absence of sensitivity data, prolonged treatment (12months) should be considered with a combination of antimicrobials. patients should be followed closely as recurrent infections are not uncommon. chief, human immunological diseases section, laboratory of clinical immunology and microbiology, niaid, nih, bethesda, md introduction: dock8 deficiency is a combined immunodeficiency characterized by eczema, recurrent sinopulmonary infections, viral skin infections, malignancy and early mortality. in recent years, liver disease and vasculopathy have been increasingly recognized as a complication of dock8 deficiency. we clinically characterized our cohort of dock8 deficient patients, with a specific focus on these newly identified areas of disease involvement. methods: chart reviews were performed on patients seen at nih with genetic and clinical diagnosis of dock8 deficiency. patients were all enrolled on irb approved niaid protocols. results: we identified 52 patients from 40 families with dock8 deficiency in our nih cohort, ranging in age from 6-44 years. of the 40 families, 17 had homozygous mutations. of the 52 patients, food allergy was diagnosed in 31 (60%), eczema in 49 (94%), and asthma in 30 (58%). chronic or recurrent viral skin infections were seen in 49/52 (94%). chronic ebv viremia by pcr positivity was seen in 18/46 patients (39%); only 2 patients were known to be ebv immune without viremia. cmv viremia was infrequent. sinopulmonary infections were common, with bronchiectasis occurring in 23 /50 (46%) with available imaging. liver disease was diagnosed in 14 (27%), with 7 having biliary tract abnormalities on imaging and stool positive for cryptosporidia; most patients with cryptosporidia were without diarrhea. the incidence of cryptosporidia is likely under-represented due to more recent availability of sensitive assays for cryptosporidia detection. other liver abnormalities included fatty liver, metastatic disease from malignancy and medication related hepatitis. vasculopathy, predominantly of the aorta and cerebral arteries, was diagnosed in 7, with patients in the last 5 years being prospectively imaged. autoimmunity was rare (5%) including autoimmune cytopenias and hypothyroidism. 36 of 50 with follow-up are alive (70%) with age range 6-44 years. of the 36 living patients, 28 (78%) have had a hsct. causes of deaths include malignancy (6), infection (1) , and hsct complications (7) . long-term follow-up of patients with hsct (up to 6 years) has revealed resolution of the infection susceptibility and eczema, no new cancers, and stabilization of vasculopathy. conclusions: in addition to the well described manifestations of dock8 deficiency including eczema, allergy, recurrent sinopulmonary infections, skin viral infections and malignancy, our cohort revealed a relatively high incidence of liver disease, frequently associated with stool positivity for cryptosporidia, as well as vasculopathy. both of these clinical manifestations should be considered during preparation for hsct as they may affect management through transplant. autoimmunity has likely been over-estimated in prior descriptions of dock8 deficiency. long-term follow-up after hsct is needed to determine the prognosis from the vasculopathy, liver disease, and malignancy risk. (166) submission id#604115 yasuhiro yamazaki 1 , stefano volpi 2 , luigi d. notarangelo 1 introduction/background: extl3 (exostosin like glycosyltransferase 3) is an exostosin family member which initiates heparan sulfate (hs) chain biosynthesis and elongation. we have reported homozygous extl3 hypomorphic mutation (r339w) as a cause of immunoosseous-dysplasia syndrome. fourteen patients who have extl3 homozygous mutation were reported so far. eight of them manifested t cell lymphopenia, and 5 presented with severe combined immunodeficiency (scid) or omenn syndrome. using patient-derived induced pluripotent stem cells (ipscs) as a model, we have previously reported that extl3 mutations affect differentiation to thymic epithelial progenitor cells as well as expansion of hematopoietic progenitor cells. consistent with the latter, previous studies have suggested that mutations in other genes involved in hs biosynthesis affect hematopoietic stem cell (hsc) differentiation. however, the exact mechanisms by which extl3 mutations affect hematopoiesis are not known. objectives: we tried to clarify gene expression difference in hscs derived from wild-type, extl3 hypomorphic and extl3 knock-out (ko) human ipscs. methods: the control bj ipsc line was engineered by crispr/cas9 gene targeting. extl3 ko ipscs were obtained which carried compound heterozygous extl3 mutations (c.1003_1004inst; c.1005_1006insgatattt). hsc differentiation was induced using the stemdiff hematopoietic kit (stemcell technologies). bulk rna from each ips cells and each differentiated cd34+cd43+cd45+ was analyzed by rna sequencing. results: as compared to control ipscs, patient-derived cells showed slightly lower capacity to generate cd34+cd43+cd45+ cells. on the other hand, extl3 ko cells showed no differentiation into cd34+ cd43+cd45+ cells. gene set enrichment analysis showed enriched expression of genes involved in hematopoietic progenitor cell differentiation, regulation of hemopoiesis, and positive regulation of hemopoiesis in both control and patient-derived cd34+cd43+cd45+ cells compared to parental ipscs. moreover, these gene sets were more abundantly enriched in control than in patient-derived cd34+cd43+cd45+ cells. the gene set of response to type i interferon was significantly enriched in control versus patient-derived cd34+cd43+cd45+ cells. conclusions: these results confirm that extl3 plays an important role for hsc homeostasis in human cells. because type 1 interferons play a role in hsc proliferation, the decreased type i interferon signature may account for the reduced number of hscs that we have previously reported upon in vitro differentiation of extl3-mutated versus control-derived ipscs. this study was supported by the division of intramural research, niaid, nih, under protocol 16-i-n139. a case of autoinflammatory syndrome with osteoporosis and specific antibody deficiency autoinflammatory syndromes are inherited disorders with an exaggerated inflammatory response with no specific trigger. the clinical phenotypes of variants of autoinflammatory syndromes may overlap. we report a case of a 13 year old male with prior diagnosis of specific antibody deficiency, periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis (pfapa) syndrome, arthralgia and moderate atopic dermatitis. he was diagnosed at 3 years of age with specific antibody deficiency based on persistently low pneumococcal titers against repeat immunizations. due to recurrent infections, he was placed on immunoglobulin replacement therapy (igrt) at 8 years of age. igrt was discontinued at 13 years of age due to full resolution in infections and patient demonstrated robust response to immunizations. patient had lifelong history of recurrent fevers (every 5 weeks) associated with pharyngitis and aphthous ulcers consistent with diagnosis of pfapa. as he became older these episodes became less frequent. last episode of fever was over a year ago. the father had similar symptoms of recurrent fevers and oral ulcers as a child but currently remains asymptomatic. paternal grandfather died of kidney disease. patient has been generally in good health until recent year with intermittent abdominal pain, arthralgia and several long bone fractures with no history of prior trauma. a bone density scan revealed osteopenia and osteoporosis with a z score of -2.2 of lumbar spine, -4.0 of left femoral neck, -3.1 of left hip. given history of familial autoinflammatory disease, and antibody deficiency genetic testing was obtained which identified a pathogenic heterozygous variant of taci and mefv c.2082g>a (p.met694lle). taci mutation has been linked to antibody deficiency syndromes. genetic study for family members is pending. the mefv gene is associated with autosomal recessive familial mediterranean fever (fmf) and has been reported in autosomal dominant fmf as well. fmf is characterized by recurrent episodes of fever associated with serositis, arthralgia, and arthritis. patients with fmf have elevation in acute phase reactants during attacks with most returning to normal levels during the episode-free periods. multiple studies have shown that patient with fmf have lower bone mineral density and zscores than the general population. inflammation in fmf is thought to be mediated by several different cytokines (il-1, il-2, il-6, il-7, il-8, il-11, il-15 and tnf-). these same cytokines play a role in osteoclast activity and bone resorption. it has been suggested chronic inflammation during acute attacks and subclinical inflammation during the disease-free period lead to bone loss and osteoporosis. regular use of colchicine, the main treatment for fmf, may slow down osteoporosis. beside careful monitoring of clinical and laboratory phenotype, genetic evaluation is an important step in distinguishing between overlapping entities and can prevent complication and promote targeted intervention. a 5 year old previously healthy boy was referred for periodic fever/ pfapa and mosquito bite hypersensitivity. eight weeks earlier he developed fever to 104f, mouth sores and exudative tonsillitis; a rapid strep screen was negative. one week later he developed moderate cervical lymphadenopathy and had a positive ebv early antigen antibody.. one month later he had several severe local reactions to mosquito bites. each manifested 6-8 cm of erythema and induration with a 1+ cm bullae which left an ulcer after rupture and healed with a hypopigmented scar. the bites were accompanied by fever to 104f for 4 days. one febrile episode was treated with low dose prednisolone for presumed pfapa, and the fever resolved within hours. his past history was positive for nasal allergy and mild asthma. his parents are not related: mom is of european-indonesian and dad european-african (creole ancestry. testing prior to this visit showed normal igg, iga and igm, elevated ige (12,000 u/l) and normal cbc. lymphocyte subsets revealed cd3+ 23% (1538/mcl), cd4+ 17% (1109/mcl), cd8+ 6% (363/ mcl), cd19+ 9% (587/mcl), nk cells 67% (4435/mcl). on examination he appeared well with height at 86th%ile and weight at 58th%ile. there was no lymphadenopathy, hepatosplenomegaly or inflammed skin lesions; there was a 1cm round scar on the right plantar surface at the site of a prior mosquito bite. laboratory studies confirmed nk lymphocytosis 64% (5459/mcl) and elevated ige (29,600 u/l). lymphoproliferation to mitogens, cd3/cd28, cmvand hsv were normal, but absent to tetanus and candida antigens. ebv antibodies reflected past infection (vca-igg+, vca-igm-, ebna+); quantitative ebv pcr was >5,000,000 copies/ml whole blood. nk cytotoxicity and cd107a expression were decreased. bone marrow nk analysis suggested conality. the patient was diagnosed with "hypersensitivity to mosquito bites with ebv-associated t-/ nk lymphoproliferation." this disorder represents a subset of chronic active ebv (caebv) that is rarely seen outside of east asia. the lack of organomegaly or lymphadenopathy with hyper-ige and nk lymphocytosis and decreased nk function support the likelihood that nk cells are the target of ebv infection in this patient. this diagnosis may be a precursor to hemophagocytosis, liver necrosis or lymphoma/leukemia, and the only curative treatment is bone marrow transplantation. the patient's sister is a 10/10 hla match. she is seropositive for past ebv infection, and she has no history of extreme reactions to mosquito bites. genetic mutations that cause familial hemophagocytic lymphohistiocytosis have not been reported in caebv, and to the best of our knowledge familial cases of this disorder have not been identified. the response to bmt in this patient is pending. introduction/background: a number of case reports have described symptomatic hypogammaglobulinemia following administration of anti-epileptic drugs (aeds), specifically lamotrigine, carbamazepine, and levetiracetam. the mechanism by which symptomatic hypogammaglobulinemia develops is unclear. we evaluated the prevalence and the clinical significance of hypogammaglobulinemia associated with use of these aeds. objectives: our aim was to characterize the prevalence of aed-induced hypogammaglobulinemia, identify specific aeds associated with hypogammaglobulinemia, and characterize the timeline to development of hypogammaglobulinemia after initiation of therapy. methods: a retrospective, multicenter, electronic medical record review spanning 18 years identified patients with hypogammaglobulinemia who were on aed therapy (lamotrigine, carbamazepine, or levetiracetam). patients were excluded if they had a pre-existing primary immunodeficiency (pid), malignancy, protein-losing enteropathy, or significant proteinuria. patients on chronic immunosuppressive therapy, those without laboratory criteria for hypogammaglobulinemia, or those on one of the aeds for less than one month were also excluded. results: of the 316 cases reviewed, 5 patients met our inclusion criteria. the median age was 35; 80% were adults, 80% were female, and 80% were white. lamotrigine was implicated in 3/5 of the cases, carbamazepine in 2/5, and levetiracetam in 1/5. tetanus and pneumococcal titers were available for 4/5 patients. of those patients, 3/4 had protective titers to both per report with responses to >70% of the serotypes. only one patient reported severe, recurrent infections while the remaining four had little to no symptoms. interestingly, the patient with severe infections did have protective titers. of the five laboratory proven hypogammaglobulinemia patients, one died of an infection, two have continued on the medication due to refractory seizures responsive only to these medications, and two are currently being tapered off of their aed. conclusion: while it appears that aed-induced hypogammaglobulinemia is quite rare, it should be considered in a patient without other secondary causes of hypogammaglobulinemia on aed therapy. many antiepileptics downregulate nfkb signaling suggestive that patients who develop symptomatic hypogammaglobulinemia may have hypomorphic mutations in the nfkb signaling pathway. (170) submission id#604503 autoimmune lymphoproliferative syndrome (alps) results from defective apoptosis of lymphocytes mediated through the fas/fas ligand (fasl) pathway. the hallmark lab finding is an expansion of t cells that express the alpha/beta t cell receptor, but lack both cd4 and cd8 (double negative t cells) in the setting of normal or elevated lymphocyte counts. patients present with chronic, nonmalignant, noninfectious lymphadenopathy or splenomegaly. for definitive diagnosis, patients need to have (1) a pathogenic mutation in fas, fas ligand or caspase 10 or (2) a defective fas-induced lymphocyte apoptosis. we describe a probable case of alps with heterozygous mutation in fas c.287a>g(p.his96arg), a variant that has not been previously reported (his lymphocyte apoptosis assay is pending). unique to this case is the patients castleman disease-like features on pathology. a 15 year-old male referred from hematology clinic presented with an 8 year history of chronic lymphadenopathy, splenomegaly, anemia, and no underlying diagnosis. malignancy had previously been excluded by bone marrow aspirate and biopsy 8 years prior. however, he had a right sided lymph node that had increased in size for the past 4 months. he was otherwise asymptomatic. a lymph node biopsy 7 years prior was reportedly normal. his exam demonstrated significant bilateral lymphadenopathy, greater on right, with an approximately 8 x 6 cm mobile right neck mass. he had splenomegaly palpated 7 cm down and across to midline. he was therefore admitted for excisional lymph node biopsy to evaluate for possible malignancy and labs were sent to evaluate for alps. labs were supportive of alps. he had elevated t cell receptor alpha beta double negative t cells (tcr a/b dntcs) in blood (10.5%). b12 level was elevated (>1000 pg/ml). plasma soluble fasl level was elevated (5517 pg/ml). interleukin-10 (il-10) and il-18 levels were elevated (88 and 909 pg/ml respectively). he had multilineage cytopenias: anemia with hgb of 9.5 g/dl and neutropenia (absolute neutrophil count of 1380 k/ul). he had hypergammaglobulinemia with an igg level of 2010 mg/dl. broad infectious work-up was negative, including hiv, quantiferon, cocci, bartonella, toxoplasma, coxiella burnetii, ebv pcr and, cmv igm. lymph node biopsy showed no evidence of malignancy. immunostains and flow cytometry showed the presence of expanded tcr a/b dntcs in the lymph node, consistent with alps. interestingly, lymph node histology showed morphologic features typical of plasma cell variant castleman disease. numerous castlemanlike follicles showed typical regressive changes with onion-skinning morphology. paracortical hyperplasia with sheets of plasma cells was noted. there was negative staining for hhv8 (a well-known cause of plasma cell variant castleman disease). the diagnosis of idiopathic multicentric hhv8-negative castleman disease was excluded by definition in the setting of alps, per evidence-based consensus criteria published in 2017. in addition, our patient did not show any symptoms typically associated with it, such as fever, night sweats, weight loss, weakness or fatigue. should his fas-induced lymphocyte apoptosis be defective (in 2 separate assays), this would confirm his alps-fas diagnosis and we would start the patient on sirolimus. head of immunology unit, children' s hospital ricardo gutierrez introduction: slc46a1 gene encodes the proto-couple folate transporter (pcft), which supports intestinal folate uptake, and participates in folate transport into the central nervous system. slc46a1 mutations cause pcft defects, resulting in low folate levels in serum and cerebrospinal fluid. hereditary folate malabsorption (hfm) is a rare, autosomal recessive disorder with pcft deficiency resulting in cerebral folate deficiency. most of the patients present megaloblastic anaemia, moderate pancytopenia in the first few months of life, failure to thrive, diarrhoea and/or later onset neurological symptoms including seizures and developmental delay. i m m u n o d e f i c i e n c y i n h f m c a n m a n i f e s t i t s e l f w i t h hypogammaglobulinemia with normal t-cell function. b-cell precursor compartment seems to be particularly vulnerable to folate deficiency in some hfm patients. this immunodeficiency can be restored with specific treatment with folic acid. aim: to describe a female patient with a homozygous pathological variation in the slc46a1 gene. results: a 17 months old girl, born of non-consanguineous parents. she started at 3 months old with diarrhoea due to rotavirus, low weight and bicytopenia with normal bone marrow aspiration. she presented low levels of folic acid 1.5ng/ml (nv 3.1-20.5 ng/ml) at first thought due to secondary to malnutrition. treatment with folic acid supplementation was administrated, improving platelets counts. at 5 months old she presented steatorrhea with severe perianal panniculitis which required surgical treatment. no germs were rescued after a skin biopsy. moreover, she suffered from a respiratory infection due to picornavirus with two episodes of pneumothorax which required intensive care. at that moment ivig treatment was administered due to hypogammaglobulinemia and clinical severity. chronic diarrhoea worsened with bloody depositions. three rectal ulcers were found in the gut biopsy. bowel inflammatory disease was suspected and mesalazine administration was started with weight improvement. furthermore, at 10 months old she presented 3 status epilepticus, with pathological eeg and normal mri; one of them related to a cmv infection, successfully treated. in the immunological evaluation igg and iga were low with normal igm and igd. the protein-antibody response was not evaluated. she presented normal lymphocyte and t cells extended populations, t cells proliferation assay, dhr, treg cells, complement, cd107a expression, alpha-fetoprotein, without autoantibodies a molecular panel testing was done by ngs and a homozygous variant in slc46a1 gene was found, causing impaired intestinal folate absorption. conclusion: hfm should be considered in the diagnosis of patients with cytopenias and hypogammaglobulinemia in order to provide specific treatment. hfm has wide clinical manifestations, not only with megaloblastic anaemia and neurological impairment but also with gastrointestinal and skin manifestations. with folate treatment, clinical and immunological defects can be normalized. introduction: multifocal epithelial hyperplasia (meh), or hecks disease, is a rare, benign infection of the mucosa caused by human papilloma virus (hpv). clinically, meh manifests as numerous painless, soft, sessile papules or plaques, and typically occurs in the labial, lingual, and buccal mucosa. meh lesions are usually associated with hpv types 13 and 32, and seen more commonly in patients of caribbean or central/south american descent. prior studies in adults have shown that tumor necrosis factor alpha (tnf) promotes hpv, and may influence duration of hpv infection. case: we present a five-year-old full term male of haitian descent referred for assessment of multiple flesh colored, papular lesions on the buccal and labial mucosa that had persisted and quantitatively increased over one year, although some lesions regressed. he had no pain or difficulty eating. medical history significant for one seizure; negative for infection. no family history of infection, immunodeficiency, consanguinity, or miscarriage. head and neck examination failed to reveal cervical lymphadenopathy, masses, or hypertrophy in the salivary glands. intraoral examination revealed multiple papular nodules, mostly flat although some were corrugated. the greatest concentration was noted on the lower left labial surface extending to the mucosal vermillion interface, not involving the vermillion or commissure region. lesions extended into the mandibular vestibule and the left buccal mucosa. no other lesions were noted on extremities, genitalia, or any other visualized mucosal surface. based on history and exam, he was diagnosed with meh. white blood cell count, neutrophils, lymphocytes, cd4 and cd8 t cell, b cell, nk cell enumeration, and immunoglobulin panel were normal for age. tetanus and streptococcus pneumoniae titers were protective. cytomegalovirus igg and igm were negative. epstein-barr virus igg was positive, igm and early antigen ab negative. serology was significant for elevated tnf (84 pg/ml; reference range <22pg/ml) while interferon gamma and interleukins 1, 2, 4, 5, 6, 8, 10, 12, 13 , and 17 were normal, as was il-2 receptor cd25. one month after the initial visit, lesions were stable and unchanged. nine-valent hpv vaccination was considered, but not administered. conclusions: meh is a rare but benign disease caused by hpv. awareness of the disease and its course is important to prevent unnecessary expanded immunodeficiency work-up and possible procedures to eliminate lesions. although mucosal immunity can be site specific, especially with hpv, our understanding of t-cell cytokine and chemokine responses to hpv in cervical and laryngeal lesions may be instructive. the mechanism which allows hpv persistence in meh is not characterized, but it likely is due to increased viral persistence and an inability for the host immune response to successfully induce viral latency and successful containment. elevated tnf levels, with normal levels of il-2, il-6, il-8, il-10, may correlate with decreased clearance of hpv and prolonged duration of meh. it remains unclear if viral persistence is the cause of, or the sequela of, increased tnf. longitudinal monitoring of cytokine (tnf, il-2, il-6, il-8, il-10) and chemokine (ccl17, ccl18, ccl19, ccl20, ccl21, and ccl22) serum concentrations may be useful biomarkers for disease resolution. introduction: autosomal dominant hyper ige (jobs) syndrome is a rare primary immunodeficiency characterized by eczema and sinopulmonary infections as well as musculoskeletal and vascular complications. as in all chronic illnesses, patient education is an ongoing need. in the rare disease population, patient education is especially important as patients must be able to explain their unique healthcare concerns in a variety of medical settings. we focused on ad-hies, due to our relatively large cohort of patients, the frequent lack of classic signs of illness often impairing diagnosis of severe infection, and the diverse nonimmunologic clinical features of this disease. objectives: we aimed to increase understanding of the clinical manifestations of ad-hies to promote earlier recognition of symptoms and to increase self-efficacy for symptom management in the adult hies population. methods: adult patients were asked to participate in a patient education project. demographic information was collected from participants. they also completed a 12-item multiple choice test about symptom recognition in ad-hies and promis self-efficacy for managing symptoms, an 8item validated survey. then, patient education handouts that focused on pulmonary symptoms, eczema, bone health, and cardiovascular complications were reviewed with the participant. six weeks later, participants were asked to repeat the 12-item test and the self-efficacy survey. the demographic information, test, and self-efficacy were collected anonymously. results: 33 participants provided demographic information, completed the test and the self-efficacy survey. of the 33 participants, 15 were male and 17 were female. participants ranged in age from 18 to 66 years. 22/33 (67%) reported looking for information about ad-hies using search engines and most patients (91%) report that they have been given information about ad-hies from a doctor. 19/33 (58%) participants identified pulmonary symptoms as the symptom that concerns them most and 10/33 (30%) participants identified more than one symptom of concern. 25 participants returned the second test and second survey. the mean test score increased from 9.08 to 10.28 with 23/25 participants achieving a score of 9/12 or higher. the self-efficacy scores were unchanged with a mean score of 50.08 before reviewing the patient education handouts and 50.13 after. conclusions: participant feedback to this project was generally positive. ad-hies patients are seeking information and an educational intervention can improve their understanding of disease. self-efficacy results were mixed and unchanged overall, but suggest that ad-hies patients manage symptoms as well as other patients with chronic illnesses. patient education should continue at each encounter. this project can be expanded to include more topics, pediatric patients, and other rare disease populations. funded by the nci contract no. introduction: bcl11b plays an important role in the development and maintenance of the immune system and the central nervous system. expression of bcl11b represses nk and myeloid factors while inducing t cell lineage genes in thymocytes at the dn2 stage. conditional loss of bcl11b expression in murine thymocytes leads to t cell deficiency while complete knockout of bcl11b was fatal within a few days of birth. recently, specific heterozygous bcl11b mutations have been reported in 11 individuals with global development delay. however, only 2 of these cases, both carrying heterozygous missense variants, had low trec values with 4 other cases having frequent infections. little is known regarding the impact of bcl11b on human nk and t cell function. methods: we identified a novel heterozygous truncating mutation in bcl11b in an infant who was first detected by trec newborn screening. she subsequently developed severe autoimmune hemolytic anemia at the age of 3 months. we used standard immunoblotting and flow cytometry methods to assess protein expression and the impact of this bcl11b mutant on t cell and nk cell development and function. results: the patient has a novel single base-pair deletion in the bcl11b gene, which is predicted to produce a truncated protein with the loss of 3 of 6 zinc finger domains in bcl11b. immunoblotting of t cell blast lysates revealed a reduced bcl11b expression in the patient consistent with the heterozygous defect in bcl11b but also generated a novel band with a smaller molecular weight that we postulate represents the truncated protein product. while mitogen responses to cona and pha were normal, both cd4+ and cd8+ t cell counts were decreased, especially cd4+ naã¯ve and cd4+cd31+ naã¯ve t cells, suggesting reduced thymic output. the function of th1 cells was skewed with reduced il-2 production but increased ifn levels after pma and ionomycin stimulation. moreover, t regulatory cell counts were below normal range. nk cell counts were normal but these were mostly cd56bright nk cells. of the few cd56dim nk cells that presented, approximately half did not express cd16, the fc receptor for adcc. perforin was only present in cd16 expressing nk cells. as such, anti-cd16 stimulation understandably led to low but not defective nk cell degranulation. function after stimulation with k562 cells was normal when controlled for nk cell counts. conclusion: we report a novel bcl11b truncating mutation with a leaky scid phenotype that manifested with t-cell lymphopenia and autoimmunity. lowered thymic-derived naã¯ve t and regulatory t cells, skewed th1 cytokine response, and incomplete nk cell development suggests that bcl11b is important for the development and differentiation of multiple lymphocyte lineages. introduction: chronic diarrhea is one of the most common gastrointestinal complaints in patients with common variable immune deficiency (cvid) and can lead to life-threatening complications such as malabsorption and malnutrition. chronic diarrhea in cvid could be caused by infections, an inflammatory bowel disease-like picture, as well as malignancy. giardia lamblia is one of the most common parasites causing diarrhea in cvid (up to 40%), and can be refractory in these patients, leading to villous atrophy, weight loss, and failure to thrive. case report: a 41-year-old female with a history of cvid presents with chronic diarrhea and significant weight loss. her cvid was diagnosed by hypogammaglobulinemia (low levels of igg, igm, and iga), inadequate responses to protein and polysaccharide-based vaccines, decreased memory b cells (cd19+cd27+ 0.5%), and recurrent sinopulmonary infections. she was started on immune globulin replacement therapy and had significant improvement in her rate of infections. four years before her presentation to our center, she developed chronic, severe diarrhea. work up revealed giardia lamblia infection on endoscopy and colonoscopy. biopsy showed intraepithelial lymphocytes, villous blunting, and atrophic gastritis with rare plasma cells concerning for non-infectious enteropathy related to her cvid, in addition to the high burden of giardia organisms. she was initially treated with metronidazole for several weeks. however, her diarrhea did not improve, and she developed significant peripheral neuropathy leading to lower extremity weakness and limited mobility. her diarrhea persisted and was associated with approximately a 20-pound weight loss. repeat endoscopy and colonoscopy two years later showed persistent high burden giardiasis of the small intestine, as well as reactive lymphocytic infiltrates and atrophic gastritis. she was treated with nitazoxanide but continued to have diarrhea, and her stool continued to show trophozoites. given the significant inflammation and the lack of response to multiple antiparasitic agents, she was referred to our center for further evaluation. she was started on oral budesonide (9 mg daily) and oral immune globulin (20 grams weekly for 12 weeks). with this regimen, she had significant improvement in her diarrhea with a 10-pound weight gain. repeat colonoscopy showed considerable improvement in inflammation and resolution of her giardia infection, though her stool antigen continues to be positive. conclusions: persistent diarrhea in our patient is most likely due to a combination of cvid enteropathy and giardiasis. a prolonged course of metronidazole and later nitazoxanide did not control her diarrhea and led to significant side effects. switching to an immunomodulatory approach significantly decreased the inflammation in her bowel and may even have helped to reduce the burden of giardia in the gut. targeting both underlying bowel inflammation as well as active infection in cvid patients with chronic diarrhea might be needed to control symptoms. introduction: sphingosine-1-phosphate (s1p) is a lipid chemoattractant that is critical for lymphocyte egress from lymphoid organs. following a s1p concentration gradient maintained by s1p lyase ubiquitously expressed in tissues, lymphocytes within lymphoid organs are drawn to efferent lymph and blood unless their s1p receptor is internalized or downregulated. owing to diminished degradation of not only s1p, but also other sphingoid bases, deleterious mutations in sgpl1 (encoding s1p lyase) perturb sphingolipid catabolism in numerous tissues. correspondingly, human s1p lyase deficiency results in multiorgan dysfunction including kidney, skin, endocrine gland, and neurologic impairment alongside expected lymphopenia. although severe t cell lymphopenia (<300 cells/microliter) rivaling that of severe combined immunodeficiency (scid) can be seen in patients with s1p lyase deficiency, no such patients have been identified by newborn screening of t cell receptor excision circle (trec) counts, which are a surrogate measure of effective t cell production. herein, we describe an infant boy with an undetectable trec count at birth who was found to have two novel, biallelic sgpl1 mutations resulting in s1p lyase deficiency. case description: a 1-day-old boy with a preceding history of fetal hydrops is born at a gestational age of 36 weeks and presents with renal failure, anasarca, and respiratory failure. trec analysis of a dried blood spot obtained at 24 hours of life reveals zero copies/microliter. subsequent peripheral blood studies show profound lymphopenia, with diminished cd3+ t (129/microliter; 96 cd4+, 27 cd8+), cd19+ b (130/microliter), and cd16/56+ natural killer (124/microliter) cell counts. recent thymic emigrants are reduced (11.3% of cd4+ t cells are cd45ra+cd31+), as is the ratio of naã¯ve-to-memory cd4+ t cells (63% cd45ra+, 37% cd45ro+). expedited whole genome sequencing identifies two novel variants in sgpl1 a paternally inherited splice site variant (c.1566+2t>c) predicted to impact a canonical splice donor site, and a maternally inherited missense change (c.854g>a; p.cys285tyr) located in a well-established functional domain of s1p. in addition to nephrotic syndrome and lymphopenia, the patient displays evidence of adrenal insufficiency and has increased plasma levels of sphingoid bases and ceramides. before further analyses could be pursued, the infant dies at 40 days of age due to ongoing complications of renal failure and eventual cardiorespiratory failure. summary: we report the first case of s1p lyase deficiency identified by newborn trec screening for scid. as sgpl1 is not included in most commercially-available, scid-tailored gene panels, s1p lyase deficiency would be missed by conventional genetic testing. therefore, analysis for variants in sgpl1 should be considered in neonates with low-to-undetectable trec counts, nephrotic syndrome, and other suggestive sequelae. w a r t s , hypogammaglobulinemia, recurrent infections, and myelokathexis) is a rare autosomal dominant primary immunodeficiency. it is caused by a defect in the gene encoding the chemokine receptor cxcr4. this receptor, along with the associated ligand cxcl12, regulates leukocyte migration. we present the case of a 40-year-old female, who presented after she self-identified the signature signs of whim syndrome in herself and multiple family members. objectives: we present the case of a 40-year-old female who presented with a history of recurrent warts, leukopenia of unknown cause, and recurrent infections as a child. as a child, she experienced multiple ear and sinus infections, along recurrent warts on her upper and lower extremities that have persisted to this day. furthermore, during a routine examination when she was 14-years-old, she had a complete blood count drawn significant for leukopenia. no further workup was undertaken at that time. when continued leukopenia was noted at the age of 30, referral to a hematologist and a bone marrow biopsy was completed. bone marrow was significant for myelokathexis with borderline hypercellular marrow for patient age (80% cellularity), and normal cell line quantity. a trial of neupoegen was undertaken, without significant improvement. her family history is significant for father and brother with both leukopenia and recurrent warts. results: genetic analysis showed a heterozygous pathogenic variant in the cxcr4 gene, c.1012_1015dup (p.ser339phe fs*6). recent complete blood count was significant for a total wbc count of 1.0 k/ul, with a differential consisting of 30% neutrophils and 57% lymphocytes. lymphocyte subsets were significant for quantitatively low cd3+, cd8+ and cd19+ subsets, with normal numbers of cd4+ and nk cells. immunoglobulin levels revealed an igg of 835 mg/dl, iga of 145 mg/ dl, and igm of 54 mg/dl; igg anti-diphtheria and tetanus titers were protective, however, none of the 23 s. pneumoniae serotype titers were > 1.3 ug/ml. mitogen (pha, cona and pwm) and antigen (candida and tetanus) stimulation of lymphocytes were normal for all stimuli. conclusions: we present the case of a 40-year-old female with a history of recurrent infections, warts, and myelokathexis. on genetic analysis, she is noted to have a pathogenic mutation of the cxcr4 gene. the substitution of a phenylalanine for a serine decreases one of the seven serine phosphorylation sites in the carboxy tail of the molecule that occurs upon binding to its ligand, cxcl12 (sdf1). additionally, the variation generates a premature stop condon terminating the remainder of the carboxy terminal amino acids including ser346-7, known to have a role in carboxy terminial beta-arrestin binding. failure to generate adequate beta-arrestin binding sites leads to prolonged cxcr4 cxcl12 interaction resulting in myelokathexis. background: lacking protective antibodies, patients with primary antibody deficiencies (pad) suffer from frequent respiratory infections leading to chronic pulmonary damage. macrolides prophylaxis has been proven effective to successfully manage chronic lung diseases as cystic fibrosis, bronchiectasis, copd. we conducted a trial to evaluate the efficacy and safety of orally low-dose azithromycin prophylaxis when added to the usual care in pad patients. methods: a 3-year, phase ii, prospective, multicenter, randomized, double-blind, placebo-controlled trial on pad patients (age 18-74 years) with chronic infection-related pulmonary disease. patients received azithromycin 250 mg or placebo once daily three-times a week for 24 months. the primary outcome was the decrease of annual episodes of respiratory exacerbations. secondary endpoints included: time to the first exacerbation, number of hospitalizations, additional doses of antibiotics, health related quality of life measures, and safety. results: forty-four patients received azithromycin and 45 patients received placebo. the mean number of exacerbations was 3â·6 per patientyear (95%ci 2â·5-4â·7) in the azithromycin arm, and 5â·2 (95%ci 4â·1-6â·4) in the placebo arm (p=0â·02). in the azithromycin group the hr for having an acute exacerbation was 0â·5 (95%ci 0,3-0â·9, p=0,03) and the hr for hospitalization was 0.5 (95%ci 0,2-1â·1) (p=0â·04). the rate of additional antibiotic treatment per patient-year was 2â·3 (95%ci 2â·1-3â·4) in the intervention and 3â·6 (95%ci 2â·9-4â·3) in placebo groups (p=0â·004). improvement in hrqofl was observed in intervention group. azithromycins safety prole was comparable with placebo. conclusion: in pad with respiratory exacerbation, azithromycin prophylaxis led to reduction of exacerbation episodes, of additional courses of antibiotics, and of risk of hospitalization. given the deleterious effects of respiratory diseases adding azithromycin to pad treatment should be considered as a valuable option. background: the autosomal-dominant hyper-ige syndrome (hies), is a primary immunodeficiency caused by mutations in signal transducer and activator of transcription 3 (stat3) that leads to defective th17 immunity. adverse reactions following 23-valent pneumococcal polysaccharide vaccine (ppsv23) have been reported in 75% of stat3-hies patients, including severe local reactions that appear to be specific to this vaccine. case report: we present the case of a six-year-old girl, second child of nonconsanguineous healthy parents, that developed an extensive inflammatory skin reaction at the vaccination site following a single dose of ppsv23. the vaccine was prescribed due to history of recurrent respiratory tract infections and an incomplete vaccine calendar with no previously administered pneumococcal vaccines. the reaction began after 2 hours with local erythema and edema at vaccination site, expanding in 48 hours to a phlyctenular lesion with no well-defined borders. within the first 3 weeks, it progressively evolved to a deep necrotic lesion that required surgical debridement. the subsequent skin defect required surgical repair with a split-thickness skin graft from her right thigh as the donor site. the complete wound healing process took about 5 months, leaving a large scar ( figure) . the patient had a longstanding history of recurrent infections with multiple hospitalizations including severe neonatal pneumonia that required respiratory support, a colon perforation with secondary peritonitis and septic shock that required a hemicolectomy at 8 months of age, recurrent oral candidiasis, recurrent pneumonias of different lobes, recurrent acute otitis media, a cervical phlegmon, three episodes of dental abscess and multiple kidney abscesses due to gram-negative bacteria treated with intravenous antibiotics and surgical drainage. family history is notable for an older sibling that died due to sudden infant death syndrome. the patients mother has large and wide nose suggestive of stat3-hies phenotype, but no history of infections. immunological work up showed mild eosinophilia (850 cells/ mm3), elevated ige (1850 mg/dl), normal igg, iga, igm and lymphocyte subsets (cd3, cd4, cd8, cd16, cd56). peripheral th17 cells were markedly decreased (0.6% vs. 3.7% of normal control). specific pneumococcal antibodies evaluated 1 month after psv23 revealed 5/10 serotypes in protective levels. high resolution thorax ct showed multilobar bronchiectasis. echocardiogram and total spine x-rays were normal. stat3-hies was suspected with a national institutes of health score of 40. a novel heterozygous missense variant in stat3 affecting the src homology 2 (sh2) domain (p.lys591glu) was found by next-generation panel sequencing. a variant in the same position (p.lys591met) has been previously reported in a hies patient (clinvar). currently, she is on monthly ivig and prophylactic antibiotics (cotrimoxazole, azithromycin and fluconazole). conclusions: the case presented raises awareness on the risk of severe local adverse reactions to ppsv23 in stat3-hies patients. the etiology of such reactions is unclear and warrants further study. the benefits and risks of immunizing stat3-hies patients with ppsv23 should be weighed carefully by medical providers. abstract (max 500 words) introduction: dock8 deficiency is a rare primary immunodeficiency characterized by susceptibility to viral infections, atopic eczema, defective t-cell activation and th17 differentiation, impaired eosinophil homeostasis and dysregulation of ige. to date, there are no reported cases from malaysia. objective: we aimed to describe the clinical, immunological profile and mutational analysis of three siblings of consanguineous parents, presented with hyper-ige and lymphopenia between the years 1998 and 2012, which were solved by mutational analysis of the second and third siblings. methods: clinical data and investigation results were collated from the medical record. scoring of the symptoms and physical examination findings using nih score was performed. t, b, nk lymphocyte subsets and serum igg, iga, igm, total ige quantification, lymphocyte proliferation test and pneumococcal specific antibody response were performed. mutational analyses were performed in freiburg, germany. result: three siblings presented at different time points over a 20-year span with raised ige levels, recurrent infections, eczema, hypereosinophilia and bronchiectasis. the nih scores for hyper-ige syndrome (hies) ranged from 39 54. we also documented two serious infections in the siblings, which were disseminated cryptococcus neoformans and salmonella sp. immunological results showed t-cell lymphopenia, defective t-cell proliferation, decreased igm, raised ige, hyper-eosinophilia and defective pneumococcal antibody responses present but not in all 3 siblings. we identified a large deletion in dock8 starting from exon 30-48 in 2 of the siblings from mutational analysis performed. we will proceed with next generation sequencing and dock8 protein assay in malaysia to further characterize the defect. conclusion: our on-going study is the first description of dock8 in a family from malaysia. the diagnosis of dock8 should be suspected in cases with raised ige levels, recurrent infections and lymphopenia, despite no warts infection in the history. this study emphasized the importance of international research collaboration and networking in solving complicated cases. the index patient presented at the age of 4 years with increased susceptibility to lower airway and gastrointestinal infections (hospital admissions 5x/year until puberty). she suffered from mumps and varicella disease despite immunization, as well as from recurrent local, partially destructive hsv infections. she was diagnosed with common variable immunodeficiency (cvid) at age 13 and started on immunoglobulin replacement therapy. following a hypoglycemic seizure at age 20, the patient was diagnosed with isolated acth insufficiency with secondary adrenal insufficiency requiring hormone substitution. during and following her first pregnancy at age 25, she suffered from recurrent bronchopneumonias including pneumocystis jirovecii infection, resulting in bronchiectases documented on chest ct at age 30. currently, chronic lung disease is severely limiting her quality of life (table 1) . her daughter was noticed to be hypogammaglobulinemic soon after birth and failed to develop antibody responses to inactivated vaccines. she was started on immunoglobulin replacement therapy. she has not suffered from severe lower airway infections, but developed alopecia totalis at age 10 and nail dystrophy. w h o l e e x o m e s e q u e n c i n g r e v e a l e d a h e t e r o z y g o u s c.2553_2554insacccgag (p.lys855profster33, nm_001077494) mutation in exon 22 of nfkb2 in both mother and daughter. this monoallelic loss-of function frameshift mutation was not found in gnomad, gvs washington or clinvar databases. as previously published, a monoallelic mutation in this c-terminal domain leads to impaired phosphorylation and subsequent reduced nuclear translocation of the nfkb2/p52 active form. pediatricians and internal specialists need to be aware of the combination of hypogammaglobulinemia, acth deficiency, immune dysregulation and ectodermal dysplasia which is unusual for cvid -possibly indicating nfkb2 deficiency. this clinical syndrome may overlap with symptoms and signs found in both apeced/ aire (ar) and eda-id/nfkbia (ad) deficiencies. besides ig and hormone replacement therapy, curative treatment with hematopoietic stem cell transplantation is a therapeutic option for patients with nfkb2 deficiency, although the experience is limited. table 1 introduction: the modes of immunoglobulin (ig) administration for primary immunodeficiency diseases (pidd) differ in pharmacokinetics, infusion parameters, and tolerability. during 3 consecutive clinical studies, a cohort of 30 patients with pidd experienced all 3 modes of administration with the same ig 10% product in sequence from intravenous (iv) to subcutaneous (sc), then to hyaluronidase-facilitated sc (ighy), providing a unique opportunity to assess each administration modality within the same patient cohort treated and observed at the same sites. here we report the rates of infections stratified by igg trough levels, and the rates of adverse events (aes) with the 3 modes of ig administration (ivig, scig, ighy) within this patient cohort. design and methods: this analysis included patients with pidd aged 4 years who participated in 3 clinical studies: in study 1 (nct00546871) patients received ivig 10% every 34 weeks followed by weekly scig 10%; in study 2 (nct00814320), patients were treated with ighy every 34 weeks; in study 3 (nct01175313; extension of study 2), patients continued with the same ighy dose. to assess a potential association between the administration route at comparable igg trough levels and the infection rate, igg trough levels were categorized as 500 <700mg/ dl, 700<900mg/dl, 900<1100mg/dl, 1100<1300 mg/dl, 1300 <1500 mg/dl and 1500 mg/dl. periods where patients had trough levels within these strata were assessed, and the infection frequency was calculated. the time periods for this analysis were 3 months for ivig and 12 months each for ighy and scig 10% (2.25 years) treatments. in order to account for differences in the frequency of administration, rates of systemic and local aes were assessed as aes/patient-year for each mode of therapy. results: for igg trough levels of <1500 mg/dl, the associated annual infection rates were lower or similar for ighy than scig ( the treatment involves the control of infections and immune dysregulation with chemotherapeutic regimens followed by definitive treatment with hematopoietic stem cell transplant (hsct). aim: to describe a female patient with a pathogenic variation in stx11 with normal cd107a expression. results: she was a 2 years old female, the 5th daughter of nonconsanguineous parents, without relevant personal or family records. she was admitted due to a prolonged febrile syndrome, lymphoproliferation, pancytopenia and hepatitis, with hhv6 rescued in bone marrow and blood. gancyclovir treatment started with good response. she was admitted one month later with similar clinical symptoms with relapsed hhv6 infection. furthermore, hemophagocytosis was found in the bone marrow and evaluation of nk cell cytotoxicity demonstrated slightly reduced cytotoxic activity. functional studies for primary fhl were performed: perforin expression and cd107a surface expression were normal. she fulfilled criteria of fhl, and treatment with gancyclovir and steroids was administered. despite this treatment, she persisted with activated macrophagic parameters, and started with hlh2014 treatment protocol. she improved the clinical symptoms and laboratory parameters, but persisted with hhv6 low viremia. three months later, when immunosupression was decreased, she was readmitted with similar clinical manifestations and added neurological symptoms (facial paralysis, abnormal movements and sleep tendency). cerebral spinal fluid was pathological with hhv6 positive rescue. immunosupresive treatment was adjusted, but hhv6 copies in blood increased markedly. foscarnet treatment was administered and immunosupression was suspended for 2 days in order to control viral infection. unfortunately the patient died 6 days later. although specific functional tests were normal, sequencing of stx11 gene by ngs revealed a homozygous variation in c.581_584deltgcc, which is a previously reported mutation responsible for fhl. conclusion: despite the fact that cd107a was normal, the strong clinical and laboratory results must keep the fhl diagnosis in mind and intensive treatment should be early administered; in order to give the patient the opportunity to achieve the curative treatment. objectives: to report and characterize the clinical course of a patient with apeced and specific antibody deficiency. methods: retrospective chart review was performed. the patient was enrolled in niaid irb-approved protocol 11-i-0187. results: the patient is a 13 year-old-girl with apeced caused by homozygous aire c.967_979del13, who manifested cmc, hypoparathyroidism, adrenal insufficiency, sjogrens-like syndrome, autoimmune hepatitis, intestinal dysfunction and autoimmune pneumonitis. she suffered from recurrent sinusitis and severe pneumonias requiring hospitalization and administration of intravenous antibiotics several times per year. at age 9, she presented to our institution with fever and cough, a computed tomography (ct) of the chest revealed bilateral pulmonary infiltrates and bronchiectasis. bronchoscopy showed mucopurulent secretions in the bilateral lower lobes with culture of the bronchoalveolar lavage fluid growing streptococcus pneumoniae. further evaluation for an underlying disorder such as primary ciliary dyskinesia and cystic fibrosis including exome sequencing and sweat chloride testing was unrevealing. quantitative immunoglobulins were normal. despite prior vaccination, specific antibody testing showed negative rubeola igg and protective levels (> 1.3 mcg/ml) to only 3 of 23 pneumococcal serotypes. lymphocyte enumeration showed normal b cell subsets. as approximately 15% of apeced patients may experience asplenia, splenic ultrasound was performed confirming the presence of a 7 cm spleen and peripheral blood smear did not reveal howell-jolly bodies. serotyping of the s. pneumoniae isolate confirmed serotype 33f, which is part of the 23-valent vaccine. follow up vaccine challenge with the 23 valent pneumococcal polysaccharide vaccine showed an inadequate response. hence, she was started on monthly immunoglobulin replacement and over the following 4 years she has experienced a single methicillin sensitive staphylococcus aureus pneumonia. she has missed very few school days and other parameters including linear growth have improved, she is now along the fifth percentile for height and along the tenth percentile for weight. although she continues to experience intermittent cough she remains active participating in sports without limitation. conclusions: we report the evaluation, treatment and outcome of a patient with apeced complicated by autoimmune pneumonitis and specific antibody deficiency. as infectious susceptibility of apeced classically pertains to the signature infectious disease, cmc, patients with invasive or recurrent infections should be evaluated for underlying immune deficiency. investigation should include assessment for asplenia, quantitative immunoglobulins and specific antibodies with response to antigens. in patients with predominate respiratory symptoms, autoimmune pneumonitis should be evaluated given the near 40% prevalence of pneumonitis observed in american apeced patients. acknowledgements: supported by dir/niaid/nih introduction: autoinflammatory diseases are genetically heterogeneous disorders of innate immunity characterized by recurrent fever, rash, and/ or serositis, which generally are considered distinct from autoimmune diseases. we report a case of a patient with lupus-like disease and a mutation of nucleotide-binding oligomerization domain-containing protein 2 (nod2 r702w, yao syndrome) suggestive of an overlap between autoinflammatory and autoimmunity processes. case presentation: a 72-year-old man was evaluated for recurrent pleural effusions, morning stiffness, erythematous rashes, and fever up to 103â°c. history was notable for hashimotos thyroiditis and multiple admissions for presumed pneumonia with recurrent bilateral lung infiltrates and pleural effusions. transbronchial biopsy showed nonspecific pneumonitis and organizing pneumonia. antinuclear and anti-dsdna antibodies were positive. he received prednisone for presumed lupus pneumonitis leading to improvement. prednisone was tapered and hydroxychloroquine was started, but his fevers, pleuritic pain and pleural effusion reoccurred. genetic testing revealed a nod2 sequent variant (r702w) associated with autoinflammatory disease. hydroxychloroquine was stopped and colchicine was added to his regimen, allowing prednisone to be tapered without recurrence of symptoms. further immunological testing revealed increased signaling through the type i interferon receptor (interferon signature). conclusion: although this patient had several clinical (serositis, arthralgia) and immunological (antinuclear and anti-dsdna antibodies, interferon signature) manifestations of lupus, his clinical presentation also was consistent with yao syndrome. in retrospect, he had been having recurrent inflammatory symptoms for many years. recent studies in both mice and humans suggest that inflammasome activation and il-1 production are involved in the pathogenesis of lupus. this case provides further support for the idea that lupus and hashimotos thyroiditis, prototypical autoimmune diseases, may have overlapping autoinflammatory features. background: the implementation of severe combined immunodeficiency (scid) newborn screening by trec assay has played a pivotal role in identifying these patients early in life. the screen has also led to the identification of infants with other immunologic abnormalities, of which the clinical implications have been unclear and there are limited data on their outcomes. objective: to review immunologic and genetic outcomes of infants referred to an immunology service of a tertiary care center with abnormal newborn scid screens. methods: we retrospectively reviewed charts of infants with positive scid screen from july 2014 to november 2018. we excluded patients who had positive screen at <36 weeks corrected gestational age. we classified outcomes into 3 groups including scid, non-scid t-cell lymphopenia (nscid-tcl) and normal t-cell count. idiopathic t-cell lymphopenia was defined as nscid-tcl (cd3+ < 2,500 cells/mcl) with negative chromosome microarray and negative whole exome sequencing/or genetic panel (either genedxâ® scid panel or invitaeâ® primary immunodeficiency panel). results: of 119 infants, 78% were male, 56% were caucasian, and 37% were african-american. fifty-four % and 46% of infants were identified by illinois and missouri screens, respectively. the mean age at initial evaluation was 22 days (4-122 days). 69% of infants had a normal tcell count (n=80) or normal repeat newborn screen (n=2), 25% had nscid-tcl, including mild (cd3+ 1,500-2,500 cells/mcl, n=20) and moderate (cd3+ 300-1,500 cells/mcl, n=10) tcl, and 6% had scid (n=5), leaky scid (n=1) or complete digeorge (n=1). genetic etiologies of nscid-tcl included 22q11 deletion (n=4), trisomy 21 (n=1), and mutations of tbx1 (n=2), foxn1 (n=1), and cd3e (n=1). three of these infants had novel variants at the time of diagnosis. secondary causes of tcl were identified in 1 infant (thoracic infantile fibrosarcoma). one infant had idiopathic tcl. eighteen infants with nscid-tcl were followed clinically without complete genetic testing performed. for scid, mutations were found in jak3 (n=2), ada (n=1), il2rg (n=1), and rag1 (n=1). the patient with leaky scid had negative whole exome sequencing. all patients with scid and leaky scid underwent hematopoietic stem cell transplantation at a median age of 5 weeks (3 weeks -4 months), with successful engraftment in all but 1 patient. of 19 idiopathic and nscid-tcl cases followed clinically, 12 had at least one follow-up visit at median age 5 months (2.6 months 2.2 years) and the majority had improved or stable lymphocyte count without serious infections requiring intravenous antibiotics, though 1 had a hospitalization for rsv infection. the mysm1 patient died after cord blood transplant from unclear etiology. our study had limitations. half of infants with nscid-tcl did not have a complete genetic workup, and only a fifth of patients with nscid-tcl were inpatients, potentially explaining the relatively low number of infants with secondary lymphopenia. conclusions: in our cohort, one-fourth of infants with abnormal scid screen had nscid-tcl. although the majority of nscid-tcl did well, approximately one-third of them had underlying genetic abnormalities associated with their t-cell lymphopenia. (189) submission id#606320 introduction: accumulation of intracellular adenosine and deoxyadenosine nucleotides (daxp) due to adenosine deaminase deficiency results in profound lymphopenia and severe combined immunodeficiency. left untreated this form of scid is uniformly fatal. while allogeneic hematopoietic cell transplant (hct) and autologous gene corrected stem cell therapy (gt) are potential cures for ada-scid , initiating enzyme replacement therapy (ert) immediately upon diagnosis regardless of definitive treatment is standard of care. hct and gt are not therapeutic options for all ada-scid patients and ert offers immediate therapeutic intervention for these patients leading to partial immune reconstitution, and durable survival in most patients treated. adagen (pegademase), approved by the fda in 1990 in the usa, is a pegylated bovine ada (nada) with the enzyme harvested from bovine intestines. this unsustainable production process led to the development of a recombinant enzyme source based on the bovine protein sequence and an improved pegylated linker by using succinimidyl carbamate (revcovitm-(elapegademase-lvlr). methods: a phase ii/iii clinical trial was performed at 5 us sites under institutional irb approval. eligible ada-scid subjects were stable on adagen and without complicating underlying conditions. demographics, medical history, lymphocyte counts, immunoglobulin levels, trough plasma ada activity and rbc daxp measurements were collected. patients were treated with adagen as a single, weekly im dose adjusted to achieve a trough plasma ada activity of > 15 mmol/hr/l and rbc daxp < 0.02 mmol/l (protocol target levels). once patients had achieved this level (3-9 weeks), a seven-day pk on adagen was done and the patients were transitioned to revcovi based on the formula for enzyme equivalent activity of 1mg revcovi = 150 units adagen. after 5 weeks on revcovi, trough ada and daxp were assessed and a seven-day pharmacokinetic study was conducted at week 9. patients were assessed periodically for clinical and laboratory values and evaluation of the study endpoints was done at week 21. subjects subsequently continued on revcovi and were assessed periodically. results: six patients, ages 16-37 entered the trial with initial adagen dosing at 7.7-42.9u/kg/wk (see table 1 ). adagen dosing was adjusted to target endpoints of ada trough activity (>15mmol/hr/l) and rbc daxp (<0.02 mml/l). patients transitioned to weekly revcovi using the aforementioned conversion formula at doses of 0.17-0.285 mg/kg/wk. the spectrum of clinical manifestations range from infections to autoimmunity and inflammation among patients with hypomorphic recombination gene 1 and 2 (rag1/2) pathogenic variants. auto-antibodies targeting cytokines ifn-alpha, ifn-omega and il-12 were reported in a large proportion of these patients and their occurrence often coincides with viral infections. we report the time of emergence and relative frequency of anti-cytokine antibodies in children and adults, and their persistence among patients with hypomorphic rag deficiency. antibodies were measured from plasma samples of patients by enzyme linked immunoassay (elisa). our rag cohort includes 28 patients with rag1 (n=17, 61%) and rag2 deficiency (n=11, 39%). antibodies targeting ifn-alpha (75%) were most common followed by il-12 and ifn-omega (40% each). two asymptomatic patients who were detected by newborn screening for scid and received hematopoietic stem cell transplantation had no detectable anti-cytokine antibodies. in the cohort of young children (ages 11 mo-7 years, n=9), all patients had detectable antibodies to ifn-alpha, prior history of severe viral infection and subsequently developed autoimmune cytopenias. other anti-cytokine antibodies were less common (ifn-omega 44%, il-12 33%). similarly, children between 10-18 yo age (n=9) also had high fraction of anti-ifn-alpha antibodies (89%) with prior history of infections (66%) and continued to have other anticytokine antibodies less commonly (ifn-omega 37%, il-12 62%). in the adult cohort (n=8, ages 25-39 years) the frequency of anti-ifnalpha anti-cytokine antibodies were lower (62%,) and il-12 and ifnomega (50% each) continued to persist. three adult patients had anticytokine (ifn-alpha, ifn-omega and il-12) antibodies tested at multiple timepoints and elevated titers persisted up to 4 years. our data demonstrates that anti-cytokine antibodies, especially those targeting ifn are frequent and emerge early in life in association with viral infections in patients with rag deficiency. a lower fraction of adult patients have detectable anti-cytokine antibodies, and maintain these over several years. anti-ifn-alpha may serve as a useful biomarker for identifying partial rag deficiency among young and adult patients with history of viral infections and autoimmune cytopenias. the role of these antibodies to cytokines is yet to be determined but a specific signature of these antibodies may help to identify an underlying immunodeficiency and initiate early definitive treatment with bone marrow transplantation. anti-cytokine antibodies appear to be a novel tool in evaluation of autoimmune diseases including rag deficiency. introduction: norovirus is one of the most common pathogens causing gastroenteritis in immunocompromised patients, often leading to chronic infection, causing villous atrophy, malabsorption, weight loss, organ failure, need for parenteral nutrition, and death. norovirus treatment in immunocompromised patients is challenging. oral immunoglobulin (poig) has been used to treat norovirus gastroenteritis with variable success. our aim in this study was to determine the outcomes of treating norovirus gastroenteritis in immunocompromised patients methods: electronic medical records were reviewed for patients with norovirus infection confirmed by rt-pcr since january 2012. our initial cohort was focused on patients with primary immunodeficiency (pid), lung, and liver transplant. data on demographics, immunological phenotype, treatment with poig, the number of bowel movements (bm), and virus clearance were collected. descriptive statistical methods were used to describe treatment outcomes. further analysis of patients immunophenotype, immunosuppression medications, and co-morbid illnesses is underway. results: twenty-six immunocompromised patients (27 norovirus infection episodes, as one patient had reinfection) were analyzed twelve females, age range 7 months-50 years. twelve patients had pid diagnosis (3 common variable immunodeficiency, 2 severe combined immunodeficiency, 1 x-linked agammaglobulinemia, 1 wiskott-aldrich syndrome, 1 digeorge syndrome, 1 hyper-igm, 1 stat3 gain-of-function, 1 nemo and 1 lymphopenia in a patient with trisomy 21), 13 patients were status-post liver transplant, and two patients were status-post lung transplant. 13 of26 patients were on ig replacement therapy at the time of the norovirus infection. the average number of bm/day in all patients was 8. 4 (range 2-20) . eight patients received poig (250-500 mg/kg) weekly for a duration from 1-12 weeks. three of those received additional nitazoxanide and 2 received ribavirin. 2/8 patients in the poig group were receiving total parenteral nutrition (tpn), and 4/19 on no treatment group received tpn. the average number of bm/day in poig before treatment was 9.5 (range 4-16), and 8.6 (range 2-20) in those who did not receive any treatment. 5 of 8 (62%) on poig vs. 11 of 19 (57%) in the no treatment group cleared the virus. the average number of weeks to return to baseline bm was 2.6 (range 1-7) in the poig group vs. 1.5 (range 3 days-5 weeks) in the no treatment group. 2 of 8 on poig continued to have chronic diarrhea that is still ongoing. conclusion: despite anecdotal reports suggesting successful use of poig in immunocompromised patients, our data did not show a significant decrease in stool output in patients treated with poig, compared to no treatment. however, poig led to a higher rate of virus clearance. a study with larger sample size might be warranted to identify the patients who benefit from poig in the context of norovirus infection and ensure the appropriate use of ig products, especially given the concerns for the national shortage of ig products. chief medical officer, novimmune sa primary hemophagocytic lymphohistiocytosis (phlh) is a life-threatening, immune regulatory disorder characterized by immune hyperactivation that is driven by high production of interferon (ifn)-. patients with hlh typically develop fever, splenomegaly, cytopenias and coagulopathy. until recently, there have been no fda approved treatments for hlh, and standard dexamethasone/etoposide-based treatment has not evolved significantly in 20+ years. emapalumab-lzsg (ni-0501) is a fully human, anti-ifn-monoclonal antibody that neutralizes ifn-and which was recently approved (november 2018) by the fda for the treatment of adult and pediatric (newborn and older) patients with phlh with refractory, recurrent, or progressive disease or intolerance with conventional hlh therapy. results of the pivotal trial supporting this approval are presented herein. methods: this open-label pivotal study (nct01818492) includes patients 18 years with a diagnosis of phlh and active disease. data presented were from 34 patients, of whom 27 had failed conventional hlh therapy prior to study entry. the initial emapalumab-lzsg dose was 1 mg/kg given intravenously every 3-4 days. subsequent doses could be increased up to 10 mg/kg based on the evolution of response parameters. dexamethasone was administered concomitantly at 5 to 10 mg/m 2 /day and could be tapered during the study. treatment duration was 8 weeks, with possible shortening to a minimum of 4 weeks, or extension up to the time of allogeneic hematopoietic stem cell transplantation (hsct). the primary efficacy endpoint was the overall response rate (orr) at end of treatment, assessed by pre-defined objective parameters, including normalization or at least 50% improvement from baseline of fever, splenomegaly, cytopenias, hyperferritinemia, fibrinogen, d-dimer, central nervous system (cns) abnormalities, and with no sustained worsening of scd25 serum levels. the primary analysis used an exact binomial test to evaluate the null hypothesis that orr be 40% at a one-sided 0.025 significance level. patients were eligible to enter an extension phase for follow-up after completing the main study (nct02069899). the data cut-off applied is july 20 2017. results: patient characteristics are summarized in table 1 and efficacy is summarized in table 2 . disease at study entry was consistent with the broad spectrum of phlh abnormalities. over 30% of patients had signs and/or symptoms of cns disease. orr was significantly higher than the pre-specified null hypothesis of 40%, meeting the primary endpoint. the response rate based on investigators clinical judgement was 70.6%. emapalumab-lzsg infusions were in general well tolerated, with mild to moderate infusion-related reactions reported in 27% of patients. the observed safety events (pre-hsct conditioning) mostly included hlh manifestations, infections or toxicities due to other administered drugs. infections caused by pathogens potentially favored by ifn-neutralization occurred in 1 patient during emapalumab-lzsg treatment (disseminated histoplasmosis), and resolved with appropriate treatment. no off-target effects were observed. conclusions: treatment with emapalumab-lzsg was able to control hlh activity with a favorable safety and tolerability profile in a very fragile population. the majority of patients proceeded to hsct with favorable outcomes. our results indicate that emapalumab-lzsg should be considered as a new therapeutic option in phlh thanks to its targeted mode of action. results: a total of 360 genes were differentially expressed between t cells of 22qds patients (n=13) and healthy controls (n=6) (log 2 fold change range (-2.0747, 15.6724)).when these 360 genes were tested for pathway enrichment, the top 5 pathways in t lymphocytes based on their p value included communication between innate and adaptive immune cells, cross talk between dendritic cells and natural killer cells, allograft rejection signaling, dendritic cell maturation, and b cell receptor signaling. the top 10 biological processes with differential expression included 36 immune response, 31 inflammatory response, 33 apoptotic process, 12 interferon gamma mediated signaling pathway, 14 nucleosome assembly, 16 defense response to virus, 8 lipopolysaccharide mediated signaling pathway, 7 positive regulation of nf-kappa b import into nucleus, 10 type i interferon signaling pathway, and 10 neutrophil chemotaxis genes. we compared gene expression between 22qds participants with low t cell counts (n=7) and 22qds participants with normal t cell counts (n=6) and found 94 genes that were differentially expressed (q<0.05) (log2 fold change range (-4.5445, 5.1297) patient began experiencing recurrent high fevers and developed splenomegaly. elevated transaminases and concern for lymphoproliferative disease prompted a splenectomy and liver biopsy. both the spleen and liver biopsy were positive for ebv but were negative for malignancy. bone marrow biopsy was unrevealing. genetic testing identified a pathogenic variant in xiap/ birc4 (1141c>t), and the patient was treated with high dose oral steroids resulting in an improvement in symptoms. subsequently, therapy with anakinra was started and steroids were tapered. during the steroid taper, he noticed a change in the vision of his left eye consistent with naion, as well as worsening of his colitis. there was loss of the inferior visual field and fundoscopic exam was significant for left optic disc swelling. oct noted superior retinal nerve fiber layer thinning. oral steroids were restarted with improvement in optic disc swelling, but without improvement or change in vision. as of his most recent exam, the patient has persistent bilateral inferior visual field defects with segmental optic nerve atrophy typical of naion. he has continued therapy with anakinra, and subsequently tapered off of prednisone; though he remains on a physiologic dose of hydrocortisone. conclusions: this case demonstrates an unreported ocular manifestation in a patient with xiap deficiency, which clinically appeared sensitive to immunomodulation. our patient is an unusual candidate for naion due to his young age, the average age of onset being the mid to late 60s, and lack of vascular risk factors. we hypothesize that his hyper-inflammatory condition contributed to irreversible vascular damage in the optic nerve head, resulting in naion. therefore, it may be useful to consider the involvement of systemic inflammatory and immune dysregulatory conditions when treating patients with atypical naion. additionally, naion should be considered in patients with xiap deficiency and sudden unilateral vision loss. the importance of de novo mutations in causing severe sporadic immune disease is well described, yet significance of such a variation in less severe and later onset of immune disease is poorly investigated. whole exome sequencing has been a powerful tool to resolve and explain the genetic basis of novel syndromes in immune related diseases. however, proving causation can be complicated due to low number of the affected individuals. we performed whole exome sequencing in a cohort of patients with noncongenital immune defects, along with detailed cellular biochemical phenotyping. we report and describe a novel non-congenital combined immune deficiency arising from a de novo gain-offunction mutation in ikbkb(c.607g>a). this gene encodes ikk2, and activates canonical nfkb signalling. cellular and biochemical studies of the proband revealed that ikk2v203i results in enhanced nf-kb signaling, as well as t and b cell functional defects. ikk2v203 is a highly-conserved residue, and to prove causation, we generated a crispr/cas9 mouse model that carry the precise orthologous missense mutation. we show that mice and humans carrying this missense mutation exhibits remarkably similar cellular and biochemical phenotypes. dysregulation in patients. total rna isolated from cryopreserved peripheral blood mononuclear cells was reverse transcribed to generate cdna. we selected four known gata2 transcriptional targets, gata1, gata2, tal1 and zfpm1 (encoding fog1) and used droplet digital pcr to quantify transcript levels normalized to the low-expressing gene tbp1. we used samples from 9 individuals with wild-type gata2 (wt), 5 known gata2 mutation patients (mut) and two individuals suspected of gata2 deficiency but without identified mutation or allelic imbalance (unk1, unk2). transcript analysis revealed significantly decreased transcript levels of gata1, gata2 and tal1 in mut pbmcs compared to wt. most wt samples had higher zfpm1 transcripts than gata2 mutated patients however it did not reach statistical significance. strikingly, we were able to use this analysis for two individuals suspected of gata2 deficiency. in the first case (unk1) a 51 yr old female with primary lymphedema, hypogammaglobulinemia, recurrent infections and possible family history of leukemia was referred for gata2 testing. no mutation was identified however it was noted that she was homozygous across the gene preventing allelic evaluation. the second patient (unk2), a 24 yr old female, had erethemya nodosa on legs, mycobacteria kansasii and cytopenias. in each of the targets analyzed, transcript levels from unk1 were lower than the wt samples and in a similar range as the gata2 mutation samples while unk2 had a profile consistent with the wt samples. we propose the use of gata2 targets as surrogate markers in cases where a mutation is not identified and allelic expression analysis is uninformative. are often under-reported and under-recognized. we sought to further understand and evaluate the prevalence, type, and association with serum immunoglobulin e (ige) for cvid patients with atopic manifestations. methods: we performed a retrospective analysis of cvid patients with atopic manifestations in the partners healthcare cvid cohort. we evaluated baseline patient characteristics, atopic diagnoses, and serum ige levels. results: in the partners cvid cohort, the average age was 52 years old (â±17) and 64% female. 92/175 (52.6%) of patients had a diagnosis of asthma, with the majority of these diagnosed by an allergist (65%) or pulmonologist (16%). eczema/atopic dermatitis was diagnosed in 47/175 patients (26%), by either an allergist (53%) or a dermatologist (8%). allergic rhinitis was diagnosed in 50/175 (28.5%) with positive skin prick testing in 52% of these patients. food allergy was diagnosed in 5 patients (2.9%). the median cohort serum ige was 7.5 iu/ml. the median serum ige was higher in patients with 2 or more atopic complications compared to those with one or less atopic condition (9 vs. 5 iu/ml), which was statistically significant (p=0.01). conclusions: we report higher rates of atopy than previously described in other cvid cohorts. consistent with previous reports, we find a low median cohort serum ige level in cvid patients compared to the general population. however, we identify a subset of patients with a predisposition towards atopy and higher ige levels within the broader characterization of cvid, and these patients may have a more specific molecular diagnosis that leads to elevated ige and atopic conditions. whole exome sequencing is underway to further evaluate this hypothesis. whim (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a primary immunodeficiency with autosomal dominant inheritance. in most patients, the genetic cause of the disease is a gain-offunction variant in c-x-c chemokine receptor type 4 (cxcr4) that results in arrest of neutrophil migration from the bone marrow. most patients develop hypogammaglobulinemia and early waning of antibody response with vaccination. however, the exact origin of aberrant humoral immunity in whim syndrome patients is yet to be clarified. here we describe a 4-year-old iraqi female with a heterozygous cxcr4 p.ser338ter variant, which is presented with haemophilus influenzae meningitis, history of tetralogy of fallot, early onset intermittent neutropenia, lymphopenia, recurrent bacterial and viral infections. immunologic evaluation revealed hypogammaglobulinemia, elevated igm level and a lack of protective vaccine titers after tetanus and prevnar vaccinations. a bone marrow biopsy was consistent with myelokathexis. immune phenotyping, functional studies and apoptosis assays were performed on peripheral blood cells by flow cytometry in our whim patient and controls. although we found that all lymphocyte compartments were reduced, naã¯ve cd4 t helper cells and switched memory b cells were predominantly affected. spontaneous apoptosis was most pronounced in b rather than t cell compartments in whim patients. in addition, naã¯ve b cells easily activated and died upon activation in vitro. cxcl12, a ligand of cxcr4, induced elevated t helper cell migration and increased actin polymerization in p.ser338ter mutant cells. we conclude that intrinsic b cell abnormalities, such as increased rate of apoptosis and altered activation, might be responsible for defective antibody response in whim patients. although most individuals effectively control herpesvirus infections, some suffer from unusually severe and/or recurrent infections requiring anti-viral prophylaxis. a subset of these patients possesses defects in nk cells, innate lymphocytes which recognize and lyse herpesvirus-infected cells; however, the exact genetic etiologies are rarely diagnosed. plcg2 encodes a signaling protein in nk cell and b cell receptor-mediated signaling. dominant-negative or gain-of-function mutations in plcg2 cause cold urticaria, antibody deficiency, or autoinflammation. however, loss-of-function mutations and plcg2 haploinsufficiency have never been reported in human disease. we examined 2 families with autosomal dominant nk cell immunodeficiency with mass cytometry and whole-exome sequencing to identify the cause of disease. we identified two novel heterozygous loss-of-function mutations inplcg2 that impaired nk cell function, including calcium flux, granule movement, and target killing. although expression of mutant plcg2 protein in vitro was normal, phosphorylation of both mutants was diminished. in contrast to plaid and aplaid, b cell function remained intact. plcg2+/-mice, as well as targeted crispr knock-in mice, also displayed impaired nk cell function with preserved b cell function, phenocopying human plcg2 haploinsufficiency. we report the first known cases of plcg2 haploinsufficiency, a clinically and mechanistically distinct syndrome from previously reported mutations. therefore, these families represent a novel disease, highlighting a role for plcg2 haploinsufficiency in herpesvirus-susceptible patients and expanding the spectrum of plcg2-related disease. we pursued genetic diagnosis, which identified bi-allelic frameshift mutations in the rag1 gene which had not been previously described: c.967delg (p.v323sfsx22) and c.1048_1075del128insaaaagagtg (p.v350kfsx47). taken together, his presentation suggested significant immune dysfunction had evolved since transplant leading to extensive pulmonary nontuberculous mycobacterial infection and possible bronchiolitis obliterans. he therefore will undergo a subsequent unconditioned cd34+ stem cell boost from his sister, the original donor, once he completes mycobacterium abscessus treatment. this case highlights the potential long-term immune dysfunction which may evolve after unconditioned allogeneic stem cell transplant for scid, in which full engraftment in all myeloid and lymphoid compartments is not expected. it also highlights the importance of guideline-driven follow-up of these patients to monitor for said dysfunction, to prevent serious infection and long-term sequelae. somatic hypermutation (shm) in the b cell receptor (bcr) heavy (igh) and light chain genes promotes affinity maturation and also mutation away from self-reactivity, therefore serves as an important peripheral tolerance checkpoint. as an example, unmutated bcr ighv4-34 genes give rise to antibodies that bind to i/i antigen on red blood cells (rbc) and may elicit cold agglutinin disease (cad), a variant of autoimmune hemolytic anemia (aiha). in case of healthy individuals, frequent shms in the i/i binding site of bcr ighv4-34 genes decrease rbc reactivity and cad. patients with primary immunodeficiencies (pid) paradoxically develop autoimmune diseases, including autoimmune cytopenias, especially aiha. it is unclear if impaired shm of bcr, in particular mutation away from i/i binding, is relevant in the development of rbc reactivity and consequently aiha in a pid background. our studies focus on pid patients with hypomorphic recombination activating gene (rag1 and 2), combined immunodeficiency phenotype and history of autoimmunity, in particular aiha (rag cid/ai). we detected increased frequency of unmutated ighv4-34 bcr in memory b cell repertoires of rag-cid/ai patients as well as elevated titer of unmutated ighv4-34 antibodies in the patients' plasma. lower level of shm likely reflect abnormal germinal center (gc) reaction. as rag1 and 2 heterotetramer primarily shapes the pre-immune t and b cell repertoire, we studied the interaction of follicular helper t cells (tfh) and naive b cells via in vitro co-culture experiment. interestingly, tfh cells from rag cid/ai patients exhibited highly activated phenotype with increased expression of cd40l and il-21 compared to healthy controls and were able to initiate exaggerated response (class switching and shm) of healthy donor naive b cells. on the contrary, in vitro activated naive b cells from rag cid/ai patients showed impaired proliferation, class switching and decreased level of shm with diminished induction of genes involved t cell co-stimulation (cd40, il-21r) and shm (aicda, repair enzymes) compared to healthy donor naive b cells indicating intrinsic defect in patient b cells. furthermore, b cells from rag cid/ai patients also showed increased apoptosis and accumulation of gamma-h2ax foci at steady state indicating reduced cellular fitness. these findings suggest that the development of aiha is a multifactorial process in partial rag deficiency. our studies highlight that impaired germinal center reaction is an important tolerance checkpoint with the inability of patient's b cells to respond to hyperactive tfh cells and introduce proper level of shm. hence, we propose that b cell fitness is compromised which impairs proper gc interaction, shm, including mutation away from self and sustains rbc reactivity in hypomorphic rag deficiency. introduction/background: the forkhead box n1 (foxn1) transcription factor is an essential regulator of t cell development, affecting the differentiation and expansion of thymic epithelial cells (tecs). autosomal recessive mutations in foxn1 cause a t-b+nk+ lymphocyte phenotype due to a thymic aplasia in conjunction with alopecia universalis and nail plate dystrophy resulting from keratinocyte dysregulation. this is a classic nude/scid (omim # 600838) phenotype. we report on the identification of two independent patients, identified through newborn screening with absent trecs and with a t-nk+b+ scid phenotype who presented with a t cell lymphopenia who had compound heterozygous mutations in foxn1. notably, these individuals had normal hair and nail beds. objectives: to determine whether distinct compound heterozygous mutations in foxn1 cause a novel t-nk+b+ phenotype in the absence of a classic nude presentation. neutralizing autoantibodies (autoabs) against cytokines increase the susceptibility for selected infections (e.g. anti-ifn-autoabs for nontuberculous mycobacteria and non-typhoid salmonella, anti-il-17-autoabs for mucocutaneous candidiasis and anti-gm-csf-auotabs for infections by cryptococcus, nocardiae and aspergillus spp). however, the role of anti-il-6-autoabs is less clear. il-6 is a key mediator of the acute-phase response and released early in bacterial infections. patients with impaired signaling or affected production of il-6 are at increased risk for severe bacterial infections. only three patients with high-titer and neutralizing anti-il-6-autoabs who suffered from severe infections caused by s. aureus, s. intermedius and e. coli have been described so far. to investigate the prevalence of anti-il-6-autoabs in patients with bacterial infections, we investigated a cohort of 350 patients and identified three further patients, all previously healthy, with neutralizing auotabs against il-6 who hardly developed an acute-phase response. the first patient suffered from life-threatening pneumonia caused by s. pneumonia, the second patient developed a submandibular abscess and septic arthritis caused by s. pyogenes and the third patient suffered from life-threatening pneumonia caused by s. aureus. we also discovered neutralizing anti-il-6-autoabs in two adults among a cohort of patients with autoimmune diseases (n = 564), in one adolescent among a cohort of obese individuals (n = 455) as well as in three mothers of neonates with impaired il-6 signaling. so far none of the later individuals developed a severe bacterial infection. this suggests that naturally occurring and neutralizing anti-il-6-autoabs are a risk factor for severe bacterial infections yet with incomplete penetrance. (215) submission id#606931 persistent transaminitis in copa syndrome 1 researcher, immunodeficiencies research unit, national institute of pediatrics, mexico city 2 social service intern, immunodeficiencies research unit, national institute of pediatrics 3 pediatrics resident, pediatrics hospital, 21st century national medical center, mexican institute of social security 4 researcher, data science department, mexican autonomous institute of technology 5 researcher, department of research methodology, national institute of pediatrics background: inborn errors of immunity constitute a heterogeneous group of over 400 individually rare congenital diseases that involve genes coding for proteins of the immune system, and which result in increased susceptibility to infection, inflammation, autoimmunity, allergy and cancer. the complexity of the diagnostic task, and the intrinsic biases and limitations of the human mind, can be aided by computational tools. among the available machine learning approaches, decision tree algorithms select the best node to split based on entropy and information gain; random forests build hundreds or thousands of decision trees randomly (bootstrapping), to improve accuracy and reduce overfitting. aim: to implement a machine learning-assisted clinical decision support system for the diagnosis of inborn errors of immunity (iei). methods: with a local database of patients with suspected iei, we built a decision tree using c4.5 dtc, and a random forest on python 3 (jupyter notebook, scikit, mathplotlib, pandas, numpy). the database was obtained by conducting an electronic search on medsys of patients with the term immunodeficiency in their electronic medical records, and then hand-picking cases in which an iei had been confirmed or ruled out. it consisted of 234 patients, of which 201 had been diagnosed with iei. we first split the dataset randomly into training (70%) and testing (30%) sets. the decision tree was tasked with classifying correctly pid or not. after running the algorithm in the training set, we evaluated in the testing set. the random forest classified all cases by majority vote into nine groups (0 to 8), according to the iuis pid group. next, we repeated the process on a larger scale with a dataset of 2,400 patients from usidnet. accuracy was assessed by out-of-bag (oob) error estimates. results: accuracy was greater than 95% for the local dataset (pid/ not, 9 groups), and for the usidnet dataset (9 groups). we provide a list of decision nodes and a diagnostic route with those questions that achieved a greater information gain and less entropy. this might help clinicians direct their interrogation and diagnostic approach of suspected iei patients. discussion: we built two classification models. decision trees lend themselves more easily to learning and deriving rules of thumb from their sequences. random forests are more robust and better suited for categoric (as opposed to binary) classification. we next want to develop a chatbot that will ask relevant questions in optimal sequence, and extract undiagnosed patients with suspected iei, based on statistical red flags. 13 researcher, immunodeficiencies research unit, national institute of pediatrics, mexico city dna repair defects are inborn errors of immunity that result in increased apoptosis and oncogenesis. dna ligase 4-deficient patients suffer from a wide range of clinical manifestations since early in life, including: microcephaly, dysmorphic facial features, growth failure, developmental delay, mental retardation; hip dysplasia, and other skeletal malformations; as well as a severe combined immunodeficiency, radiosensitivity and progressive bone marrow failure; or, they may present later in life with hematological neoplasias that respond catastrophically to chemo-and radiotherapy; or, they could be asymptomatic. we describe the clinical, laboratory and genetic features of five mexican patients with lig4 deficiency, together with a review of 36 other patients available in pubmed medline. four out of five of our patients are dead from lymphoma or bone marrow failure, with severe infection and massive bleeding; the fifth patient is asymptomatic despite a persistent cd4+ lymphopenia. most patients reported in the literature are microcephalic females with growth failure, sinopulmonary infections, hypogammaglobulinemia, very low b-cells, and radiosensitivity; while bone marrow failure and malignancy may develop at a later age. dysmorphic facial features, congenital hip dysplasia, chronic liver disease, gradual pancytopenia, lymphoma or leukemia, thrombocytopenia and gastrointestinal bleeding have been reported as well. most mutations are compound heterozygous, and all of them are hypomorphic, with two common truncating mutations accounting for the majority of patients. stem-cell transplantation after reduced intensity conditioning regimes may be curative. 1 department of laboratory medicine, clinical centre 2 immunology, allergy and rheumatology division, department of pediatrics, baylor college of medicine, texas children's hospital, houston,texas, usa 3 laboratory of clinical immunology and microbiology, fungal pathogenesis section, national institute of allergy and infectious diseases, 4 department of intramural research, national institute of allergy and infectious diseases (niaid), national institute of health, bethesda maryland, usa card9 deficiency is an autosomal recessive primary immunodeficiency known to underlay increased fungal infection susceptibility mostly presenting as invasive cns candida infections (in infancy or adulthood) and dermatophyte infections. more recently, a rare card9 variant (c.1434+1 g>c, leading to exon 11 skipping, card9del11) showed a significant protective association towards inflammatory bowel disease (ibd) when present in heterozygosity. at the nih we studied an 8-year-old male patient (p1) born to a non-consanguineous marriage who presented as an infant with recurrent/severe thrush, candida esophagitis, and an episode of tinea pedis; p1 also has mild hypogammaglobinemia (igg 500mg/dl at age 8y). p1s gdna was tested by whole exome sequencing and showed a card9 c.1434+1 g>c mutation in homozygous state. segregation analysis and sanger confirmation determined that both parents and p1s elder brother carried the same variant in heterozygosity, while his asymptomatic younger brother (p2) was also homozygous. as previously described, this variant caused card9 exon 11 deletion as determined in p1 and p2s pbmcs by cdna sequencing and by a lower molecular weight card9 protein by immunoblot evaluation. p1 and p2s pbmcs, as well as the heterozygous parents cells, showed a defective cytokine generation (tnf-, il-1, il-6 and gm-csf) in response to heat killed candida (hkc), but not to lps. while patients pbmcs failed to induce phospho-erk and phospho-p-38 upon hkc-stimulation but presented an intact response to pma+ionomycin; the parents cells responded normally to both stimuli. moreover, t-cell activation and proliferation was affected in response to hkc but not to pha in both patients, whereas the parents exhibited normal results under the same conditions. when hek293 cells were transiently transfected with wt or card9del11 vectors together with a trim62 plasmid (e3-ubiquitin ligase, naturally associated to card9), we confirmed that card9del11 failed to bind trim62 by immunoprecipitation. furthermore, malt1, bcl10 and trim62 were only co-precipitated by wt card9, but no by card9del11, strongly suggesting trim62 is an integral part of the card9/bcl10/malt1 -cbm-complex. in summary, herein we demonstrate that the card9del11 allele fails to bind trim62, and in turn is unable to conform a complete/functional cbm complex. our data also show that card9del11 acts in a dominant negative fashion in terms of cytokine generation (previously reported), but one wt allele seems sufficient to generate normal levels of hkcinduced p-erk and p-p-38, as well as t-cell proliferation. while decreased cytokine generation associated with card9del11 in heterozygosity has been described to be sufficient to protect towards ibd, other defective pathways are affected in homozygosity and likely necessary to confer increased susceptibility to fungal infections. altogether these results suggest that card9del11 acts through a gene dosage mechanism that can dissect pathways that associate ibd protection and fungal infection susceptibility. further work is warranted to explore card9del11 role, if any, in b-cell and t-cell biology. professor, endocrinology, university of michigan medical school background: acquired generalized lipodystrophy (agl) syndromes are a heterogeneous group of diseases characterized by selective dysfunction and loss of adipose tissue after birth. this causes ectopic lipid deposition and deficiency of the adipokine leptin, which promotes metabolic dysfunction through impaired glucose handling resulting in insulin-resistant diabetes mellitus, dyslipidemia and steatohepatitis. while the metabolic effects of altered adipokine secretion are known, the molecular mechanism is less clear. many agl cases are suspected to have an autoimmune etiology. effector and regulatory t cells, dendritic cells and macrophages reside in normal adipose tissue. t cells within adipose tissue highly express pd-1 and regulatory t cells express ctla4, which limits immune activation in the adipose tissue under normal circumstances. thus, inhibition of these immune checkpoints may hypothetically cause immune activation, leading to adipocyte dysfunction and autoimmune destruction. we have encountered two cases that raise clinical concern for this process. patient cases: patient 1 is a 16-year-old female who presented with failure to thrive at 6 months. she was diagnosed with insulin-resistant type 1 diabetes and hypertriglyceridemia at ages 2 and 4 years with progressive subcutaneous fat loss and low leptin levels culminating in a diagnosis of agl. her childhood clinical course was complicated by hypertrophic cardiomyopathy, hepatomegaly, autoimmune hemolytic anemia with massive splenomegaly and severe chronic diarrhea secondary to autoimmune enteropathy. she presented at 14 years with acute liver failure, thrombotic microangiopathy, nephrotic syndrome and progressive kidney insufficiency. evaluation for her multi-faceted autoimmune presentation identified a familial heterozygous pathogenic variant in the ctla4 gene (c.4_5insgttgg,p.ala2glyfster14). despite aggressive immune therapies, including ctla4-ig (abatacept), her kidney disease and enteropathy have progressed. patient 2 is a 55-year-old male diagnosed with localized malignant melanoma of the right neck in july 2014. he underwent excisional biopsy and regional lymph node dissection with negative margins. he relapsed in november 2017 and underwent a modified radical neck dissection with 1 lymph node positive for disease and received external beam radiation from january-february 2017. additionally, he was started on anti-pd-1 therapy with the humanized antibody drug pembrolizumab in april 2017 but discontinued the drug in february 2018 in the setting of toxicities including hypothyroidism. subsequently, he developed up to 7.5% weight loss with progressive loss of subcutaneous fat first in his face, then generalized to the rest of his body. in the ensuing months, imaging with pet-ct demonstrated loss of subcutaneous fat concurrent with elevations in alt and triglyceride levels plus a low leptin level consistent with agl. conclusion: these cases raise concern that inhibition of the immune checkpoints ctla4 and pd-1 may facilitate the development of agl. we hypothesize that these defects significantly increase t cell autoimmune activity in the adipose tissue and/or alter t cell metabolism resulting in agl. disorders of immune dysregulation should be considered in the etiology of agl. similarly, patients with either genetic or pharmacologic inhibition of immune checkpoints should be monitored for the development of agl with careful physical exam and periodic monitoring of glucose and triglyceride levels. background: rosai-dorfman disease (rdd; also known as sinus histiocytosis with massive lymphadenopathy) is a rare non-langerhans cell histiocytosis. it is characterized by proliferation and accumulation of activated histiocytes in affected tissues. classically, rdd presents with bilateral, non-tender, and often markedly enlarged cervical lymphadenopathy. case presentation: a 2-year-old female presented with a 6-week history of asymptomatic, persistent and bilaterally enlarged cervical lymph nodes. she was otherwise healthy with no significant past medical history. operative excision biopsy of the largest lymph node confirmed the diagnosis of rdd. three months following diagnosis, routine bloodwork revealed that she had developed lymphopenia (lymphocyte count 1.4 x 109/l). between 1-year and 2-and-a-half-years post-diagnosis, the patient was hospitalized and treated with intravenous antibiotics for 2 presumed episodes of osteomyelitis and 2 presumed episodes of lymphadenitis. given the recurrent presumed infections and persistent lymphopenia, the patient was referred to immunology for evaluation. she received a full immunologic work-up. lymphocyte immunophenotyping revealed low cd4 (288 cells/mm3) and low cd8 (228 cells/mm3) counts. the rest of her immunologic work-up was within normal limits. approximately 3-and-a-half-years post-diagnosis, the decision was made to initiate treatment for rdd. she was started on a 6-week tapering course of prednisone therapy. within 2-weeks of starting corticosteroid therapy, the lymphadenopathy had diminished, and by 6-weeks, the lymphopenia completely resolved. at her most recent clinic visit, she had been free of serious infections for more than 3-years, and her lymphocyte counts had remained stable and within normal limits for over one year. discussion: in the literature, immune system dysfunction has been reported in rdd, with both auto-antibodies and cellular immunodeficiency implicated. in this patient, the persistent lymphopenia and recurrent episodes of presumed infections appeared consistent with an immunodeficiency. given the known association of rdd with immunologic dysfunction, this was certainly a reasonable assumption; however, when these issues resolved following corticosteroid therapy, we questioned whether her clinical presentation could instead represent a manifestation of her underlying rdd. this case highlights the diagnostic challenge of differentiating between an infection and an rdd exacerbation. the episodes of presumed infections were considered probable but not confirmed with microbiologic or histopathologic specimens. the mechanism underlying lymphopenia in rdd is not clear but may involve decreased production, increased destruction, or sequestration of lymphocytes. to our knowledge, this has not been specifically studied in rdd in the past, however lymphopenia has been linked to lymphocyte maldistribution in other diseases. for example, studies have shown that experimentally altering either the surface of the lymphocyte or the environment through which the lymphocyte travels through can cause sequestration of lymphocytes in various lymphoid organs including lymph nodes. conclusion: we describe the case patient with rdd that developed persistent lymphopenia, and multiple episodes of presumed infections resulting in hospitalization and intravenous antibiotic therapy. her lymphopenia resolved and she had sustained remission of rdd following treatment with corticosteroids. we hypothesize that lymphocyte sequestration in enlarged lymph nodes may have resulted in lymphopenia. this, combined with recurrent rdd exacerbations that clinically resemble infections created a presentation that mimicked an immunodeficiency. background: there is an expanding spectrum of immunodeficiency phenotypes linked to dna repair defects, and some patients may not be diagnosed until adulthood. the most well recognized genetic defect linked to dna repair is in the gene, ataxia telangiectasia mutated (atm), which causes ataxia telangiectasia, characterized by combined immunodeficiency, neurodegeneration, radiation sensitivity, and ocular telangiectasias. however, there are several other dna repair defects associated with immunodeficiency, including some syndromic and severe combined immunodeficiency (scid) disorders. objective: we present the case of an adult patient with prolonged history of recurrent infections, facial abnormalities, and autoimmunity who was found to have radiosensitivity suggestive of a dna repair defect. methods: retrospective chart review, immunodeficiency evaluation, flow-based radiosensitivity assay, gene sequencing. results: a 68-year-old female was referred to our clinic due to a complex history of recurrent infections and immune dysregulation. the patient had a lifelong history of sinopulmonary infections and panhypogammaglobulinemia with low vaccine responses, leading to a diagnosis of common variable immunodeficiency (cvid), necessitating treatment with immunoglobulin replacement. clinical features were also notable for congenital dysmorphia (strabismus, thin and angular face, high arched palate, nasal septal defect, small mouth, missing dentition, clinodactyly, severe equinovarus, and scoliosis). she was subsequently diagnosed with autoimmune features of vasculitides requiring trial of cyclophosphamide, azathioprine, rituximab and belimumab, which was later discontinued due to neutropenia and worsening sinopulmonary and skin infections despite immunoglobulin replacement. in the course of our evaluation she was revealed to have severe b cell lymphopenia (1%), cd4 naã¯ve t cell lymphopenia, persistent iga and igm deficiency one-year post rituximab therapy, and elevated alpha fetoprotein (afp). radiosensitivity assay revealed decreased atm phosphorylation and elevated levels of h2ax 24-hours after low-dose (2gy) radiation in her lymphocyte subsets (t, b and nk cells) . due to the evidence of radiosensitivity and elevated afp levels, there was concern for an atm or other genetic defects in a dna repair pathway. therefore, a targeted (primary immunodeficiency genes) panel was pursued for genetic testing (207 genes, invitae, san francisco). the evaluation did not identify a variant in the atm gene but rather a variant of uncertain significance was identified in the chd7 gene, in exon 38, c.8440g>a (p.gly2814arg), which may be mosaic. this variant has not been reported in population databases. chd7 is typically associated with charge syndrome, and while this patient has some dysmorphic features, she is not typical for charge syndrome. currently, studies on copy number variation (cnv) and deep intronic variants in atm are pending. conclusion: dna repair defects may occur in adult patients with a primary diagnosis of cvid. our patient exhibits some phenotypic features of both a chd7 variant, and atm leading to possible abnormal dna damage responses (ddr). the exact cause of the immune deficiency in our case remains presently unsolved. this case highlights the relevance of both functional studies and genetic evaluation of complex cases of immune dysregulation, for improving our understanding of the phenotypic variability in these immunological disorders. background: womens health issues in patients with immunodeficiency are largely underrepresented in the literature. there are no studies assessing for fertility issues in patients with antibody deficiencies, and there are few sizable studies examining pregnancy and outcomes on progeny in the same cohort. the two largest studies of pregnancy in antibody deficiency, an idf survey and a study of the czech population, provide conflicting data about the safety of pregnancy for these patients. immunoglobulin replacement has been shown to be safe and beneficial in pregnancy for patients with cvid, however, dosing strategies are unguided. we sought to further understand these and other issues associated with fertility and pregnancy in a large cohort of patients with antibody deficiencies. methods: we performed a retrospective chart review of over 100 patients with icd9 and/or icd10 codes of cvid or another antibody deficiency from january 2005 to december 2018. inclusion criteria also comprised of having reached at least 16 years of age, the beginning of child bearing years. data collected included disease characteristics, comorbidities, laboratory values, and outcomes. this was followed by a phone survey to elucidate data regarding fertility, pregnancy, delivery complications, and outcomes of children. this study was irb approved. results: the current age of women included ranged from 16 to 88 years of age, currently being in childbearing years to being post-menopausal. forty percent of the women had been pregnant, delivering an average of 2 babies per woman who had been pregnant. fertility issues were not a prominent factor for women who never became pregnant. a majority of women who had babies (64%) did not receive a diagnosis of antibody deficiency until after their child bearing years. recurrent upper respiratory tract infections, bacterial sinusitis, and urinary tract infections during pregnancy were common even in those not yet diagnosed with antibody deficiency. immunoglobulin levels and dosing of intravenous and/or subcutaneous replacement were recorded for a subset of patients with recent pregnancies. the data re-enforced that increases in dosing are needed in the third trimester. cord blood igg levels were also recorded for baby and were the same or higher than the mothers most recent igg prior to delivery. it was rare for children of our patients to be diagnosed with antibody deficiency or a related condition, although cvid, hypogammaglobulinemia, combined immunodeficiency, lymphoma, rheumatoid arthritis, and other diagnoses were found. conclusion: this is the largest report of outcomes before, during, and after pregnancy for patients with antibody deficiencies in the united states. this report highlights the importance of closely monitoring women during pregnancy for recurrent infections regardless of whether a diagnosis of antibody deficiency is present. it also highlights that close monitoring of igg levels during pregnancy is necessary for women with antibody deficiencies. backgrounds: autoinflammatory diseases (aids) are a group of disorders with an inborn error of innate immunity, characterized by recurrent episodes of fever and inflammatory attacks. the spectrum of aids is expanding, but no data on clinical presentation and symptom variability exist for the iranian population for timely precise diagnosis. this study aims at establishing the first autoinflammatory registry of an iranian population focusing on the clinical and laboratory features that may help clinicians toward a better understanding and diagnosis of these disorders. methods: clinical and laboratory characteristics of patients who clinically and or genetically diagnosed with aids collected. we used the updated version of classification criteria from the eurofever registry for the clinical diagnosis. results: in our retrospective study, clinical and laboratory characteristics of the participants collected. mean age of disease onset, disease course manifestation, the mean duration of episodes, atypical symptoms, laboratory and imaging studies as well as complications, and response to treatment also reviewed. data resulted in 26 patients of whom 16 were male. their age ranged from 2 to 68 years. 5 out of 26 were genetically diagnosed. familial mediterranean fever (fmf) was the most common clinically and genetically approved diagnosis. there were also patients suspected of nlrp12 and nod2 mutations. age at disease onset differed variably and ranged from the neonatal period to adulthood. fever was present in all the participants and the duration of episodes was 1-10 days. the frequency of attacks was between 3 to more than 12 per year. some of the common clinical manifestations were as follows: myalgia or fatigue (77%), arthralgia and arthritis (70%), abdominal pain (65%), aphthous stomatitis (38%), chest pain (34%), chronic gastrointestinal symptoms (38%), skin lesion ranging from urticarial rash and severe nodular acne to pyoderma gangrenosum (50%), exudative and or erythematous pharyngitis (46%), consanguineous parents (42%), symptoms of a type of allergy (84%), lymphadenopathy (27%), splenomegaly (27%), increased acute phase reactant (54%), elevated liver function test (19%) . 10 out of 26 of the individuals reported positive family history and in one of the cases, a patient carrying the homozygous mutation in the mefv gene has shown no clinical manifestation. conclusion: this study highlights the most common manifestations of aids in the population of iranian origin and can be used as evidencebased clinical criteria for their diagnosis. background: the term benign ethnic neutropenia (ben) is used to describe patients of african/arabic descent with absolute neutrophil counts (ancs) less than 1500 cells/ul in the absence of other causes. historically, race has been used to support the diagnosis of ben, but self-reported race is notoriously imprecise. the duffy null phenotype (fya -/fyb-) is a known molecular cause of ben and may be a more reliable marker of ben than self-reported race. in addition, although the anc is known to be lower in patients with ben, the lower limit of ancs is poorly described. it is important to differentiate patients with ben from primary immunodeficiency diseases (pidd) and to recognize their expected anc values. methods: eligible subjects included patients less than 21 years seen at the university of michigan between january 2010-july 2018. duffy null (fya -/fyb-) patients were identified using electronic medical record search engine (emerse) software and search terms duffy and fyab. 105 potential subjects were identified; 67 patients met inclusion criteria including duffy null status and the absence of other conditions or medications, potentially impacting ancs. 251 unique healthy anc values were recorded from the 67 duffy null patients. age and sex matched controls were identified using emerse software with search terms tonsillectomy, department of anesthesiology and absolute neutrophil count. subjects with conditions or medications that might impact the anc or of african/arabic descent were excluded from the control group. asian and caucasian patients included as controls were presumed to be duffy null given that <1% of these populations are expected to be duffy null. 363 control subjects were identified; 134 met inclusion and exclusion criteria. statistical analysis was performed using two-sided two-sample t-test, anova and onesample t-test. results: the median age of the duffy null cases was 4.78 years (iqr: 1.68-11.48) with 61.2% (n=41) male and all of african or arabic descent. mean anc for duffy null patients was 1190 cells/ul (n=251, sd= 650) while mean anc for controls was 4300 cells/ul (n=134; sd=1600) with a mean difference between controls and duffy null cases of 3100 cells/ul (95% ci: 2950-3380; p=0.0001). the anc levels between duffy null individuals and controls were evaluated by five age categories (p=0.0001 for all age categories). however, there was no difference in anc levels between duffy null cases at different age categories (anova, p=0.14196). 54 (21.5%) duffy null cbcs had anc levels in the nonneutropenic range (>1500 cells/ul), 99 (39.4%) cbcs had mild neutropenia (1001-1500 cells/ul), 70 (27.9%) cbcs had moderate neutropenia (500-999 cells/ul), and 28 (11.2%) cbcs had severe neutropenia (<500 cells/ul). conclusions: although neutropenia can be associated with pidds and is often a sign of a compromised immune system, duffy null patients have a wide range of values that are often much lower than previously appreciated. the degree of neutropenia related to duffy null phenotype appears to persists throughout childhood and young adulthood. in the context of patients of african/arabic descent presenting with asymptomatic neutropenia, duffy null status should be assessed, and ben should be considered in the differential. complications, hypogammaglobulinemia and a unique characteristic of decreased susceptibility to enveloped viral infections. objective: to investigate the role of impaired host n-linked glycosylation on viral susceptibility to ebola virus. methods: to mimic the condition observed on cdg-iib patients, we tested in vitro three proprietary iminosugars (emergentbiosolutionsâ©), uv4b, uv001, and uv00128, which act as competitive inhibitors of -glucosidase i and ii. their ability to inhibit the trimming of n-glycans was compared to known n-glycans modifiers as castanospermine, tunicamycin, as well as the bacterial enzyme peptide-nglycosidase f (pngase-f). ebola virus envelope protein gp1 was chosen as a prototype glycoprotein, as it is heavily glycosylated with 15 nglycosylation sites. hek 293t cells were seeded at 1x10^5 cells/well in 12 well plate. after 18h, cells were transfected with pflag-ebolavirus gp1 by coupling with effecteneâ®. after 24h, cells were treated with the inhibitors and harvested 24h after treatment. trimming of n-glycans was evaluated via molecular weight assessment by western-blot. results: all three inhibitors had comparable effectiveness in inhibiting trimming of nglycans from ebola gp1 glycoprotein compared to castanospermine. a greater molecular weight shift was seen with tunicamycin and pngase f as expected. conclusions: chemical inhibition of the n-linked glycosylation pathway was successfully achieved using three new mogs inhibitors. this approach merits further investigation on potential applications on antiviral therapies. investigator, laboratory of human genetics of infectious diseases, necker branch, inserm u1163, necker enfants malades hospital, paris, france 5 head, immunodeficiencies research unit, national institute of pediatrics stat1 gof mutations are associated with infections, autoimmunity and inflammatory manifestations; the rosacea is one of the manifestations described in this disease, however, the etiology rosacea is not clearly established. the characteristics of rosacea are not described in stat gof in the different clinical series. we describe the different characteristics of rosacea in a family with 8 affected members with stat1 gof. a family with eight members with stat1 gof mutation were diagnosed through a first affected member affected with tuberculosis and onychomycosis. seven members more had a clinical history of mycobacterial, viral and fungus infections and autoimmunity disease, in all the seven, was documented the same mutation stat1gof. in six of these adults patients, we documented rosacea, it started after adolescence, it was localized in the face and/or eyes, was progressive and not ameliorated with medical treatment and caused nose deformity. rosacea has been described previously as a unique manifestation, and the etiology is not clear, an autoimmune hypothesis has been proposed. the fact that is present in patients with stat1 gof could suggest that have effectively an autoimmune component. physicians face the patients with rosacea must look for other manifestation presents in stat1 gof mutations. genetic studies in rosacea patients could evidence an new gene defect. introduction: homozygous mutations causing loss of function of the transcription factor forkhead-box n1 (foxn1) underlie autosomal recessive severe combined immunodeficiency with congenital alopecia and nail dystrophy (nude scid). affected humans, like the scid mouse, have small or absent thymus, absent or severely diminished t cells, alopecia, and nail dystrophy. infants with nude scid have had neonatal lymphopenia and severe, life-threatening infections. studies of heterozygous carriers of foxn1 mutations are limited, some having been reported with no phenotype or mild disease manifestations, such as nail dystrophy without lymphopenia or recurrent infections. objective: we describe six infants, including two brothers, with t-cell lymphopenia (tcl) following abnormal california newborn screens (nbs) for scid. each had a single heterozygous variant in foxn1. case reports: six infants (3 female, 3 male) were referred for evaluation after abnormal california nbs for scid (table 1) , with t-cell receptor excision circle (trec) counts from undetectable to 12 (normal >18). all infants were well at the time of initial evaluation. five infants with absolute cd3 t cell counts >400 cells/ul and cd4 t cell counts >250 cells/ul began evaluation as outpatients on home isolation. patient 5, with undetectable trecs, cd3 t cell count 78, and cd4 t cell count 65 was urgently admitted for inpatient evaluation and management and immediately started on antimicrobial prophylaxis. patient 5 further evaluation was significant for lymphocyte proliferation to mitogens that was initially normal but waned with time, prompting treatment with a paternal haploidentical hematopoietic cell transplant at 6 months of age. patients 3 and 5 developed neutropenia within 6 weeks of birth treated with granulocyte colony stimulating factor (gcsf). patient 3 remains well on gcsf but has had persistent growth failure under continued evaluation. patients 1, 2, 4 and 6 remain stable off antimicrobial prophylaxis, but with persistent moderate tcl. as part of an immune evaluation, patients 1 and 3-6 had gene panel testing revealing heterozygous variants in foxn1. only the variant of patient 1 (presumed shared by patient 2, his brother) was predicted to be pathogenic; patient 1 had dystrophic nails and sparse hair most evident after 2 years of age, features shared by his mother and his brother, patient 2. the other patients lack the clinical features of the previously described phenotype of nude scid. their heterozygous foxn1 variants are of unknown significance; the functional role of these variants in the patients clinical phenotype is unknown. conclusion: six infants with abnormal nbs for scid had lymphopenia and heterozygous variants in foxn1. for these infants, variation exists in level of tcl and presence of hair and nail findings. heterozygous variants of unknown significance in foxn1 have been uncovered in others, including infants with abnormal nbs for scid, highlighting the need for functional studies to address the possible role of each heterozygous foxn1 variant in congenital lymphopenia and neutropenia. more work is needed before attributing tcl to a novel foxn1 variant of unknown significance in the absence of family history, abnormal hair or nails, or functional evidence. remains poorly understood. we characterized the intestinal microbiome and metabolome in patients with cgd to determine if intestinal microbiome and metabolomic signatures could distinguish subpopulations of patients with cgd while using the metabolome to add a functional dimension to observed microbiome signatures. methods: clinical metadata and fecal samples were collected crosssectionally from healthy volunteers (hv; n=16) and patients with cgd (n=77). metabolomic profiling and 16s rrna (v4) sequencing was performed on fecal samples (total samples: 108; reads/sample: 15,254 to 191,415; median: 60,816) . results: samples from patients with cgd had distinct intestinal microbiome signatures and metabolomic profiles depending on genotype, presence of cgd-ibd and specific interventions (e.g. treatment with an elemental diet). notably, samples from patients with active cgd-ibd (compared to samples from patients without a history of cgd-ibd) had significantly different alpha-and betadiversities, and were enriched for enterococcus spp. signal transducer and activator of transcription 1 gain of function (stat1-gof) is a primary immunodeysregulatory disease in which a subset of patients have features of autoimmunity and autoinflammation. enteropathy with growth failure and nutrient wasting is a more common feature of immunodysregulation. ruxolitinib is a janus kinase-stat inhibitor that has been shown effective for the treatment of immunodysregulatory features in stat1-gof. our patient is a 13 year old male with stat1-gof (c.983a>g p.h328r) with severe total parenteral dependent enteropathy that led to growth failure (weight 28.5kg). treatment with ruxolitinib led to resolution of diarrhea, return of normal diet, and catch up growth. a dose of 12.5mg twice daily was initially started but was decreased to 12.5mg every morning and 10 mg every evening due to elevated transaminases and thrombocytopenia. over the following year the patient thrived gaining 7.5kg with normal every other day stools. despite weight gain, he remained stable on the same dose of ruxolitinib. as he outgrew his dose, he developed an increased frequency of upper respiratory infections (parainfluenza, coronavirus, rhinovirus). one year after initiation of ruxolitinib, he again developed profuse watery diarrhea that was norovirus positive (weight 36kg, bsa 0.9). he was placed on bowel rest and ruxolitinib was dose escalated with a goal of 15mg/m2/day. when he reached 15mg twice daily, enteropathy completely resolved but liver function tests began to rise. he gained weight and began thriving after 2 weeks of therapy. six months later, enteropathy is controlled, and transaminases have remained elevated (alt 88 iu/l, ast 73 iu/ml) but stable. the appropriate dose and pharmacokinetics for ruxolitinib for the treatment of immunodysregulatory symptoms in pediatric patients has not been thoroughly studied. the dose used was extrapolated from data on the use of ruxolitinib in pediatric myelofibrosis. a dose of 15mg/m2/day appears to provide the most benefit with tolerable adverse effects. this dose should be maintained in order to prevent recurrence of disease related manifestations. abstract clathrin-mediated endocytosis (cme) is the major endocytic pathway by which eukaryotic cells internalize cell-surface cargo proteins and extracellular molecules, thereby allowing for a broad range of biological processes, including cell signaling, nutrient and growth factor uptake, and cell fate and differentiation1. the fbar domain only proteins 1 and 2 (fcho1/fcho2) are involved in the initiation of clathrin coat pit formation. whether fcho1 and fcho2 are functionally redundant or have distinct functions is unclear. we report here the first cases of a severe immunodeficiency due to a genetic defect affecting cme. by using whole exome sequencing and genomic analysis of a targeted pid gene panel, we have identified biallelic loss-of-function fcho1 mutations in five patients from unrelated families of italian (p1), turkish (p2, p3, and p5) and algerian (p4) origin with severe t cell lymphopenia manifesting as recurrent and severe infections of bacterial, mycobacterial, viral and fungal origin. p3 developed ebv-associated diffuse large b cell lymphoma. three patients (p3-p5) died in childhood, whereas p1 and p2 are alive with full donor chimerism at 13 and 1.5 years after allogeneic hematopoietic stem cell transplantation, respectively and have cleared pre-transplant infections. patients p2, p3, and p4 carried homozygous frameshift mutations predicted to cause premature termination. western-blotting analysis of ha-or flag-tagged fcho1 constructs showed expression of truncated products in p2 and p3, whereas no protein was detected in p4, presumably due to mrna decay. p1 and p5 carried homozygous splice-site mutations at the invariant -1 and +1 positions, respectively, leading to skipping of exon 6 in p1's fcho1 cdna. qpcr analysis demonstrated differential expression of the fcho1 and fcho2 genes, with the former being predominantly expressed in lymphoid cells, whereas fcho2 was more abundantly expressed in fibroblasts and k562 cells. analysis of t cell activation in p2 (the only patient for whom pre-transplant pbmc were available) revealed reduced t cell proliferation. while tcr internalization in response to cd3 cross-linking was normal (consistent with recent evidence that tcr internalization occurs through a clathrin-independent pathway), chase experiments demonstrated that transferrin internalization was abolished in activated t cells from p2. we had previously reported that a missense mutation in tfrc, encoding transferrin receptor 1, impairs transferrin internalization and intracellular iron delivery, causing a combined immunodeficiency with defective t cell proliferation. our data identify the first form of severe immunodeficiency due to defects of clathrin-mediated endocytosis, and provide additional evidence in support of the critical role played by iron cellular metabolism in t cell function and homeostasis. natural history of anti-interferon-gamma autoantibody-associated immunodeficiency syndrome in thailand submission id#601826 centralized sequencing initiative at niaid: year 1 therefore, we set out to investigate the pneumococcal-specific responses of igg, igg2, iga and igm to prevnar13â® in igg subclass deficient (iggscd) patients in this study. pneumococcal responses were measured using the vacczyme pneumococcal capsular polysaccharide igg, igg2, iga and igm elisas (the binding site group, birmingham, uk) in control (n=10, median age 57 years, range 27-64) and iggscd patients (n=10, median age 55 years, range 25-65) recruited from the immunodeficiency unit at the karolinska university hospital iga and igm antibodies in response to pcv13 vaccination was observed 4 weeks post vaccination in iggscd patients (median, 2.5th and 97.5th percentile these median concentrations were lower than those observed in control patients (median, 2.5th and 97 pcv13 igg2 71 mg/l, 14-90 however, percentage changes between pre to post vaccination concentrations of igg, igg2 and iga in response to pcv13 in iggscd patients were not significantly different to the control patients u/ml vs 17.1 u/ml, respectively) iga 26 u/ml and pcv13 igm 39 u/ml) responders and non-responders of pcv13 igg iga and igm in response to pcv13 in iggscd patients were generally lower compared to the control population. these results support the fact that in addition to igg and igg2, measurement of iga and igm could also provide useful information for the clinician gain-of-function ikbkb mutation causes human combined immune deficiency submission id#606903 neutralizing anti-il-6-autoantibodies are a risk factor for pyogenic bacterial infections national institutes of health, national institutes of allergy and infectious diseases service of immunology and rheumatology, garrahan national pediatric hospital copa mutations impair er-golgi transport and cause hereditary autoimmune-mediated lung disease and arthritis copa syndrome: a novel autosomal dominant immune dysregulatory disease analysis of pulmonary features and treatment approaches in the copa syndrome expanding the phenotype of copa syndrome: a kindred with typical and atypical features the forest and the trees: machine learning to classify cases of suspected inborn errors of immunity using decision tree and random forest algorithms submission id#607035 card9î�11 gene dosage: from mono-allelic protection to ibd, to bi-allelic increased fungal infection susceptibility yamanaka d 3 , walkiewicz m 4 , lionakis m 3 and rosenzweig s 1 stim1 mutation associated with a syndrome of immunodeficiency and autoimmunity a novel hypomorphic mutation in stim1 results in a late-onset immunodeficiency clinical, histological and genetic characterisation of patients with tubular aggregate myopathy caused by mutations in stim1 gain-of-function mutation in stim1 (p.r304w) is associated with stormorken syndrome gain-of-function mutations in stim1 and orai1 causing tubular aggregate myopathy and stormorken syndrome stormorken syndrome caused by a p.r304w stim1 mutation: the first italian patient and a review of the literature by studying ecs-pre and ecs-post patients we were able to describe the bona-fide effect of gcs on the immune system in general, and t lymphocytes in particular. decreased lymphocyte/thymic output, as well as increased apoptotic tcell death underlies lymphopenia in ecs/chronic gcs-exposed patients. under such conditions, il-21 was significantly decreased in plasma and our in-vitro studies showed that il-21 replenishment was able to increase bcl2 (anti-apoptotic molecule) and bcl6 expression, and efficiently counteract the apoptotic effects of gcs. recombinant il-21 has been explored as a co-adjuvant treatment for multiple human cancers and may offer a treatment option for lymphopenia and its genetic counselor, co-director of personalized medicine, division of hematology/oncology/bmt and the institute for genomic medicine, nationwide childrens hospital 2 genetic counselor, division of hematology/oncology/bmt, nationwide children's hospital acknowledgments. genetic sequencing was kindly provided by drs. raif geha and janet chou at the division of immunology, allergy, rheumatology and dermatology, boston children's hospital, harvard medical school. the following grants are acknowledged: 1. rui 1.1001/cippt/812036 (usm) 2. bmbf 01 eo003 (freiburg) the authors would like to thank the director general of health of malaysia for permission to publish this scientific presentation. while severe viral infections may also be an initial presentation of primary immunodeficiency, an immune evaluation is not always obtained in this scenario. patients with xla have an increased susceptibility to severe enterovirus infections, manifesting as chronic meningoencephalitis, which can be fatal. the following case describes a patient with newly diagnosed xla presenting as suspected coxsackievirus and confirmed hhv-6 meningitis, pseudomonas meningitis and bacteremia. this may be the first reported new diagnosis of xla presenting with both severe bacterial and viral coinfection. case description: a 2 year old, partially vaccinated, hispanic male with a history of febrile seizures presented to the emergency room with fever, oliguria, watery diarrhea, lethargy, meningismus, ecthyma gangrenosum and lower abdominal pain. eight days prior to presentation, he was seen by his pediatrician for facial rash and low grade temperature, and was diagnosed with hand-foot-and mouth disease. he worsened on empiric antibiotics. he had no history of sinopulmonary infections. he did not attend daycare. his vaccines were delayed due to parental choice, and he had not received live vaccines (rotavirus, mmr or vzv). full sepsis evaluation was performed. csf demonstrated pleocytosis, and he was started on empiric antibiotics and transferred to picu. due to worsening abdominal pain, ct of the abdomen was performed, which was consistent with ruptured appendicitis and septic emboli at the lung bases. csf pcr panel was positive for hhv-6 and he was started on gancyclovir. csf and blood cultures subsequently grew pseudomonas aeruginosa. immune evaluation was performed. serum immunoglobulins were undetectable. in addition to iv antibiotics, he received 500 mg/kg ivig and lymphocyte subsets revealed profound b cell lymphopenia (0.23 %, 5 cells/ul). btk protein analysis revealed hemizygous btk pathogenic variant confirming the diagnosis of x-linked agammaglobulinemia. the hospital course was further complicated by brain abscesses and pyoventriculitis. he was treated with 3 additional doses of 500 mg/kg ivig and iv antibiotics. repeat mri of the brain nearly 4 weeks after admission demonstrated significant improvement. there was significant clinical recovery. he was discharged home at baseline neurological status. his igg level upon discharge home was 605 mg/dl with the plan to increase dose to 600 mg/kg per month with close monitoring. conclusion: both severe opportunistic bacterial infections and severe viral infections as the initial presentation of xla have been well reported in the literature. this case describes the first reported severe pseudomonas aeruginosa and hhv-6 co-infection in a newly diagnosed xla patient. this case further highlights the necessity for an increased index of suspicion of primary immunodeficiency in a patient who presents with a severe first infection, despite lack of recurrent infections. we present two patients with dock8 deficiency due to compound heterozygous variants including a copy number loss at chromosome band 9p24.3 spanning approximately .107 mb with partial deletion of the dock8 gene and a novel c.2603c>t (p.ser868leu) missense variant [chr9:379933 (grch37) nm_203447] in dock8. functional data is presented to support the pathogenicity of the missense change, along with a review of the literature on dock8 variants. the proband is a 14-year-old female with elevated serum ige, severe atopic dermatitis, mild persistent asthma, food allergies, and seasonal allergic rhinitis. she is currently healthy following haploidentical bone marrow transplant in june 2018. she has a 17-year-old brother with dock8 deficiency with the same compound heterozygous variants. the brother had later onset of symptoms and a milder presentation of intermittent asthma and seasonal allergic rhinitis. each of the parents is heterozygous for one of the two variants. we evaluated the pathogenicity of the c.2603c>t missense variant with western blots of dock8 protein expression, intracellular flow cytometry, and dock8 stretch assays. flow cytometry showed decreased dock8 protein expression and stretch assays revealed t cells that were stretched in collagen gels. notably, dock8 is a large gene containing 47 exons spanning 190 kb and it is relatively common to be a carrier of a rare missense change. in fact, gnomad has approximately 1500 individuals with rare (<0.002 frequency) missense alleles in dock8. therefore, it is important to demonstrate the potential pathogenicity of any given rare missense change, since few pathogenic missense variants in dock8 have been reported. of the 168 published dock8 variants listed in the human gene mutation database (hgmd) only 13 are missense. the majority are gross deletions, 97 of which were reported in hgmd. the remaining reported dock8 variants include 19 nonsense, 15 splicing, 13 small deletions (all frameshifting), 3 small insertions (all frameshifting), 2 small indels, and 5 gross insertions/duplications. this case demonstrates the relatively infrequent but important contribution of missense changes to pathogenic dock8 alleles. functional validation of missense alleles is critical in the complex evaluation of dock8 deficiency. background: hsct is the only known curative option currently for cd40l deficiency, an x-linked disorder. in cd40l deficiency and other x-linked immune deficiencies, there is an ongoing debate regarding the use of a carrier female sibling or mother as hsct donor. skewed lyonization despite complete donor chimerism has raised concerns for incomplete disease control post-hsct. no data exist regarding the efficacy of related female carrier as hsct donor for cd40l deficiency. we herein report outcomes of three patients with cd40l deficiency who underwent hsct using a related female carrier donor. method: retrospective review of patients who received hsct from carrier female related donor at three separate institutions. results: three patients with cd40l deficiency underwent hsct between 2016-2018. patient 1 had recurrent episodes of pneumocystis jiroveci pneumonia (pjp) despite being on bactrim and immunoglobulin replacement. patient 2 presented with pjp and severe neutropenia. patient 3 presented with acute respiratory failure from severe respiratory viral infections, cmvand had severe neutropenia requiring g-csf treatment. age at the time of hsct ranged from 0.5-15 yrs. all three underwent reduced toxicity hsct with busulfan and fludarabine-based preparatory regimens. two of them received matched sibling bone marrow hsct and one received tcr and cd19 depleted mobilized maternal pbsc haploidentical hsct. donor cd40l expression varied from 37% -67% on activated cd4 cells. immunoglobulin profile and lymphocyte subset were done in two of donors, they were within normal range for age, and none had significant infection history. no history of intermittent neutropenia or oral ulcers noted in donor and the absolute neutrophil count of the donor varied between 2500 6520 /l. donor age ranged from 3.2 yrs 48 years. cd34 dose ranged from 6.1 x 106 -23.1 x 106 cells/kg and cd3 dose ranged from 1 x 105 22.1 x 107 cd3+ cells/kg. gvhd prophylaxis consisted of csa/mmf (n=2) and tcr-a/b depletion and no csa (n=1). neutrophil engraftment ranged from 11-18 days and platelet engraftment ranged from 13 28 days. none of the patients developed acute or chronic gvhd. all three patients maintain full donor myeloid chimerism at the latest testing (9 months 18 months); t cell chimerism was 100% in one and mixed in two patients (91% at nine months, 80% at 12 months). all three patients had excellent t cell immune reconstitution; two patients came off immunoglobulin replacement 5 -11 months post hsct, whereas the 3rd patient is ivig dependent, though iga level was 25 mg/dl at nine months post-transplantation. latest evaluation, 9 18 months post-hsct, revealed 27% -63% cd40l expressing activated cd4 t cells, which correlated with donor cd40l expression and t-cell chimerism. conclusion: our data suggest that hsct utilizing x-linked carrier appears to be safe and results in durable engraftment with excellent humoral and cellular immune reconstitution in patients with cd40l deficiency. longer follow-up and data from a larger cohort is needed to make a definitive determination of safety and efficacy of utilizing female carrier as hsct donors in this disease. chief, immunology service, department of laboratory medicine, nih clinical center, bethesda, md, usa background: ikaros belongs to a hematopoietic-specific zinc-finger (zf) family of transcription factors. after dimerizing and dna binding to pericentric-heterochromatin (pc-hc) regions, ikaros is described as a central regulator of lymphocyte differentiation. somatic mutations/ deletions affecting ikaros n-terminal zf have been identified in b-acute lymphoblastic leukemia (all) patients, and germline n-terminal mutations were reported in cvid patients with progressive lack of b cells, hypogammaglobinemia, autoimmune diseases and b-all. methods: we performed targeted sequencing panel for known inborn errors of immunity disease-causing genes in a previously healthy male pediatric patient with burkitt lymphoma, followed by benign lymphoproliferation, thrombocytopenia and neutropenia. b-cells and immunoglobulin levels were normal. ikaros dna-binding, nuclear localization and protein binding were evaluated by emsa, fluorescence microscopy and immunoprecipitation. protein modeling was also performed. results: a novel heterozygous germline mutation in ikaros c-terminal zf6 dimerization domain (p.r502l) was detected in this patient. this mutant showed normal pc-hc localization but dna-binding was markedly reduced in terms of ikaros dimerization and multimerization. moreover, reduced wt-mutant binding was also detected. mutant/wt cotransfection experiments suggest a haploinsufficient defect. geometry based docking of wildtype ikaros predicted that r502 is within the homodimer interface and may abolish cation-pi interactions and destabilize the ikaros-zf6 dimerization domain. conclusion: a novel germline ikaros c-terminal mutation affecting homodimerization/multimerization and resulting in reduced dna binding to its dna consensus site was detected in a patient with burkitt lymphoma, benign lymphoproliferation and cytopenias. further studies are warranted to formally establish the casual connection between this genotype and phenotype.(210) submission id#606894 patricia pichilingue-reto, md 1 , prithvi raj, phd 2 , igor dozmorov, phd 3 , quan-zhen li, md, phd 4 , edward wakeland, phd 5 , nancy kelly, md 6 , maria teresa de la morena, md 7 , nicolai s. van oers, phd 8 methods: mice were generated by crispr/cas technology to genocopy the foxn1 compound heterozygous mutations identified in one of the human patients. thymopoiesis and hair follicle extrusion was analyzed in the various heterozygous and homozygous mutant mice. gene expression analyses of the hypoplastic and normal-sized thymii and the developing skin were performed. in addition, a structure-function analysis was performed with luciferase reporter assays using 9 distinct and previously unreported foxn1 mutations uncovered in patients who presented with low trecs. results: mice harboring compound heterozygous mutations in foxn1 that match the human patient phenocopy the t-b+nk+ scid phenotype with normal hair and nails. a functional characterization of the diverse foxn1 mutations suggests that the severity of the block in thymopoiesis depends on whether the mutations affect the dna binding or transactivation domains of foxn1. a 5-amino acid segment at the end of the dna binding domain appears to be essential for tec development. however, this segment is not required for normal keratinocyte functions in the skin and nail plate. gene expression comparisons are revealing key targets of foxn1 that suggest a dichotomy in its function in the thymus versus the skin. conclusions: novel compound heterozygous mutations in foxn1 are causal to a t-nk+b+ phenotype with normal hair shaft extrusion and nail plate extension. this differs from the classic nude/scid (omim # 600838) reported for individuals with autosomal recessive mutations in foxn1. assistant professor of medicine and pediatrics, department of allergy and immunology, uva introduction: copa syndrome is a recently described monogenic immunodysregulatory syndrome. the cop protein, encoded for by the copa gene, is expressed in all cell types and is involved in trafficking from the golgi complex to the endoplasmic reticulum (1) . the most common clinical features of copa syndrome are interstitial lung disease, pulmonary cysts or follicular bronchiolitis, pulmonary hemorrhage, arthritis, glomerular disease, and autoantibody development (2, 3) . atypical features of copa syndrome identified thus far include: extrapulmonary cysts in the liver and kidney, renal and neuroendocrine malignancies, autoimmune neurological disorders such as neuromyelitis optica, and infections, such as meningitis (4) . clinical case: we present a case of a 2 year-old male with copa syndrome (de novo heterozygous mutation in exon 9, c.715g>c; p.ala239pro) manifesting as lymphocytic interstitial pneumonitis, peripheral blood b-cell lymphocytosis, mediastinal lymphadenopathy and persistent transaminitis (alt and ast 100-400 u/l, nl ast<35 u/l, alt <55u/l) with normal bilirubin, alkaline phosphatase and pt/inr. the transaminitis was noted prior to diagnosis of copa syndrome, and has persisted despite seven months of therapy with pulse dose steroids, two cycles of rituximab and maintenance therapy with hydroxychloroquine and prednisone. he has had a normal ck and aldolase excluding muscle injury as a source of his transaminitis. a congenital cholestasis panel was normal. markers of autoimmune liver disease including ana, anti-liver kidney microsomal antibody and anti-smooth muscle were negative. serum ceruloplasmin and alpha-1-antitrypsin level were normal and celiac serologies, were negative. liver ultrasound was normal. a liver biopsy did not demonstrate inflammatory changes, hepatocyte necrosis, mononuclear cell infiltrates or fibrosis. nonspecific biopsy findings included occasional intraparenchymal neutrophils. it is unclear if these scattered neutrophils and the transaminitis are due to an early as yet unidentified autoimmune process, perhaps in response to hepatocellular stress exacerbated by the copa mutation. discussion: liver involvement has not been reported in copa syndrome. we describe a child with copa syndrome who has had chronic transaminitis with no clear alternative cause. if the phenotypic spectrum of copa syndrome involves the liver, it may limit immunomodulatory options for the treatment of this disease. background: in humans, biallelic stat1 lost-of-function (lof) mutations lead to a very low or complete absence of the wild-type (wt) protein. whereas, heterozygous mutations can lead to partial loss of function. these patients are susceptible to mycobacteria and herpes virus infections. on other hand, heterozygous gain-of-function (gof) mutations in the stat1 gene result in a hyperphosphorylated state where patients develop recurrent or persistent chronic mucocutaneous candidiasis (cmc), other cutaneous mycosis, bacterial infections, disseminated dimorphic fungal infections, viral infections and autoimmune disease. methods: in this study, we evaluated 4 novel stat1 mutations, three gof and one lof. in vitro, pbmcs from these patients were stimulated with ifn-and ifn-for 30, 60, and 120 minutes and levels of phospho-stat1 were measured by flow cytometry. the stat1 phosphorylation and activity (firefly and renilla luciferase activities) were evaluated in u3a-stat1 deficient cells transfected with a reporter plasmid (for luciferase), wt or mutant-stat1 plasmids. results: we observed higher levels of stat1 phosphorylation after two hours of stimulation from three gof mutations compared to wt. however, a lof mutation showed absent stat1 activation at baseline and in response to ifn-and ifn-. luciferase reporter assay confirmed gain of function and loss of function stat1 activity observed by flow cytometry. conclusions: using flow cytometry followed by a luciferase assay, we confirmed four novel stat1 mutations. measuring phosphorylation of stat1 by flow cytometry is sufficient to determine whether the stat1 mutation is disease causing. this assay can be translated to a clinically accessible test for stat1 related disease. background: variants in recombination-activating genes (rag) are common genetic causes of autosomal recessive forms combined immunodeficiencies (cid) ranging from severe combined immunodeficiency (scid), omenn syndrome (os), atypical scid (as) and cid with granulomas and/or autoimmunity (cid-g/ai). the clinical and immunological presentation is broad, ranging from severe infections secondary to near absence of t and b lymphocytes and hypogammaglobulinemia to the occurrence of autoimmunity with late manifestations with partly preserved immune subsets and near normal immunoglobulin levels and broad spectrum of autoantibodies. objective: we aim to estimate the incidence, clinical presentation, genetic variability and treatment outcome with geographic distribution of patients with the rag defects in populations inhabiting south, west and east slavic countries. due to shared ancestry, we also investigated our cohort for founder variants in rag1 and rag2 genes. methods: demographic, clinical and laboratory data were collected from rag deficient patients of slavic origin via chart review, retrospectively. results. based on the clinical and immunologic phenotype, our cohort of 80 patients from 66 families represented a wide spectrum of rag deficiencies, including scid (n=19), os (n=36), as (n=21) and cid-g/ai (n=4). sixty-six (82.5%) patients carried rag1 and 14 patients (17.5%) carried rag2 biallelic variants. we estimate that the minimal annual incidence of rag deficiency in slavic countries varies between 1 in 180,000 300,000 live birth and it may vary secondary to health care disparities in these regions. in our cohort, 70% of the patients carried rag1 p.k86vfs*33 (c.256_257delaa), either in homozygous (n=17, 26%) or compound heterozygous (n=29, 44%) form. the majority (77%) of patients with homozygous rag1 p.k86vfs*33 originated from vistula watershed area in central and eastern poland, and compound heterozygote cases distributed among all slavic countries except bulgaria. clinical and immunological presentation of homozygous rag1 p.k86vfs*33 cases was highly diverse suggestive of strong influence of other genetic and/or epigenetic factors in shaping the final phenotype. survival of rag deficient patients without hematopoietic stem cell transplant (hsct) (n=3, 8.8%) is poor and dramatically improved in the last decade with access to hsct and tailored conditioning regimens. conclusion: we propose that rag1 p.k86vfs*33 is a founder variant originating from the vistula watershed region in poland, which may explain a high proportion of homozygous cases from central and eastern poland and the presence of the variant in all slavs. our studies in cases with rag1 founder variants confirm that clinical and immunological phenotype only partially depend on the underlying genetic defect. hsct is becoming available for rag deficient patients in eastern europe with improving outcome. clinical immunologist, centre hospitalier universitaire de montrã©al (chum) background: acute gvhd following solid organ transplantation is a rare complication. intestinal and liver transplantation have the greatest risk of gvhd among solid organs due to high number of donor lymphocytes in these organs. prevalence of acute gvhd after liver transplantation is estimated to be around 0,1-2% and has a poor prognosis (1) . chronic neurological gvhd is a rare form of gvhd with three subtypes described: cerebral vasculitis, demyelinating disease and immune mediated encephalitis. acute neurological gvhd has no clear definition and is still considered a controversial entity. case presentation: a 63 year-old male underwent cadaveric liver transplantation for alcoholic cirrhosis and hepatocellular carcinoma. the donor was a 70 year-old man who died from anoxic brain injury. the receiver was induced with basiliximab and then put on prednisone, azathioprine and tacrolimus. he was readmitted 10 weeks later for myalgia, headache, fever and neutropenia. clinical state initially improved with empiric antibiotics. he then developed a skin eruption, colitis and dic. the latter was thought to be tacrolimus-induced. he was switched to cyclosporine. skin and rectosigmoid biopsies were compatible with acute gvhd. he received basiliximab and ivig and developed a refractory convulsive state. csf analysis showed elevated proteins and slight pleocytosis. cerebral mri showed non-specific white matter lesions and conventional angiography was normal. chimerism on peripheral blood was 0% but was 45% donor on csf. with the presence of chimerism on csf, evidence of cutaneous and digestive gvhd and no infectious cause, neurological gvhd was considered the most likely diagnosis. brain biopsy showed non specific change including neuropil spongiosis, microglial activation and reactive gliosis; but no signs of vasculitis or demyelinating disease. he was treated with atg, highdose systemic corticosteroids, cyclosporine, ivig and intrathecal methotrexate and corticosteroids. csf pleocytosis, proteins and chimerism improved with treatment (45% to 2% donor). no improvement was noted regarding his neurological state and he developed pancytopenia. he was then transfer to palliative care and died shortly after (4 month and a half after liver transplant). discussion: to our knowledge, there is only one prior case published of neurological gvhd following liver transplantation (2) . both patients were old, had hepatocellular carcinoma and had at least one hla match. age >50 year, hepatocellular carcinoma and shared hla antigen are known risk factors for gvhd following liver transplantation (1). our patient had only one hla match with the donor. this case is intriguing as there was a great discrepancy between blood and csf chimerism. acute neurological gvhd following transplantation is a real complication. it must be taken into consideration in patients with neurological involvement after transplant, even solid organ transplantations. introduction: hyper-igm syndrome are rare. although no data are available on the frequency of activation-induced cytidine deaminase (aid) deficiency, this disorder is estimated to affect less than 1:1,000,000 individuals. by the year 2012, 110 cases worldwide (1) with such mutation have been described. we describe a patient with hyper igm by mutation in the aicda gene. case report: mvv, 5-year-old boy, born to consanguineous parents, was referred with recurrent pneumonia, which started shortly after discontinuation of breastfeeding at 6 months old. repetitive otitis evolved with bilateral tympanic and partial hearing loss. he was submitted to adenoidectomy without improvement. immunological evaluation showed normal numbers of b and t cells with cd3+ (1290/mm3, 65%), cd4+ (547/mm3, 28%), and cd8+ (259/mm3, 13%). immunoglobulin concentrations were: igg = 138mg/dl (p97). treatment with intravenous immunoglobulin and prophylactic antibiotic was initiated and he had no infections during the follow up except for one episode of sinusitis. at 10 years of age, molecular evaluation was performed and a mutation in homozygosity in the aicda gene (omim * 605257) at position chr12: 8.757.821 was found, confirming the clinical suspicion. conclusion: the role of aid in the immunoglobulin class-switch recombination (csr) and somatic hypermutation (shm) have not been fully elucidated. summarizing within the shm and csr processes, aicda mutation can induce dna lesions in directed sequences in the s and v regions required for dna cleavage. recurrent infections and consanguinity raised the suspicion of inborn errors of immunity in this patient. the literature described late diagnosis as in the second or even the third decade of life. it was suggested that high levels of igm antibodies may provide effective defense, at least, against some infectious agents. it is important to emphasize that the impossibility to obtain genetic diagnosis did not prevent to introduce therapy. * aicda: activation induced cytidine deaminase gene patients with chronic granulomatous disease (cgd) are at risk for recurring infections and non-infectious inflammation, reduced quality of life and life expectancy. conventional treatment with life-long anti-bacterial and antifungal prophylaxis prolongs lifespan but does not eliminate the lifelong risk of infection and inflammation. allogenic stem cell transplantation is currently the only curative option for this disease. although sct with reduced intensity conditioning has improved treatment-related mortality and efficacy, it remains a matter of debate whether all patients with cgd benefit from sct, whether pre-existing infections and non-infectious inflammation are risk factors and at what age sct should be performed. we compared patients with cgd on conventional treatment with those after stem cell transplantation for their prognosis and evaluated potential risk factors for stem cell transplantation outcome followed up in six european centers. frequency of infections, inflammatory complications, hospitalizations, operations and immunomodulative/immunosuppressive therapy, height and weight were compared in patients on conventional treatment /before stem cell transplantation versus patients after sct. correlation between transplantation outcome and patient characteristics or medical history was tested. 105 patients were recruited, 55 on ct, 50 after stem cell transplantation. before/without transplantation 98% of patients suffered from at least one infection, 84,8% from inflammatory complications. patients on conventional treatment developed infection/inflammation/ hospitalization/surgery at a median of 2,28 (range [0,29-21,82] , iqr 2,79) per year, versus 9 (range , iqr 8,5) in the first year after stem cell transplantation but 0 (range [0-15], iqr 0,53) after the first year post stem cell transplantation. there was a significant decrease of all complications after stem cell transplantation (p < .05). growth improved significantly after stem cell transplantation (z-score weight -1,692 versus -0,846 (p.017), z-score height -1,906 versus -1,064 (p.029)). nevertheless, complications post stem cell transplantation are frequent: 88% of patients had at least one infection, 8% had severe acute gvhd, 12% chronic gvhd, 16% had graft rejection, 12% died. preexisting active mold infection increased the risk for complications after stem cell transplantation. in summary infections and non-infectious inflammation are common in patients with cgd on conventional treatment, their growth is significantly impaired. stem cell transplantation, if successful, significantly reduces the risk for infections and non-infectious inflammation. however, treatment related mortality of stem cell transplantation in patients with cgd remains considerable. introduction: development of a diverse t cell repertoire is essential for full immune recovery following definitive treatment for severe combined immunodeficiency (scid), whether by allogeneic hematopoietic cell transplantation (hct); autologous gene therapy (gt); or, in the case of adenosine deaminase deficiency, enzyme replacement therapy (ert). however, the time course and depth of diversity of t cell receptor rearrangements have been difficult to measure directly, necessitating estimates from total and naã¯ve t cell counts and from spectratyping, in which t cell receptor (tcr) beta chain diversity is estimated by the length distributions of cdna amplicons between a series of tcr beta chain variable (v-beta) segments that have productively recombined with the tcr beta-chain constant region. analysis of the actual sequences of rearranged tcrs could indicate more precisely the status of the t cell compartment of these patients, and might reveal oligoclonal expansion of dysregulated t cells, t cell insufficiency, or t cell exhaustion. objectives: we wished to ascertain whether deep sequencing of individual tcr v-beta rearrangements in peripheral blood could be performed sequentially following diagnosis and treatment of scid to differentiate satisfactory immune reconstitution from incomplete or skewed repertoire development that might require further cellular therapies. methods: equal amounts of total rna were obtained from peripheral blood of controls and scid patients pre-hct and at 100 d, 6 and 12 mo, and yearly post-treatment(s). cdna was used as template to semi-quantitatively amplify rearrangements at the tcr-beta locus (trb). raw sequences were filtered to remove pcr errors, and resulting fastq files were converted into fasta format (seqtk software, github, inc), filtered for productive rearrangement, and analyzed for v, d, and j gene composition and length (imgt highv-quest software). the vdj statistics file (past program) was used to calculate a shannon entropy (h) index to measure repertoire diversity, taking into account both abundance and richness of the overall repertoire; and a gini-simpson index of unevenness, measuring inequality in the relative representation of species in a given sample. graphical representations of repertoire diversity were generated by hierarchical tree maps of the trb repertoires (irepertoire software): each dot represents a unique sequence and the dot size corresponds to frequency of that sequence in the total sample. results: tcr v-beta sequence analysis of 3 scid patients (image) showed (top) baseline poor diversity due to pre-treatment ada deficiency followed by improvement to normal complexity (shannon h >7.0) after receiving peg-ada and autologous lentivirus gene therapy at age 3 m; (middle) increasing diversity in xscid after maternal t-depleted unconditioned hct, although b cells did not recover; and (bottom) failure of initial unconditioned maternal t-depleted hct in another xscid patient at 12 m, followed by autologous lentivirus gene therapy with subsequent improvement (shannon h increasing from 3.8 to 6) 12 months later. conclusions: tcr v-beta diversity sequence analysis provided a detailed assessment of repertoire diversity in response to cellular therapies for scid. this method could become a useful predictive tool to measure successful t cell immune reconstitution, both as early as 100 d and in the years following treatment. background: the stim1 (stromal interaction molecule 1) protein, encoded by the stim1 gene, is involved in calcium regulation in the endoplasmic and sarcoplasmic reticulum. pathogenic variants in this gene are associated with three different disorders. homozygous loss-of-function (lof) pathogenic variants in stim1 have been reported to cause autoimmune cytopenias, lymphoproliferation, enamel defects, anhydrosis, and iris hypoplasia. the first described cases had frequent mortality in early childhood due to recurrent life-threatening infections and development of kaposi sarcoma (1), while recently discovered cases have had more prolonged survival, though still with recurrent serious infections (2) . heterozygous gain-of-function (gof) pathogenic variants in stim1 have been associated with both tubular aggregate myopathy (tam) and stormorken syndrome. tam is a clinically heterogeneous progressive muscle disorder with a variable age of onset. muscle biopsy characteristically demonstrates tubular aggregates, with type ii muscle fiber atrophy (3) . stormorken syndrome has a phenotype that includes miosis, thrombocytopenia, intellectual disability, mild hypocalcemia, muscle fatigue, asplenia, and ichthyosis (4) . the thrombocytopenia has not been reported to be immune-mediated; rather it is due to abnormal platelet calcium regulation (5). we report a patient with stim1 pathogenic variant presenting with tam and immune-mediated thrombocytopenia, along with lymphoproliferative features, arthritis, and a mild immune deficiency. case: the patient is a 16-year-old with a history of congenital thrombocytopenia (platelets ranging 60,000-100,000) who presented with acute arthritis of bilateral hand joints after exposure to cold temperatures, which resolved with naproxen. he had back pain without muscle weakness, and preceding sore throat and general fatigue. labs were significant for leukocytosis and elevations in his inflammatory markers and creatine kinase. mri of his lower extremities was negative for inflammatory myositis, but did demonstrate bilateral hip and knee effusions, and significant inguinal lymphadenopathy and hyperintense linear signal changes in the mid-and distal femurs with patchy red marrow signal. abdominal ultrasound could not identify a definite spleen. bone marrow biopsy was negative for malignancy but significant for toxic granulation of neutrophils, evident of inflammation. alpha-beta double negative t cells were not elevated. interferon-gamma was mildly elevated. flow cytometry demonstrated normal t, b, and nk cell absolute counts. circulating antibodies against platelets (both igg and iga) were detected. on lymphocyte antigen and mitogen proliferation testing, he did not exhibit any proliferation when stimulated with tetanus toxoid even though he had been fully vaccinated against tetanus. muscle biopsy demonstrated large vacuoles consistent with tam on both light and electron microscopies. invitaes primary immunodeficiency panel identified a pathogenic variant in stim1 (c.910c>t; p.arg304trp), consistent with a diagnosis of autosomal dominant stim1-related conditions, including stormorken syndrome (6) . conclusion: this patient expands the phenotypic spectrum of stim1 related disease. based on previous evidence, gof pathogenic variants in stim1 are associated with tam and stormorken syndrome, while lof pathogenic variants in stim1 are associated with immune deficiency. however, our patient with a stim1 gof pathogenic variant has features of lymphoproliferation and immune dysregulation in addition to tam. stim1 gof pathogenic variants should be considered in the differential of patients with immune thrombocytopenia and lymphoproliferation. references: introduction / background: card11 is critical for protein binding upstream of nf-kb (nuclear factor kappa b) and mtorc1 (mammalian target of rapamycin complex 1) the signaling pathway involved in t-cell activation and inflammatory response. prior testing of card11 mutations demonstrated variable t-cell dysfunction. in vitro studies have demonstrated reduced interferon gamma cytokine production, interference of t-cell receptor (tcr) signaling, and th2 phenotype skew in t-cells with card11 defects. while homozygous mutation causes severe combined immunodeficiency deficiency, heterozygous card11 defect is associated with atopy by way of inappropriate th2 skewing. heterozygote atopy is characterized by eosinophilia, elevated ige, and severe dermatitis. despite multiple studies demonstrating in vivo consequences of card11 on t-cell function, little is known of the clinical significance. moreover, few studies have demonstrated the impact of card11 mutations on b-cell maturation and development, despite the recognized tcr and interleukin 2 signaling deficits. objectives: this case demonstrates a card11 defect that evolved from atopy to combined immunodeficiency requiring intravenous immunoglobulin therapy. it highlights the poorly understood effect of card11 mutation on t-cell function, and the downstream impact on b-cell quality. methods: 53-year-old male, with past medical history of t-cell lymphoma and no evidence of disease status post autologous stem cell transplant, was found to have card11 e57d missense mutation by genetic testing. consistent with previous literature regarding heterozygous card11 defects, the patient suffered from frequent asthma exacerbations, aeroallergen sensitivity, and eczema. lab work was consistently positive for elevated ige and eosinophilia. family history was positive for a son born with congenital molluscum, and multiple other children with recurrent infections. one child was also identified with card11 mutation. the patient had flow cytometry demonstrating 4% of circulating cells with atypical immunophenotyped cd3+ t-cells, and positive gene rearrangement studies. his qualitative immunoglobulin levels were significant for consistently low igm, but normal quantity igg. in the patients adulthood, he had recurrent bronchitis and pneumonia requiring hospitalization and intravenous antibiotics. given his recurrent infections, the patient underwent immunodeficiency evaluation. despite previous infection with herpes zoster, the patient did not have protective titers. additionally, the patient had received the pneumococcal conjugate vaccine once, and the pneumococcal polysaccharide vaccine four times. the most recent vaccination was one year prior to evaluation. despite repeated vaccinations, titers were unprotective. consequently, the patient was diagnosed with combined immunodeficiency, and initiated on intravenous immunoglobulin therapy. results: in summary, card11 defect is a cause of atopy, observed to become less severe with age. studies of card11 heterozygote mutations have demonstrated in vitro deficiencies in t-cell activation, likely secondary to skewed or decreased inflammatory cytokine production and tcr activation. our patient demonstrates that the variable t-cell dysfunction seen in vitro can have significant clinical implications evidenced by his inadequate vaccine response, and recurrent infections. his combined immunodeficiency poses a connection between card11 defects and, not only t-cell, but also b-cell function. conclusions: further studies are needed to determine deficits in t-cell and b-cell function in the setting of card11 defect, as this case suggests the clinical implications span further than atopy. genetic variants in the scaffold gene card11 cause disorders of the immune system. the clinical course and treatment depends on whether the card11 variant causes gain-or loss-of-function. however, lymphocyte immunophenotyping and proliferation assays in cells expressing card11 variants don't easily distinguish between gain-and loss-of-function. to address this challenge in variant interpretation, we used multiplexed genome editing in a lymphoma b cell line (tmd8) to generate cell populations expressing all possible singlenucleotide variants in the n-terminal 140 amino acids of card11. to assess function in each variant, we tracked its relative abundance over multiple conditions using dna sequencing. since card11 is required for survival of tmd8 lymphoma b-cells, cells expressing clinically identified gain-of-function variants grew faster relative to cells expressing other variants, even in the presence of upstream pathway inhibitors. upon evaluation of the relative abundance of each variant in genomic dna and mrna, we found that clinically identified loss-of-function variants were depleted in mrna, which could be attributed to alterations in splicing or to nonsensemediated decay. to address the impact of splicing, we modeled a newly-identified splice donor mutation (c.358+1g>a) found in two patients from one family diagnosed with combined immune deficiency, autoimmunity and atopy that was also observed in our screen. we show that the variant causes deletion of exon four and that card11 missing exon four exerts a dominant-negative effect leading to decreased nf-kb signaling and cell growth. these experiments demonstrate the utility of multiplexed functional assays for determining variant effect in clinically-relevant genes, which will improve diagnosis and treatment in patients. mutations in the rag1 and rag2 genes in humans cause a wide spectrum of phenotypes, ranging from severe combined immunodeficiency (scid) with lack of t and b cells to omenn syndrome (os), atypical scid (as) and combined immunodeficiency with granulomas and/or autoimmunity (cid-g/ai). here, we sought to investigate the molecular basis for phenotypic diversity presented in patients with various rag1 mutations. methods: we have recently described a novel flow-cytometrybased assay in which mouse rag1-/-pro-b cells containing an inverted gfp cassette flanked by recombination signal sequences (rss) are transduced with a retroviral vector expressing either wild-type or mutant human rag1 (hrag1). the green fluorescent protein expression directly relates to the activity of rag proteins, representing a quick and powerful tool to correlate between defective activity of hrag1 mutant and severity of the clinical phenotype. the genetic variants of hrag1 analyzed in this study were affecting the various domains of the protein: ring, zinc finger ring type domain (amino acids 168-283); nbr (amino acids 387-461); hbr (amino acids 531-763) and the core domain (amino acids 385-1011). using this sensitive assay, we tested the recombination activity of 27 human rag1 variants that have been reported in patients. results: we have demonstrated correlation between the recombination activity of the mutants and the in vivo clinical phenotype of patients. in particular, similarly low levels of recombination activity were observed in patients with scid and os, whereas patients with as and especially those with cid-g/ai carried mutations that retained significant residual levels of activity. conclusions: these data provide a framework to better understand the phenotypic heterogeneity of rag deficiency. here we report a case of a child with b. cepacia lymphadenitis, ultimately diagnosed with takayasu arteritis. takayasu arteritis is a large vessel vasculitis which may have a nonspecific clinical presentation in childhood possibly leading to difficulty in diagnosis. case: a 16-month-old female presented with two weeks of fever, respiratory distress, and lymphadenopathy, and was treated with ivig for presumed atypical kawasaki disease. imaging studies performed due to worsening respiratory distress revealed retropharyngeal abscess with bilateral cervical lymphadenopathy, culture-positive for prevotella oralis and melaninogenica, with improvement following incision and drainage and antibiotic therapy. recurrence of fever and respiratory distress prompted ct imaging of her neck significant for worsening lymphadenopathy. cultures from lymph node biopsy grew b. cepacia. following treatment, she was readmitted with respiratory distress requiring chronic steroid treatment and found to have candida albicans on bronchoalveloar lavage and necrotizing granulomatous inflammation on lung biopsy. an immunologic evaluation was notable for two normal dhr assays. cgd genetic panel was negative for pathogenic variants in cybb (p91), ncf1 (p47), cyba (p22), ncf2 (p67). testing was also notably negative for hiv pcr, bartonella pcr, cryptococcal antigen, histoplasma antigen, bal afb stain and mycobacterial cultures, cmv pcr, ebv pcr, anca, serial blood cultures, and sweat test. lymphocyte subsets were normal for age. mitogen stimulation test, myeloperoxidase antibody igg, serine protease3 igg, c4 level, lad panel, and cytokine panel were normal. autoimmune lymphoproliferative disorders (alps) panel was negative. whole exome sequencing demonstrated heterozygous mutations in cfi and jak3, not considered to be clinically relevant given the patients clinical picture and laboratory evaluation. the patient was then lost to follow-up for over a year. at the age of 3 years, the patient presented with fever and back pain. imaging revealed severe large vessel vasculitis involving the aorta and subclavian, vertebral, mesenteric, and renal arteries. she also had evidence of cardio-embolic strokes on brain mri. she had had no significant interval infections, and her immunologic evaluation remained unrevealing. in the context of her new vasculitis, evaluation for deficiency of ada2 (dada2) was negative. she was ultimately diagnosed with takayasu arteritis and has begun therapy with systemic corticosteroids, aspirin, and etanercept. conclusions: we describe a case of b. cepacia infection in a child without identified immunodeficiency, ultimately diagnosed with a large vessel vasculitis. the presence of b. cepacia infection warrants a thorough investigation. burkholderia has been previously associated with giant cell arteritis, another type of large vessel vasculitis, though causation has not been established. to our knowledge b. cepacia infection has not been associated with takayasu arteritis. christopher santaralas, valentine jadoul, jacqueline squire, john cannon, jessica trotter, susan aja, neil goldenberg, david graham, jennifer leiding background: chronic granulomatous disease (cgd) is a primary phagocytic immunodeficiency secondary to mutations in any of the components of nadph oxidase. in addition to infection susceptibility, patients with cgd can develop auto-inflammatory disease that is difficult to manage. metabolomics is the systematic study of small molecule biomarkers of the clinical phenotype of disease. we sought to investigate plasma metabolic profiles in cgd as we hypothesized that unique signatures may differentiate patients with cgd. methods: plasma collected from 15 subjects with cgd (9 x-linked, 4 p47phox-deficient, 2 p22phox-deficient) and 2 x-linked cgd carriers was analyzed using a targeted multiplex assay by liquid chromatography mass spectrometry (lc-ms) and simultaneously a profiling assay by lcms. sufficient signal was present for 34 metabolites. x-linked cgd and p47phox-deficient groups were sufficiently sized for multivariate and univariate analyses in metaboanalyst. twelve patients had a single time point of plasma metabolomics analysis and three had multiple time points, including one in whom both pre-and post-hematopoetic cell transplantation time points were assessed. post-hoc comparisons were also performed for those with, versus without, clinical comorbidities of autoinflammation. results: plasma from patients with x-linked and p47phox deficient cgd had a differential metabolomic signature at baseline. many metabolites as measured by ion intensity were present at high levels, particularly homocysteine, kyneurine, tryptophan, citric acid, carnitine, methionine, and adenosine. increased values of metabolites reduced to that of normal (compared to post hct). homocysteine levels were elevated among patients with (mean 1.5x105), versus without (mean 6.8x104), clinical comorbidities of auto-inflammation (i.e., colitis, lupus). baseline samples showed elevated kynurenine among all cgd patients, relative to historical normal controls (unmatched, separate analysis). patients with colitis had elevated citric acid levels that were higher among patients with (mean 2.1x106), versus without (mean 4.5x105), colitis irrespective of genotype. conclusions: preliminary data with a small patient subset suggest that patients with cgd have metabolomic signature distinguishable by phenotype. citric acid cycle metabolites are elevated in crohns disease and ulcerative colitis. based on our data, citric acid may too act as a biomarker for inflammatory bowel disease in cgd. analyzing a larger number of samples, across time points, will likely describe a metabolomics profile for cgd and identify biomarkers for auto-inflammation in cgd. no significant medical history in mother; paternal history is unknown and unavailable.no significant medical history in mother or father. rationale: ataxia telangiectasia is a disorder with variable phenotypes characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity, and cancer predisposition which may correspond to the degree of atm protein expression and/or radiosensitivity. we used in vitro cytometric assessment of atm, smc1 and h2ax phosphorylation to assess dna damage in response to radiation and found that two siblings with the same copy number gain in atm have variable clinical neurologic and immunologic phenotypes. methods: chart review and radiosensitivity assays using cytometric assessment of patm, psmc1, and h2ax expression after irradiation with 2gy. results: patient a is a 6 month old male identified after having low trecs on newborn screening, then found to have lymphopenia and elevated igm. he has diffuse cafã© au lait macules and no neurologic symptoms. his 9 year old sister, patient b, was being followed by neurology for several years for ataxia. she has selective iga deficiency, normal lymphocyte counts, lymphocyte proliferative responses, gammaglobulins, and vaccine specific antibodies. both patients have a 4 copy number gains in atm (exons 48-61). mother and father both have 3 copy number gains in atm and are healthy without neurologic symptoms or recurrent infections. both patient a and b have normal atm protein expression. phosphorylated atm, smc1, and h2ax was assessed in lymphocyte subsets (t, b, and nk cells) after low-dose irradiation to induce dna double-stranded breaks (dsbs). these parameters were assessed at 1 hour post-irradiation when they are expected to be maximal and at 24 hour post-irradiation, when under conditions of normal and effective dna repair, the phosphorylation state returns to baseline. patient a had abnormal patm and psmc1 but normal h2ax expression 1 hour and 24 hours after irradiation of t, b, and nk cells. patient b had normal patm, psmc1, and h2ax expression in t cells but abnormal patm and psmc1 expression in b and nk cells 1 hour after irradiation. patient b, however, had abnormal atm phosphorylation at 24 hours after irradiation of t, b, and nk cells.conclusions: our results indicate that a unique copy number gain in atm within a family can correspond to different clinical and immunologic phenotypes as well as variable degree of radiosensitivity. the persistence of h2ax at 24 hours post-irradiation and impaired phosphorylation of atm and smc1 at 1 hour post-irradiation demonstrates defects in dna dsb repair, and this is variably altered in different lymphocyte subsets. correlation between atm phosphorylation in lymphocytes with outcomes may be an area for future studies and particularly important in counseling patients regarding outcomes. antibodies have been implicated in both protection and pathology of dengue virus infections. however, much of this data is gathered from serum/plasma responses that is a cumulative of historical and ongoing infection. to precisely understand the role of antibodies with respect to the ongoing dengue virus infection, we employed the cutting edge approach of generating of human monoclonal antibodies from individual plasmablasts from peripheral blood of dengue patients that allows us to probe for answers at a single cell level. this method involves ex vivo single cell sorting of plasmablasts from peripheral blood of well-characterized dengue infected patient followed by single cell molecular cloning of immunoglobulin heavy-and light-variable regions into expression vectors containing the defined constant region followed by transient cotransfection of hek 293a cells with the heavy and light chain expression vectors made from genes arising from the same cell. thus far, using this powerful technology, for the first time in india, we have made 140 number of human monoclonals, of which 80 are specific to dengue and 14 neutralize dengue virus at various concentrations. all the neutralizing antibodies are dengue-envelope specific and bind the highly conserved fusion loop of the dengue virus envelope. together, with the ongoing comprehensive analysis of the b cell repertoire and somatic hypermutations, these studies provide a detailed understanding of the dengue-specific plasmablast cell response at a single cell level and create a platform for testing these antibodies for basic research, diagnostic, prophylatic and as well as therapeutic applications. surviving. six of the 13 (46.2%) surviving patients remain dependent on ig replacement despite robust donor chimerism of 99-100% and no active gvhd. all but two received rituximab pre-hsct. of the patients who are independent of ig replacement, only one (14.2%) received rituximab post-hsct, whereas 5/6 of the ig dependent patients received rituximab post-hsct. t cell immune profiling revealed that the absolute numbers of lymphocyte subsets, cd4+ naã¯ve t cells, and cd4+ recent thymic emigrants were not statistically different between ig independent and dependent patients ( figure 1 ). however, there was a marked decrease in the number of total b cells, the percentage of memory b cells (cd27+ b cells), and classswitched memory b cells (cd27+ igd-igm-cells) in ig dependent patients ( figure 1 ). t follicular helper (tfh) cell populations (cd4+cd45ra-cxcr5+pd1+) were evaluated in four patients and the frequency was similar to healthy controls (4.5+/-1.2 vs. 3.9+/-1.4%). the ability of the patients naã¯ve b cells to class-switch was assessed following exposure to il-21, anti-cd40 antibody, and anti-human igm, and revealed normal b cell class-switching and differentiation to plasmablasts ( figure 1) . additionally, t cell ability to provide b cell help was assessed by coincubating naã¯ve b cells with activated cd4+ t cells. this revealed comparable b cell class switching to that of healthy controls. conclusion: the high incidence of poor long-term functional b cell reconstitution following allogeneic hsct for xlp-1 could be related to the use of rituximab in the post-hsct setting rather than pre-hsct. normal tfh numbers and function, and ability of b-cells to class-switch in-vitro suggest that persistent hypogammaglobulinemia is these patients is unlikely from a b or t-cell intrinsic defect. the possibility of rituximab induced acquired lymph nodal stromal defect in these patients is being explored. further studies are needed to understand the biology of persistent hypogammaglobulinemia in xlp-1. additionally, due to the high incidence of persistent hypogammaglobulinemia, exposure of rituximab should be limited post-hsct. background: tandem mass spectrometry (ms/ms) has emerged as a primary platform for many clinical and newborn screening laboratories. the application of ms/ms mainly focuses on the quantification of accumulated small metabolites in plasma resulting from various metabolic defects. however, many disorders do not yield such metabolic markers and would benefit from the direct quantification of intracellular target proteins. unfortunately, the extremely low (e.g., pmol/l range) protein concentrations in blood cells limit their detection via ms/ms. in recent years, peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-srm) has emerged as a promising technique for the quantification of low abundance proteins in complex matrices, including dried blood spots (dbs). our lab has demonstrated that immuno-srm methods are able to reliably distinguish affected patients from the normal controls for wilson disease (wd), wiskott-aldrich syndrome (was), severe combined immunodeficiency (scid), and x-linked agammaglobulinemia (xla) (j. proteome res., 2017 and front. immunol., in press). these results demonstrate the utilization of immuno-srm as a sensitive platform for multiplexed quantification of signature peptides in the low pmol/l range. methods: several candidate peptides for each protein were selected based on uniqueness using in silico blast tools and lc-ms/ms response. monoclonal antibodies (mabs) were then generated for peptide enrichment from dbs. blood from normal controls, wd, xla, scid, and was patients was spotted onto filter paper, dried, and stored at -20â°c until use. proteins were extracted from dbs, digested with trypsin, and enriched using mabs bound to magnetic beads. the enriched peptides were then eluted and analyzed using srm mode with a waters xevo tq-xs. results/conclusions: to date, immuno-srm methods have been generated for wd, was, scid, xla, and cystinosis. preliminary data shows immuno-srm methods are able to reliably quantify target proteins using signature peptides and accurately distinguish affected patients from normal controls. analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case (was and btk: p = 0.0001, scid: p = 0.05). intra and inter-assay precision ranged from 11 -22% and 11 -43%, respectively, and the multiplexed assay showed a broad linear range (1.39 2000 fmol peptide) . in a blinded sample set of 42 pidd patients and 40 normal controls, immuno-srm-predicted diagnoses showed excellent agreement with clinical or genetic diagnoses. every molecularly-confirmed case of was and btk was also diagnosed by immuno-srm analysis. in addition, 62 randomly selected samples provided by the nbs laboratory of washington state were tested and peptide concentrations were found to be within normal ranges. efforts are underway to validate and incorporate peptide biomarkers for adenosine deaminase deficiency, dock8 deficiency, and ataxia telangiectasia, as well as general markers for nk cells and platelets into a single multiplexed assay. in addition, scid, was and xla samples continue to be run while we focus on reducing assay costs, time, and necessary sample input. our data herein provides proof of concept for the immuno-srm workflow to be extended to various other genetic diseases as potential multiplexed newborn screening methods.(250) submission id#617782the background: the long-term effects of glucocorticoids (gcs) on the immune system have been extensively studied in patients with different underlying conditions (e.g, malignancies or autoimmune conditions), as well as in healthy volunteers receiving short-term courses of these drugs. although these approaches provided highly relevant data, neither of them answered the unbiased/bona-fide effect of long-term gcs use on the immune system. endogenous cushing syndrome (ecs) may be caused by pituitary or ectopic acth-producing adenomas, or by tumors or hyperplasia of the adrenal cortex. patients with ecs present with different gcsdependent manifestations, including those affecting the immune system as neutrophilia and lymphopenia. when tumors are removed, most of the effects of gcs tend to progressively regress. methods: paired samples from 15 patients with ecs due to acth-producing adenomas (age range 7-16y, 8 females) were studied before (ecs-pre) and 6-12 months after tumor removal (ecs-post). extended lymphocyte phenotypes and apoptosis in different cell subsets were evaluated by flow cytometry. cytokine production (elisa) and responses, as well as their effects on cell proliferation and viability, were evaluated using cell trace violet and annexin-v staining. results: among multiple immunophenotypic changes, ecs-pre patients showed significantly reduced naã¯ve t cells and recent thymic emigrants (rte) as well as increased apoptosis in t cells when compared to themselves (ecs-post) or age matched healthy controls. moreover, significantly increased exhausted cd8 t cells were observed in ecs-pre patients. interestingly, ecs-post patients showed full cellularity recovery of t cells and rte with increased proliferation and reduced apoptosis, in addition to correction of most of the other changes evidenced. significantly lower il-21 plasma levels were also detected in ecs-pre when compared to ecspost patients. to determine the role of il-21 in an ecs-resembling condition, healthy control pbmcs were treated with gcs in-vitro and the effect of il-21 and other cytokines was tested. a significant reduction in apoptosis was observed in the il-21-treated cells that almost completely countered the pro-apoptotic effects of gcs; il-21 was also significantly more efficient than il-2, il-7, ifn-alpha and ifn-gamma in rescuing cells from apoptosis. il-21-specific upregulation of bcl2 and bcl6 expression was evidenced in these cells.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-017461-xw02c7u5 authors: kauffman, carol a. title: fungal infections date: 2009-02-02 journal: infectious disease in the aging doi: 10.1007/978-1-60327-534-7_22 sha: doc_id: 17461 cord_uid: xw02c7u5 older adults are at increased risk of developing opportunistic fungal infections because organ transplantation, intensive cancer chemotherapy regimens, and anti-tumor necrosis factor agents are now used more commonly, and because admission to an intensive care unit, which carries many risk factors for fungal infection, has become commonplace in this group. candida species are the most common cause of opportunistic fungal infections, and bloodstream infections are usually treated with fluconazole or an echinocandin antifungal agent. invasive mold infections are mostly caused by aspergillus species; in older adults, they cause primarily pulmonary and sinus infections, and they are associated with a high mortality rate. the endemic fungi, histoplasma capsulatum, coccidioides species, and blastomyces dermatitidis, cause infection when the mold form is dispersed and inhaled from the environment in those specific areas of the country in which these organisms flourish. amphotericin b is used for initial treatment of severe histoplasmosis, coccidioi­domycosis, and blastomycosis; itraconazole is the therapy of choice for most mild to moderate infections due to these endemic mycoses. serious fungal infections can be separated into two major categories: the opportunistic mycoses that include candidiasis, cryptococcosis, and invasive mold infections such as aspergillosis and zygomycosis, and the endemic mycoses, which in the united states, includes histoplasmosis, blastomycosis, and coccidioidomycosis. the fungal infections represented in these broad categories differ with respect to the characteristics of the organisms causing infection, their epidemiology, the clinical manifestations, the approach to diagnosis, and the principles guiding therapy. in response to these different groups of fungi, host defense mechanisms also differ. except in immunocompromised hosts, serious infection with the opportunistic mycoses is rare. in contrast, the endemic mycoses are true pathogens that cause disease in both healthy and compromised hosts. however, the severity of infection with the endemic mycoses is determined in part by the host's response. as the number of immunocompromised patients has risen, opportunistic fungal infections have increased dramatically in recent years. in the last decade, the elderly appear to be at increasing risk for infections with the opportunistic fungi. there are several reasons for this enhanced risk. first, with increasing realization that older adults with cancer should not be excluded because of age from intensive chemotherapeutic treatment regimens, there are more immunosuppressed older cancer patients. second, as evidence for the efficacy and safety of transplantation in this population has accrued, solid organ transplantation is now more common in patients over the age of 60. third, immunosuppressive regimens, including the use of anti-tumor necrosis factor agents, are now routine in the management of rheumatologic and dermatologic conditions often found in older adults. fourth, and possibly the most important risk factor for older adults, is the increasing role of treatment in intensive care units with the use of life-support systems, catheters, and broad-spectrum antibiotics. the increase in opportunistic infections in elderly patients is primarily due to an increase in infections with candida species. the spectrum of disease varies from localized infections such as oropharyngeal candidiasis to candidemia and disseminated candidiasis. factors that predispose older patients to the development of oropharyngeal candidiasis include xerostomia, broad-spectrum antibiotics, inhaled corticosteroids, and dentures (1) . age alone does not appear to be an independent risk factor for the development of oropharyngeal candidiasis. in older adults, the presence of systemic diseases and a multiplicity of medications frequently lead to xerostomia, which then enhances candida colonization of the mucosa. denture stomatitis due to candida species is very common (see also chapter "orofacial and odontogenic infections in the elderly"). patients who do not remove their dentures at night, and those who have poor oral hygiene, are more likely to have this manifestation of candidiasis. in contrast to other candida infections, candida vulvovaginitis is unusual in older women (2) . without estrogen stimulation, the vaginal epithelium becomes thin and atrophic, glycogen production decreases, vaginal ph rises, and colonization by candida decreases. candiduria is seen more often in older adults than in younger persons (3) . the risk factors for candiduria include diabetes mellitus, obstructive uropathy, neurogenic bladder, indwelling urinary catheters, prior surgical procedures, intensive care stay, and antibiotic therapy (3) . in older adults, many of these factors occur with increasing frequency. candida species are the fourth most common cause of nosocomial bloodstream infections. several studies have found that those over 60 constitute the majority of patients with candidemia and also have the highest mortality rates (4) . elderly patients at the highest risk are those in an intensive care unit who are on broad-spectrum antibiotics, have an indwelling central venous catheter in place, are receiving parental nutrition, require renal replacement therapy, and have had a surgical procedure. c. albicans is the species most commonly found to cause candidemia, but other species, especially c. glabrata , are an increasing problem. several studies have found that c. glabrata occurs disproportionately in older adults (5, 6) but the reasons for this have not been elucidated. in older persons, cryptococcosis is increased modestly. approximately 25% of cases of cryptococcal meningitis not associated with human immunodeficiency virus (hiv) infection are in persons over age 60. the underlying conditions most often noted are hematologic malignancy, organ transplantation, corticosteroids, and cirrhosis. however, 25-30% of patients have no overt underlying immunosuppressive condition, and many of these patients are older adults. in older patients who have cryptococcal meningitis, mortality appears to be increased (7) . although many different types of molds have been described as occasional pathogens in immnuosuppressed patients, only aspergillosis and zygomycosis will be discussed. there are hundreds of aspergillus species that are ubiquitous in the environment, but very few cause infection in humans. the most common pathogenic species are a. fumigatus and a. flavus. infection ensues when conidia (spores) are inhaled into the respiratory tract of a susceptible host. nosocomial aspergillus infections are often traced to hospital construction. depending almost entirely on the immune response of the host, a wide spectrum of infections can occur. although less common than candidiasis, aspergillus infections are life threatening in immunosuppressed patients. several forms of aspergillosis, specifically chronic necrotizing pulmonary aspergillosis and sino-orbital aspergillosis, appear to occur more often in older adults (8, 9) . zygomycosis, also known as mucormycosis, is an uncommon, but often, lethal infection; there is no age predilection. the major genera identified are rhizopus and mucor . the risk factors for zygomycosis include diabetes, hematologic malignancies with neutropenia, organ transplantation, and deferoxamine chelation therapy for iron overload (10) . because of the increased risk of myelodysplastic syndrome and subsequent need for repeated transfusions with increasing age, the latter circumstance is likely the only one in which older adults may be over-represented. white plaques on the buccal, palatal, or oropharyngeal mucosa that can easily be removed are typical of oropharyngeal candidiasis. angular cheilitis and diffuse erythema, which is often present beneath upper dentures, are also manifestations of oropharyngeal candidiasis. because typical plaques are absent, the diagnosis may be overlooked (1) . candida vaginitis usually presents with pruritus and vaginal discharge that may range from "cottage cheese-like" to thin and watery (2) . when cheesy material is absent, candida vulvovaginitis must be differentiated from atrophic vaginitis. most patients with candiduria are asymptomatic and are merely colonized (3) . fewer than 5% of patients have dysuria and frequency, and even fewer have symptoms of upper tract infection. rarely, obstructive symptoms and renal failure have been noted secondary to fungus balls composed of masses of fungi. the manifestations of systemic infection with candida species are quite varied (see table 1 ). after entering the bloodstream, either from an intravenous catheter or the gastrointestinal (gi tract), the organism disseminates widely, causing microabscesses in many organs, including eye, kidney, liver, spleen, myocardium, and brain. patients with candidemia have symptoms that are indistinguishable from those associated with bacteremia (6, 11) . some are quite ill with a sepsis picture, but others may have only unexplained fever. skin lesions occurring during the course of candidemia appear as tiny pustular lesions on an erythematous base and provide a clue to the presence of candidemia (see fig. 1 ). although the major manifestation of infection with c. neoformans is meningitis, the pathogenesis of infection begins with inhalation of the organism from the environment and subsequent pulmonary infection. the chest radiograph may show nodular infiltrates, a pleural-based mass, cavitary lesions, or diffuse infiltrates (12) (see fig. 2 ). however, most often, the pulmonary infection is asymptomatic, and clinical manifestations of cryptococcosis occur only after the organism has spread to the central nervous system. elderly patients may not have the usual symptoms of fever, headache, and cranial nerve palsies but instead can present solely with confusion without fever, nuchal rigidity, or focal neurologic findings (see table 1 ). aspergillus invasion of the upper respiratory tract leads to sinusitis and may proceed to invasion of the orbit. in patients with neutropenia, the acute onset of pain, erythema, fever, serosanguinous drainage, and proptosis is seen. in older patients who are not immunosuppressed, but who may have been on corticosteroids or are diabetic, aspergillus causes a subacute sino-orbital infection with pain, proptosis, ophthalmoplegia, and loss of vision due to invasion of the apex of the orbit (8) . most patients are thought to have a retro-orbital tumor until biopsy reveals hyphae and inflammatory debris. acute pulmonary aspergillosis in immunosuppressed patients presents with fever, pleuritic chest pain, and dyspnea and has a rapidly progressive downhill course if not treated promptly (see table 1 ). chronic necrotizing pulmonary aspergillosis, occurring mostly in middle-aged to elderly men with chronic obstructive pulmonary disease, is a subacute illness. low-dose corticosteroids and broad-spectrum antibiotics are predisposing factors for this form of aspergillosis. patients have fever, cough, purulent sputum, weight loss, and pleuritic chest pain. multilobar involvement is common, cavity formation is the rule, and extension to the pleura is frequent (see fig. 3 ). progressive pneumonia is the rule unless the diagnosis is made and appropriate therapy given. patients with zygomycosis are usually quite ill. diabetics most often have the rhinocerebral form (10) (see also chapter "infections in diabetics"). a black eschar can be seen on the palate or around the orbit, and serosanguinous material is found on endoscopic examination of the sinuses (see table 1 ). orbital invasion progresses rapidly to cavernous sinus thrombosis and can culminate with cerebral infarction. in patients with pulmonary zygomycosis, the chest radiograph shows wedge-shaped chronic necrotizing pulmonary aspergillosis in a middle-aged man with no known risk factors other than chronic obstructive pulmonary disease or nodular infiltrates, which cavitate as necrosis progresses (see fig. 4 ). localized cutaneous forms occur and generally carry a better prognosis than rhinocerebral or pulmonary zygomycosis. because of the life-threatening nature of these infections, the diagnosis of a systemic opportunistic fungal infection must be made promptly. growth in culture of opportunistic fungi is rarely difficult; cultures are usually positive within a few days. the major complicating issue is that organisms as ubiquitous in the environment as aspergillus or rhizopus can easily contaminate specimens. therefore, growth in culture must be carefully assessed as to whether it truly reflects infection (13) . confounding the diagnosis of candidiasis is the fact that candida are normal flora in the gi and genitourinary (gu) tracts and on skin, and thus, growth from samples taken from non-sterile body sites often means only colonization. however, growth of candida from blood or normally sterile body fluids is obviously significant. in contrast to the other opportunists, c. neoformans is neither common in the environment nor part of the normal flora, and thus growth of this organism in culture always reflects infection. especially in immunocompromised patients who are acutely ill, histopathologic demonstration of fungi in tissues is a very important diagnostic tool. unfortunately, fig. 4 right upper lobe zygomycosis in an elderly man who had myelodysplasia leading to dependence on transfusions and treatment with deferoxamine chelation the invasive procedures necessary to obtain lung or other tissue are often precluded in extremely ill immunosuppressed patients. for cryptococcosis, examination of cerebrospinal fluid (csf) with an india ink preparation that highlights the large capsule of c. neoformans is a quick and reliable test. antibody tests have not proved to be useful for the diagnosis of opportunistic fungal infections. detection of fungal cell wall antigens is preferred. the latex agglutination test for cryptococcal polysaccharide antigen has excellent sensitivity and specificity and is routinely performed in both serum and csf (14) . the galactomannan enzyme immunoassay detects an aspergillus -specific cell wall antigen and has proven most useful in the highest risk patients, such as stem cell transplant recipients (15) . the galactomannan assay has not been studied in patients with chronic necrotizing pulmonary aspergillosis, and it is likely that it will not be useful for sino-orbital aspergillosis. other non-culture-based systems for invasive mold infections have not proved useful thus far. treatment of oropharyngeal candidiasis with a topical agent, such as clotrimazole troches, is appropriate first-line therapy. fluconazole, 100 mg daily, should be reserved for patients with severe disease or denture stomatitis that is often difficult to treat (1) . vaginal candidiasis is easily treated with topical antifungal agents such as miconazole or clotrimazole creams. however, fluconazole, 150 mg orally as a single dose, is an attractive alternative, especially for those patients who have underlying illnesses that make topical therapy difficult to use (2, 16) . candiduria often disappears with removal of the predisposing factors, especially indwelling urethral catheters and antimicrobial agents (3) . when candiduria is persistent and shown to be causing symptoms, the most appropriate treatment is fluconazole, 200 mg daily for 14 days (16, 17) . the use of amphotericin b bladder irrigation is discouraged. amphotericin b, previously the mainstay of treatment for serious candida infections, is now rarely used this indication. currently, candidemia is treated most often with fluconazole, 400 mg/day after an initial 800 mg loading dose, or with an echinocandin (16) . three echinocandin agents are available, caspofungin, micafungin, and anidulafungin, and all three appear to have equivalent efficacy for candidemia (16) . the echinocandins are extremely safe, and they have activity against those species of candida , especially c. glabrata , that are often resistant to fluconazole (18) . all intravascular lines should be removed or replaced, and treatment should be continued for 2 weeks beyond the time that blood cultures no longer yield candida unless a focal infection is discovered that will require longer therapy. the most appropriate therapy for cryptococcal meningitis in older adults has not been specifically studied, but trials in acquired immunodeficiency syndrome (aids) patients with cryptococcal meningitis have shown that the best results are obtained when induction therapy is carried out with the combination of amphotericin b (0.7 mg/kg/day) and flucytosine (100 mg/kg/day) for at least 2 weeks, followed by consolidation therapy with fluconazole, 400 mg/day for a minimum of 10 weeks (19) . initial therapy with fluconazole alone is not adequate for patients with meningitis but has been effective for patients with isolated pulmonary cryptococcal infection (20) . in spite of appropriate therapy for meningitis, symptoms of dementia may not improve in older patients. the antifungal agent of choice for treating all forms of aspergillosis is voriconazole, an extended-spectrum azole that has been shown to be superior to amphotericin b for invasive aspergillosis (21) . this agent, which can be given either intravenously or orally, has many drug-drug interactions and is best given with the help of a clinical pharmacist or infectious diseases consultant. the echinocandins also have activity against aspergillus species, but are considered second-line therapy, available if the patient cannot tolerate voriconazole (18) . finally, amphotericin b, previously the agent of choice, can also be used for invasive aspergillosis, but toxicity is much greater than that of the azoles or the echinocandins, and it cannot be recommended for older adults. the treatment of zygomycosis involves correction of the underlying immune defect, aggressive debridement of all necrotic tissue, and antifungal treatment with a lipid formulation of amphotericin b, 5-10 mg/kg daily (22) . a new azole agent, posaconazole, has been used as salvage therapy in patients who initially had been treated with amphotericin b and offers a new option for step-down oral therapy for this devastating infection (23) . as the population of the united states ages, and as older adults remain in better health for a longer period of time, they travel more extensively, visit more exotic places, and experience different outdoor activities such as those arranged on eco-tours that increase their exposure to endemic mycoses. these fungi are found in soil or vegetation; each has its own ecological niche from which it is aerosolized and subsequently inhaled (see table 2 ). older persons may become infected while traveling in an area endemic for a certain fungus, but symptoms often appear only after they return home. older adults who spend the winter months in the desert southwest may develop symptoms of coccidioidomycosis only after returning home. a patient who consults a physician in minnesota with symptoms related to coccidioidomycosis that was acquired in southern california may be the first patient with this infection ever seen by the minnesota physician, and the correct diagnosis may not be made. several endemic mycoses have the propensity to reactivate as immunity wanes with increasing age or because of immunosuppressive medications or diseases. this reactivation event might occur in a person who retired to an area of the country outside of the endemic area for a particular fungal infection. thus, although physicians in the southwestern united states are very familiar with coccidioidomycosis, histoplasmosis or blastomycosis might be overlooked in a patient from kentucky who has retired to arizona and only then develops signs of an endemic mycosis acquired years before in kentucky. the increasing use of the anti-tumor necrosis factor agents, etanercept (enbrel), infliximab (remicade), and adalimumab (humira), for rheumatoid arthritis, inflammatory bowel disease, and several dermatological conditions in older adults has increased the risk for development of histoplasmosis and coccidioidomycosis (24, 25) . these mycoses require cell-mediated immunity to eradicate the organism, and severe disseminated infections have occurred in patients who have either become newly infected or have experienced reactivation of a prior focus of infection. hiv infection is an increasingly reported problem in the older population and constitutes another risk factor for development of either newly acquired or reactivation infection with h. capsulatum or coccidioides species (see also chapter "human immunodeficiency virus/acquired immunodeficiency syndrome"). not only is the risk higher for development of these infections, but the severity of the infection is also increased. h. capsulatum is endemic in the mississippi and ohio river valleys and throughout much of central america. it is estimated that hundreds of thousands of people are infected each year, but usually the illness is self-limited with minimal flu-like symptoms. however, severe life-threatening pneumonia and disseminated infection also occur. histoplasmosis is the only endemic mycosis in which certain manifestations are age-specific; chronic cavitary pulmonary infection and chronic progressive disseminated histoplasmosis occur predominantly in older individuals (26) . b. dermatitidis , the causative agent of blastomycosis, is found most frequently in the southeastern, south central, north central united states, and the canadian provinces of ontario and manitoba. outbreaks have occurred in groups involved in outdoor activities, but most cases are sporadic and a specific point source of infection cannot be found. for blastomycosis, there is no evidence that older individuals are at more risk for developing infection than younger persons, but the mortality does appear to be greater in those age 65 years and older (27) . as the exodus of retirees to the southwestern united states continues, first-time exposure to coccidioides species has increased in older adults. this organism proliferates in the deserts of arizona and california that are typified by flora such as the saguaro cactus. there are now known to be two species of coccidioides , c. immitis in southern california, and c. posadasii in the other areas of the southwestern united states, central america, and south america. the conidia are widely dispersed during windstorms and are highly contagious. several recent epidemics of coccidioidomycosis have occurred in arizona and southern california, and thousands more individuals have been infected (28) . two important trends have been noted recently. there has been a shift in the age of patients with symptomatic coccidioidomycosis so that the annual incidence rate for coccidioidomycosis is now highest in those age 65 years and older (29) . also, older individuals and those with diabetes are more likely to develop severe pulmonary coccidioidomycosis (30) . for reasons that have never been clarified, dark-skinned races, especially african american and filipino, are more likely to experience disseminated infection than white-skinned races. the pathogenesis of the endemic mycoses is similar in that infection starts almost always with inhalation of conidia from the mold phase of the organism in the environment. thus, pulmonary manifestations are prominent in many patients. these fungi have the propensity to silently disseminate through the bloodstream to many different organs and then cause a variety of different manifestations either at the time of the initial infection or months to years later. two forms of histoplasmosis are seen most often in older adults (see table 2 ). chronic cavitary pulmonary histoplasmosis affects mostly middle-aged and elderly men who have emphysema (31) . patients with this form of histoplasmosis have constitutional symptoms of fatigue, weakness, fever, night sweats, and weight loss. pulmonary symptoms include dyspnea, cough, sputum production, and hemoptysis. the disease is subacute to chronic in its course. upper lobe cavitary disease with extensive lower lobe fibrosis is the usual chest radiographic finding (see fig. 5 ). progressive pulmonary insufficiency and death occur unless treatment is given. another form of histoplasmosis that occurs mostly in middle-aged to elderly men is progressive disseminated disease (31) . in this form of histoplasmosis, the host is unable to eradicate the organism from parasitized macrophages, and the disease is fatal if untreated. the clinical manifestations of progressive disseminated histoplasmosis include fever, fatigue, anorexia, and weight loss. dyspnea and cough are often present, lesions on the buccal mucosa, tongue, palate, or oropharynx are common, and hepatosplenomegaly is usual. because of adrenal infiltration and destruction, the patient may also present with symptoms of addison's disease. pancytopenia and increased alkaline phosphatase are frequent, and diffuse pulmonary infiltrates are often present on chest radiograph. in older patients, pulmonary blastomycosis can mimic tuberculosis with dyspnea, cough, sputum production, fever, weight loss, and fatigue (see table 2 ). the pulmonary lesions can be mass-like and mistaken for lung cancer, cavitary, or nodular in fig. 6 ). rarely, patients develop acute overwhelming pneumonia and acute respiratory distress syndrome (ards) (33) . although blastomycosis begins in the lungs, subsequent dissemination to other organs is common. frequently, the only clinical symptom is the development of one or multiple skin lesions that are usually slowly enlarging, verrucous, and have discrete punctate areas of purulence (see fig. 7 ). osteoarticular structures are frequently involved, as is the gu tract, in which the most frequently targeted organ is the prostate. coccidioidomycosis presents in many different ways (see table 2 ). patients experiencing primary disease usually have a self-limited flu-like illness consisting of fever, cough, headache, and fatigue. patchy pneumonitis is seen on chest radiograph fig. 6 pulmonary blastomycosis initially thought to be lung cancer. bronchoscopy with biopsy showed granulomas and thick-walled budding yeasts typical of b. dermatitidis (see fig. 8 ). complications include the development of persistent thin-walled cavities and less commonly, chronic pulmonary disease (34) . the latter occurs predominantly in patients with underlying emphysema and/or diabetes mellitus (30) . diffuse pulmonary infiltrates have been noted primarily in patients who have disseminated infection and are more common in those who are immunsuppressed (35) . the organs most frequently involved with disseminated coccidioidomycosis are skin, bone, and meninges. meningitis, the most feared complication, presents with chronic headache months after the initial infection and can be especially difficult to diagnose in an elderly patient returning from the southwest to other areas of the country. the course of coccidioidal meningitis is protracted, and a successful outcome is not assured, especially in older adults. the approach to diagnosis is similar for all of the endemic mycoses. cultures obtained from the infected tissue; histopathologic or cytologic examination of tissue, body fluids, or purulent material; antibody tests; and antigen detection are variably useful for each infection. the most definitive method of diagnosis is growth of the organism, but for histoplasmosis and blastomycosis growth may take 4-6 weeks. coccidioides species usually grow on fungal or regular media within several days. coccidioides is highly contagious and is classified as a bioterrorism agent. in the laboratory setting, it must be handled under a hood using biosafety level 3 precautions. clinicians must inform the laboratory that coccidioidomycosis is a possibility to avoid transmission to technicians. histopathologic or cytologic demonstration of the organism in tissues or body fluids is extremely helpful for diagnosis, especially for those patients who are acutely ill. the typical thick-walled yeasts of b. dermatitidis , showing single broad-based buds are readily identified in cytological or calcofluor white preparations of sputum and tissue biopsies. the tiny intracellular yeast forms of h. capsulatum are best visualized in tissues using methenamine silver stains. coccidioides species are quite distinctive in tissues; the large spherules (80-100 m m) are readily identified in tissue and also in purulent drainage. serology plays an important role in the diagnosis of histoplasmosis and coccidioidomycosis (31, 36) . a positive test prompts the clinician to consider more invasive procedures such as bronchoscopy, bone marrow aspiration, or liver biopsy in order to establish a diagnosis. there are occasions when the only evidence for infection is the presence of antibodies; this is especially true of meningitis, in which both fungi are exceedingly difficult to grow but csf serology is positive. for blastomycosis, specific and sensitive antibody assays are not available. an enzyme immunoassay that detects a cell wall antigen of h. capsulatum has proved to be extremely useful for the diagnosis of disseminated histoplasmosis (37) . the sensitivity is approximately 90% in patients who have a large burden of organisms; this includes patients who have aids and those who are immunosuppressed. it is not specific, however, showing cross-reactivity with blastomycosis and coccidioidomycosis. a similar assay has been developed for b. dermatitidis . it is too early to know how useful this development will be, but it is known that false positives occur in patients with histoplasmosis (38) . treatment of the endemic mycoses is similar in regard to the antifungal agents that are used. for severe infections with any of the endemic mycoses and for those who have central nervous system involvement, amphotericin b is the agent of choice. increasingly lipid formulations of amphotericin b are used, especially in older adults who often have reduced renal function. the lipid formulations are less toxic than standard amphotericin b, but are not free of toxicity, usually require hospitalization to administer, and can be associated with severe infusion reactions. most patients will require amphotericin b therapy until they have shown clinical improvement and then step-down therapy to an azole is recommended (39, 40) . the azole antifungal agents have revolutionized the treatment of the endemic mycoses; they are much less toxic than amphotericin b, and oral administration is a benefit when treating chronic infections. ketoconazole was the first oral azole agent, but because of its toxicity and lesser efficacy, it has been supplanted by itraconazole. itraconazole is the drug of choice for histoplasmosis and blastomycosis of mild to moderate severity and for step-down therapy following amphotericin b. for coccidioidomycosis, either fluconazole or itraconazole appear to be equally efficacious (41) . the usual dosage of itraconazole is 200 mg twice daily (after a loading dose of 200 mg 3 times daily for 3 days), and the dosage for fluconazole is 400 mg daily (after a single loading dose of 800 mg). therapy generally is given for 6-12 months and sometimes longer. for those patients who have coccidioidal meningitis, fluconazole is the preferred agent because of its superior csf penetration. the dosage is 800 mg daily, and therapy must be given for life as the organism is rarely eradicated from the central nervous system (34) . absorption of itraconazole capsules is dependent on gastric acidity and the presence of food in the stomach. because older adults are more likely to be achlorhydric, absorption may be decreased. histamine (h2) receptor antagonists, proton pump inhibitors, and antacids should not be used when itraconazole capsules are prescribed. however, itraconazole oral suspension does not require food or acid for absorption and is preferred for this reason. fluconazole requires neither gastric acidity nor food for absorption. drug interactions, many of which have serious implications for older adults, are frequently encountered with the azole antifungal drugs (42) . interactions with warfarin, phenytoin, and carbamazepine occur in varying degrees with all of the azole drugs in current use. itraconazole can increase serum digoxin levels with subsequent toxicity, and fluconazole can increase the effect of oral hypoglycemics. if possible, the azoles should be avoided in patients with qt prolongation on electrocardiogram and those on other medications that prolong the qt interval. in a small percentage of mostly elderly patients, itraconazole has caused the triad of edema, hypokalemia, and hypertension. all of the azole agents have been noted to cause hepatitis, and liver enzymes tests should be followed in patients taking azole agents. in spite of these issues, the azoles are exceedingly useful in older adults with endemic mycoses. most therapy is now given in the outpatient setting, and results for most patients with infection with an endemic mycosis are excellent. oropharyngeal candidosis in the older patient treatment of vaginal candida infections a prospective multicenter surveillance study of funguria in hospitalized patients nosocomial bloodstream infections in us hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study epidemiology of candidemia: 3-year results from the emerging infections and the epidemiology of iowa organisms study candida glabrata fungemia: experience in a tertiary care center cryptococcosis in human immunodeficiency virus-negative patients in the era of effective azole therapy invasive and allergic fungal sinusitis chronic cavitary and fibrosing pulmonary and pleural aspergillosis: case series, proposed nomenclature change, and review epidemiology and outcome of zygomycosis: a review of 929 reported cases niaid mycoses study group. a prospective observational study of candidemia: epidemiology, therapy, and influences on mortality in hospitalized adult and pediatric patients pulmonary cryptococcosis in nonimmunocompromised patients the impact of culture isolation of aspergilllus species: a hospital-based survey of aspergillosis cryptococcal antigen test revisited: significance for cryptococcal meningitis therapy monitoring in a tertiary chinese hospital detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis clinical practice guidelines for the management of candidiasis: 2009 update by the infectious diseases society of america candiduria: a randomized, double-blind study of treatment with fluconazole and placebo echinocandin antifungal drugs treatment of cryptococcal meningitis associated with the acquired immunodeficiency syndrome pulmonary cryptococcosis in the immunocompetent host. therapy with oral fluconazole: a report of four cases and a review of the literature voriconazole versus amphotericin b for primary therapy of invasive aspergillosis zygomycosis: an emerging fungal pathogen with new options for management posaconazole is effective as salvage therapy in zygomycosis: a retrospective summary of 91 cases histoplasmosis after treatment with anti-tnf-(alpha) therapy increased risk of coccidioidomycosis in patients treated with tumor necrosis factor alpha antagonists fungal infections in older adults the epidemiology of blastomycosis in illinois and factors associated with death an epidemic of coccidioidomycosis in arizona associated with climatic changes risk factors for acute symptomatic coccidioidomycosis among elderly persons in arizona risk factors for severe pulmonary and disseminated coccidioidomycosis histoplasmosis: a clinical and laboratory update pulmonary blastomycosis: findings on chest radiographs in 63 patients acute respiratory distress syndrome and blastomycosis: presentation of nine cases and review of the literature coccidioidomycosis . clinical infectious diseases coccidioidomycosis in persons infected with hiv type 1 current status of serologic studies in coccidioidomycosis improvements in diagnosis of histoplasmosis antigen assay with the potential to aid in diagnosis of blastomycosis clinical practice guidelines for the management of patients with histoplasmosis: 2007 update by the infectious diseases society of america clinical practice guidelines for the management of blastomycosis: 2007 update by the infectious diseases society of america comparison of oral fluconazole and itraconazole for progressive, nonmeningeal coccidioidomycosis -a randomized, double-blind trial antibiotic therapy for geriatric patients suggested reading clinical practice guidelines for the management of blastomycosis: 2007 update by the infectious diseases society of america coccidioidomycosis . clinical infectious diseases histoplasmosis: a clinical and laboratory update zygomycosis: an emerging fungal pathogen with new options for management clinical practice guidelines for the management of candidiasis: 2009 update by the infectious diseases society of america key: cord-001521-l36f1gp7 authors: nan title: oral and poster manuscripts date: 2011-04-08 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2011.00209.x sha: doc_id: 1521 cord_uid: l36f1gp7 nan pandemic influenza h1n1 (h1n1pdm) virus of swine-origin causes mild disease, but occasionally is associated with acute respiratory distress syndrome and death. 1, 2 it is important to understand the pathogenesis of this new disease. previously we showed a comparable virus tropism and host innate immune responses between h1n1pdm and seasonal h1n1 influenza virus in the human respiratory tract, 3 however h1n1pdm virus differed from seasonal h1n1 influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection. we now compare the tropism and host responses elicited by pandemic h1n1 with that of related swine influenza viruses and a pandemic-swine reassortant virus in ex vivo and in vitro cultures of the human respiratory tract and conjunctiva. we have used recombinant virus to investigate the role of the hemagglutinin (ha) and neuraminidase (na) of h1n1pdm virus in its conjunctival tropism. these findings are relevant for understanding transmission and therapy. fragments of human conjunctiva, bronchi, and lung tissues were cut into 2-3 mm fragments within 2 h of collection and infected with influenza a viruses at a titer of 10 6 tcid 50 ⁄ ml. viruses investigated included h1n1pdm (a ⁄ hk ⁄ 415742 ⁄ 09), swine h1n2 virus (a ⁄ swine ⁄ hk ⁄ 915 ⁄ 04), which shares a common derivation for seven genes with h1n1pdm, a natural swine reassortant h1n1 (a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10), which has acquired the na gene from h1n1pdm and other swine influenza h1n1 viruses. reverse genetics derived recombinant viruses with ha and na gene segments of seasonal h1n1 and pandemic h1n1 swapped were also studied. lung fragments were cultured at 37°c in culture plates; conjunctival and bronchial biopsies were cultured in air-liquid interface at 33 and 37°c respectively. tissue fragments were infected for 1 h and incubated for 1, 24, and 48 h post infection. infectious viral yield was assessed by titration in mdck cells. the infected tissues were fixed with formalin and analyzed by immunohistochemistry for influenza antigen. cytokines profiles induced by influenza virus infected respiratory epithelial cells in vitro were measured by quantitative rt-pcr and elisa. we found comparable replication in seasonal and pandemic h1n1 viruses in human respiratory tract, while the swine influenza a ⁄ swine ⁄ hk ⁄ 4361 ⁄ 99 (h1n1) virus and a ⁄ swine ⁄ hk ⁄ 915 ⁄ 04 (h1n2) virus failed to infect and replicate in human lung ex vivo culture, but it replicated productively in human bronchus ex vivo. interestingly, the swine reassortant influenza h1n1 (a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10) virus (with the na from h1n1pdm) infected and productivity replicated in lung ex vivo and in vitro. pandemic h1n1pdm virus, but not seasonal h1n1 virus, was able to infect ex vivo cultures of human conjunctiva, suggesting subtle differences in receptor binding profile in h1n1pdm, seasonal viruses, and the swine related h1n2 viruses. using reverse genetics derived recombinant viruses, we were able to demonstrate that the ha and na segments of h1n1pdm, but not the polymerase genes, were required for the conjunctival tropism of h1n1pdm ( figure 1 ). in contrast with highly pathogenic influenza h5n1 virus, which induced high cytokine and chemokine decretion, the related swine viruses, a ⁄ swine ⁄ hk ⁄ 915 ⁄ 04 (h1n2), as well as the swine pandemic reassortant virus, a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10 (h1n1) we studied were similar to h1n1pdm and seasonal influenza viruses in their intrinsic capacity for cytokine dysregulation. collectively, our results suggest that pandemic h1n1pdm virus differs in modest but subtle ways from seasonal h1n1 virus in its intrinsic virulence for humans, findings that are in accord with the epidemiology of the pandemic to date. the ha and na gene segments are key to the conjunctival tropism manifested by the h1n1pdm virus. the pandemic reassortant influenza h1n1 (a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10) virus isolated from swine with the na from h1n1pdm shares with h1n1pdm the capacity for productive replication in lung ex vivo and in vitro. these findings are relevant for understanding transmission and therapy. isolation of influenza viruses from specimens is traditionally performed in two classical systems: embryonated chicken eggs and mdck cell culture. nevertheless, several publications are dedicated to the theme of alternative cell culture systems, which may be used for influenza virus isolation and cultivation. [1] [2] [3] this is in part because mdck cells are of animal origin, which means that they cannot be used as a proper model for estimating interactions between a human virus and a human cell culture as a host. a variety of human monolayer and suspension cell cultures have been tested on their capability to support influenza virus replication. among them, some support influenza a virus growth as well as mdck cells do, 4 others support replication of a virus, but do not enable the formation of mature viral particles, 5 whereas others show only a weak level of replication or are not permissive at all. 2 caco-2 cells, for example, represent a good substitute for mdck cells, because it has been shown that the rate of viral isolation in caco-2 cells is as effective as in mdck, and sometimes is even better. 6 the success of viral replication is determined not only by the cell culture type, but also by the virus itself. despite the accepted view that it is the type of receptor that defines the interaction between the virus and the host cell, there is evidence that it is not the only factor that predetermines the fate of the cell. 7 the fate of the infected cell can also differ. a series of articles show that apoptosis is the most probable mechanism of cell killing by influenza viruses. 8, 9 influenza a viruses of different subtypes induce apoptosis to a different extent (e.g. h3 viruses provoke more strong apoptotic response than h1 viruses do 10 ). nevertheless, it has been demonstrated that caco-2 cells do not follow the apoptotic pathway and die through necrosis. 11 the sjpl cell line also dies through necrotic pathway and not apoptosis. 12 the aim of our work was to compare growth characteristics of different flu viruses (e.g. avian, swine, and human) in various human and animal cell cultures and to evaluate their influence on cell culture growth. the parameters measured in the study were as follows: cytopathic changes of cell cultures following virus infection, hemagglutinin production, np synthesis, the dose-dependent effect of infection on cell proliferation, and the ability of viruses to induce apoptosis. influenza viruses used included: highly pathogenic avian h5n1 a ⁄ kurgan ⁄ 5 ⁄ 05, low pathogenic avian h5n1 a ⁄ gull ⁄ kostanai ⁄ 7 ⁄ 07, swine h1n1 a ⁄ swine ⁄ 1976 ⁄ 31, human h1n1v a ⁄ california ⁄ 07 ⁄ 09, human h1n1v a ⁄ saint-petersburg ⁄ 5 ⁄ 09, human h1n1 a ⁄ brisbane ⁄ 59 ⁄ 07, and human h3n2 a ⁄ brisbane ⁄ 10 ⁄ 07. the viruses were propagated in 10-days embryonated chicken eggs, the allantoic fluid was collected, the aliquots were made and stored at )70°c for further use. to evaluate tcid50 for each virus on all cell cultures, 96-well plates were used. the cells were seeded 0ae2 ml per well (concentration of 1-1ae5 · 10 cells ⁄ ml). the confluent 24-h old monolayer was used for viral inoculation. the cells were washed twice with serum-free medium, then 0ae05 ml of tenfold viral dilutions from viral aliquots were added and left for 45 min for contact at 37°c. the cells were then washed to remove the non-attached particles, and the wells were filled with tpck-trypsin (2 lg ⁄ ml)-containing medium without bovine fetal serum. the plates were observed daily for cytopathic effect, and the results were evaluated at 72 h after infection for cytopathic effect and by reaction of hemagglutination with suspension of chicken erythrocytes (0ae75%). infection of suspension cell cultures was done in centrifuge tubes. cells (concentration 3-5 · 10 5 ) were inoculated with viral dilutions (moi = 1-10). after 45 min of contact, cells were washed, resuspended in rpmi with trypsin and fetal serum, and seeded in 24-well plates (1 ml in each well). the results were fixed after 72 h, calculating the number of cells grown and estimating the rate of apoptosis by hoechst-33258 staining. 13 cells were grown in 24-well plates with seeding concentration 1 · 10 cells ⁄ ml. one millilitre of cell suspension was placed in each well, inoculated with viral dilution (moi = 1-1000) and left for 72 h. after, the cells were detached from plastic with versene and calculated in fuks-rosental camera to evaluate the number of cells. the monoclonal antibodies obtained in research institute of influenza towards viral nucleoprotein np were used following the standard protocol described in. 13 for all viruses tested, mdck turned out to be more permissive than sp cell culture. avian viruses, independently of their pathogenicity, replicated efficiently on both animal cultures tested. human h1n1 and h3n2 viruses demonstrated weaker replication in sp cells. the most significant differences were seen for swine influenza and pandemic h1n1v viruses which replicated in mdck cells at the rates comparable with other viruses, but showed poorer growth in sp cell line (see table 1 ). human cell lines displayed clear differences in their susceptibility to viruses of various origins. avian influenza viruses replicated in all cell lines except girardi heart, and the most intense replication rate was observed for ecv-304, l-41, and rd lines. a-549 and a-172 were poorly infected, as well as all suspension cell lines tested. seasonal human h1n1, as well as h3n2 viruses, replicated in all cell cultures tested, but the rate of infectivity was rather low in practically all cultures tested with the exception of rd and t-98g cell lines. strikingly, swine influenza virus and human pandemic h1n1v viruses didn't replicate well in any of human lines tested. a weak replication rate was observed in ecv-304, rd, and t-98g, but in general, human cell lines were the titers produced by swine and pandemic influenza viruses are shaded in grey. *low-pathogenic avian influenza virus; **highly-pathogenic avian influenza virus poorly susceptible to pandemic h1n1v. swine influenza virus differed because it infected weakly a-172 and girardi heart cell cultures, which was not the case for h1n1v viruses. our study has shown that all influenza viruses were able to induce apoptosis in the cell cultures tested. the degradation of chromatin found in the nucleus with hoechst-33258 staining was seen before the first symptoms of cytopathic effect (cpe) in monolayer of cells. in cell cultures where the cpe was not visible, high doses of virus still induced apoptotic response. the process of apoptosis is rather well studied in mdck cells and some other cell types, so we've focused on three human monolayer cell cultures that are relatively poorly studied: a-549, ecv-304, and flech. these cell cultures are less susceptible to viral infection, and besides, it was interesting to find out whether the viruses that do not cause any cpe do infect these cultures. a-549 turned out to be most sensitive to apoptotic response, while flech turned out to demonstrate weak reaction. time needed for apoptosis induction by different flu viruses also varied. the earliest apoptosis was noted for h5n1 and h3n2 viruses and h1n1 viruses induced apoptosis at about 20 h postinfection. it is well-known that apoptosis can be induced only by a reproducing virus, and that uv-kills viruses that are not capable of it. we tested whether swine and pandemic h1n1v viruses (that do not show cpe in these cultures) do replicate in them and induce apoptosis with the help of monoclonal antibodies against viral np. the obtained data show that they indeed do replicate in these cell cultures, as we observed np fluorescence, and that they also induce apoptosis (see table 2 ). we've shown earlier 13 thus, we've tested the ability of swine and pandemic h1n1v viruses in this aspect. it was shown that these viruses were comparable with the effect seen for seasonal h1n1 virus. moreover, swine influenza virus induced stronger apoptotic response in hemablastoid cell lines in comparison with pandemic h1n1v viruses, which also have a swine origin. we also checked the ability of flu viruses to influence monolayer cell cultures growth. the data clearly indicated that only ecv-304 endothelial line and t-98g glioblastoma line displayed cell proliferation in response to low moi. apoptosis wasn't registered in these stimulated cultures, apparently because the moi was very low. all the other monolayer cultures didn't respond to low moi by stimulation of their proliferation. interaction between an influenza virus particle and a host cell can follow several scenarios. cpe seen in infected cells is accompanied with high rates of viral particles production and leads to cell death. the death itself may be through apoptotic or necrotic pathways. 4, 8 also, infection process in low doses can stimulate cell proliferation -the effect seen for hemablastoid lines, histiocytes, peripheral blood cell lines, 14, 15 and in glioblastoma and endothelial cell lines as it was described here. considering the origin of ecv line, 16 these cells bear all the antigenic, biochemical, and physiological traits of umbilical cord and are actively used in pharmacological tests as well as glioblastoma cells; they also are of special interest for oncogenesis studies. table 2 . replication, apoptosis induction, and np synthesis of influenza viruses in a-549, ecv-304 and flech cell cultures. the numbers represent the log 10 tcid 50 ⁄ 0ae2 ml calculated by reed-muench method as described in. 17 the ()) symbol means that no cpe could be observed in any dilution and no hemagglutination could be registered. the (+) symbol means that apoptosis was observed with hoechst-33258 staining though the productive replication and production of progeny viruses in human cell lines was generally low, it is evident that viral infection does occur in these cells, even for swine and h1n1v viruses. it can be demonstrated by the presence of np de novo synthesis and by stimulation of virus-induced apoptosis. in fact, we observe a contradiction: avian influenza viruses actively reproduce in human cell lines, but we do not see their vast spreading in human population, while h1n1v viruses that hardly replicate in all human cultures tested have caused the latest pandemic. influenza viruses continue to cause problems globally in humans and their livestock, particularly poultry and pigs, as a consequence of antigenic drift and shift, resulting frequently and unpredictably in novel mutant and reassortant strains, some of which acquire the ability to cross species barriers and become pathogenic in their new hosts. long-term surveillance of influenza in migratory waterfowl in north america and europe have established the importance of anseriformes (waterfowl) and charadriiformes (gull and shorebird) in the perpetuation of all known subtypes of influenza a viruses. the available evidence suggests that each of the 16 hemagglutinin (ha) and nine neuraminidase (na) subtype combinations exist in harmony with their natural hosts, cause no overt disease, and are shed predominantly in the feces. 1, 2 in this study we determined the subtypes and prevalence of low-pathogenic influenza a viruses present on the territory of kazakhstan in 2004-2006 and further analysed the ha and na genes of these isolates in order to obtain a more detailed knowledge about the genetic variation of influenza a virus in their natural hosts. (institute for biological safety problems, gvardeiskiy, zhambyl oblast, kazakhstan)). samples that were identified as influenza a virus positive by matrix rrt-pcr were thawed, mixed with an equal volume of phosphate buffered saline containing antibiotics (penicillin 2000 u ⁄ ml, streptomycin 2 mg ⁄ ml, and gentamicin 50 lg ⁄ ml), incubated for 20 minutes at room temperature, and centrifuged at 1500 g for 15 minutes. the supernatant (0ae2 ml ⁄ egg) was inoculated into the allantoic cavity of four 9-day old embryonated hens' eggs as described in european union council directive 92 ⁄ 40 ⁄ eec. 3 embryonic death within the first 24 hour of incubation was considered as non-specific, and these eggs were discarded. after incubation at 37°c for 3 days the allantoic fluid was harvested and tested by haemagglutination (ha) assay as describe in european union council directive 92 ⁄ 40 ⁄ eec. in the cases where no influenza a virus was detected on the initial virus isolation attempt, the allantoic fluid was passaged twice in embryonated hens eggs. the number of virus passages in embryonated eggs was limited to the maximum two to limit laboratory manipulation. a sample was considered negative when the second passage ha test was negative. the subtypes of the virus isolates were determined by conventional haemagglutination inhibition (hi) test and neuraminidase inhibition (ni) test, as describe in european union council directive 92 ⁄ 40 ⁄ eec. 3 rna extraction and pcr with specific primers rna was extracted from infective allantoic fluid using rneasy mini kit (qiagene, gmbh, germany) according to the manufacturer's instructions. the rna was converted to full-length cdna using reverse transcriptase. the rt mix comprised 2ae5 ll of dmpc water, 5 ll of 5· first strand buffer (invitrogen), 0ae5 ll of 10 mm dntp mix (amersham biosciences), 2 ll of 50 mm uni12 primer, 32 u of rnaguard (amersham biosciences), 200 u of mmlv reverse transcriptase (invitrogen) and 5 ll rna solution in total volume of 25 ll. the reactions were incubated at 42°c for 60 minutes followed by inactivation of the enzyme at 95°c for 5 min. pcr amplification with ha and na gene specific primers was performed to amplify the product containing the full length ns gene. twenty-five microliter pcr-mix contained 1· platinum taq buffer (invitrogen), 200 lm dntp, 2ae5 mm mgcl 2 , 240 nm each of fw primer and rw primer, 1 u platinum taq dna polymerase (invitrogen) and 3 ll cdna. reactions were placed in a thermal cycler at 95°c for 2 min, then cycled 35 times between 95°c 20 seconds, annealing at 58°c for 60 seconds, and elongation at 72°c for 90 seconds and were finally kept at 8°c until later use. sequences of the purified pcr products were determined using gene specific primers and bigdye terminator version 3ae1 chemistry (applied biosystems, foster city, ca), according to the manufacturer's instructions. reactions were run on a abi310 tm dna analyzer (applied biosystems). sequencing was performed at least twice in each direction. after sequencing, assembly of sequences, removal of low quality sequence data, nucleotide sequence translation into protein sequence, additional multiple sequence alignments, and processing were performed with the bioedit software version 7ae0ae4ae1 with an engine based on the custal w algorithm. the phylogenetic analysis, based on complete gene nucleotide sequences were conducted using molecular evolutionary genetics analysis (mega, version 4ae0) software using neighbor joining tree inference analysis with the tamura-nei c-model, with 1000 bootstrap replications to assign confidence levels to branches. [4] [5] [6] [7] ha and na sequences obtained from genbank the ha and na gene was analyzed both with selected number of influenza isolates and in comparison with virus genes obtained from genbank were used in phylogenetic studies [22] . the nucleotide sequence data obtained in this study has been submitted to the genbank database and is available under accession numbers fj434373, fj436942ae1, fj434369, fj434370, gu982281-gu982284 for ha and fj434374, fj436943ae1, fj434371, fj434372, gu982285-gu982288 for na. avian influenza prevalence in our study h4, h5, and h13 influenza a virus subtypes were found to circulate at the same time, in the same geographic region in the kazakhstan. this finding most likely indicates the existence of a large reservoir of different influenza a viruses in kazakhstan. we analyzed the ha and na gene sequences of the eight influenza a viruses isolated in kazakhstan together with selected number of isolates, reported between year 1941 to 2008, and previously published in the genbank. 8 phylogenetic analysis of the h4 ha gene showed that all viruses separated into the american and eurasian lineages ( figure 1 ). an evolutionary tree suggests that north american isolates have diverged extensively from those circulating in other parts of the world. geographic barriers which determine flyway outlay may prevent the gene pools from extensive mixing. the lack of correlation between date of isolation and evolutionary distance suggests that different h4 ha genes co circulate in a fashion similar to avian h3 ha genes and influenza c genes, implying the absence of selective pressure by antibody that would give a significant advantage to antigenic variants. analysis of phylogenetic relationships among the ha5 ha genes reported in this study clearly shows that viruses belong to the western pacific flyway, one of the major migratory flyways in this region that have subsequently spread throughout eurasia. 9 these findings provide further evidence of the dynamic influenza virus gene pool in this region. along the western pacific migratory flyway, the influenza virus gene pool in the domestic waterfowl of southern china has 'mixed' longitudinally with viruses isolated from japan, mongolia, and siberia. however, it appears that there has also been 'mixing' latitudinally through overlapping migratory flyways, thereby facilitating interaction between the influenza virus gene pool in domestic waterfowl in the eastern and western extremities of the eurasian continent. this helps to explain the latitudinal spread of the qinghai-like (clade 2ae2) h5n1 virus in the last 2 years, while h5n1 outbreaks in korea and japan may represent the longitudinally transmitting pathway. 10 ha of subtype h13 so far has been found exclusively in shorebirds, such as gulls, and in a pilot whale (potentially a spillover from shorebirds), but not in other avian species that are natural hosts of influenza a virus, such as ducks and geese; therefore the study of the evolution of these viruses is very interesting. phylogenetic analysis h13 ha gene revealed three significantly different evolutionary lines: an american line, a european line, and a line comprising the isolates from america and eurasia. 11 further we analyzed na genes of influenza viruses (figure 2) . the na gene is important both because of its functional role in promoting the dissemination of the virus during infection, and because, like ha, it is a principal target of the immune system. it was shown that phylogeny of na genes of influenza have the same properties as hemagglutinin. na genes of kazakhstanian viruses belong to eurasian lineage of virus evolution. obtained data are important for surveillance and diagnostics because some of the lpai viruses examined in this study can infect and be shed by chickens and turkeys and may have epidemiology potential during further recombination with other influenza viruses. influenza virus is divided into different subtypes based on hemagglutinin (ha) and neuraminidase (na) on the virus surface. within each subtype, ha continues to mutate and produce immunologically distinct strains, as antigenic drift. the continuous mutation of influenza virus (iv) is important for annual epidemics and occasional pandemics of disease in humans. antigenic drift requires vaccines to be updated to correspond with the dominant epidemic strains. in humans, ivs show both antigenic drift frequently. in contrast, ivs from birds are in evolutionary stasis, 1 and they show little amino acid changes. 2, 3 the reason is that ivs in bird intestine are not subjected to strong immune selection. hemagglutinin (ha) gene of influenza a virus encodes the major surface antigen, which is the target for the protective neutralizing antibody response that is generated by infection or vaccination. in humans, influenza a viruses show antigenic drift with amino acid changes in the globular head of the ha so as to evade herd immunity of the population. on the contrary, avian influenza a viruses show evolutionary stasis in wild birds. h6 aivs have occurred frequently in chicken farms in the world. 4 although vaccination is not permitted, h6n1 aivs have circulated in taiwan for a time. 5 the seroprevalence in chicken flocks reaches about 50% in the field. h6n1 aivs invades internal organs, such as kidney and lung. 6 thus, viruses in chicken flocks are pressured into antibody selection. here, we report that h6n1 aivs in the field have showed evolutional changes instead of evolutional stasis. in response to requests from poultry farmers for diagnostic investigations of illness in poultry flocks, the authors did necropsy at the pen-site. after careful examination, tracheae were taken and kept in cold for virus isolation in the laboratory. for avian influenza virus isolation, trachea was homogenized 1:10 in tpb with antibiotics. the homogenate was frozen and thawed three times and then centrifuged at 1157 g for 15 minutes. the supernatant was passed through a 0ae45 lm filter. the homogenate was examined for the presence of virus by inoculation into five 9-to 11-day-old specific-pathogen-free (spf) chicken eggs for two passages. thirteen h6n1 aivs were isolated in this laboratory during 2000 and 2008 from different parts of taiwan. besides the viruses isolated in this laboratory, the ha sequences of 27 chicken h6n1 aivs were from the genbank. the accession numbers of hemagglutinin of aiv reference strains included in this study were as the following: g2 ⁄ 87, dq376619; g23 ⁄ 87, dq376620; 0824 ⁄ 97, dq376621; na3 ⁄ 98, dq376622; 165 ⁄ 99, dq376626; 0705 ⁄ 99, dq376624; ns2 ⁄ 99, dq376623; sp1 ⁄ 00, dq376628; 0329 ⁄ 01, dq376633; 1205 ⁄ 01, dq376630; 1212 ⁄ 01, dq376631; 1215 ⁄ 01, dq376632; 0208 ⁄ 02, dq376641; 0408 ⁄ 02, dq376642; pf1 ⁄ 02, dq376635; pf2 ⁄ 02, dq376636; pf3 ⁄ 02, dq376637; a37 ⁄ 02, dq376639; 0320 ⁄ 02, dq376638; 0107 ⁄ 02, dq376640; 0222 ⁄ 02, dq376634; 0706 ⁄ 03, dq376644; 1203 ⁄ 03, dq376645; ch1006 ⁄ 04, dq376649; 0114 ⁄ 04, dq376647; a342 ⁄ 05, dq376653 and 0204 ⁄ 05, dq376652. the viruses isolated were propagated in the allantoic cavities of 10-day-old embryonated spf eggs for 72 hour. the virus rna was extracted using qiaamp viral rna miniprep kit (qiagen) . six-week-old balb ⁄ c mice were injected emulsion intraperitoneally with 100 lg of purified and concentrated a ⁄ chicken ⁄ taiwan ⁄ 2838v ⁄ 00 (h6n1) virion with complete freund's adjuvant. every two weeks, the mice were boosted supplementary five times with 50 lg of virion in incomplete freund's adjuvant. when the mice were boosted, blood was collected from tail vein and tested by the western blot assay to check the antibody titers. the mice were then injected intraperitoneally with 50 lg of virion at week 8. five days after the last injection, the splenocytes in the mice were fused with myeloma cells (sp2 ⁄ 0-ag14). one week before fusion, the myeloma cell line was expended in dmem medium (hyclone laboratories, logan, ut) with 10% fetal bovine serum at 37°c to ensure they were in the exponential growth phase. the spleen cells from immunized mice were washed, harvested, and mixed with the previously prepared myeloma cells and fused by gradually adding 50% polyethylene glycol-1500. the resulting pellet was plated into 96 well tissue culture plates. only the fused cells grew in medium with hypoxanthine-aminopterin-thymidine (hat). with fresh medium replacement over 2 weeks, the hybridomas were ready for screening. hightiter monoclonal antibody (mab) preparations were obtained from the ascetic fluid of mice injected with the selected hybridoma clones. the antibody from mouse ascetic fluids was purified by precipitation with ammonium sulfate, then aliquoted and frozen at )70°c, avoiding repeated freezing and thawing. eventually, six mabs were obtained and named ch11-d10, eb2-b3, eb2-e5, eb2-f9, ff9-f5, and ff9-f7, respectively. the hi test was performed following a standard method. all the viruses were diluted twofold and reacted with 1% chicken erythrocytes in the v-bottomed microtiter plate by the hemagglutination test. after agglutination, four hemagglutinating units of a ⁄ chicken ⁄ taiwan ⁄ 2838v ⁄ 00 (h6n1) and ascetic fluids from the immunized mice of the six mabs were prepared for hi test. hi titers of 2 4 or more were regarded as positive. the cases submitted for diagnosis from chicken farms had respiratory signs, increase in mortality, or drop in egg production (e.g. egg production dropped from 10% to 40%). the extent of drop in egg production depended on the chicken ages. for example, the age of case 3511 was 27 weeks, a stage of increasing egg production. however, after h6n1 aiv infection, the egg production decreased 1% instead of increasing and then stayed at 65% for a week. the infected chickens showed signs of decreasing activity, anorexia from 150 g per bird to 90 g per bird, and respiratory signs. case 2829 showed infection in the second floor first and then transmitted to third and fourth floor, indicating that the virus transmitted by air or human movement. however, most cases showed air borne transmission from one flock to another in spite of enforcing restrictions of persons entering the poultry pens and changing clothes and booths. in most cases, males' mortality was higher than that of female pen mates. by comparing the sequences of ha of those h6n1 viruses, we found that amino acid changes in ha1 were higher than those in ha2, showing that antigenic changes on the globular head of ha molecule rather than randomly on the whole ha protein, indicating that h6n1 viruses in taiwan had been selected in the presence of antibody pressure. the aa residues and changes that showed yearly trends were the followings: a-5s, i19s, v30i, n35s, e39k, l45m, e54d, q66k, a94v, or t, s127n, s128r, k133n, y138d, n139t, s140i, g141d, l149v, i152v, g155e, t188n, g226s, a285v, k304e, d386n, i533m, and m535i. however, their significance on antigenic variation was previously unknown. by hemagglutinination inhibition (hi) assays, except mab ch11-d10, all other monoclonal antibodies elicited from 2838v ⁄ 00 showed different hi titers with the different h6n1 viruses (table 1 ). however, those 5 mabs showed negative hi to 2829 and 2831, the early h6n1 strains. this indicated that the epitopes recognized by those mabs were undergoing antigenic drift. introduction aquatic birds are recognized as the natural reservoirs of the influenza a virus as all known subtypes (h1-h16, n1-n9) have been found in them. phylogenetic analyses of influenza viruses found in other animals revealed that all were directly or indirectly derived from viruses resident in aquatic birds. 1 however, the prevalence, movement, and evolutionary dynamics of influenza viruses in these avian hosts have not been well defined. southern china was hypothesized to be an 'epicenter' for the generation of human pandemic influenza viruses as all major influenza pandemic viruses in the 20th century emerged from this region. 2 the ecological background that facilitates the occurrence of these pandemic influenza strains has not been fully explored. in the past two decades, four lineages, belonging to h5n1, h9n2, and h6n1 viruses, have become established and long-term endemic in different types of poultry in this region. [3] [4] [5] some of these viruses were disseminated to many countries in eurasia and africa and have continued to cause sporadic human infection, posing a persistent pandemic threat to the world. 6 in the mean time, the endemic influenza lineages have undergone extensive genetic reassortment events giving rise to many variants, dramatically increasing the genetic diversity of the influenza virus in this region. questions remain as to how and where these viruses emerged, and what were the sources of the gene segments incorporated within the novel reassortant variants of the h5n1, h9n2, and h6n1 virus lineages. to address these questions, surveillance of influenza in migratory and domestic (sentinel) ducks has been conducted since 2002 at poyang lake, the biggest fresh-water lake and the major migratory bird aggregation site in southern china. the aim of this study is to identify the prevalence, seasonality, and movement of virus between migratory and domestic ducks. migratory ducks were captured during over-wintering, from november to march. cloacal swabs and blood samples were collected from each individual bird. all birds were released after sampling. to observe the interaction between migratory ducks and domestic birds, we also sampled domestic ducks from two duck farms (designated as sentinel ducks) surrounded by rice fields and inaccessible to other types of poultry, but accessible to migratory birds. that is, the sentinel ducks share the same water body with migratory ducks and have the chance to spread viruses to each other. for sentinel ducks, sampling was conducted fortnightly, all year-round, on the two farms from august 2002 onwards. cloacal swabs and fresh fecal droppings were taken. about 200 birds were randomly sampled fortnightly from these farmed ducks. all swabs were soaked in vials containing 1ae5 ml transport medium with antibiotics and kept on ice-packs during sampling and immediately stored in )80°c freezers for further use. blood samples from migratory ducks were treated according to methods previously described. 7 serological survey and virus subtyping in migratory and sentinel ducks used hemagglutination inhibition (hi) and neuraminidase inhibition (ni) tests as previously described. 3 for isolates that were not identified by reference antisera, subtypes were determined by rt-pcr using subtype specific ha and na diagnostic primers. prevalence and seasonal patterns of influenza virus in migratory and sentinel ducks during 2002 during -2007 a total of 11 508 cloacal swabs from migratory ducks and 36 475 cloacal or fecal swabs from sentinel ducks were collected at poyang lake. from these specimens, 90 influenza isolates were obtained from migratory ducks and 1681 from sentinel ducks; isolation rates of 0ae78% and 4ae61%, respectively (table 1) . it was noted in sentinel ducks that virus occurrence formed a seasonal peak from november to february, which completely overlapped the over-wintering months of migratory ducks. this suggests that virus movement or transmission between migratory and sentinel ducks occurred during this period at poyang lake. thirty positive samples (hi titer ‡20) were identified from 715 blood samples collected during november and december in 2004. among these, 15 samples were positive to h6, 8 were positive to h9, 5 were positive to h1, and 2 were positive to h4. one serum sample was positive to both h4 and h6, which suggested co-infection of influenza virus in migratory ducks might occur in natural conditions. poyang lake, which is located in the northeastern part of jiangxi province, is the largest freshwater lake in china and is part of the eastern asia-australia migration route. 9 every year, hundreds of thousands of migratory ducks congregate at poyang lake during the migration season. 9 recent farming practice involves raising domestic waterfowl in dense populations in the poyang lake region. farmraised domestic waterfowl are allowed to feed in and share the same water body with migratory birds, thereby facilitating direct interactions between domestic waterfowl and freeranging migratory birds. this makes poyang lake an ideal site to observe the dynamics of influenza virus interactions between migratory and sentinel ducks in southern china. in our longitudinal surveillance during [2002] [2003] [2004] [2005] [2006] [2007] , the overall virus isolation rate from migratory ducks was less than 1%, which suggests a low prevalence of viral infection during the birds' southern migration. similar results have been observed in taiwan, which is also an important stopover site for migratory birds along the eastern asia-australia migration route during 10 years of surveillance. 10 the overlap in seasonal patterns of virus infection between migratory and sentinel ducks found in our study suggests that virus movement or transmission between migratory and sentinel ducks occurred during the period of time migratory birds were at poyang lake. the ha subtypes harbored in migratory and sentinel ducks were similar in our study. for migratory ducks, h4, h6, h10 were the predominant subtypes, while h3, h4, and h10 were the major subtypes in sentinel ducks. hpai h5n1 was only detected from migratory ducks in early 2005 on two sampling occasions. from phylogenetic analyses the h5n1 viruses isolated from migratory ducks were closely related to the viruses endemic in domestic poultry in southern china. 8 therefore, it appears that h5n1 viruses endemic in domestic poultry could be transmitted to migratory ducks via close contact in southern china. only lp h5 viruses were detected from sentinel ducks at poyang lake during this period. whether h5n1 virus infection was absent from sentinel ducks at poyang lake needs further investigation. serological surveys provided further evidence for the prevalence of aiv in migratory ducks at poyang lake. the serological results in 2004 did not match well with the epidemiological results during [2002] [2003] [2004] [2005] [2006] [2007] , which suggests that influenza virus infection in migratory birds could be influenced by multiple factors, such as host immune status, population size, spatial and temporal variations, and migration routes. southern china has the biggest domestic duck population in the world. our study demonstrates that dynamic interactions between migratory ducks and sentinel ducks occurred frequently throughout the surveillance period. thus, sentinel ducks could be treated as intermediate hosts between the ''real gene pool'' from migratory ducks and domestic poultry in the whole influenza virus ecosystem. a sentinel duck sampling system may be a feasible method to represent the viruses in the natural gene pool and a baseline for virus or gene interactions between migratory and domestic ducks. 11 further investigations and surveillance are required to better understand the role of the domestic duck population in facilitating virus interactions and the generation of genetic diversity. two distinct lineages of h9n2 influenza viruses represented by a ⁄ chicken ⁄ beijing ⁄ 1 ⁄ 94 (ck ⁄ bei-like) and a ⁄ quail ⁄ hong kong ⁄ g1 ⁄ 97 (g1-like) have become established and endemic in poultry in southern china. 1 these established h9n2 lineages continue evolving to generate many different reassortant variants (or genotypes) 1,2 and are causing sporadic cases of human infection. 3, 4 studies of h9n2 viruses isolated from pigs in hong kong and shandong province have also raised the possibility of reassortment with human-like viruses from pigs. 5, 6 in addition, h9n2 viruses isolated beyond the late 1990s had preferential binding with a-2,6-neuacgal human-like receptors. 7 these observations suggest that the h9n2 influenza viruses still have pandemic potential. unlike highly pathogenic h5n1 influenza viruses that have been rarely detected in the live-poultry markets in hong kong since 2002, h9n2 viruses are still frequently isolated in our surveillance program. therefore, we try to understand the continuing evolution of h9n2 viruses through genetic characterization and phylogenetic analyses of the viruses isolated in hong kong live-poultry markets from 2005 to 2009. a total of 47 421 terrestrial poultry were sampled at different live-poultry markets in the hong kong sar between january 2005 and december 2009. of those samples, 29 691 were from chickens and the others were from minor poultry species including chukar, pheasant, guinea fowl, silky chicken, and pigeon. fecal droppings, cloacal and tracheal swabs, drinking water, and environmental samples from cages were collected into transport medium. viruses were isolated in 9-to 11-day old embryonated eggs as described previously. 8 virus isolates from positive sampling occasions were selected for sequence analysis. rna extraction, cdna synthesis, and pcr were carried out as described previously. 8 dna sequencing was performed using bigdye terminator v3ae1 cycle sequencing kit on an abi 3730 dna analyzer (applied biosystems) following manufacturer's instructions. all sequences were assembled and edited with lasergene 7ae0 (dnastar, madison, wi) software. sequence alignment and residue analysis were performed with the bioedit sequence alignment editor, version 7ae0. 9 all eight gene segments of sequenced viruses were characterized and analyzed phylogenetically together with virus sequence data available in public databases. maximum-likelihood trees were constructed using garli 0ae96. 10 estimates of the phylogenies were calculated by performing 1000 neighbor-joining bootstrap replicates using paup*4ae0. 11 systematic surveillance of live-poultry in hong kong from 2005 to 2009 resulted in 1088 h9n2 isolates from 47 421 samples (overall isolation rate, 2ae3%) ( table 1 ). there were 966 strains isolated from 42 253 chicken samples (isolation rate, 2ae3%). of these viruses, four were isolated from 1556 tracheal swabs (isolation rate, 0ae3%), while 541 isolates were isolated from 29 030 cloacal or fecal swabs (isolation rate, 1ae9%). an additional 421 isolates were collected from 6926 drinking water samples (isolation rate, 6ae1%). there were 122 strains of h9n2 viruses isolated from 5168 minor poultry samples (isolation rate, 2ae4%) ( table 1) . of these viruses, only one was isolated from 1209 tracheal swabs (isolation rate, 0ae1%), whereas 72 strains of viruses were isolated from 3610 cloacal or fecal swabs (isolation rate, 2ae0%). the isolation rate in drinking water in minor poultry was again higher when compared with other sampling methods with 49 strains isolated from 297 drinking water samples (isolation rate, 16%). taken together, these findings suggest that the h9n2 viruses mainly replicated in the intestinal tract of chickens and minor poultry species. also, the high isolation rate in drinking water samples could be a sensitive indicator for monitoring the prevalence of h9n2 viruses in the field. to better understand the evolutionary pathway of h9n2 viruses in southern china, 50 representative viruses, isolated from hong kong live-poultry markets from 2005 to 2009, were sequenced and genetically characterized. phylogenetic analysis of the h9 ha gene revealed that ck ⁄ bei-like viruses were predominant and one chicken isolate had a g1-like ha gene ( figure 1 ). this is the first time the g1like h9 ha gene has been detected in chickens from livepoultry markets in hong kong. the ck ⁄ bei-like lineage is further divided into two subgroups as previously described. 1 subgroup 1 is represented by qa ⁄ st ⁄ 243 ⁄ 00 and subgroup 2 is represented by dk ⁄ hk ⁄ y280 ⁄ 97. all h9n2 viruses in this study belonged to subgroup 2 of the ck ⁄ bei-like lineage except for the virus with the g1-like ha gene. phylogenetic analysis of the na gene also showed a similar evolutionary pattern to the ha gene with all viruses clustered within the ck ⁄ bei-like lineage. these results revealed that ck ⁄ bei-like viruses are predominant in both chickens and minor poultry. all of the pb1, pa, np, ns and m genes clustered with those of h9n2 lineage viruses previously prevailing in terrestrial poultry in southern china. phylogenetic analysis of the pb2 gene revealed three different lineages; g1-like (n = 1), ck ⁄ sh ⁄ f ⁄ 98-like (n = 15), and unknown avian (n = 34). the sh ⁄ f ⁄ 98-like lineage (or f ⁄ 98-like) was previously reported in eastern china 12 and was used previously for vaccine production in an intensive vaccination program. 13 this pb2 gene lineage was also distinguishable from the ck ⁄ bei-like lineage and its presence in the viral genome may be due to reassortment between the vaccine strain and field isolates, followed by selective establishment in terrestrial poultry. gene constellation analyses of the 50 viruses revealed six genotypes. thirty-four of the viruses analyzed belonged to two genotypes, b14 and b15, which were also the prevailing reassortants found in other provinces in southern china since 2005. 2 the remaining sixteen viruses belonged to four novel genotypes that have not been identified before in this region. characterization of h9n2 influenza viruses isolated from live poultry in hong kong markets from a 5 year surveillance program revealed that ck ⁄ bei-like viruses were predominant in southern china and were continuing to evolve. two recognized and four novel genotypes were identified in this study. one characterized virus, ck ⁄ hk ⁄ nt499 ⁄ 07, had a g1like ha gene (the first time this has been detected in hong kong poultry markets) that showed a close relationship with two human h9n2 strains isolated in 2009. g1-like viruses were usually detected and caused outbreaks in chickens of middle eastern and european countries, [14] [15] [16] and minor poultry, mainly quail, in southern china. 1 whether the g1-like virus was transmitted from china to middle eastern and european countries, as the highly pathogenic h5n1 virus did in the last five years, or vice versa, is still unknown. since the ck ⁄ hk ⁄ nt499 ⁄ 07 strain clustered with other g1-like strains isolated previously in minor poultry in southern china, the g1-like viruses in chicken may be due to interspecies transmission from minor poultry species. genetic studies demonstrated that reassortants with genotypes b14 and b15 persistently occurred in either chickens or other minor poultry species from 2005 to 2009. other genotypes that were prevalent in southern china might be being gradually replaced and four novel genotypes were identified in this study. these novel genotypes were generated through reassortment of viruses with different lineages. a newly emerged f ⁄ 98-like lineage originating from eastern china is responsible for generation of some of the novel genotypes found in this study. 12 the ck ⁄ bei-like lineage is gradually being replaced by f ⁄ 98-like lineages which are becoming dominant in northern and eastern china. 12, 17 animal experiments have also demonstrated that f ⁄ 98-like viruses are more effective in replication and transmission in chickens compared with ck ⁄ bei-like viruses. 18 since the f ⁄ 98-like lineage of the pb2 gene has been introduced into southern china, this newly emerged lineage may have a higher tendency to replace the rnp genes in the circulating ck ⁄ bei-like viruses and subsequently become the endemic virus in terrestrial poultry. in vietnam, the modelling of the pandemic h1n1 progression estimates that 460 000 (260 000-740 000) pigs might be exposed to the virus on the basis of 410 000 cases among swine owners (220 000-670 000). 1 a poor level of biosecurity, high animal densities, and a mix of species could increase the risk of influenza virus flow, persistence, and emergence on swine and poultry farms. this study was set up in the red river delta, where a third of the national pig husbandry is produced. 2 the aims are to give preliminary information of the epidemiological state of swine influenza and in order to further assess the risk of infection of swiv, through cross-species transmissions from poultry to pigs. this paper will present the preliminary results on swiv and the risk factors of pig seropositivity in vietnam. a cross-sectional study was conducted in two provinces of the red river delta in april 2009. pig farms were randomly selected from nine communes representative of at risk area of avian h5n1. in each farm, pig and poultry were sampled and collected to virological and serological analyses. interviews were conducted in all farms by trained interviewees. questionnaires included closed and open questions on ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 60-78 livestock husbandry ⁄ management and household characteristics, such as herd size and structure, health history and vaccination, pig housing, watering and feeding system, reproduction, purchasing of animals, biosecurity measures, pig contact with poultry, and environmental factors. the virological detection assay was performed on pools of nasal swab specimens from pigs. we investigated whether real-time rt-pcr assay could detect gene m on pools of nasal swab specimens before attempting virus isolation from individual nasal swab specimens. the poultry and pig sera were tested against influenza type a with an enzyme-like immunosorbant assay (elisa) competition test idvetª. this commercial kit is designed to specifically detect antibodies directed against the np protein antigen of influenza type a viruses. the positive serum samples were examined in hemagglutination inhibition (hi) to determine antibody titers and subtypes. the hi test was tailored for h1, h3, and h9 subtypes in pigs and h6 and h9 subtypes in poultry. seroneutralization tests by pseudo particles were used to test the presence of antibodies directed against h5 subtype. we analysed the data for relationships between influenza a serological status (the outcome variable) and possible risk factors using r version 2ae11ae1 (r development core team). the statistical unit was the individual. initially, the quantitative variables were encoded into categorical variables according to the quartiles or median. descriptive statistics (e.g., means or medians, proportions, standard deviations) were calculated for all herd-level and commune level predictors to assist in the subsequent modeling process. we also performed the independence test among all variables to determine if variables were dependant. then, univariate analysis of potential risk factors for the pigs being positive for swiv and estimation of odds ratios were performed using generalised linear mixed models with binary outcome and logit link function for each herd-level and commune-level variable to determine which variables were individually associated with influenza a seropositivity at a significance level of p < 0ae30. herd and commune of residence were included as a random effect to account for the correlation of observations at the herd level. the third stage of the analyses included the four herdlevel variables found to be significantly (p < 0ae30) associated with influenza a seropositivity. an automatic process using all possible associations between the selected variables was computed into a mixed logistic regression models, with random effects. when two variables were collinear, as determined before, only one variable was likely to enter the multivariable model, and therefore, the selection of which collinear variable to enter the model was guided by biological plausibility and statistical significance. all of the 146 pools of nasal swabs were rt-pcr negative. the maximal possible prevalence considering perfect diagnostic tests would be of 2ae03% at a confidence level of 95%, in an infinite population within these regions (win-episcope 2ae0). six hundred-and-nine pig sera were tested in 76 nonvaccinating farms. the herd seroprevalence of swine influenza in the commune previously infected by the avian h5n1 in the red river delta raised by 17ae1% [8ae7; 25ae6] in april 2009. but among 13 seropositive farms, only four had at least two seropositive pigs. the within-herd seroprevalence is very low, and no seropositivity was detected in the majority of farms. estimates had large confidence intervals due to small sample sizes. the individual seroprevalence raised 3ae62% [1ae98; 5ae27]. the subtyping of seropositive sera is still in process. descriptive statistical analyses on five major risk factors of swiv: farm size, breeding vs. fattening, purchasing, percentage of family income, and poultry production, were conducted. based on this analysis, three types of farming systems were identified and included in mixed models ( table 1) . percentage of family income by pig production and poultry production were not differentiating factors for this typology. whereas types 1 and 2 seem to be specialized in fattening, the type 3 produces and might sell piglets on the farm site. the exploration of the different variance components indicated that the random effect variances were mainly associated with the herd, while the commune did not seem to have any effect. therefore we included in all models only the herd as a random effect. the random effect term for herd was modelled, assuming a normal distribution with a table 1 . typology of farming system type 1: large fattening farms largest scale production, with more than 40 pigs per year specialized in fattening, and purchase more than 20 pigs per year type 2: small fattening farms small scale of production, with less than 20 pigs per year specialized in fattening, and purchase less than 20 pigs per year type 3: medium breeding-fattening farms medium scale of production, with less than 40 pigs per year breeding and fattening piglets, with rare purchase common variance [$n(0,r2herd)]. 3 the univariate analyses were conducted on 22 variables and typology variables, with herd as random effect. some coefficient or confidence intervals were inconsistent because of small effectives, especially for the percentage of self-product culture or the pig freegrazing because of the lack of positive results in the dataset. the only one significant (p value < 0ae1) parameter was the percentage of pig sales in the familial annual income. surprisingly, common risk factors of swine influenza infection, such as farm size, animal movements, and sanitary parameters got low odds ratio individually (without being significant); the typology provides the hypothesis of complex interactions effects that increase the risk of infection. as shown in table 2 , the farming system type 3 got a higher seroprevalence of 6ae47% [3ae00-11ae94] and a higher risk indicator, with or = 5ae26 (p-value = 0ae18) in comparison with type 1. this finding was not significant. in the multivariate mixed model, the percentage of familial income provided by pig production was the only one significant variable, with or = 0ae22 [0ae04-1ae25]. the focus on diseased animals in the winter-time is usually required in order to increase the likelihood to isolate the virus, although the isolation rate on healthy or clinical samples never exceed 6%. 4 the season and the lack of disease reports might explain the difficulties to detect influenza viruses. additionally, the pooling method tends to decrease the isolation rate because of a dilution effect, potential presence of pcr assay inhibitors, or uneven distribution of virus in the sample. 5 our seroprevalence results must be confirmed and the subtypes identified, especially because we found only one positive animal in a few farms that could be attributed to false positive results of the elisa test (performances are not known). these preliminary results are in favor of a virus circulation at low level in the spring, but must be completed by further surveys in the winter and before the new year (têt celebration) when pig production, trade, and movement increase at their maximum. no clear prior information on the expected prevalence of swine influenza in vietnam, tests sensitivity, and speci-ficity could be obtained from literature or reliable sources. bayesian methods will be carried out in the future in order to compute prevalence and ⁄ or to estimate the probabilities of freedom. the risk factors analysis was limited by the lack of positive results. further studies are necessary to identify the at-risk season and type of farming systems at risk of swine influenza infection. however, this investigation of risk factors leads to the hypothesis that medium size breeding-fattening farms had a higher risk than large or small size fattening farms. further investigation are needed to precise this typology. the risk of swiv infection increases with a combination of three major factors. poultry production does not seem to play any role on swine infection. the generalized linear mixed model afforded to take into account all the non investigated parameters at the herd level. although we investigated the most common risk factors of swine influenza infection covering different kind of fields, the herd random effect might explain risk variations. mixed models have become a frequently used tool in epidemiology. due to software limitations, random effects are often assumed to be normally distributed. since random effects are not observed, the accuracy of this assumption is difficult to check. 6 further studies, such as case-control or cohort studies could help to identify more precisely risk factors of swine influenza seropositivity, as these study designs are more adapted than cross-sectional studies. the concept that swine are a mixing-vessel for the reassortment of influenza viruses and for the emergence of pandemic influenza viruses has been re-enforced by the emergence of the recent pandemic. 1 the pandemic h1n1 virus of 2009 (h1n1pdm) is believed to have emerged through the reassortment of north american triple reassortant and eurasian avian-like swine influenza viruses. 2 since the immediate precursor of this pandemic virus has not yet been identified, it is not possible to be definite whether the reassortment leading to the pandemic occurred in swine, but swine influenza viruses are the nearest known ancestors of each gene segment of h1n1pdm. 1, 2 the mechanisms of pandemic emergence are not clear. it is believed that the pandemics of 1957 and 1968 arose through reassortment of the pre-existing human seasonal influenza virus with avian influenza viruses, and swine have been proposed to be a possible intermediate host where such reassortment between human and avian viruses may take place. 4 the 2009 pandemic was the first to arise for over 40 years and the first to occur after the understanding that pandemics arise from animal influenza viruses. 3 systematic studies of influenza virus ecology and evolution in swine are, therefore, important in order to understand the dynamics of pandemic emergence. furthermore, since swine are the likely host within which h1n1pdm virus originated, it was predicted that this virus would readily infect swine and may reassort with endemic swine influenza viruses. these predictions have now been confirmed with reports of h1n1pdm being detected in pigs in many countries and reassortment with endemic swine influenza virus being confirmed. 5 while h1n1pdm has been genetically and antigenically stable in humans, reassortment between h1n1pdm, which is well adapted to transmission in humans, and other avian or swine viruses may lead to the origin of novel viruses posing a threat to public health. in addition to endemic swine virus lineages, avian influenza viruses such as h9n2 and highly pathogenic avian influenza (hpai) h5n1 have also been occasionally identified in pigs in parts of asia. 6, 7 it has been shown that h1n1pdm readily reassorts with h5n1 to generate viable progeny in vitro. 8 it is therefore essential to monitor the ecology, evolution, and biological characteristics of swine influenza viruses so that their continued evolution and zoonotic and pandemic potential can be monitored. there is however, a paucity of surveillance data on swine influenza viruses worldwide. this is in part related to the negative commercial consequences that may arise from detection of influenza in a swine herd leading to a major economic loss to the producer. here we outline a surveillance system that has been in place in hong kong for the last decade, based on sampling animals arriving at an abattoir in hong kong. we demonstrate the feasibility of such surveillance in an abattoir setting and compare methods used for detection influenza viruses in swine. virus isolation was carried out by inoculation into mdck cells and by allantoic inoculation in embryonated eggs as previously described. 6 virus isolates were subtyped by haemagglutination inhibition tests using specific antisera and genetically characterized by sequencing and phylogenetic analysis of the haemagglutin gene. 2, 6 virus detection by rt-pcr a subset of 1154 recent specimens was tested in parallel by real time pcr using the biorobot universal system (qiagen) that enables fully-automated viral nucleic acid extraction and downstream reaction setup in a 96-well plate format. total viral nucleic acids were extracted in a 96-well plate format with the qiaamp virus biorobot mdx kit (qiagen) on the biorobot universal system (qiagen) according to the manufacturer's instructions. briefly, 140 ll of sample was lysed in 280 ll buffer al, supplemented with 5ae6 lg carrier rna in a s block (qiagen), which placed the samples into a 96 well plate format. after protease digestion, samples were transferred to silica based membrane in 96 well plate format for binding. following two washing steps, rna was eluted in 80 ll of elution buffer (buffer ave) into a 96 well elution microplate cl (qiagen) . for the synthesis of cdna, 10 ll of purified rna was used in a 20 ll reaction containing 4 ll of 5· buffer, 0ae75 nm of each deoxynucleotide triphosphate (dntp), 10 mm dithiothreitol, 3 lg random primer, 40 u of rnaseout recombinant ribonuclease inhibitor, and 200 u of superscript iii reverse transcriptase (all from invitrogen). reactions were performed in the geneamp 9700 thermocycler (applied biosystems) with the following parameters: 60 minutes at 50°c, 15 minutes at 72°c, and soak at 4°c. subsequent to the reactions, 20 ll of cdna was diluted 1 ⁄ 10 by adding 180 ll of ae buffer (qiagen) . real-time pcr was performed using the power sybrò green pcr master mix (applied biosystems) according to the manufacturer's instructions. briefly, 5 ll of 1⁄ 10 diluted cdna was amplified in a 25 ll reaction containing 12ae5 ll of 2· power sybr green pcr master mix, 800 nm of forward primer m52c (5¢-ctt cta acc gag gtc gaa acg-3¢) and 800 nm of reverse primer m253r (5¢-agg gca ttt tgg aca aag ⁄ t cgt cta-3¢). the primers have been designed to amplify the sequences in the conserved region of influenza a virus matrix gene, thereby detecting viruses from different species including swine influenza viruses. 8 real-time pcr was performed in the abi 7500 fast system (applied biosystems) with the following cycling conditions: 10 minutes at 95°c once, 30 seconds at 95°c, and 1 minutes at 50°c for 40 cycles, followed by melting curve analysis with 15 seconds at 95°c, 1 minutes at 50°c, and 15 seconds at 95°c. in each assay, serially diluted plasmids containing the full length m gene cloned from a ⁄ vietnam ⁄ 1204 ⁄ 2004 (h5n1) were included as standards to perform absolute quantification. a manual baseline was set from cycles 3-15 and a manual cycle threshold (ct) was set at 0ae2. samples that were positive or unequivocal results from the real-time pcr were confirmed by performing gel electrophoresis on the pcr products. positive visual identification was made in the presence of the target pcr product at 244 bp in length. a total of 16 566 tracheal and 11 100 nasal swabs were processed during the years january 2002-april 2010 and yielded 393 influenza virus isolates, an overall virus isolation rate of 1ae4%. of these, 352 were subtype h1 (classical swine, eurasian avian-like swine, and triple-reassortant), 25 were human-like h3 viruses, and 16 were eurasian avianlike swine h3n2 viruses. culture in mdck cells yielded 95% of h1 subtype viruses, 100% of the human seasonal-like h3n2 viruses, and 68ae8% of the avian-like eurasian swine h3n2 viruses. culture in embryonated eggs yielded 17ae9% of the h1 subtype viruses, 0% of the human seasonal-like h3n2 viruses, and 62ae5% of eurasian avian-like swine h3n2 viruses ( figure 1 ). tracheal and nasal swabs each gave comparable overall virus isolation rates (1ae4%). however, isolation rates for human-like h3n2 viruses were 4ae8 fold higher in nasal swabs (0ae17% versus 0ae036% respectively; p = 0ae002) ( figure 1) . a parallel evaluation of rt-pcr and culture was carried out in 1154 specimens. rt-pcr detected 10 ⁄ 12 (83%) of the culture positive specimens. rt-pcr was also positive in 8 ⁄ 1154 (0ae7%) culture negative specimens, but all these specimens had very low virus load in the rt pcr tests. virus could not be cultured from these culture negative specimens even by attempts at virus re-isolation from the frozen specimen. surveillance in an abattoir setting provides an acceptable yield of influenza viruses and is a feasible method of swine influenza surveillance. sampling in a large abattoir setting allows surveillance to be carried out anonymously with no negative consequences to the supplier. the supply-chain of pigs to the hong kong abattoir involves pigs being trucked in over long distances and may provide opportunity for virus amplification during transport. thus, virus isolation rates may be lower in more vertically integrated and homogenous production and slaughter systems where less mixing of pigs occurs. our results indicate that mdck cell culture is essential for optimizing virus isolation during swine influenza surveillance. allantoic inoculation of embryonated eggs by itself is sub-optimal for isolation of swine influenza viruses. it is however possible that inoculation of embryonated eggs by the amniotic route may lead to better isolation rates than allantoic inoculation. rt-pcr detection is an alternative method for virus detection. but the additional specimens detected by rt-pcr did not yield culturable virus, even following attempts at re-isolation and sequential passage. the rt-pcr positive ⁄ virus isolation negative specimens had very low virus load, and this may be the explanation for the inability to isolate such viruses. in addition, rt-pcr did not detect all viruses isolated by culture. tracheal and nasal swabs gave comparable isolation rates with the exception of human-like h3n2 viruses which were more frequently isolated from nasal swabs. this may suggest that, in contrast to endemic swine influenza virus lineages, these human-like h3n2 viruses are less adapted to replication in the lower respiratory tract. in summary, collection of nasal or tracheal swabs in an abattoir setting together with virus isolation in mdck cells provides a feasible approach to surveillance of swine influenza viruses. kong, 2003 kong, -2010 introduction wild waterfowl are the natural reservoir of influenza a viruses (aiv), and they play an important role in the genesis of pandemic influenza. it is suggested that the 1918 pandemic virus was purely derived from avian virus, which adapted to humans and caused efficient human-to-human transmission, 1 while the pandemics of 1957 and 1968 had acquired the viral haemagglutinin, pb1 polymerase, and in 1957, the neuraminidase gene segments from the avian gene pool. 2 the major regional outbreaks of highly pathogenic avian influenza (hpai) h5n1 in asia, europe, and africa highlight the potential role played by migratory waterfowl in disseminating highly pathogenic influenza viruses. 3 therefore defining the influenza virus gene-pool in wild birds is of vital importance. surveillance was carried out 2-3 times weekly from 2003 to 2010 during the winter months of october to april in the hong kong mai po nature reserve and lok ma chau, hong kong. the hong kong mai po nature reserve and lok ma chau are along the east asia-australian flyway where a peak of more than 30 000 ducks and grebes congregate every winter. 4 fecal droppings were collected and transported in vials containing 1ae0 ml of vtm, which was prepared from m199 (9ae5 g ⁄ l), penicillin g (2 · 10 6 u ⁄ l), polymyxin b (10 · 10 6 u ⁄ l), gentamicin (250 mg ⁄ l), nystatin (0ae5 · 10 6 u ⁄ l), ofloxacin hcl (100 mg ⁄ l), and sulfamethoxazole (1 g ⁄ l). an aliquot of 100 ll from each swab sample was inoculated into the allantoic cavity of a 9-to 11-day-old chicken embryonated egg, and incubated for 3 days at 37°c. positive ha isolates were subtyped using standard antisera 5, 6 and rt-pcr was performed with the used of one-step rt-pcr assay (invitrogen) described earlier, 7 followed by sequencing on abi prism 3730xl dna analyzer. the determination of species of origin was performed by dna barcoding of the mitochondrial cyto-chrome oxidase i gene from dna extracted from the fecal droppings. 8 during the 7-year surveillance period, a total of 193 influenza viruses were isolated from 39 354 samples collected, an overall isolation rate of 0ae49%. a total of 192 isolates were obtained from 39 152 specimens collected during the winter period coinciding with the southern migration of waterfowl along the east asian flyway and one isolate obtained from 202 samples collected in spring during the period when northern migration of waterfowl took place along the east asian-australasian flyway. the isolation in hong kong was slightly lower than a similar study conducted in south korea in which the isolation rate of migratory birds was 0ae8% in 2003-2008. 9 this suggested a slightly lower prevalence of influenza virus present in hong kong as the birds migrated southwards. the viruses isolated in hong kong, representing hemagglutinin (ha) subtypes of h1-h12 and neuramidinase (na) subtypes of n1-n9, were all from wild waterfowl ( table 1) . out of the twelve ha subtypes isolated, h10 and h6 were the two subtypes that were isolated frequently every year for h10 and in six out of seven years for h6, respectively. h10 and h6 viruses accounted for 16ae7% and 22ae4% of all virus isolated, respectively. on the other hand, h3, h9, and h12 were the least prevalence (0ae5%) and were only isolated once in 7 years. of the na subtypes, n1 and n2 were isolated most often (24ae4% and 19ae2% of all isolates, respectively) and n5 was the least (0ae5%). november was the month that had the highest prevalence of influenza virus (0ae83% of samples being positive) compared to only 0ae3% in march. the subtype's variation was the most diverse in december during our 7 years of surveillance. this suggested that more of these wild migratory birds may be carrying influenza virus when they arrive in hong kong. however the continued isolation of viruses suggests continued circulation of these viruses in the vicinity of mai po. the study of dna barcoding for the mitochondrial cytochrome oxidase i gene retrieved from fecal droppings revealed that the isolates originated mainly (89ae2%) from birds of the order anseriform, family anatidae including eurasian wigeon, northern shoveler, northern pintail, common teal, and garganey. non-anseriformes which were found to have shed aiv viruses were cormorant, grey heron, and stint. none of the water samples collected from the ponds where these birds congregate were found to be positive for the virus. phylogenetic analyses of the ha gene of the lpai h5 viruses isolated in this study clustered with that of the other lpai h5 viruses isolated from hokkaido, mongolia, and siberia and were not closely related to the hpai h5n1. satellite tracking of 47 eurasian wigeons and northern pintails in dec 2008 and 2009 revealed their flyway from hong kong to as far north as eastern russia, eastern mongolia, and northern china. no hpai h5n1 viruses were isolated in this study from apparently healthy birds. however, as part of the surveillance of dead wild birds carried out by the department of agricultural, fisheries and conservation of the government of hong kong during this same period, over 50 dead wild birds were tested positive for hpai h5n1 and has been reported elsewhere. 10 our influenza surveillance in hong kong has revealed a diversity of influenza virus subtypes the migratory waterfowl infected within the region. the result of the phylogenetic analysis correlated with the findings from satellite tracking that viruses isolated in hong kong were closely related to those isolated in areas along the migratory route. no healthy bird was isolated with hpai h5n,1 although dead wild birds have been regularly found to have hpai h5n1 virus, suggesting that infected birds might not live for a long period. introduction a novel swine-origin h1n1 influenza virus emerged in mexico in april 2009 and rapidly spread worldwide, causing the first influenza pandemic of the 21st century. 1 most confirmed human cases of h1n1 ⁄ 2009 influenza have been uncomplicated and mild, but the increasing number of cases and affected persons worldwide warrant optimal prevention and treatment measures. today, almost all of the pandemic h1n1 ⁄ 2009 viruses tested are resistant to m2blockers. 2 therefore, only the neuraminidase (na) inhibitors are currently recommended for treatment of this pandemic influenza. for the control of influenza infection, the clinical use of oseltamivir has increased substantially during the 2009 pandemic. to date, the majority of tested clinical isolates have remained susceptible to na inhibitors, oseltamivir and zanamivir, 2 but oseltamivir-resistant variants with h275y na mutation (n1 numbering) have been isolated from individuals taking prophylaxis, from immunocompromised patients, and from a few community clusters. 3, 4 in view of the high prevalence of oseltamivirresistant seasonal h1n1 influenza viruses in 2007-2008, the isolation of resistant h1n1 ⁄ 2009 viruses without known oseltamivir exposure raised great concern about the transmissibility and fitness of these resistant viruses. here we studied the transmissibility of a closely matched pair of pandemic h1n1 ⁄ 2009 clinical isolates, one oseltamivir-sensitive and one resistant, in both direct contact and respiratory droplets routes among ferrets. viral fitness was evaluated by co-infecting a ferret with both the oseltamivir-sensitive and -resistant viruses. the viruses were also characterized by full genome sequencing, susceptibility to na inhibitors, and growth in mdck and mdck-siat1 cells. oseltamivir-resistant influenza a ⁄ denmark ⁄ 528 ⁄ 09 (h1n1) virus (a ⁄ dm ⁄ 528 ⁄ 09) was isolated from the throat swab of a patient who had influenza-like symptoms and received post-exposure oseltamivir prophylaxis (75 mg once daily). 5 wild-type influenza a ⁄ denmark ⁄ 524 ⁄ 09 (h1n1) virus (a ⁄ dm ⁄ 524 ⁄ 09) was isolated from a patient in the same cluster of infection as the a ⁄ dm ⁄ 528 ⁄ 09 virus. to assess growth kinetics of viruses, confluent mdck or mdck siat1 cell monolayers were infected with viruses at a multiplicity of infection (moi) of approximately 2ae0 pfu ⁄ cell (single-step) or 0ae001 pfu ⁄ cell (multi-step). supernatants were collected every 2 h or 12 h p.i. for 6 time points. a modified fluorometric assay using the fluorogenic substrate 2¢-(4-methylumbelliferyl)a-d-n-acetylneuraminic acid (munana) was used to determine viral na activity. 6 the drug concentration required to inhibit 50% of the na enzymatic activity (ic 50 ) was determined by plotting the percent inhibition of na activity as a function of compound concentration calculated in the graphpad prism 4 (la jolla, ca) software from the inhibitor-response curve. na enzyme kinetics were determined by measuring na activity every 60 seconds for 60 minutes under the same conditions as above, when all viruses were standardized to an equivalent dose of 10 6ae0 pfu ⁄ ml. the k m and v max were calculated by fitting the data to the appropriate michaelis-menten equations using nonlinear regression in the graphpad prism 4 software. young adult ferrets (4-5 months of age) were obtained from the ferret breeding program at st. jude children's research hospital. all ferrets were seronegative for influenza a h1n1 and h3n2 viruses and for influenza b viruses. for transmission studies, the donor ferrets were lightly anesthetized with isoflurane and inoculated intranasally with 10 6 tcid 50 virus in 1ae0 ml sterile pbs . after the donor ferrets were confirmed to shed virus on day 2 p.i., each donor was then housed in the same cage with two naïve direct-contact ferrets. two additional recipient ferrets were placed in an adjacent cage isolated from the donor's cage by a two layers of wire mesh (approximately 5 cm apart) that prevented physical contact but allowed the passage of respiratory droplets. ferret weight and temperature were recorded daily for 21 days. nasal washes were collected from donors and recipients on day 1, 2, 4, 6, 8, 10, 12, and 14 p.i. by flushing both nostrils with 1ae0 ml pbs, and tcid 50 titers were determined in mdck cells. serum samples were collected 3 weeks after virus inoculation, and were tested for seroconvention by hi assay. full genome sequencing revealed that the pair of h1n1 ⁄ 2009 viruses differed only at na amino acid position 275, where the pandemic a ⁄ dm ⁄ 528 ⁄ 09 virus had an h275y amino acid mutation caused by a single t-to-c nucleotide substitution at codon 275. the wild-type a ⁄ dm ⁄ 524 ⁄ 09 was susceptible to oseltamivir carboxylate (mean ic 50 : 5ae0 nm), but the a ⁄ dm ⁄ 528 ⁄ 09 carrying the h275y na mutation had ic 50 values approximately 200-300 times of the wild-type viruses (mean ic 50 : 972 nm). the ic 50 of zanamivir was comparable for both viruses and were uniformly low (mean ic 50 £ 1ae3 nm). the h275y na mutation confers resistance to oseltamivir carboxylate but did not alter susceptibility to zanamivir. to understand the impact of the h275y mutation on the na enzymatic properties, na enzyme kinetics was determined. the na of the oseltamivir-resistant virus had a slightly higher k m (mean = 55 lm) and lower v max (mean = 101 u ⁄ sec) than na of the sensitive virus (k m , mean = 80 lm; vmax, mean = 86 u ⁄ sec). the results suggested that the h275y na mutation reduced na affinity for substrate and na catalytic activity, although the function of na was not severely impaired. to further evaluate the impact of the h275y na mutation on virus growth in vitro, single-and multi-cycle growth studies of both viruses were performed in mdck and mdck-siat1 cells. in the both single-and multiple-cycle growth curves, the two viruses reached comparable levels eventually, but the initial growth of the resistant virus was significantly delayed by at least 1-2 logs in comparison to that of wild-type virus (p < 0ae05). the donor ferrets inoculated with wild-type a ⁄ dm ⁄ 524 ⁄ 09 or oseltamivir-resistant virus shed virus productively until day 6 or day 8 p.i., with a peak virus titer comparable to that of a ⁄ dm ⁄ 524 ⁄ 09 virus (table 1 ). in a ⁄ dm ⁄ 524 ⁄ 09 virus group, two of 2 direct-contact ferrets the weight loss in ferrets is the maximum percentage loss compared with the initial weight. virus shedding is indicated as number of virus-shedding animals ⁄ total number; mean peak virus titer (log 10 tcid 50 ⁄ ml) in nasal wash samples is indicated in parentheses. serum hemagglutination inhibition (hi) titer to homologous virus in ferret serum was determined on day 21 p.i. duan et al. and 1 of 2 respiratory droplet-contact ferrets were infected through virus transmission, as indicated by the virus titers and inflammatory cell counts in their nasal washes and also by sero-conversion. under identical conditions, in a ⁄ dm ⁄ 528 ⁄ 09 group, only 2 of 2 direct-contact ferrets were infected through virus transmission, but neither respiratory droplet-contact ferrets was infected, as confirmed by the absence of sero-conversion (table 1) . virus shedding in the direct-contact ferrets was lower and peaked after a longer interval in this group than in the oseltamivir-sensitive a ⁄ dm ⁄ 524 ⁄ 09 group (table 1) , but the resistant viruses appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. these results showed that an oseltamivir-resistant h275y mutant of pandemic h1n1 virus, a ⁄ dm ⁄ 528 ⁄ 09 virus could be only transmitted efficiently by direct contact. to compare the relative fitness, growth capability, and transmissibility of the sensitive and resistant h1n1 ⁄ 2009 viruses within host, a donor ferret was co-inoculated with a 1:1 ratio of the sensitive and resistant viruses, and another two naive ferrets were housed with the donor to test direct contact. during co-infection, the pattern of virus shedding and the clinical signs were similar to those in ferrets inoculated with either a ⁄ dm ⁄ 524 ⁄ 09 or a ⁄ dm ⁄ 528 ⁄ 09 virus (table 1 ). in the inoculated donor ferret, the virus population in the nasal washes remained mixed but wild-type viruses outgrew the resistant virus progressively ( figure 1 ). two of 2 direct-contact ferrets were infected through virus transmission, but only wild-type virus was detected in both direct-contact ferrets ( figure 1 ). in summary, oseltamivir-sensitive a ⁄ dm ⁄ 524 ⁄ 09 virus possessed better growth capability in the upper respiratory tract than did resistant a ⁄ dm ⁄ 528 ⁄ 09 virus, and thus had an advantage in directcontact transmission. our study determined the comparative transmissibility of two naturally circulating oseltamivir-sensitive and -resistant pandemic h1n1 ⁄ 2009 viruses; we demonstrated inefficient respiratory-droplet transmission of an oseltamivir-resistant h275y mutant of pandemic h1n1 virus among ferrets, although it retained efficient direct-contact transmission. we suggest that the lower fitness of resistant virus within the host along with its reduced na function and delayed growth in vitro may in part explain its less efficient transmission. notably, the h275y mutant of h1n1 ⁄ 2009 used in this study was the first oseltamivir-resistant h1n1 ⁄ 2009 isolate from a patient on oseltamivir prophylaxis to be characterized for transmissibility. our observation in the animal model is consistent with the epidemiological data collected from humans, which showed no evidence of predominant or continued circulation of oseltamivir-resistant viruses. 4 as this study was undertaken, additional h275y mutants of h1n1 ⁄ 2009 viruses have emerged in the absence of oseltamivir use. 7, 8 the emergence of these viruses should raise concerns as to whether resistant h1n1 ⁄ 2009 viruses will acquire greater fitness and spread worldwide as the naturally resistant h1n1 viruses did during the 2007-2008 season. two independent studies have evaluated the pathogenecity and transmission of other oseltamivir-resistant pandemic h1n1 ⁄ 2009 clinical isolates in the animal models. 9, 10 one of the studies, 9 which also used an oseltamivir-resistant virus isolated from a patient under oseltamivir prophylaxis, observed similar results as ours: although the respiratory-droplet route of transmission was not investigated, it was shown that the resistant isolate was transmitted though direct-contact route and was as virulent as wild-type virus in ferrets. 9 in another study, 10 two oseltamivir-resistant isolates were transmitted through the respiratory-droplet route in ferrets, and the dynamics of transmission were different between the two isolates. apparently, these two oseltamivir-resistant isolates were still unequal in their transmissibility and were disparate from the resistant isolate in our study. the isolation history of the two resistant isolates was unclear in this study, and this would be an important factor to understand the fitness of drug-resistant viruses. further studies with more clinical isolates of diverse isolation background are warranted to identify how these novel h275y mutants of pandemic h1n1 ⁄ 2009 virus have changed to retain their full transmissibility. taken together, all these related studies underline the necessity of continuous monitoring of drug resistance and characterization of potential evolving viral proteins. this study was supported by contract hhsn266200700005c from the national institute of allergy and infectious diseases, national institutes of pigs have been considered as hypothetical ''mixing vessels'' facilitating the genesis of pandemic influenza viruses. 1, 2 the pandemic h1n1 ⁄ 2009 virus (ph1n1 ⁄ 09) contained a very unique genetic combination and was thought to be of swine origin, as each of its eight gene segments had been found to be circulating in pig populations for more than a decade. 3 however, such a gene constellation had not been found previously in pig herds all around the world. only after its initial emergence in humans has this virus been repeatedly detected in pigs, and found to further reassort with other swine influenza virus. [3] [4] [5] a primary question remaining to be answered is whether the ph1n1 ⁄ 09-like and their genetically related viruses could become established in pig populations, thereby posing novel threats to public health. despite the fact that ph1n1 ⁄ 09 first appeared in mexico and the united states, and six of its eight gene segments were derived from the established north american triple reassortant swine influenza virus (trig), its neuraminidase (na) and matrix protein (m) genes belonged to the eurasian avian-like swine lineage (ea), which had never been detected in north america previously. 3, 6 likewise, the trig-like viruses were never reported in europe. 6 in contrast, both lineages of virus were frequently detected in asia, and reassortants between them have also been documented in recent years. 3, 5 this has given rise to a complicated ecological situation, i.e. the simultaneous prevalence of multiple genotypes of h1n1 and h1n2 viruses in pigs. 3, 5 among them, two representative reassortants showed the most similar genotypic characterization to the ph1n1 ⁄ 09 virus, the sw ⁄ hk ⁄ 915 ⁄ 2004 (h1n2) and sw ⁄ hk ⁄ 201 ⁄ 2010 (h1n1), which respectively harbor seven and six gene segments closely related to the pandemic strains. 3 , 5 to understand their in vivo characteristics and zoonotic potential, these two viruses, together with a human prototype strain and a swine ph1n1 ⁄ 09-like isolate, were chosen for a study of their pathogenicity and transmissibility in domestic pigs, ferrets, and mice. the prototype ph1n1 ⁄ 09 virus, a ⁄ california ⁄ 04 ⁄ 2009 (ca04), was provided by the world health organization collaborating centers for reference and research on influenza (atlanta, ga, usa). three ph1n1 ⁄ 09-related swine influenza viruses were isolated through our surveillance program in south china as previously described. 3, 5 the a ⁄ swine ⁄ guangdong ⁄ 106 ⁄ 2009 (h1n1, gd106) virus was a ph1n1 ⁄ 09-like swine isolate. a ⁄ swine ⁄ hong kong ⁄ 915 ⁄ 2004 (h1n2, hk915), the closest pandemic ancestor known to date, possesses an m gene derived from the ea lineage, with the other gene segments from trig viruses. 3 a ⁄ swine ⁄ hong kong ⁄ 201 ⁄ 2010 (h1n1, hk201), a recent pandemic reassortant progeny, had a ph1n1 ⁄ 09like na gene (also belonging to the ea lineage), an ea-like hemagglutinin (ha) gene, and six trig-like internal genes. 5 all viruses were propagated in madin-darby canine kidney (mdck) cells for three passages, and their titers were determined by plaque assays. all experiments with live viruses were conducted in biosafety level 3 (bsl-3) containment laboratories. pigs (4-6 week old, n = 5-6) and ferrets (5 month old, male, n = 3) were intranasally infected with 10 6 pfu of each virus, and mice (8) (9) week old, female balb ⁄ c, n = 10) with a dose of 10 4 pfu. naïve uninfected pigs (n = 2) were co-housed in the same cage with the inoculated ones from each group. body weights and clinical signs were recorded daily. virus replication was determined by titration of the virus in nasal and rectal swabs (pigs), nasal washes (ferrets), as well as from lungs and other organs (pigs and mice). seroconversion was tested by hemagglutination inhibition (hi) assays. histopathological and immunohistochemical analysis were performed as previously described. 7 statistical analysis was performed by mean analysis with pasw statistics 18 (spss inc., chicago, il, usa). the probability of a significant difference was computed using anova (analysis of variance). results were considered significant at p < 0ae05. the pathogenicity of the four viruses tested differed significantly in inoculated mice. animals infected with 10 4 pfu of hk915 experienced the most severe body weight loss (25ae1 ± 4ae7%) but started to recover after 8 days post-infection (dpi). hk201 caused similar peak body weight loss (16ae9 ± 4ae6% on 8 dpi) in mice as did ca04 (17ae3 ± 2ae4%, on 7 dpi), but the onset of clinical signs and weight loss (on 4 dpi) was 1 day later than those caused by the other three viruses. the gd106-infected group suffered the least body weight loss (6ae9 ± 1ae9%, 5 dpi) and was the earliest to recover. although all four viruses were detected in the lungs with comparable virus titers on 3 dpi (p > 0ae05), mice inoculated with gd106 consistently showed the lowest lung index (lung weight ⁄ body weight, %) on 3, 6, and 14 dpi (p < 0ae01), suggesting the slightest injury and consolidation of the lungs. in concordance with the body weight change, the lung index from the hk915 group was higher than that from any other groups on 6 and 14 dpi, indicating the marked virulence of hk915 in mice. notably, virus titer of hk201 in the nasal turbinate was lower than the other groups both on 3 and 6 dpi (p < 0ae01), but virus replication in the lower respiratory tract was either higher (in the trachea) or similar (in the lungs). observations of the body weight changes caused by infection of ph1n1 ⁄ 09 or its genetically related swine viruses in ferrets have come to a similar conclusion as that for the mouse experiment. after nasal inoculation with 10 6 pfu of each virus, all groups of ferrets experienced transient body weight loss for 2-3 days, except for those infected with gd106, which showed no significant weight loss (p > 0ae05). although ferrets from the ca04-infected group reached their peak weight loss (6ae2 ± 0ae8%, 2 dpi) one day earlier than those from the hk201 and hk915 groups, they began to regain body weight quickly thereafter. hk201-infected ferrets also recovered rapidly and their body weights reached the same level as those of the gd106-infected group at 6 dpi. comparatively, ferrets inoculated with hk915 had the most retarded body weight recovery, which did not get back to the baseline level until 11 dpi. hk201 was only detectable in the nasal wash on 2 dpi, whereas the duration of virus shedding for gd106, hk915, and ca04 was 4-6 days. by combining the data obtained from the virus titration in the mouse turbinate and ferret nasal washes, a possible conclusion can be made that hk201 may have lower transmissibility than the other three viruses. after inoculation or exposure by direct contact (physical contact) with the ph1n1 ⁄ 09 virus and its close relatives, most pigs experience no or mild symptoms, such as slight loss of appetite and inactivity. body weight loss was only recorded in pigs inoculated with hk915 during the second week post-inoculation, but not in their contact pigs or in the other groups. diarrhea was observed intermittently in each of the inoculated or contact groups throughout the experiment, and viruses could be recovered in the rectal swabs, saliva, drinking water, and environmental swabs (inner cage walls accessible to the pigs) at various time points. however, virus titers in the positive rectal swabs were just slightly above the detection limit, while those from the environment sometimes could be higher. whether these viruses can replicate in the digestive tract or were just carried-over by contaminated foods and water requires further investigation. although virus could be detected in the nasal swabs of all infected or contact animals, the lowest peak titer was from pigs inoculated or in contact with hk201 (0ae5-1ae5 log tcid50 ⁄ ml lower than the other groups), suggesting unfavorable replication in the nasal cavity for this virus. postmortem examination on 4 and 7 dpi revealed that pigs infected with hk915 had the most extensive gross lesions in the lungs, and histochemical staining of viral nucleoprotein (np) in lung tissues on 4 dpi also suggested the best replication for hk915 in the lower respiratory tract. on 11 days post-contact (dpc), all pigs exposed to the inoculated animals developed sero-conversions (hi = 80-160) except for one from the gd106 contact group. however, on 17 dpc, its hi titer reached 40, indicating slower seroconversion. this study revealed that both the 2009 pandemic h1n1 and its genetically related swine viruses could readily infect mice, ferrets, and pigs causing mild to moderate clinical symptoms. they could also transmit efficiently between pigs. when compared with the pandemic stains and its reassortant progeny (hk201), the hk915 (h1n2) virus containing the ea-like m gene in the genetic context of the trig virus showed consistently higher virulence in all three mammalian models tested, but it is still unknown what might happen if such a virus further reassorts to obtain the pandemic-like or ea-like na gene. however, our findings suggest that pigs could likely maintain the prevalence of different genotypes of pandemic-related influenza viruses, and highlight the zoonotic potential of multiple strains of swine influenza virus. pandemic influenza viruses emerge from the animal reservoirs. 1 among the three pandemics that occurred in the last century, we learned that the 1957 h2n2 and the 1968 h3n2 pandemic viruses emerged by reassortment between circulating human virus and avian-origin influenza virus(es). 1 studies on the emergence of the catastrophic 1918 spanish h1n1 virus suggest that the virus may have obtained all of its eight gene segments from the avian reservoir, 2,3 or alternatively is a reassortant between mammalian and a previously circulating human influenza virus. 4 over 40 years since the last pandemic, the first pandemic in the 21st century arose in 2009 and was caused by a swine-origin influenza virus containing a unique gene combination, with gene segments derived from the circulating north america ''triple reassortant'' (pb2, pb1, pa, ha, np, and ns) and the ''eurasian'' (na and m) swine influenza viruses. 5, 6 analysis of the pandemic h1n1 viruses failed to identify known molecular markers predictive of adaptation to humans. 6 the ''triple reassortant'' swine influenza viruses emerged in late 1990s in north america is a reassortant between classical swine (descendent of the 1918 virus after adaptation in swine population), avian, and human influenza viruses. 7 the eurasian influenza virus was originally an avian influenza virus that was introduced into the european swine population in the late 1970s. 8, 9 while incidents of zoonotic infection with triple reassortant or eurasian influenza in humans have been reported, 10, 11 sustained human-to-human transmission has never been established. these results suggest that the unique gene combination seen with the pandemic h1n1 viruses may confer its transmissibility among humans. we have carried out systematic prospective surveillance of swine influenza in southern china over that last 12 years through samples routinely collected at an abattoir in hong kong. during this time, the surveillance results suggest co-circulation of classical swine h1n1, triple reassortant h1n2, eurasian swine h1n1, and a range of reassortants between these three virus lineages. 4, 12 ferrets have been reported as a suitable model for the study of influenza transmission as they are naturally susceptible to influenza infection, exhibit similar clinical signs (including sneezing), and possess receptor distribution in the airway similar to that of humans. [13] [14] [15] to identify molecular determinants that enable sustained human-to-human transmission, we compared the pandemic virus with genetically related swine influenza viruses obtained from this surveillance program for their ability to transmit from ferret to ferret by direct contact or aerosol transmission. viruses human h3n2 influenza virus [a ⁄ wuhan ⁄ 359 ⁄ 95 (wuhan95)] and pandemic h1n1 influenza viruses [a ⁄ california ⁄ 4 ⁄ 09 (ca04)] were included for the study. swine influenza viruses that are genetically related with the pandemic h1n1 virus were selected from our surveillance system, including classical swine-like influenza virus a ⁄ sw ⁄ hk ⁄ 4167 ⁄ 99 (h1n1) (swhk4167), triple reassortant-like a ⁄ sw ⁄ arkansas ⁄ 2976 ⁄ 02 (h1n2) (swar2976), and one reassortant between triple reassortant and eurasia swine influenza viruses [a ⁄ sw ⁄ hk ⁄ 915 ⁄ 04 (h1n2) (swhk915)]. swhk915 contains seven gene segments (pb2,pb1,pa,ha,np,m,ns) closely related to the pandemic h1n1 viruses. transmissibility was tested in 4-to 6-month-old male ferrets obtained from triple f farm (sayre, pa); all ferrets were tested to have hi titer £40 against human seasonal influenza h1n1 (a ⁄ tennessee ⁄ 560 ⁄ 2009), h3n2 (a ⁄ brisbane ⁄ 10 ⁄ 2007), and influenza b (b ⁄ florida ⁄ 4 ⁄ 2006) prior the experiments. in each virus group, three ferrets were inoculated with 10 5 tcid 50 of the virus. at 1 day postinoculation (dpi), we introduced one naïve direct contact ferret to share the cage with inoculated ferret, and one naïve aerosol contact ferret into the adjacent compartment of the cage separated by a double-layered perforated divider. nasal washes were collected every other day and tested for influenza virus antigen and to determine viral titers (tcid 50 ). weight changes, temperature, and clinical signs were monitored daily. transmission is defined by detection of virus from nasal washes and ⁄ or by seroconversion (>4fold rise in the post-sera collected after 16-18 days post contact). experiments were performed in the p2+ laboratory at st. jude children's research hospital. all studies were conducted under applicable laws and guidelines and after approval from the st. jude children's research hospital animal care and use committee. at 10 5 tcid 50 inoculation dose, all viruses replicated efficiently in the ferret upper respiratory tract with peak titers detected from inoculated ferrets at 2 dpi. lower peak titers were detected from swhk4167 and swhk915 inoculated ferrets, however, the differences were not statistically significant (table 1) . tissues collected from inoculated ferrets at 3 dpi showed that pandemic h1n1 and swine influenza viruses replicated both in the upper and lower respiratory tract of the ferrets, while the replication of human seasonal influenza wuhan95 was restricted in the upper respiratory tract. direct contact transmission from inoculated donor ferrets to their cage-mates was observed for all viruses studied, albeit at different efficiency. human seasonal influenza (wuhan95) and pandemic h1n1 viruses (ca04) transmitted most efficiently via direct contact route as the virus can be detected on 2 dpi from direct contact ferrets, and the peak titers were detected on 4 dpi from direct contacts. moderate direct contact transmission efficiency was detected from swar2976 and swhk915 viruses as the virus can be detected from direct contact ferrets at 4 dpi, with peak titers detected at 4 dpi or 6 dpi. classical swine-like swhk4167 showed least efficient contact transmission as virus could be detected from all direct contacts only at 6 dpi, and the peak titer detected on 8 dpi. aerosol transmission was detected in groups of human seasonal influenza virus wuhan95 (2 ⁄ 3), pandemic h1n1 influenza virus ca04 (3 ⁄ 3), as well as swine precursor virus swhk915 (1 ⁄ 3). transmission of wuhan95 and ca04 to aerosol contacts was detected at 4 dpi or 6 dpi, while transmission of swhk915 was detected later at 8 dpi, suggesting that the swhk915 virus possessed aerosol transmission potential, but may require further adaptation to acquire efficient aerosol transmissibility. in addition to viral detection from nasal washes, we also detected viruses from the rectal swabs of ferrets inoculated or infected with pandemic h1n1 viruses (ca04) or classical swine-like virus (swhk4167), which share the common origin for the ha, np, and ns gene segments. while many of the swine influenza viruses studied were able to transmit via the direct contact route, swhk915, which shares a common genetic derivation for seven genes with h1n1pdm, possessed capacity for aerosol transmission, albeit of moderate efficiency. swhk915 differed from swine triple reassortant viruses in the origins of its m gene. it is possible that the m gene derived from eurasian avianlike swine viruses also contributes to the transmissibility of h1n1pdm influenza viruses. outbreaks of highly pathogenic avian influenza (hpai) of the h5n1 subtype are of extreme concern to global health organisations as human infection can result in severe acute respiratory distress syndrome, multi-organ failure, and coma. hpai viruses of either h5 or h7 subtypes contain a characteristic multi-basic cleavage site in the hemagglutinin glycoprotein 1 as well as other virulence factors 2 that expand the viral tropism beyond the respiratory tract of poultry. there is also emerging evidence of viral rna or antigen in multiple organs and the cns of humans infected with h5n1 that is consistent with systemic infection 3, 4 and raises the question of the role of the cleavage site in dissemination of the virus in this species. the majority of human cases with h5n1 have involved contact with sick or contaminated poultry and exposure to respiratory secretions of birds that can be inhaled and ingested. particular risk factors for h5n1 infection include bathing with sick birds, improper hand washing after handling sick birds, or slaughtering poultry. 5 viral inoculum may also be consumed directly during a variety of religious and cultural practices, such as drinking contaminated duck blood and kissing of merit release birds. h5n1 infection is lethal in 60% of human cases, and the pathogenetic mechanisms leading to this level of mortality are unclear. to date 505 cases have been reported to the who, although many more people have potentially been exposed to h5n1 through contact with infected bird populations. 6 some studies have suggested that genetic factors may predispose an individual to severe h5n1 disease, 7 but little is known about the influence of route of virus exposure on morbidity and mortality. in ferrets, an animal model frequently used to study influenza because of its similar disease profile to humans, 8 swayne et al. 9 observed that exposure to a virulent h5n1 strain a ⁄ vietnam ⁄ 1203 ⁄ 2004 by intra-gastric gavage did not lead to disease and did not generate an antibody response, whereas ferrets that experienced a more natural exposure by being fed contaminated meat developed severe signs of infection. in this study we further assessed the disease profile of h5n1 following a natural oral exposure in the ferret model. to achieve this inoculation condition, conscious ferrets voluntarily consumed a liquid inoculum of h5n1 hpai strain a ⁄ vietnam ⁄ 1203 ⁄ 2004. as a comparison anesthetised ferrets were exposed by intranasal administration of inoculum and the ensuing disease profiles of the different routes of infection were compared. eight ferrets per group were inoculated with 10 6 egg infectious dose 50 of a ⁄ vietnam ⁄ 1203 ⁄ 2004 in a volume of 500 ll that was given to the nares of anaesthetized ferrets to establish a total respiratory tract (trt) infection or voluntarily consumed by conscious ferrets to establish an oral infection. ferrets were culled at a predetermined humane endpoint that was defined as either a > 10% weight loss and ⁄ or evidence of neurological signs, discussed in 10 ; animals that did not reach the humane endpoint were euthanased on day 14 after challenge. nasal washes and oral swabs collected during the course of infection and organ homogenates were assessed for the presence of replicating virus by growth in embryonated-chicken eggs; viral loads were determined by titration on vero cells and expressed as tcid 50 . tissue samples were fixed with formalin and embedded in paraffin for sectioning. viral lesions were identified by hematoxylin and eosin staining of the sections and the presence of viral antigen in the sections was determined by staining with antibody to influenza a nucleoprotein. pre-and post-exposure antibody responses were assessed by hemagglutination-inhibition assays using irradiated a ⁄ vietnam ⁄ 1203 ⁄ 2004 virus. the majority (75%) of ferrets infected by the trt route rapidly became inactive, developed severe disease, and were euthanased at the humane endpoint following infection ( figure 1 ). ferrets infected orally had an improved chance of survival, as only 25% of animals developed severe disease (figure 1 ), and the surviving ferrets were more active than ferrets infected by the trt throughout the stage of acute infection (data not shown). the improved survival rate and wellbeing of ferrets infected orally was not a result of poor infection rates by this route, as 5 of 6 surviving ferrets developed h5 specific antibodies by day 14 post-infection, and they did not have pre-existing antibodies to h5n1 (data not shown). the two ferrets that developed severe disease after oral infection had similar disease profiles to ferrets infected by the trt route; they both progressed to a > 10% weight loss and exhibited neurological signs (data not shown). viral loads in organs of these two ferrets confirmed dissemination to extra-pulmonary sites (table 1) : replicating virus was detected at high titres in the spleen, pancreas, liver, and brain. similar findings were recorded in ferrets with trt infections in this study (not shown) and elsewhere. 10 viral load in nasal washes and oral swabs taken at days 3, 5, and 7 post-infection by the oral route did not correlate with the development of severe disease, and virus was isolated only sporadically and at low titre from the nasopharynx of these animals (data not shown). interestingly, the two ferrets with severe disease after being infected orally had no detectable viral antigen or lesions in the olfactory epithelium and bulb (table 1) , whereas 5 of 6 ferrets culled after infection by the trt route had lesions and viral antigen in both the olfactory epithelium and bulb (data not shown). trt oral 50 100 figure 1 . percentage of ferrets that survived infection after oral or trt infection. ferrets were exposed to a ⁄ vietnam ⁄ 1203 ⁄ 2004 by the total respiratory tract (trt) route (circles) or the oral route (triangles). the percentages of ferrets that survived infection are indicated at each day following challenge. ferrets exposed orally were more likely to survive h5n1 infection than ferrets exposed to the same dose of virus by the trt. the improved survival rates that were observed after an oral infection could be a consequence of low-level viral replication in the upper respiratory tract in combination with delivery of a substantial portion of the inoculum directly to the stomach where it may have been inactivated by the harsh environment of the gastro-intestinal tract. 11 most ferrets infected orally developed an h5-specific antibody response which differs from the studies of swayne et al. 6 in which ferrets gavaged with a liquid inoculum neither developed signs of disease nor an antibody response. however swayne et al. administered virus to anaesthetized ferrets by gastric gavage that would have bypassed the oropharynx. in our study virus was administered to the oral cavity directly and would have had access to the oropharynx. low level of replication at this site may have been sufficient to trigger an antibody response. the two ferrets that developed severe disease following oral infection had a similar profile of viral dissemination as ferrets infected by the trt route. differences were seen in the olfactory epithelium and bulb as lesions, and viral antigen did not occur in these sites following oral infection, although cerebral involvement was identified. one route of dissemination of h5n1 into the cns may be by transport within nerves through the olfactory bulb into the cerebrum. 12 due to the absence of lesions and antigen in these sites following oral infection the spread of virus into the brain in these two animals may be occurring through involvement of other cranial nerves or the hematagenous routes. nasal turbinates ) 3ae00 ) ) ) ) pharyngeal lymph node interactions of oseltamivir-sensitive and -resistant highly pathogenic h5n1 influenza viruses in a ferret model <2ae5 b ) + + + + olfactory epithelium nd a nd ) ) ) ) olfactory bulb nd nd ) ) ) ) trachea <2ae5 ) ) nd ) nd lung ) <2ae5 + + ) + spleen 3ae75 ) + + ) + small intestine ) ) ) ) ) + pancreas ) 3ae50 + ) + + the pandemic potential of highly pathogenic h5n1 influenza viruses remains a serious public health concern. while the neuraminidase (na) inhibitors are currently our first treatment option, the possibility of the emergence of virulent and transmissible drug-resistant h5n1 variants has important implications. clinically derived drug-resistant viruses have carried mutations that are na subtype-specific and differ with the na inhibitor used. 1 the most commonly observed mutations are h274y and n294s in the influenza a n1 na subtype (n2 numbering here and throughout the text); e119a ⁄ g ⁄ d ⁄ v and r292k in the n2 na subtype; and r152k and d198n in influenza b viruses. 2 h5n1 influenza viruses isolated from untreated patients are susceptible to the na inhibitors oseltamivir and zanamivir, 3 although oseltamivir-resistant variants with the h274y na mutation have been reported in five patients after 4,5 or before 6 drug treatment; and the isolation of two oseltamivir-resistant h5n1 viruses with n294s na mutation from an egyptian girl and her uncle after oseltamivir treatment were described. 6 the impact of drug resistance would depend on the fitness (i.e., infectivity in vitro, virulence, and transmissibility in vivo) of the drug-resistant virus. if the resistance mutation only modestly reduces the virus' biological fitness and does not impair its replication efficiency and transmissibility, the effectiveness of antiviral treatment can be significantly impaired. the recombinant wild-type h5n1 influenza a ⁄ vietnam ⁄ 1203 ⁄ 04 (vn-wt), a ⁄ turkey ⁄ 15 ⁄ 06 (tk-wt) viruses, and oseltamivir-resistant viruses with h274y na mutation (vn-h274y and tk-h274y) were generated by using the 8-plasmid reverse genetics system. susceptibility to na inhibitors was tested by using a fluorescence-based na enzyme inhibition assay with munana substrate at a final concentration of 100 lm. viral fitness was studied in vivo in a ferret model: groups of three ferrets were lightly anesthetized with isoflurane and inoculated intranasally with vn-wt, vn-h274y, or mixtures of the two at a different ratios at a dose of 10 2 pfu in 0ae5 ml pbs; they were inoculated with tk-wt, tk-h274y, or mixtures of the two at a different ratios at a dose of 10 6 pfu in 0ae5 ml pbs. respiratory signs (labored breezing, sneezing, wheezing, and nasal discharge), neurologic signs (hind-limb paresis, ataxia, torticollis, and tremor), relative inactivity index, weight, and body temperature were recorded daily. virus replication in the upper respiratory tract (urt) was determined on days 2, 4, and 6 p.i. the competitive fitness (i.e., co-inoculation of ferrets with different ratios of oseltamivir-resistant and -sensitive h5n1 viruses) was evaluated by the proportion of clones in day-6 nasal washes that contained the h274y na mutation. na mutations were analyzed by sequence analysis of individual clones ($20 clones ⁄ sample) created by ligation of purified pcr products extracted from nasal wash samples into a topo vector. introduction of the h274y na mutation conferred high resistance to oseltamivir carboxylate in vitro; the mean ic 50 of the vn-h274y and tk-h274y viruses was 3375 and 1208 times, respectively, that of the corresponding wildtype viruses. the oseltamivir ic 50 of the tk-wt virus was $16 times that of the vn-wt virus. all four recombinant h5n1 viruses were susceptible to zanamivir. introduction of the h274y na mutation reduced $90% and 60% of the na activity of vn-h274y and tk-h274y viruses, respectively, as compared to the wild-type virus activity (p < 0ae01; two-tailed t-test). all ferrets inoculated with either vn-wt or vn-h274y virus exhibited acute disease signs (high fever, marked weight loss, anorexia, extreme lethargy), rapid progression, and death by day 6-7 p.i., and no differences in clinical signs and replication in the urt of ferrets were observed between wild-type and oseltamivir-resistant viruses ( table 1) . both of the tk viruses caused milder illness than did the vn viruses, despite a much higher dose (10 6 pfu ⁄ ferret), and the tk-h274y virus caused less weight loss and fever than the tk-wt virus (table 1) . however, competitive fitness experiments revealed a disparity in the growth capacity of vn-h274y and tk-h274y viruses as compared to their wild-type counterparts: clonal analysis established the uncompromised fitness of vn-h274y virus and the impaired fitness of tk-h274y virus (table 2) . although, the trend towards an increase ⁄decrease in the frequency of the h274y na mutation relative to the wild-type was statistically significant (p > 0ae05) for two studied groups only. mutations within the na catalytic (r292k) and framework (e119a ⁄ k, i222l, h274l, n294s) sites or near the na active enzyme site (v116i, i117t ⁄ v, q136h, k150n, a250t) emerged spontaneously (without drug pressure) in both pairs of viruses (results not shown). the na substitutions i254v and e276a could exert compensatory effect on the fitness of vn-h274y and tk-h274y viruses. the lethality and continuing circulation of h5n1 influenza viruses warrants an urgent search for an optimal therapy. our results showed that the h274y na mutation affects the fitness of two h5n1 influenza viruses differently: the oseltamivir-resistant a ⁄ vietnam ⁄ 1203 ⁄ 04-like virus outgrew its wild-type counterpart, while the oseltamivir-resistant a ⁄ turkey ⁄ 15 ⁄ 06-like virus showed less fitness than its wild-type counterpart. we used a novel approach to compare the fitness of oseltamivir-sensitive and -resistant influenza viruses that included analysis of virus-virus interactions within the host (competitive fitness) during co-infection with these viruses. although mixed populations were present in the urt of ferrets on day 6 p.i., the fitness of vn-h274y virus was uncompromised as compared to that of its drug-sensitive counterpart, while that of tk-h274y virus was impaired. a minor population of na inhibitor-resistant variants may gain a replication advantage under suboptimal therapy in two ways: (i) preexisting variants less sensitive to the drug are selected from the quasispecies population, leading to an increase of the number of resistant clones, and (ii) outgrowing variants may acquire additional compensatory mutations that enhance their fitness. it is possible that use of antiviral drugs (particularly at suboptimal concentration) against mixtures of oseltamivir-resistant and sensitive viruses will promote the spread of drug-resistant variants * ferrets in all groups inoculated with a ⁄ vietnam ⁄ 1203 ⁄ 04 virus died by day 6-7 p.i. and were observed once daily for 7 days. ** results obtained from one ferret. *** by inhibiting drug-sensitive variants that are competing with them for the dominance in the infected host. the influence of multiple genes on the fitness of viruses carrying h274 na mutation cannot be excluded. in our study we focused on additional na mutations, and sequence analysis of individual na clones 7 was done to identify potential host-dependent and compensatory na mutations. we found that the na mutations e119a and n294s, which confer cross-resistance to oseltamivir and zanamivir, 1,2 can emerge spontaneously in clade 2.2 h5n1 influenza virus in ferrets. further, we observed that mutations at na catalytic (r292k) and framework (i222l and n294s) sites and in close proximity to the na enzyme active site (v116i, i117t ⁄ v, q136h, k150n, a250t) emerged without drug pressure in both pairs of h5n1 viruses. compensatory mutations in na or other genes may mitigate any fitness cost imposed by resistance mutations. our study identified six potential compensatory na changes (d103v, f132s, i254v, e276a, h296l, and f466s) that may affect the fitness of viruses with the h274y na mutation. we suggest that na mutations at residues i254v and e276a are of importance. interestingly, we observed differences in predominance of i254v and e276a na mutations in different genetic backgrounds: i254v mutation was identified in a ⁄ vietnam ⁄ 1203 ⁄ 04 (h5n1)-like and e276a in a ⁄ turkey ⁄ 15 ⁄ 06 (h5n1)-like genetic background. moreover, i254v na mutation was identified only when ferrets were inoculated with the mixtures of vn-wt and vn-h274y viruses, but not in ferrets inoculated with vn-h274y virus. none of the potential compensatory na mutations was identified in the original inoculum used to infect ferrets. the h274y na mutation causes a large shift in the position of the side chain of the neighboring e276 residue, 8 which must form a salt bridge with r224 to accommodate the large hydrophobic pentyl ether group of oseltamivir. residue i254 is located near the na active site, and although it does not alter polarity, it results in a shorter side-chain and, thus, may indirectly affect the residues in the na active site. we suggest that antigenic and genetic diversity, virulence, the degree of na functional loss, and differences in host immune response and genetic background can contribute to the observed differences in the fitness of h5n1 influenza viruses. therefore, the risk of emergence of drugresistant influenza viruses with uncompromised fitness should be monitored closely and considered in pandemic planning. this study was supported by contract hhsn266200700005c from the national institute of allergy and infectious diseases, national institutes of health, and by the american lebanese syrian associated charities (alsac). the data presented in the manuscript have been published at: govorkova ea, ilyushina na, marathe bm, mcclaren laninamivir (r-125489) is a strong na inhibitor against various influenza viruses, including oseltamivir-resistant viruses. [1] [2] [3] [4] [5] [6] we discovered a single intranasal administration of laninamivir octanoate (cs-8958), a prodrug of laninamivir, showed a superior anti-virus efficacy in mouse and ferret infection models compared to repeated administra-tion of oseltamivir and zanamivir. [2] [3] [4] 7 this suggested that cs-8958 works as a novel long-acting na inhibitor of influenza virus in vivo. a single inhalation of cs-8958 proved noninferiority in adult patients 8 and significantly superior in child patients, 9 compared to an approved dosage regimen of oseltamivir for treatment. cs-8958 has been commercially available as an inhaled drug, inavir ò , for the treatment of influenza in japan since october 2010. the long-acting characteristics of cs-8958 are explained by several reasons. first, cs-8958 was quickly hydrolyzed to an active metabolite, laninamivir, after an intranasal administration to mice, and was retained for a long time as laninamivir in target organs, such as lung and trachea. 10 however, with an intranasal administration of laninamivir, it disappeared quickly and did not demonstrate its longlasting characteristics. 10 another reason is a strong binding of laninamivir to nas of seasonal influenza viruses compared to other three na inhibitors, oseltamivir carboxylate, zanamivir, and peramivir. 3 in the following, the tight-binding ability of laninamivir to pandemic (h1n1)2009 na, as well as to the seasonal influenza virus nas, was demonstrated. in addition, we present a hypothesis of the mechanism of the long-lasting property of cs-8958 in mouse based on a localization of an enzyme that hydrolyzes cs-8958 to laninamivir. the influenza viruses, pandemic(h1n1)2009 (inf139), a ⁄ new caledonia ⁄ 20 ⁄ 99 (h1n1), a ⁄ panama ⁄ 2007 ⁄ 99 (h3n2), and b ⁄ mie ⁄ 1 ⁄ 93 were treated with excess na inhibitors, such as oseltamivir carboxylate, zanamivir, peramivir, and laninamivir, and then unbound na inhibitors were removed from the mixtures with a bio-spin column bio-gel p-6 (bio-rad laboratories, hercules, ca, usa). the na substrate, 4-methylumbelliferyl-n-acetyl-a-d-neuraminic acid (nacalai tesque, japan) was added to the virus-na inhibitor complex, and the na activities were followed for 6 hours at room temperature by measuring the fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. the enzyme which hydrolyzes cs-8958 to laninamivir was partially purified from rat lungs using ion exchange column chromatography, and almost all bands separated by an sdspolyacrylamide gel electrophoresis were identified by mass spectrometry. the gene expression profiles of the enzyme were investigated by the bioexpress database (genelogic inc., gaithersburg, md, usa). the enzyme gene cloned from mouse lung mrna was transiently expressed in cos cells. antiserum to the esterase was prepared by immunizing rabbits, and immunostaining was done using histomouse-tm-max kit (invitorgen corp., carlsbad, ca, usa) according to the manufacturer's manual. binding stability of na inhibitors to the four viruses are shown in figure 1 the enzyme that hydrolyzes cs-8958 to laninamivir in rat lungs was identified as carboxyesterase. this esterase was shown to be expressed in epithelial cells of rat lung by in situ hybridization. 11 the mouse homolog of the rat esterase was carboxylesterase 3 (ces3). the mrna of the mouse ces3 was shown to be highly expressed in lung and liver by the gene expression profile, and ces3 was also found to contain signal sequences for retention in endoplasmic reticulum (er) and golgi at the c-terminus. the cloned ces3 gene and the ces3 gene lacking the signal sequence were exogenously expressed in the cos cells. the cs-8958-hydrolyzing activity associated with the cos cells expressing ces3 was recovered from the culture sup of the cos cells expressing ces3 lacking the retention signal sequence. localization of ces3 was immunohistologically confirmed inside the airway epithelium cells of mice, which are the target cells for influenza virus infection. the long acting property of intranasal administration of cs-8958 in mice can be explained both by the long retention of laninamivir in the respiratory tract and by the stable binding of laninamivir to influenza virus na. again, stable binding of laninamivir to na of pandemic (h1n1)2009 virus was also observed similar to that of seasonal h1n1 virus. the following are speculated as the mechanisms for the long-lasting characteristics of cs-8958 in mice. we explain the mechanism by clarifying a cs-8958 hydrolyzing enzyme and its localization inside cells. the hypothesis of the mechanism is presented in figure 2 . briefly, hydrophilic laninamivir may not enter easily inside cells, whereas hydrophobic cs-8958 may enter inside cells. ces3 with er ⁄ golgi retention signal hydrolyzes octanoate of cs-8958 figure 1 . difference of binding stabilities of various na inhibitors to influenza virus neuraminidases. the na substrate was added to the influenza virus-na inhibitor complex (oseltamivir carboxylate, n; zanamivir, h; peramivir, s; laninamivir, •; distilled water, ¤), and the na reaction was followed for 360 minutes. the background (only the na substrate [d] ) is also shown. a part of data from. 3 to generate the hydrophilic drug, laninamivir, and then it is trapped inside er ⁄ golgi because of its high hydrophilicity. the glycoprotein, na, which matures in er ⁄ golgi, meets laninamivir there and efficiently makes a stable complex with it. there are some questions that remain. how does cs-8958 move from the cell membrane to er ⁄ golgi? is laninamivir indeed trapped inside er ⁄ golgi, and does it make a complex with na in mice? we are now making an attempt to clarify these concerns. in our study, we have explored the antiviral potential of two newly synthesized compounds to provide protection against the novel pandemic influenza virus h1n1 (2009) strain. the compounds were reconstituted in dimethylsulphoxide (dmso), and so the initial studies began with cytotoxicity determination of solvent on uninfected and untreated madin-darby canine kidney (mdck) cells. on obtaining an upper limit for dmso, the compounds were tested for estimation of their maximum non-toxic dose to the mdck cells. thereafter, the effective dose of the compounds was evaluated and validated by a number of assays and gene expression profiling at both nucleic acid and protein level. we found that these newly synthesized compounds possess potent inhibitory activity towards the novel pandemic influenza h1n1 (2009) virus. these findings are being evaluated in vivo for a better understanding of their inhibitory capabilities and also their effect on the host metabolism. this will be required in the course of development of new drugs for use in the prophylaxis and treatment against the influenza virus. the mdck cell line (from nccs, pune) was maintained in 1· dmem media (sigma, st. louis, mo, usa) supplemented with 10% fetal calf serum and antibiotics viz. 100 unit ⁄ ml penicillin and 100 lg ⁄ ml streptomycin at 37°c ⁄ 5% co 2 . 5 the synthesized compounds used in this study were kindly provided by the department of chemistry, university of delhi, delhi, india. the pandemic influenza h1n1 (2009) virus was isolated and propagated in the allantoic cavities of embryonated chicken eggs during the pandemic period. the virus stocks were prepared and stored at )80°c. plaque assay was performed as previously described by hui et al., 2003. 6 briefly, 0ae5 · 10 6 mdck cells ⁄ ml were seeded in six-well plates and maintained in dmem for 24 hours at 37°c ⁄ 5%co 2 . the monolayer of the cells was inoculated with serially diluted virus samples for 45 minutes at 37°c ⁄ 5%co 2 . subsequently, a mixture of agar overlay was added, and the plates were incubated at 37°c for 5 days or until formation of plaques. the plaques were visualized after removal of the agar plug and staining with 0ae1% crystal violet or neutral red solution. the virus titre was expressed as plaque forming unit (pfu) per milliliter. the in vitro cytotoxicity analysis was performed to determine the 50% cytotoxic concentration (cc 50 ) of the compounds on mdck cells. the compounds were dissolved in dimethylsulfoxide (dmso), and so a prior cytotoxicity analysis was performed to determine the toxic concentration of dmso on the cells. various concentrations of compounds were mixed with dmem containing 1% fcs before addition to the preformed monolayer of mdck cells in 96-well plates. a series of suitable controls for in vitro cc 50 determination was included in every plate, and the plates were incubated in the optimum environment for mdck cell culture. the cc 50 of test compounds was analyzed by estimation of percentage cell viability of the compound-and mocktreated mdck cells by performing a colorimetric assay using tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide (mtt) at end-point of 48 hours post-incubation. the assay was performed as described by mosman 1983. 7 briefly, mtt stock at a concentration of 0ae5 mg ⁄ ml was prepared in 1· pbs. the media was aspirated from the wells and 100 ll of mtt dye from the stock was added to each well. following incubation at 37°c ⁄ 5% co 2 for 2-4 hours, the dye was very carefully removed from the wells, and the cells were incubated with 100 ll of stop solution (dmso) per well at 37°c ⁄ 5% co 2 for 1 hour. the absorbance of the supernatants from each well was measured at 540 nm, and the percentage cell viability was calculated. madin-darby canine kidney cells were maintained overnight in a 12-well tissue culture plate at 37°c ⁄ 5% co 2 . the cells were inoculated with various virus dilutions at 37°c ⁄ 5% co 2 for 45 minutes and observed for cytopathic effect (cpe). the media from the experimental wells were aspirated after 48-72 hours of infection and were subjected to plaque assay. the percentage cell viability was determined by performing mtt assay. the results of both these tests were used to assess tcid 50 of the virus. the pre-formed monolayer of mdck cells was inoculated with the 10-fold dilution corresponding to tcid 50 of the virus for 1 hour at 37°c ⁄ co 2 . the experimental setup included control wells for the cells, virus, and compound. meanwhile, the concentrated stocks of the synthesized compounds were diluted with dmem (with1% fcs) to various concentrations within their respective cc 50 ranges. one hour post-infection, the cells were incubated with these diluted solutions. the cells were observed at various time intervals post-inoculation for cpe, and 200 ll media was collected from each experimental well for performing hemagglutination test. after 48 h, the media was collected for plaque assay and the cells were subjected to mtt cell viability assay. preformed monolayers of mdck cells were infected with virus and treated with the respective inhibitory concentration of the compounds. forty-eight to 72 hours post-incubation, total cellular rna was isolated using ribozol (amresco, solon, oh, usa) and treated with 50 lg ⁄ ml of dnase (promega, madison, usa). the concentration and quality of the rna from each well were determined by measuring their absorbance at 260 and 280 nm. one microgram of the cdna synthesized from each rna sample was used for sybr green-based real-time pcr detection of the ha gene of pandemic influenza h1n1 (2009) virus. as a control, human glyceraldehyde-3-phosphate dehydrogenase (hgapdh) was also amplified using gene specific primers. 8, 9 immunoblotting immunoblotting was performed to further validate the antiviral potential of the compounds. the experimental protocol was the same as for real time rt-pcr analysis. the cells were harvested 48 hours post-treatment with the compounds to prepare whole cell lysates in mammalian cell lysis buffer [0ae1 m nacl, 0ae01 m tris cl (ph 7ae6), 0ae001 m edta (ph 8ae0), 1 m m protease inhibitor cocktail, 100 lg ⁄ ml pmsf]. the protein concentration was determined by bca protein assay. the cell lysates were fractionated on 12% polyacrylamide for western blotting. the blot was developed using sheep monoclonal antibody (santa cruz biotechnology, ca, usa) against ha protein of influenza virus and horseradish peroxide conjugated rabbit-anti sheep igg (1:1000 dilutions) as secondary antibody. 10 the median cytotoxic concentration for compound meuh came out to be 150 lm, and that for flh was 160 lm. compounds showing potent antiviral effect on the pandemic influenza h1n1 (2009) virus propagation in madindarby canine kidney cells ( figure 1 ). the viral titres remained constant in cells treated with the compounds, while they increased in the untreated virus infected cells. ed 50 for the compounds meuh and flh were 150 and 160 lm, respectively. fifty-two percent (meuh) and 45% (flh) inhibition against the pandemic influenza h1n1 (2009) virus was achieved using ed 50 of the test compounds. both the compounds were able to reduce the rna levels of the ha gene by approximately 40-45%, whereas approximately 50% inhibition was seen when both the compounds were used in combination. similar results were obtained by the immunoblotting analysis ( figure 2 ). antiviral therapy has shown to be a promising tool in the management of various respiratory diseases, including those caused by influenza viruses. we have already shown inhibition of influenza virus replication in our earlier studies using catalytic nucleic acids, 11 which can be used as an approach in the development of new therapeutic strategy. these therapies are very useful as the influenza virus vaccines need annual renewals due to frequent genetic drifts in the viral surface proteins. in pandemic situations the existing vaccines do not provide complete protection against the novel virus as the population generally remains naïve for the newly mutated surface antigens. the antiviral drugs play an important role in the control of novel viral strains for which there are no vaccines available. however, the key obstruction in the extensive use of antiviral drugs is their cost and relative therapeutic efficacy provided. two classes of drugs were being used for treatment and control of the influenza virus infection in humans, the m2 ionchannel blockers 12, 13 (amantadine and rimantadine), which prevent viral uncoating, and the neuraminidase inhibitors 14, 15 (zanamivir and oseltmivir), which prevent the release of influenza virions from the cytoplasmic membrane. but widespread resistance to these antiviral drugs 16, 17 has limited their use. thus, novel drugs are required for the effective therapy against the emerging strains of influenza virus. the novel chemical compounds used in our study were tested for their antiviral efficacy against the pandemic influenza h1n1 (2009) virus. a reduction in the cpe in compound treated virus infected mdck cells indicated presence of antiviral activity in chemical compounds. the persistence of constant viral titers in the compound treated cells provided evidence for the interference posed by the compounds in the replication of influenza virus. inhibition in the ha gene expression further validated our hypothesis for the antiviral effect of compounds. the efficacy of these compounds in animal models is currently being validated in our laboratory. further, molecular studies are required to ameliorate the awareness regarding the mode of action of these chemical compounds against the viruses. 4 and is now licensed in japan, 5 while another, laninamivir, is being developed as an inhaled prodrug. 6 resistance to nais among circulating influenza viruses was previously low (<1% worldwide). [7] [8] [9] however, the 2007-2008 influenza season was marked by a worldwide emergence of oseltamivir-resistant seasonal influenza a (h1n1) viruses with the h275y (h274y in n2 numbering) in the na. [9] [10] [11] [12] [13] [14] the prevalence of oseltamivir resistance was even higher in the subsequent 2008-2009 influenza season with many countries reporting up to 100% oseltamivir resistance, 15 seasonal and pandemic influenza viruses collected globally between october 1, 2008 and september 30, 2009 were submitted to the who collaborating center for surveillance, epidemiology and control of influenza at the centers for disease control and prevention (cdc) in atlanta, ga, usa, and propagated in madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa). reference viruses representative of oseltamivir-sensitive and -resistant seasonal and pandemic viruses were also propagated in mdck cells. susceptibilities of virus isolates to the nais oseltamivir carboxylate (hoffman-la roche, basel, switzerland) and zanamivir (glaxosmithkline, uxbridge, uk) were assessed in the chemiluminescent ni assay using the na-star tm kit (applied biosystems, foster city, ca, usa) as previously described. 9 additionally, subsets of virus isolates were tested for susceptibility to peramivir (biocryst pharmaceuticals, birmingham, al, usa). fifty percent inhibitory concentration (ic 50 ) values were calculated using jaspr curve fitting software, an in-house program developed at cdc. curve fitting in jaspr was done using the equation: v = vmax · (1 ) ([i] ⁄ (ki + [i]))), where vmax is the maximum rate of metabolism, [i] is the inhibitor concentration, v is the response being inhibited, and ki is the ic 50 for the inhibition curve. box-and-whisker plot analyses 7 of log-transformed ic 50 s were performed for each virus type ⁄ subtype and nai using sas 9.2 software (sas institute, cary, nc, usa) to identify viruses with extreme ic 50 values (outliers). outliers were characterized based on a statistical cutoff of ic 50 greater than three interquartile ranges from the 75th percentile. outliers were subjected to genetic analysis by pyrosequencing 17 and ⁄ or conventional sequencing 18 to detect known or novel markers of nai resistance. those harboring previously characterized mutations in the na associated with nai resistance were considered drug-resistant; their descriptive statistics were determined separately from naisusceptible viruses. descriptive statistics to compute the mean, median, and standard deviation (sd), and a one-way analysis of variance were performed on original scale ic 50 data, using sas 9.2 software (sas institute) for each nai and virus among seasonal influenza a (h1n1) viruses tested for oseltamivir susceptibility (n = 1533), 1431 (93ae3%) were outliers for the drug (table 1 ) and harbored the oseltamivir-resistance conferring h275y mutation in the na. by contrast, only a small proportion (0ae7%) of tested h1n1pdm viruses (n = 2259) were resistant to oseltamivir. all influenza a (h3n2) viruses (n = 834) were sensitive to oseltamivir except for one outlier, a ⁄ ontario ⁄ rv0442 ⁄ 2009 with d151v mutation in the na, whose ic 50 of 2ae50 nm was beyond the statistical cut-value off and >10-fold the mean ic 50 for the drug (0ae24 nm). all influenza b viruses (n = 914) were sensitive to oseltamivir with exception of an outlier b ⁄ texas ⁄ 38 ⁄ 2008, with d197e (d198e in n2 numbering) mutation in the na, whose ic 50 was beyond the cut-off, but only fourfold greater than the mean ic 50 for the drug. all virus types ⁄ subtypes tested for zanamivir were sensitive to the drug (table 1) , except for some outliers among seasonal influenza a (h1n1) and a (h3n2) outliers. the seasonal influenza a (h1n1) outliers included a ⁄ thailand ⁄ 1035 ⁄ 2008 (h1n1) and a ⁄ hawaii ⁄ 20 ⁄ 2008 (h1n1), both with combined h275y and d151d ⁄ g mutations in their na. the presence of concurrent mutations at na residues h275 and d151 in seasonal influenza a (h1n1) virus isolates substantially enhances resistance to oseltamivir and peramivir and ⁄ or zanamivir, however, the changes at d151 are typically cell-derived and not present in clinical specimens. 19 influenza a (h3n2) outliers for zanamivir included a ⁄ ontario ⁄ rv0442 ⁄ 2009 with d151v mutation in the na, as well as a ⁄ maryland ⁄ 02 ⁄ 2009 and a ⁄ vladivostok ⁄ 53 ⁄ 2009 with d151g and mixed d151d ⁄ g mutations, respectively. some mild outliers for zanamivir among a (h3n2) viruses with ic 50 beyond the statistical cutoff but <10-fold mean ic 50 for the drug were also identified; their genetic analysis revealed presence of wildtype and mutant sequences at residue 151 namely, d151d ⁄ g, d151d ⁄ n, or d151d ⁄ a. mutations at residue d151 of the na are associated with reduced susceptibility to zanamivir in a (h3n2) viruses, 9 but were reported to be cell-culture derived in recent h3n2 viruses. 20 all virus isolates tested for peramivir (n = 1058) were sensitive to the drug, except for h275y variants among seasonal influenza a (h1n1) and h1n1pdm viruses, which exhibited reduced susceptibility to the drug. in addition, one influenza a (h3n2) isolate, a ⁄ ontario ⁄ rv0442 ⁄ 2009 with d151v mutation in the na, showed reduced susceptibility to peramivir. the ic 50 values determined in functional ni assays provide valuable information for detection of resistant viruses, but should not be used to draw direct correlations with drug concentrations needed to inhibit virus replication in the infected human host, as clinical data to support such inferences are inadequate. 21 nevertheless, combining elevated ic 50 values with the presence of established molecular markers of resistance in the na of virus isolates and their matching clinical specimens provides a reliable and reasonably comprehensive approach of identifying nai-resistant isolates for surveillance purposes. in this study, outliers with elevated ic 50 values for oseltamivir among seasonal influenza a (h1n1) and h1n1pdm viruses were confirmed to be oseltamivir-resistant based on the presence of the h275y mutation in the na. outliers for oseltamivir and ⁄ or zanamivir among influenza a (h3n2) viruses in this study were shown to harbor mutations at d151, which were earlier associated with reduced susceptibility to zanamivir, 9 and were cell-culture derived. 20 the effects of d151 mutations on nai susceptibility appear to be strain-specific; however, there are no conclusive supporting data and further investigations are required. outliers among the influenza a viruses in this study exhibited changes in the na, derived naturally or through cell-culture, which altered their susceptibility to nais. however, mild outliers for oseltamivir and ⁄ or zanamivir among influenza a viruses with slightly elevated ic 50 s, but without apparent changes in the na are sometimes identified. in such instances it is imperative to exclude the potential presence of influenza b among such outliers, using conclusive genetic tests such as real time pcr, since influenza b viruses exhibit higher ic 50 values for oseltamivir and zanamivir than influenza a viruses. 21 viruses exhibiting such mixes are typically excluded from statistical analyses of ic 50 s for respective drugs and virus type ⁄ subtype. establishment of a clinically relevant ic 50 cutoff value which could be used to differentiate statistical outliers from truly resistant viruses is imperative. global surveillance for nai susceptibility of influenza viruses circulating globally should be sustained to reflect the impact of seasonal and pandemic of influenza, given the limited pharmaceutical options available for control of influenza infections. nasopharyngeal swab specimens from patients with acute respiratory infection were collected at 164 influenza sentinel surveillance units (outpatient and hospital-based) all over mongolia. specimens were transported to the virology laboratory, nccd, ulaanbaatar, and rt-rt pcr positive samples were grown in a mdck cell culture according to the protocol developed by cdc. 5 6 and influenza virus gene segment 7 (m genes) sequencing (7 strains-genbank accession numbers: cy053364, cy053365, cy054547, cy054549, cy055171, cy065990, and cy065998) and influenza virus gene segment 6 (na gene) sequencing (2 strains genbank accession numbers: cy073447 and cy073448) by the standard methods with applied biosystems 3130xl genetic analyzer using primers supplied by who collaboration centers. a chemiluminescent na inhibition assay was performed with veritas microplate luminometer using the commercially available kit, na-star (applied biosystems, foster city, ca, usa), according to the manufacturers protocol. the na inhibitor susceptibility of influenza virus isolates was expressed at the concentration of na inhibitor needed to reduce na enzyme activity by 50% (ic 50 ). oseltamivir carboxylate, was provided by f. hoffman-la roche ltd (basel, switzerland). na inhibition assay data were analyzed using robosage software comparing test data with the data produced by the reference na inhibitor sensitive and resistance strains, which were provided by the who influenza collaboration center, melbourne, australia. all viruses tested were sensitive to oseltamivir with two exceptions: a seasonal influenza virus a ⁄ ulaanbaatar ⁄ 1735 ⁄ 2009(h1n1) with 137ae1 nm ic 50 value and a pandemic influenza virus a ⁄ dundgovi ⁄ 381 ⁄ 2010(h1n1) with 65ae6 nm ic 50 value ( figure 1 ). there was oseltamivir resistance detected in 2ae7% (37 ⁄ 1) of seasonal a (h1n1) and in 0ae4% (262 ⁄ 1) of a (h1n1) pdm viruses. the oseltamivirresistant viruses were collected from untreated patients. in total, 18 influenza b viruses were analyzed by na inhibition assay and all were sensitive to oseltamivir. the na of both oseltamivir-resistant strains contained h275y mutation based on the sequencing analysis. the difference in the na amino-acid sequences between the mongolian oseltamivir-resistant viruses and the respective oseltamivir-sensitive reference viruses is shown in table all 37 a(h1n1) viruses analyzed for m2 channel inhibitor resistance by pyrosequencing contained the s31n mutation and, thus, were resistant to this class of anti-influenza drugs. the segment 7 sequencing revealed that 6 seasonal a(h1n1) viruses possess the common s31n mutation. of note, a single strain a ⁄ zavkhan ⁄ 8299 ⁄ 2009(h1n1) contained an unusual s31d change in the m2 protein. our study shows that the same prevalence [2ae7% (1 ⁄ 37)] of seasonal a(h1n1) viruses with h275y mutation in 2008 ⁄ 2009 season in mongolia with the published data for 2007 ⁄ 2008 season from japan. 7, 8 however the prevalence of oseltamivir resistance in japan has dramatically increased in 2008 ⁄ 2009 season to 100% (77 ⁄ 77). the observed double mutations: h275y and d354g in a ⁄ ulaanbaatar ⁄ 1735 ⁄ 2009(h1n1) strain, which have been also found in japan in 2008 ⁄ 2009 season. 7 the patient from whom the oseltamivir resistant seasonal influenza h1n1 virus has been isolated was a 1-year-old boy, living in ulaanbaatar, the capital city, without history of using oseltamivir. the patient from whom the oseltamivir resistant a(h1n1)pdm virus was isolated was a 59 year-old man, residing in the dundgovi, the southern province, also without history of antiviral treatment. according to the who data, isolation of the pandemic viruses carrying h275y change from untreated patients has been uncommon. circulation of amantadine-resistant seasonal a (h1n1) viruses has been increasing in mongolia since 2006 ⁄ 2007 influenza season. 4 all pandemic influenza a(h1n1) strains (16) tested were resistant to m2 channel inhibitors due to the presence of the s31n mutation in the m2 protein. among seasonal a(h1n1) viruses, one contained a s31d change whereas the others had s31n, the well established marker of resistance to both amantadine and rimantadine. this is the first report of detecting the s31d change in the seasonal a(h1n1) viruses. according to the cdc data (unpublished), the s31d change conferred the drug resistance in the a(h3n2) viruses according to the virus yield reduction assay. it is essential to continue the antiviral resistance surveillance of influenza virus strains circulating in mongolia to ensure the efficiency of a proper clinical management of influenza patients. (conferred by the s31n mutation). of note, the genotype 2 and genotype 3 dual resistant viruses from asia appear to be genetically similar to those previously reported dual resistant viruses from hong kong, sar. 4, 5 the genotype 4 virus was the only dual resistant virus with a nearly complete 2c genome. oseltamivir-resistance for this virus appears to be the result of a reassortment as demonstrated by the presence of the oseltamivir-resistant clade 2b na gene. although the detection of dual resistant seasonal influenza a (h1n1) viruses is still rare, there has been an increased prevalence of dual resistance viruses during the last three seasons: 0.06% (1 of 1753 tested in 2007-2008), 1.5% (21 of 1426 in 2008-2009) , and 28% (7 of 25 in 2009-2010) (v 2 p < 0.001). while the continued circulation or co-circulation of seasonal a (h1n1) viruses is uncertain, the emergence of dual resistant influenza viruses in five countries does present a public health concern, especially since dual resistant viruses would limit the options for antiviral treatment to a single licensed antiviral drug: zanamivir. moreover, the markers of resistance seen in seasonal a (h1n1) viruses also confer resistance in the more widely circulating 2009 pandemic a (h1n1) virus. and, since the acquisition of mutations in influenza a viruses typically occur through drug selection, spontaneous mutation, or genetic reassortment with another drug resistant influenza a viruses, the detection of influenza a (h1n1) viruses that are resistant to both adamantanes and oseltamivir warrants close monitoring, even if only detected at low frequency. new antiviral agents and strategies for antiviral therapy are likely to be necessary in the future. 1 heightening concern that drug resistance will likewise become prominent in pandemic viral strains and highlighting the need for antiviral drug resistance surveillance. the h275y mutation in h1n1 neuraminidase is the most common mutation conferring resistance. however, due to the high mutation rates of viruses, new mutations can be expected that will also render viral neuraminidase less sensitive to antiviral drugs. pcr methods can be used to detect previously identified mutations; however, functional neuraminidase enzyme activity inhibition testing is necessary for detecting drug resistance that results from novel mutations. the two neuraminidase enzyme inhibition assays using either the fluorescent munana or chemiluminescent na-star ò substrate are robust tools for ni susceptibility testing. the munan-a-based assay is broadly used by many groups, including many regional health organizations for ni susceptibility testing, yet no standardized protocol or dedicated kit has been in place for this assay, making comparison of data generated between different laboratories difficult. borrowing from multiple neuraminidase inhibitor susceptibility network (nisn)-published munana-based neuraminidase assay protocols, 2 we have developed a kit-based fluorescent neuraminidase assay that offers both standardization and off-the-shelf quality-controlled reagents for ni susceptibility testing and other neuraminidase assay applications. the na-fluor tm influenza neuraminidase assay reagents and protocols were optimized in comparison to published nisn protocols according to the criteria of assay performance, ease-of-use, consideration of historically used assay conditions, reagent storage stability, and environmental impact. our optimized assay conditions consists of 100 lm munana, 33ae3 mm mes, 4 mm cacl 2 , and ph 6ae5 in a 100 ll assay volume, and performing the assay for 60 minutes at 37°c following a 30 minutes preincubation of drug with the virus. these conditions are consistent with the majority of published influenza ni screening data in publication. 2 the standard na-fluor tm assay workflow for screening viral isolates for sensitivity to nis includes first titering the viral sample by neuraminidase activity to determine optimal virus concentration to be used in subsequent ic 50 determination assays. the na-fluor tm assay is an ideal tool for titering virus based on neuraminidase activity in the viral coat. titering of viral samples prior to running the ic 50 determination assays insured that assays would be performed within the fluorescence detection dynamic range of both the assay and the fluorometric instrument being used. viral titers giving rfus in the range of 2000-5000 were used for subsequent assays. comparison to traditional munana assays a primary goal of developing a standardized munana assay was to provide a standardized protocol and set of reagents that would allow for comparison of ni surveillance data between laboratories and over time. in addition, the assay should provide data comparable to historical data sets based on traditional munana-based protocols. to insure that our newly developed na-fluor tm assay met these criteria we performed side-by-side comparisons of the na-fluor tm assay to munana-based nisn protocols, as well as our na-xtd tm and na-star ò chemiluminescent neuraminidase assays to compare assay sensitivity and dynamic range and for ni ic50 determination with multiple viral isolates. for all assay comparisons, assays were performed according to respective published protocols. for direct comparison of results, an equivalent amount of virus (and concomitant neuraminidase activity) was used for each assay. the na-fluor tm assay provides low-end sensitivity (by signal to noise ratio) and dynamic range similar to nisnpublished, munana-based protocols (data not shown). these assays all show a low-end detection of approximately 0ae002 u ⁄ well and dynamic range of 2-3 orders of magnitude when performed simultaneously side-by-side using serial dilutions of bacterial (clostridium perfringes) neuraminidase. these assays show approximately onefold less dynamic range and approximately fivefold less low-end sensitivity than chemiluminescent assays under these conditions. given the large amount of archived ni inhibition data for viral isolates over the past decade, it is very important for a standardized assay to generate data similar to established protocols so that data can be compared in relative terms. when run side-by-side, na-fluor tm assay provided oseltamivir carboxylate and zanamivir ic 50 values similar to nisn-published, munana-based protocols. ic 50 values vary somewhat for munana assays versus chemiluminescent assays depending on the viral isolate, as previously described. 3 the na-fluor tm assay also exhibited similar sensitivity for detecting ni sensitive virus compared to nisn-published fluorescent assays as shown in figure 1 . the large shift in ic 50 values between oseltamivir-sensitive and resistant virus using the na-fluor tm assay enables detection of mutant virus in mixed viral samples ( figure 2 ). this capability is critical for identifying resistant virus in clinical isolates presenting mixed populations of resistant and sensitive virus during ni susceptibility surveillance. several characteristics of the na-fluor tm assay make it an ideal assay for processing large numbers of viral isolates for ni sensitivity surveillance or for using the assay for high throughput screening for lead discovery of new antiviral reagents. the na-fluor tm assay signal was found to remain stable for up to 4 hours after stop solution addition when stored at room temperature and for several days when stored at 4°c (data not shown). ic 50 values did not change over these times, indicating that the assay is compatible with processing many samples in a short time frame. the na-fluor tm assay was also found to be highly reproducible giving a z' of 0ae78 or above indicating that the assay can be used confidently to identify nis in high throughput screening mode. 4 the assay can tolerate up to 5% dmso, a common compound delivery reagent used in high throughput screens (data not shown). we have developed a standardized na-fluor tm assay suggested protocol that gives data similar to established mun-ana protocols. however, we have also found that several protocol adaptations can be made that generate comparable data while allowing the user more flexibility in assay mode, use of additional reagents, and to meet user-specified assay time requirements. the na-fluor tm assay can be run in either the standard 60 minutes ⁄ 37°c endpoint mode described above or as real-time kinetic assay 5 with repeated reads taken over time without the addition of stop solution, which both serves to terminate neuraminidase activity and to enhance the fluorescence of the product. for typical ni-sensitive viral strains, the rate of munana substrate turnover at 37°c is linear for at least 4 hours (data not shown). as would be expected, rates of substrate turnover decrease in the presence of nis reflected in a decreased slope exhibited by real-time kinetic reads. real-time acquired rfus are typically 5-6 fold lower than rfus acquired after addition of stop solution at the same time point. ic 50 values obtained using slope analysis for real-time assays are similar to values obtained by endpoint analysis. whether run in real-time or end-point mode, the linear rate of substrate turnover allows the user to run the assay for shorter or longer assay times than the standard protocol without compromise to assay performance. the na-fluor tm assay is also compatible with standard methods used in many laboratories to inactivate virus. 6 we have shown that ni ic 50 values for multiple viral strains remain unchanged when the assay is performed in the presence of 0ae1% np-40 or 1% triton x-100 (data not shown). similar results are also obtained by adjusting the na-fluor tm stop solution to 40% ethanol prior to addition for assay termination. the assay is unaffected by phenol red concentrations present in cell culture media. we have developed a standardized munana-based fluorescent neuraminidase assay, the na-fluor tm influenza neuraminidase assay kit, which has been optimized for ni susceptibility screening. the assay provides data that can be compared to data generated using traditional munanabased protocols. the assay is economical, highly reproducible, easy to use, and environmentally friendly. the assay is flexible and amendable to user-specific adaptations including assay mode, assay timing, and reagent compatibility. trademarks ⁄ licensing ª 2010 life technologies corporation. all rights reserved. relenza is a registered trademark of glaxo to test the prophylactic potency of h5-vhhb, mice were treated intranasally with pbs, 100 lg of h5-vhhb, or negative control rsv-vhhb at 4, 24, or 48 hours before infection with one ld 50 of nibrg-14ma virus. body weight loss was monitored daily, and on day 4 mice were sacrificed to determine the viral load in the lungs. all mice that received h5-vhhb retained their original body weight, whereas those receiving pbs or rsv-vhhb gradually lost weight (data not shown). intranasal administration of h5-vhhb at 4 or 24 hours before challenge resulted in undetectable lung virus titers. when animals were treated with h5-vhhb 48 hours before challenge, virus titers were 50fold lower compared to pbs and rsv-vhhb treated mice, and three out of seven animals still had undetectable virus titers ( figure 1 ). we next determined if h5-vhhb nanobody ò could be also be used therapeutically. we administered 60 lg of this nanobody ò intranasally to mice up to 72 hours after chal-lenge with 1 ld 50 of nibrg-14ma virus. four days after challenge, animals that received h5-vhhb 4, 24, or 48 hours after challenge had significantly higher body weight (data not shown) and lower lung virus loads than control mice. although mice treated with h5-vhhb nanobody ò 72 hours after challenge were not clinically protected compared to control mice, they had significantly lower lung virus titers (figure 1 ). to identify the ha amino acid residues that are potentially involved in h5-vhh binding, escape viruses were selected by growth and plaque purification of nibrg-14ma virus in the presence of h5-vhhm or h5-vhhb nanobodies ò . the ha sequences of six independently isolated h5-vhhm escape viruses revealed substitution of a lysine by a glutamic acid residue at position 189 in ha1 (h5 numbering). in addition, two h5-vhhm escape mutants carried an n154d and four carried an n154s substitution. the three-dimensional structure of nibrg-14 ha shows that n154d ⁄ s and k189e are close to each other as part of the corresponding antigenic site b in h3 ha. 4, 5 interestingly, the n154d ⁄ s mutations remove an n-glycosylation site, which is surmised to have evolved in h5n1 ha as a strategy to mask an antigenic site. 6 escape viruses selected in the presence of h5-vhhb carried k189n (n = 4) or k189e (n = 2) substitutions. these results indicate that residues in antigenic site b, at the top of ha and very close to the receptor binding domain (rbd), are essential for neutralization of the virus by h5-vhhm ⁄ b nanobodies ò (figure 2 ). the virus titer was measured in lung homogenates prepared on day 4 after challenge. the x axis refers to the time points in hours relative to the challenge (time = 0 hours) when ha-specific nanobodies (h5-vhhb), control nanobodies (rsv-vhhb) or pbs was administered to the mice. # below detection limit, n not determined [n = 4-7 mice per condition: p values < 0ae05 (*)]. here we demonstrated that prophylactic and therapeutic treatment with llama-derived immunoglobulin single variable domain fragments is effective to control infection with h5n1 influenza virus in a mouse model. we demonstrate that pulmonary delivery is a highly effective route of administration to treat or prevent influenza virus infection. in addition, we demonstrate that a homobivalent h5-vhhb has powerful h5n1-neutralizing activity in vivo. it is important to note that we used a mouse-adapted derivative of the non-highly pathogenic nibrg-14 virus in our challenge model. nevertheless, this virus induces severe morbidity and lethality in mice. compared to conventional neutralizing monoclonal antibodies, vhhs offer the advantage that they are easy to produce in escherichia coli, typically with high yield. in addition, their small size (15 kda for a monovalent vhh) and high folding capacity allow the generation of oligovalent vhh derivatives. in vitro escape selection revealed that a k189e substitution in ha1 abolished the neutralizing effect of h5-vhhm ⁄ b. a lys or arg residue at this position is conserved in all human h5n1 virus isolates. of note, all selected escape mutants contained a glutamic acid or serine residue at position 189, which suggests that the conserved positively charged amino acid is important for neutralization by h5-vhh nanobodies ò . interestingly, escape mutants selected with h5-vhhm also carried an n154d ⁄ s co-mutation that removes an n-glycosylation site in this antigenic site of ha. the predicted n-glycosylation site at n154 in a ⁄ hong kong ⁄ 156 ⁄ 97 ha was shown to be glycosylated and may have evolved to mask an antigenic site near the rbd. 7, 8 the selected amino acid changes are located near the receptor binding site of ha. therefore, it is possible that enhanced receptor binding properties of these escape viruses contribute to or are responsible for the loss of neutralizing activity of h5-vhh nanobodies ò . 9, 10 we conclude that influenza virus neutralizing nanobodies ò have considerable potential for the treatment of h5n1 virus infections. although we focused on vhhs that presumably recognizes an epitope near the rbd, it is possible to select vhh molecules that bind to other epitopes in ha, including more conserved domains. more, a novel na (i117m) substitution was discovered in a series of specimens from a patient. for the amantadine resistance, 1113 samples were tested, and all of them were confirmed to be resistant. we collected respiratory specimens from patients who had been clinically refractory to antiviral treatment since october 2009 upon ethical approval from the relevant institutions. to investigate the resistant pattern, sequence analysis to the na and matrix (m2) genes were conducted by reverse transcription (rt)-pcr and sequencing reaction. the obtained sequences were analyzed by the influenza sequences and epitopes database, which was developed in korea. eleven patients were found to be having oseltamivir-resistant pandemic (h1n1) 2009 viruses with the h275y substitution in the viral na genes (tables 1 and 2 ). some cases were associated with oseltamivir treatment on the basis of h275y change from the oseltamivir-sensitive genotypes to oseltamivir-resistant genotypes in consecutive samples from the same patient. furthermore, a novel na (i117m) substitution that may be associated with oseltamivir resistance was detected in specimens from one patient (patient g) who had myelodysplasia and received oseltamivir and peramivir (tables 1 and 2 ). in addition, we obtained viruses from clinical specimens (patients a and c) and evaluated antiviral susceptibility by measuring the dose of oseltamivir and zanamivir required for 50% inhibition (ic 50 ) of na activity. these viruses (from patients a and c) were resistant only to oseltamivir (ic 50 713ae2 and 359ae4 nmol ⁄ l, respectively). susceptibility to zanamivir was not altered whether na contained y275 or h275 (ic 50 0ae13 and 0ae78 nmol ⁄ l, respectively). one isolate of pandemic (h1n1) 2009 virus with an oseltamivir-sensitive genotype (h275 in its na) was susceptible to oseltamivir (ic 50 1ae18 nmol ⁄ l) and zanamivir (ic 50 0ae42 nmol ⁄ l). patients with oseltamivir-resistant pandemic (h1n1) 2009 were treated during hospitalization with oseltamivir alone or with a combination of other antiviral drugs ( we found 11 patients of oseltamivir resistance with h275y mutation in the na gene of pandemic (h1n1) 2009 virus through the surveillance of patient refractory to antiviral treatment. in addition, novel amino acid change (i to m) at position 117 in the na gene, which might influence oseltamivir susceptibility, was detected in sequential specimens of a patient. these data showed that generation of oseltamivir resistance could be associated with oseltamivir treatment. therefore, it needs to strengthen the antiviral monitoring by supplementation of the clinical data including antiviral treatment. during the pandemic, oral oseltamivir was the primary antiviral medication used for treatment of hospitalized patients with ph1n1 infection. many physicians worried that clinical deterioration or failure to respond to treatment with oseltamivir was due to either oseltamivir resistance or oseltamivir failure. in the united states, two investigational intravenous (iv) nais were available during 2009-2010: peramivir through emergency use authorization and zanamivir by investigational new drug application. peramivir would be an option for patients with oseltamivir failure, but would not be appropriate for patients infected with h275y oseltamivir resistant mutants. iv zanamivir was available in limited supply, but would be appropriate for severely ill patients infected with an oseltamivir-resistant ph1n1 virus. during the pandemic, clinicians had few options for antiviral resistance testing in the united states. to respond to this need, the us centers for disease control and prevention (cdc) offered antiviral resistance testing for patients suspected to have clinical failure due to oseltamivir resistance. we describe the methods that cdc used to prioritize patients for testing during the pandemic and to detect markers for oseltamivir resistance, as well as the results from this testing. to facilitate decisions on which patients to test, we developed testing algorithms that were shared with state labora-tories, epidemiologists, and the emergency operation center at cdc. we prioritized patients who might benefit the most from antiviral testing given the inherent delay in providing antiviral results, e.g. patients who might have prolonged ph1n1 shedding. patients that were critically ill [intensive care unit (icu) admission] or patients with severe immunocompromising conditions with clinical evidence for oseltamivir treatment failure (persistent detection of virus and clinical unresponsiveness to the drug) were prioritized. in addition, we tested specimens from patients that failed oseltamivir chemoprophylaxis. standard forms with information regarding specimen and minimal clinical information were collected on all patients. all protocols were validated and approved by clinical laboratory improvement amendments, e.g. quality standards to ensure accuracy, reliability, and timeliness of patient test results. information collected on patients was deemed public health response, not research, at cdc. clinical specimens, confirmed as 2009 pandemic influenza a (h1n1), were tested for the h275y mutation in the na using pyrosequencing. 5 results were returned to sender within 24-48 hours of specimen receipt. from october 2009 until july 2010, a total of 116 specimens from 73 patients were submitted for testing. viruses from 26 (36%) of 73 patients had h275y mutation in the na in at least one submitted specimen. clinical information was available for 66 patients (table 1) . most patients had received oseltamivir for treatment prior to obtaining the specimen sent for antiviral testing. four patients received oseltamivir for chemoprophylaxis, all were immunosuppressed, and all had the h275y mutant; duration of chemoprophylaxis until ph1n1 infection was detected varied (4-39 days). among the patients with an h275y mutant who were treated with oseltamivir, the median time on oseltamivir prior to collection of specimen with h275y mutation was 18 days (range 3-36 days). three patients were part of a hospital cluster of oseltamivir-resistant virus infections and were infected with h275y mutants prior to oseltamivir treatment. 6 patients with immunocompromising conditions accounted for almost half of all patient specimens tested, but they accounted for the majority of oseltamivir-resistant ph1n1 virus infections (table 1) ; among 32 individuals with severe immunocompromising conditions and clinical failure while on oseltamivir therapy, 23 (72%) had the h275y mutant detected. among the immunosuppressed patients with an oseltamivir-resistant virus, 16 (70%) had hematologic malignancies reported. in contrast, among the subset of icu patients without immunocompromising conditions and clinical failure while on oseltamivir therapy, we found little resistance: 2 (5ae9%) of 34 icu patients had oseltamivir resistance detected. during the 2009 pandemic, we were able to provide timely and useful information to clinicians regarding suspected cases of oseltamivir resistance. our testing algorithm limited the number of specimens to specimens from the highest risk patients that would benefit the most from antiviral treatment. such an approach allowed us to offer this service without compromising our public health duties. in addition, the information we collected on patients from this service complimented our data on the national surveillance for antiviral resistance. we also performed national antiviral resistance surveillance from april 2009 to july 2010. overall, 67 resistant ph1n1 viruses were identified from april 2009 to july 2010 in the united states among 6812 tested samples, including specimens described above, surveillance specimens, and resistant viruses reported in the literature. 7 further studies to understand risk factors for oseltamivir-resistant ph1n1 infection in patients with severe immunocompromising conditions are needed. while efforts to provide antiviral testing technology and materials to state laboratories are ongoing, clinicians still have limited options for such testing. rapid and inexpensive assays that could be performed by clinical laboratories, especially those caring for immunosuppressed patients, would be useful to inform patient care. the applied biosystems ò na-xtd tm influenza neuraminidase assay kit provides the next-generation na-xtd tm 1,2-dioxetane chemiluminescent neuraminidase (na) substrate, together with all necessary assay reagents and microplates, to quantitate sensitivity of influenza virus isolates to neuraminidase inhibitors. like the na-star ò influenza neuraminidase inhibitor resistance detection kit, the na-xtd tm influenza neuraminidase assay provides highly sensitive detection of influenza neuraminidase activity. in addition, the na-xtd tm assay provides extended-glow light emission that eliminates the need for reagent injection and enables signal measurement either immediately or up to several hours after assay completion. the na-xtd tm assay is also used to quantitate influenza na activity directly in cellbased virus cultures to monitor viral growth or inhibition. global monitoring of influenza strains for resistance to neuraminidase inhibitors (nis) is essential for understanding their efficacy for seasonal, pandemic, or avian influenza, and studying the epidemiology of viral strains and resistance mutations. functional neuraminidase inhibition assays enable detection of any resistance mutation, making them extremely important for global monitoring of virus sensitivity to nis. the first-generation chemiluminescent na-star ò influenza neuraminidase inhibitor resistance detection kit has been widely used for virus ni sensitivity assays, 1-8 including identification of a ⁄ h1n1 pandemic virus resistant to oseltamivir. 9, 10 in addition, this assay has been used for identification of new ni compounds, 11 ni characterization, 12 studies of virus transmission, 13 drug delivery, 14 na quantitation of virus-like particles, 15 and cell-based virus quantitation. 16 neuraminidase assays performed with chemiluminescent 1,2-dioxetane substrates, including na-star ò and na-xtd tm substrates, typically provide 5-to-50-fold higher sensitivity by signal-to-noise ratio than assays performed with the fluorescent munana substrate. in addition, chemiluminescent assays provide linear results over 3-4 order of magnitude of neuraminidase concentration compared to 2-3 orders of magnitude with the fluorescent assay. 2 the high assay sensitivity achieved with chemiluminescent assays enables use of lower concentrations of viral stocks, and the wide assay range minimizes the need to pre-titer virus stocks prior to ic 50 determination. chemiluminescent reactions result in conversion of chemical energy to light energy, as light emission. the na-xtd tm substrate is a 1,2-dioxetane structure bearing a sialic acid cleavable group. to perform the na-xtd assay, virus dilutions (from cell culture supernatant) are pre-incubated in the presence of neuraminidase inhibitor. then na-xtd substrate is added and incubated for 30 minutes for substrate cleavage to proceed. finally, light emission is triggered upon addition of na-xtd accelerator, which provides a ph shift and a proprietary polymeric enhancer, both required for efficient light emission. chemiluminescent assays are performed in solid white microplates, and light emission is measured in a luminometer. the na-xtd tm substrate has a single structural difference from the na-star ò substrate that provides a much longer-lasting chemiluminescent signal, with a signal half-life of approximately 2 hours (not shown), compared to $10 minutes with the na-star assay, eliminating the need for luminometer instruments equipped with reagent injectors and enabling more convenient batch-mode processing of assay plates. the na-xtd tm assay kit also provides a new accelerator solution, containing a next-generation polymer enhancer, and a triton ò x-100-containing sample prep buffer providing enhanced na activity. read-time flexibility is demonstrated by determination of oseltamivir ic 50 values using data collected over 3 hours after addition of na-xtd tm accelerator. although signal intensity slowly decreases over time, the ic 50 curves and values are identical at each time point, shown using influenza b ⁄ lee ⁄ 40 ( figure 1) . triton x-100 detergent at 1% has been shown to inactivate flu virus while increasing neuraminidase activity. 17 the addition of na sample prep buffer (containing 10% triton x-100) to virus stocks (at 1 ⁄ 10 volume, achieving a final concentration of 1%) provides increase in na activity up to fourfold, but is not consistently observed, and seems to be most effective with more concentrated virus stocks. ic 50 values are unaffected by the addition of triton x-100 to the virus stock prior to virus dilution (not shown), so the assay is compatible with known virus inactivation reagents. assay sensitivity and ic 50 values determined with the na-xtd assay have been compared to those obtained with both the chemiluminescent na-star assay and the fluorescent na-fluor assay (not shown). the chemiluminescent assays provide 2-to 50-fold higher sensitivity by signal-to-noise ratio, depending on the virus strain, wider assay dynamic range, and better low-end detection limit than the fluorescent assay. the wide assay range with the chemiluminescent assays enables determination of ic 50 values over a range of virus concentrations, eliminating the need to titer virus prior to performing ic 50 determination assays. ic 50 values obtained with the na-xtd assay are nearly identical to those obtained with the na-star assay, with both oseltamivir and zanamivir neuraminidase inhibitors, and tend to be slightly lower than ic 50 values obtained with the fluorescent assay. viral na quantitation provides a convenient read-out to measure viral growth or inhibition, including inhibition in the presence of inhibitory compounds or antibodies, described as accelerated viral inhibition with na as readout assay (avina). 16 bation in the presence of varying concentrations of oseltamivir carboxylate. samples of culture media were assayed 24 hours later. quantitation of na activity with the na-xtd tm assay demonstrates inhibition of viral growth by oseltamivir carboxylate in cell culture ( figure 2 ). different volumes of culture media were assayed with the na-xtd assay, either in the culture plate or in a separate assay plate (not shown). performing the assay using the entire well contents (100 ll) reduces assay sensitivity due to the high concentration of phenol red. assaying a smaller volume of culture medium (either in culture plate or a separate assay plate) provides higher sensitivity, and enables temporal monitoring or use of remaining culture medium for other assays. the applied biosystems ò na-xtd tm influenza neuraminidase assay kit is a next-generation chemiluminescent neuraminidase assay providing high assay sensitivity and ''glow'' light emission kinetics for improved ease-ofuse. the applied biosystems ò na-fluor tm influenza neuraminidase assay kit, based on the fluorescent mun-ana substrate, has also been developed to complement the na-xtd tm and na-star ò chemiluminescence assays, for users lacking luminometer instrumentation or choosing to use fluorescence assay detection. together these kits offer: • standardized reagents and protocols • choice of detection technology • simple instrumentation requirements • high sensitivity for use with low virus concentrations • compatibility with batch-mode processing and largescale assay throughput • broad specificity of influenza detection • flexibility in assay format • additional na assay applications -cell-based viral assays, screening for new nis, detection of na from other organisms functional neuraminidase inhibition assays enable detection of any resistance mutation and are extremely important in conjunction with sequence-based screening assays for global monitoring of virus isolates for ni resistance mutations, including known and new mutations. together, these assays provide highly sensitive, convenient and versatile assay systems with standardized assay reagents, and simple assay protocols for influenza researchers. 15 over 200 000 hospitalizations and 36 000 deaths in the us annually are attributable to seasonal influenza, primarily in chronically ill persons and the elderly. 1-3 following the emergence of pandemic 2009 h1n1 influenza, severe illnesses have also been observed in children and young healthy adults. 4 the occurrences of staphylococcal and pneumococcal pneumonia complicating influenza pandemics are well described. [5] [6] [7] although temporal associations of bacterial pneumonia and influenza circulation have been reported, there is little precise data on rates of bacterial complications of seasonal or pandemic influenza. the study of bacterial lung infection has been hampered by insensitive tests for invasive disease and the difficulty of interpreting routinely obtained sputum culture results. 8, 9 procalcitonin (proct), the prohormone of calcitonin, can discriminate viral and bacterial infections. 10 this 116-aminoacid precursor protein normally produced by neuroendocrine cells of the lungs and thyroid gland was first shown to be elevated in bacterial infections in patients with pulmonary injury and pneumonitis. 11 stimuli of proct include tnf-a, endotoxin, and other bacterial products. 12 several studies indicate that bacterial infections commonly induce hyperprocalcitonemia, but that viral infections, including 2009 h1n1, are associated with only minimal increases. 10, 12, 13 of note, proct induction is attenuated by viral-induced interferon-c. 14 a meta-analysis of studies comparing proct and crp as markers for bacterial infection found that proct was more sensitive and specific than crp for differentiating bacterial from other causes of inflammation. 15, 16 therefore, we measured proct levels in patients with seasonal and pandemic influenza and compared results with conventional methods for bacterial diagnosis. adults ‡21 years of age admitted to rochester general hospital (rgh) from november 1st to june 30th for two winter seasons (2008-2010) with an admitting diagnosis compatible with acute respiratory tract infection were recruited for the study. patients were screened within 24 hours of admission, and those with prior antibiotic use, immunosupression, or pregnancy were excluded. subjects or their legal guardian provided written informed consent. the study was approved by the university of rochester and rgh research subjects review board. at enrollment demographic, clinical and laboratory information was collected. influenza testing included nosethroat swabs (nts) for rapid antigen, viral culture, and reverse transcription-polymerase chain reaction (rt-pcr) and serology. testing for bacterial pathogens included blood cultures, sputum for culture and gram stain, nts for mycoplasma pneumoniae and chlamydophila pneumoniae pcr, s. pneumoniae antigen testing, and pneumococcal serology. if patients were unable to expectorate, sputum was induced with normal saline and bronchodilators. specimens were considered adequate by the standard criteria of >25 neutrophils (pmns) and <10 epithelial cells per high power field. serum was collected at admission and hospital day 2 for proct measurements. influenza infection was defined a positive result for any of the following tests: . cloned proteins were coated on eia plates at 2 ug ⁄ ml in bicarbonate buffer. after overnight incubation, plates were washed and two-fold dilutions of serum were incubated overnight at room temperature. plates were washed and incubated with alkaline phosphatase conjugate for 3 hours, followed by substrate. a greater than or equal to fourfold rise in titer was considered evidence of infection with s. pneumoniae. urinary antigen for s. pneumoniae samples were assayed for antigen using the binax now kit. (binax inc, scarborough, me, usa). the proct was measured using time resolved amplified cryptate emission technology (kryptor pct; brahms, henningsdorf, germany). functional sensitivity is 0ae06 ng ⁄ ml (normal levels are 0ae033 ± 0ae003 ng ⁄ ml). 18 mycoplasma and chlamydia pcr real-time pcr targeting the p1 adhesion gene for m. pneumoniae and the ompa gene for c. pneumoniae was used to detect atypical bacteria. 19 results fifty-one of 529 (9ae6%) illnesses evaluated tested positive for influenza virus. of these, 20 were due to ''seasonal influenza'' (16 influenza a ⁄ h3n2 and 4 influenza b), and 31 were identified as ''pandemic influenza'' (2009 h1n1). demographics of both groups were similar: mean ages 55 ± 18 and 50 ± 11 years, respectively, and equivalent sex and racial characteristics. other than a higher incidence of underlying lung disease in the seasonal group (75% versus 45%, p = 0ae05), pre-existing medical conditions including obesity were similar. symptoms, physical findings, and discharge diagnoses did not differ, and chest radiographs (cxr) showed infiltrates in 20% and 13% of seasonal and pandemic subjects, respectively. two pandemic and one seasonal influenza patient developed respiratory failure, and none died. overall, bacterial infections were diagnosed in 8 (16%) subjects (4-seasonal and 4-pandemic), and none were bacteremic. bacterial infections included: 5-s. pneumoniae, 1-m. pneumoniae, 1-s. aureus, and 1-h. influenzae. all seasonal patients were diagnosed with asthma or bronchitis, whereas three pandemic patients had pneumonia. mean serum proct (ng ⁄ ml) levels in seasonal versus pandemic patients on admission and day 2 were: 0ae14 ± 0ae17 versus 1ae05 ± 2ae86 and 0ae17 ± 0ae17 versus 1ae39 ± 4ae37, respectively, and were not significantly different (table 1) . several patients in the pandemic group had high proct levels, and there was a trend toward more pandemic patients having admission proct values ‡0ae5 ng ⁄ ml than seasonal subjects [1 (5%) versus 6 (19%), p = 0ae22] ( figure 1a , b). of the four patients with proc-t > 1ae5 ng ⁄ ml, two had dense infiltrates on cxr, one had a peripheral wbc of 17 200 ⁄ ml with a threefold increase in s. pneumoniae antibody, and one developed respiratory failure associated with copd exacerbation. reliable sputum samples (within 6 hours of antibiotics) were collected in only 22 (43%) subjects. of these, proct was ‡0ae5 ng ⁄ ml in two with influenza alone and three associated with bacterial infection, and <0ae5 ng ⁄ ml in 12 with influenza alone and five associated with bacterial infection. in the 22 with reliable sputa and accepting the conventional bacterial diagnosis, sensitivity of a proc-t ‡ 0ae5 ng ⁄ ml for bacterial infection was 38%, specificity 86%, positive predictive value 60%, and negative predictive value 71%. notably, one patient considered to have influenza alone (proct -0ae87 ng ⁄ ml) had group a streptococcus and s. aureus in a contaminated sputum and bilateral infiltrates on cxr. three of five patients with bacterial infections and proct < 0ae25 ng ⁄ ml had a clinical diagnosis of bronchitis. mean proct values were significantly higher in patients with infiltrates versus those with atelectasis or no acute disease on cxr (2ae4 ± 4ae8 ng ⁄ ml versus 0ae36 ± 1ae2 ng ⁄ ml, p = 0ae02). combining patients with proct values ‡0ae5 ng ⁄ ml with those having positive bacterial tests, rates of bacterial infection associated with seasonal and pandemic influenza were 20% and 26%, respectively. notably, antibiotics were administered to 88% of subjects despite 73% having no acute disease on cxr. in our study, bacterial infections were diagnosed in approximately 25% of adults hospitalized with influenza with no significant difference in rates noted between seasonal and pandemic influenza infected subjects. previous reports of bacterial infection rates of 4-15% with seasonal influenza are difficult to compare with recent studies of pandemic influenza, because the latter tended to focus on more severely ill patients. [20] [21] [22] bacterial pneumonia has been suspected or diagnosed in 13-24% of patients in intensive care associated with 2009 h1n1 infection and up to 38% of patients who died. 23, 24 despite aggressive pursuit of specimens for bacterial testing, diagnoses could be confirmed in only 8 (15ae6%) of patients using conventional methodology. given the difficulty in establishing a diagnosis of bacterial infection, elevated proct values may be helpful to identify patients at high risk for invasive disease. in a study of 25 patients with severe 2009 h1n1 or bacterial infection necessitating intensive care, a threshold proct level of 0ae8 ng ⁄ ml, demonstrated 100% sensitivity and 62% specificity for bacterial infection. 25 among 38 patients with 2009 h1n1 associated pneumonia, many of whom had respiratory failure, a threshold proct value of 0ae5 ng ⁄ ml provided a sensitivity of 100% and specificity of 53% for bacterial infection. 26 access to samples from lower airways in ventilated patients in these studies may have improved recovery of bacteria and account for the different results we observed. it should be noted that none of our patients were bacteremic, which is a very strong stimulus for proct release. proct levels have been used successfully to guide therapy in community acquired pneumonia, and our data showing high proct levels in patients with infiltrates on cxr suggests proct may be most useful for excluding invasive disease. 27, 28 elevated proct levels were not observed in patients with purulent sputum and clear cxr. it is notable that a proct level of <0ae25 ng ⁄ ml did not exclude patients with bacterial bronchitis since proct has been used to guide antibiotic therapy in copd exacerbations. 29 while it could be argued that healthy patients with bacterial bronchitis do not require antibiotic treatment, physician behavior in our study indicates antibiotics are frequently prescribed. combining patients with proct values ‡0ae5 ng ⁄ ml and those with a positive bacterial test, approximately 25% in patients in our study had bacterial complications associated with influenza infection. efforts should be made to curtail antibiotic use in hemodynamically stable patients with clear cxrs. given physician discomfort regarding discontinuing antibiotics, proct measurements in combination with routine bacterial cultures should be useful tools to guide therapy. influenza, mrsa, cytokines: diagnosis, treatment, prevention -a possible strategy for outpatient care we started the antiviral treatment of influenza in humans using neuraminidase inhibitors on january 29, 1999 in a successful attempt to cure a 96-year-old patient. since then, we have used the inhalant antiviral drug zanamivir, and later (october 1, 2002) changed to the use of oseltamivir with systemic bioavailability for treating patients with influenza. after 10 years of experience with antiviral treatment of outpatients, we highlight the importance of early diagnosis and early treatment. 1 the necessity of an earliest possible diagnosis was confirmed in the pandemic of 2009. large hospitals reported that patients with an h1n1 ⁄ 09 infection had to be treated with extracorporeal membrane oxygenation. we are convinced this is due to delayed recognition of infection in most cases. valuable time is lost when the patient with a sudden onset has to be brought to a hospital for emergency treatment. the point at which the patient goes to the doctor is decisive, and this problem of timing and the delivery of early treatment is not specific to germany. in our medical office, we assessed patients with suspected influenza (to date 262 seasonal infections, and in 2009, 25 h1n1 ⁄ 09) through clinical diagnosis, 2 and then proven by point of care rapid test (quickvue; quidel, san diego, ca, usa) followed by pcr. all of the patients undergo concomitant lab tests: leukopenia, serum iron level, and the humoral inflammation status [sum of the c-reactive protein (crp) and fibrinogen levels]. because of the constant threat of a bacterial superinfection, a bacterial swab and antibiogram is carried out on every patient. in all cases positive for influenza, oseltamivir was given immediately. nowadays it is important that a double infection with influenza and mrsa must be recognized immediately and treatment started at once with antivirals and, when appropriate, with a suitable antibiotic. we pay particular attention to an extremely low iron level (signum mali ominis). in addition we monitor oxygen saturation and the course of the humoral inflammation status every 1-2 hours for every of our outpatients. among our patients with seasonal influenza, we saw 132 within 12 hours, 103 within 36 hours, and 27 within 48 hours after disease onset. for pandemic influenza, it was 12 patients within 12 hours, 11 within 24 hours, and two within 36 hours. for all patients, we measured crp < 3ae5 mg and fibrinogen < 350 mg ⁄ dl (12 hours), crp < 10 mg and fibrinogen < 450 mg ⁄ dl (36 hours), and crp > 10 mg and fibrinogen < 500 mg ⁄ dl (48 hours, only seasonal cases). antibiotics were necessary in 130 cases, heparin and oxygen administration in 27 cases. one hundred forty-eight patients had a superinfection following influenza. the most common strains were haemophilus parainfluenzae and staphylococcus aureus. the subsequent use of a suitable antibiotic was only necessary in 50% of the patients. in all cases diagnosed, treatment (including heparin and oxygen administration) and monitoring were conducted in our medical office. none of our patients (seasonal and pandemic) had to be admitted to hospital. the early decision of whether or not antiviral and antibacterial treatment is taking effect is the only way the threat of a cytokine storm can be averted. not only does the primary care physician have to be aware of the pathophysiology involved, but also the necessary diagnostic and therapeutic options have to be made available to him. the result will lead to a saving of both lives and healthcare costs. this applies both in epidemic as well as in pandemic times. today we know that influenza leaves behind a defenceless immune system, and that the proteases of s. aureus contribute to influenza associated pneumonia. mark von itzstein, who discovered neuraminidase inhibitors, emphasized the synergistic cooperation of viruses and bacteria (personal communication, 2009). mrsa and influenza viruses are posing problems worldwide. the case of a 17-year-old boy with h1n1 ⁄ 09 infection demonstrates how fatal developments can be prevented. due to his constantly recurring colds, we had already detected the mrsa colonization years earlier and had always worked on boosting his general health and resistance. both the patient and his family were included in dealing with the problem. the patient was, and is, always vaccinated early with a virosomal vaccine (baxter). during the oktoberfest in munich in september 2009, when h1n1 infections were increasingly occurring, we learned that our patient had come down with an extremely acute feverish illness. with the help of the rapid test, we diagnosed an h1n1 ⁄ 09 virus infection and started treatment with oseltamivir immediately. the humoral inflammation status, which had increased very rapidly to more than 4ae5 mg ⁄ dl within hours, was treated with the effective cotrimoxazol from the antibiogram. at the same time, the patient was heparinized. the following day the patient had no fever and was symptom-free. it was only through our early knowledge of what could develop pathophysiologically that we were in a position to make the right decision at the right time. every doctor treating outpatients can follow this procedure if he is familiar with the pathophysiology of the disease and has the available tests on hand: virus rapid test, additional laboratory parameters (leukopenia, iron), and the humoral inflammation status. the decisive factor, however, is the constant clinical alertness towards the course of every acute feverish cold with acute onset. the patient has to remain in the care of the attending physician, and the chosen treatment has to be administered and monitored. this means constant spo2 measurements and checking the humoral inflammation status every 2 hours. if a clinical worsening occurs during monitoring, the treatment regime has to be changed immediately, which means the administration of an appropriate antibiotic. this outpatient care on the part of the doctor has to be available 7 days a week so that no time will be lost. reports from the netherlands and denmark show that, with the help of this preventive strategy under the motto 'search and destroy,' the dangerous, fatal course of infections reported in germany with at least four deaths a day, can be avoided. however, the doctor has to be adequately remunerated for the elaborate amount of time this intensive outpatient care requires. with our strategy, we have moved from divergence to convergence in the care of our patients. we reported on our 10 years of clinical experience with this approach at the antivirals congress in peking. our main message was early diagnosis and early treatment. we were able to demonstrate this in 287 outpatients with seasonal influenza and 25 h1n1 ⁄ 09 outpatients. our creed is: as much outpatient care as possible and as little hospitalization as possible. virological and autopsy findings in suspected and confirmed fatal cases of 2009 h1n1 pandemic influenza in the czech republic -preliminary results influenza viruses cause substantial morbidity and mortality. pandemic influenza may have a serious impact on certain (mainly younger) age groups in comparison with seasonal flu. influenza is one of few viral infections capable of causing a pneumonia that is difficult to cure and ⁄ or leads to sudden death. the aim of this study was to analyze and compare virological and autopsy findings in patients who died with suspected or confirmed 2009 h1n1 pandemic influenza virus infection. there were 2477 virologically confirmed cases of pandemic influenza and 102 deaths in the czech republic during pandemic wave. more than 400 influenza strains belonging to the new pandemic variant were isolated in the national influenza reference laboratory. postmortem biological samples were collected from any patient who died with suspected influenza infection to test for respiratory viruses. the samples were screened for 2009 h1n1 pandemic influenza virus by real-time pcr (rt pcr), and when rt pcr positive, by virus isolation assay. no immunohistochemical staining for influenza antigen was done on the rna pcr positive cases. other important respiratory viruses such as respiratory syncytial virus, parainfluenza viruses, and adenoviruses were detected by virus isolation assay in a suitable cell culture. epidemiological analysis of postmortem histopathologic findings in the airway tissue was carried out in 57 of 61 fatal cases. virological findings were subsequently correlated with histological changes and available demographic and clinical data. statistical analysis was performed by t-test using spss software. sixty-one deaths (34 males, 27 females) were analyzed. the rna of the 2009 h1n1 pandemic influenza virus was detected by pcr in 38 cases, while 23 cases remained negative. five respiratory syncytial viruses and two adenoviruses were detected in the influenza negative group. the mean age of 38 confirmed 2009 h1n1 pandemic influenza victims was 46ae0 years, age range 7-74 years and median 47ae5 years. the mean age of 23 influenza negative victims was 55ae1 years, age range 1-89 years and median 57ae0 years. the 95% ci for the difference in the age between the two groups is )1ae7; 19ae9. the test is statistically significant at the 10% level. the obtained significance (p = 0ae07) can be explained by the relatively small size of the study group. the most common postmortem histopathologic finding in the lung tissue of the 2009 h1n1 pandemic influenza virus-positive victims was diffuse alveolar damage (often bilateral) and ⁄ or hyaline membrane formation, possibly with signs of respiratory distress syndrome (in 18, i.e., 51ae4%, of 35 autopsied patients). in the 2009 h1n1 pandemic influenza virus negatives, the most common finding was pneumonia or bronchopneumonia with the detection of various bacterial species (in 12, i.e., 54ae5% of 22 autopsied patients). the cause might be either primary bacterial infection or superinfection following primary infection with influenza virus that remained undetected. the 2009 h1n1 pandemic influenza victims were younger than the patients who died with suspected but undetected 2009 h1n1 pandemic influenza. the majority of deaths were primarily linked to rapidly developing respiratory failure. this result supports the previous reports of severe respiratory outcomes in younger age groups that are typically linked to the spread of a pandemic strain of influenza. 1 due to limited amount of pandemic vaccine, especially at the beginning of pandemic, it is advisable to assess experiences with antiviral treatment, mainly dosing, and way of antiviral administration. primers specific for each of the eight genes of pandemic h1n1 ⁄ 2009 were adopted from assays as described previously to discriminate against seasonal human h1n1 and h3n2 viral segments (table 1) . 9 the primers were allowed to cross-react specifically with the sister clade viral segments of pandemic h1n1 ⁄ 2009. 2 the method we employed in this study was a 2-step singleplex sybr green-based real-time rt-pcr. this approach helped lower the running cost of the assays and facilitated downstream molecular analyses (e.g., sequencing) by using screened cdna samples. viral rna was extracted from viral cultures or clinical samples as described 3, 10 and was converted to cdna in a universal rt-pcr. each 10 ll rt reaction containing 5ae5 ll of purified rna, 2 ll of 5· firststrand buffer (invitrogen), 100 u of superscript ii reverse transcriptase (invitrogen), 0ae1 lg of uni12 (5¢-ag-caaaagcagg-3¢), 11 0ae5 mm of deoxynucleoside triphosphates and 10 mm of dithiothreitol was incubated at 42°c for 50 minutes, followed by 72°c for 15 minutes for heat inactivation. for each segment-specific real-time pcr, the 20 ll reaction contained 1 ll of a 10-fold diluted cdna samples, 10 ll of fast sybr green master mix (applied biosystems), and 0ae5 lm of the corresponding primer pair. the thermocycling conditions of all eight segment-specific pcrs were optimized as 95°c for 20 seconds, followed by 30 cycles of 95°c for 3 seconds and 62°c for 30 seconds, and all eight assays were performed simultaneously in a 7500 sequence detection system (applied biosystems). at the end of the amplification step, pcr products went through a melting curve analysis to determine the specificity of the assay (60-95°c; temperature increment: 0ae1°c ⁄ seconds). cdna of a ⁄ california ⁄ 04 ⁄ 2009 virus was used as a positive control. robust and specific amplification was achieved in all eight segment-specific real-time rt-pcr reactions. 9 pcr product for each segment of pandemic h1n1 ⁄ 2009 yielded unique melting curve pattern with distinctive melting temperature (tm), which was not observed in negative and water controls ( figure 1 ). reactions with tm value within 2 sds of the mean tm were determined as positive. we evaluated the assays with a number of serologically confirmed human clinical samples. all pandemic h1n1 ⁄ 2009 samples (n = 67) were positive in all eight assays, while all seasonal samples (h1n1 = 38; h3n2 = 66) were negative in all assays, as expected ( figure 1 and data not shown). these results showed that no reassortant of pandemic h1n1 and seasonal viruses was present in the tested human isolates. we applied these assays to our on-going influenza virus surveillance program in swine. nasal and tracheal swab samples were collected at an abattoir in hong kong and cultured in madin darby canine kidney cells or embryonated eggs as described. 12 positive viral cultures in hemagglutination assays were tested with the established segmentspecific real-time rt-pcr assays. among 59 swine viral isolates collected from 2009 to september 2010, 10 of them were recognized as pandemic h1n1 ⁄ 2009 in all eight segments. they were confirmed to be of pandemic h1n1 ⁄ 2009 origin by subsequent full genome sequencing analyses, showing that there were interspecies transmissions of the virus from humans to pigs. 13, 14 the remaining 49 viruses had one to seven gene segments positive in the segment-specific real-time rt-pcrs. thirty of them were selected as representative samples for full genome sequencing analyses based on the genotyping data generated in our assays. they were swine h1n1 or h1n2 viruses with their gene segments derived from tr or eurasian avian-like swine lineages. it should be highlighted that all of their positive gene segments in our assays belonged to the sister groups of pandemic h1n1 ⁄ 2009. their melting curve patterns were very similar to those derived from segments of pandemic h1n1 ⁄ 2009, except for ha of tr lineage. 9 our results successfully demonstrated the use of these segment-specific real-time rt-pcrs to recognize gene segments of contemporary tr (pb2, pb1, pa, ha, np, and ns) and ea (na and m) swine viruses. 2 the ha-specific assay was able to discriminate pandemic h1n1 ⁄ 2009 from other contemporary swine viruses in the same lineage. nevertheless, to confirm the identity and to examine all the genetic variations in the viruses of interest, full genome sequencing analyses were necessary. in this study, the biggest obstacles in primer design were sequence similarity and diversity of influenza viruses. we attempted to use degenerated primers, but they were highly non-specific. the finalized non-degenerated primers crossreacted with genes from pandemic h1n1 ⁄ 2009 and its sister clade tr (pb2, pb1, pa, ha, np, and ns) and ea (na and m) swine viruses with some minor sequence mismatches. three avian (h5n1, h7n7, and h9n2) and 1 classical swine (h1n1) were also tested with our assays. all of these animal viruses were negative, except for ns gene of the classical swine virus. our segment-specific real-time rt-pcr assays might be used in high throughput genotyping. they detected pandemic h1n1 ⁄ 2009 viruses and acted as a preliminary screen-ing tool to select virus reassortants of interesting genotypes for further sequencing analyses. in fact, we identified a novel reassortant in january 2010 during the course of this study. this sw ⁄ hk ⁄ 201 ⁄ 2010 has a previously unidentified viral gene combination as shown in figure 1 . it was confirmed to be a reassortant between pandemic h1n1 ⁄ 2009 and other swine viruses in full genome sequencing characterization. it has a pandemic h1n1-like n1 gene, an ea-like h1, and the other six internal genes derived from tr swine viruses. 13, 14 the eight established real-time rt-pcrs can rapidly reveal the gene-origins of influenza viruses. we are currently using these assays in influenza surveillance in humans and other animals. it is believed that similar strategy might be applied to detect and genotype other influenza viruses and possible reassortants in the future. pandemic influenza a ⁄ h1n1 ⁄ 2009 infects millions of people around the world. a significant fraction of the world's population may also already have been exposed to the virus and, although asymptomatic, may be at least partially immune to the disease. a precise assessment of the number of people exposed to the influenza a ⁄ h1n1 ⁄ 2009 virus is epidemiologically relevant. however, assays typically used to estimate antibody titers against a particular influenza strain, namely hi and neutralization, require use of the actual virus. this seriously limits broad implementation, particularly in regions where high biosafety facilities are unavailable. we developed an elisa method for the evaluation of presence of specific 2009 h1n1 influenza virus-antibodies in serum samples. mouse anti-histidine tagged antibodies (100 ll; 5 lg ⁄ ml; abd serotec ò , uk) in pbs (ph 7ae2) were dispensed into standard 96-well plates and incubated for 12-16 hour at room temperature. excess antibody was removed by at least two successive alternate washings with pbs-tween 0ae05% and pbs. commercial blocking solution (300 ll, superblock ò t20; pierce ò , usa) was added and incubated for at least 2 hour at room temperature. after successive washing steps with pbs-tween 0ae05%, non-glycosylated histidine-tagged recombinant protein (100 ll; 10 lg ⁄ ml) was added to each well. this protein consisted of the receptor-binding domain of the hemagglutinin of the influenza a ⁄ h1n1 virus. 1,2 after 1 hour incubation, wells were washed for at least two alternating 5 minutes cycles with pbs-tween and pbs. a 1:50 dilution of the serum or plasma sample to be assayed (100 ll) was added to each well and incubated at room temperature for 1 hour. after repeated alternating 5 minutes pbs-tween 0ae05% and pbs washes, anti-human igg antibody solution (100 ll ⁄ well; 1:30 000 dilution in pbs-tween 0ae05%) marked with horse radish peroxidase (pierce ò , usa) was added and incubated for 1 hour at room temperature. after repeated alternate washes with pbs-tween 0ae05% and pbs), substrate solution (100 ll; 1-step ultra tmb-elisa; pierce ò ) was added to each well. after incubation for 15 minutes at room temperature in darkness, the enzymatic reaction was stopped by addition of 1 m h 2 so 4 (50 ll ⁄ well). yellow color produced by the enzymatic reaction was evaluated by absorbance at 450 nm in a biotek ò microplate reader (usa). blank assays using albumin in place of human sera established the elisa background signal, which was subtracted from sample absorbance signals: abs serum sample ¼ abs serum sample before correction à abs albumin sample : absorbance values were normalized based on the average signal of 103 non-exposed subjects (uninfected subjects), and expressed as normalized absorbance (abs norm ): where abs serum ample is the sample absorbance signal, abs albumin sample is the albumin control absorbance signal, abs non exposed subjects is the average absorbance signal of non-exposed subject samples. for ferret serum samples, the same basic protocol was followed, with minor modifications. an anti-igg anti-ferret polyclonal antibody preparation was used at a dilution of 1:30 000 in pbs-tween 0ae05%. 2 a recombinant receptor-binding domain of the ha of the influenza a ⁄ h1n1 ⁄ 2009 virus, expressed in escherichia coli strains, 2 was used as the elisa antigen. this 27 kda protein, designated here as ha 63-286 -rbd, contained amino acids 63-286 of the influenza a ⁄ mexico ⁄ indre4114 ⁄ 2009(h1n1) hemagglutinin. a sequence coding for a series of six histidines at the n-terminus of the protein was included in the genetic construct to allow purification using immobilized metal affinity chromatography (imac) and attachment to assay surfaces treated with anti-histidine antibodies (or alternatively co +2 or ni +2 ). a panel of four samples (kindly provided by st. jude from ferrets exposed to different influenza strains, namely h3n2, h1n1 swine, and h5n1, was also tested by the elisa method using 1:50 dilutions. protein ha 63-286 -rbd specifically and selectively recognizes antibodies from serum samples from convalescent h1n1 ⁄ 2009 influenza subjects. dubois et al. 4 demonstrated that this protein, produced in e. coli, folds properly into a 3-d structure practically indistinguishable from the analogous region in the ha of the influenza a ⁄ h1n1 ⁄ 2009 virus. ha 63-286 -rbd preserves three of the conformational immunogenic epitopes (sa, sb, and cb) described for influenza a ⁄ h1n1 hemagglutinins. 5 the recombinant protein was used as the antigen, attached through histidine tags to microplate surfaces treated with anti-histidine antibodies to discriminate between serum samples from subjects exposed and non-exposed to influenza a ⁄ h1n1 ⁄ 2009. samples collected before the pandemic onset, and therefore presumed to exhibit low specific antibody titers against influenza a ⁄ h1n1 ⁄ 2009, were analyzed by elisa using the antigen ha 63-286 -rbd. the histogram of normalized absorbance values from this sample set displayed a normal behavior with a standard deviation of 0ae57 units. only 12ae5, 9ae7, and 3ae88% of these samples exhibited normalized absorbance values higher than 1ae5, 1ae75, and 2ae0, respectively. no sample from non-exposed individuals presented an absorbance value higher than 2ae5. variability among samples from non-exposed subjects was much lower than in samples with high specific serum antibody titers from convalescent h1n1 ⁄ 2009 patients. exposure to the h1n1 ⁄ 2009 influenza virus with this elisa method can be predicted by absorbance values normalized to those of abs norm ¼ ðabs serum ample à abs albumin sample þ=ðabs non exposed subjects à abs albumin sample þ ð1þ serum from uninfected subjects. consequently, for reliable results, inclusion of samples from non-exposed subjects on every assay microplate is necessary. figure 1 shows the analysis of 20 human serum samples, including 17 samples from convalescent patients with positive diagnosis by rt-pcr. three positive (dark gray bars) and two negative controls (light gray bars) were included in the same microplate. all serum samples corresponding to convalescent subjects exhibited absorbance values 1ae75-4ae5 times higher than negative samples ( figure 1 ). normalized absorbance values above 1ae5 suggested exposure to the virus, although, a more conservative threshold value of 1ae75 units is proposed for discrimination between exposed and non-exposed subjects. the elisa method described here yields adequate reproducibility and a high signal ⁄ noise ratio within determinations in the same microplate and among different microplates. using a normalized absorbance value of 1ae75, the method was able to discriminate samples from convalescent patients, preferably after the third week of infection, and at least up to the twentyfourth week of exposure. assay sensibility was further validated against results from hi assays. a previously reported study showed that all members in a pool of fourteen samples diagnosed as positive by hi exhibited normalized absorbance values higher than 1ae5, and 85% of them exhibited normalized absorbance values higher than 2ae0. 1 in general, high hi titers (>320) were correlated with normalized absorbance values higher than 4ae0. figure 2a shows results using the ha-rbd elisa method and the hi assay on a pool of seventeen known positive serum samples corresponding to convalescent h1n1 ⁄ 2009 patients. all samples determined as positive by hi (10 samples) were also positive by elisa. while sensitivity of the hi assay was 10 ⁄ 17 = 58ae88%, the elisa method recognized all samples correctly as positive (100% sensitivity) when a threshold of 1ae5 or 1ae75 was used. figure 2b shows that sera from ferrets infected with other influenza strains (h3n2, h1n1 swine, and h5n1) showed no cross-reactivity when analyzed by elisa. in summary, the ha-rbd elisa method presented here consistently distinguished influenza a ⁄ h1n1 ⁄ 2009 infected and non-infected individuals, particularly after the third week of infection ⁄ exposure. since no actual viral particles are required, this assay can be readily implemented in any basic laboratory. in addition, should sufficient vaccine be unavailable, this elisa could determine the level of specific antibodies against the virus and presumably the extent of partial protection in a subject. therefore, the elisa protocol might allow better administration of vaccination programs during pandemic or seasonal influenza outbreaks. in april 2009, a novel h1n1 influenza virus emerged in north america and caused the first influenza pandemic of the 21st century. [1] [2] [3] [4] the 2009 pandemic h1n1 (pdmh1n1) has a unique gene constellation that was not previously identified in any species or elsewhere. it is genetically related to the triple reassortant swine h1n1 influenza viruses currently circulating in north america, with the exception of the neuraminidase (na) and matrix (m) genes, which are derived from a eurasian swine influenza virus. swine h1n1 influenza viruses were first isolated in 1930 and continued to circulate in north america with very little antigenic changes (classical swine h1n1) until 1998. since 1999, however, the antigenic make up of swine h1 viruses has shown increased diversity due to multiple reassortment events and the introduction of h1n1 genes from human influenza viruses. currently, four swine h1 clusters (a, b, c, d) are found endemic in the north american swine population. 5, 6 these swine h1 viruses show substantial antigenic drift compared to the classical swine h1 viruses. cluster d swine h1 is derived from current human h1 viruses, and there is a substantial antigenic divergence between classical swine h1 and human seasonal h1 viruses. epidemiological evidence shows a two-way transmission of influenza viruses between swine and humans, and such events lead to the emergence of the pdmh1n1 virus. 5, 7, 8 phylogenetic analysis have suggested that possible ancestors of the eight genes of pdmh1n1 were circulating in the swine population for at least 10 years prior to the emergence of the pdmh1n1 virus in humans, although the pdmh1n1 virus itself was not isolated from pigs until after the pandemic. interestingly, pdmh1n1 infections have been reported not only in humans and pigs, but also in other animal species such as turkeys, cats, ferrets, cheetahs, and dogs. [9] [10] [11] after the first report of pdmh1n1 infection in swine in canada, other countries, including argentina, australia, singapore, northern ireland, finland, iceland, england, united states, japan, and china reported outbreaks of pdmh1n1 in swine as well. 9, [12] [13] [14] the ample geographic range of pdmh1n1 outbreaks in swine, its apparent broad host range, and the possibility of two-way transmission between swine and humans poses a tremendous challenge for controlling the virus. therefore, to differentiate pdmh1n1 from other h1 strains, particularly in swine and human populations, is an important issue to ascertain the magnitude of the disease caused by the pdmh1n1. in this study, we developed an elisa assay to discriminate pdmh1n1 strains from other swine and human h1 viruses. madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa) were maintained in modified eagle's medium (mem) containing 5% fbs. a ⁄ california ⁄ 04 ⁄ 09 ⁄ h1n1 virus (ca ⁄ 04) was kindly provided by the centers for disease control and prevention (cdc), atlanta, georgia. other viruses are listed in table 1 . viruses were propagated in mdck cells and stored at )70°c until use. viruses were titrated by the reed and muench method to determine the median tissue culture infectious dose (tcid 50 ). 15 three monoclonal antibodies (3b2, 5h7, and 12f3) against ha of 2009 pandemic h1n1 were prepared in our laboratory following previously described methods (shao and perez et al., unpublished). purification and labeling of mabs mab 3b2, 5h7 and 12f3 were purified on a protein g-sepharose affinity column (upstate biotechnology, lake placid, ny, usa). biotinylation of the detection antibody in the elisa was performed using sulfo-nhs-lc-biotin (sulfosuccinimidyl-6-(biotinamido)hexanoate; pierce, rockford, il, usa) according to the manufacturer's instructions. purified 5h7 and 12f3 were selected as the capture antibody, and biotin-conjugated 3b2 was selected as the detection antibody, and hrp-conjugated streptavidin (abcom, cambridge, ma, usa) was developed using the tmb substrate system (kpl, gaithersburg, md, usa). in brief, the mixture of the purified 5h7 and 12f3 (2ae0 and 2ae2 lg ⁄ ml respectively, in carbonate ⁄ bicarbonate buffer, ph 9ae6) was coated to 96-well plates (test well, t) for 12 h at 4°c. at the same time, a control antibody was coated to 96-well plates (control well, c). after blocking the plates with 5% (w ⁄ v) non-fat milk in pbs for 1 hour at 37°c, the samples were diluted in extract buffer (1%tween-20, 0ae5%bsa in pbs) and added to the wells (100 ll ⁄ well, each sample was table 1 . specificity assay of the sandwich elisa result (t ⁄ c) added to four wells-two for t wells and two for c wellsand the mixture was incubated at 37°c for 1 hour. after four washes, 100 ll biotin-conjugated 3b2 (0ae25 lg ⁄ ml) in dilution buffer (0ae5% bsa in pbs) was added to the wells and the mixture was incubated for 1 h at 37°c. following three washes, 100 ll diluted hrp-conjugated streptavidin (62ae5 ng ⁄ ml) in dilution buffer was added to the plates. after incubation for 1 h at 37°c, the plates were washed five times, and the binding developed using the tmb substrate system for 30 minutes. the ratio of the average od 650 value of the t wells to that of the c wells (t ⁄ c) of individual samples was calculated. t ⁄ c values >1ae5 were considered positive in the sandwich elisa. we developed three monoclonal antibodies, 3b2, 5h7, and 12f3, against a prototypical pdmh1n1 strain, a ⁄ california ⁄ 04 ⁄ 2009 (h1n1) (ca ⁄ 04). these monoclonals were used to develop a rapid sandwich elisa for specific diagnosis of pdmh1n1 strains. purified 5h7 and 12f3 were used as capture antibodies, whereas the biotin-conjugated 3b2 was used as detection antibody. the sandwich elisa showed strong reaction with different pdmh1n1 strains as described in in order to evaluate if the sandwich elisa could distinguish the pdmh1n1 from other swine h1 clusters (a, b, c, d), 14 swine influenza strains spanning these clusters were tested. these viruses were first diluted 1:10 in extract buffer, and then added to the coated plates. as shown in table 1 , the t ⁄ c ratios of these viruses were <1ae5, and therefore showed negative elisa result. likewise, testing of human seasonal virus strains a ⁄ brisbane ⁄ 59 ⁄ 2007 (h1n1), a ⁄ malaya ⁄ 302 ⁄ 1954(h1n1), a ⁄ wsn ⁄ 1933 (h1n1), and a ⁄ brisbane ⁄ 10 ⁄ 2007 (h3n2) also showed negative elisa results. furthermore, the sandwich elisa showed no cross reaction with avian influenza viruses, including strains of the h2, h3, h5, h6, h7, h8, h9, h10, h11, h12, and h13 subtypes. more recently, the mutation d222g in the ha of some pdmh1n1 strains has been associated with exacerbated disease and altered receptor binding. [16] [17] [18] [19] [20] to evaluate if such mutant could be detected in our sandwich elisa, we tested a mutant of a ⁄ netherland ⁄ 602 ⁄ 2009 (h1n1) carrying the d222g mutation (engineered by reverse genetics). as described in table 1 , our elisa could still capture the d222g mutant virus and showed a positive reaction, which highlights the specificity of our assay for pdmh1n1 strains, even those with mutations. to evaluate the sensitivity of the elisa, we used the serially diluted pdmh1n1 viruses to determine the limit of detection (lod). as shown in table 2 , in our elisa the highest positive dilutions of nl ⁄ 602 and ca ⁄ 04 were 1:320 and 1:160, respectively. the lod of the sandwich elisa by tcid 50 was 3ae2 · 10 3 and 1ae5 · 10 4 tcid 50 ⁄ ml, for nl ⁄ 602 and ca ⁄ 04, respectively. it is important to note that the t ⁄ c ratio from nl ⁄ 602 and ca ⁄ 04 viruses showed clearly a dose dependent effect, while the t ⁄ c ratio of a ⁄ swine ⁄ iowa ⁄ 30 (h1n1) did not show the same dependence and was always <1ae5, corroborating the high specificity of the sandwich elisa for pdmh1n1 strains. although we did not compare our elisa with other current commercial rapid influenza detection kits, the lod of our elisa assay is similar to other commercial kits that detect human seasonal influenza virus. 21 comparison of the sandwich elisa with the ''gold standard'' -virus isolation in order to further evaluate the feasibility of the application of the elisa to clinical samples, 70 nasal wash samples 1ae58 · 10^6 )(0ae8) )(0ae8) )(0ae9) )(1ae0) )(0ae9) )(0ae8) )(1ae1) )(0ae9) -from ferrets, 56 of those previously infected with ca ⁄ 04 and shown positive by virus isolation, were tested. the samples were diluted 1:1 in extract buffer and then tested using the sandwich elisa. result showed 51 out of 56 positive samples by virus isolation were positive also by the sandwich elisa (sensitivity 90ae1%). the 14 samples tested that were negative by virus isolation were also negative in the elisa, indicating 100% specificity for our assay. these results show not only that our elisa has high compatibility with the virus culture method, but also indicates this application can be used for clinical samples. although real time rt-pcr targeting the ha gene has been used for specific diagnosis of pdmh1n1 with high sensitivity, [22] [23] [24] [25] [26] [27] it is a method that requires manipulation of the sample to extract viral rna, and it is prone to crosscontamination during the pcr steps. in this study, we described a convenient sandwich elisa based on three mabs developed against the pdmh1n1 strain. the elisa not only shows high specificity for pdmh1n1 strain, but also shows great sensitivity. the elisa could distinguish pdmh1n1 strains from human seasonal h1 and h3 viruses and, more importantly, from other swine h1 viruses. we must note that current rapid diagnostic tests cannot be used to differentiate pdmh1n1 from swine or human h1 viruses. 28 it is also worth noting that the sensitivity of commercial rapid antigen-based diagnostic tests for detecting pdmh1n1 is lower than that for human seasonal influenza viruses. 28, 29 a study by kok et al. 32 showed that sensitivity of the current rapid antigenic tests for pdmh1n1 is only 53ae4%, whereas that for seasonal influenza a is 74ae2%. chen et al. 33 developed a dot-elisa and increased the sensitivity for influenza rapid antigen detection. however, the dot-elisa developed by chen cannot distinguish among subtypes. the lod of our elisa is between 3ae2 · 10 3 to 1ae5 · 10 4 tcid50 ⁄ ml, comparable to the lod of rapid diagnostic tests for human seasonal influenza viruses. 21 compared to the ''gold standard''-virus isolation-our sandwich elisa showed 90ae1% sensitivity using ferret nasal washes. our results highlight the potential application of our sandwich elisa for the specific diagnosis of pdmh1n1 viruses. 17 the timely and reliable laboratory evidences are vital factors for field epidemiologists trying to control outbreaks of infectious diseases and for the practicing clinicians to properly manage disease cases. therefore, analysis of new detection methods in comparison to the routine ''classical'' methods is essential to select new methods to be introduced into health service practices, especially in developing countries. in this study we have compared rt-rt-pcr detection of influenza viruses and direct fluorescent-antibody assay using r-mix hybrid cells (a549&mv1lu) with the ''classical'' cell culture methods in developing country settings. in this study, we analyzed 503 nasopharyngeal swabs col the detection of influenza h1, h3, b, and pandemic influenza (h1)pdm virus-specific nucleic acids was performed by rt-rt-pcr in abi7500 fast real time pcr system using primers recommended by cdc, usa, and super-scriptô iii one-step rt-pcr and platinum ò taq dna polymerase kits (invitrogen). the cycling protocol was: 30 minutes at 50°c, 2 minutes at 95°c, and 45 cycles of 15 seconds at 95°c, 30 seconds at 55°c. 1 rapid detection of influenza infected cells has been performed by dfa using the infected hybrid cells of r-mix within 48 hours after inoculation, according to the manufacturers instruction (diagnostic hybrids, inc., usa). 2 the isolation of influenza viruses was performed on mdck cell culture by the protocol recommended by cdc, usa. 3 we detected 1891(25ae2%) influenza virus-specific nucleic acid fragments from all tested samples by rt-rt-pcr. among the positive samples, there were 25ae6% a(h1n1), 0ae4% a(h3n2), 5ae6% influenza b, and 19ae5% a(h1n1)pdm with different distributions by time series in different age-groups. inoculation of the cell lines by rt-rt-pcr positive samples selected randomly has detected influenza virus in 52ae3% (521 ⁄ 995) on mdck cell culture and 51% (77 ⁄ 151) on r-mix hybrid cell culture with varying distribution for different strains. in other words, mdck cell culture technique was better for isolation for pandemic influenza viruses and dfa using r-mix hybrid cell culture technique for detection of seasonal influenza viruses (table 1) . average times needed for the final results for different methods were: 4 hours for rt-rt-pcr, 48 hours for dfa on r-mix and 10 days for mdck cell culture with two passages at least. the peak of the seasonal influenza a virus detection occurred in the 7-8th weeks of 2009, however the pandemic influenza detection peak was observed in the 43-44th weeks of 2009 ( figure 1 ). the outbreaks by seasonal influenza viruses was observed mostly among the children of 0-15 years of age, and pandemic influenza virus outbreak was observed mostly in the adults of 16-64 years of age. the results of this study indicate that rt-rt-pcr is the most suitable method for decision makers in epidemiological and clinical settings by sensitivity and timeliness. the final results show that r-mix dfa requires 12 times longer, and by mdck cell culturing, 60 times longer periods, than by rtrt-pcr. mdck cell culture technique has a higher isolation of pandemic influenza viruses, and r-mix dfa has a greater detection rate of seasonal influenza viruses by our results. according to our study, with rtrt-pcr, the isolation of positive samples by tissue culture of influenza a viruses was 24% and influenza b viruses was 66ae5%, which is lower than in similar spanish study. 4 however our study illustrates similar results with a canadian study 5 where the sensitivity of dfa method and tissue culture technique was shown to be lower than rtrt-pcr sensitivity. as recorded by a study of american researchers, 6 r-mix hybrid and conventional cell culture techniques have had similar sensitivity, which does not match the results of our study. however, the results of our study match with the results of italian and american scientists 7, 8 where the r-mix hybrid method for seasonal influenza viruses is higher than mdck cell culture technique. background: viral kinetics is increasingly used to study influenza infectiousness. the choice of the study design, i.e. when and how many times nasal samples are to be collected in individuals depending on the sample size, is crucial to efficiently estimate the viral kinetics (vk) parameters. material and methods: we performed a model based optimal design analysis in order to determine the minimal number of nasal samples needed to be collected per subject and when to collect them in order to correctly estimate the vk parameters. the model used was a non linear mixed effect model developed with data collected from 44 patients sampled nine times in 9 days (initial design -504 samples collected), and we used d-optimization for design identification. we also computed the minimal number of participants necessary. results: considering that 25% of the influenza-like illness cases are not due to volunteer challenge studies have been used since the 60's to provide data on virus shedding from the respiratory tract during influenza infection. 1 recently, vk was studied in naturally acquired influenza infection. 2, 3 these data are invaluable to describe the natural history of influenza-infection and to compute natural history parameters such as the latent period, generation time, or the duration of infectiousness. [1] [2] [3] [4] however, among the 56 studies used in a meta-analysis about viral shedding kinetics, 1 the designs varied greatly from one to another. these differences led to variable amount of available information concerning the vk. the lack of adequate sampling leads to imprecise estimates. on the other hand, intensive sampling or over-sampling, while associated with highly informative data, may lead to unnecessary discomfort for the patient and cost to the investigator. optimal design is increasingly used to conceive studies 5 and provides cost-efficient designs. here we propose an optimised design to model vk in the case of influenza infection. we defined the number of participants, the number of samples to collect and their allocations. this design allows, at a minimum cost and discomfort, accurate vk curves and allows the natural history parameters to be well described. model a vk population model was proposed for influenza infection. this model describes with eight parameters the relations between free virus, uninfected target epithelial cells, infected epithelial cells, and early immune response. this model was built on a dataset of 44 volunteers from which nasal samples were collected once a day over 9 days. we call this dataset the ''original dataset''. three parameters, the induction of the early immune response, the virus production rate, and the virus clearance, did not show inter-individual variability and were precisely estimated (relative standard error below 10%). we considered them as fixed in this research work. five parameters were hence considered here: b the infection rate, d the infected cell mortality rate, w the effect of early immune response on virus production rate and v init the initial value of virus titre. in order to correctly estimate these parameters it is crucial to determine a design to collect informative data. optimal designs maximise the amount of information provided by the study. it involves the determination of the number and allocation of sample times per subject as well as the number of participants. 6 d-optimization is based on the maximization of the determinant of the fisher information matrix and thus minimizes the variance of the parameters. 7 we used the fedorov-wynn algorithm implemented in pfim 3.0 to maximize this determinant, 8 which implies to pre-define a set of possible sample times. with the hypothesis that the inoculation occurred at 8:00 am, we chose three possible hours (8:00, 14:00, and 20:00) for each day with respect of the sleep-time. 8 to validate the design, we simulated 100 datasets of 44 volunteers with the optimised design obtained. we then estimated the population parameters using monolix 3.1 for each of the datasets. we compared the estimated parameters obtained with the simulated datasets to the parameters used to build the optimal design. we computed the relative bias as: with n: number of successful estimations among the 100 simulated datasets. h i : parameter value obtained with the ith dataset. h: parameter value obtained with the original dataset. we also compared the observed rse from these simulations with the rse predicted by pfim and the rse obtained with the original dataset. the rse is proportional to ffiffiffi n p , where n is the population size. we can hence deduce the smallest number of participants necessary to obtain rse below 50%. where rse predicted is the highest predicted rse (here rse for w) with 44 participants and n predicted = 44 and rse min is equal to 0ae5. considering that 25% of the influenza-like illness cases are not due to influenza virus, the total number of participants should be multiplied by 1ae25. we found that the best design was when all the participants are sampled five times: three times during the second day post-inoculation at 8:00, 14:00, and 20:00 hours and twice on the third day post-inoculation at 8:00 and 20:00 ( figure 1 ). the comparison of the relative bias and rse predicted by pfim and those obtained after simulation and re-estimation of the parameters are shown in figure 2 . v init and d in a lesser extent present bias. fixed effect parameters are precisely estimated and accordingly to pfim except for v init . we found that 16 participants shedding virus or 20 participants with ili symptoms are necessary if 25% of them are not infected with influenza virus. we propose an optimised design to accurately study the vk of influenza virus with the minimal number of samples. this design is well balanced between the amount of necessary information and the precision of estimation. we found that 100 samples are necessary to precisely fit the vk curves, which is five times less than the number of samples collected in the original study. 4??? the samples should be collected during the second and third days after inoculation. yet we showed in a previous work that the incubation period lasted 1ae9 days. 4??? hence, the optimised sample times correspond to the two-first days of symptoms and this design could be applied to naturally acquired infections studies in which the inoculation time is unknown. an advantage of this design is its practicality and convenience. all samples are collected during the daytime and after the onset of symptoms. it can thus be used for studies with naturally acquired infections. the design was validated with several criteria concerning the accuracy of the estimation with the optimised design. the parameters estimates were generally satisfactory. the parameter describing the effect of the early immune response on the virus production rate was, however, less precisely estimated (predicted rse = 62%), and the initial value of the viral titre was very different of the one obtained with the original dataset (bias v init on figure 2 ). this is probably due to the fact that it was measured at day 2 post inoculation, and that the inter-individual variability is much higher than at day 0. furthermore, d (the infected cell mortality rate) seems also to be biased. this may be due to the fact that three parameters were fixed. the model used was developed from experimentally inoculated healthy volunteers with low serum haemagglutinin antibody titre and with virus inoculation time at 8:00 am. the applicability of the design to naturally acquired infection would depend on the pathogenicity of the virus as well as pre-existing immunity and the relevance of challenge method to natural influenza acquisition. 1 our design could be directly used to accurately study vk during influenza infections and would reduce the discomfort of patients and the cost of the experimentation. usefulness of a self-blown nasal discharge specimen for use with immunochromatography based influenza rapid antigen test introduction influenza rapid antigen tests (irat) have become very popular and are widely used for confirming suspected clinical diagnosis of influenza in japan. 1 most of the currently used irat that are based on immunochromatography (ic), nasopharyngeal swab, nasopharyngeal aspiration, and throat swab have been approved as specimens for japanese national health insurance purposes. but the specimen collection by these methods gives patients considerable discomfort, and sometimes appropriate specimens cannot be obtained due to patient resistance, especially by children. in the present studies, self-blown nasal discharge was used as the specimen for an irat, and the results were compared with the results of viral isolation and an identical kit primed with nasopharyngeal swab specimens for seasonal influenza viruses and pandemic (h1n1)2009 virus. patients who visited any of the 10 clinics that belong to the influenza study group of the japan physicians association in the 2006-2007 and the 2009-2010 influenza seasons with influenza-like illnesses exhibiting findings were registered after providing informed consent. a square plastic sheet of 20 · 20 cm was handed to the patient. nasal discharge was collected by blowing the nose into the plastic sheet as a specimen for irat, i.e. self-blown specimen. two nasopharyngeal swab specimens were also obtained at the same time for irat and virus isolation. self-blown specimens were obtained successfully by 152 (82ae2%) of 185 consecutive outpatients in the 2006-2007 season, as seen in table 1 the sensitivity and specificity of various influenza rapid antigen tests have been reported in various settings. [1] [2] [3] [4] direct comparison of the results is difficult because of differences in patient or influenza virus, characteristics such as age, study designs, and other features. in this study of the 2006-2007 influenza season, the sensitivity, specificity, and accuracy of the ic kit primed with nasopharyngeal swab specimens were 87ae0%, 94ae0%, and 90ae8%, respectively. these results were quite comparable to our results of the 2005-2006 season, 3 in which the overall results of other ic kits were 82ae9%, 81ae0%, and 82ae3%, respectively, indicating that the ic kit used is quite reliable. the sensitivity, specificity, and accuracy of an ic kit will vary by the method of specimen collection. in general, virus titer is considered to be highest with nasopharyngeal aspiration, lower with nasopharyngeal swabs, and lowest with throat swabs. practically, nasopharyngeal swab is the most popular. the sensitivity, specificity, and accuracy of the ic kit with self-blown discharge specimens compared well with those of an identical ic kit primed with nasopharyngeal swab specimens. for self-blown specimens, sensitivity and specificity were 87ae0% and 94ae6% for influenza a, 85ae7% and 99ae1% for influenza b, 88% and 88ae9% for pandemic (h1n1) 2009. self-blown specimens display sensitivity, specificity, and accuracy comparable to that of conventional nasopharyngeal swab specimens. there was no significant difference in sensitivity, specificity, or accuracy between self-blown specimens and nasopharyngeal swab for influenza a, influenza b, and pandemic (h1n1) 2009. these results suggest that selfblown specimens are as useful as nasal cavity swab specimens for the diagnosis of influenza in the clinical settings. nasal discharge, obviously, cannot be collected from infants incapable of blowing their own nose or patients who do not develop a nasal discharge. in this study, self-blown specimens were obtained from 82ae2% of the patients. the rate of successful collection was over 70% in the age groups of 0-6 and 7-15 years. these rates would seem to be sufficient for clinical use. the procedure of self-blown specimen collection using a plastic sheet is easy and causes no pain or discomfort. it seems to be more acceptable and safe than the other methods, especially for children. furthermore, this procedure reduces the risk of influenza transmission from patients to the medical staff members involved in sample collection. self-blown sample collection may be superior to other sample collection methods in these respects. we previously reported an inverse correlation between the amount of virus in a specimen and the time to a positive reaction. 4 in this study, there was no significant difference in the mean time to a positive between self-blown self-blown specimens enough to be examined were obtained 152 from 185 consecutive outpatients, and specimens showed a tendency to be obtained large amount from children rather than the aged. there were no statistically significant differences between the ic kit results primed with self-blown discharge and nasopharyngeal swab specimens for influenza a, influenza b and pandemic (h1n1) 2009. and nasal swab specimens, suggesting that the self-blown specimens contained sufficient viral antigen for the ic kits. the influence of the presence or absence of nasal congestion on the results of the kit was assessed. the sensitivity of selfblown specimens from patients with nasal congestion was significantly lower than that from patients without nasal congestion. it is possible that insufficient capability to blow the nose due to nasal congestion might tend to lead to false negatives. the observation that the time to positive is longer for patients with nasal congestion than for patients without nasal congestion is concordant. application of self-blown specimen collection only to appropriate patients would increase the sensitivity, which would be important in a clinical setting. we tested only two commercial antigen detection kit, the quick vue rapid sp influ kit and quicknaviô-flu (denka-seiken co., ltd). the resulting sensitivity, specificity, and accuracy of the ic kit primed with self-blown specimens were considered adequate for clinical use. to confirm the usefulness of self-blown nasal discharge specimens, further investigation is necessary using other kits and in different settings. the usefulness of a self-blown nasal discharge specimen for an influenza rapid antigen test based on immunochroma-tography was evaluated in the 2006-2007 and 2009-2010 influenza season. results suggest that self-blown nasal discharge specimens are useful as specimens for influenza rapid antigen tests based on immunochromatography for not only seasonal influenza viruses, but also pandemic (h1n1) 2009 virus. the specimen collection by the patients themselves will reduce the burden of other collection methods and the risk of infection to the medical staff. in april 2009, a mixed-origin h1n1 influenza virus was recognized as a new causative agent of influenza-like illnesses (ili) in humans. since its emergence, the virus has spread rapidly throughout the world and caused a pandemic. most commercial rapid antigen tests (rat) can detect influenza a or b viruses, but cannot specifically distinguish pandemic (h1n1) 2009 virus with seasonal influenza. recent studies have indicated that the poor performance of the rat approach and nonspecific detec-tion of the pandemic (h1n1) 2009 virus was the main obstacle to their widespread use in private clinics. 1,2 with the need for a new rapid kit with reasonable sensitivity and specificity for pandemic (h1n1) 2009 virus, we developed a new rat kit in collaboration with company, standard diagnostics, inc., (yongin-si, gyonggi, korea). monoclonal antibody (mab) against haemagglutinin (ha) of the pandemic (h1n1) 2009 virus was developed using korean isolate and applied to the new kit with the mab to seasonal influenza virus. we examined the detection limit of the kit using the serial dilution of korean pandemic virus isolate (a ⁄ korea ⁄ 01 ⁄ 2009). during december 2009, 432 clinical specimens from patients with ili were collected at 11 sentinel clinics of six provinces in korea. the specimens were tested by the new rat, and the results were compared with those of real-time reverse transcription polymerase chain reaction (rrt-pcr) by us cdc and virus isolation in mdck cell culture to determine the sensitivity and specificity for the diagnosis of pandemic (h1n1) 2009. the detection limit of the new kit against ha of a ⁄ korea ⁄ 01 ⁄ 2009 virus was confirmed to be 10 4 pfu ⁄ ml. by contrast, the detection limit against the np protein was 10 3 pfu. however, when the kit was applied to clinical specimens, no difference between the two targets was found. using rrt-pcr and viral culture as the references, the performance of the ridt is shown in table 1 . among 432 specimens, 178 were tested positive by rrt-pcr and 186 were tested positive by viral culture. among the 178 rrt-pcr confirmed cases, 122 were positive, and among the 186 viral culture confirmed cases, 120 were positive with the new rat. using rrt-pcr as the reference standard, the overall sensitivity of rat was 68ae5% (95% confidence interval (ci): 61ae7-75ae3%) and specificity was 98ae4% (ci: 96ae9-99ae9%). with viral culture as the reference, the rat sensitivity and specificity was 64ae5% (ci: 57ae6-71ae4%) and 97ae6% (ci: 95ae7-99ae5%), respectively. when analyzed by the regions tested, the sensitivity ranged between 57ae1% and 95ae5% for rrt-pcr and between 53ae3% and 87ae0% for viral culture as a reference. among 340 patients who had a record of their symptom onset and sample collection date, 86 (25ae3%) visited the clinic on the day of symptom onset, and 158 (46ae5%) visited 1 day later. when the rat performance was evaluated by day of onset, the sensitivity was lower at three or more days after the onset of symptoms; however, the sensitivity was highest at 2 days after onset and reasonable on the day of onset or at 1 day after ( table 2 ). we found that this new rat had reasonable sensitivity and high specificity compared with rrt-pcr and viral culture for detecting the pandemic (h1n1) 2009 virus. in one recent study, the sensitivity and specificity of the new rat kit was 77% and 100%, respectively, and the ha protein for pandemic (h1n1) 2009 was detected more sensitively than the np protein for influenza a virus. 3 the sensitivity and specificity of our new rat were lower than those of that study. we found that the test performance varied depending on the clinics in which the tests were performed, and this might be attributable to the persons who collected the specimens. although the clinicians were trained well for *ci, confidence interval. **ppv, positive predictive value. ***npv, negative predictive value. collecting specimens, there might be some differences in performance. the new rat kit could detect pandemic (h1n1) 2009 virus specifically. although the sensitivity was lower than those of rrt-pcr and virus culture, and negative rat results should be confirmed with more sensitive methods, this kit could be useful in sentinel clinics if used with caution. determination of infectious virus titres is central to many experiments designed to study the biology of influenza virus. assays based on the measurements of viral components, whether viral protein or nucleic acid, does not differentiate infectious virus from non-infectious or defective viral particles, which may have no infectivity or biological *three hundred and forty samples with a known date of onset and sample collection were analyzed. ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 132-158 activity. therefore the ''gold standard'' of virus measurement requires bioassays that examine the ability of viral particles to replicate and further infect other cells. titration on madin-darby canine kidney (mdck) cells in a 96 well plate format is commonly used to measure influenza virus titre. this method is labour intensive, subjective in their read out of cytopathic effect, and takes several days to obtain a result. microneutralization tests that quantitate neutralizing antibody titres and assays of drugs for antiviral activity also require 96 well based assays of residual virus infectivity. therefore, technologies that improve on the titration of infectious virus will be of great benefit. this study utilized the xcelligence system (roche applied science), which adopts microelectronic biosensor technology to monitor dynamic, real-time label free and non-invasive analysis of cellular events. 1 the system measures electronic impedance using an array of microelectrodes located at the bottom of each culture well (e-plate 96). adherent cells are attached to the sensor surface of electrode arrays, and changes in impedance can be detected and recorded. the xcelligence system can monitor cell events induced by viral infection, such as changes in cell number, adhesion, viability, morphology, and motility. measured electrode impedance is expressed as dimensionless cell index and is graphically represented using software to show the phenotypic changes of a cell population over time. the aim of this study is to demonstrate that using this platform to measure real-time cell index has potential to circumvent many of the limitations of the currently established procedures of end point titration of virus infectivity and for microneutralization assays. madin-darby canine kidney cells were propagated in growth medium consisting of minimum eagle's medium (invitrogen) supplemented with 10% fetal bovine serum (invitrogen), 0ae6 mg ⁄ l penicillin (invitrogen), and 60 mg ⁄ l streptomycin (invitrogen), with incubation at 37°c in a 5% co 2 humidified atmosphere. influenza a ⁄ hong kong ⁄ 54 ⁄ 1998 (h1n1), a seasonal influenza virus from a patient who suffered from a mild febrile illness, was propagated in mdck cells maintained in virus medium consisting of minimum eagle's medium (invitrogen) supplemented with 0ae6 mg ⁄ l penicillin (invitrogen), 60 mg ⁄ l streptomycin (invitrogen), and 2 mg ⁄ l np-tosyl-l-phenylalaninechloromethyl ketone-treated trypsin (sigma, st louis, mo, usa), with incubation at 37°c in a 5% co 2 humidified atmosphere. virus stocks were aliquoted and stored at 70°c until use, and the 50% tissue culture infectious dose (tcid 50 ) of the virus stock was determined by titration in mdck cells according to standard procedures, 2 and the tcid 50 of the stock virus was calculated by the method of reed and muench. 3 to perform a microneutralization assay, mdck cells seeded at a density of 1000 cells ⁄ well in an e-plate 96 was removed from the xcelligence system after approximately 48 hour; growth medium was then removed, cells washed, and replaced with 100 ll virus-medium. a human serum, which is known to contain high titre antibody against the h1n1 virus was heat inactivated for 30 min at 56°c, and twofold serial dilutions were performed in virus medium. the diluted serum was mixed with an equal volume of virus medium containing influenza virus at 400 tcid 50 ⁄ 100 ll. after incubation for 2 h at 37°c in a 5% co 2 humidified atmosphere, 50 ll of virus-antibody mixture was added to the mdck cells to give each well an equivalent virus dose of 100 tcid 50 . a back titration of the virus challenge dose was performed, and a cell control (free of virus) was performed in quadruplicates. after incubation at room temperature for 30 minutes, the e-plate 96 was then placed back onto the xcelligence system in the incubator and maintain at 37°c with 5% co 2 , and the cell index values were measured every 15 minutes for at least a further 72 hour. the same procedures were performed with cells seeded in conventional 96 well cell culture plates for parallel comparison with the currently used standard method. in this case, cells were examined for cytopathic effect under an inverted microscope after 3 days of infection and the lowest virus dilution, which protected the cells from viral induced cytopathic effect taken as the neutralizing end point. after 48 hour of seeding mdck cells at 1000 cells ⁄ well, standard microneutralization assay for influenza virus was performed. integral to this assay, a serial titration of the input virus at 0ae5 log 10 increments was carried out. wells infected with the undiluted virus (100 tcid 50 ⁄ well), the cell index commenced dropping at a steeper gradient than the no-virus cell control after approximately 3 hour of infection ( figure 1 ). this drop in cell index continues at a consistent slope until it flattened out when approaching zero cell index. this steep decrease in cell index with constant gradient was also observed for virus dilutions up to and including 2 log 10 (100-folds), and the profile shifted with increased time in proportion to the dilution made to the virus. virus dilutions beyond 2 log 10 have cell index profiles similar to the no virus input control, and this corresponds to the absence of cytopathic effect as determined by microscopic observation at 72 hour after infection. hence, there was a correlation between the amount of virus used for infection, the onset of the influenza virus-mediated cytopathic effect, and the steep decline in cell index. a human serum with known microneutralization antibody titre to h1n1 virus was used in this study to investigate the real time cell index changes that occur during the assay ( figure 2 ). using influenza virus treated with serum dilutions up to and including a dilution of 1:160, the cell index profile remained essentially the same as the no virus cell control, which correlates with the lack of cytopathic effects under microscopic observation at 72 hour of infection. at a serum dilution of 1:320, the steep decrease in cell index, which is characteristic of cellular cytopathic effect induced by the virus, became evident at around 50 hour post infection, and this was reduced to 40 hour when serum dilution of 1:940 was used. in contrast, for the virus -no antibody control, the onset time for this steep decrease in cell index occurs at approximately 7 hour. for both serum dilutions of 1:320 and 1:940, full cytopathic effect was observed microscopically at 72 hour of infection. from microscopic observation of cytopathic effect, according to the current standard procedures, the neutralizing titre of the human serum used in this study is at 1:160 as it is the last dilution of the serum that prevented cytopathic effect from being detected. an essential part of the microneutralization assay is to confirm the titre of the input virus (normally 100 tcid 50 ⁄well) by performing a titration assay with decreasing serial dilutions of the virus. under normal procedures, cells are examined microscopically after 72 hour of infection for sign of cytopathic effects. in the case of mdck cells, the cytopathic effect is cell death, which is indicative of the presence of live influenza virus infecting and replicating in the cells. therefore, the titre of the virus is taken as the last dilution in which cytopathic effect is present. parallel realtime cell index measurements demonstrated that for wells with cytopathic effects, the profile exhibits a steep gradient linear decrease in cell index after infection with the virus, which can be termed the ''cpe plunge.'' the time in which the cpe plunge became evident appears to be inversely proportional to the amount of virus, therefore the opportunity exists to utilize this aspect to calculate or compare quantitatively different virus concentrations. for unequivocal assignment of cytopathic effect, it normally requires 2-3 days after infecting the cells, with 3 days after infection being the standard time to read virus titration and microneutralization assays. using the real-time cell index monitoring, it is found that apparent cytopathic effect can only be observed microscopically when the cell index has dropped to near zero. as the time of onset of the ''cpe plunge'' becomes evident many hours prior to observable cytopathic effect, it is possible that the time to results can be drastically reduced after some formulation of the method. we compared the current standard method in perfoming a microneutralization assay with one utilizing the real-time cell index measurement to investigate whether this approach is able to offer better performance over the existing one. the current standard neutralization assay is the microscopic observation of antibody mediated protection from virus cytopathic effect in mdck cells. this study showed that this may also be achieved by examining the profile generated from the real-time measurements of the cell index. using real-time cell index monitoring, it is possible to detect inhibitory activity at higher dilutions of the anti-serum than can be detected by the standard microscopic observation of cytopathic effect. therefore, the realtime cell index monitoring could potentially be developed to be a more sensitive method for measuring anti-viral activity. as drug resistant strains of influenza a viruses 4 including the 2009 pandemic h1n1 are being reported, the real-time cell based monitoring system may also have the potential to be developed for use as a diagnostic platform for drug resistance assays. this study suggests that real-time cell index monitoring has the potential to substantially reduce human resources in reading results, as well as reducing time-to-result of these assays from 3 days to two. the saving could be substantial for work involving bio-hazard level 3 ⁄ 4 pathogens such as h5n1 viruses as personnel working with these organisms are require to be highly trained and experienced. in addition, the reduction in transferring plates to and from the microscope in reading cytopathic effect will substantially reduce the possibility of accidents from occurring. furthermore, the system provides objective digital data to an otherwise subjective assay method, which can improve standardization, data exchange, and hence collaboration between different laboratories. with more detailed validation and development, real-time cell index monitoring could transform the way we study and diagnose infection with pathogens such as influenza viruses. the emergence of a novel h1n1 influenza a virus of swine origin, the pandemic a(h1n1) 2009, with transmissibility from human to human in april 2009 posed pandemic con-cern and required modifications to laboratory testing protocols. a new protocol for universal detection of influenza a and b viruses and simultaneous subtyping of influenza a (h1n1) 2009 virus, composed of two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h1n1v (relab, italy), was evaluated and compared to the reference protocol recommended by who. fast set infa ⁄ infb was able to detect influenza a and b viruses circulating between 1995 and 2008 belonging to different subtypes and lineages, and no cross reactions were observed by either fast set infa ⁄ infb or fast set h1n1v. the who assay was found to have a slightly lower end-point detection limit (10 )5 dilution) in comparison to the new protocol (10 )6 ). specificity of the assays was 100% as assessed on a panel of stored clinical samples including adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, s. pneumoniae, n. meningitidis, h. influenza, and human influenza viruses. the new assay panel allows the detection, typing, and subtyping of influenza viruses as requested for diagnostic and surveillance purposes. the high sensitivity of the protocol is coupled with capacity to detect viruses presenting significant heterogeneity by fast set infa ⁄ infb and with high discriminatory ability by fast set h1n1v. a rapid and sensitive assay for the detection of influenza virus in clinical samples from subjects with ili or low respiratory tract infections is a fundamental tool for epidemiological and virological surveillance, management of hospitalized patients, and control of virus nosocomial transmission. the emergence, in april 2009, of a novel h1n1 influenza a virus of swine origin, the pandemic (a(h1n1) 2009), with transmissibility from human to human poses pandemic concern and required modifications to the laboratory testing protocols. 1 molecular diagnosis of influenza is generally achieved through a twophase process: a screening phase for the detection of virus, and the subsequent strain characterization performed by either sub-type-specific rt-pcr or entire ⁄ partial genome sequencing. 2 during a pandemic, simultaneous implementation of both the detection of influenza a and b influenza viruses and identification of the new subtype is useful for clinical and epidemiological reasons. here, we describe a new protocol including two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h1n1v (relab, italy) that allows universal detection of all influenza a viruses and, simultaneously, all subtypes that are influenza a(h1n1) 2009. specificity and clinical sensitivity of the two-one-step rt-pcrs (fast set infa ⁄ infb and fast set h1n1v; relab, italy) were evaluated by testing 306 selected specimens, including: • fifty samples collected from nasopharyngeal swabs representative of influenza viruses, belonging to differ-ent subtypes and lineages, and other respiratory viruses and bacteria circulating in italy between 1995 and 2008. • six purified a(h5n1), a(h7n7), and a(h9n2) strains, kindly supplied by alan hay, who influenza centre, london, uk. • two hundred-fifty influenza positive samples selected according to type, subtype, clade and viral concentration from >2500 specimens received by the liguria influenza reference laboratory between january 1st and december 31st, 2009. since 1995, nasopharyngeal swabs sampled from patients suspected of having contracted the influenza virus have been collected in viral transport medium, and upon arrival into the laboratory, the samples were divided in ‡3 aliquots. those not immediately processed were stored frozen at )80°c. stored samples were used for this evaluation, and all specimens were re-extracted for the study. samples collected between 1995 and 2008 included specimens positive for: no seasonal a(h1n1) have been detected since january 1st, 2009. furthermore, 1 weak positive sample using fast set infa ⁄ infb, but negative at block pcr and typing ⁄ subtyping assays was tested. the analytical sensitivity of the test under investigation was determined testing ten-fold serial dilutions of seasonal influenza a(h1n1), seasonal influenza a(h3n2), new pandemic influenza a(h1n1) 2009, and b cell culture-grown viruses. the intra-assay reproducibility was measured by testing the same a(h1n1) 2009 positive sample 15 times in the same experiment, while the inter-assay reproducibility was confirmed by testing the same samples in 3 independent experiments. to evaluate the performance of the protocol, all samples were tested using a block pcr confirmation test (seeplex ò rv12 ace detection), 3 and all specimens collected between january 1st and december 31st, 2009 and dilutions were also assayed using the recommended who ⁄ cdc protocol of real-time rtpcr for influenza a(h1n1). typing and sub typing were performed using the who protocol and ⁄ or sequencing. 4 viral rna was extracted from swabs using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's protocol. fast set infa ⁄ infb and fast set h1n1v are two multiplex one-step real time pcr assays developed and evaluated by the liguria regional reference centre for diagnosis and surveillance of influenza in collaboration with relab diagnostics. both assays contain primers and a dual-labelled hydrolysis probe that targets two regions of the matrix gene (table 1) . amplification conditions were as follows: reverse-transcription 50°c for 15 minutes, denaturation 94°c for 2 minutes, then 40 cycles of 95°c for 15 seconds, 58°c for 30 seconds. the entire amplification process extended for 110 minutes. an internal control real-time assay was also incorporated in order to detect pcr inhibition, failed extraction ⁄ pcr and technical error. the cdc realtime rtpcr (rrtpcr) protocol for detection and characterization of swine influenza includes a panel of oligonucleotide primers and dual-labelled hydrolysis (taqman ò ) probes to be used in real-time rtpcr assays for the in vitro qualitative detection and characterization of swine influenza viruses in respiratory specimens and viral cultures. this protocol recommends three primer-and-probe sets: infa, amplifying a conserved region of the matrix gene from all influenza a viruses; sw infa, designed to specifically detect the nucleoprotein (np) gene segment from all swine influenza viruses and sw h1, designed to specifically detect the hemagglutinin gene segment from a(h1n1) 2009. 5 the seeplex ò rv12 ace detection for auto-capil-lary electrophoresis is a multiplex block rt-pcr that applies dpoô (dual priming oligonucleotide) technology and is designed to detect 12 major respiratory viruses, 11 respiratory rna (influenza a and b virus, parainfluenza virus type 1, 2 and 3, respiratory syncytial virus a and b, rhinovirus a ⁄ b, coronavirus oc43 and 229e ⁄ nl63) viruses and 1 dna (adenovirus) virus, from patients' samples including nasopharyngeal aspirates, nasopharyngeal swabs and bronchoalveolar lavage. 3 conventional viral culture was performed inoculating 0ae1 ml of each specimen into mdck-siat1 seeded into 24-well plates for influenza isolation. virus detection was performed by the hemagglutination test using 0ae75% guinea pig red blood cells (rbc). specificity and clinical sensitivity results of the new protocol are reported in table 2 . fast set infa ⁄ infb was able to detect influenza a and b virus circulating between 1995 and 2008 belonging to different subtypes and lineages, and no cross-reactions were observed by either fast set infa ⁄ infb or fast set h1n1v. among specimens collected between january 1st and december 31st, 2009, all 60 fast set infa ⁄ infb and fast set h1n1v high titre positive samples resulted positive using the who ⁄ cdc assay and showing reactivity using infa and sw infa primer-andprobe sets. among 90 low titre a(h1n1) 2009 positive samples at fast set infa ⁄ infb, 28 (31ae1%) were not detected by the who ⁄ cdc assay, but were positive using seeplex ò rv12. the who ⁄ cdc sw h1 primer-and-probe set works in 96ae7% (58 ⁄ 60) and 1ae1% (1 ⁄ 90) of high and low titre a(h1n1) 2009 positive samples, respectively. all a(h3n2) strains collected during 2009 and initially detected by fast set infa ⁄ infb were confirmed after rna re-extraction by seeplex ò rv12 and who ⁄ cdc assay showing reactivity using the infa primer-and-probe set. all infa ⁄ infb were confirmed after rna re-extraction by seeplex ò rv12. one influenza a case identified by the who ⁄ cdc kit (infa primer-and-probe set, ct values: 37ae5, sw infa primerand-probe set: negative) and new protocol (a primer-andprobe set, ct values: 34ae1, a(h1n1) 2009 primer-and-probe set, ct values: 34ae6) was not detected by either seeplex ò rv12 or by who subtyping protocol and ⁄ or sequencing, suggesting a very low viral load or unspecific results by real time assays. the analysis of serial dilutions of cell culturegrown a(h1n1) 2009 showed that the detection limit of fast set infa ⁄ infb, fast set h1n1v, and seeplex ò rv12 was identical (10 )6 ) and 1log 10 lower than that using the who ⁄ cdc protocol (10 )5 ). a similar analysis with respect to a(h1n1) and a(h3n2) strains indicated that fast set infa ⁄ infb sensitivity (10 )6 and 10 )7 , respectively) was 1log 10 lower than that showed by seeplex ò rv12 (10 )5 and 10 )6 , respectively). in comparison with the new protocol, the who ⁄ cdc assays, considering infa primer-and-probe set, was found to have a slightly lower end-point detection, detecting the 10 )5 a(h1n1) and a(h3n2) dilution. also in detecting influenza b virus, fast set infa ⁄ infb sensitivity (10 )6 and 10 )7 , respectively) was 1log 10 lower than that showed by seeplex ò rv12 and the who ⁄ cdc protocol. data on intra-assay and inter-assay precision, measured as cv% of ct showed that the dispersion indices observed had values of less than 3%. since 2009 samples were detected using the new protocol that resulted negative using the who ⁄ cdc assays. the unfortunately low quantity of low titre a(h3n2) samples collected during 2009 did not allow us to highlight differences between assays fast set infa ⁄ infb, and fast set h1n1v positivity was always confirmed by seeplex ò rv12, which demonstrated high sensitivity, showing a detection limit comparable or lower when compared with those observed using the who ⁄ cdc assays. the high analytical sensitivity of seeplex ò rv12 is reported by kim 3 who observed a detection limit of 10 copies per reaction for each type ⁄ subtype of influenza viruses. the high sensitivity of the new protocol is coupled with its capacity to detect viruses presenting a significant heterogeneity by fast set infa ⁄ infb and high discriminatory ability by fast set h1n1v. fast set infa ⁄ infb was able to identify representative influenza viruses of circulating strains during the last decade belonging to different subtypes, lineages, and clusters, and fast set h1n1v primerand-probe set reacted selectively with a(h1n1) 2009 target. a recent report demonstrated that the sw infa assay is not specific to a(h1n1) 2009 and is able to detect both human and avian (h5n1) influenza a viruses and so there is the potential for misidentification. 15 high titre (ct 8ae5 and 15ae6 at fast set infa ⁄ infb) a(h5n1) viruses did not react with fast set h1n1v primer-and-probe set (data not shown). available human a(h5n1) sequences are similar within the h1n1v primer-and-probe regions, but having 4-5 mismatches in the forward primer and, more notably, two of the mismatches occurred within 9 nucleotides of the 3 end, an important determinant for primer specificity. in conclusion, this protocol can be a powerful tool in the diagnostic laboratory setting for specific simultaneous analysis of several samples in minimal time, showing enhanced sensitivity in detecting influenza viruses, and high discriminatory ability in identifying the new pandemic a(h1n1) 2009. a university-corporate partnership to enhance vaccination rates among the elderly: an example of a corporate public health care delivery public health campaigns usually rely on governmental infrastructure and finance for vaccine implementation programs. however, there are many financial and physical barriers which preclude widespread and effective vaccine administration, especially among the elderly. on an international scale, both government agencies and citizen groups have a vested interest in searching for more resourceful methods of attaining significant immunization levels (>75% of the population). in fact, it seems to have become both a grassroots civic and governmental goal, especially among developing countries. we implemented the unique strategy of enlisting the assistance of a privately-owned food market chain to address the public health issue of mass vaccination for the elderly. in this context, publix pharmacy and the university of south florida (usf) recently developed both a handbook and a training program to facilitate the administration of vaccinations. between 2008 and 2009, the publix-usf partnership resulted in administration of over thirty thousand influenza a (h3n2) vaccinations, 76% of which were given to adults over 55 years of age. consequently, vaccine administration costs were decreased by using corporate resources and bypassing overly strained municipal resources. this unique university-corporate partnership successfully delivered h3n2 vaccine to a vulnerable cross-section of society at a lower cost and with minimal side effects and morbidity. it may be safely projected that university-corporate partnerships could result in an effective method for rendering a vital service to an aging and especially vulnerable segment of the population. government policy and funding are the foundation of immunization programs on an international scale. for example, in the united states, governmental programs account for over 50% of the monetary outlay used for immunization. 1 until 2006, the global alliance for vaccines and immunizations (gavi) acted as a catalyst for implementing vaccine and immunization programs in each targeted country. 2 under the auspices of gavi-collaborations between governments, charitable organizations, and multinational health agencies (such as uncief and the who)-many countries have increased their spending for vaccination programs. however, development of financially sustainable immunization programs geared toward reaching the majority of the population are still at a nascent level of evolution. 3 the development of more innovative and costeffective approaches has become imperative in order to reach a greater number of vaccination candidates. administering the influenza vaccine only to the subpopulation of over 65 year olds would save an estimated 220 000 quality-adjusted life years in a cohort of approximately half the world's population. 4 widespread public vaccination programs are made more complex by the continuing development of newer vaccines, concomitant specialized administration costs, and the logistical challenge of conveying recipients to vaccination points of service. 1, 5 in spite of the increasing complexity of mass vaccination, cost-benefit analyses clearly favor annual influenza vaccination in the elderly population on an international scale. 4, 6 recently, in 2008, influenza vaccine administration was reported to reach between 32% and 82% of the elderly population, which denotes varying degrees of success within each particular country. 7, 8 however, there was also a report of a uniform plateau effect at around 75% of the population, beyond which additional vaccination coverage was difficult to achieve. 9 physical limitations to vaccination seem to be more insurmountable for the elderly. unfortunately, this is the population segment which could experience the most significant vaccination-associated mortality reduction. 10 we employed the unique strategy of involving the resources of publix supermarkets, a corporate food market chain, to address the public health issue of widespread vaccination for the elderly. we took advantage of recent 2005 changes in the florida statutes, which expanded the scope of pharmacists' practice to include administration of vaccines. subsequently, publix pharmacy and the university of south florida (usf) developed a handbook and training program to facilitate and enhance vaccine administration by publix pharmacists. by using proprietary pharmacists and more practical supply storage, we were able to decrease the costs of vaccine administration. the consumer was charged $10 for administration costs plus the cost of the injection itself, regardless of insurance or eligibility for governmental subsidy. although patients were initially self-selected, they were ultimately excluded if they had demonstrated prior adverse effects to influenza vaccinations or to any of the components of such vaccinations. between 2008 and 2009, the publix-usf partnership vaccinated 30 063 people against influenza a (h3n2), of which 29 404 were florida residents. the age range was 1-105 years old with a median age of 65 years old. seventysix percent of the participants were over 50 years old (see figure 1 ). within the population surveyed, the reported side effects of the vaccine in this study were not serious, but included: vertigo, cold sweats, chills, vomiting, syncope, rash, nausea, stomach pain, elevated blood pressure, injection site reaction, inflamed bursa, and bilateral thigh discomfort. participants from all socioeconomic classes were vaccinated. an income-by-zip code analysis revealed 44% of those vaccinated resided in zip code areas where the average household income was <$50 000 per year. of those remaining, 25% had an average income of $50 000-$70 000 per year, and 31% had an income of >$70 000 per year. each person vaccinated was charged ten dollars for administration costs. this represents a decrease in the administration costs ranging from one dollar to ten dollars saved per vaccine. 11, 12 conclusion this unique university-corporate partnership successfully delivered h3n2 vaccine to a high-risk population with decreased vaccine administration costs. the influenza vaccine is well-tolerated, with minimal side effects when patients who have a history of adverse reactions are excluded. we can postulate that university-corporate partnerships may indeed be effective at reaching the aging population which is a challenge in most communities. this delivery model may prove to be another tool for improving the efficiency of mass immunization by facilitating accessibility, which results in wider coverage. this model also enhances delivery of healthcare by decreasing costs of immunization regardless of whether the payer is a government, insurance company, or self-pay consumer. the gavi initiative stressed three goals for accomplishing sustainability and independence in immunization programs. 3 the goals were to: (i) mobilize additional resources from governmental and non-governmental sources; (ii) improve program efficiency to minimize additional administration resources needed; and (iii) increase the reliability of funding. empowering privately owned corporations within the community, such as food markets or pharmacies, to administer vaccines mobilizes additional resources to readily achieve the first goal of gavi. mobilizing resources of non-healthcare, corporate vaccination locations enhances accessibility due to travel convenience. in our study, participants came from all socioeconomic classes, suggesting that ease of access is independently hindering mass vaccination, and that people of all incomes are more likely engaged when access issues are eliminated. the second and third goals were also accomplished by recruiting a corporation's resources for vaccine administration (refrigeration, storage, and employees). this minimizes the money spent from vaccine program funds to support the infrastructure of immunizations, thus improving financial efficiency and sustainability. financial efficiency implies that money is spent to safely reach as large a portion of the population as possible. by using corporate storage facilities instead of paying for independent facilities, money can be spent elsewhere. more vaccines can be purchased and more money can be spent on media communications to encourage vaccination. sustainability requires the ability to fund annual vaccination programs which reach 75% of the population or greater. key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. in addition, they must minimise false alarms during a normal influenza season. we develop a method that uses historical syndromic influenza data from the existing surveillance system 'servis' 1 monitoring seasonal ili activities in scotland. we develop an algorithm based on wcr of reported ili cases to generate an alarm for pandemic influenza. wcr is defined as the ratio of the number of reported cases in a week to the number of cases reported in the previous week. from the seasonal influenza data from 13 scottish health boards, we estimate the joint probability distribution ( figure 1) we compare our method, based on our simulation study, to the mov-avg cusum and ili rate threshold methods and find it to be more sensitive and rapid. the wcr method detects pandemics in larger fraction of total runs within the same early weeks of pandemic starting than does any of the other two methods ( figure 2 ). as shown in the table, for 1% pandemic case reporting rate and detection specificity of 95%, our method is 100% sensitive and has mdt of 4 weeks, while the mov-avg cusum and ili rate threshold methods are, respectively, 97% and 100% sensitive with mdt of 5 weeks. at 99% specificity, our method remains 100% sensitive with mdt of 5 weeks. although the threshold method maintains its sensitivity of 100% with mdt of 5 weeks, sensitivity of mov-avg cusum declines to 92% with increased mdt of 6 weeks. for a two-fold decrease in the case reporting rate (0ae5%) and 99% specificity, the wcr and threshold methods, respectively, have mdt of 5 and 6 weeks with both having sensitivity close to 100%, while the mov-avg cusum method can only manage sensitivity of 77% with mdt of 6 weeks. the first cases of the 2009 pandemic were reported in scotland in the 29th week of the season. the wcr algorithm as well as the mov-avg cusum method detects the pandemic 12 weeks later in week 41. the ili threshold method detects it 1 week later in week 42. both the wcr and mov-avg cusum methods therefore outperform the ili threshold method by 1 week in the retrospective detection of the 2009 pandemic in scotland. while computationally and statistically very simple to implement, the wcr method is capable of raising alarms rapidly and sensitively for influenza pandemics against a background of seasonal influenza. although the algorithm has been developed using the servis data, it has the capacity to be used at large scale and for different disease systems where buying some early extra time is critical. more generally, we suggest that a combination of different statistical methods should be employed in generating alarms for infectious disease outbreaks. different detection methods would provide cross-checks on one another, boosting confidence in the outputs of the surveillance system as a whole. real-time evidence being created worldwide will greatly contribute to the full understanding of influenza pandemics. here we report the real-time epidemiology and virology findings of the influenza a(h1n1)2009 pandemics in mongolia. the epidemiological and virological data collected through isss of nic, nccd, mongolia (real-time information on registered ili cases and virological laboratory results are available from the weekly updates in the nic, mongolia website: http://www.flu.mn/eng/index.php?option=com_ content&task=category&sectionid=5&id=36&itemid=51) were used for analysis in relation to the previous seasonal influenza activities in the country. influenza viruses were detected in naso-pharyngeal samples from ili patients by rt-rt-pcr with applied biosystems fast real time pcr system 7500, using primers and instructions supplied by cdc, usa. 1 influenza viruses were isolated by inoculation of rt-rt-pcr-positive samples of mdck cell culture according to the standard protocol. 2 ten representative strains of a(h1n1)pdm viruses were selected for sequencing of different gene segments, namely: a ⁄ ula, and a ⁄ dundgovi ⁄ 381 ⁄ 2010. sequencing of influenza virus gene segments was performed in applied biosystems 3130xl genetic analyzer using primers and instructions supplied by cdc, usa, 3 and bioinformatic analysis was performed with abi ⁄ seqscape v.2.5 and mega4 programs. the pandemic alert in mongolia was announced by the government on april 28, 2009, just after the who announcement of the pandemic alert phase, and planned containment measures were intensified. despite intensive surveillance, no a(h1n1)pdm virus was detected in mongolia until the beginning of october 2009. around 40 suspected cases, mostly arriving from the a(h1n1)pdm epidemic countries, tested zero by rt-rt-pcr for a(h1n1)pdm virus. the first a(h1n1)pdm case detected by the routine surveillance system in ulaanbaatar city, the capital of mongolia, was confirmed by rt-rt-pcr on october 12, 2009 (41st week of 2009). the reported ili cases escalated rapidly, reached the peak in the 43-44th week of 2009, and gradually decreased thereafter ( figure 1 ). week of 2010. however, the registered ili cases increased again from the 5th week of 2010, and peaked at the 8-9th weeks of 2010. the viruses isolated during this 2nd peak were influenza b strains ( figure 1 and table 1 ). for the genetic characterization of the mongolian pandemic isolates, 3 gene segments i (pb2), 2 gene segments ii (pb1), 3 gene segments iii (pa), 9 gene segments iv (ha), 3 gene segments v (np), 4 gene segments vi (na), 7 gene segments vii (m), and 3 gene segments viii (ns) of the representative a(h1n1)pdm mongolian strains were sequenced, and all sequences have been deposited in the genbank (accession numbers: cy050844, cy0533648, cy050846, cy050845, cy065987, cy065989, cy065990, cy 065988, cy065991, cy065985, cy065984, cy065986, cy052366 , cy053365, cy054546, cy054547, cy055169, cy 055170, cy055171, cy056363, cy055308, cy057191, cy057083, cy065995, cy065997, cy065998, cy065996, cy 065999, cy065993, cy065992, cy065994, cy054548, cy054549, cy073448). all 8 genes of mongolian strains were possessing 99ae4-99ae9% similarity with the genbank deposited gene sequences of the original pandemic strain a ⁄ california ⁄ 072009(h1n1). the who declared the pandemic alert phase (phase iv) on april 27, 2009, 4 and was prompted to announce the pandemic phase (phase v) two days later. 5 after 43 days, the who declared the beginning of the pandemic peak period (phase vi) on june 11, 2009. 6 however, in mongolia, the pandemic alert period continued for 168 days. mongolia was free of the pandemic virus during the whole first wave of the pandemics in the northern hemisphere. with the confirmation of the 1st influenza a(h1n1)pdm case on october 12, 2009 in ulaanbaatar, mongolia entered into the pandemic phase (phase v), and after just 2 weeks, the registered ili cases peaked, confirming mongolia shifted into the pandemic peak period (phase vi), which i i i v i i v i v v v i i i i iii iv v vi vii viii worldwide by who in mongolia coincided with the 2nd wave of pandemics in many countries of the northern hemisphere (see, picture 1 and table 1 ). despite the relatively milder clinical manifestations, the disease burden for the health service was enormous, while the morbidity per 10 000 population at the peak period was 5-6 times higher above the upper tolerant limit, and 3-4 times higher above the seasonal influenza outbreaks. in contrast to the seasonal influenza outbreaks where over 80% of the registered ili cases have been in the age group under 15, 2 it has been observed that over 50% of the registered cases in this pandemic peak period were in the age group of 16-60. on january 18, 2010, we regarded the pandemic had entered into the post-peak period (phase vii) when the registered ili cases became lower than the upper tolerant limit, during which time mongolia experienced an influenza b outbreak. on may 24, 2010 we determined that mongolia entered the post-pandemic period (phase viii) as the influenza virus isolations were almost stopped, and after no pandemic virus detected for 3 months. who announced pandemic vii and viii phases much later. 7, 8 this first ever real-time laboratory confirmed influenza pandemics in mongolia and confirmed some variations of pandemic spread in different parts of the world. the comparison of deduced amino-acid sequence changes have shown that the mongolian strains belong to the clade 7, according to the classification of a(h1n1)pdm influenza strains suggested by m. nelson, 9 which has circulated worldwide since july 2009. this is also evidence that the 1st wave of the pandemics did not hit mongolia. the who public health research agenda for influenza 1 is aimed to support the development of evidence needed to strengthen public health guidance and actions essential for limiting the impact of influenza on individuals and populations. each stream-specific group reviewed and discussed the proposed organization, content, rationale, and global health importance of their designated research stream. specific research recommendations were made for topics within each stream: background: a syndromic surveillance system using nonclassical data sources for detection and monitoring evolution of flu and flu-like illness (ili) in djibouti is reported here as part of the preliminary report of djibouti who-copanflu international study (wcis)**. methodology: clinical reports, over-the-counter drug sales, lab diagnosis report, and health communication trends were obtained for an integrated statistical analysis. results: transition to winter is concomitant with upsurge of ili cases and ili drug sales. in addition, more rural folks manage ili infections on self medicament than through clinical consultancy. inefficient and vague data collections were observed. a successful implementation of wcis will create a platform upon which challenges faced in djibouti health department in routine surveillance will be addressed to achieve a near-real time surveillance of flu pandemic. conclusion: innovations, prompt reporting, and instituting open source syndromic surveillance system software's in resource limited environment like djibouti will enhance early detection and evolution monitoring of pandemic flu. the spanish flu in 1918 ⁄ 1919 infected and killed millions of people, and threatened to wipe humanity off the face of planet. however, the recent scenarios of influenza h1n1 (2009) pandemics' worldwide occurrence fell short of most scientific prediction on its magnitude and intensity. this dampened their confidence; they cannot state precisely as to when, how, where, and which of the spanish flu-like pandemic will occur in the future. in support of scientific community and governments, the who hasn't gone to slumber, but is reminding its member states to up their post pandemic surveillance and monitoring of influenza virus in circulation for advance preparedness in case of an outbreak. 1 despite all uncertainty around the pandemic flu h1n1 2009, there remains a common knowledge and understanding that this flu has shown a great potential to evolve and cause huge morbidity and mortality. although its future magnitude may be unpredictable, its recurring events have severe consequences on human health and the economic well being of everyone. 1 and therefore, advance planning and preparedness is critical in protecting any population in the future, especially those located in resource limited environment without universal health cover and generous disaster emergency funds. 2 . two collapsed sets of a weekly and monthly mean data (of four years period) were clustered in five categories of ili cases, drug sales, lab results, vaccine consumption, and health promotion. this was followed by a descriptive statistics analysis of cumulative weekly and monthly data to establish presence or absence of trend. time series analysis was not done due to data limitation. copanflu program: as at the time of going to press, the cohort study is at the household recruitment and inclusion phase and the study covers the djibouti city. it is in our intention to use the cohort study findings to validate or improve the niph ministry of health djibouti ili surveillance effort for better preparedness. clinical service: 83% of all health facilities are in djibouti city. of the 39ae9% (326 445) of the population that seeks medical care on influenza and influenza like illness each year, 58ae2% (187 586) and 11ae9% (38 389) of them are attended to at the city's public and private clinics, respectively ( figure 1 ). the rest are attended from the regional health centers. the majority of ili incidence sharply rise with the onset of the winter season (october to april), affecting mostly the middle age group (1-24 years). pharmaco-surveillance: 2% (99 350) of total prescriptions were antipyretic and antiflu drugs, 91ae4% (90 765) of which were consumed by peripheral regions, the non djilab diagnostics: the annual ili lab diagnosis was negligible 0ae0072% (250), which can be attributed to less equipped virology laboratories to warrant routine service utility. documented cases were from previous bouts of avian influenza that had a human incidence from 2006 and 2009. 5 with support of egypt-based naval army medical research unit three (namru 3), clinicians were motivated to sample all ili patients and submit to collaborating international reference influenza lab in cairo, egypt. vaccination: influenza vaccinations were undocumented, but at least 0ae4% (3122) of population sought the service (for yellow fever and meningitis) as mandatory travel advisory or as childhood immunization need. at the time of going to the press, there were at least 80 000 vaccine doses of h1n1 (2009) virus donations yet to be administered. health promotion and hygiene: print and audiovisual risk communication remained favorite means of reaching out to urban dwellers (70ae6%). while to the rural and nomadic population, person to person communications was the preferable means. to increasing public awareness that will encourage reporting of ili cases and entrench risk aversion health behavior that limits flu spread, who-copanflu international study djibouti has incorporated basic training on ili infection and personal hygiene by interviewers during household inclusion. improving national epidemic surveillance capacity and response under new international health regulation 6 is important for any nation, including djibouti. our finding indicates the winter season predisposes one to ili infections; they therefore opt for medical services or self medication depending on their capability and ⁄ or understanding. in djibouti, almost no city dwellers favors self medication over clinical consultation, suggesting the presence of inhibitory factors like distance from the health centers and the cost of accessing consultancy. common in the absence of universal primary health care setting, it therefore calls for active innovativeness in outbreak detection, disease reporting, and preventive medicine on the part of health authority so as to achieve good population health. 7 in respond to these, niph has turned resource limitation to a motivation instead and is working towards institutionalizing a near-real-time syndromic surveillance system as a core functional unit. it capitalizes on three major aspects within its reach: prompt accurate data generation for analysis, ehesp wcic-study input, and information technology use. prompt accurate data generation for analysis: data used in our analysis suffered from un-timeliness (weekly instead of daily basis), incompleteness (vague over-counter drug sales records), entry errors (incidence case reports), and poor collection format (most of data collection forms). use of satellite handset phones for regional health centers and mobile phones for city sentinel clinics will reduce unnecessary data delivery delays. in addition, creating awareness to data entry personnel on the importance of careful and completeness of entries is important, as is the need to reformat data collection forms to capture exact aspects of surveillance needs for relevant executable analysis. 8 besides alerting for immediate impending epidemics, these data can also be adopted for projective predictive modeling of annual epidemics, including that for influenza. 9 ehesp wcic-study input: djibouti wcic-study is complementary to the existing syndromic surveillance system, but with emphasis on flu and flu-like illness. various innovations as suggested above are used in seeking to overcome the prevailing challenges. while every attempt is made to realize its (wcic-study) objective and for global comparison, lessons learned from successful implementation will form a platform for future refined syndromic surveillance protocol as equally reported elsewhere in asian countries. 7, 10 information technology: national institute of public health djibouti has an informatics department with sufficient working pcs and personnel to execute efficient data collection and management for epidemiological analysis. however, licensing cost of near-real time syndromic surveillance software is prohibitive, but the open access software with capacity to generate custom graphs, maps, plots, and temporal-spatial analysis output for specific syndromes should make implementation a lot easier. such output for conditions like flu (or gastroenteritis) will be essential to cause prompt response of the local public health office and international partners in saving lives and suffering of djibouti people. 11 pandemic flu surveillance and preparedness requires multifaceted, interdisciplinary, and international approach whose efficiency and efficacy can only be refined over time. building on the health care system's swot for preparedness, the ehesp wcic-study promises to refine surveillance system operation and knowledge on individual's risk determinants to swine flu (h1n1) 2009 virus infection at the household level in djibouti. these efforts are ultimately creating available control options at the time of need (pandemic occurrence), and at the same time exploring investment in quality data profiling and information technology, which will include syndrome surveillance software systems like essence, ewors, or other open sourced ones. the antibody efficacy -which compares the illness frequency between those with and those without a protective level of pre-epidemic hi antibodies ( ‡1:40) -has been proposed 1 ; however, this index has rarely been used due to practical difficulties in confirming the strain-spe-cific disease corresponding to each of the vaccine-induced antibodies. we followed 114 elderly individuals residing in a nursing home, whose serum specimens were obtained before and after undergoing trivalent influenza vaccination, in 2002 ⁄ 2003 influenza season (medium-scale mixed [a ⁄ h3n2 and b] epidemic in study area, and a ⁄ h3n2 was circulating at the nursing home). 2 the serum antibody titre to each strain of influenza virus was measured by the hi method, using the same antigens as those in the vaccine. all participants' body temperatures, respiratory symptoms, other general symptoms, hospitalization, discharge, and death were recorded daily from 1 november 2002 to 30 april 2003 in a prospective manner. when the participants suffered any influenzalike symptoms, such as sudden fever ‡37ae8°c, throat swabs were collected and tested using a rapid diagnosis kit for influenza, which utilizes an immunochromatographic method. the adjusted odds ratios (or adj ) for febrile illness and kit diagnosed influenza were evaluated using multiple logistic regression models adjusting for possible confounders (i.e., age, sex, coexisting conditions, and vaccine strains). after vaccination, the proportion of subjects achieving an hi antibody titre ‡1:40 (seroprotection level) were 61ae4% (51ae8-70ae4%) for a ⁄ h1n1, 79ae8% (71ae3-86ae8%) for a ⁄ h3n2, and 26ae3% (18ae5-35ae4%) for b. during the follow-up period, the a ⁄ h3n2 strain was isolated therein, and 44 subjects experienced sudden-onset fever ( ‡37ae8°c), and eight subjects were positive for rapid diagnosis kit. patients with a seroprotection level of the hi antibody titre ( ‡1:40) had lower incidences of febrile illness (or adj , 0ae35; 95% ci, 0ae09-1ae28) and rapid kit diagnosed influenza (or adj , 0ae35; 95% ci, 0ae03-4ae64) than those with a lower titre. thus antibody efficacy (1 ) or adj ) against fever related to a ⁄ h3n2 and kit diagnosed influenza were both estimated to be 65%. although statistical significance was not detected due to limited sample size, these results lend support for the usefulness of antibody efficacy. some data presented within this manuscript was also published in hara et al. asia via a regional network from which epidemics in the temperate regions were seeded. 1 the virus isolates obtained from nasopharyngeal swab specimens from outpatients were typed and subtyped by the hemagglutination (ha) inhibition assay. 2 the emergence of a ⁄ fujian ⁄ 411 ⁄ 2002 coincided with higher levels of influenza-like illness in korea than what is typically seen at the peak of a normal season. most of the intermediates and fujian-like strains were isolated from asian countries, and the mutational events associated with the fujian strains took place in asia. closely dated phylogeny from december 26, 2001 to august 11, 2002 showed that the antigenic evolution of the h3n2 fujian strains had periods of rapid antigenic changes, equivalent to 10 amino acid changes per year ( figure 1 ). the fujian-like influenza strains were disseminated with rapid sequence variation across the antigenic sites of the ha1 domain. the antigenic evolution of the fujian strains was initiated by exceptionally rapid antigenic change that occurred in asia, which was then followed by relatively modest changes. some of the data presented in this manuscript was previously published in kang et al. we compared reactivity to the novel virus strain using haemagglutination inhibition (hi) assays performed on discarded plasma specimens left over from routine testing. samples were taken from healthy adult blood donors (>16 years) before and after the ph1n1 influenza epidemic that occurred during the southern hemisphere winter of 2009, and again prior to onset of the 2010 southern hemisphere influenza season. reactivity to the novel h1n1 2009 strain of influenza was relatively uncommon among the healthy adult population during the first australian winter wave, rising from a baseline of 12% to 22%. a further increase in the seropositive proportion from 22% to 43% was observed over the summer months, most likely attributable to immunisation. this level of immunity appears to have been sufficient to constrain the 2010 winter epidemic. together with a final serum collection, planned for late 2010, these data will aid evaluation of the extent and severity of disease in this 'second wave' of ph1n1. assessment of the extent of disease due to novel influenza a(h1n1) virus (ph1n1) during the 2009 winter outbreaks in australia was made difficult by the generally mild nature of disease. the epidemic was experienced in a staggered fashion around the country, 1 reflecting the considerable geographical distances between state and territory capital cities ( figure 1 ). differences in the intensity of case-finding during the evolving pandemic response and between jurisdictions hindered comparisons of disease burden in distinct geographical regions. 2 rates of reported hospitalisations and deaths appeared fairly similar across states 1 but, without a consistent exposure denominator, assessment of relative severity was difficult. we conducted a national serosurvey of antibody to ph1n1 using residual plasma from healthy blood donors collected before and after the 2009 epidemic to estimate ph1n1 exposure. 3 here we report the findings of that first collection, together with new data on seroprevalence of ph1n1 antibody in specimens gathered in march-april 2010. these latter samples were collected prior to onset of seasonal influenza activity to assess the impact of a national ph1n1 vaccine program conducted in spring ⁄ summer 2009 ⁄ 10 on the proportion of individuals with antibody titres deemed protective. 4 findings informed estimates of population susceptibility to ph1n1 prior to the 2010 influenza season and provided a baseline for a subsequent serosurvey that will be collected at the end of 2010 to assess the extent of exposure during the 'second wave.' tralian red cross blood service (the blood service) for dengue fever surveillance studies. these samples were used to provide a baseline estimate of prevalence of cross-reactive antibody to ph1n1 in the australian population. discarded plasma specimens, taken for virologic testing from healthy adult blood service donors, were prospectively collected at two additional timepoints for measurement of antibody to ph1n1. collection periods were as follows: approximately 120 plasma samples were randomly selected from donors in each of brisbane, hobart, melbourne, newcastle, perth, sydney, and townsville on each occasion. up to 20 specimens were identified in each of the following age strata: 16-24, 25-34, 35-44, 45-54, 55-64, and >65 years. at the last collection timepoint, there was deliberate over-sampling of the oldest and youngest age strata in which approximately 40 specimens were collected (i.e., up to 160 specimens per site). in accordance with the provisions of the national health and medical research council's national statement on ethical conduct in human research, individual consent was not required for use of these specimens, given the granting of institutional approval by the blood service human research ethics committee. reactivity of plasma against ph1n1 was measured in haemagglutination inhibition (hi) assays using turkey red blood cells (rbc). 5 egg-grown a ⁄ california ⁄ 7 ⁄ 2009 virus was purified by sucrose gradient, concentrated and inactivated with b-propiolactone, to create an influenza zonal pool preparation (a gift from csl limited). plasma samples were pretreated with receptor destroying enzyme ii (denka seiken co. ltd), 1:5 (volume ⁄ volume) and tested as previously described. 6 following 1 hour incubation, 25 ll 1% (volume ⁄ volume) of rbc was added to each well. hi was read after 30 minutes. any samples that bound to the rbc in the absence of virus were adsorbed with rbc for 1 hour and reassayed. samples in which background activity could not be eliminated by these means were excluded from the analysis. titres were expressed as the reciprocal of the highest dilution of plasma where haemagglutination was prevented. a panel of control sera and plasma samples was included in all assays. it comprised paired ferret sera pre-and postinfection with the pandemic virus or seasonal influenza a(h1n1), a(h3n2), or influenza b viruses and paired human plasma and sera collected from donors before april 2009 or after known infection with the pandemic virus or after immunisation with the australian monovalent pandemic 2009 vaccine. all assays were performed by the who collaborating centre for reference and research on influenza. for each of the three study timepoints and within each age group, the proportion of seropositive individuals (hi titres ‡40) was calculated, with exact (clopper-pearson) confidence intervals. the contribution of individual variables (age, gender) and location to seropositive status was assessed in separate multivariate logistic regression models developed to assess the post-pandemic and pre-influenza season 2010 collections. all statistical analyses were conducted in stata 10. locations of specimen collection are shown in figure 1 , together with the number of samples tested from each centre. samples with high background hi titres or discrepancies between assays were excluded at each timepoint as follows: 5 at baseline, 27 from the post-pandemic collection, and 0 in early 2010. pared with baseline was 10% overall, rising from 12% to 22% (table 1 ). the only jurisdictions in which seropositive proportions were higher in october ⁄ november than in the baseline collection were hobart [31% (95% ci 22ae3, 39ae7)], perth [24% (16ae3, 31ae7)], and sydney [24% (16ae3, 31ae7)]. in the multivariate regression model, the only jurisdiction in which exposure appeared somewhat higher than the reference population of brisbane was hobart [or 1ae83 (95% ci 0ae99, 3ae4), p = 0ae06]. a marked age effect on antibody status was observed at this timepoint, with an increase in the proportion of seropositive individuals in relation to the baseline collection only noted for those aged between 16 and 34 years (table 1) . according to the multivariate model, the youngest and oldest cohorts had similar titres, with all other groups showing significantly lower seropositive proportions than the reference population of 16-24 years [e.g. 45-54 years or 0ae36 (95% ci 0ae21, 0ae64, p < 0ae0001)]. an overall increase in the seropositive proportion from 22% to 43% was observed between october 2009 and april 2010, distributed throughout all jurisdictions ( (17, 25) ]. antibody titres prior to the 2010 influenza season rose in all age groups, but remained significantly lower among [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] year olds than in the youngest age cohort (table 1) . adjusted ors for the seropositive proportion in the multivariate model in these age groups were: 35-44 years [or 0ae49 (95% ci 0ae31, 0ae77)]; 45-54 years [or 0ae48 (0ae31, 0ae75)]. the relatively low titres observed in these groups reflected small incremental increases in the seropositive proportion across each of the time points studied, suggestive of both low rates of infection and vaccination. the rise in immunity observed across the population was most likely attributable to immunisation in the majority, given the absence of observed outbreaks and very few notified cases of ph1n1 during the period between the two plasma collections. this study suggests that, while adult exposure to ph1n1 during the 2009 southern hemisphere winter was uncommon at around 10%, vaccine uptake in the australian population over the period november 2009-may 2010 was in the order of 20%. this latter estimate is in keeping with recently published figures for adult ph1n1 vaccine coverage from a national immunisation survey conducted by the australian institute of health and welfare. 4 in that survey, vaccine coverage was significantly higher in tasmania than in other states, but mostly in those over 65 years of age, possibly in a subgroup whose health status may have differed from that of the donor population. 4 no allowance has been made in this analysis for likely waning of natural or vaccine induced immunity, possibly resulting in lower estimates of natural and ⁄ or vaccine exposure than may have occurred over the period. regardless of such intervening processes, the seropositive proportion among australian adults at the start of the 2010 winter season appeared likely to be sufficient to constrain transmission of infection in the age groups tested. this assertion has been borne out in practice, with only modest levels of influenza reported during the late and protracted 2010 season. 7 a final serum collection is planned for the end of the 2010 influenza season in australia from which to assess the level of exposure in relation to the baseline observed here. the need for epidemiologic studies such as this has been highlighted by groups such as the european centre for disease control to aid evaluation of the extent and severity of the 'second wave,' 8 known to be variable from historical reports of past pandemics in disparate populations. 9 in 2009-2010, the first wave of the swine-origin novel h1n1 flu (h1n1) pandemic swept across the world, including japan. to examine the epidemiological nature of this novel infectious disease among school children within and among small regional communities, we have carried out a complete survey on the incidence of h1n1 among school children using absentee reports provided by school health teachers in two small administrative districts (population: about 140 000 in total) in japan. we then examined the epidemiological diversity on the inci-dence of h1n1 within and among small regional communities. we investigated seventeen elementary and ten junior high schools in moroyama-town and sakado-city located in the central part of saitama prefecture. populations are: all ages, 37 015 and 100 634; elementary schools, 1873 and 5389; junior high schools, 1131 and 2568, respectively. the number of school children in each school ranges from 137 to 876. the surveillance system was built on an apache-and mysql-based web server using html, php, and java-script. 1 school health teachers enter information on children absenteeism due to school infectious diseases via web browsers at each school infirmary on a daily basis. in addition to the trend graphs shown on the web browser, detailed analyses were reported to the schools and local educational boards weekly. the basic reproduction number (r 0 ) of h1n1 was estimated according to becker. 2 agentbased modeling and simulations were also performed using a multi-paradigm simulator anylogic version 6.5 (xj technologies, st. petersburg, russia). by the end of march 2010, cumulative incidence (ci) of h1n1 among school children in moroyama and sakado reached 30% and 34%, respectively. the overall r 0 among school children in this area was 1ae43. vaccination rate of children in this area during the surveillance period was reported to be very low (<10%). there was no considerable difference between the epidemic curves in this neighboring town and city. on the other hand, in the individual schools, the cis as of the end of march 2010 scattered from 16% to 51% ( figure 1 ) even though the schools are closely located. to examine the cause of this diversity, we built an agent-based community model 3 consisted of the same numbers of agents as those of children in the actual schools and people in moroyama and sakado to simulate the infection. the ratio of probability of infection in schools and the remaining places were assumed to be 1:1 or 10:1. using a heuristic optimization scheme, we estimated the parameters for the simulations to give the overall ci of 33% (the ci as of the end of march 2010). we then performed simulations repeatedly. the cis obtained with the repetitive simulations with the assumption of higher probability of infection in schools scattered from 23% to 44%, indicating that the cis of the small population communities may vary considerably, even though all the agents were assumed to have the same susceptibility to infection at the beginning, and the other conditions were the same. the policies for surveillance ⁄ analyses ⁄ prevention of communicable diseases in local communities have generally been decided on governmental-and ⁄ or each local administrative district-basis (populations: several hundred thousands to several millions) in japan. we found the considerable variations in the cis of h1n1 for children among much smaller areas, i.e., the school districts (populations: all ages, several thousands; school children, several hundreds). we thus conclude that the granularity of surveillance ⁄ analyses ⁄ prevention should be finer than in the past to achieve the most effective policies against influenza and similar communicable diseases in the local communities. the cause of this diversity can be explained in part by the stochastic nature of infection transmission processes in the small populations shown by the agent-based simulations. we have already conducted a complete questionnaire survey for the school children and their parents to clarify the relevance of the other issues including differences in environmental factors, preventive policies (e.g., vaccination, school closures), etc., in each school. the detailed analyses will be reported elsewhere. a www-based surveillance system for transmission of infectious diseases among school children within and among small regional communities. j epidemiol 2010; 20(s1):s151. this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. the first cases of the 2009 a ⁄ h1n1v pandemic influenza were reported in mexico and the united states in april 2009. given the context of this new influenza virus and considering the likelihood of its pandemic spread, the cohorts for pandemic influenza (copanflu) international consortium was created in order to study individual and collective determinants of pandemic a ⁄ h1n1v influenza across different countries by setting up prospective cohorts of households, followed during 2 years. this study relies on the first available data from the copanflu-france project, which is part of the copanflu international consortium. we studied factors associated with elevated haemagglutination antibody titers against a/h1n1v at entry in the copanflu-france cohort. we focused in this primary analysis on the association between the titers and influenza vaccination (seasonal or pandemic) across age groups. the copanflu-france cohort was set up in fall 2009. inclusions began on december 4, 2009 and ended on july 23, 2010. households were sampled using a random telephonic design (mitofsky-waksberg method) 1 in a stratified geographical sampling scheme, aimed at including a sample of subjects representative of french general population. all household members were eligible to the cohort, without any age limit. the inclusion of a household required the participation of all members: the refusal of one or more member(s) prevented the inclusion of other members. the protocol was approved by a research ethics committee and written informed consent was obtained for all subjects. this study requires several visits to the households by nurses who collect written data with questionnaires and biological samples. during the inclusion visits, nurses collected from all subjects detailed data regarding medical history, including vaccination and preventive measures against influenza. blood samples were collected at entry and centralized. a standard hai technique was adapted to the detection and quantification of antibodies to the 2009 a ⁄ h1n1v virus. 2 the titration endpoint was the highest dilution that exhibited complete inhibition of haemagglutination in two independent readings. the lowest read dilution was 1 ⁄ 40. geometric mean titers were calculated for hai assays with the use of generalized estimating equations for interval-censored data, 3, 4 taking into account a within-household correlation. multivariate models were derived from this method to identify factors associated with elevated gmts. we defined the ''gmt ratio'' (gmtr) as the multiplicative factor applied to the gmt in presence of an explanatory variable. for qualitative explanatory variables, a gmtr of n means a predicted n-fold higher gmt for subjects exposed to the considered factor compared to others. for continuous explanatory variable, the same interpretation applies to a unit difference. the following variables were included in the multivariate models: age, history of pandemic or seasonal influenza vaccination, and history of ili. age was categorized in three groups: 0-15 years (reference group), 15-50 years and over 50 years. the definition of ili was that used by the cdc 5 : fever ‡37ae8°c and cough and ⁄ or sore throat without another known cause. history of ili was defined as an ili reported by the subject between september 12, 2009 (beginning of the influenza epidemic in france) and the date of inclusion. this preliminary analysis included 1304 subjects belonging to 544 households. results reported hereafter do not account for missing data. participating households were sized 1-7 subjects, mean size = 2ae4. in comparison, the mean size of french households is 2ae3 according to the latest national census. 6 the median age of subjects at entry was 40ae6 years [iqr: 16ae5; 58ae6] versus 34ae4 [15ae2; 52ae5] for french population. 7 the proportion of subjects reporting a history of ili since the beginning of the epidemic varied from 2ae9% for subjects over 50 years to 9ae1% for subjects below 15 years (table 1) . vaccination with the pandemic strain was the highest in subjects below 15 (16%) whereas vaccination with the seasonal strain was the highest in subjects over 50 (44ae9%). detailed data regarding vaccination is given in table 1 . this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. [8] [9] [10] [11] [12] [13] among non-vaccinated subjects, elevated gmt in the elderly may be the result of exposure to similar viruses in early life, whereas children and young adults with elevated gmt are likely to have been infected by the 2009 a ⁄ h1n1v virus. [13] [14] [15] [16] interestingly, a significant drop in the hai titer is observed during the months following vaccination with the pandemic strain. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. the reason for this association is not obvious: although we cannot discard the hypothesis of a higher incidence of a ⁄ h1n1v infections in seasonal vaccine recipients, as described by several other studies, 17-19 the main explanation may be a cross-reaction between pandemic and seasonal strains. 16, 20, 21 further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. in april 2009, the cdc alerted about the appearance of a new strain of ia h1n1 with unknown virulence. infants under 5 years old had higher risks of hospitalization, complications, and rate of death for sari. materials and methods: a cross-sectional study was executed from may to december in 2009. the sources were: mandatory reporting form of the province surveillance system, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports. inclusion criteria: children under 5 years old with diagnostic of ili or sari and confirmed cases with epidemiological nexus or laboratory confirmation (rrt-pcr, ifi). the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. results: the ili rate in infants under 5 years old was 1295ae19 ⁄ 100 000 people (95% ci 1236-1356) being higher in infants of 4 years old (1569 ⁄ 100 000 people of 4 years (95% ci 1426-1722) ( table 1) . infants had less risk of getting sick in relation to the rest of the population (rr 0ae40 [95% ci 0ae33-0ae47]) (p < 0ae05). the chance of sari in infants was 2ae89 (95% ci 2ae46-3ae39) compared to the rest of the population. the lethality rate was higher in infants under 1 year old (14 ⁄ 100 000 people [4 ⁄ 28 364]). discussion: the evidence suggests that the infants under 5 years old had lower risk of getting sick than the rest of the population, but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under 1 year old. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. in april 2009, the cdc alerted about the appearance of a new strain of ia h1n1 with unknown dissemination and virulence. in june, the world health organization declared the pandemic. 1, 2 the ili often presents an unspecific clinical picture in infants under 5 years old, from mild symptoms to sari, especially in the newborn babies. infants under 5 years old have higher risks of hospitalization, complications, and rate of death for sari. 3, 4 on may 7th, argentina declared the first imported case of ia h1n1, and by the end of the month, it announced the viral circulation in the country. the epidemiological surveillance system of the province arranged that all the patients with influenza diagnosis made by a doctor must be reported. from april 24th to november 14th, 20 212 suspected cases of ili in the province of tucumán were reported. the ili rate was 1353 ⁄ 100 000 people, and ia h1n1 comprised 40 ⁄ 100 000 people. the lethal rate of sari ia h1n1 was 306ae7 ⁄ 100 000 people (62 ⁄ 20 212). 5 the objective of this research was to determine the epidemiological characteristics of the pandemic ia h1n1 in infants under 5 years old in the province of tucumán between may and december in 2009. the province of tucumán is placed in the center of the northwest of the republic of argentina. it has a population of 1 493 488 inhabitants of which 138 821 are infants under 5 years old. the crude birth rate for 2008 was 19ae9&. the infant mortality rate was 13ae8&. respiratory pathologies in infants under 5 years old were the third cause of death in the province (12%). the public health system of the province is composed by three sectors: public, private, and welfare. with 91 health facilities as a total, the average of available beds is 3& per inhabitants and 6& per neonates. a cross-sectional study was executed from may to december in 2009 in the province of tucumán, argentina. the following sources were used: mandatory reporting form of the surveillance system of the province filled by a doctor, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports (patients with sari). inclusion criteria: • suspected case of ili: sudden appearance of fever higher than 38°c, cough, or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nausea or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea. ) were used for the analysis. the odds rations, risk ratio and 95% confidence interval were calculated to compare ambulatory with hospitalized patients, confirmed and dismissed, <5 years old and the rest of the population. it was considered significant a rate of p < 0ae05. the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. the epidemiological surveillance system of the province received 21 012 ili reports, 11ae77% (2474 ⁄ 21 012) were infants under 5 years old. twenty seven percent were dismissed (676 ⁄ 2474), and 34% (623 ⁄ 1798) of suspected cases were confirmed. the first ia h1n1 case was a child of 2 years from the province of buenos aires, in 22th epidemiological week, and the last suspected case was reported in october 26, 2009 ( figure 1 ). the ili rate in infants under 5 years old was 1295ae19 ⁄ 100 000 people (95% ci 1236-1356), being higher in infants of 4 years old (1569 ⁄ 100 000 people of 4 years, [95%ci 1426-1722]). the higher ili rates in confirmed the pandemic of ia h1n1 (2009) was detected for the first time in the province of tucumán. the evidence suggests that infants under 5 years old had lower risk of getting sick than the rest of the population (protective factor), but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under 1 year old. towns with the highest demographic density had superior proportion of cases. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. outbreak of h1n1 influenza -2009: behavior of influenza h1n1 in school children in the province of tucumá n, argentina criteria: patients treated with antiviral medication for prophylaxis, respiratory pathologies which did not justify specific medication, and incomplete forms. results: from all notifications, 6342 were cases of ili in the group aged 5-17 years old; 53% were males. the incidence rate in this group was 17ae4 per thousands of inhabitants. the 13% of laboratory samples were influenza a h1n1, 34% were confirmed as unspecific influenza, and 49% were dismissed. the school aged children group had a high risks of getting sick (r.r. 1ae78 [95% c.i. 1ae55-2ae04]), especially males. it appeared that school aged children had a protective factor for presenting sari (or 0ae66 [95% c.i. 0ae66-0ae89], p < 0ae05). the lethality rate in this group was 9ae32 ⁄ 10 thousands. headaches, myalgia, coryza, and sore throat were very common and significantly different (p < 0ae05) than the rest of the population. it was reported a decrease in the ew 28 coinciding with winter holidays (ew 27). the epidemic curve was different in males compared to females during the winter holidays. discussion: school aged children got sick more than the rest of the population, although they presented less proportions of sari. however, comorbidities were decisive in order to present sari or death. the epidemic curve was different in males compared to females. through its analysis, the beneficial effect of school closure was observed, as long as children meet the recommendation to stay home. in april 2009, different countries reported cases of influenza a h1n1; mexico reported a high mortality rate associates with this disease. 1 the world health organization (who) declared the phase 6 influenza pandemic alert on june 11. 2 several reports from different countries describe the behavior of the pandemic in school aged children. this group plays an important role in the transmission of influenza. in germany, during the summer peak, pandemic hardly spread within this group. this might be explained by the timing of the summer school holidays, which started between ew 27 and 31. since mid october, after the autumn holidays, the school-aged children began to be more affected, and the proportion increased from 16% in the initiation period to 43ae8% in the acceleration period. 3 in australia, 55% of h1n1 cases were school aged children (5-17 years), with a median age of 16 years (29% of cases were aged 13-17 years and, and 26% between 5-12 years). 4 in canada, the infection rate was highest in this group. 5 in chile, the incidence rate was 4500 ⁄ 100 000 inhabitants, although in general they had mild desease. 6 school closure can operate as a proactive measure, aimed at reducing transmission in the school and spread into the wider community, or reactive, when the high levels of absenteeism among students and staff make it impractical to continue classes. the main health benefit of proactive school closure comes from slowing down the spread of an outbreak within a given area and, thus, flattening the peak of infections. this benefit becomes especially important when the number of people requiring medical care threatens to saturate health care capacity. it has its greatest benefits when schools are closed very early in an outbreak, before 1% of the population falls ill. school closure can reduce the demand for health care by an estimated 30-50% at the peak of the pandemic under ideal conditions, but too late in the course of a community-wide outbreak, the resulting reduction in transmission is likely to be very limited. policies for school closure need to include measures that limit contact among students when they are not in school. 7 tucumán is placed in northwest argentina and has a total area of 22 524 km 2 . the population (2001 census, projection 2009) was 1 493 488 inhabitants; of wich 421 638 were 5-19 years old. the health system of the province is composed of 3 sectors: public, private, and welfare. it has a total of 91 health facilities with internement available and an average of 3 & inhabitants. influenza-like illness (ili) has seasonal and endemic behavior in this province, as evidenced by past records from the national health surveillance system and influenza sentinel surveillance unit of the province. an increase of ili was reported in 2009, with a peak in the ew 28. the objectives were: general objective to describe the behavior of the influenza a h1n1 2009 epidemic in school aged children from the province of tucumán, argentina. specific objectives • to explore the response to preventive measures by school aged population. • to assess the effect of the suspension of classes in this group. • to estimate the magnitude and severity of the disease. • to observe the effect of co-morbidities in this group. a cross-sectional study was executed from may to december 2010. data were gathered through mandatory reporting forms, wich were collected from all public and private health centers. inclusion criteria: patients with compatible symptoms with influenza a; school aged children 5-17 years old. exclusion criteria: patients treated with antiviral medication as prophylaxis, respiratory pathologies which did not justify specific antiviral medication, and incomplete forms. • suspected case of ili: cases considered by clinical criteria (fever higher than 38°c, cough or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nauseas or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea). • confirmed case: person with positive laboratory results for influenza a h1n1 or unspecificed influenza a (by laboratory results through rrt-pcr or immunofluorescence techniques). • dismissed case: by negative or different laboratory results, or different clinical evolution. • comorbidities: chronic illnesses like arterial hypertension, diabetes, asthma, recurrent obstructive bronchial syndrome (robs), smoking, chronic obstructive pulmonary disease (copd), immunosuppression, hiv ⁄ aids, cancer, nephropathy, obesity; pregnancy was also considered. data were analyzed using epi2000 software (epi infoô cdc, atlanta, eeuu). rates were calculated and rr was estimated with their respective confidence interval (ci). population data were taken from 2001 national census projections. an estimation based on the same census was used for the group between 5 and 17 years old. to observe the effects of other co-variables, the or and their ci were calculated. logistic regression was used to evaluate the influence of the comorbidities. x 2 was used to compare proportions. respiratory samples (nasopharyngeal and faryngeal swabs) were obtained. they were analyzed at influenza sentinel surveillance unit of tucumán, and ⁄ or sent to national reference laboratory dr. c. malbrán (rt-pcr). from all notifications (20 212), 6342 were cases of ili in the group aged between 5 and 17 years old, 53% (3340 ⁄ 6342) of which were males. the incidence rate was 17ae4, and it differed according to the sexes: 18ae0 males and 16ae7 females per thousands of inhabitants (p < 0ae05). of all laboratory samples (370) 13% were confirmed as influenza h1n1, 34% were confirmed as unspecificied influenza, and 49% were dismissed. the remaining percentage corresponded to the isolation of other viruses (parainfluenza, respiratory syncytial virus, and adenovirus). the school aged group had higher risk of getting sick, in relation to the rest of the population (rr 1ae78 [95% ci 1ae55-2ae04]), especially males (rr 1ae97) compared with females (rr 1ae66). the highest attack rate was observed in the capital of tucumán (42 ⁄ 1000 inhabitants). according to the rest of the population, it looked like being school aged children meant a protective factor for presenting sari (severe acute respiratory infection) (or 0ae66 [95% ci 0ae66-0ae89], p < 0ae05). the lethality rate was 9ae32 ⁄ 10 thousand. the risk of dying was low compared to other ages. persons with comorbidities had significantly higher risk of presenting sari (or 1ae8 [95% ci 1ae35-2ae58], p < 0ae05) and of dying (or 8ae1 [95% ci 19ae6-3ae34], p < 0ae05). respiratory comorbidities were the most frequent: asthma 2ae5% (162 ⁄ 6342) and 2% rors (122 ⁄ 6342). the symptoms headaches, myalgia, coryza, and sore throat were very common and significantly different (p < 0ae05) than the rest of the population. if we compared the group aged 5-17 years with 18-39 years old, the epidemic curve of the first group showed a decrease in the ew 28, coinciding with winter holidays (ew 27) (figure 1 ). there was a slight increase in the tendency when classes began, but it showed a clear declination afterwards. the analysis of rates in school aged children by ew showed a reduction of 22ae2% in males and 26ae6% in females (p < 0ae05) at ew 28. however, after the first week of winter holidays, the curve in males had a significant increased to 65ae6% compared to ew 27, reaching the highest weekly rate of the epidemic (18 ⁄ 10 000 inhabitants). the reopening of classes coincided with a significant decrease of the rate (34ae9%), from 18 to 11ae7 ⁄ 10 000 inhabitants in ew 31 (p < 0ae05). in females, the school closure coincided with a plateau-shaped curve, and the reopening with a significant decrease of 43ae4% of the rate, from 12ae1 to 6ae9 in ew 32 ( figure 2 ). the school children got sick a lot more than the rest of the population, although they presented less proportions of sari. however, comorbidities were determined in order to present sari or death. symptoms like headache, myalgia, coryza, and sore throat were considered more conducting for the definition of cases in this population in tucumán. the epidemic curve was different in males compared to females during the winter holidays. the beneficial effect of school closure was observed as long as persons met the recommendations. the difference between males compared to females during winter holidays could mean that women would have carried out social distance recommendations much better, for example, remained at home. the significant reduction after the opening of classes is a factor to be considered as an effective intervention in the declining stage of the curve. here, we report pdmh1n1 infection attack rate (iar) during the first wave of the pandemic. we used our iar estimates to infer the severity of the pandemic strain, including the age-specific proportion of infections that led to laboratory confirmation, hospitalization, intensive care unit (icu) admission, and death. [1] [2] [3] [4] part of these results are now available in ref. 5 subjects of a community study, 5-14 years old between 1 november 2008 and 31 october 2009, we conducted a cohort study of pediatric seasonal influenza vaccination and household transmission of influenza. one hundred fifty-one children aged 5-14 were recruited and provided baseline sera in november and december 2008. between september and december 2009 a further 766 children aged 5-14 were recruited and provided baseline sera for the second phase of the study. for this serologic survey, we tested the 151 sera collected before the first wave and the 766 sera collected after the first pandemic wave. written informed consent was obtained from all participants. parental consent was obtained for participants aged 15 or younger, and children between the ages of 8 and 15 gave written assent. all study protocols were approved by the institutional review board of the university of hong kong ⁄ hospital authority hong kong west cluster. age-stratified data on virologically confirmed outpatient consultations, hospitalizations, icu admissions, and deaths associated with pdmh1n1 from 29 april 2009 to 15 november 2009 were provided by the hong kong hospital authority (the e-flu database). 6 since may 2009, patients admitted with acute respiratory illnesses routinely underwent laboratory testing for pdmh1n1 virus by molecular methods. sera were tested for antibody responses to a ⁄ california ⁄ 4 ⁄ 2009 by viral microneutralization (mn). 7 most individuals infected with influenza develop antibody titers ‡1:40 by viral microneutralization after recovery. 8 we defined the pdmh1n1 seroprevalence rate as the proportion of individuals who had antibody titers ‡1:40. while mn antibody titers of ‡40 are not by themselves conclusive evidence for pdmh1n1 infection, we have assumed that the increase in cross-sectional seroprevalence between the pre-and post-first wave time periods are evidence of recent pdmhn1 infection. the iar was defined as the proportion of individuals infected by pdmh1n1 during the first wave. the case-confirmation rate (ccr), case-hospitalization rate (chr), case-icu-admission rate (cir), and case-fatality rate (cfr) were defined as the proportion of pdmh1n1 infections that led to laboratory-confirmation, hospitalization, icu admission, and death. due to containment efforts until june 29, 2009 all laboratory-confirmed cases were required to be hospitalized for isolation regardless of disease severity. as such, only surveillance data from june 30 onwards were used to estimate severity measures. we estimated the iar as the difference between the prefirst-wave and post-first-wave seroprevalence rate. we used the estimated iar as the denominator for calculating the ccr, chr, cir, and cfr. we used an age-structured sir model with 5 age classes (0-12, 13-19, 20-29, 30-59, and ‡60) to describe the transmission dynamics of pdmh1n1 in hong kong between 10 june and 15 november 2009. we assumed that the mean generation time was 2ae3 days. using the age-structured transmission model, we estimated the following transmission parameters from the serial cross-sectional serologic and hospitalization data: (i) r o , the basic reproductive number; (ii) p 1 and p 2 , the reduction in within-age-group transmission for 0-12 and 13-19 years old during summer vacation (compared to school days during september-december 2009); (iii) d r , the average time for neutralization antibodies titer to reach ‡1:40 after recovering from infection; (iv) h a , the age-specific relative susceptibility with 20-29 years old adults as the reference group. we assumed non-informative priors for all parameters and used monte carlo markov chain methods to obtain posterior distributions of the parameters. sources of specimens: [1] pediatric cohort study (2-29 april 2009 virological surveillance data suggested that the first wave of pdmh1n1 in hong kong occurred from august to october 2009. most of the laboratory-confirmed infections in this first wave occurred in individuals aged below 25 years old accounting for >72% of the lab-confirmed cases and hospitalizations, 32% of icu admissions, and 6% of deaths. taking into account a delay of 2-3 weeks for antibody titers to appear during convalescence, 8 we found that these virological surveillance data were consistent with our serial cross-sectional seroprevalence data, which indicated a sharp rise in seroprevalence among the 5-25 years old from september to november and a plateau thereafter (data not shown). among individuals aged 5-14 years, the seroprevalence rates were similar across time between pediatric outpatient subjects and pediatric cohort study subjects (data not shown). similarly, for older age groups, the seroprevalence rates were largely similar between blood donor subjects and hospital outpatient subjects (except for the 20-29 years old in november-december). this provided some evidence that despite biases in our convenience sampling scheme, the resulting serologic data provided a reasonably representative description of seroprevalence in the community. the estimated pre-and post-first-wave seroprevalence rates and the corresponding iar estimates are shown in table 1 . the severity estimates (ccr, chr, cir, and cfr) are shown in table 2 . in summary, we estimated the iar was 43ae4% among 5-14 years old, 15ae8% among 15-19 years old, 11ae8% among 20-29 years old, 4ae3% among 30-39 years old, 4ae6% among 40-49 years old, and 4ae0% among 50-59 years old. overall, we estimated a population-weighted iar of 10ae7% (9-12%) among individuals aged 5-59 years through the first wave in hong kong. ccr were around 2ae8-5ae4% among the 5-59 years old. chr were around 0ae47-0ae87% among the 5-59 years old. cir increased from 7ae9 (5ae2-12ae6) per 100 000 infections in 5-14 years old to 75 (32ae7-281) per 100 000 infections in 50-59 years old. cfr followed a similar trend with 0ae4 (0ae1-2ae3) death per 100 000 infections in 5-14 years old to 26ae5 (10ae4-109) deaths per 100 000 infections in 50-59 years old. compared to children aged 5-14, adults aged 50-59 were 9ae5 and 66 times more likely to be admitted to icu and die if infected. the best-fit age-structured transmission model gave the following parameter estimates: 1. the basic reproductive number was 1ae38 (95%ci, 1ae36-1ae41). 2. it took an average of 14 (9-21) days for recovered individuals to develop neutralization antibody titer ‡1:40. table 2 . estimated age-specific proportions of individuals with pdmh1n1 infections that were laboratory-confirmed, were hospitalized, were admitted to icu, and died. case-icu and case-fatality rates are expressed as number of episodes per 100 000 infections 3. compared to 20-29 years old, 0-12 years old children and 13-19 teenagers were 3ae7 (3ae2-4ae5) and 1ae6 (1ae3-2) times more susceptible to pdmh1n1 infection, respectively. 4. compared to 20-29 years old, 30-59 years old older adults and 60-79 years old elderly were only 0ae42 (0ae3-0ae6) and 0ae33 (0ae18-1ae5) times as susceptible as the 20-29 years old, respectively. 5. compared to the school period during september-december 2009, summer vacation reduced within-agegroup transmission by 61% (53-72%) among 0-12 years old, but only 12% (3-18%) among 13-19 years old. using computer simulations, we estimated that if preexisting seroprevalence is zero, real-time serologic monitoring with about 1000 specimens per week would allow accurate estimates of iar and severity as soon as the true iar has reached 2% (data not shown). we estimated that during the first wave in hong kong, 43ae4% of school-age children and 10ae7% of individuals aged 5-59 were infected by pdmh1n1. a serologic survey in england found similar iars in london and the west midlands. 8 both studies highlight the importance of including serologic surveys in pandemic surveillance. the geographically compact and well-mixed population in the urban environment of hong kong permits some degree of confidence in the validity of our iar and severity estimates. the completeness of the pdmh1n1 surveillance system, welldefined population denominator, and our large-scale serologic survey provide accurate numerators and denominators for the severity measures. we based severity estimates for pdmh1n1 on the iar as the denominator. in most previous studies of pdmh1n1 severity, the denominator was clinical illness attack rate, which depends on the probability of symptoms as well as medical care seeking behavior of the population. 2, 9 our estimated cirs and cfrs are broadly consistent with presanis et al.'s 2 'approach 2' severity estimates, but around 7-9 times lower than their 'approach 1' estimates. our estimates of chr are 2-10 times higher than their approach 2 estimates of symptomatic chr. however, the hospitalization-death ratio was 4253 ⁄ 27 = 164 as of november 15 in hong kong, but 996 ⁄ 53 = 19 as of june 14 in new york, 2 suggesting that the clinical threshold for admission in terms of disease severity at presentation may have been lower in hong kong. our study has a number of limitations. first, we have used antibody titers of ‡1:40 by viral microneutralization as an indicator of recent infection, correcting for pre-existing seroprevalence levels, but this may lead to underestima-tion of the iar if some infections led to antibody titers <1:40, or if some individuals with baseline titers ‡1:40 were infected. second, our estimates of the iar would be biased upwards if infection with other circulating influenza viruses led to cross-reactive antibody responses resulting in antibody titers ‡1:40. however between august and october 2009, 83% of influenza a viruses detected in hong kong were pdmh1n1, and only 3% of isolated viruses were seasonal h1n1 viruses. 10 third, a minority of severe illnesses associated with pdmh1n1 infection might not be identified by molecular detection methods, for example if admission occurred after viral shedding from the primary infection has ceased, in which case we may have underestimated the disease burden of pdmh1n1. finally, our analyses are primarily based on seroprevalence among blood donors to the hong kong red cross, who may not be representative of the whole population. we do not have detailed data on donors to compare their risk of infection with the general population, but we did observe very similar seroprevalence rates across the three groups of subjects in our study, i.e., blood donors, hospital outpatients and participants in a community cohort (data not shown). in conclusion, around 10ae7% of the population aged 5-59 and half of all school-age children in hong kong were infected during the first wave of pandemic h1n1. compared to school-children aged 5-14, older adults aged 50-59, though less likely to acquire infection, had 9ae5 and 66 times higher risk of icu-admission and death if infected. thus, although the iar of pdmh1n1 is similar to that of a seasonal epidemic, the apparently low morbidity and mortality of 2009 pandemic influenza (h1n1) appears to be due to low infection rates in older adults who had a much greater risk of severe illness if infected. the reasons why older adults appear relatively resistant to pdmh1n1 infection even though they appear to lack neutralizing antibody remains unclear. if antigenic drift or other adaptation of the pdmh1n1 virus allows these older age groups to be infected more efficiently, the morbidity and mortality of subsequent waves of the pandemic could yet become substantial. and the national institute of allergy and infectious diseases, national institutes of health (contract no. hhsn266200700005c; adb no. n01-ai-70005). the funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish. bjc reports receiving research funding from medimmune inc., a manufacturer of influenza vaccines. the authors report no other conflicts of interest. some data presented in this manuscript were previously published in wu et al. 5 it is well known that a primary goal of vaccination is to generate immunological memory against the targeted antigen to prevent disease in a vaccinated person. this ensures an accelerated immune response in the event of future contact with the pathogenic agent, such as a virus. therefore, it is very important to develop criteria for the assessment of vaccine immunogenicity by measuring both t and b memory cell levels from the vaccinated host. in contrast to inactivated influenza vaccines, live attenuated influenza vaccines (laivs) have been shown to provide primarily cellular and local immune responses. 1-3 to date, however, the hemagglutination-inhibition (hai) test (i.e. detection of serum antibodies) remains the method widely accepted for evaluation of an influenza vaccine's immunogenicity. improved understanding of the role of cellular and mucosal immunity and their contribution to protecting against severe illness caused by influenza infection has emphasized the need to reconsider methodologies used to evaluate the immunogenic impact of various influenza vaccines. such new assays need to include methods to measure local antibodies and virus-specific lymphocytes, especially in the case of live attenuated influenza vaccines, because of their potential to induce such broad-based immune responses. 4 the aim of this study was to assess the ability of new russian pandemic laivs a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) ('ultragrivak,' registered 25ae03ae2009) and a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) ('influvir,' registered 13ae10ae2009) to induce memory t-cells in naïve human subjects and to compare results to levels of hai antibodies from each subject. a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) laiv was generated by 7:1 genetic reassortment of low-pathogenic avian influenza virus a ⁄ duck ⁄ potsdam ⁄ 1406-86(h5n2) and master donor strain a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2). 5, 6 the vaccine strain contains ha gene from avian virus, as well as na and internal genes from the master donor virus. a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laiv was generated by classical (6:2) reassortment of a ⁄ california ⁄ 07 ⁄ 2009 (h1n1) with the master donor virus. 7 the vaccine strain contains ha and na genes from a 'wild-type' h1n1 strain and internal genes from the master donor virus. participants were aged 18 to 20 years and were without contra-indication of laiv vaccination. immunogenicity of a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) laiv was assessed in ten vaccinated persons and ten volunteers inoculated with a placebo (sterile physiological saline solution). immunogenicity of a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laiv was estimated in 16 vaccinated volunteers and nine volunteers inoculated with placebo. viruses or placebo were administered intranasally twice with an interval period of 21 days at a dosage of 0ae25 ml per nostril for each vaccination. physical examination, venous blood and nasal swab samples were collected at four time points during the study: (i) before vaccination (day 0); (ii) 21 days after first vaccination (day 21); (iii) 21 days after the second vaccination (day 42); and (iv) 6 weeks after the second vaccination (day 63). serum hai antibodies were measured by standard hai assay 8 using 1% human red blood cells. test antigens for the assay were a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) or a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) to match the appropriate vaccine antigen. local iga antibodies in nasal swabs were evaluated by elisa 9 using whole purified a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) or a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) viruses at 16 hau per 0ae05 ml for absorption to elisa plates. endpoint elisa titers were expressed as the highest dilution of sera that gave an optical density (od) greater than twice the mean od of six negative controls in the same assay. percentages of virus-specific cd3 + cd8 + ifn-c + and cd3 + cd4 + ifn-c + peripheral blood memory cells were determined using a flow cytometry iccs assay performed by the published method. 3 pbmcs were prepared with standard histopaque-1077 gradient centrifugation from heparinized whole blood. wilcoxon matched pair test, mann-whitney u test and the students t-test were used for statistical data analysis. prior to the first vaccination (day 0), gmts of hai antibodies to a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) and a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laivs were 1 ⁄ 5ae4 and 1 ⁄ 5ae7, respectively. in addition, gmts of siga against these specific antigens from nasal swabs were 1 ⁄ 5ae7 and 1 ⁄ 9ae1, respectively. no hai antibody titers greater than 1:40 were observed prior to vaccination. background levels of virusspecific t-cells varied significantly within groups. mean levels of virus-specific cd8 + ifnc + cells were 0ae151% to a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) and 0ae173% to a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1). for cd4 + ifnc + cells, initial levels were 0ae240% and 0ae336%, respectively. thus, background levels of virus-specific antibodies were low, but prior vaccination or virus exposure in some volunteers produced some pre-existing levels of t cells, thus they were not absolutely immunologically naïve in this sense. preexistence of h5n1-crossreactive antibodies and t-cells has been observed previously. [10] [11] [12] effect of vaccination antibody immune responses both influenza a (h5n2) and influenza a (h1n1) laivs stimulated production of serum hai antibodies and local iga antibodies in nasal swabs. following the first vaccination with influenza a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) laiv, 20% percent of volunteers exhibited seroconversion of hai antibodies; after the second vaccination, 30% of volunteers exhibited seroconversion. after the first vaccination, a 10% conversion rate of siga was observed; after the second vaccination, 60% showed conversions in levels of siga. the first vaccination with a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laiv showed 6ae3% of hai antibodies seroconversions vaccination, and 50% seroconversion after second vaccination. for local siga, those results were 18ae8% and 31ae3% following the first and second inoculation, respectively. figure 1 summarizes cellular immune responses observed in the vaccinated versus the placebo group. after the influenza a (h5n2) laiv inoculation, significant differences in both cd4 and cd8 ifnc-producing t-cells were observed at day 42 after the second vaccination (d63). these data indicate that healthy young people who never received such avian influenza vaccines and were not exposed to h5n1 wild-type viruses were able to respond to the live attenuated h5n2 influenza vaccine. after the first influenza a (h1n1) laiv vaccination, reliable increases were observed in cd8 + cells only. after the second vaccination, increases in both cd4 + and cd8 + fold changes were significantly higher in vaccinated volunteers compared to the placebo group. it is noteworthy that cellular immune responses (cd4 + and cd8 + cells) were more marked in the a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1). considering the long-term circulation of h1-subtype viruses among humans in contrast to the novelty of h5viruses, such a result would be expected. similar data were also observed following vaccination with the h5n2 laiv. after first vaccination, the percent of people with notable increases in virus-specific cd4 + and cd8 + t-cells was 20% and 10% to h5n2 and 38% and 75% to h1n1, respectively. after the second vaccination, these results were 40% and 30% to h5n2 and 69% and 69% to h1n1, respectively. importantly, a significant number of vaccinated volunteers without remarkable increases ( ‡4-fold) in hai antibodies had notable increases in cd4 + and ⁄ or cd8 + memory cells. the percent of people with notable increases in virus-specific t cells after the second vaccination among hai()) volunteers was 40% and 75% to h5n2 and h1n1, respectively. these results indicate that laivs were able to induce broadly responsive, key antiviral immune responses that would not have been detected by the hai assay alone. thus, it can be deduced that hai data alone fails to reveal important broad and specific immune responses to laiv. consequently, the hai test alone is not suitable for assessment of laiv immunogenicity. furthermore, vaccination with h5n2 laiv was able to induce cross-reactive memory t-cells to a seasonal vaccine strain, a ⁄ 17 ⁄ solomon islands ⁄ 06 ⁄ 9 (h1n1) ( table 1) . reliable increases to a (h1n1) were observed in up to 20% of volunteers. there was an inverse dependence between levels of memory t cells before and after vaccination. authors are thankful to path for the financial support of these studies. we are also thankful to jessica d'amico and dr. rick bright for their editorial review. options for the control of influenza vii background: increased susceptibility of older populations to secondary bacterial pneumonia-like infections following influenza infection has been well documented. 1 recent evidence in mouse models suggests that this increased risk from secondary bacterial infection occurs through a desensitization of the innate immune response. 2 this recent finding, however, does not account for potential differences in immune responsiveness due to age. materials and methods: to address this parameter, we used three age groups (aged, adult, and young mice) to evaluate the role of age in influenza-mediated vulnerability to secondary bacterial challenge with pseudomonas aeruginosa. all mice were evaluated for multiple parameters including: (i) survival; (ii) lung bacterial load; (iii) total lung protein content; (iv) immune cell infiltration; (v) cytokine ⁄ chemokine expression; and (vi) toll-like receptor (tlr) rna expression profiles. results: prior challenge with influenza contributed to aberrant cytokine ⁄ chemokine profiles and increased lung cellular infiltrate in response to secondary bacterial infection across all age groups, supporting a critical role for influenza infection in the alteration of immune responses to other pathogens. also similar to human influenza, these changes were exacerbated by age in mice as demonstrated by increased bacterial load, mortality, and total lung protein content (an indicator of lung damage) after p. aeruginosa challenge. conclusions: these data support a potential role for virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and the increased susceptibility of influenza virus infected mice to secondary bacterial infection. the understanding of the complex interaction of host and pathogen -and the role of age -in human influenza is critical in the development of novel therapeutics and improved vaccine approaches for influenza. our results support further examination of influenza-mediated alterations in innate immune responses in aged and non-aged animals to allow elucidation of the molecular mechanisms of influenza pathogenesis in humans. there is considerable evidence in the clinical literature to support the role of influenza infections with an enhanced risk for secondary bacterial pneumonias. [3] [4] [5] given the increased pneumonia-related morbidity and mortality in both the young and elderly populations, there is rationale for gaining a deeper understanding as to the systemic changes in the pulmonary microenvironment. although there are some recent reports that account for some of the molecular mechanisms at work in this disease process, 2 there is a paucity of experimental evidence that considers the potential effects of age. developmental changes in the immune system that occur in the aged environment have been well documented with regard to senescence of the adaptive immunity, global changes in myeloid cell function, and the establishment of a general pro-inflammatory state. 6, 7 the aim of this work was to provide evidence for the contribution of the aged immune environment to the pathology of influenza mediated secondary bacterial infections. animals used in this study were housed under conditions approved by tulane university's institutional animal use and care committee. female balb ⁄ c mice used in these studies were divided into three age groups: aged (18 months old), adult (6 months old), and young (2 months old). each age group was subdivided into two groups: influenza infected and naïve (control). mice were infected by the intranasal route with 4 · 10 5 pfu of mouse-adapted influenza a ⁄ pr ⁄ 8 ⁄ 34. clinical disease was measured by body weight changes over a 6 week period post influenza challenge, and recovery was determined as return to pre-infection weight. all mice were subsequently challenged intransally with1 · 10 7 cfu pseudomonas aeruginosa strain pao1. twenty-four hours post-pseudomonas challenge, bal with sterile pbs was performed on all mice in all groups. total rna from the cellular fraction was pooled from three experimental animals from each group. tlr mrna was detected by qrt-pcr, where expression levels were determined as relative to b-actin mrna levels. cdna was synthesized from total cellular rna from bal samples using iscript cdna synthesis kit (biorad). pcr reactions were composed of 0ae1 lg cdna forward and reverse primers according to optimized conditions and 12ae5 ll of 2 · syber green icycler supermix (biorad), in a total vol-ume of 25 ll and were run using a biorad icycler utilizing melting point determination. primers and concentrations used in this study included: mus_tlr2f: tgctttcct-gctggagattt-600 nm, mus_tlr2r: tgtaacgcaac agcttcagg-900 nm, mus_tlr3f: atatgcgcttcaa tccgttc-300 nm, mus_tlr3r: caggagcatactggt gctga-600 nm, mus_tlr4f: ggcagcaggtggaattg tat-600 nm, mus_tlr4r: aggccccagagttttgttc t-900 nm, mus_tlr5f: ctggggacccagtatgctaa-600 nm, mus_tlr5r: acagccgaagttccaagaga-900 nm, mus_tlr7f: ggagctctgtccttgagtgg-900 nm, mus_tlr7r: caaggcatgtcctaggtggt-600 nm, mus_ b-actinf: agccatgtacgtagccatcc-600 nm, mus_b-actinr: ctctcagctgtggtggtgaa-900 nm. as a measure of protein leakage into the alveolar space, total protein content in each bal was measured by bca assay of each supernatant fraction according to manufacturer's instructions (pierce). cytokine and chemokines levels were measured by multiplexed bead array (bioplex, biorad). immune cell characterization of bal was estimated by flow cytometry. lymphocyte populations were gated by forward versus side scatter and characterized as b cells (f4 ⁄ 80 ) , cd19 + ) or t cells (cd11b ) , cd4 + ). the myeloid population that is composed of macrophages, neutrophils, dendritic cells, and natural killer cells was enumerated by gating all but those found in the lymphocyte gate using forward versus side scatter plots. flow cytometry data was analyzed using flojo software (treestar). statistical analysis, where appropriate, was performed using a two-way analysis of variance (age versus influenza infection status) supported by bonferonni's correction for multiple comparisons. 8 a recent finding by didierlaurent, et al., 2 described an influenza mediated desensitization of tlr function as a primary contributor to an increase in bacterial burden when challenged after resolution of the primary influenza infection. this finding, however, was obtained using animals that were 6-8 weeks of age, where our study included two cohorts of older mice (6 months and 18 months). using whole protein content of the bal as an estimate of protein leakage into the lumen of the lung, we found elevated protein content in aged mice as compared to young and adult mice. in aged mice, a slightly lower total lung protein when comparing influenza infected to protein in the bal from influenza naïve mice challenged with p. aeruginosa (table 1) . supporting previously published studies showing a generalized pro-inflammatory cytokine environment in the aged immune system, we provide evidence for significantly (p = 0ae0465) and an increase in ifnc (p = 0ae0323) was detected. the decrease in gm-csf correlates well with a previous report that gm-csf is less prevalent in influenza resolved animals (table 1 ). 2 we also report a noticeable change in the immune cell populations with respect to b-cells, cd4 + t-cells, and the myeloid cell populations. there is a trend of increased prevalence in cd4 t-cells in the post-influenza environment across all ages. b-cell numbers also trend toward increase in influenza treated animals in young and adult animals; however, there is a noticeable decrease in the bcells in aged animals. across all age groups, there is a general decrease in frequency of cells that would normally make up the myeloid cellular fraction of the bal (macrophages, neutrophils, dendritic cells, and natural killer cells) ( table 1 ). our study also shows, as cited by others, that toll-like receptor (tlr) gene expression in the post-influenza environment is decreased in cells found in the bal 2 after both influenza and pseudomonas infection. our data support the previous finding of a reduced expression of tlr mrna in influenza-cleared mice when we measured tlr 2, 3, 5, and 7. only tlr4 showed differences with respect to age with young mice showing little or no detectable change in tlr4 mrna expression. our results show an increase in the expression across all tlrs examined in the aged mice group (table 1) irrespective of influenza infection status. these data support earlier studies performed with adult mice that showed reduced tlr mrna expression in the post-influenza environment. this study also expands the current understanding of the potential role of age in influenza mediated bacterial infection-induced mortality. the impact of these alterations in the immune microenvironment across age groups and infection status is highlighted by the ability of bacterially challenged animals to clear infection. assessment of bacterial load in the lungs of p. aeruginosa challenged mice indicated a difference in young and adult mice if previously infected with influenza virus. in aged mice, both influenza challenged and influenza-naïve mice had higher bacterial loads and less variability when comparing within the age group, supporting the risk of age alone in susceptibility to bacterial pneumonia (table 1, figure 1 ). taken together, these data support the potential role for both virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and increased the susceptibility to secondary bacterial infection that results from influenza infection in mice. these findings highlight distinct differences in the immune environment between age groups and thus reveal necessity for further examination as to the mechanisms of immunity across age with respect to current infection status. garnering a clearer understanding as to the complex interaction of host and pathogen with respect to age in influenza infections is central to the development of increased efficacy in vaccine and therapeutic strategies. prospective estimation of the effective reproduction background pandemic influenza a (h1n1) virus (ph1n1) emerged in early 2009 and rapidly spread to every continent. an urgent priority for international and national public health authorities was to estimate the transmissibility of the pandemic strain for situational awareness and to permit calibration of mitigation strategies. the basic reproductive number, r 0 , is defined as the average number of secondary cases that 1 index case generates in a completely susceptible population, and is a common measure of transmissibility. however, it is difficult to estimate r 0 without an understanding of the degree of any pre-existing immunity in the population. the effective reproductive number, r, is defined as the average number of secondary cases that 1 index case generates, and can be estimated over time (i.e. r t ). wallinga and teunis 1 described a method to estimate r t based on illness onset dates of the cases while assuming that all secondary cases would have been detected, and cauchemez et al. 2 extended the method to permit prospective estimation by adjusting for secondary cases that have not yet experienced illness onset at the time of analysis. we describe how the method can further be extended to account for reporting delays, allowing true real-time estimation of r t during an epidemic, and we illustrate the methodology on notifications of ph1n1 and associated hospitalizations in hong kong. we obtained data on all laboratory-confirmed ph1n1 infections ('cases') reported between may 1 and november 15, 2009 to the hospital authority and center for health protection in hong kong collated in the eflu database. a subset of the cases was hospitalised. the database also included information on age, sex, illness onset date, laboratory confirmation date, and contact history (for the early cases). laboratory-confirmed ph1n1 infection was a notifiable condition throughout our study period. we extended existing methods for estimating r t over time to allow for reporting delays between illness onset and notification, and between illness onset, notification, and hospitalisation for those cases that were hospitalised, where the reporting delay distribution were estimated empirically from the data. 3 we further extended the methodology to allow for imported cases (infected outside hong kong) contributing to the estimation of r t as infectors but not infectees. we used multiple imputation to allow for missing data on some symptom onset dates to make best use of all available data. 4 we used a serial interval with mean (standard deviation) of 3ae2 (1ae3) days, 5 and in sensitivity analyses, we used serial intervals with mean 2ae6 days 6 and 3ae6 days. 7 statistical analyses were performed in r version 2.9.2 (r development core team, vienna, austria). in late april 2009 following the who global alert, hong kong initiated containment protocols to attempt to delay local transmission of ph1n1 for as long as possible. these measures included screening at ports, airports, and border crossings, and enhanced surveillance for people with influenza-like illness, particularly for those who had recently returned from abroad. laboratory testing capacity was substantial due to heavy investment in local infrastructure following previous experiences with avian influenza a ⁄ h5n1 in 1997 and severe acute respiratory syndrome in 2003. laboratory-confirmed ph1n1 cases were isolated until recovery, and their close contacts were placed under quarantine for 7 days. imported cases were identified sporadically through may and early june 2009. the first case of ph1n1 not traceable to importation (i.e. a local case) was identified on june 11 and triggered a change to mitigation phase measures. some containment measures, including isolation of cases, were continued until the end of june to allow a soft transition between containment and mitigation phases. as an immediate measure to try to reduce community transmission of ph1n1, all childcare centres, kindergartens, and primary schools were proactively closed for 14 days (subsequently extended for another 7-14 days to summer vacation in early july). 8 any secondary schools in which one or more confirmed ph1n1 case was identified were reactively closed for 7 days. on june 13 the government opened eight designated flu clinics across the territory to provide free medical consultation for outpatients with influenza-like illness and free laboratory testing for ph1n1. these clinics resumed regular chronic disease services in mid-august, and laboratory testing and antiviral treatment was restricted to high risk groups in september. the various interventions are highlighted in figure 1 (a), superimposed on the epidemic curve of laboratory-confirmed ph1n1 cases and ph1n1-associated hospitalizations. around 15% of the cases were hospitalised, and this proportion increased somewhat towards the end of the epidemic. 3 figure 1(b) shows the estimates of r t based on laboratory-confirmed ph1n1 cases. the estimated r t peaked at 1ae5 on june 12, and fell below 1 between 20 june and 3 july (which was within the school closure period). r t fluctuated between 0ae8 and 1ae3 through the school summer vacations in july and august, it subsequently increased to around 1ae2-1ae3 after schools reopened in september until the epidemic peaked in late september, and then fluctuated below 1 as the epidemic declined. the trends in r t based on h1n1-associated hospitalizations were similar, although with wider confidence intervals due to the smaller number of events ( figure 1c ). the extension of the methods to allow for reporting delays avoided substantial bias in realtime estimates of r during the epidemic for the most recent 7 days, and closely tracked the final estimates of r t . 3 our results suggest that ph1n1 may have had slightly lower transmissibility in hong kong than elsewhere. for example, estimates of r t were around 1ae5-2ae0 in new zealand 9 and australia. 10 lower transmissibility in hong kong has been associated with school closures in june and july followed by summer vacations from july through august. 8 furthermore, in hong kong the influenza virus usually does not circulate after august, 11 and therefore seasonality could also be a cause for the lower r t . on the other hand, the interventions applied during the mitigation phase, such as the widespread use of antiviral treatment in hong kong and the pre-existing immunity in the ageing population in hong kong, may also be associated with lower transmissibility. there are some limitations to our work. first, we only used aggregated data, and we did not consider the heterogeneity among the cases in terms of sex and age or other factors. therefore our estimates can only provide a snapshot of the overall trend, but limited information for any specific subset of population. secondly, we did not consider the possibility that cases might be infected in hong kong and exported to other countries, which could lead to slight underestimation of the transmissibility. one has to be careful in translating the estimated r t to the effectiveness of any specific interventions, as interventions may not be the only factor influencing the transmissibility; for example, a depletion of the susceptible population during an epidemic can also be a factor for the decline in r t . 12 in conclusion, real-time monitoring of the effective reproduction number is feasible and can provide useful information to public health authorities for situational awareness and planning. in affected regions, laboratory capacity was typically focused on more severe cases, and changes in laboratory testing and notification rates meant that that case counts may not necessarily reflect the underlying epidemic. a useful alternative to case-based surveillance is surveillance of the subset of severe infections, for example hospital admissions, or icu admissions, 13 and our results show that it was feasible to monitor ph1n1-associated admissions in real-time to estimate transmissibility. influenza antigenic cartography projects influenza antigens into a two or three dimensional map based on immunological datasets, such as hemagglutination inhibition and microneutralization assays. a robust antigenic cartography can facilitate influenza vaccine strain selection since the antigenic map can simplify data interpretation through intuitive antigenic map. however, antigenic cartography construction is not trivial due to the challenging features embedded in the immunological data, such as data incom-pleteness, high noises, and low reactors. to overcome these challenges, we developed a computational method, temporal matrix completion-multidimensional scaling (mc-mds), by adapting the low rank mc concept from the movie recommendation system in netflix and the mds method from geographic cartography construction. the application on h3n2 and 2009 pandemic h1n1 influenza a viruses demonstrates that temporal mc-mds is effective and efficient in constructing influenza antigenic cartography. the web sever is available at http://sysbio.cvm. msstate.edu/antigenmap. as a segmented, negative stranded rna virus, influenza virus is notorious for rapid mutations and reassortments. the mutations on the surface glycoproteins (ha and na) of influenza viruses are called antigenic drifts, and these antigenic drift events allow the virus to evade the accumulating immunity from previous infection or vaccination and lead to seasonal influenza epidemics. a reassortment event with a novel influenza antigen may result in antigenic shift and cause influenza pandemic. for instance, the 2009 h1n1 pandemic virus is a reassortant with a swine origin ha antigen. vaccination is the primary option for reducing the effect of influenza, and identification of the right vaccine strains is the key to development of an effective vaccination program. the antigenicity of an optimal vaccine strain should match that of the epidemic strain. in influenza surveillance program, the influenza antigenic variants are generally identified by the immunological tests, such as hemagglutination inhibition (hi) assay, microneutralization (mn) assay, or elisa. these immunological assays measure the antigenic diversity between influenza viruses by comparing the reaction titers among the test antigens and reference antisera. however, data interpretation of the data from these assays is not trivial due to the embedded challenges such as data incompleteness, high noises, and low reactors. by mimicking geographic cartography, influenza antigenic cartography projects influenza antigens into a two or three dimensional map using immunological datasets. 1 antigenic cartography can simplify the data interpretation, and thus, facilitate influenza antigenic variant identification. recently, we developed a novel computational method, temporal matrix completion-multidimensional scaling (mc-mds), in antigenic cartography construction. 2 in this paper, we described the details of temporal mc-mds, especially the original concepts introduced in this method, and how they can achieve the robustness in antigenic cartography construction. our method included two integrative steps: it first reconstructs the hi matrices using low rank mc method, and then generates antigenic cartography using mds with a temporal regularization. the mc concept was adapted from the movie recommendation system in netflix and the cartography concept from geographic cartography. in 2006, netflix, an online dvd and blu-ray disc rentalby-mail and video streaming company, held a 3-year netflix prize contest (http://www.netflixprize.com/) on computational methods for improving its recommendation system. 3 in its recommendation system, netflix collected the rating data from the individuals. based on his or her renting history and the ratings in the systems (e.g., from evaluators and other renters), netflix recommendation system suggests certain movies to a renter. apparently, no individuals would be feasible to provide ratings for all of the movies, as it will take hundreds of years for a single person to rate over 50 000 movies available from netflix. thus, the resulting rating data is an incomplete matrix, and it can be as sparse as less as 1%. 4 the challenge in netflix recommendation system is a classic mc problem. [4] [5] [6] [7] [8] as the inspiration of netflix prize contest, many efficient low rank mc algorithms were developed, for instance, opt-space, 7 svt, 5 cf, 9 bellkor, 10 pf, 11 and fwls. 12 eventually, the team bellkor's pragmatic chaos won this contest. their methods combines nonlinear probe blending and linear quiz blending to come up with a predictor bigchaos. 13 matrix completion estimates the unobserved values based on the observed values. the users can refill the missing data without repeating the experiments. furthermore, mc will help reduce the noises in the data, for instance, those biases by different individuals performing experiments. in influenza antigenic characterization, hi assay is a commonly used assay for antigenic analysis, since hi assay is relatively economic and easy to perform. however, hi is labor intensive, and it is almost impossible for any individual lab to complete the hi assays for all pairs of antigens and antisera during influenza surveillance. in addition, both testing antigens and the reference antisera are dynamic. for instance, in seasonal influenza surveillance, generally only contemporary antisera are used in experiments. thus, we will have to integrate multiple hi tables in order to evaluate the overall antigenic changes for influenza vaccine strain selection. the resulting hi tables will be incomplete, and the observed entries in the integrated hi data can be as less as 3%. the completion of this matrix can be formulated as a typical mc. briefly, given the combination of hi matrix with m antigens and n antisera, the hi matrix can be represented as m m·n = (m ij ) m·n , where m ij denotes the hi values from the reaction between testing antigen i and antiserum j. the low rank mc assumes that both antigen and antiserum can be embedded into a low rank space. to be specific, the low rank mc method is to seek matrix u m·r , v n·r and a diagonal matrix r r·r , where m = u m·r r r·r (v n·r ) t . in order to achieve this goal, the optimization formulation has been employed, which can be represent as following, where e denotes the observed entries in hi matrix and g(x) is a regularization function. the eqn (1) is the standard format of a low rank mc formulation. the geographic cartography is a common technique to display the cities and their geographic distances in a map. this cartography can be generated using mds based on a geographic distance matrix. figure 1(a) shows the antigenic cartography generated using a distance matrix with seven cities, and figure 1 (b) is a map for comparison. as an analog of geographic cartography, the influenza antigenic cartography maps the influenza antigens into a two or three dimensional map based on the distance matrix generated using immunological data. this incomplete matrix can be filled through mc algorithm discussed in section mc and netflix. low reactors, non-random date incompleteness, and temporal model generally, three types of data are present in a combined hi matrix: high reactor, low reactor, and missing values. among these three data types, high reactors are the most reliable data points. the low reactors are those values present in the hi matrix as ''equal to or less than a threshold h'', where h can be 5, 10, 20, or 40. low reactors have similar values in the affinity dataset but could be from different binding settings. these low reactors are present due to the detection limits of biotechnology, and they are not reliable. both these missing values and low reactors make it very difficult to analyze and interpret antigenic correlations amongst tested antigens and reference antigens. to our best knowledge, none of the existing mc method can handle the threshold values. in addition, the non-random incompleteness of influenza immunological datasets generates an additional challenge in traditional mc methods, which are based on the assumption that the observed values are randomly distributed among the matrix. in a typical combined antigenic hi data, most of the off-diagonal entries are missing values or low reactor values. 1 in order to overcome the above issues, we incorporated a regularization function into the eqn (1), where this indicator function is only valid for those entries with low reactor values. an alternating gradient decent method is applied to solve the optimization problem in eqn (2) . in addition, a temporal mds method is proposed to project the antigens into a 2 or 3 dimensional map. x where d ij is the average distance between virus i and virus j, t i is the isolation year of virus i, d ij is the distance between virus i and virus j in cartography, d ac i is the distance between virus a and center of group i, and d c i c j is the distance between the centers of group i and group j. all the parameters are tuned by cross validation. we named this method as temporal mc-mds. by applying temporal mc-mds method in an h3n2 dataset, 2 low reactors. figure 2 (a) is a three-dimensional influenza antigenic map based on this data by using mc-mds method. the reported 11 clusters (hk68, en72, vi75, tx77, bk79, si87, be89, be92, wu95, sy97, and fu02) were displayed in the core of a spiral s-shape, and bk79 and be92 are located at the turning point of this s-shape. however, the antigenic distances between some viruses are incorrect. for example, the distance between hk68 and fu02 in the projection is 7ae1223 units, which is close to the distance between hk68 and bk79 (6ae5113 units). the main reason leading to those inaccurate distances is the unique distribution of hi datasets described in section 2.3. in comparison, with the temporal model, not only the viruses in 11 clusters have been clearly separated, but also the antigenic distances between each cluster are proportional to their isolation time interval. in this updated cartography ( figure 2b ), the antigenic distance between hk68 and fu02 is 15ae0633 units, where the distance between hk68 and fu02 is 6ae3984 units. this result suggested that the temporal information is critical for antigenic cartography construction for immunological datasets spanning a long time period. the hi data from seasonal influenza surveillance belong to this category. for seasonal influenza virus ⁄ pandemic influenza viruses within a short time span, the temporal model is probably not necessary, as there is lack of long-term immunological pressure present in the population. figure 2 (c) is an antigenic cartography generated using a hi dataset with 2009 h1n1 influenza viruses spanning from april of 2009 to june of 2009. this map demonstrates that there is lack of antigenic drifts during the first wave of this pandemic influenza as all of these viruses are mixed altogether. our limited studies on h5 and h7 avian influenza viruses suggested the temporal model is not needed for avian influenza viruses. however, extensive studies are required to investigate whether there is any special data structure present in this type of data. in this study, we described in details the concepts and applications of new computational method, temporal mc-mds for influenza antigenic cartography construction. we formulate the influenza cartography as two integrative steps: low rank mc problem from the concept of netflix movie recommendation system and mds from geographic cartography construction. in order to handle two additional challenges, including low reactor and non random distribution of antigenic data, a temporal model is incorporated into mc-mds as temporal mc-mds. our applications demonstrated that temporal mc-mds is effective in constructing influenza antigenic cartography. the three dimensional antigenic cartography for a ⁄ h3n2 seasonal influenza virus without temporal model, and the antigenic clusters were defined in ref. [2] ; (b) the three dimensional antigenic cartography for a ⁄ h3n2 seasonal influenza virus with temporal model; (c) the two dimensional antigenic cartography for 2009 a ⁄ h1n1 pandemic influenza without temporal model, and these viruses were labeled in shape by the corresponding month for them to be detected. one grid is corresponding to a twofold change in hemagglutination inhibition experiment. the mechanisms driving the three waves of infection and mortality in the uk in 1918-1919 are uncertain. although the circulation of three distinct viruses could have generated three waves of infection, 1 the virological evidence required to prove or disprove this hypothesis is lacking. social distancing, an alternate mechanism for generating fluctuations in the effective susceptible pool and therefore explaining multiple waves of infection, 2, 3 was not generally imposed in the uk as it was in the us and australia. we are therefore motivated to explore the possible role of continual population-level changes in the average protective response against the circulating virus in generating a multi-wave pandemic, within a biologically motivated deterministic model for influenza transmission. the nature and duration of protection against further infection following recovery from influenza is uncertain and depends on the mode and tempo of viral evolution, as well as the response of the cellular and humoral arms of the adaptive immune system. 4 for a given seasonal ⁄ pandemic strain, memory b-cells may generate a specific antibody response in a portion of the adult ⁄ elderly population, depending on the exposure to related antigenic sub-types. 5 however neutralising antibodies are unlikely to be a widespread immunological response to a novel (pandemic) strain. memory t-cells which recognise conserved internal viral proteins may be a more common mechanism for protection; the generation of very high levels of cytotoxic cd8 + t-cells potentially facilitates rapid viral clearance, 6, 7 and lower levels of cd8 + t-cells perhaps provide partial protection. 8 in this work we explore key drivers of multi-wave pandemics within phenomenological models that incorporate different immune response mechanisms building on existing models 9,10 incorporating the role of evolving population-level protection in multi-wave pandemics. we use weekly reports of influenza mortality rates 11 for five administrative units in the uk (blackburn, leicester, newcastle, manchester and wigan) where records from block censuses instigated by local medical officers to record the cumulative incidence of reported symptoms in each wave in a sample of 1000 or more households are also available. 10 the symptom reporting data allows us to estimate the case fatality rate and thus use the mortality time series to constrain our transmission model. furthermore, the incidence of individuals reporting symptoms in multiple waves provides information about the acquisition and loss of immunity. we extract the death rate and symptomatic (re)infection rates predicted by our model prevalence for a given set of parameters and estimate a likelihood-based on a comparison to all the death and cumulative reported incidence data assuming a negative binomial error distribution. we utilise monte carlo markov chain (mcmc) methods with parallel tempering algorithms to maximise this likelihood and obtain parameter estimates. parallel tempering -which concurrently searches for maximal likelihood parameter solutions on a set of scaled likelihood surfaces -allows for relatively rapid exploration of the parameter space. we use bayesian information criteria (combined with qualitative assessment of biological plausibility) to aid model selection. we have implemented a deterministic compartmental transmission model, which allows for a variety of phenomenological modes of protection against the pandemic virus. to facilitate this, we stratify the population into two groups; the 'experienced' population (stratum 1) who have had been exposed to an influenza virus and the 'naive' population (stratum 2) who have not. in each stratum, i hosts may be classified as either susceptible s i , exposed e1 i and e2 i , having (recovered from) a symptomatic i i (r i ), or asymptomatic a i (ra i ) infection. note that the states tq i , tq2 i , e2 i , t i , and t2 i are included so that the hosts move between the key epidemiological states with a peaked (rather than exponential) distribution of waiting times. hosts in the experienced stratum may exhibit reduced susceptibility, infectiousness, and symptomatic proportion compared to naive hosts, parameterised by e i , e s , and e a , respectively; however note that depending on the model parameters, there may be fully susceptible hosts within the experienced stratum. in addition, we assume homogeneous population mixing and a constant basic reproduction number r 0 with the force of infection: modulated by a sinusoidal seasonal term with amplitude b 1 with phase chosen to maximise transmission in the winter season. here n is the total population size, and x e is the initial fraction in the experienced strata. the proportion of symptomatic cases a and the case fatality rate l are permitted to vary from wave to wave (and given indices 1, 2 or 3 accordingly). the transmission dynamics is described by the following set of coupled ordinary differential equations. where s in,1 = p utq2 i and s in,2 = 0 in order to divert recovered infectious hosts from the naive stratum into the experienced stratum. the probabilities of gaining permanent protection are q 1 = q and q 2 = 0. the latent exposed period is fixed to be c = 1 ⁄ 1ae3 days, and the rate of recovery is parameterised by m = 1 ⁄ t inf , where t inf is the infectious period. hosts with prior sterilising protection begin in q 1 and move into s 1 at rate u q = 3 ⁄ t wq . recovered hosts (r i ) migrate back to s 1 at a rate u = 3 ⁄ t w . the state p 1 contains hosts with permanent protection. the modes of protection captured in this model are: i. permanent prior protection (beginning in state p 1 ), ii. waning prior protection (beginning in state q 1 ), iii. permanent acquired protection with probability q (moving into state p 1 ), iv. waning acquired protection with probability 1 ) q, and, v. partial prior protection (beginning in state s 1 ) resulting in reduced infectiousness (e i ), susceptibility (e s ), and symptomatic proportion (e a ). in the context of this model, 'permanent' protection refers to protection which lasts for the duration of the epidemic. here we explore the results of parameter fitting to two models which differ in the nature of the assumed pre-existing protection in the community at the beginning of the pandemic. protection hypothesis 1 assumes that the prior protection is sterilising but temporary, whilst protection hypothesis 2 assumes that the prior protection is partial but permanent and may act on susceptibility, infectiousness, and ⁄ or asymptomatic proportion. each model allows waning acquired protection and for a proportion q of the experienced population to gain permanent protection following infection. fitted parameters common to each model are t inf , b 1 , q, t w , a, l and the proportion beginning in p x i . prior protection hypothesis 1: sterilising, waning prior protection we fix x e = 1 and fit for q 1 (t = 0) ⁄ n and t wq so that protective modes i, ii, iii, and iv are enabled ( figure 1 ). it is important to note that due to the slow convergence of the mcmc chains, we cannot guarantee that our parameter estimates correspond to the global minimum. furthermore, parameter estimates can only be meaningfully interpreted for good fits to the data. due to the prediction of a fourth (unobserved) wave for the model fit to blackburn, we do not report these parameter estimates here. the fits to the leicester data are generated with the parameter set r 0 = 5ae7, a 1 = 0ae25, a 2 = 0ae65, a 3 = 0ae65, t w = 0ae28years, t wq = 0ae45 years, we fix q 1 (t = 0) ⁄ n = 0 and fit for x e , e a , e i , and e s so that protective modes i, iii, iv, and v are enabled (figure 2) . the parameters corresponding to the fit in figure 2 for leicester are r 0 = 7ae4, a 1 = 0ae09, a 2 = 0ae50, a 3 = 0ae61, t w = 0ae22 years, p 1 (t = 0) ⁄ n = 0ae01, s 2 (t = 0) ⁄ n = 0ae48, b 1 = 0ae021, t inf = 0ae94 days, q = 0ae561, e a = 0ae9966, e i = 0ae946, and e s = 0ae594. our model with protection hypothesis 1 -which, similarly to the model discussed in ref. [9] , assumes that a sub-population has waning sterilising prior protection -is able to generate multiple waves of infection via the continual replenishment of s 1 from an initially large proportion (over 40%) of hosts with prior protection in q combined with the waning of acquired immunity in around 23% of cases on a time-scale of 3 months. disease severity as measured by symptomatic proportion increases from 25% in the first wave to above 60% for the second and third waves. over a quarter of the population are initially permanently immune, and a large r 0 value of 5ae7 drives transmission in the remaining population. protection hypothesis 2 -which assumes that prior protection offers partial susceptibility and ⁄ or reduced infectiousness or symptomatic disease -performs slightly more poorly; the fit to the leicester data has an inferior likelihood (although the mortality data only likelihood is a little larger), despite the higher dimensionality of the model. nevertheless, the model fit still mirrors many characteristics of the data, particularly for leicester. we note that for this model, a 1 is very near the lower limit, corresponding to ubiquitous exposure in the first wave. in this scenario, refuelling of the susceptible pool to generate secondary and tertiary waves is still possible due to a shorter waning time of acquired protection (well within 3 months) and a lower probability of gaining permanent protection following infection, when compared with the parameter estimate for hypothesis 1. the parameter estimates suggest that approximately 50% of the population initially experiences reduced disease severity (e a $ 0ae66), but similar susceptibility and infectiousness. a larger value for r 0 $ 7ae4 is required to drive transmission despite low numbers beginning in p 1 , due to the large number of hosts who acquire temporary or permanent immunity early on in the pandemic. it is clear that, at least mathematically and perhaps biologically, there are multiple possibilities for the structure of population-level protection which are compatible with the generation of multiple pandemic waves. however, whilst the models considered here are able to explain the observed mortality and reinfection data for some patterns of infection and mortality (e.g. leicester), they are not consistently able to reproduce a pandemic which dies out after three waves across the connected populations we are studying (e.g. for blackburn). it is challenging to construct a deterministic model for the spread of disease within multiple locations in the uk in 1918, which assumes homogeneous mixing without modulation of the transmission rate by social distancing. an improved model working with these assumptions likely requires a richer structure for the host protection response than the structures we have explored thus far. we are currently seeking improved fits to the data by implementing a number of biologically defensible exten-sions to our model, including incremental immunity whereby t w increases by a factor v after each exposure to the pandemic flu, and incremental loss of prior protection whereby a increases as hosts lose their sterilising prior protection. it is important to note that the mechanism(s) generating differences in the pandemic experience recorded in geographically connected locations is an open question; true differences in demography, varying degrees of reactive social distancing, inhomogeneities in the circulation (or circulation history, i.e. prior immunity) of viral strains, stochastic variations, and ⁄ or unique socio-cultural ⁄ behavioural conditions may all contribute to this effect. the 2009 h1n1 experience in australia and elsewhere highlighted the difficulties faced by public health authorities in diagnosing infections and delivering antiviral agents (e.g. oseltamivir) as treatment for cases and prophylaxis for contacts in a timely manner. consequently, forecasts from mathematical models of the possible benefits of widespread antiviral interventions were largely unmet. we summarise results from a recently developed model that includes realworld constraints, such as finite diagnostic and antiviral distribution capacities. we find that use of antiviral agents might be capable of containing or substantially mitigating an epidemic in only a small proportion of epidemic scenarios given australia's existing public health capacities. we then introduce a statistical model that, based on just three characteristics of a hypothetical outbreak [(i) the basic reproduction number, (ii) the reduction in infectiousness of cases governments and public health agencies worldwide, spurred by outbreaks of sars and h5n1, have developed preparedness strategies to mitigate the impact of emerging infectious diseases, including pandemic influenza. pandemic response plans are presently being revised in light of the 2009 h1n1 experience. [1] [2] [3] many developed countries amassed large stockpiles of neuraminidase inhibitors (nais) with the expectation that they could be used to not only treat the most severely ill, but curb transmission in the community. without relevant field experience indicating how nais should be distributed, mathematical and computational modelling has been used to inform optimal deployment policy in a pandemic scenario. 4-10 models of population transmission were used to infer likely effects on epidemic dynamics, using data from human and animal studies of experimental infection and nai efficacy trials. in the australian (and wider) context, models indicated the potential for substantial benefit at the population level if nais were distributed in a liberal manner, targeting close contacts of indentified cases. 11 furthermore, results indicated that use of limited nai resources in this way may improve the impact of case treatment due to the effects on epidemic dynamics. 11 however, these models did not take into account logistic and other real-world constraints, such as finite diagnostic and antiviral distribution capacities, which were identified as limiting factors during the australian 2009 h1n1 pandemic response. [12] [13] [14] in particular, if using positive pcr diagnosis as a 'decision to treat' test, delays to confirmation of diagnosis, particularly once total laboratory capacity was exceeded, prevented timely delivery of nais to both cases and contacts of cases. 12 in previous work, 15 we have extended our existing models to examine how diagnostic strategies [e.g. using pcr confirmation versus syndromic influenza-like illness (ili) presentation as a decision to treat], diagnostic-capacity, and nai distribution capacity each impact on the ability to deliver an effective intervention. the model uses case severity (the proportion of infections deemed severe) to determine the overall presentation proportion, and so the ability to identify individuals eligible for nai treatment and contact prophylaxis. figure 1 (a) shows a key result from the model. for each curve shown, we simulated thousands of epidemics, sam-pling across plausible ranges of parameters describing virus, population, and intervention characteristics using a latin hypercube sampling (lhs) approach. without intervention, the proportion of the population infected either symptomatically or subclinically by the end of the epidemic is around 50%. if a syndromic strategy (ili presentation) is used to determine provision of nais as treatment and prophylaxis, excessive distribution of drug to individuals who are not infected with influenza occurs early in the epidemic. early stockpile expiry accounts for a marginal impact of the antiviral intervention on the final outbreak size, in the order of a few percent. the second strategy modelled (pcr ⁄ syndromic) is one where pcr confirmation of diagnosis is required early in the epidemic to make treatment decisions until such time as laboratory capacity is exceeded. from this point, individuals are treated on the basis of symptoms alone -during an epidemic phase in which a substantial proportion of ili presentations will be attributable to influenza. under this strategy, the intervention is able to control the outbreak in approximately 10% of the simulated epidemics given the 'base case' constraints on diagnosis and delivery assumed in the model. the results highlight that a successful antiviral intervention requires a highly sensitive diagnostic strategy in the initial stages of the epidemic and comprehensive distribution of post-exposure prophylaxis. a pcr ⁄ syndromic strategy for decision to treat and provide contacts with prophylaxis is thus optimal. the surface in figure 1(b) shows the percentage of simulation runs for the pcr ⁄ syndromic strategy that have a final population attack rate of <10% (a substantial reduction from the no intervention case of approximately 50%) as a function of pcr capacity and nai daily distribution capacity. as indicated by the arrow, the estimated australian pcr laboratory capacity appears to be sufficient, while significant benefits for the public health outcome may be achieved if logistical delivery constraints for nai distribution can be ameliorated. however, the probability that such an interventioneven with substantial increases in pcr and nai distribution capacity -would successfully mitigate an epidemic is low (12-25%), and consequently it is difficult to universally recommend an antiviral intervention. 15 in this study, we introduce a statistical model that predicts whether or not an nai distribution strategy based on a pcr ⁄ syndromic antiviral distribution policy will be successful in mitigating an epidemic. we thereby provide proof-of-principle for the design of a decision support tool that may be used by public health policy makers during an epidemic when faced with formulation of context specific nai distribution policy. synthetic data of hypothetical outbreaks and interventions were generated using the lhs simulations developed in ref. [15] . we selected a random sample of 100 outbreaks from a total of 2000 simulated epidemics (5% of model simulations). using these data, we identified independent model parameters that were most highly rank-correlated with the final attack rate. these parameters were included in a logistic regression model to assess their ability to predict whether an influenza epidemic would be successfully mitigated by an antiviral intervention (ar < 10%). model predictions were then validated against the full simulated dataset. full details of the simulation model, its structure, parameterisation and parameter distributions are available in ref. [15] . use of the lhs simulation approach, and the method of model analysis and evaluation was similar to that previously described. 16 matlab 2010a (mathworks, natick, ma, usa) was used for the analysis and statistical model fitting. table 1 shows results from our logistic regression model. key parameters sufficient to predict whether or not an outbreak may be controlled by the deployment of av agents are: 1. r 0 , the basic reproductive number of the outbreak (assigned values between 1ae35 and 1ae45 for this example). as the value of r 0 increases, the epidemic progresses more rapidly and is more difficult to control, explaining the negative correlation coefficient. 2. e t , the relative infectiousness of treated individuals (assigned values between 0ae8 and 1ae0). higher values for this parameter indicate only modest drug effects on transmission, explaining the negative correlation coefficient. 3. g, the proportion of infections that are severe (assigned values between 0ae001 and 0ae1), and which in turn determines the presenting proportion (derived values between 0ae11 and 0ae56). as the presenting proportion increases, the ability to identify and treat cases and deliver prophylaxis to contacts also rises, increasing the impact of the antiviral intervention. the roc curve (1-specificity versus sensitivity, not shown) for the logistic regression model specified in table 1 has an area under the curve of 0ae937, demonstrating that the model predicts the success of an antiviral intervention extremely well. for example, with a sensitivity of 95% we still have a specificity of approximately 80%. evaluation of the 2009 pandemic response has emphasised the need for early informed decision-making to implement proportionate disease control measures. our model identifies a low probability of successful epidemic mitigation using targeted antivirals alone (figure 1 and ref. 15 ), in distinction to results from models that fail to account for the diagnosis and delivery constraints inherent in any public health response. the decision support tool (table 1) highlights key epidemic characteristics that are predictive of a high likelihood of effective mitigation. the reproduction number was one of the earliest parameters estimated from early outbreak data during the 2009 h1n1 outbreak. 17, 18 our findings reinforce the importance of characterising epidemic severity as early and as accurately as possible, in order to inform a proportionate pandemic response. critically, a typically mild pandemic (low g), such as that experienced in 2009, is predictably difficult to contain using a targeted antiviral strategy due to the low proportion of infectious cases that present to health authorities. the relative infectiousness of treated individuals, e t , is strongly negatively correlated with successful mitigation, perhaps a surprising result given the model's underlying assumption (based on available epidemiological and human clinical trials data) that e t lies in the range [0ae8, 1]. that is, nais provided as treatment have a maximum impact of just a 20% reduction in infectiousness. however, our previous results 4 show a strong synergistic effect of treatment when overlayed on a contact prophylaxis strategy, explaining the observation here that e t is critical in determining likely success of an intervention. despite the limited impact of treatment at the individual-level, the model outcomes are highly sensitive to the value of the relative infectiousness of treated cases. it follows that determination of e t is important for predicting the population-level outcome of a control effort. a 'small' reduction (of the order approximately 10%) may be extremely valuable in terms of success of a public health control strategy, and so should not be discounted. using a mathematical model which takes into account some of the key logistic constraints that are inherent to healthcare responses, we have derived a logistic regression model for estimating the probability that an antiviral intervention based on liberal distribution of nais as treatment and prophylaxis could successfully mitigate an influenza epidemic. the model demonstrates an excellent degree of accuracy when applied to synthetic data. the choice of parameters for the regression model was restricted to those that were both highly correlated with the success of the intervention and hopefully feasible to measure during the early stages of an emerging epidemic. the model could therefore be a useful near real-time decision support tool for public health policy in the face of an influenza epidemic, although further validation on a range of synthetic data (and real-world data where available) is required. influenza to seasonal flu status to avoid overstretching the demands on healthcare services. a great deal of information has emerged as the result of the pandemic response exercises conducted by affected countries. however, uncertainties remain regarding the effectiveness of intervention measures, as well as the feasibility and the timing of their implementation. mathematical and computational models [2] [3] [4] have been used to project the outcomes of influenza outbreaks under various scenarios and epidemiological hypotheses. motivated by the events of 2009 and public health measures adopted by the taiwan cdc, we use a stochastic, individual-based simulation model 5 to study the spatio-temporal transmission characteristics of the h1n1 virus, so as to quantitatively assess the effects of early intervention strategies. our stochastic disease simulation model 5 builds upon a highly connected network of individuals interacting with each other via social contact groups. to represent the daily interactions of approximately 23 million people living in taiwan, we constructed a computer-generated mock population based on national demographic and employment statistics (to derive daily commute patterns) from the 2000 taiwan census (http://www.stat.gov.tw/). each individual is created with a set of attributes, including age, sex, residence, family structure, and social standing (employment status, etc.). based on their attributes and the time of day, each individual is assigned to miscellaneous contact groups, where the potential of interactions between any two individuals resulting in flu virus transmission occurs. such epidemiological properties are defined by empirically parameterized attributes such as basic reproduction number r 0 , transmission probability, contact probability and associated probability distributions outlining the disease's natural history. additionally, intervention measures are implemented as scheduled events that could alter control parameters during the course of a simulation run. the targeted basic reproduction number (r 0 ) in all our simulations is 1ae6, following the suggested range by who of 1ae2-1ae7. 6 as the latent ⁄ incubation and infectious periods for h1n1 have not yet been reliably ascertained, we adopt the natural history of the 1957 and 1968 pandemic influenza viruses. 2, 7 here, the latent period ranged from 1 to 3 days, with a median value of 1ae9 days. the infectious periods begin 1 day prior to symptom onset and can continue for 3-6 days, with a median value of 4ae1 days. twothirds of the infected individuals will develop clinical symptoms, and the asymptomatic cases will have half the infectious strength. the efficacy of antiviral drugs (oseltamivir) and vaccines are based on these studies. 8, 9 for the source region of the infected cases, we use the north american continent (canada, mexico and united states) with an estimated total population of 450 527 697 and an average 16 hours of flight time to taiwan. the average daily passenger number is 2489 based on the 2009 annual statistical report on tourism, tourism bureau, taiwan (http://admin.taiwan.net.tw/english/statistics/year.asp? relno=61). each simulation lasts 365 days and starts with a baseline simulation of r 0 % 1ae6 h1n1pdm outbreak at the source region. the outbreak was adjusted to approximate clinical attack rate (car) in the united states, april 2009-march 13, 2010. 10 we estimate the daily number of imported cases according to average daily passenger numbers and their probability of holding a disease status. we then apply airport exit ⁄ entry screening per corresponding success rates, by subtracting the number of identified symptomatic cases. we also consider latently infected passengers with inflight disease progression, by fitting a gamma distribution to the cumulative distribution of time to onset data with 16 hours average flight-time, as presented by pitman et al. 11 the daily imported cases are seeded according to the traveling patterns of foreign tourists and residents returning home. from the disease's natural history, we derive that roughly 50% of the infected travelers present no symptoms; the percentage increases if most symptomatic individuals elect not to travel in their condition, or are stopped by airport screening. we use the official epidemic data provided by the taiwan cdc to calibrate the simulation model and perform regression analysis on scenario parameters. this data is a close estimation of the weekly new clinical cases of h1n1pdm patients. it consists of weekly opd (outpatient department) icd-9 code 487 (influenza) tallies collected by the bureau of national health insurance, taiwanadjusted to exclude seasonal flu patients and to account for uninsured patients. we formulate our scenario settings according to 2009 events in taiwan, and establish settings to approximate the actual events. with domestic events and intervention schedules fixed in time, the start date determines the simulation outcomes and the data range for selected indicators, such as the mean car, the epidemic peak, and several significant dates for the incoming index case events. we plot the 2009 taiwan weekly h1n1 opd487 cases alongside the weekly new clinical cases from our simulation results in figure 1 . our simulations not only capture the epidemic trend, but also pick out the most likely date, may 20, for identifying the first symptomatic case at airport screening based on practical assumptions. we further analyze the effectiveness of various mitigation measures with february 6, 2009 as the empirical start date for h1n1pdm in north america. the simulation result confirms that by the time we identified the first symptomatic case at the border screening, infected cases had already made their way to the public. by our calculation, roughly four such cases had passed in each of our scenario settings, with the first case happening as early as 3 weeks before detection. figure 1 also highlights the importance of the timing for the implementation of mitigation measures; for example, a 6-day-delay of the identical intervention plan results in nearly an additional 1% of the population being infected. therefore, the rule of thumb for healthcare officials is to implement intervention measures as early as possible. in our study, we have ignored the possibility of inflight transmission and any false positive results by airport screening procedures. to assess the effectiveness of each mitigation strategy of interest and their combinations, we take the calibrated simulation model and perform 100 simulation realizations for groups of scenarios containing only those intended mitigation measures, and analyze the averaged results. for example, in the airport exit screening policy only scenario, the first imported symptomatic case can be delayed up to 2 months, and the epidemic peak can be delayed up to 13 days. as the data suggests, the exit screening policy alone has very little impact on car. combining various screening success rates for both exit and entry screening allows us to quantitatively assess their beneficial ramifications on the epidemic. for example, there is very little additional benefit between 100% and 80% suc-cess rates for entry screening policies when exit screening policies are adequate, as the enhanced border screening only delayed the epidemic peak by 1 day, and reduced car by <0ae01%. base on this result, the government should not attempt to exhaust all its resources in securing the border during a pandemic event, because the return of such a policy will be disappointing. instead, a response plan with a shifting focus on health resource allocation and the capacity of adjusting intervention strategies in line with the developing epidemic will be most effective. based on the same principle, we perform experiments with assorted scenarios, including relaxing entry screening policies after identifying the first imported symptomatic case, mass vaccination based on the actual vaccination schedule of h1n1pdm in taiwan, and altering the start dates of the vaccination schedule. our results show that with a reasonable reduction in the airport entry screening success rate, we conserve valuable healthcare resources, but loose a few days for the strategic planning and preparation of subsequent response measures. in other simulation scenarios, a national vaccination campaign has very little impact on the outcome, due to the late start of the vaccination schedule. we then explore the effect of a national vaccination campaign with various starting dates. the simulation results are illustrated in figure 2 , where the benefit of an early start date for mass vaccination is clearly demonstrated. considering a scenario with an 80% airport exit screening success rate, 80% airport entry screening success rate and 80% symptomatic case tracing success rate, the combined intervention strategy results in: a 2% reduction in car if the vaccination campaign starts in mid-november; 11% reduction if the campaign starts in mid-october; 26% reduction if the campaign starts in mid-september; and 37% reduction if the campaign starts in mid-august. in retrospect, the taiwanese government's response to h1n1pdm proved to be effective. first and foremost, it initiated enhanced border monitoring and on-board quarantine inspection as soon as the threat of a flu pandemic became clear. at the same time, the domestic preparations towards h1n1pdm were escalated, such as antiviral drug stockpiling and distribution, and vaccine acquisition. as the h1n1 cases increased worldwide, various revised plans were adopted and implemented; such as the shift from labor-extensive on-board quarantine inspection to the notifiable infectious disease reporting system and realtime outbreak and disease surveillance system in order to effectively track down symptomatic and exposed passengers, apply prophylaxis treatment and mandatory in-home quarantine. as a result, all h1n1pdm related statistics are well below the international average. in modern society, countries rely heavily on the global economy for their own prosperity. shutting down the border for any length of time is not only costly, but could have disastrous economic effects that linger long after the event is over. moreover, with nearly 50% of the infected passengers presenting no symptoms whatsoever, they are not detectable by any port authority's screening procedures, and the importation of the novel flu virus is therefore inevitable. many studies conclude that entry screening is unlikely to be effective in preventing or delaying the importation of influenza, and has negligible impact on the course of subsequent epidemic. however, these studies are based on the assumption that effective exit screening is in place. our study shows that as the exit screening success rate decreases, the sensitivity of the entry screening policy becomes more pronounced. with the same methodology, we can also study the effects of varying the length of flight time, or the disease's incubation time. lastly, the benefit of entry screening is even more crucial for a small island country such as taiwan, since all incoming traffic must go through the port authority where entry screening can be enforced. in england and wales, three waves of the pandemic struck in summer, autumn, and winter seasons of 1918-1919. although the proportion of people reporting symptoms was often greater in the first wave, 1-3 a puzzling feature was the much higher mortality in the second wave, in which 0.27% of the population died, compared with 0.03% in the out-of-season first wave and 0.10% in the third wave. 4 an obvious hypothesis to explain the changes in mortality from wave to wave would be that the 1918 virus mutated to higher virulence after the (lower mortality) first wave. although pandemic virus reconstituted from the high mortality waves has proven to have high virulence in animals, 5 it has not been possible to recover virus from the first wave in 1918 for comparative purposes. indeed it is questionable whether virulence mutation(s) occurring between wave 1 and wave 2 could have spread to so many different populations in the time-frames observed. furthermore, in all three pandemic waves, there was the same agedistribution of mortality, with more deaths occurring amongst younger adults than older adults. [1] [2] [3] this 'pandemic signature', arguably due to immune protection of older adults who were exposed to a similar virus in the years before 1890, 6, 7 suggests that the 1918-1919 viruses were at least immunologically similar in all three waves. a second hypothesis would be that the higher case fatality in the later waves was due to higher rates of complicating bacterial pneumonia, 8 to increased transmission of influenza virus in the cooler months of the year, or to other seasonal effects. 9 we have considered a third (immunological) hypothesis to explain the greatly increased mortality in waves 2 and 3. the underlying idea is that the mortality rate in the first wave was lower than in later waves because most persons were protected by prior immunity in the first wave, and that the mortality was higher in later waves because of waning of that short-lived immunity. this hypothesis builds on our earlier modelling papers suggesting that even before the first wave in 1918, military, 10 school, and urban 11 populations in england and wales apparently had (short-lived) immune protection, presumably induced by recent prior exposure to seasonal influenza. [10] [11] [12] we suggest that this short-lived strain-transcending protection was in addition to the longer-lasting immunity, presumably induced by exposures to a similar virus circulating prior to 1890, that arguably reduced pandemic mortality for older adults in 1918-9. 6,7 cumulative mortality rates attributed to pandemic influenza were available for each of the three waves in 1918-1919 for 330 populations in england and wales. 13 we have built immunological models to potentially explain the variation in mortality rates across waves and populations. to show proof of principle, we have fitted these models to mortality data from a randomly selected sub-set of twenty populations. our key assumption was that the risk of a fatal infection would be limited to persons with inadequate immunity who were being exposed to the pandemic virus for the first time. persons who were exposed and who survived an earlier wave were assumed to be protected against death in a later wave. model a and assumptions (see figure 1) before the first wave, we assumed that people could be fully susceptible (s 0 ), or partially protected (q 0 ), or fully protected (p) by prior immunity which was not necessarily specific for the new virus. we assumed that exposure to the new pandemic virus would be fatal (m) in a proportion h of fully susceptible persons who were actually exposed (e) in the relevant wave. for those surviving that first exposure, it was assumed that they would be permanently protected against death in later waves by an immune assumed that viral exposure and multiplication would induce an immune response specific for the pandemic virus that would protect them against death in that wave and in subsequent waves. in contrast, for persons with strong prior immune protection, p, the virus would not be able to multiply to induce pandemic-specific immune protection. between waves, it is assumed that due to the waning of non-specific prior immunity, persons in the p state can move to the q state, and persons in the q state can move to an s state before the next wave. the proportion (e) of susceptible persons exposed to productive infection in each population was estimated by applying the following version of the final size equation 11 to the proportion susceptible (s & q) in each wave, for each population: note: in both figures 1 and 2 , we have omitted the flows out of the q and e states that removed persons from the risk of death. parameters: s 0 = proportion fully susceptible to infection and death before wave 1; q 0 = proportion susceptible to immunising infection, but not to death from exposure in wave 1; p 0 = proportion temporarily protected against both immunising infection and death from exposure in wave 1; n 0 = proportion even more protected against both immunising infection and death from exposure in wave 1 (model b only); r 0 = basic reproduction number (the average number of secondary cases for each primary case) in a fully susceptible population; f = proportion moving from q to s between waves; g = proportion moving from p to q between waves; d = proportion moving from n to p between waves (model b); h = proportion of e that actually move to m and die. model a could provide a very good fit for the summer, autumn, and winter waves of the 1918-1919 pandemic (results not shown). however, because of the replenishment of the pool of susceptible persons over time, model a also predicted a fourth wave of influenza in the spring season of 1919. as no such wave was seen, and as we could not find parameters values for model a that did not predict a fourth wave, we must regard model a as inadequate. model b was similar to model a, but with an additional stage of prior immunity (n), which could wane to p. model b allowed us to not only fit the three observed waves, but also to fit the imputed data (zero cases) corresponding to the absent fourth wave. following earlier work, 10,11 we used a bayesian approach with markov chain monte carlo (mcmc) procedures to estimate model parameters, and we used hyper-parameters to allow for parameter variation between populations. 11 the initial conditions were specified by the parameters: p 0 , q 0 , s 0 and n 0 . from these and the other parameters, it was possible to simulate the behaviour of model a over three waves, and of model b over four waves, and to estimate the expected numbers dying in each wave in each population. we calculated the log likelihood of the observed numbers of deaths given the parameter estimates, and we used mcmc simulation to generate the posterior distributions of parameters. although we obtained an excellent fit between observed and expected numbers of deaths in each of the three waves for the 20 populations for model a, we could not find parameter values for model a that would fit the three observed waves without giving rise to a fourth wave in the spring of 1919. accordingly, in the modified model b, we allowed for an additional stage of prior immunity (figure 2) , and we fitted the model to the same data, plus imputed data corresponding to 'the absent fourth wave'. we obtained a very good fit to the three observed waves and the absent fourth wave in each population. the 95% credibility intervals for parameter estimates, derived from the posterior distributions of the hyper-parameters were: h = 0.05-0.08, s 0 = 0.015-0.032, q 0 = 0.40-0.47; n 0 = 0.2 (fixed); p 0 = 1 ) s 0 ) q 0 ) n 0 ; f = 0.33-0.44; g = 0.87-0.94; d = 0.94-0.97 and r 0 = 2.6-3.6. this analysis had allowed all parameters to vary from population to population under the constraints of the hyper-parameters. however, several of the biologically determined parameters might be expected to be more constant from population to population, whereas those dependent on mixing history and other social characteristics which vary more widely from population to population. to test this possibility, we fixed the mean values for the more biological parameters (f = 0.385; d = 0.955; g = 0.905) and estimated the 95% credibility intervals for the others as: h = 0.05-0.10; s 0 = 0.012-0.032, q 0 = 0.36-0.44; and as before n 0 = 0.2 (fixed); in a subsequent paper we will be able to provide more details of the method, the robustness of the assumptions, and the results from fitting to many more populations. this short report suggests that the observed patterns of mortality in england and wales over the three waves of the 1918-1919 influenza pandemic 4,13 can be explained by an immunological model. in particular, the lower mortality in wave one can be explained by the assumption of protective immunity antedating the first wave, arguably induced by prior exposure to seasonal influenza. 10, 11 the much greater mortality in wave two can be explained by the waning, between wave one and wave two, of that short-lived and less-specific immune protection. the somewhat lesser mortality in wave three and the 'absent fourth wave' can be explained in terms of the progressive acquisition of immunity specific to the pandemic virus. the credibility estimates for parameters are of potential interest. for example, r 0 estimates of 3.1-4.5 across different populations are consistent with our earlier findings. 10, 11 if all persons had been susceptible, such r 0 values imply that the virus would have infected most people in all populations. however, even in the first wave, the proportion susceptible, s 0 + q 0 , was <50% in all populations, so that a considerable number of persons escaped productive infection in that wave; as their immunity waned, they became susceptible to infection in the later waves. it is likely that the variation in r 0 between populations is due to different rates of population mixing. estimates for h indicate that between 5% and 10% of infections in the most susceptible persons were fatal; the higher values of h could reflect higher rates of secondary bacterial infection in the most socially disadvantaged and overcrowded populations. 4 although we have shown the plausibility of an immunological explanation for wave to wave changes in pandemic mortality, we cannot assume that our particular model is even approximately correct. nor can we exclude the possibility that the higher mortality in the later pandemic waves in 1918-1919 was because of genetic change in the virus in later waves, or because of changing rates of secondary bacterial infection 8 or seasonal effects. 9 nevertheless, there is growing evidence that the population spread of pandemic influenza, whether in 1918-1919 10, 11 or in 2009, 12, 14 can be constrained by significant prior immunity, even for viruses that are ostensibly novel. previous reports, reviewed in ref. [10, 11] , support the idea of strain-transcending immune protection, which can wane over periods of a few months. this form of protection, probably induced by recent exposure to seasonal influenza, may not be mediated by hi or neutralizing antibody. 11 in contrast, strain-specific immunity, most often mediated by hi or neutralizing antibodies can be so long-lasting that after several decades it will still provide significant protection against any closely-related virus that re-appears in the population. 14 it has not escaped our notice that although attack-rates in the h1n1 2009 pandemic were low in many countries, with generally mild symptoms, the virus did cause lifethreatening illness in a small proportion of younger affected persons. 15 it seems likely that those who were most severely affected in 2009 were doubly unlucky: they had missed out on seasonal influenza infection or vaccination in the preceding season(s), and they were born too late to have been protected by the closely-related viruses that are thought to have circulated before 1950. 14 during the early phases of the 2009 influenza pandemic in italy, real-time modeling analysis were conducted in order to estimate the impact of the pandemic. in order to evaluate the results obtained by the model we compared simulated epidemics to the estimated number of influenza-like illness (ili) collected by the italian sentinel surveillance system (influnet), showing a good agreement with the timing of the observed epidemic. by assuming in the model mitigation measures implemented in italy, the peak was expected on week 44 (95% ci: 44, 45). results were consistent with the influnet data showing that the peak in italy was reached in week 46. these predictions have proved to be a valuable support for public health policy makers for planning interventions for mitigating the spread of the pandemic. mathematical models have recently become a useful tool to analyse disease dynamics of pandemic influenza virus can-didates. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] as of april 2009, after the pandemic threat emerged worldwide, 12 it was crucial for policy makers to have early predictions on the possible spread of the 2009 pandemic influenza virus in order to support, with quantitative insight into epidemic, policy decisions. thus, after the first pandemic alert was announced by the world health organization (who) in late april 2009, a national crisis management committee headed by the minister of health was established in italy in order to provide weekly advice to the italian ministry of health. real-time analyses using an individual based model were undertaken. the transmission model was previously used for evaluating the effectiveness of the control measures adopted in the national pandemic preparedness plan 3 and for assessing the age-prioritized distribution of antiviral doses during an influenza pandemic. 2 to parameterize the transmission model, we used data derived from the national surveillance system until 17 june 2009 and estimates of key epidemiological parameters as available at that time. in order to provide a preliminary assessment of the model predictions performed during the early stages of the epidemic, we compare model predictions with surveillance data of influenza-like illness (ili) available since august 2009. after the first pandemic alert was announced by the who in late april 2009, a national active surveillance system for the 2009 pandemic influenza was set up from 28 april to july 2009. 13 however, over the period from april to october 2009, surveillance systems, laboratory testing, and diagnostic strategies have varied considerably in italy. since end of july 2009, following who recommendations, 14 the focus of surveillance activities has changed in reporting requirements, as active case-finding became unsustainable and unnecessary. for this reason, the ministry of health (ministry of health, available in italian at the website: http://www.normativasanitaria.it) requested regional health authorities to report the weekly aggregated ili cases according to a new case definition (sudden onset of acute respiratory symptoms and fever >38°c plus at least one of the following systemic symptoms: headache, malaise, chills, sweats, fatigue; plus at least one of the following respiratory symptoms: cough, sore throat, nasal obstruction). by october 2009, following the increasing number of cases, the sentinel influenza surveillance system (influ-net available at: http://www.flu.iss.it) became the official surveillance system for ili cases in italy (ministry of health, available in italian at the website: http://www. normativasanitaria.it). since 1999, influnet is routinely based on a nation-wide, voluntary sentinel network of sentinel community based physicians in the 21 regions and autonomous provinces of the country. incidence rates are, therefore, not based on consultations, but on the served population of each reporting physician each week. influ-net usually consists of an average of 830 (range 648-902) general practitioners (including physicians and pediatricians) per year, covering about 1ae5-2% of the general population, (representative for age, geographic distribution, and urbanization level) reporting ili cases (according with a specific case definition). italian influnet surveillance system is part of the european influenza surveillance scheme (eiss). a stochastic, spatially explicit, individual-based simulation model 3 was used. individuals are explicitly represented and can transmit the infection to household members, to school ⁄ work colleagues, and in the general population (where the force of infection is assumed to depend explicitly on the geographic distance). the national transmission model was coupled with a global homogeneous mixing susceptible-exposed-infectious-removed (seir) model accounting for the worldwide epidemic, which is used for determining the number of cases imported over time. regarding the epidemiological assumptions (e.g., length and shape of the infectivity period, which lead to an effective generation time of 3ae2 days), this study is consistent with refs [3, 4, 11, 15] , but for the proportion of symptomatic individuals, which is assumed to be 66ae7%. 16 the basic reproductive number of the national transmission model was set to 1ae4, according to the early estimates as obtained during the initial phase of the epidemic in mexico in a community setting. 17, 18 we initialized our simulations through the global homogeneous mixing model in such a way that 158 imported cases were generated until 7 june 2009. this gives a reliable way for fixing the time in the simulations and thus determining the timing of school closure and vaccination in the simulations. the model accounts for school closure for both summer and christmas holidays: we assumed that in these periods contacts among students decrease, while contacts in the general community increase, as in ref. [19] . we also considered scenarios accounting for partial immunity in the population. 20 in order to investigate the effects of recommendations of the ministry of health (confirmed cases coming from affected areas were isolated for 7-10 days, either in hospital or at home) established in the early phase of the pandemic (april-july 2009), we assumed that a fraction of the imported symptomatic cases were isolated on the first day after the symptoms onset. this recommendation was in place until 27 july 2009. we also assumed, according to the italian school calendar, that schools were closed from 10 june 2009 to 10 september 2009 for the summer holidays, and from 22 december 2009 to 6 january 2010 for christmas holidays. the effects of prolonged school closure were also investigated. when considering vaccination, we assumed 6 weeks for the logistical distribution of doses of pandemic vaccine. since at the time of simulation specific recommendations regarding the administration of a single dose of pandemic vaccine from ema were not available yet, we considered the administration of 2 vaccine doses 1 month apart). the pandemic vaccine was considered effective after the administration of the second dose with a vaccine efficacy of 70%. we assumed the vaccine to be administered by priority, vaccinating first the target population accounting for essential services workers (including health care workers and blood donors), pregnant women at the second or third trimester, and at risk patients (with chronic underlying conditions) younger than 65 years old. the vaccination coverage was assumed 90%. regarding antiviral treatment and prophylaxis, recommendations of the ministry of health in the initial phase of the epidemic were to administer antivirals to all confirmed cases and to their close contacts. we assumed that the surveillance system would be able to detect 90% of symptomatic cases. after 8 july 2009, recommendations changed and antiviral treatment was considered only for cases with severe complications and in case of local clusters. since it was difficult to establish the proportion of treated cases, we considered different scenarios: antiviral treatment from 0% to 30% of the symptomatic cases. consistently with ref. [3] , both treatment and prophylaxis were assumed to start 1 day after the clinical onset of symptoms in the index case. treatment was assumed to reduce infectiousness by 70%, whereas antiviral prophylaxis was assumed to reduce susceptibility to infection by 30%, infectiousness by 70%, and the occurrence of symptomatic disease by 60%. as of 26 july 2009, approximately 1238 confirmed cases have been reported to the italian surveillance system for pandemic influenza. during july, the sudden increase of ili confirmed cases suggests for sustained autochthonous transmission in italy. by analyzing the number of ili cases reported to the surveillance during the weeks from 39 to 44, we found that the exponential growth rate was 0ae832 ⁄ week and thus we estimated the national reproductive number to be r 0 = 1ae38. this estimate of the basic reproductive number supports the choice of the value adopted in the model simulations (r 0 = 1ae4). in the absence of intervention measures, the predicted cumulative attack rate was 30ae6% (95% ci: 30ae6, 30ae7), and the peak was expected on week 42 (95% ci: 41, 42) with a peak day incidence of 0ae35% (95% ci: 0ae329%, 0ae37%). by assuming case isolation, antiviral treatment, and prophylaxis to 90% of symptomatic cases until 8 july 2009, the peak was expected on week 43 (95% ci: 43, 44). when considering 33ae3% of natural immunity in the population aged more than 59 years, the peak was expected 1 week later than in the previous scenario, i.e., on week 44 (95% ci: 44, 45). to validate the model, we compared model predictions (which are based only on the available information on the early phases of the epidemic) with ili data (figure 1 ). based on model predictions, we estimated the underreporting factor of influnet ranging from 3ae3 to 3ae7, considering different scenarios. by aligning the simulations with the ili data adjusted by the underreporting factor, we can observe that almost all the points in the increasing phase of the epidemic lie within the 95% ci of the model results (both considering or not natural immunity). the decay phase of the simulated epidemics shows a small delay with respect to the ili data. when introducing single and combined mitigation measures, such as case isolation, antiviral treatment, prophylaxis, and vaccination in the model, results showed that even a low proportion of symptomatic cases treated with antiviral drugs could have led to a relevant reduction in the epidemic size (table 1) . we simulated the planned italian vaccination strategy (begun on 15 october 2009), obtaining a limited but not negligible reduction in the attack rate with respect to the scenarios accounting only for antiviral treatment. moreover, the effect of vaccination would be higher if coupled with antiviral treatment; vaccination would have no effect on delaying the peak incidence. model predictions produced in italy during the early phase of the 2009 pandemic influenza are in excellent agreement with italian surveillance data on the beginning of the epidemic (when case isolation, antiviral treatment of index cases, and antiviral prophylaxis to close contacts were implemented by the italian regional public health authorities) and are basically consistent with the influnet data during the course of the epidemic. the model has been useful for predicting the timing of the epidemic, while it has overestimated the impact of the 2009 influenza pandemic for adult and elderly individuals. however, the disalignment is probably due to the model parameterization. based on literature values, 4,5 we assumed a similar fraction of cases in the different social contexts considered in the model (namely 1 ⁄ 3 in households, 1 ⁄ 3 in schools ⁄ workplaces, and 1 ⁄ 3 in the general community), since analysis on the relative transmissibility of the virus was not carried out for any country yet. we were also able to estimate an underreporting factor for the influnet data in the range 3ae3-3ae7. if we focus our attention on the reporting factor computed by considering the total number of cases (instead of symptomatic cases), the resulting value lies in the range 18-20ae2%, which is in excellent agreement with the range estimated in ref. [21] on previous a ⁄ h1n1 influenza seasons, namely 16ae2%-21ae6%. moreover, based on our results showing that vaccinating 40% of the italian population was more than adequate to mitigate the pandemic, the ministry of health decided to stockpile a limited number of vaccines. we have also shown that starting the vaccination program in october (or later) could have had only a limited effect on reducing the impact of the epidemic, although it may have been useful to prevent a possible second wave and to protect essential workers and at-risk patients. finally, our results have shown that antiviral treatment would have been the most efficient strategy to reduce the impact of the influenza pandemic, even with a limited antiviral stockpile. a population-wide passive immunotherapy program in this paper, we assume that convalescent plasma (cp) is efficacious in treating severe cases of pandemic influenza. under this premise, we test the hypothesis that a population-wide passive immunotherapy program that collects plasma from a small percentage of convalescent individuals can harvest sufficient cp to treat a substantial percentage of severe cases during the first wave of the pandemic. the proposed program involves recruiting adults (individuals age 20-55 years) to donate blood if they have experienced influenza-like symptoms more than 2 weeks ago (to account for the time needed for neutralizing antibodies to build up). the blood samples would be screened for infectious diseases (including hiv, hbv, hcv, htlv, and syphilis, etc., as in routine blood donation screening) and neutralizing antibodies against the pandemic virus. donors whose blood samples are free of known infectious agents and contain a sufficiently high titer of neutralizing antibodies would then be invited to donate plasma by plasmapheresis or routine whole blood donation. qualified donors with higher titers may be given higher priority for plasma donation. in this paper, we use the demographic and logistical parameters of hong kong as a case study. see figure 1 for a schematic of the proposed passive immunotherapy program. we examine the following questions regarding the logistical feasibility and potential benefits of the proposed passive immunotherapy program: (i) what percentage of convalescent individuals (donor percentage) is needed in order for the program to significantly reduce pandemic mortality? (ii) how many severe cases can be offered passive immunotherapy? (iii) what are the ratelimiting factors in the supply of passive immunotherapy? (iv) what are the epidemiologic and logistical factors that determine the demand-supply balance of passive immunotherapy? a more detailed presentation of our results is now available in ref. [ 5 ] . transmission and natural history model for pandemic influenza we use an age-structured disease transmission model to simulate the spread of pandemic influenza. the natural history model is similar to that used by basta et al. 6, 7 the most important parameter in characterizing the growth of an epidemic is the basic reproductive number r 0 , which is defined as the average number of secondary cases generated by a typically infectious individual in a completely susceptible population. we consider values of r 0 between 1ae2 and 2, which is consistent with recent estimates. 6, [8] [9] [10] logistical model for the passive immunotherapy program we assume that q d (%) of 20 to 55 year-old individuals who have recovered from symptomatic infections of pandemic influenza donate their blood for screening t r = 14 days after cessation of symptoms. follow-ups of convalescent individuals infected with h1n1pdm in an ongoing clinical trial of passive immunotherapy suggested that neutralizing antibodies level reaches maximal level around 14-21 days after recovery and stays at that level for months after. 11 we assume that q s (%) of these donors are qualified for plasma donation of which q r (%) are recurrent donors who return to donate plasma every t w = 14 days. screening involves both detection of infectious agents and neutralizing antibodies against the pandemic virus. the latter is the rate-limiting step because neutralization tests of pandemic viruses can only be done in a bsl3 setting. we assume that five bsl3-trained technicians are available to test the blood specimens, each running 150 viral neutralization tests in 3 days. therefore, the capacity and turnaround time of blood screening are u s = 750 and t s = 3 days, respectively. hong kong currently has nine plasmapheresis machines which allow a maximal throughput of 162 plasma donations per day (assuming 12-hour daily operation with each donation taking 40 minutes). therefore, the capacity and turnaround time of plasmapheresis are u p = 9 and t p = 1 ⁄ 18 days, respectively. collected cp are ready for use in transfusion after final quality check, which takes t q = 2 days. we assume that r t plasma donations are required to treat one severe case on average. the expert panel of the abovementioned study of passive immunotherapy for h1n1pdm in hong kong suggested that r t < 10. we assume that p h (%) of symptomatic cases will be severe cases for whom passive immunotherapy is suitable. although p h will be smaller than the case-hospitalization rate (passive immunotherapy may not be suitable for some hospitalized cases), we assume that the two have similar ranges and consider p h ranging from 0ae1% to 1%. because each severe case requires r t plasma donations on average, demand for cp is simply r t p h times the number of symptomatic cases. therefore, r t p h can be regarded as a single parameter, which we refer to as the lumped demand parameter. we define the outcome as the percentage of severe cases that can be offered passive immunotherapy by the proposed program during the first wave of the local epidemic. we refer to this outcome as treatment coverage and denote it by q. we consider the base case scenarios assuming q r = 20% and q s = 80%. in general, the treatment coverage q increases sharply as the basic reproductive number r 0 and the lumped demand parameter r t p h decrease (figure 2a ). in particular, when r 0 is large and r t p h is small, q is very sensitive to r t p h , but insensitive to r 0 . similarly, when r 0 and r t p h are small, q is very sensitive to both. with a donor percentage of q d = 15%, the proposed program can supply passive immunotherapy to more than 82% of severe cases (q > 82%) if r 0 < 1ae4 and r t p h < 1ae5%, but <35% if r 0 > 1ae8 and r t p h > 1ae5%. in general, the treatment coverage q increases sharply as the donor percentage q d rises from 0%, but with rapidly decreasing marginal increase ( figure 2b ). when r 0 < 1ae4 and r t p h < 1ae5%, q > 67% even if q d is as low as 5%, which is comparable to the current average blood donation rate of 38ae1 donations per 1000 population in developed countries. 12 when q d is >15%, q becomes largely insensitive to further increase in q d in most scenarios. the treatment coverage q for q d = 15% is more than 81% that for q d = 50% across all values of r 0 and r t p h considered in the base case. therefore, increasing the donor percentage q d beyond 15% has a relatively small impact on cp supply. this is because increasing q d can boost supply only when plasmapheresis is not yet the supply bottleneck. for the same reason, once the donor percentage q d has reached 15%, the treatment coverage q is insensitive to further increase in q d even when the plasmapheresis and screening capacity are doubled ( figure 2b , lower panel). we conduct an extensive multivariate sensitivity analysis to test the robustness of our base case observations against uncertainties in parameter values. we generate 15 000 epidemic scenarios by randomly selecting parameter values from their plausible ranges using latin-hypercube sampling. although there are numerous model parameters, the treatment coverage q is mainly determined by three lumped parameters: (i) r t p h , which indicates the magnitude of demand; (ii) q s q d , which indicates the magnitude of supply; (iii) the initial growth rate of the epidemic r (results not shown). while the dependence of q on r t p h and q s q d is readily comprehensible, it is not obvious a priori that q depends on the natural history and transmission dynamics of the disease via only the initial epidemic growth rate. when the plasmapheresis and screening capacity are very large, the supply-demand dynamics is further simplified: the treatment coverage q depends on lumped demand parameter r t p h and the lumped supply parameter q s q d only via their ratio. finally, q becomes insensitive to q s q d when the latter increases beyond 15-20%, which is consistent with our base case observations. our results suggest that with plasmapheresis capacity similar to that in hong kong, the proposed passive immunotherapy program can supply cp transfusion to treat 67-82% of severe cases in a moderate pandemic (basic reproductive number r 0 < 1ae4, lumped demand parameter r t p h < 1ae5%) when the donor percentage is 5-15%. increasing the donor percentage beyond 15% has little additional benefit because cp supply is constrained by the capacity of plasmapheresis during most stages of the epidemic. increasing plasmapheresis capacity could significantly boost cp supply, especially when there is a substantial pool of recurrent donors to alleviate the dependence of cp supply on donor percentage. in an ongoing clinical trial of passive immunotherapy for h1n1pdm virus infection in hong kong, 20% of convalescent individuals agreed to donate their plasma for the study. therefore, the donor percentage required by the proposed passive immunotherapy program (5-15%) is likely to be feasible. in view of the logistical feasibility of such program, we recommend that further clinical studies are conducted to evaluate the safety and efficacy of passive immunotherapy as a treatment for severe cases of pandemic influenza virus infection. our study is based on the premise that cp will be efficacious in reducing morbidity and mortality associated with pandemic influenza. in theory, the polyclonal nature of neutralizing antibodies in cp would lower the probability of an escape mutant emerging in treated patients. further, besides providing neutralizing antibodies against the pandemic virus, cp also might carry antibodies to other bacterial pathogens, which might decrease the severity of coexisting bacterial infections. 4 as such, cp not only might reduce the case fatality rate but might also increase the recovery rate and shorten duration of hospitalization of severe cases. the proposed passive immunotherapy program can thus significantly reduce the burden on the healthcare system, especially the intensive care unit, which will likely be stressed, if not overloaded, at the peak of an influenza pandemic wave, hence benefiting the general public and not only those receiving passive immunotherapy. although the hypothesized efficacy of cp has yet to be proven in clinical trials, our modeling results show that a public health system similar to that in hong kong has the capacity to support a population-wide passive immunotherapy program that can supply cp treatment to a substantial percentage of the severe cases in a moderately severe pandemic. we estimate that compared to other developed countries, hong kong has a relatively low plasmapheresis capacity. our conclusions regarding donor percentage needed and rate-limiting factors remain valid for plasmapheresis capacity ranging from 50% to 400% of what we have assumed in the base case (results not shown). our conclusions are robust against uncertainties in the natural history and transmission dynamics of pandemic influenza. our sensitivity analysis shows that the outcome depends on these epidemiological characteristics only via the initial growth rate of the epidemic. as such, our results are applicable not only to pandemic influenza, but also to other emerging infectious diseases for which the time-scales of disease transmission and antibody response are similar to that for influenza virus. the three determinants of treatment coverage (the initial epidemic growth rate, the lumped demand parameter r t p h , and the lumped supply parameter q d q s ) are all readily measurable in real-time during an epidemic. therefore, our methods and results can be used as a general reference for estimating the treatment coverage of the proposed passive immunotherapy program for a given plasmapheresis capacity. background highly pathogenic h5n1 virus continues to pose a serious threat to human health and appears to have the capacity to cause severe disease in previously healthy young children and adults. at present, antiviral therapy by oseltamivir remains the mainstay for managing h5n1 patients. while early treatment improves survival, approximately 50% of patients treated within 4 days of illness still succumb to the disease. in addition to the role of viral replication, there is good evidence that the host proinflammatory responses contributes to h5n1 pathogenesis. this suggests that both antiviral and immune-modulatory drugs may have a role in therapy. we previously demonstrated that cyclooxygenase 2 (cox-2) plays a regulatory role in h5n1 hyperinduced pro-inflammatory responses, and its inhibitor has potent effects at modulating this host response. now we demonstrate that, in addition to its immune-modulatory effect, a selective cox-2 inhibitor, ns-398 has a direct antiviral effect against h5n1 infection. materials and methods human primary monocytederived macrophages or alveolar epithelial cells (a549) were pre-treated with ns-398 or drug-vehicle for 1 hour before h5n1 virus infection. h5n1 viruses at multipicity of infection (moi) of 2 was used to infect the cells. following virus adsorption for 30 mins, the virus inoculum was removed, and the cells were washed and incubated in corresponding medium with ns-398 or drug-vehicle as controls for 3, 6, 24, 48, and 72 hours post-infection. cells were harvested for rna isolation at 6 hours post-infection to study viral matrix (m) gene expression. supernatants were collected for 50% tissue culture infection dose (tcid 50 ) assay to determine the virus titers at 3, 24, 48, and 72 hours after h5n1 infection. results ns-398 was found to suppress virus gene transcription and infectious virus yield in h5n1-infected human cells. conclusion we demonstrate that a selective cox-2 inhibitor, ns-398, shows an inhibitory effect on h5n1 viral replication in addition to its immune-modulatory effect that could counter the detrimental effects of excessive proinflammatory cytokine production. the findings suggest that selective cox-2 inhibitors may be a therapeutic target for treating h5n1 disease in combination with appropriate antiviral therapy. the emergence and spread of the highly pathogenic avain influenza viruses (h5n1) in poultry and wild birds with repeated zoonotic transmission to humans has raised pandemic concern. at the time of writing, 507 human cases have been reported with 302 fatalities, an overall case fatality rate of around 60% (cumulative number of confirmed human cases of avian influenza a ⁄ (h5n1) reported to world health organization updated to 18 october 2010). our previous data demonstrated that cox-2 was markedly up-regulated in h5n1-infected primary human macrophages, and that it played a regulatory role in the h5n1hyperinduced host pro-inflammatory responses. 1 such cytokine dysregulation is proposed to be a major contributor to the pathogenesis of h5n1 disease in humans. 2 with the use of selective cox-2 inhibitors, we found that the h5n1-hyperinduced cytokine response was significantly suppressed by the drug in a dose-dependent manner. 1 selective cox-2 inhibitor is a form of a non-steroidal anti-inflammatory drug that selectively targets cox-2, and it is an inducible enzyme responsible for inflammatory process and immune response. here, we report a novel finding of a direct antiviral effect of a selective cox-2 inhibitor, ns-398, against h5n1 infection in human primary macrophages and alveolar epithelial cells. taken together with our previous findings that suggest an immuno-modulatory effect that can modulate virus driven cytokine dysregula-tion, these findings highlight a role for cox-2 and its downstream signaling as potential novel targets for adjunctive therapy of severe viral pneumonia, such as that caused by h5n1. such therapy may be combined with conventional antiviral drugs. the h5n1 virus used was a ⁄ vietnam ⁄ 3212 ⁄ 04 (3212 ⁄ 04) (h5n1), a virus from a patient with h5n1 disease in vietnam during 2004. the viruses were grown and titrated in madin-darby canine kidney cells cells as described elsewhere. 3 virus infectivity was expressed as tcid 50 . all experiments were performed in a biosafety level 3 facility. monocyte-derived macrophages: peripheral-blood leucocytes were separated from buffy coats of healthy blood donors (provided by the hong kong red cross blood transfusion service) by centrifugation on a ficoll-paque density gradient (pharmacia biotech) and purified by adherence as reported previously. 3 the research protocol was approved by the ethics committee of the university of hong kong. macrophages were seeded onto tissue culture plates in rpmi 1640 medium supplemented with 5% heat-inactivated autologous plasma. the cells were allowed to differentiate for 14 days in vitro before use in the infectious experiments. alveolar epithelial cells: a549 cells were obtained from atcc and maintained in culture using dulbecco's modified eagle medium supplemented with 10% fetal calf serum, 0.6 mg ⁄ l penicillin, and 60 mg ⁄ l streptomycin. differentiated macrophages or a549 cells were pre-treated with a selective cox-2 inhibitor, ns-398 (cayman), at concentrations as indicated or drug-vehicle for 1 hour before infection. cells were infected with h5n1 viruses at moi of 2. following virus adsorption for 30 min, the virus inoculum was removed, the cells were washed and incubated in corresponding medium with ns-398 or drug-vehicle as controls throughout the experiments. cells were harvested for rna isolation at 6 hours post-infection to study viral m gene expression. supernatants were collected for tcid 50 assay to determine the virus titers at 3, 24, 48, and 72 hours after h5n1 infection. total rna was isolated using the rneasy mini kit (qiagen) according to the manufacturer's instructions. the cdna was synthesized from mrna with poly(dt) primers and superscript iii reverse transcriptase (invitrogen). transcript expression was monitored by real-time pcr using power sybr ò green pcr master mix kit (applied biosystems) with specific primers. the fluorescence signals were measured using the 7500 real-time pcr system (applied biosystems). the specificity of the sybr ò green pcr signal was confirmed by melting curve analysis. the threshold cycle (ct) was defined as the fractional cycle number at which the fluorescence reached 10 times the standard deviation of the base-line (from cycle 2 to 10). the ratio change in target gene relative to the b-actin control gene was determined by the 2 )ddct method as described elsewhere. 4 ns-398 reduced the viral m gene expression in h5n1infected human macrophages in a dose-dependent manner ( figure 1) . similarly, production of infectious virus yield in h5n1 infected macrophages was found to be suppressed in the presence of ns-398 at 100 lm compared to vehicletreated cells (figure 2a) . a comparable effect of ns-398 was observed in h5n1-infected human alveolar epithelial cells ( figure 2b ). we have previously demonstrated that cox-2 expression was dramatically upregulated following h5n1 infection in human macrophages in vitro and in epithelial cells of lung tissue samples obtained from autopsy of patients who died of h5n1 disease. 1 this suggests that cox-2 may be an important host factor involved in h5n1 pathogenesis and also provide a possible explanation on why h5n1 virus replication is susceptible to a selective cox-2 inhibitor. cox-2 was previously reported to play an important role in the pathogenesis of other influenza a viruses. 5 an in vivo study has highlighted the importance of cox-2 in h3n2infected mice. findings showed that infection induced less severe illness and reduced mortality in cox-2 knock-out mice than in wild-type mice. on the other hand, cox-1 knock-out mice had enhanced inflammation and earlier appearance of proinflammatory cytokines in the bal fluid, whereas the inflammatory and cytokine responses were dampened in cox-2 knock-out mice. these data suggests that cox-1 and cox-2 may lead to opposite totally contrasting effects on influenza h3n2 infected mice. cox-1 deficiency is detrimental, whereas cox-2 deficiency is beneficial to the host during influenza viral infection. therefore in the present study, instead of blocking cox enzymes in general as reported by others, 5 we have chosen ns-398 that selectively block cox-2 but preserve cox-1 activity and showed that this drug significantly reduced h5n1 virus replication in a dose-dependent manner. taken together with our previous report suggesting its immuno-modulatory effects, 1 we believe that selective cox-2 inhibitors and cox-2 signaling pathways deserve investigation as a promising approach for targeting therapy in h5n1 diseases. however, a few reports have suggested the importance of cox-2 in the late stage of inflammation for the resulution of inflammation, [6] [7] [8] and this raises concern whether inhibition of cox-2 may be harmful in treating diseases related to dysregulation of host inflammatory response such as acute lung injury, 9 which is a leading cause of death in h5n1 patients. we previously looked at the autopsy samples of lung tissues from h5n1 patients and found that cox-2 expression was markedly up-regulated compared with that from persons who died of non-respiratory causes. 1 moreover, data also demonstrated that pro-inflammatory cytokines, such as tnf-a, was markedly elevated in the h5n1 infected lung autopsies. 10 taken together, with the histo-pathological findings, which showed predominant features of exudative inflammatory phase in autopsy lung samples from h5n1 patients, 11, 12 we may therefore speculate that people who had fatal h5n1 infection died during acute inflammation phase, and before the resolution could occur, especially for the cases with a short disease duration (<10-12 days). 13 in conclusion, the roles of cox-2 in both pro-inflammation and pro-resolution phases deserves detailed investi-gation. the timing of selective cox-2 inhibitor therapy in h5n1 infected patients may be extremely critical. therefore a time-dependent study using selective cox-2 inhibitors on h5n1-infected animal models will be particularly important in order to address the effectiveness of this drug in treating h5n1 disease. avian antibodies to combat potential h5n1 pandemic and seasonal influenza highly pathogenic avian influenza a virus (hpaiv) strain a ⁄ h5n1 with unprecedented spread through much of asia and parts of europe in poultry remains a serious threat to human health. passive immunization (transfer of protective immunoglobulins) offers an alternative and ⁄ or additional strategy to prevent and cure influenza. here, we report that virus-specific immunoglobulin y (igy) isolated from eggs of immunized hens provide protection in mice against lethal h5n1 virus infection by neutralization of the viruses in the lungs upon intranasal administration. importantly, chicken eggs obtained from randomly selected supermarkets and farms in vietnam, where mass poultry vaccination against a ⁄ h5n1 is mandatory, contain high levels of igy specific for a ⁄ h5n1 virus. when administered before or after the infection, igy prevented and significantly reduced replication and spread of hpaiv h5n1 and related h5n2 strains. thus, the consumable eggs readily available in markets of countries that impose poultry vaccination against a ⁄ h5n1 could offer an enormous source of valuable biological material that provides protection against a ⁄ h5n1 virus with pandemic potential. the approach could be used to control seasonal influenza. since 2004, hpaiv of the h5n1 subtype has resulted in more than 430 cases of laboratory-confirmed human infection in 15 countries with a death rate of more than 50% (http://www.who.int/csr/disease/avian_influenza/). h5n1 influenza virus remains a global threat because of its continued transmission among domestic poultry and wild birds. passive immunization (the transfer of antigen-specific antibodies (abs) to a previously non-immune recipient host) offers an alternative and ⁄ or additional countermeasure against influenza. 1 development of human monoclonal antibodies (mabs) against h5n1 influenza haemagglutinin (ha) using epstein-barr virus (ebv) immortalization of b cells isolated from patients infected with h5n1, 2 phage display, 3 humanized mabs, 4 and human recombinant abs 5 has been attempted. chickens produce a unique immunoglobulin molecule called igy that is functionally equivalent to mammalian igg. 6 igy is found in the sera of chickens and is passed from hens to the embryo via the egg yolk. 7 egg igy has been used to prevent bacterial and viral infections (see review 8 ) of the gastrointestinal tract and recently for protection against pseudomonas aeruginosa infection of the respiratory tract of patients with cystic fibrosis (cf). 9 the epidemic of hpaiv h5n1 virus has resulted in serious economic losses to the poultry industry, mostly in southeast asia. therefore, many countries including china, indonesia, thailand, and vietnam have introduced mass vaccination of poultry with h5n1 virus vaccines that controls the h5n1 epidemic to some extent. 10 chickens immunized with recombinant h5 and ⁄ or inactivated h5n1 reassortant vaccines produced a high level of virus-specific serum antibodies (abs) and were protected from h5n1 virus challenge. 11 theoretically, these abs could be found in egg yolk and separated for use in humans to prevent and cure h5n1 hpaiv infection and disease, respectively. here, we examined the possibility that igy isolated from consumable eggs available in supermarkets in vietnam, where mandatory h5n1 vaccination has been implemented, provide prophylaxis and therapy of hpaiv h5n1 infection in mice. six-to 8-week-old female balb ⁄ canncrl (h-2d) mice (charles river and jackson laboratory) and hy-line . igy abs were extracted from egg yolks as previously described. 12 the 50% egg infectious dose (eid 50 ) was determined by serial titration of virus stock in eggs, and eid 50 ⁄ ml values were calculated according to the method of reed and muench. 13 human virus stocks were grown in mdck cells as described previously ,14 with viral titers determined by standard plaque assay. the 50% tissue culture infectious dose (tcid 50 ) of virus was determined by titration in mdck cells. 15 the standard elisa was performed for detection of anti-igy in the sera of igy-immunized mice. fifty percent lethal dose (ld 50 ) titers were determined by inoculating groups of eight mice i.n. with serial 10-fold dilutions of virus as previously described. 16 for infection, ketamine-anesthetized mice were inoculated intranasally with a lethal dose with 250 pfu (5 · ld 50 ) of a ⁄ pr ⁄ 8 ⁄ 34 (h1n1) virus as previously described, 17 10 · ld 50 of vn ⁄ 1203 (h5n1) or 5 · ld 50 a ⁄ aquatic bird ⁄ korea ⁄ w81 ⁄ 2005 (h5n2) resuspended in 50 ll pbs per animal. ketamine-anesthetized mice were treated intranasally with 50 ll of igy before or after infection. mice were observed for weight loss and mortality. subsets of animals were scarified for virus titre. we found comparable hai titers in the sera and egg yolks obtained from a farm in vietnam that was participating in a national mass vaccination program. furthermore we found 90% of eggs purchased in randomly selected supermarkets in hanoi, vietnam containing h5-specific igy. the hai and vn titers of pooled egg yolk igy are comparable with those of sera obtained from hens selected randomly from the farm that underwent supervised h5n1 vaccination. in contrast, igy separated from eggs purchased in korean markets where poultry are not vaccinated against avian influenza h5n1 has no detectable h5-specific hai or vn activity. we first treated naïve mice intranasally with h5n1-specific igy before infection with hpaiv h5n1 strain, a ⁄ vietnam ⁄ 1203 ⁄ 2004, isolated from a fatal case. such treated mice displayed mild weight loss and recovered completely by the end of the first week after inoculation ( figure 1a ). when animals were treated once with h5n1specific igy after h5n1 inoculation they exhibited minimal weight loss during the first week after inoculation, and virus titers in the lungs were substantial reduced at day 3 after infection; however, 50% of treated mice succumbed to infection during the second week after inoculation ( figure 1b) . it is possible that not all the hpaiv a ⁄ h5n1 viruses were neutralized upon the single treatment with igy, and escaping viruses can spread systemically to organs outside of the lungs. these viruses may reappear in lung tissue later when specific igy is absent. indeed, vn ⁄ 1203 virus injected intravenously or into the brain can spread to the lungs. 18 to circumvent the virus escape, we administered multiple treatments with h5n1specific igy after the infection. as a result, all infected mice recovered completely by the second week post-infection ( figure 1c) , and virus titers in the lungs were substantially reduced to the level that seen in protected mice that received single prior-infection treatment ( figure 1d) . similarly, the protective efficacy of h5n1-specific igy was observed in mice infected with lethal dose of mouseadapted avian influenza virus strain a ⁄ aquatic bird ⁄ korea ⁄ w81 ⁄ 2005 (h5n2). this virus shares 94.4% nucleotide sequence homology with ha (h5) but has different na (n2) from the one used for mass immunization in vietnam (reassortant avian h5n1 influenza virus a ⁄ goose ⁄ gd ⁄ 96-derived, strain re-1). the results indicate that h5n1-specific igy isolated from eggs purchased in markets have preventive and therapeutic effects against infection with hpaiv h5n1 and the related strain h5n2. the findings suggest that while a single treatment with igy prior to lethal infection was sufficient to protect the animals from the infection, multiple treatment is required for complete therapeutic effect after infection with hpaiv such as vn ⁄ 1203 strain. we further examined the protective efficacy of igy isolated from eggs laid by hens immunized in the laboratory with heat-inactivated human influenza a ⁄ h1n1 virus, a ⁄ pr ⁄ 8 ⁄ 34. we found substantial levels of hai and vn abs in the sera and yolks derived from immunized hens. when naïve mice were administered intranasally with such anti-pr ⁄ 8 igy at 6-8 hours before or after infection with lethal dose of pr ⁄ 8 virus, they were protected from the infection or lethal disease, respectively. the virus titers in the lungs of a ⁄ pr8 specific igy-treated mice at day 3 after infection were also significantly lower than those seen in untreated mice or mice receiving normal igy. intranasal administration is the most effective route as compared to oral or peritoneal or intravenous administration for protection against lethal challenge, and the presence of virus-specific igy in bronchoalveolar lavage (bal) is required for the protection. the results provide a proof-of-concept that intranasal administration of virus-specific igy prevents influenza virus infection and cures the disease. the concept could be applied to control influenza outbreaks including seasonal and pandemic influenza. the protection was correlated with hai and vn activities of the igy and reduced virus titers in the lungs after treatments, suggesting that the protection is mediated by vn. we asked if administration of igy in the respiratory tract induces anti-igy ab response in mice. if this is the case, the next question is whether pre-existing anti-igy abs block igy-mediated protection. indeed, significant levels of anti-igy were observed in animals that received single or multiple administration of igy. when igy-immune mice were treated with virus-specific igy before or after lethal challenge, the results were identical to those obtained from treated naive mice, indicating that pre-existing anti-igy abs do not interfere with the protection mediated by virus-specific igy. consistently, incubation with anti-igy serum did not interfere with hai and vn activity of the virus-specific igy, indicating that anti-igy abs do not block virus binding by virus-specific igy (figure 2 ). the finding suggests that the igy treatment could be applied to persons who have developed anti-igy during the individuals' life, and such treatment strategy could be repeated if multiple treatment is required and ⁄ or necessary later on to protect infections with other pathogens. the approach using specific igy for prevention and therapy of hpaiv h5n1 infection offers a practical alternative to immunotherapy using convalescent plasma 19 and an additional therapeutic option to antiviral drugs since widespread drug resistance has been recently reported among influenza virus strains. igy is relatively stable. we found no change in protective activity after at least 12 months storage at 4°c, and lyophilization does not affect the activity, making production of igy practical. the use of igy immunotherapy has many advantages, since igy does not activate the human complement system or human fc-receptors, which all are well-known cell activators and mediators of inflammation. 20 we chose the water dilution method for preparation of igy. the method is simple, efficient and does not require any toxic compounds or any additives. such igy preparations by this method have been used in other human study. 9, 21 eggs are normal dietary components, so there is minimal risk of toxic side effects, except for those with egg allergy. thus, our study demonstrated that influenza virus-specific igy can be used in passive immunization that provides great help for immunocompromised patients and elderly who have weaken immune response to influenza vaccines. importantly, the consumable eggs readily available in the markets of countries that impose mandatory h5n1 vaccination offer an enormous source of valuable, affordable, and safe biological material for prevention and protection against potential h5n1 pandemic influenza. parts of the information and data presented in this manuscript were previously published in http://www.plosone.org/ article/info:doi%2f10.1371%2fjournal.pone.0010152. the polyphenol rich plant extract cystus052 is highly introduction the 2009 ⁄ 2010 h1n1influenza a virus pandemic clearly demonstrates that influenza is still a major risk for the public health. although the pandemic swine origin influenza a virus (soiv) caused only mild symptoms, the control of the outbreak still remains difficult. even as vaccine is available against this virus, the possibility of reassortment between the pandemic and a seasonal or avian a ⁄ h5n1 influenza virus strain is indeed a frightening, but a likely event. this reassortant strain might be able to transmit easily between humans causing fatal infections, and the current soiv vaccine might no longer be sufficient to protect against the reassorted virus. in such a case, we can only rely on effective antiviral drugs. today, neuraminidaseinhibitors, such as oseltamivir, represent the most common clinically approved medication against influenza a viruses. unfortunately, the frequency of reports describing the appearance of drug-resistant seasonal h1n1 and also h5n1 influenza a viruses dramatically increased in the recent past. [1] [2] [3] [4] drug resistance to the known antivirals highlights the urgent need for alternative antiviral compounds with novel defense mechanisms. recently, we have reported that a polyphenol rich plant extract, cystus052, which showed antiviral activity against influenza a viruses in cell culture and in mice. 5, 6 moreover, the antiviral activity of cy-stus052 against seasonal influenza virus and common colds was also demonstrated in humans. 7 however, the efficiency of cystus052 against soiv and a ⁄ h5n1 isolates was unknown so far. therefore, we investigated cy-stus052 effectiveness against the pandemic strain and seven natural influenza a ⁄ h5n1 isolates detected in several avian species during 2006 ⁄ 2007 avian influenza outbreak. additionally, the potency of the most common neuraminidase inhibitor oseltamivir was also investigated against these isolates. here, we show that cystus052 treatment was effective in in vitro studies against soiv and a ⁄ h5n1 influenza virus. viruses avian h5n1 isolates were originally obtained from the bavarian health and food safety authority, oberschleissheim, germany. the soiv a ⁄ hamburg ⁄ 4 ⁄ 2009 was obtained from the robert-koch-institut, berlin, germany. all h5n1 viruses were further propagated in embryonated chicken eggs or mdck ii (h1n1v) cells at the friedrich-loeffler-institut, tübingen, germany. for the cytopathological effect (cpe) inhibition screening, in accordance with sidwell, 8 mdck ii cells were infected with different viruses at moi of 0ae005. virus-infected cells were then treated with antiviral compounds cystus052 from 0ae1 to 1000 lg ⁄ ml or oseltamivir from 0ae01 nm to 1 mm. after incubation for 48 hours at 37°c and 5% co 2 , cells were fixed, and viable cells were stained with crystal violet. after extraction of crystal violet from viable cells with 100% methanol, the extinction was measured with an elisa reader. immediately before infection, mdck ii cells (8 · 10 4 cells ⁄ well) were washed with pbs and subsequently incubated with virus diluted in pbs ⁄ ba (0ae2% ba) 1 mm mgcl 2 , 0ae9 mm cacl 2 , penicillin and streptomycin to a multiplicity of infection (moi) of 0ae001 for 30 minutes at 37°c. cystus052 was added in a concentration of 50 lg ⁄ ml directly to the virus-stock and on the cell monolayer simultaneously with the infection. after 30 minutes incubation period, the inoculums were aspirated and cells were incubated with either mem or mem containing 1 lm oseltamivir. at indicated time points, supernatants were collected. infectious particles (plaque titers) in the supernatants were assessed by a plaque assay under avicel as described previously. 9 in order to investigate the antiviral potential of cy-stus052, ec 50 values based on the inhibition of the cpe on mdck ii cells were determined for cystus052 and in addition for oseltamivir. the ec 50 values for cystus052 ranged from 1ae53 to 18ae88 lg ⁄ ml. cystus052 demonstrated the highest sensitivity against the soiv, sn1 and mb1 isolates with ec 50 values below 5 lg ⁄ ml. compared to these virus strains, cystus052 showed a slightly increased ec 50 value for gsb1 (18ae88 lg ⁄ ml). in contrast the ec 50 values for bb1 and bb2 were notably elevated (65ae68 and 76ae22 lg ⁄ ml). thus, the weakest antiviral effect of cystus052 was observed against these two isolates. the ec 50 values evaluated for oseltamivir ranged from 0ae07 to 512ae76 lm ( table 1 ), indicating that bb2 (512ae76) and gsb1 (356ae92 lm) can be considered resistant against oseltamivir. to confirm these results we investigated the ability of cystus052 to block virus replication as published before. 1 as a control, virus infected cells were treated with oseltamivir as described earlier. 6 in the absence of the drugs all influenza strains showed similar growth properties (figure 1, black squares) . first progeny viruses were detectable between 8 and 20 hours post infection (figure 1, black squares) . treatment with cystus052 resulted in reduction of virus titers of all influenza virus strains (fig. 1a-h, open triangles) . surprisingly, oseltamivir failed to inhibit the replication of two h5n1 influenza virus strains (gsb1 and bb2), supporting the data of ec 50 values ( figure 1d+h , grey rhombes). we assessed the antiviral activity of cystus052 against the newly emerged soiv and seven avian h5n1 influenza viruses. cystus052 showed efficient antiviral activity against the pandemic h1n1v strain and was effective to a wide range of h5n1 viruses. furthermore, cystus052 demonstrated a broader and more efficient antiviral potential than oseltamivir. cystus052 treatment leads to a stronger reduction of progeny virus titers, and more importantly, cystus052 was effective against all tested viruses, while oseltamivir was unresponsive against two of seven a ⁄ h5n1 viruses. even though the pandemic strain in general is still sensitive to oseltamivir treatment, there are increasing numbers of reports of emerging resistant variants. the treatment with cystus052 does not result in the emergence of viral drug resistance since the mode of action is an unspecific physical binding of the virus particle that is also beneficial to reduce opportunistic bacterial infections. 5,7,10 cystus052 is an extract from a special variety of the plant cistus incanus, and it is very rich in polymeric polyphenols. 11 it is well known that polyphenols exhibit protein-binding capacity. 12 however, cystus052 exhibited no neuraminidase inhibiting activity. therefore, ingredients of cystus052 may act in a rather unspecific physical manner by interfering with the viral hemagglutinin at the surface of the virus particle as demonstrated before. 5 while this prevents binding of the virion to cellular receptors, it does not block accessibility and action of the viral neuraminidase. since, infections with influenza a viruses are still a major health burden and the options for control and treatment of the disease are limited, plant extracts such as cystus052 should be considered as a new candidate drug for a save prophylactic and therapeutic use against influenza viruses. attenuation of respiratory immune responses by antiviral neuraminidase inhibitor treatment and boost of mucosal immunoglobulin a response by co-administration of immuno-modulator clarithromycin in paediatric influenza the antiviral neuraminidase inhibitor osv and zanamivir are widely used treatment options for influenza infection and are being stockpiled in many countries. although mucosal immunity is the frontline of defense against pathogens, the effects of neuraminidase inhibitor treatment on airway mucosal immunity have not been reported. the suppression of viral rna replication and viral antigenic production by these drugs may result in a limited immune response against influenza virus. 1 macrolides, such as cam and azithromycin, have anti-inflammatory and immunomodulatory properties that are separate from their antibacterial effects. [2] [3] [4] this study examined the impact of osv treatment on immune responses in the airway mucosa and plasma in mice infected with iav and pediatric influenza patients. we also assessed the immuno-modulatory effects of cam in influenza patients who were treated with or without osv. female 3ae5-week-old weanling balb ⁄ c mice were nasally inoculated with 25 pfu of iav ⁄ pr8 ⁄ 34 h1n1 at day 0. immediately after infection, mice were given 50 lg of osv orally or vehicle at 12-hours intervals for 11 days. the levels of virus-specific siga in nws and bronchoalveolar fluids (balf) and igg in plasma were measured by elisa as reported previously. 5 a retrospective clinical study was conducted. for the study, 40 children with acute influenza were recruited and grouped according to the treatment received: 5 days treatment with osv (n = 14), cam (n = 8), osv + cam (n = 12), and untreated (n = 6). since parents in japan are well aware of the adverse effects of osv especially the neuropsychiatric complications, 6 the decision on whether to administer osv or not and to prescribe cam was made by the parents and the attending paediatricians, based on their anti-viral and immuno-modulatory activity. 3, 6 comparisons were made of the levels of siga against iav ⁄ h3n2 and iav ⁄ h1n1, total siga, in nws and disease symptoms before and after treatment. anti-ha siga and total siga in nws of patients were determined from the standard regression curves with human iga of known concentration in a human iga quantitation kit (bethy laboratories). because an affinity purified human anti-ha-specific siga standard of each influenza a subtype is not available, the relative value of anti-haspecific siga amount was expressed as unit (u). one unit was defined as the amount of one lg of human iga detected in the assay system as reported previously. 6 the concentrations of siga in individual nws were normalized by the levels of total siga (lg ⁄ ml). oseltamivir suppresses viral rna replication and viral antigenic protein production. to investigate the influence of daily treatment with osv on ha-specific mucosal and systemic immune responses, we analyzed ha-specific siga levels in nws and balf as well as igg levels in plasma at days 8 and 12 post-infection in mice treated orally with osv or methylcellulose (mc) as vehicle. the osv treated mice showed lower antibody responses in nws and balf than control mice treated with mc solution (table 1) . significantly reduced ha-specific siga responses were particularly noted in the osv group at day 12, the period of maximal mucosal siga induction. the airway secretions and plasma from mice at day 0 did not contain detectable levels of ha-specific antibodies. these findings were supported by other data whereby mice treated with osv displayed significantly lower numbers of ha-specific iga antibody-forming cells (afcs) in the nasal lamina propria, mediastinal lymph nodes, and lungs compared with mc-treated mice. these results clearly indicate that oral administration of osv downregulates ha-specific siga responses in mucosa. on the other hand, there were no significant differences in the elevated levels of ha-specific plasma iga and igg antibodies or the increased numbers of ha-specific iga and igg afcs in the spleen between osv-and mc-treated mice. taken together, these results implicated the oral administration of osv in a suppressed induction of haspecific siga responses in respiratory lymphoid tissues, although systemic ha-specific antibody responses were not significantly affected by osv. since cam up-regulates il-12, a mucosal adjuvant cytokine in the airways, and promotes the induction of siga and igg in the airway fluids of mice infected with iav, 4, 7 we assessed the impact of treatment with osv and ⁄ or cam on the levels of anti-influenza siga in nws and clinical status of influenza patients. the concentration ratio of table 1 . anti-ha-specific siga to total siga in nws was expressed as titer: anti-ha-specific siga (u ⁄ mg) ⁄ total siga (lg ⁄ mg) · 100. figure 1 shows changes in the anti-ha(h3n2) siga ratio (titer) and fold of increase in siga titer in each patient during the 5-days' treatment for the four different treatment groups. it is noteworthy that, upon admission to the hospital, the siga titers were <3 in 93% of patients. during the 5 days of treatment, rapid increases in the titers were observed in almost all patients in cam, osv + cam, and no treatment groups. in contrast, in the osv group, the anti-ha-specific siga titers remained unchanged or decreased in the majority of patients. the finding of significant low induction of anti-viral siga in the osv group was supported by the results of animal experiments. however, the addition of cam to osv augmented siga production and restored mucosal siga levels; 75% of patients treated with osv + cam showed >5-fold increase in the titers during treatment. these observations suggest that cam stimulated the local mucosal immunoresponse in the nasopharyngeal region of patients treated with osv. the prevalence of disease manifestations was also analyzed. 6 among the symptoms listed, a significant decrease in the prevalence of cough was recorded between the no treatment group and the osv + cam group and between the osv group and the osv + cam group (**p < 0ae01), despite the limited number of patients in each group. the duration of the febrile period was significantly shorter in the osv and osv + cam groups than the no treatment group. however, no significant difference was observed between the osv group and osv + cam group. it has been reported that osv does not affect the cellular immune responses, such as cytotoxic t lymphocytes and natural killer cells. 8 however, the effects of osv on mucosal immunity have not been studied so far. the present study showed that osv treatment of mice infected with iav induced insufficient protective mucosal siga responses in the respiratory tract, although treated mice showed the similar levels of systemic igg and iga antibody responses in plasma to those in mice treated with vehicle (table 1) . 1 the observed effect of osv on mucosal immunity was probably due to a suppression of viral replication and viral antigen production in the mucosal layer. these observations in mice are further supported by our clinical reports of siga in nws and balf of osv treated influenza patients. 6 the 14 membered-and 15 membered-ring macrolides have been found to possess a wide range of anti-inflammatory and immuno-modulatory properties, 2,3 and to be effective in the treatment of respiratory syncytia and iav infection. 9,10 the efficacy of low doses administered on the long term against pathogens that are insensitive to macrolides indicates a mode of action that is separate from their antibacterial activity. 2, 3, 9, 11 in the present study, we evaluated the immunomodulatory effects of cam on mucosal immune responses in pediatric influenza. a decrease in the proportion of total siga that was anti-ha-specific siga during treatment was observed in 21.4% of patients in the osv group (those represented by the dotted lines and closed diamonds in figure 1 ), whereas an increase in the proportion was observed in most patients of the other groups (except for one patient of the untreated group). despite the low or unchanged induction of anti-ha-specific siga in the majority of osv-treated patients, the additional use of cam with osv boosted the mucosal immune response and restored local mucosal siga levels. we are currently engaged in detailed immunological studies of the effects of cam and osv on the levels of mediators controlling iga class switching in nws of influenza patients and airway secretion of mice infected with iav. further studies should clarify the boost mechanisms of cam and the suppression mechanisms of osv in iga class switching. our findings suggest the risk of re-infection in patients showing a low mucosal response following osv treatment and cam effectively boosts the siga production for protection of re-infection. to date there is an urgent need to develop new antivirals against influenza. most of the molecules reported target influenza proteins that acquire rapid mutations of resistance. the development of new molecules that have a broad antiviral activity and are not subjected to influenza mutation is of particular interest. our laboratory and others recently showed that proteases can participate to the innate immune response in the airways through the activation of a family of receptors called par. in particular, through the release of interferon, par2 agonists curbed viral replication significantly in infected cells. in this study, since erk activation is crucial for virus replication, we investigated whether par2 could inhibit virus replication through inhibition of the erk pathway. results showed that while influenza a infection alone or par2 stimulation alone induced erk activation, par2 stimulation does not inhibit erk activation in influenza infected cells. thus, par2 agonists may be a potential new drug against influenza viruses that could be used in combination with other anti flu therapy such as the inhibition of the erk pathway. respiratory tract-resident proteases are key players during influenza virus type a infection. 1, 2 in addition to their direct activating effect on surface viral proteins, lung mucosal proteases can regulate cellular processes by their ability to signal through protease-activated receptors (pars). 3 after cleavage of the receptor by proteases, the new aminoterminal sequence of par binds and activates the receptor internally. these receptors are highly expressed at epithelial surfaces, in particular in the lung, where human influenza virus replicate in vivo. pars are thus directly exposed to proteases present in the airways. among the four different pars, par2 acts as an antiviral through an interferondependent pathway. 4, 5 thus, agonists of par2 are potential new drugs against a broad range of influenza viruses, which is in accordance with the broad antiviral action of interferon. however, the signalling pathway induced by par2 agonists in influenza a infected cells has still to be investigated. in this manuscript, we showed that influenza infection or activation of par2 induced erk activation, a crucial step for efficient virus replication. 6, 7 however, par2 agonists do not impaired erk activation in influenza a virus infected cells. since the pathway of par2 protection is likely to be erk-independent, the use of anti erk molecules in combination with par2 agonists maybe of potential interest in future anti-influenza therapy. influenza viruses a ⁄ wsn ⁄ 33 (h1n1) (a kind gift from nadia naffakh) was used in the present study. mdck (madin-darby canine kidney) and the human alveolar type ii a549 cell were obtained from atcc and grown as previously described. 8 for western blot analysis, the following antibodies were used: monoclonal antibody for phospho-erk1 ⁄ 2 (t202 ⁄ y204) and for erk1 ⁄ 2 antibodies from cell signaling technology (beverly, ma), horseradish peroxydase (hrp)-coupled rabbit polyclonal antibodies against mouse or rabbit igg from paris (compiègne, france). a549 cells were infected with iav at an moi of 1 in emem medium, as previously described. 9,10 at various time points post infection, cells were collected and proteins were analysed as previously described. 6,11 par2 stimulation was performed at 37°c in emem medium as previously described. 4 after infection and ⁄ or stimulation, cells were lysed in ice-cold lysis buffer. lysates were centrifuged at 12 000 g for 20 min, and total proteins of the supernatants were analyzed by western blot analysis as previously described. 12, 13 results since activation of the erk pathway is essential for efficient influenza replication, 7 we first investigated the kinetics of erk activation after influenza infection in human a549 alveolar epithelial cells. for this purpose, a549 cells were infected with influenza viruses at a moi of 1 at different time point post-infection, and activation of erk1 ⁄ 2 pathway was assessed by western blot analysis using an anti-erk antibody. results showed that erk was phosphorylated after influenza infection in a time course depen-dent manner when compared to uninfected cells. in contrast, erk phosphorylation was not observed with heatinactivated viruses, suggesting that productive infection is needed for erk activation ( figure 1a ). antibodies against erk1 ⁄ 2 were used as controls. since erk is activated after influenza infection, we then tested whether activation of par2 in uninfected cells also leads to activation of this pathway. for this purpose, a549 cells were stimulated with the selective human (h) or mouse (m) par2 agonist or a control peptide for the indicated time ( figure 1b ). when exposed to the par2 agonists and compared to controltreated cells, erk phosphorylation increased over the time course of stimulation. thus, influenza infection or stimulation of par2 without infection in a549 cells induced activation of the erk pathway at different time point post-infection. since influenza infection and par2 stimulation induced erk activation, we then investigated whether par2 could inhibit erk activation in influenza infected a549 cells. results in figure 2 showed that in influenza infected cells, par2 activation for ten minutes does not inhibit erk activation after influenza infection. thus, erk activation is not inhibited by par2 activation in influenza stimulated cells. in this manuscript, we studied the activation of the erk pathway after par2 stimulation and or influenza infection. particularly interesting is the fact that either influenza infection or par2 stimulation alone induce erk phosphorylation in a549 epithelial cells, while erk activation is not inhibited in a549 infected cells compared to uninfected ones after par2 stimulation. proteases are key factor in the pathogenicity of influenza viruses. in addition to the cleavage of ha, necessary for iav replication, extracellular proteases also play a role in the modulation of the immune system against influenza viruses through the activation of pars. particularly par2, activated by extracellular trypsin-like proteases, could inhibit virus replication through the release of interferon, 4,5 thus, strengthening the immune system via agonist peptides and providing new therapeutic potential against a broad range of influenza strains. in addition, targeting the host instead of the virus could provide a way to escape from virus resistance. 14 thus, a better understanding of how virus escapes from immune surveillance may provide new therapeutic strategies to block iav. in addition, combinations of drugs that block virus replication via different pathways are of interest. the non classical molecules hla-g maybe an interesting new target as we recently showed that it is upregulated after influenza infection, 13 and it is a well known immunotolerant molecule. 15 indeed, it inhibits the innate immune response 16 as well as the adaptive immune response. 17, 18 also, as previously suggested, the erk signal transduction cascade is also of potential interest since it is crucial for virus replication and particularly influenza replication. 6, 7 as shown here, it is unlikely that par2 protection occurs through an erkdependent pathway. thus strengthening the immune response with par2 agonists and blocking nuclear retention of the viral ribonucleoprotein complexes with inhibitors of the mek ⁄ erk pathway may be alternative combinatory approaches for influenza therapy. in addition, since those potential drugs target the host instead of the virus, this could help in the design of new antivirals molecules more resilient to iav mutations and thus to virus resistance. the initial waves of the first influenza pandemic of the 21st century have passed. in june 2009, vaccine companies estimated they could produce in 6 months almost 2.5 billion doses of pandemic vaccine. 1 instead, they actually produced only 534 million doses, of which 64% were non adjuvanted preparations. had these doses been produced with adjuvants (i.e., 3.75 lg instead of 15 lg ha per dose), an additional 1 billion doses could have been made available. yet there was public opposition to adjuvants in many countries, especially by regulatory officials in the united states. misperceptions about the safety of both adjuvanted and nonadjuvanted vaccines were widespread. added to this, shortfalls in vaccine production, delays in vaccine delivery, and the ''mildness'' of the pandemic itself meant that only a few countries achieved reasonable levels of vaccine coverage. millions of doses went unused and had to be destroyed. supplies of antiviral agents were even more limited. thus, despite the best efforts of influenza scientists, health officials, and companies, more than 90% of the world's people did not have timely access to affordable supplies of vaccines and antiviral agents. instead, they had to rely on 19th century public health ''technologies.'' given current understanding of biology in the early 21st century, they should have had -and probably could have had -something better. this report reviews evidence for an alternative approach to serious and pandemic influenza that could be used in all countries with basic health care systems. instead of confronting the influenza virus with vaccines and antiviral agents, it suggests that we might be able to modify the host response to influenza virus infection by using anti-inflammatory and immunomodulatory agents. this idea was introduced several years ago 2 and has been reviewed in several publications. [3] [4] [5] [6] [7] [8] the central importance of the host response in the 1918 pandemic, young adults had high mortality rates. ever since, influenza virologists have sought to answer the question ''why did young adults die?'' by defining the molecular characteristics of the 1918 virus that were responsible for its virulence. 8 in doing so, they have overlooked a crucial piece of clinical evidence from the 1918 pandemic: compared with young adults, children were infected more frequently with the same virus, yet they seldom died. consequently, the more important question is ''why did children live?'' this can only be explained by recognizing that children must have had a different host response to the 1918 influenza virus than adults. physicians have long recognized that for several other medical conditions, both infectious (e.g., pneumococcal bacteremia) and non-infectious (e.g., multiple trauma), children have a more benign clinical course than adults. 6, 8 a corollary of this observation is that secondary bacterial pneumonia, although commonly found in young adults in 1918, could not have been the primary cause of death. children must have had the same or higher rates of nasopharyngeal colonization with the same bacteria that were associated with pneumonia deaths in adults, yet children seldom died of secondary bacterial pneumonia. 6 if young adults died with secondary bacterial pneumonia, underlying host factors must have made them more susceptible. few people who die of influenza do so during the first few days of illness when pro-inflammatory cytokine levels are high. instead, like patients with sepsis, they usually die in the second week, when anti-inflammatory cytokines and immunosuppression dominate. 6, 8, 9 influenza deaths occur more frequently in older persons with cardiopulmonary conditions, diabetes, and renal disease, but as seen in the 2009 h1n1 pandemic, they also occur in younger adults with obesity, asthma, and in women who are pregnant. regardless of age, people with all of these conditions share one characteristic in common: they have chronic low-grade inflammation. in effect, their ''innate immune rheostats'' have been set at different, and perhaps more precarious, levels that make them more vulnerable to influenza-related complications. 10 laboratory studies of influenza virus infection confirm the importance of the host response. in several studies in mice in which the host response has been modified (e.g., cytokine knockout), survival has been improved without increasing virus replication in the lung. 5 in fact, severe disease can be induced without any influenza virus replication. for example, fatal acute lung injury has been induced in mice by inactivated (not live) h5n1 virus. 11 in this model, antiviral agents would be useless; only the host response could be responsible for disease. these observations raise the following question: could the host response be modified so patients with severe seasonal and pandemic influenza might have a better chance of surviving? influenza is associated with acute coronary syndromes, and influenza vaccination and statins reduce their occurrence. these associations led to the suggestion in 2004 that statins might be used to treat pandemic influenza. 2 other agents that might also be effective include ppara and pparc agonists (fibrates and glitazones, respectively) and ampk agonists (e.g., metformin). 5, 8 these agents have been studied in laboratory models of inflammation, sepsis, acute lung injury, ischemia ⁄ reperfusion injury, energy metabolism, mitochondrial function, and programmed cell death. the results of these studies cannot be reviewed in detail here, but the major findings for cell signaling are summarized in the table 1 . unfortunately, the results of experimental studies are not always clear cut. for example, in one study of influenza virus infected mice, il-10 was necessary for containing infection, 12 but in another study il-10 appeared to be harmful. 13 nonetheless, overall understand-ing of cell signaling pathways in influenza virus infections and the actions of statins, glitazones, fibrates, and ampk agonists strongly suggest that these agents could benefit patients with severe influenza. laboratory studies in mice infected with pr8 (h1n1) h2n2 and pandemic h1n1 viruses show that resveratrol, fibrates, glitazones, and ampk agonists reduce mortality by 30-50%, often when treatment is started 2-4 days following infection. 14-17 (resveratrol is a polyphenol found in red wine. it shares with these other agents many of the same cell signaling effects.) in h5n1-infected mice, treatment with celecoxib and mesalazine, together with zanamivir, showed better protection than zanamivir alone. 18 remarkably, these immunomodulatory agents have not increased virus replication. even more remarkable, in another model of a highly inflammatory and frequently fatal conditionhepatic ischemia ⁄ reperfusion injury -glitazone treatment ''rolled back'' the host response of ''young adult'' mice (8-10 weeks old) to that of ''children'' (3-4 weeks old). 19 this unique study suggests that immunomodulatory treatment might roll back the damaging and sometimes fatal host response of young adults with influenza to the more benign and rarely fatal response of children. several, but not all, observational studies have shown that outpatient statins decrease hospital admissions and mortality due to community-acquired pneumonia. 8 for influenza itself, preliminary evidence presented in october 2009 suggests that immunomodulatory treatment of influtable 1 . cell signaling targets that might be affected by immunomodulatory treatment of severe seasonal and pandemic influenza* down regulate pro-inflammatory cytokines (e.g., nf-kappab, tnfa, il-1, il-6) up regulate anti-inflammatory cytokines (il-10, tgfb) up regulate pro-resolution factors (lipoxin a4, resolvin e1) up regulate ho-1 and decrease tlr signaling by pamps and damps up regulate enos, downregulate inos, restore inos ⁄ enos balance and stabilize cardiovascular function decrease formation of reactive oxygen species and decrease oxidative stress improve mitochondrial function and restore mitochondrial biogenesis decrease tissue factor and its associated pro-thrombotic state stabilize the actin cytoskeleton in endothelial cells and intracellular adherins junctions, and thereby increase pulmonary barrier integrity and decrease vascular leak differentially modify caspase activation and apoptosis in epithelial and endothelial cells, macrophages, neutrophils and lymphocytes in the lung and other organs increase the bcl-2 ⁄ bax ratio in influenza virus-infected cells and prevent the apoptosis necessary for virus replication. *see references 2,5,7,8 for details. nf-kappab, nuclear factor kappab; tnfa, tumor necrosis factor alpha; tgfb, transforming growth factor beta; ho-1, heme oxygenase -1; tlr, toll-like receptor; pamp, pathogen-associated molecular pattern; damp, damage associated molecular pattern; enos, endothelial nitric oxide synthase; inos, inducible nitric oxide synthase. enza patients with severe illness could be beneficial. in a study of almost 4000 patients hospitalized with laboratoryconfirmed seasonal influenza, inpatient statin treatment reduced hospital mortality by 66%. 20 in these patients, the cell signaling effects of statin treatment, summarized in the table 1 , probably acted to reduce pulmonary infiltrates, maintain oxygenation, stabilize myocardial contractility and the peripheral circulation, reverse immunosuppression, restore mitochondrial biogenesis, and prevent multi-organ failure. achieving these clinical effects led to a decrease in mortality. because of the molecular cross-talk between statins, fibrates, glitazones, and ampk agonists, 5,8 similar clinical benefits might be expected from other members of this ''family'' of immunomodulatory agents. simvastatin, pioglitazone, and metformin are produced as inexpensive generics in developing countries. they are used throughout the world in the daily treatment of millions of patients with cardiovascular diseases and diabetes. global supplies are huge. because most people with influenza recover without specific treatment (this was true in 1918), not all patients would require immunomodulatory agents. instead, only those at risk of ards, multi-organ failure, and death would need to be treated. importantly, the cost of treatment for an individual patient would be less than $1.00 (d.s. fedson, unpublished observations). moreover, unlike vaccines they could be used on the first pandemic day. thus far, influenza scientists and the institutions that support their work (e.g., nih and cdc, national health agencies in many countries, the bill and melinda gates foundation, the welcome trust, and the world health organization) have shown little interest in immunomodulatory treatment. nonetheless, when more than 90% of the world's people have no access to influenza vaccines and antiviral agents, their physicians must have access to an effective ''option,'' especially one that might be lifesaving. research on immunomodulatory agents for influenza must involve investigators in many fields outside influenza science -those with expertise in the molecular and cell biology of inflammation, immunity, sepsis, cardiopulmonary diseases, endocrinology and metabolism, ischemia ⁄ reperfusion injury, mitochondrial function, and cell death. laboratory studies needed to identify promising treatment agents would probably cost $5-15 million (d.s. the results of these studies would inform clinical trials that critical care physicians are already eager to undertake. 21, 22 this work will be especially important for people in developing countries where critical care capacity is extremely limited and not likely to improve. 23 like critical care physicians, influenza scientists too must recognize that they cannot afford not to undertake research to determine whether generic immunomodulatory agents might be useful in managing severe seasonal and pandemic influenza. the nf-kappab-inhibitor sc75741 efficiently blocks h5n1 influenza virus propagation in vitro and in vivo without the tendency to induce resistant virus variants introduction influenza is still one of the major plagues worldwide. the appearance of highly pathogenic avian influenza (hpai) h5n1 viruses in humans and the emergence of resistant h5n1 variants against neuraminidase inhibitors highlight the need for new and amply available antiviral drugs. we and others have demonstrated that influenza virus misuses the cellular ikk ⁄ nf-kappab signalling pathway for efficient replication, suggesting that this module may be a suitable target for antiviral intervention. 1 here, we show that the novel nf-kappab inhibitor sc75741 efficiently blocks replication of influenza a viruses, including avian and human a ⁄ h5n1 isolates in vitro in concentrations that do not affect cell viability or metabolism. in a mouse infection model with hpai a ⁄ h5n1 and a ⁄ h7n7 viruses, we were able to demonstrate reduced clinical symptoms and survival of sc75741 treated mice. moreover, influenza virus was reduced in the lung of drug-treated animals. besides this direct antiviral effect, the drug also suppresses h5n1-induced overproduction of cytokines and chemokines in the lung, suggesting that it might prevent hypercytokinemia we hypothesise to be associated with pathogenesis after infections with highly pathogenic influenza viruses, such as the a ⁄ h5n1 strains. thus, a sc75741-based drug may serve as a broadly active nontoxic anti-influenza agent. to assess the number of infectious particles (plaque titers) in organs a plaque assay using avicel ò was performed in 96-well plates as described by mastrosovich and colleagues. 2 virus-infected cells were immunostained by incubating for 1 hour with a monoclonal antibody specific for the influenza a virus nucleoprotein (serotec) followed by 30 minutes incubation with peroxidase-labeled anti-mouse antibody (dianova) and 10 minutes incubation with true blueô peroxidase substrate (kpl). stained plates were scanned on a flat bed scanner and the data were acquired using microsoft ò paint software. the virus titer is given as the logarithm to the basis 10 of the mean value. the detection limit for this test was <1ae7 log 10 pfu ⁄ ml. organs of infected and control mice were homogenized and incubated over night in 1 ml trizol ò reagent (invitrogen) at 4°c. total rna isolation was performed as specified by the manufacturer (invitrogen). rna was solubilised in 50 ll rnase free water and diluted to a working concentration of 50 ng rna ⁄ ll. reverse transcription real-time pcr was performed using quantifastô sybr ò green rt-pcr kit and quantitect primer assays (qiagen) . all samples were normalized to gapdh and fold expression analyzed relative to uninfected controls. 3 ct values were obtained with the smartcycler ò (cepheid). to answer the question whether the nf-kappab inhibitor sc75741 shows antiviral properties against influenza virus, h5n1 infected mdck cells were treated with different concentrations of the inhibitor (figure 1 ). already treatment with 1 nm of sc75741 led to a reduction of viral cpe of more than 70%. almost 100% protection of cells was achieved when cells were treated with 50 lm sc75741. the results indicated that sc75741 has antiviral properties at concentrations ranging from 1 to 5 nm. we next tested whether sc75741 would also be effective in the mouse model of influenza virus infection. when h7n7 mice were treated i.v. once daily for 5 days with 5 mg ⁄ kg sc75741, survival rate of the animals increased significantly (p < 0ae05). the same results were found when h7n7 influenza virus infected mice were treated i.p. with 15 mg ⁄ kg sc75741 (data not shown). moreover, sc75741 treatment was not only effective when the inhibitor was given prior to h5n1 influenza virus infection, but also in a therapeutic setup when sc75741 was applied to the animals 4 days after infection (data not shown). since influenza virus infected mice showed increased survival after lethal infection, we next questioned whether the amount of influenza virus was reduced in the lung. therefore, we performed quantitative real-time (qrt) pcr to detect viral mrna. mice were treated with either sc75741 or the solvent, and 48 hour later the lungs were prepared to perform qrt-pcr. as shown in figure 2a the amount of viral mrna was reduced by 90% in sc75741 treated mice compared to solvent treated controls, indicating that sc75741 leads to a reduced expression of h5n1 specific mrna in the lung of infected mice. since infection of mice with h5n1 leads to hypercytekinemia, 4 we also investigated the expression of cytokines in sc75741 treated mice. as shown in figure 2b the amount of il-6 specific mrna was drastically reduced in sc75741 treated mice compared to solvent treated controls. moreover, also the expression of ip-10 was altered in sc75741 treated h5n1 influenza virus infected mice. here, roughly 90% reduction of specific mrna was detectable ( figure 2c ). thus, sc75741 leads to a reduced transcription of il-6 and ip-10 in h5n1 infected mice. there is an urgent need for new concepts to develop antiviral drugs against influenza virus. targeting cellular factors is a promising but challenging approach, and the concerns about side effects are obvious. however, it should be considered that drugs targeting viral factors, such as amantadine or oseltamivir, also exhibit a wide range of side effects in patients. thus, drug safety has to be rigorously tested in clinical trials regardless whether a drug targets a cellular or a viral factor. moreover, resistance against human h1n1 influenza viruses and highly pathogenic avian h5n1 virus strains to oseltamivir and amantadine have been reported. 5 in that respect, the strategy to target cellular factors 6,7 might be one way to ensure that new drugs against influenza virus will be useful and effective for a long time without causing the development of resistant virus variants. we were able to demonstrate that the nfkappab inhibitor sc75741 is able to reduce influenza virus activity in cell culture. moreover, the compound was also effective against highly pathogenic avian influenza viruses of the h5n1 and h7n7 subtypes in the mouse model. next to the reduction of virus sc75741 was also able to reduce h5n1-induced overproduction of cytokines and chemokines in the lung in the lung of mice after infection with h5n1. most importantly, the drug did not show any tendency to induce resistant virus variants (data not shown). thus, a sc75741based drug may serve as a broadly active non-toxic antiinfluenza agent. [1] [2] [3] [4] [5] in hong kong, the first confirmed case was a tourist from mexico reported on may 1, 2009. the local government made its first attempt to contain the spread of h1n1 in the local community by closing the metropark hotel where that tourist was staying, and quarantining 350 guests and staff for 7 days. following identification of the first local case around 6 weeks later on june 11, 2009, the government closed all kindergartens and primary schools from june 12 until early july. fever clinics were also opened, the alarm levels in hospitals were raised to the highest, and a public education campaign was implemented. previous studies of the community responses to severe acute respiratory syndrome (sars) and human-to-human h5n1 avian flu identified the importance of understanding the background perceptions of risk and psychological impact on the community. [6] [7] [8] [9] [10] in this study we investigated the psychological and behavioral responses of the general local community throughout the first wave of ph1n1, and we also examined the factors associated with greater use of preventive measures. 11 a total of 13 surveys were conducted between april and november 2009, covering the entire first wave of the ph1n1 pandemic. computer generated random-household telephone numbers from all land-based local telephone numbers covering over 98% of hong kong households were used to recruit a total of 12 965 local adults. one cantonese-speaking adult (age ‡18) was invited for interview in each selected household on the basis of a kish grid. the survey instrument was based on previous experience in sars and avian influenza projects. information, including knowledge on modes of transmission, psychological responses to pandemic influenza, preventive behaviors, attitudes towards the new vaccines and socio-demographics, was collected. informed consent was obtained prior to the interview. ethics approval was obtained from the institutional review board of the university of hong kong. descriptive statistics were weighted by sex and age based on the reference population data provided by the hong kong government census and statistics department. 12 multivariable logistic regression analyses were used to examine the association between the use of preventive measures and knowledge, perceptions and behaviors, sociodemographic characteristics, and psychological responses to pandemic influenza. multiple imputation was used to cope with a small proportion of missing data and make the best use of all available data. 13 statistical analyses were conducted in r version 2.9.1 (r development core team, vienna, austria). twelve thousand and nine hundred and sixty-five local adults were recruited throughout the study period, with a total of 127 715 telephone calls being made; the response rate among eligible participants was 69.9%. 11 hong kong entered the containment phase after the world health organization (who) announced a global alert, and policies including border screening, tracing, and quarantine of doi:10.1111/j.1750-2659.2011.00219.x www.influenzajournal.com suspected cases were implemented. hong kong transitioned to the mitigation phase on june 10, 2009 when the first local case was reported. the chronology of these and other events plus the epidemic curve of laboratory-confirmed ph1n1 cases are shown in figure 1(a) . the anxiety scores and risk perception of the respondents are shown in figure 1(b,c) . anxiety, measured by the state trait anxiety inventory, remained steady throughout the study period. in response to the announcement made by who and the unknown nature of the new virus, a higher proportion of the respondents expressed worry (more, much more, or extremely more worried than normal) if developed ili and perceived ph1n1 severity (same, more, or much more serious than sars) initially in early may 2009. fewer respondents reported worry if they developed ili as the pandemic proceeded, with a slight perturbation around the first deaths in july 2009 and a steady decline to 40.0%, while perceived severity of ph1n1 declined more dramatically after an early high. perceived risks of infection of respondents (absolute susceptibility) and risk relative to others (relative susceptibility) were also investigated and found to remain relatively stable throughout the first wave, with no indication of an increase during the period of peak ph1n1 activity in september (figure 1c) . as the first wave of ph1n1 progressed, knowledge on modes of transmission did not improve. on the contrary, later in the epidemic increasing proportions of respondents reported oral-fecal and cold weather as modes of transmission of ph1n1. around 35-40% of the respondents did not recognize direct and indirect contact or touching infected persons and contaminated objects as transmission routes for ph1n1 throughout the first wave ( figure 1d ). higher proportions of respondents avoided crowded places and rescheduled travel plans in the second half of june 2009 when local kindergartens and primary schools were closed and the first ph1n1-associated deaths were announced. social distancing measures such as avoiding crowded places and rescheduling travel plans remained stable with slightly decreasing trends thereafter. the use of hygiene measures and other social distancing strategies was relatively stable with slightly decreasing trends during the study period ( figure 2 ). female sex and older age were generally associated with greater reported use of hand hygiene measures, home disinfection, avoidance of crowded places, and rescheduling of travel plans. 11 female sex was also positively correlated with use of face masks and cough etiquette. we found a negative correlation between anxiety and use of all hand hygiene measures and cough etiquette, but a positive correlation between anxiety and use of home disinfection and (c) proportion of the respondents reporting higher worry if developed flu-like symptoms (more, much more, or extremely worried), higher perceived seriousness of h1n1 compared to sars (much more or more severe), higher probability to contract h1n1 over the next 1 month (certain, much more, or more likely), higher probability to contract h1n1 over the next 1 month compared to others outside family (certain, much more, or more likely). (d) proportion of the respondents identifying 5 possible modes of transmission as the actual modes of transmission of h1n1. social distancing measures. 11 other significant factors contributing to greater use of preventive measures were worry and knowledge. 11 greater worry was associated with higher probability of home disinfection, social distancing measures, and use of face masks. knowledge that h1n1 could be spread by indirect contact was associated all the investigated preventive measures, and knowledge that h1n1 could be spread by droplets was associated with cough etiquette, but not face masks. there were no consistent trends between all the investigated preventive measures and absolute and relative susceptibility. 11 community transmission emerged in hong kong in mid-june 2009, and prior to emergence of community transmission, perceived risk and perceived severity were high. as ph1n1 spread in hong kong, risk perception declined, even at the same time as incidence was increasing. anxiety was low throughout, at around 1.8 on the 4-point scale, compared to a maximum of 2.5 during sars on the same scale. 9 anxiety has been showed to be positively correlated to personal hygiene measures and social distancing in previous studies; 9,14 however, we found a negative correlation between anxiety and use of all hand hygiene measures, cough etiquette, and face masks, and a positive correlation between anxiety and home disinfection. 11 the differences in findings may be due to the fact that our anxiety measure was not specific to h1n1, and the score could be affected by other factors including economics. unlike hygiene measures, higher anxiety level, greater worry, and higher risk of perception were all associated with more social distancing. 6, 7, 9, 14 social distancing is the most direct strategy in avoiding infection from other people, and it is commonly observed in an outbreak that the general public avoids crowded places, travelling to other countries, and social gatherings, 9, 14 but the economic impact could be substantial. 15 as community incidence of h1n1 peaked, we did not observe any increase in use of preventive measures (figure 2) . we found that face mask use peaked at the early stage of the pandemic, while hand hygiene remained fairly constant, and the knowledge on the modes of transmission of ph1n1 did not improve over time. the lack of substantial change in preventive measures or knowledge about the modes of ph1n1 transmission in the general population suggests that community mitigation measures played little role in mitigating the impact of ph1n1 in hong kong. on the other hand, knowledge that ph1n1 could be spread by indirect contact was associated with all of the preventive measures studied. consistent with reports during the sars period, 9, 16 this study also showed that females and those of older age were more likely than others to use hygiene measures, avoid crowded places, and reschedule travel plans. this study has some limitations. first, this was a crosssectional study that was carried out at different time points, rather than a longitudinal study following the same individuals over time, and so the inferences on changes in behavior may need to be interpreted more cautiously. second, we recruited samples from all land-based local telephone numbers that cover 98% of hong kong households, but the response rate was not high enough to guarantee a representative sample, and this could be a source of selection bias. third, the responses were self-reported, and this may lead to social desirability bias in estimating knowledge, attitudes, and preventive behaviors. fourth, since the hong kong population has previously gone through unique experiences from sars in 2003 and avian flu in 1997, our results may not be comparable to other countries or settings. in conclusion, this study revealed that the ph1n1 pandemic failed to generate an increase use of preventive measures in the local community. there was no association between anxiety level and the events of the pandemic. with a relatively low mortality and morbidity rates compared to sars, ph1n1 was not a matter of concern in the hong kong community. the lack of substantial change in the use of preventive measures and improvement in knowledge on the modes of transmission of ph1n1 suggested that public health campaigns during the pandemic may not have had substantial effects on the general public. london is a major tourist destination, the seat of government and finance in the uk, and in 2012 will host much of the olympic and paralympic games. along with the rest of the global community, in 2009 and early 2010 london faced the challenges of responding to the first pandemic of the 21st century. at the time, nhs in london was composed of 72 organisations, including the london ambulance service, acute hospitals, mental health and primary care trusts, and the strategic health authority. while london's nhs is well practiced at responding to large, big bang incidents, the influenza a ⁄ h1n1v pandemic was a rising tide event that lasted many months. significant preparatory work had been undertaken prior to april 2009, which meant that the nhs in london was ready to respond. nhs london (the strategic health authority for london) led the response in partnership with local managers in all nhs organisations. the first uk cases of influenza a ⁄ h1n1v were reported in scotland on 27 april, with the first in london on 30 april. cases continued to increase, and the first wave peaked in london in july. cases reduced over the school summer holidays, but increased again when children returned to school at the start of september, and a second, smaller wave occurred. it is essential that the nhs learns from the 2009 ⁄ 10 influenza a ⁄ h1n1v pandemic to ensure it is prepared for future challenges. nhs london provided a standardised debriefing pack to all nhs organisations in the region to identify, capture, and learn lessons. each debrief event involved health and inter-agency partners to ensure all viewpoints were considered and brought together in a single local report. all local reports were compiled in an over-arching document, which brings together common themes to inform ongoing preparedness in the region. 1 the debrief process identified a number of common themes, such as the need for clear and appropriate communication, the importance of working with partners, and the benefits of strong and early leadership. however, differences between and within organisations were also highlighted; for example, some wanted more freedom for local decision making, whereas others would have preferred more stringently applied central direction. the following paragraphs considers individual areas assessed in the debrief process. command and control was in the main effective, with clear direction delivered from the national centre through nhs london to local nhs organisations. effective leadership is essential; the identification of senior local individuals to lead the response with teams of people to support them was critical. appropriate use of technology to communicate messages and coordinate command and control processes greatly aided the response. this included the development of the nhs london noon brief, a daily digest and associated web portal, and regular teleconferencing. key points are: • operational management at all levels must be considered in pandemic planning. • appointing an executive lead in each organisation was invaluable in the response. • pandemic flu planning for london must continue to be regionally led. communication is an essential component of the response to any incident. it must be clear, timely, and accurate. in the main, communication was excellent and met these criteria. one of the most challenging aspects was when messages from partner organisations differed, which occasionally led to confusion, unnecessary work, or frustration. the use of technology greatly aided communication across the region and supported the response; this included secure web sites, bluetooth, and text messaging etc. key points are: • regular internal communications and staff briefings are critical in the response to emergencies. • regular teleconferencing should be incorporated into future plans. • organisations should consider proactive and innovative methods for communicating during emergencies. robust partnership working was an essential component of pandemic preparedness work; however in the event, the a ⁄ h1n1v pandemic had little impact on sectors in london other than health. resilient communication networks between organisations, a common understanding, and the ability to make decisions were essential to the response at local level. ipcs proved an excellent mechanism to maintain local working relationships and resolve problems. clarity on the seniority of those attending these meetings and whether multi-site organisations such as mental health trusts should attend every ipc should be considered on a local and regional basis. key points are: • pandemic planning must remain part of inter-agency working. • social care resilience and planning must be embedded and integrated in health planning. 'vulnerable groups' is a universal term that covers a large and fluid group of individuals with different needs. ensuring access to healthcare during the pandemic for those who became vulnerable due to the situation, or those identified as such prior to the event, was the role of the pct in partnership with the local authorities. work continues to ensure that communication with vulnerable people is appropriate and timely in all incidents, and that organisations work together to achieve this. key points are: • planning to support the breadth of vulnerable people must continue. • pandemic preparedness for the prison sector should be further developed. • red ⁄ amber ⁄ green ratings for assessing vulnerabilities of mental health service users in an emergency should be further developed across the region. correct and appropriate usage of ppe is an essential component of reducing influenza spread, particularly in healthcare settings. london's nhs had been working towards developing local stockpiles of ppe when the pandemic commenced; however, there was little in place. the unanticipated national stockpile, while providing ppe to all organisations, was accompanied with some challenges in that it was often unfamiliar stock. key points are: • work around local stockpiling of non-standard consumables should continue. • regular training and fit testing of respirators should be embedded in all organisations. antiviral treatment was a core component of the response to influenza a ⁄ h1n1v, and was provided free of charge from a national stockpile. npfs reduced pressure on frontline nhs services once it was activated; however, there were concerns that patients could 'cheat' the system and obtain the drugs prior their clinical need. information about storage requirements of countermeasures must be clearly explained when they are delivered to frontline services, and the potential for recall into national stockpiles should be planned for. key points are: • regular exercising of local mass countermeasures centres and antiviral collection points (acps) should continue. • the use of community pharmacies as acps should be further considered in the capital. pandemic influenza vaccine uptake by healthcare workers was better than usual seasonal influenza uptake in the majority of nhs organisations, but could have been even better. this was largely due to the second pandemic wave not being as significant as expected, lack of clarity around when the vaccine would be delivered, and limited amounts being available initially. • gp-led and mass vaccination models for pandemic vaccination should be considered in local plans. • local lessons from the pandemic vaccination campaign should be applied to seasonal flu vaccination. the ability to maintain or increase capacity in response to a surge in demand, no matter what the cause, must be planned for. any of a number of situations could result in reduced staff or more patients, such as industrial action, transport disruption, disease outbreak, major incident, or poor weather. the work undertaken during planning for and responding to the pandemic will stand organisations in good stead for future disruptions. the importance of robust business continuity planning locally cannot be overlooked, as this is a key component of maintaining and increasing capacity. key points are: • local gp 'buddy schemes' should be encouraged for response to extreme pressure events. • organisations should regularly run staff skills audits so as to be aware of their overall capability for managing emergencies. • less emphasis should be placed on the use of retired staff when planning service continuity. reporting is a necessary but onerous task, and is often one of the most time-demanding parts of any incident response. it is also the aspect least likely to be tested through exercising. nhs london worked with organisations to endeavour to reduce reporting pressures, but much of this was dictated by central government. it is essential that future reporting requirements are proportional, informative, and realistic. while recognising it is not possible to predict the detail of information that may be requested, some broad assumptions can be made. key points are: • organisations should consider how they would collect and collate data from disparate parts of their organisation, rather than focussing on the detail of what that might be. • national and regional planning should consider the need for information and how this is balanced with the demand this places on organisations. • the introduction of the concept of a daily dashboard to identify areas of pressure should be incorporated into pandemic flu planning. the winter and pandemic influenza resilience assurance process undertaken in autumn 2009 was a useful process to inform planning for the first winter when the pandemic virus would be circulating in the uk. this consisted of a regional inter-agency exercise and a comprehensive review of the winter and pandemic plans of all nhs organisations in london. • regular assurance of pandemic flu preparedness should be maintained. • future resilience assurance processes should be undertaken in a timely and measured manner. • local organisations should continue to undertake regular pandemic flu exercises. the recovery period is as important as the response, but often receives minimal attention and has the potential to suffer as staff return to their normal jobs. one of the aspects that was not anticipated during the pandemic was the amount of stock (ppe, antivirals, and vaccine consumables) that would be recalled into national stockpiles. this proved particularly challenging for pcts who had to coordinate the process across their local areas. key points are: • the recovery period of an emergency must be given the same status and importance as the response. • future pandemic flu planning must include the recovery of national stockpiles of equipment and medicines. it is essential the lessons from the 2009 ⁄ 10 influenza a ⁄ h1n1v pandemic are learnt and embedded into business-as-usual and emergency response processes in preparation for the next pandemic and other incidents. even though the a ⁄ h1n1v pandemic was generally milder than previous pandemics, it still presented challenges to the nhs in london. the biggest challenge that remains is to ensure that the public and nhs staff are aware that a more virulent virus could cause significantly more illness, death, and disruption, and that we must maintain our preparedness should this happen. the influenza a ⁄ h1n1v pandemic has been a major stimulus to business continuity planning and emergency preparedness across health in london, and many of the experiences during the pandemic proved invaluable in the unusually severe weather in early 2010. it is important that this impetus and focus is maintained. changes to the nhs landscape in london will be considered in ongoing pandemic and emergency preparedness to ensure we remain as well prepared as possible for future events, particularly as london approaches the 2012 olympic and paralympic games. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. the way national health systems operate during inter-pandemic and the pandemic alert periods and the methods they use to address potential threats posed by zoonotic viruses with pandemic potential, as well as sea-sonal influenza epidemics, can clearly indicate whether the countries have enough capacities to respond adequately to unexpected influenza outbreaks. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during a pandemic scenario, the group prioritized topics and questions relating to rapid action and response. five to 10 key public health needs associated with a pandemic scenario have been identified for each of the research agenda streams: five priority public health topics were identified for a pandemic scenario as follows: • examination of host range and transmission dynamics of animal influenza viruses to guide surveillance, control strategies, and risk communication. • enhanced surveillance in animals and humans to monitor virus evolution: o early detection of novel reassortants or changes in genotype and ⁄ or phenotype related to virulence. o development of epidemiological and laboratory diagnostic tools and capacity building to optimize case finding. o develop a framework for surveillance in animals that address ethical, legal, and social barriers to intra-pandemic surveillance and reporting. • deconstruct the origins of the pandemic virus to identify factors that permitted efficient human transmission. • develop strategies to limit economic, social, and cultural disincentives of animal-based interventions to reduce intra-and inter-species transmission. • operational research to optimize risk communication in the early phases of the pandemic linked to animal husbandry and food safety. stream 2: limiting the spread of pandemic, zoonotic and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: transmissibility of influenza across the progression of infection and spectrum of disease: • relative contributions of the different modes of transmission for influenza. five priority public health topics were identified for a pandemic scenario as follows: • identification of groups at higher risk of infection and severe disease outcome through enhanced surveillance. • understanding disease severity and identification of predictors of severe outcomes. • investigation of vaccine effectiveness, especially in high risk groups in diverse geographic areas. • establishment ⁄ enhancement of pharmacovigilance, particularly for adverse events among at-risk groups. • optimization of strategies for rapid and targeted vaccine deployment. • rapid assessment to optimize acceptance of pandemic vaccine. six priority public health topics were identified for a pandemic scenario as follows: • collaboration and coordinated sharing of data, protocols, regulatory, and other implementation strategies and databases from different countries on all aspects of patient management and outcome to accelerate improvements in patient care. • development of best practices in patient management in different settings, including checklists and algorithms for clinical care and treatment, prognostic parameters, and tests to predict potential for the development of severe disease. • rapid, reliable, simple, low-cost point-of-care diagnostic tools for influenza. • best use of current antiviral drugs and optimal formulations in different target populations, such as parenteral and other routes of administration for severe infections. • use of combination therapies, including use of adjunctive therapies (e.g., use of convalescent serum and immunomodulators). • role of ongoing viral replication, host responses, and the effect of co-infections in the pathogenesis of severe disease. modern tools for early detection and monitoring of disease the group on surveillance tools concluded that the agreed topics of interest were equally applicable during a pandemic or inter-pandemic period: • studies to appraise and adapt modern technologies for early detection of influenza outbreaks in surveillance at the human-animal interface. • develop, integrate, and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems. • study efficient mechanisms on sharing data, clinical specimens, and viruses with consideration for local, ethical, legal, and research perspectives. • examine the timeliness and quality of data required for early detection from local to national and global levels for the respective stakeholders. five priority public health topics were identified for a pandemic scenario as follows: • identify environmental determinants of seasonal variation in influenza transmissibility in tropical and temperate regions. • estimate the transmission risk associated with types of contacts by comparing measured contact patterns with outbreak data. • incorporation of validated models of behavioral responses to risk and control measures in virus transmission. • development and implementation of novel technology for real-time sero-surveillance during a pandemic. • develop experimental and theoretical framework to assess host adaptation to study host receptor, antigenicity, and virulence. modern tools for strategic communication three priority public health topics were identified for a pandemic scenario as follows: • evaluate tools to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups to guide future communication efforts; develop tools and methods to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups, and thereby, guide future communication efforts. for communicating in different cultural settings, which engage and empower individuals and communities to practice and promote appropriate risk reduction measures. implementation of the identified research priorities is expected to underpin public health decision making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs and economic loss, and mitigate potential social disruption. complemented by an analogous research roadmap for a pandemic influenza scenario, the research recommendations for an interpandemic period represent a framework to provide evidence to guide public health policies on influenza control. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza 1 developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting public health needs for inter-pandemic periods according to its five key research streams: • stream 1. reducing the risk of emergence of pandemic influenza. • stream 2. limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza. • stream 3. minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza. • stream 4. optimizing the treatment of patients. • stream 5. promoting the development and application of modern public health tools. stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza inter-pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during an inter-pandemic phase, a more comprehensive approach was applied to establish research topics and prioritizing a range of questions that will build a solid foundation to guide research activities to support public health decision making. five to ten key public health needs associated with an inter-pandemic scenario have been identified for each of the research agenda streams: stream 2: limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: 1. transmissibility of influenza across the progression of infection and spectrum of disease 2. relative contributions of the different modes of transmission for influenza 3. biological, behavioral, and social host factors that influence the risk of transmission and infection 4. patterns, drivers, and mechanisms affecting the seasonality of transmission 5. viral and population factors that influence transmission and spread of different influenza types, subtypes, and strains 6. strategies to reduce the transmission of influenza in community, household, and health care settings, especially in less-resourced areas 7. impact and cost effectiveness of social measures, such as school closures, and the role of surveillance in assessing timing of these interventions 8. impact, effectiveness, and cost effectiveness of individual measures, such as isolation and quarantine 9. role of vaccination in limiting the spread of influenza and strategies for its use 10. impact of antiviral treatment and prophylaxis in reducing transmission of influenza stream 3: minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza 1. identify higher risk groups and severe disease through surveillance; disease severity and identification of predictors of severe outcomes 2. evaluate vaccination preventable disease burden and the potential impact of immunization programs through vaccine demonstration projects 3. enhancement of the properties of existing vaccines, including duration and breadth of protection, safety, immunogenicity, and dosesparing 4. development of new vaccines and vaccine platforms, especially suitable for under-resourced country settings 5. study the effectiveness of vaccine strategies to reduce disease burden in children and other high risk groups in a wide range of settings 6. improved uptake and acceptability of vaccines for both seasonal and pandemic influenza seven priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: inter-pandemic seasonal influenza scenario 1. research on the burden of severe disease with a focus on regionalspecific factors, such as the burden of tb and hiv and optimization of pandemic and management 2. development of new antiviral strategies and validation of surrogate endpoints which may aid in advancing understanding of disease progression 3. further clinical evaluation of current antiviral drugs, particularly in populations at risk 4. integration of seasonal influenza with pandemic preparedness; strengthen surveillance, health care systems, capacity, and preparedness planning 5. improving diagnostics (e.g., multiplex assays for viruses and bacteria), including antiviral resistance testing at point-of-care 6. dissemination of best practices, situation analysis, preparation for next epidemic (e.g., establish protocols for rotating stockpiles of antiviral drugs) 7. increased attention to basic science research such as studying immunomodulatory drugs five priority public health topics were identified for an inter-pandemic zoonotic influenza scenario as follows: inter-pandemic zoonotic influenza 1. antiviral susceptibility of circulating zoonotic viruses (e.g., h5, h9, h7 influenza viruses) 2. reassortment between zoonotic and human influenza viruses and the potential for inter sub-type spread of antiviral resistance and virulence modern tools for early detection and monitoring of disease the group focusing on surveillance tools concluded that the agreed topics of interest were equally applicable during both pandemic and inter-pandemic period: 1. identify modern technologies for early detection of influenza outbreaks as well as their application in surveillance at the human-animal interface 2. develop and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems 3. studies to address challenges on data, clinical specimens, and viruses sharing with consideration for local, ethical, legal, and research perspectives 4. examine the timeliness and quality of data required for early detection from local to regional, national, and global levels role of modeling in public health decision making five priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: 1. integration of genetic and epidemiological data to understand spatiotemporal spread to forecasts evolution for vaccine strain selection and to anticipate likely burden of disease 2. quantifying the relative contributions of different modes of transmission of human influenza and developing mechanistic modeling of transmission processes 3. research using data-capture technologies to characterize human contact and mobility patterns at local, regional, and global scales, and their correlation with transmission risk 4. integration of genetic, antigenic, and epidemiological analyses to optimize surveillance for newly emerging pathogens at the animal ⁄ human interface 5. identifying and quantifying human and environmental ecological, behavioral, and demographic determinants of the risk of cross-species transmission and pandemic emergence modern tools for strategic communication four priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: 1. review of evidence and experience related to health crisis communication from fields to organize knowledge and support evidencebased practice in strategic communication 2. identify and develop tools to rapidly and accurately monitor knowledge, attitudes, and practices in different population groups and guide future communication efforts 3. identify and develop communication tools and approaches for cultural settings and communities to practice and promote appropriate risk reduction measures 4. understand the potential ethical, social, economic, and political communication in crisis and develop strategies to work within constraints while maximizing opportunities complemented by an analogous research roadmap for a pandemic influenza scenario, the research topic recommendations for an inter-pandemic period represent an important outcome of joint international efforts by who, academicians, and public health experts. implementation of the identified research priorities is expected to underpin public health decision-making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs, and economic loss and mitigate potential social disruption over a medium-tolong term period. the impacts of school resumption on the incidence of pandemic (h1n1) 2009 in school students introduction school closure is one non-pharmaceutical intervention that is often suggested in pandemic preparedness plans, and it was widely implemented in pandemic (h1n1) 2009 to reduce transmission amongst school students. however, from past epidemiological studies, the effect of school closure in reducing respiratory disease transmission was inconclusive. 1 given this public health intervention causes major disruption to the education system and potentially raises childcare issues to working parents, evaluating its effect in the recent pandemic is necessary to improve future pandemic planning. in hong kong, since school closure was implemented early in the pandemic and closure was effectively continued with the commencement of summer holiday, the lack of incidence data in the absence of school closure makes it difficult to analyse its effect directly. this has prompted us to analyse the situation indirectly from the angle of school resumption after summer holiday. in hong kong, public health surveillance on pandemic (h1n1) 2009 was effective from 25th april-30th september 2009: healthcare professionals were advised to report suspected cases of infection to centre for health protection, department of health, hksar, for further laboratorial confirmation. demographics of reported cases were subsequently recorded into a computerised system (the ''e-flu'' database). following institutional approval, a dataset of all confirmed cases diagnosed from may to september 2009 was obtained, which included the age, gender, confirmation date, and notification date of each report. all cases were classified into four defined socio-economic classes by age: pre-schoolers (0-5), school students (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) , adults (20-60), and retirees ( ‡61). assuming cases had contracted infection on the earlier date between confirmation and notification, daily incidence in each age class was counted for epidemic curve construction. upon observing an unusual rise in the epidemic curve of school students when school season resumed in september, interrupted time series analysis (also known as intervention analysis) 2 was applied to obtain the statistical significance of this observation. the analysis was applied to the incidence in school students from 12th july to 26th september 2009, which covered the period from the start of summer holiday to the end of the 4th week of new school season. incidence in school students before summer holiday was deliberately dropped since not all schools were closed when the school closure policy was effective: all primary schools were closed proactively, whereas secondary schools were individually closed on a reactive basis if students were identified to have contracted the infection. school activity was formulated as a step function, which takes value from 1st september 2009 onwards (st = 0: t < 1st september, st = 1 otherwise). a range of times series models were fitted by the maximum likelihood method and aic (akakine information criterion) was used to select the one with best fit. all computations were performed in sas version 9.2. a total of 3905 (14ae8%) pre-schoolers, 13758 (52ae1%) school students, 8383 (31ae8%) adults, and 338 (1ae3%) retirees were diagnosed with the infection in the surveillance period. the epidemic curves of preschoolers, school students, and adults showed a steady rise from 11th june onwards when local transmission of pandemic influenza was identified. an upsurge in the epidemic curve of school students can be observed in early september, coinciding with the commencement of the new school year (figure 1) . interrupted time series analysis on the epidemic curve of school students returned an arima(0,1,0) model with equations: where st, yt, yt denote school activity, predicted and actual incidence in school students on day t, respectively. standard error and significance for model constants were: 60ae27 (se = 33ae5, p = 0ae08), 2ae73 (se = 3ae84, p = 0ae48). in short, the model can be interpreted as: the number of infected school students rose by 2ae73 per day on average during the entire study period, with a sharp increase by 60ae27 coming into effect when the new school year began. time series analysis showed, at the marginally significance level, that daily incidence in school students had a major increase when school season resumed. on the assumption that the increase was not caused by any change in health seeking behaviour, this result suggests that school resumption had facilitated transmission amongst school students. on the basis that school activity significantly increases incidence of pandemic influenza in school students, this study suggests closures of schools in the early phase of pandemic (h1n1) 2009 and subsequently in the summer holiday probably had a major effect in mitigating transmission amongst school students. youngsters were postulated to be major vector for transmission in pandemic (h1n1) 2009. if this were true, it would be reasonable to expect the epidemic curves of the other age classes to show a similar upsurge when one is observed in school students. the absence of such observation in the epidemic curve of hong kong suggests school students were mostly disseminating the virus amongst themselves, but not to the other age groups. in november 2009, gip convened the first global consultation on a public health research agenda for influenza to identify key research topics in each of the five main streams of public health research. during this meeting, the scientific working group (swg) of the sub-stream in ''modern tools for risk communication'' identified the requirements in research during influenza pandemics and inter-pandemic periods to provide clear, credible, and appropriate messages which meet the needs of diverse communities. the swg suggested that who hold a follow-up workshop to assess the use of modern tools related to strategic and risk communication and to further promote research in these areas. communication'' in may 2010. one of the main objectives of the meeting was to generate a roadmap of public health research priorities related to strategic and risk communication. the research roadmap was developed by the group of invited experts on the basis of an analysis of available evidence and experience on public health and health crisis communication from relevant disciplines across global regions, as well as critical assessment of existing communication methods related to influenza control in different cultural, social, and ethnic settings. the workshop consisted of a series of presentations by experts in relation to experiences and lessons learned about communication during the sars, h5n1 epidemic, and h1n1 pandemic. there were also a series of group discussions on identifying research needs for pandemic and interpandemic periods in order to strengthen the research agenda. the expert group identified important public health needs in relation to communication during pandemics as well as in the inter-pandemic times. the main topics of discussion centered on communicating issues of influenza virus transmission, the use of influenza vaccines safety and efficacy, and use of antivirals as well as definition of the severity of the pandemic and the phase changes. in this context a number of research areas were identified, which can be broadly classified into four areas: understanding of communication principles and mechanisms is associated with an array of research topics covering different subject areas. one of the key questions here relates to the link between communication and ''behaviour change'' models and their application and appropriateness for different settings. the expert group defined the term ''behaviour change'' in this context as the modification of behaviour towards better health practices that are supported by clinical and scientific evidence for personal protection against infectious diseases and other adverse health risks. research topics related to these models require understanding and differentiating information and ''behaviour change'' needs of different audience segments, such as stakeholder mapping, target audience analysis, research into behaviour motivation, social norms, and the cultural, religious, social, legal, and political barriers and enablers of particular behaviors that are beneficial in influenza control. this research area also includes the analysis of media consumption among different audiences, role models, including ways to analyse how rumours and misinformation are spread, and ways to provide evidence-based information correctly. other important areas of investigation embrace methods to communicate uncertainty, learning how to build trust while communicating about a pandemic, and understanding what needs to be done before, during, and after a pandemic in order to create the best environment for influenza pandemic communication. critical key audiences identified for more intensive analysis were health workers, religious, public health, and societal (political and community) leaders. • investigation of the role of different communication channels and communication formats for different target audiences in a pandemic, particularly for groups that are ''hard-to-reach.'' • determining effects of perceptions related to pandemic influenza (severity, susceptibility, response efficacy, self efficacy, perceived social norms) on protective behaviours in different groups. • understanding audience in terms of their knowledge, preventive activities, and reasons why engaged ⁄ not engaged. • developing mechanisms to synergies between risk communication and behavior oriented approaches in the pandemic and inter-pandemic phases. • determining social, economic, cultural, and religious factors which support behaviours to limit spread and minimize impact in different settings. • identification of the key predictors ⁄ factors that influence people's behavior among different groups and populations vis-à -vis pandemic flu behaviors. • identification of elements that contribute to trust among populations and in different settings (country, public, professional, community), particularly where trust was previously compromised. • understanding psychology of different groups regarding their response to uncertainty, and finding the best way to communicate uncertainty. the research questions in this section relate to the planning, development, and evaluation of tools that can be quickly accessed and used in a pandemic situation. these may include communication materials and channels; the setting up of key stakeholder and champion communication networks; research protocols that are ready for rapid assessment during a pandemic or new communication tools. the use and understanding of terminology and language by both lay and professional groups and communities in planning for and ⁄ or reacting to a pandemic are important areas of research. acute examples, such as the naming of the viruses or the use of the word ''pandemic,'' illustrate this need well. the research focus of this area is to look at lessons learned from the a(h1n1) 2009 pandemic and to document and evaluate case studies, both looking at best practices, challenges, and barriers that were experienced. different communication strategies need to be evaluated and models to be built not only in terms of reach, but also in terms of impact on thinking, emotional response, and behavioural modification. a key question was how to prepare communication for a pandemic and how can the pandemic communication contribute to longer term ''behavioural change.'' mathematical modelling on gauging outcomes of such ''behaviour change'' would provide strategic approaches in risk communication. this section aims to answer the question whether the modeling, mapping, and scenario planning are actually useful in the pandemic situation. the expert group agreed that the research on the above issues should use a variety of methods and engage a number of disciplines. this would include literature reviews, case studies, trials, ethnographic studies, modelling, surveys, network analysis, as well as any other useful methodology. in an inter-pandemic situation for actual behaviour under pandemic conditions. • study the synergies and develop priority research topics on strategic ⁄ risk communication for influenza under inter-pandemic situations that includes zoonotic and seasonal infections. the who public health research agenda for influenza initiated and facilitated a multi-disciplinary discussion for communication during pandemic and inter-pandemic situations. it focused on both theoretical and practical issues to improve practice and ensure the health of the public for influenza. critical areas for research were identified to build evidence in this field. it was recognized that there are extensive bodies of knowledge in a number of disciplines, 2,3 such as health promotion, behavioural psychology, social sciences, social and behaviour change communication, social marketing, and communication for development relating to these questions, and that these should be explored. outcomes of these research activities are expected to widen the evidence base which will support developing communication strategies for influenza by countries, institutions, and individuals and will, consequently, help to improve public health world-wide. abstract background: cytokine dysregulation contributes to the unusual severity of h5n1 (reviewed in 1 ). previously, we demonstrated that interferon regulatory factor 3 (irf3) and p38 map kinase (p38) signaling pathways separately contribute to the induction of pro-inflammatory cytokines and chemokines in h5n1-infected cells. 2 here we investigate the role of innate sensing receptors in the induction of these cytokines and chemokines in response to h5n1 and seasonal h1n1 infection. materials and methods: human macrophages derived from peripheral blood monocytes were infected with h5n1 (483 ⁄ 97) or seasonal h1n1 (54 ⁄ 98) viruses. the role of innate sensing receptors in cytokine and chemokine induction by h5n1 virus was investigated using transient knock-down of these receptors with sirnas. the expression of innate sensing receptors in infected cells, and as a result of paracrine activation (by virus free supernatants of infected cells) of adjacent uninfected cells were also monitored by real-time pcr and ⁄ or western blotting. the involvement of janus kinase (jak) signaling pathways in these autocrine ⁄ paracrine cascades was investigated using a jak inhibitor. results: we previously showed that tnf-alpha, ifn-beta, and ifn-lambda 1 are the key mediators directly induced by the h5n1 virus in primary human macrophages with other cytokines and chemokines being induced as part of a secondary autocrine and paracrine cascade. here we demonstrated that retinoicacid-inducible gene i (rig-i) rather than toll-like receptor 3 (tlr3) plays the predominant role in h5n1-induced cytokines and chemokines in human macrophages via the regulation of irf3 and nf-kb nuclear translocation. in addition to the effects on virus infected cells, paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the upregulation of sensing receptors. conclusions: h5n1 directly induced tnf-alpha and ifnbeta mainly via rig-i signaling, and the subsequent activa-tion and nuclear translocation of irf3 and nf-kb in human macrophages. in addition to the effects on cytokine signaling, the innate immune sensing regulators themselves were also up-regulated by h5n1 infection, much more so than by seasonal influenza infection, via jak signaling. the up-regulation of innate sensing receptors was not limited to the infected cells, but was also found in adjacent uninfected cells through paracrine feedback mechanisms. this may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. a more precise understanding of the signaling pathways triggered by h5n1 virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h5n1 influenza and also for treating other causes of acute respiratory disease syndrome. human h5n1 infection is associated with a mortality rate of more than 60%. the basis for the unusual severity of h5n1 disease has not been fully explained. cytokine dysregulation has been suggested to contribute to the disease severity of h5n1 (reviewed in 1 ). however, signaling pathways involved in the cytokine induction by h5n1 virus are not fully understood. previously, we demonstrated that irf3 and p38 map kinase (p38) are separate signaling pathways which contribute to the induction of pro-inflammatory cytokines and chemokines in h5n1-infected cells. 2 rig-i and melanoma differentiation-associated gene 5 (mda5) are important cytosolic sensors of nucleic acid of pathogens, while tlr3 and tlr8 also recognize nucleic acid species of pathogens, but they are localized at the endosomal membrane. 3 rig-i was found to be responsible for the recognition of influenza a virus infection, 4 and the transfection of vrnps induces ifn-beta expression. 5 while many studies have shown the role of rig-i in the induction of ifn-beta by influenza virus infection, the majority of these studies used either immortalized cell lines or mouse embryonic fibroblasts. there is a lack of data on the role of these innate sensing receptors in highly pathogenic avian influenza h5n1 infection in primary human cells in vitro, which are more physiologically relevant. furthermore, there is little data on the autocrine and paracrine up-regulation of these innate immune sensors following virus infection. human macrophages were obtained from peripheral blood monocytes by adhesion and differentiation in vitro for 14 days in rpmi medium supplemented with 10% autologous plasma. the cells were infected with h5n1 (483 ⁄ 97) or seasonal h1n1 (54 ⁄ 98) viruses at a moi of 2ae0. a549 cells were obtained from atcc and cultured in mem medium supplemented with 10% fcs and 1% penicillin and streptomycin. the role of innate sensing receptors in cytokine induction by h5n1 and h1n1 viruses was investigated using transient knock down of these receptors with sirnas in human macrophages as previously described 2 using specific sirnas purchased from qiagen. immunofluorescence staining assay of irf3 and nf-jb was employed to detect the nuclear translocation of these transcription factors after h5n1 infection. rabbit polyclonal antibodies against human irf3 and and nf-kb were obtained from santa cruz biotechnology. goat anti-rabbit igg antibody conjugated with alexa fluor 488 was a product of molecular probes. for investigation of paracrine effects on rig-i and tlr3 expression, culture supernatants collected from mock, 54 ⁄ 98 or 483 ⁄ 97 infected human macrophages were used to treat uninfected cells. the supernatants were first passed through a filter with 100-kda cut-off. virus particles as well as molecules with a molecular weight higher than 100 kda were retained and removed, while the filtrate was collected for treatment of uninfected cells. the expression of innate sensing receptors in infected cells and in adjacent uninfected cells following paracrine activation by virus free supernatants of infected cells was monitored by real-time pcr. the involvement of jak signaling pathways in these paracrine cascades was investigated using a jak inhibitor (calbiochem). we previously showed that tnf-alpha, ifn-beta, and ifnlambda 1 are the key mediators directly induced by the h5n1 virus in primary human macrophages with others being induced as part of a secondary autocrine and paracrine cascade. 2 in this study, we demonstrate that knockdown of rig-i or tlr3 led to the reduction of ifn-beta and tnf-alpha in human macrophages by both 54 ⁄ 98 (h1n1) and 483 ⁄ 97 (h5n1) infection. as shown in figure 1a , 483 ⁄ 97 virus induced higher level of ifn-beta mrna expression than 54 ⁄ 98 infection. cells transfected with rig-i or tlr3 sirna significantly reduced the expression of ifn-beta after 483 ⁄ 97 infection, by 63% and 29%, respectively. rig-i silencing also significantly reduced the ifn-beta expression in 54 ⁄ 98 infected cells by 52%. in contrast, silencing of mda5 or tlr8 did not suppress the induction of ifn-beta by either 54 ⁄ 98 or 483 ⁄ 97 infection; in fact, there was a slight (15%) increase of ifn-beta in cells transfected with mda5 sirna. based on these results we conclude that while both rig-i and tlr3 contribute to h5n1-induced interferon-beta induction in human macrophages, rig-i plays the dominant role. in order to investigate the relationship between these innate sensing receptors and the activation of transcription factors irf3 and nf-jb, we next measured the nuclear translocation of irf3 and nf-jb in cells with rig-i or tlr3 silencing after h5n1 infection. immunofluorescence staining assay on irf3 and nf-jb was performed and the number of cells with nuclear translocation was quantitated. the percentages of cells with nuclear translocation were plotted in figure 1b . we demonstrated that rig-i knockdown led to a significant reduction of irf3 nuclear translocation after 483 ⁄ 97 infection, whereas the nuclear translocation of nf-jb after 483 ⁄ 97 infection was significantly suppressed by rig-i or tlr3 silencing. these results suggest that the involvement of rig-i and tlr3 in the cytokine induction by 483 ⁄ 97 was via the regulation of irf3 and nf-jb nuclear translocation. since rig-i and tlr3 are important in influenza a virus-induced cytokine expression, we next explored the expression of these innate receptors in neighboring uninfected human macrophages by treating the uninfected macrophages with the filtered culture supernatants collected from mock, 54 ⁄ 98, or 483 ⁄ 97 infected macrophages. as shown in figure 2a , 483 ⁄ 97 supernatant differentially induced the mrna expression of rig-i, mda5, and tlr3 compared to 54 ⁄ 98 supernatant treated human macrophages. the induction of rig-i was higher than the induction of mda5 and tlr3. in the presence of 1 lm of jak inhibitor, the up-regulation of all three innate sensing receptors was significantly reduced showing their induction was dependent on jak activity. human lung epithelial a549 cells were also treated with the supernatants collected from macrophages infected with mock, 54 ⁄ 98, or 483 ⁄ 97 virus. differential induction of rig-i, mda5 and tlr3 by 483 ⁄ 97 supernatant compared to 54 ⁄ 98 supernatant treated cells was observed (figure 2b) . 483 ⁄ 97 supernatant dramatically induced all three innate sensing receptors, while 54 ⁄ 98 supernatant only marginally induced rig-i and mda5, but not tlr3. as in human macrophages, treatment with 1 lm of jak inhibitor caused a significant suppression of 483 ⁄ 97 supernatantinduced rig-i, mda5, and tlr3 expression in a549 cells. these results, taken together with the direct effects on virus infected cells, suggest that paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the up-regulation of innate sensing receptors. h5n1 directly induced ifn-beta ( figure 1 ) and tnf-alpha (data not shown) mainly via rig-i signaling and the consequent activation and nuclear translocation of irf3 and nf-kb in human macrophages. these results were consistent with a previous study using beas-2b cells showing the essential role of rig-i in ifn-beta reporter activity by h3n2 influenza virus infection. 4 while tlr3 also played a role in induction of ifn-beta and the activation of irf3 and nf-kb, it plays a less important role compared to rig-i. the reduction of irf3 and nf-kb activation was also confirmed with the study by le goffic 4 showing differential regulation of irf3 and nf-kb by rig-i and nf-kb can also be regulated by tlr3. in addition to the direct role of rig-i and tlr3 in sensing and signaling the presence of influenza virus, the innate immune sensing regulators were themselves also highly upregulated in both infected (data not shown) and adjacent uninfected cells by influenza virus infection. compared with seasonal h1n1 virus, the h5n1 viruses had a much more dramatic effect on inducing innate sensing receptors via jak signaling pathways activated by autocrine and paracrine mediators. the up-regulation of rig-i, mda5, and tlr3 was markedly induced by virus free culture supernatants from h5n1-infected macrophages, while supernatant from 54 ⁄ 98-infected cells induced the expression of these receptors only to a lesser degree. the soluble mediators in the virus infected cell supernatant caused paracrine upregulation of rig-i, mda5, and tlr3 in uninfected macrophages as well as human lung epithelial cells. these effects may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. taken together these results provide, at least, part of the explanation on the hyper-induction of cytokines in h5n1 infection. a more precise identification of the signaling pathways triggered by h5n1 virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h5n1 influenza and also for treating other causes of acute respiratory disease syndrome. we generated mutants of y55 (h9n2) and a ⁄ duck ⁄ hokkaido ⁄ vac generation and characterization of mutant viruses rgy55sub (h9n2), rgvac1sub (h5n1), and rgvac1ins (h5n1), which have a serial basic amino acid residues at their ha cleavage sites were generated by site-directedmutagenesis and reverse genetics. rgy55sub (h9n2) and rgvac1ins (h5n1) required trypsin to replicate in mdck cells, and showed similar levels of growth to their parental viruses (table 1) . chickens intravenously inoculated with rgy55sub (h9n2) or rgvac1ins (h5n1) did not show any signs of disease. rgvac1sub (h5n1) replicated in mdck cells without exogenous trypsin, and one of the eight chickens inoculated with the virus showed slight depression at 1 day post-infection. the h9 and h5 mutant viruses were serially passaged in the air sacs of chicks to assess their ability to acquire pathogenicity. plaque formation in mdck cells and pathogenicity in 3-day-old chicks and 4-week-old chickens are shown in table 1 . rgy55sub (h9n2) replicated in mdck cells in the absence of trypsin and killed all of the chicks after six consecutive passages. two of the eight-four-weekold chickens inoculated intravenously with rgy55sub-p8 (h9n2) died within 5 days. eventually, over 75% of the chickens intravenously infected with rgy55sub-p10 (h9n2) died by 2 days post inoculation, and its pathogenicity was comparable to that of hpaivs. 10 rgvac1sub-p1 (h5n1) was pathogenic to both chicks and 4-week-old chickens, and mortality increased after one more passage. rgvac1ins-p1 (h5n1) replicated in mdck cells in the absence of trypsin, killed all of the chicks, and caused 75% mortality among 4-week-old chickens. the lethal effect of rgvac1ins-p1 (h5n1) on chickens increased with one additional passage in the air sacs of chicks, as in the case of rgvac1sub (h5n1). to examine whether the pathogenicity of each virus via the natural route of infection correlated with that by intravenous infection or not, three 4-week-old chickens were challenged intranasally with the viruses at an eid 50 of 10 6ae5 and observed for clinical signs until day 14 post-infection (data not shown). all chickens inoculated with rgy55sub-p10 (h9n2) or its parental viruses survived without showing any clinical signs, and serum antibody responses were detected in the hi test. on the other hand, rgvac1sub-p2 (h5n1) and rgvac1ins-p2 (h5n1) were pathogenic as in the intravenous experiment, killing two of three chickens by day 11 post-inoculation. one of three chickens were not infected with rgvac1sub-p2 (h5n1) or rgvac1ins-p2 (h5n1) via intranasal route (data not shown), indicating these p2 viruses had not been completely adapted to the host. to investigate the possibility of these p2 viruses to acquire further pathogenicity for chicken, rgvac1sub-p3 (h5n1) and rgvac1ins-p3 (h5n1) were obtained from the brain homogenates of the chickens that died on 11 days post intranasal inoculation with the p2 viruses. although mortality rate of chickens inoculated with the p3 viruses was equal to that with p2 viruses, enhancement of pathogenicity was observed in intranasal inoculation study; all of the chickens inoculated with rgvac1sub-p3 (h5n1) were infected, and time to death was shortened to 4-6 days post inoculation in chickens with rgvac1ins-p3 (h5n1) (data not shown). to investigate whether tissue tropism of the viruses was involved in their pathogenicity, we determined viral titers in the tissue and blood samples from 4-week-old chickens intranasally inoculated with each virus on 3 days post infection ( table 2) . rgy55 (h9n2) and rgvac1 (h5n1) were scarcely recovered from the samples, and the mutant strains before passage showed broader tissue tropism than the parental viruses. none of the chickens inoculated with rgy55sub-p10 (h9n2) showed any signs of disease, and viruses were recovered from each of the samples except the brain and the blood. one chicken inoculated with rgvac1sub-p2 (h5n1) showed clinical signs such as depression, and viruses were recovered from virtually all of its organs and blood samples. two of three chickens inoculated with rgvac1ins-p2 (h5n1) showed disease signs, and one died 2 days post inoculation. the viruses were recovered from almost all samples of the two chickens showing signs of disease. p3 viruses were efficiently replicated in systemic organs of the chickens as compared with p2 viruses. throughout the study, the viruses were recovered from the brains of all of the chickens showing clinical signs. here, we demonstrated that the h9 influenza virus acquired intravenous pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the ha and passaged in chicks. rgy55sub-p10 (h9n2) killed 75% of chickens infected intravenously, and its pathogenicity was comparable to that of hpaivs (table 1) . however, chickens intranasally inoculated with rgy55sub-p10 (h9n2) did not show any clinical signs of disease (data not shown). these results are consistent with a previous study in chickens that found some h10 influenza viruses did not show intranasal pathogenicity although their intravenous pathogenicity index was over 1ae2, classified as hpaiv according to the definition by european union. 11 ohuchi et al. 12 reported that the insertion of additional basic amino acids into the h3 ha cleavage site resulted in intracellular proteolytic cleavage. other groups reported that h3 and h6 has tolerated amino acid mutations into their cleavage sites, and the viruses with the mutated has replicated in mdck and ⁄ or qt6 cells in the absence of trypsin. 13, 14 the results in the present study is in agreement with these, namely, cleavage-based activation by a ubiquitous protease is not restricted to the h5 and h7 has. the intranasal pathogenicity of the h9 and h5 mutants were different (data not shown), although these viruses similarly replicated in mdck cells in the absence of trypsin and killed chickens by intravenous inoculation ( table 1) . the viruses were recovered from the brain and the blood of some chickens infected with rgvac1 mutants (h5n1), and morbidity was closely associated with viral titers in the brain (table 2) . on the other hand, no viruses were recovered from the brain of chickens infected with rgy55 mutants (h9n2), explaining why rgy55sub-p10 (h9n2) did not show intranasal pathogenicity. all the viruses passaged in the air sacs of chicks killed chicken embryos by 48 hours post allantoic inoculation (data not shown). rgvac1sub-p3 (h5n1) and rgvac1ins-p3 (h5n1) were more pathogenic to chicken embryos than rgy55sub-p10 (h9n2); the allantoic fluid obtained from the embryonated eggs inoculated with the h5 viruses passaged in air sacs was turbid. it has been reported that infection of a highly pathogenic h7 virus were strictly confined to endotherial cells in chicken embryos or chickens. 15, 16 therefore, it is suggested that endotheliotropism differed between the h9 and h5 viruses passaged in air sacs and affected their intranasal pathogenicity. taken together, it is assumed that rgvac1sub-p3 (h5n1) and rgvac1ins-p3 (h5n1) showed marked intranasal pathogenicity with high levels of viremia caused by replication in vascular endothelial cells, leading to invasion of the brain. in the intravenous experiment, rgy55sub-p10 (h9n2) easily reached systemic organs, including the brain hematogenously, replicated through the cleavage of ha by a ubiquitous protease, and then exerted its pathogenicity. further study including a pathological analysis is currently underway to test this hypothesis. for all hpai viruses of subtypes h5 and h7 known to date, the cleavage of ha occurs at the c-terminal r residue in the consensus multibasic motifs, such as r-x-k ⁄ r-r with r at position p4 and k-k ⁄ r-k ⁄ t-r with k at p4, and leads to a systemic infection. early studies demonstrated that the ubiquitously expressed furin and pcs are activating proteases of hpai viruses. 1 furin and pcs cleave the consensus multi-basic motif r-x-k ⁄ r ⁄ x-r with r at position p4. 2 however, replacement of p4 r by k and a nonbasic amino acid significantly suppresses the processing activities of furin and pcs. 2 most of the type ii transmembrane serine protease identified so far recognize a single r at position p1, but the newly isolated mspl and its transcript variant tmprss13 preferentially recognize paired basic residue, particularly r and k at position p4, at the cleavage site. [3] [4] [5] thus, mspl and tmprss13 can activate various bioactive polypeptides with multibasic residue motifs, including fusogenic viral envelope glycoproteins. the present study was designed to characterize the proteolytic processing of the hpai virus ha by mspl and tmprss13 in comparison with furin. hpai virus a ⁄ crow ⁄ kyoto ⁄ 53 ⁄ 2004 (h5n1) 6 was isolated from embryonated eggs inoculated with tracheal homogenates from dead crows. then, the mutant ha sequence was constructed by changing r residue to k residue (n'-rkkr-c' to n'-kkkr-c') at the ha cleavage site by sitedirected mutagenic pcr as described. 7 we used human cell line ecv304, which expresses mspl and tmprss13 at levels below detection, and established the cells stably expressing mspl and tmprss13, such as ecv304-mspl and ecv304-tmprss13. 7 to determine the cleavage specificities of mspl ⁄ tmprss13 and furin, peptides (20 lg each) were incubated with 0ae25 mu mspl ⁄ tmprss13 for 1 hour and furin for 8 hours at 37°c, respectively. after incubation, the samples were separated by reverse-phasehigh-performance liquid chromatography (rp-hplc) with the use of a c 18 column. the elution samples were then identified by amino acid sequence analysis and by maldi-tof-ms. we analyzed the cleavability of 14-residue synthetic peptides derived from ha cleavage sites of hpai strains, such as a ⁄ chick ⁄ penn ⁄ 1370 ⁄ 83 (h5n2) 8 and a ⁄ fpv ⁄ rostock ⁄ 34 (h7n1), 9 and low pathogenic strain a ⁄ aich ⁄ 2 ⁄ 58 (h3n2). after incubation with human mspl or human furin, the digested samples were separated by rp-hplc, and peptide fragments were characterized by mass-spectrometry and protein sequencing. in contrast to the low cleavage efficiencies of the h3 ha peptide with a single r at the cleavage site ( figure 1a) , both the h5 ha peptide with the k-k-k-r motif ( figure 1b ) and the h7 ha peptide with the r-k-k-r motif ( figure 1c) were fully processed at the correct positions by mspl within 1 hour. in the case of h7 ha peptide with multiple basic residues, mspl cleaved two carboxyl-terminal sides of r in the cleavage site sequence of n'-k-k-rfl-k-k-rfl-g-c', while furin cleaved only at a single site of r with r at position p4, n'-k-k-r-k-k-rfl-g-c' in the presence of 1 mm cacl 2 . these cleavage site specificities of furin were consistent with that reported for the h5 ha peptide of hpai virus a ⁄ hong kong ⁄ 156 ⁄ 97 (h5n1) with r-k-k-r motif. 1 however, the h5 ha peptide with k at position p4 ( figure 1b) was hardly cleaved by furin under the same experimental conditions. tmprss13 showed similar results (data not shown). these findings suggest that mspl and tmprss13 cover diverse cleavage specificities, including non-susceptible specificity to furin. full length recombinant ha of hpai virus with kkkr cleavage motif was converted to mature ha subunits with membrane-fused giant cell formation in mspl or tmprss13 transfectant cells. 7 in addition, this conversion was suppressed by bowman-birk trypsin inhibitor, a membrane non-permeable highmolecular mass inhibitor against mspl ⁄ tmprss13. to test for the generation of infective virus, the conditioned media of 1-day culture of ecv304-wt and ecv304-mspl cells infected with wt and mutant hpai h5n1 viruses were inoculated into newly prepared cells and cultured for 24 hours. although spreading of wt virus infection with ha cleavage motif of r-k-k-r was detected from the conditioned medium of both ecv304-wt and ecv304-mspl cells, that of mutant virus with ha cleavage motif of k-k-k-r was only detected from the condition medium of ecv304-mspl cells. these results strongly suggest that the expression of mspl, but not furin, potentiates multicycles of hpai virus with k-k-k-r ha cleavage motif. seasonal human influenza a virus has have consensus monobasic cleavage site sequence, n'-q ⁄ e-x-rfl-g-c', and all hpai virus has have two types of cleavage site sequences with multiple basic amino acids, n'-r-k ⁄ r-k ⁄ r ⁄ x-rfl-g-c' with r at position p4 in a large number of hpai viruses and n'-k-k ⁄ r-k ⁄ t-rfl-g-c' with k at position p4 in a small number of hpai viruses. figure 1 shows furin efficiently cleaved synthetic hpai a ⁄ hong kong ⁄ 156 ⁄ 97 (h5n1) ha cleavage site peptide with the r-k-k-r motif, but hardly cleaved the hpai virus a ⁄ chick ⁄ penn ⁄ 1370 ⁄ 83 ha cleavage site peptide with the k-k-k-r motif. furthermore, cleavage of the full-length ha of hpai virus with r-k-k-r motif was detected, but cleavage of hpai virus ha with k-k-k-r motif was hardly detected in ecv304-wt cells containing furin ( figure 2 ). these substrate specificities of furin suggest that proteases other than furin and pc5 ⁄ 6 play a role in the processing of has of hpai virus with k-k ⁄ r-k ⁄ t-r cleavage motif. mspl and tmprss13 show unique cleavage site specificities of the double basic residues at the cleavage site, and r or k at position p4 greatly enhanced the efficiency, which none of the other ttsps have shown similar substrate specificities so far. furthermore, infectious and multicycle viral replication along with ha processing was also noted in genetically modified mutant recombinant live hpai virus a ⁄ crow ⁄ kyoto ⁄ 53 ⁄ 2004 (h5n1) with k-k-k-r cleavage motif in ecv304-mspl cells (figure 2) . these results were supported by the data of two cleaved peptides by mspl in figure 1c . these findings suggest that mspl has diverse cleavage specificities and may cleave ha at least two sites, although multiplicity of the mutant hpai virus was observed under the conditions. these results also suggest that mspl and tmprss13 in the membrane might potently activate the ha membrane fusion activity of hpai viruses and promote their spread. highly pathogenic avian influenza viruses replicate in various organs in birds, and the ha processing proteases might be widely distributed in these organs. indeed, tmprss13 and mspl are ubiquitously expressed in almost all human organs tested and are highly expressed in lungs, leukocytes, pancreas, spleen, and placenta. 4, 5 in addition, mspl and tmprss13 are strictly localized in the plasma membranes, suggesting that proteolytic activation of hpai virus ha occurs not only through the trans-golgi network by furin and pc5 ⁄ 6, but also on the cell surface by mspl and tmprss13. the pb1-f2 protein, which is translated from the +1 reading frame of the pb1 gene segment, has been linked to the pathogenesis of both primary viral and secondary bacterial infections in a mouse model. 1-3 a mitochondrial targeting sequence is located in the c-terminal portion of the pb1-f2 open reading frame, and expression of full length pb1-f2 has been associated with mitochondrial targeting and apoptosis in a monocyte dependent manner. 1, 4 it has been theorized that enhanced virulence could result from mitochondrial disruption with subsequent cell death mediated by pb1-f2. 4, 5 a suggested second function of the pb1-f2 protein is that it enhances immunopathology by triggering the inflammatory response. 3, 6 in earlier studies from our group, the pro-inflammatory phenotype was markedly upregulated when the pb1-f2 from the 1918 pandemic strain was expressed, arguing that this protein may be an important virulence factor for highly pathogenic pandemic viruses. 3, 6 in this report we analyze the pb1-f2 protein's contribution to pathogenesis in a mouse model, examining both inflammation and cell death. pb1-f2 proteins from a variety of epidemiologically important iav strains including all pandemic strains from the 20th century, a highly pathogenic avian influenza virus of the h5n1 subtype, and representative seasonal strains were utilized to determine the relevance to pandemic disease. we demonstrate that macrophage mediated immunopathology, but not apoptosis, are relevant functions of pb1-f2 proteins from past or potential pandemic influenza viruses. using the predicted amino acid sequences of the pb1-f2 proteins from pr8, a ⁄ brevig mission ⁄ 1 ⁄ 1918, 1 ⁄ singapore ⁄ 1 ⁄ 1957, a ⁄ hong kong ⁄ 1 ⁄ 1968, a ⁄ wuhan ⁄ 359 ⁄ 1995, and a ⁄ vietnam ⁄ 1203 ⁄ 2004, peptides from the c-terminal end were synthesized as described. 3 an additional n-terminal peptide was synthesized from the pr8 sequence as a positive control (mgqeqdtpwilstghistqk) as described. 3 a panel of viruses were reverse engineered as described 7, 8 and included laboratory strain pr8, a virus unable to express pb1-f2 (dpb1-f2 ⁄ pr8), or expressing the pb1-f2 of the 1918 pandemic strain (1918 pb1-f2 ⁄ pr8) or the truncated 1956 h1n1 strain (beij pb1-f2 ⁄ pr8). 3, 7 in addition, 7:1 reassortants encoding pb1 gene segments from a *current address: department of immunology and microbiology, university of melbourne, melbourne, vic., australia. 2004 highly pathogenic avian influenza of the h5n1 subtype (h5n1 pb1 ⁄ pr8), or from a 1995 human h3n2 strain (h3n2 pb1 ⁄ pr8) were utilized along with their isogenic deletion mutants for pb1-f2 (h5n1 dpb1-f2 ⁄ pr8 and h3n2 dpb1-f2 ⁄ pr8). cell lines and cell death assays raw264.7 cells were grown under conditions as described. 9 cells were infected with one multiplicity of infection (moi) of virus for 2-12 hours, or exposed to 50 lm (final concentration) of peptides derived from the c-terminal portion of pb1-f2 for 1 hour. cells from the supernatant and monolayers were harvested, washed, and stained with annexin (apc) and propidium iodide (pi) (becton dickinson, san jose, ca, usa), then analysed for cell death as described. 3 six-to eight week old female balb ⁄ cj mice (jackson laboratory, bar harbor, me, usa) were maintained in a biosafety level 2 facility in the animal resource center and procedures approved by the animal care and use committee at sjcrh. infectious agents and peptides were diluted in sterile pbs and administered intranasally to anesthetized mice (n = 6-10) in a volume of 100 ll (50 ll per nare) and monitored for overt signs of illness and weight loss daily. following euthanasia by co 2 inhalation, the trachea was exposed and cannulated with a 21 gauge plastic catheter (bd insyte; becton dickinson, sandy, ut, usa). bronchoalveolar lavage fluid (balf) was collected, red blood cell depleted, and cellular content analyzed via flow cytometry as described. 3 one way analysis of variance (anova) was used for multiple comparisons of cell death and cellularity of balf. a p-value of <0ae05 was considered significant for these comparisons. graphpad prism version 5.00 for windows (graphpad software, san diego, ca, usa) was utilized for all statistical analyses. to assess the contribution of pb1-f2 to inflammation, we utilized a panel of previously described reverse engineered viruses in the mouse infection model. 3, 7 the effect of pb1-f2 expression was observed clearly in the inflammatory infiltrate in response to infection in the lungs. deleting pb1-f2 from pr8 or expression of the c-terminally truncated beij pb1-f2 had a significantly reduced influx of macrophages ( figure 1a) . expression of the 1918 pb1-f2 caused similar inflammatory effects as the pr8 virus. disruption of pb1-f2 expression the virus containing the h5n1 pb1 gene segment in a pr8 background also significantly decreased the inflammatory response compared to the virus maintaining the ability to express full length pb1-f2 ( figure 1a) . however, no differences were seen that could be attributed to the 1995 h3n2 derived pb1-f2. the lungs of mice infected with the panel of pb1-f2 variant viruses were examined at 72 hours. pathologic changes typical of pr8 viral infection were observed in all lungs. these typical findings included perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris (figure 2 ). in the lungs of mice infected with pr8 or 1918 pb1-f2 ⁄ pr8, however, significantly more perivascular cuffing was noted, with a prominent increase in numbers of macrophages (figure 2a, c) . the overall number of inflammatory cells throughout the lungs, including both airways and alveoli, was quantitatively greater in these mice than in mice infected with dpb1-f2 ⁄ pr8 or beij pb1-f2 ⁄ pr8 ( figure 2b, d) . as the function and influence of pb1-f2 protein on normal viral function is not currently understood, and given the abrogation of enhanced inflammation induced by the truncated pb1-f2 beij ⁄ pr8 virus, we sought to elucidate whether the c-terminal domain of pb1-f2 could alone induce this inflammatory response. mice were exposed to a panel of peptides and were euthanized 24 hours later for collection of balf. significant influxes of macrophages into the balf were seen following exposure to c-terminal pb1-f2 peptides derived from pr8, the pandemic strains from 1918 (h1n1), 1957 (h2n2), and 1968 (h3n2), and the 2004 h5n1 virus compared to controls ( figure 1b) . similar effects were not seen with the peptide derived from a more recent h3n2 strain, a ⁄ wuhan ⁄ 359 ⁄ 1995. when peptide exposed mice were followed for morbidity for 7 days, peptides proven to induce a heightened inflammatory response correlated strongly with overt clinical signs of illness (data not shown). thus, the ability to cause lung inflammation appears to be a property of pb1-f2 proteins of viruses containing pb1 gene segments reassorted directly from the avian reservoir. the pb1-f2 protein may contribute to virulence by rendering the host cellular immune response ineffective through inducing apoptosis. 5 we sought to determine whether this was an epidemiologically important function for combating the host immune response to infection by testing the ability of pb1-f2 proteins from several different iav strains to cause cell death. we therefore infected raw264.7 cells with the panel of recombinant viruses at an moi of 1 for 2-12 hours. as has been demonstrated previously, 1,4,5 pr8 virus induces significant cell death compared to uninfected controls ( figure 1c ). when raw264.7 cells were infected with pr8 virus, necrotic death peaked 8 hours after infection. viruses lacking the c-terminal portion of pb1-f2, including the dpb1-f2 ⁄ pr8 and the beij pb1-f2 ⁄ pr8 were unable to cause cell death ( figure 1c ). in addition, expression of the 1918 pb1-f2 also did not cause significant increases in cell death over controls. expression of pb1-f2 or deletion of pb1-f2 in either an h3n2 or h5n1 pb1 gene segment background similarly did not alter the cell death phenotype. to examine additional strains for which we did not have isogenic virus pairs, we next exposed the balbcj mouse derived macrophage cell line raw264.7 to the panel of pb1-f2 peptides derived from pr8, the pandemic strains from 1918 (h1n1), 1957 (h2n2) and 1968 (h3n2), and the 2004 h5n1 for 1 hours. cell death in raw264.7 cells was caused only by the peptides derived from the laboratory strain pr8 and the peptide derived from the 1918 pandemic strain ( figure 1d ). viability was not affected by exposure of raw264.7 to peptides derived from other virus strains. we conclude from these data that the mechanism by which pb1-f2 contributes to the pathogenicity of pandemic influenza is unlikely to be through its reported ability to cause cell death. these data presented here demonstrate that the lung inflammatory response is enhanced by the influenza a virus pb1-f2 protein in a mouse model. this inflammatory response was characterized by increased cellular infiltration of macrophages into the interstitial and alveolar spaces of the lungs, as well as enhanced perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris. this augmentation was shown to be induced by pb1-f2 proteins only from those strains contributing to the formation of all pandemic strains of the 20th century and from the currently circulating, highly virulent h5n1 strains that constitute an imminent pandemic threat. the iav h1n1 strains circulating in humans since around 1950 code for a truncated pb1-f2. these viruses may lack the cterminal residues responsible for the inflammatory effects demonstrated in this publication. additionally, recently circulating h3n2 strains, in contrast to their pandemic forbear from 1968, have lost the capacity to cause pb1-f2 mediated inflammation through mutation of the c-terminus of this protein. in 2009 a novel h1n1 iav emerged from an animal reservoir and caused a human pandemic. disease burden from this strain has been considered mild 10 in contrast to the three pandemics of the 20th century. 11 the reasons for this disparity in pathogenesis are unclear. an examination of the origins of the three 20th century pandemics shows that only 2 the hemagglutinin (ha) and pb1 gene segments were reassorted directly from the avian reservoir in every case, suggesting gene products of one or both of these may be important. 12 the ha surface glycoprotein provided the antigenic novelty required for the each virus to achieve pandemic status. however, the significance of inclusion of a novel pb1 gene segment in each of the 20th century pandemics is not yet understood. we show here that the pb1-f2 of these pandemic strains contributes to virulence through induction of inflammatory responses. thus pb1-f2 may serve as a marker of the pathogenicity of pandemic strains. since the 2009 h1n1 strain codes a truncated pb1-f2 of only 11 predicted amino acids, the lack of pb1-f2 mediated inflammation may account in part for its relatively lower virulence. 13, 14 of the panel of pb1-f2 proteins studied, only that from the laboratory strain pr8 was capable of rendering responding host-immune cells ineffective by induction of cell death. we therefore hypothesize that molecular signatures specific to induction of apoptosis may have been lost through genetic mutation of the pb1-f2 gene throughout the evolution of the iavs. our findings suggest that this apoptotic function is unlikely to be important for the virulence of any of the known pandemics. rather, the inflammatory phenotype appears to be the dominant contribution of pb1-f2 to pandemic disease. influenza virus-cytokine-protease cycles are principal mechanisms of multi-organ failure in severe influenza and therapeutic approaches introduction influenza a virus is the most common infectious pathogen in humans, causing significant morbidity and mortality, particularly in infants and the elderly. mof with severe edema is observed in the advanced stage of influenza pneumonia. 1 however, the relationships amongst factors that induce vascular hyper-permeability in severe influenza remain unclear. it is reported that significant increases in levels of pro-inflammatory cytokine levels, such as tnf-a, il-6, and il-1b, affect host survival both positively and negatively. 2 the inflammatory response affects cell adhesion, permeability, apoptosis, and mitochondrial reactive oxygen species, potentially resulting in vascular dysfunction and mof. 3 in addition, iav infection up-regulates several cellular proteases including ectopic trypsin 4 and mmp-9. 5 up-regulated ectopic trypsin mediates the post-translational proteolytic cleavage of viral envelope hemagglutinin (ha), 6 which is crucial for viral entry and replication 7 and the subsequent tissue damage in various organs. 8 the aim of the this study was to define the pathogenic impact of cytokine storm in iav infection and the molecular mechanisms by which pro-inflammatory cytokines and proteases cause vascular dysfunction in animal model. weanling female mice aged 3 weeks (c57bl ⁄ 6crslc) were infected with iav ⁄ wsn ⁄ 33 (250 pfu) with and without treatment of pdtc (2.5 mg ⁄ kg), nac (10 mg ⁄ kg), and ndga (10 mg ⁄ kg). these inhibitors were administrated once daily for 4 days after infection. the levels of cytokines in tissue homogenates were measured by elisa kits. the effect of inhibitors on viral replications was determined by real-time pcr. gelatin zymography and western blotting were conducted as reported previously. 9 host cellular responses in the airway after iav infection figure 1 shows schematic view of typical biological responses in the airway of mice after iav infection. an initial response before viral proliferation is significant increases in pro-inflammatory cytokine levels. immediately after cytokine inductions, there is a marked up-regulation of ectopic trypsin along with an increase in virus titer in the airway, lung, and brain. 4 ectopic trypsin mediates the post-translational proteolytic cleavage of iav ha, which is crucial for viral entry and replication and the subsequent tissue damage in various organs. we also found that iav infection markedly induces mmp-9 and matrix degradation. 5 just after the peak of viral proliferation, the innate and adaptive immune responses of protective immunity are induced for defense and recovery, or oppositely on rare occasions, mof with vascular hyper-permeability is started into the advanced stage of influenza. the levels of tnf-a and il-6 in the lungs were increased persistently for 6 days after iav wsn infection, and that of il-1b peaked at days 4-6 post-infection (figure 2a ). since these cytokine responses are associated with activation of nf-jb and ap-1, we treated mice once daily for 4 days with anti-oxidant inhibitors: pdtc and nac against nf-jb activation, and ndga against ap-1 activation. pdtc and ndga significantly suppressed the up-regulation of tnf-a and il-1b (p < 0.001), and nac suppressed tnf-a (p < 0.001), and il-6 (p < 0.01) at day 4 post-infection. gelatin zymography showed up-regulation of ectopic trypsin and mmp-9 in mice lung, brain, and heart during infection for 4 days ( figure 2b ). trypsin and mmp-9 induction was inhibited by treatment with pdtc, nac, and ndga, probably via blockade of nf-jb and ap-1 binding in the promoter region of the genes. viral rna replication in various organs at day 4 post-infection was suppressed by more than one order of magnitude by pdtc, nac, and ndga ( figure 2c ). suppression of viral multiplication and induction of cellular factors by pdtc, nac, and ndga, significantly improved the survival of mice at day 14 post-infection, the late stage of infection ( figure 2d ). to elucidate the mechanisms underlying brain vascular dysfunction of influenza-associated encephalopathy, changes in the levels of tight-junction proteins, intracellular zonula occludens-1 (zo-1) and transmembrane occludin, and the matrix protein laminin, were analyzed by western blotting. marked reductions in the expression levels of tight-junction constituents were detected at day 4 post-infection, which were partly rescued by pdtc, nac, or ndga (figure 2e ). 9 no other tight-junction protein, claudin-5 or matrix fibronectin and type iv collagen, were affected. the present study reports several new observations: (i) proinflammatory cytokines, tnf-a, il-1b, and il-6, when up-regulated by iav infection, induce trypsin and mmp-9 expression in various organs in mice; (ii) inhibitors of nf-jb and ap-1 effectively suppress the up-regulation of proinflammatory cytokines, trypsin, and mmp-9 and improve survival rates of infected mice. based on these results, we propose the 'influenza virus-cytokine-protease cycle' hypothesis as one of the mechanisms of vascular dysfunction in mof with cytokine storm in severe influenza and influenza-associated encephalopathy. 9 the significance of pro-inflammatory hyper-cytokinemia, or 'cytokine storm,' in the pathogenesis of iav infection remains unclear. on the positive effects, cytokines promote lymphocyte activation and infiltration at the sites of infection and exert direct antiviral effects. however, on the negative effects of excess cytokines, the hyper inflammatory process evoked by viral infection may become harmful through intracellular activation of nf-jb, ap-1, and the janus kinase-signal transducers and activators of transcription signaling pathways. 3, [10] [11] [12] the in vivo experiments presented here showed that nf-jb and ap-1 inhibitors markedly suppress the expression of cytokines, trypsin, mmp-9, and viral replication, resulting in a significant increase in the survival of infected mice. furthermore, cytokines interact with mitochondria to increase the production of reactive oxygen species, resulting in the production ⁄ activation of vasodilatory mediators such as nitric oxide and bradykinin, and subsequent endothelial dysfunction and edema in various organs. 3 the molecular mechanisms underlying tight-junction disruption in endothelial cells and vascular hyper-permeability following the 'cytokine storm' remain unclear. tnfa up-regulation alters the cellular redox state, reduces the expression of four complex i subunits by increasing mitochondrial o 2 ) production and depleting atp synthesis, decreases oxygen consumption thereby resulting in mitochondrial damage, 3, 13 and increases [ca 2+ ] i 14 atp depletion dissociates zo-1 from the actin cytoskeleton and thereby increases junctional permeability. 15 endothelial dysfunction induced by 'influenza virus-cytokine-protease cycle' in the early stage of severe influenza may further affect various circulating factors, coagulation factors and complement systems, and vascular interacting cells, such as neutrophils, macrophages and lymphocytes. mof is the final outcome of metabolic and mitochondrial fuel disorder, immunosuppression, endocrine disorder, and tissue injury followed by endothelial dysfunction in many organs. another key pathway of acute lung injury in the highly pathogenic avian influenza virus h5n1and acute respiratory syndrome-corona virus infection reported recently involves oxidative stress and formation of oxidized phospholipids, which induce lung injury via toll-like receptor 4 signaling pathway. 16 in addition to these data, up-regulated trypsin and pro-inflammatory cytokines may also affect tissue destruction and immunosuppression in the late stage of iav infection. further studies are required on the role of the 'influenza virus-cytokine-protease cycle' in the pathogenesis of mof, particularly in the late stage of viral infection. though influenza a virus replication kinetics and host responses have been previously studied in umbilical vein endothelial cell or transformed endothelial cell lines, the tropism of influenza a virus including h5n1 and pandemic h1n1pdm for primary human lung microvascular endothelial cell has not been well defined. 1 in this study we employed primary human lung microvascular endothelial cells, which are more physiologically relevant for understanding pathogenesis of influenza in the lung as to obtain a better understanding of the links of endothelial cell infection to systematic virus dissemination and multiple organ involvement in severe human influenza. supernatants of cells infected at moi of two were collected for cytokine protein assays, and total rna was extracted for gene expression analysis using qpcr. we found that seasonal influenza h1n1 and h3n2 viruses initiated viral gene transcription and viral protein expression, but did not produce infectious progeny, while the highly pathogenic avian influenza h5n1 and the pandemic influenza h1n1pdm virus could replicate even with the absence of exogenous protease (figure 1) . furthermore, when compared to seasonal h1n1 and h3n2, the h5n1 virus was a more potent inducer of cytokine and chemokine including ifn-b, mcp-1, rantes, ip-10 (figure 2) , and il-6, in virus infected endothelial cells, whereas h1n1pdm induced intermediate levels of cytokine and chemokine. avian influenza h5n1 and pandemic h1n1pdm virus (but not the seasonal h1n1 and h3n2 virus) can productively replicate in human lung microvascular endothelial cells. this is likely to be of relevant to pathogenesis and provides a possible explanation for the extra-pulmonary infection seen in animal infection models. this extra-pulmonary spread may support the previous speculation and anecdotal evidence that h5n1 and h1n1pdm virus can infect the gastrointestinal tract through the virus dissemination from the infected respiratory tract as the first target cells for influenza infection. [2] [3] [4] in addition, the release of proinflammatory cytokine and chemokine induced by influenza h5n1 and h1n1pdm virus infection in lung microvascular endothelial cells may be important contributors to the pathogenesis of severe human influenza disease leading to endothelial cell dysfunction that contributes to severe pulmonary disease symptoms. during its replication, influenza virus utilizes the host cellular machinery for many aspects of its life cycle. characterization of such virus-host protein-protein interactions is a must to identify determinants of pathogenesis. the m2 ion channel protein plays a crucial role during the entry and late stages of the viral life cycle where its c-terminal domain, well conserved among influenza a viruses, is accessible to cellular machinery after fusion with endosomal membrane and during its trafficking along the secretory pathway prior to assembly and budding. 1 the aim of the study is to identify cellular interactants of m2 that play important regulatory roles during influenza infection. to identify cellular partners of m2 we performed a genome-wide yeast-two-hybrid (y2h) screening approach 2 using the cytosolic domain of m2 as bait and a human placenta random primed cdna library as prey and tested more than 60 million interactions. from the y2h screening, an interesting interaction with the human annexin a6 (anxa6) protein, 3 a member of annexin family proteins that binds to phospholipds in a ca 2+ -dependent manner, was identified. co-immunopre-cipitation of myc-tagged anxa6 and viral m2 proteins coexpressed in hek293t cells after transfection and infection confirmed the direct interaction between anxa6 and m2. we further investigated whether this interaction had any functional significance with regards to influenza life cycle. using a rna interference strategy to silence the anxa6 gene in human lung epithelial a549 cells, we observed increased progeny virus titers either in a single or multiple viral growth kinetics study, suggesting a negative regulatory role for anax6 during viral infection (figure 1 ). a novel interaction between m2 and anxa6 was identified. more functional studies are in progress to define precisely the potential negative regulatory role of this interaction during viral infection. a systematic dissection of the viral life cycle will be performed to identify the step(s) affected by the anxa6 cellular factor using specific assays such as real-time quantitative rt-pcr in a single or multiple viral growth kinetics study, cell transduction with ha-and m2-pseudotyped lentiviral particles, virion attachment and internalization assay, immunofluorescence staining of np protein as a marker of viral ribonucleoproteins localization, viral polymerase activity measurement, and viral budding observation by electron microscopy. rna extraction was achieved by qiagen biorobot ez1 prior to respiratory multiplex pcr analysis. what remained of the extracted material of each specimen was stored by refrigeration at 4°c. electronic patient records were searched for parameters, such as c-reactive protein (crp), white cell count (wcc), length of admission in days, and patient co-morbidities. patients were divided into three groups according to clinical severity: mild, moderate, and severe. the 'mild' group comprised of those admitted for three days or fewer, or not admitted at all. the 'moderate' group comprised those who required admission to hospital for more than 3 days as a result of swine flu, but who did not require admission to an intensive care unit (itu). the 'severe' group comprised those who had required itu admission. invitrogen '2· reaction mix': 0ae4 mm of each dntp + 6 mm magnesium sulphate. primer ⁄ probe mix recipe applied biosystems 7500 fast real-time pcr system, 'respiratory multiplex' program. well content 25 ll; thermocycler initial stage 50ae0°c for 15 minutes, then 95°c for 2 minutes. subsequent cycles of 95ae0°c for 15 seconds followed by 60°c for 33 seconds for 45 cycles. sequence detection software version 1.4 (applied biosystems). of 126 clinical isolates analyzed, all samples produced amplification of pdh material; 100 produced amplification of both swine flu and pdh material. human male dna (lot no. 36048611039 at 10 ng ⁄ l, applied biosystems) at concentration calculated at 16 666ae6 cells ⁄ ll was diluted from 10 )1 to 10 )4 , yielding mean average ct values of respectively 30ae10, 33ae60, 37ae78, and 37ae22. plotting log of cell number versus ct gave a y = mx + c line from which ct could be interpolated into cell numbers. for swine flu quantification, a sample of swine flu ct 19ae28 was diluted through 10 )1 to 10 )10 . it must be noted that due to variability in resultant swine flu ct values, repetitions at these dilutions were done using an rna carrier (1350 lg ⁄ l, qiagen; cat no. 1017794) in place of rnasefree water. the 10 )4 concentration was positive in nine out of 15 assays; this fraction was used in the calculation described by simmonds 2 to obtain a copy number of targets per reaction by the equation copy value = )ln(f), where f is decimal fraction of failure rate. here, f = 6 ⁄ 15 = 0ae4; )ln0ae4 = 0ae916 copies. a control curve was generated with ct values of 26ae73, 30ae02, 33ae67, and 37ae23 giving copy values of 916, 91ae6, 9ae16, and 0ae916, respectively. using excel (microsoft office, 2010), these control series were adapted into formulae to convert swine flu and pdh ct values into copy numbers of these elements per reaction. simple division derived a value for swine flu copy per pdh copy, but this was chosen to be expressed as swine flu copy number per 100 human cells. this will be referred to as the 'c' value. forty-two patients had known clinical details; average age was 29ae74, female to male ratio 63:37, and average admission length of 7 days. of the mild group (n = 26), nine cases were not admitted to hospital. of the remainder, the mean average admission length was 2ae5 days. mean average c value for all samples was 1ae49 · 10 6 , with a standard deviation of 1ae49 · 10 7 ; geometric mean was 4ae28, and median average was 6ae45. log(mean average c value) is shown for each severity group and for identified risk factors in the 'mild' severity group (figures 2a, b respectively) . in each case variation was too great to yield statistical significance. figure 1 shows the range of c values observed in the 'moderate' severity group. > -4 · 0 ; < -4 · 5 > -3 · 5 ; < -4 · 0 > -3 · 0 ; < -3 · 5 > -2 · 5 ; < -3 · 0 > -2 · 0 ; < -2 · 5 > -1 · 5 ; < -2 · 0 > -1 · 0 ; < -1 · 5 > -0 · 5 ; < -1 · 0 > -0 · 0 ; < -0 · 5 > 0 · 0 ; < 0 · 5 > 0 · 5 ; < 1 · 0 > 1 · 0 ; < 1 · 5 > 1 · 5 ; < 2 · 0 > 2 · 0 ; < 2 · 5 > 2 · 5 ; < 3 · 0 in a study by duchamp et al., 3 no significant correlation was observed between viral ct value and presence or absence of cardiaorespiratory disease, myalgia, digestive symptoms, or upper or lower respiratory tract infection (although a trend was observed towards patients presenting with signs of upper respiratory tract infection). to our knowledge, no other study has used a dual pcr for analysis of respiratory virus concentrations, and no study has attempted to correlate biochemical markers with respiratory virus concentration. the data exhibited a spectrum of c values, from values <5 · 10 )5 to over 1 · 10 8 . the three severity group standard deviations all overlapped with each other, preventing statistical significance. analysis of co-morbidities showed a high mean average c value when asthma was present (6ae05 · 10 7 ), but again this was associated with an excessive standard deviation. whereas the median average c value in the presence of asthma was higher than the overall average c value (9ae09 versus 5ae85), it was significantly lower than the median c value when no co-morbidity was documented (15ae55). there are multiple caveats that may be the cause of such variety of c values obtained. the duration between initial rna extraction and study pcr had a range of 21 to 285 days, with mean average delay of 211 days. the degradation of viral rna is an important contributor to assay variance and failure; rna degradation in clinical samples has been studied. [4] [5] [6] degradation of human dna in clinical samples may have occurred. several studies have chartered degradation of stored human dna. 2, 7 with regards to sampling, the clinical collection of throat swabs is naturally variable according to the method of the collector. a small number of bronchoalveolar lavage samples were analyzed, yet did not amplify, presumably due to rna degradation. the upper respiratory tract may be only a physical stepping stone for the virus, and take no further role in pathogenesis of severe disease (although undoubtedly is crucial for transmission). interestingly, a ferret study of pathogenesis observed that swine flu yields from the upper respiratory tract were greater than those given by ordinary seasonal h1n1, with consequently increased shedding. 8 the review by mansfield 9 cites significant findings regarding influenza pathogenesis, including the predilection of h5n1 strains for type ii pneumocyte cells and alveolar macrophages. it also highlights the limitation of knowledge through dearth of human autopsy studies; an exception is the recognition of haematophagocytic syndrome in severe cases. it is known that specific immunoglobulin is effective against establishment of infection in the upper respiratory tract, whereas specific cytotoxic t lymphocytes (ctls) are necessary for clearance of the virus from the lower respiratory tract. 10 it is also suggestive that a gap of two whole days transpires between initial infection and instigation of a specific immune response. 11 it is plausible that in the healthy individual, virus progression is confounded by efficient natural mucosal immunity, in part through good secretory immunoglobulin levels. airway inflammation associated with asthma exacerbation is known to increase both risk of respiratory viral infection and poorer outcome. it is unproven but likely that the local inflammatory processes give rise to increased virion burdens in the upper airways; however, the same effect is conceivable for epithelial cell turnover. there will likely be variance within each clinical category due to patient circumstances and clinicians' judgment of required admission. unfortunately, the duration of symptoms prior to swab collection was often omitted in the clinical notes. finally, stratification of patient group by receipt of antiviral treatment was not studied. no correlations were observed with c values and crp, wcc or admission length. trends were observed towards higher c values in 'mild' cases, but without statistical significance. the relative small study size, coupled with the intrinsic variability of the parameters studied, warrants larger, better controlled, prospective studies to elucidate clinical use of the c value for influenza illness prediction and management. in mid-april 2009 a novel variant of a(h1n1) influenza virus began to spread rapidly throughout the world, causing the first pandemic of the 21st century. the majority of the cases associated with this new virus show to be mild, but severe and fatal cases have been reported. molecular markers associated with severity have already been identified, as is the case of the mutation d222g. 1 resistant viruses to antiviral drugs have also been identified, highlighting the importance of rapid determination of the antiviral drug profile. global a(h1n1) 2009 genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. the objective of this ongoing research study was, primarily, to thoroughly characterize the genetic profile and evolution of the emergent influenza a(h1n1) 2009 virus circulating in portugal and its phenotypic expression on antiviral drugs susceptibility. the cases considered in this study were obtained from the community and from two collaborating hospitals in lisbon -a reference hospital for adults (hospital de curry cabral) and a reference hospital for children (hospital dona estefânia). the cdc real-time pcr protocol, recommended by world health organization (who), was the method used to confirmed all influenza a(h1n1) 2009 cases. from a total of 577 a(h1n1) 2009 positive cases diagnosed and confirmed, 163 were selected for this study, taking in consideration that they should cover the period of epidemic activity in portugal and include cases from persons belonging to risk groups and cases associated with more severe clinical features. ninety-six a(h1n1) 2009 strains were isolated in mdck-siat1 cells, from combined naso-oropharyngeal swabs. for the evaluation of the genetic profile of a(h1n1) 2009 virus circulating in portugal, 37 of the 96 isolates were characterized by genetic analysis of the ha, na, and mp genes. the remaining five gene segments (pb1, pb2, pa, ns, and np) were also sequenced for six of this 37 isolates. briefly, sequencing was performed according to the protocol developed by cdc and recommended by who, 2 using bigdye terminator v.1.1 technology. nucleotide sequences were determined in a dna automatic sequencer abi prism 3130xl genetic analyzer. for each genomic segment, genetic analysis was performed with lasergene v.4.05 software (dnastar inc, usa) using an average of 4-6 overlapping readings, including sense and antisense, for precise nucleotide and amino acid sequence determination. genetic mutation and phylogenetic analysis were performed by neighbor-joining method, using mega4.0 software, against published sequences from the vaccine strain (a ⁄ california ⁄ 7 ⁄ 2009) and from selected a(h1n1) 2009 strains available on gisaid epiflu database. all mutations were identified with reference to the vaccine strain genome sequence. antiviral drug susceptibility profile of a(h1n1) 2009 influenza virus circulating in portugal was evaluated both phenotypically and genotypically for nais and genotypically for amantadine. phenotypic evaluation to nais, oseltamivir and zanamivir, was performed for all 96 isolates by ic 50 determination through munana fluorescence assays. 3 genotypic evaluation was performed by searching for mutations associated with resistance to nais in all 37 na gene sequences. amantadine susceptibility profile was performed for all 96 isolates by searching on m2 sequence for the 5 molecular markers associated with resistance to this antiviral drug (l26f ⁄ i; v27a ⁄ d; a30t; s31n; g34e). genetic characterisation of the ha1 subunit of ha reveals point mutations in different strains. all 37 analysed strains present p83s and i321v mutations, which distinguish them from the vaccine strain ( figure 1a ). thirty-three of the 37 sequenced strains group in the s203t branch. this mutation is referred in the literature as being associated with the putative antigenic site ca. 4 most of these strains (19) further subgroup in the d222e branch, this mutation being associated with one loop of the receptor-binding site. 1 from the early to the late epidemic period, an increased circulation of virus carrying the mutation s203t was observed. this is in agreement with the association between this mutation and an enhanced viral fitness that is described in the literature. 5 additional mutations were also observed in a small number of virus, of which we highlight: regarding the genetic characterisation of na, the majority of strains analysed (34 of 37) presents the mutations n248d and v106i ( figure 1b) . as mutation s203t in ha gene, these two na mutations are described in the literature as associated with enhanced viral fitness. 5 the few strains not carrying these mutations have circulated in the beginning of the epidemic period. fifteen of the 37 analysed strains further subgroup in y155h branch. additionally, mutation i223v was identified in two strains. for the remaining gene segments available for the six analysed strains, the observations include: (i) no previously described virulence markers in pb2, pb1-f2, and ns1 were detected; (ii) pb1-f2 protein is present in the truncated form of 11 amino acids; (iii) the presence of mutations i123v and l122q in ns1 and v100i in np; (iv) the described association of mutation i123v in ns1 and v100i in np genes with viral fitness. phenotypic evaluation of nais susceptibility revealed the existence of three minor and two major outliers to oseltamivir ( figure 2 ). the two minor outliers exhibited a reduction of approximately twofold in the susceptibility to this antiviral drug, comparing to the baseline level, while the reduction exhibited by the two major outliers was of approximately three-and fourfold. regarding zanamivir, two minor outliers were identified with a reduction of approximately twofold in the susceptibility, compared to the baseline level. these two minor outliers (a ⁄ portugal ⁄ 17 ⁄ 2009 and a ⁄ portugal ⁄ 82 ⁄ 2009) correspond to the two major outliers identified for oseltamivir. genetic analysis revealed the presence of the mutation i223v in the na sequence of these two strains. the contribution of this mutation for the profile of reduced susceptibility identified for both nais is not known, but a mutation in the same na position (i223r) has been referred to as being associated with a reduction in nais susceptibility. 6 full genome sequence analysis of these strains shows that both strains also present the v480i mutation in pb2 gene. however, no association of this mutation with antiviral drug susceptibility is referred in the literature. concerning genetic evaluation of susceptibility to amantadine, all 96 analysed strains present a serine in position 31, which is a molecular marker of resistance to m2 inhibitors. these preliminary results allow us to discuss several points. however, the additional data that is being obtained through this ongoing study will be essential for a more complete analysis. for example, more information is needed to determine if the mutations found alter the biology and the fitness of the virus or if there are associated with an increased prevalence of the virus. the majority of the mutations identified in ha1 subunit have been detected in a(h1n1)2009 strains distributed throughout the epidemic curve, not evidencing a specific evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h1n1)2009 virus. 7 the occurrence of mutations in the position 222 of the ha1 subunit of a(h1n1)2009 virus have been described. however, more studies are needed to clarify the outcome of these mutations, as for example in patients with severe complications. it could also be relevant to investigate the presence of single and mixed variants in viruses and in clinical specimens and the possibility of these mutations affecting the binding specificity. regarding the susceptibility of a(h1n1)2009 pandemic viruses to antiviral drugs, all analysed strains were found to be resistant to amantadine. this resistant profile was not unexpected since the mp gene from this new variant had originated in the eurasian swine lineage, which is characterised by being resistant to this antiviral drug. 8 the majority of the a(h1n1)2009 strains analysed revealed to be susceptible to both nais, with only five strains exhibiting a profile of reduced susceptibility, three to oseltamivir and two to both nais. for these last two, the presence of the i223v mutation in the na sequence could explain the reduction observed, but a more complete analysis is needed to confirm this. the french national pandemic plan includes an early containment phase followed by a limitation phase. the efficacy of such a plan depends on pre-existing surveillance and laboratory networks. the grog community surveillance network and the hospital lab networks organized by the two french nics carried out the virological monitorthe efficacy of such plan depends on pre-existing influenza surveillance and laboratory networks. in france, the community surveillance is carried through the grog surveillance network. in addition, surveillance is also carried out in hospitals by the renal network. this renal network is divided in two sub-networks: the so-called h5-labs network, activated during the containment phase and the extended renal lab network activated in the limitation phase. the h5-labs have bsl-3 facilities that can be used for diagnosis purposes. as part of the national influenza surveillance system led by the french institute for public health surveillance (invs), the grog community surveillance network and the lab networks linked to the two french nics carried out the virological monitoring of the a(h1n1)2009 pandemic from the early containment phase up until the end of the pandemic phase. during the containment phase, all suspected cases were hospitalized and declared to invs. each patient was tested on the same day by specific virological diagnosis. hospital admission was not mandatory during the limitation phase, (i) the clustered cases were monitored to study transmission chains, and (ii) the circulation of the virus in the community was monitored through grog swabs collected by practitioners. the nics organized the influenza surveillance to fulfill several objectives according to the epidemiological situation. first, rt-pcr tools (influenza a m gene rt-pcr and a(h1n1)2009 specific h1 and n1 genes rt-pcrs) were developped and distributed to the lab networks on the 14th of may 2009. 1 from the early phase, the nics and the h5-lab network analyzed all the samples collected from hospitalized and community patients. during the early phase of the limitation phase, an increasing number of labs were performing the specific assays. when the pandemic wave started, all hospital labs could do the testing. results were centralised by nic and reported on a weekly basis. in addition, nics carried out the monitoring of antiviral resistance emergence (na pyrosequencing, specific h275y rt-pcr, and phenotypic assays), and real-time surveillance of genetic changes involved in virus adaptation (pb2) virulence factors or antigenic variations (ha). this sequencing was carried out by the pf8 sequencing platform of the institut pasteur. the first imported a(h1n1)2009 influenza cases were observed from the 28th of april 2009. a limited number of cases have been reported in may. local transmission could be detected end of may. clusters were observed in schools in june and in summer camps during summer. as opposed to the epidemiology of the a(h1n1)2009 virus in other european countries, no summer wave was observed in france. only a limited number of sporadic cases were reported up until october. early september, a significant number of cases presenting with influenza-like illness was reported (figure 1 ). the virological investigation of these cases showed high prevalence of rhinovirus infection. this circulation of rhinovirus was a counfounding factor of the pandemic. the pandemic wave lasted 11 weeks between mid-october and the end of december (week 43 to week 53, figure 1 ). the pandemic wave started week 42-43 in the ile-de-france area, and only week 44-45 in the rest of france. the peak was recorded week 48 ( figure 1 ). the impact of the pandemic was mainly observed in the 5-15 years group of age. overall, 1334 severe cases have been admitted to the hospital, and 308 deaths have been recorded by the end of the pandemic wave. the major impact was observed in the 15-65 years group of age (66% of deaths recorded). amongst the severe cases and the deceased cases, 20% and 16% of cases had no risk factor, respectively. these specimens, 24 279 were positives for h1n1, representing 99ae7% of total influenza virus detections. only nine brisbane-like h1n1, 62 brisbane-like h3n2, and eight b viruses have been detected in the same period of time. the weekly positive rate ranged from 0% to 48%. phylogenetic and antigenic analyses of the viruses collected during the pandemic wave did not show any emerging genetic or antigenic variants (figure 2a,b) . eight patients, all among cases presenting with severe illness, were infected by a virus harbouring the d222g mutation in the ha. 2 amongst the virus tested for antiviral susceptibility or screened for the h275y mutation by or specific rt-pcr, only 11 oseltamivir-resistant viruses related to the na h275y mutation have been detected. one of these cases also had an i223r mutation associated to a reduced sensitivity to zanamivir. all but one resistant virus were detected in treated immunocompromised patients. overall, eight patients presented a virus with the d222g mutation in the ha. all these patients had a severe infection; one of these had also a h275y mutation in the na asociated to oseltamivir resistance. the pandemic started by the end of april 2009. although the first cases recorded were as early as the 28th of april, the epidemic wave associated with a widespread spread of the virus was only recorded in october. the french population did not have to face a summer wave, as observed in north america and in numerous european countries. 3, 4 it is difficult to speculate the reasons for the lack of summer wave; the specimens collected were negative for influenza. moreover, during september, it was anticipated that school openings would be the trigger for the beginning of the pandemic wave. as a matter of fact, a significant increase of influenza-like syndromes were observed at that time, but the virological investigation carried out by the laboratories showed thta is was related to a very large epidemic of rhinovirus. 5 the epidemic circulation of other respiratory viruses can be counfounding factors for the surveillance of the influenza epidemic clinical when the survellance is only based on collection of clinical information. the starting of the pandemic wave was heterogeneous in france. the ilede-france region (paris and its suburbean area), where the population is dense, experienced an early start as compared to the rest of france. however, once the pandemic started in the rest of the county, the epidemic curves were quite similar. the peak was reached at identical times, although it may have been delayed in some remote places in france. overall, we estimate that 10% of the french population consulted for an ili presentation. the impact was mainly observed in the 5-15 years groupe of. however, this age groupe represented only a limited number of severe cases and deaths. on the other hand, the 15-65 years groupe of age, where the prevalence was not high, was the age group where the majority of severe cases and deaths was recorded (74% and 66%, respectively). 6 this data is consistent with the observational data reported by numerous other countries. 7 according to the profile of hospitalized cases, a(h1n1)2009 was more aggressive than seasonal viruses. the number of admission to the hospital was ten-fold that observed during a normal influenza epidemic. even if the mortality was limited (312 cases), the age distribution of the deceased patients was different as compared to seasonal influenza (75% mortality in <65 years of age). the lack of recordeable excess mortality has been interpreted to be the consequence of a very mild pandemic, milder than some seasonal epidemics. however, the median age of the fatal cases was much younger than those observed during the seasonal flu, leading to a mis-interpretation of the real impact of the pandemic. when the impact is measurered in loss of years of life, the impact of this pandemic is larger than seen with seasonal influenza, and is quite comparable to these of the two last pandemics. 8 the pandemic preparadness of numerous countries, the develoment of new intensive care techniques and equipment, and the large use of antivirals have reduced the overall impact of this pandemic. these are new factors that should be taken into account when evaluating the real impact of the 2009 h1n1 virus. 8 the virological monitoring of the pandemic was achieved by the community-based and hospital-based seasonal influenza networks, reminding the importance of maintining such networks. the diagnosis of influenza in most of the patients was carried out by molecular techniques. it has been clearly stated from the beginning of the pandemic that near-patient tests were lacking of susceptibility and could not be used for patient management. the distribution of a set of validated and comprehensive techniques by the two nic was very helpfull for the monitoring of the pandemic and the patients. however, this diagnostic procedure change should not preclude maintaining virus isolation that is necessary for whole genome analysis, monitoring of antigenic changes, and phenotypic testing for antiviral testing. some of the mutants that have been recorded, including viruses with antiviral resistance phenotype or genotype, could be analysed from grown virus strains. it is striking that despite a large antiviral usage, only a limited number of isolates had mutations associated to resistance. however, the frequent isolation of such resistant virus was observed in immunocompromised patients that presented severe infections and long virus shedding. 9 the impact of the pandemic is still under evaluation. sero-epidemiological analysis will be performed to asses for the real attack rate of the 2009 pandemic virus. as in other countries, it has been recorded that asymptomatic infections could be observed frequently. 10 it is quite unlikely that the impact of the pandemic was reduced by the vaccination campaign, although this vaccination started on the 12th of november, just when the pandemic started in france. it is estimated that 6 millions received the vaccination. pandemic strains of the influenza virus sporadically emerge, deviating from the regular endemic strains of seasonal influenza. in april 2009, a novel pandemic influenza virus a ⁄ h1n1 emerged, swiftly spreading across the world. immediately, domestic and international public health agencies were forced to develop containment and mitiga-tion strategies in response to the pandemic. however, the dynamics and transmission patterns of this novel virus are yet to be fully understood. simultaneously, seasonal strains of influenza (a ⁄ h1n1, a ⁄ h3n2, and b) continued to circulate in many nations. both pandemic and seasonal variants of influenza are responsible for significant morbidity and mortality. 1 to characterize the dynamics of this disease and the variation within strains, a more detailed understanding of the patterns in viral shedding during natural infection is required. the majority of data on the patterns of viral shedding during influenza infection are a result of volunteer challenge studies. 2 in these studies, volunteers are commonly screened for pre-existing immunity against the challenge strain and are of a certain demographic and age. information on the patterns of viral shedding in natural influenza infections, pandemic or seasonal, is limited but should provide greater generalizability. we describe the trends of viral shedding and clinical illness in community acquired cases of pandemic and seasonal strains of influenza. in 2008, a community-based study was conducted to analyse the effectiveness of non-pharmaceutical interventions to prevent the spread of influenza in households. 3 in 2009, a similar community-based study was initiated to collect comparative data from individuals infected with seasonal and pandemic influenza. 4 both studies were conducted with very similar protocols, involving 617 households in total. the specimens and symptom data required for this study all arise from secondary infections ascertained in these two community-based studies. the recruitment process in both studies was essentially identical. index cases were first recruited from their healthcare provider if they presented with influenza-like illness (ili). this individual would be included in the follow-up if he ⁄ she tested positive for influenza virus infection by rapid antigen test (quickvue) and was the first person in his ⁄ her household that showed signs of ili in the previous 2 weeks. follow-up consisted of three home visits that spanned approximately 7-10 days. at each home visit, nasal and throat swab (nts) specimens were collected from all household members, regardless of the presence or absence of symptoms. symptoms were recorded in daily symptom diaries provided for every household member, and digital thermometers were provided to record daily tympanic temperature. the symptoms recorded were fever ‡37ae8°c, headache, myalgia, cough, sore throat, runny nose, and phlegm. influenza virus infection and subtype was identified by reverse transcription polymerase chain reaction (rt-pcr) on the nts specimens. viral shedding was quantified from the same specimens by rt-pcr to determine viral loads, as well as by quantitative viral dilutions to determine median tissue culture infectious dose (tcid 50 ). the details concerning laboratory methods have been described in a previous study. 5 all analyses in this study focus exclusively on secondary cases; these are household contacts of recruited index cases who acquire influenza virus infection following the initial home visit. index cases generally presented with a certain threshold of illness severity requiring medical attention, whereas infections among household contacts can vary from asymptomatic to severe representing naturally acquired influenza infections. these secondary cases must be negative for influenza for their first nts specimen, and subsequently tested positive. we analysed mean viral loads measured by rt-pcr and quantitative culture by plotting by day since acute respiratory illness (ari) onset according to strain of influenza (pandemic a ⁄ h1n1, seasonal a ⁄ h1n1, seasonal a ⁄ h3n2, and seasonal b). ari is the reference time point, because the day of infection is unknown and is defined as the presence of ‡2 of the symptoms mentioned above. average symptom scores were also plotted according to ari onset and grouped into upper respiratory symptoms (sore throat and runny nose), lower respiratory symptoms (cough and phlegm), and systemic signs and symptoms (fever ‡ 37ae8°c, headache, and myalgia). mean daily tympanic temperatures were also plotted since date of ari onset and according to strain of influenza virus. all analyses were conducted using r software (version 2.10.1; r development core team). 6 a total of 617 households and 2499 individuals were followed-up in the two studies. of 1887 household con-tacts tested by rt-pcr, 153 were found to be influenza positive. among these influenza infections, 13 (8ae5%) were asymptomatic (rt-pcr positive plus 0 symptoms recorded), 20 were subclinical (rt-pcr positive plus 1 symptom recorded), and 88 presented with an onset of ari during the follow-up period. from the cases with ari onset, seven pandemic a ⁄ h1n1, 40 seasonal a ⁄ h1n1, 22 seasonal a ⁄ h3n2, and 19 seasonal b influenza virus infections were identified. the age distribution among secondary cases was observed to be largely comparable across the four strains of interest (table 1 ). there were a lower proportion of males who acquired pandemic a ⁄ h1n1 compared to the seasonal strains of the virus. cough was the most commonly reported symptoms during follow-up in cases of pandemic a ⁄ h1n1 and seasonal b, whereas runny nose was most common in seasonal a ⁄ h1n1 and a ⁄ h3n2 cases. cumulatively, fever ( ‡37ae8°c) was reported in approximately half (51%) of the secondary cases. patterns of viral shedding were analysed in a subset of 88 influenza positive individuals who recorded an onset of ari in their symptoms diaries (figure 1 ). household contacts that were asymptomatic, subclinical, or did not have an ari onset were excluded from the analysis. viral shedding in all three influenza a strains were recorded to occur on the day of ari onset or 1 day post-ari onset. following the peak, measured levels of viral shedding declined steadily to undetectable levels over 5-6 days. the trend of viral shedding in influenza b infected individuals rose 2 days before ari onset, fluctuated for around 6 days before eventually resolving. the patterns of viral shedding over time measured by quantitative viral culture were generally similar to the patterns measured by rt-pcr. the patterns of symptoms and signs were comparable in the four strains of influenza included in this study, peaking on the day or 1 day post-ari onset, and gradually declining over a period of 5-7 days. in all strains, systemic symptoms and signs were observed to resolved faster than upper and lower respiratory symptoms. the trend of tympanic temperature in each influenza strain was comparable to the respective symptom pattern. patterns of viral shedding observed in influenza a strain infections (pandemic a ⁄ h1n1, seasonal a ⁄ h1n1, and seasonal a ⁄ h3n2) were broadly similar. the pattern differed from the observed pattern of viral shedding in seasonal influenza b infections. the majority of viral shedding in influenza a strains occurred at and near ari onset, whereas there were variable amounts of viral shedding preand post-ari onset for those with influenza b. the biological reason for this difference is yet to be clarified. these differences are consistently observed regardless of laboratory method used to quantify the viral loads. it was observed that viral shedding measured by tcid 50 resolved more quickly than when measured by rt-pcr, suggesting that rt-pcr is more sensitive, but it could be detecting inactivated fragments of rna instead of active virus. the trends observed for the seasonal strains of influenza in this study were similar to those reported in literature. 2 the patterns of symptoms and signs as well as tympanic temperature in the four different strains of interest in this study were found to be comparable. these patterns closely resemble the patterns of viral shedding observed in the influenza a virus strains, but not in the influenza b virus strain. the trends of viral shedding, symptom scores, and tympanic temperature for pandemic a ⁄ h1n1 were similar to trends observed for seasonal a ⁄ h1n1 and seasonal a ⁄ h3n2 infections, suggesting that the dynamics of these viruses are largely the same. the clinical course of infection with pandemic a ⁄ h1n1 influenza virus appeared to be similar to the seasonal b influenza virus, but the patterns of viral shedding over time diverges. in general, our results suggest that the dynamics of the pandemic a ⁄ h1n1 virus were similar to the seasonal a ⁄ h1n1 and a ⁄ h3n2 viruses, and clinically similar to the seasonal b virus. this study faced sample size limitations; very few cases of pandemic a ⁄ h1n1 were detected and the secondary attack rate in general was low, though a total of 617 households were followed up. this lack of power led to the inability to analyse the differences between adult and children and other characteristics that could be correlated with amount of viral shedding. there are also biases that must be factored in during recruitment. the eligibility criteria of only healthy households could select for households with higher innate immunity. on the other hand, recruitment at health care providers can be biased towards index cases that had more severe illness that required medical attention. the strength of the study is the broad generalizability of the results due to the strict classification of secondary cases. the infections reported in this study were all community-based and should represent true natural infections. pandemic potency of the influenza virus is largely determined by its transmissibility. the first objective of this study was to model the transmission of influenza h1n1 and h5n1 viruses. at present, vaccination with laiv has been used as a widespread, effective public health measure for influenza prophylaxis. some unsubstantiated concerns have been raised about a potential possibility of reassortment of circulating influenza viruses with laiv viruses following vaccination with laiv. thus, another objective of this study was to assess the probability of pig-to-pig transmission of cold-adapted viruses and their potential reassortment with wt influenza strains. female albino guinea pigs weighing 300-350 g were inoculated intranasally with 10 5 eid 50 of virus without anaesthesia. transmission studies were then performed 24 hours after inoculation. inoculated animals were housed at 25% relative humidity and 22°c in the same cage with noninfected guinea pigs or in cages placed 3 m away from non-infected pigs. virus replication was determined by virus isolation in hen eggs and by pcr. sera were collected at 0 and 28 days post inoculation. seroconversions were assessed by routine hai test. genome composition of reassortants was monitored by rflp analysis. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated, and virus growth properties were observed following virus titration in hen eggs. when infected pigs were co-caged with non-infected (naïve) individuals, vn1203, indo ⁄ 5, a ⁄ california ⁄ 7 ⁄ 2009, and nibrg-23 were isolated in 0%, 25% 83ae3%, and 100% of contact animals, respectively. serological confirmation of virus transmission was higher than virological data (25%, 100%, 100%, and 100%, respectively). in addition, it was shown that when pigs inoculated with a ⁄ california ⁄ 7 ⁄ 2009 were co-caged with animals inoculated with nibrg-23, they got infected with both viruses ( table 1) . the ability of direct transmission of cold-adapted viruses was also investigated. data show that the a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 laiv candidate was detected in the upper respiratory tract of 87ae5% vaccinated pigs. the mdv was identified in 100% of infected animals. however, neither group of contact pigs, co-housed with the vaccinate pigs, had evidence of infection with cold-adapted viruses. in addition, none of the contact pigs had any evidence of seroconversion to the coldadapted viruses as determined by hai assay. it was also most interesting to note that pig-to-pig transmission of the highly transmittable nibrg-23 reassortant virus was not seen when pigs, vaccinated with mdv, were co-caged with animals infected with nibrg-23 virus (table 1) . this strongly implies a form of interference or protection from transmissibility that was provided by the cold-adapted virus. the results show that nibrg-23 and indo ⁄ 5 viruses were able to spread between cages over the 3 m distance (100% and 50% naïve animals were successfully infected, respectively). a ⁄ california ⁄ 7 ⁄ 2009 influenza and vn1203 viruses did not transmit between infected and non-infected guinea pigs housed in separated cages (table 1) . pigs with confirmed a ⁄ california ⁄ 7 ⁄ 2009 virus replication were also infected with nibrg-23 virus if h1n1-and h5n1-infected animals were separated by a space. thus, influenza virus transmission from h5n1-to h1n1-infected pigs has been shown, but the reverse pattern did not occur. transmission of nibrg-23 or a ⁄ california ⁄ 7 ⁄ 2009 viruses was not observed when contact pigs were first vaccinated with the mdv and housed at a 3 m distance ( table 2) . it was also shown that efficiency of transmission of nibrg-23 was much higher than of other studied h5n1 viruses; it can be transmitted between naïve guinea pigs separated from infected animals at a distance of 4-5 m (data not shown). five reassortants were isolated from animals which were infected with a ⁄ california ⁄ 7 ⁄ 2009 virus and co-caged with pigs inoculated with nibrg-23. two reassortants possessed different combinations of pr8, nibrg-23, and a ⁄ california ⁄ 7 ⁄ 2009 genes and demonstrated the non-ca ⁄ non-ts phenotype typical of wt viruses. unexpectedly, two other reassortants inherited ha gene from nibrg-23, na gene from a ⁄ california ⁄ 7 ⁄ 2009, and other genes from pr8 became ca and ts. 7:1 non-ts reassortant inherited pa gene from pr8 and seven other genes from a ⁄ california ⁄ 7 ⁄ 2009, gained ca properties. in spite of aforesaid experimental data, we cannot exclude the theoretical possibility of simultaneous infection of human host with cold-adapted and wt influenza viruses. to better understand possible consequences of such a reassortment event, we co-infected guinea pigs with a mixture of mdv and nibrg-23 viruses. nasal washes were collected and cloned by limited dilutions in hen eggs in the presence or absence of immune serum to the mdv. cloning of nasal washes without antiserum led to isolation of over 100 clones, which were all identical to the mdv (data not shown). when nasal washes were cloned in the presence of antiserum, only nine clones were isolated. genome composition analysis showed that all isolates were triple reassortants, which had inherited pb2 and na genes from mdv, pa gene from pr8, and ha gene from nibrg-23. the origin of the other gene segments (pb1, np, m, ns) in the genome of guinea pig-derived reassortants varied. reassuringly, all reassortants generated in vivo had the phenotype typical of the mdv. the severity of influenza outbreaks is partly determined by efficient spreading of the causative virus strain between human hosts. however, little is known about mechanisms underlying influenza virus transmission in humans. guinea pigs have been shown to be a suitable model for influenza transmission studies. 1 our in vivo study showed that influenza a viruses vary in their transmissibility. nib-rg-23 and indo ⁄ 5 viruses were able to transmit to naïve animals caged distantly from infected animals. in contrast, cold-adapted viruses, the same as those used for licensed laivs, showed no signs of transmission from one guinea pig to another. our study also provided evidence of a lower level of transmissibility of the novel pandemic h1n1 virus compared to the nibrg-23 and indo ⁄ 5 h5n1 strains evaluated. benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. 2 in our study, the mdv inoculated into guinea pigs appeared to interfere with and even offer protection from transmission of the highly transmissible nibrg-23 virus. the ability to immunize with the laiv and subsequently block the spread of a homologous h3n2 subtype and a heterologous h3n2 subtype influenza virus between guinea pigs has been shown. 3 interference between cold-adapted and wildtype influenza virus infection was the most likely explanation for the data observed in our study. the mdv inoculated into guinea pigs might in some way interfere with transmission of highly transmissible influenza viruses. it is believed by some that widespread use of laiv could increase the potential risk of reassortment of the vaccine strain with circulating influenza viruses immediately following vaccination. however, it was shown that any such potential reassortments would most likely lead to yet attenuated viruses. 4 our in vivo studies have shown that introduction of mdv genes into the genome of nib-rg-23 virus led to the generation of triple reassortants inherited pb2 and na genes of mdv and ha gene of h5n1 virus. all isolates possessed phenotypical markers associated with attenuation of mdv. our data suggest that even if a reassortment event of such rare occurrence between a laiv strain and a circulating virus were to occur, it would most likely lead to a reassortant that would retain highly attenuated phenotypic properties of the vaccine strain. our data strongly support the safety of laivs, especially those developed against highly transmissible h5n1 and h1n1 pandemic influenza viruses. this information builds upon databases that have clearly shown the low likelihood of transmitting an laiv, as well as the high likelihood of any field reassortment of laiv with a circulating influenza virus to retain important properties of the cold-adapted, temperature-sensitive vaccine master composition. very interestingly, we also present data that show the potential of a laiv to prevent the transmission of highly infectious influenza viruses, perhaps identifying a broader role for laiv in the overall scheme of influenza virus prophylactic use. background: schlieren imaging is a non-invasive, real-time airflow visualization technique that relies on differences in air temperatures (and the resulting changes in the refractive index) to allow exhaled human airflows to be seen clearly against the background of more-stationary, ambient air. recently, this technique, well-known to engineers, has been applied to better understand and characterize airflow behaviors associated with everyday, as well as healthcarerelated, human respiratory activities. materials and methods: as a surrogate marker for the behavior of airborne infectious agents, schlieren imaging was used to visualize the airflow patterns produced by adult human volunteers of different ages while coughing with and without the wearing of standard surgical and n95 masks. results: the cough plumes were generally similar in shape and range for all the adult volunteers used in this study. although both the surgical and n95 masks decelerated and blocked some of the forward momentum of the coughed airflows, much of the cough plume was redirected and escaped around the top, bottom, and side edges of the masks to merge with the volunteer's natural, verticallymoving thermal plume. conclusions: schlieren imaging is a safe technique for visualizing exhaled airflows from human volunteers without the need for potentially-irritant or toxic particle tracers. findings from these schlieren imaging experiments will assist the development of more effective aerosol infection control guidelines in healthcare premises where patients infected with potentially airborne infectious agents (e.g., influenza and tuberculosis) are present. these infectious agents may be transmitted to healthcare workers, other patients, and their visitors by way of exhaled airflows. with the recent influenza pandemic 1,2 and the ongoing concerns about human cases of avian influenza h5n1 infections, 3 there is now a very real concern about the potential for the aerosol transmission of respiratory pathogens. such concerns amongst staff and patients in healthcare environments have led to a greater emphasis on the understanding and control of infectious airflows. 4,5 previous visualization techniques have used potentially-toxic or irritant gas or particulate tracers with hazardous laser light sources that have precluded the use of human volunteers as subjects. instead, various forms of lung models that simulate human respiratory patterns with such particulate tracers have been used. 6, 7 schlieren imaging is a technique familiar to engineers and offers a non-invasive (i.e., no tracer required) airflow visualization method that depends only on differences in the refractive index of the warmer, human-exhaled air and the cooler ambient air. 8 the use of a simple incandescent or light-emitting diode (i.e., non-laser) light source is safe and allows human volunteers to be used as experimental subjects, where their exhaled airflows are then observed using a large, precise spherical or parabolic telescopic mirror and a camera, and are recorded for later analysis and presentation. [9] [10] [11] the analysis of these patterns of 'real-life' human airflows will be useful in optimizing aerosol infection control guidelines, which aim to reduce the transmission of airborne infectious agents to other healthcare personnel, patients, or their visitors. the images and analysis presented here have all been obtained from the large 1 m diameter parabolic mirror (figure 1 ) situated at the gas dynamics laboratory of penn state (directed by gary s. settles). this large schlie-ren imaging system has been in use for over 30 years to obtain high quality schlieren images for various engineering applications. it has only recently been applied to clinically-relevant imaging. the objective of this paper is to augment and expand upon the details of the methods and results presented in an earlier study using this same schlieren imaging system. 10 the aim of this series of studies is to visualize and capture a series of airflow images produced by coughing from adult human volunteers of different ages (24-80 years old). these included males (three of 26 years, one of 80 years of age) and females (one of 24 years, one of 30-40 years, and one of 40-50 years of age). each volunteer was tested with and without wearing either a standard surgical mask or n95 mask. more specifically, the aim was to visualize the extent and direction of leakage around the mask whilst each subject was coughing. penn state institutional approval for experiments involving human subjects was also obtained. each volunteer was asked to stand approximately 1 m in front of the schlieren mirror, facing across the surface of the mirror on one side, and to cough several times as the real-time, color image and video footage was recorded by the operator (using a nikon d90 camera; nikon inc. melville, ny, usa). this process was repeated whilst each volunteer was wearing a standard surgical mask then an n95 mask (supplied by 3mô, st paul, mn, usa). some of the schlieren images obtained from some of these volunteers have been published previously: for a 26-year old male, 9 the 24 year-old female and a 26-year old male, 10 and the 40-50 year-old female. 11 this article completes this series of schlieren images obtained from these experiments by including the images recorded for the older, 80 year-old man. generally, it was found that the shape of the cough plumes (shown in the figure as darker shadows emanating from the subject's mouth) produced by adult humans of different ages was relatively similar. cough plumes are roughly conical in shape and very turbulent, usually passing beyond the extent of the 1 m mirror (figure 2a) . a previous detailed study of one of these images measured a maximum airflow velocity of 8 m ⁄ second for an adult cough. 9 similarly, the effects of wearing surgical and n95 masks can be generalized across different ages. wearing a surgical mask allows leakage of the coughed air from the sides, top, and bottom of the mask ( figure 2b ). there is also some leakage through the mask, as indicated by the darker patches of air directly in front of the mask ( figure 2b, c) . the useful effect of the mask appears to be a deceleration and redirection of this coughed (and potentially infectious) air into the natural, upward-rising human thermal plume, which captures it and carries it upwards where it is diluted and less likely to transmit infection to others. the effects of the n95 mask are similar (i.e., deceleration and redirection), yet due to its tighter (mask-fitted) face seal, more of the coughed air appears to penetrate the front of the mask ( figure 2c ). this penetrating air is, however, also decelerated sufficiently to allow the wearer's natural thermal plume to carry it upwards. 10, 11 discussion from these series of schlieren images presented in this and other related studies, [9] [10] [11] it is clear that schlieren imaging offers a safe, non-invasive, real-time technique to visualize human exhaled airflows for all age groups. it is apparent that, at least where airflow patterns are an acceptable surrogate marker for airborne transmission risks, there are beneficial effects of wearing either type of mask, even when the mask fit is relatively poor. this is often the case when n95-style masks are purchased and used by the general public -in contrast to the situation with healthcare workers, who are often accurately fit-tested for this type of mask. the immediate significance of this can be seen when masks are bought by parents for their children. often, these will not be of pediatric size and the mask-fit will be loose. children are well-known to be major sources of infection in the community because of their relatively poor immunity to many types of infectious agents due to their young age and, therefore, limited past-exposure history. 12 these images allow infection control teams to literally see how far and how fast potentially-infectious human exhaled airflows can travel from an individual. this may have significant implications for guidance on the wearing of masks for infected staff and patients, on ward bed-spacing, as well as for the types of masks to be used in different situations. the important practical potential lies in the non-intrusive visualization of airflows associated with human volunteers, to assist in heightening the awareness amongst healthcare workers of the risks and potential for the airborne transmission of infectious agents, as well as the development of more effective aerosol infection control policies. schlieren images can be analysed more quantitatively, e.g., with the 'schlieren-piv' technique, 9, 13 though this additional quantitative data is probably more of research interest than being of immediate practical use to everyday hospital infection control teams. these are the subtypes that we have studied. clearly, the question arises as to whether the changes in antigenicity are coupled with changes in germicide susceptibility. we have employed a modified log-reduction method 3 in a cell culture system employing mdck cells 4 in serum-free ex-cellô 5 medium supplemented with trypsin. microscopic examination of cpe was the marker for infectivity together with plaque assay. we confirmed antiviral potency by using specific subtype influenza identification subtype technology, quidel quickvue ò influenza a + b test. the log inactivation and percent inactivation by bac after a 60 second contact time for the h1, h2, and h3 pandemic strains are as follows: a ⁄ swine ⁄ iowa ⁄ 12 ⁄ 30 h1n1, 3ae5 log ⁄ 99ae97%; a ⁄ swine ⁄ cal ⁄ 2009 h1n1, 4ae8 logs ⁄ 99ae998%; a ⁄ j305 ⁄ 57 ⁄ h2n2, 5 logs ⁄ 99ae999%; and a ⁄ hong kong 8 ⁄ 68 h3n2, 5ae0 logs ⁄ 99ae999% (table 1 ). comparable results of antiviral efficacy are obtained with the tcid 50 and plaque assays against all subtypes studied. when performing the plaque assay the sensitivity of virus recovery was better in the vessel with a larger surface area and overall recovery was in agreement with the potency determined by tcid 50 assay. in our plaque assay, we inoculated a ⁄ hong kong ⁄ 8 ⁄ 68 virus dilutions into two different vessels with 2 hours adsorption time: 6-well plate and t-25 flask, 9 ml inoculum per replicate. virus titers obtained were: 1ae4 · 10 6 pfu ⁄ ml from 6-well plate and 2ae2 · 10 6 pfu ⁄ ml from t-25 flask ( table 2 ). the discrepancy on virus potency can possibly be explained as: the binding of virus to host cell occurs only when virus gets a chance to interact with the cell on the monolayer during adsorption time. the percentage of virus population in the inoculum that has the opportunity to bind to the cell mainly depends on the surface area where this interaction takes place. therefore, in our experiment the plaque assay in the t-25 flask gave higher virus recovery 2ae2 10 6 versus 1ae4 · 10 6 pfu ⁄ ml. the increased virus recovery can translate into better sensitivity of the test system for disinfectant and antiviral agents. the potency of the virus used in this study was determined by tcid 50 was 5 · 10 6 tcid 50 ⁄ ml. rapid diagnostic testing for influenza (quickvue ò influenza a + b test, quidel) for aj305 versus bac was studied. the presence of influenza viral nucleoprotein a determined by quickvue kit correlated 100% with the viral infection based on by cpe in viral culture. interestingly, the inactivation of viral nucleoprotein was able to be revealed with diagnostic kit in the dilutions of virus ⁄ bac reaction mixture, which possessed prominent cytotoxic effect for the host cells in viral culture system. this type of molecular testing method is useful for interpreting antiviral efficacy against a background of cytotoxicity. these experiments are intended for the sponsor to substantiate to us fda that their antiviral substances are safe and effective. the data shows that the three hemagglutinin subtypes were highly susceptible to the quaternary ammonium compound in the short term in vitro experiment. the appearance of novel subtypes in the future can be met with the assurance that disinfectant and ⁄ or antiseptic resistance will be unlikely. certainly, from the above data, although genetic reassortment of human and swine viruses may modulate influenza pathogenesis and limit existing vaccine benefit, it is not likely be a factor in control of viruses on environmental surfaces by benzalkonium-type disinfectant ⁄ cleaning agents in community or health care environments. table 2 . comparison of viral titer obtained in different vessels using quantal tcid 50 and plaque assay methods plaque assay tcid 50 assay t-25 (25 cm 2 ) 6-well plate (9 cm 2 ) tcid 50 ⁄ ml tcid ⁄ ml 2ae2 · 10 6 pfu ⁄ ml 1ae4 · 10 6 pfu ⁄ ml 5 · 10 6 2ae5 · 10 6 options for the control of influenza vii outbreak influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. in this paper we share our experience of managing an outbreak of viral respiratory infection in an acf very early in the 2009 influenza pandemic and also describe some of the emerging issues relating to crossreacting antibodies to the pandemic (h1n1) 2009 influenza virus in the very elderly. the outbreak investigation was conducted as part of an urgent public health intervention initiated by the new south wales (nsw) department of health during the early stages of the first southern hemisphere wave of the 2009 pandemic. nose and throat swabs for nucleic acid testing (nat) plus acute and convalescent serum samples (6 weeks apart) were collected from all the residents of an acf where an influenza-like illness (ili) outbreak occurred. the investigation revealed dual outbreaks of pandemic (h1n1) 2009 influenza and rhinovirus infection. out of 28 residents, three had laboratory confirmed influenza [two with pandemic (h1n1) 2009], and 10 had rhinovirus infection on nat. testing of acute sera collected from every subject found elevated ( ‡1:40) pandemic (h1n1) 2009 hai antibody in 60% (9 ⁄ 15) subjects aged 85 years or more (born before 1925 and median age 88 years; geometric mean titre-gmt 48ae1) compared with none of the 13 residents aged under 85 years (born after 1924 and median age 79 years; gmt 10ae1, p = 0ae01). the acf was closed to visi-tors for 7 days. the symptomatic residents received treatment-dose oseltamivir, and all other residents were given oseltamivir prophylaxis. more than one virus may be circulating in an acf with an ili outbreak at any one time in winter. a significant proportion of elderly residents had pre-existing cross reacting antibody to the pandemic (h1n1) 2009, which may explain the minimal clinical impact of pandemic (h1n1) 2009 in this elderly population. influenza is one of the leading causes of infectious death in elderly people, principally due to co-morbidities and declining immune competence with age. it is the most important agent in outbreaks of respiratory illness. 1 influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. 2 the clinical presentation of influenza in residents of acfs can be subtle, with a blunted febrile response and a non-specific decline in mental and functional status. 3 residents commonly have underlying diseases that can be exacerbated by influenza infection, and in addition, they are at higher risk of serious influenza-related complications than community dwelling elderly people. 4 people aged over 65 years are also at higher risk of influenza-related death, and more than 90% of annual influenza-related mortality is usually confined to this high risk group. 5 in australia, influenza and pneumonia have sub-stantial health impacts; recorded as being the underlying causes of death for 2623 persons in 2007. 6 since the world health organization declared an influenza pandemic in june 2009, australia has suffered one of the highest rates of confirmed infection during the first southern hemisphere wave. by late october 2009 there were 187 reported deaths due to pandemic influenza in australia, 7 and to date there have been about 18 449 deaths reported worldwide. 8 although disproportionately far fewer elderly people developed clinical influenza during the current pandemic than occurs with seasonal influenza, their case-fatality rate remained substantial. 9 early in the pandemic (june 2009), we investigated a suspected pandemic influenza outbreak in a rural acf in the state of nsw, australia. the epidemiology (including virulence and clinical outcome in the elderly) of the pandemic (h1n1) 2009 virus was mostly unknown at the time of investigation, and as time passed, this investigation provided clarity on some important issues of the influenza epidemiology in the elderly population. in this paper we share our experience of managing a dual outbreak of viral respiratory infections early in the pandemic, and also describe some of the emerging issues relating to the cross-reacting antibodies to pandemic influenza in the very elderly. the outbreak investigation was conducted as part of urgent public health intervention initiated by the nsw department of heath in conjunction with the local public health unit, the national centre for immunisation research and surveillance (ncirs), and the institute of clinical pathology and medical research (a who national influenza centre). to determine the extent and cause of the outbreak, a public health research doctor (gk) was dispatched from sydney over a weekend to assist with outbreak investigation and control. on june 12th 2009, the greater southern public health unit surveillance officer (bd) received a report of a possible pandemic (h1n1) 2009 outbreak in a local acf. on investigation, it was discovered that 3 days earlier a 77 year old female resident had become generally unwell, but without specific symptoms of influenza like illness (ili). soon after, nine of the 27 co-residents (but no staff) had developed symptoms suggestive of influenza. one other resident had returned from a melbourne (victoria) hospital (where pandemic (h1n1) 2009 was known to be circulating) the previous week after surgery, but did not have ili symptoms. on june 10th, the 10 symptomatic residents had nasal swabs taken by the local doctor for influenza [including pandemic (h1n1) 2009] nucleic acid testing (nat). there was rising concern due to reports of widespread pandemic (h1n1) 2009 influenza in a local army camp just over the border in nearby victoria, where pandemic (h1n1) 2009 influenza was known to be circulating widely. on june 12th, the 77 year old lady proved nat positive for pandemic (h1n1) 2009, but none of the other samples were pandemic (h1n1) 2009 nat positive. concern arose that there might be an outbreak of pandemic (h1n1) 2009 in the facility, and that some of the swabs from other residents might be false negatives. between 14 and 15 june, after consent was obtained, directly or through next of kin in 13 demented residents, all 28 submitted to venipuncture for serology, 27 successfully, and the other 18 as yet un-swabbed residents were swabbed. basic demographic data were collected from every resident with clinical information on co-morbidities and current medication use. convalescent blood samples were collected after 4 weeks on 16th july 2009 from 23 of the 28 residents. swabs were sent to icpmr where nat for influenza a [including pandemic (h1n1) 2009] and b was performed. the acute and convalescent serum samples were tested later (in december 2009), using haemagglutination inhibition assay (hai) to detect pandemic (h1n1) 2009 antibody. 10, 11 interventions the acf was closed to visitors from 12th until 18th june. treatment of the positive case and the nine symptomatic residents, with twice daily oseltamivir, was begun on saturday june 13th, and all other residents were started on once daily oseltamivir prophylaxis. the facility manager and local general practitioner (gp) monitored patient health on a daily basis, and none had to stop oseltamivir due to adverse events. one resident with ili who was known to have moderately impaired renal function was given once daily rather than twice daily oseltamivir treatment. the age range of the residents was 58-97 years with a median of 85 years. all residents had underlying medical conditions, e.g., chronic cardiac and respiratory diseases ( table 1) testing of acute sera collected from every subject found elevated ( ‡1:40) cross-reacting hai antibody to the pandemic (h1n1) 2009 in 60% (9 ⁄ 15) of subjects aged 85 years or more (born before 1925 and median age 88 years; geometric mean titre-gmt 48ae1). however, the hai titre was consistently <1:40 and significantly lower (gmt 10ae1, p = 0ae01) in the 13 residents aged under 85 years (range 58-83 years, median 79 years) (figure 1 ). the index case (nat positive) did not show a significant raise in hai level in convalescence (going from 20 to 40). the pandemic (h1n1) 2009 case that was determined by serology was pandemic (h1n1) 2009 nat negative. to our surprise, seven of the other 18 asymptomatic residents had rhinovirus detected on extended nat (reported on june 25th), despite being asymptomatic at time of swabbing and remaining so. the original nine influenza nat negative samples were then tested and three of these were also nat positive for rhinovirus; in total, ten proved nat positive for rhinovirus (35ae7%). the serologically confirmed pandemic (h1n1) 2009 case was also positive for rhinovirus infection. of interest was that only one resident had a documented fever. this investigation illustrates some of the difficulties in managing and investigating possible influenza outbreaks in real time in the context of an influenza pandemic. 12 finding a nat positive case of pandemic (h1n1) 2009 influenza among many other symptomatic cases raised the possibility (although not the probability) that pandemic (h1n1) 2009 was the cause of the outbreak. rhinovirus infection, however, was confirmed by nat in ten residents. this outbreak illustrates that more than one virus (in this case 2 and perhaps 3) may be circulating in an acf at any one time in winter. in ili outbreaks in acfs, broad laboratory testing is recommended; nat is the most sensitive method of detecting influenza or other viruses in respiratory tract samples. 13 studies have found that the pandemic (h1n1) 2009 haemagglutinin (ha) gene is more closely related phylogenetically to the 1918 h1n1 virus and classical swine influenza a ⁄ h1n1 viruses than more recent seasonal human influenza a ⁄ h1n1 viruses. 14 it is antigenically similar to the 1918 h1n1 pandemic virus in terms of the immunodominant antibody response to haemagglutinin. [15] [16] [17] it is likely that individuals alive during the emergence and initial persistence of the 1918 pandemic virus would have higher levels of cross-reacting hai antibodies to the pandemic (h1n1) 2009, which would contribute towards better clinical protection. 18 in our investigation, 60% of the residents born before 1925 (aged 85 years or above in 2009) had pre-existing cross-reacting hai antibody to the pandemic (h1n1) 2009. in elderly populations, severe illness may be associated with organisms typically considered to be mild, such as rhinovirus. however, studies have shown that nursing home residents may be susceptible to outbreaks of rhinovirus that may cause mild to severe respiratory illness, particularly in those with a history of lung disease. one rhinovirus outbreak in a nursing home in the usa caused 12 fatalities. 19 another outbreak showed residents with underlying lung disease are more likely to have longer infection, require antibiotics, develop bronchospasm, and have difficulty breathing; two residents with underlying lung disease required emergency treatment and one died. 20 a previous influenza outbreak in a nsw aged care facility in 2006 caused significant mortality and morbidity. that outbreak resulted in 14 hospital admissions and six deaths. 21 in our investigation we have found that 17% of the residents had chronic lung disease and 64% had chronic cardiac conditions both considered as high risk for severe complications of both rhinovirus and influenza infection. however, there were no hospitalisations or deaths in our outbreak investigation. indeed only one resident developed fever, indicating that non-specific signs of illness (such as in our index case) may be the only, or early, indication of an ili. our own experience with managing other ili outbreaks has also taught us that staff of acfs may not be vigilant enough to detect fevers. in this outbreak, the nursing home staff, local gp, public health unit and the outbreak investigation team and supporting laboratory staff acted quickly and in a coordinated way. pre-existing cross-reacting antibody in the very elderly (aged ‡85 years) probably helped to limit the spread of the pandemic virus (compared to the circulation of rhinovirus) within the acf. exposure to the 1918 pandemic (or a close variant occurring before 1925) appears to be responsible for a high hai titre in the very elderly, which contributed towards better clinical protection. however, wider testing early on would have alerted us more quickly to the main cause of the outbreak. treatment and prophylactic use of oseltamivir may also have contributed to halting the spread of pandemic (h1n1) 2009 and also to symptom relief. pandemic (h1n1) 2009 influenza virus (ah1pdm) has spread worldwide since march 2009. in a paper of ah1pdm, 25% of infected individuals have experienced gastrointestinal symptoms such as diarrhea and vomiting, which is higher than that of seasonal influenza. however, little is known whether viable virus shed from stool and replication of viruses are ongoing in the gastrointestinal tract. 1,2 viral load and isolation of ah1pdm in cell culture in stool samples has been reported. 3 stool specimens were collected from 35 patients suspected to have pandemic (h1n1) 2009 infection from november 2009 through may 2010. virus isolation was conducted in cell culture by using madin-darby canine kidney (mdck) cells and taqman based rt-pcr from 10% (w ⁄ v) stool suspension in phosphate-buffered saline. taqman based rt-pcr was conducted by using primers, probes, and positive controls provided by niid (national institute of infectious diseases of japan). to confirm presence of ah1pdm viral rna, lamp (loop-mediated isothermal amplification) was used as supplemental testing. of patients, one child (case 1) submitted one nasal swab and four stool samples, another one nasal swab and two stool samples, and the other one stool sample. informed consent was obtained. strand specific rt-nested pcr was performed for only case 1 by using only one primer at the rt reaction and also assayed neu5aca2-3gal and neu5aca2-6gal binding specificity about isolated strain derived from nasal swab and stool. receptor binding specificity was performed using a solid-phase binding assay with the sialylglycopolymers (poly a-l-glutamic acid backbones containing neu5aca2-3galb1-4glcnacb-pap or neu5aca2-6galb1-4glcnacb-pap bond as described. 4 ) nucleotide sequences of the ha gene of ah1pdm viruses isolated from stool sample and nasal swab were analysed. in order to exclude the possibility of contamination, the stool samples and nasal swabs were subjected to virus isolation separately. after getting the results on the nucleotide sequence, we also confirmed no strain harboring identical sequence was isolated in our laboratory before and after the day of sample collection. ah1pdm viral rna was detected in nine (25%) of the subjects from stool samples. among nine subjects, one case (case no. 1) was positive for viral isolation. case1, a healthy 9-year-old girl, experienced fever and abdominal pain, and the others had gastrointestinal symptoms without upper respiratory symptoms. in case 1, influenza a virus was diagnosed by rapid antigen test on the day of symptom onset. viable ah1pdm virus was isolated from the stool sample and nasal swab on the second day from onset using mdck cells (table 1 ). viral load decreased gradually after symptom onset. however, viral shedding was still present 8 days after symptom onset. positive stranded rna was detected 6 days after symptom onset from the stool specimen ( figure 1 ). above two ah1pdm strains (isolated from nasal swab and stool specimen) bound exclusively to human type receptor, neu5aca2-6gal. sequence analysis demonstrated that isolated virus from stool samples was identical with that from nasal swabs in comparison of ha gene (990 bp). ah1pdm influenza virus was isolated from the stool and nasal swab samples in the same patient simultaneously by using mdck cells. our results suggests the detection of viral rna and viable ah1pdm influenza virus from stool samples may serve as a potential mode of transmission and has important implications in understanding the context of ah1pdm influenza virus. strategies to prevent transmission of influenza include use of respirators. ffp2 and n95 respirators are certified to fil-ter at least 95% of particles (0ae3 lm in diameter), and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. [1] [2] [3] we have developed a proprietary acid-polymer formulation to coat a standard ffp2 respirator with an antiviral layer. we aimed to test this coated respirator for antiviral efficacy against a range of influenza viruses. a series of tests compared the antiviral efficacy of coated and uncoated respirators in conditions designed to simulate real-life exposure to influenza by varying the route of inoculation, contact time, temperature, humidity, moisture, and contaminating substances. we also investigated whether infectious viruses could be transferred from contaminated respirator surfaces to gloves. we tested human, swine, and avian influenza viruses, including influenza a and b viruses. influenza a subtypes were the a ⁄ h1n1 2009 pandemic strain, seasonal h1n1, h5n1, h3n2, h5n9, and h2n2. in each test, suspensions of influenza viruses were prepared to 4-8 log 10 tcid 50 ⁄ ml in mem. in some tests, organic contaminants (yeast, bsa, and mucin) were added. one set of respirators was maintained at 40°c and 75% relative humidity for 24 hours before the viral challenge, and repeatedly sprayed with he-pes buffer to simulate respiratory secretions. for each test, three coated (glaxosmithkline actiprotect) and three uncoated (sperian willson easy fit) ffp2 respirator samples were inoculated with 0ae2 ml of a viral suspension, which was applied with a pipette, sprayed, or aerosolised to create airborne droplets. after 1 minute at room temperature (on a shaker), the respirator samples were assayed for the presence of infectious viruses using standard methods. 4 in one test, after a 1 minute contact time of the respirator with the virus, nitrile gloves were applied with light pressure to the outer surface of inoculated respirator samples and then assayed after 1 minute. samples were put into test medium (mem, supplemented with antibiotics [penicillin, gentamycin, or streptomycin] and amphotericin b or l-glutamine). the supernatants were vortexed, extracted, and used to prepare serial 10-fold dilutions in mem. each dilution was used to inoculate four wells of rmk cells in a multi-well plate, and these cultures were incubated and scored over 7 days for cytopathic effects, cytotoxicity, and viability. (some tests substituted mdck cells; others used inoculated embryonated chick eggs.) all tests included negative cell controls, cytotoxicity controls, and neutralisation controls. the spearman-karber formula was used to calculate viral loads as tcid 50 or eid 50 . 4 antiviral efficacy was calculated from the difference between the geometric mean loads of influenza virus on the coated and uncoated respirators after 1 minute of exposure. the viral loads applied to respirators in these experiments ranged from 5ae5 to 8ae1 log 10 tcid 50 , and were therefore high in comparison with respiratory secretions from infected patients at the peak of influenza symptoms (range 3-7 log 10 tcid 50 ). 1 tables 1-2 show that the average viral loads detected on uncoated ffp2 respirator samples remained high in all conditions tested, ranging from 3ae2 to 6ae9 log 10 tcid 50 (or 4ae5-5ae0 log 10 eid 50 ). in contrast, the average viral load on coated respirators after 1 minute of exposure ranged from below the limits of detection to £1ae5 log 10 tcid 50 (1ae0 log 10 eid 50 ). therefore, the relative antiviral efficacy of the coating ranged from ‡2ae7 to 6ae4 log 10 . table 1 shows that the relative antiviral efficacy of the coated mask remained high in simulated-use conditions such as organic contaminants and repeated saturation at high temperature and humidity. in the experiment to test transfer of viruses from respirators, the gloves applied to regular uncoated inoculated respirators had a viral load of 3ae5 log 10 eid 50 (table 1) . by contrast, no viruses were detected on either the coated respirators or the gloves applied to them. the relative reduction in contamination was therefore ‡2ae5 log 10 . ‡4ae8 log 10 viral load with organic contaminants* 6ae1 0 ae7 5 ae3 log 10 viral load after heat, moisture, and simulated secretions** 5ae1 0 ae8 4 ae3 log 10 viral load transferred to glove** 3ae5 £1ae0 ‡2ae5 log 10 eid 50 *influenza subtype was a ⁄ h5n1, and strain was vnh5n1-pr8 ⁄ cdc-rg. **influenza subtype was a ⁄ h3n2, and the strain was hong kong ⁄ 8 ⁄ 68. results are mean log 10 tcid 50 , unless specified otherwise. results are mean log 10 tcid 50 , unless specified otherwise, based on an infectivity assay in triplicate. limits of detection varied. *2009 pandemic strains. **results are mean log 10 eid 50 , based on a haemagglutinin assay in duplicate. options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 301-327 strategies to prevent transmission of influenza include use of respirators, and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings for protection against pandemic influenza. 1-3 ffp3 respirators are certified in europe to filter at least 99% of nacl particles (0ae3 lm in diameter), and ffp2 and ffp1 respirators must filter at least 95% and 80% of these particles, respectively. influenza a viruses are typically 0ae1 lm, and can be carried in aerosolised droplets smaller than 1 lm in diameter, which can disperse widely, remain airborne for 8 hours, and be inhaled deeply into the respiratory tract. 4 we have developed an acid-polymer formulation to coat the outer layer of a standard ffp2 respirator, in order to provide antiviral activity on the outer surface. we compared this coated respirator against standard ffp1, ffp2, and ffp3 respirators for filtration of aerosolised influenza viruses. the aim was to simulate protection against infectious viruses in droplets released when infected people cough and sneeze, and during aerosol-generating procedures in healthcare settings. the first assay compared three samples of coated ffp2 respirators (glaxosmithkline actiprotect) with three ffp2 controls (sperian willson easy fit). for each test, suspensions of influenza a (h3n2) at 7ae1 log 10 tcid 50 ⁄ ml in 0ae1· minimum essential medium (mem) were aerosolised with a nebulizer. the airborne droplets were introduced into a sterile chamber upstream of a respirator sample for 2 minutes, at a flow rate of 28ae3 l ⁄ minute. constant airflow was maintained for another 2 minutes after exposure to the virus. then the collection dish in the downstream sieve sampler (anderson) was assayed for infectious viruses using standard techniques. 5 briefly, serial dilutions of the collection medium (mem with 1% fbs, 5% gelatine, and 2% hepes, supplemented with antibiotics and amphotericin b) in mem + trypsin were used to inoculate madin-darby canine kidney epithelial (mdck) cells in quadruplicate in a multi-well plate. these cultures were then incubated and scored over 4-6 days for cytopathic effects, cytotoxicity, and viability. negative cell controls and cytotoxicity and neutralisation controls were also performed. the spearman-karber formula was used to calculate tcid 50 . the second assay compared five samples of coated respirators with five ffp1 controls (3m 9310) and five ffp3 controls (3m 1863). a suspension of influenza a (h1n1), at 8ae3 tcid 50 ⁄ ml, was nebulized for 1 minute and 40 seconds into the aerosol chamber, at a flow rate of 28ae3 l ⁄ minute, followed by constant airflow for 5 minutes after exposure to the virus. then the collection medium in the downstream chamber (as before, with 1% nahco 3 ) was assayed as described above. initial viral loads in the first and second assays were 8ae1 and 7ae9 log 10 tcid 50 , respectively, and were therefore high in comparison with respiratory secretions from infected patients at the peak of their influenza symptoms (range 3-7 log 10 t-cid 50 ). table 1 shows that the average viral load that passed through the uncoated ffp2 respirators in the first assay was 3ae6 log 10 tcid 50 . the average viral load that passed through the coated respirators was 1ae4 log 10 tcid 50 . therefore, for active filtration of viruses, the relative efficacy of the respirator with antiviral coating was 2ae2 log 10 greater than the uncoated respirator. for surface inactivation, the relative antiviral efficacy of the coated respirator was 3ae9 log 10. in the second study, table 2 shows that the average viral load that passed through the uncoated ffp1 respirators was 5ae2 log 10 tcid 50 . in contrast, 3ae1 log 10 tcid 50 passed through the coated ffp2 respirators. by comparison with the viral load when no respirator was present (8ae9 log 10 tcid 50 ), the ffp1 respirators reduced the viral load by 3ae7 log 10 , and the coated ffp2 by 5ae8 log 10 . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load by 2ae1 log 10 more than the ffp1 respirators. in this second study, the average viral load that passed through the uncoated ffp3 respirators was also 5ae2 log 10 tcid 50 . by comparison with the viral load when no respirator was present (8ae9 log 10 tcid 50 ), the ffp3 respirators reduced the viral load by 3ae7 log 10 . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load passing through the mask by 2ae1 log 10 more than the ffp3 respirators. table 2 also shows that the coated respirators reduced the infectious viruses remaining on the mask surfaces by 3ae8 log 10 more than the ffp1 respirators, and 3ae6 log 10 more than the ffp3 respirators. even with a very high viral challenge, the coated respirators prevented passage of at least an additional 2ae1 log 10 infectious viruses, compared with uncoated respirators. large numbers of infectious virions passed through all uncoated respirators tested. ffp3 respirators were no more effective than ffp1 respirators at blocking airborne influenza viruses. based on these in-vitro results, respirators with the antiviral coating could be expected to provide more protection than standard respirators from the risk of inhaling influenza viruses. strategies to prevent transmission of influenza include use of respiratory protection. ffp2 and n95 respirators are certified to filter at least 95% of nacl particles (0ae3 lm in diameter), and many guidelines have recommended that healthcare workers wear these respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. 1,2 we have developed a proprietary acid-polymer formulation, designed to coat a standard respirator and inactivate influenza viruses on contact. we tested this coated respirator for cytotoxicity, skin irritation, and sensitisation potential. the antiviral coating was also tested for stability and leaching under extreme environmental conditions, such as physical abrasion and simulated breathing at different temperatures, levels of humidity and co 2 , and saturation with contaminants. eight coated respirators were tested at standard relative humidity (60% rh) for 8 hours, and one at elevated humidity (80% rh) for 2 hours. four coated masks were treated with synthetic blood or oral secretions, and then tested at 30% rh for 1 hour. the sample respirators were sealed onto a mannequin head inside an airtight chamber, and air at 30°c and 5000 ppm co 2 was pumped through the masks by a cyclic breathing machine at 24 l ⁄ minute. a 37 mm glass-fibre filter was placed behind the respirator, over the mannequin's mouth opening. at the end of all tests, these filters were eluted and analysed using high-performance liquid chromatography (hplc). standard in vitro methods were used to assess the cytotoxicity of the coated polyester and uncoated polypropylene layers of the respirator (glaxosmithkline actiprotect). 3 samples were extracted in minimum essential medium (mem), supplemented with serum, penicillin, streptomycin, amphotericin b, and l-glutamine, at 37°c for 24 hours. triplicate monolayers of mouse fibroblast cells (l-929) were dosed with each extract (including a reagent control and negative and positive controls), and incubated at 37°c in 5% co 2 for 48 hours. after 48 hours of incubation with samples or controls, the monolayers of mouse fibroblast cells were examined microscopically for abnormal cell morphology or cellular degeneration. samples of the coated respirator (comprising four polypropylene layers bonded to the coated polyester outer layer) were applied under occlusive patch conditions to the skin of 51 adults. controls, including individual layers, were applied in the same way. in a separate patch test, samples of the coated polyester outer layer and controls were applied under the same conditions to 219 adults. after 24 hours, test patches and controls were removed. sites were then scored for itching, erythema, oedema, epidermal damage, and papular response after 48 and 72 hours. the patches were applied three times a week for 3 weeks. to evaluate sensitisation, test patches were applied 10-15 days later for 24 hours at different sites to the original samples. after this challenge, skin was assessed and graded for sensitisation potential after 48 and 72 hours. table 1 shows that no residues of the antiviral coating or degradation products were detected in the air that had passed through any of the eight respirators. cytotoxicity tests showed that the coated respirator material caused 10% cell lysis or toxicity, classified as slight reactivity (grade 1), and that uncoated material caused no cell lysis or toxicity (grade 0) ( table 2) . results for positive and negative controls were severe reactivity and no reaction, respectively. from the results of the two human repeat-insult patch tests, neither the coated or uncoated layers nor the fullthickness respirator fabric caused irritation (including itching, erythema, edema, vesiculation, epidermal damage, papules, or reactions beyond the patch site) or sensitisation in any of the adult volunteers at any of the time points. based on these results, in conjunction with published data on acute and repeat-dose toxicity, mutagenicity, local irritation, dermal sensitisation, and inhalation safety for all components of the antiviral coating, the potential topical or inhalation exposure to the coated antiviral respirator does not pose a safety risk. the antiviral coating is durable and stable, and stays on the outer surface of the respirator, even in extreme environmental conditions. the coated respirator is non-irritating and non-sensitising. therefore, this respirator is considered to be well-tolerated and safe for its intended use. ies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. knowing how influenza virus is transmitted at home and in school is the key to preventing its spread. at the previous two meetings of this conference, 1,3 we introduced our study of household transmission of seasonal influenza and reported our conclusion that protracted survival of the virus even after treatment increases household transmission, and is a major factor in the transmission of the virus to infants. on the other hand, during the recent pandemic, many schoolchildren developed serious respiratory tract disorders, which again highlights the significance of schoolbased transmission of the disease. in this study, we compared transmission of a new influenza strain at home and in school with that of seasonal influenza and proposed countermeasures. the for the analysis of school-based transmission, the epidemic status of seasonal influenza in 4237 children at six elementary schools over the past two seasons (2002-2003 and 2003-2004 seasons) was compared with that of pdmh1 in 1913 children at two primary schools. using observational data of school-based transmission, we also constructed a model for influenza transmission 3, 4 and evaluated the effects of factors that could affect influenza transmission (e.g., antibody prevalence, transmission rate, non-infectious latent period, infectious latent period, school closure) through the use of 1000 simulations. in this study, a diagnosis of influenza was confirmed by rapid influenza antigen detection kit. we previously reported the high sensitivity of the kits, 5-7 not only for seasonal influenza, but also for h1n1 pandemic 2009 compared to virus isolation and pcr. serum antibody was not investigated. most of the index patients were treated with oseltamivir or zanamivir, and 30 patients were treated with amatadine. no treatment was done for 16 patients. no nai therapy was done as prophylaxis within the family. the incidence of households with an initial case patient who subsequently infected another member of the household was 21ae6% (275 of 1271 households) for seasonal influenza or 18ae3% (112 of 612 households) for pdmh1. thus, the household incidence of pdmh1 was lower than that of seasonal influenza. in addition, the percentage of family members in households who were infected by initial case patients (household transmission rate) was 10ae5% (375 of 3573 individuals) for seasonal influenza or 7ae9% (141 of 1792 individuals) for pdmh1. thus, the household transmission rate was also lower for pdmh1 than that for seasonal influenza. effect of family size on household incidence and household transmission rate an analysis of the effect of family size on household incidence showed that, in families consisting of 2-7 individuals, the incidence of seasonal influenza in order of increasing family size was 3ae5%, 17ae4%, 24ae5%, 24ae2%, 32ae4%, and 42ae9%, respectively, and the incidence of pdmh1 was 0ae0%, 13ae5%, 17ae8%, 24ae7%, 42ae9%, and 33ae3%, respectively, indicating that household incidence tends to increase with increasing family size. in contrast, no definite relationship was noted between household transmission rate and family size. transmission rates for seasonal influenza in order of increasing family size were 3ae5%, 10ae6%, 11ae8%, 8ae0%, 10ae3%, and 7ae1%, respectively, or 0ae0%, 6ae7%, 7ae7%, 8ae0%, 15ae2%, and 5ae6%, respectively, for pdmh1 (shown in table 1 ). effect of age cohort of initial case patient in household on household incidence and household transmission rate an analysis of the effect of the age cohort of the initial case patient in the household on household incidence and transmission rate showed that the household incidence of seasonal influenza in c1, c2, c3, and c4 was 26ae7% (112 of 419 households), 21ae4% (66 of 308 households), 16ae3% (21 of 129 households), 9ae9% (9 of 91 households), and for m and f was 19ae6% (37 of 189 households) and 22ae5% (29 of 129 households), respectively. therefore, household incidence was the highest in c1, followed by the parents. when the initial case patient was a child, the household incidence increased with decreasing patient age. in contrast, the household incidence of pdmh1 in c1, c2, c3, and c4 was 23ae1% (36 of 156 households), 17ae3% (42 of 243 households), 8ae1% (9 of 111 households), 10ae0% (4 of 40 households), and for m and f was 30ae8% (12 of 39 households) and 39ae1% (9 of 23 households), respectively. therefore, household incidence was higher when the initial case patient was a parent, rather than a child. the household transmission rates for seasonal influenza from c1 to f were 13ae4%, 10ae1%, 6ae0%, 3ae9%, 10ae3%, and 12ae6%, respectively. therefore, as for household incidence, the highest rate (13ae4%) was observed in c1. the corresponding household transmission rates for pdmh1 were 9ae9%, 6ae9%, 4ae0%, 4ae3%, 12ae7%, and 22ae1%, respectively, with the highest transmission rates observed for infections from parents (shown in table 1 ). if the rate of individuals with a secondary infection transmitted from the initial case patient in a household is presented as a percentage of the total number of affected individuals, the rates for seasonal influenza and pdmh1 were 22ae8% (375 of 1646 individuals) and 18ae9% (142 of 753 individuals), respectively. therefore, the rate of individuals with a secondary infection was lower for pdmh1 than that for seasonal influenza. by age cohort, the corresponding rates of individuals for seasonal influenza in c1, c2, c3, and c4 were 22ae3% (120 of 539 individuals), 11ae5% (40 of 348 individuals), 12ae2% (18 of 147 individuals), 7ae1% (7 of 98 individuals), and for m and f was 42ae7% (141 of 330 individuals) and 27ae1% (48 of 177 individuals), respectively. for pdmh1, the corresponding rates in c1, c2, c3, and c4 were 24ae3% (50 of 206 individuals), 9ae3% (25 of 268 individuals), 9ae0% (11 of 122 individuals), 4ae8% (2 of 42 individuals), and for m and f was 51ae9% (42 of 81 individuals) and 35ae3% (12 of 34 individuals), respectively. these findings indicate that, especially in the case of pdmh1, most secondary infections in parents tend to be transmitted from another household member. the mean annual prevalence of seasonal influenza and the new influenza strain at the elementary schools for the two seasons was 19ae6% and 10ae3%, respectively, whereas the prevalence determined 7 days after appearance of the first case in school was 3ae9% and 4ae5%, respectively. in the recent season at the same elementary schools, however, the prevalence was a high 43ae5%. since the prevalence at 7 days after the appearance of the first case in school was already 17ae5%, these data show that the influenza virus spread quickly throughout the schools. at the schools with high transmission rates in the early period of the pandemic, new infections were confirmed even 4 days after the school closure action was taken. these findings indicate that pdmh1, the current influenza virus, has a long latent period during which it becomes infectious and spreads from infected individuals to numerous others in their vicinity. we constructed a model for influenza transmission in schools and estimated the time course of changes in the number of expected cases and the expected prevalence during the season. in this model, school children were divided into six groups depending on the stage of infection: uninfected period with no immunity, non-infectious latent period, infectious latent period, onset, post-onset infectious period, and immune period. it was assumed that schoolbased transmission occurred during the infectious latent period prior to onset and that no infections occurred during the post-onset infectious period because children were absent from school. due to the long latent period of pdmh1, the distribution of the non-infectious latent period of pdmh1 was established as (day 1, day 2, day 3, day 4) = (10%, 40%, 40%, 10%) and the distribution of the infectious period as (day 0, day 1, day 2) = (10%, 80%, 10%). when simulations were performed under these conditions using the model for school-based transmission of influenza in which children from classes with an outbreak were kept at home for 3 days, the time course of changes in the number of affected individuals actually observed and the time course of changes in the number of expected cases were determined. the expected prevalence under these conditions was 35%. to evaluate the effect of school closure, simulations were performed based on the assumption that children from affected classes were not kept at home for 3 days. it was shown that there was an increase in the expected number of cases during the 3 days corresponding to the period of actual school closure and that the expected prevalence increased to 52%. based on these findings, it was concluded that keeping children home from classes with an outbreak is an effective means of controlling the transmission of influenza in schools (shown in figure 1 ). if the transmissibility of pdmh1 virus at home is estimated based on the speed of transmission and the degree to which pdmh1 is prevalent in schools, it would be expected that the household transmission of pdmh1 is also higher than that of seasonal influenza. in fact, the opposite is the case. this paradox can be explained in two ways. 1. the number of children aged 19 or more and parents with pdmh1 influenza as a percentage of the total number of affected individuals is lower than those with seasonal influenza (20ae8% versus 37ae2%). further, although the number of parents with a secondary infection was high at home, the percentage of the total number of individuals with pdmh1 was a low 3ae8% (29 of 754 individuals), compared to that for seasonal influenza (5ae5% [91 of 1640 individuals]). in other words, adults are less susceptible to pdmh1 infections and there was a correspondingly small number of affected individuals. therefore, it was considered that the transmission rate at home was lower than that at school for this reason. 2. the percentage of households with more than one affected individual within the same family was higher for pdmh1 at 28ae5% (157 of 550 households) than for seasonal influenza at 24ae4% (309 of 1265 households). in the patients secondarily infected with pdmh1, 26ae8% of them showed symptoms of infection 10 days or more after the onset in the first patient, suggesting that they were not infected at home, and the actual household transmission was 20ae9% (115 of 550 households). therefore, although the prevalence was higher for pdmh1, it seems that household transmission was lower because households with an affected individual implemented satisfactory control measures against infection. seasonal influenza differs greatly from pdmh1 influenza in its transmissibility at home and in school. in the household transmission of pdmh1 influenza, both the household incidence and household transmission rate of pdmh1 were low compared to those for seasonal influenza. although transmission of seasonal influenza from infants to parents was marked, in the case of pdmh1, the reverse was true with transmission from parents to children being predomi-nant. it should be noted that household transmission in mothers was common in all eight seasons, suggesting the need to reconsider control measures against infection when nursing unwell family members. in the case of school-based transmission, pdmh1 was more prevalent than seasonal influenza, indicating that the virus spread quickly throughout the schools. this difference was attributed to the long infectious latent period when pdmh1 rapidly became rampant in the schools. an analysis of school-based transmission using a model for influenza transmission showed that, when 10% of the student population is infected, schools should be closed for five consecutive days in order to minimize the spread of the disease. the effectiveness of seasonal influenza vaccine in preventing pandemic and seasonal influenza infection: a randomized controlled trial introduction household transmission has been estimated to account for one-third of all influenza transmission, 1,2 and children are at high risk of spreading the disease. with reference to previous evidence, 3-15 some vaccine deployment strategies target children to prevent them from infection and transmitting influenza. 16 nevertheless, few studies evaluated the effectiveness of vaccinating children in reducing household transmission. 10, 11 during 2008-09, a pilot randomized controlled trial was conducted to investigate such effect by studying households with school age children randomized to receive trivalent inactivated seasonal influenza vaccine (tiv). 17 the monovalent vaccine against pandemic influenza a (h1n1) (ph1n1) had yet been available until the end of the first wave. various conclusions have been made as to whether seasonal influenza vaccine might possibly protect against ph1n1. [18] [19] [20] [21] [22] [23] [24] [25] we report findings on the effectiveness of tiv against ph1n1 observed in our cohort. households were screened if they expressed interest after receiving invitation letters distributed via their children's school or an existing pediatric cohort study. 26 to be eligible, the household had to include at least one child aged 6-15 years who was not allergic or hypersensitive to any of the tiv components. children known to have immunosuppressive conditions or other contraindications against tiv were also excluded. written consent and assent were obtained from participants aged above 18 years and those aged 8-17 years, respectively. proxy written consent was obtained from legal guardians or parents for participants younger than 18 years. ethical approval was obtained from the institutional review board of the university of hong kong. consented households were allocated to the tiv and placebo group (in ratio 3:2) according a code generated by block randomization with random block sizes of 5, 10, and 15. an independent nurse prepared 0. one child (study subjects) from each household in the tiv group received a single dose of tiv with one child from each household in the placebo group receiving a single dose of saline placebo. parents and legal guardians were asked to report any adverse reactions 4 days following vaccination. all participants, study nurses, and other research staff were blinded to the allocation and administration of vaccine or placebo. the vaccine allocation sequence was only disclosed to the investigators at completion of the study. serum specimens were collected from subjects shortly before (november-december 2008), one month after vaccination (december 2008-january 2009), and after the winter (april 2009) and summer influenza seasons (august-october 2009). serum specimens were obtained from household contacts at baseline and after the winter and summer influenza seasons. all household members recorded any fever ‡37.8°c, chills, headache, sore throat, cough, presence of phlegm, coryza, or myalgia daily on a symptom diary. they were also invited to report to the study hotline immediately if they experienced at least 2 of the above signs or symptoms. as a response, the study nurse would visit the households with any sick members and collect nose and throat swab from all household members. the households were also telephoned monthly or increased to fortnightly during influenza seasons to monitor for signs and symptoms and remind them to report to the hotline. supermarket or book vouchers (for children) were given to the households including us$13 for each serum specimen collected, us$6.5 for each home visit, and us$65 for completion of the study. serologically-indicated influenza infection was the primary outcome of this study. it was define as a ‡4 fold rise in antibody titer within each influenza season. other study outcomes included rt-pcr confirmed influenza virus infection, acute respiratory illness (ari) (two of any of the above listed signs or symptoms), and influenza-like illness (ili) (fever ‡37.8°c with cough or sore throat). antibody titers against the vaccine strains were obtained by testing each serum specimens by haemagluttination inhibition (hai). viral microneutralization (vn) using standard methods was found to be more sensitive than hai in detecting antibody response against a ⁄ california ⁄ 04 ⁄ 2009(h1n1) in another study conducted by our group 27 and was, therefore, used in this study. the sera was initially diluted at 1 ⁄ 10 and further tested in serial doubling dilutions. nose and throat swabs were tested by reverse transcription polymerase chain reaction (rt-pcr) for influenza a and b viruses. technical details of the laboratory methods have been reported elsewhere. 27, 28 fisher's exact test and chi-squared tests were used to compare count data including occurrence of side effects, laboratory confirmed, and clinically defined influenza infections. wilcoxon signed-rank test were used to compare the serum antibody titers between groups. exact binomial method or the wald approximation was used to estimate 95% confidence intervals where appropriate. all analyses were carried out in r version 2.8.1 (r development core team, vienna, austria). twenty-five primary and secondary schools in the district of the study clinic were invited to participate. to parents of three schools that agreed to take part and another study cohort, 3690 invitation letters were sent and 105 households were enrolled. personal referrals were made from these parents to enroll 14 additional households. among 119 enrolled households, 1 subject with history of epileptic seizure was assessed to be contra-indicated against receiving the vaccine. blood taking failed in another subject, and both of them withdrew from the study. eleven households did not complete the study. table 1 shows subject and household contacts of the tiv and placebo group were similar in demographics and prior influenza vaccination history. antibody titers before vaccination were comparable between groups (data not shown). most study subjects who received tiv showed antibody titer ‡40 against the vaccine strains 1 month after receiving tiv, and the proportion was significantly higher than those who received placebo (a ⁄ h1n1 93% in tiv versus 68% in placebo group, p < 0.01; a ⁄ h3n2 97% versus 61%, p < 0.01; b 99% versus 91%, p = 0.15). none of the study subjects had antibody titer ‡40 against ph1n1 following receipt of seasonal tiv. no serious adverse reactions were reported, and only pain at injection sites was slightly higher in tiv group (data not shown). subjects who received tiv had lower rates of serologically confirmed seasonal influenza a(h1n1) (8% versus 21%, p = 0.10), a(h3n2) (7% versus 12%, p = 0.49) and b infection (3% versus 8%, p = 0.36, although the differences were not statistically significant (table 2) . study subjects had higher rate of serologically confirmed ph1n1 infection (32% versus 17%, p = 0.09), yet it was not statistically significant. after adjusting for potential cross reactive antibody response, 31% of subjects in tiv versus 12% in placebo groups showed ph1n1 infection confirmed by either serology or rt-pcr (p = 0.04). little differences were observed for rt-pcr confirmed infection, ari, and ili in results combining the winter and summer influenza seasons. during winter season when seasonal influenza predominated, study subjects who had received tiv showed a lower tendency to develop ili (15% versus 23%, p = 0.43) or ari (46% versus 54%, p = 0.52). an opposite tendency was seen (ili 27% versus 19%, p = 0.43; ari 49% versus 44%, p = 0.68) during summer when ph1n1 predominated. however, these differences were not statistically significant. rates of ili in subjects infected with ph1n1 did not differ statistical significantly between subject who received tiv and placebo (40% versus 25%, p = 0.30). the study was not powered to detect indirect benefits to household contacts of vaccines resulting from reduced household transmission. attack rates were found to be similar between household contacts of subjects received tiv and placebo (data not shown). to examine potential factors that might affect risk of laboratory confirmed ph1n1 infection, a multivariable logistic regression model was fitted to study all subjects and their household contacts. younger participants aged below 16 years were found to have a higher risk (<16 years or = 6.60, 95% ci 2.17, 20.13; 16-45 years or = 2.53, 95% ci 0.80, 7.99, >45 or = 1.00). after adjusting for age, sex, and date of study completion, receipt of tiv for the 2008-9 influenza season was not found to affect risk of ph1n1 infection. however, participants who had laboratory confirmed seasonal influenza infection during the study period had 65% lower risk of ph1n1 infection (infected with seasonal influenza or = 0.35, 95% ci 0.14, 0.87; not infected with seasonal influenza or = 1.00). as (see table s1 for winter and summer results separately). influenza-like illness (ili) defined as temperature ‡37.8°c plus cough or sore throat; acute respiratory illness (ari) defined at least any two of fever ‡37.8°c, chills, headache, sore throat, cough, presence of phlegm, nasal congestion, runny nose, muscle or joint pain. limited by the sample size, we were not able to differentiate between the protective effect of seasonal a(h1n1) and a(h3n2) infection against ph1n1. other details of the results from the study were published elsewhere. 17 discussion a non-significantly higher rate of ph1n1 infection was observed in study subjects who received tiv compared to placebo. results from a multivariable logistic regression suggested that such a pattern might be explained by more common seasonal influenza infection in placebo group prior to the pandemic, protecting the placebo group against ph1n1. seasonal influenza infection within 3-6 months observed in our study might have conferred better cross protection than tiv against ph1n1. this resembles similar previous findings on cross protection between influenza infections in human and animal studies. [29] [30] [31] [32] [33] [34] [35] [36] however, the same phenomenon has not been observed in some studies on seasonal influenza vaccine against ph1n1. 18, 21, [23] [24] [25] apart from differences in study design and vaccine used, we speculate that a short time interval between ph1n1 and most recent seasonal influenza peak activities might be crucial for the phenomenon. hong kong is a subtropical area where the 2009 pandemic was preceded immediately by summer seasonal influenza circulation and a few months apart from the winter 2008-9 influenza peak. if cross protection from seasonal influenza lasts for only a short period, it might have waned below partial cross protection from tiv over time from last seasonal influenza infection. the current study is limited by a small sample size, and further studies are required to confirm our hypothesis. while tiv is only effective against matching strains, a universal influenza vaccine could provide better protection against the ever evolving influenza viruses. introduction immunisation of healthy, as well as high risk, children has been the focus of much recent attention both in prevention of seasonal influenza and during the 2009 h1n1 pandemic. detailed information on reactogenicity, particularly for newer vaccine formulations that include adjuvants, is limited. we recently reported results of a head-to-head comparison of two 2009 h1n1 pandemic influenza vaccines in children in the uk. 1 here we present new, detailed analyses of reactogenicity data from that study, which has important potential implications for future paediatric influenza vaccine development and use. we compared the safety, reactogenicity, and immunogenicity of two h1n1 influenza vaccines, one as03 b (tocopherol based oil in water emulsion) adjuvanted egg culture derived split virion, the other non-adjuvanted cell culture derived whole virion, given as two dose schedules 21 days apart, in a randomised, open label trial as previously reported. the study was age stratified (6 months to under 3 years & 3-12 years) to ensure adequate data in young children. age appropriate safety data (simplified for under 5 year olds) were collected for 7 days after each vaccine dose and serum was collected at enrolment & 21 days after the second dose. nine hundred-thirty seven children received vaccines as per-protocol. when comparing the two vaccines, grade 3 ( ‡50 mm) local reactions were seen more frequently following the adjuvanted than the non-adjuvanted vaccine in both age groups, after both vaccine doses. in children over 5 years old, 7ae2% versus 1ae1%, p < 0ae001, after dose one; 8ae5% versus 1ae1%, p = 0ae002, after dose two, in children under 5 years old, 1ae5% versus 0ae0%, p = 0ae06, after dose one (non significant, ns); 5ae9% versus 0ae0%, p < 0ae001 after dose two. fever ‡38°c (axillary measurement) was seen more frequently following the second dose of the adjuvanted vaccine compared to the non-adjuvanted vaccine in <5 year olds (22ae4% versus 12ae5%; p < 0ae05). looking specifically at the adjuvanted vaccine in under 5 year olds, comparing the second dose with the first, there were significantly higher rates of fever ‡38°c (axillary measurement) (22ae4% versus 8ae9%, p < 0ae001), local grade 3 ( ‡50 mm) reactions (5ae9% versus 1ae5%, p = 0ae02), pain (39ae4% versus 31ae5, p = 0ae02), use of analgesia or antipyretic medication (43ae7% versus 31ae5%, p < 0ae001), and decreased activity (31ae9% versus 20ae4%, p < 0ae001). the adjuvanted vaccine was significantly more immunogenic, most notably in the younger children. in <3 year olds, haemagglutination inhibition (hi) seroconversion rates were 98ae2% versus 80ae1%, p < 0ae001. among all general and local reactions measured, only the maximum temperature measured during the 7 days after the second dose of the adjuvanted vaccine showed a significant (positive) association with post vaccination hi titres. for each 1°c rise in temperature there was a 27% increase in titre (p < 0ae001). these reactogenicity data demonstrate a step towards the future possibility of one-dose influenza immunisation programmes for young children associated with low rates of fever and other reactions. the occurrence of fever following adjuvanted vaccine, seen particularly after a second dose in younger children, was quantitatively associated with enhanced antibody titres. this association was not seen with unadjuvanted vaccine. this apparent difference between the relatedness of the pyrogenic and immunogenic effects of the two vaccines merits further investigation. novel adjuvants appear to have the potential to overcome the relatively poor immunogenicity previously experienced with inactivated influenza vaccines in infants and young children. however, careful adjustment may be needed to optimise the balance between high protection and acceptable reaction rates. tries causing sporadic human infections. vaccination has been used as an effective public health tool for influenza prophylaxis. the goal of this study was to evaluate live attenuated influenza vaccine (laiv) vaccine candidates for subtypes h1 and h5. the attenuated phenotype of h1 and h5 laiv candidates has been proven in experiments in ovo and in vivo. in randomized clinical trials among adult volunteers, no significant adverse reactions attributable to the live vaccine occurred. our results indicate that pandemic laiv candidates were well tolerated and elicited serum, local, and cellular immune responses. the emergence and spread of highly pathogenic avian influenza h5n1 viruses in avian populations and concurrent infections in humans since 1997 has prompted efforts to develop vaccines for use in the event of an influenza pandemic. in 2009, the world faced a new h1n1 pandemic. immunization with inactivated or live vaccines is the primary measure for preventing influenza. laivs appear to be safe and efficacious, and might possibly provide broader immune responses than inactivated vaccines. our study evaluated laiv pandemic candidates as part of the global influenza pandemic preparation project outlined by the who. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated by routine technique in embryonated hen eggs. laiv and placebo were supplied by microgen (irkutsk, russia). the monovalent laiv was produced from the pandemic vaccine candidates and formulated to contain 10 7 and 10 6ae9 eid 50 per dose (0ae5 ml) of a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 and a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92, respectively. the vaccine or placebo was administered intranasally with a single-use dosing nasal sprayer. two doses were given at an interval of 21 days. one hundred-ninety healthy adults aged 18-60 years were randomly divided into groups to receive either pandemic vaccine candidates (204) or placebo (28) . subjects were informed about purposes and methods of the study and potential risks associated with participation. all participants had an hai antibody titer of £1:10 to a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) pandemic virus. in all there were 47 and 42 vaccines and 19 and 8 participants who received placebo, and were further tested for immune responses to h1n1 or h5n2 pandemic vaccine, respectively. another 29 participants vaccinated with h1n1 laiv were children between 12 to18 years old. before the children were vaccinated, their parents were advised about study and their consent was required before any child was enrolled. on the advice of the national ethics committee, we did not include a placebo group in this study. individuals were not enrolled if they had an acute illness or fever at the beginning of the study or a history of egg allergy. immune responses of subjects were assessed by routine hai test (evaluation of serum igg antibodies), elisa (evaluation of iga antibodies eluted from the nasal swabs into steril pbs), and cytokine flow cytometry assay (evaluation of virus-specific cd3 + cd4 + ifnc + and cd3 + cd8 + ifnc + peripheral blood mononuclear cells). the results of phenotypic analysis in ovo showed that pandemic vaccine candidates retained the cold adapted-temperature sensitive (ca ⁄ ts) phenotype, typical of the coldadapted parental mdv. in contrast and as expected, a ⁄ california ⁄ 07 ⁄ 2009 and a ⁄ duck ⁄ potsdam ⁄ 1406-86 parental strains had the non-ts ⁄ non-ca phenotype typical of wt viruses. the h5n2 pandemic vaccine candidate demonstrated an attenuated phenotype in mice and in java macaques and did not infect chickens. the vaccine attenuation study confirmed the attenuated phenotype of a a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 pandemic laiv candidate in mouse, ferret, and guinea pig models. the phase i ⁄ ii randomized, controlled, double-blind clinical study safety evaluation of pandemic vaccine candidates in adults clinical examination of subjects who received two doses of pandemic vaccine candidates indicated that both vaccines were well tolerated. no fever reactions were observed after the first or second vaccination. after the first vaccination, 33ae0% and 40ae0% of reactogenicity events consisting of catarrhal symptoms, such as pharyngeal irritation or hyperemia, were observed for h1n1 and h5n2 vaccine candidates, respectively. after revaccination, subjects did not report local or systemic reactions. to determine whether a serological response occurred in the cohort of immunologically naïve subjects vaccinated with pandemic vaccine candidates, hai and elisa tests were used (table 1) . post-vaccination geometrical mean titers (gmt) among 20 subjects who received two doses of h5n2 vaccine were significantly higher than pre-vaccination titers. the frequency of ‡4 fold antibody rises was significantly higher (47ae1%) after revaccination than after one dose (5ae9%). the percentage of subjects with post-vaccination serum hai titers to h5n2 ‡ 1:20 was 47ae1% and for titers ‡1:40, it was 29ae4%. no seroconversions in the placebo group were detected. the virus-specific nasal iga antibody response to vaccination after two doses of the h5n2 vaccine candidate demonstrated significant increases of ‡4 fold rise iga antibodies (65%) compared to one dose. cumulative data of h5n2 vaccination (all applied tests) showed 35% and 80% of conversions after the first and the second vaccination, respectively. increasing h5n2 vaccine virus infectivity from 10 6ae9 to 10 8ae3 eid 50 ⁄ dose lead to an enhancement of post-vaccination hai titers in vaccinees after the first vaccination to homologous h5n2 antigen from 5ae9% to 31ae0% of ‡4 fold antibody rises. values of post-vaccination serum hai antibody titers in subjects vaccinated with another pandemic vaccine candidate, a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38, also proved to be rather low. after the primary vaccination, the percentage of subjects with hai protective antibody titers ‡1:40 were 2ae1%. after revaccination, this parameter increased to 17ae0%. four-fold increases in serum hai antibody titres were four-fold conversions after the first and the second vaccination was 23ae4% and 34ae4%, respectively. elisa antibodies in nasal swabs showed had an advantage in detecting induction of local iga as compared to serum hai antibodies. after revaccination four-fold serum hai antibody conversions were 34ae4% vs. 63ae8% of iga conversions in nasal swabs, respectively. taking into account cumulative data of h1n1 vaccination (hai and elisa data), the obtained results were here and in the 42ae5% and 70ae2% of conversions after the first and the second vaccination, respectively. fourty-seven subjects were vaccinated with h1n1 laiv, and 19 who received a placebo were chosen for evaluation of cellular immune response by cytokine assays. after revaccination, the mean increases of both cd4 + and cd8 + memory cells were significantly higher in vaccinated subjects compared to the placebo group. interestingly, the same effect of vaccination was observed in vaccinees without detectable conversions of hai antibody titers. even after a single vaccination, the rate of subjects with significant increases of these cells in the blood was 37ae5% (cd8 + ) and 75% (cd4 + ). after the revaccination, the percentage of subjects with significant increases in cd8 + and in cd4 + cells was 68ae8%. immunogenicity of h1n1 pandemic vaccine candidate in children hai antibody results among children aged 12 to 18 years proved to be significantly higher when compared to adult subjects: after the first vaccination, 41ae4% of the children seroconverted; after revaccination, seroconversions reached 83ae3% ( table 2 ). the gmt rise to h1n1 vaccine with primary vaccination was 3:1; after revaccination it increased to 6:7. benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. 1 many years of laiv seasonal trials have shown excellent tolerability and low reactogenicity. [2] [3] [4] indeed, data showed that live influenza vaccines cause minimal systemic, local, and thermal reactions, generally from 0 to 3%. a different situation was observed in the cohort of immunologically naïve volunteers vaccinated with pandemic vaccines. the rate of local reactions to a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 and a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 9 vaccine candidates increased to 33ae0% and 40ae0%, respectively. after revaccination no significant local and systemic reactions were observed. this confirms, indirectly, the development of a sufficiently high level of protection after the first vaccination with pandemic laiv. the most important criterion for assessing the quality of vaccines is their estimated safety, epidemic effectiveness, and immunogenicity. however, current regulatory documentation 5 mandates that induction of serum antibodies, measured by hai, as the only criterion for a laiv immunogenicity evaluation. in addition to the standard hai assay, we determined serum (igg) and local (iga) antibodies in adult subjects vaccinated with an h1n1 pandemic vaccine candidate. evaluation of overall results obtained in these additional serological tests, as well as those from the hai assay, showed an immune response to the vaccine in the majority of subjects (42ae5% of ab seroconversions after the single vaccination and 70ae2% after revaccination, respectively). these data show that methods used to routinely measure laiv immunogenicity should be revised to include a number of additional immunological methods such as igg and iga elisa, and cytokine assays consistent with the recently updated who recommendations on laiv monitoring. these clinical studies clearly demonstrated that pandemic laiv candidates are effective at generating pandemic specific influenza immunity. a key finding from this study is that it may be practical to give the vaccine as a single dose to both children and adults. evaluation of our laiv pandemic vaccine candidates was performed as part of the global influenza pandemic preparation project outlined by the who. 6 it was considered that laiv could be produced in greater quantities and more rapidly than inactivated vaccines. together with the generation of herd immunity by laiv, this suggests that laiv implementation during the first wave of a pandemic may provide significant social, economic, and health benefits to the community. authors are thankful to path for the financial support of h1n1 pandemic vaccine study. we are grateful for the the main evolutionary mechanism of influenza viruses during inter-pandemic period is the antigenic drift, but the epidemiological picture of circulating viruses is complicated by a high level of heterogeneity of strains, even though drift does not occur, due to co-circulation of drifted and old strains or to co-circulation of viruses belonging to the same type ⁄ subtype but with different antigenic patterns. [1] [2] [3] [4] [5] [6] lack of data exists on the impact of the wide heterogeneity of circulating strains on the seroprotection and on-field effectiveness of influenza vaccine: in particular, little is known about the ability of influenza vaccine to elicit an effective immune response against isolates with few amino acid mutations with respect to vaccine strains that represent the majority of circulating viruses. mf59-adjuvanted vaccines, which are currently used for the prevention of seasonal influenza epidemics in elderly, are showed to confer higher seroprotection against homologous and drifted a(h3n2) strains than non-adjuvanted vaccines. [7] [8] [9] the broader immune response showed by mf59-adjuvanted vaccine was measured using hi and nt assays against egg-grown drifted strains representing vaccine composition changes during the following seasons, but its ability to elicit a broader immune response against circulating viruses belonging to vaccine cluster and presenting amino acid mutations onto antigenic sites or against on-field isolates not-antigenically distant from vaccine strains has not yet been investigated. showing amino acid changes onto antigenic sites in position 145 (n145k), 189 (n189k), and 227 (p227s) with respect to a ⁄ california ⁄ 7 ⁄ 04. in particular, a ⁄ genoa ⁄ 13 ⁄ 04 and a ⁄ genoa ⁄ 27 ⁄ 04 presents n126d amino acid mutation detected in clade 2 a ⁄ wyoming ⁄ 3 ⁄ 03-like viruses. 1 the ha sequences of a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 2 ⁄ 05, genoa ⁄ 11 ⁄ 05, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05 fell within the clade represented by the ha of a ⁄ califor-nia ⁄ 7 ⁄ 04; among these isolates, a ⁄ genoa ⁄ 2 ⁄ 05 and a ⁄ genoa ⁄ 11 ⁄ 05 showed antigenic site sequences very close to that of the 2005 ⁄ 06 vaccine strain, whereas ha sequences of a ⁄ genoa ⁄ 62 ⁄ 05, a ⁄ genoa ⁄ ⁄ 47 ⁄ 05 and a ⁄ genoa ⁄ 59 ⁄ 05 posses amino changes onto antigenic site a(r142k), c(g50e) and d(r208k), respectively. the ha sequences of more recent isolates fell within the clade represented by the ha of a ⁄ brisbane ⁄ 10 ⁄ 07 and characterized by the amino acid changes, relative to the ha of a a ⁄ california ⁄ 7 ⁄ 04, g50e and k140i, with the exception of a ⁄ genoa ⁄ 3 ⁄ 07, showing r142g and l157s amino acid changes present in viruses belonging to a ⁄ nepal ⁄ 921 ⁄ 06 clade. measure of genetic distance between vaccine and circulating strains was calculated as previously described by gupta. 10 two blood samples were collected from each subject, just before and 22 ± 2 day post-vaccination. all sera were stored at )20°c. all samples were tested at the laboratory of health sciences department, university of genoa, by haemagglutination-inhibition (hi) and neutralization (nt) assays, performed following the who criteria and standardised method in our laboratory, respectively. [11] [12] [13] guinea pig red blood cells were used for hi assay. all samples were assayed twice for hi and for nt. the obtained antibody titre was expressed as the reciprocal of the last sera haemagglutinating or inhibiting virus dilution. immunogenicity was determined by: geometric mean titre (gmt); mean-fold increase (mfi; ratio of post-to pre-vaccination titre); seroprotection rate (the percentage of subjects achieving an hi and nt titre ‡40 iu); and seroconversion rate (percentage of subjects with a fourfold increase in hi or nt antibody titers, providing a minimal post vaccination titer of 1:40). post-vaccination gmt was reported as ratio, with the corresponding 95% confidence interval, of gmts after vaccination with mf59-adjuvanted vaccine and with non-adjuvanted subunit vaccine. seroprotection and seroconversion rate 95% confidence interval was calculated using modified wald method. comparisons of seroconversion and seroprotection rates between subunit and mf59-adjuvanted vaccine groups have been analyzed by fischer's exact test. the results were evaluated against the committee for medicinal products for human use (chmp) criteria for approval of influenza vaccines in the elderly, which require that at least one of the following criteria be met: mfi >2; seroprotection rate >60%, or seroconversion rate >30%. furthermore, hi titres were also transformed into binary logarithms, corrected for pre-vaccination status, as described by beyer et al. 14 and were expressed as median titres, with the corresponding 25°-75°i nter-quantile range. comparisons of corrected post-vaccination titers between subunit and mf59-adjuvanted vaccine groups were analyzed by wilcoxon test. difference in immunogenicity profile between vaccine groups, expressed by ratio of different parameters, was correlated with genetic and antigenic distance between vaccine and viruses used in the study using spearman test. pre-vaccination titres were not significantly different between vaccine groups, for all 15 strains (data not shown). post-vaccination gmt ratios between mf59-adjuvanted and non-adjuvanted vaccine groups determined using hi and nt assays, with the corresponding 95% confidence interval, according to viral strain are shown in figure 1 . both vaccines met chmp requirements for mfi (>2), seroconversion (>30%), and seroprotection rate (>60%) against a ⁄ wyoming ⁄ 3 ⁄ 03-like, with the exception of a ⁄ genoa ⁄ 13 ⁄ 04 and a ⁄ california ⁄ 7 ⁄ 04-like circulating viruses and against egg-grown a ⁄ wyoming ⁄ 3 ⁄ 03, a ⁄ california ⁄ 7 ⁄ 04, and a ⁄ wisconsin ⁄ 67 ⁄ 05 strains; the immune response against a ⁄ genoa ⁄ 13 ⁄ 04 met the requirements for mfi and seroprotection rate only in mf59-adjuvanted vaccine group. requirements for mfi, seroconversion, and seroprotection rate against the a ⁄ brisbane ⁄ 10 ⁄ 07-like virus a ⁄ genoa ⁄ 2 ⁄ 07 and the a ⁄ nepal ⁄ 921 ⁄ 06-like genoa ⁄ 3 ⁄ 07 viruses and against egg-grown a ⁄ brisbane ⁄ 10 ⁄ 07 strain were reached only in subjects vaccinated with the mf59adjuvanted vaccine. a similar pattern emerged from the analysis of mfi, seroconversion and seroprotection rates using nt assays. subjects vaccinated with the mf59-adjuvanted vaccine showed significantly higher post-vaccination hi gmts against a ⁄ wyoming ⁄ 3 ⁄ 03-like, a ⁄ california ⁄ 7 ⁄ 04-like, a ⁄ nepal ⁄ 921 ⁄ 06-like and a ⁄ brisbane ⁄ 10 ⁄ 07like viruses, with the exception of a ⁄ genoa ⁄ 3 ⁄ 06, and against egg-grown a ⁄ california ⁄ 7 ⁄ 04, a ⁄ wisconsin ⁄ 67 ⁄ 05, and a ⁄ brisbane ⁄ 10 ⁄ 07 strains, compared with individuals immunized with the non-adjuvanted vaccine ( figure 1 ). the mf59-adjuvanted vaccine also induced significantly higher seroconversion and seroprotection rates against following correction for pre-vaccination status, hi titres were significantly higher for the mf59-adjuvanted vaccine group when evaluated against a ⁄ wyoming ⁄ 3 ⁄ 03-like viruses, a ⁄ brisbane ⁄ 10 ⁄ 07-like a ⁄ genoa ⁄ 2 ⁄ 07, and a ⁄ nepal ⁄ 921 ⁄ 06-like a ⁄ genoa ⁄ 3 ⁄ 07 strain ( figure 2 ). pre-vaccination titre corrected response was higher in subjects vaccinated with mf59 adjuvanted vaccine also against egg-grown a ⁄ wyoming ⁄ 3 ⁄ 03, a ⁄ california ⁄ ⁄ 7 ⁄ 04, a ⁄ wisconsin ⁄ 67 ⁄ 05, and a ⁄ brisbane ⁄ 10 ⁄ 07. among viruses more closely related to a ⁄ california ⁄ 7 ⁄ 04, subjects immunized with mf59-adjuvanted vaccine showed a significantly higher corrected titres against a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05 strains compared with the non-adjuvanted vaccine ( figure 2) . spearman test showed a clear correlation between the distances and the advantage offered by mf59 expressed by ratio between mfi, post-vaccination gmts, corrected post-vaccination median, seroconversion, and seroprotection rates calculated using hi test in the two vaccine groups. similarly, ratio between mfi, seroconversion, and seroprotection rates calculated with nt test correlated with the genetic and antigenic distance between vaccine and viruses used for the study. the ability of mf59 to enhance the immunogenicity and to elicit a broader immune response against drifted strains than non-adjuvanted vaccine is consistent with other findings reported during the last decade. [7] [8] [9] 15 in subjects vaccinated with the mf59-adjuvanted vaccine containing a ⁄ california ⁄ 7 ⁄ 04, the immune response, expressed by a number of parameters, such as crude and corrected postvaccination titers, seroconversion, and seroprotection rates calculated using hi and nt assays, is higher than that observed in individuals immunized with subunit vaccine when it is evaluated against a drifted strains, such as a ⁄ brisbane ⁄ 10 ⁄ 07-like and a ⁄ nepal ⁄ 921 ⁄ 06-like strains, and against egg-grown a ⁄ brisbane ⁄ 10 ⁄ 07 virus. for the first time in this study, the impact of heterogeneity of circulating strains antigenically close to the vaccine on the antibody response elicited by mf59-and non-adiuvanted vaccines is evaluated. immune response against viruses isolated during the 2004 ⁄ 05 season, that appear more phylogenetically close to 2004 ⁄ 05 vaccine strain a ⁄ wyoming ⁄ 3 ⁄ 03, was higher in subjects vaccinated with mf59-adiuvanted vaccine as demonstrated by higher crude and corrected post-vaccination hi titres and higher postvaccination nt titres, with the exception of a ⁄ genoa ⁄ 27 ⁄ 04, against whom the nt post-vaccination gmt is identical in mf59 and subunit vaccine groups. furthermore, hi seroconversion and seroprotection rates were higher in mf59 vaccine group when evaluated against a ⁄ genoa ⁄ 13 ⁄ 04 and a ⁄ genoa ⁄ 27 ⁄ 04. as far as the immune response against a ⁄ california ⁄ 7 ⁄ 04-like viruses, the small number of enrolled subjects did not allow appreciating differences using qualitative response indicators, but crude post-vaccination hi titres were higher in mf59 vaccine group for all the strains. interestingly, a ⁄ california ⁄ 7 ⁄ 04-like viruses with at least one amino acid change onto antigenic sites, i.e. a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05, showed a more marked difference in terms of response between the two vaccine groups. individuals immunized with mf59-adiuvanted vaccine showed higher corrected post-vaccination hi titres and post-vaccination nt titres in comparison with subjects vaccinated with plain vaccine. these response indicators were similar in the two vaccine groups when the response was evaluated against a ⁄ genoa ⁄ 2 ⁄ 05 and a ⁄ genoa ⁄ 11 ⁄ 04, which present no amino acid changes onto antigenic sites and identical hi titers respect with a ⁄ california ⁄ 7 ⁄ 04 at molecular and antigenic characterization, respectively. thus, the advantage offered by mf59 in terms of higher immunogenicity expressed by higher post-vaccination hi titres is observable also against viruses showing antigenic and molecular pattern undistinguishable from vaccine strain, but it became even more evident as the antigenic and molecular distance between vaccine and circulating strains grew. as emerged for a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05, one amino acid was a sufficient change in antigenic sites for 2-fold decrease of hi titre against homologous vaccine strain to observe 2-fold higher post-vaccination nt titers (mf59 ⁄ subunit postvaccination gmt ratio range between 1ae79 and 2ae45, figure 1) and one-dilution higher corrected post-vaccination hi titers in mf59 vaccine group ( figure 2) . finally, the correlation between the distance and the improvement offered by mf59 in terms of higher immunogenicity clearly emerged by spearman correlation analysis: it remains wellfounded both using a number of different response parameters obtained from hi and nt assays and calculating the distance by serological and genetic methods. outbreaks of h1n1pdm in pigs in commercial swine operations have been reported in several countries. in all incidents, epidemiological investigations have linked humans as the possible source of the infection to pigs. experimentally, it was established that the virus is pathogenic and transmits readily in pigs. 1 the natural outbreaks of h1n1pdm and laboratory studies underscore the threat that the virus poses to the swine industry and highlight the need for developing effective control strategies. in the united states, a trivalent live attenuated influenza vaccine (flumistò) has been licensed for use in humans since 2003. 2 in swine medicine, however, temperature-sensitive laivs are not available. currently, only inactivated vaccines are available for pigs, but they provide limited protection against antigenically diverse influenza viruses. additionally, the use of inactivated vaccines has been associated with enhanced pneumonia when immunized pigs were challenged with divergent viruses. 3 thus, the development of laivs has the potential to circumvent the drawbacks associated with commercial vaccines. with the aim of developing laiv temperature-sensitive influenza vaccines against the h1n1pdm virus, we have used reverse genetics to introduce attenuation markers in the polymerase genes of a swine-like tr h3n2 influenza virus, a ⁄ turkey ⁄ ohio ⁄ 313053 ⁄ 04 (h3n2) (ty ⁄ 04). 4 we chose this isolate because it grows well in both eggs and cell culturebased substrates, displays a broad host range, and has internal genes similar to the h1n1pdm virus. safety and efficacy studies of the ty ⁄ 04 att vaccine candidates in pigs demonstrated that this vaccine backbone is attenuated in swine and conferred sterilizing immunity upon an aggressive intratracheal challenge of pigs with the 2009 h1n1 pandemic virus. thus, introduction of genetic signatures for att in the backbone of a swine-like tr influenza virus resulted in highly attenuated and efficacious live influenza vaccines with promising applications veterinary medicine. 293-t cells and mdck cells were maintained as previously described. 5 a ⁄ turkey ⁄ ohio ⁄ 313053 ⁄ 04 (h3n2) (ty ⁄ 04) has options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 328-394 been previously described and it was kindly provided by yehia saif, ohio state university. 4 a ⁄ california ⁄ 04 ⁄ 09 (h1n1) (ca ⁄ 04) was kindly provided by the centers for disease control and prevention (cdc). generation of recombinant viruses by reverse genetics (rg) was done using a previously described method. 6 the genetic signatures for attenuation 5 were introduced into the pb2 and pb1 genes of ty ⁄ 04. ny 2:6 ty ⁄ 04 att is a 2:6 reassortant with the surface genes from the a ⁄ new york ⁄ 18 ⁄ 2009 (h1n1) virus and the ty ⁄ 04 att internal genes. all viruses were amplified in mdck cells to produce viral stocks. twenty-five pigs were divided into five groups (n = 5) and intranasally inoculated with 10 5 tcid 50 ⁄ animal of either h3n2:6ty ⁄ 04 att or with ny(h1n1)2:6ty ⁄ 04 att vaccines diluted in 2 ml of mem. two other groups were similarly inoculated with h3n2:6ty ⁄ 04 wt and h3n2:6ty ⁄ 04 rg and served as controls, whereas a fifth group was mockvaccinated with pbs alone. clinical observations were performed as previously described. 1, 7 efficacy of h1n1 ty ⁄ 04 att vaccine in pigs fourty pigs were divided in four groups (n = 10)( table 1) . group 1 was vaccinated with 10 5 tcid 50 ⁄ animal of ny(h1n1)2:6ty ⁄ 04 att through intranasal route, whereas group 2 was vaccinated intramuscularly with 2 ml of an adjuvanted uv-inactivated ca ⁄ 04 vaccine (uvadj-ca ⁄ 04). 7 group 3, non-vaccinated and challenged (nv+ca ⁄ 04), and group 4, non-vaccinated, mock-challenged (nv+mock), were also included. pigs were boosted two weeks later. fourteen days post boost (dpb), pigs from groups 1-3 were challenged intratracheally with 2 ml of 1 · 10 5 tcid 50 of ca ⁄ 04. following challenge, pigs were monitored using methods as previously described. 7 all statistical analyses were performed using graphpad prism software version 5ae00 (graphpad software inc., san diego, ca). the differences were considered statistically significant at p < 0ae05. the ty ⁄ 04 att-based vaccines are attenuated in swine pigs inoculated with wt ty ⁄ 04 viruses developed fever (>40°c) that peaked 24 hpi ( figure 1a) and shed large amounts of in nasal secretions ( figure 1b) . similarly, viral titers in bronchoalveolar lavage fluid (balf) collected at 3 dpi ranged from 10 5 to 10 6 tcid 50 ⁄ ml ( figure 1c ). at necropsy, the lungs from animals inoculated with these viruses had severe pneumonia ( figure 1d ). in contrast, none of the animals inoculated with h3n2 or h1n1 ty ⁄ 04 att viruses developed clinical signs following vaccination, indicating that the ty ⁄ 04 att viruses were safe for administration to pigs ( figure 1a) . correspondingly, there was 100-1000 fold less virus shedding from the nose of pigs vaccinated with ty ⁄ 04 att viruses as compared to unmodified ty ⁄ 04 viruses. in general, ny(h1n1)2:6ty ⁄ 04 att -vaccinated pigs shed less virus than h3n2:6 ty ⁄ 04 att inoculated pigs ( figure 1b ). in addition, viral titers in balf were significantly reduced (p < 0ae01) in ty ⁄ 04 attvaccinated pigs as compared to ty ⁄ 04 wt-infected pigs ( figure 1c ). although both vaccines caused mild gross and microscopic lesions in the lungs, the percentage of lung 0ae2 ± 0ae1* 0 ± 0* 0 ± 0* 0 ± 0* balf, bronchoalveolar lavage fluid, uvadj-ca ⁄ 04, uv-inactivated ca ⁄ 04 vaccine; nv+ca ⁄ 04, non-vaccinated, challenged positive control group; nv+mock, non-vaccinated, non-challenged negative control group. *significantly different from nv+ca ⁄ 04 control group at p < 0ae05. geometric mean hi titer against ca ⁄ 04 at the day of challenge. à percentage of macroscopic lung lesions given as mean score ± sem. § average viral titer (log 10 ) measure as tcid 50 per ml. -average viral titer (log 10 ) in balf at 5 dpc. involvement was not significantly different from mock-vaccinated pigs, corroborating the clinical findings that these vaccines are sufficiently attenuated in pigs ( figure 1d, e) . histopathologically, nasal turbinates and trachea obtained from pigs immunized with either vaccine were similar to control animals, as opposed to the wt-inoculated pigs ( figure 1e ). vaccination with h1n1 ty ⁄ 04 att-based vaccines provides sterilizing immunity against h1n1pdm in pigs the clinical performance in pigs of the h1n1 vaccines is summarized in table 1 . nv+ca ⁄ 04 animals had macroscopic pneumonia, viral replication in balf and shedding in the nose. uvadj-ca ⁄ 04 vaccine provided satisfactory protection, but this protection was not sterilizing. remarkably, animals vaccinated with ny(h1n1)2:6ty ⁄ 04 att had sterilizing immunity. in both vaccine groups there was a significant reduction (p < 0ae001) in the percentage of macroscopic lung pathology compared to the nv+ca ⁄ 04 group. control pigs had neither significant macroscopic nor microscopic lesions in the lungs. hi antibody titers measured at the day of challenge in both vaccine groups were approximately the same (table 1 ). in the present study, we developed for the first time, temperature-sensitive laiv for use in pigs. data from our safety studies showed that both the h3n2 and h1n1 ty ⁄ 04 att vaccines were attenuated in pigs. although the ty ⁄ 04 att vaccines were detected in balf samples, the level of viral replication was significantly reduced in comparison to unmodified virus and, more importantly, caused no overt clinical signs. a minimal amount of replication is likely beneficial for eliciting t-cell responses to internal genes that may provide heterologous cross-protection. one of the most challenging tasks in producing effective live attenuated vaccines is to achieve an adequate balance between safety and efficacy. by introducing the att modifications into the polymerase genes of a swine-like tr strain, this desirable balance was achieved. the vaccines were histopathologic scores of nasal turbinates, trachea and lungs at 3 dpi. ny(h1n1)2:6ty ⁄ 04 att (a virus that carries the surface genes of a ⁄ new york ⁄ 18 ⁄ 09 (h1n1) and ty ⁄ 04 att internal genes). all h3n2 viruses have their surface genes derived from ty ⁄ 04. values are shown as the mean ± sem. * p < 0ae05; **p < 0ae01; *** p < 0ae001. options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 328-394 attenuated in pigs and, more importantly, provided sterilizing immunity upon an aggressive challenge with pandemic h1n1 as opposed to an experimental ca ⁄ 04 inactivated vaccine, which elicited protective but not sterilizing immunity in all animals. in the face of influenza pandemics that have the ability to overcome the species barriers such as the 2009 h1n1, the supply of vaccines for use in agriculture could be jeopardized. our cell culture-based live att h1n1 vaccines could be an attractive alternative for this possible pandemic vaccine shortage. because the ty ⁄ 04 att live vaccines developed here are efficacious in swine, are easier to manufacture than inactivated vaccines, and do not require adjuvants, our study represents a major advance in vaccine development for the 2009 h1n1 pandemic. in conclusion, our second generation of live att influenza vaccines based on modifications of the pb2 and pb1 genes of ty ⁄ 04 retains its safety properties in vivo and can induce excellent protection against aggressive h1n1 challenges in the swine host. influenza virus is one of the most important respiratory pathogens worldwide. 1, 2 type a influenza causes an acute disease of the upper airways, and affects 20-40 million persons yearly. moreover, the threat of human influenza epidemic and pandemic has dramatically increased in recent years. 3 vaccination is one of the crucial interventions for reducing the spread and impact of influenza. the generally used parenteral inactivated influenza vaccines induce mainly systemic antibody responses and only weak cell-mediated immunity and low levels if any mucosal immunity. on the other hand, intranasal immunization with live virus can induces a broad spectrum of both systemic and mucosal antibodies, and the immune response localized in the mucosa blocks the virus even during the first phase of infection. unfortunately, the use of live vaccines is always associated with a certain risk. the development of a crossprotective vaccine against potentially pandemic strains is an essential part of the strategy to control and prevent a pandemic outbreak. we induced intrasubtypic and intersubtypic cross-protection in balb ⁄ c mice by intratracheal (it) immunization with inactivated influenza viruses together with dead delipidated bacillus firmus (dbf) as an adjuvant. ten days after the 2nd immunization dose, the mice were infected with live influenza virus b ⁄ lee ⁄ 40 lethal for mice (total infection dose corresponded to 5 · ld 50 ) or a⁄ pr 8 ⁄ 34 (total infection dose corresponded to 0ae5-5 ld 50 ). dbf adjuvant markedly increased both systemic and mucosal anti-viral antibody formation when applied together with inactivated influenza a or b viruses. 4 protective significance was tested in vivo. mice were preimmunized with 1) pbs (controls), 2) dbf alone, 3) virus alone, and 4) vir-us+dbf. influenza b virus strains b ⁄ lee and b ⁄ yamanashi 166 ⁄ 98 (58 years phylogenetically distant and antigenically substantially different, especially in terms of the main protective antigen -surface haemagglutinin) or two different influenza a subtypes -a ⁄ pr 8 ⁄ 34 (h 1 n 1 ) and a ⁄ california 7 ⁄ 04 (h 3 n 2 ) -were used (figures 1 and 2) . the mice were challenged with 5 · ld 50 of either b ⁄ lee ⁄ 40 or a ⁄ pr 8 ⁄ 34 as appropriate. all controls died. the mice treated with dbf alone died with a delay or survived, which could be explained by stimulation of innate immunity. the animals immunized with virus alone were protected against homologous strains. adjuvant immunization was cross-protective: the mice immunized with a heterologous b strain (figure 2 ) fell ill (pronounced body mass loss), but almost all survived and recovered. 5 the mice immunized with a heterologous a subtype were excellently protected (negligible weight loss and zero mortality). 6 intratracheal dbf (500 lg per mouse) given to non-immunized mice 24 hour before influenza infection eliminated the lethal effect in 40-100% of infected animals depending on infection dose (0ae5-5 ld50); in mice infected with lower than lethal doses (0ae5 ld50), weight loss was minimized or did not occur. the current mode of vaccination-induced immunity is mostly effective against a homologous strain of the virus used for vaccination. the attention is therefore focused on vaccines that are able to induce cross-protection and could be effective also in case of sudden appearance of a new virus variant. inactivated influenza viruses are known to be often insufficiently effective when used for mucosal immunization and for induction of cross-protection against drifted influenza viruses or novel subtypes. the drawback of vaccination with dead virus can be overcome by using a suitable adjuvant. mouse models were successfully immunized with vaccine containing inactivated virus in combination with cholera toxin or the escheria coli heat-labile toxin (lt). [7] [8] [9] the use of cholera toxin in humans is precluded because of its high toxicity; a number of lt mutants that retain their adjuvant activity have been prepared; these mutants were likewise tested on the mouse model and should not cause any serious side effect in humans. for this reason, current studies aim at finding a suitable and safe mucosal and systemic immune response. dbf has been shown to be a very efficient adjuvant for mucosal immunization stimulating both innate and adaptive immunity. intratracheal immunization with inactivated influenza viruses and dbf as adjuvant induced efficient and even heterosubtypic cross-protection. dbf given 24 hour before infection provided partial protection probably because of its strong stimulatory effect on the innate immunity. temperature-sensitive and cold-adapted candidates for live attenuated influenza vaccine with genomic composition of 7:1 based on highly pathogenic influenza a ⁄ h5n1 viruses with pandemic potential were generated by the replacement of six internal genes from the influenza a ⁄ puerto rico ⁄ 8 ⁄ 34 (pr8) virus from pr8-based rg-candidates for inactivated vaccine with appropriate internal genes of influenza a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) master donor virus (mdv) for russian laiv by methods of classical reassortment. all attempts to capture avian n1 neuraminidase into the genome of the mdv laiv production were ineffective. 6:2 reassortants were not generated. step by step co-infection of triple reassortants (h5n1-h1n1-h2n2) with h2n2 mdv in some cases was the only possibility to generate influenza a ⁄ h5n2 cold-adapted vaccine reassortants. difficulties in generating 6:2 reassortants could be explained by a substantial gene constellation in the genome of pr8based h5n1 reassortant viruses. strong coupling of pb2 ⁄ pr8 and avian n1 genes in a ⁄ h5n1-pr8-rg reassortants was revealed. annually updated laiv strains are generated by classical reassortment of circulating influenza viruses with well characterized, attenuated, ts ⁄ ca mdvs. resulting attenuated reassortants inherit the relevant ha and na of wild type parental virus and six internal genes of the mdv. 1 candidates for inactivated influenza vaccines based upon avian influenza viruses with pandemic potential are generally generated by reverse genetics methods. 2 in these cases, like with laiv, vaccine strains are 6:2 reassortants which possess the modified ha and na from potentially pandemic virus and six internal genes from the pr8 virus. the pr8 virus is considered to be of low virulence, i.e. attenuated, for humans, yet offers properties of high seed virus growth for influenza vaccine production. the ha of avian h5 influenza viruses with pandemic potential is engineered to remove four basic amino acid codons from the cleavage site of ha, resulting in a virus that is considered attenuated for natural hosts and safe for people. the objective of this study was to safely generate vaccine candidates for a laiv using highly pathogenic avian influenza viruses by the replacement of six internal pr8 genes in the genome of candidates for inactivated vaccine subtype h5n1 (a ⁄ h5n1-pr8-rg) with internal genes of the laiv mdv by methods of classical reassortment. len17-mdv and a ⁄ h5n1-pr8-rg virus were co-infected in embryonated chicken eggs. five rounds of selective propagation were performed, three of which were at low temperature (25°c). the production and selection of reassortants were carried out in the presence of rabbit antiserum to len17-mdv. cloning by endpoint dilution was performed in each of the last three passages. a virus sample in an open petri dish was rocked gently for 20 sec while being irradiated with a ge 15 watt germicidal lamp at a distance of 20 cm from the dish. the residual infection titer was measured by titration in embryonated chicken eggs. genome composition of reassortant viruses was monitored by rflp analysis. 3 in addition, capacity of reassortant viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) for influenza viruses was determined by virus titration in chicken eggs. reassortment of the mdv with the vn-pr or indo-pr viruses either resulted in reassortants that contained six internal genes from len17-mdv. however, all generated clones contained the na from the mdv. of ten such 7:1 reassortants based on vn-pr three reassortants had the pa gene from pr8 and one had ns gene from pr8. 6:2 reassortants from the targeted h5n1 composition were not generated. after repeated attempts, 7:1 temperature sensitive and cold adapted reassortants based on vn-pr and indo-pr viruses were obtained, but again, none had inherited the avian n1 neuraminidase (table 1) . in contrast, nibrg-23 didn't reassort with the mdv at all. twelve unsucsessful attempts to develop 6:2 or 7:1 reassortants of nibrg-23 with mdv showed that the classical reassortment procedure (cloning by limited dilutions in the presence of anti-mdv serum, followed by co-infection of equal doses of two parental viruses in eggs and two selective passages at 25°c) did not work for this virus pair. to disharmonize the incredibly strong gene constellation of nibrg-23, various modifications of the co-infection step were studied, such as: altering the nibrg-23 to mdv ratio (from 1:1 to 1: tions of anti-mdv serum alone or together with anti-pr8 serum. it was noted that even if the h5n1 to mdv ratio was 1:10 6 , the clones obtained were presumably parental h5n1 viruses without the transfer of any mdv-genes into genome of nibrg-23. in all, 234 clones were isolated, and 209 of them were identical to nibrg-23 parental virus. in nine clones, only the pa gene from mdv was included, whereas in three clones only the 'cold' ns gene was included (data not shown). using uv inactivation of nibrg-23 prior co-infection was more encouraging. after the first round of co-infection of partially uv-inactivated nibrg-23 with mdv (at ratio 1:10 2 ), reassortants that inherited several internal genes of mdv were obtained in the context of the nibrg-23 background (b3, c2, c4, d1) ( table 2 ). some of them (c2, c4, d1) were chosen for the next round of co-infection. after the second round of co-infection, c2, c4, and d1 'intermediate' reassortants with mdv (at ratio 1:1 or 1:10) 7:1 vaccine reassortants finally were obtained. live attenuated influenza vaccine is considered as one of the most promising pandemic vaccines. according to the who there is evidence that laiv might be more effective than inactivated vaccines. 4 this study attempted the safe development of laiv for potential pandemic highly pathogenic avian a ⁄ h5n1 viruses on the base of rg-reassortants for inactivated vaccine with modified h5 hemagglutinin and mdv for laiv. replacement of pr8based internal genes into genome of vn-pr and indo-pr reassortants with appropriate genes of mdv was realized by the classical reassortment procedure. difficulties were encountered in obtaining 6:2 reassortants that contained both the ha and na from the wild type avian h5n1 parental virus. in attempts to reassort the nibrg-23 with mdv, the classical reassortment procedure was unsuccessful. the challenge faced was to break an incredibly strong gene constellation of the nibrg-23 virus. partial uv-inactivation of nibrg-23 was encouraged in replacement of some pr8 internal genes with mdv genes 5 in some cases avian-human reassortant viruses with gull h13n6 and human influenza h1n1 genes were difficult to generate, and reassortants with the desired genotype of six gull virus genes with human influenza a h1 and n1 genes were not isolated despite repeated attempts. the gull pb2, np, and ns genes were not present in any of the gull-human h1n1 reassortants generated. 6 it is difficult to fully understand potential reasons for observed difficulties to reassort some avian viruses with human strains. unsuccessful attempts to develop 6:2 vaccine reassortants may be caused by an observed strong connection of pb2 and na genes in the genome of a ⁄ h5n1-pr8-rg viruses. in our attempts, each reassortant that possessed avian n1 neuraminidase inheritied pb2 gene of pr8 as well. and vice versa, the 'cold' pb2 gene always appeared to be coupled with the n2 neuraminidase of the mdv. in some cases, step by step co-infection of triple reassortants (h5n1-h1n1-h2n2) with h2n2 mdv may be the only possibility to generate a cold-adapted vaccine reassortant. our studies demonstrate unique and significant challenges that are faced in the development of influenza vaccines for avian influenza viruses with pandemic potential. such challenges must be further studied to identify methodologies to allow for rapid development and response to emerging viruses in a crisis. it is imperative that these studies be continued and expanded to identify either mechanisms of such tight gene constellations in influenza viruses produced by rg-derived vaccine strains or inability some genes of human h2n2 and avian h5n1 viruses to cross. in addition, further studies to improve the efficiency of classical reassortment processes will be conducted. during the period from 1997 to 2009, avian influenza outbreaks among humans have been registered in 15 countries of asia, europe, and africa. morbidity and mortality of humans followed the global spread of avian influenza h5n1 among wild and domestic birds, which caused great economic loss to the poultry industry in many regions including some highly developed countries. the global threat from avian influenza forced scientists to develop technologies for the production of a ⁄ h5n1 human vaccine. the development of ai a ⁄ h5n1 vaccines using strains isolated in kazakhstan and the organization of local production and creation of strategic stockpiles of effective vaccines is the an important issue for public health protection in the republic of kazakhstan. to address this, a scientific program 'influenza a ⁄ h5n1 vaccine development for public health protection in kazakhstan' was approved and financed from 2008 to 2010. in this article we give basic results of the development of a recombinant ai a ⁄ h5n1 inactivated whole virion vaccine with aluminium hydroxide as adjuvant for public health protection in kazakhstan. [1] [2] [3] the development of vaccine technology was conducted with the use of a ⁄ astanarg ⁄ 6:2 ⁄ 2009(a ⁄ h5n1) recombinant strain made of a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05(h5n1) and a ⁄ pr ⁄ 8 ⁄ 34(h1n1) strains by the reverse genetics. inactivation of virus containing allantoic fluid was carried out with the use of formalin in different concentrations. complete-ness of the virus inactivation was tested by 3-fold virus passaging in embryos. 4, 5 purification and concentration of the inactivated viruscontaining allantoic fluid was conducted with the use of ultra filtration in tangential flow, which was followed by gel filtration. then we evaluated the content of total protein, hemagglutinin, and ovalbumin in purified and concentrated material. 6 vaccine was composed of clarified and inactivated virus concentrate with the known ha dose containment, and 0ae4% aluminum hydroxide was added in 1:1 proportions. composition components and quality control of finished vaccine was determined in the stages of semi-finished product and finished biopreparation. determination of quantitative ovalbumin content was conducted by elisa applying a strip test-system chicken egg ovalbumin elisa kit cat. n 6050 (alpha diagnostic international, usa). vaccine immunogenicity was evaluated by hai micro test in u-bottom 96-well plates produced by 'costar' (usa). 7 vaccine apyrogenicity was evaluated after intravenous injection of the studied preparation to rabbits. 8, 9 for confirmation of the results vaccine series were tested for bacterial endotoxins with the use of limulus amebocyte lysate produced by charles river laboratories, inc. usa. 8 the vaccine toxicity was evaluated in white mice with body weight 18-20 gm and in rats with body weight 180-210 g both males and females according to glp principles. 9 allergenic characteristics of the inactivated vaccine was determined in white outbred mice and guinea-pigs both males and females according to 'methodic guideline for evaluation of allergenic characteristics of pharmacological substances'. 10 in the first series of experiments, we conducted work for obtaining influenza a(h5n1) recombinant strain. bidirectional expression plasmid phw_b754 with full-length sequences of ha and na gene segments of the strain a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) isolated in kazakhstan were synthesized in geneart ag, (regensburg, germany). ha gene was modified by deleting the region encoding multiple basic amino acid rrrk motif in ha cleavage site. moreover, to prevent recovery of repeating basic amino acids motif due to polymerase slide, we inserted replacements g fi t and k fi t. thus the ha cleavage site consists of the following sequence ntpqgerrrkkrglfgai ntpqtetrglfgai. the basic amino acid motif of highly pathogenic strain a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) was replaced by the sequence tetr ⁄ glf, which is characteristic of low pathogenic strains of influenza h5n1. sequence of gene coding na in the strain a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) was cloned without modifications. the other segments pb1, pb2, pa, np, m and ns were obtained from influenza virus ivr-116 and synthesized and cloned in two-forked expression plasmid phw_b754 in geneart ag company, germany. the origin of genetic segments of vaccine strain a ⁄ astanarg ⁄ 6:2 ⁄ 2009 (h5n1) is presented in table 1 . vero cell culture (134 passage) (who) was received from european cell culture collection (salisbury, wiltshire sp4 0jg, great britain). the cell culture was grown in dmem ⁄ f12 medium with the addition of 10% of fetal bovine serum and 2 mm l-glutamine. to obtain reassortant virus a ⁄ astana ⁄ 6 ⁄ 05r-6:2, vero cells were infected with correlative plasmids by way of electroporation using nucleofector ii (amaxa) equipment. infected cells were placed in 6-well plates. after 6 hour, dmem ⁄ f12 medium was changed into 4 ml of opti-pro sfm (gibco) medium adding 2 mm l-glutamine and 1lg ⁄ ml trypsin. two days after cytopathic effect appearance supernatant was collected and used for infection of spf-eggs. the virus a ⁄ astanarg ⁄ 6 ⁄ 05-6:2 was grown in chicken embryos, and then virus titer was determined in chicken embryos and madine-darby canine kidney (mdck) cell culture. the titer of two final a ⁄ astanarg6 ⁄ 05-6:2 virus stocks was 9ae1 log 10 eid 50 ⁄ ml (chicken embryos); 1ae8 log 10 tcid 50 ⁄ ml (mdck cells); ha titer 1:512. a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) virus contains motif of repeating basic amino acids in ha cleavage site. it is known that this sequence is the main determinant of ai virus pathogenicity. that is why this site was deleted in vaccine candidate strain. sequence results confirmed that influenza virus a ⁄ astanarg ⁄ 6 ⁄ 05r-6:2 strain ha gene sequence contains modified ha cleavage site and keeps mutations inserted for prevention of return to virus wild type. to confirm stability of modified ha gene sequence, five additional passages of recombinant strain a ⁄ astana rg ⁄ 6 ⁄ 05-6:2 were conducted in chicken embryos. sequencing and following phylogenetic analysis of the recombinant strain a ⁄ astana rg ⁄ 6 ⁄ 05-6:2 ha gene sequence proved the presence of modification in ha cleavage site. deletion of pathogenicity site of the obtained virus was confirmed by lethality test for chicken embryos, intravenous pathogenicity test in chicken, and in plaque-forming test with trypsin. pathogenicity test in chicken embryos showed that recombinant strain a ⁄ astanarg6 ⁄ 05-6:2 is capable of growing up to high titers without causing embryos' death. a ⁄ astanarg6 ⁄ 05-6:2 strain pathogenicity evaluation was conducted in 5-6 week-age white leghorns chicken, and this study proved that the strain a ⁄ astanarg6 ⁄ 05-6:2 (h5n1) is not virus pathogenicity inductor in chickens, which got intravenous injections of this virus (pathogenicity index is equal to 0). h5n1 strain ha cleavage site modification provides its cleavage capability only with tripsin-like proteases, which shows low level of pathogenicity. aiming at confirmation of ha cleavage site modification, we experimentally studied virus replication ability both with trypsin and without this enzyme. and we got the following results. in the plaque-forming test, a ⁄ astanarg6 ⁄ 05-6:2 strain produced plaques in mdck cells only with trypsin, proving the trypsin-dependent phenotype characteristic of low pathogenic avian influenza viruses. to prove the ha subtype antigenic analyses of a ⁄ astana ⁄ 6 ⁄ 05r-6:2 strain was conducted by means of serological methods in hemagglutinin inghibition test with the use of postinfection antisera of rabbits and rats (influenza research institute swd rams), standard serum received from cdc, atlanta, usa. hai test proved that a ⁄ astana ⁄ 6 ⁄ 05r-6:2 strain belongs to h5 subtype. furthermore, toxicity of vaccine candidate strain was evaluated by way of subcutaneous injection of viral material to balb mice. the strain appeared to be non-toxic for white mice getting subcutaneous injection of 0ae5 ml of the preparation. the conducted research showed that according to all tested characteristics, a ⁄ astana ⁄ 6 ⁄ 05r-6:2 strain can be used for influenza a ⁄ h5n1 inactivated vaccine production. according to its genetic characteristics, this strain belongs to the group of vaccine strains recommended by who for the development of influenza pre-pandemic inactivated vaccines. we determined basic cultivation parameters of the recombinant strain a ⁄ astanarg6 ⁄ 05-6:2 in 10-11 day chicken embryos. the determined parameters are the following: infection dose, 1000-10 000 eid 50 ; cultivation period, 72 hour; incubation temperature, 33°c. these cultivation parameters allow obtaining virus containing material with biological eid and hemagglutinating activity of 8ae5-9ae0 log 10 eid 50 ⁄ cm 3 and 1:512 ha titre and even higher. in the next series of experiments, we conducted research on the determination of optimal sequence of technological stages of virus clarification, concentration, and inactivation in the order of vaccine production. samples of viral material were subjected to inactivation before and after clarification and concentration. the regimen of virus inactivation by formaldehyde with final concentration of 0ae05%, period of inactivation of 3 days, temperature of inactivation medium of 4-6°c, ph of inactivation medium of 7-7ae5. on the basis of the conducted experiments we determined that the selected regimen of inactivation provides complete and irreversible inactivation of viral suspensions of the hpai strain irrespective of the kind of inactivated material. we did not observe reduction of ha activity in non-clarified viral suspensions. however, when we inactivated clarified and concentrated material, ha activity reduced by an order of magnitude. comparison of forms and sizes of virion structural elements in native (non-clarified) and formalin inactivated preparations did not reveal any significant differences. concentration of virus particles in the studied preparations was similar. the selected inactivation regimen provides obtaining completely avirulent viral suspension of the strain a ⁄ astanarg6 ⁄ 05-6:2, and it does not influence the structure of the virus. on the basis of the experiments results, we selected method of viral allantoic fluid inactivation without preliminary clarification. during further research, we tried to get highly clarified viral concentrate. this study resulted in the combined scheme, which includes clarification of inactivated viral allantoic fluid by low speed centrifugation at 4000 circulations per min for 30 minutes, filtration through membrane filters with pore diameter of 0ae45 lm, ultrafilatration ⁄ diafiltration, gel filtration in 6b sepharose, and sterilization of viral suspension through membrane filters with pore diameter of 0ae22 lm. the experiments resulted in the development of production technology of embryonic inactivated vaccine based on recombinant strain a ⁄ astanarg ⁄ 6:2 ⁄ 2009 (h5n1) contain-ing aluminium hydroxide as adjuvant. the developed influenza a ⁄ h5n1 human vaccine has the trade name kazfluvacò. its composition components are presented in table 2 . preclinical testing of the vaccine kazfluvacò was conducted according to the following parameters: general health condition of animals, change of body weight and temperature of immunised animals (for ferrets), presence of post vaccination antibodies response in sera, forming protective immune response against reassortant viruses of h5 subtype, study of acute and chronic toxicity of three experimental vaccine series in different doses and semi-finished vaccine product applying different ways of injection, study of allergic and immunotoxic characteristics of the vaccine, as well as study of pyrogenic reaction and analysis for bacterial endotoxins presence. [11] [12] [13] [14] preclinical tests of kazfluvacò vaccine safety showed that this vaccine does not have toxic effect on organisms of warm-blooded laboratory animals. double intramuscular injection of kazfluvacò vaccine in inoculative dose does not effect appearance, general health condition, behaviour of animals, their muscular strength and physical activity, does not have negative effect on biochemical parameters of blood and basic physical functions of animals organism, and does not cause pathomorphological changes. this shows the safety of the vaccine. local irritation action was not observed. the results of the vaccine allergic action study showed that the vaccine does not have allergic effect at the intravenous injection. the research also showed that the vaccine does not have negative effect on immune system of laboratory animals. research conducted on mice and ferrets showed high immunogenic activity of the vaccine at one-and two-dose regimen of injection. the research showed 100% of protective effect of kazfluvacò vaccine at two-dose injection regimen in ferrets infected by homological strain of influenza virus. the devised inactivated influenza a ⁄ h5n1 vaccine kaz-fluvacò is a safe and immunogenic biopreparation that is not worse than the overseas analogues in its immunobiological characteristics. [15] [16] [17] [18] to date the whole-virion inactivated influenza a ⁄ h5n1 vaccines of the producers such as omnivest (hungary), biken, denka seiken, kitasato institute, kaketsuken (japan), gsk biologicals (belgium), sinovac biotech (china) are registered. all of them are produced on the basis of chicken embryos and aluminum is used as an adjuvant. kazfluvacò differs from its analogues in the flowchart of the virus purification and concentration that makes possible to produce a safer preparation. 19, 20 the results of the conducted research and preclinical testing allow starting work towards implementation of phase i preclinical tests on volunteers. it is planned to conduct a randomized blind placebo-controlled phase i study on double application of kazfluvacò vaccine in increasing doses. the preparation will be administered to volunteers aged 18-60 years for assessment of its safety and immunogenicity in doses of 7ae5 and 15ae0 lg of ha. when the world health organization (who) announced the sixth phase of a ⁄ h1n1v influenza pandemic, scientists all over the world started investigation to develop technology for production of prophylactic means against the disease. having taken into consideration the threat of a pandemic for kazakhstan, the ministry of education and science of the republic of kazakhstan launched the program ''monitoring, study, and development of diagnostic, prophylactic, and therapeutic means for influenza a ⁄ h1n1.'' this paper presents the experimental data obtained at the ribsp in the course of the studies towards the development of technology for production of an inactivated a ⁄ h1n1 influenza vaccine, as well as the results of pre-clinical testing of the developed vaccine. the development of vaccine production technology was conducted with the use of who recommended vaccine strain nibrg-121xp constructed by the method of reverse genetics in the national institute for biological standards and control (nibsc, great britain). the virus was inactivated with formalin at different final concentrations, and the extent of inactivation was evaluated via threefold virus passages in developing chicken embryos. 1 the inactivated virus was purified and concentrated by the method of ultrafiltration in tangential flow followed by gel filtration. the purified and concentrated material was evaluated judging on the total protein, hemagglutinin (ha), and ovalbumin. the vaccine was prepared by pooling the purified and concentrated virus material with the certain weight content of ha and the work solution of aluminum hydroxide (0ae4%) in the ratio 1:1. the ovalbumin content was quantified in elisa with the use of the strip test system chicken egg ovalbumin elisa kit (cat. no. 6050 alpha diagnostic international, san antonio, texas, usa). weight content of the virus ha was determined according to sominina, burtseva. 2 the content of the residual formaldehyde, aluminum (al +3 ) ions, and thiomersal in the vaccine was measured according to the operating instructions. 3 the vaccine immunogenicity was assessed in the hemagglutination inhibition test, which was carried out as a microassay in 96-welled u-bottomed plates (''costar'', new york, usa). 3, 4 apyrogenicity of the vaccine was assessed post intravenous administration of the tested preparation to rabbits. 5, 6 to confirm the obtained results the vaccine batches were tested for bacterial endotoxins with use of the limulus amebocyte lysate (charles river laboratories, inc., wilmington, ma, usa). 7 the toxicity of the vaccine was assayed in white mice weighing 18-20 g and in rats weighing 180-210 g (male and female) in compliance with the principles of good laboratory practice. 8 allergenic properties of the inactivated vaccine were determined according to the ''operating instructions on assessment of allergenic properties of pharmaceutical substances'' 9 in white outbred laboratory mice and guinea-pigs of both sexes. the first step in the course of developing technology for vaccine production was to determine the major conditions for influenza virus cultivation: usage of 10-days embryonated chicken eggs at the infectious dose within 1000-10 000 eid 50 , incubation temperature (34 ± 0ae5)°c, and duration of the incubation period 72 hours. the established parameters for virus cultivation made it possible to produce virus-containing materials of infectious activity within 8ae5-9ae0 log eid 50 ⁄ cm 3 and hemagglutinating activity 1:256 and higher. in the subsequent experiments, an optimal method for virus inactivation was selected. on the basis of the experimental findings, the following conditions for inactivation of the native virus-containing material were elected: formalin of 0ae05% final concentration as an inactivating agent; inactivation period of 72 hours at temperature (4 ± 2)°c. these conditions provide the complete inactivation of the virus (nibrg-121xp strain) material, did not impact distinctly the structural organization of the virus, and did not reduce the antigenic activity. as it is well known, virus purification and concentration means very much in the development of technology for production of an inactivated whole-virion influenza vaccine. the investigation into optimization of the technological step of purification and concentration of the recombinant influenza virus nibrg-121xp strain resulted in selection of an optimal pattern including such steps as clarification of the virus suspension by filtration through membranes with pore size 0ae45 lm, virus concentration by ultrafiltration in a tangential flow, dialysis filtration in a tangential flow, gel filtration on sepharose 6b, and sterilization of the viral suspension through membrane filters with pore size 0ae22 lm. the studies conducted by the ribsp specialists resulted in the development of technology for production of the first domestic whole-virion inactivated a ⁄ h1n1 influenza vaccine with aluminum hydroxide as adjuvant and with the brand name refluvac ò . the key processing characteristics of the whole-virion inactivated a ⁄ h1n1 influenza vaccine vaccine refluvac ò are shown in table 1 . simultaneous with the performance of all process operations, the parameters such as sterility, inactivation extent, ph, vaccine specificity, total protein content, weight content of has, aluminum and formalin contents, content of thiomersal, and ovalbumin, pyrogenicity of the vaccine and its immunogenicity for mice, were optimized. the key qualitative characteristics of the designed influenza a ⁄ h1n1 vaccine refluvac ò are shown in table 2 . before implementation of phase i clinical trials on volunteers, preclinical testing of three experimental batches of refluvac for immunogenic activity and safety was carried out. it was conducted in three laboratory bases of research institutions: the toxicology institute ⁄ federal medicobiological agency, russia (st petersburg), the research institute for biological safety problems (republic of kazakhstan), and the influenza research institute ⁄ north-western branch of the russian academy of medical sciences (st petersburg), with use of different animal models (mice, rats, chinchilla rabbits, guinea-pigs, ferrets). the results of the preclinical testing are as follows: • electron microscopy of the preparation has shown that the viral particles are well dispersed and do not aggregate. the portion of whole (intact) particles is over 95%, which is evidence of virion integrity; • assessment of polypeptide composition of the vaccine refluvac by electrophoresis in 10% polyacrylamide gel with sodium dodecyl sulfate has shown the vaccine to contain both surface antigens (ha, na) and highly purified inner virion proteins (np, m1) that are typespecific antigens, so the vaccine is a preparation of full immunological value; • judging on the parameters of acute and chronic toxicity for white mice and rats of both sexes, the vaccine is a non-toxic and safe preparation; • under conditions of a chronic experiment on white mice and rats, it was found that refluvac does not produce changes in behavior, somatic, or vegetative responses; • assay of hematological and biochemical blood characteristics of white mice and rats following vaccine administration did not reveal any significant differences as compared to the animals of the control group; • refluvac does not cause allergenic and immunotoxic impact; • the vaccine refluvac does not cause local irritative effect; • refluvac is apyrogenic for laboratory animals; • the pathomorphological and hystopathological analysis did not reveal any changes due to immunization in animal organs; • testing of immunogenic characteristics of the vaccine on mice and ferrets has shown formation of hemagglutinating antibodies in animals after single administration; • refluvac induces 100% protection in immunized ferrets at their challenge with the wild-type influenza virus a ⁄ california ⁄ 07 ⁄ 2009 (h1n1v). the results of the performed preclinical testing have allowed concluding that refluvac, an inactivated whole-virion vaccine with aluminum hydroxide as adjuvant, is a safe and highly effective preparation against influenza a ⁄ h1n1v. the implemented study resulted in development of technology for production of the first domestic inactivated allantoic whole-virion influenza a ⁄ h1n1 vaccine with aluminum hydroxide as an adjuvant under the brand name refluvac ò based on the recombinant strain nibrg-121xp. the devised pandemic vaccine meets who requirements as well as requirements concerning safety and immunogenicity of the national pharmacopeias of the republic of kazakhstan and russian federation. [9] [10] [11] [12] the devised technology for vaccine production differs from the previous technologies for production of allantoic whole-virion influenza a ⁄ h1n1 vaccines in its processdependent parameters. presence of an adjuvant (aluminum hydroxide) increases significantly the vaccine immunogenicity and allows maximal reduction of the dose of the administered antigen that, in turn, results in diminished reactogenicity of the vaccine. aluminum hydroxide is an adjuvant that is most frequently used in clinical practice. 13 to date the results of the double-centered randomized study of the europe-licensed vaccine fluval p [monovalent inactivated whole-virion influenza vaccine with aluminum phosphate based on strain a ⁄ california ⁄ 07 ⁄ 2009 (h1n1) nymc x-179a (omninvest, pilisborosjeno, hungary)] that is similar to the refluvac preparation are published. the data of this research are an evidence of safety and high immunological effectiveness of the vaccine in dose 6 lg ha at single administration both in adults and elderly persons. 14 the results of the pre-clinical tests allow recommending carrying out phase 1 clinical testing of the refluvac ò vaccine for safety and immunogenicity. single immunization of volunteers with refluvac ò in doses 3ae75, 7ae50, and 15ae00 lg of ha are planned. mid 50, respectively. the study results confirm that new h1n1 laiv and h7n3 laiv candidates are safe and immunogenic and confer protection from homologues influenza virus infection in mice. the recent emergence of a new pandemic h1n1 virus and the threat of transmission of avian viruses to humans had stimulated research and development of live attenuated cold-adapted influenza vaccines against newly appeared influenza viruses. formulations of live attenuated influenza a vaccine (laiv) against pandemic influenza strains, including h1n1, h5n1, h9n2, and h7n3 are currently being tested in preclinical and phase i clinical studies. 1 the following paper describes the preclinical study of new h1n1 and h7n3 laiv candidates in mice. the study addressed the following three objectives: (i) to demonstrate that cold-adapted (ca) reassortant influenza a(h1n1) and a(h7n3) vaccine candidates are indistinguishable from the parental a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) master donor strain (mds) virus with regard to replication efficiency in upper and lower respiratory tract of mice; (ii) to demonstrate the immunogenicity of different doses of cold-adapted (ca) reassortant influenza a(h1n1) and a(h7n3) vaccine candidates in mice; and (iii) to demonstrate the protective efficacy of cold-adapted (ca) reassortant influenza a(h5n1) and a(h7n3) vaccine candidates in mice against a homologous wild-type virus challenge. the a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3) reassortant containing the ha and na genes from a ⁄ mallard ⁄ netherlands ⁄ 00 (h7n3) and six other genes from mds, the a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) reassortant containing the ha and na genes from a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) and six other genes from a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) were generated by classical genetic reassortment in embryonated chicken eggs (ec). viruses were propagated in 10days old eggs (34°c, 48 hours). fifty percent egg infectious dose (eid 50 ) titers were determined by serial titration of viruses in eggs. titers were calculated by the method of reed and muench. 2 female balb ⁄ c mice, 6-8 weeks of age were used in all experiments. mice were lightly anesthetized with ether and then inoculated intranasally (i.n.) with 50 ll of infectious virus diluted in phosphate-buffered saline (pbs). mice were inoculated with 100 mid 50 (50% mouse infectious dose) of a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1), a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3), and a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) mds. viral loads were measured in respiratory and brain tissues collected at 3 and 6 days post-infection (dpi). tissue homogenates prepared using a disruptor and clarified supernatants were titrated on eggs at permissive temperature to determine infectious concentrations. groups of animals were inoculated with 1000 mid 50 or 100 mid 50 of either h1n1 laiv or h7n3 laiv intranasally after collecting a pre-immunization blood sample. a second blood sample was collected at 28 dpi. on the same day, the animals received a second intranasal inoculation with the same virus that was used for priming at 0 dpi. to assess protection, all animals were infected 42 dpi with either 100 mid 50 of a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) or 100 mid 50 a ⁄ mallard ⁄ netherlands ⁄ 00 (h7n3) virus by the intranasal route. four animals from each group were euthanized at 45 dpi, and the respiratory and systemic organs were harvested for virus titration. a forth blood sample was collected at 56 dpi from the remaining animals. hi antibody titers were determined for individual serum samples collected on days 0, 28, 42, and 56. body weights were taken daily following challenge through day 14 postchallenge. sera were tested for hi against homologous h1n1 and h7n3 viruses. the h1n1 laiv, h7n3 laiv and h2n2 mds influenza viruses replicate in mice lungs at level 2ae1-2ae3 lgeid 50 ⁄ ml at 3 dpi (figure 1 ). at 6 dpi, replication of the viruses in the lungs decreased to 1ae6-2ae0 lgeid 50 ⁄ ml (data not shown). in contrast, the wild-type virus a ⁄ mallard ⁄ netherlands ⁄ 00 (h7n3) demonstrated high level replication in lungs -6ae4 lgeid 50 ⁄ ml. the levels of replication of studied viruses in nasal turbinates were 2ae5-3ae7 lg eid 50 ⁄ ml at 3 dpi (figure 1) , and 2ae0-2ae2 lgeid 50 ⁄ ml at 6 dpi (data not shown). there were no significant differences between the viruses in regard to replication in upper respiratory tract of mice. thus, it was shown that a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3) and a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) vaccine candidates was indistinguishable from parental a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) in terms of replication in the lungs and noses of mice at 3 and 6 dpi. no virus was found in the brain tissue of immunized mice at 3 and 6 dpi (in undiluted samples tested). thus, it was shown that a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3), a ⁄ 17 ⁄ cali-fornia ⁄ 2009 ⁄ 38 (h1n1) vaccine candidates are identical to a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) in lacking neuroivasive capacity, and all three viruses similarly fail to replicate in the brain. it was shown that all immunized animals survived after challenge with wild-type a ⁄ mallard ⁄ netherlands ⁄ 12 ⁄ 00 (h7n3) virus. the mice in vaccine groups showed no signs of morbidity. average weight changes were tracked from day 2 to day 6 in all study groups, but the changes did not exceed 5%. as shown in figure 2 , the challenge virus actively replicated in respiratory tissue taken from mock immunized animals (5ae7 lgeid 50 in the lung and 4ae2 lgeid 50 in the nose), but failed to infect the brain and spleen. on the other hand, in both h7n3 laiv vaccinated groups, all tested organs were free from presence of challenge virus. thus, immunization of mice with either 1000 mid 50 or 100 mid 50 h7n3 laiv protected the animals from the subsequent challenge infection with a homologous with wild-type h7n3 virus. both h1n1 and h7n3 laiv candidates were found to be immunogenic. after one dose of 100 mid 50 of h1n1 laiv, gmt of hi antibodies were 17ae6. one dose of 1000 mid 50 or 100 mid 50 h7n3 laiv elicited hi antibody level with gmt of 8ae7 and 6ae6, respectively. the second dose of h7n3 laiv further stimulated serum hi antibody levels to gmt 40ae0 and 23ae3, for 1000 mid 50 or 100 mid 50, respectively (data not shown). the mouse model is widely used to better understand the pathogenicity of avian influenza viruses for mammalian species, to be able to predict the pandemic potential of such viruses, and to develop improved methods for the prevention and control of the virus in a potential pandemic. 3 a subset of the h7 viruses was evaluated for the ability to replicate and cause disease in balb ⁄ c mice following intranasal administration. h7 subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication. 4 there is limited preclinical information available for laiv. thus, live monovalent vaccine against pandemic influenza virus h1n1 (influvir) was tested for acute toxicity and its effect on the systems and organs of laboratory animals. according to toxicology and necroscopy results, the live monovalent influenza vaccine influvir, when applied intranasally, was safe and was well tolerated. 5 in our current study we demonstrate that a(h1n1) and a(h7n3) laiv are indistinguishable from the parental mds virus with regards to replication kinetics in the upper and lower respiratory tract of mice. both h1n1 and h7n3 laiv candidates were immunogenic and protect mice against subsequent a challenge with the wild-type virus. live attenuated cold-adapted (ca) influenza vaccines are an effective means for the control of influenza, most likely due to their ability to induce both humoral and cellular immune responses. in our study we confirm that new h1n1 laiv and h7n3 laiv candidates are safe, immunogenic, and confer protection from influenza infection in mice. health organization (who) declared a pandemic by raising the worldwide pandemic alert level to phase 6. therefore, h1n1 inactivated monovalent vaccine formulated with our proprietary oil-in-water emulsion based adjuvant was evaluated in ferrets for its potential to induce with low antigen dose efficient, robust, and rapid protective immunity against a wild type challenge virus (a ⁄ netherlands ⁄ 602 ⁄ 2009). this adjuvant was also tested in ferrets in a h5n1 avian influenza model for its ability to induce a cross-clade immunity and cross-protection. two independent studies (a&b) were carried out with male and female outbred ferrets (musleta putorius furo) in compliance with ''guide for the care and use of laboratory animals,'' ilar recommendations and aaalac standards. ferrets used in both studies were influenza seronegative by anti-nucleoprotein elisa and by hi assay against the pandemic and seasonal strains. in study a, four groups of seven ferrets aged approximately of 6 months received one or two im vaccinations 3 weeks apart of either af03-adjuvanted (3ae8 lg of ha with af03) or unadjuvanted ( body weight loss was monitored as an indicator of disease and a mean body weight loss of 20% was recorded in the control group at day of necropsy. body weight loss was reduced to £10% and £8% in animals that had received 1 and 2 doses of either unadjuvanted or af03-adjuvanted vaccine, respectively. viral lung titration showed high levels of virus replication ( ‡4ae7 tcid50 ⁄ g tissue) in the lungs of all control ferrets 4 days after challenge. one or two administrations of unadjuvanted vaccine reduced lung viral load by 2 and 3 log 10 , respectively. interestingly, ferrets that received either one or two doses of af03-adjuvanted h1n1 2009 vaccine, showed significantly greater reduction of lung viral loads (>4 log 10 ). no virus was detected in the lungs of 6 ⁄ 7 (86%) animals immunized with a single injection of the af03-adjuvanted vaccine and in 100% of ferrets vaccinated twice. assessment of viral shedding from the upper respiratory tract showed that the af03-adjuvanted a ⁄ h1n1 monovalent vaccine was able to reduce the viral load in the nose and in the throat by 3ae8 and 3ae1 log 10 , respectively, as compared to the control group. conversely, viral loads were only slightly reduced in the nose and mostly unchanged in the throat in ferrets immunized with either one or two doses of unadjuvanted a ⁄ h1n1 monovalent vaccine. gross pathology and histology examinations revealed lung lesions consistent with influenza a ⁄ h1n1 virus infechowever, a second dose of af03-adjuvanted vaccine strongly increased hi and mn titers, which persisted for 3 months (table 2 ). antibody responses cross-reactive to heterologous clade 2.2 strain were elicited ferrets vaccinated with the af03-adjuvanted clade 2.1 vaccine. hi antibody titers ‡40 crossreactive to clade 2.2 and persistent up to d110 were observed in vaccinated animals. an inter-clade low crossreactive hi response to a clade 1 strain was only detected in a few ferrets that had been vaccinated with the af03-adjuvanted clade 2.1. all af03-adjuvanted clade 2.1 antigen vaccinated animals survived challenge either with the homologous or heterologous virus until euthanized day 5. after challenge, mean body temperature and mean body weights were monitored as indicators of disease. in the control ferrets, mean body temperature increased by 2-3°c (depending on the challenge virus strain) 24 h post challenge, with an accompanying mean body weight loss ranging from 15ae4% to 20ae7%. ferrets vaccinated with the af03-adjuvanted clade 2.1 vaccine showed a lower and delayed fever compared to control ferrets that received the same viral challenge, whereas no significant differences were observed between vaccinated animals and their respective controls upon challenge with clade 2.2 or clade 0 viruses. body weight loss was reduced in all vaccinated animals when compared to controls after challenge with either the homologous clade 2.1 strain or with one of the heterologous strains. lung virus titration showed high levels of virus replication in all control animals 5 days after homologous challenge with the clade 2.1 virus. lung viral loads of all ferrets immunized with the af03-adjuvanted clade 2.1 vaccine were reduced more than 4 log 10 . vaccination resulted in complete viral clearance from the lungs of 80% of animals assessed 5 days after challenge. as compared to controls, a reduction of the mean viral load of about 2 log 10 was observed in ferrets vaccinated with the af03-adjuvanted clade 2.1 vaccine after heterologous challenge with either the clade 0 or clade 1 virus. conversely, vaccination with af03-adjuvanted clade 2.1 vaccine did not result in reduction of lung viral loads after challenge with the clade 2.2 heterologous virus strain. titration of pharyngeal swabs showed high levels of viral shedding in all control ferrets after challenge with clade 2.1 strain, whereas virus was not detected in any vaccinated animal. similarly, 5 log 10 reduction of viral shedding was seen in vaccinated versus control ferrets following clade 1 heterologous challenge. lower reductions in viral shedding were observed after clade 2.2 challenge (2ae6 log 10 ) and clade 0 challenge (1ae4 log 10 ). gross pathology and histology revealed lung lesions consistent with influenza a ⁄ h5n1 virus infection all control animals challenged with the clade 2.1, clade 2.2 or clade 0 strains. mild to moderate lung lesions were observed in control animals following challenge with clade 1 virus. macroscopic evaluation (percentage of affected lung parenchyma) and histopathological analysis (extent and severity of alveolitis, alveolar oedema and hemorrhage) showed that lung lesions were significantly reduced in af03-adjuvanted clade 2.1 vaccinated animals after challenge with the homologous clade 2.1 virus strain as compared to controls. similarly, a reduction of the macroscopic and microscopic lung lesions was observed in vaccinated animals upon heterologous challenge with clade 2.2 and clade 0 virus strains, whereas no differences were observed between control and vaccinated animals after challenge with clade 1 virus. the results of these ferret challenge studies demonstrated that low doses of pandemic influenza vaccines formulated with an oil-in-water emulsion adjuvant, af03, elicited strong antibody responses specific to the immunizing strain. importantly, these vaccines provided protection after homologous challenge with complete virus clearance in ferret lungs and reduced viral shedding from the upper respiratory tract suggesting an ability to reduce virus transmission. moreover, af03-adjuvanted h5n1 vaccine can provide cross-protection upon challenge with different h5n1clades by preventing mortality and reducing the viral burden in the lower and the upper respiratory tract. in conclusion, the results of these studies highlighted the ability of af03-adjuvanted influenza vaccines to induce potent immune responses and full protection in ferrets against homologous challenge and suggested that protection may be mediated, at least in part, by antigenspecific humoral immunity. since 1993, outbreaks of h9n2 influenza virus infection in poultry have occurred in eurasian countries. phylogenetic and antigenic analysis of h9n2 isolates revealed that there are three sublineages, consisting of g1, g9, and korean, among ha genes of the eurasian h9n2 viruses. h9n2 viruses do not cause severe disease in poultry, but co-infection of h9n2 viruses with bacteria such as staphylococcus aureus, haemophilus paragallinarum, or attenuated coronavirus vaccine may exacerbate the disease. 1,2 h9n2 viruses were isolated from domestic pigs in china and korea and from humans with febrile respiratory illness in hong kong in 1998 kong in , 1999 kong in , and 2003 it is, thus, postulated that in the present study, h9 virus strains were analyzed antigenically and phylogenetically to select a proper h9n2 vaccine strain. inactivated whole virus particle vaccine was prepared, and its potency against h9 virus challenge was assessed in mice. viral rnas were extracted from the allantoic fluid of chicken embryos infected with viruses by using a commercial kit (trizol ls reagent; invitrogen, california, usa) and reverse-transcribed with the uni12 primer 6 and m-mlv reverse transcriptase (invitrogen). the primers used for the ha gene amplification were h9-101f 7 and h9-1341r. for phylogenetic analysis, sequence data of the genes together with those from public database were analyzed by the neighbor-joining method. 8 h9 influenza viruses were analyzed by hemagglutinationinhibition (hi) test. 9 chicken hyperimmunized antisera against seven h9 viruses were prepared according to previous report. 10 virus replication and pathogenicity against embryonated chicken eggs viruses were inoculated into 10-day-old embryonated chicken eggs and incubated for 48 hours at 35°c. ha titers and 50% egg infectious dose (eid 50 ) were measured every 12 hours post-inoculation. pathogenicity of dk ⁄ hok ⁄ 49 ⁄ 98 against embryonated chicken eggs was evaluated by mean death time (mdt) as described previously. 11 dk ⁄ hok ⁄ 49 ⁄ 98 was injected into the allantoic cavities of 10-day-old embryonated chicken eggs and propagated at 35°c for 48 hours. the virus in the allantoic fluids (512ha) was purified by differential centrifugation and sedimentation through a sucrose gradient according to previous report. 12 the concentration of protein was measured by od using ultrospec 3100 pro (amersham biosciences, tokyo, japan). the purified virus was inactivated with 0ae1% formalin at 4°c for 7 days. immunization of mice and challenge of immunized mice with hk ⁄ 1073 ⁄ 99 four-week-old female balb ⁄ c mice were purchased from japan slc, inc. (shizuoka, japan). the mice were injected subcutaneously with 10, 2, 0ae4, or 0ae08 lg proteins of inactivated dk ⁄ hok ⁄ 49 ⁄ 98 whole virus vaccine. two weeks later, the mice were boosted by subcutaneous injection with the same dose of the vaccine. control mice were injected with pbs. serum samples were tested by enzyme-linked immunosorbent assay (elisa) according to previous report. 10 one week after the second vaccination, 10 mice in each group were challenged intranasally with 30 ll of 10 6ae5 eid 50 of hk ⁄ 1073 ⁄ 99 under anesthesia. on 3 days postinfection, five mice in each group were sacrificed, and the lungs were separately homogenized to make a 10% (w ⁄ v) suspension with minimal essential medium (nissui, tokyo, japan). the virus titers of the supernatants of lung tissue homogenates were calculated in 10-day-old embryonated chicken eggs and expressed as the eid 50 ⁄ gram of tissue. the other five mice in each group were monitored for body weight for 14 days after challenge. the ha genes of 22 h9 viruses were sequenced and analyzed by the neighbor-joining method. all of the 22 h9 viruses were classified into the eurasian lineage ( figure 1) . eleven, seven, and four strains were classified in the korean, g9, and g1 sublineages, respectively. the h9 viruses of the korean and g9 sublineages were isolated from waterfowl, poultry, pigs, and humans in the east asian countries, and those of the g1 sublineage were isolated from poultry in the west asian countries. the cross-reactivity between these antisera and h9n2 viruses were analyzed by hi test. the antisera against h9 viruses belonging to the korean sublineage were broadly cross-reacted to h9 viruses belonging to the g9 and g1 sublineages. h9 viruses belonging to the korean lineage were reacted to the antisera against h9 viruses belonging to the g9 and g1 sublineage compared with h9 viruses belonging to the other sublineage (data not shown). thus, it was suggested that h9 vaccine strain should be selected from the viruses of korean sublineage to prepare for the vaccine strain of h9 viruses. dk ⁄ hok ⁄ 49 ⁄ 98 replicated efficiently in 10-day-old embryonated chicken eggs (data not shown). pathogenicity of dk ⁄ hok ⁄ 49 ⁄ 98 against embryonated chicken eggs was determined by mdt. dk ⁄ hok ⁄ 49 ⁄ 98 was low pathogenic against embryonated chicken eggs (data not shown) and was selected as an h9 vaccine strain. to assess the potency of the vaccine against h9 virus infection, mice vaccinated subcutaneously with inactivated dk ⁄ hok ⁄ 49 ⁄ 98 were challenged intra-nasally with hk ⁄ 1073 ⁄ 99. immunogenicity of the inactivated vaccine was assessed by measuring the igg antibodies in mouse sera by elisa. antibody was detected in the group of mice injected 10 lg protein after the first immunization and detected in the group of mice injected 2 lg protein after the second immunization. thus, potency of the present inactivated whole virus vaccine was demonstrated in mice. next, to assess the protective immunity of the inactivated vaccine in mice, viral titers in the lungs was determined. the virus titers in the lungs were 10 1ae5 -10 3ae7 eid 50 ⁄ g in the groups of mice injected 10, and 2 lg protein, and 10 5ae3 -10 6ae0 eid 50 ⁄ g in the other vaccinated groups. body weight reduction of mice were observed in the group of mice injected 0ae4, 0ae08 lg protein, and control groups from 3 dpi, and reached to 10% body weight loss from 4-to 6-day post-infection ( figure 2 ). this result correlates with antibody titer in mouse sera and viral titers in the lungs. these results suggest that the test h9 inactivated whole vaccine confers prevent of weight loss and reduction of virus replication against h9 influenza virus infection in mice. recently, h9n2 viruses of all of three sublineage have been isolated from wild birds and poultry in worldwide. h9n2 viruses were isolated from pigs and humans in china 3 and korea, suggesting that h9n2 virus would be a potential for a pandemic influenza virus in human population. h9n2 viruses were isolated from pigs in china and korea and were classified into the g9 and korean sublineage. in human cases, all h9n2 virus isolated from humans in china was classified into the g1 sublineage. it was suggested that h9n2 viruses isolated from pigs and humans vary in antigenicity of isolates between the korean, g9, and g1 sublineages. therefore, it is important for the preparedness of influenza pandemic to develop h9 influenza virus vaccine, which could broadly cross-react to antisera of all sublineage viruses. so, we selected the vaccine candidate strain, dk ⁄ hok ⁄ 49 ⁄ 98, which could broadly cross-react to antisera of all sublineage viruses, and which could replicate in this study, it was suggested that the test vaccine has potency to protect against challenge with h9 virus using mice for mammalian model. the challenge virus, hk ⁄ 1073 ⁄ 99, was isolated from human, replicates efficiently in mice, and shows pathogenicity in mice. the test vaccine inhibited viral replication and body weight loss in mice. whole inactivated vaccine produced protective immunity, supporting our approach of using whole virus particles for vaccine development. furthermore, whole particle virus vaccine could induce igg and mucosal iga levels after intranasal vaccination with whole particle vaccine. 13 the present results may facilitate the studies of the vaccine for future pandemic caused by h9 influenza virus in humans. tants to attempt to improve growth. to determine whether wild type h1n1pdm grew better in the novartis mdck suspension cell line (mdck33016pf) than in eggs, isolations from h1n1pdm positive clinical samples were attempted in both substrates. the isolation rate of h1n1pdm viruses was higher in mdck33016pf cells (89%) (31 ⁄ 35) compared to allantoically inoculated eggs (66%) (23 ⁄ 35) . however the yields were lower than observed with seasonal viruses. little improvement in virus yield was seen with extra passaging or dilutions of h1n1pdm viruses isolated in mdck33016pf cells. with the emergence of the swine-origin pandemic h1n1 (h1n1pdm) influenza in april 2009, 1 the need for efficient production of a suitable vaccine was a high priority. 2 virus isolates were distributed by the who for the urgent development of suitable vaccine strains early in the pandemic. vaccine viruses can be grown in embryonated chicken eggs 2 or in certified mammalian cells. 3, 4 unfortunately wildtype h1n1pdm virus strains distributed by the who grew poorly in cell lines and eggs, requiring the generation of a series of 3 conventional and 8 reverse genetics 5 derived reassortants to attempt to improve growth. from these reassortants, only the conventional egg derived reassortants nymc-x-179a and nymc-x-181 (both based on one of the earliest known viruses a ⁄ california ⁄ 7 ⁄ 2009) showed high enough growth and yield in eggs and cell culture to make them suitable for vaccine manufacture. these reassortants, while acceptable, still only gave haemagglutinin (ha) yields of approximately 60% that of seasonal h1n1 reassortants. to determine if more recent wild type h1n1pdm viruses grew better in the novartis mdck suspension cell line (mdck33016pf), h1n1pdm positive clinical samples were cultured in mdck33016pf cells and also in embryonated hen's eggs. in addition, to improve virus yields from mdck33016pf isolates, extended passaging of three wild type h1n1pdm influenza viruses was performed using various virus dilutions at each passage level. the results were assessed using various serological and molecular biology techniques and compared to viruses isolated in eggs and conventional mdck cells. h1n1pdm viruses were received at the centre from who national influenza centres, who influenza collaborating centres and other regional laboratories and hospitals in australia, new zealand, and the asia ⁄ pacific region. viruses were received as original clinical specimens consisting of nasal swabs, throat swabs, nasopharyngeal aspirates, or nasal washes that had previously been shown to be h1n1pdm positive by real time rt-pcr. these specimens were then cultured in mdck33016pf cells with serum free medium containing trypzean (optaflu) 3 and also independently inoculated into the allantoic cavity of 11 day-old embryonated hen's eggs. virus cultures in mdck33016pf cells were sampled at 48 and 72 hour and evaluated by various means including ha titres. at 72 hour, virus cultures were further passaged at varying dilutions ranging from 10 )5 to 10 )8 up to a total of 10 passages. embryonated hen's eggs were incubated at 35°c for 3 days and allantoic fluid was harvested and ha titres performed to determine whether a further passage was required in order to improve growth. the conventional reassortants were produced by a mixed infection of eggs or mdck 33016pf cells with the wild type virus and a donor virus carrying the internal genes of the a ⁄ puerto rico ⁄ 8 ⁄ 34 virus. the reassortants were obtained by sequential passages using immuno-selective antisera against the surface antigen of the donor virus to remove virus populations carrying the ha and na protein of the donor strain. 6 the reverse genetics viruses were rescued in vero cells using the 12 plasmid system. 7 both types of reassortants were generated and supplied by who collaborating centres and essential regulatory laboratories except the nvd-c-07 strain, which was produced by novartis. in this small study with recent h1n1pdm viruses, the isolation rate was higher in mdck33016pf cells (89%) (31 ⁄ 35) compared to allantoically inoculated eggs (66%) (23 ⁄ 35) . assessment of ha titres, however, showed higher ha titres in egg-isolated viruses compared to viruses isolated in mdck33016pf cells after two passages. egg generated or cell generated reassortant viruses gave higher ha titres compared to the homologous wild type viruses (table 1) . no amino acid changes were observed in mdck33016pf isolated influenza viruses compared to original specimens or viruses isolated in conventional atcc derived mdck cells, unlike egg isolated viruses which showed a number of amino acid changes, many consistent with egg adaptation mutations (table 1) . 10 viruses isolated in mdck33016pf cells grouped phylogenetically with viruses isolated in conventional atcc derived mdck cells or viruses sequenced from original clinical samples, while egg isolated viruses grouped slightly differently (data not shown). as a result of the poor growth of h1n1pdm viruses in mdck33016pf cells, serial dilutions were performed over a number of passages ( figure 1 ). based on the results obtained from the virus isolates, a ⁄ victoria ⁄ 2081 ⁄ 2009, a ⁄ wellington ⁄ 188 ⁄ 2009, and a ⁄ darwin ⁄ 2131 ⁄ 2009, a supplemental protocol was developed and used in the isolation of a ⁄ brisbane ⁄ 10 ⁄ 2010 (figure 1 ). only small differences in ha titer were seen between different dilutions, and copy number showed a similar trend to ha titer at each passage ( figure 1 ). following the supplemental protocol for the isolation of a ⁄ brisbane ⁄ 10 ⁄ 2010 results showed slightly higher ha titres with little variation between passages. the egg derived reassortants nymc-x-179a and nymc-x-181 were also assessed for growth in mdck33016pf cells and were found to be superior by ha titer to other conventional reassortants (egg or cell derived), reverse genetics derived reassortants, or wild type viruses (table 1) . two methods were used to determine the ratio of ha to other viral proteins: densiometric analysis using sds-page and reversed-phase hplc using a subtype specific standard. 11 ha content in different vaccine seeds of influenza a subtypes demonstrated that the ha content per total virus protein from the nymc h1n1pdm reassortants was significantly different to the seasonal influenza a subtypes. for the seasonal h1n1 the ratio of ha to p4 p5 p6 p7 p8 p9 p10 p3 p4 p5 p6 p7 p8 p9 p10 p3 n and m1 was ‡30%, for the h3n2 the ratio of ha to n and m1 was £30%, while for the pandemic a ⁄ h1n1, the ratio of ha to n and m1 was much lower at £20% (data not shown). the results of this study has observed the growth of a series of 2009-2010 h1n1pdm viruses in vaccine suitable mdck33016pf cells to be generally lower than what has been seen with other seasonal influenza viruses. 11 little improvement in virus yield was seen with extra passaging of h1n1pdm viruses isolated and passaged in mdck33016pf cells. passaging up to 10 times in mdck33016pf cells using dilutions ranging from 10 )5 to 10 )8 resulted in supernatants with viral ha titres ranging from 8 ha ⁄ 25 ll to 512 ha ⁄ 25 ll. the isolation rate of h1n1pdm viruses was higher in mdck33016pf cells (89%) compared to allantoically inoculated (and passaged) eggs (66%), a trend also seen in previous work with seasonal influenza viruses. 12 in contrast a study by hussain and colleagues 13 found similar rates of isolation and replication of seasonal influenza viruses in mdck cells and eggs. the virus load as determined by matrix gene copy number showed a similar trend to ha titers. two of the isolates exhibited small rises and falls in ha titer during passaging, while a third, a ⁄ victoria ⁄ 2081 ⁄ 2009 gave consistently higher titers. interestingly this virus was unable to be isolated in eggs. the ha sequences of all strains were assessed at p1, p2, p3, p4, p10 and when available compared to the original clinical sample ha sequence. mdck33016pf-isolated viruses had few if any changes in their ha amino acid sequence, while the majority of egg isolates showed 1-2 amino acid changes compared to the clinical sample, with an egg adaption change (l191i) evident in a number of them. the ha sequence of one of the better growing viruses, a ⁄ victoria ⁄ 2081 ⁄ 2009, was found to have a g155e change compared to the a ⁄ california ⁄ 7 ⁄ 2009 reference virus. this change was also seen in the virus isolated in conventional, adherent mdck cells. these viruses with g155e change when tested by hai have shown reduced reactivity with ferret antisera to a ⁄ california ⁄ 7 ⁄ 2009-like viruses, but normal reactivity with ferret antisera to h1n1pdm a ⁄ bayern ⁄ 69 ⁄ 2009-like viruses. despite this mutation all mdck33016pf derived viruses appeared to be a ⁄ california ⁄ 7 ⁄ 2009-like by hai. the h1n1pdm egg-derived reassortants (nymc x-179a and nymc x-181) when grown in mdck33016pf cells were superior to wild type h1n1pdm viruses, reverse genetics derived reassortants, and other egg-derived reassortants. the yields of haemagglutinin from the nymc h1n1pdm reassortants were still below those seen with sea-sonal h1n1 reassortants as was also seen in eggs. this trend has also been noted in other studies. 10 in summary, attempts to improve growth and yield of the h1n1pdm wild types for mdck 331016pf cells by extended passaging were not successful, and reassortants did not perform as well as seasonal h1n1 reassortants have in the past. however, using higher dilutions for the passaging of h1n1pdm viruses in mdck33016pf cells did result in higher ha titres (a ⁄ brisbane ⁄ 10 ⁄ 2010). further work is therefore required to generate pandemic h1n1 seed viruses that grow well in a variety of cell culture and egg based vaccine production systems. the aim of this study is to evaluate antibody response to influenza virus neuraminidase (na) following immunization with live attenuated influenza vaccine (laiv). we adjusted the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. (1990) to assay neuraminidase inhibition (ni) antibody in sera taken from immunized mice and from human subjects in a clinical trial. for the assay, we prepared the a(h7n1) reassortant virus containing the na of a ⁄ california ⁄ 07 ⁄ 2009 (h1n1) and the hemagglutinin (ha) of a ⁄ equine ⁄ prague ⁄ 1 ⁄ 56(h7n7). in addition, we used an na-specific igg elisa assay to test sera from immunized mice and volunteers. in mice, one dose of laiv induced ni antibody of a geometric mean titer (gmt) of 31ae7, compared to 10ae6 in the control group. gmt of ni from human subjects who received two doses of pandemic a(h1n1) were significantly higher than pre-vaccination titers. in unvaccinated human subjects, na-specific cross-reactive antibodies to pandemic a(h1n1) were detected more often than cross-reactive antibodies to ha. antibody response to influenza virus na contributes to the overall immune response to influenza and may provide partial protection against influenza infection and reduce severity of disease in the host. 1 a number of preclinical studies using purified or recombinant na have shown that various two-dose vaccine regimens in mice may significantly reduce pulmonary virus titers following viral challenge. [2] [3] [4] a plasmid dna-vaccine model demonstrated cross-reactive antibodies to human n1 in mice could provide partial protection against a lethal challenge against h5n1 or recombinant pr8 bearing the avian n1. 4 immunogenicity of current influenza vaccines, including laivs, is measured primarily as a level of strain-specific hemagglutination inhibition (hi) antibodies. 5 however, the who meeting on the role of na in inducing protective immunity against influenza infection (2008) specified a need to develop suitable assays for anti-na antibody detection to enhance influenza vaccine evaluation in preclinical and clinical studies. 6 the aim of the current study was to evaluate anti-na antibodies to pandemic a(h1n1) 2009 influenza virus following laiv immunization. the rn1 ⁄ 09-swine a(h7n1) reassortant influenza virus containing the na of a ⁄ california ⁄ 07 ⁄ 2009(h1n1) and the ha of a ⁄ equine ⁄ prague ⁄ 1 ⁄ 56(h7n7) generated by classical genetic reassortment in embryonated chicken eggs (ce). parental a ⁄ equine ⁄ prague ⁄ 1 ⁄ 56(h7n7) influenza virus was obtained from the center for disease control and prevention, atlanta, ga, usa. viruses were propagated in 10 day old ce and purified by sedimentation out of the allantoic fluid, followed by ultracentrifugation on 30-60% sucrose step gradient. for the mouse studies, 10 week old cba mice were inoculated intranasally with one dose 10 7 eid 50 ⁄ 0ae05 ml of a ⁄ 17 ⁄ california ⁄ 09 ⁄ 38(h1n1) vaccine strain or received 0ae05 ml pbs. blood samples were collected on day 15 post inoculation. healthy young adults were immunized twice, 10 or 21 days apart in the fall 2009 with a ⁄ 17 ⁄ california ⁄ 09 ⁄ 38(h1n1) laiv manufactured by microgen, irkutsk, russia. for the human studies, peripheral blood specimens were collected from volunteers before vaccination, 21 days after the first vaccination, and 21 days after the second dose of vaccine. sera from five subjects diagnosed with influenza a(h1n1) were collected in december 2009, 3 to 4 weeks post infection and kindly provided by e. vo ıtsekhovskaia from biotechnology laboratory, institute of influenza, rams. also, sera obtained in 2005 from unvaccinated vol-unteers were tested for presence of cross-reactive antibodies to a ⁄ california ⁄ 07 ⁄ 2009 (h1n1). sera were treated with a receptor-destroying enzyme from vibrio cholera (denka-seiken, tokyo, japan) and then were tested in duplicates for hemagglutination-inhibition (hi) h1 specific antibodies by standard procedures 7 using a ⁄ 17 ⁄ california ⁄ 09 ⁄ 38(h1n1) test antigen. the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. 8 was adjusted to assay ni antibody. briefly, 96-well plates (sarstedt, inc., nümbrecht, germany) were coated overnight with 150 ll of 50 lg ⁄ ml fetuin. the purified a(h7n1) reassortant virus was diluted in pbs with 1% bsa and 10 mm ca 2+ to give a four times higher optical density at 450 nm (od 450 ) compared to control wells not containing virus. fifty-microliter volumes of serially diluted serum samples were incubated with an equal volume of prediluted virus for 1 hour at 37°c. after incubation, the plates were washed and neuraminidase activity was measured by subsequently adding peroxidase-labeled lectin (2 lg ⁄ ml; sigma, st. louis, mo, usa), incubating for 1 hour at room temperature, washing the plates, and adding 100 ll of peroxidase substrate (tmb). the reaction was stopped after 5 minute by adding 100 ll of 1n sulfuric acid. od values were measured at 450 nm using the universal microplate reader (el x 800; bio-tek instruments, inc., winooski, vt, usa). the ni titers were expressed as the reciprocal dilution that gave 50% od of positive control (virus, no serum control). in addition we used an igg elisa assay 9 with 0ae5 lg ⁄ ml of purified na from a ⁄ california ⁄ 07 ⁄ 09(h1n1) to test sera from immunized mice and volunteers. data were analyzed with statistica software (version 6ae0) (statsoft, inc. tulsa, oklahoma, usa). geometric mean titers (gmt) were calculated and used to represent the antibody response. the comparisons were made within groups between pre-and postvaccinated titers (expressed as log 2 ) after first and second vaccination using wilcoxon matched pairs test. to compare multiple independent groups we used a kruskal-wallis anova with subsequent multiple pairwise comparison based on kruskal-wallis' sums of ranks. a p-value of <0ae05 was considered to be statistically significant. in mice, one dose of laiv induced antibody responses to both ha and na components of the a ⁄ california ⁄ 07 ⁄ 2009(h1n1) influenza virus vaccine (table 1) . geometric mean titers of ni antibody levels from vaccinated mice were 31ae7 and were significantly higher compared to those in unvaccinated control animals (p < 0ae02). elisa igg titers expressed as log 10 were 1ae7 compared to 1ae0 in control group. there was good correlation between antibody rises obtained using ni or elisa tests (r = 0ae7). in a study during the fall of 2009, 85% of 60 examined unvaccinated subjects were negative to pandemic a(h1n1) (hi titers £1:10). serum hi antibody titers to pandemic a(h1n1) ‡1:40 were considered to be protective against *the postvaccination gmts of hi antibodies after revaccination were higher than respective prevaccination titers (p = 0ae04) **the postvaccination gmts of ni antibodies after revaccination were higher than respective prevaccination titers (p = 0ae02) serum hi and ni antibodies to a ⁄ california ⁄ 07 ⁄ 09(h1n1) after one or two doses of pandemic laiv were evaluated in subjects who had pre-vaccination hi titers £1:10 ( table 2) . post-vaccination gmts of a(h1n1)-specific antibodies were significantly higher than pre-vaccination titers only among subjects who received two doses of laiv ( table 2 ). the frequency of subjects with ‡ fourfold rises in hi antibody titers was higher after two doses (32ae3%) compared to responses after one dose (7ae1%) although the differences were not statistically significant ( table 2 ). the highest antibody titers of hi and ni antibodies were achieved after natural infection (p < 0ae01 compared to all post-vaccination groups). all five subjects with confirmed influenza also had high levels of n1-specific igg measured by elisa using purified na as the coating antigen (data not shown). influenza ha and na surface proteins are primary targets of neutralizing antibodies that provide protection against influenza infection. the correlation of strain-specific hi antibody titers ‡1:40 to protection of 50% of the subjects against influenza infection is based on a number of reports published in 1980s. 10 serum antibodies against viral na as result of influenza infection or vaccination also can neutralize the virus from infecting cells; however, little is known about protective levels of such antibodies. to evaluate ni antibodies directed against pandemic a(h1n1) we used the reassortant a(h7n1) influenza virus with mismatched ha to avoid non-specific inhibition. we demonstrated laiv immunization effectively increased levels of ni antibody, although in smaller amounts compared to influenza infection. our data suggest that an antibody to neuraminidase, resulting from an earlier infection of the circulating seasonal influenza a(h1n1), evidently cross-reacted with the n1 of pandemic influenza virus, perhaps due to the previously reported 17% of conserved na epitopes in pandemic a(h1n1). 11 the peroxidase-linked lectin test using the reassortant a(h7n1) influenza virus was shown to be a sensitive and time effective means of revealing homologous and cross-reactive anti-na antibodies after laiv immunization or influenza infection. this could be a useful method for influenza vaccine evaluation. significant levels of anti-na antibodies detected in peripheral serum from subjects infected with wildtype h1n1 virus or with h1n1 laiv. and the cross-antibody response to ph1n1. for calculation of geometric mean titer (gmt), a titer of <10 was assigned a value of 5. statistical significance was determined by paired t-test. cross-reactive antibody response to ph1n1 in vaccinated populations of seasonal influenza virus table 1 shows the antibody response to seasonal influenza viruses and ph1n1 of participants. before vaccination, no or little antibody response to ph1n1 had been detected in all age groups. vaccination with seasonal influenza vaccines resulted in seroresponse in over 70% of subjects, except children aged 0-7 years (68%) and subjects aged of 18-59 years (69%) vaccinated with 2007-2008 season influenza vaccine and adults aged ‡60 years (59%) vaccinated with 2008-2009 season influenza vaccine. seroconversion was detected in over 40% of subjects of all ages. postvaccination to prevaccination gmt ratios for response to seasonal influenza viruses was more than 2ae5-fold. in contrast, seroresponse to a ⁄ california ⁄ 07 ⁄ 2009 after vaccination with 2007-2008 and 2008-2009 seasonal influenza vaccines were detected in only 3% and 4% of those aged 0-7 years, 3% of those aged 8-17 years, 7% and 3% of those aged 18-59 years, 7% and 5% of those aged ‡60 years, respectively. seroconversion in all participants ranged from 2% to 4%, and postvaccination to prevaccination gmt ratios were <2ae5-fold. preexisting antibody response to ph1n1 among subjects born before 1920s in china according to a recent report, people who were born from 1910 to 1929 had a preexisting immunity to ph1n1. 7 although only a very low level of cross-reactive antibody response to ph1n1 had been observed among older subjects aged more than 60 years old in china, we further analyzed these data by different age distribution of subjects, which can trace back to the previous infection that is genetically and antigenically more closely related to this new ph1n1 influenza virus. the proportion of seroresponse to ph1n1 with the titer of 40, 80, and 160 (highest titer detected from participants of all ages in this study) and the value of gmt were analyzed according to the birth decade of subjects from 1915. similarly, a peak of antibody response and the value of gmt occurred both in subjects born from 1915 to 1925 and sharply decreased afterward ( figure 1 ). the seroresponse of subjects born in and before 1925 is significantly higher than subjects born afterward (p < 0ae05). similar to recent studies in some asia countries (guangxi province of china and singapore), limited antibody response to ph1n1 had been detected in children and adults. 8, 9 but, some other studies from european countries (finland, germany, the united kingdom) and the united states reported a high proportion of older individuals aged >60 years with pre-existing cross-reactive antibodies to ph1n1, which may possibly ba a result of previous exposure to antigenically related h1n1 influenza viruses circulating in earlier decades or a lifetime of exposure to influenza a, which has resulted in broad heterosubtypic immunity among older individuals in those countries. previous infection and vaccination with a ⁄ new jersey ⁄ 76 may also contribute to the high level of cross-reactive antibody response to ph1n1 among adults older than 60 years in the us. 10, 11 the peak of the antibody response to ph1n1 in subjects born between 1915 and 1925, which is consistent with recent reports, may suggest the previous viral infections of 1918 spanish flu or closely related influenza viruses, which is before and little after the year of 1918. recent antigenic report of new ph1n1 viruses indicated that they are antigenically homogeneous among historical viruses, which are most similar to classical swine a(h1n1) viruses. a number of reviews [12] [13] [14] [15] confirmed that the 1918 virus is the likely ancestor of all four of the human and swine h1n1 and h3n2 lineages, as well as the 'extinct' h2n2 lineage. in 1930, a(h1n1) influenza viruses were first isolated from swine. they have been shown to be antigenically highly similar to the recently reconstructed human 1918 a(h1n1) virus. 16 the cellular responses may contribute to the sustaining and long term antibody response. probably, boosting by persisting antigenically related viruses in the early decades of the 20th century, may have contributed to the ability of these subjects to sustain memory b cells, 17 and it is well established that a subset of plasma cells is long-lived, and these cells contribute to durable humoral immune responses, 18 such as that observed after childhood smallpox vaccination. furthermore, t cells that recognize cross-reactive epitopes are preserved and might be enriched in the memory population; the course of each infection is influenced by the t-cell memory pool that has been laid down by a host's history of previous successive infections. 19 our study indicated that wide transmission of this new virus or any antigenically close related influenza a(h1n1) viruses may not have circulated among populations in china before the outbreak of ph1n1. our data also suggests the need for vaccination with ph1n1 vaccine in all age groups. hypo-and agammaglobulinemia patients have an impaired immune system and are particularly susceptible to bacterial infections that are normally defended against by antibodies. therefore, patients routinely receive replacement therapy with immunoglobulins isolated from healthy blood donors. 1 these patients are also prone to get viral infections, possibly due to defects in toll-like receptors 8 and 9. 2 because these patients lack an antigen specific humoral immune response, they are rarely vaccinated. the ability of hypogammaglobulinemic patients to produce a specific cell-mediated immune response upon vaccination has only been sparsely investigated. in contrast to local mucosal antibodies, vaccine-induced cell-mediated immunity is not believed to protect against pathogen entry per se, but may be sufficient to provide protection against severe disease and death following transmission of some microbes. 3, 4 the aim of this pilot study was to investigate if influenza vaccination of hypogammaglobulinemic patients can induce an influenza-specific cell-mediated immune response. we therefore vaccinated hypogammaglobulinemic patients and healthy controls with pandemic h1n1 virus vaccine and subsequently investigated the bcell and t-cell responses. the percentages of ifn-c, il-2, and tnf-a cytokine producing cd4+ th1-cells were determined, as these cytokines are important indicators of cell-mediated immunity. five a-or hypogammaglobulinemic patients were classified based on the freiburg classification 5 : patient #1 is diagnosed with x-linked agammaglobulinemia, patient #2 and #4 are in group ia, patient #3 is in group ib and patient #5 is in group ii. the monovalent egg grown split virus vaccine adjuvanted with as03 was manufactured by glaxosmithkline (gsk), belgium. the vaccine strain was produced by reassortment between influenza a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) and a ⁄ pr ⁄ 8 ⁄ 34 (h1n1) to produce a ⁄ california ⁄ 7 ⁄ 2009-like virus (x179a). the vaccine was mixed with adjuvant to contain 7ae5 lg haemagglutinin (ha) of a ⁄ california ⁄ 7 ⁄ 2009-like virus (h1n1), squalene (21ae38 mg), dl-atocopherol (23ae72 mg), and polysorbate 8 (9ae72 mg) per ml. healthy controls and hypogammaglobulinemia patients were vaccinated by intramuscular (im) injection. hypogammaglobulinemia patients received one or two vaccine doses 21 days apart. the intention was to vaccinate the hypogammaglobulinemic patients with two doses of 3ae75 lg ha, but 7ae5 lg ha was inadvertently administered to the patients as the first dose. for patient #4 this was the second dose as he had received an initial dose of 3ae75 lg ha 3 months prior to the study. patient #1, #2, and #5 received a second dose of 3ae75 lg ha. four healthy controls were immunised with one dose of 3ae75 lg ha according to norwegian national guidelines. peripheral blood mononuclear cells (pbmcs) were harvested and washed in pbs with 10% fbs. the pbmcs were resuspended in lymphocyte medium (rpmi 1640 with l-glutamine, 0ae1 mm non-essential amino acids, 10 mm hepes ph 7ae4, 1 mm sodium pyruvate, 100 iu ⁄ ml penicillin, 100 lg ⁄ ml streptomycin, 0ae25 lg ⁄ ml fungizone and 10% fbs) prior to use in the enzyme-linked immunospot (elispot) and influenza-specific cd4+ t-cell assays. serum haemagglutination inhibition antibodies were tested by a standard method using 8 ha units and 0ae7% turkey erythrocytes. all samples were tested in duplicate and the test was repeated at least two times. titres <10 were assigned a value of 5 for calculation purposes. for numeration of antibody-secreting cells (asc), an eli-spot assay was conducted as previously described 6 with the following modifications. ninety-six well elispot plates were coated with 2 lg ⁄ ml of a ⁄ california ⁄ 7 ⁄ 2009like (x179a) h1n1 virus diluted in pbs overnight at 4°c. after blocking with rpmi (10% fbs), 10 5 pbmcs were added and incubated (37°c, 5% co 2 ) for 16 hour. secreted antibodies were detected with biotinylated goat anti-human igg, iga and igm specific antibody (southern biotech, birmingham, alabama, usa), incubated for 2 hour at room temperature and developed with extravidin peroxidase and aec substrate. the numbers of spots were counted using an elispot reader (immunoscanô) and immunospot ò software. the influenza-specific cd4+ th1-cell response was measured by intracellular cytokine production of ifn-c, il-2, and tnf-a. peripheral blood mononuclear cells (10 6 per well) were incubated for 16 hour (37°c, 5% co 2 ) in 200 ll lymphocyte medium containing 1 lg ⁄ ml anti-cd28, 1 lg ⁄ ml anti-cd49d, 0ae7 lg ⁄ ml monensin, 1 lg ⁄ ml brefeldin a, (bd biosciences, franklin lakes, new jersey, usa), and the h1n1 influenza split virus vaccine x179a (either 2ae5 lg ⁄ ml or 10 lg ⁄ ml ha). basal cytokine production was determined by incubating pbmcs in lymphocyte medium without influenza virus, and the percentage of cytokine positive cells without influenza stimulation were subtracted from influenza-stimulated cells. cells were stained for cd3, cd4, cd8, ifn-c, il-2, and tnf-a (bd biosciences) as previously described. 7 finally, cells were resuspended in pbs containing 5% fbs and 0ae1% sodium azide and analysed by bd facscanto flow cytometer (300 000-500 000 cells acquired). flowjo v8ae8ae6 (tree star, ashland, oregon, usa) was used for data analysis. five to six fold lower gmts were found in the patient group as compared to the healthy controls throughout the study ( figure 1a) . the lowest hi titres were obtained in patients #1, #2, and #3, whilst patients #4 and #5 and all healthy controls fulfilled two of three european medicines agency committee for medicinal products for human use (chmp) 8 seasonal influenza vaccine licensing criteria, by obtaining an hi titre >40 and a mean geometric increase of 2ae5 between pre-and post-vaccination. thus, the hi data indicate that two vaccine doses was sufficient to induce a protective hi antibody response in two out of five of the hypogammaglobulinemia patients tested in this study. the numbers of influenza-specific iga, igg, and igm asc were tested pre-vaccination and 7 days post-vaccination with the h x179a virus. few or no ascs were detected pre-vaccination (data not shown). at 7 days post-vaccination the patient's iga, igg, and igm asc levels were significantly lower (p < 0ae01) compared to the healthy controls ( figure 1b) . but, the post-vaccination asc numbers in the patients were generally higher than at pre-vaccination stage (0-2 ascs). patient #3 had the highest iga and igg asc numbers, followed by patients #2 and #5, whilst patient #4 and #1 had few or no asc's. these results confirm that the patients are indeed hypogammaglobulinemic and that some of the patients (#1 and #4) could be agammaglobulinemic in the context of producing influenza-specific antibodies. the asc levels of patients #2, #3, and #5 were lower than those of the healthy controls, but could possibly be adequate for reducing the severity of influenza disease. the influenza-specific th1-cell response was evaluated by stimulating pbmcs with the influenza x179a virus 21, 42, and 90 days post-vaccination. stimulation of healthy control pbmcs with x179a 21 days after vaccination, induced ifn-c, il-2, and tnf-a production by an average of 0ae10%, 0ae09%, and 0ae03% cd4+ t-cells, respectively. patient #1 and #2 had higher responses than the healthy controls and stimulation with x179a induced 0ae45%, 0ae34%, and 0ae05% of t-cells from patient #2 to produce ifn-c, il-2, and tnf-a, respectively (figure 2a) . the response of patient #2 was further boosted by a second vaccine dose, which resulted in 0ae73%, 0ae54%, and 0ae25% cd4 t-cells producing ifn-c, il-2, and tnf-a, respectively at day 42 ( figure 2b ). these results show that the hypogammaglobulinemia patients studied here did not have a common impaired influenza-specific cd4+ th1 cytokine response. rather, there was a tendency towards increased responses, suggesting that the diminished antigen specific b-cell responses could induce a compensatory antigen specific th1-cell response. the results from this pilot study suggest that some hypogammaglobulinemia patients may benefit from influenza vaccination. we found very different patient responses to influenza vaccination, but some of the patients (patient #2 and #3) did mount low influenza-specific asc responses. in addition, the vaccine-induced hi antibody titres above the protective level in patient #4 and #5. these results are in accordance with previous publications, which described that polypeptide vaccines induce humoral responses in subgroups of common variable immunodeficiency patients. [9] [10] [11] in this study, we also investigated cell-mediated immunity and found the percentages of homologous and cross-reactive influenza-specific cd4+ th1-cells to be in the same range (for patient #3, #4, and #5) or higher (for patient #1 and #2) in the a-or hypogammaglobulinemic patients compared to the healthy controls. the higher response is probably due to the patients having received a vaccine dose of 7ae5 lg ha, whilst the controls received 3ae75 lg ha. in addition, the patients received a second booster dose, which influences the day 42 and 3 months responses. nonetheless, these results are the first to demonstrate that proliferation of pandemic influenza antigen specific th1cells can be induced in hypogammaglobulinemic patients. in addition, vaccination induced influenza-specific asc's in some patients. the findings are promising and provide hope that hypogammaglobulineamic patients could be vaccinated against influenza and other diseases preventable by figure 2 . peripheral blood mononuclear cells s from patients and healthy controls were isolated at day 21 (a), 42 (b), and day 90 (c) and stimulated for 17 hour with x179a virus before staining and flow cytometric analysis. the figure shows the mean ± sd frequency of influenza-specific cd4+ cytokine producing cells (%) where the basal cytokine production from unstimulated cells has been subtracted. data for the hypogammaglobulinemia patients are additionally shown as a number for each patient. **significantly higher frequency of il-2 producing cd4+ t-cells in the patients compared to the healthy controls (students t-test p < 0ae05). titres are presented as the geometric mean titre ± 95% confidence interval. elispot data (b) are presented as the mean number of influenza-specific iga, igg, and igm ascs per 100 000 peripheral blood mononuclear cells ± sem. data for the hypogammaglobulinemia patients are additionally presented by a number for each patient. *significantly higher numbers of ascs were detected in the healthy controls as compared with the hypogammaglobulinemia group (students t-test, p < 0ae01). vaccination. however, this hypothesis should be tested in larger clinical studies. the influenza virus undergoes antigenic evolution under intense immune selection pressure from herd immunity in humans through the process called antigenic drift and shift. 1,2 because of antigenic drift, yearly updating of vaccine strain is needed. a mismatch between the circulating strains and the vaccine strain in the subsequent season is often encountered, resulting in reduction of vaccine effectiveness and lack of protection from the circulating strain. 3 in order to address this, a universal influenza vaccine based on a more conserved part of the influenza virus, which is not affected by antigenic change and that is conserved across all strains, remains the ultimate goal to afford cross-protection to drifted strains as well as to other subtypes of influenza which may arise from antigenic shift. 4, 5 previous studies have investigated the potential of the m2e. 6, 7 m2e has remained highly conserved since it was first isolated in 1933. 6 several studies have examined the use of m2e as a vaccine component, using various approaches including proteins, peptides, dna vectors, and attenuated viral vectors. 6, [8] [9] [10] [11] [12] [13] although m2e is a weak antigen, by linking the protein to a carrier hepatitis b virus core particle, protection against influenza has been achieved in mice particularly when administered with an adjuvant. 3 some articles found that vaccination with m2e coupled to hbc induces protective antibodies, whereas the contribution of t cells to protection was negligible. protection induced by vaccination with m2e-hbc was weak overall and failed to prevent weight loss in vaccinated infected animals, and mice succumbed to high dose infection. 14 we aimed to address the poor immunogenicity of m2e-hbc by using igv as adjuvant. igv domain is common and conserved in the tim family. ligand binding sites of t cell immunoglobulin mucin (tim) located at igv domain. [15] [16] [17] tim function is done by anti tim antibody which recognized the ligand binding sites of igv domain. 18 tim family members share a common motif, including an igv domain. they are differentially expressed on th1 cells and th2 cells with the ability to regulate the immune system. 19, 20 the igv domain of human b7-2 is sufficient to co-stimulate t lymphocytes and induce cytokine secretion. 21 soo hoo et al. vaccinated with tim-1 antibody and inactivated influenza and found enhanced vaccine-specific immune response. 22 we report here for the first time the use of igv recombinant protein as adjuvant to immunize mice with influenza m2e-hbc. results indicated that igv can induce the strong cellular immune response and cross reaction with different subtype influenza virus antigen. target igv may be used to develop the new method for vaccination strategies. expression and purification of recombinant igv protein rna was extracted from healthy human pbmc. one-step rt-pcr (qiagen, valencia, ca, usa) was done for the amplification igv gene. the pcr product was purified and cloned into pet32a (novagen, madison, germany). the resultant construct pet32a-igv has a histidine (his) tag (6his) at the n terminus. dna sequence of the insert was determined by sequencing. igv. recombinant protein was expressed in escherichia coli and was purified on a ni column (novagen). the purified protein was examined by sds-page and western blotting. six-eight weeks female balb ⁄ c mice (institute of zoology chinese academy of sciences, china) was used for the study. mice were immunized twice intradermally with 10 ug m2e-hbc (provided by cnic, china) combined with different doses of recombinant igv protein 5, 10, 50 ug, respectively, or without igv as control. the area proximal to the tibialis anterior muscle was sterilized with 70% ethanol and different groups of mice were injected bilaterally with 5, 10, 50 ug igv plus 10 ug m2e-hbc in 50 ul phosphate buffer saline per mouse using a 1 ml syringe with attached 1 ⁄ 2¢¢ 30g needle. the immunization was given at 3 weeks intervals. four blood samples were obtained from every mouse: before immunization, after the first and second immunization, and after virus challenge by retro-orbital plexus puncture. after clotting and centrifugation, serum samples were collected and stored at )80°c prior to use for assays. mouse-adapted a ⁄ pr ⁄ 8 ⁄ 34 (h1n1), a ⁄ brisbane ⁄ 1 ⁄ 10 (h3n2), a ⁄ xinjiang ⁄ 1 ⁄ 2006 (h5n1), and a ⁄ guangzhou ⁄ 333 ⁄ 1999 (h9n2) were provided by chinese national influenza centre. nine to eleven days old embroynated specific pathogen free (spf) chicken eggs were inoculated with virus, and the eggs were incubated at 35°c for 2-3 days. the allantoic fluid was collected and purified by sucrose density gradient centrifugation, and the virus was inactivated by formaldehyde at 4°c overnight. to identify igg, igg1, igg2a against m2e, elisa assays were used. in brief, 96-well (nunc, brunei, denmark) were coated with 100 ul ⁄ well of m2e recombinant protein (provided by gene lab of ivdc, xuanwu district, beijing, china) in carbonate buffer (ph 9ae6) overnight at 4°c. immediately before use, the coated plates were incubated with blocking solution (2% bsa in pbs) for 2 h at 37°c and washed four times with pbs containing 0ae05% tween 20 (pbs-t). the serum samples were serially diluted and added in the plates. the detection color was developed by adding hrp-labeled goat anti-mouse igg, igg1, or igg2a ( figure 1) . no cross-strain response was observed in the control group. the igv adjuvented groups show splenocytes stimulation with seasonal h1n1, h3n2, h5n1, and h9n2 antigens. m2e-hbc immunization without igv showed splenocyte stimulation, but the extent was lower than animals immunized in the presence of the igv adjuvent. these data suggested that igv had enhanced effect on priming against the conserved viral antigen matrix protein and generation cross-strain immune response. influenza is a respiratory disease causing epidemics every year. h5n1 viruses and swine-origin h1n1 have also infected humans in recent years. seasonal influenza vaccine cannot cope with significant antigenic drift or with the emergence of pandemic viruses of different subtypes not contained in the vaccine. the high extent of conservation of the m2e makes it a promising immunogen. a vaccine based on coupling of the m2e peptide to an appropriate carrier may provide a universal vaccine with effectiveness and safety. m2e based vaccination induces protective antibodies not only in mice, but also in ferrets and monkeys. the carrier hepatitis b core as carrier with m2e forms a virus like particle (vlp). vaccination with m2e coupled to hbc induces protective antibody, 11 whereas the contribution of t cell protection was negligible. protection induced by vaccination with m2 coupled to hbc was weak overall. in order to improve the vaccination effect of m2e-hbc, new adjuvant igv was evaluated in combination with the m2e-hbc. the tim molecules are a recently discovered class of proteins with the ability to regulate the immune system. crystal structures of the tim molecules has revealed a unique, conserved structure with ligand-binding sites in the igv domain. to determine the potential immunostimulatory molecular properties of igv, we have evaluated immune response of the igv in combination with m2e-hbc vlp. previous papers reported that vlp immunized mice can induce the th1 and th2 immune response. 23 different adjuvant combined vlp can produce biased immune response th1 ⁄ th2 mixed immune response, or th1-preferred th1 ⁄ th2 profile. 24 thus, the response following the use of igv as a new adjuvant combined with m2e-hbc vlp needs to be evaluated. results indicated that igv combined groups showed th1 biased immune response and enhanced cross reactive t cell immune responses. this may show that igv immunized the mice and antiigv antibody can cross link the igv on t cells and enhance the cell figure 1 . t cell proliferation assay. mice were immunized twice with 50, 10, 5, 0 ug ⁄ ml igv plus m2e-hbc, respectively, and naive group was immunized with pbs. three weeks after a boosting immunization, spleens were harvested from immunized and naive mice. different subtypes of inactivated virus antigen (a) h1n1, (b) h3n2, (c) h5n1, (d) h9n2 were added and cocultured with different group splenocytes for 96 h. quick cell proliferation assay kit was used to detect the cell proliferation. the 420-480 nm absorbance was read on a plate reader. data were showed were shown as mean values. the difference between naive group and different doses igv plus m2e groups was determined using the student's t-test. all significance level is p < 0ae05. response. we also evaluated the cross-protection produced by igv combined m2e-hbc. we challenge with mouseadapted strain pr8 and prove the cross protection via reaction between the cells from the immunized animal and different subtypes of virus antigen. some subtypes of virus cannot infect the mice naturally, and therefore, virus challenge cannot be used to evaluate the effect. we co-cultured the t cells with inactivated antigen h1, h3, h5, and h9, and t cell proliferation was measured. results indicated that after immunization with igv plus m2e-hbc, the t cells show cross-protection with other subtypes. this provides evidence that igv can enhance the cross protection across subtypes. the results of this study demonstrated that recombinant igv can be useful as an adjuvant and polarize the m2e-hbc vlp immune response to a th1 profile. igv induced the m2e-hbc vlp to induce t cell proliferation and cross-reactive responses to different influenza virus subtypes. this finding represents a new direction for the promotion of cell mediated immunity in m2e based vaccine against influenza. a core european protocol, i-move, describing the methods to estimate influenza vaccine effectiveness (ive) was proposed by the european centre for disease prevention and control (ecdc) and epiconcept for the 2009-2010 season. it includes a case control method for pooled analysis based on a randomized ''systematic'' sample of swabs. 1, 2 collection of swabs using a non randomized, i.e., ''ad hoc,'' sampling strategy, left at the appreciation of sentinel practitioners, provides a greater number of cases and con-trols for ive estimation more easily than using a systematic randomized sampling strategy. the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than 5000 specimens yearly from cases of acute respiratory illness (ari), using both sampling methods. 3, 4 during the circulation of pandemic influenza 2009 viruses in france, it gave an opportunity to compare ive estimates using systematic randomized versus non systematic ''ad hoc'' sampling. influenza vaccine effectiveness was estimated by a casecontrol methodology according to ecdc i-move protocol, using on the one hand a systematic random sampling, on the other hand ''ad hoc'' non random sampling. the study was proposed to 608 primary care practitioners of the grog network (493 general practitioners and 115 pediatricians) trained to collect data and swabs. the study population was patients from the community of all ages consulting a grog practitioner for an influenza like illness (ili) and having a nasal or throat swab taken within an interval of <5 days after symptom onset. ili was defined according to the european union (eu) case definition as sudden onset of symptoms with at least one of the following four systemic symptoms: fever or feverishness, malaise, headache, myalgia; and at least one of the following three respiratory symptoms: cough, sore throat, shortness of breath. swabs were performed through usual surveillance. no ethical approval was needed, but an oral informed consent was requested. cases were excluded if they refused to participate in the study or if they were unable to give informed consent or to follow the interview in native language because of aphasia, reduced consciousness, or other reasons. an individual was considered as vaccinated against pandemic influenza if he or she reported having received a pandemic influenza vaccination during the current season, and if at least one vaccine dose occurred more than 14 days before ili onset. the study period started with the initiation of active influenza surveillance by the grog network, i.e., 15 days after the beginning of the influenza vaccination campaign, and finished at the end of the influenza period defined as the last week with at least one swab positive for influenza within the grog network. ''ad hoc'' sampling patients from which swabs were taken were selected by the grog practitioners during the study period. systematic random sampling during the same period, patients were selected at random as follows. an age-group 0-4 years (gps and pediatricians); 5-14 years (gps and pediatricians); 15-64 years (gps); 65 years or more (gps) was assigned to each practitioner, who was requested to swab the first patient of the week presenting with an ili within the pre-assigned age-group. swabs were collected in appropriate transport medium (virocult ò , viralpack ò , utm copan ò ) and sent by post to the laboratory in triple packaging following the international guidelines for the transport of infectious substances (category b, classification un 3373). laboratory confirmation of influenza was by rt-pcr to detect currently circulating influenza a (subtypes h3, seasonal and pandemic h1) and b viruses. an influenza case was defined as an ili case with a respiratory sample positive for influenza during the study period. controls were cases of ili having a swab negative for influenza during the study period. the outcome of interest is laboratory confirmed influenza. confounding factors and effects modifiers identified during the i-move preliminary study 5 were registered: risk factors, chronic diseases, severity of underlying conditions, smoking history, former vaccinations, and functional status. data on cases and controls were collected by the practitioners using a standardized questionnaire adapted from the i-move study. questionnaires were sent by the practitioners with the swab to the virology laboratory, and sent to the grog national coordination. data entry and validation were ensured by open rome through the vircases computing tool. validation steps included control of exhaustiveness of centralization of questionnaires, comparison of data entered by the labs and the national grog coordination, coherence control, and identification of missing data. analysis was done for the two sampling groups (systematic and ad hoc) on cases ⁄ controls following the european method proposed by epiconcept, using excel 2007 ª (microsoft corp. redmond, washington, usa) and stata ª . baseline characteristics of cases and controls in unmatched studies were compared using the chi-square test, fisher's exact test, or the mann-whitney test (depending on the nature of the variable and the sample size). the association between vaccination status and baseline characteristics was assessed for both case and control groups. the vaccine effectiveness was computed as ive = 1)or (odds ratio). an exact 95% confidence interval (ci) was computed around the point estimate. analysis was stratified according to age groups, time (month of onset), presence or absence of chronic disease, and previous influenza vaccination. effect modification was assessed comparing the or across the strata of the baseline characteristics. confounding factors were assessed by comparing crude and adjusted or for each baseline characteristic. a multivariable logistic regression analysis was conducted to control for negative and positive confounding factors using a complete case analysis (with records with missing data dropped) and using multiple imputation with chained equations. the complete model included age group, number of gp visits, onset week, seasonal vaccination, previous seasonal influenza vaccination, presence of chronic disease and associated hospitalizations in the previous 12 months, gender, and smoking status. variables were tested for multi-colinearity. interactions were tested using the likelihood ratio test (or wald test) and included in the model if significant at 5% level. a model with fewer variables (age group, number of gp visit, onset week, and seasonal vaccination) was also tested. several models were applied to both the ''ad hoc'' and systematic sampling groups of cases and controls. as shown in table 1 , whatever the analysis method used, the ''ad hoc'' sampling strategy led to a slightly lower estimate of ive. the ci were extended when data were missing and reduced when using multiple imputations with chained equations. however, from a statistical point of view, comparison of ''ad hoc'' versus systematic strategies is not straightforward, because ''ad hoc'' sampling is not randomized and does not allow comparisons with statistical tests using statistical distribution laws. there are more missing data with the ad hoc sampling method. this is mainly due to our validation procedure: in the case of missing data in the systematic sampling group, as required by the i-move study protocol, queries were sent to sentinel practitioners using mail and phone calls. this specific heavy workload is not usually performed during routine surveillance and has not been achieved for the ''ad hoc'' sampling group given the great number of cases and controls (2690). within the framework of the i-move study, several items were added to the grog's usual clinical form accompanying swabs (hospitalizations, number of gp visits, smoking status, help needed for bathing or walking). in 2009-2010, gps explained that this added workload was not compatible with their daily additional workload due to the pandemic situation. therefore, many of them refused to fill these new items systematically and threatened to leave the network. we thus obtained that the ''i-move items'' would be filled in for the clinical forms linked to systematic sampling, but were not in a position to obtain that for ''ad hoc'' sampling. the weekly distribution of systematic swabbing is not similar to that of ad hoc swabbing. the percentage of ad hoc swabs was higher than systematic swabs during the pandemic wave (mid-november to end of december) during which time the percentage of swabs positive for influenza was also higher ( figure 1 ). this could explain the higher rate of positive swabs within the ''ad hoc'' samples. the vaccination campaign was launched by the ministry of health on october 20, 2009, 6 and vaccination coverage increased during the surveillance period. in february, the vaccination coverage was 10ae2% in patients swabbed in the systematic group (9ae6% on imputed data) and 11ae5% [4ae4-23ae4] in the ad hoc group (11ae9% on imputed data). at the national level, vaccine coverage is estimated at 7ae95%. 7 due to the over-mediatisation of pandemic vaccination and to rumors about its poor effectiveness, overconsultation of vaccinated patients and over-swabbing of vaccinated patients in the ad hoc group are not surprising. age distribution is significantly different between our two samples (p < 0ae0001): the rate of 5-14 years old is lower in the systematic sampling group (25ae5%) than in the ad hoc sampling group (35ae7%). this can be explained by the fact that for the systematic sampling procedure, each grog practitioner had to swab the first ili patient in his assigned age group, whereas for ''ad hoc'' sampling, every grog practitioner could swab any ili patient irrespective of age. given the emphasis by health authorities and media on the burden of pandemic influenza among children and teenagers, one can hypothesize that when they were able to, sentinel practitioners focused on these age groups. gps in the ad hoc sampling scheme seem to have been more likely to select cases and further, to select vaccinated cases. those patients may have consulted earlier with specific symptoms (strong headache being more prevalent among cases). over-swabbing of patients having these symptoms in the ad hoc group is likely. the 2009-2010 pandemic influenza season was markedly different from previous ones: vaccination rate increased during and mainly after the pandemic peak; behaviors were strongly modified by unusual media hype; clinical features and risk factors might be different. it will be necessary to see if similar results are observed during a regular influenza season during which the vaccination rate increases before the epidemic peak with usual messages about vaccination and usual clinical influenza features. influenza early warning networks can estimate ive, taking into account many covariates. from a stakeholders and patients point of view, during the 2009-2010 influenza pandemic wave, there were no major discrepancies between ive estimated with an ad hoc sampling strategy, based on sentinel practitioners instinct, and ive estimated with a systematic random sampling strategy whatever the multivariable analysis methodology. although from a statistical point of view, comparison of the two strategies is not readily feasible because of the non random nature of ad hoc sampling. this latter strategy seems to result in slightly lower ive estimates, which could potentially be attributed to sentinel practitioners swabbing behavior. the ability to avoid missing data is a key point to decide which sampling method must be adopted, because ci extent depends greatly on the proportion of missing data among covariates. to match ive evaluation to surveillance networks practicality, selection of only those data essential for the study endpoint and easily collected by sentinel practitioners is paramount. it will be necessary to determine if results similar to those observed during the 2009-2010 pandemic season are found during a regular influenza season. influenza a viruses are important pathogens which remain a major cause of morbidity and mortality worldwide, and large numbers of the human population are affected every year. the first influenza pandemic in this century broke out in humans in march 2009, and it was declared to be pandemic by mid-june. as of 1 august jul 2010, the pandemic virus had caused more than 18 449 deaths worldwide, according to the world health organization (http:// www.who.int/csr/don/2010_08_06/en/index.html). the infection and spread of the pandemic influenza was reduced in part due to the use of vaccines. however, the lack of h1n1pdm vaccine early in the pandemic illustrates the need to improve vaccine production and to generate vaccines that induce stronger cross-protection. inactivated split vaccines or live attenuated influenza virus vaccines (laivs) against h1n1pdm viruses were approved for human use by the united states food and drug administration. both the inactivated vaccines and laivs are produced by creating reassortant viruses that generally contain six vrnas (pb2, pb1, pa, np, m, and ns) from a master donor strain, plus the two glycoprotein vrnas (ha and na) from a virus that antigenically matches the strain predicted to circulate in upcoming influenza season (e.g. a ⁄ ca ⁄ 07 ⁄ 2009). the reference viruses containing inactivated split virus vaccines are produced in embryonated chicken eggs, and primarily result in the production of antibodies that recognize the viral glycoproteins. both of these vaccine approaches require significant lead time for vaccine production, and modern approaches to speed preparation of vaccines and improve their efficacy is a global priority. 1, 2 the ns1 protein of influenza a virus is a multifunctional protein that plays important roles in virus replication and as potent type i ifn antagonist. 3, 4 mutations and ⁄ or deletions in ns1 typically induce stronger ifn responses by the host; those in turn suppress the replication of influenza virus 3-7 and can enhance immune recognition. [8] [9] [10] [11] in this study, we created a panel of experimental h1n1pdm ns-laiv candidates that have different deletions in the ns vrna and analyzed the vaccine potential of each ns-laiv in mice and ferrets to identify the best candidate(s). wt h1n1pdm influenza a virus a ⁄ new york ⁄ 1682 ⁄ 2009 (ny1682) was created by reverse-genetics directly from a human swab specimen collected in new york state in april 2009. 12 deletions were introduced into the ny1682 ns plasmid to create three mutant ns segments: ns1-73, ns1-126, and nsd5. nucleotides 246-482 (cdna of ns segment) and 405-482 were replaced by stop codons to generate ns1-73 and ns1-126; nucleotides 612-626 were deleted to generate nsd5, whose open reading frames for ns1 and nep were maintained. recombinant viruses were generated by co-transfection of eight reverse-genetics plasmids carrying the cdna of each gene segment into 293t ⁄ mdck cocultured monolayer adapted from hoffmann et al. 12, 13 mouse studies experiments were performed in a biosafety level 3 laboratories approved by the u.s. centers for disease control and prevention and the u.s. department of agriculture, and were conducted under approved animal care and use protocols. groups of 6-week-old female balb ⁄ cj (jackson laboratory, bar harbor, me, usa) were anesthetized with isoflurane and inoculated intranasally with 10 5 tcid 50 of each recombinant virus in 20 ll of pbs diluent, or pbs as controls. body weights and clinical symptoms of the mice were monitored daily for 10 days. nine mice in each group were euthanized on 1, 2, and 5 days post inoculation (dpi), and nasal washes and lungs were collected for virus titration by tcid 50 assay in mdck cells. at 30 dpi, 12 mice per group were challenged intranasally with 5 · 10 4 tcid 50 (100 ld 50 in 6-week-old mice) of a mouse-adapted variant of ny1682 (a ⁄ ny ⁄ 1682 ⁄ 2009-ma7) (accepted, journal of virology). disease symptoms and weights of the vaccinated mice were monitored for 10 days, and four mice from each virus group were euthanized at 3 and 6 days post challenge. lungs were removed and homogenized for virus titration by tcid 50 assay. the mice that became moribund or lost >25% of their starting body weight were euthanized for humane reasons. male fitch ferrets (triple f farms, sayre, pa, usa), 7-10 months of age and serologically negative by hemagglutination inhibition (hi) assay for currently circulating influenza viruses were used in this study. groups of 3 or 4 ferrets were inoculated intranasally with 10 6ae5 tcid 50 of one of the viruses: ny1682 wt (n = 4), ns1-73 (n = 3), ns1-126 (n = 4), or nsd5 (n = 4). ferrets were monitored for clinical signs through 14 dpi as previously described. 14 nasal washes were collected on 1, 3, 5, and 7 dpi and were titrated in mdck cells by tcid 50 assay. serum was isolated from blood collected 6ae5 weeks after immunization and used for neutralization assays. the ferrets were challenged with 10 6 pfu of a ⁄ mexico ⁄ 4482 ⁄ 2009 15 6ae5 weeks postimmunization and monitored for clinical signs of disease through 14 dpi. nasal washes were collected on 1, 3, 5, and 7 dpi, and were titrated in mdck cells by plaque assay. using reverse genetics, we created three laiv candidates weight loss of wt virus inoculated mice became evident at 3 dpi, and the mice did not recover until 8 dpi (figure 1a) . in contrast, mice inoculated with any one of the vaccine candidates had no clinical signs of disease and continued to gain weight at the same rate as did the mockinoculated mice ( figure 1a ). viral titers in the lungs of ns1-73, and ns1-126 infected mice were $100-fold lower than titers from wt virus-infected mice at all the time points analyzed (1, 2, and 5 dpi) ( figure 1b) . notably, the nsd5 laiv was cleared from the mouse lungs very rapidly, and the mean titers were $100-fold and 50 000-fold lower than the titers of the wt virus at 1 and 2 dpi, respectively ( figure 1b) . the vaccinated mice were challenged with a mouseadapted variant of ny1682 (accepted, journal of virology) on 30 dpi. no disease symptoms were observed in the mice immunized by any of the ns-laiv candidates or the wt control. in contrast, disease symptoms including ruffled fur, hunched posture, and weight loss were observed in the mock-immunized mice as early as 2 days post challenge (dpc); the symptoms progressed to severe disease, and the animals showed dramatic weight loss, became moribund, and succumbed to infection by 5 dpc (figure 1c ). high titers of virus ($10 7 tcid 50 ⁄ ml) were present in the mock-immunized mice at 3 dpc and at 6 dpc ( figure 1d ). in contrast, virus was not detected in the lungs of immunized mice ( figure 1d ). this challenge data demonstrates that all of the ns-laiv candidates, including the highly attenuated nsd5, induced sterilizing immunity that protected mice from a lethal ny1682 h1n1pdm variant. groups of ferrets were intranasally immunized with 10 6ae5 tcid 50 of each vaccine candidate or the wt virus. the titer of viruses recovered from nasal washes ranged from 10 6ae8 to 10 4ae5 tcid 50 ⁄ ml through day 5 in the wt virusinfected group, while the ns-laivs showed various degrees of attenuation (figure 2a) . the viral titer of all of the ns-laivs is at least 100-fold lower than that of wt in the nasal wash collected at 1 dpi. the ns1-73 laiv was the least attenuated in ferrets, and its replication was similar to that observed in mice. relative to the wt virus, the ns1-126 laiv showed 100-fold reduction in titer, and the nsd5 laiv was below the limit of detection (at least 1000fold reduction) at 3 dpi. sera from blood collected 6ae5 weeks after immunization was analyzed for the presence of neutralizing antibodies by micro-neutralization assays. the ns-laiv candidates all induced very strong neutralizing antibody responses (1280-5120) that were similar to the titer elicited by wt virus infection ( figure 2b ). the ferrets were challenged with 10 6 pfu of a ⁄ mexico ⁄ 4482 ⁄ 2009 (h1n1pdm) 6ae5 weeks post immunization. little disease or weight loss were observed in the naïve ferrets, and the ferrets immunized by infection with wt virus or the ns-laiv candidates didn't show any disease symptoms or weight loss. in contrast to the high titer of virus detected in the naïve ferrets through 5 dpc, the ns-laiv immunized ferrets had very low levels of a ⁄ mexico ⁄ 4482 ⁄ 2009 in their nasal washes at 1 dpc ( figure 2c ). the ferrets immunized with the ny1682 ns-laivs had $10 000-to 100 000-fold lower viral titers than did the naïve animals ( figure 2d ). in summary, the ns-laiv candidates dramatically inhibited initial replication of the h1n1pdm virus under stringent challenge conditions (10 6 pfu), and that the vaccinated animals rapidly cleared the infection (to below the limit of detection, by 3 dpc). our results demonstrate that all of the ns-laiv candidates are attenuated compared to the wt h1n1pdm virus, and the degree of attenuation is dependent on the specific ns mutation. ns1-73 was the least attenuated and does not represent a good vaccine candidate; whereas, nsd5 and ns1-126 were highly attenuated in both the mouse and ferret models. although they were markedly attenuated, they elicited strong neutralizing antibody responses and protected mice and ferrets from subsequent challenge. nsd5 has a subtle in-frame deletion (15 nt) that affects both the ns1 (residues 196-200) and nep (residues 39-43), and is analogous to a naturally attenuated variant of a normally highly pathogenic h5n1 virus (a ⁄ sw ⁄ fj ⁄ 03). 16 the analogous ns1 deletion in a ⁄ sw ⁄ fj ⁄ 03 (residues 191-195) was shown to reduce binding to host cleavage and polyadenylation specificity factor (cpsf), reduce ns1 protein stability, and enhance the type i ifn response of this h5n1 virus. 16 our study indicates that deletion of these 15nt in the ns vrna of the h1n1pdm also stimulates the host ifn response, specifically, ifn-ß, ifn-k1, ip10, and mxa (data not shown). the role of the deletion of residues 39-43 from nep has not been elucidated, but the induction of ifn and isgs by nsd5 was similar to, or slightly lower than, their induction by ns-126, suggesting that the nep mutation also has an attenuating effect that warrants future investigation. in summary, we have generated a panel of laivs directly from a swab specimen containing a new pandemic virus and analyzed their attenuation and immunogenicity in two animal models. our study demonstrates that nsd5 is a novel ns-laiv that could be used to create laivs for diverse influenza a viruses. this study also validates the use of ns-laiv candidates, which are not only highly attenuated, but they also elicit strong innate and adaptive immune responses, resulting in protection of mice from subsequent challenge with a lethal mouse-adapted variant of ny1682, and ferrets from challenge with a ⁄ mexico ⁄ 4482 ⁄ 2009 (h1n1pdm). currently, a total of approximately 300 million doses of inactivated influenza vaccine are being produced worldwide each year. one of the limitations in vaccine production is poor growth of human isolates in embryonated chicken eggs. this is essential to develop high yield seed viruses for large scale production of influenza vaccines. influenza a vaccine production utilizes high yield reassortants carrying ha and na genes from a wild type (wt) strain with generally 5-6 internal genes from the a ⁄ pr ⁄ 8 ⁄ 34 (pr8) strain, an highly egg adapted high growth donor strain. 1 influenza b vaccines, however, have been produced directly from wt strains, partly because no high yield donor analogous to pr8 has been identified. in recent years, reverse genetics has been used as an alternative means of developing high growth vaccine viruses. 2, 3 since in this plasmid-based technology, a 6:2 reassortant (six internal genes from a donor strain and two surface antigen genes from wild type strain) can be directly rescued, reverse genetics-derived reassortant viruses were expected to grow as efficiently as those derived from classical reassortment. however, reverse genetics reassortants have not produced the expected high growth for several reasons: (i) the 6:2 configuration is not always the best for virus yield, (ii) there is no process included for positive selection of adaptive mutants from quasispecies, and (iii) cell-derived viruses are not readily adapted to grow efficiently in eggs. our laboratory at new york medical college has been preparing b reassortants for several years by classical reassortment using b ⁄ lee ⁄ 40 as a donor. it has been possible to develop b reassortants, which produce higher virus yields than wt strains in eggs, and it was found that the np gene of b ⁄ lee ⁄ 40 was important in producing high yield b reassortants. 4 however, b ⁄ lee ⁄ 40 is inconsistent in providing high yield properties to b reassortants. in this study, in an attempt to find an alternative donor, we investigated the usefulness of b ⁄ panama ⁄ 45 ⁄ 90 for developing high yield b reassortants. as a wt strain, b ⁄ brisbane ⁄ 60 ⁄ 2008 was used, which is one of the recommended influenza b virus vaccine strains for the 2009 ⁄ 10 and 2010 ⁄ 11 seasons. we found that b ⁄ panama ⁄ 45 ⁄ 90 is a useful donor, and some of the resultant reassortants were considered as vaccine candidates. 5 b reassortant viruses were prepared by the classical reassortment method described by kilbourne. 1 the antiserum to b ⁄ panama ⁄ 45 ⁄ 90 hemagglutinin and neuraminidase (hana) was raised in this study by immunizing rabbits with hana isolated from b ⁄ panama ⁄ 45 ⁄ 90; purified igg was used for antibody selection. the yields of the reassortants and their corresponding parent viruses were assessed by hemagglutination assay. viral rna was extracted directly from the allantoic fluid and amplified by rt-pcr to produce cdna for analyzing the gene composition. restriction fragment length polymorphism (rflp) analyses were performed to determine the origin of each gene segment of the high yield reassortants. restriction enzyme sets for each gene segment are available upon request. in this study we investigated the usefulness of b ⁄ panama ⁄ 45 ⁄ 90 as a donor for transferring high yield phenotype. b ⁄ panama ⁄ 45 is a yamagata lineage strain with high growth phenotype (ha titer: 1024-2048). b ⁄ panama ⁄ 45 ⁄ 90 itself was a recommended b virus vaccine strain for 1989 ⁄ 90-1994 ⁄ 95 seasons. as a wt virus, a victoria lineage strain, b ⁄ brisbane ⁄ 60 ⁄ 2008, was used, which is a recommended b virus vaccine strain for use in the 2009 ⁄ 10 and 2010 ⁄ 11 seasons. reassortants were prepared according to classical reassortment protocol. after co-infection of b ⁄ panama ⁄ 45 ⁄ 90 and b ⁄ brisbane ⁄ 60 ⁄ 2008, progeny viruses carrying surface antigens (ha and na) of the vaccine strain were negatively selected by anti-b ⁄ panama hana antibodies, followed by passages without antibodies for positive selection of eggadapted viruses and finally limited dilution cloning. nymc bx-33, bx-33b, bx-33d, and r-3a are representative of resultant reassortants, which have significantly higher ha titers than the wt strain. the complete gene compositions of these reassortants were determined by rt-pcr ⁄ rflp analyses. as shown in table 1 , all of these reassortants contained the pb2 of b ⁄ panama ⁄ 45 ⁄ 90. other genes of b ⁄ panama ⁄ 45 ⁄ 90 (np of bx-33, m of bx-33b, and pb1 of bx-33d) may not be involved in the high virus yield, since no significant growth difference among these reassortants in eggs was found as assayed by hemagglutination test. accordingly, the pb2 of b ⁄ panama ⁄ 45 ⁄ 90 is considered to be the sole factor involved in the high yield phenotype donated to the vaccine strain. we previously found that the b ⁄ lee ⁄ 40 np gene was important in producing high yield b reassortants. it was of interest to examine whether b ⁄ lee ⁄ 40 np and b ⁄ panama pb2 could work together to produce even higher yields. to test this possibility, bx-33b (2:6 reassortant: pb2 and m genes from b ⁄ panama and the rest of the genes from b ⁄ brisbane) was selected and further reassorted with b ⁄ lee ⁄ 40. despite some difficulty in removing the na gene of b ⁄ lee ⁄ 40 (r-4c, 7b, 10b in table 2 ), by monitoring ha and na genes of resultant viruses after each antibody selection passage with anti b ⁄ lee ⁄ 40 hana antibodies, we were able to isolate and clone a triple reassortant, nymc bx-35, which contains the np gene from b ⁄ lee ⁄ 40 and pb2 and m genes from b ⁄ panama; the remaining genes are from b ⁄ brisbane ⁄ 60 ⁄ 2008 (table 2 ). in comparison with bx-33b, no significant growth enhancement (nor reduction) in eggs was found for bx-35 over that seen for bx-33b. nevertheless, bx-35 stably produces high virus yield and has been utilized as a seed virus for influenza b vaccine production for the 2010-2011 season by one or more vaccine manufacturers. there are contradictory reports 6-8 about the usefulness of reassortment for high yield influenza b viruses. however, we have been preparing b reassortants for several years by classical reassortment 1 using b ⁄ lee ⁄ 40 as a donor, and have been able to generate higher virus yield than wt strains. 4 in this study, we found that b ⁄ panama ⁄ 45 ⁄ 90 serves as an efficient donor in providing the high growth capacity to b ⁄ brisbane ⁄ 60 ⁄ 2008 (a recommended vaccine virus of victoria lineage for 2009 ⁄ 10-2010 ⁄ 11 seasons), and that the pb2 of b ⁄ panama ⁄ 45 ⁄ 90 is associated with the high yield phenotype. this particular strain from yamagata lineage might be useful to prepare high yield reassortants for other victoria lineage vaccine viruses. we noticed in this study that there may be segment incompatibilities between b ⁄ panama ⁄ 45 ⁄ 90 and b ⁄ brisbane ⁄ 60 ⁄ 2008. as shown in table 1 , the pa and ns genes of all the high yield reassortants examined are derived from wt, b ⁄ brisbane ⁄ 60 ⁄ 2008, not from the donor, b ⁄ panama ⁄ 45 ⁄ 90. this indicates that in this reassortment, the pa and ns genes are not replaceable with that of the donor to obtain high yield viruses. this degree of incompatibility might be common in b reassortment, resulting in low donor ⁄ wt reassortants, such as 2:6 and even 1:7 reassortants that we obtained in this study. if this is the case, reverse genetics based on 6:2 configuration may not result in generating high yield b reassortants unless a variety of donor ⁄ wt combinations are designed. one can speculate that in influenza b viruses, the surface glycoproteins (ha and na) and some of the internal proteins are functionally more closely related than in influenza a virus, as was seen in that pa and ns genes of b ⁄ brisbane ⁄ 60 ⁄ 2008 reassort together with the ha and na genes of the same parent (table 1 ). in our recent study on a reassortment between b ⁄ lee ⁄ 40 and b ⁄ panama ⁄ 45 ⁄ 90, it appeared that ha shapes overall gene constellations of the resultant reassortants, namely the reassortants tend to have more internal genes from the same parent of ha, no matter which parent's ha is selected by antibodies against the surface antigens of the other parent (data not shown). because of success in influenza a virus reassortment with pr8, it is generally believed that reassortant with 6:2 or 5:3 configuration is optimal for virus yield. this may be the case in most instances of influenza a reassortment, but is not necessarily so in b reassortment. as shown in this study, only a single donor gene is capable of improving the yield of vaccine strain by reassortment. influenza a ⁄ h1n1v has spread rapidly in all parts of the world in 2009 as a true pandemic. 1 epidemic events in russia occurred during the last week of september 2009 starting from far east region (yuzhno-sakhalinsk). kaliningrad (the western most russian city) was the second starting point of the epidemic. during october 2009 the epidemic spread over the whole russian territory. in a short period the new virus started to change genetically as it began to adapt to human populations during this pandemic (http://www.who.int; http://www.euroflu.org). in the period from may to december 2009, 1558 clinical samples (nasopharyngeal swabs and postmortem materials) of patients with influenza-like illness from different regions of russian federation were analyzed to confirm the diagnosis using real-time reverse transcription pcr (rrt-pcr). clinical nasopharyngeal swabs and bronchoalveolar lavage and post mortal fragments of trachea, lungs, bronchi, spleen from saint petersburg hospitals and 49 basic laboratories of federal influenza center were included in this study. all specimens were taken from patients with influenza-like illness or viral pneumonia. specimens were tested by rrt-pcr according to cdc protocols, i.e. using superscript iii platinum one-step qrt-pcr system (invitrogen) with primers and probes for infa, h1 seasonal, and h1sw (biosearch technologies). in addition, the test-systems 'amplisense influenza virus a ⁄ b-fl' and 'influenza virus a ⁄ h1-swine-fl' for pcr-detection, typing and subtyping of influenza viruses were also used. these test-systems are produced by central institute of epidemiology, moscow, russia and recommended by russian ministry of health as tests for influenza diagnosis. sequencing was carried out on an abi prism 3100-avant genetic analyzer (applied biosystems, usa) with bigdye terminator cycle sequencing kit. phylogenetic analysis was performed using programs vector nti 10.0 (invitrogen) and mega 4.1 (psu, usa) by maximum likelihood with the tim+i+g model for ha, and -hky+i+g model for na. evolutionary model was selected by akaike information criterion (aic) in model-test (posada, crandall, 1998). statistical reliability of tree branches was evaluated by bootstrap test (100 replications). immunohistochemical study was performed using novalink antibodies to ha and np with novocastra visualization system. influenza virus a ⁄ h1n1v rna was detected in 409 patients with severe form of influenza-like illness and 163 fatal cases. out of pcr-confirmed flu recovered cases 58% were patients under 14 years of age, 41% were aged 15-64 years, and 1% were older than 65 years. mean age of recovered patients was 15ae7 years (from 1 month to 77 years). viral rna in postmortem materials was detected mostly in lung tissue (86% of specimens) and trachea fragments (64%), and less commonly in spleen (17%). mean age of the deceased with confirmed flu (h1n1v) infection was 39ae3 years with age ranging from 7 months to 75 years. in 84% of fatal cases, influenza was complicated by viral or secondary bacterial pneumonia. median time from the onset of illness until death was 10 days. according to our data, 4% of patients died had diabetes, 4ae5% were obese, and 4% were pregnant women in the 2nd or 3rd trimester. ha and np were detected by immunohistochemical assay in lung tissue of dead patients with confirmed influenza virus a ⁄ h1n1v infection. ha and np was revealed in the endothelium of different sized blood vessels (capillaries and arterioles). these influenza virus proteins were also detected in some tissue macrophages apart from epithelium and endothelium. the localization of the two proteins was different: ha is mostly localized in cell membrane and cytoplasm, and np -mostly in the nucleus. here we present data on molecular genetic characteristics of 51 strains of pandemic virus, 40 strains obtained from clinical specimens, and 11 from post mortal ones isolated in the research institute of influenza. all the strains studied contain the s31n substitution in m2 protein, which indicates resistance to the adamantane antivirals, and have no h275y substitution in the neuraminidase, which indicates resistance to oseltamivir. the phylogenetic analysis showed that russian viruses were similar to influenza viruses a ⁄ texas ⁄ 05 ⁄ 2009 and a ⁄ california ⁄ 07 ⁄ 2009 (ha similarity 98ae9%). all russian viruses could be divided in two clusters: the first one includes viruses similar to the reference strain a ⁄ california ⁄ 07 ⁄ 2009, and the second one, which is the majority of viruses analyzed includes strains with substitutions ha s203t, na n248d, v106i, and ns i23v (figure 1 ). bootstrap support was 59. the isolates with ha s203t substitution can be classified in one of the five minor genome variants of a ⁄ h1n1v viruses found in the united states and mexico in 2009. 2 several viruses had strain-specific substitutions in antigenic sites sb and ca and the mutation d222g in ha receptor-binding site. the substitution of amino acid residue asp to gly at position 222 of ha was found in eight of eleven isolates (73%) from postmortem lung and trachea samples and two of forty isolates (5%) from nasopharyngeal swabs of patients with severe course of the disease. appearance of amino acid substitutions in the ha receptor-binding site (d187e and d222g ⁄ e) could be associated with influenza virus passaging on eggs. 3 five strains that contained g at position 222 of ha were isolated from post mortal specimens on mdck cells in this study, thereby excluding the possibility of substitution appearance hence to virus adaptation on eggs. in order to reveal genome changes in a(h1n1)v, strains isolated on the territory of russian federation during the 2009 pandemic, 67 full genome sequences from genbank, and research institute of influenza database were analyzed comparing two groups of viruses (isolated before and after 22 sept 2009). nine amino acid changes observed predominantly in late pandemic strains were found. five of them (s128p, s162n, d222g, v234i, v321i) reside in ha, two in na (i263v, n386k), two in pb2 (k340n, t588i), and one in pa (f35l). towards the end of the epidemic the viral population had demonstrated statistically certain rise in number of strains containing mutations in four genes. difference between groups was statistically significant (chisquare test, p = 0ae05). if v 2 > 3ae84, than difference between early and late strains is statistically significant. additionally fisher's test determined whether 'early strains' and 'late strains' differ significantly in the proportion of 'no mutation event' and 'mutation event' attributed to them in each particular position. all calculations were performed in fisher_tk freeware by vladimir belyaev similar to calcfisher (haseeb, 2003) fully described here (http://www.jstatsoft.org/v08/i21/paper). we have selected positions with statistically significant amino acid changes in late strains (p-value 0ae05). according to full genome analysis of influenza virus a ⁄ h1n1v 2009 strains, seven clades were distinguished, but the divergence between representatives of different clades remained small. 4 (figure 2 ). besides the strain a ⁄ perth ⁄ 12 ⁄ 2010 also contains substitution s181f in the same ha antigenic site. according to data obtained, the 2009 epidemic in russia was caused only by influenza virus a ⁄ h1n1v. unlike the previous epidemic periods when most severe influenza cases were registered among the children under 5 years and among elderly people aged over 65 years, the first wave of pandemic due to influenza virus a (h1n1)v resulted in increased level of mortality mainly among the people aged 18-53 years. though all pandemic viruses showed comparative genetic homogeneity, some evolutionary trends could be outlined. for clarification of the exact pathogenic role of mutation d222g in ha receptor binding site, further studies are necessary. full-genome analysis of influenza virus a ⁄ h1n1v strains circulating in the southern hemisphere in the new epidemic season 2010 revealed the phylogenetic subgroup distinguished by seven substitutions in inner proteins (pb2, pb1, np, ns1) and sa antigenic site of ha (n142d). the changes revealed could be caused by adaptation of the virus to an immunized human population. nasal and throat swabs (placed in 3 ml mem and frozen at )80°c until use for viral rna extraction and tissue culture inoculation) were collected from patients with febrile illness, i.e., >38ae0°c. samples were received from clinics in us embassies and us military laboratories located throughout the world since the initial who declaration of 2009 novel h1n1 outbreaks as a global pandemic on june 11, 2009. viral isolates were obtained from inoculating cultures of mdck cells with 0ae1-0ae2 ml viral suspensions collected in mem originated from patients after 2-7 days incubation. [10] [11] [12] [13] due to low viral titers in normal clinical samples, most of full viral genome sequences were derived from viral stocks obtained by tissue culturing passages (mdck, 1-2 times). viral rna was extracted from clarified supernatant fluid of nasal ⁄ throat swabs or mdck cultures using the 'charge-switch' rna extraction system based on the user manual protocol from the manufacturer (invitrogen inc., ca, usa). total rna was eluted into volume equal to original sample volume, i.e., 100 ll starting viral supernatant used to yield final 100 ll rna in molecular grade water (invotrogen inc.) and stored at )80°c until tested. generating ⁄ preparing overlapped cdnas for full genome coverage of 2009 novel h1n1 viruses by multiple rt-pcr amplifications the first step in the high-throughput sequencing pipeline for full influenza genome sequences was to establish a robust rt-pcr amplification scheme consisting 67 different rt-pcr primer pairs covering all 8 rna segments to ensure 100% amplification coverage of full viral genomes of all the incoming targeted viruses (houng, hs. 2011, submitted for publication). extracted viral rna (5 ll), derived from mostly mdck culturing stock or clinical sample containing sufficient viral load (>100 infectious units per ml) was added to primer-free rt-pcr total master mixture (10ae5 ll) for each virus followed by adding primer pair (2 ll, 3 pmole ⁄ ll per primer). rt-pcr was then performed: rt reaction through two hold-steps (50°c, 15 minutes and 94°c, 2 minutes); 35 cycling amplifications (94°c for 15 seconds, 53°c for 15 seconds, 72°c for 2ae5 minutes). specific cdna amplicons corresponding to each individual primer pair were routinely monitored and visualized by agarose gel electrophoresis. pooled 67 cdna products (2-5 lg) from each viral rt-pcr amplification run were used as sequencing substrates according to the roche 454 flx user manual and bulletins by incorporating adaptors containing individually multiplex identifier [mid]-key assigned to each individually pooled viral cdna. up to 12 different mid-keyed viral cdna were further pooled together to be clonally amplified on capture beads in water-in-oil emulsion micro-reactors (em amplifications), and pyrosequenced using one of two regions of a 40 · 75 mm picotiterplate. for each individual viral genome containing multiple assemblies (8 rna segments), we obtained sff file(s) containing raw sequencing reads from which nucleotide sequence data and phredlike quality scores were extracted. on average, 80ae0-90ae0% of 3-9 million mid-key specific nucleotides were extracted and mapped for consensus genome sequences. roche 454 gsmapper (v. 2.0 and 2.3) software was used to assemble all sequencing raw data and sff files into consensus sequences. new reference mapping projects were created to assemble each individually mid-keyed viral cdna into consensus viral sequences. one of the earliest 2009 h1n1 genomes of california origin, a ⁄ california ⁄ 04 ⁄ 2009(h1n1), deposited in genbank, was routinely employed as a reference genome sequence for most of gsmapper projects. the resultant consensus sequences obtained were further verified and validated through the ncbi annotation utility check and ultimately deposited to the ncbi influenza database, genbank. nucleotide sequences specific to each individual rna gene were aligned by the geneious pro 4.7.5 software (http://www.geneious.com). 14 trees were built based on the tamura-nei genetic distance model using the neighbor-joining method with no outgroup used via geneious pro 4.7.5. phylogenetic trees of the 2009 h1n1 genomes were constructed by importing fasta files containing specific concatenated target sequences of pb1, pb2, pa, ha, np, na, mp, and ns from each individual virus into the geneous pro software and going through the sequence assembling and tree building steps. high-throughput pyrosequencing of pooled 2009 novel h1n1 cdnas by roche 454 flx system up to 24 viral cdnas could be routinely sequenced to completion for 24 different full viral genomes from a single roche 454 flx picotiter plate by utilizing the combination of pico-titer plate's two distinct regions as well as 12 different mid-keyed adaptors. the 'shotgun' sequencing approach employed in this study is a feasible method to viral isolates (n) sequence multiple pooled 2009 h1n1 viral genomes. for each pyrosequencing experiment, approximately 800 000-1 000 000 passed key reads (single fragment per bead) were obtained that yielded readable nucleic acid sequences. among those close to a million passed key reads, only 500 000-600 000 passed key reads had an average sequencing read length of >220 bps, defined as 'long reads' (220 bps · 500 000 reads = total of 110 million bases of nucleic acid sequences) that were used to assemble into influenza genome sequences. mathematically, 110-130 million bases of raw sequencing data from each single roche 454 flx experiment would provide sufficient sequencing bases to cover 24 full genome sequences with approximate 3-400 · of sequencing depth coverage of influenza a with average genome size of 13 500 bps for the total of eight segmented rnas. so far, more than 100 full 2009 h1n1 genomes sampled worldwide have been successfully sequenced and deposited in the ncbi database by division of viral diseases, walter reed army institute of research (wrair). the bioinformatics derived from unique viral genome sequences generated from this study based on constant rt-pcr amplification scheme and identical roche 454 pyrosequencing protocols provide a reliable data set in predicting the evolutionary patterns of pandemic viruses. wrair received clinical samples from us embassies and military personnel throughout the world since the initial who announcement of 2009 novel h1n1 outbreaks. nearly equal distributions of sequenced viruses derived from three broadly categorized geographic regions, north america, central ⁄ south america, and asia ⁄ europe ⁄ africa (data not shown). besides the geographic distribution pattern of viral isolates, figure 1 displays the viral isolation time lines of all the sequenced viruses reflecting two peaks that coincided with two waves of 2009 pandemic infections, early-mid summer and fall of 2009. 15 phylogenetic trees of the eight influenza a segments of all sequenced viruses were tentatively generated. it was found that the substitution frequencies per site for the ha, na, and ns genes are at much higher rate than the other five genes, pb2, pb1, pa, np, and mp genes (data not shown). the observed higher genetic variations for ha and na genes of 2009 h1n1 are consistent with the historical genomic and epidemiological dynamics data of human influenza a revealing higher temporal fluctuations in ha and na genes. [16] [17] [18] [19] analysis of full influenza genomes containing concatenated eight complete rna segments revealed the existence of two distinctive genetic clades in circulation since the beginning of 2009 pandemic, as shown in fig-ure 2. it is noteworthy that all viruses of mexico and california origins (clade 1 shown at the top of figure 2 ) were isolated at the beginning of 2009 pandemic prior to the isolation of all other viruses belonging to the second genetic clade 2. 18, 20 discussion during the past decade, the advance of dna sequencing technology, such as development of ngs, in making full viral genome sequences readily available have enabled study of far broader and more detailed aspects of evolutionary change for any new emergent infectious pathogen. the massive sequencing capacity of roche 454 flx system allows simultaneously process and sequence millions of individual cdna molecules, in contrast to processing and sequencing individual cdna fragments by conventional sanger sequencing method. within a short period of few months since the beginning of the 2009 pandemics, wrair accomplished large number of representative 2009 h1n1 full genomes of worldwide origins via roche 454 flx system. sequencing data derived from this study illustrates a much higher genetic variation rate for ha and na genes of 2009 h1n1 that is compatible to the higher temporal fluctuation rate for ha and na genes of seasonal influenza a derived from decades of intensive monitoring and comparison studies and analyses. [16] [17] [18] [19] following the mexican and us reported cases, confirmed outbreaks of 2009 swine h1n1 rapidly proliferated and spread throughout europe, asia, africa, and south america, most probably via global airline travel. 5, 6 it seemed that new cases in the us and most cases throughout the world had been clinically mild relative to the initial reported cases in mexico. [21] [22] [23] [24] here we demonstrate through the phylogenetic relationship of sequenced 2009 h1n1 full genomes that the clinical isolates could be divided into two different clades of viruses, i.e., the clade 1 genetic group contains only viruses isolated at the beginning (march ⁄ april 2009, mexico and california) of 2009 pandemics and the rest of other viruses all belong to the 2nd genetic group, clade 2. thus, it's likely that the currently circulating 2009 h1n1 of clade 2 causing worldwide infections is genetically different from the initial 2009 h1n1 isolates that caused the early infections in mexico and california. 18, 20 introduction a pandemic influenza virus (2009 h1n1) was recently introduced into the human population. the hemagglutinin (ha) gene of 2009 h1n1 is derived from 'classical swine h1n1' virus, which likely shares a common progenitor strain with the human h1n1 virus that caused the pandemic in 1918. 1 since antigenic changes of influenza virus ha occur more slowly in swine than in humans, 2 we hypothesized that 2009 h1n1 might still retain an antigenic structure similar to that of 1918 h1n1 or the early isolates of its descendants. in this study, we compared ha antigenic structures of 2009 h1n1 and human h1n1 viruses by a molecular modeling approach to demonstrate the existence of shared epi-topes for neutralizing antibodies. we found that has of 2009 h1n1 and the 1918 h1n1 virus shared a significant number of amino acid residues in known antigenic regions. from this observation, we hypothesize that the 2009 h1n1 ha antigenic sites will be targeted by antibody-mediated selection pressure in humans in the near future. we further discuss possible directions of antigenic changes in the evolutionary process of 2009 h1n1. sequence data of ha genes modeller 9v6 was used for homology modeling of ha structures. after 100 models of the ha trimer were generated, the model was chosen by a combination of the mod-eller objective function value and the discrete optimized protein energy statistical potential score. after addition of hydrogen atoms, the model was refined by energy minimization with the minimization protocols in the accelrys discovery studio 2.1 software package using a charmm force field. steepest descent followed by conjugate gradient minimizations was carried out until the root mean square gradient was less than or equal to 0ae01 kcal ⁄ mol ⁄ a. the generalized born implicit solvent model was used to model the effects of solvation. the ha model was finally evaluated by using procheck, whatcheck, and verify-3d. custom-made programs were developed with the ruby language and used for investigating the numbers of potential n-glycosylation sites and candidate codons (cand1) in ha sequences. it is known that the h1 ha molecules have four distinct antigenic sites: sa, sb, ca, and cb. 3, 4 as a result, these sites consist of the most variable amino acids in the ha molecule of the seasonal human h1n1 viruses that have been subjected to antibody-mediated immune pressure since its emergence in 1918, although it was absent in humans from 1957 to 1976. to investigate the structures of these antigenic sites of 2009 h1n1, 3d structures of the ha molecules of sc1918, the recent seasonal human h1n1 virus (br2007), and 2009 h1n1 (ca2009) were constructed by a homology modeling approach, and compared by mapping all the amino acid residues that were distinct from those of sc1918 ha (data not shown). we found that most of these antigenic sites of br2007 ha predominantly contained altered amino acid residues if compared with sc1918. by contrast, amino acid residues at these positions were relatively conserved in ca2009 ha when compared with sc1918 ha. notably, the sa and sb sites, which contain many amino acids involved in neutralizing epitopes near the receptor binding pockets, remain almost intact ( table 1 ), suggesting that antibodies raised by natural infection with sc1918 or its antigenically related descendant viruses play a role in specific immunity against ca2009. these observations lead us to hypothesize that such antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in the human population. based on this hypothesis, we speculated that 2009 h1n1 would undergo patterns of amino acid substitutions in ha similar to those seen in seasonal human h1n1 viruses during its epidemic period (i.e. those that have been substituted since 1934) (figure 1) . we then predicted possible amino acid substitutions of 2009 h1n1 from the sequence similarity of the antigenic sites. for example, both sc1918 and ca2009 had an asn residue at position 142 in the sa site. for sc1918, the residue at this position has altered from asn to lys since 1942. combining these two facts, it seems reasonable to hypothesize that ca2009 will also undergo an amino acid substitution from asn to lys at position 142 in the future. interestingly, we found that some of the recent variants of the 2009 h1n1 virus have indeed undergone substitutions identical to those predicted in figure 1 . it is important to monitor whether such variants will be selected and survive in sustained circulation in humans. next, we analyzed the acquisition of potential n-glycosylation sites associated with antigenic changes. previously, we reported that cand1 sites, a set of three codons that require single nucleotide substitution to produce n-glycosylation sequons, were important motifs to rapidly acquire n-glycosylation sequons. 5 therefore, we investigated the number and location of potential n-glycosylation sites and cand1 sites in 2009 h1n1 ha. we found that ca2009 also had a single n-glycosylation sequon at the same position in the globular head region of ha, and lacked the multiple n-glycosylations that have been observed in the antigenic changes of the human h1n1 virus during the early epidemic of this virus. we also found that ca2009 ha possessed three cand1 sites that were present at the same position in sc1918 ha (positions of the first asn residue, 177, 179, and 184). of these, the cand1 sites with positions at 177 and 179 had actually become potential n-glycosylation sites in human h1n1 viruses. this result suggests the likelihood of additional n-glycosylation at these sites during future antigenic changes of 2009 h1n1 ha. notably, some of the recent 2009 h1n1 variants (as of march 24, 2010) have an additional n-glycosylation sequon at position 179, where the 1918 h1n1 virus readily acquired an n-glycosylation site during its circulation. the present study suggests that the antigenic structure of 2009 h1n1 ha is similar, at least in part, to that of the 1918 h1n1 ha. the 2009 and 1918 h1n1 has share unique three-codon motifs that are important to readily acquire n-glycosylation sequons in their globular head region. based on these similarities, we predicted possible amino acid substitutions that might be associated with future antigenic changes of 2009 h1n1, and confirmed that such substitutions occurred in some of the recent variants of this virus. the present study provides an insight into likely future antigenic changes in the evolutionary process of 2009 h1n1 in the human population. influenza viruses are classified into three types, a, b, and c, based upon the antigenic properties of nucleoproteins and matrix proteins. influenza a virus infects a wide range of hosts, including human, bird, swine, equine, and marine mammal species, while influenza b and c are less pathogenic than influenza a and are mainly found in humans, although there is evidence that they can also infect other species. influenza a has evolved in association with its various hosts on different continents for extended periods of time. 1 to survive as a successful pathogen, the influenza viruses have developed a number of mechanisms, including antigenic mutation and genome reassortment, to continuously evolve and evade the surveillance of the host immune systems. antigenic and genetic analyses have provided important insights into the molecular dynamics of influenza virus evolution. 2 however, a comprehensive understanding of influenza viral genetic divergence and diversity remains lacking. neuraminidase (na) is a major surface glycoprotein of influenza a and b, but is absent in influenza c. it plays a key role in virus replication through removing sialic acids from the surface of the host cell and releasing newly formed virions. influenza a viral na genes are classified into nine subtypes (na1-na9) based upon their antigenic properties, while na genes of influenza b are not classified into subtypes. furthermore, most na subtypes of influenza a have evolved into distinct lineages and sub-lineages, which correspond to specific hosts or geographical locations. in this study, we conducted large-scale analyses of influenza na sequences in order to infer their evolution and to identify lineages (or sub-lineages) of influenza a viruses. a total of 11 454 na sequences that excluded laboratory recombinant sequences were downloaded from genbank. sequences were aligned with muscle and mafft. the alignments were adjusted manually using translatorx, 3 based upon corresponding protein sequences. phylogenetic analyses were conducted using the maximum-likelihood (ml) method in raxml. 4 a set of perl scripts were written by us to facilitate this computational analysis. lineages and sub-lineages were determined based on the topology of the ml trees. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, 5 but with the following modifications: a single digit is used to represent one of the nine subtypes and a letter is used to represent a lineage; a sub-lineage is also represented using a digit; a dot is used to separate a lineage and a sublineage. for example, 2a.2 means na2 subtype, lineage a, and sub-lineage 2. the time of most recent common ancestor (tmrca) was estimated using the bayesian mcmc method in beast. 6 in all cases, we employed the gtr + u4 nucleotide substitution model, in which the first and second codon positions are allowed different rates relative to the third codon position. all data sets were analyzed under a relaxed molecular clock and the bayesian skyline population coalescent prior. the maximum clade credibility (mcc) tree across all plausible trees was computed from the beast trees using the treeannotator program, with the first 10% trees removed as burn-in. phylogenetic analysis based upon na sequences revealed two large groups corresponding to influenza a and b, respectively ( figure 1a ). within influenza a, two subgroups were found, one consisting of na1, na4, na5, and na8 and the other consisting of the remaining five subtypes. subtype na9 was found to be a sister subtype of na6, na1 being a sister subtype of na4, and na8 a sister subtype of na5. finally, each na subtype forms a distinct cluster, indicating its genetic uniqueness. influenza a and b viral na were estimated to have diverged around 2641 years ago ( figure 1b ). however, it had large 95% hpd values which ranged from 1259 years to 4299 years ago. the na subtypes of influenza a diverged from more than 1000 to several hundred years ago. the time of most recent common ancestor (tmrca) of each subtype of influenza a virus was generally recent and ranged from the calendar years 1767 to 1928 (figure 1b ). in addition, the tmrca for influenza b viral na was dated back to 1936. a total of 23 lineages were identified in influenza a (table 1) . three lineages, 1a, 1b, and 1c, were identified for na1 based upon the tree topology. linage 1a originated from avian viruses and was further divided into 5 sub-lineages: 1a.0, 1a.1, 1a.2, 1a.3, and 1a.4. linage 1b consists of north american swine influenza viruses whereas 1c is a human lineage. two large lineages, 2a and 2b, were identified in na2. lineage 2a is a human-specific lineage. interestingly, five major swine clades were observed within this lineage. lineage 2b is an avian-specific lineage, and consists of 3 sub-lineages, 2b.0, 2b.1, and 2b.2. three lineages were found in na3. lineage 3a was found in north american avian, 3b in eurasian ⁄ oceanian avian, and 3c also in avian, but it does not show any geographical pattern. for na4, na5, and na6, each was classified into 2 lineages, one found in north american avian (4a, 5a, 6a) and the other in eurasian ⁄ oceanian avian (4b, 5b, 6b). three lineages identified respectively in na7 and na8 are north american avian (7a, 8a), equine (7b, 8b), and eurasian avian (7c, 8c). na9 was also found to have 3 lineages: north american avian (9a), eurasian ⁄ oceanian avian i (9b), and eurassian ⁄ oceanian avian ii (9c), respectively. in this study, we conducted large-scale phylogeny and evolutionary analyses using influenza viral na sequences. the results showed that divergence between influenza a and b viruses occurred earlier than between any influenza a subtypes. this observation was consistent with previous findings based upon phylogenetic analysis of the ha gene, one of the most important genes related to host infection. 7 within influenza a, two sub-groups were found, one consisting of na1, 4, 5, and 8 and the other consisting of the rest of five subtypes (na2, 3, 6, 7, 9) . this observation does not agree with the result described by liu et al., 8 where na subtypes 1, 3, 4, 5, and 8 formed one group and the remaining four subtypes (na2, 6, 7, and 9) formed the other group. this difference is apparently caused by the fact that an outgroup was not used in their phylogenetic analyses. in the present study, both influenza a and b viral na sequences were included in the analysis. high bootstrap values were obtained for major groups, indicating that the inferred evolutionary relationship should be highly reliable. classification and designation of the lineages and sublineages within the influenza a virus are essential for studies of viral evolution, ecology, and epidemiology. a total of 23 lineages were identified within nine influenza a viral na subtypes and with the majority of the identified lineages found to be host or geographic specific or both. our results demonstrated a comprehensive view for the evolution of na genes and provided a framework for the inference of evolutionary history of pandemic viruses and for further exploring of viral circulations in multiple hosts. for example, the global pandemics of human h1n1 in 1918, h2n2 in 1957, the pandemic of human h3n2 virus in 1968, 9 the crisis of h5n1 hpai in hong kong in 1997, 10 and swine-origin h1n1 influenza in 2009, 11 all can be mapped onto the lineages and sub-lineages identified in this study. such information will facilitate not only identification of known genetic origins but also early detection of novel influenza a viruses. influenza viruses constantly evolve to avoid the human immune pressure in the process of antigenic drift. 1 through sequencing of viral genomes, the rates and direction of virus evolution can be observed. moreover, comparison of protein sequences allows us to determine amino acid substitutions that are related to immune pressure and antigenic drift. the creation of global influenza genetic databases, along with concurrent development of analytical tools, allows the comparison of multiple influenza virus strains. 2 the main aim of this study was to perform antigenic and genetic comparison of pandemic influenza viruses (h1n1) isolated during the 2009-2010 pandemic in ukraine and in other countries. nasopharyngeal swabs and autopsy materials collected from infected patients were received from the areas of ukraine. in addition, field isolates of influenza viruses from the 2009 ⁄ 10 season and strain specific serum were used for identification by hemagglutinin inhibition assay. influenza viruses were identified and subtyped using real-time rt-pcr analyses using cdc primers and adopted protocols. 3 sequencing was performed in two world health organization (who) influenza collaboration centers (centers for disease control and prevention, atlanta and national institute for medical research, london). hemagglutinin inhibition assay was conducted using chicken and guinea pig red blood cells following standard who protocols. 4 the all 6 ukrainian isolates of influenza viruses, which were isolated in ukraine during august-november 2009, were identified as a ⁄ california the phylogenetic analyses confirmed the evolutionary relationship between ukrainian isolates and viruses from other countries, which were isolated during the first wave of the pandemic. high genetic and antigenic conservation of pandemic influenza viruses from ukraine and other countries also were demonstrated. considering that the emergence of the novel pandemic influenza strain occurred in countries of northern hemisphere during summer, it was very interesting and significant tracking the dynamics of genetic changes in influenza viruses, which were isolated at the beginning of epidemic and those isolated during the rise of the epidemic in ukraine. influenza a virus causes moderate to severe epidemics annually and catastrophic pandemics sporadically. due to the evasiveness of the influenza virus and the nature of its genome (eight single-stranded and negative-sense rna segments), it is essential to understand the evolution of this important pathogen. 1 influenza virus evolves by two major mechanisms: mutation and reassortment. antigenic and genetic analyses have revealed partially the molecular dynamics of influenza virus evolution. 2,3 however, important questions, such as how many genotypes in the influenza a virus, remain unanswered. one of the major issues pertaining to this genotyping problem is how many lineages or sub-lineages can be determined for a subtype and according to what criteria. because of the unique structure of the influenza a viral genome, the computational genotyping methods developed for other viruses cannot be applied to the influenza virus. constructing phylogenetic trees is a powerful technique for the identification of evolutionary groupings (i.e., lineages ⁄ clades). however, for large trees, it is hard to determine how many lineages and the boundaries for each lineage. in this regard, multivariate analysis methods, such as multidimensional scaling (mds) and model-based hierarchical clustering, both taking advantage of dimension reduction and visualization, can complement conventional phylogenetic methods. 4 hemagglutinin (ha), the fastest evolving segment, is recognized as the most important gene in the influenza virus that plays a key role in viral pathogenesis. however, we have only limited knowledge of lineages and sub-lineages occurring in the hemagglutinin (ha) gene of influenza a virus, 5 although much effort has been made in assigning clades or sub-clades in highly pathogenic avian influenza (hpai) virus ha. 6 in this study, both model-based hierarchical clustering and phylogenetic methods were used for sequence analysis. one objective for this study is to explore and develop a more accurate lineage approach for further comprehensive influenza lineage and genotype analyses. a total of 11 821 hemagglutinin (ha) sequences (approximately 1778 nucleotides long), excluding laboratory recombinant sequences, were downloaded from genbank as of march, 2010. 7 sequences were aligned with muscle 8 and mafft. 9 the genetic distance matrix of all pairwise sequences was computed using the k2p model under mega 5.0. we then used the distance matrix as input to the cmdscale module in r 2.5.1 for the mds analysis. the principle coordinates resulting from mds were used for the model-based hierarchical clustering analysis, again in r 2.5.1 (the r foundation. available at: http://www.r-project.org/). the bayesian information criterion (bic) values were computed based upon ten different statistical data models -eii, vii, eei, evi, vei, vvi, eee, eev, vev, and vvv. the highest bic value was used to determine the number of clusters in the given sequence data. phylogenetic analysis was conducted using maximumlikelihood (ml) in raxml. 10 raxml uses rapid algorithms for bootstrap and maximum likelihood searches and is considered one of the fastest and most accurate phylogeny programs for large-scale sequence analysis. all the analyses were conducted on the supercomputer cluster (holland computing center, http://hcc.unl.edu/main/index.php). the trees were visualized in figtree (version 1.3.1) . lineages and sub-lineages were determined based on both the topology of the ml trees and model-based clustering results. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, 11 with the following modifications: lineage analysis was conducted for each ha subtype, which agrees with the convention of influenza virologists that 16 ha subtypes were identified in influenza a virus; ha lineages are represented with digits and letters, where the digit(s) represent one of the 16 subtypes and a letter represents a lineage; here, we present sub-lineages or sub-sub-lineages also in digits, with smaller numbers representing earlier lineages or sub-lineages within the same subtype (e.g., lineage 1 occurs earlier than lineage 2); the digit 0 is used to indicate inclusion of ancestral viruses in a lineage (or sub-lineage); a dot is used to separate lineages, sub-lineages, and sub-sub-lineages. for example, 1a.1ae2 means ha1 subtype, lineage a, and sub-lineage 1, and sub-sub-lineage 2. the sub-lineage level can be extended as necessary. the model-based clustering method corroborates commonly used phylogenetic methods in lineage and sub-lineage assignment. here we use the h12 subtype as an example to show the lineage and sub-lineage assignment. the bayesian information criterion (bic) reaches its maximum when the number of clusters for h12 equals 3, regardless of which mode we choose ( figure 1a ). therefore, based on bic, the optimal number of clusters for the h12 subtype is 3. as a result, a total of 3 clusters based upon the vvv model were identified ( figure 1b) . a significant correlation was found in lineage assignments by the phylogenetic method and the model-based hierarchical clustering method ( figure 1b,c) . lineages 12a and 12b were identified for h12, which correspond to north american avian and eurasian avian, respectively. lineage 12a was further divided into 2 sub-lineages, 12a.0, 12a.1, where 12a.0 is the ancestral sub-lineage in 12a. based on both model-based hierarchical clustering and phylogenetic analyses, a total of 41 distinct lineages were identified among 16 subtypes, averaging out to be 2ae56 lineages per subtype ( table 1 ). the majority of the identified lineages were found to be host or geographic specific or both. for example, three lineages, 1a, 1b, and 1c, were identified for ha1. lineage 1a was further divided into two sub-lineages, 1a.0 and 1a.1. the 1a.0 is swine-specific, whereas 1a.1 is a human pandemic 2009 h1n1 sub-line how to accurately identify an evolutionary lineage of influenza a viruses is challenging. one commonly used approach is molecular phylogeny, where phylogenetic trees are constructed, and the tree topology is used for lineage determination. here, we used a bayesian model-based clustering method, along with phylogenetic methods, to decide lineages and sub-lineages of influenza a viruses based upon sequence data. the results demonstrated that the modelbased clustering method corroborates phylogenetic methods and increases the accuracy of lineage assignment. one salient feature of this study is its large-scale analysis of all available influenza a hemagglutinin sequences. a total of 41 distinct lineages and 81 sub-lineages were classified; the majority of them were found to be host or geographic specific. this observation agrees largely with previous findings. 5 we are conducting further analyses of other influenza a segments 12 and expect to identify their lineages and create a comprehensive genotypes database for all influenza a viruses. such information will allow us to detect the genetic origin of newly found viruses, track their genetic changes, and identify potential genome reassortments. a hierarchical nomenclature system has been proposed and adopted for hpai ha clades and sub-clades by who influenza surveillance centers. 6 wan et al. also proposed a hierarchical approach for influenza a viral genotypes system. 13 the work presented here is one of the first steps towards the development of a nomenclature system for influenza a virus lineages (at the segment level) and genotypes (at the genome level). whether the naming system will be accepted and used by the influenza research community is more challenging than the lineage analysis itself. identification of the genetic origins of influenza a viruses will enhance our understanding the evolution and adaptation mechanisms of influenza viruses. the phylogenetic analysis is the traditional approach to identify the influenza progenitor. first, the nucleotide sequences are aligned using multiple sequence alignment methods, such as clustalw, 3 muscle, 4 and t-coffee. 5 second, phylogenetic analysis is performed on these aligned sequences to infer their evolutionary relationship using neighbor-joining (nj), 6 likelihood, or bayesian inference. 8 bootstrap analyses or computation of posterior probability are usually applied to estimate the phylogenetic uncertainty. however, this phylogenetic analysis is time consuming due to intensive computations in multiple sequence alignments and phylogenetic inferences. it is difficult to perform an analysis using this method on a large dataset, for instance, with more than 1000 taxa, as is the common case for influenza studies. alternatively, blast 9 is applied to identify the prototype genes in the database. blast determines a similarity by identifying initial short matches and starting local alignments. since influenza viral sequences have very high similarities, especially for most conserved regions, blast usually generates a large number of outputs, which will not be helpful for progenitor identification. since blast is a local sequence alignment, the results from blast may not reflect the global evolutionary information between the sequences. the blast scores cannot be used to define the evolutionary relations between viruses, especially in the context of the entire genetic pool. recently, we have developed a distance measurement method, complete composition vector (ccv), that can calculate genetic distance between influenza a viruses without performing multiple sequence alignments. 10, 11 we also adapted the minimum spanning tree (mst) clustering algorithm for influenza reassortment identification. 12 the application of this approach in the analyses of pb2 genes of influenza a virus showed that the integration of ccv and mst allows us to identify the potential progenitor genes rapidly and effectively. based on these results, here we develop a webserver called ipminer for influenza progenitor identification. ipminer can identify potential progenitors for a query sequence against all public influenza datasets within a few minutes. in order to improve the computing efficiency, 31 distance matrices were pre-computed by ccv, and they include 16 for ha (h1 to 16), 9 for na (n1 to n9), and one for each of the internal gene segments (pb2, pb1, pa, np, ns, and mp). these 31 pre-computed matrices will be updated weekly. ipminer just needs to compute the query matrices for a query sequence and sequences in the database. the standalone ccv program is also available at http://sysbio.cvm.msstate.edu/ipminer. in order to identify the influenza progenitor genes, ipminer first integrates the query matrix and a corresponding pre-computed matrix into a full distance matrix, which is then clustered by mst clustering algorithm. we adapted the threshold we measured previously in mst, u + nr, where u is the average distance and r is the standard deviation of a cluster. 12 as a result, mst will generate a hierarchical structure for the clusters. in each cluster, we will randomly select 20 viruses or 10% of the cluster size if this cluster has more than 200 viruses. ipminer will return the viruses with the smallest distances when the search reaches to the lowest level (the largest n) in this hierarchical structure. our analyses have shown that the level 5 has generally yielded good results for influenza a viruses. to visualize the overall mst structure, ipminer applies multi-dimensional scaling (mds) method to project all the viruses in the genetic pool onto a two dimensional graph, and the precursor viruses are marked in different shapes ( figure 1 ). the users can select other prototype viruses from the graph for further phylogenetic analyses. a single job with one query sequence takes <2 min. the genbank identifiers and associated genetic distances and sequence identities are displayed. the users can download the sequences for the identified precursor viruses as well as those from the prototypes viruses. in addition, for the users' convenience, ipminer generates a phylogenetic tree using nj method implemented in phylip 13 to illustrate the phylogenetic relationship among the query sequence(s), the identified progenitors, and the selected prototypes viruses. the programs in this solution package are written in java. the shell scripts are written in korn shell script in order to achieve high performance. cascading style sheets (css) are used for a consistent look across the pages. this also enables to change the overall design just by replacing the css definition file. php has been used as server side scripting and is written in java. in order to achieve high performance for computing in a genomic scale, we apply hash function or a binary tree, which enables that the precursor identification has a time complexity of o(n). for single queries, the users can visualize the results online. for batch queries of multiple sequences, the results will be sent to the users by e-mail. ipminer has been tested on microsoft internet explorer, mozilla firefox, and safari. the users need javascript to obtain full function of ipminer server. the webserver is available at http://sysbio.cvm.msstate.edu/ipminer. in summary, ipminer webserver has three major computational features for influenza progenitor identification: (i) it calculates the genetic distances through ccv and identifies the viruses with the shortest ccv distances against the query virus to be the progenitor genes; (ii) it projects influenza viruses onto a two dimensional map, which illustrates the global relationship between the progenitor genes and other viruses in the genetic pool; and (iii) it performs phylogenetic analyses between the query virus, the identified progenitor genes, and other selected prototype viruses. ipminer provides a user friendly web service for influenza progenitor identification in real time. the gisaid initiative offers an alternative to current public-domain database models in response to growing needs of the global influenza community for the sharing of genetic sequence and associated epidemiological and clinical data of all influenza strains. gisaid's publicly accessible epifluô database is governed by a unique sharing mechanism that protects the rights of the submitter, while permitting ongoing research as well as the development of medical interventions, such as drugs and vaccines. for the gisaid initiative, the max planck institute for informatics (mpii) saarbrücken, germany, has developed a web portal that is accessible at http://www.gisaid.org featuring the gisaid epifluô database that offers a unique collection of nucleotide sequence and other relevant data on influenza viruses. the database is based on software by oracle and the dante ò system by a3systems gmbh, germany. extensive metadata are also collected for most isolates. the database provides features for searching, filtering specific datasets for download, and user friendly upload functionality. to uphold gisaid's unique sharing mechanism, all users must positively identify themselves. while access is free of charge, all users agree that they will not attach any restrictions on the data, but will acknowledge both the originator of the specimen and the submitter of the data, and seek to undertake to collaborate with the submitter. all uploaded sequence data are submitted to rigorous curation by the friedrich-loeffler-institute for animal health (fli), germany. the database has been live since september 14, 2009. among its contributors are all five who collaborating centers for influenza who routinely contribute data in addition to using the epifluô database for their semiannual vaccine strain selection. to provide a complete picture of data, all data available in the public domain is routinely imported. as of october 8, 2010, the rapidly growing gisaid dataset comprises 166 989 nucleotide sequences (from 48 779 isolates) with 28 949 (from 11 857 isolates) uniquely submitted to this database. software development is underway to continually extend the spectrum of available data analysis tools. the intergovernmental process of the 62nd world health assembly specifically mentions gisaid as a publicly available database for depositing virus sequence data. starting in 2011, germany's federal ministry of food, agriculture and consumer protection will be the long-term host of the gisaid platform. the mpii will continue to develop the portal and database software and enable gisaid to act as a catalyst for the development of advanced bioinformatics software connected directly to the database. gisaid has become an indispensible resource for the international scientific community on influenza. the consortium will expand its activities and offers to catalyze research and development on a wide variety of issues pertaining to risk analysis, drug development, and therapy of influenza. options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 416-424 the pandemic h1n1 virus emerged in 2009 and spread rapidly throughout the world, principally affecting children and young adults. as this virus is new to the human population, it is important to determine if these influenza infections are more commonly associated with other respiratory pathogens compared to previously circulating influenza strains. co-infecting respiratory viruses may cause increased morbidity in individuals with pandemic h1n1, and may also be unwanted contaminants in influenza vaccines if original clinical samples containing these adventitious viruses are used to directly inoculate certified cell lines for vaccine production. to examine this issue, stored rna from 300 original clinical samples (nasal swabs, nasal aspirates, throat swabs) from australian and new zealand subjects that were collected in 2009 that were positive for pandemic h1n1 and 300 samples collected in 2008 that were positive for seasonal influenza by real time pcr assay (using the cdc, usa kits), were subjected to a resplex ii -panel version 2.0 (qiagen) pathogen screen. the resplex ii assay detects 18 common respiratory viruses, such as respiratory syncytial viruses (rsv a, b), influenza a and b viruses, parainfluenza viruses (piv1-4), human metapneumo-viruses (hmpv), coxsackieviruses ⁄ echovirus (cvev), rhinoviruses (rhv), adenoviruses (adv b, e), coronaviruses (nl63, hku1, 229e, oc43), and bocaviruses. resplex ii uses a combination of multiplex rt-pcr, hybridization of pcr onto target specific beads followed by detection using luminex-xmap technology. original clinical samples were received at the center from who national influenza centers, who influenza collaborating centers, and other regional laboratories and hospitals from australia, new zealand, and the asia ⁄ pacific region. most samples were from australia and new zealand. these samples consisted of nasal swabs, nasopharyngeal swabs, nasal washes, throat washes, and throat swabs. all samples were stored at )80°c until rna was extracted. rna was extracted from 200 ll of clinical sample using either the magnapure extraction system (roche, australia) or the qiaxtractor system (qiagen, australia) according to the manufacturer's recommendations with an elution volume of 90 ll and stored at )80°c until used. a 5 ll aliquot of rna was used to amplify the selected influenza virus gene using specific primers and probes as supplied by cdc (atlanta, usa) 1 along with super-script iii platinum one-step rt-pcr reagents (invitrogen, australia). real time pcr detection was performed on a 7500 fast system with sds software (applied biosystems, ca, usa). a cut off of a cycle threshold (c t ) of 35 or below was considered positive. resplex ii panel ver2.0 detection the qiagen molecular differential detection (mdd) system was used, which combines qiaplex amplification (multiplex rt-pcr) with detection on the liquichip 200 workstation (luminex's xmap microsphere based multiplexing system) and qiaplex mdd software according to the manufacturer's instructions. a low level cutoff was used (150) to obtain maximum sensitivity. from the 300 clinical specimens that were positive for influenza from 2008 by real time pcr, there were 18 (6%) a(h1n1) seasonal influenza viruses, 106 (35ae3%) a(h3n2) viruses, 174 (58%) b viruses, and 2 (0ae7%) viruses which were influenza a positive, but could not be typed. clinical samples from 2009 selected to study were all influenza a(h1n1) pandemic 2009 positive by real time pcr. detection of influenza virus in respiratory samples was much lower with the resplex ii assay (using a low cut off of 150 units) for pandemic influenza a virus (160 ⁄ 320; sensitivity 53ae3%) and to a lesser extent for seasonal influenza a (116 ⁄ 126; sensitivity of 92ae1%) and b viruses (161 ⁄ 174; sensitivity of 92ae5%) when compared to real time pcr. there were relatively few co-infecting respiratory viruses with either pandemic h1 infections in 2009 (5ae7%) or seasonal influenza infections in 2008 (6ae3%) ( table 1 ). the most common dual infection seen with pandemic h1n1 2009 viruses and 2008 seasonal b viruses was with cvev (7 ⁄ 18; 2009 and 5 ⁄ 14; 2008, respectively) while for 2008 a(h3) viruses there were no dominant co-infecting viruses ( table 2 ). in 2009 one case was detected with three respiratory pathogens in the same sample, a 25 year old female who had pandemic h1n1, cvev, and rhv, and in a 2008 seasonal influenza sample, one case with a triple infection was detected (bocavirus, piv2 and influenza b). the median age of subjects with co-infections was younger for both pandemic h1n1 with a median age of 10 years (range: 1 months to 27 years), compared to the full 2009 sample set which had a median age of 29 years (range: 1 months to 85 years), while for the patients from 2008 with seasonal influenza viruses with co-infections they had a median age of 6ae5 years (range: 1 months to 26 years) compared to all 2008 samples which had a median age of 16 years (range: 1 months to 84 years). there was good concordance in detecting influenza a and b in respiratory samples collected in 2008 between real time rt-pcr and the resplex ii system (100% versus >92ae1% for seasonal influenza a and b respectively). this data compares well with other studies such as li et al. 2 who found that resplex ii had 82ae8% sensitivity and 100% specificity for seasonal influenza a viruses and 90ae0% sensitivity and 100% specificity for influenza b viruses. in contrast, the present study found only 53ae3% sensitivity for the resplex ii detection of influenza a with the 2009 samples that were positive for pandemic h1n1 by real time rt-pcr. a recent study by rebbapragada et al. 3 also showed lower sensitivity for pandemic h1n1 viruses in nasopharyngeal samples with the resplex ii system (55% sensitivity and 100% specificity) compared to other commercial platforms seeplex rvp (95% sensitivity and 100% specificity) and luminex rvp (100% sensitivity and 97% specificity). interestingly the latest version of the resplex system offered by qiagen the resplex ii plus panel ruo now has a separate target for the pandemic h1n1 virus (mexico 2009). 4 in terms of detection of other respiratory viruses such as piv-1, piv-3, rsv and hmpv, high sensitivities (87ae5%, 72ae2%, 73ae2%, and 80%, respectively) and specificities (99ae7-100%) compared to taqman rt-pcr have been reported from testing of nasal wash and nasopharyngeal clinical samples. 5 in both the seasonal influenza positive 2008 and the pandemic h1n1 positive (by real time rt-pcr) clinical specimens, few other respiratory viruses were detected. only 18 of the 2008 samples had another virus detectable and one had two other viruses, while in 2009 16 out had another virus and one had two other viruses detected from a total of 300 influenza virus positive samples collected in each year. enteroviruses, coronaviruses, and parainfluenza viruses were most often found with both seasonal and pandemic infections. younger age appeared to be associated with co-infections with those subjects in 2008 with dual infections having a median age of only 10 years compared to the study groups 29 years; and similarly for 2009, the median age for subjects with dual infections was only 6ae5 years compared to the study groups' median age of 16 years. a study by chong et al. 6 on 298 nasopharyngeal swabs collected during 2007-2009 using resplex ii and luminex xtag rvp fast, they found dual respiratory virus infections in 27 ⁄ 179 (15ae1%) of samples and only 2 (0ae7%) with triple respiratory viral infections; however, these were from cases with any combination of multiple respiratory viruses not necessarily influenza, although influenza positive cases were the most common respiratory virus detected (37ae4% of all positive samples). given the low level and variety of viral co-infections along with both seasonal and pandemic influenza seen in this study, it is unlikely that influenza infections predispose subjects to particular respiratory viruses, but may still allow bacterial colonization, such as has been seen with severe and fatal cases with pandemic h1n1 with various bacteria including streptococcus pneumoniae, streptococcus pyogenes, staphylococcus aureus, or haemophilus influenzae. 7, 8 low levels of other respiratory viruses along with the finding that certain cell lines (like the mdck 33016-cells used in this study) do not propagate a number of these viruses (e.g. rsv a and b, rhinoviruses, coronaviruses), but do propagate others (e.g. parainfluenza 3) 9 should make testing for unwanted viruses that might be co-isolated with influenza viruses more focused and hence easier to detect and eliminate this isolate for future vaccine production. global influenza surveillance is one of the most important approaches to combat spread of disease. current laboratory methods for characterizing influenza are time-consuming and labor-intensive, and few viral strains undergo full characterization. 1 even fewer strains from domestic poultry and swine or from wild aquatic birds are wellcharacterized. these strains are important for global surveillance since they are thought to be the precursors to pandemic influenza strains. 2 we have designed a highthroughput global bio laboratory to address these surveillance needs. the goal of this project was to develop highspeed and high-volume laboratory capabilities for extensive surveillance and rapid and accurate detection and analysis of influenza. the workflow consists of surveillance, sample transportation, laboratory testing, data management and analysis. five robotic systems have been designed for this laboratory: sample accessioning, biobanking, screening, viral culture, and sequencing. sample accessioning logs barcodes, centrifuges, and aliquots samples are then sent to biobanking. the robotic biobank stores samples at )80°c and reformats tubes for screening. the screening system extracts rna and confirms the presence and subtype of influenza. aliquots of positive samples are sent to the viral culturing system for scale-up. finally, cultured samples are extracted and sent to the sequencing system for full genome sequencing. the sample accessioning, sequencing, and biobanking systems have been built, delivered, and validation processes are currently being completed. robotic screening and culturing systems have been fully designed and are ready to be built. a biosafety level 3-enhanced containment laboratory was built to enable the flow of samples containing highly pathogenic avian influenza viruses. in full operation, this approach to surveillance is designed to enable the sequencing of up to10 000 full virus genomes per year, more than the total of all full influenza genomes sequenced to date. the design of a robotic laboratory for influenza surveillance presents unique challenges and opportunities. before a robotic system is built, each assay is worked out on the bench top, each movement of the plates and reagents is defined, and the laboratory information management system (lims) must be able to address each step of the process. alternate assays are conceived for processes that are not automation-friendly. waste streams, worker safety, and space constraints are considered. each possibility is taken to reduce processes that have the potential to aerosolize or cross-contaminate influenza samples. instruments must be found that fit the capabilities needed. detailed specifications for each of the robotic systems were written including all the parameters listed above. once the systems are built, a long validation process takes place where the processes and instruments in each system are adjusted to function together properly. finally, a validation study is performed to ensure that the system is able to produce useful data for influenza research. the entire process takes months from start to finish for each robotic system and requires complete cooperation from a diverse team of researchers. the accessioning system logs initial sample information with the lims system. samples arrive in barcoded cryotubes. the liquid handler brings all samples up to a common volume and clarifies samples by centrifugation. samples are then transferred from screw-cap sample vials into storage plates containing 96 individually punchable storage tubes. each tube (0ae3 ml) is individually identifiable with a 2d barcode on the bottom. six archive aliquots are made, and tubes are individually weld-sealed for storage. tips for aspiration are fixed and undergo a high-pressure plasma process between each use to sterilize tips and destroy nucleic acids. samples are stored at )80°c. each module has a capacity of 600 remp plates or $60 000 samples. the automated freezer system can assemble requested samples as 96-well plates while samples remain frozen. the screening system uses magnetic bead extraction chemistry, real-time pcr, and a liquid handling system to extract samples, confirm and quantify the presence of influenza, and reformat extracted samples for input into the sequencing system. serotype of human influenza samples will be performed by real-time pcr. many samples will not have enough material for further analysis and will need to be scaled up. the culturing system combines incubators, a liquid handling platform, plate reader, and real-time pcr to culture, monitor growth, harvest, and quantify influenza. when the system is not being used for culture and scale-up, it can be used to assay previously cultured influenza samples for drug resistance. a challenge to sequencing large numbers of influenza samples is the manpower required for sample preparation. the sequencing system has the capacity to prepare up to 10 000 samples for sequencing per year for sanger sequencing. sanger sequencing was chosen because it is well-established for influenza surveillance, and automation-friendly. the system is designed to work with multiple primer sets (48, 96, 192) . robotic systems all report to the lims. each process completion, plate movement, and data point are entered and checked by an online, web-based lims. status updates, notification, reporting, and data analysis can be achieved without entering the bsl3 containment facility. routine data analysis such as determining whether a cultured sample is ready to be harvested will be performed by the lims. complex data analysis, while still requiring significant human input, will be made easier by the data-acquisition functions of the lims. the implementation of a high-throughput influenza surveillance laboratory will provide an influenza research and response capacity that far exceeds what is available today. with the addition of each new system, we add a new capability to the influenza community and new opportunities to foster partnerships and collaborations with government, foundations, businesses, and academic institutions. this laboratory will not only enable cutting edge research, but will also enable a more effective response of near real-time surveillance during a pandemic outbreak. pandemics of 1957 and 1968 were believed to arise from avian influenza viruses. 1 the tropism of avian and human seasonal influenza viruses for the human lower respiratory tract deserves investigation. the target cell types that support replication of avian influenza a viruses in the human respiratory tract in the early stages of clinical infection have not well defined. in a previous autopsy studies of human h5n1 disease, influenza a virus were found to infect alveolar epithelial cells 2 and macrophages. 3 in this study, viral infectivity and replication competence of human and high and low pathogenic avian influenza viruses were systematically investigated in the human conducting and lower respiratory tract using ex vivo organ cultures. we compared the replication kinetics of human seasonal influenza viruses (h1n1 and h3n2), low pathogenic avian influenza viruses (h9n2, h5n8) with that of the highly pathogenic h5n1 viruses isolated from human h5n1 disease. a range of human seasonal influenza a viruses of subtypes h1n1 and h3n2 viruses were included in this study from 1975 to 2009. two isolates of low pathogenic avian influenza a (lpai) (h9n2) viruses from different virus lineages isolated from poultry in hong kong in 1997, a low pathogenic influenza a (h5n8) virus isolate from wild ducks in hong kong in 2005, and two virus isolates of highly pathogenic avian influenza (hpai) a subtype h5n1 were included. fragments of human bronchi and lung were cut into multiple 2-3 mm fragments within 2 hours of collection and infected in parallel with influenza a viruses at a titer of 10 6 tcid 50 ⁄ ml and as control cultures were infected with ultraviolet light inactivated virus. these tissues fragments were infected for 1 hours and washed twice with pbs and incubated for 1, 24, and 48h at 37°c. the bronchial tissue was cultured in an air-liquid interface using sponge. viral yield was assessed by titration in mdck cells. one part of the infected tissue were fixed in formalin and processed for immunohistochemistry for influenza antigen. other part of infected tissue was homogenized and underwent rna extraction, and the expression of influenza virus matrix gene was measured by quantitative rt-pcr. human bronchus ex vivo cultures supported human seasonal influenza virus to replicate efficiently. avian influenza h9n2 virus replicated, although less efficiently than that of seasonal influenza viruses, whereas hpai h5n1 did not productively replicate in ex vivo cultures of human bronchus. this is in agreement with our previous finding in the well-differentiated bronchial epithelial cells in vitro. 4 on the other hand, human lung ex vivo cultures supported prominent productive replication of human seasonal influenza h1n1 ( figure 1a ) and hpai h5n1 ( figure 1f ) viruses. lpai, such as h9n2 ( figure 1c -d) and h5n8 ( figure 1e ), also replicated productively, but with a lower viral yield. surprisingly, the replication of human influenza h3n2 viruses ( figure 1b ) across the last three decades was greatly inhibited. there are clear differences in viral tropism of human seasonal and avian influenza viruses for replication in the human bronchus and lung. hpai h5n1 virus can infect and productively replicate in the lower lung, which may account for the severity of human h5n1 disease, but not in the conducting airways. surprisingly, there are marked differences in the replication competence of seasonal influenza viruses in ex vivo lung tissues, with influenza h1n1 viruses being able to replicate efficiently while h3n2 viruses do not. this may be related to the more strict siaa2-6gal binding preference of h3n2 viruses. on the other hand, the efficient replication of influenza h1n1 viruses in the alveolar spaces indicates factors other than tissues tropism alone play a role in the differences in disease severity between human seasonal h1n1 and avian h5n1 virus infections. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells, suggesting that viral protein(s) other than m gene-translational products facilitates the splicing of viral mrnas. the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns1 (c ⁄ ns1) and ns2 (c ⁄ ns2), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns1 and c ⁄ ns2 proteins, drastically reduced the splicing of ns mrna, raising the possibility that c ⁄ ns1 or c ⁄ ns2 enhances the splicing of viral mrnas. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns1, whereas it was reduced by co-expression of influenza a virus ns1 protein (a ⁄ ns1). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns1, whereas it was inhibited by that of a ⁄ ns1. these results suggest that influenza c virus ns1, but not a ⁄ ns1, can up-regulate the splicing of viral mrnas. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. 1, 2 the inefficient splicing of viral pre-mrnas can be understood partly by the fact that influenza a virus ns1 protein is associated with spliceosomes and inhibits pre-mrna splicing. 3, 4 cis-acting sequences in the ns1 transcript also negatively regulate splicing. 5 by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. 6 the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. 7 the yamagata ⁄ 1 ⁄ 88 strain of influenza c virus was grown in the amniotic cavity of 9-day-old embryonated hen's eggs. cos-1 and 293t cells were cultured in dulbecco's modified eagle's medium containing 10% fetal calf serum. subconfluent monolayers of cos-1 cells were transfected with pme18s containing influenza c virus m gene cdna using the lipofectamine procedure and then incubated at 37°c. total rna was extracted from both the transfected cells and cells infected with c ⁄ yamagata ⁄ 1 ⁄ 88 virus using the rneasy mini kit (qiagen). ribonuclease protection assay was performed using a ribonuclease protection assay kit rpa iii (ambion). 6 briefly, a [ 33 p]-labeled influenza c virus rna 6-specific rna probe (vrna sense) was synthesized by in vitro transcription and hybridized with the total rna at 42°c overnight. hybrids were digested with rnase a (0ae08 u) and rnase t1 (3 u) at 37°c for 30 minutes and then analyzed on a 4% polyacrylamide gel containing 4 m urea. hmv-ii cells infected with c ⁄ yamagata ⁄ 1 ⁄ 88 and cos-1 cells transfected with pme18s expressing influenza c virus ns1 were fixed with carbon tetrachloride at various times after infection and transfection, respectively. the cells were then stained by an indirect method using anti-gst ⁄ ns1 serum as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit igg (seikagaku kogyo) as the secondary antibody. the splicing efficiency of influenza c virus m gene-specific mrna (m mrna) in infected cells was higher than that in m gene-transfected cells the ratio of m1 encoded by a spliced m mrna to cm2 encoded by an unspliced m mrna in influenza c virusinfected cells was about 10 times larger than that in m gene-transfected cells. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells (figure 1 ). these data suggest that viral protein(s) other than m gene-translational products facilitates viral mrna splicing. the influenza c virus ns gene translational product may up-regulate the splicing of viral mrnas the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns1 (c ⁄ ns1) and ns2 (c ⁄ ns2), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns1 and c ⁄ ns2 proteins, drastically reduced the splicing of ns mrna, suggesting that c ⁄ ns1 or c ⁄ ns2 enhances viral mrna splicing. immunofluorescent staining showed that ns1 localized in the nucleus in the early phase of infection, and was distributed in both the nucleus and cytoplasm in the late phase of infection, raising the possibility that influenza c virus ns1 protein plays a role in viral mrna splicing that occurs in the nucleus. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns1, whereas it was reduced by co-expression of influenza a virus ns1 protein (a ⁄ ns1) (figure 2a ). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns1, though it was inhibited by that of a ⁄ ns1 ( figure 2b ). these results suggest that influenza c virus ns1, but not a ⁄ ns1, can up-regulate the splicing of viral mrnas. 7 in influenza a virus-infected cells, splicing is controlled so that the steady-state amount of spliced mrnas is only 5-10% of that of unspliced mrnas. 1, 2 the mechanisms by which influenza a virus ns pre-mrnas are poorly spliced have been investigated and the following confirmed. influenza a virus ns1 protein associates with spliceosomes and inhibits pre-mrna splicing. 3, 4 two cis-acting sequences in the ns1 transcript (positions 153-465 in the intron and positions 775-860 in the 3¢ exon region) inhibit splicing. 5 by contrast, influenza c virus m gene-specific mrna (m mrna) is efficiently spliced in influenza c virus-infected cells. 6 in this study, we examined the mechanism by which influenza c virus m mrna is efficiently spliced and the regulatory mechanism of the splicing of ns gene-specific mrna (ns mrna). the introduction of a translational pre-mature termination into the influenza c virus ns gene, thereby blocking the synthesis of influenza c virus ns1 (c ⁄ ns1) and ns2 (c ⁄ ns2) proteins, drastically reduced the splicing rate of ns mrna. we further examined whether c ⁄ ns1 potentially facilitates viral mrna splicing. the splicing rate of m mrna of influenza c virus was increased by co-expression with c ⁄ ns1, whereas it was reduced by co-expression with influenza a virus ns1 protein (a ⁄ ns1) (figure 2a ). the splicing of influenza a virus m gene-specific mrna was also increased by co-expression with c ⁄ ns1, though it was inhibited by co-expression with a ⁄ ns1 ( figure 2b ). these results suggest that influenza c virus ns1 can facilitate viral mrna splicing, but in no way inhibit it, which is in striking contrast to the inhibitory effect of influenza a virus ns1 on pre-mrna splicing. 3, 4 the mechanism for splicing enhancement by c ⁄ ns1 also remains to be determined. we speculate that c ⁄ ns1 may interact with some host proteins involved in splicing, thereby leading to an up-regulation in splicing, or that c ⁄ ns1 may bind to pre-mrna, increasing its accessibility to the spliceosome. the spliced mrna of the influenza c virus m gene encodes the m1 protein, which plays an important role in virus formation and determines virion morphology. 8, 9 therefore, it is speculated that the mechanism for efficient splicing of m mrna, which provides the m1 protein necessary for virus assembly in a redundant amount, has been maintained in the influenza c virus. by contrast, unspliced mrna from the influenza c virus m gene encodes the cm2 ion channel, which is permeable to chloride ions, 10 and also has ph-modulating activity. 11 although the role of the influenza c virus cm2 ion channel in virus replication remains to be determined, it is conceivable that the over-expression of the cm2 protein has a deleterious effect on virus replication since the fact that a high level of influenza a virus m2 protein expression inhibits the rate of intracellular transport of the influenza a virus ha protein and other integral membrane glycoproteins has been demonstrated. 12 if this is the case, efficient splicing of m mrna may control the amount of cm2 synthesized to optimize virus replication. therefore, we speculate that efficient splicing of m mrna leads to a high level of m1 expression and the reduced expression of cm2, thereby creating conditions that are optimal for virus replication. in this study, we provided evidence that c ⁄ ns1 facilitates the splicing of m mrna. furthermore, c ⁄ ns1 may regulate the splicing efficiency of its own ns mrna during infection, controlling the amount of c ⁄ ns1 and c ⁄ ns2 proteins in infected cells. c ⁄ ns2 plays an important role in the nuclear export of vrnp, and is also associated with vrnp in the later stages of infection in virus-infected cells and is incorporated into virions, 13 suggesting that c ⁄ ns2 is involved, not only in the sorting of vrnp into the assembly site, but also in virus assembly. therefore, it is likely that there is a mechanism by which an appropriate amount of c ⁄ ns2 is provided during infection to accomplish these functions. in conclusion, c ⁄ ns1, which enhances the splicing of viral mrna, may regulate both the expression level of m gene-derived m1 and cm2 proteins, and that of ns gene-derived ns1 and ns2 proteins, thereby leading to optimal virus replication. propagation of the human influenza viruses in embryonated hen's eggs always results in a selection of variants with amino acid substitutions in the hemagglutinin (ha) that affect viral receptor-binding characteristics (reviewed 1 ). brookes et al. 2 recently studied infection in pigs using the egg-grown virus that contained a mixture of the original a ⁄ california ⁄ 07 ⁄ 09 (h1n1pdm) and its two egg-adaptation mutants with single amino acid substitutions d222g and q223r (225 and 226 in h3 numbering system). only the original virus and the variant with 222g were detected in the directly inoculated animals, indicating that the variant with 223r failed to infect. only the original virus was detected in nasal secretions of contact infected pigs, suggesting that the d222g mutant failed to transmit. in contrast, there was an apparent selection of the d222g mutant in the lower respiratory tract samples from directly inoculated pigs. the d222g substitution is of a special interest as it can emerge during virus replication in humans and was associated with severe and fatal cases of pandemic influenza in 2009-2010 3-7 and 1918. 8 here we compared phenotypic properties of the original clinical isolate of h1n1pdm virus a ⁄ hamburg ⁄ 5 ⁄ 09 and its d222g and d223r mutants to explain observed effects of these mutations on virus replication in swine 2 and to predict their potential effects on virus replication in humans. a ⁄ hamburg ⁄ 5 ⁄ 09 (ham) was isolated from clinical material by two passages in mdck cells. the virus was passaged twice in 11-day-old embryonated hen's eggs and plaqued in mdck cells. the plaques were amplified in mdck cells and the sequences of the viral ha were determined. the variants with single mutation d222g and q223r were aliquoted and designated ham-e and ham-e1, respectively. the receptor-binding specificity of the viruses was assessed by assaying their binding to desialylated-resialylated peroxidase-labeled fetuin containing either a2-3-linked sialic acid (2-3-fet) or a2-6-linked sialic acid (2-6-fet). 9 in brief, viruses adsorbed in the wells of 96-well eia micro plates were incubated with serial dilutions of 2-3-fet or 2-6-fet, and the amount of bound fetuin probe was quantified by peroxidase activity. the binding data were converted to scatchard plots (a490 ⁄ c versus a490), and the association constants of the virus-fetuin complexes were determined from the slopes of these plots. viral cell tropism and replication efficiency in human airway epithelium were studied using fully differentiated cultures of human tracheo-bronchial epithelial cells (htbe). 10, 11 to determine cell tropism, cultures were infected at a moi 1, fixed 8 hours after infection, and double immuno-stained for virus antigen and cilia of ciliated cells. infected cells were counted under the microscope (100· objective with oil immersion) in the epithelial segment that included 15-30 consecutive microscopic fields containing between 5% and 20% ciliated cells relative to the total number of superficial cells. percentages of infected ciliated cells and infected non-ciliated cells relative to the total number of infected cells were calculated. ten segments per culture were analyzed and the results were averaged. to compare growth kinetics of ham and ham-e, replicate htbe cultures were infected with 200 plaque-forming units of the viruses followed by incubation at 35°c under airliquid interface conditions. at 24, 48, and 72 hours postinfection, we added dmem to the apical compartments of the cultures and incubated for 30 minutes at 35°c. the apical washes were harvested, stored at )80°c, and analyzed simultaneously for the presence of infectious virus by titration in mdck cells as described previously. 11 the non-egg-adapted h1n1pdm virus ham, similarly to the seasonal human virus a ⁄ memphis ⁄ 14 ⁄ 96 (h1n1), bound to 2-6-fet ( figure 1a ) and did not show any significant binding to 2-3-fet. this result contrasted with the binding of h1n1pdm viruses to several 2-3-specific probes in carbohydrate microarray analysis. 12 reduced avidity of virus interactions with soluble glycoprotein in solution as compared to its binding to the probe clustered on the microarray surface could account for these differences in the assay results. the d222g mutant ham-e differed from the parent virus by its ability to bind to 3-fet and by its reduced binding to 6-fet. the q223r mutant only bound to 2-3-fet, although less strongly than did the avian virus a ⁄ duck ⁄ alberta ⁄ 119 ⁄ 98 (h1n1). the viral cell tropism in htbe cultures ( figure 1b ) correlated with receptor specificity. ham and mem ⁄ 96 showed a typical human-virus-like tropism 10, 11 with preferential infection of non-ciliated cells (<5% of infected cells were ciliated). the mutant with 223r and control duck virus displayed a typical avian-virus-like tropism (preferential infection of ciliated cells). the d222g mutant displayed a cell tropism that was intermediate between those of human and avian viruses; in particular, this mutant infected significantly higher proportion of ciliated cells than ham and mem ⁄ 96. observed alteration of receptor specificity and cell tropism ( figure 1 ) suggested that egg-derived mutations can affect replication of the h1n1pdm virus in human airway epithelium. to test this, we first compared the capacity of the viruses to initiate infection in htbe cultures. replicate cultures were infected with identical doses of the viruses, fixed 8 hours post-infection, and immuno-stained for viral antigen. under these conditions, ham and ham-e infected comparable numbers of cells, whereas ham-e1 infected at least 10 times less cells (data not shown). this result indicated that the mutation q223r markedly impaired the ability of ham-e1 to infect human airway epithelial cultures. we next compared two other viruses ham and ham-e for their multi-cycle replication in htbe cultures and found that the original virus reached threefold higher peak titers 48 hours post infection than did the d222g mutant ( figure 2 ). the d222g mutation in h1n1pdm virus facilitates virus binding to 2-3-linked receptors and alters viral cell tropism in human airway epithelium. these changes could account for increased replication of the d222g mutant in the lower respiratory tract in humans 5-7 and pigs 2 and correlation of this mutation with severe pulmonary disease. [3] [4] [5] [6] [7] the d222g mutant replicates less efficiently in human airway cultures than the original virus. this finding correlates with an apparent lack of transmission of variants with 222g in humans 3 and pigs. 2 egg-derived mutation q223r abolishes virus binding to 2-6-linked receptors and strongly decreases infection in cultures of human airway epithelium. this result agrees with poor infectivity of the q223r mutant in pigs 2 and highlights potential pitfalls of using egg-adapted viruses with this mutation for the preparation of live influenza vaccines. nin-esterase-fusion (hef), nucleoprotein (np), matrix (m1) protein, cm2, and the non-structural proteins ns1 and ns2. 1,2 cm2 is the second membrane protein of the virus and is encoded by rna segment 6 (m gene). [3] [4] [5] [6] [7] [8] it is composed of three distinct domains: a 23-residue n-terminal extracellular domain, a 23-residue transmembrane domain, and a 69-residue cytoplasmic domain. 3, 9, 10 it is abundantly expressed at the plasma membranes of infected cells and is incorporated in a small amount into virions. 10,11 cm2 forms disulphide-linked dimers and tetramers, and is posttranslationally modified by n-glycosylation, palmitoylation, and phosphorylation. [10] [11] [12] analyses of a number of cm2 mutants revealed the positions of the amino acids involved in the posttranslational modifications. 10, 13 evidence was obtained that the n-glycosylation was not required for either the formation of disulfide-linked multimers or transport to the cell surface, 10 and that none of dimer-or tetramer-formation, palmitoylation or phosphorylation was essential to the transport of cm2 to the cell surface. 13 in the present study, in order to investigate the effect of cm2 palmitoylation on influenza c virus replication, we generated a cm2 palmitoylation-deficient influenza c virus, in which a cysteine at residue 65 of cm2 was mutated to alanine, and examined the viral growth and viral protein synthesis in infected cells. 293t and hmv-ii cells were maintained as described previously. 14,15 llc-mk 2 cells were maintained at 37°c in minimal essential medium with 5% foetal bovine serum and 5% calf serum. monoclonal antibodies (mabs) against the hef, np, and m1 proteins of c ⁄ ann arbor ⁄ 1 ⁄ 50 (aa ⁄ 50), and antisera against the aa ⁄ 50 virion and the cm2 protein were prepared as described previously. 3, [16] [17] [18] the seven pol i plasmids for the expression of viral rnas of aa ⁄ 50, and the nine plasmid dnas for the expression of the influenza c viral proteins were reported previously. 6, 14 plasmid dna, ppoli ⁄ cm2-acy(-), in which 995-tgt-997 of the m gene was replaced with 995-gct-997, was constructed based on ppoli ⁄ m. to generate a recombinant wild-type (rwt) virus, the above-mentioned 16 plasmids were transfected into 293t cells as described previously. 6 to rescue a mutant virus, rcm2-c65a, a recombinant influenza c virus lacking a cm2 palmitoylation site, the plasmid ppoli ⁄ cm2-acy(-), instead of ppoli ⁄ m, was transfected together with the other 15 plas-mids. at 90 hours posttransfection (p.t.), the respective culture medium of the transfected-293t cells was inoculated into the amniotic cavity of 9-day-old embryonated chicken eggs, and a stock of the recombinant virus was prepared. the infectious titres of the stocked recombinant viruses and the supernatants of recombinant-infected hmv-ii cells were determined according to the procedure reported previously. 19 radioimmunoprecipitation hmv-ii cells infected with recombinants were labeled with [ 35 s]methionine or [ 3 h]palmitic acid. cells were then disrupted and subjected to immunoprecipitation with the indicated antibodies. the immunoprecipitates obtained were then analysed by sds-page on 17ae5% gels containing 4 m urea, and processed for fluorography. 18 flotation analysis was performed according to the procedure described previously. 6 to examine whether the cm2 protein without palmitoylation is synthesized in rcm2-c65a-infected cells, hmv-ii cells infected with the recombinants were subjected to , and the lysates of the cells were immunoprecipitated with anti-cm2 serum and analysed by sds-page. as shown in figure 1 , the cm2 protein was synthesized both in the rwt-and rcm2-c65a-infected cells, but no incorporation of [ 3 h]palmitic acid into the cm2 proteins synthesized in the rcm2-c65ainfected cells was observed, indicating that cm2 in the rcm2-c65a-infected cells was not palmitoylated. the rwt or rcm2-c65a viruses were infected to hmv-ii cells at an m.o.i. of 5 and incubated at 33°c for up to 96 hours. the infectious titres (p.f.u. ⁄ ml) of rwt were approximately 5-to 10-fold higher than those of rcm2-c65a at 24-96 hours p.i. (data not shown), indicating that rwt grew more efficiently than did rcm2-c65a. thus palmitoylation of cm2 appears to have some effect on the generation of infectious virions in cultured cells. to investigate the reason(s) for the difference in growth kinetics between the two recombinants, we analysed viral proteins synthesized in the infected hmv-ii cells. pulsechase experiments of hmv-ii cells revealed no significant differences in the synthesis and maturation of the hef, np, m1, and cm2 proteins between the rwt-and rcm2-c65a-infected cells (data not shown). the infected cells pulse-labeled and chased were respectively immunoprecipitated with anti-cm2 serum in the presence of 50 mm iodoacetamide and analysed by sds-page in non-reducing condition. in both populations of infected cells, several bands corresponding to cm2a-monomer, -dimer, and -tetramer, as well as cm2b-dimer and -tetramer were detected (data not shown). these results demonstrate an absence of any significant differences between palmitoylation-deficient cm2 and authentic cm2 in terms of conformational maturation and transport in infected cells. membrane flotation analysis revealed that no significant differences in the kinetics of the hef, m1, and cm2 proteins were observed between rwt-and rcm2-c65ainfected cells (data not shown). in contrast, a slight difference in np kinetics was observed. the pulse-labeled np proteins were recovered in the bottom fractions in both rwt-and rcm2-c65a-infected cells. in the chase experiment, the amount of membrane-associated np proteins in fractions 3 and 4 was 30 % of the total np in the rwt-infected cells, which was higher than that (19%) in the rcm2-c65a-infected cells (data not shown). this finding may suggest that the affinity of the np protein, presumably representing the viral ribonucleoprotein (vrnp) complex, to the plasma membrane in the rcm2-c65ainfected cells is lower than that in rwt-infected cells, leading to the less efficient generation of infectious virions. since cm2 is structurally similar to m2, an influenza a virus membrane protein known to be involved in infectious virus production, [20] [21] [22] [23] [24] it is possible that the cytoplasmic tail of cm2 participates in the genome packaging through interaction with vrnp. in the present study, we showed that the affinity of np to the plasma membrane of rcm2-c65a-infected cells was slightly lower than that to the plasma membrane of rwt-infected cells. this observation may suggest that palmitoylation of cm2 is involved in the viral ribonucleoprotein (vrnp) incorporation, leading to efficient infectious virion generation. we hypothesize that palmitoylation contributes to proper regional structure formation in the cm2 cytoplasmic tail, which is competent to recruit vrnp efficiently into virions. alternatively, the cm2 cytoplasmic tail without palmitoylation is not likely to reach the proper conformation, resulting in reduced interaction with vrnp and less efficient generation of infectious progeny virions. the questions of if and how the m1 protein is involved in the interaction between the cm2 cytoplasmic tail and np remains to be clarified. we showed that cm2 synthesized in rcm2-c65ainfected cells was oligomerized and transported to the cell surface. this finding is consistent with the previous observation that palmitoylation is not required for the transport of cm2 to the cell surface in cm2-expressing cos-1 cells. 13 however, the use of reverse-genetics system has enabled us to conclude that the palmitoylation of cm2 is required for efficient infectious virus production. this suggests that the significance of the other posttranslational modifications of cm2 during virus replication can be clarified using recombinant viruses lacking the respective modification sites. sialic acid (sia) linked glycoproteins are the classical influenza receptors for influenza virus haemagglutinin to bind. the distribution of sia on cell surfaces is one of the determinants of host tropism, and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. previous research has shown the differences in apical versus basolateral infection and release of different influenza virus from polarized epithelial cells 1 and correlated this with sialic acid distribution in the human respiratory tract. moreover, mass spectrometric analysis was recently employed to elucidate the glycans present in the tissue in a higher resolution in human lung. 2 the objective of this study was to examine in detail the distribution of these sia-linked glycans at the cellular level by the use of confocal microscopy. human primary type i-like and type ii pneumocytes were isolated from human non-tumor lung tissue by tissue fragmentation, percoll density gradient centrifugation, and magnetic cell sorting. 1 the cells were seeded on coverslips and maintained in small airway growth medium. when confluence was reached, cell monolayers were fixed with 4% paraformaldehyde. we used the plant lectins, sambucus nigra glutinin (sna) from roche which binds to siaa2-6gal, maackia amurensis agglutinin (maa)i and maaii from vector lab, which bind the siaa2-3gal linked glycans using vector red as fluorescent chromogen. the cells were counter-stained with dapi or with fitc-conjugated antibody against endoplasmic recticulum (protein disulfideisomerase, pdi). the cells were imaged with multi-photon excitation laser scanning microscopy using zeiss 510 lsm. the optical cross-section pictures were reconstructed by zeiss lsm510 meta. we found that there was more binding of maai and ma-aii to type ii pneumocytes than type i-like pneumocytes and more overall binding of these lectins than binding of sna ( figure 1 ). in keeping with results from other polarized cells there was more binding to the apical than basolateral aspect, thus, explaining the previously published data on apical versus basolateral infection. 1 as sialic acid has been implicated in the targeting of proteins to the surface, the relative lack of sialic acid on the basolateral aspect can explain why there is little seasonal influenza virus dissemination to the systemic circulation in human infections. furthermore, though there was little binding of sna to the figure 1 . primary human type i-like and type ii pneumocytes stained with lecins (red), pdi (green), and dapi (blue) and imaged captured with confocal microscope. apical or basolateral aspects of the pneumocytes, the experimental findings of infection by influenza h3n2 virus that has a strict siaa2-6gal tropism 3 suggests that there are siaa2-6gal glycans present, which are not readily bound by the lectin sna. the in vitro model of primary human type i-like and type ii pneumocytes system formed a polarized epithelium that has a similar lectin distribution to human alveoli in vivo which demonstrated that it is a physiologically relevant model to study the tropism and pathogenesis of influenza a virus. human disease caused by highly pathogenic avian influenza (hpai) h5n1 virus is associated with fulminant viral pneumonia and mortality rates in excess of 60%. 1 cytokine dysregulation is thought to contribute to its pathogenesis. 2, 3 we previously found delayed onset of apoptosis in h5n1 infected human macrophages and, therefore, a longer survival time of the target cells for prolonged virus replication and cytokine and chemokine secretion, which may contribute to the pathogenesis of h5n1 disease in humans. 4 as bronchial and alveolar epithelial cells are target cells of influenza virus because of their proximal physiological location and interaction with macrophages, we further investigated if the differential onset of apoptosis could be found in influenza h5n1 and seasonal influenza h1n1 infected human respiratory epithelia. we dissected the apoptotic pathways triggered by influenza virus infection. seasonal influenza h1n1 virus (a ⁄ hk ⁄ 54 ⁄ 98), a low pathogenic avian influenza h9n2 lineage isolated from poultry (a ⁄ quail ⁄ hk ⁄ g1 ⁄ 97), and two virus isolates of hpai a subtype (a ⁄ hk ⁄ 483 ⁄ 97 and a ⁄ vn ⁄ 1203 ⁄ 04) were included. primary human bronchial and alveolar epithelial cells were infected with influenza viruses at moi of 2 and the cell monolayer was collected at 24, 30, and 48 hours post infection for tunel assay, and supernatant were collected for ldh assay. fragments of human lung tissues were cut into multiple 2-3 mm fragments within 2 hours of collection and infected with influenza a viruses at a titer of 10 6 tcid 50 ⁄ ml. these tissues fragments were infected for 1 hours and incubated for 48 hours at 37°c. one part of the infected tissue was fixed in formalin and processed for immunohistochemistry for influenza antigen, and the other part was homogenized and underwent rna extraction. apoptosis cdna superarray platform (sabioscience) was employed to conduct apoptosis pathway analysis. in bronchial epithelial cells, seasonal influenza h1n1 virus induced a high percentage of apoptotic cells by tunel assay at 24, 30, and 48 hours post infection with a peak of (figure 1) . a similar observation of delayed onset of apoptosis was found in influenza h5n1 and h9n2 infected alveolar epithelial cells. besides, cdna array data of ex vivo infected human lung showed that both influenza h5n1 and h1n1 virus induced trail expression compared with mock-infected tissue (approximately 15 folds) at 48 hours post infection, but influenza h3n2 virus infected lung induced significantly more trail (27 folds compared to mock infected cells), albeit with a limited viral replication ( figure 2 ). influenza h3n2 virus infected lung also elicited more tnf-alpha and fasr transcription than either h5n1 or h1n1. these observations can account for the greater apoptotic response in influenza h3n2 virus infected lung. as little impact on the expression of intrinsic pathway components was observed, it seems that the apoptotic response to influenza virus infection in lung was mainly through the extrinsic pathways. no significant changes in the expression of anti-apoptotic protein gene was found, except for a moderate induction of birc3 by influenza h3n2 virus, which may act to modulate the apoptotic response. the delayed onset of apoptosis by hpai h5n1 and low pathogenic avian influenza h9n2 virus infected respiratory epithelial cells may be a mechanism for the influenza viruses to have more prolonged replication within the human respiratory tract, and this may contribute to the pathogenesis of human disease. hemagglutination (ha) assay 10% crbc suspension was treated by 625 mu a2, 3-specific sialidase at 37°c for 15 minutes. complete elimination of a2, 3-receptor on sialidase-treated crbcs was confirmed by receptor staining and flow cytometry. ha assay of live viruses with 1% crbc or 1% sialidase-treated crbc were performed in bsl-3 facility. synthetic 3¢sln-paa-biotin(pa191), 6¢sln-paa-biotin(pa190), 6¢sln-ln-paa-biotin(pa343) was provided by the scripps research institute (tsri). as described elsewhere with some modifications, 3 generally, serial dilutions of sialyglycopolymers were coated in 96-well-flat-bottom polystryrene plates, and 32 hau live virus ⁄ well were added. alternatively, the plates were precoated with 5 lg ⁄ ml sialyglycopolymers, and then 8, 16, 32, 64, 128 hau live virus ⁄ well influenza viruses were added. rabbit antisera against a ⁄ ah ⁄ 1 ⁄ 05 diluted in pbs containing 1% bsa was added into the wells. bound antibody was detected by use of hrp-conjugated anti-rabbit igg antibody and tetramethylbenzidine substrate solution. each sample was determined in duplicates and the absorbance read at 450 nm. a total of 31 h5n1 virus strains were obtained from 2003 to 2009. the name and passage history of influenza viruses used in the study are listed in table 1 . as the same sequences of eight rna segments were detected in a ⁄ js ⁄ 1 ⁄ 07 and a ⁄ js ⁄ 2 ⁄ 07, only a ⁄ js ⁄ 2 ⁄ 07 was tested here. three amantadine-resistant variants with m2 mutation of screening of receptor-binding preference by ha assay representative results from three sets of independent experiments are shown in table 1 . complete ha with sialidasetreated crbcs, which were only with a2,6-receptors, was detected in human influenza virus (a ⁄ brisbane ⁄ 59 ⁄ 2007, h1n1) and two human h5n1 virus strains, a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08. high binding of a2,3 oligosaccharides to h5n1 viruses was detected ( figure 1a -c). and enhanced a2, 6-binding preference was also detected in a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08. the a2, 6-binding was dose dependent for sialyglycopolymers and virus titer. notably, as compared with a ⁄ gd ⁄ 1 ⁄ 06 of both short-and long-a2, 6 recognition, a ⁄ gx ⁄ 1 ⁄ 08 prefers to bind to long-a2, six oligosaccharides at low viral titer ( figure 1b,c) . however, both of them showed strong affinity to short-and long-a2, 6 oligosaccharides at high viral loads ( figure 1d ). sialoside-, galactoside-, mannoside-and sulfo-os-binding are the four types of carbohydrate-binding properties of influenza virus. 12 binding of influenza virus to the a2, 3-or a2, 6-linked sialylated glycans on cell surface is important for host range restriction, and the preference to a2, 3 of h5n1 virus limited its efficient infection in human. here, dual receptor-binding preferences were detected in a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08, which are of clade 2ae3ae4. although there is no direct evidence supporting the occurrence of human-to-human transmission in these infection events or the association between viral virulence and receptor-binding switching, viral systemic disseminations are found in the both fatal cases (data not shown). furthermore, with the introduction of clade 2ae3ae4 into the adjacent countries of china, 4 the finding of h5n1 virus with 2-6binding in human should be of concern. though h5n1 virus with human-type receptor-binding was isolated from one patient treated by oseltamivir and those viruses were with ha and ⁄ or na substitutions, 13 whether the substitutions responsible for receptor specificity switching is pre-existed or selected in human host remains unknown. our finding that three mutant viruses bearing m2 mutations of a30s, a30t, and s31n cloned from one isolate a ⁄ hb ⁄ 1 ⁄ 06 suggested it is likely that the resistant viruses emerged in the host environment. no variation was found in their ha and na sequence, and all of them show high affinity to a2-3-binding. our data suggest that the binding-specificity was not affected by the mutations on viral envelope protein m2. with the adaptation from wild aquatic birds to domestic poultry or even in human host environment, influenza virus may possess broader carbohydrate-binding spectrum or topology conformation. 11, 14 we demonstrated differential a2, 6-binding property of two human h5n1 viruses, a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08. though minor effect of short-a2, 6-binding was detected in viruses a ⁄ gx ⁄ 1 ⁄ 08 at low virus titer, both were of high affinity to long-a2, 6 glycans, even at the low titer which are rich on apical side of human upper respiratory epithelia. 11 notably, no evident binding preference switching was detected in the viruses isolated from the sporadic human infection cases at the early of 2009 in china (table 1) . however, higher affinity to the long-a2, 6 glycans was observed in bj ⁄ 1 ⁄ 09, gz ⁄ 1 ⁄ 09, and xj ⁄ 1 ⁄ 09 (data not shown). the discrepancy from the findings obtained by sialidase-treated crbc maybe associated with a limited abundance of n-linked a2-6 with long branches on crbc, as demonstrated in a recent study. 11 therefore, glycan dose-dependent binding assay is valuable and should be applied in flu surveillance. the underlying cause of the tendency is unknown, and further research on receptor-binding specificity of h5n1 viruses is required. influenza a viruses of migrating wild aquatic birds in 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virus is an oligomeric integral membrane glycoprotein structurally analogous to influenza a virus m2 and influenza b virus nb proteins characterization of a second protein (cm2) encoded by rna segment 6 of influenza c virus phosphorylation of influenza c virus cm2 protein the sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza c virus cm2 protein identification of an amino acid residue on influenza c virus m1 protein responsible for formation of the cord-like structures of the virus a human melanoma cell line highly susceptible to influenza c virus antigenic characterization of the nucleoprotein and matrix protein of influenza c virus with monoclonal antibodies construction of an antigenic map of the haemagglutinin-esterase protein of influenza c virus the synthesis of polypeptides in influenza c virus-infected cells new low-viscosity overlay medium for viral plaque assays the influenza virus m2 protein cytoplasmic tail interacts with the m1 protein and influences virus assembly at the site of virus budding the cytoplasmic tail of the influenza a virus m2 protein plays a role in viral assembly the influenza a virus m2 cytoplasmic tail is required for infectious virus production and efficient genome packaging distinct domains of the influenza a virus m2 protein cytoplasmic tail mediate binding to the m1 protein and facilitate infectious virus production influenza virus m2 ion channel protein is necessary for filamentous virion formation influenza h5n1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells das181 inhibits h5n1 influenza virus infection of human lung tissues receptor binding specificity of recent human h3n2 influenza viruses differential onset of apoptosis in avian influenza h5n1 and seasonal h1n1 virus infected human bronchial and alveolar epithelial cells: an in vitro and ex vivo study human influenza virus a ⁄ hongkong ⁄ 156 ⁄ 97 (h5n1) infection induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? proinflammatory cytokine responses induced by influenza a (h5n1) viruses in primary human alveolar and bronchial epithelial cells differential onset of apoptosis in influenza a virus h5n1-and h1n1-infected human blood macrophages avian flu: influenza virus receptors in the human airway haemagglutinin mutations responsible for the binding of h5n1 influenza a viruses to humantype receptors an avian influenza h5n1 virus that binds to a human-type receptor evolution of highly pathogenic h5n1 avian influenza viruses in vietnam between evolutionary dynamics and emergence of panzootic h5n1 influenza viruses writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h5n1) virus recent avian h5n1 viruses exhibit increased propensity for acquiring human receptor specificity a simple screening assay for receptor switching of avian influenza viruses glycan topology determines human adaptation of avian h5n1 virus hemagglutinin h5n1 chicken influenza viruses display a high binding affinity for neu5acalpha2-3galbeta1-4(6-hso3)glcnac-containing receptors a strain of human influenza a virus binds to extended but not short gangliosides as assayed by thin-layer chromatography overlay search for additional influenza virus to cell interactions avian flu: isolation of drug-resistant h5n1 virus the surface glycoproteins of h5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties this study was supported by the li ka shing foundation, the national institutes of health (niaid contract hhsn266200700005c), and the area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn266200700005c, the li ka shing foundation, and we thank all french and vietnamese field staff involved in the data collection in viet nam for their enthusiasm and support and we are grateful to the pig farmers participating in the study for their cooperation and patience. this study was a part of the gripavi project and was funded by the french ministry of foreign affairs. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn26600700005c and the area of excellence scheme of the university grants commission (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. we acknowledge the food and environmental hygiene department of hong kong for facilitating the study. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn266200700005c, the li ka shing foundation, and the area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. we gratefully acknowledge our colleagues from iiii, shantou university and skleid, hku for their excellent technical assistance. the study was supported by the rfcid commissioned study (lab#16) from research fund secretariat, food and health bureau, hong kong sar; area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06), hong kong sar; and by niaid contract (sjceirs, hhsn26620070005c), nih, usa.ferrets in all groups inoculated with a ⁄ turkey ⁄ 15 ⁄ 06 virus survived the infection and were observed once daily for 14 days. below lower limit of detection (<0ae75 log 10 eid 50 ⁄ ml).statistical cutoff of ic 50 values for nai susceptibility, determined by x 0ae75 + 3iqr. outliers with ic 50 above this cutoff and >10 times the mean ic 50 for each drug were characterized as extreme outliers; those with known drug-resistance mutations such as h275y were classified as resistant and analyzed separately. h275 wildtype, oseltamivir-susceptible isolates. h275y variants, oseltamivir-resistant virus isolates. iqr, interquartile ranges; nai, neuraminidase inhibitors. we wish to thank our collaborators in the who global influenza surveillance network and united states public health laboratories for the submission of virus isolates and clinical specimens. we also thank our colleagues from the virus reference team and the influenza sequence activity, influenza division, cdc, for their valuable technical assis-the findings and conclusions of this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention (cdc). we are indebted to yonas araya, theresa wolter, and ivan gomez-osorio for their excellent laboratory techniques and animal handling assistance. we would like to thank andrea ferrero for her laboratory managerial skills. this research was possible through funding by the cdc-hhs grant (1u01ci000355), niaid-nih grant, (r01ai052155), csrees-usda grant (1865-05523), and niaid-nih contract (hhsn266186700010c). we thank c bazzoli for advice. this work was supported by a grant from the european union fp7 project flu-modcont (no. 20160). we thank staff at seoul, incheon, daejeon, gwangju, gangwon, and jeonbuk provincial research institute of health and environments for their laboratory testing. additionally, we would like to acknowledge the contributions of participating sentinel doctors for evaluating the new rat kit. this study was supported by a grant from the korea cdc. we thank roche applied science for providing the materials and equipment for this evaluation. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn26600700005c and the area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. the authors would like express their sincere thanks to cdc, usa for supporting the routine surveillance of ili in we would like to acknowledge the australian red cross blood service (the blood service) and the australian government, which fully fund the blood service for the provision of blood products and services to the australian community. we also wish to thank the donors and staff of the blood service, who have assisted in provision of specimens for testing in this protocol, as well as the staff at the who we are grateful to liping long for her assistance in map generation. this project was supported by nih niaid 1rc1ai086830. cz is supported partially by canadian nserc postdoc fellowship. the authors thank the national investigation team based at the national institute of health (istituto superiore di sanita'), italy (in particular antonino bella, maria cristina rota, stefania salmaso) for providing their support in data collection, and the european union this study was supported in part by a grant-in-aid (21249061) and the special coordination funds for promoting science and technology of ministry of education, science, sports and culture of japan. this study was supported in part by a grant-in-aid from the ministry of education, science, and culture of japan (21249061) and the special coordination funds for promoting science and technology of mext of japan. the work described here was supported by phs grant ai-66349 (jam) and alsac. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. py, po, dw and kb are employees of glaxosmithkline. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the stud authors are thankful to path for the financial support of this research. we would like to acknowledge jessica d'amico and dr. rick bright of path for their editorial review. this study was supported by path. the authors would like to thank rick bright, jessica d'amico, and vadim tsvetnitsky for editing assistance. the we thank dr. m. enami (kanazawa university) for generously providing plasmids containing cdnas to influenza a virus m and ns genes. we also gratefully thank dr. r. sho (department of public health, yamagata university faculty of medicine) for statistical analysis. some data shown in this study have also been presented in the reference paper. 7 this work was supported in part by a grant-in-aid for scientific research from the ministry of education, culture, sports, science, and technology, japan, takeda science foundation, terumo life science foundation, and a grant-in-aid from the global coe program of the japan society for the promotion of science. we thank markus eickmann for his help in isolation and initial characterization of a ⁄ hamburg ⁄ 5 ⁄ 09 and for providing antisera against h1n1pdm. this study was supported by the european union fp6 global a(h1n1)2009 genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. in this study we analysed the genetic and antiviral drug susceptibility profiles of pandemic a(h1n1)2009 influenza virus circulating in portugal. genetic profile analysis was performed in 37 isolates to the hemagglutinin (ha), neuraminidase (na) and mp genes, and in six of these isolates the pb1, pb2, pa, np and ns genes were also analysed. antiviral drug susceptibility profile was analysed for 96 isolates, phenotypically and genotypically to neuraminidase inhibitors (nai) and genotypically to amantadine. the point mutations identified in ha, na, and mp genes of different strains do not seem to evidence an evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h1n1) 2009 virus. all analysed strains were found to be resistant to amantadine, and five of these strains exhibited a reduced susceptibility profile to nai, three only for oseltamivir and two for both inhibitors. introduction: the dynamics of pandemic influenza a ⁄ h1n1 compared to seasonal strains of influenza is not clearly understood. it is important to understand the patterns of viral shedding and symptoms over time in community-based infections.materials and methods: household infections were followed-up in two large community-based studies. patterns of viral shedding, symptoms and signs, and tympanic temperature were plotted over time and grouped according to strain for analysis.results: the patterns of viral shedding, symptoms and signs, and tympanic temperature in three influenza a strains (pandemic a ⁄ h1n1, seasonal a ⁄ h1n1, and seasonal a ⁄ h3n2) were comparable. peak viral shedding occurred close to the onset of symptoms and resolved after 6-7 days. patterns of viral shedding in influenza b virus infections differed.discussion: the patterns of viral shedding and clinical course of pandemic influenza a ⁄ h1n1 infections were broadly similar to seasonal influenza a ⁄ h1n1 and a ⁄ h3n2. only the clinical course of seasonal influenza b infections was similar to pandemic influenza a ⁄ h1n1. the dynamics of pandemic influenza a ⁄ h1n1 were observed to be largely alike to the dynamics of seasonal influenza a ⁄ h1n1 and a ⁄ h3n2. the coated respirators inactivated a broad range of influenza strains within 1 minute, including the 2009 pandemic strain and human, swine, and avian influenza viruses. antiviral effectiveness was not reduced by hot, humid conditions or repeated saturation, which might occur during prolonged use of respirators. in contrast, infectious virions were detected on the surfaces of all uncoated ffp2 respirators, and could be transferred to glove surfaces during handling of contaminated masks. growth of the viruses was monitored by ha titer using turkey red blood cells, by quantitative real time rt-pcr (qrt-pcr) to detect the influenza a matrix gene, and also by flow cytometry to detect virus positive cells using monoclonal antibodies (imagen influenza virus a and b). 8, 9 matrix gene copy number was determined using qrt-pcr and analysed using the sequence detection software on a 7500 fast system sds (applied biosystems, california, usa). further characterisation was performed through sequence analysis and the ha inhibition (hai) assay. 8 sequence analysis was performed using dnastar 8 and all sequences obtained were compared with the sequence of either the original clinical specimen if available or the conventional atcc derived mdck cell isolate. the hai assay was used to characterize the viruses against a panel of known standard reference viruses and their homologous ferret antiserum.options for the control of influenza vii abstract background: we measured the cross-reactive antibody response to pandemic h1n1 2009 in children and adults before and after vaccination with [2007] [2008] [2008] [2009] influenza season vaccines as part of the rapid public health response to the emergence of ph1n1 and to provide evidence for ph1n1 vaccination policy development in mainland china. materials and methods: archived serum specimens from previous vaccine studies were detected by hemagglutination inhibition assay. results: limited crossreactive antibody response to ph1n1 had been detected among participants of all age groups before and after they had been vaccinated with 2007-2008, 2008-2009 influenza seasonal vaccines. vaccination with seasonal influenza viruses resulted in limited seroconversion to ph1n1 in all age groups, compared with 59-94% of seroconversion to seasonal influenza viruses. but similar to recent studies, a peak of cross-reactive antibody response to ph1n1 was observed in 23% and 39% of participants born from 1915 to 1925 before and after vaccination. conclusions: in order to protect our populations in china, our study strongly suggests vaccination with ph1n1 is required in all age groups and that older populations born before 1925 may be associated with a lower infection rate of ph1n1. on april 15 and april 17, 2009, cases of ph1n1 were identified in specimens obtained from two epidemiologically unlinked patients in the united states and soon thereafter in texas and mexico. 1 since that time, the virus has spread across the globe. assessment of cross-reactive antibody response to the ph1n1 after vaccination with sea-sonal influenza vaccine was first reported from us centers of disease control and prevention (us cdc). according to their results, the seasonal influenza vaccines provided little or no protection against the ph1n1, but some degree of preexisting immunity to the virus existed, especially among adults aged ‡60 years. 2 in this study, using archived serum samples from previous vaccine studies, we measure the level of cross-reactive antibody response to ph1n1 in children and adults vaccinated intramuscularly with trivalent inactivated vaccine developed for the northern hemi serum specimens were collected and provided by provincial centers for disease control and prevention of china as a public health response to the emergence of ph1n1 exempt from human-subjects review. a total of 1308 serum samples were collected from xinjiang uygur autonomous region, yunnan, and shandong provinces. all the serum specimens were grouped by the age of subjects (0-7, 8-17, 18-59, ‡60 years) and by different influenza seasons.hemagglutination inhibition assay was performed according to standard procedures in this study. [3] [4] [5] as with h1n1 components of the vaccine, the seasonal influenza viruses used in this study were a ⁄ solomon islands ⁄ 3 ⁄ 2006 and a ⁄ brisbane ⁄ 59 ⁄ 2007. the ph1n1 influenza virus used in this study was a ⁄ california ⁄ 07 ⁄ 2009 provided by us cdc. all the viruses were propagated in specific pathogenfree embryonated chicken eggs and inactivated by 1& paraformaldehyde. the criteria 6 recommended by the european agency for the evaluation of medical product was applied for the assessment of seasonal influenza vaccine gmt, geometric mean titer; hi, hemagglutination inhibition. three weeks after boosting immunization, spleens were harvested from immunized and control mice. splenocytes were prepared by lymphocyte separation media (ez-sepô, shen zhen, china). the cells were washed and resuspended in complete rpmi-1640 containing fetal bovine serum (hyclone, logan, ut, usa), glutamax, 5 um b-me. splenocytes were cultured in vitro in the presence of inactivated h1n1, h3n2, h5n1, and h9n2 influenza virus antigen for 96 h. quick cell proliferation assay kit (biovision, san francisco, ca, usa) was used to detect the cell proliferation. the 420-480 nm absorbance was read on a plate reader. all experiments have been repeated at least three times.results are presented as mean standard error of the mean (sem). comparison of the data was performed using the student's t-test. significance was defined as a p value of <0ae05. to evaluate the adjuvant effect of recombinant igv, the anti m2e antibody subclasses was measured. igg1 and igg2a were detected after the first and second immunization ( table 1 ). the ratio of igg2a ⁄ igg1 was calculated. immunization with only m2e-hbc showed a lower igg2a ⁄ igg1 ratio <0ae5. igv combined with m2e-hbc led to a high igg2a ⁄ igg1 ratio of up to 5-15 after first and second immunization. these igg subclass distributions indicated that igv can induce a th1 immune response. to determine whether the splenocytes were stimulated in vitro with different subtypes of inactivated influenza antigen after the igv plus m2e-hbc antigen immunization, h1n1, h3n2, h5n1, h9n2 inactivated antigen was used table 1 . the serum igg, igg1, igg2a, and igg2a ⁄ igg1 ratio were measured by elisa after first and second immunization. m2e were coated on the 96 wells plate overnight, and serial dilution sera of day 7, 14, 21 after first and second immunization were added 0, 2ae5, 5, 10, 20, 40 ug ⁄ ml of igg, igg1, igg2a purified antibody were also added for obtaining the standard curve. hrp-labeled goat anti-mouse igg, igg1, or igg2a was then added, washed, and the optical density was read at 492 nm. the results were showed at mean ± sem. day after first immunization days after second immunizationoptions for the control of influenza vii the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than 5000 specimens yearly from cases of acute respiratory illness (ari), using two sampling methods: systematic randomized and non systematic ''ad hoc'' sampling. although vaccines against influenza a virus are the most effective method by which to combat infection, it is clear that their production needs to be accelerated and their efficacy improved. a panel of recombinant live attenuated human influenza a vaccines (laivs), including ns1-73, ns1-126, nsd5, were generated by rationally engineering mutations directly into the genome of a pandemic-h1n1 virus. the vaccine potential of each laiv was determined through analysis of attenuation, immunogenicity, and their ability to protect mice and ferrets. the data indicate that the novel nsd5-laiv was ideally attenuated and elicited strong protective immunity. this study also shows that attenuating mutations can be rapidly engineered into the genomes of emerging ⁄ circulating influenza a viruses in order to produce laivs. the influenza virus exhibits complicated evolutionary dynamics due to multiple reasons, such as diverse hosts, high mutation rates, and rapid replications. in this study, large-scale analyses of 11 454 influenza neuraminidase (na) sequences revealed influenza a and b na genes diverged first around 2641 years ago, and subsequently the na subtypes of influenza a emerged around 1125 years ago. all nine na subtypes of influenza a were genetically distinct from each other, with a total of 23 lineages identified. in addition, five and three sub-lineages were further identified in lineage 1a of na1 and lineage 2b of na2, respectively. the majority of lineages and sub-lineages were found to be host or geographic specific. this study provides not only a better understanding of influenza na evolution, but also a database of lineages and sub-lineages that can be used for early detection of novel genetic changes for improved influenza surveillance. although phylogenetic approaches are commonly used and often found to be powerful, how to accurately identify lineages or sub-lineages of a gene segment of the influenza a virus remains a challenging issue. in this study, we address this issue by analyzing hemagglutinin (ha) sequences using a combination of statistical and phylogenetic methods. following a hierarchical nomenclature system that uses a letter to represent a lineage and a digit for a sub-lineage, we identified 41 distinct lineages and 81 sub-lineages in all 16 ha subtypes through large-scale analyses of 11 821 influenza a hemagglutinin sequences. the majority of the lineages or sub-lineages were host or geographic specific or both. further analysis of other segments will allow us to construct a comprehensive database for influenza a lineages and genotypes, facilitating early detection of new viral strains and genome reassortments and hence improve influenza surveillance. identification of the genetic origin of influenza a viruses will facilitate understanding of the genomic dynamics, evolutionary pathway, and viral fitness of influenza a viruses. the exponential increases of influenza sequences have expanded the coverage of influenza genetic pool, thus potentially reducing the biases for influenza progenitor identification. however, these large amounts of data generate a great challenge in progenitor identification. clinical (nasopharyngeal swabs) and post-mortem materials (fragments of trachea, bronchi, lungs, spleen) were obtained from clinics and ⁄ or out-patients from st. petersburg and from 19 base virological laboratories (bvls) of the research institute of influenza in different regions of the country, which cover approximately 3 ⁄ 4 of the territory of russia. the informed consent for the bio-materials collection and studies was obtained from research subjects or from their relatives in cases of post-mortem materials. isolation of viruses was carried out in the mdck cell culture (cdc, atlanta, ga, usa) and in 10-day-old chicken embryos (e). isolation was done according the standard internationally accepted methods. 3 the reaction of hemagglutination (ha) and the inhibition of hemagglutination (hai) were performed according the who recommended standard method. 3 for the identification of epidemic isolates, we used the hyperimmune diagnostic bovine or ovine antisera annually obtained from the who reference center (cdc). for a detailed antigenic analysis we used the hyperimmune rat antisera against epidemic and reference influenza strains during the period from july 20, 2009 up to april 30, 2010, we have obtained 772 swabs from clinics and out-patients in st. petersburg and 374 swabs from the bvls. in this period, rather high incidence of lethality from pneumonia was observed, which developed on the background of the pandemic flu h1n1v. thus, we received from bvls 173 postmortem materials from 91 deceased patients which manifested pcr+ influenza h1n1v-specific rna. all materials were tested for a possibility of isolation of influenza virus h1n1v both in eggs and in mdck cells. pcr-negative materials were discarded. we isolated 229 strains of pandemic influenza from the materials collected in st. petersburg and region, which comprised 29ae7% of the total number of analyzed samples. at the same time, we did not isolate any other sub-types of influenza in the season 2009-2010 except the pandemic flu. from the swabs purchased from bvls, 47 strains were isolated, which compose 37ae9% of the pcr+ samples, and 35 strains from the post-mortem materials (43ae2% of the pcr+ samples).altogether in the season 2009-2010, we isolated, retrieved, and analyzed in hai 453 influenza strains. 91ae2% of them were pandemic strains a(h1n1)v, and only 7ae9% influenza b viruses. these data together with the epidemiologic data and the results of pcr-diagnostic provide evidence in favor of nearly mono-etiological character of epidemic season 2009-2010 in russia for pandemic influenza a(h1n1)v.though the isolation of pandemic viruses was fulfilled in two traditional model systems, in the case of pandemic 2009 virus, we could observe the tendency of preferential multiplication in embryos compared to mdck, especially in cases of post-mortem material for which chicken embryos are the preferential system of isolation.h1n1v viruses, which were isolated and passaged in mdck, even with significant ha titers, quickly lost their ha activity provided they were kept at +4°c. moreover, some other tested cell lines proved to be practically nonsensitive to the pandemic viruses h1n1v. we used hai reaction for the typing and antigenic characterization of isolated viruses. in the course of isolation of viruses in the reported period, we produced rat polyclonal antisera to the strains a ⁄ california ⁄ 07 ⁄ 09 and a ⁄ st. petersburg ⁄ 56 ⁄ 09 (h1n1)v and the antisera to the strains a ⁄ new jersey ⁄ 8 ⁄ 76 -the virus isolated during the epidemic 1976 in the united states and also of the swine origin -and to the 'swine' strains a ⁄ sw ⁄ 1976 ⁄ 31 and a ⁄ iowa ⁄ 15 ⁄ 30. the hai results of representative strains are given in figure 1 . table 1 shows that the isolated strains were homogenous in their antigenic properties and interacted with the diagnostic antiserum cdc for a(h1n1)v and also with the antisera to the strains a ⁄ california ⁄ 07 ⁄ 09 and a ⁄ st. petersburg ⁄ 56 ⁄ 09 up to 1-1 ⁄ 4 homologous titer. viruses that were isolated from post-mortem materials did not differ by their antigenic characteristics from those isolated from swabs of live patients. only two strains could be attributed to the drift-variants of the strain a ⁄ california ⁄ 07 ⁄ 09 because they reacted with the appropriate antiserum up to 1 ⁄ 8 homologous titer; these strains were a ⁄ pskov ⁄ 1 ⁄ 09 and a ⁄ belgorod ⁄ 6 ⁄ 09. it is interesting that the isolated strains reacted with the antisera to the strains a ⁄ new jersey ⁄ 8 ⁄ 76 and a ⁄ sw ⁄ 1976 ⁄ 31 to 1 ⁄ 4-1 ⁄ 8, and some particular strains even to 1 ⁄ 2 homologous titer. it is even more interesting that some pandemic isolates reacted with the antiserum to the strain iowa isolated in 1930 up to 1 ⁄ 4-1 ⁄ 8 homologous titer. despite of the fact that since the outbreak of 'swine flu' in the usa in new jersey 30 years had gone (and for the strain iowa this period is nearly 70 years) the ha of these viruses and of the pandemic influenza 2009 share some common antigenic determinants as was shown in hai.one more interesting feature of a considerable part of isolated strains is their capability to react with high titers with normal equine serum heated to 56 and to 80°c, while all the strains of swine origin isolated earlier were inhibitor-resistant ( figure 1 ). russian isolates of 2009 divided, in this respect, in two clear and approximately equal in number groups: one of them is similar to the reference strain amino acid substitutions, among them more than 30 were disclosed in antigenic sites, so the degree of similarity to this strain is 78%. a new site of glycosylation was also discovered in the position 276 of ha. 4 essential distinctions of the aminoacid sequence of ha and antigenic properties of the h1n1v strains as compared with actual circulating and vaccine strains is one of the factors that determine the pandemic potential of this new influenza virus.according to the literature, the mutation in the ha gene d222g could cause a broadening of the spectrum of receptor specificity of influenza virus by the acquisition of the capacity to bind both the residues a(2 fi 6) and a(2 fi 3) of the sialic acid of cellular receptors. 5 both types of receptors are present at the human respiratory tract, but in different parts of it, and they exist in different proportions. 6 according to the data of the european center of disease control and prevention (ecdc), the varieties g222 of the h1n1v virus were isolated in 20 countries from subjects deceased of influenza or who suffered a severe form of illness, as well as from those who sustained only a light course of influenza. 7 concerning the strains isolated in rii, this mutation was discovered in nine cases: four were isolated from live patients and five from post-mortem materials. thus, there are no convincing data at present that could prove a causal relationship of the given substitution and the aggravation of a disease course. this is in accordance with previous observations. 7 concerning the resistance of studied strains to the widely used antiviral preparations, it was shown that all tested strains possessed the substitution s31n in the m2 protein that determine the resistance to adamantanes. there was no substitution in the position 275 of neuraminidase (na), which determines the resistance to oseltamivir (h275y). these substitutions are the characteristic indices of the eurasian lineage of swine influenza viruses. 8 thus all studied russian h1n1v isolates were resistant to adamantanes (rimantadine) and sensitive to oseltamivir. respiratory clinical samples taken in 2008 and 2009 that tested positive by real time reverse transcription (rt)-pcr for seasonal influenza viruses (a and b) and pandemic h1n1 2009 respectively were assessed for other respiratory viruses using the resplex ii panel ver2.0 system distributed by qiagen. results showed that co-infections with another 16 respiratory viruses were relatively rare, with a small number of samples having another co-infecting virus present, very few samples having two other viruses detectable in their samples, and none with further viruses. this low number of co-infecting viruses and the ability of certain cell lines not to support infection with particular viruses may make primary isolation of influenza viruses in cell lines easier than might have been thought previously. cm2 is the second membrane protein of influenza c virus and is posttranslationally modified by phosphorylation, palmitoylation, n-glycosylation, and dimer ⁄ tetramer formation. in the present study, we generated rcm2-c65a, a recombinant influenza c virus lacking cm2 palmitoylation site, and examined viral growth and viral protein synthesis in the recombinant-infected cells. the rcm2-c65a virus grew less efficiently than did the wild-type virus. membrane flotation analysis of the infected cells revealed that less np was recovered in the plasma membrane fractions of the rcm2-c65a-infected cells than that in the wild-type virus-infected cells, suggesting that palmitoylation of cm2 is involved in the affinity of the ribonucleoprotein complex to the plasma membrane, leading to the efficient generation of infectious viruses. influenza c virus has seven single-stranded rna segments of negative polarity, encoding pb2, pb1, p3, haemagglutiboth the a2, 6 linkage and its topology on target cells were critical for human adaptation of influenza a viruses. the binding preference of avian flu virus h5n1 ha to the a2, 3-linked sialylated glycans is considered the major factor that limited its efficient infection in human. currently, the switch in binding-specificity of human h5n1 viruses from a2, 3 to a2, 6-glycans did naturally occur, and limited humanto-human transmission was found. to monitor their potential adaptation in the human population, receptor-binding specificity surveillance was made in china. here, the binding specificity of 32 human h5n1 virus strains isolated from 2003 to 2009 was demonstrated. dual binding preference to a2, 3 and a2, 6-glycans were found in a ⁄ guangdong ⁄ 1 ⁄ 06 and a ⁄ guangxi ⁄ 1 ⁄ 08. furthermore, both of them showed a high affinity to the long-branched a2, 6-glycans, which predominate on the upper respiratory epithelial in human. our data suggests that the existence of h5n1 virus with binding specificity to humans should be of concern.introduction via envelope glycoprotein hemagglutinin (ha), influenza viruses bind to cell-surface glycosylated oligosaccharides terminated by sialic acids (sa) where their linkage is celland species-specific. differential receptor binding preference is a host barrier for influenza virus transmission. although most h5n1 viruses have low affinity to neu5aca2, 6gal (human-type) receptor, recent findings suggested that the adaptation of h5n1 virus to human by mutations in the receptor-binding site (rbs) do indeed happen and resulted in enhanced affinity to human-type receptor. [1] [2] [3] in contrast to its putative precursor, a ⁄ gs ⁄ gd ⁄ 1 ⁄ 96, diverse genotypes were presented in currently circulating h5n1 virus, accelerating evolution and widespread occurrence. 4, 5 to date, 10 distinct phylogenetic clades (0-9) were identified based on h5n1 ha, and the confirmed human infections were caused by clade 0, 1, 2ae1, 2ae2, 2ae3, and 7. 6 in china, human h5n1 disease was mostly caused by clade2ae3ae4, which was identified in 28 isolates from 39 confirmed patients from 17 provinces since 2003. clade 7 and clade 2ae2 are responsible for the case in 2003 and 2006, respectively. two current cases of 2009 and 2010 were due to clade 2ae3ae2. now information on receptor property has been documented in some h5n1 viruses of clade 1, 2ae1, and 2ae2. [1] [2] [3] 7 little is known about h5n1 virus of clade 2ae3ae4, particularly from human.recently, a2, 3-specific sialidase-treated red blood cell (rbc) agglutination assay was developed and used for receptor specificity screening of h5n1 virus. 3, 8 the a2, 6 or a2, 3-binding preference can be distinguished by the change of hemagglutination titer reacted with rbcs and enzymatic rbcs. since fine receptor specificity existed in h5n1 viruses, 9,10 the glycan array including sulfated-, fucosylated-, linear sialosides, di-sialosides, or direct binding assay with synthetic polyacrylamide (paa)-based sialylglycopolymers was also recommended for the receptor-specificity surveillance on h5n1 viruses. furthermore, the long-branched a2, 6 sialylated glycans were currently identified to predominate on the upper respiratory epithelial in human and the recognition of this topology, 6¢sln-ln is the key determinant for the human-adaptation of influenza a virus. 11 here, we analyzed the receptor-binding specificity of human h5n1 viruses isolated in china from 2003 to 2009. since 2003, a total of 39 h5n1 infection cases were confirmed in china from 17 provinces. the pharyngeal swabs and lower airway aspirations from the patients were collected within 12 days after disease onset, maintained in viral-transport medium, and tested within 24 hours.options for the control of influenza vii